974 resultados para 2 Forms


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Background: Peroxiredoxins have diverse functions in cellular defense-signaling pathways. 2-Cys-peroxiredoxins (2-Cys-Prx) reduce H2O2 and alkyl-hydroperoxide. This study describes the purification and characterization of a genuine 2-Cys-Prx from Vigna unguiculata (Vu-2-Cys-Prx). Methods: Vu-2-Cys-Prx was purified from leaves by ammonium sulfate fractionation, chitin affinity and ion exchange chromatography. Results: Vu-2-Cys-Prx reduces H2O2 using NADPH and DTT. Vu-2-Cys-Prx is a 44 kDa (SDS-PAGE)/46 kDa (exclusion chromatography) protein that appears as a 22 kDa molecule under reducing conditions, indicating that it is a homodimer linked intermolecularly by disulfide bonds and has a pI range of 4.56-4.72; its NH2-terminal sequence was similar to 2-Cys-Prx from Phaseolus vulgaris (96%) and Populus tricocarpa (96%). Analysis by ESI-Q-TOF MS/MS showed a molecular mass/pI of 28.622 kDa/5.18. Vu-2-Cys-Prx has 8% alpha-helix, 39% beta-sheet, 22% of turns and 31% of unordered forms. Vu-2-Cys-Prx was heat stable, has optimal activity at pH 7.0, and prevented plasmid DNA degradation. Atomic force microscopy shows that Vu-2-Cys-Prx oligomerized in decamers which might be associated with its molecular chaperone activity that prevented denaturation of insulin and citrate synthase. Its cDNA analysis showed that the redox-active Cys(52) residue and the amino acids Pro(45), Thr(49) and Arg(128) are conserved as in other 2-Cys-Prx. General significance: The biochemical and molecular features of Vu-2-Cys-Prx are similar to other members of 2-Cys-Prx family. To date, only one publication reported on the purification of native 2-Cys-Prx from leaves and the subsequent analysis by N-terminal Edman sequencing, which is crucial for construction of stromal recombinant 2-Cys-Prx proteins. (C) 2012 Elsevier B.V. All rights reserved.

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Gas-phase reactions of model carbosulfonium ions (CH3-S+?=?CH2; CH3CH2-S+?=?CH2 and Ph-S+?=?CH2) and an O-analogue carboxonium ion (CH3-O+?=?CH2) with acyclic (isoprene, 1,3-butadiene, methyl vinyl ketone) and cyclic (1,3-cyclohexadiene, thiophene, furan) conjugated dienes were systematically investigated by pentaquadrupole mass spectrometry. As corroborated by B3LYP/6-311?G(d,p) calculations, the carbosulfonium ions first react at large extents with the dienes forming adducts via simple addition. The nascent adducts, depending on their stability and internal energy, react further via two competitive channels: (1) in reactions with acyclic dienes via cyclization that yields formally [4?+?2+] cycloadducts, or (2) in reactions with the cyclic dienes via dissociation by HSR loss that yields methylenation (net CH+ transfer) products. In great contrast to its S-analogues, CH3-O+?=?CH2 (as well as C2H5-O+?=?CH2 and Ph-O+?=?CH2 in reactions with isoprene) forms little or no adduct and proton transfer is the dominant reaction channel. Isomerization to more acidic protonated aldehydes in the course of reaction seems to be the most plausible cause of the contrasting reactivity of carboxonium ions. The CH2?=?CH-O+?=?CH2 ion forms an abundant [4?+?2+] cycloadduct with isoprene, but similar to the behavior of such alpha,beta-unsaturated carboxonium ions in solution, seems to occur across the C?=?C bond. Copyright (c) 2012 John Wiley & Sons, Ltd.

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Abstract Background The city of Sao Paulo has the highest AIDS case rate, with nearly 60% in Brazil. Despite, several studies involving molecular epidemiology, lack of data regarding a large cohort study has not been published from this city. Objectives This study aimed to describe the HIV-1 subtypes, recombinant forms and drug resistance mutations, according to subtype, with emphasis on subtype C and BC recombinants in the city of São Paulo, Brazil. Study design RNA was extracted from the plasma samples of 302 HIV-1-seropositive subjects, of which 211 were drug-naive and 82 were exposed to ART. HIV-1 partial pol region sequences were used in phylogenetic analyses for subtyping and identification of drug resistance mutations. The envelope gene of subtype C and BC samples was also sequenced. Results From partial pol gene analyses, 239 samples (79.1%) were assigned as subtype B, 23 (7.6%) were F1, 16 (5.3%) were subtype C and 24 (8%) were mosaics (3 CRF28/CRF29-like). The subtype C and BC recombinants were mainly identified in drug-naïve patients (72.7%) and the heterosexual risk exposure category (86.3%), whereas for subtype B, these values were 69.9% and 57.3%, respectively (p = 0.97 and p = 0.015, respectively). An increasing trend of subtype C and BC recombinants was observed (p < 0.01). Conclusion The HIV-1 subtype C and CRFs seem to have emerged over the last few years in the city of São Paulo, principally among the heterosexual population. These findings may have an impact on preventive measures and vaccine development in Brazil.

