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J Remacle

Publications and source records attributed to J Remacle.

At least 37 records · Page 2Linked to original sources
PubMed2000

Comparison between different strategies of covalent attachment of DNA to glass surfaces to build DNA microarrays.

DNA microarray is a powerful tool allowing simultaneous detection of many different target molecules present in a sample. The efficiency of the array depends mainly on the sequence of the capture probes and the way they are attached to the support. The coupling procedure must be quick, covalent, and reproducible in order to be compatible with automatic spotting devices dispensing tiny drops of liquids on the surface. We compared several coupling strategies currently used to covalently graft DNA onto a glass surface. The results indicate that fixation of aminated DNA to an aldehyde-modified surface is a choice method to build DNA microarrays. Both the coupling procedure and the hybridization efficiency have been optimized. The detection limit of human cytomegalovirus target DNA amplicons on such DNA microarrays has been estimated to be 0.01 nM by fluorescent detection.

Cytomegalovirus↗
PubMed2000

ERK activation upon hypoxia: involvement in HIF-1 activation.

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor activated by hypoxia. The HIF-1 activation transduction pathway is poorly understood. In this report, we investigated the activation of extracellular regulated kinases (ERK) in hypoxia and their involvement in HIF-1 activation. We demonstrated that in human microvascular endothelial cells-1 (HMEC-1), ERK kinases are activated during hypoxia. Using dominant negative mutants, we showed that ERK1 is needed for hypoxia-induced HIF-1 transactivation activity. Moreover, using a kinase assay and Western blot experiments, we showed that HIF-1alpha is phosphorylated in hypoxia by an ERK-dependent pathway. These results evidence the role of mitogen-activated protein kinase in the transcriptional response to hypoxia.

Blotting, Western↗
PubMed2000

Induction of replicative senescence biomarkers by sublethal oxidative stresses in normal human fibroblast.

We tested the long-term effects of sublethal oxidative stresses on replicative senescence. WI-38 human diploid fibroblasts (HDFs) at early cumulative population doublings (CPDs) were exposed to five stresses with 30 microM tert-butylhydroperoxide (t-BHP). After at least 2 d of recovery, the cells developed biomarkers of replicative senescence: loss of replicative potential, increase in senescence-associated beta-galactosidase activity, overexpression of p21(Waf-1/SDI-1/Cip1), and inability to hyperphosphorylate pRb. The level of mRNAs overexpressed in senescent WI-38 or IMR-90 HDFs increased after five stresses with 30 microM t-BHP or a single stress under 450 microM H(2)O(2). These corresponding genes include fibronectin, osteonectin, alpha1(I)-procollagen, apolipoprotein J, SM22, SS9, and GTP-alpha binding protein. The common 4977 bp mitochondrial DNA deletion was detected in WI-38 HDFs at late CPDs and at early CPDs after t-BHP stresses. In conclusion, sublethal oxidative stresses lead HDFs to a state close to replicative senescence.

Biomarkers↗
PubMed2000

Cell cycle regulation in H(2)O(2)-induced premature senescence of human diploid fibroblasts and regulatory control exerted by the papilloma virus E6 and E7 proteins.

Many biomarkers of replicative senescence appear in stress-induced premature senescence (SIPS) of human diploid fibroblasts (HDFs). The mRNA level of key cell cycle regulators was studied in H(2)O(2)-induced premature senescence of HDFs expressing or not the papillomavirus E6 and E7 proteins, which enhanced, respectively, the proteolysis of p53 and Rb. The CdKI's p21(waf-1) and p16(Ink-4a) were found overexpressed in H(2)O(2)-induced premature senescence, while p19(Ink-4d)and p27(Kip-1) were repressed. The results obtained in E6 HDFs suggest that p21(waf-1) and p16(Ink-4a) overexpressions are p53-independent, while p27(Kip-1) and p19(Ink-4d) down-regulations are p53-dependent.E6 regulated Rb, p130, p53 and p16(Ink-4a) mRNA level in non-stressing conditions, and regulated p130, p107, p53, p19(Ink-4d), p27(Kip-1) mRNA level in SIPS. SIPS modified the E6-mediated regulatory control on p107, p16(Ink-4a), p19(Ink-4d) and p27(Kip-1) mRNA level, when compared to normal conditions.E7 regulated the mRNA level of all the genes studied, in all conditions, suggesting that the Rb family or other E7-interacting proteins might modify the expression of these genes. SIPS modified strongly the E7-mediated regulatory control on p107, p16(Ink-4a), p19(Ink-4d), p27(Kip-1), p21(Waf-1) and Rb mRNA level, when compared to normal conditions. Further work is ongoing to test whether this E7-mediated regulatory control takes place through interactions with Rb or other E7-interacting proteins.

