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981.
Yuefeng Tang William M. Schneider Yang Shen Srivatsan Raman Masayori Inouye David Baker Monica J. Roth Gaetano T. Montelione 《Journal of structural and functional genomics》2010,11(4):223-232
Determination of high-quality small protein structures by nuclear magnetic resonance (NMR) methods generally requires acquisition and analysis of an extensive set of structural constraints. The process generally demands extensive backbone and sidechain resonance assignments, and weeks or even months of data collection and interpretation. Here we demonstrate rapid and high-quality protein NMR structure generation using CS-Rosetta with a perdeuterated protein sample made at a significantly reduced cost using new bacterial culture condensation methods. Our strategy provides the basis for a high-throughput approach for routine, rapid, high-quality structure determination of small proteins. As an example, we demonstrate the determination of a high-quality 3D structure of a small 8 kDa protein, E. coli cold shock protein A (CspA), using <4 days of data collection and fully automated data analysis methods together with CS-Rosetta. The resulting CspA structure is highly converged and in excellent agreement with the published crystal structure, with a backbone RMSD value of 0.5 Å, an all atom RMSD value of 1.2 Å to the crystal structure for well-defined regions, and RMSD value of 1.1 Å to crystal structure for core, non-solvent exposed sidechain atoms. Cross validation of the structure with 15N- and 13C-edited NOESY data obtained with a perdeuterated 15N, 13C-enriched 13CH3 methyl protonated CspA sample confirms that essentially all of these independently-interpreted NOE-based constraints are already satisfied in each of the 10 CS-Rosetta structures. By these criteria, the CS-Rosetta structure generated by fully automated analysis of data for a perdeuterated sample provides an accurate structure of CspA. This represents a general approach for rapid, automated structure determination of small proteins by NMR. 相似文献
982.
Alessandra Corradetti Franca Saccucci Monica Emanuelli Giorgia Vagnoni Monia Cecati Davide Sartini Stefano R. Giannubilo Andrea L. Tranquilli 《Cell stress & chaperones》2010,15(1):95-100
Mitogen-activated protein kinase (MAPK) p38α was shown to be implicated in the organogenesis of the placenta, and such placental
alteration is crucial for the development of hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome. We aimed
to analyze for the first time human placental expression of MAPK p38α in pregnancies complicated by HELLP. The placental expression
of MAPK p38α was investigated by semiquantitative polymerase chain reaction using cDNA extracted from placental tissue of
15 pregnancies with HELLP syndrome and 15 gestational age-matched controls. Seven patients with HELLP also had intrauterine
fetal growth restriction (IUGR). In placenta from pregnancy complicated by HELLP, the expression of MAPK p38α is significantly
decreased compared to the group with normal pregnancy (p < 0.001), while no difference was found between the HELLP and HELLP with IUGR subpopulations. Our study shows for the first
time that MAPK p38α is expressed in the human placenta. Pregnancies with placental dysfunction and hypertensive complications
are characterized by a significantly decreased expression of MAPK p38α. Our observations suggest that p38 MAPK signaling may
be essential in placental angiogenesis and functioning. 相似文献
983.
Chiara Bernardini Paolo Gaibani Augusta Zannoni Caterina Vocale Maria Laura Bacci Gabriela Piana Monica Forni Vittorio Sambri 《Cell stress & chaperones》2010,15(5):509-516
Treponema denticola is an oral spirochete that is associated with periodontal disease and detected occasionally in extraoral lesions associated
with systemic disorders such as cardiovascular diseases. The effect of specific bacterial products from oral treponemes on
endothelium is poorly investigated. This study analyzed the ability of components of the outer membrane of T. denticola (OMT) to induce apoptosis and heat shock proteins (HO-1 and Hsp70) in porcine aortic endothelial cells (pAECs), compared
with results obtained with classical pro-inflammatory lipopolysaccharide (LPS) treatment. Cellular apoptosis was detected
when pAECs were treated with either OMT or LPS, suggesting that OMT can damage endothelium integrity by reducing endothelial
cell vitality. Stimulation with OMT, similarly to LPS response, increased HO-1 and Hsp-70 protein expression in a time-dependent
manner, correlating with a rise in HO-1 and Hsp-70 mRNA. Collectively, these results support the hypothesis that T. denticola alters endothelial cell function. Moreover, our in vitro experiments represent a preliminary investigation to further in
vivo study using a pig model to elucidate how T. denticola leaves the initial endodontic site and participates in the development of several systemic diseases. 相似文献
984.
985.
Grady MM Wright I 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》2006,361(1474):1703-1713
One of the goals of the present Martian exploration is to search for evidence of extinct (or even extant) life. This could be redefined as a search for carbon. The carbon cycle (or, more properly, cycles) on Earth is a complex interaction among three reservoirs: the atmosphere; the hydrosphere; and the lithosphere. Superimposed on this is the biosphere, and its presence influences the fixing and release of carbon in these reservoirs over different time-scales. The overall carbon balance is kept at equilibrium on the surface by a combination of tectonic processes (which bury carbon), volcanism (which releases it) and biology (which mediates it). In contrast to Earth, Mars presently has no active tectonic system; neither does it possess a significant biosphere. However, these observations might not necessarily have held in the past. By looking at how Earth's carbon cycles have changed with time, as both the Earth's tectonic structure and a more sophisticated biology have evolved, and also by constructing a carbon cycle for Mars based on the carbon chemistry of Martian meteorites, we investigate whether or not there is evidence for a Martian biosphere. 相似文献
986.