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This Ph.D. candidate thesis collects the research work I conducted under the supervision of Prof.Bruno Samor´ı in 2005,2006 and 2007. Some parts of this work included in the Part III have been begun by myself during my undergraduate thesis in the same laboratory and then completed during the initial part of my Ph.D. thesis: the whole results have been included for the sake of understanding and completeness. During my graduate studies I worked on two very different protein systems. The theorical trait d’union between these studies, at the biological level, is the acknowledgement that protein biophysical and structural studies must, in many cases, take into account the dynamical states of protein conformational equilibria and of local physico-chemical conditions where the system studied actually performs its function. This is introducted in the introductory part in Chapter 2. Two different examples of this are presented: the structural significance deriving from the action of mechanical forces in vivo (Chapter 3) and the complexity of conformational equilibria in intrinsically unstructured proteins and amyloid formation (Chapter 4). My experimental work investigated both these examples by using in both cases the single molecule force spectroscopy technique (described in Chapter 5 and Chapter 6). The work conducted on angiostatin focused on the characterization of the relationships between the mechanochemical properties and the mechanism of action of the angiostatin protein, and most importantly their intertwining with the further layer of complexity due to disulfide redox equilibria (Part III). These studies were accompanied concurrently by the elaboration of a theorical model for a novel signalling pathway that may be relevant in the extracellular space, detailed in Chapter 7.2. The work conducted on -synuclein (Part IV) instead brought a whole new twist to the single molecule force spectroscopy methodology, applying it as a structural technique to elucidate the conformational equilibria present in intrinsically unstructured proteins. These equilibria are of utmost interest from a biophysical point of view, but most importantly because of their direct relationship with amyloid aggregation and, consequently, the aetiology of relevant pathologies like Parkinson’s disease. The work characterized, for the first time, conformational equilibria in an intrinsically unstructured protein at the single molecule level and, again for the first time, identified a monomeric folded conformation that is correlated with conditions leading to -synuclein and, ultimately, Parkinson’s disease. Also, during the research work, I found myself in the need of a generalpurpose data analysis application for single molecule force spectroscopy data analysis that could solve some common logistic and data analysis problems that are common in this technique. I developed an application that addresses some of these problems, herein presented (Part V), and that aims to be publicly released soon.

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The Ctr family is an essential part of the copper homeostasis machinery and its members share sequence homology and structural and functional features. Higher eukaryotes express two members of this family Ctr1 and Ctr2. Numerous structural and functional studies are available for Ctr1, the only high affinity Cu(I) transporter thus far identified. Ctr1 holigotrimers mediate cellular copper uptake and this protein was demonstrated to be essential for embryonic development and to play a crucial role in dietary copper acquisition. Instead very little is known about Ctr2, it bears structural homology to the yeast vacuolar copper transporter, which mediates mobilization of vacuolar copper stores. Recent studies using over-expressed epitope-tagged forms of human Ctr2 suggested a function as a low affinity copper transporter that can mediate either copper uptake from the extracellular environment or mobilization of lysosomal copper stores. Using an antibody that recognizes endogenous mouse Ctr2, we studied the expression and localization of endogenous mouse Ctr2 in cell culture and in mouse models to understand its regulation and function in copper homeostasis. By immunoblot we observed a regulation of mCtr2 protein levels in a copper and Ctr1 dependent way. Our observations in cells and transgenic mice suggest that lack of Ctr1 induces a strong downregulation of Ctr2 probably by a post-translational mechanism. By indirect immunofluorescence we observed an exclusive intracellular localization in a perinuclear compartment and no co-localization with lysosomal markers. Immunofluorescence experiments in Ctr1 null cells, supported by sequence analysis, suggest that lysosomes may play a role in mCtr2 biology not as resident compartment, but as a degradation site. In appendix a LC-mass method for analysis of algal biotoxins belonging to the family of PsP (paralytic shellfish poisoning) is described.

