Delayed angiogenesis and VEGF production in CCR2-/- mice during impaired skeletal muscle regeneration

The regulation of vascular endothelial growth factor (VEGF) levels and angiogenic events during skeletal muscle regeneration remains largely unknown. This study examined angiogenesis, VEGF levels, and muscle regeneration after cardiotoxin (CT)-induced injury in mice lacking the CC chemokine receptor 2 (CCR2). Muscle regeneration was significantly decreased in CCR2-/- mice as was the early accumulation of macrophages after injury. In both mouse strains, tissue VEGF was similar at baseline (no injections) and significantly decreased at day 3 post-CT. Tissue VEGF in wild-type (WT) mice was restored within 7 days postinjury but remained significantly reduced in CCR2-/- mice until day 21. Capillary density (capillaries/mm(2)) within regenerating muscle was maximal in WT mice at day 7 and double that of baseline muscle. In comparison, maximal capillary density in CCR2-/- mice occurred at 21 days postinjury. Maximal capillary density developed concurrent with the restoration of tissue VEGF in both strains. A highly significant, inverse relationship existed between the size of regenerated muscle fibers and capillaries per square millimeter. Although this relationship was comparable in WT and CCR2-/- animals, there was a significant decrease in the magnitude of this response in the absence of CCR2, reflecting the observation that regenerated muscle fiber size in CCR2-/- mice was only 50% of baseline at 42 days postinjury, whereas WT mice had attained baseline fiber size by day 21. Thus CCR2-dependent events in injured skeletal muscle, including impaired macrophage recruitment, contribute to restoration of tissue VEGF levels and the dynamic processes of capillary formation and muscle regeneration.     

The urokinase-type plasminogen activator receptor is not required for skeletal muscle inflammation or regeneration

The hypothesis of this study was the urokinase-type plasminogen activator receptor (uPAR) is required for accumulation of inflammatory cells in injured skeletal muscle and for efficient muscle regeneration. Expression of uPAR was elevated at 1 and 3 days after cardiotoxin-induced muscle injury in wild-type mice before returning to baseline levels. Neutrophil accumulation peaked 1 day postinjury in muscle from both wild-type (WT) and uPAR null mice, while macrophage accumulation peaked between 3 and 5 days postinjury, with no differences between strains. Histological analyses confirmed efficient muscle regeneration in both wild-type and uPAR null mice, with no difference between strains in the formation or growth of regenerating fibers, or recovery of normal morphology. Furthermore, in vitro experiments demonstrated that chemotaxis is not different between WT and uPAR null macrophages. Finally, fusion of cultured satellite cells into multinucleated myotubes was not different between cells isolated from WT and uPAR null mice. These results demonstrate that uPAR is not required for the accumulation of inflammatory cells or the regeneration of skeletal muscle following injury, suggesting uPA can act independently of uPAR to regulate events critical for muscle regeneration.     

Alterations in creatine kinase, ornithine decarboxylase, and transglutaminase during muscle regeneration

Creatine kinase (CK), transglutaminase (TGase) and ornithine decarboxylase (ODC), enzymes implicated in the regulation of growth processes, were studied during muscle regeneration subsequent to the injection of bupivacaine into rat tibialis anterior. Within 2 days, the percent BB isozyme of CK detected in the muscle was elevated 70-fold coincident with a marked decrease in total CK activity. The MB isozyme also increased and was 15-fold of control at 4-5 days postinjection. TGase activity was increased significantly to greater than 2-fold of control within 2 days of injection and significantly decreased at days 3 through 7 compared to controls. ODC activity was elevated significantly to 2- to 3-fold of control from 2-7 days after injection. These results suggest an early alteration in the expression of a coordinated battery of genes in this model of muscle degeneration-regeneration. The increased expression of MB and BB isozymes of CK in various human neuromuscular diseases may be a manifestation of an ongoing process of degeneration-regeneration.     

