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Sample records for s100 protein expression

  1. S100A10 protein expression is associated with oxaliplatin sensitivity in human colorectal cancer cells

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    Suzuki Sayo

    2011-12-01

    Full Text Available Abstract Background Individual responses to oxaliplatin (L-OHP-based chemotherapy remain unpredictable. The objective of our study was to find candidate protein markers for tumor sensitivity to L-OHP from intracellular proteins of human colorectal cancer (CRC cell lines. We performed expression difference mapping (EDM analysis of whole cell lysates from 11 human CRC cell lines with different sensitivities to L-OHP by using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS, and identified a candidate protein by liquid chromatography/mass spectrometry ion trap time-of-flight (LCMS-IT-TOF. Results Of the qualified mass peaks obtained by EDM analysis, 41 proteins were differentially expressed in 11 human colorectal cancer cell lines. Among these proteins, the peak intensity of 11.1 kDa protein was strongly correlated with the L-OHP sensitivity (50% inhibitory concentrations (P R2 = 0.80. We identified this protein as Protein S100-A10 (S100A10 by MS/MS ion search using LCMS-IT-TOF. We verified its differential expression and the correlation between S100A10 protein expression levels in drug-untreated CRC cells and their L-OHP sensitivities by Western blot analyses. In addition, S100A10 protein expression levels were not correlated with sensitivity to 5-fluorouracil, suggesting that S100A10 is more specific to L-OHP than to 5-fluorouracil in CRC cells. S100A10 was detected in cell culture supernatant, suggesting secretion out of cells. Conclusions By proteomic approaches including SELDI technology, we have demonstrated that intracellular S100A10 protein expression levels in drug-untreated CRC cells differ according to cell lines and are significantly correlated with sensitivity of CRC cells to L-OHP exposure. Our findings provide a new clue to searching predictive markers of the response to L-OHP, suggesting that S100A10 is expected to be one of the candidate protein markers.

  2. Effect of calcium-binding protein S100A8 expression on early phase of radiation pulmonary fibrosis

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    Rao Yalan; Li Ming; Cong Yue; Li Fengsheng; Chen Xiaohua; Dong Bo; Zhang Junquan; Gao Ling; Mao Bingzhi

    2008-01-01

    The study explores the expression and effect of calcium-binding protein S100A8 on early phase of radiation pulmonary fibrosis via in vivo and in vitro experiments. In vivo experiment, the thoracic regions of rats were irradiated under 20Gy 60 Co γ-rays to establish radiation pulmonary fibrosis. After irradiation, the lung specimens of the sacrificed rats were separately harvested by the ends of the first, second, and fourth weeks respectively. The protein expression of S100A8 was tested through immunohistochemistry, the mRNA expression of S100A8 and its heterodimeric S100A9 were investigated by RT-PCR method. In vitro experiment, RT-PCR method was also applied to measure the mRNA expression of S100A8 in mouse macrophage cell line RAW264.7 after γ-rays irradiation and/or lipopolysaccharide (LPS). It shows that the protein expression of S100A8 was increased in the plasma of lung macrophages samples and the mRNA expression of S100A8 and S100A9 was also increased in the lung tissue samples in four weeks after irradiation in vivo experiment. And in vitro experiment it shows that the cooperation between γ-rays and LPS can increase the mRNA expression of S100A8 in RAW264.7. These phenomena suggest that S100A8 can exert the chemotactic activity, participate in the inflammatory response, and influence the establishment of radiation pulmonary fibrosis. (authors)

  3. Expression of calcium binding protein S100 A7 (psoriasin) in laryngeal carcinoma.

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    Tiveron, Rogério Costa; de Freitas, Luiz Carlos Conti; Figueiredo, David L; Serafini, Luciano N; Mamede, Rui Celso Martins; Zago, Marco A

    2012-01-01

    Many studies have reported increased expression of S100 A7 (psoriasin) in neoplastic lesions. Among them are studies on breast carcinoma, bladder squamous cell carcinoma, skin tumors and oral cavity squamous cell carcinoma. The expression of S100 A7 has not been described for laryngeal cancer. This study aims to identify the expression of the calcium-binding protein S100 A7 and its correlation with squamous cell carcinomas of the larynx. Specimens from 63 patients were submitted to immunohistochemistry testing with antibody S100 A7. Results were classified and compared. The group with highly differentiated tumors had the highest treatment failure scores. Moderately differentiated tumors had higher treatment failure scores than poorly differentiated tumors. Higher scores were predominantly seen on stages I and II in moderately differentiated tumors, whereas score distribution was more homogeneous in advanced stage disease (III and IV). Regarding failure in treatment, the group scoring zero (3/4 complications: 75%) differed significantly from the remaining groups (13/59: 22%). S100 A7 marker was expressed in 93.7% of laryngeal cancer cases, with higher positive correlation rates in more differentiated tumors and significantly lower rates of treatment failure. Scores had no impact on survival rates.

  4. Expression of microphthalmia transcription factor, S100 protein, and HMB-45 in malignant melanoma and pigmented nevi.

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    Xia, Jianxin; Wang, Yanlong; Li, Fuqiu; Wang, Jinfeng; Mu, Yan; Mei, Xianglin; Li, Xue; Zhu, Wenjing; Jin, Xianhua; Yu, Kai

    2016-09-01

    Malignant melanoma (MM) is a type of malignant tumor, which originates from neural crest melanocytes. MM progresses rapidly and results in a high mortality rate. The present study aims to investigate the expression of microphthalmia transcription factor (MITF), the S100 protein, and HMB-45 in MM and pigmented nevi. A total of 32 MM samples (including three skin metastasis, three lymph node metastasis and two spindle cell MM samples), two Spitz nevus samples, four pigmented nevus samples and two blue nevus samples were collected. The expression levels of S100 protein, HMB-45, and MITF were observed via immunostaining. The S100 protein exhibited high positive rates in MM and pigment disorders (96.7 and 100%, respectively), but with low specificity. The S100 protein was also expressed in fibroblasts, myoepithelial cells, histocytes and Langerhans cells in normal skin samples. HMB-45 had high specificity. Its positive expression was only confined to MM cells and junctional nevus cells. Furthermore, HMB-45 was not expressed in melanocytes in the normal tissue samples around the tumor or in the benign intradermal nevus cells. MITF exhibited high specificity and high sensitivity. It was expressed in the nuclei of melanocytes, MM cells and nevus cells. It was observed to be strongly expressed in metastatic MM and spindle cell MMs. Thus, MITF may present as a specific immunomarker for the diagnosis and differential diagnosis of MM.

  5. Expression of Slug in S100β-protein-positive cells of postnatal developing rat anterior pituitary gland.

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    Horiguchi, Kotaro; Fujiwara, Ken; Tsukada, Takehiro; Yako, Hideji; Tateno, Kozue; Hasegawa, Rumi; Takigami, Shu; Ohsako, Shunji; Yashiro, Takashi; Kato, Takako; Kato, Yukio

    2016-02-01

    Among heterogeneous S100β-protein-positive (S100β-positive) cells, star-like cells with extended cytoplasmic processes, the so-called folliculo-stellate cells, envelop hormone-producing cells or interconnect homophilically in the anterior pituitary. S100β-positive cells are known, from immunohistochemistry, to emerge from postnatal day (P) 10 and to proliferate and migrate in the parenchyma of the anterior pituitary with growth. Recent establishment of S100β-GFP transgenic rats expressing specifically green fluorescent protein (GFP) under the control of the S100β-promoter has allowed us to observe living S100β-positive cells. In the present study, we first confirmed that living S100β-positive cells in tissue cultures of S100β-GFP rat pituitary at P5 were present prior to P10 by means of confocal laser microscopy and that they proliferated and extended their cytoplasmic processes. Second, we examined the expression of the Snail-family zinc-finger transcription factors, Snail and Slug, to investigate the mechanism behind the morphological changes and the proliferation of S100β-positive cells. Interestingly, we detected Slug expression in S100β-positive cells and its increase together with development in the anterior pituitary. To analyze downstream of SLUG in S100β-positive cells, we utilized specific small interfering RNA for Slug mRNAs and observed that the expression of matrix metalloprotease (Mmp) 9, Mmp14 and chemokine Cxcl12 was down-regulated and that morphological changes and proliferation were decreased. Thus, our findings suggest that S100β-positive cells express Slug and that its expression is important for subsequent migration and proliferation.

  6. Trichohyalin-like 1 protein, a member of fused S100 proteins, is expressed in normal and pathologic human skin

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    Yamakoshi, Takako [Department of Dermatology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani, Toyama 930-0194 (Japan); Makino, Teruhiko, E-mail: tmakino@med.u-toyama.ac.jp [Department of Dermatology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani, Toyama 930-0194 (Japan); Ur Rehman, Mati; Yoshihisa, Yoko [Department of Dermatology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani, Toyama 930-0194 (Japan); Sugimori, Michiya [Department of Integrative Neuroscience, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani, Toyama 930-0194 (Japan); Shimizu, Tadamichi, E-mail: shimizut@med.u-toyama.ac.jp [Department of Dermatology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani, Toyama 930-0194 (Japan)

    2013-03-01

    Highlights: ► Trichohyalin-like 1 protein is a member of the fused-type S100 protein gene family. ► Specific antibodies against the C-terminus of the TCHHL1 protein were generated. ► TCHHL1 proteins were expressed in the basal layer of the normal epidermis. ► TCHHL1 proteins were strongly expressed in tumor nests of BCC and SCC. ► The expression of TCHHL1 proteins increased in epidermis of psoriasis vulgaris. - Abstract: Trichohyalin-like 1 (TCHHL1) protein is a novel member of the fused-type S100 protein gene family. The deduced amino acid sequence of TCHHL1 contains an EF-hand domain in the N-terminus, one trans-membrane domain and a nuclear localization signal. We generated specific antibodies against the C-terminus of the TCHHL1 protein and examined the expression of TCHHL1 proteins in normal and pathological human skin. An immunohistochemical study showed that TCHHL1 proteins were expressed in the basal layer of the normal epidermis. In addition, signals of TCHHL1 proteins were observed around the nuclei of cultured growing keratinocytes. Accordingly, TCHHL1 mRNA has been detected in normal skin and cultured growing keratinocytes. Furthermore, TCHHL1 proteins were strongly expressed in the peripheral areas of tumor nests in basal cell carcinomas and squamous cell carcinomas. A dramatic increase in the number of Ki67 positive cells was observed in TCHHL1-expressing areas. The expression of TCHHL1 proteins also increased in non-cancerous hyperproliferative epidermal tissues such as those of psoriasis vulgaris and lichen planus. These findings highlight the possibility that TCHHL1 proteins are expressed in growing keratinocytes of the epidermis and might be associated with the proliferation of keratinocytes.

  7. Trichohyalin-like 1 protein, a member of fused S100 proteins, is expressed in normal and pathologic human skin

    International Nuclear Information System (INIS)

    Yamakoshi, Takako; Makino, Teruhiko; Ur Rehman, Mati; Yoshihisa, Yoko; Sugimori, Michiya; Shimizu, Tadamichi

    2013-01-01

    Highlights: ► Trichohyalin-like 1 protein is a member of the fused-type S100 protein gene family. ► Specific antibodies against the C-terminus of the TCHHL1 protein were generated. ► TCHHL1 proteins were expressed in the basal layer of the normal epidermis. ► TCHHL1 proteins were strongly expressed in tumor nests of BCC and SCC. ► The expression of TCHHL1 proteins increased in epidermis of psoriasis vulgaris. - Abstract: Trichohyalin-like 1 (TCHHL1) protein is a novel member of the fused-type S100 protein gene family. The deduced amino acid sequence of TCHHL1 contains an EF-hand domain in the N-terminus, one trans-membrane domain and a nuclear localization signal. We generated specific antibodies against the C-terminus of the TCHHL1 protein and examined the expression of TCHHL1 proteins in normal and pathological human skin. An immunohistochemical study showed that TCHHL1 proteins were expressed in the basal layer of the normal epidermis. In addition, signals of TCHHL1 proteins were observed around the nuclei of cultured growing keratinocytes. Accordingly, TCHHL1 mRNA has been detected in normal skin and cultured growing keratinocytes. Furthermore, TCHHL1 proteins were strongly expressed in the peripheral areas of tumor nests in basal cell carcinomas and squamous cell carcinomas. A dramatic increase in the number of Ki67 positive cells was observed in TCHHL1-expressing areas. The expression of TCHHL1 proteins also increased in non-cancerous hyperproliferative epidermal tissues such as those of psoriasis vulgaris and lichen planus. These findings highlight the possibility that TCHHL1 proteins are expressed in growing keratinocytes of the epidermis and might be associated with the proliferation of keratinocytes

  8. Significance of the S100A4 protein in psoriasis

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    Zibert, John R; Skov, Lone; Thyssen, Jacob P

    2010-01-01

    the expression and significance of S100A4 in psoriasis. We found significant upregulation of S100A4 in the dermis of psoriatic skin compared with normal skin. This pattern of S100A4 expression differs considerably from that of other S100 proteins, S100A7 and S100A8/9, with predominant expression in the epidermis...... of psoriasis. Furthermore, we revealed a massive release of the biologically active forms of S100A4 from psoriatic skin. Interestingly, we found stabilization (increase) of p53 in the basal layer of epidermis in close proximity to cells expressing S100A4. To examine the possible implication of S100A4...... in the pathogenesis of psoriasis, we analyzed the effect of S100A4 blocking antibodies in a human psoriasis xenograft SCID mouse model and observed a significant reduction of the epidermal thickness and impairment in cell proliferation and dermal vascularization. In conclusion, we showed strong upregulation...

  9. Expression of human protein S100A7 (psoriasin, preparation of antibody and application to human larynx squamous cell carcinoma

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    Barbieri Manuela R

    2011-11-01

    Full Text Available Abstract Background Up-regulation of S100A7 (Psoriasin, a small calcium-binding protein, is associated with the development of several types of carcinomas, but its function and possibility to serve as a diagnostic or prognostic marker have not been fully defined. In order to prepare antibodies to the protein for immunohistochemical studies we produced the recombinant S100A7 protein in E. coli. mRNA extracted from human tracheal tumor tissue which was amplified by RT-PCR to provide the region coding for the S100A7 gene. The amplified fragment was cloned in the vector pCR2.1-TOPO and sub-cloned in the expression vector pAE. The protein rS100A7 (His-tag was expressed in E. coli BL21::DE3, purified by affinity chromatography on an Ni-NTA column, recovered in the 2.0 to 3.5 mg/mL range in culture medium, and used to produce a rabbit polyclonal antibody anti-rS100A7 protein. The profile of this polyclonal antibody was evaluated in a tissue microarray. Results The rS100A7 (His-tag protein was homogeneous by SDS-PAGE and mass spectrometry and was used to produce an anti-recombinant S100A7 (His-tag rabbit serum (polyclonal antibody anti-rS100A7. The molecular weight of rS100A7 (His-tag protein determined by linear MALDI-TOF-MS was 12,655.91 Da. The theoretical mass calculated for the nonapeptide attached to the amino terminus is 12,653.26 Da (delta 2.65 Da. Immunostaining with the polyclonal anti-rS100A7 protein generated showed reactivity with little or no background staining in head and neck squamous cell carcinoma cells, detecting S100A7 both in nucleus and cytoplasm. Lower levels of S100A7 were detected in non-neoplastic tissue. Conclusions The polyclonal anti-rS100A7 antibody generated here yielded a good signal-to-noise contrast and should be useful for immunohistochemical detection of S100A7 protein. Its potential use for other epithelial lesions besides human larynx squamous cell carcinoma and non-neoplastic larynx should be explored in future.

  10. In silico analysis and verification of S100 gene expression in gastric cancer

    International Nuclear Information System (INIS)

    Liu, Ji; Li, Xue; Dong, Guang-Long; Zhang, Hong-Wei; Chen, Dong-Li; Du, Jian-Jun; Zheng, Jian-Yong; Li, Ji-Peng; Wang, Wei-Zhong

    2008-01-01

    The S100 protein family comprises 22 members whose protein sequences encompass at least one EF-hand Ca 2+ binding motif. They were involved in the regulation of a number of cellular processes such as cell cycle progression and differentiation. However, the expression status of S100 family members in gastric cancer was not known yet. Combined with analysis of series analysis of gene expression, virtual Northern blot and microarray data, the expression levels of S100 family members in normal and malignant stomach tissues were systematically investigated. The expression of S100A3 was further evaluated by quantitative RT-PCR. At least 5 S100 genes were found to be upregulated in gastric cance by in silico analysis. Among them, four genes, including S100A2, S100A4, S100A7 and S100A10, were reported to overexpressed in gastric cancer previously. The expression of S100A3 in eighty patients of gastric cancer was further examined. The results showed that the mean expression levels of S100A3 in gastric cancer tissues were 2.5 times as high as in adjacent non-tumorous tissues. S100A3 expression was correlated with tumor differentiation and TNM (Tumor-Node-Metastasis) stage of gastric cancer, which was relatively highly expressed in poorly differentiated and advanced gastric cancer tissues (P < 0.05). To our knowledge this is the first report of systematic evaluation of S100 gene expressions in gastric cancers by multiple in silico analysis. The results indicated that overexpression of S100 gene family members were characteristics of gastric cancers and S100A3 might play important roles in differentiation and progression of gastric cancer

  11. [Analysis of the mRNA expression of the S100β protein in adipocytes of patients with diabetes mellitus, type 2].

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    Hamasaki, Mike Yoshio; Hirata, Mario Hiroyuki; Hirata, Rosario Dominguez Crespo; Himelfarb, Silvia Tchernin; Campos, Leila Maria Guissoni; Nogueira, Maria Inês

    2012-10-01

    This study aims to explore the possible relationship between the expression level of S100β protein mRNA with diabetes mellitus type 2 in adipocytes from patients with this disease in comparison with normoglycemic individuals. Samples of adipose tissue of eight patients from the coronary section of the Institute Dante Pazzanese of Cardiology (IDPC), four in Group Diabetes and four of Normoglycemic group, were evaluated by RT-PCR real time. An increase around 15 times values, between the threshold cycle (ΔCt), of mRNA expression of S100β protein in adipocytes of the diabetes group was observed in comparison to the control group (p = 0.015). Our results indicate, for the first time, that there is coexistence of increased expression of the S100β and the type 2 diabetes mellitus gene.

  12. Large-scale proteomic identification of S100 proteins in breast cancer tissues

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    Cancemi, Patrizia; Di Cara, Gianluca; Albanese, Nadia Ninfa; Costantini, Francesca; Marabeti, Maria Rita; Musso, Rosa; Lupo, Carmelo; Roz, Elena; Pucci-Minafra, Ida

    2010-01-01

    Attempts to reduce morbidity and mortality in breast cancer is based on efforts to identify novel biomarkers to support prognosis and therapeutic choices. The present study has focussed on S100 proteins as a potentially promising group of markers in cancer development and progression. One reason of interest in this family of proteins is because the majority of the S100 genes are clustered on a region of human chromosome 1q21 that is prone to genomic rearrangements. Moreover, there is increasing evidence that S100 proteins are often up-regulated in many cancers, including breast, and this is frequently associated with tumour progression. Samples of breast cancer tissues were obtained during surgical intervention, according to the bioethical recommendations, and cryo-preserved until used. Tissue extracts were submitted to proteomic preparations for 2D-IPG. Protein identification was performed by N-terminal sequencing and/or peptide mass finger printing. The majority of the detected S100 proteins were absent, or present at very low levels, in the non-tumoral tissues adjacent to the primary tumor. This finding strengthens the role of S100 proteins as putative biomarkers. The proteomic screening of 100 cryo-preserved breast cancer tissues showed that some proteins were ubiquitously expressed in almost all patients while others appeared more sporadic. Most, if not all, of the detected S100 members appeared reciprocally correlated. Finally, from the perspective of biomarkers establishment, a promising finding was the observation that patients which developed distant metastases after a three year follow-up showed a general tendency of higher S100 protein expression, compared to the disease-free group. This article reports for the first time the comparative proteomic screening of several S100 protein members among a large group of breast cancer patients. The results obtained strongly support the hypothesis that a significant deregulation of multiple S100 protein members is

  13. The correlation of serum S100β protein levels and hippocampal Seladin-1 gene expression in a rat model of sporadic Alzheimer\\\\\\'s disease

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    Soheila Hosseinzadeh

    2015-11-01

    Full Text Available Background: Seladin-1 protein protects the neural cells against amyloid beta toxicity and its expression decreased in vulnerable regions of Alzheimer's disease (AD brains. On the other hand, changes in serum levels of S100 have been considered as a marker of brain damage in neurodegenerative diseases. Furthermore, this study was carried out to determine the relation between the change profile of serum S100β protein levels and hippocampal Seladin-1 gene expression in a rat model of sporadic AD. Methods: In this experimental study that established in Department of Neuroscience, School of Advanced Technologies in Medicine, Tehran University of Medical Science, from March 2011 to April 2013, 72 animals were randomly divided into control, 4, 7, 14, and 21days ICV-STZ/Saline administrated rats. Alzheimer's model was induced by intracerebroventricular (ICV injections of streptozotocin (STZ [3 mg/kg] on days 1 and 3. Serum levels of S100β and hippocampal Seladin-1 gene expression were evalu-ated in experimental groups. The initial and step-through latencies (STL were deter-mined using passive avoidance test. Results: Serum levels of S100β were significantly different between the STZ-7 day and STZ-14 day groups in comparison with the control, saline and STZ-4 day groups. As well as, there was a significant difference between the STZ-7 day group in comparison with the STZ-14 day and STZ-21 day groups (P=0.0001. Hippocampal Seladin-1 gene expression in STZ-14 day and STZ-21 day groups significantly decreased as compared to the control, saline and STZ-4 day groups (P=0.0001. However, significant correla-tion was detected between serum S100β protein decrement and Seladin-1 down regula-tion (P=0.001. Also, the STL was significantly decreased in 21 days ICV-STZ adminis-trated rats as compared to the control or saline groups (P=0.001. Conclusion: Monitoring the changes of serum S100β protein levels by relationship with changes in hippocampal Seladin-1

  14. Hornerin, an S100 family protein, is functional in breast cells and aberrantly expressed in breast cancer

    International Nuclear Information System (INIS)

    Fleming, Jodie M; Ginsburg, Erika; Oliver, Shannon D; Goldsmith, Paul; Vonderhaar, Barbara K

    2012-01-01

    Recent evidence suggests an emerging role for S100 protein in breast cancer and tumor progression. These ubiquitous proteins are involved in numerous normal and pathological cell functions including inflammatory and immune responses, Ca 2+ homeostasis, the dynamics of cytoskeleton constituents, as well as cell proliferation, differentiation, and death. Our previous proteomic analysis demonstrated the presence of hornerin, an S100 family member, in breast tissue and extracellular matrix. Hornerin has been reported in healthy skin as well as psoriatic and regenerating skin after wound healing, suggesting a role in inflammatory/immune response or proliferation. In the present study we investigated hornerin’s potential role in normal breast cells and breast cancer. The expression levels and localization of hornerin in human breast tissue, breast tumor biopsies, primary breast cells and breast cancer cell lines, as well as murine mammary tissue were measured via immunohistochemistry, western blot analysis and PCR. Antibodies were developed against the N- and C-terminus of the protein for detection of proteolytic fragments and their specific subcellular localization via fluorescent immunocytochemisty. Lastly, cells were treated with H 2 O 2 to detect changes in hornerin expression during induction of apoptosis/necrosis. Breast epithelial cells and stromal fibroblasts and macrophages express hornerin and show unique regulation of expression during distinct phases of mammary development. Furthermore, hornerin expression is decreased in invasive ductal carcinomas compared to invasive lobular carcinomas and less aggressive breast carcinoma phenotypes, and cellular expression of hornerin is altered during induction of apoptosis. Finally, we demonstrate the presence of post-translational fragments that display differential subcellular localization. Our data opens new possibilities for hornerin and its proteolytic fragments in the control of mammary cell function and breast

  15. Hornerin, an S100 family protein, is functional in breast cells and aberrantly expressed in breast cancer

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    Fleming Jodie M

    2012-06-01

    Full Text Available Abstract Background Recent evidence suggests an emerging role for S100 protein in breast cancer and tumor progression. These ubiquitous proteins are involved in numerous normal and pathological cell functions including inflammatory and immune responses, Ca2+ homeostasis, the dynamics of cytoskeleton constituents, as well as cell proliferation, differentiation, and death. Our previous proteomic analysis demonstrated the presence of hornerin, an S100 family member, in breast tissue and extracellular matrix. Hornerin has been reported in healthy skin as well as psoriatic and regenerating skin after wound healing, suggesting a role in inflammatory/immune response or proliferation. In the present study we investigated hornerin’s potential role in normal breast cells and breast cancer. Methods The expression levels and localization of hornerin in human breast tissue, breast tumor biopsies, primary breast cells and breast cancer cell lines, as well as murine mammary tissue were measured via immunohistochemistry, western blot analysis and PCR. Antibodies were developed against the N- and C-terminus of the protein for detection of proteolytic fragments and their specific subcellular localization via fluorescent immunocytochemisty. Lastly, cells were treated with H2O2 to detect changes in hornerin expression during induction of apoptosis/necrosis. Results Breast epithelial cells and stromal fibroblasts and macrophages express hornerin and show unique regulation of expression during distinct phases of mammary development. Furthermore, hornerin expression is decreased in invasive ductal carcinomas compared to invasive lobular carcinomas and less aggressive breast carcinoma phenotypes, and cellular expression of hornerin is altered during induction of apoptosis. Finally, we demonstrate the presence of post-translational fragments that display differential subcellular localization. Conclusions Our data opens new possibilities for hornerin and its

  16. Protein S100B and physical exercise

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    Álvaro Reischak Oliveira

    2010-01-01

    Full Text Available Protein S100B has been used as a peripheral biochemical marker of brain injury and/or activity. However, recent studies have demonstrated that this protein is also increased in serum after physical exercise, although the interpretation of this finding remains controversial. Although predominantly released by astrocytes in the central nervous system, extracerebral sources of protein S100B have been suggested to contribute to the increase in serum levels of this protein. However, in the case of exercises that have an impact on the brain such as boxing, elevated levels are clearly associated with brain damage. More recently, some studies have proposed that protein S100B might be released by activated adipocytes and by damaged muscle cells. If confirmed experimentally, protein S100B might be potentially useful in sports training. We are currently investigating the potential role of serum protein S100B as an indicator of muscle damage. Therefore, the objective of this review was to discuss the current knowledge about the relationship between physical exercise and serum protein S100B and its possible leakage from muscle cells injured by exercise.

  17. Andrographolide protects mouse astrocytes against hypoxia injury by promoting autophagy and S100B expression

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    Juan Du

    2018-04-01

    Full Text Available Andrographolide (ANDRO has been studied for its immunomodulation, anti-inflammatory, and neuroprotection effects. Because brain hypoxia is the most common factor of secondary brain injury after traumatic brain injury, we studied the role and possible mechanism of ANDRO in this process using hypoxia-injured astrocytes. Mouse cortical astrocytes C8-D1A (astrocyte type I clone from C57/BL6 strains were subjected to 3 and 21% of O2 for various times (0–12 h to establish an astrocyte hypoxia injury model in vitro. After hypoxia and ANDRO administration, the changes in cell viability and apoptosis were assessed using CCK-8 and flow cytometry. Expression changes in apoptosis-related proteins, autophagy-related proteins, main factors of JNK pathway, ATG5, and S100B were determined by western blot. Hypoxia remarkably damaged C8-D1A cells evidenced by reduction of cell viability and induction of apoptosis. Hypoxia also induced autophagy and overproduction of S100B. ANDRO reduced cell apoptosis and promoted cell autophagy and S100B expression. After ANDRO administration, autophagy-related proteins, S-100B, JNK pathway proteins, and ATG5 were all upregulated, while autophagy-related proteins and s100b were downregulated when the jnk pathway was inhibited or ATG5 was knocked down. ANDRO conferred a survival advantage to hypoxia-injured astrocytes by reducing cell apoptosis and promoting autophagy and s100b expression. Furthermore, the promotion of autophagy and s100b expression by ANDRO was via activation of jnk pathway and regulation of ATG5.

  18. Immunoreactivity of S100β protein in the hippocampus of chinchilla

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    Krawczyk Aleksandra

    2014-03-01

    Full Text Available The aim of the study was to investigate S100β protein in astrocytes of CA1 and CA3 areas of the hippocampus proper and the dentate gyrus with the hilus yet undefined in mature males of chinchilla. The presence of S100β was determined using indirect immunohistochemical peroxidase-antiperoxidase method with specific monoclonal antibody against this protein. Most of the S100β-positive cells were detected in the subgranular zone of the dentate gyrus and in the middle part of the hilus. In CA3 area, it was found that the most numerous cells with S100β are in stratum radiatum. In CA1 area, there were single astrocytes expressing this protein. This data demonstrates species differences and a large quantity of S100β immunoreactive cells in the subgranular zone of the dentate gyrus of chinchilla, which may be associated with structural reorganisation of the hippocampus and with neurogenesis, learning, and memorising process dependent on the hippocampus.

  19. Molecular mechanisms of Ca(2+) signaling in neurons induced by the S100A4 protein

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    Kiryushko, Darya; Novitskaya, Vera; Soroka, Vladislav

    2006-01-01

    The S100A4 protein belongs to the S100 family of vertebrate-specific proteins possessing both intra- and extracellular functions. In the nervous system, high levels of S100A4 expression are observed at sites of neurogenesis and lesions, suggesting a role of the protein in neuronal plasticity. Ext...... at the cell surface. Thus, glycosaminoglycans may act as coreceptors of S100 proteins in neurons. This may provide a mechanism by which S100 proteins could locally regulate neuronal plasticity in connection with brain lesions and neurological disorders....

  20. S100B proteins in febrile seizures

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    Mikkonen, Kirsi; Pekkala, Niina; Pokka, Tytti

    2011-01-01

    S100B protein concentrations correlate with the severity and outcome of brain damage after brain injuries, and have been shown to be markers of blood-brain barrier damage. In children elevated S100B values are seen as a marker of damage to astrocytes even after mild head injuries. S100B proteins...... may also give an indication of an ongoing pathological process in the brain with respect to febrile seizures (FS) and the likelihood of their recurrence. To evaluate this, we measured S100B protein concentrations in serum and cerebrospinal fluid from 103 children after their first FS. 33 children...

  1. Immunohistochemical localization of anterior pituitary hormones in S-100 protein-positive cells in the rat pituitary gland.

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    Kikuchi, Motoshi; Yatabe, Megumi; Tando, Yukiko; Yashiro, Takashi

    2011-09-01

    In the anterior and intermediate lobes of the rat pituitary gland, non-hormone-producing cells that express S-100 protein coexist with various types of hormone-producing cells and are believed to function as phagocytes, supporting and paracrine-controlling cells of hormone-producing cells and stem cells, among other functions; however, their cytological characteristics are not yet fully understood. Using a transgenic rat that expresses green fluorescent protein under the promoter of the S100β protein gene, we immunohistochemically detected expression of the luteinizing hormone, thyroid-stimulating hormone, prolactin, growth hormone and proopiomelanocortin by S-100 protein-positive cells located between clusters of hormone-producing cells in the intermediate lobe. These findings lend support to the hypothesis that S-100 protein-positive cells are capable of differentiating into hormone-producing cells in the adult rat pituitary gland.

  2. Expression of S100B during the innate immune of corneal epithelium against fungi invasion

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    Jie Zhang

    2016-02-01

    Full Text Available AIM: To explore the expression of S100B in corneal epithelial cells under Aspergillus stimulation both in vivo and in vitro. METHODS: Immortalized human corneal epithelial cells (HCECs were exposed to inactive Aspergillus fumigatus (A. fumigatus conidia at 0, 4, 8, 12, 16, and 24h respectively. The corneas of Wistar rats were exposed to active A. fumigatus at 0, 12, 24, 48h and the normal rat corneas were used for normal control. The mRNA level of S100B was evaluated by real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR. S100B protein expression in cornea epithelium was detected by immunohistochemical/immunocytochemical staining (IHC/ICC. RESULTS: Histopathology revealed a significant inflammatory cell infiltration in fungal keratitis human and rat cornea. Corneal epithelial cells didn’t express or rarely express S100B at baseline. A. fumigatus significantly induced S100B mRNA expression in cultured corneal epithelial cells in a time depended manner in vitro, the mRNA began to rise significantly at 8h in vitro (P<0.05 and continue to rise as time prolonged (P<0.01. In vivo, S100B mRNA level was low in the normal corneas. However, it was increased in keratitis corneas from 12h after infection (P<0.05 and reached to a peak at 24h (P<0.001. Immunochemistry revealed an obvious staining in fungal keratitis corneas as well as immortalized HCECs compared to the normal ones respectively, indicating an increased expression of S100B protein. CONCLUSION: S100B exists in corneal epithelial cells and is over-expressed under A. fumigatus stimulation. S100B may play an important role in the innate immune response of the corneal epithelium during A. fumigatus infection.

  3. Expression of S100B during the innate immune of corneal epithelium against fungi invasion

    Science.gov (United States)

    Zhang, Jie; Zhao, Gui-Qiu; Qu, Jing; Che, Cheng-Ye; Lin, Jing; Jiang, Nan; Zhao, Han; Wang, Xue-Jun

    2016-01-01

    AIM To explore the expression of S100B in corneal epithelial cells under Aspergillus stimulation both in vivo and in vitro. METHODS Immortalized human corneal epithelial cells (HCECs) were exposed to inactive Aspergillus fumigatus (A. fumigatus) conidia at 0, 4, 8, 12, 16, and 24h respectively. The corneas of Wistar rats were exposed to active A. fumigatus at 0, 12, 24, 48h and the normal rat corneas were used for normal control. The mRNA level of S100B was evaluated by real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). S100B protein expression in cornea epithelium was detected by immunohistochemical/immunocytochemical staining (IHC/ICC). RESULTS Histopathology revealed a significant inflammatory cell infiltration in fungal keratitis human and rat cornea. Corneal epithelial cells didn't express or rarely express S100B at baseline. A. fumigatus significantly induced S100B mRNA expression in cultured corneal epithelial cells in a time depended manner in vitro, the mRNA began to rise significantly at 8h in vitro (P<0.05) and continue to rise as time prolonged (P<0.01). In vivo, S100B mRNA level was low in the normal corneas. However, it was increased in keratitis corneas from 12h after infection (P<0.05) and reached to a peak at 24h (P<0.001). Immunochemistry revealed an obvious staining in fungal keratitis corneas as well as immortalized HCECs compared to the normal ones respectively, indicating an increased expression of S100B protein. CONCLUSION S100B exists in corneal epithelial cells and is over-expressed under A. fumigatus stimulation. S100B may play an important role in the innate immune response of the corneal epithelium during A. fumigatus infection. PMID:26949634

  4. Structural and functional diversification in the teleost S100 family of calcium-binding proteins

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    Korsching Sigrun I

    2008-02-01

    Full Text Available Abstract Background Among the EF-Hand calcium-binding proteins the subgroup of S100 proteins constitute a large family with numerous and diverse functions in calcium-mediated signaling. The evolutionary origin of this family is still uncertain and most studies have examined mammalian family members. Results We have performed an extensive search in several teleost genomes to establish the s100 gene family in fish. We report that the teleost S100 repertoire comprises fourteen different subfamilies which show remarkable similarity across six divergent teleost species. Individual species feature distinctive subsets of thirteen to fourteen genes that result from local gene duplications and gene losses. Eight of the fourteen S100 subfamilies are unique for teleosts, while six are shared with mammalian species and three of those even with cartilaginous fish. Several S100 family members are found in jawless fish already, but none of them are clear orthologs of cartilaginous or bony fish s100 genes. All teleost s100 genes show the expected structural features and are subject to strong negative selection. Many aspects of the genomic arrangement and location of mammalian s100 genes are retained in the teleost s100 gene family, including a completely conserved intron/exon border between the two EF hands. Zebrafish s100 genes exhibit highly specific and characteristic expression patterns, showing both redundancy and divergence in their cellular expression. In larval tissue expression is often restricted to specific cell types like keratinocytes, hair cells, ionocytes and olfactory receptor neurons as demonstrated by in situ hybridization. Conclusion The origin of the S100 family predates at least the segregation of jawed from jawless fish and some extant family members predate the divergence of bony from cartilaginous fish. Despite a complex pattern of gene gains and losses the total repertoire size is remarkably constant between species. On the expression

  5. Expression of S100 protein and protective effect of arundic acid on the rat brain in chronic cerebral hypoperfusion.

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    Ohtani, Ryo; Tomimoto, Hidekazu; Wakita, Hideaki; Kitaguchi, Hiroshi; Nakaji, Kayoko; Takahashi, Ryosuke

    2007-03-02

    S100 protein is expressed primarily by astroglia in the brain, and accumulates in and around the ischemic lesions. Arundic acid, a novel astroglia-modulating agent, is neuroprotective in acute cerebral infarction, whereas the protective effects remain unknown during chronic cerebral hypoperfusion. Rats undergoing chronic cerebral hypoperfusion were subjected to a bilateral ligation of the common carotid arteries, and were allowed to survive for 3, 7 and 14 days. The animals received a daily intraperitoneal injection of 5.0, 10.0 or 20.0 mg/kg of arundic acid, or vehicle, for 14 days. Alternatively, other groups of rats received a delayed intraperitoneal injection of 20.0 mg/kg of arundic acid or vehicle, which started from 1, 3 or 7 days after ligation and continued to 14 days. The degree of white matter (WM) lesions and the numerical density of S100 protein-immunoreactive astroglia were estimated. In the WM of rats with vehicle injections, the number of S100 protein-immunoreactive astroglia increased significantly after chronic cerebral hypoperfusion as compared to the sham-operation. A dosage of 10.0 and 20.0 mg/kg of arundic acid suppressed the numerical increase in S100 protein-immunoreactive astroglia and the WM lesions. These pathological changes were suppressed with delayed treatment up to 7 days in terms of astroglial activation, and up to 3 days in terms of the WM lesions. The protective effects of arundic acid against WM lesions were demonstrated in a dose-dependent manner, and even after postischemic treatments. These results suggest the potential usefulness of arundic acid in the treatment of cerebrovascular WM lesions.

  6. The Effect of Erythropoietin on S100 Protein Expression in Cochlea After Acoustic Overstimulation: An Experimental Study

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    Gulsen Gurgen

    2014-03-01

    Full Text Available Aim: To investigate the effect of Erythropoietin on acoustically overstimulated rat spiral ganglion neurons (SGNs using S100 protein immunostaining.Material and Method: Twenty-two Wistar albino rats were divided into three groups: healthy control group (n=7, Saline solution (n=7 and Erythropoietin injection groups (n=8. Saline solution and Erythropoietin injection groups received white noise (100 dB SPL for 3 hours. Cochlear sections were stained by silver staining technique and immunostained by S100 antibody. Results: Histochemical analysis of silver staining sections revealed normal structure and a weak staining in SGNs of healthy control group. However, dark-black cytoplasmic staining, cellular shrinkage and degeneration were detected in saline injection group. On the other hand, a few weakly stained neurons were observed in erythropoietin injection group. S100 staining demonstrated strong reaction in Schwann cells and myelin sheaths of SGNs in healthy control group (p<0.05. In saline solution injection group, Schwann cells showed moderate S100 reaction and other regions of SGNs showed weak reaction (p<0.05. In erythropoietin injection group, strong S100 expression almost similar to the healthy control group was determined, although there was an occasional decrease. Discussion: Erythropoetin may prevent noise induced SGN degeneration via protecting the Schwann cells in rat cochlea.

  7. Expressão da proteína ligadora de cálcio S100 A7 (psoriasina no carcinoma laríngeo Expression of calcium binding protein S100 A7 (psoriasin in laryngeal carcinoma

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    Rogério Costa Tiveron

    2012-08-01

    Full Text Available Muitos estudos relatam o aumento da expressão de S100 A7 (psoriasina em lesões neoplásicas. Destacam-se trabalhos em carcinoma da mama, espinocelular da bexiga, pele e cavidade oral. Não foi demonstrada expressão da S100 A7 em câncer de laringe. OBJETIVO: Identificar a expressão da proteína ligadora de cálcio S100 A7 e sua correlação com carcinomas espinocelular da laringe. MATERIAL E MÉTODOS: Amostras de tecido neoplásico de 63 pacientes foram submetidos à imunohis toquímica com o anticorpo S110 A7. Os resultados foram classificados e comparados. RESULTADOS: O grupo bem diferenciado teve a maior pontuação de falha no tratamento. O grupo moderadamente diferenciado apresentou escores mais elevados do que o grupo pouco diferenciado. Pontuações mais altas predominaram nos estágios I e II no grupo moderadamente diferenciado, enquanto a distribuição do escore foi mais homogênea em estados avançados (III e IV. Em relação às falhas no tratamento, o grupo pontuação zero (04/03 complicações: 75% diferiu significativamente da pontuação restante (13/59: 22%. CONCLUSÕES: A S100 A7 foi expressa em 93,7% dos casos de câncer de laringe, com maior positividade nos tumores mais diferenciados e taxa significativamente menor de falha no tratamento. A pontuação obtida não teve impacto sobre a sobrevivência.Many studies have reported increased expression of S100 A7 (psoriasin in neoplastic lesions. Among them are studies on breast carcinoma, bladder squamous cell carcinoma, skin tumors and oral cavity squamous cell carcinoma. The expression of S100 A7 has not been described for laryngeal cancer. OBJECTIVE: This study aims to identify the expression of the calcium-binding protein S100 A7 and its correlation with squamous cell carcinomas of the larynx. MATERIAL AND METHODS: Specimens from 63 patients were submitted to immunohistochemistry testing with antibody S100 A7. Results were classified and compared. RESULTS: The group with

  8. Expression of S100A4, ephrin-A1 and osteopontin in non-small cell lung cancer

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    Rud, Ane Kongsgaard; Lund-Iversen, Marius; Berge, Gisle; Brustugun, Odd Terje; Solberg, Steinar K; Mælandsmo, Gunhild M; Boye, Kjetil

    2012-01-01

    The metastasis-promoting protein S100A4 induces expression of ephrin-A1 and osteopontin in osteosarcoma cell lines. The aim of this study was to investigate S100A4-mediated stimulation of ephrin-A1 and osteopontin in non-small cell lung cancer (NSCLC) cell lines, and to characterize the expression of these biomarkers in primary tumor tissue from NSCLC patients. Four NSCLC cell lines were treated with extracellular S100A4, and ephrin-A1 and osteopontin expression was analyzed by real time RT-PCR and Western blotting. Immunohistochemical staining for S100A4, ephrin-A1 and osteopontin was performed on tissue microarrays containing primary tumor samples from a cohort of 217 prospectively recruited NSCLC patients, and associations with clinicopathological parameters were investigated. S100A4 induced ephrin-A1 mRNA and protein expression in adenocarcinoma, but not in squamous carcinoma cell lines, whereas the level of osteopontin was unaffected by S100A4 treatment. In primary tumors, moderate or strong immunoreactivity was observed in 57% of cases for cytoplasmic S100A4, 46% for nuclear S100A4, 86% for ephrin-A1 and 77% for osteopontin. Interestingly, S100A4 expression was associated with ephrin-A1 also in vivo, but there was no association between S100A4 and osteopontin. Expression levels of S100A4 and ephrin-A1 were significantly higher in adenocarcinomas compared to other histological subtypes, and S100A4-positive tumors were smaller and more differentiated than tumors without expression. Our findings suggest that S100A4, ephrin-A1 and osteopontin are involved in the biology of NSCLC, and further investigation of their potential use as biomarkers in NSCLC is warranted

  9. S100 Proteins As an Important Regulator of Macrophage Inflammation

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    Chang Xia

    2018-01-01

    Full Text Available The S100 proteins, a family of calcium-binding cytosolic proteins, have a broad range of intracellular and extracellular functions through regulating calcium balance, cell apoptosis, migration, proliferation, differentiation, energy metabolism, and inflammation. The intracellular functions of S100 proteins involve interaction with intracellular receptors, membrane protein recruitment/transportation, transcriptional regulation and integrating with enzymes or nucleic acids, and DNA repair. The S100 proteins could also be released from the cytoplasm, induced by tissue/cell damage and cellular stress. The extracellular S100 proteins, serving as a danger signal, are crucial in regulating immune homeostasis, post-traumatic injury, and inflammation. Extracellular S100 proteins are also considered biomarkers for some specific diseases. In this review, we will discuss the multi-functional roles of S100 proteins, especially their potential roles associated with cell migration, differentiation, tissue repair, and inflammation.

  10. Impact of S100A4 Expression on Clinicopathological Characteristics and Prognosis in Pancreatic Cancer: A Meta-Analysis

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    Shanshan Huang

    2016-01-01

    Full Text Available Background. The small Ca2+-binding protein S100A4 is identified as a metastasis-associated or metastasis-inducing protein in various types of cancer. The goal of this meta-analysis was to evaluate the relationship between S100A4 expression and clinicopathological characteristics and prognosis of patients with pancreatic cancer. Methods. A comprehensive literature search was carried out in the electronic databases PubMed and Chinese CNKI. Only the studies reporting the correlation between S100A4 expression and clinicopathological characteristics or overall survival (OS of patients with pancreatic cancer are enrolled. Extracted data was analyzed using the RevMan 5.3 software to calculate the pooled relative risks (95% confidence interval, CI for statistical analyses. Results. Seven studies including a total of 474 patients were enrolled into this meta-analysis. Negative expression of S100A4 was significantly associated with higher 3-year OS rate (RR = 3.92, 95% CI = 2.24–6.87, P<0.0001, compared to S100A4-positive cases. Moreover, negative expression of S100A4 was also related to N0 stage for lymph node metastasis (RR = 2.15, 95% CI = 1.60–2.88, P<0.0001. However, S100A4 expression was not significantly correlated with histological types and distant metastasis status. Conclusion. S100A4 expression represents a potential marker for lymph node metastasis of pancreatic cancer and a potential unfavorable factor for prognosis of patients with this disease.

  11. Overexpression of the S100A2 protein as a prognostic marker for patients with stage II and III colorectal cancer

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    MASUDA, TAIKI; ISHIKAWA, TOSHIAKI; MOGUSHI, KAORU; OKAZAKI, SATOSHI; ISHIGURO, MEGUMI; IIDA, SATORU; MIZUSHIMA, HIROSHI; TANAKA, HIROSHI; UETAKE, HIROYUKI; SUGIHARA, KENICHI

    2016-01-01

    We aimed to identify a novel prognostic biomarker related to recurrence in stage II and III colorectal cancer (CRC) patients. Stage II and III CRC tissue mRNA expression was profiled using an Affymetrix Gene Chip, and copy number profiles of 125 patients were generated using an Affymetrix 250K Sty array. Genes showing both upregulated expression and copy number gains in cases involving recurrence were extracted as candidate biomarkers. The protein expression of the candidate gene was assessed using immunohistochemical staining of tissue from 161 patients. The relationship between protein expression and clinicopathological features was also examined. We identified 9 candidate genes related to recurrence of stage II and III CRC, whose mRNA expression was significantly higher in CRC than in normal tissue. Of these proteins, the S100 calcium-binding protein A2 (S100A2) has been observed in several human cancers. S100A2 protein overexpression in CRC cells was associated with significantly worse overall survival and relapse-free survival, indicating that S100A2 is an independent risk factor for stage II and III CRC recurrence. S100A2 overexpression in cancer cells could be a biomarker of poor prognosis in stage II and III CRC recurrence and a target for treatment of this disease. PMID:26783118

  12. Interleukin-6 induces S100A9 expression in colonic epithelial cells through STAT3 activation in experimental ulcerative colitis.

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    Min Jeoung Lee

    Full Text Available BACKGROUND: Intestinal epithelium is essential for maintaining normal intestinal homeostasis; its breakdown leads to chronic inflammatory pathologies, such as inflammatory bowel diseases (IBDs. Although high concentrations of S100A9 protein and interleukin-6 (IL-6 are found in patients with IBD, the expression mechanism of S100A9 in colonic epithelial cells (CECs remains elusive. We investigated the role of IL-6 in S100A9 expression in CECs using a colitis model. METHODS: IL-6 and S100A9 expression, signal transducer and activator of transcription 3 (STAT3 phosphorylation, and infiltration of immune cells were analyzed in mice with dextran sulfate sodium (DSS-induced colitis. The effects of soluble gp130-Fc protein (sgp130Fc and S100A9 small interfering (si RNA (si-S100A9 on DSS-induced colitis were evaluated. The molecular mechanism of S100A9 expression was investigated in an IL-6-treated Caco-2 cell line using chromatin immunoprecipitation assays. RESULTS: IL-6 concentrations increased significantly in the colon tissues of DSS-treated mice. sgp130Fc or si-S100A9 administration to DSS-treated mice reduced granulocyte infiltration in CECs and induced the down-regulation of S100A9 and colitis disease activity. Treatment with STAT3 inhibitors upon IL-6 stimulation in the Caco-2 cell line demonstrated that IL-6 mediated S100A9 expression through STAT3 activation. Moreover, we found that phospho-STAT3 binds directly to the S100A9 promoter. S100A9 may recruit immune cells into inflamed colon tissues. CONCLUSIONS: Elevated S100A9 expression in CECs mediated by an IL-6/STAT3 signaling cascade may play an important role in the development of colitis.

  13. Role of S100A12 in the pathogenesis of osteoarthritis

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    Nakashima, Motoshige [Department of Orthopaedic Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan); Sakai, Tadahiro, E-mail: tadsakai@med.nagoya-u.ac.jp [Department of Orthopaedic Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan); Hiraiwa, Hideki; Hamada, Takashi; Omachi, Takaaki [Department of Orthopaedic Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan); Ono, Yohei [Department of Orthopaedic Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan); Department of Anatomy and Cell Biology, Brody School of Medicine, East Carolina University, 600 Moye Blvd., Greenville, NC 27834 (United States); Inukai, Norio [Department of Orthopaedic Surgery, Nishio Municipal Hospital, 6 Kumami-cho, Nishio 445-8510 (Japan); Ishizuka, Shinya; Matsukawa, Tetsuya; Oda, Tomoyuki; Takamatsu, Akira; Yamashita, Satoshi; Ishiguro, Naoki [Department of Orthopaedic Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan)

    2012-06-08

    Highlights: Black-Right-Pointing-Pointer This is the first report of S100A12 expression in human OA articular cartilages. Black-Right-Pointing-Pointer Exogenous S100A12 increased the production of MMP13 and VEGF in OA chondrocytes. Black-Right-Pointing-Pointer Soluble RAGE suppressed the increased production of MMP13 and VEGF. Black-Right-Pointing-Pointer p38MAPK and NF-{kappa}B inhibitors abrogated S100A12-induced MMP13 and VEGF production. Black-Right-Pointing-Pointer S100A12 may contribute to OA progression by increasing MMP13 and VEGF production. -- Abstract: S100A12 is a member of the S100 protein family, which are intracellular calcium-binding proteins. Although there are many reports on the involvement of S100A12 in inflammatory diseases, its presence in osteoarthritic cartilage has not been reported. The purpose of this study was to investigate the expression of S100A12 in human articular cartilage in osteoarthritis (OA) and to evaluate the role of S100A12 in human OA chondrocytes. We analyzed S100A12 expression by immunohistochemical staining of cartilage samples obtained from OA and non-OA patients. In addition, chondrocytes were isolated from knee cartilage of OA patients and treated with recombinant human S100A12. Real-time RT-PCR was performed to analyze mRNA expression. Protein production of matrix metalloproteinase 13 (MMP-13) and vascular endothelial growth factor (VEGF) in the culture medium were measured by ELISA. Immunohistochemical analyses revealed that S100A12 expression was markedly increased in OA cartilages. Protein production and mRNA expression of MMP-13 and VEGF in cultured OA chondrocytes were significantly increased by treatment with exogenous S100A12. These increases in mRNA expression and protein production were suppressed by administration of soluble receptor for advanced glycation end products (RAGE). Both p38 mitogen-activated protein kinase (MAPK) and nuclear factor-{kappa}B (NF-{kappa}B) inhibitors also suppressed the increases

  14. Expression of chemokine CXCL10 in dendritic-cell-like S100β-positive cells in rat anterior pituitary gland.

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    Horiguchi, Kotaro; Fujiwara, Ken; Higuchi, Masashi; Yoshida, Saishu; Tsukada, Takehiro; Ueharu, Hiroki; Chen, Mo; Hasegawa, Rumi; Takigami, Shu; Ohsako, Shunji; Yashiro, Takashi; Kato, Takako; Kato, Yukio

    2014-09-01

    Chemokines are mostly small secreted polypeptides whose signals are mediated by seven trans-membrane G-protein-coupled receptors. Their functions include the control of leukocytes and the intercellular mediation of cell migration, proliferation, and adhesion in several tissues. We have previously revealed that the CXC chemokine ligand 12 (CXCL12) and its receptor 4 (CXCR4) are expressed in the anterior pituitary gland, and that the CXCL12/CXCR4 axis evokes the migration and interconnection of S100β-protein-positive cells (S100β-positive cells), which do not produce classical anterior pituitary hormones. However, little is known of the cells producing the other CXCLs and CXCRs or of their characteristics in the anterior pituitary. We therefore examined whether CXCLs and CXCRs occurred in the rat anterior pituitary lobe. We used reverse transcription plus the polymerase chain reaction to analyze the expression of Cxcl and Cxcr and identified the cells that expressed Cxcl by in situ hybridization. Transcripts of Cxcl10 and its receptor (Cxcr3 and toll-like receptor 4, Tlr4) were clearly detected: cells expressing Cxcl10 and Tlr4 were identified amongst S100β-positive cells and those expressing Cxcr3 amongst adrenocorticotropic hormone (ACTH)-producing cells. We also investigated Cxcl10 expression in subpopulations of S100β-positive cells. We separated cultured S100β-positive cells into the round-type (dendritic-cell-like) and process-type (astrocyte- or epithelial-cell-like) by their adherent activity to laminin, a component of the extracellular matrix; CXCL10 was expressed only in round-type S100β-positive cells. Thus, CXCL10 produced by a subpopulation of S100β-positive cells probably exerts an autocrine/paracrine effect on S100β-positive cells and ACTH-producing cells in the anterior lobe.

  15. Gene expression and protein secretion of apolipoprotein B100 (ApoB100 in transition dairy cows under hot or thermoneutral environments

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    Alessandro Nardone

    2010-01-01

    Full Text Available The aim of the study was to investigate the effects of hot season on gene expression and protein secretion of ApoB100 in transition dairy cows. Hot season strongly down-regulated ApoB100 gene and protein expression. This condition and the higher circulating NEFA were responsible for the higher lipid accumulation in liver of heat-stressed transition cows.

  16. S100B protein in serum is elevated after global cerebral ischemic injury

    Institute of Scientific and Technical Information of China (English)

    Bao-di Sun; Hong-mei Liu; Shi-nan Nie

    2013-01-01

    BACKGROUND:S100B protein in patients with cardiac arrest,hemorrhagic shock and other causes of global cerebral ischemic injury will be dramatically increased.Ischemic brain injury may elevate the level of serum S100 B protein and the severity of brain damage.METHODS:This article is a critical and descriptive review on S100 B protein in serum after ischemic brain injury.We searched Pubmed database with key words or terms such as 'S100B protein', 'cardiac arrest', 'hemorrhagic shock' and 'ischemia reperfusion injury' appeared in the last five years.RESULTS:S100B protein in patients with cardiac arrest,hemorrhagic shock and other causes of ischemic brain injury will be dramatically increased.Ischemic brain injury elevated the level of serum S100 B protein,and the severity of brain damage.CONCLUSION:The level of S100 B protein in serum is elevated after ischemic brain injury,but its mechanism is unclear.

  17. Malignant peripheral nerve sheath tumor of the uterine cervix expressing both S-100 protein and HMB-45.

    Science.gov (United States)

    Kim, Na Rae; Chung, Dong-Hae; Park, Chan Yong; Ha, Seung Yeon

    2009-12-01

    A 50-year-old woman presented with a large cervical polypoid mass. Grossly, the mass occupied a substantial proportion of the cervical canal, measuring 6 cm. Histologically, the mass showed a spindle cell malignancy arranged in large fascicles that penetrated deeply into the fibromuscular wall of the cervix. The spindle cells were immunoreactive for both S-100 protein and HMB-45 antigen, but were negative for Melan-A. Electron microscopy showed that cytoplasmic processes of the spindle to oval tumor cells contained microtubules and were lined by basal lamina and abundant intercellular collagen spacing with no melanosomes in any stage. As far as we are aware, this is the ninth reported case of cervical malignant peripheral nerve sheath tumor (MPNST), and the second reported case of MPNST expressing HMB-45 antigen.

  18. [Rapid expression and preparation of the recombinant fusion protein sTNFRII-gAD by adenovirus vector system].

    Science.gov (United States)

    Lu, Yue; Liu, Dan; Zhang, Xiaoren; Liu, Xuerong; Shen, Wei; Zheng, Gang; Liu, Yunfan; Dong, Xiaoyan; Wu, Xiaobing; Gao, Jimin

    2011-08-01

    We expressed and prepared the recombinant fusion protein sTNFRII-gAD consisted of soluble TNF receptor II and the globular domain of adiponectin by Adenovirus Vector System in mammalian BHK21c022 cells. First we used the adenovirus vector containing EGFP gene (rAd5-EGFP) to infect BHK21c022 cells at different MOI (from 0 to 1 000), and then evaluated their transduction efficiency and cytotoxicity. Similarly, we constructed the replication-deficient adenovirus type 5-sTNFRII-gAD (rAd5-sTNFRII-gAD). We collected the supernatants for Western blotting to determine the optimal MOI by comparing the expression levels of sTNFRII-gAD fusion protein, 48 h after the BHK21c022 cells were infected by rAd5-sTNFRII-gAD at different MOIs (from 0 to 1 000). Then, we chose rAd5-sTNFRII-gAD at MOI 100 to infect five bottles of BHK21c022 cells in 100 mL of serum-free chemically defined media 100 mL, harvested the supernatant every 48 h for 6 times, and condense and purify sTNFRII-gAD fusion protein by ammonium sulfate salt-out and size-exclusion chromatography, respectively. Finally, we analyzed anti-TNFalpha activity of sTNFRII-gAD fusion protein on L929 cells in vitro. The results showed that the number of BHK21c022 cells expressing EGFP protein was increased significantly with the increase of MOI. However, some cells died at MOI of 1 000 while there was no significant cytotoxicity at MOI from 0 to 100. Western blotting analysis showed that the more adenoviruses, the higher expression of sTNFRII-gAD fusion protein in the supernatant with the highest expression at MOI 1 000. We successfully obtained about 11 mg bioactive and purified sTNFRII-gAD fusion protein at last. The in vitro assay demonstrated that the sTNFRII-gAD fusion protein was potent to antagonize TNFalpha's cytotoxicity to L929 cells. Put together, we established a recombinant adenovirus vector/BHK21 cell expression system, characteristic of the efficient serum-free culture and easy scaling-up.

  19. S-100 protein in the diagnosis of tuberculoid borderline tuberculoidleprosy

    International Nuclear Information System (INIS)

    Khan, A.R.

    1998-01-01

    A definitive diagnosis of tuberculoid and borderline tuberculoid leprosyis based on a demonstration of either acid-fast bacilli or nerve elementswithin the granulomas. On routine hematoxylin and eosin stains, the nervefibers are not easily identifiable. In this study, we used S-100 protein tohighlight the nerve elements and to count their numbers in leprosy andnon-leprosy granulomas. Skin biopsy specimens from 15 cases oftuberculoid/borderline tuberculoid leprosy and 14 cases belonging to othergranulomatous disease of the skin were stained with S-100 protein. Thesurface area of all the biopsies was calculated and the numbers of nervebundles stained with S-100 protein were counted in each specimen. The nervebundles were 15 per cm2 in leprosy cases, and 9.2 per cm2 in non-leprosycases. In addition, the leprosy cases showed longer nerve twigs that wereperpendicularly oriented to the skin surface. Immunostaining with S-100facilitated detection of nerve elements in tuberculoid/borderline tuberculoidleprosy. Also, an increased number of nerve elements were found in leprosygranulomas when compared with non-leprosy granulomas (P=<0.05). (author)

  20. Inflammation and pancreatic cancer: molecular and functional interactions between S100A8, S100A9, NT-S100A8 and TGFβ1.

    Science.gov (United States)

    Basso, Daniela; Bozzato, Dania; Padoan, Andrea; Moz, Stefania; Zambon, Carlo-Federico; Fogar, Paola; Greco, Eliana; Scorzeto, Michele; Simonato, Francesca; Navaglia, Filippo; Fassan, Matteo; Pelloso, Michela; Dupont, Sirio; Pedrazzoli, Sergio; Fassina, Ambrogio; Plebani, Mario

    2014-03-26

    In order to gain further insight on the crosstalk between pancreatic cancer (PDAC) and stromal cells, we investigated interactions occurring between TGFβ1 and the inflammatory proteins S100A8, S100A9 and NT-S100A8, a PDAC-associated S100A8 derived peptide, in cell signaling, intracellular calcium (Cai2+) and epithelial to mesenchymal transition (EMT). NF-κB, Akt and mTOR pathways, Cai2+ and EMT were studied in well (Capan1 and BxPC3) and poorly differentiated (Panc1 and MiaPaCa2) cell lines. NT-S100A8, one of the low molecular weight N-terminal peptides from S100A8 to be released by PDAC-derived proteases, shared many effects on NF-κB, Akt and mTOR signaling with S100A8, but mainly with TGFβ1. The chief effects of S100A8, S100A9 and NT-S100A8 were to inhibit NF-κB and stimulate mTOR; the molecules inhibited Akt in Smad4-expressing, while stimulated Akt in Smad4 negative cells. By restoring Smad4 expression in BxPC3 and silencing it in MiaPaCa2, S100A8 and NT-S100A8 were shown to inhibit NF-κB and Akt in the presence of an intact TGFβ1 canonical signaling pathway. TGFβ1 counteracted S100A8, S100A9 and NT-S100A8 effects in Smad4 expressing, not in Smad4 negative cells, while it synergized with NT-S100A8 in altering Cai2+ and stimulating PDAC cell growth. The effects of TGFβ1 on both EMT (increased Twist and decreased N-Cadherin expression) and Cai2+ were antagonized by S100A9, which formed heterodimers with TGFβ1 (MALDI-TOF/MS and co-immuno-precipitation). The effects of S100A8 and S100A9 on PDAC cell signaling appear to be cell-type and context dependent. NT-S100A8 mimics the effects of TGFβ1 on cell signaling, and the formation of complexes between TGFβ1 with S100A9 appears to be the molecular mechanism underlying the reciprocal antagonism of these molecules on cell signaling, Cai2+ and EMT.

  1. Immunohistochemical Characterization of S100A6 in the Murine Ovary

    International Nuclear Information System (INIS)

    Hanaue, Mayu; Miwa, Naofumi; Takamatsu, Ken

    2012-01-01

    S100 proteins comprise a large family of Ca 2+ -binding proteins and exhibit a variety of intra- and extracellular functions. Despite our growing knowledge about the biology of S100 proteins in some tissues such as brain and smooth muscle, little is known about S100 proteins in the normal mammalian reproductive tissue. In the present study, we investigated the distribution pattern of S100A6 (alternatively named calcyclin) in the murine ovary by immunohistochemical study using specific antibody. S100A6 was localized substantially in the cytoplasm of luteal cells, with concomitant expression of S100A11, another S100 protein, but not in the other type of cells such as oocytes, follicle epithelial cells (granulosa cells), and cells of stroma including theca interna cells in the murine ovary. S100A6-immunoreactive corpora lutea (CLs) were divided into two types: homogeneously and heterogeneously stained CLs, and possibly they may represent differentiating and mature CL, respectively. Our regression analysis revealed that expression level of S100A6 positively correlated with that of cytochrome P450 11A, a steroidogenic enzyme in the heterogeously stained CL. These results suggested that S100A6 may contribute to differentiation of steroidogenic activity of luteal cells in a synergistic manner with S100A11 by facilitating some shared functions

  2. The histone demethylase LSD1 is required for estrogen-dependent S100A7 gene expression in human breast cancer cells

    International Nuclear Information System (INIS)

    Yu, Seung Eun; Jang, Yeun Kyu

    2012-01-01

    Highlights: ► S100A7 gene is up-regulated in response to estrogen in breast cancer cells. ► Histone demethylase LSD1 can associate physically with S100A7 gene promoters. ► E2-induced S100A7 expression requires the enzymatic activity of LSD1. ► S100A7 inhibits cell proliferation, implying its tumor suppressor-like function. -- Abstract: S100A7, a member of S100 calcium binding protein family, is highly associated with breast cancer. However, the molecular mechanism of S100A7 regulation remains unclear. Here we show that long-term treatment with estradiol stimulated S100A7 expression in MCF7 breast cancer cells at both the transcriptional and translational levels. Both treatment with a histone demethylase LSD1 inhibitor and shRNA-based knockdown of LSD1 expression significantly decreased 17β-estradiol (E2)-induced S100A7 expression. These reduced E2-mediated S100A7 expression are rescued by the overexpressed wild-type LSD1 but not by its catalytically inactive mutant. Our data showed in vivo association of LSD1 with S100A7 promoters, confirming the potential role of LSD1 in regulating S100A7 expression. S100A7 knockdown increased both normal cell growth and estrogen-induced cell proliferation, suggesting a negative influence by S100A7 on the growth of cancer cells. Together, our data suggest that estrogen-induced S100A7 expression mediated by the histone demethylase LSD1 may downregulate breast cancer cell proliferation, implying a potential tumor suppressor-like function for S100A7.

  3. Olopatadine Suppresses the Migration of THP-1 Monocytes Induced by S100A12 Protein

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    2006-01-01

    Full Text Available Olopatadine hydrochloride (olopatadine is an antiallergic drug with histamine H 1 receptor antagonistic activity. Recently, olopatadine has been shown to bind to S100A12 which is a member of the S100 family of calcium-binding proteins, and exerts multiple proinflammatory activities including chemotaxis for monocytes and neutrophils. In this study, we examined the possibility that the interaction of olopatadine with S100A12 inhibits the proinflammatory effects of S100A12. Pretreatment of olopatadine with S100A12 reduced migration of THP-1, a monocyte cell line, induced by S100A12 alone, but did not affect recombinant human regulated upon activation, normal T cell expressed and secreted (RANTES-induced migration. Amlexanox, which also binds to S100A12, inhibited the THP-1 migration induced by S100A12. However, ketotifen, another histamine H 1 receptor antagonist, had little effect on the activity of S100A12. These results suggest that olopatadine has a new mechanism of action, that is, suppression of the function of S100A12, in addition to histamine H 1 receptor antagonistic activity.

  4. Participation of the oviductal s100 calcium binding protein G in the genomic effect of estradiol that accelerates oviductal embryo transport in mated rats

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    Croxatto Horacio B

    2011-05-01

    Full Text Available Abstract Background Mating changes the mechanism by which E2 regulates oviductal egg transport, from a non-genomic to a genomic mode. Previously, we found that E2 increased the expression of several genes in the oviduct of mated rats, but not in unmated rats. Among the transcripts that increased its level by E2 only in mated rats was the one coding for an s100 calcium binding protein G (s100 g whose functional role in the oviduct is unknown. Methods Herein, we investigated the participation of s100 g on the E2 genomic effect that accelerates oviductal transport in mated rats. Thus, we determined the effect of E2 on the mRNA and protein level of s100 g in the oviduct of mated and unmated rats. Then, we explored the effect of E2 on egg transport in unmated and mated rats under conditions in which s100 g protein was knockdown in the oviduct by a morpholino oligonucleotide against s100 g (s100 g-MO. In addition, the localization of s100 g in the oviduct of mated and unmated rats following treatment with E2 was also examined. Results Expression of s100 g mRNA progressively increased at 3-24 h after E2 treatment in the oviduct of mated rats while in unmated rats s100 g increased only at 12 and 24 hours. Oviductal s100 g protein increased 6 h following E2 and continued elevated at 12 and 24 h in mated rats, whereas in unmated rats s100 g protein increased at the same time points as its transcript. Administration of a morpholino oligonucleotide against s100 g transcript blocked the effect of E2 on egg transport in mated, but not in unmated rats. Finally, immunoreactivity of s100 g was observed only in epithelial cells of the oviducts of mated and unmated rats and it was unchanged after E2 treatment. Conclusions Mating affects the kinetic of E2-induced expression of s100 g although it not changed the cellular localization of s100 g in the oviduct after E2 . On the other hand, s100 g is a functional component of E2 genomic effect that accelerates egg

  5. The calcium-modulated proteins, S100A1 and S100B, as potential regulators of the dynamics of type III intermediate filaments

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    M. Garbuglia

    1999-10-01

    Full Text Available The Ca2+-modulated, dimeric proteins of the EF-hand (helix-loop-helix type, S100A1 and S100B, that have been shown to inhibit microtubule (MT protein assembly and to promote MT disassembly, interact with the type III intermediate filament (IF subunits, desmin and glial fibrillary acidic protein (GFAP, with a stoichiometry of 2 mol of IF subunit/mol of S100A1 or S100B dimer and an affinity of 0.5-1.0 µM in the presence of a few micromolar concentrations of Ca2+. Binding of S100A1 and S100B results in inhibition of desmin and GFAP assemblies into IFs and stimulation of the disassembly of preformed desmin and GFAP IFs. S100A1 and S100B interact with a stretch of residues in the N-terminal (head domain of desmin and GFAP, thereby blocking the head-to-tail process of IF elongation. The C-terminal extension of S100A1 (and, likely, S100B represents a critical part of the site that recognizes desmin and GFAP. S100B is localized to IFs within cells, suggesting that it might have a role in remodeling IFs upon elevation of cytosolic Ca2+ concentration by avoiding excess IF assembly and/or promoting IF disassembly in vivo. S100A1, that is not localized to IFs, might also play a role in the regulation of IF dynamics by binding to and sequestering unassembled IF subunits. Together, these observations suggest that S100A1 and S100B may be regarded as Ca2+-dependent regulators of the state of assembly of two important elements of the cytoskeleton, IFs and MTs, and, potentially, of MT- and IF-based activities.

  6. The metastasis-promoting S100A4 protein confers neuroprotection in brain injury

    DEFF Research Database (Denmark)

    Dmytriyeva, Oksana; Pankratova, Stanislava; Owczarek, Sylwia

    2012-01-01

    and downregulating the neuroprotective protein metallothionein I+II. We identify two neurotrophic motifs in S100A4 and show that these motifs are neuroprotective in animal models of brain trauma. Finally, we find that S100A4 rescues neurons via the Janus kinase/STAT pathway and, partially, the interleukin-10......Identification of novel pro-survival factors in the brain is paramount for developing neuroprotective therapies. The multifunctional S100 family proteins have important roles in many human diseases and are also upregulated by brain injury. However, S100 functions in the nervous system remain...... unclear. Here we show that the S100A4 protein, mostly studied in cancer, is overexpressed in the damaged human and rodent brain and released from stressed astrocytes. Genetic deletion of S100A4 exacerbates neuronal loss after brain trauma or excitotoxicity, increasing oxidative cell damage...

  7. Expression of S100A6 in Rat Hippocampus after Traumatic Brain Injury Due to Lateral Head Acceleration

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    Bo Fang

    2014-04-01

    Full Text Available In a rat model of traumatic brain injury (TBI, we investigated changes in cognitive function and S100A6 expression in the hippocampus. TBI-associated changes in this protein have not previously been reported. Rat S100A6 was studied via immunohistochemical staining, Western blot, and reverse transcription-polymerase chain reaction (RT-PCR after either lateral head acceleration or sham. Reduced levels of S100A6 protein and mRNA were observed 1 h after TBI, followed by gradual increases over 6, 12, 24, and 72 h, and then a return to sham level at 14 day. Morris water maze (MWM test was used to evaluate animal spatial cognition. TBI- and sham-rats showed an apparent learning curve, expressed as escape latency. Although TBI-rats displayed a relatively poorer cognitive ability than sham-rats, the disparity was not significant early post-injury. Marked cognitive deficits in TBI-rats were observed at 72 h post-injury compared with sham animals. TBI-rats showed decreased times in platform crossing in the daily MWM test; the performance at 72 h post-injury was the worst. In conclusion, a reduction in S100A6 may be one of the early events that lead to secondary cognitive decline after TBI, and its subsequent elevation is tightly linked with cognitive improvement. S100A6 may play important roles in neuronal degeneration and regeneration in TBI.

  8. Vildagliptin and its metabolite M20.7 induce the expression of S100A8 and S100A9 in human hepatoma HepG2 and leukemia HL-60 cells.

    Science.gov (United States)

    Asakura, Mitsutoshi; Karaki, Fumika; Fujii, Hideaki; Atsuda, Koichiro; Itoh, Tomoo; Fujiwara, Ryoichi

    2016-10-19

    Vildagliptin is a potent, orally active inhibitor of dipeptidyl peptidase-4 (DPP-4) for the treatment of type 2 diabetes mellitus. It has been reported that vildagliptin can cause hepatic dysfunction in patients. However, the molecular-mechanism of vildagliptin-induced liver dysfunction has not been elucidated. In this study, we employed an expression microarray to determine hepatic genes that were highly regulated by vildagliptin in mice. We found that pro-inflammatory S100 calcium-binding protein (S100) a8 and S100a9 were induced more than 5-fold by vildagliptin in the mouse liver. We further examined the effects of vildagliptin and its major metabolite M20.7 on the mRNA expression levels of S100A8 and S100A9 in human hepatoma HepG2 and leukemia HL-60 cells. In HepG2 cells, vildagliptin, M20.7, and sitagliptin - another DPP-4 inhibitor - induced S100A9 mRNA. In HL-60 cells, in contrast, S100A8 and S100A9 mRNAs were significantly induced by vildagliptin and M20.7, but not by sitagliptin. The release of S100A8/A9 complex in the cell culturing medium was observed in the HL-60 cells treated with vildagliptin and M20.7. Therefore, the parental vildagliptin- and M20.7-induced release of S100A8/A9 complex from immune cells, such as neutrophils, might be a contributing factor of vildagliptin-associated liver dysfunction in humans.

  9. Purification, crystallization and preliminary X-ray diffraction of human S100A15

    Energy Technology Data Exchange (ETDEWEB)

    Boeshans, Karen M. [X-ray Crystallography Facility, NIAMS, National Institutes of Health, Bethesda, MD 20892 (United States); Wolf, Ronald; Voscopoulos, Christopher [Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Gillette, William; Esposito, Dominic [Protein Expression Laboratory, Research Technology Program, National Cancer Institute, SAIC-Frederick Inc., Frederick, MD 21702 (United States); Mueser, Timothy C. [Department of Chemistry, University of Toledo, Toledo, OH 43606 (United States); Yuspa, Stuart H. [Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Ahvazi, Bijan, E-mail: ahvazib@mail.nih.gov [X-ray Crystallography Facility, NIAMS, National Institutes of Health, Bethesda, MD 20892 (United States)

    2006-05-01

    S100 proteins are differentially expressed during epithelial cell maturation, tumorigenesis and inflammation. The novel human S100A15 protein has been cloned, expressed, purified and crystallized in two crystal forms, a triclinic and a monoclinic form, which diffract to 1.7 and 2.0 Å, respectively. Human S100A15 is a novel member of the S100 family of EF-hand calcium-binding proteins and was recently identified in psoriasis, where it is significantly upregulated in lesional skin. The protein is implicated as an effector in calcium-mediated signal transduction pathways. Although its biological function is unclear, the association of the 11.2 kDa S100A15 with psoriasis suggests that it contributes to the pathogenesis of the disease and could provide a molecular target for therapy. To provide insight into the function of S100A15, the protein was crystallized to visualize its structure and to further the understanding of how the many similar calcium-binding mediator proteins in the cell distinguish their cognate target molecules. The S100A15 protein has been cloned, expressed and purified to homogeneity and produced two crystal forms. Crystals of form I are triclinic, with unit-cell parameters a = 33.5, b = 44.3, c = 44.8 Å, α = 71.2, β = 68.1, γ = 67.8° and an estimated two molecules in the asymmetric unit, and diffract to 1.7 Å resolution. Crystals of form II are monoclinic, with unit-cell parameters a = 82.1, b = 33.6, c = 52.2 Å, β = 128.2° and an estimated one molecule in the asymmetric unit, and diffract to 2.0 Å resolution. This structural analysis of the human S100A15 will further aid in the phylogenic comparison between the other members of the S100 protein family, especially the highly homologous paralog S100A7.

  10. Comparison of mRNA, Protein, and Urinary Nucleic Acid Levels of S100A8 and S100A9 between Prostate Cancer and BPH.

    Science.gov (United States)

    Yun, Seok Joong; Yan, Chunri; Jeong, Pildu; Kang, Ho Won; Kim, Ye-Hwan; Kim, Eun-Ah; Lee, Ok-Jun; Kim, Won Tae; Moon, Sung-Kwon; Kim, Isaac Yi; Choi, Yung-Hyun; Kim, Wun-Jae

    2015-07-01

    Infections and inflammation in the prostate play a critical role in carcinogenesis, and S100A8 and S100A9 are key mediators in acute and chronic inflammation. Therefore, we investigated the differences of S100A8/A9 expression between prostate cancer (CaP) and benign prostatic hyperplasia (BPH) tissues, and we evaluated the possibilities of urinary nucleic acids of S100A8/A9 as diagnostic and prognostic markers. Tissues from 132 CaP patients who underwent prostatectomy or transurethral resection and 90 BPH patients who underwent transurethral prostatectomy were assessed.sd In addition, S100A8 and S100A9 nucleic acid levels were measured in the urine of 283 CaP patients and 363 BPH controls. S100A8 and S100A9 mRNA levels were lower in CaP than BPH tissues (P BPH tissues stained more strongly for both S100A8 and S100A9 than CaP tissues (P BPH (P = 0.001 and BPH. Both were more highly expressed in patients with aggressive disease and shorter biochemical recurrence-free time. S100A8/A9 urinary cell-free nucleic acid levels correlated positively with expression levels obtained from tissue staining. Therefore, S100A8/A9 measurement in tissues and urine may have diagnostic and prognostic value in CaP.

  11. Serum S-100β protein as a biomarker for brain damage in patients with encephalopathy

    International Nuclear Information System (INIS)

    Takeda, Munekazu; Yaguchi, Arino; Yamada, Sou; Nagai, Atsushi; Yuzawa, Junji

    2008-01-01

    Cerebrospinal fluid concentrations of S-100β protein, an acidic calcium-binding protein found in astrocytes and Schwann cells, increase after central nervous system damage. Serum S-100β protein, thus, has been expected to be a biochemical marker of brain cell damage. Several reports show a relation between severity of head injury and serum S-100β protein levels, although, there are still not significant advances in the study of S-100β regarding the prediction of the clinical outcome in brain diseases. The objective of the present study was to verify S-100β as a marker for the clinical outcome in patients with encephalopathy. Serum S-100β protein concentrations (pg/ml) were measured daily using enzyme-linked immunosorbent assay (ELISA) until discharge from the intensive care unit (ICU) in 82 patients (54 men, 28 women; age 20-93 years [mean 61.0±19.2]) with moderate or severe encephalopathy. There were 50 survivors and 32 non-survivors. S-100β levels were significantly lower in survivors (240.2 pg/ml) than in non-survivors (1,594.8 pg/ml) from day 1 until ICU discharge. The electroencephalogram (EEG) and computed tomography (CT) abnormalities were correlated with S-100β levels. The optimal cut-off value at 451.2 pg/ml calculated from receiver operating characteristic (ROC) curve analysis showed the sensitivity of 80.2% and specificity of 78.1% for ICU mortality. Our results indicate that serum S-100β protein could be a useful biomarker to assess brain damage and predict prognosis in patients with encephalopathy. (author)

  12. Interplay between Trx-1 and S100P promotes colorectal cancer cell epithelial-mesenchymal transition by up-regulating S100A4 through AKT activation.

    Science.gov (United States)

    Zuo, Zhigui; Zhang, Peili; Lin, Feiyan; Shang, Wenjing; Bi, Ruichun; Lu, Fengying; Wu, Jianbo; Jiang, Lei

    2018-04-01

    We previously reported a novel positive feedback loop between thioredoxin-1 (Trx-1) and S100P, which promotes the invasion and metastasis of colorectal cancer (CRC). However, the underlying molecular mechanisms remain poorly understood. In this study, we examined the roles of Trx-1 and S100P in CRC epithelial-to-mesenchymal transition (EMT) and their underlying mechanisms. We observed that knockdown of Trx-1 or S100P in SW620 cells inhibited EMT, whereas overexpression of Trx-1 or S100P in SW480 cells promoted EMT. Importantly, S100A4 and the phosphorylation of AKT were identified as potential downstream targets of Trx-1 and S100P in CRC cells. Silencing S100A4 or inhibition of AKT phosphorylation eliminated S100P- or Trx-1-mediated CRC cell EMT, migration and invasion. Moreover, inhibition of AKT activity reversed S100P- or Trx-1-induced S100A4 expression. The expression of S100A4 was higher in human CRC tissues compared with their normal counterpart tissues and was significantly correlated with lymph node metastasis and poor survival. The overexpression of S100A4 protein was also positively correlated with S100P or Trx-1 protein overexpression in our cohort of CRC tissues. In addition, overexpression of S100P reversed the Trx-1 knockdown-induced inhibition of S100A4 expression, EMT and migration and invasion in SW620 cells. The data suggest that interplay between Trx-1 and S100P promoted CRC EMT as well as migration and invasion by up-regulating S100A4 through AKT activation, thus providing further potential therapeutic targets for suppressing the EMT in metastatic CRC. © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  13. Detection and significance of S-100 protein and NSE during mild hypothermia cardiopulmonary bypass

    International Nuclear Information System (INIS)

    Liu Xiuqin; Jin Mu; Tan Jiefang; Huang Wenqi; Chen Bingxue; Huang Weiming; Huang Xiongqing

    2001-01-01

    To observe dynamic changes of S-100 protein and NSE during mild hypothermia cardiopulmonary bypass (CPB), the venous blood samples of 25 patients with elective cardiac surgery were obtained simultaneously from the left artery and left jugular bulb before CPB(A), hypothermia period (32-35 degree C) (B) and rewarming to 36 degree C (C) during CPB, 30 minutes (D), 4-6 hours (E) and 24 hours (F) after CPB. Plasma S-100 protein concentration was determined by chemiluminescence immunoassay, and NSE level was determined by radioimmunoassay. The results showed that the levels of S-100 protein and NSE increased significantly during CPB, and NSE peaked at 30 minutes (D) after CPB. It suggested the central nervous system dysfunctions. The S-100 protein and NSE concentrations decreased gradually and retuned to normal nearly (F) after mild hypothermia CPB. It suggested that there were not obvious central nervous system dysfunctions

  14. Both Ca2+ and Zn2+ are essential for S100A12 protein oligomerization and function

    Directory of Open Access Journals (Sweden)

    Shekhtman Alexander

    2009-04-01

    Full Text Available Abstract Background Human S100A12 is a member of the S100 family of EF-hand calcium-modulated proteins that are associated with many diseases including cancer, chronic inflammation and neurological disorders. S100A12 is an important factor in host/parasite defenses and in the inflammatory response. Like several other S100 proteins, it binds zinc and copper in addition to calcium. Mechanisms of zinc regulation have been proposed for a number of S100 proteins e.g. S100B, S100A2, S100A7, S100A8/9. The interaction of S100 proteins with their targets is strongly dependent on cellular microenvironment. Results The aim of the study was to explore the factors that influence S100A12 oligomerization and target interaction. A comprehensive series of biochemical and biophysical experiments indicated that changes in the concentration of calcium and zinc led to changes in the oligomeric state of S100A12. Surface plasmon resonance confirmed that the presence of both calcium and zinc is essential for the interaction of S100A12 with one of its extracellular targets, RAGE – the Receptor for Advanced Glycation End products. By using a single-molecule approach we have shown that the presence of zinc in tissue culture medium favors both the oligomerization of exogenous S100A12 protein and its interaction with targets on the cell surface. Conclusion We have shown that oligomerization and target recognition by S100A12 is regulated by both zinc and calcium. Our present work highlighted the potential role of calcium-binding S100 proteins in zinc metabolism and, in particular, the role of S100A12 in the cross talk between zinc and calcium in cell signaling.

  15. Down-regulation of S100C is associated with bladder cancer progression and poor survival

    DEFF Research Database (Denmark)

    Memon, Ashfaque Ahmed; Sorensen, Boe Sandahl; Meldgaard, Peter

    2005-01-01

    cancer biopsy samples obtained from 88 patients followed for a median of 23 months (range, 1-97 months). RESULTS: We found a significantly lower mRNA expression of S100C in connective tissue invasive tumors (T1, P = 0.0030) and muscle invasive tumors [(T2-T4), P ...PURPOSE: The goal of this study was to identify proteins down-regulated during bladder cancer progression. EXPERIMENTAL DESIGN: By using comparative proteome analysis and measurement of mRNA, we found a significant down-regulation of S100C, a member of the S100 family of proteins, in T24 (grade 3......) as compared with RT4 (grade 1) bladder cancer cell lines. Moreover, quantification of the mRNA level revealed that decreased expression of the protein reflects a low level of transcription of the S100C gene. Based on this observation, we quantified the S100C mRNA expression level with real-time PCR in bladder...

  16. Elevated S100A9 expression in tumor stroma functions as an early recurrence marker for early-stage oral cancer patients through increased tumor cell invasion, angiogenesis, macrophage recruitment and interleukin-6 production.

    Science.gov (United States)

    Fang, Wei-Yu; Chen, Yi-Wen; Hsiao, Jenn-Ren; Liu, Chiang-Shin; Kuo, Yi-Zih; Wang, Yi-Ching; Chang, Kung-Chao; Tsai, Sen-Tien; Chang, Mei-Zhu; Lin, Siao-Han; Wu, Li-Wha

    2015-09-29

    S100A9 is a calcium-binding protein with two EF-hands and frequently deregulated in several cancer types, however, with no clear role in oral cancer. In this report, the expression of S100A9 in cancer and adjacent tissues from 79 early-stage oral cancer patients was detected by immunohistochemical staining. Although S100A9 protein was present in both tumor and stromal cells, only the early-stage oral cancer patients with high stromal expression had reduced recurrence-free survival. High stromal S100A9 expression was also significantly associated with non-well differentiation and recurrence. In addition to increasing cell migration and invasion, ectopic S100A9 expression in tumor cells promoted xenograft tumorigenesis as well as the dominant expression of myeloid cell markers and pro-inflammatory IL-6. The expression of S100A9 in one stromal component, monocytes, stimulated the aggressiveness of co-cultured oral cancer cells. We also detected the elevation of serum S100A9 levels in early-stage oral cancer patients of a separate cohort of 73 oral cancer patients. The release of S100A9 protein into extracellular milieu enhanced tumor cell invasion, transendothelial monocyte migration and angiogenic activity. S100A9-mediated release of IL-6 requires the crosstalk of tumor cells with monocytes through the activation of NF-κB and STAT-3. Early-stage oral cancer patients with both high S100A9 expression and high CD68+ immune infiltrates in stroma had shortest recurrence-free survival, suggesting the use of both S100A9 and CD68 as poor prognostic markers for oral cancer. Together, both intracellular and extracellular S100A9 exerts a tumor-promoting action through the activation of oral cancer cells and their associated stroma in oral carcinogenesis.

  17. Proton receptor GPR68 expression in dendritic-cell-like S100β-positive cells of rat anterior pituitary gland: GPR68 induces interleukin-6 gene expression in extracellular acidification.

    Science.gov (United States)

    Horiguchi, Kotaro; Higuchi, Masashi; Yoshida, Saishu; Nakakura, Takashi; Tateno, Kozue; Hasegawa, Rumi; Takigami, Shu; Ohsako, Shunji; Kato, Takako; Kato, Yukio

    2014-11-01

    S100β-positive cells, which do not express the classical pituitary hormones, appear to possess multifunctional properties and are assumed to be heterogeneous in the anterior pituitary gland. The presence of several protein markers has shown that S100β-positive cells are composed of populations such as stem/progenitor cells, epithelial cells, astrocytes and dendritic cells. Recently, we succeeded in separating S100β-positive cells into round-cell (dendritic-cell-like) and process-cell types. We also found the characteristic expression of anti-inflammatory factors (interleukin-6, Il-6) and membrane receptors (integrin β-6) in the round type. Here, we further investigate the function of the subpopulation of S100β-positive cells. Since IL-6 is also a paracrine factor that regulates hormone producing-cells, we examine whether a correlation exists among extracellular acid stress, IL-6 and hormone production by using primary cultures of anterior pituitary cells. Dendritic-cell-like S100β-positive cells notably expressed Gpr68 (proton receptor) and Il-6. Furthermore, the expression of Il-6 and proopiomelanocortin (Pomc) was up-regulated by extracellular acidification. The functional role of IL-6 and GPR68 in the gene expression of Pomc during extracellular acidification was also examined. Small interfering RNA for Il-6 up-regulated Pomc expression and that for Gpr68 reversed the down-regulation of Il-6 and up-regulated Pomc expression by extracellular acidification. Thus, S100β-positive dendritic-like cells can sense an increase in extracellular protons via GPR68 and respond by the production of IL-6 in order to suppress the up-regulation of Pomc expression.

