Detection of a soluble form of BACE-1 in human cerebrospinal fluid by a sensitive activity assay

BACKGROUND: Formation of deposits of the insoluble amyloid beta-peptide is believed to be causally related with neurodegeneration in Alzheimer disease (AD). The beta-peptide originates from a larger amyloid precursor protein (APP) by the action of proteolytic enzymes. The first proteolytic event leading to amyloid formation is the cleavage of APP by the membrane-bound aspartyl protease BACE-1, also known as memapsin-2. Inhibition of BACE-1 is thought to be a therapeutic approach to AD. Measuring BACE-1 activity in biological samples would be useful to elucidate the mechanism of AD and for development of AD drugs. METHODS: We developed a sensitive and specific activity assay for BACE-1. The assay is based on a genetically engineered proenzyme that is specifically activated by BACE-1. The resulting active enzyme is measured with a chromogenic substrate. The use of 2 coupled reactions produces a detection limit as low as 0.4 pmol/L. RESULTS: The assay detected BACE-1 activity in extracts of human brain tissue as well as, unexpectedly, in human cerebrospinal fluid (CSF). Gel electrophoresis and Western blotting identified the BACE-1 present in CSF as a truncated soluble form of the originally membrane-bound BACE-1. CONCLUSION: Detection of the soluble form of BACE-1 in CSF, a relatively easily accessible biological fluid, may be useful for monitoring the effects of drug candidates in vivo and may have diagnostic or prognostic applications.     

Cooling and response to hydrogen peroxide in human saphenous vein: role of the endothelium

In the present work we studied the responses of human saphenous vein to H2O2 and effects of moderate cooling on these responses with analysis of the role of endothelium. H2O2 (10(-7)-10(-2) M) induced concentration-dependent contraction in the intact human saphenous vein strips at both temperatures. At 28 degrees C, the maximal contraction induced by H2O2 was significantly lower than that at 37 degrees C. Compared with intact strips, the sensitivity and the maximal contraction to H2O2 were significantly enhanced in endothelium-denuded strips at 37 and 28 degrees C. However, pD2 values and maximal contractions were not significantly different in endothelium-denuded strips at different temperatures. Pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME) increased significantly the maximal contraction and sensitivity to H2O2 at 37 and 28 degrees C. The contractions increased by L-NAME were restored by the pre-incubation of l-arginine (10(-3) M) at every temperature studied. The contractile responses of intact human saphenous veins to H2O2 were reduced significantly by 10(-5) M indomethacin at both temperatures. Our results suggest that H2O2-induced contraction of human saphenous vein are mediated by its direct effect on the smooth muscle and by the generation of products of the cyclooxygenase pathway from the endothelium. Signalling pathways of these contractile effects are the same at 37 and 28 degrees C. Under normal temperature conditions, the contraction to H2O2 is possibly modulated by endothelial nitric oxide. Cooling reduces the contraction to H2O2 by increasing release of nitric oxide.     

Collagen of the normal and the varicose human saphenous vein: a biochemical study

Various types of collagen (I, III, IV, V) were identified in normal and varicose human saphenous veins using pepsin digestion and cyanogen bromide digestion followed by SDS-polyacrylamide gel electrophoresis. Varicose veins were found to have a higher collagen content than normal veins. This is consistent with the morphological fibrosis which has regularly been described. No essential differences were found in the collagen composition of dilated and apparently healthy portions of varicose veins.     

A sensitive fluorescent assay for N-acetyltransferase activity in human lymphocytes from newborns and adults.

We have developed a simplified assay for the enzyme N-acetyltransferase, based upon the loss of fluorescence after acetylation of the substrate p-aminobenzoic acid. This method is sufficiently sensitive to permit the quantitation of N-acetyltransferase activity in 10(5) human lymphocytes. Using this method, we have compared the level of N-acetyltransferase activity in lymphocytes from adult peripheral blood and from cord blood samples.     

