Analysis of the non-covalent interaction between metal ions and the cysteine-rich domain of protein kinase C eta by electrospray ionization mass spectrometry

Effect of zinc and other metal ions on the folding of the protein kinase C (PKC) surrogate peptide (PKCeta-C1B) was analyzed intact under neutral conditions by electrospray ionization mass spectrometry (ESI-MS). ESI-MS spectrum of 64ZnCl(2)-folded PKCeta-C1B clearly showed that PKCeta-C1B coordinates specifically two atoms of zinc, and that the two thiol protons are lost in each zinc ion coordinate center. 113CdCl(2)-folded PKCeta-C1B also showed stoichiometry of two cadmium atoms that was proved by addition of EDTA. The dissociation constants of zinc- and cadmium-folded PKCeta-C1B in the phorbol 12,13-dibutyrate binding (PDBu) were similar (0.66 and 0.81 nM) with different B(max) values (46.4 and 71.4%). The difference would reflect higher coordination potency of cadmium ion that was demonstrated by ESI-MS when PKCeta-C1B was folded by 1:1 mixture of zinc and cadmium ions. In contrast, 63CuCl(2)-treated PKCeta-C1B did not show any copper-coordinated peak, instead a molecular mass less than 6 mass units smaller than that of apo-PKCeta-C1B was observed. The multiple charge mass envelope of copper-treated PKCeta-C1B shifted to that of the lower mass charge state like zinc-treated PKCeta-C1B. These data suggest that the copper treatment formed three intramolecular S-S bonds to abolish the PDBu binding of PKCeta-C1B.     

Oxygen- and carbon monoxide-binding kinetic parameters for liposomal heme under semi-physiological condition

Binding and dissociation rate constants of oxygen and carbon monoxide with the meso-tetra(α,α,α,α-(o-pivalamidophenyl))porphinato iron-mono(1-lauryl-2-methylimidazole) complex incorporated into the liposomes of dimyristoylphosphatidylcholine (liposomal heme) were measured with flash-photolysis method at pH 7.0 and 20 °C. Extra large quantum yield was observed for the photo-dissociation of the oxygen adduct, while that for the CO adduct was relatively small. Rate constants for the binding and the dissociation were comparable to those of hemoglobin except for the oxygen-binding rate constant.     

Superoxide dismutase activity of Helicobacter pylori per se from 158 clinical isolates and the characteristics

We investigated the correlation between the SOD activity of Helicobacter pylori (H. pylori) and gastroduodenal diseases and the characteristics of strains exposed to oxidative stress. Two sequenced strains, 26695 and J99, and clinical isolates from 156 Japanese patients with gastroduodenal diseases such as gastric cancer (n= 59) and non-cancer (n= 97) were used. SOD activities of all 158 isolates were measured and were divided into three groups: high-SOD activity (>0.22, n= 2), moderate-SOD activity (0.15≦≦0.22, n= 16) and low-SOD activity (<0.15, n= 140). Expressions of H. pylori Fe-SOD were examined by western blotting with anti-H. pylori Fe-SOD antibody prepared inhouse, and the profiles of Fe-SOD activity were investigated by zymogram with activity staining in native-PAGE. The characteristics of strains from high-SOD and low-SOD groups were examined under oxidative stress by paraquat. The average of H. pylori SOD activity was significantly higher in the cancer group than in the non-cancer group (P < 0.05). However, irrespective of SOD activity level, the amount of Fe-SOD expressed was variable among individual strains. Zymogram revealed a single band in moderate-SOD and low-SOD strains, but multiple bands in high-SOD strains were observed. These bands were confirmed as H. pylori Fe-SOD. Under oxidative stress with paraquat, low-SOD strains were drastically eliminated without inducible SOD activity; however, high-SOD strains were still viable with increased SOD activity. This study is the first to exhibit the characteristics of high-SOD activity strains representing multiple bands in zymograms and the correlation between H. pylori SOD activity and gastric cancer.     

