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Sample records for early protein expression

  1. A Breast Tissue Protein Expression Profile Contributing to Early Parity-Induced Protection Against Breast Cancer

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    Christina Marie Gutierrez

    2015-11-01

    Full Text Available Background/Aims: Early parity reduces breast cancer risk, whereas, late parity and nulliparity increase breast cancer risk. Despite substantial efforts to understand the protective effects of early parity, the precise molecular circuitry responsible for these changes is not yet fully defined. Methods: Here, we have conducted the first study assessing protein expression profiles in normal breast tissue of healthy early parous, late parous, and nulliparous women. Breast tissue biopsies were obtained from 132 healthy parous and nulliparous volunteers. These samples were subjected to global protein expression profiling and immunohistochemistry. GeneSpring and MetaCore bioinformatics analysis software were used to identify protein expression profiles associated with early parity (low risk versus late/nulliparity (high risk. Results: Early parity reduces expression of key proteins involved in mitogenic signaling pathways in breast tissue through down regulation of EGFR1/3, ESR1, AKT1, ATF, Fos, and SRC. Early parity is also characterized by greater genomic stability and reduced tissue inflammation based on differential expression of aurora kinases, p53, RAD52, BRCA1, MAPKAPK-2, ATF-1, ICAM1, and NF-kappaB compared to late and nulli parity. Conclusions: Early parity reduces basal cell proliferation in breast tissue, which translates to enhanced genomic stability, reduced cellular stress/inflammation, and thus reduced breast cancer risk.

  2. Recombinant Protein Production from TPO Gen Cloning and Expression for Early Detection of Autoimmune Thyroid Diseases

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    Aulanni'am, Aulanni'am; Kinasih Wuragil, Dyah; Wahono Soeatmadji, Djoko; Zulkarnain; Marhendra, Agung Pramana W.

    2018-01-01

    Autoimmune Thyroid Disease (AITD) is an autoimmune disease that has many clinical symptoms but is difficult to detect at the onset of disease progression. Most thyroid autoimmune disease patients are positive with high titre of thyroid autoantibodies, especially thyroid peroxidase (TPO). The detection AITD are still needed because these tests are extremely high cost and have not regularly been performed in most of clinical laboratories. In the past, we have explored the autoimmune disease marker and it has been developed as source of polyclonal antibodies from patient origin. In the current study, we develop recombinant protein which resulted from cloning and expression of TPO gene from normal person and AITD patients. This work flows involves: DNA isolation and PCR to obtain TPO gene from human blood, insertion of TPO gene to plasmid and transformation to E. coli BL21, Bacterial culture to obtain protein product, protein purification and product analysis. This products can use for application to immunochromatography based test. This work could achieved with the goal of producing autoimmune markers with a guaranteed quality, sensitive, specific and economically. So with the collaboration with industries these devices could be used for early detection. Keywords: recombinant protein, TPO gene, Autoimmune thyroid diseases (AITD)ction of the diseases in the community.

  3. Early Changes in Costameric and Mitochondrial Protein Expression with Unloading Are Muscle Specific

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    Martin Flück

    2014-01-01

    Full Text Available We hypothesised that load-sensitive expression of costameric proteins, which hold the sarcomere in place and position the mitochondria, contributes to the early adaptations of antigravity muscle to unloading and would depend on muscle fibre composition and chymotrypsin activity of the proteasome. Biopsies were obtained from vastus lateralis (VL and soleus (SOL muscles of eight men before and after 3 days of unilateral lower limb suspension (ULLS and subjected to fibre typing and measures for costameric (FAK and FRNK, mitochondrial (NDUFA9, SDHA, UQCRC1, UCP3, and ATP5A1, and MHCI protein and RNA content. Mean cross-sectional area (MCSA of types I and II muscle fibres in VL and type I fibres in SOL demonstrated a trend for a reduction after ULLS (0.05≤P<0.10. FAK phosphorylation at tyrosine 397 showed a 20% reduction in VL muscle (P=0.029. SOL muscle demonstrated a specific reduction in UCP3 content (-23%; P = 0.012. Muscle-specific effects of ULLS were identified for linear relationships between measured proteins, chymotrypsin activity and fibre MCSA. The molecular modifications in costamere turnover and energy homoeostasis identify that aspects of atrophy and fibre transformation are detectable at the protein level in weight-bearing muscles within 3 days of unloading.

  4. Early Changes in Costameric and Mitochondrial Protein Expression with Unloading Are Muscle Specific

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    Li, Ruowei; Linnehan, Richard M.; Castells, Josiane; Tesch, Per; Gustafsson, Thomas

    2014-01-01

    We hypothesised that load-sensitive expression of costameric proteins, which hold the sarcomere in place and position the mitochondria, contributes to the early adaptations of antigravity muscle to unloading and would depend on muscle fibre composition and chymotrypsin activity of the proteasome. Biopsies were obtained from vastus lateralis (VL) and soleus (SOL) muscles of eight men before and after 3 days of unilateral lower limb suspension (ULLS) and subjected to fibre typing and measures for costameric (FAK and FRNK), mitochondrial (NDUFA9, SDHA, UQCRC1, UCP3, and ATP5A1), and MHCI protein and RNA content. Mean cross-sectional area (MCSA) of types I and II muscle fibres in VL and type I fibres in SOL demonstrated a trend for a reduction after ULLS (0.05 ≤ P < 0.10). FAK phosphorylation at tyrosine 397 showed a 20% reduction in VL muscle (P = 0.029). SOL muscle demonstrated a specific reduction in UCP3 content (−23%; P = 0.012). Muscle-specific effects of ULLS were identified for linear relationships between measured proteins, chymotrypsin activity and fibre MCSA. The molecular modifications in costamere turnover and energy homoeostasis identify that aspects of atrophy and fibre transformation are detectable at the protein level in weight-bearing muscles within 3 days of unloading. PMID:25313365

  5. Differentially expressed proteins on postoperative 3 days healing in rabbit Achilles tendon rupture model after early kinesitherapy.

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    Jialili, Ainuer; Jielile, Jiasharete; Abudoureyimu, Shajidan; Sabirhazi, Gulnur; Redati, Darebai; Bai, Jing-Ping; Bin, Liang; Duisabai, Sailike; Aishan, Jiangaguli; Kasimu, Haxiaobieke

    2011-04-01

    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. Forty-seven New Zealand white rabbits were randomized into 3 groups. Group A (immobilization group, n equal to 16) received postoperative cast immobilization; Group B (early motion group, n equal to 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 two-dimensional 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. 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, pro-alpha-1 type 1 collagen

  6. Histone Deacetylase Inhibitors Activate Tristetraprolin Expression through Induction of Early Growth Response Protein 1 (EGR1 in Colorectal Cancer Cells

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    Cyril Sobolewski

    2015-08-01

    Full Text Available The RNA-binding protein tristetraprolin (TTP promotes rapid decay of mRNAs bearing 3' UTR AU-rich elements (ARE. In many cancer types, loss of TTP expression is observed allowing for stabilization of ARE-mRNAs and their pathologic overexpression. Here we demonstrate that histone deacetylase (HDAC inhibitors (Trichostatin A, SAHA and sodium butyrate promote TTP expression in colorectal cancer cells (HCA-7, HCT-116, Moser and SW480 cells and cervix carcinoma cells (HeLa. We found that HDAC inhibitors-induced TTP expression, promote the decay of COX-2 mRNA, and inhibit cancer cell proliferation. HDAC inhibitors were found to promote TTP transcription through activation of the transcription factor Early Growth Response protein 1 (EGR1. Altogether, our findings indicate that loss of TTP in tumors occurs through silencing of EGR1 and suggests a therapeutic approach to rescue TTP expression in colorectal cancer.

  7. Pairwise protein expression classifier for candidate biomarker discovery for early detection of human disease prognosis

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    Kaur Parminder

    2012-08-01

    Full Text Available Abstract Background An approach to molecular classification based on the comparative expression of protein pairs is presented. The method overcomes some of the present limitations in using peptide intensity data for class prediction for problems such as the detection of a disease, disease prognosis, or for predicting treatment response. Data analysis is particularly challenging in these situations due to sample size (typically tens being much smaller than the large number of peptides (typically thousands. Methods based upon high dimensional statistical models, machine learning or other complex classifiers generate decisions which may be very accurate but can be complex and difficult to interpret in simple or biologically meaningful terms. A classification scheme, called ProtPair, is presented that generates simple decision rules leading to accurate classification which is based on measurement of very few proteins and requires only relative expression values, providing specific targeted hypotheses suitable for straightforward validation. Results ProtPair has been tested against clinical data from 21 patients following a bone marrow transplant, 13 of which progress to idiopathic pneumonia syndrome (IPS. The approach combines multiple peptide pairs originating from the same set of proteins, with each unique peptide pair providing an independent measure of discriminatory power. The prediction rate of the ProtPair for IPS study as measured by leave-one-out CV is 69.1%, which can be very beneficial for clinical diagnosis as it may flag patients in need of closer monitoring. The “top ranked” proteins provided by ProtPair are known to be associated with the biological processes and pathways intimately associated with known IPS biology based on mouse models. Conclusions An approach to biomarker discovery, called ProtPair, is presented. ProtPair is based on the differential expression of pairs of peptides and the associated proteins. Using mass

  8. Liver protein expression in dairy cows with high liver triglycerides in early lactation

    DEFF Research Database (Denmark)

    Sejersen, Henrik; Sørensen, Martin Tang; Larsen, Torben

    2012-01-01

    Fatty liver is a frequent subclinical health disorder in dairy cows that may lead to disorders related to the liver function. However, the effect of triglyceride (TG) accumulation on liver metabolic pathways is still unclear. The objective was, therefore, to characterize quantitative differences...... in the liver proteome between early lactation dairy cows with a low or high liver TG content. The liver proteome analysis indicated that a high liver TG content in early lactation dairy cows is associated with increased oxidation of saturated fatty acids, oxidative stress, and urea synthesis...... and decreasedoxidation of unsaturated fatty acids. Furthermore, liver gluconeogenesis is apparently not impaired by an increased liver TG content. Based on correlations between liver proteins and plasma components, we suggest that future studies investigate the sensitivity and specificity of plasma aspartate...

  9. The expression of podoplanin protein is a diagnostic marker to distinguish the early infiltration of esophageal squamous cell carcinoma.

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    Chen, Guangyong; Xu, Rui; Yue, Bing; Mei, Xue; Li, Peng; Zhou, Xiaoge; Huang, Shoufang; Gong, Liping; Zhang, Shutian

    2017-03-21

    The esophageal squamous cell carcinoma (ESCC) is usually develped from low-grade intraepithelial neoplasia (LGIEN) and high-grade intraepithelial neoplasia (HGIEN) to infiltrative squamous cell carcinoma. Till now, it remains hard to screen for infiltration at earlier stages, especially the differentiation between HGEIN and early infiltrative carcinoma. The purpose of this study is to determine a role of podoplanin in differentiating between HGEIN and early infiltrative squamous cell carcinoma. Totally 133 patients pathologically diagnosed with early ESCC and/or precancerous lesions were enrolled.The EnVision two-step IHC staining technique was applied using the monoclonal mouse anti-human Podoplanin antibody (clone number: D2-40). The expressions of PDPN protein on the basal layer of squamous epithelium lesions could be divided into three different patterns: complete type, incomplete (non-continuous) type, or missing type. A diagnosis of HGEIN can be made if the basal layer showed non-continuous or complete expression of PDPN and a diagnosis of early infiltration can be made if the expression of PDPN is completely missing. Our study confirmed that PDPN was a potential biomarker to identify the presence of early infiltrative squamous cell carcinoma.

  10. Increased expression of heat shock protein 105 in rat uterus of early pregnancy and its significance in embryo implantation

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    Hu Zhao-Yuan

    2009-03-01

    Full Text Available Abstract Background Heat shock proteins (Hsps are a set of highly conserved proteins, Hsp105, has been suggested to play a role in reproduction. Methods Spatio-temporal expression of Hsp105 in rat uterus during peri-implantation period was examined by immunohistochemistry and Western blot, pseudopregnant uterus was used as control. Injection of antisense oligodeoxynucleotides to Hsp105 into pregnant rat uteri was carried out to look at effect of Hsp105 on embryo implantation. Results Expression of Hsp105 was mainly in the luminal epithelium on day 1 of pregnancy, and reached a peak level on day 5, whereas in stroma cells, adjacent to the implanting embryo, the strongest expression of Hsp105 was observed on day 6. The immunostaining profile in the uterus was consistent with that obtained by Western blot in the early pregnancy. In contrast, no obvious peak level of Hsp105 was observed in the uterus of pseudopregnant rat on day 5 or day 6. Furthermore, injection of antisense oligodeoxynucleotides to Hsp105 into the rat uterine horn on day 3 of pregnancy obviously suppressed the protein expression as expected and reduced number of the implanted embryos as compared with the control. Conclusion Temporal and spatial changes in Hsp105 expression in pregnant rat uterus may play a physiological role in regulating embryo implantation.

  11. Temporal expression profiling of plasma proteins reveals oxidative stress in early stages of Type 1 Diabetes progression

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    Liu, Chih-Wei; Bramer, Lisa; Computational Modeling); Webb-Robertson, Bobbie-Jo; Computational Modeling); Waugh, Kathleen; Rewers, Marian J.; Zhang, Qibin; Biochemistry)

    2017-01-01

    We report that blood markers other than islet autoantibodies are greatly needed to indicate the pancreatic beta cell destruction process as early as possible, and more accurately reflect the progression of Type 1 Diabetes Mellitus (T1D). To this end, a longitudinal proteomic profiling of human plasma using TMT-10plex-based LC-MS/MS analysis was performed to track temporal proteomic changes of T1D patients (n = 11) across 9 serial time points, spanning the period of T1D natural progression, in comparison with those of the matching healthy controls (n = 10). To our knowledge, the current study represents the largest (> 2000 proteins measured) longitudinal expression profiles of human plasma proteome in T1D research. By applying statistical trend analysis on the temporal expression patterns between T1D and controls, and Benjamini-Hochberg procedure for multiple-testing correction, 13 protein groups were regarded as having statistically significant differences during the entire follow-up period. Moreover, 16 protein groups, which play pivotal roles in response to oxidative stress, have consistently abnormal expression trend before seroconversion to islet autoimmunity. Importantly, the expression trends of two key reactive oxygen species-decomposing enzymes, Catalase and Superoxide dismutase were verified independently by ELISA.

  12. Noncanonical Expression of a Murine Cytomegalovirus Early Protein CD8 T-Cell Epitope as an Immediate Early Epitope Based on Transcription from an Upstream Gene

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    Annette Fink

    2014-02-01

    Full Text Available Viral CD8 T-cell epitopes, represented by viral peptides bound to major histocompatibility complex class-I (MHC-I glycoproteins, are often identified by “reverse immunology”, a strategy not requiring biochemical and structural knowledge of the actual viral protein from which they are derived by antigen processing. Instead, bioinformatic algorithms predicting the probability of C-terminal cleavage in the proteasome, as well as binding affinity to the presenting MHC-I molecules, are applied to amino acid sequences deduced from predicted open reading frames (ORFs based on the genomic sequence. If the protein corresponding to an antigenic ORF is known, it is usually inferred that the kinetic class of the protein also defines the phase in the viral replicative cycle during which the respective antigenic peptide is presented for recognition by CD8 T cells. We have previously identified a nonapeptide from the predicted ORFm164 of murine cytomegalovirus that is presented by the MHC-I allomorph H-2 Dd and that is immunodominant in BALB/c (H-2d haplotype mice. Surprisingly, although the ORFm164 protein gp36.5 is expressed as an Early (E phase protein, the m164 epitope is presented already during the Immediate Early (IE phase, based on the expression of an upstream mRNA starting within ORFm167 and encompassing ORFm164.

  13. 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)

  14. Roles of Polypyrimidine Tract Binding Proteins in Major Immediate-Early Gene Expression and Viral Replication of Human Cytomegalovirus▿

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    Cosme, Ruth S. Cruz; Yamamura, Yasuhiro; Tang, Qiyi

    2009-01-01

    Human cytomegalovirus (HCMV), a member of the β subgroup of the family Herpesviridae, causes serious health problems worldwide. HCMV gene expression in host cells is a well-defined sequential process: immediate-early (IE) gene expression, early-gene expression, DNA replication, and late-gene expression. The most abundant IE gene, major IE (MIE) gene pre-mRNA, needs to be spliced before being exported to the cytoplasm for translation. In this study, the regulation of MIE gene splicing was investigated; in so doing, we found that polypyrimidine tract binding proteins (PTBs) strongly repressed MIE gene production in cotransfection assays. In addition, we discovered that the repressive effects of PTB could be rescued by splicing factor U2AF. Taken together, the results suggest that PTBs inhibit MIE gene splicing by competing with U2AF65 for binding to the polypyrimidine tract in pre-mRNA. In intron deletion mutation assays and RNA detection experiments (reverse transcription [RT]-PCR and real-time RT-PCR), we further observed that PTBs target all the introns of the MIE gene, especially intron 2, and affect gene splicing, which was reflected in the variation in the ratio of pre-mRNA to mRNA. Using transfection assays, we demonstrated that PTB knockdown cells induce a higher degree of MIE gene splicing/expression. Consistently, HCMV can produce more viral proteins and viral particles in PTB knockdown cells after infection. We conclude that PTB inhibits HCMV replication by interfering with MIE gene splicing through competition with U2AF for binding to the polypyrimidine tract in MIE gene introns. PMID:19144709

  15. Roles of polypyrimidine tract binding proteins in major immediate-early gene expression and viral replication of human cytomegalovirus.

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    Cosme, Ruth S Cruz; Yamamura, Yasuhiro; Tang, Qiyi

    2009-04-01

    Human cytomegalovirus (HCMV), a member of the beta subgroup of the family Herpesviridae, causes serious health problems worldwide. HCMV gene expression in host cells is a well-defined sequential process: immediate-early (IE) gene expression, early-gene expression, DNA replication, and late-gene expression. The most abundant IE gene, major IE (MIE) gene pre-mRNA, needs to be spliced before being exported to the cytoplasm for translation. In this study, the regulation of MIE gene splicing was investigated; in so doing, we found that polypyrimidine tract binding proteins (PTBs) strongly repressed MIE gene production in cotransfection assays. In addition, we discovered that the repressive effects of PTB could be rescued by splicing factor U2AF. Taken together, the results suggest that PTBs inhibit MIE gene splicing by competing with U2AF65 for binding to the polypyrimidine tract in pre-mRNA. In intron deletion mutation assays and RNA detection experiments (reverse transcription [RT]-PCR and real-time RT-PCR), we further observed that PTBs target all the introns of the MIE gene, especially intron 2, and affect gene splicing, which was reflected in the variation in the ratio of pre-mRNA to mRNA. Using transfection assays, we demonstrated that PTB knockdown cells induce a higher degree of MIE gene splicing/expression. Consistently, HCMV can produce more viral proteins and viral particles in PTB knockdown cells after infection. We conclude that PTB inhibits HCMV replication by interfering with MIE gene splicing through competition with U2AF for binding to the polypyrimidine tract in MIE gene introns.

  16. Loss of tumour-specific ATM protein expression is an independent prognostic factor in early resected NSCLC.

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    Petersen, Lars F; Klimowicz, Alexander C; Otsuka, Shannon; Elegbede, Anifat A; Petrillo, Stephanie K; Williamson, Tyler; Williamson, Chris T; Konno, Mie; Lees-Miller, Susan P; Hao, Desiree; Morris, Don; Magliocco, Anthony M; Bebb, D Gwyn

    2017-06-13

    Ataxia-telangiectasia mutated (ATM) is critical in maintaining genomic integrity. In response to DNA double-strand breaks, ATM phosphorylates downstream proteins involved in cell-cycle checkpoint arrest, DNA repair, and apoptosis. Here we investigate the frequency, and influence of ATM deficiency on outcome, in early-resected non-small cell lung cancer (NSCLC). Tissue microarrays, containing 165 formalin-fixed, paraffin-embedded resected NSCLC tumours from patients diagnosed at the Tom Baker Cancer Centre, Calgary, Canada, between 2003 and 2006, were analyzed for ATM expression using quantitative fluorescence immunohistochemistry. Both malignant cell-specific ATM expression and the ratio of ATM expression within malignant tumour cells compared to that in the surrounding tumour stroma, defined as the ATM expression index (ATM-EI), were measured and correlated with clinical outcome. ATM loss was identified in 21.8% of patients, and was unaffected by clinical pathological variables. Patients with low ATM-EI tumours had worse survival outcomes compared to those with high ATM-EI (p ATM-deficient patients may derive greater benefit from guideline-recommended adjuvant chemotherapy following surgical resection. Taken together, these results indicate that ATM loss seems to be an early event in NSCLC carcinogenesis and is an independent prognostic factor associated with worse survival in stage II/III patients.

  17. Quantitative Proteomics Analysis of Altered Protein Expression in the Placental Villous Tissue of Early Pregnancy Loss Using Isobaric Tandem Mass Tags

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    Xiaobei Ni

    2014-01-01

    Full Text Available Many pregnant women suffer miscarriages during early gestation, but the description of these early pregnancy losses (EPL can be somewhat confusing because of the complexities of early development. Thus, the identification of proteins with different expression profiles related to early pregnancy loss is essential for understanding the comprehensive pathophysiological mechanism. In this study, we report a gel-free tandem mass tags- (TMT- labeling based proteomic analysis of five placental villous tissues from patients with early pregnancy loss and five from normal pregnant women. The application of this method resulted in the identification of 3423 proteins and 19647 peptides among the patient group and the matched normal control group. Qualitative and quantitative proteomic analysis revealed 51 proteins to be differentially abundant between the two groups (≥1.2-fold, Student's t-test, P<0.05. To obtain an overview of the biological functions of the proteins whose expression levels altered significantly in EPL group, gene ontology analysis was performed. We also investigated the twelve proteins with a difference over 1.5-fold using pathways analysis. Our results demonstrate that the gel-free TMT-based proteomic approach allows the quantification of differences in protein expression levels, which is useful for obtaining molecular insights into early pregnancy loss.

  18. Expression of C-Reactive Protein and Serum Amyloid A in Early to Late Manifestations of Lyme Disease.

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    Uhde, Melanie; Ajamian, Mary; Li, Xueting; Wormser, Gary P; Marques, Adriana; Alaedini, Armin

    2016-12-01

     Infection with Borrelia burgdorferi, the causative agent of Lyme disease, triggers host immune responses that affect the clinical outcome and are a source of biomarkers with diagnostic utility. Although adaptive immunity to B. burgdorferi has been extensively characterized, considerably less information is available about the development of innate acute-phase responses in Lyme disease. Our aim in this study was to evaluate the expression of C-reactive protein (CRP) and serum amyloid A (SAA), the prototype acute-phase response proteins, in the context of the varying manifestations associated with Lyme borreliosis.  Circulating concentrations of CRP and SAA in patients with a range of early to late objective manifestations of Lyme disease and in individuals with post-treatment Lyme disease syndrome were compared with those in healthy control groups.  CRP and SAA levels were significantly elevated in early localized and early disseminated Lyme disease but not in the later stages of active infection. Levels of CRP, but not SAA, were also found to be significantly increased in patients with antibiotic-refractory Lyme arthritis and in those with post-treatment Lyme disease syndrome.  These findings indicate that circulating CRP and SAA levels are highest when the concentration of spirochetes is greatest in skin and/or blood and that levels decline after the dissemination of the organism to extracutaneous sites in subsequent stages of infection. The data also suggest that antibiotic-refractory Lyme arthritis and post-treatment Lyme disease syndrome are associated with elevated CRP responses that are driven by inflammatory mechanisms distinct from those in active infection. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  19. The induced expression of heat shock proteins as a part of the early cellular response to gamma radiation

    International Nuclear Information System (INIS)

    Stankova, K.; Ivanova, K.; Georgieva, R.; Rupova, I.; Boteva, R.

    2008-01-01

    A variety of stressful stimuli including gamma radiation can induce increase in the synthesis of heat shock proteins (Hsp). This family of molecular chaperones includes members with molecular masses ranging from 10 to 150 kDa and has been identified in all organisms from bacteria to humans. Hsp70 chaperones are very important. The present study aimed to characterize the radiation-induced changes in Hsp70 synthesis in human lymphocytes as a part of the early cellular response to gamma irradiation. The expression of Hsp70 was determined with Western blot and the radiation-induced apoptotic changes were registered by staining with fluorescent dyes. Part of the experiments were performed in the presence of the organic solvent DMSO. At low concentrations this reagent shows antioxidant activity and can reduce the level of the radiation-induced oxidant stress which determines the predominant biological effects of the ionizing radiation. Irradiation with 0.5 to 8 Gy caused statistically significant increase in the synthesis of Hsp70 which was strongest after irradiation with 4 Gy. In the range 0.5-2 Gy the enhancement of the radiation-induced synthesis of Hsp70 reached 60%. Our experimental results characterize changes in the Hsp70 synthesis after gamma irradiation as a part of the early cellular stress response in lymphocytes. (authors)

  20. Early history, discovery, and expression of Aequorea green fluorescent protein, with a note on an unfinished experiment.

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    Tsuji, Frederick I

    2010-08-01

    The bioluminescent hydromedusan jellyfish, Aequorea victoria, emits a greenish light (lambda(max) = 508 nm) when stimulated electrically or mechanically. The light comes from photocytes located along the margin of its umbrella. The greenish light depends on two intracellular proteins working in consort: aequorin (21.4 kDa) and a green fluorescent protein (27 kDa). An excited state green fluorescent protein molecule results, which, on returning to the ground state, emits a greenish light. Similarly, a green light emission may be induced in the green fluorescent protein by exposing it to ultraviolet or blue light. Because the green light can be readily detected under a fluorescence microscope, the green fluorescent protein, tagged to a protein of interest, has been used widely as a marker to locate proteins in cells and to monitoring gene expression. This article reviews the work that took place leading to the discovery, cloning, and expression of the green fluorescent protein, with a note on an unfinished experiment. (c) 2010 Wiley-Liss, Inc.

  1. Gene expression profiling reveals different molecular patterns in G-protein coupled receptor signaling pathways between early- and late-onset preeclampsia.

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    Liang, Mengmeng; Niu, Jianmin; Zhang, Liang; Deng, Hua; Ma, Jian; Zhou, Weiping; Duan, Dongmei; Zhou, Yuheng; Xu, Huikun; Chen, Longding

    2016-04-01

    Early-onset preeclampsia and late-onset preeclampsia have been regarded as two different phenotypes with heterogeneous manifestations; To gain insights into the pathogenesis of the two traits, we analyzed the gene expression profiles in preeclamptic placentas. A whole genome-wide microarray was used to determine the gene expression profiles in placental tissues from patients with early-onset (n = 7; 36 weeks) preeclampsia and their controls who delivered preterm (n = 5; 36 weeks). Genes were termed differentially expressed if they showed a fold-change ≥ 2 and q-value preeclampsia (177 genes were up-regulated and 450 were down-regulated). Gene ontology analysis identified significant alterations in several biological processes; the top two were immune response and cell surface receptor linked signal transduction. Among the cell surface receptor linked signal transduction-related, differentially expressed genes, those involved in the G-protein coupled receptor protein signaling pathway were significantly enriched. G-protein coupled receptor signaling pathway related genes, such as GPR124 and MRGPRF, were both found to be down-regulated in early-onset preeclampsia. The results were consistent with those of western blotting that the abundance of GPR124 was lower in early-onset compared with late-onset preeclampsia. The different gene expression profiles reflect the different levels of transcription regulation between the two conditions and supported the hypothesis that they are separate disease entities. Moreover, the G-protein coupled receptor signaling pathway related genes may contribute to the mechanism underlying early- and late-onset preeclampsia. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Proteomic analysis on the alteration of protein expression in the early-stage placental villous tissue of electromagnetic fields associated with cell phone exposure.

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    Luo, Qiong; Jiang, Ying; Jin, Min; Xu, Jian; Huang, He-Feng

    2013-09-01

    To explore the possible adverse effects and search for cell phone electromagnetic field (EMF)-responsive proteins in human early reproduction, a proteomics approach was employed to investigate the changes in protein expression profile induced by cell phone EMF in human chorionic tissues of early pregnancy in vivo. Volunteer women about 50 days pregnant were exposed to EMF at the average absorption rate of 1.6 to 8.8 W/kg for 1 hour with the irradiation device placed 10 cm away from the umbilicus at the midline of the abdomen. The changes in protein profile were examined using 2-dimensional electrophoresis (2-DE). Up to 15 spots have yielded significant change at least 2- to 2.5-folds up or down compared to sham-exposed group. Twelve proteins were identified- procollagen-proline, eukaryotic translation elongation factor 1 delta, chain D crystal structure of human vitamin D-binding protein, thioredoxin-like 3, capping protein, isocitrate dehydrogenase 3 alpha, calumenin, Catechol-O-methyltransferase protein, proteinase inhibitor 6 (PI-6; SerpinB6) protein, 3,2-trans-enoyl-CoA isomerase protein, chain B human erythrocyte 2,3-bisphosphoglycerate mutase, and nucleoprotein. Cell phone EMF might alter the protein profile of chorionic tissue of early pregnancy, during the most sensitive stage of the embryos. The exposure to EMF may cause adverse effects on cell proliferation and development of nervous system in early embryos. Furthermore, 2-DE coupled with mass spectrometry is a promising approach to elucidate the effects and search for new biomarkers for environmental toxic effects.

  3. Inhibition of hypoxia inducible factor-1α downregulates the expression of epithelial to mesenchymal transition early marker proteins without undermining cell survival in hypoxic lens epithelial cells.

    Science.gov (United States)

    Cammarata, Patrick R; Neelam, Sudha; Brooks, Morgan M

    2015-01-01

    The purpose of this study was to identify potential therapeutic strategies to slow down or prevent the expression of early-onset epithelial to mesenchymal transition (EMT) marker proteins (fibronectin and alpha smooth muscle actin, α-SMA) without sacrificing the synthesis and accumulation of the prosurvival protein vascular endothelial growth factor (VEGF) in cultured virally transformed human lens epithelial (HLE) cells. HLE-B3 cells, maintained in a continuous hypoxic environment (1% oxygen), were treated with SB216763, a specific inhibitor of glycogen synthase kinase-3β (GSK-3β) catalytic activity. Western blot analysis was employed to detect the cytoplasmic and nuclear levels of β-catenin, as well as the total lysate content of fibronectin and α-SMA. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of VEGF in cell culture medium. A hypoxia-inducible factor-1α (HIF-1α) translation inhibitor and an HIF-2α translation inhibitor were independently employed to evaluate the effect of hypoxia inducible factor inhibition on EMT marker protein and VEGF expression. XAV932 was used to assess the suppression of nuclear β-catenin and its downstream effect on EMT marker proteins and VEGF expression. SB216763-treated HLE-B3 cells caused marked inhibition of GSK-3β activity prompting a significant increase in the translocation of cytoplasmic β-catenin to the nucleus. The enhancement of nuclear β-catenin looked as if it positively correlated with a significant increase in the basal expression of VEGF as well as increased expression of fibronectin and α-SMA. In conjunction with SB216763, coadministration of an HIF-1α translation inhibitor, but not an HIF-2α translation inhibitor, markedly suppressed the expression of fibronectin and α-SMA without affecting VEGF levels. Treatment with XAV932 significantly reduced the level of nuclear β-catenin, but the levels of neither the EMT marker proteins nor VEGF were changed. Recently, we reported

  4. Proteomic analysis of stipe explants reveals differentially expressed proteins involved in early direct somatic embryogenesis of the tree fern Cyathea delgadii Sternb.

    Science.gov (United States)

    Domżalska, Lucyna; Kędracka-Krok, Sylwia; Jankowska, Urszula; Grzyb, Małgorzata; Sobczak, Mirosław; Rybczyński, Jan J; Mikuła, Anna

    2017-05-01

    Using cyto-morphological analysis of somatic embryogenesis (SE) in the tree fern Cyathea delgadii as a guide, we performed a comparative proteomic analysis in stipe explants undergoing direct SE. Plant material was cultured on hormone-free medium supplemented with 2% sucrose. Phenol extracted proteins were separated using two-dimensional gel electrophoresis (2-DE) and mass spectrometry was performed for protein identification. A total number of 114 differentially regulated proteins was identified during early SE, i.e. when the first cell divisions started and several-cell pro-embryos were formed. Proteins were assigned to seven functional categories: carbohydrate metabolism, protein metabolism, cell organization, defense and stress responses, amino acid metabolism, purine metabolism, and fatty acid metabolism. Carbohydrate and protein metabolism were found to be the most sensitive SE functions with the greatest number of alterations in the intensity of spots in gel. Differences, especially in non-enzymatic and structural protein abundance, are indicative for cell organization, including cytoskeleton rearrangement and changes in cell wall components. The highest induced changes concern those enzymes related to fatty acid metabolism. Global analysis of the proteome reveals several proteins that can represent markers for the first 16days of SE induction and expression in fern. The findings of this research improve the understanding of molecular processes involved in direct SE in C. delgadii. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Extensive proteomic screening identifies the obesity-related NYGGF4 protein as a novel LRP1-interactor, showing reduced expression in early Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Taddei Kevin

    2010-01-01

    Full Text Available Abstract Background The low-density lipoprotein receptor related protein 1 (LRP1 has been implicated in Alzheimer's disease (AD but its signalling has not been fully evaluated. There is good evidence that the cytoplasmic domain of LRP1 is involved in protein-protein interactions, important in the cell biology of LRP1. Results We carried out three yeast two-hybrid screens to identify proteins that interact with the cytoplasmic domain of LRP1. The screens included both conventional screens as well as a novel, split-ubiquitin-based screen in which an LRP1 construct was expressed and screened as a transmembrane protein. The split-ubiquitin screen was validated in a screen using full-length amyloid protein precursor (APP, which successfully identified FE65 and FE65L2, as well as novel interactors (Rab3a, Napg, and ubiquitin b. Using both a conventional screen as well as the split-ubiquitin screen, we identified NYGGF4 as a novel LRP1 interactor. The interaction between LRP1 and NYGGF4 was validated using two-hybrid assays, coprecipitation and colocalization in mammalian cells. Mutation analysis demonstrated a specific interaction of NYGGF4 with an NPXY motif that required an intact tyrosine residue. Interestingly, while we confirmed that other LRP1 interactors we identified, including JIP1B and EB-1, were also able to bind to APP, NYGGF4 was unique in that it showed specific binding with LRP1. Expression of NYGGF4 decreased significantly in patients with AD as compared to age-matched controls, and showed decreasing expression with AD disease progression. Examination of Nyggf4 expression in mice with different alleles of the human APOE4 gene showed significant differences in Nyggf4 expression. Conclusions These results implicate NYGGF4 as a novel and specific interactor of LRP1. Decreased expression of LRP1 and NYGGF4 over disease, evident with the presence of even moderate numbers of neuritic plaques, suggests that LRP1-NYGGF4 is a system altered

  6. Global and stage specific patterns of Krüppel-associated-box zinc finger protein gene expression in murine early embryonic cells.

    Directory of Open Access Journals (Sweden)

    Andrea Corsinotti

    Full Text Available Highly coordinated transcription networks orchestrate the self-renewal of pluripotent stem cell and the earliest steps of mammalian development. KRAB-containing zinc finger proteins represent the largest group of transcription factors encoded by the genomes of higher vertebrates including mice and humans. Together with their putatively universal cofactor KAP1, they have been implicated in events as diverse as the silencing of endogenous retroelements, the maintenance of imprinting and the pluripotent self-renewal of embryonic stem cells, although the genomic targets and specific functions of individual members of this gene family remain largely undefined. Here, we first generated a list of Ensembl-annotated KRAB-containing genes encoding the mouse and human genomes. We then defined the transcription levels of these genes in murine early embryonic cells. We found that the majority of KRAB-ZFP genes are expressed in mouse pluripotent stem cells and other early progenitors. However, we also identified distinctively cell- or stage-specific patterns of expression, some of which are pluripotency-restricted. Finally, we determined that individual KRAB-ZFP genes exhibit highly distinctive modes of expression, even when grouped in genomic clusters, and that these cannot be correlated with the presence of prototypic repressive or activating chromatin marks. These results pave the way to delineating the role of specific KRAB-ZFPs in early embryogenesis.

  7. Low Ki67/high ATM protein expression in malignant tumors predicts favorable prognosis in a retrospective study of early stage hormone receptor positive breast cancer.

    Science.gov (United States)

    Feng, Xiaolan; Li, Haocheng; Kornaga, Elizabeth N; Dean, Michelle; Lees-Miller, Susan P; Riabowol, Karl; Magliocco, Anthony M; Morris, Don; Watson, Peter H; Enwere, Emeka K; Bebb, Gwyn; Paterson, Alexander

    2016-12-27

    This study was designed to investigate the combined influence of ATM and Ki67 on clinical outcome in early stage hormone receptor positive breast cancer (ES-HPBC), particularly in patients with smaller tumors (ATM and Ki67 proteins using fluorescence and brightfield immunohistochemistry respectively, and quantified their expression with digital image analysis. Data on expression levels were subsequently correlated with clinical outcome. Remarkably, ATM expression was useful to stratify the low Ki67 group into subgroups with better or poorer prognosis. Specifically, in the low Ki67 subgroup defined as having smaller tumors and no positive nodes, patients with high ATM expression showed better outcome than those with low ATM, with estimated survival rates of 96% and 89% respectively at 15 years follow up (p = 0.04). Similarly, low-Ki67 patients with smaller tumors, 1-3 positive nodes and high ATM also had significantly better outcomes than their low ATM counterparts, with estimated survival rates of 88% and 46% respectively (p = 0.03) at 15 years follow up. Multivariable analysis indicated that the combination of high ATM and low Ki67 is prognostic of improved survival, independent of tumor size, grade, and lymph node status (p = 0.02). These data suggest that the prognostic value of Ki67 can be improved by analyzing ATM expression in ES-HPBC.

  8. Correlation Between Expression of Recombinant Proteins and Abundance of H3K4Me3 on the Enhancer of Human Cytomegalovirus Major Immediate-Early Promoter.

    Science.gov (United States)

    Soo, Benjamin P C; Tay, Julian; Ng, Shirelle; Ho, Steven C L; Yang, Yuansheng; Chao, Sheng-Hao

    2017-08-01

    Role of epigenetic regulation in the control of gene expression is well established. The impact of several epigenetic mechanisms, such as DNA methylation and histone acetylation, on recombinant protein production in mammalian cells has been investigated recently. Here we investigate the correlation between the selected epigenetic markers and five trastuzumab biosimilar-producing Chinese hamster ovary (CHO) cell lines in which the expression of trastuzumab is driven by human cytomegalovirus (HCMV) major immediate-early (MIE) promoter. We chose the producing clones in which transcription was the determinative step for the production of recombinant trastuzumab. We found that the abundance of trimethylation of histone 3 at lysine 4 (H3K4Me3) on the enhancer of HCMV MIE promoter correlated well with the relative titers of recombinant trastuzumab among the clones. Such close correlation was not observed between the recombinant protein and other epigenetic markers examined in our study. Our results demonstrate that the HCMV MIE enhancer-bound H3K4Me3 epigenetic marker may be used as the epigenetic indicator to predict the relative production of recombinant proteins between the producing CHO cell lines.

  9. 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...

  10. The equine herpesvirus-1 IR3 gene that lies antisense to the sole immediate-early (IE) gene is trans-activated by the IE protein, and is poorly expressed to a protein

    International Nuclear Information System (INIS)

    Ahn, Byung Chul; Breitenbach, Jonathan E.; Kim, Seong K.; O'Callaghan, Dennis J.

    2007-01-01

    The unique IR3 gene of equine herpesvirus 1 (EHV-1) is expressed as a late 1.0-kb transcript. Previous studies confirmed the IR3 transcription initiation site and tentatively identified other cis-acting elements specific to IR3 such as a TATA box, a 443 base pair 5'untranslated region (UTR), a 285 base pair open reading frame (ORF), and a poly adenylation (A) signal [Holden, V.R., Harty, R.N., Yalamanchili, R.R., O'Callaghan, D.J., 1992. The IR3 gene of equine herpesvirus type 1: a unique gene regulated by sequences within the intron of the immediate-early gene. DNA Seq. 3, 143-152]. Transient transfection assays revealed that the IR3 promoter is strongly trans-activated by the IE protein (IEP) and that coexpression of the IEP with the early EICP0 and IR4 regulatory proteins results in maximal trans-activation of the IR3 promoter. Gel shift assays revealed that the IEP directly binds to the IR3 promoter region. Western blot analysis showed that the IR3 protein produced in E. coli was detected by antibodies to IR3 synthetic peptides; however, the IR3 protein was not detected in EHV-1 infected cell extracts by these same anti-IR3 antibodies, even though the IR3 transcript was detected by northern blot. These findings suggest that the IR3 may not be expressed to a protein. Expression of an IR3/GFP fusion gene was not observed, but expression of a GFP/IR3 fusion gene was detected by fluorescent microscopy. In further attempts to detect the IR3/GFP fusion protein using anti-GFP antibody, western blot analysis showed that the IR3/GFP fusion protein was not detected in vivo. Interestingly, a truncated form of the GFP/IR3 protein was synthesized from the GFP/IR3 fusion gene. However, GFP/IR3 and IR3/GFP fusion proteins of the predicted sizes were synthesized by in vitro coupled transcription and translation of the fusion genes, suggesting poor expression of the IR3 protein in vivo. The possible role of the IR3 transcript in EHV-1 infection is discussed

  11. Early postnatal virus inoculation into the scala media achieved extensive expression of exogenous green fluorescent protein in the inner ear and preserved auditory brainstem response thresholds.

    Science.gov (United States)

    Wang, Yunfeng; Sun, Yu; Chang, Qing; Ahmad, Shoeb; Zhou, Binfei; Kim, Yeunjung; Li, Huawei; Lin, Xi

    2013-01-01

    Gene transfer into the inner ear is a promising approach for treating sensorineural hearing loss. The special electrochemical environment of the scala media raises a formidable challenge for effective gene delivery at the same time as keeping normal cochlear function intact. The present study aimed to define a suitable strategy for preserving hearing after viral inoculation directly into the scala media performed at various postnatal developmental stages. We assessed transgene expression of green fluorescent protein (GFP) mediated by various types of adeno-associated virus (AAV) and lentivirus (LV) in the mouse cochlea. Auditory brainstem responses were measured 30 days after inoculation to assess effects on hearing. Patterns of GFP expression confirmed extensive exogenous gene expression in various types of cells lining the endolymphatic space. The use of different viral vectors and promoters resulted in specific cellular GFP expression patterns. AAV2/1 with cytomegalovirus promoter apparently gave the best results for GFP expression in the supporting cells. Histological examination showed normal cochlear morphology and no hair cell loss after either AAV or LV injections. We found that hearing thresholds were not significantly changed when the injections were performed in mice younger than postnatal day 5, regardless of the type of virus tested. Viral inoculation and expression in the inner ear for the restoration of hearing must not damage cochlear function. Using normal hearing mice as a model, we have achieved this necessary step, which is required for the treatment of many types of congenital deafness that require early intervention. Copyright © 2013 John Wiley & Sons, Ltd.

  12. The Effects of Irradiation and Calcium-deficient Diet on the Expression of Bone Morphogenetic Protein-2/4 during Early Tooth Development

    International Nuclear Information System (INIS)

    Park, Dai Hee; Hwang, Eui Hwan; Lee, Sang Rae

    2000-01-01

    To investigate the expression of bone morphogenetic protein (BMP)-2/4 during early tooth development after irradiation and calcium-deficient diet. The pregnant three-week-old Sprague-Dawley rats were used for the study. The control group was non-irradiation/normal diet group (Group 1), and the experimental groups were irradiation/normal diet group (Group 2) and irradiation/calcium-diet group (Group 3). The abdomen of the rats at the 9th day of pregnancy were irradiated with single dose of 350 cGy. The rat pups were sacrificed at embryonic 18 days, 3 days and 14 days after delivery and the maxillae tooth germs were taken. The tissue sections of specimen were stained immunohistochemically with anti-BMP-2/4 antibody. At embryo-18 days, immunoreacivity for BMP-2/4 of the Group 1 was modetate in stratum intermedium of dental organ and weak in dental papilla and dental follicle, but that of Group 2 was weak in cell layer of dental organ, and no immunoreacivity was shown in dental papilla and dental follice of Group 2 and in all tissue components of the Group 3. At postnatal-3 days, immunoreacivity for BMP-2/4 of the Group 1 was strong in cell layer of dental organ, odontoblasts and developing alveolar bone, but that of Group of 2 and Group 3 was weak in odontoblasts and developing alveolar bone. At postnatal-14 days, immunoreacivity for BMP-2/4 of the Group 1 was strong in newly formed cementum, alveolar bone and odontoblasts, but that of Group 2 was weaker than that of Group 1. In the Group 3, tooth forming cell layer showed weak immunoreactivity, but other cell layers showed no immunoreactivity. The expression of bone morphogenetic protein (BMP)-2/4 during early tooth development was disturbed after irradiation and calcium-deficient diet.

  13. The Effects of Irradiation and Calcium-deficient Diet on the Expression of Bone Morphogenetic Protein-2/4 during Early Tooth Development

    Energy Technology Data Exchange (ETDEWEB)

    Park, Dai Hee; Hwang, Eui Hwan; Lee, Sang Rae [Dept. of Oral and Maxillofacial Radiology, College of Dentistry, Kyunghee University, Seoul (Korea, Republic of)

    2000-09-15

    To investigate the expression of bone morphogenetic protein (BMP)-2/4 during early tooth development after irradiation and calcium-deficient diet. The pregnant three-week-old Sprague-Dawley rats were used for the study. The control group was non-irradiation/normal diet group (Group 1), and the experimental groups were irradiation/normal diet group (Group 2) and irradiation/calcium-diet group (Group 3). The abdomen of the rats at the 9th day of pregnancy were irradiated with single dose of 350 cGy. The rat pups were sacrificed at embryonic 18 days, 3 days and 14 days after delivery and the maxillae tooth germs were taken. The tissue sections of specimen were stained immunohistochemically with anti-BMP-2/4 antibody. At embryo-18 days, immunoreacivity for BMP-2/4 of the Group 1 was modetate in stratum intermedium of dental organ and weak in dental papilla and dental follicle, but that of Group 2 was weak in cell layer of dental organ, and no immunoreacivity was shown in dental papilla and dental follice of Group 2 and in all tissue components of the Group 3. At postnatal-3 days, immunoreacivity for BMP-2/4 of the Group 1 was strong in cell layer of dental organ, odontoblasts and developing alveolar bone, but that of Group of 2 and Group 3 was weak in odontoblasts and developing alveolar bone. At postnatal-14 days, immunoreacivity for BMP-2/4 of the Group 1 was strong in newly formed cementum, alveolar bone and odontoblasts, but that of Group 2 was weaker than that of Group 1. In the Group 3, tooth forming cell layer showed weak immunoreactivity, but other cell layers showed no immunoreactivity. The expression of bone morphogenetic protein (BMP)-2/4 during early tooth development was disturbed after irradiation and calcium-deficient diet.

  14. The Neurokinin-1 Receptor Contributes to the Early Phase of Lipopolysaccharide-Induced Fever via Stimulation of Peripheral Cyclooxygenase-2 Protein Expression in Mice

    Directory of Open Access Journals (Sweden)

    Eszter Pakai

    2018-02-01

    Full Text Available Neurokinin (NK signaling is involved in various inflammatory processes. A common manifestation of systemic inflammation is fever, which is usually induced in animal models with the administration of bacterial lipopolysaccharide (LPS. A role for the NK1 receptor was shown in LPS-induced fever, but the underlying mechanisms of how the NK1 receptor contributes to febrile response, especially in the early phase, have remained unknown. We administered LPS (120 µg/kg, intraperitoneally to mice with the Tacr1 gene, i.e., the gene encoding the NK1 receptor, either present (Tacr1+/+ or absent (Tacr1−/− and measured their thermoregulatory responses, serum cytokine levels, tissue cyclooxygenase-2 (COX-2 expression, and prostaglandin (PG E2 concentration. We found that the LPS-induced febrile response was attenuated in Tacr1−/− compared to their Tacr1+/+ littermates starting from 40 min postinfusion. The febrigenic effect of intracerebroventricularly administered PGE2 was not suppressed in the Tacr1−/− mice. Serum concentration of pyrogenic cytokines did not differ between Tacr1−/− and Tacr1+/+ at 40 min post-LPS infusion. Administration of LPS resulted in amplification of COX-2 mRNA expression in the lungs, liver, and brain of the mice, which was statistically indistinguishable between the genotypes. In contrast, the LPS-induced augmentation of COX-2 protein expression was attenuated in the lungs and tended to be suppressed in the liver of Tacr1−/− mice compared with Tacr1+/+ mice. The Tacr1+/+ mice responded to LPS with a significant surge of PGE2 production in the lungs, whereas Tacr1−/− mice did not. In conclusion, the NK1 receptor is necessary for normal fever genesis. Our results suggest that the NK1 receptor contributes to the early phase of LPS-induced fever by enhancing COX-2 protein expression in the periphery. These findings advance the understanding of the crosstalk between NK signaling and the “cytokine-COX-2

  15. Perturbation in protein expression of the sterile salmonid hybrids between female brook trout Salvelinus fontinalis and male masu salmon Oncorhynchus masou during early spermatogenesis.

    Science.gov (United States)

    Zheng, Liang; Senda, Yoshie; Abe, Syuiti

    2013-05-01

    Most males and females of intergeneric hybrid (BM) between female brook trout (Bt) Salvelinus fontinalis and male masu salmon (Ms) Oncorhynchus masou had undeveloped gonads, with abnormal germ cell development shown by histological examination. To understand the cause of this hybrid sterility, expression profiles of testicular proteins in the BM and parental species were examined with 2-DE coupled with MALDI-TOF/TOF MS. Compared with the parental species, more than 60% of differentially expressed protein spots were down-regulated in BM. A total of 16 up-regulated and 48 down-regulated proteins were identified in BM. Up-regulated were transferrin and other somatic cell-predominant proteins, whereas down-regulated were some germ cell-specific proteins such as DEAD box RNA helicase Vasa. Other pronouncedly down-regulated proteins included tubulins and heat shock proteins that are supposed to have roles in spermatogenesis. The present findings suggest direct association of the observed perturbation in protein expression with the failure of spermatogenesis and the sterility in the examined salmonid hybrids. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Differentially expressed proteins on postoperative 3

    Directory of Open Access Journals (Sweden)

    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

  17. Hepatic expression of the GH/JAK/STAT/IGF pathway, acute-phase response signalling and complement system are affected in mouse offspring by prenatal and early postnatal exposure to maternal high-protein diet.

    Science.gov (United States)

    Vanselow, Jens; Kucia, Marzena; Langhammer, Martina; Koczan, Dirk; Rehfeldt, Charlotte; Metges, Cornelia C

    2011-12-01

    Effects of pre- and early postnatal exposure to maternal high-protein diets are not well understood. Transcription profiling was performed in male mouse offspring exposed to maternal high-protein diet during pregnancy and/or lactation to identify affected hepatic molecular pathways. Dams were fed isoenergetic diets with control (20% w/w) or high protein levels (40%). The hepatic expression profiles were evaluated by differential microarray analysis 3 days (d3) and 3 weeks (d21) after birth. Offspring from three different high-protein dietary groups, HP (d3, high-protein diet during pregnancy), HPHP (d21, high-protein diet during pregnancy and lactation) and CHP (d21, control diet during pregnancy and high-protein diet during lactation), were compared with age-matched offspring from dams fed control diet. Offspring body and liver mass of all high-protein groups were decreased. Prenatal high-protein diet affected hepatic expression of genes mapping to the acute response/complement system and the GH/JAK/STAT/IGF signalling pathways. Maternal exposure to high-protein diet during lactation affected hepatic gene expression of the same pathways but additionally affected genes mapping to protein, fatty acid, hexose and pyruvate metabolism. (1) Genes of the acute response/complement system and GH/JAK/STAT/IGF pathways were down-regulated in offspring of dams exposed to high-protein diets during pregnancy and/or lactation. (2) Genes related to nutrient and energy metabolism, however, were only affected when high-protein diet was administered during lactation. (3) Modulation of the GH/JAK/STAT/IGF pathway might be responsible for reduced body and liver masses by maternal high-protein diet.

  18. Amitriptyline induces early growth response-1 gene expression via ERK and JNK mitogen-activated protein kinase pathways in rat C6 glial cells.

    Science.gov (United States)

    Chung, Eun Young; Shin, Soon Young; Lee, Young Han

    2007-07-05

    Astrocytes play important roles in guiding the construction of the nervous system, controlling extracellular ions and neurotransmitters, and regulating CNS synaptogenesis. Egr-1 is a transcription factor involved in neuronal differentiation and astrocyte cell proliferation. In this study, we investigated whether the tricyclic antidepressant (TCA) amitriptyline induces Egr-1 expression in astrocytes using rat C6 glioma cells as a model. We found that amitriptyline increased the expression of Egr-1 in a dose- and time-dependent manner. The amitriptyline-induced Egr-1 expression was mediated through serum response elements (SREs) in the Egr-1 promoter. SREs were activated by the Ets-domain transcription factor Elk-1 through the ERK and JNK mitogen-activated protein (MAP) kinase pathways. The inhibition of the ERK and JNK MAP kinase signals attenuated amitriptyline-induced transactivation of Gal4-Elk-1 and Egr-1 promoter activity. Our findings suggest that the induction of Egr-1 expression in astrocytes may be required to attain the therapeutic effects of antidepressant drugs.

  19. Pre- and early-postnatal nutrition modify gene and protein expressions of muscle energy metabolism markers and phospholipid Fatty Acid composition in a muscle type specific manner in sheep.

    Directory of Open Access Journals (Sweden)

    Lei Hou

    Full Text Available We previously reported that undernutrition in late fetal life reduced whole-body insulin sensitivity in adult sheep, irrespective of dietary exposure in early postnatal life. Skeletal muscle may play an important role in control of insulin action. We therefore studied a range of putative key muscle determinants of insulin signalling in two types of skeletal muscles (longissimus dorsi (LD and biceps femoris (BF and in the cardiac muscle (ventriculus sinister cordis (VSC of sheep from the same experiment. Twin-bearing ewes were fed either 100% (NORM or 50% (LOW of their energy and protein requirements during the last trimester of gestation. From day-3 postpartum to 6-months of age (around puberty, twin offspring received a high-carbohydrate-high-fat (HCHF or a moderate-conventional (CONV diet, whereafter all males were slaughtered. Females were subsequently raised on a moderate diet and slaughtered at 2-years of age (young adults. The only long-term consequences of fetal undernutrition observed in adult offspring were lower expressions of the insulin responsive glucose transporter 4 (GLUT4 protein and peroxisome proliferator-activated receptor gamma, coactivator 1α (PGC1α mRNA in BF, but increased PGC1α expression in VSC. Interestingly, the HCHF diet in early postnatal life was associated with somewhat paradoxically increased expressions in LD of a range of genes (but not proteins related to glucose uptake, insulin signalling and fatty acid oxidation. Except for fatty acid oxidation genes, these changes persisted into adulthood. No persistent expression changes were observed in BF and VSC. The HCHF diet increased phospholipid ratios of n-6/n-3 polyunsaturated fatty acids in all muscles, even in adults fed identical diets for 1½ years. In conclusion, early postnatal, but not late gestation, nutrition had long-term consequences for a number of determinants of insulin action and metabolism in LD. Tissues other than muscle may account for reduced

  20. Gene expression in early stage cervical cancer

    NARCIS (Netherlands)

    Biewenga, Petra; Buist, Marrije R.; Moerland, Perry D.; van Thernaat, Emiel Ver Loren; van Kampen, Antoine H. C.; ten Kate, Fiebo J. W.; Baas, Frank

    2008-01-01

    Objective. Pelvic lymph node metastases are the main prognostic factor for survival in early stage cervical cancer, yet accurate detection methods before surgery are lacking. In this study, we examined whether gene expression profiling can predict the presence of lymph node metastasis in early stage

  1. 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.

  2. 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.

