壳聚糖酶的克隆表达与壳寡糖的制备分析

目的:克隆壳聚糖酶基因于大肠杆菌中实现高表达,制备壳寡糖。方法:以枯草芽孢杆菌总DNA为模板扩增壳聚糖酶基因(CSN),克隆至载体pET23a(+)上,转化菌株BL21(DE3)。重组子经0.5 mmol/L IPTG诱导后,SDS-PAGE和质谱检测与鉴定重组酶。酶纯化后水解壳聚糖,薄层色谱分析其水解产物。结果:质谱证明壳聚糖酶(31.5kDa)成功表达,表达量占菌体总蛋白的45%左右。纯化后重组酶浓度为900 mg/L,纯度95%、回收率85%,酶活力为10 000 U/mg。壳聚糖降解产物为壳二糖至壳四糖。结论:原核表达载体pET23a(+)-CSN构建正确,壳聚糖酶表达量与活性高,适用于水解壳聚糖制备壳寡糖。     

人血管能抑素基因的克隆、表达及其表达产物的纯化和生物活性测定

以人胎盘脐带组织为材料,提取组织总RNA,用netRTPCR方法合成人血管能抑素cDNA基因,将该cDNA克隆进pSP72载体获得重组质粒pSP72C, DNA序列分析结果与预期序列一致。用BamHⅠ和NdeⅠ双酶切,切下pSP72C上的血管能抑素cDNA,插入pET3c载体的相应位点获得重组表达质粒pETC, 转化E. coli BL21(DE3), SDSPAGE分析显示：在IPTG诱导下,血管能抑素基因获得了高效表达,表达量约占菌体总蛋白的 27.9 %,主要以包涵体形式存在。包涵体经过洗涤、裂解、蛋白复性以及Sephadex G75凝胶过滤层析等步骤后,获得了纯度达91.4 %的人血管能抑素。CAM实验证明10 μg纯化蛋白就能显著抑制鸡胚新生血管生成。     

Comparison between muscle and liver enolases and their behavior during differentiation and growth.

1. Rabbit liver enolase (EC 4.2.1.11) was purified about 200-fold and the enzyme was distinguished from crystalline muscle enolase by column isoelectrofocusing. It was found that the pI of muscle enolase was at about pH 8.8 and the pI of liver enolase was at about pH 6.7. Liver enolase was more liable to heat than muscle enolase. Anti-muscle enolase antibody did not react with liver enolase in double diffusion and immunoprecipitation tests. No substantial difference seemed to exist between muscle and liver enolases in pH optima, kinetic constants, and gel filtration. 2. It was observed by electrofocusing that the pI of rat muscle enolase was pH 7.2 to 7.9 and that of liver enolase was about pH 5.9. The main component of muscle enolase was designated as type A enolase, and liver enolase as type B enolase. Type A enolase was present in skeletal muscle and heart muscle. Type B enolase was widely distributed and present in liver, kidney, spleen, brain, lung, small intestine, and heart muscle. More acidic isozyme than type B enolase coexisted in the brain, and more basic isozyme than type A enolase, coexisted in the small intestine. A prototype of enolase in the early stage of differentiation was found to be type B enolase and, as differentiation progressed, type B decreased in muscle, while type A increased. On the other hand, liver enolase was retained as type B during differentiation. The enolase in regenerating liver was the same as in normal liver.     

Relative and combined effects of ethanol and protein deficiency on strontium and barium bone content and fecal and urinary excretion

To elucidate accumulation of minerals in human iliac arteries with aging, the content of minerals was analyzed by inductively coupled plasma atomic emission spectrometry. Bilateral common, internal, and external iliac arteries of 16 men and 8 women, ranging ages from 65 to 93 yr, were examined. It was found that an extremely high accumulation of calcium and phosphorus occurred in the common iliac artery at old age, being higher than that of the internal and external iliac arteries. It should be noted that the accumulation of calcium and phosphorus is the highest in the common iliac artery among the human arteries examined to date. Regarding sexual differences, the content of calcium and phosphorus in the common and internal iliac arteries was higher in women than in men, whereas their content in the external iliac artery was lower in women than in men.     

