Metabolism of Choline Chloride and Its Analogs in Wheat Seedlings

The incorporation rate of choline chloride and allylcholinebromide into wheat protoplasts were rapid compared with theincorporation rate of benzylcholine bromide. Choline chloridewas metabolized via two pathways: choline betaine and choline phosphorylcholine phos-phatidylcholine. Allylcholine bromidewas metabolized via only one pathway: allylcholine phosphorylallylcholine phosphatidylallylcholine, and benzylcholine bromide was notmetabolized at all. These results suggest that the stimulationof photosynthesis (Hyeon et al. 1988) by these compounds iscaused directly by these choline analogs and not by their metabolites. (Received June 29, 1989; Accepted October 20, 1989)     

Three members of the S multigene family are linked to the S locus of Brassica

Two self-incompatibility genes in Brassica, SLG and SRK (SLG encodes a glycoprotein; SRK encodes a receptor-like kinase), are included in the S multigene family. Products of members of the S multigene family have an SLG-like domain (S domain) in common, which may function as a receptor. In this study, three clustered members of the S multigene family, BcRK1, BcRL1 and BcSL1, were characterized. BcRK1 is a putative functional receptor kinase gene expressed in leaves, flower buds and stigmas, while BcRL1 and BcSL1 are considered to be pseudogenes because deletions causing frameshifts were identified in these sequences. Sequence and expression pattern of BcRK1 were most similar to those of the Arabidopsis receptor-like kinase gene ARK1, indicating that BcRK1 might have a function similar to that of ARK1, in processes such as cell expansion or plant growth. Interestingly, the region containing BcRK1, BcRL1 and BcSL1 is genetically linked to the S locus and the physical distance between SLG, SRK and the three S-related genes was estimated to be less than 610 kb. Thus the genes associated with self-incompatibility exist within a cluster of S-like genes in the genome of Brassica.     

Structure and characteristics of reassembled fluorescent protein, a new insight into the reassembly mechanisms

Bimolecular fluorescence complementation (BiFC) assay has been used widely to visualize protein-protein interactions in cells. However, there is a problem that fluorescent protein fragments have an ability to associate with each other independent of an interaction between proteins fused to the fragments. To facilitate the BiFC assay, we have attempted to determine the structure and characteristics of reassembled fluorescent protein, Venus. The anion-exchange chromatography showed an oligomer and a monomer of reassembled Venus. Our results suggested that the oligomer was formed by β-strands swapping without any serious steric clashes and was converted to the monomer. Crystal structure of reassembled Venus had an 11-stranded β-barrel fold, typical of GFP-derived fluorescent proteins. Based on the structural features, we have mutated to β-strand 7 and measured T_m values. The results have revealed that the mutation influences the thermal stability of reassembled fluorescent complex.     

NMR analyses of polysaccharide derivatives containing amine groups

Amylose, amylopectin, hydroxyethylcellulose, methylcellulose, and cellulose were reacted with diethylaminoethyl chloride HCl salt and 3-chloro-2-hydroxy-propyltrimethylammonium chloride under aqueous alkaline conditions in order to introduce tertiary amine and quaternary ammonium groups into polysaccharides. Degrees of substitution were obtained from ¹H- or ¹³C-NMR spectra of hydrolyzates, and distributions of diethylaminoethyl groups in polysaccharides were measured by ¹³C-NMR. Since amylose, amylopectin, and hydroxyethylcellulose were soluble in the reaction media, these three polysaccharides had higher reactivity for etherifications than cellulose. Methyl-cellulose, which has hydrophobic methyl groups, had as much reactivity as cellulose. Primary hydroxyl groups, C-6, of polysaccharides had the highest reactivity for diethylaminoethylation.     

Isolation of a mutant auxotrophic for L-alanine and identification of three major aminotransferases that synthesize L-alanine in Escherichia coli

For Escherichia coli, it has been assumed that L-alanine is synthesized by alanine-valine transaminase (AvtA) in conjunction with an unknown alanine aminotransferase(s). We isolated alanine auxotrophs from a prototrophic double mutant deficient in AvtA and YfbQ, a novel alanine aminotransferase, by chemical mutagenesis. A shotgun cloning experiment identified two genes, uncharacterized yfdZ and serC, that complemented the alanine auxotrophy. When the yfdZ- or serC-mutation was introduced into the double mutant, one triple mutant (avtA yfbQ yfdZ) showed alanine auxotrophy, and another (avtA yfbQ serC), prototrophy. In addition, we found that four independent alanine auxotrophs possessed a point mutation in yfdZ but not in serC. We also found that yfdZ expression was induced in minimal medium. Furthermore, yfbQ-bearing plasmid conferred the ability to excrete alanine on the mutant lacking D-amino acid dehydrogenase-encoding gene, dadA. From these results, we concluded that E. coli synthesizes L-alanine by means of three aminotransferases, YfbQ, YfdZ, and AvtA.     

