The genetic profile and molecular diagnosis of thiophanate-methyl resistant strains of Fusarium asiaticum in Japan

Fusarium asiaticum strains resistant to thiophanate-methyl were detected in four prefectures in Japan though their proportion in the total population was low in all instances. The F167Y or F200Y mutation in the β2-tubulin gene (FGSG06611.3) was detected in thiophanate-methyl resistant (TMR) strains. A PCR-based diagnostic method based on these mutations was developed and applied for all 17 TMR strains that have been detected so far in Japan. Three and 14 TMR strains were the F167Y and F200Y mutation types, respectively. Analysis by 11 variable number of tandem repeat markers showed that TMR strains from the same site had an identical genotype, while TMR strains from different sites were dissimilar. This result indicates that TMR strains did not spread clonally to the different sites.     

Morphological analysis of ghrelin and its receptor distribution in the rat pancreas

Ghrelin, a novel peptide isolated from stomach tissue of rats and humans, has been identified as the endogenous ligand for the growth hormone secretagogue receptor (GHS-R). In addition to its secretion from the stomach, ghrelin is also expressed in the hypothalamic arcuate nucleus, intestine, kidney, placenta, and pancreas. GHS-R mRNA, on the other hand, is expressed in the hypothalamus, pituitary, heart, lung, liver, pancreas, stomach, intestine, and adipose tissue. Ghrelin is considered to have important roles in feeding regulation and energy metabolism as well as in the release of growth hormone (GH). Recent physiological experiments on the pancreas have shown that ghrelin regulates insulin secretion. However, sites of action of ghrelin in the pancreas are yet to be identified. In this study, to gain insight into the role of ghrelin in rat pancreatic islets, we used immunohistochemistry to determine the localization of ghrelin and GHS-R in islet cells. Double fluorescence immunohistochemistry revealed that weak GHS-R-like immunoreactivity was found in B cells containing insulin. GHS-R immunoreactivity overlapped that of glucagon-like immunoreactive cells. Moreover, both ghrelin and GHS-R-like immunoreactivities were detected mostly in the same cells in the periphery of the islets of Langerhans. These observations suggest that ghrelin is synthesized and secreted from A cells, and acts back on A cells in an autocrine and/or paracrine manner. In addition, ghrelin may act on B cells via GHS-R to regulate insulin secretion.     

Input of orexin/hypocretin neurons revealed by a genetically encoded tracer in mice

The finding of orexin/hypocretin deficiency in narcolepsy patients suggests that this hypothalamic neuropeptide plays a crucial role in regulating sleep/wakefulness states. However, very little is known about the synaptic input of orexin/hypocretin-producing neurons (orexin neurons). We applied a transgenic method to map upstream neuronal populations that have synaptic connections to orexin neurons and revealed that orexin neurons receive input from several brain areas. These include the amygdala, basal forebrain cholinergic neurons, GABAergic neurons in the preoptic area, and serotonergic neurons in the median/paramedian raphe nuclei. Monoamine-containing groups that are innervated by orexin neurons do not receive reciprocal connections, while cholinergic neurons in the basal forebrain have reciprocal connections, which might be important for consolidating wakefulness. Electrophysiological study showed that carbachol excites almost one-third of orexin neurons and inhibits a small population of orexin neurons. These neuroanatomical findings provide important insights into the neural pathways that regulate sleep/wakefulness states.     

Role of S'1 loop residues in the substrate specificities of pepsin A and chymosin

Proteolytic specificities of human pepsin A and monkey chymosin were investigated with a variety of oligopeptides as substrates. Human pepsin A had a strict preference for hydrophobic/aromatic residues at P'1, while monkey chymosin showed a diversified preferences accommodating charged residues as well as hydrophobic/aromatic ones. A comparison of residues forming the S'1 subsite between mammalian pepsins A and chymosins demonstrated the presence of conservative residues including Tyr(189), Ile(213), and Ile(300) and group-specific residues in the 289-299 loop region near the C terminus. The group-specific residues consisted of hydrophobic residues in pepsin A (Met(289), Leu/Ile/Val(291), and Leu(298)) and charged or polar residues in chymosins (Asp/Glu(289) and Gln/His/Lys(298)). Because the residues in the loop appeared to be involved in the unique specificities of respective types of enzymes, site-directed mutagenesis was undertaken to replace pepsin-A-specific residues by chymosin-specific ones and vice versa. A yeast expression vector for glutathione-S-transferase fusion protein was newly developed for expression of mutant proteins. The specificities of pepsin-A mutants could be successfully altered to the chymosin-like preference and those of chymosin mutants, to pepsin-like specificities, confirming residues in the S'1 loop to be essential for unique proteolytic properties of the enzymes. An increase in preference for charged residues at P'1 in pepsin-A mutants might have been due to an increase in the hydrogen-bonding interactions. In chymosin mutants, the reverse is possible. The changes in the catalytic efficiency for peptides having charged residues at P'1 were dominated by k(cat) rather than K(m) values.     

