Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Several studies have implicated the extracellular matrix-degrading metalloproteinases (MMPs) as essential agents in tumor cell invasion and metastasis. In the present study, we have investigated the patterns of expression of a number of MMPs and their specific tissue inhibitors (TIMP-1 and TIMP-2) in human colonic tissue samples that represent various stages of progression from adenomas showing different degrees of dysplasia to adenocarcinomas. We assessed levels of mRNA by Northern blot analysis and the results were measured semiquantitatively by densitometry. In total, we analyzed nine adenomas of varying size and with varying degrees of dysplasia, three adenomas with adenocarcinoma (malignant polyps), and five adenocarcinomas. Although expression of MMP and TIMP mRNA was highly intercorrelated, transcripts for stromelysin 3 and TIMP-2 (high) showed the strongest relation to the neoplastic process. Detection of stromelysin 3 mRNA accompanied a diagnosis of severe dysplasia or malignancy, whereas levels of TIMP-2 (high) mRNA transcripts permitted finer distinctions on the neoplastic continuum. These data indicate changes within extracellular matrix acquired during the process of malignant transformation of human sporadic colorectal neoplasia.
Diagn Mol Pathol 1993 Jun
PMID:Expression pattern of metalloproteinases and their inhibitors changes with the progression of human sporadic colorectal neoplasia. 826 81

Tissue inhibitors of metalloproteinases (TIMPs) appear to play an important regulatory role in tissue remodelling and invasion by malignant cells. Since pregnancy involves morphological changes in existing maternal tissues, as well as a strictly controlled invasion by fetal trophoblasts, we have examined the temporal expression of TIMP-1, TIMP-2, and specific metalloproteinases in the mouse uterus, decidua, placenta, amnion, and ovaries throughout gestation by examining mRNA levels on northern and slot blots. Maximal levels of TIMP-1 mRNA were observed from day 6 to day 10 in the uterus, decidua, and placenta. In clear contrast to the early burst of TIMP-1 mRNA accumulation, the level of TIMP-2 mRNA increased steadily throughout gestation in the uterus, decidua, and amnion, while in the placenta it showed a sevenfold increase after day 14. In amnion, TIMP-1 was induced specifically on day 18. Interestingly, the normally high level of TIMP-1 mRNA seen in the ovaries of virgin mice was low during gestation, until day 18 and postpartum, when a sixfold increase over the levels in virgin ovary was observed. In contrast, ovarian TIMP-2 mRNA showed a marginal increase during gestation. The temporal pattern of 72 kDa gelatinase type A followed that of TIMP-2 in the decidua and ovary. Stromelysin-2 mRNA was detected at term only in ovary and decidua. Our data show that the temporal accumulation of TIMP-1 and TIMP-2 mRNA is precisely coordinated in each of the tissue compartments and is independently regulated during the in vivo remodeling of reproductive tissues in gestation.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Reprod Dev 1993 Jul
PMID:Temporal expression of tissue inhibitors of metalloproteinases in mouse reproductive tissues during gestation. 835 25

Matrix metalloproteinases (MMP) are present in the latent form in normal myocardium. To examine the stringent balance between MMP and tissue inhibitor of metalloproteinase (TIMP) and to determine whether MMP are secreted simultaneously and in co-ordination with their inhibitors, we analysed MMP and TIMP by immunological, isolation by gel-permeation and affinity chromatography, and enzymatic assays in tissues and extracts. Using antibodies to MMP-1 and TIMP-1, we found strong in situ staining of MMP-1 and TIMP-1 in tissues. The staining was uniform in the endo- and subendomyocardium as well as in the interstitial space. TIMP-1 was present wherever MMP-1 was localized. From the tissue extract, proteins were separated on a gel-filtration column (Sephacryl S-200) and analysed for MMP and TIMP activity by zymography as well as by using succinyl-Gly-Pro-Leu-Gly-Pro-4-amido-7-methyl coumarin (Suc-GPLGP-AMC) as a selective fluorogenic substrate for collagenase. TIMP and MMP were further purified on collagen-Sepharose affinity column. The results indicated that MMP activity was co-eluted with TIMP activity. MMP-1, MMP-2 and TIMP-1 were further analysed by Northern blot for mRNA levels in the heart, skin, lung, liver and kidney. Results suggested co-expression of MMP-1 and TIMP-1 at the transcription level in all tissues. The level of MMP-2 mRNA was specifically higher in the heart tissue, which suggests a role of MMP-2 in the integrity of cardiovascular structure. The study indicated that myocardium as well as other tissue have an endogenous inhibitory system, suggesting that the MMPs activity is co-ordinated by their inhibitors at both the gene and protein levels. Furthermore, MMP and TIMP were co-expressed and were tightly regulated in maintaining the architecture of the interstitial tissue.
J Mol Cell Cardiol 1995 Oct
PMID:Co-expression of tissue inhibitor and matrix metalloproteinase in myocardium. 857 34