  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. Herpes simplex virus induces the marked up-regulation of the zinc finger transcriptional factor INSM1, which modulates the expression and localization of the immediate early protein ICP0

    Directory of Open Access Journals (Sweden)

    Kimura Hiroshi

    2011-05-01

    Full Text Available Abstract Background Herpes simplex viruses (HSVs rapidly shut off macromolecular synthesis in host cells. In contrast, global microarray analyses have shown that HSV infection markedly up-regulates a number of host cell genes that may play important roles in HSV-host cell interactions. To understand the regulatory mechanisms involved, we initiated studies focusing on the zinc finger transcription factor insulinoma-associated 1 (INSM1, a host cell protein markedly up-regulated by HSV infection. Results INSM1 gene expression in HSV-1-infected normal human epidermal keratinocytes increased at least 400-fold 9 h after infection; INSM1 promoter activity was also markedly stimulated. Expression and subcellular localization of the immediate early HSV protein ICP0 was affected by INSM1 expression, and chromatin immunoprecipitation (ChIP assays revealed binding of INSM1 to the ICP0 promoter. Moreover, the role of INSM1 in HSV-1 infection was further clarified by inhibition of HSV-1 replication by INSM1-specific siRNA. Conclusions The results suggest that INSM1 up-regulation plays a positive role in HSV-1 replication, probably by binding to the ICP0 promoter.

  5. 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...

  6. Temporal protein expression pattern in intracellular signalling ...

    Indian Academy of Sciences (India)

    Supplementary figure 1. Protein expression dynamics observed in Experiment, Synchronous and. Asynchronous simulation. .... molecular basis for T cell suppression by IL-10: CD28-asso- ciated IL-10 receptor inhibits CD28 tyrosine ...

  7. Functional analysis of chloroplast early light inducible proteins (ELIPs)

    Energy Technology Data Exchange (ETDEWEB)

    Wetzel, Carolyn M

    2005-02-22

    The objectives of this project were to characterize gene expression patterns of early light inducible protein (ELIP) genes in Arabidopsis thaliana and in Lycopersicon esculentum, to identify knock mutants of the 2 ELIP genes in Arabidopsis, and to characterize the effects of the knockouts. Expression in Arabidopsis was studied in response to thylakoid electron transport chain (PETC) capacity, where it was found that there is a signal for expression associated with reduction of the PETC. Expression in response to salt was also studied, with different responses of the two gene copies. Knockout lines for ELIP1 and ELIP2 have been identified and are being characterized. In tomato, it was found that the single-copy ELIP gene is highly expressed in ripening fruit during the chloroplast-to-chromoplast transition. Studies of expression in tomato ripening mutants are ongoing.

  8. Localization and Differential Expression of the Krüppel-Associated Box Zinc Finger Proteins 1 and 54 in Early Mouse Development

    DEFF Research Database (Denmark)

    Albertsen, Maria; Teperek, Marta; Elholm, Grethe

    2010-01-01

    -fused reporter gene into zygotes demonstrated the intracellular distribution of ZFP1-green fluorescent protein (GFP) and ZFP54-GFP colocalized with a DNA marker in the two-cell embryo. The KRAB domain was essential to colocalize with DNA, and deletion of the KRAB domain in ZFP1-GFP and ZFP54-GFP localized...

  9. 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.

  10. 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.

  11. 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

  12. Effect of early addition of bone morphogenetic protein 5 (BMP5) to embryo culture medium on in vitro development and expression of developmentally important genes in bovine preimplantation embryos.

    Science.gov (United States)

    García, Elina V; Miceli, Dora C; Rizo, Gabriela; Valdecantos, Pablo A; Barrera, Antonio D

    2015-09-01

    Previous studies have reported that bone morphogenetic protein 5 (BMP5) is differentially expressed in the isthmus of bovine oviducts and it is present in the oviductal fluid. However, the specific action of this factor is unknown. To evaluate whether BMP5 exerts some effect during early bovine embryo development, gene expression of BMP5, BMP receptors, and the effect of exogenous BMP5 on in vitro development and expression of developmentally important genes were assessed. In experiment 1, pools of embryos at two-cell, four-cell, eight-cell, and blastocyst stages, derived from in vitro fertilization, were collected for analysis of BMP5 and BMP receptors (BMPR1A, BMPR1B, and BMPR2) messenger RNA (mRNA) expression. On the basis of previous results, in experiment 2, presumptive zygotes were cultured for the first 48 hours after insemination in CR1aa medium assaying three different treatments: (1) control (CR1aa); (2) vehicle control (CR1aa + 0.04 mM HCl), and (3) BMP5 treatment (CR1aa + 100 ng/mL of BMP5). The cleavage rate was evaluated 48 hours after insemination (Day 2), and then, embryos were transferred to CR1aa + 10% fetal bovine serum. The blastocyst rate was determined on Day 7. In experiment 3, pools of embryos at two-cell, four-cell, eight-cell, and blastocyst stages, derived from control and BMP5-treated groups, were collected for analysis of ID2 (BMP target gene), OCT4, NANOG, and SOX2 (pluripotency genes) mRNA expression. BMP5 transcripts were not detectable in any of the embryonic stages examined, whereas the relative mRNA abundance of the three BMP receptors analyzed was greater in early embryo development stages before maternal-embryonic transition, raising the possibility of a direct effect of exogenous BMPs on the embryo during the first developmental period. Although early addition of 100 ng/mL of BMP5 to the embryo culture medium had no effect on the cleavage rate, a significantly higher proportion of cleaved embryos developed to the

  13. 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...

  14. 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.

  15. Immunohistochemical expression of latent membrane protein 1 ...

    African Journals Online (AJOL)

    Methods: Archival formalin-fixed, paraffin-embedded NPC biopsies were evaluated in 23 Moroccan patients for the presence of LMP1 and p53 using immunohistochemistry (IHC). Results: No LMP1 expression was observed whereas 8 of 23 cases (34. 7%) had detectable p53 protein in the nuclei of tumor cells.

  16. Developmental expression of Drosophila Wiskott-Aldrich Syndrome family proteins

    Science.gov (United States)

    Rodriguez-Mesa, Evelyn; Abreu-Blanco, Maria Teresa; Rosales-Nieves, Alicia E.; Parkhurst, Susan M.

    2012-01-01

    Background Wiskott-Aldrich Syndrome (WASP) family proteins participate in many cellular processes involving rearrangements of the actin cytoskeleton. To the date, four WASP subfamily members have been described in Drosophila: Wash, WASp, SCAR, and Whamy. Wash, WASp, and SCAR are essential during early Drosophila development where they function in orchestrating cytoplasmic events including membrane-cytoskeleton interactions. A mutant for Whamy has not yet been reported. Results We generated monoclonal antibodies that are specific to Drosophila Wash, WASp, SCAR, and Whamy, and use these to describe their spatial and temporal localization patterns. Consistent with the importance of WASP family proteins in flies, we find that Wash, WASp, SCAR, and Whamy are dynamically expressed throughout oogenesis and embryogenesis. For example, we find that Wash accumulates at the oocyte cortex. WASp is highly expressed in the PNS, while SCAR is the most abundantly expressed in the CNS. Whamy exhibits an asymmetric subcellular localization that overlaps with mitochondria and is highly expressed in muscle. Conclusion All four WASP family members show specific expression patterns, some of which reflect their previously known roles and others revealing new potential functions. The monoclonal antibodies developed offer valuable new tools to investigate how WASP family proteins regulate actin cytoskeleton dynamics. PMID:22275148

  17. Regulation of bone morphogenetic proteins in early embryonic development

    Science.gov (United States)

    Yamamoto, Yukiyo; Oelgeschläger, Michael

    2004-11-01

    Bone morphogenetic proteins (BMPs), a large subgroup of the TGF-β family of secreted growth factors, control fundamental events in early embryonic development, organogenesis and adult tissue homeostasis. The plethora of dose-dependent cellular processes regulated by BMP signalling demand a tight regulation of BMP activity. Over the last decade, a number of proteins have been identified that bind BMPs in the extracellular space and regulate the interaction of BMPs with their cognate receptors, including the secreted BMP antagonist Chordin. In the early vertebrate embryo, the localized secretion of BMP antagonists from the dorsal blastopore lip establishes a functional BMP signalling gradient that is required for the determination of the dorsoventral or back to belly body axis. In particular, inhibition of BMP activity is essential for the formation of neural tissue in the development of vertebrate and invertebrate embryos. Here we review recent studies that have provided new insight into the regulation of BMP signalling in the extracellular space. In particular, we discuss the recently identified Twisted gastrulation protein that modulates, in concert with metalloproteinases of the Tolloid family, the interaction of Chordin with BMP and a family of proteins that share structural similarities with Chordin in the respective BMP binding domains. In addition, genetic and functional studies in zebrafish and frog provide compelling evidence that the secreted protein Sizzled functionally interacts with the Chd BMP pathway, despite being expressed ventrally in the early gastrula-stage embryo. These intriguing discoveries may have important implications, not only for our current concept of early embryonic patterning, but also for the regulation of BMP activity at later developmental stages and tissue homeostasis in the adult.

  18. Alteration of gene expression by alcohol exposure at early neurulation.

    Science.gov (United States)

    Zhou, Feng C; Zhao, Qianqian; Liu, Yunlong; Goodlett, Charles R; Liang, Tiebing; McClintick, Jeanette N; Edenberg, Howard J; Li, Lang

    2011-02-21

    We have previously demonstrated that alcohol exposure at early neurulation induces growth retardation, neural tube abnormalities, and alteration of DNA methylation. To explore the global gene expression changes which may underline these developmental defects, microarray analyses were performed in a whole embryo mouse culture model that allows control over alcohol and embryonic variables. Alcohol caused teratogenesis in brain, heart, forelimb, and optic vesicle; a subset of the embryos also showed cranial neural tube defects. In microarray analysis (accession number GSM9545), adopting hypothesis-driven Gene Set Enrichment Analysis (GSEA) informatics and intersection analysis of two independent experiments, we found that there was a collective reduction in expression of neural specification genes (neurogenin, Sox5, Bhlhe22), neural growth factor genes [Igf1, Efemp1, Klf10 (Tieg), and Edil3], and alteration of genes involved in cell growth, apoptosis, histone variants, eye and heart development. There was also a reduction of retinol binding protein 1 (Rbp1), and de novo expression of aldehyde dehydrogenase 1B1 (Aldh1B1). Remarkably, four key hematopoiesis genes (glycophorin A, adducin 2, beta-2 microglobulin, and ceruloplasmin) were absent after alcohol treatment, and histone variant genes were reduced. The down-regulation of the neurospecification and the neurotrophic genes were further confirmed by quantitative RT-PCR. Furthermore, the gene expression profile demonstrated distinct subgroups which corresponded with two distinct alcohol-related neural tube phenotypes: an open (ALC-NTO) and a closed neural tube (ALC-NTC). Further, the epidermal growth factor signaling pathway and histone variants were specifically altered in ALC-NTO, and a greater number of neurotrophic/growth factor genes were down-regulated in the ALC-NTO than in the ALC-NTC embryos. This study revealed a set of genes vulnerable to alcohol exposure and genes that were associated with neural tube

  19. 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.

  20. Contingency Table Browser - prediction of early stage protein structure.

    Science.gov (United States)

    Kalinowska, Barbara; Krzykalski, Artur; Roterman, Irena

    2015-01-01

    The Early Stage (ES) intermediate represents the starting structure in protein folding simulations based on the Fuzzy Oil Drop (FOD) model. The accuracy of FOD predictions is greatly dependent on the accuracy of the chosen intermediate. A suitable intermediate can be constructed using the sequence-structure relationship information contained in the so-called contingency table - this table expresses the likelihood of encountering various structural motifs for each tetrapeptide fragment in the amino acid sequence. The limited accuracy with which such structures could previously be predicted provided the motivation for a more indepth study of the contingency table itself. The Contingency Table Browser is a tool which can visualize, search and analyze the table. Our work presents possible applications of Contingency Table Browser, among them - analysis of specific protein sequences from the point of view of their structural ambiguity.

  1. Prion protein expression regulates embryonic stem cell pluripotency and differentiation.

    Directory of Open Access Journals (Sweden)

    Alberto Miranda

    2011-04-01

    Full Text Available Cellular prion protein (PRNP is a glycoprotein involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs. Although the physiological function of PRNP is largely unknown, its key role in prion infection has been extensively documented. This study examines the functionality of PRNP during the course of embryoid body (EB differentiation in mouse Prnp-null (KO and WT embryonic stem cell (ESC lines. The first feature observed was a new population of EBs that only appeared in the KO line after 5 days of differentiation. These EBs were characterized by their expression of several primordial germ cell (PGC markers until Day 13. In a comparative mRNA expression analysis of genes playing an important developmental role during ESC differentiation to EBs, Prnp was found to participate in the transcription of a key pluripotency marker such as Nanog. A clear switching off of this gene on Day 5 was observed in the KO line as opposed to the WT line, in which maximum Prnp and Nanog mRNA levels appeared at this time. Using a specific antibody against PRNP to block PRNP pathways, reduced Nanog expression was confirmed in the WT line. In addition, antibody-mediated inhibition of ITGB5 (integrin αvβ5 in the KO line rescued the low expression of Nanog on Day 5, suggesting the regulation of Nanog transcription by Prnp via this Itgb5. mRNA expression analysis of the PRNP-related proteins PRND (Doppel and SPRN (Shadoo, whose PRNP function is known to be redundant, revealed their incapacity to compensate for the absence of PRNP during early ESC differentiation. Our findings provide strong evidence for a relationship between Prnp and several key pluripotency genes and attribute Prnp a crucial role in regulating self-renewal/differentiation status of ESC, confirming the participation of PRNP during early embryogenesis.

  2. Intermediate filament protein nestin is expressed in developing meninges.

    Science.gov (United States)

    Yay, A; Ozdamar, S; Canoz, O; Baran, M; Tucer, B; Sonmez, M F

    2014-01-01

    Nestin is a type VI intermediate filament protein known as a marker for progenitor cells that can be mostly found in tissues during the embryonic and fetal periods. In our study, we aimed to determine the expression of nestin in meninges covering the brain tissue at different developmental stages and in the new born. In this study 10 human fetuses in different development stages between developmental weeks 9-34 and a newborn brain tissue were used. Fetuses in paraffin section were stained with H+E and nestin immunohistochemical staining protocol was performed. In this study, in the human meninges intense nestin expression was detected as early as in the 9th week of development. Intensity of this expression gradually decreased in later stages of development and nestin expression still persisted in a small population of newborn meningeal cells. In the present study, nestin positive cells gradually diminished in the developing and maturing meninges during the fetal period. This probably depends on initiation of a decrease in nestin expression and replacement with other tissue-specific intermediate filaments while the differentiation process continues. These differences can make significant contributions to the investigation and diagnosis of various pathological disorders (Tab. 1, Fig. 3, Ref. 36).

  3. The Expression of Genes Encoding Secreted Proteins in Medicago truncatula A17 Inoculated Roots

    Directory of Open Access Journals (Sweden)

    LUCIA KUSUMAWATI

    2013-09-01

    Full Text Available Subtilisin-like serine protease (MtSBT, serine carboxypeptidase (MtSCP, MtN5, non-specific lipid transfer protein (MtnsLTP, early nodulin2-like protein (MtENOD2-like, FAD-binding domain containing protein (MtFAD-BP1, and rhicadhesin receptor protein (MtRHRE1 were among 34 proteins found in the supernatant of M. truncatula 2HA and sickle cell suspension cultures. This study investigated the expression of genes encoding those proteins in roots and developing nodules. Two methods were used: quantitative real time RT-PCR and gene expression analysis (with promoter:GUS fusion in roots. Those proteins are predicted as secreted proteins which is indirectly supported by the findings that promoter:GUS fusions of six of the seven genes encoding secreted proteins were strongly expressed in the vascular bundle of transgenic hairy roots. All six genes have expressed in 14-day old nodule. The expression levels of the selected seven genes were quantified in Sinorhizobium-inoculated and control plants using quantitative real time RT-PCR. In conclusion, among seven genes encoding secreted proteins analyzed, the expression level of only one gene, MtN5, was up-regulated significantly in inoculated root segments compared to controls. The expression of MtSBT1, MtSCP1, MtnsLTP, MtFAD-BP1, MtRHRE1 and MtN5 were higher in root tip than in other tissues examined.

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

    Directory of Open Access Journals (Sweden)

    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

  5. Tlys, a newly identified Sulfolobus spindle-shaped virus 1 transcript expressed in the lysogenic state, encodes a DNA-binding protein interacting at the promoters of the early genes

    DEFF Research Database (Denmark)

    Fusco, Salvatore; She, Qunxin; Bartolucci, Simonetta

    2013-01-01

    -binding motif. DNA-binding assays demonstrated that the recombinant F55, purified from Escherichia coli, is indeed a putative transcription factor able to recognize site specifically target sequences in the promoters of the early induced T5, T6, and Tind transcripts, as well as of its own promoter. Binding...... the growth of the lysogenic host. The correponding gene f55 lies between two transcriptional units (T6 and Tind) that are upregulated upon UV irradiation. The open reading frame f55 encodes a 6.3-kDa protein which shows sequence identity with negative regulators that fold into the ribbon-helix-helix DNA....... Taking together the transcriptional analysis data and the biochemical evidences, we surmise that the protein F55 is involved in the regulation of the lysogenic state of SSV1....

  6. Early pregnancy peripheral blood gene expression and risk of preterm delivery: a nested case control study

    Directory of Open Access Journals (Sweden)

    Muhie Seid Y

    2009-12-01

    Full Text Available Abstract Background Preterm delivery (PTD is a significant public health problem associated with greater risk of mortality and morbidity in infants and mothers. Pathophysiologic processes that may lead to PTD start early in pregnancy. We investigated early pregnancy peripheral blood global gene expression and PTD risk. Methods As part of a prospective study, ribonucleic acid was extracted from blood samples (collected at 16 weeks gestational age from 14 women who had PTD (cases and 16 women who delivered at term (controls. Gene expressions were measured using the GeneChip® Human Genome U133 Plus 2.0 Array. Student's T-test and fold change analysis were used to identify differentially expressed genes. We used hierarchical clustering and principle components analysis to characterize signature gene expression patterns among cases and controls. Pathway and promoter sequence analyses were used to investigate functions and functional relationships as well as regulatory regions of differentially expressed genes. Results A total of 209 genes, including potential candidate genes (e.g. PTGDS, prostaglandin D2 synthase 21 kDa, were differentially expressed. A set of these genes achieved accurate pre-diagnostic separation of cases and controls. These genes participate in functions related to immune system and inflammation, organ development, metabolism (lipid, carbohydrate and amino acid and cell signaling. Binding sites of putative transcription factors such as EGR1 (early growth response 1, TFAP2A (transcription factor AP2A, Sp1 (specificity protein 1 and Sp3 (specificity protein 3 were over represented in promoter regions of differentially expressed genes. Real-time PCR confirmed microarray expression measurements of selected genes. Conclusions PTD is associated with maternal early pregnancy peripheral blood gene expression changes. Maternal early pregnancy peripheral blood gene expression patterns may be useful for better understanding of PTD

  7. 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.

  8. 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.

  9. Early continuous white noise exposure alters l-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit glutamate receptor 2 and gamma-aminobutyric acid type a receptor subunit beta3 protein expression in rat auditory cortex.

    Science.gov (United States)

    Xu, Jinghong; Yu, Liping; Zhang, Jiping; Cai, Rui; Sun, Xinde

    2010-02-15

    Auditory experience during the postnatal critical period is essential for the normal maturation of auditory function. Previous studies have shown that rearing infant rat pups under conditions of continuous moderate-level noise delayed the emergence of adult-like topographic representational order and the refinement of response selectivity in the primary auditory cortex (A1) beyond normal developmental benchmarks and indefinitely blocked the closure of a brief, critical-period window. To gain insight into the molecular mechanisms of these physiological changes after noise rearing, we studied expression of the AMPA receptor subunit GluR2 and GABA(A) receptor subunit beta3 in the auditory cortex after noise rearing. Our results show that continuous moderate-level noise rearing during the early stages of development decreases the expression levels of GluR2 and GABA(A)beta3. Furthermore, noise rearing also induced a significant decrease in the level of GABA(A) receptors relative to AMPA receptors. However, in adult rats, noise rearing did not have significant effects on GluR2 and GABA(A)beta3 expression or the ratio between the two units. These changes could have a role in the cellular mechanisms involved in the delayed maturation of auditory receptive field structure and topographic organization of A1 after noise rearing. Copyright 2009 Wiley-Liss, Inc.

  10. 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.

  11. 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.

  12. Changes in protein synthetic activity in early Drosophila embryos mutant for the segmentation gene Krueppel

    International Nuclear Information System (INIS)

    Bedian, V.; Summers, M.C.; Kauffman, S.A.

    1988-01-01

    We have identified early embryo proteins related to the segmentation gene Krueppel by [35S]methionine pulse labelling and two-dimensional gel electrophoresis. Protein synthesis differences shared by homozygous embryos of two Krueppel alleles when compared to heterozygous and wild-type embryos are reported. The study was extended to syncytial blastoderm stages by pulse labelling and gel analysis of single embryos, using Krueppel-specific proteins from gastrula stages as molecular markers for identifying homozygous Krueppel embryos. Localized expression of interesting proteins was examined in embryo fragments. The earliest differences detected at nuclear migration stages showed unregulated synthesis in mutant embryos of two proteins that have stage specific synthesis in normal embryos. At the cellular blastoderm stage one protein was not synthesized and two proteins showed apparent shifts in isoelectric point in mutant embryos. Differences observed in older embryos included additional proteins with shifted isoelectric points and a number of qualitative and quantitative changes in protein synthesis. Five of the proteins with altered rates of synthesis in mutant embryos showed localized synthesis in normal embryos. The early effects observed are consistent with the hypothesis that the Krueppel product can be a negative or positive regulator of expression of other loci, while blastoderm and gastrula stage shifts in isoelectric point indicate that a secondary effect of Krueppel function may involve post-translational modification of proteins

  13. Transcriptional factor PU.1 regulates decidual C1q expression in early pregnancy in human

    Directory of Open Access Journals (Sweden)

    Priyaa Madhukaran Raj

    2015-02-01

    Full Text Available C1q is the first recognition subcomponent of the complement classical pathway, which in addition to being synthesized in the liver, is also expressed by macrophages and dendritic cells. Trophoblast invasion during early placentation results in accumulation of debris that triggers the complement system. Hence, both early and late components of the classical pathway are widely distributed in the placenta and decidua. In addition, C1q has recently been shown to significantly contribute to feto-maternal tolerance, trophoblast migration, and spiral artery remodeling, although the exact mechanism remains unknown. Pregnancy in mice, genetically deficient in C1q, mirrors symptoms similar to that of human preeclampsia. Thus, regulated complement activation has been proposed as an essential requirement for normal successful pregnancy. Little is known about the molecular pathways that regulate C1q expression in pregnancy. PU.1, an Ets-family transcription factor, is required for the development of hematopoietic myeloid lineage immune cells, and its expression is tissue- specific. Recently, PU.1 has been shown to regulate C1q gene expression in dendritic cells and macrophages. Here, we have examined if PU.1 transcription factor regulates decidual C1q expression. We used immune-histochemical analysis, PCR and immunostaining to localize and study the gene expression of PU.1 transcription factor in early human decidua. PU.1 was highly expressed at gene and protein level in early human decidual cells including trophoblast and stromal cells. Surprisingly, nuclear as well as cytoplasmic PU.1 expression was observed. Decidual cells with predominantly nuclear PU.1 expression had higher C1q expression. It is likely that nuclear and cytoplasmic PU.1 localization has a role to play in early pregnancy via regulating C1q expression in the decidua during implantation.

  14. Pulmonary heat shock protein expression after exposure to a metabolically activated Clara cell toxicant: relationship to protein adduct formation

    International Nuclear Information System (INIS)

    Williams, Kurt J.; Cruikshank, Michael K.; Plopper, Charles G.

    2003-01-01

    Heat shock proteins/stress proteins (Hsps) participate in regulation of protein synthesis and degradation and serve as general cytoprotectants, yet their role in lethal Clara cell injury is not clear. To define the pattern of Hsp expression in acute lethal Clara cell injury, mice were treated with the Clara cell-specific toxicant naphthalene (NA), and patterns of expression compared to electrophilic protein adduction and previously established organellar degradation and gluathione (GSH) depletion. In sites of lethal injury (distal bronchiole), prior to organellar degradation (1 h post-NA), protein adduction is detectable and ubiquitin, Hsp 25, Hsp 72, and heme-oxygenase 1 (HO-1) are increased. Maximal Hsp expression, protein adduction, and GSH depletion occur simultaneous (by 2-3 h) with early organelle disruption. Hsp expression is higher later (6-24 h), only in exfoliating cells. In airway sites (proximal bronchiole) with nonlethal Clara cell injury elevation of Hsp 25, 72, and HO-1 expression follows significant GSH depletion (greater than 50% 2 h post-NA). This data build upon our previous studies and we conclude that (1) in lethal (terminal bronchiole) and nonlethal (proximal bronchiole) Clara cell injury, Hsp induction is associated with the loss of GSH and increased protein adduction, and (2) in these same sites, organelle disruption is not a prerequisite for Hsp induction

  15. 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 ...

  16. Fast identification of folded human protein domains expressed in E. coli suitable for structural analysis

    Directory of Open Access Journals (Sweden)

    Schlegel Brigitte

    2004-03-01

    Full Text Available Abstract Background High-throughput protein structure analysis of individual protein domains requires analysis of large numbers of expression clones to identify suitable constructs for structure determination. For this purpose, methods need to be implemented for fast and reliable screening of the expressed proteins as early as possible in the overall process from cloning to structure determination. Results 88 different E. coli expression constructs for 17 human protein domains were analysed using high-throughput cloning, purification and folding analysis to obtain candidates suitable for structural analysis. After 96 deep-well microplate expression and automated protein purification, protein domains were directly analysed using 1D 1H-NMR spectroscopy. In addition, analytical hydrophobic interaction chromatography (HIC was used to detect natively folded protein. With these two analytical methods, six constructs (representing two domains were quickly identified as being well folded and suitable for structural analysis. Conclusion The described approach facilitates high-throughput structural analysis. Clones expressing natively folded proteins suitable for NMR structure determination were quickly identified upon small scale expression screening using 1D 1H-NMR and/or analytical HIC. This procedure is especially effective as a fast and inexpensive screen for the 'low hanging fruits' in structural genomics.

  17. Identification of differentially expressed proteins in response to Pb ...

    African Journals Online (AJOL)

    In response to Pb, a total of 76 proteins, out of the 95 differentially expressed proteins, were subjected to MALDI-TOF-MS Of these, 46 identities were identified by PMF and 19 identities were identified by microsequencing. Basic metabolisms such as photosynthesis, photorespiration and protein biosynthesis in C. roseus ...

  18. 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.

  19. Major cancer protein amplifies global gene expression

    Science.gov (United States)

    Scientists may have discovered why a protein called MYC can provoke a variety of cancers. Like many proteins associated with cancer, MYC helps regulate cell growth. A new study carried out by researchers at the National Institutes of Health and colleagues

  20. Increased cystic fibrosis transmembrane conductance regulators expression and decreased epithelial sodium channel alpha subunits expression in early abortion: findings from a mouse model and clinical cases of abortion.

    Directory of Open Access Journals (Sweden)

    Min Zhou

    Full Text Available The status of the maternal endometrium is vital in regulating humoral homeostasis and for ensuring embryo implantation. Cystic fibrosis transmembrane conductance regulators (CFTR and epithelial sodium channel alpha subunits (ENaC-α play an important role in female reproduction by maintaining humoral and cell homeostasis. However, it is not clear whether the expression levels of CFTR and ENaC-α in the decidual component during early pregnancy are related with early miscarriage. CBA×DBA/2 mouse mating has been widely accepted as a classical model of early miscarriage. The abortion rate associated with this mating was 33.33% in our study. The decidua of abortion-prone CBA female mice (DBA/2 mated had higher CFTR mRNA and protein expression and lower ENaC-α mRNA and protein expression, compared to normal pregnant CBA mice (BLAB/C mated. Furthermore, increased CFTR expression and decreased ENaC-α expression were observed in the uterine tissue from women with early miscarriage, as compared to those with successful pregnancy. In conclusion, increased CFTR expression and decreased ENaC-α expression in the decidua of early abortion may relate with failure of early pregnancy.

  1. 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

  2. Distinct GAGE and MAGE-A expression during early human development indicate specific roles in lineage differentiation

    DEFF Research Database (Denmark)

    Gjerstorff, Morten; Harkness, Linda; Kassem, Moustapha

    2008-01-01

    BACKGROUND: Expression of cancer/testis-associated proteins (CTAs) has traditionally been considered to be restricted to germ cells in normal tissues and to different types of malignancies. We have evaluated the potential role of CTAs in early human development. METHODS: Using immunohistochemistry...... and RT-PCR, we investigated the expression of CTAs in differentiated human embryonic stem cells (hESC) and in late embryos and early fetuses. RESULTS: We found that melanoma antigen A (MAGE-A) family members were expressed during differentiation of hESC to embryoid bodies and in teratomas, and overlapped...... with expression of the neuroectodermal markers beta-tubulin 3, Pax6 and nestin. A widespread expression of MAGE-A was also observed in neurons of the early developing central nervous system and peripheral nerves. G antigen (GAGE) expression was present in the early ectoderm of embryos, including cells...

  3. G protein-coupled receptor 84, a microglia-associated protein expressed in neuroinflammatory conditions.

    Science.gov (United States)

    Bouchard, Caroline; Pagé, Julie; Bédard, Andréanne; Tremblay, Pierrot; Vallières, Luc

    2007-06-01

    G protein-coupled receptor 84 (GPR84) is a recently discovered member of the seven transmembrane receptor superfamily whose function and regulation are unknown. Here, we report that in mice suffering from endotoxemia, microglia express GPR84 in a strong and sustained manner. This property is shared by subpopulations of peripheral macrophages and, to a much lesser extent, monocytes. The induction of GPR84 expression by endotoxin is mediated, at least in part, by proinflammatory cytokines, notably tumor necrosis factor (TNF) and interleukin-1 (IL-1), because mice lacking either one or both of these molecules have fewer GPR84-expressing cells in their cerebral cortex than wild-type mice during the early phase of endotoxemia. Moreover, when injected intracerebrally or added to microglial cultures, recombinant TNF stimulates GPR84 expression through a dexamethasone-insensitive mechanism. Finally, we show that microglia produce GPR84 not only during endotoxemia, but also during experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. In conclusion, this study reports the identification of a new sensitive marker of microglial activation, which may play an important regulatory role in neuroimmunological processes, acting downstream to the effects of proinflammatory mediators.

  4. Expression of voltage-activated calcium channels in the early zebrafish embryo.

    Science.gov (United States)

    Sanhueza, Dayán; Montoya, Andro; Sierralta, Jimena; Kukuljan, Manuel

    2009-05-01

    Increases in cytosolic calcium concentrations regulate many cellular processes, including aspects of early development. Calcium release from intracellular stores and calcium entry through non-voltage-gated channels account for signalling in non-excitable cells, whereas voltage-gated calcium channels (CaV) are important in excitable cells. We report the expression of multiple transcripts of CaV, identified by its homology to other species, in the early embryo of the zebrafish, Danio rerio, at stages prior to the differentiation of excitable cells. CaV mRNAs and proteins were detected as early as the 2-cell stages, which indicate that they arise from both maternal and zygotic transcription. Exposure of embryos to pharmacological blockers of CaV does not perturb early development significantly, although late effects are appreciable. These results suggest that CaV may have a role in calcium homeostasis and control of cellular process during early embryonic development.

  5. Early correlation of microglial activation with enhanced tumor necrosis factor-alpha and monocyte chemoattractant protein-1 expression specifically within the entorhinal cortex of triple transgenic Alzheimer's disease mice

    Directory of Open Access Journals (Sweden)

    LaFerla Frank M

    2005-10-01

    Full Text Available Abstract Background Alzheimer's disease is a complex neurodegenerative disorder characterized pathologically by a temporal and spatial progression of beta-amyloid (Aβ deposition, neurofibrillary tangle formation, and synaptic degeneration. Inflammatory processes have been implicated in initiating and/or propagating AD-associated pathology within the brain, as inflammatory cytokine expression and other markers of inflammation are pronounced in individuals with AD pathology. The current study examines whether inflammatory processes are evident early in the disease process in the 3xTg-AD mouse model and if regional differences in inflammatory profiles exist. Methods Coronal brain sections were used to identify Aβ in 2, 3, and 6-month 3xTg-AD and non-transgenic control mice. Quantitative real-time RT-PCR was performed on microdissected entorhinal cortex and hippocampus tissue of 2, 3, and 6-month 3xTg-AD and non-transgenic mice. Microglial/macrophage cell numbers were quantified using unbiased stereology in 3xTg-AD and non-transgenic entorhinal cortex and hippocampus containing sections. Results We observed human Aβ deposition at 3 months in 3xTg-AD mice which is enhanced by 6 months of age. Interestingly, we observed a 14.8-fold up-regulation of TNF-α and 10.8-fold up-regulation of MCP-1 in the entorhinal cortex of 3xTg-AD mice but no change was detected over time in the hippocampus or in either region of non-transgenic mice. Additionally, this increase correlated with a specific increase in F4/80-positive microglia and macrophages in 3xTg-AD entorhinal cortex. Conclusion Our data provide evidence for early induction of inflammatory processes in a model that develops amyloid and neurofibrillary tangle pathology. Additionally, our results link inflammatory processes within the entorhinal cortex, which represents one of the earliest AD-affected brain regions.

  6. 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.

  7. Novel approach for transient protein expression in primary cultures of human dental pulp-derived cells.

    Science.gov (United States)

    Suguro, Hisashi; Mikami, Yoshikazu; Koshi, Rieko; Ogiso, Bunnai; Watanabe, Eri; Watanabe, Nobukazu; Honda, Masaki J; Asano, Masatake; Komiyama, Kazuo

    2011-08-01

    Transfection is a powerful method for investigating variable biological functions of desired genes. However, the efficiency of transfection into primary cultures of dental pulp-derived cells (DPDC) is low. Therefore, using a recombinant vaccinia virus (vTF7-3), which contains T7 RNA polymerase, we have established a transient protein expression system in DPDCs. In this study, we used the human polymeric immunoglobulin receptor (pIgR) cDNA as a model gene. pIgR expression by the vTF7-3 expression system was confirmed by flow cytometry analysis and Western blotting. Furthermore, exogenous pIgR protein localized at the cell surface in DPDCs and formed a secretory component (SC). This suggests that exogenous pIgR protein expressed by the vTF7-3 expression system acts like endogenous pIgR protein. These results indicate the applicability of the method for cells outgrown from dental pulp tissue. In addition, as protein expression could be detected shortly after transfection (approximately 5h), this experimental system has been used intensely for experiments examining very early steps in protein exocytosis. Copyright © 2011 Elsevier Inc. All rights reserved.

  8. Heterologous Protein Expression by Lactococcus lactis

    NARCIS (Netherlands)

    Villatoro-Hernández, J.; Kuipers, O.P.; Saucedo-Cárdenas, O.; Montes-de-Oca-Luna, R.

    2012-01-01

    This chapter describes the use of Lactococcus lactis as a safe and efficient cell factory to produce heterologous proteins of medical interest. The relevance of the use of this lactic acid bacterium (LAB) is that it is a noncolonizing, nonpathogenic microorganism that can be delivered in vivo at a

  9. 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....

  10. CURCUMIN DECREASES SPECIFICITY PROTEIN (Sp) EXPRESSION IN BLADDER CANCER CELLS

    OpenAIRE

    Chadalapaka, Gayathri; Jutooru, Indira; Chintharlapalli, Sudhakar; Papineni, Sabitha; Smith, Roger; Li, Xiangrong; Safe, Stephen

    2008-01-01

    Curcumin is the active component of tumeric, and this polyphenolic compound has been extensively investigated as an anticancer drug that modulates multiple pathways and genes. In this study, 10 – 25 µM curcumin inhibited 253JB-V and KU7 bladder cancer cell growth, and this was accompanied by induction of apoptosis and decreased expression of the proapoptotic protein survivin and the angiogenic proteins vascular endothelial growth factor (VEGF) and VEGF receptor 1 (VEGFR1). Since expression of...

  11. The 3' region of Human Papillomavirus type 16 early mRNAs decrease expression

    DEFF Research Database (Denmark)

    Vinther, J.; Rosenstierne, M.W.; Kristiansen, Karen

    2005-01-01

    Background: High risk human papillomavirus (HR-HPV) infects mucosal surfaces and HR-HPV infection is required for development of cervical cancer. Accordingly, enforced expression of the early HR-HPV proteins can induce immortalisation of human cells. In most cervical cancers and cervical cancer...... cell lines the HR-HPV double stranded DNA genome has been integrated into the host cell genome. Methods: We have used a retroviral GUS reporter system to generate pools of stably transfected HaCaT and SiHa cells. The HPV-16 early sequences that are deleted upon integration of the HPV-16 genome...

  12. Recombinant protein expression in microbial systems

    OpenAIRE

    Rosano, Germán L.; Ceccarelli, Eduardo A.

    2014-01-01

    The emergence of recombinant DNA technology during the early 70's set a revolution in molecular biology. This set of techniques was strengthened even further later on with the introduction of the polymerase chain reaction and allowed scientists to explore and understand essential life processes in an easy and straightforward way. It also marked the birth of the modern biotech industry. At that time, it was shown that eukaryotic DNA could be propagated in Escherichia coli (Morrow et al., 1974)...

  13. Deficient expression of DNA repair enzymes in early progression to sporadic colon cancer

    Science.gov (United States)

    2012-01-01

    Background Cancers often arise within an area of cells (e.g. an epithelial patch) that is predisposed to the development of cancer, i.e. a "field of cancerization" or "field defect." Sporadic colon cancer is characterized by an elevated mutation rate and genomic instability. If a field defect were deficient in DNA repair, DNA damages would tend to escape repair and give rise to carcinogenic mutations. Purpose To determine whether reduced expression of DNA repair proteins Pms2, Ercc1 and Xpf (pairing partner of Ercc1) are early steps in progression to colon cancer. Results Tissue biopsies were taken during colonoscopies of 77 patients at 4 different risk levels for colon cancer, including 19 patients who had never had colonic neoplasia (who served as controls). In addition, 158 tissue samples were taken from tissues near or within colon cancers removed by resection and 16 tissue samples were taken near tubulovillous adenomas (TVAs) removed by resection. 568 triplicate tissue sections (a total of 1,704 tissue sections) from these tissue samples were evaluated by immunohistochemistry for 4 DNA repair proteins. Substantially reduced protein expression of Pms2, Ercc1 and Xpf occurred in field defects of up to 10 cm longitudinally distant from colon cancers or TVAs and within colon cancers. Expression of another DNA repair protein, Ku86, was infrequently reduced in these areas. When Pms2, Ercc1 or Xpf were reduced in protein expression, then either one or both of the other two proteins most often had reduced protein expression as well. The mean inner colon circumferences, from 32 resections, of the ascending, transverse and descending/sigmoid areas were measured as 6.6 cm, 5.8 cm and 6.3 cm, respectively. When combined with other measurements in the literature, this indicates the approximate mean number of colonic crypts in humans is 10 million. Conclusions The substantial deficiencies in protein expression of DNA repair proteins Pms2, Ercc1 and Xpf in about 1 million

  14. Over-expression of 14-3-3zeta is an early event in oral cancer

    International Nuclear Information System (INIS)

    Matta, Ajay; Bahadur, Sudhir; Duggal, Ritu; Gupta, Siddhartha D; Ralhan, Ranju

    2007-01-01

    The functional and clinical significance of 14-3-3 proteins in human cancers remain largely undetermined. Earlier, we have reported differential expression of 14-3-3ζ mRNA in oral squamous cell carcinoma (OSCC) by differential display. The clinical relevance of 14-3-3ζ protein in oral tumorigenesis was determined by immunohistochemistry in paraffin embedded sections of oral pre-malignant lesions (OPLs), OSCCs and histologically normal oral tissues and corroborated by Western Blotting. Co-immunoprecipitation assays were carried out to determine its association with NFκB, β-catenin and Bcl-2. Intense immunostaining of 14-3-3ζ protein was observed in 61/89 (69%) OPLs and 95/120 (79%) OSCCs. Immunohistochemistry showed significant increase in expression of 14-3-3ζ protein from normal mucosa to OPLs to OSCCs (p trend < 0.001). Significant increase in expression of 14-3-3ζ protein was observed as early as in hyperplasia (p = 0.009), with further elevation in moderate and severe dysplasia, that was sustained in OSCCs. These findings were validated by Western blotting. Using Co-immunoprecipitation, we demonstrated that 14-3-3ζ protein binds to NFκB, β-catenin and Bcl-2, suggesting its involvement in cellular signaling, leading to proliferation of oral cancer cells. Our findings suggest that over-expression of 14-3-3ζ is an early event in oral tumorigenesis and may have an important role in its development and progression. Thus, 14-3-3ζ may serve as an important molecular target for designing novel therapy for oral cancer

  15. Recombinant Brucella abortus gene expressing immunogenic protein

    Energy Technology Data Exchange (ETDEWEB)

    Mayfield, J.E.; Tabatabai, L.B.

    1991-06-11

    This patent describes a synthetic recombinant DNA molecule containing a DNA sequence. It comprises a gene of Brucella abortus encoding an immunogenic protein having a molecular weight of approximately 31,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis under denaturing conditions, the protein having an isoelectric point around 4.9, and containing a twenty-five amino acid sequence from its amino terminal end consisting of Gln-Ala-Pro-Thr-Phe-Phe-Arg-Ile-Gly-Thr-Gly-Gly-Thr-Ala-Gly-Thr-Tyr-Tyr-Pro-Ile-Gly-Gly-Leu-Ile-Ala, wherein Gln, Ala, Pro, Thr, Phe, Arg, Ile, Gly, Tyr, and Leu, respectively, represent glutamine, alanine, proline, threonine, phenylalanine, arginine, isolecuine, glycine, tyrosine, and leucine.

  16. Improved means and methods for expressing recombinant proteins

    NARCIS (Netherlands)

    Poolman, Berend; Martinez Linares, Daniel; Gul, Nadia

    2014-01-01

    The invention relates to the field of genetic engineering and the production of recombinant proteins in microbial host cells. Provided is a method for enhanced expression of a recombinant protein of interest in a microbial host cell, comprising providing a microbial host cell wherein the function of

  17. Expression of green fluorescent protein (GFPuv) in Escherichia coli ...

    African Journals Online (AJOL)

    Administrator

    The recombinant green fluorescent protein (GFPuv) was expressed by transformed cells of Escherichia coli DH5-α grown in LB/amp broth at 37oC, for 8 h and 24 h. To evaluate the effectiveness of different parameters to improve the expression of GFPuv by E. coli, four variable culturing conditions were set up for assays by ...

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. Variation in Protein Intake Induces Variation in Spider Silk Expression

    Science.gov (United States)

    Blamires, Sean J.; Wu, Chun-Lin; Tso, I-Min

    2012-01-01

    Background It is energetically expensive to synthesize certain amino acids. The proteins (spidroins) of spider major ampullate (MA) silk, MaSp1 and MaSp2, differ in amino acid composition. Glutamine and proline are prevalent in MaSp2 and are expensive to synthesize. Since most orb web spiders express high proline silk they might preferentially attain the amino acids needed for silk from food and shift toward expressing more MaSp1 in their MA silk when starved. Methodology/Principal Findings We fed three spiders; Argiope aetherea, Cyrtophora moluccensis and Leucauge blanda, high protein, low protein or no protein solutions. A. aetherea and L. blanda MA silks are high in proline, while C. moluccesnsis MA silks are low in proline. After 10 days of feeding we determined the amino acid compositions and mechanical properties of each species' MA silk and compared them between species and treatments with pre-treatment samples, accounting for ancestry. We found that the proline and glutamine of A. aetherea and L. blanda silks were affected by protein intake; significantly decreasing under the low and no protein intake treatments. Glutmaine composition in C. moluccensis silk was likewise affected by protein intake. However, the composition of proline in their MA silk was not significantly affected by protein intake. Conclusions Our results suggest that protein limitation induces a shift toward different silk proteins with lower glutamine and/or proline content. Contradictions to the MaSp model lie in the findings that C. moluccensis MA silks did not experience a significant reduction in proline and A. aetherea did not experience a significant reduction in serine on low/no protein. The mechanical properties of the silks could not be explained by a MaSp1 expressional shift. Factors other than MaSp expression, such as the expression of spidroin-like orthologues, may impact on silk amino acid composition and spinning and glandular processes may impact mechanics. PMID:22363691

  3. Influence of Isoflurane on Immediate-Early Gene Expression.

    Science.gov (United States)

    Bunting, Kristopher M; Nalloor, Rebecca I; Vazdarjanova, Almira

    2015-01-01

    Anterograde amnesia is a hallmark effect of volatile anesthetics. Isoflurane is known to affect both the translation and transcription of plasticity-associated genes required for normal memory formation in many brain regions. What is not known is whether isoflurane anesthesia prevents the initiation of transcription or whether it halts transcription already in progress. We tested the hypothesis that general anesthesia with isoflurane prevents learning-induced initiation of transcription of several memory-associated immediate-early genes (IEGs) correlated with amnesia; we also assessed whether it stops transcription initiated prior to anesthetic administration. Using a Tone Fear Conditioning paradigm, rats were trained to associate a tone with foot-shock. Animals received either no anesthesia, anesthesia immediately after training, or anesthesia before, during, and after training. Animals were either sacrificed after training or tested 24 h later for long-term memory. Using Cellular Compartment Analysis of Temporal Activity by Fluorescence in situ Hybridization (catFISH), we examined the percentage of neurons expressing the IEGs Arc/Arg3.1 and Zif268/Egr1/Ngfi-A/Krox-24 in the dorsal hippocampus, primary somatosensory cortex, and primary auditory cortex. On a cellular level, isoflurane administered at high doses (general anesthesia) prevented initiation of transcription, but did not stop transcription of Arc and Zif268 mRNA initiated prior to anesthesia. On a behavioral level, the same level of isoflurane anesthesia produced anterograde amnesia for fear conditioning when administered before and during training, but did not produce retrograde amnesia when administered immediately after training. General anesthesia with isoflurane prevents initiation of learning-related transcription but does not stop ongoing transcription of two plasticity-related IEGs, Arc and Zif268, a pattern of disruption that parallels the effects of isoflurane on memory formation. Combined with

  4. Influence of isoflurane on Immediate-Early Gene expression

    Directory of Open Access Journals (Sweden)

    Kristopher M Bunting

    2016-01-01

    Full Text Available Background: Anterograde amnesia is a hallmark effect of volatile anesthetics. Isoflurane is known to affect both the translation and transcription of plasticity-associated genes required for normal memory formation in many brain regions. What is not known is whether isoflurane anesthesia prevents the initiation of transcription or whether it halts transcription already in progress. We tested the hypothesis that general anesthesia with isoflurane prevents learning-induced initiation of transcription of several memory-associated immediate-early genes (IEGs correlated with amnesia; we also assessed whether it stops transcription initiated prior to anesthetic administration.Methods: Using a Tone Fear Conditioning paradigm, rats were trained to associate a tone with foot-shock. Animals received either no anesthesia, anesthesia immediately after training, or anesthesia before, during, and after training. Animals were either sacrificed after training or tested 24 hours later for memory. Using Cellular Compartment Analysis of Temporal Activity by Fluorescence in situ Hybridization (catFISH, we examined the percentage of neurons expressing the IEGs Arc/Arg3.1 and Zif268/Egr1/Ngfi-A/Krox-24 in the dorsal hippocampus, primary somatosensory cortex, and primary auditory cortex.Results: On a cellular level, isoflurane administered at high doses (general anesthesia prevented initiation of transcription, but did not stop transcription of Arc and Zif268 mRNA initiated prior to anesthesia. On a behavioral level, the same level of isoflurane anesthesia produced anterograde amnesia for fear conditioning when administered before and during training, but did not produce retrograde amnesia when administered immediately after training. Conclusions: General anesthesia with isoflurane prevents initiation of learning-related transcription but does not stop ongoing transcription of two plasticity-related IEGs, Arc and Zif268, a pattern of disruption that parallels the

  5. Differential Protein Expression in Congenital and Acquired Cholesteatomas.

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    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

  6. Neisseria meningitidis rifampicin resistant strains: analysis of protein differentially expressed

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    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

  7. Myocardin-related transcription factor regulates Nox4 protein expression

    DEFF Research Database (Denmark)

    Rozycki, Matthew; Bialik, Janne Folke; Speight, Pam

    2016-01-01

    translocation of MRTF. Because the Nox4 promoter harbors a serum response factor/MRTF cis-element (CC(A/T)6GG box), we asked if MRTF (and thus cytoskeleton organization) could regulate Nox4 expression. We show that Nox4 protein is robustly induced in kidney tubular cells exclusively by combined application...... TGFβ/contact disruption-provoked Nox4 protein and mRNA expression, Nox4 promoter activation, and reactive oxygen species production. Mutation of the CC(A/T)6GG box eliminates the synergistic activation of the Nox4 promoter. Jasplakinolide-induced actin polymerization synergizes with TGFβ to facilitate...... MRTF-dependent Nox4 mRNA expression/promoter activation. Moreover, MRTF inhibition prevents Nox4 expression during TGFβ-induced fibroblast-myofibroblast transition as well. Although necessary, MRTF is insufficient; Nox4 expression also requires TGFβ-activated Smad3 and TAZ/YAP, two contact...

  8. An early nodulin-like protein accumulates in the sieve element plasma membrane of Arabidopsis

    DEFF Research Database (Denmark)

    Khan, Junaid A.; Wang, Qi; Sjölund, Richard D.

    2007-01-01

    Membrane proteins within the sieve element-companion cell complex have essential roles in the physiological functioning of the phloem. The monoclonal antibody line RS6, selected from hybridomas raised against sieve elements isolated from California shield leaf (Streptanthus tortuosus; Brassicaceae...... was revealed by reverse transcription-PCR of Arabidopsis leaf RNA using degenerate primers to be an early nodulin (ENOD)-like protein that is encoded by the expressed gene At3g20570. Arabidopsis ENOD-like proteins are encoded by a multigene family composed of several types of structurally related phytocyanins...... from the precursor protein, resulting in a mature peptide of approximately 15 kD that is attached to the sieve element plasma membrane via a carboxy-terminal glycosylphosphatidylinositol membrane anchor. Many of the Arabidopsis ENOD-like proteins accumulate in gametophytic tissues, whereas in both...

  9. Discovery of Cellular Proteins Required for the Early Steps of HCV Infection Using Integrative Genomics

    Science.gov (United States)

    Yang, Jae-Seong; Kwon, Oh Sung; Kim, Sanguk; Jang, Sung Key

    2013-01-01

    Successful viral infection requires intimate communication between virus and host cell, a process that absolutely requires various host proteins. However, current efforts to discover novel host proteins as therapeutic targets for viral infection are difficult. Here, we developed an integrative-genomics approach to predict human genes involved in the early steps of hepatitis C virus (HCV) infection. By integrating HCV and human protein associations, co-expression data, and tight junction-tetraspanin web specific networks, we identified host proteins required for the early steps in HCV infection. Moreover, we validated the roles of newly identified proteins in HCV infection by knocking down their expression using small interfering RNAs. Specifically, a novel host factor CD63 was shown to directly interact with HCV E2 protein. We further demonstrated that an antibody against CD63 blocked HCV infection, indicating that CD63 may serve as a new therapeutic target for HCV-related diseases. The candidate gene list provides a source for identification of new therapeutic targets. PMID:23593195

  10. 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.

  11. 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.

  12. Early signatures of regime shifts in gene expression dynamics

    Science.gov (United States)

    Pal, Mainak; Pal, Amit Kumar; Ghosh, Sayantari; Bose, Indrani

    2013-06-01

    Recently, a large number of studies have been carried out on the early signatures of sudden regime shifts in systems as diverse as ecosystems, financial markets, population biology and complex diseases. The signatures of regime shifts in gene expression dynamics are less systematically investigated. In this paper, we consider sudden regime shifts in the gene expression dynamics described by a fold-bifurcation model involving bistability and hysteresis. We consider two alternative models, models 1 and 2, of competence development in the bacterial population B. subtilis and determine some early signatures of the regime shifts between competence and noncompetence. We use both deterministic and stochastic formalisms for the purpose of our study. The early signatures studied include the critical slowing down as a transition point is approached, rising variance and the lag-1 autocorrelation function, skewness and a ratio of two mean first passage times. Some of the signatures could provide the experimental basis for distinguishing between bistability and excitability as the correct mechanism for the development of competence.

  13. Early signatures of regime shifts in gene expression dynamics

    International Nuclear Information System (INIS)

    Pal, Mainak; Pal, Amit Kumar; Ghosh, Sayantari; Bose, Indrani

    2013-01-01

    Recently, a large number of studies have been carried out on the early signatures of sudden regime shifts in systems as diverse as ecosystems, financial markets, population biology and complex diseases. The signatures of regime shifts in gene expression dynamics are less systematically investigated. In this paper, we consider sudden regime shifts in the gene expression dynamics described by a fold-bifurcation model involving bistability and hysteresis. We consider two alternative models, models 1 and 2, of competence development in the bacterial population B. subtilis and determine some early signatures of the regime shifts between competence and noncompetence. We use both deterministic and stochastic formalisms for the purpose of our study. The early signatures studied include the critical slowing down as a transition point is approached, rising variance and the lag-1 autocorrelation function, skewness and a ratio of two mean first passage times. Some of the signatures could provide the experimental basis for distinguishing between bistability and excitability as the correct mechanism for the development of competence. (paper)

  14. BAX protein expression and clinical outcome in epithelial ovarian cancer.

    Science.gov (United States)

    Tai, Y T; Lee, S; Niloff, E; Weisman, C; Strobel, T; Cannistra, S A

    1998-08-01

    Expression of the pro-apoptotic protein BAX sensitizes ovarian cancer cell lines to paclitaxel in vitro by enhancing the pathway of programmed cell death. The present study was performed to determine the relationship between BAX expression and clinical outcome in 45 patients with newly diagnosed ovarian cancer. BAX protein expression was analyzed by immunohistochemistry, and its relationship with clinical outcome was determined. Assessment of BAX mRNA transcript levels and mutational analysis of the BAX coding region were also performed. BAX protein was expressed at high levels (defined as > or = 50% of tumor cells positive) in tumor tissue from 60% of newly diagnosed patients. All patients whose tumors expressed high levels of BAX achieved a complete response (CR) to first-line chemotherapy that contained paclitaxel plus a platinum analogue, compared with 57% of patients in the low-BAX group (P = .036). After a median follow-up of 1.9 years, the median disease-free survival (DFS) of patients in the high-BAX group has not been reached, compared with a median DFS of 1.1 years for low-BAX expressors (P = .0061). BAX retained independent prognostic significance in multivariate analysis when corrected for stage and histology. BAX mRNA transcripts were easily detected in samples with low BAX protein expression, and no BAX mutations were identified. The correlation between high BAX levels and improved clinical outcome suggests that an intact apoptotic pathway is an important determinant of chemoresponsiveness in ovarian cancer patients who receive paclitaxel.

  15. DPEP1, expressed in the early stages of colon carcinogenesis, affects cancer cell invasiveness.

    Science.gov (United States)

    Toiyama, Yuji; Inoue, Yasuhiro; Yasuda, Hiromi; Saigusa, Susumu; Yokoe, Takeshi; Okugawa, Yoshinaga; Tanaka, Koji; Miki, Chikao; Kusunoki, Masato

    2011-02-01

    We investigated changes in the gene expression profile in colon cancer in order to identify gene markers that may be useful in the management of this disease. The Cancer Genome Anatomy Project was used to detect differences in gene expression between normal and cancer tissue. The overexpression of dipeptidase-1 (DPEP1) in cancer tissue was confirmed in a sample of 76 patients by real-time PCR. To identify the function of DPEP1, RNA interference (RNAi) was used to inactivate this gene in the colon cancer cell line. Immunohistochemical analysis was performed to characterize the pattern of DPEP1 expression in colon cancer. DPEP1 expression in cancer was significantly higher than that in normal tissue. However, DPEP1 expression decreased with pathological differentiation, lymph-node and distant metastasis. Patients with tumors with decreased DPEP1 expression showed a poorer prognosis, and this was also true of patients with tumors who are treated with curative intent. RNAi-mediated DPEP1 reduction in the colon cancer cell line did not result in cell proliferation or apoptosis, but was associated with an increased invasive ability. DPEP1 protein was observed on the apical side of the cancer cells, and is expressed in the early stages of carcinogenesis, even in adenomas of both sporadic colorectal cancer and familial adenomatous polyposis patients. DPEP1 expression in normal colonic mucosa is very low, but it is highly expressed in colorectal adenoma and cancer specimens and is negatively correlated with parameters of pathological aggressiveness and poor prognosis. DPEP1 is expressed in the early stages of colon carcinogenesis and affects cancer cell invasiveness.

