Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.49 (reverse transcriptase)
 31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The introduction of potent antiretroviral drugs for the treatment of patients with human immunodeficiency virus (HIV) infection has dramatically reduced the prevalence of HIV-associated neurological disorders. Such diseases can be mediated by proteolytic enzymes, i.e. matrix metalloproteinases (MMPs) and, in particular gelatinases, released from glial cells. The aim of this study was to investigate whether the antiretroviral drugs commonly used for the treatment of HIV-infected patients modulate the activity of MMPs in astrocyte and microglial cultures. Primary cultures of rat astrocyte and microglia were treated with different doses of zidovudine (AZT) or indinavir (IDV) for 20 h and simultaneously activated by exposure to lipopolysaccharide (LPS). Culture supernatants collected from astrocytes and microglia after 24 h incubation were subjected to gelatin zymography and western blot analysis for the assessment of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) protein levels. Total RNA was extracted from glial cells and used for reverse transcriptase-polymerase chain reaction for the assessment of mRNA expression. Our results indicate that both astrocyte and microglial cells constitutively express MMP-2 mRNA and protein. LPS treatment increased MMP-2 mRNA and protein expression in astrocytes, but not in microglial cells. The treatment with both AZT and IDV dose-dependently inhibited the expression of MMP-2 in astrocytes, whereas it had no effect on microglial cells. The expression of MMP-9 in both astrocytes and microglia was induced by LPS treatment and was dose-dependently inhibited by AZT and IDV treatment in LPS-stimulated astrocytes and microglia. These results raise the possibility that AZT and IDV interfere directly with MMP production in glial cells and independently from their antiviral activity, thus suggesting the possible therapeutical use in neurological diseases associated with MMPs involvement.
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PMID:Anti-HIV drugs decrease the expression of matrix metalloproteinases in astrocytes and microglia. 1466 18

The expression and activation of matrix metalloproteinases (MMPs) by tumor cells is correlated with progression to invasive and metastatic status. The purpose of this study was to examine the role of increased MMP-2 (gelatinase A) expression in prostate cancer progression utilizing human prostate PC-3 cancer cells that overexpress MMP-2 using gene transfection. PC-3 cells were transfected with pCR-3 vector only and pCR-3 MMP-2 plasmids employing the LipofectAMINE method, and stable transfectants were selected with G418. The expression of MMP-2, tissue inhibitor of metalloproteinase-2 (TIMP-2), and membrane-type MMP 1 (MT1-MMP) in PC-3 parental and transfected cells under serum-free conditions was determined by zymography, immunoblotting, immunofluorescent microscopy, Northern blotting, and/or reverse transcriptase-polymerase chain reaction (RT-PCR). MMP-2 transfected cells produced primarily the proenzyme form of MMP-2; the parental and vector control transfected PC-3 cells did not express any MMP-2 that was detectable by the methods we employed. Treatment of PC-3 MMP-2 transfected cells with Concanavalin A (Con A), in contrast to HT-1080 cells, processed only a small amount of the secreted 72-kd proenzyme to a 62-kd intermediate and a cell-associated 59-kd active form. The low level of secreted pro-MMP-2 processing induced by Con A was inhibited by serine protease inhibitors and was unaffected by cyclic adenosine monophosphate (cAMP). Immunoblotting showed that these cells produced abundant TIMP-2 and lower amounts of MT1-MMP in comparison with Con A-responding HT-1080 cells. HT-1080 cells respond to Con A by translocating MT1-MMP from intracellular localization sites to the plasma membrane, an effect not observed in PC-3 cells. The molecular basis for the low level of processing of pro-MMP-2 by PC-3 cells may be due to an overabundance of TIMP-2 and/or a low level of cell surface active MT1-MMP.
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PMID:Limited processing of pro-matrix metalloprotease-2 (gelatinase A) overexpressed by transfection in PC-3 human prostate tumor cells: association with restricted cell surface localization of membrane-type matrix metalloproteinase-1. 1476 14

