网织红百分数法测定rHuEPO体内生物学活性的研究

本文通过对网织红百分数法测定rHuEPO体内生物学活性进行研究,找出网织红细胞百分数与rHuEPO浓度线性相关的范围。从而确定了用计数网织红细胞百分数的方法测定rHuEPO体内生物学活性。提出在rHuEPO体内生物学活性测定中选择合适的稀释液至关重要。对(3,3)法测定rHuEPO体内生物学活性进行了统计,批间CV、批内CV均接近10%,单次实验FL%平均数为3126%,并提出鼠间差异是造成FL%较大的主要原因;对(3,3)法与(2,2)法测定rHuEPO体内生物学活性进行比较,发现(2,2)法较(3,3)法更适合用于rHuEPO生产过程中rHuEPO体内生物学活性的测定。     

The Ergotropic Activity Index as an Integral Indicator of the Suprasegmental Autonomic Activity

Thirty healthy males between 19 and 25 years of age were examined to develop a method for estimating the suprasegmental autonomic activity. Analysis of the data obtained revealed the most informative indices along with a factor that had positive correlations with the ergotropic autonomic activity indices and negative correlations with the trophotropic autonomic activity indices. This factor was termed the ergotropic activity index. The information content of this factor was analyzed in a thermal environment and clinically in 40 patients with autonomic crises. The effectiveness of this method was tested, and the method was judged to be sufficiently adequate for clinical practice. The procedure developed for estimating the suprasegmental autonomic activity was demonstrated to be a highly informative method for revealing the predominance of either ergotropic or trophotropic activity in healthy subjects and patients with autonomic dystonia and for forecasting heat tolerance in humans. This method can be used during physiological and neurophysiological observations and in clinical practice     

棘腹蛙消化道脂肪酶的分布及pH和温度对脂肪酶活力的影响

采用聚乙烯醇橄榄油乳化液水解法,测定了棘腹蛙消化道不同部位的脂肪酶活力,研究了pH和温度对脂肪酶活力的影响。结果显示,棘腹蛙消化道不同部位脂肪酶活力依次为回肠>直肠>十二指肠>胃>食道。pH和温度显著影响脂肪酶的活力,二者对脂肪酶活力影响的关系曲线均呈现为单峰型,食道、胃和肠道脂肪酶活力的最适pH值分别为5.0、5.0和7.5,最适温度均为35℃。     

Establishment of a standard assay method for human thrombomodulin and determination of the activity of the Japanese reference standard.

This study was undertaken to establish a standard method for determination of the activity of human (h) thrombomodulin (TM). The reactions mainly consisted of formation of a h-TM and h-thrombin complex, activation of h-protein C by the complex and digestion of substrate by activated h-protein C. Linear time-dependent formation of p -nitroaniline from the substrate, S-2366, was observed up to 12 min during measurement of the activity of urinary h-TM (uh-TM) reference material by the standard method. Therefore, 10 minutes was established as the reaction time in the standard method. In the standard method, we defined the activity of h-TM forming 0.1 micromol of p -nitroaniline per min in the reaction as 1 JRS Unit. Recombinant h-TM (rh-TM) and uh-TM reference material gave rectilinear dose-response curves within a certain range of specific activities by their original methods in the standard method. The validity of the standard method was assessed based on the coefficients of variation (CV) obtained in the various measurements of h-TM. Intra-batch precision (CV) of h-thrombin and h-protein C was 2.90% and 6.57%, respectively, in the measurement of uh-TM activity. The intra-sample, inter-day, and inter-laboratory precision (CV) was 1.30%, 1.63% and 5.02%, respectively, in the measurement of the first Japanese reference standard for h-TM coded TJRS1. In these assays, the activity of the first Japanese standard was also determined and found to be 205 JRS units per ampoule. When the stability of the Japanese reference standard was assessed by measuring of the standard stored under various thermal conditions, the predicted loss of activity assuming monomolecular degradation according to the Arrhenius equation was less than 3.0% during 103 years at -20 degrees C. These results indicate that the standard method is appropriate for determination of the activity of h-TM and that the Japanese reference standard for h-TM, whose activity was determined here, can be stored at -20 degrees C for long periods without loss of activity.     

