Isolation and sequence analysis of P450 genes from a pyrethroid resistant colony of the major malaria vector Anopheles funestus.

Pyrethroid resistance has been demonstrated in populations of Anopheles funestus from South Africa and southern Mozambique. Resistance is associated with elevated P450 monooxygenase enzymes. In this study, degenerate primers based on conserved regions of Anopheles gambiae P450 CYP4, 6 and 9 families were used to amplify genomic and cDNA templates from A. funestus. A total of 12 CYP4, 12 CYP6 and 7 CYP9 partial genes have been isolated and sequenced. BLAST results revealed that A. funestus P450s generally have a high sequence identity to A. gambiae with above 75% identity at the amino acid level. The exception is CYP9J14. The A. gambiae P450 showing highest identity to CYP9J14 exhibits only 55% identity suggesting that CYP9J14 may have arisen from a recent duplication event. Molecular phylogenetic analysis based on amino acid sequences also supported this hypothesis. Intron positions, but not size, were highly conserved between the two species. The high level of orthology that exists in the P450 gene families of these two species may facilitate the prediction of individual P450 protein function.     

Regional changes in CNS levels of the S-100 and 14-3-2 proteins during development and aging of the mouse

The levels of the S-100 and 14-3-2 proteins were determined in a number of regions of mouse brain at intervals from 1 day to 30 months of age. Both S-100 and 14-3-2 were found in measurable amounts as early as the first day of postnatal age but did not begin to accumulate rapidly in the forebrain, brain stem and cerebellum of the mouse brain until some time between the 7th and 14th days. From days 14 to 28 the levels of S-100 and 14-3-2 in each region continued to increase rapidly with the exception of the forebrain where the rate of accumulation of S-100 appeared to lag considerably behind that in the other regions. The proteins continued to accumulate at a rapid rate until approximately 6 months of age. From 6 to 30 months of age, the levels of 14-3-2 remained relatively stable in cerebellum, hippocampus and hypothalamus and appeared to decrease slightly in striatum and cerebral cortex. In the case of S-100, the level of the protein increased in all regions of brain from 6 to 30 months but the increase was most pronounced in the hippocampus, hypothalamus and striatum. The principal conclusion derived from this study is that the biochemical development and aging of the central nervous system are regionally selective processes.     

Over-expression of <Emphasis Type="Italic">CsGSTU</Emphasis> promotes tolerance to the herbicide alachlor and resistance to <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">tabaci</Emphasis> in transgenic tobacco

Glutathione transferases (GSTs) mainly catalyze the nucleophilic addition of glutathione to a large variety of hydrophobic molecules participating to the vacuole compartmentalization of many toxic compounds. In this work, the putative tolerance of transgenic tobacco plants over-expressing CsGSTU genes towards the chloroacetanilide herbicide alachlor was investigated. Our results show that the treatment with 0.0075 mg cm^-3 of alachlor strongly affects the growth of both wild type and transformed tobacco seedlings with the sole exception of the transgenic lines overexpressing CsGSTU2 isoform that are barely influenced by herbicide treatment. In order to correlate the in planta studies with enzyme properties, recombinant CsGSTs were in vitro expressed and tested for GST activity using alachlor as substrate. The recombinant GSTU2 enzyme was twice more active than GSTU1 in conjugating alachlor to GSH thus indicating that CsGSTU2 might play a crucial role in the plant defense against the herbicide. Moreover, as a consequence of the infiltration with a bacterial suspension of the P. syringae pv. tabaci, transgenic tobacco plants but not wild type plants bestowed the capability to limit toxic metabolite diffusion through plant tissues as indicated by the absence of chlorotic halos formation. Consequently, the transgenic tobacco plants described in the present study might be utilized for phytoremediation of residual xenobiotics in the environment and might represent a model for engineering plants that resist to pathogen attack.     

Draft genome sequence of Bizionia argentinensis, isolated from Antarctic surface water

A psychrotolerant marine bacterial strain, designated JUB59(T), was isolated from Antarctic surface seawater and classified as a new species of the genus Bizionia. Here, we present the first draft genome sequence for this genus, which suggests interesting features such as UV resistance, hydrolytic exoenzymes, and nitrogen metabolism.     

Effect of larval host food substrate on egg load dynamics, egg size and adult female size in four species of braconid fruit fly (Diptera: Tephritidae) parasitoids

Life history theory predicts that individuals will allocate resources to different traits so as to maximize overall fitness. Because conditions experienced during early development can have strong downstream effects on adult phenotype and fitness, we investigated how four species of synovigenic, larval-pupal parasitoids that vary sharply in their degree of specialization (niche breadth) and life history (Diachasmimorpha longicaudata, Doryctobracon crawfordi, Opius hirtus and Utetes anastrephae), allocate resources acquired during the larval stage towards adult reproduction. Parasitoid larvae developed in a single host species reared on four different substrates that differed in quality. We measured parasitoid egg load at the moment of emergence and at 24 h, egg numbers over time, egg size, and also adult size. We predicted that across species the most specialized would have a lower capacity to respond to changes in host substrate quality than wasps with a broad host range, and that within species, females that emerged from hosts that developed in better quality substrates would have the most resources to invest in reproduction. Consistent with our predictions, the more specialized parasitoids were less plastic in some responses to host diet than the more generalist. However, patterns of egg load and size were variable across species. In general, there was a remarkable degree of reproductive effort-allocation constancy within parasitoid species. This may reflect more “time-limited” rather than “egg-limited” foraging strategies where the most expensive component of reproductive success is to locate and handle patchily-distributed and fruit-sequestered hosts. If so, egg costs, independent of degree of specialization, are relatively trivial and sufficient resources are available in fly larvae stemming from all of the substrates tested.     

Characterization of Programmed Death-1 Homologue-1 (PD-1H) Expression and Function in Normal and HIV Infected Individuals

Chronic immune activation that persists despite anti-retroviral therapy (ART) is the strongest predictor of disease progression in HIV infection. Monocyte/macrophages in HIV-infected individuals are known to spontaneously secrete cytokines, although neither the mechanism nor the molecules involved are known. Here we show that overexpression of the newly described co-stimulatory molecule, PD1 homologue (PD-1H) in human monocyte/macrophages is sufficient to induce spontaneous secretion of multiple cytokines. The process requires signaling via PD-1H as cytokine secretion could be abrogated by deletion of the cytoplasmic domain. Such overexpression of PD-1H, associated with spontaneous cytokine expression is seen in monocytes from chronically HIV-infected individuals and this correlates with immune activation and CD4 depletion, but not viral load. Moreover, antigen presentation by PD-1H-overexpressing monocytes results in enhanced cytokine secretion by HIV-specific T cells. These results suggest that PD-1H might play a crucial role in modulating immune activation and immune response in HIV infection.     

Mixed-mode hydrophobic ion exchange for the separation of oligonucleotides and DNA fragments using HPLC

Hydrophobic anion exchangers were formed by cobonding both ionic and hydrophobic ligands to silica gel. These phases were used to separate single-stranded oligonucleotides and double-stranded DNA restriction fragments. By varying the ratio of n-octyldimethylsilane and either 3-chloropropyldimethylsilane or 4-chlorobutyldimethylsilane added during silanization a series of mixed-ligand or mixed-mode stationary phases was created. Concentration and ratio of bonded ligands were determined using a new gas chromatography fluorination method. Total ligand coverage was found to approach 2.1 ligands nm-2 for n-octyldimethylsilane. Bonding reproducibility for mixed-mode phases was good. Nucleic acid separations were achieved under gentle mobile phase conditions by using the stationary phase as an easily modifiable variable.