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Biomedical subjects

J Remacle

Publications and source records attributed to J Remacle.

At least 19 recordsLinked to original sources
PubMed2006

Electrical detection of DNA hybridization: three extraction techniques based on interdigitated Al/Al2O3 capacitors.

Based on interdigitated aluminum electrodes covered with Al(2)O(3) and silver precipitation via biotin-antibody coupled gold nano-labels as signal enhancement, three complementary electrical methods were used and compared to detect the hybridization of target DNA for concentrations down to the 50 pM of a PCR product from cytochrome P450 2b2 gene. Human hepatic cytochrome P450 (CYP) enzymes participate in detoxification metabolism of xenobiotics. Therefore, determination of mutational status of P450 gene in a patient could have a significant impact on the choice of a medical treatment. Our three electrical extraction procedures are performed on the same interdigitated capacitive sensor lying on a passivated silicon substrate and consist in the measurement of respectively the low-frequency inter-electrodes capacitance, the high-frequency self-resonance frequency, and the equivalent MOS capacitance between the short-circuited electrodes and the backside metallization of the silicon substrate. This study is the first of its kind as it opens the way for correlation studies and noise reduction techniques based on multiple electrical measurements of the same DNA hybridization event with a single sensor.

Aluminum↗
PubMed2005

Comparison of DNA detection methods using nanoparticles and silver enhancement.

DNA microarrays are an emerging technology for the parallel detection of DNA molecules. Fluorescent molecules are the current standard for a DNA array's optical readout but they possess some drawbacks including the stability of the dyes and the cost of the scanners. Therefore alternative labelling strategies are of considerable interests. One such strategy is the use of nanoparticles which offers several advantages in terms of stability and versatility of the detection mode. The authors present a review on the different ways DNA can be detected, mainly onto a solid support, using nanoparticle labels.

Biosensing Techniques↗
PubMed2004

DNA microarrays as a tool in toxicogenomics.

Toxicogenomics is an emerging technology that defines the use of novel genomic techniques to investigate the adverse effects of xenobiotic on gene expression. Toxicogenomics is based on the fact that most of relevant toxicological effects of a compound affect directly or indirectly the gene expression. The most common methods to profile gene expression at the transcript level are Northern Blotting and the real-time PCR. While commonly used and well accepted, these techniques are now superseded by new technologies allowing the analysis of the expression for multiple genes simultaneously. DNA microarrays are now developed for simultaneous gene analysis but inherent to such multiple assays, their quantitative aspect and their relevance for toxicogenomics have been questioned. We will review here recent studies on their use for toxicogenomics and examine the possible future of such technology in complementation with the other toxicology methods.

Animals↗
PubMed2002

CREB activation induced by mitochondrial dysfunction is a new signaling pathway that impairs cell proliferation.

We characterized a new signaling pathway leading to the activation of cAMP-responsive element-binding protein (CREB) in several cell lines affected by mitochondrial dysfunction. In vitro kinase assays, inhibitors of several kinase pathways and overexpression of a dominant-negative mutant for calcium/calmodulin kinase IV (CaMKIV), which blocks the activation of CREB, showed that CaMKIV is activated by a mitochondrial activity impairment. A high calcium concentration leading to the disruption of the protein interaction with protein phosphatase 2A explains CaMKIV activation in these conditions. Transcrip tionally active phosphorylated CREB was also found in a rho0 143B human osteosarcoma cell line and in a MERRF cybrid cell line mutated for tRNA(Lys) (A8344G). We also showed that phosphorylated CREB is involved in the proliferation defect induced by a mitochondrial dysfunction. Indeed, cell proliferation inhibition can be prevented by CaMKIV inhibition and CREB dominant-negative mutants. Finally, our data suggest that phosphorylated CREB recruits p53 tumor suppressor protein, modifies its transcriptional activity and increases the expression of p21(Waf1/Cip1), a p53-regulated cyclin-dependent kinase inhibitor.

