芦笋老茎澄清汁对链脲佐菌素致糖尿病大鼠降糖作用的研究

本文评价了芦笋老茎澄清汁(CAJ)的降血糖作用,并对其降血糖机制进行了初步探讨。腹腔注射STZ制备类似1型糖尿病大鼠模型,以0.6,1.2和2.0 g/kg体重剂量的CAJ连续灌胃21 d,监测血糖,测定糖化血清蛋白、血清胰岛素、肝糖原、脂代谢及抗氧化系统部分相关指标。结果显示,CAJ可明显降低糖尿病大鼠血清中葡萄糖、糖化血清蛋白、总胆固醇和MDA含量,并显著提高受试模型鼠的血清胰岛素水平、肝糖原含量、血清SOD活性、肝脏SOD、GSH-Px和CAT的活性。上述结果表明CAJ可明显降低糖尿病大鼠的血糖水平,刺激胰岛素分泌,调节血脂,增强抗氧化能力。     

应用氧化酶法对高糖高脂动物模型肝糖原含量的测定

目的建立氧化酶法检测肝糖原的方法。方法采用碱中和与缓冲液相配合调节水解液pH至中性,然后用氧化酶法检测肝糖原含量。结果用10μL6mol/LNaOH加入等量肝糖原水解液后,再加入4mLpH7.2磷酸盐缓冲液,可使糖原水解液pH值调为7.2,用于氧化酶法测定糖含量。结论该法操作简便易行,结果稳定、重复性好。     

新疆野生党参总黄酮体内抗氧化及抗疲劳作用研究

本论文采用对照组及新疆野生党参总黄酮低、中、高三个不同浓度剂量组灌胃小鼠25 d后对其血清与肝脏的超氧化歧化酶( Superoxide Dismutase SOD)活性、丙二醛(Maleic Dialdehyde MDA)含量及肌糖原、肝糖原、血清尿素氮水平等进行测试,并进行负重游泳实验.结果表明:新疆野生党参总黄酮能增强小鼠血清和肝脏的SOD活性,降低MDA的含量,延长小鼠的负重游泳时间,提高肝糖原、肌糖原的储备量,降低小鼠运动后血清尿素氮水平.因而,新疆野生党参黄酮类化合物具明显的抗氧化抗疲劳生理功效.     

沙棘籽渣多糖对正常及糖尿病模型动物血糖的影响

本文研究了沙棘籽渣多糖(Polysaccharides from seed residue of Hippophae rhamnoide L.,PSH)对正常小鼠及实验性2型糖尿病大鼠血糖、血脂代谢的影响.以100、200和400 mg/kg剂量的PSH连续灌胃正常小鼠20d;以50和100 mg/kg剂量的PSH连续灌胃由烟酰胺联合链脲佐菌素诱导的类似2型糖尿病大鼠3周,测定血糖、糖基化血清蛋白、血清胰岛素、血清总胆固醇、甘油三酯及肝糖原含量.结果显示:PSH对正常小鼠的血糖和血脂代谢没有明显影响;但能明显降低2型糖尿病大鼠的血清葡萄糖、总胆固醇和糖基化血清蛋白水平,同时显著增加糖尿病大鼠的血清胰岛素含量.上述结果表明:PSH在实验性2型糖尿病大鼠模型上具有降血糖和降胆固醇的活性.     

大鼠常温肝缺血再灌注肝组织化学变化的研究

本实验采用大鼠49只,常温肝缺血再灌注模型,对肝缺血不同时间及再灌注后肝组织化学活性变化进行了分组定性对比观察.结果显示:1.缺血期,肝细胞PAS反应和SDH、AKP.Mg~(++)-ATPase活性随缺血时间延长逐渐减弱,LDH活性于缺血初期有所增强,随后逐渐减弱,到缺血90min时,上述各酶组织化学活性及糖原(PAS反应)均几乎消失,而ACP活性则显著增强.2.再灌注期,肝酶组织化学活性及PAS反应的变化趋势与缺血时间长短有密切关系,缺血20、40min再灌注后,肝酶组织化学活性及糖原(PAS反应)可逐渐恢复正常水平,而缺血60、90min再灌注后,肝脏SDH、AKP、LDH、Mg~(++)-ATPase和PAS反应均消失,ACP活注进一步增强.     

