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Bernardo Celda

Publications and source records attributed to Bernardo Celda.

17 recordsLinked to original sources
PubMed2007

Structural insights into the GTPase domain of Escherichia coli MnmE protein.

The Escherichia coli MnmE protein is a 50-kDa multidomain GTPase involved in tRNA modification. Its homologues in eukaryotes are crucial for mitochondrial respiration and, thus, it is thought that the human protein might be involved in mitochondrial diseases. Unlike Ras, MnmE shows a high intrinsic GTPase activity and requires effective GTP hydrolysis, and not simply GTP binding, to be functionally active. The isolated MnmE G-domain (165 residues) conserves the GTPase activity of the entire protein, suggesting that it contains the catalytic residues for GTP hydrolysis. To explore the GTP hydrolysis mechanism of MnmE, we analyzed the effect of low pH on binding and hydrolysis of GTP, as well as on the formation of a MnmE transition state mimic. GTP hydrolysis by MnmE, but not GTP binding or formation of a complex with mant-GDP and aluminium fluoride, is impaired at acidic pH, suggesting that the chemistry of the transition state mimic is different to that of the true transition state, and that some residue(s), critical for GTP hydrolysis, is severely affected by low pH. We use a nuclear magnetic resonance (NMR)-based approach to get insights into the MnmE structure and properties. The combined use of NMR restraints and homology structural information allowed the determination of the MnmE G-domain structure in its free form. Chemical shift structure-based prediction provided a good basis for structure refinement and validation. Our data support that MnmE, unlike other GTPases, does not use an arginine finger to drive catalysis, although Arg252 may play a role in stabilization of the transition state.

Amino Acid Sequence↗
PubMed2006

Metabolite identification in human liver needle biopsies by high-resolution magic angle spinning 1H NMR spectroscopy.

High-resolution magic angle spinning (HR-MAS) 1H NMR spectroscopy of intact human liver needle biopsies has not been previously reported. HR-MAS NMR spectra collected on 17 specimens with tissue amounts between approximately 0.5 and 12 mg showed very good spectral resolution and signal-to-noise ratios. One-dimensional 1H spectra revealed many intense signals corresponding to cellular metabolites. In addition, some high molecular weight metabolites, such as glycogen and mobile fatty acids, could be observed in some spectra. Resonance assignments for 22 metabolites were obtained by combining the analysis of three different types of 1D 1H spectral editing, such as T2 filtering or the nuclear Overhauser effect and 2D TOCSY and 13C-HSQC spectra. Biochemical stability of the liver tissue during up to 16 h of magic angle spinning at 277 K was studied. Biochemical trends corresponding to the different pathologies were observed, involving free fragments of lipids among other metabolites. NMR signal intensity ratios can be useful for discrimination among non-pathological, hepatitis C affected and cirrhotic liver tissues. Overall, this work demonstrates the applicability of HR-MAS NMR spectroscopy to the biochemical characterization of needle biopsies of the human liver.

Adult↗
PubMed2006

Using the grid to analyze the pharmacokinetic modelling after contrast administration in dynamic MRI.

The analysis of the angiogenesis in hepatic lesions is an important marker of tumour aggressiveness and response to therapy. However, the quantitative analysis of this fact requires a deep knowledge of the hepatic perfusion. The development of pharmacokinetic models constitutes a very valuable tool, but it is computationally intensive. Moreover, abdominal imaging processing increases the computational requirements since the movement of the patient makes images in a time series incomparable, requiring a previous pre-processing. This work presents a Grid environment developed to deal with the computational demand of pharmacokinetic modelling. This article proposes and implements a four-level software architecture that provides a simple interface to the user and deals transparently with the complexity of Grid environment. The four layers implemented are: Grid Layer (the closest to the Grid infrastructure), the Gate-to- Grid (which transforms the user requests to Grid operations), the Web Services layer (which provides a simple, standard and ubiquitous interface to the user) and the Application Layer. An application has been developed on top of this architecture to manage the execution of multi-parametric groups of co-registration actions on a large set of medical images. The execution has been performed on the EGEE Grid infrastructure. The application is platform-independent and can be used from any computer without special requirements.

