Hydrogen peroxide induces GADD153 in Jurkat cells through the protein kinase C-dependent pathway

Growth arrest and DNA damage-inducible gene 153 (GADD153) is a CCAAT/enhancer binding protein (C/EBP) related gene and is induced in response to various stimuli including DNA damaging agents, UV irradiation, and serum starvation. In this study, we investigated which intracellular signals contribute to the expression of GADD153 mRNA in Jurkat cells in response to oxidative stress using several kinds of kinase inhibitors. GADD153 mRNA expression was immediately enhanced following hydrogen peroxide exposure and was significantly inhibited by treatment with H-7, staurosporin, and Ro-31-8220. In particular, rottlerin, a PKCdelta specific inhibitor, markedly attenuated hydrogen peroxide-induced GADD153 mRNA expression even at 1 microM. Treatment with a potent PKC activator, phorbol-12-myristate-13-acetate (PMA), augmented GADD153 mRNA in Jurkat cells in the presence of hydrogen peroxide, although PMA alone induced GADD153 mRNA marginally. Hydrogen peroxide significantly enhanced the AP-1 binding activity of the nuclear extract from Jurkat cells to the GADD153 AP-1 binding site. AP-1 binding activity was suppressed by rottlerin treatment. These findings indicate that PKC, especially PKCdelta, plays an important role in the induction of GADD153 mRNA following oxidative stress.     

A novel and potent biological antioxidant, Kinobeon A, from cell culture of safflower

Kinobeon A was originally isolated from cultured cells of safflower (Carthamus tinctorius L., Compositae). It had never previously been directly isolated from safflower or other plants, animals or microorganisms. In this report, we demonstrate the anti-oxidative effects of kinobeon A and compare the results with those two known natural antioxidants, lignan (nordihydroguaiaretic acid) and quercetin. The NADPH-induced microsomal lipid peroxidation system was employed to assess anti-oxidative effects of kinobeon A. Addition of kinobeon A to the system significantly decreased the formation of thiobarbituric acid reactive substances (TBARS) in a dose-dependent manner with effects similar to those of lignan and quercetin. Formation of TBARS was completely inhibited at 10 microM of kinobeon A. Employing the xanthine/xanthine oxidase/nitroblue tetrazolium system and the KO2/XTT system, the superoxide anion scavenging activity of kinobeon A was greater than that of lignan or quercetin. IC50 values calculated for kinobeon A in these two systems were 1 microM and 0.8 microM, respectively. Kinobeon A exerted cytoprotective effects following oxidative treatments with hydrogen peroxide, cumene hydroperoxide, menadione and xanthine oxidase (XOD). Addition of kinobeon A to the systems markedly enhanced survival ratios of Madin-Darby bovine kidney cells, while their survival significantly decreased with the oxidative treatment alone. Kinobeon A exhibited stronger effect on the cell viability than lignan or quercetin when menadion or XOD were used as inducing reagents of oxidative stress. The present study demonstrates for the first time that kinobeon A prevents oxidative stresses and could be a useful cytoprotective reagent.     

Sequence of the gene for Clostridium botulinum type B neurotoxin associated with infant botulism,expression of the C-terminal half of heavy chain and its binding activity

Previously, we demonstrated that the neurotoxin of strain 111 (111/NT) associated with type B infant botulism showed antigenic and biological properties different from that (Okra/NT) produced by a foodborne botulism-related strain, Okra. In this study, the neurotoxin genes of 111/NT and Okra/NT were amplified and the sequences determined. The nucleotide sequences of the genes for both neurotoxins possessed an open reading frame of 3873 bp that encoded 1291 amino acids. The identities of nucleotide sequences and amino acid sequences were 97.6% and 95.7%, respectively. The ratio of nonsynonymous to synonymous substitutions was 0.47. The amino acid substitutions between 111/NT and Okra/NT occurred mainly in the domain of the C-terminal half of heavy chain (H(C)) responsible for binding to its receptor complex of protein and ganglioside. To characterize the binding capability of the H(C), recombinant genes for the H(C) and two hybrid H(C) in which one half of Okra/NT was replaced by the homologous half of 111/NT were constructed and expressed in Escherichia coli. The binding activity of the recombinant H(C) of 111/NT to the protein receptor synaptotagmin II, in the presence of ganglioside GT1b, was 4.2-fold less than Okra/NT, consistent with the corresponding two NTs. The use of hybrid H(C) revealed that mutation of 23 residues in carboxy terminal half of H(C) (1029-1291) of Okra/NT could be attributed to the lower binding activity of 111/NT and thus the differences in binding affinity between the two BoNT/B.     

