Phylogeography of the red coral (Corallium rubrum): inferences on the evolutionary history of a temperate gorgonian.

The red coral Corallium rubrum (Cnidaria, Octocorallia) is an exploited, long-lived sessile species from the Mediterranean Sea and the adjacent coastline in the Atlantic Ocean. Surveys of genetic variation using microsatellites have shown that populations of C. rubrum are characterized by strong differentiation at the local scale but a study of the phylogeography of this species was still lacking. Here, we used seven polymorphic microsatellite loci, together with sequence data from an intron of the elongation factor 1 (EF1) gene, to investigate the genetic structure of C. rubrum across its geographical range in the western Mediterranean Sea and in the Adriatic Sea. The EF1 sequences were also used to analyse the consequences of demographic fluctuations linked with past environmental change. Clustering analysis with microsatellite loci highlighted three to seven genetic groups with the distinction of North African and Adriatic populations; this distinction appeared significant with AMOVA and differentiation tests. Microsatellite and EF1 data extended the isolation by distance pattern previously observed for this species at the western Mediterranean scale. EF1 sequences confirmed the genetic differentiation observed between most samples with microsatellites. A statistical parsimony network of EF1 haplotypes provided no evidence of high sequence divergence among regions, suggesting no long-term isolation. Selective neutrality tests on microsatellites and EF1 were not significant but should be interpreted with caution in the case of EF1 because of the low sample sizes for this locus. Our results suggest that recent Quaternary environmental fluctuations had a limited impact on the genetic structure of C. rubrum.

[Trypanosoma cruzi: trypomastigote forms in pleural fluid of a patient with AIDS]. / Trypanosoma cruzi: tripomastigotes en líquido pleural de un paciente con SIDA.

The presence of numerous trypomastigote forms of Trypanosoma cruzi in the pleural fluid of a patient with AIDS from Santiago del Estero, Argentina, was detected. Chagas disease is endemic in some countries of Latin America. To our knowledge, the finding of trypomastigote forms of T. cruzi in the pleural fluid has not yet been described in the literature.

Functional dynamic compartmentalization of respiratory chain intermediate substrates: implications for the control of energy production and mitochondrial diseases.

Activity defects in respiratory chain complexes are responsible for a large variety of pathological situations, including neuromuscular diseases and multisystemic disorders. Their impact on energy production is highly variable and disproportional. The same biochemical or genetic defect can lead to large differences in clinical symptoms and severity between tissues and patients, making the pathophysiological analysis of mitochondrial diseases difficult. The existence of compensatory mechanisms operating at the level of the respiratory chain might be an explanation for the biochemical complexity observed for respiratory defects. Here, we analyzed the role of cytochrome c and coenzyme Q in the attenuation of complex III and complex IV pharmacological inhibition on the respiratory flux. Spectrophotometry, HPLC-EC, polarography and enzymology permitted the calculation of molar ratios between respiratory chain components, giving values of 0.8:61:3:12:6.8 in muscle and 1:131:3:9:6.5 in liver, for CII:CoQ:CIII:Cyt c:CIV. The results demonstrate the dynamic functional compartmentalization of respiratory chain substrates, with the existence of a substrate pool that can be recruited to maintain energy production at normal levels when respiratory chain complexes are inhibited. The size of this reserve was different between muscle and liver, and in proportion to the magnitude of attenuation of each respiratory defect. Such functional compartmentalization could result from the recently observed physical compartmentalization of respiratory chain substrates. The dynamic nature of the mitochondrial network may modulate this compartmentalization and could play a new role in the control of mitochondrial respiration as well as apoptosis.

A simple-architecture fibered transmission system for dissemination of high stability 100 MHz signals.

