Relation between ESR-detectable Cu(II) and superoxide dismutase activity

The relation between ESR-detectable Cu(II) and Cu,Zn-superoxide dismutase activity was examined. The Cu(II) spin numbers per one unit of SOD were 6.26 X 10(12) (+/- 0.51 X 10(12] spins in several preparations of recombinant human Cu,Zn-SOD, native placental, and erythrocyte SOD. Measurement could be performed over a wide range of pH (4.0-10.0), preferably at temperatures below -40 degrees C. The data obtained by this method correlated well to the results obtained by the method of Fridovich et al. using the xanthine-xanthine oxidase system (correlation coefficient 0.995). The specific activity of SOD was proportional to the Cu(II) content measured by ESR, but not to the total Cu content measured by atomic absorption. This indicates that it is important to measure the Cu(II) content for determining Cu,Zn-SOD activity.     

Measurement of superoxide radicals produced by human activated neutrophils by accumulation of stable nitroxide radicals detected by MR spectroscopy

An important index of neutrophil function is the production of superoxide radicals (O2-) upon activation. Thus a development of a new adequate assay of O2- generation measurement is of great interest for phagocyte researchers. The present article considers the quantitative determination of O2- generation based on the interaction of O2- with 1-oxy-2,2,6,6-tetramethyl-4-oxypiperidine producing 4-oxo-2,2,6,6-piperidine-1-oxyl, detected by ESR. The kinetic curve of nitroxyl radical (NR) formation has a linear character. The NR formation rate after a short induction period (appr. 2 min.) approaches 3.3 X 10(-3) M/s, where cell concentration was 4 X 10(5) per ml. Hydroxylamine (3.8 mM) auto-oxidation rate is negligible as compared with activated neutrophils and is equal to 2 X 10(-9) M/s. Sensitivity NR to the presence of superoxide dismutase (SOD) came as evidence that NR formation is due O2- radicals. SOD (10(-7) M) inhibits NR formation by 90%. Hydroxylamine oxidation by O2- is an irreversible reaction--20-min incubation of activated neutrophils with NR do not influence NR concentration. The NR generation rate dependence upon the neutrophil concentration is linear in the cell concentration range from 4 X 10(5 up to 6 X 10(6) per ml. In this range a quantitative measurement of O2- production is suitable. The sensitivity of hydroxylamine assay is close to the sensitivity of chemiluminescent method, but specificity is higher, as SOD inhibits chemiluminescence only by 50%.     

SOD2-mediated effects induced by WR1065 and low-dose ionizing radiation on micronucleus formation in RKO human colon carcinoma cells

RKO36 cells exposed to either WR1065 or 10 cGy X rays show elevated SOD2 gene expression and SOD2 enzymatic activity. Cells challenged at this time with 2 Gy exhibit enhanced radiation resistance. This phenomenon has been identified as a delayed radioprotective effect or an adaptive response when induced by thiols or low-dose radiation, respectively. In this study we investigated the relative effectiveness of both WR1065 and low-dose radiation in reducing the incidence of radiation-induced micronucleus formation in binucleated RKO36 human colon carcinoma cells. The role of SOD2 in this process was assessed by measuring changes in enzymatic activity as a function of the inducing agent used, the level of protection afforded, and the inhibitory effects of short interfering RNA (SOD2 siRNA). Both WR1065 and 10 cGy X rays effectively induced a greater than threefold elevation in SOD2 activity 24 h after exposure. Cells irradiated at this time with 2 Gy exhibited a significant resistance to micronucleus formation (P < 0.05; Student's two-tailed t test). This protective effect was significantly inhibited in cells transfected with SOD2 siRNA. SOD2 played an important role in the adaptive/delayed radioprotective response by inhibiting the initiation of a superoxide anion-induced ROS cascade leading to enhanced mitochondrial and nuclear damages.     

