Evidence for variable metal-radical spin coupling in oxoferrylporphyrin cation radical complexes

Oxoferrylporphyrin cation radical complexes were generated by m-chloroperoxybenzoic acid oxidation of the chloro and trifluoromethanesulfonato complexes of tetramesitylporphyrinatoiron(III) [(TMP)Fe] and the trifluoromethanesulfonato complex of tetra(2,6-dichlorophenyl)porphyrinatoiron(III) [TPP(2,6-Cl)Fe]. Coupling between ferryl iron (S = 1) and porphyrin radical (S' = 1/2) spin systems was investigated by M?ssbauer and EPR spectroscopy. The oxoferrylporphyrin cation radical systems generated from the TMP complexes show strong ferromagnetic coupling. Analysis of the magnetic M?ssbauer spectra, using a spin Hamiltonian explicitly including a coupling tensor J, suggests an exchange-coupling constant J greater than 80 cm-1. The EPR spectra show non-zero rhombicity, the origin of which is discussed in terms of contributions from the usual zero-field effects of iron and from iron-radical spin-dipolar interaction. A consistent estimate of zero-field splitting parameter D approximately + 6 cm-1 was obtained by EPR and M?ssbauer measurements. EPR and M?ssbauer parameters are shown to be slightly dependent on solvent, but not on the axial ligand in the starting (TMP)Fe complex. In contrast to the TMP complex, the oxoferrylporphyrin cation radical system generated from [TPP(2,6-Cl)FeOSO2CF3] exhibits M?ssbauer and EPR spectra consistent with weak iron-porphyrin radical coupling of magnitude of J approximately 1 cm-1.     

Chemopreventive potential of 3-[2,6-bis(4-fluorophenyl)-3-methylpiperidin-4-ylideneamino]-2-thioxoimidazolidin-4-one on 7,12-dimethylbenz[a]anthracene (DMBA) induced hamster buccal pouch carcinogenesis

In the present work, a new bis heterocyclic compound comprising both the piperidone and thiohydantoin nuclei namely 3-[2,6-bis(4-fluorophenyl)-3-methylpiperidin-4-ylideneamino]-2-thioxoimidazolidin-4-one was synthesised and characterised with the help of mp, elemental analysis, FT-IR, MS and one-dimensional NMR (¹H and ¹³C) spectra. The inhibitory effect of 3-[2,6-bis(4-fluorophenyl)-3-methylpiperidin-4-ylideneamino]-2-thioxoimidazolidin-4-one on 7,12-dimethylbenz[a]anthracene (DMBA) induced buccal pouch carcinogenesis was investigated in Syrian male hamsters. All the hamsters that were painted with DMBA on their buccal pouches for 14 weeks developed squamous cell carcinoma. Administration of 3-[2,6-bis(4-fluorophenyl)-3-methylpiperidin-4-ylideneamino]-2-thioxoimidazolidin-4-one effectively suppressed the oral carcinogenesis initiated with the DMBA as revealed by a reduced incidence of neoplasms. Lipid peroxidation, glutathione (GSH) content and the activities of glutathione peroxidase (GPx), glutathione S-transferase (GST) were used to biomonitor the chemopreventive potential of 3-[2,6-bis(4-fluorophenyl)-3-methylpiperidin-4-ylideneamino]-2-thioxoimidazolidin-4-one. Lipid peroxidation was found to be significantly decreased, whereas GSH, GPx, GST and GGT were elevated in the oral mucosa of tumour bearing animals. Our data suggest that 3-[2,6-bis(4-fluorophenyl)-3-methylpiperidin-4-ylideneamino]-2-thioxoimidazolidin-4-one may exert its chemopreventive effects in the oral mucosa by modulation of lipid peroxidation, antioxidants and detoxification systems.     

Synthesis and mass spectra of 4-O-acetyl-1,5-anhydro-2,3,6-tri-O -(methoxycarbonylmethyl)-D-glucitol and the positional isomers of 4- O-acetyl-1,5-anhydro-di-O-(methoxycarbonylmethyl)-O-methyl-D-glucitol and 4-O-acetyl-1,5-anhydro-O-(methoxycarbonylmethyl)-di-O-methyl-D-glucitol .

