Glutathione deficiency intensifies ischaemia‐reperfusion induced cardiac dysfunction and oxidative stress

The efficacy of glutathione (GSH) in protecting ischaemia‐reperfusion (I‐R) induced cardiac dysfunction and myocardial oxidative stress was studied in open‐chest, stunned rat heart model. Female Sprague–Dawley rats were randomly divided into three experimental groups: (1) GSH‐depletion, by injection of buthionine sulphoxamine (BSO, 4 mmol kg^–1, i.p.) 24 h prior to I‐R, (2) BSO injection (4 mmol kg^–1, i.p.) in conjunction with acivicin (AT125, 0.05 mmol kg^–1, i.v.) infusion 1 h prior to I‐R, and (3) control (C), receiving saline treatment. Each group was further divided into I‐R, with surgical occlusion of the main left coronary artery (LCA) for 30 min followed by 20 min reperfusion, and sham. Myocardial GSH content and GSH : glutathione disulphide (GSSG) ratio were decreased by ?50% (P < 0.01) in both BSO and BSO + AT125 vs. C. Ischaemia‐reperfusion suppressed GSH in both left and right ventricles of C (P < 0.01) and left ventricles of BSO and BSO + AT125 (P < 0.05). Contractility (+dP/dt and –dP/dt) in C heart decreased 55% (P < 0.01) after I and recovered 90% after I‐R, whereas ±dP/dt in BSO decreased 57% (P < 0.01) with ischaemia and recovered 76 and 84% (P < 0.05), respectively, after I‐R. For BSO + AT125, ±dP/dt were 64 and 76% (P < 0.01) lower after ischaemia, and recovered only 67 and 61% (P < 0.01) after I‐R. Left ventricular systolic pressure in C, BSO and BSO + AT125 reached 95 (P > 0.05) 87 and 82% (P < 0.05) of their respective sham values after I‐R. Rate‐pressure double product was 11% (P > 0.05) and 25% (P < 0.05) lower in BSO and BSO + AT125, compared with Saline, respectively. BSO and BSO + AT125 rats demonstrated significantly lower liver GSH and heart Mn superoxide dismutase activity than C rats after I‐R. These data indicate that GSH depletion by inhibition of its synthesis and transport can exacerbate cardiac dysfunction inflicted by in vivo I‐R. Part of the aetiology may involve impaired myocardial antioxidant defenses and whole‐body GSH homeostasis.     

Aged rat hearts are not more susceptible to ischemia-reperfusion injury in vivo: role of glutathione

The current study tested the hypothesis that ischemia-reperfusion (I-R) can cause more severe myocardial dysfunction and oxidative damage in senescent rats than young adult rats. Male Fischer 344 rats at the age of 6 (adult) and 24 (old) months were subjected to an open-chest heart surgery and randomly assigned to one of the following treatments: ischemia only (I), with the occlusion of the main descending branch of the left coronary artery (LCA) for 30 min; I-R, with the release of LCA occlusion for 20 min; or sham (S) operation. Heart mechanical performance was monitored using a fluid-filled catheter inserted in the right carotid artery and advanced to the left ventricle. Ischemia caused similar reductions of left ventricle systolic pressure (LVSP) and contractility (+/-dP/dt) in adult and aged hearts. After I-R, adult hearts regained 82% (P<0.05) of the pre-ischemic LVSP, whereas the aged hearts regained 91% (P>0.05) of LVSP. There was no significant difference in the reduction of +/-dP/dt with I-R between adult and aged hearts. Old rats had lower pre-ischemic heart rate than adult rats, however, I-R caused no reduction of heart rate, and a smaller reduction of pressure-rate double product in the aged rats (10%, P>0.05) than the adult rats (23%, P<0.01). Aged rats demonstrated greater myocardial and plasma glutathione (GSH) concentrations prior to surgery, and maintained higher GSH levels and GSH:glutathione disulfide (GSSG) ratio with I-R. Aged hearts also had higher GSH peroxidase, GSH reductase and GSH sulfur-transferase activities than adult hearts, while I-R induced lipid peroxidation was similar. It is concluded that senescent hearts with intact circulatory and neural inputs are not more susceptible to I-R injury than adult hearts during myocardial I-R, partly because they have a greater GSH antioxidant protection.     

