Characterization of the F gene of contemporary mumps virus strains isolated in Japan

Mumps virus (MuV) strains isolated in Saitama Prefecture, Japan, from 1997 to 2001, were examined by analyzing the SH and the F gene nucleotide sequences. The results of the SH gene analysis showed that only genotype G was found in 2001 as well as in 2000, and that genotype J, which we proposed as a new genotype in a previous study, was from a different lineage than the genotype J described by Tecle et al. (J. Gen. Virol. 82, 2675-2680). We therefore, propose to rename the genotype as K to avoid confusion. Then, the F gene of genotypes G, H, and K strains were analyzed together with previously reported strains in this study. The results of phylogenetic analysis of the F gene nucleotide sequences showed that these strains formed a cluster as described by the SH gene analysis. Alignment of the F amino acid sequences showed that the F protein was well conserved among strains of different genotypes with a few amino acid differences. These results provide better information for the characterization of contemporary MuV strains in Japan.     

Interaction between sodium ascorbate and dopamine

The interaction between sodium ascorbate and dopamine was investigated by three different parameters: radical intensity, prooxidant action, and cytotoxicity induction. Sodium ascorbate and dopamine produced the doublet and quartet ESR signals under alkaline conditions (pH 8.0–9.5), respectively. Addition of increasing concentrations of sodium ascorbate completely scavenged the dopamine radical and replaced the latter with its own radical. Similarly, dopamine slightly, but significantly reduced the radical intensity of sodium ascorbate. These two compounds stimulated the methionine oxidation and hydrogen peroxide generation in culture medium, but in combination, their stimulation activities were weakened. Both of these two compounds dose-dependently reduced the viable cell number of human oral squamous carcinoma HSC-4 cells, and their cytotoxic activity was significantly reduced by catalase. When these two compounds were mixed together before adding to HSC-4 cells, both of their cytotoxic activities were diminished. The present study demonstrates the interaction between sodium ascorbate and dopamine, which might modify their biological activities and generation of nerve disorders such as Parkinson’s disease.     

Comparison of Cytokine Induction by Lipopolysaccharide of Bacteroides fragilis with Salmonella typhimurium in Mice

Comparison of cytokine stimulation by lipopolysaccharide (LPS) of Bacteroides fragilis and Salmonella typhimurium was done to study the early events occurring in vivo. Mice injected intraperitoneally with either LPS demonstrated endogenous production of all the cytokines studied (tumor necrosis factor-alpha, interferon-gamma and interleukin-6) within 6 hr in the bloodstream. However induction of all the cytokines by B. fragilis LPS (50 μg/mouse) was much weaker compared with S. typhimurium LPS (50 μg/mouse). Even a dose of S. typhimurium LPS 40 times smaller (1.2 μg/mouse) induced cytokines more strongly compared with B. fragilis LPS. Thus, a weak biological response to B. fragilis LPS as evidenced by chick embryo lethality, limulus lysate gelation, LD₅₀ for mice and rabbit pyrogenicity could be due to weak induction of bioactive mediators by LPS.     

Immunological Properties of TtT/M-87 Cell Line Established from Murine Pituitary Tumor-Associated Macrophages

TtT/M-87 cell is a macrophage cell line established from thyrotropic pituitary tumor tissues in mouse. In this paper, we report the immunological properties of M-87 cells as a model of tumor-associated macrophage. Contrasting with resident peritoneal macrophages, M-87 cells constitutively secreted small but significant amounts of TNF-α and IL-1α, which were detectable in both biological assays (cytotoxic activity for L929 and co-mitogenic activity for Con A-induced T cell proliferation, respectively) and ELISA, and produced larger amounts of these cytokines upon stimulation with LPS. They expressed MHC class II molecules on their cell surface without stimulation by IFN-γ. The accessory or antigen-presenting cell activity in antibody-producing response of spleen lymphocytes to sheep red blood cells was shown to be much higher in M-87 cells than normal peritoneal macrophages. In addition, when normal spleen lymphocytes were cultured with allogeneic tumor cells, such as EL-4 and S-180, in the presence of M-87 cells, lymphocytes reactive to stimulator cells were activated to manifest inhibitory effect on the tumor cell growth and also to manifest specific cytotoxic effect on the allogeneic tumor cells. These results show that M-87 cells derived from tumor-associated tissue are activated macrophages and that they are inhibitory to tumor cell growth and augmentative in the induction of T-cell-mediated immune responses.     

Computer simulation of Gumboro disease outbreak. II. Results obtained with models G-1 and G-2.

The authors conducted a computer simulation with their Models G-1 and G-2 for Gumboro disease ten times in each of the following initial conditions: (1) size of population, 50, 100, and 1,000 chickens; (2) age of housing, 1, 7, 14, and 21 days; (3) nine levels of parentally conferred immunity in one-day-old chicks; (4) four levels of virus contamination; and (5) three steps of coefficient for aggravating morbid status. Every simulation was operated up to the age when all the birds of a flock turned to be insusceptible so as to yield the daily numbers of chickens (1) susceptible, (2) diseased, (3) immunized, and (4) removed, and (5) the accumulation of diseased chickens. The innate resistance, parentally conferred immunity, virus contamination, and morbid status were expressed in such values that they could be compared with one another. As a result, Model G-2 produced a more realistic epizootic pattern than Model G-1, but both models concealed the effect of differences in size of population and in age of housing. Notwithstanding the incompleteness of the models, the computer simulation gave valuable information for a further advancement in this series of studies.     

