Efficiency,intrinsic competition and interspecific host discrimination of Copidosoma desantisi and Trichogramma evanescens,two parasitoids of Phthorimaea operculella

Trichogramma evanescens West. (Hymenoptera: Trichogrammatidae) and Copidosoma desantisi Annecke & Mynhardt (Hymenoptera: Encyrtidae) are potential parasitoids of the potato tuber moth (PTM), Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae) in Egypt. Discrimination of a parasitized host from an unparasitized host would prevent wasting of time, eggs and reduce competition with conspecifics or heterospecifics. Therefore, we evaluated interspecific host discrimination, multiparasitism and intrinsic competition between the two wasp species. In a choice test, females of T. evanescens showed high interspecific host discrimination only when they were offered 2-day-old C. desantisi parasitized and unparasitized PTM eggs. In contrast, C. desantisi showed high host discrimination and preferred unparasitized eggs to PTM eggs harboring 2-h- or 2-day-old T. evanescens’ eggs. We also evaluated the effect of different introduction sequences on the efficacy of the two wasps. Dissection data indicated that the two parasitoids had a negative impact on each other. There was a significant reduction in the total number of deposited eggs as well as total number of parasitized hosts by each parasitoid. Regarding the rearing experiment, the total number of T. evanescens-induced black eggs or C. desantisi formed mummies in combined treatments was significantly lower than in single parasitoid treatments (control). Moreover, C. desantisi was inferior and did not develop from any multiparasitized host regardless of oviposition order. It was suggested that combined release of the two wasps would not elevate rate of parasitism over that of single parasitoid treatments and competition between them would reduce their efficacy.     

Identifying modulators of hERG channel activity using the PatchXpress planar patch clamp

The authors used the PatchXpress 7000A system to measure compound activity at the hERG channel using procedures that mimicked the "gold-standard" conventional whole-cell patch clamp. A set of 70 compounds, including hERG antagonists with potencies spanning 3 orders of magnitude, were tested on hERG302-HEK cells using protocols aimed at either identifying compound activity at a single concentration or obtaining compound potency from a cumulative concentration dependence paradigm. After exposure to compounds and subsequent washout of the wells to determine reversibility of the block, blockade by a reference compound served as a quality control. Electrical parameters and voltage dependence were similar to those obtained using a conventional whole-cell patch clamp. Rank order of compound potency was also comparable to that determined by conventional methods. One exception was flunarizine, a particularly lipophilic compound. The PatchXpress accurately identified the activity of 29 moderately potent antagonists, which only weakly displace radiolabeled astemizole and are false negatives in the binding assay. Finally, no false hits were observed from a collection of relatively inactive compounds. High-quality data acquisition by PatchXpress should help accelerate secondary screening for ion channel modulators and the drug discovery process.     

Effects of iron limitation on adherence and cell surface carbohydrates of Corynebacterium diphtheriae strains

Iron limitation may cause bacterial pathogens to grow more slowly; however, it may also stimulate these microorganisms to produce greater tissue damage, given that many virulence factors are controlled by the iron supply in the environment. The present study investigated the influence of low iron availability on the expression of proteins and surface sugar residues of two toxigenic strains of Corynebacterium diphtheriae subsp. mitis and evaluated their adherence to human group B erythrocytes and HEp-2 cells. A comparison was made between bacteria grown in (i) Trypticase soy broth (TSB), (ii) TSB treated with dipyridyl to deplete free iron, and (iii) TSB enriched with FeCl(3). The effects of iron concentration on adhesive properties were different for strains 241 and CDC-E8392, of the sucrose-fermenting and non-sucrose-fermenting biotypes, respectively. Iron-limited conditions enhanced interaction of strain 241 with erythrocytes and HEp-2 cells. Inhibition assays suggested the involvement of nonfimbrial protein combination 67-72p on hemagglutination of diphtheria bacilli grown under iron-limited conditions. Conversely, iron limitation inhibited adherence to glass and expression of electron-dense material on the bacterial surface. Lectin binding assays demonstrated a reduction in the number of sialic acid residues and an increase in D-mannose and D-galactose residues on the surfaces of both strains. Thus, iron exerts a regulatory role on adhesive properties of diphtheria bacilli, and low iron availability modulates the expression of C. diphtheriae surface carbohydrate moieties. The significant changes in the degree of lectin binding specific for D-mannose, D-galactose and sialic acid residues may have an effect on binding of host cells. The expression of dissimilar microbial virulence determinants may be coordinately controlled by common regulatory systems. For C. diphtheriae, the present results imply regulation of adherence and slime production as part of a global response to iron-limited environmental conditions that includes derepression of genes for the synthesis of cytotoxin and siderophores and for transport of the Fe(III)-siderophore complexes.     

Phylogenetic and metabolic diversity of bacteria degrading aromatic compounds under denitrifying conditions,and description of Thauera phenylacetica sp. nov., Thauera aminoaromaticasp. nov., and Azoarcus buckelii sp. nov

Six strains of denitrifying bacteria isolated from various oxic and anoxic habitats on different monocyclic aromatic substrates were characterized by sequencing 16S rRNA genes, determining physiological and morphological traits, and DNA-DNA hybridization. According to these criteria, strains S100, SP and LG356 were identified as members of Thauera aromatica. Strains B5-1 and B5-2 were tentatively affiliated to the species Azoarcus tolulyticus. Strains B4P and S2 were only distantly related to each other and to other described Thauera species. These two strains are proposed as the type strains of two new species, Thauera phenylacetica sp. nov. and Thauera aminoaromaticasp. nov., respectively. By 16S rRNA gene analysis, strain U120 was highly related to the type strains of Azoarcus evansii and Azoarcus anaerobius, whereas corresponding DNA-DNA reassociation values indicated only a low degree of genomic relatedness. Based upon a low DNA similarity value and the presence of distinguishing physiological properties, strain U120 is proposed as the type strain of a new species, Azoarcus buckelii sp. nov. Almost all of the new isolates were obtained with different substrates. The highly varied substrate spectra of the isolates indicates that an even higher diversity of denitrifying bacteria degrading aromatic compounds would be discovered in the different habitats by using a larger spectrum of aromatic substrates for enrichment and isolation.     

