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Biomedical subjects

M F Counis

Publications and source records attributed to M F Counis.

At least 19 recordsLinked to original sources
PubMed1995

Involvement of DNase II in nuclear degeneration during lens cell differentiation.

The characterization of DNase II and DNase I activity was undertaken to discriminate their different roles in physiological nuclear degradation during lens fiber cell differentiation. The activity of both nucleases determined in a new assay allows to discriminate DNase II from DNase I in the same extract. In fibers, both types of nuclease activities are found and appear higher than in epithelial cells. Specific polyclonal antibodies directed against these two nucleases reveal by Western blot analysis the presence of various DNase isoforms. DNase II like-nuclease, present in fibers, is represented by three major bands (60,23, and 18 kDa), which are not detected, at least for two of them (60 and 23 kDa), in epithelial cells. DNase I like-nuclease pattern in fiber cells shows a single 32-kDa band, while several bands can be detected in epithelial cells. Immunocytochemistry studies show both nucleases present in lens cell sections. DNase II is, as usual, in cytoplasm of epithelial cells, but it appears strikingly concentrated in the nuclei of fibers. DNase I is always concentrated in nuclei of epithelial and fiber cells. DNA degradation observed in agarose gels shows that DNase II-activating medium cleaves the DNA from fiber cells more efficiently than DNase I-activating buffer. In addition, DNase II antibody is able to prevent this degradation. These results suggest a specific involvement of DNase II in nuclear degradation during lens cell differentiation.

Animals↗
PubMed1991

DNAase activities in embryonic chicken lens: in epithelial cells or in differentiating fibers where chromatin is progressively cleaved.

Lens is an organ composed of a layer of epithelial cells and a mass of fibers. During terminal differentiation, epithelial cells from the equatorial region elongate into fibers, nuclei change shape, the chromatin appears much condensed in the last step of differentiation and the DNA breaks down into nucleosomes. The pattern of DNAase activities has been recorded at different chick embryonic stages (11 and 18 days) using polyacrylamide gel electrophoresis with DNA substrate in the gel matrix. Two DNAases (30 and 40 kDa) have been observed in lens epithelia and fibers at both stages. However, the activities of both of the enzymes are augmented in fiber cells. The 30 kDa DNAase requires and Ca2+ and Mg2+ (5-15 mM) to hydrolyze the DNA substrate while the 40 kDa-activity is inhibited by added divalent cations (5-15 mM). The 30 kDa protein is inhibited by Na+ and is probably an endonuclease. Both nuclease activities probably are involved in the cleavage of fiber chromatin into nucleosomes during lens terminal differentiation, but variables such as chromatin configuration, unmasked DNA sequences, presence of cations, and pH gradients probably determine the extent of involvement of each DNAase.

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PubMed1990

Decrease of DNA per cell during development of the lens in chickens.

Developmental changes in the amount and conformation of DNA in chicken lens were studied. For this, DNA in situ in lens fiber cell nuclei of chickens was examined by microfluorometry with Hoechst 33258 (Hoe) fluorochrome. On 1 M NaCl-aided Hoe staining, by which the amount of DNA can be determined accurately, the fluorescence intensity of lens fiber cells was found to decrease with no change in that of the lens epithelial cells during development. On the contrary, on normal NaCl-free Hoe staining the fluorescence intensity of the lens cells was found to increase gradually during development. These results suggest that during development the amount of DNA in lens fiber cells decreases in association with some change in its conformation.

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PubMed1989

Increased sensitivity of various genes to endogenous DNase activity in terminal differentiating chick lens fibers.

In the lens, epithelial cells from the equatorial zone differentiate into postmitotic elongated fibers. One aspect of this differentiation is nuclear shape transformation and DNA degradation. This process is controlled by DNase activity which in fiber nuclei increases with development. DNase activity is also present in the epithelial cell nuclei which appears to be non-functional but could be activated in vitro by exogenous addition of Ca2+. We have analyzed the possible selective action of endogenous DNase on 3 genes involved in lens terminal differentiation, namely delta-crystallin, beta-tubulin and vimentin, and on 1 gene not thought to participate in this process, ovalbumin. We have compared restriction DNA patterns of these genes in nuclei isolated from 11-day-old chick embryos and incubated in Ca2+-free medium or in fresh epithelial and fiber lens tissue at 11 and 18 days of development. During incubation in vitro of 11-day fiber nuclei, there is a net increase in the sensitivity of the delta-crystallin, beta-tubulin, ovalbumin and vimentin chromatin to the endogenous DNase. The vimentin gene appears to be more stable than the beta-tubulin and delta-crystallin genes indicating a degree of specificity of the endogenous DNase activity. In the epithelial nuclei, the lens-specific genes appear to be more stable but paradoxically there is a net degradation of the ovalbumin gene. In freshly isolated tissues the 4 genes were detected in epithelial and fiber cells at 11 and 18 days. Furthermore, in the mature fibers in which the nuclei were degenerating, the latter genes were still not completely digested.

