A new crystal form of the complex between seryl-tRNA synthetase and tRNA(Ser) from Thermus thermophilus that diffracts to 2.8 A resolution.

Two distinct complexes between seryl-tRNA synthetase and tRNA(Ser) from Thermus thermophilus have been crystallized using ammonium sulphate as a precipitant. Form III crystals grow from solutions containing a 1:2.5 stoichiometry of synthetase dimer to tRNA. They are of monoclinic space group C2 with unit cell dimensions a = 211.6 A, b = 126.8 A, c = 197.1 A, beta = 132.4 degrees and diffract to about 3.5 A. Preliminary crystallographic results show that the crystallographic asymmetric unit contains two synthetase dimers. Form IV crystals grow from solutions containing a 1:1.5 stoichiometry of synthetase dimer to tRNA. They are of orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 124.5 A, b = 128.9 A, c = 121.2 A and diffract to 2.8 A resolution. Preliminary crystallographic results show that these crystals contain only one tRNA molecule bound to a synthetase dimer.     

Crystallization and preliminary analysis of the deoxyoligonucleotide d(CGTAGATCTACG)

Two crystal forms of the self-complementary DNA 12-mer d(CGTAGATCTACG) were grown by the vapour diffusion technique. Form I is in space group C2 with a = 64.8 A, b = 35.4 A, c = 24.4 A and beta = 92.2 (1 A = 0.1 nm). The crystals are grown as monoclinic blocks or hexagonal plates. There are two strands (one duplex) in the asymmetric unit. Form II crystallizes as monoclinic blocks, space group P21 with a = 64.5 A, b = 35.1 A, c = 25.2 A and beta = 91.8 degrees. This form contains four strands (2 duplexes) in the asymmetric unit. Both forms are suitable for high resolution X-ray analysis. The diffraction patterns suggest that the DNA is in a B-type conformation and that the packing in the two forms is very similar.     

Purification, crystallisation and preliminary X-ray study of haemoglobin from Crocodilis palustris and Crocodilis porosus

The unsolved three-dimensional structure of crocodile haemoglobin and its prospects as a blood substitute have led us to initiate the purification and crystallisation of haemoglobin molecules from crocodile species (Crocodilis palustris or mugger and Crocodilis porosus or salt water crocodile). The work has resulted in the prevention of polymerisation of naked haemoglobin molecules using N-ethylmaleimide or iodoacetamide. The purified monomeric haemoglobin molecule of C. porosus was crystallised in two different forms and X-ray diffraction data were collected up to 2 A resolution for both forms. Form I: a=53.62, b=53.55, c=103.77 A; beta=93.35 degrees, space group P2(1), Z=2. Form II: a=71.30, b=54.70, c=80.00 A; beta=106.4 degrees, space group P2(1), Z=2. Structure solution and rigid body refinement of form I data resulted in a model with R(free)=0.42 and R=0.35.     

Crystallization and preliminary X-ray study of a lipase from Pseudomonas glumae.

Lipase from Pseudomonas glumae has been purified and crystallized in two forms, using the hanging drop method of vapour diffusion at 4 degrees C and 15 degrees C. Both forms grew at pH 9.0 from 0.1 M-Tris buffer in the presence of 10% (v/v) acetone. Form 1 was crystallized from 27 to 29% polyethylene glycol in the presence of less than 0.5% (v/v) n-dodecyl-beta-D-glucopyranoside. Form 2 was grown from 17 to 19% ammonium sulphate in the presence of 1% n-octyl-beta-D-glucopyranoside. Form 1 is orthorhombic with space group P2(1)2(1)2(1), and cell dimensions of a = 158.1 A, b = 158.6 A, c = 63.4 A, Form 2 is tetragonal with space group P4(1)2(1)2 (or P4(3)2(1)2) and cell dimensions of a = 89.3 A, c = 180.4 A. Form 1 probably has four molecules per asymmetric unit and diffracts to at least 2.5 A. Form 2 has two molecules per asymmetric unit and diffracts to at least 3.0 A.     