Effect of cooling on the responses of human saphenous vein to fentanyl, remifentanil and sufentanil

We studied the vasodilatory effects of fentanyl, remifentanil and sufentanil on the human saphenous vein strips at 37, 32 and 28 degrees C. Fentanyl produced concentration-dependent relaxation of human saphenous vein strips precontracted with 5-hydroxytryptamine (5-HT) at every temperature studied. Compared with vein strips at 37 degrees C, relaxant responses to each one concentration of fentanyl were significantly reduced at 32 and 28 degrees C. Remifentanil relaxed vein strips in a concentration-dependent way and the relaxation for all concentrations were significantly greater at 32 and 28 degrees C compared with 37 degrees C. Sufentanil produced concentration-dependent relaxation in saphenous vein strips precontracted with 5-HT. These relaxant responses were similar at 32 degrees C compared with 37 degrees C. When bath temperature was lowered from 37 to 28 degrees C, the relaxant responses to sufentanil were significantly reduced. In summary, the present study suggests that cooling reduces the relaxation caused by fentanyl and sufentanil on human saphenous veins but augments the relaxation with remifentanil. The augmented vasodilatory effect of remifentanil with cooling may be useful on systemic vascular resistance and organ preservation under hypothermic conditions like cardiopulmonary bypass surgery.     

Diagnosis of niemann-pick disease using a simple and sensitive fluorimetric assay of sphingomyelinase activity

Phosphodiesterase activity of cultured cells was determined with bis-(4-methylumbelliferyl) phosphate as substrate. In the presence of Triton X-100 an acid component was evident and results indicated that this enzyme was identical with sphingomyelinase. Acid phosphodiesterase activity was specifically inhibited by sphingomyelin. In fibroblasts from patients with Niemann-Pick diseases types A, B and C, acid phosphodiesterase activity was deficient whereas neutral activity was normal. Neutral activity could, however, be removed by acid precipitation or by binding to DEAE-cellulose. Hence a simple and sensitive fluorimetric method is described for the assay of sphingomyelinase activity in the diagnosis of Niemann-Pick disease.     

The application of a simple, sensitive disc assay for sialyltransferase activity to particulate and soluble enzyme preparations and its use in measuring enzymatic activity in human plasma samples.

A simple assay for the determination of sialyltransferase activity is described. The method involves the isolation of the radioactive reaction product on cellulose paper discs washed with trichloroacetic acid, thereby greatly facilitating the handling of large numbers of assays. This procedure is both more accurate and more sensitive than that involving precipitation with protein denaturants and separation by centrifugation, and is applicable to both soluble and particulate enzyme preparations. The application of the method to the determination of sialytransferase in human plasma is detailed. Enzyme activity is elevated two-fold in both cancer patients and patients with non-malignant disease. This suggests that the diagnostic use of determinations of sialyltransferase activity may be limited by its non-specificity.     

Alpha adrenoceptor subtypes and receptor reserve in human versus canine saphenous vein: sensitivity to blockade by nitroglycerin

The purpose of this investigation was to determine the subtypes of alpha adrenoceptors present in human saphenous vein and to determine if there is a large receptor reserve for phenylephrine as has been demonstrated in canine saphenous vein. The subtypes of alpha adrenoceptors found in isolated human saphenous vein were determined using selective alpha-1 and alpha-2 adrenoceptor agonists and antagonists. Prazosin, a selective alpha-1 antagonist, produced a parallel shift of the concentration response curve to phenylephrine, a selective alpha-1 agonist, with no significant reduction in the maximal response. Yohimbine, a selective alpha-2 antagonist, produced a parallel shift of the concentration response curve to B-HT 920, a selective alpha-2 agonist, with no reduction in the maximal response. The pA2 values obtained for prazosin and yohimbine in human saphenous vein agreed closely with corresponding values obtained in canine saphenous vein. These results demonstrate that both alpha-1 and alpha-2 adrenoceptors exist in human saphenous vein. Phenoxybenzamine (10(-7) M), an irreversible alpha-1 adrenoceptor antagonist, markedly reduced the maximal response produced by phenylephrine, an agonist with high intrinsic activity, with no significant shift in the concentration response curve in human saphenous vein, suggesting that there was little or no alpha-1 receptor reserve for phenylephrine. The sensitivity of alpha-1 versus alpha-2 adrenoceptor-mediated vasoconstrictor responses to nitroglycerin were compared in human and canine saphenous veins. In both species, nitroglycerin blocked the vasoconstrictor response produced by stimulation of alpha-2 adrenoceptors to the same degree.(ABSTRACT TRUNCATED AT 250 WORDS)     

Toxicity of sitosterol to human umbilical vein endothelial cells in vitro.

