Mechanism of virus-induced stimulation of the hypothalamus-pituitary-adrenal axis

Increased blood levels of glucocorticoids are observed during certain viral infections. In this paper, we report data obtained from a model in rodents showing that the pituitary-adrenal axis is stimulated following inoculation of Newcastle Disease Virus (NDV). No evidence for an ectopic, lymphoid source of ACTH-like immunoreactive material capable of inducing this effect was obtained. Administration of virus-free supernatants from cocultures of human peripheral blood leukocytes with NDV also stimulated ACTH and glucocorticoid output in normal mice. This observation showed the immunological cell origin of the mediator of the hormonal effect. Pretreatment of the supernatant with anti-IL-1 sera neutralized its capacity to induce an increase in glucocorticoid and ACTH levels in blood. Furthermore, injection of IL-1 in nanogram amounts also increased ACTH and glucocorticoid blood levels. Thus, we conclude that IL-1 is the most likely mediator of the stimulation of the pituitary-adrenal axis during viral infection. The reported data are also discussed in the general context of the postulated glucocorticoid-associated immunoregulatory circuit.     

A Morphogenesis Checkpoint Monitors the Actin Cytoskeleton in Yeast

A morphogenesis checkpoint in budding yeast delays cell cycle progression in response to perturbations of cell polarity that prevent bud formation (Lew, D.J., and S.I. Reed. 1995. J. Cell Biol. 129:739– 749). The cell cycle delay depends upon the tyrosine kinase Swe1p, which phosphorylates and inhibits the cyclin-dependent kinase Cdc28p (Sia, R.A.L., H.A. Herald, and D.J. Lew. 1996. Mol. Biol. Cell. 7:1657– 1666). In this report, we have investigated the nature of the defect(s) that trigger this checkpoint. A Swe1p- dependent cell cycle delay was triggered by direct perturbations of the actin cytoskeleton, even when polarity establishment functions remained intact. Furthermore, actin perturbation could trigger the checkpoint even in cells that had already formed a bud, suggesting that the checkpoint directly monitors actin organization, rather than (or in addition to) polarity establishment or bud formation. In addition, we show that the checkpoint could detect actin perturbations through most of the cell cycle. However, the ability to respond to such perturbations by delaying cell cycle progression was restricted to a narrow window of the cell cycle, delimited by the periodic accumulation of the checkpoint effector, Swe1p.     

Crystallization and Preliminary X-Ray Analysis of Rotavirus Protein VP6

As a first step to gain insight into the structure of the rotavirus virion at atomic resolution, we report here the expression, purification, and crystallization of recombinant rotavirus protein VP6. This protein has the property of polymerizing in the form of tubular structures in solution which have hindered crystallization thus far. Using a combination of electron microscopy and small-angle X-ray scattering, we found that addition of Ca²⁺ at concentrations higher than 100 mM results in depolymerization of the tubes, leading to an essentially monodisperse solution of trimeric VP6 even at high protein concentrations (higher than 10 mg/ml), thereby enabling us to search for crystallization conditions. We have thus obtained crystals of VP6 which diffract to better than 2.4 Å resolution and belong to the cubic space group P4₁32 with a cell dimension a of 160 Å. The crystals contain a trimer of VP6 lying along the diagonal of the cubic unit cell, resulting in one VP6 monomer per asymmetric unit and a solvent content of roughly 70%.     

Algorithms and software for support of gene identification experiments

MOTIVATION: Gene annotation is the final goal of gene prediction algorithms. However, these algorithms frequently make mistakes and therefore the use of gene predictions for sequence annotation is hardly possible. As a result, biologists are forced to conduct time-consuming gene identification experiments by designing appropriate PCR primers to test cDNA libraries or applying RT-PCR, exon trapping/amplification, or other techniques. This process frequently amounts to 'guessing' PCR primers on top of unreliable gene predictions and frequently leads to wasting of experimental efforts. RESULTS: The present paper proposes a simple and reliable algorithm for experimental gene identification which bypasses the unreliable gene prediction step. Studies of the performance of the algorithm on a sample of human genes indicate that an experimental protocol based on the algorithm's predictions achieves an accurate gene identification with relatively few PCR primers. Predictions of PCR primers may be used for exon amplification in preliminary mutation analysis during an attempt to identify a gene responsible for a disease. We propose a simple approach to find a short region from a genomic sequence that with high probability overlaps with some exon of the gene. The algorithm is enhanced to find one or more segments that are probably contained in the translated region of the gene and can be used as PCR primers to select appropriate clones in cDNA libraries by selective amplification. The algorithm is further extended to locate a set of PCR primers that uniformly cover all translated regions and can be used for RT-PCR and further sequencing of (unknown) mRNA.      

