Protective effect of quercetin and rutin on photosensitized lysis of human erythrocytes in the presence of hematoporphyrin

Photosensitized hemolysis of human erythrocytes by hematoporphyrin was suppressed by flavonols such as quercetin and rutin at submillimolar concentrations. The suppression of photohemolysis was accompanied by inhibition of lipid peroxidation by the reagents. Quercetin and rutin were photooxidized in the presence of hematoporphyrin and the photooxidation was partially suppressed by 1 mM NaN3, a quencher of singlet molecular oxygen. Flavonols were also oxidized by radicals formed during degradation of lauroyl peroxide. These results indicate that flavonols can function as antioxidants in biological systems by terminating radical chain reactions and removing singlet molecular oxygen. A pharmacological function of flavonols, decrease of the increased permeability and fragility of capillary, was discussed in relation to their antioxidative functions.     

Ascorbic Acid-Dependent Regulation of Redox Levels of Chlorogenic Acid and its Isomers in the Apoplast of Leaves of Nicotiana tabacum L.

There is a question whether ascorbic acid (AA) can control redoxlevels of phenolics in the apoplast. The present study was designedto answer this question. AA, dehydroascorbic acid (DHA), chlorogenicacid (CGA) and its two structural isomers were present in theapoplast of leaves of tobacco (Nicotiana tabacum L. cv. BelW3).The levels of AA plus DHA (AA + DHA) and the ratios of AA to(AA + DHA) decreased while the levels of CGA plus its isomersincreased during leaf aging. o-Quinones of CGA plus its isomerswere found in the apoplast only in aged leaves of which apoplasticlevel of AA was nearly zero. In addition, activity of apoplasticperoxidase that could oxidize CGA and its isomers increasedduring leaf aging. From the observations, it is concluded thatAA can regulate the accumulation of the o-quinones of CGA andits isomers in the apoplast. Based on the conclusion, it isproposed that soluble peroxidase in the apoplast has two functions,namely, (i) scavenging of H₂O₂ and/or regulation of the levelof apoplastic H₂O₂ in the presence of AA, and (ii) accumulationof oxidation products of the phenolics in the absence of AA. (Received January 30, 1998; Accepted April 7, 1998)     

Analysis of endogenous pyrrolidine levels by mass fragmentography

Pyrrolidine, one of biogenic volatile amines, possesses nicotine-like synaptotropic actions on the nervous systems. In the present study, pyrrolidine levels in the tissues were examined by using mass fragmentographic technique. High concentrations of pyrrolidine were found in the seminal vesicle and lung of rabbits. Only trace amounts of pyrrolidine existed in the brain of mice and rats, although higher concentrations were detected in the brain of rabbits. In the rat brain, however, high levels of pyrrolidine were found in the pineal gland, pituitary gland and corpus striatum.     

Generation of Hydroxyl Radical in Isolated Pea Root Cell Wall, and the Role of Cell Wall-Bound Peroxidase, Mn-SOD and Phenolics in Their Production

