Plastid-to-nucleus retrograde signals are essential for the expression of nuclear starch biosynthesis genes during amyloplast differentiation in tobacco BY-2 cultured cells.

Amyloplasts, a subtype of plastid, are found in nonphotosynthetic tissues responsible for starch synthesis and storage. When tobacco (Nicotiana tabacum) Bright Yellow-2 cells are cultured in the presence of cytokinin instead of auxin, their plastids differentiate from proplastids to amyloplasts. In this program, it is well known that the expression of nucleus-encoded starch biosynthesis genes, such as ADP-Glucose Pyrophosphorylase (AgpS) and Granule-Bound Starch Synthase (GBSS), is specifically induced. In this study, we investigated the roles of plastid gene expression in amyloplast differentiation. Microarray analysis of plastid genes revealed that no specific transcripts were induced in amyloplasts. Nevertheless, amyloplast development accompanied with starch biosynthesis was drastically inhibited in the presence of plastid transcription/translation inhibitors. Surprisingly, the expression of nuclear AgpS and GBSS was significantly repressed by the addition of these inhibitors, suggesting that a plastid-derived signal(s) that reflects normal plastid gene expression was essential for nuclear gene expression. A series of experiments was performed to examine the effects of intermediates and inhibitors of tetrapyrrole biosynthesis, since some of the intermediates have been characterized as candidates for plastid-to-nucleus retrograde signals. Addition of levulinic acid, an inhibitor of tetrapyrrole biosynthesis, resulted in the up-regulation of nuclear AgpS and GBSS gene expression as well as starch accumulation, while the addition of heme showed opposite effects. Thus, these results indicate that plastid transcription and/or translation are required for normal amyloplast differentiation, regulating the expression of specific nuclear genes by unknown signaling mechanisms that can be partly mediated by tetrapyrrole intermediates.

Cloning, expression, and characterization of cytosolic sulfotransferase isozymes from Drosophila melanogaster.

We have identified four cytosolic sulfotransferase (SULT) homologs in the genome database of Drosophila melanogaster, and have designated these genes dmST1-4. Each of these four isozymes was subsequently classified into a different and novel gene family, as determined by the low amino acid sequence homology (less than 40%) between them, and also toward their vertebrate homologs. The transcripts for these four SULT homologs were detectable at all developmental stages in D. melanogaster. In addition, three of these isozymes, the exception being dmST2, were successfully expressed and purified in Escherichia coli. These recombinant dmST1, 3, and 4 products showed high sulfating activity toward phenolic compounds such as vanillin and 4-nitrophenol, but showed no activity toward typical endogenous substrates such as tyramine and serotonin. DmST4 and dmST3 showed the lowest and highest K(m) values for 3'-phosphoadenosine-5'-phosphosulfate (PAPS) respectively. DmST4 also showed low but not negligible activity toward 20-hydroxyecdysone.

A novel subpopulation lacking Oct4 expression in the testicular side population.

We characterized murine spermatogonial stem cells (SSCs) using a multi-parameter selection strategy, combining Oct4 expression determined by monitoring green fluorescent protein (GFP) expression, and the testicular side population (SP) showing weak fluorescence on Hoechst 33342 dye staining, as markers of stem cell purification. Testicular cells were collected from Oct4/GFP transgenic mice and analyzed using a fluorescence-activated cell sorter (FACS). SP was detected in testicular cell suspensions at an average rate of 0.10%. Multicolor analysis indicated that 96% of SP cells were negative for Oct4. The cells did not express SSC marker genes, but expressed Bcrp1. While the main population was 93% positive for pyronin Y staining, this was limited to 51% in SP. We found a novel subpopulation with reduced RNA content lacking Oct4 expression in testicular SP. These results suggest that the cells isolated by FACS represent a novel population of SSCs in the G0 quiescent state.

Antioxidant effects of hydroxybenzalacetones on peroxynitrite-induced lipid peroxidation in red blood cell membrane ghost and SOS response in Salmonella typhimurium TA4107/pSK1002.

Antioxidant activity of a series of hydroxybenzalacetones was determined against peroxynitrite-induced lipid peroxidation in red blood cell membrane and SOS response through DNA damage in bacterial cells. Hydroxybenzalacetone derivatives with hydroxy, methoxy, ethoxy or methyl substitution were analyzed and found to be more effective than the water-soluble vitamin E analogue Trolox. The inhibitory effect against lipid peroxidation correlated well to that against the SOS response, which is dependent on decomposition of peroxynitrite by hydroxybenzalacetones outside of the cell membrane. The antioxidant activity was shown to correlate well with the electric parameter sigma+. Electron-donating substituents with more negative sigma+ values increased the potencies. The result suggests that hydroxybenzalacetones with more electron-donating substituents will protect tissue more effectively against oxidative stress.