Reductive cleavage of fully methylated, partially O-carboxymethylated cellulose had previously been shown to produce 4-O-acetyl-1,5-anhydro-2,3,6-tri-O-methyl-, -2-O-(methoxycarbonylmethyl)-3,6-di-O-methyl-, -3-O-(methoxycarbonylmethyl)-2,6-di-O-methyl-, -6-O-(methoxycarbonylmethyl)-2,3-di-O-methyl-, -2,3-di-O-(methoxycarbonylmethyl)-6-O-methyl-, -2,6-di-O-(methoxycarbonylmethyl)-3-O-methyl-, -3,6-di-O-(methoxycarbonylmethyl)-2-O-methyl-, and -2,3,6-tri-O-(methoxycarbonylmethyl)-D-glucitol. Described herein is the independent synthesis of these derivatives, except for the first, which had been reported. In addition, their 1H-n.m.r. spectra, chemical-ionization (NH3) mass spectra, and electronionization mass spectra are tabulated.     

A comparative study of water soluble 5,10,15,20-tetrakis(2,6-dichloro-3-sulfophenyl)porphyrin and its metal complexes as efficient sensitizers for photodegradation of phenols.

5,10,15,20-Tetrakis(2,6-dichloro-3-chlorosulfophenyl)porphyrin and its tin and zinc complexes were synthesized with high yields and fully characterized. The corresponding water-soluble 5,10,15,20-tetrakis(2,6-dichloro-3-sulfophenyl)porphyrins were obtained by hydrolysis with water. An extensive photophysical study of the new water soluble porphyrinic compounds was carried out including absorption and fluorescence spectra, fluorescence quantum yields, triplet absorption spectra, triplet lifetimes, triplet and singlet oxygen quantum yields. These sensitizers were successfully used in the photodegradation of 4-chlorophenol and 2,6-dimethylphenol. A comparison is made of their efficiencies, and some mechanistic considerations are highlighted.     

Circular dichroism of N-phenylnaphthylamine derivatives complexed with the β-form of poly(L-lysine)

Complex formation of 1-anilinonaphthalene-8-sulfonate (ANS) and 2-p-toluidinonaphthalene-6-sulfonate (TNS) with the β-form of poly(L -lysine) [(β-Lys)_n] has been studied by circular dichroism (CD) and absorption spectra measurements. Not only hydrophobic interactions but also hydrogen-bonding and electrostatic interactions contribute to complex formation. The relative importance of these stabilizing factors depends on the relative position of the arylamino group and the sulfonate. For example, ionic interactions play a significant role in the binding of 1,8-ANS and 1,8-TNS, but not in the case of 2,6-TNS. The induced CD of the complexes of (β-Lys)_n with 1,8-ANS and 1,8-TNS is consistent with theoretical calculations for nonplanar conformations of these dyes, twisted in a left-handed sense. As expected for steric reasons, the dominant isomer is one in which the arylamino group is oriented away from the 8-sulfonate (α₁). The induced CD of complexes with 2,6-TNS can be accounted for by an equimolar mixture of left-handed isomers in which the arylamino group is oriented toward the 1-position (β₂) and toward the 3-position (β₁). Our results demonstrate that (β-Lys)_n is capable of chiral discrimination and suggest its general utility for CD studies of racemic anionic dyes.     

Distances of tyrosine residues from a spin-label hapten in the combining site of a specific monoclonal antibody

The nuclear magnetic resonance spectra of an Fab fragment of a monoclonal antibody specifically directed against a nitroxide spin-label hapten have been recorded at different concentrations of the hapten. The hybridoma producing this antibody was grown on deuterated phenylalanine, tryptophan, and 3,5-dideuteriotyrosine or 2,6-dideuteriotyrosine. Difference spectra--without hapten minus with hapten--were calculated for each concentration of hapten. The difference spectra reveal five well-resolved singlet proton resonance signals from tyrosine deuterated in the 3,5-positions (H 2,6 Tyr) and nine from tyrosine deuterated in the 2,6-positions (H 3,5 Tyr). The measured intensities of these signals as a function of combining site occupation have been interpreted in terms of a theory involving intrinsic line widths (T2), the hapten off-rate (k), and distances to the paramagnetic center. Good agreement with theory is found for all of the isolated proton signals. The best estimate of k is 350 s-1; distances in the range 13 to less than 9 A are calculated. Extension of this analysis to other amino acids is discussed.     

