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Biomedical subjects

J Remacle

Publications and source records attributed to J Remacle.

At least 55 records · Page 3Linked to original sources
PubMed2000

Colorimetric detection of the tuberculosis complex using cycling probe technology and hybridization in microplates.

Cycling probe technology (CPT) is a simple signal amplification method for the detection of specific target DNA sequences. CPT uses a chimeric DNA-RNA-DNA probe that is cut by RNase H when bound to its complementary target sequence. In this study, a hybridization assay was developed to detect biotinylated CPT products that result from the amplification of a Mycobacterium tuberculosis complex sequence. The chimeric probe was specifically designed to avoid the formation of secondary structures. The chosen capture probe was perfectly complementary to and was the same size as OL2, one of the two CPT products. The assay was based on the observation that a long sequence, such as the initial probe, was destabilized when bound to a small capture probe as a result of steric hindrance. The capture probe preferentially bound OL2 rather than the long initial probe. We added a prehybridization step with a helper DNA to enhance this discrimination between the two sequences. Colorimetric detection was performed using a peroxidase-streptavidin conjugate. After optimization, the non-isotopic hybridization assay allowed the detection of around 10 amol of target DNA. Besides being faster and easier to perform, this detection method was compared to electrophoresis separation and gave similar results.

Colorimetry↗
PubMed2000

Role of HIF-1 as a transcription factor involved in embryonic development, cancer progression and apoptosis (review).

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor first identified as being activated by hypoxia but also in normoxic conditions by insulin and IGF-2. It is able to induce the expression of glycolytic genes and hence the ATP production, it also regulates the expression of the angiogenic factor VEGF and stimulates erythropoiesis via EPO production. HIF-1 is a protein necessary for the normal embryonic and cardiovascular system development, but seems to be also involved in cancer progression and apoptosis. Thus, it appears that HIF-1 plays a central role in normal cellular functions and in tissue metabolism but it is also involved in pathological evolutions raising its interest as a therapeutic target. In this review, we summarize the dual role of HIF-1 as a major component of the embryo development, as well as an element of tumor progression and of anoxia-induced apoptosis.

Animals↗
PubMed1999

Growth and cytotoxic activity by Mycobacterium ulcerans in protein-free media.

The pathogenic slow-growing Mycobacterium ulcerans has, until now, been cultured in liquid media containing albumin. Here, we report the first description of use of Sauton medium and modified Reid medium, two protein-free media, in which M. ulcerans was able to grow and produce its toxin, a major virulence factor of this environmental organism which causes a skin disease commonly called Buruli ulcer. These results suggest that Sauton and modified Reid may be useful for certain fields of M. ulcerans research requiring protein-free growth conditions.

Bacterial Toxins↗
PubMed1999

Hypoxia-induced activation of HIF-1: role of HIF-1alpha-Hsp90 interaction.

The protein chaperone heat shock protein 90 (Hsp90) is a major regulator of different transcription factors such as MyoD, a basic helix loop helix (bHLH) protein, and the bHLH-Per-aryl hydrocarbon nuclear translocator (ARNT)-Sim (PAS) factors Sim and aryl hydrocarbon receptor (Ahr). The transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha), involved in the response to hypoxia, also belongs to the bHLH-PAS family. This work was aimed to investigate the putative role of Hsp90 in HIF-1 activation by hypoxia. Using a EGFP-HIF-1alpha fusion protein, co-immunoprecipitation experiments evidenced that the chimeric protein expressed in COS-7 cells interacts with Hsp90 in normoxia but not in hypoxia. We also demonstrated that Hsp90 interacts with the bHLH-PAS domain of HIF-1alpha. Moreover, Hsp90 is not co-translocated with HIF-1alpha into the nucleus. At last, we showed that Hsp90 activity is essential for HIF-1 activation in hypoxia since it is inhibited in the presence of geldanamycin. These results indicate that Hsp90 is a major regulator in HIF-1alpha activation.

Animals↗
PubMed1999

HIF1A gene transcription is dependent on a core promoter sequence encompassing activating and inhibiting sequences located upstream from the transcription initiation site and cis elements located within the 5'UTR.

