The major substrates for TAP in vivo are derived from newly synthesized proteins

The transporter associated with antigen processing (TAP) is a member of the family of ABC transporters that translocate a large variety of substrates across membranes. TAP transports peptides from the cytosol into the endoplasmic reticulum for binding to MHC class I molecules and for subsequent presentation to the immune system. Here we follow the lateral mobility of TAP in living cells. TAP's mobility increases when it is inactive and decreases when it translocates peptides. Because TAP activity is dependent on substrate, the mobility of TAP is used to monitor the intracellular peptide content in vivo. Comparison of the diffusion rates in peptide-free and peptide-saturated cells indicates that normally about one-third of all TAP molecules actively translocate peptides. However, during an acute influenza infection TAP becomes fully employed owing to the production and degradation of viral proteins. Furthermore, TAP activity depends on continuing protein translation. This implies that MHC class I molecules mainly sample peptides that originate from newly synthesized proteins, to ensure rapid presentation to the immune system.     

Identification of the lethal target of benzylpenicillin in Streptococcus faecalis by in vivo penicillin binding studies

The mode of bacterial killing by penicillins is still unknown in spite of many studies on the subject. The recent finding of multiple penicillin binding proteins (PBPs) in sensitive bacteria and the possibility of analysing the binding of the antibiotic to exponentially growing cells have provided new directions for investigating this problem. Sensitivity to lethal and other effects of penicillin varies very significantly with the conditions of growth of the cells. If PBPs were the penicillin target, changes in conditions of growth causing variations in penicillin sensitivity should be accompanied by changes in these proteins. Furthermore, if one of PBPs could be identified as the killing target, it could possibly be demonstrated to show changes in cells growing in different conditions. We show here that in Streptococcus faecalis ATCC 9790 changes in conditions of growth are accompanied by changes in PBPs. Furthermore, in the presence of the minimal dose of 14C-benzylpenicillin causing complete inhibition of cell growth, 100% of the total radioactivity is bound to a single protein (PBP 3).     

Transient association of newly synthesized unfolded proteins with the heat-shock GroEL protein

It has been suggested that newly synthesized proteins are maintained in their unfolded state by cellular ATP-driven factors which may prevent or reverse the formation of misfolded structures or promote the correct assembly of oligomeric proteins or post-translational secretion. Using a photocross-linking approach, we have identified the 20S heat-shock GroEL protein as the major cytosolic component which forms a complex with the unfolded newly synthesized pre-beta-lactamase or chloramphenicol acetyltransferase in Escherichia coli. Dissociation of these complexes is ATP-dependent. The unfolded state of pre-beta-lactamase, maintained by the transient interaction with GroEL, may be essential for the secretion of this protein.     

NMR analysis of a 900K GroEL GroES complex

Biomacromolecular structures with a relative molecular mass (M(r)) of 50,000 to 100,000 (50K 100K) have been generally considered to be inaccessible to analysis by solution NMR spectroscopy. Here we report spectra recorded from bacterial chaperonin complexes ten times this size limit (up to M(r) 900K) using the techniques of transverse relaxation-optimized spectroscopy and cross-correlated relaxation-enhanced polarization transfer. These techniques prevent deterioration of the NMR spectra by the rapid transverse relaxation of the magnetization to which large, slowly tumbling molecules are otherwise subject. We tested the resolving power of these techniques by examining the isotope-labelled homoheptameric co-chaperonin GroES (M(r) 72K), either free in solution or in complex with the homotetradecameric chaperonin GroEL (M(r) 800K) or with the single-ring GroEL variant SR1 (M(r) 400K). Most amino acids of GroES show the same resonances whether free in solution or in complex with chaperonin; however, residues 17 32 show large chemical shift changes on binding. These amino acids belong to a mobile loop region of GroES that forms contacts with GroEL. This establishes the utility of these techniques for solution NMR studies that should permit the exploration of structure, dynamics and interactions in large macromolecular complexes.     

