Studies on enzymatic reduction of aliphatic nitro compounds: Reductive dimerization of β-nitrostyrene and 1-nitro-4-phenylbutadiene

Enzymatic reduction of aliphatic nitro compounds, β-nitrostyrene (I), 1-nitro-4-phenylbutadiene (II), 1-nitro-4-phenyl-1-butene (III), 1-nitro-2-phenylethane (IV), and nitrophenylethane (V) was investigated in a xanthine oxidase-hypoxanthine system. I and II were easily reduced by the enzyme system under anaerobic conditions, but III, IV, and V resisted to the enzymatic reduction. The reduction products of I and II were isolated from the reaction mixtures and were identified as dimolecular compounds, 1,4-dinitro-2, 3-diphenylbutane and 1,4-dinitro-2,3-distyrylbutane, respectively, by mass, nuclear magnetic resonance, and infrared spectrometries, and by elementary analyses.     

Genetic and physical characterization of the ColIb plasmid using ColIb-R222 hybrids

Summary The presence of the ColIb plasmid in Escherichia coli cells inhibits the growth of bacteriophages BF23 and T5 (Ibf phenotype; inhibition of BF23 and T5 growth). To understand this abortive infection, we devised a method of isolating mutants that were defective in some ColIb phenotypes including Ibf. This method consisted of transduction of the tet (Tc^r; tetracycline resistance) or cml (Cm^r; chloramphenicol resistance) gene of plasmid R222 with phage P22 into ColIb, construction of Tc^rCm^rIbf⁺ Imm⁺ (immunity to colicin Ib) Cib^- (no production of colicin Ib) recombinants by crossing between the transductants, and isolation of deletion mutants from the recombinants by phage P1 transduction. By this procedure, pKM25-2 (Tc^rCm^sIbf^-Imm^-Cib^-) and pKM25-1 (Tc^rCm^sIbf⁺Imm⁺Cib^-) were isolated. Construction of the cleavage map of the ColIb plasmid by restriction endonucleases and comparative analyses of the DNA fragments produced from the mutant plasmids revealed that the genes determining Ibf and Imm mapped on a 4.60 Mdal HindIII fragment (H-3) and the gene determining Cib on a 1.71 Mdal EcoRI fragment (E-12).These results together with other observations (Wilkins et al. 1981; Hama personal communication) also show the approximate positions of the genes for Rep (replication), Inc (incompatibility), and Sog (suppression of dnaG) as well as Ibf, Imm, and Cib phenotypes on the cleavage map of the ColIb plasmid.Preliminary data were reported in the 1979 Annual Meeting of the Japan Molecular Biology Society (Uemura and Mizobuchi, Abst Ann Mol Biol Meet 1979, p 36)     

Inhibition of growth of bacteriophage BF23 by the ColIb plasmid: Identification of the ibfA and ibfB genes of the ColIb plasmid

Summary A 3.7 Mdal DNA fragment of plasmid ColIb which carried all the genetic information determining the growth inhibition of bacteriophage BF23 (Ibf phenotype) but not for production of colicin Ib protein (Cib) and immunity to colicin Ib (Imm) was cloned in the pBR322 vector. We also cloned the 8.7 Mdal DNA fragment that was responsible for both Cib and Imm but not for Ibf phenotype. Thus, these results clearly showed that the gene(s) determining Ibf are different from those for Cib or Imm. Dissection of the Ibf-DNA revealed that at least two genes, ibfA and ibfB, were involved in the Ibf phenotype. The ibfA gene was mapped within a 1.45 Mdal EcoRI DNA fragment (E-13); mutation of this gene by deletion or by insertion of Tn5, a kanamycin resistance transposon, resulted in the complete loss of Ibf phenotype. The ibfB gene, mapped around a 0.32 Mdal HindIII DNA fragment (H-7), was found to be active in trans to ibfA, and its function seemed to promote ibfA activity. The genetic map of the ibf genes in relation to other ColIb markers was determined as ibfB-ibfA-imm-cib. Attempts to identify the ibfA gene product in the minicell system, however, did not succeed.Preliminary results were presented in the 1980 Annual Meeting of the Japan Molecular Biology Society. (Uemura and Mizobuchi Abst Ann Mol Meet 1980, p 35)     

