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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. Matrix metalloproteinase protein expression profiles cannot distinguish between normal and early osteoarthritic synovial fluid

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    Heard Bryan J

    2012-07-01

    Full Text Available Abstract Background Osteoarthritis (OA and Rheumatoid arthritis (RA are diseases which result in the degeneration of the joint surface articular cartilage. Matrix Metalloproteinases (MMPs are enzymes that aid in the natural remodelling of tissues throughout the body including cartilage. However, some MMPs have been implicated in the progression of OA and RA as their expression levels and activation states can change dramatically with the onset of disease. Yet, it remains unknown if normal and arthritic joints demonstrate unique MMPs expression profiles, and if so, can the MMP expression profile be used to identify patients with early OA. In this study, the synovial fluid protein expression levels for MMPs 1, 2, 3, 7, 8, 9, 12 & 13, as well as those for the Tissue Inhibitors of MMPs (TIMPs 1, 2, 3, & 4 were examined in highly characterized normal knee joints, and knee joints with clinically diagnosed OA (early and advanced or RA. The purpose of this study was to determine if normal, OA, and RA patients exhibit unique expression profiles for a sub-set of MMPs, and if early OA patients have a unique MMP expression profile that could be used as an early diagnostic marker. Methods Synovial fluid was aspirated from stringently characterized normal knee joints, and in joints diagnosed with either OA (early and advanced or RA. Multiplexing technology was employed to quantify protein expression levels for 8 MMPs and 4 TIMPs in the synovial fluid of 12 patients with early OA, 17 patients diagnosed with advanced OA, 15 with RA and 25 normal knee joints. Principle component analysis (PCA was used to reveal which MMPs were most influential in the distinction between treatment groups. K – means clustering was used to verify the visual grouping of subjects via PCA. Results Significant differences in the expression levels of MMPs and TIMPs were observed between normal and arthritic synovial fluids (with the exception of MMP 12. PCA demonstrated that MMPs 2, 8

  3. Early expression of surfactant proteins D in Fusarium solani infected rat cornea.

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    Che, Cheng-Ye; Li, Xiao-Jing; Jia, Wen-Yan; Li, Na; Xu, Qiang; Lin, Jing; Wang, Qing; Jiang, Nan; Hu, Li-Ting; Zhao, Gui-Qiu

    2012-01-01

    To investigate the early expression of surfactant proteins D(SP-D) in Fusarium solani infected rat cornea. Wistar rats were divided into group A, B and C randomly. The right eyes were chosen as the experiment one. Group A was control group. Group B was not inoculated with Fusarium solani. Group C was taken as fusarium solani keratitis model. Five rats in group B and C were executed randomly at 6, 12, 24, 48 and 96 hours respectively after the experimental model being established. The expression of SP-D was assessed through immunohistochemistry and reverse transcription polymerase chain reaction(RT-PCR). RT-PCR detected that the SP-D mRNA expression was low in the corneal of normal rats and group B. The expression of fungal infected cornea increased gradually and reached the peak at 24 hours in group C. The synchronous expression of group B and C were in significant difference (Pfusarium solani infected cornea. SP-D may play a role in the early innate immunity response of the corneal resistance to Fusarium solani infection.

  4. Altered expression of heat shock proteins in embryonal carcinoma and mouse early embryonic cells.

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    Morange, M; Diu, A; Bensaude, O; Babinet, C

    1984-04-01

    In a previous paper, we have shown that in the absence of stress, mouse embryonal carcinoma cells, like mouse early embryo multipotent cells, synthesize high levels of 89- and 70-kilodalton heat shock proteins (HSP)(O. Bensaude and M. Morange, EMBO J. 2:173-177, 1983). We report here the pattern of proteins synthesized after a short period of hyperthermia in various mouse embryonal carcinoma cell lines and early mouse embryo cells. Among the various cell lines tested, two of them, PCC4-Aza R1 and PCC7-S-1009, showed an unusual response in that stimulation of HSP synthesis was not observed in these cells after hyperthermia. However, inducibility of 68- and 105-kilodalton HSP can be restored in PCC7-S-1009 cells after in vitro differentiation triggered by retinoic acid. Similarly, in the early mouse embryo, hyperthermia does not induce the synthesis of nonconstitutive HSP at the eight-cell stage, but induction of the 68-kilodalton HSP does occur at the blastocyst stage. Such a transition in the expression of HSP has already been described for Drosophila melanogaster and sea urchin embryos and recently for mouse embryos. It may be a general property of early embryonic cells.

  5. Early meiotic-specific protein expression in post-natal rat ovaries.

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    Zhang, P; Lv, L X; Xing, W J

    2010-12-01

    Recent studies in mice challenged the basic doctrine that most mammalian females lose neo-oogenesis in post-natal ovaries. In order to provide more information in other species, we examined post-natal rat ovaries by histological sections and detected the germline cell marker protein RVLG (rat vasa-like gene), BrdU (5-bromodeoxyuridine) incorporation in RVLG-expressing cells, for identification of germline cells undergoing mitosis and meiosis in the ovarian surface epithelium (OSE). We also detected the expression of early meiotic-specific proteins disruption of meiotic control 1 (DMC1), stimulated by retinoic acid gene 8 (STRA8) and synaptonemal complex protein 3 (SCP3) by immunohistochemical analysis and Western blotting, and the transcript of SCP1, SCP3 and Sporulation-specific protein 11 (SPO11) by RT-PCR in the post-natal ovarian cortex. However we failed in detecting large ovoid cells in the OSE, which may represent the putative germline stem cells (GSCs) that are supposed to sustain neo-oogenesis, and the transcription of the meiotic-specific genes SCP1, SCP3 and SPO11 by RT-PCR as well as the translation of DMC1, STRA8 and SCP3 by Western blotting. Our data support the postulation that there is no neo-oogenesis occurring in the OSE of rat post-natal ovary through meiosis of GSCs.

  6. Expression of the immediate-early gene-encoded protein Egr-1 (zif268) during in vitro classical conditioning.

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    Mokin, Maxim; Keifer, Joyce

    2005-01-01

    Expression of the immediate-early genes (IEGs) has been shown to be induced by activity-dependent synaptic plasticity or behavioral training and is thought to play an important role in long-term memory. In the present study, we examined the induction and expression of the IEG-encoded protein Egr-1 during an in vitro neural correlate of eyeblink classical conditioning. The results showed that Egr-1 protein expression as determined by immunocytochemistry and Western blot analysis rapidly increased during the early stages of conditioning and remained elevated during the later stages. Further, expression of Egr-1 protein required NMDA receptor activation as it was blocked by bath application of AP-5. These findings suggest that the IEG-encoded proteins such as Egr-1 are activated during relatively simple forms of learning in vertebrates. In this case, Egr-1 may have a functional role in the acquisition phase of conditioning as well as in maintaining expression of conditioned responses.

  7. 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.

  8. Expression of the Immediate-Early Gene-Encoded Protein Egr-1 ("zif268") during in Vitro Classical Conditioning

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    Mokin, Maxim; Keifer, Joyce

    2005-01-01

    Expression of the immediate-early genes (IEGs) has been shown to be induced by activity-dependent synaptic plasticity or behavioral training and is thought to play an important role in long-term memory. In the present study, we examined the induction and expression of the IEG-encoded protein Egr-1 during an in vitro neural correlate of eyeblink…

  9. Stress protein expression in early phase spinal cord ischemia/reperfusion injury*

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    Shanyong Zhang; Dankai Wu; Jincheng Wang; Yongming Wang; Guoxiang Wang; Maoguang Yang; Xiaoyu Yang

    2013-01-01

    Spinal cord ischemia/reperfusion injury is a stress injury to the spinal cord. Our previous studies using differential proteomics identified 21 differential y expressed proteins (n > 2) in rabbits with spinal cord ischemia/reperfusion injury. Of these proteins, stress-related proteins included protein disulfide isomerase A3, stress-induced-phosphoprotein 1 and heat shock cognate protein 70. In this study, we established New Zealand rabbit models of spinal cord ischemia/reperfusion injury by abdominal aorta occlusion. Results demonstrated that hind limb function initial y improved after spinal cord ischemia/reperfusion injury, but then deteriorated. The pathological morphology of the spinal cord became aggravated, but lessened 24 hours after reperfusion. However, the numbers of motor neurons and interneurons in the spinal cord gradual y decreased. The expression of protein disulfide isomerase A3, stress-induced-phosphoprotein 1 and heat shock cognate protein 70 was induced by ischemia/reperfusion injury. The expression of these proteins increased within 12 hours after reperfusion, and then decreased, reached a minimum at 24 hours, but subsequently increased again to similar levels seen at 6–12 hours, showing a characterization of induction-inhibition-induc-tion. These three proteins were expressed only in cytoplasm but not in the nuclei. Moreover, the expression was higher in interneurons than in motor neurons, and the survival rate of interneurons was greater than that of motor neurons. It is assumed that the expression of stress-related proteins exhibited a protective effect on neurons.

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

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

    2011-01-01

    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 ofproteomic 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: Amean 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 79unique 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-1type 1 collagen

  11. EFFECT OF ARSENICALS ON THE EXPRESSION OF CELL CYCLE PROTEINS AND EARLY SIGNALING EVENTS IN PRIMARY HUMAN KERATINOCYTES.

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    Effect of Arsenicals on the Expression of Cell Cycle Proteins and Early Signaling Events in Primary Human Keratinocytes.Mudipalli, A, Owen R. D. and R. J. Preston, Environmental Carcinogenesis Division, USEPA, RTP, NC 27711.Environmental exposure to arsenic is a m...

  12. Proteomic analysis of differential protein expression in early process of pancreatic regeneration in pancreatectomized rats

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    Ming YANG; Wei LIU; Chun-you WANG; Tao LIU; Feng ZHOU; Jing TAO; Yang WANG; Ming-tao LI

    2006-01-01

    Aim: A broad-range proteomic approach was applied to investigate the complexity of the mechanisms involved in pancreatic regeneration for identification of new targets of diabetes treatment and potential markers of pancreatic stem cells. Methods: A regeneration pancreatic model was induced by 90% partial pancreatectomy (Px) in rats. Changes in the protein expression in regenerating rat pancreas on the third day after Px, as compared with rats that received sham surgery, were analyzed by using 2-D gel electrophoresis (2-DE), mass spectrometry(MS), and mass fingerprinting. Results: 2-DE revealed 91 spots with at least 1.5-fold increases in expression at 3 d after pancreatectomy and 53 differentially expressed proteins that were identified by peptide mass fingerprinting (PMF). These included cell growth-related, lipid and energy metabolism-related, protein and amino acid metabolism-related proteins, and signal transduction proteins. Vimentin, CK8, L-plastin. hnRNP A2/B1, and AGAT are associated with embryogenesis and cell differentiation, and may be new potential pancreatic stem cells markers. Conclusion: The proteome profiling technique provided a broad-based and effective approach for the rapid assimilation and identification of adaptive protein changes during pancreas regeneration induced by pancreatectomy. Our data clarify the global proteome during the pancreatic proliferation and differentiation processes, which is important for better understanding of pancreatic regeneration and for discovering of protein biomarkers for pancreatic stem cells.

  13. 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.

  14. 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

  15. Temporal and spatial expression patterns of biomineralization proteins during early development in the stony coral Pocillopora damicornis.

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    Mass, Tali; Putnam, Hollie M; Drake, Jeana L; Zelzion, Ehud; Gates, Ruth D; Bhattacharya, Debashish; Falkowski, Paul G

    2016-04-27

    Reef-building corals begin as non-calcifying larvae that, upon settling, rapidly begin to accrete skeleton and a protein-rich skeletal organic matrix that attach them to the reef. Here, we characterized the temporal and spatial expression pattern of a suite of biomineralization genes during three stages of larval development in the reef-building coral Pocillopora damicornis: stage I, newly released; stage II, oral-aborally compressed and stage III, settled and calcifying spat. Transcriptome analysis revealed 3882 differentially expressed genes that clustered into four distinctly different patterns of expression change across the three developmental stages. Immunolocalization analysis further reveals the spatial arrangement of coral acid-rich proteins (CARPs) in the overall architecture of the emerging skeleton. These results provide the first analysis of the timing of the biomineralization 'toolkit' in the early life history of a stony coral. © 2016 The Author(s).

  16. 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.

  17. Early changes in protein expression of barley following inoculation with erysiphe graminis f. sp. hordei

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    Simons, S.P.; Somerville, S.C. (Michigan State Univ., East Lansing (USA))

    1989-04-01

    Erysiphe graminis f. sp. hordei is an obligate pathogen of barley causing the powdery mildew disease. Resistance to this disease is the product of a highly specific interaction between barley lines with specific resistance alleles and pathogen races carrying complementary avirulence alleles. Using congenic barley lines which differ at the M1-a disease reaction locus, we hope to define the early molecular events of this interaction. Accordingly, resistant and susceptible barley seedlings were labelled with {sup 35}S-methionine and examined by two-dimensional electrophoresis at two hour intervals following inoculation. Infection related changes were observed with both isolines during the four to twelve hour time period. Additional differences existed constitutively between the barley lines. These differences have been quantified. Further characterization of these proteins will yield useful markers for events preceding or coinciding with cytological responses any may lead to identification and cloning of the M1-a gene.

  18. Protein expression and oxygen consumption rate of early postmortem mitochondria relate to meat tenderness.

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    Grabež, V; Kathri, M; Phung, V; Moe, K M; Slinde, E; Skaugen, M; Saarem, K; Egelandsdal, B

    2015-04-01

    Oxygen consumption rate (OCR) of muscle fibers from bovine semimembranosus muscle of 41 animals was investigated 3 to 4 h and 3 wk postmortem. Significant relations (P meat. Tender (22.92 ± 2.2 N/cm2) and tough (72.98 ± 7.2 N/cm2) meat samples (4 samples each), separated based on their OCR measurements, were selected for proteomic studies using mitochondria isolated approximately 2.5 h postmortem. Twenty-six differently expressed proteins (P meat and 19 in tough meat. In tender meat, the more prevalent antioxidant and chaperon enzymes may reduce reactive oxygen species and prolong oxygen removal by the electron transport system (ETS). Glycolytic, Krebs cycle, and ETS enzymes were also more abundant in tender meat

  19. Altered gene expression in hippocampus and depressive-like behavior in young adult female mice by early protein malnutrition.

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    Belluscio, L M; Alberca, C D; Pregi, N; Cánepa, E T

    2016-11-01

    Perinatal development represents a critical period in the life of an individual. A common cause of poor development is that which comes from undernutrition or malnutrition. In particular, protein deprivation during development has been shown to have deep deleterious effects on brain's growth and plasticity. Early-life stress has also been linked with an increased risk to develop different psychopathologies later in life. We have previously shown that perinatal protein malnutrition in mice leads to the appearance of anxiety-related behaviors in the adulthood. We also found evidence that the female offspring was more susceptible to the development of depression-related behaviors. In the present work, we further investigated this behavior together with its molecular bases. We focused our study on the hippocampus, as it is a structure involved in coping with stressful situations. We found an increase in immobility time in the forced swimming test in perinatally malnourished females, and an alteration in the expression of genes related with neuroplasticity, early growth response 1, calcineurin and c-fos. We also found that perinatal malnutrition causes a reduction in the number of neurons in the hippocampus. This reduction, together with altered gene expression, could be related to the increment in immobility time observed in the forced swimming test. © 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

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

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

    2012-01-01

    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...

  1. Constitutively Expressed IFITM3 Protein in Human Endothelial Cells Poses an Early Infection Block to Human Influenza Viruses.

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    Sun, Xiangjie; Zeng, Hui; Kumar, Amrita; Belser, Jessica A; Maines, Taronna R; Tumpey, Terrence M

    2016-12-15

    A role for pulmonary endothelial cells in the orchestration of cytokine production and leukocyte recruitment during influenza virus infection, leading to severe lung damage, has been recently identified. As the mechanistic pathway for this ability is not fully known, we extended previous studies on influenza virus tropism in cultured human pulmonary endothelial cells. We found that a subset of avian influenza viruses, including potentially pandemic H5N1, H7N9, and H9N2 viruses, could infect human pulmonary endothelial cells (HULEC) with high efficiency compared to human H1N1 or H3N2 viruses. In HULEC, human influenza viruses were capable of binding to host cellular receptors, becoming internalized and initiating hemifusion but failing to uncoat the viral nucleocapsid and to replicate in host nuclei. Unlike numerous cell types, including epithelial cells, we found that pulmonary endothelial cells constitutively express a high level of the restriction protein IFITM3 in endosomal compartments. IFITM3 knockdown by small interfering RNA (siRNA) could partially rescue H1N1 virus infection in HULEC, suggesting IFITM3 proteins were involved in blocking human influenza virus infection in endothelial cells. In contrast, selected avian influenza viruses were able to escape IFITM3 restriction in endothelial cells, possibly by fusing in early endosomes at higher pH or by other, unknown mechanisms. Collectively, our study demonstrates that the human pulmonary endothelium possesses intrinsic immunity to human influenza viruses, in part due to the constitutive expression of IFITM3 proteins. Notably, certain avian influenza viruses have evolved to escape this restriction, possibly contributing to virus-induced pneumonia and severe lung disease in humans. Avian influenza viruses, including H5N1 and H7N9, have been associated with severe respiratory disease and fatal outcomes in humans. Although acute respiratory distress syndrome (ARDS) and progressive pulmonary endothelial damage

  2. 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.

  3. Superovulation alters embryonic poly(A)-binding protein (Epab) and poly(A)-binding protein, cytoplasmic 1 (Pabpc1) gene expression in mouse oocytes and early embryos.

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    Ozturk, Saffet; Yaba-Ucar, Aylin; Sozen, Berna; Mutlu, Derya; Demir, Necdet

    2016-03-01

    Embryonic poly(A)-binding protein (EPAB) and poly(A)-binding protein, cytoplasmic 1 (PABPC1) play critical roles in translational regulation of stored maternal mRNAs required for proper oocyte maturation and early embryo development in mammals. Superovulation is a commonly used technique to obtain a great number of oocytes in the same developmental stages in assisted reproductive technology (ART) and in clinical or experimental animal studies. Previous studies have convincingly indicated that superovulation alone can cause impaired oocyte maturation, delayed embryo development, decreased implantation rate and increased postimplantation loss. Although how superovulation results in these disturbances has not been clearly addressed yet, putative changes in genes related to oocyte and early embryo development seem to be potential risk factors. Thus, the aim of the present study was to determine the effect of superovulation on Epab and Pabpc1 gene expression. To this end, low- (5IU) and high-dose (10IU) pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotrophin (hCG) were administered to female mice to induce superovulation, with naturally cycling female mice serving as controls. Epab and Pabpc1 gene expression in germinal vesicle (GV) stage oocytes, MII oocytes and 1- and 2-cell embryos collected from each group were quantified using quantitative reverse transcription-polymerase chain reaction. Superovulation with low or high doses of gonadotropins significantly altered Epab and Pabpc1 mRNA levels in GV oocytes, MII oocytes and 1- and 2-cell embryos compared with their respective controls (Psuperovulation.

  4. Proteomics Reveals that Proteins Expressed During the Early Stage of Bacillus anthracis Infection Are Potential Targets for the Development of Vaccines and Drugs

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    Chun-Ming Huang; Craig A. Elmets; De-chu C. Tang; Fuming Li; Nabiha Yusuf

    2004-01-01

    In this review, we advance a new concept in developing vaccines and/or drugs to target specific proteins expressed during the early stage of Bacillus anthracis (an thrax) infection and address existing challenges to this concept. Three proteins (immune inhibitor A, GPR-like spore protease, and alanine racemase) initially identified by proteomics in our laboratory were found to have differential expres sions during anthrax spore germination and early outgrowth. Other studies of different bacillus strains indicate that these three proteins are involved in either germination or cytotoxicity of spores, suggesting that they may serve as potential targets for the design of anti-anthrax vaccines and drugs.

  5. 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.

  6. Time course of immediate early gene protein expression in the spinal cord following conditioning stimulation of the sciatic nerve in rats.

    Science.gov (United States)

    Bojovic, Ognjen; Panja, Debabrata; Bittins, Margarethe; Bramham, Clive R; Tjølsen, Arne

    2015-01-01

    Long-term potentiation induced by conditioning electrical stimulation of afferent fibers is a widely studied form of synaptic plasticity in the brain and the spinal cord. In the spinal cord dorsal horn, long-term potentiation is induced by a series of high-frequency trains applied to primary afferent fibers. Conditioning stimulation (CS) of sciatic nerve primary afferent fibers also induces expression of immediate early gene proteins in the lumbar spinal cord. However, the time course of immediate early gene expression and the rostral-caudal distribution of expression in the spinal cord have not been systematically studied. Here, we examined the effects of sciatic nerve conditioning stimulation (10 stimulus trains, 0.5 ms stimuli, 7.2 mA, 100 Hz, train duration 2 s, 8 s intervals between trains) on cellular expression of immediate early genes, Arc, c-Fos and Zif268, in anesthetized rats. Immunohistochemical analysis was performed on sagittal sections obtained from Th13- L5 segments of the spinal cord at 1, 2, 3, 6 and 12 h post-CS. Strikingly, all immediate early genes exhibited a monophasic increase in expression with peak increases detected in dorsal horn neurons at 2 hours post-CS. Regional analysis showed peak increases at the location between the L3 and L4 spinal segments. Both Arc, c-Fos and Zif268 remained significantly elevated at 2 hours, followed by a sharp decrease in immediate early gene expression between 2 and 3 hours post-CS. Colocalization analysis performed at 2 hours post-CS showed that all c-Fos and Zif268 neurons were positive for Arc, while 30% and 43% of Arc positive neurons were positive for c-Fos and Zif268, respectively. The present study identifies the spinal cord level and time course of immediate early gene (IEGP) expression of relevance for analysis of IEGPs function in neuronal plasticity and nociception.

  7. Expression of late viral proteins is restricted in nasal mucosal leucocytes but not in epithelial cells during early-stage equine herpes virus-1 infection.

    Science.gov (United States)

    Gryspeerdt, Annick C; Vandekerckhove, Annelies P; Baghi, Hossein Bannazadeh; Van de Walle, Gerlinde R; Nauwynck, Hans J

    2012-08-01

    Equine herpes virus (EHV)-1 replicates in the epithelial cells of the upper respiratory tract and reaches the lamina propria and bloodstream in infected mononuclear cells. This study evaluated expression of the late viral proteins gB, gC, gD and gM in respiratory epithelial and mononuclear cells using: (1) epithelial-like rabbit kidney cells and peripheral blood mononuclear cells infected with EHV-1 in vitro; (2) an equine ex vivo nasal explant system; and (3) nasal mucosa tissue of ponies infected in vivo. The viral proteins were expressed in all late-infected epithelial cells, whereas expression was not observed in infected leucocytes where proteins gB and gM were expressed in 60-90%, and proteins gC and gD in only 20% of infected cells, respectively. The results indicate that expression of these viral proteins during early-stage EHV-1 infection is highly dependent on the cell type infected.

  8. Stimulation-induced expression of immediate early gene proteins in the dorsal horn is increased in neuropathy.

    Science.gov (United States)

    Bojovic, Ognjen; Bramham, Clive R; Tjølsen, Arne

    2016-01-01

    Peripheral neuropathic pain is described as a pain state caused by an injury or dysfunction of the nervous system, and could have clinical manifestations such as hyperalgesia, allodynia and spontaneous pain. The development of neuropathic pain may depend on long-term forms of neuronal plasticity in the spinal cord (SC). Expression of the immediate early gene proteins (IEGPs) Arc, Zif268, and c-Fos are implicated in establishment of long-term potentiation (LTP) induced by conditioning stimulation (CS) of primary afferent fibres. However, the impact of the neuropathic state (Bennett's model) on CS-induced expression of IEGPs has not been studied. The aim of this study was to compare the levels of Arc, c-Fos and Zif268 immunoreactivity prior to and after conditioning stimulation in animals with developed neuropathic pain, with sham operated, non-ligated controls. Twenty-four animals were divided equally into the neuropathic and non-neuropathic groups. Neuropathic pain was induced in all animals by conducting a loose ligation of the sciatic nerve with Chromic Catgut 4.0 sutures 7 days prior to conditioning stimulation or sham operation. The loose ligation was performed by placing sutures around the sciatic nerve compressing the nerve slightly just enough to reduce but not completely diminish the perineural circulation. A state of neuropathy was confirmed by a significant decrease in mechanical withdrawal threshold measured by von Frey's fibres. Immunohistochemical analysis was performed on transverse sections obtained from the L3-L5 segments of the SC at 2 and 6h post-CS and IEGP positive cells were counted in lamina I and II of the dorsal horn. During statistical analyses, the groups were compared by means of analysis of variance (univariate general linear model). If significant differences were found, each set of animals was compared with the sham group with post hoc Tukey's multiple comparison test. Strikingly, all IEGPs exhibited a significant increase in

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

    OpenAIRE

    Cyril Sobolewski; Sandhya Sanduja; Blanco, Fernando F.; Liangyan Hu; Dixon, Dan A.

    2015-01-01

    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 HDA...

  10. Knockdown of the intraflagellar transport protein IFT46 stimulates selective gene expression in mouse chondrocytes and affects early development in zebrafish.

    Science.gov (United States)

    Gouttenoire, Jérôme; Valcourt, Ulrich; Bougault, Carole; Aubert-Foucher, Elisabeth; Arnaud, Estelle; Giraud, Lionel; Mallein-Gerin, Frédéric

    2007-10-19

    Bone morphogenetic proteins (BMPs) act as multifunctional regulators in morphogenesis during development. In particular they play a determinant role in the formation of cartilage molds and their replacement by bone during endochondral ossification. In cell culture, BMP-2 favors chondrogenic expression and promotes hypertrophic maturation of chondrocytes. In mouse chondrocytes we have identified a BMP-2-sensitive gene encoding a protein of 301 amino acids. This protein, named mIFT46, is the mouse ortholog of recently identified Caenorhabditis elegans and Chlamydomonas reinhardtii intraflagellar transport (IFT) proteins. After generation of a polyclonal antibody against mIFT46, we showed for the first time that the endogenous protein is located in the primary cilium of chondrocytes. We also found that mIFT46 is preferentially expressed in early hypertrophic chondrocytes located in the growth plate. Additionally, mIFT46 knockdown by small interfering RNA oligonucleotides in cultured chondrocytes specifically stimulated the expression of several genes related to skeletogenesis. Furthermore, Northern blotting analysis indicated that mIFT46 is also expressed before chondrogenesis in embryonic mouse development, suggesting that the role of mIFT46 might not be restricted to cartilage. To explore the role of IFT46 during early development, we injected antisense morpholino oligonucleotides in Danio rerio embryos to reduce zebrafish IFT46 protein (zIFT46) synthesis. Dramatic defects in embryonic development such as a dorsalization and a tail duplication were observed. Thus our results taken together indicate that the ciliary protein IFT46 has a specific function in chondrocytes and is also essential for normal development of vertebrates.

  11. Increased gene expression of growth associated protein-43 in skin of patients with early-stage peripheral neuropathies.

    Science.gov (United States)

    Scheytt, Sarah; Riediger, Nadja; Braunsdorf, Silvia; Sommer, Claudia; Üçeyler, Nurcan

    2015-08-15

    Growth associated protein-43 (GAP-43) is one of the neural proteins associated with nerve injury that is upregulated after nerve injury. To investigate whether GAP-43 quantification in skin biopsies would differentiate subtypes of peripheral neuropathies, we analyzed GAP-43 expression in skin from the lateral thigh and the distal leg. We prospectively enrolled 130 patients with peripheral neuropathies and compared data with healthy controls. Intraepidermal nerve fiber density (IENFD) was determined using antibodies against protein gene product 9.5 (PGP 9.5); anti-GAP-43 antibodies were applied to visualize regenerating nerve fibers. PGP 9.5 and GAP-43 gene expression was analyzed using qRT-PCR. Patients with neuropathies had a generalized reduction of IENFD and GAP-43 immunoreactive fibers compared to controls (pneuropathies. Diagnostic subgroups and neuropathic pain had no influence on skin innervation. We conclude that peripheral neuropathies lead to an initial increase in GAP-43 gene expression as a potential mechanism of regeneration, which is not sustained in neuropathies of long duration. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. The varicella-zoster virus-mediated delayed host shutoff: open reading frame 17 has no major function, whereas immediate-early 63 protein represses heterologous gene expression.

    Science.gov (United States)

    Desloges, Nathalie; Rahaus, Markus; Wolff, Manfred H

    2005-12-01

    We reported that varicella-zoster virus (VZV) causes a delayed host shutoff during its replicative cycle. VZV open reading frame 17 (ORF17) is the homologue of the herpes simplex virus (HSV) UL41 gene encoding the virion host shutoff (vhs) protein which is responsible for the shutoff effect observed in HSV-infected cells. In the present study, we demonstrated that ORF17 is expressed as a late protein during the VZV replicative cycle in different infected permissive cell lines which showed a delayed shutoff of cellular RNA. A cell line with stable expression of VZV ORF17 was infected with VZV. In these cells, VZV replication and delayed host shutoff remained unchanged when compared to normal infected cells. ORF17 was not capable of repressing the expression of the beta-gal reporter gene under the control of the human cytomegalovirus immediate-early gene promoter or to inhibit the expression of a CAT reporter gene under the control of the human GAPDH promoter, indicating that ORF17 has no major function in the VZV-mediated delayed host shutoff. To determine whether other viral factors are involved in the host shutoff, a series of cotransfection assays was performed. We found that the immediate-early 63 protein (IE63) was able to downregulate the expression of reporter genes under the control of the two heterologous promoters, indicating that this viral factor can be involved in the VZV-mediated delayed host shutoff. Other factors can be also implicated to modulate the repressing action of IE63 to achieve a precise balance between the viral and cellular gene expression.

  13. Interferon Induced Transmembrane Protein-1 Gene Expression as a Biomarker for Early Detection of Invasive Potential of Oral Squamous Cell Carcinomas.

    Science.gov (United States)

    Ramanathan, Arvind; Ramanathan, Arvind

    2016-01-01

    Early detection of malignant transformation with expression biomarkers has significant potential to improve the survival rate of patients as such biomarkers enable prediction of progression and assess sensitivity to chemotherapy. The expression of interferon inducible transmembrane protein 1 (IFITM1) has been associated with early invasion events in several carcinomas, including head and neck cancers, and hence has been proposed as a novel candidate biomarker. As the incidence of oral squamous cell carcinoma (OSCC) is highest in the Indian population, we sought to investigate: 1) the expression pattern of IFITM1 in OSCC tissue samples obtained from Indian patients of Dravidian origin; and 2) the possibility of using IFITM1 expression as a potential biomarker. Total RNA extracted from thirty eight OSCC biopsy samples was subjected to semi-quantitative RT-PCR with IFITM1 and GAPDH specific primers. Of the thirty eight OSCC samples that were analyzed, IFITM1 overexpression was identified in fifteen (39%). Seven expressed a low level, while the remainder expressed high level of IFITM1. The overexpression of IFITM1 in OSCC samples indicates that IFITM1 may be explored for the possibility of use as a high confidence diagnostic biomarker in oral cancers. To the best of our knowledge, this is the first time that IFITM1 overexpression is being reported in Indian OSCC samples.

  14. Beta-amyloid precursor protein cleavage enzyme-1 expression in adult rat retinal neurons in the early period after lead exposure

    Institute of Scientific and Technical Information of China (English)

    Jufang Huang; Kai Huang; Lei Shang; Hui Wang; Xiaoxin Yan; Kun Xiong

    2011-01-01

    Previous studies have reported that non-human primates and rodents exposed to lead during brain development may become dependent on the deposition of pre-determined β-amyloid protein (Aβ), and exhibit upregulation of β-site amyloid precursor protein expression in old age. However, further evidence is required to elucidate the precise relationship and molecular mechanisms underlying the effects of early lead exposure on excessive Aβ production in adult mammals. The present study investigated the effects of lead exposure on expression of β-amyloid precursor protein cleavage enzyme-1 (BACE-1) in the rat retina and the production of Aβ in early development, using the retina as a window for studying Alzheimer's disease. Adult rats were intraocularly injected with different doses of lead acetate (10 μmol/L, 100 μmol/L, 1 mmol/L, 10 mmol/L and 100 mmol/L). The results revealed that retinal lead concentration, BACE-1 and its cleavage products β-C-terminal fragment and retina Aβ1-40 were all significantly increased in almost all of the lead exposure groups 48 hours later in a dose-dependent manner. The only exception was the 10 μmol/L group. The distribution of BACE-1 in the retina did not exhibit obvious changes, and no distinctive increase in the activation of retinal microglia was apparent. Similarly, retinal synaptophysin expression did not exhibit any clear changes. These data suggest that lead exposure can result in the upregulation of retinal neuron BACE-1 expression in the early period of development and further increase the overproduction of Aβ1-40 in the retina. Our results provided novel insight into the molecular mechanisms underlying environmentally-induced Alzheimer's disease.

  15. Infectivity and expression of the early adenovirus proteins are important regulators of wild-type and DeltaE1B adenovirus replication in human cells.

    Science.gov (United States)

    Steegenga, W T; Riteco, N; Bos, J L

    1999-09-09

    An adenovirus mutant lacking the expression of the large E1B protein (DeltaE1B) has been reported to replicate selectively in cells lacking the expression of functionally wild-type (wt) p53. Based on these results the DeltaE1B or ONYX-015 virus has been proposed to be an oncolytic virus which might be useful to treat p53-deficient tumors. Recently however, contradictory results have been published indicating that p53-dependent cell death is required for productive adenovirus infection. Since there is an urgent need for new methods to treat aggressive, mutant p53-expressing primary tumors and their metastases we carefully examined adenovirus replication in human cells to determine whether or not the DeltaE1B virus can be used for tumor therapy. The results we present here show that not all human tumor cell lines take up adenovirus efficiently. In addition, we observed inhibition of the expression of adenovirus early proteins in tumor cells. We present evidence that these two factors rather than the p53 status of the cell determine whether adenovirus infection results in lytic cell death. Furthermore, the results we obtained by infecting a panel of different tumor cell lines show that viral spread of the DeltaE1B is strongly inhibited in almost all p53-proficient and -deficient cell lines compared to the wt virus. We conclude that the efficiency of the DeltaE1B virus to replicate efficiently in tumor cells is determined by the ability to infect cells and to express the early adenovirus proteins rather than the status of p53.

  16. Short communication: localization and expression of monocyte chemoattractant protein-1 in different subcutaneous and visceral adipose tissues of early-lactating dairy cows.

    Science.gov (United States)

    Häussler, S; Sacré, C; Friedauer, K; Dänicke, S; Sauerwein, H

    2015-09-01

    The present study aimed to examine the mRNA abundance of the monocyte chemoattractant protein-1 (MCP-1) and to localize the MCP-1 protein in different subcutaneous (s.c.) and visceral (v.c.) fat depots in high-yielding dairy cows. Early-lactating German Holstein cows (n=25) were divided into a control (CON) and a conjugated linoleic acids (CLA)-supplemented group to investigate potential effects of dietary CLA treatment on MCP-1. The MCP-1 was localized in different s.c. and v.c. adipose tissue (AT) by immunohistochemistry, whereas the mRNA abundance was investigated using quantitative PCR. Albeit the infiltration of immune cells into bovine AT has been demonstrated to be only marginal, both MCP-1 protein and mRNA could be detected in bovine AT depots. The MCP-1 protein was localized both in the cytoplasm of adipocytes and in the cytoplasm of cells from the stromal vascular fraction; however, the number of MCP-1-positive cells was low. The mRNA abundances of MCP-1 were higher in v.c. compared with s.c. AT. Moreover, neither mRNA abundance nor protein expression of MCP-1 was seriously influenced by CLA supplementation of early-lactating dairy cows.

  17. Prokaryotic Expression and Purification of Early Pregnancy Factor Protein%重组人早孕因子的表达与纯化

    Institute of Scientific and Technical Information of China (English)

    杜晓明; 张改平; 王丽; 郭军庆; 王林林; 冯华; 权凯; 王爱萍

    2011-01-01

    早孕因子(EPF)是具有免疫抑制和生长调节活性的妊娠相关蛋白,为目前最早确认妊娠的生化指标之一.为获得人早孕因子重组蛋白,通过PCR技术扩增人早孕因子基因,克隆入原核表达载体pGEX-6p-1,与GST基因相融合,构建重组表达质粒,转化大肠杆菌表达菌株BL21,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,以SDS-PAGE和Western b1ot分析重组蛋白的表达,通过GST树脂纯化表达蛋白.结果成功构建重组表达质粒pGEX6P-EPF,在大肠杆菌中诱导表达了GST-EPF融合蛋白,表达蛋白分子量为373 kDa,并能被抗GST单克隆抗体特异识别,GST亲和层析纯化,制备了EPF重组蛋白.%Early pregnancy factor (EPF) is a pregnancy-associated protein that has immune-suppressive and growth-regulatory activities. It is regarded as a biochemical index by which super-early pregnancy can be determined. The objective of the study was to express the recombinant EPF protein in Escherichia coli (E. coli). The EPF gene amplified by PCR was sub-cloned into a pGEX-6P-1 vector fused with GST gene to construct a recombinant plasmid. The recombinant plasmid was then transformed into E. coli BL21.The expression of recombinant protein was analyzed by SDS-PAGE and Western blot after induction by IPTG. The recombinant protein was purified by high-affinity GST resin chromatography. The prokaryotic expression plasmid pGEX6P-EPF for producing EPF protein was constructed and the recombinant protein with a molecular weight of 37.3 kDa was expressed successfully in E. coli, which was recognized by the anti-GST monoclonal antibody in Western blot analysis. The recombinant EPF protein was obtained after GST affinity purification.

  18. Mitochondria-targeted DsRed2 protein expression during the early stage of bovine somatic cell nuclear transfer embryo development.

    Science.gov (United States)

    Park, Hyo-Jin; Min, Sung-Hun; Choi, Hoonsung; Park, Junghyung; Kim, Sun-Uk; Lee, Seunghoon; Lee, Sang-Rae; Kong, Il-Keun; Chang, Kyu-Tae; Koo, Deog-Bon; Lee, Dong-Seok

    2016-09-01

    Somatic cell nuclear transfer (SCNT) has been widely used as an efficient tool in biomedical research for the generation of transgenic animals from somatic cells with genetic modifications. Although remarkable advances in SCNT techniques have been reported in a variety of mammals, the cloning efficiency in domestic animals is still low due to the developmental defects of SCNT embryos. In particular, recent evidence has revealed that mitochondrial dysfunction is detected during the early development of SCNT embryos. However, there have been relatively few or no studies regarding the development of a system for evaluating mitochondrial behavior or dynamics. For the first time, in mitochondria of bovine SCNT embryos, we developed a method for the visualization of mitochondria and expression of fluorescence proteins. To express red fluorescence in mitochondria of cloned embryos, bovine ear skin fibroblasts, nuclear donor, were stably transfected with a vector carrying mitochondria-targeting DsRed2 gene tagged with V5 epitope (mito-DsRed2-V5 tag) using lentivirus-mediated gene transfer because of its ability to integrate in the cell genome and the potential for long-term transgene expression in the transduced cells and their dividing cells. From western blotting analysis of V5 tag protein using mitochondrial fraction and confocal microscopy of red fluorescence using SCNT embryos, we found that the mitochondrial expression of the mito-DsRed2 protein was detected until the blastocyst stage. In addition, according to image analysis, it may be suggested possible use of the system for visualization of mitochondrial localization and evaluation of mitochondrial behaviors or dynamics in early development of bovine SCNT embryos.

  19. 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

  20. Notch2 controls prolactin and insulin-like growth factor binding protein-1 expression in decidualizing human stromal cells of early pregnancy.

    Directory of Open Access Journals (Sweden)

    Gerlinde R Otti

    Full Text Available Decidualization, the transformation of the human uterine mucosa into the endometrium of pregnancy, is critical for successful implantation and embryonic development. However, key regulatory factors controlling differentiation of uterine stromal cells into hormone-secreting decidual cells have not been fully elucidated. Hence, we herein investigated the role of the Notch signaling pathway in human decidual stromal cells (HDSC isolated from early pregnancy samples. Immunofluorescence of first trimester decidual tissues revealed expression of Notch2 receptor and its putative, membrane-anchored interaction partners Jagged1, Delta-like (DLL 1 and DLL4 in stromal cells whereas other Notch receptors and ligands were absent from these cells. During in vitro differentiation with estrogen/progesterone (E2P4 and/or cyclic adenosine monophosphate (cAMP HDSC constitutively expressed Notch2 and weakly downregulated Jagged1 mRNA and protein, measured by quantitative PCR (qPCR and Western blotting, respectively. However, increased levels of DLL1 and DLL4 were observed in the decidualizing cultures. Transfection of a Notch luciferase reporter and qPCR of the Notch target gene hairy and enhancer of split 1 (HES1 revealed an induction of canonical Notch activity during in vitro differentiation. In contrast, treatment of HDSC with a chemical Notch/γ-secretase inhibitor decreased cAMP/E2P4-stimulated Notch luciferase activity, HES1 transcript levels and mRNA expression of the decidual marker genes prolactin (PRL and insulin-like growth factor binding protein 1 (IGFBP1. Similarly, siRNA-mediated gene silencing or antibody-mediated blocking of Notch2 diminished HES1, PRL and IGFBP1 mRNA levels as well as secreted PRL protein. In summary, the data suggest that canonical, Notch2-dependent signaling plays a role in human decidualization.

  1. Early graft failure of GalTKO pig organs in baboons is reduced by expression of a human complement pathway-regulatory protein.

    Science.gov (United States)

    Azimzadeh, Agnes M; Kelishadi, Sean S; Ezzelarab, Mohamed B; Singh, Avneesh K; Stoddard, Tiffany; Iwase, Hayato; Zhang, Tianshu; Burdorf, Lars; Sievert, Evelyn; Avon, Chris; Cheng, Xiangfei; Ayares, David; Horvath, Keith A; Corcoran, Philip C; Mohiuddin, Muhammad M; Barth, Rolf N; Cooper, David K C; Pierson, Richard N

    2015-01-01

    We describe the incidence of early graft failure (EGF, defined as loss of function from any cause within 3 days after transplant) in a large cohort of GalTKO pig organs transplanted into baboons in three centers, and the effect of additional expression of a human complement pathway-regulatory protein, CD46 or CD55 (GalTKO.hCPRP). Baboon recipients of life-supporting GalTKO kidney (n = 7) or heterotopic heart (n = 14) grafts received either no immunosuppression (n = 4), or one of several partial or full immunosuppressive regimens (n = 17). Fourteen additional baboons received a GalTKO.hCPRP kidney (n = 5) or heart (n = 9) and similar treatment regimens. Immunologic, pathologic, and coagulation parameters were measured at frequent intervals. EGF of GalTKO organs occurred in 9/21 baboons (43%). hCPRP expression reduced the GalTKO EGF incidence to 7% (1/14; P organs in which EGF developed (P organ failure, and (iii) the expression of a hCPRP reduces EGF but does not prevent systemic coagulation activation. Additional strategies will be required to control coagulation activation.

  2. Huntington disease iPSCs show early molecular changes in intracellular signaling, the expression of oxidative stress proteins and the p53 pathway.

    Science.gov (United States)

    Szlachcic, Wojciech J; Switonski, Pawel M; Krzyzosiak, Wlodzimierz J; Figlerowicz, Marek; Figiel, Maciej

    2015-09-01

    Huntington disease (HD) is a brain disorder characterized by the late onset of motor and cognitive symptoms, even though the neurons in the brain begin to suffer dysfunction and degeneration long before symptoms appear. There is currently no cure. Several molecular and developmental effects of HD have been identified using neural stem cells (NSCs) and differentiated cells, such as neurons and astrocytes. Still, little is known regarding the molecular pathogenesis of HD in pluripotent cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Therefore, we examined putative signaling pathways and processes involved in HD pathogenesis in pluripotent cells. We tested naïve mouse HD YAC128 iPSCs and two types of human HD iPSC that were generated from HD and juvenile-HD patients. Surprisingly, we found that a number of changes affecting cellular processes in HD were also present in undifferentiated pluripotent HD iPSCs, including the dysregulation of the MAPK and Wnt signaling pathways and the dysregulation of the expression of genes related to oxidative stress, such as Sod1. Interestingly, a common protein interactor of the huntingtin protein and the proteins in the above pathways is p53, and the expression of p53 was dysregulated in HD YAC128 iPSCs and human HD iPSCs. In summary, our findings demonstrate that multiple molecular pathways that are characteristically dysregulated in HD are already altered in undifferentiated pluripotent cells and that the pathogenesis of HD might begin during the early stages of life.

  3. Differential expression of serum glycodelin and insulin-like growth factor binding protein 1 in early pregnancy.

    Science.gov (United States)

    Douglas, Nataki C; Thornton, Melvin H; Nurudeen, Sahadat K; Bucur, Maria; Lobo, Rogerio A; Sauer, Mark V

    2013-11-01

    This prospective study evaluated whether serum glycodelin and insulin-like growth factor binding protein 1 (IGFBP-1) predict the likelihood of embryo implantation in recipients undergoing donor egg in vitro fertilization. We measured glycodelin and IGFBP-1 at 6 points from lining check to lutenizing hormone (LH) + 31. β-Human chorionic gonadotropin levels were first measured at LH + 17. The recipients were divided into those without embryo implantation (group 1, n = 6) and those with successful implantation (group 2, n = 30). Although this is a negative study in that neither glycodelin nor IGFBP-1 alone reflected endometrial (EM) receptivity, the glycodelin/IGFBP-1 ratio on the day of blastocyst transfer was higher in recipients who achieved pregnancy (P = .05). At LH + 17, glycodelin was higher (P = .04), and IGFBP-1 was lower (P = .004) in recipients who achieved pregnancy when compared to those who did not. These observations are likely due to EM changes induced by successful embryo implantation.

  4. Shortening and intracellular Ca2+ in ventricular myocytes and expression of genes encoding cardiac muscle proteins in early onset type 2 diabetic Goto-Kakizaki rats.

    Science.gov (United States)

    Salem, K A; Adrian, T E; Qureshi, M A; Parekh, K; Oz, M; Howarth, F C

    2012-12-01

    There has been a spectacular rise in the global prevalence of type 2 diabetes mellitus. Cardiovascular complications are the major cause of morbidity and mortality in diabetic patients. Contractile dysfunction, associated with disturbances in excitation-contraction coupling, has been widely demonstrated in the diabetic heart. The aim of this study was to investigate the pattern of cardiac muscle genes that are involved in the process of excitation-contraction coupling in the hearts of early onset (8-10 weeks of age) type 2 diabetic Goto-Kakizaki (GK) rats. Gene expression was assessed in ventricular muscle with real-time RT-PCR; shortening and intracellular Ca(2+) were measured in ventricular myocytes with video edge detection and fluorescence photometry, respectively. The general characteristics of the GK rats included elevated fasting and non-fasting blood glucose and blood glucose at 120 min following a glucose challenge. Expression of genes encoding cardiac muscle proteins (Myh6/7, Mybpc3, Myl1/3, Actc1, Tnni3, Tnn2, Tpm1/2/4 and Dbi) and intercellular proteins (Gja1/4/5/7, Dsp and Cav1/3) were unaltered in GK ventricle compared with control ventricle. The expression of genes encoding some membrane pumps and exchange proteins was unaltered (Atp1a1/2, Atp1b1 and Slc8a1), whilst others were either upregulated (Atp1a3, relative expression 2.61 ± 0.69 versus 0.84 ± 0.23) or downregulated (Slc9a1, 0.62 ± 0.07 versus 1.08 ± 0.08) in GK ventricle compared with control ventricle. The expression of genes encoding some calcium (Cacna1c/1g, Cacna2d1/2d2 and Cacnb1/b2), sodium (Scn5a) and potassium channels (Kcna3/5, Kcnj3/5/8/11/12, Kchip2, Kcnab1, Kcnb1, Kcnd1/2/3, Kcne1/4, Kcnq1, Kcng2, Kcnh2, Kcnk3 and Kcnn2) were unaltered, whilst others were either upregulated (Cacna1h, 0.95 ± 0.16 versus 0.47 ± 0.09; Scn1b, 1.84 ± 0.16 versus 1.11 ± 0.11; and Hcn2, 1.55 ± 0.15 versus 1.03 ± 0.08) or downregulated (Hcn4, 0.16 ± 0.03 versus 0.37 ± 0.08; Kcna2, 0.35 ± 0

  5. Recombinant protein expression in Nicotiana.

    Science.gov (United States)

    Matoba, Nobuyuki; Davis, Keith R; Palmer, Kenneth E

    2011-01-01

    Recombinant protein pharmaceuticals are now widely used in treatment of chronic diseases, and several recombinant protein subunit vaccines are approved for human and veterinary use. With growing demand for complex protein pharmaceuticals, such as monoclonal antibodies, manufacturing capacity is becoming limited. There is increasing need for safe, scalable, and economical alternatives to mammalian cell culture-based manufacturing systems, which require substantial capital investment for new manufacturing facilities. Since a seminal paper reporting immunoglobulin expression in transgenic plants was published in 1989, there have been many technological advances in plant expression systems to the present time where production of proteins in leaf tissues of nonfood crops such as Nicotiana species is considered a viable alternative. In particular, transient expression systems derived from recombinant plant viral vectors offer opportunities for rapid expression screening, construct optimization, and expression scale-up. Extraction of recombinant proteins from Nicotiana leaf tissues can be achieved by collection of secreted protein fractions, or from a total protein extract after grinding the leaves with buffer. After separation from solids, the major purification challenge is contamination with elements of the photosynthetic complex, which can be solved by application of a variety of facile and proven strategies. In conclusion, the technologies required for safe, efficient, scalable manufacture of recombinant proteins in Nicotiana leaf tissues have matured to the point where several products have already been tested in phase I clinical trials and will soon be followed by a rich pipeline of recombinant vaccines, microbicides, and therapeutic proteins.

  6. 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.

  7. Expression of progesterone receptor membrane component 1, serpine mRNA binding protein 1 and nuclear progesterone receptor isoforms A and B in the bovine myometrium during the estrous cycle and early pregnancy.

    Science.gov (United States)

    Slonina, Dominika; Kowalik, Magdalena K; Kotwica, Jan

    2012-01-01

    The aim of this study was to investigate the (1) expression of progesterone membrane component 1 (PGRMC1), serpine mRNA binding protein 1 (SERBP1) and progesterone receptor (PR) mRNA and (2) protein expression levels of PGRMC1, SERBP1 and PR isoforms A and B in the bovine myometrium during the estrous cycle and early pregnancy. Uteri from cows on days 1-5, 6-10, 11-16 and 17-21 of the estrous cycle and weeks 3-5, 6-8 and 9-12 of pregnancy were used (n=5-6 per period). There were no changes (P>0.05) in PGRMC1 mRNA expression during the estrous cycle, while expression of SERBP1 and PR mRNA was the lowest (P0.05) in SERBP1 protein expression in cycling and pregnant cows, while the highest (P<0.05) PGRMC1 protein expression was found during weeks 3-5 of pregnancy. Similar protein expression profiles for PRA and PRB were found, and protein levels were highest on days 1-5 of the estrous cycle. From day 6 of the cycle, PRA and PRB protein expression decreased and were maintained at this lower level during pregnancy. In conclusion, our study assessed mRNA and protein expression levels of PGRMC1, SERBP1 and PR in the bovine myometrium during the estrous cycle and the first trimester of pregnancy. It is possible that progesterone (P4) affects myometrial function in a genomic and nongenomic manner.

  8. Obestatin induction of early-response gene expression in gastrointestinal and adipose tissues and the mediatory role of G protein-coupled receptor, GPR39

    NARCIS (Netherlands)

    Zhang, Jian V.; Jahr, Holger; Luo, Chin-Wei; Klein, Cynthia; Van Kolen, Kristof; Donck, Luc Ver; De, Ananya; Baart, Esther; Li, Jing; Moechars, Dieder; Hsueh, Aaron J. W.

    2008-01-01

    Obestatin was identified as a brain/gut peptide hormone encoded by the ghrelin gene and found to interact with the G protein-coupled receptor, GPR39. We investigated target cells for obestatin based on induction of an early-response gene c-fos in different tissues. After ip injection of obestatin, c

  9. Expression of nebulette during early cardiac development.

    Science.gov (United States)

    Esham, Michael; Bryan, Kourtney; Milnes, Jennifer; Holmes, William B; Moncman, Carole L

    2007-04-01

    Nebulette, a cardiac homologue of nebulin, colocalizes with alpha-actinin in the pre-myofibrils of spreading cardiomyocytes and has been hypothesized to play a critical role in the formation of the thin-filament-Z-line complex early during myofibrillogenesis. Data from mesodermal explants or whole tissue mounts of developing hearts suggest that the pattern of myofibrillogenesis in situ may differ from observations of spreading cardiomyocytes. To evaluate the role of nebulette in myofibrillogenesis, we have analyzed the expression of nebulette in chicken heart rudiments by immunoblots and immunofluorescence. We detect the 110 kDa nebulette in heart rudiments derived from stage 9-10 using the anti-nebulin mAb, N114, or polyclonal anti-nebulette Abs by immunoblotting. Immunofluorescence analysis of explants stained with anti-nebulette and anti-alpha-actinin Abs demonstrates that both proteins localize along actin filaments in punctate to continuous manner at early stages of cardiac development and later give rise to striations. In both cases, the punctate staining had a periodicity of approximately 1.0 microm indicating a pre-myofibrils distribution at the earliest time points examined. We demonstrate that nebulette is indeed associated with premyofibrils in very early stages of myofibrillogenesis and suggest that nebulette may play an important role in the formation of these structures.

  10. 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

  11. Activation of stress-activated MAP protein kinases up-regulates expression of transgenes driven by the cytomegalovirus immediate/early promoter.

    OpenAIRE

    Bruening, W; Giasson, B; Mushynski, W; Durham, H D

    1998-01-01

    The immediate/early promoter/enhancer of cytomegalovirus (CMV promoter) is one of the most commonly used promoters for expression of transgenes in eukaryotic cells. In practice, the CMV promoter is often thought of as a constitutively active unregulated promoter. However, we have observed that transcription from the CMV promoter can be up-regulated by a variety of environmental stresses. Many forms of cellular stress stimulate MAP kinase signalling pathways, resulting in activation of stress-...

  12. Effect of supplemental concentrate during the dry period or early lactation on rumen epithelium gene and protein expression in dairy cattle during the transition period.

    Science.gov (United States)

    Dieho, K; van Baal, J; Kruijt, L; Bannink, A; Schonewille, J T; Carreño, D; Hendriks, W H; Dijkstra, J

    2017-09-01

    We previously reported 2 experiments with rumen-cannulated Holstein-Friesian dairy cows showing that during the transition period, rumen papillae surface area, and fractional absorption rate of volatile fatty acids (VFA) increase after calving. However, supplemental concentrate during the dry period and rate of increase of concentrate allowance during lactation affected papillae surface area, but not VFA absorption. Here we report the changes in gene and protein expression in rumen papillae related to tissue growth and VFA utilization. The lactation experiment treatment consisted of a rapid [RAP; 1.0 kg of dry matter (DM)/d; n = 6] or gradual (GRAD; 0.25 kg of DM/d; n = 6) increase of concentrate allowance (up to 10.9 kg of DM/d), starting at 4 d postpartum (pp). The dry period experiment treatment consisted of 3.0 kg of DM/d of concentrate (n = 4) or no concentrate (n = 5) during the last 28 d of the dry period. Real-time quantitative PCR analysis of rumen papillae showed that the expression of apoptosis-related genes was neither affected by day nor its interaction with treatment for both experiments. Expression of epithelial transporter genes was not affected by day or treatment in the lactation experiment, except for NBC1. In the dry period experiment, expression of MCT1, NBC1, DRA, NHE2, NHE3, and UT-B generally decreased after calving. A day and treatment interaction was observed for ATP1A1 in the dry period experiment, with greater expression at 18 and 8 d antepartum for concentrate than no concentrate. Generally, expression of VFA metabolism-related genes was not affected by day or its interaction with treatment. In the lactation experiment, immunoblotting of 5 selected genes showed that protein expression of DRA and PCCA was greater at 16 d pp compared with 3 and 44 d pp. Expression of NHE2 was greater, and that of ATP1A1 lower, at 16 and 44 d pp compared with 3 d pp, suggesting alterations in intracellular pH regulation and sodium homeostasis. Both MCT1

  13. 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

  14. Prognostic significance of ESR1 gene amplification, mRNA/protein expression and functional profiles in high-risk early breast cancer: a translational study of the Hellenic Cooperative Oncology Group (HeCOG.

