Aminolysis of O-aryl thionobenzoates: amine basicity combines with modulation of the nature of substituents in the leaving group and thionobenzoate moiety to control the reaction mechanism.

A kinetic study is reported for aminolysis of O-Y-substituted phenyl thionobenzoates (1a-f) and O-4-nitrophenyl X-substituted thionobenzoates (2a-f) in 80 mol % H2O/20 mol % DMSO at 25.0 +/- 0.1 degrees C. The reaction proceeds through one or two intermediates (i.e., a zwitterionic tetrahedral intermediate T(+/-) and its deprotonated form T(-)) depending on the basicity difference between the nucleophile and nucleofuge, that is, the reaction proceeds through T(+/-) when the leaving aryloxide is less basic than the attacking amine, but through T(+/-) and T(-) when the leaving group is more basic than the amine. However, the reaction mechanism is not influenced by the electronic nature of the substituent X in the nonleaving group. The Hammett plot for the reactions of 2a-f with benzylamine is consisted of two intersecting straight lines, which might be interpreted as a change in the rate-determining step (RDS). However, the Yukawa-Tsuno plot for the same reactions exhibits an excellent linear correlation, indicating that the nonlinear Hammett plot is not due to a change in the RDS but caused by stabilization of the ground-state of the substrate through resonance interaction between the electron-donating substituent X and the thionocarbonyl moiety.

Light-activated indole-3-acetic acid induces apoptosis in g361 human melanoma cells.

Indole-3-acetic acid (IAA) activation by horseradish peroxidase (HRP) has been suggested as a new cancer therapy. Interestingly, we found that ultraviolet B UVB radiation also can activate IAA and produce free radicals in a dose-dependent manner. In this study, we attempted to identify the free radicals generated by UVB-irradiated IAA (IAAUVB), and to determine whether IAAUVB can induce the apoptosis of G361 human melanoma cells. Since IAA/HRP produces reactive oxygen species (ROS), we examined whether IAAUVB-generated radicals include ROS. Our results show that IAAUVB-induced free radical production is not inhibited by catalase, superoxide dismutase, or sodium formate, indicating that ROS are not generated by IAAUVB. On the other hand, IAAUVB caused lipid peroxidation, and this was blocked by Trolox, a water-soluble vitamin E derivative. Moreover, we found that IAAUVB caused apoptotic cell death and that this was inhibited by a low temperature. We further investigated IAAUVB-mediated apoptotic pathways, and found that IAAUVB causes caspase-8, Bid, caspase-3 activation, and poly (ADP-ribose) polymerase (PARP) cleavage. In addition, these apoptotic pathways were also blocked by low temperature. From these results, we propose that IAAUVB-induced free radicals cause human melanoma cell apoptosis via a death receptor-mediated apoptotic pathway.

Indole-3-acetic acid/horseradish peroxidase-induced apoptosis involves cell surface CD95 (Fas/APO-1) expression.

Recently, we showed that a combination of indole-3-acetic acid (IAA) and horseradish peroxidase (HRP) produces hydrogen peroxide (H2O2), and that this leads to the apoptosis of G361 human melanoma cells. In the present study, flow cytometric analysis confirmed that H2O2 is involved the IAA/HRP-induced apoptotic process. We also found that IAA/HRP increases cell surface CD95 (Fas/APO-1) expression, and that this is blocked by catalase treatment. Furthermore, blocking CD95 with a neutralizing antibody significantly restored IAA/HRP-induced apoptosis. In addition, the IAA/HRP-induced activations of CD95 downstream molecules, i.e., caspase-8, Bid, and caspase-3, were also inhibited by catalase. Moreover, a caspase-8 inhibitor significantly blocked IAA/HRP-induced apoptosis. These results indicate that IAA/HRP-induced apoptosis involves a CD95-initiated death receptor signaling pathway initiated by hydrogen peroxide.

Aminolyses of 4-nitrophenyl phenyl carbonate and thionocarbonate: effect of modification of electrophilic center from C=O to C=S on reactivity and mechanism.

