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)

The kidney consists of numerous functional units called nephrons. Thus, the use of individual nephron segments is essential to characterize their functional properties and to clarify the molecular basis of site-specific functions. Nephron segments can be microdissected from collagenase-treated renal slices under a stereomicroscope. A variety of intracellular ionic concentrations or membrane potential can be determined with various fluorescent probes. Fura-2/AM-loaded nephron segments reveal a transient increase of cytosolic free calcium concentrations by agonists such as angiotensin II, vasopressin, kinins, etc. To localize their receptors or to characterize their subtypes, this technique is especially beneficial, because tiny fragments of the nephron are sufficient by combination with a two-wave length microscope fluorometer. As an example, discovery of a novel vasopressin receptor (Vp) is described.
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PMID:[Usefulness of microdissection of nephron segments and fluorescent indicator for molecular biological studies of nephron functions]. 149 43

Whereas mesangial and epithelial cells from glomeruli are commonly grown in vitro, there has been a failure to isolate and propagate human glomerular capillary endothelial cells. This study defines the conditions for the reproducible isolation and growth of homogeneous monolayers of primate (baboon and human) glomerular capillary endothelial cells. Using selective media and growth factors, the following criteria were identified to optimize the isolation and proliferation of glomerular endothelial cells: (1) collagenase treatment of isolated glomeruli; (2) requirement for 20% serum, endothelial cell growth factor and heparin; (3) requirement of fibronectin as surface matrix; and (4) isolation from donors less than 60 years old, as premature senescence was directly proportional to the age of the human kidney donor. Under these conditions, primary cultures with an endothelial cell composition greater than 70% were reproducibly obtained. Homogeneous endothelial monolayers were developed from 20 of 23 human kidneys, and maintained for 5 to 10 passages, depending on the age of the kidney donor. Purification to homogeneity was achieved by patch cloning or by fluorescence-activated cell sorting. Glomerular capillary endothelial cells exhibited a cobblestone morphology at confluence, expressed factor VIII-related antigen, angiotensin converting enzyme activity, and endocytosed acetylated low-density lipoproteins. Electron microscopy revealed the presence of intracellular Weibel-Palade bodies and caveolae and microvillous projections on the luminal surface. Glomerular cells also stained positive for Ulex europaeus, a lectin characteristic of human endothelial cells. In addition, preliminary results indicate that human glomerular endothelial cells increase intracellular cyGMP in response to alpha-human 5 to 28 atrial natriuretic peptide and intracellular free calcium in response to thrombin.
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PMID:Culture of endothelial cells from baboon and human glomeruli. 150 7

A number of cells, chemotactic factors, and inflammatory mediators are implicated in the complex mechanisms underlying crystal-mediated inflammation. Interleukin-8, released from mononuclear cells that have been exposed to urate and other crystals, is a potent chemotaxin and activator of neutrophils. Experimental and clinical observations suggest that joint movements, local biomechanical factors, and previous joint damage may play a role in influencing the intensity of microcrystalline synovitis and the distribution of articular and periarticular crystal deposits in both calcium pyrophosphate dihydrate crystal deposition disease and gout. There are rare reports of extra-articular calcium pyrophosphate dihydrate crystal deposition in tendons, bursae, dura mater, and ligamentum flavum (with radiculomyelopathy) and of massive "tumoral," tophuslike, periarticular calcium pyrophosphate dihydrate crystal deposits. Synovial fluid levels of ATP, the main substrate for nucleoside triphosphate pyrophosphohydrolase ectoenzyme, which cleaves ATP-releasing inorganic pyrophosphate, are higher in patients with calcium pyrophosphate dihydrate crystal deposition disease than in those with other arthritides, and the levels correlate with inorganic pyrophosphate concentrations. Further reports of acute calcific periarthritis of the first metatarsophalangeal joint (hydroxyapatite pseudopodagra) in young women have been described. The mitogenic response of fibroblasts to stimulation with basic calcium phosphate crystals is accompanied by induction and secretion of collagenase and neutral proteases, implicating a role for the crystals in the pathogenesis of both synovial proliferation and joint damage in chronic basic calcium phosphate crystal-associated arthropathy. Subcutaneous cholesterol crystal deposition with tophus formation is extremely rare and has been described in a patient with scleroderma and calcinosis cutis.
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PMID:Calcium pyrophosphate crystal deposition disease and other crystal deposition diseases. 150 84

