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181.
Vincenzo Forgetta Gary Leveque Joana Dias Deborah Grove Robert Lyons Jr. Suzanne Genik Chris Wright Sushmita Singh Nichole Peterson Michael Zianni Jan Kieleczawa Robert Steen Anoja Perera Doug Bintzler Scottie Adams Will Hintz Volker Jacobi Louis Bernier Roger Levesque Ken Dewar 《Journal of biomolecular techniques》2013,24(1):39-49
As part of the DNA Sequencing Research Group of the Association of Biomolecular Resource Facilities, we have tested the reproducibility of the Roche/454 GS-FLX Titanium System at five core facilities. Experience with the Roche/454 system ranged from <10 to >340 sequencing runs performed. All participating sites were supplied with an aliquot of a common DNA preparation and were requested to conduct sequencing at a common loading condition. The evaluation of sequencing yield and accuracy metrics was assessed at a single site. The study was conducted using a laboratory strain of the Dutch elm disease fungus Ophiostoma novo-ulmi strain H327, an ascomycete, vegetatively haploid fungus with an estimated genome size of 30–50 Mb. We show that the Titanium System is reproducible, with some variation detected in loading conditions, sequencing yield, and homopolymer length accuracy. We demonstrate that reads shorter than the theoretical minimum length are of lower overall quality and not simply truncated reads. The O. novo-ulmi H327 genome assembly is 31.8 Mb and is comprised of eight chromosome-length linear scaffolds, a circular mitochondrial conti of 66.4 kb, and a putative 4.2-kb linear plasmid. We estimate that the nuclear genome encodes 8613 protein coding genes, and the mitochondrion encodes 15 genes and 26 tRNAs. 相似文献
182.
Pierpaolo Correale Antonio Procopio Luigi Celio Michele Caraglia Geppino Genua Vincenzo Coppola Stefano Pepe Nicola Normanno Immacolata Vecchio Giovannella Palmieri Stefania Montagnani Pierosandro Tagliaferri Angelo Raffaele Bianco 《Cancer immunology, immunotherapy : CII》1992,34(4):272-278
Summary Human melanoma cells are sensitive to the lytic activity of natural killer (NK) and lymphokine-activated killer (LAK) cells in vitro. The events resulting in tumour cell killing by lymphocytic effectors have not been completely clarified, and the same target cell determinants regulating responsiveness to immune cytolysis have not yet been identified. Indeed, changes in the differentiative status of leukemia cells as well as in the expression of major histocompatibility complex (MHC) antigens have been described to modulate sensitivity to cytotoxic effectors; moreover surface expression of adhesion factors or extracellular matrix proteins by the cancer cells can promote the activation of the cytolytic effectors and has been described to correlate with tumour cell sensitivity to cytolytic cells. We reasoned that treatment with differentiation inducers could modulate melanoma cell sensitivity to NK and LAK cells. The present study demonstrates that human melanoma GLL-19 cells, when treated with the phorbol diester phorbol 12-myristate 13-acetate (PMA) in vitro, undergo growth inhibition and neuron-like differentiation. Moreover PMA treatment induces an evident inhibition of GLL-19 cell sensitivity to NK- and LAK-mediated cytotoxicity. GLL-19 cells express constitutively MHC class I antigens. PMA treatment, however, does not modify the expression of MHC class I and class II DR antigens in human melanoma GLL-19 cells. We have finally evaluated the effects of PMA on the expression at the cell surface of adhesion factors such as ICAM-1, and extracellular matrix proteins such as collagen IV, laminin and fibronectin; we have also studied the expression of the integrin vitronectin receptor, a membrane receptor for adhesive proteins. While adhesion factors and extracellular matrix proteins appear to play an important role in the interaction between immune effector and tumour target, it can be supposed that the modulation of such membrane-associated proteins or glycoproteins induces NK and LAK resistance in cancer cells. We indeed found that PMA treatment induced in GLL-19 a marked reduction of membrane expression of collagen IV and ICAM-1; moreover PMA reduced the cell membrane expression of the integrin vitronectin receptor. On the other hand, membrane expression of fibronectin and laminin was not affected by PMA. These data indicate that the acquisition of a NK- and LAK-resistant phenotype by GLL-19 cells occurs together with cell differentiation, down-regulation of membrane expression of collagen IV, ICAM-1 and vitronectin receptor, but in the absence of changes in MHC antigens.This work has been supported by the Italian Association for Cancer Research (A. I. R. C.) and by Istituto Superiore di Sanità, Italy-USA joint program on New Therapies on Neoplasia. 相似文献
183.
