A DBL-homologous region of the yeast CLS4/CDC24 gene product is important for Ca(2+)-modulated bud assembly.

The CLS4/CDC24 is essential for the budding process of the yeast Saccharomyces cerevisiae. Disruption of the CLS4/CDC24 gene is lethal, and expression of the CLS4 product under the control of the GAL1 promoter is sufficient for cellular growth. The CLS4 product is detected in yeast cell lysate with an apparent molecular mass of 93 kD (854 amino acid residues) and shows homology with the human DBL oncogene product. Temperature-sensitive cdc24-1 mutation is located in the N-terminal portion of the protein whereas Ca(2+)-sensitive cls4-1 mutation is present after the DBL-homologous region (amino acid residues 281-518) near the putative Ca(2+)-binding site. Mutations within the DBL-homologous region are responsible for the Ca(2+)-sensitive phenotype. Thus the CLS4 gene product seems to have several functional domains within the molecule essential for bud assembly.     

Elastic behavior of zymogen granule membranes in response to changes in pH and pCa.

In the process of secretion, the membrane of secretory granules is expected to change its elastic behavior. Elastic modulus of the membrane of zymogen granules, prepared from the rat pancreas acinar cell, was measured by an osmotic swelling method. The elastic modulus of the granule membrane at pCa 8 reduced from the maximal value of 230 dyn/cm at pH 6.0 to almost zero at pH 7.5. In a cytosol of an acinar cell, calcium ions play an important role as a second messenger in secretion. The elastic modulus of the granule membrane reduced in a sigmoidal fashion at pCa between 7.0 and 6.0. This range of pCa corresponds to a physiological rise of free Ca2+ concentrations in the cell cytosol when stimulated by external secretagogues. Reduction of the elastic modulus indicates that the state of the granule membrane switches to a more flexible one in which the granule is easy to appose to the cell plasma membrane and then swell as a final step of exocytosis.     

A new electrophoretic method for the separation of disaccharides obtained by digestion of chondroitin sulfates and dermatan sulfate with chondroitinases

A new rapid and simple method has been developed for the separation of disaccharides obtained by chondroitinase digestion of chondroitin sulfates and dermatan sulfate using electrophoresis on cellulose acetate plates (Titan III cellulose acetate plates). Three disaccharides are completely separated by electrophoresis in barium acetate or calcium acetate in a short time, and less than 50 μg of glycosaminoglycan samples can be analyzed within 2 h.     

The RNA-binding protein Puf5 and the HMGB protein Ixr1 contribute to cell cycle progression through the regulation of cell cycle-specific expression of CLB1 in Saccharomyces cerevisiae

Puf5, a Puf-family RNA-binding protein, binds to 3´ untranslated region of target mRNAs and negatively regulates their expression in Saccharomyces cerevisiae. The puf5Δ mutant shows pleiotropic phenotypes including a weakened cell wall, a temperature-sensitive growth, and a shorter lifespan. To further analyze a role of Puf5 in cell growth, we searched for a multicopy suppressor of the temperature-sensitive growth of the puf5Δ mutant in this study. We found that overexpression of CLB2 encoding B-type cyclin suppressed the temperature-sensitive growth of the puf5Δ mutant. The puf5Δ clb2Δ double mutant displayed a severe growth defect, suggesting that Puf5 positively regulates the expression of a redundant factor with Clb2 in cell cycle progression. We found that expression of CLB1 encoding a redundant B-type cyclin was decreased in the puf5Δ mutant, and that this decrease of the CLB1 expression contributed to the growth defect of the puf5Δ clb2Δ double mutant. Since Puf5 is a negative regulator of the gene expression, we hypothesized that Puf5 negatively regulates the expression of a factor that represses CLB1 expression. We found such a repressor, Ixr1, which is an HMGB (High Mobility Group box B) protein. Deletion of IXR1 restored the decreased expression of CLB1 caused by the puf5Δ mutation and suppressed the growth defect of the puf5Δ clb2Δ double mutant. The expression of IXR1 was negatively regulated by Puf5 in an IXR1 3´ UTR-dependent manner. Our results suggest that IXR1 mRNA is a physiologically important target of Puf5, and that Puf5 and Ixr1 contribute to the cell cycle progression through the regulation of the cell cycle-specific expression of CLB1.     

p-aminohippuric acid transport at renal apical membrane mediated by human inorganic phosphate transporter NPT1

Organic anions are secreted into urine via organic anion transporters across the renal basolateral and apical membranes. However, no apical membrane transporter for organic anions such as p-aminohippuric acid (PAH) has yet been identified. In the present study, we showed that human NPT1, which is present in renal apical membrane, mediates the transport of PAH. The K(m) value for PAH uptake was 2.66 mM and the uptake was chloride ion sensitive. These results are compatible with those reported for the classical organic anion transport system at the renal apical membrane. PAH transport was inhibited by various anionic compounds. Human NPT1 also accepted uric acid, benzylpenicillin, faropenem, and estradiol-17beta-glucuronide as substrates. Considering its chloride ion sensitivity, Npt1 is expected to function for secretion of PAH from renal proximal tubular cells. This is the first molecular demonstration of an organic anion transport function for PAH at the renal apical membrane.     

Purification and characterization of thermostable H<Subscript>2</Subscript>O<Subscript>2</Subscript>-forming NADH oxidase from 2-phenylethanol-assimilating <Emphasis Type="Italic">Brevibacterium</Emphasis> sp. KU1309

A cytoplasmic NADH oxidase (NOX) was purified from a soil bacteria, Brevibacterium sp. KU1309, which is able to grow in the medium containing 2-phenylethanol as the sole source of carbon under an aerobic condition. The enzyme catalyzed the oxidation of NADH to NAD+ involving two-electron reduction of O2 to H2O2. The molecular weight of the enzyme was estimated to be 102 kDa by gel filtration and 57 kDa by SDS-PAGE, which indicates that the NOX was a homodimer consisting of a single subunit. The enzyme was stable up to 70 degrees C at a broad range of pH from 7 to 11. The enzyme activity increased about ten-fold with the addition of ammonium salt, while it was inhibited by Zn2+ (39%), Cu2+ (41%), Hg2+ (72%) and Ag+ (37%). The enzyme acts on NADH, but not on NADPH. The regeneration of NAD+ utilizing this enzyme made selective oxidation of mandelic acid or L: -phenylalanine possible. This thermostable enzyme is expected to be applicable as a useful biocatalyst for NAD+ recycling.     

Calcium influx in a single rat basophilic leukemia cell as revealed with a digital imaging fluorescence microscope

Using a digital imaging fluorescence microscope, we have detected a rapid transient increase in the free cytosolic calcium concentration in a single rat basophilic leukemia cell (RBL-2H3) after antigen stimulation. Calcium ions were transported very rapidly (within 1 s) after a lag time (about 10 s at 37 degrees C) from the external environment into the cytoplasm. On the basis of the present experimental results we conclude that the gradual changes in the overall fluorescence intensity observed for a cell suspension are due to the distribution of different lag times shown by different cells as to the calcium influx through membrane calcium channels.