In Vitro Activity of Actinospectacin in Human Whole Blood

A method for testing antibacterial substances in whole blood is described. The test agent for the method was actinospectacin which reportedly has good in vivo activity, approximately in the range with chloramphenicol, but relatively poor in vitro activity in the common media. In human whole blood, however, the in vitro activity compares favorably with chloramphenicol thus indicating that whole blood may predict in vivo activity better than the usual bacteriological media.     

小鼠全血中谷胱甘肽过氧化物酶活力的微量测定法

报道了用分光光度计法直接测定小鼠全血中谷胱甘肽过氧化物酶GSH-PX活力的方法.取血样10μl,423nm为测定波长,三氯醋酸为蛋白沉淀剂,在pH6.5,3min的酶反应条件下,反应剩余的谷胱甘肽和其与DTNB试剂反应生成的颜色产物成线性相关.该法灵敏度高,重复性好,所需仪器简单,可成为科研及临床研究工作中分析全血中GSH-PX的重要方法之一     

Human Serum Cholinesterase,a Tetramer

AFTER electrophoresis of human serum in starch gel¹, there are at least four bands of Cholinesterase activity. Most of the enzyme is concentrated in the band with least mobility (C₄) while three faster moving bands (C₁, C₂, C₃) are of lower molecular weight as shown by gel filtration². Although electrophoresis in Polyacrylamide reveals additional bands³, all seem to be a single genetic entity as shown by experiments on persons homozygous for the silent gene^4,5. The C₄ enzyme has a molecular weight of at least 250,000 (ref. 6) and can be assumed to be composed of more than one polypeptide chain. By preparing an enzyme of altered mobility and forming hybrids of it with the usual enzyme, we have obtained results that suggest that Cholinesterase is a tetramer.     

Determination of Natural In Vivo Noble-Gas Concentrations in Human Blood

Although the naturally occurring atmospheric noble gases He, Ne, Ar, Kr, and Xe possess great potential as tracers for studying gas exchange in living beings, no direct analytical technique exists for simultaneously determining the absolute concentrations of these noble gases in body fluids in vivo. In this study, using human blood as an example, the absolute concentrations of all stable atmospheric noble gases were measured simultaneously by combining and adapting two analytical methods recently developed for geochemical research purposes. The partition coefficients determined between blood and air, and between blood plasma and red blood cells, agree with values from the literature. While the noble-gas concentrations in the plasma agree rather well with the expected solubility equilibrium concentrations for air-saturated water, the red blood cells are characterized by a distinct supersaturation pattern, in which the gas excess increases in proportion to the atomic mass of the noble-gas species, indicating adsorption on to the red blood cells. This study shows that the absolute concentrations of noble gases in body fluids can be easily measured using geochemical techniques that rely only on standard materials and equipment, and for which the underlying concepts are already well established in the field of noble-gas geochemistry.     

Dipeptidyl-Amino-Peptidase Iv Activity in Stored Buffy Coat Smears from Human Blood

The stability of dipeptidyl-amino-peptidase IV (DAP IV) activity in lymphoid cells of buffy coat smears from human blood was studied during storage for 40 days. Fixed or unfixed smears may be stored at 20 C for up to 24 hr before a decrease in activity occurs. Storage of either fixed or unfixed smears at 4 C, -10 C and -80 C results in a significant loss of activity within 24 hr. However. the cells retain more than 85% of their DAP IV activity for up to 10 days when stored fixed at -80 C. These data underscore the importance of proper processing of slides for DAP IV staining to avoid misinterpretation of results.     