Krastanova I Sannino V Amenitsch H Gileadi O Pisani FM Onesti S 《The Journal of biological chemistry》2012,287(6):4121-4128
Cdc45 is an essential protein conserved in all eukaryotes and is involved both in the initiation of DNA replication and the progression of the replication fork. With GINS, Cdc45 is an essential cofactor of the Mcm2-7 replicative helicase complex. Despite its importance, no detailed information is available on either the structure or the biochemistry of the protein. Intriguingly, whereas homologues of both GINS and Mcm proteins have been described in Archaea, no counterpart for Cdc45 is known. Herein we report a bioinformatic analysis that shows a weak but significant relationship among eukaryotic Cdc45 proteins and a large family of phosphoesterases that has been described as the DHH family, including inorganic pyrophosphatases and RecJ ssDNA exonucleases. These enzymes catalyze the hydrolysis of phosphodiester bonds via a mechanism involving two Mn(2+) ions. Only a subset of the amino acids that coordinates Mn(2+) is conserved in Cdc45. We report biochemical and structural data on the recombinant human Cdc45 protein, consistent with the proposed DHH family affiliation. Like the RecJ exonucleases, the human Cdc45 protein is able to bind single-stranded, but not double-stranded DNA. Small angle x-ray scattering data are consistent with a model compatible with the crystallographic structure of the RecJ/DHH family members. 相似文献
184.
Locally advanced rectal cancer requires a multidisciplinary management, traditionally based on neo-adjuvant (chemo) radiotherapy, conservative surgery with total mesorectal excision and adjuvant chemotherapy. Despite effective in term of local control, this strategy is linked to a high risk of distant metastasis (up to 30%). In this context, recent published randomized phase III clinical trials have tested the potential benefits with a different sequencing and/or intensification of the standard components of the trimodal therapy.Here, we briefly assess the efficacy and discuss the clinical relevance of total neoadjuvant treatment with a focus on indications and results in the short-course radiotherapy followed by chemotherapy use for this setting of patients. Long term results and additional prospective studies are necessary to more accurately estimate the clinical benefit and further establish the role of total neoadjuvant therapy in locally advanced rectal cancer disease. 相似文献
185.
De novo protein design provides an attractive approach for the construction of models to probe the features required for the function of complex metalloproteins. These minimal models contain the essential elements believed necessary for activity of the protein. In this article, we summarize the design, structure determination, and functional properties of a family of artificial diiron proteins. 相似文献
186.
Martire M D'Amico M Panza E Miceli F Viggiano D Lavergata F Iannotti FA Barrese V Preziosi P Annunziato L Taglialatela M 《Journal of neurochemistry》2007,102(1):179-193
KCNQ2 and KCNQ3 subunits encode for the muscarinic-regulated current (I(KM)), a sub-threshold voltage-dependent K+ current regulating neuronal excitability. In this study, we have investigated the involvement of I(KM) in dopamine (DA) release from rat striatal synaptosomes evoked by elevated extracellular K+ concentrations ([K+]e) and by muscarinic receptor activation. [3H]dopamine ([3H]DA) release triggered by 9 mmol/L [K+]e was inhibited by the I(KM) activator retigabine (0.01-30 micromol/L; Emax = 54.80 +/- 3.85%; IC50 = 0.50 +/- 0.36 micromol/L). The I(KM) blockers tetraethylammonium (0.1-3 mmol/L) and XE-991 (0.1-30 micromol/L) enhanced K+-evoked [3H]DA release and prevented retigabine-induced inhibition of depolarization-evoked [3H]DA release. Retigabine-induced inhibition of K+-evoked [3H]DA release was also abolished by synaptosomal entrapment of blocking anti-KCNQ2 polyclonal antibodies, an effect prevented by antibody pre-absorption with the KCNQ2 immunizing peptide. Furthermore, the cholinergic agonist oxotremorine (OXO) (1-300 micromol/L) potentiated 9 mmol/L [K+]e-evoked [3H]DA release (Emax = 155 +/- 9.50%; EC50 = 25 +/- 1.80 micromol/L). OXO (100 micromol/L)-induced [3H]DA release enhancement was competitively inhibited by pirenzepine (1-10 nmol/L) and abolished by the M3-preferring antagonist 4-diphenylacetoxy N-methylpiperidine methiodide (1 micromol/L), but was unaffected by the M1-selective antagonist MT-7 (10-100 nmol/L) or by Pertussis toxin (1.5-3 microg/mL), which uncouples M2- and M4-mediated responses. Finally, OXO-induced potentiation of depolarization-induced [3H]DA release was not additive to that produced by XE-991 (10 micromol/L), was unaffected by retigabine (10 micromol/L), and was abolished by synaptosomal entrapment of anti-KCNQ2 antibodies. Collectively, these findings indicate that, in rat striatal nerve endings, I(KM) channels containing KCNQ2 subunits regulate depolarization-induced DA release and that I(KM) suppression is involved in the reinforcement of depolarization-induced DA release triggered by the activation of pre-synaptic muscarinic heteroreceptors. 相似文献
187.