Acute and severe hypobaric hypoxia-induced muscle oxidative stress in mice: the role of glutathione against oxidative damage

This study intended to analyze: (1) the effects of acute and severe hypoxia exposure on skeletal muscle oxidative stress and oxidative damage markers; (2) the protective role of the antioxidant glutathione against oxidative damage; and (3) the expression of heat shock protein 70 kDa (HSP70) induced by this hypoxic insult. Forty mice were divided into four groups: control + placebo (C+P), hypoxia + placebo (H+P), control + l-buthionine-[S,R]-sulfoximine (BSO, a GSH-depleting compound) (C+BSO) and hypoxia + BSO (H+BSO). Hypoxia groups were continuously exposed for 24 h to a hypobaric hypoxic environment equivalent to an altitude of 7000 m and sacrificed immediately after. Control groups were maintained at sea level during the experimental protocol. Analyzed biochemical parameters were: reduced (GSH) and oxidized (GSSG) glutathione, thiobarbituric acid reactive substances (TBARS), sulfhydryl protein groups (SH), N-acetyl--d-glucosaminidase (NAG) and HSP70 protein. Hypoxia (H+P) per se, compared to C+P, induced a significant increase in %GSSG (5.68 vs. 1.14%), TBARS (436.7 vs. 227.9 nM), NAG (4.49 vs. 3.35 U/mg) and HSP70 (178.7 vs. 100%). Compared with H+P, H+BSO showed a significant decrease in total glutathione (19.30 vs. 6.13 nmol/mg) and an additional increase in %GSSG (5.68 vs. 11.33%) and in HSP70 expression (178.7 vs. 202.2%). However, no further oxidative damage was observed in H+BSO. These data suggest that acute hypoxia per se might enhance oxidative stress; however, the glutathione system seems to have a modest role in skeletal muscle protection against hypoxia-induced oxidative stress. Moreover, hypoxia and BSO treatment is a sufficient stimulus to promote HSP70 overexpression.     

丁硫氨酸硫酸亚胺早期处理抑制单核细胞向树突状细胞的分化

 目的 研究细胞氧化-还原态对树突状细胞（DC）分化和功能的影响。 方法 细胞氧化-还原态的调控应用特异性谷胱甘肽（GSH）合成抑制剂丁硫氨酸硫酸亚胺（BSO）。自健康人外周血单个核细胞（PBMC）分离单核细胞，在含重组人粒细胞-巨噬细胞集落刺激因子和 IL-4的RPMI 1640 培养液中诱导分化 DC。将单核细胞分为 BSO 早期（培养第 1 天）用药组、BSO 晚期（培养第 5 天）用药组和不予 BSO 处理的对照组。BSO 用药剂量为终浓度 1 mmol/L，换液时补加相应剂量的 BSO。培养第 5 天，分别给予加入脂多糖（LPS）100 ng/ml（LPS刺激组）或重组人干扰素 （rhIFN- ）20 U/ml（IFN- 刺激组）或不予刺激（无刺激组）的处理；培养第 7 天收集各组悬浮细胞进行表型分析和功能检测，并在倒置显微镜下观察 BSO 早期用药组和对照组贴壁细胞。表型分析采用特异性荧光标记单抗及流式细胞仪。功能检测采用混合淋巴细胞反应试验，将经丝裂霉素C 处理的 DC（刺激细胞）与异基因非贴壁 PBMC（反应细胞）混合（刺激细胞:反应细胞分别为 1:25、1:50、1:100）培养 5 d 后，用3H-胸腺嘧啶核苷法检测淋巴细胞增殖情况。 结果 培养第 7 天，镜下观察显示 BSO早期用药组贴壁细胞明显多于对照组；流式细胞仪检测显示 BSO 早期用药组 CD86 和CD1a表达明显低于对照组，CD86 平均荧光强度（MFI）分别为311.3 ± 97.3、552.0 ± 97.9（P = 0.01），CD1a 分别为52.2 ± 22.2、121.2 ± 4.2（P = 0.03）；BSO早期用药组及对照组3H-胸腺嘧啶核苷掺入量（cpm）在刺激细胞:反应细胞＝1:25时分别为 1587 ± 1458 和9336 ± 1333（P=0.015）；加入rhIFN- 20 U/ml 刺激后二组 CD86 MFI 分别为 495.9 ± 143.9 和 800.4 ± 27.7（P = 0.06），而加入 LPS 100 ng/ml 刺激后二组CD86 MFI 分别为 1736.7 ± 375.7 和 1960.8 ± 494.5（P > 0.05）。表明单核细胞分化早期加入 BSO 对 DC 的分化和功能有抑制作用，并对 IFN- 诱导DC成熟有抑制作用。BSO 晚期用药组 DC 抗原提呈功能分子的表达及与对照组比较均无明显差异，对 LPS 或 rhIFN- 刺激的 DC成熟也无明显影响。 结论 BSO 早期持续处理能抑制单核细胞向 DC 细胞的分化，细胞内氧化-还原态水平可能是影响 DC 分化成熟的关键因子。     