    Directory of Open Access Journals (Sweden)

    George Pentheroudakis

    Full Text Available BACKGROUND: Discrepant data have been published on the incidence and prognostic significance of ESR1 gene amplification in early breast cancer. PATIENTS AND METHODS: Formalin-fixed paraffin-embedded tumor blocks were collected from women with early breast cancer participating in two HeCOG adjuvant trials. Messenger RNA was studied by quantitative PCR, ER protein expression was centrally assessed using immunohistochemistry (IHC and ESR1 gene copy number by dual fluorescent in situ hybridization probes. RESULTS: In a total of 1010 women with resected node-positive early breast adenocarcinoma, the tumoral ESR1/CEP6 gene ratio was suggestive of deletion in 159 (15.7%, gene gain in 551 (54.6% and amplification in 42 cases (4.2%, with only 30 tumors (3% harboring five or more ESR1 copies. Gene copy number ratio showed a significant, though weak correlation to mRNA and protein expression (Spearman's Rho <0.23, p = 0.01. ESR1 clusters were observed in 9.5% (57 gain, 38 amplification of cases. In contrast to mRNA and protein expression, which were favorable prognosticators, gene copy number changes did not obtain prognostic significance. When ESR1/CEP6 gene ratio was combined with function (as defined by ER protein and mRNA expression in a molecular classifier, the Gene Functional profile, it was functional status that impacted on prognosis. In univariate analysis, patients with functional tumors (positive ER protein expression and gene ratio normal or gain/amplification fared better than those with non-functional tumors with ESR1 gain (HR for relapse or death 0.49-0.64, p = 0.003. Significant interactions were observed between gene gain/amplification and paclitaxel therapy (trend for DFS benefit from paclitaxel only in patients with ESR1 gain/amplification, p = 0.066 and Gene Functional profile with HER2 amplification (Gene Functional profile prognostic only in HER2-normal cases, p = 0.029. CONCLUSIONS: ESR1 gene deletion and

  15. 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

    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...... 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......, but increased PGC1a 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...

  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

    , 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 1 1/2 years. In conclusion, early postnatal, but not late gestation......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...

  17. Differentiation, early response gene expression, and apoptosis induction in human breast tumor cells by Okadaic Acid and related inhibitors of protein phosphatases 1 and 2A. Okadaic acid effects on human breast tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Kiguchi, K.; Giometti, C.; Chubb, C.H.; Huberman, E. [Argonne National Lab., IL (United States); Fujiki, H. [National Cancer Center Research Institute, Tokyo (Japan)

    1992-08-20

    Okadaic acid (OA), a tumor promoter and an inhibitor of protein phosphatases (PPH) 1 and 2A, was tested for its ability to induce events associated with differentiation and apoptosis induction in the human MCF-7, AU-565, and MB-231 breast tumor cells. Differentiation in these cells was characterized by inhibition of cell multiplication, reactivity with monoclonal antibodies to {alpha}-lactalbumin and {beta}-casein, and the appearance of large lipid droplets; apoptosis was characterized by the appearance of cells with segmented and fragmented nuclei. In the MCF-7 cell line, OA at nanomolar concentrations elicited within 5 min an increase in the phosphorylation of a set of cellular proteins, within hours expression of the early response genes, junB, c-jun, and c-fos and within days manifestation of differentiation and apoptosis markers. Differentiation and apoptosis were also induced by dinophysistoxin-1 and calyculin A, two other tumor promoters and inhibitors of PPH 1 and 2A, but not by OA tetramethyl ether, an inactive OA derivative, or microcystin LR, a PPH 1 and 2A inhibitor that penetrates epithelial cells poorly. OA induced both differentiation and apoptosis in MB-231 cells and MCF-7, but only differentiation in AU-565 cells. Phorbol 12-myristate 13-acetate (PMA), a tumor promoter that is not an inhibitor of PPH 1 and 2A but rather an activator of protein kinase C, also induced within minutes the phosphorylation of proteins, within hours the expression of early response genes, and within days differentiation, but not apoptosis; yet PMA was able to attenuate apoptosis induced by the okadaic acid class of tumor promoters. These results indicate that OA and related agents can induce processes that result in tumor breast cell differentiation and apoptosis, and this induction is associated with their ability to inhibit PPH 1 and 2A. Yet apoptosis is not necessarily required for differentiation induction by these agents.

  18. A highly efficient pipeline for protein expression in Leishmania tarentolae using infrared fluorescence protein as marker

    Directory of Open Access Journals (Sweden)

    Mueller-Roeber Bernd

    2010-05-01

    Full Text Available Abstract Background Leishmania tarentolae, a unicellular eukaryotic protozoan, has been established as a novel host for recombinant protein production in recent years. Current protocols for protein expression in Leishmania are, however, time consuming and require extensive lab work in order to identify well-expressing cell lines. Here we established an alternative protein expression work-flow that employs recently engineered infrared fluorescence protein (IFP as a suitable and easy-to-handle reporter protein for recombinant protein expression in Leishmania. As model proteins we tested three proteins from the plant Arabidopsis thaliana, including a NAC and a type-B ARR transcription factor. Results IFP and IFP fusion proteins were expressed in Leishmania and rapidly detected in cells by deconvolution microscopy and in culture by infrared imaging of 96-well microtiter plates using small cell culture volumes (2 μL - 100 μL. Motility, shape and growth of Leishmania cells were not impaired by intracellular accumulation of IFP. In-cell detection of IFP and IFP fusion proteins was straightforward already at the beginning of the expression pipeline and thus allowed early pre-selection of well-expressing Leishmania clones. Furthermore, IFP fusion proteins retained infrared fluorescence after electrophoresis in denaturing SDS-polyacrylamide gels, allowing direct in-gel detection without the need to disassemble cast protein gels. Thus, parameters for scaling up protein production and streamlining purification routes can be easily optimized when employing IFP as reporter. Conclusions Using IFP as biosensor we devised a protocol for rapid and convenient protein expression in Leishmania tarentolae. Our expression pipeline is superior to previously established methods in that it significantly reduces the hands-on-time and work load required for identifying well-expressing clones, refining protein production parameters and establishing purification protocols

  19. 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.

  20. 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.

  1. Early gene expression changes with rush immunotherapy

    Directory of Open Access Journals (Sweden)

    Barnett Sherry

    2011-09-01

    Full Text Available Abstract Background To examine whether whole genome expression profiling could reveal changes in mRNA expression of peripheral blood mononuclear cells (PBMC from allergic patients undergoing rush immunotherapy (RIT that might be manifest within the first few months of treatment. Methods For this study, PBMC from three allergic patients undergoing RIT were assessed at four timepoints: prior to RIT, at 1 week and 7 week post-RIT, during build-up and at 4 months, after establishment of a maintenance dose. PBMC mRNA gene expression changes over time were determined by oligonucleotide microarrays using the Illumina Human-6 BeadChip Platform, which simultaneously interrogates expression profiles of > 47,000 transcripts. Differentially expressed genes were identified using well-established statistical analysis for microarrays. In addition, we analyzed peripheral blood basophil high-affinity IgE receptor (Fc epsilon RI expression and T-regulatory cell frequency as detected by expression of CD3+CD4+CD25bright cells at each timepoint using flow cytometry. Results In comparing the initial 2 timepoints with the final 2 timepoints and analyzing for genes with ≥1.5-fold expression change (p less than or equal to 0.05, BH-FDR, we identified 507 transcripts. At a 2-fold change (p less than or equal to 0.05, BH-FDR, we found 44 transcripts. Of these, 28 were up-regulated and 16 were down-regulated genes. From these datasets, we have identified changes in immunologically relevant genes from both the innate and adaptive response with upregulation of expressed genes for molecules including IL-1β, IL-8, CD40L, BTK and BCL6. At the 4 month timepoint, we noted a downward trend in Fc epsilon RI expression in each of the three patients and increased allergen-specific IgG4 levels. No change was seen in the frequency of peripheral T-regulatory cells expressed over the four timepoints. Conclusions We observed significant changes in gene expression early in peripheral

  2. In situ expression of heat-shock proteins and 3-nitrotyrosine in brains of young rats exposed to a WiFi signal in utero and in early life.

    Science.gov (United States)

    Aït-Aïssa, Saliha; de Gannes, Florence Poulletier; Taxile, Murielle; Billaudel, Bernard; Hurtier, Annabelle; Haro, Emmanuelle; Ruffié, Gilles; Athané, Axel; Veyret, Bernard; Lagroye, Isabelle

    2013-06-01

    The bioeffects of exposure to Wireless High-Fidelity (WiFi) signals on the developing nervous systems of young rodents was investigated by assessing the in vivo and in situ expression levels of three stress markers: 3-Nitrotyrosine (3-NT), an oxidative stress marker and two heat-shock proteins (Hsp25 and Hsp70). These biomarkers were measured in the brains of young rats exposed to a 2450 MHz WiFi signal by immunohistochemistry. Pregnant rats were first exposed or sham exposed to WiFi from day 6 to day 21 of gestation. In addition three newborns per litter were further exposed up to 5 weeks old. Daily 2-h exposures were performed blind in a reverberation chamber and whole-body specific absorption rate levels were 0, 0.08, 0.4 and 4 W/kg. 3-NT and stress protein expression was assayed in different areas of the hippocampus and cortex. No significant difference was observed among exposed and sham-exposed groups. These results suggest that repeated exposure to WiFi during gestation and early life has no deleterious effects on the brains of young rats.

  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. Effect of Boschniakia rossica on expression of GST-P, p53 and p21(ras)proteins in early stage of chemical hepatocarcinogenesis and its anti-inflammatory activities in rats.

    Science.gov (United States)

    Yin, Zong-Zhu; Jin, Hai-Ling; Yin, Xue-Zhe; Li, Tian-Zhu; Quan, Ji-Shu; Jin, Zeng-Nan

    2000-12-01

    AIM:To investigate the effect of Boschniakia rossica (BR) extract on expression of GST-P, p53 and p21(ras) proteins in early stage of chemical hepatocarcinogenesis in rats and its anti-inflammatory activities.METHODS:The expression of tumor marker-placental form glutathione S-transferase (GST-P), p53 and p21(ras) proteins were investigated by immunohisto-chemical techniques and ABC method. Anti-inflammatory activities of BR were studied by xylene and croton oil-induced mouse ear edema, carrageenin, histamine and hot scald-induced rat pow edema, adjuvant-induced rat arthritis and cotton pellet induced mouse granuloma formation methods.RESULTS:The 500mg/kg of BR-H2O extract frac-tionated from BR-Methanol extract had inhibitory effect on the formation of DEN-induced GST-P-positive foci in rat liver (GST-P staining was 78% positive in DEN+AAF group vs 20% positive in DEN+AAF+BR group, Panti-inflamatory effect in xylene and crotonoil induced mouse ear edema (inhibitory rates were 26%-29% and 35%-59%, respectively). BR H(2)O extract exhibited inhibitory effect in carrageenin, histamine and hot scald-induced hind paw edema and adjuvant-induced arthritis in rats and cotton pellet-induced granuloma formation in mice.CONCLUSION:BR extract exhibited inhibitory effect on formation of preneoplastic hepatic foci in early stage of rat chemical hepato-carcinogenesis.Both CH(2)Cl(2) and H(2)O extracts from BR exerted anti-inflammatory effect in rats and mice.

  5. Coevolution of gene expression among interacting proteins

    Energy Technology Data Exchange (ETDEWEB)

    Fraser, Hunter B.; Hirsh, Aaron E.; Wall, Dennis P.; Eisen,Michael B.

    2004-03-01

    Physically interacting proteins or parts of proteins are expected to evolve in a coordinated manner that preserves proper interactions. Such coevolution at the amino acid-sequence level is well documented and has been used to predict interacting proteins, domains, and amino acids. Interacting proteins are also often precisely coexpressed with one another, presumably to maintain proper stoichiometry among interacting components. Here, we show that the expression levels of physically interacting proteins coevolve. We estimate average expression levels of genes from four closely related fungi of the genus Saccharomyces using the codon adaptation index and show that expression levels of interacting proteins exhibit coordinated changes in these different species. We find that this coevolution of expression is a more powerful predictor of physical interaction than is coevolution of amino acid sequence. These results demonstrate previously uncharacterized coevolution of gene expression, adding a different dimension to the study of the coevolution of interacting proteins and underscoring the importance of maintaining coexpression of interacting proteins over evolutionary time. Our results also suggest that expression coevolution can be used for computational prediction of protein protein interactions.

  6. 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...

  7. Expression of insulin-like growth factor-1 mRNA and protein level of corpora striata in ischemic side at the early stage of middle cerebral artery ischemia/reperfusion in rhesus monkeys

    Institute of Scientific and Technical Information of China (English)

    Huanmin Gao; Rui Zhang; Yunliang Guo

    2006-01-01

    BACKGROUND: Insulin-like growth factor-I(IGF-1), as one of the important members of growth factor family,participants in the regulation of many physiological functions and behaviors, having very strong neuroprotective effect. However, the expression of IGF-1 following cerebral ischemia/reperfusion is still disputed.OBJ ECTIVE: To observe the expression of IGF-1 and protein of corpora striata in ischemic side at the early stage of middle cerebral artery ischemia/reperfusion in rhesus monkey.DESIGN: A completely randomized grouping design, controlled animal experimentSETTING: Institute of Cerebrovascular Disease, Affiliated Hospital of Medical College of Qingdao University.MATERIALS: ① Totally 17 rhesus monkeys , of either gender, aged 4 to 5 years, were enrolled . Seven rhesus monkeys observed with gene chip were randomly divided into 2 groups: sham operation group (n=3)and ischemia/reperfusion group (n=4). Ten rhesus monkeys observed with in situ hybridization and immunohistochemistry method were randomly divided into 2 groups: sham operation group (n=3)and ischemia/reperfusion group (n=7). Rhesus monkeys observed under microscope were divided into 2 groups: sham operation group (n=6) and ischamia/reperfusion group (n=11). ② Materials used in the experiment: cresyl violet (Sigma Company, America); immunohistochemical reagent kit ( Huamei Bio-engineering Company); In situ hybridization reagent kit (Boshide Bio-engineering Co. Ltd, Wuhan); 12 800 dots chip (Boxing Company,Shanghai).METHODS: This experiment was carried out at the Institute of Cerebrovascular Disease, Affiliated Hospital of Medical College of Qingdao University from January 2001 to December 2003. ① The onset area of middle cerebral artery was blocked for 2 hours, middle cerebral artery ischemia/reperfusion models were created.② After ischemia/reperfusion for 24 hours, cerebral tissue sections of rhesus monkeys were prepared and stained with cresyl violet. Image analysis was performed with 500IW

  8. High expression of Y-box-binding protein 1 correlates with poor prognosis and early recurrence in patients with small invasive lung adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Zhao S

    2016-05-01

    Full Text Available Shilei Zhao,1,* Wei Guo,1,* Jinxiu Li,1 Wendan Yu,1 Tao Guo,1 Wuguo Deng,2,3 Chundong Gu1 1The First Affiliated Hospital, Institute of Cancer Stem Cell, Lung Cancer Diagnosis and Treatment Center, Dalian Medical University, Dalian, 2Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Sun Yat-Sen University, Guangzhou, 3State Key Laboratory of Targeted Drug for Tumors of Guangdong Province, Guangzhou Double Bioproduct Inc., Guangzhou, People’s Republic of China *These authors contributed equally to this work Background: Prognosis of small (≤2 cm invasive lung adenocarcinoma remains poor, and identification of high-risk individuals from the patients after complete surgical resection of lung adenocarcinoma has become an urgent problem. YBX1 has been reported to be able to predict prognosis in many cancers (except lung adenocarcinoma that are independent of TNM (tumor, nodes, metastases staging, especially small invasive lung adenocarcinoma. Therefore, we examined the significance of YBX1 expression on prognosis and recurrence in patients with small invasive lung adenocarcinoma. Material and methods: A total of 75 patients with small invasive lung adenocarcinoma after complete resection were enrolled from January 2008 to December 2010. Immunohistochemical staining was used to detect the expression of YBX1, and receiver operating characteristic curve analysis was performed to precisely assess the overall expression of YBX1. Meanwhile, primary lesions were identified based on the International Association for the Study of Lung Cancer, the American Thoracic Society, and the European Respiratory Society’s classification of lung adenocarcinoma. The effect of different clinicopathological factors on patients’ survival was examined. Furthermore, Western blot analysis was used to show the expression of YBX1 in vitro. Results: Sensitivity and specificity of YBX1 for detecting small

  9. Coevolution of gene expression among interacting proteins

    OpenAIRE

    2004-01-01

    Physically interacting proteins or parts of proteins are expected to evolve in a coordinated manner that preserves proper interactions. Such coevolution at the amino acid-sequence level is well documented and has been used to predict interacting proteins, domains, and amino acids. Interacting proteins are also often precisely coexpressed with one another, presumably to maintain proper stoichiometry among interacting components. Here, we show that the expression levels of physically inter...

  10. 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 with bioch......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...

  11. A-TWinnipeg: Pathogenesis of rare ATM missense mutation c.6200C>A with decreased protein expression and downstream signaling, early-onset dystonia, cancer, and life-threatening radiotoxicity.

    Science.gov (United States)

    Nakamura, Kotoka; Fike, Francesca; Haghayegh, Sara; Saunders-Pullman, Rachel; Dawson, Angelika J; Dörk, Thilo; Gatti, Richard A

    2014-07-01

    We studied 10 Mennonite patients who carry the c.6200C>A missense mutation (p.A2067D) in the ATM gene, all of whom exhibited a phenotypic variant of ataxia-telangiectasia (A-T) that is characterized by early-onset dystonia and late-onset mild ataxia, as previously described. This report provides the pathogenetic evidence for this mutation on cellular functions. Several patients have developed cancer and subsequently experienced life-threatening adverse reactions to radiation (radiotoxicity) and/or chemotherapy. As the c.6200C>A mutation is, thus far, unique to the Mennonite population and is always associated with the same haplotype or haplovariant, it was important to rule out any possible confounding DNA variant on the same haplotype. Lymphoblastoid cells derived from Mennonite patients expressed small amounts of ATM protein, which had no autophosphorylation activity at ATM Ser1981, and trace-to-absent transphosphorylation of downstream ATM targets. A-T lymphoblastoid cells stably transfected with ATM cDNA which had been mutated for c.6200C>A did not show a detectable amount of ATM protein. The same stable cell line with mutated ATM cDNA also showed a trace-to-absent transphosphorylation of downstream ATM targets SMC1pSer966 and KAP1pSer824. From these results, we conclude that c.6200A is the disease-causing ATM mutation on this haplotype. The presence of at least trace amounts of ATM kinase activity on some immunoblots may account for the late-onset, mild ataxia of these patients. The cause of the dystonia remains unclear. Because this dystonia-ataxia phenotype is often encountered in the Mennonite population in association with cancer and adverse reactions to chemotherapy, an early diagnosis is important.

  12. Differential effects of Th1, monocyte/macrophage and Th2 cytokine mixtures on early gene expression for glial and neural-related molecules in central nervous system mixed glial cell cultures: neurotrophins, growth factors and structural proteins

    Directory of Open Access Journals (Sweden)

    Nedelkoska Liljana

    2007-12-01

    Full Text Available Abstract Background In multiple sclerosis, inflammatory cells are found in both active and chronic lesions, and it is increasingly clear that cytokines are involved directly and indirectly in both formation and inhibition of lesions. We propose that cytokine mixtures typical of Th1 or Th2 lymphocytes, or monocyte/macrophages each induce unique molecular changes in glial cells. Methods To examine changes in gene expression that might occur in glial cells exposed to the secreted products of immune cells, we have used gene array analysis to assess the early effects of different cytokine mixtures on mixed CNS glia in culture. We compared the effects of cytokines typical of Th1 and Th2 lymphocytes and monocyte/macrophages (M/M on CNS glia after 6 hours of treatment. Results In this paper we focus on changes with potential relevance for neuroprotection and axon/glial interactions. Each mixture of cytokines induced a unique pattern of changes in genes for neurotrophins, growth and maturation factors and related receptors; most notably an alternatively spliced form of trkC was markedly downregulated by Th1 and M/M cytokines, while Th2 cytokines upregulated BDNF. Genes for molecules of potential importance in axon/glial interactions, including cell adhesion molecules, connexins, and some molecules traditionally associated with neurons showed significant changes, while no genes for myelin-associated genes were regulated at this early time point. Unexpectedly, changes occurred in several genes for proteins initially associated with retina, cancer or bone development, and not previously reported in glial cells. Conclusion Each of the three cytokine mixtures induced specific changes in gene expression that could be altered by pharmacologic strategies to promote protection of the central nervous system.

  13. ABL Tyrosine Kinase Stimulates PUMA Protein Expression

    OpenAIRE

    Oon, Chet K

    2016-01-01

    ABL is an ubiquitously expressed non-receptor tyrosine kinase involved in multiple cellular functions including programmed cell death. Upon DNA damage, ABL has been shown to upregulate PUMA, p53 upregulated modulator of apoptosis, and causes downstream mitochondrial intrinsic apoptotic events. However, the mechanism by which ABL regulates PUMA expression remains unknown. We have shown that ABL does not change PUMA protein subcellular localization through immunofluorescence. Through protein an...

  14. Binary gene induction and protein expression in individual cells

    Directory of Open Access Journals (Sweden)

    Conolly Rory B

    2006-04-01

    Full Text Available Abstract Background Eukaryotic gene transcription is believed to occur in either a binary or a graded fashion. With binary induction, a transcription activator (TA regulates the probability with which a gene template is switched from the inactive to the active state without affecting the rate at which RNA molecules are produced from the template. With graded, also called rheostat-like, induction the gene template has continuously varying levels of transcriptional activity, and the TA regulates the rate of RNA production. Support for each of these two mechanisms arises primarily from experimental studies measuring reporter proteins in individual cells, rather than from direct measurement of induction events at the gene template. Methods and results In this paper, using a computational model of stochastic gene expression, we have studied the biological and experimental conditions under which a binary induction mode operating at the gene template can give rise to differentially expressed "phenotypes" (i.e., binary, hybrid or graded at the protein level. We have also investigated whether the choice of reporter genes plays a significant role in determining the observed protein expression patterns in individual cells, given the diverse properties of commonly-used reporter genes. Our simulation confirmed early findings that the lifetimes of active/inactive promoters and half-lives of downstream mRNA/protein products are important determinants of various protein expression patterns, but showed that the induction time and the sensitivity with which the expressed genes are detected are also important experimental variables. Using parameter conditions representative of reporter genes including green fluorescence protein (GFP and β-galactosidase, we also demonstrated that graded gene expression is more likely to be observed with GFP, a longer-lived protein with low detection sensitivity. Conclusion The choice of reporter genes may determine whether protein

  15. Analysis of correlations between protein complex and protein-protein interaction and mRNA expression

    Institute of Scientific and Technical Information of China (English)

    CAI Lun; XUE Hong; LU Hongchao; ZHAO Yi; ZHU Xiaopeng; BU Dongbo; LING Lunjiang; CHEN Runsheng

    2003-01-01

    Protein-protein interaction is a physical interaction of two proteins in living cells. In budding yeast Saccharomyces cerevisiae, large-scale protein-protein interaction data have been obtained through high-throughput yeast two-hybrid systems (Y2H) and protein complex purification techniques based on mass-spectrometry. Here, we collect 11855 interactions between total 2617 proteins. Through seriate genome-wide mRNA expression data, similarity between two genes could be measured. Protein complex data can also be obtained publicly and can be translated to pair relationship that any two proteins can only exist in the same complex or not. Analysis of protein complex data, protein-protein interaction data and mRNA expression data can elucidate correlations between them. The results show that proteins that have interactions or similar expression patterns have a higher possibility to be in the same protein complex than randomized selected proteins, and proteins which have interactions and similar expression patterns are even more possible to exist in the same protein complex. The work indicates that comprehensive integration and analysis of public large-scale bioinformatical data, such as protein complex data, protein-protein interaction data and mRNA expression data, may help to uncover their relationships and common biological information underlying these data. The strategies described here may help to integrate and analyze other functional genomic and proteomic data, such as gene expression profiling, protein-localization mapping and large-scale phenotypic data, both in yeast and in other organisms.

  16. Integral Membrane Protein Expression in Saccharomyces cerevisiae.

    Science.gov (United States)

    Boswell-Casteel, Rebba C; Johnson, Jennifer M; Stroud, Robert M; Hays, Franklin A

    2016-01-01

    Eukaryotic integral membrane proteins are challenging targets for crystallography or functional characterization in a purified state. Since expression is often a limiting factor when studying this difficult class of biological macromolecules, the intent of this chapter is to focus on the expression of eukaryotic integral membrane proteins (IMPs) using the model organism Saccharomyces cerevisiae. S. cerevisiae is a prime candidate for the expression of eukaryotic IMPs because it offers the convenience of using episomal expression plasmids, selection of positive transformants, posttranslational modifications, and it can properly fold and target IMPs. Here we present a generalized protocol and insights based on our collective knowledge as an aid to overcoming the challenges faced when expressing eukaryotic IMPs in S. cerevisiae.

  17. Biotechnology Protein Expression and Purification Facility

    Science.gov (United States)

    2003-01-01

    The purpose of the Project Scientist Core Facility is to provide purified proteins, both recombinant and natural, to the Biotechnology Science Team Project Scientists and the NRA-Structural Biology Test Investigators. Having a core facility for this purpose obviates the need for each scientist to develop the necessary expertise and equipment for molecular biology, protein expression, and protein purification. Because of this, they are able to focus their energies as well as their funding on the crystallization and structure determination of their target proteins.

  18. Fragile X mental retardation protein (FMRP) interacting proteins exhibit different expression patterns during development.

    Science.gov (United States)

    Bonaccorso, C M; Spatuzza, M; Di Marco, B; Gloria, A; Barrancotto, G; Cupo, A; Musumeci, S A; D'Antoni, S; Bardoni, B; Catania, M V

    2015-05-01

    Fragile X syndrome is caused by the lack of expression of fragile X mental retardation protein (FMRP), an RNA-binding protein involved in mRNA transport and translation. FMRP is a component of mRNA ribonucleoprotein complexes and it can interact with a range of proteins either directly or indirectly, as demonstrated by two-hybrid selection and co-immunoprecipitation, respectively. Most of FMRP-interacting proteins are RNA-binding proteins such as FXR1P, FXR2P and 82-FIP. Interestingly, FMRP can also interact directly with the cytoplasmic proteins CYFIP1 and CYFIP2, which do not bind RNA and link FMRP to the RhoGTPase pathway. The interaction with these different proteins may modulate the functions of FMRP by influencing its affinity to RNA and by affecting the FMRP ability of cytoskeleton remodeling through Rho/Rac GTPases. To better define the relationship of FMRP with its interacting proteins during brain development, we have analyzed the expression pattern of FMRP and its interacting proteins in the cortex, striatum, hippocampus and cerebellum at different ages in wild type (WT) mice. FMRP and FXR2P were strongly expressed during the first week and gradually decreased thereafter, more rapidly in the cerebellum than in the cortex. FXR1P was also expressed early and showed a reduction at later stages of development with a similar developmental pattern in these two regions. CYFIP1 was expressed at all ages and peaked in the third post-natal week. In contrast, CYFIP2 and 82-FIP (only in forebrain regions) were moderately expressed at P3 and gradually increased after P7. In general, the expression pattern of each protein was similar in the regions examined, except for 82-FIP, which exhibited a strong expression at P3 and low levels at later developmental stages in the cerebellum. Our data indicate that FMRP and its interacting proteins have distinct developmental patterns of expression and suggest that FMRP may be preferentially associated to certain proteins in

  19. Ultrastructure observation and proteomic analysis of differentially expressed proteins in hippocampus of early diabetic rats%糖尿病大鼠海马超微结构及蛋白质组学研究

    Institute of Scientific and Technical Information of China (English)

    金花; 徐晓云; 徐海元; 罗峥; 王悦

    2010-01-01

    Objective To investigate the alteration of ultrastructure and protein expression profile in hippocampus of early diabetic rats,and to explore the possible mechanism of brain damage caused by diabetes.Methods 15 male SD rats(6-8 week-old,200-250g)were randomly divided to control group and diabetes group.Diabetic rat model wag established by peritoneal injection of STZ 55 mg/kg.After 5 weeks of treatment,ultrastructure of hippocampus were observed by transmission electron microscope(TEM);twodimensional gel electrophoresis(2-DE)technology Was applied to isolate proteins in hippocampus.MALDITOF-MS was used to identify the differentially expressed protein.Results Pyknosis of nuclei and obvious swelling in end foot of astrocytes were noted in hippocampus of diabetes group under TEM.Eight differential proteins were identified by MALDI-TOF-MS:Cytochrome b-cl complex subunit,mitochondrial,14-3-3proteinβ/α,GFAP and Guanine nucleotide-binding protein G(0)subunit a were up-regulated and ATP synthase subunit d mitochondrial,Thioredoxin-dependent peroxide reductase mitochondrial,and ERP29 were down-regulated in diabetic rat:Endophilin-Al was only expressed in control group. Conclusions Pathological changes found in hippocampus of early diabetic rats may be related to abnormal energy metabolism caused by oxidative stress,and proteins related to aging and degenerative diseases are probably involved in the pathology.%目的 研究早期糖尿病大鼠海马超微结构及蛋白质表达谱变化,探寻糖尿病对中枢神经系统早期损害可能的机制.方法 将15只6~8周龄、体重200~250g雄性sD大鼠按随机数字表法分为对照组及糖尿病组.腹腔注射链脲佐菌素(STZ)55mg/kg制备糖尿病模型,5周后用透射电镜观察海马超微结构,采用双向凝胶电泳技术分离海马总蛋白,基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)鉴定差异蛋白质.结果 超微病理学观察结果:成模5周时糖尿病组海马神经

  20. The dynamics of polycomb group proteins in early embryonic nervous system in mouse and human.

    Science.gov (United States)

    Qi, Lu; Cao, Jing-Li; Hu, Yi; Yang, Ji-Gao; Ji, Yuan; Huang, Jing; Zhang, Yi; Sun, Da-Guang; Xia, Hong-Fei; Ma, Xu

    2013-11-01

    Polycomb group (PcG) proteins are transcription regulatory proteins that control the expression of a variety of genes and the antero-posterior neural patterning from early embryogenesis. Although expression of PcG genes in the nervous system has been noticed, but the expression pattern of PcG proteins in early embryonic nervous system is still unclear. In this study, we analyzed the expression pattern of PRC1 complex members (BMI-1 and RING1B) and PRC2 complex members (EED, SUZ12 and EZH2) in early embryonic nervous system in mouse and human by Western blot and Immunohistochemistry. The results of Western blot showed that EED protein was significantly up-regulated with the increase of the day of pregnancy during the early embryogenesis in mouse. BMI-1 protein level was significantly increased from the day 10 of pregnancy, when compared with the day 9 of pregnancy. But the SUZ12, EZH2 and RING1B protein level did not change significantly. From the results of Immunohistochemistry, we found that the four PcG proteins were all expressed in the fetal brain and fetal spinal cord in mouse. In human, the expression of EED, SUZ12, and EZH2 was not significantly different in cerebral cortex and sacral spinal cord, but BMI-1 and RING1B expression was enhanced with the development of embryos in early pregnancy. Collectively, our findings showed that PRC1 and PRC2 were spatiotemporally expressed in brain and spinal cord of early embryos.

  1. Designing genes for successful protein expression.

    Science.gov (United States)

    Welch, Mark; Villalobos, Alan; Gustafsson, Claes; Minshull, Jeremy

    2011-01-01

    DNA sequences are now far more readily available in silico than as physical DNA. De novo gene synthesis is an increasingly cost-effective method for building genetic constructs, and effectively removes the constraint of basing constructs on extant sequences. This allows scientists and engineers to experimentally test their hypotheses relating sequence to function. Molecular biologists, and now synthetic biologists, are characterizing and cataloging genetic elements with specific functions, aiming to combine them to perform complex functions. However, the most common purpose of synthetic genes is for the expression of an encoded protein. The huge number of different proteins makes it impossible to characterize and catalog each functional gene. Instead, it is necessary to abstract design principles from experimental data: data that can be generated by making predictions followed by synthesizing sequences to test those predictions. Because of the degeneracy of the genetic code, design of gene sequences to encode proteins is a high-dimensional problem, so there is no single simple formula to guarantee success. Nevertheless, there are several straightforward steps that can be taken to greatly increase the probability that a designed sequence will result in expression of the encoded protein. In this chapter, we discuss gene sequence parameters that are important for protein expression. We also describe algorithms for optimizing these parameters, and troubleshooting procedures that can be helpful when initial attempts fail. Finally, we show how many of these methods can be accomplished using the synthetic biology software tool Gene Designer.

  2. Salicylic acid enhances Staphylococcus aureus extracellular adhesin protein expression.

    Science.gov (United States)

    Alvarez, Lucía P; Barbagelata, María S; Cheung, Ambrose L; Sordelli, Daniel O; Buzzola, Fernanda R

    2011-11-01

    One of the virulence factors required by Staphylococcus aureus at the early stages of infection is Eap, a secreted adhesin that binds many host proteins and is upregulated by the two-component regulatory system saeRS. The S. aureus Newman strain harbors a mutation in saeS that is thought to be responsible for the high level of Eap expression in this strain. This study was designed to ascertain whether salicylic acid (SAL) affects the expression of Eap and the internalization of S. aureus into epithelial cells. The strain Newman treated with SAL exhibited increased levels of eap transcription and protein expression. Furthermore, SAL treatment increased the eap promoter activity. SAL treatment enhanced Eap expression in the Newman and in other S. aureus strains that do not carry the mutation in saeS. Internalization of S. aureus eap and sae mutants into the MAC-T epithelial cells was significantly decreased compared with the wild-type counterparts. In conclusion, we demonstrated that a low concentration of SAL increased S. aureus Eap expression possibly due to enhancement of sae. SAL may create the conditions for S. aureus persistence in the host, not only by decreasing the capsular polysaccharide expression as shown before, but also by enhancing Eap expression.

  3. Streamlined expressed protein ligation using split inteins.

    Science.gov (United States)

    Vila-Perelló, Miquel; Liu, Zhihua; Shah, Neel H; Willis, John A; Idoyaga, Juliana; Muir, Tom W

    2013-01-09

    Chemically modified proteins are invaluable tools for studying the molecular details of biological processes, and they also hold great potential as new therapeutic agents. Several methods have been developed for the site-specific modification of proteins, one of the most widely used being expressed protein ligation (EPL) in which a recombinant α-thioester is ligated to an N-terminal Cys-containing peptide. Despite the widespread use of EPL, the generation and isolation of the required recombinant protein α-thioesters remain challenging. We describe here a new method for the preparation and purification of recombinant protein α-thioesters using engineered versions of naturally split DnaE inteins. This family of autoprocessing enzymes is closely related to the inteins currently used for protein α-thioester generation, but they feature faster kinetics and are split into two inactive polypeptides that need to associate to become active. Taking advantage of the strong affinity between the two split intein fragments, we devised a streamlined procedure for the purification and generation of protein α-thioesters from cell lysates and applied this strategy for the semisynthesis of a variety of proteins including an acetylated histone and a site-specifically modified monoclonal antibody.

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

    Science.gov (United States)

    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 in two different early arthritis cohorts and 7 non-inflammatory controls was analysed using immunofluorescence to detect stromal markers CD55, CD248, fibroblast activation protein (FAP) and podoplanin. Diagnostic classification (gout, psoriatic arthritis, unclassified arthritis (UA), parvovirus associated arthritis, reactive arthritis and RA), disease outcome (resolving vs persistent) and clinical variables were determined at baseline and after follow-up, and related to the expression of stromal markers. We observed expression of all stromal markers in ST of early arthritis patients, independent of diagnosis or prognostic outcome. Synovial expression of FAP was significantly higher in patients developing early RA compared to other diagnostic groups and non-inflammatory controls. In RA FAP protein was expressed in both lining and sublining layers. Podoplanin expression was higher in all early inflammatory arthritis patients than controls, but did not differentiate diagnostic outcomes. Stromal marker expression was not associated with prognostic outcomes of disease persistence or resolution. There was no association with clinical or sonographic variables. Stromal cell markers CD55, CD248, FAP and podoplanin are expressed in ST in the earliest stage of arthritis. Baseline expression of FAP is higher in early synovitis patients who fulfil classification criteria for RA over time. These results suggest that significant fibroblast activation occurs in RA in the early window of disease.

  5. Engineering genes for predictable protein expression.

    Science.gov (United States)

    Gustafsson, Claes; Minshull, Jeremy; Govindarajan, Sridhar; Ness, Jon; Villalobos, Alan; Welch, Mark

    2012-05-01

    The DNA sequence used to encode a polypeptide can have dramatic effects on its expression. Lack of readily available tools has until recently inhibited meaningful experimental investigation of this phenomenon. Advances in synthetic biology and the application of modern engineering approaches now provide the tools for systematic analysis of the sequence variables affecting heterologous expression of recombinant proteins. We here discuss how these new tools are being applied and how they circumvent the constraints of previous approaches, highlighting some of the surprising and promising results emerging from the developing field of gene engineering.

  6. Expression and significance of P53 protein and MDM-2 protein in human gliomas

    Institute of Scientific and Technical Information of China (English)

    WANG An-liu; LIU Zhao-xia; LI Guang; ZHANG Li-wei

    2011-01-01

    Background P53 is one of the most studied tumor suppressors in the cancer research, and over 50% of human tumors carry P53 mutations. MDM-2 is amplified and/or overexpressed in a variety of human tumors of diverse tissue origin. The aim of this study was to examine the expression of P53 protein and MDM-2 protein in gliomas, and to investigate the relationship between the expression of the two proteins and the histopathological grades of glioma. The relationship between MDM-2 protein expression and P53 protein expression was also analyzed.Methods The expression of P53 protein and MDM-2 protein was immunohistochemically detected using monoclonal antibodies in 242 paraffin embedded tissues, including 30 normal brain tissues from patients with craniocerebral injury and 212 tissues from patients with primary glioma (grade Ⅰ-Ⅱ group: 5 cases of grade Ⅰ, 119 cases of grade Ⅱ; and grade Ⅲ-Ⅳ group: 53 cases of grade Ⅲ, and 35 cases of grade Ⅳ).Results The P53 positive rate was significantly higher in the glioma groups than in the control group (P <0.0001). The P53 positive rate was significantly higher in glioma tissues of grade Ⅲ-V than in glioma tissues of grade Ⅰ-Ⅱ group (P=0.001). The MDM-2 positive rate was significantly higher in glioma groups than in the control group (P <0.0001).There was no significant difference in the MDM-2 positive rate between the two glioma groups (P=0.936). The expression of P53 protein was not related to expression of MDM-2 protein (P=0.069)Conclusions Overexpression of P53 protein might be related to the occurrence and progression of glioma.Overexpression of MDM-2 protein may play an important role in glioma tumorigenesis, but may not be involved in glioma progression. The overexpression of MDM-2 protein was an early event in malignant transformation of glioma. MDM-2 may be a key player in glioma in its own right.

  7. Transgenic zebrafish recapitulating tbx16 gene early developmental expression.

    Directory of Open Access Journals (Sweden)

    Simon Wells

    Full Text Available We describe the creation of a transgenic zebrafish expressing GFP driven by a 7.5 kb promoter region of the tbx16 gene. This promoter segment is sufficient to recapitulate early embryonic expression of endogenous tbx16 in the presomitic mesoderm, the polster and, subsequently, in the hatching gland. Expression of GFP in the transgenic lines later in development diverges to some extent from endogenous tbx16 expression with the serendipitous result that one line expresses GFP specifically in commissural primary ascending (CoPA interneurons of the developing spinal cord. Using this line we demonstrate that the gene mafba (valentino is expressed in CoPA interneurons.

  8. Expression Differentiation Is Constrained to Low-Expression Proteins over Ecological Timescales.

    Science.gov (United States)

    Margres, Mark J; Wray, Kenneth P; Seavy, Margaret; McGivern, James J; Herrera, Nathanael D; Rokyta, Darin R

    2016-01-01

    Protein expression level is one of the strongest predictors of protein sequence evolutionary rate, with high-expression protein sequences evolving at slower rates than low-expression protein sequences largely because of constraints on protein folding and function. Expression evolutionary rates also have been shown to be negatively correlated with expression level across human and mouse orthologs over relatively long divergence times (i.e., ∼100 million years). Long-term evolutionary patterns, however, often cannot be extrapolated to microevolutionary processes (and vice versa), and whether this relationship holds for traits evolving under directional selection within a single species over ecological timescales (i.e., protein is predicted to be a tradeoff between the benefit of its function and the costs of its expression. Selection should drive the expression level of all proteins close to values that maximize fitness, particularly for high-expression proteins because of the increased energetic cost of production. Therefore, stabilizing selection may reduce the amount of standing expression variation for high-expression proteins, and in combination with physiological constraints that may place an upper bound on the range of beneficial expression variation, these constraints could severely limit the availability of beneficial expression variants. To determine whether rapid-expression evolution was restricted to low-expression proteins owing to these constraints on highly expressed proteins over ecological timescales, we compared venom protein expression levels across mainland and island populations for three species of pit vipers. We detected significant differentiation in protein expression levels in two of the three species and found that rapid-expression differentiation was restricted to low-expression proteins. Our results suggest that various constraints on high-expression proteins reduce the availability of beneficial expression variants relative to low-expression

  9. BMP-7 PROTEIN EXPRESSION IS DOWNREGULATED IN HUMAN DIABETIC NEPHROPATHY.

    Science.gov (United States)

    Ivanac-Janković, Renata; Ćorić, Marijana; Furić-Čunko, Vesna; Lovičić, Vesna; Bašić-Jukić, Nikolina; Kes, Petar

    2015-06-01

    Bone morphogenetic protein-7 (BMP-7) is expressed in all parts of the normal kidney parenchyma, being highest in the epithelium of proximal tubules. It protects kidney against acute and chronic injury, inflammation and fibrosis. Diabetic nephropathy is the leading cause of chronic kidney disease, and is characterized by decreased expression of BMP-7. The aim of our study was to analyze whether the expression of BMP-7 is significantly changed in advanced stages of human diabetic nephropathy. Immunohistochemical analysis of the expression of BMP-7 was performed on archival material of 30 patients that underwent renal biopsy and had confirmed diagnosis of diabetic nephropathy. Results showed that BMP-7 was differently expressed in the cytoplasm of epithelial cells of proximal tubules and podocytes among all stages of diabetic nephropathy. At early stages of diabetic nephropathy, BMP-7 was strongly positive in proximal tubules and podocytes, while low expression was recorded in the majority of samples at advanced stages. In conclusion, increased expression of BMP-7 at initial stages of diabetic nephropathy with subsequent decrease at advanced stage highlights the role of BMP-7 in the protection of kidney structure and function. Further investigations should be focused on disturbances of BMP-7 receptors and signaling pathways in patients with diabetic nephropathy.

  10. 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.

  11. Differential regulation of dentin matrix protein 1 expression during odontogenesis.

    Science.gov (United States)

    Lu, Yongbo; Zhang, Shubin; Xie, Yixia; Pi, Yuli; Feng, Jian Q

    2005-01-01

    Dentin matrix protein 1 (DMP1) is highly expressed in mineralized tooth and bone. Both in vitro and in vivo data show that DMP1 is critical for mineralization and tooth morphogenesis (growth and development). In this study, we studied Dmp1 gene regulation. The in vitro transient transfection assay identified two important DNA fragments, the 2.4- and 9.6-kb promoter regions. We next generated and analyzed transgenic mice bearing the beta-galactosidase (lacZ) reporter gene driven by the 2.4- or 9.6-kb promoter with the complete 4-kb intron 1. The 9.6-kb Dmp1-lacZ mice conferred a DMP1 expression pattern in odontoblasts identical to that in the endogenous Dmp1 gene. This is reflected by lacZ expression in Dmp1-lacZ knock-in mice during all stages of odontogenesis. In contrast, the 2.4-kb Dmp1-lacZ mice display activity in odontoblast cells only at the early stage of odontogenesis. Thus, we propose that different transcription factors regulate early or later cis-regulatory domains of the Dmp1 promoter, which gives rise to the unique spatial and temporal expression pattern of Dmp1 gene at different stages of tooth development. 2005 S. Karger AG, Basel

  12. Dietary protein intake and quality in early life

    DEFF Research Database (Denmark)

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

    2017-01-01

    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......: Recent observational and randomized controlled trials confirmed that high-protein intake in early life seems to increase early weight gain and the risk of later overweight and obesity. Recent studies have looked at the effect of different sources of protein, and especially high-animal protein intake...... 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...

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

    Directory of Open Access Journals (Sweden)

    Alberto Miranda

    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.

  14. Expression of Contractile Protein Isoforms in Microgravity

    Science.gov (United States)

    Anderson, Page A. W.

    1996-01-01

    The general objective of this experiment is to determine the effect of space flight parameters, including microgravity, on ontogenesis and embryogenesis of Japanese quail. Nine U.S. and two Russian investigators are cooperating in this study. Specific objectives of the participating scientists include assessing the gross and microscopic morphological and histological development of the embryo, as well as the temporal and spacial development of specific cells, tissues, and organs. Temporally regulated production of specific proteins is also being investigated. Our objective is to determine the effects of microgravity on developmentally programmed expression of Troponin T and I isoforms known to regulate cardiac and skeletal muscle contraction.

  15. Soluble recombinant proteins of HCCR1 and HCCR2 expression and purification in bacteria and its application for novel early diagnosis of hepatocellular carcinomas%HCCR1和HCCR2可溶性蛋白在细菌中表达、纯化及在早期肝癌诊断中的应用

    Institute of Scientific and Technical Information of China (English)

    耿东进; 陈军浩; 顾香芳; 陈蕾蕾; 张乐; 刘勇; 韩鹂

    2006-01-01

    目的评估HCCR1和HCRR2克隆、细菌表达及可溶性蛋白的纯化及应用于早期肝癌诊断的可行性.方法cDNA克隆、表达、蛋白质的纯化、Western blot分析用于评估HCCR1和HCCR2蛋白的生产,Elisa方法用检测血清抗原.结果HCCR1和HCCR2能够高水平地在细菌中表达,纯化的可溶性蛋白能用于生产抗体并应用于肝癌的早期诊断.结论HCCR1和HCCR2蛋白质可以在细菌中生产,并能成功地用于肝癌的早期诊断.%[Objective] HCCR1 and HCCR2 are the oncogenes overexpressed in hepatocellular carcinomas(HCC). In this experiment, it was evaluated that the cloning, bacterial expression, purification and characterization of soluble recombinant proteins of HCCR1 and HCCR2 for novel early diagnosis of HCC. Protein solublity was key to producing recombinant proteins for founction determination. [Method] Five methods, cDNA clone and expression,protein purification, solubility assay and western blod assay were used to evaluate production of HCCR1 and HCCR2 proteins, enzyme-linked immunosorbent assay (ELISA) was used to detect HCCR1 and HCCR2 antigens in serum.[Result] The results show that the proteins of HCCR1 and HCCR2 were expressed in Escherichia coli at high levels. [Conclusion] The recombinant proteins are soluble and may used to generate the antibody for early diagnosis of HCC. Finally, HCCR1 and HCCR2 may be useful in monitoring HCC, and as potential disease specific therapeutic targets.

  16. Ezrin is highly expressed in early thymocytes, but dispensable for T cell development in mice.

    Directory of Open Access Journals (Sweden)

    Meredith H Shaffer

    Full Text Available BACKGROUND: Ezrin/radixin/moesin (ERM proteins are highly homologous proteins that function to link cargo molecules to the actin cytoskeleton. Ezrin and moesin are both expressed in mature lymphocytes, where they play overlapping roles in cell signaling and polarity, but their role in lymphoid development has not been explored. METHODOLOGY/PRINCIPAL FINDINGS: We characterized ERM protein expression in lymphoid tissues and analyzed the requirement for ezrin expression in lymphoid development. In wildtype mice, we found that most cells in the spleen and thymus express both ezrin and moesin, but little radixin. ERM protein expression in the thymus was differentially regulated, such that ezrin expression was highest in immature thymocytes and diminished during T cell development. In contrast, moesin expression was low in early thymocytes and upregulated during T cell development. Mice bearing a germline deletion of ezrin exhibited profound defects in the size and cellularity of the spleen and thymus, abnormal thymic architecture, diminished hematopoiesis, and increased proportions of granulocytic precursors. Further analysis using fetal liver chimeras and thymic transplants showed that ezrin expression is dispensable in hematopoietic and stromal lineages, and that most of the defects in lymphoid development in ezrin(-/- mice likely arise as a consequence of nutritional stress. CONCLUSIONS/SIGNIFICANCE: We conclude that despite high expression in lymphoid precursor cells, ezrin is dispensable for lymphoid development, most likely due to redundancy with moesin.

  17. Heart Failure Protein May Signal Early Brain Damage

    Science.gov (United States)

    ... page: https://medlineplus.gov/news/fullstory_162447.html Heart Failure Protein May Signal Early Brain Damage Higher levels ... stress. Blood levels of NT-proBNP rise when heart failure worsens and fall when it gets better. Previous ...

  18. Transcriptional Factor PU.1 Regulates Decidual C1q Expression in Early Pregnancy in Human.

    Science.gov (United States)

    Madhukaran, Shanmuga Priyaa; Kishore, Uday; Jamil, Kaiser; Teo, Boon Heng Dennis; Choolani, Mahesh; Lu, Jinhua

    2015-01-01

    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 (DCs). 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 DCs 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.

  19. Microfluidic chips for protein differential expression profiling.

    Science.gov (United States)

    Armenta, Jenny M; Dawoud, Abdulilah A; Lazar, Iulia M

    2009-04-01

    Biomarker discovery and screening using novel proteomic technologies is an area that is attracting increased attention in the biomedical community. Early detection of abnormal physiological conditions will be highly beneficial for diagnosing various diseases and increasing survivability rates. Clearly, progress in this area will depend on the development of fast, reliable, and highly sensitive and specific sample bioanalysis methods. Microfluidics has emerged as a technology that could become essential in proteomics research as it enables the integration of all sample preparation, separation, and detection steps, with the added benefit of enhanced sample throughput. The combination of these advantages with the sensitivity and capability of MS detection to deliver precise structural information makes microfluidics-MS a very competitive technology for biomarker discovery. The integration of LC microchip devices with MS detection, and specifically their applicability to biomarker screening applications in MCF-7 breast cancer cellular extracts is reported in this manuscript. Loading approximately 0.1-1 microg of crude protein extract tryptic digest on the chip has typically resulted in the reliable identification of approximately 40-100 proteins. The potential of an LC-ESI-MS chip for comparative proteomic analysis of isotopically labeled MCF-7 breast cancer cell extracts is explored for the first time.

  20. Engineering Escherichia coli for Functional Expression of Membrane Proteins

    NARCIS (Netherlands)

    Ho, Franz Y; Poolman, Bert

    2015-01-01

    A major bottleneck in the characterization of membrane proteins is low yield of functional protein in recombinant expression. Microorganisms are widely used for recombinant protein production, because of ease of cultivation and high protein yield. However, the target proteins do not always obtain th

  1. Motor memory in childhood: early expression of consolidation phase gains.

    Science.gov (United States)

    Ashtamker, Lilach; Karni, Avi

    2013-11-01

    Are children faster than adults in consolidating procedural knowledge? In adults, the expression of the full benefits of motor practice requires a few hours of consolidation and sleep. Here we show that, although the processes generating the delayed gains continued beyond the first few hours post-training, children expressed significant gains as early as 1 h post-training, in the awake state. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. 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

    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......-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...... sites of F55 are included within a tandem-repeated sequence overlapping the transcription start sites and/or the B recognition element of the pertinent genes. The strongest binding was observed with the promoters of T5 and T6, and an apparent cooperativity in binding was observed with the Tind promoter...

  3. Inactivation of indispensable bacterial proteins by early proteins of bacteriophages: implication in antibacterial drug discovery.

    Science.gov (United States)

    Sau, S; Chattoraj, P; Ganguly, T; Chanda, P K; Mandal, N C

    2008-06-01

    Bacteriophages utilize host bacterial cellular machineries for their own reproduction and completion of life cycles. The early proteins that phage synthesize immediately after the entry of their genomes into bacterial cells participate in inhibiting host macromolecular biosynthesis, initiating phage-specific replication and synthesizing late proteins. Inhibition of synthesis of host macromolecules that eventually leads to cell death is generally performed by the physical and/or chemical modification of indispensable host proteins by early proteins. Interestingly, most modified bacterial proteins were shown to take part actively in phage-specific transcription and replication. Research on phages in last nine decades has demonstrated such lethal early proteins that interact with or chemically modify indispensable host proteins. Among the host proteins inhibited by lethal phage proteins, several are not inhibited by any chemical inhibitor available today. Under the context of widespread dissemination of antibiotic-resistant strains of pathogenic bacteria in recent years, the information of lethal phage proteins and cognate host proteins could be extremely invaluable as they may lead to the identification of novel antibacterial compounds. In this review, we summarize the current knowledge about some early phage proteins, their cognate host proteins and their mechanism of action and also describe how the above interacting proteins had been exploited in antibacterial drug discovery.

  4. Expression of the homeobox genes OTX2 and OTX1 in the early developing human brain

    DEFF Research Database (Denmark)

    Larsen, Karen B; Lutterodt, Melissa C; Møllgård, Kjeld

    2010-01-01

    protein was found in the subcommissural organ, pineal gland, and cerebellum. The early expression of OTX2 and OTX1 in proliferative cell layers of the human fetal brain supports the concept that these homeobox genes are important in neuronal cell development and differentiation: OTX1 primarily...... of young neurons of the deeper cortical layers. We have studied the spatial and temporal expression of the two homeobox genes OTX2 and OTX1 in human fetal brains from 7 to 14 weeks postconception by in situ hybridization and immunohistochemistry. OTX2 was expressed in the diencephalon, mesencephalon...... in the neocortex, and OTX2 in the archicortex, diencephalon, rostral brain stem, and cerebellum....

  5. 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

  6. Seedling Lethal1, a Pentatricopeptide Repeat Protein Lacking an E/E+ or DYW Domain in Arabidopsis, Is Involved in Plastid Gene Expression and Early Chloroplast Development1[C][W

    Science.gov (United States)

    Pyo, Young Jae; Kwon, Kwang-Chul; Kim, Anna; Cho, Myeon Haeng

    2013-01-01

    Chloroplasts are the site of photosynthesis and the biosynthesis of essential metabolites, including amino acids, fatty acids, and secondary metabolites. It is known that many seedling-lethal mutants are impaired in chloroplast function or development, indicating the development of functional chloroplast is essential for plant growth and development. Here, we isolated a novel transfer DNA insertion mutant, dubbed sel1 (for seedling lethal1), that exhibited a pigment-defective and seedling-lethal phenotype with a disrupted pentatricopeptide repeat (PPR) gene. Sequence analysis revealed that SEL1 is a member of the PLS subgroup, which is lacking known E/E+ or DYW domains at the C terminus, in the PLS subfamily of the PPR protein family containing a putative N-terminal transit peptide and 14 putative PPR or PPR-like motifs. Confocal microscopic analysis showed that the SEL1-green fluorescent protein fusion protein is localized in chloroplasts. Transmission electron microscopic analysis revealed that the sel1 mutant is impaired in the etioplast, as well as in chloroplast development. In sel1 mutants, plastid-encoded proteins involved in photosynthesis were rarely detected due to the lack of the corresponding transcripts. Furthermore, transcript profiles of plastid genes revealed that, in sel1 mutants, the transcript levels of plastid-encoded RNA polymerase-dependent genes were greatly reduced, but those of nuclear-encoded RNA polymerase-dependent genes were increased or not changed. Additionally, the RNA editing of two editing sites of the acetyl-CoA carboxylase beta subunit gene transcripts in the sel1 mutant was compromised, though it is not directly connected with the sel1 mutant phenotype. Our results demonstrate that SEL1 is involved in the regulation of plastid gene expression required for normal chloroplast development. PMID:24144791

  7. Effects of Losartan on expression of connexins at the early stage of atherosclerosis in rabbits

    Directory of Open Access Journals (Sweden)

    Li-ming Ruan, Wei Cai, Jun-zhu Chen, Jin-feng Duan

    2010-01-01

    Full Text Available Aim: to investigate effects of Losartan on expression of connexin 40 and 43 (Cx40 and Cx43, in arteries at the early stage of atherosclerosis in a rabbit model. Methods: A total of 28 male New Zealand white rabbits were divided into following groups: control group, high fat diet group, and Losartan group (10 mg/kg/day. Losartan was administrated in food for two weeks. Iliac arteries were obtained for immunohistochemistry, transmission electron microscopy, Western blot, and reverse transcriptase-polymerase chain reaction (RT-PCR. Results: Transmission electron microscopy revealed abundant gap junctions between neointimal smooth muscle cells (SMCs, which were markedly reduced by treatment. RT-PCR and Western blot assay showed that the mRNA and protein expression of Cx40 and Cx43 were elevated in the neointimal area at the early stage of atherosclerosis. The mRNA and protein expression of Cx43 were significantly down-regulated by losartan treatment but those of Cx40 were not markedly changed. Conclusion: Cx40 and Cx43 in the neointimal SMCs were up-regulated at the early stage of atherosclerosis. Losartan (an angiotensin-converting enzyme inhibitor could reduce neointima proliferation and down-regulate the elevated protein expression of Cx43, suggesting the rennin-angiotensin system (RAS plays an important role in the remodeling of gap junction between ventricular myocytes under pathological conditions.

