Stimulation of phospholipase A2 activity by oxygen-derived free radicals in isolated brain capillaries

An exogenous free radical generating system added to isolated brain capillaries induces degradation of phospholipids. This inductive effect reflects increased phospholipase activities as measured by fatty acid composition of various phospholipid fractions. The correlation of phospholipid degradation with stimulation of phospholipases was further investigated by using cationic amphiphilic agents, which are known to be phospholipase A2 inhibitors. The breakdown of phospholipids was inhibited by the pretreatment of isolated capillaries with these drugs.     

Pro and antioxidant responses to repeated administration of diazepam in rat brain

The role of pro/antioxidative processes during a low, subtoxic dose schedule of diazepam (3 mg/kg/day i.p.) for 7 days and its withdrawal in subcellular preparations of rat brain regions was studied in detail. The results indicated heterogeneity in the regional responses as well as in subcellular compartments. After 7 days of exposure to the drug, a decrease in the Mn-SOD activity was observed in the 3 regions studied while a significant increase in Cu/Zn-SOD activity was seen in cerebellum (CBL) and brain stem (BS) along with that of mitochondrial glutathione reductase. The post-mitochondrial fraction (PMF) showed a significant increase in GR activity in cerebrum. Enhancement of total and free thiol levels was observed in cerebrum and cerebellum whereas in BS free thiols were not enhanced. It was interesting to note that in the animals withdrawn from the drug and sacrificed after an interval of 7 days, the level of TBARS showed a highly significant increase in mitochondria of CB and CBL and 89% increase in BS. Similar trend was observed in the post-mitochondrial fractions of all the 3 regions whereas the activity of isozymes of SOD decreased (p < 0.001) in CBL and BS and to a lesser extent in CB. The GR activity was significantly decreased only in the mitochondria of cerebrum with a 34% rise in cerebellum and no change in BS. The PMFs showed a decrease in CB and CBL but a 20% rise in BS. Thus, the data show modulation of antioxidant responses during short-term administration of diazepam, and a lowering of peroxidative decomposition of polyunsaturated fatty acids of membranes. However, after withdrawal of the drug, PUFAs were found to be more vulnerable to peroxidative decomposition and changes in the antioxidant defenses were also observed, which did not come back to normal level during the study.     

Effect of guanidinoethanesulfonic acid on brain monoamines in the mouse

Guanidinoethanesulfonic acid (GES) is known to induce convulsive seizures when administered intracisternally to rabbits and cats. The effects of GES on behavior, electroencephalographic recording and brain monoamine levels were examined in mice. When GES (900 nmol) was intraventricularly injected into mice, focal clonic movements of the face, vibrissae and ears together with twitching of the limbs were observed 0.5–1 min after the injection. Hypersensitivity was observed up to 7 min after the injection, after which the mice behaved normally. GES also induced sporadic spike discharges on electrocorticogram. The levels of 5-hydroxytryptamine (5-HT) of the GES-injected mice were lower than those of the saline-injected mice in the hippocampus, diencephalon, pons-medulla oblongata and cerebellum 5 min after the injection. No changes in the norepinephrine or dopamine levels were found after the GES injection. The level of 5-hydroxyindoleacetic acid increased in the striatum and cerebellum 5 min after the GES injection. These results suggest that GES-induced convulsive activities enhance the serotonergic neuroactivity in order to suppress the convulsions.     

Effects of free radicals on cytosolic creatine kinase activities and protection by antioxidant enzymes and sulfhydryl compounds

The main purpose of this study was to investigate the effect of free radicals and experimental diabetes on cytosolic creatine kinase activity in rat heart, muscle and brain. Hydrogen peroxide decreased creatine kinase activity in a dose dependent manner which was reversed by catalase. Xanthine/xanthine oxidase, which produces superoxide anion, lowered the creatine kinase activity in the same manner whose effect was protected by superoxide dismutase. N-acetylcysteine and dithiothreitol also significantly ameliorated the effect of Xanthine/xanthine oxidase and hydrogen peroxide. Experimental diabetes of twenty-one days (induced by alloxan), also caused a similar decrease in the activity of creatine kinase. This led us to the conclusion that the decrease in creatine kinase activity during diabetes could be due to the production of reactive oxygen species. The free radical effect could be on the sulfhydryl groups of the enzyme at the active sites, since addition of sulfhydryl groups like N-acetylcysteine and dithiothreitol showed a significant reversal effect.     

