Low codon bias and high rates of synonymous substitution in Drosophila hydei and D. melanogaster histone genes

We have evaluated codon usage bias in Drosophila histone genes and have obtained the nucleotide sequence of a 5,161-bp D. hydei histone gene repeat unit. This repeat contains genes for all five histone proteins (H1, H2a, H2b, H3, and H4) and differs from the previously reported one by a second EcoRI site. These D. hydei repeats have been aligned to each other and to the 5.0-kb (i.e., long) and 4.8-kb (i.e., short) histone repeat types from D. melanogaster. In each species, base composition at synonymous sites is similar to the average genomic composition and approaches that in the small intergenic spacers of the histone gene repeats. Accumulation of synonymous changes at synonymous sites after the species diverged is quite high. Both of these features are consistent with the relatively low codon usage bias observed in these genes when compared with other Drosophila genes. Thus, the generalization that abundantly expressed genes in Drosophila have high codon bias and low rates of silent substitution does not hold for the histone genes.      

2′-and/or 3Y-Deoxy-β-L-pentofuranosyl Nucleoside Derivatives: Stereospecific Synthesis and Antiviral Activities

Abstract

Several L-enantiomers of nucleoside analogues were stereospecifically synthesized by a multi-step reaction from L-xylose and their antiviral properties were examined in vitro. Two of them, namely β-L-2′,3,′-dideoxycytidine (β-L-ddC) and its 5-fluoro derivative (β-L-FddC) were found to have potent anti-human immunodeficiency virus (HIV) and significant anti-hepatitis B virus (HBV) activities in cell cultures.     

Antibody-mediated neutralization of primary human immunodeficiency virus type 1 isolates: investigation of the mechanism of inhibition

Human immunodeficiency virus type 1 (HIV-1) neutralization occurs when specific antibodies, mainly those directed against the envelope glycoproteins, inhibit infection, most frequently by preventing the entry of the virus into target cells. However, the precise mechanisms of neutralization remain unclear. Previous studies, mostly with cell lines, have produced conflicting results involving either the inhibition of virus attachment or interference with postbinding events. In this study, we investigated the mechanisms of neutralization by immune sera and compared the inhibition of peripheral blood mononuclear cells (PBMC) infection by HIV-1 primary isolates (PI) with the inhibition of T-cell line infection by T-cell line-adapted (TCLA) strains. We followed the kinetics of neutralization to determine at which step of the viral cycle the antibodies act. We found that neutralization of the TCLA strain HIV-1MN/MT-4 required an interaction between antibodies and cell-free virions before the addition of MT-4 cells, whereas PI were neutralized even after adsorption onto PBMC. In addition, the dose-dependent inhibition of HIV-1MN binding to MT-4 cells was strongly correlated with serum-induced neutralization. In contrast, neutralizing sera did not reduce the adhesion of PI to PBMC. Postbinding inhibition was also detected for HIV-1MN produced by and infecting PBMC, demonstrating that the mechanism of neutralization depends on the target cell used in the assay. Finally, we considered whether the different mechanisms of neutralization may reflect the recognition of qualitatively different epitopes on the surface of PI and HIV-1MN or whether they reflect differences in virus attachment to PBMC and MT-4 cells.     

Analysis of a three-way race between tumor growth,a replication-competent virus and an immune response

Replication-competent viruses have the potential to overcome the delivery barrier in tumors that has plagued traditional gene-therapy approaches to cancer treatment. However, recent clinical data suggests that a cytokine-based immune response against the virus-infected tumor cells may severely limit the efficacy of the replication-competent approach. This paper generalizes our earlier spatial model to incorporate an immune response against the infected tumor cells. An approximate but accurate condition is derived for the virus—if uniformly injected throughout the tumor—to eradicate the tumor in the presence of the immune response. To validate the model using clinical data describing the temporal interaction of tumor necrosis factor and free virus in the plasma, we needed the immune response to be time-delayed and experience either saturated stimulation or second-order clearance. The resulting estimates of some unknown parameters provide some implications for the delivery of treatment.     

Delineation of pilin domains required for bacterial association into microcolonies and intestinal colonization by Vibrio cholerae

The toxin-co-regulated pilus (TCP), a type 4 pilus that is expressed by epidemic strains of Vibrio cholerae O1 and O139, is required for colonization of the human intestine. The TCP structure is assembled as a polymer of repeating subunits of TcpA pilin that form long fibres, which laterally associate into bundles. Previous passive immunization studies have suggested that the C-terminal region of TcpA is exposed on the surface of the pilus fibre and has a critical role in mediating the colonization functions of TCP. In the present study, we have used site-directed mutagenesis to delineate two domains within the C-terminal region that contribute to TCP structure and function. Alterations in the first domain, termed the structural domain, result in altered pilus stability or morphology. Alterations in the second domain, termed the interaction domain, affect colonization and/or infection by CTX-bacteriophage without affecting pilus morphology. In vitro and in vivo analyses of the tcpA mutants revealed that a major function of TCP is to mediate bacterial interaction through direct pilus-pilus contact required for microcolony formation and productive intestinal colonization. The importance of this function is supported by the finding that intragenic suppressor mutations that restore colonization ability to colonization-deficient mutants simultaneously restore pilus-mediated bacterial interactions. The alterations resulting from the suppressor mutations also provide insight into the molecular interactions between pilin subunits within and between pilus fibres.     

Rac1-induced endocytosis is associated with intracellular proteolysis during migration through a three-dimensional matrix

Transfection of Rat1 fibroblasts with an activated form of rac1 (V12rac1) stimulated cell migration in vitro compared to transfection of Rat1 fibroblasts with vector only or with dominant negative rac1 (N17rac1). To investigate the involvement of proteases in this migration, we used a novel confocal assay to evaluate the ability of the Rat1 transfectants to degrade a quenched fluorescent protein substrate (DQ-green bovine serum albumin) embedded in a three-dimensional gelatin matrix. Cleavage of the substrate results in fluorescence, thus enabling one to image extracellular and intracellular proteolysis by living cells. The Rat1 transfectants accumulated degraded substrate intracellularly. V12rac1 increased accumulation of the fluorescent product in vesicles that also labeled with the lysosomal marker LysoTracker. Treatment of the V12rac1-transfected cells with membrane-permeable inhibitors of lysosomal cysteine proteases and a membrane-permeable selective inhibitor of the cysteine protease cathepsin B significantly reduced intracellular accumulation of degraded substrate, indicating that degradation occurred intracellularly. V12rac1 stimulated uptake of dextran 70 (a marker of macropinocytosis) and polystyrene beads (markers of phagocytosis) into vesicles that also labeled for cathepsin B. Thus, stimulation of the endocytic pathways of macropinocytosis and phagocytosis by activated Rac1 may be responsible for the increased internalization and subsequent degradation of extracellular proteins.