In Utero Ethanol Exposure Elicits Oxidative Stress in the Rat Fetus

Prior studies in our laboratory have shown that exposure of cultured fetal rat hepatocytes to ethanol (E) blocks epidermal growth factor-dependent replication and that this is paralleled by cell membrane damage, mitochondrial dysfunction, membrane lipid peroxidation (LP), and enhanced generation of reactive oxygen species. These measures of E-mediated oxidative stress (OS) were mitigated by treatment with antioxidants, and cell replication could be normalized by maintaining cell glutathione (GSH) pools. We have now extended these studies to an in vivo model. Rats were administered E (4 g/kg, po) at 12-hr intervals on days 17 and 18 of gestation and killed on day 19,1 hr following a final dose of E (a total of 5 doses). Fetal and maternal brain and liver were assayed for signs of OS. The 2-day in utero E exposure increased membrane LP in fetal brain as evidenced by increased malondialdehyde (MDA) levels from 1.76 ± 0.12 se (nMol/mg protein) to 2.00 ± 0.08 (p < 0.05) and conjugated dienes from 0.230 ± 0.006 se (OD₂₃₃/mg lipid) to 0.282 ± 0.006 (p < 0.05). In fetal liver, MDA levels increased from 2.39 ± 0.08 se (nMol/mg protein) to 2.87 ± 0.08 (p < 0.05), whereas dienes differed significantly only between ad libitum controls and the E and pair-fed control groups (p < 0.05). E decreased GSH levels in fetal brain by 19%, from 19.88 ± 0.72 to 16.13 ± 1.06 (nMol/mg protein) (p < 0.05). A10% decrease in GSH was seen in fetal liver (p < 0.05). GSH in maternal brain was decreased by 44% from 47.29 ± 3.38 to 26.60 ± 2.29 (p < 0.05). Other E-related increases in these OS measures were not observed in maternal organs. E did not decrease α-tocopherol levels in fetal and maternal brain or in fetal liver (p < 0.05), whereas maternal liver α-tocopherol content was reduced by 31% (p < 0.05) by E treatment. It is concluded that maternal E consumption can induce an OS in fetal tissues that may contribute to the fetotoxic effects of E.     

Immunohistochemical characterization of hepatic malondialdehyde and 4-hydroxynonenal modified proteins during early stages of ethanol-induced liver injury

BACKGROUND: Chronic ethanol consumption is associated with hepatic lipid peroxidation and the deposition or retention of aldehyde-adducted proteins postulated to be involved in alcohol-induced liver injury. The purpose of this study was to characterize hepatocellular formation of aldehyde-protein adducts during early stages of alcohol-induced liver injury. METHODS: Female Sprague Dawley(R) rats were subjected to the intragastric administration of a low-carbohydrate/high-fat total enteral nutrition diet or a total enteral nutrition diet containing ethanol for a period of 36 days. Indexes of hepatic responses to ethanol were evaluated in terms of changes in plasma alanine aminotransferase activity, hepatic histopathologic analysis, and induction of cytochrome P-4502E1 (CYP2E1). Immunohistochemical methods were used to detect hepatic proteins modified with malondialdehyde (MDA) or 4-hydroxynonenal (4-HNE) for subsequent quantitative image analysis. RESULTS: After 36 days of treatment, rats receiving the alcohol-containing diet displayed hepatic histopathologies characterized by marked micro- and macrosteatosis associated with only minor inflammation and necrosis. Alcohol administration resulted in a 3-fold elevation of plasma alanine aminotransferase activity and 3-fold increases (p < 0.01) in hepatic CYP2E1 apoprotein and activity. Quantitative immunohistochemical analysis revealed significant (p < 0.01) 5-fold increases in MDA- and 4-HNE modified proteins in liver sections prepared from rats treated with alcohol. The MDA- or 4-HNE modified proteins were contained in hepatocytes displaying intact morphology and were colocalized primarily with microvesicular deposits of lipid. Aldehyde-modified proteins were not prevalent in parenchymal or nonparenchymal cells associated with foci of necrosis or inflammation. CONCLUSIONS: These results suggest that alcohol-induced lipid peroxidation is an early event during alcohol-mediated liver injury and may be a sensitizing event resulting in the production of bioactive aldehydes that have the potential to initiate or propagate ensuing proinflammatory or profibrogenic cellular events.     