The pathogenesis of the early development of atherosclerosis in sitosterolaemia is unknown. The effect of sitosterol on vascular endothelial cells in vitro was investigated by culturing human umbilical vein endothelial cells in the presence of up to 0.7 mmol l-1 of sitosterol. Liposomes were used to supply the high sterol concentrations. Exposure to 0.7 mmol l-1 of sitosterol for 72 h caused contraction of the endothelial cells and increased release of intracellular lactate dehydrogenase. After 96 h incubation the cells were partly detached from the substrate. At this time-point 0.35 mmol l-1 of sitosterol also caused perturbation of the endothelial cells. However, we could not confirm previous reports that tissue plasminogen activator production was enhanced by sitosterol.     

Quantification of human serum lipoprotein Lp(a): zone immunoelectrophoresis assay,a new sensitive method as compared to electroimmuno assay

This study aimed at improving the immunological procedures for the quantification of human serum lipoprotein Lp(a). Lipoprotein Lp(a) was estimated by two immunoelectrophoretic methods: electroimmunoassay and zone immunoelectrophoresis assay.Whereas the electroimmunoassay gave curvilinear calibration lines (in peak height versus concentration plots) the zone immunoelectrophoresis assay revealed linearity in the same concentration range. The results obtained are in good agreement with each other (r = 0.975). The inter-assay coefficient of variation for the zone immunoelectrophoresis assay was 12% as determined by the use of a lyophilised reference serum. Zone immunoelectrophoresis assay is recommended for the routine quantification of lipoprotein Lp(a). Although the levels for total cholesterol (p < 0.0025), HDL-cholesterol (p < 0.001) and triglycerides (p < 0.025) differed significantly between male (n = 24) and female (n = 22) individuals, no difference could be detected comparing the frequency distributions of Lp(a)-levels found for the male and the female group.     

超声引导下注射新型泡沫硬化剂治疗大隐静脉曲张

目的 观察超声引导下注射新型泡沫硬化剂治疗大隐静脉曲张的短期疗效.方法 对无深静脉及交通支静脉功能不全的重度大隐静脉曲张40条患肢(38例患者,其中2例为双下肢大隐静脉曲张),在超声引导下注射新型泡沫硬化剂Fibro-Vein对大隐静脉主干进行硬化治疗.结果 40条患肢均在超声引导下注射治疗成功,7条(7/40)大隐静脉于治疗后1个月复诊时实施了第2次注射治疗.随访1～12个月,全部患者(包括经2次治疗者)下肢活动后酸胀、乏力消失;下肢明显曲张畸形的静脉消失,其中8条(8/40)患肢远端存在局限性轻度曲张的静脉;无严重并发症发生.术后3个月复查彩色多普勒超声显示,曲张的大隐静脉较治疗前变细,22条(22/40)治疗大隐静脉的部分血管段实变,18条(18/40)静脉壁回声增强,并可见不规则、不均质中等实性回声紧密黏附于血管壁,挤压远端肢体时管腔内可见流沙样缓慢流动的血流信号,其中8条静脉(8/18)有少量反流.结论 超声引导下注射新型泡沫硬化剂治疗大隐静脉曲张方便、有效、痛苦小、无瘢痕形成、无严重并发症,可重复进行,近期疗效佳,是一种治疗与美容兼备的微创治疗方法.     