Mutation analysis of the human CYP3A4 gene 5' regulatory region: population screening using non-radioactive SSCP

Human CYP3A4 is the major cytochrome P450 isoenzyme in adult human liver and is known to metabolise many xenobiotic and endogenous compounds. There is substantial inter-individual variation in the hepatic levels of CYP3A4. Although, polymorphic mutations have been reported in the 5' regulatory region of the CYP3A4 gene, those that have been investigated so far do not appear to have any effect on gene expression. To determine whether other mutations exist in this region of the gene, we have performed a new population screen on a panel of 101 human DNA samples. A 1140 bp section of the 5' proximal regulatory region of the CYP3A4 gene, containing numerous regulatory motifs, was amplified from genomic DNA as three overlapping segments. The 300 bp distal enhancer region at -7.9kb containing additional regulatory motifs was also amplified. Mutation analysis of the resulting PCR products was carried out using non-radioactive single strand conformation polymorphism (SSCP) and confirmatory sequencing of both DNA strands in those samples showing extra SSCP bands. In addition to detection of the previously reported CYP3A4*1B allele in nine subjects, three novel alleles were found: CYP3A4*1E (having a T-->A transversion at -369 in one subject), CYP3A4*1F (having a C-->G tranversion at -747 in 17 subjects) and CYP3A4*15B containing a nine-nucleotide insertion between -845 and -844 linked to an A-->G transition at -392 and a G-->A transition in exon 6 (position 485 in the cDNA) in one subject. All the novel alleles were heterozygous. No mutations were found in the upstream distal enhancer region. Our results clearly indicate that this rapid and simple SSCP approach can reveal mutant alleles in drug metabolising enzyme genes. Detection and determination of the frequency of novel alleles in CYP3A4 will assist investigation of the relationship between genotype, xenobiotic metabolism and toxicity in the CYP3A family of isoenzymes.     

PTEN associates with the vault particles in HeLa cells

PTEN is a tumor suppressor that primarily dephosphorylates phosphatidylinositol 3,4,5-trisphosphate to down-regulate the phosphoinositide 3-kinase/Akt signaling pathway. Although the cellular functions of PTEN as a tumor suppressor have been well characterized, the mechanism by which PTEN activity is modulated by other signal molecules in vivo remains poorly understood. In searching for potential PTEN modulators through protein-protein interaction, we identified the major vault protein (MVP) as a dominant PTEN-binding protein in a yeast two-hybrid screen. MVP is the major structural component of vault, the largest intracellular ribonucleoprotein particle. Co-immunoprecipitation confirmed the interaction between PTEN and MVP in transfected mammalian cells. More importantly, we found that a significant portion of endogenous PTEN associates with vault particles in human HeLa cells. Deletion mutation analysis demonstrated that MVP binds to the C2 domain of PTEN and that PTEN interacts with MVP through its EF hand-like motif. Furthermore, the in vitro binding experiments revealed that the interaction of PTEN with MVP is Ca(2+)-dependent.     

Molecular differentiation and diversity among the California red oaks (Fagaceae; Quercus section Lobatae)

A recent epidemic of Phytopthora (Sudden Oak Death) in coastal woodlands of California is causing severe mortality in some oak species belonging to the red oak (Lobatae) group. To predict the risks of spread of this disease, an understanding of the relationships among California's red oak species and of their population genetic structure is needed. We focus here on relationships among the four species of red oak. Whereas morphological distinction of Quercus wislizeni and Quercus parvula can pose problems, Quercus kelloggii and Quercus agrifolia in pure forms are easily distinguishable from one another and from Q. wislizeni and Q. parvula in the field. However, hybrids among all species combinations are known to occur in nature and these can confound data from ecological studies. Our results revealed greatest differentiation of the deciduous Q. kelloggii, with only weak AFLP fragment differentiation of the three remaining evergreen species. The molecular data suggest a closer affinity of Q. agrifolia with Q. wislizeni and Q. parvula contrary to earlier suggestions that its origins are likely to have been with northern deciduous oaks probably through a common ancestor with Q. kelloggii. Interior and coastal populations of Q. wislizeni separated in dendrograms based on phenetic and genetic distances suggesting probable isolation in different glacial refugia. The position of Q. parvula remains ambiguous, having a closer affinity with interior populations of Q. wislizeni and with Q. agrifolia, than with coastal populations of Q. wislizeni. Mean population differentiation in Q. wislizeni was 0.18, which is somewhat higher than the average for other oak species, suggesting that range fragmentation has occurred in the past, resulting in a metapopulation structure. Our results provide evidence that introgression among these species may be causing reticulation, further confounding species separation. Whereas Phytopthora has been reported on Q. agrifolia, Q. parvula and Q. kelloggii, it has not yet been detected in natural populations of Q. wislizeni. The species relationships that our molecular data show suggest that this is more likely a result of escape due to ecological tolerances than to genetic differences.