Biotechnological potential of sulfate-reducing bacteria for transformation of nitrocellulose

The biotransformation of NC by Desulfovibrio sp. was studied. The mass of NC was decreased by 4.9-9.3%. The rate of NC transformation was between 46 and 73 mg NC per mg of bacterial protein in 10 days. Moreover, N content (%N) in the remaining NC was reduced by 2-12%. The inhibitory effect of NC was clearly expressed when the growth of D. desulfuricans 1388 in lactate/sulfate medium was initiated. The growth rate of bacteria was 1.5-fold greater when NC was not added (0.074 and 0.05 h(-1) respectively). The transformation of NC by D. desulfuricans was accompanied by the appearance of nitrate in the culture liquid, the amount of which reached the peak by the 8th day.     

Some characteristics of the insulin-induced potentiation to anaphylactoid reaction in Sprague-Dawley rats.

The insulin-induced sensitization to generalized and local anaphylactoid reaction evoked by dextran was studied in Sprague-Dawley CFY rats. The generalized reaction was shown to be potentiated by insulin given subcutaneously in a dose-related manner. The minimum effective dose was as low as 0.04 U/kg. When this dose was injected intravenously, a marked but short-lived potentiation was observed. The insulin response could be elicited throughout the whole year. The local oedema induced by subplantar injection of dextran was found to be much less sensitive to insulin. Potentiation was observed during the period from March to October, while in the intermediate months, no such effect could be seen. The seasonal refractory state to insulin was abolished by bilateral adrenalectomy, and daily pretreatment of the rats with insulin for several days. Actinomycin D prevented the restorative effect of insulin pretreatment. Sensitization by a single insulin dose to both systemic and local dextran was suppressed in rats older than 6 months, and the refractoriness was in part reversed by adrenalectomy.     

Degradative acetolactate synthase of Bacillus subtilis: purification and properties.

A degradative acetolactate synthase (acetolactate pyruvate-lyase [carboxylating], EC 4.1.3.18) from Bacillus subtilis has been partially purified and characterized. The synthesis of the enzyme was induced by growth of cells in minimal medium plus isobutyrate or acetate. The enzyme was partially purified by ammonium sulfate fractionation, gel filtration, and hydroxyapatite chromatography. The pH optimum of the purified enzyme was 7.0 in phosphate buffer. When assayed in phosphate buffer (pH 7.0), activity was stimulated by acetate and inhibited by sulfate. When assayed in acetate buffer (pH 5.8), activity was inhibited both by sulfate and phosphate. Michaelis-Menten kinetics was observed when the enzyme was assayed in phosphate buffer (pH 6.0 or 7.0), and inhibition by sulfate was competitive and activation by acetate was noncompetitive. When assayed in acetate buffer (pH 5.8), nonlinear Lineweaver-Burk plots were obtained; inhibition by phosphate appeared to be competitive and that by sulfate was of the mixed type. The approximate molecular weight of the purified enzyme was 250,000 as determined by gel filtration.     

神经营养因子GDNF编码顺序的克隆及表达产物的纯化

根据基因库中的顺序,设计了胶质细胞源神经营养因子（ＧＤＮＦ）基因的ＰＣＲ引物,以此从人基因组ＤＮＡ中扩增并克隆了ＧＤＮＦ的编码序列,经ＤＮＡ测序确认后,该片段克隆到表达质粒ｐＥＴ－３ａ中,转化大肠杆菌ＢＬ２１（ＤＥ３）．培养的重组菌经ＩＰＴＧ诱导,在Ｔ７启动子调控下表达出ｈＧＤＮＦ蛋白．经电泳分析表明ＧＤＮＦ主要存在于细菌包涵体中．从培养菌中制备包涵体,经充分洗涤,溶解于含８ｍｏｌ／Ｌ尿素的变性缓冲液中．经ＳＰ－Ｓｅｐｈａｒｏｓｅ柱层析分离,梯度洗脱,以１５％ＳＤＳ－ＰＡＧＥ检查含ＧＤＮＦ的部分．将含单体ＧＤＮＦ部分进行复性,再次用ＳＰ－Ｓｅｐｈａｒｏｓｅ离子柱分离同源二体ＧＤＮＦ．最后经ＳＤＳ－ＰＡＧＥ制备电泳纯化,纯度大于９５％．经Ｎ端测序表明序列正确．经测定,每升培养菌可得约１０ｍｇ纯化的ＧＤＮＦ．     