The hydroxyl radical produced in the apoplast has been demonstratedto facilitate cell wall loosening during cell elongation. Cellwall-bound peroxidases (PODs) have been implicated in hydroxylradical formation. For this mechanism, the apoplast or cellwalls should contain the electron donors for (i) H₂O₂ formationfrom dioxygen; and (ii) the POD-catalyzed reduction of H₂O₂to the hydroxyl radical. The aim of the work was to identifythe electron donors in these reactions. In this report, hydroxylradical (·OH) generation in the cell wall isolated frompea roots was detected in the absence of any exogenous reductants,suggesting that the plant cell wall possesses the capacity togenerate ·OH in situ. Distinct POD and Mn-superoxidedismutase (Mn-SOD) isoforms different from other cellular isoformswere shown by native gel electropho-resis to be preferably boundto the cell walls. Electron paramagnetic resonance (EPR) spectroscopyof cell wall isolates containing the spin-trapping reagent,5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO),was used for detection of and differentiation between ·OHand the superoxide radical (O₂^–·). The data obtainedusing POD inhibitors confirmed that tightly bound cell wallPODs are involved in DEPMPO/OH adduct formation. A decreasein DEPMPO/OH adduct formation in the presence of H₂O₂ scavengersdemonstrated that this hydroxyl radical was derived from H₂O₂.During the generation of ·OH, the concentration of quinhydronestructures (as detected by EPR spectroscopy) increased, suggestingthat the H₂O₂ required for the formation of ·OH in isolatedcell walls is produced during the reduction of O₂ by hydroxycinnamicacids. Cell wall isolates in which the proteins have been denaturated(including the endogenous POD and SOD) did not produce ·OH.Addition of exogenous H₂O₂ again induced the production of ·OH,and these were shown to originate from the Fenton reaction withtightly bound metal ions. However, the appearance of the DEPMPO/OOHadduct could also be observed, due to the production of O₂^–·when endogenous SOD has been inactivated. Also, O₂^–·was converted to ·OH in an in vitro horseradish peroxidase(HRP)/H₂O₂ system to which exogenous SOD has been added. Takentogether with the discovery of the cell wall-bound Mn-SOD isoform,these results support the role of such a cell wall-bound SODin the formation of ·OH jointly with the cell wall-boundPOD. According to the above findings, it seems that the hydroxycinnamicacids from the cell wall, acting as reductants, contribute tothe formation of H₂O₂ in the presence of O₂ in an autocatalyticmanner, and that POD and Mn-SOD coupled together generate ·OHfrom such H₂O₂.     

Pan-neuronal knockdown of the c-Jun N-terminal Kinase (JNK) results in a reduction in sleep and longevity in Drosophila

Sleep is a unique behavioral state that is conserved between species, and sleep regulation is closely associated to metabolism and aging. The fruit fly, Drosophila melanogaster has been used to study the molecular mechanism underlying these physiological processes. Here we show that the c-Jun N-terminal Kinase (JNK) gene, known as basket (bsk) in Drosophila, functions in neurons to regulate both sleep and longevity in Drosophila. Pan-neuronal knockdown of JNK mRNA expression by RNA interference resulted in a decrease in both sleep and longevity. A heterozygous knockout of JNK showed similar effects, indicating the molecular specificity. The JNK knockdown showed a normal arousal threshold and sleep rebound, suggesting that the basic sleep mechanism was not affected. JNK is known to be involved in the insulin pathway, which regulates metabolism and longevity. A JNK knockdown in insulin-producing neurons in the pars intercerebralis had slight effects on sleep. However, knocking down JNK in the mushroom body had a significant effect on sleep. These data suggest a unique sleep regulating pathway for JNK.     

Dual-function of thiocyanate on nitrite-induced formation of reactive nitrogen oxide species in human oral cavity: Inhibition under neutral and enhancement under acidic conditions

Salivary nitrate is reduced to nitric oxide (NO) via nitrite in the human oral cavity. The nitrite and NO formed can be transformed to reactive nitrogen oxide species (RNOS). In this investigation, RNOS formed in mixed whole saliva and its fractions were detected by the oxidation of aminophenyl fluorescein (APF) and the transformation of 3-amino-4-monomethylamino-2′,7′-difluorofluorecein (DAF-FM) to its triazol form (DAF-FMT). Nitrite-induced oxidation of APF and formation of DAF-FMT increased as pH was decreased from 7 to 5 and SCN^? inhibited the oxidation of APF and the formation of DAF-FMT around neutral pH and enhanced at pH about 5. The SCN^?-dependent inhibition was due to the suppression of salivary peroxidase and the enhancement was due to the formation of NOSCN from HNO₂ and SCN^?. It is deduced that the increase in the concentrations of nitrite and H⁺ in the oral cavity may result in the enhanced formation of RNOS.     

Characterization of a caspase-3-substrate kinome using an N- and C-terminally tagged protein kinase library produced by a cell-free system

Caspase-3 (CASP3) cleaves many proteins including protein kinases (PKs). Understanding the relationship(s) between CASP3 and its PK substrates is necessary to delineate the apoptosis signaling cascades that are controlled by CASP3 activity. We report herein the characterization of a CASP3-substrate kinome using a simple cell-free system to synthesize a library that contained 304 PKs tagged at their N- and C-termini (NCtagged PKs) and a luminescence assay to report CASP3 cleavage events. Forty-three PKs, including 30 newly identified PKs, were found to be CASP3 substrates, and 28 cleavage sites in 23 PKs were determined. Interestingly, 16 out of the 23 PKs have cleavage sites within 60 residues of their N- or C-termini. Furthermore, 29 of the PKs were cleaved in apoptotic cells, including five that were cleaved near their termini in vitro. In total, approximately 14% of the PKs tested were CASP3 substrates, suggesting that CASP3 cleavage of PKs may be a signature event in apoptotic-signaling cascades. This proteolytic assay method would identify other protease substrates.     

