Virus diversity and an outbreak of group C rotavirus among infants and children with diarrhea in Maizuru city, Japan during 2002-2003

A total of 236 fecal specimens collected from infants and children with gastroenteritis in Maizuru city, Japan from July 2002 to June 2003, were tested for the presence of rotaviruses, noroviruses, sapoviruses, astroviruses, and adenoviruses by RT-PCR, PAGE, RPHA, and latex agglutination methods. Among diarrheal viruses detected, group A rotavirus was the most prevalent (32.2%; 76 of 236) followed by norovirus GII (21.2%; 50 of 236), group C rotavirus (10.2%; 24 of 236), adenovirus (3.8%; 9 of 236), sapovirus (2.5%; 6 of 236), astrovirus (1.3%; 3 of 236), and norovirus GI (0.8%; 2 of 236), respectively. It is noteworthy that group C rotavirus infection was apparently confined only within the period of 5 months (December 2002 through April 2003). This pattern of infection implied that the outbreak of group C rotavirus in these patients, which was the first outbreak of gastroenteritis attributed to group C rotavirus in Maizuru city. Moreover, about half (12 of 24) of group C rotavirus infected cases were confined to infants and young children less than 3 years old. Another interesting feature of the study was the demonstration of the mixed infections with group C rotavirus and group A rotavirus, as well as group C rotavirus and norovirus GII in 20.8% (5 of 24) and 8.3% (2 of 24), respectively. This is the first report of gastroenteritis associated with the mixed infections with group C rotavirus and other viral enteropathogens such as norovirus. The results indicate that group C rotavirus could infect not only older children and adults but also infants and young children under 3 years old.     

Overexpression of Interleukin-15 increases susceptibility to lipopolysaccharide-induced liver injury in mice primed with Mycobacterium bovis bacillus Calmette-Guerin

Mice primed with Mycobacterium bovis bacillus Calmette-Guérin (BCG) are highly sensitive to lipopolysaccharide (LPS)-induced liver injury and lethality. We found that interleukin-15 (IL-15) transgenic (Tg) mice primed with BCG were more susceptible to LPS-induced liver injury than non-Tg mice. The numbers of CD44+ CD8+ T cells expressing intracellular gamma interferon (IFN-gamma) significantly increased in the livers of BCG-primed IL-15 Tg mice after LPS injection, and the depletion of CD8+ T cells from BCG-primed IL-15 Tg mice completely abolished the susceptibility to LPS-induced lethality. Liver T cells from BCG-primed IL-15 Tg mice produced IFN-gamma in vitro in response to LPS, which was inhibited by the addition of anti-IL-12 monoclonal antibody (MAb). In vivo treatment with anti-IL-12 MAb inhibited the appearance of CD44+ CD8+ T cells expressing intracellular IFN-gamma after LPS injection. These results suggest that the overexpression of IL-15 increases susceptibility to LPS-induced liver injury in BCG-primed mice via bystander activation of CD8+ T cells.     

Mutational screening of APP gene in patients with early-onset Alzheimer disease utilizing mismatched PCR-RFLP

To elucidate the frequency of mutations of the β/A4 amyloid protein precursor (APP) gene in early-onset Alzheimer disease, we designed a mismatched PCR-RFLP that can identify all kinds of missense mutations at codon 717 in addition to the seven kinds of known mutations at exon 17. When we screened mutations at exon 17 utilizing this method and the double missense mutations at exon 16 of the APP gene by PCR-RFLP, no cases revealed mutations of the APP gene among 13 familial and 54 sporadic cases, except one family (OS-1) that had previously been reported and used as a positive control of APP717(Val → Ile). Our results support the hypothesis that mutations in the APP gene are not major causes in early-onset Alzheimer disease.     