Animals↗
PubMed1989

Lens fiber differentiation correlated with activation of two different DNAases in lens embryonic cells.

In order to identify the different DNAases present in the lens differentiating tissue, we have used an assay which reveals their activity directly on DNA-containing gels after SDS polyacrylamide gel electrophoresis. DNAase renaturation from nuclear embryonic lens extracts does not occur after separation in 0.1% SDS polyacrylamide gel electrophoresis in contrast to that observed with purified micrococcal nuclease. When the SDS concentration in the running buffer and separating gel is decreased to 0.075%, renaturation of lens DNAase and enzyme activities are observed. Isoelectrofocusing was carried out in a polyacrylamide gel which was overlaid with an agarose gel containing DNA, permitting the visualization of the pI of DNAase activity. The presence of several DNAase isoenzymes was demonstrated in 11-day embryonic lenses. In epithelial lens nuclei, high molecular weight (MW) isoenzymes with basic pI were predominant. In post-mitotic fiber lens nuclei, two lower MW isoenzymes with acidic pI were detected as well as high MW activity with a basic pI.

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PubMed1987

Characterization of acidic and basic fibroblast growth factors in brain, retina and vitreous chick embryo.

We have purified acidic and basic fibroblast growth factors (c-aFGF, c-bFGF) from 11 day-old chick embryo brain, retina and vitreous by heparin-Sepharose chromatography and reverse phase HPLC. The analysis of their biological activity as well as their molecular weight indicates that they were analogous to basic or acidic human and bovine FGF. The ratio of c-aFGF to c-bFGF activity depended of the tissue. In brain c-aFGF represented 66% of the total mitogenic activity retained on the heparin-sepharose column and c-bFGF 34% while retina contained 16% of c-aFGF and 84% of c-bFGF; vitreous 78% of c-aFGF and 22% of c-bFGF. Like human aFGF, Heparin stimulated purified c-aFGF mitogenic activity in the absence of serum but inhibited the activity of the retina acid soluble extract, in the presence of foetal calf serum (FCS). Thus, chick embryo and adult human acidic and basic FGF respectively share the same biochemical properties. Since there are no blood vessels in chick retina or vitreous, their presence in these tissues suggests that angiogenesis is not the only role of these growth factors.

Animals↗
PubMed1987

Collagen synthesis by long-lived mRNA in embryonic chicken lens.

Lens capsule collagen synthesis by epithelial and fiber cells was examined by immunoprecipitation and collagenase digestion in embryonic and posthatch chicken eye lens. Epithelial cells and lens fibers in the process of terminal differentiation produce alpha 1 and alpha 2 type IV collagen chains. At 6 days of embryonic development in addition to the alpha 1 (IV) and alpha 2 (IV) collagen chains, lens cells produce high molecular weight collagenase-sensitive proteins not immunologically related to type IV collagen. Lens capsule collagen components have been identified in central and outer fibers isolated from 18-day embryos and from 10-day posthatch chicken eyes. At these stages, fibers which have an increasing number of picnotic nuclei still show collagen synthesis due to long-lived mRNA. Analysis of collagen synthesis by lens cells incubated with actinomycin D suggests that stabilization of collagen mRNA occurs in lens fiber cells and to a lesser extent in epithelial cells as early as 6 days of embryonic development.

Age Factors↗
PubMed1987

Effect of X-irradiation and vitamin C on DNA degradation and endogenous DNase in embryonic chick lens cells.

The lens is an organ in which epithelial cells become elongated fibers. During this process, nuclei are transformed and the DNA is degraded. In previous studies, we described an autodigestion of the chromatin in isolated fiber nuclei but not in epithelial nuclei, but the level of DNAase activity was found to be identical in both epithelial and fiber nuclei of lenses at 11 days of development. In this study, we have investigated the possibility that x-irradiation might stimulate the nuclear endogenous activity responsible for chromatin breakdown or epithelial cells to a level comparable to that observed in fiber cells. We have observed that x-irradiation does not increase the nuclear epithelial DNAase activity. Conversely, vitamin C, suspected to prevent cataract formation by protecting DNA against free radical formation, has a damaging effect on the DNA of the lens of chick embryo in vitro.

Animals↗
PubMed1986

Nuclear endogenous Ca2+-dependent endodeoxyribonuclease in differentiating chick embryonic lens fibers.

During terminal differentiation of lens epithelial cells into fiber cells, nuclei become pycnotic and DNA degradation occurs. We investigated the putative role in this process of an endogenous DNAase. After incubation of isolated nuclei of both cell types at 37 degrees C, DNAase activity was revealed by DNA size analysis on 0.3-1% neutral and alkaline agarose, one- and two-dimensional gels. This DNAase activity is more prominent in lens fiber nuclei than in epithelial nuclei at all the embryonic stages probably because of a preexisting higher concentration of divalent cations in the former. This activity is calcium or magnesium dependent in both types of nuclei.

