Effects of diacylglycerols on the structure of phosphatidylcholine bilayers: a 2H and 31P NMR study

The interaction of four diacylglycerols (DAGs) with multilamellar phospholipid bilayers consisting either of dipalmitoylphosphatidylcholine (DPPC) or of a mixture of DPPC and bovine liver phosphatidylcholine (BL-PC) extracts was investigated by a combination of 31P and 2H NMR spectrometry. We found that saturated and unsaturated long-chain DAGs induce different types of perturbations into the bilayer structure. The saturated DAGs dipalmitin and distearin induce lateral phase separation of the lipids into (i) DAG-enriched gellike domains and (ii) relatively DAG-free regions in the liquid-crystalline phase. In the latter regions, the order parameters along the fatty acyl chains of DPPC are practically identical with the control. This phase separation effect was observed in both model systems studied, and its extent is dependent upon DAG concentration and temperature. Only bilayer phases were present upon addition of dipalmitin or distearin at all concentrations and temperatures studied. The unsaturated DAGs diolein and DAG derived from egg PC (egg-DAG) affect PC bilayers in the following two ways: (i) by increasing the order parameters of the side chains, as observed for both DPPC and BL-PC model systems; (ii) by inducing nonbilayer lipid phases, as observed for BL-PC, but not DPPC. At a concentration of 25 mol % of an unsaturated DAG in mixed PC bilayers, a peak corresponding to isotropic lipid conformation appeared and increased in intensity with increase in temperature, while at 32 mol % hexagonal and bilayer phases coexisted.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enzyme reaction rate studies in electromotor neurons of the weakly electric fish Apteronotus leptorhynchus

A histochemical analysis of reaction rates of a series of enzymes was performed in electromotor neurons of the weakly electric fish Apteronotus leptorhynchus. These neurons were selected because of their functional homogeneity. The high metabolic activity of these cells as well as their large size facilitate cytophotometric analysis in cryostat sections. Sections were incubated for the activity of hexokinase, glucose-6-phosphate dehydrogenase, succinate dehydrogenase, NADPH dehydrogenase, NADPH ferrihaemoprotein reductase and beta-hydroxybutyrate dehydrogenase. All media contained polyvinyl alcohol as tissue stabilizer and Nitro BT as final electron acceptor. Measurements were performed with a Vickers M85a cytophotometer. Linear relationships between the specific formation of formazan (test minus control reaction) and incubation time were obtained for all enzymes although some reactions showed an initial lag phase or an intercept with the ordinate. The relatively high activities of hexokinase, succinate dehydrogenase and the extremely low activity of hydroxybutyrate dehydrogenase indicate that energy is mainly supplied by glycolysis. Glucose-6-phosphate dehydrogenase showed a high activity whereas NADPH reductase and dehydrogenase activity were low in electromotor neurons, indicating that the NADPH generated is largely used for biosynthesis. Despite their synchronous firing pattern activity, electromotor neurons showed a considerable heterogeneity with respect to their metabolic activity.     

Influence of lactate on isoproterenol-induced lipolysis and beta-adrenoceptors distribution in human fat cells

The influence of lactate on human adipocytes lipolysis and the possible relationship between lactate-induced metabolic effects and beta-adrenoceptor binding sites were investigated. beta-sites were identified in membranes with (125I)-cyanopindolol and in intact cells with (125I)-cyanopindolol and (3H)-CGP 12177. Lactate reduced isoproterenol-induced lipolysis in a dose-response fashion and such inhibition became significant only at 16 mmol/l lactate. Exposure of human fat cells to 16 mmol/l lactate significantly reduced beta-adrenoceptors density on crude membranes. When the binding assay was performed on intact cells using (125I)-cyanopindolol at 37 degrees C, the radioligand identified the same number of receptors, regardless of the presence of lactate in the preincubation medium. When (3H)-CGP 12177 was used, it bound to about 35% less receptors in lactate pre-treated cells than in control. Seemingly, at 37 degrees C, because of its lipophilicity, (125I)-cyanopindolol can cross the plasma membrane and bind to intracellular sites whereas, (3H)-CGP 1277, due to its hydrophilicity, identifies surface receptors only. Thus, the present in vitro study provides evidence that high levels of lactate, similar to the concentrations usually achieved in overt lactic acidosis, are able per se to inhibit human lipolysis and to redistribute beta-adrenoceptors from cell surface to a domain not accessible to hydrophilic ligands.     

Biosynthesis of glycosaminoglycan precursors: evidences for different tissue specific forms of UDP-glucose dehydrogenase

UDP-glucose dehydrogenase (UDPGDH) was extracted and partially purified from different rat tissues and the kinetic parameters and some properties of the enzyme were determined and compared. The pH optimum ranged between 8.6 and 9.4 for liver and kidney UDPGDH and between 8.4 and 8.6 for skin and lung UDPGDH. Liver and kidney enzymes showed a similar affinity for both UDPG and NAD. Lung and skin enzymes also showed similar affinity for both substrates, which differed however from that of liver and kidney UDPGDH. Both liver and kidney enzymes had a higher heat stability and a different electrophoretic mobility compared to skin and lung UDPGDH. These data suggest the existence of different tissue specific forms of the enzyme.     