We studied the mRNA expression of osteoprotegerin (OPG), receptor activator of NF-kappa B ligand (RANKL), tissue inhibitor of matrix metalloprotease (TIMP)-1 and -2, and matrix metalloprotease (MMP)-1 and -2 by human periodontal ligament (PDL) cells under intermittent tensile stress using a Flexercell Strain Unit. Analysis by reverse transcriptase-polymerase chain reaction showed that mechanical force upregulated OPG mRNA. We also demonstrated that the protein concentration of OPG in conditioned medium increased upon loading with tensile stress, as determined by enzyme-linked immunosorbent assay. TIMP-1 and -2 mRNA levels also increased, whereas levels of RANKL, MMP-1, and MMP-2 mRNA were barely affected. We further examined the effect of loading with tensile stress and addition of Salmonella abortus equi lipopolysaccharide (LPS) on the mRNA expression of PDL cells. The amount of OPG mRNA induced by mechanical strain was found to decrease with the addition of LPS to cultures. The induction of OPG mRNA expression by stretching was inhibited in the presence of indomethacin or genistein, whereas TIMP-1 mRNA expression induced by stretching was inhibited by the addition of cycloheximide, suggesting that tensile stress regulates cyclooxygenase activities, tyrosine phosphorylation, and de novo protein synthesis in PDL cells through the induction of OPG and TIMP-1 mRNA expression. These results provide evidence that the mechanical stimulus of stretching is responsible for the observed regulation of bone resorption and tissue degradation in PDL tissue.
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PMID:Periodontal ligament cells under intermittent tensile stress regulate mRNA expression of osteoprotegerin and tissue inhibitor of matrix metalloprotease-1 and -2. 1499 19

In malignant tumors the balance of matrix metalloproteinases (MMP) and tissue inhibitors of matrix metalloproteinases (TIMP) is disturbed. Radical oxygen species (ROS) and hydrogen peroxide potentially influence this balance. Therefore, we analyzed the balance of MMP and TIMP in renal cell carcinoma (RCC) specimens and cell lines. In RCC specimens MMP-2, MMP-9, TIMP-1, and TIMP-2 were immunohistochemically detected. Tumor-associated macrophages (TAM) as a potential source of ROS were characterized with an anti-CD68 antibody. Three RCC cell lines were treated with sublethal concentrations of hydrogen peroxide to simulate the effects of radical oxygen species. MMP-2 and MMP-9 protein expression was measured by zymography. mRNA expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 was assessed by quantitative reverse transcriptase polymerase chain reaction. Tumor cell-derived reactive oxygen species were measured by FACS analysis and dihydrorhodamine 123 oxidation. In RCCs the MMP and TIMP expression profile was variable. The balance between MMP and TIMP was shifted towards MMP in comparison to matched normal controls. TAM were localized in a close vicinity to MMP expresssing tumor cells. As in RCC specimens, the expression of MMP and TIMP in the analyzed RCC cell lines varied. Hydrogen peroxide induced MMP-2 and -9 mRNA and protein expression, whereas TIMP-1 and TIMP-2 mRNA levels remained unaffected in cell lines. Thus, the ratio between MMP and TIMP was shifted towards MMP. Tumor cells did not increase the production of reactive oxygen species stimulation with phorbol ester or hydrogen peroxide. In RCC the balance between MMP and TIMP is disturbed. Oxidative stress potentially increases this imbalance. TAM might be one source of hydrogen peroxide thus supporting the invasive properties of RCCs.
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PMID:The balance between MMP-2/-9 and TIMP-1/-2 is shifted towards MMP in renal cell carcinomas and can be further disturbed by hydrogen peroxide. 1506 27

Hepatic stellate cells (HSCs) were changed in their morphology, proliferative activity, and functions by culturing on type I collagen gel, as compared to the culture on polystyrene surface. HSCs have been found to produce extracellular matrix components and matrix metalloproteinases (MMPs). In this study, we have assessed the effects of several types of substrata on the expression of MMPs in HSC culture. MMP-1 expression was detectable in HSC culture on polystyrene surface and on type I collagen gel by immunofluorescence staining and reverse transcriptase-polymerase chain reaction (RT-PCR). The results from in situ zymography revealed the presence of interstitial collagenase activity around HSCs and along their cellular processes. Although proMMP-2 and proMMP-9 were detectable by gelatin zymography in the conditioned medium from both cultures using type I collagen gel and Matrigel as substratum, an active form of MMP-2 but not of MMP-9 was detected only in the culture using type I collagen as a substratum. Tissue inhibitor of metalloproteinase-2 expression was observed by RT-PCR in HSCs cultured on or in type I collagen gel, suggesting the suppression of MMP-2 activity detected in HSC culture using type I collagen. These results indicate a differential expression of MMP activity, hence the remodeling of extracellular matrix components is dependent on the substratum used for HSC culture. The HSC culture using several types of substrata appears to be a useful in vitro model to study the mechanism of extracellular matrix remodeling.
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PMID:Regulatory role of extracellular matrix components in expression of matrix metalloproteinases in cultured hepatic stellate cells. 1521 80

