Assessment and monitoring of water quality of the gulf of Morbihan, a littoral ecosystem under high anthropic pressure.

This field study is intended to propose a global methodology to assess and monitor the water quality of the gulf of Morbihan, a littoral ecosystem under increasing anthropic pressure. To this end, the Locmariaquer site, where Crassostrea gigas is extensively cultivated, was selected to perform a one-year follow-up of tissular glutathione S-transferase and acetylcholinesterase specific activities in this filter feeder organism. Calculation of an integrated index, corresponding to the ratio of the two enzymes activities, allowed to discriminate from the environmental noise, several clusters which could be representative environmental stress, potentially latent pollution. Moreover, the estrogenic activity was assessed in water samples collected at Locmariaquer and other strategic sites of the gulf. The results evidenced a low estrogenic-disrupting compound contamination of waters. Overall, this methodology produced an accurate outlook of a basal state for the gulf and could be developed in the context of a chronic monitoring of this site.

Interplay of flexibility and stability in the control of estrogen receptor activity.

Previously, we have identified an imperfect estrogen response element (rtERE) in the promoter of the rainbow trout vitellogenin gene. Although this ERE leads to a lower transcriptional activation, a better estradiol stimulation in vivo as compared to consensus ERE (EREcs) was observed. Here we examine the ability of recombinant human estrogen receptor alpha (rhERalpha) to bind DNA containing the EREcs or the natural imperfect rtERE, which contains three mismatches. At low salt concentration, whatever the ERE sequence, dissociation equilibrium constants of the specific rhERalpha-ERE complexes are similar (K(D) = 2 nM) with the same stoichiometry. As salt concentration increases from 80 to 200 mM KCl, the affinity of the rhERalpha-rtERE complex largely diminishes whereas that of rhERalpha-EREcs seems less affected. Hence the nature of the interactions stabilizing these complexes is different: more ionic in rhERalpha-rtERE as compared to rhERalpha-EREcs. Moreover, kinetic measurements showed that specific rhERalpha-ERE complexes exhibit shorter half-lives (few seconds) and that the rhERalpha-EREcs complex is more stable (33 s) than the complex that formed with rtERE (19.8 s), in accordance with equilibrium binding results. Finally, dynamic studies of rhERalpha have shown that the protein fluctuations are damped when the salt concentration increases or when bound to ERE and all the more with rtERE. The interplay of affinity, complex half-lives, and protein dynamics in the transcriptional regulation of estrogen receptor is discussed.

Metabolic imaging of tissues by infrared fiber-optic spectroscopy: an efficient tool for medical diagnosis.

Infrared fingerprints of molecules in biology contain much information on cells metabolism allowing one to distinguish between healthy and altered tissues. Here, to collect infrared signatures, we used evanescent wave spectroscopy based on an original infrared transmitting tapered glass fiber. A strict control of the fiber diameter in the tapered sensing zone allows high sensitivity and wide spectral range exploration from 800 to 3000 cm(-1). Then, merely in depositing the mouse liver biopsies on the fiber, this device has enable us to differentiate between tumorous and healthy tissues.

Bacterial swarming: a biochemical time-resolved FTIR-ATR study of Proteus mirabilis swarm-cell differentiation.

Fourier transform infrared spectroscopy was applied to the study of the differentiation process undergone by Proteus mirabilis. This bacterium exhibits a remarkable dimorphism, allowing the cells to migrate on a solid substratum in a concerted manner yielding characteristic ring patterns. We performed an in situ noninvasive analysis of biochemical events occurring as vegetative cells differentiate into elongated, multinucleate, nonseptate, and hyperflagellated swarm cells. The major findings arising from this study are (i) the real-time monitoring of flagellar filament assembly, (ii) the evidence for de novo synthesis of qualitatively different lipopolysaccharides (LPS) and/or exopolysaccharides (EPS) constituting the slime into which bacteria swarm, and (iii) the alteration in the membrane fatty acid composition with a concomitant 10 degrees C decrease in the gel/liquid crystal phase transition resulting in an elevated membrane fluidity in swarm cells at the growth temperature. The time course of events shows that the EPS-LPS syntheses are synchronous with membrane fatty acid alterations and occur about 1 h before massive flagellar filament assembly is detected. This study not only provided a time sketch of biochemical events involved in the differentiation process but also led to the identification of the major spectral markers of both vegetative and swarm cells. This identification will allow to resolve the time-space structure of P. mirabilis colonies by using infrared microscopy.

