OBJECTIVEThis study was aimed to investigate the regulatory effect of protein disulfide isomerase (PDI) on platelet GPIbα ectodomain shedding.

METHODSThe washed platelets were obtained from healthy volunteers. Platelets were incubated with PDI inhibitor bacitracin before stimulation with PMA (Phorbol-12-myristate-13-acetate), dibucaine and collagen. The N-terminal domain of GPIbα in supernatant was detected by Western blot, the GPIbα expression and the intraplatelet ROS levels were measured by flow cytometry.

RESULTSneither GC content nor GPIbα expression was changed after the washed platelets from the healthy donors were incubated only with PDI inhibitor. The washed platelets were incubated with PDI inhibitor before stimulation with different stimulin, PMA, dibucaine or collagen, and then GPIbα was cleaved and ROS levels were elevated more than that in the controls.

CONCLUSIONPDI participates in the induced GPIbα ectodomein shedding, and the effect of PDI in this process maybe depend on the change of ROS level inside platelets. These results might provide a new point of view for the platelet drug development.

Blood Platelets ; Collagen ; Flow Cytometry ; Humans ; Platelet Glycoprotein GPIb-IX Complex ; Protein Disulfide-Isomerases

OBJECTIVETo investigate the association between the pregnancy rate of intrauterine insemination (IUI) and the ERp57 expression level in donor sperm.

METHODSForty-two sperm samples were divided into 3 groups according to the pregnancy rate: Group A (n = 16, pregnancy rate = 0), B (n = 13, pregnancy rate 10%-20%) and C (n = 13, pregnancy rate > or = 20%). The optical density (OD) was normalized to the beta-tubulin band for the evaluation of the ERp57 level.

RESULTSOD (ERp57/Tubulin) was 0.95 +/- 0.24 in Group A, 1.33 +/- 0.43 in Group B and 1.33 +/- 0.39 in Group C. The ERp57 expression level was significantly lower in Group A than in B and C (P < 0.05), with no significant differences between the latter two groups.

CONCLUSIONThe ERp57 expression level in donor sperm could be used as an index to predict the pregnancy rate of IUI and to avoid IUI failure by removing low-level donor sperm.

Adult ; Female ; Humans ; Insemination, Artificial ; methods ; Male ; Pregnancy ; Pregnancy Rate ; Protein Disulfide-Isomerases ; metabolism ; Spermatozoa ; metabolism

BACKGROUND: Numerous cell surface proteins of leukemia cells such as CD33 and CD52 have been identified as diagnostic and therapeutic targets. Thus the profiling of the cell surface proteome and proteins restricted to specific leukemia(s) can provide a way to identify novel targets for leukemia diagnosis and therapy. However, there is a lack of data pertaining to the comprehensive analysis of surface membrane proteins because there are few effective strategies for profiling surface membrane proteomes. METHODS: We report on the application of quantitative proteomic techniques that incorporate affinity-capture and purification on monomeric avidin columns to identify all biotinylated cell surface proteins from leukemia cell lines. RESULTS: An analysis of a subset of biotinylated proteins among the different human leukemia cell lines using matrix-assisted laser desorption ionization and tandem mass spectrometry identified, among others, some widely expressed proteins in leukemia cells, such as CD11a, CD11c, CD18, CD31, CD44, and CD147, as well as a set of proteins identified as chaperone proteins, including HSP90, GRP78, GRP75, HSP70, HSP60 and protein disulfide isomerases. On the basis of their known functional roles, several of these proteins may participate in the progression of leukemogenesis and should be considered as potential markers of leukemia. CONCLUSION: Comprehensive profiling of the leukemia cell surface proteome provides an effective approach for the identification of commonly occurring proteins as well as proteins with restricted expression patterns to a specific cell line.
Avidin ; Cell Line ; Diagnosis ; Humans ; Leukemia* ; Membrane Proteins* ; Membranes ; Protein Disulfide-Isomerases ; Proteome ; Tandem Mass Spectrometry

Using the large scale sequencing, a novel human cDNA of 1,645 bp was screened from the cDNA library of human fetal brain. The cDNA contains an ORF encoding a 296-aa protein with a calculated molecular weight of 34.0 KD. Compared with that of current sequence databases, the putative protein was found to have 36% homology with protein-disulfide isomerase (PDI). So this cDNA was named PDI like (PDI-L) gene. Multiple tissue Northern blot analysis shows that PDI-L cDNA is expressed in heart, brain, liver, kidneys and so on. The ORF fragment of PDI-L cDNA was inserted into reconstructed pBV220 expression vehicle and its predicted expressive protein was obtained.
Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; DNA, Complementary ; genetics ; Gene Library ; Humans ; Protein Disulfide-Isomerases ; biosynthesis ; genetics