Cell Cycle↗
PubMed2000

Stress-induced premature senescence as alternative toxicological method for testing the long-term effects of molecules under development in the industry.

No alternative in vitro method exists for detecting the potential long-term genotoxic effects of molecules at subcytotoxic concentrations, in terms of days and weeks after exposure(s) to the molecule tested. A theoretical model of cellular senescence led to the concept that subcytotoxic stresses under any molecules at subcytotoxic doses, such as molecules under development in the pharmaceutical, cosmetics and food industry, might lead human fibroblasts into a state closely related to in vitro senescence. This concept was then experimentally confirmed in vitro: many biomarkers of replicative senescence of human fibroblasts were found 72 h after their exposure to various kinds of stressors used at non-cytotoxic concentrations. This phenomenon has been termed stress-induced premature senescence (SIPS). Moreover, proteomics studies have revealed that, besides their effects on the appearance of the biomarkers of senescence, sublethal stresses under a variety of stressors also lead to long-term specific changes in the expression level of proteins which are stress-specific. These changes have been coined the molecular scars of stress. The proteins corresponding to these molecular scars may be identified using the latest developments in mass spectrometry. This model of stress-induced premature senescence may be applied to the toxicological sciences when testing for the potential irreversible long-term effects of molecules on the cell fate.

Aging, Premature↗
PubMed2000

Consideration of heteroduplexes and homoduplexes for the quantification by competitive PCR of human mitochondrial DNA deletions with ageing of tissues and cells.

The purpose of this work was first to construct two internal standards for human mitochondrial DNA mt DNA corresponding respectively to the fragment resulting from the 4,977 bp common deletion (H2del) and a fragment which was never reported to be deleted (H1). Secondly, we wished to consider the possible effect of annealing between the target and corresponding internal standard which forms heteroduplexes. These experiments show that the correction of the number of copies found by competitive PCR by considering the percentage of heteroduplexes allows a more accurate quantification of the number of target copies present in mt DNA samples. The design of internal standards specific to the fragment resulting from other deletions could also help a more accurate quantification of the frequency of other mt DNA deletions as well, and reconsideration of the role of mt DNA deletions in ageing.

Aging↗
PubMed2000

Effect of venotropic drugs on the respiratory activity of isolated mitochondria and in endothelial cells.

Several drugs used in the treatment of chronic peripheral ischaemic and venous diseases, i.e. aescine, Cyclo 3, Ginkor Fort, hydroxyethylrutosides, naftidrofuryl, naphthoquinone and procyanidolic oligomers, were tested on the mitochondrial respiratory activity. The results show that all these drugs protected human endothelial cells against the hypoxia-induced decrease in ATP content. In addition, they all induced a concentration-dependent increase in respiratory control ratio (RCR) of liver mitochondria pre-incubated with the drugs for 60 min. The drugs were divided into two groups according to their effects. The first group (A), comprising aescine, Ginkor Fort, naftidrofuryl and naphthoquinone, increased RCR by decreasing state 4 respiration rate. The second group of drugs (B), comprising hydroxyethylrutosides, procyanidolic oligomers and Cyclo 3, increased RCR by increasing state 3 respiration rate. The drugs of group A were able to prevent the inhibition of complexes I and III respectively by amytal and antimycin A while the first two drugs of group B increased adenine nucleotide translocase activity. Cyclo 3 inhibited the carbonylcyanide m-chlorophenyl hydrazone (mCCP)-induced uncoupling of mitochondrial respiration. None of these seven drugs could protect complexes IV and V, respectively, from inhibition by cyanide and oligomycin. When tested on endothelial cells the drugs of group A, in contrast to group B, prevented the decrease in ATP content induced by amytal or antimycin A. The present results suggest that the protective effects on mitochondrial respiration activity by these venotropic drugs may explain their protective effect on the cellular ATP content in ischaemic conditions and some of their beneficial therapeutic effect in chronic vascular diseases.

Adenosine Triphosphate↗
PubMed2000

Identification of hypoxia-responsive messengers expressed in human microvascular endothelial cells using differential display RT-PCR.

