Mutagenicities of 2,4- and 2,6-dinitrotoluenes and their reduced products in Salmonella typhimurium nitroreductase- and O-acetyltransferase-overproducing Ames test strains

Mutagenicities of 2,4- and 2,6-dinitrotoluene (2,4-and 2,6-DNT), and reduced metabolites formed by the incubation of 2,4- and 2,6-DNT with Salmonella typhimurium TA98, were tested using S. typhimurium YG strains possessing high level of nitroreductase (NR) and/or O-acetyltransferase (OAT) activities. All compounds tested showed greatest mutagenic activities toward strains YG1041 and YG1042, which possess high levels of NR and OAT activities. The relative mutagenic activities of 2,4-DNT and its related compounds toward YG1041 and YG1042 were aminonitrotoluenes (2A4NT, 4A2NT)<2,4-DNT<2,2′-dimethyl-5,5′-dinitroazoxybenzene (2,2′-DM-5,5′-DNAOB)4-hydroxylamino-2-nitrotoluene (4HA2NT)4,4′-dimethyl-3,3′-dinitroazoxybenzene (4,4′-DM-3,3′-DNAOB), and aminonitrotoluenes (2A4NT, 4A2NT)<2,4-DNT<4HA2NT4,4′-dimethyl-3,3′-dinitroazoxybenzene (4,4′-DM-3,3′-DNAOB)<2HA4NT, respectively. In addition, the relative mutagenic activities of 2,6-DNT and its related compounds toward YG1041 and YG1042 were 2,6-DNT<2-hydroxylamino-6-nitrotoluene (2HA6NT)<2,2′-dimethyl-3,3′-dinitroazoxybenzene (2,2′-DM-3,3′-DNAOB), and 2-amino-6-nitrotoluene (2A6NT)<2,6-DNT<2HA6NT, respectively. These results, together with previous findings, suggested that aminohydroxylamino dimethylazoxybenzenes or aminohydroxylamino dimethylazobenzenes produced either by the reduction of hydroxylaminonitrotoluenes or by the reduction of dimethyl dinitroazoxybenzenes are active metabolites responsible for the mutagenic activities of 2,4- and 2,6-DNT.     

Characterization of a Calcium-Dependent Protein Kinase of Tobacco Leaves That Is Associated with the Plasma Membrane and Is Inducible by Sucrose

The plasma membrane fraction from leaves of tobacco containsa 54-kDa protein with autophosphorylation activity, and thelevel of this protein increases after feeding of leaves withsucrose [Ohto and Nakamura (1995) Plant Physiol. 109: 973].The 54-kDa autophosphorylation protein could not be releasedfrom the plasma membrane by treatment with salt or alkali butcould be efficiently solubi-lized by 1% sodium deoxycholate(NaDOC). Ion-exchange chromatography of the NaDOC-solubilizedproteins in the presence of octylglucoside separated the 54-kDaautophosphorylation protein into three peaks. The autophosphorylationactivity of the 54-kDa protein in peaks I and II increased afterfeeding of leaves with sucrose. The 54-kDa protein in the peakII fraction was enriched about 125-fold starting from the microsomalmembrane fraction. The 54-kDa protein in this fraction phosphorylatedhistone HIS in a calcium-dependent manner and cross-reactedwith an antibody against a calcium-dependent protein kinase(CDPK) of Arabidopsis thaliana. These results suggest that the54-kDa protein in the peak II fraction is a novel isoform ofCDPK which is associated with the plasma membrane and is inducibleby sucrose. (Received September 29, 1997; Accepted September 1, 1998)     

Superoxide dismutases from kidney bean leaves

Three isozymes of superoxide dismutase were found in the solubleextract of kidney bean leaves. Two of them, purified to nearhomogeneous states, were inhibited by cyanide and by the antibodyto spinach Cu, Zn-superoxide dismutase. Thus, both isozymeswere the Cu and Zn containing enzyme that has a molecular weightof 32,000 and is composed of two subunits of equal size. Anotherisozyme was insensitive to cyanide and to the antibody, butwas inactivated by acetone or heating. The cyanideinsensitiveisozyme was not inactivated by H₂O₂ showing that this isozymeis Mn-superoxide dismutase. (Received June 13, 1979; )     

