Physarum myosin light chain interacts with actin in a Ca2+-dependent manner

Actin-modulating activity was analysed with the 16,131-dalton calcium-binding light chain (CaLc, Kobayashi et al. (1988) J. Biol. Chem. 263, 305-313) of Physarum myosin, which is under an inhibitory Ca-control (Kohama and Kendrick-Jones (1986) J. Biochem. 99, 1433-1446). When skeletal muscle actin was polymerized in the presence of CaLc and Ca2+, increases in both viscosity and birefringence were reduced under high shear conditions. However, CaLc did not inhibit actin polymerization under no or low shearing forces, which was demonstrated by a variety of methods including fluorescence intensity measurements using pyrenyl actin. We propose that actin polymerized in the presence of CaLc and Ca2+ is easily fragmented under high shearing forces to produce the changes in viscosity and birefringence.     

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.     

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.     

Simulation analysis of excitation conduction in the heart: Propagation of excitation in different tissues

The normal excitation and conduction in the heart are maintained by the coordination between the dynamics of ionic conductance of each cell and the electrical coupling between cells. To examine functional roles of these two factors, we proposed a spatially-discrete model of conduction of excitation in which the individual cells were assumed isopotential. This approximation was reasoned by comparing the apparent space constant with the measured junctional resistance between myocardial cells. We used the four reconstruction models previously reported for five kinds of myocardial cells. Coupling coefficients between adjacent cells were determined quantitatively from the apparent space constants. We first investigated to what extent the pacemaker activity of the sinoatrial node depends on the number and the coupling coefficient of its cells, by using a one-dimensional model system composed of the sinoatrial node cells and the atrial cells. Extensive computer simulation revealed the following two conditions for the pacemaker activity of the sinoatrial node. The number of the sinoatrial node cells and their coupling coefficients must be large enough to provide the atrium with the sufficient electric current flow. The number of the sinoatrial node cells must be large so that the period of the compound system is close to the intrinsic period of the sinoatrial node cell. In this simulation the same sinoatrial node cells produced action potentials of different shapes depending on where they were located in the sinoatrial node. Therefore it seems premature to classify the myocardial cells only from their waveforms obtained by electrical recordings in the compound tissue. Second, we investigated the very slow conduction in the atrioventricular node compared to, for example, the ventricle. This was assumed to be due to the inherent property of the membrane dynamics of the atrioventricular node cell, or to the small value of the coupling coefficient (weak intercellular coupling), or to the electrical load imposed on the atrioventricular node by the Purkinje fibers, because the relatively small atrioventricular node must provide the Purkinje fibers with sufficient electric current flow. Relative contributions of these three factors to the slow conduction were evaluated using the model system composed of only the atrioventricular cells or that composed of the atrioventricular and Purkinje cells. We found that the weak coupling has the strongest effect. In the model system composed of the atrioventricular cells, the propagation failure was not observed even for very small values of the coupling coefficient.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Ferricyanide Induces Flowering by Suppression of Nitrate Assimilation in Lemna paucicostata 6746

Lemna paucicostata 6746, a short-day plant, produced flowerbuds even under continuous light when cultured for 3 days inferricyanide containing ammonium-free medium followed by cultureon nitrogen-rich medium (either nitrate or ammonium). Dailytreatment with ferricyanide in the absence of ammonium for morethan 8 hours, which completely inhibited nitrate reductase activitywithin 6 hours after the addition to the medium, induced daylength-independentflowering even when the ammonium-rich medium was given duringthe remaining hours. The presence of ammonium for 1 hour atthe middle of the 14-h ferricyanide treatment almost completelysuppressed floral induction. (Received March 6, 1986; Accepted June 3, 1986)     

Inhibition of Flowering in the Long-Day Plant Lemna gibba G3 by Hutner's Medium and its Reversal by Medium Modification

The long-day plant Lemna gibba G3 flowers normally in E medium(Hoagland-type medium plus 30 µM EDTA) but in 0.5 H mediumthere is no flowering. Ammonium is present in 0.5 H medium andis known to inhibit flowering in L. gibba G3, but even in NH₄⁺-free0.5 H medium there is virtually no flowering under continuouslight. Increasing the phosphate concentration of the NH₄⁺-free0.5 H medium from 1.15 ITIM to 12 or 16 mM results in substantialflowering. Decreasing the EDTA concentration from 850 µIMto 250 µM, or raising the nitrate concentration from 4mM to 12 mM, results in only a small increase in flowering.If the decrease in EDTA and increase in nitrate are combinedwith the increase in phosphate, however, the flowering responseis nearly as good as that obtained using E medium. Thus, withthese three changes the inhibitory effect of NH₄⁺free 0.5 Hmedium for flowering in L. gibba G3 is almost completely reversed In the above studies flowering was not limited by daylength.When plants were grown on E medium under an 11 hour daylengthwhere flowering is limited by daylength, decreasing the phosphateconcentration in the medium reduced flowering, but increasingthe phosphate concentration in the medium did not stimulateflowering. Thus, when flowering is limited by daylength, highphosphate will not cause flowering, but a certain level of phosphateappears to be necessary for the expression of photoinductionunder long days. (Received January 14, 1986; Accepted June 24, 1986)