Saccharomyces cerevisiae alpha1,6-mannosyltransferase has a catalytic potential to transfer a second mannose molecule

In yeast, the N-linked oligosaccharide modification in the Golgi apparatus is initiated by alpha1,6-mannosyltransferase (encoded by the OCH1 gene) with the addition of mannose to the Man(8)GlcNAc(2) or Man(9)GlcNAc(2) endoplasmic reticulum intermediates. In order to characterize its enzymatic properties, the soluble form of the recombinant Och1p was expressed in the methylotrophic yeast Pichia pastoris as a secreted protein, after truncation of its transmembrane region and fusion with myc and histidine tags at the C-terminus, and purified using a metal chelating column. The enzymatic reaction was performed using various kinds of pyridylaminated (PA) sugar chains as acceptor, and the products were separated by high performance liquid chromatography. The recombinant Och1p efficiently transferred a mannose to Man(8)GlcNAc(2)-PA and Man(9)GlcNAc(2)-PA acceptors, while Man(5)GlcNAc(2)-PA, which completely lacks alpha1,2-linked mannose residues, was not used as an acceptor. At high enzyme concentrations, a novel product was detected by HPLC. Analysis of the product revealed that a second mannose was attached at the 6-O-position of alpha1,3-linked mannose branching from the alpha1,6-linked mannose that is attached to beta1,4-linked mannose of Man(10)GlcNAc(2)-PA produced by the original activity of Och1p. Our results indicate that Och1p has the potential to transfer two mannoses from GDP-mannose, and strictly recognizes the overall structure of high mannose type oligosaccharide.     

Presence of multiple copies of capsulation loci in invasive Haemophilus influenzae type b (Hib) strains in Japan before introduction of the Hib conjugate vaccine

Despite the effectiveness of the Hib vaccine, multiple amplification of the capb locus contributes to vaccine failure. However, there has been no report on the effect of Hib locus amplification in Japan. We examined 24 Hib strains from Japanese children with invasive diseases due to Hib. Although all strains showed the same capb sequence, Southern blot analysis showed that four strains (16.7%) harbored multiple copies (more than two) of the capb locus. Careful analysis of the locus in circulating Hib strains is necessary now that the Hib vaccine has been introduced into Japan.     

Reactive Oxygen Generated by NADPH Oxidase 1 (Nox1) Contributes to Cell Invasion by Regulating Matrix Metalloprotease-9 Production and Cell Migration

A mediating role of the reactive oxygen species-generating enzyme Nox1 has been suggested for Ras oncogene transformation phenotypes including anchorage-independent cell growth, augmented angiogenesis, and tumorigenesis. However, little is known about whether Nox1 signaling regulates cell invasiveness. Here, we report that the cell invasion activity was augmented in K-Ras-transformed normal rat kidney cells and attenuated by transfection of Nox1 small interference RNAs (siRNAs) into the cells. Diphenyleneiodonium (DPI) or Nox1 siRNAs blocked up-regulation of matrix metalloprotease-9 at both protein and mRNA levels in K-Ras-transformed normal rat kidney cells. Furthermore, DPI and Nox1 siRNAs inhibited the activation of IKKα kinase and the degradation of IκBα, suppressing the NFκB-dependent matrix metalloprotease-9 promoter activity. Additionally, epidermal growth factor-stimulated migration of CaCO-2 cells was abolished by DPI and Nox1 siRNAs, indicating the requirement of Nox1 activity for the motogenic effect of epidermal growth factor. This Nox1 action was mediated by down-regulation of the Rho activity through the low molecular weight protein-tyrosine phosphatase-p190RhoGAP-dependent mechanism. Taken together, our findings define a mediating role of Nox1-generated reactive oxygen species in cell invasion processes, most notably metalloprotease production and cell motile activity.     

Bioconversion of cinnamic acid derivatives by Schizophyllum commune

To investigate the production of useful phenols from plant resources, we examined the metabolism of cinnamic acid derivatives by a wood-rotting fungus, Schizophyllum commune. Four cinnamic acid derivatives (cinnamic, p-coumaric, ferulic, and sinapic acids) were tested as substrates. Two main reactions, reduction and cleavage of the side chain, were observed. Reduction of the side chain was confirmed in cinnamic acid and p-coumaric acid metabolism. The side chain cleavage occurred in p-coumaric acid and ferulic acid metabolism but the initial reactions of these acids differed. Sinapic acid was not metabolized by S. commune. p-Hydroxybenzaldehyde accumulation was observed in the culture to which p-coumaric acid was added. This suggests that S. commune is a useful agent for transforming p-coumaric acid into p-hydroxybenzaldehyde.     