Animals↗
PubMed2002

Compact disc with both numeric and genomic information as DNA microarray platform.

The compact disc (CD) is an ideal toolfor reading, writing, and storing numeric information. It was used in this work as a support for constructing DNA microarrays suited for genomic analysis. The CD was divided into two functional areas: the external ring of the CD was used for multiparametric DNA analysis on arrays, and the inner portion was usedfor storing numeric information. Because polycarbonate and CD resins autofluoresce, a colorimetric method for DNA microarray detection was used that is well adaptedfor the fast detection necessary when using a CD reader. A double-sided CD reader was developed for the simultaneous analysis of both array and numeric data. The numeric data are engraved as pits in the CD tracks and result in the succession of 0/1, which results from the modulation of the laser reflection when one reads the edges of the pits. Another diffraction-based laser was placed above the CD for the detection of the DNA targets on the microarrays. Both readersfit easily in a PC tower. Both numeric and genomic information data were simultaneously acquired, and each array was reconstituted, analyzed, and processed for quantification by the appropriate software.

Compact Disks↗
PubMed2002

Role of the endothelium and blood stasis in the appearance of varicose veins.

Today, chronic venous insufficiency affects millions of people but the investigation of veins and of venous diseases is still very poor. Additionally, the mechanism of the occurrence of varicose veins is not understood. Blood stasis is often associated with these pathological situations and we propose that resulting ischemic conditions can trigger the endothelium to release inflammatory mediators and growth factors. On one hand, the inflammatory mediators will recruit and activate neutrophils, which then infiltrate the venous wall and damage components of the extracellular matrix. On the other hand, growth factors induce smooth muscle cell migration, proliferation and de-differentiation into the synthetic phenotype, all together leading to the formation of neointima. These processes, being repeated over time, would eventually lead to alterations of the venous wall as observed in varicose veins. Venotropic drugs are used to treat chronic venous insufficiency. They are able to increase venous tone and to decrease vein and capillary permeability but they are also able to protect the endothelial cells against ischemia. Indeed, they target complexes of the mitochondrial respiratory chain and maintain ATP production during hypoxia. Hence, the cells are resistant to ischemia and do not release inflammatory mediators and growth factors. These drugs should thus be able to prevent the alterations of the venous wall induced by blood stasis.

Endothelium, Vascular↗
PubMed2002

Role of the endothelium and blood stasis in the development of varicose veins.

Today, chronic venous insufficiency affects millions of people but the investigation of veins and of venous diseases is still very poor. Additionally, the mechanism of the occurrence of varicose veins is not understood. Blood stasis is often associated with these pathological situations and we propose that resulting ischemic conditions can trigger the endothelium to release inflammatory mediators and growth factors. On one hand, the inflammatory mediators will recruit and activate neutrophils, which then infiltrate the venous wall and damage components of the extracellular matrix. On the other hand, growth factors induce smooth muscle cell migration, proliferation and de-differentiation into the synthetic phenotype, all together leading to the formation of neointima. These processes, being repeated over time, would eventually lead to alterations of the venous wall as observed in varicose veins. Venotropic drugs are used to treat chronic venous insufficiency. They are able to increase venous tone and to decrease vein and capillary permeability but they are also able to protect the endothelial cells against ischemia. Indeed, they target complexes of the mitochondrial respiratory chain and maintain ATP production during hypoxia. Hence, the cells are resistant to ischemia and do not release inflammatory mediators and growth factors. These drugs should thus be able to prevent the alterations of the venous wall induced by blood stasis.

Blood Circulation↗
PubMed2001

Growth kinetics rather than stress accelerate telomere shortening in cultures of human diploid fibroblasts in oxidative stress-induced premature senescence.

