Adjustment of metabolism,catecholamines and β-adrenoceptors to 90 min of cycle ergometry

Adrenaline infusion of 0.1 g · kg^–1 · min^–1 in healthy volunteers results in an increase of hepatic glucose production, an increase of the absolute number of occupied -adrenoceptors and specific changes in metabolism. To compare these effects with the changes induced by an endogenous catecholamine release, we investigated healthy volunteers during cycle ergometry. After fasting at least 14 h seven healthy subjects exercised for 90 min at an intensity of 20% below their individual anaerobic threshold. The rate of glucose production as well as the turnover rates of alanine and leucine were calculated using stable isotope tracers. High and low affinity -adrenergic binding sites on lymphocytes were determined by an equilibrium binding assay with (–)¹²⁵ Iodocyanopindolol. After 90 min of cycling the rate of appearance of glucose increased significantly from means of 2.0 (SD 0.2) to 2.65 (SD 0.50) mg · kg^–1 · min^–1 with unchanged blood concentrations of glucose and lactate. The flux of the amino acids alanine and leucine decreased significantly from means of 0.91 (SD 0.21) to 0.62 (SD 0.14) mg · kg^–1 · min^–1 and from 0.40 (SD 0.05) to 0.32(SD 0.04) mg · kg^–1 · min^–1, respectively. The mean free fatty acid concentration increased significantly from 0.65 (SD 0.33) to 1.27 (SD 0.45) mmol · l^–1 during the endurance trial. The increase of glucose turnover and the decrease of amino acid flux point to a metabolic shift towards enhanced utilization of free fatty acids. Adrenaline and noradrenaline concentrations showed a moderate but significant increase from means of 0.61 (SD 0.20) to 0.99 (SD 0.36) nmol · l^–1 and from 2.27 (SD 0.75) to 3.46 (SD 0.38) nmol · 1^–1, respectively. The number of high affinity -adrenergic binding sites per cell (-adrenoceptors) nearly doubled from 770 (SD 130) to 1490 (SD 150) during 90 min of cycling. The observed endogenous plasma catecholamine concentrations were not sufficient to change significantly the relative receptor occupancy. This would seem to indicate that the aerobic exercise induced effects depended more on the absolute number of occupied -adrenoceptors than on their relative receptor occupancy. When compared to the results of the adrenaline infusion experiment the increases of the hepatic glucose production and the increase of -adrenoceptors were very similar in both groups despite ten times higher adrenaline plasma concentrations in the infusion group. This would seem to indicate that -adrenoceptors mediated effects do not correlate with catecholamine plasma concentrations.     

酪氨酸蛋白激酶抑制剂的高通量筛选模型

目的建立酪氨酸激酶抑制剂的高通量筛选模型。方法以多聚酪氨酸多肽为底物,用提取的酪氨酸激酶催化酪氨酸的磷酸化,用酶标记的单克隆抗体检测酪氨酸激酶的活性,根据待测样品对激酶活性的抑制程度,筛选酪氨酸激酶抑制剂。结果酶标板包被液的浓度为20mg·L-1PGT,使用25mg·L-1蛋白质的PTK初提物,在37℃条件下孵育60min,再使用2000倍稀释的辣根过氧化物酶标记的小鼠抗磷酸化酪氨酸单克隆抗体IgG2bk与反应产物结合、测定。同一微孔板各样品间的测定误差为0.26,微孔板间和日间的测定误差分别为0.79和0.69。Genistein对PTK的IC50为110μmol·L-1。从收集的7680个样品中,筛选出具有活性的样品16个,选中率约2‰。结论建立的酪氨酸激酶抑制剂高通量药物筛选模型,具有灵敏度高、结果稳定的特性,在每块实验上同时设立空白和对照,测定结果具有可比性。     

DOK3 PTK7在不同级别结肠腺瘤组织中的表达及意义探讨

目的分析对接蛋白3(DOK3)和蛋白酪氨酸激酶7(PTK7)在不同级别结肠腺瘤组织中的表达情况,探讨其在结肠癌的发生、发展机制中的作用。方法收集2015-01~2018-12广西壮族自治区南溪山医院经病理科确诊的结肠腺瘤伴低级别异型增生(LGD)标本22例,结肠腺瘤伴高级别异型增生(HGD)标本23例,结肠癌标本22例,瘤旁非肿瘤黏膜组织标本20例。采用免疫组织化学染色法检测不同类型标本DOK3和PTK7的表达情况。结果DOK3和PTK7均主要表达于腺上皮的胞浆和胞膜。瘤旁非肿瘤黏膜和LGD组织的DOK3高表达率均显著高于HGD和结肠癌组织(P<0.05);HGD组织的DOK3高表达率也显著高于结肠癌组织(P<0.05)。结肠癌和HGD组织的PTK7高表达率均显著高于瘤旁非肿瘤黏膜和LGD组织(P<0.05);结肠癌组织的PTK7高表达率也显著高于HGD组织(P<0.05)。结论DOK3可能是作为抑癌因子,而PTK7作为促癌因子参与结肠癌的发生、发展。     

Endoplasmic reticulum (ER) stress: hepatitis C virus induces an ER-nucleus signal transduction pathway and activates NF-kappaB and STAT-3

