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Biomedical subjects

C Briand

Publications and source records attributed to C Briand.

At least 19 recordsLinked to original sources
PubMed1992

Mechanism of binding of the new antimitotic drug MDL 27048 to the colchicine site of tubulin: equilibrium studies.

MDL 27048 [trans-1-(2,5-dimethoxyphenyl)-3-[4-(dimethylamino)phenyl]-2- methyl-2-propen-1-one] fluoresces when bound to tubulin but not in solution. This effect has been investigated and found to be mimicked by viscous solvents. Therefore, MDL 27048 appears to be a fluorescent compound whose intramolecular rotational relaxation varies as a function of microenvironment viscosity. The binding parameters of MDL 27048 to tubulin have been firmly established by fluorescence of the ligand, quenching of the protein fluorescence, and gel equilibrium chromatography. The apparent binding equilibrium constant was (2.75 +/- 0.45) x 10(6)M-1, and the binding site number was 0.81 +/- 0.12 (10 mM sodium phosphate-0.1 mM GTP, pH 7.0, at 25 degrees C). The binding is exothermic. The binding of MDL 27048 overlaps the colchicine and podophyllotoxin binding sites. Binding of MDL 27048 to the colchicine site was also measured by competition with MTC [2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one] , a well-characterized reversibly binding probe of the colchicine site [Andreu et al. (1984) Biochemistry 23, 1742-1752; Bane et al., (1984) J. Biol. Chem. 259, 7391-7398]. In contrast with close analogues of colchicine, MDL 27048 and podophyllotoxin neither affected the far-ultraviolet circular dichroism spectrum of tubulin, within experimental error, nor induced tubulin GTPase activity. Like podophyllotoxin, an excess of MDL 27048 over tubulin induced no abnormal cooperative polymerization of tubulin, which is characteristic of colchicine binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Fluorescence stopped-flow study of the interaction of tubulin with the antimitotic drug MDL 27048.

The kinetics of the binding of MDL 27048 to tubulin have been studied by fluorescence stopped flow. The binding is accompanied by a fluorescence increase. The time course can be described by a sum of two exponentials, assumed to be due to the presence of two major tubulin isoforms. The observed rate constants depend in a nonlinear way on the concentration of MDL in pseudo-first-order conditions. This concentration dependence can be described by the presence of a fast equilibrium of low affinity, followed by an isomerization of the initial complex. The dissociation kinetics have been studied by displacement experiments, in which MTC was used as a competitive ligand. The reaction enthalpy change for the first binding equilibrium and the activation energies for the forward and reverse steps of the isomerization were determined from the temperature dependence. This was possible for the two tubulin isotype populations. The kinetics of the binding of MDL to tubulin are slowed down in the presence of 3',4',5'-trimethoxyacetophenone, a fast binding analog of the colchicine A-ring, but are not influenced by the binding of tropolone methyl ether, indicating that the binding site of MDL has the A-subsite in common with colchicine, but not the C-subsite.

Animals↗
PubMed1991

Selenium: inhibition of microtubule formation and interaction with tubulin.

We have studied the interaction of Na2SeO3 with microtubule proteins and tubulin. This selenium compound inhibits the polymerization of MTP (half-inhibition occurred for Na2SeO3 10 microM), and to a lesser that of tubulin. This effect of selenite is related to the formation of disulfide bridges between tubulin sulfhydryl groups, inducing a conformational change of the protein. This is corroborated by the modified binding of colchicine and vinblastine in presence of selenium. The selenite inhibitory concentrations are similar to the toxic blood levels of selenium (40 microM).

Animals↗
PubMed1990

Thermodynamic study of the interaction of methotrexate, its metabolites, and new antifolates with thymidylate synthase: influence of FdUMP.