不同细胞破碎方法对无细胞蛋白表达系统细胞抽提物活性的影响

无细胞蛋白表达系统由于能够有效表达膜蛋白等有毒性蛋白,因此近二十年受到了关注,其蛋白表达产率有了显著的提高。细胞抽提物活性的高低是无细胞蛋白表达系统高效运行的关键,若找到简单易行的活性评估方法,将大大降低成本及时间。葡萄糖-6-磷酸脱氢酶 (glucose-6-phosphate dehydrogenase, G-6-PDH)是糖代谢的戊糖磷酸途径中的关键调控酶,该酶可以被用来评估细胞抽提物的活性。以G-6-PDH的活性为指标对无细胞蛋白表达系统中的抽提物活性进行评价,并利用G-6-PDH活性评价体系对机械破碎、高压破碎以及超声破碎三种破碎方法进行了比较,得出了三种破碎方法的最佳破碎条件。机械破碎最佳破碎条件是5 000r/min, 用直径0.1 mm玻璃珠,破碎6次;高压破碎的最佳破碎压力为1 300bar;超声破碎最佳破碎条件是功率强度为总功率的60%,破碎30次。酶活性测定结果显示机械破碎和超声破碎得到的抽提物活性比高压破碎得到的抽提物活性略高。     

酿酒酵母菌耐热性快速鉴别的方法研究

目的:找出快速有效的鉴别酵母耐热性的方法。方法:酿酒酵母菌经过不同温度热激处理后,用美兰染色计数、稀释平板计数、发酵活力直接测定法和浊度测定法对酿酒酵母菌耐热性进行了测定,同时对酵母菌的形态也进行了观察。结果:表明浊度测定的结果和发酵活力直接测定法测定的结果关系没有相关性,不便用来测定酵母菌的活力。用美兰染色计数、稀释平板计数和发酵活力直接测定法所得的结果有同样的趋势,通过相关性分析,这些方法都可以表示酵母菌的活力,但各有其特点。其中,美兰染色计数是鉴别酵母耐热性的快速有效的方法。     

Fine structural localization of peroxidase activity in the epithelium and the gland of the rat larynx

Summary Endogenous peroxidase activity was demonstrated in ciliated cells and secretory cells of the laryngeal epithelium and gland of rats, using the diaminobenzidine method for cytochemical demonstration of peroxidase activity. The intensity of peroxidase activity was greatly varied from cell to cell, but the fine structural localization of the activity was similar in various cell types. The activity was localized in cisternae of rough-surfaced endoplasmic reticulum including nuclear envelope, some vesicles and saccules of the Golgi complex, large membrane-limited granules, multivesicular bodies and probable lysosomes. In secretory cells, the activity was also found in secretory granules.The significance of peroxidase activity is not unclear, while the activity, at least a part of it, seems to be secreted into the cavity of the larynx. The possibility that peroxidase participates bactericidal mechanism, deserves further investigation.     

Determination of the carbamate kinase activity of bacteria

The activity of carbamate kinase (EC 2.5.2.2) was determined in bacteria using a simple modified procedure. Carbamoyl phosphate produced under the action of carbamate kinase carbamoylated ammonia in a reaction which was not enzyme-catalyzed yielding urea that was assayed by the colorimetric technique. The activity of carbamate kinase was found by this method in a number of microorganisms. The method can be used to study other enzymes synthesizing carbamoyl phosphate. The advantages of the method over other techniques are discussed.     

A new method for the intravital assessment of the functional activity of the membrane transporters performing the efflux of antitumor preparations from the cells of solid tumor specimens]

A new method for intravital assessment of the functional activity of anticancer drug efflux transporters in intact solid tumor specimens was developed. The method is based on the well-known approach to the transporter functional evaluation by intracellular accumulation of antitumor drugs and particularly the anthracycline antibiotic doxorubicin (Dox). The main new point of the method providing investigation of intact solid tumor specimens which markedly simplified the procedure is the fact that the intratissue and intracellular accumulation of Dox is determined not by the level of the drug in the tissue but by its fluorescence decrease in the incubation medium. To assess just the intracellular content of Dox and to estimate the transporter functional activity, investigation of the influence of membrane transporter inhibitors such as verapamil (P-gp inhibitor) and sodium azide (inhibitor of all the energy-dependent ABC transporters) on the drug fluorescence decrease in the incubation medium is stipulated. The validity of such an approach was experimentally proved with the specimens of the Ehrlich solid tumor transplants in mice (a sensitive variant of the tumor and the tumor with induced drug resistance). Biopsy specimens of human breast tumors were investigated with the new method and functional activity of various efflux transporters was revealed: (1) only P-gp, (2) both P-gp and other ABC transporters, (3) only transporters different from P-gp, (4) no functional activity of efflux transporters. The main trends of the further investigation of efflux transporter functional activity in human solid tumors possible for the first time with the use of the new method are defined.     

