Acceptor specificity of the human leukocyte alpha3 fucosyltransferase: role of FucT-VII in the generation of selectin ligands

The alpha3 fucosyltransferase, FucT-VII, is one of the key glycosyltransferases involved in the biosynthesis of the sialyl Lewis X (sLex) antigen on human leukocytes. The sialyl Lewis X antigen (NeuAcalpha(2-3)Galbeta(1-4)[Fucalpha(1-3)]GlcNAc-R) is an essential component of the recruitment of leukocytes to sites of inflammation, mediating the primary interaction between circulating leukocytes and activated endothelium. In order to characterize the enzymatic properties of the leukocyte alpha3 fucosyltransferase FucT-VII, the enzyme has been expressed in Trichoplusia ni insect cells. The enzyme is capable of synthesizing both sLexand sialyl-dimeric-Lexstructures in vitro , from 3'-sialyl-lacNAc and VIM-2 structures, respectively, with only low levels of fucose transfer observed to neutral or 3'-sulfated acceptors. Studies using fucosylated NeuAcalpha(2-3)-(Galbeta(1- 4)GlcNAc)3-Me acceptors demonstrate that FucT-VII is able to synthesize both di-fucosylated and tri-fucosylated structures from mono- fucosylated precursors, but preferentially fucosylates the distal GlcNAc within a polylactosamine chain. Furthermore, the rate of fucosylation of the internal GlcNAc residues is reduced once fucose has been added to the distal GlcNAc. These results indicate that FucT-VII is capable of generating complex selectin ligands, in vitro , however the order of fucose addition to the lactosamine chain affects the rate of selectin ligand synthesis.      

CRISPR/Cas9技术在工业微生物中的应用

近年来,通过基因编辑技术对工业微生物底盘细胞改造从而获得的优良细胞工厂,促进了农业、医学、环境、能源等领域的可持续发展,提高了人民的生活水平。微生物底盘细胞的改造离不开基因编辑,作为现阶段主要的基因编辑技术,规律间隔成簇短回文重复序列（clustered regularly interspaced short palindromic repeats,CRISPR）/Cas9系统自被发现以来,依靠其低成本、高效率等编辑优点,被广泛用于工业微生物底盘细胞的改造。本文主要简述了以CRISPR/Cas9为基础而衍伸出的各种基因编辑技术,提出了常用的工业微生物对应底盘细胞的改造策略,以期为研究者在进行微生物底盘细胞改造时选择出合适的基因编辑方法。最后指出了CRISPR基因编辑技术面临的PAM位点的依赖性、脱靶效应和应用广泛性等问题。     

日本血吸虫尾蚴头腺及钻腺的超微结构

通过电镜观察发现：日本血吸虫尾蚴的头腺为多导管的单细胞腺,由腺体部和若干突起状的短导管构成,其分泌小体呈圆形或卵圆形,平均大小为 ０． ２９ × ０． ２３μｍ,具有同心的环层膜状结构。前钻腺含有 Ａ、Ｂ两型分泌小体。 Ａ型分泌小体近似圆形,平均大小为 ０． ４６× ０． ３２μｍ,基质为电子致密的均 匀结构;Ｂ型分泌小体数量众多,略呈不规则的圆形,平均大小为１．０２×０．８２μｍ,基质为中等电子致 密并含十余个小圆形的电子透亮区。后钻腺含有椭圆形或长条状的分泌小体,平均大小为０．９７×０．６２μｍ,基质呈同质性,或伴有电子密度较深的圆形聚集物,或基质呈泡沫状。每根钻腺导管 的胞质外周具有许多沿导管长轴纵行的微管结构,在导管周围有纤维性网状构造包绕,起着对细长导管的支架作用。在导管束外周又有大量肌纤维包统而形成的鞘状结构,它对管内分泌小体的排出可能具有选择性控制作用。     

In vitro studies of the natural humoral antitumor reactivity of BALB/c and C57BL/6J mice

Summary Antitumor antibodies were produced in vitro by spleen cells harvested from 12- to 18-month-old C57BL/6J and BALB/c mice with a naturally occurring complement-dependent serum cytotoxicity for tumor cells. The antibodies were of the IgM class and had a complement-dependent cytotoxic reactivity on EL4 cells, which was not absorbed by normal thymus cells. No natural antibodies were produced by untreated spleen cells from 1- or 3-month-old mice of the same two strains, which had no natural serum reactivity. However, after a treatment with an anti-Thy-1 serum and complement before culturing, spleen cells from 3-month-old mice, but not 1-month-old mice, produced the natural antitumor cytotoxic antibodies in vitro. When antibody-producing spleen cells from 12-month-old mice were cultured in vitro together with spleen cells from the unreactive 3-month-old syngeneic mice, inhibition of the antibody production occurred. This inhibiting capacity increased between 1 and 6 months of age and was abrogated by a treatment with an anti-Thy-1 serum and complement or with an anti-Ly-2 serum, whereas the passage of the inhibiting spleen cells on an anti-IgG column did not modify the inhibiting capacity. The in vitro data confirm previous in vivo findings on the nature, specificity and systems of regulation of the natural antitumor cytotoxic antibodies.     

