Quantitative trait locus mapping reveals conservation of major and minor loci for powdery mildew resistance in four sources of resistance in mungbean [Vigna radiata (L.) Wilczek]

Powdery mildew (PM) is a common and serious disease of mungbean [Vigna radiata (L.) Wilczek]. A few quantitative trait loci (QTL) for PM resistance in mungbean have been reported. The objective of this study was to locate QTL for PM resistance in two resistant accessions V4718 and RUM5. Simple sequence repeat markers were analyzed in an F₂ population from a cross between Kamphaeng Saen 1 (KPS1; susceptible to PM) and V4718 (resistant to PM), and in F₂ and BC₁F₁ populations from a cross between Chai Nat 60 (CN60; susceptible to PM) and RUM5 (resistant to PM). Progenies of 134 F_2:3 and F_2:4 lines derived from KPS1 × V4718, and 190 F_2:3 and 74 BC₁F_1:2 lines derived from CN60 × RUM5 and CN60 × (CN60 × RUM5), respectively, were evaluated for response to PM under field conditions. Multiple interval mapping identified a major QTL on linkage group (LG) 9 and two minor QTL on LG4 for the resistance in V4718, and detected two major QTL on LG6 and LG9 and one minor QTL on LG4 for the resistance in RUM5. Comparative linkage analysis of the QTL for PM resistance in this study and in previous reports suggests that the resistance QTL on LG9 in V4718, RUM5, ATF3640 and VC6468-11-1A are the same locus or linked. One QTL on LG4 is the same in three sources (V4718, RUM5 and VC1210A). Another QTL on LG6 is the same in two sources (RUM5 and VC6468-11-1A). In addition, one QTL in V4718 on LG4 appears to be a new resistance locus. These different resistance loci will be useful for breeding durably PM-resistant mungbean cultivars.     

Synthesis and biological evaluation of pyrrolidine derivatives as novel and potent sodium channel blockers for the treatment of ischemic stroke

A novel series of pyrrolidine derivatives as Na⁺ channel blockers was synthesized and evaluated for their inhibitory effects on neuronal Na⁺ channels. Structure–activity relationship (SAR) studies of a pyrrolidine analogue 2 led to the discovery of 5e as a potent Na⁺ channel blocker with a low inhibitory action against human ether-a-go-go-related gene (hERG) channels. Compound 5e showed remarkably neuroprotective activity in a rat transient middle cerebral artery occlusion (MCAO) model, suggesting that 5e would act as a neuroprotectant for ischemic stroke.     

Dock8 interacts with Nck1 in mediating Schwann cell precursor migration

During embryonic development of the peripheral nervous system (PNS), Schwann cell precursors migrate along neuronal axons to their final destinations, where they will myelinate the axons after birth. While the intercellular signals controlling Schwann cell precursor migration are well studied, the intracellular signals controlling Schwann cell precursor migration remain elusive. Here, using a rat primary cell culture system, we show that Dock8, an atypical Dock180-related guanine-nucleotide exchange factor (GEF) for small GTPases of the Rho family, specifically interacts with Nck1, an adaptor protein composed only of Src homology (SH) domains, to promote Schwann cell precursor migration induced by platelet-derived growth factor (PDGF). Knockdown of Dock8 or Nck1 with its respective siRNA markedly decreases PDGF-induced cell migration, as well as Rho GTPase activation, in precursors. Dock8, through its unique N-terminal proline-rich motif, interacts with the SH3 domain of Nck1, but not with other adaptor proteins composed only of SH domains, e.g. Grb2 and CrkII, and not with the adaptor protein Elmo1. Reintroduction of the proline-rich motif mutant of Dock8 in Dock8 siRNA-transfected Schwann cell precursors fails to restore their migratory abilities, whereas that of wild-type Dock8 does restore these abilities. These results suggest that Nck1 interaction with Dock8 mediates PDGF-induced Schwann cell precursor migration, demonstrating not only that Nck1 and Dock8 are previously unanticipated intracellular signaling molecules involved in the regulation of Schwann cell precursor migration but also that Dock8 is among the genetically-conservative common interaction subset of Dock family proteins consisting only of SH domain adaptor proteins.     

Genetic analyses of Per.C6 cell clones producing a therapeutic monoclonal antibody regarding productivity and long-term stability

Genetic characterization of protein-producing clones represents additional value to cell line development. In the present study, ten Per.C6 clones producing a Rebmab100 monoclonal antibody were selected using two cloning methods: six clones originated from limiting dilution cloning and four by the automated colony picker ClonePix FL. A stability program was performed for 50 generations, including 4 batches distributed along the timeframe to determine specific productivity (Qp) maintenance. Four stable clones (two from limiting dilution and two from ClonePix FL) were further evaluated. The relative mRNA expression levels of both heavy chain (HC) and light chain (LC) genes were verified at generations 0, 30–35, and 50–55 of the stability program. At generations 0 and 30–35, LC gene expression level was higher than HC gene, whereas at generation 50–55, the opposite prevailed. A high correlation was observed between Qp and HC or LC mRNA expression level for all clones at each generation analyzed along the continuous culture. The mRNA stability study was performed at steady-state culture. The LC gene displayed a higher half-life and lower decay constant than HC gene, accounting for the higher observed expression level of LC mRNA in comparison to HC mRNA. Clone R6 was highlighted due its high Qp, mRNA expression levels, and mRNA stability. Besides the benefits of applying genetic characterization for the selection of stable and high-producing clones, the present study shows for the first time the correlation between Qp and HC or LC expression levels and also mRNA stability in clones derived from human cell line Per.C6(®).     

