Synthesis and conformational studies of 2-beta-chloro, 1-alpha-fluoro, and 2-beta-fluoro derivatives of 2-deoxy-N-acetyl-neuraminic acid

5-Acetamido-4,7,8,9-tetra-O-acetyl-2,3,5-trideoxy-2-fluoro-D-glycero-alp ha- and -beta-D- galacto -2- nonulosonic acid methyl esters and the beta-chloro analog were synthesized from N-acetylneuraminic acid. Their 1H- and 13C-n.m.r.spectra were completely assigned by using single-frequency decoupling, off-resonance decoupling, and spin-simulation programs. Bond angles estimated from the 1H coupling-constants indicate that all of the compounds adopt the 2C5 (L) conformation with minor conformational differences in the C3 side chain. 5-Acetamido-2,3,5,-tri-deoxy-2-fluoro-D-glycero-alpha- and -beta-D- galacto -2- nonulosonic acid and their methyl esters were also prepared.     

Nitrate reductase,nitrite reductase activities and leaf diffusive resistance in wheat under water deficit

The flag and second leaves of wheat showed physiological and biochemical differences in the normal and water stressed plants. Although the flag leaf had lower enzyme activities in the control plants, in stressed plants it could continue nitrate assimilation better than the second and other leaves. The flag leaf also exhibited a higher tendency to resume normal metabolic activity after the release of stress as is indicated by higher NRA, NiRa, lower r_ad and r_ab.     

Phase separation of the receptor for immunoglobulin E and its subunits in Triton X-114

Above its critical micelle concentration, Triton X-114 in solution forms two phases at room temperature: a lower phase containing supramicellar aggregates and an upper phase largely depleted of detergent. This property of the detergent is potentially useful for separating under mild conditions proteins that bind detergent from those that do not (Bordier, C. (1981) J. Biol. Chem. 256, 1604-1607). We studied the distribution of the receptor for immunoglobulin E (IgE) and its subunits in the two phases. IgE and IgE complexed either with intact receptors or with the alpha chains of the receptor alone are principally partitioned into the upper phase, whereas the unliganded receptor as well as the isolated alpha, and especially the beta and gamma chains of the receptor, preferentially partition into the lower detergent phase. Chromatography of IgE and of the subunits of the receptor on a hydrophobic support showed that the beta and gamma chains have a considerably greater hydrophobic surface than the alpha chains or IgE. These results indicate that the distribution of a protein in the two phases of phase-separated Triton X-114 is not an all-or-none effect based upon whether it binds detergent or not. Rather, it reflects the overall balance between the hydrophobic and hydrophilic properties of the protein's surface.     

Demonstration of bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes by monoclonal antibodies

Calmodulin-dependent cyclic nucleotide phosphodiesterase from bovine brain is found to be composed of two distinct subunits, 60,000- and 63,000-dalton polypeptides. Peptide mapping of the subunits by partial proteolysis demonstrated that the 60-kDa polypeptide is not derived from the 63-kDa species. The interaction of the enzyme with three monoclonal antibodies, A6, C1, and A2, and the analysis of immunocomplexes by sucrose density gradient centrifugation revealed that calmodulin-dependent cyclic nucleotide phosphodiesterase exists in three different forms, i.e. (a) homodiamer of 60-kDa, (b) heterodimer of 60- and 63-kDa, and (c) homodimer of 63-kDa. A6 antibody reacts with both 60- and 63-kDa polypeptides indicating that they are immunologically related. C1 and A2 antibodies react with only 60-kDa polypeptide species. By using C1 Sepharose 4B affinity column chromatography, the 63-kDa homodimer which did not bind to the column (Fraction I) was separated from the 60-kDa polypeptide containing isozymes (the heterodimer and the 60-kDa homodimer) which were retained on the column and later eluted as a mixture (Fraction II). Fraction I, the 63-kDa homodimer enzyme, has higher Vmax toward cGMP as substrate than cAMP whereas the opposite was found with Fraction II. The specific activity of Fraction II enzyme toward cAMP was approximately 500 mumol/min/mg, the highest value ever reported for brain calmodulin-dependent cyclic nucleotide phosphodiesterase preparations.     