Differential effects of adenosine A2a and A2b receptors on cardiac contractility

The mammalian myocardium expresses four adenosine receptor (AR) subtypes: A(1)AR, A(2a)AR, A(2b)AR, and A(3)AR. The A(1)AR is well known for its profound antiadrenergic effects, but the roles of other AR subtypes in modulating contractility remain inconclusive. Thus, the objective of this study was to determine the direct and indirect effects of A(2a)AR and A(2b)AR on cardiac contractility. Experiments were conducted in paced, constant pressure-perfused isolated hearts from wild-type (WT), A(2a)AR knockout (KO), and A(2b)AR KO mice. The A(2a)AR agonist CGS-21680 did not alter basal contractility or β-adrenergic receptor agonist isoproterenol (Iso)-mediated positive inotropic responses, and Iso-induced effects were unaltered in A(2a)AR KO hearts. However, A(2a)AR gene ablation resulted in a potentiation of the antiadrenergic effects mediated by the A(1)AR agonist 2-chloro-N-cyclopentyladenosine. The nonselective AR agonist 5'-N-ethylcarboxamido adenosine and the selective A(2b)AR agonist BAY 60-6583 induced coronary flow-independent increases in contractility, but BAY 60-6583 did not alter Iso-induced contractile responses. The A(1)AR antiadrenergic effect was not potentiated in A(2b)AR KO hearts. The expression of all four AR subtypes in the heart and ventricular myocytes was confirmed using real-time quantitative PCR. Taken together, these results indicate that A(2a)AR does not increase cardiac contractility directly but indirectly alters contractility by modulating the A(1)AR antiadrenergic effect, whereas A(2b)AR exerts direct contractile effects but does not alter β-adrenergic or A(1)AR antiadrenergic effects. These results indicate that multiple ARs differentially modulate cardiac function.     

New crystal forms of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Three crystal forms of the dimeric form of the enzyme ribulose-1,5-bisphosphate carboxylase from the photosynthetic bacterium Rhodospirillum rubrum have been obtained from the gene product expressed in Escherichia coli. Form A crystals formed from the quaternary complex comprising enzyme-activator carbamate-Mg2+-2'-carboxyarabinitol-1,5-bisphosphate are shown here to be devoid of ligands. In contrast, crystals of the quaternary complex formed with the hexadecameric L8S8 enzyme from spinach contain both the activator carbamate and 2'-carboxyarabinitol-1,5-bisphosphate. Form B crystals of the R. rubrum enzyme are monoclinic, space group P2(1) with cell dimensions a = 65.5 A, b = 70.6 A, c = 104.1 A and beta = 92.1 degrees, with two subunits per asymmetric unit. Rotation function calculations show a non-crystallographic 2-fold axis perpendicular to the monoclinic b-axis. Form C crystals are orthorhombic (space group P2(1)2(1)2(1)) with cell dimensions a = 79.4 A, b = 100.1 A and c = 131.0 A. The monoclinic crystal form diffracts to at least 2.0 A resolution on a conventional X-ray source.     

Two crystal forms of helix II of Xenopus laevis 5S rRNA with a cytosine bulge

The crystal structure of r(GCCACCCUG).r(CAGGGUCGGC), helix II of the Xenopus laevis 5S rRNA with a cytosine bulge (underlined), has been determined in two forms at 2.2 A (Form I, space group P4(2)2(1)2, a = b = 57.15 A and c = 43.54 A) and 1.7 A (Form II, space group P4(3)2(1)2, a = b = 32.78 A and c = 102.5 A). The helical regions of the nonamers are found in the standard A-RNA conformations and the two forms have an RMS deviation of 0.75 A. However, the cytosine bulge adopts two significantly different conformations with an RMS deviation of 3.9 A. In Form I, the cytosine bulge forms an intermolecular C+*G.C triple in the major groove of a symmetry-related duplex with intermolecular hydrogen bonds between N4C and O6G, and between protonated N3+C and N7G. In contrast, a minor groove C*G.C triple is formed in Form II with intermolecular hydrogen bonds between O2C and N2G, and between N3C and N3G with a water bridge. A partial major groove opening was observed in Form I structure at the bulge site. Two Ca2+ ions were found in Form I helix whereas there were none in Form II. The structural comparison of these two forms indicates that bulged residues can adopt a variety of conformations with little perturbation to the global helix structure. This suggests that bulged residues could function as flexible latches in bridging double helical motifs and facilitate the folding of large RNA molecules.     