Time course of increased heme oxygenase activity and expression after experimental intracerebral hemorrhage: correlation with oxidative injury

Heme oxygenase (HO) activity in tissue adjacent to an intracerebral hematoma may modulate cellular vulnerability to heme-mediated oxidative injury. Although HO-1 is induced after experimental intracerebral hemorrhage (ICH), the time course of this induction, its effect on tissue HO activity, and its association with oxidative injury markers has not been defined. We therefore quantified HO activity, HO-1 expression, tissue heme content, and protein carbonylation for 8 days after injection of autologous blood into the mouse striatum. Increased striatal HO-1 protein was observed within 24 h, peaked on day 5 at a level that was 10-fold greater than baseline, and returned to baseline by day 8; HO-2 expression was not altered. HO activity increased by only 1.6-fold at its peak on day 5, and had also returned to baseline by day 8. A significant increase in protein carbonylation was observed at 3–5 days, which also was markedly attenuated by 8 days, concomitant with a return of tissue heme to near-normal levels. These results suggest that the increase in HO activity in tissue surrounding an experimental ICH is considerably less than would be predicted based on an analysis of HO-1 expression per se . As HO-1 expression is temporally associated with increased tissue heme and increased protein carbonylation, it may be more useful as a marker of heme-mediated oxidative stress in ICH models, rather than as an index of HO activity.     

Effect of therapeutic pulsed ultrasound on parameters of oxidative stress in skeletal muscle after injury

Contusion injuries are a very common form of both athletic and non-athletic injury, that effect muscle function. Treatments to augment the normal repair and regeneration processes are important for a wide variety of patients. Therapeutic ultrasound has been claimed to promote tissue repair, especially by enhancing cell proliferation and protein synthesis. The present study aimed to investigate the effect of therapeutic pulsed ultrasound (TPU) on parameters of oxidative stress, namely thiobarbituric acid-reactive substances (TBARS), protein carbonyl content and the activities of antioxidant enzymes, catalase and superoxide dismutase (SOD), in skeletal muscle after injury. Wistar rats were submitted to an animal model of muscle (gastrocnemius) laceration. TPU was used once a day. One, three or five days after muscle laceration, the animals were killed by decapitation and oxidative stress parameters were evaluated. Serum CK levels were increased in muscle-injured animals, indicating that the laceration animal model was successful. TBARS were not altered after muscle injury, when compared to the sham group. Protein carbonyl content was increased after muscle laceration. Catalase and SOD activities were increased 1 day after muscle injury and not altered at days 3 and 5. TPU decreased TBARS levels after muscle laceration when compared to injured muscle animals without treatment. Protein carbonyl content evaluation presented similar results. It is tempting to speculate that TPU seems to protect the tissue from oxidative injury. TPU diminished catalase and SOD activities, especially on the first day following muscle laceration.     

Recovery of skeletal muscle mass after extensive injury: positive effects of increased contractile activity

The present study was designed to test the hypothesis that increasing physical activity by running exercise could favor the recovery of muscle mass after extensive injury and to determine the main molecular mechanisms involved. Left soleus muscles of female Wistar rats were degenerated by notexin injection before animals were assigned to either a sedentary group or an exercised group. Both regenerating and contralateral intact muscles from active and sedentary rats were removed 5, 7, 14, 21, 28 and 42 days after injury (n = 8 rats/group). Increasing contractile activity through running exercise during muscle regeneration ensured the full recovery of muscle mass and muscle cross-sectional area as soon as 21 days after injury, whereas muscle weight remained lower even 42 days postinjury in sedentary rats. Proliferator cell nuclear antigen and MyoD protein expression went on longer in active rats than in sedentary rats. Myogenin protein expression was higher in active animals than in sedentary animals 21 days postinjury. The Akt-mammalian target of rapamycin (mTOR) pathway was activated early during the regeneration process, with further increases of mTOR phosphorylation and its downstream effectors, eukaryotic initiation factor-4E-binding protein-1 and p70(s6k), in active rats compared with sedentary rats (days 7-14). The exercise-induced increase in mTOR phosphorylation, independently of Akt, was associated with decreased levels of phosphorylated AMP-activated protein kinase. Taken together, these results provided evidence that increasing contractile activity during muscle regeneration ensured early and full recovery of muscle mass and suggested that these beneficial effects may be due to a longer proliferative step of myogenic cells and activation of mTOR signaling, independently of Akt, during the maturation step of muscle regeneration.     