激动素、脱落酸和丙二醛对SOD活性影响及其与SOD构象和疏水性变化间的关系研究

 激动素、脱落酸和丙二醛对ＳＯＤ活性影响及其与ＳＯＤ构象和疏水性变化间的关系研究伍泽堂候万儒（四川师范学院生物系,南充６３７０００）我们在实验中曾发现激动素（ＫＴ）激活小麦ＳＯＤ活性,脱落酸（ＡＢＡ）、丙二醛（ＭＤＡ）抑制小麦超氧化物歧化酶（ＳＯＤ）活...     

发酵法制备硫酸软骨素裂解酶及其酶分离纯化的研究

以温和气单胞菌(Aeromonas sobriaYH311)为出发菌株进行发酵培养,发酵液经硫酸铵沉淀初步分离出粗酶液,将粗酶溶解并透析后再分别经过CM-Cellulose、QAE-sephadex A-50和Sephadex G-150层析柱进行逐级分纯,并跟踪测酶活,最后获得硫酸软骨素酶,经SDS-PAGE检测为一条带。此方法由粗酶至纯酶提纯倍数约186。     

Late-Glacial and Postglacial Vegetation and Associated Environment in Jianghan Plain

Pollen percentage and influx diagrams were prepared from two cores in Jianghan Plain, and studied by means of Fuzzy cluster and radiocarbon dating. This paper reports that there was a cool-temperate evergreen coniferous forest in late-glacial epoch, representing that the climate was cold and wet. In Holocene, the hypsithernaal interval took place about 9100–3500 year B. P. and the maximum was about 8000 year ago. At that time, the vegetation, was that of an evergreen and deciduous broad-leaf mixed forest, and the climate was warmer and damper than that of present. It also shows that the history of vagetational development and climate chenges in this area in the past 21900 years can be divided into five stages: (1). During 21000--10000 year B. P., the vegetation was one of the cool-temperate evergreen coniferous forest, dominated by Abies. The climate was cold and wet. (2) During 10000–9100 year B. P., the vegetation was that of the coniferous and deciduous broad-leaf mixed forest, consisting of Pinus. Quercus, Ulmus and Liquidambar, with a mild and cool climate. (3) During 9100–3500 year B. P., the mixed forest of evergreen and deciduous broad-leaf was predominant, consisting of Cyclobalanopsis, Fagus, Castanopsis, Castanea, Pterocarya, Quercus and Ulmus. The palaeoecological environment was warm and damp. (4) During 3500–2400 year B. P., there was deciduous broad-leaf, consisting of Quercus, Pterocarya, Liquidambar. and Pinus. The climate was droughter and cooler than that of stage(3). (5) During 2400 year B. P. to present, the broad-leaf forest, consisting of Quercus, Fagus, Liqui-dambar and Castanea, was dominant. The climate was warm and damp.     

胸腺素a1与复合a干扰素融合蛋白的表达及其生物学活性

实验旨在获得具有双重生物学活性的重组胸腺素a1(Thymosin alpha1, TM-a1)与复合a干扰素(IFNa-con)融合蛋白。选择大肠杆菌偏爱的密码子, 将合成的TM-a1与IFNa-con编码序列构成的融和基因克隆至大肠杆菌表达载体pET-22b(+)、在宿主菌BL21(DE3)-Codon plus-RP-X中成功表达了可溶性融合蛋白(TM-a1-IFN-con)。表达量占总蛋白的20%以上。通过硫酸铵沉淀、疏水层析、阴离子交换层析、阳离子交换层析、分子筛层析后, 产品纯度达到96%以上。采用细胞病变抑制法测定融合蛋白的抗病毒活性, 采用细胞增殖实验检测融合蛋白对小鼠脾淋巴细胞增殖的影响。结果表明, 融合蛋白的抗病毒活性优于市售的IFNa1b和IFNa2a。对小鼠脾淋巴细胞增殖的影响与市售的合成胸腺素a1相同。已有研究证实, 该融合蛋白具有良好的体外抗HBV作用, 其体外抗HBV活性比联合应用TM-a1和干扰素a强, 且细胞毒性明显低于联合应用TM-a1和干扰素a。以上结果表明, 通过大肠杆菌表达的可溶性融合蛋白(TM-a1- IFN-con), 既具有良好的干扰素a抗病毒作用, 也具有胸腺素a1促淋巴细胞增殖作用。     

Extraction and quantification of daunomycin and doxorubicin in tissues

The measurement of intracellular concentrations of the anti-cancer drug doxorubicin was performed by the application of a simple cell extraction technique combined with a rapid high-performance liquid chromatographic separation. Quantitation was done by fluorescence detection. The extraction procedure was non-degradative and the mean recovery of drug was 95%. A high drug extraction efficiency was confirmed with radiolabeled [³H]doxorubicin. The method is applicable to normal and neoplastic tissue.     

