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)

Insulin-like growth factor binding protein-3 (IGFBP-3) is degraded by a cation-dependent protease(s) present in the serum of late gestation rats. Proteolysis of IGFBP-3 results in an increase in IGF-I clearance and possibly in IGF bioavailability. Based on our previous findings that matrix metalloproteinases (MMPs) degrade IGFBP-3 in fibroblast conditioned media, we hypothesized that MMPs might be involved in the degradation of IGFBP-3 by rat pregnancy serum. In the present study, we demonstrate that tissue inhibitor of metalloproteinases (TIMP-1), a specific inhibitor of all MMPs, inhibited significantly the degradation of 125I-rhIGFBP-3 by both rat pregnancy serum and rat placental extracts. Purified human MMPs (principally MMP-1 and MMP-3) degraded IGFBP-3 in solution; MMP-3 produced a pattern of IGFBP-3 degradation products identical in size to the fragments produced by pregnancy serum. Furthermore, the combined addition of antihuman MMP-1 IgG and anti-human MMP-3 IgG to rat pregnancy serum blocked almost completely the degradation of 125I-rhIGFBP-3, suggesting that these two MMPs are the principal MMPs involved in IGFBP-3 degradation in rat pregnancy serum. Together, these data suggest that MMPs function as IGFBP-3-degrading proteases in the serum of late gestational pregnant rats.
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PMID:Proteolysis of insulin-like growth factor binding protein-3 during rat pregnancy: a role for matrix metalloproteinases. 752 35

A DNA fragment encoding IgG-binding domain B,C (PABC) was separated from protein A gene, cloned into phage M13 and modified by oligo-directed mutagenesis at the hydroxylamine-cleaved site from Asn-Gly to Asn-Ala in domain B and C, respectively. The modified PABCm gene fragment was used to construct one set of fusion expression vectors in different reading frames. Processing sequences such as those recognized by enterkinase, collagenase, thrombin, activated factor X and cleaved by the hydroxylamine, N-chlorosuccinimide etc. can be created in the fusion site. Using the above vectors, fusion proteins such as PABCm-IGF-I, -hGRF, -bGRF and their derivatives were highly expressed in E. coli. The yield of fusion proteins is over 100 mg per liter cultured by analysis of SDS-PAGE. The PABC fusion proteins can be rapidly purified by the affinity chromatography with a IgG-sepharose column.
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PMID:Fusion expression vectors for recombinant gene products processed easily and purified rapidly by affinity chromatography. 789 35

The effects of 17 beta-estradiol on the proliferation and differentiation of cultured normal human bone marrow stromal cells were investigated. Treatment of 17 beta-estradiol at the concentration of 10(-6) approximately 10(-10) M for either 48 hours or 7 days did not affect [3H] thymidine incorporation. 17 beta-estradiol (10(-8)M) treatment for 4 or 7 days also failed to stimulate alkaline phosphatase activity. Similarly, incubation with 17 beta-estradiol (10(-8) M) for 48 hours did not increase the incorporation of [3H] proline into collagenase digestible protein and noncollagen proteins and secretion of IGF-I and IGF binding proteins in human bone marrow stromal cells. Present data indicate that 17 beta-estradiol does not have a direct effect on cultured normal human bone marrow stromal cells. With previous findings that estradiol elicits few effects on normal human osteoblasts, our results strongly suggest that estrogen does not have a direct anabolic effect on normal human osteoblast lineage. Therefore, the in vivo estrogen effects may be entirely through an antiresorptive mechanism or, if any anabolic role of estrogen is present, it must be indirect and mediated by other hormones or local factors.
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PMID:Absence of a direct anabolic effect of 17 beta-estradiol on normal human bone marrow stromal cells. 803 42

