Reassortant rotaviruses containing structural proteins vp3 and vp7 from different parents induce antibodies protective against each parental serotype.

Genetic studies of reassortant rotaviruses have demonstrated that gene segments 4 and 9 each segregate with the serotype-specific neutralization phenotype in vitro. Reassortant rotaviruses derived by coinfection of MA-104 cells with the simian strain SA11 and the antigenically distinct bovine strain NCDV were used to determine which viral genes coded for proteins which induced a protective immune response in vivo. In addition, reassortant rotaviruses containing only the gene segment 4 or 9 protein products (vp3 and vp7, respectively) from SA11 or NCDV were used to determine the serotypic specificities of both vp3 and vp7 in several mammalian rotavirus strains. vp3 and vp7 from the murine strain Eb were shown to be indistinguishable from the corresponding proteins from strain SA11. Adult mice orally inoculated with strain Eb developed neutralizing antibodies to both vp3 and vp7. The two naturally occurring bovine rotavirus strains NCDV and UK were shown to contain antigenically similar vp7 but distinct vp3 proteins. Mouse dams orally immunized with a reassortant virus containing only gene 9 from NCDV passively protected their progeny against UK challenge, whereas mouse dams orally immunized with a reassortant virus containing only gene 4 from NCDV did not. Finally, we constructed reassortant viruses that immunized against rotaviruses of two distinct serotypes. SA11 X NCDV reassortants that contained vp3 and vp7 from different parents induced a protective immune response against both parental serotypes. vp3 and vp7 were independently capable of inducing a protective immune response after oral immunization. An understanding of the serotypic specificities of both vp3 and vp7 of human rotavirus isolates will be necessary for the development of successful strategies to protect infants against severe rotavirus infections.     

Glutathione in Escherichia coli is dispensable for resistance to H2O2 and gamma radiation.

Escherichia coli devoid of glutathione (because of transposon insertions in the gshA gene) has normal resistance to H2O2, cumene hydroperoxide, heat, or ionizing radiation. Intracellular glutathione thus does not protect E. coli from such lethal oxidative damage. The use of gshA::Tn10 mutants also revealed a glutathione-independent, H2O2-inducible resistance to N-ethylmaleimide.     

Structural studies on a family of cAMP-binding proteins in the nervous system of Aplysia

Five major cAMP-binding proteins that differ in size and charge have been identified in neurons of Aplysia californica by photoaffinity labeling with [32P]8-N3cAMP. These proteins, which we believe are regulatory subunits of cAMP-dependent protein kinase, all differ from the major cAMP-binding protein of buccal muscle. We have compared the structures of these proteins by peptide mapping after chemical and proteolytic cleavage. These analyses indicate that the five binding proteins from nervous tissue and the major muscle protein are closely related to each other. For example, the three neuronal proteins that are most alike and the cAMP-binding protein from muscle have a similar, if not identical, Mr 20,000 domain that contains the 8-N3cAMP-binding site; beyond this domain they diverge. All six proteins appear to belong to a family in which homologous regions have been conserved to maintain common functions. We suggest that the regions of the molecules that differ mediate special functions such as ticketing to particular compartments of the cell. Evidence for regional assortment of the cAMP-dependent protein kinases according to structural type was afforded by subcellular fractionation of Aplysia nervous tissue; photoaffinity labeling of cytoplasm, cytoskeleton, and membrane fractions demonstrated a differential distribution of the five neuronal cAMP-binding proteins. Selective phosphorylation of specific substrates could be a consequence of the compartmentation of diverse cAMP-dependent kinases.     

Preparative purification of Escherichia coli heat-stable enterotoxin

Heat-stable enterotoxin (STa) isolated from bovine Escherichia coli strains was purified to homogeneity by growing the bacterial strains in a chemically defined medium, desalting, and concentrating the culture filtrate by batch adsorption chromatography on Amberlite XAD-2 resin, batch adsorption chromatography on reversed-phase silica, and preparative reversed-phase high-performance liquid chromatography. This rapid preparative purification scheme gave high recovery yields of pure STa which exhibited biochemical homology to STa purified by more complicated procedures.     

Isolation and characterization of a factor from calf serum that promotes the pigmentation of embryonic and transformed melanocytes

A protein (Mr = 63,000) from calf serum that promotes the pigmentation of cultured chick neural crest and mouse melanoma cells has been partially isolated and characterized in this study. The stimulation of melanin synthesis in cultured cells was used to follow its activity during purification. The pigment-promoting factor was isolated by sequential column chromatography on dye-agarose matrices followed by hydroxyapatite and high pressure molecular sieve chromatography. The factor was found to stimulate melanin biosynthesis at 2-4 micrograms/ml and was specific for melanin-producing cells and their precursors. Antibodies raised in rabbits against the factor inhibited its pigment-promoting activity as well as that of whole calf serum. Enzyme-linked immunoadsorbent assays demonstrated that calf and bovine sera contain molecules that cross-react with the pigment-promoting factor. Horse, human, rat, and chicken sera, which lack the biological activity, also lacked immunological cross-reactivity. Extracts of certain tissues, particularly the submaxillary gland, were observed to be rich sources of pigment-promoting activity.     