Peroxidase-like catalytic activity of anion-exchange resins modified with metal-porphyrins in oxidative reaction of hetrocyclic amines with hydrogen peroxide.

To elucidate the peroxidase (POD)-like catalytic activity of anion-exchange resins modified with metal-tetrakis(sulfophenyl)porphine (M-TSPP(r)s), an oxidative reaction of seven mutagenic heterocyclic amines (HCAs) with hydrogen peroxide, which reaction is catalyzed by horse radish POD, was investigated in the presence of M-TSPP(r)s. Among six M-TSPP(r)s tested, Mn- and Fe-TSPP(r)s were found to have a relatively strong POD-like activity for HCAs, in particular for a typical HCA, 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ). The optimal condition for the POD-like activity was selected using Fe- and Mn-TSPP(r)s. For evaluation of an oxidation product of IQ produced in the presence of Fe-TSPP(r), the absorption, NMR and FAB-mass spectra thereof were compared with those of an oxidation product of IQ produced by horse radish POD or a chemical oxidizing agent, sodium hypochlorite. When Fe-TSPP(r) was present as a catalysts, IQ was converted into the dimmer (hydorazone type) which has no mutagenic activity in umu-test. It was revealed that Fe- and Mn-TSPP(r)s exhibit a POD-like catalytic activity in oxidative reaction of HCAs with hydrogen peroxide.

Quantitative structure-activity relationship studies for antioxidant hydroxybenzalacetones by quantum chemical- and 3-D-QSAR(CoMFA) analyses.

Antioxidant activities for a series of hydroxybenzalacetones, OH-BZ, evaluated by their inhibitory potencies against lipid peroxidation induced by gamma-ray irradiation or t-BuOOH, were analyzed quantitatively using quantum-chemical parameters calculated by semi-empirical molecular orbital (MO) calculations. The energy of the highest occupied molecular orbital (E(HOMO)) and frontier electron densities (HOMO) on the phenolic oxygen atom (F(H,O)), together with the steric parameter (E(s)) for the substituent ortho to the phenolic oxygen, showed excellent correlations. We also performed 3D-QSAR studies by using the comparative molecular field analysis (CoMFA) model. The results were compared with the corresponding classical QSAR correlations.

Quantitative structure-activity relationship analyses of antioxidant and free radical scavenging activities for hydroxybenzalacetones.

Antioxidant activities for a series of hydroxybenzalacetones, OH-BZ, were evaluated by measuring inhibitory potencies of OH-BZ against lipid peroxidation induced by t-BuOOH or gamma-irradiation. Their quantitative structure-activity relationship (QSAR) studies indicated that the activities are mainly governed by electronic and steric factors. To rationalize these results, we also performed QSAR analyses for DPPH radical scavenging activities of OH-BZ, which indicated that antioxidant and radical scavenging activities could be expressed by the same physicochemical parameters but the hydrogen bonding behavior of phenolic OH varies with the reaction medium.

Inhibitory effects of water-soluble cationic manganese porphyrins on peroxynitrite-induced SOS response in Salmonella typhimurium TA4107/pSK1002.

We have investigated the protective effects of water-soluble cationic Mn(III) porphyrins against peroxynitrite (ONOO-)-induced DNA damage in the cells of Salmonella typhimurium TA4107/pSK1002 and lipid peroxidation of red blood cell membranes. Mn(III) tetrakis (N-methylpyridinium-4-yl) porphine (TMPyP) and the brominated form, Mn(III) octabromo-tetrakis (N-methylpyridinium-4-yl) porphine (OBTMPyP) effectively reduced the damage and peroxidation induced by N-morpholino sydnonimine (SIN-1), which gradually generates ONOO- from O2*- and *NO produced through hydrolysis. Mn(III)OBTMPyP became 10-fold more active than the non-brominated form. In the presence of authentic ONOO-, the Mn(III) porphyrins were ineffective against damage and strongly enhanced lipid peroxidation, while the coexistence of ascorbic acid inhibited peroxidation. Using a diode array spectrophotometry, the reactions of Mn(III)TMPyP with authentic ONOO- and SIN-1 were measured. Mn(III)TMPyP is known to be catalytic for ONOO- decomposition in the presence of antioxidants. OxoMn(IV)TMPyP with SIN-1 was rapidly reduced back to Mn(III) without adding any oxidants. Further, in the SIN-1 system, the concentration of NO2- and NO3- were colorimetrically determined by Griess reaction based on the two-step diazotization. NO2- increased by addition of Mn(III) porphyrin and the ratio of NO2- to NO3- was 4-7 times higher than that (1.05) of SIN-1 alone. This result suggests that O2*- from SIN-1 acts as a reductant and *NO cogenerated is oxidized to NO2-, a primarily decomposition product of *NO. Under the pathological conditions where biological antioxidants are depleted and ONOO- and O2*- are extensively generated, the Mn(III) porphyrins will effectively cycle ONOO- decomposition using O2*-.