Samuele Raccosta Mauro Manno Donatella Bulone Daniela Giacomazza Valeria Militello Vincenzo Martorana Pier Luigi San Biagio 《European biophysics journal : EBJ》2010,39(6):1007-1017
The formation of protein aggregates is important in many fields of life science and technology. The morphological and mechanical
properties of protein solutions depend upon the molecular conformation and thermodynamic and environmental conditions. Non-native
or unfolded proteins may be kinetically trapped into irreversible aggregates and undergo precipitation or gelation. Here,
we study the thermal aggregation of lysozyme in neutral solutions. We characterise the irreversible unfolding of lysozyme
by differential scanning calorimetry. The structural properties of aggregates and their mechanisms of formation with the eventual
gelation are studied at high temperature by spectroscopic, rheological and scattering techniques. The experiments show that
irreversible micron-sized aggregates are organised into larger clusters according to a classical mechanism of diffusion and
coagulation, which leads to a percolative transition at high concentrations. At a smaller length scale, optical and atomic
force microscopy images reveal the existence of compact aggregates, which are the origin of the aggregation irreversibility. 相似文献
188.
Vincenzo Carelli Felice Liberatore Antonio Casini Rosanna Mondelli Alberto Arnone Italo Carelli Giuseppe Rotilio Irene Mavelli 《Bioorganic chemistry》1980,9(3):342-351
The composition and the structure of the product from the known electrochemical dimerization of the NAD+ have been conclusively demonstrated. A detailed analysis of the 1H and 13C nmr spectra has in fact led to the conclusion that the product contains three diastereoisomeric dimers of the 4,4′-tetrahydrobipyridyl type. Furthermore, the cytoplasmic fraction obtained from a standard mitochondrial preparation of rat liver has been shown to catalyze the oxygen uptake by the dimers. A 1 : 1 molar ratio of the reagents in the redox process is indicated by manometric data on oxygen uptake complemented by spectrophotometric analysis of the oxidized substrates, suggesting that H2O2 is the reduction product. NAD+ was identified as the oxidation product by an enzymatic method. 相似文献
189.
Davide Randazzo Emiliana Giacomello Stefania Lorenzini Daniela Rossi Enrico Pierantozzi Bert Blaauw Carlo Reggiani Stephan Lange Angela K. Peter Ju Chen Vincenzo Sorrentino 《The Journal of cell biology》2013,200(4):523-536
Obscurin is a large myofibrillar protein that contains several interacting modules, one of which mediates binding to muscle-specific ankyrins. Interaction between obscurin and the muscle-specific ankyrin sAnk1.5 regulates the organization of the sarcoplasmic reticulum in striated muscles. Additional muscle-specific ankyrin isoforms, ankB and ankG, are localized at the subsarcolemma level, at which they contribute to the organization of dystrophin and β-dystroglycan at costameres. In this paper, we report that in mice deficient for obscurin, ankB was displaced from its localization at the M band, whereas localization of ankG at the Z disk was not affected. In obscurin KO mice, localization at costameres of dystrophin, but not of β-dystroglycan, was altered, and the subsarcolemma microtubule cytoskeleton was disrupted. In addition, these mutant mice displayed marked sarcolemmal fragility and reduced muscle exercise tolerance. Altogether, the results support a model in which obscurin, by targeting ankB at the M band, contributes to the organization of subsarcolemma microtubules, localization of dystrophin at costameres, and maintenance of sarcolemmal integrity. 相似文献
190.
Vincenzo?Rochira Matteo?Fabbi Elena?Valassi Bruno?Madeo Cesare?CaraniEmail author 《Andrologie》2003,13(1):57-61
The role of estrogen on male reproductive function has become clearer in the last decade. During these years the study of
the effect of testosterone, estrogen or an aromatase inhibitor in hypogonadal men provided a first evidence of the effects
of estrogens in the regulation of gonadotropin secretion. At the same time, the development of a line of transgenic male mice
lacking estrogen receptor α, estrogen receptor β or aromatase gene provided further evidence about the role of estrogens not
only in the regulation of gonadotropin secretion, but also on the effects of estrogens on testicular function and development.
A confirmation of these actions of estrogens came from the observation of naturally occurring mutations of the estrogen receptor
and of the aromatase gene in human males. Based on these data it has been demonstrated that estrogens are major regulators
of gonadotropin secretion acting both at pituitary and hypotalamic level. The presence in the human reproductive structures
of estrogen receptor α, estrogen receptor β and the aromatase enzyme indicates the existence of receptor α, estrogen receptor
β or aromatase estrogen actions at this level. Anyway, the precise role of estrogens in testicular development and function
and on the regulation of human spermatogenesis has not yet been precisely clarified. 相似文献