Effect of brefeldin A on alphaherpesvirus membrane protein glycosylation and virus egress.

In this work we used brefeldin A (BFA), a specific inhibitor of export to the Golgi apparatus, to study pseudorabies virus viral glycoprotein processing and virus egress. BFA had little effect on initial synthesis and cotranslational modification of viral glycoproteins in the endoplasmic reticulum (ER), but it disrupted subsequent glycoprotein maturation and export. Additionally, single-step growth experiments demonstrated that after the addition of BFA, accumulation of infectious virus stopped abruptly. BFA interruption of virus egress was reversible. Electron microscopic analysis of infected cells demonstrated BFA-induced disappearance of the Golgi apparatus accompanied by a dramatic accumulation of enveloped virions between the inner and outer nuclear membranes and also in the ER. Large numbers of envelope-free capsids were also present in the cytoplasm of all samples. In control samples, these capsids were preferentially associated with the forming face of Golgi bodies and acquired a membrane envelope derived from the trans-cisternae. Our results are consistent with a multistep pathway for envelopment of pseudorabies virus that involves initial acquisition of a membrane by budding of capsids through the inner leaf of the nuclear envelope followed by deenvelopment and release of these capsids from the ER into the cytoplasm in proximity to the trans-Golgi. The released capsids then acquire a bilaminar double envelope containing mature viral glycoproteins at the trans-Golgi. The resulting double-membraned virus is transported to the plasma membrane, where membrane fusion releases a mature, enveloped virus particle from the cell.     

Studies on the Inoculation and Competitiveness of a Rhizobium leguminosarum Strain in Soils Containing Indigenous Rhizobia

The competitiveness of a Rhizobium leguminosarum strain was investigated at two separate locations in field inoculation studies on commercially grown peas. The soil at each location (sites I and II) contained an indigenous R. leguminosarum population of ca. 3 × 10⁴ rhizobia per g of soil. At site I it was necessary to use an inoculum concentration as large as 4 × 10⁷ CFU ml⁻¹ (2 × 10⁶ bacteria seed⁻¹) to establish the inoculum strain in the majority of nodules (73%). However, at site II the inoculum strain formed only 33% of nodules when applied at this (10⁷ CFU ml⁻¹) level. Establishment could not be further improved by increasing the inoculum concentration even as high as 10⁹ CFU ml⁻¹ (9.6 × 10⁷ bacteria seed⁻¹). The inoculum strain could be detected at both sites 19 months after inoculation. Analysis by intrinsic antibiotic resistance patterns and plasmid DNA profiles indicated that a dominant strain(s) and plasmid pool existed among the indigenous population at site II. Competition experiments were carried out under laboratory conditions between a dominant indigenous isolate and the inoculum strain. Both strains were shown to be equally competitive.     

B lymphocyte receptors and polyphosphoinositide degradation

Resting B lymphocytes can be activated and induced to proliferate by antibodies against their antigen receptors (anti-lg). We demonstrate an early increase in the level of [3H]inositol trisphosphate in [3H]inositol-labeled murine B cells, which suggests breakdown of phosphatidylinositol bisphosphate by phospholipase C. In line with this, the level of [3H]1,2-diacylglycerol was also elevated after incubation of [3H]arachidonic-acid-labeled B cells with anti-Ig. Anti-lg also caused a rapid increase in the level of cytosolic Ca2+ in B cells. In contrast, two other polyclonal B cell activators, lipopolysaccharide and phorbol myristate acetate, failed to induce any of these effects. Our results suggest that anti-lg may induce B cell growth via phosphoinositide degradation and Ca2+ mobilization, and that phorbol myristate acetate, and possibly lipopolysaccharide, bypass these initial events.     

A comparative description of mitochondrial DNA differentiation in selected avian and other vertebrate genera

Levels of mitochondrial DNA (mtDNA) sequence divergence between species within each of several avian (Anas, Aythya, Dendroica, Melospiza, and Zonotrichia) and nonavian (Lepomis and Hyla) vertebrate genera were compared. An analysis of digestion profiles generated by 13-18 restriction endonucleases indicates little overlap in magnitude of mtDNA divergence for the avian versus nonavian taxa examined. In 55 interspecific comparisons among the avian congeners, the fraction of identical fragment lengths (F) ranged from 0.26 to 0.96 (F = 0.46), and, given certain assumptions, these translate into estimates of nucleotide sequence divergence (p) ranging from 0.007 to 0.088; in 46 comparisons among the fish and amphibian congeners, F values ranged from 0.00 to 0.36 (F = 0.09), yielding estimates of P greater than 0.070. The small mtDNA distances among avian congeners are associated with protein-electrophoretic distances (D values) less than approximately 0.2, while the mtDNA distances among assayed fish and amphibian congeners are associated with D values usually greater than 0.4. Since the conservative pattern of protein differentiation previously reported for many avian versus nonavian taxa now appears to be paralleled by a conservative pattern of mtDNA divergence, it seems increasingly likely that many avian species have shared more recent common ancestors than have their nonavian taxonomic counterparts. However, estimates of avian divergence times derived from mtDNA- and protein-calibrated clocks cannot readily be reconciled with some published dates based on limited fossil remains. If the earlier paleontological interpretations are valid, then protein and mtDNA evolution must be somewhat decelerated in birds. The empirical and conceptual issues raised by these findings are highly analogous to those in the long-standing debate about rates of molecular evolution and times of separation of ancestral hominids from African apes.      

Methods for computing the standard errors of branching points in an evolutionary tree and their application to molecular data from humans and apes

Statistical methods for computing the standard errors of the branching points of an evolutionary tree are developed. These methods are for the unweighted pair-group method-determined (UPGMA) trees reconstructed from molecular data such as amino acid sequences, nucleotide sequences, restriction-sites data, and electrophoretic distances. They were applied to data for the human, chimpanzee, gorilla, orangutan, and gibbon species. Among the four different sets of data used, DNA sequences for an 895-nucleotide segment of mitochondrial DNA (Brown et al. 1982) gave the most reliable tree, whereas electrophoretic data (Bruce and Ayala 1979) gave the least reliable one. The DNA sequence data suggested that the chimpanzee is the closest and that the gorilla is the next closest to the human species. The orangutan and gibbon are more distantly related to man than is the gorilla. This topology of the tree is in agreement with that for the tree obtained from chromosomal studies and DNA-hybridization experiments. However, the difference between the branching point for the human and the chimpanzee species and that for the gorilla species and the human-chimpanzee group is not statistically significant. In addition to this analysis, various factors that affect the accuracy of an estimated tree are discussed.      

Epidemiological characteristics of platelet aggregability.

The epidemiological characteristics of platelet aggregability were established in 958 participants in the Northwick Park Heart Study. The main analyses were based on the dose of adenosine diphosphate at which primary aggregation occurred at half its maximum velocity. Aggregability increased with age in both sexes, was greater in whites than blacks (particularly among men), and tended to decrease with the level of habitual alcohol consumption. Aggregability was, however, greater in women than men and in nonsmokers than smokers. There was no relation between aggregability on the one hand and obesity, current or past oral contraceptive use, menopausal state, or blood cholesterol and triglyceride concentrations on the other. Aggregability was somewhat, though not significantly, higher in men with a history of ischaemic heart disease and in those with electrocardiographic evidence of ischaemia than in those without. There was a strong association between the plasma fibrinogen concentration and aggregability. The widely held concept of platelet aggregability and its implications is probably an oversimplification. In the prevention of thrombosis it may be as useful to consider modifying external influences on platelet behaviour, such as plasma fibrinogen concentration or thrombin production, as it is to rely solely on platelet active agents.