Changes in the Spinal Cord Proteome of an Amyotrophic Lateral Sclerosis Murine Model Determined by Differential In-gel Electrophoresis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by loss of motor neurons resulting in progressive paralysis. To date, more than 140 different mutations in the gene encoding CuZn-superoxide dismutase (SOD1) have been associated with ALS. Several transgenic murine models exist in which various mutant SOD1s are expressed. We used DIGE to analyze the changes in the spinal cord proteome induced by expression of the unstable SOD1 truncation mutant G127insTGGG (G127X) in mice. Unlike mutants used in most other models, G127X lacks SOD activity and is present at low levels, thus reducing the risk of overexpression artifacts. The mice were analyzed at their peak body weights just before onset of symptoms. Variable importance plot analysis showed that 420 of 1,800 detected protein spots contributed significantly to the differences between the groups. By MALDI-TOF MS analysis, 54 differentially regulated proteins were identified. One spot was found to be a covalently linked mutant SOD1 dimer, apparently analogous to SOD1-immunoreactive bands migrating at double the molecular weight of SOD1 monomers previously detected in humans and mice carrying mutant SOD1s and in sporadic ALS cases. Analyses of affected functional pathways and the subcellular representation of alterations suggest that the toxicity exerted by mutant SODs induces oxidative stress and affects mitochondria, cellular assembly/organization, and protein degradation.Amyotrophic lateral sclerosis (ALS)¹ is a devastating neurodegenerative disease characterized by loss of motor neurons in the motor cortex, the brainstem, and the spinal cord. This results in progressive muscular atrophy, and the patients usually succumb to respiratory failure within a few years. About 10% of ALS cases are familial (1), and in some patients the disease is linked to mutations in the CuZn-superoxide dismutase (SOD1) gene (2). SOD1 is a ubiquitously expressed antioxidant enzyme. Overall about 6% of all cases with ALS show SOD1 mutations, and more than 140 such mutations have been identified (3). The mutations confer a cytotoxic gain of function of unknown character to the enzyme (4, 5). ALS caused by mutant SOD1s shows the same spectrum of disease phenotypes as is seen in sporadic cases lacking such mutations. This suggests that the pathogenesis of ALS induced by SOD1 mutations should show significant similarities with that in sporadic disease, e.g. similar pathogenic protein alterations.ALS has been modeled in mice via transgenic overexpression of mutant SOD1s (5–8). To cause disease within the short lifespan of a mouse, the mutant SOD1s have to be expressed at rates around 25-fold higher than the rate of expression of the endogenous murine enzyme (9). Mostly structurally stable mutants have been used, resulting in up to 10-fold increases in SOD activity and 20-fold increases in SOD1 protein levels in the CNS that may cause overexpression artifacts. Overloading of mitochondria with mutant SOD1s and vacuolization have been observed in such models (10).To explore ALS pathogenesis, we studied changes in the proteome of spinal cords of SOD1 transgenic mice using DIGE. To reduce the risk of overexpression artifacts, we used mice that express the unstable human SOD1 (hSOD1) truncation mutant G127insTGGG (G127X) (7). These mice develop an aggressive form of the disease, which is of short duration, despite the fact that the mutant lacks SOD activity and the fact that the level of G127X hSOD1 protein is less than half that of the endogenous murine SOD1. The mice were studied at their peak body weights just before development of paralytic symptoms. Here we present the identity of and discuss the possible significance of 53 proteins found to be differentially regulated in ALS transgenic mice.     

The neuronal adaptor protein X11alpha interacts with the copper chaperone for SOD1 and regulates SOD1 activity

The neuronal adaptor protein X11alpha participates in the formation of multiprotein complexes and intracellular trafficking. It contains a series of discrete protein-protein interaction domains including two contiguous C-terminal PDZ domains. We used the yeast two-hybrid system to screen for proteins that interact with the PDZ domains of human X11alpha, and we isolated a clone encoding domains II and III of the copper chaperone for Cu,Zn-superoxide dismutase-1 (CCS). The X11alpha/CCS interaction was confirmed in coimmunoprecipitation studies plus glutathione S-transferase fusion protein pull-down assays and was shown to be mediated via PDZ2 of X11alpha and a sequence within the carboxyl terminus of domain III of CCS. CCS delivers the copper cofactor to the antioxidant superoxide dismutase-1 (SOD1) enzyme and is required for its activity. Overexpression of X11alpha inhibited SOD1 activity in transfected Chinese hamster ovary cells which suggests that X11alpha binding to CCS is inhibitory to SOD1 activation. X11alpha also interacts with another copper-binding protein found in neurons, the Alzheimer's disease amyloid precursor protein. Thus, X11alpha may participate in copper homeostasis within neurons.     

