Mechanism of dibucaine-induced apoptosis in promyelocytic leukemia cells (HL-60).

Dibucaine, a local anesthetic, inhibited the growth of promyelocytic leukemia cells (HL-60) without inducing arrest of the cell cycle and differentiation to granulocytes. Typical DNA fragmentation and DNA ladder formation were induced in a concentration- and time-dependent manner. The half-maximal concentration of dibucaine required to induce apoptosis was 100 microM. These effects were prevented completely by the pan-caspase inhibitor z-Val-Ala-Asp-(OMe)-fluoromethylketone (z-VAD-fmk), thereby implicating the cysteine aspartase (caspase) cascade in the process. Dibucaine activated various caspases, such as caspase-3, -6, -8, and -9 (-like) activities, but not caspase-1 (-like) activity, and induced mitochondrial membrane depolarization and the release of cytochrome c (Cyt.c) from mitochondria into the cytosol. Processing of pro-caspase-3, -8, and -9 by dibucaine was confirmed by western blot analysis. Bid, a death agonist member of the Bcl-2 family, was processed by caspases following exposure of cells to dibucaine. However, 100 microM dibucaine scarcely inhibited oxidative phosphorylation, but it induced membrane permeability transition in isolated rat liver mitochondria. Taken together, these data suggest that dibucaine induced apoptosis of HL-60 cells through activation of the caspase cascade in conjunction with Cyt.c release induced by a processed product of Bid and depolarization of the mitochondrial membrane potential.

Roles of asp126 and asp156 in the enzyme function of sphingomyelinase from Bacillus cereus.

To elucidate the roles of conserved Asp residues of Bacillus cereus sphingomyelinase (SMase) in the kinetic and binding properties of the enzyme toward various substrates and Mg2+, the kinetic data on mutant SMases (D126G and D156G) were compared with those of wild type (WT) enzyme. The stereoselectivity of the enzyme in the hydrolysis of monodispersed short-chain sphingomyelin (SM) analogs and the binding of Mg2+ to the enzyme were not affected by the replacement of Asp126 or Asp156. The pH-dependence curves of kinetic parameters (1/Km and kcat) for D156G-catalyzed hydrolysis of micellar SM mixed with Triton X-100 (1:10) and of micellar 2-hexadecanoylamino-4-nitrophenylphosphocholine (HNP) were similar in shape to those for WT enzyme-catalyzed hydrolysis. On the other hand, the curves for D126G lacked the transition observed for D156G and WT enzymes. Comparison of the values and the shape of pH-dependence curves of kinetic parameters indicated that Asp126 of WT SMase enhances the enzyme's catalytic activity toward both substrates and its binding of HNP but not SM. The deprotonation of Asp126 enhances the substrate binding and slightly suppresses the catalytic activity toward both substrates. Asp156 of WT SMase acts to decrease the binding of both substrates and the catalytic activity to HNP but not SM. From the present study and the predicted three-dimensional structure of B. cereus SMase, Asp126 was thought to be located close to the active site, and its ionization was shown to affect the catalytic activity and substrate binding.

Mg2+ binding and catalytic function of sphingomyelinase from Bacillus cereus.

The modes of Mg2+ binding to SMase from Bacillus cereus were studied on the basis of the changes in the tryptophyl fluorescence intensity. This enzyme was shown to possess at least two binding sites for Mg2+ with low and high affinities. The effects of Mg2+ binding on the enzymatic activity and structural stability of the enzyme molecule were also studied. The results indicated that the binding of Mg2+ to the low-affinity site was essential for the catalysis, but was independent of the substrate binding to the enzyme. It was also indicated that the alkaline denaturation of the enzyme was partly prevented by the Mg2+ binding, whereas no significant protective effect was observed against the denaturation by urea. The pH dependence of the kinetic parameters for the hydrolysis of micellar HNP and mixed micellar SM with Triton X-100 (1:10), catalyzed by SMase from B. cereus, was studied in the presence of a large amount of Mg2+ to saturate both the low- and high-affinity sites. The pH dependence curves of the logarithm of 1/Km for these two kinds of substrates were similar in shape to each other, and showed a single transition. On the other hand, the shapes of the pH dependence curves of the logarithm of kcat for these two kinds of substrates were different from each other. The pH dependence curve for micellar HNP showed three transitions and, counting from the acidic end of the pH region, the first and third transitions having tangent lines with slopes of +1 and -1, respectively. On the other hand, the curve for mixed micellar SM with Triton X-100 showed one large transition with a slope of +1 (the first transition) and a very small transition (the third transition). On the basis of the present results and the three-dimensional structure of bovine pancreatic DNase I, which has a primary structure similar to that of B. cereus SMase, we proposed a catalytic mechanism for B. cereus SMase based on general-base catalysis.

