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。     

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.     

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。     

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.     

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.     

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.     

Glucose and lactate turnover and gluconeogenesis in chronic metabolic acidosis and alkalosis in normal and diabetic dogs

The turnover rate of glucose, the irreversible disposal rate of lactate, and the rate of gluconeogenesis from lactate were calculated by tracer methods in four normal and four alloxan-diabetic dogs under control conditions as well as in chronic, stable metabolic acidosis and alkalosis. Acidosis was produced by feeding dogs 0.8-1 g.kg-1.day-1NH4Cl over 1 week, alkalosis was produced by feeding dogs a chloride-free diet and injections of furosemide. Mean plasma pH in the three states were 7.28 +/- 0.013, 7.40 +/- 0.024, and 7.51 +/- 0.015 in normal dogs, and 7.22 +/- 0.025, 7.42 +/- 0.009, and 7.49 +/- 0.002 in the diabetic dogs. Respective mean plasma bicarbonate levels were 14.6 +/- 0.88, 22.0 +/- 0.80, and 32.4 +/- 1.88 mequiv. in normal dogs, and 12.3 +/- 1.30, 22.6 +/- 0.66, and 35.0 +/- 1.14 mequiv. in diabetic animals. In normal dogs shifts in acid-base balance had no effect on the level of plasma glucose or the turnover rate of glucose. In diabetic dogs plasma glucose level was significantly elevated by alkalosis. Plasma lactate was positively correlated with plasma pH (r = 0.69, p less than 0.01) and was in general higher in diabetic than in normal animals. The increment in concentration was due to a decreased clearance of lactate from the plasma. The irreversible disposal rate was not changed by the acid-base status. Whereas a larger fraction of lactate removed from the plasma appeared in glucose in diabetic animals, this fraction was not changed significantly by shifts in the acid-base status.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and physicochemical and immunological analysis of chicken alpha-fetoprotein

Chicken alpha-fetoprotein was isolated from 12 to 13-day-old embryonic chicken serum by column chromatography on CM-Sephadex C-50. Hydroxyapatite and DEAE-Sephadex A-25. The purified protein was homogeneous based on polyacrylamide gel electrophoresis, immunoelectrophoresis and isoelectric focusing. The purified protein had the following physicochemical and immunological properties. (1) It was a glycoprotein with a single polypeptide chain. (2) The molecular weight of the protein was estimated at 71,000 by SDS-polyacrylamide gel electrophoresis. (3) The isoelectric point of the protein was 4.90. (4) The amino acid composition of the protein was similar to those of mammalian alpha-fetoproteins. (5) The protein showed no steroid-binding capacity. (6) It was immunologically distinct from mammalian alpha-fetoprotein. (7) No immunological cross-reaction was observed between the protein and chicken albumin.     

Sphingomyelin and ceramide-phosphoethanolamine synthesis by microsomes and plasma membranes from rat liver and brain

Pulse-chase experiments showed that phosphatidylethanolamine (PE) was the direct precursor for ceramide-phosphoethanolamine, a sphingomyelin analogue, in the same way as phosphatidylcholine was for sphingomyelin. Ceramide-phosphoethanolamine could be identified by incorporation of radioactivity from labeled PE, as well as by its stability in alkaline methanolysis and its ability to be methylated by S-adenosyl-methionine. Ceramide-phosphoethanolamine synthesis from labeled exogenous PE seemed to be independent of exogenous ceramide; it was proportional to the amount of incubated membrane, when taking into account the isotopic dilution of labeled precursor by endogenous PE. Sphingomyelin synthesis, which was demonstrated using natural PC as a substrate, was not possible using dipalmitoyl-PC. The formation of sphingomyelin and ceramide-phosphoethanolamine was demonstrated in microsomes and plasma membranes from rat brain and liver.     

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。     

ATPase in mature and differentiating phloem and xylem

A cytochemical study using a lead precipitation technique has been made of the distribution of adenosine triphosphatase (ATPase) in mature and differentiating phloem and xylem cells of Nicotiana tabacum and Pisum sativum. The sites of ATPase localization in tobacco phloem were the plasma membrane, endoplasmic reticulum, mitochondria, dictyosomes, plasmodesmata, and the dispersed P proteins of mature sieve elements. In pea phloem sieve elements ATPase was localized in the endoplasmic reticulum, but was not associated with the P proteins or plasma membranes at any stage of their differentiation. In pea transfer cells ATPase activity was associated with the endoplasmic reticulum at all stages of their differentiation and with the plasma membrane of transfer cells that had formed wall ingrowths. In xylem cells of both tobacco and pea the patterns of ATPase activity was similar. At early stages of differentiation ATPase activity was associated with the plasma membrane and the endoplasmic reticulum. At intermediate stages of differentiation ATPase activity continued to be associated with the endoplasmic reticulum, but was no longer associated with the plasma membrane. At later stages of xylem element differentiation ATPase activity was associated with disintegrating organelles and with the hydrolyzing cell walls.     

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     

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.     

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...     

