Molecular cloning and sequencing of cDNA encoding goat pre alpha-lactalbumin

In poly(A)+RNA extracted from a lactating goat mammary gland, mRNA of about 750 nucleotides was shown to encode pre alpha-lactalbumin by using in vitro translation and immunoprecipitation. From the total poly(A)+RNA, the cDNA library was constructed using the Escherichia coli plasmid pUC18; it was screened with the oligodeoxyribonucleotide probe corresponding to the amino acid sequence of Trp60-Gln65 of goat alpha-lactalbumin. A plasmid containing almost full-length cDNA of goat pre alpha-lactalbumin, pGLA-1, was identified. The cDNA insert of pGLA-1 comprises 727 base pairs and contains the signal peptide and mature protein sequence.     

Immunohistochemical localization of plasma retinolbinding protein and prealbumin in human pancreatic islets

Summary This study was undertaken, employing the immunoenzyme method, to confirm the presence of retinol-binding protein in human pancreatic islets, and to compare its distribution with that of prealbumin, insulin, glucagon, somatostatin and pancreatic polypeptide. It was found that most islet cells contained retinol-binding protein, although centrally located cells showed stronger reactivity than those in the peripheral region. The distribution of each of the five polypeptides differed from that of retinolbinding protein, indicating that these peptides did not cross-react with anti-retinol-binding protein antibody. Islet cells which contained prealbumin, on the other hand, were mostly classified as A cells. Further studies are necessary to confirm whether the islet cells produce retinol-binding protein or only store it.     

Electron spin resonance studies on a flavoprotein in neutrophil plasma membranes. Redox potentials of the flavin and its participation in NADPH oxidase

Plasma membrane fractions of stimulated and resting cells were isolated from pig blood neutrophils. The midpoint redox potential (Em) of the membrane-bound flavin was determined potentiometrically by analysis of the flavin free-radical signal by electron spin resonance (ESR) spectroscopy. In both stimulated and resting cells, a peak position of the titration curve gave an Em value of -280 mV at pH 7.0 (Em7). The flavin free radical showed an ESR spectrum at g = 2.004 with a peak to peak width of 19 G, which indicates that the redox intermediate is a neutral semiquinone. Redox titrations were anaerobically examined at 25 degrees C with NADPH in place of dithionite. Addition of NADPH to plasma membranes of stimulated cells resulted in a rapid change in potential, accompanied by the formation of the ESR signal of flavin free radical. Computer simulation of the titration points gave an ambient midpoint potential of -280 mV (Em7). In contrast, those of resting cells showed a very slow change in potential and no g = 2.00 signal formation. Power saturation behavior of the ESR signal showed a marked difference between those of stimulated and resting cells. ESR characteristics of the flavin are discussed in relation to the membrane-bound NADPH oxidase.     

The Thermostable Direct Hemolysin Gene (tdh) of Vibrio hollisae Is Dissimilar in Prevalence to and Phylogenetically Distant from the tdh Genes of Other Vibrios: Implications in the Horizontal Transfer of the tdh Gene

Vibrio hollisae strains isolated recently from patients in various locations were examined for the presence of the thermostable direct hemolysin gene (tdh) using nucleic acid hybridization and polymerase chain reaction assays. The results were consistent with the previous finding that all strains of V. hollisae carry the tdh gene. In contrast, the tdh gene has been detected in a minority of strains for other Vibrio species (V. parahaemolyticus, V. cholerae non-O1, and V. mimicus). Detailed phylogenetic analysis showed that the tdh genes of the non-V. hollisae species were very closely related to each other and that the tdh gene of V. hollisae was distantly related to the tdh genes of the non-V. hollisae species. These results and the proposed insertion sequence-mediated tdh transfer mechanism suggest that the tdh gene may have been maintained stably in V. hollisae and that the tdh genes of the non-V. hollisae species may have been involved in recent horizontal transfer.     

