Effect of magnesium on methanogenic subpopulations in a thermophilic acetate-degrading granular consortium.

The effects of Mg2+ on thermophilic (55 degrees C) granules grown on acetate in 0.2-liter upflow anaerobic sludge blanket reactors were studied. The methanogens in the granules were identified and counted by using antibody probes and the antigenic fingerprinting method. Packets of large coccoidal cells antigenically related to Methanosarcina thermophila TM-1 were scarce in the absence of Mg2+ but increased with increasing Mg2+ concentrations up to 30 mM; Methanosarcina packets immunologically related to Methanosarcina barkeri R1M3 showed a similar trend, and their numbers increased up to 100 mM Mg2+. The number of single cells antigenically related to TM-1, R1M3, and Methanosarcina mazei S-6 were scarce at low Mg2+ concentrations but increased drastically at 30 and 100 mM Mg2+. The number of rod-shaped bacteria antigenically related to Methanobacterium thermoautotrophicum GC1 and delta H was highest with no Mg2+ present, and their numbers decreased with increasing concentrations of the cation. These quantitative data, obtained by counting cells in suspensions made from disrupted granules, were confirmed by microscopic observation of the methanogenic subpopulations in thin histologic sections of the granules.     

Inhibition of human and simian immunodeficiency virus protease function by targeting Vpx-protease-mutant fusion protein into viral particles.

The human immunodeficiency virus type I (HIV-1) Vpr and HIV-2 Vpx proteins package into virions through interactions with their cognate Gag polyprotein precursor. The targeting properties of Vpr and Vpx have been exploited to incorporate foreign proteins into virions by expression as heterologous fusion molecules (X. Wu, H.-M. Liu, H. Xiao, J. Kim, P. Seshaiah, G. Natsoulis, J. D. Boeke, B. H. Hahn, and J. C. Kappes, J. Virol. 69:3389-3398, 1995). To explore the possibility of utilizing Vpx and Vpr to target dominant negative mutants of the HIV Pol proteins into virions, we fused HIV-2 Vpx with an enzymatically defective protease (PR) mutant. Using a vector system to facilitate transient coexpression with HIV provirus, Vpx-PR-mutant (VpxPR(M)) fusion protein was expressed and packaged efficiently into HIV-2 and simian immunodeficiency virus virions. Immunoblot analysis of purified virions demonstrated that the packaging of VpxPR(M) interfered with the processing of the Gag and Gag/Pol precursor proteins, similar to that of a well-characterized active-site PR inhibitor. The incomplete processing of Gag and Gag/Pol was consistent with a 25-fold reduction in virion infectivity. The coexpression of a packaging defective VpxPR(M) fusion protein with HIV-2 provirus produced virions with fully processed Gag protein, similar to wild-type virions. Importantly, virions trans complemented with a Vpx-chloramphenicol acetyltransferase fusion protein were normal with respect to the processing of Gag protein and the ability to infect and replicate in vitro. These results indicate that VpxPR(M) specifically inhibited the function of the viral protease and provide for the first time proof of principle that the incorporation of foreign proteins into virions via fusion with Vpx can inhibit HIV replication. The use of accessory proteins as vehicles to deliver deleterious proteins to virions, including dominant negative mutants of Pol proteins, may provide new opportunities for application of gene therapy-based antiretroviral strategies. The ability to package PR by expression in trans, independent of the Gag/Pol precursor, also represents a novel approach that may be exploited to study the function of the Pol proteins.     

The cost of cardiac surgery.

A study in Wessex has shown that at 1977 prices, and excluding the cost of equipment already installed in the unit, the cost of replacing a man''s aortic valve in this unit is about 1800 pounds. Nevertheless, this seems a small price to pay for return to health and full working capacity, particularly since such patients no longer need to draw social security benefits and their tax contributions will return to normal, thus probably paying for the operation within two years.     

