Biomedical subjects

F Lenfant

Publications and source records attributed to F Lenfant.

46 records · Page 3Linked to original sources

PubMed1996

[Does flumazenil correct disorders of intracardiac conduction induced by accidental intravenous injection of bupivacaine?].

The authors report the case of a patient who experienced intracardiac conduction disturbances from accidental intravenous injection of bupivacaine, which were reversed by an intravenous injection of flumazenil. Flumazenil could act either in displacing bupivacaine from its binding site, or by an interaction involving the GABAergic system, unless a specific interaction between flumazenil and bupivacaine does exist. In case of experimental confirmation of this feature, flumazenil could be proposed as a new therapeutic tool for bupivacaine-induced cardiotoxicity.

Anesthetics, Local↗

PubMed1996

Placental expression of HLA class I genes.

This article presents an overview of the more recent data dealing with the constitutive, transcriptional, and translational expression of classical class Ia and nonclassical HLA-E and -G class Ib products in the different trophoblast cell subpopulations that constitute the maternofetal interface during human pregnancy. Of particular interest is the expression of alternatively spliced HLA-G transcriptional isoforms that may be translated in membrane-bound or soluble protein products. Molecular regulatory mechanisms that may control the differential expression of class Ia and class Ib molecules, according to the cell types, state of differentiation, and stages of gestation are also examined. They may operate at the levels of transcription, translation and/or transport of proteins to the cell surface. Functional significance of the absence of detectable cell surface expression of class Ia molecules in all trophoblast cell subpopulations, and of the presence of membrane-bound HLA-G products in extravillous cytotrophoblast cells is finally questioned.

Female↗

PubMed1995

Multiple substitutions at position 104 of beta-lactamase TEM-1: assessing the role of this residue in substrate specificity.

Residue 104 is frequently mutated from a glutamic acid to a lysine in the extended-spectrum TEM beta-lactamases responsible for the resistance to third-generation cephalosporins in clinical Gram negative strains. Among class A beta-lactamases, it is the most variable residue within a highly conserved loop which delineates one side of the active site of the enzymes. To investigate the role of this residue in the extended-spectrum phenotype, it has been replaced by serine, threonine, lysine, arginine, tyrosine and proline. All these substitutions yield active enzymes, with no drastic changes in kinetic properties compared with the wild-type enzyme, except with cefaclor, but an overall improved affinity for second- and third-generation cephalosporins. Only mutant E104K exhibits a significant ability to hydrolyse cefotaxime. Molecular modelling shows that the substitutions have generally no impact on the conformation of the 101-111 loop as the side chains of residues at position 104 are all turned towards the solvent. Unexpectedly, the E104P mutant turns out to be the most efficient enzyme. All our results argue in favour of an indirect role for this residue 104 in the substrate specificity of the class A beta-lactamases. This residue contributes to the precise positioning of residues 130-132 which are involved in substrate binding and catalysis. Changing residue 104 could also modify slightly the local electrostatic potential in this part of the active site. The limited kinetic impact of the mutations at this position have to be analysed in the context of the microbiological problem of resistance to third-generation cephalosporins. Although mutation E104K improves the ability of the enzyme to hydrolyse these compounds, it is not sufficient to confer true resistance, and is always found in clinical isolates associated with at least one mutation at another part of the active site. It is the combined effect of the two mutations that synergistically enhances the hydrolytic capability of the enzyme towards third-generation cephalosporins.

Amino Acid Sequence↗

PubMed1993

Site-directed mutagenesis of beta-lactamase TEM-1. Investigating the potential role of specific residues on the activity of Pseudomonas-specific enzymes.

From sequence alignments, two groups can be defined for the carbenicillin-hydrolysing beta-lactamases (CARB enzymes). One group includes the Pseudomonas-specific enzymes PSE-1, PSE-4, CARB-3, CARB-4 and also the Proteus mirabilis GN79, for which the well-conserved residue Lys 234 in all class-A beta-lactamases is changed to an arginine residue. The second group includes the enzymes PSE-3 and AER-1 which have an arginine or a lysine residue at position 165. All these enzymes also have leucine at position 68, threonine at position 104 and glycine at position 240. We engineered these mutations into the TEM-1 beta-lactamase to study their potential role in defining the substrate profile of the CARB enzymes. The mutations K234R and E240G in TEM-1 noticeably increased the hydrolysis of carboxypenicillins relative to other penicillins by approximately sixfold and twofold, respectively. The variant E240G also demonstrated an improved rate of second-generation cephalosporin and cefotaxime hydrolysis. In contrast, the substitution of Trp165 by arginine does not extend the substrate profile to alpha-carboxypenicillins nor does it noticeably modify the kinetic behavior of the enzyme. The mutations M68L and E104T do not have a large effect on the hydrolysis rate but the mutation E104T enhances the affinity of the enzyme for third-generation cephalosporins. As the mutation K234R resulted in a severe decrease in the affinity for carboxypenicillins, the double mutant E240G/K234R was constructed in an attempt to enhance the CARB character of the enzyme. Contrary to what could be expected, the additional mutation E240G for the TEM-1 K234R enzyme increases neither the catalytic constant for the carboxypenicillins nor the affinity towards these substrates. Consequently, this study strongly suggests that the three-dimensional structures of the active site of the TEM-1 enzyme and PSE-3, PSE-4 or other related enzymes are significantly different. This probably explains the discrepancy of the substrate profile between the CARB enzymes and the TEM-1 protein variants.

Base Sequence↗

PubMed1992

Beta-lactamase TEM1 of E. coli. Crystal structure determination at 2.5 A resolution.