We report on the development of a simple-architecture fiber-based frequency distribution system used to transfer high frequency stability 100 MHz signals. This work is focused on the emitter and the receiver performances that allow the transmission of the radio-frequency signal over an optical fiber. The system exhibits a residual fractional frequency stability of 1 × 10-14 at 1 s integration time and in the low 10-16 range after 100 s. These performances are suitable to transfer the signal of frequency references such as those of a state-of-the-art hydrogen maser without any phase noise compensation scheme. As an application, we demonstrate the dissemination of such a signal through a 100 m long optical fiber without any degradation. The proposed setup could be easily extended for operating frequencies in the 10 MHz-1 GHz range.

Initiation of DNA replication by DNA polymerases from primers forming a triple helix.

Despite extensive studies on oligonucleotide-forming triple helices, which were discovered in 1957, their possible relevance in the initiation of DNA replication remains unknown. Using sequences forming triple helices, we have developed a DNA polymerisation assay by using hairpin DNA templates with a 3' dideoxynucleotide end and an unpaired 5'-end extension to be replicated. The T7 DNA polymerase successfully elongated nucleotides to the expected size of the template from the primers forming triple helices composed of 9-14 deoxyguanosine-rich residues. The triple helix-forming primer required for this reaction has to be oriented parallel to the homologous sequence of the hairpin DNA template. Substitution of the deoxyguanosine residues by N7 deazadeoxyguanosines in the hairpin of the template prevented primer elongation, suggesting that the formation of a triple helix is a prerequisite for primer elongation. Furthermore, DNA sequencing could be achieved with the hairpin template through partial elongation of the third DNA strand forming primer. The T4 DNA polymerase and the Klenow fragment of DNA polymerase I provided similar DNA elongation to the T7 polymerase-thioredoxin complex. On the basis of published crystallographic data, we show that the third DNA strand primer fits within the catalytic centre of the T7 DNA polymerase, thus underlying this new property of several DNA polymerases which may be relevant to genome rearrangements and to the evolution of the genetic apparatus, namely the DNA structure and replication processes.

What do mitochondrial diseases teach us about normal mitochondrial functions...that we already knew: threshold expression of mitochondrial defects.

This paper shows how metabolic control analysis (MCA) can help to explain two important features of mitochondrial diseases: (i) the existence of a threshold in the expression of the complex deficiencies on the respiratory flux or on ATP synthesis, i.e. the fact that it is necessary to have a large complex deficiency in order to observe a substantial decrease in these fluxes; (ii) the tissue specificity, i.e. the fact that all tissues are not affected, even if the complex deficiency is present in all of them. We also show the limits of MCA, particularly when considering the in vivo situation. However, MCA offers a new way to consider mitochondrial diseases. The fact that fluxes only slightly change, when a complex is affected, is done at the expense of great changes in intermediate metabolite concentrations; intermediate metabolites situated upstream from the deficient complex are more reduced, leading to a greater generation of free radicals. This could bring an explanation for the diseases observed in conditions where the mitochondrial rate of ATP synthesis is only slightly affected.

Cloning of murine SeGpx cDNA and synthesis of mutated GPx proteins in Escherichia coli.

Glutathione peroxidase (GPx) of mammalian cells and Escherichia coli formate dehydrogenase both contain a selenocysteine (SeCys) in their amino acid (aa) sequence. In these two enzymes, this aa is encoded by a UGA codon, which is usually a stop codon for protein synthesis. We constructed plasmids to test the synthesis of GPx in E. coli. These constructions permitted high-level production of GPx mutants, where the SeCys codon was replaced by cysteine (UGC, UGU) or serine (UCA) codons, but synthesis of selenoprotein could not be detected: our data suggest that signals used for the recognition of the UGA codon as a SeCys codon are not conserved between E. coli and mammalian cells.

Sarcolemma phospholipid structure investigated by immunogold electron microscopy and (31)P NMR spectroscopy with lanthanide ions.