The protein encoded by TXNDC5 is a member the protein disulfide isomerase family, which has disulfide isomerase activity and can act as the molecular chaperone to reduce the synthesis of abnormal proteins. Its biological functions include anti-oxidation, promoting angiogenesis, taking part in cellular inflammation, and energy metabolism, etc. Studies have demonstrated that the expression of TXNDC5 is increased in many types of tumors including cervical carcinoma, gastric carcinoma and colorectal cancer. Moreover, TXNDC5 is also closely associated with rheumatoid arthritis, diabetes, hepatic steatosis and vitiligo. This paper aims to summarize the latest progress in research on TXNDC5 in terms of biochemical function, relationship with diseases and the underlying mechanism.
Animals ; Arthritis, Rheumatoid ; enzymology ; genetics ; Diabetes Mellitus ; enzymology ; genetics ; Humans ; Neoplasms ; enzymology ; genetics ; Protein Disulfide-Isomerases ; genetics

OBJECTIVE: Protein disulfide isomerase (PDI) acts as a chaperone on the cell surface, and it has been reported that PDI is associated with the tumor cell migration and invasion. The aims of this study are to investigate the anti-migration effect of bacitracin, which is an inhibitor of PDI, and the associated factor in this process. METHODS: U87-MG glioma cells were treated with bacitracin in 1.25, 2.5, 3.75, and 5.0 mM concentrations. Western blot with caspase-3 was applied to evaluate the cytotoxicity of bacitracin. Adhesion, morphology, migration assays, and organotypic brain-slice culture were performed to evaluate the effect of bacitracin to the tumor cell. Western blot, PCR, and gelatin zymography were performed to investigate the associated factors. Thirty glioma tissues were collected following immunohistochemistry and Western blot. RESULTS: Bacitracin showed a cytotoxicity in 3rd (p<0.05) and 4th (p<0.001) days, in 5.0 Mm concentration. The cell adhesion significantly decreased and the cells became a round shape after treated with bacitracin. The migration ability, the expression of phosphorylated focal adhesion kinase (p-FAK) and matrix metalloproteinase-2 (MMP-2) decreased in a bacitracin dose- and time-dependent manner. The U87-MG cells exhibited low-invasiveness in the 2.5 mM, compared with the untreated in organotypic brain-slice culture. PDI was expressed in the tumor margin, and significantly increased with histological glioma grades (p<0.001). CONCLUSION: Bacitracin, as a functional inhibitor of PDI, decreased the phosphorylated FAK and the secreted MMP-2, which are the downstream of integrin and play a major role in cell migration and invasion, might become one of the feasible therapeutic strategies for glioblastoma.
Bacitracin* ; Blotting, Western ; Caspase 3 ; Cell Adhesion ; Cell Movement ; Focal Adhesion Protein-Tyrosine Kinases ; Gelatin ; Glioblastoma ; Glioma* ; Immunohistochemistry ; Matrix Metalloproteinase 2 ; Polymerase Chain Reaction ; Protein Disulfide-Isomerases

To test the hypothesis that in vitro protein cross-linking could be accomplished in three concerted steps: (1) a change in protein conformation; (2) formation of interchain disulfide bonds; and (3) formation of interchain isopeptide cross-links, we amplified wild and Cys/Ser mutant genes with PCR technique from E. coli BL21 cells and subcloned them into expression plasmid pTrcHisC. Recombinant proteins, which were associated with formation of inclusion bodies induced by IPTG, were purified by immobilized metal affinity chromatography (IMAC) and refolded by dialysis. In thermal unfolding and oxidative refolding experiment, wild TtdB was proved to form cross-linked dimmers/oligomers as revealed by SDS-PAGE; cross-linking intensity was obviously weakened when the loading buffer contained the reducing agent dithiothreitol (DTT). The residual cross-linking was isopeptide bonds; no dimmers/oligomers were detected when the refolding and unfolding solution contained DTT. In addition, Cys/Ser point mutation abrogated its ability to cross-link into homodimers, which showed disulfide bonds could facilitate the following formation of isopeptide bonds.
Cross-Linking Reagents ; chemistry ; Escherichia coli ; enzymology ; genetics ; Escherichia coli Proteins ; chemistry ; Hydro-Lyases ; chemistry ; genetics ; Mutation ; Protein Disulfide-Isomerases ; chemistry ; Protein Folding

