Anticarcinogenic antioxidants as inhibitors against intracellular oxidative stress

Oxidative stress has been implicated in the pathogenesis of numerous diseases, including cancer. In the present study, the protective effect of natural antioxidants, such as quercetin and tea polyphenols, on intracellular oxidative stress was studied. Here we report a novel function of quercetin and tea polyphenols, as potential inhibitors of 4-hydroxy-2-nonenal (HNE)-induced intracellular oxidative stress and cytotoxicity. In rat liver epithelial RL34 cells, a potent electrophile HNE dramatically induced the productions of reactive oxygen species (ROS), which correlated well with the reduction in cell viability. We found that quercetin and tea polyphenols, such as epigallocatechin gallate and theaflavins and their gallate esters, significantly inhibited the HNE-induced ROS production and cytotoxicity. In addition, HNE induced a transient decrease in the mitochondrial membrane potential (Δψ), which was also retarded by the antioxidants. These data suggest that the antioxidants, such as quercetin and tea polyphenols, are inhibitors against mitochondrial ROS production.     

Chronic Alterations in Monoaminergic Cells in the Locus Coeruleus in Orexin Neuron-Ablated Narcoleptic Mice

Narcolepsy patients often suffer from insomnia in addition to excessive daytime sleepiness. Narcoleptic animals also show behavioral instability characterized by frequent transitions between all vigilance states, exhibiting very short bouts of NREM sleep as well as wakefulness. The instability of wakefulness states in narcolepsy is thought to be due to deficiency of orexins, neuropeptides produced in the lateral hypothalamic neurons, which play a highly important role in maintaining wakefulness. However, the mechanism responsible for sleep instability in this disorder remains to be elucidated. Because firing of orexin neurons ceases during sleep in healthy animals, deficiency of orexins does not explain the abnormality of sleep. We hypothesized that chronic compensatory changes in the neurophysiologica activity of the locus coeruleus (LC) and dorsal raphe (DR) nucleus in response to the progressive loss of endogenous orexin tone underlie the pathological regulation of sleep/wake states. To evaluate this hypothesis, we examined firing patterns of serotonergic (5-HT) neurons and noradrenergic (NA) neurons in the brain stem, two important neuronal populations in the regulation of sleep/wakefulness states. We recorded single-unit activities of 5-HT neurons and NA neurons in the DR nucleus and LC of orexin neuron-ablated narcoleptic mice. We found that while the firing pattern of 5-HT neurons in narcoleptic mice was similar to that in wildtype mice, that of NA neurons was significantly different from that in wildtype mice. In narcoleptic mice, NA neurons showed a higher firing frequency during both wakefulness and NREM sleep as compared with wildtype mice. In vitro patch-clamp study of NA neurons of narcoleptic mice suggested a functional decrease of GABAergic input to these neurons. These alterations might play roles in the sleep abnormality in narcolepsy.     

Random Phage Display-Based Screening of Peptides that Bind to Botulinum Neurotoxin Binding Protein, Nontoxic Nonhemagglutinin

Botulinum neurotoxin (BoNT) binds to nontoxic nonhemagglutinin (NTNHA) protein in a pH-dependent manner, and yields the protease-resistant BoNT/NTNHA complex. Here, we screened short peptides that bind to the serotype D NTNHA (NTNHA-D) using random phage display technique. NTNHA was fixed onto electrode of quartz crystal microbalance (QCM) apparatus, and then the phages displaying random heptapeptides were exposed to the NTNHA-D under the acidic condition. After rinsing with acidic buffer, the released phages under the alkaline condition were collected. The binding and release of the phage were monitored by the frequency shift on the QCM. As a result of the screening, 16 were selected as peptides that bind to NTNHA-D. The selected peptides do not share any conserved sequence, but tend to be rich in basic and/or hydrophobic amino acid. This would explain the binding manner of the BoNT to the NTNHA protein.     

Hybrid Oligomer of Cyclonucleotides and Deoxynucleotides. A High Anti Left-handed Double Helical DNA Structure

Abstract

It has been shown by us that oligonucleotides containing cyclonucleosides with a high anti glycosidic conformation take left-handed, single and double helical structures (S. Uesugi, J. Yano, E. Yano and M. Ikehara, J. Am. Chem. Soc. 99, 2313 (1977) and references therein). In order to see whether DNA can adopt the high anti left-handed double helical structure or not, a self-complementary hexanucleotide containing 6,2′-O-cyclocytidine (C^○), 8,2′-O-cyclo- guanosine (G^○), deoxycytidine and deoxyguanosine, C^○G^{○dCdGC ○}G^○, was synthesized. Corresponding hexanucleotide containing only cyclonucleosides, C^○G^○C^○G^○C^○G^○, was also synthesized. Their conformation was examined by UV, CD and ¹H NMR spectroscopy. C^○G^○C^○G^○C^○G^○ forms an unusually stable, left-handed duplex. Imino proton NMR spectra and the results of nuclear Overhauser effect experiments strongly suggest that C^○G^○dCdGC^○G^○ take a left-handed double helical structure where the deoxynucleoside residues are involved in hydrogen bonding and take a high anti glycosidic conformation. Thus it is revealed that DNA could form a high anti, left-handed double helix which is different from that of Z-DNA under some constrained conditions.     

