重组人白细胞介素—2制备工艺的研究

本文对大肠杆菌表达的重组人白细胞介素-2进行了制备工艺的研究。用4mol／L脲溶解可溶性细菌蛋白后可使rIL—2包涵体纯度达70％;在变性条件下进行凝胶过滤并收集rlL－2主峰纯度可达90％;利用Cu2 氧化复性;最后用反相HPLC纯化,得到高度均一,纯度高达98％,比活达到8．0×106U／mg蛋白,且无热源,无残留DNA的rIL—2;每次反相色谱分离过程可获得400mg左右的rIL－2,得率约25％。     

SephacrylS—100制备柱有效速成装填法及其检测

随着基因工程产品不断问世，生物技术下游工作越来越得到重视。纯化基因工程产品常需要用SephacrylS－100玻璃层折枝，但进口的预装住，不仅价格高而且也不方便，自填SePhacrylS－100玻璃层析柱不仅价格低，经济实惠，而且用起来方便，随用随填，使用过程中如发现往效下降，可以再生填料，重新装柱，在实践中我们摸索出一种即快捷又行之有效的装填SephacrylS－100制备往的方法。材料与方法一、材料1．5．5XI00cm夹套玻璃柱：上海华美实验仪器厂2．SephacrylS－100凝胶：Pharmacia产口DD3．不锈钢筛网漏斗：孔径0．］54mm，浙江社浦正…     

诺卡氏菌形放线菌β-甘露聚糖酶的纯化和性质

产β－１,４－Ｄ甘露聚糖酶的诺卡氏菌形放线菌（Ｎｏｃａｒｄｉｏｆｏｒｍ ａｃｔｉｎｏｍｙｃｅｔｅｓ）菌株ＮＡ３－５４０,发酵培养７２ｈ,发酵液离心去菌体,上清经硫酸铵沉淀,９５％乙醇沉淀,ＣＭ－Ｓｅｐｈａｄｅｘ Ａ５０柱层析、羟基磷灰石柱层析、ＤＥＡＥ－纤维素离子交换及Ｓｅｐｈａｄｅｘ Ｇ－１００分子凝胶过滤柱等步骤,β－甘露聚糖酶的比活提高了１３７倍,获得凝胶电泳均一的蛋白样品。经ＳＤＳ－ＰＡＧ     

基因工程人白细胞介素－2的生物功能研究

重组白细胞介素－2(rIL－2),能维持T－淋巴细胞及CTLL-2细胞株(IL-2依赖)的增殖达一个月之久,细胞总数增加了近千倍。这种作用能被rJL-2专-的单克隆抗体破坏,显示rIL-2的专一性,rIL-2能增加天然杀伤细胞(NK)的活动达46—96％,但其剂量太高时反而起抑制作用,它能诱异淋巴因子激活的杀伤细胞(Lvmphokine Activated Killier,LAK)的生成,它还能杀伤转移性人肺癌细胞及抗NK的HL－60癌细胞株,其杀伤效率达37.06—54．84％。这说明我们获得的rIL－2在上述生物功能方面与天然lL－2相似。     

海洋细菌Bacillus sp.2—羧基还原酶的纯化和性质

从一株海洋细菌Bacillus sp,中通过硫酸铵分级盐析,Q Sepharose FF阴离子交换层析,Hydroxyapatite柱层析和Sephadex G-100凝胶过滤,分离纯化出一种3－脱氧]葡糖醛酮代谢酶,定性为2－羧基醛还原酶,以粗酶液作起始,所获样品纯度提高141．4倍,活力回收率11．4％,2－羧基醛化合物对酶是特异性很好的底物,该酶对3－脱氧葡糖醛酮的Km和2.5mmol/L,分子量约为33kD,反应最适pH为6．2,在pH5．0－8．0,温度30℃以下酶活保持稳定,适量的ED－TA,巯基乙醇或二硫苏糖醇能提高酶的活性,碘乙酸,N-乙基顺丁烯二酰亚胺均造成酶部分失活。     