A kinetic study is reported for nucleophilic substitution reactions of 4-nitrophenyl phenyl carbonate (5) and 4-nitrophenyl phenyl thionocarbonate (6) with a series of primary amines. The thiono compound 6 is less reactive than its oxygen analogue 5 toward strongly basic amines but is more reactive toward weakly basic CF3CH2NH2. The Brønsted-type plots obtained from the aminolyses of 5 and 6 are curved downwardly. The reactions are proposed to proceed through a stepwise mechanism with a change in the RDS on the basis of the curved Brønsted-type plots. The microscopic rate constants (k(1) and k(2)/k(-1) ratio) associated with the current aminolyses are consistent with the proposed reaction mechanism. The replacement of the C=O bond in 5 by a polarizable C=S group results in a decrease in the k(1) value but an increase in the k(2)/k(-1) ratio. Besides, such a modification of the electrophilic center causes a decrease in pKa degrees , defined as the pK(a) at the curvature center of curved Brønsted-type plots, but does not alter the reaction mechanism. The larger k(2)/k(-1) ratio for the reactions of 6 compared to those of 5 is proposed to be responsible for the decreased pK(a) degrees value.

Hydrogen peroxide is a mediator of indole-3-acetic acid/horseradish peroxidase-induced apoptosis.

Recently, we reported that a combination of indole-3-acetic acid (IAA) and horseradish peroxidase (HRP) induces apoptosis in G361 human melanoma cells. However, the apoptotic mechanism involved has been poorly studied. It is known that when IAA is oxidized by HRP, free radicals are produced, and since oxidative stress can induce apoptosis, we investigated whether reactive oxygen species (ROS) are involved in IAA/HRP-induced apoptosis. Our results show that IAA/HRP-induced free radical production is inhibited by catalase, but not by superoxide dismutase or sodium formate. Furthermore, catalase was found to prevent IAA/HRP-induced apoptotic cell death, indicating that IAA/HRP-produced hydrogen peroxide (H2O2) may be involved in the apoptotic process. Moreover, the antiapoptotic effect of catalase is potentiated by NADPH, which is known to protect catalase. On further investigating the IAA/HRP-mediated apoptotic pathway, we found that the IAA/HRP reaction leads to caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage, which was also blocked by catalase. Additionally, we found that IAA/HRP produces H2O2 and induces peroxiredoxin (Prx) sulfonylation. Consequently, our results suggest that H2O2 plays a major role in IAA/HRP-induced apoptosis.

Aminolysis of 2,4-dinitrophenyl X-substituted benzoates and Y-substituted phenyl benzoates in MeCN: effect of the reaction medium on rate and mechanism.

Second-order rate constants (k(N)) have been determined spectrophotometrically for the reactions of 2,4-dinitrophenyl X-substituted benzoates (1 a-f) and Y-substituted phenyl benzoates (2 a-h) with a series of alicyclic secondary amines in MeCN at 25.0 +/- 0.1 degrees C. The k(N) values are only slightly larger in MeCN than in H2O, although the amines studied are approximately 8 pK(a) units more basic in the aprotic solvent than in H2O. The Yukawa-Tsuno plot for the aminolysis of 1 a-f is linear, indicating that the electronic nature of the substituent X in the nonleaving group does not affect the rate-determining step (RDS) or reaction mechanism. The Hammett correlation with sigma- constants also exhibits good linearity with a large slope (rho(Y) = 3.54) for the reactions of 2 a-h with piperidine, implying that the leaving-group departure occurs at the rate-determining step. Aminolysis of 2,4-dinitrophenyl benzoate (1 c) results in a linear Brønsted-type plot with a beta(nuc) value of 0.40, suggesting that bond formation between the attacking amine and the carbonyl carbon atom of 1 c is little advanced in the transition state (TS). A concerted mechanism is proposed for the aminolysis of 1 a-f in MeCN. The medium change from H2O to MeCN appears to force the reaction to proceed concertedly by decreasing the stability of the zwitterionic tetrahedral intermediate (T+/-) in aprotic solvent.

Temporal changes in cerebral antioxidant enzyme activities after ischemia and reperfusion in a rat focal brain ischemia model: effect of dietary fish oil.