Biodegradation of 2,4,6-tribromophenol by Ochrobactrum sp. strain TB01

A bacterium that utilizes 2,4,6-tribromophenol (2,4,6-TBP) as sole carbon and energy source was isolated from soil contaminated with brominated pollutants. This bacterium, designated strain TB01, was identified as an Ochrobactrum species. The organism degraded 100 microM of 2,4,6-TBP within 36 h in a growing culture. In addition, it released 3 mol of bromine ions from 1 mol of 2,4,6-TBP during the complete degradation of 2,4,6-TBP in a resting cell assay. Moreover, cells grown on 2,4,6-TBP degraded 2,6-dibromophenol (2,6-DBP), 4-bromophenol (4-BP), 2,4,6-trichlorophenol (2,4,6-TCP) and phenol. Metabolic intermediates were detected in the reaction mixture of an in vitro assay for 2,4,6-TBP, and they were identified as 2,4-DBP and 2-BP. NADH was required for the debromination of 2,4,6-TBP. These results suggest that 2,4,6-TBP is converted to phenol through sequential reductive debromination reactions via 2,4-DBP and 2-BP by this strain.     

In vivo treatment of class II MHC-deficient mice with anti-TCR antibody restores the generation of circulating CD4 T cells and optimal architecture of thymic medulla

TCR ligation by the self-peptide-associated MHC molecules is essential for T cell development in the thymus, so that class II MHC-deficient mice do not generate CD4(+)CD8(-) T cells. The present results show that the administration of anti-TCR mAb into class II MHC-deficient mice restores the generation of CD4(+)CD8(-) T cells in vivo. The CD4 T cells were recovered in the thymus, peripheral blood, and the spleen, indicating that the anti-TCR treatment is sufficient for peripheral supply of newly generated CD4 T cells. Unlike peripheral CD4 T cells that disappeared within 5 wk after the treatment, CD4(+)CD8(-) thymocytes remained undiminished even after 5 wk, suggesting that CD4 T cells in the thymus are maintained separately from circulating CD4 T cells and even without class II MHC molecules. It was also found that the mass of medullary region in the thymus, which was reduced in class II MHC-deficient mice, was restored by the anti-TCR administration, suggesting that the medulla for CD4(+)CD8(-) thymocytes is formed independently of the medulla for CD4(-)CD8(+) thymocytes. These results indicate that in vivo anti-TCR treatment in class II MHC-deficient mice restores the generation of circulating CD4 T cells and optimal formation of the medulla in the thymus, suggesting that anti-TCR Ab may be useful for clinical treatment of class II MHC deficiencies.     

Construction of male and female PAC genomic libraries suitable for identification of Y-chromosome-specific clones from the liverwort, Marchantia polymorpha

Unlike higher plants, the dioecious liverwort, Marchantia polymorpha, has uniquely small sex chromosomes, with X chromosomes present only in female gametophytes and Y chromosomes only in male gametophytes. We have constructed respective genomic libraries for male and female plantlets using a P1-derived artificial chromosome (pCYPAC2). With an average insert size of approximately 90 kb, each PAC library is estimated to cover the entire genome with a probability of more than 99.9%. Male-specific PAC clones were screened for by differential hybridization using male and female genomic DNAs as separate probes. Seventy male-specific PAC clones were identified. The male specificity of one of the clones, pMM4G7, was verified by Southern hybridization and PCR analysis. This clone was indeed located on the Y chromosome as verified by fluorescence in situ hybridization (FISH). This result shows that the Y chromosome contains unique sequences that are not present either on the X chromosome or any of the autosomes. Thus, the respective male and female libraries for M. polymorpha offer an opportunity to identify key genes involved in the process of sex differentiation and this unique system of sex determination.