Oxidative modification of tryptophan 43 in the heme vicinity of the F43W/H64L myoglobin mutant

The F43W/H64L myoglobin mutant was previously constructed to investigate the effects of electron-rich tryptophan residue in the heme vicinity on the catalysis, where we found that Trp-43 in the mutant was oxidatively modified in the reaction with m-chloroperbenzoic acid (mCPBA). To identify the exact structure of the modified tryptophan in this study, the mCPBA-treated F43W/H64L mutant has been digested stepwise with Lys-C achromobacter and trypsin to isolate two oxidation products by preparative fast protein liquid chromatography. The close examinations of the (1)H NMR spectra of peptide fragments reveal that two forms of the modified tryptophan must have 2,6-disubstituted indole substructures. The (13)C NMR analysis suggests that one of the modified tryptophan bears a unique hydroxyl group in stead of the NH(2) group at the amino-terminal. The results together with mass spectrometry (MS)/MS analysis (30 Da increase in mass of Trp-43) indicate that oxidation products of Trp-43 are 2,6-dihydro-2,6-dioxoindole and 2,6-dihydro-2-imino-6-oxoindole derivatives. Our finding is the first example of the oxidation of aromatic carbons by the myoglobin mutant system.     

Inactivation of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by o-phthalaldehyde.

The two activities of chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were inactivated by o-phthalaldehyde. Absorbance and fluorescence spectra of the modified enzyme were consistent with the formation of an isoindole derivative (1 mol/mol of enzyme subunit). The inactivation of 6-phosphofructo-2-kinase by o-phthalaldehyde was faster than the inactivation of fructose-2,6-bisphosphatase, which was concomitant with the increase in fluorescence. The substrates of 6-phosphofructo-2-kinase did not protect the kinase against inactivation, whereas fructose-2,6-bisphosphate fully protected against o-phthalaldehyde-induced inactivation of the bisphosphatase. Addition of dithiothreitol prevented both the increase in fluorescence and the inactivation of fructose-2,6-bisphosphatase, but not that of 6-phosphofructo-2-kinase. It is proposed that o-phthalaldehyde forms two different inhibitory adducts: a non-fluorescent adduct in the kinase domain and a fluorescent isoindole derivative in the bisphosphatase domain. A lysine and a cysteine residue could be involved in fructose-2,6-bisphosphate binding in the bisphosphatase domain of the protein.     

Interaction of fructose 2,6-bisphosphate and AMP with fructose-1,6-bisphosphatase as studied by nuclear magnetic resonance spectroscopy

The interaction of AMP and fructose 2,6-bisphosphate with rabbit liver fructose-1,6-bisphosphatase has been investigated by proton nuclear magnetic resonance spectroscopy (1H NMR). The temperature dependence of the line widths of the proton resonances of AMP as a function of fructose-1,6-bisphosphatase concentration indicates that the nucleotide C2 proton is in fast exchange on the NMR time scale while the C8 proton is exchange limit. The exchange rate constant, koff, has been calculated for the adenine C8 proton and is 1900 s-1. Binding of fructose 6-phosphate and inorganic phosphate, or the regulatory inhibitor, fructose 2,6-bisphosphate, results in a decrease in the dissociation rate constant for AMP from fructose-1,6-bisphosphatase, as indicated by the sharpened AMP signals. A temperature dependence experiment indicates that the AMP protons are in slow exchange when AMP dissociates from the ternary complex. The rate constant for dissociation of AMP from the enzyme.AMP.fructose 2,6-bisphosphate complex is 70 s-1, 27-fold lower than that of AMP from the binary complex. These results are sufficient to explain the enhanced binding of AMP in the presence of fructose 2,6-bisphosphate and, therefore, the synergistic inhibition of fructose-1,6-bisphosphatase observed with these two regulatory ligands. Binding of fructose 2,6-bisphosphate to the enzyme results in broadening of the ligand proton signals. The effect of AMP on the binding of fructose 2,6-bisphosphate to the enzyme has also been investigated. An additional line width broadening of all the fructose 2,6-bisphosphate protons has been observed in the presence of AMP. The assignment of these signals to the sugar was accomplished by two-dimensional proton-proton correlated spectra (two-dimensional COSY) NMR. From these data, it is concluded that AMP can also affect fructose 2,6-bisphosphate binding to fructose-1,6-bisphosphatase.     