A microcalorimetric method was used for the direct study of the interaction of methotrexate, its metabolites, and new antifolates N10-propargyl-5,8-dideazafolate (CB 3717) and 2-methyl,2-desamino N10-propargyl-5,8-dideazafolate (CB 3819), with thymidylate synthase. We show that 7-hydroxymethotrexate and dideazafolates require the prior binding of dUMP or its fluorinated derivative FdUMP to bind to thymidylate synthase, as does methotrexate. Conversely, we show that methotrexate-G2 can interact directly with the enzyme alone. On the other hand, both dUMP and FdUMP exhibited a large cooperative effect on the affinity for thymidylate synthase of the inhibitors, and surprisingly, no significant difference was shown at this level between the natural substrate dUMP and its fluorinated derivative. It was demonstrated that this cooperative effect had an enthalpic origin. In the presence of FdUMP or dUMP, all the studied compounds except 7-hydroxymethotrexate exhibited a large negative enthalpy variation when binding to thymidylate synthase (from -44 to -91 kJ/mol). CB 3717 and methotrexate-G2 are competitors for the same protein binding site. Polyglutamation of methotrexate lead to compounds with higher affinity (association constants were 6.6 x 10(3) M-1 and 2.3 x 10(6) M-1 for methotrexate and methotrexate-G2 respectively) while hydroxylation has an unfavourable effect (association constant of 7-hydroxymethotrexate inferior to 500 M-1). Evidence for the influence of polyglutamation was also provided by the relatively low affinity of dideazofolates for thymidylate synthase (association constant equal to 1.4 and 1.7 x 10(7) M-1 for CB 3717 and CB 3819, respectively), whereas these compounds are known to be strong inhibitors of the enzyme in cells in their polyglutamated forms.

Calorimetry↗
PubMed1990

C-terminal cleavage of tubulin by subtilisin enhances ring formation.

Following cleavage of alpha- and beta-tubulin C termini, under mild conditions we observed that microtubule-related polymers were formed, and also that ring aggregates were abundant. These ring aggregates were clearly detected by turbidity and electron microscope studies under standard assembly conditions. It was found that removal of the C-terminal fragments of tubulin (phosphocellulose-tubulin or Weisenberg tubulin) markedly favored Mg2(+)-induced ring formation. Binding of GDP to the exchangeable nucleotide site of cleaved tubulin further enhanced ring formation. The cleaved tubulin-GDP ring aggregates could be classified into three types: aggregates without apparent order, bidimensionally ordered ring aggregates, and stacks of rings. Temperature had little effect on the formation of these ring aggregates; however, they were very sensitive to ionic strength.

Animals↗
PubMed1990

[Subcophosis and otosclerosis. Medical and surgical treatment].

The spiral ligament of the stria vascularis is nearly always damaged by otosclerotic foci when the patient is nearly totally deaf because of otosclerosis. Vascular drugs are to be prescribed in such a case, besides the Sodium Fluoride treatment. The Sodium Fluoride is, according to us, the best treatment to arrest the progression of the otosclerotic disease. Sodium fluoride will destroy the proteolytic enzymes damaging the inner ear membranes and the hair of the Corti hair cells.

Audiometry↗
PubMed1990

Activity measurements in human tissues of the methotrexate molecular target: a novel fluorometric assay.

As DHFR is the main molecular target of MTX, a widely used anticancer drug, its level in human tissues is likely to be one of the factors determining tissue sensitivity towards this drug. Forty-one biopsies were analyzed for their DHFR activity by a convenient spectrofluorometric assay developed in our laboratory; this sensitive method proved to be suitable for measurements in very small human samples. Statistical analysis of the results showed that (i) DHFR activity is not an index of tumorogenicity, at least in the cases studied, (ii) tumorous extracts contain modulators of DHFR activity.

Biopsy↗
PubMed1989

Interaction of tubulin and cellular microtubules with the new antitumor drug MDL 27048. A powerful and reversible microtubule inhibitor.

