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Gelatinase Activity (gelatinase + activity)
Selected AbstractsExpression of metalloproteinases and their tissue inhibitors in inflamed gingival biopsiesJOURNAL OF PERIODONTAL RESEARCH, Issue 5 2008L. D. R. Gonçalves Objectives:, Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are known to be involved in the periodontal disease process. Results of in vivo MMPs and TIMPs gene expressions in the gingiva, though, are still controversial. In the present study, we compared the gene expression of MMP-1, -2, -9, -13 and TIMP-1, -2 in healthy and inflamed gingiva. Methods:, 38 gingival samples were collected from gingivitis (n = 10), advanced chronic periodontitis (n = 10), generalized aggressive periodontitis (n = 8) and periodontally healthy individuals (n = 10). Total RNA isolated from those samples was subjected to reverse transcription followed by amplification by polymerase chain reaction (RT-PCR). Products were visualized in agarose gels and quantified by optical densitometry. Samples were also processed for gelatin zymography and Western blotting for MMP-2 and MMP-9 in order to assess for post-transcriptional MMP regulation at the protein level. Results:, The frequencies and levels of transcripts encoding MMPs and TIMPs were found to be not significantly different among groups (p > 0.05, Fisher's Exact and Kruskall-Wallis tests). There is a trend towards higher MMP-2 and -9 gelatinase activities in the inflamed samples, although not statistically significant. In contrast, zymography and Western blotting studies show that MMP-2 is virtually absent in the chronic periodontitis group. Conclusion:, These results could reflect a complex regulation of MMPs and TIMPs' gene expression in the course of gingival inflammation. They also reveal a great biological diversity even among individuals with similar periodontal status. [source] Enterococcus faecalis with the gelatinase phenotype regulated by the fsr operon and with biofilm-forming capacity are common in the agricultural environmentENVIRONMENTAL MICROBIOLOGY, Issue 6 2009Lilia Macovei Summary The prevalence of gelatinase activity and biofilm formation among environmental enterococci was assessed. In total, 396 enterococcal isolates from swine and cattle faeces and house flies from a cattle farm were screened for gelatinase activity. The most prevalent phenotype on Todd,Hewitt agar with 1.5% skim milk was the weak protease (WP) (72.2% of isolates), followed by the strong protease (SP) 18.7%, and no protease (NP) (9.1%). The majority of WP isolates was represented by Enterococcus hirae (56.9%), followed by Enterococcus faecium (25.9%), Enterococcus casseliflavus (10.4%), Enterococcus gallinarum (5.2%) and Enterococcus saccharolyticus (1.7%). All WP isolates were negative for gelE (gelatinase) and sprE (serine protease) as well as the fsrABDC operon that regulates the two proteases, and only four isolates (7.0%) formed biofilms in vitro. All SP isolates were Enterococcus faecalis positive for the fsrABDC, gelE, sprE genes and the majority (91.2%) formed a biofilm. Diversity of NP isolates was relatively evenly distributed among E. hirae, E. faecium, E. casseliflavus, E. gallinarum, Enterococcus durans, E. saccharolyticus and Enterococcus mundtii. All NP isolates were negative for the fsr operon and only four E. hirae (11.1%) formed a biofilm. Of further interest was the loss of the gelatinase phenotype (18.9% of isolates) from SP isolates after 4 month storage at 4,8°C and several passages of subculture. Results of reverse transcription PCR analysis indicated that mRNA was produced for all the genes in the frs operon and sequencing of the gelE gene did not reveal any significant mutations. However, gelatinase was not detectable by Western blot analysis. Our study shows that E. faecalis with the complete fsr operon and the potential to form a biofilm are relatively common in the agricultural environment and may represent a source/reservoir of clinically relevant strains. In addition, many environmental enterococci, especially E. hirae, produce an unknown WP that can