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Biomedical subjects

M Rico

Publications and source records attributed to M Rico.

At least 37 records · Page 2Linked to original sources
PubMed2000

Bacteriocin AS-48, a microbial cyclic polypeptide structurally and functionally related to mammalian NK-lysin.

The solution structure of bacteriocin AS-48, a 70-residue cyclic polypeptide from Enterococcus faecalis, consists of a globular arrangement of five alpha-helices enclosing a compact hydrophobic core. The head-to-tail union lies in the middle of helix 5, a fact that is shown to have a pronounced effect on the stability of the three-dimensional structure. Positive charges in the side chains of residues in helix 4 and in the turn linking helix 4 to helix 5 form a cluster that most probably determine its antibacterial activity by promoting pore formation in cell membranes. A similar five-helix structural motif has been found in the antimicrobial NK-lysin, an effector polypeptide of T and natural killer (NK) cells. Bacteriocin AS-48 lacks the three disulfide bridges characteristic of the saposin fold present in NK-lysin, and has no sequence homology with it. Nevertheless, the similar molecular architecture and high positive charge strongly suggest a common mechanism of antibacterial action.

Bacterial Proteins↗
PubMed2000

Structural differences between Saccharomyces cerevisiae ribosomal stalk proteins P1 and P2 support their functional diversity.

The eukaryotic acidic P1 and P2 proteins modulate the activity of the ribosomal stalk but playing distinct roles. The aim of this work was to analyze the structural features that are behind their different function. A structural characterization of Saccharomyces cerevisaie P1 alpha and P2 beta proteins was performed by circular dichroism, nuclear magnetic resonance, fluorescence spectroscopy, thermal denaturation, and protease sensitivity. The results confirm the low structure present in both proteins but reveal clear differences between them. P1 alpha shows a virtually unordered secondary structure with a residual helical content that disappears below 30 degrees C and a clear tendency to acquire secondary structure at low pH and in the presence of trifluoroethanol. In agreement with this higher disorder P1 alpha has a fully solvent-accessible tryptophan residue and, in contrast to P2 beta, is highly sensitive to protease degradation. An interaction between both proteins was observed, which induces an increase in the global secondary structure content of both proteins. Moreover, mixing of both proteins causes a shift of the P1 alpha tryptophan 40 signal, pointing to an involvement of this region in the interaction. This evidence directly proves an interaction between P1 alpha and P2 beta before ribosome binding and suggests a functional complementation between them. On a whole, the results provide structural support for the different functional roles played by the proteins of the two groups showing, at the same time, that relatively small structural differences between the two stalk acidic protein types can result in significant functional changes.

Amino Acid Sequence↗
PubMed2000

The highly refined solution structure of the cytotoxic ribonuclease alpha-sarcin reveals the structural requirements for substrate recognition and ribonucleolytic activity.

alpha-Sarcin selectively cleaves a single phosphodiester bond in a universally conserved sequence of the major rRNA, that inactivates the ribosome. The elucidation of the three-dimensional solution structure of this 150 residue enzyme is a crucial step towards understanding alpha-sarcin's conformational stability, ribonucleolytic activity, and its exceptionally high level of specificity. Here, the solution structure has been determined on the basis of 2658 conformationally relevant distances restraints (including stereoespecific assignments) and 119 torsional angular restraints, by nuclear magnetic resonance spectroscopy methods. A total of 60 converged structures have been computed using the program DYANA. The 47 best DYANA structures, following restrained energy minimization by GROMOS, represent the solution structure of alpha-sarcin. The resulting average pairwise root-mean-square-deviation is 0.86 A for backbone atoms and 1.47 A for all heavy atoms. When the more variable regions are excluded from the analysis, the pairwise root-mean-square deviation drops to 0.50 A and 1.00 A, for backbone and heavy atoms, respectively. The alpha-sarcin structure is similar to that reported for restrictocin, although some differences are clearly evident, especially in the loop regions. The average rmsd between the structurally aligned backbones of the 47 final alpha-sarcin structures and the crystal structure of restrictocin is 1.46 A. On the basis of a docking model constructed with alpha-sarcin solution structure and the crystal structure of a 29-nt RNA containing the sarcin/ricin domain, the regions in the protein that could interact specifically with the substrate have been identified. The structural elements that account for the specificity of RNA recognition are located in two separate regions of the protein. One is composed by residues 51 to 55 and loop 5, and the other region, located more than 11 A away in the structure, is the positively charged segment formed by residues 110 to 114.