Ethanol Attenuates Spatial Memory Deficits and Increases mGlu1a Receptor Expression in the Hippocampus of Rats Exposed to Prenatal Stress

Background: Although it is generally believed that chronic ethanol consumption impairs learning and memory, results obtained in experimental animals are not univocal, and there are conditions in which ethanol paradoxically improves cognitive functions. In the present work, we investigated the effects of prenatal stress and of chronic ethanol exposure during adulthood on spatial memory in rats. Methods: Rats were subjected to a prenatal stress delivered as 3 daily 45‐minute sections of restraint stress to the mothers during the last 10 days of pregnancy (PRS rats). After 7 months of ethanol exposure (ethanol 10%, oral intake), memory performances were evaluated in a spatial discrimination test in control and PRS male rats. Then, the oxidative damages and the expression of metabotropic glutamate (mGlu) receptors were assessed in their hippocampus. Results: Chronic ethanol exposure resulted in a reduced performance in a spatial recognition task in control animals. Unexpectedly, however, the same treatment attenuated spatial memory deficits in rats that had been subjected to prenatal stress. This paradigm of ethanol administration did not produce detectable signs of oxidative damage in the hippocampus in either unstressed or PRS rats. Interestingly, ethanol intake resulted in differential effects in the expression of mGlu receptor subtypes implicated in mechanisms of learning and memory. In control rats, ethanol intake reduced mGlu2/3 and mGlu5 receptor levels in the hippocampus; in PRS rats, which exhibited a constitutive reduction in the levels of these mGlu receptor subtypes, ethanol increased the expression of mGlu1a receptors but did not change the expression of mGlu2/3 or mGlu5 receptors. Conclusion: Our findings support the idea that stress‐related events occurring before birth have long‐lasting effects on brain function and behavior, and suggest that the impact of ethanol on cognition is not only dose‐ and duration‐dependent, but also critically influenced by early life experiences.     

D-半乳糖预处理大鼠患肺炎时心脏功能的改变

目的　研究肺炎对D 半乳糖 (D gal)处理大鼠心脏功能的影响。　 方法　选雄性SD大鼠 4 5只以D gal预处理 (D gal组 ) ,经气管注入细菌悬液制作肺炎模型后随机均分 3亚组 (对照组、肺炎 1d组、3d组 ;每组 15只 )进行实验 (对照组注入生理盐水 )。另有 4 5只SD大鼠以生理盐水预处理 (NS组 )后做同样分组实验。实验中观察大鼠心脏血流动力学和部分血浆参数改变。　结果　肺炎后大鼠心脏功能受损 ,左室舒张末期压 (LVEDP)升高、等容收缩期左室内压力最大上升和最大下降速率 (±dp/dtmax)降低 ,血浆肿瘤坏死因子 (TNF α)、丙二醛含量增加 ;动脉血氧分压、血氧饱和度、血浆超氧化物歧化酶 (SOD)活性降低 ;D gal组的心脏功能 ,NS 3组和D gal3组比较 ,LVED(5 3±0 9)mmHg和 (8 1± 1 4 )mmHg ,+dp/dtmax(16 5 4± 139)mmHg·s-1和 (12 91± 14 5 )mmHg·s-1;-dp/dtmin(12 6 4± 12 8)mmHg·s-1和 (85 3± 15 8)mmHg·s-1) ,血浆丙二醛含量 [(3 91± 0 85 )mmol·L-1和 (5 0 2± 1 4 5 )mmol·L-1]、SOD活性 [(44 9± 5 6 2 )U/mg蛋白和 (33 6± 4 35 )U/mg蛋白 ]改变更显著。　结论　D gal组大鼠的脂质过氧化增加其肺炎时心脏的易损性。     