Preparation of reminiscent aroma mixture of Japanese soy sauce

To prepare an aroma mixture of Japanese soy sauce by fewest components, the aroma concentrate of good sensory attributes was prepared by polyethylene membrane extraction, which could extract only the volatiles with diethyl ether. GC-MS-Olfactometry was done with the aroma concentrate, and 28 odor-active compounds were detected. Application of aroma extract dilution analysis to the separated fraction revealed high flavor dilution factors with respect to acetic acid, 4-hydroxy-2(or5)-ethyl-5(or2)-methyl-3(2H)-furanone (HEMF), 3-methyl-1-butanol (isoamyl alcohol), and 3-(methylsulfanyl)propanal (methional). A model aroma mixture containing above four odorants showed a good similarity with the aroma of the soy sauce itself. Consequently, the reminiscent aroma mixture of soy sauce was prepared in water. The ratio of acetic acid, HEMF, isoamyl alcohol, and methional was 2500:300:100:1.     

Phylogeography and population genetics of pine butterflies: Sky islands increase genetic divergence

The sky islands of southeastern Arizona (AZ) mark a major transition zone between tropical and temperate biota and are considered a neglected biodiversity hotspot. Dispersal ability and host plant specificity are thought to impact the history and diversity of insect populations across the sky islands. We aimed to investigate the population structure and phylogeography of two pine‐feeding pierid butterflies, the pine white (Neophasia menapia) and the Mexican pine white (Neophasia terlooii), restricted to these “islands” at this transition zone. Given their dependence on pines as the larval hosts, we hypothesized that habitat connectivity affects population structure and is at least in part responsible for their allopatry. We sampled DNA from freshly collected butterflies from 17 sites in the sky islands and adjacent high‐elevation habitats and sequenced these samples using ddRADSeq. Up to 15,399 SNPs were discovered and analyzed in population genetic and phylogenetic contexts with Stacks and pyRAD pipelines. Low genetic differentiation in N. menapia suggests that it is panmictic. Conversely, there is strong evidence for population structure within N. terlooii. Each sky island likely contains a population of N. terlooii, and clustering is hierarchical, with populations on proximal mountains being more related to each other. The N. menapia habitat, which is largely contiguous, facilitates panmixia, while the N. terlooii habitat, restricted to the higher elevations on each sky island, creates distinct population structure. Phylogenetic results corroborate those from population genetic analyses. The historical climate‐driven fluxes in forest habitat connectivity have implications for understanding the biodiversity of fragmented habitats.     

Gadd45a, the gene induced by the mood stabilizer valproic acid, regulates neurite outgrowth through JNK and the substrate paxillin in N1E-115 neuroblastoma cells

Valproic acid (VPA), a mood stabilizer and anticonvulsant, has a variety of neurotrophic functions; however, less is known about how VPA regulates neurite outgrowth. Here, using N1E-115 neuroblastoma cells as the model, we show that VPA upregulates Gadd45a to trigger activation of the downstream JNK cascade controlling neurite outgrowth. VPA induces the phosphorylation of c-Jun N-terminal kinase (JNK) and the substrate paxillin, while VPA induction of neurite outgrowth is inhibited by JNK inhibitors (SP600125 and the small JNK-binding peptide) or a paxillin construct harboring a Ser 178-to-Ala mutation at the JNK phosphorylation. Transfection of Gadd45a, acting through the effector MEKK4, leads to the phosphorylation of the JNK cascade. Conversely, knockdown of Gadd45a with siRNA reduces the effect of VPA. Taken together, these results suggest that upregulation of Gadd45a explains one of the mechanisms whereby VPA induces the neurotrophic effect, providing a new role of Gadd45a in neurite outgrowth.     

Characterization of subunit structural changes accompanying assembly of the bacteriophage P22 procapsid

P22 serves as a model for the assembly and maturation of icosahedral double-stranded DNA viruses. The viral capsid precursor, or procapsid, is assembled from 420 copies of a 47 kDa coat protein subunit (gp5) that is rich in beta-strand secondary structure. Maturation to the capsid, which occurs in vivo upon DNA packaging, is accompanied by shell expansion and a large increase in the level of protection against deuterium exchange of amide NH groups. Accordingly, shell maturation resembles the final step in protein folding, wherein domain packing and an exchange-protected core become more fully developed [Tuma, R., Prevelige, P. E., Jr., and Thomas, G. J., Jr. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9885-9890]. Here, we exploit recent advances in Raman spectroscopy to investigate the P22 coat protein subunit under conditions which stabilize the monomeric state, viz., in solution at very low concentrations. Under these conditions, the monomer exhibits an elongated shape, as demonstrated by small-angle X-ray scattering. Raman spectra allow the identification of conformation-sensitive marker bands of the monomer, as well as the characterization of NH exchange dynamics for comparison with procapsid and capsid shell assemblies. We show that procapsid assembly involves significant ordering of the predominantly beta-strand backbone. We propose that such ordering may mediate formation of the distinct subunit conformations required for assembly of a T = 7 icosahedral lattice. However, the monomer, like the subunit within the procapsid lattice, exhibits a moderate level of protection against low-temperature NH exchange, indicative of a nascent folding core. The environments and exchange characteristics of key side chains are also similar for the monomeric and procapsid subunits, and distinct from corresponding characteristics of the capsid subunit. The monomer thus represents a compact but metastable folding intermediate along the pathway to assembly of the procapsid and capsid.