  16. Expression of immediate-early genes in the dorsal cochlear nucleus in salicylate-induced tinnitus.

    Science.gov (United States)

    Hu, Shou-Sen; Mei, Ling; Chen, Jian-Yong; Huang, Zhi-Wu; Wu, Hao

    2016-02-01

    Spontaneous neuronal activity in dorsal cochlear nucleus (DCN) may be involved in the physiological processes underlying salicylate-induced tinnitus. As a neuronal activity marker, immediate-early gene (IEG) expression, especially activity-dependent cytoskeletal protein (Arc/Arg3.1) and the early growth response gene-1 (Egr-1), appears to be highly correlated with sensory-evoked neuronal activity. However, their relationships with tinnitus induced by salicylate have rarely been reported in the DCN. In this study, we assessed the effect of acute and chronic salicylate treatment on the expression of N-methyl D-aspartate receptor subunit 2B (NR2B), Arg3.1, and Egr-1. We also observed ultrastructural alterations in the DCN synapses in an animal model of tinnitus. Levels of mRNA and protein expression of NR2B and Arg3.1 were increased in rats that were chronically administered salicylate (200 mg/kg, twice daily for 3, 7, or 14 days). These levels returned to baseline 14 days after cessation of treatment. However, no significant changes were observed in Egr-1 gene expression in any groups. Furthermore, rats subjected to long-term salicylate administration showed more presynaptic vesicles, thicker and longer postsynaptic densities, and increased synaptic interface curvature. Alterations of Arg3.1 and NR2B may be responsible for the changes in the synaptic ultrastructure. These changes confirm that salicylate can cause neural plasticity changes at the DCN level.

  17. Abnormal expression of leiomyoma cytoskeletal proteins involved in cell migration.

    Science.gov (United States)

    Ura, Blendi; Scrimin, Federica; Arrigoni, Giorgio; Athanasakis, Emmanouil; Aloisio, Michelangelo; Monasta, Lorenzo; Ricci, Giuseppe

    2016-05-01

    Uterine leiomyomas are monoclonal tumors. Several factors are involved in the neoplastic transformation of the myometrium. In our study we focused on dysregulated cytoskeletal proteins in the leiomyoma as compared to the myometrium. Paired tissue samples of ten leiomyomas and adjacent myometria were obtained and analyzed by two‑dimensional gel electrophoresis (2-DE). Mass spectrometry was used for protein identification, and western blotting for 2-DE data validation. The values of ten cytoskeletal proteins were found to be significantly different: eight proteins were upregulated in the leiomyoma and two proteins were downregulated. Three of the upregulated proteins (myosin regulatory light polypeptide 9, four and a half LIM domains protein 1 and LIM and SH3 domain protein 1) are involved in cell migration, while downregulated protein transgelin is involved in replicative senescence. Myosin regulatory light polypeptide 9 (MYL9) was further validated by western blotting because it is considered to be a cell migration marker in several cancers and could play a key role in leiomyoma development. Our data demonstrate significant alterations in the expression of cytoskeletal proteins involved in leiomyoma growth. A better understanding of the involvement of cytoskeletal proteins in leiomyoma pathogenesis may contribute to the identification of new therapeutic targets and the development of new pharmacological approaches.

  18. EBV tegument protein BNRF1 disrupts DAXX-ATRX to activate viral early gene transcription.

    Directory of Open Access Journals (Sweden)

    Kevin Tsai

    2011-11-01

    Full Text Available Productive infection by herpesviruses involve the disabling of host-cell intrinsic defenses by viral encoded tegument proteins. Epstein-Barr Virus (EBV typically establishes a non-productive, latent infection and it remains unclear how it confronts the host-cell intrinsic defenses that restrict viral gene expression. Here, we show that the EBV major tegument protein BNRF1 targets host-cell intrinsic defense proteins and promotes viral early gene activation. Specifically, we demonstrate that BNRF1 interacts with the host nuclear protein Daxx at PML nuclear bodies (PML-NBs and disrupts the formation of the Daxx-ATRX chromatin remodeling complex. We mapped the Daxx interaction domain on BNRF1, and show that this domain is important for supporting EBV primary infection. Through reverse transcription PCR and infection assays, we show that BNRF1 supports viral gene expression upon early infection, and that this function is dependent on the Daxx-interaction domain. Lastly, we show that knockdown of Daxx and ATRX induces reactivation of EBV from latently infected lymphoblastoid cell lines (LCLs, suggesting that Daxx and ATRX play a role in the regulation of viral chromatin. Taken together, our data demonstrate an important role of BNRF1 in supporting EBV early infection by interacting with Daxx and ATRX; and suggest that tegument disruption of PML-NB-associated antiviral resistances is a universal requirement for herpesvirus infection in the nucleus.

  19. EBV Tegument Protein BNRF1 Disrupts DAXX-ATRX to Activate Viral Early Gene Transcription

    Science.gov (United States)

    Tsai, Kevin; Thikmyanova, Nadezhda; Wojcechowskyj, Jason A.; Delecluse, Henri-Jacques; Lieberman, Paul M.

    2011-01-01

    Productive infection by herpesviruses involve the disabling of host-cell intrinsic defenses by viral encoded tegument proteins. Epstein-Barr Virus (EBV) typically establishes a non-productive, latent infection and it remains unclear how it confronts the host-cell intrinsic defenses that restrict viral gene expression. Here, we show that the EBV major tegument protein BNRF1 targets host-cell intrinsic defense proteins and promotes viral early gene activation. Specifically, we demonstrate that BNRF1 interacts with the host nuclear protein Daxx at PML nuclear bodies (PML-NBs) and disrupts the formation of the Daxx-ATRX chromatin remodeling complex. We mapped the Daxx interaction domain on BNRF1, and show that this domain is important for supporting EBV primary infection. Through reverse transcription PCR and infection assays, we show that BNRF1 supports viral gene expression upon early infection, and that this function is dependent on the Daxx-interaction domain. Lastly, we show that knockdown of Daxx and ATRX induces reactivation of EBV from latently infected lymphoblastoid cell lines (LCLs), suggesting that Daxx and ATRX play a role in the regulation of viral chromatin. Taken together, our data demonstrate an important role of BNRF1 in supporting EBV early infection by interacting with Daxx and ATRX; and suggest that tegument disruption of PML-NB-associated antiviral resistances is a universal requirement for herpesvirus infection in the nucleus. PMID:22102817

  20. Differential neutrophil gene expression in early bovine pregnancy

    Directory of Open Access Journals (Sweden)

    Kizaki Keiichiro

    2013-02-01

    Full Text Available Abstract Background In food production animals, especially cattle, the diagnosis of gestation is important because the timing of gestation directly affects the running of farms. Various methods have been used to detect gestation, but none of them are ideal because of problems with the timing of detection or the accuracy, simplicity, or cost of the method. A new method for detecting gestation, which involves assessing interferon-tau (IFNT-stimulated gene expression in peripheral blood leukocytes (PBL, was recently proposed. PBL fractionation methods were used to examine whether the expression profiles of various PBL populations could be used as reliable diagnostic markers of bovine gestation. Methods PBL were collected on days 0 (just before artificial insemination, 7, 14, 17, 21, and 28 of gestation. The gene expression levels of the PBL were assessed with microarray analysis and/or quantitative real-time reverse transcription (q PCR. PBL fractions were collected by flow cytometry or density gradient cell separation using Histopaque 1083 or Ficoll-Conray solutions. The expression levels of four IFNT-stimulated genes, interferon-stimulated protein 15 kDa (ISG15, myxovirus-resistance (MX 1 and 2, and 2′-5′-oligoadenylate synthetase (OAS1, were then analyzed in each fraction through day 28 of gestation using qPCR. Results Microarray analysis detected 72 and 28 genes in whole PBL that were significantly higher on days 14 and 21 of gestation, respectively, than on day 0. The upregulated genes included IFNT-stimulated genes. The expression levels of these genes increased with the progression of gestation until day 21. In flow cytometry experiments, on day 14 the expression levels of all of the genes were significantly higher in the granulocyte fraction than in the other fractions. Their expression gradually decreased through day 28 of gestation. Strong correlations were observed between the expression levels of the four genes in the granulocyte

  1. 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

  2. Regenerating human muscle fibres express GLUT3 protein

    DEFF Research Database (Denmark)

    Gaster, M; Beck-Nielsen, H; Schrøder, H D

    2002-01-01

    The presence of the GLUT3 glucose transporter protein in human muscle cells is a matter of debate. The present study was designed to establish whether GLUT3 is expressed in mature human skeletal muscle fibres and, if so, whether its expression changes under different conditions, such as metabolic...... muscle fibres, nor did metabolic stress, training or de- and re-innervation induce GLUT3 expression, while a few GLUT3 expressing fibres were seen in some cases of polymyositis. In contrast, GLUT4 was expressed in all investigated muscle fibres. GLUT3 immunoreactivity was found in perineural...... and endoneural cells, indicating that GLUT3 is important for glucose transport into nerves through the perineurium. Taken together, these data suggest that GLUT3 expression is restricted to regenerating muscle fibres and nerves in adult human muscle. Although the significance of GLUT3 in adult human muscle...

  3. 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

  4. Studies to Prevent Degradation of Recombinant Fc-Fusion Protein Expressed in Mammalian Cell Line and Protein Characterization

    Directory of Open Access Journals (Sweden)

    Sanjukta Chakrabarti

    2016-06-01

    Full Text Available Clipping of recombinant proteins is a major issue in animal cell cultures. A recombinant Fc-fusion protein, VEGFR1(D1–D3-Fc expressed in CHOK1SV GS-KO cells was observed to be undergoing clippings in lab scale cultures. Partial cleaving of expressed protein initiated early on in cell culture and was observed to increase over time in culture and also on storage. In this study, a few parameters were explored in a bid to inhibit clipping in the fusion protein The effects of culture temperature, duration of culture, the addition of an anti-clumping agent, ferric citrate and use of protease inhibitor cocktail on inhibition of proteolysis of the Fc fusion were studied. Lowering of culture temperature from 37 to 30 °C alone appears to be the best solution for reducing protein degradation from the quality, cost and regulatory points of view. The obtained Fc protein was characterized and found to be in its stable folded state, exhibiting a high affinity for its ligand and also biological and functional activities.

  5. Kiss-1/GPR54 protein expression in breast cancer.

    Science.gov (United States)

    Papaoiconomou, Eleni; Lymperi, Maria; Petraki, Constantina; Philippou, Anastassios; Msaouel, Pavlos; Michalopoulou, Fani; Kafiri, Georgia; Vassilakos, George; Zografos, Georgios; Koutsilieris, Michael

    2014-03-01

    Numerous studies have shown that the Kiss-1 gene countervails the metastatic aptitude of several cancer cell lines and solid-tumor neoplasias. However, there still remains ambiguity regarding its role in breast cancer and literature has arisen asserting that Kiss-1 expression may be linked to an aggressive phenotype and malignant progression. Herein, we investigated the protein expression of Kiss-1 and its receptor GPR54 in breast cancer tissues compared to non-cancerous mammary tissues. Paraffin-fixed cancer tissues from 43 women with resected breast adenocarcinomas and 11 specimens derived from women suffering from fibrocystic disease, serving as controls, were immunostained with Kiss-1 and GPR54 antibodies. Kiss-1 and GPR54 protein expression levels were significantly higher in breast cancer compared to fibrocystic tissues (pbreast cancer and fibrocystic disease specimens. Kiss-1/GPR54 expression was found to be significantly higher in breast cancer compared to non-malignant mammary tissues.

  6. Transcription Factor EB Expression in Early Breast Cancer Relates to Lysosomal/Autophagosomal Markers and Prognosis.

    Science.gov (United States)

    Giatromanolaki, Alexandra; Sivridis, Efthimios; Kalamida, Dimitra; Koukourakis, Michael I

    2017-06-01

    Disrupting the autophagic balance to trigger autophagic death may open new strategies for cancer therapy. Transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and may play a role in cancer biology and clinical behavior. The expression of TFEB and the lysosomal cancer cell content (expression of lysosomal associated membrane protein 2a [LAMP2a] and cathepsin D) was studied in a series of 100 T1-stage breast carcinomas. Expression patterns were correlated with autophagy/hypoxia-related proteins, angiogenesis, and clinical outcome. The effect of hypoxic/acidic conditions on TFEB kinetics was studied in the MCF-7 cancer cell line. Overexpression of TFEB in cancer cell cytoplasm and the perinuclear/nuclear area was noted in 23 (23%) of 100 cases. High LAMP2a and cathepsin D expression was noted in 30 (30%) of 100 and 28 (28%) of 100 cases, respectively. TFEB expression was directly linked with LAMP2a (P factor 2-alpha (HIF-2α) (P = .01, r = 0.25) expression and inversely with progesterone receptor (P = .01, r = 0.22). High vascular density was directly linked with LAMP2a (P = .05, r = 0.18) and cathepsin D (P = .005, r = 0.28). In Kaplan-Meier survival analysis, TFEB and cathepsin D expression were related to an ominous prognosis (P = .001 and P = .03, respectively). In multivariate analysis, TFEB expression sustained its independent prognostic significance (P = .05, hazard ratio 2.1). In in vitro experiments, acidity triggered overexpression of TFEB and nuclear translocation. Intense TFEB expression and lysosomal biogenesis, evident in one fourth of early breast carcinomas, define poor prognosis. Tumor acidity is among the microenvironmental conditions that trigger TFEB overactivity. TFEB is a sound target for the development of lysosomal targeting therapies. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. 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

  8. SGLT2 Protein Expression Is Increased in Human Diabetic Nephropathy

    Science.gov (United States)

    Wang, Xiaoxin X.; Levi, Jonathan; Luo, Yuhuan; Myakala, Komuraiah; Herman-Edelstein, Michal; Qiu, Liru; Wang, Dong; Peng, Yingqiong; Grenz, Almut; Lucia, Scott; Dobrinskikh, Evgenia; D'Agati, Vivette D.; Koepsell, Hermann; Kopp, Jeffrey B.; Rosenberg, Avi Z.; Levi, Moshe

    2017-01-01

    There is very limited human renal sodium gradient-dependent glucose transporter protein (SGLT2) mRNA and protein expression data reported in the literature. The first aim of this study was to determine SGLT2 mRNA and protein levels in human and animal models of diabetic nephropathy. We have found that the expression of SGLT2 mRNA and protein is increased in renal biopsies from human subjects with diabetic nephropathy. This is in contrast to db-db mice that had no changes in renal SGLT2 protein expression. Furthermore, the effect of SGLT2 inhibition on renal lipid content and inflammation is not known. The second aim of this study was to determine the potential mechanisms of beneficial effects of SGLT2 inhibition in the progression of diabetic renal disease. We treated db/db mice with a selective SGLT2 inhibitor JNJ 39933673. We found that SGLT2 inhibition caused marked decreases in systolic blood pressure, kidney weight/body weight ratio, urinary albumin, and urinary thiobarbituric acid-reacting substances. SGLT2 inhibition prevented renal lipid accumulation via inhibition of carbohydrate-responsive element-binding protein-β, pyruvate kinase L, SCD-1, and DGAT1, key transcriptional factors and enzymes that mediate fatty acid and triglyceride synthesis. SGLT2 inhibition also prevented inflammation via inhibition of CD68 macrophage accumulation and expression of p65, TLR4, MCP-1, and osteopontin. These effects were associated with reduced mesangial expansion, accumulation of the extracellular matrix proteins fibronectin and type IV collagen, and loss of podocyte markers WT1 and synaptopodin, as determined by immunofluorescence microscopy. In summary, our study showed that SGLT2 inhibition modulates renal lipid metabolism and inflammation and prevents the development of nephropathy in db/db mice. PMID:28196866

  9. Gene expression profiles reveal key genes for early diagnosis and treatment of adamantinomatous craniopharyngioma.

    Science.gov (United States)

    Yang, Jun; Hou, Ziming; Wang, Changjiang; Wang, Hao; Zhang, Hongbing

    2018-04-23

    Adamantinomatous craniopharyngioma (ACP) is an aggressive brain tumor that occurs predominantly in the pediatric population. Conventional diagnosis method and standard therapy cannot treat ACPs effectively. In this paper, we aimed to identify key genes for ACP early diagnosis and treatment. Datasets GSE94349 and GSE68015 were obtained from Gene Expression Omnibus database. Consensus clustering was applied to discover the gene clusters in the expression data of GSE94349 and functional enrichment analysis was performed on gene set in each cluster. The protein-protein interaction (PPI) network was built by the Search Tool for the Retrieval of Interacting Genes, and hubs were selected. Support vector machine (SVM) model was built based on the signature genes identified from enrichment analysis and PPI network. Dataset GSE94349 was used for training and testing, and GSE68015 was used for validation. Besides, RT-qPCR analysis was performed to analyze the expression of signature genes in ACP samples compared with normal controls. Seven gene clusters were discovered in the differentially expressed genes identified from GSE94349 dataset. Enrichment analysis of each cluster identified 25 pathways that highly associated with ACP. PPI network was built and 46 hubs were determined. Twenty-five pathway-related genes that overlapped with the hubs in PPI network were used as signatures to establish the SVM diagnosis model for ACP. The prediction accuracy of SVM model for training, testing, and validation data were 94, 85, and 74%, respectively. The expression of CDH1, CCL2, ITGA2, COL8A1, COL6A2, and COL6A3 were significantly upregulated in ACP tumor samples, while CAMK2A, RIMS1, NEFL, SYT1, and STX1A were significantly downregulated, which were consistent with the differentially expressed gene analysis. SVM model is a promising classification tool for screening and early diagnosis of ACP. The ACP-related pathways and signature genes will advance our knowledge of ACP pathogenesis

  10. 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

    positive expression rate was 70.0 % in normal cervix, 73.3 % in CIN and 60.0 % in CSCC tissues, without significant difference among them (P = 0.758). PKM2 was mainly expressed in the cell nuclei, and its positive expression rate was 100.0 % in normal cervix, 93.3 % in CIN and 75.0 % in CSCC tissues, showing a difference close to statistical significance (P = 0.059) among them. There are differentially expressed proteins among normal cervix, CIN and CSCC. S100A9, eEF1A1 and PKM2 may become candidate markers for early diagnosis of cervical cancer and new targets for therapy. It also provides a basis for further studies of the mechanism for CIN developing to CSCC.

  11. Different Cells Make Different Proteins: A Laboratory Exercise Illustrating Tissue-Specific Protein Expression in Animals

    Science.gov (United States)

    Ibarguren, Izaskun; Villamarín, Antonio

    2017-01-01

    All the cells of higher organisms have the same DNA but not the same proteins. Each type of specialised cell that forms a tissue has its own pattern of gene expression and, consequently, it contains a particular set of proteins that determine its function. Here, we describe a laboratory exercise addressed to undergraduate students that aims to…

  12. 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

  13. 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.

  14. Cloning and expression analysis of a blue copperbinding protein ...

    African Journals Online (AJOL)

    Adifferentially expressed fragment EST145 was isolated by suppression subtractive hybridization (SSH) method. Using EST145 as the probe, a blue copper-binding protein gene designated as DvBCB was screened from Dasypyrum villosum cDNA Library. The DvBCB gene was 845 bp in length with an open reading frame ...

  15. Lipid transfer proteins from fruit: cloning, expression and quantification

    NARCIS (Netherlands)

    Zuidmeer, Laurian; van Leeuwen, W. Astrid; Budde, Ilona Kleine; Cornelissen, Jessica; Bulder, Ingrid; Rafalska, Ilona; Besolí, Noèlia Telléz; Akkerdaas, Jaap H.; Asero, Riccardo; Fernandez Rivas, Montserrat; Rivas, Montserrat Fernandez; Gonzalez Mancebo, Eloina; Mancebo, Eloina Gonzalez; van Ree, Ronald

    2005-01-01

    BACKGROUND: Lipid transfer proteins (LTP) are stable, potentially life-threatening allergens in fruits and many other vegetable foods. The aim of this study was to clone and express recombinant apple LTP (Mal d 3), as has previously been done for peach LTP (Pru p 3) and set up quantitative tests for

  16. Pre- and early-postnatal nutrition modify gene and protein expressions of muscle energy metabolism markers and phospholipid fatty acid composition in a muscle type specific manner in sheep

    DEFF Research Database (Denmark)

    Hou, Lei; Kongsted, Alice; Ghoreishi, S. M.

    2013-01-01

    We previously reported that undernutrition in late fetal life reduced whole-body insulin sensitivity in adult sheep, irrespective of dietary exposure in early postnatal life. Skeletal muscle may play an important role in control of insulin action. We therefore studied a range of putative key musc......, nutrition had long-term consequences for a number of determinants of insulin action and metabolism in LD. Tissues other than muscle may account for reduced whole body insulin sensitivity in adult LOW sheep....

  17. 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.

  18. Optimization of translation profiles enhances protein expression and solubility.

    Directory of Open Access Journals (Sweden)

    Anne-Katrin Hess

    Full Text Available mRNA is translated with a non-uniform speed that actively coordinates co-translational folding of protein domains. Using structure-based homology we identified the structural domains in epoxide hydrolases (EHs and introduced slow-translating codons to delineate the translation of single domains. These changes in translation speed dramatically improved the solubility of two EHs of metagenomic origin in Escherichia coli. Conversely, the importance of transient attenuation for the folding, and consequently solubility, of EH was evidenced with a member of the EH family from Agrobacterium radiobacter, which partitions in the soluble fraction when expressed in E. coli. Synonymous substitutions of codons shaping the slow-transiting regions to fast-translating codons render this protein insoluble. Furthermore, we show that low protein yield can be enhanced by decreasing the free folding energy of the initial 5'-coding region, which can disrupt mRNA secondary structure and enhance ribosomal loading. This study provides direct experimental evidence that mRNA is not a mere messenger for translation of codons into amino acids but bears an additional layer of information for folding, solubility and expression level of the encoded protein. Furthermore, it provides a general frame on how to modulate and fine-tune gene expression of a target protein.

  19. 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

  20. The Origin and Early Evolution of Membrane Proteins

    Science.gov (United States)

    Pohorille, Andrew; Schweighofter, Karl; Wilson, Michael A.

    2006-01-01

    The origin and early evolution of membrane proteins, and in particular ion channels, are considered from the point of view that the transmembrane segments of membrane proteins are structurally quite simple and do not require specific sequences to fold. We argue that the transport of solute species, especially ions, required an early evolution of efficient transport mechanisms, and that the emergence of simple ion channels was protobiologically plausible. We also argue that, despite their simple structure, such channels could possess properties that, at the first sight, appear to require markedly larger complexity. These properties can be subtly modulated by local modifications to the sequence rather than global changes in molecular architecture. In order to address the evolution and development of ion channels, we focus on identifying those protein domains that are commonly associated with ion channel proteins and are conserved throughout the three main domains of life (Eukarya, Prokarya, and Archaea). We discuss the potassium-sodium-calcium superfamily of voltage-gated ion channels, mechanosensitive channels, porins, and ABC-transporters and argue that these families of membrane channels have sufficiently universal architectures that they can readily adapt to the diverse functional demands arising during evolution.

  1. Combined protein construct and synthetic gene engineering for heterologous protein expression and crystallization using Gene Composer

    Directory of Open Access Journals (Sweden)

    Walchli John

    2009-04-01

    Full Text Available Abstract Background With the goal of improving yield and success rates of heterologous protein production for structural studies we have developed the database and algorithm software package Gene Composer. This freely available electronic tool facilitates the information-rich design of protein constructs and their engineered synthetic gene sequences, as detailed in the accompanying manuscript. Results In this report, we compare heterologous protein expression levels from native sequences to that of codon engineered synthetic gene constructs designed by Gene Composer. A test set of proteins including a human kinase (P38α, viral polymerase (HCV NS5B, and bacterial structural protein (FtsZ were expressed in both E. coli and a cell-free wheat germ translation system. We also compare the protein expression levels in E. coli for a set of 11 different proteins with greatly varied G:C content and codon bias. Conclusion The results consistently demonstrate that protein yields from codon engineered Gene Composer designs are as good as or better than those achieved from the synonymous native genes. Moreover, structure guided N- and C-terminal deletion constructs designed with the aid of Gene Composer can lead to greater success in gene to structure work as exemplified by the X-ray crystallographic structure determination of FtsZ from Bacillus subtilis. These results validate the Gene Composer algorithms, and suggest that using a combination of synthetic gene and protein construct engineering tools can improve the economics of gene to structure research.

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

    Directory of Open Access Journals (Sweden)

    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.

  3. Dietary protein intake and quality in early life

    DEFF Research Database (Denmark)

    Lind, Mads V; Larnkjær, Anni; Mølgaard, Christian

    2017-01-01

    programming. Finally, infants with catch-up growth or specific genotypes might be particularly vulnerable to high-protein intake. SUMMARY: Recent studies confirm the associations between high-protein intake during the first 2 years and later obesity. Furthermore, knowledge of the mechanisms involved......PURPOSE OF REVIEW: Obesity is an increasing problem and high-protein intake early in life seems to increase later risk of obesity. This review summarizes recent publications in the area including observational and intervention studies and publications on underlying mechanisms. RECENT FINDINGS...... seems to have an effect on obesity. Specific amino acids, such as leucine, have also been implicated in increasing later obesity risk maybe via specific actions on insulin-like growth factor I. Furthermore, additional underlying mechanisms including epigenetics have been linked to long-term obesogenic...

  4. Altered protein expression in serum from endometrial hyperplasia and carcinoma patients

    Directory of Open Access Journals (Sweden)

    Cong Qing

    2011-04-01

    Full Text Available Abstract Background Endometrial carcinoma is one of the most common gynecological malignancies in women. The diagnosis of the disease at early or premalignant stages is crucial for the patient's prognosis. To date, diagnosis and follow-up of endometrial carcinoma and hyperplasia require invasive procedures. Therefore, there is considerable demand for the identification of biomarkers to allow non-invasive detection of these conditions. Methods In this study, we performed a quantitative proteomics analysis on serum samples from simple endometrial hyperplasia, complex endometrial hyperplasia, atypical endometrial hyperplasia, and endometrial carcinoma patients, as well as healthy women. Serum samples were first depleted of high-abundance proteins, labeled with isobaric tags (iTRAQ™, and then analyzed via two-dimensional liquid chromatography and tandem mass spectrometry. Protein identification and quantitation information were acquired by comparing the mass spectrometry data against the International Protein Index Database using ProteinPilot software. Bioinformatics annotation of identified proteins was performed by searching against the PANTHER database. Results In total, 74 proteins were identified and quantified in serum samples from endometrial lesion patients and healthy women. Using a 1.6-fold change as the benchmark, 12 proteins showed significantly altered expression levels in at least one disease group compared with healthy women. Among them, 7 proteins were found, for the first time, to be differentially expressed in atypical endometrial hyperplasia. These proteins are orosomucoid 1, haptoglobin, SERPINC 1, alpha-1-antichymotrypsin, apolipoprotein A-IV, inter-alpha-trypsin inhibitor heavy chain H4, and histidine-rich glycoprotein. Conclusions The differentially expressed proteins we discovered in this study may serve as biomarkers in the diagnosis and follow-up of endometrial hyperplasia and endometrial carcinoma.

  5. Serum-dependent expression of promyelocytic leukemia protein suppresses propagation of influenza virus

    International Nuclear Information System (INIS)

    Iki, Shigeo; Yokota, Shin-ichi; Okabayashi, Tamaki; Yokosawa, Noriko; Nagata, Kyosuke; Fujii, Nobuhiro

    2005-01-01

    The rate of propagation of influenza virus in human adenocarcinoma Caco-2 cells was found to negatively correlate with the concentration of fetal bovine serum (FBS) in the culture medium. Virus replicated more rapidly at lower FBS concentrations (0 or 2%) than at higher concentrations (10 or 20%) during an early stage of infection. Basal and interferon (IFN)-induced levels of typical IFN-inducible anti-viral proteins, such as 2',5'-oligoadenylate synthetase, dsRNA-activated protein kinase and MxA, were unaffected by variation in FBS concentrations. But promyelocytic leukemia protein (PML) was expressed in a serum-dependent manner. In particular, the 65 to 70 kDa isoform of PML was markedly upregulated following the addition of serum. In contrast, other isoforms were induced by IFN treatment, and weakly induced by FBS concentrations. Immunofluorescence microscopy indicated that PML was mainly formed nuclear bodies in Caco-2 cells at various FBS concentrations, and the levels of the PML-nuclear bodies were upregulated by FBS. Overexpression of PML isoform consisting of 560 or 633 amino acid residues by transfection of expression plasmid results in significantly delayed viral replication rate in Caco-2 cells. On the other hand, downregulation of PML expression by RNAi enhanced viral replication. These results indicate that PML isoforms which are expressed in a serum-dependent manner suppress the propagation of influenza virus at an early stage of infection

  6. 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

  7. Roles of HMGA proteins in cancer: Expression, pathways, and redundancies

    Directory of Open Access Journals (Sweden)

    Giancotti V

    2016-10-01

    Full Text Available The expression of the High Mobility Group A (HMGA proteins, their participation in cancer signalling pathways, and their redundant functions have been reviewed in seven types of cancer: breast, colorectal, prostate, lung, ovarian, thyroid, and brain. The analysis of cell lines and tumours revealed an elevated level of their expression in all fully transformed cancer systems, which represents a step of the main cancer signalling pathways. In breast, colorectal, prostate, and lung cancers Wnt/β-catenin pathway is a master inducer of cell transformation in which are deeply involved HMG A1 and A2 proteins. On the other hand, IL-6/Stat3 pathway is responsible for cancer transformation in breast, lung, and prostate. The expression of HMGA1 in lung and ovarian cancers is due to an active PI3K/Akt pathway. The let-7 family of microRNA represses the expression of HMGA showing specificity by its different forms: the let-7b form is able to inhibit both proteins A1 and A2, the last also inhibited by a, c, d, and g forms. Moreover, both proteins are down-regulated by the repressor couple p53/microRNA-34a. The protein A1 and A2 participate to the Epithelial-Mesenchymal Transition cooperating with the three couples of factors Twist1/2, Snai1/2, and Zeb1/2. Through a combination of pathways, there is the simultaneous presence of high levels of both A1 and A2 together with the expression of other factors: a high co-operating efficiency is reached that supplies the tumour cells with properties of self-renewal, resistance, and invasiveness.

  8. Early Gene Expression in Wounded Human Keratinocytes Revealed by DNA Microarray Analysis

    Directory of Open Access Journals (Sweden)

    Pascal Barbry

    2006-04-01

    Full Text Available Wound healing involves several steps: spreading of the cells, migration and proliferation. We have profiled gene expression during the early events of wound healing in normal human keratinocytes with a home-made DNA microarray containing about 1000 relevant human probes. An original wounding machine was used, that allows the wounding of up to 40% of the surface of a confluent monolayer of cultured cells grown on a Petri dish (compared with 5% with a classical ‘scratch’ method. The two aims of the present study were: (a to validate a limited number of genes by comparing the expression levels obtained with this technique with those found in the literature; (b to combine the use of the wounding machine with DNA microarray analysis for large-scale detection of the molecular events triggered during the early stages of the wound-healing process. The time-courses of RNA expression observed at 0.5, 1.5, 3, 6 and 15 h after wounding for genes such as c-Fos, c-Jun, Egr1, the plasminogen activator PLAU (uPA and the signal transducer and transcription activator STAT3, were consistent with previously published data. This suggests that our methodologies are able to perform quantitative measurement of gene expression. Transcripts encoding two zinc finger proteins, ZFP36 and ZNF161, and the tumour necrosis factor α-induced protein TNFAIP3, were also overexpressed after wounding. The role of the p38 mitogen-activated protein kinase (p38MAPK in wound healing was shown after the inhibition of p38 by SB203580, but our results also suggest the existence of surrogate activating pathways.

  9. Expression of uncoupling protein 1 in bovine muscle cells.

    Science.gov (United States)

    Abd Eldaim, M A; Hashimoto, O; Ohtsuki, H; Yamada, T; Murakami, M; Onda, K; Sato, R; Kanamori, Y; Qiao, Y; Tomonaga, S; Matsui, T; Funaba, M

    2016-12-01

    Uncoupling protein 1 (Ucp1) is predominantly expressed in brown/beige adipocytes in mammals. Although myogenic cells have been suggested to commit to a brown adipocyte lineage through the induction of Prdm16 expression, Prdm16 is also expressed in skeletal muscle. Thus, we examined expression of Ucp1 in bovine myogenic cells. Considering that Ucp1 is a principle molecule that induces energy expenditure in brown/beige adipocytes, expression of Ucp1 is not preferable in beef cattle because of potential decrease in energy (fattening) efficiency. The RT-PCR analyses revealed the expression of Ucp1 in the skeletal muscle of cattle; expression levels were markedly lower than those in the brown fat of calves. Immunohistochemical analyses showed that Ucp1 surrounded muscle fibers, but not adipocytes residing in skeletal muscle. Myosatellite cells cultured in myogenic medium showed an increase in the expression levels of myogenic regulatory factors ( levels were greater in cells after myogenic culture for 12 d than in those after myogenic culture for 6 d ( bovine skeletal muscle, which suggests the necessity for further studies on Ucp1-mediated energy expenditure in bovine skeletal muscle.

  10. Differential effects of simple repeating DNA sequences on gene expression from the SV40 early promoter.

    Science.gov (United States)

    Amirhaeri, S; Wohlrab, F; Wells, R D

    1995-02-17

    The influence of simple repeat sequences, cloned into different positions relative to the SV40 early promoter/enhancer, on the transient expression of the chloramphenicol acetyltransferase (CAT) gene was investigated. Insertion of (G)29.(C)29 in either orientation into the 5'-untranslated region of the CAT gene reduced expression in CV-1 cells 50-100 fold when compared with controls with random sequence inserts. Analysis of CAT-specific mRNA levels demonstrated that the effect was due to a reduction of CAT mRNA production rather than to posttranscriptional events. In contrast, insertion of the same insert in either orientation upstream of the promoter-enhancer or downstream of the gene stimulated gene expression 2-3-fold. These effects could be reversed by cotransfection of a competitor plasmid carrying (G)25.(C)25 sequences. The results suggest that a G.C-binding transcription factor modulates gene expression in this system and that promoter strength can be regulated by providing protein-binding sites in trans. Although constructs containing longer tracts of alternating (C-G), (T-G), or (A-T) sequences inhibited CAT expression when inserted in the 5'-untranslated region of the CAT gene, the amount of CAT mRNA was unaffected. Hence, these inhibitions must be due to posttranscriptional events, presumably at the level of translation. These effects of microsatellite sequences on gene expression are discussed with respect to recent data on related simple repeat sequences which cause several human genetic diseases.

  11. Regulation of protein synthesis during sea urchin early development

    International Nuclear Information System (INIS)

    Kelso, L.C.

    1989-01-01

    Fertilization of the sea urchin egg results in a 20-40 fold increase in the rate of protein synthesis. The masked message hypothesis proposes that mRNAs are masked or unavailable for translation in the egg. We devised an in vivo assay to test this hypothesis. Our results show that masked mRNAs limit protein synthesis in the unfertilized egg. In addition, we show that protein synthesis is also regulated at the level of translational machinery. Following fertilization is a period of rapid cell divisions. This period, known as the rapid cleavage stage, is characterized by the transient synthesis of a novel set of proteins. The synthesis of these proteins is programmed by maternal mRNAs stored in the unfertilized egg. To study the behavior of these mRNAs, we prepared a cDNA library from polysomal poly (A+) RNA from 2-hour embryos. [ 32 P] labeled probes, prepared from the cDNA library, were used to monitor the levels of individual mRNAs in polysomes at fertilization and during early development

  12. 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.

  13. Nociceptive DRG neurons express muscle lim protein upon axonal injury.

    Science.gov (United States)

    Levin, Evgeny; Andreadaki, Anastasia; Gobrecht, Philipp; Bosse, Frank; Fischer, Dietmar

    2017-04-04

    Muscle lim protein (MLP) has long been regarded as a cytosolic and nuclear muscular protein. Here, we show that MLP is also expressed in a subpopulation of adult rat dorsal root ganglia (DRG) neurons in response to axonal injury, while the protein was not detectable in naïve cells. Detailed immunohistochemical analysis of L4/L5 DRG revealed ~3% of MLP-positive neurons 2 days after complete sciatic nerve crush and maximum ~10% after 4-14 days. Similarly, in mixed cultures from cervical, thoracic, lumbar and sacral DRG ~6% of neurons were MLP-positive after 2 days and maximal 17% after 3 days. In both, histological sections and cell cultures, the protein was detected in the cytosol and axons of small diameter cells, while the nucleus remained devoid. Moreover, the vast majority could not be assigned to any of the well characterized canonical DRG subpopulations at 7 days after nerve injury. However, further analysis in cell culture revealed that the largest population of MLP expressing cells originated from non-peptidergic IB4-positive nociceptive neurons, which lose their ability to bind the lectin upon axotomy. Thus, MLP is mostly expressed in a subset of axotomized nociceptive neurons and can be used as a novel marker for this population of cells.

  14. Increase of tumor necrosis factor receptor 1 expression in women with unexplained early spontaneous abortion

    Institute of Scientific and Technical Information of China (English)

    YAN Chun-fang; YU Xue-wen; JIN Hui; LI Xu

    2004-01-01

    To investigate membrane tumor necrosis factor receptor 1 protein expression level in decidua andconcentration of soluble tumor necrosis factor receptor 1 in serum in women with unexplained early spontaneous abortion,threatened abortion, and compare the levels with healthy pregnant women. Methods: Thirty-seven women with unexplainedearly spontaneous abortion, 27 women with threatened abortion, and 34 healthy pregnant women undergoing artificial abortionof pregnancy at 6 - 10 weeks of gestation were selected. Decidual samples were collected when women were undergoing arti-ficial abortion, and blood samples were collected at the same time. The level of membrane tumor necrosis factor receptor 1 indecidua was detected by flow cytometer, and the concentration of soluble tumor necrosis factor receptor 1 in sera was mea-sured with an enzyme-linked immunosorbent assay. Results: The ercentages of membrane tumor necrosis factor receptor 1positive decidual cells were 16.42 ± 7.10 Mean ± SD for women with unexplained early spontaneous abortion and 13.14 ±6.30 for healthy pregnant women ( P < 0.05). Serum oncentration of soluble tumor necrosis factor receptor 1 was signifi-cantly higher in women with unexplained early spontaneous abortion than in healthy pregnant women and in women withthreatened abortion, and no difference was found between healthy pregnant women and women with threatened abortion.Conclusion: Women with unexplained early spontaneous abortion present significantly higher expression of tumor necrosisfactor receptor 1 than healthy pregnant women, suggesting that over-expression of tumor necrosis factor receptor 1 may cont-ribute to the development of early spontaneous abortion.

  15. Correlation of gene expression and protein production rate - a system wide study

    Directory of Open Access Journals (Sweden)

    Arvas Mikko

    2011-12-01

    Full Text Available Abstract Background Growth rate is a major determinant of intracellular function. However its effects can only be properly dissected with technically demanding chemostat cultivations in which it can be controlled. Recent work on Saccharomyces cerevisiae chemostat cultivations provided the first analysis on genome wide effects of growth rate. In this work we study the filamentous fungus Trichoderma reesei (Hypocrea jecorina that is an industrial protein production host known for its exceptional protein secretion capability. Interestingly, it exhibits a low growth rate protein production phenotype. Results We have used transcriptomics and proteomics to study the effect of growth rate and cell density on protein production in chemostat cultivations of T. reesei. Use of chemostat allowed control of growth rate and exact estimation of the extracellular specific protein production rate (SPPR. We find that major biosynthetic activities are all negatively correlated with SPPR. We also find that expression of many genes of secreted proteins and secondary metabolism, as well as various lineage specific, mostly unknown genes are positively correlated with SPPR. Finally, we enumerate possible regulators and regulatory mechanisms, arising from the data, for this response. Conclusions Based on these results it appears that in low growth rate protein production energy is very efficiently used primarly for protein production. Also, we propose that flux through early glycolysis or the TCA cycle is a more fundamental determining factor than growth rate for low growth rate protein production and we propose a novel eukaryotic response to this i.e. the lineage specific response (LSR.

  16. Influence of early postmortem protein oxidation on beef quality.

    Science.gov (United States)

    Rowe, L J; Maddock, K R; Lonergan, S M; Huff-Lonergan, E

    2004-03-01

    The objective of this study was to examine the effect of early postmortem protein oxidation on the color and tenderness of beef steaks. To obtain a range of oxidation levels, the longissimus lumborum muscles (LM) from both strip loins of 20 steers fed either a finishing diet with vitamin E (1,000 IU per steer daily, minimum of 126 d [VITE]; n = 10 steers) or fed the same finishing diet without vitamin E (CON; n = 10 steers) were used. Within 24 h after slaughter, the LM muscle from each carcass was cut into 2.54-cm-thick steaks and individually vacuum packaged. Steaks from each steer were assigned to a nonirradiated group or an irradiated group. Steaks were irradiated within 26 h postmortem, and were aged at 4 degrees C for 0, 1, 3, 7, and 14 d after irradiation. Steaks from each diet/irradiation/aging time treatment were used to determine color, shear force, and degree of protein oxidation (carbonyl content). Steaks from steers fed the VITE diet had higher (P irradiation, steaks that had been irradiated had lower (P Irradiated steaks, regardless of diet, had lower a* (P irradiated steaks compared to nonirradiated steaks at 0, 1, 3, and 7 d postirradiation. Immunoblot analysis showed that vitamin E supplementation decreased the number and extent of oxidized sarcoplasmic proteins. Protein carbonyl content was positively correlated with Warner-Bratzler shear force values. These results indicate that increased oxidation of muscle proteins early postmortem could have negative effects on fresh meat color and tenderness.

  17. Differentially expressed genes in iron-induced prion protein conversion

    International Nuclear Information System (INIS)

    Kim, Minsun; Kim, Eun-hee; Choi, Bo-Ran; Woo, Hee-Jong

    2016-01-01

    The conversion of the cellular prion protein (PrP C ) to the protease-resistant isoform is the key event in chronic neurodegenerative diseases, including transmissible spongiform encephalopathies (TSEs). Increased iron in prion-related disease has been observed due to the prion protein-ferritin complex. Additionally, the accumulation and conversion of recombinant PrP (rPrP) is specifically derived from Fe(III) but not Fe(II). Fe(III)-mediated PK-resistant PrP (PrP res ) conversion occurs within a complex cellular environment rather than via direct contact between rPrP and Fe(III). In this study, differentially expressed genes correlated with prion degeneration by Fe(III) were identified using Affymetrix microarrays. Following Fe(III) treatment, 97 genes were differentially expressed, including 85 upregulated genes and 12 downregulated genes (≥1.5-fold change in expression). However, Fe(II) treatment produced moderate alterations in gene expression without inducing dramatic alterations in gene expression profiles. Moreover, functional grouping of identified genes indicated that the differentially regulated genes were highly associated with cell growth, cell maintenance, and intra- and extracellular transport. These findings showed that Fe(III) may influence the expression of genes involved in PrP folding by redox mechanisms. The identification of genes with altered expression patterns in neural cells may provide insights into PrP conversion mechanisms during the development and progression of prion-related diseases. - Highlights: • Differential genes correlated with prion degeneration by Fe(III) were identified. • Genes were identified in cell proliferation and intra- and extracellular transport. • In PrP degeneration, redox related genes were suggested. • Cbr2, Rsad2, Slc40a1, Amph and Mvd were expressed significantly.

  18. Monitoring the effects of toxic chemicals on protein expression

    International Nuclear Information System (INIS)

    Giometti, C.S.; Taylor, J.

    1987-01-01

    Two-dimensional gel electrophoresis coupled with computer-assisted image and data analysis was used to monitor protein populations for both qualitative and quantitative changes induced by exposure to chemicals. For mutagenesis studies designed to screen for heritable mutations, a computer-assisted search of the optical density data from 2DE patterns was used to look for (a) new protein spots, (b) missing protein spots and/or (c) altered expression of normal protein spots. Using this approach, 320 mice were screened for mutations induced by treatment of sires with 150 mg/kg body weight of ethylnitrosourea (ENU) and four different mutations were identified. Protein patterns from 105 offspring from untreated male mice (controls) and 369 offspring from irradiated male mice (3 Gy gamma) were also screened. No heritable mutations were found in those data sets, however. In addition, protein changes were observed in livers of animals exposed to the hepatocellular peroxisomal proliferation agents (and carcinogens) Wy-14,643 and DEHP. The de novo synthesis of a new protein by these agents was demonstrated and quantitated

  19. Expression of putative immune response genes during early ontogeny in the coral Acropora millepora.

    Directory of Open Access Journals (Sweden)

    Eneour Puill-Stephan

    Full Text Available Corals, like many other marine invertebrates, lack a mature allorecognition system in early life history stages. Indeed, in early ontogeny, when corals acquire and establish associations with various surface microbiota and dinoflagellate endosymbionts, they do not efficiently distinguish between closely and distantly related individuals from the same population. However, very little is known about the molecular components that underpin allorecognition and immunity responses or how they change through early ontogeny in corals.Patterns in the expression of four putative immune response genes (apextrin, complement C3, and two CELIII type lectin genes were examined in juvenile colonies of Acropora millepora throughout a six-month post-settlement period using quantitative real-time PCR (qPCR. Expression of a CELIII type lectin gene peaked in the fourth month for most of the coral juveniles sampled and was significantly higher at this time than at any other sampling time during the six months following settlement. The timing of this increase in expression levels of putative immune response genes may be linked to allorecognition maturation which occurs around this time in A. millepora. Alternatively, the increase may represent a response to immune challenges, such as would be involved in the recognition of symbionts (such as Symbiodinium spp. or bacteria during winnowing processes as symbioses are fine-tuned.Our data, although preliminary, are consistent with the hypothesis that lectins may play an important role in the maturation of allorecognition responses in corals. The co-expression of lectins with apextrin during development of coral juveniles also raises the possibility that these proteins, which are components of innate immunity in other invertebrates, may influence the innate immune systems of corals through a common pathway or system. However, further studies investigating the expression of these genes in alloimmune-challenged corals are

  20. Expression of putative immune response genes during early ontogeny in the coral Acropora millepora.

    Science.gov (United States)

    Puill-Stephan, Eneour; Seneca, François O; Miller, David J; van Oppen, Madeleine J H; Willis, Bette L

    2012-01-01

    Corals, like many other marine invertebrates, lack a mature allorecognition system in early life history stages. Indeed, in early ontogeny, when corals acquire and establish associations with various surface microbiota and dinoflagellate endosymbionts, they do not efficiently distinguish between closely and distantly related individuals from the same population. However, very little is known about the molecular components that underpin allorecognition and immunity responses or how they change through early ontogeny in corals. Patterns in the expression of four putative immune response genes (apextrin, complement C3, and two CELIII type lectin genes) were examined in juvenile colonies of Acropora millepora throughout a six-month post-settlement period using quantitative real-time PCR (qPCR). Expression of a CELIII type lectin gene peaked in the fourth month for most of the coral juveniles sampled and was significantly higher at this time than at any other sampling time during the six months following settlement. The timing of this increase in expression levels of putative immune response genes may be linked to allorecognition maturation which occurs around this time in A. millepora. Alternatively, the increase may represent a response to immune challenges, such as would be involved in the recognition of symbionts (such as Symbiodinium spp. or bacteria) during winnowing processes as symbioses are fine-tuned. Our data, although preliminary, are consistent with the hypothesis that lectins may play an important role in the maturation of allorecognition responses in corals. The co-expression of lectins with apextrin during development of coral juveniles also raises the possibility that these proteins, which are components of innate immunity in other invertebrates, may influence the innate immune systems of corals through a common pathway or system. However, further studies investigating the expression of these genes in alloimmune-challenged corals are needed to further

  1. Direct inhibition of RNAse T2 expression by the HTLV-1 viral protein Tax.

    Science.gov (United States)

    Polakowski, Nicholas; Han, Hongjin; Lemasson, Isabelle

    2011-08-01

    Adult T-cell leukemia (ATL) is one of the primary diseases caused by Human T-cell Leukemia Virus type 1 (HTLV-1) infection. The virally-encoded Tax protein is believed to initiate early events in the development of this disease, as it is able to promote immortalization of T-cells and transformation of other cell types. These processes may be aided by the ability of the viral protein to directly deregulate expression of specific cellular genes through interactions with numerous transcriptional regulators. To identify gene promoters where Tax is localized, we isolated Tax-DNA complexes from an HTLV-1-infected T-cell line through a chromatin immunoprecipitation (ChIP) assay and used the DNA to probe a CpG island microarray. A site within the RNASET2 gene was found to be occupied by Tax. Real-time PCR analysis confirmed this result, and transient expression of Tax in uninfected cells led to the recruitment of the viral protein to the promoter. This event correlated with a decrease in the level of RNase T2 mRNA and protein, suggesting that Tax represses expression of this gene. Loss of RNase T2 expression occurs in certain hematological malignancies and other forms of cancer, and RNase T2 was recently reported to function as a tumor suppressor. Consequently, a reduction in the level of RNase T2 by Tax may play a role in ATL development.

  2. Direct Inhibition of RNAse T2 Expression by the HTLV-1 Viral Protein Tax

    Directory of Open Access Journals (Sweden)

    Isabelle Lemasson

    2011-08-01

    Full Text Available Adult T-cell leukemia (ATL is one of the primary diseases caused by Human T-cell Leukemia Virus type 1 (HTLV-1 infection. The virally-encoded Tax protein is believed to initiate early events in the development of this disease, as it is able to promote immortalization of T-cells and transformation of other cell types. These processes may be aided by the ability of the viral protein to directly deregulate expression of specific cellular genes through interactions with numerous transcriptional regulators. To identify gene promoters where Tax is localized, we isolated Tax-DNA complexes from an HTLV-1-infected T-cell line through a chromatin immunoprecipitation (ChIP assay and used the DNA to probe a CpG island microarray. A site within the RNASET2 gene was found to be occupied by Tax. Real-time PCR analysis confirmed this result, and transient expression of Tax in uninfected cells led to the recruitment of the viral protein to the promoter. This event correlated with a decrease in the level of RNase T2 mRNA and protein, suggesting that Tax represses expression of this gene. Loss of RNase T2 expression occurs in certain hematological malignancies and other forms of cancer, and RNase T2 was recently reported to function as a tumor suppressor. Consequently, a reduction in the level of RNase T2 by Tax may play a role in ATL development.

  3. Expression, purification and crystallization of a lyssavirus matrix (M) protein

    Energy Technology Data Exchange (ETDEWEB)

    Assenberg, René [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Delmas, Olivier [UPRE Lyssavirus Dynamics and Host Adaptation, WHO Collaborating Centre for Reference and Research on Rabies, Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris CEDEX 15 (France); Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J. [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Bourhy, Hervé [UPRE Lyssavirus Dynamics and Host Adaptation, WHO Collaborating Centre for Reference and Research on Rabies, Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris CEDEX 15 (France); Grimes, Jonathan M., E-mail: jonathan@strubi.ox.ac.uk [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom)

    2008-04-01

    The expression, purification and crystallization of the full-length matrix protein from three lyssaviruses is described. The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress.

  4. Expression, purification and crystallization of a lyssavirus matrix (M) protein

    International Nuclear Information System (INIS)

    Assenberg, René; Delmas, Olivier; Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J.; Bourhy, Hervé; Grimes, Jonathan M.

    2008-01-01

    The expression, purification and crystallization of the full-length matrix protein from three lyssaviruses is described. The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6 1 22 or P6 5 22, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress

  5. Heat Shock Protein 90 (Hsp90 Expression and Breast Cancer

    Directory of Open Access Journals (Sweden)

    Christos A. Papadimitriou

    2012-09-01

    Full Text Available Hsp90 is an abundant protein in mammalian cells. It forms several discrete complexes, each containing distinct groups of co-chaperones that assist protein folding and refolding during stress, protein transport and degradation. It interacts with a variety of proteins that play key roles in breast neoplasia including estrogen receptors, tumor suppressor p53 protein, angiogenesis transcription factor HIF-1alpha, antiapoptotic kinase Akt, Raf-1 MAP kinase and a variety of receptor tyrosine kinases of the erbB family. Elevated Hsp90 expression has been documented in breast ductal carcinomas contributing to the proliferative activity of breast cancer cells; whilst a significantly decreased Hsp90 expression has been shown in infiltrative lobular carcinomas and lobular neoplasia. Hsp90 overexpression has been proposed as a component of a mechanism through which breast cancer cells become resistant to various stress stimuli. Therefore, pharmacological inhibition of HSPs can provide therapeutic opportunities in the field of cancer treatment. 17-allylamino,17-demethoxygeldanamycin is the first Hsp90 inhibitor that has clinically been investigated in phase II trial, yielding promising results in patients with HER2-overexpressing metastatic breast cancer, whilst other Hsp90 inhibitors (retaspimycin HCL, NVP-AUY922, NVP-BEP800, CNF2024/BIIB021, SNX-5422, STA-9090, etc. are currently under evaluation.

  6. Heterogeneity mapping of protein expression in tumors using quantitative immunofluorescence.

    Science.gov (United States)

    Faratian, Dana; Christiansen, Jason; Gustavson, Mark; Jones, Christine; Scott, Christopher; Um, InHwa; Harrison, David J

    2011-10-25

    Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification (Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor(1,2), and some of these sub-clones give rise to metastatic (and therefore lethal) disease. Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension(3), or on macrodissection(4). A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue(5), providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi

  7. Long interspersed element-1 protein expression is a hallmark of many human cancers.

    Science.gov (United States)

    Rodić, Nemanja; Sharma, Reema; Sharma, Rajni; Zampella, John; Dai, Lixin; Taylor, Martin S; Hruban, Ralph H; Iacobuzio-Donahue, Christine A; Maitra, Anirban; Torbenson, Michael S; Goggins, Michael; Shih, Ie-Ming; Duffield, Amy S; Montgomery, Elizabeth A; Gabrielson, Edward; Netto, George J; Lotan, Tamara L; De Marzo, Angelo M; Westra, William; Binder, Zev A; Orr, Brent A; Gallia, Gary L; Eberhart, Charles G; Boeke, Jef D; Harris, Chris R; Burns, Kathleen H

    2014-05-01

    Cancers comprise a heterogeneous group of human diseases. Unifying characteristics include unchecked abilities of tumor cells to proliferate and spread anatomically, and the presence of clonal advantageous genetic changes. However, universal and highly specific tumor markers are unknown. Herein, we report widespread long interspersed element-1 (LINE-1) repeat expression in human cancers. We show that nearly half of all human cancers are immunoreactive for a LINE-1-encoded protein. LINE-1 protein expression is a common feature of many types of high-grade malignant cancers, is rarely detected in early stages of tumorigenesis, and is absent from normal somatic tissues. Studies have shown that LINE-1 contributes to genetic changes in cancers, with somatic LINE-1 insertions seen in selected types of human cancers, particularly colon cancer. We sought to correlate this observation with expression of the LINE-1-encoded protein, open reading frame 1 protein, and found that LINE-1 open reading frame 1 protein is a surprisingly broad, yet highly tumor-specific, antigen. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  8. Manipulating heat shock protein expression in laboratory animals.

    Science.gov (United States)

    Tolson, J Keith; Roberts, Stephen M

    2005-02-01

    Upregulation of heat shock proteins (Hsps) has been observed to impart resistance to a wide variety of physical and chemical insults. Elucidation of the role of Hsps in cellular defense processes depends, in part, on the ability to manipulate Hsp expression in laboratory animals. Simple methods of inducing whole body hyperthermia, such as warm water immersion or heating pad application, are effective in producing generalized expression of Hsps. Hsps can be upregulated locally with focused direct or indirect heating, such as with ultrasound or with laser or microwave radiation. Increased Hsp expression in response to toxic doses of xenobiotics has been commonly observed. Some pharmacologic agents are capable of altering Hsps more specifically by affecting processes involved in Hsp regulation. Gene manipulation offers the ability to selectively increase or decrease individual Hsps. Knockout mouse strains and Hsp-overexpressing transgenics have been used successfully to examine the role of specific Hsps in protection against hyperthermia, chemical insults, and ischemia-reperfusion injury. Gene therapy approaches also offer the possibility of selective alteration of Hsp expression. Some methods of increasing Hsp expression have application in specialized areas of research, such cold response, myocardial protection from exercise, and responses to stressful or traumatic stimuli. Each method of manipulating Hsp expression in laboratory animals has advantages and disadvantages, and selection of the best method depends upon the experimental objectives (e.g., the alteration in Hsp expression needed, its timing, and its location) and resources available.

