Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Effects and mechanisms of FR167653, 1-[7-(4-fluorophenyl)-1,2,3,4-tetrahydro-8-(4-pyridyl)pyrazolo[5,1-c][1,2,4] triazin-2-yl]-2-phenylethanedione sulfate monohydrate, a dual inhibitor of interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha), on rat adjuvant arthritis (AA) was investigated. Complete Freund's adjuvant was used to induce AA in rats. Secondary paw swelling of AA rats was measured, and polyarthritis index was scored. Synoviocytes were separated by the method of collagenase and DNase digestion. Synoviocytes proliferation was assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. TNF-alpha, IL-1 and interleukin-10 (IL-10) production of synoviocytes was measured with ELISA. The expression of IL-10 mRNA of synoviocytes was determined using RT-PCR. There were significant secondary inflammatory reactions in AA rats, which accompanied with the decrease of body and immune organs weight simultaneously. The administration of FR167653 (4, 12, 36 mg/kg, subcutaneously (s.c.)) inhibited the inflammatory response and restored the weight of body and immune organs of AA rats. Synoviocytes proliferation of AA rats significantly increased, and the levels of TNF-alpha and IL-1 in supernatants of synoviocytes in AA rats were also elevated compared with the sham group. The administration of FR167653 (4, 12, 36 mg/kg, s.c.) reduced the above changes significantly. In contrast to TNF-alpha and IL-1, IL-10 production and the level of its mRNA of synoviocytes in AA rats were apparently decreased. FR167653 (4, 12, 36 mg/kg, s.c.) markedly increased IL-10 in synoviocytes at protein and transcription level. The results indicated that FR167653 had a beneficial effect on rats AA due to modulating inflammatory cytokines production of synoviocytes, which played a crucial role in pathogenesis of this disease.
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PMID:Effects and mechanisms of FR167653, a dual inhibitor of interleukin-1 and tumor necrosis factor, on adjuvant arthritis in rats. 1545 15

Modification of the silanophilic activity of the inner surface of the capillary wall was studied in a capillary electrophoretic system using alkylamines containing background electrolytes at acid pH. The effect of the following amine additives was investigated: (1) alkyl-alpha,omega-diamines (1,2-diaminoethane, 1,4-diaminobutane, 1,7-diaminoheptane, spermine), (2) polymeric amines (polyethylenimine, polybrene), (3) cationic amine surfactants (cetrimide, hexamethonium bromide). A seven membered test mixture of peptides (Gly-Pro-Ala, Pro-hPro, Gly-Pro-Arg, Gly-Pro-Gln, Lys-Pro-Gly, Asn-Pro-Gly, His-Pro-Gly) possessing one or more amino groups was used for selectivity evaluation. Under optimised concentration of the amine modifiers the selectivity was always improved (except for polybrene), particularly with the fast moving analytes. The best results were obtained with 1,2-diaminoethane and 1,7-diaminoheptane. On the other hand with slowly moving peaks the best separations were obtained with 1,7-diaminoheptane, hexadecyltrimethylammonium bromide and hexamethonium bromide, i.e. with modifiers possessing large aliphatic domains which are likely to be hydrophobically bonded with the separated solutes. The selectivity improvement with fast moving members of the test mixture can be ascribed to the decrease of the electroosmotic flow, while the improved separation with slowly moving peaks appears to reflect the altered interaction with the hydrophobized capillary wall. As expected the endoosmotic flow was in all cases decreased. The practical applicability of using amine based dynamic modifiers of the capillary wall was demonstrated on a natural peptide mixture (bacterial collagenase hydrolysate of collagen types I and III).
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PMID:Separation of low-molecular mass peptides by capillary electrophoresis with the use of alkylamines as dynamic coating agents at low pH. 1553 62