Angiotensin II type 1 (AT(1)) receptor blockade enhances the L-NAME-induced vasoconstriction in rat submandibular gland

The vasoregulatory role of nitric oxide (NO) and angiotensin II type 1 (AT(1)) receptors in the circulation of the submandibular gland (SMG) of rats was studied. The glandular blood flow was determined by means of laser Doppler flowmetry and rubidium isotope technique. The data obtained by these two methods correlated well (r = 0.77; P < 0.01). The AT(1) receptor antagonist candesartan (0.5 mg kg(-1), i.v.) reduced the vascular resistance in the SMG by 37 % (P < 0.05). By contrast, the NO synthase blocker L-NAME (15 mg kg(-1), i.v.) significantly increased vascular resistance in the SMG both in candesartan-treated (P < 0.001) and non-treated (P < 0.001) animals. The increase in resistance was greater (P < 0.05) after previous blockade of AT(1) receptors. These findings suggest that the AT(1) receptors have an important role in the vasoregulation of the SMG in the rat. As a result of AT(1) blockade, NO-dependent tone of glandular vessels may be enhanced significantly.     

Protective effects of the glutathione redox cycle and vitamin E on cultured fibroblasts infected by Mycoplasma pneumoniae.

The role of the glutathione (GSH) redox cycle and vitamin E as antioxidant defense systems was studied in normal human cultured skin fibroblasts infected by virulent Mycoplasma pneumoniae. In cells infected for 20 h, catalase activity was inhibited by 75% and the intracellular GSH decreased to 32% of its normal values. GSH peroxidase and oxidized glutathione (reductase activities in the infected cells were unaffected.) GSSG glutathione in the medium of the infected cells rose in accordance with the intracellular GSH decrease. The observed elevation in GSSG/GSH ratio was attributed to the increase in intracellular H2O2 content in M. pneumoniae-infected cells due to the marked inhibition in their catalase activity. The protective effect of the GSH redox cycle in infected cells was studied by depletion of cellular GSH, prior to their infection with M. pneumoniae, using buthionine sulfoximine (BSO), a selective inhibitor of gamma-glutamyl cysteine synthetase. After 16 h of incubation with BSO, the GSH levels were reduced to 38% of their normal value and recovered to 55% during 24 h after removal of the inhibitor. BSO had no effect on GSH peroxidase and catalase activities in either infected or noninfected cells. The level of malonyldialdehyde (an indicator of membrane lipid peroxidation) in BSO-treated cells infected by M. pneumoniae was 1.8 times higher than in infected controls. Cells enriched with 0.25 and 2.25 micrograms of vitamin E per mg of protein prior to their infection by M. pneumoniae revealed the following: a lesser degree of catalase inhibition, 46 and 30%, respectively, versus 64% in infected control cells that were not supplemented with vitamin E; lower levels of malonyldialdehyde, 55 and 20% increments, respectively, versus a 140% increment in infected controls; higher residual activity of lactate dehydrogenase, 76 and 96%, respectively, versus 58% in infected controls. Our data indicate that the oxidative damage induced in M. pneumoniae-infected cells due to the increase in intracellular levels of H2O2 and O2- is limited by the host cell GSH redox cycle and by supplementation with vitamin E.     

