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Biomedical subjects

J Briand

Publications and source records attributed to J Briand.

At least 19 recordsLinked to original sources
PubMed2006

Early physiological responses of Arabidopsis thaliana cells to fusaric acid: toxic and signalling effects.

Fusaric acid (FA) is a toxin produced by Fusarium species. Most studies on FA have reported toxic effects (for example, alteration of cell growth, mitochondrial activity and membrane permeability) at concentrations greater than 10(-5) m. FA participates in fungal pathogenicity by decreasing plant cell viability. However, FA is also produced by nonpathogenic Fusarii, potential biocontrol agents of vascular wilt fusaria. The aim of this study was to determine whether FA, at nontoxic concentrations, could induce plant defence responses. Nontoxic concentrations of FA were determined from cell-growth and O2-uptake measurements on suspensions of Arabidopsis thaliana cells. Ion flux variations were analysed from electrophysiological and pH measurements. H2O2 and cytosolic calcium were quantified by luminescence techniques. FA at nontoxic concentrations (i.e. below 10(-6) m) was able to induce the synthesis of phytoalexin, a classic delayed plant response to pathogen. FA could also induce rapid responses putatively involved in signal transduction, such as the production of reactive oxygen species, and an increase in cytosolic calcium and ion channel current modulations. FA can thus act as an elicitor at nanomolar concentrations.

Arabidopsis↗
PubMed1999

Mechanism of inhibition of cathepsin K by potent, selective 1, 5-diacylcarbohydrazides: a new class of mechanism-based inhibitors of thiol proteases.

The nature of the inhibition of thiol proteases by a new class of mechanism-based inhibitors, 1,5-diacylcarbohydrazides, is described. These potent, time-dependent, active-site spanning inhibitors include compounds that are selective for cathepsin K, a cysteine protease unique to osteoclasts. The 1,5-diacylcarbohydrazides are slow substrates for members of the papain superfamily with inhibition resulting from slow enzyme decarbamylation. Enzyme-catalyzed hydrolysis of 2,2'-N, N'-bis(benzyloxycarbonyl)-L- leucinylcarbohydrazide is accompanied by formation of a hydrazide-containing product and a carbamyl-enzyme intermediate that is sufficiently stable to be observed by mass spectrometry and NMR. Stopped-flow studies yield a saturation limited value of 43 s(-)(1) for the rate of cathepsin K acylation by 2,2'N, N'-bis(benzyloxycarbonyl)-L-leucinylcarbohydrazide. Inhibition potency varies among proteases tested as reflected by 2-3 orders of magnitude differences in K(i) and K(obs)/I, but all eventually form the same stable covalent intermediate. Reactivation rates are equivalent for all enzymes tested (1 x 10(-)(4) s(-)(1)), indicating hydrolysis of a common carbamyl-enzyme form. NMR spectroscopic studies with cathepsin K and 2,2'-N,N'-bis(benzyloxycarbonyl)-L-leucinylcarbohydrazide provide evidence of inhibitor cleavage to generate a covalent carbamyl-enzyme intermediate rather than a tetrahedral complex. The product Cbz-leu-hydrazide does not appear enzyme-bound after cleavage in the NMR spectra, suggesting that the stable inhibited form of the enzyme is the thioester complex. 1, 5-diacylcarbohydrazides represent a new class of unreactive cysteine protease inhibitors that share a common mechanism of action across members of the papain superfamily. Both S and S' subsite interactions are exploited in achieving high selectivity and potency.

Binding Sites↗
PubMed1999

Spin-state-selective TPPI: a new method for suppression of heteronuclear coupling constants in multidimensional NMR experiments.

A novel multidimensional NMR pulse sequence tool, spin-state-selective time-proportional phase incrementation (S(3) TPPI), is introduced. It amounts to application of different TPPIs on the two components of doublets so that their frequencies can be manipulated independently. The chief application is for suppression of large heteronuclear one-bond coupling constants in indirect dimensions of multidimensional experiments without interchanging the two transverse magnetization components of doublets as conventional decoupling does, which is advantageous when they relax at different rates such as by partial compensation of dipolar and CSA relaxation contributions. For experimental confirmation we use a sample of (15)N-labeled neural cell adhesion molecule modules 1 and 2, a protein with a molecular weight of about 20 kDa. The new tool is general and can be combined with many multidimensional NMR experiments for proteins.

