一种简单快速微量的无细胞蛋白合成系统

建立了一种简单快速微量的无细胞体系检测蛋白质合成的方法,在总体积５５μｌ的体系中加２０μｌ兔网织红细胞裂解液,在３７℃培养３０ｍｉｎ能使其中的３Ｈ－Ｌｅｕ参入量达到最大,运用该方法可以筛选出植物组织中对真核细胞蛋白质生物合成具有强烈抑制作用的单链核糖体失活蛋白（单链毒素）。     

Evidence that 5-HT2 antagonism elicits a 5-HT3-mediated increase in dopamine transmission

Amperozide, a novel atypical antipsychotic drug with few extrapyramidal side effects, is a strong serotonin₂ (5-HT₂) antagonist but has low affinity for dopamine receptors in vitro. The effect of amperozide on the dopaminergic synapse was studied with an in vivo microdialysis technique using anesthetized male Sprague-Dawley rats. Following implantation of dialysis probes into the striatum and nucleus accumbens (NuAc), amperozide was intravenously infused as six consecutive incremental doses (0.5, 0.5, 1.0, 2.0, 4.0 and 8.0 mg/kg) at intervals of 15 min. From the beginning of drug infusion, perfusates were collected in fractions every 30 min throughout a total period of 120 min. The samples were then immediately analyzed by high-performance liquid chromatography with electrochemical detection. Amperozide induced a dose-related elevation of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindolacetic acid (5-HIAA) levels in both areas.p-Chlorophenylalanine (pCPA) pretreatment abolished the production of 5-HIAA in both areas and attenuated the amperozide-induced rise of DOPAC but not of dopamine. After pretreatment with an intravenous 5-HT₃ antagonist, MDL 72222, the amperozide-induced changes in dopamine, DOPAC and 5-HIAA in both areas were lower than in the saline control group. Preliminary data showed that afterpCPA pretreatment, incremental concentrations of the 5-HT₃ agonist 1-(m-chlorophenyl)-biguanide perfused via the probe also produced significant elevation of dopamine and DOPAC levels in these two areas. Taken together, these results suggest that amperozide may directly block 5-HT₂ receptors in the striatum and NuAc, thereby enhancing 5-HT transmission. The enhanced 5-HT transmission may activate postsynpatic 5-HT₃ receptors located on the dopaminergic terminals, leading to changes in dopamine transmission in these two areas.     

Exogenous myristic acid can be partially degraded prior to activation to form acyl-acyl carrier protein intermediates and lipid A in Vibrio harveyi.

To study the involvement of acyl carrier protein (ACP) in the metabolism of exogenous fatty acids in Vibrio harveyi, cultures were incubated in minimal medium with [9,10-3H]myristic acid, and labeled proteins were analyzed by gel electrophoresis. Labeled acyl-ACP was positively identified by immunoprecipitation with anti-V. harveyi ACP serum and comigration with acyl-ACP standards and [3H]beta-alanine-labeled bands on both sodium dodecyl sulfate- and urea-polyacrylamide gels. Surprisingly, most of the acyl-ACP label corresponded to fatty acid chain lengths of less than 14 carbons: C14, C12, C10, and C8 represented 33, 40, 14, and 8% of total [3H]14:0-derived acyl-ACPs, respectively, in a dark mutant (M17) of V. harveyi which lacks myristoyl-ACP esterase activity; however, labeled 14:0-ACP was absent in the wild-type strain. 14:0- and 12:0-ACP were also the predominant species labeled in complex medium. In contrast, short-chain acyl-ACPs (< or = C6) were the major labeled derivatives when V. harveyi was incubated with [3H]acetate, indicating that acyl-ACP labeling with [3H]14:0 in vivo is not due to the total degradation of [3H]14:0 to [3H]acetyl coenzyme A followed by resynthesis. Cerulenin increased the mass of medium- to long-chain acyl-ACPs (> or = C8) labeled with [3H]beta-alanine fivefold, while total incorporation of [3H]14:0 was not affected, although a shift to shorter chain lengths was noted. Additional bands which comigrated with acyl-ACP on sodium dodecyl sulfate gels were identified as lipopolysaccharide by acid hydrolysis and thin-layer chromatography. The levels of incorporation of [3H] 14:0 into acyl-ACP and lipopolysaccharide were 2 and 15%, respectively, of that into phospholipid by 10 min. Our results indicate that in contrast to the situation in Escherichia coli, exogenous fatty acids can be activated to acyl-ACP intermediates after partial degradation in V. harveyi and can effectively label products (i.e., lipid A) that require ACP as an acyl donor.     