Estrogen regulation of nitric oxide synthesis in the procine oocyte

The endothelial type (NOS-3) of three isoforms of nitric oxide (NO) synthase occurs in porcine oocytes and granulosa cells, but the regulation of NO synthesis in oocytes remains unknown. The present study was designed to evaluate steroid control in the process of oocyte NO synthesis. Cumulus-oocyte complexes (COCs), obtained from small-sized antral follicles of immature porcine ovaries, were cultured in estrogen-deprived medium, and the effect of steroids or steroid-free porcine follicular fluids on the NO release from oocytes was investigated. Oocytes that were isolated from cultured COCs were incubated with 1 M ionomycin. The NO metabolites were identified using a NO detector-high-pressure liquid chromatography system. Oocytes from COCs cultured with 10 nM 17-estradiol (E₂) released NO in response to ionomycin, whereas progesterone and testosterone had little effect on the synthesis of NO. An inhibitor of NOS suppressed the synthesis of NO. The maximal synthesis was observed after a 15 h-culture with E₂. However, oocytes freshly obtained from antral follicles did not response to ionomycin, and the E₂ action was suppressed by the addition of steroid-free follicular fluids. Analyses of RT-PCR and Western blotting showed that E₂ did not increase NOS-3 expression. In addition, estrogen receptor was detected in oocytes and cumulus cells, and estrogen receptor was detected only in cumulus cells. These findings suggest that oocyte NOS-3 is promoted for the synthesis of NO by E₂ without increases in NOS-3 expression, but the synthesis of NO is suppressed, at least in the oocytes of early antral follicles.     

Synthesis of ceramides and cerebrosides in rat brain: comparison with synthesis of lignoceroyl-coenzyme A

The α-hydroxylation and conversion of lignoceric acid into ceramide, cerebroside, and water-soluble products in particulate fractions from rat brain was studied in the presence of sphingosine and UDP-galactose, with particular reference to the effects of CoA and the synthesis of lignoceroyl-CoA. The synthesis of lignoceroyl-CoA was found to be almost completely dependent on the addition of exogenous CoA, whereas the formation of water-soluble products, mostly glutamate, was stimulated by, but was not stringently dependent on the addition of CoA. In contrast to these two metabolic pathways, both the synthesis of ceramides and cerebrosides and α-hydroxylation were unaffected by the addition of CoA. While removal of sphingosine and UDP-galactose had drastic effects on the sphingolipid synthesis, COA did not have any effect on the removal. On the other hand, removal of sphingosine resulted in a significant increase of the synmthesis of lignoceroyl-CoA and moderate increase in the formation of water-soluble products. These observations further indicated that CoA ester formation may not be required for the synthesis of these sphingolipids, and suggest that there may be two pathways for oxidative degradation of lignoceric acid in brain—one CoA-dependent and the other CoA-independent.     

Sexual maturation of male <Emphasis Type="Italic">Bactrocera correcta</Emphasis> (Diptera: Tephritidae) and age-related responses to β-caryophyllene and methyl eugenol

Sexual maturation of male Bactrocera correcta (Bezzi) and age-related responses to β-caryophyllene (CP) and methyl eugenol (ME) were examined to identify effective attractants for sexually immature males of the species. Sexual maturation rates (100 × number of cages containing males which attained sexual maturity/total number of cages at a given day) over different ages were 0.0%, 6.7%, and 100.0% for 5-, 6-, and 13-day-old, respectively, and at all the different ages, more males were captured with CP than with ME. The capture rate (the predicted probability by logistic regression) with CP was 29.7% (30.6%) for 5-day-old males, which were still sexually immature, and 52.7% (38.2%) for 6-day-old males, for which the earliest sexual maturation was observed. In contrast, no 5-day-old males responded to ME (the probability was 12.6%), and the capture rate (the probability) of 6-day-old males was merely 0.3% (14.5%), while the rate gradually increased later. The present study revealed that certain males of B. correcta responded to CP prior to sexual maturation but not to ME under the laboratory conditions. These results imply that CP could be an effective attractant used in male annihilation techniques for B. correcta and that ME may not be useful.     

