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Biomedical subjects

H Dubois

Publications and source records attributed to H Dubois.

12 recordsLinked to original sources
PubMed2001

Polypeptide composition of an adenovirus type 5 used in cancer gene therapy.

For cancer gene therapy, a recombinant adenovirus serotype 5 named RPR/INGN201 has been constructed by susbtitution of the E1 region with human tumor suppressor gene p53. The protein components of RPR/INGN201 virions were separated by reversed-phase HPLC and were individually identified by electrospray time-of-flight mass spectrometry and N-terminal sequencing, both on intact proteins and on their proteolytic fragments after trypsin digestion. Twenty-five peptide components of the proteome (including fiber) with greater than 0.25-0.5% contribution to the protein content of the virus were identified and characterized. Fiber was confirmed to be partially glycosylated (both the non-glycosylated and the monoglycosylated states were identified), and two proteins were isolated and identified as phosphorylation derivatives, namely protein V (non-phosphorylated and monophosphorylated) and protein IIIa (mono- and diphosphorylated). This new analytical tool proved to be very useful not only for refining our current knowledge of the polypeptide repertoire of purified infectious virions but also for monitoring and very rapidly identifying structural modifications resulting from changes in the manufacturing process. It was also used successfully for the characterization of various adenoviral constructs.

Adenoviridae↗
PubMed2000

Telerehabilitation services using web-based telecommunication.

A World Wide Wide-based telerehabilitation platform has been demonstrated in a laboratory environment. This platform allows a rehabilitation provider to thoroughly evaluate the progress of a patient remotely with the same care and measurement precision that would be possible if the provider and the patient were in the same room. The platform was designed to be Web-based so that the service could be offered at the same price without regard to long distance telecommunication facility charges. The Web-based implementation allows enough bandwidth for a simultaneous video teleconference and a precision data acquisition mode even when the Web connection is a low cost analog modem computer interface at both ends of the connection.

Ambulatory Care↗
PubMed1998

[Long-term results of proximal osteocartilaginous autografts in extensive cartilagenous destruction of the knee. Apropos of 5 cases].

Five patients with a large defect in the articular cartilage at the knee joint were treated by transplantation of an autogenic osteochondral fragment. The graft was harvested from the posterior portion of the ipsilateral femoral condyle in 4 cases of osteochondritis dissecans, and from the lateral third of the patella pedicled on the patellar ligament in one case of posttraumatic necrotic collapse of the lateral tibial plateau. One patient underwent concomitant high tibial osteotomy. Two months postoperatively bony union was achieved in all cases but in one case, the grafted articular cartilage did not survive after weight bearing because of an overlooked varus deformity. At the follow-up examination (8 to 20 years) all 5 patients were asymptomatic ; the range of flexion was somewhat restricted (120 degrees) ; roentgenogram revealed slight narrowing of the articular space or at least flattening of the grafted zone and subchondral osteosclerosis. At arthroscopic exploration, the grafted zones were recognizable from the surrounding cartilage, and histologic examination of their border revealed fibrocartilage and proliferating vessels; late gonarthrosis might ensue over time. Therefore the procedure should be performed only in large osteochondral defects where neither reattachment of a loose body, nor hemiarthroplasty, nor isolated osteotomy are suitable and before degenerative changes have developed. Morever any associated varus deformity requires concomitant correction by high tibial osteotomy to relieve stress from the graft.

Adult↗
PubMed1996

Lipid-binding properties of synthetic peptide fragments of human apolipoprotein A-II.

Human apolipoprotein A-II (apo A-II) consists of three potential amphipathic helices of 17 residues each, which contribute to the lipid-binding properties of this apolipoprotein. The conformation and lipid-binding properties of these peptides, either as single-helix or as two-helix peptides, were investigated by turbidity, fluorescence, electron-microscopy and circular-dichroism measurements, and are compared in this article. The lipid affinity of shorter C-terminal segments of apo A-II was compared with those of the single-helix or two-helix peptides, to define the minimal peptide length required for stable complex formation. The properties of the apo-A-II-(13-48)-peptide were further compared with those of the same segment after deletion of the Ser31 and Pro32 residues, because the deleted apo-A-II-(13-30)-(33-48)-peptide, is predicted to form a long uninterrupted helix. The single helices of apo A-II could not form stable complexes with phospholipids, and the helix-turn-helix segment spanning residues 13-48 was not active either. The apo-A-II-(37-77)-peptide and the apo-A-II-(40-73)-peptide could form complexes with lipids, which appear as discoidal particles by negative-staining electron microscopy. The shortest C-terminal domain of apo A-II able to associate with lipids to form stable complexes was the apo-A-II-(40-73)-peptide, which consisted of the C-terminal helix, a beta-turn and part of the preceding helix. The shorter apo-A-II-(49-77)-peptide, and the helical apo-A-II-(13-30)-(33-48)-peptide, could also associate with phospholipids. The complexes formed were, however, less stable, as they dissociated outside the transition temperature range of the phospholipid. These data suggest that the C-terminal pair of helices of apo A-II, which is the most hydrophobic pair, is responsible for the lipid-binding properties of the entire protein. The N-terminal pair of helices of apo A-II at residues 13-48 does not associate tightly with lipids. The degree of internal similarity and the cooperativity between the helical segments of apo A-II is thus less pronounced than in apo A-I or apo A-IV. The N-terminal and C-terminal domains of apo A-II appear to behave as two distinct entities with regard to lipid-protein association.

