Cyst hatching in Anostraca accelerated by retinoic acid,amplified by Calcium Ionophore A23187, and inhibited by Calcium-channel blockers

Cyst hatching, under standardized conditions, of the Anostracan species Thamnocephalus platyurus and Streptocephalus dichotomus was significantly accelerated but not increased by applying the morphogen retinoic acid (RA). Cyst hatching was enhanced but not accelerated by artificially increasing the inflow of Ca²⁺ to the embryonic cells, using Calcium Ionophore A 23 187. Cyst hatching was accelerated and amplified, to a level in excess of the summed effects of each treatment, by a combined application of RA and ionophore. It was inhibited almost quantitatively by the Calcium-channel blockers Nifedipin and Verapamil. The significance of these findings is discussed.     

Assay of tannase (tanin acylhydrolase EC 3.1.1.20) by gas chromatography

A gas chromatographic method has been satisfactorily developed for the determination of gallic acid after enzymatic hydrolysis of methyl gallate by fungal tannase. The technique described herein permits a specific, quantitative analysis of the enzyme. The separate determination of both soluble and fixed tannase is described.     

Incorporation of the transmembrane hydrophobic domain of glycophorin into small unilamellar phospholipid vesicles Ion Flux studies

Human erythrocyte glycophorin is one of the best characterized integral membrane proteins. Reconstitution of the membrane-spanning hydrophobic segment of glycophorin (the tryptic insoluble peptide released when glycophorin is treated with trypsin) with liposomes results in the production of freeze-fracture intrabilayer particles of 80 Å diameter (Segrest, J.P., Gulik-Krzywicki, T. and Sardet, C. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 3294–3298), with particles appearing at or above a tryptic insoluble peptide concentration of 4 mmol per mol phosphatidylcholine. In the present study, increasing concentrations of tryptic insoluble peptide were added to sonicated small unilamellar egg phosphatidylcholine vesicles and the rate of efflux of ²²Na⁺ was examined by rapid (30 s) gel filtration on Sephadex G-50. Below a concentation of 3–5 mmol tryptic insoluble peptide/mol phosphatidylcholine, ²²Na⁺ efflux occurs at a constant slow rate at given tryptic insoluble peptide concentrations. Above a concentration of 3–5 mM, the rate of efflux is biphasic at given tryptic insoluble peptide concentrations, exhibiting both an initial fast and a subsequent slow component. On the basis of graphic and computer curve-fitting analysis, with increasing tryptic insoluble peptide concentration, the rate of the slow component reaches a plateau at a tryptic insoluble peptide concentration of 3–5 mM and remains essentially constant until much higher concentrations are reached; the fast component increases linearly with increasing tryptic insoluble peptide concentration well beyond 5 mM. The most consistent interpretation of this data is as follows. The slow ²²Na⁺ efflux component is due to perturbations of small unilamellar vesicle integrity by tryptic insoluble peptide monomers. At a tryptic insoluble peptide concentration of 3–5 mmol/mol, a critical concentration is reached following which there is intrabilayer tryptic insoluble peptide self-association. The fast ²²Na⁺ efflux component is due to the increasing presence of tryptic insoluble peptide self-associated multimers the 80-Å particles seen by freeze-fracture electron microscopy) which results in a significantly larger bilayer defect than do tryptic insoluble peptide monomers. The failure of complete saturation of efflux by the fast component is ascribed to the presence of two populations of small unilamellar vesicles, some of which contain tryptic insoluble peptide multimers and some of which do not.Addition of cholesterol to the tryptic insoluble peptide/phosphatidylcholine vesicles decreases the rate of ²²Na⁺ efflux by inhibiting primarily the fast component. Freeze-fracture electron microscopy indicates that the presence of cholesterol has no effect on the size, number or distribution of 80-Å intra-bilayer particles in the tryptic insoluble peptide/phosphatidylcholine vesicles. These results are consistent with a mechanism to explain the fast Na⁺ efflux component involving protein-lipid boundary perturbations.Efflux of ⁴⁵Ca²⁺ from phosphatidylcholine vesicles is also enhanced by incorporation of tryptic insoluble peptide, but only if divalent cations (Ca²⁺ or Mg²⁺) are present in the external bathing media as well as inside the sonicated vesicles. If monovalent Na⁺ only is present in the bathing media no ⁴⁵Ca²⁺ efflux is seen. Under conditions where ⁴⁵Ca²⁺ efflux is seen, both a fast and a slow component are present, although both appear lower than corresponding rate constants for ²²Na⁺ efflux. These results suggest a coordinated mechanism for ion efflux induced by tryptic insoluble peptide and, together with the ²²Na⁺ efflux studies, may have mechanistic implications for the transbilayer phospholipid exchange (flip-flop) suggesed to be induced at glycophorin/phospholipid interfaces (de Kruiff, B., van Zoelen, E.J.J. and van Deenen, L.L.M. (1978) Biochim. Biophys. Acta 509, 537–542).     