Results of newborn screening for galactose metabolic disorders.

A screening strategy has been used which uses the Paigen and Beutler methods for the determination of galactose and galactose-1-phosphate. A blood spot test for epimerase has also been developed. In the last 10 years, 265,019 samples from newborns have been tested by these methods. Among the 154 screening positives, we have detected seven cases of epimerase-deficient galactosaemia (Type III), seven cases of Duarte/galactosaemia heterozygotes, 48 cases of other various types of heterozygotes, four cases of persistent hypergalactosaemia, three cases of hepatitis and one case of congenital atresia of the bile duct. These results indicate that our screening system has effectively detected the infants with galactose metabolic disorders.

Effect of chlordane on hepatic mitochondrial respiration.

In order to clarify the cytotoxicity of chlordane, an industrial product used as an insecticide, its effect on oxidative phosphorylation in rat hepatic mitochondria was studied. The respiration rate, RCI and ADP/O ratios were inhibited by chlordane-related compounds; the degree of inhibition was in the descending order of trans-chlordane, cis-chlordane, heptachlor and heptachlorepoxide. Of the indices indicating various respiratory activities, state 3 respiration was the most sensitively inhibited by these compounds, suggesting that they inhibit energy transfer. However, electron transport was inhibited also by high concentrations of chlordane constituents. The inhibitory effect of the chlordane constituents on respiratory activity varied depending on the species of respiratory substrate, suggesting site-specificity of these compounds. The release of K+ ions paralleled the results of the respiratory activity study. Heptachlorepoxide, a metabolic product of heptachlor, had less effect on mitochondria than heptachlor.

Stimulative effect of chlordane on the various functions of the guinea pig leukocytes.

The effects of the chlordane-related compounds, cis-chlordane, trans-chlordane, heptachlor, and heptachlor epoxide, on stimulation responses of guinea pig polymorphonuclear leukocytes (PMNs) were examined. Treatment of PMN with these compounds stimulated superoxide (O2-) generation, altered membrane potential, and increased intracellular Ca2+ concentration ([Ca2+]i). However, there was no definite tendency among the stimulating effects of chlordane-related compounds, therefore the relationship between the effect and molecular structure of these substances remains unknown. Of these response reactions of PMN stimulated by chlordane-related compounds, stimulation of O2- generation lagged behind others. Increase in [Ca2+]i was due both to acceleration of extracellular Ca2+ penetration and to Ca2+ release from the intracellular pool. These results indicate that chlordane-related compounds stimulate PMN and suggest a causal relationship between the stimulation of O2- generation by these substances and their toxicity.

[Stimulative effect of chlordanes on guinea pig polymorphonuclear leukocytes].

To investigate the toxicity of chlordane, an organochlorine insecticide, effects of cis-Chlordane, trans-Chlordane, Heptachlor and Heptachlor epoxide were examined on stimulus responses of guinea pig polymorphonuclear leukocytes (PMNs). Results obtained were as follows. These chlordane-related compounds stimulated superoxide (O2-) generation, altered membrane potential and increased intracellular Ca2+ concentration ((Ca2+]i). As a significant tendency was not found in the stimulating effects of these compounds, the relationship between the effect and molecular structure of these substances remains unknown. Of these response reactions of PMN stimulated by chlordanes, stimulation of O2- generation lagged behind the others. Increase in [Ca2+]i was due to both acceleration of extracellular Ca2+ influx and Ca2+ release from intracellular pool. These results indicate that these chlordane-related compounds stimulate PMN and suggest a causal relationship between the stimulation of O2- generation by these substances and their toxicity.

Inhibition of metabolic response of polymorphonuclear leukocyte by biscoclaurine alkaloids.

Effects of biscoclaurine alkaloids on the various stimulus-responses of PMN, especially on the O2-. generation of PMN, were investigated. Results obtained were: cepharanthine inhibited various metabolic responses of PMN, its biological action probably being due to its membrane modifying action. Inhibition of O2-. generation by cepharanthine was stronger than any other inhibition of metabolic responses of PMN. The inhibitory effect of various biscoclaurine alkaloids on the O2-. generation of PMN was the descending order of tri-, di- and mono-ether type; the coclaurine type showed only a weak effect.