The specific effect of docosahexaenoic (DHA; C22:6 n-3), as compared to eicosapentaenoic acid (EPA; C20:5 n-3), on adrenoceptor function was investigated in cultured rat myocardial cells. The cardiomyocytes were grown for 24 h in a conventional seric medium, and then incubated for 96 h in a medium enriched with either DHA or EPA. After this treatment, the phospholipids of the DHA-treated cells contained approximately 20% of the total fatty acids as C22:6 n-3, and those of EPA-treated cells displayed a high content in C20:5 n-3 and its elongation product C22:5 n-3 (30% of total fatty acids). Additionally, the n-3/n-6 polyunsaturated fatty acid ratio was the same in the two groups of cells. These modifications were roughly similar in all the phospholipid classes. The contractions were monitored photometrically and no significant difference in basal frequency and contraction parameters could be detected. The stimulation of the beta-adrenergic receptors (isoproterenol 10(-7) M) resulted in a positive chronotropic effect, which was significantly higher in the DHA-rich cells. Conversely, the higher DHA content in the phospholipids appeared to induce a decrease in the affinity of the beta-receptors for the ligand (dihydroalprenolol) without alteration of the number of beta-receptor binding sites and provoked a significant decrease in isoproterenol-stimulated cAMP production (-19%). To investigate further these controversial data, the cardiomyocytes were treated with dibutyryl-cAMP, which elicited a positive chronotropic response significantly higher in the DHA-rich cells. The alpha-adrenergic stimulation by phenylephrine (3 x 10(-6) M) increased the spontaneous rate, but in a similar manner in the DHA- and EPA-enriched cells. Similarly, neither the alpha-adrenergic receptor binding characteristics nor the production of phosphoinositides was modulated by the membrane DNA content, although the phosphatidylinositol PUFAs were significantly altered. In conclusion, increasing the DHA content in membrane phospholipids did not affect the alpha-adrenergic system, but exerted a specific positive influence on the beta-adrenergic transduction mechanism, essentially through an increase of cAMP efficiency.
J Mol Cell Cardiol 1995 Nov
PMID:Effect of docosahexaenoic acid and eicosapentaenoic acid in the phospholipids of rat heart muscle cells on adrenoceptor responsiveness and mechanism. 859 1

Cartilage matrix protein (CMP) is a major component of different cartilages and consists of a disulfide-linked homotrimer. To test whether the C-terminal region forms a three-stranded alpha-helical coiled-coil, we synthesized a peptide, CMP-C36, corresponding to the last 36 residues of human CMP. Analytical ultracentrifugation revealed that CMP-C36 forms a homotrimer under physiological conditions. The sedimentation coefficient of 1.12 S is consistent with a rod-shaped molecule of 5.8 nm length, suggesting a lateral packing of three peptide chains. Depending on conditions, circular dichroism spectroscopy showed 75 to 96% alpha-helical content. The shapes of the spectra are characteristic for a coiled-coil structure. Thermal and guanidine-HCI-induced denaturation revealed a high degree of cooperativity and high stability. The concentration dependence of the melting temperature suggest a two-state transition. The trimer is stabilized by increasing the ionic strength above 130 mM salt, above which six ions are released upon unfolding. The peptide characteristics make it very likely that the C-terminal domain serves as the trimerization site of CMP. The two cysteine residues preceding this sequence region might stabilize the complex after assembly.
J Mol Biol 1996 Mar 15
PMID:The C-terminal domain of cartilage matrix protein assembles into a triple-stranded alpha-helical coiled-coil structure. 860 42