我国产日本医蛭水蛭素的分离和纯化

采用离子交换柱层析与凝胶过滤柱层析的方法对从我国产的日本医蛙［Hirudonipponica（whitman）］活体中提取出的唾液腺分泌物进行分离和纯化,获得了较纯净的水蛭素（Hirudin）。测定结果表明：日本医蛭唾液腺分泌物提取液中水蛭素含量为4AT－U／ml;纯化总得率为15％;纯化后的产物比活力为6708AT－U／mg蛋白。     

系统感染TMV的番茄叶片胞外四种β－N－乙酰氨基己糖苷酶的纯化和性质

系统感染ＴＭＶ的番茄叶胞外提取液经冰冻干燥浓缩、－２０℃丙酮沉淀、离子交换柱层析和凝胶柱层析纯化,获得４种β－Ｎ－乙酰氨基己糖苷酶。这些酶是由７５ｋＤ亚基构成的电荷异构体（ｃｈａｒｇｅｉｓｏｍｅｒ）,用过碘酸－Ｓｃｈｉｆｆ反应证明是糖蛋白,以Ｄ－硝基苯－Ｎ－乙酰－β－Ｄ－氨基葡萄糖苷（ｐＮＰ－β－Ｄ－ＧｌｃＮＡｃ）和ｐ－硝基苯－Ｎ－乙酰－β－Ｄ－氨基半乳糖苷（ｐＮＰ－β－Ｄ－ＧａｌＮＡｃ）为底物,这些酶具有相似的性质。Ｎ－乙酸氨基葡萄糖（ＧｌｃＮＡｃ）和Ｎ－乙酰氨基半乳糖（ＧａｌＮＡｃ）是这些酶的竞争性抑制剂,Ａｇ＋和Ｈｇ２＋是它们的强抑制剂,Ｆｅ２＋、Ｆｅ３＋和Ｃｕ２＋也抑制其活性。     

大肠杆菌表达的重组人粒细胞—巨噬细胞集落刺激因子（rhGM—CS…

对重组人粒细胞－巨噬细胞集落刺激因子（ｒｈＧＭ－ＣＳＦ）的工程菌表达产物进行纯化,经过超声破碎,包涵体抽提,凝胶层析,复性,离子交换一系列纯化步骤,终产物纯度达９８％,比活性达１０００００００ｕ／ｍｇ。     

大肠杆菌表达的重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)的纯化

对重组人粒细胞－巨噬细胞集落刺激因子（ｒｈＧＭ－ＣＳＦ）的工程菌表达产物进行纯化,经过超声破碎,包涵体抽提,凝胶层析,复性,离子交换一系列纯化步骤,终产物纯度达９８％,比活性达１０００００００ｕ／ｍｇ。     

巨噬细胞源成纤维细胞生长因子的分离纯化

为探讨二氧化硅在大鼠肺内引发的一系列纤维增生反应,试图寻找一种关键的细胞增生因子,采用高压液相色谱技术,包括凝胶过滤柱层析、离子交换柱层析以及反相Ｃ４高压液相色谱柱层析,获得一种新的巨噬细胞源成纤维细胞生长因子（ａｌｖｅｏｌａｒｍａｃｒｏｐｈａｇｅ－ｄｅｒｉｖｅｄｆｉｂｒｏｂｌａｓｔｇｒｏｗｔｈｆａｃ－ｔｏｒ,ＡＭＤＧＦ）,ＳＤＳ－ＰＡＧＥ结果表明它已达到均一的纯度,其分子量为５８０００,ｐＩ为４．７．该因子的分子量与ＭΦ在体外接受石英粉尘刺激分泌的因子的分子量明显不同．Ｎ端序列测定结果显示它与已知蛋白序列的同源性小于５０％,其刺激成纤维细胞增殖的最适浓度范围为２．０～６．０μｇ／ｍｌ,推测ＡＭＤＧＦ是矽肺纤维化病变发生、发展过程中调节成纤维细胞增殖的重要因子之一     

Conformational preferences of modified nucleoside N(2)-methylguanosine (m(2)G) and its derivative N(2), N(2)-dimethylguanosine (m(2)(2)G) occur at 26th position (hinge region) in tRNA