  18. Expression analysis of the mouse S100A7/psoriasin gene in skin inflammation and mammary tumorigenesis

    International Nuclear Information System (INIS)

    Webb, Meghan; Myal, Yvonne; Shiu, Robert; Murphy, Leigh C; Watson, Peter H; Emberley, Ethan D; Lizardo, Michael; Alowami, Salem; Qing, Gefei; Alfia'ar, Abdullah; Snell-Curtis, Linda J; Niu, Yulian; Civetta, Alberto

    2005-01-01

    The human psoriasin (S100A7) gene has been implicated in inflammation and tumor progression. Implementation of a mouse model would facilitate further investigation of its function, however little is known of the murine psoriasin gene. In this study we have cloned the cDNA and characterized the expression of the potential murine ortholog of human S100A7/psoriasin in skin inflammation and mammary tumorigenesis. On the basis of chromosomal location, phylogenetic analysis, amino acid sequence similarity, conservation of a putative Jab1-binding motif, and similarities of the patterns of mouse S100A7/psoriasin gene expression (measured by RT-PCR and in-situ hybridization) with those of human S100A7/psoriasin, we propose that mouse S100A7/psoriasin is the murine ortholog of human psoriasin/S100A7. Although mouse S100A7/psoriasin is poorly conserved relative to other S100 family members, its pattern of expression parallels that of the human psoriasin gene. In murine skin S100A7/psoriasin was significantly upregulated in relation to inflammation. In murine mammary gland expression is also upregulated in mammary tumors, where it is localized to areas of squamous differentiation. This mirrors the context of expression in human tumor types where both squamous and glandular differentiation occur, including cervical and lung carcinomas. Additionally, mouse S100A7/psoriasin possesses a putative Jab1 binding motif that mediates many downstream functions of the human S100A7 gene. These observations and results support the hypothesis that the mouse S100A7 gene is structurally and functionally similar to human S100A7 and may offer a relevant model system for studying its normal biological function and putative role in tumor progression

  19. Crystallization and preliminary X-ray analysis of human S100A13

    International Nuclear Information System (INIS)

    Imai, Fabiana Lica; Nagata, Koji; Yonezawa, Naoto; Yu, Jinyan; Ito, Eriko; Kanai, Saeko; Tanokura, Masaru; Nakano, Minoru

    2006-01-01

    Human S100A13 protein was cloned, expressed, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals obtained belonged to space group P2 1 2 1 2 1 and diffracted to a resolution of 1.8 Å. S100A13 is a member of the S100 family of EF-hand-containing calcium-binding proteins and plays an important role in the secretion of fibroblast growth factor-1 and interleukin 1α, two pro-angiogenic factors released by the endoplasmic reticulum/Golgi-independent non-classical secretory pathway. Human S100A13 was heterologously expressed in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystals diffracted X-rays from a synchrotron-radiation source to 1.8 Å resolution and the space group was assigned as primitive orthorhombic P2 1 2 1 2 1

  20. The calcium-binding protein complex S100A8/A9 has a crucial role in controlling macrophage-mediated renal repair following ischemia/reperfusion

    NARCIS (Netherlands)

    Dessing, Mark C.; Tammaro, Alessandra; Pulskens, Wilco P.; Teske, Gwendoline J.; Butter, Loes M.; Claessen, Nike; van Eijk, Marco; van der Poll, Tom; Vogl, Thomas; Roth, Johannes; Florquin, Sandrine; Leemans, Jaklien C.

    2015-01-01

    Upon ischemia/reperfusion (I/R)-induced injury, several damage-associated molecular patterns are expressed including the calcium-binding protein S100A8/A9 complex. S100A8/A9 can be recognized by Toll-like receptor-4 and its activation is known to deleteriously contribute to renal I/R-induced injury.

  1. Localization of S-100 proteins in the testis and epididymis of poultry and rabbits

    Science.gov (United States)

    Abd-Elmaksoud, Ahmed; Marei, Hany E. S.

    2014-01-01

    The present investigation was conducted to demonstrate S-100 protein in the testis and epididymis of adult chickens, Sudani ducks, pigeons, and rabbits. This study may represent the first indication for the presence of S-100 in the male reproductive organs of these species and might therefore serve as a milestone for further reports. In the testis of chickens, pigeons and rabbits, intense S-100 was seen in Sertoli cells. S-100 was also seen in the endothelial lining of blood vessels in rabbit testis. On the contrary, no S-100 reaction was detected in the Sertoli cells of Sudani ducks. In epididymis, the localization of S-100 had varied according to species studied; it was seen in the basal cells (BC) of epididymal duct in duck, non-ciliated cells of the distal efferent ductules in pigeons and ciliated cells of the efferent ductules and BC of rabbit epididymis. Conversely, S-100 specific staining was not detected in the epithelial lining of the rooster and pigeon epididymal duct as well as the principal cells of the rabbit epididymis. In conclusion, the distribution of the S-100 proteins in the testis and epididymis might point out to its roles in the male reproduction. PMID:25276477

  2. Metastasis-inducing S100A4 protein is associated with the disease activity of rheumatoid arthritis

    DEFF Research Database (Denmark)

    Oslejsková, Lucie; Grigorian, Mariam; Hulejová, Hana

    2009-01-01

    To evaluate the association between metastasis-inducing protein S100A4 and disease activity in patients with RA, and to demonstrate the effect of TNF-alpha blocking therapy on plasma levels of S100A4 in these patients.......To evaluate the association between metastasis-inducing protein S100A4 and disease activity in patients with RA, and to demonstrate the effect of TNF-alpha blocking therapy on plasma levels of S100A4 in these patients....

  3. Crystallization and preliminary X-ray analysis of human S100A13

    Energy Technology Data Exchange (ETDEWEB)

    Imai, Fabiana Lica [Department of Chemistry, Faculty of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522 (Japan); Nagata, Koji [Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Yonezawa, Naoto [Department of Chemistry, Faculty of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522 (Japan); Yu, Jinyan; Ito, Eriko; Kanai, Saeko [Graduate School of Science and Technology, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522 (Japan); Tanokura, Masaru [Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Nakano, Minoru, E-mail: mnakano@faculty.chiba-u.jp [Department of Chemistry, Faculty of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522 (Japan)

    2006-11-01

    Human S100A13 protein was cloned, expressed, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals obtained belonged to space group P2{sub 1}2{sub 1}2{sub 1} and diffracted to a resolution of 1.8 Å. S100A13 is a member of the S100 family of EF-hand-containing calcium-binding proteins and plays an important role in the secretion of fibroblast growth factor-1 and interleukin 1α, two pro-angiogenic factors released by the endoplasmic reticulum/Golgi-independent non-classical secretory pathway. Human S100A13 was heterologously expressed in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystals diffracted X-rays from a synchrotron-radiation source to 1.8 Å resolution and the space group was assigned as primitive orthorhombic P2{sub 1}2{sub 1}2{sub 1}.

  4. Mycobacterium tuberculosis HspX/EsxS Fusion Protein: Gene Cloning, Protein Expression, and Purification in Escherichia coli.

    Science.gov (United States)

    Khademi, Farzad; Yousefi-Avarvand, Arshid; Derakhshan, Mohammad; Meshkat, Zahra; Tafaghodi, Mohsen; Ghazvini, Kiarash; Aryan, Ehsan; Sankian, Mojtaba

    2017-10-01

    The purpose of this study was to clone, express, and purify a novel multidomain fusion protein of Micobacterium tuberculosis (Mtb) in a prokaryotic system. An hspX/esxS gene construct was synthesized and ligated into a pGH plasmid, E. coli TOP10 cells were transformed, and the vector was purified. The vector containing the construct and pET-21b (+) plasmid were digested with the same enzymes and the construct was ligated into pET-21b (+). The accuracy of cloning was confirmed by colony PCR and sequencing. E. coli BL21 cells were transformed with the pET-21b (+)/hspX/esxS expression vector and protein expression was evaluated. Finally, the expressed fusion protein was purified on a Ni-IDA column and verified by SDS-PAGE and western blotting. The hspX/esxS gene construct was inserted into pET-21b (+) and recombinant protein expression was induced with IPTG in E. coli BL21 cells. Various concentrations of IPTG were tested to determine the optimum concentration for expression induction. The recombinant protein was expressed in insoluble inclusion bodies. Three molar guanidine HCl was used to solubilize the insoluble protein. An HspX/EsxS Mtb fusion protein was expressed in E. coli and the recombinant protein was purified. After immunological analysis, the HspX/EsxS fusion protein might be an anti-tuberculosis vaccine candidate in future clinical trial studies.

  5. Fragile X mental retardation protein expression in Alzheimer’s disease

    Directory of Open Access Journals (Sweden)

    Abigail J Renoux

    2014-10-01

    Full Text Available The FMR1 protein product, FMRP, is an mRNA binding protein associated with translational inhibition of target transcripts. One FMRP target is the amyloid precursor protein (APP mRNA, and APP levels are elevated in Fmr1 KO mice. Given that elevated APP protein expression can elicit Alzheimer’s disease (AD in patients and model systems, we evaluated whether FMRP expression might be altered in Alzheimer’s autopsy brain samples and mouse models compared to controls. In a double transgenic mouse model of AD (APP/PS1, we found no difference in FMRP expression in aged AD model mice compared to littermate controls. FMRP expression was also similar in AD and control patient frontal cortex and cerebellum samples. Fragile X-associated tremor/ataxia syndrome (FXTAS is an age related neurodegenerative disorder caused by expanded CGG repeats in the 5’UTR of the FMR1 gene. Patients experience cognitive impairment and dementia in addition to motor symptoms. In parallel studies, we measured FMRP expression in cortex and cerebellum from three FXTAS patients and found reduced expression compared to both controls and Alzheimer’s patient brains, consistent with animal models. We also find increased APP levels in cerebellar, but not cortical, samples of FXTAS patients compared to controls. Taken together, these data suggest that a decrease in FMRP expression is unlikely to be a primary contributor to Alzheimer’s disease pathogenesis.

  6. Insulin Stimulates S100B Secretion and These Proteins Antagonistically Modulate Brain Glucose Metabolism.

    Science.gov (United States)

    Wartchow, Krista Minéia; Tramontina, Ana Carolina; de Souza, Daniela F; Biasibetti, Regina; Bobermin, Larissa D; Gonçalves, Carlos-Alberto

    2016-06-01

    Brain metabolism is highly dependent on glucose, which is derived from the blood circulation and metabolized by the astrocytes and other neural cells via several pathways. Glucose uptake in the brain does not involve insulin-dependent glucose transporters; however, this hormone affects the glucose influx to the brain. Changes in cerebrospinal fluid levels of S100B (an astrocyte-derived protein) have been associated with alterations in glucose metabolism; however, there is no evidence whether insulin modulates glucose metabolism and S100B secretion. Herein, we investigated the effect of S100B on glucose metabolism, measuring D-(3)H-glucose incorporation in two preparations, C6 glioma cells and acute hippocampal slices, and we also investigated the effect of insulin on S100B secretion. Our results showed that: (a) S100B at physiological levels decreases glucose uptake, through the multiligand receptor RAGE and mitogen-activated protein kinase/ERK signaling, and (b) insulin stimulated S100B secretion via PI3K signaling. Our findings indicate the existence of insulin-S100B modulation of glucose utilization in the brain tissue, and may improve our understanding of glucose metabolism in several conditions such as ketosis, streptozotocin-induced dementia and pharmacological exposure to antipsychotics, situations that lead to changes in insulin signaling and extracellular levels of S100B.

  7. The clinical expression of hereditary protein C and protein S deficiency: : a relation to clinical thrombotic risk-factors and to levels of protein C and protein S

    NARCIS (Netherlands)

    Henkens, C. M. A.; van der Meer, J.; Hillege, J. L.; Bom, V. J. J.; Halie, M. R.; van der Schaaf, W.

    We investigated 103 first-degree relatives of 13 unrelated protein C or protein S deficient patients to assess the role of additional thrombotic risk factors and of protein C and protein S levels in the clinical expression of hereditary protein C and protein S deficiency. Fifty-seven relatives were

  8. Downregulation of placental S100P is associated with cadmium exposure in Guiyu, an e-waste recycling town in China

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Qingying; Zhou, Taimei [Department of Preventive Medicine, Shantou University Medical College, Shantou, Guangdong 515041 (China); Xu, Xijin; Guo, Yongyong [Analytic Cytology Laboratory, Shantou University Medical College, Shantou, Guangdong 515041 (China); Zhao, Zhiguo; Zhu, Min [Department of Preventive Medicine, Shantou University Medical College, Shantou, Guangdong 515041 (China); Li, Weiqiu [Analytic Cytology Laboratory, Shantou University Medical College, Shantou, Guangdong 515041 (China); Yi, Deqing [Department of Preventive Medicine, Shantou University Medical College, Shantou, Guangdong 515041 (China); Huo, Xia, E-mail: xhuo@stu.edu.cn [Analytic Cytology Laboratory, Shantou University Medical College, Shantou, Guangdong 515041 (China)

    2011-12-01

    Excessive release of heavy metals, especially cadmium (Cd) and lead (Pb), results from primitive electronic waste (e-waste) recycling activities in Guiyu, China, and has adverse effects on the health of local infants and pregnant women. We investigated the expression of placental S100P, a Ca{sup 2+}-binding protein, as a biological indicator of heavy-metal environmental pollution in pregnant women involved in these activities and constantly exposed to Cd and Pb. We included 105 pregnant women in the study: 55 from Guiyu and 50 from Shantou, an area not involved in e-waste recycling. The placental concentrations of Cd and Pb (PCCd, PCPb) after birth were measured by graphite-furnace atomic-absorption spectrometry. S100P mRNA expression was determined by semi-quantitative RT-PCR and real-time quantitative PCR. S100P protein expression was examined by western blot analysis and immunohistochemistry. The expression of metallothionein (MT), previously found upregulated after heavy metal contamination, was used for comparison. Placentas from Guiyu women showed 62.8% higher Cd concentrations, higher MT levels, and lower S100P protein levels than placentas from Shantou women. Furthermore, PCCd was negatively correlated with S100P protein expression and positively with MT expression, with no correlation between PCPb and S100P or MT expression. The PCCd-associated downregulation of S100P in placentas from Guiyu women suggests that S100P might be an effective biological indicator in the placental response to Cd toxicity in areas of e-waste recycling.

  9. Downregulation of placental S100P is associated with cadmium exposure in Guiyu, an e-waste recycling town in China

    International Nuclear Information System (INIS)

    Zhang, Qingying; Zhou, Taimei; Xu, Xijin; Guo, Yongyong; Zhao, Zhiguo; Zhu, Min; Li, Weiqiu; Yi, Deqing; Huo, Xia

    2011-01-01

    Excessive release of heavy metals, especially cadmium (Cd) and lead (Pb), results from primitive electronic waste (e-waste) recycling activities in Guiyu, China, and has adverse effects on the health of local infants and pregnant women. We investigated the expression of placental S100P, a Ca 2+ -binding protein, as a biological indicator of heavy-metal environmental pollution in pregnant women involved in these activities and constantly exposed to Cd and Pb. We included 105 pregnant women in the study: 55 from Guiyu and 50 from Shantou, an area not involved in e-waste recycling. The placental concentrations of Cd and Pb (PCCd, PCPb) after birth were measured by graphite-furnace atomic-absorption spectrometry. S100P mRNA expression was determined by semi-quantitative RT-PCR and real-time quantitative PCR. S100P protein expression was examined by western blot analysis and immunohistochemistry. The expression of metallothionein (MT), previously found upregulated after heavy metal contamination, was used for comparison. Placentas from Guiyu women showed 62.8% higher Cd concentrations, higher MT levels, and lower S100P protein levels than placentas from Shantou women. Furthermore, PCCd was negatively correlated with S100P protein expression and positively with MT expression, with no correlation between PCPb and S100P or MT expression. The PCCd-associated downregulation of S100P in placentas from Guiyu women suggests that S100P might be an effective biological indicator in the placental response to Cd toxicity in areas of e-waste recycling.

  10. The role of S100 genes in breast cancer progression.

    LENUS (Irish Health Repository)

    McKiernan, Eadaoin

    2012-02-01

    The S100 gene family encode low molecular weight proteins implicated in cancer progression. In this study, we analyzed the expression of four S100 genes in one cohort of patients with breast cancer and 16 S100 genes in a second cohort. In both cohorts, the expression of S100A8 and S1009 mRNA level was elevated in high-grade compared to low-grade tumors and in estrogen receptor-negative compared to estrogen receptor-positive tumors. None of the S100 transcripts investigated were significantly associated with the presence of lymph node metastasis. Notably, multiple S100 genes, including S100A1, S100A2, S100A4, S100A6, S100A8, S100A9, S100A10, S100A11, and S100A14 were upregulated in basal-type breast cancers compared to non-basal types. Using Spearman\\'s correlation analysis, several S100 transcripts correlated significantly with each other, the strongest correlation has been found between S100A8 and S100A9 (r = 0.889, P < 0.001, n = 295). Of the 16 S100 transcripts investigated, only S100A11 and S100A14 were significantly associated with patient outcome. Indeed, these two transcripts predicted outcome in the cohort of patients that did not receive systemic adjuvant therapy. Based on our findings, we conclude that the different S100 genes play varying roles in breast cancer progression. Specific S100 genes are potential targets for the treatment of basal-type breast cancers.

  11. The role of S100 genes in breast cancer progression.

    LENUS (Irish Health Repository)

    McKiernan, Eadaoin

    2011-06-01

    The S100 gene family encode low molecular weight proteins implicated in cancer progression. In this study, we analyzed the expression of four S100 genes in one cohort of patients with breast cancer and 16 S100 genes in a second cohort. In both cohorts, the expression of S100A8 and S1009 mRNA level was elevated in high-grade compared to low-grade tumors and in estrogen receptor-negative compared to estrogen receptor-positive tumors. None of the S100 transcripts investigated were significantly associated with the presence of lymph node metastasis. Notably, multiple S100 genes, including S100A1, S100A2, S100A4, S100A6, S100A8, S100A9, S100A10, S100A11, and S100A14 were upregulated in basal-type breast cancers compared to non-basal types. Using Spearman\\'s correlation analysis, several S100 transcripts correlated significantly with each other, the strongest correlation has been found between S100A8 and S100A9 (r = 0.889, P < 0.001, n = 295). Of the 16 S100 transcripts investigated, only S100A11 and S100A14 were significantly associated with patient outcome. Indeed, these two transcripts predicted outcome in the cohort of patients that did not receive systemic adjuvant therapy. Based on our findings, we conclude that the different S100 genes play varying roles in breast cancer progression. Specific S100 genes are potential targets for the treatment of basal-type breast cancers.

  12. Purification and partial characterization of canine S100A12.

    Science.gov (United States)

    Heilmann, Romy M; Suchodolski, Jan S; Steiner, Jörg M

    2010-12-01

    Canine S100A12 (cS100A12) is a calcium-binding protein of the S100 superfamily of EF-hand proteins, and its expression is restricted to neutrophils and monocytes. Interaction of S100A12 with the receptor for advanced glycation end products (RAGE) has been suggested to play a central role in inflammation. Moreover, S100A12 has been shown to represent a sensitive and specific marker for gastrointestinal inflammation in humans. Only human, porcine, bovine, and rabbit S100A12 have been purified to date, and an immunoassay for the quantification of S100A12 is available only for humans. Therefore, the aim of this study was to develop a protocol for the purification of S100A12 and to partially characterize this protein in the dog (Canis lupus familiaris) as a prelude to the development of an immunologic method for its detection and quantification in canine serum and fecal specimens. Leukocytes were isolated from canine whole blood by dextran sedimentation, and canine S100A12 was extracted from the cytosol fraction of these cells. Further purification of cS100A12 comprised of ammonium sulfate precipitation, hydrophobic interaction chromatography, and strong cation- and anion-exchange column chromatography. Canine S100A12 was successfully purified from canine whole blood. The relative molecular mass of the protein was estimated at 10,379.5 and isoelectric focusing revealed an isoelectric point of 6.0. The approximate specific absorbance of cS100A12 at 280 nm was determined to be 1.78 for a 1 mg/ml solution. The N-terminal AA sequence of the first 15 residues of cS100A12 was Thr-Lys-Leu-Glu-Asp-His-X-Glu-Gly-Ile-Val-Asp-Val-Phe-His, and revealed 100% identity with the predicted protein sequence available through the canine genome project. Sequence homology for the 14 N-terminal residues identified for cS100A12 with those of feline, bovine, porcine, and human S100A12 was 78.6%. We conclude that canine S100A12 can be successfully purified from canine whole blood using the

  13. Correlation between Amitriptyline-Induced Cardiotoxic Effects and Cardiac S100b Protein in Isolated Rat Hearts

    Directory of Open Access Journals (Sweden)

    Nil Hocaoğlu

    2016-12-01

    Full Text Available Background: Amitriptyline is an important cause of mortality due to its cardiovascular toxicity. Aims: To investigate the changes in levels of cardiac S100b protein on amitriptyline-induced cardiotoxicity and also to examine the correlation between amitriptyline-induced cardiotoxic effects and cardiac S100b protein in an isolated rat heart model. Study Design: Animal experimentation, isolated heart model. Methods: After a stabilization period, isolated hearts were randomized to two groups (n=5 and n=7. In the control group, isolated hearts were subjected to an infusion of 5% dextrose for 60 minutes. In the amitriptyline group, 5.5×10-5 M amitriptyline was infused for 60 minutes to achieve amitriptyline toxicity. After the infusion period, heart tissues were removed for histological examination. Results: In comparison to control treatment, amitriptyline infusion decreased left ventricular developed pressure (LVDP, dp/dtmax and heart rate (HR and significantly prolonged QRS duration (p<0.05. The semiquantitative scores for S100b protein levels in amitriptyline-infused hearts were higher than in the control group (p<0.01. At the end of the experiment, in the amitriptyline-infused group, significant correlations were found between LVDP and S100b protein scores (r=-0.807, p=0.003 and between QRS duration and S100b protein scores (r=0.859, p=0.001. Conclusion: Our results indicate that the S100b protein may be a helpful indicator or biomarker in studying the cardiotoxic effects of amitriptyline.

  14. Differentially expressed proteins among normal cervix, cervical intraepithelial neoplasia and cervical squamous cell carcinoma.

    Science.gov (United States)

    Zhao, Q; He, Y; Wang, X-L; Zhang, Y-X; Wu, Y-M

    2015-08-01

    To explore the differentially expressed proteins in normal cervix, cervical intraepithelial neoplasia (CIN) and cervical squamous cell carcinoma (CSCC) tissues by differential proteomics technique. Cervical tissues (including normal cervix, CIN and CSCC) were collected in Department of Gynecologic Oncology of Beijing Obstetrics and Gynecology Hospital. Two-dimensional fluorescence difference in gel electrophoresis (2-D DIGE) and DeCyder software were used to detect the differentially expressed proteins. Matrix-assisted laser desorption/ionization-time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS) was used to identify the differentially expressed proteins. Western blot (WB) and immunohistochemistry (IHC) were performed to validate the expressions of selected proteins among normal cervix, CIN and CSCC. 2-D DIGE images with high resolution and good repeatability were obtained. Forty-six differentially expressed proteins (27 up-regulated and 19 down-regulated) were differentially expressed among the normal cervix, CIN and CSCC. 26 proteins were successfully identified by MALDI-TOF/TOF MS. S100A9 (S100 calcium-binding protein A9) was the most significantly up-regulated protein. Eukaryotic elongation factor 1-alpha-1 (eEF1A1) was the most significantly down-regulated protein. Pyruvate kinase isozymes M2 (PKM2) was both up-regulated and down-regulated. The results of WB showed that with the increase in the severity of cervical lesions, the expression of S100A9 protein was significantly increased among the three groups (P = 0.010). The expression of eEF1A1 was reduced but without significant difference (P = 0.861). The expression of PKM2 was significantly reduced (P = 0.000). IHC showed that protein S100A9 was mainly expressed in the cytoplasm, and its positive expression rate was 20.0 % in normal cervix, 70.0 % in CIN and 100.0 % in CSCC, with a significant difference among them (P = 0.006). eEF1A1 was mainly expressed in the cell plasma, and its

  15. Correlation of expressions of S100A8 and S100A9 and its ...

    African Journals Online (AJOL)

    (S100A9) in nasopharyngeal carcinoma (NPC) tissues and their correlation with clinical pathological characteristics and ..... receptor (RAGE), a process which also activates .... S100A8 and S100A9 between Prostate Cancer and. BPH.

  16. Molecular dynamics simulation of S100B protein to explore ligand blockage of the interaction with p53 protein

    Science.gov (United States)

    Zhou, Zhigang; Li, Yumin

    2009-10-01

    As a tumor suppressor, p53 plays an important role in cancer suppression. The biological function of p53 as a tumor suppressor is disabled when it binds to S100B. Developing the ligands to block the S100B-p53 interaction has been proposed as one of the most important approaches to the development of anti-cancer agents. We screened a small compound library against the binding interface of S100B and p53 to identify potential compounds to interfere with the interaction. The ligand-binding effect on the S100B-p53 interaction was explored by molecular dynamics at the atomic level. The results show that the ligand bound between S100B and p53 propels the two proteins apart by about 2 Å compared to the unligated S100B-p53 complex. The binding affinity of S100B and p53 decreases by 8.5-14.6 kcal/mol after a ligand binds to the interface from the original unligated state of the S100B-p53 complex. Ligand-binding interferes with the interaction of S100B and p53. Such interference could impact the association of S100B and p53, which would free more p53 protein from the pairing with S100B and restore the biological function of p53 as a tumor suppressor. The analysis of the binding mode and ligand structural features would facilitate our effort to identify and design ligands to block S100B-p53 interaction effectively. The results from the work suggest that developing ligands targeting the interface of S100B and p53 could be a promising approach to recover the normal function of p53 as a tumor suppressor.

  17. Demonstration of S-100 protein in sustentacular cells of phaeochromocytomas and paragangliomas

    DEFF Research Database (Denmark)

    Schroder, H D; Johannsen, L

    1986-01-01

    to the sustentacular cells of normal paraganglia and adrenal medulla were found in all paragangliomas and in the benign and aggressively growing phaeochromocytomas. In the two malignant tumours no positive reaction was demonstrated. In one tumour the sustentacular cells were shown to contain glial fibrillary acidic......Eighteen phaeochromocytomas, including both sporadic and familial cases, four cervical paragangliomas, two jugular paragangliomas, and one abdominal paraganglioma were examined immunohistochemically for the presence of S-100 protein. Positive staining in cells morphologically similar...... protein further supporting their Schwann cell relationship. The number of S-100 positive cells varied considerably. They demonstrated a spindle celled or elongated configuration with long slender processes. The nature of the sustentacular cell proliferation, neoplastic versus reactive, is discussed....

  18. S100A16 promotes differentiation and contributes to a less aggressive tumor phenotype in oral squamous cell carcinoma

    International Nuclear Information System (INIS)

    Sapkota, Dipak; Bruland, Ove; Parajuli, Himalaya; Osman, Tarig A.; Teh, Muy-Teck; Johannessen, Anne C.; Costea, Daniela Elena

    2015-01-01

    Altered expression of S100A16 has been reported in human cancers, but its biological role in tumorigenesis is not fully understood. This study aimed to investigate the clinical significance and functional role of S100A16 in oral squamous cell carcinoma (OSCC) suppression. S100A16 mRNA and/or protein levels were examined by quantitative RT-PCR and immunohistochemistry in whole- and laser microdissected-specimens of normal human oral mucosa (NHOM, n = 65), oral dysplastic lesions (ODL, n = 21), OSCCs (n = 132) and positive cervical nodes (n = 17). S100A16 protein expression in OSCC was examined for correlations with clinicopathological variables and patient survival. S100A16 was over-expressed and knocked-down in OSCC-derived (CaLH3 and H357) cells by employing retroviral constructs to investigate its effects on cell proliferation, sphere formation and three dimensional (3D)-organotypic invasive abilities in vitro and tumorigenesis in a mouse xenograft model. Both S100A16 mRNA and protein levels were found to be progressively down-regulated from NHOM to ODL and OSCC. Low S100A16 protein levels in OSCC significantly correlated with reduced 10-year overall survival and poor tumor differentiation. Analysis of two external OSCC microarray datasets showed a positive correlation between the mRNA expression levels of S100A16 and keratinocyte differentiation markers. CaLH3 and H357 cell fractions enriched for differentiated cells either by lack of adherence to collagen IV or FACS sorting for low p75NTR expression expressed significantly higher S100A16 mRNA levels than the subpopulations enriched for less differentiated cells. Corroborating these findings, retroviral mediated S100A16 over-expression and knock-down in CaLH3 and H357 cells led to respective up- and down-regulation of differentiation markers. In vitro functional studies showed significant reduction in cell proliferation, sphere formation and 3D-invasive abilities of CaLH3 and H357 cells upon S100A16 over-expression

  19. Leukocyte and serum S100A8/S100A9 expression reflects disease activity in ANCA-associated vasculitis and glomerulonephritis

    Science.gov (United States)

    Pepper, Ruth J; Hamour, Sally; Chavele, Konstantia-Maria; Todd, Sarah K; Rasmussen, Niels; Flint, Shaun; Lyons, Paul A; Smith, Kenneth G C; Pusey, Charles D; Cook, H Terence; Salama, Alan D

    2013-01-01

    Antineutrophil cytoplasm antibody (ANCA)–associated vasculitis (AAV) commonly results in glomerulonephritis, in which neutrophils and monocytes have important roles. The heterodimer calprotectin (S100A8/S100A9, mrp8/14) is a Toll-like receptor-4 ligand found in neutrophils and monocytes and is elevated in inflammatory conditions. By immunohistochemistry of renal biopsies, patients with focal or crescentic glomerular lesions were found to have the highest expression of calprotectin and those with sclerotic the least. Serum levels of calprotectin as measured by ELISA were elevated in patients with active AAV and the levels decreased but did not normalize during remission, suggesting subclinical inflammation. Calprotectin levels in patients with limited systemic disease increased following treatment withdrawal and were significantly elevated in patients who relapsed compared with those who did not. As assessed by flow cytometry, patients with AAV had higher monocyte and neutrophil cell surface calprotectin expression than healthy controls, but this was not associated with augmented mRNA expression in CD14+ monocytes or CD16+ neutrophils. Thus, serum calprotectin is a potential disease biomarker in patients with AAV, and may have a role in disease pathogenesis. PMID:23423260

  20. S100A7 (Psoriasin), highly expressed in Ductal Carcinoma In Situ (DCIS), is regulated by IFN-gamma in mammary epithelial cells

    International Nuclear Information System (INIS)

    Petersson, Stina; Bylander, Anna; Yhr, Maria; Enerbäck, Charlotta

    2007-01-01

    The aim of the present work was to explore signal transduction pathways used in the regulation of S100A7 (psoriasin). Members of the S100 gene family participate in many important cellular functions. Psoriasin, S100A8 (calgranulin A) and S100A9 (calgranulin B) are expressed in ductal carcinoma in situ (DCIS), as well as in the hyperproliferative skin disease, psoriasis. In the latter condition, a disturbance in the STAT pathway has recently been reported. This pathway is implicated in the regulation of IFN-gamma, widely recognized as a key cytokine in psoriasis. IFN-gamma also exerts anti-tumor action in a number of tumor cell types, including breast cancer. We therefore examined the effect of IFN-gamma and STAT-signaling on the psoriasin expression. We established a TAC2 mouse mammary epithelial cell line with tetracycline-inducible psoriasin expression (Tet-Off). Viability in cell culture was estimated using MTS assay. Protein and gene expression were evaluated by Western blotting and quantitative real-time PCR. Statistical analyses were assessed using a one-tailed, paired t-test. We report the downregulation of psoriasin by IFN-gamma in the MDA-MB-468 breast cancer cell line, as well as the downregulation of psoriasin induced by anoikis in cell lines derived from different epithelial tissues. In contrast, IFN-gamma had no suppressive effect on calgranulin A or calgranulin B. IFN-gamma is an important activator of the STAT1 pathway and we confirmed an active signaling pathway in the cell lines that responded to IFN-gamma treatment. In contrast, in the SUM190 breast carcinoma cell line, IFN-gamma did not suppress the expression of endogenous psoriasin. Moreover, a reduced phosphorylation of the STAT1 protein was observed. We showed that IFN-gamma treatment and the inhibition of the transcription factor NFkappaB had a synergistic effect on psoriasin levels. Finally, in TAC2 cells with tetracycline-induced psoriasin expression, we observed the increased viability of

  1. S100A4 amplifies TGF-β-induced fibroblast activation in systemic sclerosis

    DEFF Research Database (Denmark)

    Tomcik, Michal; Palumbo-Zerr, Katrin; Zerr, Pawel

    2015-01-01

    (SSc). METHODS: The expression of S100A4 was analysed in human samples, murine models of SSc and in cultured fibroblasts by real-time PCR, immunohistochemistry and western blot. The functional role of S100A4 was evaluated using siRNA, overexpression, recombinant protein and S100A4 knockout (S100A4...... or stimulation with recombinant S100A4 induced an activated phenotype in resting normal fibroblasts. In contrast, knockdown of S100A4 reduced the pro-fibrotic effects of TGF-β and decreased the release of collagen. S100A4(-/-) mice were protected from bleomycin-induced skin fibrosis with reduced dermal...

  2. S100A9 is a novel ligand of EMMPRIN that promotes melanoma metastasis.

    Science.gov (United States)

    Hibino, Toshihiko; Sakaguchi, Masakiyo; Miyamoto, Shoko; Yamamoto, Mami; Motoyama, Akira; Hosoi, Junichi; Shimokata, Tadashi; Ito, Tomonobu; Tsuboi, Ryoji; Huh, Nam-Ho

    2013-01-01

    The calcium-binding proteins S100A8 and S100A9 can dimerize to form calprotectin, the release of which during tissue damage has been implicated in inflammation and metastasis. However, receptor(s) mediating the physiologic and pathophysiologic effects of this damage-associated "danger signal" are uncertain. In this study, searching for candidate calprotectin receptors by affinity isolation-mass spectrometry, we identified the cell surface glycoprotein EMMPRIN/BASIGIN (CD147/BSG). EMMPRIN specifically bound to S100A9 but not S100A8. Induction of cytokines and matrix metalloproteases (MMP) by S100A9 was markedly downregulated in melanoma cells by attenuation of EMMPRIN. We found that EMMPRIN signaling used the TNF receptor-associated factor TRAF2 distinct from the known S100-binding signaling pathway mediated by RAGE (AGER). S100A9 strongly promoted migration when EMMPRIN was highly expressed, independent of RAGE, whereas EMMPRIN blockade suppressed migration by S100A9. Immunohistologic analysis of melanomas revealed that EMMPRIN was expressed at both the invasive edge of lesions and the adjacent epidermis, where S100A9 was also strongly expressed. In epidermal-specific transgenic mice, tail vein-injected melanoma accumulated in skin expressing S100A9 but not S100A8. Together, our results establish EMMPRIN as a receptor for S100A9 and suggest the therapeutic use in targeting S100A9-EMMPRIN interactions.

  3. Peptide mimetic of the S100A4 protein modulates peripheral nerve regeneration and attenuates the progression of neuropathy in myelin protein P0 null mice

    DEFF Research Database (Denmark)

    Moldovan, Mihai; Pinchenko, Volodymyr; Dmytriyeva, Oksana

    2013-01-01

    and mimicked the S100A4-induced neuroprotection in brain trauma. Here, we investigated a possible function of S100A4 and its mimetics in the pathologies of the peripheral nervous system (PNS). We found that S100A4 was expressed in the injured PNS and that its peptide mimetic (H3) affected the regeneration......, these effects were attributed to the modulatory effect of H3 on initial axonal sprouting. In contrast to the modest effect of H3 on the time course of regeneration, H3 had a long-term neuroprotective effect in the myelin protein P0 null mice, a model of dysmyelinating neuropathy (Charcot-Marie-Tooth type 1...... disease), where the peptide attenuated the deterioration of nerve conduction, demyelination and axonal loss. From these results, S100A4 mimetics emerge as a possible means to enhance axonal sprouting and survival, especially in the context of demyelinating neuropathies with secondary axonal loss...

  4. Membrane interactions of S100A12 (Calgranulin C.

    Directory of Open Access Journals (Sweden)

    Assuero F Garcia

    Full Text Available S100A12 (Calgranulin C is a small acidic calcium-binding peripheral membrane protein with two EF-hand structural motifs. It is expressed in macrophages and lymphocytes and highly up-regulated in several human inflammatory diseases. In pigs, S100A12 is abundant in the cytosol of granulocytes, where it is believed to be involved in signal modulation of inflammatory process. In this study, we investigated the interaction of the porcine S100A12 with phospholipid bilayers and the effect that ions (Ca(2+, Zn(2+ or both together have in modifying protein-lipid interactions. More specifically, we intended to address issues such as: (1 is the protein-membrane interaction modulated by the presence of ions? (2 is the protein overall structure affected by the presence of the ions and membrane models simultaneously? (3 what are the specific conformational changes taking place when ions and membranes are both present? (4 does the protein have any kind of molecular preferences for a specific lipid component? To provide insight into membrane interactions and answer those questions, synchrotron radiation circular dichroism spectroscopy, fluorescence spectroscopy, and surface plasmon resonance were used. The use of these combined techniques demonstrated that this protein was capable of interacting both with lipids and with ions in solution, and enabled examination of changes that occur at different levels of structure organization. The presence of both Ca(2+ and Zn(2+ ions modify the binding, conformation and thermal stability of the protein in the presence of lipids. Hence, these studies examining molecular interactions of porcine S100A12 in solution complement the previously determined crystal structure information on this family of proteins, enhancing our understanding of its dynamics of interaction with membranes.

  5. S100A6 - New facts and features

    Energy Technology Data Exchange (ETDEWEB)

    Lesniak, Wieslawa; Slomnicki, Lukasz P. [Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology, 02-093 Warsaw (Poland); Filipek, Anna, E-mail: a.filipek@nencki.gov.pl [Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology, 02-093 Warsaw (Poland)

    2009-12-25

    S100A6 (calcyclin) is a 10.5 kDa Ca{sup 2+}-binding protein that belongs to the S100 protein family. S100A6 contains two EF-hand motifs responsible for binding of Ca{sup 2+}. It also binds Zn{sup 2+} through not yet identified structures. Binding of Ca{sup 2+} induces a conformational change in the S100A6 molecule which in consequence increases its overall hydrophobicity and allows for interaction with target proteins. S100A6 was found in different mammalian and avian (chicken) tissues. A high level of S100A6 is observed in epithelial cells, fibroblasts and in different kinds of cancer cells. The function of S100A6 is not clear at present, but it has been suggested that it may be involved in cell proliferation, cytoskeletal dynamics and tumorigenesis. Additionally, S100A6 might have some extracellular activities. This review presents new facts and features concerning the S100A6 protein.

  6. The S100A4 Oncoprotein Promotes Prostate Tumorigenesis in a Transgenic Mouse Model

    DEFF Research Database (Denmark)

    Siddique, Hifzur R; Adhami, Vaqar M; Parray, Aijaz

    2013-01-01

    earlier showed that S100A4 expression progressively increases in prostatic tissues with the advancement of prostate cancer (CaP) in TRAMP, an autochthonous mouse model. To study the functional significance of S100A4 in CaP, we generated a heterozygously deleted S100A4 (TRAMP/S100A4(+/-)) genotype...... (intracellular and extracellular) forms. We observed that 1) the growth-promoting effect of S100A4 is due to its activation of NFκB, 2) S100A4-deficient tumors exhibit reduced NFκB activity, 3) S100A4 regulates NFκB through the RAGE receptor, and 4) S100A4 and RAGE co-localize in prostatic tissues of mice......S100A4, a calcium-binding protein, is known for its role in the metastatic spread of tumor cells, a late event of cancer disease. This is the first report showing that S100A4 is not merely a metastatic protein but also an oncoprotein that plays a critical role in the development of tumors. We...

  7. The metastasis-associated Mts1(S100A4) protein could act as an angiogenic factor

    DEFF Research Database (Denmark)

    Ambartsumian, N; Klingelhöfer, Jörg; Grigorian, M

    2001-01-01

    The involvement of Mts1(S100A4), a small Ca(2+)-binding protein in tumor progression and metastasis had been demonstrated. However, the mechanism by which mts1(S100A4) promoted metastasis had not been identified. Here we demonstrated that Mts1(S100A4) had significant stimulatory effect on the ang...

  8. Opposing Effects of Zac1 and Curcumin on AP-1-Regulated Expressions of S100A7.

    Directory of Open Access Journals (Sweden)

    Yu-Wen Chu

    Full Text Available ZAC, an encoding gene mapped at chromosome 6q24-q25 within PSORS1, was previously found over-expressed in the lower compartment of the hyperplastic epidermis in psoriatic lesions. Cytokines produced in the inflammatory dermatoses may drive AP-1 transcription factor to induce responsive gene expressions. We demonstrated that mZac1 can enhance AP-1-responsive S100A7 expression of which the encoding gene was located in PSORS4 with HaCaT keratinocytes. However, the mZac1-enhanced AP-1 transcriptional activity was suppressed by curcumin, indicating the anti-inflammatory property of this botanical agent and is exhibited by blocking the AP-1-mediated cross-talk between PSORS1 and PSORS4. Two putative AP-1-binding sites were found and demonstrated to be functionally important in the regulation of S100A7 promoter activity. Moreover, we found curcumin reduced the DNA-binding activity of AP-1 to the recognition element located in the S100A7 promoter. The S100A7 expression was found to be upregulated in the lesioned epidermis of atopic dermatitis and psoriasis, which is where this keratinocyte-derived chemoattractant engaged in the pro-inflammatory feedback loop. Understanding the regulatory mechanism of S100A7 expression will be helpful to develop therapeutic strategies for chronic inflammatory dermatoses via blocking the reciprocal stimuli between the inflammatory cells and keratinocytes.

  9. S100B Protein concentration in milk-formulas for preterm and term infants. Correlation with industrial preparation procedures.

    Science.gov (United States)

    Nigro, Francesco; Gagliardi, Luigi; Ciotti, Sabina; Galvano, Fabio; Pietri, Amedeo; Tina, Gabriella Lucia; Cavallaro, Daniela; La Fauci, Luca; Iacopino, Leonardo; Bognanno, Matteo; Li Volti, Giovanni; Scacco, Antonio; Michetti, Fabrizio; Gazzolo, Diego

    2008-05-01

    Human milk S100B protein possesses important neurotrophic properties. However, in some conditions human milk is substituted by milk formulas. The aims of the present study were: to assess S100B concentrations in milk formulas, to verify any differences in S100B levels between preterm and term infant formulas and to evaluate the impact of industrial preparation at predetermined phases on S100B content. Two different set of samples were tested: (i) commercial preterm (n = 36) and term (n = 36) infant milk formulas; ii) milk preterm (n = 10) and term infant (n = 10) formulas sampled at the following predetermined industrial preparation time points: skimmed cow milk (Time 0); after protein sources supplementation (Time 1); after pasteurization (Time 2); after spray-drying (Time 3). Our results showed that S100B concentration in preterm formulas were higher than in term ones (p 0.05) at Time 2, whereas a significant (p pasteurization but not spry-drying. New feeding strategies in preterm and term infants are therefore warranted in order to preserve S100B protein during industrial preparation.

  10. S100A14 is a novel independent prognostic biomarker in the triple-negative breast cancer subtype

    DEFF Research Database (Denmark)

    Ehmsen, Sidse; Hansen, Lea Tykgaard; Bak, Martin

    2015-01-01

    Triple-negative breast cancer (TNBC) represents a heterogeneous subgroup with generally poor outcome and lack of an effective targeted therapy. Prognostic or predictive biomarkers to guide treatment decisions for this group of patients are needed. To evaluate the potential of S100A14 protein...... as a novel biomarker in TNBC, the protein expression of S100A14 was correlated with clinical outcomes in a Pilot Sample set and a Danish cohort of predominantly TNBC patients. Kaplan-Meier analysis identified a prognostic impact of S100A14 on disease-free survival and overall survival, showing that tumors......-), had equally poor outcomes as those with tumor-positive axillary lymph nodes (N+), while TNBC/N- patients with low S100A14 expression had a significantly better disease free survival (p = 0.013). Multivariate analysis revealed that S100A14 is an independent prognostic factor for TNBC patients (p = 0...

  11. Comparison between capillary, venous and arterial levels of protein S100B in patients with severe brain pathology

    DEFF Research Database (Denmark)

    Astrand, Ramona; Romner, Bertil; Reinstrup, Peter

    2012-01-01

    of the study was to investigate the relation between capillary, venous and arterial measurements of protein S100B, primarily by determining whether capillary S100B differ from venous and if capillary S100B can predict venous S100B levels, and secondarily, if arterial S100B samples can substitute venous samples...... in severely brain-injured patients....

  12. S100B protein, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor in human milk.

    Directory of Open Access Journals (Sweden)

    Ruisong Li

    Full Text Available Human milk contains a wide variety of nutrients that contribute to the fulfillment of its functions, which include the regulation of newborn development. However, few studies have investigated the concentrations of S100B protein, brain-derived neurotrophic factor (BDNF, and glial cell line-derived neurotrophic factor (GDNF in human milk. The associations of the concentrations of S100B protein, BDNF, and GDNF with maternal factors are not well explored.To investigate the concentrations of S100B protein, BDNF, and GDNF in human milk and characterize the maternal factors associated with their levels in human milk, human milk samples were collected at days 3, 10, 30, and 90 after parturition. Levels of S100B protein, BDNF, and GDNF, and their mRNAs in the samples were detected. Then, these concentrations were compared with lactation and other maternal factors. S100B protein levels in human milk samples collected at 3, 10, 30, and 90 d after parturition were 1249.79±398.10, 1345.05±539.16, 1481.83±573.30, and 1414.39±621.31 ng/L, respectively. On the other hand, the BDNF concentrations in human milk samples were 10.99±4.55, 13.01±5.88, 13.35±6.43, and 2.83±5.47 µg/L, while those of GDNF were 10.90±1.65, 11.38±1., 11.29±3.10, and 11.40±2.21 g/L for the same time periods. Maternal post-pregnancy body mass index was positively associated with S100B levels in human milk (r = 0.335, P = 0.030<0.05. In addition, there was a significant correlation between the levels of S100B protein and BDNF (z = 2.09, P = 0.037<0.05. Delivery modes were negatively associated with the concentration of GDNF in human milk.S100B protein, BDNF, and GDNF are present in all samples of human milk, and they may be responsible for the long term effects of breast feeding.

  13. Calbindin and S100 protein expression in the developing inner ear in mice

    Czech Academy of Sciences Publication Activity Database

    Buckiová, Daniela; Syka, Josef

    2009-01-01

    Roč. 513, č. 5 (2009), s. 469-482 ISSN 0021-9967 R&D Projects: GA ČR GA309/07/1336; GA MŠk(CZ) LC554 Institutional research plan: CEZ:AV0Z50390512 Keywords : Calcium binding proteins * Immunohistochemistry * Development Subject RIV: FH - Neurology Impact factor: 3.718, year: 2009

  14. Quantitative analysis of differentially expressed saliva proteins in human immunodeficiency virus type 1 (HIV-1) infected individuals

    International Nuclear Information System (INIS)

    Zhang, Nawei; Zhang, Zhenyu; Feng, Shan; Wang, Qingtao; Malamud, Daniel; Deng, Haiteng

    2013-01-01

    Highlights: ► A high-throughput method for profiling and quantification of the differentially expressed proteins in saliva samples was developed. ► Identified that DMBT1, S100A7, S100A8, S100A9 and alpha defensin were up-regulated in saliva from HIV-1 seropositive patients. ► Established analytical strategies are translatable to the clinical setting. -- Abstract: In the present study, we have established a new methodology to analyze saliva proteins from HIV-1-seropositive patients before highly active antiretroviral therapy (HAART) and seronegative controls. A total of 593 and 601 proteins were identified in the pooled saliva samples from 5 HIV-1 subjects and 5 controls, respectively. Forty-one proteins were found to be differentially expressed. Bioinformatic analysis of differentially expressed salivary proteins showed an increase of antimicrobial proteins and decrease of protease inhibitors upon HIV-1 infection. To validate some of these differentially expressed proteins, a high-throughput quantitation method was established to determine concentrations of 10 salivary proteins in 40 individual saliva samples from 20 seropositive patients before HAART and 20 seronegative subjects. This method was based on limited protein separation within the zone of the stacking gel of the 1D SDS PAGE and using isotope-coded synthetic peptides as internal standards. The results demonstrated that a combination of protein profiling and targeted quantitation is an efficient method to identify and validate differentially expressed salivary proteins. Expression levels of members of the calcium-binding S100 protein family and deleted in malignant brain tumors 1 protein (DMBT1) were up-regulated while that of Mucin 5B was down-regulated in HIV-1 seropositive saliva samples, which may provide new perspectives for monitoring HIV-infection and understanding the mechanism of HIV-1 infectivity

  15. Quantitative analysis of differentially expressed saliva proteins in human immunodeficiency virus type 1 (HIV-1) infected individuals

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Nawei; Zhang, Zhenyu [Beijing Chaoyang Hospital Affiliated Capital Medical University, Beijing (China); Feng, Shan [MOE Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University, Beijing (China); Wang, Qingtao [Beijing Chaoyang Hospital Affiliated Capital Medical University, Beijing (China); Malamud, Daniel [NYU College of Dentistry, 345 East 24th Street, New York, NY 10010 (United States); Deng, Haiteng, E-mail: dht@mail.tsinghua.edu.cn [MOE Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University, Beijing (China)

    2013-04-24

    Highlights: ► A high-throughput method for profiling and quantification of the differentially expressed proteins in saliva samples was developed. ► Identified that DMBT1, S100A7, S100A8, S100A9 and alpha defensin were up-regulated in saliva from HIV-1 seropositive patients. ► Established analytical strategies are translatable to the clinical setting. -- Abstract: In the present study, we have established a new methodology to analyze saliva proteins from HIV-1-seropositive patients before highly active antiretroviral therapy (HAART) and seronegative controls. A total of 593 and 601 proteins were identified in the pooled saliva samples from 5 HIV-1 subjects and 5 controls, respectively. Forty-one proteins were found to be differentially expressed. Bioinformatic analysis of differentially expressed salivary proteins showed an increase of antimicrobial proteins and decrease of protease inhibitors upon HIV-1 infection. To validate some of these differentially expressed proteins, a high-throughput quantitation method was established to determine concentrations of 10 salivary proteins in 40 individual saliva samples from 20 seropositive patients before HAART and 20 seronegative subjects. This method was based on limited protein separation within the zone of the stacking gel of the 1D SDS PAGE and using isotope-coded synthetic peptides as internal standards. The results demonstrated that a combination of protein profiling and targeted quantitation is an efficient method to identify and validate differentially expressed salivary proteins. Expression levels of members of the calcium-binding S100 protein family and deleted in malignant brain tumors 1 protein (DMBT1) were up-regulated while that of Mucin 5B was down-regulated in HIV-1 seropositive saliva samples, which may provide new perspectives for monitoring HIV-infection and understanding the mechanism of HIV-1 infectivity.

  16. Expression of cancer-associated fibroblast-related proteins differs between invasive lobular carcinoma and invasive ductal carcinoma.

    Science.gov (United States)

    Park, Cheol Keun; Jung, Woo Hee; Koo, Ja Seung

    2016-08-01

    Cancer-associated fibroblasts (CAFs) are classified into various functional subtypes such as fibroblast activation protein-α (FAP-α), fibroblast specific protein-1 (FSP-1), platelet-derived growth factor receptor-α (PDGFR-α), and PDGFR-β. In this study, we compared the expression of CAF-related proteins in invasive lobular carcinoma (ILC) with those in invasive carcinoma of no special type (NST) and assessed the implications of the differences observed. Using tissue microarrays of 104 ILC and 524 invasive carcinoma (NST) cases, immunohistochemistry for CAF-related proteins [podoplanin, prolyl 4-hydroxylase, FAP-α, FSP-1/S100A4, PDGFR-α, PDGFR-β, and chondroitin sulfate proteoglycan (NG2)] was conducted. In invasive carcinoma (NST), tumor cells expressed a high level of PDGFR-α, whereas ILC tumor cells expressed high levels of podoplanin, prolyl 4-hydroxylase, FAP-α, and FSP-1/S100A4. In stromal cells of invasive carcinoma (NST), high expression levels of prolyl 4-hydroxylase, PDGFR-α, and NG2 were observed, whereas ILC stromal cells expressed high levels of FAP-α, FSP-1/S100A4, and PDGFR-β. In ILC, tumoral FSP-1/S100A4 positivity was associated with higher Ki-67 labeling index (p = 0.010) and non-luminal A type cancer (p = 0.014). Stromal PDGFR-α positivity was associated with lymph node metastasis (p = 0.011). On survival analysis of entire cases, tumoral FSP-1/S100A4 positivity (p = 0.002), stromal podoplanin positivity (p = 0.041), and stromal FSP-1/S100A4 negativity (p = 0.041) were associated with shorter disease-free survival; only tumoral FSP-1/S100A4 positivity (p = 0.044) was associated with shorter overall survival. In ILC, the expression of FAP-α and FSP-1/S100A4 was higher in both tumor and stromal cells than that observed in invasive carcinoma (NST). These results indicate that CAFs are a potential target in ILC treatment.

  17. Isolation of dendritic-cell-like S100β-positive cells in rat anterior pituitary gland.

    Science.gov (United States)

    Horiguchi, Kotaro; Fujiwara, Ken; Yoshida, Saishu; Higuchi, Masashi; Tsukada, Takehiro; Kanno, Naoko; Yashiro, Takashi; Tateno, Kozue; Osako, Shunji; Kato, Takako; Kato, Yukio

    2014-07-01

    S100β-protein-positive cells in the anterior pituitary gland appear to possess multifunctional properties. Because of their pleiotropic features, S100β-positive cells are assumed to be of a heterogeneous or even a non-pituitary origin. The observation of various markers has allowed these cells to be classified into populations such as stem/progenitor cells, epithelial cells, astrocytes and dendritic cells. The isolation and characterization of each heterogeneous population is a prerequisite for clarifying the functional character and origin of the cells. We attempt to isolate two of the subpopulations of S100β-positive cells from the anterior lobe. First, from transgenic rats that express green fluorescent protein (GFP) driven by the S100β protein promoter, we fractionate GFP-positive cells with a cell sorter and culture them so that they can interact with laminin, a component of the extracellular matrix. We observe that one morphological type of GFP-positive cells possesses extended cytoplasmic processes and shows high adhesiveness to laminin (process type), whereas the other is round in shape and exhibits low adherence to laminin (round type). We successfully isolate cells of the round type from the cultured GFP-positive cells by taking advantage of their low affinity to laminin and then measure mRNA levels of the two cell types by real-time polymerase chain reaction. The resultant data show that the process type expresses vimentin (mesenchymal cell marker) and glial fibrillary acidic protein (astrocyte marker). The round type expresses dendritic cell markers, CD11b and interleukin-6. Thus, we found a method for isolating dendritic-cell-like S100β-positive cells by means of their property of adhering to laminin.

  18. Curcumin upregulates S100 expression and improves regeneration of the sciatic nerve following its complete amputation in mice

    Directory of Open Access Journals (Sweden)

    Guo-min Liu

    2016-01-01

    Full Text Available The repair of peripheral nerve injury after complete amputation is difficult, and even with anastomosis, the rapid recovery of nerve function remains challenging. Curcumin, extracted from plants of the genus Curcuma, has been shown to have anti-oxidant and anti-inflammatory properties and to improve sciatic nerve crush injury in rats. Here, we determined whether curcumin had neuroprotective effects following complete peripheral nerve amputation injury. BALB/c mice underwent complete sciatic nerve amputation, followed by an immediate epineurium anastomosis. Mice were intragastrically administered curcumin at doses of 40 (high, 20 (moderate, and 10 mg/kg/d (low for 1 week. We found that myelin in the mice of the high- and moderate-dose curcumin groups appeared with regular shape, uniform thickness, clear boundary, and little hyperplasia surrounding the myelin. High and moderate doses of curcumin markedly improved both action potential amplitude of the sciatic nerves and the conduction velocity of the corresponding motor neurons, and upregulated mRNA and protein expression of S100, a marker for Schwann cell proliferation, in L4–6 spinal cord segments. These results suggest that curcumin is effective in promoting the repair of complete sciatic nerve amputation injury and that the underlying mechanism may be associated with upregulation of S100 expression.

  19. Expression and function of S100A8/A9 (calprotectin) in human typhoid fever and the murine Salmonella model.

    Science.gov (United States)

    De Jong, Hanna K; Achouiti, Ahmed; Koh, Gavin C K W; Parry, Christopher M; Baker, Stephen; Faiz, Mohammed Abul; van Dissel, Jaap T; Vollaard, Albert M; van Leeuwen, Ester M M; Roelofs, Joris J T H; de Vos, Alex F; Roth, Johannes; van der Poll, Tom; Vogl, Thomas; Wiersinga, Willem Joost

    2015-04-01

    Typhoid fever, caused by the Gram-negative bacterium Salmonella enterica serovar Typhi, is a major cause of community-acquired bacteremia and death worldwide. S100A8 (MRP8) and S100A9 (MRP14) form bioactive antimicrobial heterodimers (calprotectin) that can activate Toll-like receptor 4, promoting lethal, endotoxin-induced shock and multi-organ failure. We aimed to characterize the expression and function of S100A8/A9 in patients with typhoid fever and in a murine invasive Salmonella model. S100A8/A9 protein levels were determined in acute phase plasma or feces from 28 Bangladeshi patients, and convalescent phase plasma from 60 Indonesian patients with blood culture or PCR-confirmed typhoid fever, and compared to 98 healthy control subjects. To functionally characterize the role of S100A8/A9, we challenged wildtype (WT) and S100A9-/- mice with S. Typhimurium and determined bacterial loads and inflammation 2- and 5- days post infection. We further assessed the antimicrobial function of recombinant S100A8/A9 on S. Typhimurium and S. Typhi replication in vitro. Typhoid fever patients demonstrated a marked increase of S100A8/A9 in acute phase plasma and feces and this increases correlated with duration of fever prior to admission. S100A8/A9 directly inhibited the growth of S. Typhimurium and S. Typhi in vitro in a dose and time dependent fashion. WT mice inoculated with S. Typhimurium showed increased levels of S100A8/A9 in both the liver and the systemic compartment but S100A9-/- mice were indistinguishable from WT mice with respect to bacterial growth, survival, and inflammatory responses, as determined by cytokine release, histopathology and organ injury. S100A8/A9 is markedly elevated in human typhoid, correlates with duration of fever prior to admission and directly inhibits the growth of S. Typhimurium and S. Typhi in vitro. Despite elevated levels in the murine invasive Salmonella model, S100A8/A9 does not contribute to an effective host response against S

  20. Expression and function of S100A8/A9 (calprotectin in human typhoid fever and the murine Salmonella model.

    Directory of Open Access Journals (Sweden)

    Hanna K De Jong

    2015-04-01

    Full Text Available Typhoid fever, caused by the Gram-negative bacterium Salmonella enterica serovar Typhi, is a major cause of community-acquired bacteremia and death worldwide. S100A8 (MRP8 and S100A9 (MRP14 form bioactive antimicrobial heterodimers (calprotectin that can activate Toll-like receptor 4, promoting lethal, endotoxin-induced shock and multi-organ failure. We aimed to characterize the expression and function of S100A8/A9 in patients with typhoid fever and in a murine invasive Salmonella model.S100A8/A9 protein levels were determined in acute phase plasma or feces from 28 Bangladeshi patients, and convalescent phase plasma from 60 Indonesian patients with blood culture or PCR-confirmed typhoid fever, and compared to 98 healthy control subjects. To functionally characterize the role of S100A8/A9, we challenged wildtype (WT and S100A9-/- mice with S. Typhimurium and determined bacterial loads and inflammation 2- and 5- days post infection. We further assessed the antimicrobial function of recombinant S100A8/A9 on S. Typhimurium and S. Typhi replication in vitro. Typhoid fever patients demonstrated a marked increase of S100A8/A9 in acute phase plasma and feces and this increases correlated with duration of fever prior to admission. S100A8/A9 directly inhibited the growth of S. Typhimurium and S. Typhi in vitro in a dose and time dependent fashion. WT mice inoculated with S. Typhimurium showed increased levels of S100A8/A9 in both the liver and the systemic compartment but S100A9-/- mice were indistinguishable from WT mice with respect to bacterial growth, survival, and inflammatory responses, as determined by cytokine release, histopathology and organ injury.S100A8/A9 is markedly elevated in human typhoid, correlates with duration of fever prior to admission and directly inhibits the growth of S. Typhimurium and S. Typhi in vitro. Despite elevated levels in the murine invasive Salmonella model, S100A8/A9 does not contribute to an effective host response

  1. S100A8/A9 is not involved in host defense against murine urinary tract infection.

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    Mark C Dessing

    Full Text Available BACKGROUND: Inflammation is commonly followed by the release of endogenous proteins called danger associated molecular patterns (DAMPs that are able to warn the host for eminent danger. S100A8/A9 subunits are DAMPs that belong to the S100 family of calcium binding proteins. S100A8/A9 complexes induce an inflammatory response and their expression correlates with disease severity in several inflammatory disorders. S100A8/A9 promote endotoxin- and Escherichia (E. coli-induced sepsis showing its contribution in systemic infection. The role of S100A8/A9 during a local infection of the urinary tract system caused by E. coli remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the contribution of S100A8/A9 in acute urinary tract infection (UTI by instilling 2 different doses of uropathogenic E. coli transurethrally in wild type (WT and S100A9 knockout (KO mice. Subsequently, we determined bacterial outgrowth, neutrophilic infiltrate and inflammatory mediators in bladder and kidney 24 and 48 hours later. UTI resulted in a substantial increase of S100A8/A9 protein in bladder and kidney tissue of WT mice. S100A9 KO mice displayed similar bacterial load in bladder or kidney homogenate compared to WT mice using 2 different doses at 2 different time points. S100A9 deficiency had little effect on the inflammatory responses to E. Coli-induced UTI infection, as assessed by myeloperoxidase activity in bladder and kidneys, histopathologic analysis, and renal and bladder cytokine concentrations. CONCLUSIONS: We show that despite high S100A8/A9 expression in bladder and kidney tissue upon UTI, S100A8/A9 does not contribute to an effective host response against E. Coli in the urinary tract system.

  2. Protein S100B in umbilical cord blood as a potential biomarker of hypoxic-ischemic encephalopathy in asphyxiated newborns.

    Science.gov (United States)

    Zaigham, Mehreen; Lundberg, Fredrik; Olofsson, Per

    2017-09-01

    Neonatal hypoxic ischemic encephalopathy (HIE) is a devastating condition resulting from a sustained lack of oxygen during birth. The interest in identifying a relevant biomarker of HIE has thrown into limelight the role of protein S100B as a clinical diagnostic marker of hypoxic brain damage in neonates. To evaluate the diagnostic value of protein S100B, measured in umbilical cord blood immediately after birth, as a useful biomarker in the diagnosis of HIE Sarnat stages II-III as well as a marker for long-term mortality and morbidity. Protein S100B was analyzed in cord blood sampled at birth from 13 newborns later diagnosed with stage II-III HIE and compared with 21 healthy controls. S100B concentrations were related to cord artery pH, amplitude-integrated electroencephalography (aEEG), stage of HIE, and death/sequelae up to an age of 6years. Both parametric and non-parametric statistics were used with a two-sided P<0.05 considered significant. The difference in S100B concentration was marginally statistically significant between HIE cases and controls (P=0.056). Cord blood acidosis (P=0.046), aEEG pattern severity (P=0.030), HIE severity (P=0.027), and condition at 6-year follow-up (healthy/permanent sequelae/death; P=0.027) were all related to an increase in S100B concentration. Protein S100B in neonates suffering from HIE stages II-III appeared elevated in umbilical cord blood at birth. The S100B concentrations were positively associated to the severity of disease and the risk of suffering from neurodevelopmental sequelae and even death. Copyright © 2017. Published by Elsevier B.V.

  3. Combination PPARγ and RXR Agonist Treatment in Melanoma Cells: Functional Importance of S100A2

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    Joshua P. Klopper

    2010-01-01

    Full Text Available Nuclear hormone receptors, including RXR and PPARγ, represent novel therapeutic targets in melanoma. We have previously shown that the DRO subline of the amelanotic melanoma A375 responds to rexinoid and thiazolidinedione (TZD treatment in vitro and in vivo. We performed microarray analysis of A375(DRO after TZD and combination rexinoid/TZD treatment in which the calcium binding protein S100A2 had increased expression after rexinoid or TZD treatment and a synergistic increase to combination treatment. Increased S100A2 expression is dependent on an intact PPARγ receptor, but it is not sufficient to mediate the antiproliferative effects of rexinoid/TZD treatment. Over expression of S100A2 enhanced the effect of rexinoid and TZD treatment while inhibition of S100A2 expression attenuated the response to rexinoid/TZD treatment, suggesting that S100A2 is necessary for optimal response to RXR and PPARγ activation by respective ligands. In summary, we have identified potential downstream mediators of rexinoid and TZD treatment in a poorly differentiated melanoma and found that alterations in S100A2 expression affect RXR and PPARγ signaling in A375(DRO cells. These studies provide insight into potential mechanisms of tumor response or resistance to these novel therapies.

  4. The level of neuron-specific enolase and S-100 protein in the cerebrospinal fluid of patients with acute bacterial meningitis

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    A. V. Sokhan

    2016-08-01

    Full Text Available Aim. To evaluate the diagnostic and prognostic role of neuron-specific enolase (NSE and S-100 protein levels in cerebrospinal fluid (CSF of patients with acute bacterial meningitis in the course of the disease. Materials and Methods. 54 cases of acute bacterial meningitis were analyzed, among them – 26 with pneumococcal and 28 with meningococcal etiology. Patients were divided into groups depending on the severity and etiology of disease. In addition to routine laboratory methods, we analyzed the CSF levels of S-100 protein and NSE at admission and after 10 – 12 days of treatment. 12 patients with acute respiratory infections and meningism were examined as a comparison group. Results. In all patients with acute bacterial meningitis CSF NSE and protein S-100 levels were significantly higher than in the control group (P <0,05. CSF neuro specific proteins level was in direct dependence on severity of the disease, and in patients with severe disease was significantly higher than in patients with moderate severity and in the control group (P <0,01. After 10 – 12 days of treatment, the level of the NSE and S-100 protein decreased, but in severe cases was still higher than in the control group (P <0,05. Conclusions. Increased cerebrospinal fluid NSE and S100 protein levels shows the presence and value of neurons and glial cells damage in patients with acute bacterial meningitis. CSF S-100 protein and neuron-specific enolase levels help to determine the severity of neurons destruction and glial cells in patients with acute bacterial meningitis. Level of neurospecific protein is in direct proportion to the severity of the disease and is the highest in patients with severe cases (P<0,05. It confirms the diagnostic and prognostic value of CSF neurospecific protein determination in patients with bacterial meningitis.

  5. SOX10-positive cells emerge in the rat pituitary gland during late embryogenesis and start to express S100β.

    Science.gov (United States)

    Ueharu, Hiroki; Yoshida, Saishu; Kanno, Naoko; Horiguchi, Kotaro; Nishimura, Naoto; Kato, Takako; Kato, Yukio

    2018-04-01

    In the pituitary gland, S100β-positive cells localize in the neurohypophysis and adenohypophysis but the lineage of the two groups remains obscure. S100β is often observed in many neural crest-derived cell types. Therefore, in this study, we investigate the origin of pituitary S100β-positive cells by immunohistochemistry for SOX10, a potent neural crest cell marker, using S100β-green fluorescence protein-transgenic rats. On embryonic day 21.5, a SOX10-positive cell population, which was also positive for the stem/progenitor cell marker SOX2, emerged in the pituitary stalk and posterior lobe and subsequently expanded to create a rostral-caudal gradient on postnatal day 3 (P3). Thereafter, SOX10-positive cells appeared in the intermediate lobe by P15, localizing to the boundary facing the posterior lobe, the gap between the lobule structures and the marginal cell layer, a pituitary stem/progenitor cell niche. Subsequently, there was an increase in SOX10/S100β double-positive cells; some of these cells in the gap between the lobule structures showed extended cytoplasm containing F-actin, indicating a feature of migration activity. The proportion of SOX10-positive cells in the postnatal anterior lobe was lower than 0.025% but about half of them co-localized with the pituitary-specific progenitor cell marker PROP1. Collectively, the present study identified that one of the lineages of S100β-positive cells is a SOX10-positive one and that SOX10-positive cells express pituitary stem/progenitor cell marker genes.

  6. Day/night changes in serum S100B protein concentrations in acute paranoid schizophrenia.

    Science.gov (United States)

    Morera-Fumero, Armando L; Díaz-Mesa, Estefanía; Abreu-Gonzalez, Pedro; Fernandez-Lopez, Lourdes; Cejas-Mendez, Maria Del Rosario

    2017-04-03

    There are day/night and seasonal changes in biological markers such as melatonin and cortisol. Controversial changes in serum S100B protein levels have been described in schizophrenia. We aim studying whether serum S100B levels present day/night variations in schizophrenia patients and whether S100B levels are related to psychopathology. Sixty-five paranoid schizophrenic inpatients participated in the study. Psychopathology was assessed with the Positive and Negative Syndrome Scale (PANSS) at admission and discharge. Blood was drawn at 12:00 (midday) and 00:00 (midnight) hours at admission and discharge. Sixty-five healthy subjects matched by age, gender and season acted as control group. At admission and discharge patients had significantly higher serum S100B concentrations at midday and midnight than healthy subjects. At admission, patients showed a day/night variation of S100B levels, with higher S100B levels at 12:00 than at 00:00h (143.7±26.3pg/ml vs. 96.9±16.6pg/ml). This day/night difference was not present in the control group. Midday and midnight S100B at admission decreased when compared to S100B at discharge (midday, 143.7±26.3 vs. 83.0±12, midnight 96.9±16.6 vs. 68.6±14.5). There was a positive correlation between the PANSS positive subscale and S100B concentrations at admission. This correlation was not present at discharge. acute paranoid schizophrenia inpatients present a day/night change of S100B serum levels at admission that disappears at discharge. The correlation between serum S100B concentrations and the PANSS positive scores at admission as well as the decrease of S100B at discharge may be interpreted as an acute biological response to the clinical state of the patients. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  7. Protein S binding to human endothelial cells is required for expression of cofactor activity for activated protein C

    NARCIS (Netherlands)

    Hackeng, T. M.; Hessing, M.; van 't Veer, C.; Meijer-Huizinga, F.; Meijers, J. C.; de Groot, P. G.; van Mourik, J. A.; Bouma, B. N.

    1993-01-01

    An important feedback mechanism in blood coagulation is supplied by the protein C/protein S anticoagulant pathway. In this study we demonstrate that the binding of human protein S to cultured human umbilical vein endothelial cells (HUVECs) is required for the expression of cofactor activity of

  8. Metastasis-inducing S100A4 and RANTES cooperate in promoting tumor progression in mice.

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    Birgitte Forst

    2010-04-01

    Full Text Available The tumor microenvironment has been described as a critical milieu determining tumor growth and metastases. A pivotal role of metastasis-inducing S100A4 in the development of tumor stroma has been proven in animal models and verified in human breast cancer biopsies. Expression and release of S100A4 has been shown in various types of stroma composing cells, including fibroblasts and immune cells. However, the events implicated in upstream and downstream pathways regulating the activity of the extracellular S100A4 protein in the tumor milieu remain unsolved.We studied the interplay between the tumor cell-derived cytokine regulated-upon-activation, normal T-cell expressed and secreted (RANTES; CCL5 and S100A4 which were shown to be critical factors in tumor progression. We found that RANTES stimulates the externalization of S100A4 via microparticle shedding from the plasma membrane of tumor and stroma cells. Conversely, the released S100A4 protein induces the upregulation of fibronectin (FN in fibroblasts and a number of cytokines, including RANTES in tumor cells as well as stimulates cell motility in a wound healing assay. Importantly, using wild type and S100A4-deficient mouse models, we demonstrated a substantial influence of tumor cell-derived RANTES on S100A4 release into blood circulation which ultimately increases the metastatic burden in mice.Altogether, the data presented strongly validate the pro-metastatic function of S100A4 in the tumor microenvironment and define how the tumor cell-derived cytokine RANTES acts as a critical regulator of S100A4-dependent tumor cell dissemination. Additionally, for the first time we demonstrated the mechanism of S100A4 release associated with plasma membrane microparticle shedding from various cells types.

  9. Expression of S100A4 by a variety of cell types present in the tumor microenvironment of human breast cancer

    DEFF Research Database (Denmark)

    Cabezón, Teresa; Celis, Julio E; Skibshøj, Inge

    2007-01-01

    The S100A4 protein, which is involved in the metastasis process, is a member of the S100 superfamily of Ca-binding proteins. Members of this family are multifunctional signaling proteins with dual extra and intracellular functions involved in the regulation of diverse cellular processes. Several ...... interstitial fluid (TIF) as compared to their corresponding normal counterparts (NIF)....

  10. Tissue-specific posttranscriptional downregulation of expression of the S100A4(mts1) gene in transgenic animals

    DEFF Research Database (Denmark)

    Ambartsumian, N; Klingelhöfer, Jörg; Grigorian, M

    1998-01-01

    The S100A4(mts1) is a gene associated with generation of metastatic disease. In order to analyze the consequences of alteration of the pattern of expression of the S100A4(mts1) gene we obtained strains of transgenic mice bearing the S100A4(mts1) gene under the control of a ubiquitous and constitu....../or posttranslational degradation....

  11. Secretion of S100A8, S100A9, and S100A12 by Neutrophils Involves Reactive Oxygen Species and Potassium Efflux

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    Mélanie R. Tardif

    2015-01-01

    Full Text Available S100A8/A9 (calprotectin and S100A12 proinflammatory mediators are found at inflammatory sites and in the serum of patients with inflammatory or autoimmune diseases. These cytoplasmic proteins are secreted by neutrophils at sites of inflammation via alternative secretion pathways of which little is known. This study examined the nature of the stimuli leading to S100A8/A9 and S100A12 secretion as well as the mechanism involved in this alternative secretion pathway. Chemotactic agents, cytokines, and particulate molecules were used to stimulate human neutrophils. MSU crystals, PMA, and H2O2 induced the release of S100A8, S100A9, and S100A12 homodimers, as well as S100A8/A9 heterodimer. High concentrations of S100A8/A9 and S100A12 were secreted in response to nanoparticles like MSU, silica, TiO2, fullerene, and single-wall carbon nanotubes as well as in response to microbe-derived molecules, such as zymosan or HKCA. However, neutrophils exposed to the chemotactic factors fMLP failed to secrete S100A8/A9 or S100A12. Secretion of S100A8/A9 was dependent on the production of reactive oxygen species and required K+ exchanges through the ATP-sensitive K+ channel. Altogether, these findings suggest that S100A12 and S100A8/A9 are secreted independently either via distinct mechanisms of secretion or following the activation of different signal transduction pathways.

  12. The S100P/RAGE signaling pathway regulates expression of microRNA-21 in colon cancer cells.

    Science.gov (United States)

    Mercado-Pimentel, Melania E; Onyeagucha, Benjamin C; Li, Qing; Pimentel, Angel C; Jandova, Jana; Nelson, Mark A

    2015-08-19

    S100P signaling through the receptor for advanced glycation end-products (RAGE) contributes to colon cancer invasion and metastasis, but the mechanistic features of this process are obscure. Here, we investigate whether activation of S100P/RAGE signaling regulates oncogenic microRNA-21 (miR-21). We show that exogenous S100P up-regulates miR-21 levels in human colon cancer cells, whereas knockdown of S100P results in a decrease of miR-21. Furthermore, blockage of RAGE with anti-RAGE antibody suppresses S100P induction of miR-21. In addition, we found that S100P induction of miR-21 expression involves ERK and is suppressed by the MEK inhibitor U0126. Also, S100P treatment stimulates the enrichment of c-Fos, and AP-1 family members, at the miR-21 gene promoter. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  13. Attenuation of cancer-initiating cells stemness properties by abrogating S100A4 calcium binding ability in head and neck cancers.

    Science.gov (United States)

    Cheng, Li-Hao; Hung, Kai-Feng; Huang, Tung-Fu; Hsieh, Hsin-Pei; Wang, Shu-Ying; Huang, Chih-Yang; Lo, Jeng-Fan

    2016-11-29

    S100A4 is a calcium-binding protein capable of promoting epithelial-mesenchymal transition. Previously, we have demonstrated that S100A4 is required to sustain the head and neck cancer-initiating cells (HN-CICs) subpopulation. In this study, to further investigate the molecular mechanism, we established the head and neck squamous cell carcinoma (HNSCC) cell lines stably expressing mutant S100A4 proteins with defective calcium-binding sites on either N-terminal (NM) or C-terminal (CM), or a deletion of the last 15 amino-acid residues (CD). We showed that the NM, CM and CD harboring sphere cells that were enriched with HN-CICs population exhibited impaired stemness and malignant properties in vitro, as well as reduced tumor growth ability in vivo. Mechanistically, we demonstrated that mutant S100A4 proteins decreased the promoter activity of Nanog, likely through inhibition of p53. Moreover, the biophysical analyses of purified recombinant mutant S100A4 proteins suggest that both NM and CM mutant S100A4 were very similar to the WT S100A4 with subtle difference on the secondary structure, and that the CD mutant protein displayed the unexpected monomeric form in the solution phase.Taken together, our results suggest that both the calcium-binding ability and the C-terminal region of S100A4 are important for HN-CICs to sustain its stemness property and malignancy, and that the mechanism could be mediated by repressing p53 and subsequently activating the Nanog expression.

  14. Oxidative stress and S-100B protein in children with bacterial meningitis

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    Hamed Enas A

    2009-10-01

    Full Text Available Abstract Background Bacterial meningitis is often associated with cerebral compromise which may be responsible for neurological sequelae in nearly half of the survivors. Little is known about the mechanisms of CNS involvement in bacterial meningitis. Several studies have provided substantial evidence for the key role of nitric oxide (NO and reactive oxygen species in the complex pathophysiology of bacterial meningitis. Methods In the present study, serum and CSF levels of NO, lipid peroxide (LPO (mediators for oxidative stress and lipid peroxidation; total thiol, superoxide dismutase (SOD (antioxidant mediators and S-100B protein (mediator of astrocytes activation and injury, were investigated in children with bacterial meningitis (n = 40. Albumin ratio (CSF/serum is a marker of blood-CSF barriers integrity, while mediator index (mediator ratio/albumin ratio is indicative of intrathecal synthesis. Results Compared to normal children (n = 20, patients had lower serum albumin but higher NO, LPO, total thiol, SOD and S-100B. The ratios and indices of NO and LPO indicate blood-CSF barriers dysfunction, while the ratio of S-100B indicates intrathecal synthesis. Changes were marked among patients with positive culture and those with neurological complications. Positive correlation was found between NO index with CSF WBCs (r = 0.319, p Conclusion This study suggests that loss of integrity of brain-CSF barriers, oxidative stress and S-100B may contribute to the severity and neurological complications of bacterial meningitis.

  15. Thioredoxin-1 promotes colorectal cancer invasion and metastasis through crosstalk with S100P.

    Science.gov (United States)

    Lin, Feiyan; Zhang, Peili; Zuo, Zhigui; Wang, Fule; Bi, Ruichun; Shang, Wenjing; Wu, Aihua; Ye, Ju; Li, Shaotang; Sun, Xuecheng; Wu, Jianbo; Jiang, Lei

    2017-08-10

    Thioredoxin-1 (Trx-1) is a small redox-regulating protein, which plays an important role in several cellular functions. Despite recent advances in understanding the biology of Trx-1, the role of Trx-1 and its underlying signaling mechanism in colorectal cancer (CRC) metastasis have not been extensively studied. In this study, we observed that Trx-1 expression is increased in CRC tissues compared to the paired non-cancerous tissues and is significantly correlated with clinical staging, lymph node metastasis and poor survival. Overexpression of Trx-1 enhanced CRC cell invasion and metastasis in vitro and in vivo. Conversely, suppression of Trx-1 expression decreased cell invasion and metastasis in vitro and in vivo. Moreover, Trx-1 activates S100P gene transcription. S100P, in turn, promotes Trx-1 expression and nuclear localization by upregulating p-ERK1/2 and downregulating TXNIP expression. Our finding provides new insight into the mechanism of Trx-1/S100P axis in the promotion of CRC metastasis, and suggests that the Trx-1/S100P axis and their related signaling pathways could be novel targets for the treatment of metastatic CRC. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Sensitization of interferon-γ induced apoptosis in human osteosarcoma cells by extracellular S100A4

    International Nuclear Information System (INIS)

    Pedersen, Kjetil Boye; Andersen, Kristin; Fodstad, Øystein; Mælandsmo, Gunhild Mari

    2004-01-01

    S100A4 is a small Ca 2+ -binding protein of the S100 family with metastasis-promoting properties. Recently, secreted S100A4 protein has been shown to possess a number of functions, including induction of angiogenesis, stimulation of cell motility and neurite extension. Cell cultures from two human osteosarcoma cell lines, OHS and its anti-S100A4 ribozyme transfected counterpart II-11b, was treated with IFN-γ and recombinant S100A4 in order to study the sensitizing effects of extracellular S100A4 on IFN-γ mediated apoptosis. Induction of apoptosis was demonstrated by DNA fragmentation, cleavage of poly (ADP-ribose) polymerase and Lamin B. In the present work, we found that the S100A4-expressing human osteosarcoma cell line OHS was more sensitive to IFN-γ-mediated apoptosis than the II-11b cells. S100A4 protein was detected in conditioned medium from OHS cells, but not from II-11b cells, and addition of recombinant S100A4 to the cell medium sensitized II-11b cells to apoptosis induced by IFN-γ. The S100A4/IFN-γ-mediated induction of apoptosis was shown to be independent of caspase activation, but dependent on the formation of reactive oxygen species. Furthermore, addition of extracellular S100A4 was demonstrated to activate nuclear factor-κB (NF-κB). In conclusion, we have shown that S100A4 sensitizes osteosarcoma cells to IFN-γ-mediated induction of apoptosis. Additionally, extracellular S100A4 activates NF-κB, but whether these events are causally related remains unknown

  17. Os possíveis papéis da S100B na esquizofrenia Potential roles of S100B in schizophrenia

    Directory of Open Access Journals (Sweden)

    Johann Steiner

    2012-01-01

    Full Text Available CONTEXTO: Evidências científicas do aumento da concentração da proteína S100B no sangue de pacientes esquizofrênicos são muito consistentes. No passado essa informação era principalmente considerada como reflexo da disfunção astroglial ou da barreira hematoencefálica. MÉTODOS: Pesquisa de publicações no PubMed até o dia 15 de junho de 2011 visando estabelecer potenciais ligações entre a proteína S100B e as hipóteses correntes da esquizofrenia. RESULTADOS: A S100B está potencialmente associada com as hipóteses dopaminérgica e glutamatérgica. O aumento da expressão de S100B tem sido detectado em astrócitos corticais em casos de esquizofrenia paranoide, enquanto se observa uma redução da expressão em oligodendrócitos na esquizofrenia residual, dando suporte à hipótese glial. Recentemente, a hipótese da neuroinflamação da esquizofrenia tem recebido atenção crescente. Nesse sentido, a S100B pode funcionar como uma citocina secretada por células gliais, linfócitos CD8+ e células NK, levando à ativação de monócitos e microglia. Além disso, a S100B apresenta propriedades do tipo adipocina e pode estar desregulada na esquizofrenia, devido a distúrbios da sinalização de insulina, levando ao aumento da liberação de S100B e ácidos graxos do tecido adiposo. CONCLUSÃO: A expressão de S100B em diferentes tipos celulares está envolvida em muitos processos regulatórios. Atualmente, não pode ser respondido qual mecanismo relacionado à esquizofrenia é o mais importante.BACKGROUND: Scientific evidence for increased S100B concentrations in the peripheral blood of acutely ill schizophrenia patients is consistent. In the past, this finding was mainly considered to reflect astroglial or blood-brain barrier dysfunction. METHODS: Using Entrez, PubMed was searched for articles published on or before June 15, 2011, including electronic early release publications, in order to determine other potential links between S

  18. Os possíveis papéis da S100B na esquizofrenia Potential roles of S100B in schizophrenia

    Directory of Open Access Journals (Sweden)

    Johann Steiner

    2013-01-01

    Full Text Available CONTEXTO: Evidências científicas do aumento da concentração da proteína S100B no sangue de pacientes esquizofrênicos são muito consistentes. No passado essa informação era principalmente considerada como reflexo da disfunção astroglial ou da barreira hematoencefálica. MÉTODOS: Pesquisa de publicações no PubMed até o dia 15 de junho de 2011 visando estabelecer potenciais ligações entre a proteína S100B e as hipóteses correntes da esquizofrenia. RESULTADOS: A S100B está potencialmente associada com as hipóteses dopaminérgica e glutamatérgica. O aumento da expressão de S100B tem sido detectado em astrócitos corticais em casos de esquizofrenia paranoide, enquanto se observa uma redução da expressão em oligodendrócitos na esquizofrenia residual, dando suporte à hipótese glial. Recentemente, a hipótese da neuroinflamação da esquizofrenia tem recebido atenção crescente. Nesse sentido, a S100B pode funcionar como uma citocina secretada por células gliais, linfócitos CD8+ e células NK, levando à ativação de monócitos e microglia. Além disso, a S100B apresenta propriedades do tipo adipocina e pode estar desregulada na esquizofrenia, devido a distúrbios da sinalização de insulina, levando ao aumento da liberação de S100B e ácidos graxos do tecido adiposo. CONCLUSÃO: A expressão de S100B em diferentes tipos celulares está envolvida em muitos processos regulatórios. Atualmente, não pode ser respondido qual mecanismo relacionado à esquizofrenia é o mais importante.BACKGROUND: Scientific evidence for increased S100B concentrations in the peripheral blood of acutely ill schizophrenia patients is consistent. In the past, this finding was mainly considered to reflect astroglial or blood-brain barrier dysfunction. METHODS: Using Entrez, PubMed was searched for articles published on or before June 15, 2011, including electronic early release publications, in order to determine other potential links between S

  19. Expression and divalent cation binding properties of the novel chemotactic inflammatory protein psoriasin

    DEFF Research Database (Denmark)

    Vorum, H; Madsen, Peder; Rasmussen, H H

    1996-01-01

    Psoriasin is a novel chemotactic inflammatory protein that possesses weak similarity to the S100 family members of Ca(2+)-binding proteins, and that is highly up-regulated in hyperproliferative psoriatic keratinocytes. Here we have used the psoriasin cDNA to express recombinant human (rh) psorias...

  20. Metastasis-associated protein, S100A4 mediates cardiac fibrosis potentially through the modulation of p53 in cardiac fibroblasts

    DEFF Research Database (Denmark)

    Tamaki, Yodo; Iwanaga, Yoshitaka; Niizuma, Shinichiro

    2013-01-01

    Metastasis-associated protein, S100A4 is suggested as a marker for fibrosis in several organs. It also modulates DNA binding of p53 and affects its function. However, the functional role of S100A4 in the myocardium has remained unclear. Therefore, we investigated the role of S100A4 and its relati...

  1. S100A9 interaction with TLR4 promotes tumor growth.

    Directory of Open Access Journals (Sweden)

    Eva Källberg

    Full Text Available By breeding TRAMP mice with S100A9 knock-out (S100A9(-/- animals and scoring the appearance of palpable tumors we observed a delayed tumor growth in animals devoid of S100A9 expression. CD11b(+ S100A9 expressing cells were not observed in normal prostate tissue from control C57BL/6 mice but were readily detected in TRAMP prostate tumors. Also, S100A9 expression was observed in association with CD68(+ macrophages in biopsies from human prostate tumors. Delayed growth of TRAMP tumors was also observed in mice lacking the S100A9 ligand TLR4. In the EL-4 lymphoma model tumor growth inhibition was observed in S100A9(-/- and TLR4(-/-, but not in RAGE(-/- animals lacking an alternative S100A9 receptor. When expression of immune-regulating genes was analyzed using RT-PCR the only common change observed in mice lacking S100A9 and TLR4 was a down-regulation of TGFβ expression in splenic CD11b(+ cells. Lastly, treatment of mice with a small molecule (ABR-215050 that inhibits S100A9 binding to TLR4 inhibited EL4 tumor growth. Thus, S100A9 and TLR4 appear to be involved in promoting tumor growth in two different tumor models and pharmacological inhibition of S100A9-TLR4 interactions is a novel and promising target for anti-tumor therapies.

  2. S100A9 Interaction with TLR4 Promotes Tumor Growth

    Science.gov (United States)

    Källberg, Eva; Vogl, Thomas; Liberg, David; Olsson, Anders; Björk, Per; Wikström, Pernilla; Bergh, Anders; Roth, Johannes; Ivars, Fredrik; Leanderson, Tomas

    2012-01-01

    By breeding TRAMP mice with S100A9 knock-out (S100A9−/−) animals and scoring the appearance of palpable tumors we observed a delayed tumor growth in animals devoid of S100A9 expression. CD11b+ S100A9 expressing cells were not observed in normal prostate tissue from control C57BL/6 mice but were readily detected in TRAMP prostate tumors. Also, S100A9 expression was observed in association with CD68+ macrophages in biopsies from human prostate tumors. Delayed growth of TRAMP tumors was also observed in mice lacking the S100A9 ligand TLR4. In the EL-4 lymphoma model tumor growth inhibition was observed in S100A9−/− and TLR4−/−, but not in RAGE−/− animals lacking an alternative S100A9 receptor. When expression of immune-regulating genes was analyzed using RT-PCR the only common change observed in mice lacking S100A9 and TLR4 was a down-regulation of TGFβ expression in splenic CD11b+ cells. Lastly, treatment of mice with a small molecule (ABR-215050) that inhibits S100A9 binding to TLR4 inhibited EL4 tumor growth. Thus, S100A9 and TLR4 appear to be involved in promoting tumor growth in two different tumor models and pharmacological inhibition of S100A9-TLR4 interactions is a novel and promising target for anti-tumor therapies. PMID:22470535

  3. Targeting S100P Inhibits Colon Cancer Growth and Metastasis by Lentivirus-Mediated RNA Interference and Proteomic Analysis

    Science.gov (United States)

    Jiang, Lei; Lai, Yiu-Kay; Zhang, Jinfang; Wang, Hua; Lin, Marie CM; He, Ming-liang; Kung, Hsiang-fu

    2011-01-01

    S100P was recently found to be overexpressed in a variety of cancers and is considered a potential target for cancer therapy, but the functional role or mechanism of action of S100P in colon cancer is not fully understood. In the present study, we knocked down the gene expression of S100P in colon cancer cells using lentivirus-mediated RNA interference. This step resulted in significant inhibition of cancer cell growth, migration and invasion in vitro and tumor growth and liver metastasis in vivo. Moreover, S100P downstream target proteins were identified by proteomic analysis in colon cancer DLD-1 cells with deletion of S100P. Knockdown of S100P led to downregulation of thioredoxin 1 and β-tubulin and upregulation of Rho guanosine diphosphate (GDP) dissociation inhibitor α (RhoGDIA), all potential therapeutic targets in cancer. Taken together, these findings suggest that S100P plays an important role in colon tumorigenesis and metastasis, and the comprehensive and comparative analyses of proteins associated with S100P could contribute to understanding the downstream signal cascade of S100P, leading to tumorigenesis and metastasis. PMID:21327297

  4. S100A7, a novel Alzheimer's disease biomarker with non-amyloidogenic alpha-secretase activity acts via selective promotion of ADAM-10.

