Isolation and Classification of Temperature-Sensitive Mutants of Influenza B Virus

We isolated 25 temperature-sensitive mutants of B/Kanagawa/73 strain generated by mutagenesis with 5-fluorouracil and classified them into seven recombination groups by pair-wise crosses. All mutants showed a ratio of plaquing efficiency at the nonpermissive temperature (37.5 C) to the permissive temperature (32 C) of 10^–4 or less. At 37.5 C most of group I, II, and III mutants did not produce appreciable amounts of protein, but all other group mutants were protein synthesis-positive. A group VII mutant produced active hemagglutinin (HA) and neuraminidase (NA) at the nonpermissive temperature, but Group V mutants produced only active NA and were defective in the HA molecule. The other group mutants, including group IV mutants with mutation only in the NA gene (8, 10), lacked both activities at the nonpermissive temperature. One of nine influenza B virus isolates in 1989 had EOP 37.5/32 of 1/3 × 10^–2 and belonged to recombination group VII.     

Cyclopropenone-containing cysteine proteinase inhibitors. Synthesis and enzyme inhibitory activities.

By focusing on the amphiphilic properties of cyclopropenone (e.g. a good electrophile and a precursor for a stable 2pi-aromatic hydroxycyclopropenium cation), a new class of cysteine proteinase inhibitors containing a cyclopropenone moiety was designed. For the purpose of the present research, we needed to devise a new method to introduce a peptide-related moiety as a substituent on the cyclopropenone residue. We investigated the reaction of metalated cyclopropenone acetal derivatives (2, R2 = metal) with N-protected alpha-aminoaldehydes 4 to obtain the adduct 5, and succeeded in the preparation of highly potentiated cysteine proteinase inhibitors 8 after several steps transformations. They showed strong inhibitory activities only to cysteine proteinases such as calpain, papain, cathepsin B, and cathepsin L and not to serine (e.g. thrombin and cathepsin G) and aspartic proteinases (e.g. cathepsin D). Kinetic studies indicated that they are competitive inhibitors, and by the examinations of their inhibitory mechanism it became clear that they are reversible inhibitors.     

Effect of superoxide derived from lucifer yellow CH on voltage-gated currents of mouse taste bud cells

Lucifer yellow CH (LY), a fluorescent membrane-impermeable cell marker dye, has been routinely loaded into cells through recording electrodes to visualize these cells after electrophysiological investigation, without considering its pharmacological effect. Recently, we showed that the exposure of cells loaded with LY to light for microscopy produced unidentified radical species that retarded the inactivation of voltage-gated Na+ currents irreversibly (Higure Y et al. 2003). Here, we show that superoxide dismutase, an enzyme that decomposes superoxide, reverses the retardation effect, which assures that superoxide is the unidentified radical species. The estimated mean lifetime of superoxide in recording electrodes (in the absence of cytoplasm) is approximately 6 min, and hence, the Na+ currents are retarded even in the dark, when LY is exposed to light before being loaded into the cell. Superoxide has no effect on voltage-gated Cl- currents. These results show that superoxide action on ion channels is rather selective. The breakdown of superoxide inside cells and the effect of endogenous superoxide on the superoxide-susceptible channels are discussed.     

Encapsulation of lemongrass oil with cyclodextrins by spray drying and its controlled release characteristics

Inclusion of the two isomers of citral (E-citral and Z-citral), components of lemongrass oil, was investigated within the confines of various cyclodextrin (α-CD, β-CD and γ-CD) host molecules. Aqueous complex formation constants for E-citral with α-CD, β-CD and γ-CD were determined to be 123, 185, and 204 L/mol, respectively, whereas Z-citral exhibited stronger affinities (157, 206, and 253 L/mol, respectively). The binding trend γ-CD > β-CD > α-CD is a reflection of the more favorable geometrical accommodation of the citral isomers with increasing cavity size. Encapsulation of lemongrass oil within CDs was undertaken through shaking citral:CD (1:1, 1.5:1, and 2:1 molar ratio) mixtures followed by spray drying. Maximum citral retention occurred at a 1:1 molar ratio with β-CD and α-CD demonstrating the highest levels of total E-citral and Z-citral retention, respectively. Furthermore, the β-CD complex demonstrated the slowest release rate of all inclusion complex powders.     

sPAR-3, a splicing variant of PAR-3, shows cellular localization and an expression pattern different from that of PAR-3 during enterocyte polarization

PAR-3 (partitioning-defective) is a scaffold-like PDZ (postsynaptic density-95/discs large/zonula occludens-1) domain-containing protein that forms a complex with PAR-6 and atypical PKC, localizes to tight junctions, and contributes to the formation of functional tight junctions. There are several alternatively spliced isoforms of PAR-3, although their physiological significance remains unknown. In this study, we show that one of the major isoforms of PAR-3, sPAR-3, is predominantly expressed in the Caco-2 cells derived from colon carcinoma and is used as a model to investigate the events involved in the epithelial cell differentiation and cell polarity development. During the polarization of Caco-2 cells, the expression of PAR-3 increases as do those of other cell-cell junction proteins, whereas the expression of sPAR-3 decreases. Biochemical characterization revealed that sPAR-3 associates with atypical PKC, as does PAR-3. On the other hand, immunofluorescence microscopy revealed that sPAR-3 does not concentrate at the cell-cell contact region in fully polarized cells, whereas it concentrates at premature cell-cell junctions. This makes a contrast to PAR-3, which concentrates at tight junctions in fully polarized cells. These results provide evidence suggesting the difference in the role between sPAR-3 and PAR-3 in epithelial cells.     

