Characterization of a G14 equine rotavirus (strain CH3) isolated in Japan

Summary Antigenic and genomic properties of equine rotavirus strain CH3 isolated in Japan were studied by cross-neutralization tests and nucleotide sequence determination of the VP4 and VP7 genes. It was shown that the strain CH3 belongs to G14 and shares VP4 genotype with strain H2.The nucleotide sequence data reported in this paper appear in the DDBJ, EMBL and GeneBank nucleotide sequence detabases under the accession numbers D25228 (VP4 of strain CH3) and D25229 (VP7 of strain CH3).     

Macrophage plasma membrane cholesterol contributes to Brucella abortus infection of mice

Brucella abortus is a facultative intracellular bacterium capable of surviving inside macrophages. Intracellular replication of B. abortus requires the VirB complex, which is highly similar to conjugative DNA transfer systems. In this study, we show that plasma membrane cholesterol of macrophages is required for the VirB-dependent internalization of B. abortus and also contributes to the establishment of bacterial infection in mice. The internalization of B. abortus was accelerated by treating macrophages with acetylated low-density lipoprotein (acLDL). Treatment of acyl coenzyme A:cholesterol acyltransferase inhibitor, HL-004, to macrophages preloaded with acLDL accelerated the internalization of B. abortus. Ketoconazole, which inhibits cholesterol transport from lysosomes to the cell surface, inhibited the internalization and intracellular replication of B. abortus in macrophages. The Niemann-Pick C1 gene (NPC1), the gene for Niemann-Pick type C disease, characterized by an accumulation of cholesterol in most tissues, promoted B. abortus infection. NPC1-deficient mice were resistant to the bacterial infection. Molecules associated with cholesterol-rich microdomains, "lipid rafts," accumulate in intracellular vesicles of macrophages isolated from NPC1-deficient mice, and the macrophages yielded no intracellular replication of B. abortus. Thus, trafficking of cholesterol-associated microdomains controlled by NPC1 is critical for the establishment of B. abortus infection.     

Molecular epidemiological study on tetracycline resistance R plasmids in enterohaemorrhagic Escherichia coli O157:H7.

Restriction patterns obtained with EcoRI and Southern hybridization were used for the differentiation of tetracycline-resistant (Tet(r)) R plasmids in enterobaemorrhagic Escherichia coli (EHEC) O157:H7 isolates from a mass outbreak at a kindergarten in Obihiro-City, Hokkaido, Japan, 1996. Two kinds of Tet(r) R plasmids of 50 and 95 kb were detected. The 50-kb plasmids were identical to each other, while the 93-kb plasmids were of three types that were very similar to each other. The tet genes of both 50- and 95-kb R plasmids were 100% identical to the tet gene of pSC101 and all plasmids hybridized to a probe for tet. Because food-origin O157 strains were sensitive to tetracycline, we concluded that such Tet(r) R-plasmids might transfer to drug-sensitive O157 strains in the infected individuals.     

Postnatal development of orexin/hypocretin in rats

We examined developmental changes of orexins/hypocretins and their receptors (OX1R and OX2R) in the rat hypothalamus from postnatal day 0 to 10 weeks, using in situ hybridization histochemistry for the prepro-orexin, OX1R and OX2R mRNAs and immunohistochemistry for orexin-A and orexin-B. The prepro-orexin mRNA was weakly detected in the lateral hypothalamic area (LHA) from days 0 to 15. Orexin-A- and -B-like immunopositive cells and fibers were not detected from days 0 to 10, but they were observed after day 15. The prepro-orexin mRNA in the LHA markedly increased between days 15 and 20. The OX1R mRNA was detected in the ventromedial hypothalamic area (VMH) at day 0. The OX2R mRNA was not detected in the paraventricular nucleus (PVN) at days 0 and 1, but weakly observed on day 5. The OX1R mRNA in the VMH and OX2R mRNA in the PVN gradually increased throughout the postnatal period. Next, we examined the effects of milk deprivation and intraperitoneal (i.p.) administration of leptin on the hypothalamic prepro-orexin mRNA in pups. Although 24-h milk deprivation did not affect the level of the prepro-orexin mRNA at days 5 and 10, i.p. administration of leptin from days 0 to 3 caused a significant increase in the prepro-orexin mRNA on days 5 and 10. These results suggest that the development of orexins may be associated with developmental changes such as increase of leptin, weaning, feeding and sleep/wakefulness states.     

Prothrombin levels in newborn infants by the carinactivase-1 method

A recently developed method to quantitate prothrombin in plasma uses the carinactivase-1 (CA-1) method. The present study was designed to establish the reference value by the CA-1 method in the neonatal period and to explore the effect of gestational age, birth weight, concurrent diseases, and vitamin K administration on the prothrombin levels. We enrolled 78 consecutive neonates. The gestational ages ranged from 28 to 41 weeks, and the birth weight ranged from 850 to 3750 g. Twenty-nine infants had concurrent diseases, and the others (n = 49) were healthy. A 300 microL blood sample was drawn into a plastic syringe containing 60 U freeze-dried buffered heparin. Prothrombin levels did not differ between appropriate-for-date (AFD) and light-for-date (LFD) babies (p = 0.090) or between groups with and without concurrent diseases (p = 0.210). In healthy AFD babies, prothrombin levels correlated with gestational age (r = 0.465, p = 0.003) and birth weight (r = 0.458, p = 0.003). In healthy low-birth-weight infants (n = 14) and those with concurrent diseases (n = 17), the changes after vitamin K administration were not significant. The CA-1 method is of clinical use in monitoring coagulation during the early neonatal period.     

