Distinct role of Hsp70 in Drosophila hemocytes during severe hypoxia

Severe hypoxia can lead to injury and mortality in vertebrate or invertebrate organisms. Our research is focused on understanding the molecular mechanisms that lead to injury or adaptation to hypoxic stress using Drosophila as a model system. In this study, we employed the UAS-Gal4 system to dissect the protective role of Hsp70 in specific tissues in vivo under severe hypoxia. In contrast to overexpression in tissues such as muscles, heart, and brain, we found that overexpression of Hsp70 in hemocytes of flies provides a remarkable survival benefit to flies exposed to severe hypoxia for days. Furthermore, these flies were tolerant not only to severe hypoxia but also to other stresses such as oxidant stress (e.g., paraquat feeding or hyperoxia). Interestingly we observed that the better survival with Hsp70 overexpression in hemocytes under hypoxia or oxidant stress is causally linked to reactive oxygen species (ROS) reduction in whole flies. We also show that hemocytes are a major source of ROS generation, leading to injury during hypoxia, and their elimination results in a better survival under hypoxia. Hence, our study identified a protective role for Hsp70 in Drosophila hemocytes, which is linked to ROS reduction in the whole flies and thus helps in their remarkable survival during oxidant or hypoxic stress.     

Hypoxia-induced reactive oxygen species formation in skeletal muscle.

The existence of hypoxia-induced reactive oxygen species (ROS) production remains controversial. However, numerous observations with a variety of methods and in many cells and tissue types are supportive of this idea. Skeletal muscle appears to behave much like heart in that in the early stages of hypoxia there is a transient elevation in ROS, whereas in chronic exposure to very severe hypoxia there is evidence of ongoing oxidative stress. Important remaining questions that are addressed in this review include the following. Are there levels of PO2 in skeletal muscle, typical of physiological or mildly pathophysiological conditions, that are low enough to induce significant ROS production? Does the ROS associated with muscle contractile activity reflect imbalances in oxygen uptake and demand that drive the cell to a more reduced state? What are the possible molecular mechanisms by which ROS may be elevated in hypoxic skeletal muscle? Is the production of ROS in hypoxia of physiological significance, both with respect to cell signaling pathways promoting cell function and with respect to damaging effects of long-term exposure? Discussion of these and other topics leads to general conclusions that hypoxia-induced ROS may be a normal physiological response to imbalance in oxygen supply and demand or environmental stress and may play a yet undefined role in normal response mechanisms to these stimuli. However, in chronic and extreme hypoxic exposure, muscles may fail to maintain a normal redox homeostasis, resulting in cell injury or dysfunction.     

低氧运动对Nrf2基因敲除大鼠运动能力和氧化应激的影响

Nrf2可调节多种抗氧化酶的表达,Nrf2的缺失可能影响机体的运动能力,而低氧可提高机体的抗氧化能力并改善运动能力。为了考察低氧运动对Nrf2基因敲除大鼠运动能力和氧化应激的影响,本研究分别在常氧和低氧环境(12%氧浓度)中对野生型大鼠和Nrf2敲除大鼠进行4周的跑台运动。研究显示,低氧运动可提高野生型大鼠的跑台运动力竭时间,Nrf2敲除可缩短大鼠的力竭时间;低氧运动可上调大鼠的Nrf2 m RNA表达量;Nrf2敲除明显抑制HIF-1α蛋白表达,而低氧运动可上调野生型和Nrf2敲除大鼠的HIF-1α蛋白表达;Nrf2敲除大鼠的骨骼肌ROS水平明显升高,并且低氧均可降低野生型和Nrf2敲除大鼠骨骼肌ROS水平。低氧运动可上调Nrf2敲除大鼠的CAT和GSH-PX蛋白表达。苏木精和伊红(HE)染色显示,Nrf2敲除大鼠在力竭跑台运动完成后出现更严重的骨骼肌病理改变,而低氧运动可减轻骨骼肌损伤。本研究认为,Nrf2敲除导致了大鼠骨骼肌中抗氧化酶的抑制及ROS的过量累积,从而造成了骨骼肌损伤并降低了运动能力。此外,低氧可通过上调Nrf2的表达,进而激活HIF-1α及抗氧化酶活性,从而提高运动能力,并防止骨骼肌损伤。     