Effect of pulse electromagnetic radiation on erythrocyte ghosts

The pulse microwave radiation has been shown to increase the fluorescence intensity of 2-toluidinonaphthanene-6-sulfonate (2,6-TNS) and 1-anilinonaphthalene-8-sulfonate (1,8-ANS) built-in membranes of erythrocyte ghosts. In experiments with 2,6-TNS a frequency dependence of the effect of microwave radiation with maximum within the frequency range of 55-65 Hz has been found. It is suggested that the changes registered with fluorescent probes are induced by mechanical oscillations generated by the pulse microwave radiation.     

Fructose 2,6-bisphosphate and insulin stimulation of glycolysis in 3T3-L1 adipocytes

1. Insulin is able to stimulate lactate production and to enhance fructose 2,6-bisphosphate (Fru-2,6-P2) content in 3T3-L1 adipocytes. 2. Phorbol 12-myristate 13-acetate is more efficacious than insulin in rising Fru-2,6-P2 content and less effective in the stimulation of glycolysis. 3. 3T3-L1 adipocyte 6-phosphofructo-l-kinase appears to be very sensitive to exogenous Fru-2,6-P2. 4. Insulin treatment does not affect the maximum activity of 6-phosphofructo-1-kinase whereas it markedly increases the affinity of pyruvate kinase for phosphoenolpyruvate. 5. The role of Fru-2,6-P2 in the insulin induced enhancement of glycolytic flux is discussed.     

Xanthones as inhibitors of microsomal lipid peroxidation and TNF-alpha induced ICAM-1 expression on human umbilical vein endothelial cells (HUVECs)

Xanthones bearing different functionalities, namely 1-hydroxyxanthone (1), 3-hydroxyxanthone (2), 1,4-dihydroxyxanthone (3), 2,6-dihydroxyxanthone (4), 1,2-diacetoxyxanthone (5), 2,6-diacetoxyxanthone (6), 3-methoxyxanthone (7), 1,3,7-trimethoxyxanthone (8) and 1,5-dihydroxy-6-methoxyxanthone (9) were synthesised and examined for their effect on nicotinamide adenine dinucleotide phosphate (NADPH)-catalysed liver microsomal lipid peroxidation and on tumour necrosis factor-alpha (TNF-alpha) induced expression of intercellular adhesion moledule-1 (ICAM-1) on endothelial cells, with a view to establish structure-activity relationship. Hydroxy- and acetoxyxanthones showed potent inhibitory effects on NADPH-catalysed lipid peroxidation and TNF-alpha induced expression of ICAM-1 on endothelial cells.     

Induction of LacZ mutations in Muta Mouse can distinguish carcinogenic from non-carcinogenic analogues of diaminotoluenes and nitronaphthalenes

2,4-Diaminotoluene (2,4-DAT) is a liver carcinogen in rats and mice whereas 2,6-DAT is not. Both are genotoxic in vitro. Tests for mutations in transgenic mice, unscheduled DNA synthesis (UDS), DNA damage and enhancement of initiated foci in vivo have shown some discrimination between these two analogues, but only after oral administration. 1- and 2-nitronaphthalene (1- and 2-NNT) are also both genotoxic in vitro, although, unlike 2,4- and 2,6-DAT, they do not require metabolic activation. There is some evidence that 2-NNT may be able to induce liver and bladder tumours, and there is some evidence that 1-NNT is not carcinogenic to rats or mice, but none of the data are convincing. When tested for induction of LacZ mutations in Muta Mouse after topical exposure (human occupational exposure route) at their maximum tolerated doses, 2,4-DAT induced a positive response in liver and a marginal response in kidney, whereas 2,6-DAT was negative. 2-NNT also induced a positive mutagenic response in liver, and a marginal response in bladder, whereas 1-NNT was negative. Neither 2,4- nor 2,6-DAT induced mutations at the site of application (skin) as might be expected for chemicals requiring activation by liver enzymes. 2-NNT, which is a direct-acting mutagen in vitro, gave a marginal response for induced mutation at the site of application, but 1-NNT was negative. This study shows that investigation of induction of LacZ mutations after topical application in vivo can provide useful data to help discriminate potentially carcinogenic from non-carcinogenic chemicals that are mutagenic in vitro. Robust carcinogenicity data are needed to determine whether 2-NNT can induce tumours in the liver and bladder.     