Glycine betaine-assisted protein folding in a lysA mutant of Escherichia coli.

Osmoprotectants exogenously supplied to a hyperosmotic culture medium are efficiently imported and amassed by stressed cells of Escherichia coli. In addition to their evident role in the recovery and maintenance of osmotic balance, these solutes should play an important role on the behavior of cellular macromolecules, for example in the process of protein folding. Using a random chemical mutagenesis approach, a conditional lysine auxotrophic mutant was obtained. The growth of this mutant was restored by addition of either lysine or osmoprotectants including glycine betaine (GB) in the minimal medium. The growth rate increased proportionally with the augmentation of the intracellular GB concentration. The mutation was located in the lysA gene and resulted in the substitution of the Ser at position 384 by Phe of the diaminopimelate decarboxylase (DAPDC), which catalyzes the conversion of meso-diaminopimelate to L-lysine. We purified both the wild type DAPDC and the mutated DAPDC-sf and demonstrated that GB was capable of activating DAPDC-sf in vitro, thus confirming the in vivo results. Most importantly, we showed that the activation was correlated with a conformational change of DAPDC-sf. Taken together, these results show, for the first time, that GB may actively assist in vivo protein folding in a chaperone-like manner.

Probing pH and pressure effects on the apomyoglobin heme pocket with the 2'-(N,N-dimethylamino)-6-naphthoyl-4-trans-cyclohexanoic acid fluorophore.

The environmentally sensitive fluorophore 2'-(N,N-dimethylamino)-6-naphthoyl-4-trans-cyclohexanoic acid (DANCA) has been used to probe the apomyoglobin heme pocket. The unexpected polarity of this domain is generally interpreted as arising from dynamic dipolar relaxation of the peptide dipoles surrounding the heme pocket. In the present work we reexamine the photophysical properties of DANCA in a variety of solvents and complexed with apomyoglobin (apoMb) to further probe the heme pocket environment as a function of external solvent conditions. Absorption and excitation spectra in a number of solvents are consistent with the well-known pi*<--pi (LE) and pi*<--n (CT) electronic absorption transitions observed for naphthylamine derivatives. Dual emission is also a well-documented property of such derivatives. Based on the time scale of the heterogeneity in the decay of the DANCA fluorophore observed in a series of solvents, we propose that the emission properties of DANCA in apoMb are not uniquely attributable to dynamic relaxation events, but also reflect dual emission from both a long-lived, red CT state and the shorter-lived, blue LE state. The pH studies in the range of pH 5-9 of the emission properties of DANCA in apoMb support this hypothesis. They also suggest a specific interaction of DANCA with one or both of the pocket histidyl residues, which leads to a drastic static quenching and red shift of the bound DANCA fluorescence upon protonation. Similar effects are observed with increasing pressure, indicating that these two perturbations alter the DANCA-apoMb complex in a similar fashion. The pressure-induced form of the protein is distinct both energetically and structurally from the previously characterized acid intermediate, in that it is populated above pH 5 and retains a significant degree of integrity of the heme pocket.

An infrared study of 2H-bond variation in myoglobin revealed by high pressure.

A high-pressure Fourier-transform infrared technique was used to probe the evolution of 2H bonds inside the helical segments of myoglobin in relation to p2H, Tris concentration in the medium and iron-ligand nature. The analysis was focused on changes in the conformation-sensitive amide-I' band, reflecting the peptide C = O group stretching vibrations coupled to the in-plane N-2H bending and C = N stretching modes. From data obtained under high pressure, the strength of 2H bonds, inside the alpha-helical segments of the protein at atmospheric pressure, is not simply a function of p2H and salt concentration. At low Tris concentration (50 mM), the strength of these 2H bonds increases with p2H, whereas for a higher Tris concentration (100 mM) this strength is lower at p2H 7 than at p2H 6.0 or 8.5. It is also observed that the azidometmyoglobin molecule exhibits tighter intrahelical interactions and lower sensitivity to pressure than aquametmyoglobin. Information is also presented regarding interhelical interactions in relation to the solvent.

