BACKGROUND & AIMS: :D-Amino acid oxidase (DAAO), a FAD-dependent peroxisomal flavoenzyme that catalyzes oxidation of D-amino acids to hydrogen peroxide, is distributed in the spinal cord almost exclusively expressed within astrocytes. The present study aims to explore potential contributions of spinal DAAO to the development of bone cancer pain and morphine tolerance to analgesia. Tibia inoculation of carcinoma cells produced mechanical allodynia (but not heat hyperalgesia), in synchronous with induction of DAAO expression and DAAO enzymatic activity, as well as activation of spinal astrocytes marked by GFAP. Subcutaneous and intrathecal injection of the specific DAAO inhibitor CBIO (5-chloro-benzo[d]isoxazol-3-ol) blocked mechanical allodynia in a dose- and time-dependent manner in tumor-bearing rats, with maximum inhibition of 40-50 %. Multi-daily intrathecal injections of the DAAO gene silencer siRNA/DAAO also yielded anti-allodynic effects by approximately 40 % and the analgesia remained for at least 6 days. Subcutaneous injection of CBIO suppressed the production of spinal hydrogen peroxide and GFAP expression. 7-Day multiple bi-daily injections of CBIO produced anti-allodynia without inducing self-tolerance to analgesia or cross-tolerance to morphine, and concurrent injections of CBIO with morphine produced apparent additive anti-allodynia and completely prevented morphine tolerance in behaviors and spinal expression of μ-opioid receptors. Our results provide the first evidence that spinal DAAO contributes to the development of morphine tolerance to analgesia and bone cancer pain accounting for 40-50 % pain status, probably via production of hydrogen peroxide leading to activation of astrocytes. The unique characterizations of DAAO inhibitors make them a potential for the treatment of cancer pain when they are administered alone or in combination with morphine.

背景与目标： : D-氨基酸氧化酶 (DAAO) 是一种FAD依赖性过氧化物酶体黄素酶，可催化D-氨基酸氧化为过氧化氢，分布在脊髓中，几乎仅在星形胶质细胞内表达。本研究旨在探讨脊髓DAAO对骨癌疼痛和吗啡镇痛耐受性的潜在贡献。与DAAO表达和DAAO酶活性的诱导以及GFAP标记的脊髓星形胶质细胞的激活同步，胫骨接种癌细胞会产生机械性异常性疼痛 (但不会产生热痛觉过敏)。皮下和鞘内注射特定的DAAO抑制剂CBIO (5-氯-苯并 [d]isoxazol-3-ol) 以剂量和时间依赖性方式阻断荷瘤大鼠的机械性异常性疼痛，最大抑制作用为40-50%。DAAO基因沉默子siRNA/DAAO的多天鞘内注射也产生了约40% 的抗异常性作用，镇痛作用保持至少6天。皮下注射CBIO抑制了脊髓过氧化氢的产生和GFAP的表达。7天多次双日注射CBIO可产生抗异常性疼痛，而不会引起对镇痛的自我耐受性或对吗啡的交叉耐受性，同时注射CBIO与吗啡可产生明显的累加性抗异常性疼痛，并完全阻止行为和行为中的吗啡耐受性。μ 阿片受体的脊髓表达。我们的结果提供了第一个证据，表明脊髓DAAO有助于吗啡对镇痛和骨癌疼痛的耐受性的发展，这可能是通过产生过氧化氢导致星形胶质细胞活化而导致的40-50% 疼痛状态。DAAO抑制剂的独特特征使其成为单独或与吗啡联合使用时治疗癌症疼痛的潜力。

BACKGROUND & AIMS: :Although hyperuricemia has been shown to be associated with the progression of cardiovascular disorder and chronic kidney disease (CKD), there is conflicting evidence as to whether xanthine oxidase (XO) inhibitors confer organ protection besides lowering serum urate levels. In this study, we addressed the cardio-renal effects of XO inhibition in rodent CKD model with hyperuricemia. Sprague-Dawley rats underwent 5/6 nephrectomy and received a uricase inhibitor oxonic acid for 8 weeks (RK + HUA rats). In some rats, a XO inhibitor febuxostat was administered orally. Compared with control group, RK + HUA group showed a significant increase in albuminuria and renal injury. Febuxostat reduced serum uric acid as well as urinary albumin levels. Histological and immunohistochemical analysis of the kidney revealed that febuxostat alleviated glomerular, tubulointerstitial, and arteriolar injury in RK + HUA rats. Moreover, in the heart, RK + HUA showed individual myofiber hypertrophy and cardiac fibrosis, which was significantly attenuated by febuxostat. We found that renal injury and the indices of cardiac changes were well correlated, confirming the cardio-renal interaction in this model. Finally, NF-E2-related factor 2 (Nrf2) and the downstream target heme oxygenase-1 (HO-1) protein levels were increased both in the heart and in the kidney in RK + HUA rats, and these changes were alleviated by febuxostat, suggesting that tissue oxidative stress burden was attenuated by the treatment. These data demonstrate that febuxostat protects against cardiac and renal injury in RK + HUA rats, and underscore the pathological importance of XO in the cardio-renal interaction.