  9. A new essential protein discovery method based on the integration of protein-protein interaction and gene expression data

    Directory of Open Access Journals (Sweden)

    Li Min

    2012-03-01

    Full Text Available Abstract Background Identification of essential proteins is always a challenging task since it requires experimental approaches that are time-consuming and laborious. With the advances in high throughput technologies, a large number of protein-protein interactions are available, which have produced unprecedented opportunities for detecting proteins' essentialities from the network level. There have been a series of computational approaches proposed for predicting essential proteins based on network topologies. However, the network topology-based centrality measures are very sensitive to the robustness of network. Therefore, a new robust essential protein discovery method would be of great value. Results In this paper, we propose a new centrality measure, named PeC, based on the integration of protein-protein interaction and gene expression data. The performance of PeC is validated based on the protein-protein interaction network of Saccharomyces cerevisiae. The experimental results show that the predicted precision of PeC clearly exceeds that of the other fifteen previously proposed centrality measures: Degree Centrality (DC, Betweenness Centrality (BC, Closeness Centrality (CC, Subgraph Centrality (SC, Eigenvector Centrality (EC, Information Centrality (IC, Bottle Neck (BN, Density of Maximum Neighborhood Component (DMNC, Local Average Connectivity-based method (LAC, Sum of ECC (SoECC, Range-Limited Centrality (RL, L-index (LI, Leader Rank (LR, Normalized α-Centrality (NC, and Moduland-Centrality (MC. Especially, the improvement of PeC over the classic centrality measures (BC, CC, SC, EC, and BN is more than 50% when predicting no more than 500 proteins. Conclusions We demonstrate that the integration of protein-protein interaction network and gene expression data can help improve the precision of predicting essential proteins. The new centrality measure, PeC, is an effective essential protein discovery method.

  10. 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.

  11. 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)

  12. Cooperative working of bacterial chromosome replication proteins generated by a reconstituted protein expression system

    Science.gov (United States)

    Fujiwara, Kei; Katayama, Tsutomu; Nomura, Shin-ichiro M.

    2013-01-01

    Replication of all living cells relies on the multirounds flow of the central dogma. Especially, expression of DNA replication proteins is a key step to circulate the processes of the central dogma. Here we achieved the entire sequential transcription–translation–replication process by autonomous expression of chromosomal DNA replication machineries from a reconstituted transcription–translation system (PURE system). We found that low temperature is essential to express a complex protein, DNA polymerase III, in a single tube using the PURE system. Addition of the 13 genes, encoding initiator, DNA helicase, helicase loader, RNA primase and DNA polymerase III to the PURE system gave rise to a DNA replication system by a coupling manner. An artificial genetic circuit demonstrated that the DNA produced as a result of the replication is able to provide genetic information for proteins, indicating the in vitro central dogma can sequentially undergo two rounds. PMID:23737447

  13. Expression of a fatty acid-binding protein in yeast

    International Nuclear Information System (INIS)

    Scholz, H.

    1991-06-01

    The unicellular eukaryotic microorganism, Saccharomyces cerevisiae, transformed with a plasmid containing a cDNA fragment encoding bovine heart fatty acid-binding protein (H-FABP C ) under the control of the inducible yeast GAL10 promoter, expressed FABP during growth on galactose. The maximum level of immunoreactive FABP, identical in size and isoelectric point to native protein, was reached after approximately 16 hours of induction. In contrast, transcription of the gene was induced within half an hour. Both, protein and mRNA were unstable and degraded within 1 h after repression of transcription. Analysis of subcellular fractions showed that FABP was exclusively associated with the cytosol. FABP expressed in yeast cells was functional as was demonstrated by its capacity to bind long chain fatty acids in an in vitro assay. Growth of all transformants on galactose as the carbon source showed no phenotype at temperatures up to 37 deg C, but the growth of FABP-expressing cells at 37 deg C was significantly retarded. Among the biochemical effects of FABP expression on lipid metabolism is a marked reduction of chain elongation and desaturation of exogenously added 14 C-palmitic acid. This effect is most pronounced in triacylglycerols and phospholipids when cells grow at 30 deg C and 37 deg C, respectively. In an in vitro assay determining the desaturation of palmitoyl CoA by microsomal membranes cytosol with or without exo- or endogenous FABP showed the same stimulation of the reaction. The desaturation of exogenously added 14 C-stearic acid, the pattern of unlabelled fatty acids (saturated vs. unsaturated) and the distribution of exogenously added radioactive fatty acids (palmitic, stearic or oleic acid) among lipid classes was not significantly affected. Using high concentrations (1 mM) the uptake of fatty acids was first stimulated and then inhibited when FABP was expressed. (author)

  14. Expression of antimicrobial peptides and interleukin-8 during early stages of inflammation: An experimental gingivitis study.

    Science.gov (United States)

    Dommisch, H; Staufenbiel, I; Schulze, K; Stiesch, M; Winkel, A; Fimmers, R; Dommisch, J; Jepsen, S; Miosge, N; Adam, K; Eberhard, J

    2015-12-01

    In the oral cavity, the epithelial surface is constantly exposed to a number of different microorganisms that are organized in a well-structured biofilm. The aim of this study was to monitor gingival expression of antimicrobial peptides (AMPs) and interleukin-8 (IL-8) in an early gingivitis model. Experimental gingivitis was allowed to develop in healthy volunteers (n = 17). Bleeding on probing (BOP%) and gingival crevicular fluid volume (GCF) were assessed at baseline and day 1, 3, 5, 7 and 14. Expression of AMPs (human beta-defensin-2, hBD-2; CC-chemokine ligand 20, CCL20; psoriasin, pso/S100A7) and IL-8 was analyzed by immunohistochemistry in gingival biopsies. In addition, hBD-2 and IL-8 protein expression was monitored in GCF using the ELISA technology. Experimental gingivitis gradually developed with an increase in BOP scores and GCF volume over time. In GCF, elevated concentrations of hBD-2 and IL-8 were monitored at day 1, 5 and 7 (p ≤ 0.0002). Immunohistochemical analysis of gingival sections demonstrated increased staining for hBD-2 at day 3, whereas the CCL20, pso/S100A7, and IL-8 expression was increased at later time points (p gingival inflammation. Differential temporal expression for AMPs may ensure a constant antimicrobial activity against changes in the bacterial composition of the growing dental biofilm. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  15. Grizzly bear corticosteroid binding globulin: Cloning and serum protein expression.

    Science.gov (United States)

    Chow, Brian A; Hamilton, Jason; Alsop, Derek; Cattet, Marc R L; Stenhouse, Gordon; Vijayan, Mathilakath M

    2010-06-01

    Serum corticosteroid levels are routinely measured as markers of stress in wild animals. However, corticosteroid levels rise rapidly in response to the acute stress of capture and restraint for sampling, limiting its use as an indicator of chronic stress. We hypothesized that serum corticosteroid binding globulin (CBG), the primary transport protein for corticosteroids in circulation, may be a better marker of the stress status prior to capture in grizzly bears (Ursus arctos). To test this, a full-length CBG cDNA was cloned and sequenced from grizzly bear testis and polyclonal antibodies were generated for detection of this protein in bear sera. The deduced nucleotide and protein sequences were 1218 bp and 405 amino acids, respectively. Multiple sequence alignments showed that grizzly bear CBG (gbCBG) was 90% and 83% identical to the dog CBG nucleotide and amino acid sequences, respectively. The affinity purified rabbit gbCBG antiserum detected grizzly bear but not human CBG. There were no sex differences in serum total cortisol concentration, while CBG expression was significantly higher in adult females compared to males. Serum cortisol levels were significantly higher in bears captured by leg-hold snare compared to those captured by remote drug delivery from helicopter. However, serum CBG expression between these two groups did not differ significantly. Overall, serum CBG levels may be a better marker of chronic stress, especially because this protein is not modulated by the stress of capture and restraint in grizzly bears. Copyright 2010 Elsevier Inc. All rights reserved.

  16. Identification of differentially expressed proteins in vitamin B 12

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    Swati Varshney

    2015-01-01

    Full Text Available Background: Vitamin B 12 (cobalamin is a water-soluble vitamin generally synthesized by microorganisms. Mammals cannot synthesize this vitamin but have evolved processes for absorption, transport and cellular uptake of this vitamin. Only about 30% of vitamin B 12 , which is bound to the protein transcobalamin (TC (Holo-TC [HoloTC] enters into the cell and hence is referred to as the biologically active form of vitamin B 12 . Vitamin B 12 deficiency leads to several complex disorders, including neurological disorders and anemia. We had earlier shown that vitamin B 12 deficiency is associated with coronary artery disease (CAD in Indian population. In the current study, using a proteomics approach we identified proteins that are differentially expressed in the plasma of individuals with low HoloTC levels. Materials and Methods: We used isobaric-tagging method of relative and absolute quantitation to identify proteins that are differently expressed in individuals with low HoloTC levels when compared to those with normal HoloTC level. Results: In two replicate isobaric tags for relative and absolute quantitation experiments several proteins involved in lipid metabolism, blood coagulation, cholesterol metabolic process, and lipoprotein metabolic process were found to be altered in individuals having low HoloTC levels. Conclusions: Our study indicates that low HoloTc levels could be a risk factor in the development of CAD.

  17. Sperm protein 17 is expressed in the sperm fibrous sheath

    Directory of Open Access Journals (Sweden)

    Albani Elena

    2009-07-01

    Full Text Available Abstract Background Sperm protein 17 (Sp17 is a highly conserved mammalian protein characterized in rabbit, mouse, monkey, baboon, macaque, human testis and spermatozoa. mRNA encoding Sp17 has been detected in a range of murine and human somatic tissues. It was also recognized in two myeloma cell lines and in neoplastic cells from patients with multiple myeloma and ovarian carcinoma. These data all indicate that Sp17 is widely distributed in humans, expressed not only in germinal cells and in a variety of somatic tissues, but also in neoplastic cells of unrelated origin. Methods Sp17 expression was analyzed by immunocytochemistry and transmission electron microscopy on spermatozoa. Results Here, we demonstrate the ultrastructural localization of human Sp17 throughout the spermatozoa flagellar fibrous sheath, and its presence in spermatozoa during in vitro states from their ejaculation to the oocyte fertilization. Conclusion These findings suggest a possible role of Sp17 in regulating sperm maturation, capacitation, acrosomal reaction and interactions with the oocyte zona pellucida during the fertilization process. Further, the high degree of sequence conservation throughout its N-terminal half, and the presence of an A-kinase anchoring protein (AKAP-binding motif within this region, suggest that Sp17 might play a regulatory role in a protein kinase A-independent AKAP complex in both germinal and somatic cells.

  18. Complement inhibitory proteins expression in placentas of thrombophilic women Complement inhibitory proteins expression in placentas of thrombophilic women

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    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

  19. Abscisic Acid (ABA) Regulation of Arabidopsis SR Protein Gene Expression

    Science.gov (United States)

    Cruz, Tiago M. D.; Carvalho, Raquel F.; Richardson, Dale N.; Duque, Paula

    2014-01-01

    Serine/arginine-rich (SR) proteins are major modulators of alternative splicing, a key generator of proteomic diversity and flexible means of regulating gene expression likely to be crucial in plant environmental responses. Indeed, mounting evidence implicates splicing factors in signal transduction of the abscisic acid (ABA) phytohormone, which plays pivotal roles in the response to various abiotic stresses. Using real-time RT-qPCR, we analyzed total steady-state transcript levels of the 18 SR and two SR-like genes from Arabidopsis thaliana in seedlings treated with ABA and in genetic backgrounds with altered expression of the ABA-biosynthesis ABA2 and the ABA-signaling ABI1 and ABI4 genes. We also searched for ABA-responsive cis elements in the upstream regions of the 20 genes. We found that members of the plant-specific SC35-Like (SCL) Arabidopsis SR protein subfamily are distinctively responsive to exogenous ABA, while the expression of seven SR and SR-related genes is affected by alterations in key components of the ABA pathway. Finally, despite pervasiveness of established ABA-responsive promoter elements in Arabidopsis SR and SR-like genes, their expression is likely governed by additional, yet unidentified cis-acting elements. Overall, this study pinpoints SR34, SR34b, SCL30a, SCL28, SCL33, RS40, SR45 and SR45a as promising candidates for involvement in ABA-mediated stress responses. PMID:25268622

  20. Dynamic methylation and expression of Oct4 in early neural stem cells.

    Science.gov (United States)

    Lee, Shih-Han; Jeyapalan, Jennie N; Appleby, Vanessa; Mohamed Noor, Dzul Azri; Sottile, Virginie; Scotting, Paul J

    2010-09-01

    Neural stem cells are a multipotent population of tissue-specific stem cells with a broad but limited differentiation potential. However, recent studies have shown that over-expression of the pluripotency gene, Oct4, alone is sufficient to initiate a process by which these can form 'induced pluripotent stem cells' (iPS cells) with the same broad potential as embryonic stem cells. This led us to examine the expression of Oct4 in endogenous neural stem cells, as data regarding its expression in neural stem cells in vivo are contradictory and incomplete. In this study we have therefore analysed the expression of Oct4 and other genes associated with pluripotency throughout development of the mouse CNS and in neural stem cells grown in vitro. We find that Oct4 is still expressed in the CNS by E8.5, but that this expression declines rapidly until it is undetectable by E15.5. This decline is coincident with the gradual methylation of the Oct4 promoter and proximal enhancer. Immunostaining suggests that the Oct4 protein is predominantly cytoplasmic in location. We also found that neural stem cells from all ages expressed the pluripotency associated genes, Sox2, c-Myc, Klf4 and Nanog. These data provide an explanation for the varying behaviour of cells from the early neuroepithelium at different stages of development. The expression of these genes also provides an indication of why Oct4 alone is sufficient to induce iPS formation in neural stem cells at later stages.

  1. Unique expression of cytoskeletal proteins in human soft palate muscles.

    Science.gov (United States)

    Shah, Farhan; Berggren, Diana; Holmlund, Thorbjörn; Levring Jäghagen, Eva; Stål, Per

    2016-03-01

    The human oropharyngeal muscles have a unique anatomy with diverse and intricate functions. To investigate if this specialization is also reflected in the cytoarchitecture of muscle fibers, intermediate filament proteins and the dystrophin-associated protein complex have been analyzed in two human palate muscles, musculus uvula (UV) and musculus palatopharyngeus (PP), with immunohistochenmical and morphological techniques. Human limb muscles were used as reference. The findings show that the soft palate muscle fibers have a cytoskeletal architecture that differs from the limb muscles. While all limb muscles showed immunoreaction for a panel of antibodies directed against different domains of cytoskeletal proteins desmin and dystrophin, a subpopulation of palate muscle fibers lacked or had a faint immunoreaction for desmin (UV 11.7% and PP 9.8%) and the C-terminal of the dystrophin molecule (UV 4.2% and PP 6.4%). The vast majority of these fibers expressed slow contractile protein myosin heavy chain I. Furthermore, an unusual staining pattern was also observed in these fibers for β-dystroglycan, caveolin-3 and neuronal nitric oxide synthase nNOS, which are all membrane-linking proteins associated with the dystrophin C-terminus. While the immunoreaction for nNOS was generally weak or absent, β-dystroglycan and caveolin-3 showed a stronger immunostaining. The absence or a low expression of cytoskeletal proteins otherwise considered ubiquitous and important for integration and contraction of muscle cells indicate a unique cytoarchitecture designed to meet the intricate demands of the upper airway muscles. It can be concluded that a subgroup of muscle fibers in the human soft palate appears to have special biomechanical properties, and their unique cytoarchitecture must be taken into account while assessing function and pathology in oropharyngeal muscles. © 2015 Anatomical Society.

  2. 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.

  3. An inducible expression system for high-level expression of recombinant proteins in slow growing mycobacteria.

    Science.gov (United States)

    Leotta, Lisa; Spratt, Joanne M; Kong, Carlyn U; Triccas, James A

    2015-09-01

    A novel protein expression vector utilising the inducible hspX promoter of Mycobacterium tuberculosis was constructed and evaluated in this study. High-level induction of three mycobacterial antigens, comprising up to 9% of bacterial sonicate, was demonstrated in recombinant Mycobacterium bovis BCG when grown under low-oxygen tension, which serves to enhance hspX promoter activity. Recombinant proteins were efficiently purified from bacterial lysates in a soluble form by virtue of a C-terminal 6-histidine tag. Purification of the immunodominant M. tuberculosis Ag85B antigen using this system resulted in a recombinant protein that stimulated significant IFN-γ release from Ag85B-reactive T cells generated after vaccination of mice with an Ag85B-expressing vaccine. Further, the M. tuberculosis L-alanine dehydrogenase (Ald) protein purified from recombinant BCG displayed strong enzymatic activity in recombinant form. This study demonstrated that high levels of native-like recombinant mycobacterial proteins can be produced in mycobacterial hosts, and this may aid the analysis of mycobacterial protein function and the development of new treatments. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Altered expression of mitochondrial electron transport chain proteins and improved myocardial energetic state during late ischemic preconditioning

    NARCIS (Netherlands)

    J.A. Cabrera (Jesús); E.A. Ziemba (Elizabeth); L.H. Colbert (Lisa); L.B. Anderson (Lorraine); W.J. Sluiter (Wim); D.J.G.M. Duncker (Dirk); T.A. Butterick (Tammy); J. Sikora (Joseph); H.B. Ward (Herbert B.); R.F. Kelly (Rosemary); E.O. McFalls (Edward)

    2012-01-01

    textabstractAltered expression of mitochondrial electron transport proteins has been shown in early preconditioned myocardial tissue. We wished to determine whether these alterations persist in the Second Window of Protection (SWOP) and if so, whether a favorable energetic state is facilitated

  5. Expression of proteinase inhibitor II proteins during floral development in Solanum americanum.

    Science.gov (United States)

    Sin, Suk-Fong; Chye, Mee-Len

    2004-10-01

    The heterologous expression of serine proteinase inhibitor II (PIN2) proteins confers insect resistance in transgenic plants, but little is known of their endogenous roles. We have cloned two cDNAs encoding Solanum americanum PIN2 proteins, SaPIN2a and SaPIN2b. SaPIN2a is highly expressed in stem, particularly in the phloem, suggesting it could possibly regulate proteolysis in the sieve elements. When SaPIN2a was expressed in transgenic lettuce, we observed an inhibition of endogenous trypsin- and chymotrypsin-like activities. Here, we demonstrate that both SaPIN2a and SaPIN2b are expressed in floral tissues that are destined to undergo developmental programmed cell death (PCD), suggesting possible endogenous roles in inhibiting trypsin- and chymotrypsin-like activities during flower development. Northern and western blot analyses revealed that SaPIN2a and SaPIN2b mRNAs and proteins show highest expression early in floral development. In situ hybridization analysis and immunolocalization on floral sections, localized SaPIN2a and SaPIN2b mRNAs and their proteins to tissues that would apparently undergo PCD: the ovules, the stylar transmitting tissue, the stigma and the vascular bundles. Detection of PCD in floral sections was achieved using terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) analysis. Examination of the mid-style before, and 1 day after, pollination revealed that high expression of SaPIN2a and SaPIN2b in the style was inversely correlated with PCD.

  6. Choline kinase alpha and hexokinase-2 protein expression in hepatocellular carcinoma: association with survival.

    Directory of Open Access Journals (Sweden)

    Sandi A Kwee

    Full Text Available PURPOSE: Hexokinase-2 (HK2 and more recently choline kinase alpha (CKA expression has been correlated with clinical outcomes in several major cancers. This study examines the protein expression of HK2 and CKA in hepatocellular carcinoma (HCC in association with patient survival and other clinicopathologic parameters. METHODS: Immunohistochemical analysis for HK2 and CKA expression was performed on a tissue microarray of 157 HCC tumor samples. Results were analyzed in relation to clinicopathologic data from Surveillance, Epidemiology, and End-Results Program registries. Mortality rates were assessed by Kaplan-Meier estimates and compared using log-rank tests. Predictors of overall survival were assessed using proportional hazards regression. RESULTS: Immunohistochemical expression of HK2 and CKA was detected in 71 (45% and 55 (35% tumor samples, respectively. Differences in tumor HK2 expression were associated with tumor grade (p = 0.008 and cancer stage (p = 0.001, while CKA expression differed significantly only across cancer stage (p = 0.048. Increased mortality was associated with tumor HK2 expression (p = 0.003 as well as CKA expression (p = 0.03 with hazard ratios of 1.86 (95% confidence interval (CI 1.23-2.83 and 1.59 (95% CI 1.04-2.41, respectively. Similar effects on overall survival were noted in a subset analysis of early stage (I and II HCC. Tumor HK2 expression, but not CKA expression, remained a significant predictor of survival in multivariable analyses. CONCLUSION: HK2 and CKA expression may have biologic and prognostic significance in HCC, with tumor HK2 expression being a potential independent predictor of survival.

  7. Distinct patterns of gene and protein expression elicited by organophosphorus pesticides in Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Dennis William E

    2009-04-01

    Full Text Available Abstract Background The wide use of organophosphorus (OP pesticides makes them an important public health concern. Persistent effects of exposure and the mechanism of neuronal degeneration are continuing issues in OP toxicology. To elucidate early steps in the mechanisms of OP toxicity, we studied alterations in global gene and protein expression in Caenorhabditis elegans exposed to OPs using microarrays and mass spectrometry. We tested two structurally distinct OPs (dichlorvos and fenamiphos and employed a mechanistically different third neurotoxicant, mefloquine, as an out-group for analysis. Treatment levels used concentrations of chemical sufficient to prevent the development of 10%, 50% or 90% of mid-vulval L4 larvae into early gravid adults (EGA at 24 h after exposure in a defined, bacteria-free medium. Results After 8 h of exposure, the expression of 87 genes responded specifically to OP treatment. The abundance of 34 proteins also changed in OP-exposed worms. Many of the genes and proteins affected by the OPs are expressed in neuronal and muscle tissues and are involved in lipid metabolism, cell adhesion, apoptosis/cell death, and detoxification. Twenty-two genes were differentially affected by the two OPs; a large proportion of these genes encode cytochrome P450s, UDP-glucuronosyl/UDP-glucosyltransferases, or P-glycoproteins. The abundance of transcripts and the proteins they encode were well correlated. Conclusion Exposure to OPs elicits a pattern of changes in gene expression in exposed worms distinct from that of the unrelated neurotoxicant, mefloquine. The functional roles and the tissue location of the genes and proteins whose expression is modulated in response to exposure is consistent with the known effects of OPs, including damage to muscle due to persistent hypercontraction, neuronal cell death, and phase I and phase II detoxification. Further, the two different OPs evoked distinguishable changes in gene expression; about half

  8. Expression of livin protein in lung cancer and its relation with the expression of pro-caspase3 protein

    Directory of Open Access Journals (Sweden)

    Hongru LI

    2008-10-01

    Full Text Available Background and objective Livin is a novel inhibitor of apoptosis protein (IAP, recent studies showed it overexpresses in a variety of carcinomas including lung cancer and contributes much to the cancerous development. The objective of this study is to explore the expression of livin in tissues of lung cancer and its relationshipwith histological types, chemotherapy, Lymph node metastasis and to study its correlation with the expression of pro-caspase3 as well. Methods Expressions of Livin and caspase3 were detected by Western blot assay in lung cancer tissues as well as in controls. Results Livin was expressed in 15 of 27 lung cancer, significantly more than those in lung para-cancerous (1/5 or benign disease lung tissues (2/12 (P 0.05. Conclusion Livin are differently expressed in different histological types of lung cancer; High levels of livin expression do not relate to chemotherapy, lymph node metastasis (P >0.05. The levels of livin tends to be positively associated with those of accordingly pro-caspase3, it is presumed that livin could bind pro-caspase3 and suppress its activation.

  9. Modulation of brassinosteroid-regulated gene expression by jumonji domain-containing proteins ELF6 and REF6 in Arabidopsis

    OpenAIRE

    Yu, Xiaofei; Li, Li; Li, Lei; Guo, Michelle; Chory, Joanne; Yin, Yanhai

    2008-01-01

    Plant steroid hormones, brassinosteroids (BRs), are of great importance for plant growth and development. BRs signal through a cell surface receptor kinase, BRI1, and a GSK3-like kinase, BIN2, to regulate the BES1/BZR1 family of transcription factors, which directly bind to target gene promoters to activate or repress gene expression and mediate BR responses. To understand how BES1 regulates target gene expression, we identified two BES1-interacting proteins, ELF6 (early flowering 6) and its ...

  10. FAM20: an evolutionarily conserved family of secreted proteins expressed in hematopoietic cells

    Directory of Open Access Journals (Sweden)

    Cobos Everardo

    2005-01-01

    Full Text Available Abstract Background Hematopoiesis is a complex developmental process controlled by a large number of factors that regulate stem cell renewal, lineage commitment and differentiation. Secreted proteins, including the hematopoietic growth factors, play critical roles in these processes and have important biological and clinical significance. We have employed representational difference analysis to identify genes that are differentially expressed during experimentally induced myeloid differentiation in the murine EML hematopoietic stem cell line. Results One identified clone encoded a previously unidentified protein of 541 amino acids that contains an amino terminal signal sequence but no other characterized domains. This protein is a member of family of related proteins that has been named family with sequence similarity 20 (FAM20 with three members (FAM20A, FAM20B and FAM20C in mammals. Evolutionary comparisons revealed the existence of a single FAM20 gene in the simple vertebrate Ciona intestinalis and the invertebrate worm Caenorhabditis elegans and two genes in two insect species, Drosophila melanogaster and Anopheles gambiae. Six FAM20 family members were identified in the genome of the pufferfish, Fugu rubripes and five members in the zebrafish, Danio rerio. The mouse Fam20a protein was ectopically expressed in a mammalian cell line and found to be a bona fide secreted protein and efficient secretion was dependent on the integrity of the signal sequence. Expression analysis revealed that the Fam20a gene was indeed differentially expressed during hematopoietic differentiation and that the other two family members (Fam20b and Fam20c were also expressed during hematcpoiesis but that their mRNA levels did not vary significantly. Likewise FAM20A was expressed in more limited set of human tissues than the other two family members. Conclusions The FAM20 family represents a new family of secreted proteins with potential functions in regulating

  11. The expression of cytoskeleton regulatory protein Mena in colorectal lesions.

    Science.gov (United States)

    Gurzu, Simona; Jung, I; Prantner, I; Ember, I; Pávai, Z; Mezei, T

    2008-01-01

    The actin regulatory proteins Ena/VASP (Enabled/Vasodilator stimulated phosphoprotein) family is involved in the control of cell motility and adhesion. They are important in the actin-dependent processes where dynamic actin reorganization it is necessary. The deregulation of actin cycle could have an important role in the cells' malignant transformation, tumor invasion or metastasis. Recently studies revealed that the human orthologue of murine Mena is modulated during the breast carcinogenesis. In our study, we tried to observe the immunohistochemical expression of mammalian Ena (Mena) in the colorectal polyps and carcinomas. We analyzed 10 adenomatous polyps (five with dysplasia) and 36 adenocarcinomas. We used the indirect immunoperoxidase staining. BD Biosciences have provided the Mena antibody. We observed that Mena was not expressed in the normal colorectal mucosa neither in polyps without dysplasia, but its expression was very high in polyps with high dysplasia. In colorectal carcinomas, Mena marked the tumoral cells in 80% of cases. In 25% of positive cases, the intensity was 3+, in 60% 2+ and in the other 15% 1+. The Mena intensity was higher in the microsatellite stable tumors (MSS) and was correlated with vascular invasion, with intensity of angiogenesis marked with CD31 and CD105 and with c-erbB-2 and p53 expression. This is the first study in the literature about Mena expression in colorectal lesions.

  12. Effects of stress and adrenalectomy on activity-regulated cytoskeleton protein (Arc) gene expression

    DEFF Research Database (Denmark)

    Mikkelsen, Jens D; Larsen, Marianne Hald

    2006-01-01

    Activity-regulated cytoskeletal-associated protein (Arc) is an effector immediate early gene induced by novelty and involved in consolidation of long-term memory. Since activation of glucocorticoid receptors is a prerequisite for memory consolidation, we therefore aimed to study the effect of acute...... restraint stress on Arc gene expression in adrenalectomized rats. Acute stress produced a significant increase in Arc gene expression in the medial prefrontal cortex, but not in the parietal cortex or in the pyramidal cell layer of the hippocampus. The basal level of Arc mRNA in adrenalectomized animals...... was high in the medial prefrontal cortex and unaffected by acute stress in these animals. These data are consistent with the role of Arc as an integrative modulator of synaptic plasticity by emphasizing the potential role of stress and glucocorticoids in the control of Arc gene expression....

  13. Functional modules by relating protein interaction networks and gene expression.

    Science.gov (United States)

    Tornow, Sabine; Mewes, H W

    2003-11-01

    Genes and proteins are organized on the basis of their particular mutual relations or according to their interactions in cellular and genetic networks. These include metabolic or signaling pathways and protein interaction, regulatory or co-expression networks. Integrating the information from the different types of networks may lead to the notion of a functional network and functional modules. To find these modules, we propose a new technique which is based on collective, multi-body correlations in a genetic network. We calculated the correlation strength of a group of genes (e.g. in the co-expression network) which were identified as members of a module in a different network (e.g. in the protein interaction network) and estimated the probability that this correlation strength was found by chance. Groups of genes with a significant correlation strength in different networks have a high probability that they perform the same function. Here, we propose evaluating the multi-body correlations by applying the superparamagnetic approach. We compare our method to the presently applied mean Pearson correlations and show that our method is more sensitive in revealing functional relationships.

  14. 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

  15. Welfare assessment in transgenic pigs expressing green fluorescent protein (GFP)

    DEFF Research Database (Denmark)

    Huber, Reinhard C.; Remuge, Liliana; Carlisle, Ailsa

    2012-01-01

    Since large animal transgenesis has been successfully attempted for the first time about 25 years ago, the technology has been applied in various lines of transgenic pigs. Nevertheless one of the concerns with the technology—animal welfare—has not been approached through systematic assessment...... and statements regarding the welfare of transgenic pigs have been based on anecdotal observations during early stages of transgenic programs. The main aim of the present study was therefore to perform an extensive welfare assessment comparing heterozygous transgenic animals expressing GFP with wildtype animals...... months. The absence of significant differences between GFP and wildtype animals in the parameters observed suggests that the transgenic animals in question are unlikely to suffer from deleterious effects of transgene expression on their welfare and thus support existing anecdotal observations of pigs...

  16. Correlation of mammographical imaging signs with the expression of bcl-2 and bax proteins in breast cancer

    International Nuclear Information System (INIS)

    Zhang Yili; Du Hongwen; Zhang Yun; Zhang Yuelang; Kuang Fangjun; Guo Zuomin

    2004-01-01

    Objective: To discuss the correlation of mammographical imaging signs with the expression of bcl-2 and bax proteins in breast cancer for early diagnosis and forecast of its prognoses. Methods: Fifty-four breast cancers and 26 benign diseases were proved by pathologic methods and all cases underwent mammography. Immunohistochemical technique was used to measure the expression of bcl-2 and bax proteins in these tissues. The correlation of imaging signs with the expression of bcl-2 and bax proteins in breast cancer and benign lesion was analyzed. Results: The expression of bcl-2 or bax protein in the breast cancer was higher than that in breast benign diseases (χ 2 =15.116, 11.361, P 2 =10.358, 12.818, P 2 =10.996, 10.667, P 2 =10.405, P 2 =6.841, P<0.05). Conclusion: Some imaging signs of breast cancer were closely related to the expression of bcl-2 and bax proteins and these signs could reflect the biological behavior of tumor cells and prognoses. Therefore it could be helpful to the early diagnosis and treatment of breast cancer. (authors)

  17. Altered retinal microRNA expression profiles in early diabetic retinopathy: an in silico analysis.

    Science.gov (United States)

    Xiong, Fen; Du, Xinhua; Hu, Jianyan; Li, Tingting; Du, Shanshan; Wu, Qiang

    2014-07-01

    MicroRNAs (miRNAs) - as negative regulators of target genes - are associated with various human diseases, but their precise role(s) in diabetic retinopathy (DR) remains to be elucidated. The aim of this study was to elucidate the involvement of miRNAs in early DR using in silico analysis to explore their gene expression patterns. We used the streptozotocin (STZ)-induced diabetic rat to investigate the roles of miRNAs in early DR. Retinal miRNA expression profiles from diabetic versus healthy control rats were examined by miRNA array analysis. Based on several bioinformatic systems, specifically, gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, we identified signatures of the potential pathological processes, gene functions, and signaling pathways that are influenced by dysregulated miRNAs. We used quantitative real-time polymerase chain reaction (qRT-PCR) to validate six (i.e. those with significant changes in expression levels) of the 17 miRNAs that were detected in the miRNA array. We also describe the significant role of the miRNA-gene network, which is based on the interactions between miRNAs and target genes. GO analysis of the 17 miRNAs detected in the miRNA array analysis revealed the most prevalent miRNAs to be those related to biological processes, olfactory bulb development and axonogenesis. These miRNAs also exert significant influence on additional pathways, including the mitogen-activated protein and calcium signaling pathways. Six of the seventeen miRNAs were chosen for qRT-PCR validation. With the exception of a slight difference in miRNA-350, our results are in close agreement with the differential expressions detected by array analysis. This study, which describes miRNA expression during the early developmental phases of DR, revealed extensive miRNA interactions. Based on both their target genes and signaling pathways, we suggest that miRNAs perform critical regulatory functions during the early stages of DR

  18. Expression and Production of SH2 Domain Proteins.

    Science.gov (United States)

    Liu, Bernard A; Ogiue-Ikeda, Mari; Machida, Kazuya

    2017-01-01

    The Src Homology 2 (SH2) domain lies at the heart of phosphotyrosine signaling, coordinating signaling events downstream of receptor tyrosine kinases (RTKs), adaptors, and scaffolds. Over a hundred SH2 domains are present in mammals, each having a unique specificity which determines its interactions with multiple binding partners. One of the essential tools necessary for studying and determining the role of SH2 domains in phosphotyrosine signaling is a set of soluble recombinant SH2 proteins. Here we describe methods, based on a broad experience with purification of all SH2 domains, for the production of SH2 domain proteins needed for proteomic and biochemical-based studies such as peptide arrays, mass-spectrometry, protein microarrays, reverse-phase microarrays, and high-throughput fluorescence polarization (HTP-FP). We describe stepwise protocols for expression and purification of SH2 domains using GST or poly His-tags, two widely adopted affinity tags. In addition, we address alternative approaches, challenges, and validation studies for assessing protein quality and provide general characteristics of purified human SH2 domains.

  19. Expression, purification and crystallization of a lyssavirus matrix (M) protein

    Science.gov (United States)

    Assenberg, René; Delmas, Olivier; Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J.; Bourhy, Hervé; Grimes, Jonathan M.

    2008-01-01

    The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6122 or P6522, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress. PMID:18391421

  20. Mechanical stimulation increases proliferation, differentiation and protein expression in culture

    DEFF Research Database (Denmark)

    Grossi, Alberto; Yadav, Kavita; Lawson, Moira Ann

    2007-01-01

    Myogenesis is a complex sequence of events, including the irreversible transition from the proliferation-competent myoblast stage into fused, multinucleated myotubes. Myogenic differentiation is regulated by positive and negative signals from surrounding tissues. Stimulation due to stretch- or load...... to elucidate also the signaling pathway by which this mechanical stimulation can causes an increase in protein expression. When mechanically stimulated via laminin receptors on cell surface, C(2)C(12) cells showed an increase in cell proliferation and differentiation. Populations undergoing mechanical...... stimulation through laminin receptors show an increase in expression of Myo-D, myogenin and an increase in ERK1/2 phosphorylation. Cells stimulated via fibronectin receptors show no significant increases in fusion competence. We conclude that load induced signalling through integrin containing laminin...

  1. Expression of goose parvovirus whole VP3 protein and its epitopes in Escherichia coli cells.

    Science.gov (United States)

    Tarasiuk, K; Woźniakowski, G; Holec-Gąsior, L

    2015-01-01

    The aim of this study was the expression of goose parvovirus capsid protein (VP3) and its epitopes in Escherichia coli cells. Expression of the whole VP3 protein provided an insufficient amount of protein. In contrast, the expression of two VP3 epitopes (VP3ep4, VP3ep6) in E. coli, resulted in very high expression levels. This may suggest that smaller parts of the GPV antigenic determinants are more efficiently expressed than the complete VP3 gene.

  2. HER 2/neu protein expression in colorectal cancer

    International Nuclear Information System (INIS)

    Schuell, B; Gruenberger, T; Scheithauer, W; Zielinski, Ch; Wrba, F

    2006-01-01

    Conflicting data exist about the prevalence of HER-2/neu overexpression in colorectal cancer ranging from 0 to 83 %. In our study we tried to clarify the extent of expression and its relationship to clinicopathological parameters. This study involved 77 specimens of malignant colorectal cancer lesions of surgically resected patients. HER-2/neu immunohistochemistry was performed using the Hercep-Test Kit. Out of 77 specimens, 56 were Her-2/neu negative (70%), 20 (26%) showed a barely immunostaining (1+), only 1 (1%) was moderately (2+) and 2 (3%) were strongly positive (3+). Her-2/neu staining (moderately and strongly positive) was only detected in primary tumours of patients with confirmed metastases. No relationship was found between membranous HER-2 expression and patients' gender or differentiation. The median survival time of patients with positive HER-2/neu immunostaining was 21 versus 39 months in patients without HER-2/neu expression (p = 0.088). The c-erbB protein expression was observed in colorectal cancer but rarely in the therapeutic range (2+ and 3+). There was no significant association with tumour grade, gender, localization of the primary tumour or survival. These data indicate that c-erbB-2 is unlikely to play a major role in the therapeutic management of colorectal cancer

  3. Sperm protein 17 is expressed in human nervous system tumours

    International Nuclear Information System (INIS)

    Grizzi, Fabio; Baena, Riccardo Rodriguez y; Dioguardi, Nicola; Chiriva-Internati, Maurizio; Gaetani, Paolo; Franceschini, Barbara; Di Ieva, Antonio; Colombo, Piergiuseppe; Ceva-Grimaldi, Giorgia; Bollati, Angelo; Frezza, Eldo E; Cobos, E

    2006-01-01

    Human sperm protein 17 (Sp17) is a highly conserved protein that was originally isolated from a rabbit epididymal sperm membrane and testis membrane pellet. It has recently been included in the cancer/testis (CT) antigen family, and shown to be expressed in multiple myeloma and ovarian cancer. We investigated its immunolocalisation in specimens of nervous system (NS) malignancies, in order to establish its usefulness as a target for tumour-vaccine strategies. The expression of Sp17 was assessed by means of a standardised immunohistochemical procedure [(mAb/antigen) MF1/Sp17] in formalin-fixed and paraffin embedded surgical specimens of NS malignancies, including 28 neuroectodermal primary tumours (6 astrocytomas, 16 glioblastoma multiforme, 5 oligodendrogliomas, and 1 ependymoma), 25 meningeal tumours, and five peripheral nerve sheath tumours (4 schwannomas, and 1 neurofibroma),. A number of neuroectodermal (21%) and meningeal tumours (4%) were found heterogeneously immunopositive for Sp17. None of the peripheral nerve sheath tumours was immunopositive for Sp17. The expression pattern was heterogeneous in all of the positive samples, and did not correlate with the degree of malignancy. The frequency of expression and non-uniform cell distribution of Sp17 suggest that it cannot be used as a unique immunotherapeutic target in NS cancer. However, our results do show the immunolocalisation of Sp17 in a proportion of NS tumour cells, but not in their non-pathological counterparts. The emerging complex function of Sp17 makes further studies necessary to clarify the link between it and immunopositive cells

  4. 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.

  5. PSG gene expression is up-regulated by lysine acetylation involving histone and nonhistone proteins.

    Directory of Open Access Journals (Sweden)

    Soledad A Camolotto

    Full Text Available BACKGROUND: Lysine acetylation is an important post-translational modification that plays a central role in eukaryotic transcriptional activation by modifying chromatin and transcription-related factors. Human pregnancy-specific glycoproteins (PSG are the major secreted placental proteins expressed by the syncytiotrophoblast at the end of pregnancy and represent early markers of cytotrophoblast differentiation. Low PSG levels are associated with complicated pregnancies, thus highlighting the importance of studying the mechanisms that control their expression. Despite several transcription factors having been implicated as key regulators of PSG gene family expression; the role of protein acetylation has not been explored. METHODOLOGY/PRINCIPAL FINDINGS: Here, we explored the role of acetylation on PSG gene expression in the human placental-derived JEG-3 cell line. Pharmacological inhibition of histone deacetylases (HDACs up-regulated PSG protein and mRNA expression levels, and augmented the amount of acetylated histone H3 associated with PSG 5'regulatory regions. Moreover, PSG5 promoter activation mediated by Sp1 and KLF6, via the core promoter element motif (CPE, -147/-140, was markedly enhanced in the presence of the HDAC inhibitor trichostatin A (TSA. This effect correlated with an increase in Sp1 acetylation and KLF6 nuclear localization as revealed by immunoprecipitation and subcellular fractionation assays. The co-activators PCAF, p300, and CBP enhanced Sp1-dependent PSG5 promoter activation through their histone acetylase (HAT function. Instead, p300 and CBP acetyltransferase domain was dispensable for sustaining co-activation of PSG5 promoter by KLF6. CONCLUSIONS/SIGNIFICANCE: Results are consistent with a regulatory role of lysine acetylation on PSG expression through a relaxed chromatin state and an increase in the transcriptional activity of Sp1 and KLF6 following an augmented Sp1 acetylation and KLF6 nuclear localization.

  6. Expression and the antigenicity of recombinant coat proteins of tungro viruses expressed in Escherichia coli.

    Science.gov (United States)

    Yee, Siew Fung; Chu, Chia Huay; Poili, Evenni; Sum, Magdline Sia Henry

    2017-02-01

    Rice tungro disease (RTD) is a recurring disease affecting rice farming especially in the South and Southeast Asia. The disease is commonly diagnosed by visual observation of the symptoms on diseased plants in paddy fields and by polymerase chain reaction (PCR). However, visual observation is unreliable and PCR can be costly. High-throughput as well as relatively cheap detection methods are important for RTD management for screening large number of samples. Due to this, detection by serological assays such as immunoblotting assays and enzyme-linked immunosorbent assay are preferred. However, these serological assays are limited by lack of continuous supply of antibodies as reagents due to the difficulty in preparing sufficient purified virions as antigens. This study aimed to generate and evaluate the reactivity of the recombinant coat proteins of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV) as alternative antigens to generate antibodies. The genes encoding the coat proteins of both viruses, RTBV (CP), and RTSV (CP1, CP2 and CP3) were cloned and expressed as recombinant fusion proteins in Escherichia coli. All of the recombinant fusion proteins, with the exception of the recombinant fusion protein of the CP2 of RTSV, were reactive against our in-house anti-tungro rabbit serum. In conclusion, our study showed the potential use of the recombinant fusion coat proteins of the tungro viruses as alternative antigens for production of antibodies for diagnostic purposes. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. 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

  8. Early experiences mediate distinct adult gene expression and reproductive programs in Caenorhabditis elegans

    Science.gov (United States)

    Ow, Maria C.; Nichitean, Alexandra M.; Dorus, Steve; Hall, Sarah E.

    2018-01-01

    Environmental stress during early development in animals can have profound effects on adult phenotypes via programmed changes in gene expression. Using the nematode C. elegans, we demonstrated previously that adults retain a cellular memory of their developmental experience that is manifested by differences in gene expression and life history traits; however, the sophistication of this system in response to different environmental stresses, and how it dictates phenotypic plasticity in adults that contribute to increased fitness in response to distinct environmental challenges, was unknown. Using transcriptional profiling, we show here that C. elegans adults indeed retain distinct cellular memories of different environmental conditions. We identified approximately 500 genes in adults that entered dauer due to starvation that exhibit significant opposite (“seesaw”) transcriptional phenotypes compared to adults that entered dauer due to crowding, and are distinct from animals that bypassed dauer. Moreover, we show that two-thirds of the genes in the genome experience a 2-fold or greater seesaw trend in gene expression, and based upon the direction of change, are enriched in large, tightly linked regions on different chromosomes. Importantly, these transcriptional programs correspond to significant changes in brood size depending on the experienced stress. In addition, we demonstrate that while the observed seesaw gene expression changes occur in both somatic and germline tissue, only starvation-induced changes require a functional GLP-4 protein necessary for germline development, and both programs require the Argonaute CSR-1. Thus, our results suggest that signaling between the soma and the germ line can generate phenotypic plasticity as a result of early environmental experience, and likely contribute to increased fitness in adverse conditions and the evolution of the C. elegans genome. PMID:29447162

  9. Early experiences mediate distinct adult gene expression and reproductive programs in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Maria C Ow

    2018-02-01

    Full Text Available Environmental stress during early development in animals can have profound effects on adult phenotypes via programmed changes in gene expression. Using the nematode C. elegans, we demonstrated previously that adults retain a cellular memory of their developmental experience that is manifested by differences in gene expression and life history traits; however, the sophistication of this system in response to different environmental stresses, and how it dictates phenotypic plasticity in adults that contribute to increased fitness in response to distinct environmental challenges, was unknown. Using transcriptional profiling, we show here that C. elegans adults indeed retain distinct cellular memories of different environmental conditions. We identified approximately 500 genes in adults that entered dauer due to starvation that exhibit significant opposite ("seesaw" transcriptional phenotypes compared to adults that entered dauer due to crowding, and are distinct from animals that bypassed dauer. Moreover, we show that two-thirds of the genes in the genome experience a 2-fold or greater seesaw trend in gene expression, and based upon the direction of change, are enriched in large, tightly linked regions on different chromosomes. Importantly, these transcriptional programs correspond to significant changes in brood size depending on the experienced stress. In addition, we demonstrate that while the observed seesaw gene expression changes occur in both somatic and germline tissue, only starvation-induced changes require a functional GLP-4 protein necessary for germline development, and both programs require the Argonaute CSR-1. Thus, our results suggest that signaling between the soma and the germ line can generate phenotypic plasticity as a result of early environmental experience, and likely contribute to increased fitness in adverse conditions and the evolution of the C. elegans genome.

  10. Early bovine embryos regulate oviduct epithelial cell gene expression during in vitro co-culture.

    Science.gov (United States)

    Schmaltz-Panneau, Barbara; Cordova, Amanda; Dhorne-Pollet, Sophie; Hennequet-Antier, Christelle; Uzbekova, Sveltlana; Martinot, Emmanuelle; Doret, Sarah; Martin, Patrice; Mermillod, Pascal; Locatelli, Yann

    2014-10-01

    In mammals, the oviduct may participate to the regulation of early embryo development. In vitro co-culture of early bovine embryos with bovine oviduct epithelial cells (BOEC) has been largely used to mimic the maternal environment. However, the mechanisms of BOEC action have not been clearly elucidated yet. The aim of this study was to determine the response of BOEC cultures to the presence of developing bovine embryos. A 21,581-element bovine oligonucleotide array was used compare the gene expression profiles of confluent BOEC cultured for 8 days with or without embryos. This study revealed 34 differentially expressed genes (DEG). Of these 34 genes, IFI6, ISG15, MX1, IFI27, IFI44, RSAD2, IFITM1, EPSTI1, USP18, IFIT5, and STAT1 expression increased to the greatest extent due to the presence of embryos with a major impact on antiviral and immune response. Among the mRNAs at least 25 are already described as induced by interferons. In addition, transcript levels of new candidate genes involved in the regulation of transcription, modulation of the maternal immune system and endometrial remodeling were found to be increased. We selected 7 genes and confirmed their differential expression by quantitative RT-PCR. The immunofluorescence imaging of cellular localization of STAT1 protein in BOEC showed a nuclear translocation in the presence of embryos, suggesting the activation of interferon signaling pathway. This first systematic study of BOEC transcriptome changes in response to the presence of embryos in cattle provides some evidences that these cells are able to adapt their transcriptomic profile in response to embryo signaling. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. AZFa protein DDX3Y is differentially expressed in human male germ cells during development and in testicular tumours

    DEFF Research Database (Denmark)

    Gueler, B; Sonne, S B; Zimmer, J

    2012-01-01

    are believed to originate from fetal gonocytes.METHODSDDX3Y protein expression was analysed during development in different tissues by western blotting. The localization of DDX3Y in normal fetal and prepubertal testis tissue of different ages as well as in a series of distinct TGCT tissue samples (CIS......, classical seminoma, spermatocytic seminoma, teratoma and embryonal carcinoma) was performed by immunohistochemistry.RESULTSGerm cell-specific expression of DDX3Y protein was revealed in fetal prospermatogonia but not in gonocytes and not before the 17th gestational week. After birth, DDX3Y was expressed......, but not in somatically differentiated non-seminomas, consistent with its germ-cell specific function.CONCLUSIONSThe fetal germ cell DDX3Y expression suggests a role in early spermatogonial proliferation and implies that, in men with AZFa deletion, germ cell depletion may begin prenatally. The strong expression of DDX3Y...

  12. 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 ...

  13. Spatio-Temporal Gene Expression Profiling during In Vivo Early Ovarian Folliculogenesis: Integrated Transcriptomic Study and Molecular Signature of Early Follicular Growth.

    Directory of Open Access Journals (Sweden)

    Agnes Bonnet

    Full Text Available The successful achievement of early ovarian folliculogenesis is important for fertility and reproductive life span. This complex biological process requires the appropriate expression of numerous genes at each developmental stage, in each follicular compartment. Relatively little is known at present about the molecular mechanisms that drive this process, and most gene expression studies have been performed in rodents and without considering the different follicular compartments.We used RNA-seq technology to explore the sheep transcriptome during early ovarian follicular development in the two main compartments: oocytes and granulosa cells. We documented the differential expression of 3,015 genes during this phase and described the gene expression dynamic specific to these compartments. We showed that important steps occurred during primary/secondary transition in sheep. We also described the in vivo molecular course of a number of pathways. In oocytes, these pathways documented the chronology of the acquisition of meiotic competence, migration and cellular organization, while in granulosa cells they concerned adhesion, the formation of cytoplasmic projections and steroid synthesis. This study proposes the involvement in this process of several members of the integrin and BMP families. The expression of genes such as Kruppel-like factor 9 (KLF9 and BMP binding endothelial regulator (BMPER was highlighted for the first time during early follicular development, and their proteins were also predicted to be involved in gene regulation. Finally, we selected a data set of 24 biomarkers that enabled the discrimination of early follicular stages and thus offer a molecular signature of early follicular growth. This set of biomarkers includes known genes such as SPO11 meiotic protein covalently bound to DSB (SPO11, bone morphogenetic protein 15 (BMP15 and WEE1 homolog 2 (S. pombe(WEE2 which play critical roles in follicular development but other biomarkers

  14. Growing functional modules from a seed protein via integration of protein interaction and gene expression data

    Directory of Open Access Journals (Sweden)

    Dimitrakopoulou Konstantina

    2007-10-01

    Full Text Available Abstract Background Nowadays modern biology aims at unravelling the strands of complex biological structures such as the protein-protein interaction (PPI networks. A key concept in the organization of PPI networks is the existence of dense subnetworks (functional modules in them. In recent approaches clustering algorithms were applied at these networks and the resulting subnetworks were evaluated by estimating the coverage of well-established protein complexes they contained. However, most of these algorithms elaborate on an unweighted graph structure which in turn fails to elevate those interactions that would contribute to the construction of biologically more valid and coherent functional modules. Results In the current study, we present a method that corroborates the integration of protein interaction and microarray data via the discovery of biologically valid functional modules. Initially the gene expression information is overlaid as weights onto the PPI network and the enriched PPI graph allows us to exploit its topological aspects, while simultaneously highlights enhanced functional association in specific pairs of proteins. Then we present an algorithm that unveils the functional modules of the weighted graph by expanding a kernel protein set, which originates from a given 'seed' protein used as starting-point. Conclusion The integrated data and the concept of our approach provide reliable functional modules. We give proofs based on yeast data that our method manages to give accurate results in terms both of structural coherency, as well as functional consistency.

  15. Altered hypothalamic protein expression in a rat model of Huntington's disease.

    Directory of Open Access Journals (Sweden)

    Wei-na Cong

    Full Text Available Huntington's disease (HD is a neurodegenerative disorder, which is characterized by progressive motor impairment and cognitive alterations. Changes in energy metabolism, neuroendocrine function, body weight, euglycemia, appetite function, and circadian rhythm can also occur. It is likely that the locus of these alterations is the hypothalamus. We used the HD transgenic (tg rat model bearing 51 CAG repeats, which exhibits similar HD symptomology as HD patients to investigate hypothalamic function. We conducted detailed hypothalamic proteome analyses and also measured circulating levels of various metabolic hormones and lipids in pre-symptomatic and symptomatic animals. Our results demonstrate that there are significant alterations in HD rat hypothalamic protein expression such as glial fibrillary acidic protein (GFAP, heat shock protein-70, the oxidative damage protein glutathione peroxidase (Gpx4, glycogen synthase1 (Gys1 and the lipid synthesis enzyme acylglycerol-3-phosphate O-acyltransferase 1 (Agpat1. In addition, there are significant alterations in various circulating metabolic hormones and lipids in pre-symptomatic animals including, insulin, leptin, triglycerides and HDL, before any motor or cognitive alterations are apparent. These early metabolic and lipid alterations are likely prodromal signs of hypothalamic dysfunction. Gaining a greater understanding of the hypothalamic and metabolic alterations that occur in HD, could lead to the development of novel therapeutics for early interventional treatment of HD.

  16. Expression and purification of recombinant polyomavirus VP2 protein and its interactions with polyomavirus proteins

    Science.gov (United States)

    Cai, X.; Chang, D.; Rottinghaus, S.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    Recombinant polyomavirus VP2 protein was expressed in Escherichia coli (RK1448), using the recombinant expression system pFPYV2. Recombinant VP2 was purified to near homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroelution, and Extracti-Gel chromatography. Polyclonal serum to this protein which reacted specifically with recombinant VP2 as well as polyomavirus virion VP2 and VP3 on Western blots (immunoblots) was produced. Purified VP2 was used to establish an in vitro protein-protein interaction assay with polyomavirus structural proteins and purified recombinant VP1. Recombinant VP2 interacted with recombinant VP1, virion VP1, and the four virion histones. Recombinant VP1 coimmunoprecipitated with recombinant VP2 or truncated VP2 (delta C12VP2), which lacked the carboxy-terminal 12 amino acids. These experiments confirmed the interaction between VP1 and VP2 and revealed that the carboxyterminal 12 amino acids of VP2 and VP3 were not necessary for formation of this interaction. In vivo VP1-VP2 interaction study accomplished by cotransfection of COS-7 cells with VP2 and truncated VP1 (delta N11VP1) lacking the nuclear localization signal demonstrated that VP2 was capable of translocating delta N11VP1 into the nucleus. These studies suggest that complexes of VP1 and VP2 may be formed in the cytoplasm and cotransported to the nucleus for virion assembly to occur.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  18. 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

  19. Neurotoxocarosis alters myelin protein gene transcription and expression.

    Science.gov (United States)

    Heuer, Lea; Beyerbach, Martin; Lühder, Fred; Beineke, Andreas; Strube, Christina

    2015-06-01

    Neurotoxocarosis is an infection of the central nervous system caused by migrating larvae of the common dog and cat roundworms (Toxocara canis and Toxocara cati), which are zoonotic agents. As these parasites are prevalent worldwide and neuropathological and molecular investigations on neurotoxocarosis are scare, this study aims to characterise nerve fibre demyelination associated with neurotoxocarosis on a molecular level. Transcription of eight myelin-associated genes (Cnp, Mag, Mbp, Mog, Mrf-1, Nogo-A, Plp1, Olig2) was determined in the mouse model during six time points of the chronic phase of infection using qRT-PCR. Expression of selected proteins was analysed by Western blotting or immunohistochemistry. Additionally, demyelination and neuronal damage were investigated histologically. Significant differences (p ≤ 0.05) between transcription rates of T. canis-infected and uninfected control mice were detected for all analysed genes while T. cati affected five of eight investigated genes. Interestingly, 2', 3 ´-cyclic nucleotide 3'-phosphodiesterase (Cnp) and myelin oligodendrocyte glycoprotein (Mog) were upregulated in both T. canis- and T. cati-infected mice preceding demyelination. Later, CNPase expression was additionally enhanced. As expected, myelin basic protein (Mbp) was downregulated in cerebra and cerebella of T. canis-infected mice when severe demyelination was present 120 days post infectionem (dpi). The transcriptional pattern observed in the present study appears to reflect direct traumatic and hypoxic effects of larval migration as well as secondary processes including host immune reactions, demyelination and attempts to remyelinate damaged areas.