The crystal structure of beta-lactamase TEM1 from E. coli has been solved to 2.5 A resolution by X-ray diffraction methods and refined to a crystallographic R-factor of 22.7%. The structure was determined by multiple isomorphous replacement using four heavy atom derivatives. The solution from molecular replacement, using a polyalanine model constructed from the C alpha coordinates of S. Aureus PCl enzyme, provided a set of phases used for heavy atom derivatives analysis. The E. coli beta-lactamase TEM1 is made up of two domains whose topology is similar to that of the PCl enzyme. However, global superposition of the two proteins shows significant differences.

Amino Acid Sequence↗

PubMed1992

Crystallization and preliminary crystallographic data on Escherichia coli TEM1 beta-lactamase.

Two crystal forms of Gram- bacteria TEM beta-lactamase have been obtained. The tetragonal form has a very large unit cell and diffracts to 3.0 A resolution. Orthorhombic crystals, grown using ammonium sulfate and a small amount of acetone as precipitating agents, belong to space group P2(1)2(1)2(1) with cell parameters a = 43.1 A, b = 64.4 A, c = 91.2 A and diffract to 1.7 A resolution. A seeding procedure has been designed that ensures reproducibility of the crystal properties. Molecular replacement, using a model reconstructed from the C alpha co-ordinates from Staphylococcus aureus PC1 beta-lactamase, gives a solution that satisfies crystal packing constraints.

Bacterial Proteins↗

PubMed1991

Replacement of lysine 234 affects transition state stabilization in the active site of beta-lactamase TEM1.

Lysine 234 is a residue highly conserved in all beta-lactamases, except in the carbenicillin-hydrolyzing enzymes, in which it is replaced by an arginine. Informational suppression has been used to create amino acid substitutions at this position in the broad spectrum Escherichia coli beta-lactamase TEM-1, in order to elucidate the role of this residue which lies on the wall at the closed end of the active site cavity. The mutants K234R and K234T were constructed and their kinetic constants measured. Replacement of lysine 234 by arginine yields an enzyme with similar activity toward cephalosporins and most penicillins, except toward the carboxypenicillins for which the presence of the guanidine group enhances the transition state binding. The removal of the basic group in the mutant K234T yields a protein variant which retains a low activity toward penicillins, but losts drastically its ability to hydrolyze cephalosporins. Moreover, these two mutations largely decreased the affinity of the enzyme for penicillins (10-fold for K234R and 50-fold for K234T). This can be correlated with the disruption of the predicted electrostatic binding between the C3 carboxylic group of penicillins and the amine function of the lysine. Therefore, lysine 234 in the E. coli beta-lactamase TEM-1 is involved both in the initial recognition of the substrate and in transition state stabilization.

Amino Acid Sequence↗

PubMed1991

Site-directed mutagenesis on TEM-1 beta-lactamase: role of Glu166 in catalysis and substrate binding.

Class A beta-lactamases are the major cause of bacterial resistance to beta-lactam antibiotics. In these active-site serine hydrolases, glutamic acid 166 has been hypothesized to act as a general acid-base catalyst. Replacing this residue by tyrosine in TEM-1 beta-lactamase yields an enzyme the activity of which is substantially lowered and strongly dependent on pH, thus confirming the alleged role of Glu166 in catalysis. This substitution also resulted in a spectacular change in substrate profile, the mutant enzyme being more active on cephalosporins than on penicillins. In fact, the E166Y enzyme behaves much like a class C enzyme, with high affinity and low hydrolytic activity towards second and third generation cephalosporins. Glu166 therefore seems to play a major part in defining the substrate profile of class A beta-lactamases.

Binding Sites↗

PubMedDate not supplied

Probing the active site of beta-lactamase R-TEM1 by informational suppression.

Using a new extended set of 13 amber suppressors in E coli, systematic amino-acid replacements were performed at positions 104(E) and 238(G) of TEM-1 beta-lactamase from PUC19. The enzyme is tolerant to most substitutions tested at position 104. Missense revertants E104K, E104S or E104Y exhibited only minor changes in enzyme activity with respect to wild-type TEM-1. Several substitutions at position 238 resulted in a new cefotaxime hydrolysing capacity, but to an extent that did not confer cefotaxime resistance for the bacteria producing the mutated enzymes. Only when the mutations at codons 104 and 238 were combined on the same gene, did a true cefotaxime resistant phenotype appear, mimicking the situation encountered with 3rd generation cephalosporins resistant clinical isolates.

Base Sequence↗

PubMedDate not supplied

Placental HLA-G protein expression in vivo: where and what for?

In contrast to HLA-A and -B class Ia genes that are down-regulated in human trophoblast cells, HLA-G class Ib molecules are expressed in the placenta throughout gestation. In addition to extravillous cytotrophoblast that invade the decidua basalis essentially, HLA-G was also observed in endothelial cells of fetal vessels in the chorionic villi as well as in amnion cells and amniotic fluid. Both membrane-bound and soluble HLA-G isoforms have been detected. In view of the recently published functional data showing that HLA-G: (i) has the capability to bind and present peptides; (ii) is recognized by at least three different killing inhibitory receptors; and (iii) is a regulator of HLA-E expression, we can predict that such functions are likely to be exerted by extravillous cytotrophoblast. Of particular importance will be the anti-viral function of HLA-G at this materno-fetal interface, knowing that HLA-G was shown to be expressed by thymic medullary epithelial cells. In addition to these immunological functions, due to its presence on chorionic fetal endothelial cells, we hypothesize that HLA-G could also be a regulator of chorionic villous angiogenesis. Finally, soluble HLA-G isoforms may act as specific immunosuppressors during pregnancy.

Amniotic Fluid↗