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This work of thesis involves various aspects of crystal engineering. Chapter 1 focuses on crystals containing crown ether complexes. Aspects such as the possibility of preparing these materials by non-solution methods, i.e. by direct reaction of the solid components, thermal behavior and also isomorphism and interconversion between hydrates are taken into account. In chapter 2 a study is presented aimed to understanding the relationship between hydrogen bonding capability and shape of the building blocks chosen to construct crystals. The focus is on the control exerted by shape on the organization of sandwich cations such as cobalticinium, decamethylcobalticinium and bisbenzenchromium(I) and on the aggregation of monoanions all containing carboxylic and carboxylate groups, into 0-D, 1-D, 2-D and 3-D networks. Reactions conducted in multi-component molecular assemblies or co-crystals have been recognized as a way to control reactivity in the solid state. The [2+2] photodimerization of olefins is a successful demonstration of how templated solid state synthesis can efficiently synthesize unique materials with remarkable stereoselectivity and under environment-friendly conditions. A demonstration of this synthetic strategy is given in chapter 3. The combination of various types of intermolecular linkages, leading to formation of high order aggregation and crystalline materials or to a random aggregation resulting in an amorphous precipitate, may not go to completeness. In such rare cases an aggregation process intermediate between crystalline and amorphous materials is observed, resulting in the formation of a gel, i.e. a viscoelastic solid-like or liquid-like material. In chapter 4 design of new Low Molecular Weight Gelators is presented. Aspects such as the relationships between molecular structure, crystal packing and gelation properties and the application of this kind of gels as a medium for crystal growth of organic molecules, such as APIs, are also discussed.

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Diese Arbeit charakterisiert die Funktion und das Expressionmuster der beiden Zinkfinger-Homöodomänentranskriptionsfaktoren zfh1 und zfh2 von Drosophila melanogaster. Das zfh2 Gen wurde hierbei vor allem molekular charakterisiert. Es wurden eine Vielzahl möglicher Spleißformen identifiziert, welche das regulatorische Potential von Zfh2 enorm erweitern. Für Überexpressionsexperimente wurde zudem erstmalig die cDNA des längsten zfh2-Transkriptes kloniert. Durch Analysen an zfh1 Mutanten konnte gezeigt werden, dass zfh1 sowohl notwendig ist für die embryonale Entwicklung von Motoneuronen, als auch das larvale Wachstum motoneuronaler Endplatten reguliert. Wegen weit reichender pleiotroper Effekte, die zfh1 Funktionsverlustmutanten haben, war es notwendig, neben dem Einsatz hypomorpher Allele auf die Analyse genetischer Mosaike auszuweichen. Die als MARCM-Technik (Lee und Luo, 1999) bezeichnete Methode zur Erzeugung genetischer Mosaike wurde modifiziert um in dieser Arbeit erstmals für die Analyse mutanter larvaler Motoneurone eingesetzt werden zu können. Weitergehend konnte gezeigt werden, dass Zfh1 notwendig ist für die larvale Expression des Neuropeptides FMRFamid. Anhand von Sequenzvergleichen und durch Verwendung eines fmrfamid-Promoterkonstruktes (Benveniste et al., 1998) konnten Hinweise dafür gesammelt werden, dass die Zfh1-abhängige Regulation sehr wahrscheinlich direkter Natur ist. Bei fmrfamid handelt es sich somit um das erste identifizierte neurale Zielgen von Zfh1, an dem sich zudem modellhaft der molekulare Wirkmechanismus von Zfh1 erforschen lässt.

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This current work focused on the simulation of in vivo dissolution and permeation in order to be able to predict the in vivo performance of orally administered fenofibrate immediate release formulations. Therefore, the effects of the formulation surfactants on in vivo solubility and permeation of fenofibrate under physiologically relevant excipient concentrations were emphasized.rnIt was shown that the surfactant sodium dodecyl sulfate (SDS) may decrease rather than increase the solubility of fenofibrate in vivo. This was related to the interference of SDS with the vesicular system of the biorelevant medium, FaSSIFmod, and therefore its solubilization capacity. rnMoreover, in vitro permeation studies revealed that SDS concentrations inversely correlated with fenofibrate permeability. Through combination of the observed permeation and solubility data a good in vitro/in vivo correlation regarding Cmax values in humans could be established for five fenofibrate formulations which were based on the same manufacturing technique.rnBesides the experimental part, the major characteristics and their potential implementation in a dissolution/permeation device were discussed based on the promising realization of the in vitro solubility and permeation methods. rn