Subunit interaction: A molecular basis of heterosis

Acid phosphatase, a dimeric enzyme, in Drosophila malerkotliana was studied in isogenic flies to explore the molecular basis of heterosis. As the enzyme activity in heterozygotes is 34% more than that in the better parent (S/S), heterosis is indicated. Vmax, Km, and Ki values are 14.60, 3.6 X 10(-4) M, and 0.45 X 10(-4) M, respectively, for the enzyme from F/S flies and 11.80, 4.0 X 10(-4) M, and 0.37 X 10(-4) M, respectively, for the enzyme from S/S flies. Thus heterosis for enzyme activity results from a better enzyme in F/S flies. The higher efficiency and better quality of the enzyme in F/S flies were traced to the heterodimeric allozyme, present only in heterozygotes. Enzyme activity, Vmax, Km, and Ki values are 0.739, 42.1; 3.6 X 10(-4) M, and 0.50 X 10(-4) M, respectively, for the heterodimeric and 0.513, 36.8; 4.1 X 10(-4) M, and 0.37 X 10(-4) M, respectively, for the better parental homodimeric allozyme. On an equimolar basis the enzyme activity of the heterodimer is 44% higher than that of the better homodimer. The better performance of the heterodimer is probably a reflection of superior conformation resulting from interaction between component subunits (F and S polypeptides).     

Disintegration of Rhodospirillum rubrum chromatophore membrane into photoreaction units, reaction centers, and ubiquinone-10 protein with mixture of cholate and deoxycholate.

1. The membrane of Rhodospirillum rubrum chromatophores was disintegrated with mild detergents (cholate and deoxycholate) in order to study the spatial arrangement of the functional proteins in the photochemical apparatus and the electron transport system in the membrane. 2. The components solubilized from the membrane by a mixture of cholate and deoxycholate (C-DOC) were separated into four fractions by molecular-sieve chromatography in the presence of C-DOC; they were designated as F1, F2, F3, and F4 in the order of elution. The fractions were further purified by repeated molecular-sieve chromatography in the presence of C-DOC until each fraction was chromatographically homogeneous. 3. F1 appeared to be conjugated forms of F2. 4. The purified F2 was composed of a rigid complex having a weight of 7 X 10(5) daltons, containing approximately 10 different kinds of protein species with molecular weights of 3.8 X 10(4), 3.6 X 10(4), 3.5 X 10(4), 2.8 X 10(4), 2.7 X 10(4), 2.6 X 10(4), 1.3 X 10(4), 1.2 X 10(4), 1.1 X 10(4), and 1.0 X 10(4). The complex contained 33 bacteriochlorophylls, 4 iron atoms, and 90 phosphates, but no cytochrome, ubiquinone, or phospholipid. It showed the same reaction center activity as chromatophores, indicating that the complex was a unit of the photochemical apparatus (photoreaction unit). Each chromatophore of average size was estimated to possess about 24 photoreaction units. 5. The purified F3 showed an absorbance spectrum characteristic of reaction centers, and contained 3.4 bacteriochlorophylls, 2.0 bacteriopheophytins, and 1.9 acid-labile iron atoms, but no cytochrome or ubiquinone (C-DOC reaction center). It had a weight of 1.2 X 10(5) daltons, and the main components were 4 protein species with molecular weights of 2.8 X 10(4), 2.7 X 10(4), 2.6 X 10(4), and 1.0 X 10(4). 6. The purified F4 showed a molecular weight of about 11,000, and contained one mole of ubiquinone-10 per mole (ubiquinone-10 protein). 7. The reaction center activity of C-DOC reaction centers was stimulated by ubiquinone-10 protein. In addition, the reaction center oxidized reduced cytochrome c2 in the light, provided that ubiquinone-10 protein was present (photo-oxidase activity).     