The biological functions of plasma membranes depend greatly on the biophysical properties resulting from protein and phospholipid structure. We investigated the phospholipid structure of the normal sarcolemma membrane, which is known to be highly dysfunctional in myopathies. Combining electron microscopy and (31)P nuclear magnetic resonance (NMR) spectroscopy on isolated sarcolemma vesicles, we find that (i) the sarcolemma vesicles maintain the in-vivo cellular sidedness, (ii) the phospholipid mobility is close to that observed in model membranes (similar lateral diffusion coefficients and spin-lattice T(1) relaxation times). Using broad-band and magic angle spinning (31)P NMR spectroscopy with lanthanide ions (Pr(3+)), it is possible to quantify the distribution of phospholipids between internal and external membrane layers, showing that the trans-bilayer distribution is highly asymmetrical.

Simultaneous effects of local dexfenfluramine application on extracellular glutamate and serotonin levels in rat frontal cortex: a reverse microdialysis study.

We examined the effects of local perfusion by reverse dialysis of various doses of dexfenfluramine (D-fen; in mM: 2.4, 12, and 24) simultaneously on serotonin (5-HT; [5-HT]ext) and glutamate (Glu; [Glu]ext) extracellular levels in the frontal cortex of awake rats. D-fen induced a dose-dependent increase in both [5-HT]ext and [Glu]ext, the latter being Ca2+ -dependent and TTX-sensitive, while the former is not. Pretreatment with either the neurotoxin p-chloroamphetamine or the 5-HT uptake blocker fluoxetine, markedly reduced the effects of D-fen on [5-HT]ext and [Glu]ext compared to controls. This indicates that intact 5-HT nerve terminals may be required for D-fen to enter into neurones to release 5-HT by reversal of the 5-HT transporter, which then increases frontocortical [Glu]ext. Pretreatment with the Glu uptake blocker, L-trans-pyrrolidine-2,4-dicarboxylic acid (1 mM), significantly reduced by 40% the effect of D-fen's on [Glu]ext suggesting that Glu uptake sites are partially involved in this effect. These results strongly suggest that intracortical application, by reverse dialysis, of a high dose of D-fen increases frontocortical [Glu]ext by a dual mechanism of action: (1) by stimulating 5-HT release (a major indirect effect) that, in turn, facilitates the release of neuronal Glu; (2) by reversal of the glutamate transporter (a minor direct effect being Ca2+ -independent and TTX-insensitive).

Effects of temperature on carp leukocyte mitogen-induced proliferation and nonspecific cytotoxic activity.

The effect of environmental temperature on immune competence was investigated in carp subjected to abrupt changes in water temperature. The activity of nonspecific cytotoxic cells (NCC) against P815 target cells and the lymphoproliferation induced by PHA and ConA were evaluated. Low in vivo temperature (12 +/- 0.5 degrees C) enhances NCC activity and decreases the mitogen effect of PHA, respectively; by contrast high in vivo temperature (28 +/- 0.5 degrees C) is without effect on these parameters as compared to the standard temperature (20 +/- 0.5 degrees C). The stress induced by environmental temperature variation was evaluated by plasmatic cortisol measurement. Results indicate a significant increase (p < 0.05) of cortisol levels 2 hours after transfer at low but not at high temperature as compared to the standard thermic control (60.96 +/- 17.08 vs. 16.74 +/- 4.32 ng/mL, respectively). Because only approximately 20 minutes are required before carp body and environmental temperatures are identical, the same experiments were reproduced in vitro. Results show trends similar to those found in vivo. Taken together, these data reveal a direct effect of temperature on immune cellular functions and indicate that nonspecific immunity tends to offset specific immune suppression at low environmental temperature.

Microdialysis monitoring of variations in extracellular levels of serotonin, GABA and excitatory amino acids in the frontal cortex of awake rats in response to a single peripheral or central administration of dexfenfluramine.