背景与目标： : 尽管高尿酸血症已被证明与心血管疾病和慢性肾脏疾病 (CKD) 的进展有关，但关于黄嘌呤氧化酶 (XO) 抑制剂是否除了降低血清尿酸水平外还具有器官保护作用，存在相互矛盾的证据。在这项研究中，我们讨论了XO抑制在患有高尿酸血症的啮齿动物CKD模型中的心肺作用。Sprague-Dawley大鼠接受5/6肾切除术，并接受尿酸酶抑制剂oxonic酸治疗8周 (rk   +  HUA大鼠)。在某些大鼠中，口服XO抑制剂非布索坦。与对照组相比，rk   +  HUA组蛋白尿和肾损伤明显增加。非布索坦可降低血清尿酸和尿白蛋白水平。肾脏的组织学和免疫组织化学分析表明，非布司他减轻了rk   hua hua大鼠的肾小球，肾小管间质和小动脉损伤。此外，在心脏中，rk   +  HUA表现为单个肌纤维肥大和心脏纤维化，非布沙坦明显减弱。我们发现肾损伤与心脏变化的指标具有良好的相关性，从而证实了该模型中的心-肾相互作用。最终，NF-E2-related因子2 (Nrf2) 和下游靶血红素oxygenase-1 (HO-1) 蛋白在rk   +  HUA大鼠心脏和肾脏中的水平均升高，非布沙坦缓解了这些变化，提示该治疗减轻了组织氧化应激负荷。这些数据表明，非布索坦可预防rk   hua hua大鼠的心脏和肾脏损伤，并强调了XO在心肺相互作用中的病理重要性。

BACKGROUND & AIMS: :We studied whether angiotensin II (ANG II) via superoxide may contribute to retinal leukostasis and thus to the pathogenesis of retinopathies. We studied: 1) whether intravitreal ANG II induces retinal leukostasis that is altered by antioxidants or by apocynin, a NAD(P)H oxidase inhibitor and 2) whether retinal leukostasis induced by diabetes in rats is also altered by these treatments. Rats were injected intravitreally with ANG II (20 microg in 2 microl), and divided into the following three groups: 1) untreated; 2) treated with tempol doses ( approximately 3 mM/day) and N-acetylcysteine (NAC; approximately 1 g.kg(-1).day(-1)); and 3) treated with apocynin ( approximately 2 mM/day), both in the drinking water. Rats with streptozotocin-induced diabetes were similarly treated. Leukostasis was evaluated 48 h after ANG II or 2 wk after diabetes induction. ANG II increased retinal leukostasis from 0.3 +/- 0.5 to 3.7 +/- 0.4 leukocytes/ mm(2) (P < 0.01), and these changes were markedly decreased by treatment with tempol + NAC or apocynin, and also by a blocking antibody against vascular endothelial growth factor given intravitreally (P < 0.01). In addition, incubation of dihydroethidium-loaded retina sections with ANG II caused marked increase in superoxide formation. Compared with normal controls, retinal leukostasis in diabetic rats markedly increased from 0.2 +/- 0.3 to 3.8 +/- 0.1 leukocytes/mm(2) (P < 0.01). Diabetic retinal leukostasis was also decreased by treatment with tempol-NAC and normalized by apocynin. Thus increases in intravitreal ANG II can induce retinal leukostasis, which appears to be mediated via increasing superoxide generation by NAD(P)H oxidase, and by VEGF. The activity of NAD(P)H oxidase is required for leukostasis to occur in the diabetic retina.

背景与目标： : 我们研究了通过超氧化物引起的血管紧张素II (ANG II) 是否可能导致视网膜白细胞平衡，从而导致视网膜病变的发病机理。我们研究了: 1) 玻璃体内ANG II是否诱导了由抗氧化剂或apocynin (一种NAD(P)H氧化酶抑制剂) 改变的视网膜白细胞平衡，以及2) 这些治疗是否也改变了糖尿病引起的大鼠视网膜白细胞平衡。大鼠玻璃体内注射ANG II (2 microl中的20 microg)，并分为以下三组: 1) 未经治疗; 2) 用tempol剂量 (约3毫米/天) 和N-乙酰半胱氨酸 (NAC) 处理; 约1g.kg(-1) 天 (-1)); 和3) 用apocynin (约2毫米/天) 处理，两者均在饮用水中。对链脲佐菌素诱导的糖尿病大鼠进行了类似的治疗。在ANG II后48小时或糖尿病诱导后2周评估白细胞平衡。ANG II使视网膜白细胞从0.3 +/- 0.5增加到3.7 +/- 0.4白细胞/mm(2) (P <0.01)，这些变化通过tempol + NAC或apocynin治疗明显减少，并且还通过抗血管内皮生长因子的阻断抗体给予玻璃体内注射 (P <0.01)。此外，将载有二氢乙锭的视网膜切片与ANG II孵育会导致超氧化物形成显着增加。与正常对照组相比，糖尿病大鼠的视网膜白细胞平衡从0.2/0.3显着增加到3.8/0.1/mm(2) (P <0.01)。用tempol-NAC治疗也可以减少糖尿病性视网膜白细胞平衡，并通过apocynin恢复正常。因此，玻璃体内ANG II的增加可以诱导视网膜白细胞平衡，这似乎是通过NAD(P)H氧化酶和VEGF增加超氧化物生成来介导的。NAD(P)H氧化酶的活性是糖尿病视网膜中发生白细胞平衡所必需的。

