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)

Proximal tubular cells (PTC) were isolated from porcine kidney by collagenase treatment, subsequently purified on a discontinuous density gradient and finally cultured. Porcine PTC (PPTC) in primary culture expressed keratin, characteristics of epithelia and brush border specific glycoproteins (FX1A). In addition, vimentin was present. All cells were negative for the endothelial marker pal-E. Less than 0.1% expressed the Tamm-Horsfall protein, characteristic of the distal tubule, while less than 0.3% of all cells in culture expressed desmin, characteristic of connective tissue (i.e. fibroblasts) and mesangial cells. Ultrastructural analysis revealed microvilli, tight junctions and abundant mitochondrial and lysosomes, all characteristics of proximal tubular cells. Freshly isolated PPTC were validated as in vitro model to detect nephrotoxicity by studying the effect of mercuric chloride, cis-platin, p-aminophenol and the halogenated alkenes 1,2 dichlorovinyl-l-cysteine, S-(1,1-difluoro-2,2-dichloroethyl)-L-cysteine (DCDFE-cys) and the glutathione conjugate of DCDFE on viability and mitochondrial membrane potential. The cells responded, time- and dose-dependently, to the nephrotoxic compounds with a decrease in mitochondrial membrane potential and loss of viability. The sensitivity of the porcine cells in detecting toxic effects corresponded favorably with in vitro systems derived from other animals.
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PMID:Evaluation of nephrotoxicity in vitro using a suspension of highly purified porcine proximal tubular cells and characterization of the cells in primary culture. 785 34

Human fetal kidney explants can be maintained during 5 days in Leibovitz's L15, a basic serum-free medium. Because culture conditions are minimal for growth and differentiation, DNA synthesis drastically decreases during the first 48 h, but stabilizes thereafter. The addition of insulin plus transferrin significantly restores this important cellular function in kidneys of fetuses younger than 16 wk. However, renal explants from older fetuses are more difficult to culture: they respond less to growth factors and are more prone to necrosis. The objective of this study was to verify the influence of tetracycline, an antibiotic with anti-collagenase potential, on cultured kidney explants aged 17 to 20 wk. The addition of 20 micrograms/ml tetracycline did not influence DNA synthesis nor the effectiveness of insulin plus transferrin on cell proliferation. Nor did it change the activities of alkaline phosphatase and gamma-glutamyltransferase, two enzymic markers of brush border differentiation. After 5 days in L15 alone, explants often showed necrosis and an important reduction in both weight and volume. Insulin plus transferrin significantly restored these parameters to control values observed at Day 0, but evidence of necrosis was still present. Tetracycline alone markedly reduced explant necrosis resulting in a significant increase in weight and volume. The effectiveness of insulin plus transferrin on explant morphometry was not improved when tetracycline was added as third factor. These results indicate that insulin plus transferrin restores explant mass through cell proliferation, whereas tetracycline does so possibly through a reduction in extracellular matrix degradation. The two effects are not additive in cultured mid-term fetal kidneys.
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PMID:Positive influence of tetracycline on human fetal kidney in serum-free organ culture. 791 74

The Heymann nephritis antigenic complex (HNAC) consists of two components, i.e., 1) gp330, a large glycoprotein localized in coated pits of the proximal tubule and glomerular epithelium, and 2) a 44-kDa protein which is homologous to the human alpha 2-macroglobulin receptor-associated protein (RAP). To examine the biosynthesis and assembly of HNAC, tissue fragments prepared from collagenase-digested 1-day-old rat kidneys were radiolabeled, and gp330 and RAP were immunoprecipitated with specific antibodies. By electron microscopy the tubule organization was seen to be largely intact. Results obtained on the biosynthesis of a control brush border protein, dipeptidylpeptidase IV (DPPIV), showed that tubules prepared in this manner are capable of synthesis and posttranslational processing of brush border membrane proteins and thus are suitable for short-term (< 3 h) biosynthetic experiments in vitro. Results of pulse chase and digestion with endoglycosidase H (Endo H) indicated that the time required for newly synthesized gp330 to mature in the endoplasmic reticulum (ER) and transit the middle Golgi compartments [half time (t1/2) = 90 min] was significantly longer than that of DPPIV (t1/2 = 20 min). Coprecipitation and cosedimentation (sucrose velocity gradient centrifugation) experiments showed that gp330 associates with RAP very early after synthesis and that the 44-kDa protein remains associated with gp330 during its subsequent folding, oligomerization, and transport to the Golgi. These findings demonstrate that HNAC assembles in at least two steps. The first step is the association of gp330 with RAP forming a large (19.3S) heterodimer, which sediments with the thyroglobulin (mol wt = 669,000) standard. This step begins within 30 min of synthesis and is Ca2+ dependent. The second step, which occurs > 60 min after synthesis, is the formation of a larger heterooligomer, which results in a shift in size of the complex from 19.3 to 38.6S. Both steps occur before acquisition of Endo H resistance. These results indicate that HNAC consists of a large multimeric complex that is assembled in the rough ER.
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PMID:Biosynthesis of the gp330/44-kDa Heymann nephritis antigenic complex: assembly takes place in the ER. 832 89