Computer Simulation↗
PubMed2005

cDNA cloning and functional expression of jerdostatin, a novel RTS-disintegrin from Trimeresurus jerdonii and a specific antagonist of the alpha1beta1 integrin.

Jerdostatin represents a novel RTS-containing short disintegrin cloned by reverse transcriptase-PCR from the venom gland mRNA of the Chinese Jerdons pit viper Trimeresurus jerdonii. The jerdostatins precursor cDNA contained a 333-bp open reading frame encoding a signal peptide, a pre-peptide, and a 43-amino acid disintegrin domain, whose amino acid sequence displayed 80% identity with that of the KTS-disintegrins obtustatin and viperistatin. The jerdostatin cDNA structure represents the first complete open reading frame of a short disintegrin and points to the emergence of jerdostatin from a short-coding gene. The different residues between jerdostatin and obtustatin/viperistatin are segregated within the integrin-recognition loop and the C-terminal tail. Native jerdostatin (r-jerdostatin-R21) and a R21K mutant (r-jerdostatin-K21) were produced in Escherichia coli. In each case, two conformers were isolated. One-dimensional (1)H NMR showed that conformers 1 and 2 of r-jerdostatin-R21 represent, respectively, well folded and unfolded proteins. The two conformers of the wild-type and the R21K mutant inhibited the adhesion of alpha(1)-K562 cells to collagen IV with IC(50) values of 180 and 703 nm, respectively. The IC(50) values of conformers 2 of r-jerdostatin-R21 and r-jerdostatin-K21 were, respectively, 5.95 and 12.5 microm. Neither r-jerdostatin-R21 nor r-jerdostatin-K21 showed inhibitory activity toward other integrins, including alpha(IIb)beta(3), alpha(v)beta(3), alpha(2)beta(1), alpha(5)beta(1), alpha(4)beta(1), alpha(6)beta(1), and alpha(9)beta(1) up to a concentration of 24 mum. Although the RTS motif appears to be more potent than KTS inhibiting the alpha(1)beta(1) integrin, r-jerdostatin-R21 is less active than the KTS-disintegrins, strongly suggesting that substitutions outside the integrin-binding motif and/or C-terminal proteolytic processing are responsible for the decreased inhibitory activity.

Amino Acid Sequence↗
PubMed2005

Snake venom disintegrins: evolution of structure and function.

Disintegrins represent a family of polypeptides present in the venoms of various vipers that selectively block the function of integrin receptors. Here, we review our current view and hypothesis on the emergence and the structural and functional diversification of disintegrins by accelerated evolution and the selective loss of disulfide bonds of duplicated genes. Research on disintegrins is relevant for understanding the biology of viper venom toxins, but also provides information on new structural determinants involved in integrin recognition that may be useful in basic and clinical research. The role of the composition, conformation, and dynamics of the integrin inhibitory loop acting in concert with the C-terminal tail in determining the selective inhibition of integrin receptors is discussed.

Amino Acid Motifs↗
PubMed2005

Conformation and concerted dynamics of the integrin-binding site and the C-terminal region of echistatin revealed by homonuclear NMR.

