Preparation of tritiated lipopolysaccharides from Escherichia coli K12.

Tritiated lipopolysaccharide (LPS) from E. coli K12 was prepared by coupling [3H]ethanolamine to the LPS core residue ketodeoxyoctonate (KDO) via activation of its carboxylic function with N-hydroxysuccinimide or N-hydroxy-sulfosuccinimide. Specific activities of 1.5 microCi/mg and 9 microCi/mg were obtained, respectively. Experiments comparing the activity of native and derivatized LPS suggested that the preparation of the radiolabelled LPS did not alter the structural properties of E. coli K12 LPS. This probe will be useful for studying the interactions between LPS and proteins.

An inhibition enzyme-linked immunosorbent assay technique for the detection of endotoxins in proteins extracted from Escherichia coli K12 recombinant DNA.

An inhibition enzyme-linked immunosorbent assay (ELISA) was developed for the specific quantitation of rough (R) mutant E. coli K12 lipopolysaccharide (LPS). Since R-LPS binds poorly to polystyrene microplates, an LPS-BSA covalent complex was prepared following glutaraldehyde activation and used as a coating surface antigen. A 100-fold higher signal was observed using the LPS-BSA complex as solid-phase antigen instead of free LPS. The LPS detection limit obtained was 0.5 ng/ml. This test was applied to hGH extracts produced genetically engineered E. coli K12 and a good correlation was found with the LAL test. This new LPS titration technique will be useful for detecting LPS in complex mixtures and the antigen-antibody reaction will ensure the specificity of the detection.

Use of perfluorinated counter ions for the combination of ion pair HPLC and field desorption mass spectrometry. Application to the early characterization of aminoglycoside antibiotics.

The use of perfluorinated carboxylic acids counter ions is described for easy combination of ion pair HPLC and FD-MS. This technique is applied to the analysis of aminoglycoside antibiotics and to the problem of their early characterization from culture broths. Reliable molecular weight informations can be obtained with this method from minute amounts of purified products (10 micrograms) allowing formal identification in most cases.

Inactivation of lincosaminide antibiotics in Staphylococcus. Identification of lincosaminide O-nucleotidyltransferases and comparison of the corresponding resistance genes.

Resistance to lincomycin by inactivation has been detected in numerous clinical isolates of Staphylococcus; in crude extracts of Staphylococcus haemolyticus BM4610 and Staphylococcus aureus BM4611, inactivation of lincomycin and clindamycin requires the presence of a nucleoside 5'-triphosphate (ATP, GTP, CTP, or UTP) as nucleotidyl donor and Mg2+ as cofactor. The biochemical mechanism of lincosaminide inactivation was elucidated by determination of the structure of inactivated lincomycin and clindamycin by physicochemical techniques, including UV absorption spectrophotometry, 31P and 1H nuclear magnetic resonance, and periodate oxidation. In the two strains, inactivation of lincomycin gave rise to lincomycin 3-(5'-adenylate), whereas clindamycin was inactivated through its conversion to clindamycin 4-(5'-adenylate). The gene linA' encoding the 3-lincomycin, 4-clindamycin O-nucleotidyltransferase in S. aureus BM4611 has been sequenced and displays 93% homology with the gene linA encoding the 3-lincomycin, 4-clindamycin O-nucleotidyltransferase found in S. haemolyticus BM4610. The two enzymes are 161 amino acids long and differ by 14 amino acid substitutions.

Use of mixed perfluorinated ion-pairing agents as solvents in ion-pair high-performance liquid chromatography for the preparative purification of aminoglycoside antibiotics.

The possibility of using mixed ion-pair reversed-phase high-performance liquid chromatography for the preparative purification of aminoglycoside antibiotics was investigated. Retention can be satisfactorily defined in aqueous conditions but efficiency is critical. The analysis of a polynominal model allowed ion-pairing agents compositions to be designed leading to improved efficiency. Inexpensive aqueous mobile phases using volatile buffers can be defined for the satisfactory ion-pair preparative chromatography of aminoglycosides.

[The sex-trapping of Sterrha biselata (Hufn.) (Lepidoptera Geometridae, Sterrhinae) by acetoxy-1 dodecadiens-7 E, 9Z, the sex pheromone of Lobesia botrana (Schiff.) (Lepidoptera Tortricidae, Olethreutinae)]. / Sur le piégeage sexuel de Sterrha biselata (Hufn.) (Lépidoptère Geometridae, Sterrhinae) par l'acétoxy-1 dodécadiène-7 E, 9 Z phéromone sexuelle de Lobesia botrana (Schiff.) (Lépidoptère Tortricidae, Olethreutinae)

7E, 9Z dodecadienyl acetate, a sex-heromone for Lobesia botrana, is selectively attractive, in vineyards, for males of this species. By testing this compound in woods and orchards, we have found that it was also selectively attractive for males of a geometrid moth; Sterrha biselata. This is one of the first examples where a definite chemical substance is active for a geometrid species.

