Involvement of reactive oxygen species in the microsomal S-oxidation of thiobenzamide

Superoxide dismutase, catalase and methional proved capable of inhibiting the microsomal oxidation of thiobenzamide, which is most probably catalyzed by the flavin-containing monooxygenase. This indicates that excited oxygen species (e.g. X O-2,H2O2, X OH) are involved in the catalytic cycle of this enzymatic reaction. CO, which inhibits the cytochrome P-450-dependent oxygen radical formation, had no effect on the oxidation reaction, suggesting that the source of the reactive oxygen species is not the microsomal mixed-function oxidase.     

Hydroxyl radicals are not involved in NADPH dependent microsomal lipid peroxidation

Summary NADPH dependent H₂O₂ formation in microsomes in the presence of chelated iron leads to formation of hydroxyl radicals. Enhancement of hydroxyl radical generation (via ferric-EDTA or sodium azide) did not result in a concomitant increase in lipid peroxidation; rather, a decrease was observed. Moreover, the hydroxyl radical scavenger DMSO did not inhibit lipid peroxidation. This comparison of hydroxyl radical formation with lipid peroxidation suggests that hydroxyl radicals do not play a part in NADPH-dependent lipid peroxidation.     

Hydrogen peroxide and hydroxyl radical involvement in the activation of caspase-3 in chemically induced apoptosis of HL-60 cells

Apoptosis of HL-60 cells induced by actinomycin D, H7, or daunorubicin was shown to involve the activation of caspase-3-like protease, 2 h after the addition of these drugs, based on microassay of enzyme activity by high-performance liquid chromatography. Catalase and a spin trap, N-t-butyl--phenylnitrone, which effectively inhibited the apoptosis induced by these drugs, also inhibited the activation of caspase-3-like protease. These results suggest that hydrogen peroxide and the hydroxyl radical are common mediators of caspase-3 activation caused by these chemicals, with apparently different functional mechanisms. Based on mitochondrial activity determined by oxygen consumption, complexes I, II, and IV were inhibited by actinomycin D. H7 inhibited complexes I and IV, 1 and 1.5 h respectively, after the addition of the drug to HL-60 cells. Daunorubicin inhibited complex IV, 1.5 h after the addition of the drug to HL-60 cells. Inhibition of complex IV by actinomycin D, H7, and daunorubicin were almost fully restored by the addition of cytochrome c. The release to the cytosol of cytochrome c by these drugs was also demonstrated by Western blot analysis. Addition of catalase inhibited the depression of complex IV activity induced by actinomycin D and H7. These observations indicate a direct relationship between hydrogen peroxide and the release of cytochrome c during apoptosis caused by actinomycin D, H7, and daunorubicin. Received 24 November 2000; received after revision 2 January 2001; accepted 30 January 2001     

Two axes in platelet-derived growth factor signaling: tyrosine phosphorylation and reactive oxygen species

The tyrosine phosphorylation cascade is a hallmark of platelet-derived growth factor (PDGF)- induced signal transduction. The amplitude and propagation of the tyrosine phosphorylation signal relies on the balance between tyrosine kinase and tyrosine phosphatase. The tyrosine kinase is latent in the absence of stimulation, whereas the tyrosine phosphatase is highly and constitutively active. Therefore, the kinase activation should be accompanied by temporal and spatial inactivation of tyrosine phosphatase to achieve the robust amplification of tyrosine phosphorylation. For the past decade, reactive oxygen species have been receiving a great deal of attention with regard to their ability to shut down tyrosine phosphatase activities in a reversible manner. In this article, the crosstalk between tyrosine phosphorylation and reactive oxygen species in PDGF signaling is discussed. Received 2 October 2006; received after revision 13 November 2006; accepted 27 November 2006     

过氧草酰类化学发光分析研究的新进展

过氧草酰类化学发光体系具有很高的发光效率并且适于多种类型物质检测。本文对此类发光试剂在分析化学领域的最新研究进行评述,主要内容包括发光机理的研究,新试剂的合成以及在分析检测中的应用。     

Role of nitric oxide in the functional response to ischemia-reperfusion of heart mitochondria from hyperthyroid rats