Effects of carbon dioxide on the spatially separate electrogenic ion pumps and the growth rate in the hypocotyl ofVigna sesquipedalis

Carbon dioxide, introduced into the gas phase of the experimental chamber, has distinct effects on two spatially separate membrane potentials and the rate of elongation growth in hypocotyl segments ofVigna sesquipedalis Wight. Both membrane potentials (V _ps andV _px=the electric potential difference between the parenchyma symplast and the surface of the hypocotyl, and that between the parenchyma symplast and the xylem, respectively) hyperpolarized rapidly but transiently at the introduction of CO₂. Prolonged exposure of the hypocotyl to high concentrations of CO₂ (above 10%) caused depolarization of membrane potentials above the level before CO₂ introduction. When CO₂ was replaced with air, the membrane potentials exhibited a distinct depolarization response of transient nature. The growth rate of the hypocotyl segments exhibited similar responses to CO₂ as did the membrane potentials (the increase and the decrease of the growth rate were corresponded to the hyperpolarization and the depolarization, respectively), but these responses always followed the changes of the membrane potentials. The CO₂-induced maximum hyperpolarization ofV _ps and the maximum increase of the growth rate were closely correlated. All these responses were strictly dependent on aerobic metabolism. These results indicate that CO₂ may regulate elongation growth in two ways: by affecting the activity of the electrogenic ion pump via intracellular acidification, and also by acting via apoplastic acidification as a wall-loosening acid.Symbols and abbreviations V _sx electric potential difference between the surface (S) and the xylem (X) of the hypocotyl - V _px electric potential difference between the inside of a parenchyma cell (P) andX - V _ps electric potential difference betweenP andS - V _ps (CO₂, max) the maximum value of CO₂-induced hyperpolarization ofV _ps - GR(CO₂, max) the maximum value of CO₂-induced increase of the growth rate - IAA indole-3-acetic acid     

Enzymatic reduction of synthetic hemin to heme and its application to study on oxygenation in aqueous medium

The enzyme system consisting of glucose-6-phosphate, glucose-6-phosphate dehydrogenase, ferredoxin, ferredoxin-NADP-reductase, and NADP was used to reduce various synthetic iron(III) porphyrins to iron(II) in aqueous buffer (pH7.0) at 25°C. The oxygenation reactions of the thus prepared iron(II) porphyrin complexes were examined and it was found that only the iron(II) picket fence porphyrin-mono(1-lauryl-2-methylimidazole) complex incorporated in liposomes of phosphatidylcholine can form a stable oxygen adduct at 25°C in neutral aqueous medium.     

Carcinogenicity test of 50 Hz sinusoidal magnetic fields in rats

Male and female F344 rats, 48 per exposure group, were sham exposed (Group A) or exposed to 0.5 (Group B) and 5 mT (Group C) magnetic fields for two years. Animals were exposed from 5–109 weeks of age in SPF conditions according to the OECD test guideline No. 451. Average exposure was 22.6 hr/day. No significant differences in body weight and food consumption were observed between the sham and exposed groups. At the end of the exposure period, survival rates of the male rats were 73, 83, and 79%, and those of the females, 77, 79, and 75% for Groups A, B, and C, respectively, with no significant differences between groups. Differential counts of leukocytes were measured at the 52nd, 78th, and 104th weeks of exposure and no significant differences were observed between the exposure groups. All survivors were euthanized on schedule, and all the organs and tissues suspected of tumoral lesions were examined histopathologically. Incidences of mononuclear cell leukemia in the male and the female rats were 5, 4, 4 and 8, 6, 7 for Groups A, B and C, respectively; incidences of malignant lymphoma in the female rats were 0, 1 and 1. Neither significant increases nor acceleration of incidence of leukemia were observed. Incidences of brain and intracranial tumors did not increase in the exposed groups. Incidences of both benign and malignant neoplasms showed no significant difference between the exposed and sham exposed groups with one exception: fibroma of the subcutis in the male rats, which was considered not to be a statistically significant when evaluated with respect to the historical control data in our laboratory. Bioelectromagnetics 18:531–540, 1997. © Wiley-Liss, Inc.     

