Overproduction, purification, and ATPase activity of the Escherichia coli RuvB protein involved in DNA repair.

The ruvA and ruvB genes of Escherichia coli constitute an operon which belongs to the SOS regulon. Genetic evidence suggests that the products of the ruv operon are involved in DNA repair and recombination. To begin biochemical characterization of these proteins, we developed a plasmid system that overproduced RuvB protein to 20% of total cell protein. Starting from the overproducing system, we purified RuvB protein. The purified RuvB protein behaved like a monomer in gel filtration chromatography and had an apparent relative molecular mass of 38 kilodaltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which agrees with the value predicted from the DNA sequence. The amino acid sequence of the amino-terminal region of the purified protein was analyzed, and the sequence agreed with the one deduced from the DNA sequence. Since the deduced sequence of RuvB protein contained the consensus sequence for ATP-binding proteins, we examined the ATP-binding and ATPase activities of the purified RuvB protein. RuvB protein had a stronger affinity to ADP than to ATP and weak ATPase activity. The results suggest that the weak ATPase activity of RuvB protein is at least partly due to end product inhibition by ADP.     

Enhancement of the Division of Equisetum arvense Protoplasts in Culture by Activated Charcoal and Their Further Development

Protoplasts were isolated from subcultured gametophytes of Equisetumarvense by treatment with Driselase and then cultured in vitro.Addition of activated charcoal (AC) to the culture medium enhancedthe rate of cell division, as well as the survival of both protoplastsand regenerated protoplasts. However, subsequent division ofcells was not observed after one or two cycles of replicationin cultures supplemented with AC. When regenerated protoplastswere transferred to fresh medium without AC 3 to 5 weeks afterthe first plating, the transferred cells formed rhizoids anddeveloped into small, young gametophytes without the prior formationof cell clusters or calluses. Furthermore, sprophytic shootsdifferentiated from the protoplast-derived gametophytes whenthey were cultured on medium supplemented with 6-benzylaminopurine(BA). (Received April 5, 1990; Accepted July 30, 1990)     

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)     

Effects of a New Plant Growth Regulator Prohexadione Calcium (BX-112) on Shoot Elongation Caused by Exogenously Applied Gibberellins in Rice (Oryza sativa L.) Seedlings

The effects of a novel plant growth regulator (PGR) prohexadionecalcium (BX-112; calcium 3,5-dioxo-4-propionylcyclohexanecarboxylate)on shoot elongation caused by exogenously applied GA₁, GA₃,GA₄₎ GA₁₉ and GA₂₀ were investigated in rice (Oryza sativa L.cv. Nihonbare and cv. Tan-ginbozu) seedling test. Dependingon the dose, BX-112 reduced shoot elongation in both cultivarscaused by GA₁₉ and GA₂₀, but not by GA₁. When a high dose ofBX-112 (e.g. 250 ng/plant and over) was applied with GA₁, orGA₄, shoot elongation was even promoted. This promotive effect,however, was not observed with GA₃. These results suggest thatBX-112 inhibits gibberellin (GA) biosynthesis in the rice plantat the 3ß- and 2ß-hydroxylation of GAs,namely steps of activation and inactivation, respectively. (Received September 6, 1989; Accepted November 27, 1989)     

Transformation ofBacillus subtilis with the treatment by alkali cations

Summary ExposingBacillus subtilis cultures to high concentrations of alkali cations, especially K⁺, allows efficient transformation by plasmids. The method allows transformation with unfractionated plasmid DNA, monomeric plasmid DNA as well as linear plasmid DNA.B. subtilis strains, not amenable to natural transformation, were also transformed by the present method.     

Intergeneric hybridization betweenMonascus anka andAspergillus oryzae by protoplast fusion

Summary To breed industrially useful strains of a slow-growing, red-pigment-producing strain ofMonascus anka, protoplasts ofM. anka MAK1 (arg) andAspergillus oryzae AOK1 (met, thr) were fused. A mixture of protoplasts prepared from mycelia ofM. anka MAK1 treated with 2% Usukizyme and ofA. oryzae AOK1 treated with 2% Usukizyme and 0.2% NovoZym 234 was incubated with 30% (w/v) polyethylene glycol no. 6000. Heterokaryon fusants complementing the auxotrophies of both mutants were isolated on minimal medium, but segregated into red (MAK1) and white (AOK1) sectors after being cultured on a complete medium. After irradiation with UV light, the fusants gave stable heterozygous diploids that formed long white hyphae. These diploids, which had twice as much DNA in the nucleus as their parents, grew more rapidly than the parent strain YZT1, and produced ethanol earlier than the parents. Production of amylase, protease, and kojic acid by the fusants was intermediate in amount between that of the two parents.     

