Impairment of raw 264.7 macrophage function by antiarrhythmic drugs

The effect of the antiarrhythmic drugs lidocaine, quinidine and procainamide on macrophage function was investigated in RAW 264.7 mouse monocytic macrophage cell. Cells stimulated by either zymosan or phorbol ester were found to generate both superoxide (O ₂ ^– ) and H₂O₂. The production of O₂ was detected as superoxide dismutase inhibitable ferricytochrome c reduction. H₂O₂ production was monitored in both chemical and flow cytometric fluorescent assays. Although all three drugs inhibited both O₂ and H₂O₂ release in a dose dependent manner, only quinidine was found to have significant inhibitory effects. The amounts of quinidine required to cause a 50% inhibition in O₂ production in zymosan and phorbol ester stimulated cells were found to be 250 M and 300 M, respectively and the amounts required to cause one-half optimum levels of H₂O₂ production in these cells were found to be 50 M and 100 M, respectively. The effect of these drugs on O₂ producing NADPH oxidase was investigated and only procainamide was found to have a significant effect (p<0.001) in inhibiting the oxidase activity. Lidocaine and quinidine had no significant effect on the activation of the respiratory burst oxidase. A sensitive and convenient differential phagocytosis assay was devised on the basis of number of particles engulfed by individual phagocytes using flow cytometric techniques. It appears to be remarkably free of interference and was applied to investigate the role of antiarrhythmic drugs on the phagocytosis of fluorescent latex beads. All three antiarrhythmic drugs inhibited phagocytosis of latex beads in a dose dependent manner irrespective of the number of particles phagocitized by the cells. The results of these studies do not conclusively establish a mechanism of action of these drugs on the generation of O₂ and H₂O₂ by stimulated macrophages; nevertheless, it is interesting that all three drugs inhibited the phagocytic activity.     

Protective effects of lazaroids against oxygen-free radicals induced lysosomal damage

Lipid peroxidation of membranes by oxygen free radicals has been implicated in various disease states. Different antioxidants and iron chelators have been used to reduce lipid peroxidation. Lazaroids have been used for the acute treatment of central nervous system disorders such as trauma and ischemia wherein lipid peroxidative processes take place.In this study we evaluated the effect of lazaroids (U-785 18F and U-74389F) on the release of acid phosphatase activity and formation of malondialdehyde (MDA) in rat liver lyosomes subjected to exogenously generated oxygen free radicals. There was a significant increase in the acid phosphatase release and MDA formation in the presence of oxygen free radicals. This was prevented by both the lazaroids. In a separate study the effect of lazaroid U-74389F was seen on the zymosan-stimulated polymorphonuclear (PMN) leukocyte-derived chemiluminescence. The PMN leukocyte chemiluminescent activity was attenuated by the lazaroid in a dose-dependent manner. These studies suggest that lazaroids may inhibit lipid peroxidation and stabilize the membrane.     

Superoxide dismutase,catalase, and U78517F attenuate neuronal damage in gerbils with repeated brief ischemic insults

Repeated ischemic insults at one hour intervals result in more severe neuronal damage than a single similar duration insult. The mechanism for the more severe damage with repetitive ischemia is not fully understood. We hypothesized that the prolonged reperfusion periods between the relatively short ischemic insults may result in a pronounced generation of oxygen free radicals (OFRs). In this study, we tested the protective effects of superoxide dismutase (SOD) and catalase (alone or in combination), and U78517F in a gerbil model of repetitive ischemia. Three episodes (two min each) of bilateral carotid occlusion were used at one hour intervals to produce repetitive ischemia. Superoxide dismutase and catalase were infused via osmotic pumps into the lateral ventricles. Two doses of U78517F were given three times per animal, one half hour prior to each occlusion. Neuronal damage was assessed 7 days later in several brain regions using the silver staining technique. The Mann-Whitney U test was used for statistical comparison. Superoxide dismutase showed significant protection in the hippocampus (CA4), striatum, thalamus and the medial geniculate nucleus (MGN). Catalase showed significant protection in the striatum, hippocampus, thalamus, and MGN and the substantia nigra reticulata. Combination of the two resulted in additional protection in the cerebral cortex. Compared to the controls, there was little protection with a dose of 3 mg/kg of U78517F. There was significant protection with a dose of 10 mg/kg in the hippocampus (CA4), striatum, thalamus, medial geniculate nucleus and the substantia nigra reticulata. The significant protection noted with SOD, catalase or U78517F with repeated ischemia supports, the hypothesis that OFRs may play a role in neuronal damage in repeated cerebral ischemia.     