GAPDH is conformationally and functionally altered in association with oxidative stress in mouse models of amyotrophic lateral sclerosis

It is proposed that conformational changes induced in proteins by oxidation can lead to loss of activity or protein aggregation through exposure of hydrophobic residues and alteration in surface hydrophobicity. Because increased oxidative stress and protein aggregation are consistently observed in amyotrophic lateral sclerosis (ALS), we used a 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (BisANS) photolabeling approach to monitor changes in protein unfolding in vivo in skeletal muscle proteins in ALS mice. We find two major proteins, creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), conformationally affected in the ALS G93A mouse model concordant with a 43% and 41% reduction in enzyme activity, respectively. This correlated with changes in conformation and activity that were detected in CK and GAPDH with in vitro oxidation. Interestingly, we found that GAPDH, but not CK, is conformationally and functionally affected in a longer-lived ALS model (H46R/H48Q), exhibiting a 22% reduction in enzyme activity. We proposed a reaction mechanism for BisANS with nucleophilic amino acids such as lysine, serine, threonine, and tyrosine, and BisANS was found to be primarily incorporated to lysine residues in GAPDH. We identified the specific BisANS incorporation sites on GAPDH in nontransgenic (NTg), G93A, and H46R/H48Q mice using liquid chromatography-tandem mass spectrometry analysis. Four BisANS-containing sites (K52, K104, K212, and K248) were found in NTg GAPDH, while three out of four of these sites were lost in either G93A or H46R/H48Q GAPDH. Conversely, eight new sites (K2, K63, K69, K114, K183, K251, S330, and K331) were found on GAPDH for G93A, including one common site (K114) for H46R/H48Q, which is not found on GAPDH from NTg mice. These data show that GAPDH is differentially affected structurally and functionally in vivo in accordance with the degree of oxidative stress associated with these two models of ALS.     

Tumor necrosis factor-alpha promotes the accumulation of neutrophils and macrophages in skeletal muscle.

Tumor necrosis factor-alpha (TNF-alpha) has been associated with cachexia and is known to regulate multiple inflammatory cell (neutrophil and macrophage) responses. We tested the hypothesis that neutrophils and macrophages accumulate in the extensor digitorum longus (EDL) and soleus muscles of mice after chronic TNF-alpha administration. Murine recombinant TNF-alpha (approximately 100 microg x kg(-1) x day(-1)) in vehicle solution or vehicle solution alone (sham) was administered to C57BL/6 mice for 7 days via osmotic minipumps. In EDL muscles from TNF-alpha-treated mice, neutrophil and macrophage concentrations were elevated seven- and threefold, respectively, compared with sham mice. Neutrophil and macrophage concentrations were also elevated five- and twofold, respectively, in solei of TNF-alpha- relative to sham-treated mice. Treatment with TNF-alpha elevated ubiquitin content by approximately 25% relative to sham values for both the EDL and soleus muscles; however, these elevations were not statistically significant. No differences were observed between TNF-alpha- and sham-treated mice in body weight, food consumption, muscle mass, myofiber cross-sectional area, carbonyl groups, total protein content, or relative abundance of myosin heavy chain protein. Furthermore, no overt signs of muscle injury or regeneration were observed in muscles from TNF-alpha-treated mice in either the EDL or soleus muscles. These observations suggest that 7 days of TNF-alpha administration promote muscle inflammation as indicated by the accumulation of neutrophils and macrophages without overt signs of atrophy, injury, or regeneration.     

Supplementation with vitamin C and N-acetyl-cysteine increases oxidative stress in humans after an acute muscle injury induced by eccentric exercise

There has been no investigation to determine if the widely used over-the-counter, water-soluble antioxidants vitamin C and N-acetyl-cysteine (NAC) could act as pro-oxidants in humans during inflammatory conditions. We induced an acute-phase inflammatory response by an eccentric arm muscle injury. The inflammation was characterized by edema, swelling, pain, and increases in plasma inflammatory indicators, myeloperoxidase and interleukin-6. Immediately following the injury, subjects consumed a placebo or vitamin C (12.5 mg/kg body weight) and NAC (10 mg/kg body weight) for 7 d. The resulting muscle injury caused increased levels of serum bleomycin-detectable iron and the amount of iron was higher in the vitamin C and NAC group. The concentrations of lactate dehydrogenase (LDH), creatine kinase (CK), and myoglobin were significantly elevated 2, 3, and 4 d postinjury and returned to baseline levels by day 7. In addition, LDH and CK activities were elevated to a greater extent in the vitamin C and NAC group. Levels of markers for oxidative stress (lipid hydroperoxides and 8-iso prostaglandin F2alpha; 8-Iso-PGF2alpha) and antioxidant enzyme activities were also elevated post-injury. The subjects receiving vitamin C and NAC had higher levels of lipid hydroperoxides and 8-Iso-PGF2alpha 2 d after the exercise. This acute human inflammatory model strongly suggests that vitamin C and NAC supplementation immediately post-injury, transiently increases tissue damage and oxidative stress.     

