Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Proteinase-activated receptors (PARs) are a novel family of G-protein-coupled receptors. PAR2 has been implicated in inflammatory airways disease. Although fibroblasts are pathologically important in the airways, the proinflammatory role of PAR2 in these cells remains unknown. We assessed PAR expression and functionality in human primary bronchial fibroblasts (HPBFs) before assessing PAR2-mediated HPBF proliferation, cytokine production, and adhesion molecule expression. RT-PCR and flow cytometry demonstrated that HPBFs express hPAR1, hPAR2, and hPAR3, but not hPAR4. Intracellular calcium signaling in HPBFs in response to PAR agonists showed that only hPAR1 and hPAR2 were functional receptors. We used the MTT assay to assess HPBF proliferation. Of the PAR2 agonist proteinases or selective PAR2-activating peptides (PAR2-APs) tested, none stimulated HPBF proliferation, whereas thrombin was a HPBF growth factor. mRNA for IL-8 and granulocyte colony-stimulating factor (G-CSF) was upregulated after addition of SLIGKV-NH2 when assessed by RT-PCR. No significant increase in G-CSF or IL-8 protein was detected. Trypsin stimulated IL-8 and G-CSF release from HPBF in a time- and dose-dependent manner. Leupeptin and soya trypsin inhibitor abrogated trypsin-stimulated cytokine release, indicating a requirement for trypsin's proteolytic activity. Trypsin and SLIGKV-NH2 stimulated an increase in VCAM-1 expression at 12 h after treatment, which declined thereafter. PAR2-driven upregulation of VCAM-1 cell surface expression and the release of IL-8 and G-CSF from bronchial fibroblasts may be important in promoting neutrophilic airways inflammation.
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PMID:Proteinase-activated receptor2 agonists upregulate granulocyte colony-stimulating factor, IL-8, and VCAM-1 expression in human bronchial fibroblasts. 1649 82

This study demonstrates that adult human mesenchymal cells (MSC) can be encapsulated in alginate beads with a substantially retained viability (>80%) and that a Gly-Arg-Gly-Asp-Tyr (GRGDY) derivative encourages attachment and elongation to form a dense network of cells that is required for a tissue substitute. Because the availability of autologous human material is severely limited, we used and examined the beads in this study as a proxy for larger constructs. These bead constructs were assessed using phase contrast microscopy and standard histological preparations. In addition, we used a modified MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay to examine cell proliferation by dissociating the cell/alginate constructs using trisodium citrate and trypsin/EDTA. MSCs did not proliferate within the alginate-GRGDY matrix during the 2 weeks examined. These results were further substantiated by concurrent cell density measurements using a hemocytometer. In addition, the glucose consumption rate was measured and compared to that of MSCs grown in two-dimensional culture vessels, indicating steady consumption albeit at a lower level in the entrapped MSCs.
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PMID:Behavior of adult human mesenchymal stem cells entrapped in alginate-GRGDY beads. 1667 95

Proteinase-activated receptor-2 (PAR-2) is a ubiquitous surface molecule participating in many biological processes. It belongs to the family of G protein-coupled receptors activated by the site-specific proteolysis of trypsin and similar proteases. Altered function of PAR-2 has been described in different malignant tumors. In the present study, we investigated the expression of PAR-2 in breast cancer surgical specimens and the role of trypsin in breast cancer cell line MDA MB-231 proliferation and metabolism. A total of 40 surgical samples of infiltrative ductal breast cancer and breast cancer cell line were included in this study. We analyzed PAR-2 expression by immunohistochemistry, RT-PCR and western blot. Activation of PAR-2 on cell line MDA MB-231 was measured using calcium mobilization assay determined by flow cytometry. MTT cell metabolism assay and cell count analysis were used to assess the trypsin influence on breast cancer cell line MDA MB-231 proliferation. Immunohistochemical examination showed the expression of PAR-2 in all samples of breast cancer surgical specimens and high levels of cell lines which was confirmed by RT-PCR and western blot. Calcium mobilization assay corroborated the activation of PAR-2 on cell line MDA MB-231 either by trypsin or by an agonistic peptide. Cell metabolism assay and cell count analysis showed significant differences of proliferative activity of breast cancer cells dependent on the presence or absence of trypsin and serum in the culture medium. PAR-2 is expressed by high levels in infiltrative ductal breast cancer tissue specimens. PAR-2 is also strongly expressed in studied breast cancer cell lines. PAR-2 is activated by trypsin and also by agonistic peptide in the model of breast cancer cell line MDA MB-231. Activation of PAR-2 in vitro influences proliferative and metabolic activity of breast cancer cell line MDA MB-231. The action of trypsin is modified by the presence of serum which is a potential source of protease inhibitors.
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PMID:Proteinase-activated receptor-2 expression in breast cancer and the role of trypsin on growth and metabolism of breast cancer cell line MDA MB-231. 1692 62

