Transgenic plant aequorin reports the effects of touch and cold-shock and elicitors on cytoplasmic calcium

Methods for measuring plant cytoplasmic calcium using microelectrodes or microinjected fluorescent dyes are associated with extensive technical problems, so measurements have been limited to single or small groups of cells in tissue strips or protoplasts. Aequorin is a calcium-sensitive luminescent protein from the coelenterate Aequorea victoria (A. forskalea) which is formed from apoaequorin, a polypeptide of relative molecular mass approximately 22,000, and coelenterazine, a hydrophobic luminophore. Microinjected aequorin has been widely used for intracellular calcium measurement in animal cells, but its use in plants has been limited to exceptionally large cells. We show here that aequorin can be reconstituted in transformed plants and that it reports calcium changes induced by touch, cold-shock and fungal elicitors. Reconstituted aequorin is cytoplasmic and nonperturbing; measurements can be made on whole plants and a calcium indicator can be constituted in every viable cell. Now that apoaequorin can be targeted to specific organelles, cells and tissues, with the range of coelenterazines with differing calcium sensitivities and properties available, this new method could be valuable for determining the role of calcium in intracellular signalling processes in plants.     

Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin.

Introduction of Ca2+ indicators (photoproteins, fluorescent dyes) that can be trapped in the cytosolic compartment of living cells has yielded major advances in our knowledge of Ca2+ homeostasis. Ca2+ however regulates functions not only in the cytosol but also within various organelles where indicators have not yet been specifically targeted. Here we present a novel procedure by which the free Ca2+ concentration of mitochondria, [Ca2+]m, can be monitored continuously at rest and during stimulation. The complementary DNA for the Ca2+ sensitive photoprotein aequorin was fused in frame with that encoding a mitochondrial presequence. The hybrid cDNA was transfected into bovine endothelial cells and stable clones were obtained expressing variable amounts of mitochondrially targeted apoaequorin. The functional photoprotein could be reconstituted in intact cells by incubation with purified coelenterazine and [Ca2+]m could thus be monitored in situ. This allowed the unprecedented direct demonstration that agonist-stimulated elevations of cytosolic free Ca2+, [Ca2+]i, (measured in parallel with Fura-2) evoke rapid and transient increases of [Ca2+]m, which can be prevented by pretreatment with a mitochondrial uncoupler. The possibility of targeting aequorin to cellular organelles not only offers a new and powerful method for studying aspects of Ca2+ homeostasis that up to now could not be directly approached, but might also be used in the future as a tool to report in situ a variety of apparently unrelated phenomena of wide biological interest.     

Calcium transients in aequorin-injected frog cardiac muscle.

The Ca2+ -sensitive bioluminescent protein aequorin was microinjected into cells of frog atrial trabeculae to study intracellular calcium transients associated with excitation-contraction coupling. The amplitude of the aequorin signal increased with extracellular Ca2+ concentration and stimulus frequency, but decreased with stretch. Isoprenaline and acetylstrophanthidin both increased the amplitude, but had strikingly different effects on the time course of the signal.     

CD21 is a ligand for CD23 and regulates IgE production.

The molecule CD23, a low-affinity receptor for IgE (Fc epsilon R2), is a type II transmembrane molecule expressed on many haemopoietic cell types. CD23 has pleiotropic roles in the control of lymphocyte behaviour, suggesting that CD23 may interact with another ligand in addition to IgE. To identify such a CD23 ligand, we expressed and purified full-length recombinant CD23, incorporated it into fluorescent liposomes and used these as a probe. We report here that fluorescent liposomes carrying CD23 interact specifically with the cell-surface protein CD21, identified as the receptor for Epstein-Barr virus and the complement receptor-2 on B cells, some T cells and follicular dendritic cells. In addition, fluorescent CD23-liposomes were shown to bind to hamster kidney cells (BHK-21) transfected with CD21 complementary DNA. The interaction between fluorescent CD23-liposomes and B cells or CD21-transfected BHK-21 cells was specifically inhibited by anti-CD21 and anti-CD23 monoclonal antibodies. Western blotting analysis revealed that 14C-labelled liposomes carrying CD23, in contrast to anti-CD21 antibodies, reacted with a subtype of CD21 molecules. Triggering of CD21 either with an anti-CD21 antibody or with recombinant soluble CD23 was shown to increase specifically interleukin-4-induced IgE production from blood mononuclear cells. These results demonstrate that the cell-surface protein CD21 is a ligand for CD23 and that the pairing of these molecules may participate in the control of IgE production.     