Allergens↗
PubMed2000

1H NMR structural characterization of a nonmitogenic, vasodilatory, ischemia-protector and neuromodulatory acidic fibroblast growth factor.

A shortened genetically engineered form of acidic fibroblast growth factor (aFGF), that includes amino acids 28-154 of the full-length sequence (154 residues) plus Met in substitution of Leu27, does not induce cell division even though it is recognized by the cell membrane receptor, triggers the early mitogenic events, and retains the neuromodulatory, vasoactive, and cardio- and neuroprotective properties of the native full-length molecule. Taken together, these properties make this truncated aFGF a promising compound in the treatment of a wide assortment of neurological and cardiovascular pathologies where aFGF mitogenic activity is dispensable. Differences in biological activities between the shortened aFGF and the wild-type form have been attributed to lack of stability, and to the specific amino acid sequence missing at the N-terminus. Here we show that this shortened aFGF form has a three-dimensional structure even more stable than the wild-type protein at the mitogenic assay conditions; that this structure is similar to that of the wild type except at site 1 of interaction with the cell membrane receptor; that its lack of mitogenic activity cannot be attributed to the specific missing sequence; and that the vasodilatory activity of aFGF seems impaired by alterations of the three-dimensional structure of site 2 of interaction with the cell membrane receptor.

Amino Acid Sequence↗
PubMed2000

Role of haemodialysis and hepatitis c virus infection in spontaneous and induced cytokine production of patients with chronic renal disease.

Cytokines modulate general and virus infection-related host immune responses. We have investigated cytokine responses in chronic renal disease patients with regard to haemodialysis and hepatitis C virus (HCV) infection. Compared with healthy subjects with normal renal function (n=15), non-dialyzed/renal disease individuals without HCV infection (n=11) showed increased production of tumour necrosis factor (TNF)-alpha, interleukin (IL-)6, IL-10, interferon (IFN-)gamma and IL-12 by blood mononuclear cells (P<0.05). These inflammatory cytokine responses were abolished in haemodialysis patients (n=37;P<0.05), except for IL-12. This hyporesponsiveness in haemodialysis patients was more evident in stimulatory conditions, as shown by the consistent inhibition of IFN-gamma production, and the failure of exogenous IFN-gamma to prime for IL-12 inducibility (P<0.01). The disturbed cytokine response appeared to focus in the T-helper lymphocyte phenotype 1 (Th(1)) because the stimulation of IL-6 and IL-10 (Th(2)phenotype cytokines) was not impaired. The pattern of response was similar among haemodialysis patients with (n=24) or without (n=13) HCV infection. However, HCV-positive haemodialysis patients had a blunted TNF-alpha response (P<0.05) and failed to increase the stimulated IFN-gamma and IL-12 production (P<0.01) compared with chronic hepatitis C patients without renal disease (n=25). On the contrary, IL-10 stimulation was higher in HCV-positive haemodialysis patients (P<0.01). These results disclose the presence in haemodialysis patients of markedly abnormal general and HCV infection-related cytokine responses; the inhibitory alterations appear to affect predominantly the stimulated responses via the Th(1)subset and its relationship with monocyte response with possible pathogenic and therapeutic implications.

Chronic Disease↗
PubMed2000

Position effect of cross-strand side-chain interactions on beta-hairpin formation.