Mapping of functional sites on the primary structure of the contractile tail sheath protein of bacteriophage T4 by mutation analysis

In order to determine the functional roles of amino acid residues in gp18 (gp: gene product), the contractile tail sheath protein of bacteriophage T4, the mutation sites and amino acid replacements of available and newly created missense mutants with distinct phenotypes were determined. Amber mutants were also utilized for amino acid insertion by host amber suppressor cell strains. It was found that mutants that gave rise to a particular phenotype were mapped in a particular region along the polypeptide chain. Namely, all amino acid replacements in the cold-sensitive mutants (cs, which grows at 37 degrees C, but not at 25 degrees C) and the heat-sensitive mutant (hs, lose viability by incubation at 55 degrees C for 30 min) except for one hs mutant were mapped in a limited region in the C-terminal domain. On the other hand, all the temperature-sensitive mutants (ts, grow at 30 degrees C, but not at 42 degrees C) and carbowax mutants (CBW, can adsorb to the host bacterium in the presence of high concentrations of polyethylene glycol, where wild-type phage cannot) were mapped in the N-terminal protease-resistant domain, except for one ts mutant. The results suggested that the C-terminal region of gp18 is important for contraction and assembly, whereas the N-terminal protease-resistant domain constitutes the protruding part of the tail sheath.     

Population dynamics of a maternally-transmitted <Emphasis Type="Italic">Spiroplasma</Emphasis> infection in <Emphasis Type="Italic">Drosophila hydei</Emphasis>

A maternally-inherited spiroplasma endosymbiont of Drosophila hydei does not exert apparent phenotypes on both sexes of its host and is prevalent in natural populations of D. hydei. Our previous experiments using a laboratory stock of D. hydei revealed that low temperatures (such as 15°C and 18°C) dramatically lower the vertical transmission rates of this spiroplasma. Therefore, we hypothesized that, in temperate regions, the infection frequencies may decrease in cool seasons but increase in the summer season. To clarify the temporal population dynamics of the spiroplasma infection, D. hydei were collected from two Japanese populations in 2006–2008 from May to early August, representing the only period when a number of D. hydei are collectable in Japan, and examined for spiroplasma infection. Within each year, the frequency of spiroplasma infection fluctuated considerably in both populations. Consistent with our hypothesis, the infection frequency showed an increasing trend in both populations in 2007. However, the data in 2006 and 2008 did not show consistent patterns of increase. The population dynamics of spiroplasma infection may be affected but not critically determined by temperature. Moreover, despite the fluctuation within each year, the infection frequencies seemed to be stable across the years. The frequencies of spiroplasma infection in D. hydei populations may be stabilized by multiple factors. One of these factors may involve a context-dependent positive effect of spiroplasma on the fitness of D. hydei, as was recently observed in laboratory experiments.     

Persistent and high levels of Hes1 expression regulate boundary formation in the developing central nervous system

The developing central nervous system is partitioned into compartments by boundary cells, which have different properties than compartment cells, such as forming neuron-free zones, proliferating more slowly and acting as organizing centers. We now report that in mice the bHLH factor Hes1 is persistently expressed at high levels by boundary cells but at variable levels by non-boundary cells. Expression levels of Hes1 display an inverse correlation to those of the proneural bHLH factor Mash1, suggesting that downregulation of Hes1 leads to upregulation of Mash1 in non-boundary regions, whereas persistent and high Hes1 expression constitutively represses Mash1 in boundary regions. In agreement with this notion, in the absence of Hes1 and its related genes Hes3 and Hes5, proneural bHLH genes are ectopically expressed in boundaries, resulting in ectopic neurogenesis and disruption of the organizing centers. Conversely, persistent Hes1 expression in neural progenitors prepared from compartment regions blocks neurogenesis and reduces cell proliferation rates. These results indicate that the mode of Hes1 expression is different between boundary and non-boundary cells, and that persistent and high levels of Hes1 expression constitutively repress proneural bHLH gene expression and reduce cell proliferation rates, thereby forming boundaries that act as the organizing centers.     

Rapid construction of Drosophila RNAi transgenes using pRISE, a P-element-mediated transformation vector exploiting an in vitro recombination system

RNAi is a gene-silencing phenomenon mediated by double-stranded RNA (dsRNA) and has become a powerful tool to elucidate gene function. To accomplish rapid construction of transgenes expressing dsRNA in Drosophila, we developed a novel transformation vector, pRISE, which contains an inverted repeat of the attR1-ccdB-attR2 cassette for in vitro recombination and a pentameric GAL4 binding site for conditional expression. These features enabled us to construct RNAi transgenes without a complicated cloning scheme. In cultured cells and transgenic flies, pRISE constructs carrying dsRNA transgenes induced effective RNAi against an EGFP transgene and the endogenous white gene, respectively. These results indicate that pRISE is a convenient transformation vector for studies of multiple Drosophila genes for which functional information is lacking.