The cargo-binding domain regulates structure and activity of myosin 5

Myosin 5 is a two-headed motor protein that moves cargoes along actin filaments. Its tail ends in paired globular tail domains (GTDs) thought to bind cargo. At nanomolar calcium levels, actin-activated ATPase is low and the molecule is folded. Micromolar calcium concentrations activate ATPase and the molecule unfolds. Here we describe the structure of folded myosin and the GTD's role in regulating activity. Electron microscopy shows that the two heads lie either side of the tail, contacting the GTDs at a lobe of the motor domain (approximately Pro 117-Pro 137) that contains conserved acidic side chains, suggesting ionic interactions between motor domain and GTD. Myosin 5 heavy meromyosin, a constitutively active fragment lacking the GTDs, is inhibited and folded by a dimeric GST-GTD fusion protein. Motility assays reveal that at nanomolar calcium levels heavy meromyosin moves robustly on actin filaments whereas few myosins bind or move. These results combine to show that with no cargo, the GTDs bind in an intramolecular manner to the motor domains, producing an inhibited and compact structure that binds weakly to actin and allows the molecule to recycle towards new cargoes.     

Probing the calcium-induced conformational transition of troponin C with site-directed mutants

The contraction of skeletal muscle is regulated by calcium binding to troponin C (TnC). TnC consists of two spatially independent domains, each of which contains two metal ion binding sites. Calcium binding to the regulatory sites of the N-terminal domain triggers muscle contraction by a series of conformational changes. Site-directed mutagenesis offers a means of elucidating the links in this signal path between TnC and actin-myosin crossbridges. Such mapping is possible if the mutants shift the equilibrium between 'on' and 'off' states of the regulatory complex while maintaining the coupling between calcium binding and tension development. Candidate amino-acid residues for yielding this information would be in positions remote from the calcium-binding sites and from the site of development of tension. Analysis of the crystal structure of TnC and of the model of the calcium-activated molecule has enabled us to identify two such residues: Glu 57 and Glu 88. In separate experiments we have replaced each of these residues by lysines. The resulting reduction in calcium affinity indicates that these residues have a long-range effect on calcium binding. This result may reflect the formation of a salt bridge between positions 57 and 88 that is not present in the native molecule. Moreover, the level of tension recovery when the mutants are incorporated into muscle suggests that the interaction between TnC and other muscle components has also been altered. Thus, these residues may participate in the contraction signal transmission.     

多巴胺D3R蛋白同源模建及其应用

以分辨率为2.2的牛视紫红质蛋白的晶体结构为模板,采用同源模建方法,建立D3R模蛋白。对接D3R模蛋白与刺桐属配体分子,在对接的D3R蛋白的结合腔中选定一个以药物分子为质心,以半径为6的空间范围,计算此空间范围内的所有氨基酸残基与配体分子的作用能量,即残基/配体的结合能或排斥能,据此得到配体分子与受体蛋白的活性结合位点。     

The structure of ribosomal protein S5 reveals sites of interaction with 16S rRNA.

Understanding the process whereby the ribosome translates the genetic code into protein molecules will ultimately require high-resolution structural information, and we report here the first crystal structure of a protein from the small ribosomal subunit. This protein, S5, has a molecular mass of 17,500 and is highly conserved in all lifeforms. The molecule contains two distinct alpha/beta domains that have structural similarities to several other proteins that are components of ribonucleoprotein complexes. Mutations in S5 result in several phenotypes which suggest that S5 may have a role in translational fidelity and translocation. These include ribosome ambiguity or ram, reversion from streptomycin dependence and resistance to spectinomycin. Also, a cold-sensitive, spectinomycin-resistant mutant of S5 has been identified which is defective in initiation. Here we show that these mutations map to two distinct regions of the molecule which seem to be sites of interaction with ribosomal RNA. A structure/function analysis of the molecule reveals discrepancies with current models of the 30S subunit.     

膦酸盐型防垢剂的研制

研究了一种分子结构中含有较多膦酸基的膦酸盐型防垢剂．对这种防垢剂的合成条件进行了正交试验优化．按优化条件合成的防垢剂对碳酸钙垢、硫酸钙垢、硅酸钙垢均有预防作用；在最佳浓度下对碳酸钙和硫酸钙的防垢率接近１００％，对硅酸钙的防垢事可达９０％以上，这种防垢剂溶液对钢铁的腐蚀率低于油田模拟注入水，且对地层无伤害，可用吸附法设置在近井地层中使用．     

