Enhanced Expression of the Inhibitory Protein Gi2α and Decreased Activity of Adenylyl Cyclase in Lymphocytes of Abstinent Alcoholics

Ethanol exposure alters signal transduction through the adenylyl cyclase (AC) system. To elucidate the basis for this effect, we investigated the AC system in peripheral lymphocytes from abstinent alcoholic men ( n = 22), actively drinking alcoholic men ( n = 41), and nonalcoholic control men ( n = 16). Immunoblot analysis of lymphocyte membranes from abstinent alcoholics demonstrated a 3.0-fold increase in the level of G_i2α protein ( p < 0.05) compared with controls. However, levels of G_i2α protein were similar in both groups. Abstinent alcoholics had a 2.9-fold increase in G_i2α mRNA ( p < 0.001) and a 2.7-fold increase in G₂α mRNA ( p < 0.03) compared with lymphocytes from control subjects. Actively drinking alcoholics, in contrast, had unaltered G₂α protein, G_i2α protein, and G_i2α mRNA levels compared with control subjects, but did have a 1.8-fold increase ( p < 0.01) in G_i2α mRNA. Consistent with enhanced G_i2α expression, lymphocyte membranes from abstinent alcoholics had decreased basal, prostaglandin E_1-, guanosine 5'-0-(3-thiotriphosphate)-γS-, and forskolin-stimulated AC activity compared with both controls and actively drinking alcoholics ( p < 0.05). We conclude that lymphocyte AC is reduced during abstinence from alcohol and enhanced expression of the inhibitory G-protein, G_i2α, may account for this change.     

活化蛋白C对重症急性胰腺炎大鼠丝裂原活化蛋白激酶信号通路的影响

丝裂原活化蛋白激酶（MAPK）信号通路对重症急性胰腺炎（SAP）继发严重并发症起早期关键介导作用，相应抑制剂可改善SAP的病情。活化蛋白C（APC）具有改善SAP病情的作用，其具体机制尚未阐明。目的：观察APC对SAP大鼠MAPK信号通路中主要激酶的影响以及后续炎症介质的变化，为临床用药提供理论依据。方法：Sprague．DawleY大鼠诱导SAP模型后即刻静脉注射APC10μg/kg或50μg/kg。以基因芯片检测胰腺组织MAPK信号通路相关基因。以实时定量聚合酶链反应（real-timePCR）和蛋白质印迹法检测胰腺组织该通路中p38MAPK、c-Jun氨基端激酶/应激活化蛋白激酶（JNK/SAPK）、细胞外信号调节激酶（ERK）1/2mRNA、蛋白和磷酸化蛋白水平的表达，同时检测肿瘤坏死因子（TNF）-α和白细胞介素（IL）-1β蛋白的表达。结果：与APC治疗组和正常对照组相比，SAP组胰腺组织p38MAPK和JNK2mRNA呈高表达。与SAP组相比，50Ixg/kgAPC治疗组p38MAPK、JNK/SAPK蛋白/磷酸化蛋白表达水平显著降低，ERK1/2蛋白/磷酸化蛋白表达水平显著升高，TNF-α和蛋白表达水平显著降低（P均〈0．05）。APC治疗组p38MAPK、磷酸化ERK1/2和TNF-α蛋白表达水平呈剂量依赖性（P均〈0．05）。结论：APC可抑制SAP大鼠胰腺组织MAPK信号通路内p38MAPK和JNK/SAPK的表达和活化，进而抑制TNF-α和IL-1β的释放，同时上调ERK1/2的表达和活化．从而减轻胰腺组织损伤。     

