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     

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.     

Efficient release of overproduced gene products from Escherichia coli BL21(DE3) by lytic infection with newly isolated bacteriophages

Overproduced proteins from Escherichia coli BL21(DE3) were efficiently released with virulent bacteriophages. Leviviridae-like bacteriophages were isolated from soil and used to lyse BL21(DE3) cells transformed with beta-glucosidase, chitinase, or chitosanase genes. This method caused lysis of bacterial cells similar to that by conventional sonication and enabled us to effectively recover and purify the enzymes.     

Rapid measurement of phytate in raw soymilk by mid-infrared spectroscopy

The phytate content in soymilk is known to affect tofu curdling. A rapid measurement of phytate from a water extract of soybean (raw soymilk) in an early stage of tofu processing was investigated using mid-infrared spectroscopy (IR) with an ATR accessory. IR absorption of phytate was observed from 1200 cm-1 to 900 cm-1, and saccharide and protein in the extract also had IR absorption in the same region. In order to separate phytate from other components, the phytate was precipitated completely by the addition of calcium under alkaline condition (pH 11.5). The precipitate was dissolved in citrate buffer (pH 6.0) and then used for IR measurement. The absorbance at 1070 cm-1 correlated well with the phytate content of the soymilk. The measurement of phytate in raw soymilk can be done rapidly by FT-IR measurement with an ATR accessory and gives reproducible values, which can be used for the measurement of phytate content in various soybeans for tofu making.     

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.     

Expression of novel organic cation/carnitine transporter (OCTN2) in the mouse pancreas

Among the organic cation transporters, OCTN2 is identified as the most important carnitine transporter owing to the ability to transport carnitine. Although the OCTN2 is previously found in various tissues, there have been no reports showing the OCTN2 in the pancreas. In this study, we examined the expression and localization of OCTN2 in the mouse pancreas by the aid of an in situ hybridization technique and immunohistochemistry with anti-OCTN2 antibody. As a result, the OCTN2 expression was found in the A-cells for the first time. OCTN2 was not expressed in B-cells, notwithstanding that the metabolism of long-chain fatty acids, which are transported into the mitochondria with the help of carnitine, was expected for fatty acid-stimulated insulin secretion. Thus, this study suggests the possibility of carnitine uptake in the pancreatic A-cells through OCTN2 and implies the presence of carnitine transporter(s) other than OCTN2 in the B-cell.