Cloning and characterization of seven cDNAs for hyperosmolarity-responsive (HOR) genes of Saccharomyces cerevisiae

Yeast cells can respond and adapt to osmotic stress. In our attempt to clarify the molecular mechanisms of cellular responses to osmotic stress, we cloned seven cDNAs for hyperosmolarity-responsive (HOR) genes from Saccharomyces cerevisiae by a differential screening method. Structural analysis of the clones revealed that those designated HOR1, HORS, HOR4, HOR5 and HOR6 encoded glycerol-3-phosphate dehydrogenase (Gpd1p), glucokinase (Glklp), hexose transporter (Hxtlp), heat-shock protein 12 (Hsp12p) and Na⁺, K⁺, Li⁺-ATPase (Enalp), respectively. HOR2 and HOR7 corresponded to novel genes. Gpdlp is a key enzyme in the synthesis of glycerol, which is a major osmoprotectant in S. cerevisiae. Cloning of HOR1/GPD1 as a HOR gene indicates that the accumulation of glycerol in yeast cells under hyperosmotic stress is, at least in part, caused by an increase in the level of GPDH protein. We performed a series of Northern blot analyses using HOR cDNAs as probes and RNAs prepared from cells grown under various conditions and from various mutant cells. The results suggested that all the HOR genes are regulated by common signal transduction pathways. However, the fact that they exhibited certain distinct responses indicated that they might also be regulated by specific pathways in addition to the common pathways. Ca²⁺ seemed to be involved in the signaling systems. In addition, Hog1p, one of the MAP kinases in yeast, appeared to be involved in the regulation of expression of HOR genes, although its function seemed to be insufficient for the overall regulation of expression of these genes.     

Conversion of Ulva fronds to a hatchery diet for Artemia nauplii utilizing the degrading and attaching abilities of Pseudoalteromonas espejiana

An algal suspension containing protoplasmic detritus [termed, single cell detritus (SCD)] was prepared from freeze-dried fronds of Ulva and its dietary value to Artemia nauplii was tested after size fractionation. The dissolved fraction (<0.22 μm) ofthe Ulva suspension contained ca. 48% of theoriginal protein in the Ulva, but had no dietaryvalue to Artemia, which is a suspension feeder. In contrast, the fraction passing through a 100-μm meshand containing SCD of 2–14 μm in diameter, contributedto the survival of Artemia. The fraction remaining on the 100 μm mesh was further incubated with and without the bacterium Pseudoalteromonas espejiana strain AR06 FERM BP-5024. The bacterium degraded Ulva forming new SCD over 10⁶ mL ^-1 level as rapidly as by 16 h of incubation. The dietary value of Ulva for Artemia growth was elevated over four times by the incubation. The protein content of the SCD was approximately doubled by the attaching of bacteria, suggesting the enhanced Artemia growth is attributable to the combined effect of the SCD and the bacteria. Development of a hatchery diet from Ulva , a resource with a low commercial value, is suggested utilizing the degrading and attaching ability of P. espejiana. This revised version was published online in August 2006 with corrections to the Cover Date.     

The concept of biological control methods in aquaculture

Microbial techniques of biocontrol using the interaction ofmicroorganisms to repress the growth of deleterious bacteria andviruses were developed. The bacterial strain used in this work alsoimproved the growth of fishes and crustaceans. Using the conceptandprocedures of the biocontrol method described here, the aquacultureproduction became stable and evenincreased.     

Possible role of macrophage-derived soluble mediators in the pathogenesis of encephalomyocarditis virus-induced diabetes in mice.

Pancreatic islets from DBA/2 mice infected with the D variant of encephalomyocarditis (EMC-D) virus revealed lymphocytic infiltration with moderate to severe destruction of pancreatic beta cells. Our previous studies showed that the major population of infiltrating cells at the early stages of infection is macrophages. The inactivation of macrophages prior to viral infection resulted in the prevention of diabetes, whereas activation of macrophages prior to viral infection resulted in the enhancement of beta-cell destruction. This investigation was initiated to determine whether macrophage-produced soluble mediators play a role in the destruction of pancreatic beta cells in mice infected with a low dose of EMC-D virus. When we examined the expression of the soluble mediators interleukin-1 beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and inducible nitric oxide synthase (iNOS) in the pancreatic islets, we found that these mediators were clearly expressed at an early stage of insulitis and that this expression was evident until the development of diabetes. We confirmed the expression of these mediators by in situ hybridization with digoxigenin-labelled RNA probes or immunohistochemistry in the pancreatic islets. Mice treated with antibody against IL-1beta or TNF-alpha or with the iNOS inhibitor aminoguanidine exhibited a significant decrease in the incidence of diabetes. Mice treated with a combination of anti-IL-1beta antibody, anti-TNF-alpha antibody, and aminoguanidine exhibited a greater decrease in the incidence of disease than did mice treated with one of the antibodies or aminoguanidine. On the basis of these observations, we conclude that macrophage-produced soluble mediators play an important role in the destruction of pancreatic beta cells, resulting in the development of diabetes in mice infected with a low dose of EMC-D virus.     