The quantitative assay of the clustering activity of the lymphocytosis-promoting factor (pertussis toxin) of Bordetella pertussis on Chinese hamster ovary (CHO) cells

An experimental design and a statistical method for the estimation of the clustering-response activity of lymphocytosis-promoting factor (LPF) in Chinese hamster ovary cells growing in wells on a microplate were investigated. The scoring method introduced by Ipsen was adopted to express the grade of the clustering response rather than the end-point method generally used. The scoring method was validated by statistical analyses. The grade of response varied with the location of the wells on a microplate, and thus the expression of the clustering activity of a test sample in terms of the end-point may be inadequate in terms of accuracy and reproducibility. It was shown that the allocation of test samples to individual wells according to a Latin square design minimized the effect of the location of wells on the clustering response. Under such experimental conditions, a fairly precise and reproducible method for the quantification of the clustering activity was developed.     

Localization of amylase and mucins in the major salivary glands of the mouse

Summary Antibodies against murine submandibular and sublingual mucins have been raised in rabbits. Both antisera appeared to be specific. Using these antibodies, the mucins were localized in the acinar cells of the submandibular and sublingual glands respectively.The dyed amylopectin method was used to estimate the activity of amylase in the salivary glands. The enzyme was localized either by a starch-substrate film method or with antibodies against purified parotid amylase. The activity of amylase in parotid homogenates is about 1000-fold higher than that in homogenates of either submandibular or sublingual glands, in which the activity was comparable. Amylase was localized in the acinar cells of the parotid gland with both localization techniques. In the sublingual gland, amylase was found predominantly in the stroma around the acini, and there was some evidence that amylase was present in the demilune cells as well. In the submandibular gland, contradictory results were obtained with both techniques. With the starch-substrate film method, amylase activity was found in the granular convoluted tubular cells, whereas immuno-reactive amylase could only be demonstrated in the acinar cells of this gland. It is concluded that in the submandibular gland amylase and mucin are present in the same cell type.     

Histochemical localization of kallikrein-like pro-phe-arg-naphthylester esterase activity in the rat kidney

Summary In the rat kidney the presence of the kallikrein-like pro-phe-arg-naphthylester esterase activity was demonstrated by a simultaneous coupling azo dye method. The enzyme was identified as a serine-protease because it was inhibited by preincubation with diisopropyl-fluorophosphate and unaffected by sodium iodoacetate. Since kallikrein is a serine-protease and pro-phe-arg-naphthylester is a synthetic and sensitive substrate for kallikrein, the enzyme activity revealed by this method was considered to represent kallikrein, although non-kallikrein esterase activity is not totally excluded. The enzyme activity was localized mainly in the outer stripe of the outer medulla, with focal extensions primarily only in the lower half of the cortex corresponding to the medullary rays.     

Measurement of the intensity of erythropoiesis based on the RNA content of erythrocytes

The relationship between RNA content in erythrocytes and the erythropoiesis rate was studied at varying exposures as a result of which a simple method for erythropoietic activity testing was proposed. It consists in the measurement of ribonucleic acid content in erythrocytes of mice with a decreased erythropoiesis after administration of the test sample. Erythropoietic activity is determined from the magnitude found. The method suggested is marked by simplicity and higher sensitivity and thus may be applied in clinical medicine and research.     

Evaluation of a method for the determination of antibacterial activity of chitosan

A method for the determination of the antimicrobial activity of chitosan with the use of organic salts for the production of pH in the range of 5.5–8.2 was studied. The double-dilution method demonstrated the effectiveness of the determination of the antimicrobial activity of chitosan samples with different molecular weights and solubilities. It was found that the antibacterial activity increased at low pH values with increasing molecular weight, but chitosans with a molecular weight of 5–6 kDa showed higher activity at neutral and slightly alkaline pH levels. Determination of the antimicrobial activity of various chitosan samples at different pH values allowed a more reliable assessment of the potential biological activity of chitosan.     

A high-performance liquid chromatographic method for the assay of cholesterol 7 alpha-hydroxylase activity

A high-performance liquid chromatographic method has been developed for the measurement of cholesterol 7 alpha-hydroxylase activity in liver microsomes. 7 alpha-Hydroxycholesterol generated from endogenous cholesterol was derivatized with anthroyl 1-carbonitrile, chromatographed on a reverse-phase column, and detected fluorometrically. The detection limit of 7 alpha-hydroxycholesterol was 1 ng/tube. The cholesterol 7 alpha-hydroxylase activity in rat liver microsomes was assayed by this method, and the effects of some detergents and of the addition of exogenous cholesterol together with detergents on the enzyme activity were investigated. The endogenous 7 alpha- and 7 beta-hydroxycholesterol could be also measured by this method.     