WI-38 human diploid fibroblasts underwent accelerated telomere shortening (490 bp/stress) and growth arrest after exposure to four subcytotoxic 100 microM tert-butylhydroperoxide (t-BHP) stresses, with a stress at every two population doublings (PD). After subcytotoxic 160 microM H2O2 stress or five repeated 30 microM t-BHP stresses along the same PD, respectively a 322 +/- 55 and 380 +/- 129 bp telomere shortening was observed only during the first PD after stress. The percentage of cells resuming proliferation after stress suggests this telomere shortening is due to the number of cell divisions accomplished to reach confluence during the first PD after stress.

Cell Division↗
PubMed2001

Colorimetric silver detection of DNA microarrays.

Development of microarrays has revolutionized gene expression analysis and molecular diagnosis through miniaturization and the multiparametric features. Critical factors affecting detection efficiency of targets hybridization on microarray are the design of capture probes, the way they are attached to the support, and the sensitivity of the detection method. Microarrays are currently detected in fluorescence using a sophisticated confocal laser-based scanner. In this work, we present a new colorimetric detection method which is intented to make the use of microarray a powerful procedure and a low-cost tool in research and clinical settings. The signal generated with this method results from the precipitation of silver onto nanogold particles bound to streptavidin, the latter being used for detecting biotinylated DNA. This colorimetric method has been compared to the Cy-3 fluorescence method. The detection limit of both methods was equivalent and corresponds to 1 amol of biotinylated DNA attached on an array. Scanning and data analysis of the array were obtained with a colorimetric-based workstation.

Bacterial Proteins↗
PubMed2001

Development of a sensitive multi-well colorimetric assay for active NFkappaB.

The transcription factor nuclear factor kappaB (NFkappaB) is a key factor in the immune response triggered by a wide variety of molecules such as inflammatory cytokines, or some bacterial and viral products. This transcription factor represents a new target for the development of anti-inflammatory molecules, but this type of research is currently hampered by the lack of a convenient and rapid screening assay for NFkappaB activation. Indeed, NFkappaB DNA-binding capacity is traditionally estimated by radioactive gel shift assay. Here we propose a new DNA-binding assay based on the use of multi-well plates coated with a cold oligonucleotide containing the consensus binding site for NFkappaB. The presence of the DNA-bound transcription factor is then detected by anti-NFkappaB antibodies and revealed by colorimetry. This assay is easy to use, non-radioactive, highly reproducible, specific for NFkappaB, more sensitive than regular radioactive gel shift and very convenient for high throughput screening.

Animals↗
PubMed2001

Hypoxia-induced increase in intracellular calcium concentration in endothelial cells: role of the Na(+)-glucose cotransporter.

Hypoxia is a common denominator of many vascular disorders, especially those associated with ischemia. To study the effect of oxygen depletion on endothelium, we developed an in vitro model of hypoxia on human umbilical vein endothelial cells (HUVEC). Hypoxia strongly activates HUVEC, which then synthesize large amounts of prostaglandins and platelet-activating factor. The first step of this activation is a decrease in ATP content of the cells, followed by an increase in the cytosolic calcium concentration ([Ca(2+)](i)) which then activates the phospholipase A(2) (PLA(2)). The link between the decrease in ATP and the increase in [Ca(2+)](i) was not known and is investigated in this work. We first showed that the presence of extracellular Na(+) was necessary to observe the hypoxia-induced increase in [Ca(2+)](i) and the activation of PLA(2). This increase was not due to the release of Ca(2+) from intracellular stores, since thapsigargin did not inhibit this process. The Na(+)/Ca(2+) exchanger was involved since dichlorobenzamil inhibited the [Ca(2+)](i) and the PLA(2) activation. The glycolysis was activated, but the intracellular pH (pH(i)) in hypoxic cells did not differ from control cells. Finally, the hypoxia-induced increase in [Ca(2+)](i) and PLA(2) activation were inhibited by phlorizin, an inhibitor of the Na(+)-glucose cotransport. The proposed biochemical mechanism occurring under hypoxia is the following: glycolysis is first activated due to a requirement for ATP, leading to an influx of Na(+) through the activated Na(+)-glucose cotransport followed by the activation of the Na(+)/Ca(2+) exchanger, resulting in a net influx of Ca(2+).

