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M Philippe

Publications and source records attributed to M Philippe.

At least 145 records · Page 8Linked to original sources
PubMed1978

Extraction and biochemical characterization of a nuclear deoxyribonucleic acid polymerase activity in bull spermatozoa.

Bull spermatozoa heads were separated from cytoplasmic contaminants, especially mitochondria-rich middle pieces, by centrifugation through 2.4M-sucrose. DNA polymerase activity was demonstrated by incubating nuclear heads for 1 h at 37 degrees C or for 20 h at room temperature in a medium containing detergent and dithiothreitol or 2-mercaptoethanol. Optimal DNA polymerase activity was detected after extraction in a medium containing 50 mM-borate, pH9, 1 mg of soya-bean trypsin inhibitor/ml and supplemented with either 20 mM-dithiothreitol and 4% Tween 80 or 100mM-2-mercaptoethanol and 10% Tween 80. The DNA polymerase reaction was Mg2+-dependent; Mn2+ or Ca2+ could not replace Mg2+ and all four deoxynucleoside triphosphates were required for optimal activity. The polymerase activity was pH-dependent (optimum between 8.2 and 10.5) and was a function of buffer composition and also of pH values. Optimal activity was obtained with 50 mM-Na+ or 150mM-K+ and was partially lowered by N-ethylmaleimide; it was inhibited by spermidine and by salmon protamines, but was greatly stimulated by calf thymus histones. It was also resistant to actinomycin D, netropsin and ethidium bromide. The present results suggest that bull spermatozoa heads contain a beta-type DNA polymerase activity.

Animals↗
PubMed1978

Presence of two deoxyribonucleic acid polymerases in bull spermatozoa.

A DNA polymerase-endogenous template complex was isolated from nuclear heads of bull spermatozoa. The buoyant density of the complex was 1.15 g/cm 3. The sedimentation coefficient of the nuclear DNA polymerase isolated from the complex was higher at low ionic strength, but approached 3.4S when centrifuged in a medium containing 2M-KCl. Activated exogenous DNA increased polymerase activity. Only very low activities were detected with synthetic templates such as poly(A).(dT)12-18 and poly(dT).poly(A). The nuclear reaction was stimulated by 150mM-KCl and was slightly inhibited by N-ethylmaleimide; it was resistant to actinomycin D, netropsin and ethidium bromide. Another DNA polymerase, highly sensitive to ethidium bromide, was extracted from the mitochondira-rich middle-piece fraction. Its sedimentation coefficient was close to 9S, but fell to approx. 4S in high-ionic-strength medium.

Animals↗
PubMed1977

Heterogeneity of steroid hormone receptor in adult rat lung cytosol.

Chromatography on cellulose DEAE-52 columns revealed that the glucocorticoid receptor for rat lung cytosol consisted of a component in the 0.001 M prewash, revealed with synthetic steroids and natural mineralocorticoids, a second component eluted with 0.04 M PO4, labelled with triamcinolone, dexamethasone, and a third moiety in the 0.06 M PO4 region, evident with natural glucocorticoids (corticosterone, cortisol) as well as mineralocorticoids (aldosterone, deoxycorticosterone). The third component coeluted with rat blood serum transcortin in double labelled experiments. Rate lung was devoid of another component in the 0.02 M PO4 found in rat liver supernate and of the mineralocorticoid receptor evident only in rat kidney. Chromatography on Sephadex G-200 columns revealed a shift of radioactivity from a higher to a lower molecular weight region in the presence of 0.4 M KCl. Collectively, these studies indicate the subunit nature of the lung receptor as evidenced in most tissues hitherto tested. Moreover, polymorphism within a given subunit component can not be revealed by competition alone, as attempted by others, but can be revealed under selected conditions of physical separation.

Aldosterone↗
PubMed1977

In situ detection and characterization of DNA polymerase activities in the nucleus of eukaryotic cells.

We have developed a cytoenzymological method for localizing DNA polymerase activities in situ and for studying their responses to various chemical agents or environmental conditions. The incubation mixtures and the stimulatory or inhibitory agents added to these media were defined with reference to in vitro biochemical tests used to detect and to characterize DNA polymerases-alpha or -beta found in eukaryotic cells. This method has already been used to study DNA polymerase activities during cell differentiation or cell senescence. Apart from two exceptions found with lower organisms, the nuclear DNA polymerase activity was always higher under conditions which favoured the in vitro expression of DNA polymerase-beta rather than DNA polymerase-alpha. --In the various cell types studied, the cellular DNA polymerase activities were almost exclusively found in the nuclei. It is hoped that this methodology will be useful for obtaining more complete biochemical data on the intracellular localization of various DNA polymerases.