肉桂油的提取及其抑菌活性研究

本文用水蒸汽蒸馏法蒸馏经微波预处理的肉桂粉得到肉桂油,并采用滤纸片法、固相扩散法、气相扩散法研究了肉桂油对6种细菌、1种酵母、4种霉菌的抑菌活性及其最低抑菌浓度(MIC)。结果表明:肉桂油经固相和气相扩散对细菌、酵母、霉菌的抑制活性均较强,并且对真菌的抑菌活性更强一些,其次是G ,对所有供试菌种的最低抑菌浓度(MIC)<63mL/L,且真菌的MIC<细菌的MIC。     

模拟急性高原低氧对妊娠大鼠及其胎儿,新生儿肝细胞与溶酶体的影响

低气压舱模拟海拔8000m24h。观察对不同期妊娠大鼠、胎儿及新生儿肝溶酶体酶血清转氨酶、肝糖原、蛋白质和总脂水平的影响。证明:22天孕鼠肝细胞及其溶酶体损伤程度比16天孕鼠和非孕鼠严重;对16天和22天胎儿鼠肝溶酶体无明显影响,显示了胎儿肝溶酶体低氧下的高稳定性和母体对胎儿的保护作用;新生儿鼠肝细胞溶酶体对低氧的耐受性明显高于孕鼠与非孕鼠。低氧使孕鼠、胎儿、和新生儿鼠肝细胞糖原含量明显降低。新生儿诞生后肝糖原贮备极度消耗,低氧加剧这种作用。随着胎儿发育,肝蛋白质含量渐增,低氧导致全肝蛋白质量减少。无论孕鼠与非孕鼠,低氧造成肝总脂水平增高。     

急性高原性低氧对三种小哺乳动物肝脏作用的比较

人工低气压舱模拟高原低氧,观察实验动物小白鼠、豚鼠及野生动物达乌尔鼠兔,在海拔5000米及8000米24小时内的几种生理效应,并与2300米海拔对照组进行了比较。发现随着海拔高度的升高:1.3种动物体重明显下降;2.肝糖原含量明显降低:小鼠、达乌尔鼠兔与豚鼠肝糖原在8000米时分别为2300米含量的62%,35%及 9%:3.小鼠、达乌尔鼠兔及豚鼠的肝脂肪累积量依次增大;4.肝蛋白质含量减少;5.SGPT与SGOT活力升高;6.肝细胞溶酶体的酸性磷酸酶与芳基硫酸脂酶活力升高。肝组织形态学观察结果与生化检测结果一致,豚鼠在8000米海拔时,肝细胞出现气球样变,脂肪变性及灶性液化性坏死等变化。综合分析,这3种动物对极度低氧的耐受性依顺序为小鼠、达乌尔鼠兔、豚鼠。     

双歧杆菌发酵果蔬汁对小鼠抗疲劳作用的实验研究

目的检测发酵果蔬汁对小鼠的抗疲劳作用。方法将3种果蔬汁按一定比例加入双歧杆菌、保加利亚乳杆菌和嗜热链球菌,经发酵,分别以高中低3个剂量喂食小鼠30d后,进行小鼠负重游泳实验,检测肝糖原、乳酸和尿素氮等各项抗疲劳指标。结果3种果蔬汁均能显著延长小鼠负重游泳时间,提高小鼠肝糖原的储备量;降低小鼠游泳后血乳酸和尿素氮均值,增加血红蛋白含量及乳酸脱氢酶活性,减少血尿酸含量。且抗疲劳强度程度与型量声低有一定相差。结论3种发酵果蔬汁具有抗疲劳作用。     

饥饿与冬眠期牛蛙肝脏糖原含量及主要酶的活性变化

目的研究饥饿与冬眠期牛蛙肝脏糖原含量和非特异性酯酶(NSE)、碱性磷酸酶(ALP)、过氧化物酶(POX)、琥珀酸脱氢酶(SDH)的活性变化。方法应用冰冻切片、PAS染色法、酶组织化学技术及光密度定量分析。结果饥饿期肝糖原含量显著降低,冬眠期肝糖原含量与活动期无显著差异;NSE活性在活动期最高,饥饿期显著降低,冬眠期活性最低;ALP和POX活性在饥饿期均显著降低,冬眠期与活动期无显著差异;SDH活性在饥饿期和冬眠期均显著降低,活动期活性显著较高。结论饥饿和冬眠期牛蛙肝糖原含量变化不一致,其他酶类活性变化相一致,不同时期肝糖原含量和酶活性的变化与牛蛙生理状态有着较好的适应性。     