CYCLIC AMP-DEPENDENT PROTEIN KINASE ACTIVITY AND SYNAPTOSOMAL PROTEIN PHOSPHORYLATION IN THE BRAINS OF AGED RATS

The activity of soluble protein kinase and phosphorylation of endogenous synaptosomal proteins were studied in vitro, in the hippocampus and cerebral cortex of rats 3, 12, or 24 months of age. No between-age differences in the activity of cyclic AMP-dependent or independent protein kinase were detected in either brain region. The degree of stimulation by cyclic AMP and the apparent K_a, for cyclic AMP were similar at all stages. Cyclic AMP stimulated the phosphorylation of synaptosomal proteins from the cerebral cortex, hippocampus, caudate nucleus, and cerebellum of rats at all ages. There were no significant differences across age in the extent of phosphorylation of any membrane proteins in any brain region. The number and staining density of synaptosornal proteins separated by polyacrylamide gel electrophoresis were also similar at all ages. These studies indicate that the cyclic AMP-dependent phosphorylation system in the rat brain does not change during advanced aging.     

A redescription of the Middle Cambrian wormAmiskwia sagittiformis Walcott from the Burgess Shale of British Columbia

Amiskwia sagittiformis Walcott is redescribed on the basis of the five available specimens. The dorsoventrally compressed body consists of a head bearing a pair of prominent tentacles and a trunk with lateral and caudal fins. A gut with subterminal openings, cerebral ganglia, nerve cords, blood vessels and muscles have been identified with varying degrees of confidence. An active pelagic mode of life is considered probable. The rejection by earlier workers of this worm from the arrow-worms (Chaetognatha) is confirmed, but their identification of it as a nemertean (Nemertea) cannot be supported owing to the absence of critical data. The systematic position ofA. sagittiformis remains unresolved.     

The covalent attachment of multiple fluorophores to DNA containing phosphorothioate diesters results in highly sensitive detection of single-stranded DNA.

DNA fragments containing multiple internucleotidic phosphorothioate diesters, prepared by either chemical or enzymatic syntheses, are amenable to labeling with the fluorophore monobromobimane. With the incorporation of phosphorothioate diesters at each internucleotidic site, multiple fluorophores, ideally one for each nucleotide residue, can be covalently attached to the DNA fragment. The presence of multiple labels can be expected to interfere with analysis techniques, such as polyacrylamide gel electrophoresis. To avoid such problems, the fluorophores are introduced in a "postassay" fashion, that is, while the fragments are still embedded within the gel matrix. The detection limit (to the naked eye) for multiply labeled single-stranded DNA containing hundreds of base residues is in the low femtomole range. DNA containing greater than 1000 base residues can be visualized in some cases in the subfemtomole range without the use of sophisticated electronic instrumentation.     

Human immunodeficiency virus type 1 envelope gene structure and diversity in vivo and after cocultivation in vitro.

Nested-primer polymerase chain reaction (PCR) has been applied to the molecular cloning of 4.6-kb half-genome fragments of human immunodeficiency virus type 1 (HIV-1) taken directly from the peripheral blood mononuclear cells (PBMC) of an individual with neurological symptoms of HIV-1 infection. In a similar manner, gp120-coding portions of the envelope gene were cloned after PBMC from the same blood sample were cocultivated with uninfected PBMC for 28 days. The complete 1.6-kb nucleotide sequence of the gp120 gene was determined from each of 35 clones examined. Two of 13 (15%) PBMC-derived gp120 genes and 3 of 22 (14%) coculture-derived gp120 genes were defective as a result of frameshifts and an in-frame stop codon(s). Mean diversity between individual gp120-coding sequences in PBMC was fivefold greater (3.24%) than after coculture (0.65%). A predominant sequence of "strain" was found after coculture that was distinct from the diverse viral genotypes detected in vivo and therefore was selectively amplified during in vitro propagation. Multiple distinct third variable (V3) regions encoding the principal neutralizing domain of the envelope protein were detected in PBMC-derived genes, suggesting the presence of immunologic diversity of HIV env genes in vivo not reflected in the cocultured virus sample. The large size of the HIV fragments generated in this study will permit analysis of the diversity of immunologic reactivity, gene function, and pathogenicity of HIV genomes present within infected individuals, including the functional significance of the loss of diversity that occurs upon coculture.