Human heart matrix metalloproteinases (MMP) are present in the latent form and activated in the failing heart. To examine whether the MMP activation was due to gene and/or post-translational modification, we analysed tissue from 10 explanted hearts due to coronary heart disease (CHD) and five normal left atrial tissue from donor hearts. Based on in situ immunolabeling MMP-1, tissue inhibitor of metalloproteinase (TIMP-1) and collagen were co-localized in the interstitial tissue. Based on sandwich ELISA, TIMP-1 and MMP-1 levels were 37 +/- 8 ng/mg and 9 +/- 2 ng/mg in normal tissue (P < 0.01) and 12 +/- 5 ng/mg and 75 +/- 11 ng/mg in the infarcted tissue (P < 0.01), respectively. These levels suggest repression of TIMP-1 during myocardial infarction. Northern blot analysis indicated that the mRNAs for both MMP-1 and TIMP-1 were increased three-to four-fold in the infarcted tissue as compared to the normal tissue, suggesting upregulation of MMP and TIMP gene transcription following infarction. Based on in situ tissue overlay zymography, the generalized activation of MMP was observed in the interstitium of the infarcted heart. Zymographic and immunoblot analysis demonstrated the presence of one band at 66 kDa (MMP-2) in the normal tissue and several bands at 92 (MMP-9), 66 (MMP-2) and 54 kDa (MMP-1) in the infarcted heart. Incubation of the zymographic gel with metal chelator (phenanthroline) abolished bands at 92 kDa and 54 kDa but phenanthroline did not abolish the lytic band at 66 kDa. The 66 kDa band was completely abolished in the presence of phenanthroline and phenyl methyl sulfonyl fluoride (PMSF). 2D-zymographic analysis suggested that the lytic band at 66 kDa was a mixture of two neutral proteinases with different isoelectric point. Plasminogen/gelatin zymographic analysis of infarcted tissue extract indicated that the band at 66 kDa was plasmin generated due to increased expression of tissue plasminogen activator (tPA) activity. In relation to increased expression of gelatinase in the infarcted tissue, our data suggest that gelatinase B (92 kDa) is induced in diseased heart. The results suggest that tPA converts plasminogen to plasmin which, in turn, activates MMPs and inactivates TIMP-1 post-translationally following ischemic cardiomyopathy.
J Mol Cell Cardiol 1996 Jul
PMID:Post-transcriptional regulation of extracellular matrix metalloproteinase in human heart end-stage failure secondary to ischemic cardiomyopathy. 884 29

Polymorphonuclear neutrophil (PMN) migration across basement membrane is thought to be dependent on the degradation of membrane constituents. PMN gelatinase B, a metalloproteinase able to degrade type IV collagen, may be involved in this phenomenon. PMN gelatinase B is released in the extracellular medium as a latent proform and then activated, mainly by PMN elastase. We investigated the role of gelatinase B in PMN migration across a Matrigel basement membrane matrix coated onto a filter, in a Boyden chamber. The effects of gelatinase and elastase inhibitors on PMN migration in this system were tested. Chemokinesis of PMN was tested in the same Boyden chamber across a filter free of basement membrane. The agarose method was used to test the same inhibitors for effects on PMN chemotaxis. In both systems, FMLP 10(-7)M was used as a chemoattractant. Addition of 10(-8)M TIMP-1 (the preferential gelatinase B inhibitor) inhibited trans-basement membrane PMN migration by 52 +/- 6% (P<0.05), without affecting PMN chemokinesis, chemotaxis, or degranulation. Also, (Ala)(2) Pro Val chloromethyl ketone (AAPVCK) 100 micron, a specific elastase inhibitor, inhibited trans-basement membrane PMN migration by 51 +/- 8% (P<0.05), without affecting PMN chemokinesis, chemotaxis, or degranulation. The AAPVCK-TIMP combination led to a decrease in migration across Matrigel basement membrane (46 +/- 2%, P,0.05)similar to that seen with TIMP alone. AAPVCK was responsible for inhibition of gelatinase B activation, leading to a decrease in activated gelatinase from 14% to 2% of total gelatinase release (P<0.05). All these results strongly suggest that gelatinase B is a major factor of PMN migration across basement membrane and that elastase may contribute to this process by activating pro-gelatinase B.
Am J Respir Cell Mol Biol 1996 Mar
PMID:Role of gelatinase B and elastase in human polymorphonuclear neutrophil migration across basement membrane. 884 80

Extracellular matrix metalloproteinases (MMPs) are activated in dilated cardiomyopathic (DCM) hearts [Tyagi et al. (1996): Mol Cell Biochem 155:13-21]. To examine whether the MMP activation is occurring at the gene expression level, we performed differential display mRNA analysis on tissue from six dilated cardiomyopathy (DCM) explanted and five normal human hearts. Specifically, we identified three genes to be induced and several other genes to be repressed following DCM. Southern blot analysis of isolated cDNA using a collagenase cDNA probe indicated that one of the genes induced during DCM was interstitial collagenase (MMP-1). Northern blot analysis using MMP-1 cDNA probe indicated that MMP-1 was induced three- to fourfold in the DCM heart as compared to normal tissue. To analyze posttranslational expression of MMP and tissue inhibitor of matrix metalloproteinase (TIMP) we performed immunoblot, immunoassay, and substrate zymographic assays. TIMP-1 and MMP-1 levels were 37 +/- 8 ng/mg and 9 +/- 2 ng/mg in normal tissue specimens (P < 0.01) and 2 +/- 1 ng/mg and 45 +/- 11 ng/mg in DCM tissue (P < 0.01), respectively. Zymographic analysis demonstrated lytic bands at 66 kDa and 54 kDa in DCM tissue as compared to one band at 66 kDa in normal tissue. Incubation of zymographic gel with metal chelator (phenanthroline) abolished both bands suggesting activation of neutral MMP in DCM heart tissue. TIMP-1 was repressed approximately twentyfold in DCM hearts when compared with normal heart tissue. In situ immunolabeling of MMP-1 indicated phenotypic differences in the fibroblast cells isolated from the DCM heart as compared to normal heart. These results suggest disruption in the balance of myopathic-fibroblast cell ECM-proteinase and antiproteinase in ECM remodeling which is followed by dilated cardiomyopathy.
 ...
PMID:Differential gene expression of extracellular matrix components in dilated cardiomyopathy. 891 70