Rest length and compliance of non-immobilised and immobilised rabbit soleus muscle and tendon

The first aim of this study was to measure the contributions of muscle and tendon to the total compliance of resting muscle-tendon units. A second aim was to determine whether the decrease in muscle-tendon unit rest length produced by prolonged immobilisation in a shortened position is mediated primarily by adaptations of the muscle or tendon. One ankle joint from each of five rabbits was immobilised in a plantarflexed position for 14 days. The passive length-tension properties of soleus muscle fascicles and tendons from both hindlimbs were measured using a video-based tensile-testing system. In non-immobilised muscles, muscle fascicle strains exceeded tendon strains by up to four times. However, because the rest length of tendon was much greater than that of muscle fascicles, changes in tendon length accounted for nearly half of the total change in muscle-tendon unit length. The rest length of immobilised muscle-tendon units was less than that of non-immobilised muscle-tendon units from contralateral limbs. Most of this difference was attributable to a change in the rest length of the tendon; there was little change in the rest length of muscle fascicles. It is concluded that the tendon is responsible for a large part of the compliance of rabbit soleus muscle-tendon units at physiological resting tensions, and that adaptation of tendon rest length is the primary mechanism by which the rabbit soleus shortens in response to immobilisation at short lengths. Accepted: 7 May 1997     

Azaperone and azaperone-ketamine as a neuroleptic sedative and anesthetic in rats and mice

Azaperone alone and combined with ketamine were evaluated as sedative and anesthetic agents in outbred rats and mice. Using azaperone alone the duration of immobility was 1.9 to 10.8 hours for mice and 0.9 to 2.4 hours for rats. The withdrawal reflex was not eliminated from mice receiving azaperone alone; however, the withdrawal reflex was eliminated from 0.9 to 2.4 hours in rats receiving azaperone. Azaperone produced a tachypnea in rats and male mice while a depressed respiratory rate was observed in female mice. Using azaperone combined with ketamine, the duration of immobilization was 1.1 to 8.8 hours for mice and 1.3 to 6.0 hours for rats. The duration loss of the withdrawal reflex, which was used as an indication of surgical anesthesia, was 0.9 to 1.8 hours for mice and 1.0 to 6.0 hours for rats. An increase in respiratory rate was observed in rats given the combination while mice given the combination showed transient tachypnea followed by bradypnea. Overall, azaperone alone was shown to provide sedation in mice as compared to a dose dependent anesthesia in rats. The azaperone-ketamine combination produced a surgical plane of anesthesia in both rats and mice. Azaperone and the azaperone-ketamine combination appear to be a suitable alternative to sedatives and anesthetics currently used in rats and mice.     

Interaction between lactoferrin and ovotransferrin and Candida cells

Abstract The mechanism of antifungal activity of lactoferrin (Lf) and ovotransferrin (OTR) towards Candida albicans and Candida krusei was studied. In low iron-content medium, in minimal medium supplemented by 2,2'-dipyridyl, and in a medium in which Lf or OTR were separated from the culture by a dialysis membrane, the growth of C. albicans and C. krusei was proportional to the endogenous iron. Differences were observed when Lf or OTR was in contact with the fungal cells: C. albicans was inhibited, whereas C. krusei was not. Direct fluorescence indicated binding of Lf and OTR only on C. albicans surfaces, and suggested that antifungal activity is not simply related to iron deprivation, but involves interaction of the protein with the fungal surface.     