乌鲁木齐市海洛因成瘾者社区美沙酮维持治疗基线调查

目的了解进入社区美沙酮维持治疗门诊吸毒人群的吸毒行为、性行为等特征,为艾滋病防制措施的制定提供依据。方法乌鲁木齐市以社区为基础,2005年8~11月,对进入社区药物维持治疗门诊的129名戒毒人员进行问卷调查,调查其人口学、共用注射器具吸毒行为和性行为等情况。结果男性占83.1%,女性占16.2%,平均年龄为33.86±5.39岁,H IV阳性率为19.4%,HCV阳性率为68.5%,注射吸毒者中有39.2%曾与他人共用过注射针具。过去3个月使用他人用过的注射器具吸毒的25人(19.2%),共用过非直接注射器具的16人(12.4%)。过去6个月有商业性行为的8人(32.0%),有新性伙伴的27人(20.9%)。过去1个月与主要和非主要性伙伴每次性交均使用安全套的分别为30.0%(39/86)和13.1%(17/29)。在过去的6个月中,26.4%被公安抓过,22.5%贩卖过毒品;36%(9/25)为了毒品和别人发生过性行为,15.5%为了吸毒而有偷、抢、骗。目前有工作(包括固定、临时)的占48.5%。结论进入维持治疗吸毒者中存在H IV传播的高危性,迫切需要在美沙酮维持治疗的同时进行预防艾滋病的健康教育和行为干预活动。     

Ethanol Inhibition of Insulin Signaling in Hepatocellular Carcinoma Cells

Chronic ethanol toxicity impairs liver regeneration, inhibits DNA synthesis, and mutes cellular responses to growth factor stimulation. Previous studies demonstrated that the adverse effects of ethanol are mediated by inhibition of tyrosyi phosphorylation of the insulin receptor and the insulin receptor substrate-type 1 (IRS-1). However, overexpression of IRS-1 leads to increased DNA synthesis and cellular transformation due to constitutive activation of mitogen-activated protein (MAP) kinase. The present study examines the effects of ethanol on insulin signaling through IRS-1 in FOCUS hepatocellular carcinoma cells, which overexpress IRS-1, to determine whether such ceils were resistant to the inhibitory effects of ethanol. The results demonstrated that ethanol treatment (100 mM) caused 30 to 50% reductions in the levels of insulin-stimulated tyrosyi phosphorylation of the insulin receptor β-subunit, tyrosyi phosphorylation of IRS-1, phosphorylation of Erk2, association of phosphatidylinositol-3 kinase with tyrosyl-phosphorylated IRS-1, and MAP kinase and phosphatidylinositol-3 kinase activities. In contrast, ethanol treatment had no effect on epidermal growth factor-stimulated tyrosyi phosphorylation of She. Corresponding with the pronounced inhibition of MAP kinase, ethanol treatment resulted in 30 to 50% reductions in the expression levels of two important insulin-responsive genes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and proliferating cell nuclear antigen (PCNA). The findings suggest that, in FOCUS hepatocellular carcinoma cells, which overexpress IRS-1, ethanol treatment substantially inhibits IRS-1 and MAP kinase signaling and growth-associated gene expression, but has no effect on She phosphorylation, which activates p21^ras through an IRS-1 independent pathway.     

Ethanol alters opioid regulation of Ca(2+) influx through L-type Ca(2+) channels in PC12 cells

Background: Studies at the behavioral and synaptic level show that effects of ethanol on the central nervous system can involve the opioid signaling system. These interactions may alter the function of a common downstream target. In this study, we examined Ca²⁺ channel function as a potential downstream target of interactions between ethanol and μ or κ opioid receptor signaling. Methods: The studies were carried out in a model system, undifferentiated PC12 cells transfected with μ or κ opioid receptors. The PC12 cells express L‐type Ca²⁺ channels, which were activated by K⁺ depolarization. Ca²⁺ imaging was used to measure relative Ca²⁺ flux during K⁺ depolarization and the modulation of Ca²⁺ flux by opioids and ethanol. Results: Ethanol, μ receptor activation, and κ receptor activation all reduced the amplitude of the Ca²⁺ signal produced by K⁺ depolarization. Pretreatment with ethanol or combined treatment with ethanol and μ or κ receptor agonists caused a reduction in the amplitude of the Ca²⁺ signal that was comparable to or smaller than that observed for the individual drugs alone, indicating an interaction by the drugs at a downstream target (or targets) that limited the modulation of Ca²⁺ flux through L‐type Ca²⁺ channels. Conclusions: These studies provide evidence for a cellular mechanism that could play an important role in ethanol regulation of synaptic transmission and behavior through interactions with the opioid signaling.     