小蜡果皮水溶性色素的提取及光敏感性

以去离子水和不同体积分数乙醇从小蜡果皮中浸提红色素,用光谱扫描法检测该红色素的光吸收特性,比较不同浸提时间及不同体积分数乙醇对浸提效果的影响,并用暴露方式进行光敏感性测定。结果显示,小蜡果皮色素在纯水中的溶解性最好,属水溶性色素,延长浸提时间可提高色素的浸出率,但杂质质量分数也随之提高,紫外线对色素的色度影响最大,直接照射可使色素的色度大幅度下降。     

Purification of membrane proteins from Acholeplasma laidlawii by agarose suspension electrophoresis in Tween 20 and polyacrylamide and dextran gel electrophoresis in detergent-free media.

Four of the membrane proteins from Acholeplasma laidlawii that are soluble in the nonionic detergent Tween 20 have been purified by preparative electrophoretic techniques utilizing different supporting media. The last purification step for two of the major proteins was a preparative polyacrylamide gel electrophoresis performed in the absence of any detergent. The proteins were recovered by continuous elution. The purity of the fractions was examined by analytical polyacrylamide gel electrophoresis and crossed immunoelectrophoresis. Two of the minor proteins were purified by dextran gel electrophoresis as the final step, which was also performed in a detergent-free buffer. The separation was followed by scanning the dextran gel in ultraviolet light. The proteins were recovered by slicing the gel and degrading the gel slices with dextranase. The homogeneity of the fractions was checked by electroimmunoassay.     

Fragmentation of re-formation of mitotic Golgi apparatus detected by a centrifugal method

The fragmentation/re-formation process of the Golgi apparatus during mitosis was studied by flotation centrifugation in a stepwise sucrose density gradient. The mitotic Golgi fraction was obtained from Chinese hamster ovary cells synchronized with thymidine and nocodazole. The Golgi apparatus detected by a marker enzyme, galactosyltransferase, was separated into two peaks by the flotation centrifugation. The amount of the Golgi recovered at the lower density peak was less in the mitotic cells than in the interphase cells. The separation profile of the mitotic Golgi returned to that of the interphase Golgi by further incubation of the mitotic cells. The re-formation of the fragmented Golgi was inhibited by nocodazole and vinblastine, but not by actinomycin D and cycloheximide.     

Isolation and study of active ATP-dependent phosphofructokinase from apple fruits Pyrus domestica Borkh

The activity of ATP-dependent phosphofructokinase (PFK) from subepidermal tissue of apple fruits was studied. The enzyme extracted under optimal conditions was stable for 14 h at room temperature. The enzyme was partially purified by ammonium sulfate fractionation and dialysis. PFK from apple fruits was found to be ATP-, UTP-, and CTP-specific. It was inhibited by PEP, Gly-2-P, Gly-1,3-DP, and ADP. The effect of the listed inhibitors was diminished by the presence of phosphate. The activity of PFK was stimulated by magnesium cations. The activity of the enzyme in fruits of an Antonovka cultivar was higher than in the Simirenko rennet cultivar by a factor of 1.3.     