Periodontopathic bacterial endotoxin-induced tumor necrosis factor alpha production was inhibited by exercise in mice

The effects of exercise on serum interleukin-6 and tumor necrosis factor alpha levels were investigated using mice. Five-week-old female BALB/c mice (Th2-biased) and C57BL/6 mice (Th1-biased) were divided into exercise and control groups. The exercise group was exercised in a rotating basket type treadmill for 1 h (5 r.p.m.). Blood was collected and the serum was separated immediately after exercise. The serum interleukin-6 and tumor necrosis factor alpha levels were measured using an Endogen ELISA kit. Exercise significantly increased the serum interleukin-6 level in the two strains of mice (P<0.05 and P<0.01). The tumor necrosis factor alpha level was decreased in the exercise group. Next, periodontopathic bacterial endotoxin (lipopolysaccharide) was administered after exercise, and the effects of exercise on the lipopolysaccharide-induced serum interleukin-6 and tumor necrosis factor alpha levels were investigated. Exercise inhibited lipopolysaccharide-induced tumor necrosis factor alpha production, suggesting it has a defensive action against endotoxin shock.     

Investigating saccade programming in the praying mantis Tenodera aridifolia using distracter interference paradigms

To investigate the saccadic system in the mantis, I applied distracter interference paradigms. These involved presenting the mantis with a fixation target and one or several distracters supposed to affect saccades towards the target. When a single target was presented, a medium-sized target located in its lower visual field elicited higher rates of saccade response. This preference for target size and position was also observed when a target and a distracter were presented simultaneously. That is, the mantis chose and fixated the target rather than a distracter that was much smaller or larger than the target, or was located above the target. Furthermore, the mantis' preference was not affected by increasing the number of distracters. However, the presence of the distracter decreased the occurrence rate of saccade and increased the response time to saccade. I conclude that distracter interference paradigms are an effective way of investigating the visual processing underlying saccade generation in the mantis. Possible mechanisms of saccade generation in the mantis are discussed.     

A second generation of double mutant cholera toxin adjuvants: enhanced immunity without intracellular trafficking

Nasal application of native cholera toxin (nCT) as a mucosal adjuvant has potential toxicity for the CNS through binding to GM1 gangliosides in the olfactory nerves. Although mutants of cholera toxin (mCTs) have been developed that show mucosal adjuvant activity without toxicity, it still remains unclear whether these mCTs will induce CNS damage. To help overcome these concerns, in this study we created new double mutant CTs (dmCTs) that have two amino acid substitutions in the ADP-ribosyltransferase active center (E112K) and COOH-terminal KDEL (E112K/KDEV or E112K/KDGL). Confocal microscopic analysis showed that intracellular localization of dmCTs differed from that of mCTs and nCTs in intestinal epithelial T84 cells. Furthermore, both dmCTs exhibited very low toxicity in the Y1 cell assay and mouse ileal loop tests. When mucosal adjuvanticity was examined, both dmCTs induced enhanced OVA-specific immune responses in both mucosal and systemic lymphoid tissues. Interestingly, although both dmCT E112K/KDEV and dmCT E112K/KDGL showed high Th2-type and significant Th1-type cytokine responses by OVA-specific CD4+ T cells, dmCT E112K/KDEV exhibited significantly lower Th1-type cytokine responses than did nCT and dmCT E112K/KDGL. These results show that newly developed dmCTs retain strong biological adjuvant activity without CNS toxicity.     

Role of VAMP-2, VAMP-7, and VAMP-8 in constitutive exocytosis from HSY cells

We evaluated the role of VAMP-2/synaptobrevin, VAMP-7/TI-VAMP, and VAMP-8/endobrevin in exocytic pathways of HSY cells, a human parotid epithelial cell line, by coexpressing these VAMP proteins tagged with green fluorescent protein (GFP) and human growth hormone (hGH) as a secretory cargo. Exocytosis of hGH was constitutive and the fluorescent signal of hGH–GFP was observed in the Golgi area and small vesicles quickly moving throughout the cytoplasm. The cytoplasmic vesicles containing hGH overlapped well with VAMP-7-GFP, but did so scarcely with VAMP-2-GFP or VAMP-8-GFP. However, when the vesicle transport from the trans-Golgi network to the plasma membrane was arrested by incubation at 20°C for 2 h and then released by warming up to 37°C; VAMP-2-GFP and hGH were clearly colocalized together in small cytoplasmic vesicles. Neither VAMP-7-GFP nor hGH–GFP was colocalized with LAMP-1, a marker for lysosomes and late endosomes. These results suggest that (1) VAMP-2 can be one of the v-SNAREs for constitutive exocytosis; (2) VAMP-7 is involved in the constitutive exocytosis as a slow, minor v-SNARE, but not in the lysosomal transport; and (3) VAMP-8 is unlikely to be a v-SNARE for constitutive exocytosis in HSY cells.