ROOT GROWTH INHIBITING, a Rice Endo-1,4-β-d-Glucanase, Regulates Cell Wall Loosening and is Essential for Root Elongation

The molecular mechanism involved in cell wall dynamics has not been well clarified, although it is quite important for organ growth. We characterized a rice mutant, root growth inhibiting (rt), which is defective in root elongation. The rt mutant showed a severe defect in cell elongation at the root-elongating zone with additional collapse of epidermal and cortex cells at the root tip caused by the defect in the smooth exfoliation of root cap cells. Consistent with these phenotypes, expression of the RT gene, which encodes a member of the membrane-anchored endo-1,4-??-d-glucanase, was specifically localized in the root-elongating zone and at the junction between epidermal and root cap cells. The enzymatic analysis of root extracts from the wild-type and rt mutant indicated that RT hydrolyzes noncrystalline amorphous cellulose. The cellulose content was slightly increased but the crystallinity of cellulose was decreased in the rt root. In addition, the hemicellulose composition was different between wild-type and rt roots. The total extensibility was significantly lower in the rt root explants. Based on these results, we concluded that RT is involved in the disassembly of the cell wall for cell elongation in roots as well as for root cap exfoliation from the epidermal cell layer by hydrolyzing the noncrystalline amorphous cellulose fibers of cellulose microfibrils resulting in loosening of the hemicellulose and cellulose interaction.     

Modulation of plumbagin production in Plumbago zeylanica using a single-chain variable fragment antibody against plumbagin

A single-chain variable fragment antibody (scFv) against plumbagin (PL) accumulated the PL production in the hairy roots of Plumbago zeylanica. Recombinant Agrobacterium rhizogenes (ATCC 15834) containing an scFv gene against PL (PL-scFv) were obtained through triparental mating and transformed into P. zeylanica to induce PL-scFv protein in the hairy roots. Up to 40 μg recombinant PL-scFv were expressed per milligram of soluble protein in transgenic P. zeylanica hairy root cultures. The mean PL content obtained from transgenic hairy roots (12.24 μg/100 mg dry weight) exhibited 2.2 times higher than those obtained from wild-type (5.48 μg/100 mg dry weight). The high correlation between the PL-scFv expression level and PL content of the recombinant plants suggested that the PL biosynthesis pathway had been modulated by the expression of PL-scFv protein in the hairy roots of P. zeylanica.     

Colonisation of the oral cavity by periodontopathic bacteria in complete denture wearers

doi: 10.1111/j.1741‐2358.2011.00506.x Colonisation of the oral cavity by periodontopathic bacteria in complete denture wearers Objective: The purpose of this study was to investigate colonisation by periodontopathic bacteria and the sites of colonisation in elderly upper and lower complete denture wearers. We also investigated the relationship between level of oral hygiene and colonisation by periodontopathic bacteria. Materials and methods: Forty edentulous and 37 dentate volunteers participated in this study. Samples were collected from whole saliva, and levels of Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia and Fusobacterium nucleatum were determined by PCR Invader technology. Detection of these species on oral mucosal and denture surfaces was performed by PCR. Fisher’s exact test was used for the statistical analysis. Cluster analysis was employed to investigate trends in the periodontopathic bacteria flora in each sampling area. Results: Detection rates of periodontopathic bacteria in whole saliva were lower under edentulous conditions than under dentulous conditions, except for A. actinomycetemcomitans and F. nucleatum (p < 0.01). Detection rate of F. nucleatum was the highest in all areas. A positive correlation was observed between DNA quantification of P. gingivalis and number of Candida species in saliva. Cluster analysis of the test species identified two clusters. Tongue‐coating status was associated with the detection rate of all periodontopathic bacteria investigated, and denture plaque status was associated with the detection rate of T. denticola and F. nucleatum. Conclusion: Results indicate the presence of periodontopathic bacteria under edentulous conditions and that the status of oral hygiene of the mucosal or denture surfaces affects colonisation by T. denticola and F. nucleatum.     

