Studies on the biosynthesis of the antibiotic crisamicin A and carbon-13 magnetic resonance assignments.

The biosynthesis of crisamicin A, a novel dimeric isochromanequinone antibiotic from Micromonospora purpureochromogenes subsp. halotolerans has been investigated by [1-13C] and [2-13C] labeled acetate precursor feeding experiments. Analysis of the proton noise decoupled and off resonance 13C NMR spectra of 13C enriched and unenriched crisamicin A and their acetate derivatives indicated the biosynthesis via the polyketide pathway, as expected. Further analysis of the enriched spectra allowed the complete assignment of the carbon signals. Of particular interest was the establishment of the linkage between the two monomeric halves of the molecule and determination of the location of the phenolic hydroxyls.

Radioimmunoassay analysis of baculovirus granulins and polyhedrins.

Granulin and polyhedrin proteins were purified by preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis from the baculoviruses Autographa californica, Rachiplusia ou, Heliothis zea, Heliothis armigera, Trichoplusia ni, and Spodoptera frugiperda. Antisera were raised against Autographa californica (Ac) polyhedrin and Trichoplusia ni (Tn) granulin and analyzed for homologous and heterologous immunoreactivity by immunodiffusion and radioimmunoassay (RIA). Ac polyhedrin and Tn granulin antisera recognized antigenic determinants on several baculovirus polyhedrin and granulin proteins even though the heterologous proteins had different immunoreactivities when compared by competition radioimmunoassay. Antigenic differences among granulin and polyhedrin proteins were also detected by altered slopes of the competition reaction curves. Antiserum raised against Ac polyhedrin which was purified in the presence of SDS was tested by competition RIA for its ability to detect and react with native polyhedrin produced in the infected TN-368 cells. Ac polyhedrin antiserum had similar if not identical ability to bind to native polyhedrin and to polyhedrin purified in the presence of SDS.

Growth control of human foreskin fibroblasts and inhibition of extracellular sialidase activity by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid.

Normal human fibroblasts have two extreme modes of existence in culture, quiescent and proliferative. The growth and division of these cells are usually well regulated by the action of various endogenously generated stimulators and inhibitors. We have speculated that an extracellular sialidase may be involved in the regulation of growth and that inhibition of this activity might decrease or abolish cell growth. To test this hypothesis, we have incubated preconfluent cultures of fibroblasts in the presence and absence of a potent sialidase inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid. Treatment of cells with this inhibitor resulted in the inhibition of an extracellular sialidase activity for up to 24 h and had a marked growth inhibitory effect in a concentration-dependent manner. The effect of the inhibitor on cell proliferation was specific and reversible. During a chase period of 48 h after pulse labeling cells with [3H]N-acetylmannosamine and [14C]serine, there was a 15% decrease of [3H]sialic acid in the membrane-bound GM3 with 80 microM inhibitor in the medium, as compared with a 32% decrease in the controls. Our results suggest that an extracellular sialidase may participate in cell-surface modifications that accompany (or control) the changes observed when cells traverse the cell cycle, from the quiescent to the proliferative phase.

Evidence for an extrinsic immunogen in effective ribosomal vaccines from Salmonella typhimurium.

Previous work has indicated that ribosomes isolated from Salmonella typhimurium were highly immunogenic and afforded excellent protection against homologous challenge. Effective protection was obtained also when ribonucleic acid ( RNA) extracted from these ribosomes was used as a vaccine. In this investigation ribosomes prepared by another method and washed repeatedly in 1 M NH1Cl lost much of their prophylactic potency and yielded poorly protective RNA. The high-salt wash of the ribosomes was immunogenic. The RNA and the protein isolated from the salt wash of the ribosomes were effective vaccines. No intrinsic component of the ribosomes was removed by the NH4Cl wash, since the ability of both "crude" and "clean" ribosomes to function equally well in an in vitro protein synthesizing system was demonstrated. The presence of a component with toxic properties similar to those of endotoxin was found in active vaccines but not in weak ones. This was shown by the ability of effective vaccines to kill lead acetate-sensitized mice and to induce tolerance to endotoxin.

Production and characterization of monoclonal antibodies to wall-localized peroxidases from corn seedlings.

A library of 22 hybridomas, which make antibodies to soluble wall antigens from the coleoptiles and primary leaves of etiolated corn (Zea mays L.) seedlings, was raised and cloned three times by limit dilution to assure monoclonal growth and stability. Two of these hybridomas made immunoglobulin G antibodies, designated mWP3 and mWP19, which both effectively immunoprecipitated peroxidase activity from crude and partially purified preparations of wall peroxidases. Direct peroxidase-binding assays revealed that both antibodies bound enzymes with peroxidase activity. As judged by immunoblot analyses, mWP3 recognized a Mr 98,000 wall peroxidase with an isoelectric point near 4.2, and mWP19 recognized a Mr 58,000 wall peroxidase. Immunogold localization studies showed both peroxidases are predominately in cell walls.

Characterization of monoclonal antibodies to oat phytochrome by competitive radioimmunoassays and comparative immunoblots of phytochrome peptides.

A library of 50 hybridomas which make antibodies to oat phytochrome was produced from the fusion of spleen cells from immunized Balb/c mice with P3x63Ag8 myeloma cells. Hybridomas were selected in a medium containing hypoxanthine, aminopterin, and thymidine, and specific hybridomas were screened for production of antibodies to phytochrome using a solid-phase enzyme-linked immunoadsorbent assay which was antigen specific. Positive cultures were cloned three times by limit dilution to assure monoclonal growth and stability. Specificity toward phytochrome was established by Western blot analysis and immunoprecipitation. Epitope specificity of nine monoclonal antibodies was determined by competitive inhibition radioimmunoassays and/or comparative immunoblots of tryptic peptides of phytochrome.