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D Brault

Publications and source records attributed to D Brault.

At least 37 records · Page 2Linked to original sources
PubMed1996

Determination of plasma protein-bound malondialdehyde by derivative spectrophotometry.

We describe a method for the measurement of protein-bound malondialdehyde with the thiobarbituric acid reaction in human plasma using second-derivative spectrophotometry. Calibration was done by spectrum height measurement from the baseline at 532 nm. The data were compared with those obtained by using conventional absorbance and fluorimetric measurements. The results were linear from 0.2 to 80 mumol/l and the detection limit was 0.19 mumol/l. Within-run and between-run precision, evaluated by analysing pooled normal plasma, were 8 and 14% respectively. The method was tested for the influence of bilirubin, haemoglobin, glucose, urea, uric acid, sucrose and N-acetyl-neuraminic acid which interfered in the colorimetric method but not in the technique proposed here. The mean (+/-SD) malondialdehyde concentration determined in 59 healthy blood donors with the new assay was 0.34 (+/-0.14) mumol/l. This assay procedure could represent an alternative to high-performance liquid chromatography for the measurement of malondialdehyde in biological media.

Blood Proteins↗
PubMed1995

Interactions of dicarboxylic porphyrins with unilamellar lipidic vesicles: drastic effects of pH and cholesterol on kinetics.

The effect of pH on the transfer of deuteroporphyrin from dimyristoylphosphatidylcholine (DMPC) unilamellar vesicles to human serum albumin is investigated using a stopped-flow with fluorescence detection. The kinetics of this process allows for the determination of the rate constants for the porphyrin exist from the outer vesicle layer to the bulk aqueous medium (koff), and for its movement from the inner to the outer vesicle layer (kto). Both koff and kto are found to strongly depend on the pH. The observed behavior can be described by classical titration curves and is most likely due to protonation equilibria involving the two carboxylic groups of the porphyrin. A pH increase accelerates the exist of the porphyrin. The reverse effect is observed for its movement through the bilayer. The presence of cholesterol in the DMPC bilayer also strongly affects the interactions of the porphyrin with the vesicles. The rate constant kto is dramatically reduced by increasing the cholesterol content. An irregularity is noted around 10-20 mol % cholesterol. The results are discussed in relation to the preferential uptake of porphyrins by tumors, a basis of photodynamic therapy, and to possible pH-mediated relocalization of porphyrins among subcellular structures.

Cholesterol↗
PubMed1995

Comparison of the cDNA and amino acid sequences of lipoprotein lipase in eight species.

By aligning nucleotide and amino acid sequences of lipoprotein lipase in eight species (man, pig, cow, sheep, mouse, rat, guinea-pig and chicken), we found that the main domains (catalytic, N-glycosylation and putative heparin binding sites) are well conserved. The longest identical amino acid chain was encoded by a sequence between the end of exon 2 and the beginning of exon 3, emphasizing the importance of this region which encodes the beta 5-loop of the active site, among other domains. Exon 10 is entirely untranslated in the seven mammals studied here and contains species-characteristic deletions, insertions or elements rich in A or A + T. In chicken, the beginning of exon 10 is translated. These eight previously unreported alignments could be a useful tool for further studies on LPL function.

Amino Acid Sequence↗
PubMed1995

Photodynamic inactivation of cell-free HIV strains by a red-absorbing chlorin-type photosensitizer.

We have investigated the photodynamic activity of a new chlorin-type photosensitizer on a reference human immunodeficiency virus type 1 (HIV-1) strain, two wild-type HIV-1 isolates and two drug-resistant HIV-1 isolates. This chlorin was highly effective for the inactivation of free viruses, as assessed by two different quantitative cell culture assays. In the absence of blood components, all the HIV strains, including wild-type and drug-resistant mutant isolates, were totally inactivated using 30 micrograms ml-1 of chlorin and 0.75 J cm-2 of 661 nm light. Successful killing of HIV-1 strains in either plasma or whole blood was also obtained by increasing the chlorin concentration moderately. Our results demonstrate the antiviral efficiency of this chlorin, suggesting the potential application of dye-sensitized photoirradiation to decontaminate blood products.