A spectrophotometric assay of gamma-glutamylcysteine synthetase and glutathione synthetase in crude extracts from tissues and cultured mammalian cells

An assay of gamma-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase (GS) in crude extracts of cultured cells and tissues is described. It represents a novel combination of known methods, and is based on the formation of glutathione (GSH) from cysteine, glutamate and glycine in the presence of rat kidney GS for the assay of gamma-GCS, or from gamma-glutamylcysteine and glycine for the assay of GS. GSH is then quantified by the Tietze recycling method. Assay mixtures contain the gamma-glutamyl transpeptidase (GGT) inhibitor acivicin in order to prevent the degradation of gamma-glutamylcysteine and of the accumulating GSH, and dithiothreitol in order to prevent the oxidation of cysteine and gamma-glutamylcysteine. gamma-GCS and GS levels determined by this method are comparable to those determined by others. The method is suitable for the rapid determination of gamma-GCS GS in GGT-containing tissues and for the studies of induction of gamma-GCS and GS in tissue cultures.     

Development of a radiometric spot-wash assay for squalene synthase.

The principle of selective elution from a solid phase has been exploited to develop an assay for the determination of squalene biosynthesis in rat liver homogenates. Using either [1-14C]isopentenyl diphosphate as a precursor for squalene or [2-14C]farnesyl diphosphate as a direct substrate of squalene synthase, the production of radiolabeled squalene is determined after adsorption of assay mixtures onto silica gel thin-layer chromatography sheets and selective elution of the diphosphate precursors into a solution of sodium dodecyl sulfate at alkaline pH. The use of [2-14C]farnesyl diphosphate, and of an endogenous oxygen consumption system (ascorbate/ascorbate oxidase) to prevent further metabolism of squalene, allows the method to be applied as a dedicated assay for squalene synthase activity. The assay has been developed in microtiter plate format and may be deployed either in a quantitative, low-throughout mode or in a qualitative, high-through-put mode. The latter is suitable for screening to aid in the discovery of new inhibitors of squalene synthase.     

Sensitive liquid chromatographic method using fluorescence detection for the determination of estradiol 3- and 17-glucuronides in rat and human liver microsomal incubations: formation kinetics

We have developed a sensitive and specific HPLC-fluorescence assay for the determination of estradiol-3-glucuronide and estradiol-17-glucuronide in human and rat liver microsomal incubations. The method utilizes a mobile phase comprised of acetonitrile and 50 mM ammonium phosphate buffer (35:65, v/v) that is pumped though a phenyl column at 1 ml/min; the run time is less than 15 min. Calibration curves for both metabolites were linear over the range 20-4000 pmol. The intra- and inter-day coefficients of variation were <6%. In both rat and human liver microsomes, the formation of estradiol-3-glucuronide displayed atypical kinetics (consistent with activation), while estradiol-17-glucuronide formation was consistent with classical Michaelis-Menten kinetics. Overall, the assay described is a sensitive and reproducible method for the determination of estradiol glucuronides in liver microsomal preparations.     

A new neomycin phosphotransferase II solid phase assay in combination with polyacrylamide sodium dodecylsulphate gel electrophoresis

A new general method for the determination of neomycin phosphotransferase (NPT) II (EC 2.7.1.95) activity in cell extracts after separation in SDS-polyacrylamide gels is described. The enzymatic activity of NPT II is restored after SDS-polyacrylamide gel electrophoresis by incubating the gel for 3 h (20 mM Tris-HCl buffer, pH 7.4). The enzymatic activity is determined by in situ phosphorylation of aminoglycoside antibiotics bound to solid supports and brought into direct contact with the gel surface. A novel, mechanically stable, negatively charged matrix was synthesized for use in this solid phase enzyme assay and compared to phosphocellulose and carboxymethylcellulose paper. This new method allows the easy and exact determination of the molecular weight of any fusion protein with NPT II by assaying the position of the enzymatic activity in the gel and a consecutive immunological reaction following protein transfer onto nitrocellulose membranes.     

Problems associated with the assay of arylsulphatases A and B of rat tissues

A method for the separation and purification of rat liver arylsulphatases A and B by gel filtration on Sephadex G-200 is described. The properties of the A enzyme and its molecular weight are similar to those of the corresponding ox liver enzyme. The B enzymes were found to be dissimilar. The method already developed for the assay of the corresponding enzymes from human tissues was shown to be unsuitable for the assay of the enzymes of rat tissues. A method of assay was developed which permits an approximate determination of the individual rat liver enzymes in a mixture of the two, but precise determination requires prior separation of the enzymes by gel-filtration chromatography.     

