The presence of -mannosidic linkage in acidic glycopeptide from porcine thyroglobulin

The mannose residue in (Man)₁ (GlcNAc)₂-Asn obtained by a Smith degradation of the acidic glycopeptide from porcine thyroglobulin was found to be insusceptible to α-mannosidase. This residue was hydrolyzed, however, by purified β-mannosidase. After β-mannosidase treatment, the resulting (GlcNAc)₂-Asn was compared with synthetic glycosyl-asparagine derivatives. From these experiments, the core structure of the acidic glycopeptide was proposed to be β-Man-(1 → 3 or 4)-β-GlcNAc-(1 → 4)-GlcNAc-Asn.     

Identification of potential cell wall component that allows Taka-amylase A adsorption in submerged cultures of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

We observed that α-amylase (Taka-amylase A; TAA) activity in the culture broth disappeared in the later stage of submerged cultivation of Aspergillus oryzae. This disappearance was caused by adsorption of TAA onto the cell wall of A. oryzae and not due to protein degradation by extracellular proteolytic enzymes. To determine the cell wall component(s) that allows TAA adsorption efficiently, the cell wall was fractionated by stepwise alkali treatment and enzymatic digestion. Consequently, alkali-insoluble cell wall fractions exhibited high levels of TAA adsorption. In addition, this adsorption capacity was significantly enhanced by treatment of the alkali-insoluble fraction with β-glucanase, which resulted in the concomitant increase in the amount of chitin in the resulting fraction. In contrast, the adsorption capacity was diminished by treating the cell wall fraction with chitinase. These results suggest that the major component that allows TAA adsorption is chitin. However, both the mycelium and the cell wall demonstrated the inability to allow TAA adsorption in the early stage of cultivation, despite chitin content in the cell wall being identical in both early and late stages of cultivation. These results suggest the existence of unidentified factor(s) that could prevent the adsorption of TAA onto the cell wall. Such factor(s) is most likely removed or diminished from the cell wall following longer cultivation periods.     

RsgA releases RbfA from 30S ribosome during a late stage of ribosome biosynthesis

RsgA is a 30S ribosomal subunit-binding GTPase with an unknown function, shortage of which impairs maturation of the 30S subunit. We identified multiple gain-of-function mutants of Escherichia coli rbfA, the gene for a ribosome-binding factor, that suppress defects in growth and maturation of the 30S subunit of an rsgA-null strain. These mutations promote spontaneous release of RbfA from the 30S subunit, indicating that cellular disorders upon depletion of RsgA are due to prolonged retention of RbfA on the 30S subunit. We also found that RsgA enhances release of RbfA from the mature 30S subunit in a GTP-dependent manner but not from a precursor form of the 30S subunit. These findings indicate that the function of RsgA is to release RbfA from the 30S subunit during a late stage of ribosome biosynthesis. This is the first example of the action of a GTPase on the bacterial ribosome assembly described at the molecular level.     

Actin-dependent chloroplast anchoring is regulated by Ca(2+)-calmodulin in spinach mesophyll cells

Chloroplasts are actively anchored at the appropriate intracellular regions to maintain advantageous distribution patterns under specific environmental conditions. Redistribution of chloroplasts is accompanied by their de-anchoring and re-anchoring, respectively, from and to the cortical cytoplasm. In spinach mesophyll cells, high-intensity blue light and Ca(2+) treatment induced the disappearance of the meshwork-like array of actin filaments surrounding chloroplasts, which was suppressed by a calmodulin antagonist. Regulatory mechanisms of chloroplast anchoring were investigated using plasma membrane (PM) ghosts, on which the cortical cytoplasm underlying the PM was exposed. Addition of an actin-depolymerizing reagent or > 1 μM Ca(2+) induced detachment of a substantial number of chloroplasts from the PM ghosts concomitant with disordered actin organization. Calmodulin antagonists and anti-calmodulin antibodies negated the effects of Ca(2+). In addition, Ca(2+)-induced detachment of chloroplasts was no longer evident on the calmodulin-depleted PM ghosts. We propose that chloroplasts are anchored onto the cortical cytoplasm through interaction with the actin cytoskeleton, and that Ca(2+)-calmodulin-sensitized de-anchoring of chloroplasts is a critical early step in chloroplast redistribution induced by environmental stimuli.     

A simple and rapid GC/MS method for the simultaneous determination of gaseous metabolites

We modified and tuned a commercial model of a gas chromatography/mass spectrometry (GC/MS) instrument to develop a simple and rapid method for the simultaneous quantification of a variety of gas species. Using the developed method with the newly modified instrument, gas species such as H₂, N₂, O₂, CO, NO, CH₄, CO₂, and N₂O, which are common components of microbial metabolism, were accurately identified based on their retention times and/or mass-to-charge ratios (m/z) in less than 2.5 min. By examining the sensitivities and dynamic ranges for the detection of H₂, N₂, O₂, CH₄, CO₂, and N₂O, it was demonstrated that the method developed in this study was sufficient for accurately monitoring the production and the consumption of these gaseous species during microbial metabolism. The utility of the new method was demonstrated by a denitrification study with Pseudomonas aureofaciens ATCC 13985^T. This method will be suitable for a variety of applications requiring the identification of gaseous metabolites in microorganisms, microbial communities, and natural ecosystems.     

