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Biomedical subjects

M Villain

Publications and source records attributed to M Villain.

At least 19 recordsLinked to original sources
PubMed2002

Doping control for methenolone using hair analysis by gas chromatography-tandem mass spectrometry.

A sensitive, specific and reproducible method for the quantitative determination of methenolone in human hair has been developed. The sample preparation involved a decontamination step of the hair with methylene chloride. The hair sample (about 100 mg) was solubilized in 1 ml 1 M NaOH, 15 min at 95 degrees C, in presence of 1 ng testosterone-d3 used as internal standard. The homogenate was neutralized and extracted using consecutively a solid-phase (Isolute C18 eluted with methanol) and a liquid-liquid (pentane) extraction. The residue was derivatized by adding 50 microl MSTFA-NH4I-2-mercaptoethanol (1000:2:5, v/v/v), then incubated for 20 ml at 60 degrees C. A 1.5-microl aliquot of the derivatized extract was injected into the column (HP5-MS capillary column, 5% phenyl-95% methylsiloxane, 30 m x 0.25 mm I.D., 0.25 microm film thickness) of a Hewlett-Packard (Palo Alto, CA, USA) gas chromatograph (6890 Series). Methenolone was detected by its parent ion at m/z 446 and daughter ions at m/z 208 and 195 through a Finnigan TSQ 700 MS-MS system. The assay was capable of detecting 1 pg/mg of methenolone when approximately 100 mg hair material was processed. Linearity was observed for methenolone concentrations ranging from 2 to 100 pg/mg with a correlation coefficients of 0.965-0.981. Intra-day and between-day precisions at 2, 10 and 25 pg/mg were 10.9-14.1% and 13.7-16.8%, respectively, with an extraction recovery of 97.6%. The analysis of a strand of hair obtained from two bodybuilders, revealed the presence of methenolone at the concentrations of 7.3 and 8.8 pg/mg.

Anabolic Agents↗
PubMed2002

[Crime under the influence of psychoactive drugs: the problem of the duration of detection].

On a regular basis, the media presents the potential risks of the use of psycho-active compounds, including misused pharmaceuticals (flunitrazepam, GHB) or drugs of abuse (cannabis, LSD, ecstasy). Ethanol is also frequently encountered. These drugs can be used for recreational purposes by addicts or can be observed after sexual assaults (drugs spiked in food). Forensic toxicology can be involved in several situations to document impairment, such as: crime under influence, date rape, driving under influence, psychiatric disorders, determination of the cause of death... In some particular situations, it can be very cautious to investigate exposure to psycho-active drugs, due to late sampling of biological specimens. To enhance the window of detection of 3 specific drugs, the authors propose the following: 1. Use of an ultra-sensitive technique, such as GC/MS/MS/NCI for 7-aminoflunitrazepam; 2. Use of a cumulative specimen, such as sweat for GHB and 3. Use of a metabolite with a long half-life, such as ethyl glucuronide for ethanol.

Cause of Death↗
PubMed2001

Prevention and reversal of experimental autoimmune myasthenia gravis by a monoclonal antibody against acetylcholine receptor-specific T cells.

We have recently described an algorithm to design, among others, peptides with complementarity contour to autoimmune epitopes. Immunization with one such peptide resulted in a monoclonal antibody (mAb), termed CTCR8, that specifically recognized Vbeta15 containing TCR on acetylcholine receptor (AChR) alpha-chain residue 100-116-specific T cells. CTCR8 was found to label the cell surface of AChR100-116-specific T cell lines and clones, immunoprecipitate the TCR from such cells, and block their proliferative responses to AChRalpha100-116. In the present report, we have found that there is a marked reduction in IFN-gamma and no effect on IL-10 production in a CTCR8-treated AChRalpha100-116-specific T cell line. Interestingly, when AChR100-116-primed, primary T cells were stimulated with peptide and treated with CTCR8, there was once again inhibition of IFN-gamma but also marked stimulation of IL-10 production. The change in the Th1/Th2 cytokine profile was paralleled by a reduction in AChR-specific IgG2a and IgM with no effect on IgG1. Remarkably, the most profoundly inhibited Ab population was that which causes experimental autoimmune myasthenia gravis (EAMG) by reaction with the main immunogenic region (alpha61-76) of the AChR. Based on these results, CTCR8 was tested for prophylactic and therapeutic effects in EAMG. EAMG induced by immunization with purified native Torpedo AChR was both inhibited and reversed by CTCR8. These findings suggest a means to produce therapeutic mAb for the treatment of autoimmune diseases.

