Demonstration of hepatic cytosolic malic enzyme activity as a thyroid hormone sensitive physiologic parameter in a teleost, Heteropneustes fossilis bloch

Single injections of various doses (0.1, 0.25, 0.5, 5 and 20 micrograms/g) of T3 significantly increased the cytosolic malic enzyme activity (delta OD/min/mg cytosolic protein) in liver of Singi fish Heteropneustes fossilis Bloch, in a dose-dependent nature, maximum up to 5 micrograms/g dose on the 3rd day in comparison to the control. There was no difference in the enzyme activity between 5 and 20 micrograms/g of T3 doses. When the enzyme activity was expressed per mg DNA, the dose-dependent increase in the malic enzyme activity was observed upto 0.5 microgram/g of T3, whereas a fall in the enzyme activity was noticed with 5 and 20 micrograms/g of T3 doses. Lowering the dose of T3 to 0.05 microgram/g was without any effect on the malic enzyme activity (delta OD/min/mg cytosolic protein or DNA). Hepatic cytosolic protein content showed a biphasic nature of variation, significant increase with single injections of 0.05, 0.1, 0.25 and 0.5 microgram/g and a fall with 5 and 20 micrograms/g of T3 doses in comparison to the untreated control. Cycloheximide treatments of the Singi fishes counteracted both the T3-induced rise in the hepatic cytosolic malic enzyme activity (delta OD/min/mg cytosolic protein or DNA) and the hepatic cytosolic protein contents. Thiourea-treated hypothyroid fishes showed significantly decreased level of malic enzyme activity (delta OD/min/mg cytosolic protein or DNA) and cytosolic protein content in liver. A single injection of T3 at 0.25 microgram/g to the thiourea-treated fishes not only recovered but also increased the enzyme activity and cytosolic protein content above the untreated control values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reversal of insulin-induced negative cooperativity by monoclonal antibodies that stabilize the slowly dissociating ("Ksuper") state of the insulin receptor

Two monoclonal antibodies to the insulin receptor, MA-5 and MA-20, unlike other monoclonal antibodies, do not mimick the accelerating effect of insulin on the dissociation of 125I-insulin from the receptors (negative cooperativity). On the contrary, MA-5 and MA-20 markedly slow down the dissociation rate. We show now that MA-5 and MA-20 are potent antagonists of the negative cooperativity induced by insulin, and reverse the insulin-induced acceleration whether added simultaneously with insulin or after insulin. The reversal of the insulin-induced acceleration is almost immediate. These data strengthen the concept therefore that the insulin-receptor complex has access to alternative conformational states that can be stabilized by ligand-induced site-site interactions.     

Effect of dietary lipids on plasma lipoproteins and fluidity of lymphoid cell membranes in normal and leukemic mice

Mice of the GR/A strain were fed four different isocaloric semipurified diets, enriched in either (1) saturated fatty acids (palm oil), or (2) polyunsaturated fatty acids (corn oil), or (3) palm oil plus cholesterol, or (4) a fat-poor diet containing only a minimal amount of essential fatty acids. We have studied the effects of these dietary lipids on the density profile and composition of the plasma lipoproteins and on the lipid composition and fluidity of (purified) lymphoid cell membranes in healthy mice and in mice bearing a transplanted lymphoid leukemia (GRSL). Tumor development in these mice occurred in the spleen and in ascites. While the fatty acid composition of the VLDL-triacylglycerols still strongly resembled the dietary lipids, the effects of the diets decreased in the order VLDL-triacylglycerols greater than HDL-phospholipids greater than plasma membrane phospholipids. Diet-induced differences in the latter fraction were virtually confined to the content of oleic acid and linoleic acid, and they were too small to affect the membrane fluidity, as measured by fluorescence polarization using the probe 1,6-diphenyl-1,3,5-hexatriene. Healthy mice were almost irresponsive to dietary cholesterol, but in the tumor bearers, where lipoprotein metabolism has been shown to be disturbed, the cholesterol diet caused a substantial increase in the low- and very-low density regions of both blood and ascites plasma lipoproteins. The cholesterol-rich diet also increased the cholesterol/phospholipid molar ratio and lipid structural order (decreased fluidity) in GRSL ascites cell membranes, but not in the splenic GRSL cell membranes. We conclude that the composition of plasma lipoproteins and cell membrane lipids in GR/A mice is subject to exquisite homeostatic control. However, in these low-responders to dietary lipids the development of an ascites tumor may lead to increased responsiveness to dietary cholesterol. The elevated level of membrane cholesterol thus obtained in GRSL ascites cells did not affect the expression of various cell surface antigens or tumor cell growth.     