We report the development and characterization of a system of primary culture of ovine fetal hepatocytes to aid in the understanding of the cellular regulation of fetal growth and metabolism with emphasis on amino acid metabolism and insulinlike growth factor gene expression and to allow comparison to in vivo studies. Hepatocytes were isolated from late gestation fetal lambs by in situ perfusion and collagenase digestion utilizing occlusion of the ductus venosus to limit intrahepatic shunting. Hepatocytes were cultured in media modified to mimic fetal concentrations of glucose, lactate, and amino acids. Ovine fetal hepatocytes in primary culture maintain the pattern of fetal amino acid production and utilization seen across the fetal liver in vivo. Specifically, there is a net production of serine and a net utilization of glycine. Cultured ovine fetal hepatocytes specifically increase tritiated thymidine incorporation in response to insulin and insulinlike growth factor II (IGF-II). IGF-II mRNA abundance is high and IGF-I mRNA is low in cultured ovine fetal hepatocytes as in the fetal sheep liver in vivo. These data demonstrate the successful isolation of ovine fetal hepatocytes that retain some of the characteristics of the ovine fetal liver while maintained in short-term culture.
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PMID:Development of primary culture of ovine fetal hepatocytes for studies of amino acid metabolism and insulinlike growth factors. 835 68

Insulin-like growth factors I and II are peptides with a structural homology for proinsulin, and are involved in hepatocyte proliferation. IGF-I and IGF-II, however, have different metabolic roles, and their mechanisms of action are incompletely known. We hypothesized that IGF-I and IGF-II act by different signal transduction pathways. To test this hypothesis, hepatocytes from 200 g male Sprague-Dawley rats were isolated by a two-step collagenase perfusion technique and plated at a density of 10(5) cells/16 mm Primaria plate. Proliferation was measured by [3H]thymidine ([3H]thy) incorporation into DNA, and an autoradiographic nuclear labeling index (LI). To analyze signal transduction, cyclic AMP (cAMP) levels were measured 5 min after addition of reagents by a radioimmunoassay. Reagents (doses) used were: IGF-I (2 nM), IGF-II (2 nM), the inhibitory peptide somatostatin-14 (SS14) (10 nM), and the adenylyl cyclase antagonist dideoxyadenosine (DDA) (10 microM). A summary of the findings is as follows: (1) IGF-I stimulates [3H]thy, LI and cAMP accumulation. (2) IGF-II stimulates [3H]thy and LI but not cAMP; (3) IGF-I but not IGF-II effects are inhibited by SS14 and DDA. We conclude that the hepatotrophic effects of IGF-I and IGF-II occur by different mechanisms: IGF-I is cAMP-dependent, IGF-II is cAMP-independent.
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PMID:Divergent mechanisms of insulin-like growth factor I and II on rat hepatocyte proliferation. 857 Aug 60

In the pig, the corpus luteum (CL) can develop and function autonomous of pituitary gonadotropins for approximately 12 days. We hypothesized that the insulin-like growth factor (IGF) system may play an autocrine/paracrine luteotrophic role(s) during this period. In this study, we monitored the expression (i.e., steady-state levels of mRNAs) of IGF-I and IGF binding proteins (IGFBP)-2, -3, -4, -5, and -6 mRNAs in whole CL and in small and large luteal cells on Days 4-16 of the estrous cycle. CL were dissociated with collagenase, and large and small luteal cells were isolated by centrifugal elutriation. Whole CL and luteal cells were extracted to isolate total or poly(A)+ RNA, which was subjected to Northern and/or dot-blot analyses using [32P]-labeled cDNA probes for IGF-I and IGFBP-2, -3, -4, -5, and -6. Northern blots showed readily detectable transcripts for IGF-I (6.7 and 0.9 kb), IGFBP-2 (1.8 kb), IGFBP-3 (2.8 kb), IGFBP-4 (2.6 kb), and IGFBP-5 (6.0 kb), but not for IGFBP-6. IGFBP-3 and -5 transcripts were observed mainly in small luteal cells, while IGFBP-2 and -4 were seen in both cell types. Dot-blot analyses for IGF-I and IGFBP-3 mRNAs were performed on total RNA from small and large luteal cells; blots were counter-probed with 3-phosphoglyceraldehyde dehydrogenase (p-GAD) cDNA to assess RNA quantity and quality. IGF-I mRNA (ratio IGF-I:p-GAD mRNA) expression was approximately 2-fold greater in small than in large luteal cells on Days 4-10. However, steady-state levels of IGF-I mRNA in small, but not large, luteal cells decreased significantly on Days 12-16 (vs. Days 4-10). IGFBP-3 mRNA expression was significantly greater (approximately 3-fold) in small than in large luteal cells but did not vary significantly between Days 4-10 and 12-16 for either cell type. We conclude that porcine CL express mRNAs for IGF-I and IGFBP-2, -3, -4, and -5, and that while small luteal cells are the major sources of IGF-I and IGFBP-3 and -5, IGFBP-2 and -4 appear to be expressed to approximately the same extent in small and large luteal cells. These results further suggest that the IGF-I/IGF system may have autocrine/paracrine regulatory actions in CL development/function in the pig.
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PMID:Expression of the messenger ribonucleic acids for insulin-like growth factor-I and insulin-like growth factor binding proteins in porcine corpora lutea. 878 84

