Dual recognition of S 1 and S 4 pistils by S 4 sm pollen in self-incompatibility of Japanese pear (Pyrus pyrifolia Nakai)

Most cultivars of Japanese pear (Pyrus pyrifolia Nakai) exhibit gametophytic self-incompatibility controlled by a single S-locus with multiple S-haplotypes. A self-compatible (SC) cultivar, ??Osanijisseiki?? (S ₂ S ₄ ^sm ), arising by a bud mutation of ??Nijisseiki?? (S ₂ S ₄ ), has a stylar-part mutant S ₄ ^sm -haplotype, which lacks the pistil S ₄ gene, which is the S ₄ -RNase gene. To efficiently breed SC cultivars, we selected ??Nashi Chuukanbohon Nou 1 Gou?? (??NCN1??) harboring homozygous S ₄ ^sm from a self-progeny of Osanijisseiki and crossed it with ??Okusankichi?? (S ₅ S ₇ ), ??Hakkou?? (S ₄ S ₅ ), or ??Ri-14?? (S ₁ S ₂ ). Fruit set (%) was compared after self-pollination of the trees in the three progenies. All trees derived from the three progenies were predicted to be SC, except for the S ₄ S ₄ ^sm trees in the progeny of NCN1 × Hakkou. However, S ₁ S ₄ ^sm trees in the progeny of NCN1 × Ri-14 proved to be self-incompatible (SI). The pollen from Osanijisseiki was incompatible with ??Doitsu?? (S ₁ S ₂ ), but that from Nijisseiki was compatible, suggesting a possibility that the S ₄ ^sm pollen was rejected by S ₁ -harboring pistils. This possibility was clarified by crossing the pollen from NCN1 (S ₄ ^sm S ₄ ^sm ) to Doitsu, ??Imamuraaki?? (S ₁ S ₆ ), or ??Hougetsu?? (S ₁ S ₇ ), all of which proved incompatible. On the other hand, S ₄ ^sm pollen was accepted by pistils harboring the S ₂ , S ₃ , S ₅ , S ₆ , S ₇ , S ₉ , and S _k haplotypes. The dual recognition of S ₁ and S ₄ pistils by S ₄ ^sm pollen can be attributed to a mutation of the pollen S ₄ gene(s).     

Tubular Structure Induced by a Plant Virus Facilitates Viral Spread in Its Vector Insect

Rice dwarf virus (RDV) replicates in and is transmitted by a leafhopper vector in a persistent-propagative manner. Previous cytopathologic and genetic data revealed that tubular structures, constructed by the nonstructural viral protein Pns10, contain viral particles and are directly involved in the intercellular spread of RDV among cultured leafhopper cells. Here, we demonstrated that RDV exploited these virus-containing tubules to move along actin-based microvilli of the epithelial cells and muscle fibers of visceral muscle tissues in the alimentary canal, facilitating the spread of virus in the body of its insect vector leafhoppers. In cultured leafhopper cells, the knockdown of Pns10 expression due to RNA interference (RNAi) induced by synthesized dsRNA from Pns10 gene strongly inhibited tubule formation and prevented the spread of virus among insect vector cells. RNAi induced after ingestion of dsRNA from Pns10 gene strongly inhibited formation of tubules, preventing intercellular spread and transmission of the virus by the leafhopper. All these results, for the first time, show that a persistent-propagative virus exploits virus-containing tubules composed of a nonstructural viral protein to traffic along actin-based cellular protrusions, facilitating the intercellular spread of the virus in the vector insect. The RNAi strategy and the insect vector cell culture provide useful tools to investigate the molecular mechanisms enabling efficient transmission of persistent-propagative plant viruses by vector insects.     

Aldehyde dehydrogenase-2 deficiency aggravates cardiac dysfunction elicited by endoplasmic reticulum stress induction