Conformational preferences of the modified nucleosides N²-methylguanosine (m²G) and N², N²-dimethylguanosine (m₂²G) have been studied theoretically by using quantum chemical perturbative configuration interaction with localized orbitals (PCILO) method. Automated complete geometry optimization using semiempirical quantum chemical RM1, along with ab initio molecular orbital Hartree–Fock (HF-SCF), and density functional theory (DFT) calculations has also been made to compare the salient features. Single-point energy calculation studies have been made on various models of m²G26:C/A/U44 and m₂²G26:C/A/U44. The glycosyl torsion angle prefers “syn” (χ = 286°) conformation for m²G and m₂²G molecules. These conformations are stabilized by N(3)–HC2′ and N(3)–HC3′ by replacing weak interaction between O5′–HC(8). The N²-methyl substituent of (m²G26) prefers “proximal” or s-trans conformation. It may also prefer “distal” or s-cis conformation that allows base pairing with A/U44 instead of C at the hinge region. Thus, N²-methyl group of m²G may have energetically two stable s-trans m²G:C/A/U or s-cis m²G:A/U rotamers. This could be because of free rotations around C–N bond. Similarly, N², N²-dimethyl substituent of (m₂²G) prefers “distal” conformation that may allow base pairing with A/U instead of C at 44th position. Such orientations of m²G and m₂²G could play an important role in base-stacking interactions at the hinge region of tRNA during protein biosynthesis process.     

H(2)O(2)-induced O(2) production by a non-phagocytic NAD(P)H oxidase causes oxidant injury

Non-phagocytic NAD(P)H oxidases have been implicated as major sources of reactive oxygen species in blood vessels. These oxidases can be activated by cytokines, thereby generating O(2), which is subsequently converted to H(2)O(2) and other oxidant species. The oxidants, in turn, act as important second messengers in cell signaling cascades. We hypothesized that reactive oxygen species, themselves, can activate the non-phagocytic NAD(P)H oxidases in vascular cells to induce oxidant production and, consequently, cellular injury. The current report demonstrates that exogenous exposure of non-phagocytic cell types of vascular origin (smooth muscle cells and fibroblasts) to H(2)O(2) activates these cell types to produce O(2) via an NAD(P)H oxidase. The ensuing endogenous production of O(2) contributes significantly to vascular cell injury following exposure to H(2)O(2). These results suggest the existence of a feed-forward mechanism, whereby reactive oxygen species such as H(2)O(2) can activate NAD(P)H oxidases in non-phagocytic cells to produce additional oxidant species, thereby amplifying the vascular injury process. Moreover, these findings implicate the non-phagocytic NAD(P)H oxidase as a novel therapeutic target for the amelioration of the biological effects of chronic oxidant stress.     

Synthesis and structural characterization of cadmium(II) complexes with 2-(2-pyridyl)imino-N-(2-thiazolin-2-yl)thiazolidine (PyTT)

We have carried out the synthesis of the cadmium coordination compounds [Cd(NO₃)₂(PyTT)(H₂O)] (1) and [CdCl₂{(μ-Cl)₂CdCl(μ-Cl)(μ-PyTT)Cd}₂]_n (2), together with their structural determination by means of X-ray diffraction. The compounds were also characterized through elemental analysis and infrared spectroscopy. The first complex presents a distorted pentagonal bipyramidal geometry with the axial positions occupied by one oxygen atom from a water molecule and a second one from a nitrate ion which acts as a monodentate ligand, whereas the equatorial plane contains three nitrogen atoms from the organic moiety and two oxygen atoms coming from the other nitrate group, which is bidentate. The structure of the second complex consists of parallel sheets linked by van der Waals forces, each one made up of structural units [CdCl₂{(μ-Cl)₂CdCl(μ-Cl)(μ-PyTT)Cd}₂], which possesses two PyTT ligands, 10 bridging chloro ligands and 5 cadmium(II) centres belonging to three environment types: octahedral CdN₂Cl₄, octahedral CdCl₆, on which a centre of symmetry is located, and tetrahedral CdNCl₃, present in a 2:1:2 ratio.     