Cardiac hypertrophy alters myocardial response to ischaemia and reperfusion in vivo

The impact of cardiac hypertrophy on myocardial biochemical and physiological responses to ischaemia-reperfusion (I-R) was investigated in vivo. Hypertrophy was produced by aortic constriction (PH) or swimming training (TH). Open-chest rat hearts in PH, TH and a sedentary control group (SC) were subjected: (1) to ischaemia, by surgical occlusion of the main descending branch of the left coronary artery for 30 min; (2) to I-R, by releasing the occluded blood vessel for 15 min; or (3) to a sham operation. Ischaemia per se had little effect on heart oxidative and antioxidant status, or lipid peroxidation. However, I-R significantly decreased glutathione (GSH) content, increased glutathione disulfide (GSSG) content, and reduced GSH/GSSG ratio in the SC hearts. These alterations were associated with decreased activities of GSH peroxidase and GSSG reductase, and an increase in lipid peroxidation. Myocardial ATP, total adenine nucleotide content and energy charge in SC were significantly decreased after ischaemia, whereas levels of purine nucleotide derivatives, particularly adenosine, were elevated. No significant alteration of GSH status or adenine nucleotide metabolism occurred after ischaemia or I-R in hypertrophied hearts. In bodi PH and TH, glutathione content was significantly higher than in SC, whereas activities of GSH peroxidase and GSSG reductase were lower. TH rats maintained a higher heart rate (HR), peak systolic pressure, and energy charge during I-R. These data indicate that hypertrophied but well-functioned hearts may be more resistant to I-R induced disturbances of myocardial oxidative and antioxidant functions.     

大鼠脑缺血-再灌注损伤时脑组织谷氨酸转运体功能变化及地塞米松的影响

目的：探讨大鼠脑缺血-再灌注损伤时脑组织谷氨酸转运体功能及超氧化物歧化酶（SOD）活性、脂质过氧化物丙二醛（MDA）含量变化及地塞米松对其的影响。方法：采用大鼠三血管夹闭、松夹制作大鼠完全性脑缺血-再灌注损伤的模型。测定假手术对照组、脑缺血-再灌注损伤（l-R）组和I－R 地塞米松组的皮层、海马、纹状体的谷氨酸转运体功能及SOD活性、MDA含量的变化。结果：脑缺血-再灌注损伤时3个部位的谷氨酸转运体的功能均明显降低（P＜0.05．P＜0．01）、SOD活性显著降低（P＜0．05,P＜0．01）,MDA含量明显升高（P＜0．05,P＜0.01）,1－R 地塞米松组的3部位谷氨酸转运体功能与I-R组相比有明显恢复（P＜0.05,P＜0．01）,与对照组已无明显差异（P＞0.05）SOD活性回升（P＜0.05,P＜0．01）、MDA含量回降（P均＜0.05）。结论：大鼠脑缺血-再灌注损伤时脑组织三部位的谷氨酸转运体功能降低,且可能与自由基效应有关;而地塞米松则可能通过抗自由基效应使谷氨酸转运体功能恢复而发挥其抗损伤作用。     

Changes in peritoneal mesothelial cells phenotype after chronic exposure to glucose or N-acetylglucosamine

Glucose is commonly used as an osmotic solute in peritoneal dialysis fluids despite vast knowledge about deleterious peritoneal and systemic effects of that solute. N-acetylglucosamine (NAG) is a solute of the comparable size to glucose, with strong anti-inflammatory properties. We compared the chronic in vitro effect of both solutes on phenotype of peritoneal mesothelial cells. Experiments were performed of primary cultures of human peritoneal mesothelial cells, which were cultured over 4 weeks in medium supplemented either with glucose 45 mmol/L (GLU) or with NAG 45 mmol/L (NAG). Generation of reactive oxygen species (ROS) in cells was studied, as well as their ability to proliferate, synthesis of cytokines, fibronectin, and factors regulating peritoneal fibrinolysis. Cells cultured in the presence of glucose 45 mmol/L generated more ROS (+73% vs control, P < 0.01), whereas NAG did not stimulate generation of ROS. GLU caused hypertrophy of mesothelial cells (+53% vs control, P < 0.001) and prolonged their population doubling time (+16% vs control, P < 0.01); NAG did not cause significant changes in these parameters. Healing of mesothelial monolayer after mechanical injury was impaired in GLU treated cells: (-48% vs control, P < 0.001 and -40% vs NAG, P < 0.05). Synthesis of Il-6, vascular endothelial growth factor (VEGF), transforming growth factor beta (TGFbeta), and fibronectin was higher in GLU group as compared with control: + 86%, P < 0.001, +38%, P < 0.05, +51%, P < 0.001, +38%, P < 0.05, respectively. In the presence of NAG, these parameters were comparable with the control group, but at the same time NAG stimulated synthesis of hyaluronan: +116% versus control, P < 0.001 and + 96% versus GLU, P < 0.01. Treatment with GLU resulted in decline of tissue plasminogen activator/plasminogen activator inhibitor-1 (t-PA/PAI-1) ratio by 23% versus control, P < 0.001, whereas NAG increased that parameter by 43%, P < 0.01 versus control. Glucose, contrary to NAG, induces oxidative stress and proinflammatory and profibrotic changes in mesothelial cells. NAG seems to be more biocompatible osmotic solute than glucose.     