BACKGROUND & AIMS: :Until the 1970s, reactive oxygen species (ROS) were considered merely harmful by-products of aerobic respiration and the driving force behind the evolution of an array of cellular antioxidant enzymes with the purpose of rapidly metabolising ROS to minimise their oxidising effects. However, the perception that ROS are only harmful to cells has since been questioned by a burgeoning body of evidence pointing to the existence of enzymes with the dedicated function of generating ROS. NADPH oxidases represent the only known family of enzymes whose sole purpose is to generate ROS. Members of this enzyme family are expressed across mammalian and non-mammalian cells, and influence a multitude of biological functions including host defence and redox signalling. However, although ROS are deliberately generated by NADPH oxidases during normal cell physiology, the observations that their expression and activity is markedly upregulated in the blood vessel wall in a number of cardiovascular 'high-risk' states (e.g. hypertension, hypercholesterolemia) implicates them in the oxidative stress that gives rise to artery disease and ultimately heart attacks and strokes. These observations highlight the fact that NADPH oxidases are important therapeutic targets in cardiovascular disease and that, hence, there is clearly a need for the development of selective inhibitors of these enzymes. Here we highlight the structural and biochemical characteristics of the NADPH oxidase family and then comprehensively review the literature on the currently available pharmacological inhibitors of these enzymes with a particular emphasis on their mechanisms of action, isoform selectivity and therapeutic potential in cardiovascular disease.

背景与目标： : 在20世纪70年代之前，活性氧 (ROS) 仅被认为是有氧呼吸的有害副产物，也是一系列细胞抗氧化酶进化的驱动力，目的是快速代谢ROS以最大程度地减少其氧化作用。然而，自那以后，越来越多的证据表明存在具有生成ROS的专用功能的酶，从而质疑ROS仅对细胞有害的看法。NADPH氧化酶代表唯一已知的酶家族，其唯一目的是产生ROS。该酶家族的成员在哺乳动物和非哺乳动物细胞中表达，并影响多种生物学功能，包括宿主防御和氧化还原信号传导。然而，尽管ROS是在正常细胞生理过程中由NADPH氧化酶故意产生的，但在许多心血管 “高风险” 状态 (例如高血压，高胆固醇血症) 使它们参与氧化应激，从而导致动脉疾病并最终导致心脏病发作和中风。这些观察结果强调了一个事实，即NADPH氧化酶是心血管疾病的重要治疗靶标，因此，显然需要开发这些酶的选择性抑制剂。在这里，我们重点介绍了NADPH氧化酶家族的结构和生化特征，然后全面回顾了有关这些酶的当前可用药理抑制剂的文献，特别强调了它们的作用机制，同工型选择性和在心血管疾病中的治疗潜力。

BACKGROUND & AIMS: :In cytochrome c oxidase, oxido-reductions of heme a/Cu(A) and heme a3/Cu(B) are cooperatively linked to proton transfer at acid/base groups in the enzyme. H+/e- cooperative linkage at Fe(a3)/Cu(B) is envisaged to be involved in proton pump mechanisms confined to the binuclear center. Models have also been proposed which involve a role in proton pumping of cooperative H+/e- linkage at heme a (and Cu(A)). Observations will be presented on: (i) proton consumption in the reduction of molecular oxygen to H2O in soluble bovine heart cytochrome c oxidase; (ii) proton release/uptake associated with anaerobic oxidation/reduction of heme a/Cu(A) and heme a3/Cu(B) in the soluble oxidase; (iii) H+ release in the external phase (i.e. H+ pumping) associated with the oxidative (R-->O transition), reductive (O-->R transition) and a full catalytic cycle (R-->O-->R transition) of membrane-reconstituted cytochrome c oxidase. A model is presented in which cooperative H+/e- linkage at heme a/Cu(A) and heme a3/Cu(B) with acid/base clusters, C1 and C2 respectively, and protonmotive steps of the reduction of O2 to water are involved in proton pumping.