  20. Effects of Transcranial Direct Current Stimulation on Expression of Immediate Early Genes (IEG’s)

    Science.gov (United States)

    2015-12-01

    TRANSCRANIAL DIRECT CURRENT STIMULATION OF EXPRESSION OF IMMEDIATE EARLY GENES (IEG’S) Jessica...AND SUBTITLE Effects of Transcranial Direct Current Stimulation on Expression of Immediate Early Genes (IEG’s) 5a. CONTRACT NUMBER In-House 5b...community in better understanding what is occurring biologically during tDCS. 15. SUBJECT TERMS Transcranial direct current stimulation

  1. The ventralizing activity of Radar, a maternally expressed bone morphogenetic protein, reveals complex bone morphogenetic protein interactions controlling dorso-ventral patterning in zebrafish.

    Science.gov (United States)

    Goutel, C; Kishimoto, Y; Schulte-Merker, S; Rosa, F

    2000-12-01

    In Xenopus and zebrafish, BMP2, 4 and 7 have been implicated, after the onset of zygotic expression, in inducing and maintaining ventro-lateral cell fate during early development. We provide evidence here that a maternally expressed bone morphogenetic protein (BMP), Radar, may control early ventral specification in zebrafish. We show that Radar ventralizes zebrafish embryos and induces the early expression of bmp2b and bmp4. The analysis of Radar overexpression in both swirl/bmp2b mutants and embryos expressing truncated BMP receptors shows that Radar-induced ventralization is dependent on functional BMP2/4 pathways, and may initially rely on an Alk6-related signaling pathway. Finally, we show that while radar-injected swirl embryos still exhibit a strongly dorsalized phenotype, the overexpression of Radar into swirl/bmp2b mutant embryos restores ventral marker expression, including bmp4 expression. Our results suggest that a complex regulation of different BMP pathways controls dorso-ventral (DV) patterning from early cleavage stages until somitogenesis.

  2. Global gene expression analysis of early response to chemotherapy treatment in ovarian cancer spheroids

    Directory of Open Access Journals (Sweden)

    Tetu Bernard

    2008-02-01

    Full Text Available Abstract Background Chemotherapy (CT resistance in ovarian cancer (OC is broad and encompasses diverse unrelated drugs, suggesting more than one mechanism of resistance. To better understand the molecular mechanisms controlling the immediate response of OC cells to CT exposure, we have performed gene expression profiling in spheroid cultures derived from six OC cell lines (OVCAR3, SKOV3, TOV-112, TOV-21, OV-90 and TOV-155, following treatment with 10,0 μM cisplatin, 2,5 μM paclitaxel or 5,0 μM topotecan for 72 hours. Results Exposure of OC spheroids to these CT drugs resulted in differential expression of genes associated with cell growth and proliferation, cellular assembly and organization, cell death, cell cycle control and cell signaling. Genes, functionally involved in DNA repair, DNA replication and cell cycle arrest were mostly overexpressed, while genes implicated in metabolism (especially lipid metabolism, signal transduction, immune and inflammatory response, transport, transcription regulation and protein biosynthesis, were commonly suppressed following all treatments. Cisplatin and topotecan treatments triggered similar alterations in gene and pathway expression patterns, while paclitaxel action was mainly associated with induction of genes and pathways linked to cellular assembly and organization (including numerous tubulin genes, cell death and protein synthesis. The microarray data were further confirmed by pathway and network analyses. Conclusion Most alterations in gene expression were directly related to mechanisms of the cytotoxics actions in OC spheroids. However, the induction of genes linked to mechanisms of DNA replication and repair in cisplatin- and topotecan-treated OC spheroids could be associated with immediate adaptive response to treatment. Similarly, overexpression of different tubulin genes upon exposure to paclitaxel could represent an early compensatory effect to this drug action. Finally, multicellular

  3. Organ-specific gene expression: the bHLH protein Sage provides tissue specificity to Drosophila FoxA.

    Science.gov (United States)

    Fox, Rebecca M; Vaishnavi, Aria; Maruyama, Rika; Andrew, Deborah J

    2013-05-01

    FoxA transcription factors play major roles in organ-specific gene expression, regulating, for example, glucagon expression in the pancreas, GLUT2 expression in the liver, and tyrosine hydroxylase expression in dopaminergic neurons. Organ-specific gene regulation by FoxA proteins is achieved through cooperative regulation with a broad array of transcription factors with more limited expression domains. Fork head (Fkh), the sole Drosophila FoxA family member, is required for the development of multiple distinct organs, yet little is known regarding how Fkh regulates tissue-specific gene expression. Here, we characterize Sage, a bHLH transcription factor expressed exclusively in the Drosophila salivary gland (SG). We show that Sage is required for late SG survival and normal tube morphology. We find that many Sage targets, identified by microarray analysis, encode SG-specific secreted cargo, transmembrane proteins, and the enzymes that modify these proteins. We show that both Sage and Fkh are required for the expression of Sage target genes, and that co-expression of Sage and Fkh is sufficient to drive target gene expression in multiple cell types. Sage and Fkh drive expression of the bZip transcription factor Senseless (Sens), which boosts expression of Sage-Fkh targets, and Sage, Fkh and Sens colocalize on SG chromosomes. Importantly, expression of Sage-Fkh target genes appears to simply add to the tissue-specific gene expression programs already established in other cell types, and Sage and Fkh cannot alter the fate of most embryonic cell types even when expressed early and continuously.

  4. Oracle, a novel PDZ-LIM domain protein expressed in heart and skeletal muscle.

    Science.gov (United States)

    Passier, R; Richardson, J A; Olson, E N

    2000-04-01

    In order to identify novel genes enriched in adult heart, we performed a subtractive hybridization for genes expressed in mouse heart but not in skeletal muscle. We identified two alternative splicing variants of a novel PDZ-LIM domain protein, which we named Oracle. Both variants contain a PDZ domain at the amino-terminus and three LIM domains at the carboxy-terminus. Highest homology of Oracle was found with the human and rat enigma proteins in the PDZ domain (62 and 61%, respectively) and in the LIM domains (60 and 69%, respectively). By Northern hybridization analysis, we showed that expression is highest in adult mouse heart, low in skeletal muscle and undetectable in other adult mouse tissues. In situ hybridization in mouse embryos confirmed and extended these data by showing high expression of Oracle mRNA in atrial and ventricular myocardial cells from E8.5. From E9.5 low expression of Oracle mRNA was detectable in myotomes. These data suggest a role for Oracle in the early development and function of heart and skeletal muscle.

  5. Expression of Water Channel Proteins in Mesembryanthemum crystallinum1

    Science.gov (United States)

    Kirch, Hans-Hubert; Vera-Estrella, Rosario; Golldack, Dortje; Quigley, Francoise; Michalowski, Christine B.; Barkla, Bronwyn J.; Bohnert, Hans J.

    2000-01-01

    We have characterized transcripts for nine major intrinsic proteins (MIPs), some of which function as water channels (aquaporins), from the ice plant Mesembryanthemum crystallinum. To determine the cellular distribution and expression of these MIPs, oligopeptide-based antibodies were generated against MIP-A, MIP-B, MIP-C, or MIP-F, which, according to sequence and functional characteristics, are located in the plasma membrane (PM) and tonoplast, respectively. MIPs were most abundant in cells involved in bulk water flow and solute flux. The tonoplast MIP-F was found in all cells, while signature cell types identified different PM-MIPs: MIP-A predominantly in phloem-associated cells, MIP-B in xylem parenchyma, and MIP-C in the epidermis and endodermis of immature roots. Membrane protein analysis confirmed MIP-F as tonoplast located. MIP-A and MIP-B were found in tonoplast fractions and also in fractions distinct from either the tonoplast or PM. MIP-C was most abundant but not exclusive to PM fractions, where it is expected based on its sequence signature. We suggest that within the cell, MIPs are mobile, which is similar to aquaporins cycling through animal endosomes. MIP cycling and the differential regulation of these proteins observed under conditions of salt stress may be fundamental for the control of tissue water flux. PMID:10806230

  6. 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.

  7. Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli

    Directory of Open Access Journals (Sweden)

    L. Avilán

    1997-12-01

    Full Text Available We cloned the streptokinase (STK gene of Streptococcus equisimilis in an expression vector of Escherichia coli to overexpress the profibrinolytic protein under the control of a tac promoter. Almost all the recombinant STK was exported to the periplasmic space and recovered after gentle lysozyme digestion of induced cells. The periplasmic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0% ammonium sulfate, when a linear gradient was applied. Three major STK derivatives of 47.5 kDa, 45 kDa and 32 kDa were detected by Western blot analysis with a polyclonal antibody. The 32-kDa protein formed a complex with human plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a Km = 0.70 µM and kcat = 0.82 s-1, when assayed with a chromogen-coupled substrate. These results suggest that these proteins are putative fragments of STK, possibly derived from partial degradation during the export pathway or the purification steps. The 47.5-kDa band corresponded to the native STK, as revealed by peptide sequencing

  8. HTLV-1 Tax upregulates early growth response protein 1 through nuclear factor-κB signaling.

    Science.gov (United States)

    Huang, Qingsong; Niu, Zhiguo; Han, Jingxian; Liu, Xihong; Lv, Zhuangwei; Li, Huanhuan; Yuan, Lixiang; Li, Xiangping; Sun, Shuming; Wang, Hui; Huang, Xinxiang

    2017-08-01

    Human T cell leukemia virus type 1 (HTLV-1) is a complex retrovirus that causes adult T cell leukemia (ATL) in susceptible individuals. The HTLV-1-encoded oncoprotein Tax induces persistent activation of the nuclear factor-κB (NF-κB) pathway. Early growth response protein 1 (EGR1) is overexpressed in HTLV-1-infected T cell lines and ATL cells. Here, we showed that both Tax expression and HTLV-1 infection promoted EGR1 overexpression. Loss of the NF-κB binding site in the EGR1 promotor or inhibition of NF-κB activation reduced Tax-induced EGR1 upregulation. Tax mutants unable to activate NF-κB induced only slight EGR1 upregulation as compared with wild-type Tax, confirming NF-κB pathway involvement in EGR1 regulation. Tax also directly interacted with the EGR1 protein and increased endogenous EGR1 stability. Elevated EGR1 in turn promoted p65 nuclear translocation and increased NF-κB activation. These results demonstrate a positive feedback loop between EGR1 expression and NF-κB activation in HTLV-1-infected and Tax-expressing cells. Both NF-κB activation and Tax-induced EGR1 stability upregulated EGR1, which in turn enhanced constitutive NF-κB activation and facilitated ATL progression in HTLV-1-infected cells. These findings suggest EGR1 may be an effective anti-ATL therapeutic target.

  9. Comparative proteomics analysis of chronic atrophic gastritis: changes of protein expression in chronic atrophic gastritis with out Helicobacter pylori infection

    International Nuclear Information System (INIS)

    Zhang, Lin; Hou, Yanhong; Wu, Kai; Li, Dan

    2012-01-01

    Chronic atrophic gastritis (CAG) is a very common gastritis and one of the major precursor lesions of gastric cancer, one of the most common cancers worldwide. The molecular mechanism underlying CAG is unclear, but its elucidation is essential for the prevention and early detection of gastric cancer and appropriate intervention. A combination of two-dimensional gel electrophoresis and mass spectrometry was used in the present study to analyze the differentially expressed proteins. Samples from 21 patients (9 females and 12 males; mean age: 61.8 years) were used. We identified 18 differentially expressed proteins in CAG compared with matched normal mucosa. Eight proteins were up-regulated and 10 down-regulated in CAG when compared with the same amounts of proteins in individually matched normal gastric mucosa. Two novel proteins, proteasome activator subunit 1 (PSME1), which was down-regulated in CAG, and ribosomal protein S12 (RPS12), which was up-regulated in CAG, were further investigated. Their expression was validated by Western blot and RT-PCR in 15 CAG samples matched with normal mucosa. The expression level of RPS12 was significantly higher in CAG than in matched normal gastric mucosa (P < 0.05). In contrast, the expression level of PSME1 in CAG was significantly lower than in matched normal gastric mucosa (P < 0.05). This study clearly demonstrated that there are some changes in protein expression between CAG and normal mucosa. In these changes, down-regulation of PSME1 and up-regulation of RPS12 could be involved in the development of CAG. Thus, the differentially expressed proteins might play important roles in CAG as functional molecules

  10. Comparative proteomics analysis of chronic atrophic gastritis: changes of protein expression in chronic atrophic gastritis with out Helicobacter pylori infection

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Lin; Hou, Yanhong; Wu, Kai; Li, Dan [Department of Gastroenterology and Hepatology, The 309 Hospital of People' s Liberation Army, Beijing (China)

    2012-03-02

    Chronic atrophic gastritis (CAG) is a very common gastritis and one of the major precursor lesions of gastric cancer, one of the most common cancers worldwide. The molecular mechanism underlying CAG is unclear, but its elucidation is essential for the prevention and early detection of gastric cancer and appropriate intervention. A combination of two-dimensional gel electrophoresis and mass spectrometry was used in the present study to analyze the differentially expressed proteins. Samples from 21 patients (9 females and 12 males; mean age: 61.8 years) were used. We identified 18 differentially expressed proteins in CAG compared with matched normal mucosa. Eight proteins were up-regulated and 10 down-regulated in CAG when compared with the same amounts of proteins in individually matched normal gastric mucosa. Two novel proteins, proteasome activator subunit 1 (PSME1), which was down-regulated in CAG, and ribosomal protein S12 (RPS12), which was up-regulated in CAG, were further investigated. Their expression was validated by Western blot and RT-PCR in 15 CAG samples matched with normal mucosa. The expression level of RPS12 was significantly higher in CAG than in matched normal gastric mucosa (P < 0.05). In contrast, the expression level of PSME1 in CAG was significantly lower than in matched normal gastric mucosa (P < 0.05). This study clearly demonstrated that there are some changes in protein expression between CAG and normal mucosa. In these changes, down-regulation of PSME1 and up-regulation of RPS12 could be involved in the development of CAG. Thus, the differentially expressed proteins might play important roles in CAG as functional molecules.

  11. Role of alpha-crystallin, early-secreted antigenic target 6-kDa protein and culture filtrate protein 10 as novel diagnostic markers in osteoarticular tuberculosis

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    Nazia Rizvi

    2016-07-01

    Full Text Available Osteoarticular tuberculosis constitutes about 3% of all tuberculosis cases. Early and accurate diagnosis of tuberculosis is a challenging problem especially in the case of osteoarticular tuberculosis owing to the lower number of bacilli. However, an accurate and timely diagnosis of the disease results in an improved efficacy of the given treatment. Besides the limitations of conventional methods, nowadays molecular diagnostic techniques have emerged as a major breakthrough for the early diagnosis of tuberculosis with high sensitivity and specificity. Alpha-crystallin is a dominantly expressed protein responsible for the long viability of the pathogen during the latent phase under certain stress conditions such as hypoxia and nitric oxide stress. Two other proteins—early secreted antigenic target-6 and culture filtrate protein-10—show high expression in the active infective phase of Mycobacterium tuberculosis. In this article, we focus on the different proteins expressed dominantly in latent/active tuberculosis, and which may be further used as prognostic biomarkers for diagnosing tuberculosis, both in latent and active phases.

  12. Improved protein quality in transgenic soybean expressing a de novo synthetic protein, MB-16.

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    Zhang, Yunfang; Schernthaner, Johann; Labbé, Natalie; Hefford, Mary A; Zhao, Jiping; Simmonds, Daina H

    2014-06-01

    To improve soybean [Glycine max (L.) Merrill] seed nutritional quality, a synthetic gene, MB-16 was introduced into the soybean genome to boost seed methionine content. MB-16, an 11 kDa de novo protein enriched in the essential amino acids (EAAs) methionine, threonine, lysine and leucine, was originally developed for expression in rumen bacteria. For efficient seed expression, constructs were designed using the soybean codon bias, with and without the KDEL ER retention sequence, and β-conglycinin or cruciferin seed specific protein storage promoters. Homozygous lines, with single locus integrations, were identified for several transgenic events. Transgene transmission and MB-16 protein expression were confirmed to the T5 and T7 generations, respectively. Quantitative RT-PCR analysis of developing seed showed that the transcript peaked in growing seed, 5-6 mm long, remained at this peak level to the full-sized green seed and then was significantly reduced in maturing yellow seed. Transformed events carrying constructs with the rumen bacteria codon preference showed the same transcription pattern as those with the soybean codon preference, but the transcript levels were lower at each developmental stage. MB-16 protein levels, as determined by immunoblots, were highest in full-sized green seed but the protein virtually disappeared in mature seed. However, amino acid analysis of mature seed, in the best transgenic line, showed a significant increase of 16.2 and 65.9 % in methionine and cysteine, respectively, as compared to the parent. This indicates that MB-16 elevated the sulfur amino acids, improved the EAA seed profile and confirms that a de novo synthetic gene can enhance the nutritional quality of soybean.

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

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    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.

  14. Type 2 diabetes mellitus with early phase acute inflammatory protein on serum protein electrophoresis

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    ET Tuladhar

    2012-03-01

    Full Text Available Background: The onset of Type 2 diabetes has been associated with low grade systemic inflammation. The inflammatory status has been studied by measuring acute phase reactant proteins like hsCRP, α1- antitrypsin, α1-acid glycoprotein, ceruloplasmin, fibrinogen. Most of these acute phase reactants form α1 and α2 bands on electropherogram of serum proteins. The aim of this study was to evaluate inflammatory status in controlled and uncontrolled type 2 diabetes using cellulose acetate electrophoresis and to find the impact of glycemic status as indicated by HbA1c on inflammation process. Materials and Methods: Serum protein electrophoresis was done on serum samples of 60 cases of Diabetes [controlled and uncontrolled] using cellulose acetate paper technique. The electropherogram obtained was stained with Ponseu S and then quantitated using densitometer. Glycemic status was studied by HbA1c analysis. The density of α1and α2 bands in electropherogram were correlated with HbA1c level. Result: A significant increase in the percentage of α1 and α2 band proteins (0.765 and 0.716, p<0.001 were found with the increasing level of HbA1c. With cutoff of HbA1c 7% (American Diabetic Association recommended, the α1 and α2 serum proteins concentration are significantly higher (p<0.001 in uncontrolled diabetes mellitus compared to controlled diabetes mellitus Conclusion: Cellulose acetate electrophoresis of serum proteins show early phase acute inflammatory status in uncontrolled type 2 diabetes mellitus. The process of systemic inflammation worsens with uncontrolled glycemia as indicated by HbA1c. Inflammatory status should be studied adjunct to glycemic status. DOI: http://dx.doi.org/10.3126/jpn.v2i3.6024 JPN 2012; 2(3: 211-214

  15. Modulation of iridovirus-induced apoptosis by endocytosis, early expression, JNK, and apical caspase

    International Nuclear Information System (INIS)

    Chitnis, Nilesh S.; D'Costa, Susan M.; Paul, Eric R.; Bilimoria, Shaen L.

    2008-01-01

    Chilo iridescent virus (CIV) is the type species for the family Iridoviridae, which are large, isometric, cytoplasmic dsDNA viruses. We examined the mechanism of apoptosis induction by CIV. High CIV doses (CIV XS ; 400 μg/ml), UV-irradiated virus (CIV UV ; 10 μg/ml) and CVPE (CIV protein extract; 10 μg/ml) induced apoptosis in 60% of treated Choristoneura fumiferana (IPRI-CF-124T) cells. Normal doses of infectious CIV (10 μg/ml) induced apoptosis in only 10% of C. fumiferana (CF) cells. Apoptosis was inhibited by Z-IETD-FMK, an apical caspase inhibitor, indicating that CIV-induced apoptosis requires caspase activity. The putative caspase in CF cells was designated Cf-caspase-i. CIV UV or CVPE enhanced Cf-caspase-i activity by 80% at 24 h relative to mock-treated cells. Since the MAP kinase pathway induces or inhibits apoptosis depending on the context, we used JNK inhibitor SP600125 and demonstrated drastic suppression of CVPE-induced apoptosis. Thus, the JNK signaling pathway is significant for apoptosis in this system. Virus interaction with the cell surface was not sufficient for apoptosis since CIV UV particles bound to polysterene beads failed to induce apoptosis. Endocytosis inhibitors (bafilomycin or ammonium chloride) negated apoptosis induction by CIV UV , CIV XS or CVPE indicating that entry through this mode is required. Given the weak apoptotic response to infectious CIV, we postulated that viral gene expression inhibited apoptosis. CIV infection of cells pretreated with cycloheximide induced apoptosis in 69% of the cells compared to 10% in normal infections. Furthermore, blocking viral DNA replication with aphidicolin or phosphonoacetic acid suppressed apoptosis and Cf-caspase-i activity, indicating that early viral expression is necessary for inhibition of apoptosis, and de novo synthesis of viral proteins is not required for induction. We show for the first time that, in a member of the family Iridoviridae, apoptosis: (i) requires entry and

  16. SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm

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    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

  17. Attentional avoidance of fearful facial expressions following early life stress is associated with impaired social functioning.

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    Humphreys, Kathryn L; Kircanski, Katharina; Colich, Natalie L; Gotlib, Ian H

    2016-10-01

    Early life stress is associated with poorer social functioning. Attentional biases in response to threat-related cues, linked to both early experience and psychopathology, may explain this association. To date, however, no study has examined attentional biases to fearful facial expressions as a function of early life stress or examined these biases as a potential mediator of the relation between early life stress and social problems. In a sample of 154 children (ages 9-13 years) we examined the associations among interpersonal early life stressors (i.e., birth through age 6 years), attentional biases to emotional facial expressions using a dot-probe task, and social functioning on the Child Behavior Checklist. High levels of early life stress were associated with both greater levels of social problems and an attentional bias away from fearful facial expressions, even after accounting for stressors occurring in later childhood. No biases were found for happy or sad facial expressions as a function of early life stress. Finally, attentional biases to fearful faces mediated the association between early life stress and social problems. Attentional avoidance of fearful facial expressions, evidenced by a bias away from these stimuli, may be a developmental response to early adversity and link the experience of early life stress to poorer social functioning. © 2016 Association for Child and Adolescent Mental Health.

  18. Inflammatory Gene Expression in Whole Peripheral Blood at Early Stages of Sporadic Amyotrophic Lateral Sclerosis

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    Pol Andrés-Benito

    2017-10-01

    Full Text Available ObjectiveCharacterization of altered expression of selected transcripts linked to inflammation in the peripheral blood of sporadic amyotrophic lateral sclerosis (sALS patients at early stage of disease to increase knowledge about peripheral inflammatory response in sALS.MethodsRNA expression levels of 45 genes were assessed by RT-qPCR in 22 sALS cases in parallel with 13 age-matched controls. Clinical and serum parameters were assessed at the same time.ResultsUpregulation of genes coding for factors involved in leukocyte extravasation (ITGB2, INPP5D, SELL, and ICAM1 and extracellular matrix remodeling (MMP9 and TIMP2, as well as downregulation of certain chemokines (CCL5 and CXC5R, anti-inflammatory cytokines (IL10, TGFB2, and IL10RA, pro-inflammatory cytokines (IL-6, and T-cell regulators (CD2 and TRBC1 was found in sALS cases independently of gender, clinical symptoms at onset (spinal, respiratory, or bulbar, progression, peripheral leukocyte number, and integrity of RNA. MMP9 levels positively correlated with age, whereas CCR5, CCL5, and TRBC1 negatively correlated with age in sALS but not in controls. Relatively higher TNFA expression levels correlate with higher creatinine kinase protein levels in plasma.ConclusionPresent findings show early inflammatory responses characterized by upregulation of factors enabling extravasation of leukocytes and extracellular matrix remodeling in blood in sALS cases, in addition to increased TNFA levels paralleling skeletal muscle damage.

  19. Heat shock protein expression in canine malignant mammary tumours

    International Nuclear Information System (INIS)

    Romanucci, Mariarita; Marinelli, Alessia; Sarli, Giuseppe; Salda, Leonardo Della

    2006-01-01

    Abnormal levels of Heat Shock Proteins (HSPs) have been observed in many human neoplasms including breast cancer and it has been demonstrated that they have both prognostic and therapeutic implications. In this study, we evaluated immunohistochemical expression of HSPs in normal and neoplastic canine mammary glands and confronted these results with overall survival (OS), in order to understand the role of HSPs in carcinogenesis and to establish their potential prognostic and/or therapeutic value. Immunohistochemical expression of Hsp27, Hsp72, Hsp73 and Hsp90 was evaluated in 3 normal canine mammary glands and 30 malignant mammary tumours (10 in situ carcinomas, 10 invasive carcinomas limited to local structures without identifiable invasion of blood or lymphatic vessels, 10 carcinomas with invasion of blood or lymphatic vessels and/or metastases to regional lymph nodes). A semi-quantitative method was used for the analysis of the results. Widespread constitutive expression of Hsp73 and Hsp90 was detected in normal tissue, Hsp72 appeared to be focally distributed and Hsp27 showed a negative to rare weak immunostaining. In mammary tumours, a significant increase in Hsp27 (P < 0.01), Hsp72 (P < 0.05) and Hsp90 (P < 0.01) expression was observed as well as a significant reduction in Hsp73 (P < 0.01) immunoreactivity compared to normal mammary gland tissue. Hsp27 demonstrated a strong positivity in infiltrating tumour cells and metaplastic squamous elements of invasive groups. High Hsp27 expression also appeared to be significantly correlated to a shorter OS (P = 0.00087). Intense immunolabelling of Hsp72 and Hsp73 was frequently detected in infiltrative or inflammatory tumour areas. Hsp90 expression was high in all tumours and, like Hsp73, it also showed an intense positivity in lymphatic emboli. These results suggest that Hsp27, Hsp72 and Hsp90 are involved in canine mammary gland carcinogenesis. In addition, Hsp27 appears to be implicated in tumour invasiveness and

  20. Resveratrol Modulation of Protein Expression in parkin-Mutant Human Skin Fibroblasts: A Proteomic Approach

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    Daniele Vergara

    2017-01-01

    Full Text Available In this study, we investigated by two-dimensional gel electrophoresis (2-DE and mass spectrometry (MS analysis the effects of resveratrol treatment on skin primary fibroblasts from a healthy subject and from a parkin-mutant early onset Parkinson’s disease patient. Parkin, an E3 ubiquitin ligase, is the most frequently mutated gene in hereditary Parkinson’s disease. Functional alteration of parkin leads to impairment of the ubiquitin-proteasome system, resulting in the accumulation of misfolded or aggregated proteins accountable for the neurodegenerative process. The identification of proteins differentially expressed revealed that resveratrol treatment can act on deregulated specific biological process and molecular function such as cellular redox balance and protein homeostasis. In particular, resveratrol was highly effective at restoring the heat-shock protein network and the protein degradation systems. Moreover, resveratrol treatment led to a significant increase in GSH level, reduction of GSSG/GSH ratio, and decrease of reduced free thiol content in patient cells compared to normal fibroblasts. Thus, our findings provide an experimental evidence of the beneficial effects by which resveratrol could contribute to preserve the cellular homeostasis in parkin-mutant fibroblasts.

  1. Resveratrol Modulation of Protein Expression in parkin-Mutant Human Skin Fibroblasts: A Proteomic Approach

    Science.gov (United States)

    Gaballo, Antonio; Signorile, Anna; Tanzarella, Paola; Pacelli, Consiglia; Di Paola, Marco

    2017-01-01

    In this study, we investigated by two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) analysis the effects of resveratrol treatment on skin primary fibroblasts from a healthy subject and from a parkin-mutant early onset Parkinson's disease patient. Parkin, an E3 ubiquitin ligase, is the most frequently mutated gene in hereditary Parkinson's disease. Functional alteration of parkin leads to impairment of the ubiquitin-proteasome system, resulting in the accumulation of misfolded or aggregated proteins accountable for the neurodegenerative process. The identification of proteins differentially expressed revealed that resveratrol treatment can act on deregulated specific biological process and molecular function such as cellular redox balance and protein homeostasis. In particular, resveratrol was highly effective at restoring the heat-shock protein network and the protein degradation systems. Moreover, resveratrol treatment led to a significant increase in GSH level, reduction of GSSG/GSH ratio, and decrease of reduced free thiol content in patient cells compared to normal fibroblasts. Thus, our findings provide an experimental evidence of the beneficial effects by which resveratrol could contribute to preserve the cellular homeostasis in parkin-mutant fibroblasts. PMID:29138676

  2. Cellular prion protein expression is not regulated by the Alzheimer's amyloid precursor protein intracellular domain.

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    Victoria Lewis

    Full Text Available There is increasing evidence of molecular and cellular links between Alzheimer's disease (AD and prion diseases. The cellular prion protein, PrP(C, modulates the post-translational processing of the AD amyloid precursor protein (APP, through its inhibition of the β-secretase BACE1, and oligomers of amyloid-β bind to PrP(C which may mediate amyloid-β neurotoxicity. In addition, the APP intracellular domain (AICD, which acts as a transcriptional regulator, has been reported to control the expression of PrP(C. Through the use of transgenic mice, cell culture models and manipulation of APP expression and processing, this study aimed to clarify the role of AICD in regulating PrP(C. Over-expression of the three major isoforms of human APP (APP(695, APP(751 and APP(770 in cultured neuronal and non-neuronal cells had no effect on the level of endogenous PrP(C. Furthermore, analysis of brain tissue from transgenic mice over-expressing either wild type or familial AD associated mutant human APP revealed unaltered PrP(C levels. Knockdown of endogenous APP expression in cells by siRNA or inhibition of γ-secretase activity also had no effect on PrP(C levels. Overall, we did not detect any significant difference in the expression of PrP(C in any of the cell or animal-based paradigms considered, indicating that the control of cellular PrP(C levels by AICD is not as straightforward as previously suggested.

  3. Optimised 'on demand' protein arraying from DNA by cell free expression with the 'DNA to Protein Array' (DAPA) technology.

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    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. Two different protein expression profiles of oral squamous cell carcinoma analyzed by immunoprecipitation high-performance liquid chromatography.

    Science.gov (United States)

    Kim, Soung Min; Jeong, Dasul; Kim, Min Keun; Lee, Sang Shin; Lee, Suk Keun

    2017-08-08

    Oral squamous cell carcinoma (OSCC) is one of the most dangerous cancers in the body, producing serious complications with individual behaviors. Many different pathogenetic factors are involved in the carcinogenesis of OSCC. Cancer cells derived from oral keratinocytes can produce different carcinogenic signaling pathways through differences in protein expression, but their protein expression profiles cannot be easily explored with ordinary detection methods. The present study compared the protein expression profiles between two different types of OSCCs, which were analyzed through immunoprecipitation high-performance liquid chromatography (IP-HPLC). Two types of squamous cell carcinoma (SCC) occurred in a mandibular (SCC-1) and maxillary gingiva (SCC-2), but their clinical features and progression were quite different from each other. SCC-1 showed a large gingival ulceration with severe halitosis and extensive bony destruction, while SCC-2 showed a relatively small papillary gingival swelling but rapidly grew to form a large submucosal mass, followed by early cervical lymph node metastasis. In the histological observation, SCC-1 was relatively well differentiated with a severe inflammatory reaction, while SCC-2 showed severely infiltrative growth of each cancer islets accompanied with a mild inflammatory reaction. IP-HPLC analysis revealed contrary protein expression profiles analyzed by 72 different oncogenic proteins. SCC-1 showed more cellular apoptosis and invasive growth than SCC-2 through increased expression of caspases, MMPs, p53 signaling, FAS signaling, TGF-β1 signaling, and angiogenesis factors, while SCC-2 showed more cellular growth and survival than SCC-1 through the increased expression of proliferating factors, RAS signaling, eIF5A signaling, WNT signaling, and survivin. The increased trends of cellular apoptosis and invasiveness in the protein expression profiles of SCC-1 were implicative of its extensive gingival ulceration and bony destruction

  5. Modulation of PML protein expression regulates JCV infection

    International Nuclear Information System (INIS)

    Gasparovic, Megan L.; Maginnis, Melissa S.; O'Hara, Bethany A.; Dugan, Aisling S.; Atwood, Walter J.

    2009-01-01

    JC virus (JCV) is a human polyomavirus that infects the majority of the human population worldwide. It is responsible for the fatal demyelinating disease Progressive Multifocal Leukoencephalopathy. JCV binds to cells using the serotonin receptor 5-HT 2A R and α(2-6)- or α(2-3)-linked sialic acid. It enters cells using clathrin-dependent endocytosis and traffics to the early endosome and possibly to the endoplasmic reticulum. Viral DNA is then delivered to the nucleus where transcription, replication, and assembly of progeny occur. We found that the early regulatory protein large T antigen accumulates in microdomains in the nucleus adjacent to ND-10 or PML domains. This observation prompted us to explore the role of these domains in JCV infection. We found that a reduction of nuclear PML enhanced virus infection and that an increase in nuclear PML reduced infection. Infection with JCV did not directly modulate nuclear levels of PML but our data indicate that a host response involving interferon beta is likely to restrict virus infection by increasing nuclear PML.

  6. Recombinant protein production data after expression in the bacterium Escherichia coli

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    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.

  7. Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression

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    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

  8. Proteomic analysis of differential protein expression of achilles tendon in a rabbit model by two-dimensional polyacrylamide gel electrophoresis at 21 days postoperation.

    Science.gov (United States)

    Jielile, Jiasharete; Jialili, Ainuer; Sabirhazi, Gulnur; Shawutali, Nuerai; Redati, Darebai; Chen, Jiangtao; Tang, Bin; Bai, Jingping; Aldyarhan, Kayrat

    2011-10-01

    Postoperative early kinesitherapy has been advocated as an optimal method for treating Achilles tendon rupture. However, an insight into the rationale of how early kinesitherapy contributes to healing of Achilles tendon remains to be achieved, and research in the area of proteomic analysis of Achilles tendon has so far been lacking. Forty-two rabbits were randomized into control group, immobilization group, and early motion group, and received postoperative cast immobilization and early motion treatments. Achilles tendon samples were prepared 21 days following microsurgery, and the proteins were separated with two-dimensional polyacrylamide gel electrophoresis. Differentially expressed proteins were first recognized by PDQuest software, and then identified using peptide mass fingerprinting, tandem mass spectrometry, and database searching. A total of 463  ±  12, 511  ±  39, and 513  ±  80 protein spots were successfully detected in the two-dimensional polyacrylamide gels for the Achilles tendon samples of rabbits in the control group, immobilization group, and early motion group, respectively. There were 15, 8, and 9 unique proteins in these three groups, respectively, and some differentially expressed proteins were also identified in each group. It was indicated that some of the differentially expressed proteins were involved in various metabolism pathways and may play an important role in healing of Achilles tendon rupture. Postoperative early kinesitherapy resulted in differentially expressed proteins in ruptured Achilles tendon compared with those treated with postoperative cast immobilization. These differentially expressed proteins may contribute to healing of Achilles tendon rupture through a mechanobiological mechanism due to the application of postoperative early kinesitherapy.

  9. Apoptosis and cell proliferation in the development of gastric carcinomas: associations with c-myc and p53 protein expression.

    Science.gov (United States)

    Ishii, Hideaki H; Gobé, Glenda C; Pan, Wenshen; Yoneyama, Juichi; Ebihara, Yoshiro

    2002-09-01

    Patients with gastric carcinomas have a poor prognosis and low survival rates. The aim of the present paper was to characterize cellular and molecular properties to provide insight into aspects of tumor progression in early compared with advanced gastric cancers. One hundred and nine graded gastric carcinomas (early or advanced stage, undifferentiated or differentiated type) with paired non-cancer tissue were studied to define the correlation between apoptosis (morphology, terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling), cell proliferation (Ki-67 expression, morphology) and expression and localization of two proteins frequently having altered expression in cancers, namely p53 and c-myc. Overall, apoptosis was lower in early stage, differentiated and undifferentiated gastric carcinomas compared with advanced-stage cancers. Cell proliferation was comparatively high in all stages. There was a high level of p53 positivity in all stages. Only the early- and advanced-stage undifferentiated cancers that were p53 positive had a significantly higher level of apoptosis (P cancers that had either c-myc or p53-positivity. The results indicate that low apoptosis and high cell proliferation combine to drive gastric cancer development. The molecular controls for high cell proliferation of the early stage undifferentiated gastric cancers involve overexpression of both p53 and c-myc. Overexpression of p53 may also control cancer development in that its expression is associated with higher levels of apoptosis in early and late-stage undifferentiated, cancers. Copyright 2002 Blackwell Publishing Asia Pty Ltd

  10. Protein 53 expression in a mixed Labrador subcutaneous lymphoma

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    Annahita Rezaie

    2012-06-01

    Full Text Available An 11 year – old mixed female Labrador was presented with two masses in trunk and neck. The tumoral masses were excised and sent for histopathological and immunohistochemical analyses. Histopathological examination of masses revealed diffuse infiltration of small sized lymphoid cells in subcutaneous tissue which were intense around the blood vessels. More than 10% lymphoid cells were CD3 positive in the immunohistochemical staining and most of them were accumulated around vessels. Protein 53 (p53 expression was detected by brown nuclei in immunohistochemical staining. Subcutaneous lymphoma was diagnosed according to histopathological results. After 6 months the case was referred with multicentric lymphoma and based on the owner request euthanasia was performed. These findings emphasize on poor prognosis for tumors with p53 mutation.

  11. 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

  12. Transcription mapping and expression patterns of genes in the major immediate-early region of Kaposi's sarcoma-associated herpesvirus.

    Science.gov (United States)

    Saveliev, Alexei; Zhu, Fan; Yuan, Yan

    2002-08-01

    Viral immediate-early (IE) genes are the first class of viral genes expressed during primary infection or reactivation from latency. They usually encode regulatory proteins that play crucial roles in viral life cycle. In a previous study, four regions in the KSHV genome were found to be actively transcribed in the immediate-early stage of viral reactivation in primary effusion lymphoma cells. Three immediate-early transcripts were characterized in these regions, as follows: mRNAs for ORF50 (KIE-1), ORF-45 (KIE-2), and ORF K4.2 (KIE-3) (F. X. Zhu, T. Cusano, and Y. Yuan, 1999, J. Virol. 73, 5556-5567). In the present study, we further analyzed the expression of genes in these IE regions in BC-1 and BCBL-1 cells. One of the immediate-early regions (KIE-1) that encompasses ORF50 and other genes was intensively studied to establish a detailed transcription map and expression patterns of genes in this region. This study led to identification of several novel IE transcripts in this region. They include a 2.6-kb mRNA which encodes ORF48/ORF29b, a family of transcripts that are complementary to ORF50 mRNA and a novel K8 IE mRNA of 1.5 kb. Together with the IE mRNA for ORF50 which was identified previously, four immediate-early genes have been mapped to KIE-1 region. Therefore, we would designate KIE-1 the major immediate-early region of KSHV. In addition, we showed that transcription of K8 gene is controlled by two promoters, yielding two transcripts, an immediate-early mRNA of 1.5 kb and a delayed-early mRNA of 1.3 kb.

  13. Protein expression of saccharomyces cerevisiae in response to uranium exposure

    International Nuclear Information System (INIS)

    Sakamoto, Fuminori; Nankawa, Takuya; Kozai, Naofumi; Ohnuki, Toshihiko; Fujii, Tsutomu; Iefuji, Haruyuki; Francis, A.J.

    2007-01-01

    Protein expression of Saccharomyces cerevisiae grown in the medium containing 238 U (VI) and 233 U (VI) was examined by two-dimensional gel electrophoresis. Saccharomyces cerevisiae of BY4743 was grown in yeast nitrogen base medium containing glucose and glycerol 2-phosphate and 238 U of 0, 2.0, and 5.0 x 10 -4 M or 233 U of 2.5 x 10 -6 M (radioactivity was higher by 350 times than 2.0 x 10 -4 M 238 U) and 5.0 x 10 -6 M for 112 h at 30 degC. The growth of Saccharomyces cerevisiae was monitored by measuring OD 600 at 112 h after the inoculation. Uranium concentrations in the media also were measured by radiometry using a liquid scintillation counter. The growths of the yeast grown in the above media were in the following order: control>2.5 x 10 -6 M 233 U>2.0 x 10 -4 M 238 U>5.0 x 10 -6 M 233 U>5.0 x 10 -4 M 238 U. This result indicated that not only radiological but also chemical effect of U reduced the growth of the yeast. The concentrations of U in the medium containing 238 U or 233 U decreased, suggesting U accumulation by the yeast cells. The 2-D gel electrophoresis analysis showed the appearance of several spots after exposure to 238 U or to 233 U but not in the control containing no uranium. These results show that the yeast cells exposed to U express several specific proteins. (author)

  14. [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.

  15. Copper induces expression and methylation changes of early development genes in Crassostrea gigas embryos.

    Science.gov (United States)

    Sussarellu, Rossana; Lebreton, Morgane; Rouxel, Julien; Akcha, Farida; Rivière, Guillaume

    2018-03-01

    Copper contamination is widespread along coastal areas and exerts adverse effects on marine organisms such as mollusks. In the Pacific oyster, copper induces severe developmental abnormalities during early life stages; however, the underlying molecular mechanisms are largely unknown. This study aims to better understand whether the embryotoxic effects of copper in Crassostrea gigas could be mediated by alterations in gene expression, and the putative role of DNA methylation, which is known to contribute to gene regulation in early embryo development. For that purpose, oyster embryos were exposed to 4 nominal copper concentrations (0.1, 1, 10 and 20 μg L -1 Cu 2+ ) during early development assays. Embryotoxicity was monitored through the oyster embryo-larval bioassay at the D-larva stage 24 h post fertilization (hpf) and genotoxicity at gastrulation 7 hpf. In parallel, the relative expression of 15 genes encoding putative homeotic, biomineralization and DNA methylation proteins was measured at three developmental stages (3 hpf morula stage, 7 hpf gastrula stage, 24 hpf D-larvae stage) using RT-qPCR. Global DNA content in methylcytosine and hydroxymethylcytosine were measured by HPLC and gene-specific DNA methylation levels were monitored using MeDIP-qPCR. A significant increase in larval abnormalities was observed from copper concentrations of 10 μg L -1 , while significant genotoxic effects were detected at 1 μg L -1 and above. All the selected genes presented a stage-dependent expression pattern, which was impaired for some homeobox and DNA methylation genes (Notochord, HOXA1, HOX2, Lox5, DNMT3b and CXXC-1) after copper exposure. While global DNA methylation (5-methylcytosine) at gastrula stage didn't show significant changes between experimental conditions, 5-hydroxymethylcytosine, its degradation product, decreased upon copper treatment. The DNA methylation of exons and the transcript levels were correlated in control samples for HOXA1 but such

  16. 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.

  17. Expression of FGFs during early mouse tongue development

    Czech Academy of Sciences Publication Activity Database

    Du, W.; Procházka, Jan; Procházková, Michaela; Klein, O.D.

    2016-01-01

    Roč. 20, č. 2 (2016), s. 81-87 ISSN 1567-133X Institutional support: RVO:68378050 Keywords : Tongue * FGF * Expression * Papilla Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.158, year: 2016

  18. Transcriptome Sequencing, De Novo Assembly and Differential Gene Expression Analysis of the Early Development of Acipenser baeri.

    Directory of Open Access Journals (Sweden)

    Wei Song

    Full Text Available The molecular mechanisms that drive the development of the endangered fossil fish species Acipenser baeri are difficult to study due to the lack of genomic data. Recent advances in sequencing technologies and the reducing cost of sequencing offer exclusive opportunities for exploring important molecular mechanisms underlying specific biological processes. This manuscript describes the large scale sequencing and analyses of mRNA from Acipenser baeri collected at five development time points using the Illumina Hiseq2000 platform. The sequencing reads were de novo assembled and clustered into 278167 unigenes, of which 57346 (20.62% had 45837 known homologues proteins in Uniprot protein databases while 11509 proteins matched with at least one sequence of assembled unigenes. The remaining 79.38% of unigenes could stand for non-coding unigenes or unigenes specific to A. baeri. A number of 43062 unigenes were annotated into functional categories via Gene Ontology (GO annotation whereas 29526 unigenes were associated with 329 pathways by mapping to KEGG database. Subsequently, 3479 differentially expressed genes were scanned within developmental stages and clustered into 50 gene expression profiles. Genes preferentially expressed at each stage were also identified. Through GO and KEGG pathway enrichment analysis, relevant physiological variations during the early development of A. baeri could be better cognized. Accordingly, the present study gives insights into the transcriptome profile of the early development of A. baeri, and the information contained in this large scale transcriptome will provide substantial references for A. baeri developmental biology and promote its aquaculture research.

  19. Expression and proteasomal degradation of the major vault protein (MVP) in mammalian oocytes and zygotes.

    Science.gov (United States)

    Sutovsky, Peter; Manandhar, Gaurishankar; Laurincik, Jozef; Letko, Juraj; Caamaño, Jose Nestor; Day, Billy N; Lai, Liangxue; Prather, Randall S; Sharpe-Timms, Kathy L; Zimmer, Randall; Sutovsky, Miriam

    2005-03-01

    Major vault protein (MVP), also called lung resistance-related protein is a ribonucleoprotein comprising a major part (>70%) of the vault particle. The function of vault particle is not known, although it appears to be involved in multi-drug resistance and cellular signaling. Here we show that MVP is expressed in mammalian, porcine, and human ova and in the porcine preimplantation embryo. MVP was identified by matrix-assisted laser-desorption ionization-time-of-flight (MALDI-TOF) peptide sequencing and Western blotting as a protein accumulating in porcine zygotes cultured in the presence of specific proteasomal inhibitor MG132. MVP also accumulated in poor-quality human oocytes donated by infertile couples and porcine embryos that failed to develop normally after in vitro fertilization or somatic cell nuclear transfer. Normal porcine oocytes and embryos at various stages of preimplantation development showed mostly cytoplasmic labeling, with increased accumulation of vault particles around large cytoplasmic lipid inclusions and membrane vesicles. Occasionally, MVP was associated with the nuclear envelope and nucleolus precursor bodies. Nucleotide sequences with a high degree of homology to human MVP gene sequence were identified in porcine oocyte and endometrial cell cDNA libraries. We interpret these data as the evidence for the expression and ubiquitin-proteasome-dependent turnover of MVP in the mammalian ovum. Similar to carcinoma cells, MVP could fulfill a cell-protecting function during early embryonic development.

  20. Analysis of the expression and antioxidant activity of 2-Cys peroxiredoxin protein in Fasciola gigantica.

    Science.gov (United States)

    Sangpairoj, Kant; Changklungmoa, Narin; Vanichviriyakit, Rapeepun; Sobhon, Prasert; Chaithirayanon, Kulathida

    2014-05-01

    2-Cys peroxiredoxin (Prx) is the main antioxidant enzyme in Fasciola species for detoxifying hydrogen peroxide which is generated from the hosts' immune effector cells and the parasites' own metabolism. In this study, the recombinant Prx protein from Fasciola gigantica (rFgPrx-2) was expressed and purified in a prokaryotic expression system. This recombinant protein with molecular weight of 26 kDa was enzymatically active in reduction of hydrogen peroxide both in presence of thioredoxin and glutathione systems, and also protected the supercoiled plasmid DNA from oxidative damage in metal-catalyzed oxidation (MCO) system in a concentration-dependent manner. By immunoblotting, using antibody against rFgPrx-2 as probe, a native FgPrxs, whose MW at 25 kDa, was detected in all developmental stages of the parasite. Concentrations of native FgPrxs were increasing in all stages reaching highest level in adult stage. The antibody also showed cross reactivities with corresponding proteins in some cattle helminthes. Natural antibody to FgPrxs could be detected in the sera of mice at 3 and 4 weeks after infection with F. gigantica metacercariae. By immunofluorescence, FgPrxs was highly expressed in tegument and tegumental cells, parenchyma, moderately expressed in cecal epithelial cells in early, juvenile and adult worms. Furthermore, FgPrxs was also detected in the female reproductive organs, including eggs, ovary, vitelline cells, and testis, suggesting that FgPrxs might play an essential role in protecting parasite's tissues from free radical attack during their life cycle. Thus, FgPrxs is one potential candidate for drug therapy and vaccine development. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. 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.

  2. 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.

  3. Early Postoperative Low Expression of RAD50 in Rectal Cancer Patients Associates with Disease-Free Survival

    Directory of Open Access Journals (Sweden)

    Vincent Ho

    2017-11-01

    Full Text Available Background: Molecular biomarkers have the potential to predict response to the treatment of rectal cancer. In this study, we aimed to evaluate the prognostic and clinicopathological implication of RAD50 (DNA repair protein RAD50 homolog expression in rectal cancer. Methods: A total of 266 rectal cancer patients who underwent surgery and received chemo- and radiotherapy between 2000 and 2011 were involved in the study. Postoperative RAD50 expression was determined by immunohistochemistry in surgical samples (n = 266. Results: Using Kaplan–Meier survival analysis, we found that low RAD50 expression in postoperative samples was associated with worse disease free survival (p = 0.001 and overall survival (p < 0.001 in early stage/low-grade tumors. In a comparison of patients with low vs. high RAD50 expression, we found that low levels of postoperative RAD50 expression in rectal cancer tissues were significantly associated with perineural invasion (p = 0.002. Conclusion: Expression of RAD50 in rectal cancer may serve as a prognostic biomarker for long-term survival of patients with perineural invasion-positive tumors and for potential use in early stage and low-grade rectal cancer assessment.

  4. The presence of p53 influences the expression of multiple human cytomegalovirus genes at early times postinfection.

    Science.gov (United States)

    Hannemann, Holger; Rosenke, Kyle; O'Dowd, John M; Fortunato, Elizabeth A

    2009-05-01

    Human cytomegalovirus (HCMV) is a common cause of morbidity and mortality in immunocompromised and immunosuppressed individuals. During infection, HCMV is known to employ host transcription factors to facilitate viral gene expression. To further understand the previously observed delay in viral replication and protein expression in p53 knockout cells, we conducted microarray analyses of p53(+/+) and p53(-/-) immortalized fibroblast cell lines. At a multiplicity of infection (MOI) of 1 at 24 h postinfection (p.i.), the expression of 22 viral genes was affected by the absence of p53. Eleven of these 22 genes (group 1) were examined by real-time reverse transcriptase, or quantitative, PCR (q-PCR). Additionally, five genes previously determined to have p53 bound to their nearest p53-responsive elements (group 2) and three control genes without p53 binding sites in their upstream sequences (group 3) were also examined. At an MOI of 1, >3-fold regulation was found for five group 1 genes. The expression of group 2 and 3 genes was not changed. At an MOI of 5, all genes from group 1 and four of five genes from group 2 were found to be regulated. The expression of control genes from group 3 remained unchanged. A q-PCR time course of four genes revealed that p53 influences viral gene expression most at immediate-early and early times p.i., suggesting a mechanism for the reduced and delayed production of virions in p53(-/-) cells.

  5. 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.

  6. 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.

  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. 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.

  9. Kazakh therapy on differential protein expression of Achilles tendon healing in a 7-day postoperative rabbit model.

    Science.gov (United States)

    Nuerai, Shawutali; Ainuer, Jialili; Jiasharete, Jielile; Darebai, Redati; Kayrat, Aldyarhan; Tang, Bin; Jiangannur, Zheyiken; Bai, Jingping; Makabel, Bolat

    2011-12-01

    To compare the effect of cast immobilization with that of early Kiymil arkili emdew (Kazakh exercise therapy) on the post-operative healing of Achilles tendon rupture in rabbits, and to observe the influence of early Kiymil arkili emdew on the differentially expressed proteins in the healing tendon. Forty-five New Zealand white rabbits were randomly divided into three groups (Arm A: control group; Arm B: postoperative immobilization group; and Arm C: postoperative early Kiymil arkili emdew group). After tenotomy, the rabbits of the two experimental groups received microsurgery to repair the ruptured tendons, and then received either cast immobilization or early Kiymil arkili emdew treatment. Achilles tendon tissue samples were collected 7 days after the surgery, and two-dimensional gel electrophoresis and MALDI-TOF-MS technique were used to analyze differentially expressed proteins in the tendon tissue of the three Arms. A total of 462.67 +/- 11.59, 532.33 +/- 27.79, and 515.33 +/- 6.56 protein spots were detected by the two-dimensional polyacrylamide gels in the Achilles tendon samples of the rabbits in Arms A, B, and C, respectively. Nineteen differentially expressed protein spots were randomly selected from Arm C. Among them, 7 were unique, and 15 had five times higher abundance than those in Arm B. These included annexin A2, gelsolin isoforms and alpha-1 Type III collagen. It was confirmed by western blot that gelsolin isoform b, annexin A2, etc. had specific and incremental expression in Arm C. The self-protective instincts of humans were overlooked in the classical postoperative treatment for Achilles tendon rupture with cast immobilization. Kiymil arkili emdew induced the specific and incremental expression of proteins in the repaired Achilles tendon in the early healing stage in a rabbit model, compared with those treated with postoperative cast immobilization. These differentially expressed proteins may contribute to the healing of the Achilles tendon via

  10. Inhibition of iridovirus protein synthesis and virus replication by antisense morpholino oligonucleotides targeted to the major capsid protein, the 18 kDa immediate-early protein, and a viral homolog of RNA polymerase II

    International Nuclear Information System (INIS)

    Sample, Robert; Bryan, Locke; Long, Scott; Majji, Sai; Hoskins, Glenn; Sinning, Allan; Olivier, Jake; Chinchar, V. Gregory

    2007-01-01

    Frog virus 3 (FV3) is a large DNA virus that encodes ∼ 100 proteins. Although the general features of FV3 replication are known, the specific roles that most viral proteins play in the virus life cycle have not yet been elucidated. To address the question of viral gene function, antisense morpholino oligonucleotides (asMOs) were used to transiently knock-down expression of specific viral genes and thus infer their role in virus replication. We designed asMOs directed against the major capsid protein (MCP), an 18 kDa immediate-early protein (18K) that was thought to be a viral regulatory protein, and the viral homologue of the largest subunit of RNA polymerase II (vPol-IIα). All three asMOs successfully inhibited translation of the targeted protein, and two of the three asMOs resulted in marked phenotypic changes. Knock-down of the MCP resulted in a marked reduction in viral titer without a corresponding drop in the synthesis of other late viral proteins. Transmission electron microscopy (TEM) showed that in cells treated with the anti-MCP MO assembly sites were devoid of viral particles and contained numerous aberrant structures. In contrast, inhibition of 18K synthesis did not block virion formation, suggesting that the 18K protein was not essential for replication of FV3 in fathead minnow (FHM) cells. Finally, consistent with the view that late viral gene expression is catalyzed by a virus-encoded or virus-modified Pol-II-like protein, knock-down of vPol-IIα triggered a global decline in late gene expression and virus yields without affecting the synthesis of early viral genes. Collectively, these results demonstrate the utility of using asMOs to elucidate the function of FV3 proteins

  11. An unbiased expression screen for synaptogenic proteins identifies the LRRTM protein family as synaptic organizers.

    Science.gov (United States)

    Linhoff, Michael W; Laurén, Juha; Cassidy, Robert M; Dobie, Frederick A; Takahashi, Hideto; Nygaard, Haakon B; Airaksinen, Matti S; Strittmatter, Stephen M; Craig, Ann Marie

    2009-03-12

    Delineating the molecular basis of synapse development is crucial for understanding brain function. Cocultures of neurons with transfected fibroblasts have demonstrated the synapse-promoting activity of candidate molecules. Here, we performed an unbiased expression screen for synaptogenic proteins in the coculture assay using custom-made cDNA libraries. Reisolation of NGL-3/LRRC4B and neuroligin-2 accounts for a minority of positive clones, indicating that current understanding of mammalian synaptogenic proteins is incomplete. We identify LRRTM1 as a transmembrane protein that induces presynaptic differentiation in contacting axons. All four LRRTM family members exhibit synaptogenic activity, LRRTMs localize to excitatory synapses, and artificially induced clustering of LRRTMs mediates postsynaptic differentiation. We generate LRRTM1(-/-) mice and reveal altered distribution of the vesicular glutamate transporter VGLUT1, confirming an in vivo synaptic function. These results suggest a prevalence of LRR domain proteins in trans-synaptic signaling and provide a cellular basis for the reported linkage of LRRTM1 to handedness and schizophrenia.

  12. 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.

