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)

Cellular respiratory rates are normally determined by metabolic activity, but become rate limited by O2 availability if the cell O2 tension (PO2) falls below a critical value (typically 1-10 Torr). An ability to reduce metabolic activity and energy demand in response to a falling O2 availability might confer an increased resistance to a diminished O2 supply. Isolated rat hepatocytes were studied in primary culture under controlled O2 tensions. Cells were obtained by collagenase digestion and seeded into nutritive media in control and experimental spinner flasks at identical cell densities. Cells subjected to rapid reduction in PO2 (100-->0 Torr over < 40 min) exhibited undiminished O2 uptake until PO2 fell below 10 Torr. By contrast, when cell PO2 was reduced over several hours, significant decreases in O2 uptake became evident at O2 tensions as high as 70 Torr. These decreases were associated with a reduction in ATP concentration and an increase in NAD(P)H, compared with rapidly deoxygenated cells at the same PO2. No loss in cell viability was detected after 24 h at reduced PO2. The decrease in respiratory rate was associated with an increased rate of lactic acid production relative to normoxic controls. Restoration of normoxia was associated with an immediate return of O2 uptake to control levels. These results demonstrate that hepatocytes are capable of reversibly decreasing metabolic activity and O2 demand during sustained moderate reductions in PO2, via a mechanism that appears to involve an inhibition of mitochondrial function other than O2 supply limitation. This response may alter cellular susceptibility to physiological stresses including hypoxia.
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PMID:Oxygen conformance of cellular respiration in hepatocytes. 823 74

Cortisol-cortisone interconversion is catalyzed by the NADP/NADPH-dependent oxido-reductase, 11beta-hydroxysteroid dehydrogenase-1 (11betaHSD-1) and the NAD-dependent oxidase, 11betaHSD-2. Because of the importance of placental corticosteroid metabolism in dictating the amount of cortisol arriving in the fetus to regulate fetal pituitary-adrenocortical function, the present study determined whether there was a developmental change in the expression of 11betaHSD-1 and/or -2 in placental syncytiotrophoblast, the site of maternal:fetal exchange. A syncytiotrophoblast-enriched (>95%) cell fraction was isolated from baboon placentas obtained at early (day 60), mid (day 100), and late (day 165) gestation (term = day 184), and 11betaHSD-1 and -2 messenger RNA (mRNA) and protein levels were determined by Northern and Western blots. The levels (mean +/- SE) of the single 1.6-kilobase (kb) mRNA for 11betaHSD-1, expressed as a ratio to beta-actin, increased (P < 0.05) between early (0.36 +/- 0.16; n = 4) and mid (0.95 +/- 0.21; n = 11) gestation and further increased (P < 0.05) by late gestation (1.82 +/- 0.29; n = 13). Similarly, the levels of the single 1.9-kb mRNA for 11betaHSD-2 in late gestation (2.46 +/- 0.35; n = 8) were greater (P < 0.05) than respective values at mid (1.36 +/- 0.22; n = 8) and early (0.64; n = 2) gestation. The levels of 11betaHSD-1 (arbitrary densitometric units), detected as a dominant band of 34 kDa, were greater (P < 0.05) in late gestation (2.6 +/- 0.2; n = 4) than at early (1.2 +/- 0.1; n = 4) or mid (1.9 +/- 0.3; n = 4) gestation. In contrast, 11betaHSD-2 was not detected by Western blot in syncytiotrophoblast isolated by collagenase dispersion. However, immunocytochemistry revealed that 11betaHSD-2 was present in and localized to the syncytiotrophoblast layer of the baboon placenta and that expression in late gestation (n = 4) appeared to exceed that in placentas of early (n = 4) and mid (n = 4) gestation. These results indicate that both 11betaHSD-1 and 11betaHSD-2 were expressed in syncytiotrophoblasts of the baboon placenta and that the mRNA and protein levels of these two 11betaHSD enzymes increased with advancing gestation. However, because 11betaHSD-2 was not detected in syncytiotrophoblast isolated by collagenase dispersion, we suggest that the 11betaHSD-1 and -2 reside in different membrane fractions of the syncytiotrophoblast. Consequently, the estrogen-regulated change in transplacental cortisol metabolism with advancing gestation may result in a developmental change in the expression and location of the two 11betaHSD enzymes controlling cortisol-cortisone metabolism and transfer into the fetus, resulting in activation of the fetal pituitary adrenocortical system.
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PMID:Developmental increase in expression of the messenger ribonucleic acid and protein levels of 11beta-hydroxysteroid dehydrogenase types 1 and 2 in the baboon placenta. 894 Mar 99