  8. Changes in ovarian protein expression during primordial follicle formation in the hamster.

    Science.gov (United States)

    Mukherjee, Anindit; Reisdorph, Nichole; Guda, Chttibabu; Pandey, Sanjit; Roy, Shyamal K

    2012-01-02

    Although many proteins have been shown to affect the transition of primordial follicles to the primary stage, factors regulating the formation of primordial follicles remains sketchy at best. Differentiation of somatic cells into early granulosa cells during ovarian morphogenesis is the hallmark of primordial follicle formation; hence, critical changes are expected in protein expression. We wanted to identify proteins, the expression of which would correlate with the formation of primordial follicles as a first step to determine their biological function in folliculogenesis. Proteins were extracted from embryonic (E15) and 8-day-old (P8) hamster ovaries and fractionated by two-dimensional gel electrophoresis. Gels were stained with Proteosilver, and images of protein profiles corresponding to E15 and P8 ovaries were overlayed to identify protein spots showing altered expression. Some of the protein spots were extracted from SyproRuby-stained preparative gels, digested with trypsin, and analyzed by mass spectrometry. Both E15 and P8 ovaries had high molecular weight proteins at acidic, basic, and neutral ranges; however, we focused on small molecular weight proteins at 4-7 pH range. Many of those spots might represent post-translational modification. Mass spectrometric analysis revealed the identity of these proteins. The formation of primordial follicles on P8 correlated with many differentially and newly expressed proteins. Whereas Ebp1 expression was downregulated in ovarian somatic cells, Sfrs3 expression was specifically upregulated in newly formed granulosa cells of primordial follicles on P8. The results show for the first time that the morphogenesis of primordial follicles in the hamster coincides with altered and novel expression of proteins involved in cell proliferation, transcriptional regulation, and metabolism. Therefore, formation of primordial follicles is an active process requiring differentiation of somatic cells into early granulosa cells and

  9. Widely predicting specific protein functions based on protein-protein interaction data and gene expression profile

    Institute of Scientific and Technical Information of China (English)

    GAO Lei; LI Xia; GUO Zheng; ZHU MingZhu; LI YanHui; RAO ShaoQi

    2007-01-01

    GESTs (gene expression similarity and taxonomy similarity), a gene functional prediction approach previously proposed by us, is based on gene expression similarity and concept similarity of functional classes defined in Gene Ontology (GO). In this paper, we extend this method to protein-protein interaction data by introducing several methods to filter the neighbors in protein interaction networks for a protein of unknown function(s). Unlike other conventional methods, the proposed approach automatically selects the most appropriate functional classes as specific as possible during the learning process, and calls on genes annotated to nearby classes to support the predictions to some small-sized specific classes in GO. Based on the yeast protein-protein interaction information from MIPS and a dataset of gene expression profiles, we assess the performances of our approach for predicting protein functions to "biology process" by three measures particularly designed for functional classes organized in GO. Results show that our method is powerful for widely predicting gene functions with very specific functional terms. Based on the GO database published in December 2004, we predict some proteins whose functions were unknown at that time, and some of the predictions have been confirmed by the new SGD annotation data published in April, 2006.

  10. Widely predicting specific protein functions based on protein-protein interaction data and gene expression profile

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    GESTs (gene expression similarity and taxonomy similarity), a gene functional prediction approach previously proposed by us, is based on gene expression similarity and concept similarity of functional classes defined in Gene Ontology (GO). In this paper, we extend this method to protein-protein interac-tion data by introducing several methods to filter the neighbors in protein interaction networks for a protein of unknown function(s). Unlike other conventional methods, the proposed approach automati-cally selects the most appropriate functional classes as specific as possible during the learning proc-ess, and calls on genes annotated to nearby classes to support the predictions to some small-sized specific classes in GO. Based on the yeast protein-protein interaction information from MIPS and a dataset of gene expression profiles, we assess the performances of our approach for predicting protein functions to “biology process” by three measures particularly designed for functional classes organ-ized in GO. Results show that our method is powerful for widely predicting gene functions with very specific functional terms. Based on the GO database published in December 2004, we predict some proteins whose functions were unknown at that time, and some of the predictions have been confirmed by the new SGD annotation data published in April, 2006.

  11. Silencing myotubularin related protein 7 enhances proliferation and early differentiation of C2C12 myoblast.

    Science.gov (United States)

    Yuan, Zhuning; Chen, Yaosheng; Zhang, Xumeng; Zhou, Xingyu; Li, Mingsen; Chen, Hu; Wu, Ming; Zhang, Ying; Mo, Delin

    2017-03-11

    Myotubularin related protein 7 (MTMR7) is a key member of the highly conserved myotubularin related proteins (MTMRs) family, which has phosphatase activity. MTMR7 was increased during myoblast differentiation and exhibited high expression level at primary fibers formation stages in pigs. This suggests that MTMR7 may be involved in myogenesis. In our study, we investigated the roles of MTMR7 on proliferation and differentiation of C2C12 myoblasts. Knocking down MTMR7 not only enhanced myoblast early differentiation via altering the expression of Myf5, but also promoted myoblast proliferation through increasing cyclinA2 expression. The improved proliferation capacity was related to the increased phosphorylation of AKT. Taken together, our research demonstrates that MTMR7 plays an important role in proliferation and early differentiation of C2C12 myoblast.

  12. Robust expression of a bioactive mammalian protein in Chlamydomonas chloroplast

    Energy Technology Data Exchange (ETDEWEB)

    Mayfield, Stephen P

    2015-01-13

    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 of proteins/peptides, especially gut active proteins, without purification is disclosed.

  13. Strain engineering for improved expression of recombinant proteins in bacteria.

    Science.gov (United States)

    Makino, Tomohiro; Skretas, Georgios; Georgiou, George

    2011-05-14

    Protein expression in Escherichia coli represents the most facile approach for the preparation of non-glycosylated proteins for analytical and preparative purposes. So far, the optimization of recombinant expression has largely remained a matter of trial and error and has relied upon varying parameters, such as expression vector, media composition, growth temperature and chaperone co-expression. Recently several new approaches for the genome-scale engineering of E. coli to enhance recombinant protein expression have been developed. These methodologies now enable the generation of optimized E. coli expression strains in a manner analogous to metabolic engineering for the synthesis of low-molecular-weight compounds. In this review, we provide an overview of strain engineering approaches useful for enhancing the expression of hard-to-produce proteins, including heterologous membrane proteins.

  14. 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.

  15. Early postnatal testosterone predicts sex-related differences in early expressive vocabulary.

    Science.gov (United States)

    Kung, Karson T F; Browne, Wendy V; Constantinescu, Mihaela; Noorderhaven, Rebecca M; Hines, Melissa

    2016-06-01

    During the first few years of life, girls typically have a larger expressive vocabulary than boys. This sex difference is important since a small vocabulary may predict subsequent language difficulties, which are more prevalent in boys than girls. The masculinizing effects of early androgen exposure on neurobehavioral development are well-documented in nonhuman mammals. The present study conducted the first test of whether early postnatal testosterone concentrations influence sex differences in expressive vocabulary in toddlers. It was found that testosterone measured in saliva samples collected at 1-3 months of age, i.e., during the period called mini-puberty, negatively predicted parent-report expressive vocabulary size at 18-30 months of age in boys and in girls. Testosterone concentrations during mini-puberty also accounted for additional variance in expressive vocabulary after other predictors such as sex, child's age at vocabulary assessment, and paternal education, were taken into account. Furthermore, testosterone concentrations during mini-puberty mediated the sex difference in expressive vocabulary. These results suggest that testosterone during the early postnatal period contributes to early language development and neurobehavioral sexual differentiation in humans.

  16. Strain engineering for improved expression of recombinant proteins in bacteria

    OpenAIRE

    Skretas Georgios; Makino Tomohiro; Georgiou George

    2011-01-01

    Abstract Protein expression in Escherichia coli represents the most facile approach for the preparation of non-glycosylated proteins for analytical and preparative purposes. So far, the optimization of recombinant expression has largely remained a matter of trial and error and has relied upon varying parameters, such as expression vector, media composition, growth temperature and chaperone co-expression. Recently several new approaches for the genome-scale engineering of E. coli to enhance re...

  17. Calreticulin: Roles in Cell-Surface Protein Expression

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    Yue Jiang

    2014-09-01

    Full Text Available In order to perform their designated functions, proteins require precise subcellular localizations. For cell-surface proteins, such as receptors and channels, they are able to transduce signals only when properly targeted to the cell membrane. Calreticulin is a multi-functional chaperone protein involved in protein folding, maturation, and trafficking. However, evidence has been accumulating that calreticulin can also negatively regulate the surface expression of certain receptors and channels. In these instances, depletion of calreticulin enhances cell-surface expression and function. In this review, we discuss the role of calreticulin with a focus on its negative effects on the expression of cell-surface proteins.

  18. The small GTP-binding protein rab4 is associated with early endosomes

    Energy Technology Data Exchange (ETDEWEB)

    van der Sluijs, P.; Hull, M.; Mellman, I. (Yale Univ. School of Medicine, New Haven, CT (United States)); Zahraoui, A.; Tavitian, A. (INSERM U 248, Paris (France)); Goud, B. (Unite de Genetique Somatique, Paris (France))

    1991-07-15

    Small GTP-binding proteins of the rab family have been implicated as playing important roles in controlling membrane traffic on the biosynthetic and endocytic pathways. The authors demonstrate that a distinct rab protein, rab4p, is associated with the population of early endosomes involved in transferrin-receptor recycling. An antibody to human rab4p was found to detect a doublet of {approx} 24-kDa proteins on immunoblots from various cell types. Seventy-five percent of these proteins were tightly membrane bound and could be released only by detergent treatment. Upon isolation of early endosomes, late endosomes, and lysosomes, by free-flow electrophoresis and Percoll density-gradient centrifugation, most (70%) of the rab4p was found to cofractionate with early endosomes and endocytic vesicles containing {sup 125}-labeled transferrin. The rab proteins previously localized to the endoplasmic reticulum and/or Golgi apparatus were not found in these fractions. They also localized rab4p to tansferrin-receptor-containing early endosomes by immunofluorescence after expression of rab4p cDNA. The association of rab4p with early endosomes and other vesicles involved in the intracellular tansport of transferrin receptor suggests that rab4p may play a role in regulating the pathway of receptor recycling.

  19. Expression and structural analysis of membrane proteins

    OpenAIRE

    Eifler, Nora

    2006-01-01

    1.1 Membrane Proteins Between one quarter and one third of all genes in eukaryotic and prokaryotic organisms code for integral membrane proteins (IMPs) (Essen, 2002). These proteins are essential parts of biological membranes and confer various functions, such as energy conversion, transport, biosynthesis of lipids, signal transduction, or cell recognition. The enormous economical potential of membrane proteins is highlighted by the family of G-protein-coupled receptors (GPC...

  20. Genes and Proteins Differentially Expressed during In Vitro Malignant Transformation of Bovine Pancreatic Duct Cells

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    R. Jesnowski

    2007-02-01

    Full Text Available Pancreatic carcinoma has an extremely bad prognosis due to lack of early diagnostic markers and lack of effective therapeutic strategies. Recently, we have established an in vitro model recapitulating the first steps in the carcinogenesis of the pancreas. SV40 large T antigen-immortalized bovine pancreatic duct cells formed intrapancreatic adenocarcinoma tumors on k-rasmut transfection after orthotopic injection in the nude mouse pancreas. Here we identified genes and proteins differentially expressed in the course of malignant transformation using reciprocal suppression subtractive hybridization and 2D gel electrophoresis and mass spectrometry, respectively. We identified 34 differentially expressed genes, expressed sequence tags, and 15 unique proteins. Differential expression was verified for some of the genes or proteins in samples from pancreatic carcinoma. Among these genes and proteins, the majority had already been described either to be influenced by a mutated ras or to be differentially expressed in pancreatic adenocarcinoma, thus proving the feasibility of our model. Other genes and proteins (e.g., BBC1, GLTSCR2, and rhoGDlα, up to now, have not been implicated in pancreatic tumor development. Thus, we were able to establish an in vitro model of pancreatic carcinogenesis, which enabled us to identify genes and proteins differentially expressed during the early steps of malignant transformation.

  1. Expression Changes of Early Response Genes in Lung Due to High Volume Ventilation

    Institute of Scientific and Technical Information of China (English)

    WANG Yuelan; YAO Shanglong; XIONG Ping

    2005-01-01

    Summary: The expression changes of early response genes due to ventilation with high volume in adult rats in vivo were observed. Forty SD male rats were randomly divided into control and 30, 60, 90 and 120 min ventilation groups, respectively (n=8 in each group). The animals were ventilated with tidal volume of 42 ml/kg and a PEEP level of 0 cmH2O at a rate of 40 breaths per minute in room air with a ventilator was given to the small animals. The expression of Egr-1, C-jun and IL-1β mRNA and proteins was detected by RT-PCR and immunohistochemical technique, respectively. The pathological changes in lung tissues were examined by HE staining. The results indicated that the expression of Egr-1, C-jun and IL-1β mRNA was detectable at 30th min after overventilation, but there was no significant difference in comparison with that in control group until overventilation for 60 min. However, at 90 and 120 min there was a significent increase as compared with 30 min or control group (P<0.05). The expression of Egr-1, C-jun and IL-1β deteced by immunohistochemical assay also showed a similar tendency of the gradual increase. In the 120 min ventilation group, the expression intensity of Egr-1, C-jun and IL-1β proteins in lung cells was the strongest and the nuclear translocation was increased markedly in comparison with any other groups (P<0.05). HE staining suggested that the degree of lung injury was aggravated gradually with the ventialtion going on and had a similar tendency to the expression of these early response genes and proteins. The current data suggested that overventilation activated and upregulated the expression of early response genes and the expression of these genes may be taken as the early signal to predict the onset and degree of lung injury. These results may demonstrated partially that the expression of early response genes induced by the mechanical stretch is associated with biochamic lung injury.

  2. Qualitative and Quantitative Studies of Polygene Proteins Expression in Esophageal Precancerous Lesions and Esophageal Carcinoma

    Institute of Scientific and Technical Information of China (English)

    LI Chao-xia; WU Ming-yao; KUANG Li-ping

    2007-01-01

    Objective: To examine the expressions of MDM2, P53 and P27 proteins in chronic esophagitis, para-cancer mucosa and esophageal carcinoma. Methods: Immunohistochemistry was used to detect the expressions of MDM2, P53 and P27 proteins in forty-seven patients suffering from chronic esophagitis and eighty-five cases of esophageal carcinoma and corresponding para-cancer mucosa. Flow cytometry((FCM) was applied to detect the quantities of these proteins expressed in fresh tissues of 48 cases of esophageal cancer and their para-cancer tissues and 24 cases of relative normal mucosa at the surface of cutting edge. Results: Immunohistochemistry results showed that the expressions of the three studied proteins were very similar in the epithelia of chronic esophagitis and para-cancer mucosa (P>0.05). Both the qualitative and quantitative studies displayed that the P53 protein had no expression and its accumulations would appear only in the early stages of esophagus canceration while the MDM2 and P27 proteins had different degrees of expressions in cases of normal esophageal mucosa. MDM2 protein markedly increased in the advanced stages of esophageal canceration. A quantitative study showed that the expression of P27 protein had a linearity of decreasing tendency (F=9.132,P=0.002) in the course of esophageal canceration. Conclusion: Chronic esophagitis may be a precancerous lesion. Owing to the changes of the P53 and P27 proteins, we can also conclude that these occur in the early stages of esophagus oncogenesis, however the changes of MDM2 expression may occur in the advanced stage of esophageal canceration.

  3. Effects of leptin on in vitro maturation, fertilization and embryonic cleavage after ICSI and early developmental expression of leptin (Ob and leptin receptor (ObR proteins in the horse

    Directory of Open Access Journals (Sweden)

    Arrighi Silvana

    2009-10-01

    Full Text Available Abstract Background The identification of the adipocyte-derived obesity gene product, leptin (Ob, and subsequently its association with reproduction in rodents and humans led to speculations that leptin may be involved in the regulation of oocyte and preimplantation embryo development. In mice and pigs, in vitro leptin addition significantly increased meiotic resumption and promoted preimplantation embryo development in a dose-dependent manner. This study was conducted to determine whether leptin supplementation during in vitro maturation (IVM to horse oocytes could have effects on their developmental capacity after fertilization by IntraCytoplasmic Sperm Injection (ICSI. Methods Compact and expanded-cumulus horse oocytes were matured in medium containing different concentrations (1, 10, 100, 1000 ng/ml of recombinant human leptin and the effects on maturation, fertilization and embryo cleavage were evaluated. Furthermore, early developmental expression of Ob and leptin receptor (Ob-R was investigated by immunocytochemical staining. Results In expanded-cumulus oocytes, the addition of leptin in IVM medium improved maturation (74% vs 44%, for 100 ng/ml leptin-treated and control groups, respectively; P Conclusion Leptin plays a cumulus cell-mediated role in the regulation of oocyte maturation in the mare. Species-specific differences may exist in oocyte sensitivity to leptin.

  4. 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.

  5. Early expression of mannose-binding lectin 2 during Aspergillus fumigatus infection in human corneal epithelial cells

    Institute of Scientific and Technical Information of China (English)

    Cheng-Ye; Che; Jing-Fen; Zhang; Ji-Eun; Lee; Jing; Lin; Li-Ting; Hu; Nan; Jiang; Qian; Wang; Qiang; Xu; Gui-Qiu; Zhao

    2015-01-01

    AIM: To evaluate the early expression of mannose-binding lectin 2(MBL2) in human corneal epithelial cells(HCECs) infected by Aspergillus fumigatus(AF).METHODS: HCECs cultured in vitro with AF antigens and sampled at 0, 0.5, 1, 2, 4, 6 and 8h. The expression of MBL2 m RNA was evaluated by semiquantitative reverse transcription-polymerase chain reaction(RT-PCR). The expression of MBL2 protein in supernatant fluid was shown by enzyme linked immunosorbent assay(ELISA). MBL2 protein in HCECs was detected by immunocytochemistry at 0 and 24 h.RESULTS: MBL2 m RNA and protein are expressed in normal HCECs. The expression of MBL2 m RNA and protein in supernatant fluid begin to increase after being stimulated with AF antigens. The most significantly peak of MBL2 m RNA is in 2h. The protein of MBL2 in supernatant fluid decrease gradually after 0.5h. The protein in HCECs expression increase after stimulation of24 h.· CONCLUSION: MBL2 receptor expressed in normal HCECs in vitro. The stimulation by AF antigens can increase the early expression of it.

  6. Rolipram and SP600125 suppress the early increase in PTP1B expression during cerulein-induced pancreatitis in rats.

    Science.gov (United States)

    Sarmiento, Nancy; Sánchez-Bernal, Carmen; Pérez, Nieves; Sardina, José L; Mangas, Arturo; Calvo, José J; Sánchez-Yagüe, Jesús

    2010-07-01

    To analyze the expression modulation of pancreatic protein tyrosine phosphatase (PTP)1B during the development of cerulein (Cer)-induced acute pancreatitis (AP) and the effect of inhibition of type 4 phosphodiesterase and c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2 on its expression levels. Acute pancreatitis was induced in rats by subcutaneous injections of 20 microg Cer per kilogram body weight at hourly intervals, and the animals were killed at 2, 4, or 9 hours after the first injection. Neutropenia was induced with vinblastine sulfate. Phosphodiesterase and the mitogen-activated protein kinases were inhibited with rolipram and SP600125, respectively, before the induction of AP. Protein tyrosine phosphatase 1B increases its expression at the levels of both protein and messenger RNA during the early phase of Cer-induced AP. The increase in protein expression persisted along the development of the disease, and neutrophil infiltration seemed to play a central role. Rolipram and SP600125 pretreatments mostly suppressed the increase in the expression of PTP1B during the early phase of AP. Cerulein-induced AP is associated with an increase in the expression of PTP1B in its early phase. An increase in cyclic adenosine monophosphate levels in inflammatory cells and the inhibition of c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2 are able to suppress the increase in PTP1B protein level.

  7. Early childhood stuttering III: initial status of expressive language abilities.

    Science.gov (United States)

    Watkins, R V; Yairi, E; Ambrose, N G

    1999-10-01

    This investigation evaluated the expressive language abilities of 84 preschool-age children who stuttered, 62 who recovered from stuttering, and 22 who persisted in stuttering. The participants were identical to those identified in E. Yairi and N. G. Ambrose (1999) and E. Paden, E. Yairi, and N. G. Ambrose (1999). A range of lexical, morphological, and syntactic measures--calculated from spontaneous language samples of approximately 250-300 utterances in length collected relatively near stuttering onset--were used to examine the children's expressive language skills. For the purpose of analysis and comparison to normative data, children were grouped into three age intervals, in terms of the age at which they entered the study (2- to 3-year-olds, 3- to 4-year-olds, and 4- to 5-year-olds). Findings revealed similarity in the expressive language abilities of children whose stuttering persisted as opposed to abated at all age intervals. In addition, persistent and recovered stutterers displayed expressive language abilities near or above developmental expectations, based on comparison with normative data, at all age intervals. Children who entered the study at the youngest age level consistently demonstrated expressive language abilities well above normative expectations; this pattern was found for both persistent and recovered groups. These findings provide relatively limited information to assist in the early differentiation of persistence in or recovery from stuttering, but they do shed light on theoretical issues regarding the nature and character of early stuttering and potential associations with language learning.

  8. Changes in Laminin Expression Pattern during Early Differentiation of Human Embryonic Stem Cells.

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    Martin Pook

    Full Text Available Laminin isoforms laminin-511 and -521 are expressed by human embryonic stem cells (hESC and can be used as a growth matrix to culture these cells under pluripotent conditions. However, the expression of these laminins during the induction of hESC differentiation has not been studied in detail. Furthermore, the data regarding the expression pattern of laminin chains in differentiating hESC is scarce. In the current study we aimed to fill this gap and investigated the potential changes in laminin expression during early hESC differentiation induced by retinoic acid (RA. We found that laminin-511 but not -521 accumulates in the committed cells during early steps of hESC differentiation. We also performed a comprehensive analysis of the laminin chain repertoire and found that pluripotent hESC express a more diverse range of laminin chains than shown previously. In particular, we provide the evidence that in addition to α1, α5, β1, β2 and γ1 chains, hESC express α2, α3, β3, γ2 and γ3 chain proteins and mRNA. Additionally, we found that a variant of laminin α3 chain-145 kDa-accumulated in RA-treated hESC showing that these cells produce prevalently specifically modified version of α3 chain in early phase of differentiation.

  9. Changes in Laminin Expression Pattern during Early Differentiation of Human Embryonic Stem Cells.

    Science.gov (United States)

    Pook, Martin; Teino, Indrek; Kallas, Ade; Maimets, Toivo; Ingerpuu, Sulev; Jaks, Viljar

    2015-01-01

    Laminin isoforms laminin-511 and -521 are expressed by human embryonic stem cells (hESC) and can be used as a growth matrix to culture these cells under pluripotent conditions. However, the expression of these laminins during the induction of hESC differentiation has not been studied in detail. Furthermore, the data regarding the expression pattern of laminin chains in differentiating hESC is scarce. In the current study we aimed to fill this gap and investigated the potential changes in laminin expression during early hESC differentiation induced by retinoic acid (RA). We found that laminin-511 but not -521 accumulates in the committed cells during early steps of hESC differentiation. We also performed a comprehensive analysis of the laminin chain repertoire and found that pluripotent hESC express a more diverse range of laminin chains than shown previously. In particular, we provide the evidence that in addition to α1, α5, β1, β2 and γ1 chains, hESC express α2, α3, β3, γ2 and γ3 chain proteins and mRNA. Additionally, we found that a variant of laminin α3 chain-145 kDa-accumulated in RA-treated hESC showing that these cells produce prevalently specifically modified version of α3 chain in early phase of differentiation.

  10. Expression and Significance of WT1 and Betacatenin Proteins in Non-small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Feng Zhou

    2013-12-01

    by Pearson chi-square independence test (X2 = 5.915, P = 0.015, r = 0.331, indicating that the aberrant expression of WT1 protein was closely associated with that of β-catenin protein in NSCLC. Conclusions: 1. The positive expression rate of WT1 protein expression is 62.5% in NSCLC. 2. The positive expression rate of aberrant β-catenin protein is 72.9% in NSCLC. 3. There is significant association between WT1 protein and lymph node metastasis. WT1 protein expression is higher in non- lymph node metastasis group than in lymph node metastasis group (P < 0.05; and there was no significant association between WT1 protein and genders, ages, tumor sizes, pathological patterns, differentiated degrees and pTNM stagings (P > 0.05. 4. Aberrant protein of β-catenin is in reverse relevance with differentiation degrees, and the higher the differentiation degree is, the lower aberrant expression of β-catenin protein will be observed, but there is no significant association between the aberrant expression of β-catenin protein and genders, ages, tumor sizes, pathological patterns, differentiated degrees and pTNM stagings (P > 0.05. 5. There is certain relevance between WT1 protein expression and the aberrant expression of β-catenin protein in NSCLC (P < 0.05. 6. WT1 protein and β-catenin proteins may play certain roles in early events of NSCLC.

  11. Cloning and expression of special F protein from human liver

    Institute of Scientific and Technical Information of China (English)

    Shu-Ye Liu; Xin-Da Yu; Chun-Juan Song; Wei Lu; Jian-Dong Zhang; Xin-Rong Shi; Ying Duan; Ju Zhang

    2007-01-01

    AIM:To clone human liver special F protein and to express it in a prokaryotic system.METHODS:Total RNA was isolated from human liver tissue and first-strand cDNA was reverse transcribed using the PCR reverse primer. Following this,cDNA of the F protein was ligated into the clone vector pUCm-T. The segment of F protein's cDNA was subcloned into the expression vector pET-15b and transformed into E coli BL21 (DEB) pLyss. Isopropy-β-D-thiogalactoside (IPTG) was then used to induce expression of the target protein.RESULTS:The cDNA clone of human liver special F protein (1134bp) was successfully produced,with the cDNA sequence being published in Gene-bank:DQ188836. We confirmed the expression of F protein by Western blot with a molecular weight of 43 kDa. The expressed protein accounted for 40% of the total protein extracted.CONCLUSION:F protein expresses cDNA clone in a proKaryotic system,which offers a relatively simple way of producing sufficient quantities of F protein and contributes to understanding the principal biological functions of this protein.

  12. Early shutoff of host protein synthesis in cells infected with herpes simplex viruses.

    Science.gov (United States)

    Matis, J; Kúdelová, M

    2001-01-01

    Herpes simplex viruses 1 (HSV-1) and 2 (HSV-2) are capable of suppressing the host cell protein synthesis even without viral gene expression. This phenomenon is known as the early shutoff or as the virion-associated host shutoff (vhs) to emphasize that it is mediated by a component of infecting virions which is a product of the UL41 (vhs) gene. The UL41 encoded protein is a functional tegument protein also present in light (L) particles and is not essential for virus replication. The major product of UL41 gene is a 58 K phosphoprotein. At least two forms of UL41 protein differing in the extent of phosphorylation are present in HSV-1-infected cells. HSV-2 compared to HSV-1 strains display a stronger vhs phenotype. However, in superinfection experiments the less strong vhs phenotype is dominant. UL41 protein triggers disruption of polysomes and rapid degradation of all host and viral mRNAs and blocks a reporter gene expression without other HSVs proteins. The available evidence suggests that UL41 protein is either itself a ribonuclease (RNase) or a subunit of RNase that contains also one or more cellular subunits. UL41 protein is capable of interacting with a transactivator of an alpha-gene, the alpha-transinducing factor (alpha-TIF). Interaction of UL41 protein with alpha-TIF down regulates the UL41 (vhs) gene activity during lytic infection. The possible role of other viral proteins in the shutoff is discussed.

  13. Boost protein expression through co-expression of LEA-like peptide in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Shinya Ikeno

    Full Text Available The boost protein expression has been done successfully by simple co-expression with a late embryogenesis abundant (LEA-like peptide in Escherichia coli. Frequently, overexpression of a recombinant protein fails to provide an adequate yield. In the study, we developed a simple and efficient system for overexpressing transgenic proteins in bacteria by co-expression with an LEA-like peptide. The design of this peptide was based on part of the primary structure of an LEA protein that is known hydrophilic protein to suppress aggregation of other protein molecules. In our system, the expression of the target protein was increased remarkably by co-expression with an LEA-like peptide consisting of only 11 amino acid residues. This could provide a practical method for producing recombinant proteins efficiently.

  14. 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.

  15. Expression of potein complexes using multiple E. coli protein co-expression systems: a benchmarking study

    NARCIS (Netherlands)

    Busso, D.; Peleg, Y.; Folkers, G.E.; Celie, P.H.N.

    2011-01-01

    Escherichia coli (E. coli) remains the most commonly used host for recombinant protein expression. It is well known that a variety of experimental factors influence the protein production level as well as the solubility profile of over-expressed proteins. This becomes increasingly important for opti

  16. Expression, Solubilization, and Purification of Bacterial Membrane Proteins.

    Science.gov (United States)

    Jeffery, Constance J

    2016-02-02

    Bacterial integral membrane proteins play many important roles, including sensing changes in the environment, transporting molecules into and out of the cell, and in the case of commensal or pathogenic bacteria, interacting with the host organism. Working with membrane proteins in the lab can be more challenging than working with soluble proteins because of difficulties in their recombinant expression and purification. This protocol describes a standard method to express, solubilize, and purify bacterial integral membrane proteins. The recombinant protein of interest with a 6His affinity tag is expressed in E. coli. After harvesting the cultures and isolating cellular membranes, mild detergents are used to solubilize the membrane proteins. Protein-detergent complexes are then purified using IMAC column chromatography. Support protocols are included to help select a detergent for protein solubilization and for use of gel filtration chromatography for further purification.

  17. Early repeated maternal separation induces alterations of hippocampus reelin expression in rats

    Indian Academy of Sciences (India)

    Jianlong Zhang; Lina Qin; Hu Zhao

    2013-03-01

    The long-term effects of repeated maternal separation (MS) during early postnatal life on reelin expression in the hippocampus of developing rats were investigated in the present study. MS was carried out by separating Wistar rat pups singly from their mothers for 3 h a day during postnatal days (PND) 2–14. Reelin mRNA and protein levels in the hippocampus were determined using qRT-PCR and Western blotting, at PND 22, PND 60 and PND 90. MS resulted in the loss of body weight in the developing rats, and reelin mRNA and protein levels in the hippocampus generally were down-regulated over the developing period, but the reelin mRNA and protein levels in the hippocampus of 90-day-old male rats were up-regulated. These findings suggest that the long-term effects of MS on the expression levels of hippocampal reelin mRNA and protein depends on the age at which the stressed rats’ brains were collected; reelin had important implications for the maternal-neonate interaction needed for normal brain development. In conclusion, repeated MS occurring during early postnatal life may cause the alterations of hippocampal reelin expression with the increasing age of developing rats.

  18. Coffee (Coffea arabica L.) genes early expressed during infection by the rust fungus (Hemileia vastatrix).

    Science.gov (United States)

    Fernandez, Diana; Santos, Patricia; Agostini, Caroline; Bon, Marie-Claude; Petitot, Anne-Sophie; C Silva, Maria; Guerra-Guimarães, Leonor; Ribeiro, Ana; Argout, Xavier; Nicole, Michel

    2004-11-01

    SUMMARY The beverage cash crop coffee (Coffea arabica L.) is subject to severe losses caused by the rust fungus Hemileia vastatrix. In naturally resistant coffee plants, a specific hypersensitive reaction (HR) may be elicited early to stop fungal infection. To isolate host genes involved in HR, we undertook an expressed sequence tags (ESTs) analysis. Two cDNA libraries were constructed using suppression subtractive hybridization (SSH) and 527 non-redundant ESTs were generated from 784 randomly picked clones. Classification of the ESTs into several functional categories showed that more than one-quarter of the predicted proteins might encode disease resistance (R) proteins, stress- and defence-proteins, and components of signal transduction pathways. Twenty-eight differentially screened sequences (DSSs) were selected after differential hybridization of 1000 cDNA clones from each library. Investigation of the expression patterns of a subset of 13 DSSs showed higher levels of gene expression in inoculated plants compared with control plants. HR-up-regulation of transcript accumulation occurred for 9 out of the 13 genes 24 and 48 h after H. vastatrix challenge. Two genes encoded homologues of the Arabidopsis DND1 and NDR1 proteins, suggesting conservation of resistance signalling pathways in perennial plants. Other HR-regulated sequences matched receptor kinases, AP2 domain- and WRKY transcription factors, cytochromes P450, heat shock 70 proteins, glucosyltransferases and proteins of unknown function. The ESTs reported here provide a useful resource for studying coffee resistance responses and for improving C. arabica for durable disease resistance.

  19. Sonic hedgehog expression during early tooth development in Suncus murinus.

    Science.gov (United States)

    Miyado, Mami; Ogi, Hidenao; Yamada, Gen; Kitoh, Junzo; Jogahara, Takamichi; Oda, Sen-Ichi; Sato, Iwao; Miyado, Kenji; Sunohara, Masataka

    2007-11-16

    Tooth development is a highly organized process characterized by reciprocal interactions between epithelium and mesenchyme. However, the expression patterns and functions of molecules involved in mouse tooth development are unclear from the viewpoint of explaining human dental malformations and anomalies. Here, we show the expression of sonic hedgehog (Shh), a potent initiator of morphogenesis, during the early stages of tooth development in Suncus murinus. Initially, symmetrical, elongated expression of suncus Shh (sShh) was observed in the thin layer of dental epithelial cells along the mesial-distal axis of both jaws. As the dental epithelium continued to develop, sShh was strictly restricted to the predicted leading parts of the growing, invaginating epithelium corresponding to tooth primordia and enamel knots. We propose that some aspects of Shh function in tooth development are widely conserved in mammalian phylogeny.

  20. Expression of Helicobacter pylori Hsp60 protein and its immunogenicity

    Institute of Scientific and Technical Information of China (English)

    Yang Bai; Liang-Ren Li; Ji-De Wang; Ye Chen; Jian-Feng Jin; Zhao-Shan Zhang; Dian-Yuan Zhou; Ya-Li Zhang

    2003-01-01

    AIM: To express Hsp60 protein of H pylori by a constructed vector and to evaluate its immunogenicity.METHODS: Hsp60 DNA was amplified by PCR and inserted into the prokaryotie expression vector pET-22b (+), which was transformed into BL21 (DE3) E. coli strain to express recombinant protein. Immunogenicity of expressed Hsp60 protein was evaluated with animal experiments.RESULTS: DNA sequence analysis showed Hsp60 DNA was the same as GenBank's research. Hsp60 recombinant protein accounted for 27.2 % of the total bacterial protein,and could be recognized by the serum from H pylori infected patients and Balb/c mice immunized with Hsp60 itself.CONCLUSION: Hsp60 recombinant protein might become a potential vaccine for controlling and treating H pylori infection.

  1. Isoforms of the nuclear envelope protein Nurim are differentially expressed during heart development in mice.

    Science.gov (United States)

    Zhang, Wan; Bai, Tianyu; Zhang, Shuai; Xu, Shiqiang; Chen, Hengling; Li, Chenhong

    2017-09-05

    To date, transcript variants of the nuclear envelope protein Nurim and their expression profiles in mice have never been elucidated. In this study, we determined that the primary Nurim variant a was abundantly expressed in mouse heart, liver, spleen and kidney. The protein level of isoform a is initiated at an early stage of heart formation and demonstrated a significant increase in expression throughout embryonic heart development. Interestingly, Nurim b is also up-regulated from E12.5 to E18.5 in different individuals. Our research represents the first report on alternative splicing variants of mouse Nurim and their differential expression profile during embryonic development. These studies suggest a potential role for Nurim in early heart morphogenesis and should help further elucidate the function of Nurim. Copyright © 2017. Published by Elsevier B.V.

  2. Evaluation of somatic embryos of alfalfa for recombinant protein expression.

    Science.gov (United States)

    Fu, Guohua; Grbic, Vojislava; Ma, Shengwu; Tian, Lining

    2015-02-01

    Somatic embryos of alfalfa can accumulate higher levels of recombinant proteins comparing to vegetative organs. Somatic embryos may be explored as a new system for new protein production for plants. Plants have been explored via genetic engineering as an inexpensive system for recombinant protein production. However, protein expression levels in vegetative tissues have been low, which limits the commercial utilization of plant expression systems. Somatic embryos resemble zygotic embryos in many aspects and may accumulate higher levels of proteins as true seed. In this study, somatic embryo of alfalfa (Medicago sativa L.) was investigated for the expression of recombinant proteins. Three heterologous genes, including the standard scientific reporter uid that codes for β-glucuronidase and two genes of interest: ctb coding for cholera toxin B subunit (CTB), and hIL-13 coding for human interleukin 13, were independently introduced into alfalfa via Agrobacterium-mediated transformation. Somatic embryos were subsequently induced from transgenic plants carrying these genes. Somatic embryos accumulated approximately twofold more recombinant proteins than vegetative organs including roots, stems, and leaves. The recombinant proteins of CTB and hIL-13 accumulated up to 0.15 and 0.18 % of total soluble protein in alfalfa somatic embryos, respectively. The recombinant proteins expressed in somatic embryos also exhibited biological activities. As somatic embryos can be induced in many plant species and their production can be scaled up via different avenues, somatic embryos may be developed as an efficient expression system for recombinant protein production.

  3. Maltose-Binding Protein (MBP, a Secretion-Enhancing Tag for Mammalian Protein Expression Systems.

    Directory of Open Access Journals (Sweden)

    Raphael Reuten

    Full Text Available Recombinant proteins are commonly expressed in eukaryotic expression systems to ensure the formation of disulfide bridges and proper glycosylation. Although many proteins can be expressed easily, some proteins, sub-domains, and mutant protein versions can cause problems. Here, we investigated expression levels of recombinant extracellular, intracellular as well as transmembrane proteins tethered to different polypeptides in mammalian cell lines. Strikingly, fusion of proteins to the prokaryotic maltose-binding protein (MBP generally enhanced protein production. MBP fusion proteins consistently exhibited the most robust increase in protein production in comparison to commonly used tags, e.g., the Fc, Glutathione S-transferase (GST, SlyD, and serum albumin (ser alb tag. Moreover, proteins tethered to MBP revealed reduced numbers of dying cells upon transient transfection. In contrast to the Fc tag, MBP is a stable monomer and does not promote protein aggregation. Therefore, the MBP tag does not induce artificial dimerization of tethered proteins and provides a beneficial fusion tag for binding as well as cell adhesion studies. Using MBP we were able to secret a disease causing laminin β2 mutant protein (congenital nephrotic syndrome, which is normally retained in the endoplasmic reticulum. In summary, this study establishes MBP as a versatile expression tag for protein production in eukaryotic expression systems.

  4. Expression of the orexin system in the porcine uterus, conceptus and trophoblast during early pregnancy.

    Science.gov (United States)

    Smolinska, N; Kiezun, M; Dobrzyn, K; Szeszko, K; Maleszka, A; Kaminski, T

    2015-11-01

    Orexin A and B are hypothalamic peptides derived from the prepro-orexin (PPO) precursor. Orexins stimulate food intake and arousal. Those peptides bind and activate two G protein-coupled receptors: orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). Numerous authors have suggested that orexins play an important role in the regulation of the reproductive functions. The objective of the present study was to analyse the presence of and changes in the gene and protein expression pattern of the orexin system in the porcine uterus, conceptus and trophoblast (chorioallantois) during early pregnancy. In the endometrium, the highest PPO and OX1R gene expression was detected on days 15 to 16 of gestation. The OX2R mRNA content in the endometrium was higher on days 10 to 11 and 15 to 16 than on days 12 to 13 and 27 to 28. In the trophoblasts, PPO gene expression was higher on days 30 to 32 than on days 27 to 28. The highest PPO protein content in the endometrium was noted on days 12 to 13. The highest OX1R protein content in the endometrium was detected on days 10 to 11, whereas OX2R protein on days 15 to 16. In the trophoblasts, PPO and OX1R protein levels were more pronounced on days 27 to 28 than on days 30 to 32, but OX2R expression was higher on days 30 to 32. The expression of PPO, OX1R and OX2R was different in the conceptuses and trophoblasts during early pregnancy. Local orexin production and the presence of the specific orexin receptors suggest that the orexin system may participate in the control of porcine reproductive functions by exerting endocrine and auto/paracrine effects on the uterus, conceptuses and trophoblasts during early pregnancy. This study provides the first evidence for the presence of orexins and their receptors in the uteri, conceptuses and trophoblasts in pigs during early pregnancy. The local orexin system is dependent on the stage of pregnancy.

  5. 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

  6. Plant Antifreeze Proteins and Their Expression Regulatory Mechanism

    Institute of Scientific and Technical Information of China (English)

    Lin Yuan-zhen; Lin Shan-zhi; Zhang Zhi-yi; Zhang Wei; Liu Wen-feng

    2005-01-01

    Low temperature is one of the major limiting environmental factors which constitutes the growth, development,productivity and distribution of plants. Over the past several years, the proteins and genes associated with freezing resistance of plants have been widely studied. The recent progress of domestic and foreign research on plant antifreeze proteins and the identification and characterization of plant antifreeze protein genes, especially on expression regulatory mechanism of plant antifreeze proteins are reviewed in this paper. Finally, some unsolved problems and the trend of research in physiological functions and gene expression regulatory mechanism of plant antifreeze proteins are discussed.

  7. The Origin and Early Evolution of Membrane Proteins

    Science.gov (United States)

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

    2005-01-01

    Membrane proteins mediate functions that are essential to all cells. These functions include transport of ions, nutrients and waste products across cell walls, capture of energy and its transduction into the form usable in chemical reactions, transmission of environmental signals to the interior of the cell, cellular growth and cell volume regulation. In the absence of membrane proteins, ancestors of cell (protocells), would have had only very limited capabilities to communicate with their environment. Thus, it is not surprising that membrane proteins are quite common even in simplest prokaryotic cells. Considering that contemporary membrane channels are large and complex, both structurally and functionally, a question arises how their presumably much simpler ancestors could have emerged, perform functions and diversify in early protobiological evolution. Remarkably, despite their overall complexity, structural motifs in membrane proteins are quite simple, with a-helices being most common. This suggests that these proteins might have evolved from simple building blocks. To explain how these blocks could have organized into functional structures, we performed large-scale, accurate computer simulations of folding peptides at a water-membrane interface, their insertion into the membrane, self-assembly into higher-order structures and function. The results of these simulations, combined with analysis of structural and functional experimental data led to the first integrated view of the origin and early evolution of membrane proteins.

  8. Discovery of cellular proteins required for the early steps of HCV infection using integrative genomics.

    Directory of Open Access Journals (Sweden)

    Ji Hoon Park

    Full Text Available 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.

  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. Analysis of diferentially expressed protein from primary and recurrent ovarian cancer serum

    Institute of Scientific and Technical Information of China (English)

    Yuan Wang; Jin-Jin Yu; Ting Zhu; Ling Xu; Ming Xu; Yu-Zheng Huang; Hong Pu; Chun-Qing Yu

    2012-01-01

    ABSTRACT Objective:To study the value of the differentially expressed proteins from primary and recurrent ovarian cancer serum for early diagnosis of primary and recurrent ovarian cancer.Methods:WCX kit(BrukerDaltonicsGraBH) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS) technology were used to detect serum samples from49 patients with primary ovarian cancer and21 patients with recurrent disease.Results:In the mass range(Mr) from1000 to12000Da, eight differentially expressed protein peaks were screened from primary ovarian cancer serum.Among them, four protein peaks withMr1457,1857,2202, 7761 were lowly expressed and the others withMr2946,5333,5859,5901 were highly expressed. Ten diferentially expressed protein peaks were screened from recurrent ovarian cancer serum. Among them,1944,1980,2080,2661,2993,4450,4659,5359Da protein expressions were increased significantly, and1897,7868Da protein expressions were decreased significantly.The pattern of primary ovarian cancer was applied to8 early-stage ovarian cancer serum samples, and7 serum samples were successfully predicted with the accuracy of87.5%.The pattern of recurrent ovarian cancer was applied to9 without pelvic or abdominal mass recurrent ovarian cancer serum samples, and8 serum samples were successfully predicted with the accuracy of 88.9%.Conclusions:Combination ofMALDI-TOF-MS andWCX kit technology can directly screen the diferrential expressed protein from primary and recurrent ovarian cancer serum.They have clinical significance for enhancement of sensitivity and specificity of ovarian cancer diagnosis.

  11. Effects of immunosuppressive treatment on protein expression in rat kidney

    Directory of Open Access Journals (Sweden)

    Kędzierska K

    2014-09-01

    Full Text Available Karolina Kędzierska,1 Katarzyna Sporniak-Tutak,2 Krzysztof Sindrewicz,2 Joanna Bober,3 Leszek Domański,1 Mirosław Parafiniuk,4 Elżbieta Urasińska,5 Andrzej Ciechanowicz,6 Maciej Domański,1 Tomasz Smektała,2 Marek Masiuk,5 Wiesław Skrzypczak,6 Małgorzata Ożgo,6 Joanna Kabat-Koperska,1 Kazimierz Ciechanowski1 1Department of Nephrology, Transplantology, and Internal Medicine, 2Department of Dental Surgery, 3Department of Medical Chemistry, 4Department of Forensic Medicine, 5Department of Pathomorphology, Pomeranian Medical University, 6Department of Physiology, Cytobiology, and Proteomics, West Pomeranian University of Technology, Szczecin, Poland Abstract: The structural proteins of renal tubular epithelial cells may become a target for the toxic metabolites of immunosuppressants. These metabolites can modify the properties of the proteins, thereby affecting cell function, which is a possible explanation for the mechanism of immunosuppressive agents' toxicity. In our study, we evaluated the effect of two immunosuppressive strategies on protein expression in the kidneys of Wistar rats. Fragments of the rat kidneys were homogenized after cooling in liquid nitrogen and then dissolved in lysis buffer. The protein concentration in the samples was determined using a protein assay kit, and the proteins were separated by two-dimensional electrophoresis. The obtained gels were then stained with Coomassie Brilliant Blue, and their images were analyzed to evaluate differences in protein expression. Identification of selected proteins was then performed using mass spectrometry. We found that the immunosuppressive drugs used in popular regimens induce a series of changes in protein expression in target organs. The expression of proteins involved in drug, glucose, amino acid, and lipid metabolism was pronounced. However, to a lesser extent, we also observed changes in nuclear, structural, and transport proteins' synthesis. Very slight differences

  12. Paradoxical Sleep Suppresses Immediate Early Gene Expression in the Rodent Suprachiasmatic Nuclei

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    Michael J Decker

    2010-09-01

    Full Text Available Light stimulates neuronal activity with subsequent expression of the protein product of the immediate early gene, c-fos, in the Suprachiasmatic Nuclei (SCN. Non-photic stimuli is also thought to modulate activity within the SCN. Here, we sought to determine the effects intrinsic stimuli, specifically, the states of wakefulness and sleep upon c-fos protein expression in the SCN. In 16 rats, c-fos protein expression was evaluated at a fixed time of 1600 hours (subjective night, following one hour of electroencephalographically defined sleep. During sleep, as the state of paradoxical sleep (PS increased, c-fos protein expression decreased (r -0.41, p < 0.033. The PS-associated reduction of c-fos positive cells occurred equally between animals asleep in the light and those asleep in the dark. We propose a model whereby PS duration might function as a homeostatic-entraining mechanism to reduce neuronal activity within the SCN, and thereby modulate circadian rhythms during sleep.

  13. Elevated nuclear S100P expression is associated with poor survival in early breast cancer patients.

    Science.gov (United States)

    Maciejczyk, Adam; Łacko, Aleksandra; Ekiert, Marcin; Jagoda, Ewa; Wysocka, Teresa; Matkowski, Rafał; Hałoń, Agnieszka; Györffy, Balázs; Lage, Hermann; Surowiak, Paweł

    2013-04-01

    S100P - low molecular weight acidic protein has been shown to be involved in processes of proliferation, survival, angiogenesis, multidrug resistance and metastasis in various human malignancies. In breast cancer, S100P expression is associated with immortalization of neoplastic cells and aggressive tumour behaviour, indicating that this protein may have adverse prognostic value. We analyzed nuclear and cytoplasmic expression of S100P in 85 stage II breast cancer patients with a median follow up of 17 years. Immunohistochemical reactions were performed on paraffin sections of primary tumours, using monoclonal antibodies against S100P. We also studied prognostic value of S100P mRNA expression using the KM plotter which assessed the effect of 22,277 genes on survival in 2422 breast cancer patients. Moreover, the relationship was examined between expression of S100P in cells of four breast cancer cell lines and their sensitivity to the 11 most frequently applied cytotoxic drugs. Univariate and multivariate analyses showed that higher expression of nuclear S100P (S100Pn) was typical for cases of a shorter overall survival and disease-free time. KM plotter analysis showed that elevated S100P expression was specific for cases of a relapse-free survival and distant metastases-free survival. No relationship could be documented between expression of S100P and sensitivity of breast cancer cells to cytostatic drugs. We demonstrated that a high S100Pn expression level was associated with poor survival in early stage breast cancer patients. Since preliminary data indicated that expression of S100P was up-regulated by activation of glucocorticoid receptor and several agents manifested potential to activate or inhibit S100P promoter activity, this protein might become a therapy target and warrants further studies with respect to its prognostic, predictive and potentially therapeutic value.

  14. Early changes in corticospinal excitability when seeing fearful body expressions.

    Science.gov (United States)

    Borgomaneri, Sara; Vitale, Francesca; Avenanti, Alessio

    2015-09-21

    Quick inhibition of approach tendencies in response to signals of potential threats is thought to promote survival. However, little is known about the effect of viewing fearful expressions on the early dynamics of the human motor system. We used the high temporal resolution of single-pulse and paired-pulse transcranial magnetic stimulation (TMS) over the motor cortex to assess corticospinal excitability (CSE) and intracortical facilitation (ICF) during observation of happy, fearful and neutral body postures. To test motor circuits involved in approach tendencies, CSE and ICF were recorded from the first dorsal interosseous (FDI), a muscle involved in grasping, and the abductor pollicis brevis (APB), which served as a control. To test early motor dynamics, CSE and ICF were measured 70-90 ms after stimulus onset. We found a selective reduction in CSE in the FDI when participants observed fearful body expressions. No changes in ICF or in the excitability of APB were detected. Our study establishes an extremely rapid motor system reaction to observed fearful body expressions. This motor modulation involves corticospinal downstream projections but not cortical excitatory mechanisms, and appears to reflect an inhibition of hand grasping. Our results suggest a fast visuo-motor route that may rapidly inhibit inappropriate approaching actions.

  15. A spectrum of genes expressed during early stages of rice panicle and flower development

    Indian Academy of Sciences (India)

    Kumuda M. Kushalappa; Autar K. Mattoo; Usha Vijayraghavan

    2000-08-01

    To unravel gene expression patterns during rice inflorescence development, particularly at early stages of panicle and floral organ specification, we have characterized random cloned cDNAs from developmental-stage-specific libraries. cDNA libraries were constructed from rice panicles at the stage of branching and flower primordia specification or from panicles undergoing floral organogenesis. Partial sequence analysis and expression patterns of some of these random cDNA clones from these two rice panicle libraries are presented. Sequence comparisons with known DNA sequences in databases reveal that approximately sixtyeight per cent of these expressed rice genes show varying degrees of similarity to genes in other species with assigned functions. In contrast, thirtytwo per cent represent uncharacterized genes. cDNAs reported here code for potential rice homologues of housekeeping molecules, regulators of gene expression, and signal transduction molecules. They comprise both single-copy and multicopy genes, and genes expressed differentially, both spatially and temporally, during rice plant development. New rice cDNAs requiring specific mention are those with similarity to COP1, a regulator of photomorphogenesis in Arabidopsis; sequence-specific DNA binding plant proteins like AP2-domain-containing factors; genes that specify positional information in shoot meristems like leucine-rich-repeat-containing receptor kinases; regulators of chromatin structure like Polycomb domain protein; and also proteins induced by abiotic stresses.

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

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    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.

  17. A STUDY ON CYCLOOXYGENASE -2 PROTEIN EXPRESSION IN ESOPHAGEAL CAICONOGENESIS

    Institute of Scientific and Technical Information of China (English)

    王立峰; 张伟; 王吾如; 王洪平; 韩双廷; 曲平; 刘义; 李茉; 刘伯齐; 林培中

    2001-01-01

    To investigate cyclooxygenase- 2(Cox-2) protein expression in esophageal cancer and precancerous lesions. Methods: One hundred twenty biopsy specimens from esophageal carcinoma and 113 from patients with esophageal premalingnant lesions, 27 from individuals with normal esophageal mucosa and 3 from Barrett's esophagus were examined for Cox-2 protein expression by immunohistochemistry. Results: Cox-2 protein was not observed in normal esophageal squamous and glandular epithelium, hyperplasia from mild to severe dysplasia lesions and carcinoma in situ. Positive Cox-2 protein expression was found in 4 of 60 specimens of invasive squamous-cell carcinomas, 21 of 30 specimens of esophageal adenocarcinomas and in 3 of 3 Barret's esophageal tissues. Conclusion: The Cox-2 protein expression may be associated with the development of the esophageal adenocarcinomas but not esophageal squamous-cell carcinomas.

  18. Insulin influenced expression of myelin proteins in diabetic peripheral neuropathy.

    Science.gov (United States)

    Rachana, Kuruvanthe S; Manu, Mallahalli S; Advirao, Gopal M

    2016-08-26

    Diabetic peripheral neuropathy (DPN) is one of the downstream complications of diabetes. This complication is caused by the deficiency of insulin action and subsequent hyperglycemia, but the details of their pathogenesis remain unclear. Hence, it is of critical importance to understand how such hormonal variation affects the expression of myelin proteins such as myelin basic protein (MBP) and myelin associated glycoprotein (MAG) in the peripheral nerve. An earlier report from our lab has demonstrated the expression of insulin receptors (IR) in Schwann cells (SCs) of sciatic nerve. To assess the neurotrophic role of insulin in diabetic neuropathy, we studied the expression of these myelin proteins under control, DPN and insulin treated DPN subjects at developmental stages. Further, the expression of these myelin proteins was correlated with the expression of insulin receptor. Expression of myelin proteins was significantly reduced in the diabetic model compared to normal, and upregulated in insulin treated diabetic rats. Similarly, an in vitro study was also carried out in SCs grown at high glucose and insulin treated conditions. The expression pattern of myelin proteins in SCs was comparable to that of in vivo samples. In addition, quantitative study of myelin genes by real time PCR has also showed the significant expression pattern change in the insulin treated and non-treated DPN subjects. Taken together, these results corroborate the critical importance of insulin as a neurotrophic factor in demyelinized neurons in diabetic neuropathy.

