Molecular cloning of a cDNA for rat liver monoamine oxidase B

The cDNA for rat monoamine oxidase B mRNA was isolated from liver cDNA library in lambda gt11 using specific antibody and oligonucleotide probes derived from FAD-containing peptide of the enzyme. The primary structure of the protein, deduced from the nucleotide sequence, consisted of 520 amino acid residues and its molecular weight was calculated to be 58.4 kD which is in good agreement with that of the in vitro-synthesized peptide. FAD-binding site is located in the carboxy-terminal region. There is no typical structural feature common to the targeting signals for mitochondria, the periodic distribution of basic amino acids spaced by several uncharged residues, at its amino-terminal region. This region has an uninterrupted stretch of 14 hydrophobic residues.     

Oxidation of lipids. XV. Role of hydrophilic diarylamines as antioxidants in the oxidations of lipids and biological tissues

The abilities of two kinds of water-soluble diarylamines, disodium 4-chloro-2,2'-iminodibenzoate (CCA) and disodium 4-chloro-3',6'-dimethyl-2,2'-iminodibenzoate (CCM), to protect lipids, membranes and biological tissues from oxidative damages have been studied. The experimental systems studied include the oxidations of methyl linoleate micelles and soybean phosphatidylcholine (Pc) liposomal membranes in aqueous dispersions, oxidative hemolysis of rabbit erythrocytes, and the in vivo oxidative damages of biological tissues all induced by free radicals generated from an azo radical initiator. The two diarylamines functioned as moderate chain-breaking antioxidants and retarded the above oxidations.     

Tyrosinase-catalyzed binding of 3,4-dihydroxyphenylalanine with proteins through the sulfhydryl group

The cytotoxicity of catechols has been ascribed to covalent binding of the omicron-quinone oxidation products to proteins through sulfhydryl groups. The nature of the covalent binding was studied with dopaquinone formed on tyrosinase oxidation of 3,4-dihydroxyphenylalanine (DOPA). After acid hydrolysis of the reaction products, cysteinyldopas liberated (protein-bound cysteinyldopas) were determined by HPLC with electrochemical detection. When 0.1 mM DOPA was oxidized in the presence of 0.2 mM bovine serum albumin, alcohol dehydrogenase or isocitrate dehydrogenase, protein-bound cysteinyldopas were formed in yields of 5.4, 44, or 33%, respectively. The covalent binding was almost completely inhibited by 1 mM cysteine or 1 mM ascorbic acid, but 10 mM lysine had no effect. These results unambiguously demonstrate that dopaquinone can bind with proteins mostly through sulfhydryl groups.     

Bacteriocinlike killing action of a temperate bacteriophage phiBA1 of Bacillus aneurinolyticus.

A new temperate phage, phiBA1, was isolated from Bacillus aneurinolyticus, phiBA1 had an icosahedral head with a diameter of about 70 nm and a tail about 20 nm long and contained a circularly permuted, linear duplex DNA of about 38 x 106 daltons. This phage showed two activities: bacteriocin-like killing activity against five strains of B. aneurinolyticus and normal temperate phage activity against three other strains. phiBA1 killed sensitive cells by a single-hit process. After adsorption of phiBA1 to cells sensitive to killing, the content of intracellular ATP increased for the first 5 min and then gradually decreased. Phage DNA injected into the cell immediately after infection was degraded rapidly. Killing was also caused by heavily UV-irradiated phiBA1. Killing-resistant mutants showed normal adsorption of phiBA1 and normal injection of the DNA with its instantaneous restriction. Our results indicate that the killing action of phiBA1 is different from the phenomenon of abortive infection and suggest that the killing might be caused by a proteinaceous component of phiBA1.     

Comparative studies of lipoteichoic acids from several Bacillus strains.

Structural studies were carried out on lipoteichoic acids obtained from defatted cells of 10 Bacillus strains by phenol-water partition followed by chromatography on DEAE-Sephacel and Octyl-Sepharose columns. A group of the tested bacteria (group A), Bacillus subtilis, Bacillus licheniformis, and Bacillus pumilus, was shown to have a diacyl form of lipoteichoic acids which contained D-alanine, D-glucose, D-glucosamine, fatty acids, and glycerol in molar ratios to phosphorus of 0.35 to 0.69, 0.07 to 0.15 to 0.43, 0.06 to 0.11, and 0.95 to 1.18, respectively, whereas the other group (group B), Bacillus coagulans and Bacillus megaterium, had diacyl lipoteichoic acids which contained D-galactose, fatty acids, and glycerol in molar ratios to phosphorus of 0.05 to 0.42, 0.06 to 0.12, and 0.96 to 1.07, respectively. After treatment with 47% hydrogen fluoride, the lipoteichoic acids obtained from group A strains commonly gave a hydrophobic fragment, gentiobiosyl-beta (1----1 or 3)diacylglycerol, in addition to dephosphorylated repeating units, glycerol, 2-D-alanylglycerol, N-acetyl-D-glucosaminyl-alpha (1----2)glycerol, and D-alanyl-N-acetyl-D-glucosaminyl-alpha (1----2)glycerol, whereas the lipoteichoic acids from group B strains yielded diacylglycerol in addition to glycerol and D-galactosyl-alpha (1----2)glycerol. The results together with data from Smith degradations indicate that in the lipoteichoic acids of group A strains the polymer chains, made up of partially alanylated glycerol phosphate and glycosylglycerol phosphate units, are joined to the acylglycerol anchors through gentiobiose. However, in the lipoteichoic acids of group B strains, the partially galactosylated poly(glycerolphosphate) chains are believed to be directly linked to the acylglycerol anchors.     