During the preparation of a suspension of dog kidney proximal tubules by collagenase treatment, an uptake of FITC-albumin was demonstrated. This process is attributed to the activation of receptor-mediated endocytosis leading to the appearance of FITC-albumin into intracellular vesicular structures. The isolation of brush border membrane vesicles (BBMV) from the dog kidney proximal tubules in suspension by the magnesium precipitation technique leads to the copurification of a large population of endosomes. These endosomes were separated from BBM vesicles by a technique involving wheat-germ agglutinin. The enrichment in BBM markers and in bafilomycin-sensitive ATPase activity was comparable in endosomes and BBM vesicles. However, the acridine orange acidification assay showed a V-type ATPase-dependent acidification in endosomes but not in BBMV, demonstrating a different orientation of the proton pumps in these structures. SDS-PAGE analysis also showed significant differences in protein pattern of vesicles and endosomes. The most notable difference was the presence of 42-44 kDa and 20-24 kDa proteins in BBMV and their complete absence in endosomes. Western blot analysis identified these proteins as actin and RhoA, among other small proteins, respectively. Western blot experiments also demonstrated a different distribution of beta-COP, beta-adaptin, and RhoGDI in vesicles and endosomes. The morphological aspect (electron microscopy) and sedimentation of endosomes in a 50% Percoll gradient identified these structures as "heavy endosomes" (buoyant density D = 1.036 g/ml). Flow cytometry analysis of heavy endosomes purified from tubules isolated in presence of FITC-albumin showed the presence of FITC-albumin in up to 92% of these intracellular organelles. Western blot analysis using anti-FITC and anti-collagenase antibodies allowed quantification of the FITC-albumin and collagenase A in the purified endosomes. Our results indicate that heavy endosomes are formed during the preparation of the proximal tubules following activation of receptor-mediated endocytosis, probably by soluble proteins. The suspension of tubules thus offers a experimental tool to study the protein reabsorption and traffic of endosomal vesicles in the proximal tubules.
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PMID:Isolation of heavy endosomes from dog proximal tubules in suspension. 869 8

A sensitive lactate dehydrogenase (LDH) assay was modified to determine the cytolytic activity of Bacillus thuringiensis CryIC and CryIAc delta endotoxins to viable collagenase-dissociated midgut epithelial cells (MEC) from larvae of Spodoptera frugiperda and Spodoptera exigua. The MEC preparations from these Spodoptera sp. consisted predominantly of columnar cells (65-75%) and goblet cells (25-35%). Time course microscopy experiments indicated that only the columnar cells became swollen during CryIC toxin incubation. Also, comparative cytotoxicity studies were run with cell lines of nonmidgut origin established from S. frugiperda (SF21AE) and S. exigua (SEUCR1A). Optimum conditions for the cytotoxicity assay were similar for MEC and cell lines of both species, and were met in an assay in which 0.1-ml cell concentrations (8.5 +/- 0.5 x 10(4) cells) were incubated with toxin dilutions (0.01-20 micrograms) for 1 h at 24 degrees C at a final pH of 7.8. The Spodoptera sp. MEC were twofold more sensitive to CryIC (68% lysis) than CryIAc (32% lysis) at optimum toxin levels (2.5-5 micrograms). Also, the SEUCR1A cells were more sensitive (2.3-fold) to CryIC (70% lysis) than CryIAc (30% lysis) at optimum toxin levels of 5-10 micrograms. The SF21AE cells, however, were twofold less sensitive to CryIC (30% lysis) than SEUCR1A cells and response to CryIAc and CryIC was similar. Immunoblot analysis of either Spodoptera sp. MEC or brush border membrane vesicles (BBMV) identified seven CryIC binding proteins with molecular mass of 137, 120, 115, 68, 65, 63, and 45 kDa. Occasionally, a 148-kDa protein band was observed. The CryIAc toxin bound to two proteins on MEC and BBMV with molecular mass of 137 and 120 kDa.
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PMID:Cytolytic activity of Bacillus thuringiensis CryIC and CryIAc toxins to Spodoptera sp. midgut epithelial cells in vitro. 915 49

