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Biomedical subjects

M Boyer

Publications and source records attributed to M Boyer.

At least 55 records · Page 3Linked to original sources
PubMed1998

[Comparison of the results of the Etest and the method for determining minimum inhibitory concentrations in solid media for penicillin G, amoxicillin, and cefotaxime for S. pneumoniae. A multicenter study].

In 1996-1997 a multicentre study was carried out on 450 Streptococcus pneumoniae strains to compare the MICs and susceptibility categories obtained with the Etest (AB Biodisk) used under routine conditions in 22 hospital laboratories in the Rhône-Alpes region, France, with those obtained by the reference technique of agar dilution performed in a single coordinating centre. Each laboratory detected penicillin resistant pneumococci (PRP) by the oxacillin disk method (1 microgram and 5 micrograms) and determined the MICs of penicillin G (PG), amoxycillin (AMX) and cefotaxime (CTX) by the Etest. All the PRP strains were collected in the coordinating centre where MICs were carried out. The strains were classified as susceptible (S), intermediate (I) and resistant (R) according to the CASFM criteria (Comité de l'Antibiogramme de la Société Française de Microbiologie). The concordance results based on susceptibility categories are as follows: PG = 67.6%, AMX = 63.6%, CTX = 71.5%. Minor errors are as follows: PG = 31.2%, AMX = 36%, CTX = 28.5%. Major and very major errors are rare (0% to 0.6%). Agreement within 1 log2 dilution was obtained for about 80% of the strains. The minor errors results from strains clustering near the breakpoints 1 mg/l (PG) and 0.5 mg/l (AMX, CTX), and from practical difficulties in routine use of the Etest. These discrepancies may result in severe therapeutic problems. This study confirms the limits of the Etest. The authors insist on standardization and rigorous use of the Etest under routine conditions.

Amoxicillin↗
PubMed1997

HIV-1 Nef protein: purification, crystallizations, and preliminary X-ray diffraction studies.

Human immunodeficiency virus Nef protein accelerates virulent progression of AIDS by its interaction with specific cellular proteins involved in cellular activation and signal transduction. Here we report the purification and crystallization of the conserved core of HIV-1LAI Nef protein in the unliganded form and in complex with the wild-type SH3 domain of the P59fyn protein-tyrosine kinase. One-dimensional NMR experiments show that full-length protein and truncated fragment corresponding to the product of HIV-1 protease cleavage have a well-folded compact tertiary structure. The ligand-free HIV-1 Nefcore protein forms cubic crystals belonging to space group P23 with unit cell dimensions of a = b = c = 86.4 A. The Nef-Fyn SH3 cocrystals belong to the space group P6(1)22 or its enantiomorph, P6(5)22, with unit cell dimensions of a = b = 108.2 A and c = 223.7 A. Both crystal forms diffract to a resolution limit of 3.0 A resolution using synchrotron radiation, and are thus suitable for X-ray structure determination.

Crystallization↗
PubMed1997

Crystallization of monoacylated proteins: influence of acyl chain length.

The crystallization of monoacylated proteins has been investigated using a model system. Acylated derivatives of bovine pancreatic ribonuclease A, differing in their acyl chain lengths (10 to 16 carbon atoms), have been prepared using reverse micelles as microreactors. With one fatty acid moiety per polypeptide chain, covalently attached to the NH2 terminus of the protein, all the modified proteins have similar enzymatic activity and hydrodynamic radius as the native protein. Only the caprylated derivative can give crystals which diffract to high resolution. The resolved structure indicates that: (i) the protein folding is not modified by the chemical modification, (ii) the capryl moiety is not buried within the molecule but available for external interactions. Dynamic light scattering experiments on concentrated solutions show that the protein-protein interactions are dependent on acyl chain length. Proteins with the longest attached chains (14 and 16 carbon atoms) tend to self-associate through acyl group interactions.

Acylation↗
PubMed1997

Analysis of long-range structural effects induced by DNase-I interaction with actin monomeric form or complexed to CapZ.

Two fundamental properties of monomeric actin were examined in this study, ie its interaction with DNase-I, and the inhibition of endonuclease activity consecutive to the association of the two molecules. In particular, the topological independence between catalytic site of DNase-I and interface with actin, structural changes in actin monomer and the absence of conformational changes in DNase-I were described. We demonstrated a loss of flexibility of antigenic structures in actin subdomain I (ie epitopes 18-28 and 95-105) as well as modification in the exposure of Cys10 and Cys374 after DNase-I binding. Furthermore, the conformational changes induced by DNase-I into the actin molecule weakened the interaction of CapZ to its binding site located in the C-terminal region of actin monomer. These structural changes were time-dependent. When actin was cleaved in the DNase-I binding loop (sequence 38-52) at position 42 by E coli A2 strain protease, a tight DNase-I binding to split actin and the conformational changes were still observed, whereas the DNase-I inhibition activity was completely abolished. Finally, when we substitute Ca2+ by Mg2+ (ATP-Mg2+ monomeric actin) which induces a tighter conformation of actin and partially restores the inhibitory ability of split actin, long-range conformational effects of DNase-I are prevented and the ternary complex DNase-I-actin-CapZ is obtained.