MDM2与端粒保护蛋白1的相互作用及其功能

目的:探究鼠双微体2同源体(MDM2)与端粒保护蛋白1(POT1)在细胞水平是否有相互作用,及其是否发挥E3泛素化连接酶的功能。方法:首先,用蛋白酶体抑制剂MG132处理稳定表达Flag-POT1的HeLa细胞,Western印迹检测Flag-POT1的表达情况;其次,在HeLa细胞中转入外源的Myc-MDM2和Flag-POT1质粒,48 h后收集细胞,通过免疫共沉淀方法验证Myc-MDM2和Flag-POT1是否具有相互作用;再次,在稳定表达Flag-POT1的HeLa细胞中转入Myc-MDM2或MDM2 siRNA,48 h后收集细胞,Western印迹检测Flag-POT1的表达水平。结果:MG132处理后,Flag-POT1的表达量明显升高且有拖尾现象,免疫共沉淀显示Myc-MDM2和Flag-POT1具有相互作用,但无论转入Myc-MDM2还是MDM2 siRNA,Flag-POT1的表达水平没有明显变化。结论:POT1通过泛素化途径降解,MDM2与POT1具有相互作用,但MDM2不是POT1主要的E3泛素化连接酶。     

Comparison of glucose, sorbitol and fructose accumulation in lens and liver of diabetic and insulin-treated rats and mice

1. The accumulation of glucose, fructose and sorbitol was determined in the lens, liver, and blood from normal, streptozotocin-induced diabetic, and insulin-treated diabetic rats and mice. 2. Sorbitol concentration in rat lens was 10-100 times greater than that in mouse lens, with the highest concentrations in the diabetic animals. 3. Sorbitol levels in rat and mouse liver, and mouse lens were similar and increased only slightly under hyperglycemic conditions. 4. Fructose accumulation was similar in rat and mouse liver and was elevated in the diabetic mouse blood and diabetic rat lens. 5. Aldose reductase activity in rat lens was approximately 350 times that of mouse lens. 6. Lenticular sorbitol dehydrogenase activity in rats was approximately ten times that in mouse lens. 7. Administration of insulin tended to lower liver glucose and subsequent sorbitol formation in the diabetic rat and mouse.     

Transfer and maintenance of IncP and IncW group plasmids into and between extra-slow-growing Bradyrhizobium japonicum strains

Abstract IncP group plasmid pRL180 was conjugally transferred from Agrobacterium tumefaciens LBA928 into extra-slow-growing (ESG) Bradyrhizobium japonicum strains and between ESG strains, RJ17W and RJ12S. pRL180 was integrated into the chromosome of RJ12S, RJ17W and RJ19FY. ESG strains efficiently transferred pRL180 into Escherichia coli at about a 3 × 10⁻⁵ frequency. IncW group plasmid pTY97 was transferred in intergeneric matings from E. coli into ESG strains at a high frequency of 2.5 × 10⁻³; between RJ17W and RJ12S transfer was about 5.6 × 10⁻⁴. pTY97 was maintained as an R' plasmid in RJ12S. The R' plasmid was resolved upon transfer into E. coli C where only pTY97 was autonomously replicated.     

四种野生盐生植物解剖结构与抗旱耐盐性

为了解盐生植物的解剖结构与抗盐性和抗旱性的关系,以二色补血草、草木樨,艾蒿、猪毛菜为材料,通过徒手切片和显微观察了植物的叶、茎、腺毛、分泌腔、气孔、表皮毛分布和结构。结果显示:猪毛菜的气孔密度低、气孔器小,表皮毛密集,叶面积小,抗旱能力最强;艾蒿的表皮毛长、浓密,气孔密度较低、气孔较小,抗旱性较强;二色补血草表皮毛短、密集,但气孔密度较高、气孔器较大,抗旱性较差;草木樨表皮毛短、稀疏,气孔密度较高、气孔也较大,抗旱性最差;二色补血草有发达的内分泌和外分泌组织,根系吸收的大量盐份积累在分泌腔中,并通过盐腺排出叶片,是排盐植物,耐盐性强;猪毛菜具有发达的内分泌组织,有大量分泌腔,且有粘液细胞和大量薄壁细胞,是耐盐植物,耐盐性强;草木樨具有较多的盐腺,是泌盐植物,耐盐性较强;艾蒿无盐腺等分泌组织。猪毛菜可以作为盐碱地"生物脱盐器"。     

Choline derivatives and sodium fluoride protect acetylcholinesterase against irreversible inhibition and aging by DFP and paraoxon