Anionic polymerization of fluorine-containing vinyl monomers, 12. Anionic polymerization mechanism of hexafluoro-1,3-butadiene

Initiation and propagation reaction mechanisms of the anionic polymerization of hexafluoro-1,3-butadiene (HEBD) were investigated. The initiation reaction with caesium tert-butoxide was found to be completed within 5 min although the reactions were carried out at a much lower temperature than that of the polymerization reaction. The initiation reaction was, therefore, inferred to take place in an anionic fashion by adding the tert-butoxide anion to HFBD. In order to clarify the propagation reaction mechanism of HFBD which yielded a polymer with a polyvinylene structure, the polymerization reactivity of HFBD and hexafluoro-2-butyne (HFBY), the isomerization of HFBD to HFBY, and the structural difference between poly(HFBD) and poly(HFBY) were discussed. In spite of the low yield of HFBY by the isomerization reaction under polymerization conditions, higher yields of poly(HFBD) were obtained. Judging from the X-ray analysis which showed that poly(HFBD) was highly crystalline and poly(HFBY) was amorphous, poly(HFBD) might not be produced by polymerization of HFBY. An addition reaction of the propagating anion to the carbon-2 of the HFBD monomer followed by isomerization at the propagating living end to yield poly[1,2-bis(trifluoromethyl)vinylene] is proposed.     

Rapid diagnosis of human herpesvirus 6 infection by a novel DNA amplification method, loop-mediated isothermal amplification

A novel nucleic acid amplification method, termed loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions, may be a valuable tool for the rapid detection of infectious agents. LAMP was developed for human herpesvirus 6 (HHV-6), and its reliability was evaluated in this study. Although LAMP products were detected in HHV-6 B and HHV-6 A DNA, they were not detected in HHV-7 and human cytomegalovirus DNA. The sensitivity of the original HHV-6 LAMP protocol was 50 copies/tube. In order to increase the method's sensitivity, HHV-6 LAMP was modified by increasing the primer concentration. As a result of the modification, sensitivity increased to 25 copies/tube. After these initial validation studies, 13 patients with fever were tested for HHV-6 by viral isolation, serological analysis, and HHV-6 LAMP. In three of the eight patients with primary HHV-6 infection, HHV-6 DNA was detected in whole blood by the original HHV-6 LAMP protocol in not only the acute phase but also the convalescent phase. HHV-6 DNA was detected by modified HHV-6 LAMP in all eight plasma samples collected in the acute phase; however, no HHV-6 DNA was detected in plasma samples collected in the convalescent phase. Although HHV-6 DNA was detected in both the acute and convalescent phases of whole-blood samples in patients with past HHV-6 infection, it was not detected in plasma samples that did not contain latent viral DNA. Thus, detection of HHV-6 DNA in plasma by using this modified HHV-6 LAMP protocol is appropriate for diagnosis of active HHV-6 infection.     

Liberation of serotonin from rabbit blood platelets by bacterial cell walls and related compounds.

A study was made on the activity of various bacterial cell walls and peptidoglycans to liberate serotonin from rabbit blood platelets. All of the test cell walls or peptidoglycans prepared from 27 strains of 21 bacterial species were shown to cause a marked release of serotonin, regardless of differences in types of peptidoglycan and non-peptidoglycan moieties and in some biological properties. The assay made with the water-soluble "digests" of Staphylococcus epidermidis cell wall peptidoglycans, which were prepared by use of appropriate enzymes, revealed that a polymer of peptidoglycan subunits (a disaccharide-stempeptide) was definitely active in the release of serotonin, but a structural unit monomer was inactive. Among a variety of synthetic muramylpeptides and their 6-O-acyl derivatives, only 6-O-(3-hydroxy-2-docosylhexacosanoyl)-N-acetylmuramyl-L-alanyl-D-isoglutaminyl- L-lysyl-D-alanine was found to hold a strong serotonin-liberating activity.     

Co-expression in CHO cells of two muscle proteins involved in excitation-contraction coupling