An enzyme immunoassay for the quantitation of dihydropteridine reductase

A competitive, enzyme-linked immunosorbent assay for the quantitative determination of dihydropteridine reductase (DHPR) is described. This highly sensitive method can determine the content of DHPR protein in tissue preparations independently of the enzymatic activity of the protein molecule. The method involves initial incubation of samples containing soluble enzyme in microtiter plates coated with purified goat antibodies to rat DHPR and further incubation with DHPR conjugated to alkaline phosphatase. The assay is used to study the ontogeny of DHPR in rat liver.     

Spectrophotometric measurement of flavin-containing monooxygenase activity in freshly isolated rat hepatocytes and their cultures.

The determination of the mixed function flavin-containing monooxygenase activity in rat liver and in hepatocytes and their cultures by spectrophotometric measurement of the oxygenation of methimazole is complicated by an inhibition caused by some of the reagents used during this method. Optimal conditions were determined for measuring this enzyme activity in microsomal preparations of rat liver and its hepatocytes. Optimal flavin-containing monooxygenase activities were obtained for measurements performed in a 0.25 M N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine-EDTA buffer at pH 8.7 and at a methimazole concentration of 2 mM. Data are also presented which show that no interferences caused by either cytochrome P450-dependent enzymes or by the reduction of methimazole disulfide by glutathione have to be taken into account when determining methimazole oxygenation. Finally, the above assay was also used to study flavin-containing monooxygenase activity in primary monolayer cultures of hepatocytes for 6 days.     

Development of gluconeogenesis in neonatal rat liver. Effect of premature delivery

1. An assay method for the determination of phosphopyruvate carboxylase activity is described in which improved sensitivity is obtained by separation of the enzyme from interfering pyruvate kinase by zone sedimentation. 2. The molecular weight of rat liver phosphopyruvate carboxylase determined by zone sedimentation is about 68000. 3. Premature delivery of rat foetuses by uterine section results in the rapid appearance of phosphopyruvate carboxylase, but hexose diphosphatase and pyruvate carboxylase, already present in the foetal rat liver, are not significantly affected, and glucose 6-phosphatase activity is only slightly affected. 4. The rate of incorporation of [¹⁴C]pyruvate into glucose by liver slices is also greatly increased by premature delivery and there is a highly significant linear correlation between this process and the phosphopyruvate carboxylase activity.     

Characterization of tyrosylprotein sulfotransferase from rat liver and other tissues

The sulfoconjugation of tyrosyl residues is a widespread post-translational modification of biologically active peptides and proteins. In this paper we describe the characterization of a rat liver tyrosylprotein sulfotransferase that is capable of catalyzing the transfer of a sulfate moiety from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the synthetic polymer, poly-(Glu6,Ala3,Tyr1) (EAY; Mr 47,000) using a simple filter paper assay. Following sucrose density gradient centrifugation and comparison with known subcellular marker enzyme activities, rat liver tyrosylprotein sulfotransferase activity was shown to have a distribution similar to the Golgi enzyme, galactosyltransferase. Using the enriched Golgi preparation, rat liver tyrosylprotein sulfotransferase displayed a pH optimum of 6.7 and required the presence of 20 mM Mn2+ for maximal activity. Co2+ (20 mM) was able to produce 26% of the maximal stimulation observed with Mn2+, whereas other metal ions, such as Mg2+, Ca2+, and Co2+, were not effective in stimulating tyrosylprotein sulfotransferase activity. Whereas tyrosylprotein sulfotransferase activity was observed in the native membrane-bound state, EAY sulfation was maximally enhanced 3-fold when assayed in the presence of Lubrol Px. Under the optimal conditions for assaying the sulfation of EAY by a rat liver enriched Golgi fraction, significant degradation of the sulfate donor, PAPS, was observed. The addition of both NaF and 5'-AMP to the incubation mixture was found to effectively prevent PAPS degradation and increase the amount of product formed in the assay by 10-fold. Using the optimized conditions for the sulfation of EAY by rat liver tyrosylprotein sulfotransferase, membrane-bound sulfotransferase activity was also observed in the crude microsomal pellets of a variety of rat tissues, including lung, pituitary, and cerebellum, as well as in livers from different species.