Specific RNA interference in psbP genes encoded by a multigene family in Nicotiana tabacum with a short 3'-untranslated sequence

RNA interference with double-stranded RNA is a new method for the study of gene function in various organisms. In this report, we show that an inverted repeat of a short (103-bp) 3'-untranslated sequence of an isogene, 1A, of psbP genes, encoded by a small multigene family of four genes (1A, 2AF, 3F, and 5B) in Nicotiana tabacum, can specifically suppress the expression of psbP isogenes 1A and 5B with a 3'-untranslated sequence similar to a transcribed double-stranded RNA. The expression of other psbP isogenes, 2AF and 3F, was not affected, although the coding sequences of the psbP family genes are highly conserved. Consistent with this observation, small interfering RNAs were detected for the 3'-untranslated sequence used for the inverted-repeat transgene, and not for the coding sequence. These results suggest that double-stranded RNA having a 3'-untranslated sequence could be useful for an isogene-specific RNA interference of the family genes in Nicotiana tabacum.     

Sulindac sulfide is a noncompetitive gamma-secretase inhibitor that preferentially reduces Abeta 42 generation

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been known to reduce risk for Alzheimer's disease. In addition to the anti-inflammatory effects of NSAIDs to block cylooxygenase, it has been shown recently that a subset of NSAIDs selectively inhibits the secretion of highly amyloidogenic Abeta42 from cultured cells, although the molecular target(s) of NSAIDs in reducing the activity of gamma-secretase for Abeta42 generation (gamma(42)-secretase) still remain unknown. Here we show that sulindac sulfide (SSide) directly acts on gamma-secretase and preferentially inhibits the gamma(42)-secretase activity derived from the 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate-solubilized membrane fractions of HeLa cells, in an in vitro gamma-secretase assay using recombinant amyloid beta precursor protein C100 as a substrate. SSide also inhibits activities for the generation of Abeta40 as well as for Notch intracellular domain at higher concentrations. Notably, SSide displayed linear noncompetitive inhibition profiles for gamma(42)-secretase in vitro. Our data suggest that SSide is a direct inhibitor of gamma-secretase that preferentially affects the gamma(42)-secretase activity.     

Mouse germ cell-less as an essential component for nuclear integrity

A mouse homologue of the Drosophila melanogaster germ cell-less (mgcl-1) gene is expressed ubiquitously, and its gene product is localized to the nuclear envelope based on its binding to LAP2 beta (lamina-associated polypeptide 2 beta). To elucidate the role of mgcl-1, we analyzed two mutant mouse lines that lacked mgcl-1 gene expression. Abnormal nuclear morphologies that were probably due to impaired nuclear envelope integrity were observed in the liver, exocrine pancreas, and testis. In particular, functional abnormalities were observed in testis in which the highest expression of mgcl-1 was detected. Fertility was significantly impaired in mgcl-1-null male mice, probably as a result of severe morphological abnormalities in the sperm. Electron microscopic observations showed insufficient chromatin condensation and abnormal acrosome structures in mgcl-1-null sperm. In addition, the expression patterns of transition proteins and protamines, both of which are essential for chromatin remodeling during spermatogenesis, were aberrant. Considering that the first abnormality during the process of spermatogenesis was abnormal nuclear envelope structure in spermatocytes, the mgcl-1 gene product appears to be essential for appropriate nuclear-lamina organization, which in turn is essential for normal sperm morphogenesis and chromatin remodeling.     

DNA polymorphism at the ACAULIS5 locus of the wild plant Arabidopsis thaliana

Nucleotide variation in the ACL5 gene region, which encodes spermine synthase, was analyzed for 21 Arabidopsis thaliana ecotypes and one accession of Arabis gemmifera. In A. thaliana, dimorphism was also detected in the ACL5 region, as in other nuclear genes of this plant. The nucleotide diversity (pi) of the entire region, exon and intron was 0.0163, 0.0042 and 0.0293, respectively. The level of nucleotide variation in this region was among the highest of those reported for genes in this plant species. The neutrality tests of Tajima, and Fu and Li did not detect significant deviation from test assumptions for the polymorphism data. However, the HKA test indicated that the level of polymorphism in the intron was significantly high, compared with A. gemmifera. The high nucleotide variation in the intron is responsible for the high level of nucleotide variation in the entire region. These results can be explained by elevated mutation rate in the ACL5 region in the A. thaliana lineage after the two species were split.     