Echistatin is a potent antagonist of the integrins alpha(v)beta3, alpha5beta1 and alpha(IIb)beta3. Its full inhibitory activity depends on an RGD (Arg-Gly-Asp) motif expressed at the tip of the integrin-binding loop and on its C-terminal tail. Previous NMR structures of echistatin showed a poorly defined integrin-recognition sequence and an incomplete C-terminal tail, which left the molecular basis of the functional synergy between the RGD loop and the C-terminal region unresolved. We report a high-resolution structure of echistatin and an analysis of its internal motions by off-resonance ROESY (rotating-frame Overhauser enhancement spectroscopy). The full-length C-terminal polypeptide is visible as a beta-hairpin running parallel to the RGD loop and exposing at the tip residues Pro43, His44 and Lys45. The side chains of the amino acids of the RGD motif have well-defined conformations. The integrin-binding loop displays an overall movement with maximal amplitude of 30 degrees . Internal angular motions in the 100-300 ps timescale indicate increased flexibility for the backbone atoms at the base of the integrin-recognition loop. In addition, backbone atoms of the amino acids Ala23 (flanking the R24GD26 tripeptide) and Asp26 of the integrin-binding motif showed increased angular mobility, suggesting the existence of major and minor hinge effects at the base and the tip, respectively, of the RGD loop. A strong network of NOEs (nuclear Overhauser effects) between residues of the RGD loop and the C-terminal tail indicate concerted motions between these two functional regions. A full-length echistatin-alpha(v)beta3 docking model suggests that echistatin's C-terminal amino acids may contact alpha(v)-subunit residues and provides new insights to delineate structure-function correlations.

Animals↗
PubMed2004

1H and 13C HR-MAS spectroscopy of intact biopsy samples ex vivo and in vivo 1H MRS study of human high grade gliomas.

High-resolution magic angle spinning (HR-MAS) one- and two-dimensional 1H and 13C nuclear magnetic resonance (NMR) spectroscopy has been used to study intact glioblastoma (GBM) brain tumour tissue. The results were compared with in vitro chemical extract and in vivo spectra. The resolution of 1H one-dimensional, 1H TOCSY and 13C HSQC HR-MAS spectra is comparable to that obtained on perchloric extracts. 13C HSQC HR-MAS spectra have been particularly useful for the identification of 37 different metabolites in intact biopsy tumours, excluding water and DSS components. To our knowledge, this is the most detailed assignment of biochemical compounds obtained in intact human tissue, in particular in brain tumour tissue. Tissue degradation during the recording of the NMR experiment was avoided by keeping the sample at a temperature of 4 degrees C. Detailed metabolical compositions of 10 GBM (six primary, two secondary and two unclassified) were obtained. A good correlation between ex vivo and in vivo MRS has been found.

Adult↗
PubMed2003

Spectroscopic axonal damage of the right locus coeruleus relates to selective attention impairment in early stage relapsing-remitting multiple sclerosis.

Lower levels of N-acetylaspartate (NAA), a marker of axonal damage, have been found in the normal-appearing white matter (NAWM) of relapsing-remitting multiple sclerosis (RRMS) patients with low physical disability. However, its relation to the clinical status of these patients remains unclear. We explored the association between NAA levels [normalized to creatine (Cr), NAA/Cr] and a cognitive feature that is not measured by the standard scales that address functional disability [e.g. Expanded Disability Scale Score (EDSS)] in early RRMS. Given that a considerable number of RRMS patients present attentional dysfunction early in the disease and assuming a functional-anatomical oriented guide, it was hypothesized that patients with worse attentional performance would show lower NAWM NAA/Cr values in the locus coeruleus nuclei of the pontine ascendant reticular activating system. Proton magnetic resonance spectroscopy (1H-MRS) examinations with concurrent clinical evaluation were acquired for 19 RRMS patients with a mean evolution time of 24 months (range 10-60) and mild disability (EDSS 0-3.5, median = 1). 1H-MRS was obtained with spectroscopic imaging and measures were taken from the right and left hemipons. Attention was measured by means of the dichotic listening (DL) paradigm to increase the sensitivity of the testing to subtle attentional deficits. A consonant-vowel DL test was measured with and without attentional instructions. For the attentional condition, the test was digitally manipulated to cue automatically to the ear to be attended, thus allowing the obtention of both a linguistic lateralization index (LI) and an index of integrity of attentional shifts (ASI). Attentional impairment was demonstrated in 47.3% of the patients. Pontine NAA/Cr levels accounted for 39% of the ASI variability (beta = 0.65, P < 0.002) but did not relate to the LI. Moreover, when NAA/Cr levels were considered separately as left and right hemipons values in a multivariate stepwise linear regression model, the right NAA/Cr ratio alone explained 43% of the ASI variability (beta = 0.68, P < 0.001). Since the RRMS patients with greater attentional disturbances exhibited the lowest NAA/Cr levels, it is concluded that NAA provides a specific measure of pathological changes that are also relevant for cognitive functions. The use of both 1H-MRS and DL showed the connection between axonal damage at right locus coeruleus and auditive selective attention dysfunction in early-stage RRMS.

