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Biomedical subjects

M Rico

Publications and source records attributed to M Rico.

At least 55 records · Page 3Linked to original sources
PubMed1999

GHRP-6 in heifer and cow adenohypophisial cells separated by elutriation.

Previous studies have reported that the growth hormone (GH)-releasing peptide (GHRP-6), a synthetic Met-enkephalin peptide analog, stimulates GH release in vivo in a variety of species, including bovine. In the present study, the in vitro effects of GHRP-6 on bovine somatotropes separated by elutriation were analyzed as well as its interactions with the GH-releasing hormone (GHRH). The administration of GHRP-6 at doses from 10(-8) M to 10(-5) M stimulated GH release, and also 10(-9) M in cow pituitary cells, and produced maximal stimulation at 10(-6) M. The effects of GHRP-6 (10(-6) M) on GH release were shown at 1, 2, 3 and 4-h incubation (p < 0.05), except for heifer pituitary cells at 1-h incubation (p > 0.05). The GH releasing effects of either GHRH alone or GHRH+GHRP-6 were significantly more potent than that of GHRP-6 alone (p < 0.05). Contrary to what occurred in rat pituitary cells, the combined administration of 10(-6) M GHRP-6 with 10(-8) M GHRH did not result in a synergist action of GH release. Although the additive effect was significant when compared with GHRH alone (p < 0.05). The results demonstrate the existence of differences in the effect of GHRH+GHRP-6 on bovine somatotropes. These differences may reflect the physiological importance of distinct cell subpopulation, like the mammosomatotroph cells.

Aging↗
PubMed1998

Helix-stabilizing nonpolar interactions between tyrosine and leucine in aqueous and TFE solutions: 2D-1H NMR and CD studies in alanine-lysine peptides.

Interactions between side chains spaced (i,i + 3) and (i,i + 4) may explain the context dependence of helix propensities observed in different systems. Nonpolar residues with these spacings occur frequently in protein helices and stabilize isolated peptide helices. Here (i,i + 3) and (i,i + 4) nonpolar interactions between Tyr and Leu in different solution conditions are studied in detail in alanine-based peptides using 2D 1H NMR and CD spectroscopy. Helix contents analyzed using current models for helix-coil transitions yield interaction energies which demonstrate significant helix stabilization in aqueous 1 M NaCl solutions by Tyr-Leu or Leu-Tyr pairs when spaced (i,i + 4) and, to a smaller extent, when spaced (i,i + 3), comparable to those estimated for other residue pairs. The interactions persist in solutions containing TFE, a helix-stabilizing solvent believed to diminish hydrophobic interactions, but not in helix-destabilizing 6 M urea. 1H NMR resonances for all peptides and solution conditions except in 6 M urea were completely assigned. NMR data indicate that the N-terminal residues are more helical and that the N-acetyl group participates in helix formation. The two (i,i + 4) spaced pairs show the same pattern of NOE cross-peaks between the Tyr and Leu side chains, as do the two (i,i + 3) pairs in 1 M NaCl as well in TFE solutions, and correspond well with that expected for the specific Tyr-Leu pair with side-chain contacts in protein helices.

Alanine↗
PubMed1998

Characterization of pKa values and titration shifts in the cytotoxic ribonuclease alpha-sarcin by NMR. Relationship between electrostatic interactions, structure, and catalytic function.