Membrane integrity fluorescent staining is used routinely to evaluate islet viability. Results are used as one of the determining factors in islet product release criteria, and are used to assess the efficacy of different culture conditions. Recently, it has been observed that there is variation in the viability staining of freshly isolated islets based on which viability assay is used. This investigation compares three membrane integrity stains for the viability assessment of isolated human islets. Fluorescein diacetate/propidium iodide (FDA/ PI), the current standard method for assessing islet viability, demonstrates intense extracellular fluorescence, reducing the differential staining of intact islets. We further evaluated SYTO-13/ethidium bromide (SYTO/ EB) and calcein AM/ethidium homodimer (C/EthD) as alternative viability assays, and found considerable variation between FDA/PI and either SYTO/EB or C/EthD staining. Preparations of human islets were obtained from cadaveric pancreata after collagenase digestion, mechanical separation, and purification by continuous Ficoll gradient centrifugation. For each preparation, two replicate samples of 50 islets were counted for each stain, and the percent viability calculated. The results for SYTO/EB and C/EthD were nearly identical [57.6 +/- 7.3% and 57.9 +/- 7.2%, respectively (mean +/- SEM), N = 11]. FDA/PI-stained islets, however, showed consistently elevated values when compared to SYTO/EB. Accurate assessment of islet viability remains a critical determinant of islet product release. The discrepancies found between FDA/PI scoring and visual quality, compared with alternative stains, suggests that the FDA/PI stain may not be the optimal approach to assess islet viability.
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PMID:Variation in human islet viability based on different membrane integrity stains. 1556 60

In an effort to improve the zinc-chelating portion of matrix metalloproteinase (MMP) inhibitors, we have developed a family of heterocyclic zinc-binding groups (ZBGs) as alternatives to the widely used hydroxamic acid moiety. Elaborating on findings from an earlier report, we performed in vitro inhibition assays with recombinant MMP-1, MMP-2, and in a cell culture assay using neonatal rat cardiac fibroblast cells. In both recombinant and cell culture assays, the new ZBGs were found to be effective inhibitors, typically 10-100-fold more potent than acetohydroxamic acid. The toxicity of these chelators was examined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt cytotoxicity assays, which demonstrate that most of these compounds are nontoxic at concentrations of almost 100 microM. To address the possible interaction of sulfur-containing ZBGs with biological reductants, the reactivity of these chelators with 5,5'-dithiobis(2-nitrobenzoic acid) was examined. Finally, thione ZBGs were shown to be effective inhibitors of cell invasion through an extracellular matrix membrane. The data presented herein suggest these heterocyclic ZBGs are potent, nontoxic, and biocompatible compounds that show promise for incorporation into a new family of MMP inhibitors.
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PMID:Heterocyclic zinc-binding groups for use in next-generation matrix metalloproteinase inhibitors: potency, toxicity, and reactivity. 1639 44