Previous conformational analysis of 10-residue linear peptides enabled us to identify some cross-strand side-chain interactions that stabilize beta-hairpin conformations. The stabilizing influence of these interactions appeared to be greatly reduced when the interaction was located at the N- and C-termini of these 10-residue peptides. To investigate the effect of the position relative to the turn of favorable interactions on beta-hairpin formation, we have designed two 15-residue beta-hairpin forming peptides with the same residue composition and differing only in the location of two residues within the strand region. The conformational properties of these two peptides in aqueous solution were studied by 1H and 13C NMR. Differences in the conformational behavior of the two designed 15-residue peptides suggest that the influence of stabilizing factors for beta-hairpin formation, in particular, cross-strand side-chain interactions, depends on their proximity to the turn. Residues adjacent to the turn are most efficient in that concern. This result agrees with the proposal that the turn region acts as the driving force in beta-hairpin folding.

Amino Acid Sequence↗
PubMed2000

A helix-turn motif in the C-terminal domain of histone H1.

The structural study of peptides belonging to the terminal domains of histone H1 can be considered as a step toward the understanding of the function of H1 in chromatin. The conformational properties of the peptide Ac-EPKRSVAFKKTKKEVKKVATPKK (CH-1), which belongs to the C-terminal domain of histone H1(o) (residues 99-121) and is adjacent to the central globular domain of the protein, were examined by means of 1H-NMR and circular dichroism. In aqueous solution, CH-1 behaved as a mainly unstructured peptide, although turn-like conformations in rapid equilibrium with the unfolded state could be present. Addition of trifluoroethanol resulted in a substantial increase of the helical content. The helical limits, as indicated by (i,i + 3) nuclear Overhauser effect (NOE) cross correlations and significant up-field conformational shifts of the C(alpha) protons, span from Pro100 to Val116, with Glu99 and Ala117 as N- and C-caps. A structure calculation performed on the basis of distance constraints derived from NOE cross peaks in 90% trifluoroethanol confirmed the helical structure of this region. The helical region has a marked amphipathic character, due to the location of all positively charged residues on one face of the helix and all the hydrophobic residues on the opposite face. The peptide has a TPKK motif at the C-terminus, following the alpha-helical region. The observed NOE connectivities suggest that the TPKK sequence adopts a type (I) beta-turn conformation, a sigma-turn conformation or a combination of both, in fast equilibrium with unfolded states. Sequences of the kind (S/T)P(K/R)(K/R) have been proposed as DNA binding motifs. The CH-1 peptide, thus, combines a positively charged amphipathic helix and a turn as potential DNA-binding motifs.

Amino Acid Sequence↗
PubMed2000

An integrated program for evidence-based medicine in medical school.

To provide optimal care for their patients, clinicians must be able to locate and interpret the most current literature. Teaching the necessary skills to medical students is essential, if we wish to train clinicians to be able to keep up with the expansion of biomedical knowledge for their entire working lives. In this paper, we describe our school's four-year curriculum in evidence-based medicine and the performance of three senior classes on the summative evaluation exercise devised to measure the program's success.

Curriculum↗
PubMed1999

Thermodynamic analysis of the structural stability of phage 434 Cro protein.