Thiamine intestinal transport and phosphorylation : a study in vitro of potential inhibitors of small intestinal thiamine-pyrophosphokinase using a crude enzymatic preparation.

Using as enzymatic source the cytoplasmatic fraction of enterocytes isolated from the rat small intestine, thiamine-pyrophosphokinase activity was studied with a radiometric method using [thiazole-2-(14)C] thiamine. The Km value for thiamine was 2.14 X 10(-6) M and V 0.87 nmol of thiamine pyrophosphate mg-1 protein h-1. Eleven thiamine structural analogs and derivatives were assayed for their inhibitory action on the small intestine thiamine-pyrophosphokinase activity. Their Ki values were : pyrithiamine, 2.25 X 10(-6) M; thiamine monophosphate, 4 X 10(-6) M; 2'-ethylthiamine, 8 X 10(-6) M; 2'-butylthiamine, 6 X 10(-6) M; chloroethylthiamine and dimethalium, 1.5 X 10(-5) M; amprolium, 1.8 X 10(-4) M; L-582571, 1.65 X 10(-4) M; oxythiamine, 4.2 X 10(-3) M. Of the miscellaneous compounds tested (toxopyrimidine, Na-pyrophosphate, choline, L-phenylalanine, ethyl-urethane and 5-fluorouracil), none had any inhibitory action on intestinal thiamine-pyrophosphokinase activity, even if used at concentrations hundred times higher than that of labelled thiamine.     

Immunochemical analysis and protective properties of components of Pseudomonas aeruginosa mucus with different molecular weights

P. aeruginosa slime has been separated into fractions XM-300 (3 X 10(5) daltons and more), XM-100 (1 X 10(5) = 3 X 10(5) daltons), PM-30 (3 X 10(4) = 1 X 10(5) daltons) and PM-10, (1 X 10(4) = 3 X 10(4) daltons) by ultrafiltration. The high-molecular slime components (3 X 10(4) daltons and more) have been found to be serologically more active than the low-molecular components (1 X 10(4) = 3 X 10(4) daltons). As shown in experiments on mice, both high-molecular toxic and low-molecular nontoxic slime components have protective activity, but the high-molecular components are more active than the low-molecular ones. The slime components stimulate the formation of immunity to homologous and partially heterologous P. aeruginosa strains in mice. Antigenic relationship between the slime components (especially the high-molecular ones) and the corresponding lipopolysaccharides has been noted.     

In vivo radiosensitization by diethyldithiocarbamate

Diethyldithiocarbamate (DDC) has been suggested to have both radiosensitizing (due to superoxide dismutase (SOD) inhibition) and radioprotective properties. We have studied the activity of SOD up to 24 h after intratumoral administration of 50, 100, 150, and 300 mg/kg DDC in 3-methylcholanthrene-induced tumors in BALB/c mice. Maximal inhibition of SOD (8% of control) was obtained 1 h after administration of 100 mg/kg DDC. Tumor response to DDC and X irradiation was assessed using a tumor growth-delay assay, after 11 Gy 100-kVp X rays given up to 24 h after DDC administration. Radiation-induced tumor growth delay (7.11 +/- 1.76 days) was enhanced only when tumors were irradiated 2-4 h after 50 mg/kg DDC. When higher doses of DDC were used, tumor cure was noted when DDC was injected 1-6 h before irradiation. We suggest our findings are consistent with radiosensitization being due to SOD inhibition, but that if insufficient time is allowed between DDC injection and irradiation, the sensitization is masked by a radioprotective effect. We believe that further investigations as to the therapeutic potential of DDC in human patients with cancer are warranted.     

