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.     

Identification of a novel alpha-amylase by expression of a newly cloned human amy3 cDNA in yeast

A novel amylase gene (amy3) that differs in nucleotide sequence from salivary amylase gene (amy1) and pancreatic amylase gene (amy2) has been described [Tomita et al., Gene 76 (1989) 11-18], but whether this gene can ever code for an active enzyme has not been shown. We prepared cDNA of this gene from an mRNA obtained from lung carcinoid tissue, and expressed it in Saccharomyces cerevisiae under the control of an acid phosphatase promoter. The product was secreted into culture media, and showed enzymatic activity, demonstrating that this novel alpha-amylase gene (amy3) can code for a functional isozyme. We purified this enzyme, and compared its biological properties with those of salivary and pancreatic human amylases similarly expressed in yeast. We observed that the novel amylase isozyme is more heat-sensitive than others, and that its substrate specificity is different from the other two isozymes.     

Phosphotyrosine-modified proteins are concentrated at the membranes of epithelial and endothelial cells during tissue development in chick embryos

We have used high affinity polyclonal antibodies specific for phosphotyrosine (PTyr) residues to examine the localization in various chick embryonic tissues in situ of PTyr-modified proteins by immunocytochemical methods. During the period from 9 to 21 d of development, most tissues exhibit elevated levels of PTyr-modified proteins as determined by immunoblotting experiments of tissue extracts with the anti-PTyr antibodies (Maher, P. A., and E. B. Pasquale. 1988. J. Cell Biol. 106:1747-1755). By immunofluorescence labeling of semithin frozen sections, the highest concentrations of PTyr immunolabeling in all of the embryonic tissues examined were localized to the membranes of the epithelial and endothelial cells with other cells showing no detectable labeling. These results were confirmed by immunoelectron microscopic labeling, which showed particularly high concentrations of PTyr-modified proteins close to the membranes at the apical junctions. The corresponding adult tissues showed no labeling. It is proposed that these results reflect the molecular basis for the functional plasticity of epithelial and endothelial cell junctions during embryonic development.     

Purification of a receptor for formaldehyde-treated serum albumin from rat liver

A membrane-associated receptor involved in a specific uptake of formaldehyde-treated serum albumin (f-Alb) was purified from rat livers by Triton X-100 solubilization of a 105,000 X g membrane preparation and affinity chromatography on an f-Alb-Sepharose column. The purified receptor exhibited Mr = 125,000, consisting of two noncovalently linked glycoprotein components with Mr = 53,000 and Mr = 30,000, respectively. Incubation of 125I-receptor with f-Alb, but not with native albumin, resulted in a marked shift of pI value from 5.9 to 5.1, reflecting the presence of a specific ligand-receptor interaction. The receptor incorporated into liposomes displayed a saturable binding to 125I-f-Alb and the binding was effectively replaced by the presence of unlabeled f-Alb, with binding parameters being similar to those obtained from 125I-f-Alb binding to the sinusoidal liver cell membrane (Horiuchi, S., Takata, K., and Morino, Y. (1985) J. Biol. Chem. 260, 475-481). Reaction of anti-f-Alb receptor antibody with extracts of sinusoidal cells resulted in a specific precipitation of two proteins whose molecular weights were identical to those for the purified receptor. The anti-receptor IgG fraction effectively blocked 125I-f-Alb binding to the sinusoidal cell membranes. These results indicate that the purified protein represents the membrane-associated receptor which is presumably involved in a specific uptake of this ligand from the circulation.     

Genetic studies of the ribosomal proteins in Escherichia. coli. II. Altered 30s ribosomal protein component specific to spectinomycin resistant mutants

Summary Spectinomycin resistant (spc ^r) mutants were obtained by treating the cells of E. coli K12, W3637 with nitrosoguanidine. The compositions of ribosomal proteins were analyzed for six out of eleven such spc ^r-mutants with chromatography on a carboxymethyl cellulose (CMC) column. The 30s ribosomal subunit from all of the spc ^r-mutants was found to contain the altered 30-4 protein component, while no difference was detected in 50s ribosomal proteins between spc ^r and spc ^s bacteria.Abbreviations used CMC carboxymethyl cellulose - str streptomycin - spc spectinomycin     

Effect of Ethanol on the Sodium and Potassium Conductances of the Squid Axon Membrane

The effects of ethanol on squid giant axons were studied by means of the sucrose-gap technique. The membrane action potential height is moderately reduced and the duration sometimes shortened by ethanol in sea water. Voltage clamp experiments showed that ethanol in sea water reduced the maximum membrane conductances for sodium (g'_Na) and potassium (g'_K). In experiments with multiple application of ethyl alcohol to the same spot of membrane, a reduction of g'_Na to 82 per cent and of g'_K to 80 per cent of their value in sea water was brought about by 3 per cent ethanol (by volume) while 6 per cent caused a decrease of g'_Na to 59 per cent and of g'_K to 69 per cent. Ethanol has no significant effect on the steady-state inactivation of g_Na (as a function of conditioning membrane potential) or on such kinetic parameters as τ_h or the time course of turning on gi g_Na and g_K. It is concluded that ethanol mainly reduces g_Na and g_K in the Hodgkin-Huxley terminology.     

