Enhancement of Nitrogen Dioxide-Induced Lipid Peroxidation and Dna Strand Breaking by Cysteine and Glutathione

Nitrogen dioxide less than 100 ppm in air induced lipid peroxidation of liposome composed of l-palmitoyl-2-arachidonylphosphatidylcholine as assessed by thiobarbituric acid reactivity. The nitrogen dioxide-induced lipid peroxidation was enhanced by cysteine, glutathione and bovine serum albumin. While the activity of nitrogen dioxide in air to induce single strand breaks of supercoiled plasmid DNA was low, the breaking was remarkably enhanced by cysteine, glutathione and bovine serum albumin. ESR spin trapping using 5,5-dimethyl-1-pyrroline N-oxide showed that certain strong oxidant(s) were generated by interaction of nitrogen dioxide and cysteine. The spin trapping using 3,5-dibromo-4-nitrosobenzene-sulfonate suggested that sulfur-containing radicals were generated by interaction of nitrogen dioxide and cysteine or glutathione. Hence, certain sulfur-containing radicals generated by the interaction which could effectively induce lipid peroxidation and DNA strand breaks.     

Corticosteroidogenesis in the hypertrophic adrenal gland produced by betamethasone 17, 21-dipropionate in the rat fetus

Betamethasone 17, 21-dipropionate (BMDP), a potent glucocorticoid, produces adrenal hypertrophy in the rat fetus. The present study was performed to investigate the possible alterations of corticosteroidogenesis due to endogeneous substrates or exogenous pregnenolone in the incubation of homogenates of fetal hypertrophic adrenals caused by BMDP given to pregnant rats at day 19 of pregnancy.The corticosteroidal products and those levels per mg homogenate in an incubate of the hypertrophic adrenal homogenate did not differ from those of a normal adrenal. No accumulations of abnormal precursors or intermediates were found in the incubates of the hypertrophic adrenals. It is concluded from these findings that no qualitative alterations in the pathway of corticosteroidogenesis occurred in the hypertrophic adrenal glands caused by BMDP in the rat fetus. When the calculation was done per adrenal gland, the content of corticosterone in the incubate of the homogenate of the hypertrophic adrenal was remarkably higher than that found in a normal gland. This finding was compatible with the significant increase of the plasma corticosterone concentration in the fetuses with the adrenal hypertrophy caused by BMDP.     

Evidence of increased synthesis of δ-aminolevulinic acid dehydratase in experimental lead-poisoned rats

δ-Aminolevulinic acid dehydratase (porphobilinogen synthase; 5-aminolevulinate hydro-lyase, EC 4.2.1.24) was purified from rat and rabbit erythrocytes to a homogeneous state. Specific activities were 26.0 and 26.6 units/mg protein for the rat and rabbit enzymes, respectively, and their estimated molecular weight was 280 000, each consisting of 8 subunits of M_r 35 000. In order to quantitate rat δ-aminolevulinic acid dehydratase at several stages of lead-poisoning, a radioimmunoassay technique using goat antiserum against the rat enzyme was developed for the first time. This technique was specific, reproducible and high sensitive allowing determination of 1 ng enzyme. When drinking water containing 25 mM lead acetate was given daily to rats ad lib. the δ-aminolevulinic acid dehydratase activity in the blood, assayed without any pretreatment, decreased to 8% of the control level on the next day. On the contrary, the restored enzyme activity, assayed in the presence of Zn²⁺ and dithiothreitol, was greater than normal by the fourth day of lead administration in bone-marrow cells and by the ninth day in the peripheral blood. The increased activity level stayed the same from the ninth day onward. The enzyme content as determined directly by the radioimmunoassay technique at this stage was about 2-fold above that the control. There was no significant difference in the number of reticulocytes and the distribution profile of different types of reticulocytes between the lead-exposed and non-exposed rats. Therefore, the increase in the amount of δ-aminolevulinic acid dehydratase in erythrocytes of lead-poisoned rats was suggested to be due to an increased rate of synthesis in the bone-marrow cells.     