Reduction of glutathione is associated with growth restriction and enlargement of bovine pulmonary artery endothelial cells produced by transforming growth factor-beta 1.

In addition to inhibiting proliferation and causing enlargement of bovine pulmonary artery endothelial cells in culture, porcine platelet transforming growth factor-beta 1 (TGF-beta 1) (2 ng/ml) lowered glutathione (GSH) of these cells by 48% after 96 h in culture when GSH levels were normalized for cell counts. This lowering of cellular GSH was more marked when corrections were made for approximated cell volume. TGF-beta 1 produced only moderate inhibition of pulmonary artery smooth muscle cell proliferation and did not significantly reduce the GSH content of these cells, even at concentrations as high as 8 ng/ml. Elevation of GSH of endothelial cells above control levels by 0.05 mM diethylmaleate or 1 mM cystine prevented the inhibition of cellular proliferation produced by TGF-beta 1. Lowering cellular GSH levels by approximately 85% for 24 to 72 h with 0.01 mM buthionine sulfoximine (BSO) in the absence of TGF-beta 1 had no effect on proliferation or size of the endothelial cells. However, 0.01 mM BSO potentiated the inhibitory effect of TGF-beta 1 on endothelial cell proliferation and in combination with TGF-beta 1 caused cellular detachment at low endothelial cell densities. Thus, although TGF-beta 1 lowers the level of endothelial cellular GSH, this in itself does not appear to account for the inhibition of proliferation and enlargement of these cells produced by TGF-beta 1. Rather, the combination of another unidentified action of TGF-beta 1 in the presence of reduced cellular GSH likely accounts for these effects.     

Glutathione monoethyl ester protects against glutathione deficiencies due to aging and acetaminophen in mice

Our previous results indicated that glutathione (GSH) and/or cysteine (Cys) deficiency occurs in many aging tissues and also after acetaminophen (APAP) administration. The aim of this study was to investigate whether GSH monoethyl ester (GSH-OEt) can correct these deficiencies. Mice of different ages (3–31 months) through the life span were sacrificed 2 h after i.p. injection of GSH-OEt (10 mmol/kg). In separate experiments, old mice (30–31 months) received the same dose of ester 30 min before the administration of APAP (375 mg/kg) or buthionine sulfoximine (BSO, 4 mmol/kg), an inhibitor of GSH synthesis. Liver and kidney samples were analyzed for GSH and Cys by HPLC. The hepatic GSH and renal cortical GSH and Cys concentrations were about 30% lower in old mice (30–31 months) compared to mature mice (12 months). GSH-OEt corrected these aging-related decreases. APAP decreased both hepatic and renal cortical GSH and Cys concentrations in old mice, but GSH-OEt prevented these decreases. GSH-OEt also prevented the BSO-induced decreases in hepatic and renal GSH concentrations. The results demonstrated that GSH-OEt protected against GSH deficiency due to biological aging as well as APAP-induced decreases in old mice.     

Rigorous swim training impairs mitochondrial function in post-ischaemic rat heart

The effect of rigorous swim training (6 h day^?1, 5 days week^?1 for an average of 191 h) on mitochondrial respiratory function was investigated in rat heart subjected to in vivo ischaemia reperfusion (I-R). Mitochondria was isolated from the risk region of the left ventricle subjected to 60 min occlusion of the main left coronary artery followed by 30 min reperfusion. Heart weight and heart-to-body weight ratio was increased by 21 and 28% (P < 0.01), respectively, in the trained (T, n = 15) vs. control rats (C, n = 20). I-R per se showed minimal effect on heart mitochondria regardless of training status. In sham, state 4 respiration rate was 26 and 32% (P < 0.05) lower in T vs. C rats, using malate-pyruvate (M-P) and 2-oxoglutarate (OG) as substrates, respectively. Training also reduced state 3 respiration by 28% (M-P) and 50% (OG) (P < 0.01). The respiratory control index (RCI) was unaltered in T with M-P, but decreased with OG (P < 0.01). In vitro exposure to superoxide radicals severely reduced state 4 and 3 respiration and RCI, but T hearts showed greater reductions of state 4 and 3 rates than C. Mitochondria from T hearts also revealed a greater state 4 inhibition by H₂O₂ and HO^? compared with C. A lower glutathione content and a higher γ-glutamyl transpeptidase activity (P < 0.05) was observed in T vs. C. It is concluded that rigorous swim training impairs heart mitochondrial function, making them more susceptible to in vivo and in vitro oxidative stress, and that this damaging effect may be related to a diminished glutathione reserve.     