Animals↗
PubMed2001

Attenuation of very late antigen-5-mediated adhesion of bone marrow-derived mast cells to fibronectin by peptides with inverted hydropathy to EF-hands.

Release of allergic mediators from mast cells is enhanced by very late Ag (VLA)-5-mediated interaction of these cells with fibronectin. In this report, we show that VLA-5-mediated adhesion of bone marrow-derived mast cells to fibronectin can be induced by two different pathways: first, FcepsilonRI clustering, which depends on calmodulin activation and extracellular Ca(2+), and, second, by Mn(2+) stimulation, which is independent of calmodulin activation and antagonized by Ca(2+). Previous studies have shown the presence of several cation-binding domains in VLA-5 that are homologous to the calcium-binding EF-hands of calmodulin. To show a role for EF-hands of different proteins in VLA-5-mediated adhesion, we used calcium-like peptides (CALP), CALP1 and CALP2, designed to bind to EF-hands based on inverted hydropathy. CALP1 and, more potently, CALP2 inhibited FcepsilonRI-induced adhesion to fibronectin via different mechanisms. The target for the effects of CALP1 and 2 on FcepsilonRI-induced adhesion and degranulation was intracellular and likely involved calmodulin. Interestingly only CALP2 was able to inhibit Mn(2+)-induced calmodulin-independent adhesion by interfering with an extracellular target, which is probably VLA-5. We conclude that CALP1 and 2 can inhibit VLA-5-mediated adhesion of mast cells to fibronectin through binding to EF-hands of multiple proteins, and that these peptides can be used as lead compounds for the development of future therapy against allergy.

Adjuvants, Immunologic↗
PubMed2001

Covalent capture: a new tool for the purification of synthetic and recombinant polypeptides.

BACKGROUND: Purification of polypeptides and proteins derived from recombinant DNA techniques and of long synthetic polypeptides often represents a challenge. Affinity methods exist, but generally require addition of a large recognition unit to the target protein and use of expensive purification media. Use of large units is dictated by the characteristics of non-covalent complexes, where the energy necessary to form the complex derives from the sum of multiple weak energy interactions. Covalent interactions in contrast are of high energy, even when only a few bonds are formed. We decided to explore the use of the reversible covalent bond formed between N-terminal cysteine and threonine residues with an aldehyde as a method of protein purification. RESULTS: A series of test peptides with N-terminal cysteine and threonine were captured by a polyethyleneglycol-polyacrylamide resin to which an aldehyde function had been grafted. Peptides with other amino acids at the N-terminus did not interact with the resin. A recombinant polypeptide with N-terminal cysteine was purified to 90% purity in one step. Polypeptides were eluted from the resin simply by adding a hydroxylamine derivative, which reacts with aldehyde functions to form an oxime. CONCLUSIONS: Polypeptides possessing N-terminal cysteine or threonine can be easily purified using this 'covalent capture' approach.

Aldehydes↗
PubMed2001

The role of adjuvants in the efficacy of a peptide vaccine for myasthenia gravis.