    Directory of Open Access Journals (Sweden)

    Weiping Qin

    Full Text Available Alzheimer's disease (AD is the most common cause of dementia among older people. At present, there is no cure for the disease and as of now there are no early diagnostic tests for AD. There is an urgency to develop a novel promising biomarker for early diagnosis of AD. Using surface-enhanced laser desorption ionization-mass spectrometry SELDI-(MS proteomic technology, we identified and purified a novel 11.7-kDa metal- binding protein biomarker whose content is increased in the cerebrospinal fluid (CSF and in the brain of AD dementia subjects as a function of clinical dementia. Following purification and protein-sequence analysis, we identified and classified this biomarker as S100A7, a protein known to be involved in immune responses. Using an adenoviral-S100A7 expression system, we continued to examine the potential role of S100A7 in AD amyloid neuropathology in in vitro model of AD. We found that the expression of exogenous S100A7 in primary cortico-hippocampal neuron cultures derived from Tg2576 transgenic embryos inhibits the generation of beta-amyloid (Abeta(1-42 and Abeta(1-40 peptides, coincidental with a selective promotion of "non- amyloidogenic" alpha-secretase activity via promotion of ADAM (a disintegrin and metalloproteinase-10. Finally, a selective expression of human S100A7 in the brain of transgenic mice results in significant promotion of alpha-secretase activity. Our study for the first time suggests that S100A7 may be a novel biomarker of AD dementia and supports the hypothesis that promotion of S100A7 expression in the brain may selectively promote alpha-secretase activity in the brain of AD precluding the generation of amyloidogenic peptides. If in the future we find that S1000A7 protein content in CSF is sensitive to drug intervention experimentally and eventually in the clinical setting, S100A7 might be developed as novel surrogate index (biomarker of therapeutic efficacy in the characterization of novel drug agents for

  5. Segmental isotope labeling of proteins for NMR structural study using a protein S tag for higher expression and solubility

    International Nuclear Information System (INIS)

    Kobayashi, Hiroshi; Swapna, G. V. T.; Wu, Kuen-Phon; Afinogenova, Yuliya; Conover, Kenith; Mao, Binchen; Montelione, Gaetano T.; Inouye, Masayori

    2012-01-01

    A common obstacle to NMR studies of proteins is sample preparation. In many cases, proteins targeted for NMR studies are poorly expressed and/or expressed in insoluble forms. Here, we describe a novel approach to overcome these problems. In the protein S tag-intein (PSTI) technology, two tandem 92-residue N-terminal domains of protein S (PrS 2 ) from Myxococcus xanthus is fused at the N-terminal end of a protein to enhance its expression and solubility. Using intein technology, the isotope-labeled PrS 2 -tag is replaced with non-isotope labeled PrS 2 -tag, silencing the NMR signals from PrS 2 -tag in isotope-filtered 1 H-detected NMR experiments. This method was applied to the E. coli ribosome binding factor A (RbfA), which aggregates and precipitates in the absence of a solubilization tag unless the C-terminal 25-residue segment is deleted (RbfAΔ25). Using the PrS 2 -tag, full-length well-behaved RbfA samples could be successfully prepared for NMR studies. PrS 2 (non-labeled)-tagged RbfA (isotope-labeled) was produced with the use of the intein approach. The well-resolved TROSY-HSQC spectrum of full-length PrS 2 -tagged RbfA superimposes with the TROSY-HSQC spectrum of RbfAΔ25, indicating that PrS 2 -tag does not affect the structure of the protein to which it is fused. Using a smaller PrS-tag, consisting of a single N-terminal domain of protein S, triple resonance experiments were performed, and most of the backbone 1 H, 15 N and 13 C resonance assignments for full-length E. coli RbfA were determined. Analysis of these chemical shift data with the Chemical Shift Index and heteronuclear 1 H– 15 N NOE measurements reveal the dynamic nature of the C-terminal segment of the full-length RbfA protein, which could not be inferred using the truncated RbfAΔ25 construct. CS-Rosetta calculations also demonstrate that the core structure of full-length RbfA is similar to that of the RbfAΔ25 construct.

  6. The Inflammation-Related Gene S100A12 Is Positively Regulated by C/EBPβ and AP-1 in Pigs

    Directory of Open Access Journals (Sweden)

    Xinyun Li

    2014-08-01

    Full Text Available S100A12 is involved in the inflammatory response and is considered an important marker for many inflammatory diseases in humans. Our previous studies indicated that the S100A12 gene was abundant in the immune tissues of pigs and was significantly upregulated during infection with Haemophilus parasuis (HPS or porcine circovirus type 2 (PCV2. In this study, the mechanism of transcriptional regulation of S100A12 was investigated in pigs. Our results showed that S100A12, CCAAT/enhancer-binding protein beta (C/EBPβ and activator protein-1 (AP-1 genes were up-regulated in PK-15 (ATCC, CCL-33 cells when treated with LPS or Poly I: C. Additionally, the promoter activity and expression level of the S100A12 gene were significantly upregulated when C/EBPβ or AP-1 were overexpressed. We utilized electromobility shift assays (EMSA to confirm that C/EBPβ and AP-1 could directly bind the S100A12 gene promoter. We also found that the transcriptional activity and expression levels of C/EBPβ and AP-1 could positively regulate each other. Furthermore, the promoter activity of the S100A12 gene was higher when C/EBPβ and AP-1 were cotransfected than when they were transfected individually. We concluded that the S100A12 gene was cooperatively and positively regulated by C/EBPβ and AP-1 in pigs. Our study offers new insight into the transcriptional regulation of the S100A12 gene.

  7. Production and characterization of monoclonal antibodies that discriminate among individual S100 polypeptides

    International Nuclear Information System (INIS)

    Van Eldik, L.J.

    1984-01-01

    The term S100 refers to a heterogeneous fraction of low molecular weight, acidic, calcium binding proteins. The S100 fraction is a mixture of polypeptides, only some of which have been isolated and characterized. The amino acid sequences of two S100 proteins from bovine brain, S100α and S100β, have been determined. The physiological functions of the S100 proteins are not known. Although assay of immunoreactive S100 has been used clinically to screen tumors of neural origin, as an index of cell injury in various disorders, and as an index of malignancy, most of the antisera used in previous studies react with more than one protein in the S100 fraction. Even the currently available monoclonal antibodies against S100 (2-4) do not appear to measure the individual S100α and S100β components. In order to unequivocally interpret studies on the localization of S100 and its potential alterations in various disease states, and on the validity of S100 immunoreactivity as a diagnostic tool for tumor diagnosis, it would be useful to have antibodies that discriminate among the individual S100 components. The authors report here the production of monoclonal antibodies that appear to be specific for S100β

  8. A proteomic method for analysis of CYP450s protein expression changes in carbon tetrachloride induced male rat liver microsomes

    International Nuclear Information System (INIS)

    Jia Nuan; Liu Xin; Wen Jun; Qian Linyi; Qian Xiaohong; Wu Yutian; Fan Guorong

    2007-01-01

    Carbon tetrachloride (CCl 4 ) is a well-known model compound for producing chemical hepatic injury. Cytochrome P450 is an important monooxygenase in biology. We investigated the CYP450 protein expression in the in vivo hepatotoxicity of rats induced by CCl 4 . In this experiment, CCl 4 were administered to male rats, and their livers at 24 h post-dosing were applied to the proteomic analysis. Blood biochemistry and histopathology were examined to identify specific changes. At the same time, a novel acetylation stable isotopic labeling method coupled with LTQ-FTICR mass spectrometry was applied to disclose the changes of cytochrome P450 expression amounts. The quantitative proteomics method demonstrated its correlation coefficient was 0.9998 in a 100-fold dynamic range and the average ratio of the labeled peptides was 1.04, which was very close to the theoretical ratio of 1.00 and the standard deviation (S.D.) of 0.21. With this approach, 17 cytochrome P450 proteins were identified and quantified with high confidence. Among them, the expression amount of 2C11, 3A2, and 2 E1 were down-regulated, while that of 2C6, 2B2, and 2B1 were up-regulated

  9. Expression of G(alpha)(s) proteins and TSH receptor signalling in hyperfunctioning thyroid nodules with TSH receptor mutations.

    Science.gov (United States)

    Holzapfel, Hans-Peter; Bergner, Beate; Wonerow, Peter; Paschke, Ralf

    2002-07-01

    Constitutively activating mutations of the thyrotrophin receptor (TSHR) are the main molecular cause of hyperfunctioning thyroid nodules (HTNs). The G protein coupling is an important and critical step in the TSHR signalling which mainly includes G(alpha)(s), G(alpha)(i) and G(alpha)(q)/11 proteins. We investigated the in vitro consequences of overexpressing G(alpha) proteins on signalling of the wild-type (WT) or mutated TSHR. Moreover, we investigated whether changes in G(alpha) protein expression are pathophysiologically relevant in HTNs or cold thyroid nodules (CTNs). Wild-type TSH receptor and mutated TSH receptors were coexpressed with G(alpha)(s), G(alpha)(i) or G(alpha)(q)/11, and cAMP and inositol phosphate (IP) production was measured after stimulation with TSH. The expression of G(alpha)(s), G(alpha)(i) and G(alpha)(q)/11 proteins was examined by Western blotting in 28 HTNs and 14 CTNs. Coexpression of G(alpha)(s) with the WT TSH receptor in COS 7 cells significantly increased the basal and TSH-stimulated cAMP accumulation while coexpression of the G(alpha)(q) or G(alpha)11 protein significantly increased the production of cAMP and inositol triphosphate (IP(3)). The coexpression of the TSH receptor mutants (I486F, DEL613-621), known to couple constitutively to G(alpha)(s) and G(alpha)(q) with G(alpha)(s) and G(alpha)(q)/11, significantly increased the basal and stimulated cAMP and IP(3) accumulation. Coexpression of the TSH receptor mutant V556F with G(alpha)(s) only increased the basal and stimulated cAMP production while its coexpression with G(alpha)(q)/11 increased the basal and stimulated IP(3) signalling. The expression of G(alpha)(s) protein subunits determined by Western blotting was significantly decreased in 14 HTNs with a constitutively activating TSH receptor mutation in comparison with the corresponding surrounding tissue, while in 14 HTNs without TSH receptor or G(alpha)(s) protein mutation and in 14 CTNs the expression of G(alpha)(s

  10. S100A6 is a negative regulator of the induction of cardiac genes by trophic stimuli in cultured rat myocytes

    International Nuclear Information System (INIS)

    Tsoporis, J.N.; Marks, A.; Haddad, A.; O'Hanlon, D.; Jolly, S.; Parker, T.G.

    2005-01-01

    S100A6 (calcyclin), a member of the S100 family of EF-hand Ca 2+ binding proteins, has been implicated in the regulation of cell growth and proliferation. We have previously shown that S100B, another member of the S100 family, is induced postinfarction and limits the hypertrophic response of surviving cardiac myocytes. We presently report that S100A6 expression is also increased in the periinfarct zone of rat heart postinfarction and in cultured neonatal rat myocytes by treatment with several trophic agents, including platelet-derived growth factor (PDGF), the α 1 -adrenergic agonist phenylephrine (PE), and angiotensin II (AII). Cotransfection of S100A6 in cultured neonatal rat cardiac myocytes inhibits induction of the cardiac fetal gene promoters skeletal α-actin (skACT) and β-myosin heavy chain (β-MHC) by PDGF, PE, AII, and the prostaglandin F 2α (PGF 2α ), induction of the S100B promoter by PE, and induction of the α-MHC promoter by triiodothyronine (T3). By contrast, S100B cotransfection selectively inhibited only PE induction of skACT and β-MHC promoters. Fluorescence microscopy demonstrated overlapping intracellular distribution of S100B and S100A6 in transfected myocytes and in postinfarct myocardium but heterodimerization of the two proteins could not be detected by co-immunoprecipitation. We conclude that S100A6 may function as a global negative modulator of differentiated cardiac gene expression comparable to its putative role in cell cycle progression of dividing cells

  11. Study of lymphocyte sensitization to protein S-100 in the patients with cerebrovascular diseases, suffered due to Chernobyl NPP accident

    International Nuclear Information System (INIS)

    Khomenko, V.Yi.

    2004-01-01

    Among the persons with cerebrovascular diseases, suffered due to Chernobyl NPP accident two groups of patients were revealed: with DNA coloration coefficients in response to protein S-100 stimulation below and above 1. Patients with DNA coloration coefficients <1 were older, they had statistically significant lower monocytes and T-activated lymphocytes absolute counts as well as increased content of cholesterol-2 and circulating immune complexes. Charges found there suggested possible existence of different pathways of immune response to antigenic stimulation by S-100 protein

  12. S100B proteins in febrile seizures

    DEFF Research Database (Denmark)

    Mikkonen, Kirsi; Pekkala, Niina; Pokka, Tytti

    2011-01-01

    at the hospital after FS and S100B concentration in serum (r=-0.130, P=0.28) or in cerebrospinal fluid samples (r=-0.091, P=0.52). Our findings indicate that FS does not cause significant blood-brain barrier openings, and increase the evidence that these seizures are relatively harmless for the developing brain....

  13. Role of S100A1 in hypoxia-induced inflammatory response in cardiomyocytes via TLR4/ROS/NF-κB pathway.

    Science.gov (United States)

    Yu, Jiangkun; Lu, Yanyu; Li, Yapeng; Xiao, Lili; Xing, Yu; Li, Yanshen; Wu, Leiming

    2015-09-01

    S100A1 plays a crucial role in hypoxia-induced inflammatory response in cardiomyocytes. However, the role of S100A1 in hypoxia-induced inflammatory response in cardiomyocytes is still unknown. enzyme-linked immunosorbent assay (ELISA) was performed for the determination of inflammatory cytokines. Immunocytochemistry and immunofluorescence, Western blot analysis and Real-time polymerase chain reaction (RT-PCR) were conducted to assess protein or mRNA expressions. Fluorogenic probe dihydroethidium (DHE) was used to evaluate the generation of reactive oxygen species (ROS) while Hoechst 33342 staining for apoptosis. Small interfering RNA (siRNA) for S100A1 was used to evaluate the role of S100A1. The levels of ROS and inflammatory cytokine including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and IL-8 in H9c2 cells were increased remarkably by hypoxia. However, IL-37 protein or mRNA levels were decreased significantly. Both Toll-like receptor 4 (TLR4) inhibitor Ethyl (6R)-6-[N-(2-Chloro-4fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242) treatment or siRNA S100A1 downregulated TLR4 expression and inflammatory cytokine level and mRNA in H9c2 cells, as well as weakening ROS and phospho-p65 Nuclear factor (NF)-κB levels. Further, S100A1 treatment significantly reduced TNF-α protein or mRNA level whereas enhanced IL-37 protein or mRNA level, and could attenuate ROS and phospho-p65 NF-κB levels. Our results demonstrate that S100A1 can regulate the inflammatory response and oxidative stress in H9C2 cells via TLR4/ROS/NF-κB pathway. These findings provide an interesting strategy for protecting cardiomyocytes from hypoxia-induced inflammatory response. © 2015 Royal Pharmaceutical Society.

  14. Solving the crystal structure of human calcium-free S100Z: the siege and conquer of one of the last S100 family strongholds.

    Science.gov (United States)

    Calderone, V; Fragai, M; Gallo, G; Luchinat, C

    2017-06-01

    The X-ray structure of human apo-S100Z has been solved and compared with that of the zebrafish calcium-bound S100Z, which is the closest in sequence. Human apo-S100A12, which shows only 43% sequence identity to human S100Z, has been used as template model to solve the crystallographic phase problem. Although a significant buried surface area between the two physiological dimers is present in the asymmetric unit of human apo-S100Z, the protein does not form the superhelical arrangement in the crystal as observed for the zebrafish calcium-bound S100Z and human calcium-bound S100A4. These findings further demonstrate that calcium plays a fundamental role in triggering quaternary structure formation in several S100s. Solving the X-ray structure of human apo-S100Z by standard molecular replacement procedures turned out to be a challenge and required trying different models and different software tools among which only one was successful. The model that allowed structure solution was that with one of the lowest sequence identity with the target protein among the S100 family in the apo state. Based on the previously solved zebrafish holo-S100Z, a putative human holo-S100Z structure has been then calculated through homology modeling; the differences between the experimental human apo and calculated holo structure have been compared to those existing for other members of the family.

  15. Rice sHsp genes: genomic organization and expression profiling under stress and development

    Directory of Open Access Journals (Sweden)

    Grover Anil

    2009-08-01

    Full Text Available Abstract Background Heat shock proteins (Hsps constitute an important component in the heat shock response of all living systems. Among the various plant Hsps (i.e. Hsp100, Hsp90, Hsp70 and Hsp20, Hsp20 or small Hsps (sHsps are expressed in maximal amounts under high temperature stress. The characteristic feature of the sHsps is the presence of α-crystallin domain (ACD at the C-terminus. sHsps cooperate with Hsp100/Hsp70 and co-chaperones in ATP-dependent manner in preventing aggregation of cellular proteins and in their subsequent refolding. Database search was performed to investigate the sHsp gene family across rice genome sequence followed by comprehensive expression analysis of these genes. Results We identified 40 α-crystallin domain containing genes in rice. Phylogenetic analysis showed that 23 out of these 40 genes constitute sHsps. The additional 17 genes containing ACD clustered with Acd proteins of Arabidopsis. Detailed scrutiny of 23 sHsp sequences enabled us to categorize these proteins in a revised scheme of classification constituting of 16 cytoplasmic/nuclear, 2 ER, 3 mitochondrial, 1 plastid and 1 peroxisomal genes. In the new classification proposed herein nucleo-cytoplasmic class of sHsps with 9 subfamilies is more complex in rice than in Arabidopsis. Strikingly, 17 of 23 rice sHsp genes were noted to be intronless. Expression analysis based on microarray and RT-PCR showed that 19 sHsp genes were upregulated by high temperature stress. Besides heat stress, expression of sHsp genes was up or downregulated by other abiotic and biotic stresses. In addition to stress regulation, various sHsp genes were differentially upregulated at different developmental stages of the rice plant. Majority of sHsp genes were expressed in seed. Conclusion We identified twenty three sHsp genes and seventeen Acd genes in rice. Three nucleocytoplasmic sHsp genes were found only in monocots. Analysis of expression profiling of sHsp genes revealed

  16. Dynamic change of serum protein S100b and its clinical significance in patients with traumatic brain injury

    Institute of Scientific and Technical Information of China (English)

    CHEN Da-qing; ZHU Lie-lie

    2005-01-01

    Objective: To analyze the dynamic change of serum protein S100b in patients with traumatic brain injury and its clinical value in assessing brain damage. Methods: According to Glasgow coma scale (GCS), 102 cases of traumatic brain injury were divided into mild brain injury group (GCS≥13, n=31, Group A), moderate brain injury group (8S100b concentrations were analyzed by enzyme-linked immunosorbent assay (ELISA) in blood samples taken on admission, 12 h, 24 h, 48 h, 72 h and 7 days after traumatic brain injury. Results: The severe brain injury group showed significantly higher concentration of serum S100b, with earlier increase and longer duration, than the mild and moderate brain injury groups. The patients with higher S100b exhibited lower GCS scores and poor clinical prognosis. The increase in S100b could emerge before clinical image evidence indicated so. Conclusions: Serum S100b can be used as a sensitive index for assessment and prediction of traumatic brain injury severity and prognosis.

  17. Effect of poly and mono-unsaturated fatty acids on stability and structure of recombinant S100A8/A9.

    Science.gov (United States)

    Asghari, Hamideh; Chegini, Koorosh Goodarzvand; Amini, Abbas; Gheibi, Nematollah

    2016-03-01

    Recombinant pET 15b vectors containing the coding sequences S100A8 and S100A9 are expressed in Escherichia coli BL21 (DE3) and purified using Ni-NTA affinity chromatography. The structural changes of S100A8/A9 complex are analyzed upon interaction with poly/mono-unsaturated fatty acids (UFAs). The thermodynamic values, Gibbs free energy and the protein melting point, are obtained through thermal denaturation of protein both with and without UFAs by thermal scanning of protein emission using the fluorescence spectroscopy technique. The far-ultraviolet circular dichroism spectra show that all studied unsaturated fatty acids, including arachidonic, linoleic, alpha-linolenic and oleic acids, induce changes in the secondary structure of S100A8/A9 by reducing the α-helix and β-sheet structures. The tertiary structure of S100A8/A9 has fluctuations in the fluorescence emission spectra after the incubation of protein with UFAs. The blue-shift of emission maximum wavelength and the increase in fluorescence intensity of anilino naphthalene-8-sulfonic acid confirm that the partial unfolding is caused by the conformational changes in the tertiary structure in the presence of UFAs. The structural changes in S100A8/A9 and its lower stability in the presence of UFAs may be necessary for S100A8/A9 to play a biological role in the inflammatory milieu. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Protein expression analysis of inflammation-related colon carcinogenesis

    Directory of Open Access Journals (Sweden)

    Yasui Yumiko

    2009-01-01

    Full Text Available Background: Chronic inflammation is a risk factor for colorectal cancer (CRC development. The aim of this study was to determine the differences in protein expression between CRC and the surrounding nontumorous colonic tissues in the mice that received azoxymethane (AOM and dextran sodium sulfate (DSS using a proteomic analysis. Materials and Methods: Male ICR mice were given a single intraperitoneal injection of AOM (10 mg/kg body weight, followed by 2% (w/v DSS in their drinking water for seven days, starting one week after the AOM injection. Colonic adenocarcinoma developed after 20 weeks and a proteomics analysis based on two-dimensional gel electrophoresis and ultraflex TOF/TOF mass spectrometry was conducted in the cancerous and nontumorous tissue specimens. Results: The proteomic analysis revealed 21 differentially expressed proteins in the cancerous tissues in comparison to the nontumorous tissues. There were five markedly increased proteins (beta-tropomyosin, tropomyosin 1 alpha isoform b, S100 calcium binding protein A9, and an unknown protein and 16 markedly decreased proteins (Car1 proteins, selenium-binding protein 1, HMG-CoA synthase, thioredoxin 1, 1 Cys peroxiredoxin protein 2, Fcgbp protein, Cytochrome c oxidase, subunit Va, ETHE1 protein, and 7 unknown proteins. Conclusions: There were 21 differentially expressed proteins in the cancerous tissues of the mice that received AOM and DSS. Their functions include metabolism, the antioxidant system, oxidative stress, mucin production, and inflammation. These findings may provide new insights into the mechanisms of inflammation-related colon carcinogenesis and the establishment of novel therapies and preventative strategies to treat carcinogenesis in the inflamed colon.

  19. Alarmins S100A8 and S100A9 elicit a catabolic effect in human osteoarthritic chondrocytes that is dependent on Toll-like receptor 4.

    NARCIS (Netherlands)

    Schelbergen, R.F.P.; Blom, A.B.; Bosch, M.H.J. van den; Sloetjes, A.W.; Abdollahi-Roodsaz, S.; Schreurs, B.W.; Mort, J.S.; Vogl, T.; Roth, J.; Berg, W.B. van den; Lent, P.L.E.M. van

    2012-01-01

    OBJECTIVE: S100A8 and S100A9 are two Ca(2+) binding proteins classified as damage-associated molecular patterns or alarmins that are found in high amounts in the synovial fluid of osteoarthritis (OA) patients. The purpose of this study was to investigate whether S100A8 and/or S100A9 can interact

  20. The MUC4 membrane-bound mucin regulates esophageal cancer cell proliferation and migration properties: Implication for S100A4 protein

    International Nuclear Information System (INIS)

    Bruyere, Emilie; Jonckheere, Nicolas; Frenois, Frederic; Mariette, Christophe; Van Seuningen, Isabelle

    2011-01-01

    Highlights: → Loss of MUC4 reduces proliferation of esophageal cancer cells. → MUC4 inhibition impairs migration of esophageal cancer cells but not their invasion. → Loss of MUC4 significantly reduces in vivo tumor growth. → Decrease of S100A4 induced by MUC4 inhibition impairs proliferation and migration. -- Abstract: MUC4 is a membrane-bound mucin known to participate in tumor progression. It has been shown that MUC4 pattern of expression is modified during esophageal carcinogenesis, with a progressive increase from metaplastic lesions to adenocarcinoma. The principal cause of development of esophageal adenocarcinoma is the gastro-esophageal reflux, and MUC4 was previously shown to be upregulated by several bile acids present in reflux. In this report, our aim was thus to determine whether MUC4 plays a role in biological properties of human esophageal cancer cells. For that stable MUC4-deficient cancer cell lines (shMUC4 cells) were established using a shRNA approach. In vitro (proliferation, migration and invasion) and in vivo (tumor growth following subcutaneous xenografts in SCID mice) biological properties of shMUC4 cells were analyzed. Our results show that shMUC4 cells were less proliferative, had decreased migration properties and did not express S100A4 protein when compared with MUC4 expressing cells. Absence of MUC4 did not impair shMUC4 invasiveness. Subcutaneous xenografts showed a significant decrease in tumor size when cells did not express MUC4. Altogether, these data indicate that MUC4 plays a key role in proliferative and migrating properties of esophageal cancer cells as well as is a tumor growth promoter. MUC4 mucin appears thus as a good therapeutic target to slow-down esophageal tumor progression.

  1. The MUC4 membrane-bound mucin regulates esophageal cancer cell proliferation and migration properties: Implication for S100A4 protein

    Energy Technology Data Exchange (ETDEWEB)

    Bruyere, Emilie; Jonckheere, Nicolas; Frenois, Frederic [Inserm, UMR837, Jean-Pierre Aubert Research Center, Team 5 ' Mucins, Epithelial Differentiation and Carcinogenesis' , rue Polonovski, 59045 Lille Cedex (France); Universite Lille-Nord de France, 1 place de Verdun, 59045 Lille Cedex (France); Mariette, Christophe [Inserm, UMR837, Jean-Pierre Aubert Research Center, Team 5 ' Mucins, Epithelial Differentiation and Carcinogenesis' , rue Polonovski, 59045 Lille Cedex (France); Universite Lille-Nord de France, 1 place de Verdun, 59045 Lille Cedex (France); Department of Digestive and Oncological Surgery, University Hospital Claude Huriez, 1 place de Verdun, 59045 Lille Cedex (France); Van Seuningen, Isabelle, E-mail: isabelle.vanseuningen@inserm.fr [Inserm, UMR837, Jean-Pierre Aubert Research Center, Team 5 ' Mucins, Epithelial Differentiation and Carcinogenesis' , rue Polonovski, 59045 Lille Cedex (France); Universite Lille-Nord de France, 1 place de Verdun, 59045 Lille Cedex (France)

    2011-09-23

    Highlights: {yields} Loss of MUC4 reduces proliferation of esophageal cancer cells. {yields} MUC4 inhibition impairs migration of esophageal cancer cells but not their invasion. {yields} Loss of MUC4 significantly reduces in vivo tumor growth. {yields} Decrease of S100A4 induced by MUC4 inhibition impairs proliferation and migration. -- Abstract: MUC4 is a membrane-bound mucin known to participate in tumor progression. It has been shown that MUC4 pattern of expression is modified during esophageal carcinogenesis, with a progressive increase from metaplastic lesions to adenocarcinoma. The principal cause of development of esophageal adenocarcinoma is the gastro-esophageal reflux, and MUC4 was previously shown to be upregulated by several bile acids present in reflux. In this report, our aim was thus to determine whether MUC4 plays a role in biological properties of human esophageal cancer cells. For that stable MUC4-deficient cancer cell lines (shMUC4 cells) were established using a shRNA approach. In vitro (proliferation, migration and invasion) and in vivo (tumor growth following subcutaneous xenografts in SCID mice) biological properties of shMUC4 cells were analyzed. Our results show that shMUC4 cells were less proliferative, had decreased migration properties and did not express S100A4 protein when compared with MUC4 expressing cells. Absence of MUC4 did not impair shMUC4 invasiveness. Subcutaneous xenografts showed a significant decrease in tumor size when cells did not express MUC4. Altogether, these data indicate that MUC4 plays a key role in proliferative and migrating properties of esophageal cancer cells as well as is a tumor growth promoter. MUC4 mucin appears thus as a good therapeutic target to slow-down esophageal tumor progression.

  2. cDNA Cloning, expression and characterization of an allergenic 60s ribosomal protein of almond (prunus dulcis).

    Science.gov (United States)

    Abolhassani, Mohsen; Roux, Kenneth H

    2009-06-01

    Tree nuts, including almond (prunus dulcis) are a source of food allergens often associated with life-threatening allergic reactions in susceptible individuals. Although the proteins in almonds have been biochemically characterized, relatively little has been reported regarding the identity of the allergens involved in almond sensitivity. The present study was undertaken to identify the allergens of the almond by cDNA library approach. cDNA library of almond seeds was constructed in Uni-Zap XR lamda vector and expressed in E. coli XL-1 blue. Plaques were immunoscreened with pooled sera of allergic patients. The cDNA clone reacting significantly with specific IgE antibodies was selected and subcloned and subsequently expressed in E. coli. The amino acids deducted from PCR product of clone showed homology to 60s acidic ribosomal protein of almond. The expressed protein was 11,450 Dalton without leader sequence. Immunoreactivity of the recombinant 60s ribosomal protein (r60sRP) was evaluated with dot blot analysis using pooled and individual sera of allergic patients. The data showed that r60sRP and almond extract (as positive control) possess the ability to bind the IgE antibodies. The results showed that expressed protein is an almond allergen.Whether this r60sRP represents a major allergen of almond needs to be further studied which requires a large number of sera from the almond atopic patients and also need to determine the IgE-reactive frequencies of each individual allergen.

  3. Vector Development for the Expression of Foreign Proteins in the Vaccine Strain Brucella abortus S19

    Science.gov (United States)

    Comerci, Diego J.; Pollevick, Guido D.; Vigliocco, Ana M.; Frasch, Alberto C. C.; Ugalde, Rodolfo A.

    1998-01-01

    A vector for the expression of foreign antigens in the vaccine strain Brucella abortus S19 was developed by using a DNA fragment containing the regulatory sequences and the signal peptide of the Brucella bcsp31 gene. This fragment was cloned in broad-host-range plasmid pBBR4MCS, resulting in plasmid pBEV. As a reporter protein, a repetitive antigen of Trypanosoma cruzi was used. The recombinant fusion protein is stably expressed and secreted into the Brucella periplasmic space, inducing a good antibody response against the T. cruzi antigen. The expression of the repetitive antigen in Brucella neither altered its growth pattern nor generated a toxic or lethal effect during experimental infection. The application of this strategy for the generation of live recombinant vaccines and the tagging of B. abortus S19 vaccine is discussed. This is the first time that a recombinant protein has been expressed in the periplasm of brucellae. PMID:9673273

  4. Chronic sustained inflammation links to left ventricular hypertrophy and aortic valve sclerosis: a new link between S100/RAGE and FGF23.

    Science.gov (United States)

    Yan, Ling; Bowman, Marion A Hofmann

    Cardiovascular disease including left ventricular hypertrophy, diastolic dysfunction and ectopic valvular calcification are common in patients with chronic kidney disease (CKD). Both S100A12 and fibroblast growth factor 23 (FGF23) have been identified as biomarkers of cardiovascular morbidity and mortality in patients with CKD. We tested the hypothesis that human S100/calgranulin would accelerate cardiovascular disease in mice subjected to CKD. This review paper focuses on S100 proteins and their receptor for advanced glycation end products (RAGE) and summarizes recent findings obtained in novel developed transgenic hBAC-S100 mice that express S100A12 and S100A8/9 proteins. A bacterial artificial chromosome of the human S100/calgranulin gene cluster containing the genes and regulatory elements for S100A8, S100A9 and S100A12 was expressed in C57BL/6J mice (hBAC-S100). CKD was induced by ureteral ligation, and hBAC-S100 mice and WT mice were studied after 10 weeks of chronic uremia. hBAC-S100 mice with CKD showed increased FGF23 in the heart, left ventricular hypertrophy (LVH), diastolic dysfunction, focal cartilaginous metaplasia and calcification of the mitral and aortic valve annulus together with aortic valve sclerosis. This phenotype was not observed in WT mice with CKD or in hBAC-S100 mice lacking RAGE with CKD, suggesting that the inflammatory milieu mediated by S100/RAGE promotes pathological cardiac hypertrophy in CKD. In vitro, inflammatory stimuli including IL-6, TNFα, LPS, or serum from hBAC-S100 mice up regulated FGF23 mRNA and protein in primary murine neonatal and adult cardiac fibroblasts. Taken together, our study shows that myeloid-derived human S100/calgranulin is associated with the development of cardiac hypertrophy and ectopic cardiac calcification in a RAGE dependent manner in a mouse model of CKD. We speculate that FGF23 produced by cardiac fibroblasts in response to cytokines may act in a paracrine manner to accelerate LVH and diastolic

  5. Expression and purification of sea raven type II antifreeze protein from Drosophila melanogaster S2 cells.

    Science.gov (United States)

    Scotter, Andrew J; Kuntz, Douglas A; Saul, Michelle; Graham, Laurie A; Davies, Peter L; Rose, David R

    2006-06-01

    We present a system for the expression and purification of recombinant sea raven type II antifreeze protein, a cysteine-rich, C-type lectin-like globular protein that has proved to be a difficult target for recombinant expression and purification. The cDNAs encoding the pro- and mature forms of the sea raven protein were cloned into a modified pMT Drosophila expression vector. These constructs produced N-terminally His(6)-tagged pro- and mature forms of the type II antifreeze protein under the control of a metallothionein promoter when transfected into Drosophila melanogaster S2 cells. Upon induction of stable cell lines the two proteins were expressed at high levels and secreted into the medium. The proteins were then purified from the cell medium in a simple and rapid protocol using immobilized metal affinity chromatography and specific protease cleavage by tobacco etch virus protease. The proteins demonstrated antifreeze activity indistinguishable from that of wild-type sea raven antifreeze protein purified from serum as illustrated by ice affinity purification, ice crystal morphology, and their ability to inhibit ice crystal growth. This expression and purification system gave yields of 95 mg/L of fully active mature sea raven type II AFP and 9.6 mg/L of the proprotein. This surpasses all previous attempts to express this protein in Escherichia coli, baculovirus-infected fall armyworm cells and Pichia pastoris and will provide sufficient protein for structural analysis.

  6. Data set of interactomes and metabolic pathways of proteins differentially expressed in brains with Alzheimer׳s disease

    Directory of Open Access Journals (Sweden)

    Benito Minjarez

    2016-06-01

    Full Text Available Alzheimer׳s disease is one of the main causes of dementia in the elderly and its frequency is on the rise worldwide. It is considered the result of complex interactions between genetic and environmental factors, being many of them unknown. Therefore, there is a dire necessity for the identification of novel molecular players for the understanding of this disease. In this data article we determined the protein expression profiles of whole protein extracts from cortex regions of brains from patients with Alzheimer׳s disease in comparison to a normal brain. We identified 721 iTRAQ-labeled polypeptides with more than 95% in confidence. We analyzed all proteins that changed in their expression level and located them in the KEGG metabolic pathways, as well as in the mitochondrial complexes of the electron transport chain and ATP synthase. In addition, we analyzed the over- and sub-expressed polypeptides through IPA software, specifically Core I and Biomarkers I modules. Data in this article is related to the research article “Identification of proteins that are differentially expressed in brains with Alzheimer’s disease using iTRAQ labeling and tandem mass spectrometry” (Minjarez et al., 2016 [1].

  7. Efficient inhibition of tumor angiogenesis and growth by a synthetic peptide blocking S100A4-methionine aminopeptidase 2 interaction

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    Takahiro Ochiya

    Full Text Available The prometastatic calcium-binding protein, S100A4, is expressed in endothelial cells, and its downregulation markedly suppresses tumor angiogenesis in a xenograft cancer model. Given that endothelial S100A4 can be a molecular target for inhibiting tumor angiogenesis, we addressed here whether synthetic peptide capable of blocking S100A4-effector protein interaction could be a novel antiangiogenic agent. To examine this hypothesis, we focused on the S100A4-binding domain of methionine aminopeptidase 2, an effector protein, which plays a role in endothelial cell growth. Overexpression of the domain in mouse endothelial MSS31 cells reduced DNA synthesis, and the corresponding synthetic peptide (named NBD indeed interacted with S100A4 and inhibited capillary formation in vitro and new blood vessel formation in vivo. Intriguingly, a single intra-tumor administration of the NBD peptide in human prostate cancer xenografts significantly reduced vascularity, resulting in tumor regression. Mechanistically, the NBD peptide enhanced assembly of nonmuscle myosin IIA filaments along with Ser1943 phosphorylation, stimulated formation of focal adhesions without phosphorylation of focal adhesion kinase, and provoked G1/S arrest of the cell cycle. Altogether, the NBD peptide is a potent inhibitor for tumor angiogenesis, and is the first example of an anticancer peptide drug developed on the basis of an endothelial S100A4-targeted strategy.

  8. S100 chemokines mediate bookmarking of premetastatic niches

    Science.gov (United States)

    Rafii, Shahin; Lyden, David

    2010-01-01

    Primary tumours release soluble factors, including VEGF-A, TGFβ and TNFα, which induce expression of the chemokines S100A8 and S100A9 in the myeloid and endothelial cells within the lung before tumour metastasis. These chemokine-activated premetastatic niches support adhesion and invasion of disseminating malignant cells, thereby establishing a fertile habitat for metastatic tumours. PMID:17139281

  9. Effect of MgSO4 on expression of NSE and S-100 in rats brain tissue irradiated by 6 MeV electron beam

    International Nuclear Information System (INIS)

    Zhou Juying; Wang Lili; Yu Zhiying; Qin Songbing; Xu Xiaoting; Li Li; Tu Yu

    2007-01-01

    Objective: To explore the protection of magnesium sulfate (MgSO 4 ) on radiation-induced acute brain injuries. Methods: Thirty six mature Sprague-Dawley rats were randomly divided into 3 groups: blank control group, experimental control group and experimental administered group. The whole brain of SD rats of experimental control group and experimental-therapeutic group were irradiated with a dose of 20 Gy using 6 MeV electron beam. Magnesium sulfate was injected intraperitoneally into the rats of experimental-therapeutic group before and after irradiation for five times. The brain tissue were taken on days 1, 7, 14 and 30 after irradiation. Immunohistochemical method was used to detect the expressions of NSE and S-100 in brain tissue. All data were processed statistically with One-ANOVA analysis. Results: The expressions of NSE and S-100 after whole brain irradiation were time-dependent. Compared with blank control group, the expression of NSE in brains of experimental control group decreased significantly (P 4 can inhibit the expression of S-100, but induce the expression of NSE on radiation-induced acute brain injury. MgSO 4 has a protective effect on radiation-induced acute brain injury. (authors)

  10. Proteomics unveil corticoid-induced S100A11 shuttling in keratinocyte differentiation

    International Nuclear Information System (INIS)

    Dezitter, Xavier; Hammoudi, Fatma; Belverge, Nicolas; Deloulme, Jean-Christophe; Drobecq, Herve; Masselot, Bernadette; Formstecher, Pierre; Mendy, Denise; Idziorek, Thierry

    2007-01-01

    Unlike classical protein extraction techniques, proteomic mapping using a selective subcellular extraction kit revealed S100A11 as a new member of the S100 protein family modulated by glucocorticoids in keratinocytes. Glucocorticoids (GC)-induced S100A11 redistribution in the 'organelles and membranes' compartment. Microscopic examination indicated that glucocorticoids specifically routed cytoplasmic S100A11 toward perinuclear compartment. Calcium, a key component of skin terminal differentiation, directed S100A11 to the plasma membrane as previously reported. When calcium was added to glucocorticoids, minor change was observed at the proteomic level while confocal microscopy revealed a rapid and dramatic translocation of S100A11 toward plasma membrane. This effect was accompanied by strong nuclear condensation, loss of mitochondrial potential and DNA content, and increased high molecular weight S100A11 immunoreactivity, suggesting corticoids accelerate calcium-induced terminal differentiation. Finally, our results suggest GC-induced S100A11 relocalization could be a key step in both keratinocyte homeostasis and glucocorticoids side effects in human epidermis

  11. S100-A9 protein in exosomes from chronic lymphocytic leukemia cells promotes NF-κB activity during disease progression.

    Science.gov (United States)

    Prieto, Daniel; Sotelo, Natalia; Seija, Noé; Sernbo, Sandra; Abreu, Cecilia; Durán, Rosario; Gil, Magdalena; Sicco, Estefanía; Irigoin, Victoria; Oliver, Carolina; Landoni, Ana Inés; Gabus, Raúl; Dighiero, Guillermo; Oppezzo, Pablo

    2017-08-10

    Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by accumulation of clonal B lymphocytes, resulting from a complex balance between cell proliferation and apoptotic death. Continuous crosstalk between cancer cells and local/distant host environment is required for effective tumor growth. Among the main actors of this dynamic interplay between tumoral cells and their microenvironment are the nano-sized vesicles called exosomes. Emerging evidence indicates that secretion, composition, and functional capacity of exosomes are altered as tumors progress to an aggressive phenotype. In CLL, no data exist exploring the specific changes in the proteomic profile of plasma-derived exosomes from patients during disease evolution. We hereby report for the first time different proteomic profiles of plasma exosomes, both between indolent and progressive CLLs as well as within the individual patients at the onset of disease and during its progression. Next, we focus on the changes of the exosome protein cargoes, which are found exclusively in patients with progressive CLL after disease progression. The alterations in the proteomic cargoes underline different networks specific for leukemia progression related to inflammation, oxidative stress, and NF-κB and phosphatidylinositol 3-kinase/AKT pathway activation. Finally, our results suggest a preponderant role for the protein S100-A9 as an activator of the NFκB pathway during CLL progression and suggest that the leukemic clone can generate an autoactivation loop through S100-A9 expression, NF-κB activation, and exosome secretion. Collectively, our data propose a new pathway for NF-κB activation in CLL and highlight the importance of exosomes as extracellular mediators promoting tumor progression in CLL. © 2017 by The American Society of Hematology.

  12. PSAPP mice exhibit regionally selective reductions in gliosis and plaque deposition in response to S100B ablation

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    Young Keith A

    2010-11-01

    Full Text Available Abstract Background Numerous studies have reported that increased expression of S100B, an intracellular Ca2+ receptor protein and secreted neuropeptide, exacerbates Alzheimer's disease (AD pathology. However, the ability of S100B inhibitors to prevent/reverse AD histopathology remains controversial. This study examines the effect of S100B ablation on in vivo plaque load, gliosis and dystrophic neurons. Methods Because S100B-specific inhibitors are not available, genetic ablation was used to inhibit S100B function in the PSAPP AD mouse model. The PSAPP/S100B-/- line was generated by crossing PSAPP double transgenic males with S100B-/- females and maintained as PSAPP/S100B+/- crosses. Congo red staining was used to quantify plaque load, plaque number and plaque size in 6 month old PSAPP and PSAPP/S100B-/- littermates. The microglial marker Iba1 and astrocytic marker glial fibrillary acidic protein (GFAP were used to quantify gliosis. Dystrophic neurons were detected with the phospho-tau antibody AT8. S100B immunohistochemistry was used to assess the spatial distribution of S100B in the PSAPP line. Results PSAPP/S100B-/- mice exhibited a regionally selective decrease in cortical but not hippocampal plaque load when compared to PSAPP littermates. This regionally selective reduction in plaque load was accompanied by decreases in plaque number, GFAP-positive astrocytes, Iba1-positive microglia and phospho-tau positive dystrophic neurons. These effects were not attributable to regional variability in the distribution of S100B. Hippocampal and cortical S100B immunoreactivity in PSAPP mice was associated with plaques and co-localized with astrocytes and microglia. Conclusions Collectively, these data support S100B inhibition as a novel strategy for reducing cortical plaque load, gliosis and neuronal dysfunction in AD and suggest that both extracellular as well as intracellular S100B contribute to AD histopathology.

  13. Change and significance of serum inflammatory factors, NSE, S100 protein and stress hormone levels in patients with craniocerebral injury

    Directory of Open Access Journals (Sweden)

    Rui-Feng Liu

    2017-09-01

    Full Text Available Objective: To investigate the change and significance of serum inflammatory factors, neuron specific enolase (NSE, S100 protein and stress hormone levels in patients with brain diseases. Methods: A total of 115 patients with craniocerebral injury were selected as the observation group, according to the Glasgow Coma Scale (GCS, they were divided into light-sized group (n=38, middle-sized group (n=40 and severe-sized group (n=37, at the same time the other 120 healthy subjects were selected as the control group. The levels of serum inflammatory cytokines [tumor necrosis factor alpha (TNF-α and procalcitonin (PCT], neuron specific enolase (NSE, S100 protein and the stress hormone cortisol [(COR, adrenocorticotropic hormone (ACTH, β-endorphin (β-EP] of both groups were compared. Results: The levels of TNF-α, PCT, NSE, S100, COR, ACTH and β-EP in the observation group were (145.73±19.24 ng/L, (2.41±0.64 ng/mL, (38.11±12.28 ng/mL, (0.87±0.32 μg/L, (818.87±121.14 nmol/L, (107.38±13.94 ng/L, (126.74±39.04 ng/mL, which were significantly higher than control group, the difference was statistically significant; Comparison of indexes among the observation group, NF-α, PCT, NSE, S100, COR, ACTH and β-EP levels in the middle-sized group and severe-sized group were significantly higher than those in the light-sized group, and the levels in the severe-sized group were significantly higher than those of the middle-sized group, the difference was statistically significant. Conclusion: The levels of Serum inflammatory factors, NSE, S100 protein and stress hormone were significantly increased in patients with craniocerebral injury, the level was related to the degree of traumatic brain injury, which could be used as an important indicator to assess the severity of the disease.

  14. Age-related accumulation of advanced glycation end-products-albumin, S100β, and the expressions of advanced glycation end product receptor differ in visceral and subcutaneous fat

    Energy Technology Data Exchange (ETDEWEB)

    Son, Kuk Hui [Department of Thoracic and Cardiovascular Surgery, Gachon University Gil Medical Center, Gachon University, Incheon (Korea, Republic of); Son, Myeongjoo [Department of Anatomy and Cell Biology, Gachon University Graduate School of Medicine, Incheon (Korea, Republic of); Functional Cellular Networks Laboratory, Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Incheon (Korea, Republic of); Ahn, Hyosang; Oh, Seyeon; Yum, Yoonji [Functional Cellular Networks Laboratory, Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Incheon (Korea, Republic of); Choi, Chang Hu [Department of Thoracic and Cardiovascular Surgery, Gachon University Gil Medical Center, Gachon University, Incheon (Korea, Republic of); Park, Kook Yang, E-mail: kkyypark@ghil.com [Department of Thoracic and Cardiovascular Surgery, Gachon University Gil Medical Center, Gachon University, Incheon (Korea, Republic of); Byun, Kyunghee, E-mail: khbyun1@gachon.ac.kr [Department of Anatomy and Cell Biology, Gachon University Graduate School of Medicine, Incheon (Korea, Republic of); Functional Cellular Networks Laboratory, Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Incheon (Korea, Republic of)

    2016-08-19

    Visceral fat induces more inflammation by activating macrophages than subcutaneous fat, and inflammation is an underlying feature of the pathogeneses of various diseases, including cardiovascular disease and diabetes. Advanced glycation end products (AGEs), S100β, and their receptors, the receptor for advanced glycation end products (RAGE), lead to macrophage activation. However, little information is available regarding the differential accumulations of AGE-albumin (serum albumin modified by AGEs), S100β, or expressions of RAGE in different adipocyte types in fat tissues. In this study, the authors investigated whether age-related AGE-albumin accumulations S100β level, and RAGE expressions differ in subcutaneous and visceral fat tissues. Subcutaneous and visceral fat were harvested from 3- and 28-week-old rats. Macrophage activation was confirmed by Iba1 staining, and AGE-albumin accumulations and RAGE expressions were assessed by confocal microscopy. S100β were analyzed by immunoblotting. It was found that activated macrophage infiltration, AGE-albumin accumulation, and S100β in visceral fat was significantly greater in 28-week-old rats than in 3-week-old rats, but similar in subcutaneous fat. The expression of RAGE in visceral fat was much greater in 28-week-old rats, but its expression in subcutaneous fat was similar in 3- and 28-week-old rats. Furthermore, inflammatory signal pathways (NFκB, TNF-α) and proliferation pathways (FAK) in visceral fat were more activated in 28-week-old rats. These results imply that age-related AGE-albumin accumulation, S100β, and RAGE expression are more prominent in visceral than in subcutaneous fat, suggesting that visceral fat is involved in the pathogenesis of inflammation-induced diseases in the elderly. - Highlights: • The age-related AGE-albumin accumulation and S100β were more prominent in visceral than subcutaneous fat. • The age-related RAGE expression were more prominent in visceral than subcutaneous fat.

  15. Age-related accumulation of advanced glycation end-products-albumin, S100β, and the expressions of advanced glycation end product receptor differ in visceral and subcutaneous fat

    International Nuclear Information System (INIS)

    Son, Kuk Hui; Son, Myeongjoo; Ahn, Hyosang; Oh, Seyeon; Yum, Yoonji; Choi, Chang Hu; Park, Kook Yang; Byun, Kyunghee

    2016-01-01

    Visceral fat induces more inflammation by activating macrophages than subcutaneous fat, and inflammation is an underlying feature of the pathogeneses of various diseases, including cardiovascular disease and diabetes. Advanced glycation end products (AGEs), S100β, and their receptors, the receptor for advanced glycation end products (RAGE), lead to macrophage activation. However, little information is available regarding the differential accumulations of AGE-albumin (serum albumin modified by AGEs), S100β, or expressions of RAGE in different adipocyte types in fat tissues. In this study, the authors investigated whether age-related AGE-albumin accumulations S100β level, and RAGE expressions differ in subcutaneous and visceral fat tissues. Subcutaneous and visceral fat were harvested from 3- and 28-week-old rats. Macrophage activation was confirmed by Iba1 staining, and AGE-albumin accumulations and RAGE expressions were assessed by confocal microscopy. S100β were analyzed by immunoblotting. It was found that activated macrophage infiltration, AGE-albumin accumulation, and S100β in visceral fat was significantly greater in 28-week-old rats than in 3-week-old rats, but similar in subcutaneous fat. The expression of RAGE in visceral fat was much greater in 28-week-old rats, but its expression in subcutaneous fat was similar in 3- and 28-week-old rats. Furthermore, inflammatory signal pathways (NFκB, TNF-α) and proliferation pathways (FAK) in visceral fat were more activated in 28-week-old rats. These results imply that age-related AGE-albumin accumulation, S100β, and RAGE expression are more prominent in visceral than in subcutaneous fat, suggesting that visceral fat is involved in the pathogenesis of inflammation-induced diseases in the elderly. - Highlights: • The age-related AGE-albumin accumulation and S100β were more prominent in visceral than subcutaneous fat. • The age-related RAGE expression were more prominent in visceral than subcutaneous fat.

  16. EMMPRIN is associated with S100A4 and predicts patient outcome in colorectal cancer

    Science.gov (United States)

    Boye, K; Nesland, J M; Sandstad, B; Haugland Haugen, M; Mælandsmo, G M; Flatmark, K

    2012-01-01

    Background: Proteolytic enzymes and their regulators have important biological roles in colorectal cancer by stimulating invasion and metastasis, which makes these factors attractive as potential prognostic biomarkers. Methods: The expression of extracellular matrix metalloproteinase inducer (EMMPRIN) was characterised using immunohistochemistry in primary tumours from a cohort of 277 prospectively recruited colorectal cancer patients, and associations with expression of S100A4, clinicopathological parameters and patient outcome were investigated. Results: One hundred and ninety-eight samples (72%) displayed positive membrane staining of the tumour cells, whereas 10 cases (4%) were borderline positive. EMMPRIN expression was associated with shorter metastasis-free, disease-specific and overall survival in both univariate and multivariate analyses. The prognostic impact was largely confined to TNM stage III, and EMMPRIN-negative stage III patients had an excellent prognosis. Furthermore, EMMPRIN was significantly associated with expression of S100A4, and the combined expression of these biomarkers conferred an even poorer prognosis. However, there was no evidence of direct regulation between the two proteins in the colorectal cancer cell lines HCT116 and SW620 in siRNA knockdown experiments. Conclusion: EMMPRIN is a promising prognostic biomarker in colorectal cancer, and our findings suggest that it could be used in the selection of stage III patients for adjuvant therapy. PMID:22782346

  17. Long-Term Intake of Uncaria rhynchophylla Reduces S100B and RAGE Protein Levels in Kainic Acid-Induced Epileptic Seizures Rats.

    Science.gov (United States)

    Tang, Nou-Ying; Lin, Yi-Wen; Ho, Tin-Yun; Cheng, Chin-Yi; Chen, Chao-Hsiang; Hsieh, Ching-Liang

    2017-01-01

    Epileptic seizures are crucial clinical manifestations of recurrent neuronal discharges in the brain. An imbalance between the excitatory and inhibitory neuronal discharges causes brain damage and cell loss. Herbal medicines offer alternative treatment options for epilepsy because of their low cost and few side effects. We established a rat epilepsy model by injecting kainic acid (KA, 12 mg/kg, i.p.) and subsequently investigated the effect of Uncaria rhynchophylla (UR) and its underlying mechanisms. Electroencephalogram and epileptic behaviors revealed that the KA injection induced epileptic seizures. Following KA injection, S100B levels increased in the hippocampus. This phenomenon was attenuated by the oral administration of UR and valproic acid (VA, 250 mg/kg). Both drugs significantly reversed receptor potentiation for advanced glycation end product proteins. Rats with KA-induced epilepsy exhibited no increase in the expression of metabotropic glutamate receptor 3, monocyte chemoattractant protein 1, and chemokine receptor type 2, which play a role in inflammation. Our results provide novel and detailed mechanisms, explaining the role of UR in KA-induced epileptic seizures in hippocampal CA1 neurons.

  18. Chronic lithium treatment increased intracellular S100ß levels in rat primary neuronal culture.

    Directory of Open Access Journals (Sweden)

    Masoumeh Emamghoreishi

    2015-02-01

    Full Text Available S100ß a neurotrophic factor mainly released by astrocytes, has been implicated in the pathogenesis of bipolar disorder. Thus, lithium may exert its neuroprotective effects to some extent through S100ß. Furthermore, the possible effects of lithium on astrocytes as well as on interactions between neurons and astrocytes as a part of its mechanisms of actions are unknown. This study was undertaken to determine the effect of lithium on S100β in neurons, astrocytes and a mixture of neurons and astrocytes. Rat primary astrocyte, neuronal and mixed neuro-astroglia cultures were prepared from cortices of 18-day's embryos. Cell cultures were exposed to lithium (1mM or vehicle for 1day (acute or 7 days (chronic. RT-PCR and ELISA determined S100β mRNA and intra- and extracellular protein levels. Chronic lithium treatment significantly increased intracellular S100β in neuronal and neuro-astroglia cultures in comparison to control cultures (P<0.05. Acute and chronic lithium treatments exerted no significant effects on intracellular S100β protein levels in astrocytes, and extracellular S100β protein levels in three studied cultures as compared to control cultures. Acute and chronic lithium treatments did not significantly alter S100β mRNA levels in three studied cultures, compared to control cultures. Chronic lithium treatment increased intracellular S100ß protein levels in a cell-type specific manner which may favor its neuroprotective action. The findings of this study suggest that lithium may exert its neuroprotective action, at least partly, by increasing neuronal S100ß level, with no effect on astrocytes or interaction between neurons and astrocytes.

  19. Reference values for venous and capillary S100B in children

    DEFF Research Database (Denmark)

    Astrand, Ramona; Romner, Bertil; Lanke, Jan

    2011-01-01

    The current management guidelines for pediatric mild head injury (MHI) liberally recommend computed tomography (CT) and frequent admission. Serum protein S100B, currently used in management of adult head injury, has recently shown potential for reducing unnecessary CT scans after pediatric mild h...... head injury. Capillary sampling in children is commonly used when venous sampling fails or is inappropriate. We present reference values for both venous and capillary samples of protein S100B in children....

  20. Eradication of damaged keratinocytes in cutaneous lichen planus forms demonstrated by evaluation of epidermal and follicular expression of CK15, indices of apoptosis and regulatory protein S100.

    Science.gov (United States)

    Upeniece, Ilze; Groma, Valerie; Skuja, Sandra; Cauce, Vinita

    The study of cytoskeleton arrangement and its contribution to survival of cell-to-cell contacts appears to be essential for understanding of numerous cellular and tissue processes. Applying CK15, S100 labeling and TUNEL reaction to cutaneous lichen planus subtypes, we found CK15 expression in the outer and inner root sheath of hair follicles, the basal epidermal layer, and eccrine glands. Its follicular expression was decreased in nearby inflammatory infiltrates. The CK15 immunopositivity was mostly described as weak (92.3%) for lichen planus but equally subdivided into weak, moderate and strong in lichen planopilaris (2 = 32.514; df = 4; p lichen planopilaris involving the scalp: 81.2 ±10.7; 87.8 ±10.7 and 88.0 ±10.5 for the basal, spinous and upper epidermal layers, respectively. S100 positive epidermal and follicular cells did not differ in the lesions demonstrated in the study groups; still immunoreactivity was more pronounced in the scalp region of lichen planopilaris. Damage of cell-to-cell contacts was confirmed by electron microscopy. Apart from immunocyte-mediated keratinocyte death, cytoskeleton-based injury and loss of cell-to-cell and matrix contacts may be of great importance, leading to eradication of degrading cells and thus contributing to the pathogenesis of lichen planus.

  1. FAM107B is regulated by S100A4 and mediates the effect of S100A4 on the proliferation and migration of MGC803 gastric cancer cells.

    Science.gov (United States)

    Guo, Junfu; Bian, Yue; Wang, Yu; Chen, Lisha; Yu, Aiwen; Sun, Xiuju

    2017-10-01

    FAM107B expression was decreased in stomach cancer and many other kinds of cancer. The forced expression of FAM107B in HeLa cells diminished proliferation in response to growth factors, suggesting that FAM107B might play important roles in many types of cancers. But the mechanisms underlying the decreased expression of FAM107B in cancers are not clear, the functional significance needs to be further clarified. Our previous findings from cDNA microarray showed that there are 179 differentially expressed genes after S100A4 inhibition in gastric cancer cells MGC803. FAM107B was an upregulated one among them. In the present study, we confirmed that FAM107B expression was upregulated in MGC803 cells after S100A4 inhibition by qRT-PCR. We demonstrated for the first time that FAM107B was downregulated by S100A4. The results from CCK-8 and transwell assay showed that FAM107B inhibition by siRNA led to significantly increased proliferation and migrating abilities of MGC803 cells, respectively, indicating that FAM107B plays important roles in inhibiting the proliferation and migration of MGC803 cells. The rescue experiment showed that FAM107B-siRNA transfection reversed the reduced proliferation and migration abilities induced by S100A4 inhibition in the cells. These findings suggest that, as a downstream effector, FAM107B at least partly mediates the effect of S100A4 on the proliferation and migration of MGC803 cells. In conclusion, we first provide experimental evidence suggesting that FAM107B was downregulated by S100A4 in gastric cancer MGC803 cells. And FAM107B at least partially mediates the biological effect of S100A4 in the cells. © 2017 International Federation for Cell Biology.

  2. Up-regulation of metastasis-promoting S100A4 (Mts-1) in rheumatoid arthritis

    DEFF Research Database (Denmark)

    Klingelhöfer, Jörg; Senolt, Ladislav; Baslund, Bo

    2007-01-01

    To examine the involvement of the metastasis-inducing protein S100A4 (Mts-1) in the pathogenesis of rheumatoid arthritis (RA).......To examine the involvement of the metastasis-inducing protein S100A4 (Mts-1) in the pathogenesis of rheumatoid arthritis (RA)....

  3. Long-Term Intake of Uncaria rhynchophylla Reduces S100B and RAGE Protein Levels in Kainic Acid-Induced Epileptic Seizures Rats

    Directory of Open Access Journals (Sweden)

    Nou-Ying Tang

    2017-01-01

    Full Text Available Epileptic seizures are crucial clinical manifestations of recurrent neuronal discharges in the brain. An imbalance between the excitatory and inhibitory neuronal discharges causes brain damage and cell loss. Herbal medicines offer alternative treatment options for epilepsy because of their low cost and few side effects. We established a rat epilepsy model by injecting kainic acid (KA, 12 mg/kg, i.p. and subsequently investigated the effect of Uncaria rhynchophylla (UR and its underlying mechanisms. Electroencephalogram and epileptic behaviors revealed that the KA injection induced epileptic seizures. Following KA injection, S100B levels increased in the hippocampus. This phenomenon was attenuated by the oral administration of UR and valproic acid (VA, 250 mg/kg. Both drugs significantly reversed receptor potentiation for advanced glycation end product proteins. Rats with KA-induced epilepsy exhibited no increase in the expression of metabotropic glutamate receptor 3, monocyte chemoattractant protein 1, and chemokine receptor type 2, which play a role in inflammation. Our results provide novel and detailed mechanisms, explaining the role of UR in KA-induced epileptic seizures in hippocampal CA1 neurons.

  4. S100A8 and S100A9 are messengers in the crosstalk between epidermis and dermis modulating a psoriatic milieu in human skin

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Young; Jang, Sunhyae [Department of Dermatology, College of Medicine, Chungnam National University, Daejeon (Korea, Republic of); Min, Jeong-Ki; Lee, Kyungmin [Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon (Korea, Republic of); Department of Biomolecular Science, University of Science and Technology, Daejeon (Korea, Republic of); Sohn, Kyung-Cheol [Department of Dermatology, College of Medicine, Chungnam National University, Daejeon (Korea, Republic of); Lim, Jong-Soon [College of Oriental Medicine, Daejeon University, Daejeon (Korea, Republic of); Im, Myung; Lee, Hae-Eul; Seo, Young-Joon; Kim, Chang-Deok [Department of Dermatology, College of Medicine, Chungnam National University, Daejeon (Korea, Republic of); Lee, Jeung-Hoon, E-mail: jhoon@cnu.ac.kr [Department of Dermatology, College of Medicine, Chungnam National University, Daejeon (Korea, Republic of)

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer Upregulated S100A8 and/or S100A9 in psoriasis epidermis induce cytokine production. Black-Right-Pointing-Pointer Upregulated S100A8 and/or S100A9 in psoriasis epidermis induce migration of immune cells. Black-Right-Pointing-Pointer Upregulated S100A8 and/or S100A9 in psoriasis epidermis induce angiogenesis. Black-Right-Pointing-Pointer S100A8 and/or S100A9 may play a role in the crosstalk between epidermis and dermis in psoriasis. -- Abstract: S100A8 and S100A9 are members of the S100A8 protein family that exist as homodimers and heterodimers in neutrophils, monocytes, and macrophages. Recent studies have shown the pivotal roles of S100A8 and S100A9 in the propagation of inflammation and keratinocyte proliferation in psoriasis. We found significant up-regulation of S100A8 and S100A9 secretion from keratinocytes in psoriatic lesions. To mimic the in vivo secretory conditions of S100A8 and S100A9 from psoriatic epidermal keratinocytes, we used the culture medium (CM) of S100A8 and S100A8/A9 adenovirus-transduced keratinocytes to investigate the functions of S100A8 and S100A9. We detected increased levels of various pro-inflammatory cytokines in the CM, including IL-8 and TNF-{alpha}, which are involved in aggravating psoriatic skin lesions, and IL-6 and members of the CXCL family of pro-angiogenic cytokines. The CM increased immune cell migration and increased angiogenesis in human umbilical vein endothelial cells. In conclusion, we found that the upregulated production of S100A8 and S100A9 by psoriatic epidermal keratinocytes activated adjacent keratinocytes to produce several cytokines. Moreover, S100A8 and S100A9 themselves function as pro-angiogenic and chemotactic factors, generating a psoriatic milieu in skin.

  5. S100A8 and S100A9 are messengers in the crosstalk between epidermis and dermis modulating a psoriatic milieu in human skin

    International Nuclear Information System (INIS)

    Lee, Young; Jang, Sunhyae; Min, Jeong-Ki; Lee, Kyungmin; Sohn, Kyung-Cheol; Lim, Jong-Soon; Im, Myung; Lee, Hae-Eul; Seo, Young-Joon; Kim, Chang-Deok; Lee, Jeung-Hoon

    2012-01-01

    Highlights: ► Upregulated S100A8 and/or S100A9 in psoriasis epidermis induce cytokine production. ► Upregulated S100A8 and/or S100A9 in psoriasis epidermis induce migration of immune cells. ► Upregulated S100A8 and/or S100A9 in psoriasis epidermis induce angiogenesis. ► S100A8 and/or S100A9 may play a role in the crosstalk between epidermis and dermis in psoriasis. -- Abstract: S100A8 and S100A9 are members of the S100A8 protein family that exist as homodimers and heterodimers in neutrophils, monocytes, and macrophages. Recent studies have shown the pivotal roles of S100A8 and S100A9 in the propagation of inflammation and keratinocyte proliferation in psoriasis. We found significant up-regulation of S100A8 and S100A9 secretion from keratinocytes in psoriatic lesions. To mimic the in vivo secretory conditions of S100A8 and S100A9 from psoriatic epidermal keratinocytes, we used the culture medium (CM) of S100A8 and S100A8/A9 adenovirus-transduced keratinocytes to investigate the functions of S100A8 and S100A9. We detected increased levels of various pro-inflammatory cytokines in the CM, including IL-8 and TNF-α, which are involved in aggravating psoriatic skin lesions, and IL-6 and members of the CXCL family of pro-angiogenic cytokines. The CM increased immune cell migration and increased angiogenesis in human umbilical vein endothelial cells. In conclusion, we found that the upregulated production of S100A8 and S100A9 by psoriatic epidermal keratinocytes activated adjacent keratinocytes to produce several cytokines. Moreover, S100A8 and S100A9 themselves function as pro-angiogenic and chemotactic factors, generating a psoriatic milieu in skin.

  6. Presence of S100A9-positive inflammatory cells in cancer tissues correlates with an early stage cancer and a better prognosis in patients with gastric cancer

    International Nuclear Information System (INIS)

    Fan, Biao; Li, Ying-Ai; Du, Hong; Zhao, Wei; Niu, Zhao-Jian; Lu, Ai-Ping; Li, Ji-You; Ji, Jia-Fu; Zhang, Lian-Hai; Jia, Yong-ning; Zhong, Xi-Yao; Liu, Yi-Qiang; Cheng, Xiao-Jing; Wang, Xiao-Hong; Xing, Xiao-Fang; Hu, Ying

    2012-01-01

    S100A9 was originally discovered as a factor secreted by inflammatory cells. Recently, S100A9 was found to be associated with several human malignancies. The purpose of this study is to investigate S100A9 expression in gastric cancer and explore its role in cancer progression. S100A9 expression in gastric tissue samples from 177 gastric cancer patients was assessed by immunohistochemistry. The expression of its dimerization partner S100A8 and the S100A8/A9 heterodimer were also assessed by the same method. The effect of exogenous S100A9 on motility of gastric cancer cells AGS and BGC-823 was then investigated. S100A9 was specifically expressed by inflammatory cells such as macrophages and neutrophils in human gastric cancer and gastritis tissues. Statistical analysis showed that a high S100A9 cell count (> = 200) per 200x magnification microscopic field in cancer tissues was predictive of early stage gastric cancer. High S100A9-positive cell count was negatively correlated with lymph node metastasis (P = 0.009) and tumor invasion (P = 0.011). S100A9 was identified as an independent prognostic predictor of overall survival of patients with gastric cancer (P = 0.04). Patients with high S100A9 cell count were with favorable prognosis (P = 0.021). Further investigation found that S100A8 distribution in human gastric cancer tissues was similar to S100A9. However, the number of S100A8-positive cells did not positively correlate with patient survival. The inflammatory cells infiltrating cancer were S100A8/A9 negative, while those in gastritis were positive. Furthermore, exogenous S100A9 protein inhibited migration and invasion of gastric cancer cells. Our results suggested S100A9-positive inflammatory cells in gastric cancer tissues are associated with early stage of gastric cancer and good prognosis

  7. How does extracerebral trauma affect the clinical value of S100B measurements?

    DEFF Research Database (Denmark)

    Ohrt-Nissen, Søren; Friis-Hansen, Lennart; Dahl, Benny

    2011-01-01

    Background Protein S100B has proven to be a useful biomarker for cerebral damage. The predictive ability of S100B may, however, be affected by extracerebral injuries. The aim of this study was to investigate serum levels of S100B in patients with either isolated head injury (IHI), multi trauma...

  8. Low-Molecular-Weight Peptides from Salmon Protein Prevent Obesity-Linked Glucose Intolerance, Inflammation, and Dyslipidemia in LDLR-/-/ApoB100/100 Mice.

    Science.gov (United States)

    Chevrier, Geneviève; Mitchell, Patricia L; Rioux, Laurie-Eve; Hasan, Fida; Jin, Tianyi; Roblet, Cyril Roland; Doyen, Alain; Pilon, Geneviève; St-Pierre, Philippe; Lavigne, Charles; Bazinet, Laurent; Jacques, Hélène; Gill, Tom; McLeod, Roger S; Marette, André

    2015-07-01

    We previously reported that fish proteins can alleviate metabolic syndrome (MetS) in obese animals and human subjects. We tested whether a salmon peptide fraction (SPF) could improve MetS in mice and explored potential mechanisms of action. ApoB(100) only, LDL receptor knockout male mice (LDLR(-/-)/ApoB(100/100)) were fed a high-fat and -sucrose (HFS) diet (25 g/kg sucrose). Two groups were fed 10 g/kg casein hydrolysate (HFS), and 1 group was additionally fed 4.35 g/kg fish oil (FO; HFS+FO). Two other groups were fed 10 g SPF/kg (HFS+SPF), and 1 group was additionally fed 4.35 g FO/kg (HFS+SPF+FO). A fifth (reference) group was fed a standard feed pellet diet. We assessed the impact of dietary treatments on glucose tolerance, adipose tissue inflammation, lipid homeostasis, and hepatic insulin signaling. The effects of SPF on glucose uptake, hepatic glucose production, and inducible nitric oxide synthase activity were further studied in vitro with the use of L6 myocytes, FAO hepatocytes, and J774 macrophages. Mice fed HFS+SPF or HFS+SPF+FO diets had lower body weight (protein effect, P = 0.024), feed efficiency (protein effect, P = 0.018), and liver weight (protein effect, P = 0.003) as well as lower concentrations of adipose tissue cytokines and chemokines (protein effect, P ≤ 0.003) compared with HFS and HFS+FO groups. They also had greater glucose tolerance (protein effect, P < 0.001), lower activation of the mammalian target of rapamycin complex 1/S6 kinase 1/insulin receptor substrate 1 (mTORC1/S6K1/IRS1) pathway, and increased insulin signaling in liver compared with the HFS and HFS+FO groups. The HFS+FO, HFS+SPF, and HFS+SPF+FO groups had lower plasma triglycerides (protein effect, P = 0.003; lipid effect, P = 0.002) than did the HFS group. SPF increased glucose uptake and decreased HGP and iNOS activation in vitro. SPF reduces obesity-linked MetS features in LDLR(-/-)/ApoB(100/100) mice. The anti-inflammatory and glucoregulatory properties of SPF were

  9. Efficient protein production method for NMR using soluble protein tags with cold shock expression vector

    International Nuclear Information System (INIS)

    Hayashi, Kokoro; Kojima, Chojiro

    2010-01-01

    The E. coli protein expression system is one of the most useful methods employed for NMR sample preparation. However, the production of some recombinant proteins in E. coli is often hampered by difficulties such as low expression level and low solubility. To address these problems, a modified cold-shock expression system containing a glutathione S-transferase (GST) tag, the pCold-GST system, was investigated. The pCold-GST system successfully expressed 9 out of 10 proteins that otherwise could not be expressed using a conventional E. coli expression system. Here, we applied the pCold-GST system to 84 proteins and 78 proteins were successfully expressed in the soluble fraction. Three other cold-shock expression systems containing a maltose binding protein tag (pCold-MBP), protein G B1 domain tag (pCold-GB1) or thioredoxin tag (pCold-Trx) were also developed to improve the yield. Additionally, we show that a C-terminal proline tag, which is invisible in 1 H- 15 N HSQC spectra, inhibits protein degradation and increases the final yield of unstable proteins. The purified proteins were amenable to NMR analyses. These data suggest that pCold expression systems combined with soluble protein tags can be utilized to improve the expression and purification of various proteins for NMR analysis.

  10. Structural insights into calcium-bound S100P and the V domain of the RAGE complex.

    Directory of Open Access Journals (Sweden)

    Srinivasa R Penumutchu

    Full Text Available The S100P protein is a member of the S100 family of calcium-binding proteins and possesses both intracellular and extracellular functions. Extracellular S100P binds to the cell surface receptor for advanced glycation end products (RAGE and activates its downstream signaling cascade to meditate tumor growth, drug resistance and metastasis. Preventing the formation of this S100P-RAGE complex is an effective strategy to treat various disease conditions. Despite its importance, the detailed structural characterization of the S100P-RAGE complex has not yet been reported. In this study, we report that S100P preferentially binds to the V domain of RAGE. Furthermore, we characterized the interactions between the RAGE V domain and Ca(2+-bound S100P using various biophysical techniques, including isothermal titration calorimetry (ITC, fluorescence spectroscopy, multidimensional NMR spectroscopy, functional assays and site-directed mutagenesis. The entropy-driven binding between the V domain of RAGE and Ca(+2-bound S100P was found to lie in the micromolar range (Kd of ∼ 6 µM. NMR data-driven HADDOCK modeling revealed the putative sites that interact to yield a proposed heterotetrameric model of the S100P-RAGE V domain complex. Our study on the spatial structural information of the proposed protein-protein complex has pharmaceutical relevance and will significantly contribute toward drug development for the prevention of RAGE-related multifarious diseases.

  11. Efficient expression and characterization of a cold-active endo-1, 4-β-glucanase from Citrobacter farmeri by co-expression of Myxococcus xanthus protein S

    Directory of Open Access Journals (Sweden)

    Xi Bai

    2016-11-01

    Conclusions: The ProS-EglC is promising in application of various biotechnological processes because of its cold-active characterizations. This study also suggests a useful strategy for the expression of foreign proteins in E. coli using a ProS tag.

  12. Effect of low dose radiation on cell cycle and expression of its related proteins of HCT-8 cells

    International Nuclear Information System (INIS)

    Xu Ying; Ma Kewei; Li Wei; Wang Guanjun

    2009-01-01

    Objective: To study the effects of low dose radiation (LDR) on cell cycle and the expression of its related proteins of HCT-8 cells and provide theoretical basis for clinical application of LDR. Methods: Human colon carcinoma cells (HCT-8) cultivated in vitro were divided into seven groups: sham radiation group (0 mGy), LDR groups (25, 50, 75, 100 and 200 mGy) and high dose radiation group (1000 mGy). The proliferation rate was detected with the method of cell count and MTT, the ratios of G 0 /G 1 , S, G 2 /M in cell cycle were determined with flow cytometry after LDR, The cell cycle and expressions of related signal proteins were analyzed with protein assay system. Results: The results of cell count and MTT showed that there were no significant differences of proliferation rate of HCT-8 cells between 25, 50, 75, 100, 200 mGy LDR groups and sham radiation group (P>0.05); compared with high dose radiation group, there were significant differences (P 0 /G 1 phase of HCT-8 cells increased (P>0.05), the ratio of S phase decreased significantly (P 2 /M phase increased obviously (P 0 /G 1 , S, and G 2 /M phases of HCT-8 cells 48 h after radiation compared with sham radiation group (P>0.05). The protein assay result indicated that the expressions of AKt, PCNA, p27, CDK2, cyclin E, EGFR, ERK1/2, p-ERK, p-GSK-32/β in HCT-8 cells after LDR decreased compared with sham radiation group. Conclusion: LDR has no stimulating effect on HCT-8 cells. However, to some extent LDR suppress the expressions of some proteins related to proliferation and cell cycle. (authors)

  13. S100A4 interacts with p53 in the nucleus and promotes p53 degradation.

    Science.gov (United States)

    Orre, L M; Panizza, E; Kaminskyy, V O; Vernet, E; Gräslund, T; Zhivotovsky, B; Lehtiö, J

    2013-12-05

    S100A4 is a small calcium-binding protein that is commonly overexpressed in a range of different tumor types, and it is widely accepted that S100A4 has an important role in the process of cancer metastasis. In vitro binding assays has shown that S100A4 interacts with the tumor suppressor protein p53, indicating that S100A4 may have additional roles in tumor development. In the present study, we show that endogenous S100A4 and p53 interact in complex samples, and that the interaction increases after inhibition of MDM2-dependent p53 degradation using Nutlin-3A. Further, using proximity ligation assay, we show that the interaction takes place in the cell nucleus. S100A4 knockdown experiments in two p53 wild-type cell lines, A549 and HeLa, resulted in stabilization of p53 protein, indicating that S100A4 is promoting p53 degradation. Finally, we demonstrate that S100A4 knockdown leads to p53-dependent cell cycle arrest and increased cisplatin-induced apoptosis. Thus, our data add a new layer to the oncogenic properties of S100A4 through its inhibition of p53-dependent processes.

  14. Functional significance of metastasis-inducing S100A4(Mts1) in tumor-stroma interplay

    DEFF Research Database (Denmark)

    Schmidt-Hansen, Birgitte; Klingelhöfer, Jörg; Grum-Schwensen, Birgitte

    2004-01-01

    Causal implication of S100A4 in inducing metastases was convincingly shown previously. However, the mechanisms that associate S100A4 with tumor progression are not well understood. S100A4 protein, as a typical member of the S100 family, exhibits dual, intracellular and extracellular, functions. T...

  15. Efficient protein production method for NMR using soluble protein tags with cold shock expression vector

    Energy Technology Data Exchange (ETDEWEB)

    Hayashi, Kokoro [Fujifilm Corporation, Analysis Technology Center (Japan); Kojima, Chojiro, E-mail: kojima@protein.osaka-u.ac.j [Nara Institute of Science and Technology (NAIST), Graduate School of Biological Sciences (Japan)

    2010-11-15

    The E. coli protein expression system is one of the most useful methods employed for NMR sample preparation. However, the production of some recombinant proteins in E. coli is often hampered by difficulties such as low expression level and low solubility. To address these problems, a modified cold-shock expression system containing a glutathione S-transferase (GST) tag, the pCold-GST system, was investigated. The pCold-GST system successfully expressed 9 out of 10 proteins that otherwise could not be expressed using a conventional E. coli expression system. Here, we applied the pCold-GST system to 84 proteins and 78 proteins were successfully expressed in the soluble fraction. Three other cold-shock expression systems containing a maltose binding protein tag (pCold-MBP), protein G B1 domain tag (pCold-GB1) or thioredoxin tag (pCold-Trx) were also developed to improve the yield. Additionally, we show that a C-terminal proline tag, which is invisible in {sup 1}H-{sup 15}N HSQC spectra, inhibits protein degradation and increases the final yield of unstable proteins. The purified proteins were amenable to NMR analyses. These data suggest that pCold expression systems combined with soluble protein tags can be utilized to improve the expression and purification of various proteins for NMR analysis.

  16. ATM-mediated Snail Serine 100 phosphorylation regulates cellular radiosensitivity

    International Nuclear Information System (INIS)

    Boohaker, Rebecca J.; Cui, Xiaoli; Stackhouse, Murray; Xu, Bo

    2013-01-01

    Purpose: Activation of the DNA damage responsive protein kinase ATM is a critical step for cellular survival in response to ionizing irradiation (IR). Direct targets of ATM regulating radiosensitivity remain to be fully investigated. We have recently reported that ATM phosphorylates the transcriptional repressor Snail on Serine 100. We aimed to further study the functional significance of ATM-mediated Snail phosphorylation in response to IR. Material and methods: We transfected vector-only, wild-type, the Serine 100 to alanine (S100A) or to glutamic acid (S100E) substitution of Snail into various cell lines. We assessed colony formation, γ-H2AX focus formation and the invasion index in the cells treated with or without IR. Results: We found that over-expression of the S100A mutant Snail in HeLa cells significantly increased radiosensitivity. Meanwhile the expression of S100E, a phospho-mimicking mutation, resulted in enhanced radio-resistance. Interestingly, S100E could rescue the radiosensitive phenotype in ATM-deficient cells. We also found that expression of S100E increased γ-H2AX focus formation and compromised inhibition of invasion in response to IR independent of cell survival. Conclusion: ATM-mediated Snail Serine 100 phosphorylation in response to IR plays an important part in the regulation of radiosensitivity

  17. S100A8/A9 Is Not Involved in Host Defense against Murine Urinary Tract Infection

    NARCIS (Netherlands)

    Dessing, M.C.; Butter, L.M.; Teske, G.J.; Claessen, N.; van der Loos, C.M.; Vogl, T.; Roth, J.; van der Poll, T.; Florquin, S.; Leemans, J.C.

    2010-01-01

    Background: Inflammation is commonly followed by the release of endogenous proteins called danger associated molecular patterns (DAMPs) that are able to warn the host for eminent danger. S100A8/A9 subunits are DAMPs that belong to the S100 family of calcium binding proteins. S100A8/A9 complexes

  18. LPS-Toll-Like Receptor-Mediated Signaling on Expression of Protein S and C4b-Binding Protein in the Liver

    Directory of Open Access Journals (Sweden)

    Tatsuya Hayashi

    2010-01-01

    Full Text Available Protein S (PS, mainly synthesized in hepatocytes and endothelial cells, plays a critical role as a cofactor of anticoagulant activated protein C (APC. PS activity is regulated by C4b-binding protein (C4BP, structurally composed of seven α-chains (C4BPα and a β-chain (C4BPβ. In this paper, based primarily on our previous studies, we review the lipopolysaccharide (LPS-induced signaling which affects expression of PS and C4BP in the liver. Our in vivo studies in rats showed that after LPS injection, plasma PS levels are significantly decreased, whereas plasma C4BP levels first are transiently decreased after 2 to 12 hours and then significantly increased after 24 hours. LPS decreases PS antigen and mRNA levels in both hepatocytes and sinusoidal endothelial cells (SECs, and decreases C4BP antigen and both C4BPα and C4BPβ mRNA levels in hepatocytes. Antirat CD14 and antirat Toll-like receptor (TLR-4 antibodies inhibited LPS-induced NFκB activation in both hepatocytes and SECs. Furthermore, inhibitors of NFκB and MEK recovered the LPS-induced decreased expression of PS in both cell types and the LPS-induced decreased expression of C4BP in hepatocytes. These data suggest that the LPS-induced decrease in PS expression in hepatocytes and SECs and LPS-induced decrease in C4BP expression in hepatocytes are mediated by MEK/ERK signaling and NFκB activation and that membrane-bound CD14 and TLR-4 are involved in this mechanism.

  19. Expression of p53 protein in Barrett’s adenocarcinoma and adenocarcinoma of the gastric cardia and antrum

    Directory of Open Access Journals (Sweden)

    Jovanović Ivan

    2005-01-01

    Full Text Available Background/Aim. Most studies of esophageal and gastric adenocarcinomas have shown a very high rate of p53 gene mutation and/or protein overexpression, but the influence of the tumor site upon the frequency of p53 protein expression has not been evaluated (gastroesophageal junction, Barret's esophagus, and antrum. The aim of our study was to analyze the correlation between the selected clinico-pthological parameters, and p53 protein overexpression in regards to the particular tumor location. Methods. The material comprised 66 surgical specimens; 10 were Barrett’s carcinomas, 25 adenocarcinomas of the gastric cardia (type II adenocarcinoma of the esophagogastric junction - EGJ, and 31 adenocarcinomas of the antrum. Immunostaining for p53 protein was performed on formalin-fixed, paraffin-embedded tissue sections, using the alkaline phosphatase - antialkaline phosphatase (APAAP method. The cases were considered positive for p53 if at least 5% of the tumor cells expressed this protein by immunostaining. Results. There was no significant difference observed between the studied groups in regards to age, sex, Lauren’s classification and tumor differentiation. There was, however, a significant difference observed in the depth of tumor invasion between Barrrett’s adenocarcinoma and adenocarcinoma of the cardia compared with the adenocarcinoma of the antrum. Namely, at the time of surgery, both Barrett’s adenocarcinomas and adenocarcinomas of the cardia, were significantly more advanced comparing with the adenocarcinomas of the antrum. Overexpression of p53 was found in 40% (4/10 of Barrett’s adenocarcinomas, 72% (18/25 of adenocarcinoma of the cardia and 65% (20/31 of adenocarcinoma of the antrum. No significant differences in p53 expression in relation to sex, type (Lauren of tumor, depth of invasion, lymph node involvement, or tumor differentiation were observed in any of the analyzed groups of tumors. Patients with more advanced Barrett’s

  20. S100A4: a common mediator of epithelial-mesenchymal transition, fibrosis and regeneration in diseases?

    DEFF Research Database (Denmark)

    Schneider, M.; Sheikh, S.P.; Hansen, Jakob Lerche

    2008-01-01

    and neuronal injuries. Common to all these diseases is the involvement of fibrotic and inflammatory processes, i.e. processes greatly dependent on tissue remodelling, cell motility and epithelial-mesenchymal transition. Therefore, the basic biological mechanisms behind S100A4's effects are emerging. S100A4...... belongs to the S100 family of proteins that contain two Ca2+-binding sites including a canonical EF-hand motif. S100A4 is involved in the regulation of a wide range of biological effects including cell motility, survival, differentiation and contractility. S100A4 has both intracellular and extracellular...... effects. Hence, S100A4 interacts with cytoskeletal proteins and enhances metastasis of several types of cancer cells. In addition, S100A4 is secreted by unknown mechanisms, thus, paracrinely stimulating a variety of cellular responses, including angiogenesis and neuronal growth. Although many cellular...

  1. Interaction between S100P and the anti-allergy drug cromolyn

    International Nuclear Information System (INIS)

    Penumutchu, Srinivasa R.; Chou, Ruey-Hwang; Yu, Chin

    2014-01-01

    Highlights: • The interaction between S100P–cromolyn was investigated by fluorescence spectroscopy. • The interfacial residues on S100P and cromolyn contact surface were mapped by 1 H- 15 N HSQC experiments. • S100P–cromolyn complex model was generated from NMR restraints using HADDOCK program. • The stability of the S100P–cromolyn complex was studied using molecular dynamics simulations. - Abstract: The S100P protein has been known to mediate cell proliferation by binding the receptor for advanced glycation end products (RAGE) to activate signaling pathways, such as the extracellular regulated kinase (ERK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways. S100P/RAGE signaling is involved in a variety of diseases, such as cancer, metastasis, and diabetes. Cromolyn is an anti-allergy drug that binds S100P to block the interaction between S100P and RAGE. In the present study, we characterized the properties of the binding between cromolyn and calcium-bound S100P using various biophysical techniques. The binding affinity for S100P and cromolyn was measured to be in the millimolar range by fluorescence spectroscopy. NMR-HSQC titration experiments and HADDOCK modeling was employed to determine the spatial structure of the proposed heterotetramer model of the S100P–cromolyn complex. Additional MD simulation results revealed the important properties in the complex stability and conformational flexibility of the S100P–cromolyn complex. This proposed model has provided an understanding of the molecular level interactions of S100P–cromolyn complex

  2. C-reactive protein inhibits survivin expression via Akt/mTOR pathway downregulation by PTEN expression in cardiac myocytes.

    Directory of Open Access Journals (Sweden)

    Beom Seob Lee

    Full Text Available C-reactive protein (CRP is one of the most important biomarkers for arteriosclerosis and cardiovascular disease. Recent studies have shown that CRP affects cell cycle and inflammatory process in cardiac myocytes. Survivin is also involved in cardiac myocytes replication and apoptosis. Reduction of survivin expression is associated with less favorable cardiac remodeling in animal models. However, the effect of CRP on survivin expression and its cellular mechanism has not yet been studied. We demonstrated that treatment of CRP resulted in a significant decrease of survivin protein expression in a concentration-dependent manner in cardiac myocytes. The upstream signaling proteins of survivin, such as Akt, mTOR and p70S6K, were also downregulated by CRP treatment. In addition, CRP increased the protein and mRNA levels of PTEN. The siRNA transfection or specific inhibitor treatment for PTEN restored the CRP-induced downregulation of Akt/mTOR/p70S6K pathway and survivin protein expression. Moreover, pretreatment with a specific p53 inhibitor decreased the CRP-induced PTEN expression. ERK-specific inhibitor also blocked the p53 phosphorylation and PTEN expression induced by CRP. Our study provides a novel insight into CRP-induced downregulation of survivin protein expression in cardiac myocytes through mechanisms that involved in downregulation of Akt/mTOR/p70S6K pathway by expression of PTEN.

  3. Amlexanox Blocks the Interaction between S100A4 and Epidermal Growth Factor and Inhibits Cell Proliferation.

    Directory of Open Access Journals (Sweden)

    Ching Chang Cho

    Full Text Available The human S100A4 protein binds calcium, resulting in a change in its conformation to promote the interaction with its target protein. Human epidermal growth factor (EGF is the target protein of S100A4 and a critical ligand of the receptor EGFR. The EGF/EGFR system promotes cell survival, differentiation, and growth by activating several signaling pathways. Amlexanox is an anti-inflammatory and anti-allergic drug that is used to treat recurrent aphthous ulcers. In the present study, we determined that amlexanox interacts with S100A4 using heteronuclear single quantum correlation titration. We elucidated the interactions of S100A4 with EGF and amlexanox using fluorescence and nuclear magnetic resonance spectroscopy. We generated two binary models (for the S100A4-EGF and S100A4-amlexanox complexes and observed that amlexanox and EGF share a similar binding region in mS100A4. We also used a WST-1 assay to investigate the bioactivity of S100A4, EGF, and amlexanox, and found that amlexanox blocks the binding between S100A4 and EGF, and is therefore useful for the development of new anti-proliferation drugs.

