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 factors (IGF-I and IGF-II) are endocrine and autocrine factors affecting bone growth and metabolism. Binding proteins for IGFs (IGFBPs) are synthesized by the target tissues of IGF actions. Thus, IGFBPs may act as modulators for the biological functions of IGFs. We have characterized the rat IGFBPs (rIGFBPs) and studied their regulation by 17 beta-estradiol (beta E2) and human GH (hGH) in rat osteoblast-like (ROB) cell cultures. ROB cells were prepared from 19- to 20-day fetal rat calvariae by sequential collagenase digestion and studies were performed on the serum and phenol red-free conditioned medium of confluent cultures. [125I]IGF-I ligand blots showed that the major rIGFBP in the ROB is a nonglycosylated protein of 31 kilodaltons. This protein was immunoprecipitated by a specific antibody to rIGFBP-2 and messenger RNA for rIGFBP-2 was detected by RNA hybridization indicating that the rIGFBP-2 is the major rIGFBP of ROB. A minor band at 24 kilodaltons is likely to be the rat homologue of the newly isolated inhibitory IGFBP-4. The predominant glycosylated adult form of rIGFBPs of rat serum, rIGFBP-3, was undetectable. When cultures were treated with beta E2 for 2 days, there was a dose-dependent biphasic response which showed an inhibition of the rIGFBP-2 at low doses of 10(-11) to 10(-9) M and a stimulation at 10(-6) M. These changes in rIGFBP-2 parallel the changes in the endogenous IGF-I level. rIGFBP-2 level was not affected by 17 alpha-estradiol at the same concentration range. hGH, on the other hand stimulated the levels of rIGFBP-2 and rIGFBP-4 at doses ranging from 10(-11) to 10(-9) M without changing the IGF-I secretion. The alteration of the rIGFBPs by beta E2 and hGH suggests a role for these hormones in bone by modulating the biological functions of IGFs via their binding proteins.
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PMID:Further characterization of insulin-like-growth factor binding proteins in rat osteoblast-like cell cultures: modulation by 17 beta-estradiol and human growth hormone. 170 37

Insulin and insulin-like-growth-factor-I (IGF-I) receptors were partially purified from full-grown (stages V-VI) Xenopus laevis oocytes by affinity chromatography on wheat-germ agglutinin-agarose. Competitive-binding assays revealed high-affinity binding sites for both insulin and IGF-I (Kd = 2.5 x 10(-10) M and 8 x 10(-10) M respectively). However, IGF-I receptors were about 15 times more abundant than insulin receptors (22.5 x 10(11) versus 1.5 x 10(11)/mg of protein). Moreover, comparison of intact and collagenase-treated oocytes showed that most of the insulin receptors were in the oocyte envelopes, whereas IGF-I receptors were essentially at the oocyte surface. Oocyte receptors were composed of alpha-subunits of approximately 130 kDa and a doublet of beta-subunits of 95 and 105 kDa, which both had ligand-induced phosphorylation patterns compatible with IGF-I receptor beta-subunits. Accordingly, the receptor tyrosine kinase was stimulated at low IGF-I concentrations [half-maximally effective concentration (EC50) approximately 0.5-1 nM], and at higher insulin concentrations (EC50 approximately 20-50 nM). Partially purified glycoproteins from Xenopus liver and muscle contained mainly receptors of the insulin-receptor type, with alpha-subunits of 140 kDa in liver and 125 kDa in muscle, and doublets of beta-subunits of 92-98 kDa in liver and 85-94 kDa in muscle. Immunoprecipitation of receptors from oocytes, liver and muscle by receptor-specific anti-peptide antibodies suggested that the beta-subunit heterogeneity resulted from the existence of two distinct IGF-I receptors in oocytes and of two distinct insulin receptors in both liver and muscle. In the different tissues, the two receptor subtypes differed at least by their beta-subunit C-terminal region.
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PMID:Insulin and insulin-like-growth-factor-I (IGF-I) receptors in Xenopus laevis oocytes. Comparison with insulin receptors from liver and muscle. 184 19