New concepts and methods of standardizing predictive value, accuracy and incorrect diagnostic rate

OBJECTIVE: To express the value of a diagnostic test under standardized and comparable conditions. STUDY DESIGN: Four new concepts of standardizing positive predictive value (SPPV), standardizing negative predictive value (SNPV), standardizing accuracy (SAc) and standardizing an incorrect diagnostic test were developed. The theoretical positive predictive value (SPPV), theoretical negative predictive value (SNPV), theoretical accuracy (SAc) and theoretical incorrect diagnosis rate (SIDR), which are not affected by a different constituent ratio of disease and nondisease groups and are obtained under the theoretical standard condition that the sample size in the disease group equals that in the nondisease group, were defined. Based on these concepts and the principles and methods of statistics and evaluation of diagnostic tests, corresponding formulas were deduced. RESULTS: The formulas are: SPPV = a(b + d)/[a(b + d) + b(a + c)] = Se/(1 + Se - Sp), SNPV = d(a + c)/[c(b + d) + d(a + c)] = Sp/(1 - Se + Sp), SAc = [a(b + d) + d(a + c)]/[2(a + c)(b + d)] = (Se + Sp)/2, and SIDR = [b(a + c) + c(b + d)]/[2(a + c)(b + d)] = (2 - Se - Sp)/2. Here, a, b, c and d refer to the case numbers of true positives, false positives, false negatives and true negatives; Se and Sp refer, respectively, to sensitivity and specificity. CONCLUSION: SPPV, SNPV, SAc and SIDR are very useful for expressing and evaluating the value of a diagnostic test under standardized and comparable conditions.     

Crystallization and preliminary x-ray studies of spinach ribulose 1,5-bisphosphate carboxylase/oxygenase complexed with activator and a transition state analogue

Ribulose 1,5-bisphosphate carboxylase/oxygenase has been purified from spinach and crystallized by equilibrium vapor diffusion with polyethylene glycol 6000 as a precipitant. Crystals suitable for x-ray studies were obtained from a binary complex with a transition state analogue, 2-C-carboxy-D-arabinitol 1,5-bisphosphate, and a quaternary complex with 2-C-carboxy-D-arabinitol 1,5-bisphosphate, Mg2+, and HCO-3. Two forms of crystals were obtained in the presence of 2-C-carboxy-D-arabinitol 1,5-bisphosphate. Form B crystals are plates which have orthorhombic space group P2(1)2(1)2 with unit cell dimensions a = 184 A, b = 218 A, and c = 119 A. Form C crystals are tetragonal needles with space group I422 and with cell dimensions a = b = 275 A and c = 178 A. In both forms, the asymmetric unit contains half a molecule.     

