Duck hepatitis B virus can tolerate insertion, deletion, and partial frameshift mutation in the distal pre-S region.

In-frame and frameshift mutations were introduced into the pre-S region (1,212 base pairs) of duck hepatitis B virus. The in-frame mutants retained the inserted 12 nucleotides, while the frameshift mutants either reverted to wild type or exhibited a 10-nucleotide compensatory deletion downstream of the original mutation site. Thus, although duck hepatitis B virus has a compact and highly economical genome organization, it can replicate despite alterations of up to 9 amino acid codons in the pre-S and P open reading frames.     

Effects of MPTP, MPP+ and paraquat on mitochondrial potential and oxidative stress

The effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-phenylpyridinium (MPP+) and 1,1-dimethyl-4,4-bipyridinium (paraquat) upon the electrical potential across the plasma and mitochondrial membranes within synaptosomes has been investigated. MPTP selectively depressed plasma membrane potential while MPP+ specifically reduced mitochondrial potential. The structurally similar compound paraquat had no effect on either membrane potential. Enhancement of the lipid peroxidative activity with an Fe-ADP complex depressed both potentials. Paraquat effected increased peroxidative activity in brain homogenates that was less pronounced than that due to Fe-ADP. MPTP reduced basal but stimulated Fe-ADP enhanced peroxidation. The mechanisms underlying the toxicity of MPP+ are likely to differ from those of paraquat, primarily involving impaired mitochondrial function rather than increased oxidative stress.     

Metabolism of Pyrene by the Basidiomycete Crinipellis stipitaria and Identification of Pyrenequinones and Their Hydroxylated Precursors in Strain JK375

The metabolism of pyrene, a polycyclic aromatic hydrocarbon, by submerged cultures of the basidiomycete Crinipellis stipitaria was studied. After incubation for 68 h at 25°C in a 20-liter fermentor with complex medium and 20 mg of pyrene per liter, five metabolites were detected. The compounds were isolated by preparative high-performance liquid chromatography on RP18 and DIOL gels. By UV, infrared, and ¹H nuclear magnetic resonance spectroscopy and mass spectrometry, 1-hydroxypyrene, 1,6-dihydroxypyrene, 1,8-dihydroxypyrene, 1,6-pyrenequinone, and 1,8-pyrenequinone were identified. 1,6- and 1,8-dihydroxypyrene were obtained from fungal cultures for the first time. The formation of these metabolites was confirmed by investigations with [4,5,9,10-¹⁴C]pyrene.     

Cell cycle dependent distribution of a centrosomal antigen at the perinuclear MTOC or at the kinetochores of higher plant cells

Compelling evidence has been obtained in favour of the idea that the nuclear surface of higher plant cells is a microtubule-nucleating and/or organizing site (MTOC), in the absence of defined centrosomes. How these plant MTOC proteins are redistributed and function during the progression of the cell cycle remains entirely unknown. Using a monoclonal antibody (mAb 6C6) raised against isolated calf thymus centrosomes and showing apparent reaction with the plant nuclear surface, we followed the targeted antigen distribution during mitosis and meiosis of higher plants. Immunoblot analysis of protein fractions from Allium root meristematic cell extracts probed with mAb 6C6 reveals a polypeptide of an apparent Mr of 78000. In calf centrosome extracts, a polypeptide of comparable molecular mass is found in addition to a major antigen of Mr 180000 after mAb 6C6 immunoblotting. During mitotic initiation, the plant antigen is prominent on the periphery of the prophase nucleus. When the nuclear envelope breaks down, the antigen suddenly becomes associated with the centromere-kinetochores until late anaphase. In telophase, when the nuclear envelope is being reconstructed, it is no longer detected at the kinetochores but is solely associated again with the nuclear surface. This antigen displays a unique spatial and temporal distribution, which may reflect the pathway of plant protein(s) between the nuclear surface and the kinetochores under cell cycle control. So far, such processes have not been described in higher plant cells. These observations shed light on the putative activity of the plant kinetochore as a protein transporter. They also suggest that a plant centrosome-like antigen may have different cytoskeletal related functions depending on cell cycle regulated changes in its subcellular distribution.Abbreviations mAb monoclonal antibody - MSB microtubule stabilizing buffer - TBS Tris buffered saline - MTOC microtubule organizing centre     

Monogènes Dactylogyridae parasites de Cyprinidae du genreBarbus d'Afrique du Nord