(Mg-ATP)-dependent self-assembly of molecular chaperone GroEL

The important Escherichia coli heat-shock protein GroEL of relative molecular mass 57,259 is a typical molecular chaperone. It possesses ATPase activity and interacts in ATP-driven reactions with non-folded proteins to stimulate their correct folding and/or assembly by preventing the formation of improper protein structures or aggregates. As GroEL is isolated and functions as a 20-25S tetradecameric particle (GroELp), the question arises--what is the mechanism of its own assembly? Here we show the (Mg-ATP)-dependent self-stimulation ('self-chaperoning') in vitro of GroELp reassembly from its monomeric state.     

Heat shock in Escherichia coli alters the protein-binding properties of the chaperonin groEL by inducing its phosphorylation.

When bacterial or eukaryotic cells are exposed to high temperatures or other harsh conditions, they respond by synthesis of a specific set of heat-shock proteins. Certain heat-shock proteins such as groEL, called 'chaperonins', can prevent misfolding and promote the refolding and proper assembly of unfolded polypeptides generated under harmful conditions. We report here a new aspect of the heat-shock response in Escherichia coli: at high temperatures a fraction of groEL becomes modified covalently, altering its interaction with unfolded proteins. The heat-modified form can be eluted with ATP from an unfolded protein more easily than normal groEL. The critical heat-induced modification seems to be phosphorylation, which is reversed on return to low temperature. Treatment of the modified groEL with phosphatases caused its apparent size, charge and binding properties to resemble those of the unmodified form. Thus during heat shock some groEL is reversibly phosphorylated, which allows its ATP-dependent release from protein substrates in the absence of its usual cofactor (groES), and probably promotes the repair of damaged polypeptides.     

Different conformations for the same polypeptide bound to chaperones DnaK and GroEL.

The proteins DnaK (hsp70) and GroEL (cpn60) from Escherichia coli are prototypes of two classes of molecular chaperones conserved throughout evolution. The analysis of transferred nuclear Overhauser effects in two-dimensional NMR spectra is ideally suited to determine chaperone-bound conformations of peptides. The peptide vsv-C (amino-acid sequence KLIGVLSSLFRPK) stimulates the ATPase of BiP and Hsc70 (ref. 3) and the intrinsic ATPase of DnaK. The affinity of the vsv-C peptide for DnaK is greatly reduced in the presence of ATP. Here we analyse transferred nuclear Overhauser effects and show that the peptide is in an extended conformation while bound to DnaK but is helical when bound to GroEL. NMR also indicates that the mobility of the peptide backbone is reduced more by binding to DnaK than by binding to GroEL, whereas the side chains are less mobile when bound to GroEL.     

Cyclin specificity in the phosphorylation of cyclin-dependent kinase substrates

Cell-cycle events are controlled by cyclin-dependent kinases (CDKs), whose periodic activation is driven by cyclins. Different cyclins promote distinct cell-cycle events, but the molecular basis for these differences remains unclear. Here we compare the specificity of two budding yeast cyclins, the S-phase cyclin Clb5 and the M-phase cyclin Clb2, in the phosphorylation of 150 Cdk1 (Cdc28) substrates. About 24% of these proteins were phosphorylated more efficiently by Clb5-Cdk1 than Clb2-Cdk1. The Clb5-specific targets include several proteins (Sld2, Cdc6, Orc6, Mcm3 and Cdh1) involved in early S-phase events. Clb5 specificity depended on an interaction between a hydrophobic patch in Clb5 and a short sequence in the substrate (the RXL or Cy motif). Phosphorylation of Clb5-specific targets during S phase was reduced by replacing Clb5 with Clb2 or by mutating the substrate RXL motif, confirming the importance of Clb5 specificity in vivo. Although we did not identify any highly Clb2-specific substrates, we found that Clb2-Cdk1 possessed higher intrinsic kinase activity than Clb5-Cdk1, enabling efficient phosphorylation of a broad range of mitotic Cdk1 targets. Thus, Clb5 and Clb2 use distinct mechanisms to enhance the phosphorylation of S-phase and M-phase substrates.     