  19. Clinical significance of PHPT1 protein expression in lung cancer

    Institute of Scientific and Technical Information of China (English)

    XU An-jian; XIA Xiang-hou; DU Song-tao; GU Jun-chao

    2010-01-01

    Background in our previous studies, we found the expression of 14-kD phosphohistidine phosphatase (PHPT1) was associated with lung cancer cells migration and invasion, and PHPT1 mRNA expression level in lung cancer tissues clinically correlated with lymph node metastasis. in the present study, we aimed to further investigate the expression of PHPT1 protein in lung cancer.Methods Expression of PHPT1 protein in tissue samples from 146 lung cancers and 30 normal tissues adjacent to lung cancers was assessed using immunohistochemical method. Fisher's exact test was used to analyze expression patterns of PHPT1 protein in these tissue types. Meanwhile, we studied the correlation between expression of PHPT1 protein and clinicopathological features in lung cancer.Results Significantly higher expression levels of PHPT1 protein were found in lung cancer samples (53.42%) than in normal tissues adjacent to lung cancer (23.33%) (P=0.003). Fisher's exact test showed that lung cancer stage positively correlated with expression of PHPT1 protein (P=0.02), and lung cancer samples with lymph node metastasis showed higher PHPT1 protein expression (P=0.016) than the samples without lymph node metastasis.Conclusions The results of this study agree with findings from our previous study of PHPT1 mRNA expression in lung cancer tissues, and strongly suggest that PHPT1 protein is closely associated with the carcinogenesis and metastasis of lung cancer. Thus, therapy targeting PHPT1 (inhibition or silencing) could be potentially benefited for lung cancer patients.

  20. Stable protein expression in mammalian cells using baculoviruses.

    Science.gov (United States)

    Lackner, Andreas; Kreidl, Emanuel; Peter-Vörösmarty, Barbara; Spiegl-Kreinecker, Sabine; Berger, Walter; Grusch, Michael

    2012-01-01

    The baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) has been widely used in biotechnology for protein expression in insect cells. Baculoviruses use arthropods as their natural hosts and are unable to replicate in mammalian cells. However, AcMNPV is able to enter many mammalian cell types and can be used for transgene expression if engineered to contain suitable expression cassettes. In this chapter, we describe the construction and application of a recombinant baculovirus containing a bicistronic expression cassette that can be used for stable protein expression in mammalian cells. As an example, the generation of glioblastoma and hepatocellular carcinoma cell lines stably expressing green fluorescent protein after puromycin selection is shown.

  1. Translational control of protein synthesis: the early years.

    Science.gov (United States)

    Lodish, Harvey F

    2012-10-19

    For the past fifty-five years, much of my research has focused on the function and biogenesis of red blood cells, including the cloning and study of many membrane proteins such as glucose and anion transporters and the erythropoietin receptor. We have also elucidated the mechanisms of membrane insertion, folding, and maturation of many plasma membrane and secreted proteins. Despite all of this work and more, I remain extremely proud of our very early work on the regulation of mRNA translation: work on bacteriophage f2 RNA in the 1960s and on translation of α- and β-globin mRNAs in the early 1970s. Using techniques hopelessly antiquated by today's standards, we correctly elucidated many important aspects of translational control, and I thought readers would be interested in learning how we did these experiments.

  2. Chemometrics of differentially expressed proteins from colorectal cancer patients

    Institute of Scientific and Technical Information of China (English)

    Lay-Chin Yeoh; Saravanan Dharmaraj; Boon-Hui Gooi; Manjit Singh; Lay-Harn Gam

    2011-01-01

    AIM: To evaluate the usefulness of differentially expressed proteins from colorectal cancer (CRC) tissues for differentiating cancer and normal tissues. METHODS: A Proteomic approach was used to identify the differentially expressed proteins between CRC and normal tissues. The proteins were extracted using Tris buffer and thiourea lysis buffer (TLB) for extraction of aqueous soluble and membrane-associated proteins, respectively. Chemometrics, namely principal component analysis (PCA) and linear discriminant analysis (LDA), were used to assess the usefulness of these proteins for identifying the cancerous state of tissues. RESULTS: Differentially expressed proteins identified were 37 aqueous soluble proteins in Tris extracts and 24 membrane-associated proteins in TLB extracts. Based on the protein spots intensity on 2D-gel images, PCA by applying an eigenvalue > 1 was successfully used to reduce the number of principal components (PCs) into 12 and seven PCs for Tris and TLB extracts, respectively, and subsequently six PCs, respectively from both the extracts were used for LDA. The LDA classification for Tris extract showed 82.7% of original samples were correctly classified, whereas 82.7% were correctly classified for the cross-validated samples. The LDA for TLB extract showed that 78.8% of original samples and 71.2% of the cross-validated samples were correctly classified. CONCLUSION: The classification of CRC tissues by PCA and LDA provided a promising distinction between normal and cancer types. These methods can possibly be used for identification of potential biomarkers among the differentially expressed proteins identified.

  3. Temporal dynamics of immediate early gene expression during cellular consolidation of spatial memory.

    Science.gov (United States)

    Barry, Daniel N; Commins, Sean

    2017-06-01

    The consolidation of newly acquired memories on a cellular level is thought to take place in the first few hours following learning. This process is dependent on de novo protein synthesis during this time, which ultimately leads to long-term structural and functional neuronal changes and the stabilisation of a memory trace. Immediate early genes (IEGs) are rapidly expressed in neurons following learning, and previous research has suggested more than one wave of IEG expression facilitates consolidation in the hours following learning. We analysed the expression of Zif268, c-Fos and Arc protein in a number of brain regions involved in spatial learning either 90min, 4h or 8h following training in the Morris water maze task. Consistent with the role of IEGs in the earliest stages of consolidation, a single wave of expression was observed in most brain regions at 90min, however a subsequent wave of expression was not observed at 8h. In fact, Zif268 expression was observed to fall below the levels of naïve controls at this time-point in the medial prefrontal and perirhinal cortices. This may be indicative of synaptic downscaling in these regions in the hours following learning, and an important marker of the consolidation of spatial memory. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Protein Biomarkers for the Early Detection of Breast Cancer

    Directory of Open Access Journals (Sweden)

    David E. Misek

    2011-01-01

    Full Text Available Advances in breast cancer control will be greatly aided by early detection so as to diagnose and treat breast cancer in its preinvasive state prior to metastasis. For breast cancer, the second leading cause of cancer-related death among women in the United States, early detection does allow for increased treatment options, including surgical resection, with a corresponding better patient response. Unfortunately, however, many patients' tumors are diagnosed following metastasis, thus making it more difficult to successfully treat the malignancy. There are, at present, no existing validated plasma/serum biomarkers for breast cancer. Only a few biomarkers (such as HER-2/neu, estrogen receptor, and progesterone receptor have utility for diagnosis and prognosis. Thus, there is a great need for new biomarkers for breast cancer. This paper will focus on the identification of new serum protein biomarkers with utility for the early detection of breast cancer.

  5. Regulation of chick early B-cell factor-1 gene expression in feather development.

    Science.gov (United States)

    El-Magd, Mohammed Abu; Sayed-Ahmed, Ahmed; Awad, Ashraf; Shukry, Mustafa

    2014-05-01

    The chick Ebf1 (early B-cell factor-1) gene is a member of a novel family of helix loop helix transcription factors. The expression profile, regulation and significance of this gene have been extensively studied in lymphatic, nervous, adipose and muscular tissues. However, cEbf1 expression, regulation and function in the feather of chick embryo have not yet been investigated. cEbf1 expression was first detected throughout the mesenchymal core of some few feather placodes (D7-D7.5). After feathers became mature and grew distally (D9 and D10), the mesenchymal expression of cEbf1 became confined to the caudal margin of the proximal half of all formed feather buds. Because this dynamic pattern of expression resembles that of Sonic Hedgehog (Shh) protein and bone morphogenetic protein (Bmp4) plus the crucial role of these two major signals in feather development, we hypothesized that cEbf1 expression in the feather may be regulated by Shh and Bmp4. In a feather explant culture system, Shh signals are necessary to initiate and maintain cEbf1 expression in the posterior half of the feather bud, while Bmp4 is crucial for the initial cEbf1 expression in the anterior half of the feather bud. Inhibition of Shh, not only down-regulates cEbf1, but also changes the morphology of feather buds, which become irregular and fused. This is the first study to demonstrate that cEbf1 expression in the feather bud is under the control of Shh and Bmp4 signals and that expression may play a role in the normal development of feathers.

  6. Expression of a Carrot Antifreeze Protein Gene in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    Ma Xinyu; Shen Xin; Lu Cunfu

    2003-01-01

    The recombinant expression vectorpET43. lb-AFP, which contains full encoding region of a carrot 36 kD antifreeze protein (AFP) gene was constructed. The recombinant was transformed into expression host carrying T7 RNA polymerase gene (DE3 lysogen) and induced by 1 mmol. L-1 IPTG (isopropyl-β-D-thiogalactoside) to express 110 kD polypeptide of AFP fusion protein.The analysis of product solubility revealed that pET43. 1b-AFP was predominately soluble, and the expressed amount reached the maximum after the IPTG treatment for 3 h.

  7. 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

  8. Expression of Early Light Induced Protein in Grapevine and Pea, under Different Conditions and its Relation with Photoinhibition Expresión de una Proteína inducida Tempranamente por Luz en Vid y Arveja Bajo Diferentes Condiciones y su Relación con la Fotoinhibición

    Directory of Open Access Journals (Sweden)

    Maritza Berti

    2012-09-01

    Full Text Available Early light induced proteins (ELIP are a type of proteins which are expressed before than other chloroplast proteins in the presence of light. These proteins have been studied in a large number of annual species such as pea (Pisum sativum L., barley (Hordeum vulgare L. and Arabidopsis sp. In perennials plants the studies about ELIPs are very scarce. The possible photoprotective function of the ELIPs has motivated the interest in investigating the presence of this type of proteins in a perennial plant such as grapevine (Vitis vinifera L. and if their characteristics differ from those found in annual plants. In this paper a comparative study was conducted on the ELIPs expression in grapevine and pea to investigate whether there are differences regarding to temperature and light intensity conditions necessary for maximum ELIP expression in each species and for studying in each case the relationship between ELIP expression and photoinhibition degree. The results of this study showed that the maximum ELIPs expression was reached from 25 °C and 1000 µmol PAR m-2 s-1 in both species. Above these values the expression remained constant. Regarding the temperature and light intensity effect on the photoinhibition degree, it was observed that temperature produced inverse relation in grapevine but no relation with pea. On the other hand, the light intensity produced direct relation in both grapevine and pea. The light intensity effect on ELIP expression suggests that these proteins may have a photorepairing role of the photosynthetic system, but the effect of temperature on the ELIP expression in short-term stress may be associated rather to the optimum conditions for their synthesis.Las proteínas tempranamente inducidas por luz (ELIP se expresan antes que otras proteínas del cloroplasto en presencia de luz. Estas proteínas han sido estudiadas en un gran número de especies anuales tales como arveja (Pisum sativum L., cebada (Hordeum bulgare L. y

  9. A gene expression atlas of early craniofacial development.

    Science.gov (United States)

    Brunskill, Eric W; Potter, Andrew S; Distasio, Andrew; Dexheimer, Phillip; Plassard, Andrew; Aronow, Bruce J; Potter, S Steven

    2014-07-15

    We present a gene expression atlas of early mouse craniofacial development. Laser capture microdissection (LCM) was used to isolate cells from the principal critical microregions, whose development, differentiation and signaling interactions are responsible for the construction of the mammalian face. At E8.5, as migrating neural crest cells begin to exit the neural fold/epidermal ectoderm boundary, we examined the cranial mesenchyme, composed of mixed neural crest and paraxial mesoderm cells, as well as cells from adjacent neuroepithelium. At E9.5 cells from the cranial mesenchyme, overlying olfactory placode/epidermal ectoderm, and underlying neuroepithelium, as well as the emerging mandibular and maxillary arches were sampled. At E10.5, as the facial prominences form, cells from the medial and lateral prominences, the olfactory pit, multiple discrete regions of underlying neuroepithelium, the mandibular and maxillary arches, including both their mesenchymal and ectodermal components, as well as Rathke's pouch, were similarly sampled and profiled using both microarray and RNA-seq technologies. Further, we performed single cell studies to better define the gene expression states of the early E8.5 pioneer neural crest cells and paraxial mesoderm. Taken together, and analyzable by a variety of biological network approaches, these data provide a complementing and cross validating resource capable of fueling discovery of novel compartment specific markers and signatures whose combinatorial interactions of transcription factors and growth factors/receptors are responsible for providing the master genetic blueprint for craniofacial development.

  10. Analysis of interferon gamma protein expression in zebrafish (Danio rerio).

    Science.gov (United States)

    Yoon, Sohye; Alnabulsi, Ayham; Wang, Ting Yu; Lee, Po Tsang; Chen, Tzong-Yueh; Bird, Steve; Zou, Jun; Secombes, Christopher J

    2016-10-01

    IFN-γ is a major effector cytokine, produced to induce type I immune responses. It has been cloned in several fish species including zebrafish, however to date few studies have looked at IFN-γ protein expression and bioactivity in fish. Hence, the current study focused on developing a monoclonal antibody (moAb) against zfIFN-γ. We show that the zfIFN-γ moAb specifically recognises E. coli produced recombinant IFN-γ protein and zfIFN-γ produced in transfected HEK293 cells, by Western blot analysis. Next we analysed the production of the native protein following expression induced by PHA stimulation of leukocytes in vitro or antigen re-stimulation in vivo. We show the IFN-γ protein is produced as a dimer, and that a good correlation exists between transcript expression levels and protein levels.

  11. 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 m

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

    African Journals Online (AJOL)

    EB

    2013-09-03

    Sep 3, 2013 ... Department of Pathology, Ibn Rochd University Medical Center, Casablanca, ... NPC is a characteristic tumor displaying epidemiological, genetic and regional distribution properties that ... expression and histological type, age and sex distributions was ..... according to geographical location and we could.

  13. Prokaryotic expression of f protein from PPRV and characterization of its polyclonal antibody.

    Science.gov (United States)

    Wang, Qiuxia; Dou, Yongxi; Yang, Xiangfang; Meng, Xuelian; Zhai, Junjun; Zhu, Xueliang; Luo, Xuenong; Chen, Lei; Cai, Xuepeng

    2013-02-01

    The goal of this study was to evaluate the specificity of a polyclonal antibody against the F protein from Peste des petits ruminants virus (PPRV). A pET30a/F prokaryotic expression vector was successfully constructed and its recombinant protein was expressed. The result of Western blot analysis showed that the fusion protein pET30a/F possessed good immunoreactivity and the purified recombinant protein was then used as the antigen to raise anti-pET30a/F polyclonal antibody in rabbits. The polyclonal antibody titer against the recombinant F protein was confirmed by indirect ELISA, and the protein's specificity against pET30/F polyclonal antibody was confirmed by both Western blot and indirect immunofluorescence assay in transfected cells. In short, we obtained the high-level expression of recombinant F protein as well as high titers of rabbit polyclonal antibody specificity against F protein in pCAGGS/F transfected cells. This special polyclonal antibody offers a valuable and useful tool for further study of the pathogenesis of PPRV early infection and the structural and functional characterization of PPRV F protein.

  14. Expression of Yes-associated protein modulates Survivin expression in primary liver malignancies.

    Science.gov (United States)

    Bai, Haibo; Gayyed, Mariana F; Lam-Himlin, Dora M; Klein, Alison P; Nayar, Suresh K; Xu, Yang; Khan, Mehtab; Argani, Pedram; Pan, Duojia; Anders, Robert A

    2012-09-01

    Hepatocellular carcinoma and intrahepatic cholangiocarcinoma account for 95% of primary liver cancer. For each of these malignancies, the outcome is dismal; incidence is rapidly increasing, and mechanistic understanding is limited. We observed abnormal proliferation of both biliary epithelium and hepatocytes in mice after genetic manipulation of Yes-associated protein, a transcription coactivator. Here, we comprehensively documented Yes-associated protein expression in the human liver and primary liver cancers. We showed that nuclear Yes-associated protein expression is significantly increased in human intrahepatic cholangiocarcinoma and hepatocellular carcinoma. We found that increased Yes-associated protein levels in hepatocellular carcinoma are due to multiple mechanisms including gene amplification and transcriptional and posttranscriptional regulation. Survivin, a member of the inhibitors-of-apoptosis protein family, has been reported as an independent prognostic factor for poor survival in both hepatocellular carcinoma and intrahepatic cholangiocarcinoma. We found that nuclear Yes-associated protein expression correlates significantly with nuclear Survivin expression for both intrahepatic cholangiocarcinoma and hepatocellular carcinoma. Furthermore, using mice engineered to conditionally overexpress Yes-associated protein in the liver, we found that Survivin messenger RNA expression depends upon Yes-associated protein levels. Our findings suggested that Yes-associated protein contributes to primary liver tumorigenesis and likely mediates its oncogenic effects through modulating Survivin expression.

  15. Early expression of KCC2 in rat hippocampal cultures augments expression of functional GABA synapses.

    Science.gov (United States)

    Chudotvorova, Ilona; Ivanov, Anton; Rama, Sylvain; Hübner, Christian A; Pellegrino, Christophe; Ben-Ari, Yehezkel; Medina, Igor

    2005-08-01

    The development of GABAergic synapses is associated with an excitatory to inhibitory shift of the actions of GABA because of a reduction of [Cl-]i. This is due to a delayed postnatal expression of the K+ -Cl- cotransporter KCC2, which has low levels at birth and peaks during the first few postnatal weeks. Whether the expression of the cotransporter and the excitatory to inhibitory shift have other consequences on the operation of GABA(A) receptors and synapses is not yet known. We have now expressed KCC2 in immature neurones at an early developmental stage and determined the consequences on the formation of GABA and glutamate synapses. We report that early expression of the cotransporter selectively enhances GABAergic synapses: there is a significant increase of the density of GABA(A) receptors and synapses and an increase of the frequency of GABAergic miniature postsynaptic currents. The density of glutamate synapses and frequency of AMPA miniature postsynaptic currents are not affected. We conclude that the expression of KCC2 and the reduction of [Cl-]i play a critical role in the construction of GABAergic networks that extends beyond the excitatory to inhibitory shift of the actions of GABA.

  16. Performance benchmarking of four cell-free protein expression systems.

    Science.gov (United States)

    Gagoski, Dejan; Polinkovsky, Mark E; Mureev, Sergey; Kunert, Anne; Johnston, Wayne; Gambin, Yann; Alexandrov, Kirill

    2016-02-01

    Over the last half century, a range of cell-free protein expression systems based on pro- and eukaryotic organisms have been developed and have found a range of applications, from structural biology to directed protein evolution. While it is generally accepted that significant differences in performance among systems exist, there is a paucity of systematic experimental studies supporting this notion. Here, we took advantage of the species-independent translation initiation sequence to express and characterize 87 N-terminally GFP-tagged human cytosolic proteins of different sizes in E. coli, wheat germ (WGE), HeLa, and Leishmania-based (LTE) cell-free systems. Using a combination of single-molecule fluorescence spectroscopy, SDS-PAGE, and Western blot analysis, we assessed the expression yields, the fraction of full-length translation product, and aggregation propensity for each of these systems. Our results demonstrate that the E. coli system has the highest expression yields. However, we observe that high expression levels are accompanied by production of truncated species-particularly pronounced in the case of proteins larger than 70 kDa. Furthermore, proteins produced in the E. coli system display high aggregation propensity, with only 10% of tested proteins being produced in predominantly monodispersed form. The WGE system was the most productive among eukaryotic systems tested. Finally, HeLa and LTE show comparable protein yields that are considerably lower than the ones achieved in the E. coli and WGE systems. The protein products produced in the HeLa system display slightly higher integrity, whereas the LTE-produced proteins have the lowest aggregation propensity among the systems analyzed. The high quality of HeLa- and LTE-produced proteins enable their analysis without purification and make them suitable for analysis of multi-domain eukaryotic proteins.

  17. Small-scale expression of proteins in E. coli.

    Science.gov (United States)

    Zerbs, Sarah; Giuliani, Sarah; Collart, Frank

    2014-01-01

    Proteins participate in virtually every cellular activity, and a knowledge of protein function is essential for an understanding of biological systems. However, protein diversity necessitates the application of an array of in vivo and in vitro approaches for characterization of the functional and biochemical properties of proteins. Methods that enable production of proteins for in vitro studies are critical for determination of the molecular, kinetic, and thermodynamic properties of these molecules. Ideally, proteins could be purified from the original source; however, the native host is often unsuitable for a number of reasons. Consequently, systems for heterologous protein production are commonly used to produce large amounts of protein. Heterologous expression hosts are chosen using a number of criteria, including genetic tractability, advantageous production or processing characteristics (secretion or posttranslational modifications), or economy of time and growth requirements. The subcloning process also provides an opportunity to introduce purification tags, epitope tags, fusions, truncations, and mutations into the coding sequence that may be useful in downstream purification or characterization applications. Bacterial systems for heterologous protein expression have advantages in ease of use, cost, short generation times, and scalability. These expression systems have been widely used by high-throughput protein production projects and often represent an initial experiment for any expression target. Escherichia coli has been studied for many years as a model bacterial organism and is one of the most popular hosts for heterologous protein expression (Terpe, 2006). Its protein production capabilities have been intensively studied, and the ease of genetic manipulation in this organism has led to the development of strains engineered exclusively for use in protein expression. These resources are widely available from commercial sources and public repositories

  18. 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...

  19. 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

  20. Patterns of fluorescent protein expression in Scleractinian corals.

    Science.gov (United States)

    Gruber, David F; Kao, Hung-Teh; Janoschka, Stephen; Tsai, Julia; Pieribone, Vincent A

    2008-10-01

    Biofluorescence exists in only a few classes of organisms, with Anthozoa possessing the majority of species known to express fluorescent proteins. Most species within the Anthozoan subgroup Scleractinia (reef-building corals) not only express green fluorescent proteins, they also localize the proteins in distinct anatomical patterns.We examined the distribution of biofluorescence in 33 coral species, representing 8 families, from study sites on Australia's Great Barrier Reef. For 28 of these species, we report the presence of biofluorescence for the first time. The dominant fluorescent emissions observed were green (480-520 nm) and red (580-600 nm). Fluorescent proteins were expressed in three distinct patterns (highlighted, uniform, and complementary) among specific anatomical structures of corals across a variety of families. We report no significant overlap between the distribution of fluorescent proteins and the distribution of zooxanthellae. Analysis of the patterns of fluorescent protein distribution provides evidence that the scheme in which fluorescent proteins are distributed among the anatomical structures of corals is nonrandom. This targeted expression of fluorescent proteins in corals produces contrast and may function as a signaling mechanism to organisms with sensitivity to specific wavelengths of light.

  1. Early changes in gene expression profiles of hepatic GVHD uncovered by oligonucleotide microarrays.

    Science.gov (United States)

    Ichiba, Tamotsu; Teshima, Takanori; Kuick, Rork; Misek, David E; Liu, Chen; Takada, Yuichiro; Maeda, Yoshinobu; Reddy, Pavan; Williams, Debra L; Hanash, Samir M; Ferrara, James L M

    2003-07-15

    The liver, skin, and gastrointestinal tract are major target organs of acute graft-versus-host disease (GVHD), the major complication of allogeneic bone marrow transplantation (BMT). In order to gain a better understanding of acute GVHD in the liver, we compared the gene expression profiles of livers after experimental allogeneic and syngeneic BMT using oligonucleotide microarray. At 35 days after allogeneic BMT when hepatic GVHD was histologically evident, genes related to cellular effectors and acute-phase proteins were up-regulated, whereas genes largely related to metabolism and endocrine function were down-regulated. At day 7 after BMT before the development of histologic changes in the liver, interferon gamma (IFN-gamma)-inducible genes, major histocompatibility (MHC) class II molecules, and genes related to leukocyte trafficking had been up-regulated. Immunohistochemistry demonstrated that expression of IFN-gamma protein itself was increased in the spleen but not in hepatic tissue. These results suggest that the increased expression of genes associated with the attraction and activation of donor T cells induced by IFN-gamma early after BMT is important in the initiation of hepatic GVHD in this model and provide new potential molecular targets for early detection and intervention of acute GVHD.

  2. Controlled expression of enhanced green fluorescent protein and hepatitis B virus precore protein in mammalian cells

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A novel tetracycline regulation expression system was used to regulate the expression of enhanced green fluorescent protein (EGFP) and hepatitis B virus precore protein in the mammalian cell lines with lipofectAMINE. Flow cytometry assays showed that application of the system resulted in about 18-fold induction of EGFP expression in CHO cell lines and 5-fold induction in SSMC-7721 cells and about 2-fold in the HEK293 cells. Furthermore, the effective use of this system for the controlled expression of HBV precore protein gene in hepatocellular carcinoma cells was tested.

  3. 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.

  4. Prolonged morphine administration alters protein expression in the rat myocardium

    OpenAIRE

    Drastichova Zdenka; Skrabalova Jitka; Neckar Jan; Kolar Frantisek; Novotny Jiri

    2011-01-01

    Abstract Background Morphine is used in clinical practice as a highly effective painkiller as well as the drug of choice for treatment of certain heart diseases. However, there is lack of information about its effect on protein expression in the heart. Therefore, here we aimed to identify the presumed alterations in rat myocardial protein levels after prolonged morphine treatment. Methods Morphine was administered to adult male Wistar rats in high doses (10 mg/kg per day) for 10 days. Protein...

  5. 丹参静脉滴注对早期糖尿病肾病患者外周血自噬相关蛋白表达的影响%Effect of Danshen Powder Injection on the Expression of Autophagy-related Protein in the Pe-ripheral Blood of Patients with Early-stage of Diabetic Nephropathy

    Institute of Scientific and Technical Information of China (English)

    解加泳; 朱强; 高建民; 陈伟; 徐燕

    2016-01-01

    Obj ective]To study the effect of Danshen powder inj ection on the expression of autophagy-re-lated protein in the peripheral blood of patients with early-stage of diabetic ephropathy.[Methods]Sixty-one patients with early diabetic nephropathy were divided into control group and treatment group according to the patients'willing.Both of the two groups were treated with glimepiride tablets 2 mg once a day,while the treat-ment group were given with Danshen powder injection 1200mg plus xylitol 250 mL intravenous drip once a day additionally.The treatment course covered 14 d,and two consecutive courses of treatment were conducted. The patients of two groups were detected before and after 24 h ,the urinary albumin excretion amount (UAER),24 h urine total protein (Upro),serum creatinine (SCr)and blood urea nitrogen (BUN)and glo-merular filtration rate (eGFR)and peripheral blood of autophagy associated protein p62 and LC3 expression levels were compared.[Results]The 24h UAER,24 h Upro,BUN,SCr and eGFR in the control group had no statistical significance compared with those before treatment (P >0.05),but after treatment,the UAER, 24hUpro、BUN、SCr and eGFR of the observation group was significantly decreased compared with those be-fore treatment (P 0.05);the expression level of LC3 mRNA of the treatment group was significantly increased compared to that before treatment (P 0.05),但观察组治疗后的 UAER、24hUpro、BUN、SCr 和 eGFR 较治疗前显著降低(P 0.05),观察组治疗后LC3 mRNA 表达水平较治疗前显著升高(P <0.05),p62抗体阳性率较治疗前显著降低(P <0.05)。【结论】丹参静脉滴注对早期DN有肾脏保护作用,其作用机制可能与对自噬的干预作用有关。

  6. Climbazole increases expression of cornified envelope proteins in primary keratinocytes.

    Science.gov (United States)

    Pople, J E; Moore, A E; Talbot, D C S; Barrett, K E; Jones, D A; Lim, F L

    2014-10-01

    Dandruff is a troubling consumer problem characterized by flaking and pruritus of the scalp and is considered a multifactorial condition with sebum, individual susceptibility and the fungus Malassezia all thought to play a part. The condition is commonly treated with shampoo products containing antifungal ingredients such as zinc pyrithione and climbazole. It is hypothesized that these ingredients may be delivering additional scalp skin benefits besides their antifungal activity helping to relieve dandruff effectively. The objective of this study was to evaluate the anti-dandruff ingredient climbazole for potential skin benefits using genomics and in vitro assays. Microarray analysis was performed to profile gene expression changes in climbazole-treated primary human keratinocyte cells. Results were independently validated using qPCR and analysis of protein expression using ELISA and immunocytochemistry. Microarray analysis of climbazole-treated keratinocytes showed statistically significant expression changes in genes associated with the gene ontology groups encompassing epidermal differentiation, keratinization, cholesterol biosynthesis and immune response. Upregulated genes included a number encoding cornified envelope proteins such as group 3 late-cornified envelope proteins, LCE3 and group 2 small-proline-rich proteins, SPRR2. Protein analysis studies of climbazole-treated primary keratinocytes using ELISA and immunocytochemistry were able to demonstrate that the increase in gene transcripts translated into increased protein expression of these cornified envelope markers. Climbazole treatment of primary keratinocytes results in an upregulation in expression of a number of genes including those encoding proteins involved in cornified envelope formation with further studies demonstrating this did translate into increased protein expression. A climbazole-driven increase in cornified envelope proteins may improve the scalp skin barrier, which is known to be weaker

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

    NARCIS (Netherlands)

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

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

  8. Immunohistochemical analysis of p53 and ras p21 expression in colorectal adenomas and early carcinomas.

    Science.gov (United States)

    Ieda, S; Watatani, M; Yoshida, T; Kuroda, K; Inui, H; Yasutomi, M

    1996-01-01

    To further investigate whether multiple genetic changes are involved in the development of colorectal cancer, we performed an immunohistochemical analysis of p53 and ras p21 protein expression in 139 specimens of colorectal adenoma with varying degrees of dysplasia, 57 specimens of early cancer with an adenomatous component, and 12 specimens of superficial early cancer without any adenomatous component. Positive p53 staining was found in 15% of the adenomas with moderate dysplasia and in 42% of the adenomas with severe dysplasia or intramucosal carcinoma (IMCA). Positive immunostaining of p53 was observed to be significantly correlated with the degree of dysplasia and the depth of invasion, as was the expression of ras p21. However, a closer correlation was observed with the increasing size of the adenomas. Furthermore, p53 staining was positive in 42% of the 12 superficial early cancer specimens, while ras staining was positive in only 1 specimen (8%). These results indicate that p53 gene overexpression may play some biological role in both the adenoma-to-carcinoma sequence and in de novo cancer development, whereas ras p21 expression may not be as involved in de novo cancer development as in the malignant conversion of colorectal adenomas.

  9. Proteomic analysis of Clostridium thermocellum core metabolism: relative protein expression profiles and growth phase-dependent changes in protein expression

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    Rydzak Thomas

    2012-09-01

    Full Text Available Abstract Background Clostridium thermocellum produces H2 and ethanol, as well as CO2, acetate, formate, and lactate, directly from cellulosic biomass. It is therefore an attractive model for biofuel production via consolidated bioprocessing. Optimization of end-product yields and titres is crucial for making biofuel production economically feasible. Relative protein expression profiles may provide targets for metabolic engineering, while understanding changes in protein expression and metabolism in response to carbon limitation, pH, and growth phase may aid in reactor optimization. We performed shotgun 2D-HPLC-MS/MS on closed-batch cellobiose-grown exponential phase C. thermocellum cell-free extracts to determine relative protein expression profiles of core metabolic proteins involved carbohydrate utilization, energy conservation, and end-product synthesis. iTRAQ (isobaric tag for relative and absolute quantitation based protein quantitation was used to determine changes in core metabolic proteins in response to growth phase. Results Relative abundance profiles revealed differential levels of putative enzymes capable of catalyzing parallel pathways. The majority of proteins involved in pyruvate catabolism and end-product synthesis were detected with high abundance, with the exception of aldehyde dehydrogenase, ferredoxin-dependent Ech-type [NiFe]-hydrogenase, and RNF-type NADH:ferredoxin oxidoreductase. Using 4-plex 2D-HPLC-MS/MS, 24% of the 144 core metabolism proteins detected demonstrated moderate changes in expression during transition from exponential to stationary phase. Notably, proteins involved in pyruvate synthesis decreased in stationary phase, whereas proteins involved in glycogen metabolism, pyruvate catabolism, and end-product synthesis increased in stationary phase. Several proteins that may directly dictate end-product synthesis patterns, including pyruvate:ferredoxin oxidoreductases, alcohol dehydrogenases, and a putative

  10. Expression and purification of splicing proteins from mammalian cells.

    Science.gov (United States)

    Allemand, Eric; Hastings, Michelle L

    2014-01-01

    Pre-mRNA splicing is a complex process that is carried out by a large ribonucleoprotein enzyme, termed the spliceosome, which comprises up to 200 proteins. Despite this complexity, the role of individual spliceosomal proteins in the splicing reaction has been successfully investigated using cell-free assays. In many cases, the splicing factor of interest must be expressed and purified in order to study its function in vitro. Posttranslational modifications such as phosphorylation, methylation, acetylation, and ubiquitination of splicing factors are important for activity. Thus, their purification from mammalian cells presents numerous advantages. Here, we describe a method for expression and purification of splicing proteins from mammalian cells.

  11. Loss of disabled-2 expression is an early event in esophageal squamous tumorigenesis

    Institute of Scientific and Technical Information of China (English)

    Kumar Anupam; Chatopadhyay Tusharkant; Siddhartha Datta Gupta; Ralhan Ranju

    2006-01-01

    AIM: Disabled-2 (D4B2) is a candidate tumor-suppressor gene identified in ovarian cancer that negatively influences mitogenic signal transduction of growth factors and blocks ras activity. In a recent study, we observed down-regulation of DAB2 transcripts in ESCCs using cDNA microarrays. In the present study, we aimed to determine the clinical significance of loss of DAB2protein in esophageal tumorigenesis, hypothesizing that DAB2 promoter hypermethylation-mediated gene silencing may account for loss of the protein.METHODS: DAB2 expression was analyzed by immunohistochemistry in 50 primary esophageal squamous cell carcinomas (ESCCs), 30 distinct hyperplasia, 15 dysplasia and 10 non-malignant esophageal tissues. To determine whether promoter hypermethylation contributes to loss of DAB2 expression in ESCCs, methylation status of DAB2 promoter was analyzed in DAB2 immuno-negative tumors using methylation-specific PCR.RESULTS: Loss of DAB2 protein was observed in 5/30 (17%) hyperplasia, 10/15 (67%) dysplasia and 34/50 (68%) ESCCs. Significant loss of DAB2 protein was observed from esophageal normal mucosa to hyperplasia, dysplasia and invasive cancer (Ptrend < 0.001).Promoter hypermethylation of DAB2 was observed in 2of 10 (20%) DAB2 immuno-negative ESCCs.CONCLUSION: Loss of DAB2 protein expression occurs in early pre-neoplastic stages of development of esophageal cancer and is sustained down the tumorigenic pathway. Infrequent DAB2 promoter methylation in ESCCs suggests that epigenetic gene silencing is only one of the mechanisms causing loss of DAB2 expression in ESCCs.

  12. Smad3 Deficiency Ameliorates Hepatic Fibrogenesis through the Expression of Senescence Marker Protein-30, an Antioxidant-Related Protein

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    Da-Hee Jeong

    2013-12-01

    Full Text Available Smad3 is a key mediator of the transforming growth factor (TGF-β1 signaling pathway that plays central role in inflammation and fibrosis. In present study, we evaluated the effect of Smad3 deficiency in Smad3−/− mice with carbon tetrachloride (CCl4-induced liver fibrosis. The animals were received CCl4 or olive oil three times a week for 4 weeks. Histopathological analyses were performed to evaluate the fibrosis development in the mice. Alteration of protein expression controlled by Smad3 was examined using a proteomic analysis. CCl4-induced liver fibrosis was rarely detected in Smad3−/− mice compared to Smad3+/+. Proteomic analysis revealed that proteins related to antioxidant activities such as senescence marker protein-30 (SMP30, selenium-binding proteins (SP56 and glutathione S-transferases (GSTs were up-regulated in Smad3−/− mice. Western blot analysis confirmed that SMP30 protein expression was increased in Smad3−/− mice. And SMP30 levels were decreased in CCl4-treated Smad3+/+ and Smad3−/− mice. These results indicate that Smad3 deficiency influences the proteins level related to antioxidant activities during early liver fibrosis. Thus, we suggest that Smad3 deteriorate hepatic injury by inhibitor of antioxidant proteins as well as mediator of TGF-β1 signaling.

  13. 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

    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...

  14. Protein dynamics modulated electron transfer kinetics in early stage photosynthesis.

    Science.gov (United States)

    Kundu, Prasanta; Dua, Arti

    2013-01-28

    A recent experiment has probed the electron transfer kinetics in the early stage of photosynthesis in Rhodobacter sphaeroides for the reaction center of wild type and different mutants [Science 316, 747 (2007)]. By monitoring the changes in the transient absorption of the donor-acceptor pair at 280 and 930 nm, both of which show non-exponential temporal decay, the experiment has provided a strong evidence that the initial electron transfer kinetics is modulated by the dynamics of protein backbone. In this work, we present a model where the electron transfer kinetics of the donor-acceptor pair is described along the reaction coordinate associated with the distance fluctuations in a protein backbone. The stochastic evolution of the reaction coordinate is described in terms of a non-Markovian generalized Langevin equation with a memory kernel and Gaussian colored noise, both of which are completely described in terms of the microscopics of the protein normal modes. This model provides excellent fits to the transient absorption signals at 280 and 930 nm associated with protein distance fluctuations and protein dynamics modulated electron transfer reaction, respectively. In contrast to previous models, the present work explains the microscopic origins of the non-exponential decay of the transient absorption curve at 280 nm in terms of multiple time scales of relaxation of the protein normal modes. Dynamic disorder in the reaction pathway due to protein conformational fluctuations which occur on time scales slower than or comparable to the electron transfer kinetics explains the microscopic origin of the non-exponential nature of the transient absorption decay at 930 nm. The theoretical estimates for the relative driving force for five different mutants are in close agreement with the experimental estimates obtained using electrochemical measurements.

  15. Protein expression analysis of inflammation-related colon carcinogenesis

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    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.

  16. Altered expression of nuclear matrix proteins in etoposide induced apoptosis in HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The events of cell death and the expression of nuclear matrix protein(NMP)have been investigated in a promyelocytic leukemic cell line HL-60 induced with etoposide.By means of TUNEL assay,the nuclei displayed a characteristic morphology change,and the amount of apoptotic cells increased early and reached maximun about 39% after treatment with etoposide for 2 h.Nucleosomal DNA fragmentation was observed after treatment for 4 h.The morphological change of HL-60 cells,thus,occurred earlier than the appearance of DNA ladder.Total nuclear matrix proteins were analyzed by 2-dimensional gel electrophoresis.Differential expression of 59 nuclear matrix proteins was found in 4 h etoposide treated cells.Western blotting was then performed on three nuclear matrix acssociated proteins,PML,HSC70 and NuMA.The expression of the suppressor PML protein and heat shock protein HSC70 were significantly upregulated after etoposide treatment,while NuMA,a nuclear mitotic apparatus protein,was down regulated.These results demonstrate that significant biochemical alterations in nuclear matrix proteins take place during the apoptotic process.

  17. 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

  18. Variation in protein intake induces variation in spider silk expression.

    Directory of Open Access Journals (Sweden)

    Sean J Blamires

    Full Text Available 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

  19. 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.

  20. 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

  1. Identification of Differentially Expressed Serum Proteins in Infectious Purpura Fulminans

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    Ting He

    2014-01-01

    Full Text Available Purpura fulminans (PF is a life-threatening hemorrhagic condition. Because of the rarity and randomness of the disease, no improvement in treatment has been made for a long time. In this study, we assessed the serum proteome response to PF by comparing serum proteins between healthy controls and PF patient. Liquid chromatography with tandem mass spectrometry (LC-MS/MS approach was used after depleting 6 abundant proteins of serum. In total, 262 proteins were confidently identified with 2 unique peptides, and 38 proteins were identified significantly up- (≥2 or downregulated (≤0.5 based on spectral counting ratios (SpCPF/N. In the 38 proteins with significant abundance changes, 11 proteins were previously known to be associated with burn or sepsis response, but 27 potentially novel proteins may be specifically associated with PF process. Two differentially expressed proteins, alpha-1-antitrypsin (SERPINA1 and alpha-2 antiplasmin (SERPINF2, were validated by Western blot. This is the first study where PF patient and healthy controls are compared in a proteomic study to elucidate proteins involved in the response to PF. This study provides an initial basis for future studies of PF, and the differentially expressed proteins might provide new therapeutic targets to decrease the mortality of PF.

  2. Translational Modulation of Proteins Expressed from Bicistronic Vectors

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    Prasun J. Mishra

    2009-11-01

    Full Text Available Bicistronic vectors are useful tools for exogenous expression of two gene products from a single promoter element; however, reduced expression of protein from the second cistron compared with the first cistron is a common limitation to this approach. To overcome this limitation, we explored use of dihydrofolate reductase (DHFR complementary DNA encoded in bicistronic vectors to induce a second protein of interest by methotrexate (MTX treatment. Previous studies have demonstrated that levels of DHFR protein and DHFR fusion protein can be induced translationally following MTX treatment of cells. We demonstrated that in response to MTX treatment, DHFR partner protein in a bicistronic construct is induced for longer periods of time when compared with endogenous DHFR and DHFR fusion protein, in vitro and in vivo. Using rapamycin pretreatment followed by MTX treatment, we also devised a strategy to modulate levels of two proteins expressed from a bicistronic construct in a cap-independent manner. To our knowledge, this is the first report demonstrating that levels of proteins in DHFR-based bicistronic constructs can be induced and modulated using MTX and rapamycin treatment.

  3. Prolonged morphine administration alters protein expression in the rat myocardium

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    Drastichova Zdenka

    2011-11-01

    Full Text Available Abstract Background Morphine is used in clinical practice as a highly effective painkiller as well as the drug of choice for treatment of certain heart diseases. However, there is lack of information about its effect on protein expression in the heart. Therefore, here we aimed to identify the presumed alterations in rat myocardial protein levels after prolonged morphine treatment. Methods Morphine was administered to adult male Wistar rats in high doses (10 mg/kg per day for 10 days. Proteins from the plasma membrane- and mitochondria-enriched fractions or cytosolic proteins isolated from left ventricles were run on 2D gel electrophoresis, scanned and quantified with specific software to reveal differentially expressed proteins. Results Nine proteins were found to show markedly altered expression levels in samples from morphine-treaded rats and these proteins were identified by mass spectrometric analysis. They belong to different cell pathways including signaling, cytoprotective, and structural elements. Conclusions The present identification of several important myocardial proteins altered by prolonged morphine treatment points to global effects of this drug on heart tissue. These findings represent an initial step toward a more complex view on the action of morphine on the heart.

  4. Proteomic analysis of changes in protein expression in serum from animals exposed to paraquat.

    Science.gov (United States)

    Kim, Yong-Sik; Jung, Hana; Gil, Hyo-Wook; Hong, Sae-Yong; Song, Ho-Yeon

    2012-12-01

    Paraquat (PQ) poisoning remains a major public health concern in many countries. Extensive research has focused on finding early diagnostic biomarkers of acute PQ poisoning. In order to investigate the characterization of diagnostic biomarkers in PQ poisoning, we utilized proteomic analysis using serum from rats exposed to PQ, and we identified 8 differentially expressed proteins from over 500 protein spots. The expression of apolipoprotein E (ApoE), preprohaptoglobin (Pphg), a precursor of haptoglobin (Hp), and complement component 3 (C3) proteins was greatly induced by PQ exposure while the expression of fibrinogen γ-chain (FGG) and Ac-158 was dramatically reduced. To further investigate the possibility of ApoE, Pphg and FGG as useful diagnostic biomarkers of PQ poisoning, western blot and qRT-PCR analyses were conducted using cell lines as well as rat and human sera. The expression levels of ApoE, Hp and FGG were significantly altered in the presence of PQ in both rat and human serum suggesting that these proteins may be appropriate candidate molecular biomarkers for the early diagnosis of acute PQ intoxication.

  5. Negative and positive auto-regulation of BMP expression in early eye development.

    Science.gov (United States)

    Huang, Jie; Liu, Ying; Filas, Benjamen; Gunhaga, Lena; Beebe, David C

    2015-11-15

    Previous results have shown that Bone Morphogenetic Protein (BMP) signaling is essential for lens specification and differentiation. How BMP signals are regulated in the prospective lens ectoderm is not well defined. To address this issue we have modulated BMP activity in a chicken embryo pre-lens ectoderm explant assay, and also studied transgenic mice, in which the type I BMP receptors, Bmpr1a and Acvr1, are deleted from the prospective lens ectoderm. Our results show that chicken embryo pre-lens ectoderm cells express BMPs and require BMP signaling for lens specification in vitro, and that in vivo inhibition of BMP signals in the mouse prospective lens ectoderm interrupts lens placode formation and prevents lens invagination. Furthermore, our results provide evidence that BMP expression is negatively auto-regulated in the lens-forming ectoderm, decreasing when the tissue is exposed to exogenous BMPs and increasing when BMP signaling is prevented. In addition, eyes lacking BMP receptors in the prospective lens placode develop coloboma in the adjacent wild type optic cup. In these eyes, Bmp7 expression increases in the ventral optic cup and the normal dorsal-ventral gradient of BMP signaling in the optic cup is disrupted. Pax2 becomes undetectable and expression of Sfrp2 increases in the ventral optic cup, suggesting that increased BMP signaling alter their expression, resulting in failure to close the optic fissure. In summary, our results suggest that negative and positive auto-regulation of BMP expression is important to regulate early eye development.

  6. Salivary proteins and early childhood caries: A gel electrophoretic analysis

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    Sumati Bhalla

    2010-01-01

    Full Text Available Background: Early childhood caries (ECC is a common disease process that afflicts a large proportion of the child population worldwide. Extensive research in past indicates that it is the result of bacterial infection, also influenced by host and dietary factors. Current caries research seeks to identify risk factors as well as natural oral defenses that may protect against or prevent caries development. Saliva, in spite of being the strongest defense system, still has a wide array of properties and proteins whose role is yet not clearly known. Aim: To compare the resting human whole salivary characteristics in children with ECC and those who are caries free. Settings and Design: The study was conducted over a period of 9 months in 4- to 6-year-old 100 children comprising two groups - 50 with ECC and 50 caries free. Materials and Methods: The whole salivary flow rate, pH, mean protein concentration, and the electrophoretic profile of salivary proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE were compared among both groups. Statistical Analysis: The SPSS (version 11.0 software package was used to conduct the chi-square, Fisher′s exact and Pearson′s chi-square tests to compare the data. Results: On gel electrophoresis, there was a significant difference among both groups with caries-free subjects having a higher number of proline-rich protein bands, substantiating the protective role of this protein. A significantly higher number of glycoprotein bands were observed in the whole saliva of subjects with ECC. A significant inverse correlation between the mean protein concentration and the whole salivary flow rate was observed in both groups.

  7. Construction, Expression and Purification of SUMO1-GST Fusion Protein

    Institute of Scientific and Technical Information of China (English)

    QIAO Xiao-fang; FANG Xue-dong; LIU Jun

    2011-01-01

    Sumoylation is an important protein modification discovered recently. SUMO(small ubiquitin-related modifier) pathway regulates the protein stability and transcriptional activity with a 12-kDa small molecular protein,SUMO, ligated to the target protein. The purification of SUMO proteins is a key step to reveal their function. The purpose of this study was to construct the recombinant SUMO1 gene cloned to a pGEX-4T-1 vector to express and purify the SUMO1-GST fusion protein in Escherichia coli. First, the full length DNA sequence of SUMO1 gene was amplified by PCR and was ligated to pMD18-T vector. Then the SUMO1 gene was subcloned to pGEX-4T-1 prokaryotic expression vector between BamHI and XhoI sites, and transformed in Escherichia coli DH5a cells. The right colonies were identified by restrictive enzyme digestion and sequencing. The correct rebombinant plasmid of pGEX-4T-1-SUMO1 was transformed in Escherichia coli BL21 cells and then induced by IPTG(isopropyl-β-D-lthiogalacto-pyranoside) to express the SUMO1-GST fusion protein. The highly purified SUMOl-GST(glutathione S-transferase) fusion protein was obtained by affinity chromatography. Finally, the properties of SUMO1-GST fusion protein were confirmed by Coomassie brilliant blue strain and Western blot analysis. The recombinant plasmid of pGEX-4T-1-SUMO 1 was successfully constructed, and SUMO1-GST fusion proteins were successfully expressed.

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

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    Eneour Puill-Stephan

    Full Text Available BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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

  9. Loss of fragile histidine triad protein expression in inflammatory bowel disease

    Institute of Scientific and Technical Information of China (English)

    Chun-Mei Xu; Chuan-Hu Qiao

    2006-01-01

    AIM: To investigate the expression of fragile histidine triad (FHIT) protein in 64 patients with ulcerative colitis (UC) and Crohn's disease (CD), and its relation with clinicopathological data.METHODS: Rabbit-anti-FHIT antibody was used to detect FHIT protein expression in 64 formalin-fixed,paraffin-embedded tissue specimens of inflammatory bowel disease (IBD) by citrate-microwave-streptavidin (SP)-HRP immunohistochemical method.RESULTS: The positive FHIT protein expression was 22.79% ± 16.16%, 42.14% ± 16.82% in active and remittent phases of UC, 36.07% ± 19.23% in CD, and 57.05% ± 8.86% in normal colon mucosa. Statistically significant differences in FHIT protein expression were observed between the active and remittent phases of UC, between the active phase of UC and normal colon mucosa, as well as between the remittent phase of UC and normal colon mucosa, and between CD and normal colon mucosa.CONCLUSION: Our results show that FHIT protein expression is completely absent or reduced in IBD,suggesting that the FHIT gene might be associated with the oncogenesis and progression of IBD, an early event from inflammatory conditions to carcinoma in IBD.

  10. Involvement of skeletal muscle protein, glycogen, and fat metabolism in the adaptation on early lactation of dairy cows.

    Science.gov (United States)

    Kuhla, Björn; Nürnberg, Gerd; Albrecht, Dirk; Görs, Solvig; Hammon, Harald M; Metges, Cornelia C

    2011-09-02

    During early lactation, high-yielding dairy cows cannot consume enough feed to meet nutrient requirements. As a consequence, animals drop into negative energy balance and mobilize body reserves including muscle protein and glycogen for milk production, direct oxidation, and hepatic gluconeogenesis. To examine which muscle metabolic processes contribute to the adaptation during early lactation, six German Holstein cows were blood sampled and muscle biopsied throughout the periparturient period. From pregnancy to lactation, the free plasma amino acid pattern imbalanced and plasma glucose decreased. Several muscle amino acids, as well as total muscle protein, fat, and glycogen, and the expression of glucose transporter-4 were reduced within the first 4 weeks of lactation. The 2-DE and MALDI-TOF-MS analysis identified 43 differentially expressed muscle protein spots throughout the periparturient period. In early lactation, expression of cytoskeletal proteins and enzymes involved in glycogen synthesis and in the TCA cycle was decreased, whereas proteins related to glycolysis, fatty acid degradation, lactate, and ATP production were increased. On the basis of these results, we propose a model in which the muscle breakdown in early lactation provides substrates for milk production by a decoupled Cori cycle favoring hepatic gluconeogenesis and by interfering with feed intake signaling.

  11. THE EXPRESSION OF BCL-2 AND BAX PROTEINS IN GASTRIC CARCINOMA AND PRECANCEROUS LESIONS

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To investigate the variance of expression of bcl-2 and bax genes in the genesis of gastric carcinoma as well as their relationship. Methods Thirty-five cases of early-stage gastric carcinoma and Twenty-four cases of chronic atrophic gastritis were studied by immunohistochemical method. Results There were no statistical differences of bcl-2 expression levels between gastric carcinoma and atypical hyperplasia or paracancerous intestinalepithelial metaplasia(IEM)(P>0. 05). There were statistical differences of bcl-2 expression between normal epithelial tissues (or non-cancerous IEM) and the other three groups (P<0.05),but no statistical difference between the normal epithelial and the non-cancerous IEM group was observed (P>0. 05). The expressions of bax protein were found in the normal epithelial and the other groups in varying degrees,but there were no statistical differences between either two of the groups (P>0.05). The bcl-2/bax ratio was higher in early-stage gastric carcinoma,atypical hyperplasia and paracancerous intestinai-metaplasia than in the non-cancerous intestinal-metaplasia (P<0. 05) and normal epithelial tissues(P<0. 01). Conclusion The abnormal expression of bcl-2 protein and bax protein ,especially the increased bcl-2/bax ratio, probably play an important role in the course of carcinogenesis of gastric carcinoma.

  12. Expression of genes encoding extracellular matrix proteins: a macroarray study.

    Science.gov (United States)

    Futyma, Konrad; Miotła, Paweł; Różyńska, Krystyna; Zdunek, Małgorzata; Semczuk, Andrzej; Rechberger, Tomasz; Wojcierowski, Jacek

    2014-12-01

    Endometrial cancer (EC) is one of the most common gynecological malignancies in Poland, with well-established risk factors. Genetic instability and molecular alterations responsible for endometrial carcinogenesis have been systematically investigated. The aim of the present study was to investigate, by means of cDNA macroarrays, the expression profiles of genes encoding extracellular matrix (ECM) proteins in ECs. Tissue specimens were collected during surgical procedures from 40 patients with EC, and control tissue was collected from 9 patients with uterine leiomyomas. RNA was isolated and RT-PCR with radioisotope-labeled cDNA was performed. The levels of ECM protein gene expression in normal endometrial tissues were compared to the expression of these genes in EC specimens. Statistically significant differences in gene expression, stratified by clinical stage of the ECs, were detected for aggrecan, vitronectin, tenascin R, nidogen and two collagen proteins: type VIII chain α1 and type XI chain α2. All of these proteins were overexpressed in stage III endometrial carcinomas compared to levels in stage I and II uterine neoplasms. In conclusion, increased expression of genes encoding ECM proteins may play an important role in facilitating accelerated disease progression of human ECs.

  13. Expression of SKP2 Protein in Lung Carcinoma

    Institute of Scientific and Technical Information of China (English)

    ZHAO Jun; YANG Chun-lu; ZHANG Huan; DING Wei-zhong; LIU Zhi-ping; LIU Jing-yi

    2008-01-01

    Objective:To study the expressive characteristics of SKP2 protein in lung carcinoma and its implication for prognosis.Methods:The expression of SKP2 protein was detected in 89 non small cell lung carcinoma,13 small cell lung carcinoma,10 lung benign lesion tissues by Tissue Chip and Immunohistochemistry technology.Results:The positive rate of SKP2 protein staining was(23.52±13.57)% in non small cell lung carcinoma and (53.85+12.26)% in small cell lung carcinoma,which were significantly higher than(2.91±1.27)% in lung benign lesion tissues.It was highest in small cell lung carcinoma and lowest in lung benign lesion tissues,with a significant difference between them(P=0.000).The expressive level of SKP2 protein in lung carcinoma tissues was closely related to cell differentiation,lymph node metastasis and pathological types,but not to age,sex,smoking history,tumor site and size,and TNM staging.The survival analysis revealed that the 5-year survival rate of lung carcinoma patients was lower in SKP2 protein positive expression group than that in negative expression group(P1=0.003/0.002;r=-0.275,P2=0.005).Conclusion:The positive expression of SKP2 protein is higher in lung carcinoma than in lung benign lesion tissues.in particular,much higher in small cell lung carcinoma.In lung carcinoma,its expressive level was closely related to cell differentiation,lymph node metastasis and pathological types.Moreover,it may be an independent factor to prognosis of patients with lung carcinoma.

  14. 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.