  4. The Murine Natural Cytotoxic Receptor NKp46/NCR1 Controls TRAIL Protein Expression in NK Cells and ILC1s

    Directory of Open Access Journals (Sweden)

    Sam Sheppard

    2018-03-01

    Full Text Available Summary: TRAIL is an apoptosis-inducing ligand constitutively expressed on liver-resident type 1 innate lymphoid cells (ILC1s and a subset of natural killer (NK cells, where it contributes to NK cell anti-tumor, anti-viral, and immunoregulatory functions. However, the intrinsic pathways involved in TRAIL expression in ILCs remain unclear. Here, we demonstrate that the murine natural cytotoxic receptor mNKp46/NCR1, expressed on ILC1s and NK cells, controls TRAIL protein expression. Using NKp46-deficient mice, we show that ILC1s lack constitutive expression of TRAIL protein and that NK cells activated in vitro and in vivo fail to upregulate cell surface TRAIL in the absence of NKp46. We show that NKp46 regulates TRAIL expression in a dose-dependent manner and that the reintroduction of NKp46 in mature NK cells deficient for NKp46 is sufficient to restore TRAIL surface expression. These studies uncover a link between NKp46 and TRAIL expression in ILCs with potential implications in pathologies involving NKp46-expressing cells. : Sheppard et al. find that mice deficient in the activating receptor NCR1/NKp46 (Ncr1−/− fail to express the apoptosis-inducing ligand TRAIL at the surface of group 1 innate lymphoid cells (ILC1s. Keywords: NK cell, natural killer cell, NKp46, ILC1, TRAIL, IL-15, IL-2

  5. S100A11 promotes human pancreatic cancer PANC-1 cell proliferation and is involved in the PI3K/AKT signaling pathway.

    Science.gov (United States)

    Xiao, Mingbing; Li, Tao; Ji, Yifei; Jiang, Feng; Ni, Wenkai; Zhu, Jing; Bao, Baijun; Lu, Cuihua; Ni, Runzhou

    2018-01-01

    S100A11, a member of S100 calcium-binding protein family, is associated with the numerous processes of tumorigenesis and metastasis. In the present study, the role of S100A11, and its possible underlying mechanisms in cell proliferation, apoptosis and cell cycle distribution in human pancreatic cancer were explored. Immunohistochemical analyses of S100A11 and phosphorylated (p)-AKT serine/threonine kinase (AKT) were performed in 30 resected specimens from patients with pancreatic cancer. PANC-1 cells were transfected with pcDNA3.1-S100A11 or treated with 50 µmol/l LY294002 for 48 h. Cell proliferation was determined using a cell counting kit-8 assay, whereas apoptosis and cell cycle distribution were determined by flow cytometry analysis. The mRNA and protein levels of S100A11, and AKT were determined using semi quantitative reverse transcription-polymerase chain reaction and western blot analyses, respectively. Pearson correlation analysis revealed that the expression levels of S100A11 and p-AKT were positively correlated (r, 0.802; PPANC-1 cell proliferation and reduced the percentage of early apoptotic cells. Flow cytometric analysis indicated that the proportion of PANC-1 cells in the S phase was significantly elevated and cell percentage in the G0/G1 phase declined in response to S100A11 overexpression (all PPANC-1 cell proliferation, promoted apoptosis and caused G1/S phase arrest in PANC-1 cells (all PPANC-1 cells through the upregulation of the PI3K/AKT signaling pathway. Thus, S100A11 may be considered as a novel drug target for targeted therapy of pancreatic cancer.

  6. S100A4 and its role in metastasis – computational integration of data on biological networks.

    Science.gov (United States)

    Buetti-Dinh, Antoine; Pivkin, Igor V; Friedman, Ran

    2015-08-01

    Characterising signal transduction networks is fundamental to our understanding of biology. However, redundancy and different types of feedback mechanisms make it difficult to understand how variations of the network components contribute to a biological process. In silico modelling of signalling interactions therefore becomes increasingly useful for the development of successful therapeutic approaches. Unfortunately, quantitative information cannot be obtained for all of the proteins or complexes that comprise the network, which limits the usability of computational models. We developed a flexible computational framework for the analysis of biological signalling networks. We demonstrate our approach by studying the mechanism of metastasis promotion by the S100A4 protein, and suggest therapeutic strategies. The advantage of the proposed method is that only limited information (interaction type between species) is required to set up a steady-state network model. This permits a straightforward integration of experimental information where the lack of details are compensated by efficient sampling of the parameter space. We investigated regulatory properties of the S100A4 network and the role of different key components. The results show that S100A4 enhances the activity of matrix metalloproteinases (MMPs), causing higher cell dissociation. Moreover, it leads to an increased stability of the pathological state. Thus, avoiding metastasis in S100A4-expressing tumours requires multiple target inhibition. Moreover, the analysis could explain the previous failure of MMP inhibitors in clinical trials. Finally, our method is applicable to a wide range of biological questions that can be represented as directional networks.

  7. Expression of small heat shock proteins from pea seedlings under gravity altered conditions

    Science.gov (United States)

    Talalaev, Alexandr S.

    2005-08-01

    A goal of our study was to evaluate the stress gene expression in Pisum sativum seedlings exposed to altered gravity and temperature elevation. We investigate message for the two inducible forms of the cytosolic small heat shock proteins (sHsp), sHsp 17.7 and sHsp 18.1. Both proteins are able to enhance the refolding of chemically denatured proteins in an ATP- independent manner, in other words they can function as molecular chaperones. We studied sHsps expression in pea seedlings cells by Western blotting. Temperature elevation, as the positive control, significantly increased PsHsp 17.7 and PsHsp 18.1 expression. Expression of the housekeeping protein, actin was constant and comparable to unstressed controls for all treatments. We concluded that gravitational perturbations incurred by clinorotation did not change sHsp genes expression.

  8. Interaction between S100P and the anti-allergy drug cromolyn

    Energy Technology Data Exchange (ETDEWEB)

    Penumutchu, Srinivasa R. [Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan (China); Chou, Ruey-Hwang [Graduate Institute of Cancer Biology and Center for Molecular Medicine, China Medical University, Taichung 404, Taiwan (China); Department of Biotechnology, Asia University, Taichung 413, Taiwan (China); Yu, Chin, E-mail: cyu.nthu@gmail.com [Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan (China); The Key Laboratory for Chemical Biology of Fujian Province, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China)

    2014-11-21

    Highlights: • The interaction between S100P–cromolyn was investigated by fluorescence spectroscopy. • The interfacial residues on S100P and cromolyn contact surface were mapped by {sup 1}H-{sup 15}N HSQC experiments. • S100P–cromolyn complex model was generated from NMR restraints using HADDOCK program. • The stability of the S100P–cromolyn complex was studied using molecular dynamics simulations. - Abstract: The S100P protein has been known to mediate cell proliferation by binding the receptor for advanced glycation end products (RAGE) to activate signaling pathways, such as the extracellular regulated kinase (ERK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways. S100P/RAGE signaling is involved in a variety of diseases, such as cancer, metastasis, and diabetes. Cromolyn is an anti-allergy drug that binds S100P to block the interaction between S100P and RAGE. In the present study, we characterized the properties of the binding between cromolyn and calcium-bound S100P using various biophysical techniques. The binding affinity for S100P and cromolyn was measured to be in the millimolar range by fluorescence spectroscopy. NMR-HSQC titration experiments and HADDOCK modeling was employed to determine the spatial structure of the proposed heterotetramer model of the S100P–cromolyn complex. Additional MD simulation results revealed the important properties in the complex stability and conformational flexibility of the S100P–cromolyn complex. This proposed model has provided an understanding of the molecular level interactions of S100P–cromolyn complex.

  9. Functional heterologous protein expression by genetically engineered probiotic yeast Saccharomyces boulardii.

    Directory of Open Access Journals (Sweden)

    Lauren E Hudson

    Full Text Available Recent studies have suggested the potential of probiotic organisms to be adapted for the synthesis and delivery of oral therapeutics. The probiotic yeast Saccharomyces boulardii would be especially well suited for this purpose due to its ability, in contrast to probiotic prokaryotes, to perform eukaryotic post translational modifications. This probiotic yeast thus has the potential to express a broad array of therapeutic proteins. Currently, however, use of wild type (WT S. boulardii relies on antibiotic resistance for the selection of transformed yeast. Here we report the creation of auxotrophic mutant strains of S. boulardii that can be selected without antibiotics and demonstrate that these yeast can express functional recombinant protein even when recovered from gastrointestinal immune tissues in mice. A UV mutagenesis approach was employed to generate three uracil auxotrophic S. boulardii mutants that show a low rate of reversion to wild type growth. These mutants can express recombinant protein and are resistant in vitro to low pH, bile acid salts, and anaerobic conditions. Critically, oral gavage experiments using C57BL/6 mice demonstrate that mutant S. boulardii survive and are taken up into gastrointestinal immune tissues on a similar level as WT S. boulardii. Mutant yeast recovered from gastrointestinal immune tissues furthermore retain expression of functional recombinant protein. These data show that auxotrophic mutant S. boulardii can safely express recombinant protein without antibiotic selection and can deliver recombinant protein to gastrointestinal immune tissues. These auxotrophic mutants of S. boulardii pave the way for future experiments to test the ability of S. boulardii to deliver therapeutics and mediate protection against gastrointestinal disorders.

  10. Functional heterologous protein expression by genetically engineered probiotic yeast Saccharomyces boulardii.

    Science.gov (United States)

    Hudson, Lauren E; Fasken, Milo B; McDermott, Courtney D; McBride, Shonna M; Kuiper, Emily G; Guiliano, David B; Corbett, Anita H; Lamb, Tracey J

    2014-01-01

    Recent studies have suggested the potential of probiotic organisms to be adapted for the synthesis and delivery of oral therapeutics. The probiotic yeast Saccharomyces boulardii would be especially well suited for this purpose due to its ability, in contrast to probiotic prokaryotes, to perform eukaryotic post translational modifications. This probiotic yeast thus has the potential to express a broad array of therapeutic proteins. Currently, however, use of wild type (WT) S. boulardii relies on antibiotic resistance for the selection of transformed yeast. Here we report the creation of auxotrophic mutant strains of S. boulardii that can be selected without antibiotics and demonstrate that these yeast can express functional recombinant protein even when recovered from gastrointestinal immune tissues in mice. A UV mutagenesis approach was employed to generate three uracil auxotrophic S. boulardii mutants that show a low rate of reversion to wild type growth. These mutants can express recombinant protein and are resistant in vitro to low pH, bile acid salts, and anaerobic conditions. Critically, oral gavage experiments using C57BL/6 mice demonstrate that mutant S. boulardii survive and are taken up into gastrointestinal immune tissues on a similar level as WT S. boulardii. Mutant yeast recovered from gastrointestinal immune tissues furthermore retain expression of functional recombinant protein. These data show that auxotrophic mutant S. boulardii can safely express recombinant protein without antibiotic selection and can deliver recombinant protein to gastrointestinal immune tissues. These auxotrophic mutants of S. boulardii pave the way for future experiments to test the ability of S. boulardii to deliver therapeutics and mediate protection against gastrointestinal disorders.

  11. Can S100B predict cerebral vasospasms in patients suffering from subarachnoid hemorrhage?

    Directory of Open Access Journals (Sweden)

    Moshgan eAmiri

    2013-06-01

    Full Text Available Background: Protein S100B has proven to be a useful biomarker for cerebral damages. Increased levels of serum and CSF S100B have been shown in patients suffering subarachnoid hemorrhage, severe head injury and stroke. In patients with subarachnoid hemorrhage, the course of S100B levels has been correlated with neurological deficits and outcome. Cerebral vasospasm is a major contributor to morbidity and mortality. The primary aim of this study was to investigate the potential of S100B protein as a predictor of cerebral vasospasm in patients with severe subarachnoid hemorrhage.Methods: Patients with SAH, Fisher grade 3 and 4, were included in the study. Five samples of CSF and serum S100B were collected from each patient. The first sample (baseline sample was drawn within the first three days following ictus and the following four samples, once a day on days 5 to 8, with day of ictus defined as day 1. Clinical suspicion of cerebral vasospasm confirmed by computed tomography angiography was used to diagnose cerebral vasospasm.Results: A total of 18 patients were included. Five patients (28 % developed cerebral vasospasm, two (11 % developed ventriculitis. There were no significant differences between S100B for those with and without vasospasm. Serum S100B levels in patients with vasospasm were slightly lower within the first 5 days following ictus, compared to patients without vasospasm. Two out of 5 patients had elevated and increasing serum S100B prior to vasospasm. Only one showed a peak level of S100B one day before vasospasm could be diagnosed. Due to the low number of patients in the study, statistical significance could not be reached. Conclusion: Neither serum nor CSF S100B can be used as predictor of cerebral vasospasm in patients suffering from subarachnoid hemorrhage.

  12. Os possíveis papéis da S100B na esquizofrenia

    Directory of Open Access Journals (Sweden)

    Johann Steiner

    2013-01-01

    Full Text Available CONTEXTO: Evidências científicas do aumento da concentração da proteína S100B no sangue de pacientes esquizofrênicos são muito consistentes. No passado essa informação era principalmente considerada como reflexo da disfunção astroglial ou da barreira hematoencefálica. MÉTODOS: Pesquisa de publicações no PubMed até o dia 15 de junho de 2011 visando estabelecer potenciais ligações entre a proteína S100B e as hipóteses correntes da esquizofrenia. RESULTADOS: A S100B está potencialmente associada com as hipóteses dopaminérgica e glutamatérgica. O aumento da expressão de S100B tem sido detectado em astrócitos corticais em casos de esquizofrenia paranoide, enquanto se observa uma redução da expressão em oligodendrócitos na esquizofrenia residual, dando suporte à hipótese glial. Recentemente, a hipótese da neuroinflamação da esquizofrenia tem recebido atenção crescente. Nesse sentido, a S100B pode funcionar como uma citocina secretada por células gliais, linfócitos CD8+ e células NK, levando à ativação de monócitos e microglia. Além disso, a S100B apresenta propriedades do tipo adipocina e pode estar desregulada na esquizofrenia, devido a distúrbios da sinalização de insulina, levando ao aumento da liberação de S100B e ácidos graxos do tecido adiposo. CONCLUSÃO: A expressão de S100B em diferentes tipos celulares está envolvida em muitos processos regulatórios. Atualmente, não pode ser respondido qual mecanismo relacionado à esquizofrenia é o mais importante.

  13. The expression and genetic immunization of chimeric fragment of Hantaan virus M and S segments

    International Nuclear Information System (INIS)

    Zhang Fanglin; Wu Xingan; Luo Wen; Bai Wentao; Liu Yong; Yan Yan; Wang Haitao; Xu Zhikai

    2007-01-01

    Hemorrhagic fever with renal syndrome (HFRS), which is characterized by severe symptoms and high mortality, is caused by hantavirus. There are still no effective prophylactic vaccines directed to HFRS until now. In this research, we fused expressed G2 fragment of M segment and 0.7 kb fragment of S segment. We expect it could be a candidate vaccine. Chimeric gene G2S0.7 was first expressed in prokaryotic expression system pGEX-4T. After inducing expressed fusion proteins, GST-G2S0.7 was induced and its molecular weight was about 100 kDa. Meanwhile, the fusion protein kept the activity of its parental proteins. Further, BALB/c mice were vaccinated by the chimeric gene. ELISA, cell microculture neutralization test in vitro were used to detect the humoral immune response in immunized BALB/c mice. Lymphocyte proliferation assay was used to detect the cellular immune response. The results showed that the chimeric gene could simultaneously evoke specific antibody against nucleocapsid protein (NP) and glycoprotein (GP). And the immunized mice of every group elicited neutralizing antibodies with different titers. But the titers were low. Lymphocyte proliferation assay results showed that the stimulation indexes of splenocytes of chimeric gene to NP and GP were significantly higher than that of control. It suggested that the chimeric gene of Hantaan virus containing G2 fragment of M segment and 0.7 kb fragment of S segment could directly elicit specific anti-Hantaan virus humoral and cellular immune response in BALB/c mice

  14. Investigation of mRNA expression for secreted frizzled-related protein 2 (sFRP2) in chick embryos.

    Science.gov (United States)

    Lin, Chung-Tien; Lin, Yu-Ting; Kuo, Tzong-Fu

    2007-08-01

    The roles of secreted frizzled-related protein 2 (sFRP2) in organ development of vertebrate animals are not well understood. We investigated expression of sFRP2 during embryogenesis of Arbor Acre broiler chicken eggs. Expression of sFRP2 was detected in the folds and lateral layer of developing brains. The sFRP2 signals in the developing eye were marked as a circle along the orbit. In younger embryos on days 3-6, the sFRP2 signals were consistent with growth of the sclerotome, suggesting that sFRP2 may be associated with somite development. Furthermore, with the exception of bones, sFRP2 mRNA was detectable in the interdigital tissue of embryos older than eight days as the limbs matured. This revealed that sFRP2 might play a role in myogenesis. In situ hybridization was also used to analyze the expression of sFRP2 in day 3-10 chick embryos. Signals were expressed in the gray matter of the developing brain coelom, including the optic lobe, metencephalon, myelencephalon, mesencephalon and diencephalon. The developing eyes contained an intercellular distribution of sFRP2 in the pigmented layer of the retina and photoreceptors. Furthermore, sFRP2 was expressed in the mantle layer of the neural tube and notochord. Based on these findings, it seems reasonable to suggest that sFRP2 may play an active role in embryogenesis, especially in development of the neural system, eyes, muscles and limbs.

  15. The role of the metastasis promoting protein S100A4 during EMT in mammary gland epithelial cells

    OpenAIRE

    Rognlien, Vibeke Wethe

    2013-01-01

    Master i biomedisin Breast cancer is one of the greatest contributors to mortality among the different cancer types in the female population of the western world each year. An increasing degree of evidence state that the S100A4 protein, which has been identified in several tumors of different origins and has proven to be associated with a poor patient prognosis, might have an important role in a process which induces carcinoma cells of the breast to gain a more motile and invas...

  16. S100 calcium binding protein B as a biomarker of delirium duration in the intensive care unit – an exploratory analysis

    Directory of Open Access Journals (Sweden)

    Khan BA

    2013-12-01

    Full Text Available Babar A Khan,1–3 Mark O Farber,1 Noll Campbell,2–5 Anthony Perkins,2,3 Nagendra K Prasad,6 Siu L Hui,1–3 Douglas K Miller,1–3 Enrique Calvo-Ayala,1 John D Buckley,1 Ruxandra Ionescu,1 Anantha Shekhar,1 E Wesley Ely,7,8 Malaz A Boustani1–3 1Indiana University School of Medicine, 2Indiana University Center for Aging Research, 3Regenstrief Institute, Inc., 4Wishard Health Services, Indianapolis, 5Department of Pharmacy Practice, Purdue University College of Pharmacy, West Lafayette, 6Indiana University Melvin and Bren Simon Cancer Center, Indianapolis, IN, 7Vanderbilt University School of Medicine, 8VA Tennessee Valley Geriatric Research Education Clinical Center (GRECC, Nashville, TN, USA Background: Currently, there are no valid and reliable biomarkers to identify delirious patients predisposed to longer delirium duration. We investigated the hypothesis that elevated S100 calcium binding protein B (S100β levels will be associated with longer delirium duration in critically ill patients. Methods: A prospective observational cohort study was performed in the medical, surgical, and progressive intensive care units (ICUs of a tertiary care, university affiliated, and urban hospital. Sixty-three delirious patients were selected for the analysis, with two samples of S100β collected on days 1 and 8 of enrollment. The main outcome measure was delirium duration. Using the cutoff of <0.1 ng/mL and $0.1 ng/mL as normal and abnormal levels of S100β, respectively, on day 1 and day 8, four exposure groups were created: Group A, normal S100β levels on day 1 and day 8; Group B, normal S100β level on day 1 and abnormal S100β level on day 8; Group C, abnormal S100β level on day 1 and normal on day 8; and Group D, abnormal S100β levels on both day 1 and day 8. Results: Patients with abnormal levels of S100β showed a trend towards higher delirium duration (P=0.076; Group B (standard deviation (7.0 [3.2] days, Group C (5.5 [6.3] days, and Group D

  17. Evaluating the prognosis and degree of brain injury by combined S-100 protein and neuron specific enolase determination

    Institute of Scientific and Technical Information of China (English)

    Xihua Wang; Xinding Zhang

    2006-01-01

    Background:S-100 and neuron specific enolase(NSE)possess the characteristics of specific distribution in brain and relative stable content.Some studies suggest that combined detection of the both is of very importance for evaluating the degree of brain injury.OBJECTIVE: To observe the changes of S-100 protein and NSE levels at different time points after acute brain injury,and evaluate the values of combined detection detection of the both for different injury degrees,pathological changes and prognosis.DESIGN: Case-control observation SETTING: Department of Neurosurgery,Second Affiliated Hospital,Lanzhou University.PARTICIPANTS:Thirty-four inpatients with brain injury,19 males and 15 females,aged 15 to 73 years.who received treatment between September 2005 and May 2006 in the Department of Neurosurgery. Second Affiliated Hospital,Lanzhou University,were recruited.The patients were admitted to hospital at 24 hours after brain injury.After admission,skull CT confirmed that they suffered from brain injury.Following Glasgow coma score(GCS)on admission,the patients were assigned into 3 groups:severe group(GCS 3 to 8 points,n=15).moderate group(GCS 9 to 12 points,n=8)and mild group(GCS 13 to 15 points,n=11).Following Glasgow outcome scale(GOS)at 3 months after brain injury,the patients were assigned into good outcome group (GOS 4 to 5 points,good recovery and moderate disability included,n=19)and poor outcome group(GOS 1 to 3 points,severe disability,vegetative state and death,n=15).Ten subjects who received health examination concurrently were chosen as normal control group,including 6 males and 4 females,aged(45.4±14.3)years.In our laboratory,the normal level of NSE was≤15.2 ng/L,and that of S100 was≤0.105 μg/L.METHODS:①Blood samples of control group were collected when the subjects received health examination Blood samples of patients with brain injury were collected at 24 hours,3,7 and 14 days after injury.According to the instructions of NSE and S-100 kits

  18. Expression of cbsA encoding the collagen-binding S-protein of Lactobacillus crispatus JCM5810 in Lactobacillus casei ATCC 393T

    NARCIS (Netherlands)

    Martínez, B.; Sillanpää, J.; Smit, E.; Korhonen, T.K.; Pouwels, P.H.

    2000-01-01

    The cbsA gene encoding the collagen-binding S-layer protein of Lactobacillus crispatus JCM5810 was expressed in L. casei ATCC 393T. The S-protein was not retained on the surface of the recombinant bacteria but was secreted into the medium. By translational fusion of CbsA to the cell wall sorting

  19. Differential protein expression in tears of patients with primary open angle and pseudoexfoliative glaucoma.

    Science.gov (United States)

    Pieragostino, Damiana; Bucci, Sonia; Agnifili, Luca; Fasanella, Vincenzo; D'Aguanno, Simona; Mastropasqua, Alessandra; Ciancaglini, Marco; Mastropasqua, Leonardo; Di Ilio, Carmine; Sacchetta, Paolo; Urbani, Andrea; Del Boccio, Piero

    2012-04-01

    Primary open angle (POAG) and pseudoexfoliative glaucoma (PXG) are the most common primary and secondary forms of glaucoma, respectively. Even though the patho-physiology, aqueous humor composition, risk factors, clinical features, therapy and drug induced ocular surface changes in POAG and PXG have been widely studied, to date information concerning tear protein characterization is lacking. Tears are a source of nourishment for ocular surface tissues and a vehicle to remove local waste products, metabolized drugs and inflammatory mediators produced in several ophthalmic diseases. In glaucoma, the proteomic definition of tears may provide insights concerning patho-physiology of the disease and ocular surface modifications induced by topical therapy. Our study aimed at characterizing protein patterns in tears of patients with medically controlled POAG and PXG. A comparative tears proteomic analysis by label-free LC-MS(E) highlighted differences in the expression of several proteins in the two glaucoma sub-types and control subjects, highlighting inflammation pathways expressed in both diseases. Results were independently reconfirmed by SDS-PAGE and linear MALDI-TOF MS, validating altered levels of Lysozyme C, Lipocalin-1, Protein S100, Immunoglobulins and Prolactin Inducible Protein. Moreover, we found a differential pattern of phosphorylated Cystatin-S that distinguishes the two pathologies. The most relevant results suggest that in both pathologies there may be active inflammation pathways related to the disease and/or induced by therapy. We show, for the first time, tear protein patterns expressed under controlled intraocular pressure conditions in POAG and PXG subjects. These findings could help in the understanding of molecular machinery underlying these ophthalmologic diseases, resulting in early diagnosis and more specific therapy.

  20. Serum S100B: A possible biomarker for severity of obstructive sleep apnea

    Directory of Open Access Journals (Sweden)

    Eman Riad

    2017-10-01

    Conclusion: Serum S100B protein was significantly elevated in OSA patients and its serum levels correlated with the severity of the disease. Increased serum S100B could indicat brain injury and could be a potential serum biomarker for detection of early neurological complications in OSA patients that could improve the management and care of these patients.

  1. Efficient Expression of Acetylcholine-Binding Protein from Aplysia californica in Bac-to-Bac System

    Directory of Open Access Journals (Sweden)

    Bo Lin

    2014-01-01

    Full Text Available The Bac-to-Bac baculovirus expression system can efficiently produce recombinant proteins, but the system may have to be optimized to achieve high-level expression for different candidate proteins. We reported here the efficient expression of acetylcholine-binding proteins from sea hares Aplysia californica (Ac-AChBP and a convenient method to monitor protein expression level in this expression system. Three key factors affecting expression of Ac-AChBP were optimized for maximizing the yield, which included the cell density, volume of the infecting baculovirus inoculums, and the culturing time of postinfection. We have found it to reach a high yield of ∼5 mg/L, which needs 55 h incubation after infection at the cell density of 2 × 106 cells/mL with an inoculum volume ratio of 1 : 100. The optimized expression system in this study was also applied for expressing another protein Ls-AChBP from Lymnaea stagnalis successfully. Therefore, this established method is helpful to produce high yields of AChBP proteins for X-ray crystallographic structural and functional studies.

  2. Ethylene-induced senescence-related gene expression requires protein synthesis

    International Nuclear Information System (INIS)

    Lawton, K.A.; Raghothama, K.G.; Woodson, W.R.

    1990-01-01

    We have investigated the effects of inhibiting protein synthesis on the ethylene-induced expression of 3 carnation senescence-related genes, pSR5, pSR8, and pSR12. Treatment of preclimacteric carnation petal discs with 1μg/ml of cycloheximide, a cytoplasmic protein synthesis inhibitor, for 3h inhibited protein synthesis by >80% as quantitated by the incorporation of [35S]methionine into protein. Pre-treatment of petal discs with cycloheximide prevented ethylene-induced SR transcript accumulation. Cycloheximide treatment of petal discs held in air did not result in increased levels of SR mRNA. These results indicate that ethylene does not interact with pre-formed factors but rather that the activation of SR gene expression by ethylene is mediated by labile protein factor(s) synthesized on cytoplasmic ribosomes. Experiments are currently underway to determine if cycloheximide exerts its effect at the transcriptional or post-transcriptional level

  3. Immunohistochemical Expression of P53 Protein in Cutaneous Basal Cell Carcinoma: A Clinicopathological Study of 66 Cases

    Directory of Open Access Journals (Sweden)

    Vladim and iacute;r Barto and scaron;

    2016-12-01

    Full Text Available Objective: Nuclear expression of p53 protein is associated with a biological behavior in a variety of human malignancies. In cutaneous basal cell carcinoma (BCC, however, many studies have provided conflicting results in this regard. We aimed to determine whether there is relationship between p53 expression and different histologic subtypes of BCC, and whether it may indicate tumor aggressiveness. Materials and Methods: Biopsy samples from 66 cutaneous BCCs from 57 patients were collected. P53 expression was demonstrated by immunohistochemical staining using the anti-p53 antibody. Among them, 52 cases were also evaluated for Ki-67 antigen. Results: Immunoreactivity of p53 protein varied in the range of 0 to 100% of total tumor tissue (mean value 46.0%. The expression exceeding 5% of cancer tissue (positive staining was found in 54 BCCs (81.8%. Within this group, there were 25 cases (37.9% with low and 29 cases (43.9% with high expression. In superficial, superficial-nodular, nodular, nodular-infiltrative and infiltrative BCCs, p53 protein positivity was found in 100% (8/8, 80% (8/10, 70.4% (19/27, 88.2% (15/17 and 100% (4/4, respectively. We did not reveal a significant correlation between the extent of p53 protein expression and BCC subtypes except for nodular BCC, in which a number of negative cases (8/27, 29.6% were just above the threshold of statistical significance (P = 0.04. After merging cancers into non-aggressive and aggressive growth phenotype, no association with expression of p53 protein was found. There was no relationship between p53 protein expression and topographical sites after they have been gathered into sun-exposed and sun-protected locations. We did not observe any association between expression of p53 protein and Ki-67 antigen. Conclusion: In cutaneous BCC, the expression of p53 protein does not seem to reflect a biological behavior and tumor aggressiveness. Therefore, in a routine dermatopathological practice

  4. EF-hands at atomic resolution: The structure of human psoriasin (S100A7) solved by MAD phasing

    DEFF Research Database (Denmark)

    Brodersen, Ditlev Egeskov; Etzerodt, Michael; Madsen, Peder Søndergaard

    1998-01-01

    psoriasin reveals that this protein, in contrast to other S100 proteins with known structure, is not likely to strongly bind more than one calcium ion per monomer. The present study contradicts the idea that calcium binding induces large changes in conformation, as suggested by previously determined......The S100 family consists of small acidic proteins, belonging to the EF-hand class of calcium-binding proteins. They are primarily regulatory proteins, involved in cell growth, cell structure regulation and signal transduction. Psoriasin (S100A7) is an 11.7 kDa protein that is highly upregulated...... in the epidermis of patients suffering from the chronic skin disease psoriasis. Although its exact function is not known, psoriasin is believed to participate in the biochemical response which follows transient changes in the cellular Ca2+ concentration. RESULTS: The three-dimensional structure of holmium...

  5. Circulating S100B and Adiponectin in Children Who Underwent Open Heart Surgery and Cardiopulmonary Bypass

    Directory of Open Access Journals (Sweden)

    Alessandro Varrica

    2015-01-01

    Full Text Available Background. S100B protein, previously proposed as a consolidated marker of brain damage in congenital heart disease (CHD newborns who underwent cardiac surgery and cardiopulmonary bypass (CPB, has been progressively abandoned due to S100B CNS extra-source such as adipose tissue. The present study investigated CHD newborns, if adipose tissue contributes significantly to S100B serum levels. Methods. We conducted a prospective study in 26 CHD infants, without preexisting neurological disorders, who underwent cardiac surgery and CPB in whom blood samples for S100B and adiponectin (ADN measurement were drawn at five perioperative time-points. Results. S100B showed a significant increase from hospital admission up to 24 h after procedure reaching its maximum peak (P0.05 have been found all along perioperative monitoring. ADN/S100B ratio pattern was identical to S100B alone with the higher peak at the end of CPB and remained higher up to 24 h from surgery. Conclusions. The present study provides evidence that, in CHD infants, S100B protein is not affected by an extra-source adipose tissue release as suggested by no changes in circulating ADN concentrations.

  6. Heterogeneity of proteins expressed by Brazilian Sporothrix schenckii isolates.

    Science.gov (United States)

    Fernandes, Geisa Ferreira; Do Amaral, Cristiane Candida; Sasaki, Alexandre; Godoy, Patrício Martinez; De Camargo, Zoilo Pires

    2009-12-01

    The profiles of proteins present in the exoantigens of Brazilian Sporothrix schenckii isolates were studied and compared by electrophoresis (SDS-PAGE). Thirteen isolates from five different regions of Brazil (1,000 to 2,000 km apart) and ten from a more limited region (200 to 400 km apart within the state of São Paulo) were cultured in Sabouraud, M199 and minimum (MM) media. Qualitative and quantitative differences in the expression of proteins, which varied according to the medium and the isolate, were observed. Fractions with the same MW but varying in intensity were detected, as well as fractions present in 1 isolate but absent in others. Dendrograms were constructed and isolates grouped based on the fractions obtained, irrespective of the intensity. The results showed that Brazilian S. schenckii isolates express different protein profiles, a feature also present in isolates from a more restricted region. The exoantigens were found to have a maximum of 15 protein fractions, ranging in MW from 19-220 KDaltons depending on the medium used for the cultures. These data show the great heterogeneity of Brazilian S. schenckii protein expression.

  7. Pentamidine blocks the interaction between mutant S100A5 and RAGE V domain and inhibits the RAGE signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Ching Chang, E-mail: ccjwo@yahoo.com.tw [Department of Chemistry, National Tsing Hua University, No. 101, Section 2, Kuang-Fu Road, Hsinchu 30013, Taiwan (China); Chou, Ruey Hwang, E-mail: rhchou@mail.cmu.edu.tw [Graduate Institute of Cancer Biology and Center for Molecular Medicine, China Medical University, No.91, Hsueh-Shih Road, Taichung 40402, Taiwan (China); Yu, Chin, E-mail: cyu.nthu@gmail.com [Department of Chemistry, National Tsing Hua University, No. 101, Section 2, Kuang-Fu Road, Hsinchu 30013, Taiwan (China)

    2016-08-19

    The human S100 protein family contains small, dimeric and acidic proteins that contain two EF-hand motifs and bind calcium. When S100A5 binds calcium, its conformation changes and promotes interaction with the target protein. The extracellular domain of RAGE (Receptor of Advanced Glycation End products) contain three domains: C1, C2 and V. The RAGE V domain is the target protein of S100A5 that promotes cell survival, growth and differentiation by activating several signaling pathways. Pentamidine is an apoptotic and antiparasitic drug that is used to treat or prevent pneumonia. Here, we found that pentamidine interacts with S100A5 using HSQC titration. We elucidated the interactions of S100A5 with RAGE V domain and pentamidine using fluorescence and NMR spectroscopy. We generated two binary models—the S100A5-RAGE V domain and S100A5-Pentamidine complex—and then observed that the pentamidine and RAGE V domain share a similar binding region in mS100A5. We also used the WST-1 assay to investigate the bioactivity of S100A5, RAGE V domain and pentamidine. These results indicated that pentamidine blocks the binding between S100A5 and RAGE V domain. This finding is useful for the development of new anti-proliferation drugs. - Highlights: • The interaction between mS100A5–RAGE V was investigated by fluorescence spectroscopy. • The interfacial residues on mS100A5–RAGE V and mS100A5–pentamidine contact surface were mapped by {sup 1}H-{sup 15}N HSQC experiments. • mS100A5–RAGE V and mS100A5–pentamidine complex models were generated from NMR restraints using HADDOCK program. • The bioactivity of the mS100A5–RAGE V and mS100A5–pentamidine complex was studied using WST-1 assay.

  8. GFAP and S100B in the acute phase of mild traumatic brain injury

    NARCIS (Netherlands)

    Metting, Z.; Wilczak, N.; Rodiger, L. A.; Schaaf, J. M.; van der Naalt, J.

    Objective: The biomarkers glial fibrillary acid protein (GFAP) and S100B are increasingly used as prognostic tools in severe traumatic brain injury (TBI). Data for mild TBI are scarce. This study aims to analyze the predictive value of GFAP and S100B for outcome in mild TBI and the relation with

  9. Arabidopsis mRNA polyadenylation machinery: comprehensive analysis of protein-protein interactions and gene expression profiling

    Directory of Open Access Journals (Sweden)

    Mo Min

    2008-05-01

    Full Text Available Abstract Background The polyadenylation of mRNA is one of the critical processing steps during expression of almost all eukaryotic genes. It is tightly integrated with transcription, particularly its termination, as well as other RNA processing events, i.e. capping and splicing. The poly(A tail protects the mRNA from unregulated degradation, and it is required for nuclear export and translation initiation. In recent years, it has been demonstrated that the polyadenylation process is also involved in the regulation of gene expression. The polyadenylation process requires two components, the cis-elements on the mRNA and a group of protein factors that recognize the cis-elements and produce the poly(A tail. Here we report a comprehensive pairwise protein-protein interaction mapping and gene expression profiling of the mRNA polyadenylation protein machinery in Arabidopsis. Results By protein sequence homology search using human and yeast polyadenylation factors, we identified 28 proteins that may be components of Arabidopsis polyadenylation machinery. To elucidate the protein network and their functions, we first tested their protein-protein interaction profiles. Out of 320 pair-wise protein-protein interaction assays done using the yeast two-hybrid system, 56 (~17% showed positive interactions. 15 of these interactions were further tested, and all were confirmed by co-immunoprecipitation and/or in vitro co-purification. These interactions organize into three distinct hubs involving the Arabidopsis polyadenylation factors. These hubs are centered around AtCPSF100, AtCLPS, and AtFIPS. The first two are similar to complexes seen in mammals, while the third one stands out as unique to plants. When comparing the gene expression profiles extracted from publicly available microarray datasets, some of the polyadenylation related genes showed tissue-specific expression, suggestive of potential different polyadenylation complex configurations. Conclusion An

  10. Evaluation of GO-based functional similarity measures using S. cerevisiae protein interaction and expression profile data

    Directory of Open Access Journals (Sweden)

    Du LinFang

    2008-11-01

    Full Text Available Abstract Background Researchers interested in analysing the expression patterns of functionally related genes usually hope to improve the accuracy of their results beyond the boundaries of currently available experimental data. Gene ontology (GO data provides a novel way to measure the functional relationship between gene products. Many approaches have been reported for calculating the similarities between two GO terms, known as semantic similarities. However, biologists are more interested in the relationship between gene products than in the scores linking the GO terms. To highlight the relationships among genes, recent studies have focused on functional similarities. Results In this study, we evaluated five functional similarity methods using both protein-protein interaction (PPI and expression data of S. cerevisiae. The receiver operating characteristics (ROC and correlation coefficient analysis of these methods showed that the maximum method outperformed the other methods. Statistical comparison of multiple- and single-term annotated proteins in biological process ontology indicated that genes with multiple GO terms may be more reliable for separating true positives from noise. Conclusion This study demonstrated the reliability of current approaches that elevate the similarity of GO terms to the similarity of proteins. Suggestions for further improvements in functional similarity analysis are also provided.

  11. Hypoxia Mediated Release of Endothelial Microparticles and Increased Association of S100A12 with Circulating Neutrophils

    Directory of Open Access Journals (Sweden)

    Rebecca V. Vince

    2009-01-01

    Full Text Available Microparticles are released from the endothelium under normal homeostatic conditions and have been shown elevated in disease states, most notably those characterised by endothelial dysfunction. The endothelium is sensitive to oxidative stress/status and vascular cell adhesion molecule-1 (VCAM-1 expression is upregulated upon activated endothelium, furthermore the presence of VCAM-1 on microparticles is known. S100A12, a calcium binding protein part of the S100 family, is shown to be present on circulating leukocytes and is thought a sensitive marker to local inflammatory process, which may be driven by oxidative stress. Eight healthy males were subjected to breathing hypoxic air (15% O2, approximately equivalent to 3000 metres altitude for 80 minutes in a temperature controlled laboratory and venous blood samples were processed immediately for VCAM-1 microparticles (VCAM-1 MP and S100A12 association with leukocytes by flow cytometry. A pre-hypoxic blood sample was used for comparison. Both VCAM-1 MP and S100A12 association with neutrophils were significantly elevated post hypoxic breathing later declining to levels observed in the pre-test samples. A similar trend was observed in both cases and a correlation may exist between these two markers in response to hypoxia. These data offer evidence using novel markers of endothelial and circulating blood responses to hypoxia.

  12. Faecal S100A12 as a non-invasive marker distinguishing inflammatory bowel disease from irritable bowel syndrome

    NARCIS (Netherlands)

    Kaiser, T; Langhorst, J; Wittkowski, H; Becker, K; Friedrich, A W; Rueffer, A; Dobos, G J; Roth, J; Foell, D

    2007-01-01

    OBJECTIVE: S100A12 is a pro-inflammatory protein that is secreted by granulocytes. S100A12 serum levels increase during inflammatory bowel disease (IBD). We performed the first study analysing faecal S100A12 in adults with signs of intestinal inflammation. METHODS: Faecal S100A12 was determined by

  13. Simvastatin enhances bone morphogenetic protein receptor type II expression

    International Nuclear Information System (INIS)

    Hu Hong; Sung, Arthur; Zhao, Guohua; Shi, Lingfang; Qiu Daoming; Nishimura, Toshihiko; Kao, Peter N.

    2006-01-01

    Statins confer therapeutic benefits in systemic and pulmonary vascular diseases. Bone morphogenetic protein (BMP) receptors serve essential signaling functions in cardiovascular development and skeletal morphogenesis. Mutations in BMP receptor type II (BMPR2) are associated with human familial and idiopathic pulmonary arterial hypertension, and pathologic neointimal proliferation of vascular endothelial and smooth muscle cells within small pulmonary arteries. In severe experimental pulmonary hypertension, simvastatin reversed disease and conferred a 100% survival advantage. Here, modulation of BMPR2 gene expression by simvastatin is characterized in human embryonic kidney (HEK) 293T, pulmonary artery smooth muscle, and lung microvascular endothelial cells (HLMVECs). A 1.4 kb BMPR2 promoter containing Egr-1 binding sites confers reporter gene activation in 293T cells which is partially inhibited by simvastatin. Simvastatin enhances steady-state BMPR2 mRNA and protein expression in HLMVEC, through posttranscriptional mRNA stabilization. Simvastatin induction of BMPR2 expression may improve BMP-BMPR2 signaling thereby enhancing endothelial differentiation and function

  14. Thioredoxin 1 regulation of protein S-desulfhydration

    Directory of Open Access Journals (Sweden)

    Youngjun Ju

    2016-03-01

    Full Text Available The importance of H2S in biology and medicine has been widely recognized in recent years, and protein S-sulfhydration is proposed to mediate the direct actions of H2S bioactivity in the body. Thioredoxin 1 (Trx1 is an important reducing enzyme that cleaves disulfides in proteins and acts as an S-denitrosylase. The regulation of Trx1 on protein S-sulfhydration is unclear. Here we showed that Trx1 facilitates protein S-desulfhydration. Overexpression of Trx1 attenuated the basal level and H2S-induced protein S-sulfhydration by direct interaction with S-sulfhydrated proteins, i.e., glyceraldehyde 3-phosphate dehydrogenase and pyruvate carboxylase. In contrast, knockdown of Trx1 mRNA expression by short interfering RNA or blockage of Trx1 redox activity with PX12 or 2,4-dinitrochlorobenzene enhanced protein S-sulfhydration. Mutation of cysteine-32 but not cysteine-35 in the Trp–Cys32–Gly–Pro–Cys35 motif eliminated the binding of Trx1 with S-sulfhydrated proteins and abolished the S-desulfhydrating effect of Trx1. All these data suggest that Trx1 acts as an S-desulfhydrase.

  15. Several genes encoding ribosomal proteins are over-expressed in prostate-cancer cell lines: confirmation of L7a and L37 over-expression in prostate-cancer tissue samples.

    Science.gov (United States)

    Vaarala, M H; Porvari, K S; Kyllönen, A P; Mustonen, M V; Lukkarinen, O; Vihko, P T

    1998-09-25

    A cDNA library specific for mRNA over-expressed in prostate cancer was generated by subtractive hybridization of transcripts originating from prostatic hyperplasia and cancer tissues. cDNA encoding ribosomal proteins L4, L5, L7a, L23a, L30, L37, S14 and S18 was found to be present among 100 analyzed clones. Levels of ribosomal mRNA were significantly higher at least in one of the prostate-cancer cell lines, LNCaP, DU-145 and PC-3, than in hyperplastic tissue, as determined by slot-blot hybridization. Furthermore, L23a- and S14-transcript levels were significantly elevated in PC-3 cells as compared with those in the normal prostate epithelial cell line PrEC. Generally, dramatic changes in the mRNA content of the ribosomal proteins were not detected, the most evident over-expression being that of L37 mRNA, which was 3.4 times more abundant in LNCaP cells than in hyperplastic prostate tissue. The over-expression of L7a and L37 mRNA was confirmed in prostate-cancer tissue samples by in situ hybridization. Elevated cancer-related expression of L4 and L30 has not been reported, but levels of the other ribosomal proteins are known to be increased in several types of cancers. These results therefore suggest that prostate cancer is comparable with other types of cancers, in that a larger pool of some ribosomal proteins is gained during the transformation process, by an unknown mechanism.

  16. Identification of Protein-Protein Interactions with Glutathione-S-Transferase (GST) Fusion Proteins.

    Science.gov (United States)

    Einarson, Margret B; Pugacheva, Elena N; Orlinick, Jason R

    2007-08-01

    INTRODUCTIONGlutathione-S-transferase (GST) fusion proteins have had a wide range of applications since their introduction as tools for synthesis of recombinant proteins in bacteria. GST was originally selected as a fusion moiety because of several desirable properties. First and foremost, when expressed in bacteria alone, or as a fusion, GST is not sequestered in inclusion bodies (in contrast to previous fusion protein systems). Second, GST can be affinity-purified without denaturation because it binds to immobilized glutathione, which provides the basis for simple purification. Consequently, GST fusion proteins are routinely used for antibody generation and purification, protein-protein interaction studies, and biochemical analysis. This article describes the use of GST fusion proteins as probes for the identification of protein-protein interactions.

  17. Influence of energy balance on the antimicrobial peptides S100A8 and S100A9 in the endometrium of the post-partum dairy cow

    Science.gov (United States)

    Swangchan-Uthai, Theerawat; Chen, Qiusheng; Kirton, Sally E; Fenwick, Mark A; Cheng, Zhangrui; Patton, Joe; Fouladi-Nashta, Ali A; Wathes, D Claire

    2013-01-01

    Uterine inflammation occurs after calving in association with extensive endometrial remodelling and bacterial contamination. If the inflammation persists, it leads to reduced fertility. Chronic endometritis is highly prevalent in high-yielding cows that experience negative energy balance (NEB) in early lactation. This study investigated the effect of NEB on the antimicrobial peptides S100A8 and S100A9 in involuting uteri collected 2 weeks post partum. Holstein-Friesian cows (six per treatment) were randomly allocated to two interventions designed to produce mild or severe NEB (MNEB and SNEB) status. Endometrial samples were examined histologically, and the presence of neutrophils, macrophages, lymphocytes and natural killer cells was confirmed using haematoxylin and eosin and immunostaining. SNEB cows had greater signs of uterine inflammation. Samples of previously gravid uterine horn were used to localise S100A8 and S100A9 by immunohistochemistry. Both S100 proteins were present in bovine endometrium with strong staining in epithelial and stromal cells and in infiltrated leucocytes. Immunostaining was significantly higher in SNEB cows along with increased numbers of segmented neutrophils. These results suggest that the metabolic changes of a post-partum cow suffering from NEB delay uterine involution and promote a chronic state of inflammation. We show that upregulation of S100A8 and S100A9 is clearly a key component of the early endometrial response to uterine infection. Further studies are warranted to link the extent of this response after calving to the likelihood of cows developing endometritis and to their subsequent fertility. PMID:23533291

  18. GILT expression in B cells diminishes cathepsin S steady-state protein expression and activity

    OpenAIRE

    Phipps-Yonas, Hannah; Semik, Vikki; Hastings, Karen Taraszka

    2012-01-01

    MHC class II-restricted Ag processing requires protein degradation in the endocytic pathway for the activation of CD4+ T cells. Gamma-interferon-inducible lysosomal thiol reductase (GILT) facilitates Ag processing by reducing protein disulfide bonds in this compartment. Lysosomal cysteine protease cathepsin S (CatS) contains disulfide bonds and mediates essential steps in MHC class II-restricted processing, including proteolysis of large polypeptides and cleavage of the invariant chain. We so...

  19. [S100A7 promotes the metastasis and epithelial-mesenchymal transition on HeLa and CaSki cells].

    Science.gov (United States)

    Tian, T; Hua, Z; Wang, L Z; Wang, X Y; Chen, H Y; Liu, Z H; Cui, Z M

    2018-02-25

    Objective: To elucidate the impact of over-expression of S100A7 on migration, invasion, proliferation, cell cycle, and epithelial-mesenchymal transition (EMT) in human cervical cancer HeLa and CaSki cells. Methods: (1) Immunohistochemistry of SP was used to examine the expression of S100A7 in 40 cases of squamous cervical cancer tissues and 20 cases of normal cervical tissues. (2) The vectors of pLVX-IRES-Neo-S100A7 and pLVX-IRES-Neo were used to transfect human cervical cancer HeLa and CaSki cells, and the positive clones were screened and identified. Next, transwell migration assay, cell counting kit-8 (CCK-8) assay and fluorescence activating cell sorter (FACS) were used to detect the effect of S100A7-overexpression on the migration, invasion, proliferation and cell cycle of cervical cancer cells. Furthermore, western blot was performed to observe the expression of epithelial marker (E-cadherin) and mesenchymal markers (N-cadherin, vimentin, and fibronectin) of EMT. Results: (1) S100A7 expression was significantly higher in cervical squamous cancer tissues (median 91.6) than that in normal cervical tissues (median 52.1; Z=- 2.948, P= 0.003) . (2) Stable S100A7-overexpressed cells were established using lentiviral-mediated gene delivery in HeLa and CaSki cells. S100A7 was detected by real-time quantitative reverse transcription PCR, S100A7 mRNA of S100A7-overexpressed cells were 119±3 and 177±16, increased significantly compared with control groups of median ( Pcells, the number of S100A7-overexpressed HeLa and CaSki cells that passed the transwell membrane assay were increased significanatly (572±51 vs 337±25, PHeLa and CaSki cells that passed the transwell membrane were respectively 441±15 and 110±14, elevated significantly compared with control cells (156±21 and 59±7; Pcell cycle progression of HeLa and CaSki cells ( P> 0.05) . Expression of E-cadherin was dramatically decreased, while N-cadherin, vimentin, and fibronectin increased in S100A7

  20. Brain injury markers (S100B and NSE) in chronic cocaine dependents

    OpenAIRE

    Kessler, Felix Henrique Paim; Woody, George; Portela, Luís Valmor Cruz; Tort, Adriano Bretanha Lopes; De Boni, Raquel; Peuker, Ana Carolina Wolf Baldino; Genro, Vanessa; Diemen, Lísia von; Souza, Diogo Onofre Gomes de; Pechansky, Flavio

    2007-01-01

    OBJECTIVE: Studies have shown signs of brain damage caused by different mechanisms in cocaine users. The serum neuron specific enolase and S100B protein are considered specific biochemical markers of neuronal and glial cell injury. This study aimed at comparing blood levels of S100B and NSE in chronic cocaine users and in volunteers who did not use cocaine or other illicit drugs. METHOD: Twenty subjects dependent on cocaine but not on alcohol or marijuana, and 20 non-substance using controls ...

  1. Efficient expression of SRK intracellular domain by a modeling-based protein engineering.

    Science.gov (United States)

    Murase, Kohji; Hirano, Yoshinori; Takayama, Seiji; Hakoshima, Toshio

    2017-03-01

    S-locus protein kinase (SRK) is a receptor kinase that plays a critical role in self-recognition in the Brassicaceae self-incompatibility (SI) response. SRK is activated by binding of its ligand S-locus protein 11 (SP11) and subsequently induced phosphorylation of the intracellular kinase domain. However, a detailed activation mechanism of SRK is still largely unknown because of the difficulty in stably expressing SRK recombinant proteins. Here, we performed modeling-based protein engineering of the SRK kinase domain for stable expression in Escherichia coli. The engineered SRK intracellular domain was expressed about 54-fold higher production than wild type SRK, without loss of the kinase activity, suggesting it could be useful for further biochemical and structural studies. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Neuroprotective Effect of Uncaria rhynchophylla in Kainic Acid-Induced Epileptic Seizures by Modulating Hippocampal Mossy Fiber Sprouting, Neuron Survival, Astrocyte Proliferation, and S100B Expression.

    Science.gov (United States)

    Liu, Chung-Hsiang; Lin, Yi-Wen; Tang, Nou-Ying; Liu, Hsu-Jan; Hsieh, Ching-Liang

    2012-01-01

    Uncaria rhynchophylla (UR), which is a traditional Chinese medicine, has anticonvulsive effect in our previous studies, and the cellular mechanisms behind this are still little known. Because of this, we wanted to determine the importance of the role of UR on kainic acid- (KA-) induced epilepsy. Oral UR for 6 weeks can successfully attenuate the onset of epileptic seizure in animal tests. Hippocampal mossy fiber sprouting dramatically decreased, while neuronal survival increased with UR treatment in hippocampal CA1 and CA3 areas. Furthermore, oral UR for 6 weeks significantly attenuated the overexpression of astrocyte proliferation and S100B proteins but not γ-aminobutyric acid A (GABA(A)) receptors. These results indicate that oral UR for 6 weeks can successfully attenuate mossy fiber sprouting, astrocyte proliferation, and S100B protein overexpression and increase neuronal survival in KA-induced epileptic rat hippocampus.

  3. The Biochemistry and Regulation of S100A10: A Multifunctional Plasminogen Receptor Involved in Oncogenesis

    Directory of Open Access Journals (Sweden)

    Patricia A. Madureira

    2012-01-01

    Full Text Available The plasminogen receptors mediate the production and localization to the cell surface of the broad spectrum proteinase, plasmin. S100A10 is a key regulator of cellular plasmin production and may account for as much as 50% of cellular plasmin generation. In parallel to plasminogen, the plasminogen-binding site on S100A10 is highly conserved from mammals to fish. S100A10 is constitutively expressed in many cells and is also induced by many diverse factors and physiological stimuli including dexamethasone, epidermal growth factor, transforming growth factor-α, interferon-γ, nerve growth factor, keratinocyte growth factor, retinoic acid, and thrombin. Therefore, S100A10 is utilized by cells to regulate plasmin proteolytic activity in response to a wide diversity of physiological stimuli. The expression of the oncogenes, PML-RARα and KRas, also stimulates the levels of S100A10, suggesting a role for S100A10 in pathophysiological processes such as in the oncogenic-mediated increases in plasmin production. The S100A10-null mouse model system has established the critical role that S100A10 plays as a regulator of fibrinolysis and oncogenesis. S100A10 plays two major roles in oncogenesis, first as a regulator of cancer cell invasion and metastasis and secondly as a regulator of the recruitment of tumor-associated cells, such as macrophages, to the tumor site.

  4. 100 Gbps PCI-Express Readout for the LHCb Upgrade

    CERN Document Server

    Durante, Paolo; Schwemmer, Rainer; Marconi, Umberto; Balbi, Gabriele; Lax, Ignazio

    2015-01-01

    We present a new data acquisition system under development for the next upgrade of the LHCb experiment at CERN. We focus in particular on the design of a new common readout board, the PCIe40, and on the viability of PCI-Express as an interconnect technology for high speed readout. We describe a new high-performance DMA controller for data acquisition, implemented on an FPGA, coupled with a custom software module for the Linux kernel. Lastly, we describe how these components can be leveraged to achieve a throughput of 100 Gbit/s per readout board.

  5. A synthetic arabinose-inducible promoter confers high levels of recombinant protein expression in hyperthermophilic archaeon Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Peng, Nan; Deng, Ling; Mei, Yuxia

    2012-01-01

    Despite major progresses in genetic studies of hyperthermophilic archaea, recombinant protein production in these organisms always suffers from low yields and a robust expression system is still in great demand. Here we report a versatile vector that confers high levels of protein expression...... to remove the peptide tags from expressed recombinant proteins. While pEXA employed an araS promoter for protein expression, pSeSD utilized P(araS-SD), an araS derivative promoter carrying an engineered ribosome-binding site (RBS; a Shine-Dalgarno [SD] sequence). We found that P(araS-SD) directed high...... levels of target gene expression. More strikingly, N-terminal amino acid sequencing of recombinant proteins unraveled that the protein synthesized from pEXA-N-lacS lacked the designed 6×His tag and that translation initiation did not start at the ATG codon of the fusion gene. Instead, it started...

  6. A plasma coagulation assay for an activated protein C-independent anticoagulant activity of protein S

    NARCIS (Netherlands)

    van Wijnen, M.; van 't Veer, C.; Meijers, J. C.; Bertina, R. M.; Bouma, B. N.

    1998-01-01

    To study the physiological importance of the activated protein C (APC)-independent anticoagulant activity of protein S, we developed an assay specific for this activity. The ability of protein S to prolong the clotting time in an APC-independent way was expressed as the ratio of the clotting time in

  7. ERG protein expression over time

    DEFF Research Database (Denmark)

    Berg, Kasper Drimer; Brasso, Klaus; Thomsen, Frederik Birkebæk

    2015-01-01

    AIMS: We evaluated the consistency in ERG protein expression from diagnostic specimens through rebiopsies to radical prostatectomies in patients with clinically localised prostate cancer to investigate the validity of ERG status in biopsies. METHODS: ERG expression was assessed by immunohistochem......AIMS: We evaluated the consistency in ERG protein expression from diagnostic specimens through rebiopsies to radical prostatectomies in patients with clinically localised prostate cancer to investigate the validity of ERG status in biopsies. METHODS: ERG expression was assessed...

  8. High-throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli

    Science.gov (United States)

    Bruni, Renato

    2014-01-01

    Recently, several structural genomics centers have been established and a remarkable number of three-dimensional structures of soluble proteins have been solved. For membrane proteins, the number of structures solved has been significantly trailing those for their soluble counterparts, not least because over-expression and purification of membrane proteins is a much more arduous process. By using high throughput technologies, a large number of membrane protein targets can be screened simultaneously and a greater number of expression and purification conditions can be employed, leading to a higher probability of successfully determining the structure of membrane proteins. This unit describes the cloning, expression and screening of membrane proteins using high throughput methodologies developed in our laboratory. Basic Protocol 1 deals with the cloning of inserts into expression vectors by ligation-independent cloning. Basic Protocol 2 describes the expression and purification of the target proteins on a miniscale. Lastly, for the targets that express at the miniscale, basic protocols 3 and 4 outline the methods employed for the expression and purification of targets at the midi-scale, as well as a procedure for detergent screening and identification of detergent(s) in which the target protein is stable. PMID:24510647

  9. 4EBP-Dependent Signaling Supports West Nile Virus Growth and Protein Expression.

    Science.gov (United States)

    Shives, Katherine D; Massey, Aaron R; May, Nicholas A; Morrison, Thomas E; Beckham, J David

    2016-10-18

    West Nile virus (WNV) is a (+) sense, single-stranded RNA virus in the Flavivirus genus. WNV RNA possesses an m7 GpppN m 5' cap with 2'- O -methylation that mimics host mRNAs preventing innate immune detection and allowing the virus to translate its RNA genome through the utilization of cap-dependent translation initiation effectors in a wide variety of host species. Our prior work established the requirement of the host mammalian target of rapamycin complex 1 (mTORC1) for optimal WNV growth and protein expression; yet, the roles of the downstream effectors of mTORC1 in WNV translation are unknown. In this study, we utilize gene deletion mutants in the ribosomal protein kinase called S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein (4EBP) pathways downstream of mTORC1 to define the role of mTOR-dependent translation initiation signals in WNV gene expression and growth. We now show that WNV growth and protein expression are dependent on mTORC1 mediated-regulation of the eukaryotic translation initiation factor 4E-binding protein/eukaryotic translation initiation factor 4E-binding protein (4EBP/eIF4E) interaction and eukaryotic initiation factor 4F (eIF4F) complex formation to support viral growth and viral protein expression. We also show that the canonical signals of mTORC1 activation including ribosomal protein s6 (rpS6) and S6K phosphorylation are not required for WNV growth in these same conditions. Our data suggest that the mTORC1/4EBP/eIF4E signaling axis is activated to support the translation of the WNV genome.

  10. 4EBP-Dependent Signaling Supports West Nile Virus Growth and Protein Expression

    Directory of Open Access Journals (Sweden)

    Katherine D. Shives

    2016-10-01

    Full Text Available West Nile virus (WNV is a (+ sense, single-stranded RNA virus in the Flavivirus genus. WNV RNA possesses an m7GpppNm 5′ cap with 2′-O-methylation that mimics host mRNAs preventing innate immune detection and allowing the virus to translate its RNA genome through the utilization of cap-dependent translation initiation effectors in a wide variety of host species. Our prior work established the requirement of the host mammalian target of rapamycin complex 1 (mTORC1 for optimal WNV growth and protein expression; yet, the roles of the downstream effectors of mTORC1 in WNV translation are unknown. In this study, we utilize gene deletion mutants in the ribosomal protein kinase called S6 kinase (S6K and eukaryotic translation initiation factor 4E-binding protein (4EBP pathways downstream of mTORC1 to define the role of mTOR-dependent translation initiation signals in WNV gene expression and growth. We now show that WNV growth and protein expression are dependent on mTORC1 mediated-regulation of the eukaryotic translation initiation factor 4E-binding protein/eukaryotic translation initiation factor 4E-binding protein (4EBP/eIF4E interaction and eukaryotic initiation factor 4F (eIF4F complex formation to support viral growth and viral protein expression. We also show that the canonical signals of mTORC1 activation including ribosomal protein s6 (rpS6 and S6K phosphorylation are not required for WNV growth in these same conditions. Our data suggest that the mTORC1/4EBP/eIF4E signaling axis is activated to support the translation of the WNV genome.

  11. Effect of polysaccharides from Angelica sinensis on Bcl-2 and Bax protein expression of irradiated liver cells

    International Nuclear Information System (INIS)

    Sun Yuanlin; Tang Jian; Gu Xiaohong; Li Deyuan

    2009-01-01

    Objective: To investigate the effect of polysaccharides from Angelica sinensis (ASP3) on Bcl-2 and Bax protein expression of irradiated liver cells from mice. Methods: Bcl-2 and Bax protein expression of liver cells in vitro exposed to 2.0 Gy rays were examined by using immunohistochemistry method. Results: The expression of apoptosis-accelerating protein Bax in the irradiation group was enhanced obviously (70.83%), while apoptosis inhibiting protein Bcl-2 tended to decline (55.60%), with the statistically significant difference (P <0.01) compared with that of the control. ASP3 pretreatment could regulate Bcl-2 and Bax protein expression of liver cells, inhibiting Bax protein expression(64.14/58.37%) and increasing Bcl-2 protein expression(59.21%/ 67.45%). The differences between the high dosage (100 mg/L of ASP3) and the irradiation group were statistically significant (P<0.05). Conclusions: ASP3 pretreatment could prohibit the apoptosis of radiation- damaged liver cells due to abnormal expression of Bcl-2 and Bax, and reduce the cell apoptosis by increasing Bcl-2/Bax protein expression so as to enhance the radiation endurance of liver cells. (authors)

  12. Genome-wide identification of heat shock proteins (Hsps) and Hsp interactors in rice: Hsp70s as a case study.

    Science.gov (United States)

    Wang, Yongfei; Lin, Shoukai; Song, Qi; Li, Kuan; Tao, Huan; Huang, Jian; Chen, Xinhai; Que, Shufu; He, Huaqin

    2014-05-07

    Heat shock proteins (Hsps) perform a fundamental role in protecting plants against abiotic stresses. Although researchers have made great efforts on the functional analysis of individual family members, Hsps have not been fully characterized in rice (Oryza sativa L.) and little is known about their interactors. In this study, we combined orthology-based approach with expression association data to screen rice Hsps for the expression patterns of which strongly correlated with that of heat responsive probe-sets. Twenty-seven Hsp candidates were identified, including 12 small Hsps, six Hsp70s, three Hsp60s, three Hsp90s, and three clpB/Hsp100s. Then, using a combination of interolog and expression profile-based methods, we inferred 430 interactors of Hsp70s in rice, and validated the interactions by co-localization and function-based methods. Subsequent analysis showed 13 interacting domains and 28 target motifs were over-represented in Hsp70s interactors. Twenty-four GO terms of biological processes and five GO terms of molecular functions were enriched in the positive interactors, whose expression levels were positively associated with Hsp70s. Hsp70s interaction network implied that Hsp70s were involved in macromolecular translocation, carbohydrate metabolism, innate immunity, photosystem II repair and regulation of kinase activities. Twenty-seven Hsps in rice were identified and 430 interactors of Hsp70s were inferred and validated, then the interacting network of Hsp70s was induced and the function of Hsp70s was analyzed. Furthermore, two databases named Rice Heat Shock Proteins (RiceHsps) and Rice Gene Expression Profile (RGEP), and one online tool named Protein-Protein Interaction Predictor (PPIP), were constructed and could be accessed at http://bioinformatics.fafu.edu.cn/.

  13. Neuroprotective Effect of Uncaria rhynchophylla in Kainic Acid-Induced Epileptic Seizures by Modulating Hippocampal Mossy Fiber Sprouting, Neuron Survival, Astrocyte Proliferation, and S100B Expression

    Directory of Open Access Journals (Sweden)

    Chung-Hsiang Liu

    2012-01-01

    Full Text Available Uncaria rhynchophylla (UR, which is a traditional Chinese medicine, has anticonvulsive effect in our previous studies, and the cellular mechanisms behind this are still little known. Because of this, we wanted to determine the importance of the role of UR on kainic acid- (KA- induced epilepsy. Oral UR for 6 weeks can successfully attenuate the onset of epileptic seizure in animal tests. Hippocampal mossy fiber sprouting dramatically decreased, while neuronal survival increased with UR treatment in hippocampal CA1 and CA3 areas. Furthermore, oral UR for 6 weeks significantly attenuated the overexpression of astrocyte proliferation and S100B proteins but not γ-aminobutyric acid A (GABAA receptors. These results indicate that oral UR for 6 weeks can successfully attenuate mossy fiber sprouting, astrocyte proliferation, and S100B protein overexpression and increase neuronal survival in KA-induced epileptic rat hippocampus

  14. Bcl-2 protein expression is associated with p27 and p53 protein expressions and MIB-1 counts in breast cancer

    International Nuclear Information System (INIS)

    Tsutsui, Shinichi; Yasuda, Kazuhiro; Suzuki, Kosuke; Takeuchi, Hideya; Nishizaki, Takashi; Higashi, Hidefumi; Era, Shoichi

    2006-01-01

    Recent experimental studies have shown that Bcl-2, which has been established as a key player in the control of apoptosis, plays a role in regulating the cell cycle and proliferation. The aim of this study was to investigate the relationship between Bcl-2 and p27 protein expression, p53 protein expression and the proliferation activity as defined by the MIB-1 counts. The prognostic implication of Bcl-2 protein expression in relation to p27 and p53 protein expressions and MIB-1 counts for breast cancer was also evaluated. The immunohistochemical expression of Bcl-2 protein was evaluated in a series of 249 invasive ductal carcinomas of the breast, in which p27 and p53 protein expressions and MIB-1 counts had been determined previously. The Bcl-2 protein expression was found to be decreased in 105 (42%) cases. A decreased Bcl-2 protein expression was significantly correlated with a nuclear grade of III, a negative estrogen receptor, a decreased p27 protein expression, a positive p53 protein expression, positive MIB-1 counts and a positive HER2 protein expression. The incidence of a nuclear grade of III and positive MIB-1 counts increased as the number of abnormal findings of Bcl-2, p27 and p53 protein expressions increased. A univariate analysis indicated a decreased Bcl-2 protein expression to be significantly (p = 0.0089) associated with a worse disease free survival (DFS), while a multivariate analysis indicated the lymph node status and MIB-1 counts to be independently significant prognostic factors for the DFS. The Bcl-2 protein expression has a close correlation with p27 and p53 protein expressions and the proliferation activity determined by MIB-1 counts in invasive ductal carcinoma of the breast. The prognostic value of Bcl-2 as well as p27 and p53 protein expressions was dependent on the proliferation activity in breast cancer

  15. Bcl-2 protein expression is associated with p27 and p53 protein expressions and MIB-1 counts in breast cancer

    Directory of Open Access Journals (Sweden)

    Nishizaki Takashi

    2006-07-01

    Full Text Available Abstract Background Recent experimental studies have shown that Bcl-2, which has been established as a key player in the control of apoptosis, plays a role in regulating the cell cycle and proliferation. The aim of this study was to investigate the relationship between Bcl-2 and p27 protein expression, p53 protein expression and the proliferation activity as defined by the MIB-1 counts. The prognostic implication of Bcl-2 protein expression in relation to p27 and p53 protein expressions and MIB-1 counts for breast cancer was also evaluated. Methods The immunohistochemical expression of Bcl-2 protein was evaluated in a series of 249 invasive ductal carcinomas of the breast, in which p27 and p53 protein expressions and MIB-1 counts had been determined previously. Results The Bcl-2 protein expression was found to be decreased in 105 (42% cases. A decreased Bcl-2 protein expression was significantly correlated with a nuclear grade of III, a negative estrogen receptor, a decreased p27 protein expression, a positive p53 protein expression, positive MIB-1 counts and a positive HER2 protein expression. The incidence of a nuclear grade of III and positive MIB-1 counts increased as the number of abnormal findings of Bcl-2, p27 and p53 protein expressions increased. A univariate analysis indicated a decreased Bcl-2 protein expression to be significantly (p = 0.0089 associated with a worse disease free survival (DFS, while a multivariate analysis indicated the lymph node status and MIB-1 counts to be independently significant prognostic factors for the DFS. Conclusion The Bcl-2 protein expression has a close correlation with p27 and p53 protein expressions and the proliferation activity determined by MIB-1 counts in invasive ductal carcinoma of the breast. The prognostic value of Bcl-2 as well as p27 and p53 protein expressions was dependent on the proliferation activity in breast cancer.

  16. Downregulation of ATM Gene and Protein Expression in Canine Mammary Tumors.

    Science.gov (United States)

    Raposo-Ferreira, T M M; Bueno, R C; Terra, E M; Avante, M L; Tinucci-Costa, M; Carvalho, M; Cassali, G D; Linde, S D; Rogatto, S R; Laufer-Amorim, R

    2016-11-01

    The ataxia telangiectasia mutated (ATM) gene encodes a protein associated with DNA damage repair and maintenance of genomic integrity. In women, ATM transcript and protein downregulation have been reported in sporadic breast carcinomas, and the absence of ATM protein expression has been associated with poor prognosis. The aim of this study was to evaluate ATM gene and protein expression in canine mammary tumors and their association with clinical outcome. ATM gene and protein expression was evaluated by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry, respectively, in normal mammary gland samples (n = 10), benign mammary tumors (n = 11), nonmetastatic mammary carcinomas (n = 19), and metastatic mammary carcinomas (n = 11). Lower ATM transcript levels were detected in benign mammary tumors and carcinomas compared with normal mammary glands (P = .011). Similarly, lower ATM protein expression was observed in benign tumors (P = .0003), nonmetastatic mammary carcinomas (P ATM gene or protein levels were detected among benign tumors and nonmetastatic and metastatic mammary carcinomas (P > .05). The levels of ATM gene or protein expression were not significantly associated with clinical and pathological features or with survival. Similar to human breast cancer, the data in this study suggest that ATM gene and protein downregulation is involved in canine mammary gland tumorigenesis. © The Author(s) 2016.

  17. Involvement of proinflammatory S100A9/A8 in the atherocalcinosis of aortic valves

    Directory of Open Access Journals (Sweden)

    R. А. Moskalenko

    2017-04-01

    Full Text Available According to the results of the Euro-Heart Survey on Vascular Heart Disease the most common pathology is nonrheumatic aortic stenosis, it is also called as calcific aortic valve stenosis (CAVS, as in its pathogenesis the process of biomineralization of valve cusps and ring plays the main role. The aim of the work is the immunohistochemical study of mineralized tissue of aortic heart valves, which are affected by atherocalcinosis. Materials and methods. 30 samples of mineralized aortic valves (I group and 10 samples of aortic valve without evidence of biomineralization (II group -– control were studied. Immunohistochemical study of expression of collagen (Collagen I, CD68, myeloperoxidase (MPO, calgranulin A (S100A8, calgranulin B (S100A9, caspase 3 (Casp 3 and osteopontin (OPN was conducted in AV tissue of both groups. Results. In CAV tissues the fibrillar component (collagen I growths was found, but the quantitative and qualitative compositions of CD68+ circulating inflammatory cells are not significantly different from the control group. CAVs contain much more MPO+ -cells (p <0.001 in comparison to the group of AVs without biomineralization. Our data show a significant increase of the S100A9 and OPN expression in the mineralized tissue of AVs (p < 0.01. Also, a higher expression level of Casp3 and MPO was found in CAVs (p < 0.05. Comparing the first and the second groups of AVs connection between the expression of S100A8 was not determined. Conclusion. High Casp 3 expression confirms the increased level of cell elimination in the CAVs tissue, which is obviously connected with the impact of high local concentrations of S100A9. These facts can contribute to the development of pathological biomineralization of AV. Since osteopontin inhibits the hydroxyapatite formation by binding to the surface of the crystals, its hyperproduction is a counteracting factor against biomineralization in AV tissue.

  18. Expression of the calcium-binding proteins MRP8 and MRP14 in monocytes is regulated by a calcium-induced suppressor mechanism.

    OpenAIRE

    Roth, J; Goebeler, M; Wrocklage, V; van den Bos, C; Sorg, C

    1994-01-01

    MRP8 and MRP14 are two calcium-binding proteins of the S-100 family the expression of which is restricted to distinct stages of monocytic differentiation. Heteromeric MRP8/MRP14 complexes have been shown to represent their biologically active forms. However, it is not as yet clear whether biochemical modification of complexes, or regulation on the transcriptional level, are responsible for the control of MRP8/MRP14 expression. Employing Western-blot analysis and metabolic labelling we have de...

  19. Holder pasteurization affects S100B concentrations in human milk.

    Science.gov (United States)

    Peila, Chiara; Coscia, Alessandra; Bertino, Enrico; Li Volti, Giovanni; Galvano, Fabio; Visser, Gerard H A; Gazzolo, Diego

    2018-02-01

    Donor milk (DM) represents an important nutrition source for high-risk newborns. Holder pasteurization (HoP) is the most recommended procedure for DM treatment, providing a good compromise between microbiological safety and biological quality. HoP was previously shown to affect DM cytokines, growth factors and hormones levels, whilst no data concerning the possible effects of HoP on neurobiomarkers (NB) are available. Therefore, our study investigated whether the concentration in DM of a well-known NB involved in brain development/damage, namely S100B, changes due to HoP. We conducted a pretest-test study in 11 mothers, whose DM samples were sub-divided into two parts: the first was immediately frozen (-80 °C); the second was pasteurized with Holder method before freezing. S100B DM levels were measured using a commercially available immunoluminometric assay. S100B protein was detected in all milk samples. Results showed significant differences between groups (p pasteurization stresses and the need to develop new storage techniques to preserve the biological quality of human milk.

  20. Effect of 17-allylamino-17-demethoxygeldanamycin (17-AAG) on Akt protein expression is more effective in head and neck cancer cell lineages that retain PTEN protein expression.

    Science.gov (United States)

    Pontes, Flávia Sirotheau C; Pontes, Hélder A R; de Souza, Lucas L; de Jesus, Adriana S; Joaquim, Andrea M C; Miyahara, Ligia A N; Fonseca, Felipe P; Pinto Junior, Décio S

    2018-03-01

    The aim of this study was to evaluate the expression of Akt, PTEN, Mdm2 and p53 proteins in three different head and neck squamous cell carcinoma (HNSCC) cell lines (HN6, HN19 and HN30), all of them treated with epidermal growth factor (EGF) and 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of Hsp90 protein. Immunofluorescence and western blot were performed in order to analyze the location and quantification, respectively, of proteins under the action 17-AAG and EGF. Treatment with EGF resulted in increased levels of Akt, PTEN and p53 in all cell lineages. The expression of Mdm2 was constant in HN30 and HN6 lineages, while in HN19 showed slightly decreased expression. Under the action 17-AAG, in HN6 and HN19, the expression of PTEN and p53 proteins was suppressed, while Akt and Mdm2 expression was reduced. Finally, in the HN30 cell lineage were absolute absence of expression of Akt, Mdm2 and p53 and decreased expression of PTEN. These data allow us to speculate on the particular utility of 17-AAG for HNSCC treatment through the inhibition of Akt protein expression, especially in the cases that retain the expression of PTEN protein. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. Case report: Extreme levels of serum S-100B in a patient with chronic subdural hematoma

    Directory of Open Access Journals (Sweden)

    Malin Elisabet Persson

    2012-12-01

    Full Text Available The protein S-100B is a biomarker increasingly used within neurosurgery and neurointensive care. As a relatively sensitive, yet unspecific, indicator of CNS pathology, potential sources of error must be clearly understood when interpreting serum S-100B levels. This case report studied the course of a 46-year-old gentleman with a chronic subdural haemorrhage, serum S-100B levels of 22 μg/L and a history of malignant melanoma. Both intra- and extra-cranial sources of S-100B are evaluated and imply an unclear contribution of several sources to the total serum concentration. Potential sources of error when interpreting serum concentrations of S-100B are discussed

  2. EXPRESSION AND SIGNIFICANCE OF ERK PROTEIN IN HUMAN BREAST CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    张秀梅; 李柏林; 宋敏; 宋继谒

    2004-01-01

    Objective: To investigate the expression of ERK and p-ERK protein in human breast cancer and their corresponding tissue, to assess the significance of ERK signal pathway in tumorigenesis and progression of breast carcinoma. Methods: 40 breast cancer cases were used in S-P immunohistochemistry technique and Western Blot study. Results: The expression of ERK1, ERK2, and p- ERK protein levels increased remarkably in breast cancer tissues in comparison to normal tissues (P<0.01). The expression was upregulated by 1.32-, 1.53-and 4.27-fold, respectively. The overexpressions of ERK1, ERK2, and p- ERK proteins were obviously correlated with clinical stage of breast cancer. Protein levels of ERK and p-ERK were higher in stage III patients than in stage I and stage II patients (P<0.05). These proteins were strongly related with axillary lymph node metastasis of breast cancer, but not correlated with histopathological type and status of ER and PR of breast cancer. Expression of ERK1, and ERK2, protein showed a positive linear correlation. Conclusion: ERK signal transduction pathway is a key factor during human breast tumorigenesis and breast cancer progression.

  3. Evidence supporting a role for astrocytes in the regulation of cognitive flexibility and neuronal oscillations through the Ca2+ binding protein S100β.

    Science.gov (United States)

    Brockett, Adam T; Kane, Gary A; Monari, Patrick K; Briones, Brandy A; Vigneron, Pierre-Antoine; Barber, Gabriela A; Bermudez, Andres; Dieffenbach, Uma; Kloth, Alexander D; Buschman, Timothy J; Gould, Elizabeth

    2018-01-01

    The medial prefrontal cortex (mPFC) is important for cognitive flexibility, the ability to switch between two task-relevant dimensions. Changes in neuronal oscillations and alterations in the coupling across frequency ranges have been correlated with attention and cognitive flexibility. Here we show that astrocytes in the mPFC of adult male Sprague Dawley rats, participate in cognitive flexibility through the astrocyte-specific Ca2+ binding protein S100β, which improves cognitive flexibility and increases phase amplitude coupling between theta and gamma oscillations. We further show that reduction of astrocyte number in the mPFC impairs cognitive flexibility and diminishes delta, alpha and gamma power. Conversely, chemogenetic activation of astrocytic intracellular Ca2+ signaling in the mPFC enhances cognitive flexibility, while inactivation of endogenous S100β among chemogenetically activated astrocytes in the mPFC prevents this improvement. Collectively, our work suggests that astrocytes make important contributions to cognitive flexibility and that they do so by releasing a Ca2+ binding protein which in turn enhances coordinated neuronal oscillations.

  4. Genome-Wide Characterization of Heat-Shock Protein 70s from Chenopodium quinoa and Expression Analyses of Cqhsp70s in Response to Drought Stress.

    Science.gov (United States)

    Liu, Jianxia; Wang, Runmei; Liu, Wenying; Zhang, Hongli; Guo, Yaodong; Wen, Riyu

    2018-01-23

    Heat-shock proteins (HSPs) are ubiquitous proteins with important roles in response to biotic and abiotic stress. The 70-kDa heat-shock genes ( Hsp70s ) encode a group of conserved chaperone proteins that play central roles in cellular networks of molecular chaperones and folding catalysts across all the studied organisms including bacteria, plants and animals. Several Hsp70s involved in drought tolerance have been well characterized in various plants, whereas no research on Chenopodium quinoa HSPs has been completed. Here, we analyzed the genome of C. quinoa and identified sixteen Hsp70 members in quinoa genome. Phylogenetic analysis revealed the independent origination of those Hsp70 members, with eight paralogous pairs comprising the Hsp70 family in quinoa. While the gene structure and motif analysis showed high conservation of those paralogous pairs, the synteny analysis of those paralogous pairs provided evidence for expansion coming from the polyploidy event. With several subcellular localization signals detected in CqHSP70 protein paralogous pairs, some of the paralogous proteins lost the localization information, indicating the diversity of both subcellular localizations and potential functionalities of those HSP70s. Further gene expression analyses revealed by quantitative polymerase chain reaction (qPCR) analysis illustrated the significant variations of Cqhsp70s in response to drought stress. In conclusion, the sixteen Cqhsp70 s undergo lineage-specific expansions and might play important and varied roles in response to drought stress.

  5. Monocyte activation, brain-derived neurotrophic factor (BDNF), and S100B in bipolar offspring: a follow-up study from adolescence into adulthood.

    Science.gov (United States)

    Mesman, Esther; Hillegers, Manon Hj; Ambree, Oliver; Arolt, Volker; Nolen, Willem A; Drexhage, Hemmo A

    2015-02-01

    There is increasing evidence that both immune and neurochemical alterations are involved in the pathogenesis of bipolar disorder; however, their precise role remains unclear. In this study, we aimed to evaluate neuro-immune changes in a prospective study on children of patients with bipolar disorder. Bipolar offspring, from the prospective Dutch bipolar offspring study (n = 140), were evaluated cross-sectionally within a longitudinal context at adolescence, young adulthood, and adulthood. We examined the expression of 44 inflammation-related genes in monocytes, the cytokines pentraxin 3 (PTX3), chemokine ligand 2 (CCL2), and interleukin-1β (IL-1β), and brain-derived neurotrophic factor (BDNF) and S100 calcium binding protein B (S100B) in the serum of bipolar offspring and healthy controls. During adolescence, bipolar offspring showed increased inflammatory gene expression in monocytes, high serum PTX3 levels, but normal CCL2 levels. BDNF levels were decreased, while S100B levels were normal. During young adulthood, monocyte activation remained, although to a lesser degree. Serum PTX3 levels remained high, and signs of monocyte migration became apparent through increased CCL2 levels. BDNF and S100B levels were not measured. At adulthood, circulating monocytes had lost their activation state, but CCL2 levels remained increased. Both BDNF and S100B were now increased. Abnormalities were independent of psychopathology state at all stages. This study suggests an aberrant neuro-immune state in bipolar offspring, which followed a dynamic course from adolescence into adulthood and was present irrespective of lifetime or future mood disorders. We therefore assumed that the aberrant neuro-immune state reflects a general state of vulnerability for mood disorders rather than being of direct predictive value. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  6. Shared features of S100B immunohistochemistry and cytochrome oxidase histochemistry in the ventroposterior thalamus and lateral habenula in neonatal rats.

    Science.gov (United States)

    Muneoka, Katsumasa; Funahashi, Hisayuki; Ogawa, Tetsuo; Whitaker-Azmitia, Patricia M; Shioda, Seiji

    2012-10-01

    The ventroposterior thalamus and the habenular nuclei of the epithalamus are relevant to the monoaminergic system functionally and anatomically. The glia-derived S100B protein plays a critical role in the development of the nervous system including the monoaminergic systems. In this study, we performed an immunohistochemical study of glia-related proteins including S100B, serotonin transporter, and microtubule-associated protein 2, as well as cytochrome oxidase histochemistry in neonatal rats. Results showed the same findings for S100B immunohistochemistry between the ventroposterior thalamus and the lateral habenula at postnatal day 7: intense staining in cell bodies of astrocytes, diffusely spread immunoproduct in the intercellular space, and S100B-free areas as well as a strong reaction to cytochrome oxidase histochemistry. Further common features were the scarcity of glial fibrillary acidic protein-positive astrocytes and the few apoptotic cells observed. The results of the cytochrome oxidase reaction suggested that S100B is released actively into intercellular areas in restricted brain regions showing high neuronal activity at postnatal day 7. Pathology of the ventroposterior thalamus and the habenula is suggested in mental disorders, and S100B might be a key factor for investigations in these areas. Copyright © 2012 ISDN. Published by Elsevier Ltd. All rights reserved.

  7. CIP2A protein expression in high-grade, high-stage bladder cancer

    International Nuclear Information System (INIS)

    Huang, Lisa P; Savoly, Diana; Sidi, Abraham A; Adelson, Martin E; Mordechai, Eli; Trama, Jason P

    2012-01-01

    Bladder cancer is one of the most common cancers in the United States. Numerous markers have been evaluated for suitability of bladder cancer detection and surveillance. However, few of them are acceptable as a routine tool. Therefore, there exists a continuing need for an assay that detects the presence of bladder cancer in humans. It would be advantageous to develop an assay with a protein that is associated with the development of bladder cancer. We have identified the cancerous inhibitor of PP2A (CIP2A) protein as a novel bladder cancer biomarker. In this study, Western blot analysis was used to assess the expression level of CIP2A protein in bladder cancer cell lines and bladder cancer patient tissues (n = 43). Our studies indicated CIP2A protein was abundantly expressed in bladder cancer cell lines but not in nontumor epithelial cell lines. Furthermore, CIP2A was specifically expressed in transitional cell carcinoma (TCC) of the bladder tumor tissues but not in adjacent nontumor bladder tissue. Our data showed that CIP2A protein detection in high-grade TCC tissues had a sensitivity of 65%, which is 3.4-fold higher than that seen in low-grade TCC tissues (19%). The level of CIP2A protein expression increased with the stage of disease (12%, 27%, 67%, and 100% for pTa, pT1, pT2, and pT3 tumor, respectively). In conclusion, our studies suggest that CIP2A protein is specifically expressed in human bladder tumors. CIP2A is preferentially expressed in high-grade and high-stage TCC tumors, which are high-risk and invasive tumors. Our studies reported here support the role of CIP2A in bladder cancer progression and its usefulness for the surveillance of recurrence or progression of human bladder cancer

  8. Expression, secretion and antigenic variation of bacterial S-layer proteins

    NARCIS (Netherlands)

    Boot, H.J.; Pouwels, P.H.

    1996-01-01

    The function of the S-layer, a regularly arranged structure on the outside of numerous bacteria, appears to be different for bacteria living in different environments. Almost no similarity exists between the primary sequences of S-proteins, although their amino acid composition is comparable.

  9. Optimized expression in Pichia pastoris eliminates common protein contaminants from subsequent His-tag purification.

    Science.gov (United States)

    Chen, Yong; Li, Yang; Liu, Peng; Sun, Qun; Liu, Zhu

    2014-04-01

    A weakness of using immobilized metal affinity chromatography (IMAC) to purify recombinant proteins expressed in Pichia pastoris is the co-purification of native proteins that exhibit high affinities for Ni-IMAC. We have determined the elution profiles of P. pastoris proteins and have examined the native proteins that co-purify when eluting with 100 mM imidazole. Four major contaminants were identified: mitochondrial alcohol dehydrogenase isozyme III (mADH), nucleotide excision repair endonuclease, and the hypothetical proteins TPHA_0L01390 and TDEL_0B02190 which are homologous proteins derived from Tetrapisispora phaffii and Torulaspora delbrueckii, respectively. A new P. pastoris expression strain was engineered that eliminated the predominant contaminant, mADH, by gene disruption. The total amount of protein contaminants was reduced by 55 % without effecting cell growth. The present study demonstrates the feasibility of using a proteomic approach to facilitate bioprocess optimization.

  10. Hypoxic-induced stress protein expression in rat cardiac myocytes

    International Nuclear Information System (INIS)

    Howard, G.; Geoghegan, T.E.

    1986-01-01

    Mammalian stress proteins can be induced in cells and tissues exposed to a variety of conditions including hyperthermia and diminished O 2 supply. The authors have previously shown that the expression of three stress proteins (71, 85, and 95 kDa) was induced in cardiac tissue from mice exposed to hypoxic conditions. The expression of mRNAs coding for the 85 and 95 kDa proteins increase with time of exposure to hypoxia, while the mRNA coding for the 71 kDa protein is transiently induced. The authors extended these studies to investigate the expression of stress proteins in isolated rat cardiac myocytes. Freshly prepared myocytes were exposed to control, hypoxic, anoxic, or heat-shock environments for up to 16 h. The proteins were then labeled for 6 hours with [ 35 S]methionine. Analysis of the solubilized proteins by SDS-PAGE and autoradiography showed that there was a 6-fold increase in synthesis of the 85 kDa protein upon exposure to hypoxia but not heat-shock conditions. The 71 kDa protein was present at high levels in both control and treated myocyte protein preparations, and presumably had been induced during the isolation procedure. Total RNA isolated from intact rat heart and isolated myocytes was compared by cell-free translation analysis and showed induction of RNAs coding for several stress proteins in the myocyte preparation. The induced proteins at 85 and 95 kDa have molecular weights similar to reported cell stress and/or glucose-regulated proteins

  11. Correlation of serum S100B protein with depressive episode of bipolar disorder and its prognosis%血清S100B蛋白与双相障碍抑郁发作及其预后的相关性研究

    Institute of Scientific and Technical Information of China (English)

    张载福; 杨帆; 王卫平; 施波; 赵俊雄; 吕望强; 喻跃国; 贾玉萍; 张晨

    2017-01-01

    目的·探讨血清S100B蛋白水平与双相障碍抑郁发作及其预后的相关性.方法·根据美国精神病协会《精神障碍诊断与统计手册》第4版(DSM-Ⅳ)诊断标准,入组双相障碍抑郁发作患者80例(病例组)以及健康对照者42名(对照组).病例组患者采用随机数字表法进入碳酸锂联合喹硫平治疗组(喹硫平组)及碳酸锂合并改良无抽搐电休克治疗组(MECT组);治疗前及治疗4周末分别测定2组血清S100B水平并评定汉密尔顿抑郁量表(HAMD).结果·经过4周随访,喹硫平组共完成36例,MECT组完成31例.病例组治疗前血清S100B水平显著高于对照组(P=0.000);治疗后,喹硫平组与MECT组患者血清S100B水平均较治疗前显著下降,HAMD评分均较治疗前显著降低(P=0.000);PearSon相关分析显示病例组治疗前后血清S100B变化水平与HAMD评分变化值呈正相关(r=0.33,P=0.013).结论·S100B可能与双相障碍抑郁发作以及预后有关.%Objective · To explore the correlation of serum S100B protein with depressive episode of bipolar disorder (BD) and its prognosis.Methods· Based on BD criteria of Diagnostic and Statistical Manual of Mental Disorders 4th edition (DSM-Ⅳ),80 patients with depressive episode of BD (case group) and 42 healthy controls (control group) were enrolled.Patients were randomly assigned into quetiapine group who were treated with lithium and quetiapine and modified electroconvulsive therapy (MECT) group who received lithium and MECT.The serum S100B level and Hamilton Rating Scale for Depression (HAMD) were assayed before and after 4-week treatment.Results· The serum S100B levels before treatment in patients with depressive episode of BD were significantly higher than those in healthy controls (P=0.000).The levels of S100B in both drug and MECT groups decreased after 4-week treatment.The HAMD score after treatment significantly decreased than that before treatment (P=0.000).Pearson correlation analysis

  12. RNA-binding protein VICKZ is expressed in a germinal center associated pattern among lymphoma subtypes

    DEFF Research Database (Denmark)

    Natkunam, Y.; Vainer, G.; Zhao, S.C.

