Jean-Paul Brouard

A Novel Peptidoglycan Cross-linking Enzyme for a β-Lactam-resistant Transpeptidation Pathway

The β-lactam antibiotics remain the most commonly used to treat severe infections. Because of structural similarity between the β-lactam ring and the d-alanyl4-d-alanine5 extremity of bacterial cell wall precursors, the drugs act as suicide substrates of the dd-transpeptidases that catalyze the last cross-linking step of cell wall assembly. Here, we show that this mechanism of action can be defeated by a novel type of transpeptidase identified for the first time by reverse genetics in aβ-lactam-resistant mutant of Enterococcus faecium. The enzyme, Ldtfm, catalyzes in vitro the cross-linking of peptidoglycan subunits in a β-lactam-insensitive ld-transpeptidation reaction. The specificity of Ldtfm for the l-lysyl3-d-alanine4 peptide bond of tetrapeptide donors accounts for resistance because the substrate does not mimic β-lactams in contrast to d-alanyl4-d-alanine5 in the pentapeptide donors required for dd-transpeptidation. Ldtfm homologues are encountered sporadically among taxonomically distant bacteria, indicating that ld-transpeptidase-mediated resistance may emerge in various pathogens.

Aslfm, the D-aspartate ligase responsible for the addition of D-aspartic acid onto the peptidoglycan precursor of Enterococcus faecium.

d-Aspartate ligase has remained the last unidentified peptide bond-forming enzyme in the peptidoglycan assembly pathway of Gram-positive bacteria. Here we show that a two-gene cluster of Enterococcus faecium encodes aspartate racemase (Racfm) and ligase (Aslfm) for incorporation of d-Asp into the side chain of the peptidoglycan precursor. Aslfm was identified as a new member of the ATP-grasp protein superfamily, which includes a diverse set of enzymes catalyzing ATP-dependent carboxylate-amine ligation reactions. Aslfm specifically ligated the β-carboxylate of d-Asp to the ϵ-amino group of l-Lys in the nucleotide precursor UDP-N-acetylmuramyl-pentapeptide. d-iso-Asparagine was not a substrate of Aslfm, indicating that the presence of this amino acid in the peptidoglycan of E. faecium results from amidation of the α-carboxyl of d-Asp after its addition to the precursor. Heterospecific expression of the genes encoding Racfm and Aslfm in Enterococcus faecalis led to production of stem peptides substituted by d-Asp instead of l-Ala2, providing evidence for the in vivo specificity and function of these enzymes. Strikingly, sequencing of the cross-bridges revealed that substitution of l-Ala2 by d-Asp is tolerated by the d,d-transpeptidase activity of the penicillin-binding proteins both in the acceptor and in the donor substrates. The Aslfm ligase appears as an attractive target for the development of narrow spectrum antibiotics active against multiresistant E. faecium.

Isolation and structure of cyclosenegalins A and B, novel cyclopeptides from the seeds of Annona senegalensis

Two new cyclopeptides, cyclosenegalin A, cyclo(Pro1-Gly2-Leu3-Ser4-Ala5-Val6-Thr7-) (1) and cyclosenegalin B, cyclo(Pro1-Gly2-Tyr3-Val4-Tyr5-Pro6-Pro7-Val8-) (2), have been isolated from the methanol extract of the seeds of Annona senegalensis Pers., along with the known cyclic peptide, glabrin A. The structures were elucidated on the basis of the MS/MS fragmentation, using a Q-TOF mass spectrometer equipped with an ESI source, chemical degradation and extensive 2D-NMR.

Thymosiosterol and Δ24 thymosiosterol, new sterols from the sponge Thymosiopsis sp.

Abstract Two new sterols were isolated from the sponge Thymosiopsis sp. and their structure elucidated by 2D NMR spectral data.

New indolines derivatives from strychnobrasiline, modulators of chloroquine resistance in Plasmodium falciparum

Abstract an unexpected molecular rearrangement in lithium aluminium hydride reduction of the indole alkaloid strychnobrasiline was observed. The resulting derivatives obtained were evaluated as modulators of chloroquine resistance in Plasmodium falciparum.

Two pyran type glycosides from saponaria and dianthus

Abstract 2,3-Dihydro 4- O -β-D-glucopyranosyl 3-hydroxy-2-methyl-4 H -pyran was isolated from Saponaria officinallis and its relative configuration and conformation determined. A new glycoside, isomeric with the former but having a different configuration at C-4, has been isolated from Dianthus barbatus and D. deltoides .

Composition and Antimicrobial Activity of Leaf Oils of Vepris leandriana H. Perr.

Abstract Field-distilled and laboratory distilled leaf oils of Vepris leandriana (Rutaceae) were analyzed by GC and GC/MS. The field-distilled oil was found to contain α-terpinene (9.0%), neral (19.5%), geranial (27.0%) and citronellol (33.6%), while the laboratory-distilled oil contained α-terpinene (4.7%), neral (21.8%), geranial (33.0%) and citronellol (33.2%). The field-distilled oil was examined for antimicrobial activity. It was found to possess greater antimicrobial activity than chloramphenicol.

Clearance of Amyloid-β Peptide by Neuronal and Non-Neuronal Cells: Proteolytic Degradation by Secreted and Membrane associated Proteases

Deposition of amyloid-beta peptide (Abeta) in the brain is an early and invariant feature of all forms of Alzheimers disease (AD). As for all proteins or peptides, the steady-state level of Abeta peptide is determined not only by its production, but also by its degradation. So, overactive proteases involved in generating Abeta from amyloid precursor protein or underactive Abeta-degrading enzymes could lead to abnormal Abeta deposition in the brain. Since in the sporadic forms of AD (90% of all AD cases) an impaired clearance of Abeta appears to be at the origin of its aggregation and tissue deposition, we have investigated its proteolytic degradation by several neuronal and non-neuronal cells. In this report, we show that these cell types exhibit a similar profile of Abeta-degradation by cell-surface and secreted proteases which were respectively characterized as metallo- and serine proteases. By using a combination of the liquid chromatography/on-line mass spectrometry, we demonstrate that: (i)-the membrane associated protease(s) hydrolizes Abeta40 essentially at Lys(28) Gly(29), Phe(19) Phe(20) and Val(18) Phe(19) bonds; and (ii)-the secreted protease(s) cleaves the generating fragments Abeta (1-28), Abeta (1-19), Abeta (1-18) at His(14) Gln(15) bond and also Abeta (1-28) at Phe(20) Ala(21) and Asp(23) Val(24) sites. This is the first time our results define a proteolytic degradation process of Abeta40 that appears to be independent of the cell type and may represent a general pattern of its enzymatic clearance.

Two pyran type glycosides from Tunica prolifera

Abstract Two new glycosides allied to dianthoside have been isolated from Tunica prolifera . Their structures were determined to be those of the 3-glucoside

n-Alkanes, friédéline et neooleana 3 (5) 12 diene chez les végétaux

ResumeLe present travail resume les donnees anterieures sur la repartition des n-alkanes et de la friedeline chez les vegetaux. Ces composes ont ete recherches par Chromatographie en phase gazeuse et spectrometrie de masse dans 91 especes choisies dans tous les groupes botaniques. Les resultats ont ete discutes au point de vue taxinomique. Le neooleana 3 (5) 12 diene, nouveau triterpenoide naturel a ete rencontre dans Euonymus, Davidia et Diospyros.