  15. Expression of Extracellular Superoxide Dismutase Protein in Diabetes

    Directory of Open Access Journals (Sweden)

    Chul Han Kim

    2013-09-01

    Full Text Available Background Diabetes is characterized by chronic hyperglycemia, which can increase reactiveoxygen species (ROS production by the mitochondrial electron transport chain. The formationof ROS induces oxidative stress and activates oxidative damage-inducing genes in cells. Noresearch has been published on oxidative damage-related extracellular superoxide dismutase(EC-SOD protein levels in human diabetic skin. We investigated the expression of EC-SOD indiabetic skin compared with normal skin tissue in vivo.Methods The expression of EC-SOD protein was evaluated by western blotting in 6 diabeticskin tissue samples and 6 normal skin samples. Immunohistochemical staining was also carriedout to confirm the EC-SOD expression level in the 6 diabetic skin tissue samples.Results The western blotting showed significantly lower EC-SOD protein expression in thediabetic skin tissue than in the normal tissue. Immunohistochemical examination of EC-SODprotein expression supported the western blotting analysis.Conclusions Diabetic skin tissues express a relatively small amount of EC-SOD protein andmay not be protected against oxidative stress. We believe that EC-SOD is related to the alteredmetabolic state in diabetic skin, which elevates ROS production.

  16. Raf-1 kinase inhibitory protein expression in thyroid carcinomas.

    Science.gov (United States)

    Kim, Hyun-Soo; Kim, Gou Young; Lim, Sung-Jig; Kim, Youn Wha

    2010-12-01

    Raf-1 kinase inhibitory protein (RKIP) has been implicated in several fundamental signal transduction pathways that control cellular growth, differentiation, apoptosis and migration. RKIP is reduced in a variety of human carcinomas, but RKIP expression in thyroid carcinomas has not been analyzed at the protein level. In this study, we examined the immunohistochemical expression of RKIP in various subtypes of thyroid carcinoma. Immunostaining for RKIP was performed on 104 cases of primary thyroid carcinoma (40 papillary, 29 follicular, 11 medullary, 11 poorly differentiated, and 13 anaplastic carcinomas) and 26 cases of nodal metastatic tumor (17 papillary, 4 medullary, and 5 anaplastic carcinomas). Normal thyroid tissue and all cases of follicular, papillary, and medullary carcinomas showed uniform, strong cytoplasmic immunoreactivity for RKIP. With the exception of one case, poorly differentiated carcinomas also revealed strong RKIP expression. In contrast, RKIP expression was completely absent in all anaplastic carcinomas. The transition zone from the differentiated carcinoma component (strong RKIP expression) to the anaplastic carcinoma component (no RKIP expression) demonstrated a completely opposite pattern of RKIP immunoreactivity. This reduction of RKIP expression in anaplastic carcinoma was statistically significant (P carcinomas showed uniform, strong cytoplasmic RKIP immunoreactivity, in contrast, in metastatic anaplastic carcinomas, RKIP expression was completely absent. RKIP expression is significantly reduced in anaplastic thyroid carcinoma as compared to other subtypes of thyroid carcinoma. Further studies are necessary to elucidate the precise mechanism of RKIP action in anaplastic thyroid carcinoma.

  17. Expression and biochemical characterization of recombinant human epididymis protein 4.

    Science.gov (United States)

    Hua, Ling; Liu, Yunhui; Zhen, Shuai; Wan, Deyou; Cao, Jiyue; Gao, Xin

    2014-10-01

    Whey acidic proteins (WAP) belong to a large gene family of antibacterial peptides that perform critical immune system functions. The function of human epididymis protein 4 (HE4), a 124-amino acid long polypeptide that has two whey acidic protein four-disulfide core (WFDC) domains, is not well studied. Here, a fusion gene encoding the HE4 protein fused to an IgG1 Fc domain was constructed. The recombinant HE4 protein was expressed as a secretory protein in Pichia pastoris and mammalian HEK293-F cells and was subsequently purified. Our data suggested that the HE4 protein produced by these two expression systems bound to both gram-negative and gram-positive bacteria, but demonstrated slightly inhibitory activity towards the growth of Staphylococcus aureus. Moreover, HE4 exhibited proteinase inhibitory activity towards trypsin, elastase, matrix metallopeptidase 9, and the secretory proteinases from Bacillus subtilis. The effects of glycosylation on the biochemical characterization of HE4 were also investigated. LC-ESI-MS glycosylation analysis showed that the high-mannose glycosylated form of HE4 expressed by P. pastoris has lower biological activity when compared to its complex-glycosylated form produced from HEK293-F cells. The implications of this are discussed, which may be provide theoretical basis for its important role in the development of cancer and innate immune system.

  18. Expression of Escherichia coli cspA during early exponential growth at 37 °C.

    Science.gov (United States)

    Brandi, Anna; Pon, Cynthia L

    2012-01-25

    CspA is a small (7.4 kDa) nucleic acid binding protein of Escherichia coli whose expression is stimulated after cold-stress but whose level is also extraordinarily high during the early phase of growth of non-stressed cells. In this study the relationship existing between cspA transcription/translation on the one hand and the acquisition of critical mass for cell division and chromosome replication, on the other, in stationary phase cells subjected to a nutritional up-shift at 37 °C has been analyzed. Measurements of optical density and viable counts, pulse-chase, real-time PCR and immunodetection experiments, as well as cytofluorimetric and DNA duplication analyses show that synthesis of new CspA molecules at 37 °C is not only restricted to the lag phase ensuing the nutritional up-shift, but continues also during the first stages of logarithmic growth, when cells have already started dividing; although the early synthesized molecules are diluted by the following cell divisions and new synthesis occurs at an extremely low level, cspA mRNA and CspA continue to be present. A possible explanation for the apparent paradox that cspA is activated not only following cold stress, but also under non-stress and other stress conditions which entail a down-regulation of bulk gene expression and protein synthesis is presented.

  19. Protein Tyrosine Phosphatase PRL2 Mediates Notch and Kit Signals in Early T Cell Progenitors.

    Science.gov (United States)

    Kobayashi, Michihiro; Nabinger, Sarah C; Bai, Yunpeng; Yoshimoto, Momoko; Gao, Rui; Chen, Sisi; Yao, Chonghua; Dong, Yuanshu; Zhang, Lujuan; Rodriguez, Sonia; Yashiro-Ohtani, Yumi; Pear, Warren S; Carlesso, Nadia; Yoder, Mervin C; Kapur, Reuben; Kaplan, Mark H; Daniel Lacorazza, Hugo; Zhang, Zhong-Yin; Liu, Yan

    2017-04-01

    The molecular pathways regulating lymphoid priming, fate, and development of multipotent bone marrow hematopoietic stem and progenitor cells (HSPCs) that continuously feed thymic progenitors remain largely unknown. While Notch signal is indispensable for T cell specification and differentiation, the downstream effectors are not well understood. PRL2, a protein tyrosine phosphatase that regulates hematopoietic stem cell proliferation and self-renewal, is highly expressed in murine thymocyte progenitors. Here we demonstrate that protein tyrosine phosphatase PRL2 and receptor tyrosine kinase c-Kit are critical downstream targets and effectors of the canonical Notch/RBPJ pathway in early T cell progenitors. While PRL2 deficiency resulted in moderate defects of thymopoiesis in the steady state, de novo generation of T cells from Prl2 null hematopoietic stem cells was significantly reduced following transplantation. Prl2 null HSPCs also showed impaired T cell differentiation in vitro. We found that Notch/RBPJ signaling upregulated PRL2 as well as c-Kit expression in T cell progenitors. Further, PRL2 sustains Notch-mediated c-Kit expression and enhances stem cell factor/c-Kit signaling in T cell progenitors, promoting effective DN1-DN2 transition. Thus, we have identified a critical role for PRL2 phosphatase in mediating Notch and c-Kit signals in early T cell progenitors. Stem Cells 2017;35:1053-1064.

  20. Early Exercise Affects Mitochondrial Transcription Factors Expression after Cerebral Ischemia in Rats

    Directory of Open Access Journals (Sweden)

    Yongshan Hu

    2012-02-01

    Full Text Available Increasing evidence shows that exercise training is neuroprotective after stroke, but the underlying mechanisms are unknown. To clarify this critical issue, the current study investigated the effects of early treadmill exercise on the expression of mitochondrial biogenesis factors. Adult rats were subjected to ischemia induced by middle cerebral artery occlusion followed by reperfusion. Expression of two genes critical for transcriptional regulation of mitochondrial biogenesis, peroxisome proliferator-activated receptor coactivator-1 (PGC-1 and nuclear respiratory factor-1 (NRF-1, were examined by RT-PCR after five days of exercise starting at 24 h after ischemia. Mitochondrial protein cytochrome C oxidase subunit IV (COX IV was detected by Western blot. Neurological status and cerebral infarct volume were evaluated as indices of brain damage. Treadmill training increased levels of PGC-1 and NRF-1 mRNA, indicating that exercise promotes rehabilitation after ischemia via regulation of mitochondrial biogenesis.

  1. Expression of Structural Protein E2 of Hepatitis C Virus

    Institute of Scientific and Technical Information of China (English)

    GUO Tai-lin; YE Lin-bo; MAO Can-quan

    2005-01-01

    The E2 glycoprotein is one of the structural components of the hepatitis C virus (HCV) virion. It elicits production of neutralising antibodies against the virus, and is involved in viral morphogenesis. The protein is considered as a major candidate for anti- HCV vaccine. Despitethis, little is known about this protein. Previous studies have focused on the and functional analysis of the glycosylated forms. This report describes expression of the E2 (recE2) in different forms and in different expression systems in Escherichia coli cells and in mammalian cells in order to obtain enough protein efficiently in vitro, in addition we also analysed the usage of rare codons in the genes of E2 and CORE. All results have shown that great efforts should be made to improve the expression efficiency of E2 in bacteria or mammalian cells.

  2. Expression and Purification of SARS Coronavirus Membrane Protein

    Institute of Scientific and Technical Information of China (English)

    戴五星; 雷明军; 吴少庭; 陈智浩; 梁靓; 潘晖榕; 秦莉; 高士同; 袁仕善; 张仁利

    2004-01-01

    To construct a recombinant plasmid Pet23a-M, the gene encoding severe acute respiratory syndrome (SARS) coronavirus membrane protein was amplified by RT-PCR and cloned into the expression plasmid Pet23a. Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis revealed that the cloned DNA sequence was the same as that reported. The re combinants were transformed into Escherichia coli (E. Coli) BL21 (DE3) and induced by Isopropylβ-D-thiogalactopyranoside (IPTG). The expression of 27 kD (1 kD=0. 992 1 ku) protein was detected by SDS-PAGE and pured by metal chelated chromatography. Results of Western-blot showed that this expressed protein could react with antibodies in sera of SARS patients during convalescence. This provided the basis for the further study on SARS virus vaccine and diagnostic agents.

  3. Expression of protein-coding genes embedded in ribosomal DNA

    DEFF Research Database (Denmark)

    Johansen, Steinar D; Haugen, Peik; Nielsen, Henrik

    2007-01-01

    Ribosomal DNA (rDNA) is a specialised chromosomal location that is dedicated to high-level transcription of ribosomal RNA genes. Interestingly, rDNAs are frequently interrupted by parasitic elements, some of which carry protein genes. These are non-LTR retrotransposons and group II introns...... that encode reverse transcriptase-like genes, and group I introns and archaeal introns that encode homing endonuclease genes (HEGs). Although rDNA-embedded protein genes are widespread in nuclei, organelles and bacteria, there is surprisingly little information available on how these genes are expressed....... Exceptions include a handful of HEGs from group I introns. Recent studies have revealed unusual and essential roles of group I and group I-like ribozymes in the endogenous expression of HEGs. Here we discuss general aspects of rDNA-embedded protein genes and focus on HEG expression from group I introns...

  4. Using ion exchange chromatography to purify a recombinantly expressed protein.

    Science.gov (United States)

    Duong-Ly, Krisna C; Gabelli, Sandra B

    2014-01-01

    Ion exchange chromatography (IEX) separates molecules by their surface charge, a property that can vary vastly between different proteins. There are two types of IEX, cation exhange and anion exchange chromatography. The protocol that follows was designed by the authors for anion exchange chromatography of a recombinantly expressed protein having a pI of 4.9 and containing two cysteine residues and one tryptophan residue, using an FPLC system. Prior to anion exchange, the protein had been salted out using ammonium sulfate precipitation and partially purified via hydrophobic interaction chromatography (see Salting out of proteins using ammonium sulfate precipitation and Use and Application of Hydrophobic Interaction Chromatography for Protein Purification). Slight modifications to this protocol may be made to accommodate both the protein of interest and the availability of equipment.

  5. The expression and significance of p53 protein and Ki-67 protein in pterygium

    Directory of Open Access Journals (Sweden)

    Ljubojević Vesna

    2016-01-01

    Full Text Available Background/Aim. Pterygium is considered to be a degenerative disease of the conjunctiva, however, the presence of tumor markers in pterygium reinforces the hypothesis that this lesion is similar to tumor. Inactivation of p53 function removes an obstacle to increased proliferation. Factors affecting the prevalence of p53 expression in pterygium deserve investigation. The aim of the study was to investigate the expression of p53 and Ki-67 proteins in pterygium and normal conjunctiva, the effects of gender and age on p53 expression, and the relationship between the expression of p53 and Ki-67 proteins. Methods. A total of 34 samples of pterygium and 34 samples of the normal conjunctiva were analyzed. The samples were studied by immunohistochemistry using antibodies against p53 and Ki-67. Results. Totally 15 (44% samples of pterygia were p53 positive. Correlations between the expression of p53 protein and sex, and age were not established. The number of Ki-67 positive cells in pterygium (9.74% was significantly higher than the number of Ki-67 positive cells in the normal conjunctiva (1.74%, (p = 0.001. Between the expression of p53 protein and Ki-67 protein in pterygium there was a significant positive correlation (p = 0.000. Conclusion. The prevalence of p53 positive samples of pterygium was 44%. The influence of sex and age on p53 protein expression in pterygium was not found. The increased proliferative acivity was present in the epithelium of pterygium. The expression of Ki-67 protein is associated with the expression of p53 protein in pterygium. The findings of our study support the thesis of pterygium as tissue growth disorder.

  6. Expression Profiles of Epithelial-Mesenchymal Transition-Associated Proteins in Epithelial Ovarian Carcinoma

    Directory of Open Access Journals (Sweden)

    Mi-Kyung Kim

    2014-01-01

    Full Text Available Epithelial-mesenchymal transition (EMT has been suggested to contribute to tumor progression and acquisition of therapeutic resistance. To assess the clinical significance of EMT-associated proteins, we evaluated the expression of Snail and Slug, the key regulators of EMT, in the primary ovarian cancer samples (n=103 by immunohistochemistry. Snail was differentially expressed according to the histologic subtype (P=0.001 and was predominantly expressed in serous and endometrioid types. In the serous and endometrioid adenocarcinomas, the expression of Snail remained high across the stage and grade, suggesting its role in the early phase of carcinogenesis. However, the expression of Snail and Slug was not related to chemoresistance and poor prognosis and did not serve as independent predictive or prognostic marker.

  7. Global absolute quantification reveals tight regulation of protein expression in single Xenopus eggs.

    Science.gov (United States)

    Smits, Arne H; Lindeboom, Rik G H; Perino, Matteo; van Heeringen, Simon J; Veenstra, Gert Jan C; Vermeulen, Michiel

    2014-09-01

    While recent developments in genomic sequencing technology have enabled comprehensive transcriptome analyses of single cells, single cell proteomics has thus far been restricted to targeted studies. Here, we perform global absolute protein quantification of fertilized Xenopus laevis eggs using mass spectrometry-based proteomics, quantifying over 5800 proteins in the largest single cell proteome characterized to date. Absolute protein amounts in single eggs are highly consistent, thus indicating a tight regulation of global protein abundance. Protein copy numbers in single eggs range from tens of thousands to ten trillion copies per cell. Comparison between the single-cell proteome and transcriptome reveal poor expression correlation. Finally, we identify 439 proteins that significantly change in abundance during early embryogenesis. Downregulated proteins include ribosomal proteins and upregulated proteins include basal transcription factors, among others. Many of these proteins do not show regulation at the transcript level. Altogether, our data reveal that the transcriptome is a poor indicator of the proteome and that protein levels are tightly controlled in X. laevis eggs.

  8. Express your LOV: an engineered flavoprotein as a reporter for protein expression and purification.

    Directory of Open Access Journals (Sweden)

    Jayde A Gawthorne

    Full Text Available In this work, we describe the utility of Light, Oxygen, or Voltage-sensing (LOV flavoprotein domains from plant phototropins as a reporter for protein expression and function. Specifically, we used iLOV, an enhanced and more photostable variant of LOV. A pET-based plasmid for protein expression was constructed, encoding a C terminal iLOV-octahistidine (His8-tag and a HRV 3C protease cleavage recognition site. Ten different proteins, with various sub-cellular locations, were cloned into the plasmid, creating iLOV-His8 tag fusions. To test protein expression and how iLOV could be used as a reporter, the proteins were expressed in three different cell lines, in four different culture media, at two different temperatures. To establish whether the presence of the iLOV tag could have an impact on the functionality, one of the proteins, EspG, was over-expressed and purified. EspG is an "effector" protein normally produced by enterohemorrhagic E. coli strains and "injected" into host cells via the T3SS. We tested functionality of EspG-iLOV fusion by performing functional studies of EspG in mammalian host cells. When EspG-iLOV was microinjected into the host cell, the Golgi apparatus was completely disrupted as had previously been observed for EspG.

  9. Early Epigenetic Downregulation of microRNA-192 Expression Promotes Pancreatic Cancer Progression.

    Science.gov (United States)

    Botla, Sandeep K; Savant, Soniya; Jandaghi, Pouria; Bauer, Andrea S; Mücke, Oliver; Moskalev, Evgeny A; Neoptolemos, John P; Costello, Eithne; Greenhalf, William; Scarpa, Aldo; Gaida, Matthias M; Büchler, Markus W; Strobel, Oliver; Hackert, Thilo; Giese, Nathalia A; Augustin, Hellmut G; Hoheisel, Jörg D

    2016-07-15

    Pancreatic ductal adenocarcinoma (PDAC) is characterized by very early metastasis, suggesting the hypothesis that metastasis-associated changes may occur prior to actual tumor formation. In this study, we identified miR-192 as an epigenetically regulated suppressor gene with predictive value in this disease. miR-192 was downregulated by promoter methylation in both PDAC and chronic pancreatitis, the latter of which is a major risk factor for the development of PDAC. Functional studies in vitro and in vivo in mouse models of PDAC showed that overexpression of miR-192 was sufficient to reduce cell proliferation and invasion. Mechanistic analyses correlated changes in miR-192 promoter methylation and expression with epithelial-mesenchymal transition. Cell proliferation and invasion were linked to altered expression of the miR-192 target gene SERPINE1 that is encoding the protein plasminogen activator inhibitor-1 (PAI-1), an established regulator of these properties in PDAC cells. Notably, our data suggested that invasive capacity was altered even before neoplastic transformation occurred, as triggered by miR-192 downregulation. Overall, our results highlighted a role for miR-192 in explaining the early metastatic behavior of PDAC and suggested its relevance as a target to develop for early diagnostics and therapy. Cancer Res; 76(14); 4149-59. ©2016 AACR.

  10. SPINK 1 Protein Expression and Prostate Cancer Progression

    Science.gov (United States)

    Flavin, Richard; Pettersson, Andreas; Hendrickson, Whitney K.; Fiorentino, Michelangelo; Finn, Stephen; Kunz, Lauren; Judson, Gregory L.; Lis, Rosina; Bailey, Dyane; Fiore, Christopher; Nuttall, Elizabeth; Martin, Neil E.; Stack, Edward; Penney, Kathryn L.; Rider, Jennifer R.; Sinnott, Jennifer; Sweeney, Christopher; Sesso, Howard D.; Fall, Katja; Giovannucci, Edward; Kantoff, Philip; Stampfer, Meir; Loda, Massimo; Mucci, Lorelei A.

    2014-01-01

    Purpose SPINK1 over-expression has been described in prostate cancer and is linked with poor prognosis in many cancers. The objective of this study was to characterize the association between SPINK1 over-expression and prostate cancer specific survival. Experimental Design The study included 879 participants in the US Physicians’ Health Study and Health Professionals Follow–Up Study, diagnosed with prostate cancer (1983 – 2004) and treated by radical prostatectomy. Protein tumor expression of SPINK1 was evaluated by immunohistochemistry on tumor tissue microarrays. Results 74/879 (8%) prostate cancer tumors were SPINK1 positive. Immunohistochemical data was available for PTEN, p-Akt, pS6, stathmin, androgen receptor (AR) and ERG (as a measure of the TMPRSS2:ERG translocation). Compared to SPINK1 negative tumors, SPINK1 positive tumors showed higher PTEN and stathmin expression, and lower expression of AR (p<0.01). SPINK1 over-expression was seen in 47 of 427 (11%) ERG negative samples and in 19 of 427 (4%) ERG positive cases (p=0.0003). We found no significant associations between SPINK1 status and Gleason grade or tumor stage. There was no association between SPINK1 expression and biochemical recurrence (p=0.56). Moreover, there was no association between SPINK1 expression and prostate cancer mortality (there were 75 lethal cases of prostate cancer during a mean of 13.5 years follow-up [HR 0.71 (95% confidence interval 0.29–1.76)]). Conclusions Our results suggest that SPINK1 protein expression may not be a predictor of recurrence or lethal prostate cancer amongst men treated by radical prostatectomy. SPINK1 and ERG protein expression do not appear to be entirely mutually exclusive, as some previous studies have suggested. PMID:24687926

  11. Expression and export: recombinant protein production systems for Aspergillus.

    Science.gov (United States)

    Fleissner, André; Dersch, Petra

    2010-07-01

    Several Aspergillus species, in particular Aspergillus niger and Aspergillus oryzae, are widely used as protein production hosts in various biotechnological applications. In order to improve the expression and secretion of recombinant proteins in these filamentous fungi, several novel genetic engineering strategies have been developed in recent years. This review describes state-of-the-art genetic manipulation technologies used for strain improvement, as well as recent advances in designing the most appropriate engineering strategy for a particular protein production process. Furthermore, current developments in identifying bottlenecks in the protein production and secretion pathways are described and novel approaches to overcome these limitations are introduced. An appropriate combination of expression vectors and optimized host strains will provide cell factories customized for each production process and expand the great potential of Aspergilli as biotechnology workhorses to more complex multi-step industrial applications.

  12. Decreased Expression of GPER1 Gene and Protein in Goiter

    Directory of Open Access Journals (Sweden)

    Raquel Weber

    2015-01-01

    Full Text Available Goiter is more common in women, suggesting that estrogen could be involved in its physiopathology. The presence of classical estrogen receptors (ERα and ERβ has been described in thyroid tissue, suggesting a direct effect of estrogen on the gland. A nonclassic estrogen receptor, the G-protein-coupled estrogen receptor (GPER1, has been described recently in several tissues. However, in goiter, the presence of this receptor has not been studied yet. We investigated GPER1 gene and protein expressions in normal thyroid and goiter using reverse transcription quantitative polymerase chain reaction (RT-qPCR and Western blot, respectively. In normal thyroid (n=16 and goiter (n=19, GPER1 gene was expressed in all samples, while GPER1 protein was expressed in all samples of normal thyroid (n=15 but in only 72% of goiter samples (n=13. When comparing GPER1 gene and protein levels in both conditions, gene expression and protein levels were higher in normal thyroid than in goiter, suggesting a role of this receptor in this condition. Further studies are needed to elucidate the role of GPER1 in normal thyroid and goiter.

  13. p53 and MDM2 protein expression in actinic cheilitis.

    Science.gov (United States)

    de Freitas, Maria da Conceição Andrade; Ramalho, Luciana Maria Pedreira; Xavier, Flávia Caló Aquino; Moreira, André Luis Gomes; Reis, Sílvia Regina Almeida

    2008-01-01

    Actinic cheilitis is a potentially malignant lip lesion caused by excessive and prolonged exposure to ultraviolet radiation, which can lead to histomorphological alterations indicative of abnormal cell differentiation. In this pathology, varying degrees of epithelial dysplasia may be found. There are few published studies regarding the p53 and MDM2 proteins in actinic cheilitis. Fifty-eight cases diagnosed with actinic cheilitis were histologically evaluated using Banóczy and Csiba (1976) parameters, and were subjected to immunohistochemical analysis using the streptavidin-biotin method in order to assess p53 and MDM2 protein expression. All studied cases expressed p53 proteins in basal and suprabasal layers. In the basal layer, the nuclei testing positive for p53 were stained intensely, while in the suprabasal layer, cells with slightly stained nuclei were predominant. All cases also tested positive for the MDM2 protein, but with varying degrees of nuclear expression and a predominance of slightly stained cells. A statistically significant correlation between the percentage of p53 and MDM2-positive cells was established, regardless of the degree of epithelial dysplasia. The expression of p53 and MDM2 proteins in actinic cheilitis can be an important indicator in lip carcinogenesis, regardless of the degree of epithelial dysplasia.

  14. p53 and MDM2 protein expression in actinic cheilitis

    Directory of Open Access Journals (Sweden)

    Maria da Conceição Andrade de Freitas

    2008-12-01

    Full Text Available Actinic cheilitis is a potentially malignant lip lesion caused by excessive and prolonged exposure to ultraviolet radiation, which can lead to histomorphological alterations indicative of abnormal cell differentiation. In this pathology, varying degrees of epithelial dysplasia may be found. There are few published studies regarding the p53 and MDM2 proteins in actinic cheilitis. Fifty-eight cases diagnosed with actinic cheilitis were histologically evaluated using Banóczy and Csiba (1976 parameters, and were subjected to immunohistochemical analysis using the streptavidin-biotin method in order to assess p53 and MDM2 protein expression. All studied cases expressed p53 proteins in basal and suprabasal layers. In the basal layer, the nuclei testing positive for p53 were stained intensely, while in the suprabasal layer, cells with slightly stained nuclei were predominant. All cases also tested positive for the MDM2 protein, but with varying degrees of nuclear expression and a predominance of slightly stained cells. A statistically significant correlation between the percentage of p53 and MDM2-positive cells was established, regardless of the degree of epithelial dysplasia. The expression of p53 and MDM2 proteins in actinic cheilitis can be an important indicator in lip carcinogenesis, regardless of the degree of epithelial dysplasia.

  15. Interfacial polymerization for colorimetric labeling of protein expression in cells.

    Directory of Open Access Journals (Sweden)

    Jacob L Lilly

    Full Text Available Determining the location of rare proteins in cells typically requires the use of on-sample amplification. Antibody based recognition and enzymatic amplification is used to produce large amounts of visible label at the site of protein expression, but these techniques suffer from the presence of nonspecific reactivity in the biological sample and from poor spatial control over the label. Polymerization based amplification is a recently developed alternative means of creating an on-sample amplification for fluorescence applications, while not suffering from endogenous labels or loss of signal localization. This manuscript builds upon polymerization based amplification by developing a stable, archivable, and colorimetric mode of amplification termed Polymer Dye Labeling. The basic concept involves an interfacial polymer grown at the site of protein expression and subsequent staining of this polymer with an appropriate dye. The dyes Evans Blue and eosin were initially investigated for colorimetric response in a microarray setting, where both specifically stained polymer films on glass. The process was translated to the staining of protein expression in human dermal fibroblast cells, and Polymer Dye Labeling was specific to regions consistent with desired protein expression. The labeling is stable for over 200 days in ambient conditions and is also compatible with modern mounting medium.

  16. Interfacial polymerization for colorimetric labeling of protein expression in cells.

    Science.gov (United States)

    Lilly, Jacob L; Sheldon, Phillip R; Hoversten, Liv J; Romero, Gabriela; Balasubramaniam, Vivek; Berron, Brad J

    2014-01-01

    Determining the location of rare proteins in cells typically requires the use of on-sample amplification. Antibody based recognition and enzymatic amplification is used to produce large amounts of visible label at the site of protein expression, but these techniques suffer from the presence of nonspecific reactivity in the biological sample and from poor spatial control over the label. Polymerization based amplification is a recently developed alternative means of creating an on-sample amplification for fluorescence applications, while not suffering from endogenous labels or loss of signal localization. This manuscript builds upon polymerization based amplification by developing a stable, archivable, and colorimetric mode of amplification termed Polymer Dye Labeling. The basic concept involves an interfacial polymer grown at the site of protein expression and subsequent staining of this polymer with an appropriate dye. The dyes Evans Blue and eosin were initially investigated for colorimetric response in a microarray setting, where both specifically stained polymer films on glass. The process was translated to the staining of protein expression in human dermal fibroblast cells, and Polymer Dye Labeling was specific to regions consistent with desired protein expression. The labeling is stable for over 200 days in ambient conditions and is also compatible with modern mounting medium.

  17. Nanog suppresses the expression of vasa by directly regulating nlk1 in the early zebrafish embryo.

    Science.gov (United States)

    Liu, Yanhua; Xue, Weiwei; Zhu, Lin; Ye, Ding; Zhu, Xiaoqin; Wang, Huannan; Sun, Yonghua; Deng, Fengjiao

    2017-07-28

    Nanog is a homeodomain transcription factor that is essential for maintenance of pluripotency and self-renewal of embryonic stem cells (ESCs). In the present study, we demonstrate that zebrafish Nanog (zNanog) directly binds to the promoter region of zebrafish nlk1 (znlk1) by ChIP-Seq analysis and that it up-regulates the expression of znlk1 in fibroblast-like embryonic cells of Danio rerio (ZEM-2S cells) and in zebrafish embryos at 30% epiboly both at the mRNA and protein levels. In addition, compared with control (MO-C) embryos at 30% epiboly, the mRNA and protein expression of vasa and the numbers of vasa-positive cells were increased in embryos injected with zNanog morpholino (MO-zNanog). Further, injection of znlk1 mRNA into zNanog-depleted embryos restored the expression of vasa and the number of vasa-positive cells. These data indicated that zNanog up-regulates the expression of znlk1 through directly binding to the znlk1 promoter, thereby suppressing the expression of vasa. Vasa is a marker gene for PGCs. Our results suggest that zNanog plays a role in restraint of PGC cell number through regulating the expression of znlk1 in the early embryonic development. The current results provide fundamental information to support further investigation regarding the regulatory mechanism of zNanog during the development of PGCs. Copyright © 2017. Published by Elsevier B.V.

  18. Gene expression profiles deciphering leaf senescence variation between early- and late-senescence cotton lines.

    Directory of Open Access Journals (Sweden)

    Xiangqiang Kong

    Full Text Available Leaf senescence varies greatly among genotypes of cotton (Gossypium hirsutium L, possibly due to the different expression of senescence-related genes. To determine genes involved in leaf senescence, we performed genome-wide transcriptional profiling of the main-stem leaves of an early- (K1 and a late-senescence (K2 cotton line at 110 day after planting (DAP using the Solexa technology. The profiling analysis indicated that 1132 genes were up-regulated and 455 genes down-regulated in K1 compared with K2 at 110 DAP. The Solexa data were highly consistent with, and thus were validated by those from real-time quantitative PCR (RT-PCR. Most of the genes related to photosynthesis, anabolism of carbohydrates and other biomolecules were down-regulated, but those for catabolism of proteins, nucleic acids, lipids and nutrient recycling were mostly up-regulated in K1 compared with K2. Fifty-one differently expressed hormone-related genes were identified, of which 5 ethylene, 3 brassinosteroid (BR, 5 JA, 18 auxin, 8 GA and 1 ABA related genes were up-regulated in K1 compared with K2, indicating that these hormone-related genes might play crucial roles in early senescence of K1 leaves. Many differently expressed transcription factor (TF genes were identified and 11 NAC and 8 WRKY TF genes were up-regulated in K1 compared with K2, suggesting that TF genes, especially NAC and WRKY genes were involved in early senescence of K1 leaves. Genotypic variation in leaf senescence was attributed to differently expressed genes, particularly hormone-related and TF genes.

  19. Recombinant protein expression by targeting pre-selected chromosomal loci

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    Krömer Wolfgang

    2009-12-01

    Full Text Available Abstract Background Recombinant protein expression in mammalian cells is mostly achieved by stable integration of transgenes into the chromosomal DNA of established cell lines. The chromosomal surroundings have strong influences on the expression of transgenes. The exploitation of defined loci by targeting expression constructs with different regulatory elements is an approach to design high level expression systems. Further, this allows to evaluate the impact of chromosomal surroundings on distinct vector constructs. Results We explored antibody expression upon targeting diverse expression constructs into previously tagged loci in CHO-K1 and HEK293 cells that exhibit high reporter gene expression. These loci were selected by random transfer of reporter cassettes and subsequent screening. Both, retroviral infection and plasmid transfection with eGFP or antibody expression cassettes were employed for tagging. The tagged cell clones were screened for expression and single copy integration. Cell clones producing > 20 pg/cell in 24 hours could be identified. Selected integration sites that had been flanked with heterologous recombinase target sites (FRTs were targeted by Flp recombinase mediated cassette exchange (RMCE. The results give proof of principle for consistent protein expression upon RMCE. Upon targeting antibody expression cassettes 90-100% of all resulting cell clones showed correct integration. Antibody production was found to be highly consistent within the individual cell clones as expected from their isogenic nature. However, the nature and orientation of expression control elements revealed to be critical. The impact of different promoters was examined with the tag-and-targeting approach. For each of the chosen promoters high expression sites were identified. However, each site supported the chosen promoters to a different extent, indicating that the strength of a particular promoter is dominantly defined by its chromosomal context

  20. hTERT protein expression is independent of clinicopathological parameters and c-Myc protein expression in human breast cancer

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    Meligonis G

    2005-01-01

    Full Text Available Abstract Background Telomerase is a ribonucleoprotein enzyme that synthesises telomeres after cell division and maintains chromosomal length and stability thus leading to cellular immortalisation. The hTERT (human telomerase reverse transcriptase subunit seems to be the rate-limiting determinant of telomerase and knowledge of factors controlling hTERT transcription may be useful in therapeutic strategies. The hTERT promoter contains binding sites for c-Myc and there is some experimental and in vitro evidence that c-Myc may increase hTERT expression. We previously reported no correlation between c-Myc mRNA expression and hTERT mRNA or telomerase activity in human breast cancer. This study aims to examine the correlation between hTERT expression as determined by immunohistochemistry and c-Myc expression, lymph node status, and tumour size and grade in human breast cancer. Materials and methods The immunohistochemical expression of hTERT and c-Myc was investigated in 38 malignant breast tumours. The expression of hTERT was then correlated with the lymph node status, c-Myc expression and other clinicopathological parameters of the tumours. Results hTERT expression was positive in 27 (71% of the 38 tumours. 15 (79% of 19 node positive tumours were hTERT positive compared with 11 (63% of 19 node negative tumours. The expression was higher in node positive tumours but this failed to reach statistical significance (p = 0.388. There was no significant association with tumour size, tumour grade or c-Myc expression. However, hTERT expression correlated positively with patients' age (correlation coefficient = 0.415, p = 0.0097. Conclusion hTERT protein expression is independent of lymph node status, tumour size and grade and c-Myc protein expression in human breast cancer

  1. Effects of ischemic preconditioning on cyclinD1 expression during early ischemic reperfusion in rats

    Institute of Scientific and Technical Information of China (English)

    Fang-Gang Cai; Jian-Sheng Xiao; Qi-Fa Ye

    2006-01-01

    AIM: To observe the effect of ischemic preconditioning on cyclinD1 expression in rat liver cells during early ischemic reperfusion.METHODS: Fifty-four SD rats were randomly divided into ischemic preconditioning group (IP), ischemia/reperfusion group (IR) and sham operation group (SO). The IP and IR groups were further divided into four sub-groups (n = 6). Sham operation group (SO)served as the control group (n = 6). A model of partial liver ischemia/reperfusion was used, in which rats were subjected to liver ischemia for 60 min prior to reperfusion. The animals in the IP group underwent ischemic preconditioning twice for 5 min each time prior to the ischemia/reperfusion challenge. After 0, 1, 2, and 4 h of reperfusion, serum and liver tissue in each group were collected to detect the level of serum ALT, liver histopathology and expression of cyclinD1 mRNA and protein. Flow cytometry was used to detect cell cycle as the quantity indicator of cell regeneration.RESULTS: Compared with IR group, IP group showed asignificantly lower ALT level in 1 h to 4 h sub-groups (P< 0.05). Proliferation index(PI) indicated by the S-phase and G2/M-phase ratio [(S+G2/M)/(G0/G1+S+G2/M)] was significantly increased in IP group at 0 and 1 h (26.44± 7.60% vs 18.56 ± 6.40%,41.87 ± 7.27% vs 20.25 ±6.70%, P < 0.05). Meanwhile, cyclinD1 protein expression could be detected in IP group. But in IR group, cyclinD1 protein expression occurred 2 h after reperfusion. The expression of cyclinD1 mRNA increased significantly in IP group at 0 and 1 h (0.568 ± 0.112 vs 0.274 ± 0.069, 0.762± 0.164 vs 0.348 ± 0.093, P < 0.05).CONCLUSION: Ischemic preconditioning can protect liver cells against ischemia/reperfusion injury, which may be related to cell proliferation and expression of cyclinD1 during early ischemic reperfusion.

  2. Expression profiling identifies genes expressed early during lint fibre initiation in cotton.

    Science.gov (United States)

    Wu, Yingru; Machado, Adriane C; White, Rosemary G; Llewellyn, Danny J; Dennis, Elizabeth S

    2006-01-01

    Cotton fibres are a subset of single epidermal cells that elongate from the seed coat to produce the long cellulose strands or lint used for spinning into yarn. To identify genes that might regulate lint fibre initiation, expression profiles of 0 days post-anthesis (dpa) whole ovules from six reduced fibre or fibreless mutants were compared with wild-type linted cotton using cDNA microarrays. Numerous clones were differentially expressed, but when only those genes that are normally expressed in the ovule outer integument (where fibres develop) were considered, just 13 different cDNA clones were down-regulated in some or all of the mutants. These included: a Myb transcription factor (GhMyb25) similar to the Antirrhinum Myb AmMIXTA, a putative homeodomain protein (related to Arabidopsis ATML1), a cyclin D gene, some previously identified fibre-expressed structural and metabolic genes, such as lipid transfer protein, alpha-expansin and sucrose synthase, as well as some unknown genes. Laser capture microdissection and reverse transcription-PCR were used to show that both the GhMyb25 and the homeodomain gene were predominantly ovule specific and were up-regulated on the day of anthesis in fibre initials relative to adjacent non-fibre ovule epidermal cells. Their spatial and temporal expression pattern therefore coincided with the time and location of fibre initiation. Constitutive overexpression of GhMyb25 in transgenic tobacco resulted in an increase in branched long-stalked leaf trichomes. The involvement of cell cycle genes prompted DNA content measurements that indicated that fibre initials, like leaf trichomes, undergo DNA endoreduplication. Cotton fibre initiation therefore has some parallels with leaf trichome development, although the detailed molecular mechanisms are clearly different.

  3. Inactivation of Tor proteins affects the dynamics of endocytic proteins in early stage of endocytosis

    Indian Academy of Sciences (India)

    Brandon Tenay; Evin Kimberlin; Michelle Williams; Juliette Denise; Joshua Fakilahyel; Kyoungtae Kim

    2013-06-01

    Tor2 is an activator of the Rom2/Rho1 pathway that regulates -factor internalization. Since the recruitment of endocytic proteins such as actin-binding proteins and the amphiphysins precedes the internalization of -factor, we hypothesized that loss of Tor function leads to an alteration in the dynamics of the endocytic proteins. We report here that endocytic proteins, Abp1 and Rvs167, are less recruited to endocytic sites not only in tor2 but also tor1 mutants. Furthermore, we found that the endocytic proteins Rvs167 and Sjl2 are completely mistargeted to the cytoplasm in tor1tor2ts double mutant cells. We also demonstrate here that the efficiency of endocytic internalization or scission in all tor mutants was drastically decreased. In agreement with the Sjl2 mislocalization, we found that in tor1tor2ts double mutant cells, as well as other tor mutant cells, the overall PIP2 level was dramatically increased. Finally, the cell wall chitin content in tor2ts and tor1tor2ts mutant cells was also significantly increased. Taken together, both functional Tor proteins, Tor1 and Tor2, are essentially required for proper endocytic protein dynamics at the early stage of endocytosis.

  4. Expression profile analysis of early fruit development in iaaM-parthenocarpic tomato plants

    Directory of Open Access Journals (Sweden)

    Spena Angelo

    2009-07-01

    Full Text Available Abstract Background Fruit normally develops from the ovary after pollination and fertilization. However, the ovary can also generate seedless fruit without fertilization by parthenocarpy. Parthenocarpic fruit development has been obtained in tomato (Solanum lycopersicum by genetic modification using auxin-synthesising gene(s (DefH9-iaaM; DefH9-RI-iaaM expressed specifically in the placenta and ovules. Findings We have performed a cDNA Amplified Fragment Length Polymorphism (cDNA-AFLP analysis on pre-anthesis tomato flower buds (0.5 cm long collected from DefH9-iaaM and DefH9-RI-iaaM parthenocarpic and wild-type plants, with the aim to identify genes involved in very early phases of tomato fruit development. We detected 212 transcripts differentially expressed in auxin-ipersynthesising pre-anthesis flower buds, 65 of them (31% have unknown function. Several differentially expressed genes show homology to genes involved in protein trafficking and protein degradation via proteasome. These processes are crucial for auxin cellular transport and signaling, respectively. Conclusion The data presented might contribute to elucidate the molecular basis of the fruiting process and to develop new methods to confer parthenocarpy to species of agronomic interest. In a recently published work, we have demonstrated that one of the genes identified in this screening, corresponding to #109 cDNA clone, regulates auxin-dependent fruit initiation and its suppression causes parthenocarpic fruit development in tomato.

  5. Identification of c-Yes expression in the nuclei of hepatocellular carcinoma cells: involvement in the early stages of hepatocarcinogenesis.

    Science.gov (United States)

    Nonomura, Takako; Masaki, Tsutomu; Morishita, Asahiro; Jian, Gong; Uchida, Naohito; Himoto, Takashi; Izuishi, Kunihiko; Iwama, Hisakazu; Yoshiji, Hitoshi; Watanabe, Seishiro; Kurokohchi, Kazutaka; Kuriyama, Shigeki

    2007-01-01

    It is thought that the subcellular distribution of Src-family tyrosine kinases, including c-Yes binding to the cellular membrane, is membranous and/or cytoplasmic. c-Yes protein tyrosine kinase is known to be related to malignant transformation. However, the expression patterns of c-Yes in hepatocellular carcinoma (HCC) remains unknown. In the present study, we report that c-Yes is expressed not only in the membrane and cytoplasm, but also in the nuclei of cancer cells in some human HCC tissues and in a human HCC cell line. We examined the expression and localization of c-Yes in human HCC cell lines (HLE, HLF, PLC/PRF/5 and Hep 3B) by Western blotting and immunohistochemical analyses; we also examined the expression of c-Yes by immunohistochemistry and Western blotting in the tissues of various liver diseases, including 39 samples from HCC patients. We used an antibody array to detect proteins that bind to nuclear c-Yes in PLC/PRF/5 cell line. c-Yes was found to be expressed in the membranes and cytoplasm of HLE, HLF and Hep 3B HCC cells; it was also detected in the nuclei in addition to the membranes and cytoplasm of PLC/PRF/5 HCC cells. HCC with nuclear c-Yes was detected in 5 of 39 cases (13.0%), and nuclear c-Yes expression was not detected in normal, chronic hepatitis or cirrhotic livers. All HCCs with nuclear c-Yes expression were well-differentiated, small tumors at the early stages. In the PLC/PRF/5 cell line, the nuclear localization of c-Yes with cyclin-dependent kinase 1 was confirmed by a protein antibody array. In conclusion, nuclear c-Yes expression was found in cancer cells at the early stages of hepatocarcinogenesis, suggesting that nucleus-located c-Yes may be a useful marker to detect early-stage HCC.

  6. Protein expression on Cr resistant microorganism using electrophoresis method

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    SAJIDAN

    2009-01-01

    Full Text Available Fatmawati U, Suranto, Sajidan. 2009. Protein expression on Cr resistant microorganism using electrophoresis method. Nusantara Bioscience 1: 31-37. Hexavalent chromium (Cr(VI is known as toxic heavy metals, so the need is reduced to Cr(III is much less toxicity. Pseudomonas aeruginosa, Pseudomonas putida, Klebsiella pneumoniae, Pantoea sp. and Saccharomyces cerevisiae are resistant Cr(VI microorganism and have ability to reduce Cr(VI. The aim of this research is to know ability of microorganism to reduce Cr(VI and to know protein band pattern between Cr(VI resistant microorganism and non resistant microorganism which inoculated on LB broth. SDS-PAGE was used to indentify protein expression. While, Cr(VI concentration was identified by 1.5 diphenylcarbazide method. The quantitative data was analyzed by two factorial ANOVA that continued with DMRT at 1% level test. The qualitative data i.e. protein expression analyzed by relative mobility (Rf. The results showed that the ability of microorganisms to reduce Cr(VI at initial concentration of 0.5 ppm, 1 ppm, 5 ppm and 10 ppm may vary, the average percentage of the ability of each microorganism in reducing Cr(VI is P. putida (65% > S. cerevisiae (64.45% >. P. aeruginosa (60.73% > Pantoea sp. (50.22% > K. pneumoniae (47.82% > without microorganisms (34.25%. The adding microorganisms have significantly influenced toward reduction of Cr(VI. The SDS-PAGE shows that protein expression between resistant and not resistant microorganisms are no different, but resistant microorganisms have more protein (protein band is thicker.

  7. Using Green and Red Fluorescent Proteins to Teach Protein Expression, Purification, and Crystallization

    Science.gov (United States)

    Wu, Yifeng; Zhou, Yangbin; Song, Jiaping; Hu, Xiaojian; Ding, Yu; Zhang, Zhihong

    2008-01-01

    We have designed a laboratory curriculum using the green and red fluorescent proteins (GFP and RFP) to visualize the cloning, expression, chromatography purification, crystallization, and protease-cleavage experiments of protein science. The EGFP and DsRed monomer (mDsRed)-coding sequences were amplified by PCR and cloned into pMAL (MBP-EGFP) or…

  8. Increased expression of peroxiredoxin 1 and identification of a novel lipid-metabolizing enzyme in the early phase of liver ischemia reperfusion injury

    NARCIS (Netherlands)

    Wilson, Claire H.; Zeile, Susanne; Chataway, Tim; Nieuwenhuijs, Vincent B.; Padbury, Robert T. A.; Barritt, Greg J.

    2011-01-01

    Warm ischemia reperfusion (IR) injury of the liver is associated with changes in the expression and/or post-translational modification of numerous proteins. Only a few of these have been identified. We used 2-D DIGE to identify cytosolic proteins altered in the early stage of IR in an established ra

  9. Breast Cancer Resistance Protein Expression and 5-Fluorouracil Resistance

    Institute of Scientific and Technical Information of China (English)

    JIAN-HUI YUAN; ZHI-XIONG ZHUANG; JIN-QUAN CHENG; LONG-YUAN JIANG; WEI-DONG JI; LIANG-FENG GUO; JIAN-JUN LIU; XING-YUN XU; JING-SONG HE; XIAN-MING WANG

    2008-01-01

    To filtrate breast cancer resistance protein (BCRP)-mediated resistant agents and to investigate clinical relationship between BCRP expression and drug resistance. Methods MTT assay was performed to filtrate BCRP-mediated resistant agents with BCRP expression cell model and to detect chemosensitivity of breast cancer tissue specimens to these agents. A high performance liquid chromatography (HPLC) assay was established, and was used to measure the relative dose of intracellular retention resistant agents. RT-PCR and immununohistochemistry (IHC) were employed to investigate the BCRP expression in breast cancer tissue specimens. Results MTT assay showed that the expression of BCRP increased with the increasing resistance of 5-fluorouracil (5-Fu) (P=0.8124, P<0.01). Condusion Resistance to 5-Fu can be mediated by BCRP. Clinical chemotherapy for breast cancer patients can be optimized based on BCRP-positive expression.

  10. 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...... stress (obesity, obese non-insulin-dependent diabetes mellitus), hypertrophy (training), de- and reinnervation (amyotrophic lateral sclerosis) or regeneration (polymyositis). We used an immunohistochemical approach to detect and localise GLUT3. GLUT3 immunoreactivity was not detectable in adult skeletal...... 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...

  11. Visualizing changes in circuit activity resulting from denervation and reinnervation using immediate early gene expression.

    Science.gov (United States)

    Temple, Meredith D; Worley, Paul F; Steward, Oswald

    2003-04-01

    We describe a novel strategy to evaluate circuit function after brain injury that takes advantage of experience-dependent immediate early gene (IEG) expression. When normal rats undergo training or are exposed to a novel environment, there is a strong induction of IEG expression in forebrain regions, including the hippocampus. This gene induction identifies the neurons that are engaged during the experience. Here, we demonstrate that experience-dependent IEG induction is diminished after brain injury in young adult rats (120-200 gm), specifically after unilateral lesions of the entorhinal cortex (EC), and then recovers with a time course consistent with reinnervation. In situ hybridization techniques were used to assess the expression of the activity-regulated cytoskeleton-associated protein Arc at various times after the lesion (4, 8, 12, 16, or 30 d). One group of rats was allowed to explore a complex novel environment for 1 hr; control operated animals remained in their home cage. In unoperated animals, exposure to the novel environment induced Arc mRNA levels in most pyramidal neurons in CA1, in many pyramidal neurons in CA3, and in a small number of dentate granule cells. This characteristic pattern of induction was absent at early time points after unilateral EC lesions (4 and 8 d) but recovered progressively at later time points. The recovery of Arc expression occurred with approximately the same time course as the reinnervation of the dentate gyrus as a result of postlesion sprouting. These results document a novel approach for quantitatively assessing activity-regulated gene expression in polysynaptic circuits after trauma.

  12. Secreted Proteins Defy the Expression Level-Evolutionary Rate Anticorrelation.

    Science.gov (United States)

    Feyertag, Felix; Berninsone, Patricia M; Alvarez-Ponce, David

    2017-03-01

    The rates of evolution of the proteins of any organism vary across orders of magnitude. A primary factor influencing rates of protein evolution is expression. A strong negative correlation between expression levels and evolutionary rates (the so-called E-R anticorrelation) has been observed in virtually all studied organisms. This effect is currently attributed to the abundance-dependent fitness costs of misfolding and unspecific protein-protein interactions, among other factors. Secreted proteins are folded in the endoplasmic reticulum, a compartment where chaperones, folding catalysts, and stringent quality control mechanisms promote their correct folding and may reduce the fitness costs of misfolding. In addition, confinement of secreted proteins to the extracellular space may reduce misinteractions and their deleterious effects. We hypothesize that each of these factors (the secretory pathway quality control and extracellular location) may reduce the strength of the E-R anticorrelation. Indeed, here we show that among human proteins that are secreted to the extracellular space, rates of evolution do not correlate with protein abundances. This trend is robust to controlling for several potentially confounding factors and is also observed when analyzing protein abundance data for 6 human tissues. In addition, analysis of mRNA abundance data for 32 human tissues shows that the E-R correlation is always less negative, and sometimes nonsignificant, in secreted proteins. Similar observations were made in Caenorhabditis elegans and in Escherichia coli, and to a lesser extent in Drosophila melanogaster, Saccharomyces cerevisiae and Arabidopsis thaliana. Our observations contribute to understand the causes of the E-R anticorrelation. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  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. Expression of Prokaryotic Integral Membrane Proteins in E. coli.

    Science.gov (United States)

    Love, James D

    2017-01-01

    Production of prokaryotic membrane proteins for structural and functional studies in E. coli can be parallelized and miniaturized. All stages from cloning, expression, purification to detergent selection can be investigated using high-throughput techniques to rapidly and economically find tractable targets.

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

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    Nikita Abraham

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

  16. Heterologous expression of membrane proteins: choosing the appropriate host.

    Directory of Open Access Journals (Sweden)

    Florent Bernaudat

    Full Text Available BACKGROUND: Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. METHODOLOGY/PRINCIPAL FINDINGS: The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals, functions (transporters, receptors, enzymes and topologies (between 0 and 13 transmembrane segments. The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. CONCLUSIONS/SIGNIFICANCE: Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein.

  17. Heterologous expression of membrane proteins: choosing the appropriate host.

    Science.gov (United States)

    Bernaudat, Florent; Frelet-Barrand, Annie; Pochon, Nathalie; Dementin, Sébastien; Hivin, Patrick; Boutigny, Sylvain; Rioux, Jean-Baptiste; Salvi, Daniel; Seigneurin-Berny, Daphné; Richaud, Pierre; Joyard, Jacques; Pignol, David; Sabaty, Monique; Desnos, Thierry; Pebay-Peyroula, Eva; Darrouzet, Elisabeth; Vernet, Thierry; Rolland, Norbert

    2011-01-01

    Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein. © 2011 Bernaudat et al.

  18. NR2F6 Expression Correlates with Pelvic Lymph Node Metastasis and Poor Prognosis in Early-Stage Cervical Cancer

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    Chunhao Niu

    2016-10-01

    Full Text Available Background: There is an abnormal expression of nuclear receptor subfamily 2 group F member 6 (NR2F6 in human cancers such as breast cancer, colon cancer, and acute myelogenous leukemia. However, its clinical significance in cervical cancer has not been established. We explored NR2F6 expression and its clinicopathological significance in early-stage cervical cancer. Methods: NR2F6 expression in cervical cancer cell lines and cervical cancer tissues was determined by Western blotting, real-time PCR, and immunochemistry (IHC. NR2F6 expression in 189 human early-stage cervical cancer tissue samples was evaluated using IHC. The relevance between NR2F6 expression and early-stage cervical cancer prognosis and clinicopathological features was determined. Results: There was marked NR2F6 mRNA and protein overexpression in the cervical cancer cells and clinical tissues compared with an immortalized squamous cell line and adjacent noncancerous cervical tissues, respectively. In the 189 cervical cancer samples, NR2F6 expression was positively related to International Federation of Gynecology and Obstetrics (FIGO stage (p = 0.006, squamous cell carcinoma antigen (p = 0.006, vital status (p < 0.001, tumor recurrence (p = 0.001, chemotherapy (p = 0.039, and lymph node metastasis (p < 0.001. Overall and disease-free survival was shorter in patients with early-stage cervical cancer and higher NR2F6 levels than in patients with lower levels of NR2F6. Univariate and multivariate analysis determined that NR2F6 was an independent prognostic factor of survival in early-stage cervical cancer. Conclusions: Taken together, our findings suggest that high NR2F6 expression predicts pelvic lymph node metastasis, tumor recurrence and poor prognosis in early-stage cervical cancer. NR2F6 might be a novel prognostic biomarker and potential therapeutic target of cervical cancer.

  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, CMA; VANDERMEER, J; HILLEGE, JL; BOM, VJJ; HALIE, MR; van der Schaaf, W

    1993-01-01

    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. Optimization of translation profiles enhances protein expression and solubility.

    Science.gov (United States)

    Hess, Anne-Katrin; Saffert, Paul; Liebeton, Klaus; Ignatova, Zoya

    2015-01-01

    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.

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

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    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.

  2. Raman microscopy of bladder cancer cells expressing green fluorescent protein

    Science.gov (United States)

    Mandair, Gurjit S.; Han, Amy L.; Keller, Evan T.; Morris, Michael D.

    2016-11-01

    Gene engineering is a commonly used tool in cellular biology to determine changes in function or expression of downstream targets. However, the impact of genetic modulation on biochemical effects is less frequently evaluated. The aim of this study is to use Raman microscopy to assess the biochemical effects of gene silencing on T24 and UMUC-13 bladder cancer cell lines. Cellular biochemical information related to nucleic acid and lipogenic components was obtained from deconvolved Raman spectra. We show that the green fluorescence protein (GFP), the chromophore that served as a fluorescent reporter for gene silencing, could also be detected by Raman microscopy. Only the gene-silenced UMUC-13 cell lines exhibited low-to-moderate GFP fluorescence as determined by fluorescence imaging and Raman spectroscopic studies. Moreover, we show that gene silencing and cell phenotype had a greater effect on nucleic acid and lipogenic components with minimal interference from GFP expression. Gene silencing was also found to perturb cellular protein secondary structure in which the amount of disorderd protein increased at the expense of more ordered protein. Overall, our study identified the spectral signature for cellular GFP expression and elucidated the effects of gene silencing on cancer cell biochemistry and protein secondary structure.