Resumé Quinze nouvelles espèces de Monogènes Dactylogyridae sont décrites chez quinze espèces deBarbus (Teleostei, Cyprinidae) appartenant aux sous-genresB. (Barbus) etB. (Labeobarbus) en Afrique du Nord. Les barbeaux examinés proviennent des différents bassins hydrographiques du Maroc et d'une localité nommée Hamman Bourgiba en Tunisie. Dans cette dernière région, le genreBarbus n'est représenté que par une seule espèce:Barbus (B.) callensis. Au Maroc, on en dénombre actuellement quatorze dont quatre appartiennent au sous-genreLabeobarbus: il s'agit deBarbus (L.) fritschii; B. (L.) harteti; B. (L.) paytonii etB. (L.) reinii. Les dix espèces appartenant au sous-genreBarbus sont:Barbus (B.) figuiensis; B. (B.) ksibi; B. (B.) lepineyi; B. (B.) magniatlantis; B. (B.) massaensis; B. (B.) moulouyensis; B. (B.) nasus; B. (B.) pallaryi; B. (B.) setivemensis etB. (B.) issenensis.Chaque sous-genre possède son propre pool parasitaire, à l'exception deDactylogyrus marocanus n. sp., recontré sur des espèces appartenant aux deux sous-genres (B. (L.) fritschii, B. (L.) paytonii, B. (L.) harteti, B. (L.) reinii, B. (L.) nasus, B. (B.) setivimensis, B. (B.) ksibi). Sur les cinqDactylogyrus parasitant lesLabeobarbus, trois présentent une spécificité stricte vis à vis de leur hôte. Il s'agit deDactylogyrus reinii n. sp. surB. (L.) reinii; D. volutus n. sp. etD. zatensis n. sp. surB. (L.) fritschii. Les espècesD. oumiensis n. sp. etD. kulindrii n. sp. présentent une spécifité stenoxène et parasitent respectivementB. (L.) harteti, B. (L.) paytonii, B. (L.) reinii etB. (L.) fritschii, B. (L.) reinii.Nous avons recontré neufDactylogyrus chez les poisson-hôtes appartenent au sous-genreBarbus. Six d'entre eux ont une spécificité oïoxène; ce sont:D. guirensis n. sp.,D. atlasensis n. sp. etD. draaensis n. sp. surB. (B.) pallaryi; D. borjensis n. sp. surB. (B.) nasus etD. heteromorphus n. sp. etD. tunisiensis n. sp. surB. (B.) callensis. Les trois autres parasites ont un spectre d'hôtes plus large. Il s'agit deD. ksibii n. sp. recontré chezB. (B.) ksibi, B. (B.) setivimensis etB. (B.) magniatlantis; D. ksibioïdes n. sp. recontré chezB. (B.) setivimensis etB. (B.) moulouyensis. L'espèceD. fimbriphallus n. sp. stenoxène, se recontre chez les poisson-hôtes du versant Sud de l'Atlas et de la façade méditerranéenne à savoir:B. (B.) figuiensis, B. (B.) lepineyi, B. (B.) massaensis, B. (B.) moulouyensis, B. (B.) pallaryi etB. (B.) issenensis.Le rôle des Dactylogyridae en tant que marqueurs biogéographiques, phylogénétiques et taxonomiques est discuté à partir de la composition spécifique des communautés de Monogènes rencontrés et de leurs différents types morphologiques.

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Fifteen new species of the Dactylogyridae (Monogenea) parasitic on fifteen species of barbels (Barbus) from North Africa (Teleostei, Cyprinidae) are described. The fishes studied belong to two subgenera,B. (Labeobarbus) andB. (Barbus), collected from various hydrographical basins of Morocco and from the Hamman Bourgiba locality in Tunisia. In the latter area, the genusBarbus is represented by onlyBarbus (Barbus) callensis. In Morocco, fourteen species are listed, four of which belong to the subgenusLabeobarbus; these areBarbus (L.) fritschii; B. (L.) harteti; B. (L.) paytonii andB. (L.) reinii. The other ten species belong to the subgenusBarbus: these areBarbus (B.) figuiensis; B. (B.) ksibi; B. (B.) lepineyi; B. (B.) magniatlantis; B. (B.) massaensis; B. (B.) moulouyensis; B. (B.) nasus; B. (B.) pallaryi; B. (B.) setivimensis andB. (B.) issenensis. Each of the two subgenera has its unique parasitic fauna, except forDactylogyrus marocanus n. sp. collected on species belonging to both subgenera (B. (L.) fritschii, B. (L.) paytonii, B. (L.) harteti, B. (L.) reinii, B. (B.) nasus, B. (B.) setivimensis andB. (B.) ksibi). Of the five monogeneans found onLabeobarbus, three appear to be specific to one host: they areDactylogyrus reinii n. sp. onB. (L.) reinii, andD. volutus n. sp. andD. zatensis n. sp. onB. (L.) fritschii. D. kulindrii n. sp. parasitisedB. (L.) reinii andB. (L.) fritschii; andD. oumiensis n. sp. occurred onB. (L.) reinii, B. (L.) paytonii andB. (L.) harteti. NineDactylogyrus species were found in fishes belonging to the subgenusBarbus. Six of them have an oïoxenous specificity: these areD. guirensis n. sp.,D. atlasensis n. sp. andD. draaensis n. sp. onB. (B.) pallaryi; D. borjensis n. sp. onB. (B.) nasus andD. heteromorphus n. sp. andD. tunisiensis n. sp. on(B.) callensis. These other three have a wider range of hosts: they areD. ksibii n. sp. collected fromB. (B.) ksibi, B. (B.) setivimensis andB. (B.) magniatlantis, andD. ksibioïdes n. sp. found onB. (B.) setivimensis andB. (B.) moulouyensis. D. fimbriphallus n. sp. is a characteristic parasite of fishes from the southern side of the Atlas mountains and the Mediterranean coast (B. (B.) figuiensis, B. (B.) lepineyi, B. (B.) massaensis, B. (B.) moulouyensis, B. (B.) pallaryi andB. (B.) issenensis).The role of dactylogyrids as biogeographical phylogenetic and taxonomic indicators is discussed in relation to the specific structure of the monogenean communities and the different morphological types found.

