家蚕系统表达的重组人丁酰胆碱酯酶的生化性质

研究家蚕系统中高效表达的重组人丁酰胆碱酯酶(rhBChE)的生化性质并与天然人丁酰胆碱酯酶(nhBChE)进行比较。采用丁酰胆碱酯酶活性测定,抑制剂及重活化剂作用,免疫印迹等方法。实验结果表明,rhBChE及nhBChE在底物亲和力、抑制剂敏感性及可重活化性、稳定性、对抗体的反应性等方面均有相似的生化性质,重组人丁酰胆碱酯酶具有天然人丁酰胆碱酯酶的功能。但rhBChE的糖基化修饰程度较低,用于人体时应予考虑     

Transport of Wild-Type and Recombinant Nucleopolyhedroviruses by Scavenging and Predatory Arthropods

Abstract Wild-type and recombinant nucleopolyhedroviruses (NPVs) were compared in their capability to be transported over limited distances by the predator Podisus maculiventris (Say) and scavengers Sarcophaga bullata (Parker) and Acheta domesticus (Linnaeus) in Trichoplusia ni (Hübner) larvae infesting collards in a greenhouse microcosm. Viruses tested were variants of Autographa californica (Speyer) NPV (AcNPV): wild-type virus (AcNPV.WT), AcNPV expressing a scorpion toxin (AcNPV.AaIT), and AcNPV expressing juvenile hormone esterase (AcJHE.SG). Podisus maculiventris transported AcNPV.WT and S. bullata transported AcNPV.WT and AcNPV.AaIT. Prevalence and transport of AcNPV.WT were greater than those of AcNPV.AaIT and AcJHE.SG, regardless of whether the nontarget organism carriers were present or absent. Podisus maculiventris and S. bullata transported recombinant and wild-type NPVs at a rate of up to 62.5 cm/day, and A. domesticus transported wild-type NPV at 125 cm/day. The infected host insects, T. ni, undoubtedly contributed to viral transport in the current research. In every experiment, both the wild-type and recombinant virus spread to some degree in the plots without predators or scavengers. The relative amounts of NPVs that accumulated in soil, as indicated by bioassay mortality percentages, generally exhibited spatial patterns similar to those of T. ni mortality due to NPV on the collards plants. Thus, the predator and scavengers in the current research demonstrated some capacity to transport wild-type as well as recombinant viruses at significant rates in a greenhouse microcosm. Received: 6 December 1999; Accepted: 29 February 2000; Online Publication: 12 May 2000     

腹腔注射重组腺病毒诱导的免疫反应

为研究重组腺病毒接种实验动物后的免疫反应性,利用RT-PCR方法,从HAV的RNA中克隆了结构蛋白基因插入穿梭质粒pXCX2Not I,通过磷酸钙-DNA共沉淀技术,将复制缺陷型腺病毒载体与线形化的pXCX2 CMV-HAV共转染293细胞.一系列检测方法证明产生了重组腺病毒rAdHAV.纯化后的rAdHAV滴度为1×109TCID50/mL,腹腔注射免疫昆明种小白鼠后,可诱导产生抗HAV IgG和HAV中和抗体.复制缺陷型腺病毒可作为发展基因工程病毒疫苗载体的有效系统.     

Genetic Association of Butyrylcholinesterase with Major Depressive Disorder

Biochemical Genetics - Major depressive disorder (MDD) is characterized as clinical depression, which primarily affects the mood and behaviour of an individual. In the present study...     

腹腔注射重组腺病毒诱导的免疫反应

为研究重组腺病毒接种实验动物后的免疫反应性,利用ＲＴ－Ｐ　Ｃ　Ｒ方法,从ＨＡＶ的ＲＮＡ中克隆了结构蛋白基因插入穿梭质粒ｐＸＣＸ２Ｎｏｔ　Ｉ,通过磷酸钙－ＤＮＡ共　沉淀技术,将复制缺陷型腺病毒载体与线形化的ｐＸＣＸ２－ＣＭＶ－ＨＡＶ共转染２９３细胞。一系列检测方法证明产生了重组腺病毒ｒＡｄＨＡＶ。纯化后的ｒＡｄＨＡＶ滴度为１×１０９ＴＣＩＤ５０／ｍＬ　,腹腔注射免疫昆明种小白鼠后,可诱导产生抗ＨＡＶ　ＩｇＧ和ＨＡＶ中和抗体。复制缺陷型腺病毒　可作为发展基因工程病毒疫苗载体的有效系统。     