Magnetic Resonance Spectroscopy↗
PubMed1998

Simultaneous and independent rotations with arbitrary flip angles and phases for I, ISalpha, and ISbeta spin systems.

A new pulse sequence element for simultaneous and independent rotations with arbitrary flip angles and phases for isolated I, ISalpha, and ISbeta resonances without the use of selective radiofrequency pulses is introduced and experimentally demonstrated. S is a directly attached heteronucleus either at natural abundance or isotopically enriched. This pulse sequence element, dubbed TIG-BIRD (triselective independent gyrations BIRD), generalizes earlier elements like BIRD, TANGO, BANGO, and BIG-BIRD, the latter of which allows for arbitrary selection of flip angles and phases for I and IS spin systems without discriminating between ISalpha and ISbeta resonances. For ISalpha and ISbeta spin systems it also generalizes the spin-state-selective excitation (S3E) element selectively exciting only one of the ISalpha or ISbeta resonances. TIG-BIRD is a nonselective addition to the NMR toolkit which effects the equivalent of three independent selective rotations for I, ISalpha, and ISbeta resonances.

Hydrocarbons, Iodinated↗
PubMed1997

Design of potent and selective human cathepsin K inhibitors that span the active site.

Potent and selective active-site-spanning inhibitors have been designed for cathepsin K, a cysteine protease unique to osteoclasts. They act by mechanisms that involve tight binding intermediates, potentially on a hydrolytic pathway. X-ray crystallographic, MS, NMR spectroscopic, and kinetic studies of the mechanisms of inhibition indicate that different intermediates or transition states are being represented that are dependent on the conditions of measurement and the specific groups flanking the carbonyl in the inhibitor. The species observed crystallographically are most consistent with tetrahedral intermediates that may be close approximations of those that occur during substrate hydrolysis. Initial kinetic studies suggest the possibility of irreversible and reversible active-site modification. Representative inhibitors have demonstrated antiresorptive activity both in vitro and in vivo and therefore are promising leads for therapeutic agents for the treatment of osteoporosis. Expansion of these inhibitor concepts can be envisioned for the many other cysteine proteases implicated for therapeutic intervention.

Binding Sites↗
PubMed1996

Inhibition of HIV infection by pseudopeptides blocking viral envelope glycoprotein-mediated membrane fusion and cell death.

The RP dipeptide motif is highly conserved in the third hypervariable region (V3 loop) of the extracellular envelope glycoprotein of different types of HIV isolates. In view of this, we have designed and synthesized a construction referred to as "template assembled synthetic peptide" (TASP), in which a lysine-rich short polypeptide was used as a template to covalently anchor arrays of tripeptides, such as RPR, RPK, or KPR. The pentavalent presentation, 5(RPR)-, 5(RPK)-, or 5(KPR)-TASP, molecules manifested maximum inhibitory activity on HIV infection with a 50% inhibitory concentration value of 1-5 microM, respectively. Structure and inhibitory-activity relationship studies using analogs of 5(KPR)-TASP indicated that the positively charged side chains of the K and R residues in the tripeptide molecules are critical for the optimal inhibitory activity of the pentavalent construct. Interestingly, replacement of L-amino acid residues by D-amino acids or reduction of the peptide bond between the first two amino acids of the tripeptide generated peptide-TASP analogs active at sub-microM, concentrations. The anti-HIV action of the peptide-TASP constructs is specific, since they inhibit infection of several types of CD4-expressing cells by HIV-1 Lai and HIV-2 EHO but not by the simian SIV-mac isolate. Our results suggest that these inhibitors block three post-CD4 binding functions of the HIV envelope glycoproteins, mediation of viral entry, syncytium formation, and triggering cell death by apoptosis. As the peptide-TASP derivatives with unnatural amino acid sequences in the tripeptide moiety retain full inhibitory activity, they should provide potent protease-resistant peptide inhibitors as potential therapeutic agents for treatment of AIDS patients.