We have characterized the binding of trans-1-(2,5-dimethoxyphenyl)-3-[4-(dimethylamino)phenyl]-2-methyl-2- propen- 1-one (MDL 27048) to purified procine brain tubulin, and the inhibition of microtubule assembly by this compound in vitro and using cultured cells. Binding measurements were performed by difference absorption and fluorescence spectroscopy. MDL 27048 binds to one site/tubulin heterodimer with an apparent equilibrium constant Kb = (2.8 +/- 0.8) X 10(6) M-1 (50 mM 2-(N-morpholino)ethanesulfonic acid, 1 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, 0.5 mM MgCl2, 0.1 mM GTP buffer, pH 6.7, at 25 degrees C). Podophyllotoxin displaced the binding of MDL 27048, suggesting an overlap with the colchicine-binding site. Assembly of purified tubulin into microtubules was inhibited by substoichiometric concentrations of MDL 27048, which also induced a slow depolymerization of preassembled microtubules. The cytoplasmic microtubules of PtK2 cells were disrupted in a concentration and time-dependent manner by MDL 27048, as observed by indirect immunofluorescence microscopy. Maximal depolymerization took place with 2 X 10(-6) M MDL 27048 in 3 h. When the inhibitor was washed off from the cells, fast microtubule assembly (approximately 8 min) and complete reorganization of the cytoplasmic microtubule network (15-30 min) were observed. MDL 27048 also induced mitotic arrest in SV40-3T3 cell cultures. Due to all these properties, this anti-tumor drug constitutes a new and potent microtubule inhibitor, characterized by its specificity and reversibility.

Animals↗
PubMed1988

Influence of the guanine nucleotide phosphorylation state and of Mg2+ ions on the interaction of vinzolidine/tubulin 6 S: a fluorescence quenching study.

The binding of the new vincaalkaloid vinzolidine to tubulin 6 S was investigated by using fluorescence quenching methods. The value of the apparent equilibrium binding constant was found to depend on the phosphorylation state of the guanine nucleotide bound to the tubulin exchangeable site (E-site), with Ka values of 4.9 X 10(4) and 8.19 X 10(4) M-1 for GTP- and GDP-tubulin, respectively. The effect of Mg2+ ions on this binding was more important on GTP-tubulin than on GDP-tubulin, and might be related to the existence of Mg2+ site(s) independent of the nucleotide.

Animals↗
PubMed1988

Vertigo in postoperative follow-up of otosclerosis.

Causes of vertigo after otosclerosis surgery were studied postoperatively and in long-term follow-up examinations. Pressure and mobility changes in the posterior labyrinth fluids, enzymatic process, and decrease in blood supply at the time of operation appear to be the major causes. Methods of detection, avoiding, and managing vertigo are presented.

Follow-Up Studies↗
PubMed1988

[Sound injuries after surgery of otosclerosis].

Surgery for otosclerosis means that the sound protection due to the fixation of the footplate does not exist anymore. As the Corti hair cells have been fragilized by proteolytic enzymes, it is absolutely necessary to reconstruct the sound mechanism in order to maintain the vibration of the perilymph absolutely within acoustical physiologic limit of movement. The means to realize this reconstruction are presented in this paper, as well as the results.

Hearing Loss, Noise-Induced↗
PubMed1987

A flow microcalorimetric method for enzyme activity measurements: application to dihydrofolate reductase.

A flow microcalorimetric method was developed for the analysis of enzymatic activities in crude tissue homogenates. It can be applied whenever a heat exchange is involved in an enzymatic reaction. The consequent sensitivity obviously depends on the enthalpy variation observed. Dihydrofolate reductase was chosen as an example; this enzyme is the molecular target of methotrexate, a widely used anticancer agent. This calorimetric method, whose sensitivity limit is 1.48 X 10(-4) units of dihydrofolate reductase per milliliter of reactant medium, allows enzyme activity measurements in tissues with low dihydrofolate reductase levels. A few examples of measurements in animal tissues are given. These measurements are of some interest; indeed, increased activity and increased levels of this enzyme are two of the mechanisms which may explain resistance to methotrexate.

Animals↗
PubMed1987

Effects of spirogermanium on the in vitro assembly and disassembly of brain microtubules.

The inhibition of microtubule proteins (MTP) assembly by Spirogermanium (SP, 1.25-100 microM) has been studied. Assembly at 37 degrees C was monitored by turbidity measurements and electron microscopy. For SP in 1:1 protein-drug ratio the inhibition of assembly was 50%. Addition of 12.5 microM SP to microtubules induced spontaneous disassembly. SP had less effect on the assembly of pure tubulin (tubulin 6S). Complete inhibition of assembly induced by glycerol and Mg2+ was found with 250 microM and the ratio of SP to tubulin to obtain 50% inhibition was higher than with MTP.

