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M Coca-Prados

Publications and source records attributed to M Coca-Prados.

86 records · Page 5Linked to original sources
PubMed1988

Na+/H+ exchange regulates intracellular pH in a cell clone derived from bovine pigmented ciliary epithelium.

The regulation of intracellular pH (pHi) was monitored in a virus-transformed cell clone derived from bovine ciliary body exhibiting characteristics of pigmented ciliary epithelium. Data were obtained from confluent monolayers grown on plastic coverslips in nominally bicarbonate-free media using the pH-sensitive absorbance of 5- (and 6-) carboxy-4',5'-dimethylfluorescein. Under resting conditions, pHi averaged 6.98 +/- 0.01 (SEM; n = 57). When cells were acid loaded by briefly exposing them to Ringer containing NH4+ and then withdrawing the NH4+, pHi spontaneously regained its initial value. In the presence of 1 mM amiloride or in the absence of Na+, this process was blocked, indicating the involvement of an Na+/H+ exchanger in the regulation of pHi after an acid load. Removing Na+ during resting conditions decreased cytoplasmatic pH. This acidification could be slowed by amiloride, which is evidence for reversal of the Na+/H+ countertransport exchanging intracellular Na+ for extracellular protons. Application of 1 mM amiloride during steady state led to a slow acidification. Thus the Na+/H+ exchanger is operative during resting conditions extruding protons, derived from cellular metabolism, or from downhill leakage into the cell. Addition of Na+ to Na+ -depleted cells led to an alkalinization, which was sensitive to amiloride, with an IC50 of about 20 microM. This alkalinization was attributed to the Na+/H+ exchanger and exhibited saturation kinetics with increasing Na+ concentrations, with an apparent KM of 29.6 mM Na+. It is concluded that Na+/H+ exchange regulates pHi during steady state and after an acid load.

Amiloride↗
PubMed1987

Evidence that the alpha and alpha (+)isoforms of the catalytic subunit of (Na+, K+)-ATPase reside in distinct ciliary epithelial cells of the mammalian eye.

Polyclonal antibodies against the canine kidney (Na+,K+)-ATPase were used to examine the localization and distribution of this protein in intact ciliary processes (CP) from bovine eyes by indirect immunofluorescence. The basolateral surface of non-pigmented (NPE) and pigmented (PE) ciliary epithelial cells was found to be stained specifically for the (Na+,K+)-ATPase. Immunoblot analysis of intact CP, separated PE and NPE cells by density gradients and cultured ciliary epithelial cells, revealed two forms of the catalytic subunit of the (Na+,K+)-ATPase: the alpha and alpha (+). The alpha (+) form was enriched in NPE cells while alpha was in PE cells.

Animals↗
PubMed1986

Growth of non-pigmented ciliary epithelial cells in serum-free hormone-supplemented media.

Bovine non-pigmented ciliary epithelial (NPE) cells are cultured following a dissecting procedure that preserves the original cellular polarity. Cell growth of primary cultures of NPE cells is supported by hormonally defined serum-free culture media and an artificial substrate with high binding affinity to attach cells in culture. Cell proliferation of primary cultures of bovine NPE cells was limited to 3-5 weeks. Nerve growth factor (NGF) was found to have a profound effect on the cellular junctions of growing NPE cells when added to the culture media. Nerve growth factor induced the formation abundant microfilament bundles associated with junctional complexes of undetermined nature between adjacent cells. Electron microscopy of NPE in culture shows a high intracellular transport activity with abundant coated pits and clathrin-coated vesicles, a complex network of microfilaments, microtubules and intermediate filaments associated with desmosomes.

Actin Cytoskeleton↗
PubMed1986

Transformation of human ciliary epithelial cells by simian virus 40: induction of cell proliferation and retention of beta 2-adrenergic receptors.

Ciliary epithelial cells derived from human eye were successfully propagated through many generations after transformation with simian virus 40. The cell clone 8-SVHCE was isolated and characterized by immunoprecipitation and pharmacological studies that demonstrated the presence of several functional properties observed in the parent cells of this tissue. Immunoprecipitation revealed the presence of large tumor (T) antigen, and Southern blot analysis showed the incorporation of viral DNA into high molecular weight ciliary epithelial cell DNA. The presence of beta-adrenergic receptors was demonstrated by direct binding of a radiolabeled antagonist, [125I]iodopindolol, to membrane preparations of 8-SVHCE cells (Kd = 41.8 pM and Bmax = 67.1 fmol/mg of protein). Competition experiments with [125I]iodopindolol and selective drugs suggested that the receptors are of the beta 2-adrenergic subtype. Studies of catecholamine-stimulated cellular cAMP production and of isoproterenol-dependent protein phosphorylation of vimentin in 8-SVHCE indicated the functional conservation of beta-adrenergic receptor-mediated processes that are thought to be important in the regulation of aqueous humor production by the ciliary epithelium in vivo.

Adenylyl Cyclases↗
PubMed1985

Regulation of protein phosphorylation of the intermediate-sized filament vimentin in the ciliary epithelium of the mammalian eye.