This study investigated the neuroprotective effects of dietary supplementation of fish oil on both brain infarction and the activities of antioxidant enzymes. Male Sprague-Dawley rats (4-weeks old) were divided into two groups and received either a regular diet (RD) or a fish-oil-supplemented diet (FOD) for 6 weeks prior to middle cerebral artery (MCA) occlusion. The infarction volume of the brain was calculated using image analysis after staining. Antioxidant enzymes were measured before ischemia (BI), after 2 h of ischemia (AI) and after 24 h (24hR), 48 h (48hR) and after 7 days (7dR) of reperfusion. The infarction volume of the brain was significantly smaller in the FOD group than in the RD group after 24 h of reperfusion (p<0.05). Before ischemia, the levels of lipid peroxide and the glutathione peroxidase (GPx) activity were higher in the FOD group than in the RD group. During reperfusion, the catalase (CAT) activity in the FOD group remained at the preischemia level until after 48 h of reperfusion, while those in the RD group did not. The Mn-superoxide dismutase (SOD) activity and GPx activity were higher in the FOD group than in the RD group only after 2 h of ischemia. In the fatty acid analysis, the ratio of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were higher in the FOD group than in the RD group (p<0.05). Our results demonstrate that supplementing the diet with fish oil could decrease the cerebral infarction volume following ischemia and reperfusion (I/R) partly by working directly as an antioxidant and partly by modulating antioxidant enzyme activities.

Cellular senescence induced loss of stem cell proportion in the skin in vitro.

BACKGROUND: It is known that cellular senescence could affect culture results. A previous study on K19 found that the loss of stem cell proportion is the reason for difficulties experienced when culturing aged keratinocytes. But the situation is unclear, because K19 is not generally accepted as an epidermal stem cell marker. OBJECTIVE: The aim of this study was to investigate the effects of cellular senescence caused by chronological aging or by repeated subcultures. METHODS: The effects of cellular senescence were investigated using monolayer cultures of keratinocytes and reconstructed epidermis. We prepared keratinocytes from donors of different ages and by repeated subcultures. Flow cytometric analysis was performed using alpha6 integrin and CD71 antibodies, and candidate keratinocyte stem cell proportions were separated according to reactivities to these antibodies. Living skin equivalents (LSEs) were reconstructed using keratinocytes from child, adult and elderly donors. RESULTS: Flow cytometric analysis showed a decrease in the candidate stem cell proportion in an age- or culture passage-dependent manner. LSE experiments showed that a reconstructed epidermis using child's keratinocytes was well formed compared to epidermis reconstructed using an elderly donor's keratinocytes. Different expression of proliferation markers was also observed according to donor age. CONCLUSION: Our results showed that cellular senescence by chronological aging or repeated sub-culture induced the loss of candidate stem cell proportion in keratinocyte cultures. This seems to be the reason why it is difficult to culture keratinocytes from the elderly or by repeatedly culturing keratinocytes in vitro.

Oxidation of indole-3-acetic acid by horseradish peroxidase induces apoptosis in G361 human melanoma cells.

The combination of indole-3-acetic acid (IAA) and horseradish peroxidase (HRP) has recently been proposed as a novel cancer therapy. However, the mechanism underlying the cytotoxic effect involved is substantially unknown. Here, we show that IAA/HRP treatment induces apoptosis in G361 human melanoma cells, whereas IAA or HRP alone have no effect. It is known that IAA produces free radicals when oxidized by HRP. Because oxidative stress could induce apoptosis, we measured the production of free radicals at varying concentrations of IAA and HRP. Our results show that IAA/HRP produces free radicals in a dose-dependent manner, which are suppressed by ascorbic acid or (-)-epigallocatechin gallate (EGCG). Furthermore, antioxidants prevent IAA/HRP-induced apoptosis, indicating that the IAA/HRP-produced free radicals play an important role in the apoptotic process. In addition, IAA/HRP was observed to activate p38 mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK), which are almost completely blocked by antioxidants. We further investigated the IAA/HRP-mediated apoptotic pathways, and found that IAA/HRP activates caspase-8 and caspase-9, leading to caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage. These events were also blocked by antioxidants, such as ascorbic acid or EGCG. Thus, we propose that IAA/HRP-induced free radicals lead to the apoptosis of human melanoma cells via both death receptor-mediated and mitochondrial apoptotic pathways.

Significant and differential acceleration of dephosphorylation of the insecticides, paraoxon and parathion, caused by alkali metal ethoxides.

In the reaction of paraoxon with alkali metal ethoxides, ion-paired EtO-M+ species are more reactive than the dissociated EtO- with the reactivity order EtO-Li+ EtO-Na+ > EtO-K+ > EtO-, while in the reaction of parathion, the reactivity follows the order EtO-K+ > EtO- > EtO-Na+ > EtO-Li+.