Does endothelin-1 participate in the exercise-induced changes of blood flow distribution of muscles in humans?

Maeda, Seiji, Takashi Miyauchi, Michiko Sakane, MakotoSaito, Shinichi Maki, Katsutoshi Goto, and Mitsuo Matsuda. Does endothelin-1 participate in the exercise-induced changes of blood flowdistribution of muscles in humans? J. Appl.Physiol. 82(4): 1107-1111, 1997.Endothelin-1(ET-1) is an endothelium-derived potent vasoconstrictor peptide thatpotentiates contractions to norepinephrine in human vessels. Wepreviously reported that the circulating plasma concentration of ET-1is significantly increased after exercise (S. Maeda, T. Miyauchi, K. Goto, and M. Matsuda. J. Appl.Physiol. 77: 1399-1402, 1994). Tostudy the roles of ET-1 during and after exercise, we investigatedwhether endurance exercise affects the production of ET-1 in thecirculation of working muscles and nonworking muscles. Male athletesperformed one-leg cycle ergometer exercise of 30-min duration atintensity of 110% of their individual ventilatory threshold. Plasmaconcentrations of ET-1 in both sides of femoral veins (veins in theworking leg and nonworking leg) and in the femoral artery (artery inthe nonworking leg) were measured before and afterexercise. The plasma ET-1 concentration in the femoralvein in the nonworking leg was significantly increased after exercise,whereas that in femoral vein in the working leg was not changed. Thearteriovenous difference in ET-1 concentration was significantlyincreased after exercise in the circulation of the nonworking leg butnot of the working leg, which suggests that the production of ET-1 wasincreased in the circulation of the nonworking leg by exercise. Thepresent study also demonstrated that the plasma norepinephrineconcentrations were elevated by exercise in the femoral veins of boththe working and nonworking legs, suggesting that the sympathetic nerveactivity was augmented in both legs during exercise. Therefore, thepresent study demonstrates the possibility that the increase inproduction of ET-1 in nonworking muscles may cause vasoconstriction andhence decrease blood flow in nonworking muscles through its directvasoconstrictive action or through an indirect effect of ET-1 toenhance vasoconstrictions to norepinephrine and that these responsesmay be helpful in increasing blood flow in workingmuscles. We propose that endogenous ET-1 contributes tothe exercise-induced redistribution of blood flow in muscles.

     

Multiple genes encoding 2,3-dihydroxybiphenyl 1,2-dioxygenase in the gram-positive polychlorinated biphenyl-degrading bacterium Rhodococcus erythropolis TA421, isolated from a termite ecosystem.

Rhodococcus erythropolis TA421 was isolated from a termite ecosystem and is able to degrade a wide range of polychlorinated biphenyl (PCB) congeners. Genetic and biochemical analyses of the PCB catabolic pathway of this organism revealed that there are four different bphC genes (bphC1, bphC2, bphC3, and bphC4) which encode 2,3-dihydroxybiphenyl dioxygenases. As determined by Southern hybridization, none of the bphC genes exhibits homology to any other bphC gene. bphC1, bphC2, and bphC4 encode enzymes that have narrow substrate specificities and cleave the first aromatic ring in the meta position. In contrast, bphC3 encodes a meta cleavage dioxygenase with broad substrate specificity. Asturias et al. have shown that the closely related organism Rhodococcus globerulus P6 contains three different bphC genes (bphC1, bphC2, and bpHC3) which encode meta cleavage dioxygenases. The data suggest that there is a diverse family of bphC genes which encode PCB meta cleavage dioxygenases in members of the genus Rhodococcus.     