Animals↗
PubMed1986

Chromatin condensation and terminal differentiation process in embryonic chicken lens in vivo and in vitro.

During embryonic chick lens differentiation, the epithelial cells become transformed into elongated fibres. Concomitantly, the fibre nuclei undergo degeneration and high molecular weight (HMW) DNA breaks down due to nuclear endodeoxyribonuclease activity. An electronmicroscopic study of lens epithelial and fibre nuclei was made at different stages of chick embryonic development, both in vivo and in vitro. The in vitro conditions are conducive to the expression of endogenous endodeoxyribonuclease activity in fibres. In both conditions we observed condensation of chromatin. The organization of some nuclear material into distinct linear arrays followed by streaming of nuclear material into the cytoplasm is recorded only in vitro. Such a condition may lead to acceleration of the process of aging in lens fibres.

Animals↗
PubMed1985

Nuclear ADP-ribosylation in the chick lens during embryonic development.

Nuclear ADP-ribosyltransferase is present in cells from the chick lens throughout embryonic development. The activity does not decrease when the cells become post-mitotic and commence terminal differentiation but declines slowly in both epithelia and fibre cells. At all stages studied the enzyme retains its ability to be activated by DNA strand breaks induced either by X-irradiation or by the action of an endogenous endonuclease. There is no correlation between the enzyme activity or the levels of its substrate NAD+ and the changes in DNA repair capacity which have been observed during the development of the lens.

Animals↗
PubMed1985

DNA repeat size in chick embryonic lens epithelium, lens fiber, brain and liver cell nuclei.

The DNA repeat size is determined by micrococcal nuclease digestion kinetics and subsequent electrophoresis of the products among various chick embryonic tissues. The repeat size is found to be not significantly different from 193 to 197 bp, for brain and liver at 11 days and for lens epithelium and fiber at different embryonic stages. However, the pattern of micrococcal digestion seems to reveal an overall chromatin modification as a function of development in the lens fibers.

Animals↗
PubMed1984

Changes in the DNA breakage and crystallin synthesis of embryonic chicken lenses cultured in a tryptophan-deficient medium.

In vitro experiments were performed in order to understand the biochemistry of tryptophan-deficient cataract. Eleven-day-old embryonic chick lenses were cultured in vitro for 3 hr, one and three days in a tryptophan-deficient medium. DNA breakage was followed on sucrose gradient and water-soluble protein synthesis was analysed by SDS-PAGE coupled with fluorography. A medium lacking tryptophan delays the DNA degradation and decreases the synthesis of all soluble proteins including the crystallins.

Animals↗
PubMed1981

DNA polymerase, DNA ligase, and thymidine kinase activity in chicken lens, related to DNA X-ray lesion repair.

The activity of DNA polymerases alpha, beta, and gamma; DNA ligases I and II; and thymidine kinase in chicken lenses is determined. These enzymes are present in embryonic intact lenses freshly isolated at 6 days and 11 days of development and in lenses isolated at 11 days of development and cultured for three days. They are also found in both epithelium and fibers when separated at 10 days of embryonic development and in the epithelium of 141/2-month-old hen lenses. In the anucleate mature hen lens fibers, the only detected enzyme is thymidine kinase. Previous results showed that repair of X-ray irradiated DNA was total in 11-day-old embryonic lenses while repair was not detected at six days. On the other hand, repair was very much impaired when the 11-day-old lenses were cultured for three days. Enzymic analyses suggest that the defect in DNA repair and the spontaneous DNA degradation observed in chick lenses at certain embryonic stages, are not due to the absence of any of the above enzymic activities. Alternative explanations are discussed.

Animals↗
PubMed1980

Cycloheximide effect on DNA degradation and delta-crystallin synthesis in terminally differentiating lens cells.

Low concentrations of a protein synthesis inhibitor, cycloheximide, were added throughout the process of in vitro differentiation of 11-day old embryonic chick lens cells. We found with low concentrations of cycloheximide (0.01 to 0.03 microgram/ml, 3 days of culture), that there was an almost complete delay of DNA degradation as observed on alkaline sucrose gradient. Identical concentrations and exposure time had no blocking effect on increased delta-crystallin synthesis as detected by immunoprecipitation and electrophoresis. Higher concentrations of cycloheximide (0.1 to 1 microgram/ml) showed a marked effect on DNA size and a net inhibition on delta-crystallin synthesis. Thus a selective effect of low doses of cycloheximide was observed on terminal differentiation suggesting that there was not a relationship between DNA degradation and delta-crystallin synthesis in these short term experiments. The investigations of minor proteins could be of interest as they may have a crucial role in intact nuclei cataracts.

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