Heat-shock proteins DnaK and GroEL facilitate export of LacZ hybrid proteins in E. coli

The use of lacZ gene fusions, producing a hybrid protein containing an amino terminus specified by a target gene fused to the functional carboxy terminus of beta-galactosidase, has facilitated the study of protein targeting in various organisms. One of the best characterized fusions in Escherichia coli is phi(lamB-lacZ)42-1(Hyb), which produces a hybrid protein with the signal sequence and 181 N-terminal amino acids of the exported protein LamB, attached to LacZ. In common with other LacZ hybrids, the LamB-LacZ(42-1) protein is poorly exported from E. coli, conferring a Lac+ phenotype. beta-Galactosidase activity decreases markedly when cells producing the LamB-LacZ protein are grown at 42 degrees C or when a heat-shock response is induced at lower temperatures by overproducing heat-shock factor RpoH3, indicating the LacZ hybrids are being efficiently targeted to the cell envelope. We now report that the heat-shock proteins DnaK and GroEL can, in sufficient amounts, decrease beta-galactosidase activity and facilitate the export of lacZ-hybrid proteins.     

Successive action of DnaK, DnaJ and GroEL along the pathway of chaperone-mediated protein folding.

The main stress proteins of Escherichia coli function in an ordered protein-folding reaction. DnaK (heat-shock protein 70) recognizes the folding polypeptide as an extended chain and cooperates with DnaJ in stabilizing an intermediate conformational state lacking ordered tertiary structure. Dependent on GrpE and ATP hydrolysis, the protein is then transferred to GroEL (heat-shock protein 60) which acts catalytically in the production of the native state. This sequential mechanism of chaperone action may represent an important pathway for the folding of newly synthesized polypeptides.     

Identification of in vivo interaction between rabbit hemorrhagic disease virus capsid protein and minor structural protein

We investigated the ability of the rabbit hemorrhagic disease virus(RHDV) capsid protein(VP60) to interact specifically with the minor structural protein VP10,using an in vivo cell-based CheckMate Mammalian Two-Hybrid System.RHDV VP60 protein interacted specifically with VP10.Immunofluorescence analysis and co-immunoprecipitation with specific antibodies revealed the existence of biologically important VP60/VP10 complexes.However,when VP60 was divided into two fragments,the interaction between VP60 and VP10 was impaired dramatically.These results will be helpful for further investigating the mechanism of RHDV particle assembly.     

分子伴侣GroEL对青霉素G酰化酶亚基折叠的影响

采用Tac启动子控制表达质粒，在不同的宿主细胞中表达了青霉素G酰化酶（PAC），检测这些菌株所表达的PAC活性，分析细胞内分子伴侣GroEL含量，PAC翻译后加工为α，β亚基的状况，以及它们之间的关系，结果表明：质粒pKK-SP在不同宿主中表达时，翻译后加工状况有明显差异，单位质量细胞所表达的PAC活性与翻译后加工效率相关，且与细胞内分子伴侣GroEL在菌体总蛋白中含量正相关，同时也阐明了亚基的折叠成为翻译后加工过程的限制步骤，细胞内分子伴侣GroEL有助于PAC亚基的折叠和稳定。     

固体表面化学发光基质背景降低的研究

对固体表面化学发光法固体基质的发光背景进行了考察．提出了降低背景的方法，以ＮａＯＨ与水的质量比值为１．５％溶液、１０－２ｍｏｌ·Ｌ－１ＥＤＴＡ溶液或其混合溶液浸泡滤纸２４ｈ，滤纸背景的降低分别为６０％，７９％及８２％．     

堆肥体系中有机物动态模拟模型研究进展

堆肥作为生活垃圾处理的一项重要手段,以其无害化程度高、减量化效果明显,以及可以最大限度地实现生活垃圾处理资源化的特点,被越来越多的国家和地区广泛利用。该文的研究目的在于归纳描述堆肥动态变化的模型,分析模型中的关键环境参数,从而借助模型对堆肥过程的有机质动态变化获得更清晰的理解,并预测堆肥产品的质量。为此,文中概述了堆肥模型中环境参数的影响,并列举了目前几种实用的有机质模型。研究结果表明,大多数的堆肥模型一般都用于描述和预测干物质、温度、湿度、氧气和二氧化碳的动态变化过程。随着堆肥模型领域的发展,关于有机质的生化降解以及微生物的作用等方面的研究逐渐展开。     