The electrostatic behavior of titrating groups in alpha-sarcin was investigated using 1H NMR spectroscopy. A total of 209 chemical shift titration curves corresponding to different protons in the molecule were determined over the pH range of 3.0-8.5. Nonlinear least-squares fits of the data to simple relationships derived from the Henderson-Hasselbalch equation led to the unambiguous determination of pKa values for all glutamic acid and histidine residues, as well as for the C-terminal carboxylate and most of the aspartic acids in the free enzyme. The ionization constants of catalytically relevant histidines, His50 and His137, and glutamic acid, Glu96, in the alpha-sarcin-2'-GMP complex were also determined. The pKa values of 15 ionizable groups (C-carboxylate, six aspartic acids, four glutamic acids, and four histidines) were found to be close to their normal values. On the other hand, a number of side chain groups, including those in the active center, showed pKa values far from their intrinsic values. Thus, the pKa values for active site residues His50, Glu96, and His137 were 7.7, 5.2, and 5.8 in the free enzyme and 7.6, approximately 4.8, and 6.8 in the alpha-sarcin-2'-GMP complex, respectively. The pKa values and the activity profile against ApA, as a function of pH, are in agreement with the proposed enzymatic mechanism (in common with RNase T1 and the family of the microbial ribonucleases), in which Glu96 and His137 act as a general base and general acid, respectively. In almost all microbial ribonucleases, a Phe-His interaction is present, which affects the pKa of one of the His residues at the active site (His137). The absence of this interaction in alpha-sarcin would explain the lower pKa value of this His residue, and provides an explanation for the decreased RNase activity of this protein as compared to those of other microbial ribonucleases.

Aspergillus↗
PubMed1998

Solution structure of acidic fibroblast growth factor bound to 1,3, 6-naphthalenetrisulfonate: a minimal model for the anti-tumoral action of suramins and suradistas.

Recent data show that anti-angiogenesis may provide a promising route to treat cancer. Fibroblast growth factors (FGFs) are powerful angiogenic polypeptides, whose mitogenic activity requires the presence of heparin-like compounds. It has been shown that angiogenesis promoted by FGFs on inhibition by monoclonal antibodies and antisense targeting can also inhibit tumour growth. Derivatives of suramin, a polysulfonated binaphthyl urea and binaphthylsulfonated derivatives of distamycin, suradistas, constitute an important group of potential anti-cancer agents. These compounds compete with heparin in forming tight complexes with FGFs. This inhibits the recognition of these growth factors by their tyrosine kinase membrane receptors thereby suppressing their angiogenic activity. Here we show that 1,3,6-naphthalenetrisulfonate, a common chemical function of the suramins and suradistas with the highest anti-angiogenic activity inhibits the mitogenic activity of acidic fibroblast growth factor, and that this inhibition is relieved by increasing concentrations of heparin in the assay. We have also solved the three-dimensional structure in solution of the protein complexed to this compound. The structural data provide clues that may help in understanding the inhibitory effect of suramins and suradistas, and could contribute to the development of new anti-tumoral drugs.

Amino Acid Sequence↗
PubMed1998

Beta structure motif recognition by anti-gliadin antibodies in coeliac disease.

A 20-amino acid synthetic peptide from the N-terminal region of gamma3 avenin yields a surprisingly strong reactivity with anti-gliadin antibodies (AGA) of coeliac sera, comparable to that of a gliadin extract. In contrast, a low reactivity is observed with five similar peptides derived from alpha-gliadin, gamma70 and omega1 secalins. Circular dichroism studies of these peptides show that the avenin peptide displays the highest beta-turn content (30%), while other peptides yield much lower values. In agreement with circular dichroism data, nuclear magnetic resonance data point to the presence of a beta-turn in the avenin peptide DPSEQ segment, a sequence with a high statistical beta-turn preference. A strong linear dependence between AGA reactivity and beta-turn content was observed for these peptides, indicating for the first time a role of beta-turn motifs in anti-gliadin antibodies recognition in coeliac disease. This suggests that circulating AGA in coeliac patients comprise not only linear but also conformational antibodies against beta-turn motifs. Polyclonal antibodies raised against the avenin peptide containing beta-turn motifs react by immunoblotting with all gliadin, hordein and secalin proteins, which are rich in beta-turn conformations, despite that their primary structures are unrelated to that of the peptide.

Antibodies↗
PubMed1998

Transrectal sonography in the assessment of vaginal pathology: a preliminary study.