Apoptosis and Bcl-2 Protein Expression in Experimental Alcoholic Liver Disease in the Rat

We used the intragastric feeding rat model to investigate the relationship between severity of alcoholic liver injury, apoptosis, bcl-2 protein expression, and lipid peroxidation. Rats were fed ethanol with different dietary fats (saturated fat, corn oil, and fish oil) for a 1-month period. Apoptosis was evaluated using an immunohistochemical method, and flow cytometry. Bcl-2 protein concentrations in liver were evaluated by Western blot analysis and lipid peroxidation by measurement of conjugated diems. Pathological changes (fatty liver, necrosis, and inflammation) were present in com oil-ethanol and fish oil-ethanol groups only. The highest number of apoptotic cells were seen in the group of rats exhibiting her Injury. The fish oil-ethanol-fed group had the highest concentrations of bcl-2 protein; this protein was localized in the bile duct epithelial and inflammatory cells. A significant correlation was seem between bcl-2 protein assessed densitometrically and the number of inflammatory cells/mm² ( r = 0.78, p < 0.02) and conjugated diene levels ( r = 0.82, p < 0.01). Increased numbers of apoptotic cells were seen in rats developing ethanol-induced pathological liver injury. Increased bcl-2 protein concentrations are associated with the presence of inflammatory cells and lipid peroxidation.     

Iron-induced lipid peroxidation and inhibition of proliferation in Ehrlich ascites tumor cells

Summary The purpose of this study was to find further experimental evidence for the postulated negative association between the extent of lipid peroxidation in tumor cells and their proliferative behavior. After incubation of Ehrlich ascites tumor cells at 37°C for 30 min with increasing concentrations of Fe(II) histidinate (Fe/His) the following parameters were determined: the formation of lipid hydroperoxides was measured fluorimetrically after reaction with dichlorofluorescein; 4-hydroxynonenal was determined by reversed-phase high-pressure chromatography after derivatization with dinitrophenylhydrazine; as a third parameter of lipid peroxidation the formation of 2-thiobarbituric-acid-reactive substances was determined. The proliferative activity was determined by measuring the growth rate in vivo after reimplantation i.p. of the tumor cells into mice. Trypan-blue exclusion tests for viability were performed before reimplantation. The reliability of the trypan-blue exclusion tests was checked by comparing the results with another parameter of viability, the release of the cytosolic enzyme lactate dehydrogenase. The concentration both of lipid hydroperoxides and of 2-thiobarbituric-acid-reactive substances showed a biphasic dependence on the concentration of Fe/His with maximal increase at iron concentrations of 0.25 mM and 0.1mM respectively. 4-Hydroxynonenal, in contrast, showed a continuous increase up to 41.1 nM (corresponding to 0.58 pmol/10⁹ cells) with increasing iron concentration in the range from 0.1 mM to 0.6 mM. The total number of tumor cells, when determined 5 days after reimplantation, continuously decreased with increasing iron concentration, showing half-maximal inhibition at about 0.22 mM Fe. The exclusion of the trypan-blue dye was unaffected by the presence of iron at any concentration used. Similarly, iron had no influence on the release of lactate dehydrogenase. The results support the hypothesis that 4-hydroxynonenal may act as an inhibiting messenger between endogenic lipid peroxidation and proliferation.Parts of this paper were presented at scientific meetings (Kink et al. 1986; Schaur 1987; Hammer et al. 1987, 1988)This work was supported by the Association for International Cancer Research, UK     