背景与目标： : 在细胞色素c氧化酶中，血红素a/Cu(A) 和血红素a3/Cu(B) 的氧化还原与酶中酸/碱基团的质子转移协同连接。预计Fe(a3)/Cu(B) 处的H/e协同连接涉及限于双核中心的质子泵机制。还提出了一些模型，这些模型涉及在血红素a (和Cu (a)) 的协同H/e键的质子泵送中的作用。将提出以下观察结果 :( i) 将可溶性牛心脏细胞色素c氧化酶中的分子氧还原为H2O的质子消耗; (ii) 与可溶性氧化酶中血红素a/Cu(A) 和血红素a3/Cu(B) 的厌氧氧化/还原相关的质子释放/吸收; (iii) 与氧化 (R->O转变) 相关的外部相 (即H泵) 中的H释放，膜重建的细胞色素c氧化酶的还原 (O->R跃迁) 和全催化循环 (R->O->R跃迁)。提出了一个模型，其中血红素A/Cu (a) 和血红素a3/Cu(B) 分别具有酸/碱簇C1和C2的协同H/e键，以及将O2还原为水的质子运动步骤参与质子泵送。

BACKGROUND & AIMS: BACKGROUND:Nitric oxide (NO) is an important signal molecule in many types of cells and tissues. Efficiently balanced NO production was noted to play an important role in the healing of burns. However, the exact pathophysiological role of NO in burns and its potent relation with clinical and laboratory parameters has not been elucidated. METHODS:A cohort of 23 burn patients followed for 5 days were enrolled. NO, antioxidant capacity (AC), NO synthase (NOS) activity and xanthine oxidase (XO) activity were indirectly determined by fluorophotometer. Multiple regression against total burn surface area (TBSA), age, weight, height, proximity of septic episode, hemoglobin, white blood cells, percent of neutrophils, platelets, glucose, urea, potassium, sodium and albumin was performed. RESULTS:Elevation of NO, XO and AC levels is observed from day 2 (p<0.00001), day 4 (p=0.005) and day 6 (p=0.036), respectively. At the end of follow-up period (day 6), NO production was found to independently correlate with TBSA, glucose levels and percent of neutrophils (p=0.0004), AC with age, hemoglobin and glucose levels (p=0.012), and NOS with proximity of septic episode and glucose levels (p=0.027). CONCLUSIONS:NO production exerts its prophylactic effect from the first 24h after burn, and is independently correlated with severe injury, enhanced neutrophil motivation and augmented glucose levels, thus possibly representing a response to stress. This need might trigger induction of XO and salvage of antioxidants, as suggested by their rise at a later stage. These data underline that an effort to compromise stress and to administer antioxidants could be a priority in the treatment of these patients.

背景与目标：

BACKGROUND & AIMS: :The production of reactive oxygen species (ROS) by neutrophils has a vital role in defence against a range of infectious agents, and is driven by the assembly of a multi-protein complex containing a minimal core of five proteins: the two membrane-bound subunits of cytochrome b(558) (gp91(phox) and p22(phox)) and three soluble factors (GTP-Rac, p47(phox) and p67(phox) (refs 1, 2). This minimal complex can reconstitute ROS formation in vitro in the presence of non-physiological amphiphiles such as SDS. p40(phox) has subsequently been discovered as a binding partner for p67(phox) (ref. 3), but its role in ROS formation is unclear. Phosphoinositide-3-OH kinases (PI(3)Ks) have been implicated in the intracellular signalling pathways coordinating ROS formation but through an unknown mechanism. We show that the addition of p40(phox) to the minimal core complex allows a lipid product of PI(3)Ks, phosphatidylinositol 3-phosphate (PtdIns(3)P), to stimulate specifically the formation of ROS. This effect was mediated by binding of PtdIns(3)P to the PX domain of p40(phox). These results offer new insights into the roles for PI(3)Ks and p40(phox) in ROS formation and define a cellular ligand for the orphan PX domain.

背景与目标： : 嗜中性粒细胞产生活性氧 (ROS) 在防御一系列感染因子方面具有至关重要的作用，并且是由包含五种蛋白质的最小核心的多蛋白复合物的组装驱动的: 细胞色素b的两个膜结合亚基 (558) (gp91(phox) 和p22(phox)) 和三个可溶性因子 (gtp-rac，p47(phox) 和p67(phox) (参考文献1，2)。这种最小复合物可以在非生理两亲物如SDS的存在下在体外重建ROS的形成。p40(phox) 随后被发现作为p67(phox) 的结合伴侣 (参考文献3)，但是它在ROS形成中的作用尚不清楚。Phosphoinositide-3-OH激酶 (PI(3)Ks) 已与协调ROS形成的细胞内信号通路有关，但通过未知的机制。我们表明，将p40(phox) 添加到最小核心复合物中可以使PI(3)Ks的脂质产物,磷脂酰肌醇3-磷酸 (PtdIns(3)P)，具体地刺激ROS的形成。这种作用是通过PtdIns(3)P与p40(phox) 的PX结构域的结合来介导的。这些结果为PI(3)Ks和p40(phox) 在ROS形成中的作用提供了新的见解，并为孤儿定义了细胞配体PX域。

BACKGROUND & AIMS: :The anthracycline anticancer agents daunorubicin (DAUN) and doxorubicin (DOX) are reduced by different NADPH-dependent cytosolic reductases into their corresponding alcohol metabolites daunorubicinol (DAUNol) and doxorubicinol (DOXol), which have been implicated in the development of chronic cardiomyopathy. To better understand the individual importance of each enzyme in the reduction and to provide deeper insight into the binding at atomic level we performed molecular docking and dynamics simulations of DAUN and DOX into the active sites of human carbonyl reductase 1 (CBR1) and human aldehyde reductase (AKR1A1). Such simulations evidenced a different behavior between the reductases with respect to DAUN and DOX suggesting major contribution of CBR1 in the reduction. The results are in agreement with available experimental data and for each enzyme and anthracycline pair provided the identification of key residues involved in the interactions. The structural models that we have derived could serve as a useful tool for structure-guided drug design studies.

