Correlation between bilayer destabilization and activity enhancement by diacylglycerols in reconstituted Ca-ATPase vesicles

Using the reconstituted Ca-ATPase vesicles as a model system, we demonstrated that the presence of 1,2-dioleoyl-sn-glycerol (diolein) in the membrane introduces a pronounced enhancement in the Ca-transport function of Ca-ATPase, while the 1,2-dipalmitoyl-sn-glycerol (dipalmitin) does not. We also found by both 31P NMR and freeze-fraction electron microscopy that diolein destabilized lipid bilayers to a greater extent than did dipalmitin. We conclude that the tendency of diacylglycerols to destabilize the phospholipid bilayer is related to their capacity to enhance the activity of the membrane calcium pump.     

Identity of a cytosolic neutral cholesterol esterase in rat liver with the bile salt stimulated cholesterol esterase in pancreas

A neutral cholesterol esterase has been purified to homogeneity from the cytosolic fraction of rat liver. The 105,000 x g supernatant fraction of rat liver was applied to a DEAE-cellulose column to isolate a partially purified fraction of hepatic cholesterol esterase. Immunoblot analysis of the partially purified liver fraction with the anti-porcine pancreatic cholesterol esterase IgG demonstrated a single band with a molecular weight of 67,000. The hepatic protein was then isolated by immunoaffinity chromatography technique using a column constructed with antibodies prepared against the pancreatic cholesterol esterase. Characterization of the hepatic cholesterol esterase revealed that the hepatic enzyme shared antigenic epitopes with the pancreatic cholesterol esterase and was similarly activated by addition of bile salt such as taurocholate. Moreover, amino-terminal sequencing analysis of the hepatic cholesterol esterase showed an identical sequence with the pancreatic enzyme. Taken together, these results showed that the cholesterol esterases in the liver and the pancreas are very similar and possibly identical proteins.     

Functional RelBE-Family Toxin-Antitoxin Pairs Affect Biofilm Maturation and Intestine Colonization in Vibrio cholerae

Toxin–antitoxin (TA) systems are small genetic elements that typically encode a stable toxin and its labile antitoxin. These cognate pairs are abundant in prokaryotes and have been shown to regulate various cellular functions. Vibrio cholerae, a human pathogen that is the causative agent of cholera, harbors at least thirteen TA loci. While functional HigBA, ParDE have been shown to stabilize plasmids and Phd/Doc to mediate cell death in V. cholerae, the function of seven RelBE-family TA systems is not understood. In this study we investigated the function of the RelBE TA systems in V. cholerae physiology and found that six of the seven relBE loci encoded functional toxins in E. coli. Deletion analyses of each relBE locus indicate that RelBE systems are involved in biofilm formation and reactive oxygen species (ROS) resistance. Interestingly, all seven relBE loci are induced under the standard virulence induction conditions and two of the relBE mutants displayed a colonization defect, which was not due to an effect on virulence gene expression. Although further studies are needed to characterize the mechanism of action, our study reveals that RelBE systems are important for V. cholerae physiology.     

Effects of sodium butyrate on human colonic adenocarcinoma cells. Induction of placental-like alkaline phosphatase

We have examined the effects of the "differentiating agent," sodium butyrate, on the induction of alkaline phosphatase in human colonic tumor cell line LS174T. Culture of these cells in the presence of 2 mM butyrate caused this activity to increase from less than 0.0001 unit/mg of protein to greater than 0.7 unit/mg of protein over an 8-day period. This induction proceeded in a nonlinear fashion with a lag time of 2-3 days occurring before enzymatic activity began to rise. These increases in activity were accompanied by elevations in the content of a placental-like isozyme of alkaline phosphatase as demonstrated by "Western" immunoblots. Dome formation, indicative of differentiation in cultured cells, also required 3 days treatment with butyrate before becoming evident. The rate of biosynthesis of the enzyme, examined using metabolic labeling with L-[35S]methionine and immunoprecipitation, was found to increase continuously between days 2 and 6 of butyrate treatment. "Northern" blot analysis indicated that treatment of these cells with butyrate caused greater than 20-fold induction of a 2700-base mRNA that hybridized to a cDNA probe for placental alkaline phosphatase. The mRNA for alkaline phosphatase produced by these cells upon butyrate treatment was approximately 300-400 bases smaller than the mRNA for alkaline phosphatase found in placenta. Human small intestine also contained two mRNAs that hybridized relatively weakly with the placental alkaline phosphatase probe. These results indicate that a placental alkaline phosphatase-like protein and mRNA are induced by butyrate in LS174T cells with a time course consistent with cellular differentiation preceding induction.     

Polygalacturonase activity and variation in ripening of papaya fruit with tissue depth and heat treatment

Effects of tissue position (viz. outer vs inner mesocarp) and heat treatment (48°C, 20 min) on variations in polygalacturonase (EC 3.2.1.15 and EC 3.2.1.67) activity and ripening of fruits of Carica papaya L. cv. Backcross Solo were investigated. Polygalacturonase activity increased during ripening concomitantly with an increase in tissue softness and soluble polyuronide level. Throughout ripening, inner mesocarp tissue was softer and contained higher polygalacturonase activity than outer mesocarp tissue. Titratable acidity as well as ß-galactosidase (EC 3.2.1.23) activity also increased during ripening; however, unlike polygalacturonase, their level or activity was lower in inner than in outer mesocarp. Ascorbic acid could partially account for the increase in titratable acidity during ripening but contributed very little to the differences in titratable acid levels between outer and inner mesocarp. Heat treatment had no effect on either fruit softness or titratable acidity, but it markedly reduced the increase in ascorbic acid and polygalacturonase activity during ripening. Ripening, as reflected by changes in tissue softness and polygalacturonase activity, progressed outwardly from the interior towards the exterior of the fruit. The effect of heat treatment in suppressing polygalacturonase activity was relatively greater in inner than in outer mesocarp, suggesting that sensitivity of the enzyme to heat treatment may vary with stage of ripeness of the tissue.