复方保肝宁对次氮基三醋酸铁溶液致HSC-T6细胞氧化应激的防护作用

目的:探讨复方保肝宁对次氮基三醋酸铁溶液(FeNTA)致肝星状细胞系(HSC-T6)细胞氧化应激反应的影响.方法:用复方保肝宁给正常大鼠灌胃,制备药物血清.建立FeNTA所致HSC-T6细胞氧化应激脂质过氧化模型,观察保肝宁药物血清作用后过氧化氢(H2O2)、丙二醛(MDA)、谷胱甘肽(GSH)、谷胱甘肽过氧化物酶(GSH-Px)含量的变化.结果:FeNTA与HSC-T6共同培养1小时、3小时、5小时、24小时后脂质过氧化物(H2O2、MDA)生成均增多,抗氧化能力(GSH-Px、GSH)均下降(5小时最为显著).复方保肝宁药物血清可抑制HSC氧化应激反应过程中H2O2、MDA含量,同时能不同程度升高GSH-Px、GSH水平.结论:复方保肝宁能降低FeNTA所致HSC氧化应激反应时的脂质过氧化水平.对HSC氧化应激的防护作用,减弱HSC的自分泌放大效应,可能是复方保肝宁抗肝纤维化的主要机制之一.     

4-羟基-2-壬烯酸诱导培养的主动脉内皮细胞DNA损伤

研究 4 羟基 2 壬烯酸对体外培养的主动脉内皮细胞作用 ,以探讨动脉粥样硬化的发病机理。采用单细胞凝胶电泳检测DNA损伤 ,对体外培养的主动脉内皮细胞在不同浓度 4 羟基 2 壬烯酸作用下产生的DNA损伤进行检测。结果发现 ,体外培养的主动脉内皮细胞分别用 5 μmol L、10 μmol L和 15 μmol L 4 羟基 2 壬烯酸处理 10h后彗星试验的尾距分别为 32 .8± 1.1、4 4 .3± 1.0和 74 .6± 1.0 ,与未用 4 羟基 2 壬烯酸处理的正常对照组彗星试验的尾距 (6 .0± 0 .7)比较 ,差异有显著性 (P <0 .0 0 1) ,经 1μmol L 4 羟基 2 壬烯酸处理后的主动脉内皮细胞彗星试验的尾距为 11.3± 0 .9,与正常对照组比较 ,两者之间差异无显著性 (P >0 .0 5 )。结果提示 ,4 羟基 2 壬烯酸可导致体外培养的主动脉内皮细胞的DNA损伤 ,并随着 4 羟基 2 壬烯酸的浓度增高DNA损伤加剧。     

Dose-dependent effect of ethanol on hepatic oxidative stress and interleukin-6 production after burn injury in the mouse

BACKGROUND: Burned patients with detectable blood alcohol levels (BAL) show an elevated mortality rate. Interleukin (IL)-6 and reactive oxygen species (ROS) production is stimulated independently by alcohol and burn injury. The aim of the study was to determine whether increasing levels of alcohol differentially enhance the hepatic production of IL-6 and ROS after burn in a murine model of dorsal scald injury. Groups of mice received either saline or alcohol intraperitoneally to reach a BAL of 100 mg/dl or 300 mg/dl at the time of burn (15% total body surface scald) or sham injury. RESULTS: Burn injury alone resulted in a low mortality rate at 24 hr after injury as did the burn group with a BAL of 100 mg/dl (15%), whereas 57% of the mice burned with a BAL of 300 mg/dl did not survive (p = 0.02). Twenty-four hours after burn or sham injury, IL-6 levels were measured by enzyme-linked immunosorbent assay in serum and liver. In the saline-treated group, IL-6 circulating and hepatic levels rose after burn injury (p < 0.03). Circulating IL-6 levels in sham mice increased 1.5-fold in the group with a BAL of 100 mg/dl and 3-fold in those with a BAL of 300 mg/ml (p = 0.005 versus burn-injured, saline-treated). IL-6 hepatic production after burn injury was higher in the mice with a BAL of 300 mg/dl than in those with a BAL of 100 mg/dl and the saline-treated group (p = 0.001). Among the burned mice, alcohol exposure increased hepatic ROS production, measured by lipid peroxidation and protein oxidation, in a dose-dependent manner. CONCLUSIONS: Alcohol enhances in a dose-dependent manner the hepatic production of IL-6 induced by burn injury through the modulation of oxidative stress. The increased mortality rate of mice exposed to alcohol and burn injury may be due to the adverse effect on immune function induced by IL-6 elevation.     

