François Vanlinden

Cardiac natriuretic peptides for diagnosis and risk stratification in heart failure: influences of left ventricular dysfunction and coronary artery disease on cardiac hormonal activation.

Cardiac natriuretic peptides are activated in heart failure. However, their diagnostic and prognostic values have not been compared under the routine conditions of an outpatient practice.

Metabolism of the TMA group of antigens during the growth cycle of mycobacteria

The TMA (thermostable macromolecular antigens) group includes A60 ofMycobacterium tuberculosis and A7 ofM. leprae, active components of tuberculin and lepromin. We have previously described the purification and composition of A60, and its ability to elicit immune reactions of humoral and cellular type. In the present work, the intracellular and extracellular distribution and composition of A60 have been traced, as a function of the replication cycle, in static surface cultures ofM. bovis. In exponentially-growing mycobacteria, most A60 was present in the cytoplasm and had a high protein/polysaccharide ratio: this ratio, as well as the level of cytoplasmic A60, decreased after cessation of cell proliferation. The A60 fraction located within the cell wall increased during the stationary phase, but its protein/polysaccharide ratio underwent minor changes. A release of cellular polypeptides and polysaccharides into the extracellular fluid occurred during the declining and lysing phases: a fraction of it was represented by A60. This explains the practice of old tuberculin preparation by autoclaving filtrates of autolysed mycobacterial cultures. The pattern of an A60-like antigen in shaken homogeneous cultures ofM. smegmatis was similar (most antigen present in cytoplasm during growth, increase of the wall fraction in stationary phase, and extracellular release during the declining phase).

Subcellular localisation and sedimentation behaviour of antigen 60 from Mycobacterium bovis BCG

Preparation, composition and immunological properties of A60 of Mycobacterium bovis BCG were previously described (Cocito and Vanlinden 1986). The present study focused on the intracellular distribution of this antigen. Fractionation of mycobacterial homogenates by ultracentrifugation indicated that most of A60 was present within the cytoplasm. Some of the antigen was located within the cell wall, from which it was released by extraction with alkali. Submission of cytoplasm to high speed centrifugation caused A60 to cosediment with ribosomes; however, dissociation of ribosomes in low-Mg buffer did not alter the sedimentation pattern of A60. Labelled A60, after ultracentrifugation in sucrose density gradients without Mg2+, was distributed throughout the entire gradient: treatment of (125I)A60 with urea or detergents produced a peak of radioactivity located in the upper part of the gradient. It is concluded that A60 is represented by a heterogeneous family of molecules of increasing sizes: polymerization being enhanced by Mg2+ and reversibly prevented by urea. Some or all of the biological properties hitherto attributed to ribosomal particles may, in fact, be due to their contamination with cosedimented A60.

Composition and immunoreactivity of the A60 complex and other cell fractions from Mycobacterium bovis BCG.

Surface static cultures of Mycobacterium bovisBCG contained cells embedded in an extracellular matrix, whose mechanical removal yielded free cells that were pressure disrupted and fractionated into cytoplasm and walls. Cell envelopes were either mechanically disrupted or extracted with detergents. Intracellular and extracellular fractions were analysed for proteins, polysaccharides, and antigen 60 (A60), a major complex immunodominant in tuberculosis. A60 was present in extracellular matrix, cytoplasm and walls: it represented a substantial portion of the proteins and polysaccharides of these fractions. While the protein/polysaccharide ratio varied according to the origin of A60 preparations, the electrophoretic patterns of A60 proteins (which accounted for the immunogenicity of the complex) remained unchanged. Western blots pointed to the proteins present within the 29–45 kDa range as the A60 components endowed with the highest immunogenicity level. Since the most heavily stained protein bands in SDS‐PAGE patterns were located outside the region best recognized by antisera, a striking discordance was found between concentration and immunogenicity patterns of A60 proteins. The electrophoretic patterns of A60‐ and non‐A60‐proteins from cytoplasm were also different. A60 complexes in dot blots and some electrophoresed A60 proteins reacted with monoclonal antibodies directed against lipoarabinomannan (LAM), a highly immunogenic polymer of cell envelope. This contaminating compound was removed from A60 with organic solvents and detergents. SDS‐PAGE and Western blot patterns of proteins from delipidated A60 were similar to those of native A60 proteins.