1. Insulin and insulin-like growth factor I (IGF-I) receptors were studied in bovine chromaffin cells isolated from the medulla by collagenase digestion and kept in primary culture. 2. Specific 125I-labelled insulin binding increased with time in culture with no significant change in the dissociation constant, Kd approximately 0.3 nM. Insulin was nearly 100-fold more potent than IGF-I in displacing 125I-labelled insulin. 3. Affinity crosslinking and SDS gel electrophoresis revealed increased binding of 125I-labelled insulin and 125I-IGF-I with time in culture, the densities of the labelling indicating relatively a much higher expression of IGF-I than insulin receptors in the cells. The apparent molecular weight of both the hormone binding subunits were 135,000, suggesting that the insulin and IGF-I receptors in the adrenal medulla are of the peripheral types. 4. Both receptors thus appeared to be affected by the collagenase treatment but with a subsequent recovery when cells were kept in culture.
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PMID:Binding of insulin and insulin-like growth factor I in bovine chromaffin cells in primary culture. 185 Jul 3

Adipose tissue is a major, nonglandular site for the aromatization of androgens to estrogens. In this tissue, the aromatase activity resides primarily in the stromal cells, and we have used cultures of stromal cells to study the effects of insulin and insulin-like growth factor I (IGF-I) on aromatase activity. Adipose tissue, obtained during indicated surgery, was digested with collagenase, and the stromal cells were isolated and cultured. Aromatase activity was determined by measuring the tritiated water (3H2O) in the medium after incubating stromal cells with [1 beta-3H]androstenedione. Insulin and IGF-I had no effect on the aromatase activity in cultured adipose stromal cells at concentrations of 10 to 1,000 microU/ml. However, insulin (100 to 1,000 microU/ml) and IGF-I (500 ng/ml) markedly attenuated the stimulatory effect of (Bu)2cAMP, but significantly augmented the dexamethasone-stimulated aromatase activity. The greater effects of IGF-I compared with the effect of insulin are compatible with both effects being mediated through the IGF-I compared with the effect of insulin are compatible with both effects being mediated through the IGF-I receptor. In addition, the effects of insulin in attenuating the aromatase activity in adipose tissue could potentiate its role in hyperandrogenic syndromes in women.
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PMID:Aromatase activity in human adipose tissue stromal cells: effect of growth factors. 196 38

The purpose of the present study was to test the influence of donor age on islet isolation yield in swines. Large White pigs of 10 months (group 1, n = 10) and 2-3 years (group 2, n = 5) were used. A modification of the automated method for human islet isolation was used for pig islet separation. After ductal injection of a collagenase solution (Seromed, type C-1000) the islets were separated through a continuous digestion process. In group 1 it was possible to obtain an average of 636,100 islets (volume = 476.1 mm3, insulin content = 101.4 Units). In group 2 the average total islet number was 799,800 representing a volume of 1190.6 mm3 (Insulin content 210 Units). Total islet number was not significantly different in the two groups, however when islet volume and total insulin content were considered, older animals allowed a significantly higher yield (P less than 0.01 for volume and P less than 0.05 for insulin content). These findings indicate that it is possible to separate large number of pig islets from both young (10 months) and older (2-3 years) animals and that selection of donor pigs will improve islet isolation yield. These results may be of assistance in the definition of requirements for more effective islet isolation in swines.
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PMID:Selection of donors significantly improves pig islet isolation yield. 208 80