The objective of this study was to determine whether a fragment(s) of type II collagen can induce cartilage degradation. Fragments generated by cyanogen bromide (CB) cleavage of purified bovine type II collagen were separated by HPLC. These fragments together with selected overlapping synthetic peptides were first analysed for their capacity to induce cleavage of type II collagen by collagenases in chondrocyte and explant cultures of healthy adult bovine articular cartilage. Collagen cleavage was measured by immunoassay and degradation of proteoglycan (mainly aggrecan) was determined by analysis of cleavage products of core protein by Western blotting. Gene expression of matrix metalloproteinases MMP-13 and MMP-1 was measured using Real-time PCR. Induction of denaturation of type II collagen in situ in cartilage matrix with exposure of the CB domain was identified with a polyclonal and monoclonal antibodies that only react with this domain in denatured but not native type II collagen. As well as the mixture of CB fragments and peptide CB12, a single synthetic peptide CB12-II (residues 195-218), but not synthetic peptide CB12-IV (residues 231-254), potently and consistently induced in explant cultures at 10 microM and 25 microM, in a time, cell and dose dependent manner, collagenase-induced cleavage of type II collagen accompanied by upregulation of MMP-13 expression but not MMP-1. In isolated chondrocyte cultures CB12-II induced very limited upregulation of MMP-13 as well as MMP-1 expression. Although this was accompanied by concomitant induction of cleavage of type II collagen by collagenases, this was not associated by aggrecan cleavage. Peptide CB12-IV, which had no effect on collagen cleavage, clearly induced aggrecanase specific cleavage of the core protein of this proteoglycan. Thus these events involving matrix molecule cleavage can importantly occur independently of each other, contrary to popular belief. Denaturation of type II collagen with exposure of the CB12-II domain was also shown to be much increased in osteoarthritic human cartilage compared to non-arthritic cartilage. These observations reveal that peptides of type II collagen, to which there is increased exposure in osteoarthritic cartilage, can when present in sufficient concentration induce cleavage of type II collagen (CB12-II) and aggrecan (CB12-IV) accompanied by increased expression of collagenases. Such increased concentrations of denatured collagen are present in adult and osteoarthritic cartilages and the exposure of chondrocytes to the sequences they encode, either in soluble or more likely insoluble form, may therefore play a role in the excessive resorption of matrix molecules that is seen in arthritis and development.
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PMID:Peptides of type II collagen can induce the cleavage of type II collagen and aggrecan in articular cartilage. 1691 30

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

Gene therapy with insulin-like growth factor-1 (IGF-1) increases matrix production and enhances chondrocyte proliferation and survival in vitro. The purpose of this study was to determine whether arthroscopically-grafted chondrocytes genetically modified by an adenovirus vector encoding equine IGF-1 (AdIGF-1) would have a beneficial effect on cartilage healing in an equine femoropatellar joint model. A total of 16 horses underwent arthroscopic repair of a single 15 mm cartilage defect in each femoropatellar joint. One joint received 2 x 10(7) AdIGF-1 modified chondrocytes and the contralateral joint received 2 x 10(7) naive (unmodified) chondrocytes. Repairs were analysed at four weeks, nine weeks and eight months after surgery. Morphological and histological appearance, IGF-1 and collagen type II gene expression (polymerase chain reaction, in situ hybridisation and immunohistochemistry), collagen type II content (cyanogen bromide and sodium dodecyl sulphate-polyacrylamide gel electrophoresis), proteoglycan content (dimethylmethylene blue assay), and gene expression for collagen type I, matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, aggrecanase-1, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and TIMP-3 were evaluated. Genetic modification of chondrocytes significantly increased IGF-1 mRNA and ligand production in repair tissue for up to nine weeks following transplantation. The gross and histological appearance of IGF-1 modified repair tissue was improved over control defects. Gross filling of defects was significantly improved at four weeks, and a more hyaline-like tissue covered the lesions at eight months. Histological outcome at four and nine weeks post-transplantation revealed greater tissue filling of defects transplanted with genetically modified chondrocytes, whereas repair tissue in control defects was thin and irregular and more fibrous. Collagen type II expression in IGF-1 gene-transduced defects was increased 100-fold at four weeks and correlated with increased collagen type II immunoreaction up to eight months. Genetic modification of chondrocytes with AdIGF-1 prior to transplantation improved early (four to nine weeks), and to a lesser degree long-term, cartilage healing in the equine model. The equine model of cartilage healing closely resembles human clinical cartilage repair. The results of this study suggest that cartilage healing can be enhanced through genetic modification of chondrocytes prior to transplantation.
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PMID:Genetic modification of chondrocytes with insulin-like growth factor-1 enhances cartilage healing in an equine model. 1754 Jul 57