Fractionation of the ribosome inactivating protein preparations with triazine dyes

Aspergillins are ribosome-inactivating proteins (RIPs), isolated from several strains of Aspergillus. The interaction between Cibacron Blue F3GA and two members of this family, alpha sarcin and mitogillin, and other RIPs of type I, was studied. Alpha sarcin retention depended on pH and ionic strength. By chromatography on Affi-Gel Blue in mild experimental conditions, mitogillin and PAP-I did not interact with the dye, whereas 40% of alpha sarcin and 70-90% of briodin, RTA and gelonin were recovered in the bound fraction. In all cases, the major fraction showed a higher toxicity level in protein synthesis inhibition assays. The unbound alpha sarcin, conjugated with the anti-ovarian carcinoma monoclonal antibody MOv17, showed on OVCA 432 a cytotoxicity which was 900 times higher than that exerted by the alpha sarcin alone.     

RNA聚合酶Ⅲ启动子的结构与功能

ＲＮＡ聚合酶Ⅲ (ｐｏｌⅢ )是真核生物催化合成ｔＲＮＡ和 5ＳｒＲＮＡ及一些核小ＲＮＡ和胞质ＲＮＡ必需的酶。ＲＮＡ聚合酶Ⅲ能够识别ｔＲＮＡ基因中一些高度保守的特征性ＤＮＡ序列而启动下游ＤＮＡ的转录 ,这些序列称为ＲＮＡ聚合酶Ⅲ启动子。ＲＮＡ聚合酶Ⅲ启动子不仅广泛存在于真核细胞中ｔＲＮＡ、Ｕ6核小ＲＮＡ(ＳＮＲ6 )等的基因中 ,也是病毒催化合成一些小片段ＲＮＡ如腺病毒ＶＡＲＮＡ所必需的。ＲＮＡ聚合酶Ⅲ启动子因其能在体内外高效快速地转录某些小片段基因 ,已被人们广泛地应用于核酶和反义ＲＮＡ技术中 ,在众多的ＲＮＡ…     

集胞藻Synechocystissp.PCC6803利用葡萄糖生长与光合能量转化的关系

用PAM叶绿素荧光仪测定了饥饿细胞、光自养细胞和混合营养细胞的叶绿素荧光 ,并对 3种类型细胞的荧光参数 :PSⅡ实际光化学效率 φⅡ和还原型质醌Q-A 进行了比较。用双重转盘磷光机测定了光自养细胞和混合营养细胞的毫秒延迟发光。根据叶绿素荧光动力学分析和毫秒延迟发光的结果及光合电子传递抑制剂 3,4_二氯苯基二甲脲 (DCMU)、二溴百里香醌 (DBMIB)对集胞藻 6 80 3(Synechocystissp .PCC 6 80 3)混合营养生长影响进行了分析 ,集胞藻 6 80 3混合营养培养的生长速率显著高于光自养培养的原因可能在于一是外源葡萄糖没有抑制反而是促进了混合营养细胞的光自养生长 ,二是呼吸基质向质醌库提供电子 ,使光合机构的能量转化加强 ,从而促进了集胞藻6 80 3细胞的利用葡萄糖的合成代谢。     

基因重组大肠杆菌表达类人胶原蛋白诱导条件优化研究

为了提高重组大肠杆菌BL21高密度发酵生产类人胶原蛋白的产量,分别研究了诱导时机、乙酸浓度、诱导强度以及补氮方式对外源基因表达的影响,并对各因素进行了优化。采用补料-分批式发酵,初始装液体积为6L。最终细胞密度可达到84.3g／L,类人胶原蛋白的表达量为14.6g/L。结果表明:在对数生长的中后期进行诱导,尽量减少乙酸的积累量,42℃诱导3h后降温至39℃继续诱导,并采用每隔10min补36ml补料氮溶液的周期操作方式,有利于细胞的生长和外源蛋白的表达。