  13. Protein needs early in life and long-term health

    DEFF Research Database (Denmark)

    Michaelsen, Kim F.; Greer, Frank R

    2014-01-01

    foods have been introduced. At this age, mean protein intake is ∼3 times as high as the physiologic requirement, but some children receive 4-5 times their physiologic requirement. Protein from cow milk constitutes a main part of protein intake in toddlers and seems to have a specific effect on insulin......-like growth factor I concentrations and growth. Meat has a high protein content, but the small amounts of meat needed to ensure good iron status have less impact on total protein intake. The difference in protein intake between breastfed and formula-fed infants is likely to play a role in the difference...... between breastfed and formula-fed infants. There is emerging evidence that high protein intake during the first 2 y of life is a risk factor for later development of overweight and obesity. It therefore seems prudent to avoid a high protein intake during the first 2 y of life. This could be accomplished...

  14. Resistin in Dairy Cows: Plasma Concentrations during Early Lactation, Expression and Potential Role in Adipose Tissue

    Science.gov (United States)

    Reverchon, Maxime; Ramé, Christelle; Cognié, Juliette; Briant, Eric; Elis, Sébastien; Guillaume, Daniel; Dupont, Joëlle

    2014-01-01

    Resistin is an adipokine that has been implicated in energy metabolism regulation in rodents but has been little studied in dairy cows. We determined plasma resistin concentrations in early lactation in dairy cows and investigated the levels of resistin mRNA and protein in adipose tissue and the phosphorylation of several components of insulin signaling pathways one week post partum (1 WPP) and at five months of gestation (5 MG). We detected resistin in mature bovine adipocytes and investigated the effect of recombinant bovine resistin on lipolysis in bovine adipose tissue explants. ELISA showed that plasma resistin concentration was low before calving, subsequently increasing and reaching a peak at 1 WPP, decreasing steadily thereafter to reach pre-calving levels at 6 WPP. Plasma resistin concentration was significantly positively correlated with plasma non esterified fatty acid (NEFA) levels and negatively with milk yield, dry matter intake and energy balance between WPP1 to WPP22. We showed, by quantitative RT-PCR and western blotting, that resistin mRNA and protein levels in adipose tissue were higher at WPP1 than at 5 MG. The level of phosphorylation of several early and downstream insulin signaling components (IRβ, IRS-1, IRS-2, Akt, MAPK ERK1/2, P70S6K and S6) in adipose tissue was also lower at 1 WPP than at 5 MG. Finally, we showed that recombinant bovine resistin increased the release of glycerol and mRNA levels for ATGL (adipose triglyceride lipase) and HSL (hormone-sensitive lipase) in adipose tissue explants. Overall, resistin levels were high in the plasma and adipose tissue and were positively correlated with NEFA levels after calving. Resistin is expressed in bovine mature adipocytes and promotes lipid mobilization in adipose explants in vitro. PMID:24675707

  15. Resistin in dairy cows: plasma concentrations during early lactation, expression and potential role in adipose tissue.

    Directory of Open Access Journals (Sweden)

    Maxime Reverchon

    Full Text Available Resistin is an adipokine that has been implicated in energy metabolism regulation in rodents but has been little studied in dairy cows. We determined plasma resistin concentrations in early lactation in dairy cows and investigated the levels of resistin mRNA and protein in adipose tissue and the phosphorylation of several components of insulin signaling pathways one week post partum (1 WPP and at five months of gestation (5 MG. We detected resistin in mature bovine adipocytes and investigated the effect of recombinant bovine resistin on lipolysis in bovine adipose tissue explants. ELISA showed that plasma resistin concentration was low before calving, subsequently increasing and reaching a peak at 1 WPP, decreasing steadily thereafter to reach pre-calving levels at 6 WPP. Plasma resistin concentration was significantly positively correlated with plasma non esterified fatty acid (NEFA levels and negatively with milk yield, dry matter intake and energy balance between WPP1 to WPP22. We showed, by quantitative RT-PCR and western blotting, that resistin mRNA and protein levels in adipose tissue were higher at WPP1 than at 5 MG. The level of phosphorylation of several early and downstream insulin signaling components (IRβ, IRS-1, IRS-2, Akt, MAPK ERK1/2, P70S6K and S6 in adipose tissue was also lower at 1 WPP than at 5 MG. Finally, we showed that recombinant bovine resistin increased the release of glycerol and mRNA levels for ATGL (adipose triglyceride lipase and HSL (hormone-sensitive lipase in adipose tissue explants. Overall, resistin levels were high in the plasma and adipose tissue and were positively correlated with NEFA levels after calving. Resistin is expressed in bovine mature adipocytes and promotes lipid mobilization in adipose explants in vitro.

  16. Expression of bone morphogenetic proteins and Msx genes during root formation.

    Science.gov (United States)

    Yamashiro, T; Tummers, M; Thesleff, I

    2003-03-01

    Like crown development, root formation is also regulated by interactions between epithelial and mesenchymml tissues. Bone morphogenetic proteins (BMPs), together with the transcription factors Msx1 and Msx2, play important roles in these interactions during early tooth morphogenesis. To investigate the involvement of this signaling pathway in root development, we analyzed the expression patterns of Bmp2, Bmp3, Bmp4, and Bmp7 as well as Msx1 and Msx2 in the roots of mouse molars. Bmp4 was expressed in the apical mesenchyme and Msx2 in the root sheath. However, Bmps were not detected in the root sheath epithelium, and Msx transcripts were absent from the underlying mesenchyme. These findings indicate that this Bmp signaling pathway, required for tooth initiation, does not regulate root development, but we suggest that root shape may be regulated by a mechanism similar to that regulating crown shape in cap-stage tooth germs. Msx2 expression continued in the epithelial cell rests of Malassez, and the nearby cementoblasts intensely expressed Bmp3, which may regulate some functions of the fragmented epithelium.

  17. Retinoid-induced expression and activity of an immediate early tumor suppressor gene in vascular smooth muscle cells.

    Directory of Open Access Journals (Sweden)

    Jeffrey W Streb

    2011-04-01

    Full Text Available Retinoids are used clinically to treat a number of hyper-proliferative disorders and have been shown in experimental animals to attenuate vascular occlusive diseases, presumably through nuclear receptors bound to retinoic acid response elements (RARE located in target genes. Here, we show that natural or synthetic retinoids rapidly induce mRNA and protein expression of a specific isoform of A-Kinase Anchoring Protein 12 (AKAP12β in cultured smooth muscle cells (SMC as well as the intact vessel wall. Expression kinetics and actinomycin D studies indicate Akap12β is a retinoid-induced, immediate-early gene. Akap12β promoter analyses reveal a conserved RARE mildly induced with atRA in a region that exhibits hyper-acetylation. Immunofluorescence microscopy and protein kinase A (PKA regulatory subunit overlay assays in SMC suggest a physical association between AKAP12β and PKA following retinoid treatment. Consistent with its designation as a tumor suppressor, inducible expression of AKAP12β attenuates SMC growth in vitro. Further, immunohistochemistry studies establish marked decreases in AKAP12 expression in experimentally-injured vessels of mice as well as atheromatous lesions in humans. Collectively, these results demonstrate a novel role for retinoids in the induction of an AKAP tumor suppressor that blocks vascular SMC growth thus providing new molecular insight into how retiniods may exert their anti-proliferative effects in the injured vessel wall.

  18. Early prostate cancer antigen expression in predicting presence of prostate cancer in men with histologically negative biopsies.

    Science.gov (United States)

    Hansel, D E; DeMarzo, A M; Platz, E A; Jadallah, S; Hicks, J; Epstein, J I; Partin, A W; Netto, G J

    2007-05-01

    Early prostate cancer antigen is a nuclear matrix protein that was recently shown to be expressed in prostate adenocarcinoma and adjacent benign tissue. Previous studies have demonstrated early prostate cancer antigen expression in benign prostate tissue up to 5 years before a diagnosis of prostate carcinoma, suggesting that early prostate cancer antigen could be used as a potential predictive marker. We evaluated early prostate cancer antigen expression by immunohistochemistry using a polyclonal antibody (Onconome Inc., Seattle, Washington) on benign biopsies from 98 patients. Biopsies were obtained from 4 groups that included 39 patients with first time negative biopsy (group 1), 24 patients with persistently negative biopsies (group 2), 8 patients with initially negative biopsies who were subsequently diagnosed with prostate carcinoma (group 3) and negative biopsies obtained from 27 cases where other concurrent biopsies contained prostate carcinoma (group 4). Early prostate cancer antigen staining was assessed by 2 of the authors who were blind to the group of the examined sections. Staining intensity (range 0 to 3) and extent (range 1 to 3) scores were assigned. The presence of intensity 3 staining in any of the blocks of a biopsy specimen was considered as positive for early prostate cancer antigen for the primary outcome in the statistical analysis. In addition, as secondary outcomes we evaluated the data using the proportion of blocks with intensity 3 early prostate cancer antigen staining, the mean of the product of staining intensity and staining extent of all blocks within a biopsy, and the mean of the product of intensity 3 staining and extent. Primary outcome analysis revealed the proportion of early prostate cancer antigen positivity to be highest in group 3 (6 of 8, 75%) and lowest in group 2 (7 of 24, 29%, p=0.04 for differences among groups). A relatively higher than expected proportion of early prostate cancer antigen positivity was present in

  19. 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.

  20. 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.

  1. Non-invasive identification of protein biomarkers for early pregnancy diagnosis in the cheetah (Acinonyx jubatus.

    Directory of Open Access Journals (Sweden)

    Diana C Koester

    Full Text Available Approximately 80% of cheetahs living in typical zoological collections never reproduce. In more than 60% of breedings, the female is confirmed to ovulate, but parturition fails to occur. It is unknown if these non-pregnant intervals of elevated progesterone (deemed luteal phases are conception failures or a pregnancy terminating in embryonic/fetal loss. There have been recent advances in metabolic profiling and proteome analyses in many species with mass spectrometry used to identify 'biomarkers' and mechanisms indicative of specific physiological states (including pregnancy. Here, we hypothesized that protein expression in voided cheetah feces varied depending on pregnancy status. We: 1 identified the expansive protein profile present in fecal material of females; and 2 isolated proteins that may be candidates playing a role in early pregnancy establishment and diagnosis. Five hundred and seventy unique proteins were discovered among samples from pregnant (n = 8, non-pregnant, luteal phase (n = 5, and non-ovulatory control (n = 5 cheetahs. Four protein candidates were isolated that were significantly up-regulated and two were down-regulated in samples from pregnant compared to non-pregnant or control counterparts. One up-regulated candidate, immunoglobulin J chain (IGJ; an important component of the secretory immune system was detected using a commercially available antibody via immunoblotting. Findings revealed that increased IGJ abundance could be used to detect pregnancy successfully in >80% of 23 assessed females within 4 weeks after mating. The discovery of a novel fecal pregnancy marker improves the ability to determine reproductive, especially gestational, status in cheetahs managed in an ex situ insurance and source population.

  2. Non-invasive identification of protein biomarkers for early pregnancy diagnosis in the cheetah (Acinonyx jubatus).

    Science.gov (United States)

    Koester, Diana C; Wildt, David E; Maly, Morgan; Comizzoli, Pierre; Crosier, Adrienne E

    2017-01-01

    Approximately 80% of cheetahs living in typical zoological collections never reproduce. In more than 60% of breedings, the female is confirmed to ovulate, but parturition fails to occur. It is unknown if these non-pregnant intervals of elevated progesterone (deemed luteal phases) are conception failures or a pregnancy terminating in embryonic/fetal loss. There have been recent advances in metabolic profiling and proteome analyses in many species with mass spectrometry used to identify 'biomarkers' and mechanisms indicative of specific physiological states (including pregnancy). Here, we hypothesized that protein expression in voided cheetah feces varied depending on pregnancy status. We: 1) identified the expansive protein profile present in fecal material of females; and 2) isolated proteins that may be candidates playing a role in early pregnancy establishment and diagnosis. Five hundred and seventy unique proteins were discovered among samples from pregnant (n = 8), non-pregnant, luteal phase (n = 5), and non-ovulatory control (n = 5) cheetahs. Four protein candidates were isolated that were significantly up-regulated and two were down-regulated in samples from pregnant compared to non-pregnant or control counterparts. One up-regulated candidate, immunoglobulin J chain (IGJ; an important component of the secretory immune system) was detected using a commercially available antibody via immunoblotting. Findings revealed that increased IGJ abundance could be used to detect pregnancy successfully in >80% of 23 assessed females within 4 weeks after mating. The discovery of a novel fecal pregnancy marker improves the ability to determine reproductive, especially gestational, status in cheetahs managed in an ex situ insurance and source population.

  3. RRE-dependent HIV-1 Env RNA effects on Gag protein expression, assembly and release

    International Nuclear Information System (INIS)

    López, Claudia S.; Sloan, Rachel; Cylinder, Isabel; Kozak, Susan L.; Kabat, David; Barklis, Eric

    2014-01-01

    The HIV-1 Gag proteins are translated from the full-length HIV-1 viral RNA (vRNA), whereas the envelope (Env) protein is translated from incompletely spliced Env mRNAs. Nuclear export of vRNAs and Env mRNAs is mediated by the Rev accessory protein which binds to the rev-responsive element (RRE) present on these RNAs. Evidence has shown there is a direct or indirect interaction between the Gag protein, and the cytoplasmic tail (CT) of the Env protein. Our current work shows that env gene expression impacts HIV-1 Gag expression and function in two ways. At the protein level, full-length Env expression altered Gag protein expression, while Env CT-deletion proteins did not. At the RNA level, RRE-containing Env mRNA expression reduced Gag expression, processing, and virus particle release from cells. Our results support models in which Gag is influenced by the Env CT, and Env mRNAs compete with vRNAs for nuclear export. - Highlights: • At the protein level, full-length HIV-1 Env alters Gag protein expression. • HIV-1 Env RNA expression reduces Gag levels and virus release. • Env RNA effects on Gag are dependent on the RRE. • RRE-containing Env RNAs compete with vRNAs for nuclear export

  4. 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.

  5. In vitro protein expression: an emerging alternative to cell-based approaches.

    Science.gov (United States)

    He, Mingyue

    2011-04-30

    Protein expression remains a bottleneck in the production of proteins. Owing to several advantages, cell-free translation is emerging as an alternative to cell-based methods for the generation of proteins. Recent advances have led to many novel applications of cell-free systems in biotechnology, proteomics and fundamental biological research. This special issue of New Biotechnology describes recent advances in cell-free protein expression systems and their applications. Copyright © 2010 Elsevier B.V. All rights reserved.

  6. Regulation of c-myc and c-fos mRNA levels by polyomavirus: distinct roles for the capsid protein VP1 and the viral early proteins

    International Nuclear Information System (INIS)

    Zullo, J.; Stiles, C.D.; Garcea, R.L.

    1987-01-01

    The levels of c-myc, c-fos, and JE mRNAs accumulate in a biphasic pattern following infection of quiescent BALB/c 3T3 mouse cells with polyomavirus. Maximal levels of c-myc and c-fos mRNAs were seen within 1 hr and were nearly undetectable at 6 hr after infection. At 12 hr after infection mRNA levels were again maximal and remained elevated thereafter. Empty virions (capsids) and recombinant VP 1 protein, purified from Escherichia coli, induced the early but not the late phase of mRNA accumulation. Virions, capsids, and recombinant VP 1 protein stimulated [ 3 H]thymidine nuclear labeling and c-myc mRNA accumulation in a dose-responsive manner paralleling their affinity for the cell receptor for polyoma. The second phase of mRNA accumulation is regulated by the viral early gene products, as shown by polyomavirus early gene mutants and by a transfected cell line (336a) expressing middle tumor antigen upon glucocorticoid addition. These results suggest that polyomavirus interacts with the cell membrane at the onset of infection to increase the levels of mRNA for the cellular genes associated with cell competence for DNA replication, and subsequently these levels are maintained by the action of the early viral proteins

  7. Osmotic stress changes the expression and subcellular localization of the Batten disease protein CLN3.

    Directory of Open Access Journals (Sweden)

    Amanda Getty

    Full Text Available Juvenile CLN3 disease (formerly known as juvenile neuronal ceroid lipofuscinosis is a fatal childhood neurodegenerative disorder caused by mutations in the CLN3 gene. CLN3 encodes a putative lysosomal transmembrane protein with unknown function. Previous cell culture studies using CLN3-overexpressing vectors and/or anti-CLN3 antibodies with questionable specificity have also localized CLN3 in cellular structures other than lysosomes. Osmoregulation of the mouse Cln3 mRNA level in kidney cells was recently reported. To clarify the subcellular localization of the CLN3 protein and to investigate if human CLN3 expression and localization is affected by osmotic changes we generated a stably transfected BHK (baby hamster kidney cell line that expresses a moderate level of myc-tagged human CLN3 under the control of the human ubiquitin C promoter. Hyperosmolarity (800 mOsm, achieved by either NaCl/urea or sucrose, dramatically increased the mRNA and protein levels of CLN3 as determined by quantitative real-time PCR and Western blotting. Under isotonic conditions (300 mOsm, human CLN3 was found in a punctate vesicular pattern surrounding the nucleus with prominent Golgi and lysosomal localizations. CLN3-positive early endosomes, late endosomes and cholesterol/sphingolipid-enriched plasma membrane microdomain caveolae were also observed. Increasing the osmolarity of the culture medium to 800 mOsm extended CLN3 distribution away from the perinuclear region and enhanced the lysosomal localization of CLN3. Our results reveal that CLN3 has multiple subcellular localizations within the cell, which, together with its expression, prominently change following osmotic stress. These data suggest that CLN3 is involved in the response and adaptation to cellular stress.

  8. Cloning, Expression and Purification of the Recombinant HIV-1 Tat-Nef Fusion Protein in Prokaryotic Expression System

    Directory of Open Access Journals (Sweden)

    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.

  9. Low birthweight is associated with specific changes in muscle insulin-signalling protein expression

    DEFF Research Database (Denmark)

    Ozanne, SE; Jensen, CB; Tingey, KJ

    2005-01-01

    muscle in a human cohort and a rat model. METHODS: We recruited 20 young men with low birthweight (mean birthweight 2702+/-202 g) and 20 age-matched control subjects (mean birthweight 3801+/-99 g). Biopsies were obtained from the vastus lateralis muscle and protein expression of selected insulin......-signalling proteins was determined. Rats used for this study were male offspring born to dams fed a standard (20%) protein diet or a low (8%) protein diet during pregnancy and lactation. Protein expression was determined in soleus muscle from adult offspring. RESULTS: Low-birthweight subjects showed reduced muscle...... expression of protein kinase C (PKC)zeta, p85alpha, p110beta and GLUT4. PKCzeta, GLUT4 and p85 were also reduced in the muscle of rats fed a low-protein diet. Other proteins studied were unchanged in low-birthweight humans and in rats fed a low-protein diet when compared with control groups. CONCLUSIONS...

  10. Porcine bladder acellular matrix (ACM): protein expression, mechanical properties

    Energy Technology Data Exchange (ETDEWEB)

    Farhat, Walid A [Department of Surgery, Division of Urology, University of Toronto and Hospital for Sick Children, Toronto, ON M5G 1X8 (Canada); Chen Jun; Haig, Jennifer; Antoon, Roula; Litman, Jessica; Yeger, Herman [Department of Developmental and Stem Cell Biology, Research Institute, Hospital for Sick Children, Toronto, ON M5G 1X8 (Canada); Sherman, Christopher [Department of Anatomic Pathology, Sunnybrook and Women' s College Health Sciences Centre, Toronto, ON (Canada); Derwin, Kathleen [Department of Biomedical Engineering, Lerner Research Institute and Orthopaedic Research Center, Cleveland Clinic Foundation, Cleveland, OH (United States)], E-mail: walid.farhat@sickkids.ca

    2008-06-01

    Experimentally, porcine bladder acellular matrix (ACM) that mimics extracellular matrix has excellent potential as a bladder substitute. Herein we investigated the spatial localization and expression of different key cellular and extracellular proteins in the ACM; furthermore, we evaluated the inherent mechanical properties of the resultant ACM prior to implantation. Using a proprietary decellularization method, the DNA contents in both ACM and normal bladder were measured; in addition we used immunohistochemistry and western blots to quantify and localize the different cellular and extracellular components, and finally the mechanical testing was performed using a uniaxial mechanical testing machine. The mean DNA content in the ACM was significantly lower in the ACM compared to the bladder. Furthermore, the immunohistochemical and western blot analyses showed that collagen I and IV were preserved in the ACM, but possibly denatured collagen III in the ACM. Furthermore, elastin, laminin and fibronectin were mildly reduced in the ACM. Although the ACM did not exhibit nucleated cells, residual cellular components (actin, myosin, vimentin and others) were still present. There was, on the other hand, no significant difference in the mean stiffness between the ACM and the bladder. Although our decellularization method is effective in removing nuclear material from the bladder while maintaining its inherent mechanical properties, further work is mandatory to determine whether these residual DNA and cellular remnants would lead to any immune reaction, or if the mechanical properties of the ACM are preserved upon implantation and cellularization.

  11. Porcine bladder acellular matrix (ACM): protein expression, mechanical properties

    International Nuclear Information System (INIS)

    Farhat, Walid A; Chen Jun; Haig, Jennifer; Antoon, Roula; Litman, Jessica; Yeger, Herman; Sherman, Christopher; Derwin, Kathleen

    2008-01-01

    Experimentally, porcine bladder acellular matrix (ACM) that mimics extracellular matrix has excellent potential as a bladder substitute. Herein we investigated the spatial localization and expression of different key cellular and extracellular proteins in the ACM; furthermore, we evaluated the inherent mechanical properties of the resultant ACM prior to implantation. Using a proprietary decellularization method, the DNA contents in both ACM and normal bladder were measured; in addition we used immunohistochemistry and western blots to quantify and localize the different cellular and extracellular components, and finally the mechanical testing was performed using a uniaxial mechanical testing machine. The mean DNA content in the ACM was significantly lower in the ACM compared to the bladder. Furthermore, the immunohistochemical and western blot analyses showed that collagen I and IV were preserved in the ACM, but possibly denatured collagen III in the ACM. Furthermore, elastin, laminin and fibronectin were mildly reduced in the ACM. Although the ACM did not exhibit nucleated cells, residual cellular components (actin, myosin, vimentin and others) were still present. There was, on the other hand, no significant difference in the mean stiffness between the ACM and the bladder. Although our decellularization method is effective in removing nuclear material from the bladder while maintaining its inherent mechanical properties, further work is mandatory to determine whether these residual DNA and cellular remnants would lead to any immune reaction, or if the mechanical properties of the ACM are preserved upon implantation and cellularization

  12. Porcine bladder acellular matrix (ACM): protein expression, mechanical properties.

    Science.gov (United States)

    Farhat, Walid A; Chen, Jun; Haig, Jennifer; Antoon, Roula; Litman, Jessica; Sherman, Christopher; Derwin, Kathleen; Yeger, Herman

    2008-06-01

    Experimentally, porcine bladder acellular matrix (ACM) that mimics extracellular matrix has excellent potential as a bladder substitute. Herein we investigated the spatial localization and expression of different key cellular and extracellular proteins in the ACM; furthermore, we evaluated the inherent mechanical properties of the resultant ACM prior to implantation. Using a proprietary decellularization method, the DNA contents in both ACM and normal bladder were measured; in addition we used immunohistochemistry and western blots to quantify and localize the different cellular and extracellular components, and finally the mechanical testing was performed using a uniaxial mechanical testing machine. The mean DNA content in the ACM was significantly lower in the ACM compared to the bladder. Furthermore, the immunohistochemical and western blot analyses showed that collagen I and IV were preserved in the ACM, but possibly denatured collagen III in the ACM. Furthermore, elastin, laminin and fibronectin were mildly reduced in the ACM. Although the ACM did not exhibit nucleated cells, residual cellular components (actin, myosin, vimentin and others) were still present. There was, on the other hand, no significant difference in the mean stiffness between the ACM and the bladder. Although our decellularization method is effective in removing nuclear material from the bladder while maintaining its inherent mechanical properties, further work is mandatory to determine whether these residual DNA and cellular remnants would lead to any immune reaction, or if the mechanical properties of the ACM are preserved upon implantation and cellularization.

  13. Protein nutrition and metabolism during early development of the chick embryo

    International Nuclear Information System (INIS)

    Klein, N.W.

    1976-01-01

    Cultures of intact early chick embryos have been used as a model system in which to study the nutrition and metabolism of proteins during early embryonic development. Previous studies have shown that these embryos require nutrient proteins for growth and development. The protein requirement was found to be specific in that at least two proteins were essential; one a transferrin (either conalbumin or yolk transferrin) and the other either ovalbumin or lipovitellin. Variations in the quantity or type of protein provided in the medium altered the growth of embryo regions through regionally specific changes in protein breakdown. This was confirmed through protein synthetic studies with isolated polyribosomes. More recently such variations in protein nutrition have been shown also to affect the actual patterns of proteins synthesized by regions of the embryo. These observed responses to protein nutrition have been difficult to reconcile with our observation that proteins as such did not reach the embryo proper but were first degraded to amine acids within the yolk-sac membrane. Studies on the synthesis of serum proteins by the yolk-sac membrane have provided a possible explanation in that the relative synthesis of individual serum proteins was dramatically influenced by the protein composition of the culture medium. We are currently attempting to demonstrate that serum proteins are indeed the mediators of the response of embryos to protein nutrition. (author)

  14. 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.

  15. Baculovirus IE2 Stimulates the Expression of Heat Shock Proteins in Insect and Mammalian Cells to Facilitate Its Proper Functioning.

    Science.gov (United States)

    Tung, Hsuan; Wei, Sung-Chan; Lo, Huei-Ru; Chao, Yu-Chan

    2016-01-01

    Baculoviruses have gained popularity as pest control agents and for protein production in insect systems. These viruses are also becoming popular for gene expression, tissue engineering and gene therapy in mammalian systems. Baculovirus infection triggers a heat shock response, and this response is crucial for its successful infection of host insect cells. However, the viral protein(s) or factor(s) that trigger this response are not yet clear. Previously, we revealed that IE2-an early gene product of the baculovirus-could form unique nuclear bodies for the strong trans-activation of various promoters in mammalian cells. Here, we purified IE2 nuclear bodies from Vero E6 cells and investigated the associated proteins by using mass spectrometry. Heat shock proteins (HSPs) were found to be one of the major IE2-associated proteins. Our experiments show that HSPs are greatly induced by IE2 and are crucial for the trans-activation function of IE2. Interestingly, blocking both heat shock protein expression and the proteasome pathway preserved the IE2 protein and its nuclear body structure, and revived its function. These observations reveal that HSPs do not function directly to assist the formation of the nuclear body structure, but may rather protect IE2 from proteasome degradation. Aside from functional studies in mammalian cells, we also show that HSPs were stimulated and required to determine IE2 protein levels, in insect cells infected with baculovirus. Upon inhibiting the expression of heat shock proteins, baculovirus IE2 was substantially suppressed, resulting in a significantly suppressed viral titer. Thus, we demonstrate a unique feature in that IE2 can function in both insect and non-host mammalian cells to stimulate HSPs, which may be associated with IE2 stabilization and lead to the protection of the its strong gene activation function in mammalian cells. On the other hand, during viral infection in insect cells, IE2 could also strongly stimulate HSPs and

  16. CXCL10 Decreases GP73 Expression in Hepatoma Cells at the Early Stage of Hepatitis C Virus (HCV Infection

    Directory of Open Access Journals (Sweden)

    Yuan Liu

    2013-12-01

    Full Text Available Golgi protein 73 (GP73, which is up-regulated in hepatocellular carcinoma (HCC, has recently been identified as a novel serum marker for HCC diagnosis. Several reports also noted the increased levels of GP73 expression in chronic liver disease in patients with acute hepatitis of various etiologies, chronic Hepatitis C virus (HCV infection and alcoholic liver disease. The molecular mechanisms of GP73 expression in HCV related liver disease still need to be determined. In this study, we aimed to evaluate the effect of HCV infection on GP73 expression. GP73 was highly expressed in Huh7, Hep3B, 293T and HUVEC cells, and was low-expressed in HepG2 cells. HCV infection led to down-regulation of GP73 in Huh7 and HepG2/CD81 cells at the early stage of infection. CXCL10 decreased GP73 expression in Huh7 and HepG2 cells. Up-regulation of GP73 was noted in hepatocytes with cytopathic effect at advanced stage of HCV infection, and further research is needed to determine the unknown factors affecting GP73 expression. In conclusion, our study provided additional evidence for the roles of GP73 in liver disease.

  17. Intratumoral heterogeneity of Ki67 expression in early breast cancers exceeds variability between individual tumours

    NARCIS (Netherlands)

    Focke, Cornelia M.; Decker, Thomas; van Diest, Paul J.

    2016-01-01

    Aims: Regional differences in proliferative activity are commonly seen within breast cancers, but little is known on the extent of intratumoral heterogeneity of Ki67 expression. Our aim was to study the intratumoral heterogeneity of Ki67 expression in early breast cancers and its association with

  18. Stromal cell markers are differentially expressed in the synovial tissue of patients with early arthritis

    NARCIS (Netherlands)

    Choi, Ivy Y.; Karpus, Olga N.; Turner, Jason D.; Hardie, Debbie; Marshall, Jennifer L.; de Hair, Maria J. H.; Maijer, Karen I.; Tak, Paul P.; Raza, Karim; Hamann, Jörg; Buckley, Christopher D.; Gerlag, Danielle M.; Filer, Andrew

    2017-01-01

    Previous studies have shown increased expression of stromal markers in synovial tissue (ST) of patients with established rheumatoid arthritis (RA). Here, ST expression of stromal markers in early arthritis in relationship to diagnosis and prognostic outcome was studied. ST from 56 patients included

  19. Changes in expression of c-Fos protein following cocaine-cue extinction learning.

    Science.gov (United States)

    Nic Dhonnchadha, B Á; Lovascio, B F; Shrestha, N; Lin, A; Leite-Morris, K A; Man, H Y; Kaplan, G B; Kantak, K M

    2012-09-01

    Extinguishing abnormally strengthened learned responses to cues associated with drugs of abuse remains a key tactic for alleviating addiction. To assist in developing pharmacotherapies to augment exposure therapy for relapse prevention, investigation into neurobiological underpinnings of drug-cue extinction learning is needed. We used regional analyses of c-Fos and GluR2 protein expression to delineate neural activity and plasticity that may be associated with cocaine-cue extinction learning. Rats were trained to self-administer cocaine paired with a light cue, and later underwent a single 2h extinction session for which cocaine was withheld but response-contingent cues were presented (cocaine-cue extinction). Control groups consisted of rats yoked to animals self-administering cocaine and receiving saline non-contingently followed by an extinction session, or rats trained to self-administer cocaine followed by a no-extinction session for which levers were retracted, and cocaine and cues were withheld. Among 11 brain sites examined, extinction training increased c-Fos expression in basolateral amygdala and prelimbic prefrontal cortex of cocaine-cue extinguished rats relative to both control conditions. In dorsal subiculum and infralimbic prefrontal cortex, extinction training increased c-Fos expression in both cocaine-cue and saline-cue extinguished rats relative to the no-extinction control condition. GluR2 protein expression was not altered in any site examined after extinction or control training. Findings suggest that basolateral amygdala and prelimbic prefrontal cortex neurons are activated during acquisition of cocaine-cue extinction learning, a process that is independent of changes in GluR2 abundance. Other sites are implicated in processing the significance of cues that are present early in extinction training. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Levetiracetam attenuates hippocampal expression of synaptic plasticity-related immediate early and late response genes in amygdala-kindled rats

    Directory of Open Access Journals (Sweden)

    Watson William P

    2010-01-01

    Full Text Available Abstract Background The amygdala-kindled rat is a model for human temporal lobe epilepsy and activity-dependent synaptic plasticity. Hippocampal RNA isolated from amygdala-kindled rats at different kindling stages was analyzed to identify kindling-induced genes. Furthermore, effects of the anti-epileptic drug levetiracetam on kindling-induced gene expression were examined. Results Cyclooxygenase-2 (Cox-2, Protocadherin-8 (Pcdh8 and TGF-beta-inducible early response gene-1 (TIEG1 were identified and verified as differentially expressed transcripts in the hippocampus of kindled rats by in situ hybridization and quantitative RT-PCR. In addition, we identified a panel of 16 additional transcripts which included Arc, Egr3/Pilot, Homer1a, Ania-3, MMP9, Narp, c-fos, NGF, BDNF, NT-3, Synaptopodin, Pim1 kinase, TNF-α, RGS2, Egr2/krox-20 and β-A activin that were differentially expressed in the hippocampus of amygdala-kindled rats. The list consists of many synaptic plasticity-related immediate early genes (IEGs as well as some late response genes encoding transcription factors, neurotrophic factors and proteins that are known to regulate synaptic remodelling. In the hippocampus, induction of IEG expression was dependent on the afterdischarge (AD duration. Levetiracetam, 40 mg/kg, suppressed the development of kindling measured as severity of seizures and AD duration. In addition, single animal profiling also showed that levetiracetam attenuated the observed kindling-induced IEG expression; an effect that paralleled the anti-epileptic effect of the drug on AD duration. Conclusions The present study provides mRNA expression data that suggest that levetiracetam attenuates expression of genes known to regulate synaptic remodelling. In the kindled rat, levetiracetam does so by shortening the AD duration thereby reducing the seizure-induced changes in mRNA expression in the hippocampus.

  1. Differential expression of proteins and phosphoproteins during larval metamorphosis of the polychaete Capitella sp. I

    Directory of Open Access Journals (Sweden)

    Qian Pei-Yuan

    2011-09-01

    Full Text Available Abstract Background The spontaneous metamorphosis of the polychaete Capitella sp. I larvae into juveniles requires minor morphological changes, including segment formation, body elongation, and loss of cilia. In this study, we investigated changes in the expression patterns of both proteins and phosphoproteins during the transition from larvae to juveniles in this species. We used two-dimensional gel electrophoresis (2-DE followed by multiplex fluorescent staining and MALDI-TOF mass spectrometry analysis to identify the differentially expressed proteins as well as the protein and phosphoprotein profiles of both competent larvae and juveniles. Results Twenty-three differentially expressed proteins were identified in the two developmental stages. Expression patterns of two of those proteins were examined at the protein level by Western blot analysis while seven were further studied at the mRNA level by real-time PCR. Results showed that proteins related to cell division, cell migration, energy storage and oxidative stress were plentifully expressed in the competent larvae; in contrast, proteins involved in oxidative metabolism and transcriptional regulation were abundantly expressed in the juveniles. Conclusion It is likely that these differentially expressed proteins are involved in regulating the larval metamorphosis process and can be used as protein markers for studying molecular mechanisms associated with larval metamorphosis in polychaetes.

  2. Differential expression of proteins and phosphoproteins during larval metamorphosis of the polychaete Capitella sp. I

    KAUST Repository

    Chandramouli, Kondethimmanahalli

    2011-09-03

    Background: The spontaneous metamorphosis of the polychaete Capitella sp. I larvae into juveniles requires minor morphological changes, including segment formation, body elongation, and loss of cilia. In this study, we investigated changes in the expression patterns of both proteins and phosphoproteins during the transition from larvae to juveniles in this species. We used two-dimensional gel electrophoresis (2-DE) followed by multiplex fluorescent staining and MALDI-TOF mass spectrometry analysis to identify the differentially expressed proteins as well as the protein and phosphoprotein profiles of both competent larvae and juveniles.Results: Twenty-three differentially expressed proteins were identified in the two developmental stages. Expression patterns of two of those proteins were examined at the protein level by Western blot analysis while seven were further studied at the mRNA level by real-time PCR. Results showed that proteins related to cell division, cell migration, energy storage and oxidative stress were plentifully expressed in the competent larvae; in contrast, proteins involved in oxidative metabolism and transcriptional regulation were abundantly expressed in the juveniles.Conclusion: It is likely that these differentially expressed proteins are involved in regulating the larval metamorphosis process and can be used as protein markers for studying molecular mechanisms associated with larval metamorphosis in polychaetes. © 2011 Chandramouli et al; licensee BioMed Central Ltd.

  3. Differential expression of proteins and phosphoproteins during larval metamorphosis of the polychaete Capitella sp. I

    KAUST Repository

    Chandramouli, Kondethimmanahalli; Soo, Lisa; Qian, Pei-Yuan

    2011-01-01

    Background: The spontaneous metamorphosis of the polychaete Capitella sp. I larvae into juveniles requires minor morphological changes, including segment formation, body elongation, and loss of cilia. In this study, we investigated changes in the expression patterns of both proteins and phosphoproteins during the transition from larvae to juveniles in this species. We used two-dimensional gel electrophoresis (2-DE) followed by multiplex fluorescent staining and MALDI-TOF mass spectrometry analysis to identify the differentially expressed proteins as well as the protein and phosphoprotein profiles of both competent larvae and juveniles.Results: Twenty-three differentially expressed proteins were identified in the two developmental stages. Expression patterns of two of those proteins were examined at the protein level by Western blot analysis while seven were further studied at the mRNA level by real-time PCR. Results showed that proteins related to cell division, cell migration, energy storage and oxidative stress were plentifully expressed in the competent larvae; in contrast, proteins involved in oxidative metabolism and transcriptional regulation were abundantly expressed in the juveniles.Conclusion: It is likely that these differentially expressed proteins are involved in regulating the larval metamorphosis process and can be used as protein markers for studying molecular mechanisms associated with larval metamorphosis in polychaetes. © 2011 Chandramouli et al; licensee BioMed Central Ltd.

  4. 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

  5. Temporal expression of HIV-1 envelope proteins in baculovirus-infected insect cells: Implications for glycosylation and CD4 binding

    International Nuclear Information System (INIS)

    Murphy, C.I.; Lennick, M.; Lehar, S.M.; Beltz, G.A.; Young, E.

    1990-01-01

    Three different human immunodeficiency virus type I (HIV-1) envelope derived recombinant proteins and the full length human CD4 polypeptide were expressed in Spodoptera frugiperda (Sf9) cells. DNA constructs encoding CD4, gp120, gp160, and gp160 delta were cloned into the baculovirus expression vector pVL941 or a derivative and used to generate recombinant viruses in a cotransfection with DNA from Autographa californica nuclear polyhedrosis virus (AcMNPV). Western blotting of cell extracts of the recombinant HIV-1 proteins showed that for each construct two major bands specifically reacted with anti-HIV-1 envelope antiserum. These bands corresponded to glycosylated and nonglycosylated versions of the HIV proteins as determined by 3H-mannose labeling and tunicamycin treatment of infected cells. A time course of HIV envelope expression revealed that at early times post-infection (24 hours) the proteins were fully glycosylated and soluble in nonionic detergents. However, at later times postinfection (48 hours), expression levels of recombinant protein reached a maximum but most of the increase was due to a rise in the level of the nonglycosylated species, which was largely insoluble in nonionic detergents. Thus, it appears that Sf9 cells cannot process large amounts of glycosylated recombinant proteins efficiently. As a measure of biological activity, the CD4 binding ability of both glycosylated and nonglycosylated recombinant HIV envelope proteins was tested in a coimmunoprecipitation assay. The results showed that CD4 and the glycosylated versions of recombinant gp120 or gp160 delta specifically associated with one another in this analysis. Nonglycosylated gp120 or gp160 delta proteins from tunicamycin-treated cultures did immunoprecipitate with anti-HIV-1 antiserum but did not interact with CD4

  6. Gene Expression Patterns during the Early Stages of Chemically Induced Larval Metamorphosis and Settlement of the Coral Acropora millepora

    Science.gov (United States)

    Siboni, Nachshon; Abrego, David; Motti, Cherie A.; Tebben, Jan; Harder, Tilmann

    2014-01-01

    The morphogenetic transition of motile coral larvae into sessile primary polyps is triggered and genetically programmed upon exposure to environmental biomaterials, such as crustose coralline algae (CCA) and bacterial biofilms. Although the specific chemical cues that trigger coral larval morphogenesis are poorly understood there is much more information available on the genes that play a role in this early life phase. Putative chemical cues from natural biomaterials yielded defined chemical samples that triggered different morphogenetic outcomes: an extract derived from a CCA-associated Pseudoalteromonas bacterium that induced metamorphosis, characterized by non-attached metamorphosed juveniles; and two fractions of the CCA Hydrolithon onkodes (Heydrich) that induced settlement, characterized by attached metamorphosed juveniles. In an effort to distinguish the genes involved in these two morphogenetic transitions, competent larvae of the coral Acropora millepora were exposed to these predictable cues and the expression profiles of 47 coral genes of interest (GOI) were investigated after only 1 hour of exposure using multiplex RT–qPCR. Thirty-two GOI were differentially expressed, indicating a putative role during the early regulation of morphogenesis. The most striking differences were observed for immunity-related genes, hypothesized to be involved in cell recognition and adhesion, and for fluorescent protein genes. Principal component analysis of gene expression profiles resulted in separation between the different morphogenetic cues and exposure times, and not only identified those genes involved in the early response but also those which influenced downstream biological changes leading to larval metamorphosis or settlement. PMID:24632854

  7. Cloning, Expression and Purification of the Recombinant HIV-1 Tat-Nef Fusion Protein in Prokaryotic Expression System

    OpenAIRE

    Somayeh Kadkhodayan; Shiva Irani; Seyed Mehdi Sadat; Fatemeh Fotouhi; Azam Bolhassani

    2016-01-01

    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 confi...

  8. Predicting protein-protein interactions in Arabidopsis thaliana through integration of orthology, gene ontology and co-expression

    Directory of Open Access Journals (Sweden)

    Vandepoele Klaas

    2009-06-01

    Full Text Available Abstract Background Large-scale identification of the interrelationships between different components of the cell, such as the interactions between proteins, has recently gained great interest. However, unraveling large-scale protein-protein interaction maps is laborious and expensive. Moreover, assessing the reliability of the interactions can be cumbersome. Results In this study, we have developed a computational method that exploits the existing knowledge on protein-protein interactions in diverse species through orthologous relations on the one hand, and functional association data on the other hand to predict and filter protein-protein interactions in Arabidopsis thaliana. A highly reliable set of protein-protein interactions is predicted through this integrative approach making use of existing protein-protein interaction data from yeast, human, C. elegans and D. melanogaster. Localization, biological process, and co-expression data are used as powerful indicators for protein-protein interactions. The functional repertoire of the identified interactome reveals interactions between proteins functioning in well-conserved as well as plant-specific biological processes. We observe that although common mechanisms (e.g. actin polymerization and components (e.g. ARPs, actin-related proteins exist between different lineages, they are active in specific processes such as growth, cancer metastasis and trichome development in yeast, human and Arabidopsis, respectively. Conclusion We conclude that the integration of orthology with functional association data is adequate to predict protein-protein interactions. Through this approach, a high number of novel protein-protein interactions with diverse biological roles is discovered. Overall, we have predicted a reliable set of protein-protein interactions suitable for further computational as well as experimental analyses.

  9. Protein expression based multimarker analysis of breast cancer samples

    International Nuclear Information System (INIS)

    Presson, Angela P; Horvath, Steve; Yoon, Nam K; Bagryanova, Lora; Mah, Vei; Alavi, Mohammad; Maresh, Erin L; Rajasekaran, Ayyappan K; Goodglick, Lee; Chia, David

    2011-01-01

    Tissue microarray (TMA) data are commonly used to validate the prognostic accuracy of tumor markers. For example, breast cancer TMA data have led to the identification of several promising prognostic markers of survival time. Several studies have shown that TMA data can also be used to cluster patients into clinically distinct groups. Here we use breast cancer TMA data to cluster patients into distinct prognostic groups. We apply weighted correlation network analysis (WGCNA) to TMA data consisting of 26 putative tumor biomarkers measured on 82 breast cancer patients. Based on this analysis we identify three groups of patients with low (5.4%), moderate (22%) and high (50%) mortality rates, respectively. We then develop a simple threshold rule using a subset of three markers (p53, Na-KATPase-β1, and TGF β receptor II) that can approximately define these mortality groups. We compare the results of this correlation network analysis with results from a standard Cox regression analysis. We find that the rule-based grouping variable (referred to as WGCNA*) is an independent predictor of survival time. While WGCNA* is based on protein measurements (TMA data), it validated in two independent Affymetrix microarray gene expression data (which measure mRNA abundance). We find that the WGCNA patient groups differed by 35% from mortality groups defined by a more conventional stepwise Cox regression analysis approach. We show that correlation network methods, which are primarily used to analyze the relationships between gene products, are also useful for analyzing the relationships between patients and for defining distinct patient groups based on TMA data. We identify a rule based on three tumor markers for predicting breast cancer survival outcomes

  10. Expression of phosphorylated raf kinase inhibitor protein (pRKIP) is a predictor of lung cancer survival

    International Nuclear Information System (INIS)

    Huerta-Yepez, Sara; Chia, David; Bonavida, Benjamin; Goodglick, Lee; Yoon, Nam K; Hernandez-Cueto, Angeles; Mah, Vei; Rivera-Pazos, Clara M; Chatterjee, Devasis; Vega, Mario I; Maresh, Erin L; Horvath, Steve

    2011-01-01

    Raf-1 kinase inhibitor protein (RKIP) has been reported to negatively regulate signal kinases of major survival pathways. RKIP activity is modulated in part by phosphorylation on Serine 153 by protein kinase C, which leads to dissociation of RKIP from Raf-1. RKIP expression is low in many human cancers and represents an indicator of poor prognosis and/or induction of metastasis. The prognostic power has typically been based on total RKIP expression and has not considered the significance of phospho-RKIP. The present study examined the expression levels of both RKIP and phospho-RKIP in human lung cancer tissue microarray proteomics technology. Total RKIP and phospho-RKIP expression levels were similar in normal and cancerous tissues. phospho-RKIP levels slightly decreased in metastatic lesions. However, the expression levels of phospho-RKIP, in contrast to total RKIP, displayed significant predictive power for outcome with normal expression of phospho-RKIP predicting a more favorable survival compared to lower levels (P = 0.0118); this was even more pronounced in more senior individuals and in those with early stage lung cancer. This study examines for the first time, the expression profile of RKIP and phospho-RKIP in lung cancer. Significantly, we found that phospho-RKIP was a predictive indicator of survival

  11. End-Binding Protein 1 (EB1) Up-regulation is an Early Event in Colorectal Carcinogenesis

    Science.gov (United States)

    Stypula-Cyrus, Yolanda; Mutyal, Nikhil N.; Cruz, Mart Angelo Dela; Kunte, Dhananjay P.; Radosevich, Andrew J.; Wali, Ramesh; Roy, Hemant K.; Backman, Vadim

    2014-01-01

    End-binding protein (EB1) is a microtubule protein that binds to the tumor suppressor adenomatous polyposis coli (APC). While EB1 is implicated as a potential oncogene, its role in cancer progression is unknown. Therefore, we analyzed EB1/APC expression at the earliest stages of colorectal carcinogenesis and in the uninvolved mucosa ("field effect") of human and animal tissue. We also performed siRNA-knockdown in colon cancer cell lines. EB1 is up-regulated in early and field carcinogenesis in the colon, and the cellular/nano-architectural effect of EB1 knockdown depended on the genetic context. Thus, dysregulation of EB1 is an important early event in colon carcinogenesis. PMID:24492008

  12. Loss of co-ordinate expression of progesterone receptors A and B is an early event in breast carcinogenesis.

    Science.gov (United States)

    Mote, P A; Bartow, S; Tran, N; Clarke, C L

    2002-03-01

    Progesterone receptor (PR) mediates the effects of progesterone in mammary tissues and plays a crucial role in normal breast development and in breast cancer. PR proteins are expressed as two isoforms, PRA and PRB, that have different capacities to activate target genes, yet it is unknown whether progesterone action in normal and malignant breast is mediated by PRA and/or PRB. This study determines the relative expression of PRA and PRB in normal breast and in benign, premalignant and malignant archival breast lesions by dual immunofluorescent histochemistry. In normal breast and in proliferative disease without atypia (PDWA) PRA and PRB were co-expressed within the same cells in comparable amounts, implicating both isoforms in progesterone action. In atypical lesions, however, there was a significant increase in predominant expression of PRA or PRB, with lesion progression from the normal state to malignancy. PR isoform predominance, especially PRA predominance, was evident in a high proportion of ductal carcinomas in situ (DCIS) and invasive breast lesions. In the normal breast and in PDWA, the relative expression of PRA and PRB in adjacent cells was homogenous. There was a significant increase in cell-to-cell heterogeneity of PR isoform expression in ADH and DCIS lesions and in the majority of breast cancers. Heterogeneous cell-to-cell expression of PR isoforms occurred prior to overall predominant expression of one isoform in premalignant breast lesions, demonstrating that loss of control of relative PRA:PRB expression is an early event in the development of breast cancer. PRA:PRB ratios within a breast lesion are likely to be important as both markers and effectors of tumor growth and development, and progressively aberrant PR isoform expression may play a role in the etiology of breast cancer.

  13. Maternal low protein diet and postnatal high fat diet increases adipose imprinted gene expression

    Science.gov (United States)

    Maternal and postnatal diet can alter Igf2 gene expression and DNA methylation. To test whether maternal low protein and postnatal high fat (HF) diet result in alteration in Igf2 expression and obesity, we fed obese-prone Sprague-Dawley rats 8% (LP) or 20% (NP) protein for 3 wk prior to breeding and...

  14. Evolved Lactococcus lactis Strains for Enhanced Expression of Recombinant Membrane Proteins

    NARCIS (Netherlands)

    Martinez Linares, Daniel; Geertsma, Eric R.; Poolman, Bert

    2010-01-01

    The production of complex multidomain (membrane) proteins is a major hurdle in structural genomics and a generic approach for optimizing membrane protein expression is still lacking. We have devised a selection method to isolate mutant strains with improved functional expression of recombinant

  15. Tuning protein expression using synonymous codon libraries targeted to the 5' mRNA coding region

    DEFF Research Database (Denmark)

    Goltermann, Lise; Borch Jensen, Martin; Bentin, Thomas

    2011-01-01

    intermediate expression levels of green fluorescent protein in Escherichia coli. At least in one case, no apparent effect on protein stability was observed, pointing to RNA level effects as the principal reason for the observed expression differences. Targeting a synonymous codon library to the 5' coding...

  16. C-fos protein expression in central nervous system. Effects of acute whole-body irradiation

    International Nuclear Information System (INIS)

    Martin, C.; Chollat, S.; Mahfoudi, H.; Lambert, F.; Baille Le Crom, V.; Fatome, M.

    1995-01-01

    Study of c-Fos protein expression in the rat striatum after gamma or (neutron-gamma) irradiation was carried on. c-Fos protein is expressed one hour after gamma exposure at the dose of 15 Gy but specificity of the response must be verified. (author)

  17. Spatiotemporal distribution of PAX6 and MEIS2 expression and total cell numbers in the ganglionic eminence in the early developing human forebrain

    DEFF Research Database (Denmark)

    Larsen, Karen B; Lutterodt, Melissa C; Laursen, Henning

    2010-01-01

    The development of the human neocortex is a complex and highly regulated process involving a time-related expression of many transcription factors including the homeobox genes Pax6 and Meis2. During early development, Pax6 is expressed in nuclei of radial glia cells in the neocortical proliferative...... in the same time window. We demonstrate by in situ hybridization and immunohistochemistry that the two homeobox genes are expressed during early fetal brain development in humans. PAX6 mRNA and protein were located in the proliferative zones of the neocortex and in single cells in the cortical preplate at 7...... in the proliferative zones of the human fetal neocortex and a higher expression of MEIS2 than PAX6 was observed in these areas at 9 fetal weeks. Further, MEIS2 was expressed at a very high level in the developing ganglionic eminence and at a more moderate level in the cortical plate....

  18. 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.

  19. Identification and Expression Profiles of Six Transcripts Encoding Carboxylesterase Protein in Vitis flexuosa Infected with Pathogens

    Directory of Open Access Journals (Sweden)

    Md. Zaherul Islam

    2016-08-01

    Full Text Available Plants protect themselves from pathogen attacks via several mechanisms, including hypersensitive cell death. Recognition of pathogen attack by the plant resistance gene triggers expression of carboxylesterase genes associated with hypersensitive response. We identified six transcripts of carboxylesterase genes, Vitis flexuosa carboxylesterase 5585 (VfCXE5585, VfCXE12827, VfCXE13132, VfCXE17159, VfCXE18231, and VfCXE47674, which showed different expression patterns upon transcriptome analysis of V. flexuosa inoculated with Elsinoe ampelina. The lengths of genes ranged from 1,098 to 1,629 bp, and their encoded proteins consisted of 309 to 335 amino acids. The predicted amino acid sequences showed hydrolase like domains in all six transcripts and contained two conserved motifs, GXSXG of serine hydrolase characteristics and HGGGF related to the carboxylesterase family. The deduced amino acid sequence also contained a potential catalytic triad consisted of serine, aspartic acid and histidine. Of the six transcripts, VfCXE12827 showed upregulated expression against E. ampelina at all time points. Three genes (VfCXE5585, VfCXE12827, and VfCXE13132 showed upregulation, while others (VfCXE17159, VfCXE18231, and VfCXE47674 were down regulated in grapevines infected with Botrytis cinerea. All transcripts showed upregulated expression against Rhizobium vitis at early and later time points except VfCXE12827, and were downregulated for up to 48 hours post inoculation (hpi after upregulation at 1 hpi in response to R. vitis infection. All tested genes showed high and differential expression in response to pathogens, indicating that they all may play a role in defense pathways during pathogen infection in grapevines.

  20. 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.

  1. Expression of immediate-early genes in the inferior colliculus and auditory cortex in salicylate-induced tinnitus in rat.

    Science.gov (United States)

    Hu, S S; Mei, L; Chen, J Y; Huang, Z W; Wu, H

    2014-03-12

    Tinnitus could be associated with neuronal hyperactivity in the auditory center. As a neuronal activity marker, immediate-early gene (IEG) expression is considered part of a general neuronal response to natural stimuli. Some IEGs, especially the activity-dependent cytoskeletal protein (Arc) and the early growth response gene-1 (Egr-1), appear to be highly correlated with sensory-evoked neuronal activity. We hypothesize, therefore, an increase of Arc and Egr-1 will be observed in a tinnitus model. In our study, we used the gap prepulse inhibition of acoustic startle (GPIAS) paradigm to confirm that salicylate induces tinnitus-like behavior in rats. However, expression of the Arc gene and Egr-1 gene were decreased in the inferior colliculus (IC) and auditory cortex (AC), in contradiction of our hypothesis. Expression of N-methyl d-aspartate receptor subunit 2B (NR2B) was increased and all of these changes returned to normal 14 days after treatment with salicylate ceased. These data revealed long-time administration of salicylate induced tinnitus markedly but reversibly and caused neural plasticity changes in the IC and the AC. Decreased expression of Arc and Egr-1 might be involved with instability of synaptic plasticity in tinnitus.

  2. Expression of immediate-early genes in the inferior colliculus and auditory cortex in salicylate-induced tinnitus in rat

    Directory of Open Access Journals (Sweden)

    S.S. Hu

    2014-03-01

    Full Text Available Tinnitus could be associated with neuronal hyperactivity in the auditory center. As a neuronal activity marker, immediate-early gene (IEG expression is considered part of a general neuronal response to natural stimuli. Some IEGs, especially the activity-dependent cytoskeletal protein (Arc and the early growth response gene-1 (Egr-1, appear to be highly correlated with sensory-evoked neuronal activity. We hypothesize, therefore, an increase of Arc and Egr-1 will be observed in a tinnitus model. In our study, we used the gap prepulse inhibition of acoustic startle (GPIAS paradigm to confirm that salicylate induces tinnitus-like behavior in rats. However, expression of the Arc gene and Egr-1 gene were decreased in the inferior colliculus (IC and auditory cortex (AC, in contradiction of our hypothesis. Expression of N-methyl d-aspartate receptor subunit 2B (NR2B was increased and all of these changes returned to normal 14 days after treatment with salicylate ceased. These data revealed long-time administration of salicylate induced tinnitus markedly but reversibly and caused neural plasticity changes in the IC and the AC. Decreased expression of Arc and Egr-1 might be involved with instability of synaptic plasticity in tinnitus.

  3. 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

  4. Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris

    International Nuclear Information System (INIS)

    Clark, Lindsay; Zahm, Jacob A.; Ali, Rustam; Kukula, Maciej; Bian, Liangqiao; Patrie, Steven M.; Gardner, Kevin H.; Rosen, Michael K.; Rosenbaum, Daniel M.

