Decrease of nitrate biosynthesis in scorbutic mutant rats unable to synthesize ascorbic acid

The effect of ascorbic acid deficiency on the urinary excretion of nitrate was investigated using a mutant strain of rats (osteogenic disorder syndrome rats; ODS rats) unable to synthesize ascorbic acid. The amount of urinary nitrate excreted by ODS rats with or without ascorbic acid supplementation were measured before and after the intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS). Urinary nitrate excretion increased markedly after LPS injection. Urinary nitrate excretion by ODS rats not supplied with ascorbic acid was significantly less than that of those supplied with ascorbic acid both before and after LPS injection. These results show that ascorbic acid enhances both LPS-stimulated and constitutive nitrate production in vivo.     

Integration of Agrobacterium T-DNA into a tobacco chromosome: Possible involvement of DNA homology between T-DNA and plant DNA

Summary We established tobacco tumour cell lines from crown galls induced by Agrobacterium. Restriction fragments containing T-DNA/plant DNA junctions were cloned from one of the cell lines, which has a single copy of the T-DNA in a unique region of its genome. We also isolated a DNA fragment that contained the integration target site from nontransformed tobacco cells. Nucleotide sequence analyses showed that the right and left breakpoints of the T-DNA mapped ca. 7.3 kb internal to the right 25 by border and ca. 350 by internal to the left border respectively. When the nucleotide sequences around these breakpoints were compared with the sequence of the target, significant homology was seen between the region adjacent to the integration target site and both external regions of the T-DNA breakpoints. In addition, a short stretch of plant DNA in the vicinity of the integration site was deleted. This deletion seems to have been promoted by homologous recombination between short repeated sequences that were present on both sides of the deleted stretch. Minor rearrangements, which included base substitutions, insertions and deletions, also took place around the integration site in the plant DNA. These results, together with previously reported results showing that in some cases sequences homologous to those in T-DNA are present in plant DNA regions adjacent to left recombinational junctions, indicate that sequence homology between the incoming T-DNA and the plant chromosomal DNA has an important function in T-DNA integration. The homology may promote close association of both termini of a T-DNA molecule on a target sequence; then TDNA may in some cases be integrated by a mechanism at least in part analogous to homologous recombination.Shogo Matsumoto is on leave from Biochemical Research Institute, Nippon Menard Cosmetic Co., Ltd, Ogaki, Gifu-ken 503, Japan     

Manganese-mediated oxidative damage of cellular and isolated DNA by isoniazid and related hydrazines: non-Fenton-type hydroxyl radical formation.

The mechanism by which hydrazines induce damage to cellular and isolated DNA in the presence of metal ions has been investigated by pulsed-field gel electrophoresis (PFGE), DNA sequencing methods, and the ESR spin-trapping technique. For the detection of single-strand breaks by PFGE, an experimental procedure with alkali treatment has been designed. Isoniazid, hydrazine, and phenylhydrazine induced DNA single- and double-strand breaks in cells pretreated with Mn(II), whereas iproniazid did not. With isolated 32P-DNA, isoniazid produced DNA damage in the presence of Cu(II), Mn(II), or Mn(III). Iproniazid damage isolated DNA only in the presence of Cu(II). The Cu(II)-mediated DNA damage by isoniazid or iproniazid is due to active oxygen species other than hydroxyl free radical (.OH), presumably the Cu(I)-peroxide complex. Cleavage of isolated DNA by isoniazid plus Mn(II) occurred without marked site specificity. The DNA damage was inhibited by .OH scavengers and superoxide dismutase (SOD) but not by catalase, suggesting the involvement of .OH formed via O2- but not via H2O2. Consistently, in ESR experiments .OH formation was observed during Mn(II)-catalyzed autoxidation of isoniazid, and the .OH formation was inhibited by SOD, but not by catalase. Iproniazid plus Mn(II) produced no or little .OH. We propose a reaction mechanism for the .OH formation without a H2O2 intermediate during manganese-catalyzed autoxidation of hydrazine. The present and previous data raise the possibility that hydrazines plus Mn(II)-induced cellular DNA damage may occur, at least in part, through the non-Fenton-type reaction.     