Predominant synthesis of 5-hydroxyeicosatetraenoic acid by a cloned mastocytoma P-815 line, 2-E-6 cells

Since mouse mast tumor P-815 cells produce the slow reacting substance of anaphylaxis, their 5-lipoxygenase activity was examined by determining the conversion of arachidonic acid to 5-hydroxyeicosatetraenoic acid (HETE). Mast tumor cells from mouse ascites fluid synthesized 12-HETE as a major and 5-HETE as a minor metabolite. Once the cells were transferred to an in vitro culture system, the predominant synthesis of 12-HETE was abolished and synthesis of 5-HETE was greater than that of 12-HETE. 2-E-6 cells, obtained by cloning the tumor cells, synthesized a negligible amount of 12-HETE, but produced a large amount of 5-HETE. When the 2-E-6 cells were inoculated into mice and harvested again from the ascites fluid, their ratio of 5-HETE to 12-HETE synthesis was similar to that of normal mouse peritoneal cells; that is, 12-HETE synthesis was much greater than 5-HETE synthesis. It is concluded that the predominant synthesis of 12-HETE in mast tumor cells was derived from natural peritoneal cells, which have very high 12-lipoxygenase activity. The cloned mastocytoma, 2-E-6 cells, should be useful in investigating regulation of 5-lipoxygenase activity.     

RseP (YaeL), an Escherichia coli RIP protease, cleaves transmembrane sequences

Escherichia coli RseP (formerly YaeL) is believed to function as a 'regulated intramembrane proteolysis' (RIP) protease that introduces the second cleavage into anti-sigma(E) protein RseA at a position within or close to the transmembrane segment. However, neither its enzymatic activity nor the substrate cleavage position has been established. Here, we show that RseP-dependent cleavage indeed occurs within predicted transmembrane sequences of membrane proteins in vivo. Moreover, RseP catalyzed the same specificity proteolysis in an in vitro reaction system using purified components. Our in vivo and in vitro results show that RseP can cleave transmembrane sequences of some model membrane proteins that are unrelated to RseA, provided that the transmembrane region contains residues of low helical propensity. These results show that RseP has potential ability to cut a broad range of membrane protein sequences. Intriguingly, it is nevertheless recruited to the sigma(E) stress-response cascade as a specific player of RIP.     

Effects of humid heat exposure in later sleep segments on sleep stages and body temperature in humans

This study sought to investigate the effects of humid heat exposure in later sleep segments on sleep stages and body temperature in humans. The subjects were eight healthy males, from whom informed consent had been obtained. The experiments were carried out under three different sets of conditions: a control climate [air temperature (Ta)=26°C, relative humidity (RH)=50%] (C); a humid heat climate (Ta=32°C, RH=80%) (H); and a humid heat exposure in later sleep segments (C for the first 3 h 45 min, followed by a 30-min transition to H, which was then maintained for the last 3 h 45 min) (C–H). Electroencephalogram, EOG, and mental electromyogram, rectal temperature (Tre), and skin temperature (Tsk) were continuously measured. The total amount of wakefulness was significantly increased in H compared to C–H or C. Compared to C, wakefulness in C–H and H was significantly increased during later sleep segments. Tre and mean Tsk were significantly higher in H than in C–H or C. In C–H, Tsk and Tre increased to levels equal to those observed in H after Ta and RH increase. Whole body sweat loss was significantly lower in C–H and C than in H. These results suggest that humid heat exposure in the later sleep segment reduces thermal load as compared to full-night humid heat exposure. In daily life, the use of air conditioning in the initial sleep hours can protect sleep and thermoregulation.     

Mannose-binding lectin is a regulator of inflammation that accompanies myocardial ischemia and reperfusion injury

The mannose-binding lectin (MBL), a circulating pattern recognition molecule, recognizes a wide range of infectious agents with resultant initiation of the complement cascade in an Ab-independent manner. MBL recognizes infectious non-self and altered self in the guise of apoptotic and necrotic cells. In this study, we demonstrate that mice lacking MBL, and hence are devoid of MBL-dependent lectin pathway activation but have fully active alternative and classical complement pathways, are protected from cardiac reperfusion injury with resultant preservation of cardiac function. Significantly, mice that lack a major component of the classical complement pathway initiation complex (C1q) but have an intact MBL complement pathway, are not protected from injury. These results suggest that the MBL-dependent pathway of complement activation is a key regulator of myocardial reperfusion ischemic injury. MBL is an example of a pattern recognition molecule that plays a dual role in modifying inflammatory responses to sterile and infectious injury.     

An efficient system to establish multiple embryonic stem cell lines carrying an inducible expression unit

The growing use of mouse embryonic stem (ES) cells in research emphasizes their importance in studies of molecular mechanisms that maintain pluripotency and direct cellular differentiation. Although systems for regulatable transgene expression are essential for fine analysis of cellular processes at the molecular level, a strategy for the establishment of multiple ES cell lines carrying any of these systems has not yet been described. Here, we report our development of the ROSA-TET system, an effective system for the establishment of multiple ES cell lines carrying a tetracycline (Tc)-regulatable transgene at the Gt (ROSA)26asSor (ROSA26) locus. This system contains a knock-in step of a construct carrying both loxP and its mutant sequences into the ROSA26 locus, followed by a subsequent exchange step that introduces a cDNA to be Tc-regulated to the locus using the recombinase-mediated cassette exchange reaction. Both steps are demonstrated to give desired clones with high efficiency, suggesting that this system can be introduced readily into any ES cell lines, leading to the simultaneous establishment of multiple cell lines carrying different Tc-regulated cDNAs. We believe that use of this system will strongly accelerate molecular biological research using ES cells.