Highly repeated DNA sequences and systematics of malagasy primates

We have analyzed highly repeated DNA sequences of Malagasy lemurs with restriction enzymes, Southern blotting and nucleotide sequencing to assess relatedness among repeated DNA fragments from different species. We have focused our studies on two prosimian families:Lemuridae andCheirogaleidae. Our results have allowed us to draw the following conclusions: confirmation of the separation based on molecular biology data ofEulemur species from theLemur catta/Hapalemur group; classification ofVarecia in a genus clearly separated from the otherLemuridae genera; confirmation of the specific status ofHapalemur aureus; confirmation of cytogenetic data in favour of the subdivision of the familyCheirogaleidae into two subfamilies:Cheirogaleinae andPhanerinae. Finally a cladogram has been constructed by numbering all the highly repeated DNA fragments (obtained by Southern blotting) and analyzing the inferred systematic relationships between the Lemuridae and Cheirogaleidae species.     

Studies on the turnover of exogenous mannose-terminal glucocerebrosidase in rat liver lysosomes

We have cloned the full coding cDNA sequence of chicken annexin V and of a mutant lacking 8 amino acid residues of the N-terminal tail for prokaryotic expression. Both proteins were synthesized in Escherichia coli upon induction with isopropyl thio-β-D-galactoside, and were purified following two different protocols: one based on the ability of these proteins to interact reversibly with liposomes in the presence of calcium, and the other based on two sequential ion-exchange chromatographic steps. Spectroscopical analysis of recombinant annexin V revealed that binding of calcium did not change the circular dichroism spectra indicating no significant changes on the secondary structure; however, a conformational change affecting the exposition to the solvent of the tryptophan residue 187 was detected by analysis of fluorescence emission spectra. Recombinant annexin V binds with high affinity to collagen types II and X, and with lower affinity to collagen type I in a calcium-independent manner. Heat denaturing of collagen decreases this interaction while pepsin-treatment of collagen almost completely abolishes annexin V binding. Mutated annexin V interacts with collagen in a similar way as the nonmutated recombinant protein, indicating that the N-terminal tail of annexin V is not essential for collagen binding.     

Oxidative events in neuronal and glial cell-enriched fractions of rat cerebral cortex

The aim of this work was to investigate how neurons and glial cells separated from rat brain cortex respond to “in vitro” oxidative stress induced by incubation of the cellular fractions in the presence of prooxidant mixtures; in addition, the endogenous enzymatic antioxidant capacity of the purified fractions was investigated. Neuronal and glial cell-enriched fractions were obtained from rat cerebral cortex following passages of the tissue through meshes and centrifugations. The following parameters were evaluated: antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPx), and glucose-6-phosphate dehydrogenase (G6PDH); lipid peroxidation products (TBARS) prior to (basal) and after (iron-stimulated) incubation with a mixture of iron and ascorbic acid; intracellular production of reactive oxygen species (ROS) using a fluorescent probe, dichlorofluorescin-diacetate, in basal, iron-stimulated, and menadione stimulated conditions. SOD and GSHPx activities showed no significant changes between neurons and glia, whereas CAT and G6PDH activities were found to be significantly lower in glia than in neurons. TBARS levels were significantly lower in the glial fraction than in neurons, both in basal and iron-stimulated conditions. ROS production showed no differences between neurons and glia in both basal and menadione-stimulated conditions. Iron-stimulation produced a marked increase in ROS production, limited to the neuronal fraction, with the glial values being similar to the basal ones. Our conclusion is that glia and neurons isolated from rat cerebral cortex show a similar pattern of the most important antioxidant enzymes and of their basal ROS production, whereas glia is more resistant in “oxidative stress” conditions.     

Effect of manganese on the development of glial cells cultured from prenatally alcohol exposed rats

Maternal alcohol abuse is known to produce retardation in brain maturation and brain functions. Using cultured glial cells as a model system to study these effects of alcohol we found an alcohol antagonizing property for manganese (Mn). Mn was added to the alcohol diet (MnCl₂ 25 mg/l of 20% v/v ethanol) of pregnant rats. Glial cells were cultured during 4 weeks from cortical brain cells of pups born to these mothers. Several biochemical parameters were examined: protein levels, enzymatic markers of glial cell maturation (enolase and glutamine synthetase), superoxide dismutase a scavenger of free radicals produced during alcohol degradation. The results were compared to appropriate controls. A beneficent effect of Mn was observed for the pups weight which was no more significantly different from the control values. Protein levels, enolase and glutamine synthetase activities were increased mainly during the proliferative period when Mn was added to the alcohol diet compared to the only alcohol treated animals. This Mn effect was not found for superoxide dismutase in cultured glial cells but exists in the total brain of the 2 week-old offspring. In the total 2 and 4 week-old brain the alcohol induced decrease of enolase and glutamine synthetase was also antagonized by the Mn suplementation. Our data suggest that Mn may act as a factor overcoming at least partially some aspects of alcohol induced retardation of nerve cell development.     

Bioconversion potential of plant enzymes for the production of pharmaceuticals

Plant enzymes are able to catalyze regio- and stereospecific reactions. Freely suspended and immobilized plant cells as well as enzyme preparations can therefore be applied for the production of pharmaceuticals by bioconversion, as such or in combination with chemical syntheses. This review paper deals with bioconversions of added precursors from natural or synthetic origin by several biocatalytic systems.     

