Measurement of mouse anti-phospholipid antibodies to solid-phase microspheres by both flow cytofluorometry and Alcian blue-pretreated microtitre plates in an ELISA.

Conventional solid-phase immunoassays measuring interactions between anti-phospholipid antibodies and phospholipids are generally characterized by problems of reproducibility and high levels of non-specific binding. Here we describe two immunoassays based on the use of phospholipids in the form of solid-phase microspheres to measure the presence of anti-phospholipid antibodies in sera. Following the production of antibodies in mice against liposomes containing lipid A, we show that flow cytofluorometric analysis provides a reproducible and sensitive way to detect anti-phospholipid antibodies. We also present a sensitive, rapid and reproducible enzyme-linked immunosorbent assay (ELISA) using Alcian blue pretreated microtitre plates and solid-phase microspheres as coating antigen. This ELISA permitted the detection of antibodies to 1/1000 dilution, while untreated plates gave negative results. Such modified ELISA procedures may be applicable to other types of molecule exhibiting solid-phase binding problems e.g. synthetic peptides (J. Immunol. Methods 175 (1994) 131-135).

Relationship between valine, fatty acids, and spiramycin biosynthesis in Streptomyces ambofaciens.

Spiramycin biosynthesis in Streptomyces ambofaciens was stimulated in the presence of valine or by sequential addition of some short-chain fatty acids to a culture medium containing an ammonium salt as source of nitrogen. Acetate kinase and acetyl-CoA carboxylase, enzymes that catalysed the formation of precursors of spiramycin biosynthesis (acetyl-CoA and malonyl-CoA), were detected during the active growth and antibiotic production phases. In this latter phase a higher level of acetyl-CoA carboxylase activity was observed with valine (1.02 mumol.min-1.mg protein-1) than with ammonium (0.05 mumol.min-1.mg protein-1) as nitrogen source, while the evolution and the level of acetate kinase activity were the same in both media. Successive addition of acetate and isobutyrate stimulated highly and weakly the acetyl-CoA carboxylase and acetate kinase activity, respectively.

Influence of short-chain fatty acids on the production of spiramycin by Streptomyces ambofaciens.

The addition of short-chain fatty acids stimulates the production of spiramycin by Streptomyces ambofaciens cultivated on dextrins and ammonium chloride. The fatty acids were activated by two enzymatic systems. The first system (acyl-CoA synthetases) was present only during the exponential phase. The second system (acylkinases coupled with acylphosphotransferases) was synthesized during the growth phase and during the stationary phase, in which spiramycin production started. Short-chain fatty acids induced the synthesis of acylkinases and acylphosphotransferases. Added at the beginning of cultures, they increased the specific activity of these enzymes during the exponential growth phase. Added at the early stationary phase, the specific activity of these enzymes and of the spiramycin production increased. Excess ammonium in the culture considerably lowered the specific activity of acylkinases synthesized in the stationary phase, when spiramycin production started. This ammonium effect can be reduced by the addition of short-chain fatty acids.

Hypervariability, a new phenomenon of genetic instability, related to DNA amplification in Streptomyces ambofaciens.

The wild-type strain Streptomyces ambofaciens DSM 40697 exhibits a high degree of genetic instability. Pigment-defective colonies were observed in the progeny of wild-type colonies at a frequency of about 0.01. While only 13% of these pigment-defective colonies gave rise to homogeneous progeny exhibiting the mutant parental phenotype, 87% of the mutant colonies gave rise to hetergeneous progeny without a preponderant phenotype. This new phenomenon of instability was called hypervariability. In addition, 21% of the mutant strains arising in hypervariable progeny contained highly reiterated DNA sequences, while amplified DNA sequences could be detected in neither stable pigment-defective mutant clones nor in wild-type clones. These results indicate a frequent association between genetic instability and hypervariability and a frequent association between hypervariability and amplification of DNA sequences.