A number of amidine derivatives (3a-i) were synthesized by condensation of cyanopyridine and cyanopyrazine with sulfonylhydrazides in the presence of sodium methoxide. 2-Acetylpyridine and 4-acetylpyridine were condensed with sulfonylhydrazides by microwave irradiation in solid phase to give corresponding hydrazones (5a-d). Indole-3-carboxaldehyde was condensed with sulfonylhydrazides by refluxing in acetic acid to give corresponding condensation product (5e and f). All the compounds, that is, 3a-i and 5a-f were purified by crystallization or by column chromatography. Structures of all the synthesized compounds are supported by correct IR, (1)H NMR, mass spectral and analytical data. Anti-inflammatory activity evaluation was carried out using carrageenin-induced paw oedema assay and compounds 3e,f and 5e exhibited good anti-inflammatory activity, that is 52%, 37% and 38% at 50 mg/kg po, respectively. Analgesic activity evaluation was carried out using acetic acid writhing assay and compounds 3a,c,e and 5f showed good analgesic activity, that is, 50%, 50%, 50% and 60% at 50 mg/kg po, respectively. 相似文献
987.
Revealing domain structure through linker-scanning analysis of the murine leukemia virus (MuLV) RNase H and MuLV and human immunodeficiency virus type 1 integrase proteins 下载免费PDF全文
Puglia J Wang T Smith-Snyder C Cote M Scher M Pelletier JN John S Jonsson CB Roth MJ 《Journal of virology》2006,80(19):9497-9510
988.
Maia LF Soares MR Valente AP Almeida FC Oliveira AC Gomes AM Freitas MS Schneemann A Johnson JE Silva JL 《The Journal of biological chemistry》2006,281(39):29278-29286
The gamma(1)-peptide is a 21-residue lipid-binding domain from the non-enveloped Flock House virus (FHV). Unlike enveloped viruses, the entry of non-enveloped viruses into cells is believed to occur without membrane fusion. In this study, we performed NMR experiments to establish the solution structure of a membrane-binding peptide from a small non-enveloped icosahedral virus. The three-dimensional structure of the FHV gamma(1)-domain was determined at pH 6.5 and 4.0 in a hydrophobic environment. The secondary and tertiary structures were evaluated in the context of the capacity of the peptide for permeabilizing membrane vesicles of different lipid composition, as measured by fluorescence assays. At both pH values, the peptide has a kinked structure, similar to the fusion domain from the enveloped viruses. The secondary structure was similar in three different hydrophobic environments as follows: water/trifluoroethanol, SDS, and membrane vesicles of different compositions. The ability of the peptide to induce vesicle leakage was highly dependent on the membrane composition. Although the gamma-peptide shares some structural properties to fusion domains of enveloped viruses, it did not induce membrane fusion. Our results suggest that small protein components such as the gamma-peptide in nodaviruses (such as FHV) and VP4 in picornaviruses have a crucial role in conducting nucleic acids through cellular membranes and that their structures resemble the fusion domains of membrane proteins from enveloped viruses. 相似文献
989.
The Src homology 2 (SH2) domain of interleukin-2 tyrosine kinase (Itk) is a critical component of the regulatory apparatus controlling the activity of this immunologically important enzyme. To gain insight into the structural features associated with the activated form of Itk, we have solved the NMR structure of the SH2 domain bound to a phosphotyrosine-containing peptide (pY) and analyzed changes in trans-hydrogen bond scalar couplings ((3h)J(NC')) that result from pY binding. Isomerization of a single prolyl imide bond in this domain is responsible for simultaneous existence of two distinct SH2 conformers. Prolyl isomerization directs ligand recognition: the trans conformer preferentially binds pY. The structure of the SH2/pY complex provides insight into the ligand specificity; the BG loop in the ligand-free trans SH2 conformer is pre-arranged for optimal contacts with the pY+3 residue of the ligand. Analysis of (3h)J(NC') couplings arising from hydrogen bonds has revealed propagation of structural changes from the pY binding pocket to the CD loop containing conformationally heterogeneous proline as well as to the alphaB helix, on the opposite site of the domain. These findings offer a structural framework for understanding the roles of prolyl isomerization and pY binding in Itk regulation. 相似文献
990.
Frøkjaer-Jensen C Kindt KS Kerr RA Suzuki H Melnik-Martinez K Gerstbreih B Driscol M Schafer WR 《Journal of neurobiology》2006,66(10):1125-1139
Voltage-gated calcium channels (VGCCs) serve as a critical link between electrical signaling and diverse cellular processes in neurons. We have exploited recent advances in genetically encoded calcium sensors and in culture techniques to investigate how the VGCC alpha1 subunit EGL-19 and alpha2/delta subunit UNC-36 affect the functional properties of C. elegans mechanosensory neurons. Using the protein-based optical indicator cameleon, we recorded calcium transients from cultured mechanosensory neurons in response to transient depolarization. We observed that in these cultured cells, calcium transients induced by extracellular potassium were significantly reduced by a reduction-of-function mutation in egl-19 and significantly reduced by L-type calcium channel inhibitors; thus, a main source of touch neuron calcium transients appeared to be influx of extracellular calcium through L-type channels. Transients did not depend directly on intracellular calcium stores, although a store-independent 2-APB and gadolinium-sensitive calcium flux was detected. The transients were also significantly reduced by mutations in unc-36, which encodes the main neuronal alpha2/delta subunit in C. elegans. Interestingly, while egl-19 mutations resulted in similar reductions in calcium influx at all stimulus strengths, unc-36 mutations preferentially affected responses to smaller depolarizations. These experiments suggest a central role for EGL-19 and UNC-36 in excitability and functional activity of the mechanosensory neurons. 相似文献