人穿孔素羧基端肽段的表达纯化与活性鉴定

穿孔素,即成孔蛋白（pore forming protein,PFP),其溶细胞作用与免疫调节和自身免疫病以及其它多种疾病过程中的免疫性病理损伤相关。为得到足够量的PFP建立与之相关的免疫学研究手段用于基础和临床研究,在已克隆人PFP cDNA的基础上,用基因工程方法表达了人PFP C端124个氨基酸肽段（hPFP-C),并通过谷胱甘肽琼脂糖新和层析获得纯化的GST/hPFP-C融合蛋白,经凝血酶酶切和再次北极和层析去除GST部分,得到了纯化的hPFP-C蛋白。纯化的hPFP-C蛋白与兔红细胞共育,呈现钙依赖的溶血活性。     

A novel three primers PCR (TP-PCR) method to obtain recombinant DNA molecule independent of restriction enzyme

In this note, we report a novel and efficient three primers PCR (TP-PCR) method to rapidly generate recombinant DNA molecule at precise junction between two arbitrary DNA fragments. TP-PCR method is characterized by its reaction system with two templates and three primers, which can produce a recombinant DNA molecule in one PCR reaction. The main advantages of this method are the independence of sequences at the recombination site, the rapidness, and the easy establishment of adequate conditions. This method has been successfully applied to constructing a fusion protein gene, sck gene.     

The CED-4-homologous protein FLASH is involved in Fas-mediated activation of caspase-8 during apoptosis

Fas is a cell-surface receptor molecule that relays apoptotic (cell death) signals into cells. When Fas is activated by binding of its ligand, the proteolytic protein caspase-8 is recruited to a signalling complex known as DISC by binding to a Fas-associated adapter protein. A large new protein, FLASH, has now been identified by cloning of its complementary DNA. This protein contains a motif with oligomerizing activity whose sequence is similar to that of the Caenorhabditis elegans protein CED-4, and another domain (DRD domain) that interacts with a death-effector domain in caspase-8 or in the adapter protein. Stimulated Fas binds FLASH, so FLASH is probably a component of the DISC signalling complex. Transient expression of FLASH activates caspase-8, whereas overexpression of a truncated form of FLASH containing only one of its DRD or CED-4-like domains does not allow activation of caspase-8 and Fas-mediated apoptosis to occur. Overexpression of full-length FLASH blocks the anti-apoptotic effect of the adenovirus protein E1B19K. FLASH is therefore necessary for the activation of caspase-8 in Fas-mediated apoptosis.     

重组人甲状旁腺激素(1-34)在大肠杆菌中的表达与纯化

将化学合成的rhPTH(1-34)基因用PCR扩增后,克隆至表达载体pET-35b( ),使rhPTH(1—34)融合于纤维素结合结构域(CBDdos)的羧基端,并得到高效表达．融合蛋白经纤维素树脂亲和层析纯化后,经Factor Xa裂解释放出rhPTH(1-34),再通过纤维素树脂亲和层析、C4反向高效液相色谱纯化得到rhPTH(1-34)纯品．每升培养液可获取3mg高纯度的rhPTH(1-34)．经质谱测定,所得样品的分子量为4117．0Da,与rhPTH(1—34)理论分子量一致．     

Evolutionary origin of a calcium-dependent protease by fusion of genes for a thiol protease and a calcium-binding protein?

Calcium-dependent protease (calcium protease) is apparently involved in a variety of cellular processes. Here we have attempted to clarify the role and regulatory mechanism of calcium protease by analysing its structure. The complete primary structure of calcium protease (relative molecular mass (Mr) 80,000 (80K), 705 amino acids) was deduced from the nucleotide sequence of cloned complementary DNA. The protein contains four distinct domains, and we have observed a marked similarity between the second and fourth domains and the papain-like thiol proteases and calmodulin-like calcium-binding proteins, respectively. This finding suggests that calcium protease arose from the fusion of genes for proteins of completely different function and evolutionary origin. Further, it provides functional insight into cellular regulatory mechanisms mediated by Ca2+ through calcium-binding proteins.     

两种Cu(Ⅱ)配合物的合成和结构表征

选取1,9-二(2-氟代苯基)-2,5,8一三氮杂壬烷与铜(Ⅱ)的苯甲酸盐和硝酸盐,合成了两个新的配合物[Cu(C18H23F2N3)(C7H5O2)(H2O)](C7H5O2)·2(H2O)(1)和[Cu(C18H23F2N3)(NO3)2]·(H2O)1/2(2),并获得其单晶,测定了晶体结构.单晶衍射数据显示化合物(1)属三斜晶系Pi空间群,化合物(2)属单斜晶系Cc空间群.     