Amino Acid Sequence↗
PubMed1996

Purification, stabilization, and crystallization of a modular protein: Grb2.

We report here the purification and the crystallization of the modular protein Grb2. The protein was expressed as a fusion with glutathione-S-transferase and purified by affinity chromatography on glutathione agarose. It was apparent from reverse phase chromatography that the purified protein was conformationally unstable. Instability was overcome by the addition of 100 mM arginine to the buffers. Because Grb2 appeared to be extremely sensitive to oxidation, crystallization experiments were performed with a dialysis button technique involving daily addition of fresh DTT to the reservoirs. The presence of 8 to 14% glycerol was necessary to obtain monocrystals. These results are discussed in relation with the modular nature of Grb2.

Adaptor Proteins, Signal Transducing↗
PubMed1994

The human galanin receptor: ligand-binding and functional characteristics in the Bowes melanoma cell line.

The human galanin receptor has been characterized pharmacologically from the Bowes melanoma cell line. Using porcine [125I]galanin as the radioligand, a single population of non-interacting high-affinity binding sites (KD = 0.05 +/- 0.01 nM; Bmax = 135 +/- 7 fmol/mg protein) was demonstrated. Human galanin peptide competitively inhibited the specific binding of [125I]galanin (IC50 = 0.35 +/- 0.13 nM) and decreased the forskolin-stimulated cAMP production (EC50 = 0.46 +/- 0.05 nM) with a maximal inhibition of 63 +/- 2% at 10(-7) M. Rat and porcine galanin peptides and the chimeric peptides M15, M35, M32, M40 and C7 also dose-dependently inhibited the forskolin-stimulated cAMP production, while the fragment porcine galanin-(3-29) and [D-Trp2]galanin were found to be inactive. The specific binding of [125I]galanin was decreased in a dose-dependent manner by GTP and the cAMP response was inhibited by the pertussis toxin, suggesting the activation of a G-protein dependent process. The Bowes cell line thus appears to be a relevant tool for the study of human galanin receptor.

Animals↗
PubMed1989

Coenzyme binding by 3-hydroxybutyrate dehydrogenase, a lipid-requiring enzyme: lecithin acts as an allosteric modulator to enhance the affinity for coenzyme.

The role of phospholipid in the binding of coenzyme, NAD(H), to 3-hydroxybutyrate dehydrogenase, a lipid-requiring membrane enzyme, has been studied with the ultrafiltration binding method, which we optimized to quantitate weak ligand binding (KD in the range 10-100 microM). 3-Hydroxybutyrate dehydrogenase has a specific requirement of phosphatidylcholine (PC) for optimal function and is a tetramer quantitated both for the apodehydrogenase, which is devoid of phospholipid, and for the enzyme reconstituted into phospholipid vesicles in either the presence or absence of PC. We find that (i) the stoichiometry for NADH and NAD binding is 0.5 mol/mol of enzyme monomer (2 mol/mol of tetramer); (ii) the dissociation constant for NADH binding is essentially the same for the enzyme reconstituted into the mixture of mitochondrial phospholipids (MPL) (KD = 15 +/- 3 microM) or into dioleoyl-PC (KD = 12 +/- 3 microM); (iii) the binding of NAD+ to the enzyme-MPL complex is more than an order of magnitude weaker than NADH binding (KD approximately 200 microM versus 15 microM) but can be enhanced by formation of a ternary complex with either 2-methylmalonate (apparent KD = 1.1 +/- 0.2 microM) or sulfite to form the NAD-SO3- adduct (KD = 0.5 +/- 0.1 microM); (iv) the binding stoichiometry for NADH is the same (0.5 mol/mol) for binary (NADH alone) and ternary complexes (NADH plus monomethyl malonate); (v) binding of NAD+ and NADH together totals 0.5 mol of NAD(H)/mol of enzyme monomer, i.e., two nucleotide binding sites per enzyme tetramer; and (vi) the binding of nucleotide to the enzyme reconstituted with phospholipid devoid of PC is weak, being detected only for the NAD+ plus 2-methylmalonate ternary complex (apparent KD approximately 50 microM or approximately 50-fold weaker binding than that for the same complex in the presence of PC). The binding of NADH by equilibrium dialysis or of spin-labeled analogues of NAD+ by EPR spectroscopy gave complementary results, indicating that the ultrafiltration studies approximated equilibrium conditions. In addition to specific binding of NAD(H) to 3-hydroxybutyrate dehydrogenase, we find significant binding of NAD(H) to phospholipid vesicles. An important new finding is that the nucleotide binding site is present in 3-hydroxybutyrate dehydrogenase in the absence of activating phospholipid since (a) NAD+, as the ternary complex with 2-methylmalonate, binds to the enzyme reconstituted with phospholipid devoid of PC and (b) the apodehydrogenase, devoid of phospholipid, binds NADH or NAD-SO3- weakly (half-maximal binding at approximately 75 microM NAD-SO3- and somewhat weaker binding for NADH).(ABSTRACT TRUNCATED AT 400 WORDS)

