Enterobacter kobei sp. nov., a new species of the family Enterobacteriaceae resembling Enterobacter cloacae

The name Enterobacter kobei sp. nov. is proposed for a group of organisms referred to as NIH Group 21 at the National Institute of Health, Tokyo. The members of this species are Gram-negative, motile rods conforming to the definition of the family Enterobacteriaceae. The DNA relatedness of 23 strains of NIH Group 21 to the representative proposed as the type strain of this species averaged 82% at 70°C, whereas the relatedness to other species within the family Enterobacteriaceae was less than 42%. Because the phenotypic resemblance to Enterobacter cloacae is very close and the DNA relatedness (12–42%) is closer to species of the genus Enterobacter than to other species of the family, the members of NIH Group 21 were placed in the genus Enterobacter. Close phenotypic and genetic relationships were also found between NIH Group 21 and a member of a group of organisms referred to as Enteric Group 69 at the Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, USA. It is suggested that the latter could be regarded as a subspecific rank of E. kobei, though this is subject to study of further strains. The majority of strains of E. kobei were isolated from clinical specimens. A culture of the type strain (NIH 1485-79) has been deposited in the Japan Collection of Microorganisms as JCM 8580. Received: 22 March 1996 / Accepted: 19 April 1996     

The novel kappa-opioid receptor agonist TRK-820 suppresses the rewarding and locomotor-enhancing effects of morphine in mice

The effects of the novel kappa-opioid receptor agonist TRK-820 on the rewarding and locomotor-enhancing effects of morphine were investigated in mice. Morphine (1-5 mg/kg, s.c.) caused a dose-related preference for the drug-associated place. In contrast, TRK-820 (0.003-0.03 mg/kg, s.c.) did not produce a significant preference for either compartment of the test box. In combination studies, co-injection of TRK-820 (0.01 and 0.03 mg/kg, s.c.) with morphine significantly suppressed the morphine (5 mg/kg, s.c.)-induced place preference, and this effect of TRK-820 was antagonized by pretreatment with nor-BN1 (3 mg/kg, s.c.), a selective kappa-opioid receptor antagonist. TRK-820 also suppressed morphine-induced hyperlocomotion, and this suppression was also blocked by nor-BNI. These results suggest that TRK-820 suppresses the rewarding and locomotor-enhancing effects of morphine through the activation of kappa-opioid receptors. Thus, we propose that TRK-820 may be useful for controlling pain while reducing undesirable side-effects.     

Expression of host genes in influenza virus infected cells

The NS1 protein of influenza virus shuts off host gene expression by inhibiting the polyadenylation-site cleavage of host pre-mRNAs, resulting in a general decline in cellular protein synthesis. On the other hand, an activation of several host genes related to host antiviral defense such as interferon- alpha/beta, MxA, 2',5'-oligoadenylate synthetase, and Fas occures upon infection. Therefore, balance of the shut-off and the activation of cellular genes during virus growth may be crucial in determining the outcome of infection. To obtain a comprehensive view of the global effects of influenza virus infection on human respiratory epithelial cells at the cytoplasmic mRNA level, we performed oligo DNA microarray analysis using GeneChip arrays (Affymetrix). In NCl-H292 cells infected with A/Udorn/72 virus, more than 4-fold increase of expression level was observed for 164 genes at 12 h pi. Approximately 60% of the virus-stimulated genes (VSGs) were also stimulated with interferon-beta treatment and contained the genes known to possess antiviral activity. Interestingly, majority of the VSGs were stimulated before induction of interferons, suggesting that the stimulation of the VSGs during early phase of infection is not mediated by interferons, but it is triggered from within by the virus infection.     

Susceptibility gene for non-obstructive azoospermia located near HLA-DR and -DQ loci in the HLA class II region

The technical developments and expanded indications for testicular sperm extraction (TESE) with intracytoplasmic sperm injection (ICSI) provide great advantages for patients with non-obstructive azoospermia. Such success, however, also means that genetic abnormalities in non-obstructive azoospermia can be transmitted to the next generation, demonstrating the importance of being able to understand the genetic background of non-obstructive azoospermia. We have previously reported that human leukocyte antigens (HLA)-A33 and -B44 in the HLA class I region and the HLA-DRB1*1302 allele in the HLA class II region are linked to susceptibility to non-obstructive azoospermia in Japanese men. However, strong linkage of HLA-DRB1*1302 with HLA-A33 and -B44 is also evident in the Japanese population. Thus, uncertainty prevails as to whether the HLA class I or class II molecule is more directly associated with non-obstructive azoospermia. In the present study, we performed association analysis with 21 polymorphic microsatellite markers identified near the HLA genes to map the gene involved in the development of non-obstructive azoospermia more precisely. Microsatellite markers located in the HLA class I region or the class III region showed no statistically significant association with this disorder, although once again the HLA-A33 and -B44 alleles showed a significant association. In contrast, some of the microsatellite markers in the HLA class II region and at the HLA-DRB1 and -DQB1 loci displayed strong associations with non-obstructive azoospermia. Taken together, our previous and present data suggest that the critical region for development of non-obstructive azoospermia is near the HLA-DRB1 and -DQB1 segments in the HLA class II region.     

Utility of NucleoCounter for the chondrocyte count in the collagenase digest of human native cartilage

In cartilage tissue engineering, viable cell numbers should be correctly counted in the collagenase digest of the biopsied cartilage. However, this is a difficult task due to the presence of matrix debris, cell ghosts and their aggregates. To search for the correct cell counting method in this situation, we evaluated the utility of an automatic cell counting device, the NucleoCounter, and compared it with conventional staining using the LIVE/DEAD® kit. We first measured the cell numbers of a standard chondrocyte sample by the NucleoCounter, which showed a high accuracy (R² = 0.9999) and reproducibility (%CV: 2.00–8.66). We then calculated the cell numbers and viability in some collagenase digests of native cartilage using either the NucleoCounter or LIVE/DEAD® kit, revealing that the total cell numbers, viable ones and viability were highly correlated between them (R² = 0.9601, 0.9638 and 0.917, respectively). However, both the intrapersonal and interpersonal variabilities in the NucleoCounter was significantly decreased to about 1/20–1/5, compared to that of the LIVE/DEAD® kit. The NucleoCounter was regarded as a useful tool for simple, rapid, and highly reproducible cell counts, which may not only provide constant experimental data in a certain laboratory, but also contribute to the high reproducibility of the clinical results of cartilage tissue engineering among multiple institutions.