Differential regulation of phenylalanine ammonia-lyase genes during anthocyanin synthesis and by transfer effect in carrot cell suspension cultures

Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) catalyses the first step in the phenylpropanoid pathway and is induced during differentiation and by various stimuli. In carrot ( Daucus carota L. cv. Kurodagasun) suspension culture cells, PAL is slowly induced during anthocyanin synthesis which occurs in a medium lacking 2,4-dichlo-rophenoxyacetic acid and is also induced rapidly and transiently by transferring and diluting cells to fresh medium. Analyses of nucleotide sequences derived from PAL cDNAs revealed that the PAL mRNAs induced by transfer were transcribed from different carrot PAL genes than the PAL mRNAs induced during anthocyanin synthesis. Northern blotting using probes derived from 3'non-coding regions for PAL cDNAs confirmed that different PAL genes were induced during anthocyanin synthesis and after transfer. Induction of different PAL genes occurs in response to differences in the induction trigger.     

Atrial natriuretic peptide and angiotensin II binding sites in cerebral capillaries of spontaneously hypertensive rats

1. We carried out investigations on specific atrial natriuretic peptide (ANP) and angiotensin II (ANG) binding sites in capillaries isolated from the cerebral cortex of spontaneously hypertensive rats (SHR), an animal model of human essential hypertension, and also from Wistar Kyoto rats (WKY). 2. In an equilibrium binding study done in the presence of increasing concentrations of the radiolabeled ligands, the binding of 125I-rat alpha-ANP (1-28) [ANF-(99-126)] (125I-rANP) and 125I-ANG (5-L-isoleucine) (125I-ANG) to the cerebral capillaries was single and of a high affinity. 3. The maximum binding capacity (Bmax) and dissociation constant (Kd) in the 125I-rANP binding of 20-week-old, hypertensive SHR was significantly lower than in age-matched, normotensive WKY. Conversely, a significant increase in the Bmax of 125I-ANG binding of adult SHR was observed, with a significant decrease in the Kd. 4. There was no differences in the Bmax of 125I-rANP and 125I-ANG binding between 4-week-old, prehypertensive SHR and age-matched WKY. However, there was a significant decrease in the Kd of 125I-rANP binding of SHR. 5. As a dramatic change in the binding kinetics of 125I-rANP and 125I-ANG was noted in the cerebral capillaries of adult sustained-hypertensive SHR, the possibility that ANP and ANG play a role in the etiology of dysfunction of the blood-brain barrier complicated with hypertension, by interacting with specific receptors, would have to be considered.     

Overexpression of the gene encoding HMG-CoA reductase in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> for production of prenyl alcohols

To develop microbial production method for prenyl alcohols (e.g., (E,E)-farnesol (FOH), (E)-nerolidol (NOH), and (E,E,E)-geranylgeraniol (GGOH)), the genes encoding enzymes in the mevalonate and prenyl diphosphate pathways were overexpressed in Saccharomyces cerevisiae, and the resultant transformants were evaluated as to the production of these alcohols. Overexpression of the gene encoding hydroxymethylglutaryl (HMG)-CoA reductase was most effective among the genes tested. A derivative of S. cerevisiae ATCC 200589, which was selected through screening, was found to be the most suitable host for the production. On cultivation of the resultant transformant, in which the HMG-CoA reductase gene was overexpressed, in a 5-liter bench-scale jar fermenter for 7 d, the production of FOH, NOH, and GGOH reached 145.7, 98.8, and 2.46 mg/l, respectively.