Loss of the Inducible Hsp70 Delays the Inflammatory Response to Skeletal Muscle Injury and Severely Impairs Muscle Regeneration

Skeletal muscle regeneration following injury is a highly coordinated process that involves transient muscle inflammation, removal of necrotic cellular debris and subsequent replacement of damaged myofibers through secondary myogenesis. However, the molecular mechanisms which coordinate these events are only beginning to be defined. In the current study we demonstrate that Heat shock protein 70 (Hsp70) is increased following muscle injury, and is necessary for the normal sequence of events following severe injury induced by cardiotoxin, and physiological injury induced by modified muscle use. Indeed, Hsp70 ablated mice showed a significantly delayed inflammatory response to muscle injury induced by cardiotoxin, with nearly undetected levels of both neutrophil and macrophage markers 24 hours post-injury. At later time points, Hsp70 ablated mice showed sustained muscle inflammation and necrosis, calcium deposition and impaired fiber regeneration that persisted several weeks post-injury. Through rescue experiments reintroducing Hsp70 intracellular expression plasmids into muscles of Hsp70 ablated mice either prior to injury or post-injury, we confirm that Hsp70 optimally promotes muscle regeneration when expressed during both the inflammatory phase that predominates in the first four days following severe injury and the regenerative phase that predominates thereafter. Additional rescue experiments reintroducing Hsp70 protein into the extracellular microenvironment of injured muscles at the onset of injury provides further evidence that Hsp70 released from damaged muscle may drive the early inflammatory response to injury. Importantly, following induction of physiological injury through muscle reloading following a period of muscle disuse, reduced inflammation in 3-day reloaded muscles of Hsp70 ablated mice was associated with preservation of myofibers, and increased muscle force production at later time points compared to WT. Collectively our findings indicate that depending on the nature and severity of muscle injury, therapeutics which differentially target both intracellular and extracellular localized Hsp70 may optimally preserve muscle tissue and promote muscle functional recovery.     

Angiogenesis gene expression in murine endothelial cells during post-pneumonectomy lung growth

Background

Human umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) attenuate hyperoxic neonatal lung injury primarily through anti-inflammatory effects. We hypothesized that intratracheal transplantation of human UCB-derived MSCs could attenuate Escherichia coli (E. coli)-induced acute lung injury (ALI) in mice by suppressing the inflammatory response.

Methods

Eight-week-old male ICR mice were randomized to control or ALI groups. ALI was induced by intratracheal E. coli instillation. Three-hours after E. coli instillation, MSCs, fibroblasts or phosphate-buffered saline were intratracheally administered randomly and survival was analyzed for 7 days post-injury. Lung histology including injury scores, myeloperoxidase (MPO) activity, and protein levels of interleukin (IL)-1α, IL-1β, IL-6, tumor necrosis factor (TNF)-α, and macrophage inflammatory protein (MIP)-2 as well as the wet-dry lung ratio and bacterial counts from blood and bronchoalveolar lavage (BAL) were evaluated at 1, 3, and 7 days post-injury. Levels of inflammatory cytokines in the lung were also profiled using protein macroarrays at day 3 post-injury which showed peak inflammation.

Results

MSC transplantation increased survival and attenuated lung injuries in ALI mice, as evidenced by decreased injury scores on day 3 post-injury and reduced lung inflammation including increased MPO activity and protein levels of IL-1α, IL-1β, IL-6, TNF-α, and MIP-2 on day 3 and 7 post-injury. Inflammatory cytokine profiles in the lungs at day 3 post-injury were attenuated by MSC transplantation. MSCs also reduced the elevated lung water content at day 3 post-injury and bacterial counts in blood and BAL on day 7 post-injury.

Conclusions

Intratracheal transplantation of UCB-derived MSCs attenuates E. coli-induced ALI primarily by down-modulating the inflammatory process and enhancing bacterial clearance.     