  3. 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

  4. A comparative approach to understanding tissue-specific expression of uncoupling protein 1 expression in adipose tissue

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    Andrew eShore

    2013-01-01

    Full Text Available The thermoregulatory function of brown adipose tissue (BAT is due to the tissue-specific expression of uncoupling protein 1 (UCP1 which is thought to have evolved in early mammals. We report that a CpG island close to the UCP1 transcription start site is highly conserved in all 29 vertebrates examined apart from the mouse and xenopus. Using methylation sensitive restriction digest and bisulphite mapping we show that the CpG island in both the bovine and human is largely un-methylated and is not related to differences in UCP1 expression between white and brown adipose tissue. Tissue-specific expression of UCP1 has been proposed to be regulated by a conserved 5’ distal enhancer which has been reported to be absent in marsupials. We demonstrate that the enhancer, is also absent in 5 eutherians as well as marsupials, monotremes, amphibians and fish, is present in pigs despite UCP1 having become a pseudogene, and that absence of the enhancer element does not relate to brown adipose tissue-specific UCP1 expression. We identify an additional putative 5’ regulatory unit which is conserved in 14 eutherian species but absent in other eutherians and vertebrates, but again unrelated to UCP1 expression. We conclude that despite clear evidence of conservation of regulatory elements in the UCP1 5’ untranslated region, this does not appear to be related to species or tissues-specific expression of UCP1.

  5. Transient expression and cellular localization of recombinant proteins in cultured insect cells

    Science.gov (United States)

    Heterologous protein expression systems are used for production of recombinant proteins, interpretation of cellular trafficking/localization, and for the determination of biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for ...

  6. Inhibition of hepatitis C virus protein expression by RNA interference.

    Science.gov (United States)

    Sen, Adrish; Steele, Robert; Ghosh, Asish K; Basu, Arnab; Ray, Ranjit; Ray, Ratna B

    2003-10-01

    Hepatitis C virus (HCV) is a serious human pathogen and an estimated 170 million people are infected worldwide. Current therapeutic regimens have shown limited efficacy against selected genotypes of the virus. The phenomenon of RNA interference can be used to selectively block homologous genes post-transcriptionally, and has revolutionized approaches to study gene function. In this report, we have demonstrated that small interfering RNAs (siRNAs) targeted against NS5A of HCV genotype 1a specifically inhibit NS5A RNA and protein expression in a human hepatoma (HepG2) cell line. Expression of endogenous alpha-actin and the ds-RNA activated serine/threonine kinase-PKR were unaltered, demonstrating that the inhibitory effect observed from siRNA was specific to the HCV NS5A protein. We next examined whether siRNA directed against NS5A could inhibit core protein expression, the first gene product synthesized in virus infected cells due to its localization at the 5' end of the HCV polyprotein. For this purpose, a full-length cDNA clone from HCV (H77, genotype 1a) was used, and results indicated that the introduction of NS5A targeted siRNA resulted in an inhibition of NS5A and core protein expression. Moreover, we observed that this siRNA effectively inhibited NS5A mediated activation of the IL-8 promoter. Taken together, our results demonstrated that siRNA was effective in inhibiting HCV protein expression, and may have therapeutic potential to limit HCV replication in chronically infected patients.

  7. Protein expression profiling during chick retinal maturation: a proteomics-based approach

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    Stitt Alan W

    2008-12-01

    Full Text Available Abstract Background The underlying pathways that drive retinal neurogenesis and synaptogenesis are still relatively poorly understood. Protein expression analysis can provide direct insight into these complex developmental processes. The aim of this study was therefore to employ proteomic analysis to study the developing chick retina throughout embryonic (E development commencing at day 12 through 13, 17, 19 and post-hatch (P 1 and 33 days. Results 2D proteomic and mass spectrometric analysis detected an average of 1514 spots per gel with 15 spots demonstrating either modulation or constitutive expression identified via MS. Proteins identified included alpha and beta-tubulin, alpha enolase, B-creatine kinase, gamma-actin, platelet-activating factor (PAF, PREDICTED: similar to TGF-beta interacting protein 1, capping protein (actin filament muscle Z line, nucleophosmin 1 (NPM1, dimethylarginine dimethylaminohydrolase, triosphoaphate isomerase, DJ1, stathmin, fatty acid binding protein 7 (FABP7/B-FABP, beta-synuclein and enhancer of rudimentary homologue. Conclusion This study builds upon previous proteomic investigations of retinal development and represents the addition of a unique data set to those previously reported. Based on reported bioactivity some of the identified proteins are most likely to be important to normal retinal development in the chick. Continued analysis of the dynamic protein populations present at the early stages and throughout retinal development will increase our understanding of the molecular events underpinning retinogenesis.

  8. High-yield expression of recombinant SARS coronavirus nucleocapsid protein in methylotrophic yeast Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    Ru-Shi Liu; Kun-Yu Yang; Jian Lin; Yi-Wei Lin; Zhi-Hong Zhang; Jun Zhang; Ning-Shao Xia

    2004-01-01

    AIM: Nucleocapsid (N) protein plays an important role in reproduction and pathological reaction of severe acute respiratory syndrome (SARS) coronavirus (SCoV), theantigenicity of the protein is better than spike (S) protein.This study was to find a highly specific and antigenic recombinant SCoV nucleocapsid (rSCoVN) protein, and to provide a basis for further researches on early diagnosis of SARS.METHODS: Full length cDNA of SCoV nucleocapsid (SCoVN)protein was amplified through polymerase chain reaction (PCR) and cloned into yeast expression vector pPIC3.5K to construct plasmid of pPIC3.5K-SCoVN. The plasmid was linearized and then transformed into Pichia pastoris (P. pastoris) GS115 (HisMut+) by electroporation. His+Mut+recombinant strains were identified by PCR and cultivated on MM/MD plates. The influence of different factors on biomass and rSCoVN protein production during induction phase, such as various induction media, dissolved oxygen (DO) and different final concentrations of methanol, was subsequently studied. The expression level and activation were detected by SDS-PAGE and Western-blot respectively.RESULTS: All of the recombinants were His+Mut+ aftertransformation of P. pastoriswith linearized plasmids. The BMMY medium was optimal for recombinant ScoVN (rSCoVN)protein expression and growth of the recombinant strains.The final optimal concentration of methanol was 20 mL/L,the DO had a significant effect on rSCoVN protein expression and growth of recombinant strains. The rSCoVN protein expressed in recombinant strains was about 8% of the total cell protein, 520 mg/L of rSCoVN protein was achieved,and a maximum cell ,A at 600 nm of 62 was achieved in shake flask culture. The rSCoVN protein had a high specificity against mouse-anti-SARS-CoVN-mAb and SARS positive sera, but had no cross-reaction with normal human serum.The biological activity of rSCoVN expressed in P. pastoris was about 4-fold higher than that expressed in E.coliwhen the same rSCoVN protein

  9. Grape seed extract inhibits VEGF expression via reducing HIF-1alpha protein expression.

    Science.gov (United States)

    Lu, Jianming; Zhang, Keqiang; Chen, Shiuan; Wen, Wei

    2009-04-01

    Grape seed extract (GSE) is a widely consumed dietary supplement that has antitumor activity. Here, we have investigated the inhibitory effect of GSE on the expression of vascular endothelial growth factor (VEGF) and the mechanism underlying this action. We found that GSE inhibited VEGF messenger RNA (mRNA) and protein expression in U251 human glioma cells and MDA-MB-231 human breast cancer cells. GSE inhibited transcriptional activation of the VEGF gene through reducing protein but not mRNA expression of hypoxia-inducible factor (HIF) 1alpha. The inhibitory effect of GSE on HIF-1alpha expression was mainly through inhibiting HIF-1alpha protein synthesis rather than promoting protein degradation. Consistent with this result, GSE-suppressed phosphorylation of several important components involved in HIF-1alpha protein synthesis, such as Akt, S6 kinase and S6 protein. Furthermore, in the MDA-MB-231 tumor, we found that GSE treatment inhibited the expression of VEGF and HIF-1alpha and the phosphorylation of S6 kinase without altering the subcellular localization of HIF-1alpha, correlating with reduced vessel density and tumor size. Depletion of polyphenol with polyvinylpyrrolidone abolished the inhibitory activity of GSE, suggesting a water-soluble fraction of polyphenol in GSE is responsible for the inhibitory activity. Taken together, our results indicate that GSE inhibits VEGF expression by reducing HIF-1alpha protein synthesis through blocking Akt activation. This finding provides new insight into the mechanisms of anticancer activity of GSE and reveals a novel molecular mechanism underlying the antiangiogenic action of GSE.

  10. Spatial-temporal expressions of Crumbs and Nagie oko and their interdependence in zebrafish central nervous system during early development.

    Science.gov (United States)

    Zou, Jian; Wen, Yi; Yang, Xiaojun; Wei, Xiangyun

    2013-12-01

    A vast number of apicobasal polarity proteins play essential roles in the polarization and morphogenesis of the neuroepithelia. Crumbs (Crb) type I transmembrane cell-cell adhesion proteins are among these proteins. Five crb genes have been identified in zebrafish. However, their expressional and functional differences during early neural development remain to be fully elucidated. Here, we study the spatial-temporal expression patterns and functions of Crb1, Crb2a, and Crb2b in the central nervous system (CNS) during the neurulation period. We show that: 1, the optic vesicle and undifferentiated retinal neuroepithelium only express Crb2a; 2, Crb1 and Crb2a expressions overlap extensively in the undifferentiated neural tube epithelium; 3, Crb2b expression is the weakest of the three and is restricted to the ventral-most regions of the anterior CNS; and 4, Nok and Crb proteins require each other for their apical localization in neuroepithelium. The commencements of Crb1, Crb2a, and Crb2b expressions follow a spatial-temporal spread from anterior to posterior and from ventral to dorsal and lag behind that of adherens junction components, such as ZO-1 and actin bundles. Genetic and morpholino suppression analyses suggest that in regions where these Crb expressions overlap, they are functionally redundant in maintaining apicobasal polarity of the undifferentiated neuroepithelium.

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

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    Jeremy A. Kroemer

    2015-01-01

    Full Text Available 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.

  12. Morphine Withdrawal Modifies Prion Protein Expression in Rat Hippocampus

    Science.gov (United States)

    Mattei, Vincenzo; Martellucci, Stefano; Santilli, Francesca; Manganelli, Valeria; Garofalo, Tina; Candelise, Niccolò; Caruso, Alessandra; Sorice, Maurizio; Scaccianoce, Sergio

    2017-01-01

    The hippocampus is a vulnerable brain structure susceptible to damage during aging and chronic stress. Repeated exposure to opioids may alter the brain so that it functions normally when the drugs are present, thus, a prolonged withdrawal might lead to homeostatic changes headed for the restoration of the physiological state. Abuse of morphine may lead to Reacting Oxygen Species-induced neurodegeneration and apoptosis. It has been proposed that during morphine withdrawal, stress responses might be responsible, at least in part, for long-term changes of hippocampal plasticity. Since prion protein is involved in both, Reacting Oxygen Species mediated stress responses and synaptic plasticity, in this work we investigate the effect of opiate withdrawal in rats after morphine treatment. We hypothesize that stressful stimuli induced by opiate withdrawal, and the subsequent long-term homeostatic changes in hippocampal plasticity, might modulate the Prion protein expression. Our results indicate that abstinence from the opiate induced a time-dependent and region-specific modification in Prion protein content, indeed during morphine withdrawal a selective unbalance of hippocampal Prion Protein is observable. Moreover, Prion protein overexpression in hippocampal tissue seems to generate a dimeric structure of Prion protein and α-cleavage at the hydrophobic domain. Stress factors or toxic insults can induce cytosolic dimerization of Prion Protein through the hydrophobic domain, which in turn, it stimulates the α-cleavage and the production of neuroprotective Prion protein fragments. We speculate that this might be the mechanism by which stressful stimuli induced by opiate withdrawal and the subsequent long-term homeostatic changes in hippocampal plasticity, modulate the expression and the dynamics of Prion protein. PMID:28081197

  13. Novel Implant Coating Agent Promotes Gene Expression of Osteogenic Markers in Rats during Early Osseointegration

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    Kostas Bougas

    2012-01-01

    Full Text Available The aim of this study was to evaluate the early bone response around laminin-1-coated titanium implants. Forty-five rats distributed in three equally sized groups were provided with one control (turned and one test (laminin-1-coated implant and were sacrificed after 3, 7, and 21 days. Real-time reverse-transcriptase polymerase chain reaction was performed for osteoblast markers (alkaline phosphatase, runt-related transcription factor 2, osteocalcin, type I collagen, and bone morphogenic protein 2, osteoclast markers (cathepsin K and tartrate-resistant acid phosphatase, inflammation markers (tumor necrosis factor α, interleukin 1β and interleukin 10, and integrin β1. Bone implant contact (BIC and bone area (BA were assessed and compared to the gene expression. After 3 days, the expression of bone markers was higher for the control group. After 7 days, the expression of integrin β1 and osteogenic markers was enhanced for the test group, while cathepsin K and inflammation markers were down-regulated. No significant differences in BIC or BA were detected between test and control at any time point. As a conclusion, implant coating with laminin-1 altered gene expression in the bone-implant interface. However, traditional evaluation methods, as histomorphometry, were not adequately sensitive to detect such changes due to the short follow-up time.

  14. Lysine-specific demethylase 1 expression in zebrafish during the early stages of neuronal development

    Institute of Scientific and Technical Information of China (English)

    Aihong Li; Yong Sun; Changming Dou; Jixian Chen; Jie Zhang

    2012-01-01

    Lysine-specific demethylase 1 (Lsd1) is associated with transcriptional coregulation via the modulation of histone methylation. The expression pattern and function of zebrafish Lsd1 has not, however, been studied. Here, we describe the pattern of zebrafish Lsd1 expression during different development stages. In the zebrafish embryo, lsd1 mRNA was present during the early cleavage stage, indicating that maternally derived Lsd1 protein is involved in embryonic patterning. During embryogenesis from 0 to 48 hours post-fertilization (hpf), the expression of lsd1 mRNA in the embryo was ubiquitous before 12 hpf and then became restricted to the anterior of the embryo (particularly in the brain) from 24 hpf to 72 hpf. Inhibition of Lsd1 activity (by exposure to tranylcypromine) or knockdown of lsd1 expression (by morpholino antisense oligonucleotide injection) led to the loss of cells in the brain and to a dramatic downregulation of neural genes, including gad65, gad75, and reelin, but not hey1. These findings indicate an important role of Lsd1 during nervous system development in zebrafish.

  15. Fluoride alters type I collagen expression in the early stages of odontogenesis.

    Science.gov (United States)

    Maciejewska, I; Spodnik, J H; Domaradzka-Pytel, B; Sidor-Kaczmarek, J; Bereznowski, Z

    2006-11-01

    Fluoride alters the expression and post-translational modifications of extracellular matrix proteins in dentin. The aim of our study was to determine the effects of fluoride on type I collagen expression during the early stages of tooth germ development in rats. Pregnant dams were divided into three groups and fed a standard diet. From the fifth day of pregnancy the three groups received tap water with, respectively, trace amounts of fluoride (C), a low fluoride concentration (FL) or and a high fluoride concentration (FH). Changes in type I collagen expression and distribution were evaluated. The expression of type I collagen was restricted to the extracellular spaces of cells of mesenchymal origin. In the youngest animals the most intense immunoreactivity for type I collagen was detected in predentin of the FL group. Although the intensity of immunostaining increased in proportion to the age of the animals, the largest increase in the groups investigated was detected in the FL group. We concluded that a low concentration of fluoride can act as a stimulator of type I collagen deposition in the extracellular matrix of dentin, while high concentrations of fluoride have an opposite effect, acting as an inhibitor of type I collagen formation in dentin.

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

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    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

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

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    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.

  18. Expression of Recombinant Pregnancy-associated Plasma Protein-A

    Institute of Scientific and Technical Information of China (English)

    CHENG; Bin-yan; LI; Zi-ying; ZHANG; Xue-feng; LIU; Yi-bing

    2013-01-01

    Pregnancy-associated plasma protein-A(PAPP-A)is producted by the syntrophoblast tissue of the placenta and decidual cells.It belongs to macromolecular glycoprotein.PAPP-A is a sensitive serum marker of Down’s syndrome and has clinical valuable in the early identification of acute coronary syndrome(ACS).According to the structure of PAPP-A,PAPP-A DNA is divided into five segments(S1-S5)for

  19. Aberrant expression of krüppel-like factor 6 protein in colorectal cancers

    Institute of Scientific and Technical Information of China (English)

    Yong-Gu Cho; Byung-Jun Choi; Jae-Whie Song; Su-Young Kim; Suk-Woo Nam; Sug-Hyung Lee; Nam-Jin Yoo; Jung-Young Lee; Won-Sang Park

    2006-01-01

    AIM: To investigate whether kr(U)ppel-like factor 6 (KLF6)plays an important role in the development and/or progression of colorectal cancer.METHODS: A total of 123 formalin-fixed and paraffinembedded colorectal cancer specimens were analyzed by immunohistochemistry using tissue microarray for the expression of KLF6 protein. The specimens were collected over a 3-year period in the laboratories at our large teaching hospital in Seoul, Republic of Korea. The correlation of KLF6 expression with clinicopathologic parameters was analyzed by x2 test and Bartholomew test.RESULTS: Normal colonic epithelium showed weak to moderate expression of KLF6, whereas reduced KLF 6expression or loss of KLF6 expression was seen in 45(36.6%) of the 123 colorectal carcinoma specimens.Interestingly, aberrant expression of KLF6 was detected in 25 (43.1%) of 58 cases with metastasis to regional lymph node and in 31 (47.0%) of 66 tumors more than 5 cm in size. Statistically, loss of KLF6 expression was significantly associated with tumor size (P<0.05).However, there was no significant correlation between KLF6 expression and Dukes' stage (Bartholomew test,P> 0.05), tumor location and lymph node metastasis (x2test, P> 0.05).CONCLUSION: Loss of KLF6 expression may be a common and early event in colorectal carcinogenesis.

  20. 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.

  1. The Role of Bromodomain Proteins in Regulating Gene Expression

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    Michael F. Duffy

    2012-05-01

    Full Text Available Histone modifications are important in regulating gene expression in eukaryotes. Of the numerous histone modifications which have been identified, acetylation is one of the best characterised and is generally associated with active genes. Histone acetylation can directly affect chromatin structure by neutralising charges on the histone tail, and can also function as a binding site for proteins which can directly or indirectly regulate transcription. Bromodomains specifically bind to acetylated lysine residues on histone tails, and bromodomain proteins play an important role in anchoring the complexes of which they are a part to acetylated chromatin. Bromodomain proteins are involved in a diverse range of functions, such as acetylating histones, remodeling chromatin, and recruiting other factors necessary for transcription. These proteins thus play a critical role in the regulation of transcription.

  2. Expression of early growth response factor-1 in rats with cerulein-induced acute pancreatitis and its significance

    Institute of Scientific and Technical Information of China (English)

    Lan-Bo Gong; Li He; Yang Liu; Xue-Qing Chen; Bo Jiang

    2005-01-01

    AIM: To observe the expressions of early growth response factor-1 (Egr-L) and tissue factor (TF) in rats with cerulein-induced acute pancreatitis and to explore its significance.METHODS: A large dose of cerulein was used to create the experimental acute pancreatitis model in rats. The changes of Egr-1 mRNA and protein in rats were observed during 30 min to 4 h after the treatment and immunohistochemical method was used to observe the localized expression of Egr-1 in tissues. In addition to the mRNA expression of Egr-1 target gene, TF was also observed. A blank control group, and a bombesinadministered group were used for comparison.RESULTS: After the stimulation of a large dose of cerulein,the rats showed typical inflammatory changes of acute pancreatitis. Thirty minutes after the stimulation, the mRNA expression of Egr-1 in the pancreatic tissue reached its peak and then declined, while the expression of Egr-1protein reached its peak 2 h after the stimulation.Histologically, 2 h after the stimulation, almost all pancreatic acinar cells had the expression of Egr-1 protein,which was focused in the nuclei. The mRNA expression of TF occurred 1 h after the stimulation and gradually increased within 4 h. However, a large dose of bombesin only stimulated the pancreatic tissue to produce a little mRNA expression of Egr-1 and no mRNA expression of Egr-1 protein and TF.CONCLUSION: Egr-1 as a pro-inflammatory transcription factor may play an important role in the pathogenesis of acute pancreatitis by modulating the expression of TF.

  3. Deep sequencing and expression of microRNAs from early honeybee (Apis mellifera embryos reveals a role in regulating early embryonic patterning

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    Zondag Lisa

    2012-11-01

    Full Text Available Abstract Background Recent evidence supports the proposal that the observed diversity of animal body plans has been produced through alterations to the complexity of the regulatory genome rather than increases in the protein-coding content of a genome. One significant form of gene regulation is the contribution made by the non-coding content of the genome. Non-coding RNAs play roles in embryonic development of animals and these functions might be expected to evolve rapidly. Using next-generation sequencing and in situ hybridization, we have examined the miRNA content of early honeybee embryos. Results Through small RNA sequencing we found that 28% of known miRNAs are expressed in the early embryo. We also identified developmentally expressed microRNAs that are unique to the Apoidea clade. Examination of expression patterns implied these miRNAs have roles in patterning the anterior-posterior and dorso-ventral axes as well as the extraembryonic membranes. Knockdown of Dicer, a key component of miRNA processing, confirmed that miRNAs are likely to have a role in patterning these tissues. Conclusions Examination of the expression patterns of novel miRNAs, some unique to the Apis group, indicated that they are likely to play a role in early honeybee development. Known miRNAs that are deeply conserved in animal phyla display differences in expression pattern between honeybee and Drosophila, particularly at early stages of development. This may indicate miRNAs play a rapidly evolving role in regulating developmental pathways, most likely through changes to the way their expression is regulated.

  4. Expression of Tyrosine Hydroxylase is Negatively Regulated Via Prion Protein.

    Science.gov (United States)

    da Luz, Marcio Henrique Mello; Glezer, Isaias; Xavier, Andre Machado; da Silva, Marcelo Alberti Paiva; Pino, Jessica Monteiro Volejnik; Zamith, Thiago Panaro; Vieira, Taynara Fernanda; Antonio, Bruno Brito; Antunes, Hanna Karen Moreira; Martins, Vilma Regina; Lee, Kil Sun

    2016-07-01

    Cellular prion protein (PrP(C)) is a glycoprotein of the plasma membrane that plays pleiotropic functions by interacting with multiple signaling complexes at the cell surface. Recently, a number of studies have reported the involvement of PrP(C) in dopamine metabolism and signaling, including its interactions with tyrosine hydroxylase (TH) and dopamine receptors. However, the outcomes reported by independent studies are still debatable. Therefore in this study, we investigated the effects of PrP(C) on the TH expression during the differentiation of N2a cells with dibutyryl-cAMP, a well-known cAMP analog that activates TH transcription. Upon differentiation, TH was induced with concomitant reduction of PrP(C) at protein level, but not at mRNA level. shRNA-mediated PrP(C) reduction increased the basal level of TH at both mRNA and protein levels without dibutyryl-cAMP treatment. This phenotype was reversed by re-expression of PrP(C). PrP(C) knockdown also potentiated the effect of dibutyryl-cAMP on TH expression. Our findings suggest that PrP(C) has suppressive effects on TH expression. As a consequence, altered PrP(C) functions may affect the regulation of dopamine metabolism and related neurological disorders.

  5. Early Evolution of Vertebrate Mybs: An Integrative Perspective Combining Synteny, Phylogenetic, and Gene Expression Analyses.

    Science.gov (United States)

    Campanini, Emeline B; Vandewege, Michael W; Pillai, Nisha E; Tay, Boon-Hui; Jones, Justin L; Venkatesh, Byrappa; Hoffmann, Federico G

    2015-10-15

    The genes in the Myb superfamily encode for three related transcription factors in most vertebrates, A-, B-, and c-Myb, with functionally distinct roles, whereas most invertebrates have a single Myb. B-Myb plays an essential role in cell division and cell cycle progression, c-Myb is involved in hematopoiesis, and A-Myb is involved in spermatogenesis and regulating expression of pachytene PIWI interacting RNAs, a class of small RNAs involved in posttranscriptional gene regulation and the maintenance of reproductive tissues. Comparisons between teleost fish and tetrapods suggest that the emergence and functional divergence of the Myb genes were linked to the two rounds of whole-genome duplication early in vertebrate evolution. We combined phylogenetic, synteny, structural, and gene expression analyses of the Myb paralogs from elephant shark and lampreys with data from 12 bony vertebrates to reconstruct the early evolution of vertebrate Mybs. Phylogenetic and synteny analyses suggest that the elephant shark and Japanese lamprey have copies of the A-, B-, and c-Myb genes, implying their origin could be traced back to the common ancestor of lampreys and gnathostomes. However, structural and gene expression analyses suggest that their functional roles diverged between gnathostomes and cyclostomes. In particular, we did not detect A-Myb expression in testis suggesting that the involvement of A-Myb in the pachytene PIWI interacting RNA pathway is probably a gnathostome-specific innovation. We speculate that the secondary loss of a central domain in lamprey A-Myb underlies the functional differences between the cyclostome and gnathostome A-Myb proteins. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  6. Bcl-2/Bax protein and mRNA expression in yak (Bos grunniens) placentomes.

    Science.gov (United States)

    Fan, JiangFeng; Yu, SiJiu; Cui, Yan; Xu, Gengquan; Wang, Libin; Pan, Yangyang; He, Honghong

    2017-07-29

    Placental function is complex and influenced by various factors; furthermore, it depends on a delicate balance between cell proliferation, cell differentiation, and cell death. Bcl-2 and Bax proteins are key apoptosis regulators and are considered to play an important role in the maintenance of both dynamic balance and integrity of many tissues. Changes in Bcl-2 and Bax expressions have been described during different developmental stages in normal human placentas. Studies furthermore indicated several pathological placental changes to be related to abnormal Bcl-2 and Bax expressions. In the present study, we investigated both expression and distribution of Bcl-2 and Bax in yak placentas. For this, we collected placentas of 35 yaks at different stages of pregnancy as well as cotyledonary villi of four postpartum yaks. Protein and mRNA expressions of both Bcl-2 and Bax were investigated via immunohistochemistry, Western blot, and real-time PCR. Immunoreactive Bcl-2 protein was mainly localized near the fetal villous trophoblast at various gestational stages and post-partum. The Positive Index (PI) of Bcl-2 protein expression significantly decreased with increasing gestational age. Early during pregnancy (≤2 months), the Bax protein was widely distributed in the fetal villous trophoblast layer, the maternal caruncular crypt epithelium, and the stroma. Subsequently, the Bax protein distribution gradually concentrated in the fetal villous trophoblast layer. The staining intensity of Bax increased from the 3rd month to the prepartum of gestation. The PI reached a minimum of 9.4 ± 2.2 in fetal chorionic villi (FCV) and 1.3 ± 0.8 in maternal caruncular crypts (MCC) of the three months group. Both Bcl-2 and Bax had maximum immunoreactivity in the fetal villous trophoblast layer of placentas collected form postpartum yaks (with PIs of 36.6 ± 5.7 and 38.2 ± 4.8, respectively). Protein and mRNA expression of Bcl-2 and Bax investigated via Western blot and real

  7. Expression patterns of CREB binding protein (CREBBP) and its methylated species during zebrafish development.

    Science.gov (United States)

    Batut, Julie; Duboé, Carine; Vandel, Laurence

    2015-01-01

    Proper embryonic development requires a fine-tuned control of gene expression, which is achieved in part through the activity of transcription coactivators or corepressors. The nuclear coactivator cAMP-response element-binding protein (CREB) binding protein (CREBBP or CBP) interacts with numerous transcription factors and thereby plays a key role in various signaling pathways. Interestingly, in cell-based studies CREBBP activity is modulated by post-translational modifications such as methylation on arginine residues which is catalyzed by coactivator-associated arginine methyltransferase 1 (CARM1). However, whether and where CREBBP, and in particular its methylated forms, are expressed during development in vertebrates has not been addressed so far. Here, we analyzed the expression of the two crebbp genes (crebbpa & crebbpb) during zebrafish development using both RT-qPCR and in situ hybridization. We found that while crebbpa expression is higher in posterior, caudal nascent somites during somitogenesis, crebbpb accumulates in anterior, rostral, and more mature somites. In addition, crebbpa mRNA is enriched in the central myotome at 24 hpf indicating that its expression is spatially and temporally controlled. We next characterized the expression of CREBBP protein from blastula to gastrula stages by immunohistochemistry. We found that while CREBBP is clearly cytoplasmic in the early blastula, it becomes both cytoplasmic and nuclear at 30% epiboly before turning mainly nuclear during gastrulation. Of interest, CREBBP methylated species appear to be mainly nuclear from 30% epiboly to 6-somite stage. This suggests that methylation may regulate CREBBP import to the nucleus during zebrafish development and could therefore participate in the control of early developmental processes.

  8. Expression and Signiifcance of WT1 and Beta-catenin Proteins in Non-small Cell Lung Cancer

    Institute of Scientific and Technical Information of China (English)

    Zhou Feng; Mao Guoxin

    2013-01-01

    =5.915, P=0.015, r=0.331), indicating that the aberrant expression of WT1 protein was closely associated with that ofβ-catenin protein in NSCLC. Conclusions:1. The positive expression rate of WT1 protein expression is 62.5% in NSCLC. 2. The positive expression rate of aberrant β-catenin protein is 72.9% in NSCLC. 3. There is signiifcant association between WT1 protein and lymph node metastasis. WT1 protein expression is higher in non-lymph node metastasis group than in lymph node metastasis group (P0.05). 4. Aberrant protein ofβ-catenin is in reverse relevance with differentiation degrees, and the higher the differentiation degree is, the lower aberrant expression of β-catenin protein will be observed, but there is no significant association between the aberrant expression of β-catenin protein and genders, ages, tumor sizes, pathological patterns, differentiated degrees and pTNM stagings (P>0.05). 5. There is certain relevance between WT1 protein expression and the aberrant expression of β-catenin protein in NSCLC (P < 0.05). 6. WT1 protein andβ-catenin proteins may play certain roles in early events of NSCLC.

  9. Bacteria and protozoa differentially modulate the expression of Rab proteins.

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    Elsa Seixas

    Full Text Available Phagocytic cells represent an important line of innate defense against microorganisms. Uptake of microorganisms by these cells involves the formation of a phagosome that matures by fusing with endocytic compartments, resulting in killing of the enclosed microbe. Small GTPases of the Rab family are key regulators of vesicular trafficking in the endocytic pathway. Intracellular pathogens can interfere with the function of these proteins in order to subvert host immune responses. However, it is unknown if this subversion can be achieved through the modulation of Rab gene expression. We compared the expression level of 23 distinct Rab GTPases in mouse macrophages after infection with the protozoan Plasmodium berghei, and the bacteria Escherichia coli and Salmonella enterica. We found that P. berghei induces an increase in the expression of a different set of Rab genes than E. coli and S. enterica, which behaved similarly. Strikingly, when one of the Rab proteins whose expression was increased by P. berghei, namely Rab14, was silenced, we observed a significant increase in the phagocytosis of P. berghei, whereas Rab14 overexpression led to a decrease in phagocytosis. This suggests that the parasite might induce the increase of Rab14 expression for its own advantage. Similarly, when Rab9a, whose expression was increased by E. coli and S. enterica, was silenced, we observed an increase in the phagocytosis of both bacterial species, whereas Rab9a overexpression caused a reduction in phagocytosis. This further suggests that the modulation of Rab gene expression could represent a mechanism of immune evasion. Thus, our study analyzes the modulation of Rab gene expression induced by bacteria and protozoa and suggests that this modulation could be necessary for the success of microbial infection.

  10. Genome-wide identification and analysis of rice genes preferentially expressed in pollen at an early developmental stage.

    Science.gov (United States)

    Nguyen, Tien Dung; Moon, Sunok; Nguyen, Van Ngoc Tuyet; Gho, Yunsil; Chandran, Anil Kumar Nalini; Soh, Moon-Soo; Song, Jong Tae; An, Gynheung; Oh, Sung Aeong; Park, Soon Ki; Jung, Ki-Hong

    2016-09-01

    Microspore production using endogenous developmental programs has not been well studied. The main limitation is the difficulty in identifying genes preferentially expressed in pollen grains at early stages. To overcome this limitation, we collected transcriptome data from anthers and microspore/pollen and performed meta-expression analysis. Subsequently, we identified 410 genes showing preferential expression patterns in early developing pollen samples of both japonica and indica cultivars. The expression patterns of these genes are distinguishable from genes showing pollen mother cell or tapetum-preferred expression patterns. Gene Ontology enrichment and MapMan analyses indicated that microspores in rice are closely linked with protein degradation, nucleotide metabolism, and DNA biosynthesis and regulation, while the pollen mother cell or tapetum are strongly associated with cell wall metabolism, lipid metabolism, secondary metabolism, and RNA biosynthesis and regulation. We also generated transgenic lines under the control of the promoters of eight microspore-preferred genes and confirmed the preferred expression patterns in plants using the GUS reporting system. Furthermore, cis-regulatory element analysis revealed that pollen specific elements such as POLLEN1LELAT52, and 5659BOXLELAT5659 were commonly identified in the promoter regions of eight rice genes with more frequency than estimation. Our study will provide new sights on early pollen development in rice, a model crop plant.

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

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    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.

  12. Detection of gene expression pattern in the early stage after spinal cord injury by gene chip

    Institute of Scientific and Technical Information of China (English)

    刘成龙; 靳安民; 童斌辉

    2003-01-01

    Objective: To study the changes of the gene expression pattern of spinal cord tissues in the early stage after injury by DNA microarray (gene chip). Methods: The contusion model of rat spinal cord was established according to Allen's falling strike method and the gene expression patterns of normal and injured spinal cord tissues were studied by gene chip. Results: The expression of 45 genes was significantly changed in the early stage after spinal cord injury, in which 22 genes up-regulated and 23 genes down-regulated. Conclusions: The expression of some genes changes significantly in the early stage after spinal cord injury, which indicates the complexity of secondary spinal cord injury.

  13. Transgenic expression of green fluorescent protein in mouse oxytocin neurones.

    Science.gov (United States)

    Young, W S; Iacangelo, A; Luo, X Z; King, C; Duncan, K; Ginns, E I

    1999-12-01

    Routine targeting of neurones for expression of exogenous genes would facilitate our ability to manipulate their internal milieu or functions, providing insight into physiology of neurones. The magnocellular neurones of the paraventricular and supraoptic nuclei of the hypothalamus have been the objects of limited success by this approach. Here we report on the placement of the enhanced green fluorescent protein (eGFP) coding sequence at various locations within an oxytocin transgene. Placement within the first exon yielded little to no expression, whereas placement in the third exon (as an in-frame fusion with the carboxyl terminus of the oxytocin preprohormone) resulted in cell-specific expression of eGFP in oxytocin neurones. Furthermore, placement of the eGFP sequence downstream of a picornavirus internal ribosomal entry site (IRES), also in the third exon, allowed expression of the eGFP as a separate protein. Other coding sequences should now be amenable to expression within oxytocin neurones to study their physiology.

  14. Stepwise optimization of a low-temperature Bacillus subtilis expression system for "difficult to express" proteins.

    Science.gov (United States)

    Welsch, Norma; Homuth, Georg; Schweder, Thomas

    2015-08-01

    In order to improve the overproduction of "difficult to express" proteins, a low-temperature expression system for Bacillus subtilis based on the cold-inducible promoter of the desaturase-encoding des gene was constructed. Selected regulatory DNA sequence elements from B. subtilis genes known to be cold-inducible were fused to different model genes. It could be demonstrated that these regulatory elements are able to mediate increased heterologous gene expression, either by improved translation efficiency or by higher messenger RNA (mRNA) stability. In case of a cold-adapted β-galactosidase from Pseudoalteromonas haloplanktis TAE79A serving as the model, significantly higher expression was achieved by fusing its coding sequence to the so-called "downstream box" sequence of cspB encoding the major B. subtilis cold-shock protein. The combination of this fusion with a cspB 5'-UTR stem-loop structure resulted in further enhancement of the β-galactosidase expression. In addition, integration of the transcription terminator of the B. subtilis cold-inducible bkd operon downstream of the target genes caused a higher mRNA stability and enabled thus a further significant increase in expression. Finally, the fully optimized expression system was validated by overproducing a B. subtilis xylanase as well as an α-glucosidase from Saccharomyces cerevisiae, the latter known for tending to form inclusion bodies. These analyses verified the applicability of the engineered expression system for extracellular and intracellular protein synthesis in B. subtilis, thereby confirming the suitability of this host organism for the overproduction of critical, poorly soluble proteins.

  15. Heat-shock protein expression in canine corneal wound healing.

    Science.gov (United States)

    Peterson, Cornelia W M; Carter, Renee T; Bentley, Ellison; Murphy, Christopher J; Chandler, Heather L

    2016-05-01

    Heat-shock proteins, particularly the 70-kDa member (Hsp70), have been implicated in facilitating wound healing in multiple tissues. Expression and localization of three HSPs were assessed in normal and wounded canine corneas to elucidate a role in epithelial healing. Paraffin-embedded normal corneas, acute and repeatedly abraded corneas, and keratectomies of spontaneous chronic corneal epithelial defects (SCCEDs) were subjected to routine immunohistochemistry for Hsp27, 47, and 70 expression. Ex vivo corneal defects were created and treated with anti-HSPs or IgG controls, and wound healing was monitored. Primary cultures of canine corneal stromal fibroblasts and corneal epithelial cells were treated with exogenous Hsp70, and an artificial wound was created in vitro to monitor restoration of the monolayer. Normal canine corneas exhibited constitutive expression of all HSPs evaluated. Inducible expression was demonstrated in acutely wounded tissues, and expression in the chronically abraded corneas was relocalized. All HSP expression was below the limits of detection in the epithelium of SCCED samples. Inhibition of HSPs in culture resulted in delayed wound healing when compared to controls. Hsp70-treated fibroblasts demonstrated significantly (P healing response, and suppressed expression may contribute to the pathophysiology of nonhealing defects. © 2015 American College of Veterinary Ophthalmologists.

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

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    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.

  17. Generation of transgenic dogs that conditionally express green fluorescent protein.

    Science.gov (United States)

    Kim, Min Jung; Oh, Hyun Ju; Park, Jung Eun; Kim, Geon A; Hong, So Gun; Jang, Goo; Kwon, Mo Sun; Koo, Bon Chul; Kim, Teoan; Kang, Sung Keun; Ra, Jeong Chan; Ko, Chemyong; Lee, Byeong Chun

    2011-06-01

    We report the creation of a transgenic dog that conditionally expresses eGFP (enhanced green fluorescent protein) under the regulation of doxycycline. Briefly, fetal fibroblasts infected with a Tet-on eGFP vector were used for somatic cell nuclear transfer. Subsequently reconstructed oocytes were transferred to recipients. Three clones having transgenes were born and one dog was alive. The dog showed all features of inducible expression of eGFP upon doxycycline administration, and successful breeding resulted in eGFP-positive puppies, confirming stable insertion of the transgene into the genome. This inducible dog model will be useful for a variety of medical research studies.

  18. Transcriptional Modulation of Heat-Shock Protein Gene Expression

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    Anastasis Stephanou

    2011-01-01

    Full Text Available Heat-shock proteins (Hsps are molecular chaperones that are ubiquitously expressed but are also induced in cells exposed to stressful stimuli. Hsps have been implicated in the induction and propagation of several diseases. This paper focuses on regulatory factors that control the transcription of the genes encoding Hsps. We also highlight how distinct transcription factors are able to interact and modulate Hsps in different pathological states. Thus, a better understanding of the complex signaling pathways regulating Hsp expression may lead to novel therapeutic targets.

  19. Transcriptional modulation of heat-shock protein gene expression.

    Science.gov (United States)

    Stephanou, Anastasis; Latchman, David S

    2011-01-01

    Heat-shock proteins (Hsps) are molecular chaperones that are ubiquitously expressed but are also induced in cells exposed to stressful stimuli. Hsps have been implicated in the induction and propagation of several diseases. This paper focuses on regulatory factors that control the transcription of the genes encoding Hsps. We also highlight how distinct transcription factors are able to interact and modulate Hsps in different pathological states. Thus, a better understanding of the complex signaling pathways regulating Hsp expression may lead to novel therapeutic targets.

  20. 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.

  1. 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 durin

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

    Directory of Open Access Journals (Sweden)

    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.

  4. Recombinant Dragline Silk-Like Proteins-Expression and Purification.

    Science.gov (United States)

    Gaines, William A; Marcotte, William R

    2011-03-01

    Spider dragline silk is a proteinaceous fiber with impressive physical characteristics making it attractive for use in advanced materials. The fiber is composed of two proteins (spidroins MaSp1 and MaSp2), each of which contains a large central repeat array flanked by non-repetitive N- and C-terminal domains. The repeat arrays appear to be largely responsible for the tensile properties of the fiber, suggesting that the N- and C-terminal domains may be involved in self-assembly. We recently isolated the MaSp1 and MaSp2 N-terminal domains from Nephila clavipes and have incorporated these into mini-silk genes for expression in transgenic systems. Current efforts involve the development of expression vectors that will allow purification using a removable affinity tag for scalable protein purification.

  5. Disposable bioreactors for inoculum production and protein expression.

    Science.gov (United States)

    Eibl, Regine; Löffelholz, Christian; Eibl, Dieter

    2014-01-01

    Disposable bioreactors have been increasingly implemented over the past ten years. This relates to both R & D and commercial manufacture, in particular, in animal cell-based processes. Among the numerous disposable bioreactors which are available today, wave-mixed bag bioreactors and stirred bioreactors are predominant. Whereas wave-mixed bag bioreactors represent the system of choice for inoculum production, stirred systems are often preferred for protein expression. For this reason, the authors present protocols instructing the reader how to use the wave-mixed BIOSTAT CultiBag RM 20 L for inoculum production and the stirred UniVessel SU 2 L for recombinant protein production at benchtop scale. All methods described are based on a Chinese hamster ovary (CHO) suspension cell line expressing the human placental secreted alkaline phosphatase (SEAP).

  6. Puerarin enhances superoxide dismutase activity and inhibits RAGE and VEGF expression in retinas of STZ-induced early diabetic rats

    Institute of Scientific and Technical Information of China (English)

    Fang Chen; Hong-Quan Zhang; Jun Zhu; Kai-Yang Liu; Hong Cheng; Guo-Li Li; Shan Xu; Wei-Hong Lv; Zheng-Gao Xie

    2012-01-01

    Objective:To investigate the effects of puerarin on the activity of superoxide dismutase (SOD), and expressions of advanced glycation end-product (AGE) receptor (RAGE) and vascular endothelial growth factor (VEGF) in retinas of streptozotocin (STZ)-induced early diabetic rats. Methods: Diabetic rat models were established by inducing diabetes via intra-peritoneal injection of STZ. Rats were randomly divided into normal (control), diabetic (DM), and DM+puerarin groups. After intra-gastric administration of puerarin (500 mg/kg/day for 4 weeks), levels of SOD and malondialdehyde (MDA) were determined in serum and retina. mRNA and protein expression levels of RAGE and VEGF in retinas were determined by real-time polymerase chain reaction (RT-PCR) (mRNA) and Western blot analysis (protein levels). Results:There was significantly lower SOD activity and significantly higher MDA in serum and retinas of the DM group compared with the two other groups (P<0.05). After treatment with puerarin, SOD activity increased and MDA content decreased in this group (P<0.05). mRNA and protein expression levels of RAGE and VEGF in the DM group were significantly higher than those of the other groups (P<0.05), and decreased after puerarin treatment (P<0.05). Conclusions: Puerarin is able to enhance SOD activity, and inhibit RAGE and VEGF expressions in retinas of STZ-induced early diabetic rats.

  7. Splice Isoforms of Phosducin-like Protein Control the Expression of Heterotrimeric G Proteins*

    Science.gov (United States)

    Gao, Xueli; Sinha, Satyabrata; Belcastro, Marycharmain; Woodard, Catherine; Ramamurthy, Visvanathan; Stoilov, Peter; Sokolov, Maxim

    2013-01-01

    Heterotrimeric G proteins play an essential role in cellular signaling; however, the mechanism regulating their synthesis and assembly remains poorly understood. A line of evidence indicates that the posttranslational processing of G protein β subunits begins inside the protein-folding chamber of the chaperonin containing t-complex protein 1. This process is facilitated by the ubiquitously expressed phosducin-like protein (PhLP), which is thought to act as a CCT co-factor. Here we demonstrate that alternative splicing of the PhLP gene gives rise to a transcript encoding a truncated, short protein (PhLPs) that is broadly expressed in human tissues but absent in mice. Seeking to elucidate the function of PhLPs, we expressed this protein in the rod photoreceptors of mice and found that this manipulation caused a dramatic translational and posttranslational suppression of rod heterotrimeric G proteins. The investigation of the underlying mechanism revealed that PhLPs disrupts the folding of Gβ and the assembly of Gβ and Gγ subunits, events normally assisted by PhLP, by forming a stable and apparently inactive tertiary complex with CCT preloaded with nascent Gβ. As a result, the cellular levels of Gβ and Gγ, which depends on Gβ for stability, decline. In addition, PhLPs evokes a profound and rather specific down-regulation of the Gα transcript, leading to a complete disappearance of the protein. This study provides the first evidence of a generic mechanism, whereby the splicing of the PhLP gene could potentially and efficiently regulate the cellular levels of heterotrimeric G proteins. PMID:23888055

  8. Label-Free Proteome Analysis of Plasma from Patients with Breast Cancer: Stage-Specific Protein Expression

    Science.gov (United States)

    Lobo, Marina Duarte Pinto; Moreno, Frederico Bruno Mendes Batista; Souza, Gustavo Henrique Martins Ferreira; Verde, Sara Maria Moreira Lima; Moreira, Renato de Azevedo; Monteiro-Moreira, Ana Cristina de Oliveira

    2017-01-01

    Breast cancer is one of the most commonly diagnosed types of cancer among women. Breast cancer mortality rates remain high probably because its diagnosis is hampered by inaccurate detection methods. Since changes in protein expression as well as modifications in protein glycosylation have been frequently reported in cancer development, the aim of this work was to study the differential expression as well as modifications of glycosylation of proteins from plasma of women with breast cancer at different stages of disease (n = 30) compared to healthy women (n = 10). A proteomics approach was used that depleted albumin and IgG from plasma followed by glycoprotein enrichment using immobilized Moraceae lectin (frutalin)-affinity chromatography and data-independent label-free mass spectrometric analysis. Data are available via ProteomeXchange with identifier PXD003106. As result, 57,016 peptides and 4,175 proteins among all samples were identified. From this, 40 proteins present in unbound (PI—proteins that did not interact with lectin) and bound (PII—proteins that interacted with lectin) fractions were differentially expressed. High levels of apolipoprotein A-II were detected here that were elevated significantly in the early and advanced stages of the disease. Apolipoprotein C-III was detected in both fractions, and its level was increased slightly in the PI fraction of patients with early-stage breast cancer and expressed at higher levels in the PII fraction of patients with early and intermediate stages. Clusterin was present at higher levels in both fractions of patients with early and intermediate stages of breast cancer. Our findings reveal a correlation between alterations in protein glycosylation, lipid metabolism, and the progression of breast cancer. PMID:28210565

  9. Transcriptional Modulation of Heat-Shock Protein Gene Expression

    OpenAIRE

    Anastasis Stephanou; Latchman, David S.

    2011-01-01

    Heat-shock proteins (Hsps) are molecular chaperones that are ubiquitously expressed but are also induced in cells exposed to stressful stimuli. Hsps have been implicated in the induction and propagation of several diseases. This paper focuses on regulatory factors that control the transcription of the genes encoding Hsps. We also highlight how distinct transcription factors are able to interact and modulate Hsps in different pathological states. Thus, a better understanding of the complex sig...

  10. Transcriptional modulation of heat-shock protein gene expression.

    OpenAIRE

    A. Stephanou; Latchman, D S

    2011-01-01

    Heat-shock proteins (Hsps) are molecular chaperones that are ubiquitously expressed but are also induced in cells exposed to stressful stimuli. Hsps have been implicated in the induction and propagation of several diseases. This paper focuses on regulatory factors that control the transcription of the genes encoding Hsps. We also highlight how distinct transcription factors are able to interact and modulate Hsps in different pathological states. Thus, a better understanding of the complex sig...

  11. Expression of 16 Nitrogenase Proteins within the Plant Mitochondrial Matrix

    Science.gov (United States)

    Allen, Robert S.; Tilbrook, Kimberley; Warden, Andrew C.; Campbell, Peter C.; Rolland, Vivien; Singh, Surinder P.; Wood, Craig C.

    2017-01-01

    The industrial production and use of nitrogenous fertilizer involves significant environmental and economic costs. Strategies to reduce fertilizer dependency are required to address the world's increasing demand for sustainable food, fibers, and biofuels. Biological nitrogen fixation, a process unique to diazatrophic bacteria, is catalyzed by the nitrogenase complex, and reconstituting this function in plant cells is an ambitious biotechnological strategy to reduce fertilizer use. Here we establish that the full array of biosynthetic and catalytic nitrogenase (Nif) proteins from the diazotroph Klebsiella pneumoniae can be individually expressed as mitochondrial targeting peptide (MTP)-Nif fusions in Nicotiana benthamiana. We show that these are correctly targeted to the plant mitochondrial matrix, a subcellular location with biochemical and genetic characteristics potentially supportive of nitrogenase function. Although Nif proteins B, D, E, F, H, J, K, M, N, Q, S, U, V, X, Y, and Z were all detectable by Western blot analysis, the NifD catalytic component was the least abundant. To address this problem, a translational fusion between NifD and NifK was designed based on the crystal structure of the nitrogenase MoFe protein heterodimer. This fusion protein enabled equimolar NifD:NifK stoichiometry and improved NifD expression levels in plants. Finally, four MTP-Nif fusion proteins (B, S, H, Y) were successfully co-expressed, demonstrating that multiple components of nitrogenase can be targeted to plant mitochondria. These results establish the feasibility of reconstituting the complete componentry for nitrogenase in plant cells, within an intracellular environment that could support the conversion of nitrogen gas into ammonia. PMID:28316608

  12. Expression of 16 Nitrogenase Proteins within the Plant Mitochondrial Matrix.

    Science.gov (United States)

    Allen, Robert S; Tilbrook, Kimberley; Warden, Andrew C; Campbell, Peter C; Rolland, Vivien; Singh, Surinder P; Wood, Craig C

    2017-01-01

    The industrial production and use of nitrogenous fertilizer involves significant environmental and economic costs. Strategies to reduce fertilizer dependency are required to address the world's increasing demand for sustainable food, fibers, and biofuels. Biological nitrogen fixation, a process unique to diazatrophic bacteria, is catalyzed by the nitrogenase complex, and reconstituting this function in plant cells is an ambitious biotechnological strategy to reduce fertilizer use. Here we establish that the full array of biosynthetic and catalytic nitrogenase (Nif) proteins from the diazotroph Klebsiella pneumoniae can be individually expressed as mitochondrial targeting peptide (MTP)-Nif fusions in Nicotiana benthamiana. We show that these are correctly targeted to the plant mitochondrial matrix, a subcellular location with biochemical and genetic characteristics potentially supportive of nitrogenase function. Although Nif proteins B, D, E, F, H, J, K, M, N, Q, S, U, V, X, Y, and Z were all detectable by Western blot analysis, the NifD catalytic component was the least abundant. To address this problem, a translational fusion between NifD and NifK was designed based on the crystal structure of the nitrogenase MoFe protein heterodimer. This fusion protein enabled equimolar NifD:NifK stoichiometry and improved NifD expression levels in plants. Finally, four MTP-Nif fusion proteins (B, S, H, Y) were successfully co-expressed, demonstrating that multiple components of nitrogenase can be targeted to plant mitochondria. These results establish the feasibility of reconstituting the complete componentry for nitrogenase in plant cells, within an intracellular environment that could support the conversion of nitrogen gas into ammonia.

  13. Hypoxia decreases podocyte expression of slit diaphragm proteins

    Directory of Open Access Journals (Sweden)

    Lu H

    2012-07-01

    Full Text Available Hong Lu,1 Gaurav Kapur,1 Tej K Mattoo,1 William D Lyman1,21Carman and Ann Adams Department of Pediatrics, 2Children's Research Center of Michigan, Children's Hospital of Michigan, Detroit, MI, USABackground: Chronic hypoxia contributes to progressive tubulointerstitial injury and, consequently, renal failure. However, the effect of hypoxia on glomerular podocytes, which are integral to the slit diaphragm complex and responsible for selectivity of the glomerular filtration barrier, has not been completely determined. Methods: Conditionally immortalized mouse podocyte cells were exposed to hypoxic (1% O2 or normoxic (room air conditions for 24, 48, or 72 hours, after which cell viability was determined by MTT assay. Cells were stained with podocin and phalloidin to determine podocin and intracellular actin distribution. Expression of synaptopodin, CD2-associated protein (CD2AP, NcK, transforming growth factor-β1 (TGF-β1, hypoxia-inducible factor (HIF-1α were evaluated by real-time polymerase chain reaction.Results: Podocytes exposed to hypoxia had significantly reduced viability at 48 (87% and 72 hours (66%. There was disarrangement of intracellular filament actin by phalloidin staining, a 30% weaker fluorescence intensity by podocin staining, significantly reduced expression of synaptopodin (12%, CD2AP (42%, NcK (38%, and increased expression of TGF-β1 and P-ERK after hypoxia treatment.Conclusion: Podocyte exposure to hypoxia leads to reduced viability and SD protein expression, which may explain persistent and/or increasing proteinuria in patients with progressive renal failure. Increased expression of TGF-β1 and P-ERK is associated with apoptosis and fibrosis, which could be the link between hypoxia and glomerular injury.Keywords: podocytes, hypoxia, slit-diaphragm proteins

  14. Differential expression of ribosomal proteins in myelodysplastic syndromes.

    Science.gov (United States)

    Rinker, Elizabeth B; Dueber, Julie C; Qualtieri, Julianne; Tedesco, Jason; Erdogan, Begum; Bosompem, Amma; Kim, Annette S

    2016-02-01

    Aberrations of ribosomal biogenesis have been implicated in several congenital bone marrow failure syndromes, such as Diamond-Blackfan anaemia, Shwachman-Diamond syndrome and Dyskeratosis Congenita. Recent studies have identified haploinsufficiency of RPS14 in the acquired bone marrow disease isolated 5q minus syndrome, a subtype of myelodysplastic syndromes (MDS). However, the expression of various proteins comprising the ribosomal subunits and other proteins enzymatically involved in the synthesis of the ribosome has not been explored in non-5q minus MDS. Furthermore, differences in the effects of these expression alterations among myeloid, erythroid and megakaryocyte lineages have not been well elucidated. We examined the expression of several proteins related to ribosomal biogenesis in bone marrow biopsy specimens from patients with MDS (5q minus patients excluded) and controls with no known myeloid disease. Specifically, we found that there is overexpression of RPS24, DKC1 and SBDS in MDS. This overexpression is in contrast to the haploinsufficiency identified in the congenital bone marrow failure syndromes and in acquired 5q minus MDS. Potential mechanisms for these differences and aetiology for these findings in MDS are discussed.

  15. Expression of DNA-dependent protein kinase in human granulocytes

    Institute of Scientific and Technical Information of China (English)

    Annahita SALLMYR; Anna MILLER; Aida GABDOULKHAKOVA; Valentina SAFRONOVA; Gunnel HENRIKSSON; Anders BREDBERG

    2004-01-01

    Human polymorphonuclear leukocytes (PMN) have been reported to completely lack of DNA-dependent protein kinase (DNA-PK) which is composed of Ku protein and the catalytic subunit DNA-PKcs, needed for nonhomologous end-joining (NHEJ) of DNA double-strand breaks. Promyelocytic HL-60 cells express a variant form of Ku resulting in enhanced radiation sensitivity. This raises the question if low efficiency of NHEJ, instrumental for the cellular repair of oxidative damage, is a normal characteristic of myeloid differentiation. Here we confirmed the complete lack of DNAPK in P MN protein extracts, and the expression of the truncated Ku86 variant form in HL-60. However, this degradation of DNA-PK was shown to be due to a DNA-PK-degrading protease in PMN and HL-60. In addition, by using a protease-resistant whole cell assay, both Ku86 and DNA-PKcs could be demonstrated in PMN, suggesting the previously reported absence in PMN of DNA-PK to be an artefact. The levels of Ku86 and DNA-PKcs were much reduced in PMN, as compared with that of the lymphocytes, whereas HL-60 displayed a markedly elevated DNA-PK concentration.In conclusion, our findings provide evidence of reduced, not depleted expression of DNA-PK during the mature stages of myeloid differentiation.