Adenosine Triphosphate↗
PubMed2001

PGF(2alpha), a prostanoid released by endothelial cells activated by hypoxia, is a chemoattractant candidate for neutrophil recruitment.

Despite increasing evidence supporting the involvement of neutrophils in ischemic and postischemic damages, the mechanisms underlying the early recruitment of these cells are not completely understood. In this report, the effects of conditioned media from hypoxic endothelial cells on neutrophil chemotaxis were investigated by biochemical and morphological studies. We showed that conditioned media collected from several endothelial cell origins submitted to hypoxia as well as ischemic rat liver perfusion liquids have a chemotactic activity for neutrophils. The role of various chemoattractant molecules like HETEs, platelet-activating factor, and cytokines such as interleukin-8 and interleukin-1 was examined in the same model. Chemotactic peptide contribution was ruled out as boiled conditioned media still trigger chemotaxis. However, cell treatment with cyclooxygenase inhibitors, neutralization of PGF(2alpha) biological activity with polyclonal antibodies, and the neutrophil preincubation with a specific PGF(2alpha) antagonist, all dramatically inhibited neutrophil chemotaxis. A strong chemoattractant effect of pure exogenous PGF(2alpha) or of a synthetic analog was also observed. The major effect of PGF(2alpha) on neutrophil chemotaxis was confirmed ex vivo in a rat liver perfusion ischemic model. These results suggest that PGF(2alpha), a prostanoid abundantly released by the endothelium of hypoxic or ischemic tissues, is a chemoattractant molecule that might be involved in the early recruitment of neutrophils in ischemic organs.

Animals↗
PubMed2001

Synthesis and structural analysis of the copper(II) complexes of N2-(2-pyridylmethyl)-2-pyridinecarboxamide.

Previous investigations of the potential of metal-organic compounds as inhibitors of human immunodeficiency virus type I protease (HIV-1 PR) showed that the copper(II) complex diaqua [bis(2-pyridylcarbonyl)amido] copper(II) nitrate dihydrate and the complex bis[N2-(2,3,6-trimethoxybenzyl)-4-2-pyridinecarboxamide] copper(II) behaved as inhibitors of HIV-1 PR. In a search for similar readily accessible ligands, we synthesised and studied the structural properties of N2-(2-pyridylmethyl)-2-pyridinecarboxamide (L) copper(II) complexes. Three different crystal structures were obtained. Two were found to contain ligand L simultaneously in a tridentate and bidentate conformation [Cu(L(tri)L(bi))]. The other contained two symmetry-related ligands, coordinated through the pyridine nitrogen and the amide oxygen atoms [Cu(L(bi))(2)]. A search of the Cambridge Structural Database indicated that L(tri) resulting from nitrogen bound amide hydrogen metal substitution is favoured over chelation through the amide oxygen atom. In our case, we calculated that the conformation of L(tri) is 11 kcal/mol more favourable than that of L(bi). ESI-MS experiments showed that the Cu(L(bi))(2) structure could not be observed in solution, while Cu(L(tri)L(bi))-related complexes were indeed present. The lack of protease inhibition of the pyridine carboxamide copper(II) complexes was explained by the fact that the Cu(L(bi)L(tri)) complex could not fit into the HIV-1 active site.

Catalytic Domain↗
PubMed2001

Quantitative assay for group M (subtype A-H) and group O HIV-1 RNA detection in plasma.

