Gene structure of human thrombomodulin, a cofactor for thrombin-catalyzed activation of protein C

The gene coding for human thrombomodulin, a thrombin receptor on endothelial cells and a cofactor for the activation of anticoagulant protein C zymogen, was isolated from a human genomic library by employing human thrombomodulin cDNA as a probe. The nucleotide sequences of the gene and the adjacent 5' and 3' flanking regions were then determined. The nucleotide sequence of this gene with approximately 3.7 kilobase pairs was identical to that of the cDNA, indicating that the gene for human thrombomodulin is free of introns. Hybridization data showed that there is only a single thrombomodulin gene in the human genome.     

Gelatinases of metastatic cell lines of murine colonic carcinoma as detected by substrate-gel electrophoresis

Gelatinases were detected in the conditioned medium of murine colonic carcinoma cells by SDS-polyacrylamide gel electrophoresis using gels copolymerized with gelatin. Several gelatinase activities differing in molecular weight were detected but the major activities migrated with molecular weights of 60,000 and 95,000. The enzymes did not hydrolyze bovine serum albumin or casein, and required calcium for activity. All of the gelatinase activities were inhibited by EDTA, 1,10-phenanthroline and dithiothreitol but not by N-ethylmaleimide and phenylmethylsulfonyl fluoride. The 95,000 dalton gelatinase was separated from the 60,000 dalton gelatinase by affinity chromatography on Ricinus communis agglutinin-agarose, and the former activity was markedly increased in highly metastatic cell lines as compared with its activity in poorly metastatic cell lines.     

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.     

Chromosomal localization of Sau3A repetitive DNA revealed by in situ hybridization

The Sau3A DNA family consists of unique alphoid human repetitive DNA which is prone to be excised from the chromosomes and exhibits restriction fragment length polymorphism. We studied the chromosomal localization of the DNA by in situ hybridization using cultured normal human lymphocytes. Under standard hybridization conditions, the sequence hybridized with the centromeric regions of chromosomes 1, 2, 4, 11, 15, 17, 18, 19 and X, but under high stringency hybridization conditions, it hybridized with the centromeric regions of chromosomes 1, 17 and X, and particularly chromosome 11. Based on these results, we discuss the evolutionary relationship among the sequences of the Sau3A DNA family.     

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.     

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.