Study of pro-angiogenic activity of astilbin on human umbilical vein endothelial cells in vitro and zebrafish in vivo

Astilbin is a dihydroflavonol natural product isolated from a variety of food and medicinal herbs (e.g. Smilax glabra Roxb.), and its mechanism of action in vascular pharmacology remains unclear. The aim of this study was to investigate the pro-angiogenic effects of astilbin and its putative mechanism of action. Briefly, our in vitro studies showed a dose-dependent ability of astilbin to increase the ability of HUVECs to proliferate and migrate, and undergo cell invasion and tube formation. Moreover, astilbin significantly increased the expression levels of several major proteins involved in the angiogenesis pathway, e.g. PI3K, Akt, p38 and ERK1/2. Our in vivo studies demonstrated the ability of astilbin to significantly restore the blood vessel loss induced by VRI in a VRI-induced vascular insufficiency zebrafish model. In conclusion, in this study we first demonstrate that astilbin exhibits pro-angiogenic activity in HUVECs and VRI-induced vascular insufficient zebrafish, possibly through the activation of the PI3K/Akt and MAPK/ERK dependent signaling pathways. These findings suggest that astilbin could be further developed as a potential agent in the prevention or treatment of insufficient angiogenesis related diseases in the future.

Pro-angiogenic activity of astilbin on endothelial cells in vitro and zebrafish in vivo.     

体外培养下可持续分泌人血管抑素和内皮抑素基因工程细胞对血管生成的抑制效应

背景:以往实验构建了可持续分泌人血管抑素和内皮抑素的基因工程细胞,即人血管抑素/293细胞和人内皮抑素293细胞,并已证明可持续分泌人血管抑素蛋白和人内皮抑素蛋白.但尚不了解其分泌物是否能发挥血管生成的抑制作用.目的:观察以基因工程技术构建的人血管抑素/293细胞和人内皮抑素,293细胞对鸡胍尿囊膜血管新生的抑制效应.设计、时间及地点:随机对照动物实验,于2006-0612007-01在解放军总医院老年医学研究所细胞生物学研究室完成.材料:Hek/293细胞为人胚胎肾细胞,由解放军军事医学科学院提供.人血管抑素/293细胞和人内皮抑素/293细胞由课题组前期构建.SPF级鸡胚购自北京维通利华实验动物有限公司.方法:将鸡胚消毒后,置于37℃无菌恒温箱中孵育5 d,在距胚头1 cm两条卵黄静脉之间的卵壳投影部位磨切暴露尿囊膜,制成假气室,用无菌滤纸封闭窗口.制成鸡胚尿囊膜模型,备用.用灭菌蒸馏水配制体积分数为0.5%甲基纤维素溶液,制成甲基纤维素小杯.卵壳开窗后第3天,分别吸取浓缩的293细胞、人血管抑素/293细胞、人内皮抑素/293细胞培养上清液各20 μ L.或人血管抑素/293细胞和人内皮抑素/293细胞培养上清液各10 μ L,加于甲基纤维素小杯中,将其置于鸡胚 尿囊膜的两条大血管之间的无血管区.主要观察指标:观察尿囊膜血管生成的变化.结果:共培养9 d时,与单纯293细胞上清液组相比,人血管抑素/293细胞上清液或人内皮抑素/293细胞上清液组,甲基纤维素小杯内及邻近区的鸡胚尿囊膜血管明显发育不良,血管分布稀疏,数量明显减少,其一级血管数量盟未明显减少,但其管径较细,而二级血管和二级以下的血管数量明显减少、管径明显变细,伸入尿囊膜边缘区的血管数量减少,管径明显变细.与人血管抑素/293细胞组或人内皮抑素/293细胞组相比,人血管抑素/293细胞+人内皮抑素/293细胞组的一级血管数量和管径未见明显变化,而其二级血管和三级以下血管数量明显减少,血管树消失.结论:人血管抑素/293细胞和人内皮抑素/293细胞的分泌物对鸡胚尿囊膜的血管新生均具有明显的抑制作用,且联合用药抑制作用增强.     

