Microheterogeneity in yeast invertase.

Yeast external invertase (EC 3.2.1.25), a glycoenzyme consisting of equal parts by weight of protein and mannan, has been found to contain covalently bound phosphate. Three preparations (from two yeast strains) had mannose/PO4 ratios of 31-35, equivalent to 24-27 PO4 residues per mol of enzyme, while a fourth had only 7 PO4 residues per mol. From one of the high-PO4 enzymes, approx. 69% of the phosphorus was recovered as mannose 6-phosphate. No correlation was found between invertase activity and phosphorus content. The PO4 contents of the invertases exceeded those of the cell wall mannans from the respective yeasts. Thus, contamination of the invertases by cell wall phosphomannan is unlikely. Electrofocusing of the low-PO4 invertase yielded four components with pI values from 3.96 to 4.40, and yeast internal invertase (a mannan- and PO4-free, cytoplasmic isozyme) was isoelectric at approx. pH 4.5. The high-PO4 invertase was considerably more heterogeneous, with two major species of pI 3.65 and 3.32 and a highly acidic component of pI smaller than 2.7; however, the mannose/PO4 ratio of each species was approximately the same. PO4-gradient elution from hydroxyapatite resolved the high-PO4 invertase into five isozymes of increasing acidity and mannan content. Since the mannose/PO4 ratios of these invertase species are constant, the increase in the mannan/protein (and, therefore PO4/protein ratio is apparently responsible for the microheterogeneity of phosphoinvertase.23

Lomofungin, an inhibitor of ribonucleic acid synthesis in yeast protoplasts: its effect on enzyme formation.

Lomofungin, an antibiotic active against fungi, yeasts, and bacteria, rapidly inhibits synthesis of ribonucleic acid (RNA) but not protein by protoplasts of Saccharomyces strain 1016. With 40 mug of lomofungin/ml, RNA synthesis was almost completely halted after 10 min of incubation; protein synthesis continued for at least 40 min. Since lomofungin inhibits isolated RNA polymerases from yeast, but not in vitro protein synthesis, it is concluded that the primary action of lomofungin in yeast protoplasts is on RNA synthesis. Examination of the pulse-labeled RNA indicated that biosynthesis of both ribosomal precursor RNAs and messenger RNAs was severely inhibited after the protoplasts were incubated with lomofungin for 5 min, whereas formation of small-molecular-weight RNA (4 to 5s) was only slightly affected. Under these conditions, lomofungin almost completely prevented induction of alpha-glucosidase. Once the protoplasts had been induced, further production of the enzyme was not impaired by lomofungin until after 30 min of incubation, but was rapidly halted by cycloheximide (4 mug/ml). Lomofungin inhibition of invertase formation by protoplasts actively synthesizing the enzyme also became evident only after a lag of about 30 to 40 min, although synthesis was promptly halted by cycloheximide. These observations suggest the existence of relatively long-lived specific messenger RNAs for these enzymes.

Lomofungin, an inhibitor of deoxyribonucleic acid-dependent ribonucleic acid polymerases.

Lomofungin, an antibiotic active against fungi, yeasts, and bacteria, was found to be a potent inhibitor of purified Escherichia coli deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerase. It prevents RNA synthesis by a direct interaction with the polymerase and not with the template or substrate; chain elongation is halted promptly. Three DNA-dependent RNA polymerases isolated from Saccharomyces strain 1016 were also sensitive to the antibiotic. Lomofungin does not appear to react generally with proteins, as bovine serum albumin did not prevent the inhibitory effect and numerous other enzymes were not affected by lomofungin.

Antibody responses after immunization with polyvalent pneumococcal vaccine containing 10, 25, or 50 micrograms of 14 polysaccharide types per dose.

Immune responses were determined to three different doses of a pneumococcal vaccine which contained 10, 25, or 50 micrograms of 14 polysaccharide types (1, 3, 4, 6A, 6B, 7F, 8, 9N, 12F, 14, 18C, 19F, 20, and 23F) per dose. Pre- and 4-weeks postvaccination sera were examined by radioimmunoassay. Ratios of geometric mean titers (GMT) showed a greater than threefold increase of antibodies to each type of polysaccharide regardless of the dose of antigen. The post-GMT was highest to 12 of 14 types in recipients of the 25-micrograms vaccine. Subjects in the group receiving a 25-micrograms dose of each antigen showed more two- and fourfold titer increases than did subjects in the group receiving 10- and 50-micrograms doses, but, in general, seroconversion rates were similar in all the groups. All GMT values in the 25-micrograms vaccine group were well above what is currently considered to be the protective level of antibody. Local reactions, overall, were low in all groups. Pain, induration, and tenderness at the site of injection, which were greatest in recipients of the 50-micrograms dose/antigen, were progressively lower with the 25- and 10-micrograms dose/antigen.

Characterization of Vero cells.

A cell line used in the production of biologicals should be free of infectious agents, and 'described with respect to cytogenetic characteristics and tumorigenicity'. Vero, a continuous cell line derived from a normal African green monkey kidney, was examined for the presence of retroviruses and for tumorigenic potential. We were unable to detect the presence of retroviruses by reverse transcriptase assay, electron microscopy or hybridization of cellular genomic DNA with Mason-Pfizer monkey virus DNA probes. In addition, passage 156 Vero cells did not form progressively growing tumors in nude mice or grow with high efficiency in soft agarose.

A mutation present in the amino terminus of Sabin 3 poliovirus VP1 protein is attenuating.

The attenuated phenotype of Sabin 3 poliovirus compared with its neurovirulent progenitor strain has been largely accounted for by mutations in the genome at positions 472 and 2034 (G. D. Westrop, K. A. Wareham, D. M. A. Evans, G. Dunn, P. D. Minor, D. I. Magrath, F. Taffs, S. Marsden, M. A. Skinner, G. C. Schild, and J. W. Almond, J. Virol. 63:1338-1344, 1989). By sequencing vaccine virus RNA, we recently identified another Sabin 3-specific mutation at position 2493 (U----C), which predicts an Ile----Thr change at the sixth residue of VP1 (C. Weeks-Levy, J. M. Tatem, S. J. DiMichele, W. Waterfield, A. F. Georgiu, and S. J. Mento, Virology 185:934-937, 1991). Viruses generated by using cDNAs which represent the vaccine sequence (LED3) and a derivative (VR318) possessing a single base change to the wild-type nucleotide (U) at 2493 were used to determine the impact of the 2493 mutation on virus phenotype. The VP1 proteins of LED3 and VR318 viruses were distinguishable by denaturing electrophoretic analysis. LED3 produced smaller plaques in Vero cells than VR318 virus did. Neurovirulence testing of these cDNA-derived viruses in monkeys demonstrated that the 2493 mutation in LED3 virus is attenuating.