Biochemical and immunological analysis of human immunodeficiency virus gag gene products p17 and p24.

Human immunodeficiency virus (HIV) p24 was purified to homogeneity and subjected to NH2-terminal sequencing. The sequence determined perfectly corresponded to the amino acid sequence predicted from the nucleotide sequence of a middle portion of the HIV first open frame: the gag gene. Edman degradation of purified HIV p17 revealed instead a blocked NH2 terminus. Hybridomas secreting monoclonal antibodies to p24 and p17 were developed and used to immunologically characterize these two HIV gag gene products. They identified two gag precursor polyproteins in the cytoplasm of HIV-infected cells: Pr53gag, which corresponds to the primary translational product, and Pr39gag, which corresponds to an intermediate product of cleavage of Pr53gag. These monoclonal antibodies allowed us also to study posttranslational modification of HIV p24 and p17. p24 was found to be phosphorylated, which is a very unusual feature for a major retroviral core protein. p17 was found to be myristylated, as are all NH2-terminal gag proteins of the known human retroviruses.     

Tracing paternal ancestry in mice, using the Y-linked, sex-determining locus, Sry

The molecular evolution of mammalian Y-linked DNA sequences is of special interest because of their unique mode of inheritance: most Y- linked sequences are clonally inherited from father to son. Here we investigate the use of Y-linked sequences for phylogenetic inference. We describe a comparative analysis of a 515-bp region from the male sex- determining locus, Sry, in 22 murine rodents (subfamily Murinae, family Muridae), including representatives from nine species of Mus, and from two additional murine genera--Mastomys and Hylomyscus. Percent sequence divergence was < 0.01% for comparisons between populations within a species and was 0.19%-8.16% for comparisons between species. Our phylogenetic analysis of 12 murine taxa resulted in a single most- parsimonius tree that is highly concordant with phylogenies based on mitochondrial DNA and allozymes. A total evidence tree based on the combined data from Sry, mitochondrial DNA, and allozymes supports (1) the monophyly of the subgenus Mus, (2) its division into a Palearctic group (M. musculus, M. domesticus, M. spicilegus, M. Macedonicus, and M. spretus) and an Oriental group (M. cookii++, M. cervicolor, and M. caroli), and (3) sister-group relationships between M. spicilegus and M. macedonicus and between M. cookii and M. cervicolor. We argue that Y- chromosome DNA sequences represent a valuable new source of characters for phylogenetic inference.      

Active and passive cation transport and L antigen hertogeneity in low potassium sheep red cells

Several lines of experimental evidence are presented suggesting that the L antigens in low potassium (LK) sheep red cells are associated with separate Na(+)K(+) pump flux is distinct from the action of anti-L(l) on K(+) leak flux, implying that K(+) leak transport sites may not be converted into active pumps by the L antiserum. Treatment of LK red cells with trypsin completely abolished both the stimulation of K(+) pump flux and the enhancement of the rate of ouabain binding brought about by anti- L. That this effect is due to a total destruction of the L(p) determinant associated with the LK pump was evident from the complete failure of anti-L(p) to bind to trypsinized LK red cells. The L(p) antigen can be effectively protected against the trypsin attack by prior incubation with anti-L, indicating that the sites for antibody binding and trypsin action may be closely adjacent at the structural level. Trypsin treatment, however, did not interfere with anti-L(l) reducing ouabain insensitive K(+) leak influx, nor did it prevent binding of anti-L(ly), the hemolytically active L antibody which is probably identical with anti-L(l). The functional independence of the L(p) and L(l) sites was documented by the observation that anti-L(l) still reduced K(+) leak influx in LK cells with experimentally induced high potassium concentrations, at which K(+) pump flux is fully suppressed, whether or not anti-L(p) was binding to the L(p) antigen associated with the LK pump.     

B cell activation during HIV-1 infection. II. Cell-to-cell interactions and cytokine requirement

This study examined the mechanisms underlying the intense activation of HIV-1-specific B cells observed in peripheral blood of HIV-1-infected subjects. Spontaneous in vitro synthesis of anti-HIV-1 antibodies, as well as total Ig production, were dramatically reduced by accessory cell, but not T cell removal. This fall was counteracted by addition of rIL-6, but not other cytokines, to monocyte-depleted cultures; moreover, antisera against IL-6 suppressed spontaneous anti-HIV-1 antibody synthesis in a dose-dependent manner. Although IL-6 apparently sustained HIV-1-specific B cell activation, no increase in serum IL-6 levels was observed; PBMC from seropositive subjects did not produce increased amounts of IL-6 in vitro, compared to seronegative controls, both spontaneously and in the presence of LPS stimulation; finally, no constitutive expression of IL-6 gene could be documented in freshly isolated PBMC. These findings indicate that IL-6 may play a central role in HIV-1-specific B cell activation in seropositive patients, and further stress the importance of this cytokine during HIV-1 infection.     

