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81.
Cinzia Ciccacci Carlo Perricone Fulvia Ceccarelli Sara Rufini Davide Di Fusco Cristiano Alessandri Francesca Romana Spinelli Enrica Cipriano Giuseppe Novelli Guido Valesini Paola Borgiani Fabrizio Conti 《PloS one》2014,9(11)
Background
Systemic lupus erythematosus (SLE) is an autoimmune disease with complex pathogenesis in which genes and environmental factors are involved. We aimed at analyzing previously identified loci associated with SLE or with other autoimmune and/or inflammatory disorders (STAT4, IL10, IL23R, IRAK1, PSORS1C1, HCP5, MIR146a, PTPN2, ERAP1, ATG16L1, IRGM) in a sample of Italian SLE patients in order to verify or confirm their possible involvement and relative contribution in the disease.Materials and methods
Two hundred thirty-nine consecutive SLE patients and 278 matched healthy controls were enrolled. Study protocol included complete physical examination, and clinical and laboratory data collection. Nineteen polymorphisms were genotyped by allelic discrimination assays. A case-control association study and a genotype-phenotype correlation were performed.Results
STAT4 was the most associated gene [P = 3×10−7, OR = 2.13 (95% CI: 1.59–2.85)]. IL10 confirmed its association with SLE [rs3024505: P = 0.02, OR = 1.52 (95% CI: 1.07–2.16)]. We describe a novel significant association between HCP5 locus and SLE susceptibility [rs3099844: P = 0.01, OR = 2.06 (95% CI: 1.18–3.6)]. The genotype/phenotype correlation analysis showed several associations including a higher risk to develop pericarditis with STAT4, and an association between HCP5 rs3099844 and anti-Ro/SSA antibodies.Conclusions
STAT4 and IL10 confirm their association with SLE. We found that some SNPs in PSORS1C1, ATG16L1, IL23R, PTPN2 and MIR146a genes can determine particular disease phenotypes. HCP5 rs3099844 is associated with SLE and with anti-Ro/SSA. This polymorphism has been previously found associated with cardiac manifestations of SLE, a condition related with anti-Ro/SSA antibodies. Thus, our results may provide new insights into SLE pathogenesis. 相似文献82.
The 5-phospho-α-D-ribose 1-diphosphate (PRPP) metabolite plays essential roles in several biosynthetic pathways, including histidine, tryptophan, nucleotides, and, in mycobacteria, cell wall precursors. PRPP is synthesized from α-D-ribose 5-phosphate (R5P) and ATP by the Mycobacterium tuberculosis prsA gene product, phosphoribosylpyrophosphate synthase (MtPRS). Here, we report amplification, cloning, expression and purification of wild-type MtPRS. Glutaraldehyde cross-linking results suggest that MtPRS predominates as a hexamer, presenting varied oligomeric states due to distinct ligand binding. MtPRS activity measurements were carried out by a novel coupled continuous spectrophotometric assay. MtPRS enzyme activity could be detected in the absence of P(i). ADP, GDP and UMP inhibit MtPRS activity. Steady-state kinetics results indicate that MtPRS has broad substrate specificity, being able to accept ATP, GTP, CTP, and UTP as diphosphoryl group donors. Fluorescence spectroscopy data suggest that the enzyme mechanism for purine diphosphoryl donors follows a random order of substrate addition, and for pyrimidine diphosphoryl donors follows an ordered mechanism of substrate addition in which R5P binds first to free enzyme. An ordered mechanism for product dissociation is followed by MtPRS, in which PRPP is the first product to be released followed by the nucleoside monophosphate products to yield free enzyme for the next round of catalysis. The broad specificity for diphosphoryl group donors and detection of enzyme activity in the absence of P(i) would suggest that MtPRS belongs to Class II PRS proteins. On the other hand, the hexameric quaternary structure and allosteric ADP inhibition would place MtPRS in Class I PRSs. Further data are needed to classify MtPRS as belonging to a particular family of PRS proteins. The data here presented should help augment our understanding of MtPRS mode of action. Current efforts are toward experimental structure determination of MtPRS to provide a solid foundation for the rational design of specific inhibitors of this enzyme. 相似文献
83.