Crystal structure of form II of cholesteryl palmitelaidate at 295 K

Form II for cholesteryl palmitelaidate (trans-9-hexadecenoate) (C43H74O2) is monoclinic P2(1) with a = 12.745(3), b = 9.006(2), c = 18.153(4) A, beta = 96.63 (2) degrees, Z = 2. The X-ray crystal structure of form II has been determined from 2506 reflections of which 2126 gave (F greater than 2 sigma). The data up to sin theta/lambda = 0.44A-1 (Dmin = 1.14 A) were measured with CuK alpha radiation from a sealed tube. These were supplemented up to sin theta/lambda = 0.52 A-1 (Dmin = 0.96 A) by measurements on the same crystal using a rotating anode X-ray source. The electron density was diffuse in the ester chain and the atoms of the cholesteryl tail were found to be disordered. The tail and the chain atoms were refined by restrained least squares methods to give R = 0.087 and Rw = 0.10 for reflections with F greater than 2 sigma. Crystal forms I and II represent two standard structure types already characterized for fatty acid esters of cholesterol. In form II, the ester chain is almost fully extended as is also the case for one of the two independent molecules (A) in form I. In form II, the chains pack loosely together for most of their length. M.s. amplitudes of thermal vibration for the chain C-atoms are almost uniform along the entire chain (approximately 0.25 A2 at 295 K). In form I, the proximal part of the A chain is surrounded by rigid cholesteryl groups. In this region, C-atom m.s. amplitudes are much reduced (approximately 0.10 A2) but they increase to about 0.5 A2 at the distal end of the chain where packing is very loose.     

Cardiovascular alpha1-adrenoceptor subtypes: functions and signaling

alpha-Adrenoceptors (alpha1AR) are G protein-coupled receptors and include alpha1A, alpha1B, and alpha1D subtypes corresponding to cloned alpha1a, alpha1b, and alpha1d, respectively. alpha1AR mediate several cardiovascular actions of sympathomimetic amines such as vasoconstriction and cardiac inotropy, hypertrophy, metabolism, and remodeling. alpha1AR subtypes are products of separate genes and differ in structure, G protein-coupling, tissue distribution, signaling, regulation, and functions. Both alpha(1A)AR and alpha(1B)AR mediate positive inotropic responses. On the other hand, cardiac hypertrophy is primarily mediated by alpha(1A)AR. The only demonstrated major function of alpha(1D)AR is vasoconstriction. alpha1AR are coupled to phospholipase C, phospholipase D, and phospholipase A2; they increase intracellular Ca2+ and myofibrillar sensitivity to Ca2+ and cause translocation of specific phosphokinase C isoforms to the particulate fraction. Cardiac hypertrophic responses to alpha1AR agonists might involve activation of phosphokinase C and mitogen-activated protein kinase via Gq x alpha1AR subtypes might interact with each other and with other receptors and signaling mechanisms.     

Fascin在结肠癌中表达的临床病理学研究

目的:Fascin是一种肌动蛋白结合蛋白,在多种上皮性肿瘤中高表达并与肿瘤侵袭有关。在本研究中观察fascin在结肠癌中表达及探讨其临床病理意义,为其在结肠癌早期诊断和预后预测方面的应用提供依据。方法:应用免疫组织化学技术检测Fascin在结肠肿瘤中的表达,并分析其表达的结肠癌临床病理意义。结果:Fascin在癌旁肠粘膜、腺瘤、腺癌中表达有显著性差异,其中癌旁肠粘膜组和腺瘤组阳性表达率无显著性差异(X2=0.344,P0.05),腺癌组阳性表达率显著高于癌旁粘膜组(X2=8.492,P0.0,1),腺癌组阳性表达率显著高于腺瘤组(X2=7.450,P0.01)。Fascin表达与结肠癌患者的年龄、性别、肿瘤浸润肠壁深度、淋巴结转移、分化程度无显著相关性,而在中-晚期病例(III/IV期)中的表达率显著高于早期病例(P0.05)。Fascin表达阳性病例的生存期显著高于Fascin表达阴性病例(P0.022)。结论:Fascin表达与结肠癌发生和预后密切相关,可能作为结肠癌早期诊断和预后的标志物。     

Variation and Phylogenetic Relationships Between Polygonatum odoratum and P. cyrtonema