Mechanism of selectivity in ion-pair high-performance liquid chromatography of aminoglycoside antibiotics using perfluorinated pairing ions.

The behaviour of perfluorinated carboxylic acids as pairing ions for the chromatography of the aminoglycoside antibiotics was studied. As with perhydrogenated pairing ions, the capacity factor can be modified according to the percentage of organic modifier and the nature and concentration of perfluorinated pairing ion in the mobile phase. The special selectivity effect observed with trifluoroacetic acid was investigated and interpreted as a concerted mechanism involving ionic and hydrophobic interactions.

Comparative studies of lipopolysaccharide and exopolysaccharide from a virulent strain of Pseudomonas solanacearum and from three avirulent mutants.

The composition of the Pseudomonas solanacearum lipolysaccharide (LPS) was found to be similar to that described for the LPS of enterobacteria. The lipid A contained fatty acids and glucosamine in a molar ratio of 5:2. The LPS fraction contained 2-keto-3-deoxyoctulosonic acid, L-glycero-D-mannoheptose, hexoses (glucose, rhamnose, and glucosamine), and a pentose (xylose). The LPSs from the wild-type strain (GMI1000), from the spontaneous rough mutant (GMI2000), and from their respective acridine orange-resistant (Acrr) mutants (GMI1178 and GMI2179) contained the same component sugars in their polysaccharide moieties, but the relative amounts of each sugar varied greatly. Spontaneous mutation to the rough type was characterized by a decrease in the ratio of rhamnose to glucose, whereas a reverse effect was seen for the acridine orange resistance mutation from the parent strains (GMI1000 and GMI2000) to the respective mutant strains (GMI1178 and GMI2179). The exopolysaccharide (EPS) from GMI1000 was found to be composed of two fractions: a heteropolysaccharide (galactosamine, glucose, and rhamnose) excluded from Sephadex G-50 and an additional glucan with a lower molecular weight. Strains GMI1000 and GMI1178 produced comparable amounts of EPS, GMI2179 synthesized less EPS, and GMI2000 produced no detectable EPS. High-pressure liquid chromatography and 13C nuclear magnetic resonance analyses revealed some differences between these EPSs. The glucan fraction seemed to be the major component of the EPS from GMI2179, whereas GMI1000 and GMI1178 EPSs contained both fractions and appeared to differ in the structures of their heteropolysaccharide fractions. Viscosity measurements confirmed differences between whole EPSs produced by the three strains.

Stabilization and enhancement of interleukin-2 in vitro bioactivity by new carriers: supramolecular biovectors.

Human recombinant interleukin-2 can be associated and released from supramolecular biovectors (SMBVs), consisting of particles made of polymerized polysaccharides. The particles are substituted with phosphate residues and contain bound lipid molecules (palmitic acid) buried near their surfaces. The association of IL-2 with SMBVs modifies its in vitro bioactivity. SMBVs prolong the growth of IL-2-dependent cells, enhance IL-2 proliferative activity and restore the activity of impaired IL-2. These properties mainly depend on the presence of lipids linked to the SMBV and on both the degree of acylation and the SMBV: IL-2 ratio. SMBVs are therefore good candidates for the stabilization and enhancement of the biological activity of IL-2.

A new family of carriers (biovectors) enhances the immunogenicity of rabies antigens.

Biovectors (BV) are a new family of protein carriers. They are nanoparticles of polymerized polysaccharides substituted with phosphate residues and surrounded by covalently bound lipid molecules (palmitic acid). The effect of BV was tested on the immunogenicity of rabies antigens. Biovectors enhanced the production of antibody induced by both rabies glycoprotein and ribonucleoprotein. Moreover, they enhanced the protective activity of an experimental rabies vaccine composed of inactivated and purified virus. The isotype profile of antibody produced in vivo was not modified when BV were mixed with rabies antigens. To clarify the mechanism of the adjuvant/ immunostimulation effect of BV, two types of approach were used: (1) analysis of the antibody response when antigen and BV were injected separately; (2) determination of the nature of cells involved in the proliferation in vitro of murine splenocytes in the presence of BV. The enhancing effect of BV on antibody production was highest when mixed with antigens. In vitro BV induced the proliferation of B cells. These findings suggest that BV have immunostimulating properties in addition to their probable depot and/or antigen-presentation effect which explain in part their adjuvant activity.