Polverini E Cugini G Annoni F Abbruzzetti S Viappiani C Gensch T 《Biochemistry》2006,45(16):5111-5121
The interaction of nile red (NR) with apomyoglobin (ApoMb) in the native (pH 7) and molten globule (pH 4) states was investigated using experimental and computational methods. NR binds to hydrophobic locations in ApoMb with higher affinity (K(d) = 25 +/- 5 microM) in the native state than in the molten globule state (K(d) = 52 +/- 5 microM). In the molten globule state, NR is located in a more hydrophobic environment. The dye does not bind to the holoprotein, suggesting that the binding site is located at the heme pocket. In addition to monitoring steady-state properties, the fluorescence emission of NR is capable of tracking submillisecond, time-resolved structural rearrangements of the protein, induced by a nanosecond pH jump. Molecular dynamics simulations were run on ApoMb at neutral pH and at pH 4. The structure obtained for the molten globule state is consistent with the experimentally available structural data. The docking of NR with the crystal structure shows that the ligand binds into the binding pocket of the heme group, with an orientation bringing the planar ring system of NR to overlap with the position of two of the heme porphyrin rings in Mb. The docking of NR with the ApoMb structure at pH 4 shows that the dye binds to the heme pocket with a slightly less favorable binding energy, in keeping with the experimental K(d) value. Under these conditions, NR is positioned in a different orientation, reaching a more hydrophobic environment in agreement with the spectroscopic data. 相似文献
84.
Tomás Pellizzaro Pereira Fernanda Plucani do Amaral Pamela Dall’Asta Fábio Cristiano Angonesi Brod Ana Carolina Maisonnave Arisi 《Molecular biotechnology》2014,56(7):660-670
The plant growth promoting bacteria Herbaspirillum seropedicae SmR1 is an endophytic diazotroph found in several economically important crops. Considering that methods to monitor the plant–bacteria interaction are required, our objective was to develop a real-time PCR method for quantification of PGPB H. seropedicae in the rhizosphere of maize seedlings. Primer pairs were designed, and their specificity was verified using DNA from 12 different bacterial species. Ten standard curves of qPCR assay using HERBAS1 primers and tenfold serial dilutions of H. seropedicae SmR1 DNA were performed, and PCR efficiency of 91 % and correlation coefficient of 0.99 were obtained. H. seropedicae SmR1 limit of detection was 101 copies (corresponding to 60.3 fg of bacterial DNA). qPCR assay using HERBAS1 was used to detect and quantify H. seropedicae strain SmR1 in inoculated maize roots, cultivated in vitro and in pots, harvested 1, 4, 7, and 10 days after inoculation. The estimated bacterial DNA copy number per gram of root was in the range 107–109 for plants grown in vitro and it was around 106 for plants grown in pots. Primer pair HERBAS1 was able to quantify H. seropedicae SmR1, and this assay can be useful for monitoring plant–bacteria interaction. 相似文献
85.
Gomes CC Moreira LM Santos VJ Ramos AS Lyon JP Soares CP Santos FV 《Genetics and molecular biology》2011,34(1):116-121
The use of artificial implants provides a palliative or permanent solution for individuals who have lost some bodily function through disease, an accident or natural wear. This functional loss can be compensated for by the use of medical devices produced from special biomaterials. Titanium alloy (Ti-6Al-4V) is a well-established primary metallic biomaterial for orthopedic implants, but the toxicity of the chemical components of this alloy has become an issue of concern. In this work, we used the MTT assay and micronucleus assay to examine the cytotoxicity and genotoxicity, respectively, of an extract obtained from this alloy. The MTT assay indicated that the mitochondrial activity and cell viability of CHO-K1 cells were unaffected by exposure to the extract. However, the micronucleus assay revealed DNA damage and an increase in micronucleus frequency at all of the concentrations tested. These results show that ions released from Ti-6Al-4V alloy can cause DNA and nuclear damage and reinforce the importance of assessing the safety of metallic medical devices constructed from biomaterials. 相似文献
86.
