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V Robert

Publications and source records attributed to V Robert.

At least 73 records · Page 4Linked to original sources
PubMed1998

Direct monitoring of the calcium concentration in the sarcoplasmic and endoplasmic reticulum of skeletal muscle myotubes.

Direct monitoring of the free Ca2+ concentration in the sarcoplasmic reticulum (SR) was carried out in rat skeletal myotubes transfected with a specifically targeted aequorin chimera (srAEQ). Myotubes were also transfected with a chimeric aequorin (erAEQ) that we have demonstrated previously is retained in the endoplasmic reticulum (ER). Immunolocalization analysis showed that although both recombinant proteins are distributed in an endomembrane network identifiable with immature SR, the erAEQ protein was retained also in the perinuclear membrane. The difficulty of measuring [Ca2+] in 100-1000 microM range was overcome with the use of the synthetic coelenterazine analogue, coelenterazine n. We demonstrate that the steady state levels of [Ca2+] measured with srAEQ is around 300 microM, whereas that measured with erAEQ is significantly lower, i.e. around 200 microM. The effects of caffeine, high KCl, and nicotinic receptor stimulation, in the presence or absence of external calcium or after blockade of the Ca-ATPase, were investigated with both chimeras. The kinetics of [Ca2+] changes revealed by the erAEQ were similar, but not identical, neither quantitatively nor qualitatively, to those monitored with the srAEQ, indicating that at this stage of muscle development, differences exist between SR and ER in their mechanisms of Ca2+ handling. The functional implications of these findings are discussed.

Aequorin↗
PubMed1998

De novo synthesis of arginine and ornithine from citrulline in human colon carcinoma cells: metabolic fate of L-ornithine.

In human colon carcinoma cells (HT-29 cells), L-arginine is the common precursor of L-ornithine which generates polyamines strictly necessary for cellular growth, and nitric oxide which has a strong antiproliferative activity. We show here that proliferative HT-29 cells possess the capacity for de novo synthesis of L-arginine from L-citrulline, but not from L-ornithine. L-Ornithine is apparently not an L-arginine precursor due to the absence of any detectable ornithine carbamoyltransferase activity. In contrast, the newly synthesized L-arginine was competent for urea and thus L-ornithine production in a context of a high putrescine production in the ornithine decarboxylase pathway and a low degradation of this polyamine in the diamine oxidase pathway. However, cells grown in an arginine-free culture medium containing added L-citrulline were unable to reach confluency. Furthermore, the low amount of nitric oxide produced from L-arginine by these cells was apparently not involved in the control of cell growth since inhibition of nitric oxide synthase activity was without effect. On the other hand, the capacity of more differentiated and less proliferative HT-29 cells for de novo L-arginine synthesis from L-citrulline was increased. It is concluded that L-citrulline is a precursor of L-arginine and L-ornithine in proliferative HT-29 cells and that the metabolic fate of L-ornithine in these cells is mainly devoted to polyamine synthesis. The similarity between differentiated HT-29 cells and the enterocytes of newborn animals in terms of L-arginine metabolism is finally discussed.

Animals↗
PubMed1998

Alpha-difluoromethylornithine (DFMO) as a potent arginase activity inhibitor in human colon carcinoma cells.

Alpha-difluoromethylornithine (DFMO) is commonly used as a specific ornithine decarboxylase (ODC, EC4.1.1.17) irreversible inhibitor. ODC is the enzyme responsible for polyamine biosynthesis, which has been shown to be strictly necessary for cell proliferation. In HT-29 Glc-/+ cells, L-arginine is the major precursor of these molecules through the sequential actions of arginase, which leads to L-ornithine generation and ODC. L-ornithine, a substrate for ODC, retroinhibits arginase. Since DFMO is an ornithine analogue, we searched for a direct effect of this agent upon arginase. The flux of L-arginine through arginase in intact cells was inhibited by 51+/-11% by 10 mM of DFMO whereas 10 mM of L-valine, a known potent arginase inhibitor, inhibited this flux by 73+/-6%. DFMO equilibrated between extracellular and intercellular spaces and, when used at 10-mM concentration, was without effect on L-arginine net uptake. Measurement of arginase activity in HT-29 cell homogenates with increasing concentrations of DFMO and L-arginine led to an inhibition with a calculated Ki (inhibitory constant) equal to 3.9+/-1.0 mM. L-ornithine was less effective than DFMO in inhibiting arginase activity. Bovine liver arginase, used as another source of the enzyme, was also severely inhibited by DFMO. The inhibitory effect of DFMO upon arginase, one step upstream of the ODC reaction in the metabolic conversion of L-arginine to polyamines, is of potential physiological importance, since it could alter the production of ornithine and thus its metabolism in pathways other than the ODC pathway.