Production of optically active 2,3-butanediol by Bacillus polymyxa

Bacillus polymyxa produces (R, R)-2,3-butanediol from a variety of carbohydrates. Other metabolites are also produced including acetoin, acetate, lactate, and ethanol. The excretion of each metabolite was found to depend on the relative availability of oxygen to the culture. When the relative oxygen uptake rate was high, enhanced yields of acetate and acetoin were noted. At an intermediate oxygen availability, the butanediol yield was maximal. When the availability of oxygen was more restricted, higher yields of lactate and ethanol occurred. The cells appeared to regulate themselves such that energy generation is optimal subject to the constraint that the cells do not produce more reducing equivalents than can be oxidized by the electron transport system. The dependence of each product yield on the relative oxygen availability was determined, and this knowledge was used to carry out a fed-batch fermentation that attained a final butanediol concentration of over 40 g/L in 50 h.     

Antidiabetic sulfonylureas control action potential properties in heart cells via high affinity receptors that are linked to ATP-dependent K+ channels

Both avian and mammalian heart cells have high affinity receptors for antidiabetic sulfonylureas. The biochemical identification of these receptors has been carried out with [3H]glibenclamide. The Kd values for the most potent sulfonylureas, such as glibenclamide itself, are in the nanomolar range. Comparative studies of structure-function relationships indicate high similarities of binding properties between the sulfonylurea receptors in cardiac cells and insulinoma cells, respectively. The duration of the action potential of guinea pig cardiac cells was drastically reduced by decreasing intracellular ATP concentrations by perfusion or by blockade of oxidative phosphorylation. Glibenclamide was found to restore normal or nearly normal action potential properties in [ATP]in-depleted cardiac cells. Single channel recording using the patch-clamp technique has shown that this effect is associated with high affinity blockade of ATP-sensitive K+ channels by sulfonylureas.     

Calcium transport systems in the LLC-PK1 renal epithelial established cell line

ATP-dependent calcium uptake was measured in membrane vesicles prepared from the renal epithelial LLC-PK1 established cell line. The relative contribution of the nonmitochondrial versus the mitochondrial calcium uptake is larger in LLC-PK1 cell homogenates than in homogenates from renal cortex. Two types of calcium pump, characterized by the formation of calcium-dependent phosphointermediates of 135 kDa and 115 kDa, were found in membrane fractions from LLC-PK1 cells. The 135 kDa calcium pump was also detected by 125I-labelled calmodulin overlay. Although the subcellular localization in LLC-PK1 cell membranes could not be unambiguously determined, it is conceivable that the 135 kDa and the 115 kDa molecules represent the plasma membrane calcium pump and the endoplasmic reticulum calcium pump respectively, in agreement with what was found for renal cortex preparations. Extravesicular sodium partially inhibits ATP-driven calcium uptake in a plasma-membrane-enriched fraction of the LLC-PK1 cells. The effect is potentiated by a vesicle inside-negative membrane potential. Although the effect is less pronounced than in renal cortex basal-lateral membranes, this observation suggests that an Na+-Ca2+ exchange mechanism is also present in LLC-PK1 cells. ATP-dependent calcium uptake in nonmitochondrial intracellular stores was investigated, using saponin-permeabilized cells. Permeabilized LLC-PK1 cells lowered the free calcium concentration in the medium to less than 0.4 microM. More than 60% of the accumulated calcium can be released by addition of inositol 1,4,5-trisphosphate. Our data indicate that the LLC-PK1 cell line can be successfully used as model system for the study of renal calcium handling.     

Secretion of alpha-amylase and multiple forms of glucoamylase by the yeast Trichosporon pullulans

Trichosporon pullulans IGC 3488 produced extracellular alpha-amylase and glucoamylase activities when grown in batches in a medium containing corn steep liquor and soluble starch or corn starch. alpha-Amylase, unlike glucoamylase activity, was secreted biphasically. For both amylases the maximum concentration was found in stationary phase cultures. The amylolytic enzymes, previously concentrated by ammonium sulfate precipitation, were separated into a glucoamylase fraction and an alpha-amylase fraction by Ultrogel AcA 54 gel filtration. Pullulanase activity was located in the glucoamylase fraction, whereas cyclodextrinase activity was restricted to the alpha-amylase fraction. Isoamylase and alpha-glucosidase were not detected. Electrophoretic analysis showed that alpha-amylase activity was due to a single protein. Glucoamylase, however, occurred in multiple forms. The four glucoamylases and the alpha-amylase were glycoproteins.     