Key residues involved in calcium-binding motifs in EGF-like domains

Many extracellular proteins with diverse functions contain domains similar to epidermal growth factor (EGF), a number of which have a consensus Asp/Asn, Asp/Asn, Asp*/Asn*, Tyr/Phe (where the asterisk denotes a beta-hydroxylated residue). These include the coagulation factors IX and X, proteins with two EGF-like domains, the first of which contains the consensus residues. The first EGF-like domain of human factor IX contains a calcium-binding site, which is believed to be responsible for one of the high-affinity sites detected in this protein. Similar results have been obtained for bovine factor X. We have now used protein engineering and 1H-NMR techniques to investigate the importance of individual consensus residues for ligand binding. Measurement of a calcium-dependent Tyr 69 shift in the isolated first EGF-like domain from human factor IX demonstrates that Asp 47, Asp 49, and Asp 64 are directly involved in this binding. Gln 50, whose importance has previously been overlooked, is also involved in this binding. Two mutations in this domain, Asp 47----Glu, and Asp 64----Asn, present in patients with haemophilia B, reduce calcium binding to the domain greater than 4-fold and greater than 1,000-fold, respectively. Furthermore, the defective calcium binding of Asn 64 can be partially rescued by the compensatory mutation Gln 50----Glu. This latter mutation, when introduced singly more than doubles the affinity of the domain for calcium. This study thus defines residues involved in a new type of calcium-binding site and provides strong circumstantial evidence for calcium-binding motifs in many extracellular proteins, including the developmentally important proteins of Drosophila, notch, delta and crumbs.     

内蒙古盐碱湖一株编码细菌视紫红质的嗜盐古菌bop基因全序列克隆及表达

细菌视紫红质(Bacteriorhodopsin,BR)是由bop基因编码的唯一膜蛋白,具有光推动质子泵的作用,为生命活动提供所需要的能量。为了研究高表达且具有生物活性的BR蛋白,应用PCR扩增技术,从内蒙古额吉淖尔盐碱湖分离得到的一株嗜盐古菌中扩增得到bop基因全序列;并构建了具有His6标签的原核表达载体p ET28a-IM-1,转化到大肠杆菌BL21(DE3),用IPTG诱导表达BR;并对表达产物进行SDS-PAGE鉴定分析。结果显示获得的bop基因全序列开放阅读框为672 bp,编码223个氨基酸,含有重组质粒p ET28a-IM-1的阳性菌株在IPTG诱导下高效表达了分子量约为25 k Da的BR蛋白。同源性分析表明该BR蛋白结构在古菌与细菌中具有相对较高的保守性,这与其质子泵功能密切相关。该BR蛋白为新发现的细菌视紫红质,其基因序列已递交Gen Bank,其登录号为KT873301。     

Intracellular Ca indicator Quin-2 inhibits Ca2+ inflow via Na/Ca exchange in squid axon

Until recently, intracellular free calcium has been amenable to measurement and investigation only in cells large enough to permit either microinjection of a suitable Ca sensor such as a aequorin or arsenazo III or insertion of a Ca-sensitive microelectrode. This constraint on cell size was removed by the development of the fluorescent Ca2+ -sensitive dye Quin-2 and its acetoxymethyl ester, which can be introduced into a wide range of cell types. A major requirement of any intracellular Ca2+ indicator is that it should not disturb intracellular Ca2+ homeostasis and Quin-2 is generally considered to be satisfactory in this respect. We now report that injection of Quin-2 into squid (Loligo forbesi) axons can almost completely abolish one component of Ca2+ entry--intracellular Na+ (Nai)-dependent Ca2+ inflow, which occurs via Na/Ca exchange. Mixtures of Ca and Quin-2 that buffer an ionized Ca2+ at close to physiological concentrations also block Nai-dependent Ca2+ influx but these same mixtures fail to block the extracellular Na+ (Na0)-dependent extrusion of Ca2+, showing that Quin-2 acts specifically on Ca2+ inflow.     

Measurement of the intracellular free calcium concentration in salamander rods

Measurement of the free calcium concentration within a photo-receptor outer segment has been considered an important aim since the proposal by Hagins and Yoshikami that the primary event in phototransduction is a release of Ca (2+) inside the cell. More recent evidence has cast doubt on the calcium hypothesis, and the observations of Yau and Nakatani and Matthews et al. suggest that the internal Ca (2+) concentration ([Ca (2+)]i), may decrease after a flash of light. In the present study we have measured [Ca (2+)]i directly by using a new method for incorporating the Ca-sensitive photoprotein aequorin into an isolated rod. We report that the light response is accompanied by a decrease in [Ca (2+)]i, caused by the closure of light-sensitive channels which are the main route for Ca (2+) entry into the outer segment. Of the Ca (2+) entering through light-sensitive channels, about 95% is sequestered by a rapid and reversible buffering mechanism. Calcium is removed from the cell by an electrogenic pump in which 3 Na (+) ions are exchanged for each Ca (2+); the pump is highly active and the free Ca (2+) in the cell declines with a time constant of ~0.5 s after a flash of light.