重组沙蚕激酶的诱导表达和分离纯化

将已构建好的表达载体pGEX-4T-2SAK 转化大肠杆菌E.coli BL21(DE3),经IPTG诱导表达融合的沙蚕激酶蛋白,通过GST树脂亲和吸附和凝血酶酶切及冷冻干燥等步骤进行分离纯化,用SDS-PAGE分析要重组的目的蛋白及纯化后的蛋白.纯化后的沙蚕激酶经纤维平板法测定具有溶栓活性.冷冻干燥品经SDS-PAGE分析表明达到电泳纯.     

Interaction of Human Butyrylcholinesterase Variants with Bambuterol and Terbutaline

Bambuterol, a dimethylcarbamate, carbamoylates butyrylcholinesterase (BChE; EC 3.1.1.8). The carbamoylated enzyme is not very stable and the final product of the two-step hydrolysis is a bronchodilator drug, terbutaline (1-(3,5-dihydroxyphenyl)-2-t-butylaminoethanol sulphate). Both bambuterol and terbutaline inhibit BChE, but their affinities differ in human serum BChE variants (U, A, F, K and S) due to their positive charge. Bambuterol inhibition rate constants for the homozygous usual (UU), Kalow (KK), fluoride-resistant (FF) or atypical (AA) variant ranged from 4.4 to 0.085?min-1?μM-1. Terbutaline showed competitive reversible inhibition for all BChE variants. The dissociation constants for UU, FF and AA homozygotes were 0.18, 0.31 and 3.3?mM, respectively. The inhibition rate or dissociation constants for heterozygotes were distributed between the respective constants for the corresponding homozygotes. A 50-fold difference in inhibition between the UU and AA enzyme might affect terbutaline release in humans. The affinity of all studied BChE variants for terbutaline was low, which suggests that terbutaline originating from bambuterol hydrolysis should not affect the hydrolysis of bambuterol by BChE.     

Identification of an opd (Organophosphate Degradation) Gene in an Agrobacterium Isolate

We isolated a bacterial strain, Agrobacterium radiobacter P230, which can hydrolyze a wide range of organophosphate (OP) insecticides. A gene encoding a protein involved in OP hydrolysis was cloned from A. radiobacter P230 and sequenced. This gene (called opdA) had sequence similarity to opd, a gene previously shown to encode an OP-hydrolyzing enzyme in Flavobacterium sp. strain ATCC 27551 and Brevundimonas diminuta MG. Insertional mutation of the opdA gene produced a strain lacking the ability to hydrolyze OPs, suggesting that this is the only gene encoding an OP-hydrolyzing enzyme in A. radiobacter P230. The OPH and OpdA proteins, encoded by opd and opdA, respectively, were overexpressed and purified as maltose-binding proteins, and the maltose-binding protein moiety was cleaved and removed. Neither protein was able to hydrolyze the aliphatic OP malathion. The kinetics of the two proteins for diethyl OPs were comparable. For dimethyl OPs, OpdA had a higher k_cat than OPH. It was also capable of hydrolyzing the dimethyl OPs phosmet and fenthion, which were not hydrolyzed at detectable levels by OPH.     

重组谷糠源过氧化物酶的克隆、表达及逆转结肠癌化疗耐药活性

本课题组前期首次从谷糠中获得具抗肿瘤活性的过氧化物酶FMBP。为了该蛋白质后期在体外的规模化生产,本研究以谷子cDNA为模板,利用基因工程手段构建了pMal-s-FMBP融合质粒,并优化诱导条件进行原核表达与纯化,最终获得纯度大于90 %的具抗肿瘤活性的重组FMBP（Re-FMBP）。研究发现,Re-FMBP可以逆转HCT-8/5-Fu耐药细胞对5-Fu的耐药性,耐药逆转倍数达到9.6倍。通过Annexin V/碘化丙啶双染色流式细胞仪检测显示：Re-FMBP能够诱导HCT-8/5-Fu耐药细胞发生凋亡;通过罗丹明123染色,流式细胞仪检测结果显示,Re-FMBP处理后,HCT-8/5-Fu耐药细胞内的荧光强度增强。说明Re-FMBP能够增加5-Fu药物在HCT-8/5-Fu耐药细胞内的蓄积。结果揭示,Re-FMBP蛋白可通过抑制耐药细胞增殖,诱导耐药细胞凋亡,抑制耐药细胞对5-Fu药物外排功能来增加HCT-8/5-Fu耐药细胞对5-Fu药物的敏感性。因此,谷糠来源的Re-FMBP蛋白可以作为肿瘤化疗辅助药物来开发。     