Protective Effect of Japanese Green Tea Extract on Gnotobiotic Mice Infected with an Escherichia coli O157:H7 Strain

We examined the effect of Japanese green tea extract (JGTE) on enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection in a gnotobiotic mouse model. Gnotobiotic mice inoculated with an EHEC strain developed neurologic and systemic symptoms, usually culminating in death. In contrast, none of mice receiving dietary JGTE showed clinical signs or death. This report describes the effect of JGTE, which includes the inhibition of bacterial growth in vivo. The Shiga-like toxin (SLT) level in the feces of the JGTE diet group was significantly lower than that of the control group.     

Structure and expression of cDNA for D-amino acid oxidase active against cephalosporin C from Fusarium solani

D-Amino acid oxidase (DAO) was extracted and purified from cultured mycelia of Fusarium solani M-0718 (FERM P-2688). The enzyme was able to oxidatively deaminate cephalosporin C to 7-beta-(5-carboxy-5-oxopentanamido)cephalosporanic acid. Ninety-eight amino acid residues of the F. solani DAO were determined by sequence analysis of 9 peptides derived from Acromobacter protease I digests of the protein. Complementary DNAs encoding F. solani DAO were isolated from the F. solani cDNA library by hybridization with synthetic oligonucleotide probes corresponding to the partial amino acid sequences. Analysis of the nucleotide sequences of the clones revealed a 1,186-nucleotide sequence with a 5'-terminal untranslated region of 41 nucleotides, an open reading frame of 1,083 nucleotides that encoded 361 amino acids, and a 3'-terminal untranslated region of 62 nucleotides. The amino acid sequence of F. solani DAO had 25% homology to that of porcine kidney DAO [EC 1.4.3.3] and 37% homology to that of Trigonopsis variabilis DAO. The constructed plasmid overproduced F. solani DAO in Escherichia coli. The recombinant DAO had almost the same molecular activity as the native DAO against cephalosporin C.     

Structural elucidation of minor components of peptidyl antibiotic P168s (leucinostatins) by tandem mass spectrometry.

Tandem mass spectrometry with a four-sector type mass spectrometer was used to elucidate the structures of minor components of the peptidyl antibiotic P168s (leucinostatins). As N-terminal fragments, ions by B-type cleavage were dominant, while V-type cleavages were observed along with X, Y, and Z types as C-terminal ions. The V-type ions were predominant in the cleavages of the amino terminals of leucyl and hydroxyleucyl residues. The structures of several minor components could be deduced from the tandem mass spectra.     

Determination of disulfide bond arrangement in bombyxin-IV,an insulin superfamily peptide from the silkworm,Bombyx mori,by combination of thermolysin digestion of natural peptide and selective synthesis of disulfide bond isomers

The mode of disulfide linkages in bombyxin-IV, an insulin superfamily peptide consisting of A- and B-chains, was determined as A6–A11, A7–B10, and A20–B22. An intermolecular bond of A20–B22 was identified by sequencing and mass spectrometric analysis of the fragments generated by thermolysin digestion of natural bombyxin-IV. The mode of the remaining two bridges was determined by chemical and selective synthesis of three possible disulfide bond isomers of bombyxin-IV. A- and B-chains were synthesized by solid-phase method, and three disulfide bonds were bridged stepwise and in a fully controlled manner. Retention time on reversed-phase high-performance liquid chromatography (HPLC), thermolysin digests, and biological activity of the synthetic [A6–A11, A7–B10, A20–B22-cystine]-bombyxin-IV revealed that it was identical with the natural bombyxin-IV. Two other isomers with respect to disulfide bond arrangement, [A6–A7, A11–B10, A20–B22-cystine]- and [A6–B10, A7–A11, A20–B22-cystine]-bombyxin-IVs, were distinguishable from the natural one by use of HPLC, thermolysin digestion, and bioassay.