Enhancement of NMDA-mediated responses by cyanide

The effect of cyanide on NMDA-activated ion current and MK801 binding was studied in cultured rat hippocampal neurons. In microfluorometric analysis using fura-2, removal of extracellular Mg²⁺ resulted in a five-fold increase in NMDA-induced peak of [Ca²⁺]_i. One mM NaCN enhanced the peak NMDA responses in the presence, but not in the absence of extracellular Mg²⁺. Cyanide enhanced the immediate rise in [Ca²⁺]_i produced by NMDA, followed over a 1–5 min period by a gradual increase of [Ca²⁺]_i. Similar results were obtained in whole-cell patch clamp recordings from hippocampal neurons. One mM KCN enhanced the NMDA-activated current in the presence, but not in the absence of extracellular Mg²⁺. This effect was independent of cyanide-mediated metabolic inhibition since the recording pipette contained ATP (2 mM). In binding assays NaCN (1 mM) increased the binding affinity of [³H]MK-801 to rat forebrain membranes in the presence of Mg²⁺, whereas in the absence of Mg²⁺, NaCN did not influence binding. These results indicate that cyanide enhances NMDA-mediated Ca²⁺ influx and inward current by interacting with the Mg²⁺ block of the NMDA receptor. The effect of cyanide can be explained by an initial interaction with the Mg²⁺ block of the NMDA receptor/ionophore which appears to be energy-independent, followed by a gradual increase in Ca²⁺ influx resulting from cellular energy reserve depletion.Abbreviations NMDA N-Methyl-D-Aspartate - EAA excitatory amino acid - MK-801 (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohept-5,10-imine maleate     

MPEG－SOD对急性低氧下鼠左心功能的保护作用

单甲氧基聚乙二醇（ＭＰＥＧ）活化后与超氧化物歧化酶（ＳＯＤ）偶联,经纯化后得到ＭＰＥＧ－ＳＯＤ,鉴定其分子量约为７．０×１０５Ｄａｔｉｏｎ。其在血液循环中酶活性半衰期超过３０ｈ,远大于天然ＳＯＤ（６－１０ｍｉｎ）。ＳＤ雄性大鼠分三组．对照组（ｎ＝８．由股静脉注入０．２ｍｌ生理盐水）、ＮａｔｉｖｅＳＯＤ组（ｎ＝８,注以０．２ｍｌ生理盐水配的８００ＵＳＯＤ）和ＭＰＥＧ－ＳＯＤ组（ｎ＝９,注以８００ＵＭＰＥＧ－ＳＯＤ）。低氧前三组鼠的心率（ＨＲ）、左室内峰压（ＬＶＰ）、左室压微分（ＬＶ±ｄｐ／ｄｔｍａｘ）和股动脉压（ＡＰ）均无统计学差别。在模拟６０００—６５００ｍ高度急性低氧１．８．１４ｍｉｎ记录上述各项指标,结果如下：除ＨＲ外,ＮａｔｉｖｅＳＯＤ组与对照组各指标均无统计学差别,ＭＰＥＧ－ＳＯＤ组的各项指标均明显高于对照组。表明ＭＰＥＧ－ＳＯＤ对急性低氧导致的左心室力学指标的减弱有明显保护作用,提示超氧自由基（Ｏ２－）在急性低氧导致左心室功能损伤中起重要作用,即可能是由Ｏ２－及其衍生的其它自由基损害心肌细胞生物膜系统所致。     

Intrinsic pKas of ionizable residues in proteins: An explicit solvent calculation for lysozyme