Myasthenia gravis (MG) and its animal model, experimental autoimmune (EA) MG, are caused by interference with neuromuscular transmission by autoantibodies against the nicotinic acetylcholine receptor (AChR) on muscle. Previously, we have shown that two peptides, denoted RhCA 67-16 and RhCA 611-001, designed to be complementary in structure to the main immunogenic region and the dominant Lewis rat T cell epitope (alpha-chain residues 100-116) of the AChR, respectively, are effective vaccines that prevent EAMG in rats by inducing antiidiotypic/clonotypic antibodies (Ab) and lowering levels of AChR Ab. These studies employed keyhole limpet hemocyanin (KLH) as a carrier and complete Freunds adjuvant (CFA). In advance of a clinical trial the present study tested the efficacy of RhCA 611-001 when combined with different adjuvants that are approved for use in humans. Adjuvants chosen for comparison were incomplete Freunds adjuvant (IFA) and aluminum hydroxide (Alum). As a second goal we evaluated diphtheria toxin (DT) as an alternative carrier protein to KLH. Alum was found to be an effective adjuvant, particularly when used with the peptide conjugated to DT. This combination of carrier and adjuvant provided protection against EAMG comparable with that observed with CFA and KLH. Using enzyme-linked immunosorbent assays for Ab against RhCA 611-001, it was found that disease protection is qualitatively, but not quantitatively, related to the anti-peptide Ab response. Our results demonstrate a vaccine formulation that should be useful in the first soon-to-be-conducted clinical trials of peptide vaccines to specifically correct aberrant T and B cell responses in an autoimmune disease.

Adjuvants, Immunologic↗
PubMed2001

Inhibitory effect of a complementary peptide on ulceration in the alkali-injured rabbit cornea.

PURPOSE: Two tripeptide chemoattractants, acetyl-proline-glycine-proline (Ac-PGP) and methyl-proline-glycine-proline (Me-PGP), are the primary triggers for early neutrophil invasion into the alkali-injured cornea. In the present study the effectiveness of a complementary peptide designed to inhibit the PGP chemoattractants (arginine-threonine-arginine [RTR] tetrameric peptide) and an apo A-1 mimicking peptide (5F) was investigated in the alkali-injured rabbit eye. METHODS: (L)-RTR tetramer, (D)-RTR tetramer, and 5F were tested in vitro for their effects on neutrophil polarization. Synthetic 5F was also tested in vitro for its effect on the neutrophil respiratory burst. In the alkali-injured rabbit eye model, the right corneas of 48 rabbits were exposed to 1 N NaOH for 35 seconds. Sixteen animals were randomly assigned to each of three groups: phosphate-buffered saline (PBS) control; 800 microM RTR (dextrorotatory) tetramer in PBS alternating each hour with 1.5 mM RTR (levorotatory) tetramer in PBS; and 12 microM 5F in PBS. One topical drop of each substance was administered hourly (14 times per day) for 33 days. The experiment was continued until day 42 with no additional drops administered. RESULTS: (L)-RTR tetramer and (D)-RTR tetramer inhibited neutrophil polarization activated by the PGP chemoattractants in vitro. Synthetic 5F did not inhibit neutrophil polarization in the presence of Ac-PGP or the respiratory burst of neutrophils in the presence of a metabolic stimulant derived from alkali-degraded corneas. During the entire animal experiment, statistically fewer ulcers occurred in the RTR tetramer group than in the PBS control group (43.8% vs. 87.5%, P = 0.0046). The frequency of ulceration in the 5F group (68.8%) was not significantly different from the PBS control group. CONCLUSIONS: The reduction in the frequency of corneal ulceration by the RTR tetramer possibly resulted from its complementary binding to Ac-PGP and Me-PGP in the cornea shortly after alkali injury, leading to a reduction in the early and late infiltration of neutrophils. RTR tetramer appears to hold enough promise to warrant additional study as a therapeutic drug for the alkali-injured eye.

Animals↗
PubMed2000

Cellular uptake and in situ binding of a peptide agonist for calmodulin.

We have used the method of inverted hydropathy to develop peptides that interact with EF hands of calmodulin (CaM). Previously we have shown these peptides specifically interact with their desired target in a productive manner, in that they activated CaM in the absence of Ca(2+). Therefore, we sought to determine whether these peptides would enter cells, remain intact, and interact with CaM in the interior of the cell. Using several techniques we have demonstrated cellular uptake, stability, and an intracellular interaction with CaM with fluorescein-labeled and radiolabeled peptides in Jurkat T cells. The results suggest that these peptides may be useful in the study and the manipulation of Ca(2+)-mediated pathways in cells.

Animals↗
PubMed2000

De novo design of peptides targeted to the EF hands of calmodulin.

