Inhibition of lipolysis by hydrocarbons and fatty alcohols

The hydrolysis of long-chain triglyceride by pancreatic lipase (EC 3.1.1.3) is inhibited by hydrophobic solutes that are dissolved in the fat. Solutes tested included n-alkanes (C10-C16), aromatic and chlorinated aromatic hydrocarbons (including a PCB and DDT), n-alcohols (C10-C16), and cholesterol. Except for cholesterol, which stimulated lipolysis at low concentrations, all compounds produced roughly similar inhibition curves that followed the pattern of a typical Langmuir adsorption isotherm (Mattson, F. H., R. A. Volpenhein, and L. Benjamin, 1970. J. Biol. Chem. 245: 5335-5340). According to this interpretation, hydrophobic solutes dissolved within fat droplets partition between the interior oil phase and the surface monolayer where lipolysis occurs. Although the aromatic and chlorinated aromatic hydrocarbons were approximately 25% more inhibitory than the long-chain aliphatic hydrocarbons, as a single class, hydrocarbons were 7-10 times weaker inhibitors of lipolysis than fatty alcohols. In contrast to the alcohols whose inhibitory action may involve several mechanisms, the hydrocarbons behaved like simple dilution inhibitors; i.e., at 50% inhibition the mass ratio of hexadecane to triglyceride was 0.42. The lack of a chain length effect indicates that the hydrocarbons are not adsorbed at the interface but interdigitate the triglyceride molecules and align parallel to the lipid acyl chains. Inhibition by hydrophobic solutes was not reversed by the presence of 4 mM taurodeoxycholate and pancreatic procolipase or colipase.     

Identification of a common molecular basis for combined 17α-hydroxylase/17,20-lyase deficiency in two Mennonite families

Summary During the course of studies to characterize mutations of the CYP17 gene that cause the 17-hydroxylase-deficient form of congenital adrenal hyperplasia we have discovered two ostensibly unrelated Mennonite families in which affected individuals are homozygous for the same mutation. The defect is a four-base duplication in exon 8 of the CYP17 gene, which alters the reading frame encoding the C-terminal 26 animo acids of cytochrome P450₁₇.     

Oxidative damage to sarcoplasmic reticulum Ca2+-pump induced by Fe2+/H2O2/ascorbate is not mediated by lipid peroxidation or thiol oxidation and leads to protein fragmentation

The major protein in the sarcoplasmic reticulum (SR) membrane is the Ca²⁺ transporting ATPase which carries out active Ca²⁺ pumping at the expense of ATP hydrolysis. The aim of this work was to elucidate the mechanisms by which oxidative stress induced by Fenton's reaction (Fe²⁺ + H₂O₂ HO· + OH^–+ Fe³⁺) alters the function of SR. ATP hydrolysis by both SR vesicles (SRV) and purified ATPase was inhibited in a dose-dependent manner in the presence of 0–1.5 MM H₂O₂ plus 50 M Fe²⁺ and 6 mM ascorbate. Ca²⁺ uptake carried out by the Ca²⁺-ATPase in SRV was also inhibited in parallel. The inhibition of hydrolysis and Ca²⁺ uptake was not prevented by butylhydroxytoluene (BHT) at concentrations which significantly blocked formation of thiobarbituric acid-reactive substances (TBARS), suggesting that inhibition of the ATPase was not due to lipid peroxidation of the SR membrane. In addition, dithiothreitol (DTT) did not prevent inhibition of either ATPase activity or Ca²⁺ uptake, suggesting that inhibition was not related to oxidation of ATPase thiols. The passive efflux of ⁴⁵Ca²⁺ from pre-loaded SR vesicles was greatly increased by oxidative stress and this effect could be only partially prevented (ca 20%) by addition of BHT or DTT. Trifluoperazine (which specifically binds to the Ca²⁺-ATPase, causing conformational changes in the enzyme) fully protected the ATPase activity against oxidative damage. These results suggest that the alterations in function observed upon oxidation of SRV are mainly due to direct effects on the Ca²⁺-ATPase. Electrophoretic analysis of oxidized Ca²⁺-ATPase revealed a decrease in intensity of the silver-stained 110 kDa Ca²⁺-ATPase band and the appearance of low molecular weight peptides (MW < 100 kDa) and high molecular weight protein aggregates. Presence of DTT during oxidation prevented the appearance of protein aggregates and caused a simultaneous increase in the amount of low molecular weight peptides. We propose that impairment of function of the Ca²⁺-pump may be related to aminoacid oxidation and fragmentation of the protein.Abbreviations AcP acetylphosphate - BHT butylhydroxytoluene - DTT dithiothreitol - Hepes 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid - SDS sodium dodecyl sulfate - SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate - SR sarcoplasmic reticulum - SRV sarcoplasmic reticulum vesicles - TBA thiobarbituric acid - TBARS thiobarbituric acid-reactive substances - TFP trifluoperazine     

Intrinsic fluorescence as a probe of structure-function relationships in Ca2+-transport ATPases

Applications of intrinsic fluorescence measurements in the study of Ca²⁺-transport ATPases are reviewed. Since the initial reports showing that the fluorescence emission was sensitive to Ca²⁺ binding, a substantial amount of work has focused on the use of both steady-state and time-resolved fluorescence spectroscopy to investigate structure-function relationships in sarcoplasmic reticulum and plasma membrane Ca²⁺-ATPases. These studies have revealed ligand-induced conformational changes, as well as provided information on protein-protein, protein-solvent and/or protein-lipid interactions in different functional states of these proteins. The main results of these studies, as well as possible future prospects are discussed.     