Effects of transforming growth factor (TGF)-beta, epidermal growth factor (EGF), insulin, 1, 25-dihydroxyvitamin D3 (1, 25 (OH)2D3), and parathyroid hormone (PTH) on the proliferation and differentiation of clonal dental pulp cells of rats were investigated. Interaction between growth factors (TGF-beta and EGF) and two hormones insulin and 1, 25 (OH)2D3, which have been noticed to accelerate the differentiation of the cells, were also studied, and the following results were obtained: 1) TGF-beta decreased alkaline phosphatase (ALPase) activity in a dose-dependent manner, and the inhibitory effect was not blocked by indomethacin, suggesting that the effect of TGF-beta on the cells may not be mediated by prostaglandins. Inhibitory effects of ALPase antagonists (L-phenylalanine, L-homoarginine, levamisole) on the activity were not affected by TGF-beta. TGF-beta showed no evident effect on the DNA synthesis (incorporation of [3H] thymidine) and collagen synthesis (incorporation of [2, 3-3H] proline into the collagenase-digestible protein) of the cells. 2) EGF stimulated the incorporation of [3H] thymidine and inhibited the ALPase activity. The inhibitory effect was not blocked by indomethacin, indicating that the EGF effect is not mediated by prostaglandins. Collagen synthesis was significantly inhibited by EGF. 3) Insulin showed a weak but significant inhibition of the DNA synthesis. Insulin increased the ALPase activity evidently, and accelerated the collagen synthesis significantly. 4) The vitamin 1, 25 (OH)2D3 significantly increased the ALPase activity though no significant changes were observed in the DNA synthesis and collagen synthesis. 5) PTH had no evident effect on the DNA synthesis and ALPase activity, but did tend to accelerate the collagen synthesis. 6) A study on the interaction between insulin and EGF or TGF-beta revealed that the acceleration of DNA synthesis induced by EGF was inhibited when the factor was combined with insulin, and the increase in ALPase activity elicited by insulin was inhibited by EGF and weakened by TGF-beta significantly when these factors were added simultaneously with the insulin. Or viewed another way, the inhibitory effect of EGF or TGF-beta on the ALPase activity was antagonized by insulin. The accelerative action of insulin on collagen synthesis was antagonized by EGF and potentiated by TGF-beta. 7) A study on the interaction between 1, 25 (OH)2D3 and EGF or TGF-beta revealed that 1, 25 (OH)2D3 inhibited the accelerating effect of EGF on the DNA synthesis and that the increasing effect of 1, 25 (OH)2D3 on ALPase activity was strongly inhibited by EGF.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:[Effects of various growth factors and hormones on clonal rat pulp cells]. 213 79

High yields of human hepatocytes (up to 23 X 10(6) viable cells/g) were obtained from small surgical liver biopsies (1 to 3 g) by a two-step collagenase microperfusion method. Cell viability was about 95%, attachment efficiency of hepatocytes seeded on fibronectin-coated plates was 80% within 1 h after plating, and cells survived for about 2 wk in serum-free Ham's F12 containing 0.2% bovine serum albumin, 10(-8) M insulin, and 10(-8) M dexamethasone. To evaluate the metabolism of human hepatocytes in serum-free conditions, we measured their most characteristic biochemical functions and compared them to those reported for human liver. After 24 h in culture, glycogen content was 1250 +/- 177 nmol glucose/mg cell protein and remained stable for several days. Gluconeogenesis from lactate in hormone-free media was (3.50 +/- 0.17 nmol glucose.mg-1.min-1) similar to that reported for human liver. Insulin at 10(-8) M activated glycolysis (X1.40) and glycogenesis (X1.34), and glucagon at 10(-9) M stimulated gluconeogenesis (X1.35) and glycogenolysis (X2.18). Human hepatocytes synthesized albumin, transferrin, fibrinogen, alpha 1-antitrypsin, alpha 1-antichymotrypsin, alpha 1-acid glycoprotein, haptoglobin, alpha 2-macroglobulin, and plasma fibronectin and excreted them to the culture medium. Maximum protein synthesis was stimulated by 10(-9) M dexamethasone. Basal urea synthesis oscillated between 2.5 and 3.5 nmol.mg-1 cell protein.min-1, about 5 times the value estimated for human liver. Cytochrome P-450 decreased in culture but it was still 20% of freshly isolated hepatocytes by Day 5 in culture. In addition, ethoxycumarin-O-deethylase and aryl hydrocarbon hydroxylase could be induced in vitro by treatment with methyl cholanthrene. Glutathione levels were similar to those reported for human liver (35 nmol.mg-1). The results of our work show that adult human hepatocytes obtained from small surgical biopsies and cultured in chemically defined conditions express their most important metabolic functions to an extent that is similar to that reported for adult human liver.
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PMID:Culture of human hepatocytes from small surgical liver biopsies. Biochemical characterization and comparison with in vivo. 215 94