The Density of Monoamine Oxidase B Sites Is Not Altered in the Postmortem Brain of Alcoholics

The status of the enzyme monoamine oxidase B (MAO-B) was directly evaluated in the postmortem brain from 20 alcoholics and 23 matched controls. The density of MAO-B sites was quantified by the specific binding of the selective inhibitor [³H]Ro 19-6327 (lazabemide) (8 nM) to cortical membranes. A positive correlation between age at death and MAO-B density was observed in the total sample ( r = 0.37, p = 0.015). The density of MAO-B in alcoholics (B_max= 1,263 ± 131 fmol/mg of protein) was not different form that in control (B_max= 1,131 ± 96 fmol/mg of protein). Ethanol in vitro inhibited [³H]Ro 19-6327 binding, with similar potency in membranes form alcoholics ( K _i= 280 ± 13 mM) and controls ( K _i= 338 ± 84 mM). The present results in brain tissue contrast with previous reports of decreased MAO-B enzymatic activity in platelets of alcoholics, but strongly agree with recent genetic studies on MAO-B status in alcoholism.     

Vitamin B6 Metabolism and Binding to Proteins in the Blood of Alcoholic and Nonalcoholic Men

Blood obtained from nonalcoholic and alcoholic subjects was incubated with 100 nm [³H]pyridoxine to study its uptake and metabolism by erythrocytes and the binding of vitamin B₆ metabolites to proteins in plasma and erythrocytes. Erythrocytes of the alcoholics accumulated tritium faster than those of the controls; however, they contained the same total amount of tritiated compounds by 15 min. After incubation for 30 min, the erythrocytes had converted most of the pyridoxine to pyridoxal phosphate and pyridoxal. Pyridoxal-P remained in the erythrocytes, and ?40% of the pyridoxal diffused into the plasma. [³H]Pyridoxal and [³H]pyridoxal-P levels in the erythrocytes and plasma of the alcoholics were similar to those in the controls. However, dialyzed hemolysates of the alcoholics had more [³H]pyridoxal and a lower percentage of [³H]pyridoxal-P than those of the controls. The total concentration of plasma pyridoxal-P was lower in the alcoholics than in the controls and did not change upon incubation of whole blood with pyridoxine or upon dialysis. The erythrocytes of the alcoholics and controls had similar concentrations of pyridoxal-P that increased 2.5-fold upon incubation of whole blood with pyridoxine for 30 min and returned to the initial concentrations upon dialysis. The amount of [³H]pyridoxal and [³H]pyridoxal-P bound to protein was assessed by treating hemolysate and plasma samples with borohydride before dialysis. More ³H was bound to protein in the erythrocytes than in the plasma. The amount of protein-bound ³H in the erythrocytes of the alcoholics was lower than that of the controls, whereas the amount of protein-bound ³H in plasma was similar in both groups. It is concluded that less of the pyridoxal-P and pyridoxal was protein bound, and the pyridoxal-P was more susceptible to hydrolysis to pyridoxal by phosphatases in the erythrocytes of the alcoholics compared with the controls.     

Association of Plasma Retinol Binding Protein-4 (RBP4) and Sonographic Grading of Fatty Liver in Obese Iranian Children

Background

The prevalence of obesity and its related comorbidities, such as fatty liver, in children is increasing worldwide mostly due to changes in diet and life-style. Many serological markers have been suggested for screening of fatty liver but investigations for finding more reliable factors are still in progress.

Objectives

This study aimed to investigate the correlation between the level of retinol binding protein-4 (RBP4) in the serum and sonographic grading of fatty-liver severity in obese Iranian children.