Actins↗
PubMed1996

Biochemical evidence for the presence of an unconventional actin protein in a prokaryotic organism.

The ubiquity of actin, like the functional diversity of many associated proteins, raises a question concerning diversification of motility mechanisms and thus the emergence of an elementary functional system. Our aim was to investigate, in particular, mobiles prokaryotics cells as Synechocystis lacking cilia and flagella, search for actin essential properties and then locate the molecular behaviours. Here we report the presence and purification of a 56-kDa (apparent molecular weight) prokaryotic protein that polymerizes to form filaments, activates myosin Mg(++)-ATPase activity, inhibits DNase-1 activity and affords close antigenic homology to skeletal actin. This protein was found to be associated with thylakoid membranes and extracted in the presence of Triton X-100.

Actins↗
PubMed1996

Interleukin-2 secretion by transduced and unselected BDL-2 lymphoma results in increased survival in mice with previously established disseminated disease.

The transfer and expression of cytokine genes into malignant cells to provide a more effective tumor response has shown promise. The majority of murine models in which tumor vaccination strategies have been tested have utilized selected and expanded clones of tumor cells, which is impractical clinically. In a model of murine B lineage lymphoma (BDL-2), we compared the effectiveness of tumor vaccines composed of a) a BDL-2 clone established by G-418 resistance following transduction with the LIL2SN retrovirus and screened for maximal IL-2 secretion, b) a syngeneic fibroblast line transduced with LIL2SN and screened for G-418 resistance and IL-2 expression, which was co-injected with the parental line, and c) a heterogeneous (unselected) population of BDL-2 cells transduced with the MFG/IL2 virus, reported to provide enhanced expression of cytokine genes and minimize the need for selection. Testing of splenocytes derived from vaccinated animals reveals that injections of BDL-2 expressing IL-2 results in an increased capacity of splenocytes to kill BDL-2 in vitro, compared to vaccination of BDL-2 alone or in combination with IL-2 secreting fibroblasts. We show that a vaccine composed of MFG/IL2 transduced, unselected BDL-2 cells is equivalent or superior to a clone derived from LIL2SN transduction in prolonging survival of animals with previously established tumor. These studies provide evidence that transduction of tumor with MFG based vectors without in vitro selection leads to expression of high levels of IL-2 and can impact the survival of animals with disseminated tumor.

Animals↗
PubMed1996

Latex allergy and the student with spina bifida.

This article discusses the potential problem of latex allergy in the school, particularly among students with spina bifida. The origin of the problem, its symptoms, and prevention and management of latex allergy in the school environment are described. The individual roles of school administrator, school nurse, parent, student and other service providers are listed within an Individual Healthcare Plan (IHP). The IHP is proposed as a guide to schools to assist with identification of latex allergy reactions as well as response procedures if an allergic reaction is identified.

Child↗
PubMed1995

Two identical hydrophobic clusters are present on the same actin monomer: interaction between one myosin subfragment-1 and two actin monomers.

Two-dimensional hydrophobic clusters analysis (HCA) was used to compare the distribution of hydrophobic clusters along various actin sequence. HCA-deduced patterns were not altered by amino-acid variations throughout the evolution of actin and we observed similar hydrophobic motifs comprising myosin subfragment-1 ATP-independent binding sites. HCA suggested the presence of two groups of identical hydrophobic motifs (A1 and A2) which bound on each side of the S1 (63 kDa-31 kDa) connecting segment in relation with two actin monomers. This connection is important in communications between actin- and nucleotide-binding sites. We postulate that some relation and message between the two motifs A1 and A2 take place through myosin subfragment-1 (63 kDa-31 kDa) connecting segment.

Actins↗
PubMed1994

Interaction of skeletal-muscle myosin subfragment-1 with the actin-(338-348) peptide.