Partikelgestalt des Spargel-Virus 2 (asparagus virus 2) (Kurze Mitteilung)

Zwei Kernpolyedervirus‐Isolate der Kohleule aus Deutschland (MbKPV‐D) und Moldawien (MbKPV‐Ki) wurden im Biotest geprüft und der Genotyp mit Hilfe der Restriktionsenzym‐Fragmentanalyse (REN) untersucht. Beide Isolate ergaben eine gleiche biologische Aktivität. Die REN‐Profile zeigten weitestgehende Übereinstimmung in der DNA‐Struktur. Geringe Unterschiede wurden in den Profilen der Eco RI‐ und Hind III‐ Schnitte gefunden. Die erhaltenen REN‐Profile stimmen im wesentlichen mit den für ein niederländisches MbKPV‐Isolate beschriebenen Bandenmustern überein.     

Stereospecific formation of alpha-hydroxycarboxylato oxo peroxo complexes of vanadium(V). Crystal structure of (NBu4)2[V2O2(O2)2(L-lact)2] x 2H2O and (NBu4)2[V2O2(O2)2(D-lact)(L-lact)] x 2H2O

An overview of structurally characterized alpha-hydroxycarboxylatodioxo- and alpha-hydroxycarboxylatooxoperoxovanadates(V) is presented and the geometric parameters of the V2O2 bridging core are discussed. The first case of a stereospecific formation of oxoperoxovanadates(V) is reported: The crystal structures of the isomeric compounds (NBu4)2[V2O2(O2)2(L-lact)2] x 2H2O and (NBu4)2[V2O2(O2)2(D-lact)(L-lact)] x 2H2O (lact = C3H4O3(2-), the anion of the lactic acid) differ mainly in the arrangement of the V2O2 core and in mutual orientation of the V=O bonds. The complexes with achiral ligands adopt the same structural type as the complexes formed from a racemic mixture of a chiral ligand, while the structure obtained using an enantiopure L,L-hydroxycarboxylate is different.     

Synthesis,spectral and magnetical characterization of monomeric [Cu(2-NO2bz)2(3-pyme)2(H2O)2] and polymeric [Cu{3,5-(NO2)2bz}2(3-pyme)2]n

Two new complexes of composition [Cu(2-NO₂bz)₂(3-pyme)₂(H₂O)₂] (1) and/or [Cu{3,5-(NO₂)₂bz}₂(3-pyme)₂] (2) (3-pyme = 3-pyridylmethanol, ronicol or 3-pyridylcarbinol, 2-NO₂bz = 2-nitrobenzoate and 3,5-(NO₂)₂bz = 3,5-dinitrobenzoate) have been prepared and studied by elemental analysis, electronic, infrared and EPR spectroscopy, magnetic susceptibility measurements and the structure of both complexes has been solved. Complex (1) shows an unusual molecular type of structure consisting of the [Cu(2-NO₂bz)₂(3-pyme)₂(H₂O)₂] molecules held together by hydrogen bonds and van der Waals interactions. Complex (2) exhibits a polymeric chain-like structure [Cu{3,5-(NO₂)₂bz}₂(3-pyme)₂]_n with copper atoms doubly bridged by two 3-pyridylmethanol molecules and the polymeric molecules are held together by van der Waals interactions. Complex (1) exhibits a magnetic moment μ_eff = 1.84 B.M. at 300 K that remains nearly constant within the temperature region (5–300 K). Further cooling results in lowering the magnetic moment to μ_eff = 1.82 B.M. at 1.8 K. The magnetic susceptibility temperature dependence obeys Curie–Weiss law with Curie constant of 0.423 cm³ K mol⁻¹ and with Weiss constant of −0.06 K. The magnetic moment of (2) exhibits a small increase with a decrease in the temperature (μ_eff = 1.80 B.M. at 300 K and μ_eff = 1.85 B.M. at 1.8 K) with Curie constant of 0.409 cm³ K mol⁻¹ and with Weiss constant of +1.1 K, which can indicate a very weak ferromagnetic interaction between the copper atoms within the chain. Applying the molecular field model resulted in obtaining zJ′ values −0.08 cm⁻¹ for complex (1), and −0.07 cm⁻¹ for complex (2), respectively, that could characterize intermolecular and interchain interactions transmitted through π–π stacking.