    2005-01-01

    and tumorigenesis/metastasis. We generated an antibody that recognizes all three isoforms of VICKZ protein and characterized its expression in normal lymphoid tissue and in lymphoma subtypes. In normal tonsils, VICKZ protein showed a germinal center-specific pattern of expression with staining localized...... to the cytoplasm. Among 868 non-Hodgkin and Hodgkin lymphomas tested by immunohistochemistry on tissue microarrays, staining for VICKZ protein was present in 76% (126/165) of follicular lymphoma, 78% (155/200) of DLBCL, 90% (9/10) of mediastinal large B-cell lymphoma, and 100% (2/2) of Burkitt lymphoma. A subset...... protein in lymphoma subtypes suggests a potential utility for VICKZ in the identification of subgroups of DLBCL associated with different prognoses....

  13. Cellular responses to the expression of unstable secretory proteins in the filamentous fungus Aspergillus oryzae.

    Science.gov (United States)

    Yokota, Jun-Ichi; Shiro, Daisuke; Tanaka, Mizuki; Onozaki, Yasumichi; Mizutani, Osamu; Kakizono, Dararat; Ichinose, Sakurako; Shintani, Tomoko; Gomi, Katsuya; Shintani, Takahiro

    2017-03-01

    Filamentous fungi are often used as cell factories for recombinant protein production because of their ability to secrete large quantities of hydrolytic enzymes. However, even using strong transcriptional promoters, yields of nonfungal proteins are generally much lower than those of fungal proteins. Recent analyses revealed that expression of certain nonfungal secretory proteins induced the unfolded protein response (UPR), suggesting that they are recognized as proteins with folding defects in filamentous fungi. More recently, however, even highly expressed endogenous secretory proteins were found to evoke the UPR. These findings raise the question of whether the unfolded or misfolded state of proteins is selectively recognized by quality control mechanisms in filamentous fungi. In this study, a fungal secretory protein (1,2-α-D-mannosidase; MsdS) with a mutation that decreases its thermostability was expressed at different levels in Aspergillus oryzae. We found that, at moderate expression levels, wild-type MsdS was secreted to the medium, while the mutant was not. In the strain with a deletion for the hrdA gene, which is involved in the endoplasmic reticulum-associated degradation pathway, mutant MsdS had specifically increased levels in the intracellular fraction but was not secreted. When overexpressed, the mutant protein was secreted to the medium to a similar extent as the wild-type protein; however, the mutant underwent hyperglycosylation and induced the UPR. Deletion of α-amylase (the most abundant secretory protein in A. oryzae) alleviated the UPR induction by mutant MsdS overexpression. These findings suggest that misfolded MsdS and unfolded species of α-amylase might act synergistically for UPR induction.

  14. Brain injury markers (S100B and NSE) in chronic cocaine dependents Marcadores de lesão cerebral (S100B e NSE) em dependentes crônicos de cocaína

    OpenAIRE

    Felix Henrique Paim Kessler; George Woody; Luís Valmor Cruz Portela; Adriano Bretanha Lopes Tort; Raquel De Boni; Ana Carolina Wolf Baldino Peuker; Vanessa Genro; Lísia von Diemen; Diogo Onofre Gomes de Souza; Flavio Pechansky

    2007-01-01

    OBJECTIVE: Studies have shown signs of brain damage caused by different mechanisms in cocaine users. The serum neuron specific enolase and S100B protein are considered specific biochemical markers of neuronal and glial cell injury. This study aimed at comparing blood levels of S100B and NSE in chronic cocaine users and in volunteers who did not use cocaine or other illicit drugs. METHOD: Twenty subjects dependent on cocaine but not on alcohol or marijuana, and 20 non-substance using controls ...

  15. Protein S is protective in pulmonary fibrosis.

    Science.gov (United States)

    Urawa, M; Kobayashi, T; D'Alessandro-Gabazza, C N; Fujimoto, H; Toda, M; Roeen, Z; Hinneh, J A; Yasuma, T; Takei, Y; Taguchi, O; Gabazza, E C

    2016-08-01

    Essentials Epithelial cell apoptosis is critical in the pathogenesis of idiopathic pulmonary fibrosis. Protein S, a circulating anticoagulant, inhibited apoptosis of lung epithelial cells. Overexpression of protein S in lung cells reduced bleomycin-induced pulmonary fibrosis. Intranasal therapy with exogenous protein S ameliorated bleomycin-induced pulmonary fibrosis. Background Pulmonary fibrosis is the terminal stage of interstitial lung diseases, some of them being incurable and of unknown etiology. Apoptosis plays a critical role in lung fibrogenesis. Protein S is a plasma anticoagulant with potent antiapoptotic activity. The role of protein S in pulmonary fibrosis is unknown. Objectives To evaluate the clinical relevance of protein S and its protective role in pulmonary fibrosis. Methods and Results The circulating level of protein S was measured in patients with pulmonary fibrosis and controls by the use of enzyme immunoassays. Pulmonary fibrosis was induced with bleomycin in transgenic mice overexpressing human protein S and wild-type mice, and exogenous protein S or vehicle was administered to wild-type mice; fibrosis was then compared in both models. Patients with pulmonary fibrosis had reduced circulating levels of protein S as compared with controls. Inflammatory changes, the levels of profibrotic cytokines, fibrosis score, hydroxyproline content in the lungs and oxygen desaturation were significantly reduced in protein S-transgenic mice as compared with wild-type mice. Wild-type mice treated with exogenous protein S showed significant decreases in the levels of inflammatory and profibrotic markers and fibrosis in the lungs as compared with untreated control mice. After bleomycin infusion, mice overexpressing human protein S showed significantly low caspase-3 activity, enhanced expression of antiapoptotic molecules and enhanced Akt and Axl kinase phosphorylation as compared with wild-type counterparts. Protein S also inhibited apoptosis of alveolar

  16. Expression of the Major Vault Protein (MVP) and Cellular Vault Particles in Fish.

    Science.gov (United States)

    Margiotta, Alyssa L; Bain, Lisa J; Rice, Charles D

    2017-11-01

    Cellular vaults are ubiquitous 13 mega Da multi-subunit ribonuceloprotein particles that may have a role in nucleocytoplasmic transport. Seventy percent of the vault's mass consists of a ≈100 kDa protein, the major vault protein (MVP). In humans, a drug resistance-associated protein, originally identified as lung resistance protein in metastatic lung cancer, was ultimately shown to be the previously described MVP. In this study, a partial MVP sequence was cloned from channel catfish. Recombinant MVP (rMVP) was used to generate a monoclonal antibody that recognizes full length protein in distantly related fish species, as well as mice. MVP is expressed in fish spleen, liver, anterior kidney, renal kidney, and gills, with a consistent expression in epithelial cells, macrophages, or endothelium at the interface of the tissue and environment or vasculature. We show that vaults are distributed throughout cells of fish lymphoid cells, with nuclear and plasma membrane aggregations in some cells. Protein expression studies were extended to liver neoplastic lesions in Atlantic killifish collected in situ at the Atlantic Wood USA-EPA superfund site on the southern branch of the Elizabeth River, VA. MVP is highly expressed in these lesions, with intense staining at the nuclear membrane, similar to what is known about MVP expression in human liver neoplasia. Additionally, MVP mRNA expression was quantified in channel catfish ovarian cell line following treatment with different classes of pharmacological agents. Notably, mRNA expression is induced by ethidium bromide, which damages DNA. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:1981-1992, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  17. Huperzine A, but not tacrine, stimulates S100B secretion in astrocyte cultures.

    Science.gov (United States)

    Lunardi, Paula; Nardin, Patrícia; Guerra, Maria Cristina; Abib, Renata; Leite, Marina Concli; Gonçalves, Carlos-Alberto

    2013-04-09

    The loss of cholinergic function in the central nervous system contributes significantly to the cognitive decline associated with advanced age and dementias. Huperzine A (HupA) is a selective inhibitor of acetylcholinesterase (AChE) and has been shown to significantly reduce cognitive impairment in animal models of dementia. Based on the importance of astrocytes in physiological and pathological brain activities, we investigated the effect of HupA and tacrine on S100B secretion in primary astrocyte cultures. S100B is an astrocyte-derived protein that has been proposed to be a marker of brain injury. Primary astrocyte cultures were exposed to HupA, tacrine, cholinergic agonists, and S100B secretion was measured by enzyme-linked immunosorbent assay (ELISA) at 1 and 24h. HupA, but not tacrine, at 100μM significantly increased S100B secretion in astrocyte cultures. Nicotine (at 100 and 1000μM) was able to stimulate S100B secretion in astrocyte cultures. Our data reinforce the idea that AChE inhibitors, particularly HupA, do not act exclusively on the acetylcholine balance. This effect of HupA could contribute to improve the cognitive deficit observed in patients, which are attributed to cholinergic dysfunction. In addition, for the first time, to our knowledge, these data indicate that S100B secretion can be modulated by nicotinic receptors, in addition to glutamate, dopamine and serotonin receptors. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Evolved Escherichia coli strains for amplified, functional expression of membrane proteins.

    Science.gov (United States)

    Gul, Nadia; Linares, Daniel M; Ho, Franz Y; Poolman, Bert

    2014-01-09

    The major barrier to the physical characterization and structure determination of membrane proteins is low protein yield and/or low functionality in recombinant expression. The enteric bacterium Escherichia coli is the most widely employed organism for producing recombinant proteins. Beside several advantages of this expression host, one major drawback is that the protein of interest does not always adopt its native conformation and may end up in large insoluble aggregates. We describe a robust strategy to increase the likelihood of overexpressing membrane proteins in a functional state. The method involves fusion in tandem of green fluorescent protein and the erythromycin resistance protein (23S ribosomal RNA adenine N-6 methyltransferase, ErmC) to the C-terminus of a target membrane protein. The fluorescence of green fluorescent protein is used to report the folding state of the target protein, whereas ErmC is used to select for increased expression. By gradually increasing the erythromycin concentration of the medium and testing different membrane protein targets, we obtained a number of evolved strains of which four (NG2, NG3, NG5 and NG6) were characterized and their genome was fully sequenced. Strikingly, each of the strains carried a mutation in the hns gene, whose product is involved in genome organization and transcriptional silencing. The degree of expression of (membrane) proteins correlates with the severity of the hns mutation, but cells in which hns was deleted showed an intermediate expression performance. We propose that (partial) removal of the transcriptional silencing mechanism changes the levels of proteins essential for the functional overexpression of membrane proteins. © 2013.

  19. Myeloid-Related Protein-14/MRP-14/S100A9/Calgranulin B is Associated with Inflammation in Proliferative Diabetic Retinopathy.

    Science.gov (United States)

    Abu El-Asrar, Ahmed M; Alam, Kaiser; Siddiquei, Mohammad M; Van den Eynde, Kathleen; Mohammad, Ghulam; De Hertogh, Gert; Opdenakker, Ghislain

    2018-01-01

    To investigate the expression of the leukocyte proteins myeloid-related protein (MRP)-8 and MRP-14 in proliferative diabetic retinopathy (PDR) and the effect of MRP-8/MRP-14 (calprotectin) heterodimer on induction of proinflammatory factors in human retinal microvascular endothelial cells (HRMEC). Epiretinal membranes from 20 patients with PDR and 10 patients with proliferative vitreoretinopathy (PVR), vitreous fluid samples from PDR and non-diabetic subjects and HRMEC were studied by immunohistochemistry and Western blot analysis. MRP-14 expression was localized in endothelial cells, leukocytes and myofibroblasts in all PDR membranes. MRP-8 expression was limited to intravascular leukocytes in 42% of the studied membranes. In PVR membranes, MRP-14 was expressed in leukocytes and myofibroblasts, whereas MRP-8 immunoreactivity was limited to leukocytes. MRP-14 was significantly upregulated in vitreous from PDR patients. MRP-8/MRP-14 (calprotectin) increased expression of intercellular adhesion molecule-1, but attenuated vascular cell adhesion molecule-1 expression in HRMEC. Increased MRP-14 levels are associated with inflammation in PDR.

  20. Cloning, periplasmic expression, purification and structural characterization of human ribosomal protein L10

    International Nuclear Information System (INIS)

    Pereira, Larissa Miranda

    2009-01-01

    The ribosomal protein L10 (RP L10) is a strong candidate to be included in the class of tumor suppressor proteins. This protein, also denominated as QM, is known to participate in the binding of ribosomal subunits 60S and 40S and the translation of mRNAs. It has a molecular weight that varies between 24 and 26 kDa and an isoelectric point of (pI) 10.5. The sequence of the protein QM is highly conserved in mammals, plants, invertebrates, insects and yeast which indicates its critical functions in a cell. As a tumor suppressor, RP L10 has been studied in strains of Wilm's tumor (WT-1) and tumor cells in the stomach, where was observed a decrease in the amount of its mRNA. More recently, the RP L10 was found in low amounts in the early stages of prostate adenoma and showed some mutation in ovarian cancer, what indicates its role as a suppressor protein in the development of these diseases. It has also been described that this protein interacts with c-Jun and c-Yes inhibiting growth factors and consequently, cell division. This work has an important role on the establishment of soluble expression of QM to give base information for further studies on expression that aim to evaluate the specific regions where it acts binding the 60S and 40S ribosomal subunits and translation, as well as its binding to proto-oncogenes. The cDNA for QM protein was amplified by PCR and cloned into periplasmic expression vector p3SN8. The QM protein was expressed in E. coli BL21 (DE3) in the region of cytoplasm and periplasm, the best condition was obtained from the expression of the recombinant plasmid QM p1813 Q M at 25 degree C or 30 degree C, the soluble protein was obtained with small amounts of contaminants. The assays of secondary structure showed that the QM protein is predominantly alpha-helix, but when it loses the folding, this condition changes and the protein is replaced by β- sheet feature. (author)

  1. The predictive nature of transcript expression levels on protein expression in adult human brain.

    Science.gov (United States)

    Bauernfeind, Amy L; Babbitt, Courtney C

    2017-04-24

    Next generation sequencing methods are the gold standard for evaluating expression of the transcriptome. When determining the biological implications of such studies, the assumption is often made that transcript expression levels correspond to protein levels in a meaningful way. However, the strength of the overall correlation between transcript and protein expression is inconsistent, particularly in brain samples. Following high-throughput transcriptomic (RNA-Seq) and proteomic (liquid chromatography coupled with tandem mass spectrometry) analyses of adult human brain samples, we compared the correlation in the expression of transcripts and proteins that support various biological processes, molecular functions, and that are located in different areas of the cell. Although most categories of transcripts have extremely weak predictive value for the expression of their associated proteins (R 2 values of < 10%), transcripts coding for protein kinases and membrane-associated proteins, including those that are part of receptors or ion transporters, are among those that are most predictive of downstream protein expression levels. The predictive value of transcript expression for corresponding proteins is variable in human brain samples, reflecting the complex regulation of protein expression. However, we found that transcriptomic analyses are appropriate for assessing the expression levels of certain classes of proteins, including those that modify proteins, such as kinases and phosphatases, regulate metabolic and synaptic activity, or are associated with a cellular membrane. These findings can be used to guide the interpretation of gene expression results from primate brain samples.

  2. The LIKE system, a novel protein expression toolbox for Bacillus subtilis based on the liaI promoter

    Science.gov (United States)

    2012-01-01

    Background Bacillus subtilis is a very important Gram-positive model organism of high biotechnological relevance, which is widely used as a host for the production of both secreted and cytoplasmic proteins. We developed a novel and efficient expression system, based on the liaI promoter (PliaI) from B. subtilis, which is under control of the LiaRS antibiotic-inducible two-component system. In the absence of a stimulus, this promoter is kept tightly inactive. Upon induction by cell wall antibiotics, it shows an over 100-fold increase in activity within 10 min. Results Based on these traits of PliaI, we developed a novel LiaRS-controlled gene expression system for B. subtilis (the “LIKE" system). Two expression vectors, the integrative pLIKE-int and the replicative pLIKE-rep, were constructed. To enhance the performance of the PliaI-derived system, site-directed mutagenesis was employed to optimize the ribosome binding site and alter its spacing to the initiation codon used for the translational fusion. The impact of these genetic modifications on protein production yield was measured using GFP as a model protein. Moreover, a number of tailored B. subtilis expression strains containing different markerless chromosomal deletions of the liaIH region were constructed to circumvent undesired protein production, enhance the positive autoregulation of the LiaRS system and thereby increase target gene expression strength from the PliaI promoter. Conclusions The LIKE protein expression system is a novel protein expression system, which offers a number of advantages over existing systems. Its major advantages are (i) a tightly switched-off promoter during exponential growth in the absence of a stimulus, (ii) a concentration-dependent activation of PliaI in the presence of suitable inducers, (iii) a very fast but transient response with a very high dynamic range of over 100-fold (up to 1,000-fold) induction, (iv) a choice from a range of well-defined, commercially available

  3. Proteomic Identification of Differentially Expressed Proteins during Alfalfa (Medicago sativa L.) Flower Development.

    Science.gov (United States)

    Chen, Lingling; Chen, Quanzhu; Zhu, Yanqiao; Hou, Longyu; Mao, Peisheng

    2016-01-01

    Flower development, pollination, and fertilization are important stages in the sexual reproduction process of plants; they are also critical steps in the control of seed formation and development. During alfalfa ( Medicago sativa L.) seed production, some distinct phenomena such as a low seed setting ratio, serious flower falling, and seed abortion commonly occur. However, the causes of these phenomena are complicated and largely unknown. An understanding of the mechanisms that regulate alfalfa flowering is important in order to increase seed yield. Hence, proteomic technology was used to analyze changes in protein expression during the stages of alfalfa flower development. Flower samples were collected at pre-pollination (S1), pollination (S2), and the post-pollination senescence period (S3). Twenty-four differentially expressed proteins were successfully identified, including 17 down-regulated in pollinated flowers, one up-regulated in pollinated and senesced flowers, and six up-regulated in senesced flowers. The largest proportions of the identified proteins were involved in metabolism, signal transduction, defense response, oxidation reduction, cell death, and programmed cell death (PCD). Their expression profiles demonstrated that energy metabolism, carbohydrate metabolism, and amino acid metabolism provided the nutrient foundation for pollination in alfalfa. Furthermore, there were three proteins involved in multiple metabolic pathways: dual specificity kinase splA-like protein (kinase splALs), carbonic anhydrase, and NADPH: quinone oxidoreductase-like protein. Expression patterns of these proteins indicated that MAPK cascades regulated multiple processes, such as signal transduction, stress response, and cell death. PCD also played an important role in the alfalfa flower developmental process, and regulated both pollination and flower senescence. The current study sheds some light on protein expression profiles during alfalfa flower development and

  4. Primary Screening for Proteins Differentially Expressed in the Myocardium of a Rat Model of Acute Methamphetamine Intoxication

    Directory of Open Access Journals (Sweden)

    Guoqiang Qu

    2016-01-01

    Full Text Available The mechanism of myocardial injury induced by the cardiovascular toxicity of methamphetamine (MA has been shown to depend on alterations in myocardial proteins caused by MA. Primary screening of the expression of myocardial proteins in a rat model of MA intoxication was achieved by combining two-dimensional electrophoresis and mass spectrometry analyses, which revealed a total of 100 differentially expressed proteins. Of these, 13 displayed significantly altered expression. Moreover, Western blotting and real-time reverse transcription quantitative polymerase chain reaction analyses of several relative proteins demonstrated that acute MA intoxication lowers protein expression and mRNA transcription of aldehyde dehydrogenase-2 and NADH dehydrogenase (ubiquinone 1 alpha subcomplex subunit 10. In contrast, MA intoxication elevated the protein expression and mRNA transcription of heat shock protein family B (small member 1. By combining behavioral assessments of experimental rat models with the histological and pathological changes evident in cardiomyocytes, a mechanism accounting for MA myocardial toxicity was suggested. MA alters the regulation of gene transcription and the subsequent expression of certain proteins that participate in myocardial respiration and in responding to oxidative stress, resulting in myocardial dysfunction and structural changes that affect the functioning of the cardiovascular system.

  5. Beneficial Immune Effects of Myeloid-Related Proteins in Kidney Transplant Rejection

    NARCIS (Netherlands)

    Rekers, N. V.; Bajema, I. M.; Mallat, M. J. K.; Petersen, B.; Anholts, J. D. H.; Swings, G. M. J. S.; van Miert, P. P. M. C.; Kerkhoff, C.; Roth, J.; Popp, D.; van Groningen, M. C.; Baeten, D.; Goemaere, N.; Kraaij, M. D.; Zandbergen, M.; Heidt, S.; van Kooten, C.; de Fijter, J. W.; Claas, F. H. J.; Eikmans, M.

    2016-01-01

    Acute rejection is a risk factor for inferior long-term kidney transplant survival. Although T cell immunity is considered the main effector in clinical acute rejection, the role of myeloid cells is less clear. Expression of S100 calcium-binding protein A8 (S100A8) and S100A9 was evaluated in 303

  6. S(+)-ibuprofen destabilizes MYC/MYCN and AKT, increases p53 expression, and induces unfolded protein response and favorable phenotype in neuroblastoma cell lines.

    Science.gov (United States)

    Ikegaki, Naohiko; Hicks, Sakeenah L; Regan, Paul L; Jacobs, Joshua; Jumbo, Amina S; Leonhardt, Payton; Rappaport, Eric F; Tang, Xao X

    2014-01-01

    Neuroblastoma is a common pediatric solid tumor that exhibits a striking clinical bipolarity: favorable and unfavorable. The survival rate of children with unfavorable neuroblastoma remains low among all childhood cancers. MYCN and MYC play a crucial role in determining the malignancy of unfavorable neuroblastomas, whereas high-level expression of the favorable neuroblastoma genes is associated with a good disease outcome and confers growth suppression of neuroblastoma cells. A small fraction of neuroblastomas harbors TP53 mutations at diagnosis, but a higher proportion of the relapse cases acquire TP53 mutations. In this study, we investigated the effect of S(+)-ibuprofen on neuroblastoma cell lines, focusing on the expression of the MYCN, MYC, AKT, p53 proteins and the favorable neuroblastoma genes in vitro as biomarkers of malignancy. Treatment of neuroblastoma cell lines with S(+)-ibuprofen resulted in a significant growth suppression. This growth effect was accompanied by a marked decrease in the expression of MYC, MYCN, AKT and an increase in p53 expression in neuroblastoma cell lines without TP53 mutation. In addition, S(+)-ibuprofen enhanced the expression of some favorable neuroblastoma genes (EPHB6, CD44) and genes involved in growth suppression and differentiation (EGR1, EPHA2, NRG1 and SEL1L). Gene expression profile and Ingenuity pathway analyses using TP53-mutated SKNAS cells further revealed that S(+)-ibuprofen suppressed molecular pathways associated with cell growth and conversely enhanced those of cell cycle arrest and the unfolded protein response. Collectively, these results suggest that S(+)-ibuprofen or its related compounds may have the potential for therapeutic and/or palliative use for unfavorable neuroblastoma.

  7. Expression of a novel stress-inducible protein, sestrin 2, in rat glomerular parietal epithelial cells

    Science.gov (United States)

    Hamatani, Hiroko; Sakairi, Toru; Takahashi, Satoshi; Watanabe, Mitsuharu; Maeshima, Akito; Ohse, Takamoto; Pippin, Jeffery W.; Shankland, Stuart J.; Nojima, Yoshihisa

    2014-01-01

    Sestrin 2, initially identified as a p53 target protein, accumulates in cells exposed to stress and inhibits mammalian target of rapamycin (mTOR) signaling. In normal rat kidneys, sestrin 2 was selectively expressed in parietal epithelial cells (PECs), identified by the marker protein gene product 9.5. In adriamycin nephropathy, sestrin 2 expression decreased in PECs on day 14, together with increased expression of phosphorylated S6 ribosomal protein (P-S6RP), a downstream target of mTOR. Sestrin 2 expression was markedly decreased on day 42, coinciding with glomerulosclerosis and severe periglomerular fibrosis. In puromycin aminonucleoside nephropathy, decreased sestrin 2 expression, increased P-S6RP expression, and periglomerular fibrosis were observed on day 9, when massive proteinuria developed. These changes were transient and nearly normalized by day 28. In crescentic glomerulonephritis, sestrin 2 expression was not detected in cellular crescents, whereas P-S6RP increased. In conditionally immortalized cultured PECs, the forced downregulation of sestrin 2 by short hairpin RNA resulted in increased expression of P-S6RP and increased apoptosis. These data suggest that sestrin 2 is involved in PEC homeostasis by regulating the activity of mTOR. In addition, sestrin 2 could be a novel marker of PECs, and decreased expression of sestrin 2 might be a marker of PEC injury. PMID:25056347

  8. Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF.

    Science.gov (United States)

    Cantu-Bustos, J Enrique; Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Galbraith, David W; McEvoy, Megan M; Zarate, Xristo

    2016-05-01

    Production of recombinant proteins in Escherichia coli has been improved considerably through the use of fusion proteins, because they increase protein solubility and facilitate purification via affinity chromatography. In this article, we propose the use of CusF as a new fusion partner for expression and purification of recombinant proteins in E. coli. Using a cell-free protein expression system, based on the E. coli S30 extract, Green Fluorescent Protein (GFP) was expressed with a series of different N-terminal tags, immobilized on self-assembled protein microarrays, and its fluorescence quantified. GFP tagged with CusF showed the highest fluorescence intensity, and this was greater than the intensities from corresponding GFP constructs that contained MBP or GST tags. Analysis of protein production in vivo showed that CusF produces large amounts of soluble protein with low levels of inclusion bodies. Furthermore, fusion proteins can be exported to the cellular periplasm, if CusF contains the signal sequence. Taking advantage of its ability to bind copper ions, recombinant proteins can be purified with readily available IMAC resins charged with this metal ion, producing pure proteins after purification and tag removal. We therefore recommend the use of CusF as a viable alternative to MBP or GST as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. S100A4 and BMP-2 Co-Dependently Induce Vascular Smooth Muscle Cell Migration via pERK and Chloride Intracellular Channel 4 (CLIC4)

    Science.gov (United States)

    Spiekerkoetter, Edda; Guignabert, Christophe; de Jesus Perez, Vinicio; Alastalo, Tero-Pekka; Powers, Janine M; Wang, Lingli; Lawrie, Allan; Ambartsumian, Noona; Schmidt, Ann-Marie; Berryman, Mark; Ashley, Richard H; Rabinovitch, Marlene

    2009-01-01

    Rationale S100A4/Mts1 is implicated in motility of human pulmonary artery smooth muscle cells (hPASMC), through an interaction with the receptor for advanced glycation end products (RAGE). Objective We hypothesized that S100A4/Mts1-mediated hPASMC motility might be enhanced by loss of function of bone morphogenetic protein (BMP) receptor (R) II, observed in pulmonary arterial hypertension (PAH). Methods and Results Both S100A4/Mts1 (500ng/ml) and BMP-2 (10ng/ml) induce migration of hPASMCS in a novel co-dependent manner, in that the response to either ligand is lost with anti-RAGE or BMPRII siRNA. Phosphorylation of ERK is induced by both ligands and is required for motility by inducing MMP2 activity, but phosphoERK1/2 is blocked by anti-RAGE and not by BMPRII siRNA. In contrast, BMPRII siRNA, but not anti-RAGE, reduces expression of intracellular chloride channel 4 (CLIC4), a scaffolding molecule necessary for motility in response to S100A4/Mts1 or BMP-2. Reduced CLIC4 expression does not interfere with S100A4/Mts1 internalization or its interaction with myosin heavy chain IIA (MHCIIA), but does alter alignment of MHCIIA and actin filaments creating the appearance of vacuoles. This abnormality is associated with reduced peripheral distribution and/or delayed activation of RhoA and Rac1, small GTPases required for retraction and extension of lamellipodiae in motile cells. Conclusions Our studies demonstrate how a single ligand (BMP-2 or S100A4/Mts1) can recruit multiple cell surface receptors to relay signals that coordinate events culminating in a functional response, i.e., cell motility. We speculate that this carefully controlled process limits signals from multiple ligands, but could be subverted in disease. PMID:19713532

  10. Diagnostic accuracy of S100B urinary testing at birth in full-term asphyxiated newborns to predict neonatal death.

    Directory of Open Access Journals (Sweden)

    Diego Gazzolo

    Full Text Available BACKGROUND: Neonatal death in full-term infants who suffer from perinatal asphyxia (PA is a major subject of investigation, since few tools exist to predict patients at risk of ominous outcome. We studied the possibility that urine S100B measurement may identify which PA-affected infants are at risk of early postnatal death. METHODOLOGY/PRINCIPAL FINDINGS: In a cross-sectional study between January 1, 2001 and December 1, 2006 we measured S100B protein in urine collected from term infants (n = 132, 60 of whom suffered PA. According to their outcome at 7 days, infants with PA were subsequently classified either as asphyxiated infants complicated by hypoxic ischemic encephalopathy with no ominous outcome (HIE Group; n = 48, or as newborns who died within the first post-natal week (Ominous Outcome Group; n = 12. Routine laboratory variables, cerebral ultrasound, neurological patterns and urine concentrations of S100B protein were determined at first urination and after 24, 48 and 96 hours. The severity of illness in the first 24 hours after birth was measured using the Score for Neonatal Acute Physiology-Perinatal Extension (SNAP-PE. Urine S100B levels were higher from the first urination in the ominous outcome group than in healthy or HIE Groups (p1.0 microg/L S100B had a sensitivity/specificity of 100% for predicting neonatal death. CONCLUSIONS/SIGNIFICANCE: Increased S100B protein urine levels in term newborns suffering PA seem to suggest a higher risk of neonatal death for these infants.

  11. Methylation status and protein expression of RASSF1A in breast cancer patients.

    Science.gov (United States)

    Hagrass, Hoda A; Pasha, Heba F; Shaheen, Mohamed A; Abdel Bary, Eman H; Kassem, Rasha

    2014-01-01

    Recently genetics and epigenetics alterations have been found to be characteristic of malignancy and hence can be used as targets for detection of neoplasia. RAS association domain family protein 1A (RASSF1A) gene hypermethylation has been a subject of interest in recent researches on cancer breast patients. The aim of the present study was to evaluate whether RASSF1A methylation status and RASSF1A protein expression are associated with the major clinico-pathological parameters. One hundred and twenty breast cancer Egyptian patients and 100-control subjects diagnosed with benign lesions of the breast were enrolled in this study. We evaluated RASSF1A methylation status in tissue and serum samples using Methyl specific PCR together with RASSF1A protein expression in tissues by immunohistochemistry. Results were studied in relation to known prognostic clinicopathological features in breast cancer. Frequency of RASSF1A methylation in tissues and serum were 70 and 63.3 % respectively and RASSF1A protein expression showed frequency of 46.7 %. There was an association between RASSF1A methylation in tissues, serum and loss of protein expression in tissues with invasive carcinoma, advanced stage breast cancer, L.N. metastasis, ER/PR and HER2 negativity. RASSF1A methylation in serum showed high degree of concordance with methylation in tissues (Kappa = 0.851, P < 0.001). RASSF1A hypermethylation in tissues and serum and its protein expression may be a valid, reliable and sensitive tool for detection and follow up of breast cancer patients.

  12. Homologous high-throughput expression and purification of highly conserved E coli proteins

    Directory of Open Access Journals (Sweden)

    Duchmann Rainer

    2007-06-01

    Full Text Available Abstract Background Genetic factors and a dysregulated immune response towards commensal bacteria contribute to the pathogenesis of Inflammatory Bowel Disease (IBD. Animal models demonstrated that the normal intestinal flora is crucial for the development of intestinal inflammation. However, due to the complexity of the intestinal flora, it has been difficult to design experiments for detection of proinflammatory bacterial antigen(s involved in the pathogenesis of the disease. Several studies indicated a potential association of E. coli with IBD. In addition, T cell clones of IBD patients were shown to cross react towards antigens from different enteric bacterial species and thus likely responded to conserved bacterial antigens. We therefore chose highly conserved E. coli proteins as candidate antigens for abnormal T cell responses in IBD and used high-throughput techniques for cloning, expression and purification under native conditions of a set of 271 conserved E. coli proteins for downstream immunologic studies. Results As a standardized procedure, genes were PCR amplified and cloned into the expression vector pQTEV2 in order to express proteins N-terminally fused to a seven-histidine-tag. Initial small-scale expression and purification under native conditions by metal chelate affinity chromatography indicated that the vast majority of target proteins were purified in high yields. Targets that revealed low yields after purification probably due to weak solubility were shuttled into Gateway (Invitrogen destination vectors in order to enhance solubility by N-terminal fusion of maltose binding protein (MBP, N-utilizing substance A (NusA, or glutathione S-transferase (GST to the target protein. In addition, recombinant proteins were treated with polymyxin B coated magnetic beads in order to remove lipopolysaccharide (LPS. Thus, 73% of the targeted proteins could be expressed and purified in large-scale to give soluble proteins in the range of 500

  13. Signaling system in Porphyromonas gingivalis based on a LuxS protein.

    Science.gov (United States)

    Chung, W O; Park, Y; Lamont, R J; McNab, R; Barbieri, B; Demuth, D R

    2001-07-01

    The luxS gene of quorum-sensing Vibrio harveyi is required for type 2 autoinducer production. We identified a Porphyromonas gingivalis open reading frame encoding a predicted peptide of 161 aa that shares 29% identity with the amino acid sequence of the LuxS protein of V. harveyi. Conditioned medium from a late-log-phase P. gingivalis culture induced the luciferase operon of V. harveyi, but that from a luxS insertional mutant did not. In P. gingivalis, the expression of luxS mRNA was environmentally controlled and varied according to the cell density and the osmolarity of the culture medium. In addition, differential display PCR showed that the inactivation of P. gingivalis luxS resulted in up-regulation of a hemin acquisition protein and an arginine-specific protease and reduced expression of a hemin-regulated protein, a TonB homologue, and an excinuclease. The data suggest that the luxS gene in P. gingivalis may function to control the expression of genes involved in the acquisition of hemin.

  14. Increased expression of TLR9 associated with pro-inflammatory S100A8 and IL-8 in diabetic wounds could lead to unresolved inflammation in type 2 diabetes mellitus (T2DM) cases with impaired wound healing.

    Science.gov (United States)

    Singh, Kanhaiya; Agrawal, Neeraj K; Gupta, Sanjeev K; Sinha, Pratima; Singh, Kiran

    2016-01-01

    Type 2 diabetes mellitus (T2DM) is characterized by persistent hyperglycemia which causes a chain of abrupt biochemical and physiological changes. Immune dys-regulation is the hallmark of T2DM that could contribute to prolonged inflammation causing transformation of wounds into non-healing chronic ulcers. Toll like receptor -9 (TLR9) is a major receptor involved in innate immune regulation. TLR9 activation induces release of pro-inflammatory molecules like S100A8 and interleukin-8 (IL-8) by myeloid cells causing migration of myeloid cells to the site of inflammation. We hypothesized that pro-inflammatory S100A8 and IL-8 proteins could cause persistent inflammation in chronic wounds like diabetic foot ulcer (DFU) and may contribute to impaired wound healing in T2DM patients. Expression of TLR9 and its downstream effector molecules S100A8, and IL-8 were analyzed in chronic diabetic wound and non-diabetic control wound tissue samples by semiquantitative reverse transcriptase - polymerase chain reaction (RT-PCR), quantitative RT-PCR, western blot and immunofluorescence. CD11b(+)CD33(+) myeloid cells were analyzed by flow cytometry. TLR9 message and protein were higher in diabetic wounds compared to control wounds (p=0.03, t=2.21 for TLR9 mRNA; p=diabetic wounds (p=0.003, t=3.1 for S100A8 mRNA; p=0.04, t=2.04 for IL-8). CD11b(+) CD33(+) myeloid cells were decreased in T2DM as compared to non-diabetic controls (p=0.001, t=3.6). DFU subjects had higher levels of CD11b(+) CD33(+) myeloid cells as compared to non-DFU T2DM control (p=0.003, t=2.8). Infection in the wound microenvironment could be the cause of increase in CD11b(+)CD33(+) myeloid cells in DFU (p=0.03, t=2.5). The up-regulation of myeloid cell-derived pro-inflammatory molecules S100A8 and IL-8 in combination with lower levels of CD11b(+) CD33(+) myeloid cells may cause the impairment of wound healing in T2DM subjects leading to chronic ulcers. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Differentially expressed proteins on postoperative 3

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    Jialili Ainuer

    2011-04-01

    Full Text Available 【Abstract】Objectives: Surgical repair of Achilles tendon (AT rupture should immediately be followed by active tendon mobilization. The optimal time as to when the mobilization should begin is important yet controversial. Early kinesitherapy leads to reduced rehabilitation period. However, an insight into the detailed mechanism of this process has not been gained. Proteomic technique can be used to separate and purify the proteins by differential expression profile which is related to the function of different proteins, but research in the area of proteomic analysis of AT 3 days after repair has not been studied so far. Methods: Forty-seven New Zealand white rabbits were randomized into 3 groups. Group A (immobilization group, n=16 received postoperative cast immobilization; Group B (early motion group, n=16 received early active motion treatments immediately following the repair of AT rupture from tenotomy. Another 15 rabbits served as control group (Group C. The AT samples were prepared 3 days following the microsurgery. The proteins were separated employing twodimensional polyacrylamide gel electrophoresis (2D-PAGE. PDQuest software version 8.0 was used to identify differentially expressed proteins, followed by peptide mass fingerprint (PMF and tandem mass spectrum analysis, using the National Center for Biotechnology Information (NCBI protein database retrieval and then for bioinformatics analysis. Results: A mean of 446.33, 436.33 and 462.67 protein spots on Achilles tendon samples of 13 rabbits in Group A, 14 rabbits in Group B and 13 rabbits in Group C were successfully detected in the 2D-PAGE. There were 40, 36 and 79 unique proteins in Groups A, B and C respectively. Some differentially expressed proteins were enzyme with the gel, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS. We successfully identified 9 and 11 different proteins in Groups A and B, such as GAPDH, phosphoglycerate kinase 1

  16. Expression, Delivery and Function of Insecticidal Proteins Expressed by Recombinant Baculoviruses

    Science.gov (United States)

    Kroemer, Jeremy A.; Bonning, Bryony C.; Harrison, Robert L.

    2015-01-01

    Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification. PMID:25609310

  17. p100, a precursor of NF-κB2, inhibits c-Rel and reduces the expression of IL-23 in dendritic cells

    International Nuclear Information System (INIS)

    Mise-Omata, Setsuko; Obata, Yuichi; Doi, Takahiro S.

    2014-01-01

    Highlights: • The deficiency of p100 enhances c-Rel-, not RelA-, dependent cytokine expression. • p100 associates with c-Rel in the steady state but dissociates after LPS stimulation. • The deficiency of p100 enhances the nuclear translocation of c-Rel. • p100 negatively regulates the c-Rel function. - Abstract: Nuclear factor κB regulates various genes involved in the immune response, inflammation, cell survival, and development. NF-κB activation is controlled by proteins possessing ankyrin repeats, such as IκBs. A precursor of the NF-κB2 (p52) subunit, p100, contains ankyrin repeats in its C-terminal portion and has been found to act as a cytoplasmic inhibitor of RelA in the canonical pathway of NF-κB activation. Here, we demonstrate that p100 also suppresses c-Rel function in dendritic cells. Expression of the p19 and p40 subunits of IL-23, a c-Rel-dependent cytokine, was enhanced in p100-deficient cells, although expression of a RelA-dependent cytokine, TNF-α, was reduced. Nuclear translocation of c-Rel was enhanced in p100-deficient cells. p100, and not the processed p52 form, associated with c-Rel in the steady state and dissociated immediately after lipopolysaccharide stimulation in wild-type dendritic cells. Four hours after the stimulation, p100 was newly synthesized and associated with c-Rel again. In cells expressing both c-Rel and RelA, c-Rel is preferentially suppressed by p100

  18. Escherichia coli Protein Expression System for Acetylcholine Binding Proteins (AChBPs.

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    Nikita Abraham

    Full Text Available Nicotinic acetylcholine receptors (nAChR are ligand gated ion channels, identified as therapeutic targets for a range of human diseases. Drug design for nAChR related disorders is increasingly using structure-based approaches. Many of these structural insights for therapeutic lead development have been obtained from co-crystal structures of nAChR agonists and antagonists with the acetylcholine binding protein (AChBP. AChBP is a water soluble, structural and functional homolog of the extracellular, ligand-binding domain of nAChRs. Currently, AChBPs are recombinantly expressed in eukaryotic expression systems for structural and biophysical studies. Here, we report the establishment of an Escherichia coli (E. coli expression system that significantly reduces the cost and time of production compared to the existing expression systems. E. coli can efficiently express unglycosylated AChBP for crystallography and makes the expression of isotopically labelled forms feasible for NMR. We used a pHUE vector containing an N-terminal His-tagged ubiquitin fusion protein to facilitate AChBP expression in the soluble fractions, and thus avoid the need to recover protein from inclusion bodies. The purified protein yield obtained from the E. coli expression system is comparable to that obtained from existing AChBP expression systems. E. coli expressed AChBP bound nAChR agonists and antagonists with affinities matching those previously reported. Thus, the E. coli expression system significantly simplifies the expression and purification of functional AChBP for structural and biophysical studies.

  19. [High-level expression of heterologous protein based on increased copy number in Saccharomyces cerevisiae].

    Science.gov (United States)

    Zhang, Xinjie; He, Peng; Tao, Yong; Yang, Yi

    2013-11-04

    High-level expression system of heterologous protein mediated by internal ribosome entry site (IRES) in Saccharomyces cerevisiae was constructed, which could be used for other applications of S. cerevisiae in metabolic engineering. We constructed co-expression cassette (promoter-mCherry-TIF4631 IRES-URA3) containing promoters Pilv5, Padh2 and Ptdh3 and recombined the co-expression cassette into the genome of W303-1B-A. The URA3+ transformants were selected. By comparing the difference in the mean florescence value of mCherry in transformants, the effect of three promoters was detected in the co-expression cassette. The copy numbers of the interested genes in the genome were determined by Real-Time PCR. We analyzed genetic stability by continuous subculturing transformants in the absence of selection pressure. To verify the application of co-expression cassette, the ORF of mCherry was replaced by beta-galactosidase (LACZ) and xylose reductase (XYL1). The enzyme activities and production of beta-galactosidase and xylose reductase were detected. mCherry has been expressed in the highest-level in transformants with co-expression cassette containing Pilv5 promoter. The highest copy number of DNA fragment integrating in the genome was 47 in transformants containing Pilv5. The engineering strains showed good genetic stability. Xylose reductase was successfully expressed in the co-expression cassette containing Pilv5 promoter and TIF4631 IRES. The highest enzyme activity was 0. 209 U/mg crude protein in the transformants WIX-10. Beta-galactosidase was also expressed successfully. The transformants that had the highest enzyme activity was WIL-1 and the enzyme activity was 12.58 U/mg crude protein. The system mediated by Pilv5 promoter and TIF4631 IRES could express heterologous protein efficiently in S. cerevisiae. This study offered a new strategy for expression of heterologous protein in S. cerevisiae and provided sufficient experimental evidence for metabolic engineering

  20. Expression of P-glycoprotein, multidrug resistance-associated protein, glutathione-S-transferase pi and p53 in canine transmissible venereal tumor

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    Daniel G. Gerardi

    2014-01-01

    Full Text Available The overexpression of proteins P-glycoprotein (P-gp, multidrug resistance-associated protein (MRP1, mutant p53, and the enzyme glutathione-S-transferase (GSTpi are related to resistance to chemotherapy in neoplasms. This study evaluated the expression of these markers by immunohistochemistry in two groups of canine TVT, without history of prior chemotherapy (TVT1, n=9 and in TVTs presented unsatisfactory clinical response to vincristine sulfate (TVT2, n=5. The percentage of specimens positively stained for P-gp, MRP1, GSTpi and p53 were, respectively 88.8%, 0%, 44.5% and 22.2% in TVT1 and 80%, 0%, 80% and 0% in TVT2. In TVT1, one specimen presented positive expression for three markers and four specimens for two markers. In TVT2, three specimens expressed P-gp and GSTpi. In conclusion, the canine TVTs studied expressed the four markers evaluated, but just P-gp and GSTpi were significantly expressed, mainly at cytoplasm and cytoplasm and nuclei, respectively, either before chemotherapy as after vincristine sulfate exposure. Future studies are needed to demonstrate the function of these two markers in conferring multidrug resistance (MDR or predict the response to chemotherapy in canine TVT.

  1. Analysis of the Sarcocystis neurona microneme protein SnMIC10: protein characteristics and expression during intracellular development.

    Science.gov (United States)

    Hoane, Jessica S; Carruthers, Vernon B; Striepen, Boris; Morrison, David P; Entzeroth, Rolf; Howe, Daniel K

    2003-07-01

    Sarcocystis neurona, an apicomplexan parasite, is the primary causative agent of equine protozoal myeloencephalitis. Like other members of the Apicomplexa, S. neurona zoites possess secretory organelles that contain proteins necessary for host cell invasion and intracellular survival. From a collection of S. neurona expressed sequence tags, we identified a sequence encoding a putative microneme protein based on similarity to Toxoplasma gondii MIC10 (TgMIC10). Pairwise sequence alignments of SnMIC10 to TgMIC10 and NcMIC10 from Neospora caninum revealed approximately 33% identity to both orthologues. The open reading frame of the S. neurona gene encodes a 255 amino acid protein with a predicted 39-residue signal peptide. Like TgMIC10 and NcMIC10, SnMIC10 is predicted to be hydrophilic, highly alpha-helical in structure, and devoid of identifiable adhesive domains. Antibodies raised against recombinant SnMIC10 recognised a protein band with an apparent molecular weight of 24 kDa in Western blots of S. neurona merozoites, consistent with the size predicted for SnMIC10. In vitro secretion assays demonstrated that this protein is secreted by extracellular merozoites in a temperature-dependent manner. Indirect immunofluorescence analysis of SnMIC10 showed a polar labelling pattern, which is consistent with the apical position of the micronemes, and immunoelectron microscopy provided definitive localisation of the protein to these secretory organelles. Further analysis of SnMIC10 in intracellular parasites revealed that expression of this protein is temporally regulated during endopolygeny, supporting the view that micronemes are only needed during host cell invasion. Collectively, the data indicate that SnMIC10 is a microneme protein that is part of the excreted/secreted antigen fraction of S. neurona. Identification and characterisation of additional S. neurona microneme antigens and comparisons to orthologues in other Apicomplexa could provide further insight into the

  2. Proteomic identification of differentially expressed proteins during alfalfa (Medicago sativa L. flower development

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    Lingling Chen

    2016-10-01

    Full Text Available Flower development, pollination, and fertilization are important stages in the sexual reproduction process of plants; they are also critical steps in the control of seed formation and development. During alfalfa (Medicago sativa L. seed production, some distinct phenomena such as a low seed setting ratio, serious flower falling, and seed abortion commonly occur. However, the causes of these phenomena are complicated and largely unknown. An understanding of the mechanisms that regulate alfalfa flowering is important in order to increase seed yield. Hence, proteomic technology was used to analyze changes in protein expression during the stages of alfalfa flower development. Flower samples were collected at pre-pollination (S1, pollination (S2, and the post-pollination senescence period (S3. Twenty-four differentially expressed proteins were successfully identified, including 17 down-regulated in pollinated flowers, one up-regulated in pollinated and senesced flowers, and six up-regulated in senesced flowers. The largest proportions of the identified proteins were involved in metabolism, signal transduction, defense response, oxidation reduction, cell death, and programmed cell death (PCD. Their expression profiles demonstrated that energy metabolism, carbohydrate metabolism, and amino acid metabolism provided the nutrient foundation for pollination in alfalfa. Furthermore, there were three proteins involved in multiple metabolic pathways: dual specificity kinase splA-like protein (kinase splALs, carbonic anhydrase (CA, and NADPH: quinone oxidoreductase-like protein (NQOLs. Expression patterns of these proteins indicated that MAPK cascades regulated multiple processes, such as signal transduction, stress response, and cell death. PCD also played an important role in the alfalfa flower developmental process, and regulated both pollination and flower senescence. The current study sheds some light on protein expression profiles during alfalfa flower

  3. Characterization of Three Different Unusual S-Layer Proteins from Viridibacillus arvi JG-B58 That Exhibits Two Super-Imposed S-Layer Proteins

    Science.gov (United States)

    Günther, Tobias J.; Raff, Johannes; Pollmann, Katrin

    2016-01-01

    Genomic analyses of Viridibacillus arvi JG-B58 that was previously isolated from heavy metal contaminated environment identified three different putative surface layer (S-layer) protein genes namely slp1, slp2, and slp3. All three genes are expressed during cultivation. At least two of the V. arvi JG-B58 S-layer proteins were visualized on the surface of living cells via atomic force microscopy (AFM). These S-layer proteins form a double layer with p4 symmetry. The S-layer proteins were isolated from the cells using two different methods. Purified S-layer proteins were recrystallized on SiO2 substrates in order to study the structure of the arrays and self-assembling properties. The primary structure of all examined S-layer proteins lack some features that are typical for Bacillus or Lysinibacillus S-layers. For example, they possess no SLH domains that are usually responsible for the anchoring of the proteins to the cell wall. Further, the pI values are relatively high ranging from 7.84 to 9.25 for the matured proteins. Such features are typical for S-layer proteins of Lactobacillus species although sequence comparisons indicate a close relationship to S-layer proteins of Lysinibacillus and Bacillus strains. In comparison to the numerous descriptions of S-layers, there are only a few studies reporting the concomitant existence of two different S-layer proteins on cell surfaces. Together with the genomic data, this is the first description of a novel type of S-layer proteins showing features of Lactobacillus as well as of Bacillus-type S-layer proteins and the first study of the cell envelope of Viridibacillus arvi. PMID:27285458

  4. Use of genomics to identify bacterial undecaprenyl pyrophosphate synthetase: cloning, expression, and characterization of the essential uppS gene.

    Science.gov (United States)

    Apfel, C M; Takács, B; Fountoulakis, M; Stieger, M; Keck, W

    1999-01-01

    The prenyltransferase undecaprenyl pyrophosphate synthetase (di-trans,poly-cis-decaprenylcistransferase; EC 2.5.1.31) was purified from the soluble fraction of Escherichia coli by TSK-DEAE, ceramic hydroxyapatite, TSK-ether, Superdex 200, and heparin-Actigel chromatography. The protein was labeled with the photolabile analogue of the farnesyl pyrophosphate analogue (E, E)-[1-3H]-(2-diazo-3-trifluoropropionyloxy)geranyl diphosphate and was detected on a sodium dodecyl sulfate-polyacrylamide gel as a protein with an apparent molecular mass of 29 kDa. This protein band was cut out from the gel, trypsin digested, and subjected to matrix-assisted laser desorption ionization mass spectrometric analysis. Comparison of the experimental data with computer-simulated trypsin digest data for all E. coli proteins yielded a single match with a protein of unassigned function (SWISS-PROT Q47675; YAES_ECOLI). Sequences with strong similarity indicative of homology to this protein were identified in 25 bacterial species, in Saccharomyces cerevisiae, and in Caenorhabditis elegans. The homologous genes (uppS) were cloned from E. coli, Haemophilus influenzae, and Streptococcus pneumoniae, expressed in E. coli as amino-terminal His-tagged fusion proteins, and purified over a Ni2+ affinity column. An untagged version of the E. coli uppS gene was also cloned and expressed, and the protein purified in two chromatographic steps. We were able to detect Upp synthetase activity for all purified enzymes. Further, biochemical characterization revealed no differences between the recombinant untagged E. coli Upp synthetase and the three His-tagged fusion proteins. All enzymes were absolutely Triton X-100 and MgCl2 dependent. With the use of a regulatable gene disruption system, we demonstrated that uppS is essential for growth in S. pneumoniae R6.

  5. Expression Levels and Localizations of DVL3 and sFRP3 in Glioblastoma

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    Anja Kafka

    2017-01-01

    Full Text Available The expression patterns of critical molecular components of Wnt signaling, sFRP3 and DVL3, were investigated in glioblastoma, the most aggressive form of primary brain tumors, with the aim to offer potential biomarkers. The protein expression levels and localizations in tumor tissue were revealed by immunohistochemistry and evaluated by the semiquantitative method and immunoreactivity score. Majority of glioblastomas had moderate expression levels for both DVL3 (52.4% and sFRP3 (52.3%. Strong expression levels were observed in 23.1% and 36.0% of samples, respectively. DVL3 was localized in cytoplasm in 97% of glioblastomas, of which 44% coexpressed the protein in the nucleus. sFRP3 subcellular distribution showed that it was localized in the cytoplasm in 94% of cases. Colocalization in the cytoplasm and nucleus was observed in 50% of samples. Wilcox test indicated that the domination of the strong signal is in connection with simultaneous localization of DVL3 protein in the cytoplasm and the nucleus. Patients with strong expression of DVL3 will significantly more often have the protein in the nucleus (P=6.33×10−5. No significant correlation between the two proteins was established, nor were their signal strengths correlated with epidemiological parameters. Our study contributes to better understanding of glioblastoma molecular profile.

  6. Increased Levels of S100A8/A9 in Patients with Peritonsillar Abscess: A New Promising Diagnostic Marker to Differentiate between Peritonsillar Abscess and Peritonsillitis.

    Science.gov (United States)

    Spiekermann, Christoph; Russo, Antonella; Stenner, Markus; Rudack, Claudia; Roth, Johannes; Vogl, Thomas

    2017-01-01

    Peritonsillar abscess (PTA) is a very frequent reason for urgent outpatient consultation and otolaryngological hospital admission. Early, correct diagnosis and therapy of peritonsillar abscess are important to prevent possible life-threatening complications. Based on physical examinations, a reliable differentiation between peritonsillar cellulitis and peritonsillar abscess is restricted. A heterodimeric complex called calprotectin consists of the S100 proteins A8 and A9 (S100A8/A9) and is predominantly expressed not only in monocytes and neutrophils but also in epithelial cells. Due to its release by activated phagocytes at local sites of inflammation, we assumed S100A8/A9 to be a potential biomarker for peritonsillar abscess. We examined serum and saliva of patients with peritonsillitis, acute tonsillitis, peritonsillar abscess, and healthy controls and found significantly increased levels of S100A8/A9 in patients with PTA. Furthermore, we could identify halitosis, trismus, uvula edema, and unilateral swelling of the arched palate to be characteristic symptoms for PTA. Using a combination of these characteristic symptoms and S100A8/A9 levels, we developed a PTA score as an objective and appropriate tool to differentiate between peritonsillitis and peritonsillar abscess with a sensitivity of 92% and specificity of 93%.

  7. Increased Levels of S100A8/A9 in Patients with Peritonsillar Abscess: A New Promising Diagnostic Marker to Differentiate between Peritonsillar Abscess and Peritonsillitis

    Directory of Open Access Journals (Sweden)

    Christoph Spiekermann

    2017-01-01

    Full Text Available Peritonsillar abscess (PTA is a very frequent reason for urgent outpatient consultation and otolaryngological hospital admission. Early, correct diagnosis and therapy of peritonsillar abscess are important to prevent possible life-threatening complications. Based on physical examinations, a reliable differentiation between peritonsillar cellulitis and peritonsillar abscess is restricted. A heterodimeric complex called calprotectin consists of the S100 proteins A8 and A9 (S100A8/A9 and is predominantly expressed not only in monocytes and neutrophils but also in epithelial cells. Due to its release by activated phagocytes at local sites of inflammation, we assumed S100A8/A9 to be a potential biomarker for peritonsillar abscess. We examined serum and saliva of patients with peritonsillitis, acute tonsillitis, peritonsillar abscess, and healthy controls and found significantly increased levels of S100A8/A9 in patients with PTA. Furthermore, we could identify halitosis, trismus, uvula edema, and unilateral swelling of the arched palate to be characteristic symptoms for PTA. Using a combination of these characteristic symptoms and S100A8/A9 levels, we developed a PTA score as an objective and appropriate tool to differentiate between peritonsillitis and peritonsillar abscess with a sensitivity of 92% and specificity of 93%.

  8. Hepatocellular carcinoma-associated mesenchymal stem cells promote hepatocarcinoma progression: role of the S100A4-miR155-SOCS1-MMP9 axis.

    Science.gov (United States)

    Yan, Xin-Long; Jia, Ya-Li; Chen, Lin; Zeng, Quan; Zhou, Jun-Nian; Fu, Chun-Jiang; Chen, Hai-Xu; Yuan, Hong-Feng; Li, Zhi-Wei; Shi, Lei; Xu, Ying-Chen; Wang, Jing-Xue; Zhang, Xiao-Mei; He, Li-Juan; Zhai, Chao; Yue, Wen; Pei, Xue-Tao

    2013-06-01

    Cancer-associated mesenchymal stem cells (MSCs) play a pivotal role in modulating tumor progression. However, the interactions between liver cancer-associated MSCs (LC-MSCs) and hepatocellular carcinoma (HCC) remain unreported. Here, we identified the presence of MSCs in HCC tissues. We also showed that LC-MSCs significantly enhanced tumor growth in vivo and promoted tumor sphere formation in vitro. LC-MSCs also promoted HCC metastasis in an orthotopic liver transplantation model. Complementary DNA (cDNA) microarray analysis showed that S100A4 expression was significantly higher in LC-MSCs compared with liver normal MSCs (LN-MSCs) from adjacent cancer-free tissues. Importantly, the inhibition of S100A4 led to a reduction of proliferation and invasion of HCC cells, while exogenous S100A4 expression in HCC cells resulted in heavier tumors and more metastasis sites. Our results indicate that S100A4 secreted from LC-MSCs can promote HCC cell proliferation and invasion. We then found the expression of oncogenic microRNA (miR)-155 in HCC cells was significantly up-regulated by coculture with LC-MSCs and by S100A4 ectopic overexpression. The invasion-promoting effects of S100A4 were significantly attenuated by a miR-155 inhibitor. These results suggest that S100A4 exerts its effects through the regulation of miR-155 expression in HCC cells. We demonstrate that S100A4 secreted from LC-MSCs promotes the expression of miR-155, which mediates the down-regulation of suppressor of cytokine signaling 1, leading to the subsequent activation of STAT3 signaling. This promotes the expression of matrix metalloproteinases 9, which results in increased tumor invasiveness. S100A4 secreted from LC-MSCs is involved in the modulation of HCC progression, and may be a potential therapeutic target. (HEPATOLOGY 2013). Copyright © 2013 American Association for the Study of Liver Diseases.

  9. Proteins Differentially Expressed in the Pancreas of Hepatic Alcohol Dehydrogenase-Deficient Deer Mice Fed Ethanol For 3 Months.

    Science.gov (United States)

    Bhopale, Kamlesh K; Amer, Samir M; Kaphalia, Lata; Soman, Kizhake V; Wiktorowicz, John E; Shakeel Ansari, Ghulam A; Kaphalia, Bhupendra S

    2017-07-01

    The aim of this study was to identify differentially expressed proteins in the pancreatic tissue of hepatic alcohol dehydrogenase-deficient deer mice fed ethanol to understand metabolic basis and mechanism of alcoholic chronic pancreatitis. Mice were fed liquid diet containing 3.5 g% ethanol daily for 3 months, and differentially expressed pancreatic proteins were identified by protein separation using 2-dimensional gel electrophoresis and identification by mass spectrometry. Nineteen differentially expressed proteins were identified by applying criteria established for protein identification in proteomics. An increased abundance was found for ribosome-binding protein 1, 60S ribosomal protein L31-like isoform 1, histone 4, calcium, and adenosine triphosphate (ATP) binding proteins and the proteins involved in antiapoptotic processes and endoplasmic reticulum function, stress, and/or homeostasis. Low abundance was found for endoA cytokeratin, 40S ribosomal protein SA, amylase 2b isoform precursor, serum albumin, and ATP synthase subunit β and the proteins involved in cell motility, structure, and conformation. Chronic ethanol feeding in alcohol dehydrogenase-deficient deer mice differentially expresses pancreatic functional and structural proteins, which can be used to develop biomarker(s) of alcoholic chronic pancreatitis, particularly amylase 2b precursor, and 60 kDa heat shock protein and those involved in ATP synthesis and blood osmotic pressure.

  10. Protein stability: a crystallographer’s perspective

    International Nuclear Information System (INIS)

    Deller, Marc C.; Kong, Leopold; Rupp, Bernhard

    2016-01-01

    An understanding of protein stability is essential for optimizing the expression, purification and crystallization of proteins. In this review, discussion will focus on factors affecting protein stability on a somewhat practical level, particularly from the view of a protein crystallographer. Protein stability is a topic of major interest for the biotechnology, pharmaceutical and food industries, in addition to being a daily consideration for academic researchers studying proteins. An understanding of protein stability is essential for optimizing the expression, purification, formulation, storage and structural studies of proteins. In this review, discussion will focus on factors affecting protein stability, on a somewhat practical level, particularly from the view of a protein crystallographer. The differences between protein conformational stability and protein compositional stability will be discussed, along with a brief introduction to key methods useful for analyzing protein stability. Finally, tactics for addressing protein-stability issues during protein expression, purification and crystallization will be discussed

  11. Protein stability: a crystallographer’s perspective

    Energy Technology Data Exchange (ETDEWEB)

    Deller, Marc C., E-mail: mdeller@stanford.edu [Stanford University, Shriram Center, 443 Via Ortega, Room 097, MC5082, Stanford, CA 94305-4125 (United States); Kong, Leopold [National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH), Building 8, Room 1A03, 8 Center Drive, Bethesda, MD 20814 (United States); Rupp, Bernhard [k.-k. Hofkristallamt, 91 Audrey Place, Vista, CA 92084 (United States); Medical University of Innsbruck, Schöpfstrasse 41, A-6020 Innsbruck (Austria)

    2016-01-26

    An understanding of protein stability is essential for optimizing the expression, purification and crystallization of proteins. In this review, discussion will focus on factors affecting protein stability on a somewhat practical level, particularly from the view of a protein crystallographer. Protein stability is a topic of major interest for the biotechnology, pharmaceutical and food industries, in addition to being a daily consideration for academic researchers studying proteins. An understanding of protein stability is essential for optimizing the expression, purification, formulation, storage and structural studies of proteins. In this review, discussion will focus on factors affecting protein stability, on a somewhat practical level, particularly from the view of a protein crystallographer. The differences between protein conformational stability and protein compositional stability will be discussed, along with a brief introduction to key methods useful for analyzing protein stability. Finally, tactics for addressing protein-stability issues during protein expression, purification and crystallization will be discussed.

  12. Changes in Hepatic TRβ Protein Expression, Lipogenic Gene Expression, and Long-Chain Acylcarnitine Levels During Chronic Hyperthyroidism and Triiodothyronine Withdrawal in a Mouse Model.

    Science.gov (United States)

    Ohba, Kenji; Sinha, Rohit Anthony; Singh, Brijesh Kumar; Iannucci, Liliana Felicia; Zhou, Jin; Kovalik, Jean-Paul; Liao, Xiao-Hui; Refetoff, Samuel; Sng, Judy Chia Ghee; Leow, Melvin Khee-Shing; Yen, Paul Michael

    2017-06-01

    Thyroid hormone (TH) has important roles in regulating hepatic metabolism. It was previously reported that most hepatic genes activated by a single triiodothyronine (T3) injection became desensitized after multiple injections, and that approximately 10% of target genes did not return to basal expression levels after T3 withdrawal, despite normalization of serum TH and thyrotropin (TSH) levels. To determine the possible mechanism(s) for desensitization and incomplete recovery of hepatic target gene transcription and their effects on metabolism, mRNA and/or protein expression levels of key regulators of TH action were measured, as well as metabolomic changes after chronic T3 treatment and withdrawal. Adult male mice were treated with daily injections of T3 (20 μg/100 g body weight) for 14 days followed by the cessation of T3 for 10 days. Livers were harvested at 6 hours, 24 hours, and 14 days after the first T3 injection, and at 10 days after withdrawal, and then analyzed by quantitative reverse transcription polymerase chain reaction, Western blotting, and metabolomics. Although TH receptor (TRα and TRβ) mRNAs decreased slightly after chronic T3 treatment, only TRβ protein decreased before returning to basal expression level after withdrawal. The expression of other regulators of TH action was unchanged. TRβ protein expression was also decreased in adult male monocarboxylate transporter-8 (Mct8)-knockout mice, an in vivo model of chronic intrahepatic hyperthyroidism. Previously, increased hepatic long-chain acylcarnitine levels were found after acute TH treatment. However, in this study, long-chain acylcarnitine levels were unchanged after chronic T3, and paradoxically increased after T3 withdrawal. Pathway analyses of the previous microarray results showed upregulation of lipogenic genes after acute T3 treatment and withdrawal. Phosphorylation of acetyl-CoA carboxylase also decreased after T3 withdrawal. Decreased hepatic TRβ protein expression occurred

  13. Recombinant Expression Screening of P. aeruginosa Bacterial Inner Membrane Proteins

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    Jeffery Constance J

    2010-11-01

    Full Text Available Abstract Background Transmembrane proteins (TM proteins make up 25% of all proteins and play key roles in many diseases and normal physiological processes. However, much less is known about their structures and molecular mechanisms than for soluble proteins. Problems in expression, solubilization, purification, and crystallization cause bottlenecks in the characterization of TM proteins. This project addressed the need for improved methods for obtaining sufficient amounts of TM proteins for determining their structures and molecular mechanisms. Results Plasmid clones were obtained that encode eighty-seven transmembrane proteins with varying physical characteristics, for example, the number of predicted transmembrane helices, molecular weight, and grand average hydrophobicity (GRAVY. All the target proteins were from P. aeruginosa, a gram negative bacterial opportunistic pathogen that causes serious lung infections in people with cystic fibrosis. The relative expression levels of the transmembrane proteins were measured under several culture growth conditions. The use of E. coli strains, a T7 promoter, and a 6-histidine C-terminal affinity tag resulted in the expression of 61 out of 87 test proteins (70%. In this study, proteins with a higher grand average hydrophobicity and more transmembrane helices were expressed less well than less hydrophobic proteins with fewer transmembrane helices. Conclusions In this study, factors related to overall hydrophobicity and the number of predicted transmembrane helices correlated with the relative expression levels of the target proteins. Identifying physical characteristics that correlate with protein expression might aid in selecting the "low hanging fruit", or proteins that can be expressed to sufficient levels using an E. coli expression system. The use of other expression strategies or host species might be needed for sufficient levels of expression of transmembrane proteins with other physical

  14. Prokaryotic expression of chicken interferon-γ fusion protein and its effect on expression of poultry heat shock protein 70 under heat stress.

    Science.gov (United States)

    Sun, Jinhua; Chen, Yinglin; Qin, Feiyue; Guan, Xueting; Xu, Wei; Xu, Liangmei

    2017-06-01

    Interferons have attracted considerable attention due to their vital roles in the host immune response and low induction of antibiotic resistance. In this study, total RNA was extracted from spleen cells of chicken embryos inoculated with Newcastle disease vaccine, and the full-length chicken interferon-γ (ChIFN-γ) gene was amplified by RT-PCR. The full complementary DNA sequence of the ChIFN-γ gene was 495 bp long and was cloned into the prokaryotic expression vector pProEX™HT b . The plasmid was transformed into Escherichia coli DH5α and the expression of ChIFN-γ was induced by isopropyl β-D-1-thiogalactopyranoside. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis and Western blot results showed the expressed fusion protein had a molecular weight of approximately 18 kDa and was recognized by an anti-His mAb. Moreover, ChIFN-γ was found to demonstrate anti-viral activity in vitro. To test the in vivo function of ChIFN-γ in broilers under heat stress, a total of 100 broilers were randomly assigned to either a control group or a treated group, in which they were hypodermically injected with recombinant ChIFN-γ. Results demonstrated ChIFN-γ affects the messenger RNA expression levels of heat shock protein 70 (HSP70) in the heart and lung tissues, and decreases the concentration of HSP70 in serum. Therefore, we conclude recombinant ChIFN-γ can reduce heat stress to some extent in vivo. © 2016 Japanese Society of Animal Science.

  15. Heme oxygenase-1 and abscisic acid effects MAPK´s gene expression in soybean seeds

    International Nuclear Information System (INIS)

    Giacometti, R.; Santa Cruz, D.; Noriega, G.; Balestrasse, K.

    2012-01-01

    In soybean previous studies enabled the identification of MAPK3 and 6 whose activity is enhanced within the signaling pathway leading to defense reactions. In this study the effects of different compounds related to hemeoxygenase (HO-1) biosynthesis on mitogen-activated protein kinase (MAPK’s) genes expression in soybean seeds were tested. To this end, 20μM hemine, 22μM ZnPPIX, 0.5mM furidine or 100μM 8-bromoguanosine 3',5'-cyclic monophosphate (8Br) were added to pre-hydrated seeds for 5 days. MAPK’s genes expression was enhanced in seeds treated with hemine. This result indicates that heme catabolism could be involved in the signaling mediated by this cascade pathway. To confirm this hypothesis experiments were carried out in the precsence of ZnPPIX, a potent irreversible HO-1 inhibitor. In this case, no gene induction was observed. On the other hand, 8Br, a cGMP analog, induced HO-1 gene expression but did not modulate MAPK’s, indicating that this effect could not be mediated by cGMP. When the action of furidine, an abscisic acid inhibitor, was tested a diminution of HO-1 gene expression was observed. In this regard, MAPK’s showed a different response, being MAPK6 the only transcript that showed a diminished respect to controls, while MAPK3 mRNA as well as MAPKK1 was enhanced. These results were confirmed by western blotting and activity determinations. (authors)

  16. Molecular characterization and expression analysis of three homoeologous Ta14S genes encoding 14-3-3 proteins in wheat (Triticum aestivum L.

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    Xinguo Wang

    2016-06-01

    Full Text Available The purpose of this study was to characterize Ta14S homoeologs and assess their functions in wheat seed development. The genomic and cDNA sequences of three Ta14S homoeologous genes encoding 14-3-3 proteins were isolated. Sequence analysis revealed that the three homoeologs consisted of five exons and four introns and were very highly conserved in the coding regions and in exon/intron structure, whereas the cDNA sequences were variable in the 5′ and 3′-UTR. The three genes, designated as Ta14S-2A, Ta14S-2B and Ta14S-2D, were located in homoeologous group 2 chromosomes. The polypeptide chains of the three Ta14S genes were highly similar. These genes were most homologous to Hv14A from barley. Real-time quantitative PCR indicated that the three Ta14S genes were differentially expressed in different organs at different developmental stages and all exhibited greater expression in primary roots of 1-day-old germlings than in other tissues. Comparison of the expression patterns of the three homoeologous genes at different times after pollination also revealed that their expression was developmentally regulated. The transcription of Ta14S-2B was clearly higher during seed germination, whereas expressions of Ta14S-2A and Ta14S-2D were up-regulated at the beginning of seed imbibition (0–12 h, but declined thereafter. The results suggested that the three Ta14S homoeologous genes have regulatory roles in seed development and germination.

  17. Expression of transcellular and paracellular calcium and magnesium transport proteins in renal and intestinal epithelia during lactation.

    Science.gov (United States)

    Beggs, Megan R; Appel, Ida; Svenningsen, Per; Skjødt, Karsten; Alexander, R Todd; Dimke, Henrik

    2017-09-01

    Significant alterations in maternal calcium (Ca 2+ ) and magnesium (Mg 2+ ) balance occur during lactation. Ca 2+ is the primary divalent cation mobilized into breast milk by demineralization of the skeleton and alterations in intestinal and renal Ca 2+ transport. Mg 2+ is also concentrated in breast milk, but the underlying mechanisms are not well understood. To determine the molecular alterations in Ca 2+ and Mg 2+ transport in the intestine and kidney during lactation, three groups of female mice consisting of either nonpregnant controls, lactating mice, or mice undergoing involution were examined. The fractional excretion of Ca 2+ , but not Mg 2+ , rose significantly during lactation. Renal 1-α hydroxylase and 24-OHase mRNA levels increased markedly, as did plasma 1,25 dihydroxyvitamin D levels. This was accompanied by significant increases in intestinal expression of Trpv6 and S100g in lactating mice. However, no alterations in the expression of cation-permeable claudin-2, claudin-12, or claudins-15 were found in the intestine. In the kidney, increased expression of Trpv5 and Calb1 was observed during lactation, while no changes in claudins involved in Ca 2+ and Mg 2+ transport (claudin-2, claudin-14, claudin-16, or claudin-19) were found. Consistent with the mRNA expression, expression of both calbindin-D 28K and transient receptor potential vanilloid 5 (TRPV5) proteins increased. Colonic Trpm6 expression increased during lactation, while renal Trpm6 remained unaltered. In conclusion, proteins involved in transcellular Ca 2+ and Mg 2+ transport pathways increase during lactation, while expression of paracellular transport proteins remained unchanged. Increased fractional Ca 2+ excretion can be explained by vitamin D-dependent intestinal hyperabsorption and bone demineralization, despite enhanced transcellular Ca 2+ uptake by the kidney. Copyright © 2017 the American Physiological Society.

  18. The E4 protein; structure, function and patterns of expression

    Energy Technology Data Exchange (ETDEWEB)

    Doorbar, John, E-mail: jdoorba@nimr.mrc.ac.uk

    2013-10-15

    The papillomavirus E4 open reading frame (ORF) is contained within the E2 ORF, with the primary E4 gene-product (E1{sup ∧}E4) being translated from a spliced mRNA that includes the E1 initiation codon and adjacent sequences. E4 is located centrally within the E2 gene, in a region that encodes the E2 protein′s flexible hinge domain. Although a number of minor E4 transcripts have been reported, it is the product of the abundant E1{sup ∧}E4 mRNA that has been most extensively analysed. During the papillomavirus life cycle, the E1{sup ∧}E4 gene products generally become detectable at the onset of vegetative viral genome amplification as the late stages of infection begin. E4 contributes to genome amplification success and virus synthesis, with its high level of expression suggesting additional roles in virus release and/or transmission. In general, E4 is easily visualised in biopsy material by immunostaining, and can be detected in lesions caused by diverse papillomavirus types, including those of dogs, rabbits and cattle as well as humans. The E4 protein can serve as a biomarker of active virus infection, and in the case of high-risk human types also disease severity. In some cutaneous lesions, E4 can be expressed at higher levels than the virion coat proteins, and can account for as much as 30% of total lesional protein content. The E4 proteins of the Beta, Gamma and Mu HPV types assemble into distinctive cytoplasmic, and sometimes nuclear, inclusion granules. In general, the E4 proteins are expressed before L2 and L1, with their structure and function being modified, first by kinases as the infected cell progresses through the S and G2 cell cycle phases, but also by proteases as the cell exits the cell cycle and undergoes true terminal differentiation. The kinases that regulate E4 also affect other viral proteins simultaneously, and include protein kinase A, Cyclin-dependent kinase, members of the MAP Kinase family and protein kinase C. For HPV16 E1{sup

  19. RAC1 P29S regulates PD-L1 expression in melanoma

    Science.gov (United States)

    Vu, Ha Linh; Rosenbaum, Sheera; Purwin, Timothy J.; Davies, Michael A.; Aplin, Andrew E.

    2015-01-01

    Summary Whole exome sequencing of cutaneous melanoma has led to the detection of P29 mutations in RAC1 in 5–9% of samples, but the role of RAC1 P29 mutations in melanoma biology remains unclear. Using reverse phase protein array analysis to examine the changes in protein/phospho-protein expression, we identified cyclin B1, PD-L1, Ets-1, and Syk as being selectively upregulated with RAC1 P29S expression and downregulated with RAC1 P29S depletion. Using the melanoma patient samples in TCGA, we found PD-L1 expression to be significantly increased in RAC1 P29S patients compared to RAC1 WT as well as other RAC1 mutants. The finding that PD-L1 is upregulated suggests that oncogenic RAC1 P29S may promote suppression of the antitumor immune response. This is a new insight into the biological function of RAC1 P29S mutations with potential clinical implications as PD-L1 is a candidate biomarker for increased benefit from treatment with anti-PD1 or anti-PD-L1 antibodies. PMID:26176707

  20. Using the 2A Protein Coexpression System: Multicistronic 2A Vectors Expressing Gene(s) of Interest and Reporter Proteins.

    Science.gov (United States)

    Luke, Garry A; Ryan, Martin D

    2018-01-01

    To date, a huge range of different proteins-many with cotranslational and posttranslational subcellular localization signals-have been coexpressed together with various reporter proteins in vitro and in vivo using 2A peptides. The pros and cons of 2A co-expression technology are considered below, followed by a simple example of a "how to" protocol to concatenate multiple genes of interest, together with a reporter gene, into a single gene linked via 2As for easy identification or selection of transduced cells.

  1. Elevated expression of ribosomal protein genes L37, RPP-1, and S2 in the presence of mutant p53.

    Science.gov (United States)

    Loging, W T; Reisman, D

    1999-11-01

    The wild-type p53 protein is a DNA-binding transcription factor that activates genes such as p21, MDM2, GADD45, and Bax that are required for the regulation of cell cycle progression or apoptosis in response to DNA damage. Mutant forms of p53, which are transforming oncogenes and are expressed at high levels in tumor cells, generally have a reduced binding affinity for the consensus DNA sequence. Interestingly, some p53 mutants that are no longer effective at binding to the consensus DNA sequence and transactivating promoters containing this target site have acquired the ability to transform cells in culture, in part through their ability to transactivate promoters of a number of genes that are not targets of the wild-type protein. Certain p53 mutants are therefore considered to be gain-of-function mutants and appear to be promoting proliferation or transforming cells through their ability to alter the expression of novel sets of genes. Our goal is to identify genes that have altered expression in the presence of a specific mutant p53 (Arg to Trp mutation at codon 248) protein. Through examining differential gene expression in cells devoid of p53 expression and in cells that express high levels of mutant p53 protein, we have identified three ribosomal protein genes that have elevated expression in response to mutant p53. Consistent with these findings, the overexpression of a number of ribosomal protein genes in human tumors and evidence for their contribution to oncogenic transformation have been reported previously, although the mechanism leading to this overexpression has remained elusive. We show results that indicate that expression of these specific ribosomal protein genes is increased in the presence of the R248W p53 mutant, which provides a mechanism for their overexpression in human tumors.

  2. Multiple growth hormone-binding proteins are expressed on insulin-producing cells

    DEFF Research Database (Denmark)

    Møldrup, A; Billestrup, N; Thorn, N A

    1989-01-01

    The insulin-producing rat islet tumor cell line, RIN-5AH, expresses somatogen binding sites and responds to GH by increased proliferation and insulin production. Affinity cross-linking shows that RIN-5AH cells contain two major GH-binding subunits of Mr 100-130K (110K), which appear to exist as d....... It is concluded that the RIN-5AH cells have multiple GH-binding proteins which may mediate signals for either proliferation and/or insulin production....

  3. Predictable tuning of protein expression in bacteria

    DEFF Research Database (Denmark)

    Bonde, Mads; Pedersen, Margit; Klausen, Michael Schantz

    2016-01-01

    We comprehensively assessed the contribution of the Shine-Dalgarno sequence to protein expression and used the data to develop EMOPEC (Empirical Model and Oligos for Protein Expression Changes; http://emopec.biosustain.dtu.dk). EMOPEC is a free tool that makes it possible to modulate the expressi...

  4. Evolutionary Divergence of Gene and Protein Expression in the Brains of Humans and Chimpanzees.

    Science.gov (United States)

    Bauernfeind, Amy L; Soderblom, Erik J; Turner, Meredith E; Moseley, M Arthur; Ely, John J; Hof, Patrick R; Sherwood, Chet C; Wray, Gregory A; Babbitt, Courtney C

    2015-07-10

    Although transcriptomic profiling has become the standard approach for exploring molecular differences in the primate brain, very little is known about how the expression levels of gene transcripts relate to downstream protein abundance. Moreover, it is unknown whether the relationship changes depending on the brain region or species under investigation. We performed high-throughput transcriptomic (RNA-Seq) and proteomic (liquid chromatography coupled with tandem mass spectrometry) analyses on two regions of the human and chimpanzee brain: The anterior cingulate cortex and caudate nucleus. In both brain regions, we found a lower correlation between mRNA and protein expression levels in humans and chimpanzees than has been reported for other tissues and cell types, suggesting that the brain may engage extensive tissue-specific regulation affecting protein abundance. In both species, only a few categories of biological function exhibited strong correlations between mRNA and protein expression levels. These categories included oxidative metabolism and protein synthesis and modification, indicating that the expression levels of mRNA transcripts supporting these biological functions are more predictive of protein expression compared with other functional categories. More generally, however, the two measures of molecular expression provided strikingly divergent perspectives into differential expression between human and chimpanzee brains: mRNA comparisons revealed significant differences in neuronal communication, ion transport, and regulatory processes, whereas protein comparisons indicated differences in perception and cognition, metabolic processes, and organization of the cytoskeleton. Our results highlight the importance of examining protein expression in evolutionary analyses and call for a more thorough understanding of tissue-specific protein expression levels. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular

  5. Systematic review and meta-analysis of circulating S100B blood levels in schizophrenia.

    Directory of Open Access Journals (Sweden)

    Katina Aleksovska

    Full Text Available S100B is a calcium-binding protein secreted in central nervous system from astrocytes and other glia cells. High blood S100B levels have been linked to brain damage and psychiatric disorders. S100B levels have been reported to be higher in schizophrenics than healthy controls. To quantify the relationship between S100B blood levels and schizophrenia a systematic literature review of case-control studies published on this topic within July 3rd 2014 was carried out using three bibliographic databases: Medline, Scopus and Web of Science. Studies reporting mean and standard deviation of S100B blood levels both in cases and controls were included in the meta-analysis. The meta-Mean Ratio (mMR of S100B blood levels in cases compared to controls was used as a measure of effect along with its 95% Confidence Intervals (CI. 20 studies were included totaling for 994 cases and 785 controls. Schizophrenia patients showed 76% higher S100B blood levels than controls with mMR = 1.76 95% CI: 1.44-2.15. No difference could be found between drug-free patients with mMR = 1.84 95%CI: 1.24-2.74 and patients on antipsychotic medication with mMR = 1.75 95% CI: 1.41-2.16. Similarly, ethnicity and stage of disease didn't affect results. Although S100B could be regarded as a possible biomarker of schizophrenia, limitations should be accounted when interpreting results, especially because of the high heterogeneity that remained >70%, even after carrying out subgroups analyses. These results point out that approaches based on traditional categorical diagnoses may be too restrictive and new approaches based on the characterization of new complex phenotypes should be considered.

  6. Serum S100B levels after meningioma surgery: A comparison of two laboratory assays

    Directory of Open Access Journals (Sweden)

    Weiniger Carolyn F

    2008-09-01

    Full Text Available Abstract Background S100B protein is a potential biomarker of central nervous system insult. This study quantitatively compared two methods for assessing serum concentration of S100B. Methods A prospective, observational study performed in a single tertiary medical center. Included were fifty two consecutive adult patients undergoing surgery for meningioma that provided blood samples for determination of S100B concentrations. Eighty samples (40 pre-operative and 40 postoperative were randomly selected for batch testing. Each sample was divided into two aliquots. These were analyzed by ELISA (Sangtec and a commercial kit (Roche Elecsys® for S100B concentrations. Statistical analysis included regression modelling and Bland-Altman analysis. Results A parsimonious linear model best described the prediction of commercial kit values by those determined by ELISA (y = 0.045 + 0.277*x, x = ELISA value, R2 = 0.732. ELISA measurements tended to be higher than commercial kit measurements. This discrepancy increased linearly with increasing S100B concentrations. At concentrations above 0.7 μg/L the paired measurements were consistently outside the limits of agreement in the Bland-Altman display. Similar to other studies that used alternative measurement methods, sex and age related differences in serum S100B levels were not detected using the Elecsys® (p = 0.643 and 0.728 respectively. Conclusion Although a generally linear relationship exists between serum S100B concentrations measured by ELISA and a commercially available kit, ELISA values tended to be higher than commercial kit measurements particularly at concentrations over 0.7 μg/L, which are suggestive of brain injury. International standardization of commercial kits is required before the predictive validity of S100B for brain damage can be effectively assessed in clinical practice.

  7. Effect of salvia miltiorrhiza and ligustrazine hydrochloride injection combined with hydroxyethyl starch injection on serum BNP, Hcy, MMP-2, S100B protein and hemorheology in patients with acute cerebral watershed infarction

    Directory of Open Access Journals (Sweden)

    Dong Chen

    2017-09-01

    Full Text Available Objective: To study the effect of salvia miltiorrhiza and ligustrazine hydrochloride injection combined with hydroxyethyl starch injection on serum BNP, Hcy, MMP-2, S100B protein and hemorheology in patients with acute cerebral watershed infarction. Methods: A total of 90 patientswith acute cerebral watershed infarction in our hospital from August 2014 to December 2016 were enrolled in this study. The subjects were divided into the control group (n=45 and the treatment group (n=45 randomly. The control group was treated with hydroxyethyl starch injection, the treatment group was treated withsalvia miltiorrhiza and ligustrazine hydrochloride injection combined with hydroxyethyl starch injection, and both the two groups were treated for 2 weeks. The serum BNP, Hcy, MMP-2, S100B protein and hemorheology of the two groups before and after treatments were compared. Results: There were no significantly differences of the serum BNP, Hcy, MMP-2, S100B protein and hemorheology of the two groups before treatment. The serum BNP, Hcy, MMP-2, S100B proteinlevels of the two groups after treatment were significantly lower than before treatment, and that of the treatment group after treatment were significantly lower than the control group. The PV, Lr, Mr, Hr and RE of the two groups after treatment were significantly lower than before treatment, and that of the treatment group after treatment were significantly lower than the control group. Conclusion: Salvia miltiorrhiza and ligustrazine hydrochloride injection combined with hydroxyethyl starch injectioncan significantlyimprovetheneurological function and hemorheology, reduce inflammation of the patients with acute cerebral watershed infarction, and it was worthy clinical application.

  8. Can Melan-A replace S-100 and HMB-45 in the evaluation of sentinel lymph nodes from patients with malignant melanoma?

    Science.gov (United States)

    Kucher, Cynthia; Zhang, Paul J; Acs, Geza; Roberts, Shelley; Xu, Xiaowei

    2006-09-01

    The sentinel lymph node (SLN) biopsy has become an increasingly important procedure used in the primary staging of malignant melanoma. However, micrometastases in a lymph node can be easily missed on routine H&E-stained sections. Therefore, S-100 and HMB-45 IHC stains are standardly performed on grossly negative SLNs for detection of metastatic melanoma. Each of these IHC markers, however, is not ideal. The authors investigated whether the newer IHC marker Melan-A would improve the detection of metastatic melanoma in SLN biopsies. Forty lymph nodes previously diagnosed with metastatic melanoma were retrospectively evaluated for S-100, HMB-45, and Melan-A expression. In addition, 42 SLN biopsies for metastatic melanoma detection were prospectively collected and evaluated for S-100, HMB-45, and Melan-A expression. All lymph nodes with metastatic melanoma from the retrospective study demonstrated S-100 reactivity. Five of the lymph nodes with metastatic melanoma from the retrospective study failed to express either HMB-45 or Melan-A, all of which displayed a desmoplastic morphology. One of the metastases positive for S-100 and HMB-45 failed to show reactivity with Melan-A (3%). The prospective study found 10 lymph nodes from 42 cases to be positive for metastatic melanoma, which were positive for S-100 (100%). Nine of the involved lymph nodes were positive for HMB-45(90%), and nine were positive for Melan-A (90%). Melan-A, although very specific, cannot replace the use of S-100 and HMB-45 for the detection of metastatic melanoma in SLNs. It can, however, substitute for HMB-45 with equally good results.

  9. Protein Expression Analyses at the Single Cell Level

    Directory of Open Access Journals (Sweden)

    Masae Ohno

    2014-09-01

    Full Text Available The central dogma of molecular biology explains how genetic information is converted into its end product, proteins, which are responsible for the phenotypic state of the cell. Along with the protein type, the phenotypic state depends on the protein copy number. Therefore, quantification of the protein expression in a single cell is critical for quantitative characterization of the phenotypic states. Protein expression is typically a dynamic and stochastic phenomenon that cannot be well described by standard experimental methods. As an alternative, fluorescence imaging is being explored for the study of protein expression, because of its high sensitivity and high throughput. Here we review key recent progresses in fluorescence imaging-based methods and discuss their application to proteome analysis at the single cell level.

  10. Comparison of hippocampal G protein activation by 5-HT(1A) receptor agonists and the atypical antipsychotics clozapine and S16924.