PURPOSE: We assessed the value of transrectal sonography (TRUS) in the diagnosis of vaginal abnormalities. METHODS: Six women between 16 and 77 years old underwent TRUS with a biplanar probe equipped with 5-MHz axial sector and longitudinal linear transducers. Sagittal and axial images of the bladder, vagina, and urethra were obtained. Sonographic findings were compared with those from surgery. RESULTS: TRUS showed hematocolpos in 3 patients and cysts in 3 patients. Surgery confirmed the findings. Pathologic examination revealed 2 Gartner's cysts and 1 vaginal inclusion cyst. CONCLUSIONS: TRUS represents a valid diagnostic tool for the assessment of vaginal abnormalities because it accurately visualizes lesions and clearly shows anatomic relationships.

Adolescent↗
PubMed1998

Sequence-specific 1H assignment and secondary structure of the bacteriocin AS-48 cyclic peptide.

The bacteriocin AS-48 is a cationic peptide (7149 Da) having a broad antimicrobial spectrum, encoded by the 68 kb conjugative plasmid pMB2 from Enterococcus faecalis S-48. It is a unique peptide since it has a cyclic structure, which is achieved by the formation of a tail-head peptide bond after ribosomal synthesis (Gálvez et al., 1989; Martínez-Bueno et al., 1994; Samyn et al., 1994). Preliminary CD and calorimetric studies (data not shown) pointed towards a highly helical and very stable three dimensional structure. All the information gathered until now indicates that the target of AS-48 is the cytoplasmic membrane in which it opens channels or pores, leading to dissipation of the proton motive force and cell death, which in some cases is also followed by bacterial lysis (Gálvez et al., 1991). This peptide is a suitable tool for studying protein-membrane interactions, and it also offers promising perspectives for biotechnological applications. Knowledge of the 3D structure of AS-48 is a first step in the conduct of further structure-function studies. Here we report the complete 1H NMR assignment of its proton resonances together with the resulting secondary structure pattern as prerequisites for the determination of a high-resolution 3D solution structure.

Amino Acid Sequence↗
PubMed1998

Using coupling with the Gibbs sampler to assess convergence in animal models.

The coupling method was investigated as a method to assess the convergence of the Gibbs sampler when drawing marginal inferences with an animal model. This method is based on the output of two Markov chains with different starting values but the same conditional deviates. The coupling method shows that the Gibbs sampler has an exponential convergence when the variance components are assumed to be known. All the variables in the model have the same rate of convergence, and it is closely related with the largest eigenvalue of a matrix derived from the coefficient matrix of the mixed model equations. Models including the variance components as unknowns showed a rate of convergence equal for all the variables, and this rate of convergence was approximately exponential. The coupling method provides an estimation of the convergence at the current iteration, and it does not require a post-Gibbs analysis as do the single chain-based methods. The coupling method is less computationally demanding than multiple chain methods, because only two chains are required to assess convergence.

Analysis of Variance↗
PubMed1998

[The usefulness of a selective disk-broth media for the detection of group B streptococci in the vagina].

BACKGROUND: The detection of pregnant group B streptococci (GBS) carriers allows intrapartum administration of antibiotic prophylaxis to these women and prevents perinatal infection by this microorganism. The aim of this study was to determine the usefulness of a selective media to detect GBS in the vagina based on the disk-broth method. MATERIAL AND METHODS: One thousand six hundred five vaginal swabs were seeded in blood agar with colistin and nalidixic acid (NDA) and in a selective disk-broth tube consisting of 2 ml of Mueller-Hinton broth with 5% serum and a disk of 30 micrograms of amikacin which was reseeded at 24 hours of incubation in blood agar with NDA. RESULTS: GBS was isolated by either of the two methods in 209 samples (13.0%); in 160 (9.9%) in the initial dish of blood agar with NDA and in 205 (12.7%) in the subculture of the selective disk-broth media. CONCLUSIONS: The selective disk-broth media used in this study is a simple method which allows detection of GBS in vaginal samples of women with GBS cultures in a negative agar blood dish.