  16. Expression of P16 protein and Bcl-2 protein in malignant eyelid tumors

    Institute of Scientific and Technical Information of China (English)

    牛膺筠; 周占宇; 刘夫玲; 王红云

    2002-01-01

    Objective To investigate the relationship between P16 gene (the tumor suppressor gene) and the bcl-2 gene (the apoptosis inhibitor gene) and the incidence and development of malignant eyelid tumors. Methods The streptavidin-biotin-peroxidase complex immunohistochemistry method was used to study the expression of P16 gene and the bcl-2 gene in 96 cases of malignant eyelid tumors. Results Among the 96 cases, there were 40 basal cell carcinomas (BCCs), 33 squamous carcinomas and 23 sebaceous carcinoma, with P16 protein positive (nuclear staining) rates 70%, 54.6% and 56.5%, respectively. The P16 positive rate was negatively correlated with the degree of tumor histological differentiation, and the rate difference between the high differentiated carcinomas was significant (P<0.05). Positive Bcl-2 protein expression was detected in the cytoplasm. All 40 BCC cases were Bcl-2 positive, and nearly all of the tumor cells showed positive cytoplasmic expression, while in the 33 squamous cell carcinoma cases only one showed positive focal reaction, and the staining in the other 32 cases was relatively faint. None of the 23 sebaceous carcinomas expressed Bcl-2. Conclusions The expression of the P16 protein was related to the occurrence and degree of differentiation of malignant eyelid tumors. The overexpression of the Bcl-2 protein suggests that suppression of apoptosis might play a role in the tumorigenesis of BCC.

  17. PEX11β induces peroxisomal gene expression and alters peroxisome number during early Xenopus laevis development

    Directory of Open Access Journals (Sweden)

    Damjanovski Sashko

    2011-04-01

    Full Text Available Abstract Background Peroxisomes are organelles whose roles in fatty acid metabolism and reactive oxygen species elimination have contributed much attention in understanding their origin and biogenesis. Many studies have shown that de novo peroxisome biogenesis is an important regulatory process, while yeast studies suggest that total peroxisome numbers are in part regulated by proteins such as Pex11, which can facilitate the division of existing peroxisomes. Although de novo biogenesis and divisions are likely important mechanisms, the regulation of peroxisome numbers during embryonic development is poorly understood. Peroxisome number and function are particularly crucial in oviparous animals such as frogs where large embryonic yolk and fatty acid stores must be quickly metabolized, and resulting reactive oxygen species eliminated. Here we elucidate the role of Pex11β in regulating peroxisomal gene expression and number in Xenopus laevis embryogenesis. Results Microinjecting haemagglutinin (HA tagged Pex11β in early embryos resulted in increased RNA levels for peroxisome related genes PMP70 and catalase at developmental stages 10 and 20, versus uninjected embryos. Catalase and PMP70 proteins were found in punctate structures at stage 20 in control embryos, whereas the injection of ectopic HA-Pex11β induced their earlier localization in punctate structures at stage 10. Furthermore, the peroxisomal marker GFP-SKL, which was found localized as peroxisome-like structures at stage 20, was similarly found at stage 10 when co-microinjected with HA-Pex11β. Conclusions Overexpressed Pex11β altered peroxisomal gene levels and induced the early formation of peroxisomes-like structures during development, both of which demonstrate that Pex11β may be a key regulator of peroxisome number in early Xenopus embryos.

  18. Gene and protein expressions of P28gank in rat with liver regeneration

    Institute of Scientific and Technical Information of China (English)

    Jian-Min Qin; Xiao-Yong Fu; Shen-Jing Li; Shu-Qin Liu; Jin-Zhang Zeng; Xiu-Hua Qiu; Meng-Chao Wu; Hong-Yang Wang

    2003-01-01

    AIM: To observe the gene and protein expression changes of p28GANK in regenerating liver tissues, and to reveal the biological function of p28GANK on the regulation of liver regeneration.METHODS: One hundred and thirty two adult male Sprague-Dawley rats were selected, weighing 200-250 g,and divided randomly into sham operation (SO) group and partial hepatectomy (PH) group. Each group had eleven time points: 0, 2, 6, 12, 24, 30, 48, 72, 120, 168 and 240 h,six rats were in each time point. The rats were undergone 70 % PH under methoxyflurane anesthesia by resection of the anterior and left lateral lobes of the liver. SO was conducted by laparotomy plus slight mobilization of the liver without resection. Liver specimens were collected at the indicated time points after PH or SO. The expression level of p28GANK mRNA was determined by Northern blot as well as at protein level via immunohistochemical staining.The expressions of p28GANK mRNA in these tissues were analyzed by imaging analysis system of FLA-2000 FUJIFILM and one way analysis of variance. The protein expressions of p28GANK in these tissues were analyzed with Fromowitz'method and Rank sum test.RESULTS: The expression of p28GANK mRNA in bhe regenerating liver tissues possessed two transcripts, which were 1.5 kb and 1.0 kb. There was a significantly different expression patterns of p28GANK mRNA between SO and PH groups (P<0.01). The expression of p28GANK mRNA increased 2 h after PH, the peak time was 72 h (SO group: 163.83±1.4720; PH group: 510.5±17.0499, P<0.01). There was a significant difference in the 1.5 kb transcript, which decreased gradually after 72 hours. The protein expression of p28GANK was mainly in the cytoplasm of regenerating hepatocytes, and increased near the central region 24 h after PH, and became strongly positive at 48 h (+++, vs the other time points P<0.05),but decreased 72 h after PH.CONCLUSION: The expression of p28GANK mRNA increases in the early stage of rat liver regeneration, the

  19. 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.

  20. Efficient expression and purification of biologically active human cystatin proteins.

    Science.gov (United States)

    Chauhan, Sakshi; Tomar, Raghuvir S

    2016-02-01

    Cystatins are reversible cysteine protease inhibitor proteins. They are known to play important roles in controlling cathepsins, neurodegenerative disease, and in immune system regulation. Production of recombinant cystatin proteins is important for biochemical and function characterization. In this study, we cloned and expressed human stefin A, stefin B and cystatin C in Escherichia coli. Human stefin A, stefin B and cystatin C were purified from soluble fraction. For cystatin C, we used various chaperone plasmids to make cystatin C soluble, as it is reported to localize in inclusion bodies. Trigger factor, GroES-GroEL, DnaK-DnaJ-GrpE chaperones lead to the presence of cystatin C in the soluble fraction. Immobilized metal affinity chromatography, glutathione sepharose and anion exchange chromatography techniques were employed for efficient purification of these proteins. Their biological activities were tested by inhibition assays against cathepsin L and H3 protease.

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

    Science.gov (United States)

    Doorbar, John

    2013-10-01

    The papillomavirus E4 open reading frame (ORF) is contained within the E2 ORF, with the primary E4 gene-product (E1^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^E4 mRNA that has been most extensively analysed. During the papillomavirus life cycle, the E1^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^E4, these kinases regulate one of

  2. Identification of differentially expressed proteins in vitamin B 12

    Directory of Open Access Journals (Sweden)

    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.

  3. 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.

  4. Expression of parathyroid hormone-related protein in ameloblastomas.

    Science.gov (United States)

    Ohtsuru, Mitsunobu

    2005-12-01

    Parathyroid hormone-related protein (PTHrP) was first discovered as a causative protein for hypercalcemia, which is often seen in the malignant tumor. PTHrP binds to the parathyroid hormone 1 receptor (PTH1R) for signal transduction. PTHrP-PTH1R interactions were associated with bone resorption. The present study, therefore, sought to clarify the expression of PTHrP, parathyroid hormone (PTH) and PTH1R in ameloblastoma, using RT-PCR (N = 8), immunohistochemistry (N = 23) and ELISA (N = 11) techniques. PTHrP and B-actin mRNA were detected in the all samples. Expression of PTHrP was also seen in all of the 23 cases in ameloblastoma by immunohistochemistry. There was a significant difference in PTHrP concentration by ELISA between typical unicystic type and solid type including unicystic type 3 (p = 0.0427). Only one exhibited the weak expression of PTH1R mRNA. PTH1R was observed on osteoblasts in bone around the tumor but no expression was observed on ameloblastoma cells in tumor parenchyma by immunohistochemistry. PTH was not detected in ameloblastoma by RT-PCR, immunohistochemistory as well as ELISA. In addition, hypercalcemia and increase of serum PTHrP level was observed in one case of 8 ameloblastomas. It was suggested that PTHrP level may be associated with local bone infiltration and hypercalcemia in ameloblastoma.

  5. Myocardin-related transcription factor regulates Nox4 protein expression

    DEFF Research Database (Denmark)

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

    2016-01-01

    TGFβ-induced expression of the NADPH oxidase Nox4 is essential for fibroblast-myofibroblast transition. Rho has been implicated in Nox4 regulation, but the underlying mechanisms are largely unknown. Myocardin-related transcription factor (MRTF), a Rho/actin polymerization-controlled coactivator...... 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...... of contact uncoupling and TGFβ. Nox4 knockdown abrogates epithelial-myofibroblast transition-associated reactive oxygen species production. Laser capture microdissection reveals increased Nox4 expression in the tubular epithelium also during obstructive nephropathy. MRTF down-regulation/inhibition suppresses...

  6. Conservation and divergence of plant LHP1 protein sequences and expression patterns in angiosperms and gymnosperms.

    Science.gov (United States)

    Guan, Hexin; Zheng, Zhengui; Grey, Paris H; Li, Yuhua; Oppenheimer, David G

    2011-05-01

    Floral transition is a critical and strictly regulated developmental process in plants. Mutations in Arabidopsis LIKE HETEROCHROMATIN PROTEIN 1 (AtLHP1)/TERMINAL FLOWER 2 (TFL2) result in early and terminal flowers. Little is known about the gene expression, function and evolution of plant LHP1 homologs, except for Arabidopsis LHP1. In this study, the conservation and divergence of plant LHP1 protein sequences was analyzed by sequence alignments and phylogeny. LHP1 expression patterns were compared among taxa that occupy pivotal phylogenetic positions. Several relatively conserved new motifs/regions were identified among LHP1 homologs. Phylogeny of plant LHP1 proteins agreed with established angiosperm relationships. In situ hybridization unveiled conserved expression of plant LHP1 in the axillary bud/tiller, vascular bundles, developing stamens, and carpels. Unlike AtLHP1, cucumber CsLHP1-2, sugarcane SoLHP1 and maize ZmLHP1, rice OsLHP1 is not expressed in the shoot apical meristem (SAM) and the OsLHP1 transcript level is consistently low in shoots. "Unequal crossover" might have contributed to the divergence in the N-terminal and hinge region lengths of LHP1 homologs. We propose an "insertion-deletion" model for soybean (Glycine max L.) GmLHP1s evolution. Plant LHP1 homologs are more conserved than previously expected, and may favor vegetative meristem identity and primordia formation. OsLHP1 may not function in rice SAM during floral induction.

  7. Sequential expression, activity and nuclear localization of prolyl oligopeptidase protein in the developing rat brain.

    Science.gov (United States)

    Hannula, Mirva J; Männistö, Pekka T; Myöhänen, Timo T

    2011-01-01

    Prolyl oligopeptidase (POP) is a serine protease that hydrolyzes peptides shorter than 30-mer. Some evidence has recently been obtained that POP can generate protein-protein interactions and therefore participate in various physiological and pathological events. Several studies have reported that POP may be involved in neurogenesis since its activity increases during development and can be found in the nucleus of proliferating tissues. In cell cultures, POP has been shown to be localized in the nucleus, but only early in the development, since during maturation it is moved to the cytosol. We have now studied for the first time the expression of POP protein, its enzymatic activity and nuclear localization in vivo in the developing rat brain. We observed that enzymatic activity of POP is highest on embryonic day 18 while the protein amounts reach their peak at birth. Furthermore, POP is located in the nucleus only early in the development but is transferred to the cytosol already before parturition. Our in vivo results confirm the previous cell culture results supporting the role of POP in neurogenesis. A discordance of antenatal protein amounts and enzymatic activities is suggesting a tight regulation of POP activity and possibly even a nonhydrolytic role at that stage.

  8. Sweeping away protein aggregation with entropic bristles: intrinsically disordered protein fusions enhance soluble expression.

    Science.gov (United States)

    Santner, Aaron A; Croy, Carrie H; Vasanwala, Farha H; Uversky, Vladimir N; Van, Ya-Yue J; Dunker, A Keith

    2012-09-18

    Intrinsically disordered, highly charged protein sequences act as entropic bristles (EBs), which, when translationally fused to partner proteins, serve as effective solubilizers by creating both a large favorable surface area for water interactions and large excluded volumes around the partner. By extending away from the partner and sweeping out large molecules, EBs can allow the target protein to fold free from interference. Using both naturally occurring and artificial polypeptides, we demonstrate the successful implementation of intrinsically disordered fusions as protein solubilizers. The artificial fusions discussed herein have a low level of sequence complexity and a high net charge but are diversified by means of distinctive amino acid compositions and lengths. Using 6xHis fusions as controls, soluble protein expression enhancements from 65% (EB60A) to 100% (EB250) were observed for a 20-protein portfolio. Additionally, these EBs were able to more effectively solubilize targets compared to frequently used fusions such as maltose-binding protein, glutathione S-transferase, thioredoxin, and N utilization substance A. Finally, although these EBs possess very distinct physiochemical properties, they did not perturb the structure, conformational stability, or function of the green fluorescent protein or the glutathione S-transferase protein. This work thus illustrates the successful de novo design of intrinsically disordered fusions and presents a promising technology and complementary resource for researchers attempting to solubilize recalcitrant proteins.

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

    Directory of Open Access Journals (Sweden)

    Przemysław Krzysztof Wirstlein

    2012-10-01

    Full Text Available Factors controlling complement activation appear to exert a protective effect on pregnancy. This is
    particularly important in women with thrombophilia. The aim of this study was to determine the transcript and
    protein levels of complement decay-accelerating factor (DAF and membrane cofactor protein (MCP in the
    placentas of women with acquired and inherited thrombophilia. Also, we assessed immunohistochemistry staining
    of inhibitors of the complement cascade, DAF and MCP proteins, in the placentas of thrombophilic women.
    Placentas were collected from eight women with inherited thrombophilia and ten with acquired thrombophilia.
    The levels of DAF and MCP transcripts were evaluated by qPCR, the protein level was evaluated by Western
    blot. We observed a higher transcript (p < 0.05 and protein (p < 0.001 levels of DAF and MCP in the placentas
    of thrombophilic women than in the control group. DAF and MCP were localized on villous syncytiotrophoblast
    membranes, but the assessment of staining in all groups did not differ. The observed higher expression level of
    proteins that control activation of complement control proteins is only seemingly contradictory to the changes
    observed for example in the antiphospholipid syndrome. However, given the hitherto known biochemical changes
    associated with thrombophilia, a mechanism in which increased expression of DAF and MCP in the placentas is
    an effect of proinflammatory cytokines, which accompanies thrombophilia, is probable.Factors controlling complement activation appear to exert a protective effect on pregnancy. This is
    particularly important in women with thrombophilia. The aim of this study was to determine the transcript and
    protein levels of complement decay-accelerating factor (DAF and membrane cofactor protein (MCP in the
    placentas of women with acquired and inherited thrombophilia. Also, we assessed immunohistochemistry

  10. Temporal expression of bacterial proteins instructs host CD4 T cell expansion and Th17 development.

    Directory of Open Access Journals (Sweden)

    Seung-Joo Lee

    2012-01-01

    Full Text Available Pathogens can substantially alter gene expression within an infected host depending on metabolic or virulence requirements in different tissues, however, the effect of these alterations on host immunity are unclear. Here we visualized multiple CD4 T cell responses to temporally expressed proteins in Salmonella-infected mice. Flagellin-specific CD4 T cells expanded and contracted early, differentiated into Th1 and Th17 lineages, and were enriched in mucosal tissues after oral infection. In contrast, CD4 T cells responding to Salmonella Type-III Secretion System (TTSS effectors steadily accumulated until bacterial clearance was achieved, primarily differentiated into Th1 cells, and were predominantly detected in systemic tissues. Thus, pathogen regulation of antigen expression plays a major role in orchestrating the expansion, differentiation, and location of antigen-specific CD4 T cells in vivo.

  11. Effect of IFN-γ/IL-10 on IL-10 Receptor Expression of Human Decidual Stromal Cells in Early Pregnancy

    Institute of Scientific and Technical Information of China (English)

    Chang-ying XING; Sui-qi GUI

    2006-01-01

    Objective To study the role of IFN-γ /IL-10 cytokines on IL-10 receptor gene and protein expression of human decidual stromal cells(DSC) in human early pregnancy in vitro.Methods Human DSC was isolated and cultured in vitro, and the expression of IL-10R1 and IL-10R2 gene was analyzed after cells had been treated with TH2-type cytokines IL-10 and TH1-type cytokines IFN-γ within 60 min with semiquantitative reverse transcriptase-PCR, then the influence of IL-10 and IFN-γ on expression of IL-10R protein was examined by first trimester DSC using flow cytometry. In addition,the vitality of DSC was detected by MTT.Results IL-10R1 mRNA levels of DSC treated with IL-10 (10 ng/ml) reached the peak level within 15 min, and were significantly lower at 30 min, then were not detected at 45 min. The expression of IL-10R1 were induced to moderate level by IFN-γ(10 ng/ml)within 30 min, and reduced to undetected levels at 60 min. There was no significant difference of IL-10R2 expression (P>0.05) between treated and not with the abovementioned cytokines. The IL-10R protein expression and vitality of DSC were significantly enhanced by IL-10 (10 ng/ml) and IFN-γ (10 ng/ml) which treated DSC 48 h (P<0. 05).Conclusion IL-10 and IFN-γ may play an important role of biologic function in early pregnancy by influencing IL-10R expression of DSC.

  12. 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.

  13. Identification of differentially expressed serum proteins in gastric adenocarcinoma☆

    Science.gov (United States)

    Subbannayya, Yashwanth; Mir, Sartaj Ahmad; Renuse, Santosh; Manda, Srikanth S.; Pinto, Sneha M.; Puttamallesh, Vinuth N.; Solanki, Hitendra Singh; Manju, H.C.; Syed, Nazia; Sharma, Rakesh; Christopher, Rita; Vijayakumar, M.; Kumar, K.V. Veerendra; Prasad, T.S. Keshava; Ramaswamy, Girija; Kumar, Rekha V.; Chatterjee, Aditi; Pandey, Akhilesh; Gowda, Harsha

    2015-01-01

    Gastric adenocarcinoma is an aggressive cancer with poor prognosis. Blood based biomarkers of gastric cancer have the potential to improve diagnosis and monitoring of these tumors. Proteins that show altered levels in the circulation of gastric cancer patients could prove useful as putative biomarkers. We used an iTRAQ-based quantitative proteomic approach to identify proteins that show altered levels in the sera of patients with gastric cancer. Our study resulted in identification of 643 proteins, of which 48 proteins showed increased levels and 11 proteins showed decreased levels in serum from gastric cancer patients compared to age and sex matched healthy controls. Proteins that showed increased expression in gastric cancer included inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), Mannose-binding protein C (MBL2), sex hormone-binding globulin (SHBG), insulin-like growth factor-binding protein 2 (IGFBP2), serum amyloid A protein (SAA1), Orosomucoid 1 (ORM1) and extracellular superoxide dismutase [Cu–Zn] (SOD3). We used multiple reaction monitoring assays and validated elevated levels of ITIH4 and SAA1 proteins in serum from gastric cancer patients. Biological significance Gastric cancer is a highly aggressive cancer associated with high mortality. Serum-based biomarkers are of considerable interest in diagnosis and monitoring of various diseases including cancers. Gastric cancer is often diagnosed at advanced stages resulting in poor prognosis and high mortality. Pathological diagnosis using biopsy specimens remains the gold standard for diagnosis of gastric cancer. Serum-based biomarkers are of considerable importance as they are minimally invasive. In this study, we carried out quantitative proteomic profiling of serum from gastric cancer patients to identify proteins that show altered levels in gastric cancer patients. We identified more than 50 proteins that showed altered levels in gastric cancer patient sera. Validation in a large cohort of well

  14. 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

  15. lin28 proteins promote expression of 17∼92 family miRNAs during amphibian development.

    Science.gov (United States)

    Warrander, Fiona; Faas, Laura; Kovalevskiy, Oleg; Peters, Daniel; Coles, Mark; Antson, Alfred A; Genever, Paul; Isaacs, Harry V

    2016-01-01

    Lin28 proteins are post-transcriptional regulators of gene expression with multiple roles in development and the regulation of pluripotency in stem cells. Much attention has focussed on Lin28 proteins as negative regulators of let-7 miRNA biogenesis; a function that is conserved in several animal groups and in multiple processes. However, there is increasing evidence that Lin28 proteins have additional roles, distinct from regulation of let-7 abundance. We have previously demonstrated that lin28 proteins have functions associated with the regulation of early cell lineage specification in Xenopus embryos, independent of a lin28/let-7 regulatory axis. However, the nature of lin28 targets in Xenopus development remains obscure. Here, we show that mir-17∼92 and mir-106∼363 cluster miRNAs are down-regulated in response to lin28 knockdown, and RNAs from these clusters are co-expressed with lin28 genes during germ layer specification. Mature miRNAs derived from pre-mir-363 are most sensitive to lin28 inhibition. We demonstrate that lin28a binds to the terminal loop of pre-mir-363 with an affinity similar to that of let-7, and that this high affinity interaction requires to conserved a GGAG motif. Our data suggest a novel function for amphibian lin28 proteins as positive regulators of mir-17∼92 family miRNAs. © 2015 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc.

  16. Expression and Characterization of Human Heart Type Fatty Acid Binding Protein in Pichia Pastoris

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    H-FABP is regarded as a tissue-specific protein existing only in myocardial cells. It is released from the cardiac tissue and gets into the plasma when a heart attack occurs; the myocardial infarction is a good case in point. As a result, the detection of H-FABP will be an early and important biomarker for the disease concerned. The objective of the study is to prepare the recombinant H-FABP by aeukaryotic expression system, pichia, to produce the protein mimicking natural H-FABP, as an immunogen for the production of the specific antibody. A gene fragment encoding H-FABP was cloned in the expressing vector pPICZα, after sequencing. The recombinant plasmid was transformed into the competent cells of the X-33 strain by means of electroporation. The expression of the target peptide indueed by methanol was screened by means of Western blotting, with the available MAb( Clone 6B6 ). Highly expressive engineer strains were obtained. The production of recombinant H-FABP under induction was about 0.7 g/L, with an Mr of 14.5 kDa and recognized by a commercially available MAb (Clone 6B6). The recombinant vector was successfully constructed. Following this, H-FABP was expressed in X-33, and it would become the source of the preparation of specific antibodies, to develop diagnostic kits.

  17. Differential gene expression of fatty acid binding proteins during porcine adipogenesis.

    Science.gov (United States)

    Samulin, Johanna; Berget, Ingunn; Lien, Sigbjørn; Sundvold, Hilde

    2008-10-01

    Four different subtypes of fatty acid binding proteins i.e. liver-type FABP1, heart/muscle-type FABP3, adipocyte-type FABP4 and epithelial/epidermal-type FABP5 are expressed in adipose tissue. However, only the regulatory role of FABP4 in adipogenesis has been thoroughly investigated. To increase the knowledge on possible roles of these FABP subtypes in preadipocyte differentiation, gene expression patterns were examined during adipogenesis in pig (Sus scrofa). FABP1 expression was induced in proliferating cells, whereas FABP3, FABP4 and FABP5 expression increased throughout preadipocyte differentiation. Interestingly, the FABP4 and FABP5 expression increased early in the differentiation, followed by FABP3 later in the differentiation process. This indicates a role of FABP4 and FABP5 in intracellular fatty acid transport during initiation of differentiation, whereas, FABP3 likely is involved in the transport of fatty acids during intermediate stages of adipogenesis. In this study we demonstrate that FABP3, FABP4 and FABP5 expression is correlated with that of the peroxisome proliferator-activated receptors alpha and gamma (PPARA and PPARG). Altogether, this suggests a role of FABP1 during cell proliferation, whereas a coordinated expression of FABP3, FABP4 and FABP5 together with that of PPARA, PPARG1 and PPARG2 might be critical for the metabolic regulation during porcine adipogenesis.

  18. Changes of Survivin mRNA and Protein Expression during Paclitaxel Treatment in Breast Cancer Cells

    Institute of Scientific and Technical Information of China (English)

    XIONG Huihua; YU Shiying; ZHUANG Liang; XIONG Hua

    2007-01-01

    In order to investigate the role of antiapoptosis gene, survivin in the resistance to palcitaxel, the expression of survivin mRNA and protein in the process of paclitaxel treatment in breast cancer cell line MCF-7 was detected. MCF-7 cells were incubated with paclitaxel at different concentrations. The growth inhibition rate of MCF-7 was investigated by tetrazolium bromide (MTT) colorimetry. The change of apoptosis was detected by Annexin-V/PI methods. The changes in the expression of survivin mRNA and protein were studied by reverse transcription polymerase chain reaction (RT-PCR) and Western-blot assay respectively. The growth inhibition rate of MCF-7 was increased in a concentration- and time-dependent manner. Paclitaxel of higher concentration could effectively induce apoptosis in MCF-7 cells after 48 h, while the expression of survivin was increased at early time (within 6 h) and decreased after 24 h regardless of treatment concentrations of paclitaxel. It suggested that tumor cells might evade the paclitaxel-induced cell cycle arrest and apoptosis by increasing the level of survivin at early treatment time.

  19. Molecular characterization and gene expression of juvenile hormone binding protein in the bamboo borer, Omphisa fuscidentalis.

    Science.gov (United States)

    Ritdachyeng, Eakartit; Manaboon, Manaporn; Tobe, Stephen S; Singtripop, Tippawan

    2012-11-01

    Juvenile hormone (JH) plays an important role in many physiological processes in insect development, diapause and reproduction. An appropriate JH titer in hemolymph is essential for normal development in insects. Information concerning its carrier partner protein, juvenile hormone binding protein (JHBP), provides an alternative approach to understanding how JH regulates metamorphosis. In this study, we cloned and sequenced the Omphisa juvenile hormone binding protein (OfJHBP). The full-length OfJHBP cDNA sequence is comprised of 849 nucleotides with an open reading frame of 726bp encoding 242 amino acids. The molecular mass of the protein was estimated to be 26.94kDa. The deduced protein sequence of OfJHBP showed moderate homology with the lepidopteran, Heliothis virescens JHBP (52% amino acid identity) and lower homology with the Bombyx mori JHBP (45%) and the Manduca sexta JHBP (44%). The OfJHBP was expressed mainly in the fat body. OfJHBP transcripts in the fat body was moderately high during 3rd, 4th and 5th instars, then rapidly increased, reaching a peak during early diapause. The expression remained high in mid-diapause, then decreased in late-diapause until the pupal stage. Both juvenile hormone analog (JHA), methoprene, 20-hydroxyecdysone (20E) exhibited a similar stimulatory pattern in OfJHBP expression of diapausing larvae. OfJHBP mRNA levels gradually increased and showed a peak of gene expression on the penultimate, then declined to low levels in the pupal stage. For in vitro gene expression, both of JHA and 20E induced OfJHBP mRNA expression in fat body. Fat body maintenance in vitro in the presence of 0.1μg/50μl JHA induced OfJHBP mRNA expression to high levels within the first 30min whereas 0.1μg/50μl 20E induced gene expression at 120min. To study the synergistic effect of these two hormones, fat body was incubated in vitro with 0.1μg/50μl JHA or 0.1μg/50μl 20E or a combination of both hormone for 30min. Induction of OfJHBP expression by

  20. Amino-terminal sequence of adenovirus type 2 proteins: hexon, fiber, component IX, and early protein 1B-15K

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, C.W.; Lewis, J.B.

    1980-07-15

    The partial amino-terminal amino acid sequence was determined for four adenovirus 2 proteins: hexon, fiber, component IX, and early protein E1B-15K. A comparison of these sequences with the nucleotide sequences of the region of the genome encoding each of these proteins has identified the initiation sites for protein synthesis. Each protein is initiated at the AUG codon nearest the 5' end of its mRNA. The initiating methionine is retained by fiber and component IX while it is removed from hexon and protein E1B-15K.

  1. Expression of P53 protein after exposure to ionizing radiation

    Science.gov (United States)

    Salazar, A. M.; Salvador, C.; Ruiz-Trejo, C.; Ostrosky, P.; Brandan, M. E.

    2001-10-01

    One of the most important tumor suppressor genes is p53 gene, which is involved in apoptotic cell death, cell differentiation and cell cycle arrest. The expression of p53 gene can be evaluated by determining the presence of P53 protein in cells using Western Blot assay with a chemiluminescent method. This technique has shown variabilities that are due to biological factors. Film developing process can influence the quality of the p53 bands obtained. We irradiated tumor cell lines and human peripheral lymphocytes with 137Cs and 60Co gamma rays to standardize irradiation conditions, to compare ionizing radiation with actinomycin D and to reduce the observed variability of P53 protein induction levels. We found that increasing radiation doses increase P53 protein induction while it decreases viability. We also conclude that ionizing radiation could serve as a positive control for Western Blot analysis of protein P53. In addition, our results show that the developing process may play an important role in the quality of P53 protein bands and data interpretation.

  2. A molecular clock regulates angiopoietin-like protein 2 expression.

    Science.gov (United States)

    Kadomatsu, Tsuyoshi; Uragami, Shota; Akashi, Makoto; Tsuchiya, Yoshiki; Nakajima, Hiroo; Nakashima, Yukiko; Endo, Motoyoshi; Miyata, Keishi; Terada, Kazutoyo; Todo, Takeshi; Node, Koichi; Oike, Yuichi

    2013-01-01

    Various physiological and behavioral processes exhibit circadian rhythmicity. These rhythms are usually maintained by negative feedback loops of core clock genes, namely, CLOCK, BMAL, PER, and CRY. Recently, dysfunction in the circadian clock has been recognized as an important foundation for the pathophysiology of lifestyle-related diseases, such as obesity, cardiovascular disease, and some cancers. We have reported that angiopoietin-like protein 2 (ANGPTL2) contributes to the pathogenesis of these lifestyle-related diseases by inducing chronic inflammation. However, molecular mechanisms underlying regulation of ANGPTL2 expression are poorly understood. Here, we assess circadian rhythmicity of ANGPTL2 expression in various mouse tissues. We observed that ANGPTL2 rhythmicity was similar to that of the PER2 gene, which is regulated by the CLOCK/BMAL1 complex. Promoter activity of the human ANGPTL2 gene was significantly induced by CLOCK and BMAL1, an induction markedly attenuated by CRY co-expression. We also identified functional E-boxes in the ANGPTL2 promoter and observed occupancy of these sites by endogenous CLOCK in human osteosarcoma cells. Furthermore, Cry-deficient mice exhibited arrhythmic Angptl2 expression. Taken together, these data suggest that periodic expression of ANGPTL2 is regulated by a molecular clock.

  3. A molecular clock regulates angiopoietin-like protein 2 expression.

    Directory of Open Access Journals (Sweden)

    Tsuyoshi Kadomatsu

    Full Text Available Various physiological and behavioral processes exhibit circadian rhythmicity. These rhythms are usually maintained by negative feedback loops of core clock genes, namely, CLOCK, BMAL, PER, and CRY. Recently, dysfunction in the circadian clock has been recognized as an important foundation for the pathophysiology of lifestyle-related diseases, such as obesity, cardiovascular disease, and some cancers. We have reported that angiopoietin-like protein 2 (ANGPTL2 contributes to the pathogenesis of these lifestyle-related diseases by inducing chronic inflammation. However, molecular mechanisms underlying regulation of ANGPTL2 expression are poorly understood. Here, we assess circadian rhythmicity of ANGPTL2 expression in various mouse tissues. We observed that ANGPTL2 rhythmicity was similar to that of the PER2 gene, which is regulated by the CLOCK/BMAL1 complex. Promoter activity of the human ANGPTL2 gene was significantly induced by CLOCK and BMAL1, an induction markedly attenuated by CRY co-expression. We also identified functional E-boxes in the ANGPTL2 promoter and observed occupancy of these sites by endogenous CLOCK in human osteosarcoma cells. Furthermore, Cry-deficient mice exhibited arrhythmic Angptl2 expression. Taken together, these data suggest that periodic expression of ANGPTL2 is regulated by a molecular clock.

  4. 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...

  5. Glycolipid transfer protein expression is affected by glycosphingolipid synthesis.

    Directory of Open Access Journals (Sweden)

    Matti A Kjellberg

    Full Text Available Members of the glycolipid transfer protein superfamily (GLTP are found from animals and fungi to plants and red micro-alga. Eukaryotes that encode the glucosylceramide synthase responsible for the synthesis of glucosylceramide, the precursor for most glycosphingolipids, also produce GLTPs. Cells that does not synthesize glucosylceramide neither express GLTPs. Based on this genetic relationship there must be a strong correlation between the synthesis of glucosylceramide and GLTPs. To regulate the levels of glycolipids we have used inhibitors of intracellular trafficking, glycosphingolipid synthesis and degradation, and small interfering RNA to down-regulate the activity of glucosylceramide synthase activity. We found that GLTP expression, both at the mRNA and protein levels, is elevated in cells that accumulate glucosylceramide. Monensin and brefeldin A block intracellular vesicular transport mechanisms. Brefeldin A treatment leads to accumulation of newly synthesized glucosylceramide, galactosylceramide and lactosylceramide in a fused endoplasmic reticulum-Golgi complex. On the other hand, inhibiting glycosphingolipid degradation with conduritol-B-epoxide, that generates glucosylceramide accumulation in the lysosomes, did not affect the levels of GLTP. However, glycosphingolipid synthesis inhibitors like PDMP, NB-DNJ and myriocin, all decreased glucosylceramide and GLTP below normal levels. We also found that an 80% loss of glucosylceramide due to glucosylceramide synthase knockdown resulted in a significant reduction in the expression of GLTP. We show here that interfering with membrane trafficking events and simple neutral glycosphingolipid synthesis will affect the expression of GLTP. We postulate that a change in the glucosylceramide balance causes a response in the GLTP expression, and put forward that GLTP might play a role in lipid directing and sensing of glucosylceramide at the ER-Golgi interface.

  6. Common and specific signatures of gene expression and protein-protein interactions in autoimmune diseases.

    Science.gov (United States)

    Tuller, T; Atar, S; Ruppin, E; Gurevich, M; Achiron, A

    2013-03-01

    The aim of this study is to understand intracellular regulatory mechanisms in peripheral blood mononuclear cells (PBMCs), which are either common to many autoimmune diseases or specific to some of them. We incorporated large-scale data such as protein-protein interactions, gene expression and demographical information of hundreds of patients and healthy subjects, related to six autoimmune diseases with available large-scale gene expression measurements: multiple sclerosis (MS), systemic lupus erythematosus (SLE), juvenile rheumatoid arthritis (JRA), Crohn's disease (CD), ulcerative colitis (UC) and type 1 diabetes (T1D). These data were analyzed concurrently by statistical and systems biology approaches tailored for this purpose. We found that chemokines such as CXCL1-3, 5, 6 and the interleukin (IL) IL8 tend to be differentially expressed in PBMCs of patients with the analyzed autoimmune diseases. In addition, the anti-apoptotic gene BCL3, interferon-γ (IFNG), and the vitamin D receptor (VDR) gene physically interact with significantly many genes that tend to be differentially expressed in PBMCs of patients with the analyzed autoimmune diseases. In general, similar cellular processes tend to be differentially expressed in PBMC in the analyzed autoimmune diseases. Specifically, the cellular processes related to cell proliferation (for example, epidermal growth factor, platelet-derived growth factor, nuclear factor-κB, Wnt/β-catenin signaling, stress-activated protein kinase c-Jun NH2-terminal kinase), inflammatory response (for example, interleukins IL2 and IL6, the cytokine granulocyte-macrophage colony-stimulating factor and the B-cell receptor), general signaling cascades (for example, mitogen-activated protein kinase, extracellular signal-regulated kinase, p38 and TRK) and apoptosis are activated in most of the analyzed autoimmune diseases. However, our results suggest that in each of the analyzed diseases, apoptosis and chemotaxis are activated via

  7. Dynamic expression pattern of kinesin accessory protein in Drosophila

    Indian Academy of Sciences (India)

    Ritu Sarpal; Krishanu Ray

    2002-09-01

    We have identified the Drosophila homologue of the non-motor accessory subunit of kinesin-II motor complex. It is homologous to the SpKAP115 of the sea urchin, KAP3A and KAP3B of the mouse, and SMAP protein in humans. In situ hybridization using a DmKAP specific cRNA probe has revealed a dynamic pattern of expression in the developing nervous system. The staining first appears in a subset of cells in the embryonic central nervous system at stage 13 and continues till the first instar larva stage. At the third instar larva stage the staining gets restricted to a few cells in the optic lobe and in the ventral ganglion region. It has also stained a subset of sensory neurons from late stage 13 and till the first instar larva stage. The DmKAP expression pattern in the nervous system corresponds well with that of Klp64D and Klp68D as reported earlier. In addition, we have found that the DmKAP gene is constitutively expressed in the germline cells and in follicle cells during oogenesis. These cells are also stained using an antibody to KLP68D protein, but mRNA in situ hybridization using KLP64D specific probe has not stained these cells. Together these results proved a basis for further analysis of tissue specific function of DmKAP in future.

  8. Stimulation of the human CTP:phosphoethanolamine cytidylyltransferase gene by early growth response protein 1.

    Science.gov (United States)

    Zhu, Lin; Johnson, Christa; Bakovic, Marica

    2008-10-01

    Change in phosphoethanolamine pool size in tumor tissues is an important indicator of tumor prognosis and drug therapy efficacy. Phosphoethanolamine is the substrate of the regulatory enzyme CTP:phosphoethanolamine cytidylyltransferase (ECT) in the de novo biosynthesis of phosphatidylethanolamine (PE). Metabolic labeling with [14C]ethanolamine revealed a reduced ECT activity in MCF-7 breast cancer cells, which led to an accumulation of phosphoethanolamine and a decrease in PE synthesis in comparison with MCF-10A mammary epithelial cells. The enhanced ECT activity in MCF-10A cells was due to significantly elevated CTP:phosphoethanolamine cytidylyltransferase gene (PCYT2) expression, at the level of promoter activity, mRNA, and protein content. The early growth response protein 1 (EGR1) could account for most of the elevated ECT activity in MCF-10A cells relative to MCF-7 cells, as evidenced by promoter-luciferase reporter assays, gel-shift analyses, and by alterations in the EGR1 gene expression. In MCF-7 cells, EGR1 is present at lower levels and the basal PCYT2 promoter activity is maintained by proximal CAAT and GC regions and by elevated nuclear NFkappaB activity. Together, these data demonstrate that EGR1 is an important transcriptional stimulator of the human PCYT2 and that conditions that modify EGR1 also affect the function of ECT and consequently PE synthesis.

  9. Ribosomal protein S6 associates with alphavirus nonstructural protein 2 and mediates expression from alphavirus messages.

    Science.gov (United States)

    Montgomery, Stephanie A; Berglund, Peter; Beard, Clayton W; Johnston, Robert E

    2006-08-01

    Although alphaviruses dramatically alter cellular function within hours of infection, interactions between alphaviruses and specific host cellular proteins are poorly understood. Although the alphavirus nonstructural protein 2 (nsP2) is an essential component of the viral replication complex, it also has critical auxiliary functions that determine the outcome of infection in the host. To gain a better understanding of nsP2 function, we sought to identify cellular proteins with which Venezuelan equine encephalitis virus nsP2 interacted. We demonstrate here that nsP2 associates with ribosomal protein S6 (RpS6) and that nsP2 is present in the ribosome-containing fractions of a polysome gradient, suggesting that nsP2 associates with RpS6 in the context of the whole ribosome. This result was noteworthy, since viral replicase proteins have seldom been described in direct association with components of the ribosome. The association of RpS6 with nsP2 was detected throughout the course of infection, and neither the synthesis of the viral structural proteins nor the presence of the other nonstructural proteins was required for RpS6 interaction with nsP2. nsP1 also was associated with RpS6, but other nonstructural proteins were not. RpS6 phosphorylation was dramatically diminished within hours after infection with alphaviruses. Furthermore, a reduction in the level of RpS6 protein expression led to diminished expression from alphavirus subgenomic messages, whereas no dramatic diminution in cellular translation was observed. Taken together, these data suggest that alphaviruses alter the ribosome during infection and that this alteration may contribute to differential translation of host and viral messages.

  10. Expression of mammalian DT-diaphorase in Escherichia coli: purification and characterization of the expressed protein.

    Science.gov (United States)

    Ma, Q; Wang, R; Yang, C S; Lu, A Y

    1990-12-01

    A full-length cDNA clone, pKK-DTD4, complementary to rat liver cytosolic DT-diaphorase [NAD(P)H:quinone oxidoreductase (EC 1.6.99.2)] mRNA was expressed in Escherichia coli. The pKK-DTD4 cDNA was obtained by extending the 5'-end sequence of a rat liver DT-diaphorase cDNA clone, pDTD55, to include an ATG initiation codon and the NH2-terminal codons using polymerase chain reaction (PCR). Restriction sites for EcoRI and HindIII were incorporated at the 5'- and 3'-ends of the cDNA, respectively, by the PCR reaction. The resulting full-length cDNA was inserted into an expression vector, pKK2.7, at the EcoRI and HindIII restriction sites. E. coli strain AB1899 was transformed with the constructed expression plasmid, and DT-diaphorase was expressed under the control of the tac promotor. The expressed DT-diaphorase exhibited high activity of menadione reduction and was inhibited by dicumarol at a concentration of 10(-5)M. After purification by Cibacron Blue affinity chromatography, the expressed enzyme migrated as a single band on 12.5% sodium dodecyl sulfate-polyacrylamide gel with a molecular weight equivalent to that of the purified rat liver cytosolic DT-diaphorase. The purified expressed protein was recognized by polyclonal antibodies against rat liver DT-diaphorase on immunoblot analysis. It utilized either NADPH or NADH as electron donor at equal efficiency and displayed high activities in reduction of menadione, 1,4-benzoquinone, and 2,6-dichlorophenolindophenol which are typical substrates for DT-diaphorase. The expressed DT-diaphorase exhibited a typical flavoprotein spectrum with absorption peaks at 380 and 452 nm. Flavin content determination showed that it contained 2 mol of FAD per mole of the enzyme. Edman protein sequencing of the first 20 amino acid residues at the NH2 terminus of the expressed protein indicated that the expressed DT-diaphorase is not blocked at the NH2 terminus and has an alanine as the first amino acid. The remaining 19 amino acid

  11. Monotreme lactation protein is highly expressed in monotreme milk and provides antimicrobial protection.

    Science.gov (United States)

    Enjapoori, Ashwantha Kumar; Grant, Tom R; Nicol, Stewart C; Lefèvre, Christophe M; Nicholas, Kevin R; Sharp, Julie A

    2014-09-22

    Monotremes (platypus and echidna) are the descendants of the oldest ancestor of all extant mammals distinguished from other mammals by mode of reproduction. Monotremes lay eggs following a short gestation period and after an even briefer incubation period, altricial hatchlings are nourished over a long lactation period with milk secreted by nipple-less mammary patches located on the female's abdomen. Milk is the sole source of nutrition and immune protection for the developing young until weaning. Using transcriptome and mass spectrometry analysis of milk cells and milk proteins, respectively, a novel Monotreme Lactation Protein (MLP) was identified as a major secreted protein in milk. We show that platypus and short-beaked echidna MLP genes show significant homology and are unique to monotremes. The MLP transcript was shown to be expressed in a variety of tissues; however, highest expression was observed in milk cells and was expressed constitutively from early to late lactation. Analysis of recombinant MLP showed that it is an N-linked glycosylated protein and biophysical studies predicted that MLP is an amphipathic, α-helical protein, a typical feature of antimicrobial proteins. Functional analysis revealed MLP antibacterial activity against both opportunistic pathogenic Staphylococcus aureus and commensal Enterococcus faecalis bacteria but showed no effect on Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, and Salmonella enterica. Our data suggest that MLP is an evolutionarily ancient component of milk-mediated innate immunity absent in other mammals. We propose that MLP evolved specifically in the monotreme lineage supporting the evolution of lactation in these species to provide bacterial protection, at a time when mammals lacked nipples.

  12. Monotreme Lactation Protein Is Highly Expressed in Monotreme Milk and Provides Antimicrobial Protection

    Science.gov (United States)

    Enjapoori, Ashwantha Kumar; Grant, Tom R.; Nicol, Stewart C.; Lefèvre, Christophe M.; Nicholas, Kevin R.; Sharp, Julie A.

    2014-01-01

    Monotremes (platypus and echidna) are the descendants of the oldest ancestor of all extant mammals distinguished from other mammals by mode of reproduction. Monotremes lay eggs following a short gestation period and after an even briefer incubation period, altricial hatchlings are nourished over a long lactation period with milk secreted by nipple-less mammary patches located on the female’s abdomen. Milk is the sole source of nutrition and immune protection for the developing young until weaning. Using transcriptome and mass spectrometry analysis of milk cells and milk proteins, respectively, a novel Monotreme Lactation Protein (MLP) was identified as a major secreted protein in milk. We show that platypus and short-beaked echidna MLP genes show significant homology and are unique to monotremes. The MLP transcript was shown to be expressed in a variety of tissues; however, highest expression was observed in milk cells and was expressed constitutively from early to late lactation. Analysis of recombinant MLP showed that it is an N-linked glycosylated protein and biophysical studies predicted that MLP is an amphipathic, α-helical protein, a typical feature of antimicrobial proteins. Functional analysis revealed MLP antibacterial activity against both opportunistic pathogenic Staphylococcus aureus and commensal Enterococcus faecalis bacteria but showed no effect on Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, and Salmonella enterica. Our data suggest that MLP is an evolutionarily ancient component of milk-mediated innate immunity absent in other mammals. We propose that MLP evolved specifically in the monotreme lineage supporting the evolution of lactation in these species to provide bacterial protection, at a time when mammals lacked nipples. PMID:25245409

  13. 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.

  14. 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.

  15. Identification of differentially expressed proteins and phosphorylated proteins in rice seedlings in response to strigolactone treatment.

    Directory of Open Access Journals (Sweden)

    Fangyu Chen

    Full Text Available Strigolactones (SLs are recently identified plant hormones that inhibit shoot branching and control various aspects of plant growth, development and interaction with parasites. Previous studies have shown that plant D10 protein is a carotenoid cleavage dioxygenase that functions in SL biosynthesis. In this work, we used an allelic SL-deficient d10 mutant XJC of rice (Oryza sativa L. spp. indica to investigate proteins that were responsive to SL treatment. When grown in darkness, d10 mutant seedlings exhibited elongated mesocotyl that could be rescued by exogenous application of SLs. Soluble protein extracts were prepared from d10 mutant seedlings grown in darkness in the presence of GR24, a synthetic SL analog. Soluble proteins were separated on two-dimensional gels and subjected to proteomic analysis. Proteins that were expressed differentially and phosphoproteins whose phosphorylation status changed in response to GR24 treatment were identified. Eight proteins were found to be induced or down-regulated by GR24, and a different set of 8 phosphoproteins were shown to change their phosphorylation intensities in the dark-grown d10 seedlings in response to GR24 treatment. Analysis of these proteins revealed that they are important enzymes of the carbohydrate and amino acid metabolic pathways and key components of the cellular energy generation machinery. These proteins may represent potential targets of the SL signaling pathway. This study provides new insight into the complex and negative regulatory mechanism by which SLs control shoot branching and plant development.

  16. Structure and expression of a novel compact myelin protein – Small VCP-interacting protein (SVIP)

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Jiawen [Department of Neurology, Vanderbilt University School of Medicine (United States); Peng, Dungeng [Department of Biochemistry, Vanderbilt University School of Medicine (United States); Voehler, Markus [Center for Structural Biology, Vanderbilt University (United States); Sanders, Charles R. [Department of Biochemistry, Vanderbilt University School of Medicine (United States); Center for Structural Biology, Vanderbilt University (United States); Li, Jun, E-mail: jun.li.2@vanderbilt.edu [Department of Neurology, Vanderbilt University School of Medicine (United States); Tennessee Valley Healthcare System (TVHS) – Nashville VA (United States)

    2013-10-11

    Highlights: •SVIP (small p97/VCP-interacting protein) co-localizes with myelin basic protein (MBP) in compact myelin. •We determined that SVIP is an intrinsically disordered protein (IDP). •The helical content of SVIP increases dramatically during its interaction with negatively charged lipid membrane. •This study provides structural insight into interactions between SVIP and myelin membranes. -- Abstract: SVIP (small p97/VCP-interacting protein) was initially identified as one of many cofactors regulating the valosin containing protein (VCP), an AAA+ ATPase involved in endoplasmic-reticulum-associated protein degradation (ERAD). Our previous study showed that SVIP is expressed exclusively in the nervous system. In the present study, SVIP and VCP were seen to be co-localized in neuronal cell bodies. Interestingly, we also observed that SVIP co-localizes with myelin basic protein (MBP) in compact myelin, where VCP was absent. Furthermore, using nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopic measurements, we determined that SVIP is an intrinsically disordered protein (IDP). However, upon binding to the surface of membranes containing a net negative charge, the helical content of SVIP increases dramatically. These findings provide structural insight into interactions between SVIP and myelin membranes.

  17. Fatty acid binding protein 7 and n-3 poly unsaturated fatty acid supply in early rat brain development.

    Science.gov (United States)

    Maximin, Elise; Langelier, Bénédicte; Aïoun, Josiane; Al-Gubory, Kaïs H; Bordat, Christian; Lavialle, Monique; Heberden, Christine

    2016-03-01

    Fatty acid binding protein 7 (FABP7), abundant in the embryonic brain, binds with the highest affinity to docosahexaenoic acid (DHA) and is expressed in the early stages of embryogenesis. Here, we have examined the consequences of the exposure to different DHA levels and of the in utero depletion of FABP7 on early rat brain development. Neurodevelopment was evaluated through the contents of two proteins, connexin 43 (Cx43) and cyclin-dependent kinase 5 (CDK5), both involved in neuroblast proliferation, differentiation, and migration. The dams were fed with diets presenting different DHA contents, from deficiency to supplementation. DHA brain embryos contents already differed at embryonic day 11.5 and the differences kept increasing with time. Cx43 and CDK5 contents were positively associated with the brain DHA levels. When FABP7 was depleted in vivo by injections of siRNA in the telencephalon, the enhancement of the contents of both proteins was lost in supplemented animals, but FABP7 depletion did not modify phospholipid compositions regardless of the diets. Thus, FABP7 is a necessary mediator of the effect of DHA on these proteins synthesis, but its role in DHA uptake is not critical, although FABP7 is localized in phospholipid-rich areas. Our study shows that high contents of DHA associated with FABP7 are necessary to promote early brain development, which prompted us to recommend DHA supplementation early in pregnancy.

  18. 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.

  19. Loss of Pnn expression results in mouse early embryonic lethality and cellular apoptosis through SRSF1-mediated alternative expression of Bcl-xS and ICAD.

    Science.gov (United States)

    Leu, Steve; Lin, Yen-Ming; Wu, Chu-Han; Ouyang, Pin

    2012-07-01

    Pinin (Pnn), a serine/arginine-rich (SR)-related protein, has been shown to play multiple roles within eukaryotic cells including cell-cell adhesion, cell migration, regulation of gene transcription, mRNA export and alternative splicing. In this study, an attempt to generate mice homozygously deficient in Pnn failed because of early embryonic lethality. To evaluate the effects of loss of Pnn expression on cell survival, RNA interference experiments were performed in MCF-7 cells. Depletion of Pnn resulted in cellular apoptosis and nuclear condensation. In addition, nuclear speckles were disrupted, and expression levels of SR proteins were diminished. RT-PCR analysis showed that alternative splicing patterns of SRSF1 as well as of apoptosis-related genes Bcl-x and ICAD were altered, and expression levels of Bim isoforms were modulated in Pnn-depleted cells. Cellular apoptosis induced by Pnn depletion was rescued by overexpression of SRSF1, which also restored generation of Bcl-xL and functionless ICAD. Pnn expression is, therefore, essential for survival of mouse embryos and the breast carcinoma cell line MCF-7. Moreover, Pnn depletion, modulated by SRSF1, determines cellular apoptosis through activation of the expression of pro-apoptotic Bcl-xS transcripts.

  20. Entamoeba histolytica calreticulin: an endoplasmic reticulum protein expressed by trophozoites into experimentally induced amoebic liver abscesses.

    Science.gov (United States)

    González, Enrique; de Leon, Maria del Carmen García; Meza, Isaura; Ocadiz-Delgado, Rodolfo; Gariglio, Patricio; Silva-Olivares, Angelica; Galindo-Gómez, Silvia; Shibayama, Mineko; Morán, Patricia; Valadez, Alicia; Limón, Angelica; Rojas, Liliana; Hernández, Eric G; Cerritos, René; Ximenez, Cecilia

    2011-02-01

    Entamoeba histolytica calreticulin (EhCRT) is remarkably immunogenic in humans (90-100% of invasive amoebiasis patients). Nevertheless, the study of calreticulin in this protozoan is still in its early stages. The exact location, biological functions, and its role in pathogenesis are yet to be fully understood. The aim of the present work is to determine the location of EhCRT in virulent trophozoites in vivo and the expression of the Ehcrt gene during the development of experimentally induced amoebic liver abscesses (ALA) in hamsters. Antibodies against recombinant EhCRT were used for the immunolocalization of EhCRT in trophozoites through confocal microscopy; immunohistochemical assays were also performed on tissue sections of ALAs at different times after intrahepatic inoculation. The expression of the Ehcrt gene during the development of ALA was estimated through both in situ RT-PCR and real-time RT-PCR. Confocal assays of virulent trophozoites showed a distribution of EhCRT in the cytoplasmic vesicles of different sizes. Apparently, EhCRT is not exported into the hepatic tissue. Real-time RT-PCR demonstrated an over-expression of the Ehcrt gene at 30 min after trophozoite inoculation, reaching a peak at 1-2 h; thereafter, the expression fell sharply to its original levels. These results demonstrate for the first time in an in vivo model of ALA, the expression of Ehcrt gene in E. histolytica trophozoites and add evidence that support CRT as a resident protein of the ER in E. histolytica species. The in vivo experiments suggest that CRT may play an important role during the early stages of the host-parasite relationship, when the parasite is adapting to a new environment, although the protein seems to be constitutively synthesized. Moreover, trophozoites apparently do not export EhCRT into the hepatic tissue in ALA.

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

    DEFF Research Database (Denmark)

    Michaelsen, Kim F.; Greer, Frank R

    2014-01-01

    The objective of this review was to summarize selected health aspects of protein intake during the first 2 y of life. During this period there is a marked increase in protein intake from an intake of ∼5% of energy from protein (PE%) in an exclusively breastfed infant to ∼15 PE% when complementary...... 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...... by decreasing the upper allowable limit of the protein content of infant formulas for the first year of life and limiting the intake of cow milk in the second year of life....

  2. Cystic fibrosis transmembrane conductance regulator protein expression in the male excretory duct system during development.