High-resolution solution structure of siamycin II: Novel amphipathic character of a 21-residue peptide that inhibits HIV fusion

Summary The 21-amino acid peptides siamycin II (BMY-29303) and siamycin I (BMY-29304), derived from Streptomyces strains AA3891 and AA6532, respectively, have been found to inhibit HIV-1 fusion and viral replication in cell culture. The primary sequence of siamycin II is CLGIGSCNDFAGCGYAIVCFW. Siamycin I differs by only one amino acid; it has a valine residue at position 4. In both peptides, disulfide bonds link Cys¹ with Cys¹³ and Cys⁷ with Cys¹⁹, and the side chain of Asp⁹ forms an amide bond with the N-terminus. Siamycin II, when dissolved in a 50:50 mixture of DMSO and H₂O, yields NOESY spectra with exceptional numbers of cross peaks for a peptide of this size. We have used 335 NOE distance constraints and 13 dihedral angle constraints to generate an ensemble of 30 siamycin II structures; these have average backbone atom and all heavy atom rmsd values to the mean coordinates of 0.24 and 0.52 Å, respectively. The peptide displays an unusual wedge-shaped structure, with one face being predominantly hydrophobic and the other being predominantly hydrophilic. Chemical shift and NOE data show that the siamycin I structure is essentially identical to siamycin II. These peptides may act by preventing oligomerization of the HIV transmembrane glycoprotein gp41, or by interfering with interactions between gp41 and the envelope glycoprotein gp120, the cell membrane or membrane-bound proteins [Frèchet, D. et al. (1994) Biochemistry, 33, 42–50]. The amphipathic nature of siamycin II and siamycin I suggests that a polar (or apolar) site on the target protein may be masked by the apolar (or polar) face of the peptide upon peptide/protein complexation.Abbreviations ABNR adopted basis Newton Raphson - AIDS acquired immunodeficiency syndrome - CW continuous wave - DMSO dimethylsulfoxide - DQF-COSY two-dimensional double-quantum-filtered correlation spectroscopy - HIV human immunodeficiency virus - HSQC heteronuclear single-quantum coherence - NOE nuclear Overhauser enhancement - NOESY two-dimensional nuclear Overhauser enhancement spectroscopy - ppm parts per million - P.E.-COSY two-dimensional primitive exclusive correlation spectroscopy - REDAC redundant dihedral angle constraint - rf radio frequency - rmsd root-mean-square difference - SIV simian immunodeficiency virus - sw spectral width - _m mixing time - TOCSY two-dimensional total correlation spectroscopy - TSP trimethylsilyl-2,2,3,3-²H₄-propionate - 2D two-dimensional     

Self-Nonself Recognition Activity Extracted from Self-Sterile Eggs of the Ascidian, Ciona intestinalis

Eggs of the hermaphrodite, self-sterile ascidian, Ciona intestinalis , were washed with acid seawater (pH 3.2), and the washing solution was then adjusted to pH 8.2. This solution was found to inhibit only the binding of non-autologous sperm to the vitelline coat (VC) of eggs, indicating that it contained self-nonself recognition activity. This activity was heat-stable and insensitive to trypsin, but was destroyed by V-8 protease and α-glucosidase. Both the hydrophobic and hydrophilic components of a lyophilized powder of the extract showed allo-recognizing activity. On TLC, the hydrophobic components gave a major spot of glucose (Glc) and a peptide spot(s) containing mainly glutamic acid and/or glutamine (Glx). The glucosyl conjugate was purified by HPLC and shown to block sperm-egg binding to various extents. Individual peptide subfractions had no inhibitory activity, but in combination they showed inhibitory activity. These findings suggest that the acid extract of Ciona eggs contains a Glc-enriched nonspecific inhibitor of sperm-egg binding, which could be the primary effector of self-incompatibility, and Glx-enriched modulators, which serve as acceptors of allo-sperm. The cooperative interactions of these components may be responsible for the diversity of allo-recognition in Ciona gametes.