Isolation and sequencing of a cDNA clone encoding the 85 kDa human lysosomal sialoglycoprotein (hLGP85) in human metastatic pancreas islet tumor cells.

A full length cDNA for a human lysosomal membrane sialoglycoprotein (hLGP85) was isolated as a probe of the cDNA of rat LGP85 (rLGP85) from the cDNA library prepared from total mRNA of QGP-1NL cells, a human pancreatic islet tumor cell with a high metastatic activity. The deduced amino acid sequence shows that hLGP85 consists of 478 amino acid residues (MW. 54,289). The protein has 10 putative N-glycosylation sites and 2 hydrophobic regions at the NH2- and near the COOH-termini, respectively. Thus, both domains probably constitute putative transmembrane domains. It exhibits 86% and 79% sequence similarities in amino acids and nucleic acids to rat lysosomal membrane sialoglycoprotein (rLGP85), respectively. The protein contained the short cytoplasmic tail at the COOH-terminus which does not form the glycine-tyrosine sequence (GY motif), the so-called lysosomal targetting signal.     

Effects of abdominal pressure on venous return: abdominal vascular zone conditions

The effects of changes in abdominal pressure (Pab) on inferior vena cava (IVC) venous return were analyzed using a model of the IVC circulation based on a concept of abdominal vascular zone conditions analogous to pulmonary vascular zone conditions. We hypothesized that an increase in Pab would increase IVC venous return when the IVC pressure at the level of the diaphragm (Pivc) exceeds the sum of Pab and the critical closing transmural pressure (Pc), i.e., zone 3 conditions, but reduce IVC venous return when Pivc is below the sum of Pab and Pc, i.e., zone 2 conditions. The validity of the model was tested in 12 canine experiments with an open-chest IVC bypass. An increase in Pab produced by phrenic stimulation increased the IVC venous return when Pivc-Pab was positive but decreased the IVC venous return when Pivc - Pab was negative. The value of Pivc - Pab that separated net increases from decreases in venous return was 1.00 +/- 0.72 (SE) mmHg (n = 6). An increase in Pivc did not influence the femoral venous pressure when Pivc was lower than the sum of Pab and a constant, 0.96 +/- 0.70 mmHg (n = 6), consistent with presence of a waterfall. These results agreed closely with the predictions of the model and its computer simulation. The abdominal venous compartment appears to function with changes in Pab either as a capacitor in zone 3 conditions or as a collapsible Starling resistor with little wall tone in zone 2 conditions.     

Cyclization reaction catalyzed by branching enzyme.

The action of branching enzyme (EC 2.4.l.l8) from Bacillus stearothermophilus on amylose was analyzed. The enzyme reduced the molecular size of amylose without increasing the reducing power. This result could not be explained by the normal branching reaction model. When the product was treated with glucoamylase (an exo++-type amylase), a resistant component remained. The glucoamylase-resistant component was easily digested by an endo-type alpha-amylase or by isoamylase plus glucoamylase. These results suggested that the glucoamylase-resistant component was a cyclic glucan composed of alpha-1,4- and alpha-l,6-glucosidic linkages. In other words, it was suggested that branching enzyme catalyzed cyclization of the alpha-l,4-glucan chain of the amylose molecule to form an alpha-l,6-glucosidic linkage, thereby forming two smaller molecules. Mass spectrometry also supported the cyclic nature of the product.     

Isolation of Serratia marcescens mutants which could overproduce and excrete Escherichia coli alkaline phosphatase and beta-lactamase

Serratia marcescens mutants, which excrete Escherichia coli alkaline phosphatase (APase) encoded by the plasmid-bearing phoA gene, were isolated after mutagenesis by N-methyl--nitro-N-nitrosoguanidine. These mutants produced two to four times as much APase as did the parent strain under a phosphate-limiting condition, and more than 70% of the enzyme was released into the culture medium. In addition, overproduction and excretion of beta-lactamase was observed in these mutants.