    Science.gov (United States)

    Marcorelles, Pascale; Gillet, Danièle; Friocourt, Gaëlle; Ledé, Françoise; Samaison, Laura; Huguen, Geneviève; Ferec, Claude

    2012-03-01

    Sterility due to bilateral destruction in utero or in early infancy resulting in congenital absence of the vas deferens is the rule in male patients with cystic fibrosis. To understand the developmental pattern of this anomaly, the microscopic morphology of the male excretory system was analyzed during development and the expression of the cystic fibrosis transmembrane conductance regulator protein was explored by immunohistochemistry. We observed that cystic fibrosis fetuses had no excretory ducts agenesis or obstruction until 22 weeks of gestation. However, a focal inflammatory pattern and mucinous plugs in the oldest cystic fibrosis case suggested a disruptive mechanism. Immunolabeling of cytoplasmic epithelial cystic fibrosis transmembrane conductance regulator protein was demonstrated in all cystic fibrosis and control cases with a similar pattern of expression of the protein between age-matched controls and cystic fibrosis cases. At midgestation, an apical intensification appeared in both cystic fibrosis and control cases and was stable during the remainder of fetal life. No gradient of intensity could be detected between the different segments of the excretory tract. These findings are different from those reported in adults. The absence of any morphologic anomaly until 22 weeks of gestation, the focal destruction of the epithelial structures during the second trimester, and the chronological pattern of expression of cystic fibrosis transmembrane conductance regulator are of interest for a better understanding of the pathophysiology of this disease.

  3. A Simple Method for Immunohistochemical Staining of Zebrafish Brain Sections for c-fos Protein Expression.

    Science.gov (United States)

    Chatterjee, Diptendu; Tran, Steven; Shams, Soaleha; Gerlai, Robert

    2015-12-01

    Immediate early genes (IEGs) are transcription factors whose own transcription is initiated rapidly, for example, in the brain in response to environmental stimuli. c-fos is an IEG often used as a marker of neuronal activation. c-fos mRNA expression has started to be quantified and localized in the zebrafish brain following environmental manipulations but analysis of the expression of c-fos protein in the zebrafish brain has rarely been attempted. Here, we describe an immunofluorescence staining method for quantifying c-fos protein expression in different regions of the zebrafish brain. In addition, we expose zebrafish to caffeine, a positive control for c-fos activation in the brain. To confirm cell nucleus specific binding of the c-fos antibody, we counterstained brain sections with the nuclear fluorescent stain DAPI. Furthermore, we describe a method for reducing background autofluorescence often observed in zebrafish brain tissue. Our analysis showed that exposure to caffeine increased the number of c-fos protein-positive cells in specific zebrafish brain regions detected by the immunofluorescence method. Our results demonstrate the feasibility of immunofluorescence-based methods in the analysis of neuronal activation in the zebrafish brain, and reinforce the utility of the zebrafish in behavioral neuroscience research.

  4. Functional analysis of expressed peptides that bind yeast STE proteins.

    Science.gov (United States)

    Caponigro, Giordano; Abedi, Majid; Kamb, Alexander

    2003-08-15

    Peptides are potentially useful for target validation and other reverse genetic applications. For instance, if a specific protein is susceptible to peptide inhibition, it may have a higher probability of being vulnerable to small molecules. We used the yeast two-hybrid technique to identify and study peptide binders for three yeast proteins involved in pheromone response: Ste11p, Ste18p, and Ste50p. A subset of peptide binders was shown to inhibit pheromone response in cells using two different functional assays. In addition, we utilized a variant of the yeast two-hybrid method to examine relative binding affinities based on competitive interactions in yeast. Our results suggest that binding affinity and inhibitory potency of peptides do not correlate perfectly and that peptide-protein interactions can be complex and unpredictable. Taken together these results suggest that while peptides are useful as in vivo inhibitors of protein function, caution must be exercised when choosing peptides for further studies and when inferring affinities from expression phenotypes.

  5. Measuring ERCC1 protein expression in cancer specimens

    DEFF Research Database (Denmark)

    Smith, David Hersi; Fiehn, Anne-Marie Kanstrup; Fogh, Louise

    2014-01-01

    Platinum chemotherapy remains part of standard therapies in the management of a variety of cancers. Severe side effects and a high degree of resistance to platinum drugs have led numerous researchers to search for predictive biomarkers, which could aid in identifying patients that are the most...... likely to respond to therapy. The ERCC1-ERCC4 endonuclease plays a critical role in the repair of platinum-DNA damage and has widely been studied in relation to sensitivity to platinum chemotherapy. The standard method to evaluate ERCC1 protein expression is through the use of immunohistochemistry...

  6. Expressed protein ligation for metalloprotein design and engineering.

    Science.gov (United States)

    Clark, Kevin M; van der Donk, Wilfred A; Lu, Yi

    2009-01-01

    Metalloproteins contain highly specialized metal-binding sites that are designed to accept specific metal ions to maintain correct function. Although many of the sites have been modified with success, the relative paucity of functional group availability within proteinogenic amino acids can sometimes leave open questions about specific functions of the metal binding ligands. Attaining a more thorough analysis of individual amino acid function within metalloproteins has been realized using expressed protein ligation (EPL). Here we describe our recent efforts using EPL to incorporate nonproteinogenic cysteine and methionine analogues into the type 1 copper site found in Pseudomonas aeruginosa azurin.

  7. Early gene expression in Pseudomonas fluorescens exposed to a polymetallic solution.

    Science.gov (United States)

    Gómez-Sagasti, María T; Becerril, José M; Epelde, Lur; Alkorta, Itziar; Garbisu, Carlos

    2015-02-01

    The molecular response of Pseudomonas fluorescens cells exposed to a mixture of heavy metals remains largely unknown. Here, we studied the temporal changes in the early gene expression of P. fluorescens cells exposed to three doses of a polymetallic solution over two exposure times, through the application of a customized cDNA microarray. At the lowest metal dose (MD/4), we observed a repression of the Hsp70 chaperone system, MATE and MFS transporters, TonB membrane transporter and histidine kinases, together with an overexpression of metal transport (ChaC, CopC), chemotaxis and glutamine synthetase genes. At the intermediate metal dose (MD), several amino acid transporters, a response regulator (CheY), a TonB-dependent receptor and the mutT DNA repair gene were repressed; by contrast, an overexpression of genes associated with the antioxidative stress system and the transport of chelates and sulfur was observed. Finally, at the highest metal dose (4MD), a repression of genes encoding metal ion transporters, drug resistance and alginate biosynthesis was found, together with an overexpression of genes encoding antioxidative proteins, membrane transporters, ribosomal proteins, chaperones and proteases. It was concluded that P. fluorescens cells showed, over exposure time, a highly complex molecular response when exposed to a polymetallic solution, involving mechanisms related with chemotaxis, signal transmission, membrane transport, cellular redox state, and the regulation of transcription and ribosomal activity.

  8. Complexity of Gene Expression Evolution after Duplication: Protein Dosage Rebalancing

    Directory of Open Access Journals (Sweden)

    Igor B. Rogozin

    2014-01-01

    Full Text Available Ongoing debates about functional importance of gene duplications have been recently intensified by a heated discussion of the “ortholog conjecture” (OC. Under the OC, which is central to functional annotation of genomes, orthologous genes are functionally more similar than paralogous genes at the same level of sequence divergence. However, a recent study challenged the OC by reporting a greater functional similarity, in terms of gene ontology (GO annotations and expression profiles, among within-species paralogs compared to orthologs. These findings were taken to indicate that functional similarity of homologous genes is primarily determined by the cellular context of the genes, rather than evolutionary history. Subsequent studies suggested that the OC appears to be generally valid when applied to mammalian evolution but the complete picture of evolution of gene expression also has to incorporate lineage-specific aspects of paralogy. The observed complexity of gene expression evolution after duplication can be explained through selection for gene dosage effect combined with the duplication-degeneration-complementation model. This paper discusses expression divergence of recent duplications occurring before functional divergence of proteins encoded by duplicate genes.

  9. Increased heat shock protein expression after stress in Japanese quail.

    Science.gov (United States)

    Hoekstra, K A; Iwama, G K; Nichols, C R; Godin, D V; Cheng, K M

    1998-12-01

    Heat shock proteins (HSPs) have been shown to provide information on the biological impact of environmental stress to organisms, yet none have investigated the HSP response to stress in birds. Japanese quail were exposed to seven different stressors (mild restraint, loud noise, inescapable irritation, cold temperature, isolation in darkness, and two stressful social situations) and expression of HSP30, 60, 70, and 90 in heart, liver, lung, kidney and gonads was examined. Tonic Immobility (TI) tests were also conducted to assess whether the stressors increased fear response. Increased expression of HSP70 was found in the myocardial tissue of birds exposed to loud noise, inescapable irritation, cold temperature, and isolation in darkness. Increased expression of other HSPs was not apparent in the heart or any of the other all tissues examined. Longer TI was observed only in birds exposed to the noise stress. Evidence is presented that a fairly wide range of stressors caused increased expression of HSP70 in the Japanese quail myocardial tissue and that HSPs may provide useful biomarkers for the study of environmental stress in birds.

  10. Expression analysis of Shh signaling members in early stages of chick lung development.

    Science.gov (United States)

    Moura, Rute Silva; Silva-Gonçalves, Carla; Vaz-Cunha, Patrícia; Correia-Pinto, Jorge

    2016-10-01

    Lung organogenesis is guided by epithelial-mesenchymal interactions that coordinate cellular events responsible for the formation of the respiratory system. Several signaling pathways have been implicated in this process; among them, sonic hedgehog (Shh) signaling has emerged as a crucial regulator of branching morphogenesis in the mammalian lung. Canonical Shh signaling requires the presence of patched (Ptch) and smoothened (Smo) transmembrane receptors in order to induce the activation of glioblastoma (Gli) zinc finger transcription factors that are the true effectors of the pathway. Signal transduction is finely regulated by Ptch1, Gli, and Hhip (hedgehog-interacting protein). The present work characterizes, for the first time, the expression pattern of shh, ptch1, smo, gli1, and hhip in early stages of the embryonic chick lung. In situ hybridization studies revealed that these genes are expressed in the same cellular compartments as their mammalian counterparts, although their proximo-distal distribution is slightly changed. Moreover, the molecular interactions between fibroblast growth factor (FGF) and Shh signaling pathway were assessed, in vitro, by grafting beads soaked in SU5402 (an FGF receptor inhibitor). In the chick lung, Shh signaling seems to have some features that are species specific since shh is not a downstream target of FGF signaling. Nonetheless and despite the observed differences, these findings suggest a role for Shh signaling in the epithelial-mesenchymal interactions that control chick lung morphogenesis.

  11. 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.

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

    Directory of Open Access Journals (Sweden)

    Frezza Eldo E

    2006-01-01

    Full Text Available Abstract Background 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. Methods 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,. Results 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. Conclusion 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.

  13. Analysis of cDNA sequence, protein structure and expression of parotid secretory protein in pig

    Institute of Scientific and Technical Information of China (English)

    YIN Haifang; FAN Baoliang; ZHAO Zhihui; LIU Zhaoliang; FEI Jing; LI Ning

    2003-01-01

    Parotid secretory protein (PSP) secreted abundantly in saliva, whose function is related with the anti-bacterial effect. The PSP cDNA has been isolated from pig parotid glands by 3′ and 5′ rapid amplification of cDNA end (RACE),based on the conserved signal peptide region among the known mammalian PSP. Theresult of homologous comparison shows that pig PSP and human PSP shares the high identity at the level of the primary, secondary and tertiary protein structure. A search for functionally significant protein motifs revealed a unique amino acid sequence pattern consisting of the residues Leu-X(6)-Leu-X(6)-Leu- X(7)-Leu-X(6)-Leu-X(6)-Leu near the amino-terminal portion of the protein, which is important to its function. RT-PCR, Dot blot and Northern blot analysis demonstrated that PSP was strongly expressed in parotid glands, but not in other tissues.

  14. Expression of fragile X mental retardation protein and Fmr1 mRNA during folliculogenesis in the rat.

    Science.gov (United States)

    Ferder, Ianina; Parborell, Fernanda; Sundblad, Victoria; Chiauzzi, Violeta; Gómez, Karina; Charreau, Eduardo H; Tesone, Marta; Dain, Liliana

    2013-04-01

    Fragile X mental retardation protein (FMRP) belongs to a small family of RNA-binding proteins. Its absence or inactivity is responsible for fragile X syndrome, the most common cause of inherited mental retardation. Despite its ubiquitous expression, FMRP function and expression remain almost understudied in non-neuronal tissues, though previous studies on germline development during oogenesis may suggest a special function of this protein also in ovarian tissue. In addition, the well-documented association of FMR1 premutation state with fragile X-related premature ovarian insufficiency adds interest to the role of FMRP in ovarian physiology. The aim of the present work was to investigate the expression of Fmr1 mRNA and its protein, FMRP, at different stages of rat follicular development. By immunohistochemical studies we demonstrated FMRP expression in granulosa, theca and germ cells in all stages of follicular development. In addition, changes in Fmr1 expression, both at the protein and mRNA levels, were observed. FMRP levels increased upon follicular development while preantral and early antral follicles presented similar levels of Fmr1 transcripts with decreased expression in preovulatory follicles. These observations suggest that Fmr1 expression in the ovary is regulated at different and perhaps independent levels. In addition, our results show expression of at least four different isoforms of FMRP during all stages of follicular growth with expression patterns that differ from those observed in brain and testis. Our study shows a regulated expression of Fmr1, both at mRNA and protein levels, during rat follicular development.

  15. Down-expression of tumor protein p53-induced nuclear protein 1 in human gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Pei-Hong Jiang; Yoshiharu Motoo; Stéphane Garcia; Juan Lucio Iovanna; Marie-Josèphe Pébusque; Norio Sawabu

    2006-01-01

    AIM: Overexpression of tumor protein p53-induced nuclear protein 1 (TP53INP1) induces G1 cell cycle arrest and increases p53-mediated apoptosis. To clarify the clinical importance of TP53INP1, we analyzed TP53INP1and p53 expression in gastric cancer.METHODS: TP53INP1 and p53 expression were examined using immunohistochemistry in 142 cases of gastric cancer. The apoptosis of gastric cancer cells was analyzed using the TUNEL method. The relationship between the expression of TP53INP1 and clinicopathological factors was statistically analyzed.RESULTS: TP53INP1 was expressed in 98% (139/142cases) of non-cancerous gastric tissues and was downexpressed in 64% (91/142 cases) of gastric cancer lesions from the same patients. TP53INP1 expression was significantly decreased (43.9%) in poorly differentiated adenocarcinoma compared with well or moderately differentiated adenocarcinoma (81.6%).Cancers invading the submucosa or deeper showed lower positively (59.1%) compared with mucosal cancers (85.2%). Decrease or loss of TP53INP1 expression was significantly correlated with lymphatic invasion (54.3%vs 82.0% without lymphatic invasion) and node-positive patients (31.3% vs 68.3% in node-negative patients).P53 was expressed in 68 (47.9%) patients of gastric cancer, whereas it was absent in normal gastric tissues.A significant association was also observed between TP53INP1 status and the level of apoptosis in tumor cells: the apoptotic index in TP53INP1-positive tissues was significantly higher than that in TP53INP1-negative portions. Finally, when survival data were analyzed,loss of TP53INP1 expression had a significant effect in predicting a poor prognosis (P= 0.0006).CONCLUSION: TP53INP1-positive rate decreases with the progression of gastric cancer. TP53INP1 protein negativity is significantly associated with aggressive pathological phenotypes of gastric cancer. TP53INP1is related to the apoptosis of gastric cancer cells. The decreased expression of the TP53INP1 protein may

  16. 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.

  17. Genetic characterization of early maturing maize hybrids (Zea mays L.) obtained by protein and RAPD markers

    National Research Council Canada - National Science Library

    Bauer, Iva; Mladenovic-Drinic, Snezana; Filipovic, Milomir; Konstantinov, Kosana

    2005-01-01

    .... The objective of our study was to characterize set of early maturing maize hybrids with protein and RAPD markers and to compare this clasification with their pedigree information. RAPD markers gave significantly higher rate of polymorphism than protein markers. Better corelation was found among pedigree information and protein markers.

  18. Targeting Cell Surface Proteins in Molecular Photoacoustic Imaging to Detect Ovarian Cancer Early

    Science.gov (United States)

    2013-07-01

    10-1-0422 TITLE: Targeting Cell Surface Proteins in Molecular Photoacoustic Imaging to Detect Ovarian Cancer Early PRINCIPAL...DATES COVERED 1 July 2010 - 30 June 2013 4. TITLE AND SUBTITLE Targeting Cell Surface Proteins in Molecular 5a. CONTRACT NUMBER Photoacoustic ...upon request). Aim 2) Prioritize ovarian cancer-associated surface proteins for their utility as molecular photoacoustic imaging targets and

  19. 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.

  20. Early secretory antigenic target protein-6/culture filtrate protein-10 fusion protein-specific Th1 and Th2 response and its diagnostic value in tuberculous pleural effusion

    Institute of Scientific and Technical Information of China (English)

    戈启萍

    2013-01-01

    Objective To detect the Th1 and Th2 cell percentage in pleural effusion mononuclear cells (PEMCs) stimulated by early secretory antigenic target protein-6 (ESAT-6) /culture filtrate protein-10 (CFP-10) fusion protein (E/C) with flow cytometry (FCM) ,and to explore the local antigen specific Th1 and Th2 response and

  1. Early Childhood Stuttering III: Initial Status of Expressive Language Abilities.

    Science.gov (United States)

    Watkins, Ruth V.; Yairi, Ehud; Ambrose, Nicoline Grinager

    1999-01-01

    A study compared the expressive language abilities of 22 preschool children whose stuttering persisted and 62 who recovered over a four-year period. Findings revealed similarity in the abilities of children whose stuttering persisted as opposed to abated at all ages. All stutterers displayed abilities near or above developmental expectations. (CR)

  2. Follow-Up of Children with Early Expressive Phonology Disorders.

    Science.gov (United States)

    Lewis, Barbara A.; Freebairn, Lisa A.; Taylor, H. Gerry

    2000-01-01

    Fifty-two children identified at ages 4 and 6 as having an expressive phonology disorder were followed to the third and fourth grades. Children with a phonology disorder along with other language problems performed more poorly than the others on measures of phoneme awareness, language, reading decoding, reading comprehension, and spelling.…

  3. Differential protein expression in heart in UT-B null mice with cardiac conduction defects.

    Science.gov (United States)

    Yu, Hao; Meng, Yan; Wang, Li-Shun; Jin, Xian; Gao, Li-Fang; Zhou, Lei; Ji, Kun; Li, Yang; Zhao, Li-Juan; Chen, Guo-Qiang; Zhao, Xue-Jian; Yang, Baoxue

    2009-02-01

    Cardiac conduction defects were found in transgenic mice deficient in urea transporter UT-B. To investigate the molecular mechanisms of the conduction defects caused by UT-B deletion, we studied the protein expression profiles of heart tissue (comprising most conduction system) in wild-type versus UT-B null mice at different ages. By two-dimensional electrophoresis-based comparative analysis, we found that more than dozen proteins were modulated (>two-fold) in the myocardium of UT-B null mice. Out of these modulated proteins, troponin T (TNNT2) presented significant changes in UT-B null mice at early stage prior to the development of P-R interval elongation, while the change of atrial natriuretic peptide (ANP) occurred only at late stage in UT-B null mice that had the AV block. These data indicate that UT-B deletion caused the dynamic expression regulation of TNNT2 and ANP, and these proteins may provide new clues to investigate the molecular events involved in cardiac conduction.

  4. Maternal High Fat Diet Affects Offspring's Vitamin K-Dependent Proteins Expression Levels.

    Directory of Open Access Journals (Sweden)

    Stuart Lanham

    Full Text Available Studies suggest bone growth & 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 Gas6, in bone and vascular development. This study extends the analysis of VKDPs previously conducted in 6 week old offspring, into offspring of 30 weeks of age, to assess the longer term effects of a maternal and postnatal high fat (HF diet on VKDP expression. Overall a HF maternal diet and offspring diet exacerbated the bone changes observed. Sex specific and tissue specific differences were observed in VKDP expression for both aorta and femoral tissues. In addition, significant correlations were observed between femoral OCN, Periostin Gas6, and Vkor expression levels and measures of femoral bone structure. Furthermore, MGP, OCN, Ggcx and Vkor expression levels correlated to mass and fat volume, in both sexes. In summary the current study has highlighted the importance of the long-term effects of maternal nutrition on offspring bone development and the correlation of VKDPs to bone structure.

  5. Real time imaging of mRNA expression dynamics in live cells using protein complementation methods

    Science.gov (United States)

    Meller, Amit

    2009-03-01

    Traditional methods for mRNA quantification in cells, such as northern blots, quantitative PCR or microarrays assays, require cell lysis and therefore do not preserve its dynamics. These methods cannot be used to probe the spatio-temporal localization of mRNA in cells, which provide useful information for a wide range biomolecular process, including RNA metabolizim, expression kinetics and RNA interference. To probe mRNA dynamics in live prokaryotic and eukaryotic cells, we develop a method, which exploit the strong affinity of the eukaryotic initiation factor 4A (eIF4A) to specific RNA aptamers. Two parts of the eIF4A are fused to a split Green Fluorescence Protein (GFP), and are expressed in the cells at high abundance. However, only when the RNA apatmer is also present, the two protein parts complement and become fluorescent. Thus, the fluorescent background remains low, allowing us to directly image the expression of mRNA molecules in live e-coli cells from its early onset, over hours. We find that the expression kinetics can be classified in one out of at least three forms, which also display distinct spatial distributions. I will discuss the possible biological origin for these distributions and their time evolution.

  6. Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum.

    Science.gov (United States)

    Watanabe, Masahito; Kobayashi, Mirina; Nagaya, Masaki; Matsunari, Hitomi; Nakano, Kazuaki; Maehara, Miki; Hayashida, Gota; Takayanagi, Shuko; Sakai, Rieko; Umeyama, Kazuhiro; Watanabe, Nobuyuki; Onodera, Masafumi; Nagashima, Hiroshi

    2015-01-01

    Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36-37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum.

  7. Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum

    Science.gov (United States)

    WATANABE, Masahito; KOBAYASHI, Mirina; NAGAYA, Masaki; MATSUNARI, Hitomi; NAKANO, Kazuaki; MAEHARA, Miki; HAYASHIDA, Gota; TAKAYANAGI, Shuko; SAKAI, Rieko; UMEYAMA, Kazuhiro; WATANABE, Nobuyuki; ONODERA, Masafumi; NAGASHIMA, Hiroshi

    2015-01-01

    Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36–37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum. PMID:25739316

  8. A novel naturally occurring tandem promoter in modified vaccinia virus ankara drives very early gene expression and potent immune responses.

    Directory of Open Access Journals (Sweden)

    Sonia T Wennier

    Full Text Available Modified vaccinia virus Ankara (MVA has been shown to be suitable for the generation of experimental vaccines against cancer and infectious diseases, eliciting strong humoral and cellular immune responses. In viral vectored vaccines, strong recombinant antigen expression and timing of expression influence the quantity and quality of the immune response. Screening of synthetic and native poxvirus promoters for strong protein expression in vitro and potent immune responses in vivo led to the identification of the MVA13.5L promoter, a unique and novel naturally occurring tandem promoter in MVA composed of two 44 nucleotide long repeated motifs, each containing an early promoter element. The MVA13.5L gene is highly conserved across orthopoxviruses, yet its function is unknown. The unique structure of its promoter is not found for any other gene in the MVA genome and is also conserved in other orthopoxviruses. Comparison of the MVA13.5L promoter activity with synthetic poxviral promoters revealed that the MVA13.5L promoter produced higher levels of protein early during infection in HeLa cells and particularly in MDBK cells, a cell line in which MVA replication stops at an early stage before the expression of late genes. Finally, a recombinant antigen expressed under the control of this novel promoter induced high antibody titers and increased CD8 T cell responses in homologous prime-boost immunization compared to commonly used promoters. In particular, the recombinant antigen specific CD8 T cell responses dominated over the immunodominant B8R vector-specific responses after three vaccinations and even more during the memory phase. These results have identified the native MVA13.5L promoter as a new potent promoter for use in MVA vectored preventive and therapeutic vaccines.

  9. Antigenic assessment of a recombinant human CD90 protein expressed in prokaryotic expression system.

    Science.gov (United States)

    Yousefi-Rad, Narges; Shokrgozar, Mohammad Ali; Behdani, Mahdi; Moradi-Kalbolandi, Shima; Motamedi-Rad, Mahdieh; Habibi-Anbouhi, Mahdi

    2015-12-01

    Cluster of Differentiation 90 (CD90, Thy-1) has been proposed as one of the most important biomarkers in several cancer cells including cancer stem cells (CSCs). CD90 is considered as a potential normal stem cell and CSCs biomarker and also has been identified in lung cancer stem cells, hepatocellular carcinoma cells and high-grade gliomas. Using eukaryotic host systems involves complex procedures and frequently results in low protein yields. The expression of recombinant proteins in Escherichia coli is comparatively easier than eukaryotic host cells. The potential of large scale production of recombinant protein has made this system an economic production platform. In this study we expressed the extra-membrane domain of human CD90 (exCD90) antigen (Gln15-Cys130) in E. coli expression host cells. The epitope integrity of purified recombinant antigen was confirmed by antibody-antigen interaction using 5E10 anti-CD90 monoclonal antibody and binding study through ELISA and florescent staining of CD90(+) cells in a flow cytometry experiment. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. 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.

  11. 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.

  12. 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.

  13. Co-expression of Fos immunoreactivity in protein kinase (PKCγ)-positive neurones : quantitative analysis of a brain region involved in learning

    NARCIS (Netherlands)

    Ambalavanar, R.; Zee, E.A. van der; Bolhuis, J.J.; McCabe, B.J.; Horn, G.

    1993-01-01

    The expression of the gamma protein kinase C isoenzyme (PKCγ) and of the c-fos immediate early gene protein product Fos in the intermediate and medial hyperstriatum ventrale (IMHV) of day-old chicks was determined immunocytochemically. Previous research has shown that (a) there is a learning-related

  14. Dynamic transition of Dnmt3b expression in mouse pre- and early post-implantation embryos.

    Science.gov (United States)

    Hirasawa, Ryutaro; Sasaki, Hiroyuki

    2009-01-01

    The de novo DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the creation of DNA methylation patterns in mouse development. Dnmt3b is more highly expressed in early developmental stages than Dnmt3a, and is thought to have an important role in the epigenetic gene regulation during early embryogenesis. Previous reports suggest that Dnmt3b is expressed preferentially in the embryonic lineage, but less in the extra-embryonic lineage, in early post-implantation embryos. However, it is unclear when this lineage-specific differential expression is established. Here we demonstrate that Dnmt3b shows a dynamic expression change during pre- and early post-implantation development. Contrary to the expectation, Dnmt3b is preferentially expressed in the trophectoderm rather than the inner cell mass at the mid blastocyst stage. Subsequently, the spatial Dnmt3b expression gradually changes during pre- and early post-implantation development, and finally Dnmt3b expression is settled in the embryonic lineage at the epiblast stage. The findings are consistent with the role for Dnmt3b in cell-lineage specification and the creation of lineage-specific DNA methylation patterns.

  15. 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.

  16. Biological Analysis of HSV-1 Immediate-early Proteins ICPO, ICP22,and ICP27 in Neuro-blastoma Cells

    Institute of Scientific and Technical Information of China (English)

    Lei WANG; Yan-chun CHE; Wei CUN; Wei-zhong LI; Yun LIAO; Long-ding LIU; Qi-han LI

    2008-01-01

    The three immediate-early proteins of HSV-1, ICPO, ICP22, and ICP27, have specific and pivotal functions in transcriptional activation and inhibition, multiple regulatory and control processes of viral genes. In this paper, the expression and localization of these three proteins were studied in neuroblastoma cells using biochemical assays, and their possible and potential interactive functions are discussed. The data show that the three proteins are localized in different structures, specifically in the PML-NB-associated structure, which is a specific nuclear structure composed of many protein molecules and bound tightly to the nuclear matrix in neuroblastoma cells. The results suggest that the activating and suppressive functions of ICPs are mostly dependent on their transcriptional and regulatory roles, including the PML-NB-associated structure.

  17. Expression and phosphorylation of neurofilament protein in different neuronal tissues

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The neurofilament proteins (NFPs) from different neuronal tissues including Alzheimer and Huntington disease gray matter, rat brain gray, white matter and spinal cord were separated biochemically into two major fractions. A systematic investigation on the distribution, expression and phosphorylation of NFPs in those fractions was undertaken in the present study. It was found that only non-phosphorylated NF-H and NF-M, but not NF-L subunit were detected in Alzheimer brain gray matter high speed supernatant, whereas all neurofilament subunits including non-phosphorylated and phosphorylated were measured in high speed pellet fraction of the same tissue. The hyperphosphorylation of NF-H and NF-M in Alzheimer brain was shown by phosphorylation dependent monoclonal antibodies SMI31 and SMI34. This hyperphosphorylation was confirmed by non-phosphorylation dependent antibody SMI32 with dephosphosphorylation of the samples. Furthermore, an increased amount of NF-H, NH-M and NF-L, detected by SMI33 and NR4 respectively, was also observed in Alzheimer samples, in which the elevation in NF-L was significant. A significantly different immunoblot patterns in distribution, expression and phosphorylation were determined in various position of the neural system and alternative fractions. To our best knowledge, this is the first data shown definite abnormality of NFPs in Alzheimer disease. The information obtained in the present study will be extremely valuable in further study of the proteins both in physiological and pathological conditions.

  18. 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.

  19. Osteopontin is expressed in the mouse uterus during early pregnancy and promotes mouse blastocyst attachment and invasion in vitro.

    Directory of Open Access Journals (Sweden)

    Qian-Rong Qi

    Full Text Available Embryo implantation into the maternal uterus is a decisive step for successful mammalian pregnancy. Osteopontin (OPN is a member of the small integrin-binding ligand N-linked glycoprotein family and participates in cell adhesion and invasion. In this study, we showed that Opn mRNA levels are up-regulated in the mouse uterus on day 4 and at the implantation sites on days 5 and 8 of pregnancy. Immunohistochemistry localized the OPN protein to the glandular epithelium on day 4 and to the decidual zone on day 8 of pregnancy. OPN mRNA and proteins are induced by in vivo and in vitro decidualization. OPN expression in the endometrial stromal cells is regulated by progesterone, a key regulator during decidualization. As a secreted protein, the protein level of OPN in the uterine cavity is enriched on day 4, and in vitro embryo culturing has indicated that OPN can facilitate blastocyst hatching and adhesion. Knockdown of OPN attenuates the adhesion and invasion of blastocysts in mouse endometrial stromal cells by suppressing the expression and enzymatic activity of matrix metalloproteinase-9 in the trophoblast. Our data indicated that OPN expression in the mouse uterus during early pregnancy is essential for blastocyst hatching and adhesion and that the knockdown of OPN in mouse endometrial stroma cells could lead to a restrained in vitro trophoblast invasion.

  20. 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.

  1. 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

    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.

  2. Expression of aquaporin 1, 5 and 9 in the ovarian follicles of cycling and early pregnant pigs.

    Science.gov (United States)

    Skowronska, A; Mlotkowska, P; Eliszewski, M; Nielsen, S; Skowronski, M T

    2015-01-01

    Aquaporins (AQPs) are water channel proteins responsible for water homeostasis and important for proper functioning of all body systems, including reproductive structures. This study was designed to determine their localization and quantitative changes in the pig ovary during different stages of the estrous cycle and early pregnancy. The expression of AQP 1, 5 and 9 proteins was determined by immunocytochemistry and Western blot analyses. AQP1 was found in the plasma membranes of capillary endothelium, AQP5 - in the plasma membranes of granulosa cells of developing follicles and flattened follicle cells of the primordial follicles, and AQP9 - in granulosa cells of the developing follicles. In the cyclic pigs, the expression of AQP1 and 5 proteins was the highest on Days 18-20, but did not change significantly between Days 2-4, 10-12 and 14-16 of the cycle. In pregnant pigs (Days 14-16 and 30-32), the expression of AQP1 and 5 did not change and was similar to that observed during Days 10-12 and 14-16. In turn, AQP9 expression did not change between all studied periods. In conclusion, studied AQP are localized in different cells populations, the endothelial and granulosa cells, and AQP1 and 5 seem to be crucial for follicular development in pigs.

  3. 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

    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...

  4. Differential expression of murine adult hemoglobins in early ontogeny

    Energy Technology Data Exchange (ETDEWEB)

    Wawrzyniak, C.J.; Lewis, S.E.; Popp, R.A.

    1985-01-01

    A hemoglobin mutation is described that permits study of the expression of the two adult ..beta..-globin genes throughout fetal and postnatal development. Mice with a mutation at the Hbb/sup s/, ..beta..-globin locus, were used to study the relative levels of ..beta..-s2major and ..beta..-sminor globins specified by the mutant Hbb/sup s2/ haplotype during development. At 11.5 days of gestation ..beta..-sminor comprised over 80% and ..beta..-s2major under 20% of the adult beta-globin. The relative level of ..beta..-sminor decreased through fetal development; at birth ..beta..-sminor represented 33.7% of the ..beta..-globin. The adult values of 71.0% ..beta..-s2major and 29.0% ..beta..-sminor globin are expressed in mice six days after birth. Because the two ..beta..-globin genes are expressed in mice of the Hbb/sup 2s/ haplotype, both the ..beta..-smajor and ..beta..-sminor genes must be expressed in mice of the Hbb/sup s/ haplotype. Expression of the ..beta..-sminor gene is elevated to 35.6% in Hbb/sup s2/ mice that have been bled repeatedly. Thus, the 5' ..beta..-s2major and 3' ..beta..-sminor genes of the Hbb/sup s2/ haplotype and, presumably the 5' ..beta..-smajor and 3' ..beta..-sminor genes of the Hbb/sup s/ haplotype, are regulated independently and are homologous to the 5' ..beta..-dmajor and 3' ..beta..-dminor genes of the Hbb/sup d/ haplotype. Mice of the Hbb/sup s2/ haplotype are better than mice of the Hbb/sup d/ haplotytpe for studying the mechanisms of hemoglobin switching because the Hbb/sup s2/ each of the three embryonic and two adult hemoglobins can be separated by electrophoresis. 17 refs., 3 figs.

  5. Early-onset gastric cancers have a different molecular expression profile than conventional gastric cancers

    NARCIS (Netherlands)

    A.N.A. Milne; R. Carvalho; F.M. Morsink; A.R. Musler; W.W.J. de Leng; A. Ristimaki; G.J.A. Offerhaus

    2006-01-01

    Many studies examine the molecular genetics of gastric cancer, but few look at young patients in particular and there is no comparison of molecular expression between early-onset gastric cancer (<= 45 years old) and conventional gastric cancers. Expression of cycloxygenase-2 (COX-2) is elevated in g

  6. Expression of p53 protein in pituitary adenomas

    Directory of Open Access Journals (Sweden)

    Oliveira M.C.

    2002-01-01

    Full Text Available Inactivating mutations of TP53, a tumor suppressor gene, are associated with abnormal cell proliferation. Although p53 expression is common in many human malignancies, p53 protein has seldom been evaluated in pituitary tumors. When detected, the percentage of p53-positive cells is low, and, in general, it is exclusive for invasive lesions. The aim of the present study was to use immunohistochemistry to determine the presence of p53 protein in pituitary adenomas from tumor samples of 163 surgeries performed in 148 patients (40% male, 60% female. In 35% of the cases the adenoma was nonfunctional, while in the others it was associated with PRL, GH and/or ACTH endocrine hypersecretion syndrome. Macroadenomas were observed in 83.2% of the cases with available neuroimage evaluation, of which 28% invaded the cavernous, sphenoid and/or ethmoidal sinus, bone, third ventricle or subfrontal lobe. p53 protein was detected in 2/148 patients (1.3%. Immunohistochemistry was positive for PRL and GH in these cases. Due to the high percentage of invasive pituitary adenomas found in our study, the low frequency of p53 detection suggests that it is inadequate as a routine marker for aggressiveness and as a predictive factor of tumor behavior.

  7. A hemocyte-expressed fibrinogen-related protein gene (LvFrep) from the shrimp Litopenaeus vannamei: Expression analysis after microbial infection and during larval development.

    Science.gov (United States)

    Coelho, Jaqueline da Rosa; Barreto, Cairé; Silveira, Amanda da Silva; Vieira, Graziela Cleusa; Rosa, Rafael Diego; Perazzolo, Luciane Maria

    2016-09-01

    Fibrinogen-related proteins (FREPs) comprise a large family of microbial recognition proteins involved in many biological functions in both vertebrate and invertebrate animals. By taking advantage of publicly accessible databases, we have identified a FREP-like homolog in the most cultivated penaeid shrimp, Litopenaeus vannamei (LvFrep). The obtained sequence showed a conserved fibrinogen-related domain (FReD) and displayed significant similarities to FREP-like proteins from other invertebrates and to ficolins from crustaceans. The expression of LvFrep appeared to be limited to circulating hemocytes. Interestingly, LvFrep gene expression was induced in shrimp hemocytes only in response to a Vibrio infection but not to the White spot syndrome virus (WSSV). Moreover, LvFrep transcript levels were detected early in fertilized eggs, suggesting the participation of this immune-related gene in the antimicrobial defenses during shrimp development.

  8. 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

    , 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...... at first only in the nuclei of Ap spermatogonia, then also in the cytoplasm similarly to that seen after puberty. In CIS cells, DDX3Y was highly expressed and located predominantly in the nuclei. In invasive TGCT, significant DDX3Y expression was found in seminomas of the classical and spermatocytic type......, 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...

  9. Expression pattern of protein kinase C δ during mouse embryogenesis

    Directory of Open Access Journals (Sweden)

    Carracedo Sergio

    2013-01-01

    Full Text Available Abstract Background The members of the protein kinase C (PKC family consist of serine/threonine kinases classified according to their regulatory domain. Those that belong to the novel PKC subfamily, such as PKCδ, are dependent on diacylglycerol but not Calcium when considering their catalytic activity. Although several studies have shown the importance of PKCδ in different cellular events in health and disease, the overall in vivo distribution of this PKC isoform during development is still lacking. Through Lac Z and antibody staining procedures, we show here the in vivo expression of PKCδ during mouse embryogenesis. Results Ganglia were the domains with most prominent expression of PKCδ in most of the stages analysed, although PKCδ could also be detected in heart and somites at earlier stages, and cartilage primordium and skin among other sites in older embryos. Conclusions The strong expression of PKCδ in ganglia during murine development shown in this study suggests a significant role of this isoform as well as redundancy with other PKCs within the nervous system, since PKCδ deficient mice develop normally.

  10. Real-time quantification of protein expression at the single-cell level via dynamic protein synthesis translocation reporters.

    Science.gov (United States)

    Aymoz, Delphine; Wosika, Victoria; Durandau, Eric; Pelet, Serge

    2016-04-21

    Protein expression is a dynamic process, which can be rapidly induced by extracellular signals. It is widely appreciated that single cells can display large variations in the level of gene induction. However, the variability in the dynamics of this process in individual cells is difficult to quantify using standard fluorescent protein (FP) expression assays, due to the slow maturation of their fluorophore. Here we have developed expression reporters that accurately measure both the levels and dynamics of protein synthesis in live single cells with a temporal resolution under a minute. Our system relies on the quantification of the translocation of a constitutively expressed FP into the nucleus. As a proof of concept, we used these reporters to measure the transient protein synthesis arising from two promoters responding to the yeast hyper osmolarity glycerol mitogen-activated protein kinase pathway (pSTL1 and pGPD1). They display distinct expression dynamics giving rise to strikingly different instantaneous expression noise.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  13. Evaluation of the influenza A replicon for transient expression of recombinant proteins in mammalian cells.

    Science.gov (United States)

    Krammer, Florian; Pontiller, Jens; Tauer, Christopher; Palmberger, Dieter; Maccani, Andreas; Baumann, Martina; Grabherr, Reingard

    2010-10-11

    Recombinant protein expression in mammalian cells has become a very important technique over the last twenty years. It is mainly used for production of complex proteins for biopharmaceutical applications. Transient recombinant protein expression is a possible strategy to produce high quality material for preclinical trials within days. Viral replicon based expression systems have been established over the years and are ideal for transient protein expression. In this study we describe the evaluation of an influenza A replicon for the expression of recombinant proteins. We investigated transfection and expression levels in HEK-293 cells with EGFP and firefly luciferase as reporter proteins. Furthermore, we studied the influence of different influenza non-coding regions and temperature optima for protein expression as well. Additionally, we exploited the viral replication machinery for the expression of an antiviral protein, the human monoclonal anti-HIV-gp41 antibody 3D6. Finally we could demonstrate that the expression of a single secreted protein, an antibody light chain, by the influenza replicon, resulted in fivefold higher expression levels compared to the usually used CMV promoter based expression. We emphasize that the influenza A replicon system is feasible for high level expression of complex proteins in mammalian cells.

  14. 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

  15. Chronic morphine treatment enhances sciatic nerve stimulation-induced immediate early gene expression in the rat dorsal horn.

    Science.gov (United States)

    Bojovic, Ognjen; Bramham, Clive R; Tjølsen, Arne

    2015-01-01

    Synaptic plasticity is a property of neurons that can be induced by conditioning electrical stimulation (CS) of afferent fibers in the spinal cord. This is a widely studied property of spinal cord and hippocampal neurons. CS has been shown to trigger enhanced expression of immediate early gene proteins (IEGPs), with peak increases observed 2 hour post stimulation. Chronic morphine treatment has been shown to promoteinduce opioid-induced hyperalgesia, and also to increase CS-induced central sensitization in the dorsal horn. As IEGP expression may contribute to development of chronic pain states, we aimed to determine whether chronic morphine treatment affects the expression of IEGPs following sciatic nerve CS. Changes in expression of the IEGPs Arc, c-Fos or Zif268 were determined in cells of the lumbar dorsal horn of the spinal cord. Chronic Morphine pretreatment over 7 days led to a significant increase in the number of IEGP positive cells observed at both 2 h and 6 h after CS. The same pattern of immunoreactivity was obtained for all IEGPs, with peak increases occurring at 2 h post CS. In contrast, morphine treatment alone in sham operated animals had no effect on IEGP expression. We conclude that chronic morphine treatment enhances stimulus-induced expression of IEGPs in the lumbar dorsal horn. These data support the notion that morphine alters gene expression responses linked to nociceptive stimulation and plasticity.

  16. Early Growth Response gene 1 (Egr-1) regulates HSV-1 ICP4 and ICP22 gene expression

    Institute of Scientific and Technical Information of China (English)

    Gautam R Bedadala; Rajeswara C Pinnoji; Shao-Chung V Hsia

    2007-01-01

    The molecular mechanisms mediating herpes simplex virus type 1 (HSV-1) gene silencing during latent infection are not clear. Five copies of early growth response gene 1 (Egr-1) binding elements were identified in the intron of HSV-1 ICP22 (infected cell protein No. 22) gene, leading to the hypothesis that Egr-1 binds to the viral genome and regulates the viral gene expression. Transient co-transfection assays indicated that Egr-1 negatively regulated the transcription of both full-length and intron-removed ICP22 promoters. The same assays also revealed that Egr-1 repressed ICP4 (infected cell protein No. 4) promoter activity in a dose-dependent manner but showed less inhibition when the intron was removed.Histone deacetylation was not involved in this regulation since histone deacetylase inhibitor trichostatin A did not exhibit any effect on Egr-1-mediated repression. Chromatin immunoprecipitation assays showed that Egr-1 reduced the binding of Sp1 to the promoters and that the co-repressor Nab2 (NGFI-A/EGR1-binding protein) was recruited to the proximity of ICP4 in the presence of Egr-1. These results suggested that the multi functional transcription factor Egr-1 can repress HSV-1 immediate-early gene expression through the recruitment of co-repressor Nab2 and reduction of Sp1 occupancy,and thus may play a critical role in HSV-1 gene silencing during latency.

  17. Global gene analysis of oocytes from early stages in human folliculogenesis shows high expression of novel genes in reproduction.

    Science.gov (United States)

    Markholt, S; Grøndahl, M L; Ernst, E H; Andersen, C Yding; Ernst, E; Lykke-Hartmann, K

    2012-02-01

    The pool of primordial follicles in humans is laid down during embryonic development and follicles can remain dormant for prolonged intervals, often decades, until individual follicles resume growth. The mechanisms that induce growth and maturation of primordial follicles are poorly understood but follicles once activated either continue growth or undergo atresia. We have isolated pure populations of oocytes from human primordial, intermediate and primary follicles using laser capture micro-dissection microscopy and evaluated the global gene expression profiles by whole-genome microarray analysis. The array data were confirmed by qPCR for selected genes. A total of 6301 unique genes were identified as significantly expressed representing enriched specific functional categories such as 'RNA binding', 'translation initiation' and 'structural molecule activity'. Several genes, some not previously known to be associated with early oocyte development, were identified with exceptionally high expression levels, such as the anti-proliferative transmembrane protein with an epidermal growth factor-like and two follistatin-like domains (TMEFF2), the Rho-GTPase-activating protein oligophrenin 1 (OPHN1) and the mitochondrial-encoded ATPase6 (ATP6). Thus, the present study provides not only a technique to capture and perform transcriptome analysis of the sparse material of human oocytes from the earliest follicle stages but further includes a comprehensive basis for our understanding of the regulatory factors and pathways present during early human folliculogenesis.

  18. Direct interactions of early and late assembling division proteins in Escherichia coli cells resolved by FRET.

    Science.gov (United States)

    Alexeeva, Svetlana; Gadella, Theodorus W J; Verheul, Jolanda; Verhoeven, Gertjan S; den Blaauwen, Tanneke

    2010-07-01

    The bacterial cell division machinery is organized in the so-called divisome composed of highly dynamic but low abundant interacting (membrane-bound) proteins. In order to elucidate the molecular interactions between these proteins, we developed a robust background-insensitive quantitative spectral unmixing method for estimating FRET efficiencies at near endogenous protein levels using fluorescent protein fusions. The assembly of the division machinery of Escherichia coli occurs in two steps that are discrete in time: first the FtsZ-ring and the so-called early localizing proteins that together seem to prepare the division assembly at midcell. Subsequently, the late localizing protein complexes that contain the peptidoglycan-synthesizing proteins PBP1B and FtsI (PBP3) are recruited to the division site, which initiates septation. Physical interactions were observed between members within each group but also between the early and late localizing proteins strongly suggesting that these proteins despite their differential localization in time are linked at the molecular and functional level. Interestingly, we find FtsN, one of the latest proteins in the divisome assembly, interacting with late assembling proteins FtsI and FtsW, but also with early (proto-ring) protein ZapA. This is in line with the recently described role of FtsN in divisome stabilization including the proto-ring elements.

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

    Science.gov (United States)

    2010-01-01

    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. PMID:20105316

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

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    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. Developmental expression of odorant-binding proteins and chemosensory proteins in the embryos of Locusta migratoria.

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    Yu, Yanxue; Zhang, Shangan; Zhang, Long; Zhao, Xingbo

    2009-06-01

    We have investigated the development of chemosensilla and the secretion of odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) in the embryo of Locusta migratoria manilensis. We first report the changes of each sensillum in embryo just preceding hatch in detail and show that different sensilla have different developmental processes. Trichogen cells are first involved in forming the structure of pegs, and then, after retraction, they start secreting OBPs and CSPs in the sensillar lymph. The synthesis of LmigOBP1 starts during the embryogenesis about 0.5 h preceding hatching, specifically in sensilla trichodea and basiconica of the antenna. LmigOBP2, instead, was only found in the outer sensillum lymph (oSl) of sensilla chaetica of the antenna, while we could not detect LmigOBP3 in any type of sensilla of the antenna. The ontogenesis of CSPs in the embryos is similar to that of OBPs. Expression of CSPI homolog in Locusta migratoria is detected using the antiserum raised against SgreCSPI. CSPI is specifically expressed in the outer sensillum lymph of sensilla chaetica of the antenna, and anti-LmigCSPII dose not label any sensilla of the embryos. These data indicate that in locusts, OBPs and CSPs follow different temporal expression patterns, and also that OBPs are expressed in different types of sensilla.

  2. 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

  3. MicroRNA Expression Profiles Related to Early Stage Murine Concanavalin A-Induced Hepatitis

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    Hong-Yu Jia

    2014-06-01

    Full Text Available Background: Fulminant hepatitis is a severe liver disease characterized by massive hepatocyte necrosis and clinical signs of liver failure. This study explores the expression profile of microRNAs, which are regulators of a number of pathophysiological processes, during the early stage of concanavalin A (Con A-induced hepatitis. Methods: Balb/c mice were given ConA injections to induce fulminant hepatitis. miRNA expression profiling in liver tissues was carried out by microarray analysis. The differentially expressed miRNAs were subjected to time sequence profile analysis, gene-miRNA regulatory network analysis, and gene ontology-miRNA regulatory network analysis. Results: Eleven miRNAs among multiClass were found to be significantly differentially expressed between liver tissue in early stage fulminant hepatitis and normal control liver tissue. Mmu-miR-133a was the most differentially expressed with the strongest regulatory ability, regulating 47 mRNAs. Mmu-miR-10a was the most highly expressed in the microRNA-GO-Network and also exerted a strong regulatory ability. The expression profiles of miR-133a and miR-10a were verified by RT-PCR. Conclusions: These results show that, in the early stage, ConA-induced fulminant hepatitis induces a distinct miRNA expression profile. This differential miRNA expression profile may provide pathogenic clues and potential diagnostic and prognostic markers in acute and severe liver disease.

  4. Differentially expressed protein markers in human submandibular and sublingual secretions.

    Science.gov (United States)

    Hu, Shen; Denny, Patricia; Denny, Paul; Xie, Yongming; Loo, Joseph A; Wolinsky, Lawrence E; Li, Yang; McBride, Jim; Ogorzalek Loo, Rachel R; Navazesh, Mavash; Wong, David T

    2004-11-01

    Proteome analysis of secretions from individual salivary glands is important for understanding the health of the oral cavity and pathogenesis of certain diseases. However, cross-contamination of submandibular (SM) and sublingual (SL) glandular secretions can occur. The close anatomic relationship of the SM and SL ductal orifices can lead to such contamination. Additionally, these glands may share common ducts. To insure the purity of SM/SL secretions for proteomic analysis, it is important to develop unique biomarkers which could be used to verify the integrity of the individual glandular saliva. In this study, a proteomics approach based on mass spectrometry and gel electrophoresis techniques was utilized to identify and verify a set of proteins (cystatin C, calgranulin B and MUC5B mucin), which are differentially expressed in SM/SL secretions. SM/SL fluids were obtained from nine healthy subjects. Cystatin C was found to be an SM-selective protein as it was found in all SM fluids but not detected in two SL fluids. MUC5B mucin and calgranulin B, on the other hand, were found to be SL-selective proteins. All SL samples contained MUC5B mucin, whereas MUC5B mucin was not detected in four SM samples. Eight of the SL samples contained calgranulin B; however, calgranulin B was absent in eight SM samples. This set of protein markers, especially calgranulin B, can be used to determine the purity of SM/SL samples, and therefore identify potential individuals who do not exhibit cross-contaminated SM/SL secretions, an important requirement for subsequent proteome analysis of pure SM and SL secretions.

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

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    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.

  6. 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.

  7. Involvement of FGF and BMP family proteins and VEGF in early human kidney development.

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    Carev, Dominko; Saraga, Marijan; Saraga-Babic, Mirna

    2008-07-01

    The spatial and temporal pattern of the appearance of the fibroblast growth factor proteins (FGF-8 and FGF-10), the bone morphogenetic proteins (BMP-2/4 subfamily and BMP-7) and the vascular endothelial growth factor protein (VEGF) was investigated in the human mesonephros and metanephros of the 5-9 week-old conceptuses. In the mesonephros, both FGF's and BMP's were found in all structures and their expression slightly decreased in the early fetal period. VEGF positivity appeared in all mesonephric structures, and increased in the fetal period coincidently with formation of the mesonephric blood vessel network. In the metanephros, FGF-8 first appeared only in the metanephric mesenchyme, but from the 7th week on, its reactivity increased and spread to other metanephric structures. FGF-10 positive cells appeared in all metanephric structures already in the 5th week, and slightly intensified with progression of development. Cell survival and nephrogenesis in the permanent kidney might be associated with the appearance of both growth factors. Both BMP-2/4 and BMP-7 displayed a similar pattern of reactivity in all metanephric structures, and their reactivity intensified with advancing development. Alterations in their pattern of appearance might lead to the formation of small and dysplastic kidneys. Already in the earliest developmental stages, VEGF protein appeared in all metanephric structures. At later stages, VEGF showed more intense reaction in the collecting system than in the differentiating nephrons and interstitium. Due to VEGF involvement in vasculogenesis and angiogenesis, abnormal VEGF appearance might lead to impaired formation of the blood vessel network in the human permanent kidney.

  8. 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.

  9. 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.

  10. Gene expression patterns during the early stages of chemically induced larval metamorphosis and settlement of the coral Acropora millepora.

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    Nachshon Siboni

    Full Text Available 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.

  11. Cardiac protein kinases: the cardiomyocyte kinome and differential kinase expression in human failing hearts

    OpenAIRE

    Fuller, Stephen J.; Osborne, Sally A.; Leonard, Sam J.; Hardyman, Michelle A.; Vaniotis, George; Allen, Bruce G.; Sugden, Peter H.; Clerk, Angela

    2015-01-01

    Aims. Protein kinases are potential therapeutic targets for heart failure, but most studies of cardiac protein kinases derive from other systems, an approach that fails to account for specific kinases expressed in the heart and the contractile cardiomyocytes. We aimed to define the cardiomyocyte kinome (i.e. the protein kinases expressed in cardiomyocytes) and identify kinases with altered expression in human failing hearts. Methods and Results. Expression profiling (Affymetrix microarrays) d...

  12. Localized rbp4 expression in the yolk syncytial layer plays a role in yolk cell extension and early liver development

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    Korzh Vladimir

    2007-10-01

    Full Text Available Abstract Background The number of genes characterized in liver development is steadily increasing, but the origin of liver precursor cells and the molecular control of liver formation remain poorly understood. Existing theories about formation of zebrafish visceral organs emphasize either their budding from the endodermal rod or formation of independent anlage followed by their later fusion, but none of these is completely satisfactory in explaining liver organogenesis in zebrafish. Results Expression of a gene encoding the retinol binding protein 4 (Rbp4 was analyzed in zebrafish. rbp4, which is expressed mainly in the liver in adults, was shown to be expressed in the yolk syncytial layer (YSL during early embryogenesis. At 12–16 hpf rbp4 expression was restricted to the ventro-lateral YSL and later expanded to cover the posterior YSL. We demonstrated that rbp4 expression was negatively regulated by Nodal and Hedgehog (Hh signalling and positively controlled by retinoic acid (RA. Knockdown of Rbp4 in the YSL resulted in shortened yolk extension as well as the formation of two liver buds, which could be due to impaired migration of liver progenitor cells. rbp4 appears also to regulate the extracellular matrix protein Fibronectin1 (Fn1 specifically in the ventro-lateral yolk, indicating a role of Fn1 in liver progenitor migration. Since exocrine pancreas, endocrine pancreas, intestine and heart developed normally in Rbp4 morphants, we suggest that rbp4 expression in the YSL is required only for liver development. Conclusion The characteristic expression pattern of rbp4 suggests that the YSL is patterned despite its syncytial nature. YSL-expressed Rbp4 plays a role in formation of both yolk extension and liver bud, the latter may also require migration of liver progenitor cells.

  13. 3-hydroxi-anthranilic acid is early expressed in stroke

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    A. Mangas

    2016-12-01

    Full Text Available Using an immunohistochemical technique, we have studied the distribution of 3-OH-anthranilic acid (3-HAA in the rat brain. Our study was carried out in control animals and in rats in which a stroke model (single transient middle cerebral artery occlusion was performed. A monoclonal antibody directed against 3-HAA was also developed. 3-HAA was exclusively observed in the infarcted regions (ipsilateral striatum/cerebral cortex, 2, 5 and 21 days after the induction of stroke. In control rats and in the contralateral side of the stroke animals, no immunoreactivity for 3-HAA was visualized. Under pathological conditions (from early phases of stroke, we reported for the first time the presence of 3-HAA in the mammalian brain. By double immunohistochemistry, the coexistence of 3-HAA and GFAP was observed in astrocytes. The distribution of 3-HAA matched perfectly with the infarcted regions. Our findings suggest that, in stroke, 3-HAA could be involved in the tissue damage observed in the infarcted regions, since it is well known that 3-HAA exerts cytotoxic effects.