The Marfan syndrome (MFS), a relatively common autosomal dominant disorder of connective tissue, is caused by mutations in the gene for fibrillin-1 (FBN1). Fibrillin-1 is the main component of the 10- to 12-nm microfibrils that together with elastin form elastic fibers found in tissues such as the aortic media. Recently, FBN1 mutations have been shown to increase the susceptibility of fibrillin-1 to proteolysis in vitro, and other findings suggest that up-regulation of matrix metalloproteinases (MMP), as well as fragmentation of microfibrils, could play a role in the pathogenesis of MFS. In the present work, we have investigated the influence of fibrillin-1 fragments on the expression of MMP-1, MMP-2, and MMP-3 in a cell culture system. Cultured human dermal fibroblasts were incubated with several different recombinant fibrillin-1 fragments. The expression level of MMP-1, MMP-2, and MMP-3, was determined by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), and the concentration of the corresponding proteins was estimated by quantitative Western blotting. Our results establish that treatment of cultured human dermal fibroblasts with recombinant fibrillin-1 fragments containing the arginine-glycine-aspartic acid (RGD) integrin-binding motif of fibrillin-1 induces up-regulation of MMP-1 and MMP-3. A similar effect was seen upon stimulation with a synthetic RGD peptide. The expression of MMP-2 was not influenced by treatment. Our results suggest the possibility that fibrillin fragments could themselves have pathogenic effects by leading to up-regulation of MMPs, which in turn may be involved in the progressive breakdown of microfibrils thought to play a role in MFS.
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PMID:RGD-containing fibrillin-1 fragments upregulate matrix metalloproteinase expression in cell culture: a potential factor in the pathogenesis of the Marfan syndrome. 1551 94

The aim in this study was to investigate whether our experimental model for stroke therapy, flushing the ischemic territory with saline prior to reperfusion, could ameliorate disruption of microvascular integrity by reducing matrix metalloproteinase (MMP) expression during reperfusion. Stroke in Sprague Dawley rats (n = 42) was induced by a 2-h right middle cerebral artery (MCA) occlusion using a novel intraluminal hollow filament. Prior to reperfusion, 24 of the ischemic rats received 6ml isotonic saline at 37 degrees C infused into the ischemic area through the filament. Brain edema was determined by comparing the percentage difference in brain volume between the right and left (contralateral to stroke site) hemispheres, while the expressions of MMP-2 and -9 mRNA were analyzed by real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR). A significant (p < 0.01) brain edema, determined by an increased brain volume of 19 +/- 4%, and overexpression of the mRNA encoding MMPs, determined by increased relative mRNA level ratio, were found in ischemic rats. The brain damage, in terms of brain edema (4 +/- 1%) and overexpression of MMPs, was significantly (p < 0.05) ameliorated as a result of saline flushing into the ischemic territory prior to reperfusion. This study has enhanced our understanding of the causal mechanisms by which the neuroprotective effect of ischemic area "flushing" can be achieved.
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PMID:Reduced brain edema and matrix metalloproteinase (MMP) expression by pre-reperfusion infusion into ischemic territory in rat. 1553 Oct 84