重组小鼠血管内皮生长因子在毕赤酵母菌中的表达和纯化

目的：建立毕赤酵母重组小鼠血管内皮生长因子（mVEGF）的制备方法,为研究mVEGF的生物活性、抗原性等提供基础。方法：通过全基因合成方法获得编码mVEGF的基因片段,将其克隆至表达载体pPICZaA上,电转化整合到毕赤酵母GS115基因组中,用甲醇诱导表达目的蛋白,表达上清经硫酸铵沉淀、SephadexG25柱脱盐、阳离子交换层析三步纯化获得目的蛋白;用还原型和非还原型SDS-PAGE检测目的蛋白的聚体状态,用Westelqq印迹验证纯化蛋白;通过PNGaseF酶切分析目的蛋白的N-糖基化修饰;通过人脐静脉内皮细胞（HUVEC）增殖实验检测目的蛋白的生物活性。结果：获得mVEGF的重组毕赤酵母表达菌株,SDS-PAGE分析可见GSll5表达的重组mVEGF在还原状态下表观相对分子质量约为20×10^3,在非还原状态下约为40×10^3;经Western印迹检测,这些条带均为目的蛋白条带,能被兔抗mVEGF抗体特异性结合,PNGase F酶切后相对分子质量降至18×10^3左右,证明目的蛋白发生了Ⅳ-糖基化修饰;细胞测活实验表明,mVEGF具有刺激HUVEC增殖的生物活性。结论：利用毕赤酵母菌制备了具有生物活性的重组mVEGF。     

Anatomical variations of the arterial pattern in the right hemiliver

The arterial supply to the right hemiliver was studied in 80 liver casts. The arteries were divided into 10 groups according to their origin and branching pattern. The right hemiliver was supplied by one artery in 96% of cases and by two arteries in 4%. When there was only one artery it originated from the proper hepatic artery in 73/77 cases and from the superior mesenteric artery in 4/77 cases. The replacing right hepatic artery which originated from the superior mesenteric vessel supplied the whole right hemiliver in 5% of cases. The incomplete replacing right hepatic artery which supplied only a part of the right hemiliver was found in 4% of cases. The anterior section (segments 5 and 8) was supplied by one artery in 61%, by two arteries in 30% and by three arteries in 9% of cases. The posterior section (segments 6 and 7) was supplied by one artery in 66%, by two arteries in 31% and by three arteries in 3% of cases. Segments 5 and 7 were predominantly supplied by one artery, whereas segments 6 and 8 by two arteries.     

猪胎盘脂多糖的分离纯化及其对脾淋巴细胞的诱导作用

 健康猪胎盘经酶解,提取,氯仿-正丁醇除杂蛋白与5'-磷酸二酯酶除核酸,再经透析,乙醇沉淀,DEAE-Dextran-Gel A-25层析制得纯品猪胎盘脂多糖(简称P-脂多糖)。分析结果表明,P-脂多糖是一种类酸性粘多糖与脂质的共价结合物,分子量约为75kD,分子中含11种单糖组成,其中半乳糖含量最高,其次为岩藻糖,氨基已糖和已糖醛酸。体外实验发现P-脂多糖能明显诱导小鼠淋巴细胞的增殖,强烈地促进淋巴细胞的DNA合成,刺激指数约为9.3,最适刺激浓度50～60μg/mL,最适刺激时间48小时,同时能明显促进淋巴细胞的蛋白质合成。实验还证实,P-脂多糖对脾淋巴细胞的增殖诱导作用并不是通过依赖于白间素-2(IL-2)途径,而是通过增强淋巴细胞IgM的表达,表明P-脂多糖是一种促B-淋巴细胞分裂剂。     

The molecular weight and properties of a neutral metallo-endopeptidase from rabbit kidney brush border

1. Some properties of a brush-border neutral endopeptidase purified from rabbit kidney were investigated. The peptidase was assayed by its ability to hydrolyse [¹²⁵I]iodoinsulin B chain. 2. The enzyme was found to be homogeneous when studied in the analytical ultracentrifuge and stained as a single glycoprotein band after electrophoresis in polyacrylamide gels. 3. The molecular weight was estimated by gel filtration in columns of Sephadex G-200, by polyacrylamide-gel electrophoresis in the presence of 2-mercapto-ethanol and sodium dodecyl sulphate and by sedimentation equilibrium in the ultra-centrifuge. The estimates fell within the range 87000–96000. The mean from two sedimentation equilibrium experiments was 93000, though this estimate may be slightly inflated because of the carbohydrate component of the enzyme. No evidence of dissociation into smaller subunits was obtained in the presence of thiol, sodium dodecyl sulphate or guanidine hydrochloride. 4. The endopeptidase was maximally active at pH6.0, although in phosphate buffer, which was strongly inhibitory, an optimum above pH8 was observed. 5. The enzyme was not affected by di-isopropyl phosphofluoridate nor by several thiol reagents. It was, however, strongly inhibited by many thiols and by EDTA and other chelating agents. 6. Although activity of the EDTA-treated enzyme could be partially restored by various bivalent metal ions, the optimum concentration for its reactivation by Zn²⁺ was lower than that for other ions. This metal was detected in the enzyme preparation by atomic absorption spectrophotometry in an amount equivalent to approximately one atom/mol. 7. The enzyme is the only endopeptidase shown to be located in the kidney brush border and is the first mammalian example of a neutral Zn²⁺- activated endopeptidase to be characterized.     