BACKGROUND/AIMS: Protein disulfide isomerase (PDI) has been implicated in the survival and progression of some cancer cells, by compensating for endoplasmic reticulum stress by upregulating the protein-folding capacity. However, its prognostic role in patients with hepatocellular carcinoma (HCC) has not been investigated. METHODS: We collected HCC tissues from 83 HCC patients who underwent surgical resection for an immunohistochemical study of PDI. Overall survival (OS) was measured from the date of surgical resection until the date of death from any cause. Radiological progression was evaluated using the modified Response Evaluation Criteria in Solid Tumors in an independent radiological assessment. RESULTS: PDI expression was found to be increased in human HCC compared to adjacent nontumor tissues. Increased immunopositivity for PDI was associated with a high Edmondson-Steiner grade (p = 0.028). Univariate analysis of patients who had undergone surgical resection for HCC showed that tumor PDI upregulation is a significant risk factor for poor OS (p = 0.016; hazard ratio [HR], 1.980) and time to progression (TTP; p = 0.007; HR, 1.971). Multivariate analyses revealed that high PDI expression was an independent predictor of a shorter TTP (p = 0.015; HR, 1.865) and poor OS (p = 0.012; HR, 2.069). CONCLUSIONS: Upregulated PDI expression is associated with aggressive clinicopathological features of HCC; thus, PDI might serve as an independent prognostic factor and a potential therapeutic target for HCC patients.
Carcinoma, Hepatocellular/*enzymology/pathology ; Female ; Humans ; Kaplan-Meier Estimate ; Liver Neoplasms/*enzymology/pathology ; Male ; Middle Aged ; Prognosis ; Protein Disulfide-Isomerases/*metabolism ; Retrospective Studies ; Tumor Markers, Biological/metabolism

OBJECTIVETo investigate the role of TXNDC5 in serum starvation-induced proliferation inhibition of HeLa cell.

METHODSTXNDC5 was either over-expressed or knocked down by small interfering RNA (siRNA) in HeLa cells which were then cultured in conventional medium or serum starvation medium. The protein level of TXNDC5 was evaluated by Western blot analysis. The mRNA level of TXNDC5 was measured by quantitative real-time PCR. Cell growth rate was determined by cell proliferation assay kit (MTS method). Cell cycle distribution and apoptosis were detected by flow cytometry.

RESULTSSerum starvation mildly reduced the mRNA level of TXNDC5 (P<0.05), but dramatically increased the protein level of TXNDC5 in HeLa cells. The stability of TXNDC5 mRNA remained unchanged. Cycloheximide abolished the serum starvation-induced up-regulation of TXNDC5 protein. Over-expression of TXNDC5 had no effect on cell proliferation. However, suppression of TXNDC5 attenuated the proliferation inhibition of HeLa cell induced by serum starvation (P<0.05), increased the proportion of cells in S phase (P<0.05), but had no effect on cell apoptosis.

CONCLUSIONTXNDC5 mediates serum starvation-induced proliferation inhibition of HeLa cell.

Apoptosis ; Cell Cycle ; Cell Proliferation ; Culture Media ; chemistry ; Gene Knockdown Techniques ; HeLa Cells ; Humans ; Protein Disulfide-Isomerases ; genetics ; metabolism ; Serum ; chemistry

OBJECTIVETo explore the effects of gastrin on the expression of 1,25(OH)2D3-membrane associated rapid response steroid (1,25D3-MARRS) binding protein in rat intestinal epithelium.

METHODSSD rats received intraperitoneal injections of gastrin, omeprazole or physiological saline. The protein expression of 1,25D3-MARRS binding protein in SD rat intestinal was determined with Western blotting and immunohistochemistry, and its mRNA levels determined by RT-PCR. The serum calcium and phosphate levels in the rats were also detected.

RESULTSImmunohistochemistry showed that 1,25D3-MARRS binding protein was expressed mainly in the nuclei, cytoplasm and membrane of the intestinal epithelial cells. Both the protein and mRNA expression levels of 1,25D3-MARRS binding protein were up-regulated after treatments with gastrin and omeprazole (P<0.05), but the serum calcium and phosphate concentrations showed no obvious increase.

CONCLUSION1,25D3-MARRS binding protein, which is widely expressed with versatile functionalities, is regulated by gastrin and shows high potentials in the study of gastrointestinal diseases.

Animals ; Calcitriol ; metabolism ; Epithelial Cells ; drug effects ; metabolism ; Gastrins ; pharmacology ; Intestines ; cytology ; drug effects ; Male ; Protein Disulfide-Isomerases ; metabolism ; Rats ; Rats, Sprague-Dawley