Diffusion of autoinducer is involved in regulation of the Vibrio fischeri luminescence system.

The enzymes for luminescence in Vibrio fischeri are induced by the accumulation of a species-specific metabolite (autoinducer) in the culture medium. Tritium-labeled autoinducer was used to study the mechanism of autoinduction. When 3H-autoinducer was added to suspensions of V. fischeri or Escherichia coli, cellular concentrations equaled external concentrations. For V. fischeri, equilibration of 3H-autoinducer was rapid (within 20 s), and greater than 90% of the cellular tritium remained in unmodified autoinducer. When V. fischeri or E. coli cells containing 3H-autoinducer were transferred to autoinducer-free buffer, 85 to 99.5% of the radiotracer escaped from the cells, depending on the strain. Concentrations of autoinducer as low as 10 nM, which is equivalent to 1 or 2 molecules per cell, were sufficient for induction, and the maximal response to autoinducer occurred at about 200 nM. If external autoinducer concentrations were decreased to below 10 nM after induction had commenced, the induction response did not continue. Based on this study, a model for autoinduction is described wherein autoinducer association with cells is by simple diffusion and binding of autoinducer to its active site is reversible.     

Cellular systems for the study of the biosynthesis of polyamines and ethylene,as well as of virus multiplication

Leaves of Chinese cabbage from healthy plants or from those infected with turnip yellow mosaic virus yield protoplasts which convert methionine to protein, S-adenosylmethionine, decarboxylated S-adenosylmethionine, spermidine, spermine and 1-aminocyclopropane-1-carboxylate. The enzyme spermidine synthase is entirely cytosolic and has been purified extensively. An inhibitor of this enzyme, dicyclohexylamine, blocks spermidine synthesis in intact protoplasts, and in so doing stimulates spermine synthesis. Aminoethoxyvinylglycine blocks the conversion of S-adenosylmethionine to 1-aminocyclopropane-1-carboxylate, the precursor to ethylene, in protoplasts. This inhibitor markedly stimulates the synthesis of both spermidine and spermine. Essentially all the protoplasts obtained from new leaves of plants infected 7 days earlier are infected. On incubation, such protoplasts convert exogenous methionine to viral protein and viral spermidine whose specific radioactivity is twice that of total cell spermidine. Exogeneous spermidine is also converted to cell putrescine and viral spermidine and spermine. Normal and virus-infected cells are being studied for their content of phenolic acid amides of the polyamines.     

Photochemical reactions in interstellar grains photolysis of co,NH3, and H2O

A simulation of the organic layer accreted onto interstellar dust particles was prepared by slow deposition of a CO:NH₃:H₂O gas mixture on an Al block at 10K, with concomitant irradiation with vacuum UV. The residues were analyzed by GC-MS, HPLC, and near IR; a reaction pathway leading from NH₃ to complex alcohol, fatty acid, and amide products in 27 stages is postulated. The astronomical relevance and significance of the observations are discussed.     

Interaction of vasoactive intestinal peptide with mouse lymphocytes: specific binding and the modulation of mitogen responses

Binding of radioiodinated vasoactive intestinal peptide (VIP) to mouse lymphocytes has been investigated. Specific cell binding of 125I-VIP was demonstrated with lymphocytes from mesenteric lymph nodes, subcutaneous lymph nodes, spleen, and Peyer's patches. The binding of VIP by these cells was accounted for by VIP binding sites upon T cells rather than non-T cells. In the presence of VIP, the in vitro response of lymphocytes to the T cell mitogens concanavalin A (Con A) and phytohemagglutinin (PHA) was inhibited in a dose-dependent fashion, whereas that to the B cell mitogen lipopolysaccharide (LPS) was not. There was a close correlation between the potency of VIP and some structurally related peptides for inhibition of 125I-VIP binding and the effect of those peptides on T cell mitogen responses. These observations demonstrate that mouse T lymphocytes have specific VIP receptors and that VIP can modulate the response of T cells to mitogenic stimulation. VIP may be an important immunoregulatory molecule, and may be implicated in the regulation of T cell function in mucosal tissues innervated by VIP-containing neurons.     

Ethanol and the γ-Aminobutyric Acid-Benzodiazepine Receptor Complex

Abstract: Ethanol appears to enhance γ-aminobutyric acid (GABA)-mediated synaptic transmission. Using radioligand binding techniques, we investigated the possibility that the GABA-benzodiazepine receptor complex is the site responsible for this effect. Ethanol at concentrations up to 100 m M failed to alter binding of [³H]flunitrazepam (FNZ), [³H]Ro 15-1788, or [³H]methyl-γ-carboline-3-carboxylate (MBCC) to benzodiazepine receptors, or of [³H]muscimol to GABA receptors in rat brain membranes. Scatchard analyses of the binding of these radioligands at 4°C and 37°C revealed no significant effects of 100 m M ethanol on receptor affinity or number. A variety of drugs as well as chloride ion increased binding of [³H]FNZ and/or [³H]muscimol, but these influences were not modified by ethanol. These findings indicate that ethanol probably potentiates GABAergic neurotransmission at a signal transduction site beyond the GABA-benzodiazepine receptor complex.