Hypoxia inducible factor-1 (HIF-1) is a transcription factor composed of two subunits, HIF-1alpha and ARNT, which is activated under hypoxia. HIF-1alpha mRNA is expressed constitutively in a wide variety of cell types, whereas in some others HIF1A gene expression is upregulated by hypoxia. In this report, we show that in endothelial cells (HMEC-1) the HIF-1alpha mRNA expression level is the same in both normoxia and hypoxia. Deletion analysis experiments of the HIF1A promoter showed that in hypoxia HIF1A gene expression is upregulated through a short sequence located next to the transcription initiation site. We also show that in hypoxia another sequence located upstream from the +1 initiation site plays an inhibitory role on HIF1A transcription in HMEC-1 but not in hepatoma cells and brings back this expression level to that observed in normoxia. Finally, we demonstrate that HIF1A gene transcription is dependent on Sp1 binding sites and that the 5'UTR sequence also contains other important cis-acting elements.

5' Untranslated Regions↗
PubMed1999

Protection of mitochondrial respiration activity by bilobalide.

Mitochondria alteration is an early event in ischemia-induced damage, and its prevention improves tissue survival upon reperfusion. Adenine translocase and complex I activities are rapidly affected by ischemia. Ginkgo biloba extract demonstrates anti-ischemic properties attributable to the terpenoid fraction, mainly due to the presence of bilobalide. The mechanism of the protection afforded by bilobalide is not yet known. In this work, the effects of bilobalide on mitochondrial respiration were investigated. Mitochondria isolated from rats treated with bilobalide (2 to 8 mg/kg) showed a dose-dependent increase in the respiratory control ratio, due to a lower oxygen consumption during state 4. Bilobalide also decreased the sensitivity of oxygen consumption to inhibition of complex I by Amytal or to inhibition of complex III by antimycin A or myxothiazol. There was no protection of complexes IV and V. It also increased the activity of complex I but not of adenine translocase. Similar effects were also obtained in vitro when control mitochondria were preincubated for 1 hr with 0.8 microg/mL bilobalide. Treatment of the rats with 8 mg/kg bilobalide also prevented the ischemia-induced decrease in state 3 of the mitochondrial respiration and thus the decrease in RCR. The protective effect of bilobalide on cellular ATP content observed under ischemic conditions can be correlated with the above observations. By protecting complex I and III activities, bilobalide allows mitochondria to maintain their respiratory activity under ischemic conditions as long as some oxygen is present, thus delaying the onset of ischemia-induced damage. This mechanism provides a possible explanation for the anti-ischemic properties of bilobalide and of Ginkgo biloba extract in therapeutic interventions.

Animals↗
PubMed1999

Increase in circulating endothelial cells in patients with primary chronic venous insufficiency: protective effect of Ginkor Fort in a randomized double-blind, placebo-controlled clinical trial.

One possible mechanism that accounts for the alterations observed in varicose veins is the activation of endothelial cells by ischemia occurring in the leg veins during blood stasis and the cascade of reactions that follows. Because in vitro data suggest that endothelium alteration is a key event in the development of the pathology, it was important to confirm this hypothesis in patients. We used the number of circulating endothelial cells detached from the vascular wall as a criterion of the endothelium injury. We first compared the number of circulating endothelial cells (CECs) in patients with chronic venous insufficiency (CVI) with those of a control population. A twofold increase in the CEC count (1,001+/-127 CEC/ml of plasma compared with 514+/-82 CECs/ml) was observed in CVI patients, which indeed suggests an alteration of the endothelium in this disease. Second, the protective effect of a venotropic drug, Ginkgo biloba extract, troxerutine, and heptaminol (Ginkor Fort), was tested by a randomized double-blind, placebo-controlled clinical trial. In the active-treatment group, the mean values of the CEC count decreased by 14.5% after a 4-week treatment, whereas in the placebo group, the decrease was less (8.4%). The decrease from week 0 to the end of treatment was significantly higher in the active-treatment group than in the placebo group. These results confirm the important role of the endothelium alterations in the development of varicose veins and suggest a potential beneficial action of a venotropic drug on the venous wall.

Adult↗
PubMed1999

Effect of Ruscus extract and hesperidin methylchalcone on hypoxia-induced activation of endothelial cells.