This report describes the use of the concept of inversion of hydropathy patterns to the de novo design of peptides targeted to a predetermined site on a protein. Eight- and 12-residue peptides were constructed with the EF hands or Ca(2+)-coordinating sites of calmodulin as their anticipated points of interaction. These peptides, but not unrelated peptides nor those with the same amino acid composition but a scrambled sequence, interacted with the two carboxyl-terminal Ca(2+)-binding sites of calmodulin as well as the EF hands of troponin C. The interactions resulted in a conformational change whereby the 8-mer peptide-calmodulin complex could activate phosphodiesterase in the absence of Ca(2+). In contrast, the 12-mer peptide-calmodulin complex did not activate phosphodiesterase but rather inhibited activation by Ca(2+). This inhibition could be overcome by high levels of Ca(2+). Thus, it would appear that the aforementioned concept can be used to make peptide agonists and antagonists that are targeted to predetermined sites on proteins such as calmodulin.

Amino Acid Sequence↗
PubMed2000

A peptide vaccine that prevents experimental autoimmune myasthenia gravis by specifically blocking T cell help.

Myasthenia gravis (MG) and its animal model, experimental autoimmune (EA) MG, are caused by T cell-dependent autoantibodies that react with the nicotinic acetylcholine receptor (AChR) on muscle and interfere with neuromuscular transmission. Thus, selective inactivation of CD4(+) AChR-specific T helper cells should lower AChR Ab levels and ameliorate disease. In the Lewis rat model of EAMG, alpha chain residues 100-116 of the AChR represent the dominant T cell epitope, which is important in helping Ab responses to this autoantigen. In the present report, we have applied a new design technique that requires no knowledge of Ag receptor sequences on errant T cells in order to develop a synthetic peptide vaccine against T cells reactive with the aforementioned T cell epitope. Immunization with the peptide 1) induced polyclonal and monoclonal Ab, which inhibited AChR 100-116 stimulation of AChR-sensitized lymphocytes and recognized Vbeta15 containing T cell receptors on AChR 100-116-specific T cell lines and clones; 2) lowered AChR Ab levels; 3) reduced the loss of muscle AChR; and 4) lessened the incidence and severity of EAMG. These findings suggest a new strategy for the functional abrogation of epitope-specific T cells that could have potential application to human autoimmune diseases.

Amino Acid Sequence↗
PubMed2000

A new type of Ca(2+) channel blocker that targets Ca(2+) sensors and prevents Ca(2+)-mediated apoptosis.

Calmodulin (CaM), as well as other Ca(2+) binding motifs (i.e., EF hands), have been demonstrated to be Ca(2+) sensors for several ion channel types, usually resulting in an inactivation in a negative feedback manner. This provides a novel target for the regulation of such channels. We have designed peptides that interact with EF hands of CaM in a specific and productive manner. Here we have examined whether these peptides block certain Ca(2+)-permeant channels and inhibit biological activity that is dependent on the influx of Ca(2+). We found that these peptides are able to enter the cell and directly, as well as indirectly (through CaM), block the activity of glutamate receptor channels in cultured neocortical neurons and a nonselective cation channel in Jurkat T cells that is activated by HIV-1 gp120. As a consequence, apoptosis mediated by an influx of Ca(2+) through these channels was also dose-dependently inhibited by these novel peptides. Thus, this new type of Ca(2+) channel blocker may have utility in controlling apoptosis due to HIV infection or neuronal loss due to ischemia.

Amino Acid Sequence↗
PubMed2000

Synthetic complementary peptides inhibit a neutrophil chemoattractant found in the alkali-injured cornea.