N-terminal amino acid sequence of human urinary prokallikrein

The N-terminal amino acid sequences of human urinary prokallikrein and kallikrein have been determined. Their amino acid sequences are as follows. (Formula; see text) The results showed that prokallikrein comprises an additional seven amino acids at the amino terminus of the kallikrein, of which the sequence is (H2N)Ala-Pro-Pro-Ile-Gln-Ser-Arg(COOH). Comparison of the structure of this peptide with those of other proteins revealed extensive sequence identity with the propeptide portions of rat and mouse tissue kallikreins, that were predicted from the preproenzyme-encoded nucleotide sequences. The amino acid sequence of the peptide was also highly homologous to that of the propeptide portion of EGF-binding protein, that was predicted from the nucleotide sequence, and that of the alpha-subunit of NGF. The N-terminal amino acid sequence of kallikrein was completely identical to the reported one (Lottspeich, F., et al. (1979) Hoppe-Seyler's Z. Physiol. Chem. 360, 1947-1950) and shows considerable amino acid sequence homology with the porcine and rat pancreatic kallikreins. As far as the present results are concerned, it is strongly indicated that the inactive kallikrein in human urine is a tissue type prokallikrein which is activated on the release of the N-terminal peptide consisting of seven amino acids.     

Complete resistance to challenges with Hymenolepis nana cysticercoids derived from mouse, rat and beetle in mice

and 1986. Complete resistance to challenges with Hymenolepis nana cysticercoids derived from mouse, rat and beetle in mice. International Journal for Parasitology 16: 623–628. When BALB/c and dd strains of mice were given eggs of Hymenolepis nana, they all became completely resistant not only to challenge with mouse-derived cysticercoids but also to challenges with rat-derived and beetle-derived cysticercoids. Serum IgG antibodies at 47–60 days post egg inoculation reacted strongly with these three different host-derived cysticercoids when examined by IFA test, but IgA and IgM isotypes reacted very weakly. Antibodies of infected mouse sera (IgG, IgM and IgA were examined) reacted not only with the protoscolex (scolex of the excysted juvenile) but also with the outer cyst wall. By contrast, uninfected mouse sera and immune sera prepared seven days post cysticercoid inoculation did not react at all. Antigens of both cyst wall and protoscolex appeared to be of parasite origin and not of host origin, and appeared similar in parasites from the different host species.     

Suppressors of the secY24 mutation: identification and characterization of additional ssy genes in Escherichia coli

We previously reported (Shiba et al., J. Bacteriol. 160:696-701, 1984) the isolation and characterization of the mutation (ssy) that suppresses the protein export defect due to the secY24(Ts) mutation and causes cold-sensitive growth of Escherichia coli. This report describes more systematic isolation of ssy mutations. Among temperature-resistant revertants of the secY24 mutant, 65 mutants were found to be cold sensitive. These cold-sensitive mutations have been classified by genetic mapping. Twenty-two mutations fell into the ssyA class previously described. The remaining mutations were located at five new loci: ssyB at 9.5 min between tsx and lon; ssyD around 3 min; ssyE at 72.5 min near secY; ssyF at 20.5 min within rpsA; and ssyG at 69.0 min near argG. Two predominant classes, ssyA and ssyB, are probably affected in protein synthesis at the elongation step, whereas the ssyF mutant contained an altered form of ribosomal protein S1 (the gene product of rpsA). These cold-sensitive ssy mutations which suppress secY24 may define genes whose function is somehow involved in the secY-dependent protein secretion mechanism. However, the existence of multiple suppressor loci makes it unlikely that all of these genes specify additional components of the export machinery. A delicate balance may exist between the systems for synthesizing and exporting proteins.     

Spiral conformation of Vibrio cholerae as determined by scanning electron microscopy of elongated cells induced by cephalexin treatment.

The elongated cells of Vibrio spp. induced by cephalexin treatment were examined by scanning electron microscopy. The results showed that Vibrio cholerae has a twisted cell body and a right-handed spiral conformation and that the cell bodies of V. parahaemolyticus and V. alginolyticus are straight rather than curved.     

Studies on glucosyltransferase and endogenous glucosyl acceptor in Bacillus cereus AHU 1030 membranes

A glucosyltransferase, extracted from the membranes of Bacillus cereus AHU 1030 with Tris-HCl buffer containing 0.1% Triton X-100 at pH 9.5, was separated from an endogenous glucosyl acceptor by chromatography on DEAE-Sepharose CL-6B subsequent to chromatography on Sepharose 6B. Structural analysis data showed that the glucosyl acceptor was a glycerol phosphate polymer linked to beta-gentiobiosyl diglyceride. The enzyme catalyzed the transfer of glucosyl residues from UDP-glucose to C-2 of the glycerol residues of repeating units of the acceptor. On the other hand, a lipoteichoic acid which contained 0.3 D-alanine residue per phosphorus was isolated from the cells by phenol treatment at pH 4.6. Except for the presence of D-alanine, this lipoteichoic acid had the same structure as the glucosyl acceptor. The rate of glucosylation observed with the D-alanine-containing lipoteichoic acid as the substrate was less than 40% of that observed with the D-alanine-free lipoteichoic acid, indicating that the ester-linked D-alanine in the lipoteichoic acid interferes with the action of the glucosyltransferase. The enzyme also catalyzed glucosylation of poly(glycerol phosphate) which was synthesized in the reaction of a separate enzyme fraction with CDP-glycerol. Thus, it is likely that the glucosyltransferase functions in the synthesis of cell wall teichoic acid.