Oxidative stress contributes to vascular endothelial dysfunction in heart failure

Congestive heart failure (HF) is characterized by inadequate nitric oxide (NO) production in the vasculature. Because NO is degraded by oxygen radicals, we hypothesized that NO is degraded faster in HF from inadequate peripheral arterial antioxidant reserves. HF was induced in male Sprague-Dawley rats by left coronary artery ligation. Vascular endothelial function was evaluated by measuring the NO-mediated vasorelaxation response to acetylcholine (ACh; 10(-9)-10(-4) M) in excised aortas. This was repeated with the free radical generator pyrogallol (20 microM) and again with pyrogallol and superoxide dismutase (SOD; 60 U/ml). Aortic and myocardial SOD activity was also determined. ACh-induced vasorelaxation was reduced in HF (n = 9) compared with normal control rats (n = 11; P < 0.001). Pyrogallol further reduced vasorelaxation in HF: 74 +/- 11% at 10(-4) M ACh versus 58 +/- 10% in normal control rats (P < 0.004). There was a trend (P = 0.06) toward reduced SOD activity in HF aortas. In conclusion, altered NO-dependent vasorelaxation in HF is in part due to excessive degradation of NO and is likely related to reduced vascular SOD activity.     

Characterization of leukotriene C4 synthetase in mouse peritoneal exudate cells

Cell lysates of mouse peritoneal macrophages, in the presence of reduced glutathione, converted leukotriene LTA4 to LTC4, and neither LTD4 nor LTE4 was detected. Therefore, like cultured rat basophilic leukemia cells (RBL cells), the peritoneal macrophage contains LTC4 synthetase and appears to contain little, if any, gamma-glutamyl transpeptidase. When LTA4 was added to subcellular fractions of mouse macrophage lysate, the highest specific activity of LTC4 synthetase (nmol LTC4/mg protein per 10 min) was associated with the particulate or membrane fractions (i.e., 10(4) and 10(5) X g pellets). The 10(5) X g supernatant contains approx. 1% of the specific activity and 6% of the total LTC4 synthetase activity compared with that of the 10(5) X g pellet. Conversely, the 10(5) X g supernatant had four-times more specific activity and 19-times more total GSH S-transferase activity than did the 10(5) X g pellet when evaluated using 1-chloro-2,4-dinitrobenzene (DNCB) as the substrate. LTA4 was converted to LTC4 by the membrane enzyme LTC4 synthetase in a dose-dependent manner at low LTA4 concentrations (3-50 microM) and reached a plateau of approx. 30 microM LTA4 using the macrophage 10(5) X g pellet as an enzyme source. The apparent Km value of LTC4 synthetase for LTA4 was estimated to be 5 microM based on Lineweaver-Burk plots. Enzyme in the 10(5) X g supernatant produced negligible quantities of LTC4 (1% or less of the particulate fractions) over a wide range of LTA4 concentrations. However, an enzyme in the 10(5) X g supernatant fraction presumed to be GSH S-transferase effectively catalyzes the conjugation of glutathione (GSH) with the aromatic compound DNCB. The apparent Km value of GSH S-transferase for DNCB was estimated to be 1.0-1.5 mM. On the other hand, enzyme from the membrane fraction (i.e., 10(5) X g pellet) catalyzed this reaction at a negligible rate over a wide range of DNCB concentrations. The apparent Km value of LTC4 synthetase for GSH was estimated to be 0.36 mM and the corresponding Km value estimated for the glutathione S-transferase was 0.25-0.76 mM. These values indicate similar kinetics for GSH utilization by both enzymes. These Km values are also significantly lower than the intracellular GSH levels of 2 to 5 mM. Therefore, it is suggested that the substrate limiting LTC4 synthetase activity is LTA4 and not GSH. Our results indicate that LTC4 synthetase from mouse peritoneal macrophages is a particulate or membrane-bound enzyme, as was reported by Bach et al.(ABSTRACT TRUNCATED AT 400 WORDS)     

