Specific Interaction of a 25-kilodalton Cellular Protein,a 40S Ribosomal Subunit Protein,with the Internal Ribosome Entry Site of Hepatitis C Virus Genome

Translation initiation of hepatitis C virus (HCV) RNA is controlled by an internal ribosome entry site (IRES) contained in 5 noncoding region (NCR) and in several nucleotides of the coding region. The ability of a 25-kilodalton cellular protein (p25) to bind the HCV 5 NCR is correlated with the efficiency of translation initiation of HCV RNA, indicating that this protein plays a critical role in HCV translation (S. Fukushi, C. Kurihara, N. Ishiyama, F. B. Hoshino, A. Oya, and K. Katayama, J Virol 71, 1662–1666, 1997). We have extended the study for identification of the IRES region required for p25 binding. For this purpose, we have performed UV cross-linking competition analyses using 5- or 3- deleted mutants of the HCV 5 NCR as competitor RNAs for binding of p25 to wild-type HCV 5 NCR. Competitor RNAs lacking nucleotides (nt) 47–74 or nt 279–331 did not inhibit p25 binding to the HCV IRES, indicating that these regions are necessary for interaction of the p25 and HCV IRES. Since p25 binding was not observed in the IRES elements of encephalomyocarditis virus and poliovirus in UV cross-linking competition analyses, the p25 binding may be specific for the HCV IRES. p25 bound to the HCV IRES was detected when a purified 40S ribosomal subunit was used for UV cross-linking experiment, indicating that p25 is one of 40S ribosomal subunit proteins. These results reveal an unique interaction between the 40S ribosomal subunit and HCV IRES to contribute to translation initiation of the HCV genome.     

Effect of solvent evaporation rate on microphase-separated structure of segmented poly(urethane-urea) prepared by solution casting

A useful instrument for polymer film preparation by solution casting was employed in this study. It enabled us to control the solvent evaporation rate of the polymer solution. By using this instrument, the aggregation of hard segments in segmented poly(urethane-urea) (SPUU) was investigated. SPUU was prepared from poly(tetramethy1ene oxide), 4,4′-diphenylmethane diisocyanate and ethylenediamine. The effect of solvent evaporation rate on the microphase-separated structure of SPUU was elucidated by dynamic mechanical analysis, tensile test, differential scanning calorimetry analysis, small-angle X-ray scattering measurement, IR and IR dichroism analyses. The aggregation of hard segments in SPUU was observed to be affected considerably by the solvent evaporation rate of the cast film during the preparation. It was found that the slower the solvent evaporation rate, the higher the aggregation of hard segments to form rigid hard segment domains in SPUU. Nine months after casting, this casting effect still remained on the aggregation state of hard segments of SPUU films, although the interdomain spacing was not influenced by its rate.     

Lon,a stress-induced ATP-dependent protease,is critically important for systemic Salmonella enterica serovar typhimurium infection of mice

Studies on the pathogenesis of Salmonella enterica serovar Typhimurium infections in mice have revealed the presence of two prominent virulence characteristics-the invasion of the nonphagocytic cells to penetrate the intestinal epithelium and the proliferation within host phagocytic cells to cause a systemic spread and the colonization of host organs. We have recently demonstrated that the ATP-dependent Lon protease of S. enterica serovar Typhimurium negatively regulates the efficiency of invasion of epithelial cells and the expression of invasion genes (A. Takaya et al., J. Bacteriol. 184:224-232, 2002). This study was performed to reveal the contribution of the Lon protease to the virulence of S. enterica serovar Typhimurium in mice. Determination of 50% lethal doses for the lon disruption mutant and wild-type strain revealed that the mutant was highly attenuated when administered either orally or intraperitoneally to BALB/c mice. The mutant was also found to be able to reach extraintestinal sites but unable to proliferate efficiently within the spleen and cause lethal systemic disease of mice. Macrophage survival assays revealed that the lon disruption mutant could not survive or proliferate within murine macrophages. In addition, the mutant showed extremely increased susceptibility to hydrogen peroxide, which contributes to the bactericidal capacity of phagocytes. The mutant also showed increased sensitivity to acidic conditions. Taken together, the impaired ability of the lon disruption mutant to survive and grow in macrophages could be due to the enhanced susceptibility to the oxygen-dependent killing mechanism associated with respiratory burst and the low phagosomal pH. These results suggest that the Lon protease is essentially involved in the systemic infection of mice with S. enterica serovar Typhimurium, which can be fatal. Of further interest is the finding that the lon disruption mutant persists in the BALB/c mice for long periods without causing an overwhelming systemic infection.     

Accumulation of filamentous tau in the cerebral cortex of human tau R406W transgenic mice

Missense mutations of the tau gene cause autosomal dominant frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), an illness characterized by progressive personality changes, dementia, and parkinsonism. There is prominent frontotemporal lobe atrophy of the brain accompanied by abundant tau accumulation with neurofibrillary tangles and neuronal cell loss. Using a hamster prion protein gene expression vector, we generated several independent lines of transgenic (Tg) mice expressing the longest form of the human four-repeat tau with the R406W mutation associated with FTDP-17. The TgTauR406W 21807 line showed tau accumulation beginning in the hippocampus and amygdala at 6 months of age, which subsequently spread to the cortices and subcortical areas. The accumulated tau was phosphorylated, ubiquitinated, conformationally changed, argyrophilic, and sarcosyl-insoluble. Activation of GSK-3beta and astrocytic induction of mouse tau were observed. Astrogliosis and microgliosis correlated with prominent tau accumulation. Electron microscopic examination revealed the presence of straight filaments. Behavioral tests showed motor disturbances and progressive acquired memory loss between 10 to 12 months of age. These findings suggested that TgTauR406W mice would be a useful model in the study of frontotemporal dementia and other tauopathies such as Alzheimer's disease (AD).     