Adult↗
PubMed2003

Concerted motions of the integrin-binding loop and the C-terminal tail of the non-RGD disintegrin obtustatin.

Obtustatin is a potent and selective inhibitor of the alpha1beta1 integrin in vitro and of angiogenesis in vivo. It possesses an integrin recognition loop that harbors, in a lateral position, the inhibitory 21KTS23 motif. We report an analysis of the dynamics of the backbone and side-chain atoms of obtustatin by homonuclear NMR methods. Angular mobility has been calculated for 90 assigned cross-peaks from 22 off-resonance rotating frame nuclear Overhauser effect spectroscopy spectra recorded at three magnetic fields. Our results suggest that the integrin binding loop and the C-terminal tail display concerted motions, which can be interpreted by hinge effects. Among the integrin-binding motif, threonine 22 and serine 23 exhibit the lowest and the highest side-chain flexibility, respectively. It is noteworthy that the side chain of threonine 22 is not solvent-exposed, although based on synthetic peptides it appears to be the most critical residue for the inhibitory activity of obtustatin on the binding of integrin alpha1beta1 to collagen IV. Instead, the side chain of threonine 22 is oriented toward the loop center and hydrogen-bonded to residues Thr25 and Ser26. This network of interactions explains the restrained mobility of threonine 22 and suggests that its functional importance lies in maintaining the active conformation of the alpha1beta1 inhibitory loop.

Alanine↗
PubMed2003

NMR solution structure of the non-RGD disintegrin obtustatin.

The solution structure of obtustatin, a novel non-RGD disintegrin of 41 residues isolated from Vipera lebetina obtusa venom, and a potent and selective inhibitor of the adhesion of integrin alpha(1)beta(1) to collagen IV, has been determined by two-dimensional nuclear magnetic resonance. Almost the whole set of chemical shifts for 1H, 13C and 15N were assigned at natural abundance from 2D homonuclear and heteronuclear 500 MHz, 600 MHz and 800 MHz spectra at pH 3.0 recorded at 298 K and 303 K. Final structural constraints consisted of 302 non-redundant NOE (95 long-range, 60 medium, 91 sequential and 56 intra-residue), four disulfide bond distances, five chi1 dihedral angles and four hydrogen bonds. The 20 conformers with lowest total energy had no NOE violations greater than 0.35A or dihedral angle violations greater than 12 degrees. The average root-mean-square deviation (RMSD) for backbone atoms of all residues among the 20 conformers was 1.1A and 0.6A for the 29 best-defined residues. Obtustatin lacks any secondary structure. Compared to all known disintegrin structures in which the RGD motif is located at the apex of an 11 residue hairpin loop, the active KTS tripeptide of obtustatin is oriented towards a side of its nine residue integrin-binding loop. The C-terminal tail is near to the active loop, and these two structural elements display the largest atomic displacements due to local conformational disorder. Double cross-peaks for W20, Y28 and H27 in the aromatic region of TOCSY spectra, local RMSD values for these residues, and positive cross-peaks in a ROESY spectrum (600 MHz, 100 ms mixing time), suggest that these residues act as a hinge allowing for the overall flexibility of the entire integrin-binding loop. These distinct structural features, along with its different electrostatic surface potential in relation to other known disintegrins, may confer to obtustatin its reported alpha(1)beta(1) integrin inhibitory selectivity.