Histopathological effects of tannic acid on the midgut epithelium of some aquatic Diptera larvae

The impact of tannins on larval Nematocera was investigated by an extensive survey of the relative toxicity of tannic acid in Diptera larvae representative of mosquito communities from alpine hydrosystems (Culicidae, Chaoboridae, Chironomidae, and Simuliidae) together with a nonindigenous vector competent Culicidae species. Bioassays indicate that exposure to tannic acid at concentrations from 0.25 to 4 mM is deleterious for Culex pipiens, Simulium variegatum, and Chironomus annularius, but not for Aedes, Anopheles, Culiseta, and Chaoborus species. Histopathological observations reveal that, among the target organs of tannic acid, mainly the midgut epithelium is affected by treatment. However, the extent of degeneration varies according to the taxon, the duration of the treatment, and the concentrations assayed. The vulnerability of epithelial cells differs among cell types, clear cells of the anterior midgut showing symptoms of intoxication before dark cells of the posterior midgut. The toxic effects of tannic acid are discussed, particularly in comparison to those of insecticidal bacteria, in order to evaluate the potential for use of tannins in the regulation of larval populations of dipteran pests.     

Recent evolutionary history of American Onchocerca volvulus, based on analysis of a tandemly repeated DNA sequence family

Polymerase chain reaction (PCR) products were characterized for a repeated sequence family (designated "O-150") of the human filarial parasite Onchocerca volvulus. In phylogenetic inferences, the O-150 sequences clustered into closely related groups, suggesting that concerted evolution maintains sequence homology in this family. Using a novel mathematical model based on a nested application of an analysis of variance, we demonstrated that African rainforest and savannah strain parasite populations are significantly different. In contrast, parasites collected in the New World are indistinguishable from African savannah strains of O. volvulus. This finding supports the hypothesis that onchocerciasis was recently introduced into the New World, possibly as a result of the slave trade.      

Raffinose increases sporulation and enterotoxin production by Clostridium perfringens type A.

Replacement of starch with raffinose in Duncan and Strong sporulation medium improved percent sporulation in six of eight strains tested. Enterotoxin concentration in cell extracts was increased in the case of four of five known enterotoxin-positive strains. With strain NCTC 10240, levels of 0.3, 0.4, and 0.5% raffinose produced the highest enterotoxin concentration 300 to 320 micrograms of enterotoxin per mg of cell extract protein. At a level of 0.4% raffinose the highest enterotoxin concentration in cell extracts of NCTC 10240 occurred after 8 h of growth in Duncan and Strong medium. Enterotoxin produced in the presence of starch or raffinose by three separate strains all migrated at similar Rm by polyacrylamide gel electrophoresis.     

Immunoregulation mediated by the sympathetic nervous system.

A postulated immunoregulatory role for the autonomous nervous system was explored utilizing several in vivo and in vitro approaches. Local surgical denervation of the spleen in rats and general chemical sympathectomy by 6-hydroxydopamine combined with adrenalectomy yielded a similar removal of restraint expressed as enhancement in the number of PFC in response to immunization. Noradrenaline and the synthetic α-agonist clonidine which are, respectively, natural and artificial effector molecules of the sympathetic nervous system each strongly suppressed the in vitro induced immune response of murine spleen cells to SRBC. Further, radiometric-enzymatic assay of noradrenaline in the splenic pulp revealed a decrease in the content of this neurotransmitter just preceding the exponential phase of the immune response to SRBC (Days 3 and 4) in this site. Taken together, these findings point to a dynamic immunoregulatory relationship between the immune and sympathetic nervous system.     

Synthesis of 1,3-β-Glucanases in Saccharomyces cerevisiae During the Mitotic Cycle, Mating, and Sporulation

Upon fractionating Saccharomyces cerevisiae asynchronous cultures by sucrose density gradient centrifugation in a zonal rotor and examining the exo-1,3-beta-glucanase and deoxyribonucleic acid content of the cells, a periodic step increase in the activity of this enzyme was observed, indicating a discontinuous pattern of synthesis or activation of exo-1,3-beta-glucanase during the mitotic cycle at the transition from the S to the G(2) phase. Similar results were obtained for endo-1,3-beta-glucanase by assaying activity against oxidized laminarin in permeabilized cells, suggesting that the synthesis of endo-1,3-beta-glucanase is controlled in the same way. When a and alpha strains were mated, the specific activity of cell extracts against laminarin, oxidized laminarin, and pustulan remained constant while zygote formation was taking place. However, when growth resumed, active synthesis of 1,3-beta-glucanases took place as shown by the occurrence of a significant increase in the specific activity against the three substrates. Specific changes in the level of glucan degradative enzymes, not observed in a haploid parental strain, occurred when the diploid S. cerevisiae AP-1 was induced to sporulate. The sporulation process triggered the activation of first the pustulan degradative capacity and then the capacity to hydrolyze oxidized laminarin. The specific activity against this substrate was 10 times higher than that against pustulan.