The effects of a single dexfenfluramine (D-fen) administration on the release of endogenous serotonin (5-hydroxytryptamine, 5-HT), excitatory (glutamate, Glu, aspartate, Asp) and inhibitory (gamma-aminobutyric acid, GABA) amino acids from the frontal cortex were studied by using in vivo microdialysis in freely-moving rats. Extracellular levels of these neurotransmitters were measured with HPLC coupled to electrochemical detection or with capillary electrophoresis coupled to laser-induced fluoresence detection (CE-LIFD). In a first study, single intraperitoneal administration of D-fen (0.5, 1.3, 5 and 10 mg/kg) increased extracellular 5-HT levels in a dose-dependent manner (maximal increase by 982% over baseline for the highest dose) while changes in Glu, Asp or GABA never reached statistical significance. In a second study, 73 nM of D-fen applied locally through the frontocortical dialysis probe, at a flow rate of 1.5 microliters/min in 30 microliters of perfusion fluid for 20 min, increased extracellular 5-HT and Asp levels [the maximal increases were to 1804% and 280% of the respective basal values (100%)] without altering extracellular levels of Glu and GABA. Thus, the order of magnitude of the changes induced by systemic administration or local infusion of D-fen on frontocortical extracellular levels of several neurotransmitters (5-HT > > Asp > GABA = Glu) demonstrate that D-fen, an indirect serotoninergic agonist, mainly increases 5-HT release while producing slight (Asp) or no (Glu, GABA) short-term in vivo variations in amino acid extracellular levels in the rat frontal cortex.

Effects of repeated systemic administration of d-Fenfluramine on serotonin and glutamate release in rat ventral hippocampus: comparison with methamphetamine using in vivo microdialysis.

We compared the effects of three systemic injections of various doses of d-Fenfluramine, an indirect serotonergic agonist (1.3, 5 or 10 mg/kg), to those of a known neurotoxin, methamphetamine (METH, at a 7.5 mg/kg dose), given i.p. at 2-h intervals, simultaneously on extracellular levels of glutamate [Gluext] and 5-HT [5-HText] in the ventral hippocampus (VHPC) using in vivo microdialysis in conscious rats. METH markedly increased both [Gluext] (+77% over the control value in saline-treated rats) and [5-HText] (around +250% over controls) in the VHPC. d-Fenfluramine, at all the doses studied, induced gradual increases of [5-HText] in the VHPC (between +125% and +417% over control values), but did not modify [Gluext]. These data highlight marked in vivo differences between METH and d-Fenfluramine in their effects on extracellular levels of 5-HT and Glu in the rat ventral hippocampus following their repeated systemic administration.

Effect of pulsed magnetic fields on triglyceride and cholesterol levels in plasma of rats.

BACKGROUND: Liver is a crucial organ in metabolism. For instance liver is the main source of circulating lipoproteins. METHODS: In this paper cholesterol and triglyceride plasma levels were measured in male rats previously exposed to pulsed magnetic fields (PMF) used in therapy. Rats underwent a one-hour exposure to a 6 mT 12 Hz PMF. RESULTS: Twenty-four hours after the end of the exposure to the PMF the rats' livers were heavier, cholesterol and triglyceride plasma levels decreased. All these variations were significantly different according to a variance ratio test as was a rebound in triglyceride level 48 hours after the end of the exposure. Normal values were observed 48 and 96 hours after the end of exposure respectively for cholesterol and triglycerides. CONCLUSIONS: These alterations may be due to a reversible accumulation of either triglycerides or of their precursors in liver following acute exposure to a 12 Hz PMF.

The effect of a pulsed magnetic field and that of methyl-silane triol on galactosamine induced hepatitis among rats.

Liver cell necrosis was induced in rats by a galactosamine injection. Cell death was due to an increase of Ca++ intracellular levels and was also under the control of genes. Rats were then either exposed or not to a 6 mT 100 HZ pulsed magnetic field (PMF) and they either received or not methylsilane-triol injections. Animals were sacrificed twenty-seven hours after a galactosamine injection. On the one hand it appeared from transaminase levels that the PMF increased the number of animals which were sensitized to galactosamine but decreased transaminase levels. On the other hand PMF decreased the protective effect of MST against galactosamine. We may suggest that PMF should be considered as an additional cellular signal received through genes which would determine the evolution towards or against apoptosis according to the age of the cell itself but also the Ca++ intracellular level.