Oxidative stress in septic shock and disseminated intravascular coagulation

Oxidative stress results from an oxidant/antioxidant imbalance, an excess of oxidants and/or a depletion of antioxidants. A considerable body of recent evidence suggests that oxidant stress plays a major role in several aspects of septic shock and disseminated intravascular coagulation (DIC), and it is the subject of this review. Immunohistochemical and biochemical evidence demonstrate the significant role of reactive oxygen species (ROS) in endotoxic and hemorrhagic shock, and in endothelial injury associated with DIC syndrome. Initiation of lipid peroxidation, direct inhibition of mitochondrial respiratory chain enzymes, inactivation of glyceraldehyde-3-phosphate dehydrogenase, inhibition of membrane Na+/K+ ATP-ase activity, inactivation of membrane sodium channels, and other oxidative protein modifications contribute to the cytotoxic effect of ROS. In addition, reactive oxygen species are potent triggers of DNA strand breakage, with subsequent activation of the nuclear enzyme poly-ADP ribosyl synthetase, with eventual severe energy depletion of the cells. Pharmacological evidence suggests that the peroxynitrite-poly-ADP ribosyl synthetase pathway contributes to the cellular injury in shock and endothelial injury. Treatment with superoxide dismutase mimetics (SODms), which selectively mimic the catalytic activity of the human superoxide dismutase enzymes, have been shown to prevent in vivo shock and the cellular energetic failure associated with shock.     

Intracellular generation of reactive oxygen species by contracting skeletal muscle cells

The aim of this work was to examine the intracellular generation of reactive oxygen species in skeletal muscle cells at rest and during and following a period of contractile activity. Intracellular generation of reactive oxygen species was examined directly in skeletal muscle myotubes using 2',7'-dichlorodihydrofluorescein (DCFH) as an intracellular probe. Preliminary experiments confirmed that DCFH located to the myotubes but was readily photoxidizable during repeated intracellular fluorescence measurements and strategies to minimize this were developed. The rate of oxidation of DCFH did not change significantly over 30 min in resting myotubes, but was increased by approximately 4-fold during 10 min of repetitive, electrically stimulated contractile activity. This increased rate was maintained over 10 min following the end of the contraction protocol. DCF fluorescence was distributed evenly throughout the myotube with no evidence of accumulation at any specific intracellular sites or localization to mitochondria. The rise in DCF fluorescence was effectively abolished by treatment of the myotubes with the intracellular superoxide scavenger, Tiron. Thus these data appear to represent the first direct demonstration of a rise in intracellular oxidant activity during contractile activity in skeletal muscle myotubes and indicate that superoxide, generated from intracellular sites, is the ultimate source of oxidant(s) responsible for the DCFH oxidation.     

Free radicals and muscle fatigue: Of ROS, canaries, and the IOC

Skeletal muscle fibers continually generate reactive oxygen species (ROS) at a slow rate that increases during muscle contraction. This activity-dependent increase in ROS production contributes to fatigue of skeletal muscle during strenuous exercise. Existing data suggest that muscle-derived ROS primarily act on myofibrillar proteins to inhibit calcium sensitivity and depress force. Decrements in calcium sensitivity and force are acutely reversible by dithiothreitol, a thiol-selective reducing agent. These observations suggest that thiol modifications on one or more regulatory proteins are responsible for oxidant-induced losses during fatigue. More intense ROS exposure leads to losses in calcium regulation that mimic pathologic changes and are not reversible. Studies in humans, quadrupeds, and isolated muscle preparations indicate that antioxidant pretreatment can delay muscle fatigue. In humans, this phenomenon is best defined for N-acetylcysteine (NAC), a reduced thiol donor that supports glutathione resynthesis. NAC has been shown to inhibit fatigue in healthy adults during electrical muscle activation, inspiratory resistive loading, handgrip exercise, and intense cycling. These findings identify ROS as endogenous mediators of muscle fatigue and highlight the importance of future research to (a) define the cellular mechanism of ROS action and (b) develop antioxidants as novel therapeutic interventions for treating fatigue.     

Oxidant stress during simulated ischemia primes cardiomyocytes for cell death during reperfusion

Ischemia-reperfusion injury induces oxidant stress, and the burst of reactive oxygen species (ROS) production after reperfusion of ischemic myocardium is sufficient to induce cell death. Mitochondrial oxidant production may begin during ischemia prior to reperfusion because reducing equivalents accumulate and promote superoxide production. We utilized a ratiometric redox-sensitive protein sensor (heat shock protein 33 fluorescence resonance energy transfer (HSP-FRET)) to assess oxidant stress in cardiomyocytes during simulated ischemia. HSP-FRET consists of the cyan and yellow fluorescent protein fluorophores linked by the cysteine-containing regulatory domain from bacterial HSP-33. During ischemia, ROS-mediated oxidation of HSP-FRET was observed, along with a decrease in cellular reduced glutathione levels. These findings were corroborated by measurements using redox-sensitive green fluorescent protein, another protein thiol ratiometric sensor, which became 93% oxidized by the end of simulated ischemia. However, cell death did not occur during ischemia, indicating that this oxidant stress is not sufficient to induce death before reperfusion. However, interventions that attenuate ischemic oxidant stress, including antioxidants or scavengers of residual O(2) that attenuate/prevent ROS generation during ischemia, abrogated cell death during simulated reperfusion. These findings reveal that, in isolated cardiomyocytes, sublethal H(2)O(2) generation during simulated ischemia regulates cell death during simulated reperfusion, which is mediated by the reperfusion oxidant burst.     