Several voltage-gated ion channels are modulated by clinically relevant doses of general anesthetics. However, the structural basis of this modulation is not well understood. Previous work suggested that n-alcohols and inhaled anesthetics stabilize the closed state of the Shaw2 voltage-gated (Kv) channel (K-Shaw2) by directly interacting with a discrete channel site. We hypothesize that the inhibition of K-Shaw2 channels by general anesthetics is governed by interactions between binding and effector sites involving components of the channel's activation gate. To investigate this hypothesis, we applied Ala/Val scanning mutagenesis to the S4-S5 linker and the post-PVP S6 segment, and conducted electrophysiological analysis to evaluate the energetic impact of the mutations on the inhibition of the K-Shaw2 channel by 1-butanol and halothane. These analyses identified residues that determine an apparent binding cooperativity and residue pairs that act in concert to modulate gating upon anesthetic binding. In some instances, due to their critical location, key residues also influence channel gating. Complementing these results, molecular dynamics simulations and in silico docking experiments helped us visualize possible anesthetic sites and interactions. We conclude that the inhibition of K-Shaw2 by general anesthetics results from allosteric interactions between distinct but contiguous binding and effector sites involving inter- and intrasubunit interfaces. 相似文献
87.
Eduardo Vilanova Cristiano Coutinho Guilherme Maia Paulo A. S. Mourão 《Cell and tissue research》2010,340(3):523-531
Marine sponges (Porifera) display an ancestral type of cell-cell adhesion, based on carbohydrate-carbohydrate interaction.
The aim of the present work was to investigate further details of this adhesion by using, as a model, the in vitro aggregation
of dissociated sponge cells. Our results showed the participation of sulfated polysaccharides in this cell-cell interaction,
as based on the following observations: (1) a variety of sponge cells contained similar sulfated polysaccharides as surface-associated
molecules and as intracellular inclusions; (2) 35S-sulfate metabolic labeling of dissociated sponge cells revealed that the majority (two thirds) of the total sulfated polysaccharide
occurred as a cell-surface-associated molecule; (3) the aggregation process of dissociated sponge cells demanded the active
de novo synthesis of sulfated polysaccharides, which ceased as cell aggregation reached a plateau; (4) the typical well-organized
aggregates of sponge cells, known as primmorphs, contained three cell types showing sulfated polysaccharides on their cell
surface; (5) collagen fibrils were also produced by the primmorphs in order to fill the extracellular spaces of their inner
portion and the external layer surrounding their entire surface. Our data have thus clarified the relevance of sulfated polysaccharides
in this system of in vitro sponge cell aggregation. The molecular basis of this system has practical relevance, since the
culture of sponge cells is necessary for the production of molecules with biotechnological applications. 相似文献
88.
89.
Cristiano S. Mota Maria G. Rivas Carlos D. Brondino Isabel Moura José J. G. Moura Pablo J. González Nuno M. F. S. A. Cerqueira 《Journal of biological inorganic chemistry》2011,16(8):1255-1268
Metal-dependent formate dehydrogenases (Fdh) from prokaryotic organisms are members of the dimethyl sulfoxide reductase family
of mononuclear molybdenum-containing and tungsten-containing enzymes. Fdhs catalyze the oxidation of the formate anion to
carbon dioxide in a redox reaction that involves the transfer of two electrons from the substrate to the active site. The
active site in the oxidized state comprises a hexacoordinated molybdenum or tungsten ion in a distorted trigonal prismatic
geometry. Using this structural model, we calculated the catalytic mechanism of Fdh through density functional theory tools.
The simulated mechanism was correlated with the experimental kinetic properties of three different Fdhs isolated from three
different Desulfovibrio species. Our studies indicate that the C–H bond break is an event involved in the rate-limiting step of the catalytic cycle.
The role in catalysis of conserved amino acid residues involved in metal coordination and near the metal active site is discussed
on the basis of experimental and theoretical results. 相似文献
90.
Cavallari N Frigato E Vallone D Fröhlich N Lopez-Olmeda JF Foà A Berti R Sánchez-Vázquez FJ Bertolucci C Foulkes NS 《PLoS biology》2011,9(9):e1001142
The circadian clock is synchronized with the day-night cycle primarily by light. Fish represent fascinating models for deciphering the light input pathway to the vertebrate clock since fish cell clocks are regulated by direct light exposure. Here we have performed a comparative, functional analysis of the circadian clock involving the zebrafish that is normally exposed to the day-night cycle and a cavefish species that has evolved in perpetual darkness. Our results reveal that the cavefish retains a food-entrainable clock that oscillates with an infradian period. Importantly, however, this clock is not regulated by light. This comparative study pinpoints the two extra-retinal photoreceptors Melanopsin (Opn4m2) and TMT-opsin as essential upstream elements of the peripheral clock light input pathway. 相似文献