Determination of monosaccharides and sugar alcohols in tissues from diabetic rats by high-performance liquid chromatography with pulsed amperometric detection.

A sensitive and simple high-performance liquid chromatographic method has been developed to determine the concentration of monosaccharides and sugar alcohols in animal tissues. Five neutral monosaccharides (D-glucose, D-galactose, D-mannose, D-fructose, and D-ribose) and three neutral sugar alcohols (myo-inositol, glycerol, and D-sorbitol) predominate in the renal cortices and sciatic nerves of rats. These monosaccharides and sugar alcohols were extracted with distilled water, purified by deproteinization with ethanol, a Sep-Pak C18 cartridge, and columns of Dowex 50W-X8 and Amberlite CG-400, then separated on Ca2+ and Pb2+ cation-exchange columns, eluted with deionized distilled water at 80 degrees C, and detected using integrated pulsed amperometry. About 10 pmol of each sugar was detectable with a signal-to-noise ratio of 10:1. D-Glucose, D-fructose, D-sorbitol, and D-mannose were higher in both the renal and sciatic tissues of diabetic rats than in those of normal animals. D-Ribose and glycerol were higher in the renal cortex of diabetic animals.     

Termination of endothelin signaling: Role of nitric oxide

Cellular mechanisms responsible for the termination of ET-1 signal are poorly understood. In order to examine the hypothesis that nitric oxide serves as a physiological brake of ET- 1 signaling, Chinese hamster ovary (CHO) cells stably transfected with the ET_A receptor cDNA (CHO-ET) were studied. CHO-ET responded to ET-1 with robust [Ca²⁺], transients and developed a long-lasting homologous desensitization. Donors of nitric oxide (NO), 3-morpholino-sydnonimine HCl(SIN-1), or sodium nitroprusside (SNP) reduced the amplitude of these responses, accelerated the rate of [Ca²⁺], recovery, and counteracted the development of homologous desensitization by a cyclic GMP-independent mechanism, suggesting an alternative mode for NO modulation of ET-1 responses. Stimulation of CHO-ET cells with mastoparan, a wasp venom acting directly on G proteins (bypassing receptor activation), was inhibited by NO, revealing a postreceptoral target for NO-induced modulation of [Ca²⁺] mobilization. Using a lys⁹-biotinylated ET-1 (ET-1 [BtK⁹]), binding sites were “mapped” in CHO-ET cells. Receptor-ligand complexes did not exhibit spontaneous dissociation during 60min observations. Quantitative fluorescence microscopy revealed that SNP or SIN-1 caused a rapid, concentration-dependent, and reversible dissociation of biotinylated ET- 1 from ET_A receptor (EC₅₀ = 75 μM and 6 μM, respectively), an effect that was not mimicked by 8-bromo-cyclic GMP. “Sandwich” co-culture of endothelial cells with CHO-ET showed that activation of NO production by endothelial cells similarly resulted in dissociation of ET-1 [BtK⁹] from ET_A receptors. We hypothesize that NO plays a role in physiological termination of ET-1 signalling by dual mechanisms: (1) displacement of bound ET-1 from its receptor, thus preventing homologous desensitization, and (2) interference with the postreceptoral pathway for [Ca²⁺] mobilization, hence inhibiting end-responses to ET-1. © 1994 Wiley-Liss, Inc.     

Transmembrane signal transduction by the Escherichia coli osmotic sensor, EnvZ: intermolecular complementation of transmembrane signalling

Summary
The Escherichia coli regulatory proteins, EnvZ and OmpR, are crucially involved in expression of the outer membrane proteins OmpF/OmpC in response to the medium osmolarity. The EnvZ protein is presumably a membrane-located osmotic sensor (or signal transducer), which exhibits both kinase and phosphatase activities specific for the OmpR protein. To examine the functional importance of the membrane-spanning segments (named TM1 and TM2) of EnvZ molecules in transmembrane signalling, a set of EnvZ mutants, each having amino acid substitutions within the membrane-spanning regions, was characterized in terms of both their in vivo phenotype and in vitro catalytic activities. One of them, characterized further, has an amino acid change (Pro-41 to Ser or Leu) In TM1, and appeared to be defective in its phosphatase activity but not in its kinase activity. This EnvZ mutant conferred a phenotype of OmpF⁻/OmpC-constitutive. For this EnvZ(P41S or P41L) mutant, a set of intragenic suppressors, each exhibiting a wild-type phenotype of OmpF⁺/OmpC⁺, was isolated. These suppresor mutants were revealed to have an additional amino acid change within either TM1 or TM2. Furthermore, they exhibited restored phosphatase activity (i.e., both kinase⁺ and phosphatase⁺ activities). It was further demonstrated that one of the suppressors, EnvZ(Arg-180 to Trp in TM2), was able to suppress the defects in both the in vivo phenotype and the in vitro catalytic activities caused by EnvZ(P41S), through intermolecular complementation. These results are best interpreted as meaning that an intimate intermolecular interaction between the membrane–spanning segments of EnvZ is crucial for transmembrane signalling per se in response to an external osmotic stimulus.     

Effect of exchangeable aluminium on the reduction of potato scab

The severity of the incidence of the fungal disease, potato scab, varies with different soil groups at the same soil pH. At a soil pH of 5.3, potato scab is easily controlled in soils of western Hokkaido (soil group A) by simply decreasing soil pH, but in soils from eastern Hokkaido (soil group B) it is not so easily controlled. The difference appears to be due to higher levels and exchangeable aluminium in Group A.Addition of sufficient aluminium or ferrous sulfate to a group B soil decreased the incidence of potato scab in a field experiment. Higher levels of aluminium sulfate depressed crop production. It is concluded that aluminium ions control the incidence of potato scab in acid soils. It is suggested that, in soils with low exchange acidity Y1, potato scab can be controlled by adding sufficient aluminium to increase their exchange acidity Y1 to above 7–8.     

Acid-induced changes in the in vivo wall-yielding properties of hypocotyl sections of Vigna unguiculata

Elongation growth of hypocotyl sections of Vigna unguiculata under xylem perfusion was significantly enhanced when acid was applied by acid-aerosol to an abraded hypocotyl surface in the air. The in vivo wall extensibility (φ) and the effective turgor (Pⁱ– Y), both of which were determined by the pressure-jump method, increased during acid-induced growth as observed in IAA-induced growth. The intracellular pressure (Pⁱ), however, decreased significantly at the beginning of acid-induced growth whereas Pⁱ scarcely changed in IAA-induced growth. This result indicates that protons increase the effective turgor by decreasing the yield threshold as IAA does. There seems to be no essential difference between proton and auxin in the effects on the in vivo mechanical properties of the surface cell wall.