Metabolic response of polymorphonuclear leukocyte to arachidonic and linoleic acids.

Various stimuli act on polymorphonuclear leukocytes (PMN), activating membrane-bound phospholipase A2 and C, and diglyceride lipase and then liberating unsaturated fatty acids (USFAs). These liberated USFAs are immediately metabolized through various metabolic pathways such as cyclooxygenase, lipoxygenase, phosphatidylinositol metabolism etc. It is possible that the metabolic intermediates of these pathways reveal various physiological actions. This work was undertaken to clarify whether stimuli on PMN depend on these USFAs themselves or on their oxidation products. The following results were obtained: 1. USFAs such as arachidonate and linoleate stimulate PMN, accelerating superoxide (O2) generation, depolarization of membrane potential and increase in [Ca2+]i. 2. Oxidation products of USFAs have no stimulative effect on PMN. The decrease in the stimulative effect of these USFAs following their oxidation is proportional to the quantitative decrease in non-oxidized linoleate. 3. USFAs accelerate membrane permeability of Ca2+, and their oxidation products enhance non-specific membrane permeability in proportion to the formation of monohydroxy compound. These results suggest that stimulative effects of USFAs on PMN do not depend on their oxidation products but on unoxidized fatty acids. Furthermore, among the oxidation products of the USFAs, monohydroxy compound acts as a strong perturber of membrane and accelerates membrane permeability.

Effect of tricyclic drugs on mitochondrial membrane.

The effects of tricyclic drugs (clomipramine, imipramine, chlorpromazine and promethazine) on isolated liver mitochondria of rats were examined. All the drugs tested accelerated state 4 respiration. Their stimulative potency at concentrations below 100 microM was in the order of chlorpromazine greater than clomipramine greater than imipramine, promethazine. On state 3 respiration, the chlorine containing drugs had an inhibitive effect at high concentrations, while the other drugs seemed to have a slightly stimulative effect. These drugs stimulated latent ATPase activity of mitochondria. Clomipramine and chlorpromazine inhibited 2, 4-dinitrophenol-stimulated ATPase activity in a dose-dependent fashion. Imipramine also inhibited 2, 4-dinitrophenol-stimulated ATPase activity at high concentrations. Promethazine, however, had almost no effect. All the drugs induced potassium release from mitochondrial vesicles, and their potency was in the order of clomipramine greater than chlorpromazine greater than imipramine greater than promethazine. These results suggest that clomipramine, imipramine, chlorpromazine and promethazine cause impediments in both mitochondrial respiration and ion compartmentation, and that the chlorine containing drugs are more toxic than others on the functions of the mitochondrial membrane.

Effects of environmental pollutants on the mitochondrial and red cell membranes.

Most environmental pollutants affect biological membranes more or less, resulting in an increase in permeability of mitochondrial and red cell membranes, which causes changes in potassium compartmentation, and also impairment of oxidative phosphorylation of mitochondria. Authors classified various environmental pollutants from the mode of action on biological membranes.

Classification of potentially toxic chemicals based on their effects on mitochondrial respiration.

The classification of potentially toxic chemicals including environmental pollutants was made with respect to state 3 and state 4 respiration of mitochondria. The concentration of certain metals for 50% inhibition of respiratory control index (RCI; state 3/state 4) was lower than that of organic compounds tested. Various chemicals including environmental pollutants were classified into four groups by combination of effects on state 3 and state 4 respiration.

The effects of echinatin and its related compounds on the mitochondrial energy transfer reaction.

To investigate the mechanism by which various biological action of licorice root are brought about, the effects of echinatin as a small constituent of Glycyrrhiza echinata and several related compounds on mitochondrial energy transfer reactions were examined. The results obtained were as follows: 1) Echinatin, 4'-hydroxychalcone, chalcone and 3,4'-dihydroxychalcone at a low concentration cause deterioration of respiratory control and oxidative phosphorylation of isolated rat liver mitochondria. 2) Chalcone and 4'-hydroxychalcone stimulate both latent and DNP-ATPase activity of mitochondria. Echinatin inhibits DNP-ATPase activity while stimulating range latent ATPase activity in the low concentration. 3) Chalcone and 4'-hydroxychalcone induce a rapid potassium release from mitochondrial vesicles, while echinatin and 3,4'-dihydroxychalcone have lesser effect than the former two substances. From these results, it can be concluded that echinatin and several related compounds disturb the mitochondrial energy transfer reactions and membrane permeability.