Animals↗
PubMed1998

Differential inhibitory effects of three nitric oxide donors on ornithine decarboxylase activity in human colon carcinoma cells.

Ornithine decarboxylase (ODC, EC 4.1.1.17) is the enzyme responsible for the synthesis of polyamines, which are absolutely necessary for cell proliferation. In the present work, we tested the effects of 3 nitric oxide (NO) donors, namely, sodium nitroprusside (SNP), (Z)-1-(N-methyl-N-[6-(N-methylammoniohexyl)amino] diazen-1-ium-1,2-diolate (MAHMA/NO) and 1,1-diethyl-2-hydroxy-2-nitroso-hydrazine sodium (DEA/NO), on ODC activity in human-colon carcinoma cells (HT-29). SNP was the most effective inhibitor of ODC activity with a concentration of 8 micromol/L inducing 50% inhibition of basal activity. The effect of SNP was reversed by haemoglobin (Hb), but not by GSH or L-cysteine (CYS). Very little of the SNP in solution was degraded into nitrite, but the presence of cellular homogenate increased the production of nitrite. MAHMA/NO and DEA/NO were much less effective than SNP as ODC inhibitors, since the concentrations of these agents which induce 50% inhibition of basal activity were 20- to 60-fold higher than that of SNP. The effects of MAHMA/NO and DEA/NO were not reversed by haemoglobin. In solution, these latter 2 agents were totally degraded into nitrites. In conclusion, SNP on the one hand and MAHMA/NO and DEA/NO on the other appeared to release different NOx species with different efficiency on ODC activity.

Dose-Response Relationship, Drug↗
PubMed1998

Identification of human p53 mutations with differential effects on the bax and p21 promoters using functional assays in yeast.

Recent studies have suggested that a rare class of p53 mutants found in tumours has a subtle transcriptional defect affecting bax induction but not p21 induction. We have therefore developed simple functional assays in yeast which can be used to identify these mutants. Analysis of 51 different mutations observed in human tumours showed that all mutants tested scored as mutant with the bax reporter strain but nine scored as wild-type with the p21 reporter strain. These results, which can be explained by the lower affinity of the p53 protein for the bax site, may suggest that p21 is not the key target of p53 mutations in tumours. Since p21 status has recently been shown to modulate the chemotherapeutic and radiotherapeutic sensitivities of cancerous cells, the functional assays described here may have important clinical implications.

Base Sequence↗
PubMed1998

Myocardial production of aldosterone and corticosterone in the rat. Physiological regulation.

Increasing evidence suggests that mineralo- and glucocorticoids modulate cardiovascular homeostasis via the effects of circulating components generated within the adrenals but also through local synthesis. The aim of this study was to assess the existence of such a steroidogenic system in heart. Using the quantitative reverse transcriptase-polymerase chain reaction, the terminal enzymes of corticosterone and aldosterone synthesis (11beta-hydroxylase and aldosterone synthase, respectively) were detected in the rat heart. This pathway was shown to be physiologically active, since production of aldosterone, corticosterone, and their precursor, deoxycorticosterone, was detected in both the homogenate and perfusate of isolated rat hearts using radioimmunoassay after Celite column chromatography. Perfusion of angiotensin II or adrenocorticotropin for 3 h increased aldosterone and corticosterone production and decreased deoxycorticosterone, suggesting that aldosterone and corticosterone are formed within the isolated heart from a locally present substrate. Chronic regulation of this intracardiac system was then examined. As in adrenals cardiac 11beta-hydroxylase and aldosterone-synthase mRNAs were independently regulated by 1 week's treatment with either low sodium and high potassium diet (which increased aldosterone synthase mRNA level only), angiotensin II (which raised level of both mRNAs), or adrenocorticotropin (which stimulated the 11beta-hydroxylase gene exclusively). Changes in cardiac steroid levels during treatment were not directly related to their plasma levels suggesting independent regulating mechanisms. This study, therefore, provides the first evidence for the existence of an endocrine cardiac steroidogenic system in rat heart and emphasizes its potential physiological and pathological relevance.