    Science.gov (United States)

    Newman-Tancredi, A; Rivet, J-M; Cussac, D; Touzard, M; Chaput, C; Marini, L; Millan, M J

    2003-09-01

    This study employed [(35)S]guanosine 5'- O-(3-thiotriphosphate) ([(35)S]GTPgammaS) binding to compare the actions of antipsychotic agents known to stimulate cloned, human 5-HT(1A) receptors with those of reference agonists at postsynaptic 5-HT(1A) receptors. In rat hippocampal membranes, the following order of efficacy was observed (maximum efficacy, E(max), values relative to 5-HT=100): (+)8-OH-DPAT (85), flesinoxan (62), eltoprazine (60), S14506 (59), S16924 (48), buspirone (41), S15535 (22), clozapine (22), ziprasidone (21), pindolol (7), p-MPPI (0), WAY100,635 (0), spiperone (0). Despite differences in species and tissue source, the efficacy and potency (pEC(50)) of agonists (with the exception of clozapine) correlated well with those determined previously at human 5-HT(1A) receptors expressed in Chinese hamster ovary (CHO) cells. In contrast, clozapine was more potent at hippocampal membranes. The selective antagonists p-MPPI and WAY100,635 abolished stimulation of binding by (+)8-OH-DPAT, clozapine and S16924 (p-MPPI), indicating that these actions were mediated specifically by 5-HT(1A) receptors. Clozapine and S16924 also attenuated 5-HT- and (+)8-OH-DPAT-stimulated [(35)S]GTPgammaS binding, consistent with partial agonist properties. In [(35)S]GTPgammaS autoradiographic studies, 5-HT-induced stimulation, mediated through 5-HT(1A) receptors, was more potent in the septum (pEC(50) approximately 6.5) than in the dentate gyrus of the hippocampus (pEC(50) approximately 5) suggesting potential differences in coupling efficiency or G protein expression. Though clozapine (30 and 100 microM) did not enhance [(35)S]GTPgammaS labelling in any structure, S16924 (10 micro M) modestly increased [(35)S]GTPgammaS labelling in the dentate gyrus. On the other hand, both these antipsychotic agents attenuated 5-HT (10 microM)-stimulated [(35)S]GTPgammaS binding in the dentate gyrus and septum. In conclusion, clozapine, S16924 and ziprasidone act as partial agonists for G

  11. Amyloid protein-mediated differential DNA methylation status regulates gene expression in Alzheimer’s disease model cell line

    International Nuclear Information System (INIS)

    Sung, Hye Youn; Choi, Eun Nam; Ahn Jo, Sangmee; Oh, Seikwan; Ahn, Jung-Hyuck

    2011-01-01

    Highlights: ► Genome-wide DNA methylation pattern in Alzheimer’s disease model cell line. ► Integrated analysis of CpG methylation and mRNA expression profiles. ► Identify three Swedish mutant target genes; CTIF, NXT2 and DDR2 gene. ► The effect of Swedish mutation on alteration of DNA methylation and gene expression. -- Abstract: The Swedish mutation of amyloid precursor protein (APP-sw) has been reported to dramatically increase beta amyloid production through aberrant cleavage at the beta secretase site, causing early-onset Alzheimer’s disease (AD). DNA methylation has been reported to be associated with AD pathogenesis, but the underlying molecular mechanism of APP-sw-mediated epigenetic alterations in AD pathogenesis remains largely unknown. We analyzed genome-wide interplay between promoter CpG DNA methylation and gene expression in an APP-sw-expressing AD model cell line. To identify genes whose expression was regulated by DNA methylation status, we performed integrated analysis of CpG methylation and mRNA expression profiles, and identified three target genes of the APP-sw mutant; hypomethylated CTIF (CBP80/CBP20-dependent translation initiation factor) and NXT2 (nuclear exporting factor 2), and hypermethylated DDR2 (discoidin domain receptor 2). Treatment with the demethylating agent 5-aza-2′-deoxycytidine restored mRNA expression of these three genes, implying methylation-dependent transcriptional regulation. The profound alteration in the methylation status was detected at the −435, −295, and −271 CpG sites of CTIF, and at the −505 to −341 region in the promoter of DDR2. In the promoter region of NXT2, only one CpG site located at −432 was differentially unmethylated in APP-sw cells. Thus, we demonstrated the effect of the APP-sw mutation on alteration of DNA methylation and subsequent gene expression. This epigenetic regulatory mechanism may contribute to the pathogenesis of AD.

  12. Oral Uncaria rhynchophylla (UR) reduces kainic acid-induced epileptic seizures and neuronal death accompanied by attenuating glial cell proliferation and S100B proteins in rats.

    Science.gov (United States)

    Lin, Yi-Wen; Hsieh, Ching-Liang

    2011-05-17

    Epilepsy is a common clinical syndrome with recurrent neuronal discharges in cerebral cortex and hippocampus. Here we aim to determine the protective role of Uncaria rhynchophylla (UR), an herbal drug belong to Traditional Chinese Medicine (TCM), on epileptic rats. To address this issue, we tested the effect of UR on kainic acid (KA)-induced epileptic seizures and further investigate the underlying mechanisms. Oral UR successfully decreased neuronal death and discharges in hippocampal CA1 pyramidal neurons. The population spikes (PSs) were decreased from 4.1 ± 0.4 mV to 2.1 ± 0.3 mV in KA-induced epileptic seizures and UR-treated groups, respectively. Oral UR protected animals from neuronal death induced by KA treatment (from 34 ± 4.6 to 191.7 ± 48.6 neurons/field) through attenuating glial cell proliferation and S100B protein expression but not GABAA and TRPV1 receptors. The above results provide detail mechanisms underlying the neuroprotective action of UR on KA-induced epileptic seizure in hippocampal CA1 neurons. Crown Copyright © 2011. Published by Elsevier Ireland Ltd. All rights reserved.

  13. BIOMARKERS S100B AND NSE PREDICT OUTCOME IN HYPOTHERMIA-TREATED ENCEPHALOPATHIC NEWBORNS

    Science.gov (United States)

    Massaro, An N.; Chang, Taeun; Baumgart, Stephen; McCarter, Robert; Nelson, Karin B.; Glass, Penny

    2014-01-01

    Objective To evaluate if serum S100B protein and neuron specific enolase (NSE) measured during therapeutic hypothermia are predictive of neurodevelopmental outcome at 15 months in children with neonatal encephalopathy (NE). Design Prospective longitudinal cohort study Setting A level IV neonatal intensive care unit in a free-standing children’s hospital. Patients Term newborns with moderate to severe NE referred for therapeutic hypothermia during the study period. Interventions Serum NSE and S100B were measured at 0, 12, 24 and 72 hrs of hypothermia. Measurements and Main Reseults Of the 83 infants were enrolled, fifteen (18%) died in the newborn period. Survivors were evaluated by the Bayley Scales of Infant Development (BSID-II) at 15 months of age. Outcomes were assessed in 49/68 (72%) survivors at a mean age of 15.2±2.7 months. Neurodevelopmental outcome was classified by BSID-II Mental (MDI) and Psychomotor (PDI) Developmental Index scores, reflecting cognitive and motor outcomes respectively. Four-level outcome classifications were defined a priori: normal= MDI/PDI within 1SD (>85), mild= MDI/PDI <1SD (70–85), moderate/severe= MDI/PDI <2SD (<70), or died. Elevated serum S100B and NSE levels measured during hypothermia were associated with increasing outcome severity after controlling for baseline and soceioeconomic characteristics in ordinal regression models. Adjusted odds ratios for cognitive outcome were: S100B 2.5 (95% CI 1.3–4.8) and NSE 2.1 (1.2–3.6); for motor outcome: S100B 2.6 (1.2–5.6) and NSE 2.1 (1.2–3.6). Conclusions Serum S100B and NSE levels in babies with NE are associated with neurodevelopmental outcome at 15 months. These putative biomarkers of brain injury may help direct care during therapeutic hypothermia. PMID:24777302

  14. Differential Protein Expressions in Virus-Infected and Uninfected Trichomonas vaginalis.

    Science.gov (United States)

    He, Ding; Pengtao, Gong; Ju, Yang; Jianhua, Li; He, Li; Guocai, Zhang; Xichen, Zhang

    2017-04-01

    Protozoan viruses may influence the function and pathogenicity of the protozoa. Trichomonas vaginalis is a parasitic protozoan that could contain a double stranded RNA (dsRNA) virus, T. vaginalis virus (TVV). However, there are few reports on the properties of the virus. To further determine variations in protein expression of T. vaginalis , we detected 2 strains of T. vaginalis ; the virus-infected (V + ) and uninfected (V - ) isolates to examine differentially expressed proteins upon TVV infection. Using a stable isotope N-terminal labeling strategy (iTRAQ) on soluble fractions to analyze proteomes, we identified 293 proteins, of which 50 were altered in V + compared with V - isolates. The results showed that the expression of 29 proteins was increased, and 21 proteins decreased in V + isolates. These differentially expressed proteins can be classified into 4 categories: ribosomal proteins, metabolic enzymes, heat shock proteins, and putative uncharacterized proteins. Quantitative PCR was used to detect 4 metabolic processes proteins: glycogen phosphorylase, malate dehydrogenase, triosephosphate isomerase, and glucose-6-phosphate isomerase, which were differentially expressed in V + and V - isolates. Our findings suggest that mRNA levels of these genes were consistent with protein expression levels. This study was the first which analyzed protein expression variations upon TVV infection. These observations will provide a basis for future studies concerning the possible roles of these proteins in host-parasite interactions.

  15. Marginal and joint distributions of S100, HMB-45, and Melan-A across a large series of cutaneous melanomas.

    Science.gov (United States)

    Viray, Hollis; Bradley, William R; Schalper, Kurt A; Rimm, David L; Gould Rothberg, Bonnie E

    2013-08-01

    The distribution of the standard melanoma antibodies S100, HMB-45, and Melan-A has been extensively studied. Yet, the overlap in their expression is less well characterized. To determine the joint distributions of the classic melanoma markers and to determine if classification according to joint antigen expression has prognostic relevance. S100, HMB-45, and Melan-A were assayed by immunofluorescence-based immunohistochemistry on a large tissue microarray of 212 cutaneous melanoma primary tumors and 341 metastases. Positive expression for each antigen required display of immunoreactivity for at least 25% of melanoma cells. Marginal and joint distributions were determined across all markers. Bivariate associations with established clinicopathologic covariates and melanoma-specific survival analyses were conducted. Of 322 assayable melanomas, 295 (91.6%), 203 (63.0%), and 236 (73.3%) stained with S100, HMB-45, and Melan-A, respectively. Twenty-seven melanomas, representing a diverse set of histopathologic profiles, were S100 negative. Coexpression of all 3 antibodies was observed in 160 melanomas (49.7%). Intensity of endogenous melanin pigment did not confound immunolabeling. Among primary tumors, associations with clinicopathologic parameters revealed a significant relationship only between HMB-45 and microsatellitosis (P = .02). No significant differences among clinicopathologic criteria were observed across the HMB-45/Melan-A joint distribution categories. Neither marginal HMB-45 (P = .56) nor Melan-A (P = .81), or their joint distributions (P = .88), was associated with melanoma-specific survival. Comprehensive characterization of the marginal and joint distributions for S100, HMB-45, and Melan-A across a large series of cutaneous melanomas revealed diversity of expression across this group of antigens. However, these immunohistochemically defined subclasses of melanomas do not significantly differ according to clinicopathologic correlates or outcome.

  16. S100β-Positive Cells of Mesenchymal Origin Reside in the Anterior Lobe of the Embryonic Pituitary Gland.

    Directory of Open Access Journals (Sweden)

    Kotaro Horiguchi

    Full Text Available The anterior and intermediate lobes of the pituitary gland develop through invagination of the oral ectoderm and as they are endocrine tissues, they participate in the maintenance of vital functions via the synthesis and secretion of numerous hormones. We recently observed that several extrapituitary cells invade the anterior lobe of the developing pituitary gland. This raised the question of the origin(s of these S100β-positive cells, which are not classic endocrine cells but instead comprise a heterogeneous cell population with plural roles, especially as stem/progenitor cells. To better understand the roles of these S100β-positive cells, we performed immunohistochemical analysis using several markers in S100β/GFP-TG rats, which express GFP in S100β-expressing cells under control of the S100β promoter. GFP-positive cells were present as mesenchymal cells surrounding the developing pituitary gland and at Atwell's recess but were not present in the anterior lobe on embryonic day 15.5. These cells were negative for SOX2, a pituitary stem/progenitor marker, and PRRX1, a mesenchyme and pituitary stem/progenitor marker. However, three days later, GFP-positive and PRRX1-positive (but SOX2-negative cells were observed in the parenchyma of the anterior lobe. Furthermore, some GFP-positive cells were positive for vimentin, p75, isolectin B4, DESMIN, and Ki67. These data suggest that S100β-positive cells of extrapituitary origin invade the anterior lobe, undergoing proliferation and diverse transformation during pituitary organogenesis.

  17. S100β-Positive Cells of Mesenchymal Origin Reside in the Anterior Lobe of the Embryonic Pituitary Gland.

    Science.gov (United States)

    Horiguchi, Kotaro; Yako, Hideji; Yoshida, Saishu; Fujiwara, Ken; Tsukada, Takehiro; Kanno, Naoko; Ueharu, Hiroki; Nishihara, Hiroto; Kato, Takako; Yashiro, Takashi; Kato, Yukio

    2016-01-01

    The anterior and intermediate lobes of the pituitary gland develop through invagination of the oral ectoderm and as they are endocrine tissues, they participate in the maintenance of vital functions via the synthesis and secretion of numerous hormones. We recently observed that several extrapituitary cells invade the anterior lobe of the developing pituitary gland. This raised the question of the origin(s) of these S100β-positive cells, which are not classic endocrine cells but instead comprise a heterogeneous cell population with plural roles, especially as stem/progenitor cells. To better understand the roles of these S100β-positive cells, we performed immunohistochemical analysis using several markers in S100β/GFP-TG rats, which express GFP in S100β-expressing cells under control of the S100β promoter. GFP-positive cells were present as mesenchymal cells surrounding the developing pituitary gland and at Atwell's recess but were not present in the anterior lobe on embryonic day 15.5. These cells were negative for SOX2, a pituitary stem/progenitor marker, and PRRX1, a mesenchyme and pituitary stem/progenitor marker. However, three days later, GFP-positive and PRRX1-positive (but SOX2-negative) cells were observed in the parenchyma of the anterior lobe. Furthermore, some GFP-positive cells were positive for vimentin, p75, isolectin B4, DESMIN, and Ki67. These data suggest that S100β-positive cells of extrapituitary origin invade the anterior lobe, undergoing proliferation and diverse transformation during pituitary organogenesis.

  18. Sequence analysis and over-expression of ribosomal protein S28 ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-06-03

    Jun 3, 2009 ... protein S28 gene (RPS28) from the Giant Panda. Wan-ru Hou1* .... Materials. Skeletal muscle was collected from a dead Giant Panda at the Wo- ... synthesis. ... 72°C for 3 min in the first cycle and the anneal temperature decea- ..... laboratory manual 2nd ed., Cold Spring Harbor Laboratory Press, Cold.

  19. A systematic review of the biomarker S100B: implications for sport-related concussion management.

    Science.gov (United States)

    Schulte, Stefanie; Podlog, Leslie W; Hamson-Utley, J Jordan; Strathmann, Frederick G; Strüder, Heiko K

    2014-01-01

    Elevated levels of the astroglial protein S100B have been shown to predict sport-related concussion. However, S100B levels within an athlete can vary depending on the type of physical activity (PA) engaged in and the methodologic approach used to measure them. Thus, appropriate reference values in the diagnosis of concussed athletes remain undefined. The purpose of our systematic literature review was to provide an overview of the current literature examining S100B measurement in the context of PA. The overall goal is to improve the use of the biomarker S100B in the context of sport-related concussion management. PubMed, SciVerse Scopus, SPORTDiscus, CINAHL, and Cochrane. We selected articles that contained (1) research studies focusing exclusively on humans in which (2) either PA was used as an intervention or the test participants or athletes were involved in PA and (3) S100B was measured as a dependent variable. We identified 24 articles. Study variations included the mode of PA used as an intervention, sample types, sample-processing procedures, and analytic techniques. Given the nonuniformity of the analytical methods used and the data samples collected, as well as differences in the types of PA investigated, we were not able to determine a single consistent reference value of S100B in the context of PA. Thus, a clear distinction between a concussed athlete and a healthy athlete based solely on the existing S100B cutoff value of 0.1 μg/L remains unclear. However, because of its high sensitivity and excellent negative predictive value, S100B measurement seems to have the potential to be a diagnostic adjunct for concussion in sports settings. We recommend that the interpretation of S100B values be based on congruent study designs to ensure measurement reliability and validity.

  20. Comparative temporospatial expression profiling of murine amelotin protein during amelogenesis.

    Science.gov (United States)

    Somogyi-Ganss, Eszter; Nakayama, Yohei; Iwasaki, Kengo; Nakano, Yukiko; Stolf, Daiana; McKee, Marc D; Ganss, Bernhard

    2012-01-01

    Tooth enamel is formed in a typical biomineralization process under the guidance of specific organic components. Amelotin (AMTN) is a recently identified, secreted protein that is transcribed predominantly during the maturation stage of enamel formation, but its protein expression profile throughout amelogenesis has not been described in detail. The main objective of this study was to define the spatiotemporal expression profile of AMTN during tooth development in comparison with other known enamel proteins. A peptide antibody against AMTN was raised in rabbits, affinity purified and used for immunohistochemical analyses on sagittal and transverse paraffin sections of decalcified mouse hemimandibles. The localization of AMTN was compared to that of known enamel proteins amelogenin, ameloblastin, enamelin, odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4. Three-dimensional images of AMTN localization in molars at selected ages were reconstructed from serial stained sections, and transmission electron microscopy was used for ultrastructural localization of AMTN. AMTN was detected in ameloblasts of molars in a transient fashion, declining at the time of tooth eruption. Prominent expression in maturation stage ameloblasts of the continuously erupting incisor persisted into adulthood. In contrast, amelogenin, ameloblastin and enamelin were predominantly found during the early secretory stage, while odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4 expression in maturation stage ameloblasts paralleled that of AMTN. Secreted AMTN was detected at the interface between ameloblasts and the mineralized enamel. Recombinant AMTN protein did not mediate cell attachment in vitro. These results suggest a primary role for AMTN in the late stages of enamel mineralization. Copyright © 2011 S. Karger AG, Basel.

  1. A hybrid approach to protein differential expression in mass spectrometry-based proteomics

    KAUST Repository

    Wang, X.

    2012-04-19

    MOTIVATION: Quantitative mass spectrometry-based proteomics involves statistical inference on protein abundance, based on the intensities of each protein\\'s associated spectral peaks. However, typical MS-based proteomics datasets have substantial proportions of missing observations, due at least in part to censoring of low intensities. This complicates intensity-based differential expression analysis. RESULTS: We outline a statistical method for protein differential expression, based on a simple Binomial likelihood. By modeling peak intensities as binary, in terms of \\'presence/absence,\\' we enable the selection of proteins not typically amenable to quantitative analysis; e.g. \\'one-state\\' proteins that are present in one condition but absent in another. In addition, we present an analysis protocol that combines quantitative and presence/absence analysis of a given dataset in a principled way, resulting in a single list of selected proteins with a single-associated false discovery rate. AVAILABILITY: All R code available here: http://www.stat.tamu.edu/~adabney/share/xuan_code.zip.

  2. Development of a reverse genetics system to generate a recombinant Ebola virus Makona expressing a green fluorescent protein

    Energy Technology Data Exchange (ETDEWEB)

    Albariño, César G., E-mail: calbarino@cdc.gov; Wiggleton Guerrero, Lisa; Lo, Michael K.; Nichol, Stuart T.; Towner, Jonathan S.

    2015-10-15

    Previous studies have demonstrated the potential application of reverse genetics technology in studying a broad range of aspects of viral biology, including gene regulation, protein function, cell entry, and pathogenesis. Here, we describe a highly efficient reverse genetics system used to generate recombinant Ebola virus (EBOV) based on a recent isolate from a human patient infected during the 2014–2015 outbreak in Western Africa. We also rescued a recombinant EBOV expressing a fluorescent reporter protein from a cleaved VP40 protein fusion. Using this virus and an inexpensive method to quantitate the expression of the foreign gene, we demonstrate its potential usefulness as a tool for screening antiviral compounds and measuring neutralizing antibodies. - Highlights: • Recombinant Ebola virus (EBOV) derived from Makona variant was rescued. • New protocol for viral rescue allows 100% efficiency. • Modified EBOV expresses a green fluorescent protein from a VP40-fused protein. • Modified EBOV was tested as tool to screen antiviral compounds and measure neutralizing antibodies.

  3. Development of a reverse genetics system to generate a recombinant Ebola virus Makona expressing a green fluorescent protein

    International Nuclear Information System (INIS)

    Albariño, César G.; Wiggleton Guerrero, Lisa; Lo, Michael K.; Nichol, Stuart T.; Towner, Jonathan S.

    2015-01-01

    Previous studies have demonstrated the potential application of reverse genetics technology in studying a broad range of aspects of viral biology, including gene regulation, protein function, cell entry, and pathogenesis. Here, we describe a highly efficient reverse genetics system used to generate recombinant Ebola virus (EBOV) based on a recent isolate from a human patient infected during the 2014–2015 outbreak in Western Africa. We also rescued a recombinant EBOV expressing a fluorescent reporter protein from a cleaved VP40 protein fusion. Using this virus and an inexpensive method to quantitate the expression of the foreign gene, we demonstrate its potential usefulness as a tool for screening antiviral compounds and measuring neutralizing antibodies. - Highlights: • Recombinant Ebola virus (EBOV) derived from Makona variant was rescued. • New protocol for viral rescue allows 100% efficiency. • Modified EBOV expresses a green fluorescent protein from a VP40-fused protein. • Modified EBOV was tested as tool to screen antiviral compounds and measure neutralizing antibodies

  4. Developmental expression of a cell surface protein involved in sea urchin skeleton formation

    International Nuclear Information System (INIS)

    Farach, M.C.; Valdizan, M.; Park, H.R.; Decker, G.L.; Lennarz, W.J.

    1986-01-01

    The authors have previously used a monoclonal antibody (1223) to identify a 130 Kd cell surface protein involved in skeleton formation is sea urchin embryos. In the current study the authors have examined the expression of the 1223 antigen over the course of development of embryos of two species, Strongylocentrotus purpuratus and Lytechinus pictus. The 130 Kd protein is detected in S. purp eggs on immunoblots. Labeling with [ 3 H] leucine and immunoaffinity chromatography show that it also is synthesized shortly after fertilization. Immunofluroescence reveals that at this early stage the 1223 antigen is uniformly distributed on all of the cells. Synthesis decreases to a minimum by the time of hatching (18 h), as does the total amount of antigen present in the embryo. A second period of synthesis commences at the mesenchyme blastula stage, when the spicule-forming primary mesenchyme cells (PMCs) have appeared. During this later stage, synthesis and cell surface expression are restricted to the PMCs. In contrast to S. purp., in L. pictus the 130 Kd protein does not appear until the PMCs are formed. Hybrid embryos demonstrate a pattern of expression of the maternal species. These results suggest that early expression of 1223 antigen in S. purp. is due to utilization of maternal transcripts present in the egg. In both species later expression in PMCs appears to be the result of cell-type specific synthesis, perhaps encoded by embryonic transcripts

  5. Expression of multidrug resistance proteins in retinoblastoma

    Directory of Open Access Journals (Sweden)

    Swati Shukla

    2017-11-01

    Full Text Available AIM: To elucidate the mechanism of multidrug resistance in retinoblastoma, and to acquire more insights into in vivo drug resistance. METHODS: Three anticancer drug resistant Y79 human RB cells were generated against vincristine, etoposide or carboplatin, which are used for conventional chemotherapy in RB. Primary cultures from enucleated eyes after chemotherapy (PCNC were also prepared. Their chemosensitivity to chemotherapeutic agents (vincristine, etoposide and carboplatin were measured using MTT assay. Western blot analysis was performed to evaluate the expression of p53, Bcl-2 and various multidrug resistant proteins in retinoblastoma cells. RESULTS: Following exposure to chemotherapeutic drugs, PCNC showed less sensitivity to drugs. No significant changes observed in the p53 expression, whereas Bcl-2 expression was found to be increased in the drug resistant cells as well as in PCNC. Increased expression of P-glycoprotein (P-gp was observed in drug resistant Y79 cells; however there was no significant change in the expression of P-gp found between primary cultures of primarily enucleated eyes and PCNC. Multidrug resistance protein 1 (Mrp-1 expression was found to be elevated in the drug resistant Y79 cells as well as in PCNC. No significant change in the expression of lung resistance associated protein (Lrp was observed in the drug resistant Y79 cells as well as in PCNC. CONCLUSION: Our results suggest that multidrug resistant proteins are intrinsically present in retinoblastoma which causes treatment failure in managing retinoblastoma with chemotherapy.

  6. Expression of multidrug resistance proteins in retinoblastoma.

    Science.gov (United States)

    Shukla, Swati; Srivastava, Arpna; Kumar, Sunil; Singh, Usha; Goswami, Sandeep; Chawla, Bhavna; Bajaj, Mandeep Singh; Kashyap, Seema; Kaur, Jasbir

    2017-01-01

    To elucidate the mechanism of multidrug resistance in retinoblastoma, and to acquire more insights into in vivo drug resistance. Three anticancer drug resistant Y79 human RB cells were generated against vincristine, etoposide or carboplatin, which are used for conventional chemotherapy in RB. Primary cultures from enucleated eyes after chemotherapy (PCNC) were also prepared. Their chemosensitivity to chemotherapeutic agents (vincristine, etoposide and carboplatin) were measured using MTT assay. Western blot analysis was performed to evaluate the expression of p53, Bcl-2 and various multidrug resistant proteins in retinoblastoma cells. Following exposure to chemotherapeutic drugs, PCNC showed less sensitivity to drugs. No significant changes observed in the p53 expression, whereas Bcl-2 expression was found to be increased in the drug resistant cells as well as in PCNC. Increased expression of P-glycoprotein (P-gp) was observed in drug resistant Y79 cells; however there was no significant change in the expression of P-gp found between primary cultures of primarily enucleated eyes and PCNC. Multidrug resistance protein 1 (Mrp-1) expression was found to be elevated in the drug resistant Y79 cells as well as in PCNC. No significant change in the expression of lung resistance associated protein (Lrp) was observed in the drug resistant Y79 cells as well as in PCNC. Our results suggest that multidrug resistant proteins are intrinsically present in retinoblastoma which causes treatment failure in managing retinoblastoma with chemotherapy.

  7. Expression of multidrug resistance proteins in retinoblastoma

    Science.gov (United States)

    Shukla, Swati; Srivastava, Arpna; Kumar, Sunil; Singh, Usha; Goswami, Sandeep; Chawla, Bhavna; Bajaj, Mandeep Singh; Kashyap, Seema; Kaur, Jasbir

    2017-01-01

    AIM To elucidate the mechanism of multidrug resistance in retinoblastoma, and to acquire more insights into in vivo drug resistance. METHODS Three anticancer drug resistant Y79 human RB cells were generated against vincristine, etoposide or carboplatin, which are used for conventional chemotherapy in RB. Primary cultures from enucleated eyes after chemotherapy (PCNC) were also prepared. Their chemosensitivity to chemotherapeutic agents (vincristine, etoposide and carboplatin) were measured using MTT assay. Western blot analysis was performed to evaluate the expression of p53, Bcl-2 and various multidrug resistant proteins in retinoblastoma cells. RESULTS Following exposure to chemotherapeutic drugs, PCNC showed less sensitivity to drugs. No significant changes observed in the p53 expression, whereas Bcl-2 expression was found to be increased in the drug resistant cells as well as in PCNC. Increased expression of P-glycoprotein (P-gp) was observed in drug resistant Y79 cells; however there was no significant change in the expression of P-gp found between primary cultures of primarily enucleated eyes and PCNC. Multidrug resistance protein 1 (Mrp-1) expression was found to be elevated in the drug resistant Y79 cells as well as in PCNC. No significant change in the expression of lung resistance associated protein (Lrp) was observed in the drug resistant Y79 cells as well as in PCNC. CONCLUSION Our results suggest that multidrug resistant proteins are intrinsically present in retinoblastoma which causes treatment failure in managing retinoblastoma with chemotherapy. PMID:29181307

  8. Evidence for the association of the S100beta gene with low cognitive performance and dementia in the elderly

    DEFF Research Database (Denmark)

    Lambert, J-C; Ferreira, S; Gussekloo, J

    2007-01-01

    independent populations. Moreover, we detected a significant association of this SNP with increased risk of developing dementia or Alzheimer's disease (AD) in six independent populations, especially in women and in the oldest. Furthermore, we characterised a new primate-specific exon within intron 2 (the...... corresponding mRNA isoform was called S100beta2). S100beta2 expression was increased in AD brain compared with controls, and the rs2300403 SNP was associated with elevated levels of S100beta2 mRNA in AD brains, especially in women. Therefore, this genetic variant in S100beta increases the risk of low cognitive...

  9. Nucleic acid programmable protein array a just-in-time multiplexed protein expression and purification platform.

    Science.gov (United States)

    Qiu, Ji; LaBaer, Joshua

    2011-01-01

    Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. 2D proteome analysis initiates new Insights on the Salmonella Typhimurium LuxS protein

    Directory of Open Access Journals (Sweden)

    Vanderleyden Jos

    2009-09-01

    Full Text Available Abstract Background Quorum sensing is a term describing a bacterial communication system mediated by the production and recognition of small signaling molecules. The LuxS enzyme, catalyzing the synthesis of AI-2, is conserved in a wide diversity of bacteria. AI-2 has therefore been suggested as an interspecies quorum sensing signal. To investigate the role of endogenous AI-2 in protein expression of the Gram-negative pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium, we performed a 2D-DIGE proteomics experiment comparing total protein extract of wildtype S. Typhimurium with that of a luxS mutant, unable to produce AI-2. Results Differential proteome analysis of wildtype S. Typhimurium versus a luxS mutant revealed relatively few changes beyond the known effect on phase 2 flagellin. However, two highly differentially expressed protein spots with similar molecular weight but differing isoelectric point, were identified as LuxS whereas the S. Typhimurium genome contains only one luxS gene. This observation was further explored and we show that the S. Typhimurium LuxS protein can undergo posttranslational modification at a catalytic cysteine residue. Additionally, by constructing LuxS-βla and LuxS-PhoA fusion proteins, we demonstrate that S. Typhimurium LuxS can substitute the cognate signal peptide sequences of β-lactamase and alkaline phosphatase for translocation across the cytoplasmic membrane in S. Typhimurium. This was further confirmed by fractionation of S. Typhimurium protein extracts, followed by Western blot analysis. Conclusion 2D-DIGE analysis of a luxS mutant vs. wildtype Salmonella Typhimurium did not reveal new insights into the role of AI-2/LuxS in Salmonella as only a small amount of proteins were differentially expressed. However, subsequent in depth analysis of the LuxS protein itself revealed two interesting features: posttranslational modification and potential translocation across the cytoplasmic membrane. As

  11. Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression

    Directory of Open Access Journals (Sweden)

    Perera Rajika L

    2004-12-01

    Full Text Available Abstract Background In the search for generic expression strategies for mammalian protein families several bacterial expression vectors were examined for their ability to promote high yields of soluble protein. Proteins studied included cell surface receptors (Ephrins and Eph receptors, CD44, kinases (EGFR-cytoplasmic domain, CDK2 and 4, proteases (MMP1, CASP2, signal transduction proteins (GRB2, RAF1, HRAS and transcription factors (GATA2, Fli1, Trp53, Mdm2, JUN, FOS, MAD, MAX. Over 400 experiments were performed where expression of 30 full-length proteins and protein domains were evaluated with 6 different N-terminal and 8 C-terminal fusion partners. Expression of an additional set of 95 mammalian proteins was also performed to test the conclusions of this study. Results Several protein features correlated with soluble protein expression yield including molecular weight and the number of contiguous hydrophobic residues and low complexity regions. There was no relationship between successful expression and protein pI, grand average of hydropathicity (GRAVY, or sub-cellular location. Only small globular cytoplasmic proteins with an average molecular weight of 23 kDa did not require a solubility enhancing tag for high level soluble expression. Thioredoxin (Trx and maltose binding protein (MBP were the best N-terminal protein fusions to promote soluble expression, but MBP was most effective as a C-terminal fusion. 63 of 95 mammalian proteins expressed at soluble levels of greater than 1 mg/l as N-terminal H10-MBP fusions and those that failed possessed, on average, a higher molecular weight and greater number of contiguous hydrophobic amino acids and low complexity regions. Conclusions By analysis of the protein features identified here, this study will help predict which mammalian proteins and domains can be successfully expressed in E. coli as soluble product and also which are best targeted for a eukaryotic expression system. In some cases

  12. Epithelial Cell Damage Activates Bactericidal/Permeability Increasing-Protein (BPI Expression in Intestinal Epithelium

    Directory of Open Access Journals (Sweden)

    Arjun Balakrishnan

    2017-08-01

    Full Text Available As the first line of defense against invading pathogen, intestinal epithelium produces various antimicrobial proteins (AMP that help in clearance of pathogen. Bactericidal/permeability-increasing protein (BPI is a 55 kDa AMP that is expressed in intestinal epithelium. Dysregulation of BPI in intestinal epithelium is associated with various inflammatory diseases like Crohn’s Disease, Ulcerative colitis, and Infectious enteritis’s. In this paper, we report a direct correlation between intestinal damage and BPI expression. In Caco-2 cells, we see a significant increase in BPI levels upon membrane damage mediated by S. aureus infection and pore-forming toxins (Streptolysin and Listeriolysin. Cells detect changes in potassium level as a Danger-associated molecular pattern associated with cell damage and induce BPI expression in a p38 dependent manner. These results are further supported by in vivo findings that the BPI expression in murine intestinal epithelium is induced upon infection with bacteria which cause intestinal damage (Salmonella Typhimurium and Shigella flexneri whereas mutants that do not cause intestinal damage (STM ΔfliC and STM ΔinvC did not induce BPI expression. Our results suggest that epithelial damage associated with infection act as a signal to induce BPI expression.

  13. TRPM4 protein expression in prostate cancer

    DEFF Research Database (Denmark)

    Berg, Kasper Drimer; Soldini, Davide; Jung, Maria

    2016-01-01

    BACKGROUND: Transient receptor potential cation channel, subfamily M, member 4 (TRPM4) messenger RNA (mRNA) has been shown to be upregulated in prostate cancer (PCa) and might be a new promising tissue biomarker. We evaluated TRPM4 protein expression and correlated the expression level.......79-2.62; p = 0.01-0.03 for the two observers) when compared to patients with a lower staining intensity. CONCLUSIONS: TRPM4 protein expression is widely expressed in benign and cancerous prostate tissue, with highest staining intensities found in PCa. Overexpression of TRPM4 in PCa (combination of high...

  14. Protein stability: a crystallographer’s perspective

    Science.gov (United States)

    Deller, Marc C.; Kong, Leopold; Rupp, Bernhard

    2016-01-01

    Protein stability is a topic of major interest for the biotechnology, pharmaceutical and food industries, in addition to being a daily consideration for academic researchers studying proteins. An understanding of protein stability is essential for optimizing the expression, purification, formulation, storage and structural studies of proteins. In this review, discussion will focus on factors affecting protein stability, on a somewhat practical level, particularly from the view of a protein crystallographer. The differences between protein conformational stability and protein compositional stability will be discussed, along with a brief introduction to key methods useful for analyzing protein stability. Finally, tactics for addressing protein-stability issues during protein expression, purification and crystallization will be discussed. PMID:26841758

  15. Response to Infliximab in Crohn’s Disease: Genetic Analysis Supporting Expression Profile

    Directory of Open Access Journals (Sweden)

    Luz María Medrano

    2015-01-01

    Full Text Available Substantial proportion of Crohn’s disease (CD patients shows no response or a limited response to treatment with infliximab (IFX and to identify biomarkers of response would be of great clinical and economic benefit. The expression profile of five genes (S100A8-S100A9, G0S2, TNFAIP6, and IL11 reportedly predicted response to IFX and we aimed at investigating their etiologic role through genetic association analysis. Patients with active CD (350 who received at least three induction doses of IFX were included and classified according to IFX response. A tagging strategy was used to select genetic polymorphisms that cover the variability present in the chromosomal regions encoding the identified genes with altered expression. Following genotyping, differences between responders and nonresponders to IFX were observed in haplotypes of the studied regions: S100A8-S100A9 (rs11205276*G/rs3014866*C/rs724781*C/rs3006488*A; P=0.05; G0S2 (rs4844486*A/rs1473683*T; P=0.15; TNFAIP6 (rs11677200*C/rs2342910*A/rs3755480*G/rs10432475*A; P=0.10; and IL11 (rs1126760*C/rs1042506*G; P=0.07. These differences were amplified in patients with colonic and ileocolonic location for all but the TNFAIP6 haplotype, which evidenced significant difference in ileal CD patients. Our results support the role of the reported expression signature as predictive of anti-TNF outcome in CD patients and suggest an etiological role of those top-five genes in the IFX response pathway.

  16. Geminivirus vectors for high-level expression of foreign proteins in plant cells.

    Science.gov (United States)

    Mor, Tsafrir S; Moon, Yong-Sun; Palmer, Kenneth E; Mason, Hugh S

    2003-02-20

    Bean yellow dwarf virus (BeYDV) is a monopartite geminivirus that can infect dicotyledonous plants. We have developed a high-level expression system that utilizes elements of the replication machinery of this single-stranded DNA virus. The replication initiator protein (Rep) mediates release and replication of a replicon from a DNA construct ("LSL vector") that contains an expression cassette for a gene of interest flanked by cis-acting elements of the virus. We used tobacco NT1 cells and biolistic delivery of plasmid DNA for evaluation of replication and expression of reporter genes contained within an LSL vector. By codelivery of a GUS reporter-LSL vector and a Rep-supplying vector, we obtained up to 40-fold increase in expression levels compared to delivery of the reporter-LSL vectors alone. High-copy replication of the LSL vector was correlated with enhanced expression of GUS. Rep expression using a whole BeYDV clone, a cauliflower mosaic virus 35S promoter driving either genomic rep or an intron-deleted rep gene, or 35S-rep contained in the LSL vector all achieved efficient replication and enhancement of GUS expression. We anticipate that this system can be adapted for use in transgenic plants or plant cell cultures with appropriately regulated expression of Rep, with the potential to greatly increase yield of recombinant proteins. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 430-437, 2003.

  17. Parts Characterization for Tunable Protein Expression

    DEFF Research Database (Denmark)

    Klausen, Michael Schantz; Sommer, Morten Otto Alexander

    2018-01-01

    Flow-seq combines flexible genome engineering methods with flow cytometry-based cell sorting and deep DNA sequencing to enable comprehensive interrogation of genotype to phenotype relationships. One application is to study the effect of specific regulatory elements on protein expression. Construc......Flow-seq combines flexible genome engineering methods with flow cytometry-based cell sorting and deep DNA sequencing to enable comprehensive interrogation of genotype to phenotype relationships. One application is to study the effect of specific regulatory elements on protein expression...

  18. Soluble endothelial protein C receptor (sEPCR) is likely a biomarker of cancer-associated hypercoagulability in human hematologic malignancies

    International Nuclear Information System (INIS)

    Ducros, Elodie; Mirshahi, Shah Soltan; Faussat, Anne-Marie; Mirshahi, Pezhman; Dimicoli, Sophie; Tang, Ruoping; Pardo, Julia; Ibrahim, Jdid; Marie, Jean-Pierre; Therwath, Amu; Soria, Jeannette; Mirshahi, Massoud

    2012-01-01

    Elevated plasma level of soluble endothelial protein C receptor (sEPCR) may be an indicator of thrombotic risk. The present study aims to correlate leukemia-associated hypercoagulability to high level plasma sEPCR and proposes its measurement in routine clinical practice. EPCR expressions in leukemic cell lines were determined by flow cytometry, immunocytochemistry, and reverse transcription polymerase chain reaction (RT-PCR). EPCR gene sequence of a candidate cell line HL-60 was also determined. Plasma samples (n = 76) and bone marrow aspirates (n = 72) from 148 patients with hematologic malignancies and 101 healthy volunteers were analyzed by enzyme-linked immunosorbent assay (ELISA) via a retrospective study for sEPCR and D-dimer. All leukemic cell lines were found to express EPCR. Also, HL-60 EPCR gene sequence showed extensive similarities with the endothelial reference gene. All single nucleotide polymorphisms (SNPs) originally described and some new SNPs were revealed in the promoter and intronic regions. Among these patients 67% had plasma sEPCR level higher than the controls (100 ± 28 ng/mL), wherein 16.3% patients had experienced a previous thrombotic event. These patients were divided into: group-1 (n = 45) with amount of plasmatic sEPCR below 100 ng/mL, group-2 (n = 45) where the concentration of sEPCR was between 100 and 200, and group-3 (n = 20) higher than 200 ng/mL. The numbers of thrombotic incidence recorded in each group were four, six, and eight, respectively. These results reveal that EPCR is expressed not only by a wide range of human malignant hematological cells but also the detection of plasma sEPCR levels provides a powerful insight into thrombotic risk assessment in cancer patients, especially when it surpasses 200 ng/mL

  19. Differential Protein Expression in Congenital and Acquired Cholesteatomas.

    Directory of Open Access Journals (Sweden)

    Seung-Ho Shin

    Full Text Available Congenital cholesteatomas are epithelial lesions that present as an epithelial pearl behind an intact eardrum. Congenital and acquired cholesteatomas progress quite differently from each other and progress patterns can provide clues about the unique origin and pathogenesis of the abnormality. However, the exact pathogenic mechanisms by which cholesteatomas develop remain unknown. In this study, key proteins that directly affect cholesteatoma pathogenesis are investigated with proteomics and immunohistochemistry. Congenital cholesteatoma matrices and retroauricular skin were harvested during surgery in 4 patients diagnosed with a congenital cholesteatoma. Tissue was also harvested from the retraction pocket in an additional 2 patients during middle ear surgery. We performed 2-dimensional (2D electrophoresis to detect and analyze spots that are expressed only in congenital cholesteatoma and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS to separate proteins by molecular weight. Protein expression was confirmed by immunohistochemical staining. The image analysis of 2D electrophoresis showed that 4 congenital cholesteatoma samples had very similar protein expression patterns and that 127 spots were exclusively expressed in congenital cholesteatomas. Of these 127 spots, 10 major spots revealed the presence of titin, forkhead transcription activator homolog (FKH 5-3, plectin 1, keratin 10, and leucine zipper protein 5 by MALDI-TOF/MS analysis. Immunohistochemical staining showed that FKH 5-3 and titin were expressed in congenital cholesteatoma matrices, but not in acquired cholesteatomas. Our study shows that protein expression patterns are completely different in congenital cholesteatomas, acquired cholesteatomas, and skin. Moreover, non-epithelial proteins, including FKH 5-3 and titin, were unexpectedly expressed in congenital cholesteatoma tissue. Our data indicates that congenital cholesteatoma origins

  20. Evolution, diversification and expression of KNOX proteins in plants

    Directory of Open Access Journals (Sweden)

    Jie eGao

    2015-10-01

    Full Text Available The KNOX (KNOTTED1-like homeobox transcription factors play a pivotal role in leaf and meristem development. The majority of these proteins are characterized by the KNOX1, KNOX2, ELK and homeobox domains whereas the proteins of the KNATM family contain only the KNOX domains. We carried out an extensive inventory of these proteins and here report on a total of 394 KNOX proteins from 48 species. The land plant proteins fall into two classes (I and II as previously shown where the class I family seems to be most closely related to the green algae homologs. The KNATM proteins are restricted to Eudicots and some species have multiple paralogs of this protein. Certain plants are characterized by a significant increase in the number of KNOX paralogs; one example is Glycine max. Through the analysis of public gene expression data we show that the class II proteins of this plant have a relatively broad expression specificity as compared to class I proteins, consistent with previous studies of other plants. In G. max, class I protein are mainly distributed in axis tissues and KNATM paralogs are overall poorly expressed; highest expression is in the early plumular axis. Overall, analysis of gene expression in G. max demonstrates clearly that the expansion in gene number is associated with functional diversification.

  1. 1.8 Å structure of murine GITR ligand dimer expressed in Drosophila melanogaster S2 cells

    International Nuclear Information System (INIS)

    Chattopadhyay, Kausik; Ramagopal, Udupi A.; Nathenson, Stanley G.; Almo, Steven C.

    2009-01-01

    1.8 Å X-ray crystal structure of mouse GITRL expressed in D. melanogaster S2 cells shows an identical ‘strand-exchanged’ dimeric assembly similar to that observed previously for the E. coli-expressed protein. Glucocorticoid-induced TNF receptor ligand (GITRL), a prominent member of the TNF superfamily, activates its receptor on both effector and regulatory T cells to generate critical costimulatory signals that have been implicated in a wide range of T-cell immune functions. The crystal structures of murine and human orthologs of GITRL recombinantly expressed in Escherichia coli have previously been determined. In contrast to all classical TNF structures, including the human GITRL structure, murine GITRL demonstrated a unique ‘strand-exchanged’ dimeric organization. Such a novel assembly behavior indicated a dramatic impact on receptor activation as well as on the signaling mechanism associated with the murine GITRL costimulatory system. In this present work, the 1.8 Å resolution crystal structure of murine GITRL expressed in Drosophila melanogaster S2 cells is reported. The eukaryotic protein-expression system allows transport of the recombinant protein into the extracellular culture medium, thus maximizing the possibility of obtaining correctly folded material devoid of any folding/assembly artifacts that are often suspected with E. coli-expressed proteins. The S2 cell-expressed murine GITRL adopts an identical ‘strand-exchanged’ dimeric structure to that observed for the E. coli-expressed protein, thus conclusively demonstrating the novel quaternary structure assembly behavior of murine GITRL

  2. 1.8 Å structure of murine GITR ligand dimer expressed in Drosophila melanogaster S2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Chattopadhyay, Kausik [Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Ramagopal, Udupi A. [Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Nathenson, Stanley G., E-mail: nathenso@aecom.yu.edu [Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Almo, Steven C., E-mail: nathenso@aecom.yu.edu [Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461 (United States)

    2009-05-01

    1.8 Å X-ray crystal structure of mouse GITRL expressed in D. melanogaster S2 cells shows an identical ‘strand-exchanged’ dimeric assembly similar to that observed previously for the E. coli-expressed protein. Glucocorticoid-induced TNF receptor ligand (GITRL), a prominent member of the TNF superfamily, activates its receptor on both effector and regulatory T cells to generate critical costimulatory signals that have been implicated in a wide range of T-cell immune functions. The crystal structures of murine and human orthologs of GITRL recombinantly expressed in Escherichia coli have previously been determined. In contrast to all classical TNF structures, including the human GITRL structure, murine GITRL demonstrated a unique ‘strand-exchanged’ dimeric organization. Such a novel assembly behavior indicated a dramatic impact on receptor activation as well as on the signaling mechanism associated with the murine GITRL costimulatory system. In this present work, the 1.8 Å resolution crystal structure of murine GITRL expressed in Drosophila melanogaster S2 cells is reported. The eukaryotic protein-expression system allows transport of the recombinant protein into the extracellular culture medium, thus maximizing the possibility of obtaining correctly folded material devoid of any folding/assembly artifacts that are often suspected with E. coli-expressed proteins. The S2 cell-expressed murine GITRL adopts an identical ‘strand-exchanged’ dimeric structure to that observed for the E. coli-expressed protein, thus conclusively demonstrating the novel quaternary structure assembly behavior of murine GITRL.

  3. Subject-specific increases in serum S-100B distinguish sports-related concussion from sports-related exertion.

    Science.gov (United States)

    Kiechle, Karin; Bazarian, Jeffrey J; Merchant-Borna, Kian; Stoecklein, Veit; Rozen, Eric; Blyth, Brian; Huang, Jason H; Dayawansa, Samantha; Kanz, Karl; Biberthaler, Peter

    2014-01-01

    The on-field diagnosis of sports-related concussion (SRC) is complicated by the lack of an accurate and objective marker of brain injury. To compare subject-specific changes in the astroglial protein, S100B, before and after SRC among collegiate and semi-professional contact sport athletes, and compare these changes to differences in S100B before and after non-contact exertion. Longitudinal cohort study. From 2009-2011, we performed a prospective study of athletes from Munich, Germany, and Rochester, New York, USA. Serum S100B was measured in all SRC athletes at pre-season baseline, within 3 hours of injury, and at days 2, 3 and 7 post-SRC. Among a subset of athletes, S100B was measured after non-contact exertion but before injury. All samples were collected identically and analyzed using an automated electrochemiluminescent assay to quantify serum S100B levels. Forty-six athletes (30 Munich, 16 Rochester) underwent baseline testing. Thirty underwent additional post-exertion S100B testing. Twenty-two athletes (16 Rochester, 6 Munich) sustained a SRC, and 17 had S100B testing within 3 hours post-injury. The mean 3-hour post-SRC S100B was significantly higher than pre-season baseline (0.099±0.008 µg/L vs. 0.058±0.006 µg/L, p = 0.0002). Mean post-exertion S100B was not significantly different than the preseason baseline. S100B levels at post-injury days 2, 3 and 7 were significantly lower than the 3-hour level, and not different than baseline. Both the absolute change and proportional increase in S100B 3-hour post-injury were accurate discriminators of SRC from non-contact exertion without SRC (AUC 0.772 and 0.904, respectively). A 3-hour post-concussion S100B >0.122 µg/L and a proportional S100B increase of >45.9% over baseline were both 96.7% specific for SRC. Relative and absolute increases in serum S100B can accurately distinguish SRC from sports-related exertion, and may be a useful adjunct to the diagnosis of SRC.

  4. Subject-specific increases in serum S-100B distinguish sports-related concussion from sports-related exertion.

    Directory of Open Access Journals (Sweden)

    Karin Kiechle

    Full Text Available The on-field diagnosis of sports-related concussion (SRC is complicated by the lack of an accurate and objective marker of brain injury.To compare subject-specific changes in the astroglial protein, S100B, before and after SRC among collegiate and semi-professional contact sport athletes, and compare these changes to differences in S100B before and after non-contact exertion.Longitudinal cohort study.From 2009-2011, we performed a prospective study of athletes from Munich, Germany, and Rochester, New York, USA. Serum S100B was measured in all SRC athletes at pre-season baseline, within 3 hours of injury, and at days 2, 3 and 7 post-SRC. Among a subset of athletes, S100B was measured after non-contact exertion but before injury. All samples were collected identically and analyzed using an automated electrochemiluminescent assay to quantify serum S100B levels.Forty-six athletes (30 Munich, 16 Rochester underwent baseline testing. Thirty underwent additional post-exertion S100B testing. Twenty-two athletes (16 Rochester, 6 Munich sustained a SRC, and 17 had S100B testing within 3 hours post-injury. The mean 3-hour post-SRC S100B was significantly higher than pre-season baseline (0.099±0.008 µg/L vs. 0.058±0.006 µg/L, p = 0.0002. Mean post-exertion S100B was not significantly different than the preseason baseline. S100B levels at post-injury days 2, 3 and 7 were significantly lower than the 3-hour level, and not different than baseline. Both the absolute change and proportional increase in S100B 3-hour post-injury were accurate discriminators of SRC from non-contact exertion without SRC (AUC 0.772 and 0.904, respectively. A 3-hour post-concussion S100B >0.122 µg/L and a proportional S100B increase of >45.9% over baseline were both 96.7% specific for SRC.Relative and absolute increases in serum S100B can accurately distinguish SRC from sports-related exertion, and may be a useful adjunct to the diagnosis of SRC.

  5. Screening and large-scale expression of membrane proteins in mammalian cells for structural studies.

    Science.gov (United States)

    Goehring, April; Lee, Chia-Hsueh; Wang, Kevin H; Michel, Jennifer Carlisle; Claxton, Derek P; Baconguis, Isabelle; Althoff, Thorsten; Fischer, Suzanne; Garcia, K Christopher; Gouaux, Eric

    2014-11-01

    Structural, biochemical and biophysical studies of eukaryotic membrane proteins are often hampered by difficulties in overexpression of the candidate molecule. Baculovirus transduction of mammalian cells (BacMam), although a powerful method to heterologously express membrane proteins, can be cumbersome for screening and expression of multiple constructs. We therefore developed plasmid Eric Gouaux (pEG) BacMam, a vector optimized for use in screening assays, as well as for efficient production of baculovirus and robust expression of the target protein. In this protocol, we show how to use small-scale transient transfection and fluorescence-detection size-exclusion chromatography (FSEC) experiments using a GFP-His8-tagged candidate protein to screen for monodispersity and expression level. Once promising candidates are identified, we describe how to generate baculovirus, transduce HEK293S GnTI(-) (N-acetylglucosaminyltransferase I-negative) cells in suspension culture and overexpress the candidate protein. We have used these methods to prepare pure samples of chicken acid-sensing ion channel 1a (cASIC1) and Caenorhabditis elegans glutamate-gated chloride channel (GluCl) for X-ray crystallography, demonstrating how to rapidly and efficiently screen hundreds of constructs and accomplish large-scale expression in 4-6 weeks.

  6. Autoimmune Regulator (AIRE) Is Expressed in Spermatogenic Cells, and It Altered the Expression of Several Nucleic-Acid-Binding and Cytoskeletal Proteins in Germ Cell 1 Spermatogonial (GC1-spg) Cells.

    Science.gov (United States)

    Radhakrishnan, Karthika; Bhagya, Kongattu P; Kumar, Anil Tr; Devi, Anandavalli N; Sengottaiyan, Jeeva; Kumar, Pradeep G

    2016-08-01

    Autoimmune regulator (AIRE) is a gene associated with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). AIRE is expressed heavily in the thymic epithelial cells and is involved in maintaining self-tolerance through regulating the expression of tissue-specific antigens. The testes are the most predominant extrathymic location where a heavy expression of AIRE is reported. Homozygous Aire-deficient male mice were infertile, possibly due to impaired spermatogenesis, deregulated germ cell apoptosis, or autoimmunity. We report that AIRE is expressed in the testes of neonatal, adolescent, and adult mice. AIRE expression was detected in glial cell derived neurotrophic factor receptor alpha (GFRα)(+) (spermatogonia), GFRα(-)/synaptonemal complex protein (SCP3)(+) (meiotic), and GFRα(-)/Phosphoglycerate kinase 2 (PGK2)(+) (postmeiotic) germ cells in mouse testes. GC1-spg, a germ-cell-derived cell line, did not express AIRE. Retinoic acid induced AIRE expression in GC1-spg cells. Ectopic expression of AIRE in GC1-spg cells using label-free LC-MS/MS identified a total of 371 proteins that were differentially expressed. 100 proteins were up-regulated, and 271 proteins were down-regulated. Data are available via ProteomeXchange with identifier PXD002511. Functional analysis of the differentially expressed proteins showed increased levels of various nucleic-acid-binding proteins and transcription factors and a decreased level of various cytoskeletal and structural proteins in the AIRE overexpressing cells as compared with the empty vector-transfected controls. The transcripts of a select set of the up-regulated proteins were also elevated. However, there was no corresponding decrease in the mRNA levels of the down-regulated set of proteins. Molecular function network analysis indicated that AIRE influenced gene expression in GC1-spg cells by acting at multiple levels, including transcription, translation, RNA processing, protein transport, protein

  7. The Arabidopsis TOR Kinase Specifically Regulates the Expression of Nuclear Genes Coding for Plastidic Ribosomal Proteins and the Phosphorylation of the Cytosolic Ribosomal Protein S6.

    Science.gov (United States)

    Dobrenel, Thomas; Mancera-Martínez, Eder; Forzani, Céline; Azzopardi, Marianne; Davanture, Marlène; Moreau, Manon; Schepetilnikov, Mikhail; Chicher, Johana; Langella, Olivier; Zivy, Michel; Robaglia, Christophe; Ryabova, Lyubov A; Hanson, Johannes; Meyer, Christian

    2016-01-01

    Protein translation is an energy consuming process that has to be fine-tuned at both the cell and organism levels to match the availability of resources. The target of rapamycin kinase (TOR) is a key regulator of a large range of biological processes in response to environmental cues. In this study, we have investigated the effects of TOR inactivation on the expression and regulation of Arabidopsis ribosomal proteins at different levels of analysis, namely from transcriptomic to phosphoproteomic. TOR inactivation resulted in a coordinated down-regulation of the transcription and translation of nuclear-encoded mRNAs coding for plastidic ribosomal proteins, which could explain the chlorotic phenotype of the TOR silenced plants. We have identified in the 5' untranslated regions (UTRs) of this set of genes a conserved sequence related to the 5' terminal oligopyrimidine motif, which is known to confer translational regulation by the TOR kinase in other eukaryotes. Furthermore, the phosphoproteomic analysis of the ribosomal fraction following TOR inactivation revealed a lower phosphorylation of the conserved Ser240 residue in the C-terminal region of the 40S ribosomal protein S6 (RPS6). These results were confirmed by Western blot analysis using an antibody that specifically recognizes phosphorylated Ser240 in RPS6. Finally, this antibody was used to follow TOR activity in plants. Our results thus uncover a multi-level regulation of plant ribosomal genes and proteins by the TOR kinase.

  8. Resistance Risk Assessment of Spodoptera frugiperda (Lepidoptera: Noctuidae) and Diatraea saccharalis (Lepidoptera: Crambidae) to Vip3Aa20 Insecticidal Protein Expressed in Corn.

    Science.gov (United States)

    Bernardi, Oderlei; Bernardi, Daniel; Amado, Douglas; Sousa, Renan S; Fatoretto, Julio; Medeiros, Fernanda C L; Conville, Jared; Burd, Tony; Omoto, Celso

    2015-12-01

    Transgenic Agrisure Viptera 3 corn that expresses Cry1Ab, Vip3Aa20, and EPSPS proteins and Agrisure Viptera expressing Vip3Aa20 are used for control of Spodoptera frugiperda (J.E. Smith) and Diatraea saccharalis (F.) in Brazil. To support a resistance management program, resistance risk assessment studies were conducted to characterize the dose expression of Vip3Aa20 protein and level of control against these species. The Vip3Aa20 expression in Agrisure Viptera 3 and Agrisure Viptera decreased from V6 to V10 stage of growth. However, Vip3Aa20 expression in Agrisure Viptera 3 at V6 and V10 stages was 13- and 16-fold greater than Cry1Ab, respectively. The Vip3Aa20 expression in lyophilized tissue of Agrisure Viptera 3 and Agrisure Viptera diluted 25-fold in an artificial diet caused complete larval mortality of S. frugiperda and D. saccharalis. In contrast, lyophilized tissue of Bt11 at the same dilution does not provide complete mortality of these species. Agrisure Viptera 3 and Agrisure Viptera also caused a high level of mortality against S. frugiperda and D. saccharalis. Moreover, 100% mortality was observed for S. frugiperda larvae (neonates through fifth-instar larvae) when fed in corn with the Vip trait technology. Viptera corn achieves a high level of control against S. frugiperda and D. saccharalis providing a high dose, which is an important determination to support the refuge strategy for an effective resistance management program. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  9. Long-term functional impairment of hemopoietic progenitor cells engineered to express the S1 catalytic subunit of pertussis toxin.

    Science.gov (United States)

    Bonig, Halvard; Rohmer, Laurence; Papayannopoulou, Thalia

    2005-06-01

    A large body of data suggests that pertussis toxin (PTX)-sensitive G protein signals in mature and immature hemopoietic cells control their migration patterns in vitro and in vivo. These effects were derived after treatment of cells or animals with PTX. To circumvent several inherent problems of PTX holotoxin treatment, we expressed the S1 catalytic activity of PTX, thus blocking Gi protein signaling, in 32D murine myeloid progenitor cells and in primary human CD34+ cells, and studied its functional consequences. S1 was expressed using viral vectors. Effects of Gi protein blockade on proliferation, migration, adhesion, and gene expression were tested in vitro. S1 expression was nontoxic for the cells; expression and function were stable long-term and not overridden by compensatory mechanisms. S1-transduced 32D cells and primary CD34+ cells migrated poorly and did not contract their cytoskeleton upon treatment with the chemoattractant stromal cell-derived factor -1 (SDF-1), similar to the phenotype induced by PTX treatment. Gene expression studies comparing S1-transduced and control 32D cells uncovered four genes, expression of which was regulated by Gi protein blockade. Of interest, although SDF-1 signaling was inhibited, comparison between SDF-1-treated and untreated cells suggests that SDF-1 stimulation does not depend on de novo gene expression in these cells. Furthermore, when injected into nonobese diabetic/severe combined immunodeficient mice, seeding of S1-expressing 32D cells to bone marrow was largely blocked. Expression of S1 is an effective approach for studying long-term functional consequences of Gi protein blockade in hemopoietic cells in vitro and in vivo.

  10. HMB-45, S-100, NK1/C3, and MART-1 in metastatic melanoma.

    Science.gov (United States)

    Zubovits, Judit; Buzney, Elizabeth; Yu, Lawrence; Duncan, Lyn M

    2004-02-01

    The diagnosis of melanoma metastatic to lymph node remains a difficult problem given its histological diversity. We examined the staining patterns of S-100, NK1/C3, HMB-45, and MART-1 (DC10) in melanoma metastases to lymph nodes. Immunohistochemical stains were performed on tissue sections of 126 formalin-fixed lymph nodes from 126 patients with an established diagnosis of metastatic melanoma. A total of 98% of cases (123 of 126) stained positive for S-100, 93% (117 of 125) stained positive for NK1/C3, 82% (103 of 126) stained positive for MART-1, and 76% (95 of 125) stained positive for HMB-45. The distribution and intensity of staining varied among these markers. A diffuse staining pattern, defined as >50% of tumor cells stained, was observed in 83% of MART-1-positive cases but in only 56% of S-100-positive cases, 48% of NK1/C3-positive cases, and 34% of HMB-45-positive cases. A maximally intense signal was almost always observed for MART-1 (83% of positive cases) but was rarely observed for NK1/C3 (20%). S-100 and HMB-45 showed maximally intense staining in 50% and 54% of cases, respectively. S-100 and NK1/C3 stained both histiocytes and melanocytes, whereas MART-1 and HMB-45 stained only melanocytes. Seventy-eight cases (63%) stained positive for all 4 markers, 17 cases (14%) stained for all markers except HMB-45, 13 cases (10%) stained for all markers except MART-1, 6 cases (5%) stained only with S-100 and NK1/C3, 4 cases (3%) stained only with S-100 and HMB-45, and 2 cases stained for all markers except S-100. One case each stained for the following: only S-100, only S-100 and HMB-45, and all markers except NK1/C3. One case exhibited absence of staining for any of these markers. We demonstrate that lymph node metastases of melanoma are heterogeneous with regard to tumor marker expression. S-100 and NK1/C3 were the most sensitive stains for detecting metastatic melanoma; however, they both also stain other nontumor cells in lymph nodes. MART-1 did not stain

  11. A novel bidirectional expression system for simultaneous expression of both the protein-coding genes and short hairpin RNAs in mammalian cells

    International Nuclear Information System (INIS)

    Hung, C.-F.; Cheng, T.-L.; Wu, R.-H.; Teng, C.-F.; Chang, W.-T.

    2006-01-01

    RNA interference (RNAi) is an extremely powerful and widely used gene silencing approach for reverse functional genomics and molecular therapeutics. In mammals, the conserved poly(ADP-ribose) polymerase 2 (PARP-2)/RNase P bidirectional control promoter simultaneously expresses both the PARP-2 protein and RNase P RNA by RNA polymerase II- and III-dependent mechanisms, respectively. To explore this unique bidirectional control system in RNAi-mediated gene silencing strategy, we have constructed two novel bidirectional expression vectors, pbiHsH1 and pbiMmH1, which contained the PARP-2/RNase P bidirectional control promoters from human and mouse, for simultaneous expression of both the protein-coding genes and short hairpin RNAs. Analyses of the dual transcriptional activities indicated that these two bidirectional expression vectors could not only express enhanced green fluorescent protein as a functional reporter but also simultaneously transcribe shLuc for inhibiting the firefly luciferase expression. In addition, to extend its utility for the establishment of inherited stable clones, we have also reconstructed this bidirectional expression system with the blasticidin S deaminase gene, an effective dominant drug resistance selectable marker, and examined both the selection and inhibition efficiencies in drug resistance and gene expression. Moreover, we have further demonstrated that this bidirectional expression system could efficiently co-regulate the functionally important genes, such as overexpression of tumor suppressor protein p53 and inhibition of anti-apoptotic protein Bcl-2 at the same time. In summary, the bidirectional expression vectors, pbiHsH1 and pbiMmH1, should provide a simple, convenient, and efficient novel tool for manipulating the gene function in mammalian cells

  12. Validation of an enzyme-linked immunosorbent assay (ELISA) for the measurement of canine S100A12.

    Science.gov (United States)

    Heilmann, Romy M; Cranford, Shannon M; Ambrus, Andy; Grützner, Niels; Schellenberg, Stefan; Ruaux, Craig G; Suchodolski, Jan S; Steiner, Jörg M

    2016-03-01

    Canine S100 calcium-binding protein A12 (cS100A12) shows promise as biomarker of inflammation in dogs. A previously developed cS100A12-radioimmunoassay (RIA) requires radioactive tracers and is not sensitive enough for fecal cS100A12 concentrations in 79% of tested healthy dogs. An ELISA assay may be more sensitive than RIA and does not require radioactive tracers. The purpose of the study was to establish a sandwich ELISA for serum and fecal cS100A12, and to establish reference intervals (RI) for normal healthy canine serum and feces. Polyclonal rabbit anti-cS100A12 antibodies were generated and tested by Western blotting and immunohistochemistry. A sandwich ELISA was developed and validated, including accuracy and precision, and agreement with cS100A12-RIA. The RI, stability, and biologic variation in fecal cS100A12, and the effect of corticosteroids on serum cS100A12 were evaluated. Lower detection limits were 5 μg/L (serum) and 1 ng/g (fecal), respectively. Intra- and inter-assay coefficients of variation were ≤ 4.4% and ≤ 10.9%, respectively. Observed-to-expected ratios for linearity and spiking recovery were 98.2 ± 9.8% (mean ± SD) and 93.0 ± 6.1%, respectively. There was a significant bias between the ELISA and the RIA. The RI was 49-320 μg/L for serum and 2-484 ng/g for fecal cS100A12. Fecal cS100A12 was stable for 7 days at 23, 4, -20, and -80°C; biologic variation was negligible but variation within one fecal sample was significant. Corticosteroid treatment had no clinically significant effect on serum cS100A12 concentrations. The cS100A12-ELISA is a precise and accurate assay for serum and fecal cS100A12 in dogs. © 2016 American Society for Veterinary Clinical Pathology.

  13. Mucosal Immunogenicity of Genetically Modified Lactobacillus acidophilus Expressing an HIV-1 Epitope within the Surface Layer Protein.

    Directory of Open Access Journals (Sweden)

    Akinobu Kajikawa

    Full Text Available Surface layer proteins of probiotic lactobacilli are theoretically efficient epitope-displaying scaffolds for oral vaccine delivery due to their high expression levels and surface localization. In this study, we constructed genetically modified Lactobacillus acidophilus strains expressing the membrane proximal external region (MPER from human immunodeficiency virus type 1 (HIV-1 within the context of the major S-layer protein, SlpA. Intragastric immunization of mice with the recombinants induced MPER-specific and S-layer protein-specific antibodies in serum and mucosal secretions. Moreover, analysis of systemic SlpA-specific cytokines revealed that the responses appeared to be Th1 and Th17 dominant. These findings demonstrated the potential use of the Lactobacillus S-layer protein for development of oral vaccines targeting specific peptides.

  14. Tyrosinase expression in malignant melanoma, desmoplastic melanoma, and peripheral nerve tumors

    DEFF Research Database (Denmark)

    Boyle, Jenny L; Haupt, Helen M; Stern, Jere B

    2002-01-01

    of tyrosinase expression in the differential diagnosis of melanoma, desmoplastic melanoma, and peripheral nerve sheath tumors. DESIGN: Immunoreactivity for tyrosinase, HMB-45 (anti-gp100 protein), S100 protein, CD34, and vimentin was studied in 70 tumors, including 15 melanomas (5 desmoplastic, 4 amelanotic, 6...... at 121 degrees C. RESULTS: All melanomas demonstrated positive immunostaining for tyrosinase, HMB-45, and S100 protein. Immunoreactivity for HMB-45 was generally stronger than that for tyrosinase in amelanotic lesions and significantly stronger in 1 of the desmoplastic lesions. The 4 pigmented...... neurofibromas were focally positive for tyrosinase, but did not stain for HMB-45. The pigmented schwannoma was focally positive for both tyrosinase and HMB-45. The malignant peripheral nerve sheath tumors, dermatofibrosarcoma protuberans, and dermatofibromas were nonreactive for tyrosinase and HMB-45...

  15. Development of a simple measurement method for GluR2 protein expression as an index of neuronal vulnerability

    Directory of Open Access Journals (Sweden)

    Chihiro Sugiyama

    2015-01-01

    Full Text Available In vitro estimating strategies for potential neurotoxicity are required to screen multiple substances. In a previous study, we showed that exposure to low-concentrations of some chemicals, such as organotin, decreased the expression of GluR2 protein, which is a subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA-type glutamate receptors, and led to neuronal vulnerability. This result suggested that GluR2 decreases as an index of neuronal cell sensitivity and vulnerability to various toxic insults. Accordingly, we developed a versatile method that is a large scale determination of GluR2 protein expression in the presence of environmental chemicals by means of AlphaLISA technology. Various analytical conditions were optimized, and then GluR2 protein amount was measured by the method using AlphaLISA. The GluR2 amounts were strongly correlated with that of measured by western blotting, which is currently used to determine GluR2 expression. An ideal standard curve could be written with the authentic GluR2 protein from 0 ng to 100 ng. Subsequently, twenty environmental chemicals were screened and nitenpyram was identified as a chemical which lead to decrease in GluR2 protein expression. This assay may provide a tool for detecting neurotoxic chemicals according to decreases in GluR2 protein expression.

  16. The study on highly expressed proteins as a function of an elevated ultraviolet radiation in the copepod, Tigriopus japonicus

    Science.gov (United States)

    Zubrzycki, Igor Z.; Lee, Seunghan; Lee, Kanghyun; Wiacek, Magdalena; Lee, Wonchoel

    2012-06-01

    The objective of the study was to analyze constantlyhighly expressed proteins as a function of elevated midultraviolet (UVB, 280-315 nm) radiation in Tigriopus japonicus sensu lato ( T. japonicus s.l). We also analyzed associations between kinetics of radiation avoidance, measured as a covered distance per time unit, and highly expressed proteins. The obtained results indicate an increase in T. japonicus s.l. mobility between the control (no radiation) and mild UV radiation levels (15 kJ·m-2). Two-dimensional gel electrophoresis combined with MALDI-MS-MS resulted in 2D protein map comprising of 686 protein spots, of which 19 were identified as highly expressed proteins across all experimental conditions. Obtained results indicate that calpain, vitellogenin, and collagenase are housekeeping protein that are expressed at a constant level independently of environmental changes and that adoption of a locomotive system for the avoidance of a UV source may be, at least partially, supported by hepatopancreas-driven metabolism.

  17. The proteome response to amyloid protein expression in vivo.

    Directory of Open Access Journals (Sweden)

    Ricardo A Gomes

    Full Text Available Protein misfolding disorders such as Alzheimer, Parkinson and transthyretin amyloidosis are characterized by the formation of protein amyloid deposits. Although the nature and location of the aggregated proteins varies between different diseases, they all share similar molecular pathways of protein unfolding, aggregation and amyloid deposition. Most effects of these proteins are likely to occur at the proteome level, a virtually unexplored reality. To investigate the effects of an amyloid protein expression on the cellular proteome, we created a yeast expression system using human transthyretin (TTR as a model amyloidogenic protein. We used Saccharomyces cerevisiae, a living test tube, to express native TTR (non-amyloidogenic and the amyloidogenic TTR variant L55P, the later forming aggregates when expressed in yeast. Differential proteome changes were quantitatively analyzed by 2D-differential in gel electrophoresis (2D-DIGE. We show that the expression of the amyloidogenic TTR-L55P causes a metabolic shift towards energy production, increased superoxide dismutase expression as well as of several molecular chaperones involved in protein refolding. Among these chaperones, members of the HSP70 family and the peptidyl-prolyl-cis-trans isomerase (PPIase were identified. The latter is highly relevant considering that it was previously found to be a TTR interacting partner in the plasma of ATTR patients but not in healthy or asymptomatic subjects. The small ubiquitin-like modifier (SUMO expression is also increased. Our findings suggest that refolding and degradation pathways are activated, causing an increased demand of energetic resources, thus the metabolic shift. Additionally, oxidative stress appears to be a consequence of the amyloidogenic process, posing an enhanced threat to cell survival.

  18. Expression of Tight Junction Protein Claudin-1 in Human Crescentic Glomerulonephritis

    Directory of Open Access Journals (Sweden)

    Ryo Koda

    2014-01-01

    Full Text Available The origin of crescent forming cells in human glomerulonephritis (GN remains unknown. Some animal studies demonstrated that parietal epithelial cells of Bowman’s capsule (PECs were the main component of proliferating cells and PEC-specific tight junction protein claudin-1 was expressed in crescentic lesions. We investigated the expression of claudin-1 in human GN. Immunohistochemistry for claudin-1 was performed on 17 kidney biopsy samples with crescent formation. Colocalization of claudin-1 with intracellular tight junction protein ZO-1 was also evaluated by immunofluorescence double staining. Claudin-1 is expressed mainly at the cell to cell contact site of proliferating cells in cellular crescentic lesions in patients with these forms of human GN. Small numbers of crescent forming cells showed extrajunctional localization of claudin-1. Colocalization of claudin-1 with ZO-1 was found at cell to cell contact sites of adjacent proliferating cells. In control samples, staining of claudin-1 was positive in PECs, but not in podocytes. Our findings suggest that claudin-1 contributes to crescent formation as a component of the tight junction protein complex that includes ZO-1. Co-localization of claudin-1 with ZO-1 implies the formation of functional tight junction complexes in crescentic lesions to prevent the interstitial damage caused by penetration of filtered molecules from Bowman’s space.

  19. MMP-9, uPA and uPAR proteins expression and its prognostic significance in esophageal squamous cell carcinoma treated by radiotherapy

    International Nuclear Information System (INIS)

    Zhu Shuchai; Wang Yafei; Su Jingwei; Wang Yuxiang; Shen Wenbin; Li Juan

    2008-01-01

    Objective: To explore the the prognostic significance of MMP-9, uPA and uPAR protein expression and its relationship with clinical-pathologic factors in esophageal squamous cell carcinoma treated by radiotherapy. Methods: MMP-9, uPA and uPAR protein expression was measured in 59 esophageal carcinomas and 41 peri-carcinoma tissues with immunohistochemistry. The relationship between the protein expression and the clinical-pathological parameters was analyzed, and the prognostic factors in esophageal squamous cell carcinoma treated by radiotherapy alone was evaluated. Results: The rates of positive expression of MMP-9, uPA and uPAR were 85%, 76% and 78% in esophageal carcinoma and 39%, 49% and 44% in peri-carcinoma tissues (χ 2 =22.54, 8.04 and 12.18; P=0.000,0.005 and 0.000). The rates of positive expression of MMP-9 was 79% and 100% when the depth of tumor invasion was ≤2 cm and >2 cm(P= 0.048), respectively. The expression of uPA was significantly correlated with the status of fat interspace between the esophageal lesion and the vertebra in CT scanning image. When the fat interspace existed and disappeared, the rates of strong positive expression was 44% and 70%, respectively (χ 2 =4.21, P=0.040). The positive expression rate of uPA was significantly correlated with distant metastasis, which was 100% in patients with distant metastasis and 68.89% in those without distant metastasis(χ 2 =4.12, P=0.042). The positive expression rate of MMP-9, uPA and uPAR did not affect the prognosis and the short-term result of esophageal carcinoma treated by radiotherapy alone. Conclusions: The protein expression of MMP-9, uPA and uPAR may correlate with local infiltration and distant metastasis in esophageal squamous cell carcinoma. Protein expression may not influence the prognosis of esophageal carcinoma treated by radio therapy, though long time followed-up is still needed. (authors)

  20. Expression of endogenous proteins in maize hybrids in a multi-location field trial in India.

    Science.gov (United States)

    Gutha, Linga R; Purushottam, Divakar; Veeramachaneni, Aruna; Tigulla, Sarita; Kodappully, Vikas; Enjala, Chandana; Rajput, Hitendrasinh; Anderson, Jennifer; Hong, Bonnie; Schmidt, Jean; Bagga, Shveta

    2018-05-17

    Genetically modified (GM) crops undergo large scale multi-location field trials to characterize agronomics, composition, and the concentration of newly expressed protein(s) [herein referred to as transgenic protein(s)]. The concentration of transgenic proteins in different plant tissues and across the developmental stages of the plant is considered in the safety assessment of GM crops. Reference or housekeeping proteins are expected to maintain a relatively stable expression pattern in healthy plants given their role in cellular functions. Understanding the effects of genotype, growth stage and location on the concentration of endogenous housekeeping proteins may provide insight into the contribution these factors could have on transgenic protein concentrations in GM crops. The concentrations of three endogenous proteins (actin, elongation factor 1-alpha, and glyceraldehyde 3-phosphate dehydrogenase) were measured in several different maize hybrids grown across multiple field locations over 2 years. Leaf samples were collected from healthy plants at three developmental stages across the growing seasons, and protein concentrations were quantified by indirect enzyme-linked immunosorbent assay (ELISA) for each protein. In general, the concentrations of these three endogenous proteins were relatively consistent across hybrid backgrounds, when compared within one growth stage and location (2-26%CV), whereas the concentrations of proteins in the same hybrid and growth stage across different locations were more variable (12-64%CV). In general, the protein concentrations in 2013 and 2014 show similar trends in variability. Some degree of variability in protein concentrations should be expected for both transgenic and endogenous plant-expressed proteins. In the case of GM crops, the potential variation in protein concentrations due to location effects is captured in the current model of multi-location field testing.

  1. Ligand Binding Domain Protein in Tetracycline-Inducible Expression

    African Journals Online (AJOL)

    Purpose: To investigate tetracycline-inducible expression system for producing clinically usable, highquality liver X receptor ligand-binding domain recombinant protein. Methods: In this study, we have expressed and purified the recombinant liver X receptor β-ligand binding domain proteins in E. coli using a tetracycline ...

  2. Extracellular S100A4(mts1) stimulates invasive growth of mouse endothelial cells and modulates MMP-13 matrix metalloproteinase activity

    DEFF Research Database (Denmark)

    Schmidt-Hansen, Birgitte; Ornås, Dorte; Grigorian, Mariam

    2004-01-01

    with the transcriptional modulation of genes involved in the proteolytic degradation of extracellular matrix (ECM). Treatment of SVEC 4-10 with the S100A4 protein leads to the transcriptional activation of collagenase 3 (MMP-13) mRNA followed by subsequent release of the protein from the cells. Beta-casein zymography...... demonstrates enhancement of proteolytic activity associated with MMP-13. This observation indicates that extracellular S100A4 stimulates the production of ECM degrading enzymes from endothelial cells, thereby stimulating the remodeling of ECM. This could explain the angiogenic and metastasis...

  3. [Expression of c-jun protein after experimental rat brain concussion].

    Science.gov (United States)

    Wang, Feng; Li, Yong-hong

    2010-02-01

    To observe e-jun protein expression after rat brain concussion and explore the forensic pathologic markers following brain concussion. Fifty-five rats were randomly divided into brain concussion group and control group. The expression of c-jun protein was observed by immunohistochemistry. There were weak positive expression of c-jun protein in control group. In brain concussion group, however, some neutrons showed positive expression of c-jun protein at 15 min after brain concussion, and reach to the peak at 3 h after brain concussion. The research results suggest that detection of c-jun protein could be a marker to determine brain concussion and estimate injury time after brain concussion.

  4. Rationally designed, heterologous S. cerevisiae transcripts expose novel expression determinants

    Science.gov (United States)

    Ben-Yehezkel, Tuval; Atar, Shimshi; Zur, Hadas; Diament, Alon; Goz, Eli; Marx, Tzipy; Cohen, Rafael; Dana, Alexandra; Feldman, Anna; Shapiro, Ehud; Tuller, Tamir

    2015-01-01

    Deducing generic causal relations between RNA transcript features and protein expression profiles from endogenous gene expression data remains a major unsolved problem in biology. The analysis of gene expression from heterologous genes contributes significantly to solving this problem, but has been heavily biased toward the study of the effect of 5′ transcript regions and to prokaryotes. Here, we employ a synthetic biology driven approach that systematically differentiates the effect of different regions of the transcript on gene expression up to 240 nucleotides into the ORF. This enabled us to discover new causal effects between features in previously unexplored regions of transcripts, and gene expression in natural regimes. We rationally designed, constructed, and analyzed 383 gene variants of the viral HRSVgp04 gene ORF, with multiple synonymous mutations at key positions along the transcript in the eukaryote S. cerevisiae. Our results show that a few silent mutations at the 5′UTR can have a dramatic effect of up to 15 fold change on protein levels, and that even synonymous mutations in positions more than 120 nucleotides downstream from the ORF 5′end can modulate protein levels up to 160%–300%. We demonstrate that the correlation between protein levels and folding energy increases with the significance of the level of selection of the latter in endogenous genes, reinforcing the notion that selection for folding strength in different parts of the ORF is related to translation regulation. Our measured protein abundance correlates notably(correlation up to r = 0.62 (p=0.0013)) with mean relative codon decoding times, based on ribosomal densities (Ribo-Seq) in endogenous genes, supporting the conjecture that translation elongation and adaptation to the tRNA pool can modify protein levels in a causal/direct manner. This report provides an improved understanding of transcript evolution, design principles of gene expression regulation, and suggests simple

  5. Synthetic cold-inducible promoter enhances recombinant protein accumulation during Agrobacterium-mediated transient expression in Nicotiana excelsior at chilling temperatures.

    Science.gov (United States)

    Gerasymenko, I M; Sheludko, Y V

    2017-07-01

    To exploit cold-inducible biochemical processes beneficial for foreign mRNA transcription, translation and storage, as well as protein product stability, during Agrobacterium-mediated transient expression. The efficiency of three different 5'-regulatory sequences to achieve transient expression of the GFP-based reporter gene under chilling conditions (6-8 °C since the 3rd day post inoculation) was compared. We studied the upstream sequences of a cold-inducible Arabidopsis thaliana cor15a gene, the core element of 35S CaMV promoter fused to the TMV omega 5'-UTR, and the synthetic promoter including the 35S core sequence and two binding sites for cold-inducible CBF transcription factors (P_DRE::35S). Cultivation of plants transiently expressing reporter gene under control of the synthetic P_DRE::35S promoter under chilling conditions since the 3rd dpi led to the reliably higher reporter accumulation as compared to the other tested regulatory sequences under chilling or greenhouse conditions. Reporter protein fluorescence under chilling conditions using P_DRE::35S reached 160% as compared to the transient expression in the greenhouse. Period of transient expression considerably extended if plants were cultivated at chilling temperature since the 3rd dpi: reporter protein fluorescence reached its maximum at the 20th dpi and was detected in leaves up to the 65th dpi. The enhanced protein accumulation at low temperature was accompanied by the prolonged period of corresponding mRNA accumulation. Transient expression under chilling conditions using synthetic cold-inducible promoter enhances target protein accumulation and may decrease greenhouse heating expenses.

  6. Diversity of Histologic Patterns and Expression of Cytoskeletal Proteins in Canine Skeletal Osteosarcoma.

    Science.gov (United States)

    Nagamine, E; Hirayama, K; Matsuda, K; Okamoto, M; Ohmachi, T; Kadosawa, T; Taniyama, H

    2015-09-01

    Osteosarcoma (OS), the most common bone tumor, includes OS of the head (OSH) and appendicular OS (OSA). In dogs, it is classified into 6 histologic subtypes: osteoblastic, chondroblastic, fibroblastic, telangiectatic, giant cell, and poorly differentiated. This study investigated the significance of the histologic classification relevant to clinical outcome and the histologic and immunohistochemical relationships between pleomorphism and expression of cytoskeletal proteins in 60 cases each of OSH and OSA. Most neoplasms exhibited histologic diversity, and 64% of OS contained multiple subtypes. In addition to the above 6 subtypes, myxoid, round cell, and epithelioid subtypes were observed. Although the epithelioid subtypes were observed in only OSH, no significant difference in the frequency of other subtypes was observed. Also, no significant relevance was observed between the clinical outcome and histologic subtypes. Cytokeratin (CK) was expressed in both epithelioid and sarcomatoid tumor cells in various subtypes, and all CK-positive tumor cells also expressed vimentin. Vimentin and α-smooth muscle actin (SMA) were expressed in all subtypes. A few SMA-positive spindle-shaped tumor cells exhibited desmin expression. Glial fibrillary acidic protein-positive tumor cells were observed in many subtypes, and some of these cells showed neurofilament expression. Although OSH exhibited significantly stronger immunoreactivity for SMA than OSA, no significant difference in other cytoskeletal proteins was observed. Some tumor cells had cytoskeletal protein expression compatible with the corresponding histologic subtypes, such as CK in the epithelioid subtype and SMA in the fibroblastic subtype. Thus, canine skeletal OS is composed of pleomorphic and heterogenous tumor cells as is reflected in the diversity of histologic patterns and expression of cytoskeletal proteins. © The Author(s) 2015.

  7. Recombinant protein production data after expression in the bacterium Escherichia coli

    Directory of Open Access Journals (Sweden)

    J. Enrique Cantu-Bustos

    2016-06-01

    Full Text Available Fusion proteins have become essential for the expression and purification of recombinant proteins in Escherichia coli. The metal-binding protein CusF has shown several features that make it an attractive fusion protein and affinity tag: "Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF" (Cantu-Bustos et al., 2016 [1]. Here we present accompanying data from protein expression experiments; we tested different protein tags, temperatures, expression times, cellular compartments, and concentrations of inducer in order to obtain soluble protein and low formation of inclusion bodies. Additionally, we present data from the purification of the green fluorescent protein (GFP tagged with CusF, using Ag(I metal affinity chromatography.

  8. 100 Gbps PCI-Express readout for the LHCb upgrade

    International Nuclear Information System (INIS)

    Durante, P.; Neufeld, N.; Schwemmer, R.; Balbi, G.; Marconi, U.

    2015-01-01

    We present a new data acquisition system under development for the next upgrade of the LHCb experiment at CERN. We focus in particular on the design of a new generation of readout boards, the PCIe40, and on the viability of PCI-Express as an interconnect technology for high speed readout. We show throughput measurements across the PCI-Express bus, on Altera Stratix 5 devices, using a DMA mechanism and different synchronization schemes between the FPGA and the readout unit. Finally we discuss hardware and software design considerations necessary to achieve a data throughput of 100 Gbps in the final readout board

  9. Use of Donkey Milk in Children with Cow’s Milk Protein Allergy

    Directory of Open Access Journals (Sweden)

    Paolo Polidori

    2013-05-01

    Full Text Available Human breast milk is the best nutritional support that insures the right development and influences the immune status of the newborn infant. However, when it is not possible to breast feed, it may be necessary to use commercial infant formulas that mimic, where possible, the levels and types of nutrients present in human milk. Despite this, some formula-fed infant develops allergy and/or atopic disease compared to breast-fed infants. Cow’s milk allergy can be divided into immunoglobulin IgE mediated food allergy and non-IgE-mediated food allergy. Most infants with cow’s milk protein allergy (CMPA develop symptoms before 1 month of age, often within 1 week after introduction of cow’s milk-based formula. Donkey milk may be considered a good substitute for cow’s milk in feeding children with CMPA since its composition is very similar to human milk. Donkey milk total protein content is low (1.5–1.8 g/100 g, very close to human milk. A thorough analysis of the donkey milk protein profile has been performed in this study; the interest was focused on the milk proteins considered safe for the prevention and treatment of various disorders in humans. The content of lactoferrin, lactoperoxidase and lysozyme, peptides with antimicrobial activity, able to stimulate the development of the neonatal intestine, was determined. Donkey milk is characterized by a low casein content, with values very close to human milk; the total whey protein content in donkey milk ranges between 0.49 and 0.80 g/100 g, very close to human milk (0.68–0.83 g/100 g. Among whey proteins, α-lactalbumin average concentration in donkey milk is 1.8 mg/mL. The results of this study confirmed the possibility of using donkey milk in feeding children with CMPA.

  10. Myonuclear domain size and myosin isoform expression in muscle fibres from mammals representing a 100,000-fold difference in body size.

    Science.gov (United States)

    Liu, Jing-Xia; Höglund, Anna-Stina; Karlsson, Patrick; Lindblad, Joakim; Qaisar, Rizwan; Aare, Sudhakar; Bengtsson, Ewert; Larsson, Lars

    2009-01-01

    This comparative study of myonuclear domain (MND) size in mammalian species representing a 100,000-fold difference in body mass, ranging from 25 g to 2500 kg, was undertaken to improve our understanding of myonuclear organization in skeletal muscle fibres. Myonuclear domain size was calculated from three-dimensional reconstructions in a total of 235 single muscle fibre segments at a fixed sarcomere length. Irrespective of species, the largest MND size was observed in muscle fibres expressing fast myosin heavy chain (MyHC) isoforms, but in the two smallest mammalian species studied (mouse and rat), MND size was not larger in the fast-twitch fibres expressing the IIA MyHC isofom than in the slow-twitch type I fibres. In the larger mammals, the type I fibres always had the smallest average MND size, but contrary to mouse and rat muscles, type IIA fibres had lower mitochondrial enzyme activities than type I fibres. Myonuclear domain size was highly dependent on body mass in the two muscle fibre types expressed in all species, i.e. types I and IIA. Myonuclear domain size increased in muscle fibres expressing both the beta/slow (type I; r = 0.84, P fast IIA MyHC isoform (r = 0.90; P muscle fibre type, independent of species. However, myosin isoform expression is not the sole protein determining MND size, and other protein systems, such as mitochondrial proteins, may be equally or more important determinants of MND size.

