Gap Junctions Mediate Glucose Transport Between GLUT1-Positive and -Negative Cells in the Spiral Limbus of the Rat Cochlea

To elucidate the role of the spiral limbus in glucose transport in the cochlea, we analyzed the expression and localization of GLUT1, connexin26, connexin30, and occludin in the spiral limbus of the rat cochlea. GLUT1 and occludin were detected in blood vessels. GLUT1, connexin26, connexin30, and occludin were also expressed in fibrocytes just basal to the supralimbal lining cells. Connexin26 and connexin30 were present among not only these GLUT1-positive fibrocytes but also GLUT1-negative fibrocytes. In vivo glucose imaging using 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-6-deoxyglucose (6-NBDG, MW 342) together with Evans Blue Albumin (EBA, MW 68,000) showed that 6-NBDG was rapidly distributed throughout the spiral limbus, whereas EBA was localized only in the vessels. Moreover, the gap junctional uncoupler heptanol inhibited the distribution of 6-NBDG. These findings suggest that gap junctions play an important role in glucose transport in the spiral limbus, i.e., that gap junctions mediate glucose transport from GLUT1-positive fibrocytes to GLUT1-negative fibrocytes in the spiral limbus.     

Purification of a fibrolast-inhibitory factor from Actinobacillus actinomycetemcomitans Y4

Abstract A factor showing inhibitory activity against human gingival fibrolasts was extracted from the cytosol fraction of Actinobacillus actinomycetemcomitans Y4. The activity markedly inhibited the proliferation of human gingival fibrolasts, but had no effect on cell viability or gross morphology. No such activity was found in cytosol fractions from either Porphyromonas gingivalis 381 or Escherichia coli HB101. The extract from A. actinomycetemcomitans Y4 was then purified by anion-exchange chromatography, hydroxyapatite chromatography and gel-filtration chromatography to give a single band on SDS-PAGE with an apparent molecular mass of 65 kDa. The purification ratio was 183-fold with a recovery rate of 5% compared with the crude extract (starting material) when the activity was assessed by direct cell counts.     

Effect of molar ratio of fluorescent dye to IgG on the detection of lymphocyte surface immunoglobulins by immunofluorescence

Labeled antibodies with different F/P molar ratios of FITC to protein (F/P molar ratio) were used for the detection of surface immunoglobulin (S-Ig) of human and mouse lymphocytes by membrane immunofluorescence, and the following results were obtained. 1. The percentage of S-Ig bearing cells increased markedly when labeled anti-human H- or L-chains antibodies were used with higher F/P molar ratios. The investigation of frozen kidney sections of mice injected with human immunoglobulin revealed that such an increase of the positive ratio in S-Ig was caused by increased non-specific adsorption of the fraction of labeled antibody with a high F/P molar ratio. 2. This non-specific adsorption phenomenon was observed at various intensities in materials from different species; materials from mcie showed less non-specific adsorption than those from humans. 3. It was possible to exclude reactivity with an Fc receptor using the top one third of the supernatant of labeled antibody centrifuged at 150,000 for 30 min.     

Characterization of a polysaccharide component of lipopolysaccharide from Pseudomonas aeruginosa IID 1008 (ATCC 27584) as D-rhamnan

Structural studies were carried out on a rhamnose-rich polysaccharide isolated from the O-polysaccharide fraction of lipopolysaccharide in Pseudomonas aeruginosa IID 1008 (ATCC 27584) after destruction of the major O-specific chain by alkaline treatment. The isolated polysaccharide contained rhamnose, 3-O-methyl-6-deoxyhexose, glucose, xylose, alanine, galactosamine and phosphorus in a molar ratio of 67:6.9:4.3:2.1:1.1:1.0:4.1. Data from analysis involving Smith degradation, methylation, 1H-NMR spectroscopy and optical rotation measurement showed that the polysaccharide was built up of three moieties, a rhamnan chain composed of about 70 D-rhamnose residues, the core chain and an oligosaccharide chain comprising 3-O-methyl-6-deoxyhexose, xylose, rhamnose and probably glucose. The repeating unit of the rhamnan chain was indicated to have the following structure:----3)D-Rha(alpha 1----3)D-Rha(alpha 1----2)D-Rha(alpha 1----. This structure is identical with that proposed previously for the repeating unit of the side chain of lipopolysaccharide from plant pathogenic bacteria Pseudomonas syringae pv. morsprunorum C28 [Smith, A.R.W., Zamze, S.E., Munro, S.M., Carter, K. J. and Hignett, R.C. (1985) Eur. J. Biochem. 149, 73-78].     

Evolutionary trends of the hairy-faceSaguinus in terms of the dental and cranial morphology

Shape variations in the dentition and the cranium were analyzed for sevenSaguinus forms of the hairy-face tamarin by applying the factor analysis method. The results obtained for the dental and cranial measurements were almost consistent with each other. The magnitude of the difference in shape factors between theS. nigricollis group and theS. midas group is appreciably larger than that between the former group and theS. mystax group. If the ancestral geographic centre of origin is postulated as being within the region which is inhabited by the livingS. nigricollis group, the morphological distances between any pairs of groups correlate well with the geographic distances between them. Concerning the dental and cranial morphologies, the physical changes in the three species group probably took place in two directions; that is, from theS. nigricollis group to theS. mystax group, and from theS. nigricollis group to theS. midas group. The forms belonging to each species group are more closely related to each other, with the exception ofS. imperator in theS. mystax group. The uniqueness ofS. imperator was clearly demonstrated by factor analysis and distance analysis. In theS. mystax group, although still hypothetical,S. imperator may have been related only through the basic ancestral stock toS. labiatus andS. mystax.     

