In vivo oxidation of carboxyl-labelled cyclic fatty acids formed from linoleic and linolenic acids in the rat

Cyclic fatty acid monomers (CFAM), which occur from the intramolecular cyclisation of linoleic and linolenic acids, are subsequently present in some edible oils and are suspected to induce metabolic disorders. One may suggest that the presence of a ring would alter the ability of the organism to oxidise these molecules. In order to test this hypothesis, we assessed the oxidative metabolism of CFAM in rats. For this purpose, rats were force-fed from 1.5 to 2.6 MBq of [1-(14)C]-linoleic acid, [1-(14)C]-linolenic acid, [1-(14)C]-CFAM-18:2 or [1-(14)C]-CFAM-18:3, and 14CO2 production was monitored for 24 h. The animals were then sacrificed and the radioactivity was determined in different tissues. No significant differences in 14CO2 production were found 24 h after the administration of CFAM and their respective precursors. Our data clearly demonstrate that, at least for the first beta-oxidation cycle, CFAM are oxidised in a similar way as both essential fatty acids.     

Effects of iron and oxygen species scavengers on Listeria spp. chemiluminescence

Listeria monocytogenes and Listeria innocua are able, under certain conditions, to produce chemiluminescence (CL), which is amplified by luminol. Kinetic studies of CL by L. monocytogenes and L. innocua show a close parallelism between CL and growth curves during the exponential phase, with a maximum of CL reached just before entrance of bacteria into the stationary phase. CL is tightly correlated with the release of oxygen compounds. The reactive oxygen species scavengers tryptophan, mannitol, and tiron, as well as cellobiose and high temperature, were assessed with regard to CL in the two Listeria species. Only tiron strongly reduced the CL emitted by L. monocytogenes and L. innocua. On the other hand, charcoal pretreatment of the growth medium inhibited the CL, whereas ferric citrate strongly increased the CL of L. monocytogenes and L. innocua. These data suggest that iron and superoxide radical are implicated in the CL produced by these bacteria, but this phenomenon is not correlated to virulence.     

Attenuated total reflection IR spectroscopy as a tool to investigate the orientation and tertiary structure changes in fusion proteins

Membrane fusion proceeds via a merging of two lipid bilayers and a redistribution of aqueous contents and bilayer components. It involves transition states in which the phospholipids are not arranged in bilayers and in which the monolayers are highly curved. Such transition states are energetically unfavourable since biological membranes are submitted to strong repulsive hydration electrostatic and steric barriers. Viral membrane proteins can help to overcome these barriers. Viral proteins involved in membrane fusion are membrane associated and the presence of lipids restricts drastically the potential of methods (RMN, X-ray crystallography) that have been used successfully to determine the tertiary structure of soluble proteins. We describe here how IR spectroscopy allows to solve some of the problems related to the lipid environment.The principles of the method, the experimental setup and the preparation of the samples are briefly described. A few examples illustrate how attenuated total reflection Fourier-transform IR (ATR-FTIR) spectroscopy can be used to gain information on the orientation and the accessibility to the water phase of the fusogenic domain of viral proteins. Recent developments suggest that the method could also be used to detect changes located in the membrane domains and to identify intermediate structural states involved in the fusion process.     

Polydnavirus genome: integrated vs. free virus

Polydnaviruses are unique because of their obligatory association with thousands of parasitoid wasp species from the braconid and ichneumonid families of hymenopterans. PDVs are injected into the parasitized hosts and are essential for parasitism success. However, polydnaviruses are also unique because of their genome composed of multiple dsDNA segments. Cytological evidence has recently confirmed the results of genetic and molecular analyses indicating that PDV segments were integrated in the wasp genome. Moreover a phylogenetic study performed using the age of available fossils to calibrate the molecular clock indicated that the polydnaviruses harboured by braconid wasps have resided within the wasp genome for approximately 70 million years. In the absence of horizontal transmission, the evolution of the PDV genomes has been driven exclusively by the reproductive success they have offered the wasps. The consequences of this particular selection pressure can be observed in the gene content of certain PDV genomes from which increasing sequence data are available. Molecular mechanisms already identified could be involved in the acquisition and loss of genes by the PDV genomes and lead us to speculate on the definition of the virus genome.     

Maturation-dependent changes of angiotensin receptor expression in fowl

An angiotensin (ANG) receptor homologous to the type 1 receptor (AT1) has been cloned in chickens (cAT1). We investigated whether cAT1 expression in various tissues shows maturation/age-dependent changes. cAT1 mRNA levels detected in renal glomeruli [in situ hybridization (ISH)] and kidney extract (RT-PCR) are significantly (P < 0.01) higher in 19-day embryos (EB) than in chicks (CH, 2-3 wk) and pullets/cockerels (PL/CK, 14-16 wk). The levels in adrenal glands (concentrated in subcapsular regions) are high in EB and further increased in CH and PL/CK. cAT1 mRNA is also detectable in smooth muscle (SM)/adventitia of EB and CH aorta and in the adventitia, but not SM, from PL/CK aortas. The endothelia from small arteries and arterioles, but not from aorta, express cAT1 mRNA (ISH). In all age groups, ANG II induces profound endothelium-dependent relaxation of abdominal aorta, partly (37-47%) inhibitable (P < 0.01) by Nomega-nitro-l-arginine methyl ester (l-NAME, 10(-4) M), suggesting the presence of ANG receptor in endothelium. l-NAME-resistant ANG II relaxation, examined in a limited number of EB or CH aortas, was reduced by 125 mM K+ or apamin plus charybdotoxin. The results suggest that 1) cAT1 is present in kidney, adrenal gland, and vascular endothelium (heterogeneity exists among arteries) of EB, CH, and PL/CK, and in aortic SM/adventitia of EB/CH but only in adventitia of PL/CK; 2) levels of cAT1 gene expression change during maturation in a tissue-specific manner; and 3) ANG II-induced relaxation may be partly attributable to nitric oxide and potassium channel activation.     

