Purification and characterization of α-glucosidases produced by Saccharomyces in response to three distinct maltose genes

α-Glucosidases or maltases (EC 3.2.1.20) were purified to electrophoretic homogeneity from a respective strain of Sacchromyces cerevisiae which carries a single MAL gene, either MALα, MALβ or MALγ, using gluconate-Sepharose affinity chromography and isoelectrofocusing. Of these maltases, two types of maltase were obtained from the MALγ strain, the pI values of which were 5.6 and 5.9. From the MALα and MALβ strain was obtained only one type of maltase with the pI at 5.6 which was identical to one of the maltases from the MALγ strain. These four maltases possessed the same properties, except for pI. They were monomers with molecular weights of between 66 000 and 67 000. With regard to the substrate specificity, they hydrolyzed maltose and sucrose exclusively but not α-methulglucoside nor maltooligosaccharide. They did not differ in immunological properties.     

Continuous-flow NMR culture system for mammalian cells

A continuous-flow NMR culture system for mammalian cells has been developed on which ³¹P-NMR experiments under complete and strictly physiologic conditions have been performed. Observations on the response of the cellular metabolism to stresses such as starvation, low temperature and changes in environmental pH monitored by ³¹P-NMR are reported. The response of the intracellular pH relative to the external pH of the growth medium is studied. We find that under the experimental conditions used there exists a ΔpH varying between less than 0.2 and more than 0.6 pH units. These results are compatible with those obtained using other techniques.     

The discovery of diazetidinyl diamides as potent and reversible inhibitors of monoacylglycerol lipase (MAGL)

Monoacylglycerol lipase (MAGL) has emerged as an attractive drug target because of its important role in regulating the endocannabinoid 2-arachidonoylglycerol (2-AG) and its hydrolysis product arachidonic acid (AA) in the brain. Herein, we report the discovery of a novel series of diazetidinyl diamide compounds 6 and 10 as potent reversible MAGL inhibitors. In addition to demonstrating potent MAGL inhibitory activity in the enzyme assay, the thiazole substituted diazetidinyl diamides 6d–l and compounds 10 were also effective at increasing 2-AG levels in a brain 2-AG accumulation assay in homogenized rat brain. Furthermore, selected compounds have been shown to achieve good brain penetration after oral administration in an animal study.     

Transport protein synthesis in non-growing yeast cells

Addition of a metabolizable substrate (glucose, ethanol and, to a degree, trehalose) to non-growing baker's yeast cells causes a boost of protein synthesis, reaching maximum rate 20 min after addition of glucose and 40–50 min after ethanol or trehalose addition. The synthesis involves that of transport proteins for various solutes which appear in the following sequence: H⁺, l-proline, sulfate, l-leucine, phosphate, α-methyl-d-glucoside, 2-aminoisobutyrate. With the exception of the phosphate transport system, the K_t of the synthesized systems is the same as before stimulation. Glucose is usually the best stimulant, but ethanol matches it in the case of sulfate and exceeds it in the case of proline. This may be connected with ethanol's stimulating the synthesis of transport proteins both in mitochondria and in the cytosol while glucose acts on cytosolic synthesis alone. The stimulation is often repressed by ammonium ions (leucine, proline, sulfate, H⁺), by antimycin (proline, trehalose, sulfate, H⁺), by iodoacetamide (all systems tested), and by anaerobic preincubation (leucine, proline, trehalose, sulfate). It is practically absent in a respiration-deficient petite mutant, only little depressed in the op₁ mutant lacking ADP/ATP exchange in mitochondria, but totally suppressed (with the exception of transport of phosphate) in a low-phosphorus strain. The addition of glucose causes a drop in intracellular inorganic monophosphate by 30%, diphosphate by 45%, ATP by 70%, in total amino acids by nearly 50%, in transmembrane potential (absolute value) by about 50%, an increase of high-molecular-weight polyphosphate by 65%, of total cAMP by more than 100%, in the endogenous respiration rate by more than 100%, and a change of intracellular pH from 6.80 to 7.05. Ethanol caused practically no change in ATP, total amino acids, endogenous respiration, intracellular pH or transmembrane potential; a slight decrease in inorganic monophosphate and diphosphate and a sizeable increase in high-molecular-weight polyphosphate. The synthesis of the various transport proteins thus appears to draw its energy from different sources and with different susceptibility to inhibitors. It is much more stimulated in facultatively aerobic species (Saccharomyces cerevisiae, Endomyces magnusii) than in strictly aerobic ones (Rhodotorula glutinis, Candida parapsilosis) where an inhibition of transport activity is often observed after preincubation with metabolizable substrates.     

Impact on energy metabolism of quantitative and functional cyclosporine-induced damage of kidney mitochondria

In this study we have measured, under experimental conditions which maintained efficient coupling, respiratory intensity, respiratory control, oxidative phosphorylation capacity and protonmotive force. Succinate cytochrome-c reductase and cytochrome-c oxidase activities were also studied. These investigations were carried out using kidney mitochondria from cyclosporine-treated rats (in vivo studies) and from untreated rats in the presence of cyclosporine (in vitro studies). Inhibition of respiratory intensity by cyclosporine did not exceed 21.1% in vitro and 15.9% in vivo. Since there was no in vitro inhibition of succinate cytochrome-c reductase and cytochrome-c oxidase activities, the slowing of electron flow observed can be interpreted as a consequence of an effect produced by cyclosporine between cytochromes b and c₁. Cyclosporine had no effect on respiratory control either in vitro or in vivo. Statistically significant inhibition of the oxidative phosphorylation was observed both in vitro (6.6%) and in vivo (12.1%). Moreover, cyclosporine did not induce any change of membrane potential either in vivo or in vitro. Our findings show that cyclosporine is neither a protonophore, nor a potassium ionophore. In cyclosporine-treated rats we noticed a decrease of protein in subcellular fraction, including the mitochondrial fraction. The role of the inhibition respiratory characteristics by cyclosporine in nephrotoxicity in vivo must take account of these two parameters: inhibition of the respiratory characteristics measured in vitro and diminution of mitochondrial protein in cyclosporine-treated rats.     

A novel array-based assay of in situ tissue transglutaminase activity in human umbilical vein endothelial cells

Transglutaminases (TGs), a family of calcium-dependent transamidating enzymes, are involved in functions such as apoptosis and inflammation and play a role in autoimmune diseases and neurodegenerative disorders. In this study, we describe a novel array-based approach to rapidly determine in situ TG activity in human umbilical vein endothelial cells and J82 human bladder carcinoma cells. Amine arrays were fabricated by immobilizing 3-aminopropyltrimethoxysilane on glass slides. The assay was specific and highly reproducible. The average coefficient of variation betweens spots was 2.6% (n = 3 arrays), and the average correlation coefficients between arrays and between arrays/reactions were 0.998 and 0.976, respectively (n = 3 arrays). The assay was successfully applied to detect changes in TG activity induced by maitotoxin and to analyze inhibition of the TG activation with cystamine and monodansyl cadaverine. In addition, the assay demonstrated that intracellular reactive oxygen species regulate the maitotoxin-induced activation of TG. Thus, the array-based in situ TG activity assay constitutes a rapid and high-throughput approach to investigating the roles of TGs in cell signaling.