Oxygen toxicity in the nervous tissue: Comparison of the antioxidant defense of rat brain and sciatic nerve

Nervous tissue, central and peripheral, is, as any other, subject to variations in oxygen tension, and to the attack of different xenobiotics; these situations may promote the generation of activated oxygen species of free radical character. Results are presented showing that the content of total glutathione (GSH) in brain is 10-fold that found in the sciatic nerve of the rat (2620 vs. 261 nmol/g wet weight, respectively). The existence of a relatively high superoxide dismutase activity in peripheral nervous tissue, when compared with brain or liver, in combination with the DT-diaphorase activity detected in the sciatic nerve might represent an effective defense mechanism against quinone toxicity, as is also discussed. Nervous tissue, both central and peripheral lack Se-independent GSH peroxidase activity. Finally, the activities of other glutathione-related enzymes studied in the sciatic nerve are very low, when compared with the central nervous tissue, thus suggesting a higher susceptibility of peripheral tissue to oxidative stress damage, since GSH concentration and/or any GSH-related enzymatic activities, e.g. GSH peroxidase or glutathione disulfide reductase, might become limiting.     

Variation in proton affinity of the guanidino group between free and blocked arginine

Summary. In this paper, the analog of arginine residues in peptides was synthesized and characterized by ESI-MS/MS (electrospray ionization with tandem mass spectrometry), ³¹P NMR, ¹H NMR, IR and high-resolution mass spectrometry. When the Todd reaction activity of the guanidino group in free arginine and the arginine peptide analog were compared, it was found that the proton affinity of the guanidino group was decreased when both the N- and the C-terminal were blocked. As a result, the guanidino group of arginine residues in peptides could be phosphorylated under the Todd reaction condition, but not the free arginine. This result was further proved by the theoretical calculation of their proton affinity.     

Immuno-detection of aluminium and aluminium induced conformational changes in calmodulin - implications in Alzheimer's disease

Studies were conducted to ascertain any involvement of free radical mediated prooxidative processes in different brain regions following diazopam administration. A significant decrease in TBA reactive substance formation was observed in cerebral cortex, cerebellum and brain stem regions after single doses of 1.5, 3 and 6 mg/kg b.wt. For further studies rats were given diazepam (i.p.) at 3 mg/kg body weight dose and sacrificed after 1 h to follow changes in the pro/antioxidant status. An enhancement in the TBARS formation was found in the mitochondrial fractions from cerebral cortex and brain stem. This effect was highest in brain stem being 107% as compared to controls. In the post mitochondrial fraction, cerebellum showed 49% enhancement whereas decreased formation of thiobarbituric acid reactive substances was observed in cerebral cortex and brain stem. Isozymes of superoxide dismutase showed a decrease in activity which was region dependent. Even though, total thiols were not significantly altered, free thiols showed depletion in cerebellum (39.8%) and brain stem (50%). Glutathione reductase activity was also decreased in cerebellum and brain stem. The results indicate that a single dose of diazepam causes free radical mediated changes and the modulatory response of antioxidant defences appears to be region specific.     

Oxidative stress in Ca2+‐induced membrane permeability transition in brain mitochondria