Substance P Hydrolysis by Human Serum Cholinesterase

Highly purified human serum cholinesterase (EC 3.1.1.8, also known as pseudocholinesterase and butyrylcholinesterase) had peptidase activity toward substance P. Digestion of substance P was monitored by high performance liquid chromatography, which separated three product peptides. The cleavages occurred sequentially. The first peptide to appear as Arg1-Pro2. The Km for this hydrolysis was 0.3 mM; maximum activity was 7.9 nmol min-1 mg-1 of protein, which corresponded to a turnover number of 0.6 min-1. A second cleavage yielded Lys3-Pro4. A third cleavage occurred at the C-terminal, where the amide was removed from Met11 to yield a peptide containing residues 5-11. Both the peptidase and esterase activities of the enzyme were completely inhibited by the anticholinesterase agent, diisopropylfluorophosphate. Substance P inhibited the hydrolysis of benzoylcholine (a good ester substrate) with a KI of 0.17 mM, indicating that substance P interacted with cholinesterase rather than with a trace contaminant. Peptidase and amidase activities for serum cholinesterase are novel activities for this enzyme. It was demonstrated previously that the related enzyme acetylcholinesterase (EC 3.1.1.7) catalyzed the hydrolysis of substance P, but at entirely different cleavage sites from those reported in the present work. Since butyrylcholinesterase is present in brain and muscle, as well as in serum, it may be involved in the physiological regulation of substance P.     

Determination of Thymidine Phosphorylase Activity in Human Blood Cells and Fibroblasts by a Nonradiochemical Assay Using Reversed-Phase High-Performance Liquid Chromatography

In this study, we demonstrated that the highest activity of thymidine phosphorylase (TP) was found in peripheral blood mononuclear (PBM) cells followed by that of thrombocytes and granulocytes whereas no activity of TP could be detected in erythrocytes. The activity of TP in leukocytes proved to be intermediate compared to the TP activity observed in PBM cells and granulocytes. The activity of TP also was readily detectable in human fibroblasts.     

Functional Activity of Blood Polymorphonuclear Leukocytes as an Oxidative Stress Biomarker in Human Subjects

In the present work, we studied the role of polymorphonuclear leukocytes (PMN) in aged individuals and coronary heart disease (CHD)-bearing patients, two physiopathological processes associated with overproduction of reactive oxygen species (ROS). The effects of antioxidant supplementation on the functional activity of PMN from CHD patients were also determined. The function of PMNs was evaluated by measuring of phagocytosis, killing activity, and ROS production. Luminol amplified chemiluminescence (CL) was used to estimate ROS production by stimulated PMNs. Total cholesterol and the LDL-cholesterol fraction from CHD patients were found to be higher than those recommended, returning to normal levels after antioxidant therapy. PMN CL of CHD patients was found to be higher than the associated control groups. Antioxidant therapy administrated to CHD patients lead to an increase in the killing activity accompanied by a decrease in PMN CL of these subjects. The study also showed that killing activity of PMN from human subjects over 60 years was significantly lower than the activity measured in younger subjects. PMN CL produced after stimulation was found to be positively correlated with the increasing age of human subjects (r = .946, p < .01).     

Pentoxifylline Pretreatment Enhances the Oxidative Burst Activity of Human Peripheral Blood Mononuclear Cells

The effect of pentoxifylline pretreatment on the lucigenin-augmented chemiluminescence and dismutase-inhibitable superoxide production of human neutrophils and mononuclear cells (MNCs) was studied. Pentoxifylline at 20–2,000 μg/ml enhanced the lucigenin-augmented chemiluminescence (118–165% of the control, P < 0.01) of phorbol myristate acetate (PMA)-stimulated MNC. Pentoxifylline at 20–2,000 μg/ml increased the MNC superoxide production, i.e., 142–171% of the control (P < 0.05) using PMA stimulation and 145–159% of the control (P < 0.01) using opsonized zymosan stimulation. In contrast, pentoxifylline (up to 2,000 μg/ml) did not influence the lucigenin-augmented chemiluminescence and superoxide production of human neutrophils, stimulated by either PMA or opsonized zymosan. These results suggest that pentoxifylline is an immunomodulator and may have potential usefulness in the enhancement of immune defenses in compromised hosts.     