Aggregatibacter actinomycetemcomitans induces detachment and death of human gingival epithelial cells and fibroblasts via elastase release following leukotoxin‐dependent neutrophil lysis

Aggregatibacter actinomycetemcomitans is considered to be associated with periodontitis. Leukotoxin (LtxA), which destroys leukocytes in humans, is one of this bacterium's major virulence factors. Amounts of neutrophil elastase (NE), which is normally localized in the cytoplasm of neutrophils, are reportedly increased in the saliva of patients with periodontitis. However, the mechanism by which NE is released from human neutrophils and the role of NE in periodontitis is unclear. In the present study, it was hypothesized that LtxA induces NE release from human neutrophils, which subsequently causes the breakdown of periodontal tissues. LtxA‐treatment did not induce significant cytotoxicity against human gingival epithelial cells (HGECs) or human gingival fibroblasts (HGFs). However, it did induce significant cytotoxicity against human neutrophils, leading to NE release. Furthermore, NE and the supernatant from LtxA‐treated human neutrophils induced detachment and death of HGECs and HGFs, these effects being inhibited by administration of an NE inhibitor, sivelestat. The present results suggest that LtxA mediates human neutrophil lysis and induces the subsequent release of NE, which eventually results in detachment and death of HGECs and HGFs. Thus, LtxA‐induced release of NE could cause breakdown of periodontal tissue and thereby exacerbate periodontitis.     

Subnuclear localization and antitransforming activity of N-myc:beta-galactosidase fusion proteins.

N-myc expression is under stage- and tissue-specific regulation in mammalian development, but its function is totally unknown. We sought agents to block N-myc activity in order to infer from the effect the possible function of N-myc in the apparently complex processes. As candidates for such agents, we tested fusion genes encoding N-myc:beta-galactosidase fusion proteins for their effects on the formation of transformed foci of rat embryo primary fibroblasts as the result of transfection with N-myc and activated H-ras. One of the gene constructs very efficiently antagonized N-myc activity, as assessed by its effect on focus formation, but did not appreciably affect cell viability. The product of this gene was not only targeted to the nucleus but also accumulated in subnuclear loci which may represent the sites where normal N-myc proteins reside. The occurrence of antagonistic effect at a low stoichiometric ratio suggested that the fusion protein gene competed with the N-myc gene in a fashion analogous to a dominant negative mutation.     

MiR-376c Down-Regulation Accelerates EGF-Dependent Migration by Targeting GRB2 in the HuCCT1 Human Intrahepatic Cholangiocarcinoma Cell Line

MicroRNA miR-376c was expressed in normal intrahepatic biliary epithelial cells (HIBEpiC), but was significantly suppressed in the HuCCT1 intrahepatic cholangiocarcinoma (ICC) cell line. The biological significance of the down-regulation of miR-376c in HuCCT1 cells is unknown. We hypothesized that miR-376c could function as a tumor suppressor in these cells. To test this hypothesis, we sought the targets of miR-376c, and characterized the effect of its down-regulation on HuCCT1 cells. We performed proteomic analysis of miR-376c-overexpressing HuCCT1 cells to identify candidate targets of miR-376c, and validated these targets by 3′-UTR reporter assay. Transwell migration assays were performed to study the migratory response of HuCCT1 cells to miR-376c overexpression. Furthermore, microarrays were used to identify the signaling that were potentially involved in the miR-376c-modulated migration of HuCCT1. Finally, we assessed epigenetic changes within the potential promoter region of the miR-376c gene in these cells. Proteomic analysis and subsequent validation assays showed that growth factor receptor-bound protein 2 (GRB2) was a direct target of miR-376c. The transwell migration assay revealed that miR-376c significantly reduced epidermal growth factor (EGF)-dependent cell migration in HuCCT1 cells. DNA microarray and subsequent pathway analysis showed that interleukin 1 beta and matrix metallopeptidase 9 were possible participants in EGF-dependent migration of HuCCT1 cells. Bisulfite sequencing showed higher methylation levels of CpG sites upstream of the miR-376c gene in HuCCT1 relative to HIBEpiC cells. Combined treatment with the DNA-demethylating agent 5-aza-2′-deoxycytidine and the histone deacetylase inhibitor trichostatin A significantly upregulated the expression of miR-376c in HuCCT1 cells. We revealed that epigenetic repression of miR-376c accelerated EGF-dependent cell migration through its target GRB2 in HuCCT1 cells. These findings suggest that miR-376c functions as a tumor suppressor. Since metastasis is the major cause of death in ICC, microRNA manipulation could lead to the development of novel anti-cancer therapy strategies for ICC.     