Mitochondrial permeability transition (PT) is a non-selective inner membrane permeabilization, typically promoted by the accumulation of excessive quantities of Ca(2+) ions in the mitochondrial matrix. This phenomenon may contribute to neuronal cell death under some circumstances, such as following brain trauma and hypoglycemia. In this report, we show that Ca(2+)-induced brain mitochondrial PT was stimulated by Na(+) (10 mM) and totally prevented by the combination of ADP and cyclosporin A. Removal of Ca(2+) from the mitochondrial suspension by EGTA or inhibition of Ca(2+) uptake by ruthenium red partially reverted the dissipation of the membrane potential associated with PT. Ca(2+)-induced brain mitochondrial PT was significantly inhibited by the antioxidant catalase, indicating the participation of reactive oxygen species in this process. An increased detection of reactive oxygen species, measured through dichlorodihydrofluorescein oxidation, was observed after mitochondrial Ca(2+) uptake. Ca(2+)-induced dichlorodihydrofluorescein oxidation was enhanced by Na(+) and prevented by ADP and cyclosporin A, indicating that PT enhances mitochondrial oxidative stress. This could be at least in part a consequence of the extensive depletion in NAD(P)H that accompanied this Ca(2+)-induced mitochondrial PT. NADPH is known to maintain the antioxidant function of the glutathione reductase/peroxidase and thioredoxin reductase/peroxidase systems. In addition, the occurrence of mitochondrial PT was associated with membrane lipid peroxidation. We conclude that PT further increases Ca(2+)-induced oxidative stress in brain mitochondria leading to secondary damage such as lipid peroxidation.     

脑部乳酸供能的方式

脑部存在星形胶质细胞-神经元的乳酸穿梭方式供能。单羧酸转运蛋白2(monocarboxylate transporter 2,MCT2)和MCT4的细胞特异性分布与乳酸穿梭的方向存在密切联系。MCT2和MCT4分别集中于神经元突触后膜致密区与星形胶质细胞终足膜,可能有利于N-甲基-D-天冬氨酸(N-methyl-D-asparticacid,NMDA)受体抑制的解除及循环中乳酸穿越血脑屏障。神经元和星形胶质细胞均能利用乳酸或血糖作为氧化底物。神经元中,乳酸是比血糖更为优势的氧化底物,其原因有待研究。脑部乳酸不仅来源于血糖酵解,还来源于血液循环中的乳酸及星形胶质细胞的糖原酵解。     

Paraquat-Induced Free Radical Reaction in Mouse Brain Microsomes

Paraquat has been implicated as an environmental toxin which may induce the syndrome of Parkinson's disease after exposure to this agent. However, the biochemical mechanism by which paraquat causes cell death and neurodegeneration has not been extensively studied. Paraquat was rapidly taken up by nerve terminals isolated from mouse cerebral cortices. It induced lipid peroxidation in a concentration dependent manner in the presence of NADPH and ferrous ion. The maximal stimulation effect was obtained at a paraquat concentration around 100 M and the K_mvalue for paraquat was 46.7 M. The lipid peroxidation required microsomal enzymes. Antioxidants, such as superoxide dismutase, catalase and promethazine significantly inhibited paraquat-induced lipid peroxidation. Due to its structural similarity to the pyridinium compound MPP⁺(N-methyl-4-phenyl pyridium ion), it may be taken up by dopamine neurons and cause lipid peroxidation and cell death resulting in the manifestation of Parkinsonian syndrome.     

Reoxygenation Injury of Human Brain Capillary Endothelial Cells

1. Many studies have demonstrated that endothelial cells from several species can generate oxygen free radicals when subjected to anoxia and reoxygenation. However, due to the heterogeneity of the endothelium within different organs and species, the effects of superoxide dismutase (SOD), catalase, and allopurinol on reoxygenated cultured cells remain quite controversial.2. This review outlines the possible sources of oxygen free radicals within brain endothelial cells.3. We examine the aspects of the effects of SOD catalase and allopurinol on cultured human brain capillary endothelial cells upon reoxygenation.4. Also, we introduce briefly a method of culturing human brain capillary endothelial cells and present our experimental results on the effects of SOD, catalase, and allopurinol in these cultured cells following anoxia and reoxygenation.     