Animals↗
PubMed1977

[Deep hypothermia on the infant: physiopathology and technics of ECC].

In 81 operations for correction of infants's cardiopathies, authors used, associated with E.C.C., a deep hypothermia allowing a circulatory arrest of an average duration of 52 minutes, according to the technics described by BARRAT-BOYES in 1971. From this experience, authors study the modifications brought to the organism by this hypothermia, and discuss the technical aspects in pre, per and post-operative periods. Mortality of this series is of 13,5 p. 100. It is in relation with the cardiopathy or it's correction, without anu possibility or directly charge the technique of hypothermia in its determinism. The early mortality includes a bilateral phrenic paralysis, an air embolism, three septic complications and two neurologic complications probably related to a poor thermic repartition. Advantages of this technique concern the possibility to operate in a bloodless field and a diminution of E.C.C. time.

Coronary Vessel Anomalies↗
PubMed1976

Further characterization of a DNA polymerase activity in mouse sperm nuclei.

The presence of a nuclear DNA polymerase in mouse sperm from adult testes has been confirmed and the properties of this enzyme further investigated. This activity was shown to be greatly enhanced by treating the spermatozoa with methanol or ethanol before incubation in the reaction medium or by their addition in small amounts to this medium. It was protected against degradation by nuclear proteases by adding soybean trypsin inhibitor and was stimulated by ATP. It was found to be Mg2+ dependent (optimum concentration: 7.5 mM), DNA dependent, and all four deoxynucleoside triphosphates were needed for optimal reaction. The radioactive acid-precipitable product of polymerization was not eliminated by organic solvents, nor by pronase, ribonuclease or by nuclease S1; however, it was converted to a large extent to acid-soluble products by pancreatic deoxyribonuclease. Since it was only partially solubilized by Triton X-100, it therefore did not appear to be preferentially associated with the nuclear membranes. The activity recovered after incubation depended also on the pH (optimum at pH 8.3) and did not work well in a medium for DNA polymerase alpha. The temperature for maximum incorporation of nucleotides was found to be 32 degrees C and, under our conditions, the reaction was linear for 30 min. The DNA polymerase activity was inhibited by low and high concentrations of KCl. It was not lowered by N-ethylmaleimide or p-hydroxymercuribenzoate; urea slightly stimulated the reaction and this stimulation was reversed by subsequent treatment with N-ethylmaleimide. Actinomycin D (40 mug/ml), ethidium bromide (25--50 muM), netropsin (5--50 mug/ml), and spermidine (0.5--2.5 mM) lowered the polymerization of DNA precursors. The nuclear enzyme could shift from the endogenous template to activated exogenous calf thymus DNA, the resulting nuclear radioactivity being reduced. The endogenous DNP template ability was not increased by deoxyribonuclease activation according to the method of Aposhian and Kornberg (J. Biol. Chem. (1962) 237, 519--525) suggesting that the amount of DNA polymerase associated with chromatin was probably limiting the reaction. The DNA polymerase activity detected in mouse sperm nuclei has numerous properties of low molecular weight DNA polymerases (DNA polymerase beta) reported in several eukaryotic organisms.

Animals↗
PubMed1976

In situ detection of a DNA-polymerase activity in the nuclei of mouse spermatozoa.

A nuclear DNA-polymerase activity has been detected in situ in mouse spermatozoa with a cytochemical method. The acid-insoluble radioactive product obtained after incubation in a mixture containing all four deoxyribonucleoside 5'-triphosphates, Mg++,KCl and dithiothreitol (DTT) was completely removed by DNAse but was insensitive to RNAse and pronase action. The polymerization reaction did not take place in the presence of only one nucleotide and was dramatically reduced when actinomycin D was present; it did not seem to be inhibited markedly by N-ethylmaleimide (NEM) or by parahydroxymercuribenzoate (PHMB) but was sensitive to high concentrations of KCl.--The reaction depended strictly on the presence of nuclear DNA as template since treatment with pancreatic DNAae before the DNA-polymerase assay completely prevented the appearance of any radioactivity.

Animals↗