金桂与丹桂醇溶性色素的提取及其稳定性比较

用不同体积分数的乙醇从金桂和丹桂的花瓣中浸提所含的花色苷类色素,用光谱扫描法检测该色素的光吸收特性,比较不同乙醇体积分数、不同浸提时间对色素浸提效果的影响,并进行光敏感性试验和酸碱影响试验。结果显示,金桂和丹桂的花色素用体积分数90%的乙醇浸提具较高的浸出率;延长浸提时间可提高色素浸出率;紫外线直射可使色素的色度大幅度下降,日光灯和散射光也会导致色度在一定程度上的下降;色素在酸性溶液中具一定的稳定性,但在碱性溶液中表现出不稳定性。     

Effect of osmotic shock on tetracycline resistance in Escherichia coli bearing an R-factor

1. Escherichia coli with an R-factor conferring resistance to tetracycline was induced to high-degree resistance by pre-exposure to the antibiotic. The degree of resistance was drastically lowered by subjecting the cells to osmotic shock. 2. Resistance to tetracycline was rapidly restored by incubating the shocked cells in a glucose-salts medium containing shock proteins prepared from tetracycline-sensitive and -resistant cells. Resistance was also restored by incubating the cells in a complex medium without shock protein. 3. The initial recovery of resistance was followed by a secondary fall in resistance when the cells were cultured in complex medium; this secondary fall was largely prevented by the addition of a low concentration (10mug/ml) of tetracycline to cells. The secondary fall was significantly less in shocked E. coli cells harbouring a mutant R-factor in which tetracycline resistance is largely constitutive. 4. Tetracycline resistance was also transiently depressed by treating R-factor-bearing cells with EDTA in tris buffer. 5. The significance of these results in relation to the mechanism of tetracycline resistance in R-factor-bearing cells is discussed.     

SOX4单克隆抗体的制备及其在肿瘤细胞表达分析中的应用

应用分子生物学技术, 构建了含SOX4编码序列的原核表达载体, 在大肠杆菌 DH5a中获得了GST-SOX4融合蛋白的可溶性表达。应用谷胱甘肽-Sepharose 4B对重组蛋白进行了纯化, 利用纯化的融合蛋白免疫小鼠, 制备了可特异性识别SOX4的单克隆抗体。通过间接 ELISA 法鉴定了抗体的效价为1 × 10-5, Western blotting 分析证实了抗体的特异性。结果显示, 该抗体可识别细胞内外源性过表达及内源性的SOX4蛋白。在培养细胞系、小鼠不同组织中, SOX4蛋白的表达存在显著的差异。本研究制备的SOX4单克隆抗体具有良好的特异性, 为进一步研究SOX4在肿瘤发生中的作用提供了重要的工具。     

The regulation of respiration and growth of potato tuber callus by endogenous ethylene. Suppression of ethylene action by 2,5-norbornadiene

The growth (fresh and dry weight increase) of potato tuber ( Solanum tuberosum L. cv. Bintje) callus discs was stimulated by incubation in air with 500 ppm 2,5-norbornadiene (NBD, a competitive inhibitor of ethylene action) and inhibited by incubation in air with 4 000 ppm NBD. Ethylene formation by the callus was stimulated by NBD. The development of the alternative pathway, measured in isolated mitochondria was inhibited by NBD in a concentration-dependent way. The alternative pathway capacity, measured in vivo, was inhibited by 4 000 ppm NBD, but not by 500 ppm. Uninhibited in vivo respiration, which consists of cytochrome path activity and alternative path activity, was stimulated by the treatment with 500 ppm NBD. The main contribution to this stimulation was made by the cytochrome pathway. In 4 000 ppm NBD-treated callus, uninhibited respiration seemed to be unaffected as a consequence of an inhibited cytochrome path activity, which was compensated by a stimulated alternative path activity. Both in 500 and 4 OIK) ppm NBD-treated callus the alternative path activity in vivo was stimulated.
The regulatory role for endogenous ethylene in potato tuber callus is discussed in relation to: 1) The induction of respiratory pathways, 2) the supply of reduction equivalents in vivo and 3) growth.     