Effect of Recombinant hPTEN Gene Expression on PDGF Induced VSMC Proliferation

Human phosphatase and tensin homolog (hPTEN) gene was expressed in vascular smooth muscle cells (VSMCs) to study its effect on VSMC proliferation induced in platelet-derived growth factor (PDGF) conditioned medium. After G418 selection, MTT assay was conducted to examine transfected VSMC proliferation induced in human PDGF conditioned medium. We successfully constructed eukaryotic expression vector pcDNA4/myc-His-PTEN and transferred into VSMC cells. We report that in vitro proliferation of VSMC was inhibited in PTEN transfected VSMCs induced in PDGF conditioned medium. RT-PCR and Western blot results indicated significantly high levels of protein kinase B-PKB and nuclear factor kappa B mRNA and protein, respectively, in PDGF group as compared with the control group.     

Alcohol Induced Reversal of Enantioselectivity in a Lipase Catalyzed Resolution of 2-Chloropropionic Acid

Alcohol induced reversal of enantioselectivity in the esterification of 2-chloropropionic acid using lipase from Candida cylindracea has been investigated. It was found that an alcohol having substituents both at the α- and the β-carbon preferentially esterified the S-acid, while a straight chain alcohol preferentially esterified the R-acid. Intermediate enantioselectivities were obtained with alcohols having substituents either at the α- or the β-carbon, but still in favor of the R-acid. An acyl binding domain composed of three subsites is proposed for this lipase; one for the hydrocarbon chain, a second for a methyl substituent and a third for an electronegative substituent.     

Transfection of Reaggregating Embryonic Chicken Retinal Cells with an Antisense 5'-DNA Butyrylcholinesterase Expression Vector Inhibits Proliferation and Alters Morphogenesis

Abstract: The function of the enzyme butyrylcholinesterase (BChE) in the developing and mature brain is still unclear. We have inserted 577 bp of the 5' upstream region plus 106 bp of the exon 1 of the rabbit BChE gene in reverse orientation under control of an SV40 early promoter derivative in an expression vector. This vector was introduced by calcium phosphate-mediated transfection into embryonic chicken retina cells during the first days of reaggregation culture. Depending on the retinal origin, the transfected cell population forms histotypic retina-like spheres, so-called rosetted or stratified retinospheroids. We show that antisense 5'-BChE gene expression decreased the steady-state mRNA level of BChE and the translation of the BChE protein, inhibited proliferation, and accelerated histogenesis in both cellular systems. The pronounced effects of antisense 5'-BChE transfection of spheroids document a key role of BChE during the early reaggregation process of retinal cells, most likely by regulating their growth and differentiation.     

Synchronization of Cultured Vascular Smooth Muscle Cells Following Reversal of Quiescence Induced by Treatment with the AntioxidantN-Acetylcysteine

Smooth muscle cell (SMC) proliferation plays an important role in the pathogenesis of vascular diseases such as atherosclerosis and postangioplasty restenosis. Recently we demonstrated the thiol antioxidantN-acetylcysteine (NAC) inhibits constitutive NF-κB/Rel activity and growth of vascular SMCs. Here we show that treatment of human and bovine aortic SMC with the thiol antioxidant NAC causes cells to exit the cell cycle and remain quiescent as determined by a greatly reduced incorporation of [³H]thymidine and G₀/G₁DNA content. Removal of NAC from the culture medium stimulates SMCs to synchronously reenter the cell cycle as judged by induction of cyclin D1 and B-mybgene expression during mid and late G₁phase, respectively, and induction of histone gene expression and [³H]thymidine incorporation during S phase. The time course of cyclin D1, B-myb,and histone gene expression after NAC removal was similar to that of serum-deprived cells induced to resume cell cycle progression by the addition of fetal bovine serum to the culture medium. Taken together, these results indicate that NAC treatment causes SMCs to enter a reversible G₀quiescent, growth-arrested state. Thus, NAC provides an important new method for synchronizing SMCs in culture.     