    2015-01-01

    13 C Methyl TROSY NMR spectroscopy has emerged as a powerful method for studying the dynamics of large systems such as macromolecular assemblies and membrane proteins. Specific 13 C labeling of aliphatic methyl groups and perdeuteration has been limited primarily to proteins expressed in E. coli, preventing studies of many eukaryotic proteins of physiological and biomedical significance. We demonstrate the feasibility of efficient 13 C isoleucine δ1-methyl labeling in a deuterated background in an established eukaryotic expression host, Pichia pastoris, and show that this method can be used to label the eukaryotic protein actin, which cannot be expressed in bacteria. This approach will enable NMR studies of previously intractable targets

  5. Changes in diapause related gene expression pattern during early embryonic development in HCl-treated eggs of bivoltine silkworm Bombyx mori (Lepidoptera: Bombycidae

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    Sirigineedi Sasibhushan

    2013-02-01

    Full Text Available Investigation of differential expression of diapause related genes (five metabolic, five heat shock protein and one translational regulatory in HCl-treated (non-diapause and untreated (diapause eggs of B. mori during early embryogenesis (up to 48h following oviposition revealed the up-regulation of sorbitol dehydrogenase upon HCl treatment, indicating increased glycogen synthesis for further embryonic development but, down-regulation of phosphofructo kinase gene expression after 18h of oviposition indicating an arrest of glycerol and sorbitol conversion. The expression of poly A binding protein gene expression was higher upon HCl treatment, revealing the initiation of translation. The expression levels of other genes analyzed did not vary significantly, except for Hsp90 and Hsp40, which were up-regulated on acid treatment until 18h. Thus, Sorbitoldehydrogenase and phosphofructo kinasegenes have a crucial role in diapause termination as evidenced by HCl treatment, while the other genes did not have major roles.

  6. Amplification and protein overexpression of cyclin D1: Predictor of occult nodal metastasis in early oral cancer.

    Science.gov (United States)

    Noorlag, Rob; Boeve, Koos; Witjes, Max J H; Koole, Ronald; Peeters, Ton L M; Schuuring, Ed; Willems, Stefan M; van Es, Robert J J

    2017-02-01

    Accurate nodal staging is pivotal for treatment planning in early (stage I-II) oral cancer. Unfortunately, current imaging modalities lack sensitivity to detect occult nodal metastases. Chromosomal region 11q13, including genes CCND1, Fas-associated death domain (FADD), and CTTN, is often amplified in oral cancer with nodal metastases. However, evidence in predicting occult nodal metastases is limited. In 158 patients with early tongue and floor of mouth (FOM) squamous cell carcinomas, both CCND1 amplification and cyclin D1, FADD, and cortactin protein expression were correlated with occult nodal metastases. CCND1 amplification and cyclin D1 expression correlated with occult nodal metastases. Cyclin D1 expression was validated in an independent multicenter cohort, confirming the correlation with occult nodal metastases in early FOM cancers. Cyclin D1 is a predictive biomarker for occult nodal metastases in early FOM cancers. Prospective research on biopsy material should confirm these results before implementing its use in routine clinical practice. © 2016 Wiley Periodicals, Inc. Head Neck 39: 326-333, 2017. © 2016 Wiley Periodicals, Inc.

  7. Gene expression profiling reveals underlying molecular mechanisms of the early stages of tamoxifen-induced rat hepatocarcinogenesis

    International Nuclear Information System (INIS)

    Pogribny, Igor P.; Bagnyukova, Tetyana V.; Tryndyak, Volodymyr P.; Muskhelishvili, Levan; Rodriguez-Juarez, Rocio; Kovalchuk, Olga; Han Tao; Fuscoe, James C.; Ross, Sharon A.; Beland, Frederick A.

    2007-01-01

    Tamoxifen is a widely used anti-estrogenic drug for chemotherapy and, more recently, for the chemoprevention of breast cancer. Despite the indisputable benefits of tamoxifen in preventing the occurrence and re-occurrence of breast cancer, the use of tamoxifen has been shown to induce non-alcoholic steatohepatitis, which is a life-threatening fatty liver disease with a risk of progression to cirrhosis and hepatocellular carcinoma. In recent years, the high-throughput microarray technology for large-scale analysis of gene expression has become a powerful tool for increasing the understanding of the molecular mechanisms of carcinogenesis and for identifying new biomarkers with diagnostic and predictive values. In the present study, we used the high-throughput microarray technology to determine the gene expression profiles in the liver during early stages of tamoxifen-induced rat hepatocarcinogenesis. Female Fisher 344 rats were fed a 420 ppm tamoxifen containing diet for 12 or 24 weeks, and gene expression profiles were determined in liver of control and tamoxifen-exposed rats. The results indicate that early stages of tamoxifen-induced liver carcinogenesis are characterized by alterations in several major cellular pathways, specifically those involved in the tamoxifen metabolism, lipid metabolism, cell cycle signaling, and apoptosis/cell proliferation control. One of the most prominent changes during early stages of tamoxifen-induced hepatocarcinogenesis is dysregulation of signaling pathways in cell cycle progression from the G 1 to S phase, evidenced by the progressive and sustained increase in expression of the Pdgfc, Calb3, Ets1, and Ccnd1 genes accompanied by the elevated level of the PI3K, p-PI3K, Akt1/2, Akt3, and cyclin B, D1, and D3 proteins. The early appearance of these alterations suggests their importance in the mechanism of neoplastic cell transformation induced by tamoxifen

  8. The p66(Shc adaptor protein controls oxidative stress response in early bovine embryos.

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    Dean H Betts

    Full Text Available The in vitro production of mammalian embryos suffers from high frequencies of developmental failure due to excessive levels of permanent embryo arrest and apoptosis caused by oxidative stress. The p66Shc stress adaptor protein controls oxidative stress response of somatic cells by regulating intracellular ROS levels through multiple pathways, including mitochondrial ROS generation and the repression of antioxidant gene expression. We have previously demonstrated a strong relationship with elevated p66Shc levels, reduced antioxidant levels and greater intracellular ROS generation with the high incidence of permanent cell cycle arrest of 2-4 cell embryos cultured under high oxygen tensions or after oxidant treatment. The main objective of this study was to establish a functional role for p66Shc in regulating the oxidative stress response during early embryo development. Using RNA interference in bovine zygotes we show that p66Shc knockdown embryos exhibited increased MnSOD levels, reduced intracellular ROS and DNA damage that resulted in a greater propensity for development to the blastocyst stage. P66Shc knockdown embryos were stress resistant exhibiting significantly reduced intracellular ROS levels, DNA damage, permanent 2-4 cell embryo arrest and diminished apoptosis frequencies after oxidant treatment. The results of this study demonstrate that p66Shc controls the oxidative stress response in early mammalian embryos. Small molecule inhibition of p66Shc may be a viable clinical therapy to increase the developmental potential of in vitro produced mammalian embryos.

  9. Development of Conformation Independent Computational Models for the Early Recognition of Breast Cancer Resistance Protein Substrates

    Directory of Open Access Journals (Sweden)

    Melisa Edith Gantner

    2013-01-01

    Full Text Available ABC efflux transporters are polyspecific members of the ABC superfamily that, acting as drug and metabolite carriers, provide a biochemical barrier against drug penetration and contribute to detoxification. Their overexpression is linked to multidrug resistance issues in a diversity of diseases. Breast cancer resistance protein (BCRP is the most expressed ABC efflux transporter throughout the intestine and the blood-brain barrier, limiting oral absorption and brain bioavailability of its substrates. Early recognition of BCRP substrates is thus essential to optimize oral drug absorption, design of novel therapeutics for central nervous system conditions, and overcome BCRP-mediated cross-resistance issues. We present the development of an ensemble of ligand-based machine learning algorithms for the early recognition of BCRP substrates, from a database of 262 substrates and nonsubstrates compiled from the literature. Such dataset was rationally partitioned into training and test sets by application of a 2-step clustering procedure. The models were developed through application of linear discriminant analysis to random subsamples of Dragon molecular descriptors. Simple data fusion and statistical comparison of partial areas under the curve of ROC curves were applied to obtain the best 2-model combination, which presented 82% and 74.5% of overall accuracy in the training and test set, respectively.

  10. Can early protein restriction induce the development of binge eating?

    Science.gov (United States)

    Fechine, Madge Farias; Borba, Tássia Karin; Cabral-Filho, José Eulálio; Bolaños-Jiménez, Francisco; Lopes-de-Souza, Sandra; Manhães-de-Castro, Raul

    2016-04-01

    We tested the hypothesis that perinatal undernourishment is a factor for binge eating. At 52 days rats born from dams fed on 17% protein (Control) or 8% protein (Undernourished) were distributed into four groups, two of which continued to be fed ad libitum chow and two were submitted to three consecutive Restricted/Refeeding (R/R) cycles. According to the following schedule: Control Naïve (from mothers fed 17% protein/no restriction phase); Control Restricted (from mothers fed 17% protein/restriction phase); Undernourished Naïve (from mothers fed 8% protein/no restriction phase); and Undernourished Restricted (from mothers fed 8% protein/restriction phase). Each cycle consisted of a restriction phase (in the first four days 40% of the mean daily individual chow intake was offered for consumption), followed by a refeeding phase (4 days of chow ad libitum). After the three cycles, all animals were subjected to a feeding test (chow diet and palatable food ad libitum for 24h). During the feeding test, the Undernourished Restricted demonstrated rebound hyperphagia during 2, 4 and 6h. These results suggest the perinatal undernourishment cannot contribute to a binge eating phenotype. Copyright © 2016. Published by Elsevier B.V.

  11. Engineering of kinase-based protein interacting devices: active expression of tyrosine kinase domains

    KAUST Repository

    Diaz Galicia, Miriam Escarlet

    2018-05-01

    Protein-protein interactions modulate cellular processes in health and disease. However, tracing weak or rare associations or dissociations of proteins is not a trivial task. Kinases are often regulated through interaction partners and, at the same time, themselves regulate cellular interaction networks. The use of kinase domains for creating a synthetic sensor device that reads low concentration protein-protein interactions and amplifies them to a higher concentration interaction which is then translated into a FRET (Fluorescence Resonance Energy Transfer) signal is here proposed. To this end, DNA constructs for interaction amplification (split kinases), positive controls (intact kinase domains), scaffolding proteins and phosphopeptide - SH2-domain modules for the reading of kinase activity were assembled and expression protocols for fusion proteins containing Lyn, Src, and Fak kinase domains in bacterial and in cell-free systems were optimized. Also, two non-overlapping methods for measuring the kinase activity of these proteins were stablished and, finally, a protein-fragment complementation assay with the split-kinase constructs was tested. In conclusion, it has been demonstrated that features such as codon optimization, vector design and expression conditions have an impact on the expression yield and activity of kinase-based proteins. Furthermore, it has been found that the defined PURE cell-free system is insufficient for the active expression of catalytic kinase domains. In contrast, the bacterial co-expression with phosphatases produced active kinase fusion proteins for two out of the three tested Tyrosine kinase domains.

  12. 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.

  13. 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

  14. Expression and purification of recombinant proteins in Escherichia coli tagged with a small metal-binding protein from Nitrosomonas europaea.

    Science.gov (United States)

    Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Zarate, Xristo

    2016-02-01

    Escherichia coli is still the preferred organism for large-scale production of recombinant proteins. The use of fusion proteins has helped considerably in enhancing the solubility of heterologous proteins and their purification with affinity chromatography. Here, the use of a small metal-binding protein (SmbP) from Nitrosomonas europaea is described as a new fusion protein for protein expression and purification in E. coli. Fluorescent proteins tagged at the N-terminal with SmbP showed high levels of solubility, compared with those of maltose-binding protein and glutathione S-transferase, and low formation of inclusion bodies. Using commercially available IMAC resins charged with Ni(II), highly pure recombinant proteins were obtained after just one chromatography step. Proteins may be purified from the periplasm of E. coli if SmbP contains the signal sequence at the N-terminal. After removal of the SmbP tag from the protein of interest, high-yields are obtained since SmbP is a protein of just 9.9 kDa. The results here obtained suggest that SmbP is a good alternative as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. A Protein Disulfide Isomerase Gene Fusion Expression System That Increases the Extracellular Productivity of Bacillus brevis

    Science.gov (United States)

    Kajino, Tsutomu; Ohto, Chikara; Muramatsu, Masayoshi; Obata, Shusei; Udaka, Shigezo; Yamada, Yukio; Takahashi, Haruo

    2000-01-01

    We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system. PMID:10653729

  16. Probing Early Misfolding Events in Prion Protein Mutants by NMR Spectroscopy

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    Gregor Ilc

    2013-08-01

    Full Text Available The post-translational conversion of the ubiquitously expressed cellular form of the prion protein, PrPC, into its misfolded and pathogenic isoform, known as prion or PrPSc, plays a key role in prion diseases. These maladies are denoted transmissible spongiform encephalopathies (TSEs and affect both humans and animals. A prerequisite for understanding TSEs is unraveling the molecular mechanism leading to the conversion process whereby most α-helical motifs are replaced by β-sheet secondary structures. Importantly, most point mutations linked to inherited prion diseases are clustered in the C-terminal domain region of PrPC and cause spontaneous conversion to PrPSc. Structural studies with PrP variants promise new clues regarding the proposed conversion mechanism and may help identify “hot spots” in PrPC involved in the pathogenic conversion. These investigations may also shed light on the early structural rearrangements occurring in some PrPC epitopes thought to be involved in modulating prion susceptibility. Here we present a detailed overview of our solution-state NMR studies on human prion protein carrying different pathological point mutations and the implications that such findings may have for the future of prion research.

  17. Phospholipase D family member 4, a transmembrane glycoprotein with no phospholipase D activity, expression in spleen and early postnatal microglia.

    Directory of Open Access Journals (Sweden)

    Fumio Yoshikawa

    Full Text Available BACKGROUND: Phospholipase D (PLD catalyzes conversion of phosphatidylcholine into choline and phosphatidic acid, leading to a variety of intracellular signal transduction events. Two classical PLDs, PLD1 and PLD2, contain phosphatidylinositide-binding PX and PH domains and two conserved His-x-Lys-(x(4-Asp (HKD motifs, which are critical for PLD activity. PLD4 officially belongs to the PLD family, because it possesses two HKD motifs. However, it lacks PX and PH domains and has a putative transmembrane domain instead. Nevertheless, little is known regarding expression, structure, and function of PLD4. METHODOLOGY/PRINCIPAL FINDINGS: PLD4 was analyzed in terms of expression, structure, and function. Expression was analyzed in developing mouse brains and non-neuronal tissues using microarray, in situ hybridization, immunohistochemistry, and immunocytochemistry. Structure was evaluated using bioinformatics analysis of protein domains, biochemical analyses of transmembrane property, and enzymatic deglycosylation. PLD activity was examined by choline release and transphosphatidylation assays. Results demonstrated low to modest, but characteristic, PLD4 mRNA expression in a subset of cells preferentially localized around white matter regions, including the corpus callosum and cerebellar white matter, during the first postnatal week. These PLD4 mRNA-expressing cells were identified as Iba1-positive microglia. In non-neuronal tissues, PLD4 mRNA expression was widespread, but predominantly distributed in the spleen. Intense PLD4 expression was detected around the marginal zone of the splenic red pulp, and splenic PLD4 protein recovered from subcellular membrane fractions was highly N-glycosylated. PLD4 was heterologously expressed in cell lines and localized in the endoplasmic reticulum and Golgi apparatus. Moreover, heterologously expressed PLD4 proteins did not exhibit PLD enzymatic activity. CONCLUSIONS/SIGNIFICANCE: Results showed that PLD4 is a non

  18. Maternal high-fat diet and offspring expression levels of vitamin K-dependent proteins.

    Science.gov (United States)

    Lanham, S A; Cagampang, F R; Oreffo, R O C

    2014-12-01

    Studies suggest that bone growth and development and susceptibility to vascular disease in later life are influenced by maternal nutrition during intrauterine and early postnatal life. There is evidence for a role of vitamin K-dependent proteins (VKDPs) including osteocalcin, matrix Gla protein, periostin, and growth-arrest specific- protein 6, in both bone and vascular development. We have examined whether there are alterations in these VKDPs in bone and vascular tissue from offspring of mothers subjected to a nutritional challenge: a high-fat diet during pregnancy and postnatally, using 6-week-old mouse offspring. Bone site-specific and sex-specific differences across femoral and vertebral bone in male and female offspring were observed. Overall a high-fat maternal diet and offspring diet exacerbated the bone changes observed. Sex-specific differences and tissue-specific differences were observed in VKDP levels in aorta tissue from high-fat diet-fed female offspring from high-fat diet-fed mothers displaying increased levels of Gas6 and Ggcx compared with those of female controls. In contrast, differences were seen in VKDP levels in femoral bone of female offspring with lower expression levels of Mgp in offspring of mothers fed a high-fat diet compared with those of controls. We observed a significant correlation in Mgp expression levels within the femur to measures of bone structure of the femur and vertebra, particularly in the male offspring cohort. In summary, the current study has highlighted the importance of maternal nutrition on offspring bone development and the correlation of VKDPs to bone structure.

  19. The Influence of Early Protein Energy Malnutrition on Subsequent Behavior and Intellectual Performance.

    Science.gov (United States)

    Gupta, Sarita

    1990-01-01

    Protein-energy malnutrition in early childhood, as seen in many developing countries, influences subsequent behavior and intellectual performance. These impairments are associated with further reduction in fine motor skills and academic performance. (Author)

  20. Interferon-inducible protein 10 (IP-10) is associated with viremia of early HIV-1 infection in Korean patients.

    Science.gov (United States)

    Lee, SoYong; Chung, Yoon-Seok; Yoon, Cheol-Hee; Shin, YoungHyun; Kim, SeungHyun; Choi, Byeong-Sun; Kim, Sung Soon

    2015-05-01

    Cytokines/chemokines play key roles in modulating disease progression in human immunodeficiency virus (HIV) infection. Although it is known that early HIV-1 infection is associated with increased production of proinflammatory cytokines, the relationship between cytokine levels and HIV-1 pathogenesis is not clear. The concentrations of 18 cytokines/chemokines in 30 HIV-1 negative and 208 HIV-1 positive plasma samples from Korean patients were measured by the Luminex system. Early HIV-1 infection was classified according to the Fiebig stage (FS) based on the characteristics of the patients infected with HIV-1. Concentrations of interleukin-12 (IL-12), interferon-inducible protein-10 (IP-10), macrophage inflammatory protein-1α (MIP-1α) and regulated upon activation, normal T cells expressed and secreted (RANTES) were increased significantly during the early stage of HIV-1 infection (FS II-IV) compared with the HIV-1-negative group. Of these cytokines, an elevated level of IP-10 was the only factor to be correlated positively with a higher viral load during the early stages of HIV-1 infection (FS II-IV) in Koreans (R = 0.52, P IP-10 may be an indicator for HIV-1 viremia and associated closely with viral replication in patients with early HIV-1 infection. © 2015 Wiley Periodicals, Inc.

  1. Immunohistochemical expression profiles of mucin antigens in salivary gland mucoepidermoid carcinoma: MUC4- and MUC6-negative expression predicts a shortened survival in the early postoperative phase.

    Science.gov (United States)

    Honjo, Kie; Hiraki, Tsubasa; Higashi, Michiyo; Noguchi, Hirotsugu; Nomoto, Mitsuharu; Yoshimura, Takuya; Batra, Surinder K; Yonezawa, Suguru; Semba, Ichiro; Nakamura, Norifumi; Tanimoto, Akihide; Yamada, Sohsuke

    2018-02-01

    In mucoepidermoid carcinoma (MEC), the most common salivary gland carcinoma, there is a lack of novel prognostic markers, but post-operative early recurrence strongly affects the clinical course and a poor outcome. It is critical to predict which MEC patients are prone to develop recurrence/metastases. Mucins play pivotal roles in influencing cancer biology, thus affecting cell differentiation, adhesion, carcinoma invasion, aggressiveness and/or metastatic potential. Our aim is to elucidate the significance of expression profiles for mucins, particularly MUC4 and MUC6, and their correlations with various clinicopathological features and recurrence in salivary gland MECs. We performed immunohistochemical analyses on patients with surgically resected primary MEC using antibodies against mucin core proteins MUC4/8G7 and MUC6/CLH5 in 73 paraffin-embedded samples. Recurrence was noted in 15 of 73 (20.5%) patients. MUC4 or MUC6 expression was considered to be negative when <30% or 0% of the MEC cells showed positive staining, respectively. MUC4- and/or MUC6-negative expression respectively and variably showed a significant relationship to pathological tumor high-grade, the presence of lymphovascular invasion, lymph node metastasis and/or tumor-related death. In addition, MUC4 showed significantly negative co-expression with MUC6. Kaplan-Meier analyses revealed that not only single MUC4/6-negative expression but also the combination of both predicted significantly shorter disease-free and disease-specific survivals in MECs, especially within the first two years postoperatively. Therefore, each mucin plays a pivotal role in the pathogenesis of MEC progression. The detection of MUC4 and/or MUC6 might be a powerful parameter in the clinical management of MECs in the early postsurgical phase.

  2. 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

  3. Expression and purification of toxic anti-breast cancer p28-NRC chimeric protein

    OpenAIRE

    Soleimani, Meysam; Mirmohammad-Sadeghi, Hamid; Sadeghi-Aliabadi, Hojjat; Jahanian-Najafabadi, Ali

    2016-01-01

    Background: Chimeric proteins consisting of a targeting moiety and a cytotoxic moiety are now under intense research focus for targeted therapy of cancer. Here, we report cloning, expression, and purification of such a targeted chimeric protein made up of p28 peptide as both targeting and anticancer moiety fused to NRC peptide as a cytotoxic moiety. However, since the antimicrobial activity of the NRC peptide would intervene expression of the chimeric protein in Escherichia coli, we evaluated...

  4. Evaluation of Selected Borrelia burgdorferi lp54 Plasmid-Encoded Gene Products Expressed during Mammalian Infection as Antigens To Improve Serodiagnostic Testing for Early Lyme Disease

    Science.gov (United States)

    Weiner, Zachary P.; Crew, Rebecca M.; Brandt, Kevin S.; Ullmann, Amy J.; Schriefer, Martin E.; Molins, Claudia R.

    2015-01-01

    Laboratory testing for the diagnosis of Lyme disease is performed primarily by serologic assays and is accurate for detection beyond the acute stage of the infection. Serodiagnostic assays to detect the early stages of infection, however, are limited in their sensitivity, and improvement is warranted. We analyzed a series of Borrelia burgdorferi proteins known to be induced within feeding ticks and/or during mammalian infection for their utility as serodiagnostic markers against a comprehensive panel of Lyme disease patient serum samples. The antigens were assayed for IgM and IgG reactivity in line immunoblots and separately by enzyme-linked immunosorbent assay (ELISA), with a focus on reactivity against early Lyme disease with erythema migrans (EM), early disseminated Lyme neuroborreliosis, and early Lyme carditis patient serum samples. By IgM immunoblotting, we found that recombinant proteins BBA65, BBA70, and BBA73 reacted with early Lyme EM samples at levels comparable to those of the OspC antigen used in the current IgM blotting criteria. Additionally, these proteins reacted with serum samples from patients with early neuroborreliosis and early carditis, suggesting value in detecting early stages of this disease progression. We also found serological reactivity against recombinant proteins BBA69 and BBA73 with early-Lyme-disease samples using IgG immunoblotting and ELISA. Significantly, some samples that had been scored negative by the Centers for Disease Control and Prevention-recommended 2-tiered testing algorithm demonstrated positive reactivity to one or more of the antigens by IgM/IgG immunoblot and ELISA. These results suggest that incorporating additional in vivo-expressed antigens into the current IgM/IgG immunoblotting tier in a recombinant protein platform assay may improve the performance of early-Lyme-disease serologic testing. PMID:26376927

  5. Evaluation of Selected Borrelia burgdorferi lp54 Plasmid-Encoded Gene Products Expressed during Mammalian Infection as Antigens To Improve Serodiagnostic Testing for Early Lyme Disease.

    Science.gov (United States)

    Weiner, Zachary P; Crew, Rebecca M; Brandt, Kevin S; Ullmann, Amy J; Schriefer, Martin E; Molins, Claudia R; Gilmore, Robert D

    2015-11-01

    Laboratory testing for the diagnosis of Lyme disease is performed primarily by serologic assays and is accurate for detection beyond the acute stage of the infection. Serodiagnostic assays to detect the early stages of infection, however, are limited in their sensitivity, and improvement is warranted. We analyzed a series of Borrelia burgdorferi proteins known to be induced within feeding ticks and/or during mammalian infection for their utility as serodiagnostic markers against a comprehensive panel of Lyme disease patient serum samples. The antigens were assayed for IgM and IgG reactivity in line immunoblots and separately by enzyme-linked immunosorbent assay (ELISA), with a focus on reactivity against early Lyme disease with erythema migrans (EM), early disseminated Lyme neuroborreliosis, and early Lyme carditis patient serum samples. By IgM immunoblotting, we found that recombinant proteins BBA65, BBA70, and BBA73 reacted with early Lyme EM samples at levels comparable to those of the OspC antigen used in the current IgM blotting criteria. Additionally, these proteins reacted with serum samples from patients with early neuroborreliosis and early carditis, suggesting value in detecting early stages of this disease progression. We also found serological reactivity against recombinant proteins BBA69 and BBA73 with early-Lyme-disease samples using IgG immunoblotting and ELISA. Significantly, some samples that had been scored negative by the Centers for Disease Control and Prevention-recommended 2-tiered testing algorithm demonstrated positive reactivity to one or more of the antigens by IgM/IgG immunoblot and ELISA. These results suggest that incorporating additional in vivo-expressed antigens into the current IgM/IgG immunoblotting tier in a recombinant protein platform assay may improve the performance of early-Lyme-disease serologic testing. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  6. High frequency of tumor cells with nuclear Egr-1 protein expression in human bladder cancer is associated with disease progression

    International Nuclear Information System (INIS)

    Egerod, Frederikke Lihme; Bartels, Annette; Fristrup, Niels; Borre, Michael; Ørntoft, Torben F; Oleksiewicz, Martin B; Brünner, Nils; Dyrskjøt, Lars

    2009-01-01

    Egr-1 (early growth response-1 transcription factor) has been proposed to be involved in invasion and metastasis processes of human bladder cancer, but Egr-1 protein expression levels in human bladder cancer have not been investigated. In the present study we investigated the expression levels of Egr-1 protein in early stages of human bladder cancer and correlated it to later progression. Expression of Egr-1 protein in human bladder cancer was examined by immunohistochemistry, on a tissue microarray constructed from tumors from 289 patients with non-muscle invasive urothelial bladder cancer. The frequency of tumor cells with nuclear Egr-1 immunolabelling correlated to bladder cancer stage, grade and to later progression to muscle-invasive bladder cancer (T2-4). Stage T1 tumors exhibited significantly higher frequencies of tumor cells with nuclear Egr-1 immunolabelling than Ta tumors (P = 0.001). Furthermore, Kaplan-Meier survival analysis showed that a high frequency of tumor cells with nuclear Egr-1 immunolabelling was significantly associated with a higher risk of progression to stage T2-4 (log-rank test, P = 0.035). Tumor cells with nuclear Egr-1 immunolabelling were found to localize at the tumor front in some of the tumor biopsies. The results from this study support a potential involvement of Egr-1 in the progression from non-muscle invasive bladder cancers to muscle invasive bladder cancer

  7. Effect of early postnatal exposure to valproate on neurobehavioral development and regional BDNF expression in two strains of mice.

    Science.gov (United States)

    Bath, Kevin G; Pimentel, Tiare

    2017-05-01

    Valproate has been used for over 30years as a first-line treatment for epilepsy. In recent years, prenatal exposure to valproate has been associated with teratogenic effects, limiting its use in women that are pregnant or of childbearing age. However, despite its potential detrimental effects on development, valproate continues to be prescribed at high rates in pediatric populations in some countries. Animal models allow us to test hypotheses regarding the potential effects of postnatal valproate exposure on neurobehavioral development, as well as identify potential mechanisms mediating observed effects. Here, we tested the effect of early postnatal (P4-P11) valproate exposure (100mg/kg and 200mg/kg) on motor and affective development in two strains of mice, SVE129 and C57Bl/6N. We also assessed the effect of early valproate exposure on regional BDNF protein levels, a potential target of valproate, and mediator of neurodevelopmental outcomes. We found that early life valproate exposure led to significant motor impairments in both SVE129 and C57Bl/6N mice. Both lines of mice showed significant delays in weight gain, as well as impairments in the righting reflex (P7-8), wire hang (P17), open field (P12 and P21), and rotarod (P25 and P45) tasks. Interestingly, some of the early locomotor effects were strain- and dose-dependent. We observed no effects of valproate on early markers of anxiety-like behavior. Importantly, early life valproate exposure had significant effects on regional BDNF expression, leading to a near 50% decrease in BDNF levels in the cerebellum of both strains of mice, while not impacting hippocampal BDNF protein levels. These observations indicate that postnatal exposure to valproate may have significant, and region-specific effects, on neural and behavioral development, with specific consequences for cerebellar development and motor function. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Screening and large-scale expression of membrane proteins in mammalian cells for structural studies

    OpenAIRE

    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

    2014-01-01

    Structural, biochemical and biophysical studies of eukaryotic membrane proteins are often hampered by difficulties in over-expression 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 productio...

  9. Radioautographic study of protein synthesis during early embryogenesis of Leptimotarsa decemlineata Say (Coleoptera)

    International Nuclear Information System (INIS)

    Maisonhaute, Claude

    1976-01-01

    Protein synthesis in early embryonic stages of the Colorado beetle has been investigated by radioautography. Radioactive precursor (L. Leucine-3 H) was injected in eggs. At the stage of blastoderm formation amino-acid incorporation decreases sharply: at late blastula stage, incorporation reaches the same levels as during early cleavage, and at gastrula stage becomes higher. Nuclear protein synthesis is first detected during blastoderm formation and increases at gastrula stage [fr

  10. Dual-function vector for protein expression in both mammalian cells and Xenopus laevis oocytes

    DEFF Research Database (Denmark)

    Jespersen, Thomas; Grunnet, M; Angelo, K

    2002-01-01

    Both Xenopus laevis oocytes and mammalian cells are widely used for heterologous expression of several classes of proteins, and membrane proteins especially, such as ion channels or receptors, have been extensively investigated in both cell types. A full characterization of a specific protein wil...

  11. Evolved Escherichia coli Strains for Amplified, Functional Expression of Membrane Proteins

    NARCIS (Netherlands)

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

    2014-01-01

    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

  12. Expression and analysis of exogenous proteins in epidermal cells.

    Science.gov (United States)

    Dagnino, Lina; Ho, Ernest; Chang, Wing Y

    2010-01-01

    In this chapter we review protocols for transient transfection of primary keratinocytes. The ability to transfect primary epidermal cells regardless of their differentiation status allows the biochemical and molecular characterization of multiple proteins. We review methods to analyze exogenous protein abundance in transfected keratinocytes by immunoblot and immunoprecipitation. We also present protocols to determine the subcellular distribution of these proteins by indirect immunofluorescence microscopy approaches.

  13. 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

  14. The effect of HCV Core protein on the expression of miR-150

    Directory of Open Access Journals (Sweden)

    Sayad Khanizadeh

    2016-09-01

    Full Text Available Background : Hepatitis C virus (HCV is considered as one of the major pathogenic agents of chronic liver diseases. Previous studies have shown that HCV proteins can interaction with gene regulatory networks such as microRNAs. The aim of this study was to investigate the effect of HCV core protein on the expression of miR-150 in a cell culture model. Materials and Methods: Plasmids expressing full HCV core protein was transfected into Huh7 cell lines while a GFP expressing plasmid employed as negative control. Subsequently, total RNA extracted and Real-Time PCR performed to measure the expression level of miR-150 expression. Moreover, trypan blue exclusion assay was performed to investigate the effect of core protein on cell viability. Results: The gene expression analysis of miR-150 in Huh7 cells showed that endogenous HCV core protein could significantly down regulation of miR-150 when compared to GFP control plasmid and normal cells (P<0.01. Beside, core protein induced no significant proliferative or cytotoxic effects on hepatic cells as determined by trypan blue exclusion assay (P<0.05. Conclusion: Our study suggests that HCV core protein can led to down regulation of miR-150 expression. This data revealed that HCV protein interactions with cell regulatory machinery may contribute to pathogenesis of chronic liver diseases.

  15. Coat Protein Mutations That Alter the Flux of Morphogenetic Intermediates through the ϕX174 Early Assembly Pathway.

    Science.gov (United States)

    Blackburn, Brody J; Li, Shuaizhi; Roznowski, Aaron P; Perez, Alexis R; Villarreal, Rodrigo H; Johnson, Curtis J; Hardy, Margaret; Tuckerman, Edward C; Burch, April D; Fane, Bentley A

    2017-12-15

    Two scaffolding proteins orchestrate ϕX174 morphogenesis. The internal scaffolding protein B mediates the formation of pentameric assembly intermediates, whereas the external scaffolding protein D organizes 12 of these intermediates into procapsids. Aromatic amino acid side chains mediate most coat-internal scaffolding protein interactions. One residue in the internal scaffolding protein and three in the coat protein constitute the core of the B protein binding cleft. The three coat gene codons were randomized separately to ascertain the chemical requirements of the encoded amino acids and the morphogenetic consequences of mutation. The resulting mutants exhibited a wide range of recessive phenotypes, which could generally be explained within a structural context. Mutants with phenylalanine, tyrosine, and methionine substitutions were phenotypically indistinguishable from the wild type. However, tryptophan substitutions were detrimental at two sites. Charged residues were poorly tolerated, conferring extreme temperature-sensitive and lethal phenotypes. Eighteen lethal and conditional lethal mutants were genetically and biochemically characterized. The primary defect associated with the missense substitutions ranged from inefficient internal scaffolding protein B binding to faulty procapsid elongation reactions mediated by external scaffolding protein D. Elevating B protein concentrations above wild-type levels via exogenous, cloned-gene expression compensated for inefficient B protein binding, as did suppressing mutations within gene B. Similarly, elevating D protein concentrations above wild-type levels or compensatory mutations within gene D suppressed faulty elongation. Some of the parental mutations were pleiotropic, affecting multiple morphogenetic reactions. This progressively reduced the flux of intermediates through the pathway. Accordingly, multiple mechanisms, which may be unrelated, could restore viability. IMPORTANCE Genetic analyses have been

  16. Lack of A-factor production induces the expression of nutrient scavenging and stress-related proteins in Streptomyces griseus.

    Science.gov (United States)

    Birkó, Zsuzsanna; Swiatek, Magdalena; Szájli, Emília; Medzihradszky, Katalin F; Vijgenboom, Erik; Penyige, András; Keseru, Judit; van Wezel, Gilles P; Biró, Sándor

    2009-10-01

    The small gamma-butyrolactone A-factor is an important autoregulatory signaling molecule for the soil-inhabiting streptomycetes. Starvation is a major trigger for development, and nutrients are provided by degradation of the vegetative mycelium via a process of programmed cell death, reusing proteins, nucleic acids, and cell wall material. The A-factor regulon includes many extracellular hydrolases. Here we show via proteomics analysis that many nutrient-scavenging and stress-related proteins were overexpressed in an A-factor non-producing mutant of Streptomyces griseus B-2682. Transcript analysis showed that this is primarily due to differential transcription of the target genes during early development. The targets include proteins relating to nutrient stress and environmental stress and an orthologue of the Bacillus sporulation control protein Spo0M. The enhanced expression of these proteins underlines the stress that is generated by the absence of A-factor. Wild-type developmental gene expression was restored to the A-factor non-producing mutant by the signaling protein Factor C in line with our earlier observation that Factor C triggers A-factor production.

  17. Early Programming by Protein Intake: The Effect of Protein on Adiposity Development and the Growth and Functionality of Vital Organs

    Directory of Open Access Journals (Sweden)

    Veronica Luque

    2015-01-01

    Full Text Available This article reviews the role of protein intake on metabolic programming early in life. The observations that breastfeeding in infancy reduces the risk of being overweight and obese later in life and the differences in the protein content between formula milk and human milk have generated the early protein hypothesis. The present review focuses on a mechanistic approach to programmed adiposity and the growth and development of other organs by protein intake in infancy, which may be mediated by branched-chain amino acids, insulin, and insulin-like growth factor 1 via the mammalian target of rapamycin. Observational studies and clinical trials have shown that lowering the protein content in infant and follow-on formulas may reduce the risk of becoming obese later in life. The recent body of evidence is currently being translated into new policies. Therefore, the evolution of European regulatory laws and recommendations by expert panels on the protein content of infant and follow-on formulas are also reviewed. Research gaps, such as the critical window for programming adiposity by protein intake, testing formulas with modified amino acids, and the long-term consequences of differences in protein intake on organ functionality among well-nourished infants, have been identified.

  18. A New Strain Collection for Improved Expression of Outer Membrane Proteins

    Directory of Open Access Journals (Sweden)

    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.

  19. 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.

  20. 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

  1. Differential maturation of rhythmic clock gene expression during early development in medaka (Oryzias latipes).

    Science.gov (United States)

    Cuesta, Ines H; Lahiri, Kajori; Lopez-Olmeda, Jose Fernando; Loosli, Felix; Foulkes, Nicholas S; Vallone, Daniela

    2014-05-01

    One key challenge for the field of chronobiology is to identify how circadian clock function emerges during early embryonic development. Teleosts such as the zebrafish are ideal models for studying circadian clock ontogeny since the entire process of development occurs ex utero in an optically transparent chorion. Medaka (Oryzias latipes) represents another powerful fish model for exploring early clock function with, like the zebrafish, many tools available for detailed genetic analysis. However, to date there have been no reports documenting circadian clock gene expression during medaka development. Here we have characterized the expression of key clock genes in various developmental stages and in adult tissues of medaka. As previously reported for other fish, light dark cycles are required for the emergence of clock gene expression rhythms in this species. While rhythmic expression of per and cry genes is detected very early during development and seems to be light driven, rhythmic clock and bmal expression appears much later around hatching time. Furthermore, the maturation of clock function seems to correlate with the appearance of rhythmic expression of these positive elements of the clock feedback loop. By accelerating development through elevated temperatures or by artificially removing the chorion, we show an earlier onset of rhythmicity in clock and bmal expression. Thus, differential maturation of key elements of the medaka clock mechanism depends on the developmental stage and the presence of the chorion.

  2. In vivo extracellular matrix protein expression by human periodontal ...

    African Journals Online (AJOL)

    It is well known that the orthodontic force applied to teeth generates a series of events that remodel the periodontal ligament (PDL). Extracellular matrix proteins (ECM) are described as molecular regulators of these events. However, the exact contribution of these proteins in human PDL modeling by orthodontic force ...

  3. Expression of melanin and insecticidal protein from Rhodotorula ...

    African Journals Online (AJOL)

    SERVER

    2006-02-16

    Feb 16, 2006 ... In this paper, the isolation of E. coli transformants capable of producing both ... The crystal protein gene is located on the chromosome as well as on a ... levels of foreign protein include alterations in cells size and growth rate ...

  4. Synthesis of uterine endometrial proteins during early diestrus in the cyclic and pregnant dog, and after estrogen and progesterone treatment.

    Science.gov (United States)

    Buhi, W C; Thatcher, M J; Shille, V M; Alvarez, I M; Lannon, A P; Johnson, J

    1992-09-01

    The objectives of this study were to identify and characterize dog uterine endometrial proteins synthesized de novo in explant culture during early luteal phase, to examine distribution of these proteins prior to the embryo's entering the uterus and during its free-floating period prior to implantation, and to examine regulation of endometrial proteins by estrogen and progesterone (P4) treatments. Uterine endometrium was collected from cyclic and pregnant bitches on diestrus Days 3, 7, and 10 as determined by loss of cornification of vaginal epithelium, and from ovariectomized dogs after treatment with corn oil, estrogen, P4, or estrogen followed by 1 or 2 wk of P4. Tissue was incubated in an explant culture system in the presence of [3H]leucine or [35S]methionine. The rate of incorporation of [3H]leucine into nondialyzable macromolecules indicated no significant change in rates of incorporation by status (pregnant vs. nonpregnant), day, or steroid treatment. Uterine endometrial-conditioned culture medium, analyzed by two-dimensional SDS-PAGE and fluorography, revealed a complex array of at least ten proteins or protein complexes in cyclic and pregnant bitches. No difference in protein pattern was detected by status; however, differences in distribution were apparent by day of cycle or early pregnancy. Two major proteins, cP5 (M(r) 54,686) and cP6 (M(r) 23,010) appeared to be differentially expressed. Expression of cP5, maximal on diestrus Day 3, decreased as the cycle or pregnancy progressed to diestrus Day 10. In contrast, expression of cP6, a minor protein on diestrus Day 3, appeared to be up-regulated for each status to Day 10, with increased intensity and multiple isoelectric and molecular-weight variants. In ovariectomized steroid-treated dogs, two-dimensional SDS-PAGE showed that pattern and distribution of specific proteins were affected by treatment. Acidic protein cP1 (M(r) 87,600), synthesized after corn oil and P4 treatment, was suppressed with

  5. Schisandrin B protects PC12 cells by decreasing the expression of amyloid precursor protein and vacuolar protein sorting 35★

    Science.gov (United States)

    Yan, Mingmin; Mao, Shanping; Dong, Huimin; Liu, Baohui; Zhang, Qian; Pan, Gaofeng; Fu, Zhiping

    2012-01-01

    PC12 cell injury was induced using 20 μM amyloid β-protein 25–35 to establish a model of Alzheimer's disease. The cells were then treated with 5, 10, and 25 μM Schisandrin B. Methylthiazolyldiphenyl-tetrazolium bromide assays and Hoechst 33342 staining results showed that with increasing Schisandrin B concentration, the survival rate of PC12 cells injured by amyloid β-protein 25–35 gradually increased and the rate of apoptosis gradually decreased. Reverse transcription-PCR, immunocytochemical staining and western blot results showed that with increasing Schisandrin B concentration, the mRNA and protein expression of vacuolar protein sorting 35 and amyloid precursor protein were gradually decreased. Vacuolar protein sorting 35 and amyloid precursor protein showed a consistent trend for change. These findings suggest that 5, 10, and 25 μM Schisandrin B antagonizes the cellular injury induced by amyloid β-protein 25–35 in a dose-dependent manner. This may be caused by decreasing the expression of vacuolar protein sorting 35 and amyloid precursor protein. PMID:25745458

  6. Differential protein expression in alligator leukocytes in response to bacterial lipopolysaccharide injection.

    Science.gov (United States)

    Merchant, Mark; Kinney, Clint; Sanders, Paige

    2009-12-01

    Blood was collected from three juvenile alligators (Alligator mississippiensis) before, and again 24h after, injection with bacterial lipopolysaccharide (LPS). The leukocytes were collected from both samples, and the proteins were extracted. Each group of proteins was labeled with a different fluorescent dye and the differences in protein expression were analyzed by two dimensional differential in-gel expressions (2D-DIGE). The proteins which appeared to be increased or decreased by treatment with LPS were selected and analyzed by MALDI-TOF to determine mass and LC-MS/MS to acquire the partial protein sequences. The peptide sequences were compared to the NCBI protein sequence database to determine homology with other sequences from other species. Several proteins of interest appeared to be increased upon LPS stimulation. Proteins with homology to human transgelin-2, fish glucose-6-phosphate dehydrogenase, amphibian α-enolase, alligator lactate dehydrogenase, fish ubiquitin-activating enzyme, and fungal β-tubulin were also increased after LPS injection. Proteins with homology to fish vimentin 4, murine heterogeneous nuclear ribonucleoprotein A3, and avian calreticulin were found to be decreased in response to LPS. In addition, five proteins, four of which were up-regulated (827, 560, 512, and 650%) and one that exhibited repressed expression (307%), did not show homology to any protein in the database, and thus may represent newly discovered proteins. We are using this biochemical approach to isolate and characterize alligator proteins with potential relevant immune function.

  7. Decreased SAP expression in T cells from patients with SLE contributes to early signaling abnormalities and reduced IL-2 production

    Science.gov (United States)

    Karampetsou, Maria P.; Comte, Denis; Kis-Toth, Katalin; Terhorst, Cox; Kyttaris, Vasileios C.; Tsokos, George C.

    2016-01-01

    T cells from patients with systemic lupus erythematosus (SLE) display a number of functions including increased early signaling events following engagement of the T cell receptor (TCR). Signaling lymphocytic activation molecule family (SLAMF) cell surface receptors and the X-chromosome-defined signaling lymphocytic activation molecule-associated protein (SAP) adaptor are important in the development of several immunocyte lineages and modulating immune response. Here we present evidence that SAP protein levels are decreased in T cells and in their main subsets isolated from 32 women and 3 men with SLE independently of disease activity. In SLE T cells the SAP protein is also subject to increased degradation by a caspase-3. Forced expression of SAP in SLE T cells simultaneously heightened IL-2 production, calcium (Ca2+) responses and tyrosine phosphorylation of a number of proteins. Exposure of normal T cells to SLE serum IgG, known to contain anti-CD3/TCR antibodies, resulted in SAP downregulation. We conclude that SLE T cells display reduced levels of the adaptor protein SAP probably as a result of continuous T cell activation and degradation by caspase-3. Restoration of SAP levels in SLE T cells corrects the overexcitable lupus T cell phenotype. PMID:27183584

  8. Comparative transcriptional and translational analysis of leptospiral outer membrane protein expression in response to temperature.

    Science.gov (United States)

    Lo, Miranda; Cordwell, Stuart J; Bulach, Dieter M; Adler, Ben

    2009-12-08

    Leptospirosis is a global zoonosis affecting millions of people annually. Transcriptional changes in response to temperature were previously investigated using microarrays to identify genes potentially expressed upon host entry. Past studies found that various leptospiral outer membrane proteins are differentially expressed at different temperatures. However, our microarray studies highlighted a divergence between protein abundance and transcript levels for some proteins. Given the abundance of post-transcriptional expression control mechanisms, this finding highlighted the importance of global protein analysis systems. To complement our previous transcription study, we evaluated differences in the proteins of the leptospiral outer membrane fraction in response to temperature upshift. Outer membrane protein-enriched fractions from Leptospira interrogans grown at 30 degrees C or overnight upshift to 37 degrees C were isolated and the relative abundance of each protein was determined by iTRAQ analysis coupled with two-dimensional liquid chromatography and tandem mass spectrometry (2-DLC/MS-MS). We identified 1026 proteins with 99% confidence; 27 and 66 were present at elevated and reduced abundance respectively. Protein abundance changes were compared with transcriptional differences determined from the microarray studies. While there was some correlation between the microarray and iTRAQ data, a subset of genes that showed no differential expression by microarray was found to encode temperature-regulated proteins. This set of genes is of particular interest as it is likely that regulation of their expression occurs post-transcriptionally, providing an opportunity to develop hypotheses about the molecular dynamics of the outer membrane of Leptospira in response to changing environments. This is the first study to compare transcriptional and translational responses to temperature shift in L. interrogans. The results thus provide an insight into the mechanisms used by L

  9. Postexercise nutrient intake enhances leg protein balance in early postmenopausal women

    DEFF Research Database (Denmark)

    Holm, Lars; Esmarck, Birgitte; Suetta, Charlotte

    2005-01-01

    BACKGROUND: We investigated the effect of nutrient administration after a session of resistance exercise on muscle protein kinetics in six healthy, early postmenopausal women, in a crossover design of random and double-blinded administration of protein and carbohydrate (PC) or placebo (NON). METH...

  10. Expression of melanin and insecticidal protein from Rhodotorula ...

    African Journals Online (AJOL)

    Both the salmon/red melanin and the insecticidal producing genes of Rhodotorula glutinis was successfully expressed in Escherichia coli using plasmid pZErO-1. This work suggests that in Rhodotorula species melanin and insecticidal toxin are co-expressed and therefore possibly co-evolved. Keywords: Rhodotorula ...

  11. Immunohistochemical analysis of Clara cell secretory protein expression in a transgenic model of mouse lung carcinogenesis

    International Nuclear Information System (INIS)

    Hicks, Sarah M.; Vassallo, Jeffrey D.; Dieter, Matthew Z.; Lewis, Cindy L.; Whiteley, Laurence O.; Fix, Andrew S.; Lehman-McKeeman, Lois D.

    2003-01-01

    Immunohistochemical methods have been widely used to determine the histogenesis of spontaneous and chemically-induced mouse lung tumors. Typically, antigens for either alveolar Type II cells or bronchiolar epithelial Clara cells are studied. In the present work, the morphological and immunohistochemical phenotype of a transgenic mouse designed to develop lung tumors arising from Clara cells was evaluated. In this model, Clara cell-specific transformation is accomplished by directed expression of the SV40 large T antigen (TAg) under the mouse Clara cell secretory protein (CC10) promoter. In heterozygous mice, early lesions at 1 month of age consisted of hyperplastic bronchiolar epithelial cells. These progressed to adenoma by 2 months as proliferating epithelium extended into adjacent alveolar spaces. By 4 months, a large portion of the lung parenchyma was composed of tumor masses. Expression of constitutive CC10 was diminished in transgenic animals at all time points. Only the occasional cell or segment of the bronchiolar epithelium stained positively for CC10 by immunohistochemistry, and all tumors were found to be uniformly negative for staining. These results were corroborated by Western blotting, where CC10 was readily detectable in whole lung homogenate from nontransgenic animals, but not detected in lung from transgenic animals at any time point. Tumors were also examined for expression of surfactant apoprotein C (SPC), an alveolar Type II cell-specific marker, and found to be uniformly negative for staining. These results indicate that, in this transgenic model, expression of CC10, which is widely used to determine whether lung tumors arise from Clara cells, was reduced and subsequently lost during Clara cell tumor progression

  12. [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.

  13. Positive muscle protein net balance and differential regulation of atrogene expression after resistance exercise and milk protein supplementation

    DEFF Research Database (Denmark)

    Reitelseder, Søren; Agergaard, Jakob; Doessing, Simon

    2014-01-01

    Purpose Resistance exercise and amino acid availability are positive regulators of muscle protein net balance (NB). However, anabolic responses to resistance exercise and protein supplementation deserve further elucidation. The purpose was to compare intakes of whey, caseinate (both: 0.30 g/kg lean...... body mass), or a non-caloric control after heavy resistance exercise on protein turnover and mRNA expressions of forkhead homeobox type O (FOXO) isoforms, muscle RING finger 1 (MuRF1), and Atrogin1 in young healthy males. Methods Protein turnover was determined by stable isotope-labeled leucine...

  14. Expression and affinity purification of recombinant proteins from plants

    Science.gov (United States)

    Desai, Urvee A.; Sur, Gargi; Daunert, Sylvia; Babbitt, Ruth; Li, Qingshun

    2002-01-01

    With recent advances in plant biotechnology, transgenic plants have been targeted as an inexpensive means for the mass production of proteins for biopharmaceutical and industrial uses. However, the current plant purification techniques lack a generally applicable, economic, large-scale strategy. In this study, we demonstrate the purification of a model protein, beta-glucuronidase (GUS), by employing the protein calmodulin (CaM) as an affinity tag. In the proposed system, CaM is fused to GUS. In the presence of calcium, the calmodulin fusion protein binds specifically to a phenothiazine-modified surface of an affinity column. When calcium is removed with a complexing agent, e.g., EDTA, calmodulin undergoes a conformational change allowing the dissociation of the calmodulin-phenothiazine complex and, therefore, permitting the elution of the GUS-CaM fusion protein. The advantages of this approach are the fast, efficient, and economical isolation of the target protein under mild elution conditions, thus preserving the activity of the target protein. Two types of transformation methods were used in this study, namely, the Agrobacterium-mediated system and the viral-vector-mediated transformation system. Copyright 2002 Elsevier Science (USA).

  15. 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.

  16. Strategies for production of active eukaryotic proteins in bacterial expression system

    Institute of Scientific and Technical Information of China (English)

    Orawan Khow; Sunutcha Suntrarachun

    2012-01-01

    Bacteria have long been the favorite expression system for recombinant protein production. However, the flaw of the system is that insoluble and inactive proteins are co-produced due to codon bias, protein folding, phosphorylation, glycosylation, mRNA stability and promoter strength. Factors are cited and the methods to convert to soluble and active proteins are described, for example a tight control of Escherichia coli milieu, refolding from inclusion body and through fusion technology.

  17. Changes in protein expression in p53 deleted spontaneous thymic lymphomas

    DEFF Research Database (Denmark)

    Honoré, Bent; Vorum, Henrik; Pedersen, Anders Elm

    2004-01-01

    with the protein expression in p53+/+ and p53-/- thymocytes. Only a minority (13 proteins) of the quantitatively changed proteins were common for the two thymic lymphoma cell lines, suggesting that the p53 deficiency mainly results in genetic dysfunctions which are individual for a given tumor. Two of the detected...... structure containing motifs of the glyoxalase-bleomycin resistance protein family (MDR) as deduced from the cDNA....

  18. 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.

  19. 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.

  20. Post-Transcriptional Control of Gene Expression in Mouse Early Embryo Development: A View from the Tip of the Iceberg

    Directory of Open Access Journals (Sweden)

    Claudio Sette

    2011-04-01

    Full Text Available Fertilization is a very complex biological process that requires the perfect cooperation between two highly specialized cells: the male and female gametes. The oocyte provides the physical space where this process takes place, most of the energetic need, and half of the genetic contribution. The spermatozoon mostly contributes the other half of the chromosomes and it is specialized to reach and to penetrate the oocyte. Notably, the mouse oocyte and early embryo are transcriptionally inactive. Hence, they fully depend on the maternal mRNAs and proteins stored during oocyte maturation to drive the onset of development. The new embryo develops autonomously around the four-cell stage, when maternal supplies are exhausted and the zygotic genome is activated in mice. This oocyte-to-embryo transition needs an efficient and tightly regulated translation of the maternally-inherited mRNAs, which likely contributes to embryonic genome activation. Full understanding of post-transcriptional regulation of gene expression in early embryos is crucial to understand the reprogramming of the embryonic genome, it might help driving reprogramming of stem cells in vitro and will likely improve in vitro culturing of mammalian embryos for assisted reproduction. Nevertheless, the knowledge of the mechanism(s underlying this fundamental step in embryogenesis is still scarce, especially if compared to other model organisms. We will review here the current knowledge on the post-transcriptional control of gene expression in mouse early embryos and discuss some of the unanswered questions concerning this fascinating field of biology.

  1. [Correlation between RNA Expression Level and Early PMI in Human Brain Tissue].

    Science.gov (United States)

    Lü, Y H; Ma, K J; Li, Z H; Gu, J; Bao, J Y; Yang, Z F; Gao, J; Zeng, Y; Tao, L; Chen, L

    2016-08-01

    To explore the correlation between the expression levels of several RNA markers in human brain tissue and early postmortem interval (PMI). Twelve individuals with known PMI (range from 4.3 to 22.5 h) were selected and total RNA was extracted from brain tissue. Eight commonly used RNA markers were chosen including β -actin, GAPDH, RPS29, 18S rRNA, 5S rRNA, U6 snRNA, miRNA-9 and miRNA-125b, and the expression levels were detected in brain tissue by real-time fluorescent quantitative PCR. The internal reference markers with stable expression in early PMI were screened using geNorm software and the relationship between its expression level and some relevant factors such as age, gender and cause of death were analyzed. RNA markers normalized by internal reference were inserted into the mathematic model established by previous research for PMI estimation using R software. Model quality was judged by the error rate calculated with estimated PMI. 5S rRNA, miRNA-9 and miRNA-125b showed quite stable expression and their expression levels had no relation with age, gender and cause of death. The error rate of estimated PMI using β -actin was 24.6%, while GAPDH was 41.0%. 5S rRNA, miRNA-9 and miRNA-125b are suitable as internal reference markers of human brain tissue owing to their stable expression in early PMI. The expression level of β -actin correlates well with PMI, which can be used as an additional index for early PMI estimation. Copyright© by the Editorial Department of Journal of Forensic Medicine

  2. miR-24 and miR-205 expression is dependent on HPV onco-protein expression in keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    McKenna, Declan J., E-mail: dj.mckenna@ulster.ac.uk [Biomedical Sciences Research Institute, University of Ulster, Coleraine, Co. Derry BT52 1SA (United Kingdom); Centre for Cancer Research and Cell Biology, School of Medicine, Dentistry and Biomedical Science, Queen' s University Belfast, Belfast BT9 7BL (United Kingdom); Patel, Daksha, E-mail: d.patel@qub.ac.uk [Centre for Cancer Research and Cell Biology, School of Medicine, Dentistry and Biomedical Science, Queen' s University Belfast, Belfast BT9 7BL (United Kingdom); McCance, Dennis J., E-mail: d.mccance@qub.ac.uk [Centre for Cancer Research and Cell Biology, School of Medicine, Dentistry and Biomedical Science, Queen' s University Belfast, Belfast BT9 7BL (United Kingdom)

    2014-01-05

    A screen of microRNA (miRNA) expression following differentiation in human foreskin keratinocytes (HFKs) identified changes in several miRNAs, including miR-24 and miR-205. We investigated how expression of Human Papilloma Virus Type-16 (HPV16) onco-proteins E6 and E7 affected expression of miR-24 and miR-205 during proliferation and differentiation of HFKs. We show that the induction of both miR-24 and miR-205 observed during differentiation of HFKs is lost in HFKs expressing E6 and E7. We demonstrate that the effect on miR-205 is due to E7 activity, as miR-205 expression is dependent on pRb expression. Finally, we provide evidence that miR-24 effects in the cell may be due to targeting of cyclin dependent kinase inhibitor p27. In summary, these results indicate that expression of both miR-24 and miR-205 are impacted by E6 and/or E7 expression, which may be one mechanism by which HPV onco-proteins can disrupt the balance between proliferation and differentiation in keratinocytes. - Highlights: • miR-24 and miR-205 are induced during keratinocyte differentiation. • This induction is lost in keratinocytes expressing HPV onco-proteins E6 and E7. • miR-205 is dependent upon pRb expression. • miR-24 targets p27 in cycling keratinocytes.