Variations of electrostatic interactions in myoglobin probed by UVRR spectroscopy. Effect of iron ligand and pH on tryptophan and tyrosine vibrations.

The present work investigates the variations of electrostatic interactions within the myoglobin molecule associated with azide heme binding and pH variations. Far ultraviolet (223 nm) resonance Raman spectroscopy of the tryptophan and tyrosine residues, along with acid-base titration measurements, have been used to monitor variations in the protein matrix. With previously determined mode assignments, it is shown that the Trp and Tyr residues of the globin moiety are influenced by the charge spatial distribution. Upon ligand binding or under various pH conditions, the polar interactions inside the protein appear to be modulated by the electric field generated by the charge array. It is concluded that the binding site properties of myoglobin can be modulated by the charge spatial distribution within the protein, even in the absence of measurable conformational changes of the bulk.

Relationship between protein/solvent proton exchange and progressive conformation and fluctuation changes in hemoglobin.

The pH dependence of the tertiary structural changes and the quaternary reorganization of the alpha beta interface of oxyhemoglobin in solution has been previously observed in our laboratory. The present work aims to establish whether or not these progressive structural changes with pH result from proton exchange between the protein and the solvent. We therefore have used infrared spectroscopy, acid/base titration and 1H/2H exchange to assess the effect of external proton concentration on the structural and dynamic properties of the hemoglobin molecule in solution. This study is also performed on the carbonmonoxy form since the affinity of hemoglobin for CO is much higher than for oxygen. The present findings demonstrate that affinity changes are closely related to protein fluctuations, as well as structural modifications. An increased affinity, induced either by ligand replacement or pH variation, is associated with the constrained chains exhibiting a lower degree of fluctuation, together with a looser alpha beta interface association.

[Multicenter study of the sensitivity to beta-lactam antibiotics of 1,707 strains of Pseudomonas aeruginosa isolated at a general hospital]. / Etude multicentrique de la sensibilité aux bêtalactamines de 1,707 souches de Pseudomonas aeruginosa isolées en hôpital général.

1,707 non replicate clinical strains of Pseudomonas aeruginosa from non teaching hospitals were investigated. Beta-lactam antibiotics were tested by agar disk diffusion method. Variance analysis revealed a significant interaction between strains and experimental laboratories. The bacterial population was grouped according to the diameters of inhibition zones of antibiogram, into five phenotypes. Pseudomonas aeruginosa is frequently isolated from urines and pulmonary sampling, rarely from blood cultures. Resistance to beta-lactams increased significantly with the duration of hospitalisation, and age of patients. Clinical strains are significantly more resistant in blood cultures, and for strains isolated from reanimation. 84% of strains are susceptible to ticarcillin.

Changes of fatty acid composition of phospholipids and lipid structural order in rat liver mitochondrial membrane subsequent to galactosamine intoxication. Effect of clofibrate.

Since it has been earlier reported that D-galactosamine induces an inhibition of palmitoylcarnitine transferase I and a depletion of mitochondrial phospholipids which were both prevented by clofibrate, an evaluation of the effects of these drugs on mitochondrial fatty acid composition was made. Galactosamine does not alter the fatty acid pattern of these fatty acids whereas clofibrate induces a 2-fold increase in monounsaturated/saturated fatty acids ratio and a 10-fold decrease of the 20:4 (n - 6)/20:3 (n - 6) ratio in phosphatidylcholine. These alterations suggest an increase of delta 9-desaturation and a decrease of delta 5-desaturation. To determine whether the drug-induced changes in mitochondrial phospholipids has an effect on the physical properties of the membrane, the lipid structural order of mitochondrial preparations was studied using the lipophilic probes DPH and TMA-DPH. Mitochondrial isolated either from galactosamine- or clofibrate-treated rats showed a decrease in fluorescence polarization, indicating an overall decrease in lipid structural order. This alteration is more drastic when both drugs are administered. This phenomenon suggests drastic changes in the bulk phase of inner mitochondrial membrane lipids after treatments and could explain the altered kinetic properties of palmitoylcarnitine transferase I.

