Effect of Two Opiine Parasitoids (Hymenoptera: Braconidae) Introduced for Fruit Fly Control on a Native Hawaiian Tephritid,Trupanea dubautiae(Diptera: Tephritidae)

Diachasmimorpha longicaudata(Ashmead) andPsyttalia fletcheri(Silvestri) are opiine parasitoids introduced into Hawaii for control of the Oriental fruit fly,Bactrocera dorsalis(Hendel) and the melon fly,Bactrocera cucurbitae(Coquillett), respectively. Both species have recently been mass-reared and released for research in augmentative biocontrol programs. Laboratory and field sleeve cage experiments were conducted to investigate the potential impact of mass-producedD. longicaudataandP. fletcherion a native Hawaiian tephritid,Trupanea dubautiae(Bryan), infesting the flowerheads of the native composite shrubDubautia raillardioidesHillebrand. Gravid females ofD. longicaudataandP. fletcheriwere confined with bloomingD. raillardioidesflowerheads infested with late instarT. dubautiae.BothD. longicaudataandP. fletcherilacked positive oviposition responses toT. dubautiaelarvae in infested flowerheads and caused neither parasitism nor mortality to the flies. However, when larvae were removed from the flowerheads and presented in screened dishes containing artificial diet of the parasitoids' normal rearing hosts (B. dorsalisandB. cucurbitae), bothD. longicaudataandP. fletcherireadily oviposited in the test larvae. Oviposition byD. longicaudatadid not significantly affect the percentage pupation ofT. dubautiae,but did reduce the emergence of adult flies. Oviposition byP. fletcherisignificantly reduced both pupation and adult fly emergence. All progeny of both parasitoid species died as eggs or first-instar larvae. Results from our experiments demonstrate that biological control programs targeted against frugivorous tephritid pests byD. longicaudataandP. fletcherihave no harmful impact on flowerhead-infestingT. dubautiae.     

3H-Dihydromorphine binding in brain regions of young and aged rats

Opiate receptor-ligand binding was investigated in brains of young and aged female F344 rats. A significant reduction in the density of binding sites for ³H-dihydromorphine was observed in the thalamus and midbrain of aged rats. Evidence of two binding sites was observed in the anterior cortex of young rats, whereas aged rats exhibited only a single site. The occurrence of pituitary tumors in the old female rats did not affect ³H-dihydromorphine binding. The highest density of dihydromorphine binding sites was found in the diencephalon, and the lowest density in the hippocampus. Receptor densities in the anterior cortex, striatum, amygdala, frontal poles and midbrain were intermediate, and few, if any, high affinity binding sites for dihydromorphine were found in the pons-medulla or posterior cortex.     

Identification of lipocalin-2 as a PKCδ phosphorylation substrate in neutrophils

Background

PKCδ expressed in neutrophils is implicated in promoting reperfusion injury after ischemic stroke. To understand the molecular and cellular actions of PKCδ, we employed a chemical-genetics approach to identify PKCδ substrates in neutrophils.

Results

We recently generated knock-in mice endogenously expressing analog-specific PKCδ (AS-PKCδ) that can utilize ATP analogs as phosphate donors. Using neutrophils isolated from the knock-in mice, we identified several PKCδ substrates, one of which was lipocalin-2 (LCN2), which is an iron-binding protein that can trigger apoptosis by reducing intracellular iron concentrations. We found that PKCδ phosphorylated LCN2 at T115 and this phosphorylation was reduced in Prkcd^−/− mice. PKCδ colocalized with LCN2 in resting and stimulated neutrophils. LCN2 release from neutrophils after cerebral ischemia was reduced in PKCδ null mice.

Conclusions

These findings suggest that PKCδ phosphorylates LCN2 and mediates its release from neutrophils during ischemia-reperfusion injury.     

Antiadhesive Properties of Abelmoschus esculentus (Okra) Immature Fruit Extract against Helicobacter pylori Adhesion

Background

Traditional Asian and African medicine use immature okra fruits (Abelmoschus esculentus) as mucilaginous food to combat gastritis. Its effectiveness is due to polysaccharides that inhibit the adhesion of Helicobacter pylori to stomach tissue. The present study investigates the antiadhesive effect in mechanistic detail.