We have examined the effects of arachidonic acid (AA) and some of its metabolites on progesterone (P4) and oxytocin (OT) release by corpora lutea obtained from Holstein heifers at day 8 of the estrous cycle (Day 0 = estrus). The luteal cells were dispersed with collagenase and small and large cells were separated by unit gravity sedimentation and flow cytometry. After an 18-hr preincubation period, the cells were incubated in the presence of various treatments for 1 hr, followed by a 23-hr incubation period with no treatment. OT was secreted by the large, but not by the small, luteal cells into the incubation medium. AA elicited a significant (P less than 0.05) release of OT from the large cells and P4 from both the large and small cells within 1 hr of incubation, having a specific effect at a concentration of 10 microM. Larger doses (25 and 100 microM) of AA adversely affected the cell viability. Phospholipases A2 (0.5 unit/ml) and C (0.05 unit/ml) and calcium ionophore A23187 (0.1 microM) stimulated OT release from the large cells to the same extent as AA (10 microM). Inhibition of the AA cyclooxygenase metabolic pathway by indomethacin did not affect AA-induced release of OT and P4, although exogenous prostaglandins F2 alpha and I2 (5-25 ng/ml) stimulated the release of OT. Lipoxygenase products of AA (hydroxyeicosatetraenoic acid and leukotrienes; 25 ng/ml) also stimulated OT release. Inhibition of the lipoxygenase metabolic pathway by nordihydroguaiaretic acid abolished AA-induced release of both OT and P4. These results suggest that intracellular accumulation of free AA may modulate secretory functions in the bovine corpora lutea, including OT and P4 release.
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PMID:Role of arachidonic acid and its metabolites in the regulation of progesterone and oxytocin release from the bovine corpus luteum. 152 4

Dietary copper deficiency has been shown to reduce copper-dependent superoxide dismutase (SOD) activity and to increase lipid peroxidation in rats. Circulating reduced glutathione (GSH) concentrations are elevated in copper-deficient (CuD) rats, which suggests an increased GSH synthesis or decreased degradation, perhaps as an adaptation to the oxidative stress of copper deficiency. GSH synthesis was examined in isolated hepatocytes from CuD rats. Isolated hepatocytes were prepared by collagenase perfusion and incubated in Krebs-Henseleit bicarbonate buffer, pH 7.4, 10 mM glucose, 2.5 mM Ca2+ in the presence and absence of 1.0 mM buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis. Cell viability was assessed by trypan blue exclusion. GSH and oxidized glutathione (GSSG) were measured by the glutathione reductase recycling assay. Copper deficiency depressed hepatocyte Cu by greater than 90% and increased intracellular GSH by 41-117% over the 3-h incubation, with a two- to threefold increase in the rate of intracellular GSH synthesis. Intracellular GSSG values were minimally influenced by CuD, with a constant mol% GSSG. Extracellular total glutathione (GSH + 2GSSG) synthesis was increased by approximately 33%. Both intracellular GSH and extracellular total glutathione synthesis were inhibited by BSO. The pattern of food consumption in CuD rats, meal fed versus ad libitum fed, had no effect on glutathione synthesis. The results indicate an increased hepatic GSH synthesis as a response to dietary copper deficiency and suggest an interrelationship between the essential nutrients involved in oxyradical metabolism.
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PMID:Glutathione production in copper-deficient isolated rat hepatocytes. 155 18