We investigated the role of nitric oxide (NO) in the mitochondrial derangement associated with the functional response to ischemia-reperfusion of hyperthyroid rat hearts. Mitochondria were isolated at 3000 g from hearts subjected to ischemia-reperfusion, with or without N-nitro-L-arginine (L-NNA, an NO synthase inhibitor). During reperfusion, hyperthyroid hearts displayed tachycardia and low functional recovery. Their mitochondria exhibited O₂ consumption similar to euthyroid controls, while H₂O₂ production, hydroperoxide, protein-bound carbonyl and nitrotyrosine levels, and susceptibility to swelling were higher. L-NNA blocked the reperfusion tachycardic response and increased inotropic recovery in hyperthyroid hearts. L-NNA decreased mitochondrial H₂O₂ production and oxidative damage, and increased respiration and tolerance to swelling. Such effects were higher in hyperthyroid preparations. These results confirm the role of mitochondria in ischemia-reperfusion damage, and strongly suggest that NO overproduction is involved in the high mitochondrial dysfunction and the low recovery of hyperthyroid hearts from ischemia-reperfusion. L-NNA also decreased protein content and cytochrome oxidase activity of a mitochondrial fraction isolated at 8000 g. This and previous results suggest that the above fraction contains, together with light mitochondria, damaged mitochondria coming from the heaviest fraction, which has the highest cytochrome oxidase activity and capacity to produce H₂O₂. Therefore, we propose that the high mitochondrial susceptibility to swelling, favoring mitochondrial population purification from H₂O₂-overproducing mitochondria, limits hyperthyroid heart oxidative stress.Received 24 March 2004; received after revision 9 June 2004; accepted 5 July 2004     

Role of active oxygen species in diethyldithiocarbamate-induced gastric ulcer in the rat

Summary Diethyldithiocarbamate, an inhibitor of Cu,Zn-superoxide dismutase, was recently found to be ulcerogenic in the rat stomach, and active oxygen species were found to be responsible for its ulcerogenicity. To clarify which active oxygen species play a role in ulcerogenesis, the effects of various scavengers and iron-chelators were studied. As superoxide dismutase and catalase reduced the ulcerogenesis induced by diethyldithiocarbamate, the superoxide radical and hydrogen peroxide were considered to play a pathogenic role in this ulcer model.     

Role of active oxygen species in diethyldithiocarbamate-induced gastric ulcer in the rat

Diethyldithiocarbamate, an inhibitor of Cu,Zn-superoxide dismutase, was recently found to be ulcerogenic in the rat stomach, and active oxygen species were found to be responsible for its ulcerogenicity. To clarify which active oxygen species play a role in ulcerogenesis, the effects of various scavengers and iron-chelators were studied. As superoxide dismutase and catalase reduced the ulcerogenesis induced by diethyldithiocarbamate, the superoxide radical and hydrogen peroxide were considered to play a pathogenic role in this ulcer model.     

Functional and biochemical characteristics of mitochondrial fractions from rat liver in cold-induced oxidative stress

We determined characteristics of rat liver mitochondrial fractions, resolved at 1000 (M₁), 3000 (M₃), and 10,000 g (M₁₀) after 2 and 10 days cold exposure. In all groups, the M₁ fraction exhibited the highest oxidative capacity, oxidative damage, H₂O₂ production rate, and susceptibility to stress conditions, and the lowest antioxidant levels. Cold exposure increased cytochrome oxidase activity in all fractions and succinate-supported O₂ consumption in the M₁ and M₁₀ fractions during state 3 and state 4 respiration, respectively. With succinate, the H₂O₂ release rate increased in all fractions during state 4 and state 3 respiration, whereas with pyruvate/malate, it increased only during state 4 respiration. Increases in tissue mitochondrial proteins caused a faster H₂O₂ flow from the mitochondrial to cytosolic compartment, which was limited by the reduction in the M₁ fraction. Despite increased liposoluble antioxidant levels, cold also caused enhanced oxidative damage and susceptibility to oxidative challenge and Ca²⁺-induced swelling in all fractions. These changes leading to elimination of H₂O₂-overproducing mitochondria avoided excessive tissue damage. We propose that triiodothyronine, whose levels increase in the cold environment, brings about the biochemical changes producing oxidative damage and those limiting its extent.Received 16 July 2004; received after revision 27 September 2004; accepted 18 October 2004     

Effects of ovarian steroids on superoxide dismutase activity in the rat brain

The levels of manganese superoxide dismutase (MnSOD) and copper-zinc superoxide dismutase (CuZnSOD) were determined in appropriate subcellular fractions prepared from whole brain homogenates of cycling and long-term (3 week) ovariectomized (OVX) Wistar rats, and were compared to the levels found in corresponding samples prepared from OVX rats treated with progesterone (P) or estradiol 17B-benzoate (EB). The activity of both SODs was steady during the estrous cycle, except at proestrus, when MnSOD activity was elevated significantly. Bilateral ovariectomy resulted three weeks later in an increase of the MnSOD activity even higher than that recorded at proestrus. High post-castration MnSOD activity was lowered profoundly by exogenous P (2 mg) or EB (0.5 g), given s.c. to OVX animals 2 h or 24 h before sacrifice. Neither removal of the ovaries nor the hormone treatments affected the activity of CuZnSOD. These results suggest suppressive effects of ovarian steroids on MnSOD activity in the rat brain.     