Preventive effect of clofibrate on carnitine palmitoyltransferase I inhibition and mitochondrial membrane phospholipid depletion induced by galactosamine.

We have previously reported that a D-galactosamine injection induces a decrease of carnitine palmitoyltransferase I activity correlated with a depletion of total phospholipid content in the mitochondrial membrane. The impact of a short-term clofibrate treatment on these membrane alterations is investigated, i.e., the kinetic properties of carnitine palmitoyltransferase I, including its sensitivity to malonyl-CoA and mitochondrial membrane content of the various phospholipids. A 4-day clofibrate treatment increases by 42% the apparent Km value of carnitine palmitoyltransferase I for palmitoyl-CoA, while the sensitivity of the enzyme to malonyl-CoA appears slightly decreased. Simultaneously, the cardiolipin content is increased by 70% in the mitochondrial membrane, whereas the phosphatidylethanolamine and phosphatidylcholine contents remain almost unaffected. This 4-day clofibrate treatment prevents the inhibition of carnitine palmitoyltransferase I activity subsequent to galactosamine administration but induces an increase in the apparent Km value for palmitoyl-CoA and a decrease of the sensitivity of the enzyme to malonyl-CoA. The contents of phospholipids which are decreased by galactosamine (phosphatidylcholine, -21%; phosphatidylethanolamine, -29%; cardiolipin, -40%) regain the control values when galactosamine administration is preceded by a clofibrate treatment. The data suggest that the clofibrate treatment counteracts the inhibition of activity of carnitine palmitoyltransferase I through the maintenance of mitochondrial membrane integrity.

Effect of D-galactosamine in vitro on [U-14C]palmitate oxidation, triacylglycerol synthesis and secretion in isolated hepatocytes.

Isolated rat hepatocytes were used to study in vitro effects of 10 mM D-galactosamine (GalN) on hepatic fatty acids metabolism. At this concentration, membrane integrity and biochemical competence (i.e., gluconeogenesis and ureogenesis) remained unaffected. Protein synthesis and secretion, as measured by the incorporation of [U-14C]leucine into total and medium protein, was significantly inhibited when incubated for more than 2 h. GalN activated the incorporation of [U-14C]palmitate into triacylglycerols and depressed its utilization in the formation of labelled ketone bodies and 14CO2. Hepatocytes isolated from fasted rats exposed to GalN in vitro did not show any variation in prelabelled triacylglycerol secretion. GalN induced a rapid inhibition of prelabelled triacylglycerol secretion by hepatocytes isolated from fed rats in which this secretion occurred to a larger extent than in hepatocytes isolated from fasted rats. The data reported here suggest that GalN induces a rise of triacylglycerol synthesis by inhibiting the palmitate oxidation pathway and a decrease of triacylglycerol secretion through an early derangement of the secretory pathway.

Carnitine palmitoyltransferase I. Inhibition by D-galactosamine and role of phospholipids.

Palmitate oxidation by liver mitochondria from rats treated with D-galactosamine (GalN) was markedly inhibited, 3 h after administration. The mitochondrial defect responsible for this inhibition was shown to be an inhibition of the activity of palmitoylcarnitine transferase I (EC 2.3.1.21). Apparent Km of the enzyme remained unchanged whereas apparent V was reduced by 30%. Addition of 10 mM GalN did not impair the activity of palmitoylcarnitine transferase I in mitochondria isolated from normal rats. Inhibition of palmitoylcarnitine biosynthesis by GalN treatment was completely reversed by phospholipid supply. At this stage of intoxication, mitochondrial phospholipid content was decreased whereas incorporation of [14C]palmitate into phospholipids in isolated hepatocytes was drastically inhibited: the phosphatidylcholine/phosphatidylethanolamine ratio was reduced by 33%. The results obtained from these studies show that the depletion of the phospholipid membrane content could account for the altered functional activity of palmitoylcarnitine transferase I.