Mitochondrial aldehyde dehydrogenase-2 (ALDH2) has been characterized as an important mediator of endogenous cytoprotection in the heart. This study was designed to examine the role of ALDH2 knockout (KO) in the regulation of cardiac function after endoplasmic reticulum (ER) stress. Wild-type (WT) and ALDH2 KO mice were subjected to a tunicamycin challenge, and the echocardiographic property was examined. Protein levels of six items--78 kDa glucose-regulated protein (GRP78), phosphorylation of eukaryotic initiation factor 2 subunit α (p-eIF2α), CCAAT/enhancer-binding protein homologous protein (CHOP), phosphorylation of Akt, p47(phox) nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and 4-hydroxynonenal--were determined by using Western blot analysis. Cytotoxicity and apoptosis were estimated using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay and caspase-3 activity, respectively. ALDH2 deficiency exacerbated cardiac contractile dysfunction and promoted ER stress after ER stress induction, manifested by the changes of ejection fraction and fractional shortening. In vitro study revealed that tunicamycin significantly upregulated the levels of GRP78, p-eIF2α, CHOP, p47(phox) NADPH oxidase and 4-hydroxynonenal, which was exacerbated by ALDH2 knockdown and abolished by ALDH2 overexpression, respectively. Overexpression of ALDH2 abrogated tunicamycin-induced dephosphorylation Akt. Inhibition of phosphatidylinositol 3-kinase using LY294002 did not affect ALDH2-conferred protection against ER stress, although LY294002 reversed the antiapoptotic action of ALDH2 associated with p47(phox) NADPH oxidase. These results suggest a pivotal role of ALDH2 in the regulation of ER stress and ER stress-induced apoptosis. The protective role of ALDH2 against ER stress-induced cell death was probably mediated by Akt via a p47(phox) NADPH oxidase-dependent manner. These findings indicate the critical role of ALDH2 in the pathogenesis of ER stress in heart disease.     

Efficient uptake of recombinant α-galactosidase A produced with a gene-manipulated yeast by Fabry mice kidneys

To economically produce recombinant human α-galactosidase A (GLA) with a cell culture system that does not require bovine serum, we chose methylotrophic yeast cells with the OCH1 gene, which encodes α-1,6-mannosyltransferase, deleted and over-expressing the Mnn4p (MNN4) gene, which encodes a positive regulator of mannosylphosphate transferase, as a host cell line. The enzyme (yr-hGLA) produced with the gene-manipulated yeast cells has almost the same enzymological parameters as those of the recombinant human GLA produced with cultured human fibroblasts (agalsidase alfa), which is currently used for enzyme replacement therapy for Fabry disease. However, the basic structures of their sugar chains are quite different. yr-hGLA has a high content of phosphorylated N-glycans and is well incorporated into the kidneys, the main target organ in Fabry disease, where it cleaves the accumulated glycosphingolipids. A glycoprotein production system involving this gene-manipulated yeast cell line will be useful for the development of a new enzyme replacement therapy for Fabry disease.     

Fluorescence changes of rhodamine 6G associated with changes in membrane potential in synaptosomes

The intensity of rhodamine 6G fluorescence was found to be a useful scale for measuring the membrane potential in synaptosomes. The fluorescence of rhodamine 6G in synaptosomal suspensions increases with depolarization in the synaptosomes induced by the replacement of cations in the medium or by the addition of agents known to depolarize the membrane potential. Considering the character of the dye, we have derived an equation which gives the relation between the fluorescence intensity of the dye and the membrane potential. The change in membrane potential (diffusion potential) of synaptosomes was calculated using the equation. The calculated membrane potential was proportional to the logarithm of the K⁺ concentration above 20 mM, and the slope of membrane potential against log[K⁺] was about 52 mV per decade of concentration. The permeability ratio ($\text{P}{}_{\mathrm{<mtext>X</mtext>}}\text{P}{}_{\mathrm{<mtext>K</mtext>}}$; the ratio of the permeability constants of a given cation, X⁺, and K⁺) was estimated from the calculated membrane potential.     

Glucose enhances collectrin protein expression in insulin-producing MIN6 β cells

Collectrin is a novel target gene of hepatocyte nuclear factor-1α in pancreatic β-cells and controls insulin exocytosis. Although glucose is known to stimulate the expression of genes of the insulin secretory pathway, there is no information on how glucose regulates collectrin expression. We investigated the effects of glucose on the expression of collectrin in MIN6 β-cell line. Glucose, in a dose-dependent manner, increased collectrin protein levels without changing collectrin mRNA levels and protein stability, indicating that glucose stimulation of collectrin protein expression is primarily mediated at a translational level. Although mannose and pyruvate also increased collectrin protein expression level, neither 2-deoxyglucose, mitochondrial fuels leucine and glutamate, sulphonylurea nor Ca²⁺ channel blockers, mimicked the effects of glucose. These data indicate the involvement of mitochondrial TCA cycle intermediates, distal to pyruvate, in the regulation of collectrin protein expression in β-cells.