Amino Acid Sequence↗
PubMed2003

NMR study of hexanucleotide d(CCGCGG)2 containing two triplet repeats of fragile X syndrome.

Long repeated stretches of d(CCG) and tri-nucleotide are crucial mutations that cause hereditary forms of mental retardation (fragile X-syndrome). Moreover, the alternating (CG) di-nucleotide is one of the candidates for Z-DNA conformation. Solution NMR structure of d(CCGCGG)(2) has been solved and is discussed. The determined NMR solution structure is a distorted highly bent B-DNA conformation with increased flexibility in both terminal residues. This conformation differs significantly from the Z-DNA tetramer structure reported for the same hexamer in the crystal state at similar ionic strength by Malinina and co-workers. Crystal structure of d(CCGCGG)(2) at high salt concentration includes a central alternating tetramer in Z-DNA conformation, while the initial cytosine swings out and forms a Watson-Crick base-pair with the terminal guanine of a symmetry-related molecule. In solution, NMR data for sugar ring puckering combined with restrained molecular dynamics simulations starting from a Z-DNA form show that terminal furanose residues could adopt the conformation required for aromatic bases swinging out. Therefore, tetramer formation could be considered possible once the hexanucleotide had previously adopted the Z-DNA form. This work gives some insight into correlations between anomalous crystal structures and their accessibility in the solution state.

Computer Simulation↗
PubMed2003

Study of electrostatic potential surface distribution of wild-type plastocyanin Synechocystis solution structure determined by homonuclear NMR.

Plastocyanin is a small (approximately 10 kDa), type I blue copper protein that works as an electron donor to photosystem I from cytochrome f in both chloroplast systems and in some strains of cyanobacteria. Comparative studies of the kinetic mechanisms of plastocyanins in different organisms show that the electron transfer from photosystem I happens by simple collision in cyanobacteria but through a intermediate transition complex in green algae and superior plants. Previous work has proved that this effect cannot be explained by structural variations across the different plastocyanins but it can be explained by differences in the electrostatic potential distribution at the protein surface. In that case, minor conformational errors at the amino acid side chain level may imply an important effect in the electrostatic potential distribution calculation. In this work we present a high resolution study of side chain conformation by homonuclear NMR for the reduced wild-type plastocyanin Synechocystis using intensity ratios for 2D-NOESY and 2D-H,H-TOCSY cross peaks at different mixing times. We also present the corresponding comparison with different plastocyanin structures and the effect in the electrostatic potential distribution at the protein surface. We discuss the importance of indirect J-coupling information from TOCSY-type experiments as complement for intraresidue distances derived from NOESY experiments in the determination of side chain orientation and stereo-specific assignments.

Cyanobacteria↗
PubMed2003

Amino acid sequence and homology modeling of obtustatin, a novel non-RGD-containing short disintegrin isolated from the venom of Vipera lebetina obtusa.

Disintegrins represent a group of cysteine-rich peptides occurring in Crotalidae and Viperidae snake venoms, and are potent antagonists of several integrin receptors. A novel disintegrin, obtustatin, was isolated from the venom of the Vipera lebetina obtusa viper, and represents the first potent and selective inhibitor of the binding of integrin alpha(1)beta(1) to collagen IV. The primary structure of obtustatin contains 41 amino acids and is the shortest disintegrin described to date. Obtustatin shares the pattern of cysteines of other short disintegrins. However, in contrast to known short disintegrins, the integrin-binding loop of obtustatin is two residues shorter and does not express the classical RGD sequence. Using synthetic peptides, a KTS motif was identified as the integrin-binding sequence. A three-dimensional model of obtustatin, built by homology-modeling structure calculations using different templates and alignments, strongly indicates that the novel KTS motif may reside at the tip of a flexible loop.

Amino Acid Sequence↗