Adult↗
PubMed1998

Effect of growth hormone-releasing peptide 1-6 on GH secretion-stimulated by GHRH and pyridostigmine in lambs.

The GHRP-6 seems to act at a pituitary site, activating different intracellular messenger pathways from those utilized by GHRH, and at the hypothalamic level where receptors for GHRP-6 have been demonstrated. This study examines the effect of GHRP-6 on GH secretion in vitro and in vivo. Lamb adenohypophysial cell cultures were subjected to a challenge with 1) 10 nM GHRH-1-29; 2) 1 microM GHRP-6; and 3) 10 nM GHRH plus 1 microM GHRP-6. Both peptides released GH, GHRP-6 being less potent than GHRH, and the GH response to GHRH and GHRP-6 not being synergistic, without statistically significant differences between GHRH+GHRP-6 and GHRH alone. In in vivo studies, six lambs received 15 microg/kg GHRH (1-29) or 15 microg/kg GHRH plus 10 microg/kg GHRP-6 and six other lambs received 100 microg/kg GHRP-6 or 3 mg/kg pyridostigmine plus 100 microg/kg GHRP-6. The results have shown that the combination of GHRH plus GHRP-6, at low doses, causes higher GH peak (p < 0.05) and higher GH area under curve (p < 0.05) with respect to administration of GHRH alone. The administration of GHRP-6 plus pyridostigmine produced in a stronger GH response to GHRP-6 (100 microg/kg) in the amplitude of the GH peak (p < 0.001) and in the area under the GH response curve (p < 0.001). The complementary interactions of GHRP-6 with GHRH or pyridostigmine in releasing GH seem to indicate independent actions of these compounds. These results suggest that GHRP-6 potentiates the GH secretion stimulated by GHRH at the hypothalamic level, in these animals.

Animals↗
PubMed1997

NMR solution structure of the 205-316 C-terminal fragment of thermolysin. An example of dimerization coupled to partial unfolding.

The solution structure of the C-terminal fragment 205-316 of thermolysin has been determined by 1H-NMR methods. The fragment forms a dimer in which each subunit has two different regions: the largely disordered N-terminal segment 205-260 and the structurally well-defined segment 261-316. The structured part of each subunit is composed of three helices and is largely coincident with the corresponding region in the solution structure of the dimer formed by the shorter fragment 255-316, which in turn coincides with the crystallographic structure of intact thermolysin. As with the fragment 255-316, the subunit interface is highly hydrophobic and coincides topologically with the one between the segment 255-316 and the rest of the protein in the intact enzyme. A fourth helix (residues 235-246), present in the segment 205-316 of native thermolysin, is mostly disordered in the dimer formed by the fragment 205-316. The location of the fourth helix in the native structure of intact thermolysin does not allow the formation of the dimer interface observed in the solution structure of the fragment 255-316. Under the NMR conditions, dimer formation is energetically more favorable than the dissociated monomers. The latter, based on calorimetric data, was proposed to have partial structure in the region 205-254 as in native thermolysin. Thus, it appears that the assembly of the dimer would require an initial unfolding in the region 205-254 of the monomer.

Amino Acid Sequence↗
PubMed1997

Amide hydrogen exchange and internal dynamics in the chemotactic protein CheY from Escherichia coli.

The backbone internal dynamics of the wild-type 129 amino acid alpha/beta parallel protein CheY and its double mutant F14N/P110G are analysed here by the hydrogen-exchange method. The F14N mutation is known to stabilise the protein and to accelerate refolding while P110G is destabilising and accelerates unfolding. We first assigned and characterised the double mutant by nuclear magnetic resonance (NMR), to try and discover any possible conformational change induced by the two mutations. The main difference between the two proteins is a favourable N-capping interaction of the newly introduced Asn14 side-chain at the beginning of the first alpha-helix (alpha-helix A). Second, we have measured the exchange rates in the wild-type and mutant CheY. In the first case the observed protection factors are slightly dispersed around an average value. According to their distribution in the structure, protein stability is highest on one face of the central beta-sheet, in the surroundings of the main hydrophobic core formed by side-chains of residues in beta-strands I, II and III and helices A and E. The mutations in the double mutant protein affect two distinct subdomains differently (from beta-strand I to III and from alpha-helix C to the end). In the second subdomain the number of protected protons is reduced with respect to those in the wild-type. This differential behaviour can be explained by a selective decrease in stability of the second folding subdomain produced by the P110G mutation and the opposite effect in the first subdomain, produced by the F14N mutation. alpha-Helix A, which is involved together with beta-strands I and III in the folding nucleus of CheY, shows the largest protection factors in both proteins.