An efficient method for the rapid establishment of Epstein-Barr virus immortalization of human B lymphocytes

Abstract. Several methods have been developed for the immortalization of B lymphocytes by Epstein-Barr virus (EBV). We developed an efficient method which reduces the time from culture initiation to immortalization and cryopreservation. Two infections of EBV to lymphocytes, and the use of phorbol ester-induced EBV stock significantly improved immortalization efficiency and reduced the time between initiation and immortalization and cryopreservation. The resulting cell bank was used to produce DNA for genetic studies focusing on the genes involved in immune and autistic disorders.     

Hydrophobic substrate utilisation by the yeast Yarrowia lipolytica, and its potential applications

The alkane-assimilating yeast Yarrowia lipolytica degrades very efficiently hydrophobic substrates such as n-alkanes, fatty acids, fats and oils for which it has specific metabolic pathways. An overview of the oxidative degradation pathways for alkanes and triglycerides in Y. lipolytica is given, with new insights arising from the recent genome sequencing of this yeast. This includes the interaction of hydrophobic substrates with yeast cells, their uptake and transport, the primary alkane oxidation to the corresponding fatty alcohols and then by different enzymes to fatty acids, and the subsequent degradation in peroxisomal beta-oxidation or storage into lipid bodies. Several enzymes involved in hydrophobic substrate utilisation belong to multigene families, such as lipases/esterases (LIP genes), cytochromes P450 (ALK genes) and peroxisomal acyl-CoA oxidases (POX genes). Examples are presented demonstrating that wild-type and genetically engineered strains of Y. lipolytica can be used for alkane and fatty-acid bioconversion, such as aroma production, for production of SCP and SCO, for citric acid production, in bioremediation, in fine chemistry, for steroid biotransformation, and in food industry. These examples demonstrate distinct advantages of Y. lipolytica for their use in bioconversion reactions of biotechnologically interesting hydrophobic substrates.     

Comparison of quantitative and molecular variation in agroforestry populations of the shea tree (Vitellaria paradoxa C.F. Gaertn) in Mali

In this study we investigated the within- and between-population genetic variation using microsatellite markers and quantitative traits of the shea tree, Vitellaria paradoxa, an important agroforestry tree species of the Sudano–Sahelian region in Africa. Eleven populations were sampled across Mali and in northern Côte d’Ivoire. Leaf size and form and growth traits were measured in a progeny test at the nursery stage. Eight microsatellites were used to assess neutral genetic variation. Low levels of heterozygosity were recorded (1.6–3.0 alleles/locus; H_E = 0.25–0.42) and the fixation index (F_IS = −0.227–0.186) was not significantly different from zero suggesting that Hardy–Weinberg equilibrium is encountered in all populations sampled. Quantitative traits exhibited a strong genetic variation between populations and between families within populations. The degree of population differentiation of the quantitative traits (Q_ST = 0.055–0.283, Q_STmean = 0.189) strongly exceeds that in eight microsatellite loci (F_ST = −0.011–0.142, F_STmean = 0.047). Global and pairwise F_ST values were very low and not significantly different from zero suggesting agroforestry practices are amplifying gene flow (Nm = 5.07). The population means for quantitative traits and the rainfall variable were not correlated, showing variation was not linked with this climatic cline. It is suggested that this marked differentiation for quantitative traits, independent of environmental clines and despite a high gene flow, is a result of local adaptation and human selection of shea trees. This process has induced high linkage disequilibrium between underlying loci of polygenic characters.     

In vitro metabolism of rumenic acid in bovine liver slices

Ruminant products are the major source of CLA for humans. However, during periods of fat mobilization, the liver might play an important role in CLA metabolism which would limit the availability of the latter for muscles and milk. In this context, rumenic acid (cis-9, trans-11 CLA) metabolism in the bovine liver (n = 5) was compared to that of oleic acid (n = 3) by using the in vitro liver slice method. Liver slices were incubated for 17 h in a medium containing 0.75 mM of FA mixture and 55 microM of either [1-(14)C] rumenic acid or [1-(14)C] oleic acid at 37 degrees C under an atmosphere of 95% O(2)-5% CO(2). Rumenic acid uptake by liver slices was twice (P = 0.009) that of oleic acid. Hepatic oxidation of both FA (> 50% of incorporated FA) led essentially to the production of acid-soluble products and to a lower extent to CO(2) production. Rumenic acid was partly converted (> 12% of incorporated rumenic acid) into conjugated C18:3. CLA and its conjugated derivatives were mainly esterified into polar lipids (71.7%), whereas oleic acid was preferentially esterified into neutral lipids (59.8%). Rumenic acid secretion as part of VLDL particles was very low and was one-fourth lower than that of oleic acid. In conclusion, rumenic acid was highly metabolized by bovine hepatocytes, especially by the oxidation pathway and by its conversion into conjugated C18:3 for which the biological properties need to be elucidated.