In order to identify new genes overexpressed in endothelial cells exposed to hypoxia, differential display RT-PCR was performed on total RNA extracted from human microvascular endothelial cells incubated under hypoxia or under normoxic conditions. Northern blot and reverse Northern blot analyses were used to confirm the results. Sequences corresponding to tissue inhibitor of metalloproteinase-1, prostate tumor inducing factor-1, enolase-alpha and prothymosin-alpha were evidenced as overexpressed in hypoxia. These results were confirmed by Western blot and immunofluorescence experiments. Moreover, several elements homologous to partial sequences of cDNA (expressed sequence tag) were also identified, as well as unknown cDNA sequences. The present study suggests that hypoxia can change the expression of numerous genes in endothelial cells, and that mRNA differential display is useful for cloning known and unknown hypoxia-responsive genes.

Cell Hypoxia↗
PubMed2000

Appearance of biomarkers of in vitro ageing after successive stimulation of WI-38 fibroblasts with IL-1alpha and TNF-alpha: senescence associated beta-galactosidase activity and morphotype transition.

Sublethal oxidative stresses increase the proportions of human fibroblasts positive for senescence associated beta-galactosidase activity and accelerate the transition in the fibroblast morphotypes characterising fibroblast ageing. Stimulation of fibroblasts with TNF-alpha or IL-1alpha transiently increases the production of reactive oxygen species (ROS) in human fibroblasts. Here we propose that repeated stimulation of WI-38 fibroblasts with TNF-alpha or IL-1alpha can generate enough ROS to accelerate the transition in the fibroblast morphotypes and increase the proportion of cells positive for senescence associated beta-galactosidase activity. The involvement of ROS is suggested by experiments where the stimulation of fibroblasts with TNF-alpha or IL-1alpha are performed in the presence of N-acetylcysteine which increases the intracellular antioxidant potential. It is proposed that the decrease in the proportions of morphotypes I and II, and the increase in the proportions of morphotypes III to VI observed after successive stimulation with TNF-alpha or IL1-alpha is attributed to an increased ROS production occurring during the stimulation.

Acetylcysteine↗
PubMed2000

A model for the complex between the hypoxia-inducible factor-1 (HIF-1) and its consensus DNA sequence.

Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor activated by hypoxia. When activated, HIF-1 mediates the differential expression of genes such as erythropoietin and Vascular Endothelial Growth Factor (VEGF) during hypoxia. It is composed of two different subunits, HIF-1alpha and ARNT (Aryl Receptor Nuclear Translocator). These two subunits belong to the bHLH (basic Helix-Loop-Helix) PAS (Per, Ahr/ARNT, Sim) family. The bHLH domain of these factors is responsible for dimerization through the two helices and for DNA binding through their basic domain. In this work, we used various methods of molecular modeling in order to develop a 3D structure for the HIF-1 bHLH domain bound to its DNA consensus sequence. Firstly, the 3D structure of the bHLH domain of both subunits based on their amino acid sequence was defined. Secondly, we compared this model with data from known crystal structures of basic leucine zipper-DNA and bHLH-DNA complexes in order to determine a potential canvas for HIF-1. Thirdly, we performed a manual approach of the HIF-1 bHLH domain onto the DNA recognition site using this canvas. Finally, the protein-DNA complex 3D structure was optimized using a Monte Carlo program called MONTY. The model predicted a pattern of interactions between amino acids and DNA bases which reflect for ARNT what is experimentally observed among different X-ray structures of other bHLH transcription factors possessing the H (His), E (Glu), R (Arg) triad, as ARNT does. On the other hand, only the Arg residue is conserved in HIF- 1alpha. We propose from this model that a serine replaces the histidine while an alanine and a lysine also make contacts with DNA. From these results, we postulate that the specificity of HIF-1 toward its DNA sequence could be driven by the HIF-1alpha subunit. The predicted model will be verified by X-Ray currently ongoing.

Alanine↗
PubMed2000

Biochemical and genetic evidence for phospholipase C activity in Mycobacterium ulcerans.

This study reports the existence of phospholipase C and D enzymatic activities in Mycobacterium ulcerans cultures as determined by use of thin-layer chromatography to detect diglycerides in hydrolysates of radiolabeled phosphatidylcholine. M. ulcerans DNA sequences homologous to the genes encoding phospholipase C in Mycobacterium tuberculosis and Pseudomonas aeruginosa were identified by sequence analysis and DNA-DNA hybridization. Whether or not the phospholipase C and D enzymes of M. ulcerans plays a role in the pathogenesis of the disease needs further investigation.

DNA, Bacterial↗
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↗