Methodology

A standardized aqueous fresh extract (Okra FE) from immature okra fruits was used for a quantitative in vitro adhesion assay with FITC-labled H. pylori J99, 2 clinical isolates, AGS cells, and fluorescence-activated cell sorting. Bacterial adhesins affected by FE were pinpointed using a dot-blot overlay assay with immobilized Lewis^b, sialyl-Lewis^a, H-1, laminin, and fibronectin. ¹²⁵I-radiolabeled Okra FE polymer served for binding studies to different H. pylori strains and interaction experiments with BabA and SabA. Iron nanoparticles with different coatings were used to investigate the influence of the charge-dependence of an interaction on the H. pylori surface.

Principal findings

Okra FE dose-dependently (0.2 to 2 mg/mL) inhibited H. pylori binding to AGS cells. FE inhibited the adhesive binding of membrane proteins BabA, SabA, and HpA to its specific ligands. Radiolabeled compounds from FE bound non-specifically to different strains of H. pylori, as well as to BabA/SabA deficient mutants, indicating an interaction with a still-unknown membrane structure in the vicinity of the adhesins. The binding depended on the charge of the inhibitors. Okra FE did not lead to subsequent feedback regulation or increased expression of adhesins or virulence factors.

Conclusion

Non-specific interactions between high molecular compounds from okra fruits and the H. pylori surface lead to strong antiadhesive effects.     

Distribution and Abundance of Mymarid Parasitoids (Hymenoptera: Mymaridae) of Sophonia rufofascia Kuoh and Kuoh (Homoptera: Cicadellidae) in Hawaii

The abundance of mymarid parasitoids attacking the two-spotted leafhopper (Sophonia rufofascia [Kuoh and Kuoh]), a polyphagous pest recently adventive to Hawaii, was monitored using yellow sticky cards deployed in several areas on the islands of Kauai and Hawaii. The yellow cards captured Chaetomymar sp. nr bagicha Narayanan, Subba Rao, & Kaur and Schizophragma bicolor (Dozier), both adventive species, and Polynema sp. Haliday, which is endemic to Hawaii (Hymenoptera: Mymaridae). The former two species were most abundant at all sites. On Kauai, there was a negative correlation between the captures of C. sp. nr bagicha and those of Polynema sp. Throughout the season, the increase in parasitoid numbers generally followed the increase in leafhopper numbers. C. sp. nr. bagicha and S. bicolor showed distinct habitat preferences. Removal of Myrica faya Aiton, an invasive weed that is a highly preferred two-spotted leafhopper host, decreased the overall numbers of captured parasitoids, but led to a twofold increase in the ratio of trapped parasitoids/hosts in weed-free areas.     

Protein kinase C epsilon regulates gamma-aminobutyrate type A receptor sensitivity to ethanol and benzodiazepines through phosphorylation of gamma2 subunits

Ethanol enhances gamma-aminobutyrate (GABA) signaling in the brain, but its actions are inconsistent at GABA(A) receptors, especially at low concentrations achieved during social drinking. We postulated that the epsilon isoform of protein kinase C (PKCepsilon) regulates the ethanol sensitivity of GABA(A) receptors, as mice lacking PKCepsilon show an increased behavioral response to ethanol. Here we developed an ATP analog-sensitive PKCepsilon mutant to selectively inhibit the catalytic activity of PKCepsilon. We used this mutant and PKCepsilon(-/-) mice to determine that PKCepsilon phosphorylates gamma2 subunits at serine 327 and that reduced phosphorylation of this site enhances the actions of ethanol and benzodiazepines at alpha1beta2gamma2 receptors, which is the most abundant GABA(A) receptor subtype in the brain. Our findings indicate that PKCepsilon phosphorylation of gamma2 regulates the response of GABA(A) receptors to specific allosteric modulators, and, in particular, PKCepsilon inhibition renders these receptors sensitive to low intoxicating concentrations of ethanol.     

nPKCepsilon, a P2Y2-R downstream effector in regulated mucin secretion from airway goblet cells