Glutamate Dehydrogenase in Human Brain: Regional Distribution and Properties

Glutamate dehydrogenase (GDH) activity was studied in 17 regions of six human brains. Duration and conditions of the postmortem period did not affect enzyme activity. Specific activity ranged between 103 and 377 nmoles/min/mg protein at 25 degrees C and it was 10-fold higher than that found in leukocytes. Apart from exclusively white matter regions (corpus callosum and centrum ovale), there was a moderate regional distribution (2.5-fold variation), with highest values in the inferior olive and hypothalamus, and lowest in the cerebellum and lenticular nucleus. With alpha-ketoglutarate (alpha-KG), NADH, or NH4+ as variable substrate, the apparent Km values in human brain were Km alpha-KG = 1.9 X 10(-3) M, KmNADH = 0.21 X 10(-3) M, and KmNH4+ = 28 X 10(-3) M, and in leukocytes they were Km alpha-KG = 1.7 X 10(-3) M, KmNADH = 0.24 X 10(-3) M, and KmNH4+ = 28 X 10(-3) M. The effects of cofactors, inhibitor, and pH were similar in brain and leukocyte GDH.     

Regulation of aortic CuZn-superoxide dismutase with copper. Effects in vivo.

Cu2Zn2-superoxide dismutase (CuZn-SOD) was purified from chicken liver. The liver enzyme had a subunit Mr of 16900 and contained equimolar amounts of copper and zinc [0.26% (w/w) for each]. Aortic CuZn-SOD had the same Mr as estimated by gel filtration and cross-reacted with antibodies to the liver enzyme. Both enzymes were inhibited by 1.0 mM-NaCN. Within 24-72 h after hatching, total SOD activity in aorta rose 3-fold over the day-1 level and stayed elevated for 10 days. With low dietary copper, the total SOD activity rose as before, but then decayed progressively to non-detectable levels in 10 days. Both the cyanide-sensitive (CuZn-SOD) and insensitive (mangano-SOD) activities fell, but not at the same rate. When the 10-day-old deficient chicks were injected with 0.5 mumol of CuSO4 intraperitoneally, SOD activity in aorta was restored to control levels in about 8 h. Despite non-measurable SOD activity in aorta, extracts from the 15-day-old-deficient-chick tissue contained as much, or slightly more, immunoreactive CuZn-SOD protein as age-matched control tissue. The data show clearly that dietary copper regulates SOD activity in the aortas of young developing animals. They further suggest that a copper deficiency suppresses CuZn-SOD activity without inhibiting synthesis or accumulation of the CuZn protein in this tissue.     

Mechanism of action of leukotrienes on the guinea pig bronchus

The pharmacological activity of leukotrienes (LT) A4, C4, D4, E4, and histamine was investigated on guinea pig upper and lower bronchi. The contractions of the upper bronchi to histamine, LTA4, C4 and D4 were enhanced by cyclooxygenase inhibitors aspirin (1.67 X 10(-5) and 1.67 X 10(-6) M) and indomethacin (2.8 X 10(-6) and 2.8 X 10(-5) M) whereas the responses to LTE4 were not affected. The myotropic activity of the lower bronchi to all agonists were either very slightly or not at all modified by the presence of cyclooxygenase inhibitors. The thromboxane synthetase inhibitor OKY-046 (1.77 X 10(-5) and 1.77 X 10(-6) M) did not change the responses of higher bronchi to the agonists which suggested that the response of the upper bronchi may be mediated by prostaglandins but not by thromboxanes. The responses of the lower bronchi to leukotrienes A4, C4, D4 and E4 were inhibited by compound OKY-046. Blockade of thromboxane receptors together with inhibition of lipoxygenases by compound L-655,240 (2.53 X 10(-8) to 2.53 X 10(-5) M) had a slight effect on the stimulation of upper and lower bronchi by leukotrienes and histamine. The compound FPL-55712 (1.92 X 10(-6) and 1.92 X 10(-5) M) strongly reduced the contractions of the upper and lower bronchi to leukotrienes but did not affect the responses to histamine. These results suggest that the contractile effects of leukotrienes on upper bronchi is modulated by bronchorelaxant prostaglandins whereas the responses of the lower bronchi are mediated by thromboxanes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of guinea pig polymorphonuclear leukocytes with soluble stimulators leads to nonrandom distribution of NADPH oxidase in the plasma membrane