STAT3 couples with 14-3-3σ to regulate BCR signaling,B-cell differentiation,and IgE production

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Conjugation of acetaldehyde with cysteinylglycine,the first metabolite in glutathione breakdown byγ-glutamyltranspeptidase

Glutathione and its metabolites were examined for reactivity to acetaldehyde. When acetaldehyde was incubated with glutathione alone, there was only a slight decrease of acetaldehyde, while an apparently equimolar reaction between acetaldehyde and free sulfhydryl was observed with the addition of -glutamyltranspeptidase. Cysteinylglycine, the first metabolite in the glutathione breakdown by -glutamyltranspeptidase, showed a rapid and equimolar reactivity to acetaldehyde and such was comparable to the reaction seen withl-cysteine ord-penicillamine. In light of the chemical structure, cysteinylglycine probably conjugates with acetaldehyde to form thiazolidinecarboxylic acid derivatives, 2-methyl-thiazolidine-4-carbonyl-glycine, and if so, the alteration of glutathione metabolism by acetaldehyde during ethanol intoxication warrants further attention.     

Motor cortical activity in a memorized delay task

Summary Two rhesus monkeys were trained to move a handle on a two-dimensional (2D) working surface in directions specified by a light at the plane. They first captured with the handle a light on the center of the plane and then moved the handle in the direction indicated by a peripheral light (cue signal). The signal to move (go signal) was given by turning off the center light. The following tasks were used: (a) In the non-delay task the peripheral light was turned on at the same time as the center light went off. (b) In the memorized delay task the peripheral light stayed on for 300 ms and the center light was turned off 450–750 ms later. Finally, (c) in the non-memorized delay task the peripheral light stayed on continuously whereas the center light went off 750–1050 ms after the peripheral light came on. Recordings in the arm area of the motor cortex (N= 171 cells) showed changes in single cell activity in all tasks. In both delay tasks, the neuronal population vector calculated every 20 ms after the onset of the peripheral light pointed in the direction of the upcoming movement, which was instructed by the cue light. Moreover, the strength of the population signal showed an initial peak shortly after the cue onset in both the memorized and non-memorized delay tasks but it maintained a higher level during the memorized delay period, as compared to the non-memorized task. These results indicate that the motor cortex is involved in encoding and holding in memory directional information concerning a visually cued arm movement and that these processes can be visualized using neuronal population vector analysis.     

Actin assembly is a crucial factor for superoxide anion generation from adherent human eosinophils

BACKGROUND: Cellular adhesion is crucial for eosinophil effector functions. OBJECTIVE: We sought to elucidate the role of the actin cytoskeleton in cellular adhesion and superoxide anion generation by human eosinophils. METHODS: Eosinophils were stimulated with platelet-activating factor (PAF) or complement component 5a on human serum albumin-coated plates with or without an actin-polymerization inhibitor, cytochalasin B (CB), or cytochalasin D (CD). Superoxide anion generation was measured on the basis of reduction of absorbance associated with cytochrome c.2 Eosinophil adhesion was assessed on the basis of eosinophil protein X content in adherent cells. Transient stimulus-induced increase of intracellular calcium and translocation of protein kinase C (PKC) betaII, PKC delta, PKC zeta, and p47 phagocyte oxidase (a component of nicotinamide adenine dinucleotide phosphate oxidase) were also investigated. RESULTS: CB, CD, or antibodies against CD18 (the beta2 chain of integrin, alphaMbeta2) inhibited stimulus-induced eosinophil superoxide anion generation. Stimulus-induced eosinophil adhesion was unaltered by CB, whereas it was significantly suppressed by CD or anti-CD18 antibodies. Transient PAF-induced intracellular calcium increase was also unaffected by CB or CD, but stimulus-induced eosinophil shape changes and translocation of PKCs and p47 phagocyte oxidase to the cell membrane region were completely inhibited by CB. PAF-induced eosinophil degranulation was inhibited by CB, CD, or anti-CD18 antibodies, whereas complement component 5-induced degranulation was not suppressed by CB. CONCLUSION: By itself, beta2 integrin-dependent cellular adhesion is not sufficient for promoting eosinophil effector function. Adequate actin assembly is required for eosinophil adhesion and also for full superoxide anion generation in eosinophils.     

Follow-up study in a patient with Setleis syndrome

We report on an 8-year-old Japanese boy with Setleis syndrome. The patient had a very characteristic “coarse” facial appearance, bitemporal “forceps marks,” skin aplasia, sparse hair, and skin hypo- and hy-perpigmentation. He also had previously undescribed manifestations, including an aberrant hair pattern of the forehead, linear skin lesions on the forehead, short palpebral fissures, a small skin tag on the right cheek, cone-shaped teeth, and pectus carinatum. Dermatoglyphic studies documented aberrant distal palmar creases (simian crease variant), 8 arches, and reduced total finger ridge count. When serial photographs were reviewed, his facial characteristics became more obvious with increasing age. © 1995 Wiley-Liss, Inc.