HIV-1 Populations in Semen Arise through Multiple Mechanisms

HIV-1 is present in anatomical compartments and bodily fluids. Most transmissions occur through sexual acts, making virus in semen the proximal source in male donors. We find three distinct relationships in comparing viral RNA populations between blood and semen in men with chronic HIV-1 infection, and we propose that the viral populations in semen arise by multiple mechanisms including: direct import of virus, oligoclonal amplification within the seminal tract, or compartmentalization. In addition, we find significant enrichment of six out of nineteen cytokines and chemokines in semen of both HIV-infected and uninfected men, and another seven further enriched in infected individuals. The enrichment of cytokines involved in innate immunity in the seminal tract, complemented with chemokines in infected men, creates an environment conducive to T cell activation and viral replication. These studies define different relationships between virus in blood and semen that can significantly alter the composition of the viral population at the source that is most proximal to the transmitted virus.     

Identification and characterization of two functional domains of the hemolysin translocator protein HlyD

Secretion of Escherichia coli hemolysin is mediated by a sec-independent pathway which requires the products of at least three genes, hlyB, hlyD and tolC. Two regions of HlyD were studied. The first region (region A), consisting of the 33-amino acid, C-terminal part of the HlyD protein, is predicted to form a potential helix-loop-helix structure. This sequence is conserved among HlyD analogues of similar transport systems of other bacterial species. Using site-directed mutagenesis, we showed that the amino acids Leu475, Glu477 and Arg478 of this region are essential for HlyD function. The last amino acid of HlyD, Arg478, is possibly involved in the release of the HlyA protein, since cells bearing a hlyD gene mutant at this position produce similar amounts of HlyA to the wild-type strain, but most of the protein remains cell-associated. Competition experiments between wild-type and mutant HlyD proteins indicate that region A interacts directly with a component of the secretion apparatus. The second region of HIyD (region B), located between amino acids Leul27 and Leu170, is highly homologous to the otherwise unrelated outer membrane protein TolC. Deletion of this region abolishes secretion of hemolysin. This sequence of HlyD also seems to interact with a component of the hemolysin secretion machinery since a hybrid HIyD protein carrying the corresponding TolC sequence, although inactive in the transport of HlyA, is able to displace wild-type HlyD from the secretion apparatus.     

Biochemical characterization of recombinant Drosophila type 1 serine/threonine protein phosphatase (PP1c) produced in Pichia pastoris.

The methylotrophic yeast Pichia pastoris was used to express Drosophila melanogaster type 1beta serine/threonine phosphoprotein phosphatase catalytic subunit (PP1beta9C). A construct encoding PP1beta9C with a short NH(2)-terminal fusion including six histidine residues was introduced into the X-33 and KM71H strains of P. pastoris by homologous recombination. Recombinant protein was purified from cell free extracts 24 h after methanol induction. PP1beta9C was purified to a specific activity of 12,077 mU/mg by a three-step purification method comprising (NH(4))(2)SO(4)-ethanol precipitation followed by Ni(2+)-agarose affinity chromatography and Mono Q anion-exchange chromatography. This purification scheme yielded approximately 80 microg of active, soluble PP1beta9C per 1 L of culture. In contrast to recombinant PP1beta9C overexpressed in bacteria, which differs from native PP1c in several biochemical criteria including the requirement for divalent cations, sensitivity to vanadate, and p-nitrophenyl phosphate (pNPP) phosphatase activity, recombinant PP1beta9C produced in P. pastoris has native-like properties. P. pastoris thus provides a reliable and convenient system for the production of active, native-like recombinant PP1beta9C.     

Age of A2 Horizon Charcoal and Forest Structure near Porto Trombetas,Pará, Brazil1

To study the structure and composition of old‐growth forest in the Saracá‐Taquera National Forest near Porto Trombetas, Brazil, we established 36 0.25 ha plots and described the vegetation. We collected charcoal from the A2 soil horizon of each plot for radiocarbon dating. Although fires have been very rare in this forest during historic times, the presence of charcoal in these soils indicates fire at some earlier period. The ages (conventional radiocarbon age adjusted to 1997) of the charcoal ranged from 177 to 1547 years. These ages, however, did not correlate significantly with any of several measures of biodiversity or stand characteristics. The relative uniformity of the current old‐growth forest indicates that either the prehistoric fires were of such low intensity that they had little long‐term effect on the vegetation or that the present stands have progressed to near steady state.     

Water relations of Picea abies. II. Determination of the apoplastic water content and other water relations parameters of needles by means of capillary microcryoscopy and the pressure-volume

Using the pressure volume analysis (PV analysis) on the shoots of Norway spruce (Picea abies [L.] Karst.) and the here presented capillary microcryoscopy of the needle press sap of the same shoots, it was possible to determine the amount of apoplastic water in the needles (W_an) as well as in the defoliated shoots (W_as). Additionally, the bulk osmotic pressure at full water saturation in the symplast of the needles and defoliated shoots (π_on and π_os) was determined. The dependence of the bulk-averaged turgor pressure (P_t) on the water content and the relationship between the bulk modulus of elasticity of the needles (?_n) and the bulk-averaged needle turgor pressure (P_tn) was shown with help of the PV analysis on the whole shoots and defoliated shoots. The study was conducted at the end of the vegetation period in 1987 and during winter 1988. The proportion of W_an in the total needle water content (W_tn) was 14% in September 1987 and 12.5% in March 1988. The respective percentage of W_as in W_ts were 27% and 25%. The amount of apoplastic water depended on the ratio of the dry weight of defoliated shoots to the dry weight of the whole shoots. A standard mean value for the amount of W_an in the total water content of the shoots (W_t) was therefore not possible. The bulk osmotic pressure at full water saturation in the needle symplasts was –1.9 MPa in September 1987 and –2.2 MPa in winter 1988. The respective values of the bulk osmotic pressures in the symplast of the defoliated shoots (π_os) were –1.5 MPa and –1.7 MPa. Thus π_on was 0.1 MPa lower and π_os 0.3–0.4 MPa higher than the average osmotic pressure during full water saturation in the symplast of the whole shoots (π_o). The relation between bulk-averaged turgor pressure and water content showed that during water loss P_tn dropped more rapidly than the turgor pressure of defoliated shoots (P_ts). Consequently the needles were less elastic than the defoliated shoots. The turgor values of whole shoots followed an intemediate course between P_tn and P_ts. The flat course of P_ts seems to be the main reason for the often observed “plateau” of ψ-isotherms of whole shoots near full turgor.     

Sixteen Decisions

Sixteen Decisions. 2000. 58 minutes, color. video by Gayle Ferraro. For more information, contact University of California Extension, Center for Media and Independent Learning, 2000 Center Street, Suite 400, Berkeley, CA 94704.