Ionizing radiation induces mitochondrial reactive oxygen species production accompanied by upregulation of mitochondrial electron transport chain function and mitochondrial content under control of the cell cycle checkpoint

Whereas ionizing radiation (Ir) instantaneously causes the formation of water radiolysis products that contain some reactive oxygen species (ROS), ROS are also suggested to be released from biological sources in irradiated cells. It is now becoming clear that these ROS generated secondarily after Ir have a variety of biological roles. Although mitochondria are assumed to be responsible for this Ir-induced ROS production, it remains to be elucidated how Ir triggers it. Therefore, we conducted this study to decipher the mechanism of Ir-induced mitochondrial ROS production. In human lung carcinoma A549 cells, Ir (10 Gy of X-rays) induced a time-dependent increase in the mitochondrial ROS level. Ir also increased mitochondrial membrane potential, mitochondrial respiration, and mitochondrial ATP production, suggesting upregulation of the mitochondrial electron transport chain (ETC) function after Ir. Although we found that Ir slightly enhanced mitochondrial ETC complex II activity, the complex II inhibitor 3-nitropropionic acid failed to reduce Ir-induced mitochondrial ROS production. Meanwhile, we observed that the mitochondrial mass and mitochondrial DNA level were upregulated after Ir, indicating that Ir increased the mitochondrial content of the cell. Because irradiated cells are known to undergo cell cycle arrest under control of the checkpoint mechanisms, we examined the relationships between cell cycle and mitochondrial content and cellular oxidative stress level. We found that the cells in the G2/M phase had a higher mitochondrial content and cellular oxidative stress level than cells in the G1 or S phase, regardless of whether the cells were irradiated. We also found that Ir-induced accumulation of the cells in the G2/M phase led to an increase in cells with a high mitochondrial content and cellular oxidative stress level. This suggested that Ir upregulated mitochondrial ETC function and mitochondrial content, resulting in mitochondrial ROS production, and that Ir-induced G2/M arrest contributed to the increase in the mitochondrial ROS level by accumulating cells in the G2/M phase.     

Site-specific isotope labeling of long RNA for structural and mechanistic studies

A site-specific isotope labeling technique of long RNA molecules was established. This technique is comprised of two simple enzymatic reactions, namely a guanosine transfer reaction of group I self-splicing introns and a ligation with T4 DNA ligase. The trans-acting group I self-splicing intron with its external cofactor, 'isotopically labeled guanosine 5'-monophosphate' (5'-GMP), steadily gave a 5'-residue-labeled RNA fragment. This key reaction, in combination with a ligation of 5'-remainder non-labeled sequence, allowed us to prepare a site-specifically labeled RNA molecule in a high yield, and its production was confirmed with (15)N NMR spectroscopy. Such a site-specifically labeled RNA molecule can be used to detect a molecular interaction and to probe chemical features of catalytically/structurally important residues with NMR spectroscopy and possibly Raman spectroscopy and mass spectrometry.     

Characterization of Lactose Utilization and β-Galactosidase in Lactobacillus brevis KB290, the Hetero-Fermentative Lactic Acid Bacterium

Unlike dairy lactic acid bacteria, Lactobacillus brevis cannot ferment milk. We characterized the lactose utilization by L. brevis KB290. In a carbohydrate fermentation assay using API 50 CHL, we showed during 7?days L. brevis did not ferment lactose. L. brevis grew to the stationary phase in 2?weeks in MRS broth containing lactose as the carbon source. L. brevis slowly consumed the lactose in the medium. L. brevis hydrolyzed lactose and a lactose analog, o-nitrophenyl-β-d-galactopyranoside (ONPGal). This β-galactosidase activity for ONPGal was not repressed by glucose, galactose, fructose, xylose, or maltose showing the microorganism may not have carbon catabolite repression. We purified the L. brevis β-galactosidase using ammonium sulfate precipitation and several chromatographies. The enzyme’s molecular weight is estimated at 72 and 37?kDa using SDS-PAGE analysis. The N-terminal amino acid sequence of the larger protein was 90?% similar to the sequence of the putative β-galactosidase (YP_796339) and the smaller protein was identical to the sequence of the putative β-galactosidase (YP_796338) in L. brevis ATCC367. This suggests the enzyme is a heterodimeric β-galactosidase. The specific activity of the purified enzyme for lactose is 55?U/mg. We speculate inhibition of lactose transport delays the lactose utilization in L. brevis KB290.