Separation of four class II molecules from DR2 and DRw6 homozygous B lymphoid cell lines

Four human class 11 molecules, one FA, one DC1, and two DR-like molecules, were isolated from DR2 and DRw6 homozygous cell lines by means of a variety of monoclonal antibodies and were compared with each other by two-dimensional (2-D) gel electrophoresis. Anti-DR2 or anti-DR3 + 5 + w6 sera immunoprecipitated two distinct light chains (L1 and L2) and one heavy chain (H1) from a DR2 or DRw6 homozygous cell line, respectively. One or both of these two class II molecules were also immunoprecipitated by DR-specific monoclonal antibodies and were considered to constitute a DR family of molecules. Three DC1-specific monoclonal antibodies, SDR4.1, Tu22, and PLM5, immunoprecipitated a set of heavy (H2) and light (L3) chains distinct from those of two DR-like molecules. The heavy chains of the DC1 antigens from DR2 and DRw6 cell lines were indistinguishable, whereas the light chains were structurally distinct from each other. A fourth molecule, FA, was identified by a novel monoclonal antibody and was also detected by two additional antibodies, Tu39 and SG171, that blocked the SB-specific T-cell proliferative response. The FA light chain (L4) was distinct from those of the former three antigens on both cell lines, whereas the FA heavy chain was indistinguishable from the DC1 heavy chain (H2) in this 2-D gel analysis. Thus, four light chains and two heavy chains were isolated from both DR2 and DRw6 homozygous cell lines. A possible gene-antigen organization of the DC-like antigens was also discussed.     

A simplified immunofluorescence technique for antibody to varicella-zoster membrane antigen (FAMA)

Indirect fluorescent antibody to varicella-zoster membrane antigens (FAMA) was measured by a new technique. The procedure gives rapid, sensitive and accurate results and is suitable for use in diagnosis or screening of susceptibility to varicella-zoster virus (VZV) infection. The test procedure was simplified by using Terasaki tissue culture plates for the reaction and for direct observation by fluorescence microscopy. Preparations of VZV-infected Vero cells stored in liquid nitrogen could be used as antigen in this FAMA-test.     

Cloning and nucleotide sequences of the linear DNA killer plasmids from yeast.

The linear DNA killer plasmids (pGKL1 and pGKL2) isolated from a Kluyveromyces lactis killer strain are also maintained and expressed its killer character in Saccharomyces cerevisiae. After these killer plasmid DNAs isolated from S. cerevisiae were treated with alkali, four terminal fragments from each plasmid DNAs were cloned separately. Using these and other cloned DNA fragments, the terminal nucleotide sequences of pGKL2 and the complete nucleotide sequence of pGKL1 were determined. The inverted terminal repetitions of 202 bp and 182 bp were found in pGKL1 and pGKL2, respectively. The pGKL1 sequence showed an extremely high A + T content of 73.2% and it contained five large open reading frames. The largest of these open reading frame was suggested to code for a membrane-bound precursor of glycoprotein subunit of the killer toxin.     

Behavior of vesicular stomatitis virus glycoprotein in mouse LM cells with modified membrane-phospholipids

LM cells in which the membrane phospholipids had been modified with choline analogues were infected with vesicular stomatitis virus. The choline analogues tested were choline, N,N'-dimethylethanolamine, N-monomethylethanolamine and ethanolamine. These modifications per se did not affect the syntheses of individual viral proteins. The viral glycoprotein was detected in the plasma membranes of all the modified cells by pronase digestion in pulse-chase experiments, but the amount of glycoprotein susceptible to proteolysis varied, decreasing in these modified cells in the following order: N,N'-dimethylethanolamine- greater than choline- greater than N-monomethylethanolamine- greater than ethanolamine-treated cells. After a 4-h chase, glycoprotein was mainly distributed in the plasma membranes of cells modified with N,N'-dimethylethanolamine, whereas it was found in both the microsomes and plasma membranes of cells modified with other analogues. Fairly large amounts of glycoprotein were also found in the soluble fraction of ethanolamine-treated cells, but not in that of choline- or N,N'-dimethylethanolamine-treated cells. More precise experiments on the behaviour of glycoprotein with a short period of chase strongly suggested that migration of glycoprotein from the microsomes to the plasma membranes was fastest in cells modified with N,N'-dimethylethanolamine and slowest in cells modified with ethanolamine. Membrane lipid modifications also resulted in release of different numbers of progeny virions from the cells, release of virions from the cells decreasing in the following order: N,N'-dimethylethanolamine- greater than choline- greather N-monomethylethanolamine- greater than ethanolamine-treated cells. These results indicate that modification of membrane phospholipids influences not only the insertion of glycoprotein into the microsomes and its migration to the plasma membranes, but also the production of progeny virions.