Inhibitory-action of Virginiamycin Components On Cell-free Systems for Polypeptide Formation From Bacillus-subtilis

Although virginiamycin components VM and VS are known to exert in vivo a synergistic inhibition of bacterial growth and viability, in cell-free systems only VM has proven active. In the present work, the in vivo and in vitro activities of VM and VS on Bacillus subtilis have been compared.Peptide formation in homogenates of bacteria previously incubated with either VM or VS was found strongly repressed; the 2 components acted synergistically. Ribosomes were fully responsible for this effect, as shown by mixed reconstitution experiments. On the other hand, cytoplasm from control bacteria disrupted in 10 mM Mg2+ buffer was refractory to in vitro inhibition by virginiamycin, whereas ribosomes prepared in 1 mM Mg2+ were sensitive to VM. VS was inactive on poly(U)-directed poly(phenylalanine) formation, and displayed some activity on the poly(A)-poly(lysine) system. In a cell-free system from Bacillus subtilis infected with phage 2C, both VM and VS were active and blocked synergistically protein synthesis in vitro. When the host cells were incubated with VS and the corresponding homogenate was then treated with VM, a complete inhibition of protein synthesis was observed. The present work, thus, describes the techniques for investigating the in vivo and in vitro action of synergimycins on the same organism, and for reproducing in vitro the synergistic interaction of type A and B components previously observed only in vivo.

Action of Virginiamycin-m On the Stability of Different Ribosomal Complexes To Ultracentrifugation

It was previously shown that virginiamycin M produces in vivo an accumulation of pressure-sensitive (60 S) ribosomes, and in vitro an inactivation of the donor and acceptor sites of peptidyl transferase. The latter action, however, is expected to cause the accumulation in vivo of ribosome complexes carrying acylated tRNA species: such complexes are usually endowed with pressure resistance. However, present data indicate that poly(U).ribosome complexes carrying Phe-tRNA, Ac-Phe-tRNA or Ac-Phe-Phe-tRNA at either the A or the P site become pressure-sensitive after exposure to virginiamycin M in vitro. It is known also that uncoupled EF-G GTPase is stimulated by P-site-bound unacylated tRNA, not by the acylated species. Our data show, however, a stimulation of EF-G GTPase, when ribosomal complexes carrying Ac-Phe-tRNA or Ac-Phe-Phe-tRNA at the P site are incubated with virginiamycin M. The interpretation proposed to account for all these findings is that complexes carrying A- and P-site-bound aminoacyl-tRNA derivatives, which undergo a stable interaction with the peptidyl transferase, are endowed with ultracentrifugal stability, whereas complexes with unacylated tRNA (which does not interact with the enzyme) are pressure-sensitive. By inactivating the donor and acceptor sites of peptidyltransferase, virginiamycin M causes aminoacyl-tRNA.ribosome complexes to mimic tRNA.ribosome complexes in their pressure-lability and competence in EF-G GTPase stimulation. This interpretation is supported by the finding that the ribosome-promoted protection of aminoacyl-tRNA against spontaneous hydrolysis is suppressed by virginiamycin M.

Composition and Immunogenicity of the Polysaccharide Components of the Thermostable Macromolecular Antigen Group of Mycobacterial Antigens

The thermostable macromolecular antigen (TMA) group includes major components of the mycobacterial cell envelope and cytoplasm, which elicit humoral and cellular immune reactions, and seems to play important roles in infectious diseases. The best known member of this group, antigen A60 of Mycobacterium bovis BCG, was previously shown to contain three moieties of polysaccharides, free lipids, and polypeptides. In this work, the TMA polysaccharides of three pathogenic mycobacteria (M. avium, M. bovis and M. paratuberculosis) have been analyzed by coupled gas chromatography-mass spectrometry. In all cases the cores of the TMA complexes were represented by branched glucans of high molecular mass (about 106 daltons), for which structural models have been proposed. The immunogenicity of the polysaccharide components from the three TMA was verified with several immunological procedures (immunodiffusion and immunoelectrophoresis of the antigen, and immunoblotting of the corresponding electrofocused immunoglobulins). All tests tallied in showing a negligible immunogenicity of the glucans examined (inability to produce, upon injection, the synthesis of specific immunoglobulins), thus pointing to the protein moiety of TMA as the one responsible for the high immunoreactivity of the complexes.

Inhibition of Protein-synthesis in Chloroplasts From Plant-cells By Virginiamycin

Abstract The light-driven incorporation of amino acids by isolated spinach chloroplasts is inhibited by the M component (VM) and not by the S component (VS) of virginiamycin. This inhibitory effect is partially reversible. In chloroplast extracts, poly(U)-directed polyphenylalanine formation is strongly inhibited by VM and not by VS. The in vivo synergistic effect of VM and VS observed in bacteria and algae, does not occur in isolated chloroplasts and chloroplast extracts.