Structure of flexible filamentous plant viruses

Flexible filamentous viruses make up a large fraction of the known plant viruses, but in comparison with those of other viruses, very little is known about their structures. We have used fiber diffraction, cryo-electron microscopy, and scanning transmission electron microscopy to determine the symmetry of a potyvirus, soybean mosaic virus; to confirm the symmetry of a potexvirus, potato virus X; and to determine the low-resolution structures of both viruses. We conclude that these viruses and, by implication, most or all flexible filamentous plant viruses share a common coat protein fold and helical symmetry, with slightly less than 9 subunits per helical turn.     

Increased intracellular Ca2+ and SR Ca2+ load contribute to arrhythmias after acidosis in rat heart. Role of Ca2+/calmodulin-dependent protein kinase II

Returning to normal pH after acidosis, similar to reperfusion after ischemia, is prone to arrhythmias. The type and mechanisms of these arrhythmias have never been explored and were the aim of the present work. Langendorff-perfused rat/mice hearts and rat-isolated myocytes were subjected to respiratory acidosis and then returned to normal pH. Monophasic action potentials and left ventricular developed pressure were recorded. The removal of acidosis provoked ectopic beats that were blunted by 1 muM of the CaMKII inhibitor KN-93, 1 muM thapsigargin, to inhibit sarcoplasmic reticulum (SR) Ca(2+) uptake, and 30 nM ryanodine or 45 muM dantrolene, to inhibit SR Ca(2+) release and were not observed in a transgenic mouse model with inhibition of CaMKII targeted to the SR. Acidosis increased the phosphorylation of Thr(17) site of phospholamban (PT-PLN) and SR Ca(2+) load. Both effects were precluded by KN-93. The return to normal pH was associated with an increase in SR Ca(2+) leak, when compared with that of control or with acidosis at the same SR Ca(2+) content. Ca(2+) leak occurred without changes in the phosphorylation of ryanodine receptors type 2 (RyR2) and was blunted by KN-93. Experiments in planar lipid bilayers confirmed the reversible inhibitory effect of acidosis on RyR2. Ectopic activity was triggered by membrane depolarizations (delayed afterdepolarizations), primarily occurring in epicardium and were prevented by KN-93. The results reveal that arrhythmias after acidosis are dependent on CaMKII activation and are associated with an increase in SR Ca(2+) load, which appears to be mainly due to the increase in PT-PLN.     

New substrate analogues of human serotonin N-acetyltransferase produce in situ specific and potent inhibitors.

Melatonin is synthesized by an enzymatic pathway, in which arylalkylamine (serotonin) N-acetyltransferase catalyzes the rate-limiting step. A previous study reported the discovery of bromoacetyltryptamine (BAT), a new type of inhibitor of this enzyme. This compound is the precursor of a potent bifunctional inhibitor (analogue of the transition state), capable of interfering with both the substrate and the cosubstrate binding sites. This inhibitor is biosynthesized by the enzyme itself in the presence of free coenzyme A. In the present report, we describe the potency of new N-halogenoacetyl derivatives leading to a strong in situ inhibition of serotonin N-acetyltransferase. The new concept behind the mechanism of action of these precursors was studied by following the biosynthesis of the inhibitor from tritiated-BAT in a living cell. The fate of tritiated-phenylethylamine (PEA), a natural substrate of the enzyme, in the presence or absence of [(3)H]BAT was also followed, leading to their incorporation into the reaction product or the inhibitor (N-acetyl[(3)H]PEA and coenzyme A-S[(3)H]acetyltryptamine, respectively). The biosynthesis of this bifunctional inhibitor derived from BAT was also followed by nuclear magnetic resonance during its catalytic production by the pure enzyme. In a similar manner we studied the production of another inhibitor generated from N-[2-(7-hydroxynaphth-1-yl)ethyl]bromoacetamide. New derivatives were also screened for their capacity to inhibit a purified enzyme, in addition to enzyme overexpressed in a cellular model. Some of these compounds proved to be extremely potent, with IC(50)s of approximately 30 nM. As these compounds, by definition, closely resemble the natural substrates of arylalkylamine N-acetyltransferase, we also show that they are potent ligands at the melatonin receptors. Nevertheless, these inhibitors form a series of pharmacological tools that could be used to understand more closely the inhibition of pineal melatonin production in vivo.     

Polyoxometalates--a new class of potent ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) inhibitors

Polyoxotungstates were identified as potent inhibitors of NTPDases1, 2, and 3. The most potent compound was K(6)H(2)[TiW(11)CoO(40)], exhibiting K(i) values of 0.140 microM (NTPDase1), 0.910 microM (NTPDase2), and 0.563 microM (NTPDase3). One of the compounds, (NH(4))(18)[NaSb(9)W(21)O(86)], was selective for NTPDases2 and 3 versus NTPDase1. NTPDase inhibition might contribute to the described biological effects of polyoxometalates, including their anti-cancer activity.     

Conserved and non-conserved loci of the glucagon gene in old world ruminating ungulates

The homology and diversification of genomic sequence encoding glucagon gene among native Egyptian buffalos, camel and sheep were tested using cattle as model. Oligodeoxynucleotide primers designed from the available GenBank data were used for PCR probing of the glucagon gene encoding sequence at different loci. The DNA oligomer probes were constructed to flank either the whole gene encoding sequence or different intra-gene encoding sequences. The PCR products were visualized using agarose gel electrophoresis. All species showed a same size band of prepro-glucagon when PCR was used to amplify the whole gene encoding sequence. In contrary, amplifications of different intra-gene loci failed to give the same results. The results indicated variable degrees of diversity among old world ruminating ungulates in the glucagon gene encoding sequence. Compared with other ruminants, the variation appears predominantly in camel. Surprisingly, the similarity in size between both amplification products of whole gene encoding sequence and the proposed size of glucagon cDNA definitely excludes the possibility of large intervening introns spanning the genomic sequence of the glucagon gene in these species. This indicates that, in contrast to other tested mammals, the glucagon gene includes an essentially full-length copy of glucagon mRNA. The study revealed a possible new aspect of glucagon gene evolution in order to correlate its corresponding protein function among different ruminant species.     

Toxicity and inhibition of feeding and tunneling response of naphthalene and 10 derivatives on the formosan subterranean termite (Isoptera: Rhinotermitidae)

In laboratory choice tests with either filter paper treatment or sand treatment, naphthalene and 10 derivatives were evaluated for their effects on the behavior of the Formosan subterranean termite, Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae). After 24-h exposure, workers avoided 50-microg/cm2-treated filter paper with 1'- and 2'-acetonaphthone. By day 8, feeding activity on filter paper treated with 1- and 2-methoxynaphthalene, 1'- and 2'-acetonaphthone were significantly reduced compared with the controls. Naphthalene and six other derivatives were not effective at 50-microg/cm2-treated filter paper. After 12-d exposure, tunnels constructed in sand treated with 1- and 2-methoxynaphthalene (0-3 cm2), 1'- and 2'-acetonaphthone (2-9 cm2), 2-naphthalene methanol (14-19 cm2), or 2-isopropylnaphthalene (16-19 cm2) at the rate of 100 mg/kg sand were significantly less than the controls (34 cm2). Consumption on filter paper placed on sand treated with 1- and 2-methoxynaphthalene and 1'- and 2'-acetonaphthone was significantly reduced (0-7 mg) compared with the controls (33-54 mg). 1-Methoxynaphthalene was the only treatment that caused significantly less feeding activity in the untreated sand chamber (0-6 mg) compared with the control (63-128 mg).     

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.