The Discovery,Distribution, and Evolution of Viruses Associated with Drosophila melanogaster

Drosophila melanogaster is a valuable invertebrate model for viral infection and antiviral immunity, and is a focus for studies of insect-virus coevolution. Here we use a metagenomic approach to identify more than 20 previously undetected RNA viruses and a DNA virus associated with wild D. melanogaster. These viruses not only include distant relatives of known insect pathogens but also novel groups of insect-infecting viruses. By sequencing virus-derived small RNAs, we show that the viruses represent active infections of Drosophila. We find that the RNA viruses differ in the number and properties of their small RNAs, and we detect both siRNAs and a novel miRNA from the DNA virus. Analysis of small RNAs also allows us to identify putative viral sequences that lack detectable sequence similarity to known viruses. By surveying >2,000 individually collected wild adult Drosophila we show that more than 30% of D. melanogaster carry a detectable virus, and more than 6% carry multiple viruses. However, despite a high prevalence of the Wolbachia endosymbiont—which is known to be protective against virus infections in Drosophila—we were unable to detect any relationship between the presence of Wolbachia and the presence of any virus. Using publicly available RNA-seq datasets, we show that the community of viruses in Drosophila laboratories is very different from that seen in the wild, but that some of the newly discovered viruses are nevertheless widespread in laboratory lines and are ubiquitous in cell culture. By sequencing viruses from individual wild-collected flies we show that some viruses are shared between D. melanogaster and D. simulans. Our results provide an essential evolutionary and ecological context for host–virus interaction in Drosophila, and the newly reported viral sequences will help develop D. melanogaster further as a model for molecular and evolutionary virus research.     

Laser nephelometer evaluation of factor IX.

Laser Nephelometry is a technique which allows the evaluation of the concentration of several serum proteins and clotting factors. By means of this technique it is also possible to study the kinetic of the reaction between antigen and antibody. In a few instances the method was also applied in the characterization of abnormal molecules. We developed assays for the measurement of Factor IX antigen and the results were compared with those obtained by conventional immunological methods such as rocket immunoelectrophoresis. Plasmas from patients with haemophilia B, on coumarin treatment, with liver cirrhosis were studied. A standard reference curve was obtained using pooled normal plasma. The factor IX levels obtained by laser nephelometer correlated fairly well with those obtained by electroimmunoassay.     

Identification of dominant bacterial phylotypes in a cadmium-treated forest soil

The presence of heavy metals in soils can lead to changes in microbial community structure, characterized by the dominance of groups that are able to tolerate contamination. Such groups may provide good microbial indicators of heavy-metal pollution in soil. Through terminal restriction fragment length polymorphism (T-RFLP) profiling, changes in the bacterial community structure of an acidic forest soil that had been incubated with cadmium (Cd) for 30 days were investigated. T-RFLP revealed, in particular, three operational taxonomic units (OTUs) strongly dominating in relative abundance in the contaminated soil. By cloning of the amplified 16S rRNA genes and partial sequencing of 25 clones, these three dominant OTUs were phylogenetically characterized. One dominant OTU in the cadmium-contaminated soil was derived from Betaproteobacteria, genus Burkholderia, and the other two were from uncultured members of the class Actinobacteria, closely related to the genus Streptomyces. To confirm T-RFLP data, four primers were designed on the basis of this study's dominant sequences, targeting the OTUs corresponding to Burkholderia or Actinobacteria. Real-time PCR showed that Burkholderia target sequences were more abundant in cadmium-treated soil (7.8 x 10(7)+/- 3.0 x 10(7) targets g(-1) soil) than in untreated soil (4.0 x 10(6)+/- 8.9 x 10(5) targets g(-1) soil). It was concluded that the genus Burkholderia includes species that may be particularly dominant under cadmium contamination.     

The prevalence of AIDS, AIDS related complex and HIV seropositivity in a large population of patients with congenital clotting disorders

We have investigated up to the beginning of 1987 114 patients with congenital clotting disorders. 84 had received plasma and/or clotting factors concentrates. 18 out of 84 (21%) had leukopenia, thrombocytopenia, or both. 64 out of 84 (76%) had been infected by hepatitis B virus. The great majority of them (62 out 64) developed adequate immunity (anti Hbs antibodies). Despite this, 47 out 84 (57%) showed persistently elevated transaminases. 17 out of 84 (20%) had HIV-seropositivity. Among them, 7 are free of symptoms related to such a virus up to present time, 8 developed AIDS-related complex and 2 had the full-blown AIDS and died. Non significant difference in HIV seroconversion or its clinical manifestations was noted depending on the administration of factor VIII concentrates versus prothrombin complex concentrates. In contrast, plasma administration appeared to be associated with a lower risk of viral transmission. No abnormality was observed in patients who had never received haemoderivatives, except the presence of anti Hbs antibodies in 1 of them.     