Thermodynamic parameters describing the phage 434 Cro protein have been determined by calorimetry and, independently, by far-UV circular dichroism (CD) measurements of isothermal urea denaturations and thermal denaturations at fixed urea concentrations. These equilibrium unfolding transitions are adequately described by the two-state model. The far-UV CD denaturation data yield average temperature-independent values of 0.99 +/- 0.10 kcal mol(-)(1) M(-)(1) for m and 0.98 +/- 0.05 kcal mol(-)(1) K(-)(1) for DeltaC(p)()(,U), the heat capacity change accompanying unfolding. Calorimetric data yield a temperature-independent DeltaC(p)()(,U) of 0.95 +/- 0.30 kcal mol(-)(1) K(-)(1) or a temperature-dependent value of 1.00 +/- 0.10 kcal mol(-)(1) K(-)(1) at 25 degrees C. DeltaC(p)()(,U) and m determined for 434 Cro are in accord with values predicted using known empirical correlations with structure. The free energy of unfolding is pH-dependent, and the protein is completely unfolded at pH 2.0 and 25 degrees C as judged by calorimetry or CD. The stability of 434 Cro is lower than those observed for the structurally similar N-terminal domain of the repressor of phage 434 (R1-69) or of phage lambda (lambda(6)(-)(85)), but is close to the value reported for the putative monomeric lambda Cro. Since a protein's structural stability is important in determining its intracellular stability and turnover, the stability of Cro relative to the repressor could be a key component of the regulatory circuit controlling the levels and, consequently, the functions of the two proteins in vivo.

Bacteriophage lambda↗
PubMed1999

Hydrogen exchange in ribonuclease A and ribonuclease S: evidence for residual structure in the unfolded state under native conditions.

Two-dimensional NMR spectroscopy has been used to monitor the exchange of backbone amide protons in ribonuclease A (RNase A) and its subtilisin-cleaved form, ribonuclease S (RNase S). Exchange measurements at two different pH values (5.4 and 6.0) show that the exchange process occurs according to the conditions of the EX2 limit. Differential scanning calorimetry measurements have been carried out in 2H2O under conditions analogous to those used in the NMR experiments in order to determine the values of DeltaCp, DeltaHu and Tm, corresponding to the thermal denaturation of both proteins. For the amide protons of a large number of residues in RNase A, the free energies at 25 degreesC for exchange competent unfolding processes are much lower than the calorimetric denaturation free energies, thus showing that exchange occurs through local fluctuations in the native state. For 20 other protons, the cleavage reaction had approximately the same effect on the exchange rate constants than on the equilibrium constant for unfolding, indicating that those protons exchange by global unfolding. There is a good agreement between the residues to which these protons belong and those involved in the putative folding nucleation site identified by quench-flow NMR studies. The unfolding free energies of the slowest exchanging protons, DeltaGex, as evaluated from exchange data, are much larger than the calorimetric free energies of unfolding, DeltaGu. Given the agreement between DeltaDeltaGex(A-S), the difference in free energy from exchange for a given proton of the two proteins, and DeltaDeltaGu(A-S), the difference in the calorimetric free energy of the two proteins, the discrepancy indicates that the intrinsic exchange rates in the unfolded state of those protons cannot be approximated by those measured in short unstructured peptides and, consequently, exchange for those protons in RNase A and S must occur through a rather structured denatured state.

Amino Acid Sequence↗
PubMed1999

Texture segregation shows only a very small lower-hemifield advantage.

Possible hemifield differences in texture segregation were investigated for both simple (Fourier, linear) and complex (non-Fourier, second-order) texture channels. There was only a very small lower-field advantage for texture segregation, consistent with the notion that the major processing in texture segregation is quite low level, perhaps V1. Complex-channel tasks do not show larger hemifield asymmetries than do simple-channel tasks, which suggests that the processes in complex texture channels are not higher level than those in simple.

Adolescent↗
PubMed1999

In vitro effect of amantadine and interferon alpha-2a on hepatitis C virus markers in cultured peripheral blood mononuclear cells from hepatitis C virus-infected patients.