In Vitro Susceptibility of Wistar Rat Platelets to Hydrogen Peroxide and AAPH-Induced Oxidative Stress

Hydroxyl and peroxyl radicals are biologically active species because of their likelihood to damage cellular constituents. An in vitro study on Wistar rats was conducted to investigate the influence of hydrogen peroxide (H₂O₂) and 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) on platelets and compare the vulnerability of platelets to oxidative stress (OS) induced by these two free radical initiators. Isolated platelets were divided into controls (without free radical initiators; n = 5) and experimentals (with free radical initiators; n = 5). Different concentrations (0.5, 1.0 and 2.0) of free radical initiators H₂O₂ and AAPH were used to treat the platelets and incubated for 5, 15 and 30 min. Biomarkers such as platelet aggregation, superoxide generation, lipid peroxidation (thiobarbituric acid reactive substances, conjugate dienes), protein oxidation (protein carbonyls, sulfhydryls) and antioxidant enzymes were assessed. In H₂O₂ and AAPH treated platelets, though OS was observed at concentrations of 0.5 and 1.0 mM, platelets could tolerate the oxidative insult. Treatment of platelets with 2.0 mM H₂O₂ demonstrated the onset of irreversible changes in platelets as observed in the results of increased superoxide generation and lipid peroxidation products. In 2.0 mM AAPH platelets, the oxidative damage was evident as indicated through increased aggregation, superoxide generation and conjugate dienes and lower protein sulfhydryls. Platelets were more susceptible to AAPH than H₂O₂, as AAPH acted on both lipids and proteins whereas H₂O₂ acted only on lipids. This study gives insight on platelet survival under different OS situations.     

Chronic Alcohol-Induced Alterations in the Pancreatic Secretory Control Mechanisms

Chronic alcohol ingestion appears to increase susceptibility of the pancreas to pancreatitis through multiple mechanisms. The aim of the current study was to determine the effect of chronic low- and high-dose alcohol consumption on the neurohormonal control of the exocrine pancreas in rats. Male Wistar rats were fed Lieber DeCarli liquid control-, low-, and high-dose alcohol diets for 3 months. Pancreatic exocrine secretion was measured under basal and 2-deoxy-d-glucose (2-DG)-, CCK-, bethanechol-, or meal-stimulated conditions while on chronic alcohol diets and after 2-DG or CCK stimulation during alcohol withdrawal in awake rats. Chronic alcohol ingestion was associated with a dose-related inhibition of basal pancreatic protein secretion, which was reversed upon alcohol withdrawal. Low-dose alcohol feeding had no effect on bethanechol-stimulated pancreatic secretion but altered 2-DG-stimulated pancreatic secretion. In chronic high-dose alcohol rats, meal- and bethanechol-stimulated protein secretion was significantly potentiated during early and late phases. The response to CCK appeared to be disinhibited, whereas the response to 2-DG was uniformly blunted. Upon withdrawal of low-dose alcohol, the response to 2-DG was potentiated, whereas with the withdrawal of high-dose alcohol, the response to CCK was potentiated. Adaptation to chronic alcohol consumption differs depending on the alcohol dose. The most significant effects were seen after high-dose alcohol withdrawal, with apparent loss of central inhibitory regulation combined with exaggerated response at the acinar cell level. This combination of factors could increase susceptibility to acute alcoholic pancreatitis through a hyperstimulation mechanism.     

