Purification and characterization of alpha 1-thiol proteinase inhibitor and its identity with kinin- and fragment 1.2-free high molecular weight kininogen

1. alpha 1-Thiol proteinase inhibitor (alpha 1 TPI) purified from outdated human plasma was a glycoprotein with Mr 83,000 and was composed of heavy and light chains held together with a disulfide bond. 2. The data on amino acid composition, amino terminal sequence of the light chain and carboxyl terminal sequences of the heavy and light chains indicate that alpha 1 TPI is identical with kinin- and fragment 1.2-free HMW kininogen. 3. Purified human plasmin generated a derivative having the same molecular weight (Mr 83,000), same subunit structure (heavy and light chains) and same inhibitory capacity as alpha 1 TPI from HMW kininogen and kinin-free HMW kininogen. This indicated the possibility that alpha 1 TPI is derived from HMW kininogen by plasmin.     

Genetic differentiation between laboratory lines of the musk shrew (Suncus murinus,insectivora) based on restriction endonuclease cleavage patterns of mitochondrial DNA

The cleavage patterns of mitochondrial DNA (mtDNA) by 17 restriction endonucleases were compared between eight lines of musk shrews derived from different wild-caught stocks. Enzymatic digestion byBamHI,PvuII,XbaI, andXhoI showed a cleavage pattern common to all lines that were from five Japanese islands (Nag, Ize, OKI, TKU, and Tr), Bangladesh (BAN), Sri Lanka (SRI), and Java (Bog), and every line lacked cleavage sites forSalI andSmaI. Different cleavage patterns were detected by the remaining 11 enzymes. Within the BAN line, the presence of at least two types of mtDNAs was proved by six enzymes and was not contradictory to the maternal pedigrees going up to the wild ancestors of the stock. More than 30 cleavage sites of the shrew mtDNA were mapped by double-digestion methods. Nucleotide diversities of mtDNA were calculated from these maps and were estimated to be less than 0.5% among Japanese and Bog lines but to be 3.8% between BAN and the other seven lines and 2.3% within the BAN line. These results indicate that BAN shrews differentiate from the other lines to the intersubspecific extent reported in mice previously.     

Effect of Bacteriophage Ghost Infection on Protein Synthesis in Escherichia coli

The rate of protein synthesis by Escherichia coli markedly decreased within 1 min after phage T4 infection, whereas a complete cessation of protein synthesis was observed within at least 25 sec after T4 ghost infection. The cellular level of amino acids and aminoacyl-transfer ribonucleic acid (tRNA) did not change drastically upon infection with ghosts, indicating that the inhibition of protein synthesis took place at a step(s) beyond aminoacyl-tRNA formation. The host messenger RNA remained intact and still bound to ribosomes shortly after ghost infection. Kinetic studies of the effect of ghosts on host protein synthesis revealed that nascent peptide chains on ribosomes were not released upon ghost infection.     

Enzymic alanylation of an isoxazolinone glucoside by legume seedling extracts

Enzymic synthesis of the natural product β-(2-β-d-glucopyranosyl-3-isoxazolin-5-on-4-yl) alanine is described, using the natural isoxazolinone glucoside and O-acetyl-l-serine as substrates and extracts from seedlings as enzyme preparations. Lathyrus odoratus extracts show a higher activity than those of Pisum sativum, Citrullus vulgaris and Leucaena leucocephala.     

(−)-N-ethylcytisine,a lupin alkaloid from the flowers of Echinosophora koreensis

A new lupin alkaloid, (?)-N-ethylcytisine, was isolated from the fresh flowers of Echinosophora koreensis. Its structure has been confirmed b     

A bromine compound isolated from human cerebrospinal fluid

A new bromine compound with properties characteristic of a ketone was isolated from human cerebrospinal fluid. It was degraded by alkali treatment producting acetic acid, glycoli acid, oxalic acid, HBr and 1-methylheptanol. By this treatment, a part of the compound was converted to di-1-methylheptyl-2,5-dioxocyclohexane-1,4-dicarboxylate. These degradation products were also obtained from synthetic 1-methylheptyl-γ-bromoacetoacetate by alkali treatment. 1-methylheptyl-γ-bromoacetoacetate and realted substances were synthesized and compared with the isolated bromine compound in chemical properties, infrared spectra, spectra of nuclear magnetic resonance, and elemental analysis, etc. These results showed that the isolated bromine compound corresponded well to 1-methylheptyl-γ-bromoacetoacetate (synonym of 2-octyl-γ-bromoacetoacetate).     

Derivatization of epoxy-activated agarose with various carbohydrates for the preparation of stable and high-capacity affinity adsorbents: Their use for affinity chromatography of carbohydrate-binding proteins

Two types of affinity adsorbents for lectins were prepared by new simple procedures. Both types of adsorbents had high ligand concentration and chemically stable linkage between ligand and Sepharose 4B. Oligosaccharide ligands were coupled by reductive amination with sodium cyanoborohydride to amino-Sepharose 4B prepared by amination of epoxy-activated Sepharose 4B. The glycamyl-Sepharose 4B thus obtained had particularly high adsorption capacities for lectins; lactamyl-Sepharose 4B, 58 mg/l ml of gel for peanut lectin; maltamyl-Sepharose 4B, 146 mg/ml for concanavalin A; and tetra-N-acetylchitotetraamyl-Sepharose 4B, 36 mg/ml for wheat germ agglutinin. Hexosamine was coupled by the aid of carbodiimide to carboxyl-Sepharose 4B prepared by succinylation of amino-Sepharose 4B. Galactosamine-Sepharose 4B adsorbed 145 mg soybean agglutinin/l ml gel. The columns turned from a semitransparent white to a milky white as they were saturated with lectins.     

Cloning of the blasticidin S deaminase gene (BSD) from Aspergillus terreus and its use as a selectable marker for Schizosaccharomyces pombe and Pyricularia oryzae

Aspergillus terreus produces a unique enzyme, blasticidin S deaminase, which catalyzes the deamination of blasticidin S (BS), and in consequence confers high resistance to the antibiotic. A cDNA clone derived from the structural gene for BS deaminase (BSD) was isolated by transforming Escherichia coli with an Aspergillus cDNA expression library and directly selecting for the ability to grow in the presence of the antibiotic. The complete nucleotide sequene of BSD was determined and proved to contain an open reading frame of 393 bp, encoding a polypeptide of 130 amino acids. Comparison of its nulceotide sequence with that of bsr, the BS deaminase gene isolated from Bacillus cereus, indicated no homology and a large difference in codon usage. The activity of BSD expressed in E. coli was easily quantified by an assay based on spectrophotometric recording. The BSD gene was placed in a shuttle vector for Schizosaccharomyces pombe, downstream of the SV40 early region promoter, and this allowed direct selection with BS at high frequency, following transformation into the yeast. The BSD gene was also employed as a selectable marker for Pyricularia oryzae, which could not be transformed to BS resistance by bsr. These results promise that the BSD gene will be useful as a new dominant selectable marker for eukaryotes.     

Mapping of the human C3G gene coding a guanine nucleotide releasing protein for Ras family to 9q34.3 by fluorescence in situ hybridization

C3G, a human guanine nucleotide releasing protein for Ras protein, was mapped to human chromosome 9q34.3 by fluorescence in situ hybridization with Rbanded chromosomes. C3G was originally identified as one of the CRK-binding proteins, similar to c-ab1 (9q34.1). Our result suggests that the downstream factors of Crk are localized in close proximity on chromosome 9.