Insulin and IGF-I receptor binding were characterized in cardiac muscle cells isolated from the brown trout, Salmo trutta fario. Cardiomyocyte suspensions obtained by perfusion of ventricles with collagenase showed a high degree of viability as judged by trypan blue exclusion, LDH leakage, and morphology. Specific insulin binding was 2.88 +/- 0.28%/10 mg cells after overnight incubation at 4 degrees. Scatchard analysis indicated the presence of high affinity insulin binding sites with an apparent dissociation constant (Kd) of 0.285 +/- 0.043 nM and a binding site density of 1. 61 +/- 0.19 x 10(8)/mg cells. Specificity of insulin binding was determined by displacing labeled insulin with increasing concentrations of IGF-I, and the Kd value obtained was 4.77 +/- 2.82 nM, 17-fold higher than Kd values for displacement of insulin tracer by nonlabeled insulin. The percentage of IGF-I specific binding (6.70 +/- 1.42%/10 mg cells), affinity (Kd = 0.163 +/- 0.023 nM), and binding site density (4.00 +/- 1.13 x 10(8)/mg cells) were higher than those of insulin. Displacement curves of labeled IGF-I with nonlabeled insulin (Kd = 33.6 +/- 9.9 nM), indicated a high specificity of the IGF-I binding site. High concentrations of cold insulin and IGF-I were able to decrease markedly the specific binding to their own receptor. Incubation with cold IGF-I also induced a diminution in insulin binding in agreement with the lower specificity of the insulin receptor. These data suggest that insulin and IGF-I are able to down-regulate their own receptor number in cardiac muscle cells. The present results demonstrate that the isolated cardiac myocyte preparation from brown trout is a useful model for studying insulin and IGF-I binding in fish heart tissue.
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PMID:Insulin and IGF-I binding in isolated trout cardiomyocytes. 881 93

Insulin-like growth factors (IGF)-I and -II are presumed to act as autocrine regulators of bone formation. Recently, we demonstrated that IGF-I and -II inhibit bone collagen degradation and collagenase-3 synthesis in osteoblast cultures. Therefore, we tested the autocrine role of IGFs in the endogenous expression of collagenase-3 in cultures of osteoblast-enriched cells from 22-day fetal rat calvariae (Ob cells). Steady-state messenger RNA (mRNA) levels were determined by Northern blot analysis and collagenase concentrations in the culture medium were determined by Western immunoblot. Basal level collagenase-3 transcripts decreased in Ob cell cultures, coinciding with an increase in IGF-I and -II protein levels. Removal of the conditioned medium modestly increased collagenase-3 mRNA levels and restored the ability of exogenously added IGF-I to repress collagenase-3 transcripts. IGF neutralizing antibodies and IGF binding proteins-2 and -3 in excess increased and sustained collagenase mRNA, heterogeneous nuclear RNA, and protease levels in Ob cell cultures. In conclusion, IGF-I and -II are autocrine repressors of collagenase-3 synthesis, and this effect may contribute to their actions on the maintenance of a normal bone collagen matrix.
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PMID:Autocrine down-regulation of collagenase-3 in rat bone cell cultures by insulin-like growth factors. 889 31

