Molecular Evolution of Hemagglutinin (H) Gene in Measles Virus Genotypes D3, D5, D9, and H1

We studied the molecular evolution of H gene in four prevalent Asian genotypes (D3, D5, D9, and H1) of measles virus (MeV). We estimated the evolutionary time scale of the gene by the Bayesian Markov Chain Monte Carlo (MCMC) method. In addition, we predicted the changes in structure of H protein due to selective pressures. The phylogenetic tree showed that the first division of these genotypes occurred around 1931, and further division of each type in the 1960–1970s resulted in four genotypes. The rate of molecular evolution was relatively slow (5.57×10⁻⁴ substitutions per site per year). Only two positively selected sites (F476L and Q575K) were identified in H protein, although these substitutions might not have imparted significant changes to the structure of the protein or the epitopes for phylactic antibodies. The results suggested that the prevalent Asian MeV genotypes were generated over approximately 30–40 years and H protein was well conserved.     

Micropropagation of Cypripedium macranthos var. rebunense through Protocorm-like Bodies Derived from Mature Seeds

Cypripedium macranthos var. rebunense is the most famous terrestrial orchid in Japan, since the variety has large beautiful yellowish-white flowers and is threatened with extinction. Establishment of an efficient method for micropropagation is urgently needed. When imbibed mature seeds of the orchid, that had been pre-chilled at 4°C for 3 months, were sown onto Hyponex-peptone medium that contained both α-naphthaleneacetic acid (NAA) and cytokinin, protocorm-like bodies (PLBs) were formed from germinated seeds. Although the growth of PLBs was very slow, plantlets were easily regenerated from the PLBs on hormone-free medium. The PLBs were subcultured eight times along 2 years without loss of ability to regenerate plantlets, and one aggregate of PLBs (ca. 5 mm in diameter) produced ca. 10 plants within a year. A reduction of commercial value through a large-scale micropropagation by this method will be able to prevent illegal collection from the wild populations.     

Presence of H type 3/4 chains of ABO histo-blood group system in serous cells of human submandibular gland and regulation of their expression by the secretor gene (FUT2).

We have investigated by immunochemistry the distribution of H Type 3/4 chains of the ABO histo-blood group system in human submandibular gland using a monoclonal anti-H MBr1 antibody specific for H Type 3/4 chains, and have found the expression of H Type 3/4 chains was mainly in the serous cells. Serous cells from secretors were stained by MBr1 but not by anti-A and anti-B antibodies, whereas serous cells from nonsecretors exhibited a negative reaction with MBr1. Mucous cells were not stained by MBr1. Only a few striated duct cells showed a weak reaction with anti-H MBr1. These results suggested that the H Type 3/4 chains were distributed predominantly in the serous cells of the human submandibular gland and that secretor Type alpha(1,2)fucosyltransferase (Se enzyme) controlled the synthesis of H Type 3/4 chains in vivo. Saliva also contained H Type 3/4 chains, which were controlled by the secretor gene (FUT2). The differences in the distributions of H Type 1, H Type 2, and H Type 3/4 chains of the ABO histo blood group system in the submandibular gland are discussed.     

Cu/Zn‐ and Mn‐superoxide dismutases are specifically up‐regulated in neurons after focal brain injury

In previous studies, we have demonstrated that damaged neurons within a boundary area around necrosis fall into delayed cell death due to the cytotoxic effect of microglial nitric oxide (NO), and are finally eliminated by activated microglia. In contrast, neurons in a narrow surrounding region nearby this boundary area remain alive even though they may encounter cytotoxic NO. To investigate the mechanism by which neurons tolerate this oxidative stress, we examined the in vitro and in vivo expression levels of superoxide dismutase (SOD) under pathological conditions. Results from our in situ hybridization and immunohistochemical studies showed up‐regulation of Cu/Zn‐SOD only in neurons outside the boundary area, whereas up‐regulation of Mn‐SOD was detected in both neurons and glial cells in the same region. In vitro experiments using rat PC12 pheochromocytoma and C6 glioma cell lines showed that induction of both Cu/Zn‐ and Mn‐SOD mRNA could only be detected in PC12 cells after treatment with NO donors, while a slight induction of Mn‐SOD mRNA alone could be seen in C6 glioma cells. The mechanism of resistance toward oxidative stress therefore appears to be quite different between neuronal and glial cells. It is assumed that these two types of SOD might play a critical role in protecting neurons from NO cytotoxicity in vivo, and the inability of SOD induction in damaged neurons seems to cause their selective elimination after focal brain injury. © 2000 John Wiley & Sons, Inc. J Neurobiol 45: 39–46, 2000     

Synthesis, antifungal, haemolytic and cytotoxic activities of a series of bis(alkylpyridinium)alkanes

A series of bis(alkylpyridinium)alkanes with a twelve carbon spacer between the positive charges was synthesised and their antifungal activity has been investigated. Compounds with 2-pentyl, 4-pentyl, 4-hexyl, 4-octyl, 4-propylbenzene, 3,4-dipentyl, 4-(5′-nonyl) and 3-methyl,4-pentyl head groups were the most potent antifungal agents with MICs in the range of 1.4–2.7 μM against reference strains of both Cryptococcus neoformans and Candida albicans.     

Specific expression of Neu2 type B in mouse thymus and the existence of a membrane-bound form in COS cells

Neu2 mRNA from the mouse thymus, as we have reported [K. Kotani, A. Kuroiwa, T. Saito, Y. Matsuda, T. Koda, S. Kijimoto-Ochiai, Cloning, chromosomal mapping, and characteristic 5′-UTR sequence of murine cytosolic sialidase, Biochem. Biophys. Res. Commun. 286 (2001) 250-258], has a novel sequence at the 5′ terminus that shows the ability to encode 6 extra amino acids in the N-terminus than that of the muscle. In this paper, we analyzed the cDNA and EST database and found the five types of alternative splicing of Neu2 mRNA: A, B, C, D and N. We studied the expression of these types in the immune tissues and found that the thymus expressed only type B. We constructed 2 types of plasmid that encode long (B) or short (C) form of Neu2 protein, and transfected them into COS7 cells to study them under the same conditions. We found that 30-40% of the both forms of Neu2 activity was located in the crude membrane-fraction, and hydrolyzed ganglioside effectively, while both soluble fraction showed particular behavior with substrate specificity. Microscopic study by active staining with X-NANA showed that they located not only in the cytoplasm but also in areas surrounding the nucleus and in the peripheral ruffled spot.