A light addressable potentiometric sensor was used to measure acetylcholinesterase (AChE) activity in order to evaluate the protective effects of quaternary compounds and NaF against enzyme phosphorylation and aging by two organophosphates. The use of the immobilized AChE made possible the quick removal of reagents (i.e., organophosphate, 2-pralidoxime, and protectant), thereby permitting accurate determination of AChE activity before and after phosphorylation and aging. Paraoxon was 15-fold more potent in inhibiting AChE than DFP, while the percent aging following phosphorylation by diiso-propylfluorophosphate (DFP) was much higher. Sodium fluoride (NaF), the most effective protectant against phosphorylation and aging, and the quaternary ammonium compounds reduced significantly AChE inhibition by DFP and paraoxon, to similar degrees. Even though the percent AChE activity that was lost to aging was reduced by these agents, aging as a percent of phosphorylated AChE was not reduced. Thus, their major effect was in reducing the percent AChE phosphorylation, which consequently resulted in reduction of total aged AChE. The finding that quaternary ammonium compounds protect against phosphorylation is consonant with the proposed presence of the active site of AChE in an aromatic gorge.     

翦股颖和高羊茅对铜、铅吸收及耐受性

测定了高羊茅和翦股颖两种草坪植物在Cu、Pb污染下发芽率、叶绿素、净光合速率和SOD的变化,以及两种草对Cu、Pb的吸收.结果表明, 重金属污染对高羊茅种子的萌发产生显著影响.高浓度Pb污染以及Pb、Cu复合污染对高羊茅的叶绿素影响很大,Cu单独污染对高羊茅叶绿素的抑制作用较小.翦股颖的叶绿素变化不显著.在3个Pb处理及350 mg·kg^-1 Cu处理下,翦股颖的SOD活性均高于对照,而只有在700 mg·kg^-1Pb 和350 mg·kg^-1 Cu+1 100 mg·kg^-1 Pb处理下高羊茅的SOD活性才升高.高羊茅的净光合速率受Cu污染影响较小,而受Pb污染影响较大.在Cu、Pb单独污染条件下,翦股颖净光合速率均呈下降趋势.从生理角度来看,翦股颖对Cu、Pb污染的耐受性强于高羊茅,而高羊茅对Cu污染的耐受性强于Pb.高羊茅Cu吸收量大,但Cu吸收量不随污染浓度升高而升高.翦股颖对土壤Pb的吸收能力较强, 且Pb吸收量随污染浓度的增加而增加.翦股颖和高羊茅可分别用于Pb和Cu污染土壤的治理.     

A rapid and sensitive method for the determination of hypusine in proteins and its distribution and developmental changes

A simple and sensitive method for determining hypusine in proteins was developed. A greater part of amino acids in the acid hydrolysate of proteins was separated from hypusine by treatment with an ion-exchange resin. The sample containing partially purified hypusine was then analyzed by high-performance liquid chromatography using the post-column derivatization method with o-phthalaldehyde. The recovery rate of hypusine through the overall procedure was more than 95%. Using this method, the distribution and developmental changes of hypusine in proteins were determined. The amino acid was found in proteins of all examined organs of rat. Its concentration was 5–40 nmol/g protein. The subcellular distribution in rat liver was also determined. About 60% of total amount of hypusine was present in the proteins of cytoplasmic and microsomal fractions and its relative concentration was high in the proteins of microsome and lysosome and low in mitochondria. In developing rat, the concentration of hypusine in the brain proteins was relatively high during the first 2 or 3 weeks of postnatal life and then decreased until adulthood. Its concentration in the liver proteins was highest at birth and then decreased continuously to the adult level.     

The absorption and translocation of diclofop-methyl and amitrole in wheat and oat roots

The absorption and translocation of diclofop-methyl (methyl 2-[4(2',4'-dichlorophenoxy)phenoxy]propanoate) was examined by using a specially designed treatment apparatus that separated excised roots or roots of seedlings into four zones. [¹⁴C]-Diclofop-methyl was absorbed along the entire root length of both wheat ( Triticum aestivum L.) and oat ( Avena sativa L.). In both species, absorption was greatest in the apical region of the root. Absorption by the apical region of wheat roots was more than three times greater than the basal portions, and more than twice as great as the apical region of oat roots. Less than 5% of the absorbed diclofop-methyl was translocated in both wheat and oat roots. Diclofop-methyl and diclofop(2-[4(2',4'-dichlorophenoxy)phenoxy]propanoic acid) were the predominant translocated forms. The absorption and translocation of amitrole (3-amino-1,2,4-triazole) were also examined. Amitrole was absorbed along the entire length of wheat roots and translocated primatily in the basipetal direction. The usefulness of the specially designed apparatus for biochemical and physiological studies is discussed.