背景与目标： : 蒽环类抗癌剂柔红霉素 (DAUN) 和阿霉素 (DOX) 被不同的NADPH依赖性胞质还原酶还原成其相应的醇代谢产物柔红霉素 (DAUNol) 和阿霉素 (DOXol)，这与慢性心肌病的发展有关。为了更好地了解每种酶在还原中的个体重要性并提供对原子水平结合的更深入的了解，我们进行了DAUN和DOX到人羰基还原酶1 (CBR1) 和人的活性位点的分子对接和动力学模拟醛还原酶 (AKR1A1)。这样的模拟证明了还原酶之间相对于DAUN和DOX的不同行为，表明CBR1在还原中的主要贡献。结果与可用的实验数据一致，并且对于每种酶和蒽环类药物对，都提供了相互作用中涉及的关键残基的鉴定。我们得出的结构模型可以作为结构指导药物设计研究的有用工具。

BACKGROUND & AIMS: :Endoplasmic reticulum (ER) thiol oxidases initiate a disulfide relay to oxidatively fold secreted proteins. We found that combined loss-of-function mutations in genes encoding the ER thiol oxidases ERO1α, ERO1β, and PRDX4 compromised the extracellular matrix in mice and interfered with the intracellular maturation of procollagen. These severe abnormalities were associated with an unexpectedly modest delay in disulfide bond formation in secreted proteins but a profound, 5-fold lower procollagen 4-hydroxyproline content and enhanced cysteinyl sulfenic acid modification of ER proteins. Tissue ascorbic acid content was lower in mutant mice, and ascorbic acid supplementation improved procollagen maturation and lowered sulfenic acid content in vivo. In vitro, the presence of a sulfenic acid donor accelerated the oxidative inactivation of ascorbate by an H(2)O(2)-generating system. Compromised ER disulfide relay thus exposes protein thiols to competing oxidation to sulfenic acid, resulting in depletion of ascorbic acid, impaired procollagen proline 4-hydroxylation, and a noncanonical form of scurvy.

背景与目标： : 内质网 (ER) 硫醇氧化酶启动二硫键中继氧化折叠分泌的蛋白质。我们发现，编码ER硫醇氧化酶ERO1α，ERO1β 和PRDX4的基因的联合功能丧失突变损害了小鼠的细胞外基质，并干扰了前胶原的细胞内成熟。这些严重的异常与分泌蛋白中二硫键形成的意外适度延迟有关，但前胶原4-羟基脯氨酸含量大大降低了5倍，并且ER蛋白的半胱氨酰亚砜酸修饰增强。突变小鼠的组织抗坏血酸含量较低，补充抗坏血酸可改善胶原蛋白的成熟并降低体内的亚砜酸含量。在体外，亚磺酸供体的存在加速了H(2)O(2) 生成系统对抗坏血酸的氧化失活。因此，受损的ER二硫键中继使蛋白质硫醇暴露于竞争氧化为亚磺酸，导致抗坏血酸耗竭，前胶原脯氨酸4-羟基化受损以及坏血病的非规范形式。

BACKGROUND & AIMS: :Although primary care clinicians have developed considerable expertise in managing patients with major depressive disorder, and a range of treatment strategies is currently available, some patients still fail to reach remission. Two strategies have fallen out of common use: treating patients with monoamine oxidase inhibitors (MAOIs) and subgrouping patients by diagnosis when selecting antidepressant treatment. Monoamine oxidase inhibitors became less popular because other treatments were perceived to be safer and easier to use. However, a newer transdermal formulation of an MAOI that limits the need for the dietary restrictions of oral MAOIs may make it worthwhile to consider using this class of medication in patients who have failed several treatment trials. Although adverse events due to patients' diets are less likely with the transdermal MAOI, clinicians should still be alert for drug interactions and observe recommended washout periods. Patients who may benefit from MAOI treatment include those with treatment-resistant depression, atypical depression, anxiety, or anergic bipolar depression and those who have experienced intolerable metabolic or sexual side effects with other medications.