Pre-junctional inhibitory action of prostaglandin E2 on excitatory neuro-effector transmission in the human bronchus

The effects of prostaglandin E₂ (PGE₂) and indomethacin on excitatory neuro-effector transmission in the human bronchus were investigated by tension recording and microelectrode methods. PGE₂ (10⁻¹⁰–10⁻⁹M) suppressed the amplitude of twitch contractions and excitatory junction potentials (e.j.ps) evoked by field stimulation at a steady level of basal tension obtained by the combined application of indomethacin (10⁻⁵M) and FPL55712 (10⁻⁶M). In doses over 10⁻⁸M, PGE₂ reduced the muscle tone and dose-dependently suppressed the amplitude of twitch contractions. Indomethacin (10⁻⁵ or 5 × 10⁻⁵M) reduced the muscle tone and enhanced the amplitude of twitch contractions and e.j.ps evoked by field stimulation in the presence of FPL55712. PGE₂ (10⁻⁹M) had no effect on the post-junctional response of smooth muscle cells to exogenously applied acetylcholine (ACh) (4 × 10⁻⁷M). However, indomethacin (10⁻⁵M) significantly enhanced the ACh-induced contraction of the human bronchus. These results indicate that PGE₂ in low concentrations has a pre-junctional action to inhibit excitatory neuro-effector transmission in addition to a post-junctional action, presumably by suppressing transmitter release from the vagus nerve terminals in the human bronchial tissues.     

Typing of Verotoxins by DNA Colony Hybridization with Poly- and Oligonucleotide Probes,a Bead-Enzyme-Linked Immunosorbent Assay,and Polymerase Chain Reaction

To identify the type of Verotoxins (VT) produced by Verocytotoxin-producing Escherichia coli (VTEC), a sensitive bead-enzyme-linked immunosorbent assay and polymerase chain reaction with common and specific primers to various VTs (VT1, VT2, VT2vha, VT2vhb, and VT2vp1) were developed. Together with colony hybridization tests with oligo- and polynucleotide probes, these methods were applied to VTEC isolates to type the VT produced. The toxin types of 26 of 37 strains were identified, but the reaction profiles in assays of the remaining 11 strains suggested the existence of new VT2 variants. The application of these identification procedures may be useful as a tool for clinical and epidemiological studies of VTEC infection.     

Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho.

The small GTP binding protein Rho is implicated in cytoskeletal responses to extracellular signals such as lysophosphatidic acid to form stress fibers and focal contacts. Here we have purified a Rho-interacting protein with a molecular mass of approximately 164 kDa (p164) from bovine brain. This protein bound to GTPgammaS (a non-hydrolyzable GTP analog).RhoA but not to GDP.RhoA or GTPgammaS.RhoA with a mutation in the effector domain (RhoAA37).p164 had a kinase activity which was specifically stimulated by GTPgammaS.RhoA. We obtained the cDNA encoding p164 on the basis of its partial amino acid sequences and named it Rho-associated kinase (Rho-kinase). Rho-kinase has a catalytic domain in the N-terminal portion, a coiled coil domain in the middle portion and a zinc finger-like motif in the C-terminal portion. The catalytic domain shares 72% sequence homology with that of myotonic dystrophy kinase and the coiled coil domain contains a Rho-interacting interface. When COS7 cells were cotransfected with Rho-kinase and activated RhoA, some Rho-kinase was recruited to membranes. Thus it is likely that Rho-kinase is a putative target serine/threonine kinase for Rho and serves as a mediator of the Rho-dependent signaling pathway.     

Aflatoxin and cyclopiazonic acid production by a sclerotium-producing Aspergillus tamarii strain.

The production of aflatoxins B1 and B2 by Aspergillus tamarii (subgenus Circumdati section Flavi) is reported for the first time. The fungus was isolated from soil collected from a tea (Camellia sinensis) field in Miyazaki Prefecture, Japan. Three single-spore cultures, NRRL 25517, NRRL 25518, and NRRL 25519, were derived from subcultures of the original isolate 19 (MZ2). Each of these single-spore cultures of A. tamarii produced aflatoxins B1 and B2 and cyclopiazonic acid, as well as black, pear-shaped sclerotia. The demonstration of aflatoxin production by A. tamarii is examined in connection with A. tamarii phylogenetic relationships, chemical ecology, and potential use in food fermentations.     

Analysis of the multi-stem clump structure ofLitsea japonica Juss. growing in a coastal dwarf forest

The multi-stem clump structure of a coastal dwarf forest dominated byLitsea japonica Juss. was investigated in order to clarify the sprouting characteristics and self-maintenance of clumps by stem alternation. The size and age distribution of multi-stem clumps were analyzed using cumulative relative frequency curves.L. japonica had a large number of stems and an even height distribution or young age-biased distribution of stems within a clump. These results indicated the sequential flushing of sprouts at high frequency. Height distribution within a clump ofL. japonica was relatively even compared to other species. This clump structure suggested the stable self-maintenance of individuals in all ranges of size and age without disturbances. It originated specific sprouting characteristics as a response to the severe stress of salty wind.Ardisia sieboldii Miq. had few stems within a clump. Although the stem height distribution of large individuals tended to be even, most clumps had a large size-biased distribution of stem height which indicated simultaneous sprouting. From this structure, sprouts of this species were thought to be of less significance in the stable self-maintenance of individuals thanL. Japonica.