To clarify the pathogenesis of allergic pulmonary diseases by analysis of the reactivity of human lung mast cells, we established a method of dispersion and purification of mast cells from human lung tissue. This method consisted of 4 steps; 1) mincing by scissors, 2) enzymatic treatment by a pronase-chymopapain and collagenase-elastase mixture, 3) percoll centrifugation and 4) exclusion of adherent cells. Using this method, dispersed human lung mast cells were obtained with 38.8% purity and more than 95% viability. These mast cells contained 4.1 pg of histamine per cell, which showed these cells had mild spontaneous histamine release after treatment. The mast cells released histamine in a dose-dependent manner after treatment with calcium ionophore A 23187 and anti-IgE, and these phenomena were dependent on extracellular calcium ions. However, the cells did not release histamine with less than 100 micrograms/ml of compound 48/80. These results indicate that the human lung mast cells obtained by this method are useful to make immunological and pharmacological analyses in allergic lung diseases.
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PMID:[Purification and reactivity of human lung mast cells]. 157 35

Laminin self-assembles in vitro into a polymer by a reversible, entropy-driven and calcium-facilitated process dependent upon the participation of the short arm globular domains. We now find that this polymer is required for the structural integrity of the collagen-free basement membrane of cultured embryonal carcinoma cells (ECC) and for the supramolecular organization and anchorage of laminin in the collagen-rich basement membrane of the Engelbreth-Holm-Swarm tumor (EHS). First, low temperature and EDTA induced the dissolution of ECC basement membranes and released approximately 80% of total laminin from the EHS basement membrane. Second, laminin elastase fragments (E4 and E1') possessing the short arm globules of the B1, B2, and A chains selectively acted as competitive ligands that dissolved ECC basement membranes and displaced laminin from the EHS basement membrane into solution. The fraction of laminin released increased as a function of ligand concentration, approaching the level of the EDTA-reversible pool. The smaller (approximately 20%) residual pool of EHS laminin, in contrast, could only be effectively displaced by E1' and E4 if the collagenous network was first degraded with bacterial collagenase. The supramolecular architecture of freeze-etched and platinum/carbon replicated reconstituted laminin gel polymer, ECC, and collagenase-treated EHS basement membranes were compared and found to be similar, further supporting the biochemical data. We conclude that laminin forms a network independent of that of type IV collagen in basement membranes. Furthermore, in the EHS basement membrane four-fifths of laminin is anchored strictly through noncovalent bonds between laminin monomers while one-fifth is anchored through a combination of these bonds and laminin-collagen bridges.
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PMID:Laminin forms an independent network in basement membranes. 157 69

A gamete lytic enzyme (GLE) of Chlamydomonas reinhardtii is a zinc metalloprotease and mediates digestion of the cell walls of the two mating-type gametes during mating as a necessary prelude to cell fusion. The nucleotide sequence analysis of a cDNA revealed that GLE is synthesized in a preproenzyme form, a 638-amino acid polypeptide (Mr, 69,824) with a 28-amino acid signal peptide, a 155-amino acid propolypeptide, and a 455-amino acid mature polypeptide (Mr, 49,633). A potential site for autocatalytic activation was contained within the propolypeptide and a zinc binding site found within the mature polypeptide; both sites were highly homologous to those in mammalian collagenase. A putative calcium binding site was present in the near C-terminal region of the mature GLE. Both propolypeptide and mature polypeptide had potential sites for asparagine-linked glycosylation, and the Arg-(Pro)3 and Arg-(Pro)2 motifs, which are known to exist in hydroxyproline-rich glycoproteins of the Chlamydomonas cell wall. Northern blot analysis revealed that steady-state levels of the 2.4-kilobase GLE mRNA increased during growth and mitotic cell division in the vegetative cell cycle and also increased markedly during gametogenesis under nitrogen-starved conditions.
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PMID:Primary structure and expression of a gamete lytic enzyme in Chlamydomonas reinhardtii: similarity of functional domains to matrix metalloproteases. 158 6