The effects of amantadine (1-5 microM) and interferon alpha (IFNalpha)-2a alone (1000 IU/ml) and combined, have been studied in cultured peripheral blood mononuclear cells (PBMC) from 15 chronic hepatitis C patients and ten healthy donors. Amantadine itself did not affect cell viability and had minor effects on the response to mitogens by PBMC. Four patients (27%), but no donors, had hepatitis C virus (HCV) core and NS3-specific proliferative responses. Amantadine suppressed these responses in all cases and its antiproliferative effect was greater than that of IFNalpha (Mann-Whitney's U-test: P < 0.05 in both cases). All PBMC cultures from patients, but none from donors, were HCV RNA positive. Amantadine alone or combined with IFNalpha dose-dependently reduced HCV RNA content in individual PBMC (Wilcoxon's signed rank test: 1 microM, P < 0.05; 2 microM, P < 0.02; and 5 microM, P = 0.16) with respect to untreated cultures. In addition, 7, 13 and 20% of PBMC cultures became HCV RNA negative with 2 microM amantadine alone, IFNalpha alone and their combination, respectively. Finally, in contrast to IFNalpha, amantadine did not modify expression of 2',5'-oligoadenylate synthetase activity or the spontaneous or mitogen-stimulated IFNgamma and interleukin 10 production. In conclusion, these effects in PBMC from HCV patients suggest that the amantadine/IFNalpha combination might be considered a therapeutic option for treating chronic hepatitis C patients.

2',5'-Oligoadenylate Synthetase↗
PubMed1999

The turn sequence directs beta-strand alignment in designed beta-hairpins.

A previous NMR investigation of model decapeptides with identical beta-strand sequences and different turn sequences demonstrated that, in these peptide systems, the turn residues played a more predominant role in defining the type of beta-hairpin adopted than cross-strand side-chain interactions. This result needed to be tested in longer beta-hairpin forming peptides, containing more potentially stabilizing cross-strand hydrogen bonds and side-chain interactions that might counterbalance the influence of the turn sequence. In that direction, we report here on the design and 1H NMR conformational study of three beta-hairpin forming pentadecapeptides. The design consists of adding two and three residues at the N- and C-termini, respectively, of the previously studied decapeptides. One of the designed pentadecapeptides includes a potentially stabilizing R-E salt bridge to investigate the influence of this interaction on beta-hairpin stability. We suggest that this peptide self-associates by forming intermolecular salt bridges. The other two pentadecapeptides behave as monomers. A conformational analysis of their 1H NMR spectra reveals that they adopt different types of beta-hairpin structure despite having identical strand sequences. Hence, the beta-turn sequence drives beta-hairpin formation in the investigated pentadecapeptides that adopt beta-hairpins that are longer than the average protein beta-hairpins. These results reinforce our previous suggestion concerning the key role played by the turn sequence in directing the kind of beta-hairpin formed by designed peptides.

Amino Acid Sequence↗
PubMed1999

Helicity of alpha(404-451) and beta(394-445) tubulin C-terminal recombinant peptides.

We have investigated the solution conformation of the functionally relevant C-terminal extremes of alpha- and beta-tubulin, employing the model recombinant peptides RL52alpha3 and RL33beta6, which correspond to the amino acid sequences 404-451(end) and 394-445(end) of the main vertebrate isotypes of alpha- and beta-tubulin, respectively, and synthetic peptides with the alpha-tubulin(430-443) and beta-tubulin(412-431) internal sequences. Alpha(404-451) and beta(394-445) are monomeric in neutral aqueous solution (as indicated by sedimentation equilibrium), and have circular dichroism (CD) spectra characteristic of nearly disordered conformation, consistent with low scores in peptide helicity prediction. Limited proteolysis of beta(394-445) with subtilisin, instead of giving extensive degradation, resulted in main cleavages at positions Thr409-Glu410 and Tyr422-Gln423-Gln424, defining the proteolysis resistant segment 410-422, which corresponds to the central part of the predicted beta-tubulin C-terminal helix. Both recombinant peptides inhibited microtubule assembly, probably due to sequestration of the microtubule stabilizing associated proteins. Trifluoroethanol (TFE)-induced markedly helical CD spectra in alpha(404-451) and beta(394-445). A substantial part of the helicity of beta(394-445) was found to be in the CD spectrum of the shorter peptide beta(412-431) with TFE. Two-dimensional 1H-NMR parameters (nonsequential nuclear Overhauser effects (NOE) and conformational C alphaH shifts) in 30% TFE permitted to conclude that about 25% of alpha(404-451) and 40% of beta(394-451) form well-defined helices encompassing residues 418-432 and 408-431, respectively, flanked by disordered N- and C-segments. The side chains of beta(394-451) residues Leu418, Val419, Ser420, Tyr422, Tyr425, and Gln426 are well defined in structure calculations from the NOE distance constraints. The apolar faces of the helix in both alpha and beta chains share a characteristic sequence of conserved residues Ala,Met(+4),Leu(+7),Tyr(+11). The helical segment of alpha(404-451) is the same as that described in the electron crystallographic model structure of alphabeta-tubulin, while in beta(394-451) it extends for nine residues more, supporting the possibility of a functional coil --> helix transition at the C-terminus of beta-tubulin. These peptides may be employed to construct model complexes with microtubule associated protein binding sites.