Blood↗
PubMed1995

Homozygous deletion of exon 9 causes lipoprotein lipase deficiency: possible intron-Alu recombination.

We studied a homozygous deletion in the lipoprotein lipase gene at the molecular level. Comprising the end of intron 8, the whole of exon 9, and about two-thirds of intron 9, this 2.136-kb deletion caused complete lipoprotein lipase deficiency and severe hypertriglyceridemia (type I hyperlipoproteinemia). Intron 9 of a normal control subject was also sequenced in order to define the exact borders of the deletion. Up to now, only the first 0.721 kb of intron 9 had been sequenced. Thus the complete sequence of intron 9 (3.090 kb) is now available. Three Alu sequences were characterized in the normal intron 9, while the proband had only the third complete Alu sequence. The first Alu sequence was located in the deleted region, and only the left arm of the second was present, as the deletion began near its center. A stem-loop structure involving a 14-nt region towards the end of intron 8 and an Alu sequence in intron 9 might have led to the deletion. Sequence analysis showed that the three Alu sequences belonged to the 40-million-year-old Alu-Sa subclass.

Adult↗
PubMed1994

Kinetic and equilibrium studies of porphyrin interactions with unilamellar lipidic vesicles.

The interaction of deuteroporphyrin with dimyristoylphosphatidylcholine unilamellar vesicles of various sizes (ranging from 38 to 222 nm) has been studied using a stopped flow with fluorescence detection. Beside the kinetics of porphyrin incorporation into vesicles, the transfer of porphyrin from vesicles to human serum albumin has been investigated both experimentally and theoretically. The effects of both vesicle and albumin concentrations indicate that the transfer proceeds through the aqueous phase. It is governed by the rate of incorporation of porphyrin into the outer vesicle hemileaflet (kon), by the exit to the bulk aqueous medium (koff), and by the association (kas) and dissociation (kdis) constants relative to albumin. In both systems studied, a slower transbilayer flip-flop accounts for the biphasic character of the kinetics. This model is strongly supported by the effects of vesicle size, temperature, and cholesterol. The dependence of kon on the vesicle size indicates that the incorporation is diffusion controlled. The constant koff is found to be closely coupled to the phase state of the bilayer. The transbilayer flip-flop rate constant is approximately the same in both directions (approximately 0.4 s-1 at 32 degrees C and pH 7.4). It is strongly affected by the presence of cholesterol in vesicles and by the temperature, with a sharp enhancement around the phase transition. With the exception of very small vesicles obtained by sonication, no influence of the vesicle size on the flip-flop rate was observed. An accelerating effect of tetrahydrofuran, used to improve the solubility of porphyrin, has been noted. Steady-state measurements and kinetics results were in excellent agreement. The interest of systems involving albumin as a scavenger to extract important rate constants, is emphasized.

Deuteroporphyrins↗
PubMed1994

Validation of the in vivo CD1 mouse splenocyte micronucleus test.

In order to validate the in vivo micronucleus test in mouse splenocytes using the cytokinesis block method, 14 compounds with various mechanisms of action were tested: three direct alkylating agents (mitomycin C, ethylnitrosourea, beta-propiolactone), seven indirect alkylating agents (cyclophosphamide, benzo[a]pyrene, diethylnitrosamine, dimethylnitrosamine, 4-aminophenol, 4-aminobiphenyl, 1,1-dimethylhydrazine), two intercalating agents (acridine orange, ethidium bromide) and two spindle poisons (vincristine, colchicine). Male mice were dosed once with the compound, and spleen samples were taken 2 or 14 days after treatment. A significant increase in the binucleated micronucleated splenocyte rate was observed with all the alkylating and intercalating agents at at least one sampling time. In contrast, no increase in the binucleated micronucleated splenocyte rate was observed with the spindle poisons. In conclusion, under these experimental conditions, this in vivo test seems appropriate for the detection of clastogenic compounds including compounds that cannot be detected in the bone marrow micronucleus test. The limit of this test, as expected, is the lack of detection of aneugenic compounds.