山梨糖脱氢酶在脱氮副球菌中的表达

目的:在脱氮副球菌PD1222中表达山梨糖脱氢酶(SDH)。方法:从质粒pMD-18T上复制氨苄西林抗性基因Ampr,从酮古龙酸菌中复制SDH基因sdh,先后酶切连接到pIND4质粒上,构建pIND4-Ampr-sdh穿梭质粒;再把pIND4-Ampr-sdh电转入大肠杆菌S17-1λpir作为供体菌,脱氮副球菌PD1222为受体菌进行双亲本接合转移;挑取壮观霉素和氨苄西林双抗平板上的接合子进行培养,菌液PCR复筛接合子,测序鉴定,通过DCIP法和非变性聚丙烯酰胺凝胶电泳法检测阳性克隆的SDH活性。结果:构建的质粒pIND4-Ampr-sdh成功转入脱氮副球菌PD1222中,SDH获得表达并检测到其蛋白活性。结论:实现了SDH在脱氮副球菌中的表达,为在脱氮副球菌中研究SDH的下游电子传递链奠定了基础。     

Isolation and study of active ATP-dependent phosphofructokinase from apple fruits Pyrus domestica Borkh

The activity of ATP-dependent phosphofructokinase (PFK) from subepidermal tissue of apple fruits was studied. The enzyme extracted under optimal conditions was stable for 14 h at room temperature. The enzyme was partially purified by ammonium sulfate fractionation and dialysis. PFK from apple fruits was found to be ATP-, UTP-, and CTP-specific. It was inhibited by PEP, Gly-2-P, Gly-1,3-DP, and ADP. The effect of the listed inhibitors was diminished by the presence of phosphate. The activity of PFK was stimulated by magnesium cations. The activity of the enzyme in fruits of an Antonovka cultivar was higher than in the Simirenko rennet cultivar by a factor of 1.3.     

The Effects of Plant Growth Substances on Rind-Regeneration after Girdling in Solanum melongena cv. esculantum

This paper deals with the effects of four plant growth substances, ic. IAA, NAA, 2,4-D and GA and their different concentration on rind-regeneration after girdling in Solanum melongen var. esculantum. The formation of callus was promoted by IAA, NAA and GA, but retarded by 2,4-D in early stage. The initiation of vascular cambium in callus was retarded by all these substances. However, an increase in amount of xylem was promoted by IAA at low concentrations. The different concentrations of NAA and GA affected a decrease in amount of xylem. The formation of "bundled" vascular tissue was impelled by NAA, GA and 2,4-D. The initiation of phellogen was promoted by IAA and NAA at high concentrtion. In addition, the nest-like tracheid mass was induced in callus by IAA and NAA frequently.     

小鼠pEGFP-Hoxa11真核表达载体的构建及表达

目的 构建和鉴定Hoxa11和EGFP双基因共表达真核载体.方法 采用DNA重组技术,将目的 基因Hoxa11克隆至含有报告基因EGFP的pEGFP-N1真核表达载体中,构建的真核表达载体pEGFP-Hoxa11经PCR,双酶切及基因测序鉴定;转染至CHO细胞,荧光显微镜下观察重组质粒的表达,提取细胞蛋白Western印迹检测蛋白表达.结果 pEGFP-Hoxa11重组质粒构建成功.构建的真核表达载体pEGFP-Hoxa11能在CHO细胞中有效表达.结论 成功构建了共表达Hoxa11和EGFP的真核表达载体,并能在CHO细胞中有效表达.为进一步研究Hoxa11的功能提供实验基础.