Amino Acid Sequence↗
PubMed1999

De novo design of a monomeric three-stranded antiparallel beta-sheet.

Here we describe the NMR conformational study of a 20-residue linear peptide designed to fold into a monomeric three-stranded antiparallel beta-sheet in aqueous solution. Experimental and statistical data on amino acid beta-turn and beta-sheet propensities, cross-strand side-chain interactions, solubility criteria, and our previous experience with beta-hairpins were considered for a rational selection of the peptide sequence. Sedimentation equilibrium measurements and NMR dilution experiments provide evidence that the peptide is monomeric. Analysis of 1H and 13C-NMR parameters of the peptide, in particular NOEs and chemical shifts, and comparison with data obtained for two 12-residue peptides encompassing the N- and C-segments of the designed sequence indicates that the 20-residue peptide folds into the expected conformation. Assuming a two-state model, the exchange kinetics between the beta-sheet and the unfolded peptide molecules is in a suitable range to estimate the folding rate on the basis of the NMR linewidths of several resonances. The time constant for the coil-beta-sheet transition is of the order of several microseconds in the designed peptide. Future designs based on this peptide system are expected to contribute greatly to our knowledge of the many factors involved in beta-sheet formation and stability.

Amino Acids↗
PubMed1999

Folding propensities of synthetic peptide fragments covering the entire sequence of phage 434 Cro protein.

The phage 434 Cro protein, the N-terminal domain of its repressor (R1-69) and that of phage lambda (lambda6-85) constitute a group of small, monomeric, single-domain folding units consisting of five helices with striking structural similarity. The intrinsic helix stabilities in lambda6-85 have been correlated to its rapid folding behavior, and a residual hydrophobic cluster found in R1-69 in 7 M urea has been proposed as a folding initiation site. To understand the early events in the folding of 434 Cro, and for comparison with R1-69 and lambda6-85, we examined the conformational behavior of five peptides covering the entire 434 Cro sequence in water, 40% (by volume) TFE/water, and 7 M urea solutions using CD and NMR. Each peptide corresponds to a helix and adjacent residues as identified in the native 434 Cro NMR and crystal structures. All are soluble and monomeric in the solution conditions examined except for the peptide corresponding to the 434 Cro helix 4, which has low water solubility. Helix formation is observed for the 434 Cro helix 1 and helix 2 peptides in water, for all the peptides in 40% TFE and for none in 7 M urea. NMR data indicate that the helix limits in the peptides are similar to those in the native protein helices. The number of side-chain NOEs in water and TFE correlates with the helix content, and essentially none are observed in 7 M urea for any peptide, except that for helix 5, where a hydrophobic cluster may be present. The low intrinsic folding propensities of the five helices could account for the observed stability and folding behavior of 434 Cro and is, at least qualitatively, in accord with the results of the recently described diffusion-collision model incorporating intrinsic helix propensities.

Amino Acid Sequence↗