The present paper is a report on combined cytological and taxonomical studies of Polygonatum odoratum and P. cyrtonema. Based on the extensive field observation and mass collection for some years, the authors made a detailed comparison of the variation pattern of morphology、cytology as well as geographic distribution between forms of P. odoratum and P. cyrtonema. The results show that: Form I: Stem angular, leaves elliptic, peduncles with one, rarely two flowers, perianth 1.7-2.0 cm long, stamens adnate to perianth in the middle part or a little lower. The karyotype formula is 2n=20=12m+8sm (2SAT); Form II: Stem angular, leaves narrow-oblong, peduncles with 2-3 flowers, perianth 2.8-3.2 cm long, the position of stamens on perianth is above the middle part. It has the karyotype 2n=20=8m+1osm(4SAT)+2st; Form III: Stem terete, leaves oblong-lanceolate, peduncles with 3- 10 flowers, perianth 1.5- 2.0cmlong, stamens are adnate to perianth also above the middle part. In this form, two kinds of karyotypes are found: 2n = 22 = 6m+8sm+8st and 2n = 20 = 4m+6sm (2SAT) +10st; Form IV: Stem terete, leaves elliptic, peduncles with 1-4 flowers, perianth 2.3-2.8 cmlong, stamens adnate to perianth above the middle part. The karyotype formulae are 2n = 20 = 4m+14sm (2SAT)+2st and 2n = 20 = 10m+10sm (2SAT); Form V: Stem terete, leaves oblong-lanceolate, peduncles with 3-5 flowers, perianth less than 2.0cm long, stamens adnate to perianth above the middle part. It is of the karyotype 2n=22=4m+8sm(2SAT) +10st. It is pointed out that the P. odoratum collected from the Qinling Range may be a hybrid or an intermediate in the evolutionary process from P. odoratum to P. cyrtonema. The differences between the different forms of P. cyrtonema are obvious and stable, and are probably derived from the chromosomal variation. The different forms of P. cyrtonema may have evolvedfrom P. odoratum along different courses.     

Exome Capture Sequencing of Adenoma Reveals Genetic Alterations in Multiple Cellular Pathways at the Early Stage of Colorectal Tumorigenesis

Most of colorectal adenocarcinomas are believed to arise from adenomas, which are premalignant lesions. Sequencing the whole exome of the adenoma will help identifying molecular biomarkers that can predict the occurrence of adenocarcinoma more precisely and help understanding the molecular pathways underlying the initial stage of colorectal tumorigenesis. We performed the exome capture sequencing of the normal mucosa, adenoma and adenocarcinoma tissues from the same patient and sequenced the identified mutations in additional 73 adenomas and 288 adenocarcinomas. Somatic single nucleotide variations (SNVs) were identified in both the adenoma and adenocarcinoma by comparing with the normal control from the same patient. We identified 12 nonsynonymous somatic SNVs in the adenoma and 42 nonsynonymous somatic SNVs in the adenocarcinoma. Most of these mutations including OR6X1, SLC15A3, KRTHB4, RBFOX1, LAMA3, CDH20, BIRC6, NMBR, GLCCI1, EFR3A, and FTHL17 were newly reported in colorectal adenomas. Functional annotation of these mutated genes showed that multiple cellular pathways including Wnt, cell adhesion and ubiquitin mediated proteolysis pathways were altered genetically in the adenoma and that the genetic alterations in the same pathways persist in the adenocarcinoma. CDH20 and LAMA3 were mutated in the adenoma while NRXN3 and COL4A6 were mutated in the adenocarcinoma from the same patient, suggesting for the first time that genetic alterations in the cell adhesion pathway occur as early as in the adenoma. Thus, the comparison of genomic mutations between adenoma and adenocarcinoma provides us a new insight into the molecular events governing the early step of colorectal tumorigenesis.     

Crystallization and preliminary X-ray analysis of the lactose-specific phosphocarrier protein IIAlac of the phosphoenolpyruvate: sugar phosphotransferase system from Staphylococcus aureus.