Inhibition of the First Step in Synthesis of the Mycobacterial Cell Wall Core,Catalyzed by the GlcNAc-1-phosphate Transferase WecA,by the Novel Caprazamycin Derivative CPZEN-45

Because tuberculosis is one of the most prevalent and serious infections, countermeasures against it are urgently required. We isolated the antitubercular agents caprazamycins from the culture of an actinomycete strain and created CPZEN-45 as the most promising derivative of the caprazamycins. Herein, we describe the mode of action of CPZEN-45 first against Bacillus subtilis. Unlike the caprazamycins, CPZEN-45 strongly inhibited incorporation of radiolabeled glycerol into growing cultures and showed antibacterial activity against caprazamycin-resistant strains, including a strain overexpressing translocase-I (MraY, involved in the biosynthesis of peptidoglycan), the target of the caprazamycins. By contrast, CPZEN-45 was not effective against a strain overexpressing undecaprenyl-phosphate–GlcNAc-1-phosphate transferase (TagO, involved in the biosynthesis of teichoic acid), and a mutation was found in the tagO gene of the spontaneous CPZEN-45-resistant strain. This suggested that the primary target of CPZEN-45 in B. subtilis is TagO, which is a different target from that of the parent caprazamycins. This suggestion was confirmed by evaluation of the activities of these enzymes. Finally, we showed that CPZEN-45 was effective against WecA (Rv1302, also called Rfe) of Mycobacterium tuberculosis, the ortholog of TagO and involved in the biosynthesis of the mycolylarabinogalactan of the cell wall of M. tuberculosis. The outlook for WecA as a promising target for the development of antituberculous drugs as a countermeasure of drug resistant tuberculosis is discussed.     

Endogenous abscisic acid is involved in methyl jasmonate-induced reactive oxygen species and nitric oxide production but not in cytosolic alkalization in Arabidopsis guard cells

We recently demonstrated that endogenous abscisic acid (ABA) is involved in methyl jasmonate (MeJA)-induced stomatal closure in Arabidopsis thaliana. In this study, we investigated whether endogenous ABA is involved in MeJA-induced reactive oxygen species (ROS) and nitric oxide (NO) production and cytosolic alkalization in guard cells using an ABA-deficient Arabidopsis mutant, aba2-2, and an inhibitor of ABA biosynthesis, fluridon (FLU). The aba2-2 mutation impaired MeJA-induced ROS and NO production. FLU inhibited MeJA-induced ROS production in wild-type guard cells. Pretreatment with 0.1 μM ABA, which does not induce stomatal closure in the wild type, complemented the insensitivity to MeJA of the aba2-2 mutant. However, MeJA induced cytosolic alkalization in both wild-type and aba2-2 guard cells. These results suggest that endogenous ABA is involved in MeJA-induced ROS and NO production but not in MeJA-induced cytosolic alkalization in Arabidopsis guard cells.     

Effects of Detergents on Lysis of Micrococcus lysodeikticus by Lysozyme

The effects of detergents on the lysozyme-catalyzed hydrolysis of Micrococcus lysodeikticus cells were investigated by changing the concentration of Na-phosphate buffer and pH in the presence or absence of sucrose. Also, a parallel study of the hydrolysis of glycolchitin by lysozyme was conducted and compared to the lytic reaction. Electron microscopy was utilized to follow the changes in cell morphology during the various treatments.

None of the detergents changed turbidity of the cell suspension. However, they did affect the change in turbidity during lysis in unique ways. SDS, which is an anionic detergent, inhibited lysozyme activity and its addition to the reaction mixture caused a rapid and large decrease in the turbidity. Brij 35 and Triton X-100, which are non-ionic detergents, did not inhibit lysozyme activity, but their presence in the reaction mixture changed the rate of turbidity change. Apparently non-ionic detergents disrupt only the protoplast, while anionic detergents disrupt both the protoplast and the damaged cell. The lytic mechanism of M. lysodeikticus by lysozyme was discussed in detail.     

Chitin Coated Cellulose as an Adsorbent of lysozyme like Enzymes Preparation and Properties

As a new adsorbent of lysozyme-like enzymes, chitin coated (CC-)cellulose was prepared. CC-cellulose was stable and had good flow properties for use in column chromatography. Affinity chromatography with CC-cellulose showed that 3~5 mg of lysozyme/ml resin was adsorbed specifically and desorbed quantitatively under mild conditions. The utilities of the method of affinity chromatography with CC-cellulose are discussed.     

Studies on Erythorbic Acid Production by Fermentation

Screening was carried out for erythorbic acid (EA)-producing strains from about 5,000 newly isolated fungi and bacteria. Penicillium notatum FY 115 was screened out as most powerful EA producer. Only Penicillium, but no other genera, was obtained as EA producers from our screening program. Monospore selections and mutagenic treatments succeeded to elevate the yield of EA over 40% to glucose supplied. Various cultural conditions were studied, and pH change during fermentation process was proved to be most important for favorable EA production. Over 80% yield could be obtained when washed mycelium was used in dilute glucose solution.

Abundant accumulation of EA by the strain FY 115, Penicillium sp., in fermentation broth was studied, and EA, both free and Na-salt, was obtained as crystal in the yield of about 45% to glucose supplied, in the media of 8% glucose by jar fermentor, in considering the inhibitory effect of some metal ion.

Extraction processes were improved to elevate the yield and was developed the continuous multi-bed extraction system of anion-exchange resin, which resulted in the yield of 90.9% of EA from fermentation broth in sum total.