Patients and Methods

This case-control, double-blind study involved 51 obese children aged between five and 17 years as the case group. In addition, 35 healthy lean children with no liver problems were selected as the control group. Plasma RBP4 (using an ELISA), serum triglycerides (TG), low-density-lipoproteins (LDL), high-density-lipoproteins (HDL), total-cholesterol (Chol), and body mass index (BMI) were measured. Grading the severity of the fatty liver condition was done by an expert radiologist in the case group.

Results

RBP4 levels in obese children (19 482.9 ± 3 302.2 pg/ml) were higher than those found in the lean control group (14 295.68 ± 2 381.3 pg/ml) (P < 0.05). In the obese patients, RBP4 levels showed a significant correlation with the grade of fatty liver and BMI (P < 0.05).

Conclusions

It was found that the level of RBP4 had a strong correlation with the severity of fatty liver. Therefore, RBP4 may be considered as a useful, noninvasive predictive biomarker of intrahepatic lipid content in obese children prior to using radiological investigations. In particular, abdominal sonography, for the evaluation of intrahepatic lipid content in obese patients, as the sensitivity of a sonography is decreased due to the increased thickness of the abdominal wall as a result of fat deposits.     

炎性小体在脑缺血中的作用

固有免疫在脑缺血后炎性损伤中发挥重要作用,其中炎性小体被认为是一个关键因素.炎性小体是一种大分子蛋白质复合物,可识别各种病原体相关分子模式和损伤相关分子模式介导免疫炎性反应.研究显示,脑缺血或脑缺血再灌注可诱导NLRP1和NLRP3炎性小体激活.文章对炎性小体的结构、活化、调控以及在脑缺血中的作用进行了综述.     

自发性高血压大鼠心肌细胞蛋白激酶C活性的变化及其与心肌细胞凋亡的关系

目的 :研究自发性高血压大鼠 (SHR)心肌细胞蛋白激酶 C(PKC)活性的动态变化及其与心肌细胞调亡的关系。　　方法 :将 17只 SHR分为 3组 :1月龄 SHR组 (n=6 )、10月龄 SHR组 (n=6 )和 18～ 2 0月龄 SHR组 (n=5 )。每组均等数按鼠龄、体重及雌雄配对配予 WKY大鼠做对照。采用同位素法、电镜技术和末端脱氧核糖核苷酸转移酶介导的脱氧三磷酸尿苷缺口末端标记法检测 SHR左心室心肌细胞 PKC活性和心肌细胞凋亡的变化。采用直线回归分析确定PKC活性与心肌细胞凋亡的关系。　　结果 :11月龄 SHR组和 18～ 2 0月龄 SHR组心肌细胞膜 PKC活性显著低于同龄 WKY大鼠组 ,而心肌细胞凋亡却显著高于同龄 WKY大鼠组 (P均 <0 .0 1) ;但 10月龄 SHR组心肌细胞膜 PKC活性显著高于同龄 WKY大鼠组、1月龄 SHR组和 18～ 2 0月龄 SHR组而心肌细胞凋亡却显著低于同龄 WKY大鼠组 (P均 <0 .0 1) ;各组间心肌细胞浆 PKC活性无显著性差异 (P均 >0 .0 5 )。 2 SHR心肌细胞膜 PKC活性与心肌细胞凋亡指数呈显著负相关 (P<0 .0 5 ) ,但心肌细胞浆 PKC活性与心肌细胞凋亡指数不相关 (P>0 .0 5 )。　　结论 :心肌细胞膜 PKC活性降低可能与 SHR心肌细胞凋亡增加和充血性心力衰竭相关 ,而心肌细胞膜 PKC活性增加可能与 SHR心肌细胞凋亡减少和     