Induction of heme oxygenase-1 expression by root canal sealers in human gingival fibroblasts is augmented by oxidative stress

Heme oxygenase-1 (HO-1) is known as a stress-inducible protein and functions as an antioxidant enzyme. It has been shown that HO-1 is induced rapidly by a variety of chemical and physical stimuli. However, little is known about the induction of cellular signaling events after cell exposure to root canal sealers. The aim of this study was to investigate the effects of zinc oxide-eugenol-based root canal sealer N2 and epoxy resin-based root canal sealer Topseal on the expression of HO-1 protein in cultured human gingival fibroblasts (HGFs). The results showed that both N2 and Topseal were cytotoxic to HGFs in a concentration-dependent manner (p < 0.05). The exposure of quiescent HGFs to N2 and Topseal resulted in the induction of HO-1 protein expression in a time-dependent manner (p < 0.05). Furthermore, to determine whether oxidative stress could modulate HO-1 expression in HGFs by root canal sealers, HGFs were pretreated with glutathione (GSH) synthesis precursor 2-oxothiazolidine-4-carboxylic acid (OTZ) and GSH synthesis inhibitor buthionine sulfoximine (BSO) for 24 h. The pretreatment with OTZ decreased the N2-induced HO-1 protein level by approximately 32% (p < 0.05). However, BSO enhanced the N2-induced HO-1 protein level by up to twofold (p < 0.05). Similar results were found by Topseal. The pretreatment with OTZ decreased the Topseal-induced HO-1 protein level by approximately 12% (p < 0.05). However, BSO enhanced the Topseal-induced HO-1 protein level by up to 1.7-fold (p < 0.05). Taken together, HO-1 expression might be one signal transduction pathway linked to the induction of stress-inducible protein by root canal sealers. In addition, the activation of HO-1 is augmented by oxidative stress. Factors that induce GSH synthesis may appear useful in preventing oxidative damage mediated by root canal sealers.     

Inhibition of glutathione synthesis of Ascaris suum by buthionine sulfoximine

Abstrac t We investigated the effect of dl-buthionine-S,R-sulfoximine (BSO), a selective glutathione (GSH)-depleting agent, on the GSH synthesis of Ascaris suum. The GSH concentrations of the reproductive and muscle tissues of A. suum were determined to be 8.5±0.3 and 14.3±1.3 (n=3) nmol/mg protein, respectively. After treatment of the parasites with 10 μM BSO for 24 h, the GSH content of the reproductive tissue of A. suum was totally depleted as compared with that of untreated controls. However, the GSH levels of the muscle tissue were reduced to only 50% after treatment of the worms for 24 h with 10 μM BSO. Exogenous GSH had no significant effect on the GSH level of the parasites when the worms were incubated for 4 h in RPMI 1640 medium supplemented with 1 mM GSH. In the presence of exogenous GSH, BSO was less effective in depleting the GSH levels of the parasites, which may indicate that the parasites can replenish their GSH levels. GSH depletion, which has been discussed as being therapeutically effective when normal and tumor cells or parasites have markedly different requirements for GSH, may have applications in the development of drugs against nematode infections. Received: 7 September 1995 / Accepted: 15 November 1995     