Transferrin and ferritin endocytosis and recycling in guinea-pig reticulocytes

Transferrin and ferritin endocytosis and exocytosis by guinea-pig reticulocytes were studied using incubation with pronase at 4 degrees C to distinguish internalized and membrane-bound protein. Internalization of both transferrin and ferritin occurred in a time- and temperature-dependent fashion. Transferrin endocytosis was more rapid than that of ferritin. Transferrin binding to receptors was not altered, but transferrin endocytosis was decreased in the presence of ferritin. Iron accumulation from transferrin was inhibited by ferritin to a greater extent than could be accounted for by the decreased rate of endocytosis. In pulse-chase experiments, almost all of the transferrin was released intact from reticulocytes, but only about 50% of the total internalized ferritin was released, of which 85% was intact. The endocytosis of transferrin by rabbit reticulocytes was 2- to 2.5-times faster than guinea-pig reticulocytes. These data suggest that ferritin and transferrin are internalized by receptor-mediated endocytosis, possibly involving the same coated pits and vesicles, but that the proteins are recycled only partly in common.     

重组超耐热酸性α-淀粉酶的分离纯化及其性质研究

基因工程菌所产生的重组超耐热酸性α-淀粉酶,通过超滤浓缩、脱盐和聚丙烯酰胺垂直板凝胶电泳进行纯化,得到电泳纯的超耐热酸性α-淀粉酶,纯化倍数为11.7,活力回收率为29.8%。用SDSPAGE测得该酶的分子量为55kD,酶的等电点pI(室温)为5.0,以可溶性淀粉为底物的Km值为1.12gL,用硫酸酚法测得其含糖量为15.4%。该酶的最适反应温度为95℃,最适反应pH值为4.5。在pH4.0～7.0室温放置48h酶活没有变化,110℃保温1h残留60%活力。Cr3 、Fe2 、Cu2 抑制酶的活性,Ca2 对酶活无影响。EDTA和DTT对酶的活性无影响。     

Synthesis and turnover of cerebrosides and phosphatidylserine of myelin and microsomal fractions of adult and developing rat brain.

The synthesis and turnover of cerebrosides and phospholipids was followed in microsomal and myelin fractions of developing and adult rat brains after an intracerebral injection of [U-14C]serine. The kinetics of incorporation of radioactivity into microsomal and myelin cerebrosides indicate the possibility of a precursor-product relationship between cerebrosides of these membranes. The specific radioactivity of myelin cerebrosides was corrected for the deposition of newly formed cerebrosides in myelin. Multiphasic curves were obtained for the decline in specific radioactivity of myelin and microsomal cerebrosides, suggesting different cerebroside pools in these membranes. The half-life of the fast turning-over pool of cerebrosides of myelin was 7 and 22 days for the developing and adult rat brain respectively. The half-life of the slowly turning-over pool of myelin cerebrosides was about 145 days for both groups of animals. The half-life of the rapidly turning-over microsomal cerebrosides was calculated to be 20 and 40 h for the developing and adult animals respectively. The half-life of the intermediate and slowly turning-over microsomal cerebrosides was 11 and 60 days respectively, for both groups of animals. The amount of incorporation of radioactivity into microsomal cerebrosides from L-serine was greatly decreased in the adult animals, and greater amounts of the precursor were directed towards the synthesis of phosphatidylserine. In the developing animals, considerable amounts of cerebrosides were synthesized from L-serine, besides phosphatidylserine. The time-course of incorporation indicated that a precursor-product relationship exists between microsomal and myelin phosphatidylserine. The half-life of microsomal phosphatidylserine was calculated to be about 8 h for the fast turning-over pool in both groups of animals.     

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.     

Separation of outer and cytoplasmic membranes of Fibrobacter succinogenes and membrane and glycogen granule locations of glycanases and cellobiase.

The outer membrane (OM) of Fibrobacter succinogenes was isolated by a combination of salt, sucrose, and water washes from whole cells grown on either glucose or cellulose. The cytoplasmic membrane (CM) was isolated from OM-depleted cells after disruption with a French press. The OM and membrane vesicles isolated from the extracellular culture fluid of cellulose-grown cells had a higher density, much lower succinate dehydrogenase activity, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles different from those of the CM. The OM from both glucose- and cellulose-grown cells and the extracellular membrane vesicles from cellulose-grown cultures exhibited higher endoglucanase, xylanase, and acetylesterase activities than the CM and other cell fractions. Endoglucanase 2 was absent from the isolated OM fractions of glucose- and cellulose-grown cells and from the extracellular membrane vesicles of cellulose-grown cells but was present in the CM and intracellular glycogen granule fractions, while endoglucanase 3 was enriched in the OM. Cellobiosidase was located primarily in the periplasm as previously reported, while cellobiase was mainly present in the glycogen granule fraction of glucose-grown cells and in a nongranular glycogen and CM complex in cellulose-grown cells. The cellobiase was not eluted from glycogen granules by cellobiose, maltose, and maltotriose nor from either the granules or the cell membranes by nondenaturing detergents but was eluted from both glycogen granules and cell membranes by high concentrations of salts. The eluted cellobiase rebound almost quantitatively when diluted and mixed with purified glycogen granules but exhibited a low affinity for Avicel cellulose. Thus, we have documented a method for isolation of OM from F. succinogenes, identified the OM origin of the extracellular membrane vesicles, and located glycanases and cellobiase in membrane and glycogen fractions.     

Growth and survival studies of psychrophilic and mesophilic yeasts.

The effect of temperature on the growth of members of five genera of yeasts was studied in one glucose-containing and two glucose-free media. The maximum growth rate was seen in the glucose-containing medium, and the minimum growth was in either of the two glucose-free media depending upon the organism. Data obtained by optical density measurements was supported by total cell counts. The highest survival at the restrictive temperatures was within 5 degrees C of the optimum temperature for a particular organism. Among the temperatures other than the optimum, the highest growth rate and cell yield was obtained at a temperature 5 degrees C below or above the optimum.