Tumor promoter chloroform is a potent protein kinase C activator

The major interaction site for tumor-promoting phorbol esters is the calcium-activated, phospholipid-dependent protein kinase (protein kinase C), a key-element in signal transduction. Binding of phorbol esters results in enzyme activation which mediates, at least in part, the action of these agents. We have investigated the effects of tumor promoter chloroform on protein kinase C activity. Like thrombin and 12-O-tetradecanoylphorbol-13-acetate (TPA), chloroform was able to activate protein kinase C in intact rabbit platelets. In addition, chloroform stimulated enzyme activity as well as TPA binding capacity in cell-free system. Scatchard analysis of the data has shown that chloroform increased the number of phorbol ester binding sites. Structurally related compounds, carbon tetrachloride and methylene chloride, activated the enzyme similarly.     

Metabolism of N6,O2'-[3H]dibutyryl cyclic adenosine 3',5'-monophosphate and macromolecular interactions of the products perfused rat liver.

Mitochondria from liver, kidney, brain, and skeletal muscle metabolized acetaldehyde. Acetaldehyde oxidation by liver and kidney mitochondria was maximal at low levels of acetaldehyde and was sensitive to rotenone, suggesting the involvement of a NAD⁺-dependent aldehyde dehydrogenase with a high affinity for acetaldehyde. Acetaldehyde oxidation was stimulated 50% by ADP, suggesting that, in state 4, reoxidation of NADH is rate limiting for acetaldehyde oxidation. In state 4, acetaldehyde oxidation was decreased by NAD⁺-dependent substrates, as well as by succinate and ascorbate. The inhibition by the latter two substrates was prevented by ADP, dinitrophenol, valinomycin, and gramicidin, but not by oligomycin. Since these compounds are linked to energy transduction and utilization, the data suggest that the inhibition is mediated via energy-dependent reversed electron transport. In state 3, all of these substrates caused considerably less inhibition of acetaldehyde oxidation, suggesting that the activity of aldehyde dehydrogenase, and not of NADH reoxidation, is probably rate limiting for acetaldehyde oxidation. The ionophores valinomycin and gramicidin stimulated acetaldehyde oxidation to a greater extent than ADP. These ionophores also stimulated acetaldehyde oxidation in the presence of ADP. Stimulation by valinomycin occurred in the presence of monovalent cations transported by this ionophore, e.g., K⁺, Rb⁺, Cs⁺. Stimulation by gramicidin also occurred in the presence of these cations, but did not occur with Na⁺ or Li⁺. Na⁺ prevents the stimulation of acetaldehyde oxidation, which occurs in the presence of gramicidin and K⁺. The stimulation by valinomycin and gramicidin was energy dependent and required the presence of a permeant anion. In the absence of an ionophore, potassium phosphate had no effect on acetaldehyde oxidation. These data suggest that the oxidation of acetaldehyde by rat liver and kidney mitochondria is influenced by the oxidation-reduction state of the mitochondria and by the cationic environment. With brain and muscle mitochondria, the rate of acetaldehyde oxidation increased two- to threefold as the concentration of acetaldehyde was raised from 0.167 to 0.50 mm. Acetaldehyde oxidation in these mitochondria was also sensitive; to rotenone, indicating dependence on NAD⁺. ADP, valinomycin, gramicidin, and succinate, compounds which either increased or decreased the rate of acetaldehyde oxidation by liver and kidney mitochondria, had no effect on acetaldehyde oxidation by muscle or brain mitochondria. In state 4, mitochondria from Becker-transplantable hepatocellular carcinoma HC-252 oxidized acetaldehyde at the same rate as liver mitochondria. However, in the presence of ADP, dinitrophenol, valinomycin and gramicidin, the rate of acetaldehyde oxidation by the tumor mitochondria was two to three times greater than that of liver mitochondria, suggesting the presence of a more active; acetaldehyde-oxidizing system in tumor than in liver mitochondria.     

Oscillating levels of adenylate and guanylate cyclase activities in rat embryo fibroblasts stimulated to divide.

Basal activities of membrane-bound adenylate and guanylate cyclase were determined in confluent rat embryo cells stimulated to proliferate by either the renewal of serum-supplemented growth medium or the addition of a mitogen, the 12-0-tetradecanoyl-phorbol-13-acetate (TPA). A transient increase in guanylate cyclase activity was observed within minutes following either treatment while adenylate cyclase activity either abruptly declined in serum-stimulated cells or remained unaffected in TPA-treated cells. In response to both mitogenic treatment, adenylate and guanylate cyclase activities varied reciprocally throughout the pre-replicative phase up to DNA synthesis. The lower levels of guanylate over adenylate activity ratio occurred prior to the onset of the replicative phase whereas the higher levels were coincident with DNA synthesis. A similar pattern of oscillating levels of sodium-fluoride-stimulated adenylate and lubrol-treated guanylate cyclase activities was observed.     