Phenol content, acidic peroxidase and IAA-oxidase during somatic embryogenesis in Theobroma cacao L.

Calli were induced in cacao cotyledon explants on a half-strength Murashige and Skoog medium containing 6 × 10-2 g m-3 saccharose and various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) with kinetin (kin), benzylaminopurine (BAP) or 2-isopentenylphosphate (2-iP). Experiments were carried out on two clones of cacao differing in their susceptibility to black pod disease. The highest percentage of explants forming callus and the most rapid callus development were obtained with 10-6 g m-3 2,4-D and 0.5× 10-6 g m-3 kin. Somatic embryogenesis and rhizogenesis were induced by transferring 3-week-old callus in a half strength Murashige and Skoog medium containing 3 × 10-2 g m-3 saccharose and NAA or IBA in the 0 to 5 × 10-6 g m-3 concentration range. No differentiation could be observed when the medium was supplemented with kin or BAP. The conversion of callus into somatic embryos and roots was accompanied by a drop in phenol content and an increase in peroxidase and IAA-oxidase activities. Moreover, cell differentiation was characterized by the persistence in the callus of one acidic soluble isoperoxidase which was not detected in nondifferentiating callus. Although some differences were noticed between the clones, alterations responsible for cell differentiation were the same in both genotypes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of lithium on hepatic drug-metabolizing enzymes of protein-deficient rats

Protein deficiency was produced by feeding synthetic 8%-protein diet. Lithium carbonate at the dose level of 1.1g/kg diet was administered to normal and protein-deficient rats for a period of one mo. A significant inhibition in the levels of cytochrome (cyt) P₄₅₀, cyt b₅, glutathione (GSH), glutathione S-transferase (GST) and glutathione peroxidase (GPx), but an increase in γ-glutamyl transpeptidase (γ-GT), was observed in low-protein LP-fed rats. Lithium treatment to normal rats caused no significant change in the activities of cyt P₄₅₀, cyt b₅, GST, and GSH levels, whereas there was elevation in the activities of γ-GT and GPx and suppression in glutathione reductase (GRd) activity. Lithium administration to LP-fed rats resulted in significant increases in the hepatic γ-GT and GPx activities.     

Influence des variations de la composition faunistique d'une population zooplanctonique sur l'expression des activités enzymatiques digestives au cours d'un cycle nycthéméral

Diel changes in the digestive enzyme activities of a Zooplankton population, captured with a Regent type net (700 μm mesh size) have been studied. During the period considered, the population was dominated by the appendicularian Oikopleura dioica Fol and several species of copepods. A clear rhythmicity was observed for most carbohydrases (laminarinase, amylase, cellulase, disaccharidases) but not for proteases. Changes in population composition explained a significant part of the enzymatic variability, although part of it still corresponded to nutritional rhythmicity. These results stress the importance of the physical and biological conditions of the environment when considering metabolic rhythmic changes at the population or the individual level.     

Molecular evidence using enzyme and RAPD markers for sympatric evolution in British species of Tetramesa (Hymenoptera: Eurytomidae)

Some species of the insect genus Tetramesa (Hymenoptera: Eurytomidae), which has a world‐wide distribution, are morphologically very similar, both in the adult and larval stages. In the British Isles, there are 37 recorded species, all of which feed on grasses as larvae and are largely host specific. Some form galls on their hosts; others do not. We used a range of enzyme and random amplified polymorphic DNA (RAPD) markers to investigate a complex of five cryptic species occurring sympatrically in the UK, collected from seven sites in mainland England and Wales: T. calamagrostidis (von Schlechtendal), T. longicornis (Walker) and T. petiolata (Walker) infesting different grass hosts, and T. hyalipennis (Walker) s.l. comprising two‐host adapted forms (labelled 1 and 2) reared from the grasses Elymus repens and E. farctus, respectively. Nine soluble enzyme systems (some known to be polymorphic in other insects) and 37 RAPD primers allowed taxonomic separation of the species. However, whilst RAPD markers were able to discriminate between the two host‐adapted forms of T. hyalipennis, enzyme markers (producing phenotypic profiles in the absence of genetic crosses) could not. Upon calculating genetic distances for the RAPD data from which a cladogram of Euclidean distances (relatedness) was produced along with multivariate analysis of the data, T. longicornis was shown to be the most ‘basal’ species, and most related to T. hyalipennis s.l.; T. calamagrostidis and T. petiolata were found to be more distantly related to these species but most closely related to each other. The two forms of T. hyalipennis s.l. appear to be the most closely related of any of the species investigated, probably diverging the most recently. From this data, and since the populations examined were all sympatric without obvious physical barriers to reproduction, it can be concluded that some degree of sympatric evolution has occurred, most obviously in the case of the host‐adapted forms of T. hyalipennis. If so, this complex of species could be another rare example of sympatric speciation in insects. Further research using more sophisticated molecular markers such as microsatellites, amplified fragment length polymorphic markers (AFLPs) and DNA sequencing (e.g. of mtDNA and ribosomal DNA regions), in conjunction with behavioural studies, are required to further elucidate this interesting species group. © 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 83 , 509–525.