hydrolyse casein but does not contribute to biofilm formation. The stability of the gelatinase phenotype in E. faecalis and its regulation will require additional studies. [source] HIGH PREVALENCE OF EXTENDED-SPECTRUM ,-LACTAMASES ESCHERICHIA COLI AND VANCOMYCIN-RESISTANT ENTEROCOCCI ISOLATES FROM CHICKEN PRODUCTS.JOURNAL OF FOOD SAFETY, Issue 1 2010A PROBLEM OF PUBLIC HEALTH ABSTRACT Twenty-nine chicken products were acquired from different supermarkets in Portugal during September to December 2007 and were analyzed for extended-spectrum ,-lactamases (ESBL) Escherichia coli and vancomycin-resistant enterococci (VRE). Cefotaxime-resistant E. coli isolates were recovered in 27 of 29 chicken samples representing 93% of the analyzed samples. The highest percentages of resistance (more than 50% of the isolates) were detected for ampicillin, nalidixic acid and tetracycline. VRE isolates were detected in 17 of 29 samples of chicken origin (59%) and were identified as Enterococcus durans (n = 15) and E. faecium (n = 2) with the highest percentages of resistance being detected for erythromycin, tetracycline, ampicillin and ciprofloxacin. Seven E. durans and the two E. faecium isolates recovered from chicken wings, gizzard and skin show gelatinase activity. The high rate of colonization of chicken products by these bacteria supports other studies suggesting that the food chain could be a source of ESBL and VRE colonization in humans representing a public health problem. PRACTICAL APPLICATIONS The data indicate that chicken products may be contaminated with a high prevalence of extended-spectrum ,-lactamases Escherichia coli and vancomycin-resistant enterococci (VRE). It is important to mention that the isolates present a diversity of phenotypes of antimicrobial resistance, and half of the VRE isolates show also gelatinase activity, indicating that these animals may be a reservoir of bacteria showing virulence and increased resistance to antimicrobial agents, raising special concerns about their transmission to humans through the food chain. [source] Chemically modified tetracyclines stimulate matrix metalloproteinase-2 production by periodontal ligament cellsJOURNAL OF PERIODONTAL RESEARCH, Issue 5 2006M. M. Bildt Background and Objective:, The purpose of this study was to investigate the effects of chemically modified tetracyclines (CMTs) on the production of gelatinases [matrix metalloproteinase (MMP)-2 and -9] by human periodontal ligament (PDL) cells, and on the activity of recombinant gelatinases. Material and Methods:, Human PDL cells were cultured with CMT-1, -3, -5, -7 or -8 in concentrations of 0, 1, 5, 10, 20, 50, 100, 200 and 500 µm. Gelatin zymography was used to determine MMP-2 and -9 production of the cells. The amount of DNA present in the cultures was analyzed using a fluorescent assay. The cytotoxicity of the CMTs was also determined. Recombinant human MMP-2 and -9 were incubated with the CMTs (0,500 µm) and their activity was analyzed using an internally quenched fluorogenic substrate. Results:, MMP-2 production was stimulated up to sevenfold by CMT-1, -3, -7 and -8 at low concentrations (10,200 µm). No significant amounts of MMP-9 were produced. In contrast, MMP-2 and -9 activity was reduced by ,,10,40-fold at higher concentrations (200,500 µm). CMT-5 had no effect on the production or on the activity of MMP-2 and -9. Only CMT-3 and -8 had cytotoxic effects on the PDL cells at the highest concentrations. Conclusion:, Surprisingly, CMTs are able to stimulate MMP-2 production at relatively low concentrations. However, at higher concentrations they exert a much stronger inhibitory effect on gelatinase activity. A possible stimulatory effect of CMTs on MMP production should be considered in their clinical use. [source] Development of a Model Bladder Extracellular Matrix Combining Disulfide Cross-Linked Hyaluronan with Decellularized Bladder TissueMACROMOLECULAR BIOSCIENCE, Issue 8 2006Allison L. Brown Abstract Summary: In this work we investigate the feasibility of modifying porcine-derived BAM to include HA with a view to developing a model, artificial extracellular