Strain and sex differences in the response of mice to drugs that induce protoporphyria: role of porphyrin biosynthesis and removal

A hepatic green pigment, inhibitory toward ferrochelatase, has been isolated from the liver of mice treated with griseofulvin, isogriseofulvin, or 3,5-diethoxycarbonyl-1,4-dihydrocollidine and has been shown to exhibit identical chromatographic characteristics to authentic N-methyl protoporphyrin. All four possible structural isomers have been demonstrated, and each drug produced primarily the same isomer. N-Methyl protoporphyrin has also been found in very small amounts in the liver of untreated mice, but the isomeric composition appeared to differ from that of the drug-induced N-methyl protoporphyrin. Intraperitoneal administration of 3,5-diethoxy-carbonyl-1,4-dihydrocollidine to female C3H/He/Ola and NIH/Ola inbred mice produced a marked dose-related loss of hepatic ferrochelatase activity, which was identical in magnitude in the two strains. Induction of hepatic 5-aminolevulinate synthase (ALA-S), and accumulation of liver protoporphyrin, however, were greater in C3H/He/Ola mice. The strain difference in ALA-S response was most marked when inhibition of ferrochelatase (the "specific" effect of the drug) was maximal, and this suggests that a genetic variation exists in the sensitivity of ALA-S to a second drug action, the so-called nonspecific action, which is shared by many lipid-soluble compounds. Male mice of three strains accumulated greater amounts of hepatic protoporphyrin than females after treatment with griseofulvin, yet no significant difference was found between the two sexes in the extent of ferrochelatase inhibition. Stimulation of ALA-S activity was slightly greater in males, but when porphyria was very marked, ALA-S activities were significantly lower in this sex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of {beta}-galactoside {alpha}2,6 sialyltransferase blocks synthesis of polysialic acid in Xenopus embryos

Polysialic acid is a developmentally regulated carbohydratestructure found on neural cell adhesion molecules (NCAM). Expressionof ß-galactoside 2,6-sialyltransferase in Xenopusembryos, by injection of mRNA, prevents the polysialylationof NCAM, presumably by introducing a different type of sugarlinkage that terminates chain elongation. Abnormalities in neuraldevelopment result from this treatment, but in general the bodyplan of the injected embryos is not severely affected. The resultsprovide evidence that the mis-expression of glycosyltransferasescan be used to interfere with the normal pattern of glycosylationin whole organisms. glycosylation NCAM polysialic acid Xenopus development     

Olfactory cytochrome P-450. Studies with suicide substrates of the haemoprotein.

1. The olfactory epithelium of male hamsters has been found to be extremely active in the cumene hydroperoxide-supported oxidation of tetramethylphenylenediamine, and this peroxidase activity has been shown to be cytochrome P-450-dependent. 2. The interaction of a series of suicide substrates of cytochrome P-450 with the hepatic and olfactory mono-oxygenase systems has been assessed by determination of peroxidase, 7-ethoxycoumarin O-de-ethylase (ECOD) and 7-ethoxyresorufin O-de-ethylase (EROD) activities after treatment in vivo with these compounds. Chloramphenicol, OOS-trimethylphosphorothiolate and two dihydropyridines [DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine) and 4-ethyl DDC (3,5-diethoxycarbonyl-4-ethyl-1,4-dihydro-2,6-dimethylpyridine)] all caused similar percentage inhibitions of hepatic and olfactory activities, but the absolute amounts of enzymic activity lost were considerably greater in the latter tissue. In contrast, halothane had little effect upon hepatic cytochrome P-450-dependent reactions, whereas it severely inhibited those of the olfactory epithelium. 3. The time course of loss and recovery of hepatic and olfactory peroxidase, ECOD and EROD activities after a single dose of 4-ethyl DDC was studied. The rates of loss of activity observed were very similar, irrespective of tissue or reaction examined. In the olfactory epithelium, all three activities recovered concurrently and at a rate similar to that of the hepatic peroxidase activity. In contrast, the hepatic de-ethylation of 7-ethoxycoumarin and 7-ethoxy-resorufin recovered significantly more rapidly. 4. It is suggested that this behaviour is due to 4-ethyl DDC acting not only as a suicidal inhibitor but also as an inducer of certain forms of cytochrome P-450 in the liver; in the olfactory epithelium, however, inactivation, but not induction, occurs. Classical inducing agents were reported to have no effect upon olfactory cytochrome P-450, and in the present study neither phenobarbitone nor beta-naphthoflavone treatment had any effect upon olfactory cytochrome P-450-dependent reactions, although it induced those of the liver.     

Detection of fos protein during osteogenesis by monoclonal antibodies.

We have generated monoclonal antibodies by using a synthetic peptide corresponding to amino acid positions 4 to 17 of the human fos protein. The antibodies detected both v- and c-fos proteins by immunoprecipitation, immunoblotting, and indirect immunofluorescence. The monoclonal antibodies not only identified the fos protein complex with the cellular 39-kilodalton protein, but also recognized the modified forms of the mouse, rat, and human fos proteins. In day-17 rat embryos, nuclear-staining fos protein could be identified in the cartilage by immunohistochemical staining.     

Molecular cloning of lactose genes in dairy lactic streptococci: the phospho-beta-galactosidase and beta-galactosidase genes and their expression products

The mesophilic (S. lactis and S. cremoris) and thermophilic (S. thermophilus) dairy lactic streptococci, which are used in industrial dairy fermentations, contain two different lactose hydrolysing enzymes, a phospho-beta-galactosidase and a beta-galactosidase. The central role of these enzymes in the pathways used for lactose transport and degradation is discussed along with their properties and distributions in lactic streptococci. In addition, recent results on the cloning, expression and sequence organization of the genes for the mesophilic phospho-beta-galactosidase and thermophilic beta-galactosidase are reviewed. Original data are presented concerning heterologous gene expression in the study of lactose hydrolysis in lactic streptococci. These include 1) the purification of the S. lactis phospho-beta-galactosidase from an overproducing Escherichia coli, and 2) the expression of the E. coli beta-galactosidase (lacZ) gene in S. lactis employing a lactic streptococcal expression vector.