In vitro metabolism of progesterone and 17 alpha-hydroxyprogesterone in the testis of the rainbow trout, Salmo gairdneri Rich., at different stages of spermatogenesis.

Rainbow trout testicular fragments were incubated at 8 different stages of the spermatogenetic cycle in the presence of tritiated progesterone or 17 alpha-hydroxyprogesterone. This paper describes the potential enzymatic activities involved in the delta 4 route leading to the production of "major" derivatives. 1) A 5-reductase converted progesterone into 5 alpha (beta)-pregnane-3,20-dione. 2) A 20 beta-oxydoreductase converted 17 alpha-hydroxyprogesterone into 17 alpha-hydroxy-20 beta-dihydroprogesterone. At some stages this synthesis reached or exceeded androgen production. 3) 20 beta-oxydoreductase, 17-20 desmolase and 17 beta-oxydoreductase are proposed as putative enzymatic steps in the regulation and modulation of steroidogenic metabolism in the trout testis. 4) The physiological role of the different major steroids synthesized by the testis in vitro is discussed, and the hypothesis of the involvement of 17 alpha-hydroxy-20 beta-dihydroprogesterone in the control of germ cell mitotic activity and/or feedback on the hypothalamo-pituitary system is examined.

Influence of previous diuretic intake on the humoral and hormonal profile of idiopathic oedema.

Three groups of women underwent water loading tests: normal subjects, idiopathic oedema patients who had taken no medication for at least 3 months, and a second oedema group with recent diuretic intake. Idiopathic oedema in drug-free patients was characterized by an abnormal capillary permeability, lower basal protein values, a dramatic drop in urinary output in the upright position due to reduced glomerular filtration, enhanced reabsorption of sodium and water and stimulation of aldosterone and antidiuretic hormone (AVP) secretions. In these idiopathic oedema cases, osmolar AVP regulation was disrupted but AVP control by plasma volume was maintained. In the basal state, patients with recent diuretic intake were characterized by a gain in body weight and by depletion of plasma volume and plasma potassium. In these subjects, urinary output in the upright posture was as insufficient as in drug-free patients but was due to higher sodium reabsorption. Renal insensitivity to AVP action was also observed. Osmolar regulation and volume regulation of AVP were both disrupted in these cases with recent diuretic intake.

Antihypertensive and hormonal effects of single oral doses of captopril and nifedipine in essential hypertension.

In a single-dose crossover study Captopril (SQ 14225), 1 mg/kg body weight, and Nifedipine (Bay a 1040) 20 mg were administered orally to 12 hospitalized patients with essential hypertension (Stage 1 or 2, W.H.O.). Both drugs significantly reduced blood pressure, but each dose acted differently: the mean maximum arterial pressure reduction was faster and greater with Nifedipine than with Captopril: -23 +/- 2% at 37 +/- 15 min and -17 +/- 1% at 86 +/- 25 min, respectively. Captopril inhibited angiotensin II and aldosterone production, but did not accelerate heart rate or stimulate vasopressin release. Nifedipine stimulated vasopressin release and increased heart rate, but the renin angiotensin aldosterone system was not significantly affected. The blood pressure reduction was related to the initial level of activation of the renin angiotensin system only for Captopril. The blood pressure reduction induced by one drug was not related to that produced by the other in the same patient.

In vitro effect of a fish gonadotropin on aromatase and 17 beta-hydroxysteroid dehydrogenase activities in the ovary of the rainbow trout (Salmo gairdneri Rich.).

Aromatase activity has been studied in the rainbow trout ovary in a perifusion system. During early vitellogenesis, as in other stages of the annual cycle, the ovaries were capable of aromatizing androstenedione and testosterone. Aromatase activity increased from the postovulatory stages to exogenous vitellogenesis. When highly purified salmon glycoprotein maturational gonadotropin was added to the medium (50 ng/ml), the androstenedione/testosterone ratio was reduced by half. This indicates a stimulation of 17 beta-hydroxysteroid dehydrogenase activity favourizing the formation of testosterone. At a concentration of 50, 150 or 300 ng/ml, the gonadotropin inhibited between 13 and 38 p. 100 of the aromatase enzymatic activity.