Angiogenesis and inflammation in skeletal muscle in response to ascites tumor in mice

This study addresses the interaction between Ehrlich ascites tumor and skeletal abdominal muscle, presenting quantitative analysis of ascites-induced angiogenesis and inflammation in this tissue of mice bearing-tumor. Time-dependent changes in the muscle (cellular activity, angiogenesis, inflammation and cytokines production) were assessed by morphometric, functional, and biochemical parameters at days 1, 4 and 8 after i.p. inoculation of Ehrlich tumor cells (2.5 x 10(7)). The number of cells stained with AgNOR technique (argyrophilic nucleolar organizer region) in the muscle, together with MTS assay used as markers of cellular activity increased progressively in parallel with the out flow rate of sodium fluorescein (blood flow index), hemoglobin content (vascular index) and VEGF production. Likewise, the inflammatory process in the muscle, as assessed by myeloperoxidase (MPO) and n-acethylglucosaminidase (NAG) activities and the levels of the chemokines, keratinocyte-derived chemokine (CXC1-3/KC) and macrophage-chemoattractant protein (CCL2/MCP-1) increased with tumor development. The combination of techniques used to describe angiogenesis and inflammation in a muscle model system has proved to be suited for quantitative measurements of microvascular changes and cellular infiltration occurring in the abdominal muscle wall of ascites-bearing mice. This study holds potential for investigating events and mechanisms associated with skeletal muscle response to neoplasic stimulus.     

Caffeic acid phenethyl ester improves oxidative organ damage in rat model of thermal trauma

Severe burn injuries cause functional impairment in distant internal organs. Although this mechanism is not clear, it is possible that free radical toxicity plays an important role. Research in animals and clinical studies have shown that there is a close relationship between a lipid peroxidative reaction and secondary pathological changes following thermal injury. It has been demonstrated that antioxidant treatment prevents oxidative tissue damage associated with thermal trauma. This study was designed to determine the possible protective effect of caffeic acid phenethyl ester (CAPE) treatment against oxidative damage in the kidney and lung induced by thermal injury. Rats were decapitated either 1, 3 or 7 days after burn injury. CAPE was administered intraperitoneally immediately after thermal injury. Kidney and lung tissues were taken for the determination of malondialdehyde (MDA) level, myeloperoxidase (MPO), catalase (CAT), superoxide dismutase (SOD) and xanthine oxidase (XO) activities. Severe skin thermal injury caused a significant decrease in SOD and CAT activities, as well as significant increases in MDA level, XO and MPO activities in tissues during the postburn period. Treatment of rats with CAPE (10 micromol/kg) significantly elevated the decreased SOD and CAT activities, while it decreased MDA levels and MPO as well as XO activity.     

Effect of heparin and antivenom on skeletal muscle damage produced by Bothrops jararacussu venom

We examined the effect of treatment with heparin and polyvalent antivenom on mice muscle Extensor digitorum longus (EDL) regeneration, after damage induced by injection of Bothrops jararacussu crude venom over the muscle of the right posterior limb. The mice were separated into groups and each group received treatment, by intravenous route with either high molecular weight heparin (H), low molecular weight heparin (LMWH), polyvalent antivenom (PAV) or with the combination of PAV plus H or PAV plus LMWH at 15 minutes and 4 hours after the injection of the venom. Myotoxicity was measured by the increase in plasma creatine kinase (CK) activity at two hours after the injection of the venom. The histological changes in EDL at 1, 3, 7 and 21 days after the injection of the venom were analyzed by light microscopy. In each group the normal and regenerated muscle fibers were quantified using Scion Image computer program. We also evaluated in vitro, the influence of these substances in the proteolytic and phospholipase activities of the venom. Heparins decreased the proteolytic activity of the venom but did not affect its phospholipase activity. However the PAV antagonized both activities. PAV and its combinations showed antimyotoxic activity, according to the magnitude of CK plasma levels. At 21 days the regeneration was observed in all animals, also in those that received only the venom. All treatments, except LMWH, promote a significant increase in the number of muscle fibers.     

Effects of caffeic acid phenethyl ester on endotoxin-induced cardiac stress in rats: a possible mechanism of protection

Endotoxins (lipopolysaccharides; LPS) are known to cause multiple organ failure, including myocardial dysfunction. The present study aimed to investigate the mechanism of caffeic acid phenethyl ester (CAPE) protection against LPS-induced cardiac stress. Rats were allocated into three groups; group 1 served as a normal control group, group 2 (LPS) received a single intraperitoneal injection of LPS (10 mg/kg), group 3 (LPS + CAPE) was injected intraperitoneally with CAPE (10 mg/kg/day; solubilized in saline containing 20% tween 20) throughout a period of 10 days prior to LPS injection. Rats were maintained 4 h before sacrifice. Caffeic acid phenethyl ester pretreatment normalized LPS-enhanced activities of serum creatine kinase (CK) and lactate dehydrogenase (LDH) as well as glutathione peroxidase (GPx), and myeloperoxidase (MPO) in cardiac tissue. A significant reduction of the elevated levels of serum tumor necrosis factor-alpha (TNF-α) as well as serum and cardiac nitrite/nitrate (NOx) ) was achieved after CAPE pretreatment. CAPE also restored malondialdelyde (MDA), reduced glutathione (GSH), and cytosolic calcium (Ca2+ ) levels in the heart. A marked induction of cardiac heme oxygenase-1 (HO-1) protein level was detected in CAPE-pretreated group. Whereas, LPS-induced reduction of adenosine triphosphate (ATP) and phosphocreatine (PCr) levels was insignificantly changed. Conclusively, the early treatment with CAPE maintained antioxidant defences, reduced oxidative injury, cytokine damage, and inflammation but did not markedly improve energy status in cardiac tissue. The beneficial effect of CAPE might be mediated, at least in part, by the superinduction of HO-1.     