背景与目标： : 尽管初级保健临床医生在管理重度抑郁症患者方面已经发展了相当多的专业知识，并且目前有一系列治疗策略，但一些患者仍然无法达到缓解。两种策略已不再常用: 用单胺氧化酶抑制剂 (MAOIs) 治疗患者，并在选择抗抑郁药治疗时通过诊断对患者进行分组。单胺氧化酶抑制剂变得不那么受欢迎，因为其他治疗被认为更安全，更容易使用。但是，较新的MAOI透皮制剂限制了口服MAOI的饮食限制，可能使考虑在几项治疗试验失败的患者中使用此类药物是值得的。尽管透皮MAOI不太可能因患者饮食引起的不良事件，但临床医生仍应警惕药物相互作用并观察建议的冲洗期。可能从MAOI治疗中受益的患者包括患有难治性抑郁症，非典型抑郁症，焦虑或无性双相抑郁症的患者，以及患有其他药物无法忍受的代谢或性副作用的患者。

BACKGROUND & AIMS: :Glucose oxidase (GOD) is an oxidoreductase catalyzing the reaction of glucose and oxygen to peroxide and gluconolacton (EC 1.1.3.4.). GOD is a widely used enzyme in biotechnology. Therefore the production of monoclonal antibodies and antibody fragments to GOD are of interest in bioanalytics and even tumor therapy. We describe here the generation of a panel of monoclonal antibodies to native and heat inactivated GOD. One of the hybridomas, E13BC8, was used for cloning of a single chain antibody (scFv). This scFv was expressed in Escherichia coli XL1-blue with the help of the vector system pOPE101. The scFv was isolated from the periplasmic fraction and detected by western blotting. It reacts specifically with soluble active GOD but does not recognize denatured GOD adsorbed to the solid phase. The same binding properties were also found for the monoclonal antibody E13BC8.

背景与目标： : 葡萄糖氧化酶 (GOD) 是一种氧化还原酶，可催化葡萄糖和氧气与过氧化物和葡萄糖醇的反应 (EC 1.1.3.4。)。上帝是生物技术中广泛使用的酶。因此，向上帝生产单克隆抗体和抗体片段在生物分析甚至肿瘤治疗中都很有意义。我们在这里描述了针对天然和热灭活上帝的一组单克隆抗体的产生。杂交瘤之一E13BC8用于单链抗体 (scFv) 的克隆。该scFv在载体系统pope101的帮助下在大肠杆菌XL1-blue中表达。从周质部分中分离出scFv，并通过蛋白质印迹法检测。它与可溶性活性神特别反应，但不识别吸附在固相上的变性神。单克隆抗体E13BC8也发现了相同的结合特性。

BACKGROUND & AIMS: :We have shown previously that rats subjected to tourniquet shock develop an acute form of remote organ injury of the liver that is both Kupffer cell (KC) and polymorphonuclear (PMN) leukocyte dependent. Circulating plasma xanthine oxidase (XO) has been shown to be responsible for the development of endothelial dysfunction and for remote organ injury of the lung and intestine after ischemia-reperfusion protocols. We now hypothesize that XO is released from rat hind limbs upon reperfusion and that it is responsible for KC and PMN leukocyte activation in this shock model. Our results show that about 30% of rat gastrocnemius muscle xanthine dehydrogenase (XD) is converted to XO during the 5-h tourniquet period and that it is released into the femoral vein within 10 min of reperfusion. Total muscle xanthine oxidoreductase activity (XO + XD) decreases within 30 min of reperfusion and is paralleled by a corresponding increase in femoral vein lactic dehydrogenase. In addition, liver tissue XO increases significantly within 30 min of reperfusion without a corresponding conversion of endogenous XD. Conversion of hepatic XD becomes evident 60 min after reperfusion is initiated, as does XO, and alanine aminotransferase (ALT) release into the hepatic vein, presumably from damaged hepatocytes as a consequence of oxidative stress. Tissue myeloperoxidase activity also increases significantly after the 60-min reperfusion period. That XO mediates KC and PMN activation is supported by the following observations: a) the close relationships between plasma XO and the time courses of tumor necrosis factor-alpha TNFalpha release into the hepatic vein and colloidal carbon clearance by KCs; b) that colloidal carbon clearance, TNFalpha and ALT release, loss of tissue free thiols, lipid peroxidation (TBARS), and liver infiltration by PMN neutrophils can also be induced by the administration of exogenous XO to normal rats; and c) pretreatment of rats with allopurinol inhibits KC activation and liver leukocyte infiltration. These results suggest that XO, released from the ischemic limb on reperfusion, is taken up by the liver were it mediates KC and PMN neutrophil activation and thus contributes to the development of multiple system organ failure after hind limb reperfusion.