In the present work, we examined the feasibility of using cardosins, plant aspartic-proteinases from Cynara cardunculus L., to isolate cells from rat embryonic brain. Using morphological and functional assays, we compared cell cultures obtained with cardosins with those prepared with a well-established trypsin protocol. Cardosins and trypsin dissociation produced cells with similar yield, viability, and GABA release in response to a depolarizing stimulus. However, cardosins-dissociated cells appeared to recover faster in culture, as assessed by the MTT-test and by the number and length of neurtites, suggesting that cardosins are less aggressive to neurons than trypsin. This feature might be helpful for research and medical purposes requiring fast manipulations of cells.
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PMID:Cardosins: a new and efficient plant enzymatic tool to dissociate neuronal cells for the establishment of cell cultures. 1709 9

Because hESC (human embryonic stem cells) are 'social cells' that require co-operative interactions and intimate physical contact with each other, it is absolutely essential to dissociate hESC colonies into cellular clumps rather than into a single-cell suspension during serial passage. The present study compared two commonly used protocols for dissociating hESC colonies. The first protocol involved mild enzymatic treatment with collagenase type IV (1 mg/ml) for approx. 5-10 min, prior to mechanical dissociation into cellular clumps through manual scraping with a plastic pipette tip. The second protocol involved a short duration of exposure (2-3 min) to low concentrations of trypsin (0.05%), followed by gentle pipetting. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay was used to compare the recovery of viable cells after dissociating hESC colonies with these two protocols, before and after conventional freeze-thawing with 10% (v/v) DMSO. Besides undifferentiated hESC, the randomly differentiated fibroblastic progenies of hESC at various passages (P0-P4), together with an immortalized cell line (CRL-1486), were also utilized to compare the two protocols. The results demonstrated that the second protocol (trypsinization with gentle pipetting) is much less detrimental to cellular viability than is the first protocol (collagenase treatment with scratching). This in turn translated to higher freeze-thaw survival rates. It is hypothesized that scratching after collagenase treatment (first protocol) somehow induces physical damage to the cells, thereby leading to a lower recovery of viable cells, both before and after freeze-thawing.
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PMID:Mechanical dissociation of human embryonic stem cell colonies by manual scraping after collagenase treatment is much more detrimental to cellular viability than is trypsinization with gentle pipetting. 1711 76

Methyl tert-butyl ether (MTBE) is a class of synthetic organic chemical. In the USA, MTBE pollution is regarded as a serious environmental problem. The objective of the present study was to investigate the cytotoxic effects and oxidative stress induced by MTBE in isolated rat spermatogenic cells. In cytotoxic experiments, spermatogenic cells isolated from the testes of adult Sprague-Dawley rats by a mechanical procedure without the use of trypsin were incubated with medium alone (control), 0.5, 5, 50 mm MTBE, respectively, for 6, 12 and 18 h. MTT assay, staining with fluorescein diacetate (FDA) and propidium iodide (PI) and flow cytometric analyses were used. In oxidative stress experiments, the spermatogenic cells were incubated with medium alone (control) and with 0.5, 50 microm, 5 mm MTBE. For 1, 2, 6, 12, 18 h incubation, ROS production was tested using a 2',7'-dichlorofluorescein diacetate (DCHF-DA) probe; for 1, 3, 6, 12, 18 h incubation, cytosolic superoxide dismutase (SOD) and extracellular SOD (SOD(EX)) activity was assessed; and for 18 h incubation, lipid peroxidation was assessed. The results showed that MTBE at high doses significantly decreased the spermatogenic cell viability and increased plasma membrane damage and the ratio of necrotic cells compared with the control. Assessment of the MTBE-induced oxidative stress revealed that MTBE increased the production of reactive oxygen species (ROS) and enhanced lipid peroxidation. In addition, although SOD(EX) activity increased at a high dose level, cytosolic SOD activity decreased. These results suggest that an increase of MTBE-induced ROS production and an enhancement of membrane lipid peroxidation may play an important role in its cytotoxicity in isolated rat spermatogenic cells.
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PMID:Methyl tert-butyl ether (MTBE)-induced cytotoxicity and oxidative stress in isolated rat spermatogenic cells. 1717 68