Cortical substrates for exploratory decisions in humans

Decision making in an uncertain environment poses a conflict between the opposing demands of gathering and exploiting information. In a classic illustration of this 'exploration-exploitation' dilemma, a gambler choosing between multiple slot machines balances the desire to select what seems, on the basis of accumulated experience, the richest option, against the desire to choose a less familiar option that might turn out more advantageous (and thereby provide information for improving future decisions). Far from representing idle curiosity, such exploration is often critical for organisms to discover how best to harvest resources such as food and water. In appetitive choice, substantial experimental evidence, underpinned by computational reinforcement learning (RL) theory, indicates that a dopaminergic, striatal and medial prefrontal network mediates learning to exploit. In contrast, although exploration has been well studied from both theoretical and ethological perspectives, its neural substrates are much less clear. Here we show, in a gambling task, that human subjects' choices can be characterized by a computationally well-regarded strategy for addressing the explore/exploit dilemma. Furthermore, using this characterization to classify decisions as exploratory or exploitative, we employ functional magnetic resonance imaging to show that the frontopolar cortex and intraparietal sulcus are preferentially active during exploratory decisions. In contrast, regions of striatum and ventromedial prefrontal cortex exhibit activity characteristic of an involvement in value-based exploitative decision making. The results suggest a model of action selection under uncertainty that involves switching between exploratory and exploitative behavioural modes, and provide a computationally precise characterization of the contribution of key decision-related brain systems to each of these functions.     

DNA在不同衬底表面的固定

分别将DNA梳直在APS处理的云母和巯基乙胺处理的金膜表面,讨论了巯基乙胺浓度对金膜表面吸附DNA的影响.AFM图像表明,DNA能够有效吸附在处理过的两种衬底表面,经过盖玻片拉伸后,DNA能沿一定方向延伸,有较好的定向性.实验发现:处理金膜的巯基乙胺最佳浓度为5 mM,在该浓度下,DNA分布均匀,易于寻找,且巯基乙胺对金膜的破坏作用较小.     

新疆绵羊种布鲁氏菌GroEL基因的克隆及序列分析

参考牛种布鲁氏菌的GroEL（热休克蛋白）基因设计引物,扩增出新疆绵羊种布鲁氏菌GroEL基因片段,将其片段克隆到T载体上测序。结果表明：新疆源布鲁氏菌GroEL基因片段长1641bp,编码546个氨基酸,与羊种（B．melitensis）、猪种（B．suis）以及牛种（B．abortus）GroEL基因的核苷酸序列同源性分别为99．88％、99．82％、99．88％,推导的氨基酸序列同源性在99％以上,显示了很强的保守性。     

葡萄糖转运蛋白1（GLUT-1）抑制剂BAY-876体内活性的正电子发射型计算机断层显像（PET）检测

目前,葡萄糖转运蛋白(glucose transporter,GLUT)不仅是细胞摄取葡萄糖的功能与物质保障,同时也是恶性肿瘤抗肿瘤药物治疗的新型干预靶点。建立GLUT抑制剂的体内活性评价方法具有重要意义。利用正电子发射型计算机断层显像检测仪(positron emission computed tomography,PET)对小动物肝细胞癌(hepatocellular carcinoma,HCC)肿瘤模型进行PET检测,建立GLUT抑制剂的小动物体内活性检测方法。在HCC细胞系中检测GLUT-1的表达,选取GLUT-1表达水平最高的细胞系接种BalB/c免疫缺陷动物皮下,形成免疫缺陷动物皮下肿瘤模型。连续三日口服灌胃给药,给予动物5 mg/kg的GLUT-1抑制剂BAY-876后,进行PET检测。动物行尾静脉注射200μCi的核素探针~(18)F-FDG,约30 min后进行PET检测。在此基础上,使用盖革计数器检测动物肿瘤与血液的放射性强度比较。在所选细胞系中,MHCC-97H细胞中GLUT-1的表达显著高于其他细胞系,BAY-876治疗动物能够显著抑制MHCC-97H皮下肿瘤组织对18F-FDG的摄取。GLUT-1抑制剂BAY-876能够显著抑制HCC细胞对葡萄糖的摄取,成功建立了利用PET检测GLUT抑制剂动物体内活性的PET检测方法。