Guinea pig polymorphonuclear leukocytes (PMN) were briefly activated with soluble stimulators such as sodium myristate (SM) or phorbol myristate acetate (PMA) and then disrupted by the nitrogen cavitation method to study the subcellular distribution of NADPH oxidase, which is responsible for O2 - generation. Fc-receptor and 5'-nucleotidase activities were measured as plasma membrane markers. 1) The homogenate was first fractionated by differential centrifugation. The O2- -generating activity of PMN activated either by SM or PMA was recovered in a 2 X 10(4) g pellet which contained a large amount of granules and about 50% of the plasma membrane markers, but not in a 1 X 10(5) g pellet which consisted of plasma membranes and few granules. 2) Further separation of the 2 X 10(4) g pellet from PMA-activated PMN was attempted by an iso-osmotic Percoll density gradient centrifugation. The O2- -generating activity was recovered in light fractions in which plasma membrane markers were found, but neither in specific nor in azurophil granules. The 1 X 10(5) g pellet showed a similar distribution of the plasma membrane markers to that of the 2 X 10(4) g pellet, except that the peak of the O2- -generating activity was much smaller on an identical density gradient. The results showed that NADPH oxidase is located in the plasma membranes precipitated by centrifugation at 2 X 10(4) X g but not in the ones precipitated at 1 X 10(5) X g. The results suggest that the plasma membrane of activated PMN has a mosaic distribution of NADPH oxidase.     

Noradrenaline modulates the immunity of white shrimp Litopenaeus vannamei

The total haemocyte count (THC), phenoloxidase activity, respiratory burst, superoxide dismutase (SOD) activity, phagocytic activity and clearance efficiency in response to pathogen Vibrio alginolyticus were measured when the white shrimp Litopenaeus vannamei (18.4 +/- 1.2 g) were injected individually with noradrenaline at 10(-8), 10(-7) and 10(-6) mol shrimp(-1). For the shrimp that received noradrenaline at 10(-8), 10(-7) and 10(-6) mol shrimp(-1), the THC decreased by 15%, 21% and 32%, phenoloxidase activity decreased by 15%, 31% and 31%, respiratory burst decreased by 13%, 21% and 32%, and SOD activity decreased by 46%, 56% and 55%, respectively, after 2 h. The phagocytic activity and clearance efficiency of shrimp that received noradrenaline at either dose decreased significantly after 2 h. The THC, phenoloxidase activity, respiratory burst, SOD activity, phagocytic activity and clearance efficiency returned to normal values after 4, 4, 8, 24, 16 and 8 h, respectively, in the shrimp that received noradrenaline at either dose. In another experiment, L. vannamei which had received noradrenaline at 10(-8), 10(-7) and 10(-6) mol shrimp(-1) were challenged after 1h by injection with V. alginolyticus at 1.0 x 10(5) colony-forming units (cfu)shrimp(-1) and then placed in seawater of 20 per thousand. The cumulative mortality of shrimp that received noradrenaline at either dose was significantly higher than that of shrimp that received saline after 4 h, and at the termination of the experiment (48 h after the challenge). It is therefore concluded that noradrenaline administration at 10(-6) mol shrimp(-1) or less causes immune modulation of L. vannamei.     

Manganese superoxide dismutase (SOD2)-mediated delayed radioprotection induced by the free thiol form of amifostine and tumor necrosis factor alpha

