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     

Bacterial Plasmids in Antarctic Natural Microbial Assemblages

Samples of psychrophilic and psychrotrophic bacteria were collected from sea ice, seawater, sediments, and benthic or ice-associated animals in McMurdo Sound, Antarctica. A total of 155 strains were isolated and tested for the presence of plasmids by DNA agarose gel electrophoresis. Thirty-one percent of the isolates carried at least one kind of plasmid. Bacterial isolates taken from sediments showed the highest plasmid incidence (42%), and isolates from seawater showed the lowest plasmid incidence (20%). Plasmids were significantly more frequent in the strains which had been first isolated from low-nutrient media (46%) than in the strains which had been isolated from high-nutrient media (25%). Multiple forms of plasmids were observed in two-thirds of the plasmid-carrying strains. A majority of the plasmids detected were estimated to have a mass of 10 megadaltons or less. Among 48 plasmid-carrying strains, 7 showed antibiotic resistance. It is concluded that bacterial plasmids are ubiquitous in natural microbial assemblages of the pristine marine ecosystem of Antarctica.     

Effects of taurine and γ-aminobutyric acid on akinesia and analgesia induced by D-Ala2-Met-enkephalinamide in rats

Effects of taurine or γ-aminobutyric acid (GABA) on akinesia and analgesia induced by D-Ala²-Met-enkephalinamide were investigated in rats. Administration of taurine (dose range: 2.375×10^?2 M–9.5×10^?2 M/10 μl) into the left lateral ventricle 10 min prior to the injection of D-Ala²-Met-enkephalinamide (50 μg/10 μl) produced a dose-dependent reduction in the duration of akinesia and to some extent of analgesia, as estimated at 30 min and 60 min following the enkephalinamide injection; at the first estimation-time (10 min), taurine did not alter the duration of akinesia or that of analgesia. The median effective dose (ED₅₀) for akinesia determined at 60 min after D-Ala²-Met-enkephalinamide was 5 times greater and that for analgesia assessed at the same time was 1.7 times greater in taurine-treated rats than the respective doses in control animals. Administration of GABA under similar experimental conditions produced a dose-dependent reduction in the duration of analgesia from the initial estimation time (10 min) following the injection of D-Ala²-Met-enkephalinamide. The ED₅₀ for analgesia determined at 30 min after D-Ala²-Met-enkephalinamide was 3 times greater in GABA-treated rats than in control animals. Unlike the effects of taurine, GABA did not alter the duration of akinesia. Neither the duration of akinesia nor that of analgesia was modified by taurine or GABA alone in rats tested 9 min after the injection of each amino acid. These findings suggest that taurine may promote a recovery from both akinesia and analgesia, while GABA decreases only the analgesia induced by D-Ala²-Met-enkephalinamide.     

Growth-related changes in color and sex inHalichoeres poecilopterus

Coloration and sex change were studied in a temperate wrasseHalichoeres poecilopterus in the central part of the Seto Inland Sea, Japan. 1,270 examples, 45–179 mm SL, were collected from May to December both in 1983 and 1984. The species is a diandric, protogynous hermaphrodite, and has three color patterns: pale color type (A), brilliant color type (B) and intermediate color type (AB). A-fish were less than 142 mm SL and consisted of primary males (42.6%), females (55.4%), secondary males (0.3%) and fish with transitional gonads (1.7%). A-females changed their color to B, through AB, in the size range 101–131 mm SL. A-primary males changed their color to B, through AB, in the size range 103–134 mm SL. B-fish consisted of primary males (38.6%), secondary males (54.6%) and fish with transitional gonads (6.8%). The majority of females changed their sex to male in the size range 98–131 mm SL.     

Verbascoside production by plant cell cultures

Verbascoside was found to be produced in all calli derived from eleven species that contained the compound in their leaves. Cell suspension cultures were also established in three species, i.e., Leucosceptrum japonicum f. barbinerve, Syringa josikaea, and Sy. vulgaris, all of which were found to produce verbascoside at more than 1 g/l. Of the three species, suspension cultures of L. japonicum f. barbinerve showed rapid growth and the highest yield of verbascoside (1.89 g/l). In these cultures, the effects of major salt concentration in B5 medium on cell growth and verbascoside production were examined. Maximum cell growth and maximum verbascoside production were both achieved by reducing the major salt concentration to half that of the original medium.     

Agrobacterium-mediated transformation and regeneration of kiwi fruit

Summary Genetically transformed kiwi fruit (Actinidia deliciosa) plants were obtained from hypocotyl and stem segments co-cultured with Agrobacterium tumefaciens strain EHA101 harboring a binary vector, pLAN411 or pLAN421, which contained the neomycin phosphotransferase II (nptII) gene and the -glucuronidase (GUS) gene. After co-culturing with the A. tumefaciens, the hypocotyl or stem segments were cultured on a selection medium containing 25g/ml kanamycin and 500g/ml Claforan. After one month in culture, shoots had regenerated from the cuttings. Green shoots were analyzed for NPTII activity and GUS activity. Eighty-five percent of the green shoots examined expressed the nptII and GUS genes. GUS histochemical assays revealed strong GUS expression in guard cells, mesophyll cells, and trichomes.     