N-methyl-2-anilino-6-naphthalenesulfonyl hydrazine (2,6-mansyl hydrazine): A new sensitive fluorometric reagent for carbonyl compounds

An almost quantitative synthesis of N-methyl-2-anilino-6-naphthalenesulfonyl hydrazine (2,6-mansyl hydrazine) from sulfonyl chloride and hydrazinlum hydroxide is described. The 2,6-mansyl chloride was prepared by different methods from 6-hydroxynaphthalene-2-sulfonic acid (overall yield: 69%). The N- and the O-mansylation of suitable compounds (e.g., amines, amino acids, and phenolic steroids) with 2,6-mansyl chloride and the preparation of oxosteroid-2,6-mansyl hydrazones are deseribed, and the derivatives obtained, their uv spectra, and methods for their thin-layer chromatographic separation are compared with the corresponding data for dansylated compounds.     

2,6-二异丙基苯酚逆转嗅球切除抑郁模型大鼠电休克后的Tau蛋白过度磷酸化和认知障碍

通过观察2, 6-二异丙基苯酚对电休克后嗅球切除抑郁模型大鼠学习记忆和Tau蛋白过度磷酸化的影响,探讨兴奋性氨基酸受体拮抗剂对Tau蛋白过度磷酸化的调节及两者对抑郁大鼠学习记忆的影响,为改善学习记忆障碍的神经心理学机制研究和临床干预性治疗提供实验依据．按随机单位组2×2析因设计设置2个干预因素,即电休克干预(两水平：无处置、施行一个疗程电休克)和2, 6-二异丙基苯酚干预(两水平：腹腔注射5 ml 生理盐水或5 ml 2, 6-二异丙基苯酚100 mg/kg)的所有组合．选24周龄健康雄性Sprague-Dawley大鼠建立嗅球切除抑郁模型,将32只24周龄模型大鼠随机分为4个实验组(n=8):Ⅰ组(腹腔注射5 ml 2, 6-二异丙基苯酚100 mg/kg)、Ⅱ组(腹腔注射5 ml 2, 6-二异丙基苯酚100 mg/kg +施行电休克1个疗程)、Ⅲ组(腹腔注射5 ml 生理盐水)、Ⅳ组(腹腔注射5 ml 生理盐水+施行电休克1个疗程)．全部电休克处置结束24 h内开始Morris水迷宫检测,之后留取海马组织．高效液相色谱法检测神经递质谷氨酸(Glu)在海马组织中的含量;免疫组化SP法和蛋白质印迹法检测Tau-5(总Tau蛋白)、p-PHF1^Ser396/404、p-AT8^Ser199/202、p-12E8^Ser262、GSK-3β^1H8和PP-2A在海马组织神经元中的表达．电休克和2, 6-二异丙基苯酚均可造成大鼠学习记忆障碍,即延长逃避潜伏期并缩短空间探索时间,两者的影响呈相减效果．电休克可明显增加海马中神经递质谷氨酸(Glu)的浓度,2, 6-二异丙基苯酚可降低海马中神经递质Glu的浓度,且两者有相减效果．电休克和2, 6-二异丙基苯酚对海马总Tau蛋白和PP-2A蛋白的表达无明显影响．电休克可增加海马中磷酸化Tau蛋白和GSK-3β^1H8蛋白的表达;2, 6-二异丙基苯酚可减少海马中磷酸化Tau蛋白和GSK-3β^1H8蛋白的表达;两者的影响均呈相减效果．实验结果表明,电休克导致海马Glu浓度升高,通过上调GSK-3β^1H8增加海马Tau蛋白的磷酸化程度导致学习记忆功能障碍,而2, 6-二异丙基苯酚则可通过降低海马Glu浓度下调GSK-3β1H8的表达,从而减缓Tau蛋白的磷酸化程度以改善ECT后的学习记忆．     