Effects of progesterone and estradiol benzoate on superoxide dismutase activity in the brain of male rats

The activities of mitochondrial, manganese-containing superoxide dismutase (MnSOD) and cytoplasmic, copper-zinc-containing superoxide dismutase (CuZnSOD) were measured in subcellular fractions of whole brain homogenates prepared from intact and gonadectomized (GDX) male rats, untreated or treated subcutaneously (sc) with a single dose of 2 mg progesterone (P) and/or 5 g estradiol benzoate (EB). Neither MnSOD nor CuZnSOD was affected by the removal of the testes. Similarly, CuZnSOD activity was steady following systemic administration of P and/or EB to intact and GDX animals 2 h or 24 h prior to sacrifice. On the other hand, both P and EB suppressed MnSOD in the brain of either intact or GDX rats. These results suggest involvement of P and EB in the control of MnSOD activity in the brain of male rats.     

On the mechanism of the involvement of endothelium in reactive hyperemia

Experiments on anesthetized dogs and on vascular test-preparations demonstrated that reactive hyperemia (RH) was accompanied by the appearance of vasodilator in the blood, and that the level increased with the duration of occlusion of the artery. Removal of the endothelium of the part of the vascular bed studied using saponin, decreased the RH and relaxation of a test-preparation. A rise of pressure in the vascular bed, and a decrease in the deformability of the endothelium resulting from pretreatment with dimerized glutaraldehyde, affected both the hyperemia and the reaction of the vascular preparation in a similar way. It was concluded that the RH resulted from the secretion of vasoactive substances by the endothelium in response to a fall in intravascular pressure.     

Involvement of a sperm aminopeptidase in fertilization of the sea urchin

Summary Inhibitory efficiencies of bestatin methyl ester and its nine analogs for sea urchin sperm aminopeptidase activity were similar to the efficiency of the same compounds as inhibitors either of sperm respiration or of fertilization. This suggests that a sperm aminopeptidase plays a role in fertilization in the sea urchin, possibly through its involvement in sperm respiration.Acknowledgment. We are grateful to Dr Wataru Tanaka of Nippon kayaku co. for his gifts of bestatin and its analogs. We are also indebted to Mrs Akiko Tsuchida-Watanabe for her technical assitance, and to the staff of Asamushi Marine Biological Station, Tohoku University, where part of this work was carried out. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.     

Age-related lipid peroxidation in the digestive gland of mussels: The role of the antioxidant defence systems

Summary The main cellular defence systems against free radical-mediated oxidative stress are significantly reduced in the dige⁺ive gland of aged (>10 years old) compared to younger (2–4 years old) mussels (Mytilus edulis L.). Moreover, the concentration of lipid peroxidation products (malondialdehyde) is increased in the same age group with respect to younger animals. The obtained data indicate that an impairment of the antioxidant defence systems would render the older animals more susceptible to peroxidative stress, thus supporting the general significance of the free radical theory of aging.     

Microdialysis study of ischemia-induced hydroxyl radicals in the canine heart

A new experimental approach for spin-trapping of oxygen radicals in a selected region of the heart in situ is described. This approach is based on microdialysis, and it permits the detection of oxygen radicals in conditions of local ischemia and restoration of normal blood flow. Increased hydroxyl radical generation in an ischemic area of canine myocardium, as a result of 40 min local occlusion, has been studied.     

Involvement of penaeidins in defense reactions of the shrimp Litopenaeus stylirostris to a pathogenic vibrio

The present study reports for the first time the involvement of an antimicrobial peptide in the defense reactions of a shrimp infected by a pathogenic Vibrio, Vibrio penaeicida. New members of the penaeidin family were characterized in the shrimp Litopenaeus stylirostris by RT-PCR and RACE-PCR from hemocyte total RNAs, and by mass spectrometry detection and immunolocalization of mature peptides in shrimp hemocytes. In infected shrimps, bacteria and penaeidin distribution colocalized in the gills and the lymphoid organ that represented the main infected sites. Moreover, the shrimp immune response to infection involved massive hemocyte recruitment to infection sites where released penaeidin may participate in the isolation and elimination of the bacteria, We show that the ability of the shrimps to circumvent shrimp infections is closely related to a recovery phase based on the hematopoietic process.Received 25 November 2003; received after revision 8 January 2004; accepted 21 January 2004     

Involvement of thiols in the induction of inward current induced by silver in frog skeletal muscle membrane

Exposure of voltage-clamped frog skeletal muscle fibres to silver caused a maintained inward current which could be carried by Ca²⁺, Mg²⁺ or Na⁺. Inorganic Ca²⁺ channel blockers and dithiothreitol (SH reducing agent) diminished this current, but a Na⁺ channel blocker did not. Thus, silver activates the Ca²⁺ channel by acting on SH groups in a Ca²⁺ channel protein.