Antineoplastic Agents↗
PubMed1987

A novel fluorometric assay for quantitative analysis of dihydrofolate reductase activity in biological samples.

In an effort to study the level of dihydrofolate reductase (DHFR), the main molecular target of antifolate drugs, in healthy and malignant tissues of human origin, a new and convenient fluorometric enzymatic assay has been developed. The technique measures the overall decrease in fluorescence emission at 454 nm (lambda ex = 342 nm) due to the contributions from coenzyme oxidation and substrate reduction. This technique was developed by using an enzyme purified from beef liver. All criteria of quality were checked: sensitivity, reproducibility and specificity made it suitable for low activity measurements. It was successfully applied to human tissue crude extracts.

Animals↗
PubMed1987

Influence of time and chloride ions on the interaction of cisplatin with human albumin in-vitro.

The interaction of cis-dichlorodiammineplatinum (II) (cisplatin) with human serum albumin (HSA), dissolved in phosphate buffer with or without sodium chloride (0.1 M) has been examined at pH 7.4 and mu = 0.154. Equal volumes of cisplatin and HSA solutions were incubated at 37 degrees C for various times and filterable platinum concentrations versus time measured by flameless atomic absorption spectrophotometry. Binding kinetics differed depending on the buffer solutions used and on the time elapsing between cisplatin dissolution and outset of incubation with HSA. Experimental data were fitted to a theoretical equation used to calculate the number of nucleophilic sites per HSA molecule. Titrations of the HSA sulphydryl group content before and after incubation with a cisplatin solution were made, from which it was shown that the lone SH-group of the HSA macromolecule is involved in cisplatin binding. We also studied HSA's sensitivity towards denaturing agents when it was complexed with cisplatin. This sensitivity was decreased upon cisplatin binding. Also, the binding capacities of HSA and the HSA-Pt(II) complex to both tryptophan and warfarin were compared to determine the possible influence of cisplatin upon the binding to HSA of other drugs; this influence was negligible.

Chlorides↗
PubMed1986

In vitro mechanism study of microtubule assembly inhibition by cis-dichlorodiammine-platinum(II).

The inhibitions of microtubule protein (MTP) and tubulin 6S polymerizations by cis-dichlorodiammine-platinum(II) (CDDP) have been investigated by turbidity measurements and electron microscopy. For 2.5 X 10(-4) M CDDP after 40 min contact time at 27 degrees, the inhibition was 60% for MTP (1.2 mg/ml) and nearly 90% for tubulin 6S (1.2 mg/ml). Microtubules were not present after a 1 hr contact time at 27 degrees with 2.5 X 10(-4) M CDDP. Free sulfhydryl group determinations with 5,5'-dithio-bis-(2-nitrobenzoate) (DTNB) showed that 20.10 (+/- 0.05) sulfhydryl groups were found per tubulin dimer. In the presence of excess CDDP, this number was reduced to 17.74 (+/- 0.05) after a 1 hr contact time at 27 degrees. By using CDDP-tubulin dialysis assays, the CDDP-tubulin complex formation was found to be an irreversible reaction through a covalent binding at the sulfhydryl group sites. By the DEAE filter paper method, CDDP was shown to slightly decrease vinca-alkaloid and colchicine bindings to tubulin likely by inducing a conformational change of the protein.

Animals↗
PubMed1986

pH-Stat titration method for dihydrofolate reductase activity measurements: application to determination of substrate Michaelis constant and antifolate inhibition constant.

A pH-Stat titration method was developed for measuring dihydrofolate reductase (DHFR) activity; this method permits detection of very low DHFR activities corresponding to 100 pmol of substrate reduced per minute. This value is about ten times lower than those observed using the classical spectrophotometric method. This sensitivity makes it possible to measure the DHFR in crude tissue extracts. With beef liver DHFR, Michaelis constants for the cofactor NADPH and the natural substrate determined by this method were 1.9 +/- 0.3 X 10(-5) and 8.5 +/- 0.5 X 10(-7) M, respectively. The inhibition constant of methotrexate, a competitive inhibitor of dihydrofolate, was 3.4 +/- 1.3 X 10(-11) M.

Animals↗