The Pleistocene lake deposits of the NE Baza Basin (Spain): salinity variations and ostracod succession

The Guadix-Baza Basin in Spain covers an area of approximately 3000 km² and yields a sedimentary sequence ranging from Lower Miocene to Pleistocene. Twenty five meters of Lower Pleistocene lacustrine sediments have been located in the NE part of the Basin at about 1000 meters in altitude. This sequence which overlies dolomitic mud flat deposits consists of limestones, calcareous and dolomitic mudstones, dolostones, silty clays, sands and gravels. Salinity fluctuations and short dry episodes, related to lake level oscillations, have been recorded by textural, mineralogical and faunal changes throughout the sequence. Ostracods, which are the most commonly encountered fossils, permit to detect recurrent changes in water salinity and regime, and solute composition. The faunal changes indicate an alternation of slightly saline and bicarbonate-rich water (when ostracods and gastropods occur) with a saline NaCl-dominant water (in which ostracods, Cerastoderma bivalves and non-marine foraminifers are found).The frequent and recurrent hydrochemical changes in the Baza Basin in the Early Pleistocene point to a climate of high contrast like in the Mediterranean region today but with a greater availability of water within the system compared to the present situation in the area.     

Steroid-bovine serum albumin conjugates: molecular characterization and their interaction with androgen and estrogen receptors

Conjugates of testosterone-3-carboxymethyloxime (T-3-CMO), testosterone-17-hemisuccinate (T-17-HS), 17 beta-estradiol-6-carboxymethyloxime (E-6-CMO), or 17 beta-estradiol-17-hemisuccinate (E-17-HS) and bovine serum albumin (BSA) with varying steroid:protein ratios were prepared using the mixed anhydride method. Dialysis followed by molecular filtration yielded monomer steroid-BSA conjugates with a molecular weight of 70,000 dalton, and polymer conjugates with molecular weights of 140,000 dalton and higher. When conjugates were prepared with increasing initial steroid:BSA molar ratios the ratio of the obtained conjugates increased, in parallel with a decrease in the relative amount of monomers and an increase in the mean molecular size of polymers. The molecular properties of these conjugates were studied further by polyacrylamide gel electrophoresis (PAGE) in native and denaturing conditions. In native PAGE the monomer fractions showed one main band with a mobility slightly lower than BSA and a faint band corresponding with BSA-dimers. The polymer fractions consisted of a heterogeneous population of protein oligomers with molecular weights varying from 140,000 to over a million dalton. In the presence of sodium dodecylsulphate part of the polymers dissociated into monomers. In buffered aqueous solutions the bulk of the conjugate preparation retained its molecular size and composition, although the generated covalent bonds were found to be liable to spontaneous hydrolysis. Steroid-protein conjugates were shown to contain appreciable amounts of non protein-bound steroids. Binding of T-BSA to androgen receptors in rat ventral prostate cytosol was assayed using LH-20 chromatography and sucrose gradient centrifugation analysis. Binding of E-BSA to estrogen receptors was analysed with rat uterus cytosol using the dextran coated charcoal assay and the sucrose gradient centrifugation technique. Relative binding affinities (RBA) were analyzed in competition experiments using radiolabeled ligands. It was found that the molecular size of the conjugate does not influence its interaction with steroid receptors. Steroid coupled via the 17-position show a higher RBA to receptors than the T-3 or E-6 derivatives. The RBA of T-3-BSA, T-3-CMO, T-17-BSA and T-17-HS appeared to be very low, i.e. between 0.1 and 1.7% of the RBA of dihydrotestosterone. Consequently, high concentrations of conjugate are required to saturate androgen receptor binding sites. Under these conditions involvement of type II and eventually type III binding sites, which show less ligand specificity and lower affinity, may be anticipated preventing exclusive detection of androgen receptors.(ABSTRACT TRUNCATED AT 400 WORDS)