背景与目标： : 我们先前已经表明，遭受止血带休克的大鼠会发生急性形式的肝脏远程器官损伤，该损伤既是Kupffer细胞 (KC) 又是多形核 (PMN) 白细胞依赖性。循环血浆黄嘌呤氧化酶 (XO) 已被证明是导致内皮功能障碍的发生以及缺血再灌注方案后肺和肠的远程器官损伤的原因。现在，我们假设XO在再灌注后从大鼠后肢释放，并且在该休克模型中，XO负责KC和PMN白细胞的激活。我们的结果表明，在5小时止血带期间，约30% 的大鼠腓肠肌黄嘌呤脱氢酶 (XD) 转化为XO，并在再灌注后10分钟内释放到股静脉。总肌肉黄嘌呤氧化还原酶活性 (XO XD) 在再灌注后30分钟内降低，并与股静脉乳酸脱氢酶的相应增加平行。此外，肝组织XO在再灌注后30分钟内显着增加，而内源性XD没有相应的转化。再灌注开始后60分钟肝XD的转化变得明显，XO也是如此，丙氨酸氨基转移酶 (ALT) 释放到肝静脉中，可能是由于氧化应激而受损的肝细胞。再灌注60分钟后，组织髓过氧化物酶活性也显着增加。以下观察结果支持XO介导KC和PMN激活: a) 血浆XO与肿瘤坏死因子-α TNFalpha释放到肝静脉和KCs的胶体碳清除的时间过程之间的密切关系; b) 胶体碳清除，TNFalpha和ALT释放，向正常大鼠施用外源性XO也可以诱导组织游离硫醇的丢失，脂质过氧化 (TBARS) 和PMN中性粒细胞的肝脏浸润; c) 用别嘌醇预处理大鼠可抑制KC活化和肝白细胞浸润。这些结果表明，XO在再灌注时从缺血肢体释放，通过介导KC和PMN中性粒细胞激活而被肝脏吸收，从而有助于后肢再灌注后多系统器官衰竭的发展。

BACKGROUND & AIMS: :R-(-)-Deprenyl (deprenyl, selegiline), a monoamine oxidase B (MAO-B) inhibitor, delays progression of Parkinson's disease. This action could be mediated by inhibition of MAO-B but there may also be unrelated mechanisms. Direct neuroprotective and antiapoptotic actions of deprenyl have previously been observed in vitro. Here we describe an antineurotoxic action of deprenyl which is independent of direct neuronal effects. We employed a previously described assay in which human neuroblastoma SH-SY5Y cells are exposed to cell-free supernatants of stimulated human monocytic THP-1 cells. Deprenyl reduced the secretion of neurotoxic products by such stimulated cells in a concentration-dependent manner, while the MAO inhibitors iproniazid, isocarboxazid, nialamide, tranylcypromine, phenelzine, and clorgyline were without effect. No antineurotoxic action was observed when deprenyl was added directly to SH-SY5Y cells. Messenger RNAs for MAO-A and MAO-B were not detected in THP-1 cells by reverse transcriptase-polymerase chain reaction analysis of total RNA extracts. Such mRNAs were easily detected in extracts of SH-SY5Y cells under comparable conditions. MAO enzymatic activity was also undetectable in THP-1 cell lysates, while it was readily observed in SH-SY5Y cells. It was concluded that the effect of deprenyl on THP-1 cells was not mediated by MAO and that deprenyl itself was not protecting neurons. These data suggest that deprenyl may have utility in neurodegenerative diseases due to its antineurotoxic actions.

背景与目标： : R-(-)-异戊二烯基 (Deprenyl，司来吉兰)，一种单胺氧化酶B (mao-b) 抑制剂，可延缓帕金森氏病的进展。这种作用可以通过抑制mao-b来介导，但也可能存在不相关的机制。先前已在体外观察到异戊二烯基的直接神经保护和抗凋亡作用。在这里，我们描述了异戊二烯基的抗神经毒性作用，该作用与直接神经元作用无关。我们采用了先前描述的测定，其中人神经母细胞瘤SH-SY5Y细胞暴露于受刺激的人单核THP-1细胞的无细胞上清液。异戊二烯基以浓度依赖性方式减少了此类刺激细胞对神经毒性产物的分泌，而MAO抑制剂异丙嗪，异卡索嗪，烟酰胺，反苯环丙胺，苯乙嗪和氯代林则无效。当将异戊二烯基直接添加到SH-SY5Y细胞中时，没有观察到抗神经毒性作用。通过总RNA提取物的逆转录酶-聚合酶链反应分析，未在THP-1细胞中检测到mao-a和mao-b的信使RNA。在可比较的条件下，在SH-SY5Y细胞的提取物中容易地检测到这种mrna。在THP-1细胞裂解物中也无法检测到MAO酶活性，而在SH-SY5Y细胞中很容易观察到MAO酶活性。结论是，deprenyl对THP-1细胞的作用不是由MAO介导的，并且deprenyl本身不能保护神经元。这些数据表明，由于其抗神经毒性作用，deprenyl可能在神经退行性疾病中有用。

