Midkine Inhibits Caspase-Dependent Apoptosis via the Activation of Mitogen-Activated Protein Kinase and Phosphatidylinositol 3-Kinase in Cultured Neurons

Midkine (MK) is a new member of the heparin-binding neurotrophic factor family. MK plays important roles in development and carcinogenesis and has several important biological effects, including promotion of neurite extension and neuronal survival. However, the mechanism by which MK exerts its neurotrophic actions on neurons has not been elucidated to date. We have established an apoptosis induction system by serum deprivation in primary neuronal cultures isolated from mouse cerebral cortices. Neuronal apoptosis induced by serum deprivation was accompanied by the activation of caspase-3. MK, when added into the culture medium, inhibited the induction of apoptosis and activation of caspase-3 in a dose-dependent manner. Extracellular signal-regulated kinase (ERK) and Akt were not activated by serum deprivation, whereas ERK and Akt were rapidly activated by addition of MK. In addition, the trophic actions of MK of suppressing apoptosis and suppressing the activation of caspase-3 were abolished by concomitant treatment with PD98059, a specific inhibitor of mitogen-activated protein kinase kinase, and with wort-mannin or LY294002, specific inhibitors of phosphatidyl-inositol 3-kinase (PI 3-kinase). These PI 3-kinase inhibitors also inhibited the activation of ERK in response to MK, demonstrating a link between ERK and the caspase-3 pathway that is modulated by the PI 3-kinase activation. These results indicate that the ERK cascade plays a central role in MK-mediated neuronal survival via inhibition of caspase-3 activation.     

Otolith development and daily increment formation in larvae of the Kabyabya,a Malawian cyprinid, <Emphasis Type="Italic">Opsaridium tweddleorum</Emphasis>

Features of the sagitta, asteriscus, and lapillus of laboratory-hatched larvae of the Kabyabya, Opsaridium tweddleorum, were investigated until day 33 after hatching. The sagitta was round until day 17, subsequently becoming arrowhead-shaped with the development of anterior and posterior rostra. Increments in the sagitta increased at the rate of one per day before rostrum formation. However, they were occasionally overestimated in rostrum sections as a result of subdaily increment occurrence. Furthermore, both anterior and posterior rostra were fragile, being thin and platelike, and were often damaged during the extraction and grinding processes. The lapillus was also round until day 17, thereafter becoming fan-shaped. Increments in the lapillus were distinctive from the core to the margin, usually increasing at the rate of one per day after hatching. The asteriscus appeared in fish larger than 9.15 mm in standard length (from day 17), being oval with a somewhat ambiguous core, causing difficulty in discerning the first increment. These features indicated that the lapillus was the most appropriate otolith part for daily increment analysis in this species, although the sagitta was useful before rostrum formation.     

Synthesis of 24-Epibrassinolide-related Compounds with Plant Growth-promoting Activity

2-Mono- and 2,3-diacetate derivatives of (22R,23R)- and (22S,23S)-24-epibrassinolides, and (22R,23R)- and (22S,23S)-2-deoxy-24-epibrassinolides were synthesized. They were found to possess weak biological activity in a rice-lamina inclination test.     

A Single Amino Acid in the M1 Protein Responsible for the Different Pathogenic Potentials of H5N1 Highly Pathogenic Avian Influenza Virus Strains

Two highly pathogenic avian influenza virus strains, A/duck/Hokkaido/WZ83/2010 (H5N1) (WZ83) and A/duck/Hokkaido/WZ101/2010 (H5N1) (WZ101), which were isolated from wild ducks in Japan, were found to be genetically similar, with only two amino acid differences in their M1 and PB1 proteins at positions 43 and 317, respectively. We found that both WZ83 and WZ101 caused lethal infection in chickens but WZ101 killed them more rapidly than WZ83. Interestingly, ducks experimentally infected with WZ83 showed no or only mild clinical symptoms, whereas WZ101 was highly lethal. We then generated reassortants between these viruses and found that exchange of the M gene segment completely switched the pathogenic phenotype in both chickens and ducks, indicating that the difference in the pathogenicity for these avian species between WZ83 and WZ101 was determined by only a single amino acid in the M1 protein. It was also found that WZ101 showed higher pathogenicity than WZ83 in mice and that WZ83, whose M gene was replaced with that of WZ101, showed higher pathogenicity than wild-type WZ83, although this reassortant virus was not fully pathogenic compared to wild-type WZ101. These results suggest that the amino acid at position 43 of the M1 protein is one of the factors contributing to the pathogenicity of H5N1 highly pathogenic avian influenza viruses in both avian and mammalian hosts.     

An Efficient Method Using Gluconacetobacter europaeus To Reduce an Unfavorable Flavor Compound,Acetoin, in Rice Vinegar Production

Gluconacetobacter europaeus, one of the microorganisms most commonly used for vinegar production, produces the unfavorable flavor compound acetoin. Since acetoin reduction is important for rice vinegar production, a genetic approach was attempted to reduce acetoin produced by G. europaeus KGMA0119 using specific gene knockout without introducing exogenous antibiotic resistance genes. A uracil-auxotrophic mutant with deletion of the orotate phosphoribosyltransferase gene (pyrE) was first isolated by positive selection using 5-fluoroorotic acid. The pyrE disruptant designated KGMA0704 (ΔpyrE) showed 5-fluoroorotic acid resistance. KGMA0704 and the pyrE gene were used for further gene disruption experiments as a host cell and a selectable marker, respectively. Targeted disruption of aldC or als, which encodes α-acetolactate decarboxylase or α-acetolactate synthase, was attempted in KGMA0704. The disruption of these genes was expected to result in a decrease in acetoin levels. A disruption vector harboring the pyrE marker within the targeted gene was constructed for double-crossover recombination. The cells of KGMA0704 were transformed with the exogenous DNA using electroporation, and genotypic analyses of the transformants revealed the unique occurrence of targeted aldC or als gene disruption. The aldC disruptant KGMA4004 and the als disruptant KGMA5315 were cultivated, and the amount of acetoin was monitored. The acetoin level in KGMA4004 culture was significantly reduced to 0.009% (wt/vol) compared with KGMA0119 (0.042% [wt/vol]), whereas that of KGMA5315 was not affected (0.037% [wt/vol]). This indicates that aldC disruption is critical for acetoin reduction. G. europaeus KGMA4004 has clear application potential in the production of rice vinegar with less unfavorable flavor.