Involvement of oxygen-depending processes in the pathogenesis of hepatic reperfusion injury

The disbalance between reactive oxygen species generation and antioxidant defensive factors importantly contributes in the development of hepatic reperfusion damage. Changes in blood oxygen transport during hepatic ischemia/reperfusion are involved in its development. Ischemia-induced oxyhemoglobin dissociation curve shift rightwards also exists during the hepatic reperfusion and thereby may facilitate the free radical attacks against the liver. The least disorders in blood oxygen transport, prooxidant-antioxidant balance and hepatic morpho-functional state were observed during the postischemic period under the conditions of moderate hypoxia. The protective effect of L-arginine during the hepatic ischemia-reperfusion may be partially due to the changed hemoglobin function and thereby tissue oxygen delivery and to the keeping of body prooxidant-antioxidant balance. The development of new pharmacological tools to modify the blood oxygen transport and generate the optimal nitric oxide amounts may be a promising strategy for correction of reperfusion injury.     

Allantoin formation and urate and glutathione exchange in human muscle during submaximal exercise.

Seven males performed two exhaustive cycling bouts (EX1 and EX2) at a work-rate of 90% of maximal oxygen uptake, separated by 60 min. During EX1 there was a significant accumulation of urate (from 0.16 +/- 0.02 to 0.27 +/- 0.03 micromol/kg d.w.) and allantoin (from 0.39 +/- 0.05 to 0.69 +/- 0.14 micromol/kg d.w.) in the muscle. An uptake of urate was observed in early recovery from EX1 (0-9 min: 486 +/- 136 micromol; p <.05). There was no exchange of total glutathione or cysteine over the muscle either during or after exercise, and muscle and plasma total glutathione remained unaltered (p <.05). The glycogen levels were lowered by 40% at the onset of EX2, yet the level of oxidative stress in EX1 and EX2 was similar as evidenced by a similar increase in muscle allantoin in both exercise bouts. The data suggest that urate is utilized as antioxidant in human skeletal muscle and that reactive oxygen species are formed in muscle during intense submaximal exercise. No net exchange of glutathione appears to occur over the muscle either at rest, during exercise or in recovery. Moreover, when an exhaustive exercise bout is repeated with lowered glycogen levels, the level of oxidative stress is not different than that of the first bout.     

Oxidative stress and redox regulation of phospholipase D in myocardial disease

Oxidative stress may be viewed as an imbalance between reactive oxygen species (ROS) and oxidant production and the state of glutathione redox buffer and antioxidant defense system. Recently, a new paradigm of redox signaling has emerged whereby ROS and oxidants can function as intracellular signaling molecules, where ROS- and oxidant-induced death signal is converted into a survival signal. It is now known that oxidative stress is involved in cardiac hypertrophy and in the pathogenesis of cardiomyopathies, ischemic heart disease and congestive heart failure. Phospholipase D (PLD) is an important signaling enzyme in mammalian cells, including cardiomyocytes. PLD catalyzes the hydrolysis of phosphatidylcholine to produce phosphatidic acid (PA). Two mammalian PLD isozymes, PLD1 and PLD2 have been identified, characterized and cloned. The importance of PA in heart function is evident from its ability to stimulate cardiac sarcolemmal membrane and sarcoplasmic reticular Ca2+-related transport systems and to increase the intracellular concentration of free Ca2+ in adult cardiomyocytes and augment cardiac contractile activity of the normal heart. In addition, PA is also considered an important signal transducer in cardiac hypertrophy. Accordingly, this review discusses a role for redox signaling mediated via PLD in ischemic preconditioning and examines how oxidative stress affects PLD in normal hearts and during different myocardial diseases. In addition, the review provides a comparative account on the regulation of PLD activities in vascular smooth muscle cells under conditions of oxidative stress.     

Calcium-independent phospholipase A2 modulates cytosolic oxidant activity and contractile function in murine skeletal muscle cells.

