Effects of Hypoxia on the Catecholamine Release, Ca2+ Uptake, and Cytosolic Free Ca2+ Concentration in Cultured Bovine Adrenal Chromaffin Cells

The purpose of the present study is to clarify the effects of hypoxia on catecholamine release and its mechanism of action. For this purpose, using cultured bovine adrenal chromaffin cells, we examined the effects of hypoxia on high (55 mM) K(+)-induced increases in catecholamine release, in cytosolic free Ca2+ concentration ([Ca2+]i), and in 45Ca2+ uptake. Experiments were carried out in media preequilibrated with a gas mixture of either 21% O2/79% N2 (control) or 100% N2 (hypoxia). High K(+)-induced catecholamine release was inhibited by hypoxia to approximately 40% of the control value, but on reoxygenation the release returned to control levels. Hypoxia had little effect on ATP concentrations in the cells. In the hypoxic medium, [Ca2+]i (measured using fura-2) gradually increased and reached a plateau of approximately 1.0 microM at 30 min, whereas the level was constant in the control medium (approximately 200 nM). High K(+)-induced increases in [Ca2+]i were inhibited by hypoxia to approximately 30% of the control value. In the cells permeabilized by digitonin, catecholamine release induced by Ca2+ was unaffected by hypoxia. Hypoxia had little effect on basal 45Ca2+ uptake into the cells, but high K(+)-induced 45Ca2+ uptake was inhibited by hypoxia. These results suggest that hypoxia inhibits high K(+)-induced catecholamine release and that this inhibition is mainly the result of the inhibition of high K(+)-induced increases in [Ca2+]i subsequent to the inhibition of Ca2+ influx through voltage-dependent Ca2+ channels.     

Dermaphis coccidiformis sp. nov. (Hemiptera), an aphid species with asymmetrically sclerotized apterae and “winter alates”

Dermaphis coccidiformis sp. nov. (Hormaphidinae: Nipponaphidini) is described from Japan. Apterous adults of the species were found between winter buds (or between a winter bud and a leaf petiole) of the evergreen oaks Quercus glauca, Q. myrsinifolia and Q. salicina. Their morphology is peculiar in that their tergites are heavily sclerotized only in the part that seems to have been exposed to sunlight. The new species is also peculiar in that nymphs to be alates (sexuparae) were found on the upper surfaces of leaves of the host oak only during winter, from December to March or early April, before the bud break of the oak. Our molecular phylogenetic analysis indicated that the new species is closely related to Dermaphis spp., therefore it was placed in the genus. The analysis incidentally indicated that “Dinipponaphis” autumna, a monoecious species forming galls on Distylium racemosum, was included in the clade of the genus Dermaphis, and therefore it was transferred to this genus.     

Identification of a major glucose transporter in Flavobacterium johnsoniae: Inhibition of F. johnsoniae colony spreading by glucose uptake

Many members of the phylum Bacteroidetes, such as Flavobacterium johnsoniae, can glide over a solid surface: an ability called gliding motility. It can be usually observed on agar plates as thin, flat, spreading colonies with irregular, feathery edges; this phenomenon is called colony spreading. Colony spreading of F. johnsoniae on 1.5% agar plates containing poor nutrients is dose‐dependently inhibited by addition of D‐glucose, as previously reported. Accordingly, here, we created mutants (by transposon mutagenesis) that partially suppressed glucose‐mediated inhibition of colony spreading. Among the isolates, we found that one had a transposon insertion in Fjoh_4565, tentatively named mfsA, which encodes a major facilitator superfamily (MFS) transporter previously shown to be required for growth on glucose, N‐acetyl‐glucosamine, and chitin. We constructed an mfsA deletion mutant and found that the mutant showed no glucose‐mediated acceleration of growth or glucose uptake. The mfsA gene complemented the phenotype of a glucose‐negative Escherichia coli. These results suggest that the mfsA gene encodes the sole MFS transporter of glucose in F. johnsoniae and that glucose uptake is partially required for the glucose‐mediated inhibition of F. johnsoniae colony spreading.