Oxidative Stress in the Dahl Salt-Sensitive Hypertensive Rat

Oxidative stress has been proposed as important in the pathogenesis of hypertension. Measurement of 8-iso prostaglandin F_2α(8-ISO) is introduced for evaluating oxidative stress in vivo. 8-ISO is the major urinary metabolite of F₂-isoprostanes and is formed nonenzymatically from the attack of superoxide radicals on arachidonic acid. We examined the oxidative stress level in the Dahl salt-sensitive (Dahl-S) rats and the Dahl salt-resistant (Dahl-R) rats. Dahl-S and Dahl-R rats were fed either a high salt diet (8% NaCl; HS) or low salt diet (0.3% NaCl; LS) for 3 weeks, and systolic blood pressure (SBP) and 24-hr urinary excretion of 8-ISO (U-8-ISO) were measured. In Dahl-S rats, the high salt diet induced hypertension (139 ± 3 mmHg in LS versus 186 ± 2 mmHg in HS, p < .05) and significantly increased the U-8-ISO (24.9 ± 3.6 ng/24 hr in LS versus 63.2 ± 14.6 ng/24 hr in HS, p < .05). No significant difference in blood pressure or U-8-ISO was observed between high-salt and low-salt treated Dahl-R rats. U-8-ISO concentration was correlated with SBP in all four experimental groups (r = 0.866). Moreover, urinary 8-hydroxy-2′-deoxyguanosine (U-8-OHdG), which is one of the most commonly used markers for evaluation of oxidative stress, was higher in Dahl-S-8% rats than in Dahl-S-0.3% rats (136.1 ± 48.4 ng/24 hr in LS versus 322.8 ± 46.7 ng/24 hr in HS, p < .05), and U-8-OHdG was correlated with SBP (r = 0.681) in Dahl-S rats. These results suggest oxygen radicals are involved in the pathogenesis of hypertension.     

不同浓度脂联素对心肌细胞氧化应激损伤后GRP78及Caspase-12表达的影响及意义

目的 通过原代培养SD大鼠的乳鼠心肌细胞建立H_2O_2心肌细胞氧化应激损伤模型,观察脂联素对心肌细胞氧化应激所致内质网应激的保护作用.方法 采用酶消化法原代培养乳鼠心肌细胞,倒置相差显微镜下观察细胞生长状态,通过α-肌动蛋白免疫荧光法对培养的心肌细胞进行鉴定.选用原代培养3～4天的心肌细胞,随机分为对照组、H_2O_2组、H_2O_2+10 mg/L脂联素组、H_2O_2+20 mg/L脂联素组和H_2O_2+30 mg/L脂联素组.实验终止后,在倒置相差显微镜下观察心肌细胞形态的变化,采用化学比色法测定乳酸脱氢酶的释放,通过流式细胞术来检测心肌细胞的凋亡,用RT-PCR与western Blotting方法检测内质网应激指标GRP78和Caspase-12的表达.结果 与对照组相比,给予H_2O_2后,细胞凋亡率显著增加(70.7%±6.4%比1.0%±0.6%,P<0.05),LDH释放增加(1411.5 ±189.7 U/L比353.3 ±50.3 U/L,P<0.05),内质网伴侣蛋白GRP78以及Caspage-12在mRNA(分别为1.25±0.50比0.18 ±0.10和1.32±0.15比0.26±0.06)及蛋白水平(分别为0.92±0.50比0.37±0.10和1.24 ±0.50比0.51±0.01)表达增加(P<0.05),30 mg/L脂联素预处理后给予H_2O_2,可较大程度地逆转上述指标变化,细胞凋亡率显著下降(43.6%±3.8%),LDH释放减少(686.7±61.1 U/L),内质网伴侣蛋白GRP78以及Cagpase-12在mRNA(分别为0.56±0.03和0.83±0.04)及蛋白水平(分别为0.66±0.03和0.64±0.03)表达减少(P<0.05).结论 氧化应激使GRP78和Caspase-12表达增强,启动内质网应激,脂联素可以通过减轻内质网应激逆转H_2O_2所致的心肌细胞损伤及凋亡作用,对心肌细胞有保护作用.     