Development of filamentous green algae in the benthic algal community in a littoral sand-beach zone of Lake Biwa

Temporal changes of biomass and dominant species in benthic algal communities were investigated in a littoral sand-beach zone in the north basin of Lake Biwa from December 1999 to September 2000. Chlorophyll-a amounts of benthic algal communities per unit area of the sandy sediments rapidly increased from late April to June. Increases in biomass of the benthic algal communities are considered to result from the propagation of filamentous green algae Oedogonium sp. and Spirogyra sp. The cell numbers of filamentous green algae and chlorophyll-a amounts of benthic algal communities at depths of 30 and 50cm at a station protected by a breakwater in May were significantly higher than those of a station exposed directly to wave activity. Thus, the biomass accumulation of the benthic algal communities seems to be regulated strongly by wave disturbance. The development of filamentous green algae may contribute to the increase in biomass of the benthic algal community and to the changes in seasonal patterns of biomass in the sand-beach zone of Lake Biwa. We consider that the development of the filamentous green algal community in the littoral zone of Lake Biwa is the result of eutrophication.     

Effect of Selective Brain Hypothermia on Regional Cerebral Blood Flow and Tissue Metabolism Using Brain Thermo-Regulator in Spontaneously Hypertensive Rats

To investigate the effect of selective hypothermia of the brain (brain cooling) on regional cerebral blood flow and tissue metabolism, we have developed a brain thermo-regulator. Brain temperature was modulated by a water-cooled metallic plate placed on the surface of the rats' scalp to get the appropriate brain temperature precisely with ease. Regional cerebral blood flow and brain temperature were measured simultaneously using a Teflon-coated platinum electrode and thermocouple probe inserted stereotaxically into the parietal cortex and thalamus in spontaneously hypertensive rats. Experimental forebrain ischemia was induced by the occlusion of bilateral common carotid artery under normo- and hypothermic brain condition, and the supratentorial brain tissue metabolites were measured enzymatically after 60 min of forebrain ischemia. When cortical temperature was set to hypothermia, cortical blood flow was significantly lowered by 40% at 30°C and 20% at 33°C as compared with that at 36°C (p < 0.0001 and p < 0.05, respectively). Thalamic blood flow was also significantly reduced by 20% when cortical temperature was set to 30°C as compared with 36°C (p < 0.05). There were no significant differences in arterial blood pressure and gas parameters throughout these experiments. In the rats with selective brain hypothermia (30°C) immediately after the induction of cerebral ischemia, the level of brain ATP concentration after 60 min of ischemia was significantly higher than that in normothermia rats (36°C) (p < 0.05). Our findings indicate that: 1) the metallic plate brain thermo-regulator is useful in small animal experiments; 2) regional brain temperature regulates regional cerebral blood flow; and 3) selective brain hypothermia, even started after the forebrain ischemia, ameliorates the derangement of brain metabolism, suggesting its effectiveness as a cytoprotective strategy.     

Regulation of reactive oxygen species by Atm is essential for proper response to DNA double-strand breaks in lymphocytes

The ataxia telangiectasia-mutated (ATM) gene plays a pivotal role in the maintenance of genomic stability. Although it has been recently shown that antioxidative agents inhibited lymphomagenesis in Atm(-/-) mice, the mechanisms remain unclear. In this study, we intensively investigated the roles of reactive oxygen species (ROS) in phenotypes of Atm(-/-) mice. Reduction of ROS by the antioxidant N-acetyl-l-cysteine (NAC) prevented the emergence of senescent phenotypes in Atm(-/-) mouse embryonic fibroblasts, hypersensitivity to total body irradiation, and thymic lymphomagenesis in Atm(-/-) mice. To understand the mechanisms for prevention of lymphomagenesis, we analyzed development of pretumor lymphocytes in Atm(-/-) mice. Impairment of Ig class switch recombination seen in Atm(-/-) mice was mitigated by NAC, indicating that ROS elevation leads to abnormal response to programmed double-strand breaks in vivo. Significantly, in vivo administration of NAC to Atm(-/-) mice restored normal T cell development and inhibited aberrant V(D)J recombination. We conclude that Atm-mediated ROS regulation is essential for proper DNA recombination, preventing immunodeficiency, and lymphomagenesis.