In the present study, a peptide having antioxidant properties was isolated from bullfrog skin protein, Rana catesbeiana Shaw. Bullfrog skin protein was hydrolyzed using alcalase, neutrase, pepsin, papain, alpha-chymotrypsin and trypsin. Antioxidant activities of respective hydrolysates were evaluated using lipid peroxidation inhibition assay and direct free radical scavenging activity by using electron spin resonance (ESR) spectrometer. Among hydrolysates, alcalase derived hydrolysate exhibited the highest antioxidant activities than those of other enzyme hydrolysates. In order to purity a peptide having potent antioxidant properties, alcalase hydrolysate was separated using consecutive chromatographic methods on a Hiprep 16/10 DEAE FF anion exchange column, Superdex Peptide 10/300 GL gel filtration column and highan octadecylsilane (ODS) C18 reversed phase column. Finally, a potent antioxidative peptide was isolated and its sequence was identified to be LEELEEELEGCE (1487 Da) by Q-TOF ESI mass spectroscopy. This antioxidant peptide from bullfrog skin protein (APBSP) inhibited lipid peroxidation higher than that of alpha-tocopherol as positive control and efficiently quenched different sources of free radicals: DPPH radical (IC(50)=16.1 microM), hydroxyl radical (IC(50)=12.8 microM), superoxide radical (IC(50)=34.0 microM) and peroxyl radical (IC(50)=32.6 microM). Moreover, MTT assay showed that this peptide does not exert any cytotoxicity on human embryonic lung fibroblasts cell line (MRC-5).
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PMID:Free radical scavenging activity of a novel antioxidative peptide purified from hydrolysate of bullfrog skin, Rana catesbeiana Shaw. 1751 26

In this study, a 15-mer phage display peptide library was employed to pan against human rotavirus immobilized on solid phase. 4 different peptides were selected and could bind with rotavirus particles specifically. Plaque reduction neutralization test and MTT analysis results indicated that 3 of the peptides can inhibit rotavirus infecting in vitro. A peptide which sequence is QSNPIHIITNTRNHP showed the best efficiency--93% neutralization infectivity. Two other peptides, A and B, showed 40% and 50% neutralization infectivity respectively. Amino sequence analysis results indicate the 3 peptides containing 2 conserved motifs: SNPIHII and NIP. No putative trypsin hydrolysis site was found in C peptide, however, 4 and 3 potential sites were found in A and B peptides respectively. Using trypsin inhibitor, both A and B peptides showed the similar antiviral effect as that of C peptide. It suggests that the intactness of the 2 conserved motifs play an important role in counteracting virus infection. According to the results of this study, peptide C is hopeful to be exploited as an antiviral peptide drug.
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PMID:[Screening for peptides of anti-rotavirus by phage-displayed technique]. 1757 83

To observe the growth of rat bone marrow mesenchymal cells (BMMCs) on decellular bovine pericardia in vitro and to investigate the effect of proteins pre-coating on cells retention and proliferation, bovine pericardia were decellularized using trypsin, DNase and Triton X-100 respectively. Then three proteins (fibronetin (FN), gelatin, collagen I) were coated on the surfaces of the bovine pericardia separately. BMMCs were harvested from rat thighbone marrow , then expanded and seeded onto decellular bovine pericardia with the proteins pre-coated . Decelluar bovine pericardia without coating were used as controls. The retention and growth of BMMSCs were observed by Hochest staining and analyzed by MTT method. It was shown that the retention and proliferation of BMMCs on FN group and gelatin group were significantly enhanced comparing with those on collagen I group and control group (P < 0.001). There was no significant difference between FN group and gelatin group (P > 0.05), nor between collagen I group and control group (P > 0.05). We conclude that the retention and proliferation of seeding cells on FN and gelatin could be significantly improved on decellular bovine pericardia (DBP) but not on collagen I.
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PMID:[Effect of proteins pre-coating on bone marrow mesenchymal cells retention and proliferation on decellular bovine pericardium]. 1771 67

The nucleoside analogue cordycepin (3 '-deoxyadenosine, 3 '-dA), one of the components of Cordyceps militaris, has been shown to inhibit the growth of various tumor cells. However, the probable mechanism is still obscure. In this study, the inhibition of cell growth and changes in protein expression induced by cordycepin were investigated in BEL-7402 cells. Using the MTT assay and flow cytometry, we found that cordycepin inhibits cell viability and induces apoptosis in BEL-7402 cells. Additionally, the proteins were separated using two-dimensional polyacrylamide gel electrophoresis, and eight proteins were found to be significantly affected by cordycepin compared to untreated control; among them, two were downregulated and six were upregulated. Of the eight proteins, six were identified with peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) after in-gel trypsin digestion. These proteins are involved in various aspects of cellular metabolism. It is suggested that the effect of cordycepin on the growth of tumor cells is significantly related to the metabolism-associated protein expression induced by cordycepin.
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PMID:Proteomic detection of changes in protein expression induced by cordycepin in human hepatocellular carcinoma BEL-7402 cells. 1880 93


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