  3. miR-24 and miR-205 expression is dependent on HPV onco-protein expression in keratinocytes

    International Nuclear Information System (INIS)

    McKenna, Declan J.; Patel, Daksha; McCance, Dennis J.

    2014-01-01

    A screen of microRNA (miRNA) expression following differentiation in human foreskin keratinocytes (HFKs) identified changes in several miRNAs, including miR-24 and miR-205. We investigated how expression of Human Papilloma Virus Type-16 (HPV16) onco-proteins E6 and E7 affected expression of miR-24 and miR-205 during proliferation and differentiation of HFKs. We show that the induction of both miR-24 and miR-205 observed during differentiation of HFKs is lost in HFKs expressing E6 and E7. We demonstrate that the effect on miR-205 is due to E7 activity, as miR-205 expression is dependent on pRb expression. Finally, we provide evidence that miR-24 effects in the cell may be due to targeting of cyclin dependent kinase inhibitor p27. In summary, these results indicate that expression of both miR-24 and miR-205 are impacted by E6 and/or E7 expression, which may be one mechanism by which HPV onco-proteins can disrupt the balance between proliferation and differentiation in keratinocytes. - Highlights: • miR-24 and miR-205 are induced during keratinocyte differentiation. • This induction is lost in keratinocytes expressing HPV onco-proteins E6 and E7. • miR-205 is dependent upon pRb expression. • miR-24 targets p27 in cycling keratinocytes

  4. 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.

  5. Early Involvement of Death-Associated Protein Kinase Promoter Hypermethylation in the Carcinogenesis of Barrett's Esophageal Adenocarcinoma and Its Association with Clinical Progression

    Directory of Open Access Journals (Sweden)

    Doerthe Kuester

    2007-03-01

    Full Text Available Esophageal Barrett's adenocarcinoma (BA develops through a multistage process, which is associated with the transcriptional silencing of tumor-suppressor genes by promoter CpG island hypermethylation. In this study, we explored the promoter hypermethylation and protein expression of proapoptotic deathassociated protein kinase (DAPK during the multistep Barrett's carcinogenesis cascade. Early BA and paired samples of premalignant lesions of 61 patients were analyzed by methylation-specific polymerase chain reaction and immunohistochemistry. For the association of clinicopathological markers and protein expression, an immunohistochemical tissue microarray analysis of 66 additional BAs of advanced tumor stages was performed. Hypermethylation of DAPK promoter was detected in 20% of normal mucosa, 50% of Barrett's metaplasia, 53% of dysplasia, and 60% of adenocarcinomas, and resulted in a marked decrease in DAPK protein expression (P < .01. The loss of DAPK protein was significantly associated with advanced depth of tumor invasion and advanced tumor stages (P < .001. Moreover, the severity of reflux esophagitis correlated significantly with the hypermethylation rate of the DAPK promoter (P < .003. Thus, we consider DAPK inactivation by promoter hypermethylation as an early event in Barrett's carcinogenesis and suggest that a decreased protein expression of DAPK likely plays a role in the development and progression of BA.

  6. 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.

  7. 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

  8. Classification, expression pattern and comparative analysis of sugarcane expressed sequences tags (ESTs encoding glycine-rich proteins (GRPs

    Directory of Open Access Journals (Sweden)

    Fusaro Adriana

    2001-01-01

    Full Text Available Since the isolation of the first glycine-rich proteins (GRPs in plants a wealth of new GRPs have been identified. The highly specific but diverse expression pattern of grp genes, taken together with the distinct sub-cellular localization of some GRP groups, clearly indicate that these proteins are involved in several independent physiological processes. Notwithstanding the absence of a clear definition of the role of GRPs in plant cells, studies conducted with these proteins have provided new and interesting insights into the molecular biology and cell biology of plants. Complexly regulated promoters and distinct mechanisms for the regulation of gene expression have been demonstrated and new protein targeting pathways, as well as the exportation of GRPs from different cell types have been discovered. These data show that GRPs can be useful as markers and/or models to understand distinct aspects of plant biology. In this paper, the structural and functional features of these proteins in sugarcane (Saccharum officinarum L. are summarized. Since this is the first description of GRPs in sugarcane, special emphasis has been given to the expression pattern of these GRP genes by studying their abundance and prevalence in the different cDNA-libraries of the Sugarcane Expressed Sequence Tag (SUCEST project . The comparison of sugarcane GRPs with GRPs from other species is also discussed.

  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. Cpt1a gene expression in peripheral blood mononuclear cells as an early biomarker of diet-related metabolic alterations

    Directory of Open Access Journals (Sweden)

    Rubén Díaz-Rúa

    2016-11-01

    Full Text Available Background: Research on biomarkers that provide early information about the development of future metabolic alterations is an emerging discipline. Gene expression analysis in peripheral blood mononuclear cells (PBMC is a promising tool to identify subjects at risk of developing diet-related diseases. Objective: We analysed PBMC expression of key energy homeostasis-related genes in a time-course analysis in order to find out early markers of metabolic alterations due to sustained intake of high-fat (HF and high-protein (HP diets. Design: We administered HF and HP diets (4 months to adult Wistar rats in isocaloric conditions to a control diet, mainly to avoid overweight associated with the intake of hyperlipidic diets and, thus, to be able to characterise markers of metabolically obese normal-weight (MONW syndrome. PBMC samples were collected at different time points of dietary treatment and expression of relevant energy homeostatic genes analysed by real-time reverse transcription-polymerase chain reaction. Serum parameters related with metabolic syndrome, as well as fat deposition in liver, were also analysed. Results: The most outstanding results were those obtained for the expression of the lipolytic gene carnitine palmitoyltransferase 1a (Cpt1a. Cpt1a expression in PBMC increased after only 1 month of exposure to both unbalanced diets, and this increased expression was maintained thereafter. Interestingly, in the case of the HF diet, Cpt1a expression was altered even in the absence of increased body weight but correlated with alterations such as higher insulin resistance, alteration of serum lipid profile and, particularly, increased fat deposition in liver, a feature characteristic of metabolic syndrome, which was even observed in animals fed with HP diet. Conclusions: We propose Cpt1a gene expression analysis in PBMC as an early biomarker of metabolic alterations associated with MONW phenotype due to the intake of isocaloric HF diets, as

  11. Prokineticin 1 protein expression is a useful new prognostic factor for human sporadic colorectal cancer.

    Science.gov (United States)

    Nakazawa, Toshiyuki; Goi, Takanori; Hirono, Yasuo; Yamaguchi, Akio

    2015-05-01

    Hematogenous metastasis, regarded as closely related to angiogenic growth factors, is associated with colorectal cancer prognosis. The angiogenic growth factor prokineticin 1 (PROK1) has been cloned from endocrine cells. However, its protein expression in human malignant tumors has not been studied. The current study established the anti-PROK1 monoclonal antibody (mAb) and examined the relationship between the expression of PROK1 protein and human colorectal cancer. The expression of PROK1 protein was assessed in 620 resected sporadic colorectal cancer tissue samples by immunohistochemical staining with in-house-developed human PROK1 mAb to investigate the relationship of PROK1 expression to clinicopathologic factors, recurrence, and survival rate and to evaluate its prognostic significance. The expression of PROK1 protein was detected in 36 % (223/620) of human primary colorectal cancer lesions but no in the healthy mucosa adjacent to the colorectal cancer lesions. According to the clinicopathologic examinations, the frequency of positive PROK1 expression was significantly higher in cases with serosal invasion, lymphatic invasion, venous invasion, lymph node metastasis, liver metastasis, hematogenous metastasis, and higher stage disease. The recurrence rate and prognosis for patients with PROK1 expression-positive lesions were significantly worse. In the Cox proportional hazard model, PROK1 expression was an independent prognostic factor. The expression of PROK1 protein was identified for the first time as a new prognostic factor in colorectal cancer.

  12. Cloning and expression of Toxoplasma gondii tachyzoite P22 protein

    African Journals Online (AJOL)

    Jane

    2011-08-01

    Aug 1, 2011 ... Expressd protein was purified by affinity chromatography and confirmed by western blot ... Key words: Toxoplasma gondii, cloning, recombinant P22. INTRODUCTION. Toxoplasma gondii ..... an ELISA Assay. Iran. J. Immunol.

  13. Adaption of Saccharomyces cerevisiae expressing a heterologous protein

    DEFF Research Database (Denmark)

    Krogh, Astrid Mørkeberg; Beck, Vibe; Højlund Christensen, Lars

    2008-01-01

    Production of the heterologous protein, bovine aprotinin, in Saccharomyces cerevisiae was shown to affect the metabolism of the host cell to various extent depending on the strain genotype. Strains with different genotypes, industrial and laboroatory, respectively, were investigated. The maximal...

  14. Differential expression of speckled POZ protein, SPOP: Putative ...

    Indian Academy of Sciences (India)

    2014-05-01

    May 1, 2014 ... In other mouse tissues and human cancer cell lines analysed, only low SPOP ... speckled POZ protein; SRC-3, steroid receptor co-activator-3; TNF, tumour necrosis factor; ...... complexity of primary human prostate cancer.

  15. Ligand Binding Domain Protein in Tetracycline-Inducible Expression ...

    African Journals Online (AJOL)

    binding domain proteins in E. coli using a tetracycline inducible system. To allow for ... development of molecular ligands with improved therapeutic windows. Keywords: Nuclear receptor ..... functional recombinant cannabinoid receptor CB2 in ...

  16. In vivo extracellular matrix protein expression by human periodontal ...

    African Journals Online (AJOL)

    ONOS

    2010-08-23

    Aug 23, 2010 ... Extracellular matrix proteins (ECM) are described as molecular regulators of these events. ..... zation and adhesive interaction of cells (Yamada, 1983). .... periodontal ligament fibroblasts after simulation of orthodontic force.

  17. Increased tolerance to two oomycete pathogens in transgenic tobacco expressing pathogenesis-related protein 1a.

    OpenAIRE

    Alexander, D; Goodman, R M; Gut-Rella, M; Glascock, C; Weymann, K; Friedrich, L; Maddox, D; Ahl-Goy, P; Luntz, T; Ward, E

    1993-01-01

    Expression of pathogenesis-related protein 1a (PR-1a), a protein of unknown biochemical function, is induced to high levels in tobacco in response to pathogen infection. The induction of PR-1a expression is tightly correlated with the onset of systemic acquired resistance (SAR), a defense response effective against a variety of fungal, viral, and bacterial pathogens. While PR-1a has been postulated to be involved in SAR, and is the most highly expressed of the PR proteins, evidence for its ro...

  18. Ectopic expression of the erythrocyte band 3 anion exchange protein, using a new avian retrovirus vector

    DEFF Research Database (Denmark)

    Fuerstenberg, S; Beug, H; Introna, M

    1990-01-01

    into protein. Using the Escherichia coli beta-galactosidase gene cloned into the vector as a test construct, expression of enzyme activity could be detected in 90 to 95% of transfected target cells and in 80 to 85% of subsequently infected cells. In addition, a cDNA encoding the avian erythrocyte band 3 anion...... exchange protein has been expressed from the vector in both chicken embryo fibroblasts and QT6 cells and appears to function as an active, plasma membrane-based anion transporter. The ectopic expression of band 3 protein provides a visual marker for vector function in these cells....

  19. Analysis of disease-associated protein expression using quantitative proteomics—fibulin-5 is expressed in association with hepatic fibrosis.

    Science.gov (United States)

    Bracht, Thilo; Schweinsberg, Vincent; Trippler, Martin; Kohl, Michael; Ahrens, Maike; Padden, Juliet; Naboulsi, Wael; Barkovits, Katalin; Megger, Dominik A; Eisenacher, Martin; Borchers, Christoph H; Schlaak, Jörg F; Hoffmann, Andreas-Claudius; Weber, Frank; Baba, Hideo A; Meyer, Helmut E; Sitek, Barbara

    2015-05-01

    Hepatic fibrosis and cirrhosis are major health problems worldwide. Until now, highly invasive biopsy remains the diagnostic gold standard despite many disadvantages. To develop noninvasive diagnostic assays for the assessment of liver fibrosis, it is urgently necessary to identify molecules that are robustly expressed in association with the disease. We analyzed biopsied tissue samples from 95 patients with HBV/HCV-associated hepatic fibrosis using three different quantification methods. We performed a label-free proteomics discovery study to identify novel disease-associated proteins using a subset of the cohort (n = 27). Subsequently, gene expression data from all available clinical samples were analyzed (n = 77). Finally, we performed a targeted proteomics approach, multiple reaction monitoring (MRM), to verify the disease-associated expression in samples independent from the discovery approach (n = 68). We identified fibulin-5 (FBLN5) as a novel protein expressed in relation to hepatic fibrosis. Furthermore, we confirmed the altered expression of microfibril-associated glycoprotein 4 (MFAP4), lumican (LUM), and collagen alpha-1(XIV) chain (COL14A1) in association to hepatic fibrosis. To our knowledge, no tissue-based quantitative proteomics study for hepatic fibrosis has been performed using a cohort of comparable size. By this means, we add substantial evidence for the disease-related expression of the proteins examined in this study.

  20. Differential protein expression in maize (Zea mays) in response to ...

    African Journals Online (AJOL)

    Jane

    2011-07-27

    Jul 27, 2011 ... Accepted 25 May, 2011. Maize (Zea mays) is a major food stable in sub-Saharan Africa. .... has investigated differential expression at the proteome level, comparing this ..... GK, Jwa NS (2001). Characterization of rice (Oryza.

  1. 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.

  2. Unique protein expression signatures of survival time in kidney renal clear cell carcinoma through a pan-cancer screening.

    Science.gov (United States)

    Han, Guangchun; Zhao, Wei; Song, Xiaofeng; Kwok-Shing Ng, Patrick; Karam, Jose A; Jonasch, Eric; Mills, Gordon B; Zhao, Zhongming; Ding, Zhiyong; Jia, Peilin

    2017-10-03

    In 2016, it is estimated that there will be 62,700 new cases of kidney cancer in the United States, and 14,240 patients will die from the disease. Because the incidence of kidney renal clear cell carcinoma (KIRC), the most common type of kidney cancer, is expected to continue to increase in the US, there is an urgent need to find effective diagnostic biomarkers for KIRC that could help earlier detection of and customized treatment strategies for the disease. Accordingly, in this study we systematically investigated KIRC's prognostic biomarkers for survival using the reverse phase protein array (RPPA) data and the high throughput sequencing data from The Cancer Genome Atlas (TCGA). With comprehensive data available in TCGA, we systematically screened protein expression based survival biomarkers in 10 major cancer types, among which KIRC presented many protein prognostic biomarkers of survival time. This is in agreement with a previous report that expression level changes (mRNAs, microRNA and protein) may have a better performance for prognosis of KIRC. In this study, we also identified 52 prognostic genes for KIRC, many of which are involved in cell-cycle and cancer signaling, as well as 15 tumor-stage-specific prognostic biomarkers. Notably, we found fewer prognostic biomarkers for early-stage than for late-stage KIRC. Four biomarkers (the RPPA protein IDs: FASN, ACC1, Cyclin_B1 and Rad51) were found to be prognostic for survival based on both protein and mRNA expression data. Through pan-cancer screening, we found that many protein biomarkers were prognostic for patients' survival in KIRC. Stage-specific survival biomarkers in KIRC were also identified. Our study indicated that these protein biomarkers might have potential clinical value in terms of predicting survival in KIRC patients and developing individualized treatment strategies. Importantly, we found many biomarkers in KIRC at both the mRNA expression level and the protein expression level. These

  3. Tight junction protein ZO-2 expression and relative function of ZO-1 and ZO-2 during mouse blastocyst formation

    International Nuclear Information System (INIS)

    Sheth, Bhavwanti; Nowak, Rachael L.; Anderson, Rebecca; Kwong, Wing Yee; Papenbrock, Thomas; Fleming, Tom P.

    2008-01-01

    Apicolateral tight junctions (TJs) between epithelial cells are multiprotein complexes regulating membrane polarity and paracellular transport and also contribute to signalling pathways affecting cell proliferation and gene expression. ZO-2 and other ZO family members form a sub-membranous scaffold for binding TJ constituents. We investigated ZO-2 contribution to TJ biogenesis and function during trophectoderm epithelium differentiation in mouse preimplantation embryos. Our data indicate that ZO-2 is expressed from maternal and embryonic genomes with maternal ZO-2 protein associated with nuclei in zygotes and particularly early cleavage stages. Embryonic ZO-2 assembled at outer blastomere apicolateral junctional sites from the late 16-cell stage. Junctional ZO-2 first co-localised with E-cadherin in a transient complex comprising adherens junction and TJ constituents before segregating to TJs after their separation from the blastocyst stage (32-cell onwards). ZO-2 siRNA microinjection into zygotes or 2-cell embryos resulted in specific knockdown of ZO-2 mRNA and protein within blastocysts. Embryos lacking ZO-2 protein at trophectoderm TJs exhibited delayed blastocoel cavity formation but underwent normal cell proliferation and outgrowth morphogenesis. Quantitative analysis of trophectoderm TJs in ZO-2-deficient embryos revealed increased assembly of ZO-1 but not occludin, indicating ZO protein redundancy as a compensatory mechanism contributing to the mild phenotype observed. In contrast, ZO-1 knockdown, or combined ZO-1 and ZO-2 knockdown, generated a more severe inhibition of blastocoel formation indicating distinct roles for ZO proteins in blastocyst morphogenesis

  4. Alterations in expression of senescence marker protein-30 gene by 3,3',5-triiodo-L-thyronine (T3).

    Science.gov (United States)

    Sar, Pranati; Rath, Bandita; Subudhi, Umakanta; Chainy, Gagan Bihari Nityananda; Supakar, Prakash Chandra

    2007-09-01

    Thyroid hormone (T3) is essential for normal development, differentiation, and metabolic balance of the body. A toxic dose of T(3) in animals increases the basal metabolic rate and reactive oxygen species production, resulting more oxidative stress through Ca(2+) influx to cytoplasm. Senescence Marker Protein-30 (SMP30) is preferentially expressed in the liver and protects cells against various injuries by enhancement of Ca(2+) efflux to either extra cellular space or intraorganellar spaces through membrane Ca(2+) pump activity. In this paper we report an alteration in the level of SMP30 gene expression using RT-PCR and western blot analysis in T(3) treated female Wistar rats. The results indicate that there is an induction of SMP30 expression during early hours of T(3 )treatment and it declines in severe hyperthyroidism. Therefore, we speculate that SMP30 is regulated by T(3) and might play a protective role in hyperthyroidism.

  5. Nuclear and cellular expression data from the whole 16-cell stage Arabidopsis thaliana embryo and a cell type-specific expression atlas of the early Arabidopsis embryo

    NARCIS (Netherlands)

    Palovaara, J.P.J.

    2017-01-01

    SuperSeries contain expression data from the nuclei of cell types involved in patterning events, with focus on root apical stem cell formation, at 16-cell stage, early globular stage and late globular stage in the early Arabidopsis embryo (atlas). Expression data comparing nuclear and cellular RNA

  6. Del-1 Expression as a Potential Biomarker in Triple-Negative Early Breast Cancer.

    Science.gov (United States)

    Lee, Soo Jung; Lee, Jeeyeon; Kim, Wan Wook; Jung, Jin Hyang; Park, Ho Yong; Park, Ji-Young; Chae, Yee Soo

    2018-01-01

    A differential diagnostic role for plasma Del-1 was proposed for early breast cancer (EBC) in our previous study. We examined tumoral Del-1 expression and analyzed its prognostic impact among patients with EBC. Del-1 mRNA expression was assessed in breast epithelial and cancer cells. Meanwhile, the tumoral expression of Del-1 was determined based on tissue microarrays and immunohistochemistry results from 440 patients. While a high Del-1 mRNA expression was found in all the breast cancer cell lines, the expression was significantly higher in MDA-MB-231. Tumoral expression of Del-1 was also significantly associated with a negative expression of estrogen receptor or progesterone receptor, and low expression of Ki-67, particularly in the case of triple-negative breast cancer (TNBC) (p breast cancer cell lines exhibited Del-1 expression, the expression rate and intensity were specifically prominent in TNBC. In addition, based on its relationship to an unfavorable histology and worse survival trend, Del-1 could act as a molecular target in TNBC patients. © 2018 S. Karger AG, Basel.

  7. Identification and functional characterization of a novel monotreme- specific antibacterial protein expressed during lactation.

    Directory of Open Access Journals (Sweden)

    Swathi Bisana

    Full Text Available Monotremes are the only oviparous mammals and exhibit a fascinating combination of reptilian and mammalian characters. They represent a component of synapsidal reproduction by laying shelled eggs which are incubated outside the mother's body. This is accompanied by a prototherian lactation process, marking them as representatives of early mammals. The only extant monotremes are the platypus, and the short- and long- beaked echidnas, and their distributions are limited to Australia and New Guinea. Apart for a short weaning period, milk is the sole source of nutrition and protection for the hatchlings which are altricial and immunologically naive. The duration of lactation in these mammals is prolonged relative to the gestational length and period of incubation of eggs. Much of the development of monotreme young occurs in the non-sterile ex-utero environment. Therefore the role of milk in the growth, development and disease protection of the young is of significant interest. By sequencing the cDNA of cells harvested from monotreme milk, we have identified a novel monotreme- specific transcript, and the corresponding gene was designated as the EchAMP. The expression profile of this gene in various tissues revealed that it is highly expressed in milk cells. The peptides corresponding to the EchAMP protein have been identified in a sample of echidna milk In silico analysis indicated putative antimicrobial potential for the cognate protein of EchAMP. This was further confirmed by in vitro assays using a host of bacteria. Interestingly, EchAMP did not display any activity against a commensal gut floral species. These results support the hypothesis of enhancement of survival of the young by antimicrobial bioactives of mammary gland origin and thus emphasize the protective, non- nutritional role of milk in mammals.

  8. A systematic analysis of the early transcribed membrane protein family throughout the life cycle of Plasmodium yoelii.

    Science.gov (United States)

    MacKellar, Drew C; Vaughan, Ashley M; Aly, Ahmed S I; DeLeon, Sasha; Kappe, Stefan H I

    2011-11-01

    The early transcribed membrane proteins (ETRAMPs) are a family of small, highly charged transmembrane proteins unique to malaria parasites. Some members of the ETRAMP family have been localized to the parasitophorous vacuole membrane that separates the intracellular parasite from the host cell and thus presumably have a role in host-parasite interactions. Although it was previously shown that two ETRAMPs are critical for rodent malaria parasite liver-stage development, the importance of most ETRAMPs during the parasite life cycle remains unknown. Here, we comprehensively identify nine new etramps in the genome of the rodent malaria parasite Plasmodium yoelii, and elucidate their conservation in other malaria parasites. etramp expression profiles are diverse throughout the parasite life cycle as measured by RT-PCR. Epitope tagging of two ETRAMPs demonstrates protein expression in blood and liver stages, and reveals differences in both their timing of expression and their subcellular localization. Gene targeting studies of each of the nine uncharacterized etramps show that two are refractory to deletion and thus likely essential for blood-stage replication. Seven etramps are not essential for any life cycle stage. Systematic characterization of the members of the ETRAMP family reveals the diversity in importance of each family member at the interface between host and parasite throughout the developmental cycle of the malaria parasite. © 2011 Blackwell Publishing Ltd.

  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. Expression and significance of Bax protein in model of radiation injury in mouse skin

    International Nuclear Information System (INIS)

    Feng Yizhong; Mo Yahong

    2002-01-01

    Objective: The study is to find some valuable criteria for diagnosis and treatment of radiation injury in skin. Methods: The expression of Bax protein was studied by SP immunohistochemistry in 40 cases of model of radiation injury in mouse skin. Their relationship relating to radiation dose was also investigated. Results: The expression rates of Bax were 30%, 30%, 70%, 70% in 5 Gy group, 15 Gy group, 30 Gy group, 45 Gy group respectively. There was no significant correlation between the expression of Bax and radiation groups. Conclusions: The experiment shows that radiation can increase the expression of Bax protein which might be related to poor healing in radiation skin injury

  11. Changes in UCP expression in tissues of Zucker rats fed diets with different protein content.

    Science.gov (United States)

    Masanés, R M; Yubero, P; Rafecas, I; Remesar, X

    2002-09-01

    The effect of dietary protein content on the uncoupling proteins (UCP) 1, 2 and 3 expression in a number of tissues of Zucker lean and obese rats was studied. Thirty-day-old male Zucker lean (Fa/?) and obese (fa/fa) rats were fed on hyperproteic (HP, 30% protein), standard (RD, 17% protein) or hypoproteic (LP, 9% protein) diets ad libitum for 30 days. Although dietary protein intake affected the weights of individual muscles in lean and obese animals, these weights were similar. In contrast, huge differences were observed in brown adipose tissue (BAT) and liver weights. Lean rats fed on the LP diet generally increased UCP expression, whereas the HP group had lower values. Obese animals, HP and LP groups showed higher UCP expression in muscles, with slight differences in BAT and lower values for UCP3 in subcutaneous adipose tissue. The mean values of UCP expression in BAT of obese rats were lower than in their lean counterpart, whereas the expression in skeletal muscle was increased. Thus, expression of UCPs can be modified by dietary protein content, in lean and obese rats. A possible thermogenic function of UCP3 in muscle and WAT in obese rats must be taken into account.

  12. 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.

  13. A Guide to Transient Expression of Membrane Proteins in HEK-293 Cells for Functional Characterization

    KAUST Repository

    Ooi, Amanda Siok Lee

    2016-07-19

    The human embryonic kidney 293 (HEK-293) cells are commonly used as host for the heterologous expression of membrane proteins not least because they have a high transfection efficiency and faithfully translate and process proteins. In addition, their cell size, morphology and division rate, and low expression of native channels are traits that are particularly attractive for current-voltage measurements. Nevertheless, the heterologous expression of complex membrane proteins such as receptors and ion channels for biological characterization and in particular for single-cell applications such as electrophysiology remains a challenge. Expression of functional proteins depends largely on careful step-by-step optimization that includes the design of expression vectors with suitable identification tags, as well as the selection of transfection methods and detection parameters appropriate for the application. Here, we use the heterologous expression of a plant potassium channel, the Arabidopsis thaliana guard cell outward-rectifying K+ channel, AtGORK (At5G37500) in HEK-293 cells as an example, to evaluate commonly used transfection reagents and fluorescent detection methods, and provide a detailed methodology for optimized transient transfection and expression of membrane proteins for in vivo studies in general and for single-cell applications in particular. This optimized protocol will facilitate the physiological and cellular characterization of complex membrane proteins.

  14. A Guide to Transient Expression of Membrane Proteins in HEK-293 Cells for Functional Characterization

    KAUST Repository

    Ooi, Amanda Siok Lee; Wong, Aloysius Tze; Esau, Luke; Lemtiri-Chlieh, Fouad; Gehring, Christoph A

    2016-01-01

    The human embryonic kidney 293 (HEK-293) cells are commonly used as host for the heterologous expression of membrane proteins not least because they have a high transfection efficiency and faithfully translate and process proteins. In addition, their cell size, morphology and division rate, and low expression of native channels are traits that are particularly attractive for current-voltage measurements. Nevertheless, the heterologous expression of complex membrane proteins such as receptors and ion channels for biological characterization and in particular for single-cell applications such as electrophysiology remains a challenge. Expression of functional proteins depends largely on careful step-by-step optimization that includes the design of expression vectors with suitable identification tags, as well as the selection of transfection methods and detection parameters appropriate for the application. Here, we use the heterologous expression of a plant potassium channel, the Arabidopsis thaliana guard cell outward-rectifying K+ channel, AtGORK (At5G37500) in HEK-293 cells as an example, to evaluate commonly used transfection reagents and fluorescent detection methods, and provide a detailed methodology for optimized transient transfection and expression of membrane proteins for in vivo studies in general and for single-cell applications in particular. This optimized protocol will facilitate the physiological and cellular characterization of complex membrane proteins.

  15. The Chromatin Protein DUET/MMD1 Controls Expression of the Meiotic Gene TDM1 during Male Meiosis in Arabidopsis.

    Science.gov (United States)

    Andreuzza, Sébastien; Nishal, Bindu; Singh, Aparna; Siddiqi, Imran

    2015-09-01

    Meiosis produces haploid cells essential for sexual reproduction. In yeast, entry into meiosis activates transcription factors which trigger a transcriptional cascade that results in sequential co-expression of early, middle and late meiotic genes. However, these factors are not conserved, and the factors and regulatory mechanisms that ensure proper meiotic gene expression in multicellular eukaryotes are poorly understood. Here, we report that DUET/MMD1, a PHD finger protein essential for Arabidopsis male meiosis, functions as a transcriptional regulator in plant meiosis. We find that DUET-PHD binds H3K4me2 in vitro, and show that this interaction is critical for function during meiosis. We also show that DUET is required for proper microtubule organization during meiosis II, independently of its function in meiosis I. Remarkably, DUET protein shows stage-specific expression, confined to diplotene. We identify two genes TDM1 and JAS with critical functions in cell cycle transitions and spindle organization in male meiosis, as DUET targets, with TDM1 being a direct target. Thus, DUET is required to regulate microtubule organization and cell cycle transitions during male meiosis, and functions as a direct transcription activator of the meiotic gene TDM1. Expression profiling showed reduced expression of a subset comprising about 12% of a known set of meiosis preferred genes in the duet mutant. Our results reveal the action of DUET as a transcriptional regulator during male meiosis in plants, and suggest that transcription of meiotic genes is under stagewise control in plants as in yeast.

  16. Early dengue virus protein synthesis induces extensive rearrangement of the endoplasmic reticulum independent of the UPR and SREBP-2 pathway.

    Directory of Open Access Journals (Sweden)

    José Peña

    Full Text Available The rearrangement of intracellular membranes has been long reported to be a common feature in diseased cells. In this study, we used dengue virus (DENV to study the role of the unfolded protein response (UPR and sterol-regulatory-element-binding-protein-2 (SREBP-2 pathway in the rearrangement and expansion of the endoplasmic reticulum (ER early after infection. Using laser scanning confocal and differential interference contrast microscopy, we demonstrate that rearrangement and expansion of the ER occurs early after DENV-2 infection. Through the use of mouse embryonic fibroblast cells deficient in XBP1 and ATF6, we show that ER rearrangement early after DENV infection is independent of the UPR. We then demonstrate that enlargement of the ER is independent of the SREBP-2 activation and upregulation of 3-hydroxy-3-methylglutaryl-Coenzyme-A reductase, the rate-limiting enzyme in the cholesterol biosynthesis pathway. We further show that this ER rearrangement is not inhibited by the treatment of DENV-infected cells with the cholesterol-inhibiting drug lovastatin. Using the transcription inhibitor actinomycin D and the translation elongation inhibitor cycloheximide, we show that de novo viral protein synthesis but not host transcription is necessary for expansion and rearrangement of the ER. Lastly, we demonstrate that viral infection induces the reabsorption of lipid droplets into the ER. Together, these results demonstrate that modulation of intracellular membrane architecture of the cell early after DENV-2 infection is driven by viral protein expression and does not require the induction of the UPR and SREBP-2 pathways. This work paves the way for further study of virally-induced membrane rearrangements and formation of cubic membranes.

  17. Transgenic nude mouse with green fluorescent protein expression-based human glioblastoma multiforme animal model with EGFR expression and invasiveness.

    Science.gov (United States)

    Tan, Guo-Wei; Lan, Fo-Lin; Gao, Jian-Guo; Jiang, Cai-Mou; Zhang, Yi; Huang, Xiao-Hong; Ma, Yue-Hong; Shao, He-Dui; He, Xue-Yang; Chen, Jin-Long; Long, Jian-Wu; Xiao, Hui-Sheng; Guo, Zhi-Tong; Diao, Yi

    2012-08-01

    Previously, we developed an orthotopic xenograft model of human glioblastoma multiforme (GBM) with high EGFR expression and invasiveness in Balb/c nu/nu nude mice. Now we also developed the same orthotopic xenograft model in transgenic nude mice with green fluorescent protein (GFP) expression. The present orthotopic xenografts labeled by phycoerythrin fluorescing red showed high EGFR expression profile, and invasive behavior under a bright green-red dual-color fluorescence background. A striking advantage in the present human GBM model is that the change of tumor growth can be observed visually instead of sacrificing animals in our further antitumor therapy studies.

  18. Cloning of zebrafish Mustn1 orthologs and their expression during early development.

    Science.gov (United States)

    Camarata, Troy; Vasilyev, Aleksandr; Hadjiargyrou, Michael

    2016-11-15

    Mustn1 is a small nuclear protein that is involved in the development and regeneration of the musculoskeletal system. Previous work established a role for Mustn1 in myogenic and chondrogenic differentiation. In addition, recent evidence suggests a potential role for Mustn1 in cilia function in zebrafish. A detailed study of Mustn1 expression has yet to be conducted in zebrafish. As such, we report herein the cloning of the zebrafish Mustn1 orthologs, mustn1a and mustn1b, and their expression during zebrafish embryonic and larval development. Results indicate a 44% nucleotide identity between the two paralogs. Phylogenetic analysis further confirmed that the Mustn1a and 1b predicted proteins were highly related to other vertebrate members of the Mustn1 protein family. Whole mount in situ hybridization revealed expression of both mustn1a and 1b at the 7-somite stage through 72hpf in structures such as Kupffer's vesicle, segmental mesoderm, head structures, and otic vesicle. Additionally, in 5day old larva, mustn1a and 1b expression is detected in the neurocranium, otic capsule, and the gut. Although both were expressed in the neurocranium, mustn1a was localized in the hypophyseal fenestra whereas mustn1b was found near the posterior basicapsular commissure. mustn1b also displayed expression in the ceratohyal and ceratobranchial elements of the pharyngeal skeleton. These expression patterns were verified temporally by q-PCR analysis. Taken together, we conclude that Mustn1 expression is conserved in vertebrates and that the variations in expression of the two zebrafish paralogs suggest different modes of molecular regulation. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. NDRG4, an early detection marker for colorectal cancer, is specifically expressed in enteric neurons

    NARCIS (Netherlands)

    Vaes, N.; Lentjes, M. H. F. M.; Gijbels, M. J.; Rademakers, G.; Daenen, K. L.; Boesmans, W.; Wouters, K. A. D.; Geuzens, A.; Qu, X.; Steinbusch, H. P. J.; Rutten, B. P. F.; Baldwin, S. H.; Sharkey, K. A.; Hofstra, R. M. W.; van Engeland, M.; Vanden Berghe, P.; Melotte, V.

    2017-01-01

    Background: Promoter methylation of N-myc Downstream-Regulated Gene 4 (NDRG4) in fecal DNA is an established early detection marker for colorectal cancer (CRC). Despite its connection to CRC, NDRG4 is predominantly studied in brain and heart, with little to no knowledge about its expression or role

  20. B3GNT3 Expression Is a Novel Marker Correlated with Pelvic Lymph Node Metastasis and Poor Clinical Outcome in Early-Stage Cervical Cancer

    Science.gov (United States)

    Niu, Chunhao; Song, Libing; Zhang, Yanna

    2015-01-01

    Background The β1,3-N-acetylglucosaminyltransferase-3 gene (B3GNT3) encodes a member of the B3GNT family that functions as the backbone structure of dimeric sialyl-Lewis A and is involved in L-selectin ligand biosynthesis, lymphocyte homing and lymphocyte trafficking. B3GNT3 has been implicated as an important element in the development of certain cancers. However, the characteristics of B3GNT3 in the development and progression of cancer remain largely unknown. Thus, our study aimed to investigate the expression pattern and the prognostic value of B3GNT3 in patients with early-stage cervical cancer. Methods The mRNA and protein levels of B3GNT3 expression were examined in eight cervical cancer cell lines and ten paired cervical cancer tumors, using real-time PCR and western blotting, respectively. Immunohistochemistry (IHC) was used to analyze B3GNT3 protein expression in paraffin-embedded tissues from 196 early-stage cervical cancer patients. Statistical analyses were applied to evaluate the association between B3GNT3 expression scores and clinical parameters, as well as patient survival. Results B3GNT3 expression was significantly upregulated in cervical cancer cell lines and lesions compared with normal cells and adjacent noncancerous cervical tissues. In the 196 cases of tested early-stage cervical cancer samples, the B3GNT3 protein level was positively correlated with high risk TYPES of human papillomavirus (HPV) infection (P = 0.026), FIGO stage (P cervical cancer patients. Conclusions Our study demonstrated that elevated B3GNT3 expression is associated with pelvic lymph node metastasis and poor outcome in early-stage cervical cancer patients. B3GNT3 may be a novel prognostic marker and therapeutic target for the treatment of cervical cancer. PMID:26709519

  1. Diagnostic value of determination of amount of urinary excretion of proteins for early diabetic nephropathy

    International Nuclear Information System (INIS)

    Li Zhuocheng; Chen Jianxiong; Yan Dewen

    2007-01-01

    Objective: To investigate the value of detection of changes of the amount of usinary excretion of albumin, β 2 -m, Tamm- Horsfall protein and α 1 -m for diagnosis of early diabetic nephropathy. Methods: The amounts of 24h urinary excretion of albumin, β 2 -m, Tamm-Horsfall protein and α 1 -m were determined with RIA in 78 patients with diabetes mellitus and 40 controls. Results: The amounts of 24h urinary excretion of albumin, β 2 -m, α 1 -m in patients with diabetes mellitus were significantly higher than those in controls (P<0.01 ), while the amount of Tamm-Horsfall protein was significantly lower (P<0.01). Among the diabetic patients, the changes of the amount of protein excretion were more pronounced in those with advanced impairment of renal function. Conclusion: Determination of amount of urinary excretion of proteins was helpful for diagnosis and assessment of early diabetic nephropathy. (authors)

  2. The TIM Barrel Architecture Facilitated the Early Evolution of Protein-Mediated Metabolism.

    Science.gov (United States)

    Goldman, Aaron David; Beatty, Joshua T; Landweber, Laura F

    2016-01-01

    The triosephosphate isomerase (TIM) barrel protein fold is a structurally repetitive architecture that is present in approximately 10% of all enzymes. It is generally assumed that this ubiquity in modern proteomes reflects an essential historical role in early protein-mediated metabolism. Here, we provide quantitative and comparative analyses to support several hypotheses about the early importance of the TIM barrel architecture. An information theoretical analysis of protein structures supports the hypothesis that the TIM barrel architecture could arise more easily by duplication and recombination compared to other mixed α/β structures. We show that TIM barrel enzymes corresponding to the most taxonomically broad superfamilies also have the broadest range of functions, often aided by metal and nucleotide-derived cofactors that are thought to reflect an earlier stage of metabolic evolution. By comparison to other putatively ancient protein architectures, we find that the functional diversity of TIM barrel proteins cannot be explained simply by their antiquity. Instead, the breadth of TIM barrel functions can be explained, in part, by the incorporation of a broad range of cofactors, a trend that does not appear to be shared by proteins in general. These results support the hypothesis that the simple and functionally general TIM barrel architecture may have arisen early in the evolution of protein biosynthesis and provided an ideal scaffold to facilitate the metabolic transition from ribozymes, peptides, and geochemical catalysts to modern protein enzymes.

  3. Expression, purification and biochemical characterization of a single-stranded DNA binding protein from Herbaspirillum seropedicae.

    Science.gov (United States)

    Vernal, Javier; Serpa, Viviane I; Tavares, Carolina; Souza, Emanuel M; Pedrosa, Fábio O; Terenzi, Hernán

    2007-05-01

    An open reading frame encoding a protein similar in size and sequence to the Escherichia coli single-stranded DNA binding protein (SSB protein) was identified in the Herbaspirillum seropedicae genome. This open reading frame was cloned into the expression plasmid pET14b. The SSB protein from H. seropedicae, named Hs_SSB, was overexpressed in E. coli strain BL21(DE3) and purified to homogeneity. Mass spectrometry data confirmed the identity of this protein. The apparent molecular mass of the native Hs_SSB was estimated by gel filtration, suggesting that the native protein is a tetramer made up of four similar subunits. The purified protein binds to single-stranded DNA (ssDNA) in a similar manner to other SSB proteins. The production of this recombinant protein in good yield opens up the possibility of obtaining its 3D-structure and will help further investigations into DNA metabolism.

  4. Sialotranscriptomics of Rhipicephalus zambeziensis reveals intricate expression profiles of secretory proteins and suggests tight temporal transcriptional regulation during blood-feeding.

    Science.gov (United States)

    de Castro, Minique Hilda; de Klerk, Daniel; Pienaar, Ronel; Rees, D Jasper G; Mans, Ben J

    2017-08-10

    Ticks secrete a diverse mixture of secretory proteins into the host to evade its immune response and facilitate blood-feeding, making secretory proteins attractive targets for the production of recombinant anti-tick vaccines. The largely neglected tick species, Rhipicephalus zambeziensis, is an efficient vector of Theileria parva in southern Africa but its available sequence information is limited. Next generation sequencing has advanced sequence availability for ticks in recent years and has assisted the characterisation of secretory proteins. This study focused on the de novo assembly and annotation of the salivary gland transcriptome of R. zambeziensis and the temporal expression of secretory protein transcripts in female and male ticks, before the onset of feeding and during early and late feeding. The sialotranscriptome of R. zambeziensis yielded 23,631 transcripts from which 13,584 non-redundant proteins were predicted. Eighty-six percent of these contained a predicted start and stop codon and were estimated to be putatively full-length proteins. A fifth (2569) of the predicted proteins were annotated as putative secretory proteins and explained 52% of the expression in the transcriptome. Expression analyses revealed that 2832 transcripts were differentially expressed among feeding time points and 1209 between the tick sexes. The expression analyses further indicated that 57% of the annotated secretory protein transcripts were differentially expressed. Dynamic expression profiles of secretory protein transcripts were observed during feeding of female ticks. Whereby a number of transcripts were upregulated during early feeding, presumably for feeding site establishment and then during late feeding, 52% of these were downregulated, indicating that transcripts were required at specific feeding stages. This suggested that secretory proteins are under stringent transcriptional regulation that fine-tunes their expression in salivary glands during feeding. No open

  5. Regulation of early signaling and gene expression in the α-particle and bystander response of IMR-90 human fibroblasts

    Directory of Open Access Journals (Sweden)

    Hei Tom K

    2010-07-01

    Full Text Available Abstract Background The existence of a radiation bystander effect, in which non-irradiated cells respond to signals from irradiated cells, is well established. To understand early signaling and gene regulation in bystander cells, we used a bio-informatics approach, measuring global gene expression at 30 minutes and signaling pathways between 30 minutes and 4 hours after exposure to α-particles in IMR-90 fibroblasts. Methods We used whole human genome microarrays and real time quantitative PCR to measure and validate gene expression. Microarray analysis was done using BRB-Array Tools; pathway and ontology analyses were done using Ingenuity Pathway Analysis and PANTHER, respectively. We studied signaling in irradiated and bystander cells using immunoblotting and semi-quantitative image analysis. Results Gene ontology suggested signal transduction and transcriptional regulation responding 30 minutes after treatment affected cell structure, motility and adhesion, and interleukin synthesis. We measured time-dependent expression of genes controlled by the NF-κB pathway; matrix metalloproteinases 1 and 3; chemokine ligands 2, 3 and 5 and interleukins 1β, 6 and 33. There was an increased response of this set of genes 30 minutes after treatment and another wave of induction at 4 hours. We investigated AKT-GSK3β signaling and found both AKT and GSK3β are hyper-phosphorylated 30 minutes after irradiation and this effect is maintained through 4 hours. In bystander cells, a similar response was seen with a delay of 30 minutes. We proposed a network model where the observed decrease in phosphorylation of β-catenin protein after GSK3β dependent inactivation can trigger target gene expression at later times after radiation exposure Conclusions These results are the first to show that the radiation induced bystander signal induces a widespread gene expression response at 30 minutes after treatment and these changes are accompanied by modification of

  6. Suppression of lipin-1 expression increases monocyte chemoattractant protein-1 expression in 3T3-L1 adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, Nobuhiko, E-mail: ntkhs@hoku-iryo-u.ac.jp [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1-1-1 Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510 (Japan); Yoshizaki, Takayuki [Innovation Center, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890-0065 (Japan); Hiranaka, Natsumi; Suzuki, Takeshi [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Yui, Tomoo; Akanuma, Masayasu; Oka, Kazuya [Department of Fixed Prosthodontics and Oral Implantology, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Kanazawa, Kaoru [Department of Dental Anesthesiology, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Yoshida, Mika; Naito, Sumiyoshi [Department of Clinical Laboratory, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Fujiya, Mikihiro; Kohgo, Yutaka [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1-1-1 Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510 (Japan); Ieko, Masahiro [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)

    2011-11-11

    Highlights: Black-Right-Pointing-Pointer Lipin-1 affects lipid metabolism, adipocyte differentiation, and transcription. Black-Right-Pointing-Pointer Adipose lipin-1 expression is reduced in obesity. Black-Right-Pointing-Pointer Lipin-1 depletion using siRNA in 3T3-L1 adipocytes increased MCP-1 expression. Black-Right-Pointing-Pointer Lipin-1 is involved in adipose inflammation. -- Abstract: Lipin-1 plays a crucial role in the regulation of lipid metabolism and cell differentiation in adipocytes. Expression of adipose lipin-1 is reduced in obesity, and metabolic syndrome. However, the significance of this reduction remains unclear. This study investigated if and how reduced lipin-1 expression affected metabolism. We assessed mRNA expression levels of various genes related to adipocyte metabolism in lipin-1-depleted 3T3-L1 adipocytes by introducing its specific small interfering RNA. In lipin-1-depleted adipocytes, mRNA and protein expression levels of monocyte chemoattractant protein-1 (MCP-1) were significantly increased, although the other genes tested were not altered. The conditioned media from the cells promoted monocyte chemotaxis. The increase in MCP-1 expression was prevented by treatment with quinazoline or salicylate, inhibitors of nuclear factor-{kappa}B activation. Because MCP-1 is related to adipose inflammation and systemic insulin resistance, these results suggest that a reduction in adipose lipin-1 in obesity may exacerbate adipose inflammation and metabolism.

  7. Suppression of lipin-1 expression increases monocyte chemoattractant protein-1 expression in 3T3-L1 adipocytes

    International Nuclear Information System (INIS)

    Takahashi, Nobuhiko; Yoshizaki, Takayuki; Hiranaka, Natsumi; Suzuki, Takeshi; Yui, Tomoo; Akanuma, Masayasu; Oka, Kazuya; Kanazawa, Kaoru; Yoshida, Mika; Naito, Sumiyoshi; Fujiya, Mikihiro; Kohgo, Yutaka; Ieko, Masahiro

    2011-01-01

    Highlights: ► Lipin-1 affects lipid metabolism, adipocyte differentiation, and transcription. ► Adipose lipin-1 expression is reduced in obesity. ► Lipin-1 depletion using siRNA in 3T3-L1 adipocytes increased MCP-1 expression. ► Lipin-1 is involved in adipose inflammation. -- Abstract: Lipin-1 plays a crucial role in the regulation of lipid metabolism and cell differentiation in adipocytes. Expression of adipose lipin-1 is reduced in obesity, and metabolic syndrome. However, the significance of this reduction remains unclear. This study investigated if and how reduced lipin-1 expression affected metabolism. We assessed mRNA expression levels of various genes related to adipocyte metabolism in lipin-1-depleted 3T3-L1 adipocytes by introducing its specific small interfering RNA. In lipin-1-depleted adipocytes, mRNA and protein expression levels of monocyte chemoattractant protein-1 (MCP-1) were significantly increased, although the other genes tested were not altered. The conditioned media from the cells promoted monocyte chemotaxis. The increase in MCP-1 expression was prevented by treatment with quinazoline or salicylate, inhibitors of nuclear factor-κB activation. Because MCP-1 is related to adipose inflammation and systemic insulin resistance, these results suggest that a reduction in adipose lipin-1 in obesity may exacerbate adipose inflammation and metabolism.

  8. Expression and characterization of insulin growth factor-I-enhanced green fluorescent protein fused protein as a tracer for immunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Shi Ruina [College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China); Huang Yong [College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China); Wang Dan [College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China); Zhao Meiping [College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China); Li Yuanzong [College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China)]. E-mail: yzli@pku.edu.cn

    2006-09-25

    The insulin-like growth factor-I (IGF-I) is an important polypeptide hormone under investigation for body metabolism study and for doping detection. Here, we describe for the first time the expression of a recombinant fusion protein of IGF-I and the enhanced green fluorescent protein (EGFP). The genetic fusion approach enables preparation of conjugates with 1:1 stoichiometry and homogeneous structure. The fused protein (EGFP-IGF-I) was expressed as a soluble protein in cytoplasm of Escherichia coli and its fluorescence and immunoreaction properties were thoroughly characterized. Finally, we demonstrated the utility of the EGFP-IGF-I fusion protein for the fluorescence immunoassay of IGF-1. The linear range of the assay is 1.6 x 10{sup -8} to 2.0 x 10{sup -6} M with a detection limit of 1.6 x 10{sup -8} M. To our knowledge, this is the first time that EGFP has been used as a quantitative label in a fusion protein to develop a quantitative assay for IGF-I. Furthermore, the use of genetically engineered fusion proteins, which combine peptide hormones with fluorescent protein, can lead to a new labeling approach to a number of bioanalytical applications.

  9. Expression and characterization of insulin growth factor-I-enhanced green fluorescent protein fused protein as a tracer for immunoassay

    International Nuclear Information System (INIS)

    Shi Ruina; Huang Yong; Wang Dan; Zhao Meiping; Li Yuanzong

    2006-01-01

    The insulin-like growth factor-I (IGF-I) is an important polypeptide hormone under investigation for body metabolism study and for doping detection. Here, we describe for the first time the expression of a recombinant fusion protein of IGF-I and the enhanced green fluorescent protein (EGFP). The genetic fusion approach enables preparation of conjugates with 1:1 stoichiometry and homogeneous structure. The fused protein (EGFP-IGF-I) was expressed as a soluble protein in cytoplasm of Escherichia coli and its fluorescence and immunoreaction properties were thoroughly characterized. Finally, we demonstrated the utility of the EGFP-IGF-I fusion protein for the fluorescence immunoassay of IGF-1. The linear range of the assay is 1.6 x 10 -8 to 2.0 x 10 -6 M with a detection limit of 1.6 x 10 -8 M. To our knowledge, this is the first time that EGFP has been used as a quantitative label in a fusion protein to develop a quantitative assay for IGF-I. Furthermore, the use of genetically engineered fusion proteins, which combine peptide hormones with fluorescent protein, can lead to a new labeling approach to a number of bioanalytical applications

  10. Gene expression changes as markers of early lapatinib response in a panel of breast cancer cell lines

    LENUS (Irish Health Repository)

    O’Neill, Fiona

    2012-06-18

    AbstractBackgroundLapatinib, a tyrosine kinase inhibitor of HER2 and EGFR and is approved, in combination with capecitabine, for the treatment of trastuzumab-refractory metastatic breast cancer. In order to establish a possible gene expression response to lapatinib, a panel of breast cancer cell lines with varying sensitivity to lapatinib were analysed using a combination of microarray and qPCR profiling.MethodsCo-inertia analysis (CIA), a data integration technique, was used to identify transcription factors associated with the lapatinib response on a previously published dataset of 96 microarrays. RNA was extracted from BT474, SKBR3, EFM192A, HCC1954, MDAMB453 and MDAMB231 breast cancer cell lines displaying a range of lapatinib sensitivities and HER2 expression treated with 1 μM of lapatinib for 12 hours and quantified using Taqman RT-PCR. A fold change ≥ ± 2 was considered significant.ResultsA list of 421 differentially-expressed genes and 8 transcription factors (TFs) whose potential regulatory impact was inferred in silico, were identified as associated with lapatinib response. From this group, a panel of 27 genes (including the 8 TFs) were selected for qPCR validation. 5 genes were determined to be significantly differentially expressed following the 12 hr treatment of 1 μM lapatinib across all six cell lines. Furthermore, the expression of 4 of these genes (RB1CC1, FOXO3A, NR3C1 and ERBB3) was directly correlated with the degree of sensitivity of the cell line to lapatinib and their expression was observed to “switch” from up-regulated to down-regulated when the cell lines were arranged in a lapatinib-sensitive to insensitive order. These included the novel lapatinib response-associated genes RB1CC1 and NR3C1. Additionally, Cyclin D1 (CCND1), a common regulator of the other four proteins, was also demonstrated to observe a proportional response to lapatinib exposure.ConclusionsA panel of 5 genes were determined to be differentially

  11. High SRPX2 protein expression predicts unfavorable clinical outcome in patients with prostate cancer

    Science.gov (United States)

    Zhang, Meng; Li, Xiaoli; Fan, Zhirui; Zhao, Jing; Liu, Shuzheng; Zhang, Mingzhi; Li, Huixiang; Goscinski, Mariusz Adam; Fan, Huijie; Suo, Zhenhe

    2018-01-01

    Background Sushi repeat-containing protein X-linked 2 (SRPX2) is overexpressed in a variety of different tumor tissues and correlated with poor prognosis in patients. Little research focuses on the role of SRPX2 expression in prostate cancer (PCa), and the clinicopathological significance of the protein expression in this tumor is relatively unknown. However, our previous transcriptome data from those cancer stem-like cells indicated the role of SRPX2 in PCa. Materials and methods In this study, RT-PCR and Western blotting were firstly used to examine the SRPX2 expression in three PCa cell lines including LNCaP, DU145, and PC3, and then SRPX2 protein expression was immunohistochemically investigated and statistically analyzed in a series of 106 paraffin-embedded PCa tissue specimens. Results Significantly lower levels of SRPX2 expression were verified in the LNCaP cells, compared with the expression in the aggressive DU145 and PC3 cells, in both mRNA and protein levels. Immunohistochemically, there were variable SRPX2 protein expressions in the clinical samples. Moreover, high levels of SRPX2 expression in the PCa tissues were significantly associated with Gleason score (P=0.008), lymph node metastasis (P=0.009), and distant metastasis (P=0.021). Furthermore, higher levels of SRPX2 expression in the PCa tissues were significantly associated with shorter overall survival (OS) (P<0.001). Conclusion Our results demonstrate that SRPX2 is highly expressed in aggressive PCa cells in vitro, and its protein expression in PCa is significantly associated with malignant clinical features and shorter OS, strongly indicating its prognostic value in prostate cancers. PMID:29881288

  12. Cloning, expression, purification and characterization of Leishmania tropica PDI-2 protein

    Directory of Open Access Journals (Sweden)

    Ali Dina

    2016-01-01

    Full Text Available In Leishmania species, protein disulfide isomerase (PDI is an essential enzyme that catalyzes thiol-disulfide interchange. The present work describes the isolation, cloning, sequencing and expression of the pdI-2 gene. Initially, the gene was amplified from L. tropica genomic DNA by PCR using specific primers before cloning into the expression vector pET-15b. The construct pET/pdI-2 was transformed into BL21(DE3 cells and induced for the protein expression. SDS-PAGE and western blot analysis showed that the expressed protein is about 51 kDa. Cloned gene sequence analysis revealed that the deduced amino acid sequence showed significant homology with those of several parasites PDIs. Finally, recombinant protein was purified with a metal-chelating affinity column. The putative protein was confirmed as a thiol - disulfide oxidoreductase by detecting its activity in an oxidoreductase assay. Assay result of assay suggested that the PDI-2 protein is required for both oxidation and reduction of disulfide bonds in vitro. Antibodies reactive with this 51 kDa protein were detected by Western blot analysis in sera from human infected with L. tropica. This work describes for the first t