PURPOSE: We have previously presented evidence that the neutrophil chemoattractant, N-acetyl-proline-glycine-proline (N-acetyl-PGP), triggers the initial polymorphonuclear leukocyte (PMN) invasion into the alkali-injured eye. In this study, sense-antisense methodology was used to develop novel complementary peptides to be potential inhibitors of N-acetyl-PGP. METHODS: The polarization assay was used to measure the potential chemotactic response of PMNs to synthetic N-acetyl-PGP, the ultrafiltered tripeptide chemoattractants obtained from alkali-degraded rabbit corneas, or leukotriene B4 (LTB4). Inhibition was expressed as the peptide concentration producing 50% inhibition (ID50) of polarization. Five complementary peptides were tested as potential inhibitors of N-acetyl-PGP: arginine-threonine-arginine (RTR), RTR-glycine-glycine (RTRGG), RTR dimer, RTR tetramer, and alanine-serine-alanine (ASA) tetramer. In addition, the RTR tetramer and both monomeric peptides (RTR and RTRGG) were separately tested for inhibition of the ultrafiltered tripeptide chemoattractants or LTB4. RESULTS: The complementary RTR tetrameric peptide was a powerful antagonist of N-acetyl-PGP-induced PMN polarization (ID50 of 200 nM). The RTR dimer was much less potent (ID50 of 105 microM). Both monomeric peptides, RTR and RTRGG, were only antagonistic at millimolar concentrations. The ASA tetramer showed no capacity to inhibit N-acetyl-PGP. The RTR tetramer also inhibited PMN activation by the ultrafiltered tripeptide chemoattractants (ID50 of 30 microM) but had no effect on LTB4. CONCLUSIONS: A complementary peptide (RTR) was designed which is an effective inhibitor of the neutrophil chemoattractant, N-acetyl-PGP. The potency of the RTR complementary peptide is dramatically enhanced by tetramerization. Inhibition of N-acetyl-PGP by complementary peptides offers great promise for control of the inflammatory response in the alkali-injured eye.

Animals↗
PubMed1999

Conformation of the regulatory domain of cardiac muscle troponin C in its complex with cardiac troponin I.

Calcium activation of fast striated muscle results from an opening of the regulatory N-terminal domain of fast skeletal troponin C (fsTnC), and a substantial exposure of a hydrophobic patch, essential for Ca(2+)-dependent interaction with fast skeletal troponin I (fsTnI). This interaction is obligatory to relieve the inhibition of strong, force-generating actin-myosin interactions. We have determined intersite distances in the N-terminal domain of cardiac TnC (cTnC) by fluorescence resonance energy transfer measurements and found negligible increases in these distances when the single regulatory site is saturated with Ca(2+). However, in the presence of bound cardiac TnI (cTnI), activator Ca(2+) induces significant increases in the distances and a substantial opening of the N-domain. This open conformation within the cTnC.cTnI complex has properties favorable for the Ca(2+)-induced interaction with an additional segment of cTnI. Thus, the binding of cTnI to cTnC is a prerequisite to achieve a Ca(2+)-induced open N-domain similar to that previously observed in fsTnC with no bound fsTnI. This role of cardiac TnI has not been previously recognized. Our results also indicate that structural information derived from a single protein may not be sufficient for inference of a structure/function relationship.

Animals↗
PubMed1999

CFTR chloride channel regulation by an interdomain interaction.

The cystic fibrosis gene encodes a chloride channel, CFTR (cystic fibrosis transmembrane conductance regulator), that regulates salt and water transport across epithelial tissues. Phosphorylation of the cytoplasmic regulatory (R) domain by protein kinase A activates CFTR by an unknown mechanism. The amino-terminal cytoplasmic tail of CFTR was found to control protein kinase A-dependent channel gating through a physical interaction with the R domain. This regulatory activity mapped to a cluster of acidic residues in the NH(2)-terminal tail; mutating these residues proportionately inhibited R domain binding and CFTR channel function. CFTR activity appears to be governed by an interdomain interaction involving the amino-terminal tail, which is a potential target for physiologic and pharmacologic modulators of this ion channel.

Adenosine Triphosphate↗
PubMed1999

An analysis of the binding of repressor protein ModE to modABCD (molybdate transport) operator/promoter DNA of Escherichia coli.