Amides↗
PubMed1997

The exchangeable yeast ribosomal acidic protein YP2beta shows characteristics of a partly folded state under physiological conditions.

The eukaryotic acidic ribosomal P proteins, contrary to the standard r-proteins which are rapidly degraded in the cytoplasm, are found forming a large cytoplasmic pool that exchanges with the ribosome-bound proteins during translation. The native structure of the P proteins in solution is therefore an essential determinant of the protein-protein interactions that take place in the exchange process. In this work, the structure of the ribosomal acidic protein YP2beta from Saccharomyces cerevisiae has been investigated by fluorescence spectroscopy, circular dichroism (CD), nuclear magnetic resonance (NMR), and sedimentation equilibrium techniques. We have established the fact that YP2beta bears a 22% alpha-helical secondary structure and a noncompact tertiary structure under physiological conditions (pH 7.0 and 25 degrees C); the hydrophobic core of the protein appears to be solvent-exposed, and very low cooperativity is observed for heat- or urea-induced denaturation. Moreover, the 1H-NMR spectra show a small signal dispersion, and virtually all the amide protons exchange with the solvent on a very short time scale, which is characteristic of an open structure. At low pH, YP2beta maintains its secondary structure content, but there is no evidence for tertiary structure. 2,2,2-Trifluoroethanol (TFE) induces a higher amount of alpha-helical structure but also disrupts any trace of the remaining tertiary fold. These results indicate that YP2beta may have a flexible structure in the cytoplasmic pool, with some of the characteristics of a "molten globule", and also point out the physiological relevance of such flexible protein states in processes other than protein folding.

Amino Acid Sequence↗
PubMed1997

Three-dimensional solution structure and stability of phage 434 Cro protein.

1H NMR resonances of the phage 434 Cro protein were assigned using standard 2D NMR methods, and its solution structure determined using 867 distance constraints in distance geometry (DIANA) calculations ultimately refined by restrained molecular dynamics (GROMOS). In the 20 best NMR structures, the average pairwise backbone and heavy atom RMSDs are 0.63 +/- 0.14 and 1.53 +/- 0.15 A, respectively, for the structurally well-defined residues 4-65. Residues 1-3 and 66-71 at the N- and C-termini are structurally disordered. The region 4-65 includes five alpha-helices and tight turns which define the hydrophobic core of the protein. The backbone and heavy atom RMSDs for residues 4-65 are 0.92 +/- 0.12 and 1.99 +/- 0.12 A, respectively, for the NMR versus the crystal structures, but there are significant differences in the side-chain conformations and solvent accessibilities for some core residues. Analytical ultracentrifugation experiments confirm that 434 Cro is monomeric even at the high NMR concentrations. 434 Cro folding under NMR solution conditions is two-state as indicated by coincident urea denaturation curves from circular dichroism and intrinsic fluorescence measurements. They yield values for 434 Cro stability which show good correspondence to the free energy for global unfolding determined by NMR hydrogen exchange measurements for the slowest exchanging amide protons.

Amino Acid Sequence↗
PubMed1997

Non-native local interactions in protein folding and stability: introducing a helical tendency in the all beta-sheet alpha-spectrin SH3 domain.

