The role of site-specific N-glycosylation in secretion of soluble forms of rabies virus glycoprotein

Rabies virus glycoprotein is important in the biology and pathogenesis of neurotropic rabies virus infection. This transmembrane glycoprotein is the only viral protein on the surface of virus particles, is the viral attachment protein that facilitates virus uptake by the infected cell, and is the target of the host humoral immune response to infection. The extracellular domain of this glycoprotein has N- glycosylation sequons at Asn37, Asn247, and Asn319. Appropriate glycosylation of these sequons is important in the expression of the glycoprotein. Soluble forms of rabies virus glycoprotein were constructed by insertion of a stop codon just external to the transmembrane domain. Using site-directed mutagenesis and expression in transfected eukaryotic cells, it was possible to compare the effects of site-specific glycosylation on the cell-surface expression and secretion of transmembrane and soluble forms, respectively, of the same glycoprotein. These studies yielded the surprising finding that although any of the three sequons permitted cell surface expression of full-length rabies virus glycoprotein, only the N-glycan at Asn319 permitted secretion of soluble rabies virus glycoprotein. Despite its biological and medical importance, it has not yet been possible to determine the crystal structure of the full-length transmembrane form of rabies virus glycoprotein which contains heterogeneous oligosaccharides. The current studies demonstrate that a soluble form of rabies virus glycoprotein containing only one sequon at Asn319 is efficiently secreted in the presence of the N-glycan processing inhibitor 1-deoxymannojirimycin. Thus, it is possible to purify a conformationally relevant form of rabies virus glycoprotein that contains only one N-glycan with a substantial reduction in its microheterogeneity. This form of the glycoprotein may be particularly useful for future studies aimed at elucidating the three-dimensional structure of this important glycoprotein.      

Babesia microti: Biochemistry and function of hamster erythrocytes infected from a human source

Babesia microti, a protozoan parasite of mammalian erythrocytes was obtained from the blood of an infected human and maintained in golden hamsters, in which a parasitemia of 70% was obtained regularly. The hamsters' response—a subacute, hemolytic anemia—was studied with regard to oxygen affinity and red cell organic phosphate content. In addition, the reduced glutathione status of infected erythrocytes was observed because of the possible importance of this metabolite to parasite growth and red cell integrity. Infected animals developed a severe anemia with reticulocytosis; there occurred a 4-mm decrease in whole blood oxygen affinity without any change in erythrocytes' 2,3-diphosphoglycerate levels. The glutathione content of the infected animals' erythrocytes increased twofold during the course of the infection. In uninfected animals, in which anemia and reticulocytosis had been produced by bleeding, all changes seen in infected animals were reproduced. It was concluded that the changes in the infected animals were due to the anemia and reticulocytosis alone, and that the parasite played no role in these changes apart from being a cause of anemia and reticulocytosis.     

Trypanosoma cruzi: role of the immune response in the natural resistance of inbred strains of mice.

Nine inbred strains of mice were challenged with 10⁴ or 10⁵ trypomastigotes of the Brazil strain of Trypanosoma cruzi. A spectrum of resistance was evident ranging from highly susceptible strains, e.g., C3H, which developed high parasitemias and died within 3 to 4 weeks, to resistant strains, e.g., C57BL/10, which developed low parasitemias and survived. Impairment of the immune system in resistant C57BL/10 mice by X-irradiation, splenectomy, or treatment with silica led to high, often fatal parasitemias. Athymic nude mice, in particular, attained exceptionally high parasitemias before dying. The immune response appears to be necessary for survival and to play a role in the natural resistance of some mouse strains by effectively eliminating parasites and minimizing parasitemia. Using congenic strains of mice, it was shown that the principal genetic determinant of resistance is not associated with their H-2 haplotype.     

Correlated rates of synonymous site evolution across plant genomes

Synonymous substitution rates have been shown to vary among evolutionary lineages of both nuclear and organellar genes across a broad range of taxonomic groups. In animals, rate heterogeneity does not appear to be correlated across nuclear and mitochondrial genes. In this paper, we contrast substitution rates in two plant groups and show that grasses evolve more rapidly than palms at synonymous sites in a mitochondrial, a nuclear, and a plastid gene. Furthermore, we show that the relative rates of synonymous substitution between grasses and palms are similar at the three loci. The correlation in synonymous substitution rates across genes is particularly striking because the three genes evolve at very different absolute rates. In contrast, relative rates of nonsynonymous substitution are not conserved among the three genes.      

SITES OF CYTOPLASMIC RIBONUCLEOPROTEIN-FILAMENT ASSEMBLY IN RELATION TO HELICAL BODY FORMATION IN AXENIC TROPHOZOITES OF ENTAMOEBA HISTOLYTICA

Axenic trophozoites of Entamoeba histolytica showed increased logarithmic growth but absence of "chromatoid" material (stacked helical arrays of ribonucleoprotein [RNP]) when grown in an all-liquid monophasic culture. Organisms grown in a liquid overlay on a semisolid slant (biphasic medium) showed slow logarithmic growth and the presence of chromatoid material. Chromatoid material accumulated in the rapidly growing trophozoites from monophasic culture during treatment with the Vinca alkaloid, vinblastine. Many of the glycogen-free regions of vinblastine-treated trophozoites as well as, to a lesser degree, of normal cells grown in monophasic and biphasic cultures, contained free ribosomes and randomly oriented 60 A filaments. As ribonucleoprotein assumed the packed helical configuration, areas consisting of parallel, packed filaments could be detected adjacent to and continuous with the ordered RNP arrays. This arrangement could be visualized most frequently in vinblastine-treated trophozoites grown in monophasic cultures. Depending on the tilt of the section with respect to the longitudinal axis of individual helices, 60 A filamentous material could be demonstrated associated with the RNP helices. Localization of ribonucleoprotein precursors was followed by means of high resolution radioautography with uridine-³H and cytidine-³H. With a short (30-min) pulse, label could be visualized only over the glycogen-free areas containing free ribosomes and filaments. With 60-min pulses, label could also be seen over the packed helical arrays. With 30-min pulses followed by a 60-min cold chase, label was seen chiefly over RNP helices. It is postulated that the areas containing ribosomes and filaments represent sites of assembly of the RNP helices possibly on a filament protein column. The possibility that the final helical configuration may be due to a property of this protein is suggested.