  11. Effects of immunosuppressive treatment on protein expression in rat kidney

    Directory of Open Access Journals (Sweden)

    Kędzierska K

    2014-09-01

    Full Text Available Karolina Kędzierska,1 Katarzyna Sporniak-Tutak,2 Krzysztof Sindrewicz,2 Joanna Bober,3 Leszek Domański,1 Mirosław Parafiniuk,4 Elżbieta Urasińska,5 Andrzej Ciechanowicz,6 Maciej Domański,1 Tomasz Smektała,2 Marek Masiuk,5 Wiesław Skrzypczak,6 Małgorzata Ożgo,6 Joanna Kabat-Koperska,1 Kazimierz Ciechanowski1 1Department of Nephrology, Transplantology, and Internal Medicine, 2Department of Dental Surgery, 3Department of Medical Chemistry, 4Department of Forensic Medicine, 5Department of Pathomorphology, Pomeranian Medical University, 6Department of Physiology, Cytobiology, and Proteomics, West Pomeranian University of Technology, Szczecin, Poland Abstract: The structural proteins of renal tubular epithelial cells may become a target for the toxic metabolites of immunosuppressants. These metabolites can modify the properties of the proteins, thereby affecting cell function, which is a possible explanation for the mechanism of immunosuppressive agents' toxicity. In our study, we evaluated the effect of two immunosuppressive strategies on protein expression in the kidneys of Wistar rats. Fragments of the rat kidneys were homogenized after cooling in liquid nitrogen and then dissolved in lysis buffer. The protein concentration in the samples was determined using a protein assay kit, and the proteins were separated by two-dimensional electrophoresis. The obtained gels were then stained with Coomassie Brilliant Blue, and their images were analyzed to evaluate differences in protein expression. Identification of selected proteins was then performed using mass spectrometry. We found that the immunosuppressive drugs used in popular regimens induce a series of changes in protein expression in target organs. The expression of proteins involved in drug, glucose, amino acid, and lipid metabolism was pronounced. However, to a lesser extent, we also observed changes in nuclear, structural, and transport proteins' synthesis. Very slight differences

  12. The XylS/Pm regulator/promoter system and its use in fundamental studies of bacterial gene expression, recombinant protein production and metabolic engineering.

    Science.gov (United States)

    Gawin, Agnieszka; Valla, Svein; Brautaset, Trygve

    2017-07-01

    The XylS/Pm regulator/promoter system originating from the Pseudomonas putida TOL plasmid pWW0 is widely used for regulated low- and high-level recombinant expression of genes and gene clusters in Escherichia coli and other bacteria. Induction of this system can be graded by using different cheap benzoic acid derivatives, which enter cells by passive diffusion, operate in a dose-dependent manner and are typically not metabolized by the host cells. Combinatorial mutagenesis and selection using the bla gene encoding β-lactamase as a reporter have demonstrated that the Pm promoter, the DNA sequence corresponding to the 5' untranslated end of its cognate mRNA and the xylS coding region can be modified and improved relative to various types of applications. By combining such mutant genetic elements, altered and extended expression profiles were achieved. Due to their unique properties, obtained systems serve as a genetic toolbox valuable for heterologous protein production and metabolic engineering, as well as for basic studies aiming at understanding fundamental parameters affecting bacterial gene expression. The approaches used to modify XylS/Pm should be adaptable for similar improvements also of other microbial expression systems. In this review, we summarize constructions, characteristics, refinements and applications of expression tools using the XylS/Pm system. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  13. The prognostic value of serum S100B in patients with cutaneous melanoma: a meta-analysis.

    Science.gov (United States)

    Mocellin, Simone; Zavagno, Giorgio; Nitti, Donato

    2008-11-15

    S100B protein detected in the serum of patients with cutaneous melanoma has been long reported as a prognostic biomarker. However, no consensus exists on its implementation in the routine clinical setting. This study aimed to comprehensively and quantitatively summarize the evidence on the suitability of serum S100B to predict patients' survival. Twenty-two series enrolling 3393 patients with TNM stage I to IV cutaneous melanoma were reviewed. Standard meta-analysis methods were applied to evaluate the overall relationship between S100B serum levels and patients' survival (meta-risk). Serum S100B positivity was associated with significantly poorer survival (hazard ratio [HR] = 2.23, 95% CI: 1.92-2.58, p < 0.0001). Between-study heterogeneity was significant, which appeared to be related mainly to dissemination bias and the inclusion of patients with stage IV disease. Considering stage I to III melanoma (n = 1594), the meta-risk remained highly significant (HR = 2.28, 95% CI: 1.8-2.89; p < 0.0001) and studies' estimates were homogeneous. Subgroup analysis of series reporting multivariate survival analysis supported S100B as a prognostic factor independent of the TNM staging system. Our findings suggest that serum S100B detection has a clinically valuable independent prognostic value in patients with melanoma, with particular regard to stage I-III disease. Further investigation focusing on this subset of patients is justified and warranted before S100B can be implemented in the routine clinical management of melanoma. (c) 2008 Wiley-Liss, Inc.

  14. Effects of electroacupuncture at 2 and 100 Hz on rat type 2 diabetic neuropathic pain and hyperalgesia-related protein expression in the dorsal root ganglion.

    Science.gov (United States)

    He, Xiao-Fen; Wei, Jun-Jun; Shou, Sheng-Yun; Fang, Jian-Qiao; Jiang, Yong-Liang

    To investigate the analgesic effects of electroacupuncture (EA) at 2 and 100 Hz on type 2 diabetic neuropathic pain (DNP) and on the expressions of the P2X3 receptor and calcitonin gene-related peptide (CGRP) in the dorsal root ganglion (DRG). Rat type 2 DNP was induced by a high calorie and high sugar diet fed for 7 weeks, plus a single intraperitoneal injection of streptozotocin (STZ) after 5 weeks. EA at 2 and 100 Hz was carried out once every day after 7 weeks for 7 consecutive days. Body weight, serum fasting insulin (FINS), fasting blood glucose (FBG), insulin sensitivity index (ISI), and paw withdrawal latency (PWL) were measured. The expressions of L4-L6 DRG P2X3 receptors and CGRP were assessed by immunofluorescence. Type 2 DNP was successfully induced as shown by the increased body weight, FINS, and FBG, as well as the reduced ISI and PWL. Expressions of P2X3 receptors and CGRP in L4-L6 DRGs increased. EA at both 2 and 100 Hz relieved type 2 DNP, but the analgesic effect of EA was stronger at 2 Hz. P2X3 receptor expression decreased in L4-L6 DRGs following EA at 2 Hz and in L5 and L6 DRGs following EA at 100 Hz. EA at both 2 and 100 Hz down-regulated CGRP overexpression in L4-L6 DRGs. These findings indicate that EA at 2 Hz is a good option for the management of type 2 DNP. The EA effect may be related to its down-regulation of the overexpressions of the DRG P2X3 receptors and CGRP in this condition.

  15. Six1 induces protein synthesis signaling expression in duck myoblasts mainly via up-regulation of mTOR

    Directory of Open Access Journals (Sweden)

    Haohan Wang

    2016-03-01

    Full Text Available Abstract As a critical transcription factor, Six1 plays an important role in the regulation of myogenesis and muscle development. However, little is known about its regulatory mechanism associated with muscular protein synthesis. The objective of this study was to investigate the effects of overexpression ofSix1 on the expression of key protein metabolism-related genes in duck myoblasts. Through an experimental model where duck myoblasts were transfected with a pEGFP-duSix1 construct, we found that overexpression of duckSix1 could enhance cell proliferation activity and increase mRNA expression levels of key genes involved in the PI3K/Akt/mTOR signaling pathway, while the expression of FOXO1, MuRF1and MAFbx was not significantly altered, indicating thatSix1 could promote protein synthesis in myoblasts through up-regulating the expression of several related genes. Additionally, in duck myoblasts treated with LY294002 and rapamycin, the specific inhibitors ofPI3K and mTOR, respectively, the overexpression of Six1 could significantly ameliorate inhibitive effects of these inhibitors on protein synthesis. Especially, the mRNA expression levels of mTOR and S6K1 were observed to undergo a visible change, and a significant increase in protein expression of S6K1 was seen. These data suggested that Six1plays an important role in protein synthesis, which may be mainly due to activation of the mTOR signaling pathway.

  16. G-protein α-subunit expression, myristoylation, and membrane association in COS cells

    International Nuclear Information System (INIS)

    Mumby, S.M.; Gilman, A.G.; Heukeroth, R.O.; Gordon, J.I.

    1990-01-01

    Myristolyation of seven different α subunits of guanine nucleotide-binding regulatory proteins (G proteins) was examined by expressing these proteins in monkey kidney COS cells. Metabolic labeling studies of cells transfected with cytomegalovirus-based expression vectors indicated that [ 3 H]myristate was incorporated into α i1 , α i2 , α i3 , α 0 , and α 1 , and α z but not α s subunits. The role of myristoylation in the association of α subunits with membranes was analyzed by site-directed mutagenesis and by substitution of myristate with a less hydrophobic analog, 10-(propoxy)decanoate (11-oxamyristate). Myristoylation of α 0 was blocked when an alanine residue was substituted for its amino-terminal glycine, as was association of the protein with membranes. Substitution of the myristoyl group with 11-oxamyristate affected the cellular distribution of a subset of acylated α subunits. The results are consistent with a model wherein the hydrophobic interaction of myristate with the bilayer permits continued association of the protein with the plasma membrane when G-protein α subunits dissociated from βγ

  17. The characterization of a novel S100A1 binding site in the N-terminus of TRPM1

    Czech Academy of Sciences Publication Activity Database

    Jirků, M.; Lánský, Z.; Bednárová, Lucie; Šulc, M.; Monincová, Lenka; Majer, Pavel; Vyklický, L.; Vondrášek, Jiří; Teisinger, J.; Boušová, Kristýna

    2016-01-01

    Roč. 78, Sep (2016), s. 186-193 ISSN 1357-2725 Institutional support: RVO:61388963 Keywords : TRPM1 channel * binding site * calcium-binding protein S100A1 * steady-state fluorescence anisotropy * molecular modeling * circular dichroism Subject RIV: CE - Biochemistry Impact factor: 3.505, year: 2016

  18. Potential role of S100A8 in skin rejuvenation with the 1064-nm Q-switched Nd:YAG laser.

    Science.gov (United States)

    Qin, Yan; Qin, Xiaofeng; Xu, Peng; Zhi, Yuanting; Xia, Weili; Dang, Yongyan; Gu, Jun; Ye, Xiyun

    2018-04-01

    The 1064-nm Q-switched Nd:YAG laser is demonstrated to be effective for non-ablative skin rejuvenation, but the molecular mechanism by which dermis responses to laser-induced damage and initiates skin remodeling is still unclear. HaCaT cells and 3T3 skin fibroblasts were irradiated with the 1064-nm Q-switched Nd:YAG laser at the different doses. Then, cells were collected and lysed for PCR and Western blot analysis. Cell viability was detected by Cell Counting Kit-8 (CCK-8) before and after laser irradiation. The expressions of S100A8, advanced glycosylation end product-specific receptor (RAGE) and inflammatory cytokines in two cell lines were markedly upregulated after laser treatments. The PCR, Western blot, and ELISA analysis showed the significant increase of type I and III procollagen in the 3T3 cells treated with the 1064-nm laser. Interestingly, si S100A8 effectively inhibited the expression of cytokines and collagen, while S100A8 treatments significantly increased them. P-p38 and p-p65 levels were also elevated after the 1064-nm laser irradiation, which is positively related with S100A8. Cell viability and reactive oxygen species (ROS) levels were not changed, while the content of superoxidase dismutase (SOD) in two cells was increased after laser irradiation. Our results demonstrated that the overexpression of S100A8 induced by the 1064-nm laser irradiation triggered inflammatory reactions in skin cells. The inflammatory microenvironment and improvement of skin antioxidant capacity contribute to new collagen synthesis in the skin cells. Thus, S100A8 was required for laser-induced new collagen synthesis in skin cells. p38/MAPK and NF-κB signal pathways were involved in S100A8-mediated inflammatory reactions in response to laser irradiation.

  19. Scutellaria barbata attenuates diabetic retinopathy by preventing retinal inflammation and the decreased expression of tight junction protein

    Directory of Open Access Journals (Sweden)

    Xi-Yu Mei

    2017-06-01

    Full Text Available AIM: To observe the attenuation of ethanol extract of Herba Scutellaria barbata (SE against diabetic retinopathy (DR and its engaged mechanism. METHODS: C57BL/6J mice were intraperitoneally injected with streptozotocin (STZ, 55 mg/kg for 5 consecutive days to induce diabetes. The diabetic mice were orally given with SE (100, 200 mg/kg for 1mo at 1mo after STZ injection. Blood-retinal barrier (BRB breakdown was detected by using Evans blue permeation assay. Real-time polymerase chain reaction (RT-PCR, Western blot and immunofluorescence staining were used to detect mRNA and protein expression. Enzyme-linked immunosorbent assay (ELISA was used to detect serum contents of tumor necrosis factor-α (TNF-α and interleukin (IL-1β. RESULTS: SE (100, 200 mg/kg reversed the breakdown of BRB in STZ-induced diabetic mice. The decreased expression of retinal claudin-1 and claudin-19, which are both tight junction (TJ proteins, was reversed by SE. SE decreased the increased serum contents and retinal mRNA expression of TNF-α and IL-1β. SE also decreased the increased retinal expression of intercellular cell adhesion molecule-1 (ICAM-1. SE reduced the increased phosphorylation of nuclear factor kappa B (NFκB p65 and its subsequent nuclear translocation in retinas from STZ-induced diabetic mice. Results of Western blot and retinal immunofluorescence staining of ionized calcium-binding adapter molecule 1 (Iba1 demonstrated that SE abrogated the activation of microglia cells in STZ-induced diabetic mice. CONCLUSION: SE attenuates the development of DR by inhibiting retinal inflammation and restoring the decreased expression of TJ proteins including claudin-1 and claudin-19.

  20. Transient Co-Expression of Post-Transcriptional Gene Silencing Suppressors for Increased in Planta Expression of a Recombinant Anthrax Receptor Fusion Protein

    Directory of Open Access Journals (Sweden)

    Kittipong Rattanaporn

    2011-08-01

    Full Text Available Potential epidemics of infectious diseases and the constant threat of bioterrorism demand rapid, scalable, and cost-efficient manufacturing of therapeutic proteins. Molecular farming of tobacco plants provides an alternative for the recombinant production of therapeutics. We have developed a transient production platform that uses Agrobacterium infiltration of Nicotiana benthamiana plants to express a novel anthrax receptor decoy protein (immunoadhesin, CMG2-Fc. This chimeric fusion protein, designed to protect against the deadly anthrax toxins, is composed of the von Willebrand factor A (VWA domain of human capillary morphogenesis 2 (CMG2, an effective anthrax toxin receptor, and the Fc region of human immunoglobulin G (IgG. We evaluated, in N. benthamiana intact plants and detached leaves, the expression of CMG2-Fc under the control of the constitutive CaMV 35S promoter, and the co-expression of CMG2-Fc with nine different viral suppressors of post-transcriptional gene silencing (PTGS: p1, p10, p19, p21, p24, p25, p38, 2b, and HCPro. Overall, transient CMG2-Fc expression was higher on intact plants than detached leaves. Maximum expression was observed with p1 co-expression at 3.5 days post-infiltration (DPI, with a level of 0.56 g CMG2-Fc per kg of leaf fresh weight and 1.5% of the total soluble protein, a ten-fold increase in expression when compared to absence of suppression. Co-expression with the p25 PTGS suppressor also significantly increased the CMG2-Fc expression level after just 3.5 DPI.

  1. Transient co-expression of post-transcriptional gene silencing suppressors for increased in planta expression of a recombinant anthrax receptor fusion protein.

    Science.gov (United States)

    Arzola, Lucas; Chen, Junxing; Rattanaporn, Kittipong; Maclean, James M; McDonald, Karen A

    2011-01-01

    Potential epidemics of infectious diseases and the constant threat of bioterrorism demand rapid, scalable, and cost-efficient manufacturing of therapeutic proteins. Molecular farming of tobacco plants provides an alternative for the recombinant production of therapeutics. We have developed a transient production platform that uses Agrobacterium infiltration of Nicotiana benthamiana plants to express a novel anthrax receptor decoy protein (immunoadhesin), CMG2-Fc. This chimeric fusion protein, designed to protect against the deadly anthrax toxins, is composed of the von Willebrand factor A (VWA) domain of human capillary morphogenesis 2 (CMG2), an effective anthrax toxin receptor, and the Fc region of human immunoglobulin G (IgG). We evaluated, in N. benthamiana intact plants and detached leaves, the expression of CMG2-Fc under the control of the constitutive CaMV 35S promoter, and the co-expression of CMG2-Fc with nine different viral suppressors of post-transcriptional gene silencing (PTGS): p1, p10, p19, p21, p24, p25, p38, 2b, and HCPro. Overall, transient CMG2-Fc expression was higher on intact plants than detached leaves. Maximum expression was observed with p1 co-expression at 3.5 days post-infiltration (DPI), with a level of 0.56 g CMG2-Fc per kg of leaf fresh weight and 1.5% of the total soluble protein, a ten-fold increase in expression when compared to absence of suppression. Co-expression with the p25 PTGS suppressor also significantly increased the CMG2-Fc expression level after just 3.5 DPI.

  2. Serum levels of psoriasin (S100A7) and koebnerisin (S100A15) as potential markers of atherosclerosis in patients with psoriasis.

    Science.gov (United States)

    Awad, S M; Attallah, D A; Salama, R H; Mahran, A M; Abu El-Hamed, E

    2018-04-01

    Psoriasin (S100A7) and koebnerisin (S100A15) are proinflammatory proteins upregulated in psoriasis, but their relation to atherosclerosis remains unclear. To evaluate the role of serum psoriasin and koebnerisin as possible markers for subclinical atherosclerosis in patients with psoriasis. Serum levels of psoriasin and koebnerisin were measured by ELISA in 45 patients with psoriasis and in 45 healthy controls (HCs). Intima-media thickness (IMT) of the right and left common carotid arteries was measured to detect the presence of subclinical atherosclerosis. Clinical severity of psoriasis was estimated using the Psoriasis Area and Severity Index (PASI). Compared with HCs, patients with psoriasis had significantly higher levels of psoriasin (26.61 ± 22.45 ng/mL vs. 6.31 ± 1.68 ng/mL, P  0.01) and serum koebnerisin (r = 0.48, P psoriasis with subclinical atherosclerosis had higher serum levels of koebnerisin compared with patients without subclinical atherosclerosis (P = 0.04), which was not observed for psoriasin (P = 0.94). Serum psoriasin and koebnerisin correlate with IMT, underlining their value as a potential link between psoriasis and atherosclerosis. In particular, koebnerisin seems to be a useful marker of subclinical atherosclerosis in patients with psoriasis. © 2018 British Association of Dermatologists.

  3. Differential protein expression in mussels Mytilus galloprovincialis exposed to nano and ionic Ag

    International Nuclear Information System (INIS)

    Gomes, Tânia; Pereira, Catarina G.; Cardoso, Cátia; Bebianno, Maria João

    2013-01-01

    Highlights: •Different protein expression profiles between tissues and Ag forms. •Ag NPs and Ag + presented different mechanisms of toxic action. •Ag NPs toxicity is mediated by oxidative stress-induced cell signalling cascades. •New biomarkers for Ag NPs were proposed, i.e. MVP, ras partial and precol-P. -- Abstract: Ag NPs are one of the most commonly used NPs in nanotechnology whose environmental impacts are to date unknown and the information about bioavailability, mechanisms of biological uptake and toxic implications in organisms is scarce. So, the main objective of this study was to investigate differences in protein expression profiles in gills and digestive gland of mussels Mytilus galloprovincialis exposed to Ag NPs and Ag + (10 μg L −1 ) for a period of 15 days. Protein expression profiles of exposed gills and digestive glands were compared to those of control mussels using two–dimensional electrophoresis to discriminate differentially expressed proteins. Different patterns of protein expression were obtained for exposed mussels, dependent not only on the different redox requirements of each tissue but also to the Ag form used. Unique sets of differentially expressed proteins were affected by each silver form in addition to proteins that were affected by both Ag NPs and Ag + . Fifteen of these proteins were subsequently identified by MALDI–TOF–TOF and database search. Ag NPs affected similar cellular pathways as Ag + , with common response mechanisms in cytoskeleton and cell structure (catchin, myosin heavy chain), stress response (heat shock protein 70), oxidative stress (glutathione s-transferase), transcriptional regulation (nuclear receptor subfamily 1G), adhesion and mobility (precollagen-P) and energy metabolism (ATP synthase F0 subunit 6 and NADH dehydrogenase subunit 2). Exposure to Ag NPs altered the expression of two proteins associated with stress response (major vault protein and ras partial) and one protein involved in

  4. Differential protein expression in mussels Mytilus galloprovincialis exposed to nano and ionic Ag

    Energy Technology Data Exchange (ETDEWEB)

    Gomes, Tânia; Pereira, Catarina G.; Cardoso, Cátia; Bebianno, Maria João, E-mail: mbebian@ualg.pt

    2013-07-15

    Highlights: •Different protein expression profiles between tissues and Ag forms. •Ag NPs and Ag{sup +} presented different mechanisms of toxic action. •Ag NPs toxicity is mediated by oxidative stress-induced cell signalling cascades. •New biomarkers for Ag NPs were proposed, i.e. MVP, ras partial and precol-P. -- Abstract: Ag NPs are one of the most commonly used NPs in nanotechnology whose environmental impacts are to date unknown and the information about bioavailability, mechanisms of biological uptake and toxic implications in organisms is scarce. So, the main objective of this study was to investigate differences in protein expression profiles in gills and digestive gland of mussels Mytilus galloprovincialis exposed to Ag NPs and Ag{sup +} (10 μg L{sup −1}) for a period of 15 days. Protein expression profiles of exposed gills and digestive glands were compared to those of control mussels using two–dimensional electrophoresis to discriminate differentially expressed proteins. Different patterns of protein expression were obtained for exposed mussels, dependent not only on the different redox requirements of each tissue but also to the Ag form used. Unique sets of differentially expressed proteins were affected by each silver form in addition to proteins that were affected by both Ag NPs and Ag{sup +}. Fifteen of these proteins were subsequently identified by MALDI–TOF–TOF and database search. Ag NPs affected similar cellular pathways as Ag{sup +}, with common response mechanisms in cytoskeleton and cell structure (catchin, myosin heavy chain), stress response (heat shock protein 70), oxidative stress (glutathione s-transferase), transcriptional regulation (nuclear receptor subfamily 1G), adhesion and mobility (precollagen-P) and energy metabolism (ATP synthase F0 subunit 6 and NADH dehydrogenase subunit 2). Exposure to Ag NPs altered the expression of two proteins associated with stress response (major vault protein and ras partial) and one

  5. Enhanced green fluorescent protein is a nearly ideal long-term expression tracer for hematopoietic stem cells, whereas DsRed-express fluorescent protein is not.

    Science.gov (United States)

    Tao, Wen; Evans, Barbara-Graham; Yao, Jing; Cooper, Scott; Cornetta, Kenneth; Ballas, Christopher B; Hangoc, Giao; Broxmeyer, Hal E

    2007-03-01

    Validated gene transfer and expression tracers are essential for elucidating functions of mammalian genes. Here, we have determined the suitability and unintended side effects of enhanced green fluorescent protein (EGFP) and DsRed-Express fluorescent protein as expression tracers in long-term hematopoietic stem cells (HSCs). Retrovirally transduced mouse bone marrow cells expressing either EGFP or DsRed-Express in single or mixed dual-color cell populations were clearly discerned by flow cytometry and fluorescence microscopy. The results from in vivo competitive repopulation assays demonstrated that EGFP-expressing HSCs were maintained nearly throughout the lifespan of the transplanted mice and retained long-term multilineage repopulating potential. All mice assessed at 15 months post-transplantation were EGFP positive, and, on average, 24% total peripheral white blood cells expressed EGFP. Most EGFP-expressing recipient mice lived at least 22 months. In contrast, Discosoma sp. red fluorescent protein (DsRed)-expressing donor cells dramatically declined in transplant-recipient mice over time, particularly in the competitive setting, in which mixed EGFP- and DsRed-expressing cells were cotransplanted. Moreover, under in vitro culture condition favoring preservation of HSCs, purified EGFP-expressing cells grew robustly, whereas DsRed-expressing cells did not. Therefore, EGFP has no detectable deteriorative effects on HSCs, and is nearly an ideal long-term expression tracer for hematopoietic cells; however, DsRed-Express fluorescent protein is not suitable for these cells.

  6. [Association between S100B gene polymorphisms and hand, foot and mouth disease caused by enterovirus 71 infection].

    Science.gov (United States)

    Li, Jing; Shan, Ruo-Bing; Liu, Rui-Hai; Xu, Ying-Jun; Qu, Ni-Yan; Pan, Gui-Mei; Zhang, Na; Yang, Na; Chen, Zhen-Zhen; Zhang, Wen-Xiang; Li, Zi-Pu

    2017-08-01

    To investigate the association between rs9722 polymorphisms in the S100B gene and hand, foot and mouth disease (HFMD) caused by enterovirus 71. A total of 124 HFMD children with enterovirus 71 infection were enrolled as subjects, and 56 healthy children were enrolled as control group. The rs9722 polymorphisms in the S100B gene were detected for both groups, and the serum level of S100B protein was measured for 74 HFMD children. The rs9722 locus of the S100B gene had three genotypes, CC, CT, and TT, and the genotype frequencies were in accordance with Hardy-Weinberg equilibrium. Compared with the control group, the HFMD group had significant increases in the frequencies of TT genotype and T allele (Penterovirus 71 infection had significantly higher frequencies of TT genotype and T allele than those with moderate or mild HFMD (Penterovirus 71 infection.

  7. Junction region of EWS-FLI1 fusion protein has a dominant negative effect in Ewing’s Sarcoma in vitro

    International Nuclear Information System (INIS)

    Jully, Babu; Vijayalakshmi, Ramshankar; Gopal, Gopisetty; Sabitha, Kesavan; Rajkumar, Thangarajan

    2012-01-01

    Ewing’s sarcoma is a malignancy characterized by a specific 11:22 chromosomal translocation which generates a novel EWS-FLI1 fusion protein functioning as an aberrant transcription factor. In the present study, we have further characterized the junction region of the EWS-FLI1 fusion protein. In-silico model of EWS-FLI1 fusion protein was analysed for ligand binding sites, and a putative region (amino acid (aa) 251–343 of the type 1 fusion protein) in the vicinity of the fusion junction was cloned and expressed using bacterial expression. The recombinant protein was characterized by Circular Dichroism (CD). We then expressed aa 251–280 ectopically in Ewing’s sarcoma cell-line and its effect on cell proliferation, tumorigenicity and expression of EWS-FLI1 target genes were analysed. Our modelling analysis indicated that Junction region (aa 251–343) encompasses potential ligand biding sites in the EWS-FLI1 protein and when expressed in bacteria was present as soluble form. Ectopically expressing this region in Ewing’s sarcoma cells inhibited tumorigenicity, and EWS-FLI1 target genes indicating a dominant negative biological effect. Junction region can be exploited further as target for drug development in future to specifically target EWS-FLI1 in Ewing’s Sarcoma

  8. XL-100S microprogrammable processor

    International Nuclear Information System (INIS)

    Gorbunov, N.V.; Guzik, Z.; Sutulin, V.A.; Forytski, A.

    1983-01-01

    The XL-100S microprogrammable processor providing the multiprocessor operation mode in the XL system crate is described. The processor meets the EUR 6500 CAMAC standards, address up to 4 Mbyte memory, and interacts with 7 CAMAC branchas. Eight external requests initiate operations preset by a sequence of microcommands in a memory of the capacity up to 64 kwords of 32-Git. The microprocessor architecture allows one to emulate commands of the majority of mini- or micro-computers, including floating point operations. The XL-100S processor may be used in various branches of experimental physics: for physical experiment apparatus control, fast selection of useful physical events, organization of the of input/output operations, organization of direct assess to memory included, etc. The Am2900 microprocessor set is used as an elementary base. The device is made in the form of a single width CAMAC module

  9. Aberrant intermediate filament and synaptophysin expression is a frequent event in malignant melanoma: an immunohistochemical study of 73 cases.

    Science.gov (United States)

    Romano, Ryan C; Carter, Jodi M; Folpe, Andrew L

    2015-08-01

    Malignant melanomas are known to express vimentin, among other intermediate filaments. Though anomalous keratin expression by malignant melanoma has been reported, its frequency is not well-established and this phenomenon is not well-known. We have seen in consultation a number of malignant melanomas with anomalous expression of keratin, other intermediate filaments, or synaptophysin, and therefore studied a large group of primary and metastatic melanomas to determine the frequency of these events. About 73 cases of malignant melanoma (22 primaries and 51 metastases) from 71 patients (51 male, 20 female; mean 59 years, range 17-87 years) were retrieved from our archives. Prior diagnoses were confirmed by re-review of hematoxylin and eosin sections and relevant (e.g., S100 protein, HMB45, Melan-A, and tyrosinase) immunohistochemical studies. Available sections were immunostained for keratin (OSCAR and AE1/AE3 antibodies), desmin, neurofilament protein, glial fibrillary acidic protein, synaptophysin, and chromogranin A. Not all cases could be tested for all markers. Cases were predominantly epithelioid (48/73, 66%) or spindle cell/desmoplastic (25/73, 34%). S100 protein, Melan-A, HMB45, and tyrosinase were positive in 60/65 (92%), 34/64 (53%), 30/60 (50%), 25/48 (52%) of cases, respectively. All five S100-protein-negative cases expressed at least one of the other melanocytic markers: Melan-A (two of four, 50%), HMB45 (two of three, 67%), and tyrosinase (one of two, 50%). All cases expressed at least one melanocytic marker. Cases were positive for keratin (OSCAR, 17/61, 28%; AE1/AE3, 16/40, 40%), desmin (11/47, 24%), neurofilament protein (5/31, 16%), glial fibrillary acidic protein (3/32, 9%), and synaptophysin (10/34, 29%), typically only in a minority of cells. Chromogranin was negative (0/32, 0%). Altogether 9/73 cases (12%) showed expression of >1 intermediate filament. All S100-protein-negative melanomas showed anomalous intermediate filament expression (keratin

  10. Altered gravity causes the changes in the proteins NoA100 in plant cell nucleoli

    Science.gov (United States)

    Sobol, Margarita A.; Gonzalez-Camacho, Fernando; Kordyum, Elizabeth L.; Medina, Francisco Javier

    2005-08-01

    A nucleolar protein homologous to the mammalian nucleolin and to the onion nucleolin-like protein NopA100 was detected in nuclear soluble protein fraction from Lepidium sativum root meristematic cells, using the specific silver staining method and the cross-reaction with the anti-NopA100 antibody. In 2D Western blots of soluble nuclear fraction, NopA100 was revealed as a smear extending through a certain range of pI. In extracts obtained from seedlings grown under clinorotation, the extension of the pI range was shorter than in the stationary control indicating a lower phosphorylation of the protein. This suggests that altered gravity causes a decrease in the rate of nucleolar activity.

  11. Overexpression of pucC improves the heterologous protein expression level in a Rhodobacter sphaeroides expression system.

    Science.gov (United States)

    Cheng, L; Chen, G; Ding, G; Zhao, Z; Dong, T; Hu, Z

    2015-04-27

    The Rhodobacter sphaeroides system has been used to express membrane proteins. However, its low yield has substantially limited its application. In order to promote the protein expression capability of this system, the pucC gene, which plays a crucial role in assembling the R. sphaeroides light-harvesting 2 complex (LH2), was overexpressed. To build a pucC overexpression strain, a pucC overexpression vector was constructed and transformed into R. sphaeroides CQU68. The overexpression efficiency was evaluated by quantitative real-time polymerase chain reaction. A well-used reporter β-glucuronidase (GUS) was fusion-expressed with LH2 to evaluate the heterologous protein expression level. As a result, the cell culture and protein in the pucC overexpression strain showed much higher typical spectral absorption peaks at 800 and 850 nm compared with the non-overexpression strain, suggesting a higher expression level of LH2-GUS fusion protein in the pucC overexpression strain. This result was further confirmed by Western blot, which also showed a much higher level of heterologous protein expression in the pucC overexpression strain. We further compared GUS activity in pucC overexpression and non-overexpression strains, the results of which showed that GUS activity in the pucC overexpression strain was approximately ten-fold that in the non-overexpression strain. These results demonstrate that overexpressed pucC can promote heterologous protein expression levels in R. sphaeroides.

  12. Complement inhibitory proteins expression in placentas of thrombophilic women Complement inhibitory proteins expression in placentas of thrombophilic women

    Directory of Open Access Journals (Sweden)

    Przemysław Krzysztof Wirstlein

    2012-10-01

    Full Text Available Factors controlling complement activation appear to exert a protective effect on pregnancy. This is
    particularly important in women with thrombophilia. The aim of this study was to determine the transcript and
    protein levels of complement decay-accelerating factor (DAF and membrane cofactor protein (MCP in the
    placentas of women with acquired and inherited thrombophilia. Also, we assessed immunohistochemistry staining
    of inhibitors of the complement cascade, DAF and MCP proteins, in the placentas of thrombophilic women.
    Placentas were collected from eight women with inherited thrombophilia and ten with acquired thrombophilia.
    The levels of DAF and MCP transcripts were evaluated by qPCR, the protein level was evaluated by Western
    blot. We observed a higher transcript (p < 0.05 and protein (p < 0.001 levels of DAF and MCP in the placentas
    of thrombophilic women than in the control group. DAF and MCP were localized on villous syncytiotrophoblast
    membranes, but the assessment of staining in all groups did not differ. The observed higher expression level of
    proteins that control activation of complement control proteins is only seemingly contradictory to the changes
    observed for example in the antiphospholipid syndrome. However, given the hitherto known biochemical changes
    associated with thrombophilia, a mechanism in which increased expression of DAF and MCP in the placentas is
    an effect of proinflammatory cytokines, which accompanies thrombophilia, is probable.Factors controlling complement activation appear to exert a protective effect on pregnancy. This is
    particularly important in women with thrombophilia. The aim of this study was to determine the transcript and
    protein levels of complement decay-accelerating factor (DAF and membrane cofactor protein (MCP in the
    placentas of women with acquired and inherited thrombophilia. Also, we assessed immunohistochemistry

  13. Expression and roles of pannexins in ATP release in the pituitary gland.

    Science.gov (United States)

    Li, Shuo; Bjelobaba, Ivana; Yan, Zonghe; Kucka, Marek; Tomic, Melanija; Stojilkovic, Stanko S

    2011-06-01

    Pannexins are a newly discovered three-member family of proteins expressed in the brain and peripheral tissues that belong to the superfamily of gap junction proteins. However, in mammals pannexins do not form gap junctions, and their expression and function in the pituitary gland have not been studied. Here we show that the rat pituitary gland expresses mRNA and protein transcripts of pannexins 1 and 2 but not pannexin 3. Pannexin 1 was more abundantly expressed in the anterior lobe, whereas pannexin 2 was more abundantly expressed in the intermediate and posterior pituitary. Pannexin 1 was identified in corticotrophs and a fraction of somatotrophs, the S100-positive pituicytes of the posterior pituitary and AtT-20 (mouse pituitary adrenocorticotropin-secreting cells) and rat immortalized pituitary cells secreting prolactin, whereas pannexin 2 was detected in the S100-positive folliculostellate cells of the anterior pituitary, melanotrophs of the intermediate lobe, and vasopressin-containing axons and nerve endings in the posterior lobe. Overexpression of pannexins 1 and 2 in AtT-20 pituitary cells enhanced the release of ATP in the extracellular medium, which was blocked by the gap junction inhibitor carbenoxolone. Basal ATP release in At-T20 cells was also suppressed by down-regulating the expression of endogenous pannexin 1 but not pannexin 2 with their short interfering RNAs. These results indicate that pannexins may provide a pathway for delivery of ATP, which is a native agonist for numerous P2X cationic channels and G protein-coupled P2Y receptors endogenously expressed in the pituitary gland.

  14. AR-v7 protein expression is regulated by protein kinase and phosphatase

    Science.gov (United States)

    Li, Yinan; Xie, Ning; Gleave, Martin E.; Rennie, Paul S.; Dong, Xuesen

    2015-01-01

    Failure of androgen-targeted therapy and progression of castration-resistant prostate cancer (CRPC) are often attributed to sustained expression of the androgen receptor (AR) and its major splice variant, AR-v7. Although the new generation of anti-androgens such as enzalutamide effectively inhibits AR activity, accumulating pre-clinical and clinical evidence indicates that AR-v7 remains constitutively active in driving CRPC progression. However, molecular mechanisms which control AR-v7 protein expression remain unclear. We apply multiple prostate cancer cell models to demonstrate that enzalutamide induces differential activation of protein phosphatase-1 (PP-1) and Akt kinase depending on the gene context of cancer cells. The balance between PP-1 and Akt activation governs AR phosphorylation status and activation of the Mdm2 ubiquitin ligase. Mdm2 recognizes phosphorylated serine 213 of AR-v7, and induces AR-v7 ubiquitination and protein degradation. These findings highlight the decisive roles of PP-1 and Akt for AR-v7 protein expression and activities when AR is functionally blocked. PMID:26378044

  15. Transduced PEP-1-ribosomal protein S3 (rpS3) ameliorates 12-O-tetradecanoylphorbol-13-acetate-induced inflammation in mice

    International Nuclear Information System (INIS)

    Ahn, Eun Hee; Kim, Dae Won; Kang, Hye Won; Shin, Min Jae; Won, Moo Ho; Kim, Joon; Kim, Dong Joon; Kwon, Oh-Shin; Kang, Tae-Cheon; Han, Kyu Hyung; Park, Jinseu; Eum, Won Sik; Choi, Soo Young

    2010-01-01

    This study investigated the preventive effect of ribosomal protein S3 (rpS3) on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema in mice. A cell permeable expression vector PEP-1-rpS3 was constructed. Topical application of the vector markedly inhibited TPA-induced expression levels of cyclooxygenase-2 (COX-2) and pro-inflammatory cytokines. Application of PEP-1-rpS3 also resulted in a significant reduction in the activation of nuclear factor-kappa B (NF-kB) and mitogen-activated protein kinase (MAPK) in TPA-treated ears. These results indicate that PEP-1-rpS3 inhibits inflammatory response cytokines and enzymes by blocking NF-kB and MAPK, prompting the suggestion that PEP-1-rpS3 can be used as a therapeutic agent against skin inflammation.

  16. Genome-wide screens for expressed hypothetical proteins

    DEFF Research Database (Denmark)

    Madsen, Claus Desler; Durhuus, Jon Ambæk; Rasmussen, Lene Juel

    2012-01-01

    A hypothetical protein (HP) is defined as a protein that is predicted to be expressed from an open reading frame, but for which there is no experimental evidence of translation. HPs constitute a substantial fraction of proteomes of human as well as of other organisms. With the general belief that...... that the majority of HPs are the product of pseudogenes, it is essential to have a tool with the ability of pinpointing the minority of HPs with a high probability of being expressed....

  17. Characterization and Oral Delivery of Proinsulin-Transferrin Fusion Protein Expressed Using ExpressTec

    Directory of Open Access Journals (Sweden)

    Yu-Sheng Chen

    2018-01-01

    Full Text Available Proinsulin-transferrin fusion protein (ProINS-Tf has been designed and successfully expressed from the mammalian HEK293 cells (HEK-ProINS-Tf. It was found that HEK-ProINS-Tf could be converted into an activated form in the liver. Furthermore, HEK-ProINS-Tf was demonstrated as an extra-long acting insulin analogue with liver-specific insulin action in streptozotocin (STZ-induced type 1 diabetic mice. However, due to the low production yield from transfected HEK293 cells, there are other interesting features, including the oral bioavailability, which have not been fully explored and characterized. To improve the protein production yield, an alternative protein expression system, ExpressTec using transgenic rice (Oryza sativa L., was used. The intact and active rice-derived ProINS-Tf (ExpressTec-ProINS-Tf was successfully expressed from the transgenic rice expression system. Our results suggested that, although the insulin-like bioactivity of ExpressTec-ProINS-Tf was slightly lower in vitro, its potency of in vivo blood glucose control was considerably stronger than that of HEK-ProINS-Tf. The oral delivery studies in type 1 diabetic mice demonstrated a prolonged control of blood glucose to near-normal levels after oral administration of ExpressTec-ProINS-Tf. Results in this report suggest that ExpressTec-ProINS-Tf is a promising insulin analog with advantages including low cost, prolonged and liver targeting effects, and most importantly, oral bioactivity.

  18. SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm

    Directory of Open Access Journals (Sweden)

    Lobstein Julie

    2012-05-01

    Full Text Available Abstract Background Production of correctly disulfide bonded proteins to high yields remains a challenge. Recombinant protein expression in Escherichia coli is the popular choice, especially within the research community. While there is an ever growing demand for new expression strains, few strains are dedicated to post-translational modifications, such as disulfide bond formation. Thus, new protein expression strains must be engineered and the parameters involved in producing disulfide bonded proteins must be understood. Results We have engineered a new E. coli protein expression strain named SHuffle, dedicated to producing correctly disulfide bonded active proteins to high yields within its cytoplasm. This strain is based on the trxB gor suppressor strain SMG96 where its cytoplasmic reductive pathways have been diminished, allowing for the formation of disulfide bonds in the cytoplasm. We have further engineered a major improvement by integrating into its chromosome a signal sequenceless disulfide bond isomerase, DsbC. We probed the redox state of DsbC in the oxidizing cytoplasm and evaluated its role in assisting the formation of correctly folded multi-disulfide bonded proteins. We optimized protein expression conditions, varying temperature, induction conditions, strain background and the co-expression of various helper proteins. We found that temperature has the biggest impact on improving yields and that the E. coli B strain background of this strain was superior to the K12 version. We also discovered that auto-expression of substrate target proteins using this strain resulted in higher yields of active pure protein. Finally, we found that co-expression of mutant thioredoxins and PDI homologs improved yields of various substrate proteins. Conclusions This work is the first extensive characterization of the trxB gor suppressor strain. The results presented should help researchers design the appropriate protein expression conditions using

  19. 100 Gb/s single VCSEL data transmission link

    DEFF Research Database (Denmark)

    Rodes Lopez, Roberto; Estaran Tolosa, Jose Manuel; Li, Bomin

    2012-01-01

    100 Gb/s optical fiber transmission link with a single 1.5 um VCSEL has been experimentally demonstrated using 4-level pulse amplitude modulation.......100 Gb/s optical fiber transmission link with a single 1.5 um VCSEL has been experimentally demonstrated using 4-level pulse amplitude modulation....

  20. Expression and Purification of Z Protein from Junín Virus

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    S. E. Goñi

    2010-01-01

    Full Text Available Arenaviridae comprises 23 recognized virus species with a bipartite ssRNA genome and an ambisense coding strategy. The virions are enveloped and include nonequimolar amounts of each genomic RNA species, designated L and S, coding for four ORFs (N, GPC, L, and Z. The arenavirus Junín (JUNV is the etiological agent of Argentine Hemorrhagic Fever, an acute disease with high mortality rate. It has been proposed that Z is the functional counterpart of the matrix proteins found in other negative-stranded enveloped RNA viruses. Here we report the optimized expression of a synthetic gene of Z protein, using three expression systems (two bacterial and a baculoviral one. One of these recombinant proteins was used to generate antibodies. A bioinformatic analysis was made where Z was subdivided into three domains. The data presented contributes methodologies for Z recombinant production and provides the basis for the development of new experiments to test its function.

  1. Natural selection in avian protein-coding genes expressed in brain.

    Science.gov (United States)

    Axelsson, Erik; Hultin-Rosenberg, Lina; Brandström, Mikael; Zwahlén, Martin; Clayton, David F; Ellegren, Hans

    2008-06-01

    The evolution of birds from theropod dinosaurs took place approximately 150 million years ago, and was associated with a number of specific adaptations that are still evident among extant birds, including feathers, song and extravagant secondary sexual characteristics. Knowledge about the molecular evolutionary background to such adaptations is lacking. Here, we analyse the evolution of > 5000 protein-coding gene sequences expressed in zebra finch brain by comparison to orthologous sequences in chicken. Mean d(N)/d(S) is 0.085 and genes with their maximal expression in the eye and central nervous system have the lowest mean d(N)/d(S) value, while those expressed in digestive and reproductive tissues exhibit the highest. We find that fast-evolving genes (those which have higher than expected rate of nonsynonymous substitution, indicative of adaptive evolution) are enriched for biological functions such as fertilization, muscle contraction, defence response, response to stress, wounding and endogenous stimulus, and cell death. After alignment to mammalian orthologues, we identify a catalogue of 228 genes that show a significantly higher rate of protein evolution in the two bird lineages than in mammals. These accelerated bird genes, representing candidates for avian-specific adaptations, include genes implicated in vocal learning and other cognitive processes. Moreover, colouration genes evolve faster in birds than in mammals, which may have been driven by sexual selection for extravagant plumage characteristics.

  2. Differentially expressed proteins in human breat cancer cells sensitive andresistant to paclitaxel

    Czech Academy of Sciences Publication Activity Database

    Pavlíková, N.; Bartoňová, I.; Dinčáková, L.; Halada, Petr; Kovář, J.

    2014-01-01

    Roč. 45, č. 2 (2014), s. 822-830 ISSN 1019-6439 R&D Projects: GA MZd NT13679 Institutional support: RVO:61388971 Keywords : paclitaxel * cancer * breaast cells * expressed proteins * cytokeratin 18 Subject RIV: EC - Immunology Impact factor: 3.025, year: 2014

  3. IGF-1 modulates gene expression of proteins involved in inflammation, cytoskeleton, and liver architecture.

    Science.gov (United States)

    Lara-Diaz, V J; Castilla-Cortazar, I; Martín-Estal, I; García-Magariño, M; Aguirre, G A; Puche, J E; de la Garza, R G; Morales, L A; Muñoz, U

    2017-05-01

    Even though the liver synthesizes most of circulating IGF-1, it lacks its receptor under physiological conditions. However, according to previous studies, a damaged liver expresses the receptor. For this reason, herein, we examine hepatic histology and expression of genes encoding proteins of the cytoskeleton, extracellular matrix, and cell-cell molecules and inflammation-related proteins. A partial IGF-1 deficiency murine model was used to investigate IGF-1's effects on liver by comparing wild-type controls, heterozygous igf1 +/- , and heterozygous mice treated with IGF-1 for 10 days. Histology, microarray for mRNA gene expression, RT-qPCR, and lipid peroxidation were assessed. Microarray analyses revealed significant underexpression of igf1 in heterozygous mice compared to control mice, restoring normal liver expression after treatment, which then normalized its circulating levels. IGF-1 receptor mRNA was overexpressed in Hz mice liver, while treated mice displayed a similar expression to that of the controls. Heterozygous mice showed overexpression of several genes encoding proteins related to inflammatory and acute-phase proteins and underexpression or overexpression of genes which coded for extracellular matrix, cytoskeleton, and cell junction components. Histology revealed an altered hepatic architecture. In addition, liver oxidative damage was found increased in the heterozygous group. The mere IGF-1 partial deficiency is associated with relevant alterations of the hepatic architecture and expression of genes involved in cytoskeleton, hepatocyte polarity, cell junctions, and extracellular matrix proteins. Moreover, it induces hepatic expression of the IGF-1 receptor and elevated acute-phase and inflammation mediators, which all resulted in liver oxidative damage.

  4. Activation of p44/42 in Human Natural Killer Cells Decreases Cell-surface Protein Expression: Relationship to Tributyltin-induced alterations of protein expression

    Science.gov (United States)

    Dudimah, Fred D.; Abraha, Abraham; Wang, Xiaofei; Whalen, Margaret M.

    2010-01-01

    Tributyltin (TBT) activates the mitogen activated protein kinase (MAPK), p44/42 in human natural killer (NK) cells. TBT also reduces NK cytotoxic function and decreases the expression of several NK-cell proteins. To understand the role that p44/42 activation plays in TBT-induced loss of NK cell function, we have investigated how selective activation of p44/42 by phorbol 12-myristate 13-acetate (PMA) affects NK cells. Previously we showed that PMA caused losses of lytic function similar to those seen with TBT exposures. Here we examined activation of p44/42 in the regulation of NK-cell protein expression and how this regulation may explain the protein expression changes seen with TBT exposures. NK cells exposed to PMA were examined for levels of cell-surface proteins, granzyme mRNA, and perforin mRNA expression. The expression of CD11a, CD16, CD18, and CD56 were reduced, perforin mRNA levels were unchanged and granzyme mRNA levels were increased. To verify that activation of p44/42 was responsible for the alterations seen in CD11a, CD16, CD18, and CD56 with PMA, NK cells were treated with the p44/42 pathway inhibitor (PD98059) prior to PMA exposures. In the presence of PD98059, PMA caused no decreases in the expression of the cell-surface proteins. Results of these studies indicate that the activation of p44/42 may lead to the loss of NK cell cytotoxic function by decreasing the expression of CD11a, CD16, CD18, and CD56. Further, activation of p44/42 appears to be at least in part responsible for the TBT-induced decreases in expression of CD16, CD18, and CD56. PMID:20883105

  5. The frequency of p53, Ki67, CD99 and Fli-1 protein expression in paraffin-embedded tissue blocks in Ewing’s sarcoma

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    Bagheri Hossein-Abadi Z

    2011-06-01

    Full Text Available "n Normal 0 false false false EN-US X-NONE AR-SA MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Arial; mso-bidi-theme-font:minor-bidi;} Background: Ewing sarcoma family tumors (ESFTs are among the most malignant tumors in children and young adults. ESFTs include Ewing sarcoma (ES and peripheral primitive neuroectodermal tumors (pPNETs. As there seemed to be few studies on the molecular biology of ESFTs, we investigated the frequency of CD99, Ki67, p53 and Fli-1 protein expression in 15 Iranian patients with ESFTs. In addition, the correlation between expression rate of these proteins and various clinical factors, including age, sex and survival was computed."n"nMethods: The expression of the aforesaid proteins was studied by immunohisto-chemistry in formalin-fixed and paraffin-embedded blocks of 15 ESFTs specimens. Stained sections were classified according to the percentage of stained tumor cells."n"nResults: The results showed the membrane expression of CD99 protein in all of the specimens. The nuclear expression of Fli-1 protein was observed in 86.7% and the over-expression of p53 nuclear protein was seen in 53.3% of the specimens. The expression rate of Ki67 protein was 60%. Although a significant correlation was not shown between the expression levels of Ki67, p53 or Fli-1 proteins with age, sex or survival of the patients, there was a significant

  6. Cloning, Expression and Purification of the Recombinant HIV-1 Tat-Nef Fusion Protein in Prokaryotic Expression System

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    Somayeh Kadkhodayan

    2016-07-01

    Full Text Available Abstract Background: Nef is one of the HIV-1 critical proteins, because it is essential for viral replication and AIDS disease progression and induction of immune response against it can partially inhibit viral infection. Moreover, a domain of the HIV-1 Trans-Activator of Transcription (Tat, 48-60 aa could act as a cell penetrating peptide (CPP. In current study, cloning and expression of Tat-Nef fusion protein was performed in E. coli for the first time. The protein expression was confirmed by western blot analysis and was purified using reverse staining method. Materials and Methods: In this experimental study, primarily, cloning of Tat-Nef fusion gene was done in pGEX6p2 expression vector. Then, the expression of Tat-Nef recombinat protein in E.coli BL21 (DE3 strain was performed by using IPTG inducer. The protein expression was confirmed by SDS-PAGE and western blotting using anti-Nef monoclonal antibody. Then, the recombinant fusion protein was purified from gel using reverse staining method. Results: The results of PCR analysis and enzyme digestion showed a clear band of ~ 726 bp in agarose gel indicating the correct Tat-Nef fusion cloning in pGEX6p2 prokaryotic expression vector. In addition, a 54 kDa band of Tat-Nef on SDS-PAGE revealed Tat-Nef protein expression that western blot analysis using anti-Nef monoclonal antibody confirmed it. Conclusion: The purified Tat-Nef recombinant fusion protein will be used as an antigen for protein vaccine design against HIV infection.

  7. Lung metastasis fails in MMTV-PyMT oncomice lacking S100A4 due to a T-cell deficiency in primary tumors

    DEFF Research Database (Denmark)

    Grum-Schwensen, Birgitte; Klingelhöfer, Jörg; Grigorian, Mariam

    2010-01-01

    significantly reduced the metastatic burden in lungs of PyMT-induced mammary tumors. In S100A4(+/+) PyMT mice, massive leukocyte infiltration at the site of the growing tumor at the stage of malignant transition was associated with increased concentration of extracellular S100A4 in the tumor microenvironment......Interactions between tumor and stroma cells are essential for the progression of cancer from its initial growth at a primary site to its metastasis to distant organs. The metastasis-stimulating protein S100A4 exerts its function as a stroma cell-derived factor. Genetic depletion of S100A4...... monolayers. In vivo, the presence of S100A4(+/+), but not S100A4(-/-), fibroblasts significantly stimulated the attraction of T lymphocytes to the site of the growing tumor. Increased levels of T cells were also observed in the premetastatic lungs of tumor-bearing mice primed to metastasize by S100A4...

  8. Nimodipine accelerates the postnatal development of parvalbumin and S-100β immunoreactivity in the rat brain

    NARCIS (Netherlands)

    Buwalda, Bauke; Naber, Riet; Nyakas, Csaba; Luiten, Paul G.M.

    1994-01-01

    The effects of chronic maternal perinatal nimodipine treatment on the immunocytochemical distribution of the Ca2+-binding proteins parvalbumin (PV) and S-100β in neocortex and hippocampus were studied at the age of postnatal day (PD) 5, 7, 10, 14 and 20. The Ca2+ antagonist nimodipine (1000 ppm BAY

  9. Enzyme-treated asparagus extract promotes expression of heat shock protein and exerts antistress effects.

    Science.gov (United States)

    Ito, Tomohiro; Maeda, Takahiro; Goto, Kazunori; Miura, Takehito; Wakame, Koji; Nishioka, Hiroshi; Sato, Atsuya

    2014-03-01

    A novel enzyme-treated asparagus extract (ETAS) has been developed as a functional material produced from asparagus stem. Studies were conducted to determine the effect of ETAS on heat shock protein 70 (HSP70) expression and alleviation of stress. HeLa cells were treated with ETAS, and HSP70 mRNA and protein levels were measured using a reverse transcription-polymerase chain reaction (RT-PCR) assay and an enzyme-linked immunosorbent assay (ELISA), respectively. ETAS showed significant increases in HSP70 mRNA at more than 0.125 mg/mL and the protein at more than 1.0 mg/mL. The antistress effect was evaluated in a murine sleep-deprivation model. A sleep-deprivation stress load resulted in elevation of blood corticosterone and lipid peroxide concentrations, while supplementation with ETAS at 200 and 1000 mg/kg body weight was associated with significantly reduced levels of both stress markers, which were in the normal range. The HSP70 protein expression level in mice subjected to sleep-deprivation stress and supplemented with ETAS was significantly enhanced in stomach, liver, and kidney, compared to ETAS-untreated mice. A preliminary and small-sized human study was conducted among healthy volunteers consuming up to 150 mg/d of ETAS daily for 7 d. The mRNA expression of HSP70 in peripheral leukocytes was significantly elevated at intakes of 100 or 150 mg/d, compared to their baseline levels. Since HSP70 is known to be a stress-related protein and its induction leads to cytoprotection, the present results suggest that ETAS might exert antistress effects under stressful conditions, resulting from enhancement of HSP70 expression. © 2014 Institute of Food Technologists®

  10. Robust expression of a bioactive mammalian protein in chlamydomonas chloroplast

    Science.gov (United States)

    Mayfield, Stephen P.

    2010-03-16

    Methods and compositions are disclosed to engineer chloroplast comprising heterologous mammalian genes via a direct replacement of chloroplast Photosystem II (PSII) reaction center protein coding regions to achieve expression of recombinant protein above 5% of total protein. When algae is used, algal expressed protein is produced predominantly as a soluble protein where the functional activity of the peptide is intact. As the host algae is edible, production of biologics in this organism for oral delivery or proteins/peptides, especially gut active proteins, without purification is disclosed.

  11. Expression of multiple proteins in transgenic plants

    Science.gov (United States)

    Vierstra, Richard D.; Walker, Joseph M.

    2002-01-01

    A method is disclosed for the production of multiple proteins in transgenic plants. A DNA construct for introduction into plants includes a provision to express a fusion protein of two proteins of interest joined by a linking domain including plant ubiquitin. When the fusion protein is produced in the cells of a transgenic plant transformed with the DNA construction, native enzymes present in plant cells cleave the fusion protein to release both proteins of interest into the cells of the transgenic plant. Since the proteins are produced from the same fusion protein, the initial quantities of the proteins in the cells of the plant are approximately equal.

  12. Interaction of an S100A9 gene variant with saturated fat and carbohydrates to modulate insulin resistance in 3 populations of different ancestries

    Science.gov (United States)

    BACKGROUND: S100 calcium binding protein A9 (S100A9) has previously been identified as a type 2 diabetes (T2D) gene. However, this finding requires independent validation and more in depth analyses in other populations and ancestries. OBJECTIVES: We aimed to replicate the associations between an S10...

  13. Effect of crude oil petroleum hydrocarbons on protein expression of the prawn Macrobrachium borellii.

    Science.gov (United States)

    Pasquevich, M Y; Dreon, M S; Gutierrez Rivera, J N; Vázquez Boucard, C; Heras, H

    2013-05-01

    Hydrocarbon pollution is a major environmental threat to ecosystems in marine and freshwater environments, but its toxicological effect on aquatic organisms remains little studied. A proteomic approach was used to analyze the effect of a freshwater oil spill on the prawn Macrobrachium borellii. To this aim, proteins were extracted from midgut gland (hepatopancreas) of male and female prawns exposed 7 days to a sublethal concentration (0.6 ppm) of water-soluble fraction of crude oil (WSF). Exposure to WSF induced responses at the protein expression level. Two-dimensional gel electrophoresis (2-DE) revealed 10 protein spots that were differentially expressed by WSF exposure. Seven proteins were identified using MS/MS and de novo sequencing. Nm23 oncoprotein, arginine methyltransferase, fatty aldehyde dehydrogenase and glutathione S-transferase were down-regulated, whereas two glyceraldehyde-3-phosphate dehydrogenase isoforms and a lipocalin-like crustacyanin (CTC) were up-regulated after WSF exposure. CTC mRNA levels were further analyzed by quantitative real-time PCR showing an increased expression after WSF exposure. The proteins identified are involved in carbohydrate and amino acid metabolism, detoxification, transport of hydrophobic molecules and cellular homeostasis among others. These results provide evidence for better understanding the toxic mechanisms of hydrocarbons. Moreover, some of these differentially expressed proteins would be employed as potential novel biomarkers. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. The role of secreted frizzled-related protein 2 expression in prostate cancer.

    LENUS (Irish Health Repository)

    O'Hurley, Gillian

    2012-02-01

    AIMS: Improved prostate cancer (PCa)-specific biomarkers are urgently required to distinguish between indolent and aggressive disease, in order to avoid overtreatment. In this study, we investigated the prostatic tissue expression of secreted frizzled-related protein (SFRP)-2. METHODS AND RESULTS: Following immunohistochemical analysis on PCa tissue microarrays with samples from 216 patients, strong\\/moderate SFRP-2 expression was observed in epithelial cells of benign prostatic hyperplasia, and negative\\/weak SFRP-2 expression was observed in the majority of tumour epithelia. However, among Gleason grade 5 carcinomas, 40% showed strong\\/moderate SFRP-2 expression and 60% showed negative SFRP-2 expression in epithelial cells. Further microscopic evaluation of Gleason grade 5 tumours revealed different morphological patterns, corresponding with differential SFRP-2 expression. The first subgroup (referred to as Type A) appeared to have a morphologically solid growth pattern, whereas the second subgroup (referred to as Type B) appeared to have a more diffuse pattern. Furthermore, 100% (4\\/4) of Type A patients experienced biochemical recurrence, as compared with 0% (0\\/6) of Type B patients. CONCLUSIONS: These results imply: (i) that there is a loss of SFRP-2 expression from benign to malignant prostate glands; and (ii) differential SFRP-2 expression among two possible subgroups of Gleason grade 5 tumours.

  15. High-level transient expression of recombinant protein in lettuce.

    Science.gov (United States)

    Joh, Lawrence D; Wroblewski, Tadeusz; Ewing, Nicholas N; VanderGheynst, Jean S

    2005-09-30

    Transient expression following agroinfiltration of plant tissue was investigated as a system for producing recombinant protein. As a model system, Agrobacterium tumefaciens containing the beta-glucuronidase (GUS) gene was vacuum infiltrated into lettuce leaf disks. Infiltration with a suspension of 10(9) colony forming units/mL followed by incubation for 72 h at 22 degrees C in continuous darkness produced a maximum of 0.16% GUS protein based on dry tissue or 1.1% GUS protein based on total soluble protein. This compares favorably to expression levels for commercially manufactured GUS protein from transgenic corn seeds. A. tumefaciens culture medium pH between 5.6 and 7.0 and surfactant concentrations lettuce to produce GUS protein more rapidly, but final levels did not exceed the GUS production in leaves incubated in continuous darkness after 72 h at 22 degrees C. The kinetics of GUS expression during incubation in continuous light and dark were represented well using a logistic model, with rate constants of 0.30 and 0.29/h, respectively. To semi-quantitatively measure the GUS expression in large numbers of leaf disks, a photometric enhancement of the standard histochemical staining method was developed. A linear relationship with an R2 value of 0.90 was determined between log10 (% leaf darkness) versus log10 (GUS activity). Although variability in expression level was observed, agroinfiltration appears to be a promising technology that could potentially be scaled up to produce high-value recombinant proteins in planta. Copyright 2005 Wiley Periodicals, Inc

  16. Correlation of human S100A12 (EN-RAGE) and high-sensitivity C-reactive protein as gingival crevicular fluid and serum markers of inflammation in chronic periodontitis and type 2 diabetes.

    Science.gov (United States)

    Pradeep, A R; Martande, Santosh S; Singh, Sonender Pal; Suke, Deepak Kumar; Raju, Arjun P; Naik, Savitha B

    2014-04-01

    The aim of the present study was to evaluate the levels and correlation of human S100A12 and high-sensitivity C-reactive protein (hs-CRP) in gingival crevicular fluid (GCF) and serum in chronic periodontitis (CP) subjects with and without type 2 diabetes mellitus (DM). A total of 44 subjects were divided into three groups: group 1 had 10 periodontally healthy subjects, group 2 consisted of 17 CP subjects and group 3 had 17 type 2 DM subjects with CP. GCF and serum levels of human S100A12 and hs-CRP were quantified using enzyme-linked immunosorbent assay and immunoturbidimetric analysis, respectively. The clinical outcomes evaluated were gingival index, probing depth and clinical attachment level and the correlations of the two inflammatory mediators with clinical parameters were evaluated. Both human S100A12 and hs-CRP levels increased from group 1 to group 2 to group 3. The GCF and serum values of both these inflammatory mediators correlated positively with each other and with the periodontal parameters evaluated (p < 0.05). Human S100A12 and hs-CRP can be considered as possible GCF and serum markers of inflammatory activity in CP and DM.

  17. S100ß and fibroblast growth factor-2 are present in cultured Schwann cells and may exert paracrine actions on the peripheral nerve injury S100ß e fator de crescimento de fibroblasto-2 estão presentes nas células de Schwann cultivadas e exercem ações parácrinas na lesão do nervo

    Directory of Open Access Journals (Sweden)

    Tatiana Duobles

    2008-12-01

    Full Text Available PURPOSE: The neurotrophic factor fibroblast growth factor-2 (FGF-2, bFGF and Ca++ binding protein S100ß are expressed by the Schwann cells of the peripheral nerves and by the satellite cells of the dorsal root ganglia (DRG. Recent studies have pointed out the importance of the molecules in the paracrine mechanisms related to neuronal maintenance and plasticity of lesioned motor and sensory peripheral neurons. Moreover, cultured Schwann cells have been employed experimentally in the treatment of central nervous system lesions, in special the spinal cord injury, a procedure that triggers an enhanced sensorymotor function. Those cells have been proposed to repair long gap nerve injury. METHODS: Here we used double labeling immunohistochemistry and Western blot to better characterize in vitro and in vivo the presence of the proteins in the Schwann cells and in the satellite cells of the DRG as well as their regulation in those cells after a crush of the rat sciatic nerve. RESULTS: FGF-2 and S100ß are present in the Schwann cells of the sciatic nerve and in the satellite cells of the DRG. S100ß positive satellite cells showed increased size of the axotomized DRG and possessed elevated amount of FGF-2 immunoreactivity. Reactive satellite cells with increased FGF-2 labeling formed a ring-like structure surrounding DRG neuronal cell bodies.Reactive S100ß positive Schwann cells of proximal stump of axotomized sciatic nerve also expressed higher amounts of FGF-2. CONCLUSION: Reactive peripheral glial cells synthesizing FGF-2 and S100ß may be important in wound repair and restorative events in the lesioned peripheral nerves.OBJETIVO: O fator neurotrófico fator de crescimento de fibroblastos-2 (FGF-2, bFGF e a proteína ligante de Ca++ S100ß são expressos pelas células de Schwann dos nervos e por células satélites do gânglio da raiz dorsal (GRD. Estudos recentes indicam a importância das moléculas nos mecanismos parácrinos relacionados

  18. Oral Administration of Recombinant Saccharomyces boulardii Expressing Ovalbumin-CPE Fusion Protein Induces Antibody Response in Mice.

    Science.gov (United States)

    Bagherpour, Ghasem; Ghasemi, Hosnie; Zand, Bahare; Zarei, Najmeh; Roohvand, Farzin; Ardakani, Esmat M; Azizi, Mohammad; Khalaj, Vahid

    2018-01-01

    Saccharomyces boulardii , a subspecies of Saccharomyces cerevisiae , is a well-known eukaryotic probiotic with many benefits for human health. In the present study, a recombinant strain of S. boulardii was prepared to use as a potential oral vaccine delivery vehicle. In this sense, a ura3 auxotroph strain of S. boulardii CNCM I-745 (known as S. cerevisiae HANSEN CBS 5926, Yomogi ® ) was generated using CRISPR/Cas9 methodology. Then a gene construct encoding a highly immunogenic protein, ovalbumin (OVA), was prepared and transformed into the ura3 - S. boulardii . To facilitate the transport of the recombinant immunogen across the intestinal barrier, a claudin-targeting sequence from Clostridium perfringens enterotoxin (CPE) was added to the C-terminus of the expression cassette. The recombinant S. boulardii strain expressing the OVA-CPE fusion protein was then administered orally to a group of mice, and serum IgG and fecal IgA levels were evaluated by ELISA. Our results demonstrated that anti-OVA IgG in serum significantly increased in test group ( P boulardii or PBS), and the fecal IgA titer was significantly higher in test group ( P boulardii strain expressing the similar construct lacking C-terminal CPE was also administered orally. The result showed an increased level of serum IgG in group receiving yeasts expressing the CPE negative construct compared to control groups; however, the fecal IgA levels did not increase significantly. In conclusion, our findings indicated that the yeast S. boulardii , as a delivery vehicle with possible immunomodulatory effects, and c-CPE, as a targeting tag, synergistically assist to stimulate systemic and local immunity. This proposed recombinant S. boulardii system might be useful in the expression of other antigenic peptides, making it as a promising tool for oral delivery of vaccines or therapeutic proteins.

  19. Multiplexed expression and screening for recombinant protein production in mammalian cells

    Directory of Open Access Journals (Sweden)

    McCafferty John

    2006-12-01

    Full Text Available Abstract Background A variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner. Results A nested PCR approach was used to clone 21 extracellular domains of mouse receptors as CD4 fusions within a mammalian GATEWAY expression vector system. Following transient transfection, HEK-293E cells grown in 2 ml cultures in 24-deep well blocks showed similar growth kinetics, viability and recombinant protein expression profiles, to those grown in 50 ml shake flask cultures as judged by western blotting. Following optimisation, fluorescent dot blot analysis of transfection supernatants was shown to be a rapid method for analysing protein expression yielding similar results as western blot analysis. Addition of urea enhanced the binding of glycoproteins to a nitrocellulose membrane. A good correlation was observed between the results of a plate based small scale transient transfection dot blot pre-screen and successful purification of proteins expressed at the 50 ml scale. Conclusion The combination of small scale multi-well plate culture and dot blotting described here will allow the multiplex analysis of different mammalian expression experiments enabling a faster identification of high yield expression constructs or conditions prior to large scale protein production. The methods for parallel GATEWAY cloning and expression of multiple constructs in cell

  20. Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps

    Directory of Open Access Journals (Sweden)

    Überla Klaus

    2007-06-01

    Full Text Available Abstract Background Proteins of human and animal viruses are frequently expressed from RNA polymerase II dependent expression cassettes to study protein function and to develop gene-based vaccines. Initial attempts to express the G protein of vesicular stomatitis virus (VSV and the F protein of respiratory syncytial virus (RSV by eukaryotic promoters revealed restrictions at several steps of gene expression. Results Insertion of an intron flanked by exonic sequences 5'-terminal to the open reading frames (ORF of VSV-G and RSV-F led to detectable cytoplasmic mRNA levels of both genes. While the exonic sequences were sufficient to stabilise the VSV-G mRNA, cytoplasmic mRNA levels of RSV-F were dependent on the presence of a functional intron. Cytoplasmic VSV-G mRNA levels led to readily detectable levels of VSV-G protein, whereas RSV-F protein expression remained undetectable. However, RSV-F expression was observed after mutating two of four consensus sites for polyadenylation present in the RSV-F ORF. Expression levels could be further enhanced by codon optimisation. Conclusion Insufficient cytoplasmic mRNA levels and premature polyadenylation prevent expression of RSV-F by RNA polymerase II dependent expression plasmids. Since RSV replicates in the cytoplasm, the presence of premature polyadenylation sites and elements leading to nuclear instability should not interfere with RSV-F expression during virus replication. The molecular mechanisms responsible for the destabilisation of the RSV-F and VSV-G mRNAs and the different requirements for their rescue by insertion of an intron remain to be defined.

  1. A small asparagine-rich protein required for S-allele-specific pollen rejection in Nicotiana.

    Science.gov (United States)

    McClure, B; Mou, B; Canevascini, S; Bernatzky, R

    1999-11-09

    Although S-locus RNases (S-RNases) determine the specificity of pollen rejection in self-incompatible (SI) solanaceous plants, they alone are not sufficient to cause S-allele-specific pollen rejection. To identify non-S-RNase sequences that are required for pollen rejection, a Nicotiana alata cDNA library was screened by differential hybridization. One clone, designated HT, hybridized strongly to RNA from N. alata styles but not to RNA from Nicotiana plumbaginifolia, a species known to lack one or more factors necessary for S-allele-specific pollen rejection. Sequence analysis revealed a 101-residue ORF including a putative secretion signal and an asparagine-rich domain near the C terminus. RNA blot analysis showed that the HT-transcript accumulates in the stigma and style before anthesis. The timing of HT-expression lags slightly behind S(C10)-RNase in SI N. alata S(C10)S(C10) and is well correlated with the onset of S-allele-specific pollen rejection in the style. An antisense-HT construct was prepared to test for a role in pollen rejection. Transformed (N. plumbaginifolia x SI N. alata S(C10)S(C10)) hybrids with reduced levels of HT-protein continued to express S(C10)-RNase but failed to reject S(C10)-pollen. Control hybrids expressing both S(C10)-RNase and HT-protein showed a normal S-allele-specific pollen rejection response. We conclude that HT-protein is directly implicated in pollen rejection.

  2. Molecular cloning and expression of the transformation sensitive epithelial marker stratifin. A member of a protein family that has been involved in the protein kinase C signalling pathway

    DEFF Research Database (Denmark)

    Leffers, H; Madsen, Peder; Rasmussen, H H

    1993-01-01

    tissues showed that polypeptides comigrating with proteins 9124, 9125 and 9126 are ubiquitous and highly expressed in the brain. Stratifin, however, was present only in cultured epithelial cells and was most abundant in fetal and adult human tissues enriched in stratified squamous keratinising epithelium......We have identified a family of abundant acidic human keratinocyte proteins with apparent molecular masses ranging between 30,000 and 31,100 (isoelectric focussing sample spot proteins 9109 (epithelial marker stratifin), 9124, 9125, 9126 and 9231 in the master two-dimensional gel database of human...

  3. Expression of human ferredoxin and assembly of the [2Fe-2S] center in Escherichia coli

    International Nuclear Information System (INIS)

    Coghlan, V.M.; Vickery, L.E.

    1989-01-01

    A cDNA fragment encoding human ferredoxin, a mitochondrial [2Fe-2S] protein, was introduced into Escherichia coli by using an expression vector based on the approach of Nagai and Thogersen. Expression was under control of the λP L promoter and resulted in production of ferredoxin as a cleavable fusion protein with an amino-terminal fragment derived from bacteriophage λcII protein. The fusion protein was isolated from the soluble fraction of induced cells and was specifically cleaved to yield mature recombinant ferredoxin. The recombinant protein was shown to be identical in size to ferredoxin isolated from human placenta (13,546 Da) by NaDodSO 4 /PAGE and partial amino acid sequencing. E. coli cells expressing human ferredoxin were brown in color, and absorbance and electron paramagnetic resonance spectra of the purified recombinant protein established that the [2Fe-2S]center was assembled and incorporated into ferredoxin in vivo. Recombinant ferredoxin was active in steroid hydroxylations when reconstituted with cytochromes P-450 sec and P-450 11β and exhibited rates comparable to those observed for ferredoxin isolated from human placenta. This expression system should be useful in production of native and structurally altered forms of human ferredoxin for studies of ferredoxin structure and function

  4. Intracellular expression of IRF9 Stat fusion protein overcomes the defective Jak-Stat signaling and inhibits HCV RNA replication

    Directory of Open Access Journals (Sweden)

    Balart Luis A

    2010-10-01

    Full Text Available Abstract Interferon alpha (IFN-α binds to a cell surface receptor that activates the Jak-Stat signaling pathway. A critical component of this pathway is the translocation of interferon stimulated gene factor 3 (a complex of three proteins Stat1, Stat2 and IRF9 to the nucleus to activate antiviral genes. A stable sub-genomic replicon cell line resistant to IFN-α was developed in which the nuclear translocation of Stat1 and Stat2 proteins was prevented due to the lack of phosphorylation; whereas the nuclear translocation of IRF9 protein was not affected. In this study, we sought to overcome defective Jak-Stat signaling and to induce an antiviral state in the IFN-α resistant replicon cell line by developing a chimera IRF9 protein fused with the trans activating domain (TAD of either a Stat1 (IRF9-S1C or Stat2 (IRF9-S2C protein. We show here that intracellular expression of fusion proteins using the plasmid constructs of either IRF9-S1C or IRF9-S2C, in the IFN-α resistant cells, resulted in an increase in Interferon Stimulated Response Element (ISRE luciferase promoter activity and significantly induced HLA-1 surface expression. Moreover, we show that transient transfection of IRF9-S1C or IRF9-S2C plasmid constructs into IFN-α resistant replicon cells containing sub-genomic HCV1b and HCV2a viruses resulted in an inhibition of viral replication and viral protein expression independent of IFN-α treatment. The results of this study indicate that the recombinant fusion proteins of IRF9-S1C, IRF9-S2C alone, or in combination, have potent antiviral properties against the HCV in an IFN-α resistant cell line with a defective Jak-Stat signaling.

  5. High-level expression of soluble recombinant proteins in Escherichia coli using an HE-maltotriose-binding protein fusion tag.

    Science.gov (United States)

    Han, Yingqian; Guo, Wanying; Su, Bingqian; Guo, Yujie; Wang, Jiang; Chu, Beibei; Yang, Guoyu

    2018-02-01

    Recombinant proteins are commonly expressed in prokaryotic expression systems for large-scale production. The use of genetically engineered affinity and solubility enhancing fusion proteins has increased greatly in recent years, and there now exists a considerable repertoire of these that can be used to enhance the expression, stability, solubility, folding, and purification of their fusion partner. Here, a modified histidine tag (HE) used as an affinity tag was employed together with a truncated maltotriose-binding protein (MBP; consisting of residues 59-433) from Pyrococcus furiosus as a solubility enhancing tag accompanying a tobacco etch virus protease-recognition site for protein expression and purification in Escherichia coli. Various proteins tagged at the N-terminus with HE-MBP(Pyr) were expressed in E. coli BL21(DE3) cells to determine expression and solubility relative to those tagged with His6-MBP or His6-MBP(Pyr). Furthermore, four HE-MBP(Pyr)-fused proteins were purified by immobilized metal affinity chromatography to assess the affinity of HE with immobilized Ni 2+ . Our results showed that HE-MBP(Pyr) represents an attractive fusion protein allowing high levels of soluble expression and purification of recombinant protein in E. coli. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. The presence of two S-layer-protein-encoding genes is conserved among species related to Lactobacillus acidophilus

    NARCIS (Netherlands)

    Boot, H.J.; Kolen, C.P.A.M.; Pot, B.; Kersters, K.; Pouwels, P.H.

    1996-01-01

    Previously we have shown that the type strain of Lactobacillus acidophilus possesses two S-protein-encoding genes, one of which is silent, on a chromosomal segment of 6 kb. The S-protein-encoding gene in the expression site can be exchanged for the silent S-protein-encoding gene by inversion of this

  7. Frame-Insensitive Expression Cloning of Fluorescent Protein from Scolionema suvaense

    Directory of Open Access Journals (Sweden)

    Yuki Horiuchi

    2018-01-01

    Full Text Available Expression cloning from cDNA is an important technique for acquiring genes encoding novel fluorescent proteins. However, the probability of in-frame cDNA insertion following the first start codon of the vector is normally only 1/3, which is a cause of low cloning efficiency. To overcome this issue, we developed a new expression plasmid vector, pRSET-TriEX, in which transcriptional slippage was induced by introducing a DNA sequence of (dT14 next to the first start codon of pRSET. The effectiveness of frame-insensitive cloning was validated by inserting the gene encoding eGFP with all three possible frames to the vector. After transformation with one of these plasmids, E. coli cells expressed eGFP with no significant difference in the expression level. The pRSET-TriEX vector was then used for expression cloning of a novel fluorescent protein from Scolionema suvaense. We screened 3658 E. coli colonies transformed with pRSET-TriEX containing Scolionema suvaense cDNA, and found one colony expressing a novel green fluorescent protein, ScSuFP. The highest score in protein sequence similarity was 42% with the chain c of multi-domain green fluorescent protein like protein “ember” from Anthoathecata sp. Variations in the N- and/or C-terminal sequence of ScSuFP compared to other fluorescent proteins indicate that the expression cloning, rather than the sequence similarity-based methods, was crucial for acquiring the gene encoding ScSuFP. The absorption maximum was at 498 nm, with an extinction efficiency of 1.17 × 105 M−1·cm−1. The emission maximum was at 511 nm and the fluorescence quantum yield was determined to be 0.6. Pseudo-native gel electrophoresis showed that the protein forms obligatory homodimers.

  8. DMBT1 expression distinguishes anorectal from cutaneous melanoma

    DEFF Research Database (Denmark)

    Helmke, Burkhard Maria; Renner, Marcus; Poustka, Annemarie

    2009-01-01

    tumours 1 (DMBT1) in cases of primary anorectal malignant melanoma and CM. METHODS AND RESULTS: Expression analyses of classical immunohistochemical markers (S100, HMB45, Melan A and MiTF) and of the protein DMBT1 were carried out in 27 cases of primary anorectal malignant melanoma and 26 cases of CM. All...

  9. In situ protein expression in tumour spheres: development of an immunostaining protocol for confocal microscopy

    International Nuclear Information System (INIS)

    Weiswald, Louis-Bastien; Guinebretière, Jean-Marc; Richon, Sophie; Bellet, Dominique; Saubaméa, Bruno; Dangles-Marie, Virginie

    2010-01-01

    Multicellular tumour sphere models have been shown to closely mimic phenotype characteristics of in vivo solid tumours, or to allow in vitro propagation of cancer stem cells (CSCs). CSCs are usually characterized by the expression of specific membrane markers using flow cytometry (FC) after enzymatic dissociation. Consequently, the spatial location of positive cells within spheres is not documented. Confocal microscopy is the best technique for the imaging of thick biological specimens after multi-labelling but suffers from poor antibody penetration. Thus, we describe here a new protocol for in situ confocal imaging of protein expression in intact spheroids. Protein expression in whole spheroids (150 μm in diameter) from two human colon cancer cell lines, HT29 and CT320X6, has been investigated with confocal immunostaining, then compared with profiles obtained through paraffin immunohistochemistry (pIHC) and FC. Target antigens, relevant for colon cancer and with different expression patterns, have been studied. We first demonstrate that our procedure overcomes the well-known problem of antibody penetration in compact structures by performing immunostaining of EpCAM, a membrane protein expressed by all cells within our spheroids. EpCAM expression is detected in all cells, even the deepest ones. Likewise, antibody access is confirmed with CK20 and CD44 immunostaining. Confocal imaging shows that 100% of cells express β-catenin, mainly present in the plasma membrane with also cytoplasmic and nuclear staining, in agreement with FC and pIHC data. pIHC and confocal imaging show similar CA 19-9 cytoplasmic and membranar expression profile in a cell subpopulation. CA 19-9 + cell count confirms confocal imaging as a highly sensitive method (75%, 62% and 51%, for FC, confocal imaging and pIHC, respectively). Finally, confocal imaging reveals that the weak expression of CD133, a putative colon CSC marker, is restricted to the luminal cell surface of colorectal cancer acini

  10. Comparative Immunohistochemical Analysis of Ochratoxin A Tumourigenesis in Rats and Urinary Tract Carcinoma in Humans; Mechanistic Significance of p-S6 Ribosomal Protein Expression

    Directory of Open Access Journals (Sweden)

    Sarah Pinder

    2012-09-01

    Full Text Available Ochratoxin A (OTA is considered to be a possible human urinary tract carcinogen, based largely on a rat model, but no molecular genetic changes in the rat carcinomas have yet been defined. The phosphorylated-S6 ribosomal protein is a marker indicating activity of the mammalian target of rapamycin, which is a serine/threonine kinase with a key role in protein biosynthesis, cell proliferation, transcription, cellular metabolism and apoptosis, while being functionally deregulated in cancer. To assess p-S6 expression we performed immunohistochemistry on formalin-fixed and paraffin-embedded tumours and normal tissues. Marked intensity of p-S6 expression was observed in highly proliferative regions of rat renal carcinomas and a rare angiosarcoma, all of which were attributed to prolonged exposure to dietary OTA. Only very small OTA-generated renal adenomas were negative for p-S6. Examples of rat subcutaneous fibrosarcoma and testicular seminoma, as well as of normal renal tissue, showed no or very weak positive staining. In contrast to the animal model, human renal cell carcinoma, upper urinary tract transitional cell carcinoma from cases of Balkan endemic nephropathy, and a human angiosarcoma were negative for p-S6. The combined findings are reminiscent of constitutive changes in the rat tuberous sclerosis gene complex in the Eker strain correlated with renal neoplasms, Therefore rat renal carcinogenesis caused by OTA does not obviously mimic human urinary tract tumourigenesis.

  11. A New Strain Collection for Improved Expression of Outer Membrane Proteins

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    Ina Meuskens

    2017-11-01

    Full Text Available Almost all integral membrane proteins found in the outer membranes of Gram-negative bacteria belong to the transmembrane β-barrel family. These proteins are not only important for nutrient uptake and homeostasis, but are also involved in such processes as adhesion, protein secretion, biofilm formation, and virulence. As surface exposed molecules, outer membrane β-barrel proteins are also potential drug and vaccine targets. High production levels of heterologously expressed proteins are desirable for biochemical and especially structural studies, but over-expression and subsequent purification of membrane proteins, including outer membrane proteins, can be challenging. Here, we present a set of deletion mutants derived from E. coli BL21(DE3 designed for the over-expression of recombinant outer membrane proteins. These strains harbor deletions of four genes encoding abundant β-barrel proteins in the outer membrane (OmpA, OmpC, OmpF, and LamB, both single and in all combinations of double, triple, and quadruple knock-outs. The sequences encoding these outer membrane proteins were deleted completely, leaving only a minimal scar sequence, thus preventing the possibility of genetic reversion. Expression tests in the quadruple mutant strain with four test proteins, including a small outer membrane β-barrel protein and variants thereof as well as two virulence-related autotransporters, showed significantly improved expression and better quality of the produced proteins over the parent strain. Differences in growth behavior and aggregation in the presence of high salt were observed, but these phenomena did not negatively influence the expression in the quadruple mutant strain when handled as we recommend. The strains produced in this study can be used for outer membrane protein production and purification, but are also uniquely useful for labeling experiments for biophysical measurements in the native membrane environment.

  12. Protein samples for NMR: expression and analysis without purification, and stabilization by covalent cyclization

    International Nuclear Information System (INIS)

    Otting, G.; Ozawa, K.; Prosselkov, P.; Williams, N.K.; Dixon, N.E.; Liepinsh, E.

    2002-01-01

    Full text: A modified cell-free in vitro expression system was established for the expression of milligram quantities of protein per mL reaction medium. Expression levels of the E coli cytoplasmic peptidyl-prolyl cis-trans isomerase, PpiB, in 0 6 mL reaction medium were sufficient for the direct recording of clean 15N-HSQC spectra without chromatographic purification or sample concentration steps, using a 600 MHz NMR spectrometer with cryoprobe. Besides providing a route to high-throughput sample preparation, in vitro expression systems are known to be highly economic in their utilization of selectively labelled ammo acids. Using dual-selective labelling with 15N- and 13C-labelled amino acids, the 15N-HSQC cross peaks of strategically selected ammo acids can readily be identified and monitored for their response to the presence of ligand molecules, again without sample purification. 2) The N-terminal domain of E coli DnaB is a protein of ca 110 residues with a structured core composed of 6 helices. Additional segments of 10 residues each at the N- and C-termini are highly mobile. Both ends are close in space and can be linked together in a covalent peptide bond using intern technology. The core structures of linear (lin-DnaB-N) and cyclized (cz-DnaB-N) protein are conserved, as evidenced by superimposable NOESY spectra and chemical shifts. The linker segment in cz-DnaB-N is mobile as shown by 1H-15N NOEs. Yet, the cyclic protein melts about 10 degrees higher than the linear version. A stabilization free energy of ca 2 kcal/mol is in agreement with predictions based on the reduced entropy in the unfolded state. Amide proton exchange rates are much slower in the cyclic protein and reveal cooperative exchange through total, global unfolding at a rate of once every 100 minutes in the linear protein

  13. Impact of High-Level Expression of Heterologous Protein on Lactococcus lactis Host.

    Science.gov (United States)

    Kim, Mina; Jin, Yerin; An, Hyun-Joo; Kim, Jaehan

    2017-07-28

    The impact of overproduction of a heterologous protein on the metabolic system of host Lactococcus lactis was investigated. The protein expression profiles of L. lactis IL1403 containing two near-identical plasmids that expressed high- and low-level of the green fluorescent protein (GFP) were examined via shotgun proteomics. Analysis of the two strains via high-throughput LC-MS/MS proteomics identified the expression of 294 proteins. The relative amount of each protein in the proteome of both strains was determined by label-free quantification using the spectral counting method. Although expression level of most proteins were similar, several significant alterations in metabolic network were identified in the high GFP-producing strain. These changes include alterations in the pyruvate fermentation pathway, oxidative pentose phosphate pathway, and de novo synthesis pathway for pyrimidine RNA. Expression of enzymes for the synthesis of dTDP-rhamnose and N -acetylglucosamine from glucose was suppressed in the high GFP strain. In addition, enzymes involved in the amino acid synthesis or interconversion pathway were downregulated. The most noticeable changes in the high GFP-producing strain were a 3.4-fold increase in the expression of stress response and chaperone proteins and increase of caseinolytic peptidase family proteins. Characterization of these host expression changes witnessed during overexpression of GFP was might suggested the metabolic requirements and networks that may limit protein expression, and will aid in the future development of lactococcal hosts to produce more heterologous protein.

  14. Abnormal histopathology, fat percent and hepatic apolipoprotein A I and apolipoprotein B100 mRNA expression in fatty liver hemorrhagic syndrome and their improvement by soybean lecithin.

    Science.gov (United States)

    Song, Yalu; Ruan, Jiming; Luo, Junrong; Wang, Tiancheng; Yang, Fei; Cao, Huabin; Huang, Jianzhen; Hu, Guoliang

    2017-10-01

    To investigate the etiopathogenesis of fatty liver hemorrhagic syndrome (FLHS) and the protective effects of soybean lecithin against FLHS in laying hens, 135 healthy 300-day-old Hyline laying hens were randomly divided into groups: control (group 1), diseased (group 2), and protected (group 3). Each group contained 45 layers with 3 replicates. The birds in these 3 groups were fed a control diet, a high-energy/low-protein (HELP) diet or the HELP diet supplemented with 3% soybean lecithin instead of maize. The fat percent in the liver was calculated. Histopathological changes in the liver were determined by staining, and the mRNA expression levels of apolipoproteinA I (apoA I) and apolipoprotein B100 (apoB100) in the liver were determined by RT-PCR. The results showed that the fat percent in the liver of group 2 was much higher (P steatosis in the liver cell on d 30 and 60. The mRNA expression levels of apoA I and apoB100 in the livers were variable throughout the experiment. The expression level of apoA I in group 2 significantly decreased on d 60 (P < 0.05); the expression level of apoB100 slightly increased on d 30 in group 2, while it sharply decreased on d 60. Compared to group 1, the expression level of apoB100 showed no significant difference in group 3 (P < 0.05). This study indicated that FLHS induced pathological changes and abnormal expression of apoA I and apoB100 in the livers of laying hens and that soybean lecithin alleviated these abnormal changes. © 2017 Poultry Science Association Inc.

  15. Fos and jun proteins are specifically expressed during differentiation of human keratinocytes.

    Science.gov (United States)

    Mehic, Denis; Bakiri, Latifa; Ghannadan, Minoo; Wagner, Erwin F; Tschachler, Erwin

    2005-01-01

    Activator protein 1 (AP-1) proteins play key roles in the regulation of cell proliferation and differentiation. In this study we investigated the expression of Fos and Jun proteins in different models of terminal differentiation of human keratinocytes and in skin from psoriasis patients. All Jun and Fos proteins, with the exception of FosB, were efficiently expressed in keratinocytes in monolayer cultures. In contrast, in normal epidermis as well as in organotypic epidermal cultures, the expression pattern of AP-1 proteins was dependent on the differentiation stage. Fos proteins were readily detected in nuclei of keratinocytes of basal and suprabasal layers. JunB and JunD were expressed in all layers of normal epidermis. Interestingly, expression of c-Jun started suprabasally, then disappeared and became detectable again in distinct cells of the outermost granular layer directly at the transition zone to the stratum corneum. In psoriatic epidermis, c-Jun expression was prominent in both hyperproliferating basal and suprabasal keratinocytes, whereas c-Fos expression was unchanged. These data indicate that AP-1 proteins are expressed in a highly specific manner during terminal differentiation of keratinocytes and that the enhanced expression of c-Jun in basal and suprabasal keratinocytes might contribute to the pathogenesis of psoriasis.