The intermediate-sized filaments of vimentin-type (Mr = 57,000) have been identified biochemically and immunochemically as a major cytoskeleton component in the ciliary epithelium of the mammalian eye. When human or rabbit ciliary processes, or cultured ciliary epithelial-derived cells were incubated in serum-free medium containing [32P]orthophosphate and any of the following agents: 1) beta-adrenergic agonists (isoproterenol or epinephrine), 2) direct activators of adenylate cyclase (cholera toxin or forskolin), 3) analogs of cyclic AMP (8-Br-cAMP), or 4) prostaglandin E1, the phosphorylation of vimentin was significantly enhanced. The maximal enhancement ranged, in vivo and in vitro, from about 3-fold in human to 5-fold in rabbit, with either 1 mM 8-Br-cAMP or 0.1 microM forskolin. Phosphorylation of vimentin increased in the presence of beta-adrenergic agonists and could be blocked by the antiglaucoma beta-adrenergic antagonist timolol. The alpha-adrenergic agonist phenylephrine had no effect on phosphorylation of vimentin. Indirect immunofluorescence microscopy using a monoclonal antibody, anti-vimentin, allowed the localization of vimentin filaments in cultured ciliary epithelial cells. Treatment of these cells in culture with the catecholamine hormone, isoproterenol (1 microM), resulted in a profound reorganization of vimentin filaments. This may be correlated with the enhanced levels of phosphorylated vimentin observed upon increasing cellular cyclic AMP.

Animals↗
PubMed1985

Separation of bovine pigmented ciliary epithelial cells by density gradient and further characterization in culture.

Dissociated pigmented epithelial (PE) cells from ciliary processes (CP) of bovine eyes were separated by isopycnic centrifugation in metrizamide gradients and further cultivated. The level of cellular contamination during separation of PE by non-pigmented epithelial (NPE) cells was estimated to be 5-10% and by non-epithelial cells (i.e. endothelial, fibroblasts and blood cells) less than 1%. Bovine PE cells were found to have different serum requirements than NPE to grow in vitro. PE cells were actively stimulated to proliferate in the presence of fetal calf serum for a few generations. Short-term culture of NPE was only obtained in media containing a low concentration of L-tyrosine and no serum. Two morphological markers, namely desmosomes and intermediate-sized filaments (IF) of the vimentin type, were found by immunolabeling to be coexpressed in cultured PE and were used as criteria to define the epithelial identity of these cells. The present study was unable to detect any tyrosinase activity in cultured PE cells.

Animals↗
PubMed1984

Human ciliary epithelia in monolayer culture.

The ciliary epithelia of human (one to 12 months old) ciliary processes were isolated by trypsin and EDTA, cultured in Dulbecco's Modified Eagle Medium (DMEM) with 5% fetal calf serum and examined by phase and electron microscopy. The primary cultures were maintained for three to four months. Only a few non-pigmented epithelia adhered and none of them proliferated. After the first passage the culture seemed to consist of only the pigmented epithelia. Most cells were densely pigmented at first, then became less pigmented during successive proliferations. Half of the cells remained densely pigmented after the first subculture, another half remained less pigmented. The cells started to lose their pigment granules at four to six weeks in culture. After three months of culture, the cell sheets became entirely unpigmented. In thin section, most of the pigment granules in the cells at two weeks in culture were pre-melanosomes, and half of them were at the earliest stage of pre-melanosomes. Monolayer cells possessed basement membranes. At 14 weeks in culture, most cells established an apparent polarity, contained well-developed Golgi apparatus and rough endoplasmic reticulum and intermediate filaments, but no pigment granules. A bundle of intermediate filaments was found in the perinuclear cytoplasm. Multilayer cells presented a typical apex-to-apex and base-to-base configuration , and the extracellular material was detected only in the base-to-base intercellular spaces. Our culture system provided differentiated cells derived from the pigment epithelia of human ciliary processes.

Cells, Cultured↗
PubMed1983

Ciliary epithelia of the mammalian eye in cultured explants.

The ultrastructural characteristics of ciliary epithelium from bovine, pigmented rabbit, and fetal albino rabbit were studied in cultured explants. The tips of ciliary processes were cultured in plastic dishes with Dulbecco Modified Eagle Medium (DMEM) containing 5% fetal bovine serum. More than half of the explants adhered to the plastic culture dish, and epithelial cells spread as monolayers within a few days. Initially the explant contains two layers, the outer (nonpigmented cells) and the inner (pigmented cells). Later the explant exhibits three layers: 1) outermost lightly pigmented flattened cells, 2) an outer layer of non-pigmented cells, and 3) an inner layer of densely pigmented cuboidal cells. The cells of the outermost layer are continuous with the cells of the inner layer. A narrow space lies between the outermost layer and the outer layer. The columnar cells in the outer layer contain well developed organelles but no pigment granules; they possess a basement membrane, lateral interdigitations, and junctional complexes near their apices. Numerous focal junctions and some ciliary channel-like structures were detected between the columnar cells of the outer layer and the cuboidal cells of the inner layer. The cuboidal cells of the inner layer are filled with pigment granules. These observations suggest that the columnar cells of the outer layer are nonpigmented epithelium, the cuboidal cells of the inner layer are pigmented epithelium, and the flattened cells in the outermost layer are derived from pigmented epithelium.