Additions a la faune de sélaciens fossilesdu Maroc: 1: Sur la présence des genres Heptranchias,Alopias etOdontorhytis dans l'Yprésien des Ouled Abdoun

Recent researches in the phosphat-bearing basins ofMorocco have allowed to collect new selachian forms. In this paper, three Ypresian genera from Ouled Abdoun basin, new for Morocco, are described: Heptranchias howellii (Reed, 1946), Alopias denticulatus nov. sp. and Odontorhytis pappenheimiBöhm, 1926. The re-discovery of the genus Odontorhytis allows to state precisely its morphological dental features and to affirm that it is well a selachian and not a teleostean, as it was thought. The stratigraphic distribution of some species is stated precisely.     

Androgen on the estrogen receptor I — Binding and in vivo nuclear translocation

In the immature rat uterus, high concentrations of androgens competed specifically with estradiol on the estrogen receptor (RE). This competition was stereospecific for C₁₉ steroids bearing a 17β and/or 3 hydroxyl group. Very low affinity ligands, such as testosterone, could not compete with estradiol at equilibrium but decreased the association rate of estradiol on its receptor. High doses (> 0.4mg) of 5 α aihydrotestosterone provoked $\text{in vivo}$ as $\text{in vitro}$ the nuclear translocation of RE. The nuclear receptor thus formed displayed the same 5.2 S sedimentation constant as that induced by estradiol. We conclude that the weak affinity binding of androgens to the estrogen receptor is sufficient to induce its nuclear translocation $\text{in vivo}$ provided androgen concentration is high enough in uterus to occupy the estradiol binding site. Conversely, progesterone which does not bind RE could not provoke its nuclear translocation.     

Observations nouvelles sur les otolithes des Téléostéens du Calcaire Grossier (Eocène du bassin de Paris)

An important new collection of otoliths from theCalcaire Grossier of the Paris Basin, as well as a critical review of already published material is studied and reveals the presence of 56 species. Five of these are new: Muraenesox spatulus, Genus Synodontidarum intermedius, Genus Ophidiidarum spinosus, Chanda bohlkei and Mene sekharani. The assemblage is indicative of tropical to subtropical marine and neritic environment with solid bottom. Biogeographically, it shows affinities with the recent Indo-pacific fauna. The association of the sites at Fercourt and Château-Rouge is clearly different from the one found at the sites of Condé-en-Brie and Damery, as it is less littoral than the second association mentioned.     

Studies on an unstable phenotype induced by UV irradiation

Unstable clones excreting L-lysine into their growth medium are obtained at a very high frequency following UV irradiation in both haploid and diploid strains of Saccharomycopsis lipolytica, provided they carry a mutation affecting the first enzyme of the lysine pathway and confering resistance to end product inhibition. The phenotype can be stabilized in some sublines; it appears as dominant and coupled with a decrease in spore viability. Excretion in batch cultures is confined to the end of the exponential phase, and seems not to consist in a simple release of the lysine pool content.     

Use of an inhibitor to identify members of the hormone-sensitive lipase family

Hormone-sensitive lipase (HSL) contributes importantly to the mobilization of fatty acids from the triacylglycerols stored in adipocytes, which provide the main source of energy in mammals. On the basis of amino acid sequence alignments and three-dimensional structures, this enzyme was previously found to be a suitable template for defining a family of serine carboxylester hydrolases. In this study, the HSL family members are characterized rather on the basis of their inhibition by 5-methoxy-3-(4-phenoxyphenyl)-3H-[1,3,4]oxadiazol-2-one (compound 7600). This compound inhibits mammalian HSL as well as other HSL family members, such as EST2 from the thermophilic eubacterium Alicyclobacillus acidocaldarius and AFEST from the hyperthermophilic archaeon Archaeoglobus fulgidus. Various carboxylester hydrolases that are not members of the HSL family were found not to be inhibited by compound 7600 under the same experimental conditions. These include nonlipolytic hydrolases such as Torpedo californica acetylcholinesterase and pig liver esterase, as well as lipolytic hydrolases such as human pancreatic lipase, dog gastric lipase, Thermomyces lanuginosus lipase, and Bacillus subtilis LipA. When vinyl esters were used as substrates, the residual activity of HSL, AFEST, and EST2 decreased with an increase in compound 7600 concentration in the incubation mixture. The inhibitor concentration at which the enzyme activity decreased to 50% after incubation for 5 min was 70, 20, and 15 nM with HSL, AFEST, and EST2, respectively. Treating EST2 and AFEST with the inhibitor resulted in an increase in the molecular mass, as established by performing matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis. This increase in the molecular mass, which corresponds approximately to the molecular mass of the inhibitor, indicates that a covalent enzyme-inhibitor complex has been formed. Surface-enhanced laser desorption ionization time-of-flight mass spectrometry analysis of a trypsin digest of AFEST treated with the inhibitor or not treated showed the occurrence of an increase in the molecular masses of the "GESAGG"-containing peptide, which is compatible with the formation of a covalent complex with the inhibitor.