Expression of the modABCD operon in Escherichia coli, which codes for a molybdate-specific transporter, is repressed by ModE in vivo in a molybdate-dependent fashion. In vitro DNase I-footprinting experiments identified three distinct regions of protection by ModE-molybdate on the modA operator/promoter DNA, GTTATATT (-15 to -8; region 1), GCCTACAT (-4 to +4; region 2), and GTTACAT (+8 to +14; region 3). Within the three regions of the protected DNA, a pentamer sequence, TAYAT (Y = C or T), can be identified. DNA-electrophoretic mobility experiments showed that the protected regions 1 and 2 are essential for binding of ModE-molybdate to DNA, whereas the protected region 3 increases the affinity of the DNA to the repressor. The stoichiometry of this interaction was found to be two ModE-molybdate per modA operator DNA. ModE-molybdate at 5 nM completely protected the modABCD operator/promoter DNA from DNase I-catalyzed hydrolysis, whereas ModE alone failed to protect the DNA even at 100 nM. The apparent K(d) for the interaction between the modA operator DNA and ModE-molybdate was 0.3 nM, and the K(d) increased to 8 nM in the absence of molybdate. Among the various oxyanions tested, only tungstate replaced molybdate in the repression of modA by ModE, but the affinity of ModE-tungstate for modABCD operator DNA was 6 times lower than with ModE-molybdate. A mutant ModE(T125I) protein, which repressed modA-lac even in the absence of molybdate, protected the same region of modA operator DNA in the absence of molybdate. The apparent K(d) for the interaction between modA operator DNA and ModE(T125I) was 3 nM in the presence of molybdate and 4 nM without molybdate. The binding of molybdate to ModE resulted in a decrease in fluorescence emission, indicating a conformational change of the protein upon molybdate binding. The fluorescence emission spectra of mutant ModE proteins, ModE(T125I) and ModE(Q216*), were unaffected by molybdate. The molybdate-independent mutant ModE proteins apparently mimic in its conformation the native ModE-molybdate complex, which binds to a DNA sequence motif of TATAT-7bp-TAYAT.

Amino Acid Sequence↗
PubMed1999

The activation sequence of thrombospondin-1 interacts with the latency-associated peptide to regulate activation of latent transforming growth factor-beta.

One of the primary points of regulation of transforming growth factor-beta (TGF-beta) activity is control of its conversion from the latent precursor to the biologically active form. We have identified thrombospondin-1 as a major physiological regulator of latent TGF-beta activation. Activation is dependent on the interaction of a specific sequence in thrombospondin-1 (K412RFK415) with the latent TGF-beta complex. Platelet thrombospon-din-1 has TGF-beta activity and immunoreactive mature TGF-beta associated with it. We now report that the latency-associated peptide (LAP) of the latent TGF-beta complex also interacts with thrombospondin-1 as part of a biologically active complex. Thrombospondin.LAP complex formation involves the activation sequence of thrombospondin-1 (KRFK) and a sequence (LSKL) near the amino terminus of LAP that is conserved in TGF-beta1-5. The interactions of LAP with thrombospondin-1 through the LSKL and KRFK sequences are important for thrombospondin-mediated activation of latent TGF-beta since LSKL peptides can competitively inhibit latent TGF-beta activation by thrombospondin or KRFK-containing peptides. In addition, the association of LAP with thrombospondin-1 may function to prevent the re-formation of an inactive LAP.TGF-beta complex since thrombospondin-bound LAP no longer confers latency on active TGF-beta. The mechanism of TGF-beta activation by thrombospondin-1 appears to be conserved among TGF-beta isoforms as latent TGF-beta2 can also be activated by thrombospondin-1 or KRFK peptides in a manner that is sensitive to inhibition by LSKL peptides.

Amino Acid Sequence↗
PubMed1999

[Intra-orbital decompression of the optic nerve by opening the peri-optic meninges in the treatment of severe optic neuropathies complicating benign intracranial hypertension].

Papilledema from benign intracranial hypertension can cause severe loss of visual acuity and visual field, with an optic neuropathy. We report a study of 5 patients with benign intracranial hypertension, and severe visual loss (visual field loss and visual acuity reduced to 1/10 or less) not improved by medical therapy (acetazolamide). We performed unilateral orbital decompression of the optic nerve sheath. No major operative complication was noted. Follow-up ranged from 11 months to 23 months, with an average of sixteen months. Visual function improvement was noted within 3 months after operation in 4 patients (for 2 eyes on the surgical side improvement of 2/10 and 5/10, for 3 eyes on the opposite surgical side average improvement of 4.6/10). The other patient showed improvement, but no significant. Optic nerve sheath decompression in benign intracranial hypertension seems to be a safe procedure and a therapeutic option in the management of raised intracranial pressure complicated by optic neuropathy with severe visual loss.

Acetazolamide↗