After collagenase digestion and Percoll density gradient centrifugation of human renal tissue, tubular epithelial cells of the proximal and the distal segments were isolated with an immunomagnetic method using MACS microbeads. To enrich proximal tubular (PT) cells we used a monoclonal antibody (mAb) against aminopeptidase M (APM, CD 13), specific of the proximal tubule. Distal tubular (DT) cells were isolated through a mAb recognizing Tamm-Horsfall glycoprotein (THG), a specific antigen for the thick ascending limb and the early distal convoluted tubule. Cells of the proximal primary isolate were histochemically strongly positive for aminopeptidase M (98.6%), however, cells of the distal portion were negative (98.7%). Ultrastructural analysis of PTC primary isolates revealed highly preserved brush border microvilli, well-developed endocytosis apparati and numerous mitochondria, whereas DTC primary isolates showed smaller cells with basolateral invaginations and less apical microvilli. Characterization by immunofluorescence indicated the coexpression of cytokeratin and vimentin, whereas staining for desmin, smooth muscle actin, a fibroblast-specific marker and von Willebrand factor was negative. Cultured PT and DT cells displayed different adenylate cyclase responsiveness to hormonal stimulation. PTH (10(-6) M) increased cAMP production in distal cells up to 32.8-fold of the basal level and in proximal only up to 3.5-fold (10(-8) M, DT 14.4x and PT 2.25x). Calcitonin stimulated adenylate cyclase in DT in a dose dependent fashion (10(-6) M, 4.3x; 10(-8) M, 2.25x), whereas only a low calcitonin response was found in PT cells (10(-6) M, 1.6x; 10(-8) M, 1.4x). AVP (10(-6) M) activated the distal cAMP-production only up to 1.9x of the basal level, but the proximal cAMP-production was negligible (only 1.3x the basal level). The data of this study indicate the proximal and distal tubule origin of the cultured cells that were isolated according to their segment-specific antigens.
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PMID:Isolation of proximal and distal tubule cells from human kidney by immunomagnetic separation. Technical note. 935 Jun 55

Using Brown Norway (BN) rats, we isolated and characterized the tubular basement membrane (TBM) antigens that are immunologically common to humans. The renal basement membrane (RBM) of BN rat, as an antigen source, was solubilized with 8 M urea instead of collagenase followed by extraction with 0.5 M NaCl. On frozen section-immunohistochemistry, the autoantibody obtained from BN rats, which had been immunized with human RBM and showed tubulointerstitial nephritis, bound to the TBM, the basement membrane of the Bowman's capsule, and the brush border of the proximal tubules, but not to the GBM of the normal BN rat kidney. Nephritogenic antigens were isolated by immunoaffinity chromatography using Sepharose-bound purified autoantibody. By Western blot analysis of the eluate, bands with molecular weight of 200 kDa and 180 kDa were positively reacted to anti-FX1A (brush border antigen) antibody and were apparently different from the major bands with molecular weight of 145 kDa and 130 kDa. The bands with molecular weight of 145 and 130 kDa showed major cross reactivity with antibodies to fibronectin and laminin. In contrast with these high molecular weight (HMW) bands, the major 60 kDa band with three minor bands showed no reactivity with any type of antibody tested. These results indicated that the non-enzymatic solubilization of RBM is one of the possible procedures for isolating the HMW form of antigens. These antigens may be epitopically modified pre-existing constitutions of the basement membrane and may play a role in the induction of tubulointerstitial nephritis.
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PMID:Isolation and characterization of tubular basement membrane antigen common to humans and rats. 985 23