Molecular dynamics simulations of triclinic hen egg white lysozyme in aqueous solution were performed to calculate the intrinsic pK_as of 14 ionizable residues. An all-atom model was used for both solvent and solute, and a single 180 ps simulation in conjunction with a Gaussian fluctuation analysis method was used. An advantage of the Gaussian fluctuation method is that it only requires a single simulation of the system in a reference state to calculate all the pK_as in the protein, in contrast to multiple simulations for the free energy perturbation method. pK_int shifts with respect to reference titratable residues were evaluated and compared to results obtained using a finite difference Poisson-Boltzmann (FDPB) method with a continuum solvent model; overall agreement with the direction of the shifts was generally observed, though the magnitude of the shifts was typically larger with the explicit solvent model. The contribution of the first solvation shell to the total charging free energies of the titratable groups was explicitly evaluated and found to be significant. Dielectric shielding between pairs of titratable groups was examined and found to be smaller than expected. The effect of the approximations used to treat the long-range interactions on the pK_int shifts is discussed. © 1994 Wiley-Liss, Inc.     

Influence of some exogenous amino acids on the production of maize embryogenic callus and on endogenous amino acid content

The effects of four exogenous amino acids (proline, glycine, asparagine and serine) on the production of maize embryogenic callus and on its endogenous amino acid content have been investigated. For this purpose, an established embryogenic line of Type 1 callus from the inbred W64Ao2 has been used. From the results it may be concluded that a concentration of proline exceeding 6 mM is negative for the production of embryogenic callus. When proline is eliminated from the medium, other amino acids tested in certain concentrations yield a percentage of embryogenic callus production that exceeds or equals that of proline. The endogenous free proline content in embryogenic callus is significantly higher than that in non-embryogenic callus regardless of proline presence in the medium. The only exception are the glycine-containing media, in which endogenous free alanine of embryogenic callus increases at the expense of endogenous free proline. This study suggest a positive role of endogenous free proline or alanine accumulation in the embryogenic callus production which might be related to an adaptation to the metabolic changes produced by in vitro culture and embryogenesis induction. Furthermore, these results indicate that treatments with amino acids that are different from proline can be used to improve the efficiency of embryogenic callus production from well established maize callus cultures.Abbreviations Ala alanine - Asn asparagine - 2,4-d 2,4-dichlorophenoxyacetic acid - EC embryogenic callus - nEC non-embryogenic callus - Gaba gamma-aminobutyric acid - Glu glutamic acid - Gly glycine - Pro proline - Ser serine     

Contrasting effects of PACAP and carbachol on [Ca]i and inositol phosphates in human neuroblastoma NB-OK-1 cells

The effects of PACAPs on [Ca²⁺]_i were compared to those of carbachol in human neuroblastoma NB-OK-1 cells. PACAP(1–27) and PACAP(1–38) increased [Ca²⁺]_i in a biphasic manner: a transient rise and a secondary plateau. The transient phase reflected the mobilization of [Ca²⁺]_i pool(s) via the inositol phosphate pathway. The modest sustained plateau required extracellular Ca²⁺. Carbachol also increased [Ca²⁺]_i in a biphasic manner, but it mobilized intracellular Ca²⁺ pool(s) with a higher efficacy than PACAPs, then greatly increased Ca²⁺ entry, this being accompanied by a more marked and prolonged elevation of IP₃ and IP₄ than with PACAPs. It is likely that cAMP-mediated phosphorylations due to PACAPs facilitated desensitization at the PACAP receptor-phospholipase C level, so that there was less Ca²⁺ handling through PACAP receptors than with muscarinic M₁ receptors.     

Oxidative processes as indicators of chemical stress in marine bivalves

Deleterious effects of environmental contaminants could be due to enhanced prooxidant forces overcoming antioxidant defences. Before practical biomarkers based on free radical biology will be generally accepted and validated in situ, additional research is required concerning normal physiological and environmental influences on the relevant systems. The aims of this study were to evaluate in situ the importance of oxyradical production in the presence and absence of pollutants and to characterize some antioxidant systems in Mytilus edulis L. Specimens of M. edulis L. were transplanted from a reference site (Franquelin) to Baie Comeau (Baie des Anglais), on the North shore of the St. Lawrence maritime estuary, where are found aluminium and pulp and paper plants. An oxidative stress was observed in mussels submitted to a chronic exposure in the polluted environment. Variations of pro-and anti-oxidant molecules involved in oxidative processes were related in part to seasonal and physico-chemical influences. Catalase activity, malondialdehyde and glutathione concentrations will be useful as biomarkers of stress in situ since they react to anthropogenic influence and to abiotic factors such as emersion period and temperature.