Differences in sleep-induced hypoxia between A/J and DBA/2J mouse strains

In obstructive sleep apnea, hypoxic ventilatory sensitivity may affect the degree of hypoxic stress and sleep disruption that occurs in response to upper airway obstruction. We induced (1) sleep-induced hypoxia (SIH) or (2) sleep fragmentation (SF) without hypoxia for 5 days (12-hour light/dark cycle) in two inbred mouse strains with low (A/J) and high (DBA/2J) hypoxic ventilatory sensitivities. During SIH, the time to arousal (26.4 +/- 1.1 vs. 21.3 +/- 1.5 seconds, p<0.025) and the severity of hypoxic exposure (nadir FIO2: 11.5 +/- 0.4 vs. 13.6 +/- 0.1%, p<0.002) was greater in A/J than DBA/2J mice. Furthermore, A/J mice had a greater frequency of hypoxic events (640 +/- 29 vs. 368 +/- 33 events per 24 hours, p<0.001) and total sleep time (47.5 +/- 2.8% vs. 26.5 +/- 2.4% per 24 hours, p<0.0001) during SIH than DBA/2J mice. In contrast, the event characteristics and total sleep time during SF were the same in both strains. Furthermore, in the light phase, both strains showed a longer (p<0.01) time to arousal during SIH and SF compared with the dark phase. We conclude that genetic background can influence respiratory events and sleep architecture during SIH and that the arousal threshold is subject to circadian variation. Our data imply that individuals with low hypoxic sensitivity may be at a greater risk for hypoxia-related complications of obstructive sleep apnea.     

Serum DJ-1 level is positively associated with improvements in some aspects of metabolic syndrome in Japanese women through lifestyle intervention

DJ-1 is a protein that is associated with Parkinson disease and cancer, and the reduction of DJ-1 function and expression is also thought to be a cause of diabetes and hypertension. However, little is known about the association between the plasma concentration of DJ-1 and risk of metabolic syndrome. We hypothesized that a lifestyle intervention would increase serum DJ-1 and that up-regulated DJ-1 functions will result in the prevention of metabolic syndrome. The objective of our study is to examine whether the level of serum DJ-1 is associated with the risk of metabolic syndrome. Therefore, to reveal the association between DJ-1 and metabolic syndrome, this study investigated lifestyle intervention in a control group (n = 37) and intervention group (n = 45). The results showed that body mass index, body fat ratio, waist-hip ratio, waist circumference, blood pressure, and plasma glucose level were improved in the intervention group, as compared with those in the control group. Furthermore, serum levels of DJ-1 were increased in the intervention group, when compared with those in the control group. These results suggest that serum DJ-1 is increased by lifestyle intervention and that increased serum DJ-1 prevents metabolic syndrome. Thus, the level of serum DJ-1 will become one of the indexes for the risk of metabolic syndrome.     

Suppression of immunoglobulin production in human peripheral blood mononuclear cells by monocytes via secretion of heavy-chain ferritin

In vitro antigen stimulation of peripheral blood mononuclear cells (PBMCs) does not induce immunoglobulin (Ig) production. However, pretreatment of PBMCs with l-leucyl-l-leucine methyl ester (LLME) prior to in vitro stimulation removes the suppression of Ig production. In the present study, we attempted to identify the target cells of LLME and determine the mechanisms by which Ig production in PBMCs is suppressed. We found that CD14⁺ monocytes are involved in the suppression of Ig production in PBMCs. Furthermore, we confirmed that heavy-chain ferritin derived from CD14⁺ monocytes suppresses Ig production in PBMCs, possibly through iron sequestration.     

Isolation of two forms of growth hormone secreted from eel pituitaries in vitro

Two forms of growth hormone (GH) were purified by chromatofocusing of medium from cultured Japanese eel (Anguilla japonica) pituitaries. The pituitaries were organ-cultured in Eagle's minimum essential medium with Earle's salts. Following polyacrylamide gel electrophoresis of the medium at pH 9.5, two prominent bands were seen with Rf 0.36 and 0.29; they were designated as eGHI and eGHII, respectively. Seven-hundred fifty milliliters of medium, in which 260 pituitaries were cultured for 6-10 weeks, was concentrated by DIAFLO membrane (YM-5) and subjected to gel filtration on a Sephadex G-75 column and to chromatofocusing on a PBE-94 column. eGHI and II were finally purified by gel filtration on a Sephadex G-75 column, yielding 2.0 mg of eGHI and 1.3 mg of eGHII. Both eGHI and eGHII were equipotent to ovine GH in promoting growth of juvenile rainbow trout. The putative GH-producing cells in the proximal pars distalis of the eel pituitary were stained specifically with antisera raised against eGHI or eGHII; no cross-reactivity was seen in the follicular prolactin cells in the rostral pars distalis. As determined by gel isoelectric focusing, eGHI and eGHII have isoelectric points of 6.3 and 6.7, respectively. Identical molecular masses of 23,000 Da were determined by sodium dodecyl sulfate gel electrophoresis. Their amino acid compositions strongly resembled each other; comparison of the partial N-terminal amino acids indicates that sequence 1 to 36 of GHII is exactly the same as 4 to 39 of GHI.