Alkylating Agents↗
PubMed1993

Kinetics of incorporation of porphyrins into small unilamellar vesicles.

The kinetics of hematoporphyrin or deuteroporphyrin incorporation in egg phosphatidylcholine small unilamellar vesicles have been investigated by fluorescence stopped-flow measurements. The processes can be described by a fast equilibrium. The on-rate constant is nearly diffusion controlled regardless of the compound used and the pH. The affinity of these porphyrins for the vesicles is merely governed by the exit rate which depends on the structure of the porphyrin and on its charge determined by pH.

Deuteroporphyrins↗
PubMed1993

Interactions of dicarboxylic porphyrins with membranes in relation to their ionization state.

The interactions of dicarboxylic porphyrins with membrane systems are discussed with particular emphasis on the effect of the charge of the porphyrin and the nature of the side-chains. The incorporation of hematoporphyrin or related dicarboxylic porphyrins within small unilamellar vesicles as membrane models is favored by a decrease of the pH in the range of physiological pH values. This effect might play an important role in the retention of porphyrins by tumors, which are more acidic than normal tissues. Kinetics studies also show that the partition of the porphyrin between the lipidic bilayer and the aqueous phase is governed by its release rate rather than by its incorporation rate.

Animals↗
PubMed1992

Sequence of rat lipoprotein lipase-encoding cDNA.

A rat lipoprotein lipase (LPL)-encoding cDNA (LPL) has been entirely sequenced and compared to the sequences of all the LPL cDNAs reported in other species. As expected, high homology was found between the coding exons. The putative catalytic triad, Ser132, Asp156, His241, according to human numbering, is conserved in rat. As is the case in mouse, an Asn444 present in human LPL is also missing. The major divergences between human, mouse and rat LPLs were observed in the untranslated exon 10, where (i) the rat cDNA exhibits a 157-bp insertion and an 81-bp deletion relative to human; (ii) neither the B1 repeat nor the homopurine stretch reported in mouse can be recognized, and (iii) the rat cDNA displays several A+T-rich stretches.

Amino Acid Sequence↗
PubMed1992

Uptake and retention of Photofrin by cultivated human lymphoblastic cells (Reh6): preferential affinity of the cells for a minor component demonstrated by normal phase chromatography.

The uptake of Photofrin by the human cultivated lymphoblastic cell line Reh6 was studied using normal phase high performance liquid chromatography (HPLC) techniques. Relative cellular uptake of eight fractions (uptake/amount of component initially present in the incubation solution) was determined. After 4 h of incubation, protoporphyrin and a small fraction (denoted 4) were incorporated to a greater relative extent than the other fractions. Weakly incorporated components (hematoporphyrin and aggregate-like components) were better retained by cells than the hydrophobic monomeric porphyrins (protoporphyrin and hydroxyethylvinyldeuteroporphyrin). Thus, any benefit gained from a higher uptake was mostly cancelled by a fast release--a situation observed for all fractions except for fraction 4, which displayed both high uptake and good cellular retention. This pattern was not modified when Photofrin concentration or serum percentage was changed. Fraction 4 was further resolved using a gradient system on normal silica. A single component appeared to be mostly responsible for the favorable properties presented by fraction 4, i.e. high uptake and retention within cells. This component was found to correspond to a late eluted peak in the typical reverse-phase HPLC profile of Photofrin. These results emphasize the possible role of minor Photofrin components.

Antineoplastic Agents↗
PubMed1992

[Genes of the lipase family: comparison of nucleic and proteinic sequences].