A quantitative HIV-1 test is described based on a competitive RT-PCR assay combined with a sandwich hybridization as a detection system. The internal RNA standard (IS) was designed specifically to be competitive during the amplification and during the hybridization step. Sample viral load determination was carried out with one RT-PCR in the presence of 10(3) IS copies. The HIV-1 copy number was calculated by reference to an external standard curve performed on known and increasing amounts of the reference HIV-1 (Ref HIV-1) RNA co-amplified with a constant amount of the IS RNA. The assay had a linear range from 10(1) to 10(6) HIV-1 copies. HIV-1 strains belonging to the different subtypes from group M, but also group O, were all detected. Absolute quantification of purified HIV-1 RNA copies gave identical results as the AMPLICOR HIV-1 Monitor assay. The quantification of patient's samples was evaluated according to different criteria such as dynamic range, sensitivity, efficacy of material recovery, reproducibility and convenience of sample handling. The microplate format of the assay combined with the colorimetric detection provides a convenient tool and fulfills the requirement for routine molecular diagnostic laboratories.

HIV Infections↗
PubMed2001

RT-PCR-ELOSA tests on pooled sample units for the detection of virus Y in potato tubers.

A highly sensitive RT-PCR protocol able to detect potato virus Y (PVY) in pooled sample units (tubers) was developed. PVY-specific primers selected in the coat protein gene were found to amplify a 359 bp fragment from diluted crude extract of infected tubers. For the detection of the amplification products, a colorimetric detection procedure in microtiter plates was established. The amplicons are hybridized between a covalently linked capture probe and a specific biotinylated detection probe ELOSA tests. This detection method detects at least 50 pg of virus per reaction for the four cultivars tested. The RT-PCR-ELOSA assay was adapted to pooled units in order to increase the sample size while reducing the number of tests.

DNA Primers↗
PubMed2001

Is the effect of interleukin-1 on glutathione oxidation in cultured human fibroblasts involved in nuclear factor-kappaB activation?

Our understanding of the interleukin-1 (IL-1) signaling molecular mechanisms has recently made considerable progress, with the discovery of the IL-1 receptor-associated kinase and the downstream enzymatic cascade that leads to the activation of nuclear factor-kappaB (NF-kappaB). IL-1 signaling and especially NF-kappaB activation are thought to be redox-sensitive, even though the precise nature and the molecular targets of the oxidants/antioxidants involved remain largely unknown. Here, we investigated the possible role of cellular oxidized/reduced glutathione (GSSG/GSH) balance in IL-1 signaling. We describe a quantitative method based on capillary electrophoresis designed to assay both intracellular GSH and GSSG in adhering fibroblasts. This method allows the GSSG/GSH balance to be followed during IL-1 stimulation. Our data show that IL-1 induces rapid and transient oxidation of intracellular glutathione in human fibroblasts. Using various antioxidants, including pyrrolidine dithiocarbamate and curcumin, we were unable to show a direct relationship between this IL-1-induced glutathione oxidation and NF-kappaB activation. Of the five antioxidants tested, only curcumin was able to inhibit IkappaBalpha degradation upstream and, hence, NF-kappaB DNA-binding activity and NF-kappaB-dependent expression of IL-6 downstream.

Antioxidants↗
PubMed2001

Consensus PCR and microarray for diagnosis of the genus Staphylococcus, species, and methicillin resistance.

We propose the use of DNA microarray for the discrimination of homologous products after a single PCR amplification with consensus primers. The method was applied to Staphylococcus identification. The femA nucleotide sequences, which are phylogenetically conserved among the staphylococci, were first amplified using a consensus primer pair together with the mecA sequence, a molecular marker for methicillin resistance. Products were then identified on a glass array. The microarray contained five selective DNA capture probes for the simultaneous and differential identification of the five most clinically relevant staphylococcal species (S. aureus, S. epidermidis, S. haemolyticus, S. hominis, and S. saprophyticus), while a consensus capture probe could detect all femA sequences, allowing the identification of the genus Staphylococcus. The mecA sequence hybridized to a specific capture probe. The identification was univocal because only a single capture probe had to be present for each sequence to be identified. The hybridization and identification processes were completed in less than 2 h. Current results demonstrate that low-density microarrays are powerful multigenotypic post-PCR analyzers and could compete with conventional bacteria identification.

Bacterial Proteins↗