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The Gaussian-2, Gaussian-3, complete basis set- (CBS-) QB3, and CBS-APNO methods have been used to calculate ΔH° and ΔG° values for neutral clusters of water, (H2O)n, where n = 2−6. The structures are similar to those determined from experiment and from previous high-level calculations. The thermodynamic calculations by the G2, G3, and CBS-APNO methods compare well against the estimated MP2(CBS) limit. The cyclic pentamer and hexamer structures release the most heat per hydrogen bond formed of any of the clusters. While the cage and prism forms of the hexamer are the lowest energy structures at very low temperatures, as temperature is increased the cyclic structure is favored. The free energies of cluster formation at different temperatures reveal interesting insights, the most striking being that the cyclic trimer, cyclic tetramer, and cyclic pentamer, like the dimer, should be detectable in the lower troposphere. We predict water dimer concentrations of 9 × 1014 molecules/cm3, water trimer concentrations of 2.6 × 1012 molecules/cm3, tetramer concentrations of approximately 5.8 × 1011 molecules/cm3, and pentamer concentrations of approximately 3.5 × 1010 molecules/cm3 in saturated air at 298 K. These results have important implications for understanding the gas-phase chemistry of the lower troposphere.

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The title compound, C(34)H(24)Cl(4)N(4)O(8)S, is a linear penta-cyclic system formed of two substituted benzoxazinyl groups fused to 2-n-butyl-tetra-hydro-thio-phene. The oxazine ring, which is fused to the n-butyl-substituted side of the thio-phene ring, is in a boat conformation. The other fused oxazine ring and the tetra-hydro-thiene ring are each in an envelope conformation. The bridgehead C atom alpha to both the S and N atoms forms the flap of each envelope. This results in a twist of the penta-cyclic system such that the dihedral angle between the terminal dichloro-benzene rings is 82.92 (8)°. In the crystal, inversion-related mol-ecules form a weakly hydrogen-bonded dimer, with two C-H⋯O inter-actions between an H atom on the oxazine ring and an amide O atom. Additionally, C-H⋯O inter-actions occur between an H atom on a screw-related nitro-benzene ring and an O atom on the nitro-benzene ring of one mol-ecule. One of the Cl atoms and the butyl group are disordered over two sets of sites with occupancy ratios of 0.94 (2):0.06 (2) and 0.624 (4):0.376 (4), respectively.

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Reticulate eruptions of vascular origin may represent an underlying arterial, venous, microvascular or combined pathology. In the presence of arterial pathology, individual rings are centred around ascending arterial vessels that supply the corresponding area of skin within an arterial hexagon that clinically presents with a blanched centre. Confluence of multiple arterial hexagons generates a stellate (star-like) pattern. In the presence of a primary venous pathology, individual rings correspond to the underlying reticular veins forming multiple venous rings. Focal involvement of a limited number of vessels presents with a branched (racemosa) configuration while a generalized involvement forms a reticulate (net-like) pattern. 'Livedo' refers to the colour and not the pattern of the eruption. Primary livedo reticularis (Syn. cutis marmorata) is a physiological response to cold and presents with a diffuse blanchable reticulate eruption due to vasospasm of the feeding arteries and sluggish flow and hyperviscosity in the draining veins. Livedo reticularis may be secondary to underlying conditions associated with hyperviscosity of blood. Livedo racemosa is an irregular, branched eruption that is only partially-blanchable or non-blanchable and always signifies a pathological process. Retiform purpura may be primarily inflammatory with secondary haemorrhage or thrombohaemorrhagic, as seen in disseminated intravascular coagulopathy.

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We carry out some computations of vector-valued Siegel modular forms of degree two, weight (k, 2) and level one, and highlight three experimental results: (1) we identify a rational eigenform in a three-dimensional space of cusp forms; (2) we observe that non-cuspidal eigenforms of level one are not always rational; (3) we verify a number of cases of conjectures about congruences between classical modular forms and Siegel modular forms. Our approach is based on Satoh's description of the module of vector-valued Siegel modular forms of weight (k, 2) and an explicit description of the Hecke action on Fourier expansions. (C) 2013 Elsevier Inc. All rights reserved.