Vertebrates' plasmatic apolipoproteins and a few number of lipases in their metabolism present sequence homologies. They are grouped in genes families. The four exons apolipoproteins gene family includes nine human genes: the divergence rate of their sequences allows to place the first ancestral gene very high in the phylogenetic tree of the evolution. However, a more recent duplication of apolipoprotein C-I gene dating from 40 millions years, may be a phylogenetic marker for the radiation of Monkeys. Pancreatic lipase and isoforms, lipoprotein-lipase and hepatic triacylglycerol-lipase form by their homologies a "superfamily" of genes, which also includes yolk proteins of Dipterians eggs. Sequence homologies of PL, LPL and HL are analysed and compared with multiple alignments of amino-acids and nucleotides on spreadsheets. From these comparisons we may characterize four classes of phylogenetic markers: 1) repetitive DNA sequence (Alu, B1, PRE-1) appeared during Mammals evolution, 2) short insertions or deletions (within N-terminal domain) and a gene conversion in guinea-pig lineage, 3) a progressive reduction of intron number during the lipases evolution, 4) several duplications of genes which have produced the five genes of this superfamily currently known in the human genome.

Amino Acid Sequence↗
PubMed1992

[Lipoprotein lipase deficiencies].

Lipoprotein lipase (LPL) is an enzyme which plays a major role in the metabolism of circulating triglyceride-rich lipoproteins. It hydrolyzes chylomicron and VLDL triglycerides, thereby delivering fatty acids to tissues for storage or oxidation. In order to gain insight into the molecular basis of LPL deficiency, the structure of the LPL gene (ten exons and nine introns spanning about 30 kb) is first set out in relation to the different domains of the LPL protein. There is a high sequence homology between the aminoacids of LPL and of other lipases, such as hepatic triglyceride lipase (HL) and pancreatic lipase (PL). The PL catalytic triad Ser132, Asp156, His241 is also present in LPL. Absence of LPL activity can result from absence of LPL protein synthesis (Brunzell class I), or from the synthesis of an LPL protein devoid of enzymatic activity consequently to a mutation (class II). LPL can also be unable to bind to endothelial cells--a defect combined with deficient enzymatic activity--(class III). Among the known mutations of the LPL gene (such as nonsense, frameshift, abnormality in intron-exon junction, deletion, duplication) resulting in pathological cases, the most frequent are punctual mutations located mainly in exons 4, 5 and 6, leading to the substitution of an aminoacid for another in essential domains of LPL. The combined deficiency LPL + HL has also been described. The study of the abnormalities of the LPL gene, known only since the years 1990-1991, allows not only to better understand the pathology of LPL deficiencies, but also to point out which aminoacids play a major role in LPL activity.

DNA Mutational Analysis↗
PubMed1991

In vitro uptake of dicarboxylic porphyrins by human atheroma. Kinetic and analytical studies.

Human atheromatous aorta segments as well as presumably disease-free control aorta were obtained at autopsy. They were incubated with solutions of various purified dicarboxylic porphyrins including hematoporphyrin (HP) and hydroxyethylvinyldeuteroporphyrin (HVD), and with solutions of Photofrin. Selective labelling of the atheroma was shown by macroscopic and microscopic observations of the characteristic porphyrin fluorescence associated with the atheromatous plaques. The time dependence of the uptake, monitored by absorption spectrophotometry or by high performance liquid chromatography, was inferred from the disappearance of the porphyrins in the incubation medium. Significant binding was observed in the absence of albumin or serum proteins. The uptake of HP was less than that of the more hydrophobic compounds HVD or Photofrin when these porphyrins were used alone. The presence of albumin or serum drastically reduces atheroma labelling. Some competition between HP and HVD for binding sites is also seen. The present results do indicate that hydrophobic porphyrins have an intrinsic affinity for atheroma and that they can be taken up through passive processes. Taking into account previous data on animal models (Photochem. Photobiol. (1989), 731-737), it appears however that, in vivo, interactions with proteins and pharmacokinetics will primarily determine plaque labelling.

Aorta↗