A Ds-insertion mutant of OSH6 (Oryza sativa Homeobox 6) exhibits outgrowth of vestigial leaf-like structures, bracts, in rice

OSH6 (Oryza sativa Homeobox6) is an ortholog of lg3 (Liguleless3) in maize. We generated a novel allele, termed OSH6-Ds, by inserting a defective Ds element into the third exon of OSH6, which resulted in a truncated OSH6 mRNA. The truncated mRNA was expressed ectopically in leaf tissues and encoded the N-terminal region of OSH6, which includes the KNOX1 and partial KNOX2 subdomains. This recessive mutant showed outgrowth of bracts or produced leaves at the basal node of the panicle. These phenotypes distinguished it from the OSH6 transgene whose ectopic expression led to a “blade to sheath transformation” phenotype at the midrib region of leaves, similar to that seen in dominant Lg3 mutants. Expression of a similar truncated OSH6 cDNA from the 35S promoter (35S::ΔOSH6) confirmed that the ectopic expression of this product was responsible for the aberrant bract development. These data suggest that OSH6-Ds interferes with a developmental mechanism involved in bract differentiation, especially at the basal nodes of panicles. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Novel 1H-pyrrolo[2,3-c]pyridines as acid pump antagonists (APAs)

A series of 1H-pyrrolo[2,3-c]pyridines as acid pump antagonists (APAs) was synthesized and the inhibitory activities against H⁺/K⁺ ATPase isolated from hog gastric mucosa were determined. After elaborating on substituents at N1, C5, and C7 position of 1H-pyrrolo[2,3-c]pyridine scaffold, we have observed that compounds 14f and 14g are potent APAs with H⁺/K⁺ ATPase IC₅₀ = 28 and 29 nM, respectively.     

Integrative Functional Genomics of Hepatitis C Virus Infection Identifies Host Dependencies in Complete Viral Replication Cycle

Recent functional genomics studies including genome-wide small interfering RNA (siRNA) screens demonstrated that hepatitis C virus (HCV) exploits an extensive network of host factors for productive infection and propagation. How these co-opted host functions interact with various steps of HCV replication cycle and exert pro- or antiviral effects on HCV infection remains largely undefined. Here we present an unbiased and systematic strategy to functionally interrogate HCV host dependencies uncovered from our previous infectious HCV (HCVcc) siRNA screen. Applying functional genomics approaches and various in vitro HCV model systems, including HCV pseudoparticles (HCVpp), single-cycle infectious particles (HCVsc), subgenomic replicons, and HCV cell culture systems (HCVcc), we identified and characterized novel host factors or pathways required for each individual step of the HCV replication cycle. Particularly, we uncovered multiple HCV entry factors, including E-cadherin, choline kinase α, NADPH oxidase CYBA, Rho GTPase RAC1 and SMAD family member 6. We also demonstrated that guanine nucleotide binding protein GNB2L1, E2 ubiquitin-conjugating enzyme UBE2J1, and 39 other host factors are required for HCV RNA replication, while the deubiquitinating enzyme USP11 and multiple other cellular genes are specifically involved in HCV IRES-mediated translation. Families of antiviral factors that target HCV replication or translation were also identified. In addition, various virologic assays validated that 66 host factors are involved in HCV assembly or secretion. These genes included insulin-degrading enzyme (IDE), a proviral factor, and N-Myc down regulated Gene 1 (NDRG1), an antiviral factor. Bioinformatics meta-analyses of our results integrated with literature mining of previously published HCV host factors allows the construction of an extensive roadmap of cellular networks and pathways involved in the complete HCV replication cycle. This comprehensive study of HCV host dependencies yields novel insights into viral infection, pathogenesis and potential therapeutic targets.     

Olfactory attraction to aggregation pheromone is mediated by disti-flagellum of antennal segments in Riptortus pedestris

Riptortus pedestris (Hemiptera: Alydidae) is a serious pest of soybean and sweet persimmon and uses male produced aggregation pheromone, (E)-2-hexenyl (Z)-3-hexenoate, (E)-2-hexenyl (E)-2-hexenoate, and tetradecyl isobutyrate to facilitate food location and recognition by conspecifics. Using electroantennogram (EAG) and greenhouse bioassay, we determined which antennal segment is involved in the detection of their aggregation pheromone. In the first EAG test using individual antennal segments, significant EAG responses to 1:1:1 mixture of the aggregation pheromone were observed only from the disti-flagellum segments of both male and female antennae at both pheromone doses tested (1 µg and 100 µg). In the following EAG tests using gradually removed antennal segment(s), EAG response was still maintained when the distal half of a disti-flagellum was surgically removed, while EAG response was lost when whole segment of disti-flagellum or other whole segments were gradually removed from intact antenna of both sexes. In greenhouse experiment, removing one or both segment(s) of disti-flagellum from male or female antennae resulted in significant reduction in their attraction to the aggregation pheromone. Together, these findings support that the disti-flagellum of R. pedestris houses olfactory neurons associated with attraction to their aggregation pheromone.     