Paradoxical role of lipid metabolism in heart function and dysfunction

The naturally occurring flavonoid, quercetin, in the presence of Cu(II) and molecular oxygen caused breakage of calf thymus DNA, supercoiled pBR322 plasmid DNA and single stranded M13 phage DNA. In the case of the plasmid, the product(s) were relaxed circles or a mixture of these and linear molecules depending upon the conditions. For the breakage reaction, Cu(II) could be replaced by Fe(III) but not by other ions tested [Fe(II), Co(II), Ni(II), Mn(II) and Ca(II)]. Structurally related flavonoids, rutin, galangin, apigenin and fisetin were effective or less effecive than quercetin in causing DNA breakage. In the case of the quercetin-Cu(II) reaction, Cu(I) was shown to be essential intermediate by using the Cu(1)-sequestering reagent, bathocuproine. By using Job plots we established that, in the absence of DNA, five Cu(II) ions were reduced by one quercetin molecule; in contrast two ions were reduced per quercetin molecule in the DNA breakage reaction. Equally neocuproine inhibited the DNA breakage reaction. The involvement of active oxygen in the reaction was established by the inhibition of DNA breakage by superoxide dismutase, iodide, mannitol, formate and catalase (the inhibition was complete in the last case). The strand scission reaction was shown to account for the biological activity of quercetin as assayed by bacteriophage inactivation. From these data we propose a mechanism for the DNA strand scission reaction of quercetin and related flavonoids.     

A role for glutamate in growth and invasion of primary brain tumors

The vast majority of primary brain tumors derive from glial cells and are collectively called gliomas. While, they share some genetic mutations with other cancers, they do present with a unique biology and have developed adaptations to meet specific biological needs. Notably, glioma growth is physically restricted by the skull, and, unless normal brain cells are destroyed, tumors cannot expand. To overcome this challenge, glioma cells release glutamate which causes excitotoxic death to surrounding neurons, thereby vacating room for tumor expansion. The released glutamate also explains peritumoral seizures which are a common symptom early in the disease. Glutamate release occurs via system X^c, a cystine–glutamate exchanger that releases glutamate in exchange for cystine being imported for the synthesis of the cellular antioxidant GSH. It protects tumor cells from endogenously produced reactive oxygen and nitrogen species but also endows tumors with an enhanced resistance to radiation- and chemotherapy. Pre-clinical data demonstrates that pharmacological inhibition of system X^c causes GSH depletion which slows tumor growth and curtails tumor invasion in vivo . An Food and Drug Administration approved drug candidate is currently being introduced into clinical trials for the treatment of malignant glioma.     

Estimates of Gibbs free energies of formation of chlorinated aliphatic compounds

The Gibbs free energy of formation of chlorinated aliphatic compounds was estimated with Mavrovouniotis' group contribution method. The group contribution of chlorine was estimated from the scarce data available on chlorinated aliphatics in the literature, and found to vary somewhat according to the position of chlorine in the molecule. The resulting estimates of the Gibbs free energy of formation of chlorinated aliphatic compounds indicate that both reductive dechlorination and aerobic mineralization of these compounds can yield sufficient energy to sustain microbial growth.     

Antiprion compounds that reduce PrPSc levels in dividing and stationary-phase cells

During prion diseases, a normally benign, host protein, denoted PrP^C, undergoes alternative folding into the aberrant isoform, PrP^Sc. We used ELISA to identify and confirm hits in order to develop leads that reduce PrP^Sc in prion-infected dividing and stationary-phase mouse neuroblastoma (ScN2a-cl3) cells. We tested 52,830 diverse small molecules in dividing cells and 49,430 in stationary-phase cells. This led to 3100 HTS and 970 single point confirmed (SPC) hits in dividing cells, 331 HTS and 55 confirmed SPC hits in stationary-phase cells as well as 36 confirmed SPC hits active in both. Fourteen chemical leads were identified from confirmed SPC hits in dividing cells and three in stationary-phase cells. From more than 682 compounds tested in concentration–effect relationships in dividing cells to determine potency (EC₅₀), 102 had EC₅₀ values between 1 and 10 μM and 50 had EC₅₀ values of <1 μM; none affected cell viability. We observed an excellent correlation between EC₅₀ values determined by ELISA and Western immunoblotting for 28 representative compounds in dividing cells (R² = 0.75; p <0.0001). Of the 55 confirmed SPC hits in stationary-phase cells, 23 were piperazine, indole, or urea leads. The EC₅₀ values of one indole in stationary-phase and dividing ScN2a-cl3 cells were 7.5 and 1.6 μM, respectively. Unexpectedly, the number of hits in stationary-phase cells was ～10% of that in dividing cells. The explanation for this difference remains to be determined.     