Phylogenetic analysis and pathogenicity of tick-borne encephalitis virus from Japan and far-east Russia

Phylogenetic analysis of tick-borne encephalitis (TBE) virus revealed that Hokkaido strain of TBE virus evolved several hundreds years ago in far-east Russia. TBE virus strains in Irkutsk area were identified as Siberian subtype of TBE virus. BHK-cell adapted mutant of TBE virus showed lower neuro-invasive virulence in mice than parent virus. The mutant carried one amino acid substitution in envelope protein which resulted in increase of positive charge of the protein. The mutant-infected mice showed lower virus titers in bloods and spleens than the parent-infected mice. Infectious c-DNA clone of TBE virus Hokkaido strain was successfully generated and was applied to examine the neurovirulence in mice. One amino acid change in envelope protein and 2 amino acid changes in Ns5 protein showed a synergistic effect on reduced neurovirulence in mice.     

Enhanced neurovirulence of borna disease virus variants associated with nucleotide changes in the glycoprotein and L polymerase genes

Borna disease virus (BDV) infection produces a variety of clinical diseases, from behavioral illnesses to classical fatal encephalitis (i.e., Borna disease [BD]). Since the genomes of most BDV isolates differ by less than 5%, host factors are believed responsible for much of the reported variability in disease expression. The contribution of BDV genomic differences to variation in BD expression is largely unexplored. Here we compared the clinical outcomes of rats infected with one of two related BDV variants, CRP3 or CRNP5. Compared to rats inoculated with CRP3, adult and newborn Lewis rats inoculated with CRNP5 had more severe and rapidly fatal neurological disease, with increased damage to the hippocampal pyramidal neurons and rapid infection of brain stem neurons. To identify possible virus-specific contributions to the observed variability in disease outcome, the genomes of CRP3 and CRNP5 were sequenced. Compared to CRP3, there were four nucleotide changes in the CRNP5 variant, two each in the G protein and in the L polymerase, resulting in four amino acid changes. These results suggest that small numbers of genomic differences between BDV variants in the G protein and/or L polymerase can contribute to the variability in BD outcomes.     

Galectin-3 phosphorylation is required for its anti-apoptotic function and cell cycle arrest.

Galectin-3, a beta-galactoside-binding protein, is implicated in cell growth, adhesion, differentiation, and tumor progression by interactions with its ligands. Recent studies have revealed that galectin-3 suppresses apoptosis and anoikis that contribute to cell survival during metastatic cascades. Previously, it has been shown that human galectin-3 undergoes post-translational signaling modification of Ser(6) phosphorylation that acts as an "on/off" switch for its sugar-binding capability. We questioned whether galectin-3 phosphorylation is required for its anti-apoptotic function. Serine to alanine (S6A) and serine to glutamic acid (S6E) mutations were produced at the casein kinase I phosphorylation site in galectin-3. The cDNAs were transfected into a breast carcinoma cell line BT-549 that innately expresses no galectin-3. Metabolic labeling revealed that only wild type galectin-3 undergoes phosphorylation in vivo. Expression of Ser(6) mutants of galectin-3 failed to protect cells from cisplatin-induced cell death and poly(ADP-ribose) polymerase from degradation when compared with wild type galectin-3. The non-phosphorylated galectin-3 mutants failed to protect cells from anoikis with G(1) arrest when cells were cultured in suspension. In response to a loss of cell-substrate interactions, only cells expressing wild type galectin-3 down-regulated cyclin A expression and up-regulated cyclin D(1) and cyclin-dependent kinase inhibitors, i.e. p21(WAF1/CIP1) and p27(KIP1) expression levels. These results demonstrate that galectin-3 phosphorylation regulates its anti-apoptotic signaling activity.     

Release characteristics of flavor from spray-dried powder in boiling water and during rice cooking

The release characteristics of flavor in boiling water and the flavor retention in the rice after cooking were investigated by using spray dried powder in encapsulated in or emulsified with d-limonene or ethyl n-hexanoate in cyclodextrin and maltodextrin, or in gum arabic and maltodextrin. The behavior of flavor release into the boiling water was well simulated by Avrami's equation. The retention of d-limonene and ethyl n-hexanoate in cooked rice was correlated in each case with the flavor amount of spray-dried powder added.