Amino Acid Sequence↗
PubMed1995

Internal mobility of cyclic RGD hexapeptides studied by 13C NMR relaxation and the model-free approach.

The internal mobility of three isomeric cyclic RGD hexapeptides designed to contain two beta-turns in defined positions, cyclo(Arg-Gly-Asp-Gly-D-Pro-Pro) (I), cyclo(Arg-Gly-Asp-D-Pro-Gly-Pro) (II) and cyclo(Arg-Gly-Asp-D-Pro-Pro-Gly) (III), have been studied by 13C NMR longitudinal and transverse relaxation experiments and measurements of steady-state heteronuclear (1H)-13C NOE enhancement with 13C at natural abundance. The data were interpreted according to the model-free formalism of Lipari and Szabo, which is usually applied to data from macromolecules or larger sized peptides with overall rotational correlation times exceeding 1 ns, to yield information about internal motions on the 10-100 ps time scale. The applicability of the model-free analysis with acceptable uncertainties to these small peptides, with overall rotational correlation times slightly below 0.3 ns, was demonstrated for this specific instance. Chemical exchange contributions to T2 from slower motions were also identified in the process. According to the order parameters obtained for its backbone alpha-carbon atoms, II has the most rigid backbone conformation on the 10-100 ps time scale, and I the most flexible. This result coincides with the results of earlier NMR-constrained conformational searches, which indicated greatest uncertainty in the structure of I and least in II.

Animals↗
PubMed1994

Conformational study of eight-membered diazocine turn mimics by two-dimensional NMR spectroscopy.

The eight-membered ring conformations of two diazocine turn mimics, methyl-[2,5-dioxo-3-(S)-(3-omega-tosyl-guanidino-propyl)-4-methyl-octahy dro- 1,4-diazocin-1-yl]acetate (I) and methyl-[2,5-dioxo-3-(S)-(3-omega-tosyl-guanidino-propyl)-octahydro-1,4- diazocin-1-yl]acetate (II), were determined using torsion angle constraints derived from 3J(C,H) coupling constants extracted from 13C-filtered TOCSY spectra with 13C in natural abundance. For I, the torsion angle constraints derived from 3J(C,H) coupling constants were in agreement with torsion angle constraints derived from 3J(H,H) coupling constants extracted from a P.E. COSY spectrum. Similar 3J(C,H) coupling constants were found for I and II, and they shared an identical eight-membered ring conformation characterized by two cis-amide bonds and a staggered conformation of the trimethylene group in which the H3 proton is proximal to both the H6 and H8 protons.

Azocines↗
PubMed1993

Presence of proteins recognized by mammalian cytochrome P-450 antibodies in Euglena gracilis.

We have attempted to probe three microsomal cytochrome P-450 isozymes in Euglena gracilis using immunochemical methods. They cross-react with anti-rat cytochrome P4502C11, cytochrome P4502E, and cytochrome P4502B. Activities of alkoxyphenoxazone dealkylation have been tested in living cells. In untreated cultures, the amount of proteins recognized by anti-cytochrome P4502C11 or anti-cytochrome P4502E is high. Phenobarbital treatment increased the levels of microsomal proteins recognized by antibody to cytochrome P4502B, as well as dealkylases of pentoxyresorufin, but decreased the level of proteins recognized by anti-cytochrome P450C11 or cytochrome P4502E. These results suggest that these unicellular algae may contain different isozymes of microsomal cytochromes P-450, comparable to those in mammalian liver. They are cytochrome P-450 equivalents of mammalian isoenzymes 2C, 2E and 2B. However, we could not demonstrate ethanol induction of cytochrome P-450 equivalent to isoenzyme 2E. Its role in xeno- or endobiotic metabolism remains to be elucidated.