Role of the proteasome in ethanol-induced liver pathology

The ubiquitin-proteasome system has come to be known as a vital constituent of mammalian cells. The proteasome is a large nonlysosomal enzyme that acts in concert with an 8.5 kDa polypeptide called ubiquitin and a series of conjugating enzymes, known as E1, E2 and E3, that covalently bind multiple ubiquitin moieties in a polyubiquitin chain to protein substrates in a process called ubiquitylation. The latter process targets protein substrates for unfolding and degradation by the 26S proteasome. This enzyme system specifically recognizes and degrades polyubiquitylated proteins, many of which are key proteins involved in cell cycle regulation, apoptosis, signal transduction, and antigen presentation. The 26S proteasome contains a cylinder-shaped 20S catalytic core that, itself, degrades proteins in an ATP- and ubiquitin-independent manner. The 20S form is actually the predominant enzyme form in mammalian cells. Proteolysis by the constitutive 20S proteasome is vital in removing oxidized, misfolded and otherwise modified proteins. Such degradation is critical as a means of cellular detoxification, as intracellular accumulation of damaged and misfolded proteins is potentially lethal. Studies have shown that inhibition of proteasome activity can lead to cell death. Ethanol and its metabolism cause partial inhibition of the proteasome. This leads to a number of pleiotropic effects that can affect a variety of cellular processes. This critical review describes important aspects of ethanol metabolism and its influence on the proteasome. The review will summarize recent findings on: (1) the interactions between the proteasome and the ethanol metabolizing enzyme, CYP2E1; (2) the dynamics of proteasome inhibition by ethanol in animal models and cultured cells; (3) ethanol-elicited suppression of proteasome activity and its effect on signal transduction; (4) The role of proteasome inhibition in cytokine production by liver cells; and (5) ethanol elicited suppression of peptide hydrolysis and the potential effects on antigen presentation. While the principal focus is on alcohol-induced liver injury, the authors foresee that the findings presented in this review will prompt further research on the role of this proteolytic system in other tissues injured by excessive alcohol consumption.     

Long-Term Ethanol Exposure Impairs Neuronal Differentiation of Human Neuroblastoma Cells Involving Neurotrophin-Mediated Intracellular Signaling and in Particular Protein Kinase C

Background: Revealing the molecular changes in chronic ethanol‐impaired neuronal differentiation may be of great importance for understanding ethanol‐related pathology in embryonic development but also in the adult brain. In this study, both acute and long‐term effects of ethanol on neuronal differentiation of human neuroblastoma cells were investigated. We focused on several aspects of brain‐derived neurotrophic factor (BDNF) signaling because BDNF activates the extracellular signal‐regulated kinase (ERK) cascade, promoting neuronal differentiation including neurite outgrowth. Methods: The effects of ethanol exposure on morphological differentiation, cellular density, neuronal marker proteins, basal ERK activity, and ERK responsiveness to BDNF were measured over 2 to 4 weeks. qRT‐PCR and Western blotting were performed to investigate the expression of neurotrophin receptor tyrosin kinase B (TrkB), members of the ERK‐cascade, protein kinase C (PKC) isoforms and Raf‐Kinase‐Inhibitor‐Protein (RKIP). Results: Chronic ethanol interfered with the development of a neuronal network consisting of cell clusters and neuritic bundles. Furthermore, neuronal and synaptic markers were reduced, indicating impaired neuronal differentiation. BDNF‐mediated activation of the ERK cascade was found to be continuously impaired by ethanol. This could not be explained by expressional changes monitored for TrkB, Raf‐1, MEK, and ERK. However, BDNF also activates PKC signaling which involves RKIP, which finally leads to ERK activation as well. Therefore, we hypothesized that ethanol impairs this branch of BDNF signaling. Indeed, both PKC and RKIP were significantly down‐regulated. Conclusions: Chronic ethanol exposure impaired neuronal differentiation of neuroblastoma cells and BDNF signaling, particularly the PKC‐dependent branch. RKIP, acting as a signaling switch at the merge of the PKC cascade and the Raf/MEK/ERK cascade, was associated with neuronal differentiation and significantly reduced in ethanol treatment. Moreover, PKC expression itself was even more strongly reduced. In contrast, members of the Raf‐1/MEK/ERK cascade were less affected and the observed changes were not associated with impaired differentiation. Thus, reduced RKIP and PKC levels and subsequently reduced positive feedback on ERK activation provide an explanation for the striking effects of long‐term ethanol exposure on BDNF signal transduction and neuronal differentiation, respectively.