Tolbutamide and phenytoin hydroxylations by cDNA-expressed human liver cytochrome P4502C9

A human cytochrome P4502C9 cDNA clone has been isolated from a human liver bacteriophage Lambda gt11 library using oligonucleotide probes. Expression of the 1762 base pair cDNA in COS cells demonstrated that the encoded enzyme has a molecular mass of 55 kDa as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The expressed enzyme catalysed the methylhydroxylation of tolbutamide with an apparent Km of 131.7 microM, similar to that observed in human liver microsomes. P4502C9 also catalysed the 4-hydroylation of phenytoin, and inhibition experiments demonstrated that phenytoin was a competitive inhibitor of tolbutamide hydroxylation with an apparent Ki of 19.1 microM. Sulphaphenazole was a potent inhibitor of the expressed enzyme with respect to both tolbutamide and phenytoin hydroxylations. These data demonstrate that a single isozyme can catalyse the hydroxylations of both tolbutamide and phenytoin, and suggest that both reactions are mediated by the same isozyme(s) of cytochrome P450 in human liver.     

Monoclonal antibodies specific to transforming polyproteins encoded by independent isolates of feline sarcoma virus.

Hybridomas secreting monoclonal antibodies directed against polyprotein gene products of the Gardner, Snyder-Theilen, and McDonough strain of feline sarcoma virus have been isolated. Antibody produced by one hybridoma recognizes immunological determinants localized within a feline leukemia virus gag gene structural component (p15) common to polyproteins encoded by each feline sarcoma virus isolate while antibody produced by a second is specific for p30 determinants unique to P170gag-fms. Additional hybridomas secrete antibody directed against v-fes specific determinants common to the Gardner and Snyder-Theilen feline sarcoma virus-encoded polyproteins and to v-fms determinants unique to P170gas-fms polyprotein. GA P110gas-fes and ST P85gas-fes immunoprecipitated by antibody directed against p15 exhibit readily detectable levels of protein kinase activity but lack such activity when precipitated by antibody specific for their acquired sequence (v-fes) components. P170gas-fms immunoprecipitated by monoclonal antibody to either p15 or p30 lacks detectable levels of autophosphorylation but represents a substrate for the GA P110gag-fes and ST P85gag-fes enzymatic activities. These findings argue that the v-fes-associated protein kinase represents an intrinsic property of the v-fes gene product and recognizes tyrosine acceptor sites within polyprotein gene products of all three strains of feline sarcoma virus.     

Ovipositional deterrents in Dacus oleae

The deterrent substances diverting D. oleae females from ovipositing on already attacked olives are contained, at least partly, in the juice which trickles from the oviposition wounds. Surprisingly, the water fraction of the olive juice had limited deterrent activity. The principal deterrent stimuli are present in the oil fraction. Acetophenone and benzaldehyde are likely to be involved.Some liposoluble volatile substances can be deterrent to the females as vapours. The deterrent power of olives containing D. oleae larvae is probably linked to these substances.Most deterrent hydro-soluble substances had two contiguous hydroxyls, characterized by a comparative acidity and located in the ortho position in diphenols and somewhat analogously in glycolic acid.Amongst orthodiphenols, Pyrocatechol-the simplest compound-was the most active substance.

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Résumé Les jus d'olive qui sourt des blessures dues à la ponte, contient des substances inhibitrices qui empêchent les attaques ultérieures de la mouche de l'olivier.Une telle activité était attribuée à des substances hydrosolubles et, en particuliet, au dihydroxyphenil ethanol.On a testé l'activité inhibitrice des fractions huileuse et acqueuse obtenues par centrifugation de la pulpe des olives, rendue homogène.De facon inattendue, la fraction huileuse s'estavérée posséder une activité inhibitrice environ 20 fois supérieure à celle de la fraction acqueuse.Les différences d'activités inhibitrices d'huile d'olives mûres ou non, ou d'huiles commerciales, permet de penser que l'activité inhibitrice principale est liée à des substances liposolubles présentes dans l'huile.En outre, on a constaté que la pulpe d'olives, broyée, présente une action inhibitrice en l'absence de contact direct avec les femelles. Des substances inhibitrices volatiles sont aussi présentes dans l'huile.Des inhibiteurs volatiles qui se libèrent des tissus lacérés par les larves, peuvent expliquer l'activité inhibitrice des olives attaquées durant la période de développement des larves. Les substances inhibitrices dont la femelle couvre la surface cireuse des fruits, sont, en effet, facilement éliminées par la pluie.Parmi les substances liposolubles dont on connaît la présence dans l'huile, l'aceto-phénone et le benzaldehyde, possèdent un pouvoir inhibiteur prononcé.Les substances hydrolsolubles jouent un rôle mineur dans l'ensemble des stimuli inhibiteurs. Le pouvoir inhibiteur relatif est lié à deux hydroxyles contigus à une fonction acide présente en position ortho dans les diphénols, ceci par analogie avec l'acide glycolique qui possède un hydroxyle en caractérisé par une certaine acidité.Le plus actif orthodiphénol s'est avéré être la pyrocatéchine. Aussi une part des inhibiteurs liposolubles sont des substances liées au biochimisme des phénols.