Solution conformation of a synthetic fragment of human pituitary growth hormone. Two-dimensional NMR of an alpha-helical dimer

Circular dichroism and two-dimensional NMR spectra indicate that a peptide fragment consisting of the first 28 residues from the N-terminus of human growth hormone (hGH 1-28) has considerable alpha-helical structure. The peptide, (1) H-Phe-Pro-Thr-Ile-Pro-Leu-Ser-Arg-Leu-Phe-Asp-Asn-Ala-Met-Leu-Arg-Ala-Hi s-Arg- Leu-His-Gln-Leu-Ala-Phe-Asp-Thr-Tyr-OH (28), was synthesized on an automated peptide synthesizer using the Merrifield solid-phase method. The peptide can be modeled as an amphiphilic helix, and the unusual stability of the alpha-helix in aqueous solution is suggested to be attributable to formation of a dimer of alpha-helices. Most of the 1H NMR signals were assigned through pure absorption phase COSY/NOESY and single- and double-relay COSY 2D NMR spectra by using the sequential assignment methodology. The NOEs were large and negative, suggesting that the peptide was not a random coil and that it existed in solution primarily as a large, fairly rigid macromolecule, consistent with the dimer structure. A network of N alpha Hi-N alpha Hi+1 NOESY crosspeaks is observed from residues 13 to 18 as are several other crosspeaks which indicate that the peptide has considerable alpha-helical structure between residues 8 and 24. In addition, gel filtration of the peptide is consistent with a dimer structure, presumably involving packing of the two hydrophobic faces of the amphiphilic alpha-helices.     

Cloning and expression of alpha-amylase from Bacillus amyloliquefaciens in a stable plasmid vector in Lactobacillus plantarum

Lactobacillus plantarum is used in a wide range of agricultural and food fermentations. In this paper we report the introduction of alpha-amylase into the organism from Bacillus amyloliquefaciens on a stable recombinant plasmid. The genetically manipulated organism grew on MRSB medium supplemented with starch and it may be a prototype for the development of lactobacilli able to use an increased range of substrates in commercial fermentations.     

Reversible cell damage by T-cell perforins. Calcium influx and propidium iodide uptake into K562 cells in the absence of lysis.

The non-lethal effects of the lymphocyte-derived pore-forming toxin perforin on the human erythroleukaemia cell line K562 were investigated. By using the fluorescent Ca2+ indicator fura-2, perforin was shown to cause intracellular Ca2+ concentration to rise transiently into the micromolar range in the absence of cell death. By fluorescence-activated cell sorting it was demonstrated that K562 cells took up the membrane-impermeant nuclear stain propidium iodide (PI) when exposed to non-lethal doses of perforin. The permeability to PI was short-lived, confirming the transience of the perforin pore. Analogies with non-lethal effects and recovery processes occurring in nucleated cells exposed to the membrane-attack complex of complement are drawn.     

Patterns of [13N]ammonium uptake and assimilation by Frankia HFPArl3

Nitrogen-starved cells of Frankia strain HFPArl3 incorporated [¹³N]-labeled ammonium into glutamine serine (glutamate, alanine, aspartate), after five-minute radioisotope exposures. High initial endogenous pools of glutamate were reduced, while total glutamine increased, during short term NH _inf4 ^sup+ incubation. Preincubation of cells in methionine sulfoximine (MSX) resulted in [¹³N]glutamine reduced by more than 80%, while [¹³N]glutamate and [¹³N]alanine levels increased. The results suggest that glutamine synthetase is the primary enzyme of ammonium assimilation, and that glutamate dehydrogenase and alanine dehydrogenase may also function in ammonium assimilation at low levels. Efflux of [¹³N]serine and lesser amounts of [¹³N]glutamine was detected from the Frankia cells. The identity of both Ser and Gln in the extracellular compartment was confirmed with gas chromatography/mass spectrometry. Serine efflux may be of significance in nitrogen transfer in Frankia.Abbreviations Pthr phosphothreonine - Aad -amino-adipate - MSX methionine sulfoximine     

Crystal and solution structures of the oligonucleotide d(ATGCGCAT)2: a combined X-ray and NMR study.

A combined crystal-structure determination and NMR analysis of the octanucleotide d(ATGCGCAT)2 is reported. The X-ray analysis shows that the structure is A-form duplex in crystal state. The NMR study shows that in solution this sequence is B-type. The conformational results from each technique are presented in detail. The implications of these findings in terms of conformational flexibility and ligand binding are discussed.     

Distribution of the stimulatory GTP-binding proteins, Gs and Golf, within olfactory neuroepithelium

Several lines of evidence suggest that, for certain odorants,olfactory signal transduction may be mediated by a stimulatoryG-protein coupled adenylate cyclase cascade. Two stimulatoryG-proteins, G_olf and G_s, are expressed in olfactory tissue.To evaluate their relative contributions to the process of odorantsignal transduction, specific antisera were used to determinethe distribution and relative abundance of G_olf and G_s in ratolfactory neuroepithelium and olfactory sensory cilia. Theseanalyses demonstrate that (1) Golf is far more abundant thanG_s in olfactory neuroepithelium and (2) G_olf is essentiallythe only stimulatory G-protein present in olfactory sensorycilia. ¹Present address: Gene Expression Laboratory, The Salk Institute,PO Box 85800, San Diego, CA 92138, USA     

Relationship of alcohol production to lipid composition of yeast in a continuous flow bioreactor

Saccharomyces cerevisiae was cultivated in a controlled aerated, dual-stage (column), continuous flow bioreactor in a hybrid free-cell and immobilized-cell state. The yeast cells maintained an ethanol concentration of 58-64 and 91-98 g/L in stages I and II, respectively. The lipid composition of the cells cultivated under these conditions was correlated to the effects of aeration by interrupting the aeration on days 113 and 266 of continuous operation. Under conditions of aeration or nonaeration, an alternating increase and decrease in the contents of squalene, sterols, and fatty acids of the respiratory-competent and -deficient unattached free cells was observed. The cellular free lipid compositions of the immobilized cells in the aerated and nonaerated conditions were similar and characteristic of respiratory-deficient cells with the exception of the immobilized cells exposed to a higher ethanol concentration (stage II). These cells contained a broader range of sterol components and increased levels of unsaturated fatty acids than immobilized cells at a lower ethanol concentration (stage I). The neutral lipid to phospholipid ratio decreased for respiratory-deficient cells with phosphatidylethanolamine and phosphatidylinositol being the principal phospholipids. The data demonstrated the essentiality of the hybrid bioreactor design for continuous long term performance and the importance of maintaining specific yeast lipid constituents for continuous high alcohol productivity.