Aromatic and aliphatic mono- and bis-nitroxides: A study on their radical scavenging abilities

Nitroxide radicals are an emerging class of interesting compounds with versatile antioxidant and radioprotective properties. All literature studies have so far concentrated on compounds bearing only one nitroxide function. Here, we now investigate and compare the radical scavenging behaviour and antioxidant activity of aromatic indolinonic and aliphatic piperidine bis-nitroxides, i.e compounds bearing two nitroxide functions. Their corresponding mono-derivatives were also studied for comparison. Radical scavenging activity was investigated using EPR and UV–Vis spectroscopy by following spectral changes in acetonitrile of the nitroxides in the presence of alkyl and peroxyl radicals generated, respectively, under anoxic or aerobic conditions from thermal decomposition of AMVN [2,2′-azobis(2,4-di-methylvaleronitrile)]. Antioxidant activity of the nitroxides was evaluated by monitoring conjugated dienes (CD) formation during methyl linoleate micelles peroxidation and by measuring carbonyl content in oxidized bovine serum albumin (BSA). The results show that: (a) each nitroxide moiety in bis-nitroxides scavenges radicals independent of each other; (b) aliphatic nitroxides do not scavenge peroxyl radicals, at least under the experimental conditions used here, whereas indolinonic aromatic ones do: their stoichiometric number is 1.14 and 2.17, respectively, for mono- and bis-derivatives; (c) bis-nitroxides are roughly twice more efficient at inhibiting lipid peroxidation compared to their corresponding mono-derivatives. Although this study provides only comparative information on the relative radical-scavenging abilities of mono- and bis-nitroxides, it helps in understanding further the interesting reactivity of these compounds especially with regards to peroxyl radicals where many controversies in the literature exist.     

Differential ozone sensitivity interferes with cadmium stress in poplar clones

Information on plant responses to combined ozone and cadmium stresses are scarce and limited to herbaceous species. In this research, two poplar clones (I-214 and Eridano), differently sensitive to O₃, were grown for 5 weeks in pots supplied with 0, 53.5, and 160.5 mg(Cd) kg^?1 (soil d.m.) and then exposed to 15-d O₃ fumigation (0.06 mm³ dm^?3, 5 h a day). The effects of the two stressors, alone or in combination, on Cd, Ca, Fe, and Zn accumulation in above-nad below-ground organs, photosynthesis, leaf pigments, and accumulation of H₂O₂ and NO were investigated. Cadmium induced a reduction in stomatal conductance and a significant accumulation of H₂O₂ and NO in both clones nad negatively affected the carotenoid content in I-214. Ozone, on the other hand, counteracted Cd accumulation in the above-ground organs and significantly increased the xanthophyll de-epoxidation state indicating photoinhibition in O₃-treated plants. Surprisingly, O₃ alone or in combination with Cd decreased H₂O₂ accumulation in I-214. The NO production was generally stimulated by Cd, whereas it decreased following O₃ exposure in I-214. The overall data indicate that Cd and O₃ induced clone specific responses. Moreover, when they were applied in combination, antagonistic rather than synergistic effects were observed.     

Glutamate 59 is critical for transport function of the amino acid cotransporter KAAT1

KAAT1 is a neutral amino acid transporter activated by K⁺ or by Na⁺ (9). The protein shows significant homology with members of the Na⁺/Cl^–-dependent neurotransmitter transporter super family. E59G KAAT1, expressed in Xenopus oocytes, exhibited a reduced leucine uptake [20–30% of wild-type (WT)], and kinetic analysis indicated that the loss of activity was due to reduction of V_max and apparent affinity for substrates. Electrophysiological analysis revealed that E59G KAAT1 has presteady-state and uncoupled currents larger than WT but no leucine-induced currents. Site-directed mutagenesis analysis showed the requirement of a negative charge in position 59 of KAAT1. The analysis of permeant and impermeant methanethiosulfonate reagent effects confirmed the intracellular localization of glutamate 59. Because the 2-aminoethyl methanethiosulfonate hydrobromid inhibition was not prevented by the presence of Na⁺ or leucine, we concluded that E59 is not directly involved in the binding of substrates. N-ethylmaleimide inhibition was qualitatively and quantitatively different in the two transporters, WT and E59G KAAT1, having the same cysteine residues. This indicates an altered accessibility of native cysteine residues due to a modified spatial organization of E59G KAAT1. The arginine modifier phenylglyoxal effect supports this hypothesis: not only cysteine but also arginine residues become more accessible to the modifying reagents in the mutant E59G. In conclusion, the results presented indicate that glutamate 59 plays a critical role in the three-dimensional organization of KAAT1. amino acid transport; structure/function; amino acid modifiers; Manduca sexta     

Relation between fitness tests and match performance in elite Italian soccer referees

This study examined the relation between field-test results and match performance in elite Italian soccer referees. Subjects (n = 22) were all experienced elite-level referees enrolled in the Commissione Arbitri Nazionali (CAN) and thus officiating in the Serie A and B Italian championships. Referees were, on separate occasions, tested for fitness (50-m, 200-m, and 12-minute run tests) and observed a minimum of 1 and a maximum of 3 times (n = 39) during Serie A matches. Match analyses were performed considering 11 match activity categories. Analyses of correlations were performed considering 50-m, 200-m, and 12-minute run test performances as independent variables and total distance, maximal speed distance (runs performed at speeds faster than 24 km.h-1), and high-intensity activity distance (runs performed at speeds faster than 18 km.h-1, high intensity activity [HIA]) as dependent variables. Statistical significance was set at p 相似文献