Summary Ryanodine receptors and dihydropyridine receptors are located opposite each other at the junctions between sarcoplasmic reticulum and either the surface membrane or the transverse tubules in skeletal muscle. Ryanodine receptors are the calcium release channels of the sarcoplasmic reticulum and their cytoplasmic domains form the feet, connecting sarcoplasmic reticulum to transverse tubules. Dihydropyridine receptors are L-type calcium channels that act as the voltage sensors of excitation-contraction coupling: they sense surface membrane and tranverse tubule depolarization and induce opening of the sarcoplasmic reticulum release channels. In skeletal muscle, ryanodine receptors are arranged in extensive arrays and dihydropyridine receptors are grouped into tetrads, which in turn are associated with the four subunits of ryanodine receptors. The disposition allows for a direct interaction between the two sets of molecules.CHO cells were stably transformed with plasmids for skeletal muscle ryanodine receptors and either the skeletal dihydropyridine receptor, or a skeletal-cardiac dihydropyridine receptor chimera (CSk3) which can functionally substitute for the skeletal dihydropyridine receptor, in addition to plasmids for the ₂, and subunits. RNA blot hybridization gave positive results for all components. Immunoblots, ryanodine binding, electron microscopy and exposure to caffeine show that the expressed ryanodine receptors forms functional tetrameric channels, which are correctly inserted into the endoplasmic reticulum membrane, and form extensive arrays with the same spacings as in skeletal muscle. Since formation of arrays does not require coexpression of dihydropyridine receptors, we conclude that self-aggregation is an independent property of ryanodine receptors. All dihydropyridine receptor-expressing clones show high affinity binding for dihydropyridine and immunolabelling with antibodies against dihydropyridine receptor. The presence of calcium currents with fast kinetics and immunolabelling for dihydropyridine receptors in the surface membrane of CSk3 clones indicate that CSk3-dihydropyridine receptors are appropriately targeted to the cell's plasmalemma. The expressed skeletal-type dihydropyridine receptors, however, remain mostly located within perinuclear membranes. In cells coexpressing functional dihydropyridine receptors and ryanodine receptors, no junctions between feet-bearing endoplasmic reticulum elements and surface membrane are formed, and dihydropyridine receptors do not assemble into tetrads. A separation between dihydropyridine receptors and ryanodine receptors is not unique to CHO cells, but is found also in cardiac muscle, in muscles of invertebrates and, under certain conditions, in skeletal muscle. We suggest that failure to form junctions in co-transfected CHO cell may be due to lack of an essential protein necessary either for the initial docking of the endoplasmic reticulum to the surface membrane or for maintaining the interaction between dihydropyridine receptors and ryanodine receptors. We also conclude that formation of tetrads requires a close interaction between dihydropyridine receptors and ryanodine receptors.     

Experimental arthritis induced by continuous infusion of IL-8 into rabbit knee joints.

To determine the roles of IL-8 in inflammatory synovitis, examination was made of the results of continuously injecting human recombinant IL-8 into the knee joints of New Zealand while rabbits. Recombinant human IL-8 was infused continuously into the joint cavity at 75 ng/h for 14 days by a polypropylene catheter connected to a mini-osmotic pump implanted in each rabbit. Infiltration of inflammatory cells into joint cavity and histopathological changes in synovial tissue were examined at 7 and 14 days following the start of infusion. The continuous infusion of IL-8 for 14 days led to severe arthritis characterized by apparent erythema and joint pain, the accumulation of leucocytes, infiltration of mononuclear cells in synovial tissue, and marked hypervascularization in the synovial lining layer. IL-8 may be a factor which can contribute to the inflammatory process of chronic arthritis by mediating leucocyte recruitment and hypervascularization in inflamed joints.     

Pathophysiology of blood pressure variability in patients with chronic renal failure under maintenance hemodialysis

The rise of blood pressure is negatively related with the glomerullar filtration rate(GFR) in patients with terminal renal failure. Hypertension may be a mechanism to maintain renal blood flow and GFR constant by the increased driving force of blood to the kidney. Elevated levels of a so-called third factor, now designated as endogenous digitalis, are found in those patients. The most likely candidate of the endogenous digitalis is ouabain, which causes hypertension with chronic administration. On the other hand, extreme hypotension often occurs during maintenance hemodialysis, and since hemodynamic alterations closely resemble endotoxin shock, the involvement of nitric oxide(NO) over-production has been suggested. When we measured nitrate anion as the final metabolite of NO, the concentration was significantly higher in patients with marked hypotension during hemodialysis than those without hypotension. Since reflex tachycardia was not observed during hypotension, we speculated that those patients had autonomic disturbances, and assessed autonomic function by heart rate spectral analysis. Although the high frequency spectral power, regarded as the vagal tone, was not significantly different between the groups, low/high frequency spectral power ratio, which was thought to be a sympathetic component, was significantly lower in patients with hypotension during hemodialysis than that in patients without hypotension. We speculated that NO synthase may be induced by the stimuli to monocytes by tubes and dialyser membrane made of synthetic materials leading to the over production of NO during and after regular hemodialysis. Thus, cytokines may be the mediator of the induction of NO synthase. Dilated capacitance vessels decrease the venous return to the heart, which may be the direct cause of dialysis-induced hypotension.