Expression and characterization of glutamate decarboxylase from <Emphasis Type="Italic">Lactobacillus brevis</Emphasis> HYE1 isolated from <Emphasis Type="Italic">kimchi</Emphasis>

A putative gene (gadlbhye1) encoding glutamate decarboxylase (GAD) was cloned from Lactobacillus brevis HYE1 isolated from kimchi, a traditional Korean fermented vegetable. The amino acid sequences of GADLbHYE1 showed 48% homology with the GadA family and 99% identity with the GadB family from L. brevis. The cloned GADLbHYE1 was functionally expressed in Escherichia coli using inducible expression vectors. The expressed recombinant GADLbHYE1 was successfully purified by Ni–NTA affinity chromatography, and had a molecular mass of 54 kDa with optimal hydrolysis activity at 55 °C and pH 4.0. Its thermal stability was determined to be higher than that of other GADs from L. brevis, based on its melting temperature (75.18 °C). Kinetic parameters including K_m and V_max values for GADLbHYE1 were 4.99 mmol/L and 0.224 mmol/L/min, respectively. In addition, the production of gamma-aminobutyric acid in E. coli BL21 harboring gadlbhye1/pET28a was increased by adding pyridoxine as a cheaper coenzyme.     

Determination of the penicillin acylase activity of E. coli cultures using a method of gas-liquid chromatography]

Penicillinacylase activity was determined in E. coli by the product of benzylpenicillin destruction, i.e. phenylacetic acid formed under the effect of the enzyme. The determination was performed on a chromatograph. The immobile phase consisted of 10 per cent of ethylenglycol edipate on chromosorb A, modified with 2 per cent H3PO4. Nitrogen was used as the gaseous carrier. The method is rapid and handy for mass testing of the cultures with a purpose of detecting penicillinacylase-producing strains. It provided reliable determination of penicillinacylase in the cultures producing simultaneously beta-lactamase, another penicillin-destroying enzyme.     

Minor contribution of cytosolic Ca2+ transients to the pacemaker rhythm in guinea pig sinoatrial node cells

The question of the extent to which cytosolic Ca(2+) affects sinoatrial node pacemaker activity has been discussed for decades. We examined this issue by analyzing two mathematical pacemaker models, based on the "Ca(2+) clock" (C) and "membrane clock" (M) hypotheses, together with patch-clamp experiments in isolated guinea pig sinoatrial node cells. By applying lead potential analysis to the models, the C mechanism, which is dependent on potentiation of Na(+)/Ca(2+) exchange current via spontaneous Ca(2+) release from the sarcoplasmic reticulum (SR) during diastole, was found to overlap M mechanisms in the C model. Rapid suppression of pacemaker rhythm was observed in the C model by chelating intracellular Ca(2+), whereas the M model was unaffected. Experimental rupturing of the perforated-patch membrane to allow rapid equilibration of the cytosol with 10 mM BAPTA pipette solution, however, failed to decrease the rate of spontaneous action potential within ～30 s, whereas contraction ceased within ～3 s. The spontaneous rhythm also remained intact within a few minutes when SR Ca(2+) dynamics were acutely disrupted using high doses of SR blockers. These experimental results suggested that rapid disruption of normal Ca(2+) dynamics would not markedly affect spontaneous activity. Experimental prolongation of the action potentials, as well as slowing of the Ca(2+)-mediated inactivation of the L-type Ca(2+) currents induced by BAPTA, were well explained by assuming Ca(2+) chelation, even in the proximity of the channel pore in addition to the bulk cytosol in the M model. Taken together, the experimental and model findings strongly suggest that the C mechanism explicitly described by the C model can hardly be applied to guinea pig sinoatrial node cells. The possible involvement of L-type Ca(2+) current rundown induced secondarily through inhibition of Ca(2+)/calmodulin kinase II and/or Ca(2+)-stimulated adenylyl cyclase was discussed as underlying the disruption of spontaneous activity after prolonged intracellular Ca(2+) concentration reduction for >5 min.