HPLC retention behavior of poly-aromatic-hydrocarbons on aminopropyl silica gels modified with Cu(II)- and Ni(II)-phthalocyanine derivatives in non-polar eluent.

A comparative study was conducted to elucidate the mechanism underlying the separation of poly-aromatic-hydrocarbons (PAHs) and related compounds thereof on a column packed with silica gels modified with Ni(II)- or Cu(II)-phthalocyanine derivatives (PCS) (Ni- or Cu-PCS(D) column) and commercially available PYE and NPE columns with a non-polar eluent, such as n-hexane. It has been revealed that the dominant interaction responsible to the separation of PAHs on the Cu-PCS(D) and the PYE columns with n-hexane is the pi-pi interaction; however, in the separation of PAHs having 4 rings such as pyrene on the Ni-PCS(D) column, participation of pi-d interaction was indicated. The predominant role of pi-pi interaction in the separation of PAHs of less than three rings on the Ni-PCS(D) column was demonstrated using anthracene. All the columns possessed planar recognition ability and were estimated to be potentially useful in the separation and the analysis of PAHs.

pi-Electron interaction of PAHs with anion-exchange silica gels modified with anionic metal-porphine and -phthalocyanine derivatives as HPLC stationary phase for preparative column in organic solvents.

To develop easy-to-prepare stationary phases for HPLC, we investigated anion-exchange silica gels, Nucleosil 5SB (Nuc), modified with metal-porphines and -phthalocyanines (M-P). The modified silica gels (M-P(N)) were evaluated for the availability as a stationary phase of HPLC for the separation of pi-electron-rich polyaromatic hydrocarbons (PAHs) in polar and non-polar eluents. Separation ability of silica gels modified with Cu-phthalocyanine derivative (Cu-PCS(N)) was comparable to that of the silica gels binding Cu-PCS through sulfonamide bonds; however, the latter requires troublesome procedures for the preparation. The PAHs tested interact with Cu-PCS(N) in non-polar organic eluents through their pi-electrons similarly as in the case of the PYE column((R)), in which interaction with PAHs was reported to be only the pi-pi-electron interaction.

Flow injection analysis of hydrogen peroxide using glass-beads modified with manganese(III)-tetra(4-carboxyphenyl)porphine derivative and its analytical application to the determination of serum glucose.

A flow injection analysis system of hydrogen peroxide was developed. The present system is based on measuring of the absorbance of a quinoid dye formed by the following reaction catalyzed by peroxidase: Phenol + 4-Aminoantipyrine + 2H2O2 --> Peroxidase --> Quinoid dye + 4H2O. A column packed with aminopropyl-glass beads modified with amanganese(III)-tetra(4-carboxyphenyl)porphine derivative (Mn-TCPP(G) column), which has peroxidase-like activity, was used in place of an immobilized peroxidase column in the above reaction. The linear range of the calibration curve was 0.4-80 microg/ml hydrogen peroxide. The relative standard deviation of this system was 2.97% (n = 100, 10 microg/ml hydrogen peroxide 20 microl injection). The Mn-TCPP(G) column has sufficiently stability for the continuous injection of hydrogen peroxide untill 100 times. The advantageous feature of the Mn-TCPP(G) column was a less-electrostatic interaction between the mother glass beads and the anionic chromogen or quinoid dye formed and the stability in terms of the storage, temperature and moisture. The determination of serum glucose was achieved by attaching an immobilized glucose oxidase column to this system without deproteinization.

Flow injection analysis of ascorbic acid using carriers modified with metal-porphine as oxidative solid catalyses.

To investigate one of practical applications of the supports modified with metal-porphines as artificial solid-catalysts, columns into which the supports were packed were supplied to catalytic columns for a flow injection analysis (FIA) system for determination of ascorbic acid (AsA) by the following reactions: AsA+O(2)-->dehydoroAsA+H(2)O(2),H(2)O(2)+chromogen-->2H(2)O+Dye. Among the columns tested, the column containing silica gels modified with Co-tetrakis(carboxyphenyl)porphine catalyzed most rapidly the oxidation reaction of AsA that is accompanied by the formation of hydrogen peroxide. The resulting hydrogen peroxide was determined by FIA system equipped with the column containing glass beads modified with Mn-tetrakis(carboxyphenyl)porphine, which gave a linear calibration curve and large peak-areas of the range corresponding to AsA concentration between 0.2 and 10 micromol/ml. The results indicated that some supports modified with metal-porphine would be applicable to the FIA for AsA as the solid catalyses which function as if the immobilized enzymes.