The IIA constituent of the lactose permease from Staphylococcus aureus has been crystallized in two different forms. Crystals of form I have been grown from polyethylene glycol 4000 with beta-octyl glucoside. They diffract to 3.0 A resolution and belong to space group C2 with unit cell dimensions a = 141.7 A, b = 130.7 A, c = 96.5 A and beta = 96.2 degrees. Form II crystals have been obtained from a solution containing polyethylene glycol 400, ammonium sulfate and manganese chloride. They diffract to at least 2.8 A resolution and belong to space group P2(1)2(1)2(1) with unit cell dimensions a = 89.9 A, b = 101.5 A and c = 90.9 A.     

Crystallization and preliminary x-ray diffraction studies of a psychrophilic iron superoxide dismutase from Pseudoalteromonas haloplanktis

The Antarctic eubacterium Pseudoalteromonas haloplanktis (Ph) produces a cold-active iron superoxide dismutase (SOD). PhSOD is a homodimeric enzyme, that displays a high catalytic activity even at low temperature. Using hanging-drop vapour-diffusion technique, PhSOD has been successfully crystallized in two different crystal forms. Both crystal forms are monoclinic with space group P2(1) and diffract to 2.1 A resolution. Form I has unit-cell parameters a=45.49A b=103.63A c=50.37A beta=108.2 degrees and contains a homodimer in the asymmetric unit. Form II has unit-cell parameters a=50.48A b=103.78A c=90.25A beta=103.8 degrees and an asymmetric unit containing two PhSOD homodimers. Structure determination has been achieved using molecular replacement. The crystallographic study of this cold-adapted enzyme could contribute to the understanding of the molecular mechanisms of cold-adaptation and of the high catalytic efficiency at low temperature.     

Binding of IKe gene 5 protein to polynucleotides. Fluorescence binding experiments of IKe gene 5 protein and mutual cooperativity of IKe and M13 gene 5 proteins

Fluorescence studies of the binding of IKe gene 5 protein to various polynucleotides were performed to obtain insight into the question as to what extent the binding characteristics of the gene 5 proteins of the IKe and M13 phages resemble and/or differ from each other. The fluorescence of IKe gene 5 protein is quenched 60% upon binding to most polynucleotides. At moderate salt concentrations, i.e., below 1 M salt, the binding stoichiometry is 4.0 +/- 0.5 nucleotides per IKe gene 5 protein monomer. The affinity of the protein for homopolynucleotides depends strongly on sugar and base type; in order of increasing affinities we find poly(rC) less than poly(dA) less than poly(rA) less than poly(dI) less than poly(rU) less than poly(dU) less than poly(dT). For most polynucleotides studied, the affinity depends linearly on the salt concentration: [d log (Kint omega)]/(d log [M+]) = -3. The binding is highly cooperative. The cooperativity parameter omega, as deduced from protein titration curves, is 300 +/- 150 and appears independent of the type of polynucleotide studied. Estimation of this binding parameter from salt titrations of gene 5 protein-polynucleotide complexes results in systematically higher values. A comparison of the binding data of the IKe and M13 gene 5 proteins shows that the fluorescence quenching, stoichiometry, order of binding affinities, and cooperativity in the binding are similar for both proteins. From this it is concluded that at least the DNA binding grooves of both proteins must show a close resemblance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding pathway determines norepinephrine selectivity for the human β1AR over β2AR

Beta adrenergic receptors (βARs) mediate physiologic responses to the catecholamines epinephrine and norepinephrine released by the sympathetic nervous system. While the hormone epinephrine binds β₁AR and β₂AR with similar affinity, the smaller neurotransmitter norepinephrine is approximately tenfold selective for the β₁AR. To understand the structural basis for this physiologically important selectivity, we solved the crystal structures of the human β₁AR bound to an antagonist carazolol and different agonists including norepinephrine, epinephrine and BI-167107. Structural comparison revealed that the catecholamine-binding pockets are identical between β₁AR and β₂AR, but the extracellular vestibules have different shapes and electrostatic properties. Metadynamics simulations and mutagenesis studies revealed that these differences influence the path norepinephrine takes to the orthosteric pocket and contribute to the different association rates and thus different affinities.Subject terms: X-ray crystallography, Molecular biology