matrix for the study of bladder cell-matrix interactions. HA-DPTH was incorporated into BAM disks and then cross-linked oxidatively to a disulfide containing hydrogel. Disks were seeded with bladder smooth muscle cells (BSMC) and UEC under three culture configurations and incubated for 3, 7, and 14 d. At each time point, matrix contraction was measured, and media supernatants assayed for cell-secreted gelatinase activity. To evaluate cell adherence and organization, triple immunofluorescent labeling of cell nuclei, actin cytoskeleton, and focal contacts was performed. HA-modified BAM exhibited a significant increase in matrix contraction and induced a higher level of cell-secreted gelatinase activity compared to unmodified BAM. Immunofluorescent labeling demonstrated that BSMCs remained adherent to both scaffold types over time. The distribution and organization of the cytoskeleton and focal contacts did not appear to be altered by the presence of HA. Interestingly, cellular infiltration into modified BAM was evident by 7 d and continued beyond 14 d, while BSMCs seeded onto unmodified BAM remained localized to the surface out to 14 d, with minimal infiltration evident only at day 28. These differences in cell infiltration support the gelatinase activity results. Increases in cell migration and matrix proteolysis in the presence of HA may be contributing factors toward BAM remodeling leading to increased matrix contraction with time. The model ECM developed in this work will be utilized for future studies aimed at elucidating the mechanisms controlling key remodeling events associated with bladder repair. Matrix contraction of cell-seeded BAM scaffolds. [source] Overexpression of cyclin D2 is associated with increased in vivo invasiveness of human squamous carcinoma cellsMOLECULAR CARCINOGENESIS, Issue 3 2002Shao Chen Liu Abstract Overexpression of cyclin D2 was studied in 10 human squamous cell carcinoma lines, to establish whether this gene plays a role in tumor progression. We found that those cell lines that overexpressed cyclin D2 (CCND2) had the most invasive in vivo behavior. The invasive ability of the cell lines was determined by evaluating the penetration of carcinoma cells into the tracheal wall in an in vivo assay with de-epithelialized tracheas transplanted into the subcutaneous tissue of nude mice. From five cell lines that exhibited low invasive ability, we selected two that had very little CCND2 expression (SCC9 and SCC15), to evaluate whether CCND2 gene transfer would increase the invasive behavior. After confirming the successful transfer of CCND2 by Northern, Western, and kinase-activity assays, we assessed the in vivo invasive behavior of the CCND2 -transfected cells and their respective vector alone,transfected controls. The cell lines containing the transferred CCND2 gene had a significantly higher invasive ability than respective controls. This was accompanied by a moderate increase in gelatinase activity. In addition, the in vitro proliferative abilities, under normal culture conditions, of the parental CCND2 - transfected and vector alone,transfected cells were found to be similar, as was the in vivo labeling index of Ki-67 in the tracheal transplants. These results indicated that the overexpression of CCND2 in squamous cell carcinoma lines modulates cell proliferation after induced quiescence and also has a powerful enhancing effect on in vivo aggressive growth behavior. © 2002 Wiley-Liss, Inc. [source] Interferon-, Inhibits Metalloproteinase Activity and Cytotrophoblast Cell MigrationAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 1 2010Vanina Andrea Fontana Citation Fontana VA, Sanchez M, Cebral E, Calvo JC. Interferon-, inhibits metalloproteinase activity and cytotrophoblast cell migration. Am J Reprod Immunol 2010; 64: 20,26 Problem, Establishment of a successful pregnancy relies on a complex fetal,mother communication that starts with the embryo adhering and invading the endometrium. This requires remodeling of extracellular matrix, performed by metalloproteinases. Cytokines, such as interferon-, (IFN-,), play a role in