Purification of a fibrolast-inhibitory factor from Actinobacillus actinomycetemcomitans Y4

Abstract A factor showing inhibitory activity against human gingival fibrolasts was extracted from the cytosol fraction of Actinobacillus actinomycetemcomitans Y4. The activity markedly inhibited the proliferation of human gingival fibrolasts, but had no effect on cell viability or gross morphology. No such activity was found in cytosol fractions from either Porphyromonas gingivalis 381 or Escherichia coli HB101. The extract from A. actinomycetemcomitans Y4 was then purified by anion-exchange chromatography, hydroxyapatite chromatography and gel-filtration chromatography to give a single band on SDS-PAGE with an apparent molecular mass of 65 kDa. The purification ratio was 183-fold with a recovery rate of 5% compared with the crude extract (starting material) when the activity was assessed by direct cell counts.     

Feedbacks between nutrient cycling and vegetation predict plant species coexistence and invasion

To investigate the role of species‐specific litter decomposability in determining plant community structure, we constructed a theoretical model of the codevelopmental dynamics of soil and vegetation. This model incorporates feedback between vegetation and soil. Vegetation changes the nutrient conditions of soil by affecting mineralization processes; soil, in turn, has an impact on plant community structure. The model shows that species‐level traits (decomposability, reproductive and competitive abilities) determine whether litter feedback effects are positive or negative. The feedback determines community‐level properties, such as species composition and community stability against invasion. The model predicts that positive feedback may generate multiple alternative steady states of the plant community, which differ in species richness or community composition. In such cases, the realized state is determined by initial abundance of co‐occurring species. Further, the model shows that the importance of species‐level traits depends on environmental conditions such as system fertility.     

Requirement of caspase and p38MAPK activation in zinc-induced apoptosis in human leukemia HL-60 cells.

Zinc (Zn), an endogenous regulator of apoptosis, and has abilities both to induce apoptosis and inhibit the induction of apoptosis via the modulation of caspase activity. Due to the multifunctions of Zn, the intracellular Zn level is strictly regulated by a complex system in physiological and pathological conditions. The commitment of Zn to the regulation of apoptosis is not fully understood. In the present study, we investigated the role of intracellular Zn level in the induction of apoptosis in human leukemia cells (HL-60 cells) using a Zn ionophore [pyrithione (Py)]. Treatment of HL-60 cells with Zn for 6 h in the presence of Py (1 micro m) exhibited cytotoxicity in a Zn dose-dependent manner (25-200 micro m). Necrotic cells, assayed by trypan blue permeability, increased in number in a Zn dose-dependent fashion (50-100 micro m), but the appearance of apoptotic cells, assayed by formation of a DNA ladder and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling method, peaked at 25 micro m, suggesting the dependence of intracellular Zn level on the execution of apoptosis. In fact, treatment with Py resulted in increases in intracellular Zn levels, and N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine, a cell-permeable Zn chelator, inhibited DNA ladder formation induced by Py/Zn treatment (1 micro m Py and 25 micro m Zn). Py/Zn treatment activated the caspases, as assessed by the proteolysis of poly(ADP-ribose) polymerase (PARP), which is a substrate of caspase, and activated p38 mitogen-activated protein kinase (p38MAPK), which is a transducer of apoptotic stimuli to the apparatus of the apoptosis execution. Z-Asp-CH2-DCB, a broad-spectrum inhibitor of caspase, attenuated proteolysis of PARP and DNA ladder formation by Py/Zn, indicating that apoptosis induced by Py/Zn is mediated by caspase activation. The p38MAPK-specific inhibitor SB203580 also inhibited induction of apoptosis by Py/Zn. Although SB203580 suppressed the proteolysis of PARP, Z-Asp-CH2-DCB did not inhibit the phosphorylation of p38MAPK, raising the possibility that apoptosis triggered by Py/Zn might be mediated by the p38MAPK/caspase pathway.     

Effects of Exogenous Amino Acids on Iron Uptake in Relation to their Effects on Photoperiodic Flowering in Lemna paucicostata 6746

The flowering of Lemna paucicostata 6746 grown on 14-h photoperiodwas enhanced by the addition of high concentrations of ironto the medium, which also increased the endogenous iron concentration.The addition of asparagine, aspartate, glutamate, -alanine,glycine or serine to the medium also increased the endogenousiron level, resulting in the promotion of flowering. In contrast,the addition of cysteine, cystine, glutamine, arginine, threonineor phenylalanine lowered the endogenous iron level, resultingin the inhibition of flowering. Glycine and asparagine added to the medium during an inductive96-h dark period did not promote iron uptake and had no effecton flowering, but when added during the subsequent 120-h lightperiod, they promoted both iron uptake and flowering response.The increase in the endogenous iron level seems to favor floraldevelopment rather than induction of photoperiodic floweringof Lemna paucicostata 6746. (Received September 8, 1986; Accepted March 31, 1987)