Overexpression of inducible 70-kDa heat shock protein in mouse attenuates skeletal muscle damage induced by cryolesioning

Heat shock protein expression is elevated upon exposure to a variety of stresses and limits the extent of stress-induced damage. To investigate the putative role of inducible 70-kDa heat shock protein (HSP70) in skeletal muscle damage and regeneration, soleus and tibialis anterior (TA) muscles from HSP70-overexpressing transgenic mice were subjected to cryolesioning and analyzed after 1, 10, and 21 days. Histological analysis showed that the muscles from both HSP70 and wild-type mice treated with radicicol (a HSP inducer) had decreased necrosis after cryolesioning compared with controls. The decrease in muscle fiber cross-sectional area in both soleus and TA muscles in 10 days postlesioning was attenuated in HSP70 mice compared with wild-type mice. Glutathione peroxidase activity was increased 1 day after cryolesioning in both HSP70 and control mice and remained elevated for up to 21 days. Immunodetection of neuronal cell adhesion molecule (a satellite cell marker) and developmental/neonatal MHC were significantly lower in cryolesioned HSP70-overexpressing mice than in cryolesioned controls. These results suggest that HSP70 protects skeletal muscle against injury and radicicol might be useful as a skeletal muscle protective agent. regeneration; radicicol; transgenic mouse; myoprotection     

A novel approach for screening the proteome for changes in protein conformation

Changes in surface hydrophobicity are generally considered as a sensitive indicator for monitoring the structural alterations of proteins that are often associated with changes in function. Currently, no technique has been developed to screen a complex mixture of proteins for changes in the conformation of specific proteins. In this study, we adapted a UV photolabeling approach, using an apolar fluorescent probe, 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (BisANS), to monitor changes in surface hydrophobic domains in either purified rhodanese or skeletal muscle cytosolic proteins by urea-induced unfolding or in response to in vitro metal-catalyzed oxidation. Using two-dimensional polyacrylamide gel electrophoresis (2D PAGE), we identified two specific proteins in skeletal muscle cytosol that exhibited a marked loss of incorporation of BisANS after exposure to in vitro oxidative stress: creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found that the activities of both enzymes were also reduced significantly in response to oxidative stress. We then determined if this method could detect changes in surface hydrophobicity in specific proteins arising from oxidative stress generated in vivo by muscle denervation. A loss in surface hydrophobic domains in CK and GAPDH was again observed as measured by the BisANS photoincorporation approach. In addition, the CK and GAPDH activity in denervated muscle was markedly reduced. These data demonstrate for the first time that this assay can screen a complex mixture of proteins for alterations in surface hydrophobic domains of individual proteins.     

Protective effect of aqueous garlic extract against oxidative organ damage in a rat model of thermal injury

Oxygen free radicals have been implicated in mediating various pathological processes including burn-induced organ damage. This study was designed to determine the possible protective effect of aqueous garlic extract against oxidative organ damage distant from the original burn wound. Under ether anaesthesia, rats were subjected to severe skin scald injury covering 30% of total body surface area. Rats were decapitated either 2 h or 24 h after burn injury. Aqueous garlic extract (1 ml/kg) was administered i.p. immediately after burn injury. In the 24-h burn group injection was repeated once more (at 12 hour) following the burn injury. Liver, intestine and lung tissues were taken for the determination of malondialdehyde (MDA) and glutathione (GSH) levels, myeloperoxidase (MPO) activity and protein oxidation (PO). Burn injury caused a significant decrease in GSH level, and significant increases in MDA and PO levels, and MPO activity at post-burn 2 and 24 hours. Since garlic extract reversed these oxidant responses it seems likely that garlic extract protects tissues against oxidative damage.