Animals↗
PubMed1993

Conformational mobility in cyclic oligopeptides.

Analysis of two isomeric cyclic hexapeptides of composition (Asp, Arg, Gly2, Pro, D-Pro) by a nuclear Overhauser effect constrained distance geometry conformation search yielded a narrowly defined backbone conformation for one and considerable ambiguity about the conformation in part of the other. Preliminary 13C relaxation studies of these peptides suggest that it is possible that this difference may correspond to a physical difference in internal mobility. In connection with this observation, other experimental evidence bearing on the backbone conformational mobility of cyclic oligopeptides with 4-10 residues, frequently considered to have well-defined backbones, is reviewed. Conformational heterogeneity involving rotation of a peptide bond plane relative to the overall ring plane is identified as a common phenomenon. Nuclear magnetic resonance line-shape studies at temperatures down to 200 K can detect backbone motions with activation free energy barriers down to about 10 kcal/mole, but conformational exchange with lower barriers, though detectable in other ways, will not be obvious from nmr spectra alone.

Amino Acid Sequence↗
PubMed1992

Use of a microbial model for the determination of drug effects on cell metabolism and energetics: study of citrulline-malate.

Euglena gracilis can be used as a microbial model to study the effect of drugs on lactate metabolism and gluconeogenetic synthesis. The cell growth and metabolism have been characterized in a 33 mM lactate medium, non-supplemented or supplemented by dl-malate or by l-citrulline alone or by the compound formed by the stoichiometric combination of the two components: the citrulline-malate (Stimol). The malate of the complex accelerated the ammonium disappearance, while the citrulline facilitated the lactate consumption. A synergistic action of the complex, by comparison with the additive effects of the individual components, on most of the parameters studied was detected. A remarkable resistance to anoxia, and a quicker recovery under aeration of the cells supplemented with CM, were evident: after carbonation for 2 min the total nucleotides in the medium were increased by 44 per cent with an unchanged energy charge; and after a prolonged (20 min) anoxia followed by an aeration, the capacities of the cells to synthesize ATP in the presence of excesses of both ADP and phosphate were two-fold higher in Stimol treated cells than in control.

Adenosine Triphosphate↗
PubMed1992

Microsomal ethanol-oxidizing system in Euglena gracilis. Similarities between Euglena and mammalian cell systems.

1. ADH activity of Euglena grown with 50 mM ethanol decreased, but MEOS activity increased with a corresponding increase in the total amount of cytochrome P-450. 2. Phenobarbital treatment increased the total amount of cytochrome P-450. 3. CO and KCN, cytochrome P-450 ligands, diminished acetaldehyde formed from ethanol oxidation by MEOS. 4. The amounts of NAD(P)H cytochrome c reductases and cytochrome b5 type, components of microsomal monooxygenase reaction, have been spectrophotometrically measured. 5. NAD(P)H cytochrome c reductases activities were induced by phenobarbital. 6. DMSO, an inhibitor of rabbit MEOS, inhibited O2 consumption (11-20%) by Euglena grown with an ethanol, but not a lactate medium. 7. These studies indicate the presence of cytochrome P-450-dependent MEOS in Euglena similar to that in the mammalian hepatic cell.

Alcohol Dehydrogenase↗
PubMed1991

Citrulline-malate effect on microsome phospholipids and cytochrome P450 in Euglena grown with ethanol.

This study indicates for the first time the presence of cytochrome P450 in the microsomes of Euglena grown in lactate medium and substantiates the use of Euglena as a hepatic cell model. Similar effects of ethanol on Euglena and on rat hepatic microsomes were demonstrated: (i) decrements in the quantities of FA per milligram of proteins; (ii) increases in the proportions of PE; (iii) decreases in the proportions of PC; and (iv) production of cytochrome P450, degraded in P420. The citrulline-malate reestablishes in the microsomes the phospholipid environment and the cytochrome P450 concentration. These findings illustrate that the complex acts on the lipid peroxidation via the changes in cytochrome P450 activity.

Animals↗