The effect of nisin on Listeria monocytogenes in culture medium and long-life cottage cheese

M.A.S.S. FERREIRA AND B.M. LUND. 1996. The sensitivity to nisin of 27 strains of Listeria monocytogenes , four of L. innocua and one of L. ivanovii was estimated at pH 6.8 and pH 5.5. Strains of L. monocytogenes showed differences in sensitivity which were not correlated with serotype. Strains of L. innocua were as resistant as the most resistant strains of L. monocytogenes , whereas the strain of L. ivanovii was relatively sensitive. Two of the most resistant strains of L. monocytogenes multiplied in aerated liquid medium adjusted to pH 5.0 with HCl, incubated at 20°C; nisin, 500 IU ml^-1, prevented multiplication and caused death. Following inoculation of a resistant strain into long-life cottage cheese, pH 4.6–4.7, the number of viable L. monocytogenes decreased approximately 10-fold during storage at 20°C for 7 d; addition of nisin, 2000 IU g^-1, to the cottage cheese increased the rate of inactivation to approximately a 1000-fold decrease in 3 d.     

Somatic embryogenesis,organogenesis and callus growth kinetics of flax

The effects of plant growth regulators (PGR) on calli induction, morphogenesis and somatic embryogenesis of flax were studied. The organogenic and callus formation capacity were assessed for different types of source explants. Root and shoot explants were equally good material for calli production but the former produced calli without shoot regeneration capacity. Under the experimental conditions tested, 2,4-dichlorophenoxyacetic acid (2,4-D) + zeatin was the most efficient PGR combination on calli induction and biomass production. The calli were green but with no rhizogenic capacity. In contrast, and at similar concentrations, indole-3-butyric acid (IBA) + kinetin induced white or pale green friable calli with a good root regeneration capacity (60%). A factorial experiment with different combinations of 2,4-D + zeatin + gibberellic acid (GA₃) levels revealed that the direction of explant differentiation was determined by specific PGR interactions and concentrations. The results from these experiments revealed that the morphogenetic pathway (shoot versus root differentiation) can be manipulated on flax explants by raising the 2,4-D level from 0.05 to 3.2 mg l^?1 in the induction medium. The induction and development of somatic embryos from flax explants was possible in a range of 2,4-D + zeatin concentrations surrounding 0.4 mg l^?1 2,4-D and 1.6 mg l^?1 zeatin, the most efficient growth regulator combination.     

Patterns of divergence during evolution of alpha 1-proteinase inhibitors in mammals

alpha 1-Proteinase inhibitor (alpha 1-PI), a member of the serine proteinase inhibitor superfamily, has a primary role in controlling neutrophil elastase activity within the mammalian circulation. Several studies have indicated that the reactive center region of alpha 1-PI, the amino acid sequence of which is critical to recognition of and binding to target proteinases, is highly divergent within and among species. This appears to be a consequence of accelerated rates of evolution that may have been driven by positive Darwinian selection. In order to examine this and other features of alpha 1-PI evolution in more detail, we have isolated and sequenced cDNAs representing alpha 1- PI mRNAs of the mouse species Mus saxicola and Mus minutoides and have compared these with a number of other mammalian alpha 1-PI mRNAs. Relative to other mammalian mRNAs, the extent of nonsynonymous substitution is generally high throughout the alpha 1-PI mRNA molecule, indicating greater overall rates of amino acid substitution. Within and among mouse species, the 5'-half of the mRNA, but not the 3'-half, has been homogenized by concerted evolution. Finally, the reactive center is under diversifying or positive Darwinian selection in murid rodents (rats, mice) and guinea pigs yet is under purifying selection in primates and artiodactyls. The significance of these findings to alpha 1-PI function and the possible selective forces driving evolution of serpins in general are discussed.      

Digestive enzymes in midgut cells,endo-and ectoperitrophic contents,and peritrophic membranes of Spodoptera frugiperda (lepidoptera) larvae

In the midgut of Spodoptera frugiperda larvae, subcellular fractionation data suggest that aminopeptidase and part of amylase, carboxypeptidase A, dipeptidase, and trypsin are bound to the microvillar membranes; that major amounts of soluble dipeptidase, cellobiase, and maltase are trapped in the cell glycocalyx; and finally that soluble carboxypeptidase, amylase, and trypsin occur in intracellular vesicles. Most luminal acetylglucosaminidase is soluble and restricted to the ectoperitrophic contents. Aminopeptidase occurs in minor amounts bound to membranes both in the ectoperitrophic contents and incorporated in the peritrophic membrane. Amylase, carboxypeptidase A, and trypsin are found in minor amounts in the ectoperitrophic contents (both soluble and membrane-bound) and in major amounts in the peritrophic membrane with contents. Part of the activities recovered in the last mentioned contents corresponds to enzyme molecules incorporated in the peritrophic membrane. The results suggest that initial digestion is carried out in major amounts by enzymes in the endoperitrophic space and, in minor amounts, by enzymes immobilized in the peritrophic membrane. Intermediate and final digestion occur at the ectoperitrophic space or at the surface of midgut cells. The results also lend support to the hypothesis that amylase and trypsin are derived from membrane-bound forms, are released in soluble form by a microapocrine mechanism, and are partly incorporated into the peritrophic membrane. © 1994 Wiley-Liss, Inc.