The post-exercise oxidative stress is depressed by acetylsalicylic acid

In order to assess whether oxidative stress occurs after fatiguing dynamic contractions of a small forearm muscle group, we estimated the kinetics of changes in some of its biomarkers (thiobarbituric acid reactive substances or TBARS; plasma reduced ascorbic acid or RAA; erythrocyte reduced glutathione or GSH). We also tested the hypothesis that acetylsalicylic acid (ASA) may compete with endogenous radical targets, attenuating the post-exercise oxidative stress. Seven male subjects successively performed a 3-min dynamic handgrip exercise with the dominant and then the contralateral forearm. Blood samples were taken from an antecubital vein in each exercising forearm. Biochemical analyses, including the concentration measurements of lactic acid, potassium, and oxidative stress markers were performed at rest and then during the 30-min period of recovery following each exercise. The same day, exercises were repeated after ingestion of a single dose (10 mg/kg) of ASA, and the same exercises were performed after a 3-day ASA treatment (30 mg/kg/day). In control condition, the changes in TBARS, RAA and GSH were already significant immediately after the end of the forearm exercise. They culminated after 5 min, and control values were recovered by a 30-min rest period. We verified that repeated bouts failed to alter the post-exercise variations. ASA did not modify the lactic acid production significantly, though the 3-day ASA treatment significantly reduced the efflux of potassium (-74%, P < 0.05), and the post-exercise variations of TBARS (-45%, P < 0.01), RAA (-44%, P < 0.01) and GSH (-48%, P < 0.01). These results suggest that the dynamic handgrip exercise is a good model for studying the post-exercise oxidative stress and also that ASA seems to offer an efficient protection against oxidative stress and the changes in membrane permeability to potassium.     

大鼠肢体缺血-再灌注后肾内HO-1表达的变化及意义

目的:观察肢体缺血-再灌注(I-R)后肾组织内诱导型血红素氧合酶(HO-1)表达的变化及意义。方法:夹闭大鼠双侧股动脉根部4h、开放2-24h复制肢体I-R模型。反转录-多聚酶链反应检测肾内HO-1mRNA表达的变化;免疫组化染色法标记HO-1蛋白的组织分布;用锌原卟啉抑制HO-1活性后,光镜观察肾组织的病理变化。结果:肢体I-R后肾组织HO-1mRNA表达水平明显高于各对照组,再灌注18h表达至峰值,再灌至24h仍显著高于各对照组(P＜0.01);肢体I-R组近曲小管、髓袢粗段小管上皮细胞内出现密集的颗粒状HO-1免疫阳性产物;抑制HO-1活性使肢体I-R所引发的肾组织损伤明显加重。结论:肢体I-R后肾小管上皮细胞HO-1基因表达上调,所诱生的HO-1对肾组织具有保护效应。     

Controlled comparison of original vented aerobic fan medium with new nonvented BacT/ALERT FA medium for culturing blood

To evaluate the performance of BacT/ALERT FA (FA) medium, a new aerobic BacT/ALERT FAN (FAN) medium (Organon Teknika Corporation, Durham, N.C.) that does not require the added cost and inconvenience of a venting unit, we inoculated blood specimens from adult patients with suspected sepsis into an original FAN aerobic culture bottle and an FA bottle. Of 7,745 blood culture sets containing both bottles, 5,256 (68%) met the criteria for adequacy of filling. A total of 466 isolates judged to represent the causes of true infections were recovered from 276 patients; 271 isolates were recovered from both bottles, 82 were recovered from the FAN bottle only, and 113 were recovered from the FA bottle only (P < 0.05). More Burkholderia cepacia isolates (P < 0.01), Candida albicans isolates (P < 0.001), Cryptococcus neoformans isolates (P < 0.01), yeasts overall (P < 0.001), and total microorganisms (P < 0.05) were recovered from FA bottles. Of cultures found to be positive within the first 72 h of incubation, the mean times to detection were almost identical for FAN (20.4 h) and FA (20.7 h) bottles. Of 263 isolates that caused monomicrobic episodes of bloodstream infections, 180 were detected in both bottles, 32 were detected in FAN bottles only, and 51 were detected in FA bottles only (P < 0.05). Of 186 isolates considered to be contaminants, 63 were detected in both media, 64 were detected in FAN bottles only, and 59 were detected in FA bottles only (P was not significant). The number of false-positive results were comparable: 69 (1.3%) in FAN bottles and 56 (1.1%) in FA bottles. However, there were 14 isolates with false-negative results (6 yeasts, 6 nonfermenters, and 1 isolate each of Propionibacterium acnes and coagulase-negative staphylococci) in FAN bottles, whereas there were none in FA bottles. On the basis of these results, we conclude that the new nonvented FA bottle is superior to the original vented FAN medium for the recovery of B. cepacia and yeasts, especially C. albicans and C. neoformans, and is comparable to FAN medium for other microorganisms.     