Phospholipase A2 (PLA2) activity supports production of reactive oxygen species (ROS) by mammalian cells. In skeletal muscle, endogenous ROS modulate the force of muscle contraction. We tested the hypothesis that skeletal muscle cells constitutively express the calcium-independent PLA2 (iPLA2) isoform and that iPLA2 modulates both cytosolic oxidant activity and contractile function. Experiments utilized differentiated C2C12 myotubes and a panel of striated muscles isolated from adult mice. Muscle preparations were processed for measurement of mRNA by real-time PCR, protein by immunoblot, cytosolic oxidant activity by the dichlorofluorescein oxidation assay, and contractile function by in vitro testing. We found that iPLA2 was constitutively expressed by all muscles tested (myotubes, diaphragm, soleus, extensor digitorum longus, gastrocnemius, heart) and that mRNA and protein levels were generally similar among muscles. Selective iPLA2 blockade by use of bromoenol lactone (10 microM) decreased cytosolic oxidant activity in myotubes and intact soleus muscle fibers. iPLA2 blockade also inhibited contractile function of unfatigued soleus muscles, shifting the force-frequency relationship rightward and depressing force production during acute fatigue. Each of these changes could be reproduced by selective depletion of superoxide anions using superoxide dismutase (1 kU/ml). These findings suggest that constitutively expressed iPLA2 modulates oxidant activity in skeletal muscle fibers by supporting ROS production, thereby influencing contractile properties and fatigue characteristics.     

Acute hypoxia and hypoxic exercise induce DNA strand breaks and oxidative DNA damage in humans.

The present study investigated the effect of a single bout of exhaustive exercise on the generation of DNA strand breaks and oxidative DNA damage under normal conditions and at high-altitude hypoxia (4559 meters for 3 days). Twelve healthy subjects performed a maximal bicycle exercise test; lymphocytes were isolated for analysis of DNA strand breaks and oxidatively altered nucleotides, detected by endonuclease III and formamidipyridine glycosylase (FPG) enzymes. Urine was collected for 24 h periods for analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a marker of oxidative DNA damage. Urinary excretion of 8-oxodG increased during the first day in altitude hypoxia, and there were more endonuclease III-sensitive sites on day 3 at high altitude. The subjects had more DNA strand breaks in altitude hypoxia than at sea level. The level of DNA strand breaks further increased immediately after exercise in altitude hypoxia. Exercise-induced generation of DNA strand breaks was not seen at sea level. In both environments, the level of FPG and endonuclease III-sensitive sites remained unchanged immediately after exercise. DNA strand breaks and oxidative DNA damage are probably produced by reactive oxygen species, generated by leakage of the mitochondrial respiration or during a hypoxia-induced inflammation. Furthermore, the presence of DNA strand breaks may play an important role in maintaining hypoxia-induced inflammation processes. Hypoxia seems to deplete the antioxidant system of its capacity to withstand oxidative stress produced by exhaustive exercise.     

ROS in the local and systemic pathogenesis of COPD

An imbalance between oxidants and antioxidants is proposed in the pathogenesis of COPD. Potential alterations responsible for an imbalance in oxidant production and intra- and extracellular antioxidant defense systems are discussed with respect to COPD-related changes in the pulmonary compartment. In line with the current view of COPD as a disease with multiple systemic consequences, there is increasing evidence that imbalances in the redox milieu extend beyond the diseased lung in COPD patients. Skeletal muscle dysfunction is often observed in COPD and may result from imbalances in the redox environment of skeletal muscle. Potential triggers of oxidative stress in the muscle compartment include inflammation and hypoxia, and local sources of reactive oxygen and nitrogen species are discussed, as well the mechanisms by which skeletal muscle trophical state, contractility and fatigability may be affected by oxidative stress, resulting in skeletal muscle dysfunction.     

The effects of exercise load during development on oxidative stress levels and antioxidant potential in adulthood

The objective of this study was to elucidate the impact of physical activity during the growth period as well as on oxidative stress and antioxidative potential in adulthood. The experimental animals used were four-week old male Wistar rats, which were randomly divided into three groups. The exercise loads were as follows: control (CON), treadmill exercise (TE), and jumping exercise (JE). The exercise was performed at the same time of day, at a frequency of five days per week, for eight weeks. Derivatives of reactive oxygen metabolites (d-ROSs) and biological antioxidant potential (BAP) were measured during periods of rest prior to commencement of the experiment and after the experiment. Analysis was conducted using a Wilcoxon signed-rank test and Schaffer’s multiple comparison procedure and the significance level was set at p?相似文献