Animals↗
PubMed1982

Intracellular forms of simian virus 40 nucleoprotein complexes. IV. Micrococcal nuclease digestion.

The structures of DNAs present in various intracellular forms of simian virus 40 (SV40) nucleoprotein complexes were analyzed by micrococcal nuclease digestion. The results showed that the 70S SV40 chromatin was completely sensitive to nuclease digestion, whereas CsCl gradient-purified mature virion was completely resistant. Virion assembly intermediates with different degrees of virion maturation showed intermediate resistance, and three products were found: nucleosomal DNA fragments, representing the fraction of intermediates that were sensitive to nuclease; linear SV40 genome-sized DNA, representing the more mature intermediates that contained one or limited defects in the capsid shell; and supercoiled SV40, which was derived from mature virions. These digestion products, however, remained associated with capsid shells after nuclease digestion. These results were consistent with the model in which maturation of the SV40 virion is achieved through the organization of capsid proteins that accumulate around SV40 chromatin. Mild digestion of SV40 nucleoprotein complexes with micrococcal nuclease revealed the difference in nucleosome repeat length between SV40 chromatin and virion assembly intermediates. A novel DNA fragment of about 75 nucleotides was observed early in nuclease digestion.

Animals↗
PubMed1980

Enzymatic analysis of 5-methylcytosine content in eukaryotic DNA. Study of intracellular Simian Virus 40 DNA.

An enzymatic method is described for the analysis of the content of 5-methylcytosine in the DNA of eukaryotic cells. The conventional acid hydrolysis procedure was found to result in variable deamination of 5-methylcytosine into thymine, making the exact quantitation difficult. We applied the enzymatic method for the analysis of a simple eukaryotic viral genome. Viral and cell DNA in Simian Virus 40-infected monkey kidney cells was labeled with [methyl-3H]methionine in vivo and their 5-methylcytosine content was analyzed. The content of 5-methylcytosine per thymine in the total intracellular viral DNA was found to be less than 10(-4) of the host DNA. The implication of this result with regard to eukaryotic gene expression is discussed.

5-Methylcytosine↗
PubMed1980

Intracellular forms of simian virus 40 nucleoprotein complexes. III. Study of histone modifications.

The modification patterns of histones present in various forms of intracellular simian virus 40 nucleoprotein complexes were analyzed by acetic acid-urea-polyacrylamide gel electrophoresis. The results showed that different viral nucleoprotein complexes contain different histone patterns. Simian virus 40 chromatin, which contains the activities for the synthesis of viral RNA and DNA, exhibits a histone modification pattern similar to that of the host chromatin. However, virion assembly intermediates and mature virions contain highly modified histones. Pulse-chase experiments with [3H]lysine showed that the newly incorporated histones in the virion assembly intermediates were already highly modified. The majority of in vivo acetylation activity of histones occurred on the 70S simian virus 40 chromatin as analyzed by pulse-labeling with [3H]acetate. These results and our previous analysis of the virion assembly pathway suggest that three stages are involved in the packaging of simian virus 40 chromatin into the mature virion: (i) modification of histones, (ii) accumulation of capsid protein around the chromatin with highly modified histones, and (iii) organization of capsid proteins into salt-resistant shells. The role of histone modification in virion assembly is discussed.

Acetylation↗
PubMed1979

Intracellular forms of simian virus 40 nucleoprotein complexes. I. Methods of isolation and characterization in CV-1 cells.

A new method was developed for isolation of intracellular forms of simian virus 40 (SV40) nucleoprotein complexes from SV40-infected CV-1 cells late in the infectious cycle. In contrast to the Triton extraction method, which yields only a 60-70S complex, this new procedure yielded three forms of SV40 nucleoprotein complexes: complex I, complex II, and the nature virion (V). The three nucleoprotein complexes differed in physical as well as biochemical properties. Complex I, which is only a small portion of the total SV42 nucleoprotein complexes late during infection, was active in synthesizing both SV40-specific DNA and RNA. Pulse-labeling experiments suggest the following metabolic pathway: I leads to II leads to V. Conversion of complex I to II occurred shortly after the completion of SV40 DNA replication and resulted in the inactivation of the biosynthetic activities of I.

Animals↗
PubMed1979

Intracellular forms of simian virus 40 nucleoprotein complexes. II. Biochemical and electron microscopic analysis of simian virus 40 virion assembly.

The simian virus 40 virion assembly process was studied with pulse-labeling kinetics of virion proteins, CsCl gradient analysis, electron microscopy, and low-salt gel electrophoresis. The results obtained are consistent with the model of gradual addition and organization of capsid proteins around simian virus 40 chromatin. Empty virions, as observed in the CsCl gradient by previous workers, were found to be the dissociation product of immature virus. Histone H1 was found in simian virus 40 chromatin and virion assembly intermediates but not in the mature virion banding at 1.34 g/ml in the CsCl gradient.

Capsid↗