Photoreaction generating active oxygens of In(3+)-tetrakis(4-methylpyridyl)-porphine in the presence of albumins.

The complex formation of In(3+)-tetrakis(4-N-methylpyridyl)-porphine (In-TMPyP) with albumin was studied by resonance Raman spectroscopy. Albumin coordinated to In(3+) through the -S(-) group(s). The photoreaction was investigated using the visible spectral change and In-TMPyP-thiourea complex was used as a model. It was demonstrated that the complex in a weak basic solution (pH 8.5) is excited by light and the excited complex converts oxygen to superoxide anion, which finally cleavages the porphine ring of In-TMPyP.

Quantum chemical- and 3-D-QSAR (CoMFA) studies of benzalacetones and 1,1,1-trifluoro-4-phenyl-3-buten-2-ones.

The inhibitory effect (IC(50)) of the title compounds on UV-induced mutagenesis in Escherichia coli WP2uvrA was analyzed quantitatively by using various quantum chemical descriptors and also by the CoMFA method: both approaches provided results of similar quality. The activity was shown to be increased by electron-withdrawing substituents and also by hydrogen-bonding between 2-hydroxy group and the bio-component.

Induction of SOS response in Salmonella typhimurium TA4107/pSK1002 by peroxynitrite-generating agent, N-morpholino sydnonimine.

Salmonella typhimurium TA4107/pSK1002 strain was used to measure the SOS response induced by peroxynitrite. The parent strain TA4107 (oxydelta1[oxydelta(oxyR argH)1]) is sensitive to oxidative stress and the plasmid of pSK1002 carries a fused gene umuC'-'lacZ, in which umu and lacZ genes are involved in the induction of mutagenesis and beta-galactosidase activity, respectively. Therefore, the level of SOS response was monitored via beta-galactosidase activity. A bolus addition of authentic peroxynitrite (0.3-0.6 mM) increased about eight times the enzyme activity. In N-morpholino sydnonimine (SIN-1), which produces peroxynitrite from superoxide and nitric oxide generated through hydrolysis, addition of over 1mM SIN-1 induced four-five-fold activity. The SIN-1-induced SOS response was scarcely influenced by superoxide dismutase (SOD), catalase or a combination of both, removing the possibility of induction by superoxide, hydrogen peroxide and hydroxyl radical. Two types of peroxynitrite scavengers, mannitol (type I) and glutathione (type II), decreased the response. Mannitol showed a constant inhibition (70%) at a concentration up to 20 mM, exhibiting kinetics that are zero-order in mannitol and first-order in peroxynitrite. On the other hand, glutathione sharply reduced the response dependent on concentration up to 2 mM (90%), indicating second-order kinetics, first-order in both glutathione and peroxynitrite. Dihydrorhodamine (DHR)123, which traps peroxynitrite in a molar ratio of 1:1, efficiently inhibited the SOS response. These effects suggest that peroxynitrite, generated gradually from SIN-1, penetrates through the cell membrane, damages the DNA and induces the SOS response. This strain can thus, be used in screening of antioxidants against peroxynitrite-induced DNA damage in cells.

Quantitative structure--activity relationships of antimutagenic benzalacetones and 1,1,1-trifluoro-4-phenyl-3-buten-2-ones.

The antimutagenic activities (IC(50)) of benzalacetones (BZ) and 1,1,1-trifluo-4-phenyl-3-buten-2-ones (TF) against UV-induced mutagenesis in Escherichia coli WP2s(uvrA trpE) were quantitatively analyzed in terms of physicochemical parameters by regression analyses. Structural requirements for maximal potency were derived from the results of quantitative structure--activity relationship (QSAR) analyses: (1) ring substituents should be electron-withdrawing; (2) 2-OH substituents incapable of intramolecular hydrogen-bonding notably increase the potency; and (3) replacement of CH(3) group by CF(3) in the side chain enhances the activity. Contrary to our expectations, the best correlation lacked hydrophobic effects. Antimutagenic activities against gamma-induced mutagenesis in Salmonella typhimurium TA2638 were also studied for some derivatives in the BZ series, where, in addition to electronic and hydrogen-bonding factors, a hydrophobic term was also significant. Physicochemical meanings of the derived correlations are discussed.