Insulin acts directly as a mitogen on isolated embryonic tissues but has been shown to be inactive at physiological concentrations on several fetal cell types. Since insulin availability is obligatory for optimal fetal growth, we have investigated its mitogenic actions on chondrocytes isolated from the epiphyseal growth plates of fetal lambs. Chondrocytes were isolated from the proximal tibial growth plate of lamb fetuses between 40 and 130 days gestation using collagenase and were cultured in monolayer before use between passages 2 and 6. The synthesis of DNA was assessed from the incorporation of [3H]thymidine after incubation in medium supplemented with glucose (0.7 mM-25 mM) with or without insulin (0.08 nM-167 nM). Increase in cell number was assessed after incubation with test medium for up to 8 days. Insulin substantially increased both DNA synthesis, and cell number, compared to control incubations with a biphasic dose response; an initial 3- to 5-fold increase in DNA synthesis occurring at approximately 1 nM insulin with a second response seen at approximately 50 nM. Within the physiological range of concentrations insulin was only 50% as active as insulin-like growth factor I (IGF I), but was 15 times more active than IGF II. Similar effects of insulin were observed throughout the fetal age range, although the DNA synthetic rate in basal medium declined with both fetal age and cell passage number. The mitogenic actions of insulin were glucose-dependent and were maximal in the presence of 2.7 mM glucose. Insulin did not cause any change in chondrocyte cell cycle duration. Chondrocytes released immunoreactive IGF II but no detectable IGF I. While exposure to insulin concentrations of approximately 50 nM or greater resulted in a statistically significant increase in IGF II release from chondrocytes, no change in IGF II release was seen in response to physiological insulin concentrations. However, exposure of cells to a blocking monoclonal antibody against human IGF I, McAb SM 1.2, which also negates the mitogenic actions of IGF II, consistently reduced insulin-stimulated DNA synthesis suggesting that IGF II presence may be necessary for optimal insulin action. Combination experiments using maximal concentrations of IGF I (13.3 nM) and increasing amounts of insulin (0.16 nM-1.67 nM) showed additive effects on DNA synthesis, suggesting that each hormone was acting through distinct receptor populations. We conclude that insulin, at physiological concentrations, may exert direct growth-promoting actions at the epiphyseal growth plates of the fetal lamb throughout gestation.
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PMID:Insulin is a mitogen for isolated epiphyseal growth plate chondrocytes from the fetal lamb. 218 20

Insulin secretion in response to glucose, glucose-stimulated insulin biosynthesis and insulin content was studied in pancreatic islets freshly isolated from male Wistar rats (150-200 g) with galactosamine-induced hepatitis. Animals were sacrificed by decapitation 3, 6, 12 and 24 hours after a single intraperitoneal injection of 500 mg/kg of galactosamine. Isolated islets prepared by collagenase method were perifused in Swim's medium with 20 mM glucose at 37 degrees C up to 30 minutes. Samples were taken at 2-10 min intervals for insulin assay. Insulin biosynthesis was assessed by the incorporation of [3H]-leucine into immunoprecipitable products (insulin and proinsulin) in pancreatic islets after 120 min incubation with 20 mM glucose. Glucose-stimulated insulin secretion was significantly increased at 6, 12 and 24 hours following the administration of galactosamine compared to control. The rate of insulin biosynthesis was stimulated to 170, 138 and 185% of control level 3, 6 and 12 hours after galactosamine-treatment, respectively. Significant increase in insulin content of islets was found 24 hours after galactosamine treatment, following the increased insulin biosynthesis. The present results indicate that pancreatic B cell function is activated in early stage of acute liver injury.
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PMID:Increase in glucose-stimulated insulin release and insulin biosynthesis in isolated pancreatic islets from D-galactosamine-treated rats. 219 63

We recently described autoantibodies that stimulate the release of insulin from pancreatic beta-cells both in vitro and in vivo. The aim of this study was to establish whether islet cell-stimulating antibodies (ICSTAs) also increase islet cell preproinsulin mRNA content. Wistar rat islets, isolated by collagenase digestion, were exposed to 2.7 and 11.1 mM glucose. Insulin release increased 10-fold in response to the higher glucose concentration, and dot-blot analysis of islet mRNA with a rat preproinsulin cDNA probe showed a concomitant increase in mRNA levels. The globulin fractions of four test serums, three from patients with type I (insulin-dependent) diabetes and one from a patient with the insulin autoimmune syndrome, showed clear (5- to 8-fold) stimulation of insulin release. The nonglobulin fractions of these serums and both fractions of three control serums failed to stimulate secretion of insulin. The insulin mRNA content of islets incubated with the ICSTA globulin fractions was greatly increased compared with levels observed in islets treated with control serum globulin fractions. We conclude that ICSTAs not only can stimulate the release of insulin but also increase the preproinsulin mRNA content of islet cells.
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PMID:Increased preproinsulin mRNA in pancreatic islets incubated with islet cell-stimulating antibodies from serums of type I diabetic patients. 221 69


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