Sugar phosphorylation modulates ADP inhibition of maize mitochondrial hexokinase

The preference of maize ( Zea mays L.) mitochondrial hexokinase (EC 2.7.1.1.) for glucose and fructose and the ADP regulation were evaluated. The Michaelis-Menten constants (K_m) varied between 0.02 and 0.09 m M for glucose and from 2 to 6 m M for fructose as substrates. The value of V_max was five times higher in the presence of glucose as compared with fructose in membrane-bound enzyme preparations. It was shown that ADP produced from the reaction inhibits the hexokinase activity (K_i=20–50 μ M ). However, the inhibition was very specific for adenine nucleotide. Only a small inhibition was observed when 1 m M of UDP, CDP or GDP was included in the assay medium. Nevertheless, the ADP inhibition was observed only when glucose was phosphorylated. In assay conditions where fructose serves as substrate, the affinity for ADP decreased by 10-fold (K_i varied between 500 and 1  000 μ M ). These kinetics properties were also observed in partially purified soluble enzyme preparations. These data suggest that the type of hexose bound to the catalytic site modulates the ADP control of maize mitochondrial hexokinase.     

Cyclosporin A inhibits protein kinase C activity: a contributing mechanism in the development of nephrotoxicity?

Cyclosporin A modifies many intracellular functions in a variety of different cells. This study investigated the potential interaction between cyclosporin A and protein kinase C, as a possible mechanism for the development of nephrotoxicity. The activity of protein kinase C, in the cytosol of renal epithelial cells, was shown to be significantly inhibited in a dose-dependent manner by CSA. Activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate (phorbol ester) in rat mesangial cells in culture leads to an increase in PGE2 release. Phorbol ester stimulated PGE2 release was significantly inhibited by cyclosporin A. These results would suggest that intracellular site of action of cyclosporin A, in producing alterations in intracellular function and toxicity, may be at the level of protein kinase C.     

Glucose metabolism during embryogenesis of the hard tick Boophilus microplus

Glucose metabolism plays an essential role in the physiology and development of almost all living organisms. In the present study we investigated glucose metabolism during the embryogenesis of the hard tick Boophilus microplus. An increase in glucose and glycogen content during the embryonic development of B. microplus was detected and shown to be due to the high enzyme activity of both gluconeogenesis and glycolytic pathways. Glucose 6-phosphate (G-6P), formed by hexokinase, is driven mainly to pentose-phosphate pathway, producing fundamental substrates for cellular biosynthesis. We detected an increase in glucose 6-phosphate dehydrogenase and pyruvate kinase activities after embryo cellularization. Accumulation of key metabolites such as glycogen and glucose was monitored and revealed that glycogen content decreases from day 1 up to day 6, as the early events of embryogenesis take place, and increases after the formation of embryo cellular blastoderm on day 6. Glucose and guanine (a sub-product of amino acids degradation in arachnids) accumulate almost concomitantly. The activity of phosphoenolpyruvate carboxykinase was increased after embryo cellularization. Taken together these data indicate that glycogen and glucose, formed during B. microplus embryogenesis after blastoderm formation, are produced by intense gluconeogenesis.     

Detection of gunshot residues on cadaveric skin using sodium rhodizonate and a counterstain

We report a staining method for cadaveric tissue using sodium rhodizonate as a skin marker for gunshot residues and a counterstain for the surrounding connective tissue. We studied six well preserved subjects who had died of close range gunshot injury. Skin fragments were removed from the bullet entrance hole including both the disrupted area and adjacent macroscopically intact tissue. Because microscopic examination of postmortem material is difficult after histomorphologic alterations already have occurred as a consequence of postmortem tissue changes, it is necessary to use a staining method that, while detecting gunshot residues, can also make skin cell constituents recognizable from both qualitative and quantitative perspectives. Triphenylmethane dyes (acid fuchsin, aniline blue WS, light green SF yellowish, brilliant green and ethyl green) have proven appropriate for the purpose.