Structural bases for the interaction and stabilization of the human amino acid transporter LAT2 with its ancillary protein 4F2hc

Heteromeric amino acid transporters (HATs) are the unique example, known in all kingdoms of life, of solute transporters composed of two subunits linked by a conserved disulfide bridge. In metazoans, the heavy subunit is responsible for the trafficking of the heterodimer to the plasma membrane, and the light subunit is the transporter. HATs are involved in human pathologies such as amino acidurias, tumor growth and invasion, viral infection and cocaine addiction. However structural information about interactions between the heavy and light subunits of HATs is scarce. In this work, transmission electron microscopy and single-particle analysis of purified human 4F2hc/L-type amino acid transporter 2 (LAT2) heterodimers overexpressed in the yeast Pichia pastoris, together with docking analysis and crosslinking experiments, reveal that the extracellular domain of 4F2hc interacts with LAT2, almost completely covering the extracellular face of the transporter. 4F2hc increases the stability of the light subunit LAT2 in detergent-solubilized Pichia membranes, allowing functional reconstitution of the heterodimer into proteoliposomes. Moreover, the extracellular domain of 4F2hc suffices to stabilize solubilized LAT2. The interaction of 4F2hc with LAT2 gives insights into the structural bases for light subunit recognition and the stabilizing role of the ancillary protein in HATs.Heteromeric amino acid transporters (HATs) are composed of two subunits, a heavy (SLC3 family) and a light subunit [SLC7 or L-type amino acid transporter (LAT) family] linked by a conserved disulfide bridge (1). HATs are amino acid exchangers (1), and this transport activity resides in the light subunit (2). The heavy subunit (either 4F2hc or rBAT) is essential for trafficking of the holotransporter to the plasma membrane (3, 4). In mammals, six transporters heterodimerize with 4F2hc, and only one heterodimerizes with rBAT. The rBAT/b^0,+AT complex is a dimer of heterodimers in which the light subunit is required for proper rBAT folding and stability (5, 6). In contrast, 4F2hc-associated transporters are simple heterodimers (6), and possible stabilizing roles of the two subunits in the biogenesis of the heterodimer have not been described.HATs have major impacts on human health and are involved directly in amino acidurias (cystinuria and lysinuric protein intolerance), tumor cell growth, glioma invasion, Kaposi’s sarcoma-associated herpesvirus infection, and cocaine relapse (1). In addition to the role of HATs in amino acid transport, 4F2hc heterodimers mediate β1- and β3-integrin signaling (7).Structural information about HATs is scarce (1). The heavy subunits are type II membrane N-glycoproteins with a single transmembrane domain (TMD), an intracellular N terminus, and a large extracellular C terminus with sequence homology with bacterial α-amylases. Indeed, the atomic structure of the extracellular domain (ED) of human 4F2hc (4F2hc-ED) is similar to that of bacterial glucosidases [i.e., a triose phosphate isomerase barrel, (βα)₈, (subdomain A) and eight antiparallel β-strands (subdomain C)] but lacks glucosidase activity (8). The conserved cysteine residue participating in the intersubunit disulfide bridge is located between the single TMD and 4F2hc-ED. Physical and functional interaction of 4F2hc with integrins has been mapped to the TMD and cytosolic N-terminal domain (7, 9), whereas 4F2hc-ED is necessary for functional heterodimerization with the light subunit (9, 10). The light subunits are nonglycosylated proteins and have a 12-TMD topology with intracellular N and C termini (1). The conserved cysteine residue involved in the intersubunit disulfide bridge is located between TMD3 and TMD4 (11). Structure–function studies (12, 13) suggest that the structures of the light subunit of HATs should be similar to those of remote bacterial amino acid/polyamine/organocation (APC) transporters (∼20% amino acid sequence identities), which present the LeuT-fold (14–17). In contrast, there is no structural information about the interaction between the two HAT subunits. In the present study, we show that information from transmission electron microscopy (TEM) and single-particle analysis (SPA) of human 4F2hc/L-type amino acid transporter 2 (LAT2) heterodimers is compatible with 4F2hc-ED interacting with the extracellular loops of LAT2. Docking analyses and crosslinking experiments indicated a location of 4F2hc-ED almost completely covering the external face of LAT2. Moreover, 4F2hc increases the stability of detergent-solubilized LAT2, allowing functional reconstitution of the heterodimer into proteoliposomes. The interaction of 4F2hc-ED with LAT2 provides insights regarding the structural bases for recognition of the light subunit and the stabilization of this complex and other 4F2hc associated transporters.     