BACKGROUND & AIMS: :Reduced insulin sensitivity is a key factor in the pathogenesis of type 2 diabetes and hypertension. Skeletal muscle insulin resistance is particularly important for its major role in insulin-mediated glucose disposal. Angiotensin II (ANG II) is integral in regulating blood pressure and plays a role in the pathogenesis of hypertension. In addition, we have documented that ANG II-induced skeletal muscle insulin resistance is associated with generation of reactive oxygen species (ROS). However, the linkage between ROS and insulin resistance in skeletal muscle remains unclear. To explore potential mechanisms, we employed the transgenic TG(mRen2)27 (Ren-2) hypertensive rat, which harbors the mouse renin transgene and exhibits elevated tissue ANG II levels, and skeletal muscle cell culture. Compared with Sprague-Dawley normotensive control rats, Ren-2 skeletal muscle exhibited significantly increased oxidative stress, NF-kappaB activation, and TNF-alpha expression, which were attenuated by in vivo treatment with an angiotensin type 1 receptor blocker (valsartan) or SOD/catalase mimetic (tempol). Moreover, ANG II treatment of L6 myotubes induced NF-kappaB activation and TNF-alpha production and decreased insulin-stimulated Akt activation and GLUT-4 glucose transporter translocation to plasma membranes. These effects were markedly diminished by treatment of myotubes with valsartan, the antioxidant N-acetylcysteine, NADPH oxidase-inhibiting peptide (gp91 ds-tat), or NF-kappaB inhibitor (MG-132). Similarly, NF-kappaB p65 small interfering RNA reduced NF-kappaB p65 subunit expression and nuclear translocation and TNF-alpha production but improved insulin-stimulated phosphorylation (Ser(473)) of Akt and translocation of GLUT-4. These findings suggest that NF-kappaB plays an important role in ANG II/ROS-induced skeletal muscle insulin resistance.

背景与目标： : 胰岛素敏感性降低是2型糖尿病和高血压发病的关键因素。骨骼肌胰岛素抵抗因其在胰岛素介导的葡萄糖处置中的主要作用而特别重要。血管紧张素II (ANG II) 在调节血压中不可或缺，并在高血压的发病机理中起作用。此外，我们已经记录了ANG II诱导的骨骼肌胰岛素抵抗与活性氧 (ROS) 的产生有关。然而，骨骼肌中ROS与胰岛素抵抗之间的联系仍不清楚。为了探索潜在的机制，我们使用了转基因TG(mRen2)27 (Ren-2) 高血压大鼠，该大鼠具有小鼠肾素转基因并表现出升高的组织ANG II水平和骨骼肌细胞培养。与Sprague-Dawley正常血压对照组大鼠相比，Ren-2骨骼肌表现出明显增加的氧化应激，NF-κ b活化和TNF-α 表达，这些作用通过血管紧张素1型受体阻滞剂 (缬沙坦) 或SOD/过氧化氢酶模拟物 (tempol) 的体内处理而减弱。此外，ANG II处理L6肌管可诱导NF-κ b活化和TNF-α 产生，并减少胰岛素刺激的Akt活化和GLUT-4葡萄糖转运蛋白向质膜的转运。通过用缬沙坦，抗氧化剂N-乙酰半胱氨酸，NADPH氧化酶抑制肽 (gp91 ds-tat) 或NF-κ b抑制剂 (MG-132) 处理肌管，这些作用显着减弱。类似地，NF-κ b p65小干扰RNA降低NF-κ b p65亚基表达和核易位和TNF-α 产生，但改善Akt的胰岛素刺激磷酸化 (Ser(473)) 和GLUT-4的易位。这些发现表明，NF-κ b在ANG II/ROS诱导的骨骼肌胰岛素抵抗中起重要作用。

BACKGROUND & AIMS: :The effects of styrene on mitochondrial monoamine oxidase (MAO) activity in rat and monkey brains were compared in vitro. After preincubation at 25 degrees C for 20 min with 1 mM styrene monomer MAO-A activity in monkey brain was inhibited potently using 5-HT (for MAO-A substrate), but MAO-B activity in monkey brain and platelets were slightly inhibited using beta-PEA (for MAO-B substrate). Styrene monomer also competitively inhibited MAO-A activity in a dose-dependent manner. MAO-A in monkey brain was inhibited by styrene in ascending order of potency: styrene trimer>styrene dimer>styrene monomer. In contrast styrene monomer slightly inhibited both MAO-A and MAO-B activities in rat brain mitochondria. In the present study styrene monomer potently inhibits MAO-A activity, but not MAO-B activity, in monkey brain mitochondria in vitro. These results indicate the inhibiting action of styrene differs depending on animal species and MAO isoforms.

背景与目标： : 比较了苯乙烯对大鼠和猴脑线粒体单胺氧化酶 (MAO) 活性的影响。在25 ℃ 用毫米苯乙烯单体预孵育20分钟后，使用5-HT (对于mao-a底物) 有效地抑制了猴脑中的mao-a活性，但是使用beta-PEA (对于mao-b底物) 略微抑制了猴脑和血小板中的mao-b活性。苯乙烯单体还以剂量依赖性方式竞争性抑制mao-a活性。苯乙烯对猴脑中的mao-a的抑制作用按效力顺序依次为: 苯乙烯三聚体> 苯乙烯二聚体> 苯乙烯单体。相反，苯乙烯单体会稍微抑制大鼠脑线粒体中的mao-a和mao-b活性。在本研究中，苯乙烯单体可在体外抑制猴脑线粒体中的mao-a活性，但不能抑制mao-b活性。这些结果表明，苯乙烯的抑制作用因动物种类和MAO亚型而异。