葡萄糖转运蛋白1在肝癌组织中的表达与缺氧诱导因子1α表达的关系及临床意义

[摘要]：目的：探讨肝细胞癌组织中葡萄糖转运蛋白1（Glut1）和缺氧诱导因子1α（HIF-1α）的表达情况及其临床意义，并分析两者的相关性。方法：用免疫组织化学Envision法检测45例肝细胞癌、11例肝正常组织中Glut1和HIF-1α的表达。结果：Glut1和HIF-1α在肝癌组织中的阳性率明显高于正常肝组织（P＜0.05)。在肝癌组织中Glut1和HIF-1α的表达均与肿瘤大小、临床分期、有无淋巴结转移、有无门静脉癌栓有关（P＜0.05)。肝癌组织中Glut1表达越强，HIF-1α表达也越强，两者的表达呈正相关(r=0.583,P＜0.01)。结论：Glut1和HIF-1α的异常表达与肝癌的发生、发展有一定的关系。可能存在HIF-1α通过上调Glut1的表达促进肝细胞癌的发生的机理。     

Neutrophil adherence to human endothelium in vitro occurs by CDw18 (Mo1, MAC-1/LFA-1/GP 150,95) glycoprotein-dependent and -independent mechanisms

Components of the CDw18 leukocyte surface glycoprotein complex (Mo1/LFA-1/GP 150,95 or MAC-1, LFA-1 family) are required for some adhesion-related functions of human neutrophils (PMNs). We evaluated the ability of monoclonal antibodies (MoAb) directed against specific determinants on the CDw18 glycoproteins to inhibit neutrophil adherence to cultured human endothelial cells (EC) stimulated by a variety of agonists, including thrombin and leukotriene C4, which induce the EC-dependent adhesion of PMNs. MoAb 60.3, an antibody that binds to an epitope common to the 3 heterodimer subunits of the neutrophil CDw18 complex, potently inhibited (90-100%) the rapid (5-30 minute) adherence response stimulated by N-formyl-methyionyl-leucyl-phenylalanine, leukotriene B4, platelet-activating factor, phorbol myristate acetate, Ionophore A23187, and tumor necrosis factor. MoAbs directed against epitopes on the alpha polypeptide of the CD11b (Mol, MAC-1) heterodimer also inhibited PMN adherence to EC and to cell-free surfaces induced by these agonists. In contrast, the anti-CDw18 MoAbs had a trivial effect on maximal EC-dependent neutrophil adherence stimulated by thrombin and leukotriene C4, and incompletely inhibited PMN adherence induced by these agonists under submaximal conditions. These findings indicate that there is an alternative mechanism for neutrophil adherence, presumably resulting from molecular alterations of the EC surface, that does not require the PMN CDw18 glycoproteins. They also suggest that the inability to adhere to endothelium may not completely account for the defect in chemotaxis that is observed in vivo in neutrophils that are deficient in the CDw18 complex.     

Characterisation of a highly sensitive troponin I assay and its application to a cardio-healthy population

Abstract Background: Abbott Diagnostics have developed a new highly sensitive troponin I (hs-TnI) assay. We have assessed its analytical characteristics and applied the assay to a population of apparently cardio-healthy persons. Methods: We assessed imprecision, bias compared to the previous generation assay, matrix effects, and interferences and applied the assay to an apparently healthy population, deriving the 99th percentile limit of the distribution of values in reference populations for men and women separately. Results: The dynamic range of the assay was ranged from 0.5-50,000 ng/L (pg/mL). The 10% CV was at a concentration of 3.9 ng/L, and the 20% CV was at a concentration of 1.8 ng/L. The new and current version of the TnI assay were highly correlated [slope: 0.98 (95%CI:0.88-1.07), y-intercept:1.20 (95%CI:-2.35-4.75) r2=0.99]. The 99th percentile limit of the distribution of values in a reference population was different for males and females: for males 14.0 ng/L and for females 11.1 ng/L and at these concentrations the assay CV was 5.0%. TnI was detectable in nearly all patient samples from the healthy reference population (98.6%). Conclusions: This new hs-TnI assay is able to measure to an order of magnitude lower than the current generation TnI assay from the same manufacturer. With TnI being detectable in nearly all apparently healthy subject samples this suggests that TnI presence does not always indicate cardiomyocyte necrosis.     

Evaluation of a rapid, sensitive and specific assay for the determination of collagenolytic activity in biological samples.

Several methods for the determination of collagenolytic activity were compared from the point of view of sensitivity, selectivity, simplicity and practical value for large numbers of biological samples. A labelled collagen substrate was prepared using [3H]acetic anhydride. The specificity of the assay as well as conditions allowing an optimum detection limit were investigated. The influence of low temperatures, lyophilisation and salt concentration on Clostridium histolyticum collagenase have been investigated.     