Predicting efficacy of dipeptidyl peptidase‐4 inhibitors in patients with type 2 diabetes: Association of glycated hemoglobin reduction with serum eicosapentaenoic acid and docosahexaenoic acid levels

This study was initiated to identify clinical and dietary parameters that predict efficacy of dipeptidyl peptidase‐4 inhibitors. A total of 72 untreated Japanese patients with type 2 diabetes who received DPP‐4 inhibitors (sitagliptin, alogliptin or vildagliptin) for 4 months were examined for changes of glycated hemoglobin (HbA_1c) and body mass index (BMI), and self‐administered 3‐day food records, as well as serum levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). DPP‐4 inhibitors significantly reduced HbA_1c (before initiation of DPP‐4 inhibitors 7.2 ± 0.7%, 4 months after initiation of DPP‐4 inhibitors 6.7 ± 0.6% [paired t‐test, P < 0.01 vs before]). Multiple regression analysis showed that changes of HbA_1c were significantly correlated with baseline HbA_1c, as well as estimated intake of fish. Furthermore, changes of HbA_1c were significantly correlated with serum levels of EPA (r = −0.624, P < 0.01) and DHA (r = −0.577, P < 0.01). HbA_1c reduction by DPP‐4 inhibitors is significantly correlated with estimated intake of fish and serum levels of EPA and DHA. (J Diabetes Invest, doi: 10.1111/j.2040‐1124.2012.00214.x, 2012)     

老年冠心病患者血清RBP4水平与氧化应激、颈动脉粥样硬化的关系研究

目的：探讨冠心病患者血清视黄醇结合蛋白4（retinal binding protein 4, RBP4）水平的变化,及其与氧化应激、颈动脉粥样硬化的关系。方法：选取老年冠心病患者100例及健康体检者100例,采用酶联免疫吸附法测定血清RBP4水平,黄嘌吟氧化酶法及硫代巴比妥酸显色法分别测定血清超氧化物歧化酶活性和丙二醛水平,高分辨彩色血管多普勒超声仪测量颈动脉粥样斑块面积和内-中膜厚度（IMT）。结果：冠心病组血清RBP4（35.10±5.76 vs. 27.82±4.90 ng/mL）和丙二醛（14.21±1.22 vs. 9.89±1.64 ?mol/L）水平,以及颈动脉粥样斑块面积（15.27±1.16 vs. 10.13±2.53 mm2）、IMT（1.19±0.13 vs. 1.03±0.16 mm）均明显高于健康体检者组（P<0.01）,血清超氧化物歧化酶活性（76.49±13.82 vs. 93.29±12.11 kU/L,P<0.01）则明显低于健康体检者组。血清RBP4水平与丙二醛（R = 0.486）、颈动脉粥样斑块面积（R = 0.354）及IMT（R = 0.388）呈正相关（P<0.05）,与血清超氧化物歧化酶活性呈负相关（R = 0.343,P<0.05）。结论：老年冠心病患者血清RBP4水平明显升高,并且升高的RBP4水平与氧化应激损伤及颈动脉粥样硬化程度呈正相关。     

Oxidative Stress Status in the Transition of Hypertrophy to Heart Failure

In response to a variety of stressful conditions such as pressure overload, volume overload, myocardial infarction and different types of cardiomyopathies, the heart undergoes an adaptation process during which it grows in size, a phenomenon referred to as hypertrophy. During this compensatory phase, the heart is better able to meet the increased work demand. However, if left untreated for a prolonged period, it can advance into the decompensated hypertrophic or the failing stage. It is now clear that transition of heart hypertrophy to the failure stage involves a number of abnormalities which include ionic imbalances, impairment in energy production and utilization, altered calcium metabolism and defects in the contractile proteins. More recently, alterations in free radicals and antioxidant reserve have been identified in the hypertrophy as well as failing stages. Discussion in this review is focussed on the role of oxidative stress in both compensated and decompensated stages, in various animal models of heart failure. Some clinical data on oxidative stress in heart failure patients is also reviewed. It is suggested that an increase in oxidative stress may play a role in the pathogenesis of heart failure.