The relative importance of secondary structure interactions versus tertiary interactions for stabilising and guiding the folding process is a matter for discussion. Phenomenological models of protein folding assign an important role to local contacts in protein folding and stability. On the other hand, simplistic lattice simulations find that secondary structure is mainly the product of protein compaction and that optimisation of folding speed seems to require small contributions of local contacts to the stability of the folded state. To examine the extent to which secondary structure propensities influence protein folding and stability, we have designed mutations that introduce a strong non-native helical propensity in the first 19 residues of the alpha-spectrin SH3 domain. The mutant proteins have the same three-dimensional structure as the wild-type, but they are less stable and have less co-operative folding transitions. There seems to be a relationship between the non-native helical propensity and the compaction of the denatured state. This suggests that in the denatured ensemble under native conditions there is a significant proportion of compact structures with non-native secondary structures. Our results demonstrate that non-local interactions can overcome strong non-native secondary structure propensities and, more important, that optimisation of folding speed and co-operativity requires the latter to be relatively small.

Amino Acid Sequence↗
PubMed1997

Characterisation of the isolated Che Y C-terminal fragment (79-129)--Exploring the structure/stability/folding relationship of the alpha/beta parallel protein Che Y.

To gain insight into how the three-dimensional structure, stability and folding of the protein Che Y are related to one another, we have performed a conformational analysis of a long fragment of this protein, encompassing its C-terminal 51 residues (79-129). This fragment consists of residues in the beta-strands 4 and 5 and alpha-helices 4 and 5 of native Che Y. The study has been performed by two-dimensional NMR and far-ultraviolet circular dichroism in aqueous solution and in 30% (by vol.) trifluoroethanol/ water at 273 K and 298 K. We observe little structure for this fragment in aqueous solution which could be due to low helical populations in the regions corresponding to helices 4 and 5. Within the limits of the residual helical structure experimentally detected, helix 4 appears to extend beyond the N-terminus observed in the native structure by over four residues belonging to the preceding loop. In 30% trifluoroethanol the helical content of both helices increase and helix 4 extends further to include the preceding beta-strand 4. None of the long-range NOEs present in native Che Y are observed under the explored experimental conditions. The conformational shifts of the H(alpha) protons within the alpha-helices of fragment 79-129 are identical to those of shorter synthetic peptides corresponding to the isolated alpha-helices. Thus, the fragment 79-129 appears to behave as an open chain with low local helical populations. The very low intrinsic ability for structure formation displayed by this region of Che Y at pH 2.5 suggests that in the folded protein this region could be mainly stabilised by interactions with the N-terminal Che Y region. This is in accordance with the contact map of Che Y, which shows that the strongest non-local contacts of C-terminal residues are with residues of the N-terminal region, while those within the C-terminal region are very weak. More importantly, the relationship appears to be possibly extended to the folding properties of the protein, since the C-terminal region is not structurally formed in the folding transition state of Che Y but in the final steps of the folding.

Amino Acid Sequence↗
PubMed1997

1H-NMR analysis of CD3-epsilon reveals the presence of turn-helix structures around the ITAM motif in an otherwise random coil cytoplasmic tail.

The conformation adopted in solution by the cytoplasmic tail of CD3-epsilon has been analyzed by 1H-nmr. The cytoplasmic tail is mostly random coil expect for the amino acids conforming the immunoreceptor tyrosine-based activation motif (ITAM), YxxL/IxxxxxxxY xxL. Although the N-terminal Y xxL sequence of the motif is poorly folded, adopting 6-residue turn-like conformations with the Tyr side chain in two different orientations, the C-terminal Y xxL sequence is placed in a more complex structure involving a set of nonclassical alpha-helix turns and beta-turns that comprises 11 amino acids. This structure is not modified by phosphorylation of the tyrosine residue. The differences in the conformation adopted around the two tyrosines of the ITAM motif suggest that they may play different roles pertaining to either binding signal transducing proteins or, alternatively, proteins involved in other processes such as endoplasmic reticulum location.

Amino Acid Sequence↗