Peroxynitrite and hydroxyl radicals are potent initiators of DNA single strand breakage, which is an obligatory stimulus for the activation of the nuclear enzyme poly(ADP-ribose)synthetase (PARS). Rapid activation of PARS depletes the intracellular concentration of its substrate, NAD+, slowing the rate of glycolysis, electron transport and ATP formation. This process can result in acute cell dysfunction and cell necrosis. Accordingly, inhibitors of PARS protect against cell death under these conditions. In addition to the direct cytotoxic pathway regulated by DNA injury and PARS activation, PARS also appears to modulate the course of inflammation by regulating the expression of a number of genes, including the gene for intercellular adhesion molecule 1, collagenase and the inducible nitric oxide synthase. The research into the role of PARS in inflammatory conditions is now supported by novel tools, such as novel, potent inhibitors of PARS, and genetically engineered animals lacking the gene for PARS. In vivo data demonstrate that inhibition of PARS protects against various forms of inflammation, including zymosan or endotoxin induced multiple organ failure, arthritis, allergic encephalomyelitis, and diabetic islet cell destruction. Pharmacological inhibition of PARS may be a promising novel approach for the experimental therapy of various forms of inflammation.
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PMID:Role of poly(ADP-ribose)synthetase in inflammation. 968 9

The purpose of this study was to determine if exacerbation of apoptosis precedes liver injury during chronic exposure of rats to alcohol. After 7 weeks of feeding an alcohol- or dextrin-containing liquid diet, the animals were treated with gram-negative bacterial lipopolysaccharide (1 mg x kg(-1) body weight, intravenously) or sterile saline and sacrificed 3 hr after the treatment. Alanine:2-oxoglutarate aminotransferase (ALT) and lactate:NAD oxidoreductase [lactate dehydrogenase (LDH)] were measured in plasma. The caudate lobe of the liver was resected for histology, while the rest of the organ was perfused with collagenase to isolate hepatocytes, Kupffer cells (KCs), and sinusoidal endothelial cells (SECs) by centrifugal elutriation. Hepatocyte mitochondria were isolated by differential centrifugation of the cell homogenate. Reduced and oxidized glutathione (GSH and GSSG) in isolated hepatocytes and hepatocyte mitochondria, and malondialdehyde in hepatocytes were assayed. Caspase-3 activity and Fas ligand mRNA expression were determined in hepatocytes, KCs, and SECs. Plasma ALT and LDH activity, liver histology, GSH, GSSG and their ratio, and malondialdehyde content were not affected by alcohol treatment Caspase-3 activity was significantly increased in alcohol-treated rats in all three cell types, with the lowest response observed in hepatocytes and the highest in KCs. Fas ligand mRNA expression, which had the highest level in SECs, followed by KCs and hepatocytes, was not affected by alcohol administration. Lipopolysaccharide had the following effects: an increase in ALT in both pair- and alcohol-fed rats, and LDH only in alcohol-fed rats, a decrease in GSH + GSSG levels in both mitochondria and hepatocytes, an elevation of malondialdehyde content in hepatocytes, a raise in caspase-3 activity in all groups and cell types, and an augmentation of Fas ligand expression in hepatocytes and KCs, but not in SECs. These data suggest that, during chronic alcohol consumption, an exacerbated apoptosis precedes alcohol-induced liver injury.
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PMID:Modulation of caspase-3 activity and Fas ligand mRNA expression in rat liver cells in vivo by alcohol and lipopolysaccharide. 1006 67