     

Physical interactions among flavonoid enzymes in snapdragon and torenia reveal the diversity in the flavonoid metabolon organization of different plant species

Flavonoid metabolons (weakly‐bound multi‐enzyme complexes of flavonoid enzymes) are believed to occur in diverse plant species. However, how flavonoid enzymes are organized to form a metabolon is unknown for most plant species. We analyzed the physical interaction partnerships of the flavonoid enzymes from two lamiales plants (snapdragon and torenia) that produce flavones and anthocyanins. In snapdragon, protein–protein interaction assays using yeast and plant systems revealed the following binary interactions: flavone synthase II (FNSII)/chalcone synthase (CHS); FNSII/chalcone isomerase (CHI); FNSII/dihydroflavonol 4‐reductase (DFR); CHS/CHI; CHI/DFR; and flavonoid 3′‐hydroxylase/CHI. These results along with the subcellular localizations and membrane associations of snapdragon flavonoid enzymes suggested that FNSII serves as a component of the flavonoid metabolon tethered to the endoplasmic reticulum (ER). The observed interaction partnerships and temporal gene expression patterns of flavonoid enzymes in red snapdragon petal cells suggested the flower stage‐dependent formation of the flavonoid metabolon, which accounted for the sequential flavone and anthocyanin accumulation patterns therein. We also identified interactions between FNSII and other flavonoid enzymes in torenia, in which the co‐suppression of FNSII expression was previously reported to diminish petal anthocyanin contents. The observed physical interactions among flavonoid enzymes of these plant species provided further evidence supporting the long‐suspected organization of flavonoid metabolons as enzyme complexes tethered to the ER via cytochrome P450, and illustrated how flavonoid metabolons mediate flower coloration. Moreover, the observed interaction partnerships were distinct from those previously identified in other plant species (Arabidopsis thaliana and soybean), suggesting that the organization of flavonoid metabolons may differ among plant species.     

Distribution of enteric neural peptide YY in the dog gastrointestinal tract

Various regions of the dog gastrointestinal tract were investigated for the distribution of peptide YY (PYY) neurons using immunocytochemistry and radioimmunoassay. PYY neurons that encircled non-PYY-immunoreactive neurons were mainly observed in the myenteric plexus from the stomach to the colon. There was more PYY-like immunoreactivity in the muscle layer of the stomach and ileum than in the other intestines. The results of high performance liquid chromatography revealed that neural PYY-immunoreactive substance is identical to authentic PYY. PYY was not localized in the cholinergic neurons. These results indicate that PYY, as a neuropeptide, is involved in the regulation of gastrointestinal function.     

Lp3/Hapln3, a novel link protein that co-localizes with versican and is coordinately up-regulated by platelet-derived growth factor in arterial smooth muscle cells.

Link proteins (LPs) belong to the link-module superfamily, which can stabilize and enhance the binding of lecticans to hyaluronan. We report here the identification and characterization of a novel rat link protein gene (Lp3/Hapln3). The deduced protein sequence shares the typical modular elements of link proteins and has an estimated mass of 39 kDa. Examination of the rat genomic DNA sequence revealed that Lp3/Hapln3 and aggrecan genes were paired on chromosome 1q31. Another LP gene and the lectican gene were also paired at a different locus, as they are in the human and mouse genomes. Immunohistochemical analysis showed the prominent expression of Lp3/Hapln3 in the smooth muscle tissues of the vascular wall and gastrointestinal tract. Further comparative studies revealed that Lp3/Hapln3 was well co-localized with versican around the smooth muscle cells of blood vessels but not around endothelial cells. In vitro experiments using primary cultured rat arterial smooth muscle cells (ASMCs) demonstrated the coordinated up-regulation of Lp3/Hapln3 and versican by platelet-derived growth factor (PDGF). These data were supported by in vivo studies of a mechanical vascular injury model in mice. Altogether, our results suggest that Lp3/Hapln3 is involved, together with versican and hyaluronan, in the formation of the pericellular matrix of vascular smooth muscle cells.     