     

Artificial mosaic protein containing antigenic epitopes of hepatitis E virus.

A synthetic gene encoding an artificial polypeptide composed of antigenic epitopes of the hepatitis E virus (HEV) proteins was constructed from short oligodeoxyribonucleotides by using PCR. The polypeptide comprises a mosaic of three antigenically active dominant regions from the protein encoded by open reading frame 2 (ORF2), one antigenically active region from the protein encoded by ORF3 of the Burmese HEV strain, and one antigenically active region from the protein encoded by ORF3 of the Mexican HEV strain. The mosaic protein was expressed in Escherichia coli as a chimera with glutathione S-transferase or beta-galactosidase. Guinea pig sera containing antibodies to the corresponding HEV synthetic peptides were used to demonstrate by Western immunoblot analysis and enzyme immunoassay the presence and accessibility of all HEV-specific antigenic epitopes introduced into the mosaic protein. Both the glutathione S-transferase and beta-galactosidase hybrid proteins were analyzed by using a panel of human anti-HEV-positive and -negative sera. The data obtained strongly indicate a diagnostic potential for the mosaic protein.     

Volume regulation in human fibroblasts: role of Ca2+ and 5-lipoxygenase products in the activation of the Cl− efflux

Trypsinized human skin fibroblasts in suspension perform regulatory volume decrease (RVD) after cell swelling in hypotonic medium. During RVD, ³⁶Cl^– efflux is dramatically increased and the cell membrane is depolarized, indicating the activation of Cl^– channels. This activation of Cl^– channels depends on extracellular as well as on intracellular Ca²⁺. The swelling-induced Cl^– efflux and the RVD response are inhibited by the 5-lipoxygenase inhibitor ETH 615-139. Finally, following hypotonic treatment, cellular pH decreases. The pH decrease does not involve the Cl^–/HCO ₃ ^– exchange because it is independent of the external Cl^– concentration.T. Mastrocola was recipient of a scientific fellowship from the Italian Consiglio Nazionale delle Ricerche (C.N.R.). This work was supported by Progetto Finalizzato Ingegneria Genetica, C.N.R., Roma, and by the Danish Natural Research Council.     

Regulation of taurine transport in Ehrlich ascites tumor cells

Summary Taurine influx is inhibited and taurine efflux accelerated when the cell membrane of Ehrlich ascites tumor cells is depolarized. Taurine influx is inhibited at acid pH partly due to the concomitant depolarization of the cell membrane partly due to a reduced availability of negatively charged free carrier. These results are in agreement with a 2Na, 1Cl, 1taurine cotransport system which is sensitive to the membrane potential due to a negatively charged empty carrier. Taurine efflux from Ehrlich cells is stimulated by addition of LTD4 and by swelling in hypotonic medium. Cell swelling in hypotonic medium is known to result in stimulation of the leukotriene synthesis and depolarization of the cell membrane. The taurine efflux, activated by cell swelling, is dramatically reduced when the phospholipase A₂ is inhibited indirectly by addition of the anti-calmodulin drug pimozide, or directly by addition of RO 31-4639. The inhibition is in both cases lifted by addition of LTD₄. The swelling-induced taurine efflux is also inhibited by addition of the 5-lipoxygenase inhibitors ETH 615-139 and NDGA. It is concluded that the swelling-induced activation of the taurine leak pathway involves a release of arachidonic acid from the membrane phospholipids and an increased oxidation of arachidonic acid into leukotrienes via the 5-lipoxygenase pathway. LTD₄ seems to act as a second messenger for the swelling induced activation of the taurine leak pathway either directly or indirectly via its activation of the Cl^– channels, i.e., via a depolarization of the cell membrane.