Effect of the proteinase isolated from a fungus on the kallikrein-kinin system

Carboxyl proteinase (CP) with the isoelectric point of 6.3-6.4 was isolated from a fungus at the Laboratory of Enzymology of the All-Union Research Technological Institute of Antibiotics and Enzymes and its effect on the kallikrein-kinin system and trypsin caseinolytic activity was studied. Four lots of the preparation with the activity of 1116 to 2300 milk coagulating units per 1 mg were used. The kininogenase activity of kallikrein, bradykinin and trypsin was determined with the routine biological procedures and the trypsin caseinolytic activity was determined with the Kunitz method and the diffusion method on casein-containing agar. It was shown that CP inhibited the kininogenase activity of kallikrein in the secretion of the salivary glands of man and crystalline trypsin in aqueous media and blood serum. It also inactivated the bradykinin constrictor effect on the smooth muscle tissue of the uterus horn in rats. CP had a capacity for inhibiting the caseinolytic activity of crystalline trypsin. Possible use of CP in treatment of patients with diseases accompanied by impairment of the kallikrein-kinin system (increased activity) is discussed.     

Distribution of oxytocic and vasopressor activity in the rat diencephalon

The oxytocic and vasopressor activity was studied in five 1 mm thick, horizontal sections of the rat diencephalon. The diencephalon was cut frozen in dry ice. The sections obtained from identical parts of the diencephalon of 10 rats were homogenized together in 0.9% NaCl solution acidified with glacial acetic acid. The homogenate was heated to 100 degrees C for 5 min and centrifuged. The oxytocic activity of extracts was determined in vitro by, the method of Holton using the rat myometrium. The vasopressor activity was determined in vivo recording blood pressure in the carotid artery of rat by the method of Dekańaski. Oxytocic activity was found in all five sections of diencephalon and vasopressor activity in only two sections. The first section included the median eminence and ventral hypothalamus together with the supraoptic nucleus, the second section included the the dorsal hypothalamus with paraventricular nucleus, the third section--the ventral thalamus, the fourth section--the middle part of thalamus, the fifth section--the dorsal thalamus.     

Determination of the epinephrine-refractory hypoglycaemic activity of whole-cell pertussis vaccine in mice

A new assay method has been developed for the quantitative estimation of the inhibitory effect of pertussis vaccine on epinephrine-induced hyperglycaemia in mice. The statistical analysis of the assay was based on logarithm-transformed estimates of the blood glucose levels. The method was sufficiently sensitive to detect the activity of 0.004 millilitre of commercial combined diphtheria-tetanus-whole cell pertussis vaccine. The estimated common variance was as small as 0.0034 and the assay was highly reproducible. Among commercial vaccines there was a significant difference in activity. The activity of a stock pertussis vaccine was inactivated by 5 mM glutaraldehyde at 37 degrees C for 30 min, but resisted treatment with 40 mM formaldehyde at 37 degrees C for 5 days. The extent of inactivation with the chemicals was calculated by a parallel line assay as the activity relative to that of untreated control pertussis vaccine.     

Analysis of the role of adrenergic receptors in the regulation of small intestine motor activity in sheep

In sheep with chronic fistulae of the small intestine and rumen the participation of alpha- and beta-adrenergic receptors in the regulation of the motor activity of the small intestine was studied by the method of pharmacological analysis. The movements of the fistulated parts of the alimentary tract were recorded by the balloon method. Slow intravenous infusion of isoprenaline inhibited the contractions of the small intestine. This inhibitory effect of isoprenaline was abolished by propranolol. Intravenous phenylephrine inhibited the motor activity of this intestinal part as well. The effect of phenylephrine was abolished by pretreatment with dihydroergotamine. In the small intestine of sheep stimulation of the alpha and beta adrenergic receptors decrease the motor activity of intestine.     

赤芍总苷抗氧化活性及对大鼠肝星状细胞增殖的影响

目的:研究赤芍总苷体外抗氧化活性及对大鼠肝星状细胞HSC-T6细胞增殖的影响.方法:采用DPPH法测定赤芍总苷体外清除DPPH自由基及抗氧化活性,根据清除率曲线,确定EC50值;采用MTT法考察赤芍总苷对大鼠肝星状细胞HSC-T6细胞增殖的影响,确定起效剂量,并考察其量效关系.结果:赤芍总苷对DPPH自由基具有较强清除作用,其对DPPH的清除率的回归方程为y=7.4432x0.6111(r=.9967),EC50值为6.64mg/L;赤芍总苷对HSC-T6的增殖具有明显的抑制作用,给药浓度为1.152mg/mL时,其对HSC-T6细胞增殖的抑制率可达39.240%.结论:赤芍总苷具有较强的抗氧化活性,对肝星状细胞的增殖有一定抑制作用.