食管鳞状细胞癌患者细胞分化抑制因子-1和低氧诱导因子-1α的表达及意义

目的 研究细胞分化抑制因子-1（Id-1）、缺氧诱导因子-1α（HIF-1α）在食管鳞状细胞癌中的表达及其相关性,探讨其在食管鳞状细胞癌发生发展中的意义.方法 选取食管鳞状细胞癌60例,正常食管组织40例,应用免疫组化SP法分别检测Id-1、HIF-1α在两组中的表达,分析其与食管鳞状细胞癌临床病理特征的关系.结果 Id-1和HIF-1 0在食管鳞状细胞癌组织中表达率分别为81.7％和73.3％,与正常组比较差异有统计学意义（P＜0.05）,且Id-1和HIF-1α的表达与食管鳞状细胞癌的浸润深度、TNM分期及淋巴结转移有关,并随着浸润深度的加深、临床分期的提高及淋巴结的转移阳性率增高,差异有统计学意义（P＜0.05）.在食管鳞状细胞癌组织中,Id-1和HIF-1α的表达呈正相关（r=0.396,P=0.002）.结论 Id-1和HIF-1d与食管鳞状细胞癌的发生发展密切相关,,联合检测可作为判断食管鳞状细胞癌浸润和转移的重要指标.     

Activation of Akt1 by human 5-hydroxytryptamine (serotonin)1B receptors is sensitive to inhibitors of MEK.

Akt1/protein kinase B and the mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase 1 (ERK1) and ERK2 have been shown to promote cell survival in a cell-specific manner. Since many receptors activate both pathways, inhibitors are commonly used to study the relative role of each pathway. In the present study, we examined the effects of PD098059 and U0126, two structurally dissimilar inhibitors of MAP kinase kinase (MEK1/2), on the activation of ERK and Akt stimulated by human 5-hydroxytryptamine(1B) (serotonin) (5-HT1B) receptors. Surprisingly, pathways for activation of both ERK and Akt were found to be sensitive to the two MEK inhibitors at concentrations commonly used to selectively inhibit the activation of ERK. Both compounds caused complete inhibition of phosphorylation of ERK and a maximal 60% inhibition of 5-HT1B receptor-mediated phosphorylation of Akt. Inhibition of Akt activation required almost complete inhibition of ERK. Transfection with cDNA for activated forms of MEK1/2 caused increased phosphorylation of ERK but not of Akt, demonstrating that independent activation of MEK/ERK was insufficient for activation of Akt. Therefore, it is not clear whether inhibition of activation of Akt resulted from selective inhibition of MEK or from additional actions on other unidentified common pathways. Nevertheless, our findings that PD098059 and U0126 inhibit activation of Akt at commonly used concentrations demonstrate that in at least some systems, these compounds inhibit activation of both ERK and Akt, and cannot be used to discern the relative roles of each pathway in mediating cellular responses.     

Intravenous anesthetics inhibit human paraoxonase-1 (PON1) activity in vitro and in vivo

ObjectivesHere we evaluated the in vitro and in vivo effects of the intravenous anesthetics, etomidate, propofol, and ketamine, on the activity of human serum paraoxonase (hPON1).Design and methodshPON1 was purified from human serum using simple chromatographic methods, including DEAE–Sephadex anion exchange and Sephadex G-200 gel filtration chromatography.ResultsThe three anesthetics dose-dependently decreased in vitro hPON1 activity. Inhibition mechanisms are: etomidate was noncompetitive, propofol was competitive, and ketamine was uncompetitive. In vivo studies were performed on five patients for each drug. PON1 was significantly inhibited by 0.3 mg/kg etomidate (p < 0.05), 2 mg/kg propofol (p < 0.001), and 1 mg/kg ketamine (p < 0.05) for up to 5 min following intravenous administration.ConclusionsOur results showed that anesthetics significantly inhibit hPON1 activity, both in vitro and in vivo, with rank order etomidate > propofol > ketamine in vitro, and propofol > etomidate > ketamine in vivo.