Physical properties and function of phallolysin

Phallolysin, a mixture of two to three cytolytic proteins (all of Mr 34 000), has been isolated from Amanita phalloides mushrooms and purified to homogeneity (specific activity 24 000 hemolytic units/mg of protein). After separation by isoelectric focusing, the amino acid composition of two of these proteins has been determined. They are rich in water-soluble amino acids and contain one tryptophan residue each, but no cysteine or methionine. Mr was determined to be 34 000 in the native form as well as under denaturing conditions, indicating that the native proteins exist as monomers. Many of the physical properties of phallolysin are strikingly similar to those of staphylococcal alpha-toxin, e.g., molecular weight, existence of multiple forms, pI values, amino acid composition, and thermolability (60 degrees C). Pure phallolysin allowed us to prepare a radioactively labeled toxin. Labeling was achieved by reaction with formaldehyde, followed by reduction with sodium [3H]borohydride. With the labeled toxin (specific activity 7-14 Ci/mmol, ca. 60% biological activity), we investigated its binding to human A2 erythrocytes. We determined the number of receptors on these cells (2 X 10(4) per cell) as well as their affinity to the toxin (KD = 4 X 10(-9) M). In studies on the mechanism of cytolytic activity, we were able to distinguish at least three sequential events: binding of the toxin to human erythrocytes, K+ release, and membrane rupture (hemoglobulin release). These steps could be characterized by different kinetics as well as by different temperature dependencies. Again, the kinetic data for phallolysin are very closely related to those obtained for staphylococcal alpha-toxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calorimetric measurement of free energy utilization by Nitrosomonas and Nitrobacter

Summary Calorimetric estimates of the utilization efficiency of the free-energy derived from substrate oxidation by cell suspensions of two nitrifying bacteria, Nitrosomonas and Nitrobacter, provided two ranges of values: 11 to 27% and 15 to 51%, respectively. About 15 to 30% of the utilized free-energy is used for driving endergonic reactions other than CO₂ fixation, probably the synthesis of polyphosphates.The molar heat of substrate oxidation does not seem to be influenced by the age of cells harvested during growth or by the length of the incubation period during which cells have been kept in a buffer suspension in a starved condition. The loss of respiratory activity measured either by oxygen uptake or heat evolution in the presence of the specific substrate, nitrite or ammonium, decreases according to kinetics which are influenced by the aerobiosis of the suspension. The viability of the starved cells decreases in a way which is similar to that of the respiratory activity. It seemed impossible to obtain cells which had lost their viability but kept the ability to oxidize their substrate.Two inhibitors of the respiratory chain, quinacrine and cyanide, are without effect on the molar heat of substrate oxidation and consequently on the free-energy utilization efficiency. 2.4 dinitrophenol did decrease the rate of heat evolution during substrate oxidation at concentrations at which the rate of oxygen uptake was not depressed, with the consequences that free-energy efficiency was apparently increased.     

L'excursion en Provence (Sud-Est de la France) de la Societe Internationale de Phytosociologie

Conclusions La Provence apparaît, en définitive, comme un territoire des plus variés aux divers points de vue climatique, édaphique, biotique.C'est une région d'autant plus favorable aux études phytosociologiques que la variété des milieux s'y double d'une brutalité souvent étonnante des contrastes, conduisant à la juxtaposition parfois extrêmement précise d'associations végétales très différentes, dont les limites peuvent alors être cartographiquement figurées avec une très suffisante exactitude.Cette vigueur des contrastes est surtout marquée en Provence calcaire où les divers termes des séries évolutives dérivant du Quercetum ilicis climacique ou y conduisant, se juxtaposent souvent sans transition de quelque importance.Les contrastes sont cependant moins accusés en Provence cristalline et dans la Basse-Vallée du Rhône.En Crau comme en Camargue, les associations végétales se juxtaposent souvent en ceintures parallèles ou concentriques — elles s'individualisent alors facilement —, souvent en mosaïques irrégulières, au grè des variations locales des deux facteurs déterminants: l'eau en Crau, le sel en Camargue.En Provence cristalline, les Maures étant surtout faites de roches se délitant facilement (grès, micaschistes et phyllades) sous un climat chaud et humide, le sol se reconstitue facilement après sa destruction partielle par l'érosion; les contrastes sont moins tranchés; on observe des transitions nombreuses entre les divers termes des séries évolutives, dont les limites ne peuvent être fixées qu'approximativement. Nous y avons notamment abordé le problème de la forêt de chêne-liège dont la signification est loin d'être connue, non seulement en Provence mais sur toute l'étendue de son aire, en Afrique du Nord, en Espagne, au Portugal, en France méridionale, en Italie et dans les Iles de la Méditerranée occidentale.En provence cristalline, il est certain que l'homme en a considérablement assuré l'extension, directement pour l'utilisation du liège, indirectement par la coupe et le feu, et, en bien des points de la Méditerranée occidentale, le chêne-liège se présente comme une essence cultivée.Par ailleurs rien ne permet de mettre en doute son indigénat. Mais, dans les conditions actuelles du milieu en Provence cristalline:le chêne vert est possible partout;dans les conditions naturelles de la concurrence vitale le chêne vert l'emporte sur le chêne-liège.Si, donc, la forêt originelle a comporté le chêne-liège, ce ne peut être qu'à l'état d'essence secondaire et seulement en quelques points où une faible couverture forestière lui permettait de cohabiter avec le chêne vert.Les documents cartographiques au I/20 000^e tendent à montrer, d'aileurs que, dans les conditions naturelles de reconstitution de la forêt ou dans le peuplement naturel des sols neufs (Isthme de Giens), c'est la chênaie de chêne vert, non la chênaie de chêne-liège qui s'installe.Les grandes unités phytosociologiques de la Provence paraissent être maintenant convenablement définies floristiquement; il convient donc d'en poursuivre l'étude écologique, dont certains aspects seulement les plus apparents—mais peut-être pas toujours les plus actifs-, ont été abordés.Notons enfin la démonstration faite, au cours de l'excursion, de la parfaite application possible des méthodes phytosociologiques à l'étude des groupements marins et la nécessité, particulièrement apparente en milieu marin, de considérer tout autant les animaux que les végétaux. L'avenir est bien aux biocénoses dont les grands traits ont été mis en évidence, quant à la côte de la presqu'île de Giens, par Roger Molinier.Reçu par la rédaction le 20. XII. 1958.avec la collaboration de ROGER MOLINIER pour la partie marine et G. TALLON pour la Camargue.     