Connexin 43 Is Necessary for Salivary Gland Branching Morphogenesis and FGF10-induced ERK1/2 Phosphorylation

Cell-cell interaction via the gap junction regulates cell growth and differentiation, leading to formation of organs of appropriate size and quality. To determine the role of connexin43 in salivary gland development, we analyzed its expression in developing submandibular glands (SMGs). Connexin43 (Cx43) was found to be expressed in salivary gland epithelium. In ex vivo organ cultures of SMGs, addition of the gap junctional inhibitors 18α-glycyrrhetinic acid (18α-GA) and oleamide inhibited SMG branching morphogenesis, suggesting that gap junctional communication contributes to salivary gland development. In Cx43^−/− salivary glands, submandibular and sublingual gland size was reduced as compared with those from heterozygotes. The expression of Pdgfa, Pdgfb, Fgf7, and Fgf10, which induced branching of SMGs in Cx43^−/− samples, were not changed as compared with those from heterozygotes. Furthermore, the blocking peptide for the hemichannel and gap junction channel showed inhibition of terminal bud branching. FGF10 induced branching morphogenesis, while it did not rescue the Cx43^−/− phenotype, thus Cx43 may regulate FGF10 signaling during salivary gland development. FGF10 is expressed in salivary gland mesenchyme and regulates epithelial proliferation, and was shown to induce ERK1/2 phosphorylation in salivary epithelial cells, while ERK1/2 phosphorylation in HSY cells was dramatically inhibited by 18α-GA, a Cx43 peptide or siRNA. On the other hand, PDGF-AA and PDGF-BB separately induced ERK1/2 phosphorylation in primary cultured salivary mesenchymal cells regardless of the presence of 18α-GA. Together, our results suggest that Cx43 regulates FGF10-induced ERK1/2 phosphorylation in salivary epithelium but not in mesenchyme during the process of SMG branching morphogenesis.     

Purification and properties of Cellvibrio gilvus cellobiose phosphorylase

The cellobiose phosphorylase (EC 2.4.1.20) of Cellvibrio gilvus, which is an endocellular enzyme, has been purified 196-fold with a recovery of 11% and a specific activity of 27.4 mumol of glucose 1-phosphate formed/min per mg of protein. The purification procedure includes fractionation with protamine sulphate, and hydroxyapatite and DEAE-Sephadex A-50 chromatography. The enzyme appears homogeneous on polyacrylamide-gel electrophoresis, and a molecular weight of 280 000 was determined by molecular-sieve chromatography. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed a single band and mol.wt. 72 000, indicating that cellobiose phosphorylase consists of four subunits. The enzyme had a specificity for cellobiose, requiring Pi and Mg2+ for phosphorylation, but not for cellodextrin, gentibiose, laminaribiose, lactose, maltose, kojibiose and sucrose. The enzyme showed low thermostability, an optimum pH of 7.6 and a high stability in the presence of 2-mercaptoethanol or dithiothreitol. The Km values for cellobiose and Pi were 1.25 mM and 0.77 mM respectively. Nojirimycin acted as a powerful pure competitive inhibitor (with respect to cellobiose) of the enzyme (Ki = 45 microM). Addition of thiol-blocking agents to the enzyme caused 56% inhibition at 500 microM-N-ethylmaleimide and 100% at 20 microM-p-chloromercuribenzoate.     

Quantification of two variant strains contained in freeze-dried Japanese BCG vaccine preparation by real-time PCR.

Japanese bacillus Calmette-Guerin (BCG) vaccine preparation contains two types of variant strains, Type I, which has a 22-base-pair deletion in the RD16 region, and Type II, which has an identical sequence to those of other BCG strains. In this study, we established a method to quantify the percentage of variant strain Type II contained in freeze-dried BCG product with real-time PCR. With this method we examined the master seed lot Tokyo 172, two secondary seed lots, Tokyo 172-1 and Tokyo 172-2, which were produced from Tokyo 172, and four commercial lots produced form Tokyo 172-1. Tokyo 172, Tokyo 172-1, and Tokyo 172-2 contained 55.1%, 19.5%, and 3.6% of Type II variant strain, respectively. Commercial lots contained 1.5%, 4.5%, 7.4%, and 4.3% of Type II variant strain, respectively. These results indicated that the two variant strains contained in the master seed lot continued to coexist in subsequently produced lots with a decrease in population of variant strain Type II. This method would be useful for quality control of commercial Japanese BCG vaccine preparations.