BACKGROUND: Ruscus aculeatus extract and the flavonoid hesperidin methylchalcone (HMC) are drugs used in the treatment of chronic venous insufficiency. METHODS: In the present study, we investigated their effects on the activation of endothelial cells by hypoxia, a condition which mimics venous blood stasis. RESULTS: We observed that Ruscus extract was able to inhibit the activation of endothelial cells by hypoxia: the decrease in ATP content, the activation of phospholipase A2 as well as the subsequent increase in neutrophil adherence with a maximal protection obtained at 50 microg/ml. HMC was also able to inhibit the hypoxia-induced decrease in ATP content. Furthermore, the effects of Ruscus extract and of HMC on this decrease seem to be additive. CONCLUSIONS: The biochemical mechanism evidenced in this work might explain some of the beneficial therapeutic effects of these products in the treatment of chronic venous insufficiency patients.

Adenosine Triphosphate↗
PubMed1998

Effect of aescine on hypoxia-induced neutrophil adherence to umbilical vein endothelium.

Although venous stasis due to blood stagnation in lower limbs has been recognised as an important etiological factor for the development of varicose veins, the mechanism linking this ischemic situation to the modifications of the venous wall in varicose veins is still unclear. There is evidence that the activation of the endothelium during blood stasis and its subsequent cascade of interactions with other cell types could alter the structure of the vein wall and could possibly be at the origin of the disease. While phlebotonic drugs are often used to improve symptoms in chronic venous insufficiency, their precise mechanism of action is not well understood. We now tested aescine (Reparil i.v. form) in an ex vivo model which mimics this situation, i.e., perfused human umbilical vein exposed to hypoxic conditions. To study the effect of aescine on neutrophil activation and adhesion to the endothelium, human umbilical veins were incubated under hypoxic conditions with or without aescine and the interactions between the endothelium and neutrophil-like cells, HL60, were investigated. We observed that a large number of HL60 became adherent to the endothelium of veins after 2 h hypoxia and that these adherent HL60 were activated: they released high amounts of superoxide anion and of leukotriene B4. Aescine (250 ng/ml or 0.22 microM) was shown to markedly inhibit HL60 adherence to hypoxic endothelium. By decreasing the number of adherent HL60, aescine also decreased the subsequent production of superoxide anion and of leukotriene B4. Scanning electron microscopy confirmed the increased HL60 adherence to the endothelium, as well as the inhibitory effect of aescine. These results support results of in vitro studies on isolated endothelial cells in which aescine was shown to inhibit the hypoxia-induced activation of endothelial cells and the subsequent increased adherence of neutrophils. In vivo, the activated and infiltrated leukocytes release free radicals, chemotactic molecules such as leukotriene B4 and proteases which then can degrade the extracellular matrix. These processes could contribute to alterations of the venous wall similar to those observed in varicose veins. By maintaining an intact endothelium during in vivo blood stasis in the lower limbs and preventing neutrophil recruitment, adherence and activation, aescine could prevent the resulting alterations of the venous wall. These results could explain at least in part the potential benefit of the drug in the prevention of venous insufficiency.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1998

Effect of hydroxyethylrutosides on hypoxial-induced neutrophil adherence to umbilical vein endothelium.

A clinically available mixture of hydroxyethylrutosides (HR) was examined as a protector against endothelial cell activation by hypoxia in perfused human umbilical vein. The results showed that 500 micrograms/mL HR totally inhibited the adherence of human unstimulated neutrophils to the endothelium of umbilical vein incubated in hypoxic conditions. This inhibition was confirmed by a morphological study performed by scanning electron microscopy. In addition, neutrophils adherent to the hypoxic umbilical vein endothelium became activated, as evidence by the increased release of superoxide anions and synthesis of leukotriene B4. These processes could also be inhibited by HR. In conclusion, the results of this study suggest that the improvement in venous insufficiency observed clinically with HR could, in part, be the result of their ability to inhibit the recruitment and activation of neutrophils by endothelium activated during blood stasis.

Cell Adhesion↗
PubMed1998

Effect of Ginkor Fort on hypoxia-induced neutrophil adherence to human saphenous vein endothelium.