Comparison of growth properties of carrot hairy root in various bioreactors

Summary Growth properties of carrot hairy root cells in various bioreactors were investigated. A turbine-blade reactor and an immobilized rotating drum reactor were found to be advantageous for the hairy root culture because of a high oxygen transfer coefficient (k in L a). After 30 days of culture, 10 g/l of dry hairy root cells were obtained in both bioreactors and maximum growth rates (V _m ) were found to be 0.63 and 0.61 g/l per day for the turbine-blade reactor and immobilized rotating drum reactor, respectively. Specific growth rates () at various cultivation times were observed to be linearly proportional to X/k _l a for both bioreactor configurations where X is the cell concentration. The estimated specific oxygen uptake rate of 0.34 mmol O₂/g dry cells per hour compares fairly well with an experimental value of 0.3.     

Expression of pGKL killer 28K subunit in Saccharomyces cerevisiae: identification of 28K subunit as a killer protein.

Saccharomyces cerevisiae and other yeast cells harboring the linear double stranded (ds) DNA plasmids pGKL1 and pGKL2 secrete a killer toxin consisting of 97K, 31K and 28K subunits into the culture medium (EMBO J. 5, 1995-2002 (1986), Nucleic Acids Res., 15, 1031-1046 (1987]. The 28K subunit of the killer toxin was successfully expressed in S. cerevisiae when it was cloned on a circular plasmid with its putative promoter region replaced with that of S. cerevisiae chromosomal genes. The expression of the 28K subunit of the killer toxin in killer-sensitive cells resulted in the death of the host cells. This killing activity by the 28K subunit was prevented by the expression of the killer immunity, indicating that the killing activity of the killer toxin complex was carried out by the 28K subunit. Although the 28K subunit was synthesized as a intact precursor protein with its own signal sequence, it was not secreted into the culture medium but remained in the host cells. This indicated that 28K subunit killed host cells from inside of the cells rather than from outside. We further suggested that 28K killer subunit without 97K and 31K subunits did not kill the killer-sensitive cells from outside.     

Potassium channels from NG108-15 neuroblastoma-glioma hybrid cells. Primary structure and functional expression from cDNAs

The complete amino acid sequences of two potassium channel proteins from NG108-15 neuroblastoma-glioma hybrid cells have been deduced by cloning and sequencing the cDNAs. One of these proteins (NGK2) is structurally more closely related to the Drosophila Shaw gene product than to the Shaker and Shab gene products, whereas the other (NGK1) is identical with a rat brain potassium channel protein (BK2) which is more closely related to the Drosophila Shaker gene product. mRNAs derived from both the cloned cDNAs, when injected into Xenopus oocytes, direct the formation of functional potassium channels with properties of delayed rectifiers.     

Cloning of the Membrane-Bound Aldehyde Dehydrogenase Gene of Acetobacter polyoxogenes and Improvement of Acetic Acid Production by Use of the Cloned Gene

A genomic clone bank of Acetobacter polyoxogenes NBI1028 constructed in Escherichia coli by use of the expression vector pUC18 was screened with antibody raised against membrane-bound aldehyde dehydrogenase (ALDH; 75 kilodaltons [kDa]) from A. polyoxogenes NBI1028. A clone that synthesized a 41-kDa protein cross-reactive with anti-ALDH antibody was isolated. For cloning of the full-length ALDH structural gene, a cosmid gene bank was screened by Southern blot hybridization with the cloned DNA as a probe, and subcloning from the positive cosmid clone was performed with shuttle vector pMV24. Plasmid pAL25, containing the full-length ALDH structural gene, was isolated and expressed in both E. coli and Acetobacter aceti to produce a fused protein (78 kDa) with a short NH₂-terminal β-galactosidase peptide. pAL25 conferred ALDH production on a mutant of A. aceti lacking the enzyme activity. Transformation of A. aceti subsp. xylinum NBI2099 with pAL25 caused 2- and 1.4-fold increases in the production rate and in the maximum concentration of acetic acid in submerged fermentation, respectively.     

Expression of active alpha-3 subunit of rat brain Na+,K+-ATPase from the messenger RNA injected into Xenopus oocytes

Cloned cDNA encoding the so-far uncharacterized alpha-3 subunit of rat brain Na+,K+-ATPase (Hara et al. (1987) J. Biochem. 102, 43-58, Shull et al. (1986) Biochemistry 25, 8125-8132) was incorporated into a vector carrying the SP6 promoter. The mRNA produced in vitro was injected into Xenopus oocytes with the mRNA encoding the Na+,K+-ATPase beta subunit of Torpedo electroplax. Increased Na+,K+-ATPase activity in the oocyte membrane was observed. This newly expressed activity was inhibited by ouabain (Ki = 1.5 x 10(-7) M), suggesting that the alpha-3 subunit of rat brain Na+,K+-ATPase is a highly ouabain-sensitive catalytic subunit.