Adrenal Glands↗
PubMed1998

Effects of L-valine on growth and polyamine metabolism in human colon carcinoma cells.

HT-29 cells, originating from a human colon carcinoma, can proliferate in standard culture conditions with an absolute requirement for polyamines. The major precursor provided in the culture medium for polyamine biosynthesis is L-arginine. L-Arginine conversion to L-ornithine by arginase is followed by stepwise conversion of this latter amino acid to putrescine, spermidine and spermine. The aim of the present work was to document the consequences of a total inhibition of L-arginine flux through arginase, resulting in a decreased L-ornithine availability, on HT-29 cell proliferation and polyamine metabolism. L-Valine, a known arginase inhibitor, when used at a high concentration, i.e., 100 mM, inhibits L-arginine flux through arginase almost totally. The addition in the culture medium of 100 mM L-valine or 50 mM NaCl used to mimic the L-valine induced increase in medium osmolality both reduced equally cellular growth. Cell viability, protein synthesis or oxidative metabolism measured in isolated cells were unaffected by the L-valine treatment, suggesting that decreased proliferation was not associated with an acute toxic effect of this aminoacid, but was rather due to the increase in the medium osmolality. L-Valine treated cells displayed an altered polyamine metabolism when compared with control cells grown in the absence of the amino acid. After 4 days of treatment with 100 mM L-valine, L-ornithine flux through ornithine decarboxylase was significantly higher as well as putrescine and spermidine cellular uptakes in treated cells. However, the changes in polyamine metabolism led to similar polyamine cell contents in untreated and L-valine treated cells. In conclusion, we propose that the observed alterations of polyamine metabolism may reflect an adaptative response of HT-29 cells to the presence of L-valine which contribute together with the low amount of L-ornithine present in the culture medium to polyamine homeostasis.

Arginine↗
PubMed1998

[Malaria transmission in the rural zone of Niakhar, Senegal].

The anopheline bioecology and the malaria transmission were studied from January to December 1995 in three villages of the sahelian rural area of Niakhar, Senegal. This area of 29000 inhabitants, has been for several decades, a regional observatory for population and health. The three methods used for collecting mosquitoes were the collection at larval stages, the all night human biting collection, and the pyrethrum spray catch in houses during afternoons. The anophelines collected were, by numerical importance: Anopheles arabiensis, An. rufipes, An. gambiae, An. pharoensis, An. funestus and An. coustani. In the An. gambiae complex, An. arabiensis represented 97% of man biting females and 98% of half gravid resting females (difference not significant); the other reminding species of this complex was always An. gambiae. These two species belonging to the An. gambiae complex were responsible for the totality of the transmission. The anthropophilic index, obtained from half gravid indoor resting An. gambiae s.l., was 83%. The annual biting rate of An. gambiae s.l. varied from 512 to 1558 bites per man per night, depending on the villages. Vectors were observed all year long but their densities were low during the dry season. Vector population presented a notable increase due to the rains, with a maximum of about 10 bites per man per night in September or at the beginning of October; during September the biting rate represented 48% of the annual biting rate. The sporozoitic index of An. gambiae s.l., obtained by ELISA revealing the circumsporozoite protein, was 1.6% for human biting females and 1.8% for half-gravid resting females (difference not significant). Plasmodium falciparum was the only plasmodial species observed among infected anophelines. The annual transmission in the vo villages representative of the Niakhar area were 9 and 12 bites of infected anophelines per man, occurring mainly from August to October. In the third village, not representative of the area regarding permanent breeding places, the transmission was 26 bites of infected anopheline per man per year. These results were discussed in the Senegambian and sahelian contexts.

Animals↗
PubMed1998

Age grading Anopheles arabiensis: their gorging and surviving responses using a membrane feeding system.

Literature on artificial membrane feeding has traditionally supported the notion that mosquitoes have to be enough old to present a high gorging rate and enough young to present a high surviving rate. In order to know the best mosquito age to perform a Plasmodium sp. experimental infection revealed by oocyst examination, Anopheles arabiensis of known ages were fed on baudruche membrane with fresh human blood thermostated at 37 degrees C. The response was evaluated in term of gorging after ten minutes in contact with the membrane feeder and, for engorged females, in term of survival eight days after the engorgement. Two strains of mosquitoes were used, one wild, collected at larval stages in the city of Dakar and the other, originating from the same place but reared in our insectary since three years. The two strains showed their highest gorging rate at 3-4 days after emergence (39.4% for the wild strain and 63.3% for the insectary strain). The survival rate of the wild strain was highest at 2-3-4 days after emergence (64.8%) and those of the insectary strain at four days (65.6%). In conclusion, the gorging mosquitoes would have to be aged of 3-4 days after emergence for the wild strain and of four days for the insectary strain in order to obtain eight days after the engorging the best output of fed and surviving mosquitoes.