We examined and compared the actions of IGF-I and -II on the release of insulin from isolated, intact rat islets of Langerhans within a perifusion system. Islets were isolated from adult male rats by collagenase digestion and Ficoll gradient separation, and were maintained in tissue culture for 48 h before perifusion. Following an equlibration period, islets were perifused with medium containing 2.7 mM glucose from 0 to 30 min. and 2.7, 11.1 or 16.7 mM glucose from 30 to 90 min. All chambers then received medium with 2.7 mM glucose from 90 to 120 min. Various doses (6.7-53 nM) of IGF-1, des(1-3) IGF-I or IGF-II were given either as a pulse between 30 and 35 min, or continuously from 30 to 90 min. Insulin was measured in effluent medium by RIA. When 11.1 mM glucose was administered after 30 min an immediate increase in insulin release occurred, from a baseline of 1-3 pmol/fraction to approximately 7 pmol/ fraction. The elevated rate of release was maintained until 90 min, and fell when the glucose concentration was lowered. Glucose at 16.7 mM was a less effective insulin secretogogue than was 11.1 mM. When islets received a pulse infusion of IGF-I (13.3 nM) at 30 min in the presence of 11.1 mM glucose, a statistically significant increase (p < 0.005) in insulin release occurred, of approximately 10 pmol/fraction in excess of that seen with glucose alone. The IGF-1-stimulated insulin release was still higher than controls at 115 min. When the concentration of IGF-I was altered between 6.7 nM and 53 nM, maximum insulin release was achieved with 13.3 nM IGF I, both lower and higher concentrations being less effective. A significant inhibition of insulin release occurred with 53 nM IGF-I compared with glucose alone. IGF-II (13.3 nM) did not significantly increase insulin release, while 53 nM IGF-II significantly inhibited release of insulin relative to controls. Des(1-3) IGF-I (13.3 nM), which has a reduced binding affinity for IGF-binding proteins (IGFBPs), administered with 11.1 mM glucose caused an immediate increase in insulin release, which fell to control values within 30 min. Western ligand blot analysis identified four IGFBP species in perifused islets, of 46 kDa, 35 kDa, 28 kDa, and 19 kDa respectively, of which the 28 kDa species was identified immunologically as IGFBP-1. When IGF-I was administered continuously from 30 to 90 min it inhibited glucose-stimulated insulin release at all concentrations used. The results suggest that under perfusion conditions, IGF-I can act both as a potent insulin secretogogue, augmenting the actions of glucose, and as an inhibitor of insulin release, depending on concentration and kinetics of administration.
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PMID:IGF-I has a dual effect on insulin release from isolated, perifused adult rat islets of Langerhans. 913 65

Knee laxity has been shown to increase during human pregnancy, and the laxity of the rabbit medial collateral ligament also increases during pregnancy. To determine whether the changes in tissue function could be related to alterations in the regulation of gene expression for a subset of relevant molecules in ligaments, RNA was isolated from the medial collateral(MCL) and anterior cruciate(ACL) ligaments of first time pregnant adolescent rabbits. Levels of mRNA for matrix molecules (collagen types I and III and the proteoglycans biglycan, decorin, versican and lumican), proteinases and inhibitors (collagenase, urokinase, PAI-1 and TIMP-1, -2 and -3), growth factors (bFGF, IGF-I, TGF-beta1 and ET-1), cytokines (IL-1beta and TNF) and enzymes responsible for important tissue mediators (COX-2 and iNOS) were assessed by semi-quantitative RT-PCR. In the MCL, levels of transcripts for all of the matrix molecules, growth factors and TIMPs 1 and 2 were significantly depressed at 29 days of pregnancy compared to age-matched non-pregnant controls. In contrast, transcripts for PAI-1 were elevated during pregnancy, while those for collagenase (MMP-1), urokinase, TIMP-3, IL-1beta, TNF, COX-2 and iNOS were not statistically altered. mRNA transcript levels rebounded by 7 days post-partum for most genes studied, indicating that the changes were rapidly reversible. For some molecules, transcript levels were again depressed at 18 days post-partum, indicating that regulatory mechanisms were still not stabilized. Analysis of mRNA from the ACL also revealed changes in the pattern of gene expression, with some similarities and differences from the MCL noted. These results indicate that pregnancy induces reversible changes in mRNA for matrix molecules in ligaments, but differences in responsiveness exist between different ligaments. The complexity of the changes observed indicates that there is probably no simple cause and effect relationship between laxity changes and the molecular alterations during pregnancy.
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PMID:Pregnancy induces complex changes in the the pattern of mRNA expression in knee ligaments of the adolescent rabbit. 962 50


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