Spontaneous intracerebral hemorrhage (ICH) is one of the most devastating subtypes of stroke. Since angiogenesis is a fundamental process to brain development and repair by new blood vessel formation from pre-existing ones, mediated by numerous angiogenic factors including vascular endothelial growth factor (VEGF), the goal of the present work is to establish whether there is cerebral angiogenesis in rat brains with collagenase-induced ICH. Investigations were also performed to evaluate whether ICH alters expression of VEGF and its receptors Flt-1 and Flk-1. ICH was induced on adult male Sprague-Dawley rats by stereotactic injection of collagenase type VII into right globus pallidus. Angiogenesis was identified by hematoxylin-eosin stain and double immunolabeling method, and expression of VEGF and the receptors was evaluated by immunohistochemistry and quantitative real time reverse transcription-polymerase chain reaction. New vessels appeared around the hematoma and extended into it from 7 days, and 5-Bromo-2-Deoxyuridine-labeled nuclei in cerebral endothelial cells resided around the hematoma and the labeling peaked from 7 to 14 days. Expression of VEGF, Flt-1 and Flk-1 was observed in cerebral endothelial cells at the hemorrhagic basal ganglion, and increases of their mRNA persisted to 28 days. These findings suggest that ICH can induce cerebral angiogenesis and upregulation of VEGF, Flt-1 and Flk-1 and that modulation of angiogenesis via altering expression of VEGF and its receptors may be a potential strategy for promoting ICH repair.
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PMID:Cerebral angiogenesis after collagenase-induced intracerebral hemorrhage in rats. 1788 90

The acinar cell culture plays a very important role in research of pancreatic pathophysiology. The aim of this study was to establish a long-term culture of human (foetal) pancreatic acinar cells in standardized nutrient media with supplements. Acinar cells were prepared from pancreatic tissues obtained from aborted foetus (> or =35 weeks) with no prior pancreatic complications by collagenase digestion and cultured using different media and supplements. The purity and phenotype of acinar cells was confirmed by various staining techniques and FACS. The acinar cell proliferation was determined at different time intervals by Bromo-deoxyuridine (BrdU) incorporation, and metabolic enzyme activity was analysed. The acini could be cultured and maintained in Ham's F-12 K/M199 media in the presence of 5% BSA, 0.1 mg/ml STI, 10 ng/ml EGF, and 10% FCS with the same morphological appearance as that of freshly prepared for 12 days with maximum viability of 80-85% and formation of monolayer without extracellular matrix. A significant BrdU incorporation of acinar cells in primary culture was observed which was maximum (105%) at day four. Higher amylase and lipase activity was seen in freshly isolated acinar cells which decreased with time of the culture. The established human pancreatic acinar cell culture may act as an excellent model to study exocrine dysfunction or pancreatitis in response to acinar cell injury.
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PMID:Primary culture of pancreatic (human) acinar cells. 1824 27

Nonsteroidal antiinflammatory drugs are widely used to treat sports-related tendon injuries or tendinopathy. This study was designed to investigate the effect of ibuprofen on expressions of types I and III collagen, as well as collagen-degrading enzymes including matrix metalloproteinase (MMP)-1, -2, -8, -9, and -13. Rat Achilles tendon cells were treated with ibuprofen and then underwent MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Reverse transcription-polymerase chain reaction was used to evaluate mRNA expressions of types I and III collagen, MMP-1, -2, -8, -9, and -13. Protein expressions of types I and III collagen, MMP-1, -8, and -13 were determined by Western blot analysis. Gelatin zymography was used to evaluate the enzymatic activities of MMP-2 and MMP-9. The results revealed that ibuprofen upregulated expressions of MMP-1, -8, -9, and -13, both at mRNA and protein levels. There was no effect of ibuprofen on mRNA and protein expressions of types I and III collagen. Gelatin zymography revealed that the enzymatic activity of MMP-9 was upregulated after ibuprofen treatment. In conclusion, ibuprofen upregulates the expressions of collagenases including MMP-1, -8, -9, and -13 without affecting the expressions of types I and III collagen. These findings suggest a molecular mechanism potentially accounting for the inhibition of tendon healing by ibuprofen.
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PMID:Ibuprofen upregulates expressions of matrix metalloproteinase-1, -8, -9, and -13 without affecting expressions of types I and III collagen in tendon cells. 1984 88


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