Animals↗
PubMed1998

Ecology of larval mosquitoes, with special reference to Anopheles arabiensis (Diptera: Culcidae) in market-garden wells in urban Dakar, Senegal.

The urban area of Dakar, Senegal, contains > 5,000 market-garden wells that provide permanent sites for mosquito larvae, in particular Anopheles arabiensis Patton, the major vector of malaria. A study of the bioecology of mosquito larvae was conducted over 1 yr with a monthly visit to 48 of these wells. Overall, 9,589 larvae were collected of which 80.1% were Culicinae and 11.9% Anophelinae. Larvae from stages III and IV (n = 853) were identified to 10 species. An. arabiensis represented 86% of the anophelines collected and An. ziemanni Grunberg 14%. The most common Culicinae species included Aedeomyia africana Neveu-Lemaire, Culex quinquefasciatus Say, and Mimomyia splendens Theobald. Maximum anopheline abundance was observed at the end of the dry season in June, whereas maximum Culicinae abundance was observed at the end of the rainy season in September. Most wells (67%) did not harbor any An. arabiensis larvae and in the remaining 33% the larval abundance was low, averaging 0.54 larvae in stages III-IV per tray sample. To identify factors that determine the abundance of larvae in these wells, a co-inertia (multivariate) analysis was carried out to account for physicochemical variables (depth, turbidity, temperature, pH, conductivity, Na+, Cl-, HCO3-, CO3--, and NO3- concentrations) and biological variables (abundance of mosquito species, predators [e.g., fish, Dytiscidae, Notonectidae, odonates], molluscs [Bulinus and Biomphalaria], and surface plants [water lettuce, Lemna, and filamentous algae]). The co-inertia analysis indicated that the abundance of An. arabiensis was associated with Cx. quinquefasciatus and Cx. decens for the physiochemical data but was not associated with other mosquito species for floro-faunistic data. The conditions associated with abundant An. arabiensis were warm temperature (28-30 degrees C), clear and not too deep water (< 0.5 m), elevated concentrations of HCO3- and CO3--, low concentrations of NO3- and NaCl, low populations of larvivorous fish and invertebrate predators (notably odonates), the presence of water lettuce, and an absence of Lemna. These results indicate that many contributing factors influence the ecology of the immature stages of An. arabiensis.

Animals↗
PubMed1998

In vivo left ventricular function and collagen expression in aldosterone/salt-induced hypertension.

Cardiac fibrosis is linked to aldosterone-induced hypertension, but the effects on in vivo left ventricular (LV) function are not established. We studied the relations between in vivo LV function and aldosterone/salt cardiac fibrosis. Adult guinea pigs (GPs) were treated for 3 months with an aldosterone infusion and high-salt diet. This treatment induced arterial hypertension (+35%) and moderate LV hypertrophy (LVH; +60%) without right ventricular (RV) hypertrophy. Echo-Doppler LV assessment demonstrated unaltered cardiac output, stroke volume, or LV relaxation. Type I collagen messenger RNA (mRNA) was significantly increased in both ventricles (LV, +48%; RV, +77%) and accompanied by a significant increase in total collagen deposition (LV, from 0.52% in controls to 4.4% in treated GPs; RV, from 0.82 to 5.5% in treated GPs). Plasma norepinephrine levels increased 2.6-fold (p < 0.01) and correlated with the increase in collagen deposition in both ventricles. Collagen content was not correlated with hypertension or LVH. We conclude that aldosterone administration induces cardiac collagen accumulation and a sympathetic stimulation, which might preserve systolic and diastolic function.

Aldosterone↗
PubMed1998

[Dynamics of Plasmodium falciparum gametocytemia as a function of therapeutic response to chloroquine in a mesoendemic malaria zone].