Hormonal regulation of JP29 in the epidermis during larval development and metamorphosis in the tobacco hornworm,Manduca sexta

Previous studies have shown that the larval epidermis of the tobacco hornworm, Manduca sexta, contains a 29 kDa nuclear protein (JP29) that binds pothoaffinity analogs of juvenile hormone (JH), but does not bind JH I with high affinity. We now find that JP29 is also associated with the insecticyanin granules, and we show that JP29 mRNA is regulated in a complex fashion by both 20-hydroxyecdysone (20E) and JH. Studies with day 2 fourth instar larval epidermis in vitro showed that a molting concentration 12 μg/ml) of 20E caused the disappearance of JP29 mRNA, irrespective of the presence or absence of JH; this effect was dependent on the concentration of 20E (ED₅₀=200 ng/ml). The reappearance of JP29 mRNA around the time of ecdysis required the presence of JH at head capsule slippage (HCS), since little appeared in larvae allatectomized about 6 h before HCS unless JH I was applied at the time of HCS. Maintenance of JP29 mRNA in fifth instar epidermis also required the continued presence of JH in both isolated abdomens and in vitro. Culture of either day 1 or day 2 fifth instar epidermis without hormones for 24 h caused decline of JP29 mRNA, which was accelerated by 20E in a concentration-dependent manner (ED₅₀ = 30 and 10 ng/ml 20E respectively). When day 2 epidermis was exposed to 500 ng/ml 20E for 24 h to cause pupal commitment, JP29 mRNA disappeared. Neither methoprene nor JH I (in either the presence or the absence of the esterase inhibitor O-ethyl, S-phenyl phosphamidethiolate [EPPAT]) was able to prevent this loss, although both slowed its rate. The mRNA for the larval cuticle protein LCP14 was found to be regulated similarly to that for JP29 by 20E, but differently by JH. The JP29 protein was relatively long-live, persisting after the disappearance of its mRNA for at least 19 h during the larval molt and for more than 24 h in vitro. Although trace amounts of JP29 are found for the first 12 h after pupal ecdysis, injection of 5 μg JH II into pupae during the critical period to cause the synthesis of a second pupal cuticle had no effect on the amount of JP29 present. Thus, although the presence of JP29 in larval epidermis is associated with and dependent on JH, high amounts are not associated with the “status quo” action of JH on the pupa. The role of this protein consequently remains obscure. Arch. Insect Biochem. Physiol. 34:409–428, 1997. © 1997 Wiley-Liss, Inc.     

A Novel Subtype of Prostacyclin Receptor in the Central Nervous System

Recently, in the course of our search for the prostacyclin receptor in the brain, we found a novel subtype, designated as IP2, which was finely discriminated by use of the specific ligand (15R)-16-m-tolyl-17,18,19,20-tetranorisocarbacyclin (15R-TIC) and specifically localized in the rostral part of the brain. In the present study, the tritiated compound 15R-[15-(3)H]TIC was synthesized and utilized for more specific research on IP2. The specificity of binding to rat brain regions was confirmed by use of several prostacyclin derivatives including 15S-TIC. Mapping of 15R- and 15S-[3H]TIC binding in adjacent pairs of frozen sections of rat brain demonstrated a quite similar pattern of distribution in almost all rostral brain regions, indicating that the regions may contain only the IP2 subtype. On the other hand, 15R-[3H]TIC binding was very faint as compared with 15S-[3H]TIC binding in the caudal medullary region. High densities of 15R-[3H]TIC binding sites were shown in the dorsal part of the lateral septal nucleus, thalamic nuclei, limbic structures, and some of the cortical regions. Scatchard plot analysis showed two components of high-affinity 15R-[3H]TIC binding in the rostral regions, one with a K(D) value at approximately 1 nM and the other with approximately 30 nM. These results strengthen our previous finding that a different subtype of prostacyclin receptor is expressed in the CNS, and the map with 15R-[3H]TIC obtained here could guide further studies on the molecular and functional properties of the IP2.