Purification and Characterization of the Egg Jelly Macromolecules, Sialoglycoprotein and Fucose Sulfate Glycoconjugate, of the Sea Urchin Hemicentrotus Pulcherrimus

A sialoglycoprotein and a fucose sulfate glycoconjugate (FSG) were purified from egg jelly of the sea urchin Hemicentrotus pulcherrimus . Sialoglycoprotein which consisted of sialic acid (90%, w/w) and protein (10%, w/w) did not cause induction of the acrosome reaction and sperm isoagglutination. FSG which contained one mol sulfate/mol fucose possessed 2.0 times protein to fucose by weight. The proteins in intact FSG were separated to two major (258 kDa and 237 kDa) and one minor (120 kDa) proteins by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of 2-mercaptoethanol (2-ME) while the proteins could not be separated by HPLC in the presence of 0.1% SDS or SDS-PAGE without 2-ME. However, after carboxymethylation of FSG, two major (260 kDa and 240 kDa) proteins and two minor (140 kDa and 135 kDa) proteins were separated from the fucose sulfate moiety by HPLC in the presence of 0.1% SDS or SDS-PAGE without 2-ME. When FSG was first carboxymethylated with non-radioactive iodoacetic acid and then reduced with 2-ME and finally carboxymethylated with ¹⁴C-iodoacetic acid, the most of radioactivity was detected in 140 kDa and 135 kDa proteins. Carboxymethylted-FSG was less potent than intact FSG in induction of the acrosome reaction. Fucoidan, a fucose sulfate polymer, did not induce the acrosome reaction.     

Effects of pantolactone and butyrolaectone on the pleiotropic phenotypes of lon mutants and on thermal induction of the SOS phenomena in a tif mutant of Escherichia coli K12

Hydrophobic and charge-charge interactions of Salmonella typhimirium and Serratia marcescens were determined and related to their content of fimbriae and lipopolysaccharide (LPS). The cell surface structures were characterized with hydrophobic interaction chromatography (HIC), electrostatic interaction chromatography (ESIC) and particle electrophoresis measurements. The degree of interaction at the air-water interface was tested using a monolayered lipid film applied to an aqueous surface. The cell surface hydrophobicity of S. typhimurium in the presence of fimbriae was less in smooth than in rought bacteria. Examination of a series of rough mutants of S. typhimurium indicates that reduction of the O-side chain and core oligosaccharides was correlated with increased cell hydrophobicity. The enrichment factors at the air-water interface were significantly higher for fimbriated than for non-fimbriated S. typhimurium cells. Fimbriated S. marcescens cells were less hydrophobic and adhered to a lesser degree at the air-water surface than non-fimbriated counterparts. Electrophoretic measurements and adsorption to ion exchangers gives different information about the surface charge of bacteria. The latter technique gives the interaction between localized charged surfaces.Abbreviations HIC hydrophobic interaction chromatography - ESIC electrostatic interaction chromatography - LPS lipopolysaccharide - PBS phosphate buffered saline solution     

Antioxidative effect of α-tocopherol incorporation into lecithin liposomes on ascorbic acid-Fe2+-induced lipid peroxidation

The antioxidative effect of α-tocopherol incorporated into lecithin liposomes was studied. Lipid peroxidation of liposome membranes, assayed as malondialdehyde production, was catalyzed by ascorbic acid and Fe²⁺. The peroxidation reaction, which did not involve the formation of singlet oxygen, superoxide, hydrogen peroxide, or a hydroxyl radical, was inhibited by α-tocopherol and a model compound of α-tocopherol, 2,2,5,7,8-pentamethyl-6-hydroxy-chroman (TMC), but not by phytol, α-tocopherylquinone, or α-tocopheryl acetate. One mole of α-tocopherol completely prevented peroxidation of about 100 moles of polyunsaturated fatty acid. Decrease in membrane fluidity by lipid peroxidation, estimated as increase of fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) embedded in the membrane, was also inhibited by α-tocopherol and TMC, reflecting their antioxidant functions. Cholesterol did not act as an antioxidant, even when incorporated in large amount into the liposome membranes, but it increased the antioxidative efficiency of α-tocopherol. When a mixture of liposomes with and without α-tocopherol was incubated with Fe²⁺ and ascorbic acid, α-tocopherol did not protect the liposomes not containing α-tocopherol from peroxidation. However, preincubation of the mixture, or addition of Triton X-100 allowed the α-tocopherol to prevent peroxidation of the liposomes not containing α-tocopherol. In contrast, in similar experiments, liposomes containing TMC prevented peroxidation of those without TMC without preincubation. Tocopherol in an amount so small as to exhibit only a slight antioxidative effect was oxidized when incorporated in egg lecithin liposomes, but it mostly remained unoxidized when incorporated in dipalmitoyllecithin liposomes, indicating that oxygen activated by ascorbic acid-Fe²⁺ does not oxidize α-tocopherol directly. Thus, decomposition of α-tocopherol may be caused by its interaction with peroxy and/or alkoxyl radicals generated in the process of lipid peroxidation catalyzed by Fe²⁺ and ascorbic acid.     

Oligouridylates as a template for nonenzymatic synthesis of oligoadenylates

Summary Chemical polymerization of adenosine-5-phosphorimidazolide was conducted in the presence of oligouridylate templates. Oligo U with chain length more than eight served as a template and facilitated oligoadenylate formation. No template activity was observed when oligo U up to a hexamer was used. These results correlate with thermal transition temperatures of oligo U-pA complexes.