Patients with Plasmodium falciparum infections were selected with an in vivo chloroquine sensitivity assay in a mesoendemic area of Senegal. Gametocytemia was studied in relation to asexual parasite responses classified as drug-sensitive or showing RI or RII resistance. Gametocyte prevalence and density appeared significantly higher in resistant infections than in sensitive ones. These observations were made on children as well as on adults. The period between first clinical symptom appearance and treatment, varying from 0 to 5 days, was not linked to asexual parasite responses, but was significantly and positively correlated to gametocyte prevalence and density observed seven days after beginning the treatment. The comparison between these results and those previously observed in a hypoendemic area suggests that the dynamic of gametocytemia is independent of the endemic level. These observations illustrate that the dissemination of chimioresistant malaria can be reinforced--via the gametocyte stage--by selective chloroquine pressure.

Adolescent↗
PubMed1998

[The microwave oven for drying thick blood smears. Benefits and limits for microscopic observation of Plasmodium falciparum trophozoites and gametocytes].

The micro-wave oven permits a rapid drying of thick blood smears classically used for parasitological malaria diagnosis. In order to evaluate this type of drying on the microscopic reading, a study was carried out in an hyperendemic area of malaria with 382 asymptomatic volunteers of all ages. Two thick smears were made for each volunteer, one immediately dried with a micro-wave oven for one minute, the second dried in the open air without any intervention. A single microscopist examined all the thick smears. The observation of Plasmodium falciparum trophozoites differed significantly between the two methods, the prevalence was 50% by normal drying versus 41% by the micro-wave oven. The geometric mean of the trophozoite number for positive thick smears was significantly lower with the micro-wave oven. When the parasitological density with normal drying was lower than 200 trophozoites/microliter of blood, 54% of results were wrongly negative with the micro-wave oven. On the other hand, the observation of P. falciparum gametocytes was significantly facilitated after drying with the micro-wave oven; the prevalence was 8% with normal drying versus 12% by micro-wave oven. In conclusion, the use of the micro-wave oven for the drying of thick smears is not recommended for malaria diagnosis although it improves the observation and counting of P. falciparum gametocytes.

Animals↗
PubMed1997

Molecular characteristics of cocaine-induced cardiomyopathy in rats.

Cocaine abuse induces severe cardiomyopathy. To investigate the molecular effects of acute and prolonged administration of cocaine, mRNAs encoding markers of either mechanical overload, as atrial natriuretic factor (ANF) and alpha- and beta-myosin heavy chains, or fibrosis as type I and III procollagens, were quantitated in the left ventricle of rats 4 h after one injection of cocaine (40 mg/kg, n = 7), or 14 (n = 15) and 28 days (n = 10) after chronic infusion of cocaine (40 mg/kg per day). Plasma cocaine and benzylecgonine concentrations were both significantly augmented during the infusion while plasma levels of triiodothyronine and thyroxine were lowered. Acute injection of cocaine induced ANF gene expression. Cocaine treatment during 28 days resulted in left ventricular hypertrophy (+ 20% after 24 days, P < 0.05) with normal blood pressure, associated with an accumulation of mRNAs encoding ANF and type I and III collagens (+66% and +55%, P < 0.05). Such a chronic treatment also induced a shift from the alpha- to the beta-myosin heavy chain gene expression (-40% and +50%, P < 0.05). In conclusion, cocaine activates markers of both hemodynamic overload and fibrosis. Such an activation may result from direct and/or indirect effects of the drug such as myocardial ischemia, mechanical overload and/or hypothyroidism.

Animals↗
PubMed1997

Comparison of three sampling methods of man-biting anophelines in order to estimate the malaria transmission in a village of south Cameroon.

In order to get an accurate measure of malaria transmission by mean of human bait attraction, three methods of catches for human-bail mosquitoes were compared in a Cameroonian village of high anopheline density. These three methods were 1) the classical human landing catches where the man was in the same time bait and catcher, 2) the single-nets which acted as a trap and 3) the double-nets where the outer net acted as a trap and the inner avoided mosquito bites. The anopheline densities per man with human landings were 1.6 time higher than those obtained with single-nets, these later being 4.7 times higher than those obtained with double-nets. In the three methods, the results were similar for the anophelines species catched and for their respective proportions. The samples of Anopheles nili and An. gambiae had comparative parity rates and sporozoitic indexes. From these results, in order to estimate the malaria transmission it can be envisaged to change the standard human-bait catch for the human-baited single bed-net catch; on the contrary the double-net have to be discarded because of their poor results.

Animals↗