Production,morphology, and cytogenetic analysis of Elymus caninus (Agropyron caninum) x Triticum aestivum F1 hybrids and backcross-1 derivatives

Summary Intergeneric hybrids were produced between common wheat, Triticum aestivum (2n=6x=42, AABBDD) and wheatgrass, Etymus caninus (Agropyron caninum) (2n=4x=28, SSHH) — the first successful report of this cross. Reciprocal crosses and genotypes differed for percent seed set, seed development and F₁ hybrid plant production. With E. caninus as the pollen parent, there was no hybrid seed set. In the reciprocal cross, seed set was 23.1–25.4% depending upon wheat genotype used. Hybrid plants were produced only by rescuing embryos 12–13 days post pollination with cv Chinese Spring as the wheat parent. Kinetin in the medium facilitated embryo germination but inhibited root development and seedling growth. The hybrids were vigorous, self sterile, and intermediate between parents. These had expected chromosome number (2n=5x=35, ABDSH), very little chromosome pairing (0.51 II, 0.04 III) and some secondary associations. The hybrids were successfully backcrossed with wheat. Chromosome number in the BC₁ derivatives varied 54–58 with 56 as the modal class. The BC₁ derivatives showed unusually high number of rod bivalents or reduced pairing of wheat homologues. These were sterile and BC₂ seed was produced using wheat pollen.     

Isolation of the cDNA for human prostaglandin H synthase

Prostaglandin H Synthase (PGHS, cyclooxygenase) is a 67 kd protein which catalyzes the first step in prostaglandin synthesis. The primary amino acid sequence and the molecular mechanisms regulating expression are unknown. We report here isolation of a cDNA clone for the enzyme from human vascular endothelial cells for use in such studies. High titre, polyclonal antiserum against PGHS was developed in rabbits. The antiserum was monospecific, reacted with cyclooxygenase on Western blots at a limiting dilution of 1:500,000 and immunoprecipitated cyclooxygenase synthesized by in vitro translation of PGHS messenger RNA. It was used to screen a lambda gt11 cDNA expression library from human endothelial cells. Three positive clones were isolated. Following plaque purification, one clone reacted strongly with two other polyclonal antisera independently raised against highly purified cyclooxygenase and the aspirin-acetylated enzyme. Western blot analysis confirmed production of a large approximately 180 kd fusion protein of cyclooxygenase and beta-galactosidase. The cDNA insert of approximately 2.2 kilo base pairs was excised and subcloned into plasmid pUC8. A 24 nucleotide DNA probe, synthesized according to the amino acid sequence of the aspirin-acetylation site of cyclooxygenase, hybridized strongly with the 2.2 kbp cDNA insert. It is concluded that the 2.2 kbp cDNA insert represents a cDNA clone for human cyclooxygenase, which also expresses the aspirin-acetylation site. This is the first reported isolation of the cDNA for this enzyme, and will facilitate further studies on the primary sequence and on the regulation of the enzyme at the molecular level.     

Solvation properties of ubiquinone-10 in solvents of different polarity

The solvation properties of ubiquinone-10 and ubiquinol-10 in a wide variety of solvents of polarity varying from alkanes to water are reported. Greatest solubility is observed in solvents of intermediate polarity and particularly where low polarity is combined with a pronounced tendency to interact with the benzoquinone substituent of the ubiquinone molecule. This includes solvents like chloroform and benzene. Ubiquinone-10 is somewhat less polar than ubiquinol-10 as judged by comparative solubilities of the two molecules. Proton-NMR chemical shift measurements and aggregation studies in selected solvents indicate that in ubiquinone-10 in the liquid phase and in solution in hydrocarbons like dodecane the molecules have a preferred association possibly involving stacking of the benzoquinone rings. Surface balance studies indicated that the surface-active character of ubiquinone-10 is relatively weak and only in a comparatively polar and highly structured solvent, formamide, was there evidence of an effect on surface tension of the solvent. The critical micelle concentratiom in this solvent was estimated to be about 5 M on the basis of surface tension measurements. Ubiquinone-10 is well known to form virtually insoluble monolayers at the air/water interface. Studies of the partition of ubiquinone-10 in binary mixtures of solvents suggest that the interaction of the benzoquinone ring substituent with structured polar solvents is considerably weaker than the internal cohesion between molecules of the solvent. No evidence on the basis of wide-angle X-ray diffraction measurements was obtained to indicate that solvent molecules were a component of the crystal lattice of ubiquinone-10 that had precipitated from solvent mixtures.     

Effects of the application of chemical additives to desiccated flax on retting

Summary Application of chemical additives to alter the rate of retting of desiccated flax stems was successful. Treatment with ethylenediaminetetra-acetic acid (EDTA) and urea increased the rate of retting. Increases, in the population of fungal colonisers were observed on urea-treated stems but not after EDTA treatment. Enhanced PG activities were detected in stems treated with EDTA and maimum PL and xylanase activities were detected in stems treated with urea. Urea treated stems produced relatively finer fibres compared to fibres from EDTA treated stems of controls.