implantation and could affect the success of pregnancy. Method of study, Using JEG-3 cell line as model, we cultured the cells in the presence or absence of IFN-, and determined the activities of MMP-2 and MMP-9 using zymography and the secretion of leptin using Western blot. Results, Interferon-, inhibits gelatinase activity from MMP-2 and MMP-9 in a dose-dependent manner, reducing the secretion of leptin (not because of a general inhibition on protein synthesis) and impairs cell migration on Matrigel. Conclusion, Our results correlate with previous reports from our laboratory indicating that IFN- , is deleterious for mouse embryo outgrowth, having an effect on metalloproteinases activity as well as leptin secretion. [source] Neutrophil gelatinase,associated lipocalin is expressed in osteoarthritis and forms a complex with matrix metalloproteinase 9ARTHRITIS & RHEUMATISM, Issue 10 2007Kalpana Gupta Objective Expression of matrix metalloproteinase 9 (MMP-9) is up-regulated in osteoarthritis (OA) and usually presents as multiple bands when synovial fluid (SF) from OA patients is analyzed by zymography. Among these bands is an ,125,130,kd band for high molecular weight (HMW) gelatinase, which has not been characterized. This study was undertaken to characterize the HMW MMP activity in OA SF. Methods MMP activity in OA SF was determined by gelatin zymography. Recombinant MMPs were used to identify MMP activity on the zymogram. Western immunoblotting, immunoprecipitation, and immunodepletion analyses were performed using antibodies specific for human MMP-9 and human neutrophil gelatinase,associated lipocalin (NGAL). Human cartilage matrix degradation was determined by dimethylmethylene blue assay. Results Zymographic analysis showed that the HMW gelatinase in OA SF comigrated with a purified NGAL,MMP-9 complex. Results of Western immunoblotting showed that the HMW gelatinase was also recognized by antibodies specific for human NGAL or human MMP-9. These same antibodies also immunoprecipitated the HMW gelatinase activity from OA SF. The NGAL,MMP-9 complex was reconstituted in vitro in gelatinase buffer. In the presence of NGAL, MMP-9 activity was stabilized; in the absence of NGAL, rapid loss of MMP-9 activity occurred. MMP-9,mediated release of cartilage matrix proteoglycans was significantly higher in the presence of NGAL (P < 0.05). Conclusion Our findings demonstrate that the HMW gelatinase activity in OA SF represents a complex of NGAL and MMP-9. The ability of NGAL to protect MMP-9 activity is relevant to cartilage matrix degradation in OA and may represent an important mechanism by which NGAL may contribute to the loss of cartilage matrix proteins in OA. [source] Therapeutic and Analysis Model of Intrahepatic Metastasis Reflects Clinical Behavior of Hepatocellular CarcinomaCANCER SCIENCE, Issue 2 2002Shigeaki Sawada This study was designed to establish an intrahepatic metastasis model to investigate the biology and therapy of hepatocellular carcinoma (HCC) in mice. A fragment of mouse HCC tumor CBO140C12 was orthotopically implanted into the mouse liver. The number of intrahepatic metastatic colonies and the volume of the implanted tumor increased in a time-dependent manner. At 28 days after fragment implantation, all mice showed intrahepatic metastasis. Intravenous administrations of cisplatin and doxorubicin at 7 and 21 days after the implantation significantly sup- pressed the growth of the primary tumor nodule, but tended to inhibit intrahepatic metastasis. However, a marked decrease of body weight was observed during the experiment. On the other hand, an inhibitor of matrix metalloproteinases (MMPs), ONO,4817, decreased the gelatinase activity of MMP,9 secreted by CBO140C12 cells, and significantly reduced the number of colonies of intrahepatic metastasis when administered orally. Our established model, which is focused on intrahepatic metastasis, is suitable for evaluating the therapeutic effect of HCC and for analyzing intrahepatic metastasis, because this model reflects the clinical features of HCC and all the steps of tumor metastasis. [source] | ||||||||||