Allosteric Regulation↗
PubMed1989

An enzymic assay for uric acid in serum and urine compared with HPLC.

We evaluated a colorimetric method for the assay of uric acid in serum or urine, which utilises a Trinder chromogenic system modified by the inclusion of 2,4,6-tribromo-3-hydroxybenzoic acid for oxidative coupling to p-aminophenazone. Colour development (Amax: 512 nm) is complete within five minutes. Measurement of a sample blank is not needed. The procedure involves pre-incubation with ascorbic acid oxidase and detergent to eliminate interference by ascorbic acid and to abolish turbidity due to lipaemia; this pretreatment was effective up to 1.14 mmol/l ascorbate and up to at least 25 mmol/l triacylglycerol. Interference by icteric sera was insignificant up to about 170 mumol/l bilirubin. The method is linear up to at least 1428 mumol/l. In human serum and urine the procedure correlates well with HPLC and the uricase p-aminophenazone method on the SMAC analyser. Within-run and between-run imprecisions of the enzymic test were higher than for HPLC, but did not exceed 1.2% (CV) and 2.5% (CV), respectively.

Chemical Phenomena↗
PubMed1986

Cyanylation of 3-hydroxybutyrate dehydrogenase. The "essential" sulfhydryl group is not involved in catalysis.

3-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme with an absolute requirement of lecithin for function. The enzyme contains two sulfhydryl groups per monomer. Modification of the more reactive sulfhydryl group with N-ethylmaleimide resulted in inactivation of the enzyme and modification of coenzyme-binding characteristics [McIntyre, J. O., Fleer, E. A. M. and Fleischer, S. (1984) Biochemistry 23, 5135-5141]. The present study further investigates the function of the sulfhydryl groups by utilizing chemical derivatization techniques. The reactive sulfhydryl was derivatized first with 3,3'-dithiobis(6-nitrobenzoic acid) (Ellman's reagent) to form the S-(carboxynitrophenylthio) derivative which could then be replaced with cyanide to form the S-cyanylated enzyme. We find that derivatizing the essential sulfhydryl group leads to some loss of activity. The effect appears to be steric since a larger derivatizing group gives greater loss of activity. The normal enzyme is inhibited approximately 50% in excess substrate. Derivatization of the reactive sulfhydryl group results in loss of this substrate inhibition, the modified enzyme being at least three-fold more active at high substrate concentrations; the activity increases from 18% to 54% and from 1% to 4% of maximal activity for the S-cyanylated and S-(carboxynitrophenylthio) enzyme derivatives, respectively. Cyanylation results in complete loss of fluorescence energy transfer from tryptophan to NADH at low salt concentration but is normal in the presence of 100mM NaCl. However, the binding constant of the coenzyme is decreased only several-fold in the cyanylated enzyme as studied by fluorescence quenching. The cyanylated enzyme formed tight ternary complexes (spin-labeled NADH-monomethylmalonate) (spin-labeled NAD-sulfite) similar to that formed by the normal enzyme. The spin label is highly immobilized, but the hyperfine splitting values differ somewhat from the normal enzyme. We conclude that the reactive sulfhydryl is close to the active site of 3-hydroxybutyrate dehydrogenase but is not involved in the catalytic mechanism.

Animals↗