The binding of GRP (gastrin-releasing peptide) to mouse pancreatic islets was studied. Binding of 100 pM 125I-GRP to collagenase-prepared isolated islets at 22 degrees C was one-half maximal after 15 min and maximal at 60 min. At 60 min, total binding was 1.62% of total radioactivity per 50 islets; nonspecific binding (presence of 1 microM unlabeled GRP-1-27) was 0.05-0.61% of total radioactivity. GRP binds specifically to a high-affinity site (Kd1 = 0.81 nM; Bmax1 = 12.8 fmol/50 islets). The specific binding is saturable. Hormones with the intact C-terminus of GRP-1-27, such as N-acetyl-GRP-20-27 and neuromedin C (GRP-18-27), possess the same inhibition curve as GRP-1-27. GRP-1-16, with a cleaved C-terminus, does not inhibit binding of 125I-GRP. However, hormones that virtually are not structurally related to GRP, such as eledoisin, galanin, and VIP (vasoactive intestinal peptide) do not compete for GRP binding. The rank order of GRP analogs such as GRP-1-27, N-acetyl-GRP-20-27, and GRP-1-16 is similar though not identical with respect to inhibition of 125I-GRP binding and insulin secretory potency. We found that 1 and 10 nM GRP-1-27, at a stimulatory glucose concentration, increases the breakdown of phosphatidylinositol to Ins-1,4,5-P3, the biological relevant isomer of Ins-P3; 10 nM GRP-1-27 is effective even at a nonstimulatory glucose concentration in this respect. In a virtually Ca(2+)-free medium, 5 nM GRP-1-27 increases the 45Ca2+ efflux from 45Ca(2+)-prelabeled islets. These data indicate that (a) specific binding sites for GRP are present in mouse pancreatic islets; (b) GRP superimposes the maximal insulinotropic effect of glucose; and (c) Ins-1,4,5-P3 is probably involved as a second messenger in the biological effects of GRP-1-27, which is underlined by the efflux of Ca2+ from intracellular stores but is not a sufficient signal by itself.
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PMID:Gastrin-releasing peptide: binding and functional studies in mouse pancreatic islets. 159 56

This report introduces a novel method to load indo-1 "free acid" selectively into the cytosol of cardiac myocytes, presumably by diffusion through momentarily permeable gap junction sites during mechanical dissociation after low-Ca2+ collagenase treatment. Calibration of indo-1 fluorescence in these cells has been accomplished after subtracting average autofluorescence (AF) from time-matched non-indo-loaded cells, taking into account apparent changes in cell AF due to indo-1. There is wide variation in the degree of uncertainty of individual intracellular Ca2+ concentration ([Ca2+]i) determinations among cells, related principally to differences in cellular indo-1 content, to nonlinear aspects of the [Ca2+]-to-fluorescence ratio relationship, and to the uncertainty in the AF subtraction. Consequently, a quantitative estimate of uncertainty also may be employed in formulating weighted estimates of cytosolic [Ca2+]i. The following [Ca2+]i values in rat ventricular cells (nM; in 1 mM bathing extracellular Ca2+ concentration, 25 degrees C) are given as weighted means +/- 95% confidence intervals (unweighted values in parentheses): 138 +/- 5 (136 +/- 6, n = 44) in quiescent cells, 435 +/- 74 (482 +/- 76, n = 43) at the [Ca2+]i-transient peak during 0.5 Hz steady-state stimulation, and 760 +/- 124 (1,027 +/- 250, n = 42) at the [Ca2+]i-transient peak, postrest. Moreover, these peak [Ca2+]i values fall near the steepest portion of the force-Ca2+ curve (from intact cardiac muscle), consistent with sensitive inotropic regulation and maximal contractile reserve.
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PMID:Novel technique to load indo-1 free acid into single adult cardiac myocytes to assess cytosolic Ca2+. 162 51


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