Midgut epithelial cells were isolated from fifth-instar Pseudaletia unipuncta larvae by collagenase treatment of midgut tissue, and cultured in TNM-FH medium. Long-term continuous culture and maintenance of midgut cells were achieved with P. unipuncta armyworm intestinal cells. Several cells lines were obtained from these P. unipuncta primary cultures, and they have been subcultured and maintained for over 24 mo. The three major midgut cell types were present in the cultures, including stem (regenerative), columnar, and goblet cells. In vitro morphogenesis and differentiation of columnar and goblet cells from stem cells were observed. There appeared to be a cycle of cell death of goblet and columnar cells followed by their replacement from stem cells every 7-8 wk. After approximately six passages, the cell density in T-flasks appeared to be somewhat constant, reaching 10(3)-10(4) cells per milliliter of medium. The columnar cells are round to rectangular in shape and possess a brush border, while the goblet cells have a classic flask-like shape with a central cavity. Peritrophic membrane-like secretions were observed in all the culture flasks. Infection of these cells with multiply embedded nucleopolyhedrovirus was confirmed, and we conclude that these midgut cells can be used as an in vitro model system to study early events in baculovirus infection.
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PMID:Primary and continuous midgut cell cultures from Pseudaletia unipuncta (Lepidoptera: Noctuidae). 1151 67

A method for producing large quantities of pure proximal tubule cells is described. The procedure involves collagenase perfusion to prepare kidney cells that are separated by centrifugal elutriation to produce a purified cell preparation of proximal tubule cells (PCS). The elutriation-purified cells were treated with a rabbit polyclonal antibody to rat gamma-glutamyl transpeptidase (GGT). The resulting antibody-labelled cells were then incubated with Dynabeads coated with a sheep anti-rabbit IgG antibody. The Dynabeads, which are monodispersed polystyrene beads with a magnetic ferrite core, bind specifically to the antibody-labelled cells. The cell-bead complexes were then harvested with a magnet and washed twice to remove cells not labelled with antibody. The procedure is simple and rapid, and can be used to produce 20-25 x 10(6) cells. The cells exhibit a well defined brush border membrane with increased GGT and alkaline phosphatase enzyme activities. Concomitant with this is a four-fold decrease in hexokinase activity, which demonstrates that contaminating distal and loop of Henle tubule cells have been removed.
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PMID:Immunomagnetic purification of rat proximal kidney cells. 2069 84

Renal proximal tubule fragments (RPT) were prepared from young-adult, male F-344 rats by deferoxamine/collagenase perfusion and evaluated as a potential model for mechanistic studies and screening, using known nephrotoxins. Chloroform and S-(1,2- dichlorovinyl )- l - cysteine (DCVC) produced depressed O(2) consumption rates (basal and/or nystatin-stimulated) and lactate dehydrogenase (LDH) release during 8-hr incubations at 0.5 mg RPT protein/ml. Cytochrome P-450 inhibitors piperonyl butoxide and metyrapone were either without effect or potentiated chloroform-induced toxicity. DCVC was more cytotoxic to RPT than to rat hepatocytes. The cytotoxic potency for cephalothin relative to cefazolin decreased as RPT content in the medium was increased to 3.0 mg protein/ml, giving a rank order more in accord with results reported in vivo. Cephalosporins markedly depressed brush border alkaline phosphatase (ALP) activity, without affecting gamma-glutamyltranspeptidase activity; the effect on ALP was less sensitive to the RPT level. Acetaminophen (25 mm) and p-aminophenol (1.0 mm) induced LDH release without ALP depression and inhibited mitochondrial respiration. These results in general corresponded well with in vivo responses and indicate that this RPT system may be valuable for studies of chemical-induced nephrotoxicity.
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PMID:Studies of nephrotoxic agents in an improved renal proximal tubule system. 2070 4


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