Molecular Cloning and Characterization of Protein Kinase C from the Sea Urchin Lytechinus pictus

Protein kinase C (PKC) has been shown to play a role in events involved in fertilization such as activation of the Na⁺/H⁺antiporter and an NADPH dependent oxidase. In addition, it is involved in cell fate programming later in development of the sea urchin embryo. In order to further address the role of PKC in sea urchin development, we have screened a Lytechinus pictus ovary tissue cDNA library and identified one clone for sea urchin protein kinase C (suPKC1). This clone encodes a deduced protein with a molecular mass of 72.4 kDa, which shows strong homology to invertebrate and mammalian protein kinase C (PKC) sequences. PKC has been partially purified from eggs of L. pictus. This kinase activity has been shown to be dependent upon phosphatidylserine, diacylglycerol and Ca²⁺. In agreement with this biochemical data, suPKC1 has a C2 or Ca²⁺-binding domain suggesting its activity would be Ca²⁺-dependent. Polyclonal antibodies raised against peptides of the suPKC1 sequence recognize an antigen of approximately 71 kDa in DE52 fractions that contain PKC activity; this reactivity is not observed in fractions that lack PKC activity. Using a ribonuclease protection assay, we have demonstrated the presence of suPKC1 message throughout developmental stages of the sea urchin embryo.     

Dynamic approaches to the mechanism of photosynthesis

An account of the author's life and scientific research is presented. Two main lines of research have been pursued: (1) Studies on the physiological aspect of photosynthesis started from experiments with crops under field conditions and then extended to the study of photosynthesis in nature; and (2) studies on the mechanism of photophosphorylation and related problems which began with the measurement of quantum requirement of photophosphorylation. This work led to the discovery of the high energy state of phosphorylation and many other interesting findings. In recent years, efforts have been made to study the operation and regulation of photosynthetic apparatus with a view to link the above-mentioned lines of research together.Written at the invitation of Govindjee.     

Purification and characterization of recombinant human pro-urokinase produced in silkworm using a baculovirus vector

Summary A recombinant baculovirus (BmNPV-pk2) was constructed by inserting the human pro-urokinase(pro-UK) cDNA into the genome of baculovirus Bombyx mori nuclear polyhedrosis virus (BmNPV) adjacent to the strong polyhedrin promoter. The recombinant virus replicated in silkworm larvae, which synthesized 30g pro-UK/ml in the haemolymph within 4 days post-infection. Purification to near homogeneity was accomplished by fractional precipitation with ammonium sulphate and immunoaffinity chromatography with an overall yield of 23% and a specific activity of 100,000IU/mg in fibrin plate assay. This purified product was comprised of a single chain protein with approximately Mr. 50kDa as determined by SDS-PAGE gel. The N-Terminal amino acids sequence revealed that the secretion signal of pro-UK was correctly processed.     

转化生长因子α－绿脓杆菌外毒素融合蛋白TGFα－PE40高效表达质粒的构建和表达

利用基因工程技术,将质粒pYX382用xbaI和EcoRl切下插入的TGFa—PE40融合 基因片段-连接到可表达载体pCB604的XbaⅠ／EcoR Ⅰ位点中,构建成新的重组质粒p2x—TP1。P2x—TP1转化E.coli BL2l感受志菌后,在ⅠPTG诱导下Ⅰpp启动子转录表达TGFα—PE40融合蛋白。表达产物主要以包涵体形式沉积在细胞内。融合蛋白表达量的高低与诱导时的细胞密度,诱导的温度以及培养基有关。在一定范围内与诱导剂的剂量以及诱导时间的长短无关。P2x—TPl重组质粒在工程菌中的表达TGFα－PE40融合蛋白量约为50mg／L。