Induction of tomato stress protein mRNAs by ethephon, 2,6-dichloroisonicotinic acid and salicylate

To study the possible involvement of plant hormones in the synthesis of stress proteins in tomato upon inoculation with Cladosporium fulvum, we investigated the induction of mRNAs encoding PR proteins and ethylene biosynthesis enzymes by ethephon, 2,6-dichloroisonicotinic acid (INA) and salicylic acid (SA) by northern blot analysis. Ethephon slightly induced some but not all mRNAs encoding intra- and extracellular PR proteins. INA induced all PR protein mRNAs analysed, except for intracellular chitinase and extracellular PR-4. SA induced all PR protein mRNAs analyzed, except for intracellular chitinase and osmotin. None of the inducers affected the expression of ACC synthase mRNA, whereas all three induced ethylene-forming enzyme (EFE) mRNA.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme - HR hypersensitive response - INA 2,6-dichloroisonicotinic acid - PR pathogenesis-related - SA salicylic acid - SAR systemic acquired resistance     

The interaction of fructose 2,6-bisphosphate and AMP with rat hepatic fructose 1,6-bisphosphatase

The binding of the inhibitory ligands fructose 2,6-bisphosphate and AMP to rat liver fructose 1,6-bisphosphatase has been investigated. 4 mol of fructose-2,6-P2 and 4 mol of AMP bind per mol of tetrameric enzyme at pH 7.4. Fructose 2,6-bisphosphate exhibits negative cooperatively as indicated by K'1 greater than K'2 greater than K'3 greater than or equal to K'4 and a Hill plot, the curvature of which indicates K'2/K'1 less than 1, K'3/K'2 less than 1, and K'4/K'3 = 1. AMP binding, on the other hand, exhibits positive cooperativity as indicated by K'1 less than K'2 less than K'3 less than K'4 and an nH of 2.05. Fructose 2,6- and fructose 1,6-bisphosphates enhance the binding of AMP as indicated by an increase in the intrinsic association constants. At pH 9.2, where fructose 2,6-bisphosphate and AMP inhibition of the enzyme are diminished, fructose 2,6-bisphosphate binds with a lower affinity but in a positively cooperative manner, whereas AMP exhibits half-sites reactivity with only 2 mol of AMP bound per mol of tetramer. Ultraviolet difference spectroscopy confirmed the results of these binding studies. The site at which fructose 2,6-bisphosphate binds to fructose 1,6-bisphosphatase has been identified as the catalytic site on the basis of the following. 1) Fructose 2,6-bisphosphate binds with a stoichiometry of 1 mol/mol of monomer; 2) covalent modification of the active site with acetylimidazole inhibits fructose 2,6-bisphosphate binding; and 3) alpha-methyl D-fructofuranoside-1,6-P2 and beta-methyl D-fructofuranoside-1,6-P2, substrate analogs, block fructose 2,6-bisphosphate binding. We propose that fructose 2,6-bisphosphate enhances AMP affinity by binding to the active site of the enzyme and bringing about a conformational change which may be similar to that induced by AMP interaction at the allosteric site.     

Metabolism of 2,6-dimethylnaphthalene by flavobacteria.

Flavobacteria that were able to grow on 2,6-dimethylnaphthalene (2,6-DMN) were isolated from soil. Most were able to oxidize a broad range of aromatic hydrocarbons after growth on 2,6-DMN at rates comparable to that of the oxidation of 2,6-DMN itself. One small group was neither able to grow on naphthalene nor able to oxidize this compound after growth on 2,6-DMN, but metabolized 2,6-DMN by a pathway which converged with that previously described for naphthalene metabolism in pseudomonads. These organisms could also grow on salicylate or methylsalicylate, and in so doing, early enzymes for 2,6-DMN metabolism were induced.