Leydig cells were isolated from the perch testes belonging to the pre-spawning stage by collagenase treatment and mechanical separation followed by percoll gradient. They were incubated in vitro either for 5 h or at different times in the absence (control) or presence of piscine gonadotropin (GTH, 2 microg (1 x 10(6) cells)(-1)) or 3,5,3'-triiodothyronine (T3, 50 ng (1 x 10(6) cells(-1)) or T3-induced protein (TIP, 2 microg (1 x 10(6) cells)(-1)). 3Beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase (3beta-HSD) activity was determined by the conversion of [3H]delta5-dehydroepiandrosterone (DHEA) to [3H]delta4-androstenedione or [3H]delta5-pregnenolone to [3H]delta4-progesterone (P4) or by spectrophotometric estimation of NADH formation from NAD. T3 significantly increased (P < 0.01) both delta5-DHEA to delta4-androstenedione and delta5-pregnenolone to delta4-P4 conversion in Leydig cells indicating stimulation of 3beta-HSD activity. T3 stimulation of 3beta-HSD activity could be inhibited by cycloheximide (50 microg ml(-1)) suggesting the involvement of T3-induced protein (TIP) which was isolated and purified earlier in this laboratory from goat Leydig cells [15]. Addition of TIP or GTH significantly stimulated Leydig cell 3beta-HSD activity (P < 0.01). However, there was a difference between TIP and GTH stimulation in time kinetic study where TIP enhanced 3beta-HSD activity at 1 h (P < 0.05), reached its peak at 3 h (P < 0.01) and then plateaued till 8 h. GTH, on the other hand, did not show any stimulation of 3beta-HSD activity for 2 h, stimulation was marked only at 3 h (P < 0.05), reached a peak at 6 h (P < 0.01) and then leveled off. Determination of Km and Vmax of the enzyme showed an increase in the velocity of reaction by GTH with unaltered Km. TIP increased both velocity and affinity of the enzyme. GTH significantly increased the synthesis of 3beta-HSD protein at 3 h (P < 0.01) reaching maximal stimulation at 6 h which clearly coincided with the enzyme activity. In contrast, TIP had no effect on 3beta-HSD protein synthesis, but its direct addition to 3beta-HSD enzyme preparation in vitro caused significant augmentation of the enzyme activity (P < 0.01) suggesting thereby its modulatory effect on the enzyme. Results, therefore, show that although both T3 and GTH stimulated perch testicular Leydig cell 3beta-HSD activity, T3 effect was not direct but mediated via TIP and there is a clear distinction between GTH and TIP stimulation. GTH increased the enzyme activity by stimulating 3beta-HSD protein synthesis while TIP acts directly on the enzyme modulating it from less active to more active state.
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PMID:Differential regulation of Leydig cell 3beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase activity by gonadotropin and thyroid hormone in a freshwater perch, Anabas testudineus (Bloch). 1062 32

A method for the preparation of intact rat hepatocytes in high yield was first described in 1969. The procedure involved digestion of hepatic tissue by perfusion of the liver with crude collagenase; later, purified collagenase without other enzymic additions was shown to be ineffective. Recently it has been discovered that the combination of purified collagenase plus elastase is superior to crude collagenase in that it consistently provides high yields of undamaged hepatocytes. The isolated hepatocyte preparation has proved particularly useful for the study of mechanisms responsible for long-range interactions within the cell. These can be studied over prolonged time courses and in the presence of graded concentrations of specific inhibitors. Studies of this kind have demonstrated a close relationship between cytoplasmic metabolic flows and mitochondrial forces and have also revealed that the cytoplasmic and mitochondrial free NAD-linked redox potentials are maintained by energy-dependent reactions.
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PMID:The isolated hepatocyte preparation: 30 years on. 1081 14

Degradation of type I collagen by collagenases is an important part of extracellular remodeling. To understand the role of the hinge region of fibroblast collagenase in its collagenolytic activity, we individually substituted the 10 conserved amino acid residues at positions 264, 266, 268, 296, 272, 277, 284, 289, 307, and 313 in this region of the enzyme by their corresponding residues in MMP-3, a noncollagenolytic matrix metalloproteinase. The general proteolytic and triple helicase activities of all of the enzymes were determined, and their abilities to bind to type I collagen were assessed. Among the mutants, only G272D mutant enzyme exhibited a significant change in type I collagenolysis. The alteration of the Gly(272) to Asp reduced the collagenolytic activity of the enzyme to 13% without affecting its general proteolytic activity, substrate specificity, or the collagen binding ability. The catalytic efficiency of the G272D mutant for the triple helical peptide substrate [C(6)-(GP- Hyp)(4)GPL(Mca)GPQGLRGQL(DPN)GVR(GP-HYP)(4)-NH(2)](3) and the peptide substrate Mca-PLGL(Dpa)AR-NH(2) and its dissociation constant for the triple helical collagen were similar to that of the wild type enzyme, indicating that the presence of this residue in fibroblast collagenase is particularly important for the efficient cleavage of type I collagen. Gly(272) is evidently responsible for the hinge-bending motion that is essential for allowing the COOH-terminal domain to present the collagen to the active site.
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PMID:Unexpected crucial role of residue 272 in substrate specificity of fibroblast collagenase. 1201 Oct 42