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We prospectively investigated the potential of positron emission tomography (PET) using the somatostatin receptor (SSTR) analogue ⁶⁸Ga-DOTATATE and 2-deoxy-2[¹⁸F]fluoro-D-glucose (¹⁸F-FDG) in diffuse parenchymal lung disease (DPLD). Twenty-six patients (mean age 68.9 ± 11.0 years) with DPLD were recruited for ⁶⁸Ga-DOTATATE and ¹⁸F-FDG combined PET/high-resolution computed tomography (HRCT) studies. Ten patients had idiopathic pulmonary fibrosis (IPF), 12 patients had nonspecific interstitial pneumonia (NSIP), and 4 patients had other forms of DPLD. Using PET, the pulmonary tracer uptake (maximum standardized uptake value [SUV(max)]) was calculated. The distribution of PET tracer was compared to the distribution of lung parenchymal changes on HRCT. All patients demonstrated increased pulmonary PET signal with ⁶⁸Ga-DOTATATE and ¹⁸F-FDG. The distribution of parenchymal uptake was similar, with both tracers corresponding to the distribution of HRCT changes. The mean SUV(max) was 2.2 ± 0.7 for ⁶⁸Ga-DOTATATE and 2.8 ± 1.0 (t-test, p  =  .018) for ¹⁸F-FDG. The mean ⁶⁸Ga-DOTATATE SUV(max) in IPF patients was 2.5 ± 0.9, whereas it was 2.0 ± 0.7 (p  =  .235) in NSIP patients. The correlation between ⁶⁸Ga-DOTATATE SUV(max) and gas transfer (transfer factor of the lung for carbon monoxide [TLCO]) was r  =  -.34 (p  =  .127) and r  =  -.49 (p  =  .028) between ¹⁸F-FDG SUV(max) and TLCO. We provide noninvasive in vivo evidence in humans showing that SSTRs may be detected in the lungs of patients with DPLD in a similar distribution to sites of increased uptake of ¹⁸F-FDG on PET.

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In adult skeletal muscle, abluminal sprouting or longitudinal splitting of capillaries can be initiated separately by muscle overload and elevated microcirculation shear stress respectively. In the present study, gene and protein expression patterns associated with the different forms of angiogenesis were examined using a targeted gene array (Superarray), validated by quantitative RT (reverse transcription)-PCR and immunoblots. Sprouting angiogenesis induced large changes in expression levels in genes associated with extracellular matrix remodelling, such as MMP-2 (matrix metalloproteinase-2), TIMP (tissue inhibitor of metalloproteinases), SPARC (secreted protein, acidic and rich in cysteine) and thrombospondin. Changes in neuropilin, midkine and restin levels, which may underpin changes in endothelial morphology, were seen during splitting angiogenesis. Up-regulation of VEGF (vascular endothelial growth factor), Flk-1, angiopoietin-2 and PECAM-1 (platelet/endothelial cell adhesion molecule-1) was seen in both forms of angiogenesis, representing a common angiogenic response of endothelial cells. In conclusion, the present study demonstrates that general angiogenic signals from growth factors can be influenced by the local microenvironment resulting in differing forms of capillary growth to produce a co-ordinated expansion of the vascular bed.

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In the tsetse fly, the protozoan parasite Trypanosoma congolense is covered by a dense layer of glycosylphosphatidylinositol (GPI)-anchored molecules. These include a protease-resistant surface molecule (PRS), which is expressed by procyclic forms early in infection, and a glutamic acid- and alanine-rich protein (GARP), which appears at later stages. Since neither of these surface antigens is expressed at intermediate stages, we investigated whether a GPI-anchored protein of 50 to 58 kDa, previously detected in procyclic culture forms, might constitute the coat of these parasites. We therefore partially purified the protein from T. congolense Kilifi procyclic forms, obtained an N-terminal amino acid sequence, and identified its gene. Detailed analyses showed that the mature protein consists almost exclusively of 13 heptapeptide repeats (EPGENGT). The protein is densely N glycosylated, with up to 13 high-mannose oligosaccharides ranging from Man(5)GlcNAc(2) to Man(9)GlcNAc(2) linked to the peptide repeats. The lipid moiety of the glycosylphosphatidylinositol is composed of sn-1-stearoyl-2-lyso-glycerol-3-HPO(4)-1-(2-O-acyl)-d-myo-inositol. Heavily glycosylated proteins with similar repeats were subsequently identified in T. congolense Savannah procyclic forms. Collectively, this group of proteins was named T. congolense procyclins to reflect their relationship to the EP and GPEET procyclins of T. brucei. Using an antiserum raised against the EPGENGT repeat, we show that T. congolense procyclins are expressed continuously in the fly midgut and thus form the surface coat of cells that are negative for both PRS and GARP.