Isolation and characterization of a polynucleotide phosphorylase from Bacillus amyloliquefaciens.

Bacillus amyloliquefaciens BaM-2 produces large amounts of extracellular enzymes, and the synthesis of these proteins appears to be dependent upon abnormal ribonucleic acid metabolism. A polynucleotide phosphorylase (nucleoside diphosphate:polynucleotide nucleotidyl transferase) was identified, purified, and characterized from this strain. The purification scheme involved cell disruption, phase partitioning, differential (NH4)2SO4 solubilities, agarose gel filtration, and diethylaminoethyl-Sephadex chromatography. The purified enzyme demonstrated the reactions characteristic of polynucleotide phosphorylase: polymerization, phosphorolysis, and inorganic phosphate exchange with the beta-phosphate of a nucleotide diphosphate. The enzyme was apparently primer independent and required a divalent cation. The reactions for the synthesis of the homopolyribonucleotides, (A)n and (G)n, were optimized with respect to pH and divalent cation concentration. The enzyme is sensitive to inhibition by phosphate ion and heparin and is partially inhibited by rifamycin SV and synthetic polynucleotides.     

Paternal age and Down syndrome.

The frequency of Down syndrome (DS) in infants of older fathers has been examined in two sets of data. The effect of maternal age was controlled by single years of age. Lack of tight control has been an important weakness of other studies on this subject. Data obtained in metropolitan Atlanta by an intensive case-ascertainment program showed no overall excess of DS infants born to older fathers. Nor was there evidence of such an effect in recent birth certificate data made available by the National Center for Health Statistics. The Atlanta data suggest an increased number of DS infants born to older fathers who had children by women less than or equal to 34 years. However, there was a small deficiency of DS infants born to older fathers by women greater than or equal to 35 years. The possibility of a paternal-age effect remains open, but the available data suggest that, if it exists, it is quite small.     

Analysis of migratory behavior of neural crest and fibroblastic cells in embryonic tissues

The precise migration of neural crest cells is apparently controlled by their environment. We have examined whether the embryonic tissue spaces in which crest cells normally migrate are sufficient to account for the pattern of crest cell distribution and whether other migratory cells could also distribute themselves along these pathways. To this end, we grafted a variety of cell types into the initial crest cell migratory pathway in chicken embryos. These cell types included (a) undifferentiated neural crest cells isolated from cultured neural tubes, intact crest from cranial neural folds, and crest derivatives (pigment cells and spinal ganglia); (b) normal embryonic fibroblastic cells from somite, limb bud, lateral plate, and heart ventricle; and (c) a transformed fibroblastic cell line (Sarcoma 180). Crest cells or their derivatives grafted into the crest migratory pathway all distributed normally, although in contrast to the result when neural tubes were graftedin situ, fewer cells were observed in the epithelium and few or none were localized in the nascent spinal ganglia. Grafted quail somite cells contributed to normal somitic structures and did not migrate extensively in the chicken host. Other fibroblasts did not migrate along cranial or trunk crest pathways, or invade adjacent tissues, but remained intact at the graft site. Sarcoma 180 cells, however, distributed themselves along the normal trunk crest pathway. Cranial and trunk crest cells and crest derivatives grafted ectopically in the limb bud or somite also dispersed, and were found along the ventral migratory pathway. Fibroblastic cells grafted into ectopic sites again remained intact and did not invade host tissue. We conclude (1) that neural crest cells and their derivatives are highly motile and invasive in their normal pathway, as well as in unfamiliar embryonic environments; and (2) that the crest pathway does not act solely to direct neural crest cells, since at least one transformed cell can follow the crest migratory route.     

Chemical relaxation studies on the horse liver alcohol dehydrogenase system.

Chemical relaxation studies on the system horse liver alcohol dehydrogenase, nicotinamide adenine dinucleotide, and ethanol were conducted observing fluorescence changes between 400 and 500 nm. Temperature-jump experiments were performed at pH 6.5, 7.0, 8.0, and 9.0; concentration-jump experiments at pH 9.0. The reciprocal of the slowest relaxation time was found to be linearly dependent upon the enzyme concentration for relatively low enzyme concentrations, as predicted earlier. Use of the wide pH-range necessitated expression of the four apparent dissociation constants of the catalytic reaction cycle in terms of pH-independent constants. The system was described in terms of only one (or two) catalysis-linked protons not associated with the electron transfer. Protonic steps in a buffered system are in rapid equilibrium, too fast to be measured with the equipment available. Assuming only two of the four bimolecular reaction steps in the four-step cycle are fast compared to the remaining two, six cases may be considered with six expressions for the reciprocal of the slowest relaxation time. Comparison with the experimental data revealed that the bimolecular reaction steps governing the slowest relaxation time change with pH. Above the effective time resolution of the temperature-lump instrument with fluorescence detection (0.1 msec) only one other relaxation time was detectable and only at pH 9. This relaxation time, found to be independent of the concentration of all reactants within experimental error (r = 10 +/- 5 msec), is most likely due to an interconversion among ternary complexes.     