Photoaffinity labelling of nuclear steroid 5 alpha-reductase of rat ventral prostate

In order to get more information on the molecular structure of the rat prostatic 5 alpha-reductase (3-oxo-5 alpha-steroid: NADP+ 4-ene-oxidoreductase, EC 1.3:1.22) a systematic photoaffinity labelling study has been performed. To irreversibly freeze the status quo of interaction, either testosterone, the physiological ligand, or diazo-MAPD (21-diazo-4-methyl-4-aza-5 alpha-pregnane-3,20-dione), a specific 5 alpha-reductase inhibitor, was irradiated with isolated nuclei or with purified nuclear membranes or with solubilized nuclear membrane proteins and checked for optimal labelling conditions. The principal substances covalently labelled were phospholipids and at a minor ratio proteins. Analysis by SDS-PAGE and autoradiofluorography revealed two labelled polypeptides with molecular weights of 20 kDa and 26 kDa. The following evidence indicates that these polypeptides might be derived from the enzyme 5 alpha-reductase: both proteins are labelled only when specific ligands for 5 alpha-reductase are used; binding can be reduced by the addition of an excess of unlabelled ligand; enzyme activity is irreversibly suppressed when irradiated in the presence of these ligands; only subcellular fractions containing 5 alpha-reductase reveal the labelled proteins; in all 5 alpha-reductase containing preparations with increasing specific activity, independent of the polypeptide pattern, the same proteins are labelled.     

The tryptophan residues of aspartate transcarbamylase: site-directed mutagenesis and time-resolved fluorescence spectroscopy.

Aspartate transcarbamylase (EC 2.1.3.2) contains two tryptophan residues in position 209 and 284 of the catalytic chains (c) and no such chromophore in the regulatory chains (r). Thus, as a dodecamer [(c3)2(r2)3] the native enzyme molecule contains 12 tryptophan residues. The present study of the regulatory conformational changes in this enzyme is based on the fluorescence properties of these intrinsic probes. Site-directed mutagenesis was used in order to differentiate the respective contributions of the two tryptophans to the fluorescence properties of the enzyme and to identify the mobility of their environment in the course of the different regulatory processes. Each of these tryptophan residues gives two independent fluorescence decays, suggesting that the catalytic subunit exists in two slightly different conformational states. The binding of the substrate analog N-phosphonacetyl-L-aspartate promotes the same fluorescence signal whether or not the catalytic subunits are associated with the regulatory subunits, suggesting that the substrate-induced conformational change of the catalytic subunit is the essential trigger for the quaternary structure transition involved in cooperativity. The binding of the substrate analog affects mostly the environment of tryptophan 284, while the binding of the activator ATP affects mostly the environment of tryptophan 209, confirming that this activator acts through a mechanism different from that involved in homotropic cooperativity.     

A new species of Lernanthropus De Blainville, 1822 (Copepoda: Lernanthropidae) parasitic on Menticirrhus ophicephalus (Jenyns) (Teleostei: Sciaenidae) from the Peruvian coast

Lernanthropus huamani n. sp. (Copepoda: Lernanthropidae), a parasite of the Peruvian sciaenid fish Menticirrhus ophicephalus (Jenyns), is described and illustrated. The new species differs from all other species of Lernanthropus by a combination of characters, including the dorsal plate, legs and other appendages. L. guacoldae Villalba & Fernández, 1984 is considered a synonym of L. pacificus Oliva & Duran, 1982.     

Enhanced immunocytochemical staining sensitivity in formalin-fixed and paraffin-embedded material by simultaneous use of PAP and ABC

The simultaneous use of two immunoperoxidase (IP) methods; e.g. the PAP (Peroxidase-antiperoxidase) and ABC (Avidin-Biotin) to localize a single antigen enhances the sensitivity of polyclonal antibodies (FVIII/RAg and laminin) or monoclonal antibodies (MABs) (against human B-, T-cells and macrophages) in routinely formalin fixed and in paraffin embedded material. The increased sensitivity was not accompanied by loss of specificity. With antibodies against Factor VIII related antigen (FVIII/RAg) and laminin the increased staining intensity of blood vessels were demonstrated in experimental rat transplantation tumors. The similar enhancement of immunocytochemical reactions was observed with biopsies of human brain tumors, where the monoclonal antibodies against white blood cells were used. The staining results were of comparable intensity as in snapfrozen tissue or after special embedding procedure for immunocytochemical purposes acc. to Bolton and Mesnard formol sucrose gum, sucrose paraffin; FSGSP).