An in vitro study on the role of metal catalyzed oxidation in glyeation and crosslinking of collagen

The present investigation was carried out to understand the effect of metal catalyzed oxidation on glycation and crosslinking of collagen. Tail tendons obtained from rats weighing 200-225 g were incubated with glucose (250 mM) and increasing concentrations of copper ions (5, 25, 50 and 100 M) under physiological conditions of temperature and pH. Early glycation, crosslinking and late glycation (fluorescence) of collagen samples were analyzed periodically. Early glycation was estimated by phenol sulfuric acid method, and the crosslinking was assessed by pepsin and cyanogen bromide digestion. A concentrationdependent effect of metal ions on the rate of glycation and crosslinking of collagen was observed. Tendon collagen incubated with glucose and 100 M copper ions showed 80% reduction in pepsin digestion within seven days, indicating extensive crosslinking, whereas collagen incubated with glucose alone for the same period showed only 7% reduction. The presence of metal ions in the incubation medium accelerated the development of Maillard reaction fluorescence on collagen, and the increase was dependent on the concentration of metal ions used. The metal chelator Diethylene triamine penta-acetate significantly prevented the increase in collagen crosslinking by glucose and copper ions. Free radical scavengers benzoate and mannitol effectively prevented the increased crosslinking and browning of collagen by glucose. The results indicate that the metal catalyzed oxidation reactions play a major role in the crosslinking of collagen by glucose. It is also suggested that the prevention of increased oxidative stress in diabetes may prevent the accelerated advanced glycation and crosslinking of collagen.     

A role for the magnetic field in the radiation-induced efflux of calcium ions from brain tissue in vitro

Two independent laboratories have demonstrated that electromagnetic radiation at specific frequencies can cause a change in the efflux of calcium ions from brain tissue in vitro. In a local geomagnetic field (LGF) at a density of 38 microTesla (microT), 15- and 45-Hz electromagnetic signals (40 Vp-p/m in air) have been shown to induce a change in the efflux of calcium ions from the exposed tissues, whereas 1- and 30-Hz signals do not. We now show that the effective 15-Hz signal can be rendered ineffective when the LGF is reduced to 19 microT with Helmholtz coils. In addition, the ineffective 30-Hz signal becomes effective when the LGF is changed to +/- 25.3 microT or to +/- 76 microT. These results demonstrate that the net intensity of the LGF is an important variable. The results appear to describe a resonance-like relationship in which the frequency of the electromagnetic field that can induce a change in efflux is proportional to a product of LGF density and an index, 2n + 1, where n = 0,1. These phenomenological findings may provide a basis for evaluating the apparent lack of reproducibility of biological effects caused by low-intensity extremely-low-frequency (ELF) electromagnetic signals. In future investigations of this phenomenon, the LGF vector should be explicitly described. If the underlying mechanism involves a general property of tissue, then research conducted in the ambient electromagnetic environment (50/60 Hz) may be subjected to unnoticed and uncontrolled influences, depending on the density of the LGF.     

Electrochemical Properties As A Function of Ph for the Benzotriazine DI-N-Oxides

The electrochemistry of five benzotriazine di-N-oxides has been examined by cyclic voltammetry and differential pulse and dc polarographies as a function of pH. Between the pH range 8.5 and 2 the trend to less negative potentials with lowering of pH can be described by an equation of the type Ep = - apH + b. Comparison has been made with the mono- and zero-N-oxides which were found to show virtually identical trends in electron affinity with pH. The general electrochemical characteristics for the di- and mono-N-oxides under acidic conditions were found to be comparable with the zero-N-oxide. This was particularly the case on repeat scanning in the cyclic voltammetric mode. The redox mechanism involved reduction by a 4-electron addition step and subsequent loss of the A-oxide group(s) yielding the intact benzotriazine heterocycle. The helerocycle was also redox active, involving a reversible 2-electron reduction. for the di-N-oxides these two stages could be identified as separate processes at alkaline pH, but ony a single step at acidic values. The mono-N-oxide in which the electrochemical behaviour was dominated by the triazine, showed only a single reduction step, although the single N-oxide group was redox active.