This study was performed to evaluate the effects of Ginkor Fort, a venotropic drug composed of Ginkgo biloba extract, troxerutine, and heptaminol, on neutrophil adherence to the endothelium of saphenous veins. When saphenous veins were incubated 2 h in hypoxic conditions, they showed a five- to sixfold increase in neutrophil adherence to the endothelium. Ginkor Fort at 0.3 mg/ml was able to inhibit this increase by 69%. These results were confirmed by observations in scanning electron microscopy. Ginkor Fort also inhibited the subsequent activation of these neutrophils, as evidenced by the inhibition of superoxide anion release. The biochemical mechanism of this inhibition of neutrophil adherence was studied on endothelial cells in culture. We observed that Ginkor Fort was able to inhibit the different steps of the activation of endothelial cells by hypoxia: the activation of phospholipase A2 and the decrease in adenosine triphosphate (ATP) content. By preventing the first step of the activation cascade, the decrease in ATP content, Ginkor Fort blocks the subsequent increase in neutrophil adherence as well as neutrophil activation. The biochemical mechanism evidenced in this work might explain the beneficial effect of this drug in the treatment of patients with chronic venous insufficiency.

Cell Adhesion↗
PubMed1998

Quantitative determination of CMV DNA using a combination of competitive PCR amplification and sandwich hybridization.

A quantitative PCR method is proposed that combines the use of a competitive internal standard with the sandwich hybridization of the products. The variability of the PCR efficiency was corrected using a specifically designed internal standard, competitive not only for the PCR amplification, but also for the hybridization on capture probes fixed onto microwells. The design of such standard gave a dynamic range extending from 30-1 million copies of target DNA when the internal standard copy number was fixed to 1000 using a simple colorimetric detection. The assay was independent from the number of PCR cycles, which indicates a true competition between the standard and the template DNA. The assay was developed for a cytomegalovirus (CMV) DNA sequence and is illustrated by the quantification of CMV in a culture sample.

Base Sequence↗
PubMed1998

Role of PECAM-1 in the adherence of PMN to hypoxic endothelial cells.

Hypoxia induces an increase in PMN adherence to endothelial cells for which an interaction between ICAM-1 and CD18/CD11b has been demonstrated. Since PECAM-1 has been shown to be involved in PMN transmigration through the endothelium and to increase the binding capacity of leukocyte CD18/CD11b, the role of this molecule in the hypoxia-induced PMN adherence was investigated. Hypoxia did not change the total surface expression of PECAM-1 on HUVEC and did not change the cell-cell border localization of this molecule as TNF-alpha did. In addition, blocking anti-PECAM-1 antibodies could not inhibit the increased adherence of unstimulated human PMN to hypoxia-incubated HUVEC while anti-ICAM-1 partially inhibited this process. These results indicate that PECAM-1 is probably not involved in the hypoxia-induced PMN adherence to endothelial cells.

Antibodies, Monoclonal↗
PubMed1997

Comparison between microwell and bead supports for the detection of human cytomegalovirus amplicons by sandwich hybridization.

In this study, we compared the efficiency of capture DNA probes covalently bound onto magnetic beads or microplates for their hybridization with target human cytomegalovirus (HCMV) DNA amplicons. Polystyrene supports were first aminated by wet chemistry to allow covalent grafting of the capture probes. The level of amines grafted was three times higher on beads than on microwells. Increasingly higher sizes of capture probes were fixed on both supports and the best reaction yield ranged from 300 to 500 fmol. The sizes of the capture and detection probes were optimized in order to obtain high target DNA hybridization yield. Long capture probes were more accessible than short ones to the target, with faster kinetics of hybridization obtained on beads than on microplates. Sensitivity of the hybridization assay was then determined with a nonisotopic method and the detection limit found was 30 amol of HCMV amplicons on both supports. HCMV DNA extracted from clinical samples were amplified by PCR. The resulting amplicons were then analyzed using the optimized sandwich hybridization assay discussed here. The results perfectly fitted with the qualitative conclusions obtained after a nested PCR analyzed on agarose gel.

Colorimetry↗
PubMed1997

Effects of antioxidant enzyme modulations on interleukin-1-induced nuclear factor kappa B activation.