Airway goblet cell mucin secretion is controlled by agonist activation of P2Y(2) purinoceptors, acting through Gq/PLC, inositol-1,4,5-trisphosphate (IP(3)), diacylglycerol, Ca(2+) and protein kinase C (PKC). Previously, we showed that SPOC1 cells express cPKCalpha, nPKCdelta, nPKCepsilon, and nPKCeta; of these, only nPKCdelta translocated to the membrane in correlation with mucin secretion (Abdullah LH, Bundy JT, Ehre C, Davis CW. Am J Physiol Lung Physiol 285: L149-L160, 2003). We have verified these results and pursued the identity of the PKC effector isoform by testing the effects of altered PKC expression on regulated mucin release using SPOC1 cell and mouse models. SPOC1 cells overexpressing cPKCalpha, nPKCdelta, and nPKCeta had the same levels of ATPgammaS- and phorbol-1,2-myristate-13-acetate (PMA)-stimulated mucin secretion as the levels in empty retroviral vector expressing cells. Secretagogue-induced mucin secretion was elevated only in cells overexpressing nPKCepsilon (14.6 and 23.5%, for ATPgammaS and PMA). Similarly, only SPOC1 cells infected with a kinase-deficient nPKCepsilon exhibited the expected diminution of stimulated mucin secretion, relative to wild-type (WT) isoform overexpression. ATPgammaS-stimulated mucin secretion from isolated, perfused mouse tracheas was diminished in P2Y(2)-R null mice by 82% relative to WT mice, demonstrating the utility of mouse models in studies of regulated mucin secretion. Littermate WT and nPKCdelta knockout (KO) mice had nearly identical levels of stimulated mucin secretion, whereas mucin release was nearly abolished in nPKCepsilon KO mice relative to its WT littermates. We conclude that nPKCepsilon is the effector isoform downstream of P2Y(2)-R activation in the goblet cell secretory response. The translocation of nPKCdelta observed in activated cells is likely not related to mucin secretion but to some other aspect of goblet cell biology.     

Efficacy of clone fingerprinting methodologies

With the development of new high-information content fingerprinting techniques for constructing BAC-based physical maps, physical map construction is accelerating and it is important to determine which methodologies work best. In a recent publication (Z. Xu et al., 2004, Genomics 84:941-951), Xu et al. evaluated five different techniques (one agarose-based and four using multiple enzymes) and concluded that a two-enzyme technique was superior. In addition, they found that no benefit was gained from fingerprinting more than 10x coverage. In this paper we report our own extensive simulation results, which lead to contrasting conclusions. Our data indicate that the five-enzyme method known as SNaPshot is the most effective and that the assembly can in fact be significantly improved with greater than 10x coverage.     

KRAS Prenylation Is Required for Bivalent Binding with Calmodulin in a Nucleotide-Independent Manner

Deregulation of KRAS4b signaling pathway has been implicated in 30% of all cancers. Membrane localization of KRAS4b is an essential step for the initiation of the downstream signaling cascades that guide various cellular mechanisms. KRAS4b plasma membrane (PM) binding is mediated by the insertion of a prenylated moiety that is attached to the terminal carboxy-methylated cysteine, in addition to electrostatic interactions of its positively charged hypervariable region with anionic lipids. Calmodulin (CaM) has been suggested to selectively bind KRAS4b to act as a negative regulator of the RAS/mitogen-activated protein kinase (MAPK) signaling pathway by displacing KRAS4b from the membrane. However, the mechanism by which CaM can recognize and displace KRAS4b from the membrane is not well understood. In this study, we employed biophysical and structural techniques to characterize this mechanism in detail. We show that KRAS4b prenylation is required for binding to CaM and that the hydrophobic pockets of CaM can accommodate the prenylated region of KRAS4b, which might represent a novel CaM-binding motif. Remarkably, prenylated KRAS4b forms a 2:1 stoichiometric complex with CaM in a nucleotide-independent manner. The interaction between prenylated KRAS4b and CaM is enthalpically driven, and electrostatic interactions also contribute to the formation of the complex. The prenylated KRAS4b terminal KSKTKC-farnesylation and carboxy-methylation is sufficient for binding and defines the minimal CaM-binding motif. This is the same region implicated in membrane and phosphodiesterase6-δ binding. Finally, we provide a structure-based docking model by which CaM binds to prenylated KRAS4b. Our data provide new insights into the KRAS4b-CaM interaction and suggest a possible mechanism whereby CaM can regulate KRAS4b membrane localization.