  16. Changes in protein expression in testes of L2 strain Taiwan country chickens in response to acute heat stress.

    Science.gov (United States)

    Wang, Shih-Han; Cheng, Chuen-Yu; Chen, Chao-Jung; Chen, Hsin-Hsin; Tang, Pin-Chi; Chen, Chih-Feng; Lee, Yen-Pai; Huang, San-Yuan

    2014-07-01

    Heat stress causes a decrease of fertility in roosters. Yet, the way acute heat stress affects protein expression remains poorly understood. This study investigated differential protein expression in testes of the L2 strain of Taiwan country chickens following acute heat stress. Twelve 45-week-old roosters were allocated into four groups, including control roosters kept at 25 °C, roosters subjected to 38 °C acute heat stress for 4 hours without recovery, with 2 hours of recovery, and with 6 hours of recovery. Testis samples were collected for morphologic assay and protein analysis. Some of the differentially expressed proteins were validated by Western blot and immunohistochemistry. Abnormal and apoptotic spermatogenic cells were observed at 2 hours of recovery after acute heat stress, especially among the spermatocytes. Two-dimensional difference gel electrophoresis revealed that 119 protein spots were differentially expressed in chicken testes following heat stress, and peptide mass fingerprinting revealed that these spots contained 92 distinct proteins. In the heat-stressed samples, the heat shock proteins, chaperonin containing t-complex, and proteasome subunits were downregulated, and glutathione S-transferase, transgelin, and DJ-1 were upregulated. Our results demonstrate that acute heat stress impairs the processes of translation, protein folding, and protein degradation, and thus results in apoptosis and interferes with spermatogenesis. On the other hand, the increased expression of antioxidant enzymes, including glutathione S-transferase and DJ-1, may attenuate heat-induced damage. These findings may have implications for breeding chickens that can tolerate more extreme conditions. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Experimental study of inhibitory effects of diallyl trisulfide on the growth of human osteosarcoma Saos-2 cells by downregulating expression of glucose-regulated protein 78

    Directory of Open Access Journals (Sweden)

    Zhang Y

    2018-01-01

    Full Text Available Yue Zhang,1,* Wen-Peng Xie,1,* Yong-Kui Zhang,2 Yi-Qiang Chen,3 Dong-Li Wang,2 Gang Li,2 Dong-Hui Guan2 1First Clinical Medical College, Shandong University of Traditional Chinese Medicine, Jinan, People’s Republic of China; 2Department of Orthopedics, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, People’s Republic of China; 3Department of Orthopedics, The First People’s Hospital of Taian City, Taian, People’s Republic of China *These authors contributed equally to the paper Background: Diallyl trisulfide (DATS is a natural organic sulfur compound isolated from garlic that has good anticancer activity according to many previous reports. There are many studies pointing out that DATS can downregulate expression of the glucose-regulated protein 78 (GRP78, which is associated with poor prognosis and drug resistance in various types of human cancers. However, it remains unknown whether DATS has the same effect on human osteosarcoma cells. This study attempted to clarify the potential molecular mechanisms of the action of DATS in human osteosarcoma Saos-2 cells.Methods: We used an inverted phase microscope and immunofluorescent staining to observe the morphological changes of Saos-2 cells after being cultured in different concentrations of DATS (0, 25, 50, and 100 µM for 24 h, or for four time periods (24, 48, 72, and 96 h in the same DATS concentration (50 µM. Quantitative real-time polymerase chain reaction and Western blot were used to detect the expression level of GRP78 mRNA and proteins in Saos-2 cells. GRP78 expression was suppressed in Saos-2 cells by utilizing small-interfering RNA, and the cells were subsequently used to study the anti-proliferative effects of DATS treatment.Results: The expression level of GRP78 mRNA and proteins was significantly downregulated due to the increased concentration and effective times of DATS (P<0.05. In addition, there were significant associations between GRP78

  18. Expression of the human blood coagulation protein factor XIIIa in Saccharomyces cerevisiae: dependence of the expression levels from host-vector systems and medium conditions.

    Science.gov (United States)

    Bröker, M; Bäuml, O; Göttig, A; Ochs, J; Bodenbenner, M; Amann, E

    1991-03-01

    The human blood coagulation protein Factor XIIIa (FXIIIa) was expressed in Saccharomyces cerevisiae employing Escherichia coli-yeast shuttle vectors based on a 2-mu plasmid. Several factors affecting high production yield of recombinant FXIIIa were analysed. The use of the regulatable GAL-CYC1 hybrid promoter resulted in higher FXIIIa expression when compared with the constitutive ADCI promoter. Screening for suitable yeast strains for expression of FXIIIa under the transcriptional control of the GAL-CYC1 hybrid promoter revealed a broad spectrum of productivity. No obvious correlation between the expression rate and the genetic markers of the strains could be identified. The medium composition markedly influenced the FXIIIa expression rates. The expression of FXIIIa was strictly regulated by the carbon source. Glucose as the only sugar and energy source repressed the synthesis of FXIIIa, whereas addition of galactose induced FXIIIa expression. Special feeding schemes resulted in a productivity of up to 100 mg FXIIIa/l in shake flasks.

  19. Characterization and expression of genes encoding three small heat shock proteins in Sesamia inferens (Lepidoptera: Noctuidae).

    Science.gov (United States)

    Sun, Meng; Lu, Ming-Xing; Tang, Xiao-Tian; Du, Yu-Zhou

    2014-12-12

    The pink stem borer, Sesamia inferens (Walker), is a major pest of rice and is endemic in China and other parts of Asia. Small heat shock proteins (sHSPs) encompass a diverse, widespread class of stress proteins that have not been characterized in S. inferens. In the present study, we isolated and characterized three S. inferens genes that encode members of the α-crystallin/sHSP family, namely, Sihsp21.4, Sihsp20.6, and Sihsp19.6. The three cDNAs encoded proteins of 187, 183 and 174 amino acids with calculated molecular weights of 21.4, 20.6 and 19.6 kDa, respectively. The deduced amino acid sequences of the three genes showed strong similarity to sHSPs identified in other lepidopteran insects. Sihsp21.4 contained an intron, but Sihsp20.6 and Sihsp19.6 lacked introns. Real-time quantitative PCR analyses revealed that Sihsp21.4 was most strongly expressed in S. inferens heads; Whereas expression of Sihsp20.6 and Sihsp19.6 was highest in eggs. The three S. inferens sHSP genes were up-regulated during low temperature stress. In summary, our results show that S. inferens sHSP genes have distinct regulatory roles in the physiology of S. inferens.

  20. Characterization and Expression of Genes Encoding Three Small Heat Shock Proteins in Sesamia inferens (Lepidoptera: Noctuidae

    Directory of Open Access Journals (Sweden)

    Meng Sun

    2014-12-01

    Full Text Available The pink stem borer, Sesamia inferens (Walker, is a major pest of rice and is endemic in China and other parts of Asia. Small heat shock proteins (sHSPs encompass a diverse, widespread class of stress proteins that have not been characterized in S. inferens. In the present study, we isolated and characterized three S. inferens genes that encode members of the α-crystallin/sHSP family, namely, Sihsp21.4, Sihsp20.6, and Sihsp19.6. The three cDNAs encoded proteins of 187, 183 and 174 amino acids with calculated molecular weights of 21.4, 20.6 and 19.6 kDa, respectively. The deduced amino acid sequences of the three genes showed strong similarity to sHSPs identified in other lepidopteran insects. Sihsp21.4 contained an intron, but Sihsp20.6 and Sihsp19.6 lacked introns. Real-time quantitative PCR analyses revealed that Sihsp21.4 was most strongly expressed in S. inferens heads; Whereas expression of Sihsp20.6 and Sihsp19.6 was highest in eggs. The three S. inferens sHSP genes were up-regulated during low temperature stress. In summary, our results show that S. inferens sHSP genes have distinct regulatory roles in the physiology of S. inferens.

  1. Oral Administration of Recombinant Saccharomyces boulardii Expressing Ovalbumin-CPE Fusion Protein Induces Antibody Response in Mice

    Directory of Open Access Journals (Sweden)

    Ghasem Bagherpour

    2018-04-01

    Full Text Available Saccharomyces boulardii, a subspecies of Saccharomyces cerevisiae, is a well-known eukaryotic probiotic with many benefits for human health. In the present study, a recombinant strain of S. boulardii was prepared to use as a potential oral vaccine delivery vehicle. In this sense, a ura3 auxotroph strain of S. boulardii CNCM I-745 (known as S. cerevisiae HANSEN CBS 5926, Yomogi® was generated using CRISPR/Cas9 methodology. Then a gene construct encoding a highly immunogenic protein, ovalbumin (OVA, was prepared and transformed into the ura3- S. boulardii. To facilitate the transport of the recombinant immunogen across the intestinal barrier, a claudin-targeting sequence from Clostridium perfringens enterotoxin (CPE was added to the C-terminus of the expression cassette. The recombinant S. boulardii strain expressing the OVA-CPE fusion protein was then administered orally to a group of mice, and serum IgG and fecal IgA levels were evaluated by ELISA. Our results demonstrated that anti-OVA IgG in serum significantly increased in test group (P < 0.001 compared to control groups (receiving wild type S. boulardii or PBS, and the fecal IgA titer was significantly higher in test group (P < 0.05 than control groups. In parallel, a recombinant S. boulardii strain expressing the similar construct lacking C-terminal CPE was also administered orally. The result showed an increased level of serum IgG in group receiving yeasts expressing the CPE negative construct compared to control groups; however, the fecal IgA levels did not increase significantly. In conclusion, our findings indicated that the yeast S. boulardii, as a delivery vehicle with possible immunomodulatory effects, and c-CPE, as a targeting tag, synergistically assist to stimulate systemic and local immunity. This proposed recombinant S. boulardii system might be useful in the expression of other antigenic peptides, making it as a promising tool for oral delivery of vaccines or therapeutic

  2. Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells.

    Science.gov (United States)

    Fabrick, Jeffrey A; Hull, J Joe

    2017-04-20

    Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

  3. Expression of the innate defense receptor S5D-SRCRB in the urogenital tract

    DEFF Research Database (Denmark)

    Miro-Julia, C.; Escoda-Ferran, C.; Carrasco, E.

    2014-01-01

    S5D-SRCRB is a novel mouse secretory glycoprotein belonging to the ancient and highly conserved scavenger receptor cysteine-rich superfamily of protein receptors. Available evidence indicates that S5D-SRCRB interacts with conserved microbial cell wall components, as well as with some endogenous...... proteins, and presents a restricted tissue expression pattern. This study further analyzes the expression of S5D-SRCRB along the mouse urogenital tract. Immunohistochemical staining for S5D-SRCRB was observed in spermatocytes from seminiferous tubules and in the epithelial surface from urethra and bladder......-SRCRB was observed in the apical border of terminally differentiated IC. Colocalization of S5D-SRCRB with galectin-3 (Gal-3) was also observed in kidney and bladder, but not in testis, supporting concurrent biochemical studies demonstrating the carbohydrate-dependent interaction of Gal-3 and S5D-SRCRB. Furthermore...

  4. Prolonged calorie restriction downregulates skeletal muscle mTORC1 signaling independent of dietary protein intake and associated microRNA expression

    Directory of Open Access Journals (Sweden)

    Lee M Margolis

    2016-10-01

    Full Text Available Short-term (5-10 days calorie restriction (CR downregulates muscle protein synthesis, with consumption of a high protein-based diet attenuating this decline. Benefit of increase protein intake is believed to be due to maintenance of amino acid-mediated anabolic signaling through the mechanistic target of rapamycin complex 1 (mTORC1, however, there is limited evidence to support this contention. The purpose of this investigation was to determine the effects of prolonged CR and high protein diets on skeletal muscle mTORC1 signaling and expression of associated microRNA (miR. 12-wk old male Sprague Dawley rats consumed ad libitum (AL or calorie restricted (CR; 40% adequate (10%, AIN-93M or high (32% protein milk-based diets for 16 weeks. Body composition was determined using dual energy X-ray absorptiometry and muscle protein content was calculated from muscle homogenate protein concentrations expressed relative to fat-free mass to estimate protein content. Western blot and RT-qPCR were used to determine mTORC1 signaling and mRNA and miR expression in fasted mixed gastrocnemius. Independent of dietary protein intake, muscle protein content was 38% lower (P < 0.05 in CR compared to AL. Phosphorylation and total Akt, mTOR, rpS6 and p70S6K were lower (P < 0.05 in CR versus AL, and total rpS6 was associated with muscle protein content (r = 0.64, r2 = 0.36. Skeletal muscle miR expression was not altered by either energy or protein intake. This study provides evidence that chronic CR attenuates muscle protein content by downregulating mTORC1 signaling. This response is independent of skeletal muscle miR and dietary protein.

  5. Use of a protein engineering strategy to overcome limitations in the production of "Difficult to Express" recombinant proteins.

    Science.gov (United States)

    Hussain, Hirra; Fisher, David I; Abbott, W Mark; Roth, Robert G; Dickson, Alan J

    2017-10-01

    Certain recombinant proteins are deemed "difficult to express" in mammalian expression systems requiring significant cell and/or process engineering to abrogate expression bottlenecks. With increasing demand for the production of recombinant proteins in mammalian cells, low protein yields can have significant consequences for industrial processes. To investigate the molecular mechanisms that restrict expression of recombinant proteins, naturally secreted model proteins were analyzed from the tissue inhibitors of metalloproteinase (TIMP) protein family. In particular, TIMP-2 and TIMP-3 were subjected to detailed study. TIMP proteins share significant sequence homology (∼50% identity and ∼70% similarity in amino acid sequence). However, they show marked differences in secretion in mammalian expression systems despite this extensive sequence homology. Using these two proteins as models, this study characterized the molecular mechanisms responsible for poor recombinant protein production. Our results reveal that both TIMP-2 and TIMP-3 are detectable at mRNA and protein level within the cell but only TIMP-2 is secreted effectively into the extracellular medium. Analysis of protein localization and the nature of intracellular protein suggest TIMP-3 is severely limited in its post-translational processing. To overcome this challenge, modification of the TIMP-3 sequence to include a furin protease-cleavable pro-sequence resulted in secretion of the modified TIMP-3 protein, however, incomplete processing was observed. Based on the TIMP-3 data, the protein engineering approach was optimized and successfully applied in combination with cell engineering, the overexpression of furin, to another member of the TIMP protein family (the poorly expressed TIMP-4). Use of the described protein engineering strategy resulted in successful secretion of poorly (TIMP-4) and non-secreted (TIMP-3) targets, and presents a novel strategy to enhance the production of "difficult" recombinant

  6. Protein expression of Myt272-3 recombinant clone and in silico ...

    African Journals Online (AJOL)

    Purpose: To investigate the expression of Myt272-3 recombinant protein and also to predict a possible protein vaccine candidate against Mycobacterium tuberculosis. Methods: Myt272-3 protein was expressed in pET30a+-Myt272-3 clone. The purity of the protein was determined using Dynabeads® His-Tag Isolation ...

  7. Molecular cloning and expression of a novel keratinocyte protein (psoriasis-associated fatty acid-binding protein [PA-FABP]) that is highly up-regulated in psoriatic skin and that shares similarity to fatty acid-binding proteins

    DEFF Research Database (Denmark)

    Madsen, Peder; Rasmussen, H H; Leffers, H

    1992-01-01

    termed PA-FABP (psoriasis-associated fatty acid-binding protein). The deduced sequence predicted a protein with molecular weight of 15,164 daltons and a calculated pI of 6.96, values that are close to those recorded in the keratinocyte 2D gel protein database. The protein comigrated with PA......-FABP as determined by 2D gel analysis of [35S]-methionine-labeled proteins expressed by transformed human amnion (AMA) cells transfected with clone 1592 using the vaccinia virus expression system and reacted with a rabbit polyclonal antibody raised against 2D gel purified PA-FABP. Structural analysis of the amino...... with epidermal growth factor (EGF), pituitary extract, and 10% fetal calf serum] revealed a strong up-regulation of PA-FABP, psoriasin, calgranulins A and B, and a few other proteins that are highly expressed in psoriatic skin. The levels of these proteins exceeded by far those observed in non-cultured normal...

  8. Human Cementum Protein 1 induces expression of bone and cementum proteins by human gingival fibroblasts

    International Nuclear Information System (INIS)

    Carmona-Rodriguez, Bruno; Alvarez-Perez, Marco Antonio; Narayanan, A. Sampath; Zeichner-David, Margarita; Reyes-Gasga, Jose; Molina-Guarneros, Juan; Garcia-Hernandez, Ana Lilia; Suarez-Franco, Jose Luis; Chavarria, Ivet Gil; Villarreal-Ramirez, Eduardo; Arzate, Higinio

    2007-01-01

    We recently presented evidence showing that a human cementoblastoma-derived protein, named Cementum Protein 1 (CEMP1) may play a role as a local regulator of cementoblast differentiation and cementum-matrix mineralization. This protein was shown to be expressed by cementoblasts and progenitor cells localized in the periodontal ligament. In this study we demonstrate that transfection of CEMP1 into human gingival fibroblasts (HGF) induces mineralization and expression of bone and cementum-matrix proteins. The transfected HGF cells had higher alkaline phosphatase activity and proliferation rate and they expressed genes for alkaline phosphatase, bone sialoprotein, osteocalcin, osteopontin, the transcription factor Runx2/Cbfa1, and cementum attachment protein (CAP). They also produced biological-type hydroxyapatite. These findings indicate that the CEMP1 might participate in differentiation and mineralization of nonosteogenic cells, and that it might have a potential function in cementum and bone formation

  9. Cloning and expression of Tenebrio molitor antifreeze protein in Escherichia coli.

    Science.gov (United States)

    Yue, Chang-Wu; Zhang, Yi-Zheng

    2009-03-01

    A novel antifreeze protein cDNA was cloned by RT-PCR from the larva of the yellow mealworm Tenebrio molitor. The coding fragment of 339 bp encodes a protein of 112 amino acid residues and was fused to the expression vectors pET32a and pTWIN1. The resulted expression plasmids were transformed into Escherischia coli strains BL21 (DE3), ER2566, and Origami B (DE3), respectively. Several strategies were used for expression of the highly disulfide-bonded beta-helix-contained protein with the activity of antifreeze in different expression systems. A protocol for production of refolded and active T. molitor antifreeze protein in bacteria was obtained.

  10. Expression of G-protein inwardly rectifying potassium channels (GIRKs in lung cancer cell lines

    Directory of Open Access Journals (Sweden)

    Schuller Hildegard M

    2005-08-01

    Full Text Available Abstract Background Previous data from our laboratory has indicated that there is a functional link between the β-adrenergic receptor signaling pathway and the G-protein inwardly rectifying potassium channel (GIRK1 in human breast cancer cell lines. We wanted to determine if GIRK channels were expressed in lung cancers and if a similar link exists in lung cancer. Methods GIRK1-4 expression and levels were determined by reverse transcription polymerase chain reaction (RT-PCR and real-time PCR. GIRK protein levels were determined by western blots and cell proliferation was determined by a 5-bromo-2'-deoxyuridine (BrdU assay. Results GIRK1 mRNA was expressed in three of six small cell lung cancer (SCLC cell lines, and either GIRK2, 3 or 4 mRNA expression was detected in all six SCLC cell lines. Treatment of NCI-H69 with β2-adrenergic antagonist ICI 118,551 (100 μM daily for seven days led to slight decreases of GIRK1 mRNA expression levels. Treatment of NCI-H69 with the β-adrenergic agonist isoproterenol (10 μM decreased growth rates in these cells. The GIRK inhibitor U50488H (2 μM also inhibited proliferation, and this decrease was potentiated by isoproterenol. In the SCLC cell lines that demonstrated GIRK1 mRNA expression, we also saw GIRK1 protein expression. We feel these may be important regulatory pathways since no expression of mRNA of the GIRK channels (1 & 2 was found in hamster pulmonary neuroendocrine cells, a suggested cell of origin for SCLC, nor was GIRK1 or 2 expression found in human small airway epithelial cells. GIRK (1,2,3,4 mRNA expression was also seen in A549 adenocarcinoma and NCI-H727 carcinoid cell lines. GIRK1 mRNA expression was not found in tissue samples from adenocarcinoma or squamous cancer patients, nor was it found in NCI-H322 or NCI-H441 adenocarcinoma cell lines. GIRK (1,3,4 mRNA expression was seen in three squamous cell lines, GIRK2 was only expressed in one squamous cell line. However, GIRK1 protein

  11. HMB-45 may be a more sensitive maker than S-100 or Melan-A for immunohistochemical diagnosis of primary oral and nasal mucosal melanomas.

    Science.gov (United States)

    Yu, Chuan-Hang; Chen, Huang-Hsu; Liu, Chia-Ming; Jeng, Yung-Ming; Wang, Jeng-Tzung; Wang, Yi-Ping; Liu, Bu-Yuan; Sun, Andy; Chiang, Chun-Pin

    2005-10-01

    Primary mucosal melanomas (MMs) of the head and neck are a rare entity. Melanomas with characteristic melanin-pigmented tumor cells are easy to diagnose, but those without melanin-pigmented tumor cells, amelanotic melanomas, are difficult to identify and need immunohistochemistry (IHC) to confirm the final diagnosis. In this study, we examined the expression of three melanocytic differentiation markers, HMB-45, S-100, and Melan-A in primary oral and nasal MMs. We tried to evaluate whether HMB-45, S-100, and Melan-A were useful for diagnosis of primary oral and nasal MMs and to find out which marker was the best of the three. This study used IHC to examine the expression of HMB-45, S-100, and Melan-A in 17 formalin-fixed paraffin-embedded specimens of primary oral and nasal MMs. The staining intensities (SIs) and labeling indices (LIs) of HMB-45, S-100, and Melan-A in 17 MMs were calculated and compared between any two markers. Immunostaining results showed that the positive rate was 94% (16 of 17) for HMB-45, 88% (15 of 17) for S-100, and 71% (12 of 17) for Melan-A in 17 MMs. The SI of HMB-45 was significantly higher than that of S-100 (P = 0.0011) or of Melan-A (P = 0.0034). In addition, the mean LI of Melan-A (59 +/- 43%) was significantly lower than that of HMB-45 (83 +/- 28%, P = 0.0065) or of S-100 (79 +/- 33%, P = 0.0237). Our results indicate that both HMB-45 and S-100 show a high positive rate and LI in MMs and therefore may be good markers for immunohistochemical diagnosis of primary oral and nasal MMs. In addition, HMB-45 may be a more sensitive marker than S-100 because HMB-45 shows a significantly higher SI than S-100 in this study.

  12. Expression of a hantavirus N protein and its efficacy as antigen in immune assays

    Directory of Open Access Journals (Sweden)

    L.T.M. Figueiredo

    2008-07-01

    Full Text Available Hantavirus cardiopulmonary syndrome (HCPS has been recognized as an important public heath problem. Five hantaviruses associated with HCPS are currently known in Brazil: Juquitiba, Araraquara, Laguna Negra-like, Castelo dos Sonhos, and Anajatuba viruses. The laboratory diagnosis of HCPS is routinely carried out by the detection of anti-hantavirus IgM and/or IgG antibodies. The present study describes the expression of the N protein of a hantavirus detected in the blood sample of an HCPS patient. The entire S segment of the virus was amplified and found to be 1858 nucleotides long, with an open reading frame of 1287 nucleotides that encodes a protein of 429 amino acids. The nucleotide sequence described here showed a high identity with the N protein gene of Araraquara virus. The entire N protein was expressed using the vector pET200D and the Escherichia coli BL21 strain. The expression of the recombinant protein was confirmed by the detection of a 52-kDa protein by Western blot using a pool of human sera obtained from HCPS patients, and by specific IgG detection in five serum samples of HCPS patients tested by ELISA. These results suggest that the recombinant N protein could be used as an antigen for the serological screening of hantavirus infection.

  13. Neisseria meningitidis rifampicin resistant strains: analysis of protein differentially expressed

    Directory of Open Access Journals (Sweden)

    Schininà Maria

    2010-09-01

    Full Text Available Abstract Background Several mutations have been described as responsible for rifampicin resistance in Neisseria meningitidis. However, the intriguing question on why these strains are so rare remains open. The aim of this study was to investigate the protein content and to identify differential expression in specific proteins in two rifampicin resistant and one susceptible meningococci using two-dimensional electrophoresis (2-DE combined with mass spectrometry. Results In our experimental conditions, able to resolve soluble proteins with an isoelectric point between 4 and 7, twenty-three proteins have been found differentially expressed in the two resistant strains compared to the susceptible. Some of them, involved in the main metabolic pathways, showed an increased expression, mainly in the catabolism of pyruvate and in the tricarboxylic acid cycle. A decreased expression of proteins belonging to gene regulation and to those involved in the folding of polypeptides has also been observed. 2-DE analysis showed the presence of four proteins displaying a shift in their isoelectric point in both resistant strains, confirmed by the presence of amino acid changes in the sequence analysis, absent in the susceptible. Conclusions The analysis of differentially expressed proteins suggests that an intricate series of events occurs in N. meningitidis rifampicin resistant strains and the results here reported may be considered a starting point in understanding their decreased invasion capacity. In fact, they support the hypothesis that the presence of more than one protein differentially expressed, having a role in the metabolism of the meningococcus, influences its ability to infect and to spread in the population. Different reports have described and discussed how a drug resistant pathogen shows a high biological cost for survival and that may also explain why, for some pathogens, the rate of resistant organisms is relatively low considering the

  14. Interaction of avian infectious bronchitis virus S1 protein with heat ...

    African Journals Online (AJOL)

    The interaction between S1 and HSP47 was verified by colocalization experiment and co-immunoprecipitation of HeLa cell lysates expressing both proteins. The mapping studies localized the critical S1 sequences for this interaction to amino acids 340-470. Based on these results, we speculate that HSP47 is a functional ...

  15. Protein S Protects against Podocyte Injury in Diabetic Nephropathy.

    Science.gov (United States)

    Zhong, Fang; Chen, Haibing; Xie, Yifan; Azeloglu, Evren U; Wei, Chengguo; Zhang, Weijia; Li, Zhengzhe; Chuang, Peter Y; Jim, Belinda; Li, Hong; Elmastour, Firas; Riyad, Jalish M; Weber, Thomas; Chen, Hongyu; Wang, Yongjun; Zhang, Aihua; Jia, Weiping; Lee, Kyung; He, John C

    2018-05-01

    Background Diabetic nephropathy (DN) is a leading cause of ESRD in the United States, but the molecular mechanisms mediating the early stages of DN are unclear. Methods To assess global changes that occur in early diabetic kidneys and to identify proteins potentially involved in pathogenic pathways in DN progression, we performed proteomic analysis of diabetic and nondiabetic rat glomeruli. Protein S (PS) among the highly upregulated proteins in the diabetic glomeruli. PS exerts multiple biologic effects through the Tyro3, Axl, and Mer (TAM) receptors. Because increased activation of Axl by the PS homolog Gas6 has been implicated in DN progression, we further examined the role of PS in DN. Results In human kidneys, glomerular PS expression was elevated in early DN but suppressed in advanced DN. However, plasma PS concentrations did not differ between patients with DN and healthy controls. A prominent increase of PS expression also colocalized with the expression of podocyte markers in early diabetic kidneys. In cultured podocytes, high-glucose treatment elevated PS expression, and PS knockdown further enhanced the high-glucose-induced apoptosis. Conversely, PS overexpression in cultured podocytes dampened the high-glucose- and TNF- α -induced expression of proinflammatory mediators. Tyro3 receptor was upregulated in response to high glucose and mediated the anti-inflammatory response of PS. Podocyte-specific PS loss resulted in accelerated DN in streptozotocin-induced diabetic mice, whereas the transient induction of PS expression in glomerular cells in vivo attenuated albuminuria and podocyte loss in diabetic OVE26 mice. Conclusions Our results support a protective role of PS against glomerular injury in DN progression. Copyright © 2018 by the American Society of Nephrology.

  16. Rhythmic expression of DEC2 protein in vitro and in vivo.

    Science.gov (United States)

    Sato, Fuyuki; Muragaki, Yasuteru; Kawamoto, Takeshi; Fujimoto, Katsumi; Kato, Yukio; Zhang, Yanping

    2016-06-01

    Basic helix-loop-helix (bHLH) transcription factor DEC2 (bHLHE41/Sharp1) is one of the clock genes that show a circadian rhythm in various tissues. DEC2 regulates differentiation, sleep length, tumor cell invasion and apoptosis. Although studies have been conducted on the rhythmic expression of DEC2 mRNA in various tissues, the precise molecular mechanism of DEC2 expression is poorly understood. In the present study, we examined whether DEC2 protein had a rhythmic expression. Western blot analysis for DEC2 protein revealed a rhythmic expression in mouse liver, lung and muscle and in MCF-7 and U2OS cells. In addition, AMP-activated protein kinase (AMPK) activity (phosphorylation of AMPK) in mouse embryonic fibroblasts (MEFs) exhibited a rhythmic expression under the condition of medium change or glucose-depleted medium. However, the rhythmic expression of DEC2 in MEF gradually decreased in time under these conditions. The medium change affected the levels of DEC2 protein and phosphorylation of AMPK. In addition, the levels of DEC2 protein showed a rhythmic expression in vivo and in MCF-7 and U2OS cells. The results showed that the phosphorylation of AMPK immunoreactivity was strongly detected in the liver and lung of DEC2 knockout mice compared with that of wild-type mice. These results may provide new insights into rhythmic expression and the regulation between DEC2 protein and AMPK activity.

  17. Induction of Ski Protein Expression upon Luteinization in Rat Granulosa Cells

    Directory of Open Access Journals (Sweden)

    Hyun Kim

    2012-05-01

    Full Text Available Ski protein is implicated in proliferation/differentiation in a variety of cells. We had previously reported that Ski protein is present in granulosa cells of atretic follicles, but not in preovulatory follicles, suggesting that Ski has a role in apoptosis of granulosa cells. The alternative fate of granulosa cells other than apoptosis is to differentiate to luteal cells; however, it is unknown whether Ski is expressed and has a role in granulosa cells undergoing luteinization. Thus, the aim of the present study was to locate Ski protein in the rat ovary during luteinizationto predict the possible role of Ski. In order to examine the expression pattern of Ski protein along with the progress of luteinization, follicular growth was induced by administration of equine chorionic gonadtropin to immature female rats, and luteinization was induced by human chorionic gonadtropin treatment to mimic luteinizing hormone (LH surge. While no Ski-positive granulosa cells were present in preovulatory follicle, Ski protein expression was induced in response to LH surge, and was maintained after the formation of the corpus luteum (CL. Though Ski protein is absent in granulosa cells of preovulatory follicle, its mRNA (c-Ski was expressed and the level was unchanged even after LH surge. Taken together, these results demonstrated that Ski protein expression is induced in granulosa cells upon luteinization, and suggests that its expression is regulated post-transcriptionally.

  18. [Molecular cloning and expression of Nattokinase gene in Bacillus subtilis].

    Science.gov (United States)

    Liu, B Y; Song, H Y

    2002-05-01

    In order to characterize biochemically the nattokinase,the nucleotide sequence of the nattokinase gene was amplified from the chromosomal DNA of B.subtilis (natto) by PCR. The expression plasmid pBL NK was constructed and was used to transform Bacillus subtilis containing a chromosomal deletion in its subtilisin gene. The supernatant of the culture was collected after 15 h culture. The target proteins were identified by SDS-PAGE. Nattokinase was purified by a method including ultrafiltration, Sephacryl S-100 gel filtration and S-Sepharose ion-exchange chromatography, and 100 mg of purified nattokinase was obtained from one liter of culture. The purity of the protein and the specific activity were 95% and 12 000 u/mg (compared to tPA), respectively.

  19. Genome engineering for improved recombinant protein expression in Escherichia coli.

    Science.gov (United States)

    Mahalik, Shubhashree; Sharma, Ashish K; Mukherjee, Krishna J

    2014-12-19

    A metabolic engineering perspective which views recombinant protein expression as a multistep pathway allows us to move beyond vector design and identify the downstream rate limiting steps in expression. In E.coli these are typically at the translational level and the supply of precursors in the form of energy, amino acids and nucleotides. Further recombinant protein production triggers a global cellular stress response which feedback inhibits both growth and product formation. Countering this requires a system level analysis followed by a rational host cell engineering to sustain expression for longer time periods. Another strategy to increase protein yields could be to divert the metabolic flux away from biomass formation and towards recombinant protein production. This would require a growth stoppage mechanism which does not affect the metabolic activity of the cell or the transcriptional or translational efficiencies. Finally cells have to be designed for efficient export to prevent buildup of proteins inside the cytoplasm and also simplify downstream processing. The rational and the high throughput strategies that can be used for the construction of such improved host cell platforms for recombinant protein expression is the focus of this review.

  20. Thyroid hormone affects secretory activity and uncoupling protein-3 expression in rat harderian gland.

    Science.gov (United States)

    Chieffi Baccari, Gabriella; Monteforte, Rossella; de Lange, Pieter; Raucci, Franca; Farina, Paola; Lanni, Antonia

    2004-07-01

    The effects of T(3) administration on the rat Harderian gland were examined at morphological, biochemical, and molecular levels. T(3) induced hypertrophy of the two cell types (A and B) present in the glandular epithelium. In type A cells, the hypertrophy was mainly due to an increase in the size of the lipid compartment. The acinar lumina were filled with lipoproteic substances, and the cells often showed an olocrine secretory pattern. In type B cells, the hypertrophy largely consisted of a marked proliferation of mitochondria endowed with tightly packed cristae, the mitochondrial number being nearly doubled (from 62 to 101/100 microm(2)). Although the average area of individual mitochondria decreased by about 50%, the total area of the mitochondrial compartment increased by about 80% (from 11 to 19/100 microm(2)). This could be ascribed to T(3)-induced mitochondrial proliferation. The morphological and morphometric data correlated well with our biochemical results, which indicated that mitochondrial respiratory activity is increased in hyperthyroid rats. T(3), by influencing the metabolic function of the mitochondrial compartment, induces lipogenesis and the release of secretory product by type A cells. Mitochondrial uncoupling proteins 2 and 3 were expressed at both mRNA and protein levels in the euthyroid rat Harderian gland. T(3) treatment increased the mRNA levels of both uncoupling protein 2 (UCP2) and UCP3, but the protein level only of UCP3. A possible role for these proteins in the Harderian gland is discussed.

  1. Subcellular localization of Bombyx mori ribosomal protein S3a and ...

    African Journals Online (AJOL)

    USER

    2010-04-05

    Apr 5, 2010 ... In the present study, using a BV/PH-Bms3a-EGFP, we found that Bombyx mori ribosomal protein S3a. (BmS3a) with EGFP fused to its C-terminal, was predominantly localized in the cytoplasm of B. mori cells. Subsequently, to investigate the effect of BmS3a over-expression on BmNPV infection both at the.

  2. Brain injury markers (S100B and NSE in chronic cocaine dependents Marcadores de lesão cerebral (S100B e NSE em dependentes crônicos de cocaína

    Directory of Open Access Journals (Sweden)

    Felix Henrique Paim Kessler

    2007-06-01

    Full Text Available OBJECTIVE: Studies have shown signs of brain damage caused by different mechanisms in cocaine users. The serum neuron specific enolase and S100B protein are considered specific biochemical markers of neuronal and glial cell injury. This study aimed at comparing blood levels of S100B and NSE in chronic cocaine users and in volunteers who did not use cocaine or other illicit drugs. METHOD: Twenty subjects dependent on cocaine but not on alcohol or marijuana, and 20 non-substance using controls were recruited. Subjects were selected by consecutive and non-probabilistic sampling. Neuron specific enolase and S100B levels were determined by luminescence assay. RESULTS: Cocaine users had significantly higher scores than controls in all psychiatric dimensions of the SCL-90 and had cognitive deficits in the subtest cubes of WAIS and the word span. Mean serum S100B level was 0.09 ± 0.04 µg/l among cocaine users and 0.08 ± 0.04 µg/l among controls. Mean serum neuron specific enolase level was 9.7 ± 3.5 ng/l among cocaine users and 8.3 ± 2.6 ng/l among controls. CONCLUSIONS: In this first study using these specific brain damage markers in cocaine users, serum levels of S100B and neuron specific enolase were not statistically different between cocaine dependent subjects and controls.OBJETIVO: Estudos têm demonstrado sinais de lesão cerebral causadas por diferentes mecanismos em usuários de cocaína. A enolase sérica neurônio-específica e a proteína S100B são consideradas marcadores bioquímicos específicos de lesão neuronal e glial. Este estudo objetivou comparar os níveis sangüíneos de S100B e enolase sérica neurônio-específica em usuários crônicos de cocaína e em voluntários que não usam cocaína ou outras drogas ilícitas. MÉTODO: Vinte sujeitos dependentes de cocaína, mas não dependentes de álcool, maconha ou outra droga, e 20 sujeitos controles não usuários de drogas foram recrutados. Os sujeitos foram selecionados por

  3. Optimised 'on demand' protein arraying from DNA by cell free expression with the 'DNA to Protein Array' (DAPA) technology.

    Science.gov (United States)

    Schmidt, Ronny; Cook, Elizabeth A; Kastelic, Damjana; Taussig, Michael J; Stoevesandt, Oda

    2013-08-02

    We have previously described a protein arraying process based on cell free expression from DNA template arrays (DNA Array to Protein Array, DAPA). Here, we have investigated the influence of different array support coatings (Ni-NTA, Epoxy, 3D-Epoxy and Polyethylene glycol methacrylate (PEGMA)). Their optimal combination yields an increased amount of detected protein and an optimised spot morphology on the resulting protein array compared to the previously published protocol. The specificity of protein capture was improved using a tag-specific capture antibody on a protein repellent surface coating. The conditions for protein expression were optimised to yield the maximum amount of protein or the best detection results using specific monoclonal antibodies or a scaffold binder against the expressed targets. The optimised DAPA system was able to increase by threefold the expression of a representative model protein while conserving recognition by a specific antibody. The amount of expressed protein in DAPA was comparable to those of classically spotted protein arrays. Reaction conditions can be tailored to suit the application of interest. DAPA represents a cost effective, easy and convenient way of producing protein arrays on demand. The reported work is expected to facilitate the application of DAPA for personalized medicine and screening purposes. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. [Eukaryotic Expression and Immunogenic Research of Recombination Ebola Virus Membrane Protein Gp-Fc].

    Science.gov (United States)

    Zhang, Xiaoguang; Yang, Ren; Wang, Jiao; Wang, Xuan; Hou, Mieling; An, Lina; Zhu, Ying; Cao, Yuxi; Zeng, Yi

    2016-01-01

    We used 293 cells to express the recombinant membrane protein of the Ebola virus. Then, the immunogenicity of the recombinant protein was studied by immunized BALB/c mice. According to the codon use frequency of humans, the gene encoding the extracellular domain of the Ebola virus membrane protein was optimized, synthesized, and inserted into the eukaryotic expression plasmid pXG-Fc to construct the human IgG Fc and Ebola GP fusion protein expression plasmid pXG-modGP-Fc. To achieve expression, the fusion protein expression vector was transfected into high-density 293 cells using transient transfection technology. The recombinant protein was purified by protein A affinity chromatography. BALB/c mice were immunized with the purified fusion protein, and serum antibody titers evaluated by an indirect enzyme-linked immunosorbent assay (ELISA). Purification and analyses of the protein revealed that the eukaryotic expression vector could express the recombinant protein GP-Fc effectively, and that the recombinant protein in the supernatant of the cell culture was present as a dimer. After immunization with the purified recombinant protein, a high titer of antigen-specific IgG could be detected in the serum of immunized mice by indirect ELISA, showing that the recombinant protein had good immunogenicity. These data suggest that we obtained a recombinant protein with good immunogenicity. Our study is the basis for development of a vaccine against the Ebola virus and for screening of monoclonal antibodies.

  5. Evaluation of outer dense fiber-1 and -2 protein expression in asthenozoospermic infertile men

    Directory of Open Access Journals (Sweden)

    Silvia W. Lestari

    2015-07-01

    Full Text Available Background: Most of male infertility are caused by defect in sperm motility (asthenozoospermia. The molecular mechanism of low sperm motility in asthenozoospermic patients has not been fully understood. Sperm motility is strongly related to the axoneme structure which is composed of microtubules and supported by outer dense fiber (ODF and fibrous sheath (FS protein. The objective of this study was to characterize the ODF (ODF1 and ODF2 expression in asthenozoospermic infertile male and control normozoospermic fertile male.Methods: Asthenozoospermic samples (n=18 were collected from infertile patients at Andrology Lab, Cipto Mangunkusumo Hospital Jakarta and control were taken from normozoospermic fertile donor (n=18. After motility analyses by computer-assisted sperm analysis (CASA, semen were divided into two parts, for Western blot and for immunocytochemistry analysis. Antibody against ODF1 and ODF2 protein were used in both analyses.Results: Analysis of ODF1 protein expression showed bands with molecular weight of ~30 kDa and ODF2 ~85 kDa. The mean band intensity of ODF1 and ODF2 protein were lower in the asthenozoospermic group (AG compared to normozoospermic group (NG. Moreover, both ODF proteins were less intense and less localized in the AG than NG. Sperm motility was lower in AG, compared to control NG, i.e. average path velocity (VAP = 32.07 ± 7.03 vs 37.58 ± 8.73 µm/s, p = 0.455; straight line velocity (VSL = 24.17 ± 6.90 vs 27.61 ± 4.50 µm/s, p = 0.317 and curvilinear velocity (VCL = 45.68 ± 7.91 vs 55.55 ± 16.40 µm/s, p = 0.099.Conclusion: There is down-regulation of ODF1 and ODF2 protein expression and less-compact localization in AG sperm compared to the NG. These changes might have caused disturbances in the sperm motility as observed in this study.

  6. A Generic Protocol for Intracellular Expression of Recombinant Proteins in Bacillus subtilis.

    Science.gov (United States)

    Phan, Trang; Huynh, Phuong; Truong, Tuom; Nguyen, Hoang

    2017-01-01

    Bacillus subtilis (B. subtilis) is a potential and attractive host for the production of recombinant proteins. Different expression systems for B. subtilis have been developed recently, and various target proteins have been recombinantly synthesized and purified using this host. In this chapter, we introduce a generic protocol to express a recombinant protein in B. subtilis. It includes protocols for (1) using our typical expression vector (plasmid pHT254) to introduce a target gene, (2) transformation of the target vector into B. subtilis, and (3) evaluation of the actual expression of a recombinant protein.

  7. Expression of membrane-associated proteins within single emulsion cell facsimiles.

    Science.gov (United States)

    Chanasakulniyom, Mayuree; Martino, Chiara; Paterson, David; Horsfall, Louise; Rosser, Susan; Cooper, Jonathan M

    2012-07-07

    MreB is a structural membrane-associated protein which is one of the key components of the bacterial cytoskeleton. Although it plays an important role in shape maintenance of rod-like bacteria, the understanding of its mechanism of action is still not fully understood. This study shows how segmented flow and microdroplet technology can be used as a new tool for biological in vitro investigation of this protein. In this paper, we demonstrate cell-free expression in a single emulsion system to express red fluorescence protein (RFP) and MreB linked RFP (MreB-RFP). We follow the aggregation and localisation of the fusion protein MreB-RFP in this artificial cell-like environment. The expression of MreB-RFP in single emulsion droplets leads to the formation of micrometer-scale protein patches distributed at the water/oil interface.

  8. Connecting protein and mRNA burst distributions for stochastic models of gene expression

    International Nuclear Information System (INIS)

    Elgart, Vlad; Jia, Tao; Fenley, Andrew T; Kulkarni, Rahul

    2011-01-01

    The intrinsic stochasticity of gene expression can lead to large variability in protein levels for genetically identical cells. Such variability in protein levels can arise from infrequent synthesis of mRNAs which in turn give rise to bursts of protein expression. Protein expression occurring in bursts has indeed been observed experimentally and recent studies have also found evidence for transcriptional bursting, i.e. production of mRNAs in bursts. Given that there are distinct experimental techniques for quantifying the noise at different stages of gene expression, it is of interest to derive analytical results connecting experimental observations at different levels. In this work, we consider stochastic models of gene expression for which mRNA and protein production occurs in independent bursts. For such models, we derive analytical expressions connecting protein and mRNA burst distributions which show how the functional form of the mRNA burst distribution can be inferred from the protein burst distribution. Additionally, if gene expression is repressed such that observed protein bursts arise only from single mRNAs, we show how observations of protein burst distributions (repressed and unrepressed) can be used to completely determine the mRNA burst distribution. Assuming independent contributions from individual bursts, we derive analytical expressions connecting means and variances for burst and steady-state protein distributions. Finally, we validate our general analytical results by considering a specific reaction scheme involving regulation of protein bursts by small RNAs. For a range of parameters, we derive analytical expressions for regulated protein distributions that are validated using stochastic simulations. The analytical results obtained in this work can thus serve as useful inputs for a broad range of studies focusing on stochasticity in gene expression

  9. Expression analysis on 14-3-3 proteins in regenerative liver following partial hepatectomy

    Directory of Open Access Journals (Sweden)

    Deming Xue

    2017-11-01

    Full Text Available Abstract 14-3-3 proteins play a vital part in the regulation of cell cycle and apoptosis as signaling integration points. During liver regeneration, the quiescent hepatocytes go through hypertrophy and proliferation to restore liver weight. Therefore, we speculated that 14-3-3 proteins regulate the progression of liver regeneration. In this study, we analyzed the expression patterns of 14-3-3 proteins during liver regeneration of rat to provide an insight into the regenerative mechanism using western blotting. Only four isoforms (γ, ε, σ and τ/θ of the 14-3-3 proteins were expressed in regenerative liver after partial hepatectomy (PH. The dual effects, the significant down-regulation of 14-3-3ε and the significant up-regulation of 14-3-3τ/θ at 2 h after PH, might play particularly important roles in S-phase entry. The significant peaks of 14-3-3σ at 30 h and of ε and τ/θ at 24 h might be closely related not only to the G2/M transition but also to the size of hepatocytes. Possibly, the peak of 14-3-3ε expression seen at 168 h plays critical roles in the termination of liver regeneration by inhibiting cellular proliferation.

  10. Expression of small leucine-rich proteoglycans in rat anterior pituitary gland.

    Science.gov (United States)

    Horiguchi, Kotaro; Syaidah, Rahimi; Fujiwara, Ken; Tsukada, Takehiro; Ramadhani, Dini; Jindatip, Depicha; Kikuchi, Motoshi; Yashiro, Takashi

    2013-01-01

    Proteoglycans are components of the extracellular matrix and comprise a specific core protein substituted with covalently linked glycosaminoglycan chains. Small leucine-rich proteoglycans (SLRPs) are a major family of proteoglycans and have key roles as potent effectors in cellular signaling pathways. Research during the last two decades has shown that SLRPs regulate biological functions in many tissues such as skin, tendon, kidney, liver, and heart. However, little is known of the expression of SLRPs, or the characteristics of the cells that produce them, in the anterior pituitary gland. Therefore, we have determined whether SLRPs are present in rat anterior pituitary gland. We have used real-time reverse transcription with the polymerase chain reaction to analyze the expression of SLRP genes and have identified the cells that produce SLRPs by using in situ hybridization with a digoxigenin-labeled cRNA probe. We have clearly detected the mRNA expression of SLRP genes, and cells expressing decorin, biglycan, fibromodulin, lumican, proline/arginine-rich end leucine-rich repeat protein (PRELP), and osteoglycin are located in the anterior pituitary gland. We have also investigated the possible double-staining of SLRP mRNA and pituitary hormones, S100 protein (a marker of folliculostellate cells), desmin (a marker of capillary pericytes), and isolectin B4 (a marker of endothelial cells). Decorin, biglycan, fibromodulin, lumican, PRELP, and osteoglycin mRNA have been identified in S100-protein-positive and desmin-positive cells. Thus, we conclude that folliculostellate cells and pericytes produce SLRPs in rat anterior pituitary gland.

  11. Parasitization by Scleroderma guani influences protein expression in Tenebrio molitor pupae.

    Science.gov (United States)

    Zhu, Jia-Ying; Wu, Guo-Xing; Ze, Sang-Zi; Stanley, David W; Yang, Bin

    2014-07-01

    Ectoparasitoid wasps deposit their eggs onto the surface and inject venom into their hosts. Venoms are chemically complex and they exert substantial impact on hosts, including permanent or temporary paralysis and developmental arrest. These visible venom effects are due to changes in expression of genes encoding physiologically relevant proteins. While the influence of parasitization on gene expression in several lepidopterans has been reported, the molecular details of parasitoid/beetle relationships remain mostly unknown. This shortcoming led us to pose the hypothesis that envenomation by the ectoparasitic ant-like bethylid wasp Scleroderma guani leads to changes in protein expression in the yellow mealworm beetle Tenebrio molitor. We tested our hypothesis by comparing the proteomes of non-parasitized and parasitized host pupae using iTRAQ-based proteomics. We identified 41 proteins that were differentially expressed (32↑- and 9↓-regulated) in parasitized pupae. We assigned these proteins to functional categories, including immunity, stress and detoxification, energy metabolism, development, cytoskeleton, signaling and others. We recorded parallel changes in mRNA levels and protein abundance in 14 selected proteins following parasitization. Our findings support our hypothesis by documenting changes in protein expression in parasitized hosts. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Glucose-regulated protein 78 regulates the expression of mitochondrial genesis proteins in HBV-related hepatocellular carcinoma: a clinical analysis

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    LI Yaping

    2017-10-01

    Full Text Available ObjectiveTo investigate the expression of glucose-regulated protein 78 (GRP78 in HBV-related hepatocellular carcinoma (HBV-HCC and its association with clinicopathological features, as well as its regulatory effect on mitochondrial genesis proteins in hepatoma cells, and to provide a basis for new strategies for the prevention and treatment of HCC. MethodsTissue samples were collected from 54 patients with HBV-HCC, and immunohistochemistry and Western blot were used to measure the expression of GRP78, Lon, TFAM, and cytochrome C oxidase Ⅳ (COXⅣ. The expression of GRP78 in hepatoma cells was interfered by siRNA, and then the expression of GRP78, Lon, mitochondrial transcription factor A (TFAM, and COX Ⅳ was measured. Quantitative real-time PCR was used to measure the level of mitochondrial DNA (mtDNA in clinical specimens and HCC cells after GRP78 expression was interfered with. A statistical analysis was performed for clinical and experimental data. The t-test was used for comparison of continuous data between groups, the Fisher′s exact test was used for comparison of categorical data between groups, and the Kaplan-Meier method was used for survival analysis. Results Compared with the adjacent tissues, HBV-HCC tissues had significantly higher expression of GRP78 and Lon (t=9.135 and 5523, both P<0.0001 and significantly lower expression of the mitochondrial genesis proteins TFAM and COX Ⅳ and mtDNA level (t=2.765, 4260, and 12.280, P=0.011, <0.001, and <0.001. There were significant increases in the expression of the mitochondrial genesis proteins TFAM and COX Ⅳ and mtDNA level after the interference with GRP78 expression in hepatoma cells (all P<0.05. There were significant differences in the expression of GRP78 between patients with different numbers of tumors, patients with and without portal vein tumor thrombus, and patients with different tumor stages (P=0.016, 0.003, and 0.045. The patients with low GRP78

  13. Increased expression of Myosin binding protein H in the skeletal muscle of amyotrophic lateral sclerosis patients

    KAUST Repository

    Conti, Antonio

    2014-01-01

    Amyotrophic lateral sclerosis (ALS) is a severe and fatal neurodegenerative disease of still unknown pathogenesis. Recent findings suggest that the skeletal muscle may play an active pathogenetic role. To investigate ALS\\'s pathogenesis and to seek diagnostic markers, we analyzed skeletal muscle biopsies with the differential expression proteomic approach. We studied skeletal muscle biopsies from healthy controls (CN), sporadic ALS (sALS), motor neuropathies (MN) and myopathies (M). Pre-eminently among several differentially expressed proteins, Myosin binding protein H (MyBP-H) expression in ALS samples was anomalously high. MyBP-H is a component of the thick filaments of the skeletal muscle and has strong affinity for myosin, but its function is still unclear. High MyBP-H expression level was associated with abnormal expression of Rho kinase 2 (ROCK2), LIM domain kinase 1 (LIMK1) and cofilin2, that might affect the actin-myosin interaction. We propose that MyBP-H expression level serves, as a putative biomarker in the skeletal muscle, to discriminate ALS from motor neuropathies, and that it signals the onset of dysregulation in actin-myosin interaction; this in turn might contribute to the pathogenesis of ALS. © 2013 Elsevier B.V.

  14. Major vault protein/lung resistance-related protein (MVP/LRP) expression in nervous system tumors.

    Science.gov (United States)

    Sasaki, Tsutomu; Hankins, Gerald R; Helm, Gregory A

    2002-01-01

    Lung resistance-related protein (LRP) was identified as the major vault protein (MVP), the main component of multimeric vault particles. It functions as a transport-associated protein that can be associated with multidrug resistance. In previous studies, expression of MVP/LRP has been documented in tumors of various types. In general, good correlations have been reported for expression of MVP/LRP and decreased sensitivity to chemotherapy and poor prognosis. MVP/LRP expression has been documented in glioblastomas, but its expression in nervous system tumors in general has not been well characterized. Immunohistochemistry using anti-human MVP/LRP antibody (LRP-56) was performed on formalin-fixed, paraffin-embedded archival tissue from 69 primary central nervous system tumors. Expression of MVP/LRP was observed in 81.2% (56/69) of primary nervous system tumors, including astrocytomas (11/13), oligodendrogliomas (1/2), oligoastrocytomas (5/5), ependymoma (1/1), meningiomas (35/45), schwannomas (2/2), and neurofibroma (1/1). Various degrees and distributions of immunoreactivity to MVP/ LRP were observed. Neither the presence nor the degree of immunoreactivity to MVP/LRP showed any correlation with either tumor grade or the presence of brain invasion.

  15. Transcript and protein expression profile of PF11_0394, a Plasmodium falciparum protein expressed in salivary gland sporozoites

    Directory of Open Access Journals (Sweden)

    Schlarman Maggie S

    2012-03-01

    Full Text Available Abstract Background Plasmodium falciparum malaria is a significant problem around the world today, thus there is still a need for new control methods to be developed. Because the sporozoite displays dual infectivity for both the mosquito salivary glands and vertebrate host tissue, it is a good target for vaccine development. Methods The P. falciparum gene, PF11_0394, was chosen as a candidate for study due to its potential role in the invasion of host tissues. This gene, which was selected using a data mining approach from PlasmoDB, is expressed both at the transcriptional and protein levels in sporozoites and likely encodes a putative surface protein. Using reverse transcription-polymerase chain reaction (RT-PCR and green fluorescent protein (GFP-trafficking studies, a transcript and protein expression profile of PF11_0394 was determined. Results The PF11_0394 protein has orthologs in other Plasmodium species and Apicomplexans, but none outside of the group Apicomplexa. PF11_0394 transcript was found to be present during both the sporozoite and erythrocytic stages of the parasite life cycle, but no transcript was detected during axenic exoerythrocytic stages. Despite the presence of transcript throughout several life cycle stages, the PF11_0394 protein was only detected in salivary gland sporozoites. Conclusions PF11_0394 appears to be a protein uniquely detected in salivary gland sporozoites. Even though a specific function of PF11_0394 has not been determined in P. falciparum biology, it could be another candidate for a new vaccine.

  16. Immunohistochemical diagnosis of malignant melanoma of the conjunctiva and uvea: comparison of the novel antibody against melan-A with S100 protein and HMB-45

    DEFF Research Database (Denmark)

    Heegaard, Steffen; Jensen, O.A.; Prause, J.U.

    2000-01-01

    ophthalmology, A103, conjunctiva, gp100, HMB-45, malignant melanoma, MART-1, melan-A, S100, uvea......ophthalmology, A103, conjunctiva, gp100, HMB-45, malignant melanoma, MART-1, melan-A, S100, uvea...

  17. Novel leukocyte protein, Trojan, differentially expressed during thymocyte development.

    Science.gov (United States)

    Petrov, Petar; Motobu, Maki; Salmi, Jussi; Uchida, Tatsuya; Vainio, Olli

    2010-04-01

    "Trojan" is a novel cell surface protein, discovered from chicken embryonic thymocytes on the purpose to identify molecules involved in T cell differentiation. The molecule is predicted as a type I transmembrane protein having a Sushi and two fibronectin type III domains and a pair of intracellular phosphorylation sites. Its transcript expression is specific for lymphoid tissues and the presence of the protein on the surface of recirculating lymphocytes and macrophages was confirmed by immunofluorescence analysis. In thymus, about half of the double negative (CD4(-) CD8(-)) and CD8 single positive and the majority of CD4 single positive cells express Trojan with a relatively high intensity. However, only a minority of the double positive (CD4(+) CD8(+)) cells are positive for Trojan. This expression pattern, similar to that of some proteins with anti-apoptotic and function, like IL-7Ralpha, makes Trojan an attractive candidate of having an anti-apoptotic role. Copyright 2010 Elsevier Ltd. All rights reserved.

  18. Expression of Hepatitis C Virus Core and E2 antigenic recombinant proteins and their use for development of diagnostic assays.

    Science.gov (United States)

    Ali, Amjad; Nisar, Muhammad; Idrees, Muhammad; Rafique, Shazia; Iqbal, Muhammad

    2015-05-01

    Early diagnosis of HCV infection is based on detection of antibodies against HCV proteins using recombinant viral antigens. The present study was designed to select, clone and express the antigenic regions of Core and E2 genes from local HCV-3a genotype and to utilize the antigenic recombinant proteins (Core & E2) to develop highly sensitive, specific and economical diagnostic assays for detection of HCV infection. The antigenic sites were determined within Core and E2 genes and were then cloned in pET-28a expression vector. The right orientation of the desired inserted fragments of Core and E2 were confirmed via sequencing prior to expression and were then transformed in BL21 (DE3) pLysS strains of E. coli and induced with 0.5mM Isopropyl-b-D-thiogalactopyranoside (IPTG) for the production of antigenic recombinant proteins. The produced truncated antigens were then purified by Nickel affinity chromatography and were confirmed by western blotting, immunoblotting and enzyme-linked immunosorbent assay (ELISA). The expressed Core and E2 recombinant antigens were used to develop immunoblotting assay for the detection of anti-HCV antibodies in sera. With immunoblotting, a total of 93-HCV infected sera and 35-HCV negative individuals were tested for the presence of anti-HCV antibodies to the Core and E2 antigens. Recombinant antigen showed 100% reactivity against HCV infected sera, with no cross reactivity against HCV-negative sera. The immunoblot assay mixture of recombinant antigens (Core+E2) showed a strong reaction intensity in the test area (TA) as compared to the individual truncated Core and E2 recombinant antigens. In the in-house ELISA assay, mixed Core and E2 recombinant antigens showed 100% reactivity against a standardized panel of 150-HCV-positive sera and non reactivity against a standardized panel of 150 HCV-negative sera while also being non reactive to sera positive for other viral infections. The antigenic recombinant antigens also were tested for the

  19. Effect of zeranol on expression of apoptotic and cell cycle proteins in murine placentae

    International Nuclear Information System (INIS)

    Wang, Yanfei; Li, Lu; Wang, C.C.; Leung, Lai K.

    2013-01-01

    Highlights: • Zeranol administered at 1–100 mg/kg/day demonstrated an increasing trend of fetal resorption and early delivery in mice. • Placental expression of Cdk2 and 4, Cyclin D1 and Bcl-xL were reduced mostly in mice treated with ≥10 mg zeranol/kg/day. • Increased pErk-1 and 2 were also observed in the placentae of zeranol-treated mice. - Abstract: Mycotoxins are chemicals produced by fungus and many of them are toxic to humans. Zeranol is a mycotoxin used to promote growth in cattle in North America; yet such a practice draws concern about the residual compound in meat in European countries. In the present study, the toxicity of zeranol was tested in a mouse model for reproduction. Pregnant ICR mice were given p.o. daily doses of zeranol at 0, 1, 10, 100 mg/kg for 4 days (from E13.5 to E16.5). Increased rates of fetal resorption at late gestation (E17.5) and preterm birth (< E18.5) were observed in mice treated with zeranol. The apparent factors causing these perinatal conditions were subsequently investigated. Perturbation of cell death or proliferation-related proteins might deter the growth and maintenance of the placentae, and the subsequent fetal resorption and preterm birth. Placental tissue isolated from pregnant mice at E17.5 showed that the expressions of Cdk2 and 4, Cyclin D1 and Bcl-xL were reduced in zeranol-treatment groups. The downregulations might signify growth or maintenance failure in the placentae. Furthermore, reduction in the signaling proteins Erk-1/2 in the placentae could trigger the decrease in the cell cycle/apoptosis proteins. In addition, relaxin is associated with preterm labor. An increase in placental Relaxin-1 expression could also contribute to early delivery in this study. Result of the current study suggested that exposure to zeranol might introduce adverse effect in pregnancy

  20. Ethanol extracts of black pepper or turmeric down-regulated SIRT1 protein expression in Daudi culture cells.

    Science.gov (United States)

    Nishimura, Yuri; Kitagishi, Yasuko; Yoshida, Hitomi; Okumura, Naoko; Matsuda, Satoru

    2011-01-01

    SIRT1 is a mammalian candidate molecule involved in longevity and diverse metabolic processes. The present study aimed to determine the effects of certain herbs and spices on SIRT1 expression. Human cell lines Daudi, Jurkat, U937 and K562 were cultured in RPMI-1640. Herb and spice powders were prepared and the supernatants were collected. RT-PCR was used to quantify the expression level of the gene. Protein samples were then analyzed by Western blotting. Western blotting revealed the down-regulation of SIRT1 protein expression in Daudi cells treated with extracts of black pepper or turmeric. On the other hand, the effect on the SIRT1 gene expression examined by reverse transcription polymerase chain reaction was unaltered. In conclusion, component(s) of certain herbs and spices may induce the down-regulation of SIRT1 protein.

  1. Expression of Iron-Related Proteins Differentiate Non-Cancerous and Cancerous Breast Tumors

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    Sara Pizzamiglio

    2017-02-01

    Full Text Available We have previously reported hepcidin and ferritin increases in the plasma of breast cancer patients, but not in patients with benign breast disease. We hypothesized that these differences in systemic iron homeostasis may reflect alterations in different iron-related proteins also play a key biochemical and regulatory role in breast cancer. Thus, here we explored the expression of a bundle of molecules involved in both iron homeostasis and tumorigenesis in tissue samples. Enzyme-linked immunosorbent assay (ELISA or reverse-phase protein array (RPPA, were used to measure the expression of 20 proteins linked to iron processes in 24 non-cancerous, and 56 cancerous, breast tumors. We found that cancerous tissues had higher level of hepcidin than benign lesions (p = 0.012. The univariate analysis of RPPA data highlighted the following seven proteins differentially expressed between non-cancerous and cancerous breast tissue: signal transducer and transcriptional activator 5 (STAT5, signal transducer and activator of transcription 3 (STAT3, bone morphogenetic protein 6 (BMP6, cluster of differentiation 74 (CD74, transferrin receptor (TFRC, inhibin alpha (INHA, and STAT5_pY694. These findings were confirmed for STAT5, STAT3, BMP6, CD74 and INHA when adjusting for age. The multivariate statistical analysis indicated an iron-related 10-protein panel effective in separating non-cancerous from cancerous lesions including STAT5, STAT5_pY694, myeloid differentiation factor 88 (MYD88, CD74, iron exporter ferroportin (FPN, high mobility group box 1 (HMGB1, STAT3_pS727, TFRC, ferritin heavy chain (FTH, and ferritin light chain (FTL. Our results showed an association between some iron-related proteins and the type of tumor tissue, which may provide insight in strategies for using iron chelators to treat breast cancer.

  2. Negative regulation of parathyroid hormone-related protein expression by steroid hormones

    International Nuclear Information System (INIS)

    Kajitani, Takashi; Tamamori-Adachi, Mimi; Okinaga, Hiroko; Chikamori, Minoru; Iizuka, Masayoshi; Okazaki, Tomoki

    2011-01-01

    Highlights: → Steroid hormones repress expression of PTHrP in the cell lines where the corresponding nuclear receptors are expressed. → Nuclear receptors are required for suppression of PTHrP expression by steroid hormones, except for androgen receptor. → Androgen-induced suppression of PTHrP expression appears to be mediated by estrogen receptor. -- Abstract: Elevated parathyroid hormone-related protein (PTHrP) is responsible for humoral hypercalcemia of malignancy (HHM), which is of clinical significance in treatment of terminal patients with malignancies. Steroid hormones were known to cause suppression of PTHrP expression. However, detailed studies linking multiple steroid hormones to PTHrP expression are lacking. Here we studied PTHrP expression in response to steroid hormones in four cell lines with excessive PTHrP production. Our study established that steroid hormones negatively regulate PTHrP expression. Vitamin D receptor, estrogen receptor α, glucocorticoid receptor, and progesterone receptor, were required for repression of PTHrP expression by the cognate ligands. A notable exception was the androgen receptor, which was dispensable for suppression of PTHrP expression in androgen-treated cells. We propose a pathway(s) involving nuclear receptors to suppress PTHrP expression.

  3. Differential expression of pancreatitis-associated protein and thrombospondins in arterial versus venous tissues.

    Science.gov (United States)

    Szasz, Theodora; Eddy, Susan; Paulauskis, Joseph; Burnett, Robert; Ellekilde, Merete; Iovanna, Juan L; Watts, Stephanie W

    2009-01-01

    Arteries and veins modulate cardiovascular homeostasis and contribute to hypertension pathogenesis. Functional differences between arteries and veins are based upon differences in gene expression. To better characterize these expression patterns, and to identify candidate genes that could be manipulated selectively in the venous system, we performed whole genome expression profiling of arteries and veins. We used the CodeLink platform and the major artery (thoracic aorta) and vein (caudal vena cava) of the rat. The most prominent difference was pancreatitis-associated protein (PAP1), expressed 64-fold higher in vena cava versus aorta. Expression of mRNA for thrombospondins (TSP-1, TSP-4) was greater than 5-fold higher in veins versus arteries. Higher mRNA expression of TSP-1, TSP-2, TSP-4 and PAP1 in vena cava versus aorta was confirmed by PCR. Immunohistochemical analysis of tissue sections qualitatively confirmed a higher expression of these proteins in vena cava versus aorta. This is the first gene array study of adult rat arterial and venous tissues, and also the first study to report differences in inflammatory genes between arteries and veins. Data from these studies may provide novel insights into the genetic basis for functional differences between arteries and veins in health and disease. Copyright 2009 S. Karger AG, Basel.

  4. Bcl-2 Protein Expression in Egyptian Acute Myeloid Leukemia

    International Nuclear Information System (INIS)

    El-Shakankiry, N.; El-Sayed, Gh.M.M.; El-Maghraby, Sh.; Moneer, M.M.

    2009-01-01

    Objective: The primary cause of treatment failure in acute myeloid leukemia (AML) is the emergence of both resistant disease and early relapse. The bcl-2 gene encodes a 26-kDa protein that promotes cell survival by blocking programmed cell death (apoptosis). In the present study, bcl-2 protein expression was evaluated in newly diagnosed AML patients and correlated with the induction of remission and overall survival (OS), in an attempt to define patients who might benefit from modified therapeutic strategies. Patients and methods: Pretreatment cellular bcl-2 protein expression was measured in bone marrow samples obtained from 68 patients of newly diagnosed acute myeloid leukemia and 10 healthy controls by western blotting. Results: The mean bcl-2 protein expression was significantly higher in patients (0.68610.592) compared to controls (0.313±0.016) (p=0.002). The overall survival for patients with mean bcl-2 expression of less, and more than or equal to 0.315, was 67% and 56%, respectively, with no significant difference between the two groups 0»=0.86). Conclusion: Even though we did not observe a significant difference in overall survival between patients with high and low levels of bcl-2, modulation of this protein might still be considered as an option for enhancing the effectiveness of conventional chemotherapy.

  5. Cross-talk between amyloidogenic proteins in type-2 diabetes and Parkinson’s disease

    OpenAIRE

    Horvath, Istvan; Wittung-Stafshede, Pernilla

    2016-01-01

    Protein assembly into ordered so-called amyloid fibers is a process that promotes several neurodegenerative disorders, such as Alzheimer’s and Parkinson’s disease (PD). Also type-2 diabetes (T2D) is a disease involving amyloid formation, although it occurs in the pancreas. Since the protein that forms amyloids in PD, α-synuclein (aS), is also expressed in the pancreas, we investigated whether it could affect aggregation of the peptide involved in T2D, and vice versa. Using in vitro methods an...

  6. High glutamate attenuates S100B and LDH outputs from rat cortical slices enhanced by either oxygen-glucose deprivation or menadione.

    Science.gov (United States)

    Demircan, Celaleddin; Gül, Zülfiye; Büyükuysal, R Levent

    2014-07-01

    One hour incubation of rat cortical slices in a medium without oxygen and glucose (oxygen-glucose deprivation, OGD) increased S100B release to 6.53 ± 0.3 ng/ml/mg protein from its control value of 3.61 ± 0.2 ng/ml/mg protein. When these slices were then transferred to a medium containing oxygen and glucose (reoxygenation, REO), S100B release rose to 344 % of its control value. REO also caused 192 % increase in lactate dehydrogenase (LDH) leakage. Glutamate added at millimolar concentration into the medium decreased OGD or REO-induced S100B release and REO-induced LDH leakage. Alpha-ketoglutarate, a metabolic product of glutamate, was found to be as effective as glutamate in decreasing the S100B and LDH outputs. Similarly lactate, 2-ketobutyrate and ethyl pyruvate, a lipophilic derivative of pyruvate, also exerted a glutamate-like effect on S100B and LDH outputs. Preincubation with menadione, which produces H2O2 intracellularly, significantly increased S100B and LDH levels in normoxic medium. All drugs tested in the present study, with the exception of pyruvate, showed a complete protection against menadione preincubation. Additionally, each OGD-REO, menadione or H2O2-induced mitochondrial energy impairments determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining and OGD-REO or menadione-induced increases in reactive oxygen substances (ROS) determined by 2,7-dichlorofluorescin diacetate (DCFH-DA) were also recovered by glutamate. Interestingly, H2O2-induced increase in fluorescence intensity derived from DCFH-DA in a slice-free physiological medium was attenuated significantly by glutamate and alpha-keto acids. All these drug actions support the conclusion that high glutamate, such as alpha-ketoglutarate and other keto acids, protects the slices against OGD- and REO-induced S100B and LDH outputs probably by scavenging ROS in addition to its energy substrate metabolite property.

  7. Expression Patterns and Identified Protein-Protein Interactions Suggest That Cassava CBL-CIPK Signal Networks Function in Responses to Abiotic Stresses.

    Science.gov (United States)

    Mo, Chunyan; Wan, Shumin; Xia, Youquan; Ren, Ning; Zhou, Yang; Jiang, Xingyu

    2018-01-01

    Cassava is an energy crop that is tolerant of multiple abiotic stresses. It has been reported that the interaction between Calcineurin B-like (CBL) protein and CBL-interacting protein kinase (CIPK) is implicated in plant development and responses to various stresses. However, little is known about their functions in cassava. Herein, 8 CBL ( MeCBL ) and 26 CIPK ( MeCIPK ) genes were isolated from cassava by genome searching and cloning of cDNA sequences of Arabidopsis CBL s and CIPK s. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis showed that the expression levels of MeCBL and MeCIPK genes were different in different tissues throughout the life cycle. The expression patterns of 7 CBL and 26 CIPK genes in response to NaCl, PEG, heat and cold stresses were analyzed by quantitative real-time PCR (qRT-PCR), and it was found that the expression of each was induced by multiple stimuli. Furthermore, we found that many pairs of CBLs and CIPKs could interact with each other via investigating the interactions between 8 CBL and 25 CIPK proteins using a yeast two-hybrid system. Yeast cells co-transformed with cassava MeCIPK24, MeCBL10 , and Na + /H + antiporter MeSOS1 genes exhibited higher salt tolerance compared to those with one or two genes. These results suggest that the cassava CBL-CIPK signal network might play key roles in response to abiotic stresses.

  8. The C-terminal random coil region tunes the Ca²⁺-binding affinity of S100A4 through conformational activation.

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    Annette Duelli

    Full Text Available S100A4 interacts with many binding partners upon Ca2+ activation and is strongly associated with increased metastasis formation. In order to understand the role of the C-terminal random coil for the protein function we examined how small angle X-ray scattering of the wild-type S100A4 and its C-terminal deletion mutant (residues 1-88, Δ13 changes upon Ca2+ binding. We found that the scattering intensity of wild-type S100A4 changes substantially in the 0.15-0.25 Å-1 q-range whereas a similar change is not visible in the C-terminus deleted mutant. Ensemble optimization SAXS modeling indicates that the entire C-terminus is extended when Ca2+ is bound. Pulsed field gradient NMR measurements provide further support as the hydrodynamic radius in the wild-type protein increases upon Ca2+ binding while the radius of Δ13 mutant does not change. Molecular dynamics simulations provide a rational explanation of the structural transition: the positively charged C-terminal residues associate with the negatively charged residues of the Ca2+-free EF-hands and these interactions loosen up considerably upon Ca2+-binding. As a consequence the Δ13 mutant has increased Ca2+ affinity and is constantly loaded at Ca2+ concentration ranges typically present in cells. The activation of the entire C-terminal random coil may play a role in mediating interaction with selected partner proteins of S100A4.

  9. Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli

    DEFF Research Database (Denmark)

    Sørensen, Hans; Mortensen, Kim

    2005-01-01

    Pure, soluble and functional proteins are of high demand in modern biotechnology. Natural protein sources rarely meet the requirements for quantity, ease of isolation or price and hence recombinant technology is often the method of choice. Recombinant cell factories are constantly employed...... molecular tools available. In spite of all these qualities, expression of recombinant proteins with E. coli as the host often results in insoluble and/or nonfunctional proteins. Here we review new approaches to overcome these obstacles by strategies that focus on either controlled expression of target...... for the production of protein preparations bound for downstream purification and processing. Eschericia coli is a frequently used host, since it facilitates protein expression by its relative simplicity, its inexpensive and fast high density cultivation, the well known genetics and the large number of compatible...

  10. Marker Protein Expression Combined With Expression Heterogeneity is a Powerful Indicator of Malignancy in Acral Lentiginous Melanomas.

    Science.gov (United States)

    Cintra Lopes Carapeto, Fernando; Neves Comodo, Andréia; Germano, Andressa; Pereira Guimarães, Daiane; Barcelos, Denise; Fernandes, Mariana; Landman, Gilles

    2017-02-01

    Samples of acral lentiginous melanomas (ALMs) were obtained from the Department of Pathology at Escola Paulista de Medicina-Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazil. Demographic, clinical, and follow-up data were obtained from the charts of Hospital São Paulo. From 2 tissue microarrays containing 60 nevi and quadruplicate samples of ≥1.0-mm of 49 ALM, sections were stained to evaluate SCF, KIT, BRAF, CYCLIND1, MYC, and PTEN immunohistochemical protein expression. Nevi and ALM from 2006 to 2010 were reviewed and collected. All specimens were in the vertical growth phase, and histopathological parameters indicated that tumors were at an advanced stage at diagnosis. Average tumor thickness was 6.95 mm, 63% were ulcerated, average mitotic index was 5 mitotic cells per mm, and 43% were at Clark's level V. Compared with nevi, the χ test showed that ALM significantly correlated with SCF protein expression (P = 0.001) and expression heterogeneity (P < 0.000). Similar findings were observed for KIT (P = 0.005, P = 0.003, respectively), MYC (P < 0.000, P < 0.000), and PTEN (P = 0.005, P < 0.000). Malignancy did not correlate with BRAF and CYCLIN D1 expression (P = 0.053 and P = 0.259, respectively), but it did significantly correlate with their heterogeneous expression (P < 0.000, P = 0.024, respectively). Combined protein expression had an odds ratio of greater malignancy when BRAF and MYC were positive and/or heterogeneously expressed (OR of 78 and 95, respectively). We show that marker protein expression, when combined with heterogeneous expression as shown by immunohistochemistry, is a powerful indicator of malignancy in ALMs, especially, when protein pairs are combined.

  11. A phycocyanin·phellandrene synthase fusion enhances recombinant protein expression and β-phellandrene (monoterpene) hydrocarbons production in Synechocystis (cyanobacteria).

    Science.gov (United States)

    Formighieri, Cinzia; Melis, Anastasios

    2015-11-01

    Cyanobacteria can be exploited as photosynthetic platforms for heterologous generation of terpene hydrocarbons with industrial applications. Transformation of Synechocystis and heterologous expression of the β-phellandrene synthase (PHLS) gene alone is necessary and sufficient to confer to Synechocystis the ability to divert intermediate terpenoid metabolites and to generate the monoterpene β-phellandrene during photosynthesis. However, terpene synthases, including the PHLS, have a slow Kcat (low Vmax) necessitating high levels of enzyme concentration to enable meaningful rates and yield of product formation. Here, a novel approach was applied to increase the PHLS protein expression alleviating limitations in the rate and yield of β-phellandrene product generation. Different PHLS fusion constructs were generated with the Synechocystis endogenous cpcB sequence, encoding for the abundant in cyanobacteria phycocyanin β-subunit, expressed under the native cpc operon promoter. In one of these constructs, the CpcB·PHLS fusion protein accumulated to levels approaching 20% of the total cellular protein, i.e., substantially higher than expressing the PHLS protein alone under the same endogenous cpc promoter. The CpcB·PHLS fusion protein retained the activity of the PHLS enzyme and catalyzed β-phellandrene synthesis, yielding an average of 3.2 mg product g(-1) dry cell weight (dcw) versus the 0.03 mg g(-1)dcw measured with low-expressing constructs, i.e., a 100-fold yield improvement. In conclusion, the terpene synthase fusion-protein approach is promising, as, in this case, it substantially increased the amount of the PHLS in cyanobacteria, and commensurately improved rates and yield of β-phellandrene hydrocarbons production in these photosynthetic microorganisms. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  12. The expression and function of hsp30-like small heat shock protein genes in amphibians, birds, fish, and reptiles.

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    Heikkila, John J

    2017-01-01

    Small heat shock proteins (sHSPs) are a superfamily of molecular chaperones with important roles in protein homeostasis and other cellular functions. Amphibians, reptiles, fish and birds have a shsp gene called hsp30, which was also referred to as hspb11 or hsp25 in some fish and bird species. Hsp30 genes, which are not found in mammals, are transcribed in response to heat shock or other stresses by means of the heat shock factor that is activated in response to an accumulation of unfolded protein. Amino acid sequence analysis revealed that representative HSP30s from different classes of non-mammalian vertebrates were distinct from other sHSPs including HSPB1/HSP27. Studies with amphibian and fish recombinant HSP30 determined that they were molecular chaperones since they inhibited heat- or chemically-induced aggregation of unfolded protein. During non-mammalian vertebrate development, hsp30 genes were differentially expressed in selected tissues. Also, heat shock-induced stage-specific expression of hsp30 genes in frog embryos was regulated at the level of chromatin structure. In adults and/or tissue culture cells, hsp30 gene expression was induced by heat shock, arsenite, cadmium or proteasomal inhibitors, all of which enhanced the production of unfolded/damaged protein. Finally, immunocytochemical analysis of frog and chicken tissue culture cells revealed that proteotoxic stress-induced HSP30 accumulation co-localized with aggresome-like inclusion bodies. The congregation of damaged protein in aggresomes minimizes the toxic effect of aggregated protein dispersed throughout the cell. The current availability of probes to detect the presence of hsp30 mRNA or encoded protein has resulted in the increased use of hsp30 gene expression as a marker of proteotoxic stress in non-mammalian vertebrates. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. The small heat shock proteins from Acidithiobacillus ferrooxidans: gene expression, phylogenetic analysis, and structural modeling

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    Ribeiro Daniela A

    2011-12-01

    Full Text Available Abstract Background Acidithiobacillus ferrooxidans is an acidophilic, chemolithoautotrophic bacterium that has been successfully used in metal bioleaching. In this study, an analysis of the A. ferrooxidans ATCC 23270 genome revealed the presence of three sHSP genes, Afe_1009, Afe_1437 and Afe_2172, that encode proteins from the HSP20 family, a class of intracellular multimers that is especially important in extremophile microorganisms. Results The expression of the sHSP genes was investigated in A. ferrooxidans cells submitted to a heat shock at 40°C for 15, 30 and 60 minutes. After 60 minutes, the gene on locus Afe_1437 was about 20-fold more highly expressed than the gene on locus Afe_2172. Bioinformatic and phylogenetic analyses showed that the sHSPs from A. ferrooxidans are possible non-paralogous proteins, and are regulated by the σ32 factor, a common transcription factor of heat shock proteins. Structural studies using homology molecular modeling indicated that the proteins encoded by Afe_1009 and Afe_1437 have a conserved α-crystallin domain and share similar structural features with the sHSP from Methanococcus jannaschii, suggesting that their biological assembly involves 24 molecules and resembles a hollow spherical shell. Conclusion We conclude that the sHSPs encoded by the Afe_1437 and Afe_1009 genes are more likely to act as molecular chaperones in the A. ferrooxidans heat shock response. In addition, the three sHSPs from A. ferrooxidans are not recent paralogs, and the Afe_1437 and Afe_1009 genes could be inherited horizontally by A. ferrooxidans.

  14. Exploiting translational coupling for the selection of cells producing toxic recombinant proteins from expression vectors.

    Science.gov (United States)

    Tagliavia, Marcello; Cuttitta, Angela

    2016-01-01

    High rates of plasmid instability are associated with the use of some expression vectors in Escherichia coli, resulting in the loss of recombinant protein expression. This is due to sequence alterations in vector promoter elements caused by the background expression of the cloned gene, which leads to the selection of fast-growing, plasmid-containing cells that do not express the target protein. This phenomenon, which is worsened when expressing toxic proteins, results in preparations containing very little or no recombinant protein, or even in clone loss; however, no methods to prevent loss of recombinant protein expression are currently available. We have exploited the phenomenon of translational coupling, a mechanism of prokaryotic gene expression regulation, in order to select cells containing plasmids still able to express recombinant proteins. Here we designed an expression vector in which the cloned gene and selection marker are co-expressed. Our approach allowed for the selection of the recombinant protein-expressing cells and proved effective even for clones encoding toxic proteins.

  15. [(35)S]-GTPgammaS autoradiography reveals alpha(2) adrenoceptor-mediated G-protein activation in amygdala and lateral septum.

    Science.gov (United States)

    Newman-Tancredi, A; Chaput, C; Touzard, M; Millan, M J

    2000-04-03

    alpha(2)-adrenoceptor-mediated G-protein activation was examined by [(35)S]-GTPgammaS autoradiography. In alpha(2)-adrenoceptor-rich regions (amygdala, lateral septum), noradrenaline stimulated [(35)S]-GTPgammaS binding. These actions were abolished by the selective alpha(2) antagonist, atipamezole. Conversely, in caudate nucleus, which expresses few alpha(2) receptors, noradrenaline-induced stimulation was not inhibited by atipamezole, suggesting that it is not mediated by alpha(2)-adrenoceptors.

  16. The Human Antimicrobial Protein Calgranulin C Participates in Control of Helicobacter pylori Growth and Regulation of Virulence.

    Science.gov (United States)

    Haley, Kathryn P; Delgado, Alberto G; Piazuelo, M Blanca; Mortensen, Brittany L; Correa, Pelayo; Damo, Steven M; Chazin, Walter J; Skaar, Eric P; Gaddy, Jennifer A

    2015-07-01

    During infectious processes, antimicrobial proteins are produced by both epithelial cells and innate immune cells. Some of these antimicrobial molecules function by targeting transition metals and sequestering these metals in a process referred to as "nutritional immunity." This chelation strategy ultimately starves invading pathogens, limiting their growth within the vertebrate host. Recent evidence suggests that these metal-binding antimicrobial molecules have the capacity to affect bacterial virulence, including toxin secretion systems. Our previous work showed that the S100A8/S100A9 heterodimer (calprotectin, or calgranulin A/B) binds zinc and represses the elaboration of the H. pylori cag type IV secretion system (T4SS). However, there are several other S100 proteins that are produced in response to infection. We hypothesized that the zinc-binding protein S100A12 (calgranulin C) is induced in response to H. pylori infection and also plays a role in controlling H. pylori growth and virulence. To test this, we analyzed gastric biopsy specimens from H. pylori-positive and -negative patients for S100A12 expression. These assays showed that S100A12 is induced in response to H. pylori infection and inhibits bacterial growth and viability in vitro by binding nutrient zinc. Furthermore, the data establish that the zinc-binding activity of the S100A12 protein represses the activity of the cag T4SS, as evidenced by the gastric cell "hummingbird" phenotype, interleukin 8 (IL-8) secretion, and CagA translocation assays. In addition, high-resolution field emission gun scanning electron microscopy (FEG-SEM) was used to demonstrate that S100A12 represses biogenesis of the cag T4SS. Together with our previous work, these data reveal that multiple S100 proteins can repress the elaboration of an oncogenic bacterial surface organelle. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  17. Calcium affecting protein expression in longan under simulated acid rain stress.

    Science.gov (United States)

    Pan, Tengfei; Li, Yongyu; Ma, Cuilan; Qiu, Dongliang

    2015-08-01

    Longan (Dimocarpus longana Lour. cv. Wulongling) of uniform one-aged seedlings grown in pots were selected to study specific proteins expressed in leaves under simulated acid rain (SiAR) stress and exogenous Ca(2+) regulation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results showed that there was a protein band specifically expressed under SiAR of pH 2.5 stress for 15 days with its molecular weight of about 23 kD. A 17 kD protein band specifically expressed after SiAR stress 5 days. Compared with pH 2.5, the pH 3.5 of SiAR made a less influence to protein expression. Two-dimensional electrophoresis (2-DE) results showed that six new specific proteins including C4 (20.2 kD pI 6.0), F (24 kD pI 6.35), B3 (22.3 kD pI 6.35), B4 (23.5 kD pI 6.5), C5 (21.8 kD pI 5.6), and C6 (20.2 kD pI 5.6) specifically expressed. C4 always expressed during SiAR stress. F expressed under the stress of pH 2.5 for 15 days and expressed in all pH SiAR stress for 20 days. The expression of proteins including B3, C5, and C6 was related to pH value and stress intensity of SiAR. The expression of B4 resulted from synergistic effects of SiAR and Ca. The expression of G1 (Mr 19.3 kD, pI 4.5), G2 (Mr 17.8 kD, pI 4.65), G3 (Mr 16.6 kD, pI 4.6), and G4 (Mr 14.7 kD, pI 4.4) enhanced under the treatment of 5 mM ethylene glycol tetraacetic acid (EGTA) and 2 mM chlorpromazine (CPZ). These proteins showed antagonistic effects and might be relative to the Ca-calmodulin (Ca-CaM) system of longan in response to SiAR stress.

  18. Serum immune-related proteins are differentially expressed during hibernation in the American black bear.

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    Brian A Chow

    Full Text Available Hibernation is an adaptation to conserve energy in the face of extreme environmental conditions and low food availability that has risen in several animal phyla. This phenomenon is characterized by reduced metabolic rate (∼25% of the active basal metabolic rate in hibernating bears and energy demand, while other physiological adjustments are far from clear. The profiling of the serum proteome of the American black bear (Ursus americanus may reveal specific proteins that are differentially modulated by hibernation, and provide insight into the remarkable physiological adaptations that characterize ursid hibernation. In this study, we used differential gel electrophoresis (DIGE analysis, liquid chromatography coupled to tandem mass spectrometry, and subsequent MASCOT analysis of the mass spectra to identify candidate proteins that are differentially expressed during hibernation in captive black bears. Seventy serum proteins were identified as changing by ±1.5 fold or more, out of which 34 proteins increased expression during hibernation. The majority of identified proteins are involved in immune system processes. These included α2-macroglobulin, complement components C1s and C4, immunoglobulin μ and J chains, clusterin, haptoglobin, C4b binding protein, kininogen 1, α2-HS-glycoprotein, and apoplipoproteins A-I and A-IV. Differential expression of a subset of these proteins identified by proteomic analysis was also confirmed by immunodetection. We propose that the observed serum protein changes contribute to the maintenance of the hibernation phenotype and health, including increased capacities for bone maintenance and wound healing during hibernation in bears.

  19. Extracellular ATP drives breast cancer cell migration and metastasis via S100A4 production by cancer cells and fibroblasts.

    Science.gov (United States)

    Liu, Ying; Geng, Yue-Hang; Yang, Hui; Yang, Han; Zhou, Yan-Ting; Zhang, Hong-Quan; Tian, Xin-Xia; Fang, Wei-Gang

    2018-05-04

    Our previous work has demonstrated that extracellular ATP is an important pro-invasive factor, and in this study, we tapped into a possible mechanism involved. We discovered that ATP could upregulate both the intracellular expression and secretion of S100A4 in breast cancer cells and fibroblasts. Apart from stimulating breast cancer cell motility via intracellular S100A4, ATP enhanced the ability of breast cancer cells to transform fibroblasts into cancer-associated fibroblast (CAF)-like cells, which in turn secreted S100A4 to further promote cancer cell motility. Both apyrase and niclosamide treatments could inhibit metastasis of inoculated tumors to lung, liver and kidney in mice model, and CAFs from these treated tumors exhibited weakened migration-stimulating capacity for breast cancer cells. Collectively, our data indicate that extracellular ATP promotes the interactions between breast cancer cells and fibroblasts, which work collaboratively via production of S100A4 to exacerbate breast cancer metastasis. Copyright © 2018. Published by Elsevier B.V.

  20. Cortisol, Interleukins and S100B in Delirium in the Elderly

    Science.gov (United States)

    van Munster, Barbara C.; Bisschop, Peter H.; Zwinderman, Aeilko H.; Korevaar, Johanna C.; Endert, Erik; Wiersinga, W. Joost; van Oosten, Hannah E.; Goslings, J. Carel; de Rooij, Sophia E. J. A.

    2010-01-01

    In independent studies delirium was associated with higher levels of cortisol, interleukin(IL)s, and S100B. The aim of this study was to simultaneously compare cortisol, IL-6, IL-8, and S100B levels in patients aged 65 years and older admitted for hip fracture surgery with and without delirium. Cortisol, IL-6, IL-8, and S100B were assayed in…