Expression of a mammalian aquaporin 3 homolog in the anterior pituitary gonadotrophs of the tree frog, <Emphasis Type="Italic">Hyla japonica</Emphasis>

Aquaporins (AQPs) are a family of water channel proteins that play a major role in maintaining water homeostasis in various organisms. Several AQPs have been identified in the tree frog, Hyla japonica. Of these, AQP-h3BL, which is expressed in the basolateral membrane of the epithelial cells, is a homolog of mammalian AQP3. Using immunohistochemistry and in situ RT-PCR, we have demonstrated that AQP-h3BL is expressed in the anterior pituitary gonadotrophs of the tree frog but not in the other hormone-producing cells of the anterior pituitary. In gonadotrophs labeled for luteinizing hormone subunit-β (LHβ), AQP-h3BL protein was found to reside in the plasma membrane, the nuclear membrane and the cytoplasm. Double-labeling of AQP-h3BL mRNA and LHβ protein revealed that AQP-h3BL mRNA is expressed in the gonadotrophs. Following stimulation by gonadotropin-releasing hormone (GnRH), the label for AQP-h3BL localized in the plasma membrane became more intense, concomitant with the transport of LHβ-positive materials to the plasma membrane. These developments coincided with a decrease in the labeling density in the cytoplasm and near the nuclear membrane, suggesting that the latter localizations may function as “storage area“ for AQP-h3BL. Immunoelectron microscopy also confirmed these localizations of AQP-h3BL protein. Based on these results, we suggest that AQP-h3BL protein in the frog gonadotrophs is involved in the formation of secretory granules, the swelling and increase in the volume of the granules and exocytosis.     

Effect of prior vaccination with a seasonal trivalent influenza vaccine on the antibody response to the influenza pandemic H1N1 2009 vaccine: a randomized controlled trial

Vaccination with the non-adjuvanted split-virion A/California/7/2009 influenza vaccine (pandemic H1N1 2009 vaccine) began in October 2009 in Japan. The present study was designed to assess the effect of prior vaccination with a seasonal trivalent influenza vaccine on the antibody response to the pandemic H1N1 2009 vaccine in healthy adult volunteers. One hundred and seventeen participants aged 22 to 62 were randomly assigned to two study groups. In Group 1 (the priming group), participants were first vaccinated with the seasonal trivalent influenza vaccine followed by two separate one-dose vaccinations of the pandemic H1N1 2009 vaccine, whereas in Group 2 (the non-priming group), the participants were first vaccinated with one dose of the pandemic H1N1 2009 vaccine, followed by simultaneous vaccination of the seasonal trivalent vaccine and the second dose of the pandemic H1N1 2009 vaccine. The participants in Group 2 had a seroprotection rate (SPR) of 79.7% and a seroconversion rate (SCR) of 79.7% in the hemagglutination-inhibition test after the first dose of the pandemic H1N1 2009 vaccine, indicating that the pandemic H1N1 2009 vaccine is sufficiently immunogenic. On the other hand, the participants of Group 1 had a significantly weaker antibody response, with a SPR of 60.8% and a SCR of 58.5%. These results indicate that prior vaccination with the seasonal trivalent influenza vaccine inhibits the antibody response to the pandemic H1N1 2009 vaccine. Therefore, the pandemic H1N1 2009 vaccine should be administered prior to vaccination with the seasonal trivalent influenza vaccine.     

Losartan improves aortic endothelium-dependent relaxation via proline-rich tyrosine kinase 2/Src/Akt pathway in type 2 diabetic Goto-Kakizaki rats

In diabetic states, endothelial dysfunction is related to vascular complications. We hypothesized that insulin-induced relaxation and the associated proline-rich tyrosine kinase 2 (Pyk2)/Src/Akt pathway would be abnormal in aortas from the Goto-Kakizaki (GK) type 2 diabetic rat, which exhibits hyperglycemia/insulin resistance, and that losartan treatment of such rats (25 mg·kg(-1)·day(-1) for 2 wk) would correct these abnormalities. Endothelium-dependent relaxation was by measuring isometric force in helical strips of aortas from four groups, each of 30 rats: normal Wistar (control), GK (diabetic), losartan-treated normal, and losartan-treated GK. Pyk2, Src, and Akt/endothelial nitric oxide synthase (eNOS) signaling-pathway protein levels and activities were assayed mainly by Western blotting and partly by immunohistochemistry. In GK (vs. age-matched control) aortas, various insulin-stimulated levels [nitric oxide production and the phosphorylations of eNOS at Ser(1177), of Akt at Thr(308), of phosphoinositide-dependent kinase-1 (PDK1) at Ser(241), of Src at Tyr(416), and of Pyk2 at Tyr(579)] were all significantly decreased and unaffected by either Src inhibitor (PP2) or Pyk2 inhibitor (AG17), while the insulin-stimulated levels of insulin receptor substrate (IRS)-1 phosphorylation at Ser(307), total-eNOS, and total-Akt were significantly increased. Losartan treatment normalized these altered levels. The insulin-stimulated phosphorylation levels of Src/PDK1/Akt/eNOS, but not of Pyk2, were decreased by PP2 in control and losartan-treated GK, but not in GK, aortas. These results suggest that in the GK diabetic aorta increased phospho-IRS-1 (at Ser(307)) and decreased Pyk2/Src activity inhibit insulin-induced stimulation of the PDK/Akt/eNOS pathway. The observed increase in phospho-IRS-1 (at Ser(307)) may result from increased angiotensin II activity.     

Single-embryo metabolomics and systematic prediction of developmental stage in zebrafish

Metabolites, the end products of gene expression in living organisms, are tightly correlated with an organism's development and growth. Thus, metabolic profiling is a potentially important tool for understanding the events that have occurred in cells, tissues, and individual organisms. Here, we present a method for predicting the developmental stage of zebrafish embryos using novel metabolomic non-target fingerprints of "single-embryos". With this method, we observed the rate of development at different temperatures. Our results suggest that this method allows us to analyse the condition, or distinguish the genotype, of single-embryos before expression of their ultimate phenotype.