Recombinant amyloid beta-peptide production by coexpression with an affibody ligand

Background

The isolation and production of human monoclonal antibodies is becoming an increasingly important pursuit as biopharmaceutical companies migrate their drug pipelines away from small organic molecules. As such, optimization of monoclonal antibody technologies is important, as this is becoming the new rate-limiting step for discovery and development of new pharmaceuticals. The major limitations of this system are the efficiency of isolating hybridoma clones, the process of stabilizing these clones and optimization of hybridoma cell secretion, especially for large-scale production. Many previous studies have demonstrated how perturbations in the aqueous environment can impact upon cell biology. In particular, radio frequency (RF) irradiation of solutions can have dramatic effects on behavior of solutions, cells and in particular membrane proteins, although this effect decays following removal of the RF. Recently, it was shown that nanoparticle doping of RF irradiated water (NPD water) produced a stabilized aqueous medium that maintained the characteristic properties of RF irradiated water for extended periods of time. Therefore, the ordering effect in water of the RF irradiation can now be studied in systems that required prolonged periods for analysis, such as eukaryotic cell culture. Since the formation of hybridoma cells involves the formation of a new membrane, a process that is affected by the surrounding aqueous environment, we tested these nanoparticle doped aqueous media formulations on hybridoma cell production.

Results

In this study, we tested the entire process of isolation and production of human monoclonal antibodies in NPD water as a means for further enhancing human monoclonal antibody isolation and production. Our results indicate an overall enhancement of hybridoma yield, viability, clonability and secretion. Furthermore, we have demonstrated that immortal cells proliferate faster whereas primary human fibroblasts proliferate slower in NPD water.

Conclusion

Overall, these studies indicate that NPD water can enhance cell proliferation, clonability and secretion. Furthermore, the results support the hypothesis that NPD water is effectively composed of stable microenvironments.     

Monoclonal Antibodies Allow Precipitation of Esterasic but Not Peptidasic Activities Associated with Butyrylcholinesterase

Commercially available and affinity-purified butyrylcholinesterases isolated from human serum were examined for their esterasic activity and their ability to hydrolyze various neuropeptides, including neurotensin, substance P, and leucine-enkephalin. The three pools that displayed the lowest esterasic activities were shown to hydrolyze neurotensin with the same HPLC degradative pattern. By contrast, noticeable qualitative and quantitative discrepancies were observed when hydrolyses of substance P and leucine-enkephalin by these three butyrylcholinesterase pools were studied. The pool that exhibited the highest esterasic activity appeared to be homogeneously constituted by 90- and 180-kDa protein bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and was totally unable to hydrolyze these three neuropeptides. This suggested that the three other butyrylcholinesterase preparations could be contaminated by exogenous peptidases. This was confirmed by means of three distinct monoclonal antibodies directed toward human serum butyrylcholinesterase. The three IgG-purified fractions precipitated the esterasic activity, whereas they failed to precipitate the neuropeptide-hydrolyzing activities whatever the substrate examined. Altogether, these results demonstrate that peptidases associated with butyrylcholinesterase are contaminating enzymes that cannot be considered as intrinsic activities of this enzyme.     

Reversal of the Myosin Power Stroke Induced by Fast Stretching of Intact Skeletal Muscle Fibers

Force generation and movement in skeletal muscle result from a cyclical interaction of overlapping myosin and actin filaments that permits the free energy of ATP hydrolysis to be converted into mechanical work. The rapid force recovery that occurs after a step release imposed on a muscle is thought to result from a synchronized tilting of myosin lever arms toward a position of lower free energy (the power stroke). We investigated the power stroke mechanism in intact muscle fibers of Rana esculenta using a fast stretch to detach forcibly cross-bridges. Stretches were applied either with or without a conditioning step release. Cross-bridge rupture tension was not significantly influenced by the release, whereas sarcomere elongation at the rupture point increased immediately after the release and returned to the prerelease condition within 15-20 ms, following a slower time course compared to the recovery of tension. These observations suggest that the rupture force of a bridge is unaltered by a conditioning release, but rupture must first be preceded by a power stroke reversal, which restores the prepower stroke state. The sarcomere extension at the rupture point indicates both the extent of this power stroke reversal and the time course of strained bridge replenishment.