Differential Regulation of Intracellular Signaling Systems by Sodium Fluoride in Rat Glioma Cells

Abstract: We investigated the rapid and slow effects of NaF on intracellular signaling systems such as Ca²⁺ homeostasis and cyclic GMP (cGMP) generation in rat glioma C6 cells, using the Ca²⁺-sensitive dye fura-2 and cGMP enzyme immunoassay. We found that the following: (a) NaF enhanced cGMP generation in a concentration-dependent manner. This enhancement was abolished by pretreatment with 100 µ M BAPTA tetraacetoxymethyl ester or in the presence of W-7 in a concentration-dependent manner. N ^G-Monomethyl- l -arginine (NMMA), a competitive inhibitor of nitric oxide synthase (NOS), also inhibited the NaF-induced generation of cGMP. These results suggest that NaF-induced cGMP generation occurs via a calcium/calmodulin- and NOS-dependent pathway. (b) The basal intracellular Ca²⁺ concentration ([Ca²⁺]_i) was transiently greater at 1 and 3 h after pretreatment with NaF. W-7 and W-13 antagonized the increase in [Ca²⁺]_i, whereas NMMA had little effect. This suggests that the NaF-induced change in basal [Ca²⁺]_i was mediated by a calmodulin-dependent pathway but was independent of a NOS-sensitive pathway. (c) The serotonin (5-HT)-induced intracellular mobilization of Ca²⁺ was reduced by pretreating the cells with NaF. The reduction in Ca²⁺ mobilization was antagonized by genistein, a tyrosine kinase inhibitor. W-7, W-5, and H-8 had no effect. Results suggest that NaF differentially regulates the cGMP generation, basal [Ca²⁺]_i, and 5-HT_2A receptor function in C6 glioma cells.     

Three-Dimensional Labeling Program for Elucidation of the Geometric Properties of Biological Particles in Three-Dimensional Space

For all biological particles such as cells or cellular organelles, there are three-dimensional coordinates representing the centroid or center of gravity. These coordinates and other numerical parameters such as volume, fluorescence intensity, surface area, and shape are referred to in this paper as geometric properties, which may provide critical information for the clarification ofin situmechanisms of molecular and cellular functions in living organisms. We have established a method for the elucidation of these properties, designated the three-dimensional labeling program (3DLP). Algorithms of 3DLP are so simple that this method can be carried out through the use of software combinations in image analysis on a personal computer. To evaluate 3DLP, it was applied to a 32-cell-stage sea urchin embryo, double stained with FITC for cellular protein of blastomeres and propidium iodide for nuclear DNA. A stack of optical serial section images was obtained by confocal laser scanning microscopy. The method was found effective for determining geometric properties and should prove applicable to the study of many different kinds of biological particles in three-dimensional space.     

Prosaposin Facilitates Sciatic Nerve Regeneration In Vivo

Abstract: Prosaposin, a multifunctional protein, is the precursor of saposins, which activate sphingolipid hydrolases. In addition to acting as a precursor for saposins, prosaposin has been shown to rescue hippocampal CA1 neurons from lethal ischemic damage in vivo and to promote neurite extension of neuroblastoma cells in vitro. Here we show that prosaposin, when added to a collagen-filled nerve guide after sciatic nerve transection in guinea pigs, increased dramatically the number of regenerating nerve fibers within the guide. To identify the target neurons of prosaposin during peripheral nerve regeneration, we determined the degree of atrophy and chromatolysis of neurons in the spinal anterior horn and dorsal root ganglia on the prosaposin-treated and untreated side. The effect of prosaposin on large spinal neurons and small neurons of the dorsal root ganglion was more conspicuous. Subsequent immunohistochemistry demonstrated that the atrophy of cholinergic large neurons in the anterior horn is prevented to significant extent by prosaposin treatment. These findings suggest that prosaposin promotes peripheral nerve regeneration by acting on α-motor neurons in the anterior horn and on small sensory neurons in the dorsal root ganglion. The present study raises the possibility of using prosaposin as a tool for the treatment of peripheral nerve injuries.