Human hepatitis C virus (HCV) is the leading cause of chronic hepatitis, which often results in liver cirrhosis and hepatocellular carcinoma. The HCV RNA genome codes for at least ten proteins. The HCV non-structural protein 5A (NS5A) has generated considerable interest due to its effect on interferon sensitivity via binding and inactivating the cellular protein kinase, PKR. It has been shown that NS5A engages in the endoplasmic reticulum (ER)-nucleus signal transduction pathway. The expression of NS5A in the ER induces an ER stress ultimately leading to the activation of STAT-3 and NF-kappaB. This pathway is sensitive to inhibitors of Ca(2+) uptake in the mitochondria (ruthenium red), Ca(2+) chelators (TMB-8, EGTA-AM), and antioxidants (PDTC, NAC, Mn-SOD). The inhibitory effect of protein tyrosine kinase (PTK) inhibitors indicates the involvement of PTK in NF-kappaB activation by NS5A. This implicates an alternate pathway of NF-kappaB activation by NS5A. The actions of NS5A have also been studied in the context of an HCV subgenomic replicon inducing a similar intracellular event. Thus, activation of NF-kappaB leads to the induction of cellular genes, which are largely antiapoptotic in function. These studies suggest a potential function of NS5A in inducing chronic liver disease and hepatocellular carcinoma associated with HCV infection.     

酪氨酸激酶抑制剂的进展与评价

目的:探讨酪氨酸激酶抑制剂的进展与临床应用评价。方法:采用国内外专业文献进行综述与评估。结果及结论:蛋白酪氨酸激酶是一类具有酪氨酸激酶活性的蛋白质,近年来其进展迅速,上市和临床应用广泛,其抑制肿瘤细胞的损伤修复、可使肿瘤细胞分裂阻滞。成为研发高效、低毒、特异性强的新型抗癌药的重要方向。     

痹颗粒对RA-MsPGN大鼠肾组织PTK活性的影响

目的探讨痹颗粒对RA-MsPGN大鼠模型肾组织PTK活性的影响,治疗RA-MsPGN的作用机制。方法按照Phospho Safe TM ExtR Action Reagent试剂盒提取蛋白质,按照K-LISA PTK Screening Kit试剂盒测定蛋白酪氨酸激酶(PTK)活性,并根据检测试剂盒提供的标准品直线回归方程计算PTKs的含量。结果RA-MsPGN模型大鼠与正常组相比肾组织PTK含量显著升高(P<0.001),雷公藤多苷片和痹颗粒各剂量组均能显著降低大鼠肾组织PTK含量(P<0.05或0.01)。结论痹颗粒对RA-MsPGN大鼠肾组织PTK活性有明显抑制作用,其治疗作用有可能是通过对PTK的作用而实现的。     

PRK联合PTK治疗近视伴颗粒状角膜营养不良（附1例报告）

目的介绍准分子激光角膜切削术（PRK）联合准分子激光治疗性角膜切削术（PTK）治疗近视伴颗粒状角膜营养不良患者的方法及疗效。方法先用PTK模式削除患者浅层病变角膜组织后,再用PRK模式对近视进行治疗。比较术前、术后患者角膜透明性和视力情况。结果经手术治疗后患者角膜混浊较术前明显减少．术后视力恢复至1．0。结论PRK联合PTK治疗近视伴颗粒状角膜营养不良具有良好的疗效,但远期疗效还需长期观察。     

PTK787在SCID小鼠-人AML模型中的抗骨髓血管生成作用研究

目的 观察酪氨酸激酶抑制剂PTK787在SCID小鼠-人急性髓系白血病模型中对血血管内皮生长因子受体-2mRNA (VEGFR-2mRNA)、外周血CD33阳性表达率以及骨髓微血管密度的影响,探讨PTK787抗急性髓系白血病的作用机制.方法 (1) 采用RT-PCR法分析30例不同组别之间SCID小鼠VEGFR-2mRNA水平的差异.(2) 利用流式细胞仪检测外周血CD33表达.(3) 采用SP免疫组化法,用兔抗人vWF (von Wille-brand factor)多克隆抗体标记骨髓内皮细胞,比较不同组别小鼠之间的骨髓微血管密度(MVD)的差异.结果 (1) 患病组SCID小鼠外周血VEGFR-2mRNA灰度扫描比为(0.3257±0.0709),高于正常组(0.0826±0.0315) (P<0.05),治疗组为(0.1411±0.0404),与正常组比较(P<0.05).(2) 外周血白细胞CD33的阳性率,治疗组(9.41±1.35%)及患病组(15.08±2.90%)与正常组(0.81±0.11%)比较,P值均<0.01,差异有统计学意义;治疗组与患病组比较,P<0.05,差异有统计学意义.(3) 患病组SCID小鼠中骨髓微血管数为(20.4±3.2)个/HP,显著高于正常组(14.2±2.1)个/HP(P<0.001),治疗组为(15.6±2.4)个/HP,与正常组相似(P>0.05).结论 PTK787在体内可以有效的抑制血管生成,一定程度具有抗白血病细胞增生的作用.