RKO36 cells, a subclone of RKO colorectal carcinoma cells that have been stably transfected with the pCMV-EGFP2Xho vector, were grown to confluence and then exposed to either the radioprotector WR-1065, i.e. the active thiol form of amifostine, for 30 min at doses of 40 microM and 4 mM or the cytokine tumor necrosis factor alpha (TNFalpha, TNFA) for 30 min at a concentration of 10 ng/ml and then washed. Total protein was isolated as a function of time up to 32 h after these treatments. Both doses of WR-1065 as well as the concentration of TNFalpha used were effective in elevating intracellular levels of the antioxidant protein SOD2 (also known as MnSOD) at least 15-fold over background levels as determined by Western blot analysis, while measured SOD2 activity was elevated between 5.5- and 6.9-fold. SOD2 reached a maximal level 24 h and 20 h after WR-1065 and TNFalpha treatments, respectively. The antioxidant proteins catalase and glutathione peroxidase (GPX) were also monitored over the 32-h period. In contrast to the robust changes observed in intracellular levels of SOD2 as a function of time after exposure of cells to WR-1065, catalase levels were elevated only 2.6-fold over background as determined by Western blot analysis, while GPX activity was unaffected by WR-1065 exposure. GPX protein levels were extremely low in cells, and analysis of GPX activity using a spectrophotometric method based on the consumption of reduced NADPH also revealed no measurable change as a function of WR-1065 or TNFalpha exposure. RKO36 cells either were irradiated with X rays in the presence of either 40 microM or 4 mM WR-1065 or 10 ng/ml TNFalpha or were irradiated 24 or 20 h later, respectively, when SOD2 protein levels were most elevated. The concentrations and exposure conditions used for WR-1065 and TNFalpha were not cytotoxic and had no effect on plating efficiencies or cell survival compared to untreated controls. No protection or sensitization was observed for cells irradiated in the presence of 40 microM WR-1065 or TNFalpha. Survival was elevated 1.90-fold for cells irradiated in the presence of 4 mM WR-1065. When RKO36 cells were irradiated with 2 Gy 24 h after 40 microM or 4 mM WR-1065 and 20 h after TNFalpha treatments when SOD2 levels were the most increased, survival was elevated 1.42-, 1.48- and 1.36-fold, respectively. This increased survival represents a SOD2-mediated delayed radioprotective effect. SOD2 appears to be an important antioxidant gene whose inducible expression is an important element in adaptive cellular responses in general, and the delayed radioprotective effect in particular. It can be induced by a range of agents including cytoprotective nonprotein thiols such as WR-1065 and pleiotropic cytokines such as TNFalpha.     

Purification and characterization of a thermostable MnSOD from the thermophilic fungus Chaetomium thermophilum

A thermostable superoxide dismutase (SOD) from the culture supernatant of a thermophilic fungus Chaetomium thermophilum strain CT2 was purified to homogeneity by fractional ammonium sulfate precipitation, ion-exchange chromatography on DEAE-sepharose, phenyl-sepharose hydrophobic interaction chromatography. The pure SOD had a specific activity of 115.77 U/mg of protein and was purified 7.49-fold, with a yield of 14.4%. The molecular mass of a single band of the enzyme was estimated to be 23.5 kDa, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using gel filtration on Sephacryl S-100, the molecular mass was estimated to be 94.4 kDa, indicating that this enzyme was composed of four identical subunits of 23.5 kDa each. The SOD was found to be inhibited by NaN3, but not by KCN and H2O2. Atomic absorption spectrophotometric analysis showed that the content of Mn was 2.05 microg/mg of protein and Fe was not detected in the purified enzyme. These results suggested that the SOD in C. thermophilum was the manganese superoxide dismutase type. N-terminal amino acid sequencing (10 residues) was KX (X is uncertain) TLPDLKYD. The N-terminal amino acid sequencing homologies to other MnSod also indicated that it was a manganese-containing superoxide dismutase. The SOD exhibited maximal activity at pH 7.5 and optimum temperature at 60 C. It was thermostable at 50 and 60 C and retained 60% activity after 60 min at 70 C. The half-life of the SOD at 80 C was approximately 25 min and even retained 20% activity after 30 min at 90 C.     

Benzylamine oxidase activity in rat embryo and placenta at organogenesis

Benzylamine oxidase, characterized by its sensitivity to semi-carbazide (1 X 10(-2) mol/l) inhibition and its insensitivity to deprenyl (4 X 10(-4) mol/l) inhibition, is present in rat placenta during days 10-15 of the gestation period and in the embryo at days 11-15. The activity represents a substantial part of the overall activity observed with p-chlorobenzylamine as substrate. A much higher activity is present in the placenta than in the embryo.