Regulation of blood glucose is a fundamental homeostasis in the body. Insulin is released from pancreatic beta-cells in response to changes in blood glucose, the defect of which leads to impaired insulin secretion and diabetes mellitus. Pancreatic beta-cells that release insulin occupy approximately 70% of the islet cells, while alpha, delta, PP-cells are also present in islets. Therefore, analysis of beta-cells is a direct approach for studying mechanisms of insulin secretion. Insulin secretion is regulated by cytosolic Ca(2+) in beta-cells, and its concentration and localization can be measured in real time by fluorescence imaging using indicators. Glucose metabolism is assessed by measurements of NAD(P)H by its autofluorescence. Furthermore, beta-cells are equipped with ion channels, which transduce glucose-evoked metabolic signals to electric signals. Electrophysiological analysis by patch clamp techniques detects the activity of various ion channels and membrane potential. Several beta-cell lines including HIT, MIN6, INS1, RIN, and beta TC are used; however, they do not necessarily retain normal responsiveness to glucose. Therefore, analysis of physiologic functions of beta-cells requires the use of acutely isolated or primary cultured beta-cells from normal animals. The methods for preparation of islets and single beta-cells using collagenase can be applied to a variety of animals of small to large sizes, which can produce islets and beta-cells with physiological responsiveness to glucose. These primary cultured beta-cells can be used for elucidating signal transduction mechanisms, and evaluating effects of drugs, providing excellent tools for physiological, pharmacological, and disease-oriented studies.
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PMID:[Methods for preparation and functional analysis of islet beta-cells]. 1550

Reactive oxygen species (ROS) are regulators of redox-sensitive cell signaling pathways. In osteoarthritis, human interleukin-1beta is implicated in cartilage destruction through an ROS-dependent matrix metalloproteinase production. To determine the molecular source of ROS production in the human IL-1beta (hIL-1beta)-sensitive chondrocyte immortalized cell line C-20/A4, transfected cells were constructed that overexpress NAD(P)H oxidases. First, RT-PCR analysis showed that the C-20/A4 cell line expressed Nox2, Nox4, p22( phox ), and p67( phox ), but not p47( phox ). It was found that ROS production by C-20/A4 chondrocytes does not depend on PMA and ionomycin activation. This indicates that Nox2 was not involved in the production of ROS. In C- 20/A4 cells that overexpress Nox4, hIL-1beta stimulated ROS production three times more than the normal production of C-20/A4 cells. Moreover, there was a fourfold increase in the production of collagenase (MMP-1) by chondrocytes that overexpress Nox4. Interestingly, MMP-1 production in cells that overexpress Nox2 was not sensitive to hIL-1beta. These data suggest that under hIL-1beta stimulation, C-20/A4 chondrocytes produce MMP-1 through a Nox4-mediated, ROS-dependent pathway.
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PMID:NAD(P)H oxidase activity of Nox4 in chondrocytes is both inducible and involved in collagenase expression. 1698 5

Ciliary neurotrophic factor (CNTF) belongs to the cytokine family and increases neuron differentiation and/or survival. Pancreatic islets are richly innervated and express receptors for nerve growth factors (NGFs) and may undergo neurotypic responses. CNTF is found in pancreatic islets and exerts paracrine effects in neighboring cells. The aim of this study was to investigate possible effects of CNTF on neonatal rat pancreatic islet differentiation and/or survival. For this purpose, we isolated pancreatic islets from neonatal rats (1-2 days old) by the collagenase method and cultured for 3 days in RPMI medium with (CNTF) or without (CTL) 1 nM CNTF. Thereafter, glucose-stimulated insulin secretion (RIA), general metabolism by (NAD(P)H production; MTS), glucose metabolism ((14)CO(2) production), gene (RT-PCR), protein expression (western blotting), caspase-3 activity (Asp-Glu-Val-Asp (DEVD)), and apoptosis (DNA fragmentation) were analyzed. Our results showed that CNTF-treated islets demonstrated reduced glucose-induced insulin secretion. CNTF treatment did not affect glucose metabolism, as well as the expression of mRNAs and proteins that are crucial for the secretory process. Conversely, CNTF significantly increased mRNA and protein levels related to cell survival, such as Cx36, PAX4, and BCL-2, reduced caspase-3 activity, and islet cells apoptosis, suggesting that CNTF does not affect islet cell differentiation and, instead, acts as a survival factor reducing apoptosis by increasing the expression of the anti-apoptotic BCL-2 protein and decreasing caspase-3 activity.
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PMID:Ciliary neurotrophic factor promotes survival of neonatal rat islets via the BCL-2 anti-apoptotic pathway. 1791 7


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