The data presented here confirm and provide further experimental evidence that rabbit skeletal-muscle myosin subfragment-1 (S-1) binds to the postulated actin-(338-348) hydrophobic segment [Kabsch, Mannherz, Suck, Pai and Holmes (1990) Nature (London) 347, 37-44] with high affinity in the absence and presence of MgATP. The apparent dissociation constant of the S-1 interaction (5.5 x 10(-7) M) with the actin-(338-348) peptide was of the same order of magnitude as that of the actin-(18-28) binding site (2 x 10(-6) M). In similar conditions, fragmented (27 kDa-50 kDa-20 kDa) S-1 also bound to the peptide. Antibodies directed to the vicinal sequence 348-358 were rapidly eliminated from actin by S-1 interaction and weakened S-1 binding to monomeric or filamentous actin. The antigenic site (348-358) is located very close to the C-terminal S-1-binding site (360-369) and encompasses some residues (Leu-349 and Phe-352) included in the hydrophobic S-1-binding region [Schröder, Manstein, Jahn, Holden, Rayment, Holmes and Spudich (1993) Nature (London) 364, 171-174]. It was observed that anti-[actin-(348-358)] antibodies were also unable to decrease actomyosin ATPase activity, in contrast with previous results obtained with anti-[actin-(18-28)] antibodies [Adams and Reisler (1993) Biochemistry 32, 5051-5056]. The hydrophobic actin-(338-348) peptide used in considerable excess was unable to perturb acto-S-1 and S-1 activities in contrast with results obtained with the N-terminal actin peptide [Kôgler, Moir, Trayer and Ruegg (1991) FEBS Lett. 294, 31-34].

Actins↗
PubMed1994

Purification and characterization of an acetyl esterase from Aspergillus niger.

Optimized acetyl esterase enzyme production conditions using Aspergillus niger ATCC 10864 in 14-L fermentation jars were determined to be 33 degrees C, 1.5 vvm aeration, and 300 rpm agitation without pH control. The acetyl esterase was purified by precipitation in 60-80% saturation in ammonium sulfate. The pellet was applied directly to a Pharmacia high-load Phenyl Sepharose column for hydrophobic interaction chromatography and purified to homogeneity in two steps. Stability and kinetic characteristics of the acetyl esterase were determined over a pH range of 4.0-7.5 and from 4 to 45 degrees C. At temperatures > 25 degrees C, stability was superior at pH values < 5.0. The temperature activity optimum was 35 degrees C, and the pH optimum was 7.0. The Vmax was determined to be 46,700 U/mg protein, and the Km was 0.023M p-nitrophenyl acetate at pH 6.5 in 0.2M phosphate buffer at 35 degrees C. The mol wt of the enzyme was 35,000 dalton by size-exclusion chromatography and SDS gel electrophoresis. The N-terminal amino acid sequence and the glycosylation composition were also determined.

Acetylesterase↗
PubMed1993

Localization of two myosin-subfragment-1 binding contacts in the 96-132 region of actin subdomain-1.

Many direct observations and indirect experimental approaches have pin-pointed two segments (sequences 1-28 and 360-372) in actin subdomain-1 which bind to myosin subfragment-1. In a previous investigation [Labbé, J. P., Méjean, C., Benyamin, Y. & Roustan, C. (1990) Biochem. J. 271, 407-413], we have observed competition between myosin subfragment-1 and anti-actin antibodies specific to epitopes including Thr103. A multisite interface model has also been proposed to take into account myosin-head binding to the N-terminal and C-terminal regions and to more central 40-113 sequence of actin. In the present study, two limited actin segments encompassing residues 96-103 and 112-125 were identified as myosin-head-binding sites. Myosin subfragment-1 competed for monomeric actin with the antibodies directed against sequences 96-105 and 114-120 and its binding to the tryptic 96-113 and synthetic 112-125 actin peptides was prevented by magnesium pyrophosphate but not by calcium pyrophosphate. In the presence of ATP-Mg2+, myosin subfragment-1 was dissociated by filamin from its complex with monomeric actin or with peptide 105-120. Contact points of filamin on actin were previously located in the 105-120 and 360-372 actin sequences [Méjean, C., Lebart, M. C., Boyer, M., Roustan, C. & Benyamin, Y. (1992) Eur. J. Biochem. 209, 555-562]. The in vitro inhibitory effect of filamin on actin-activated Mg2+-ATPase would thus be explained by this competition. Furthermore, the (27-kDa-50-kDa-20-kDa) trypsin-split myosin subfragment-1 which could no longer be activated by actin, did not bind at all to the two sites located in the 96-125 region, but it still interacted with the 360-372 segment. Our results regarding the position of the myosin head on actin monomers in rigor conditions provide evidence on the presence of two topologically independent contact points in the myosin-head/actin interface. One group exposed residues in the 1-7, 21-29, 77-95 and 96-103 actin segment, another, on the opposite side of subdomain-1, included residues from 112-125 and 360-372 sequences.

Actins↗