Exposure of mice to pulsed magnetic fields: long-term observations.

This work was undertaken to check the effects of 460 Hz pulsed magnetic fields (PMF) on the growth curves of mice, and to observe any possible long term effect. Effects on the growth curves have already been shown in young female AKR mice. Eight-week-old Swiss female mice underwent, until death, a twice weekly a one-hour-exposure to a 460 Hz 6 mT PMF. They initially showed slower than that of the controls, but it later became faster, 460 Hz 9 mT PMF always kept the growth curves of the exposed mice under those of the controls. However, no difference with controls was noticed when the mice were exposed after the thirtieth week. Moreover, there was no difference in the survival or in pathological aspects between the exposed mice and controls.

Effect of a pulsed magnetic field and of first cold-pressure sunflower oil on mice.

In previous studies it has been shown that exposure of mice to a 12-Hz 6 mT unipolar square pulsed magnetic field (PMF) suppressed the excess of weight due to application of 1st cold-pressure sunflower oil. This time we considered the effect of oil and/or PMF on the growing curves lifespans of mice. The exposure took place for 30 min 5 days a week, from the 7th week of life to death. The results are 1) a broken slope in the growing curves from the 125th day of aging: the exposed mice were lighter than the controls, keeping the differences between the growing curves needed a repeated exposure all life long; 2) a significant increase in the lifespan of the controls which received oil versus the controls which received water; 3) an increase in the lifespan of the exposed mice versus the non-exposed control batches. On one hand it has been reported that essential polyunsaturated fatty acids found in first cold-pressure sunflower oil played a prominent role in membrane structures and in immune equilibrium. On the other hand, it was shown that oscillating electric fields could activate Na+K+-ATPase.

Effect of pulsed magnetic fields on cholesterol and tryglyceride levels in rats study of field intensity and length of exposure.

In a previous work a decrease in cholesterol and triglyceride plasma levels was observed in rats 24 hours after their exposure to a 12 Hz 6 mT pulsed magnetic field (PMF). This time, a study of intensity effects of a 12 Hz PMF for a sixty-minute exposure and of length of exposure for a 12 Hz 6 mT PMF took place. Non-linear effect-dose relationships were observed for the PMF intensity as well as for the length of exposure used. The highest decreases in cholesterol and triglyceride levels were obtained after to a sixty-minute exposure with 1.5 mT and 12 mT.

Mitochondrial energetic and AKT status mediate metabolic effects and apoptosis of metformin in human leukemic cells.

Previous reports demonstrate that metformin, an anti-diabetic drug, can decrease the risk of cancer and inhibit cancer cell growth. However, its mechanism in cancer cells is still unknown. Metformin significantly blocks cell cycle and inhibits cell proliferation and colony formation of leukemic cells. However, the apoptotic response to metformin varies. Furthermore, daily treatment with metformin induces apoptosis and reduces tumor growth in vivo. While metformin induces early and transient activation of AMPK, inhibition of AMPKα1/2 does not abrogate anti-proliferative or pro-apoptotic effects of metformin. Metformin decreases electron transport chain complex I activity, oxygen consumption and mitochondrial ATP synthesis, while stimulating glycolysis for ATP and lactate production, pentose phosphate pathway for purine biosynthesis, fatty acid metabolism, as well as anaplerotic and mitochondrial gene expression. Importantly, leukemic cells with high basal AKT phosphorylation, glucose consumption or glycolysis exhibit a markedly reduced induction of the Pasteur effect in response to metformin and are resistant to metformin-induced apoptosis. Accordingly, glucose starvation or treatment with deoxyglucose or an AKT inhibitor induces sensitivity to metformin. Overall, metformin elicits reprogramming of intermediary metabolism leading to inhibition of cell proliferation in all leukemic cells and apoptosis only in leukemic cells responding to metformin with AKT phosphorylation and a strong Pasteur effect.