Fine structure of seminiferous tubules in antarctic seals

Summary The fine structure of seminiferous tubules from 5 crabeater, 2 leopard and 2 Ross seals showed that during the nonbreeding season the tubules were essentially similar in possessing spermatogenic and Sertoli cells. However, the tubules of leopard and Ross seals had more primary and secondary spermatocytes and spermatids than the crabeater seals. In general, the tubules were devoid of spermatozoa. The spermatids showed stages of maturation such as Golgi phase of acrosome formation, acrosomal cap formation and condensation of nuclei. Some spermatids degenerated in tubules. Both maturing and degenerating spermatids were closely associated with Sertoli cells. Junctional complexes with plaques of filaments were observed between Sertoli cells and the spermatogenic cells. Sertoli cells, irregular and polygonal, contained highly convoluted nuclei, strands of rough endoplasmic reticulum, smooth endoplasmic reticulum, Golgi complexes, small mitochondria, variable amounts of lipid droplets, lysosomes, lipofuscin granules and highly plicated plasma membranes. In brief, the spermatogenic activity had practically ceased in the testes and the animals probably secreted low levels of testosterone during the nonbreeding season.This research was supported in part by National Science Foundation Grants G.U. 30270 and G.U. 29829X from the Office of Polar Program and by NIH Grant 5 R01 AM11-376     

Glucocorticoid inhibition of fsh-induced estrogen production in cultured rat granulosa cells

The effects of glucocorticoids on the steroidogenesis of ovarian granulosa cells were investigated. Cortisol and dexamethasone inhibited the increase in aromatase activity induced by FSH in cultured rat granulosa cells. In the same cultures progesterone production was stimulated to a maximum of 167% of the control level. This differential effect of glucocorticoids on estrogen and progesterone production by the granulosa cells indicates that glucocorticoids exert specific inhibition of the induction of aromatase by FSH and do not cause a general suppression of granulosa cell activity. In contrast to their inhibition of the FSH induction of aromatase enzymes, glucocorticoids did not interfere with the activity of pre-existing aromatase enzymes. In granulosa cells containing fun aromatase activity, treatment with cortisol and dexamethasone did not inhibit aromatization of androstenedione to estrogens whereas two known aromatase inhibitors (dihydrotestosterone and 4-androstene-3, 6, 17-trione) were effective. These results indicate that the glucocorticoids exert a selective inhibition of the FSH-induction of aromatase activity in rat granulosa cells by a mechanism other than directly interfering with the aromatization reaction.     

Purification and properties of mammalian toxins from the venom of the Brazilian scorpion Tityus serrulatus Lutz and Mello

The water-soluble part of the dried venom from the scorpion, Tityus serrulatus Lutz and Mello (range, Southeastern Brazil), showed 16 polypeptide bands on polyacrylamide gel electrophoresis. This material exhibited toxic and hyaluronidase activity but no phospholipase, phosphodiesterase, protease, or fibrinolytic activity. Fractionation on glycinamide-treated Sephadex G-50 afforded three protein fractions, which were non-toxic, equitoxic, and three times more toxic than the water-soluble venom. Subsequent separation of the toxic fractions on carboxymethyl-cellulose with phosphate buffers furnished five toxic components, which were further purified on carboxymethyl-cellulose with a salt gradient in acetate buffer. Toxin γ, the major and most basic toxin, is a 62-residue protein that, unlike other scorpion toxins, contains methionine. Automated Edman degradation showed the amino-terminal sequence to be H-Lys-Glu-Gly-Tyr-Leu-Met-Asp-His-Glu-Gly-Cys-Lys-Leu-Ser-Cys-Phe-Ile-Arg-Pro-Ser-Gly-Tyr-Cys-Gly-Arg-Glu-Cys-Gly-Ile-. Toxin γ is the first example of a fifth structural type of mammalian toxin from scorpion venom. Its amino-terminal sequence shows greater homology with toxins similar to Centruroides suffusus suffusus toxin III and Androctonus australis toxin II than with toxins similar to A. australis toxin I or Bhutus occitanus tunetanus toxin I.     

Molecular analysis of rearrangements in human ribosomal RNA gene clones

Human placental DNA, enriched for ribosomal sequences, was cloned in the phage vector lambda Charon 16A. Recombinants containing 28S rDNA sequences were isolated, and all were found to have deletions in the insert and/or vector DNA. Electron microscopic analysis was used to map the deletions and provide evidence that unstable forms of the recombinants can revert to the original vector or undergo further rearrangements. Specific deletions are manifested as previously unreported plaque phenotypes.     

Maximum likelihood estimation of growth yields

This work is concerned with statistical methods to estimate yield and maintenance parameters associated with microbial growth. For a given dilution rate, an experimenter typically measures substrate concentration, oxygen utilization rate, the rate of carbon dioxide evolution, and biomass concentration. These correlated response variables each contain information about the maintenance and yield parameters of interest. A maximum likelihood estimator which combines this correlated information for the yield and maintenance parameters is proposed, evaluated, and tested on literature data. Both point and interval estimators are considered.