Nuclear factor kappa B (NF-kappa B) is a potent and pleiotropic transcription factor that can be activated by a wide variety of inducers, including interleukin-1 (IL-1). Although the detailed activation mechanism of NF-kappa B is still under investigation, it requires both phosphorylation and degradation of its inhibitory subunit I kappa B and the presence of an oxidative environment. In this study, we systematically evaluated the influence of glutathione peroxidase, glutathione reductase and catalase on IL-1-induced NF-kappa B activation by analysing the effect of specific inhibitors of these enzymes. For the three antioxidant enzymes mentioned, their inhibition correlated with an overactivation of NF-kappa B, particularly for glutathione peroxidase. Inversely, we tested the response of glutathione peroxidase-transfected cells on NF-kappa B activation, which was lower as compared with the parental cells. Furthermore, interleukin-6 production also correlated perfectly with the reduced level of NF-kappa B activation is these experiments. The results clearly show that NF-kappa B activation is, strongly dependent on the antioxidant potential of the cells, especially on the activity of reduced glutathione-dependent enzymes such as glutathione peroxidase. The results support the hypothesis that the level of the oxidised glutathione:reduced glutathione ratio and the activity of intracellular antioxidant enzymes play a major role in NF-kappa B tine tuning.

Amitrole↗
PubMed1997

Identification of lysine 74 in the pyruvate binding site of alanine dehydrogenase from Bacillus subtilis. Chemical modification with 2,4,6-trinitrobenzenesulfonic acid, n-succinimidyl 3-(2-pyridyldithio)propionate, and 5'-(p-(fluorosulfonyl)benzoyl)adenosine.

L-Alanine dehydrogenase from Bacillus subtilis was inactivated with two different lysine-directed chemical reagents, i.e. 2,4, 6-trinitrobenzenesulfonic acid and N-succinimidyl 3-(2-pyridyldithio)propionate. In both cases, the inactivation followed pseudo first-order kinetics, with a 1:1 stoichiometric ratio between the reagent and the enzyme subunits. Partial protection of the active site from inactivation could be obtained by each of the substrates, NADH or pyruvate, but complete protection could only be achieved in the presence of the ternary complex E.NADH. pyruvate. The nucleotide analogue of NADH, 5'-(p-(fluorosulfonyl)benzoyl)adenosine was also used for affinity labeling of the enzyme active site. Differential peptide mapping, performed both in the presence and in the absence of the substrates, followed by reversed phase high performance liquid chromatography separation, diode-array analysis, mass spectrometry, and N-terminal sequencing of the resulting peptides, allowed the identification of lysine 74 in the active site of the enzyme. This residue, which is conserved among all L-alanine dehydrogenases, is most likely the residue previously postulated to be necessary for the binding of pyruvate in the active site. Surprisingly, this residue and the surrounding conserved residues are not found in amino acid dehydrogenases like glutamate, leucine, phenylalanine, or valine dehydrogenases, suggesting that A-stereospecific amino acid dehydrogenases such as L-alanine dehydrogenase could have evolved apart from the B-stereospecific amino acid dehydrogenases.

Adenosine↗
PubMed1997

Comparison of three luminescent assays combined with a sandwich hybridization for the measurement of PCR-amplified human cytomegalovirus DNA.

Identification of human tissue contaminated by cytomegalovirus is currently carried out by PCR amplification followed by measurement of the amplicons. Three luminescent detection systems undertaken after sandwich hybridization of the amplicons were compared. The sandwich hybridization takes place between a covalent linked capture probe, bound onto a plastic 96-well plate, and a biotinylated or digoxigenin-labeled detection probe. The three non-isotopic luminescent detection systems need either streptavidin-conjugated peroxydase or streptavidin-conjugated pyruvate kinase or antibodies conjugated with alkaline phosphatase. Detection of the enzymes was carried out by measurement of light emission in the presence of, respectively, luminol for peroxidase or dioxethane for alkaline phosphatase. The kinase assay was carried out not only in the presence of its substrates, ADP and phospho-enol pyruvate, but also of luciferase, which converts the produced ATP into light. The method was found to be sensitive, with the luciferase bioluminescent assay with the production of a long lasting signal. Amplicons from eight clinical samples were detected by this combination of sandwich hybridization and the three luminescent assays. The results were comparable with nested PCR for the identification of positive samples. The same correlation was obtained with 45 clinical samples using only the pyruvate kinase detection system. The high performance of these assays is given by the specificity of the sandwich hybridization combined with the sensitivity of the luminescent detection systems.

Cytomegalovirus↗