TRPM2 channel protective properties of N-acetylcysteine on cytosolic glutathione depletion dependent oxidative stress and Ca2+ influx in rat dorsal root ganglion

N-acetylcysteine (NAC) is a thiol-containing (sulphydryl donor) antioxidant, which contributes to regeneration of glutathione (GSH) and also acts through a direct reaction with free radicals. Thiol depletion has been implicated in the neurobiology of sensory neurons and pain. We reported recently an activator role of intracellular GSH depletion on calcium influx through transient receptor potential melastatin-like 2 (TRPM2) channels in rat dorsal root ganglion (DRG). NAC may have a protective role on calcium influx through regulation of TRPM2 channels in the neurons. Therefore, we tested the effects of NAC on TRPM2 channel currents in cytosolic GSH depleted DRG in rats. DRG neurons were freshly isolated from rats and the neurons were incubated for 24 h with buthionine sulfoximine (BSO). In whole-cell patch clamp experiments, TRPM2 currents in the DRG incubated with BSO were gated by H(2)O(2). TRPM2 channels current densities, cytosolic free Ca(2+) content, and lipid peroxidation values in the neurons were higher in H(2)O(2) and BSO + H(2)O(2) group than in controls; however GSH and GSH peroxidase (GSH-Px) values were decreased. BSO + H(2)O(2)-induced TRPM2 channel gating was totally inhibited by extracellular NAC and partially inhibited by 2-aminoethyl diphenylborinate. GSH-Px activity, lipid peroxidation and GSH levels in the DRG neurons were also modulated by NAC. In conclusion, we observed a modulator role of NAC on Ca(2+) influx through a TRPM2 channel in intracellular GSH depleted DRG neurons. NAC incubation before BSO exposure appears to be more protective than NAC incubation after BSO exposure. Since cytosolic thiol group depletion is a common feature of neuropathic pain, our findings are relevant to the etiology and treatment of pain neuropathology in DRG neurons.     

肾缺血再灌注大鼠蛋白尿的生成及NO的作用

目的:探讨肾缺血再灌注(I-R)损伤时排出尿蛋白的量和质的变化以及一氧化氮(NO)在其中的作用。方法:在肾I-R大鼠模型上,用考马斯亮蓝比色法测定尿蛋白含量,SDS-PAGE分析尿液中蛋白种类的改变,硝酸还原酶法测定肾组织NO含量。并观察硝普钠(SNP)、L-硝基精氨酸(L-NNA)和氨基胍(AG)对尿蛋白的影响。结果:肾I-R损伤导致大鼠产生明显蛋白尿(P＜0.01),而且出现了分子量大于白蛋白(66kD)的蛋白质,肾I-R后肾组织的NO的增多与尿蛋白排出量密切相关(r=0.704,P＜0.01),SNP使I-R大鼠尿蛋白排出增多,AG、L-NNA使I-R大鼠尿蛋白排出减少。结论:肾I-R导致肾小球滤过膜通透性增加及蛋白尿产生,NO是蛋白尿形成的重要因素之一。     

Male accessory sex gland secretions affect oocyte Ca2+ oscillations during in-vitro fertilization in golden hamsters.

To evaluate the effect of male accessory sex gland secretions on Ca2+ oscillations of oocytes, epididymal or ejaculated spermatozoa recovered from uteri were used to inseminate oocytes. Ca2+ oscillations were measured by Fura 2 fluorescence imaging (F340/F380). We showed that although Ca2+ oscillations induced by ejaculated spermatozoa had a pattern similar to those induced by epididymal spermatozoa, the amplitude of the first Ca2+ transient in the former group was significantly higher (P < 0.05) and the duration was significantly longer (P < 0.01). Oocytes inseminated with ejaculated spermatozoa recovered from uteri from males had ampullary glands or ventral prostates removed showed significantly lower Ca2+ oscillations compared to the controls (P < 0.05, P < 0.01 respectively). Moreover, the relative area of the first Ca2+ transient in treatment groups was significantly smaller than the control. In addition, a significantly higher percentage of oocytes (52%) inseminated by spermatozoa from males with all accessory sex glands removed showed non-oscillatory Ca2+ transients, compared to the controls (5%, P < 0.05). These results indicate that accessory sex gland secretions can affect Ca2+ oscillations. The differences between Ca2+ oscillations induced by epididymal and uterine spermatozoa from males with all accessory sex glands removed suggest that uterine factors may also influence this process.