Carnosol, a constant constituent of Rosmarinus officinalis extracts, is a phenolic diterpene shown to have antioxidant and anticarcinogen properties. In our studies, carnosol inhibited the invasion of highly metastatic mouse melanoma B16/F10 cells in vitro. First, the antimetastatic potentials of carnosol were examined by soft agar colony formation assay. Second, carnosol dose-dependently inhibited B16/F10 cell migration and invasion by in vitro transwell assay. Third, the decreasing activity of metalloproteinase was observed by zymographic assay. The result revealed that the treatment of carnosol could diminish the activity of MMP-9 more than MMP-2. Next, we analyzed the amounts of MMP-9 and MMP-2 proteins in the cells. The data indicated MMP-9 protein was also suppressed by carnosol in the same manner. In accordance with the above data, the results of reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed a reduced level of MMP-9 mRNA. Furthermore, carnosol significantly inhibited the tyrosine phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, AKT, p38, JNK and inhibition of activation of transcription factors NFkappa-B and c-Jun. These results lead us to conclude that carnosol could restrict the invasive ability of B16/F10 mouse melanoma cells by reducing MMP-9 expression and activity through suppressing (ERK) 1/2, AKT, p38, and JNK signaling pathway and inhibition of NF-kappaB and AP-1 binding activity. Taken together, these results indicate that carnosol targets MMP-mediated cellular events in cancer cells and provides a new mechanism for its anticancer activity.
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PMID:Carnosol inhibits the invasion of B16/F10 mouse melanoma cells by suppressing metalloproteinase-9 through down-regulating nuclear factor-kappa B and c-Jun. 1562 74

A cranial suture consists of neural-crest derived cells and matrices between mineralized skull bones. Little is known regarding the involvement of matrix metalloproteinases (MMPs) in the degradation of extracellular matrix of cranial sutures. In the postnatal rat model, the posterior frontal suture (PFS) undergoes complete ossification between P12-P22, whereas the sagittal suture (SS) remains patent. The present study utilized reverse transcriptase-polymerase chain reaction (RT-PCR) to explore the expression of MMP-1 and MMP-2 genes in the PFS and SS in P8 and P32 rats, and also to determine whether these MMP genes are modulated by exogenous mechanical forces. RNA was isolated from P8 and P32 normal PFS and SS each by pooling sutural specimens from 14 to 20 rats. RT-PCR analysis and semi-quantitative luminosity demonstrated the expression of MMP-1 and MMP-2 genes in the patent P8 PFS, P8 SS, and P32 SS, but no apparent MMP-2 expression in the physiologically ossified P32 PFS. Exogenous cyclic forces applied to the maxilla at 1000 mN and 4 Hz elicited corresponding cyclic bone strain waveforms with peak strain of 134.14+/-38.15 muepsilon (mean+/-S.D.) for the PFS, and 28.35+/-10.86 muepsilon for the SS in P32 rats. These cyclic forces delivered for 20 min/d over 2 consecutive days induced the expression of MMP-2 gene in the physiologically fused P32 PFS that was not expressed without mechanical stresses. Taken together, these data suggest potentially important roles of MMP genes in the postnatal development of cranial sutures, and their susceptibility to mechanical stresses.
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PMID:Expression of matrix metalloproteinase genes in the rat intramembranous bone during postnatal growth and upon mechanical stresses. 1565 46

Rapamune, an inhibitor of the mammalian target of rapamycin, exhibits antiproliferative actions and is increasingly used as adjuvant therapy with calcineurin inhibitors. This study investigated the effect of Rapamune on functional and molecular markers in a rat model of calcineurin inhibitor-induced graft dysfunction. Prograf (6 mg), with or without addition of Rapamune (1 mg), was administered to salt-depleted male rats (n = 6/group). Urinary protein excretion and serum creatinine were measured. Rats were culled at 28 days, and messenger RNA expression of TGF-beta, MMP-2, MMP-9, TIMP-1, and collagen III was evaluated with reverse transcriptase polymerase chain reaction. Serum creatinine increased with Prograf (P = .01), but not Rapamune (P = .69) treatment, compared to controls at 28 days. The combination of Rapamune and Prograf produced a rise in serum creatinine at 7 (P = .007) and 14 (P = .01) days, but this was not observed at later time points. Urinary protein excretion was unaltered by any drug or combination. While confirming a synergistic effect of Rapamune and calcineurin inhibitors on renal function, these results suggest that sole therapy with Prograf produces inhibition of fibrotic gene expression. Rapamune alone has no deleterious effect on gene expression but addition of Rapamune cancels out the beneficial effects of Prograf.
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PMID:Prograf produces a molecular environment favoring antifibrosis, an effect reversed by the addition of rapamune. 1580 77


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