人外周血淋巴细胞体外诱导培养前后细胞表型与细胞毒活性相关性研究

为了分析体外细胞因子诱导培养CIK细胞过程中细胞表型的变化与其杀瘤活性的相关性及为临床过继免疫治疗提供实验依据,本研究采用体外诱导方法扩增培养正常人外周血淋巴细胞及单个核细胞,应用流式细胞术测定培养前、培养第7天和第14天的CD3~+等15种不同表型细胞百分率的变化,用CCK-8试剂检测第7天和第14天的细胞毒活性。结果显示,扩增培养后T细胞活化表型的表达和细胞毒活性在第7天最强,与其细胞表型CD3~+CD25~+、CD3~+CD28~+、CD3~+CD25~+CD28~+、CD3~+CD4~+呈正相关(P<0.05),与CD3~+CD45RA~+CD45RO~+呈负相关(P<0.05)。本研究表明测定培养细胞活化相关表型可以间接监测其杀瘤能力,为临床CIK细胞过继免疫治疗的应用提供实验依据。     

对肝炎患者进行胆碱酯酶活性监测的临床意义分析

目的:血清胆碱酯酶活性(CHE)是反应肝细胞损害的灵敏指标,对于肝炎疾病的临床应用具有重大的价值,评估血清胆碱酯酶的活性变化是本文的主要目的.方法:对各种类型肝炎患者的CHE活性采用医院引进的国外先进的全自动生化分析仪进行检测,并对患病肝炎组与正常组进行胆碱酯酶活性的对照(P＜0.01).结果:三组肝炎患者在接触时间、基础疾病、血清肌酸激酶(CK)、昏迷时间等有差异有显著性(P＜0.01),在年龄、性别、中毒原因方面没有显著性差异(P＞0.05).迟发性脑病组和无迟发性脑病组在年龄、基础疾病、并发症、接触时间、昏迷时间、CK方面差异有显著性(P＜0.01);而在性别、中毒原因方面差异无显著性(P＞0.05).结论:最终结果表明,CHE活性检测对于判定肝炎患者的预后具有重大的价值和作用,应得到充分的重视,而广泛、有效的应用于临床.     

重组人半乳凝集素-1的原核表达、纯化及生物活性检测

目的:在大肠杆菌中表达半乳凝集素-1(galectin-1),并进行纯化及生物活性检测。方法:将人半乳凝集素-1基因克隆至带有His融合标签的原核表达载体pQE-30上,转化大肠杆菌M15,经IPTG诱导表达,表达产物经亲和层析纯化后,进行Western印迹鉴定,并用红细胞凝集试验检测其生物学活性。结果:双酶切鉴定和核苷酸序列测定表明重组表达质粒pQE-30-Galectin-1构建正确;重组蛋白的表达量约占菌体总蛋白的50%,主要以可溶形式表达,纯化后蛋白纯度达95%以上,且具有良好的红细胞凝集活性。结论:在大肠杆菌中表达了重组人半乳凝集素-1,且具有良好的生物活性。     

灵芝多糖对人脐血LAK细胞活性的影响

本文研究了灵芝多糖（ＧＬＰ）对人脐血ＬＡＫ（ＣＢ—ＬＡＫ）细胞活性的影响,结果发现,单独ＧＬＰ能刺激人脐血单个核细胞（ＣＢＭＣ）增殖,但不能诱导ＬＡＫ活性,当与５０ｕ／ｍｌｒＩＬ—２伍用时,可增殖ＣＢ—ＬＡＫ细胞诱导活性,不同剂量ＧＬＰ（０．５—１００μｇ／ｍｌ）影响作用不同,以１０μｇ／ｍｌ浓度最好．在不同浓度ｒＩＬ—２（１０—１００ｕ／ｍｌ）诱导ＣＢ—ＬＡＫ细胞过程中加入ＧＬＰ（１０μｇ／ｍｌ）,可明显提高细胞增殖能力,减少ｒＩＬ—２用量。ＧＬＰ亦能促进效应阶段ＣＢ—ＬＡＫ细胞对Ｒａｊｉ肿瘤靶细胞的杀伤作用（Ｐ＜０．００１）。由此看出,ＧＬＰ具有增强ＣＢ—ＬＡＫ细胞活性的作用,是一很好的生物反应调节剂（ＢＲＭ）,有必要进行深入的研究。