Cytoplasmic malate dehydrogenase from ovine liver Echinococcus granulosus protoscolices was purified 22-fold by QAE- and SP-Sephadex chromatography. The pH optimum of the enzyme was 8.0 in either Tris-HCI or barbital buffer. The Km values of oxaloacetate and NADH were 0.400 +/- 0.018 and 0.410 +/- 0.038 mM, respectively. The enzyme lost about 90% of its activity when heated for 2 min at 65 degrees C. A 61.4% inhibition of the enzyme was noted at 4 mM concentration of diethyl pyrocarbonate. A 3 mM concentration of fructose 1,6-diphosphate inhibited the enzyme by 76.5%. The inhibition was non-competitive with respect to NADH with a Ki value of 0.85 mM. A 75% inhibition of the enzyme was noted at 1 mM concentration of mebendazole that inhibited the enzyme upon competing with NADH with a Ki value of 0.176 mM. A 2-mM concentration of citrate almost doubled the enzyme activity. The enzyme was inhibited at high concentrations of either substrate. The enzyme was not inhibited by p-hydroxymercuribenzoate or fumarate. The enzyme was absolutely specific for NADH as a cofactor. The properties of this enzyme are compared with those of the enzyme from the host liver, the cyst fluid and some other animal sources. The results are discussed in terms of the differences among the properties of the host liver, the cyst fluid and the protoscolices enzymes. The biochemical basis for the use of mebendazole in the treatment of echinococcosis is also elucidated.
Comp Biochem Physiol B Biochem Mol Biol 1996 Apr
PMID:Partial purification and kinetic properties of cytoplasmic malate dehydrogenase from ovine liver Echinococcus granulosus protoscolices. 892 42

Heparin has been shown to stimulate angiogenesis in the border zones surrounding infarcted myocardium. Matrix metalloproteinases (MMP), which are involved in extracellular matrix (ECM) organization, have also been shown to be activated. Cholesterol is required for receptor signaling in the plasma membrane, but a role of MMPs for cholesterol in ECM remodeling has not yet been shown. To examine whether heparin and cholesterol induce MMP and tissue inhibitor of metalloproteinase (TIMP) in human heart fibroblast (HHF) cells, confluent HHF cells were treated with cholesterol (100 microM) or heparin (20 microM). MMP activity was measured using zymography and TIMP was measured by Western blot analysis. The number of HHF cells, measured by a hemocytometer, increased after heparin or cholesterol treatment. Gelatinase A (MMP-2) activity increased in heparin treated cells, and the TIMP-1 level increased in cholesterol-treated cells. Based on Northern blot analysis, we observed that both MMP-1 and MMP-2 were induced at the gene transcription level by heparin and that TIMP-1 was induced by cholesterol. To examine whether the effects of heparin and cholesterol were due to Ca2+ mobilization, we carried out Ca2+ transient assays using FURA-2/AM as a fluorescence probe in HHF cells. Heparin induced a slow rise in the Ca2+ transient with a slow decay, and cholesterol induced a rapid rise with a slow reversal to the baseline calcium level. This suggested that the effect of heparin on Ca2+ release from HHF may be secondary to the receptor binding on the cell membrane but that cholesterol may have a direct effect. Protein kinase inhibitor and Ca2+-channel blocker have been shown to inhibit MMP expression. To examine whether the effect of heparin on MMP expression is mediated through the collagenase promoter activity, we carried out gel-shift assays using a 21-oligonucleotide analogue to the MMP-1 promoter sequence. Results suggested that the increase in MMP promoter activity by heparin is due to a specific transcription factor binding to MMP-1 promoter sequence. The effect of cholesterol on fibroblast cell proliferation is due in part to the tissue inhibitor. This study demonstrated the role of heparin and cholesterol in ECM remodeling and has implications for angiogenesis and athersclerosis, respectively.
J Mol Cell Cardiol 1997 Jan
PMID:Differential regulation of extracellular matrix metalloproteinase and tissue inhibitor by heparin and cholesterol in fibroblast cells. 904 53


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