Mercedes Campillo

An epidemiological comparison of pain complaints in the general population of Catalonia (Spain)

Epidemiological studies help to establish the health status in a country and allow a better allocation of economic resources. This survey estimated pain prevalence in Catalonia (Spain), analysed its relationship with demographic variables and evaluated pain-associated disabilities. The study was carried out in 1964 adults via phone interviews asking about any pain complaint they experienced in the last 6 months, regardless of its intensity and duration. Overall pain prevalence was 78.6%, significantly lower in men, with a trend to decrease with age. Back (50.9%), head (42%) and legs (36.8%) were the most affected locations. Less educated people reported, in general, higher prevalences. Pain described to be most annoying was related to musculoskeletal disease (26.2%) and migraines (16.5%). Pain was either very severe or unbearable in 33% of the sample, with women and older people reporting higher intensities. Personal and social activities were affected in 25.4% of cases and in 10.4% they became virtually impossible. Both the limitation of activity and the need for bed rest, which occurred in 19.6% of those who suffered pain, were more common amongst unemployed people, whereas 10.2% of workers had to take days off work due to pain, and 3.3% were fully incapacitated by it. In conclusion, the prevalence of pain was clearly higher among women, with an inverse relationship to age. Back pain and headaches were most prevalent and pain was rated as very severe to unbearable in one third of the patients. Pain-associated disabilities were a frequent finding. The present survey reports that pain is a substantial problem in the Catalonian population and generally reflects the characteristics of data previously reported in Anglo-saxon and Scandinavian countries.

Glycoprotein hormone receptors: determinants in leucine-rich repeats responsible for ligand specificity

Glycoprotein hormone receptors [thyrotropin (TSHr), luteinizing hormone/chorionic gonadotropin (LH/CGr), follicle stimulating hormone (FSHr)] are rhodopsin‐like G protein‐coupled receptors with a large extracellular N‐terminal portion responsible for hormone recognition and binding. In structural models, this ectodomain is composed of two cysteine clusters flanking nine leucine‐rich repeats (LRRs). The LRRs form a succession of β‐strands and α‐helices organized into a horseshoe‐shaped structure. It has been proposed that glycoprotein hormones interact with residues of the β‐strands making the concave surface of the horseshoe. Gain‐of‐function homology scanning of the β‐strands of glycoprotein hormone receptors allowed identification of the critical residues responsible for the specificity towards human chorionic gonadotropin (hCG). Substitution of eight or two residues of the LH/CGr into the TSHr or FSHr, respectively, resulted in constructs displaying almost the same affinity and sensitivity for hCG as wild‐type LH/CGr. Molecular dynamics simulations and additional site‐directed mutagenesis provided a structural rationale for the evolution of binding specificity in this duplicated gene family.

The Role of Internal Water Molecules in the Structure and Function of the Rhodopsin Family of G Protein‐Coupled Receptors

Membrane receptors coupled to guanine nucleotide-binding regulatory proteins (commonly known as G protein-coupled ACHTUNGTRENNUNGreceptors, GPCRs) constitute one of the most attractive pharmaceutical targets, as around 40% of clinically prescribed drugs and 25% of the top-selling drugs act at these receptors. GPCRs are receptors for sensory signals of external origin such as odors, pheromones, or tastes; and for endogenous signals such as neurotransmitters, (neuro)peptides, divalent cations, proteases, glycoprotein hormones, and purine ligands. Phylogenetic analyses of the human genome have permitted GPCR sequences to be classified into five main families: rhodopsin (class A or family 1), secretin (class B or family 2), glutamate (class C or family 3), adhesion, and frizzled/ taste2. Specialized databases of GPCRs can be found at http://www.gpcr.org/7tm, http://gris.ulb.ac.be/, and http:// www.iuphar-db.org. Due to the low natural abundance of GPCRs and the difficulty in producing and purifying recombinant protein, only one member of this family, rhodopsin, the photoreceptor protein of rod cells, has been crystallized so far. Five structural models of inactive rhodopsin are available at the Protein Data Bank, at resolutions of 2.8 A (PDB IDs: 1F88 and 1HZX), 2.65 A (1GZM), 2.6 A (1L9H), and 2.2 A (1U19). Structural models of rhodopsin photointermediates such as bathorhodopsin (2G87), lumirhodopsin (2HPY), metarhodopsin I, and a photoactivated deprotonated intermediate reminiscent of metarhodopsin II (2I37) are also available. Rhodopsin is formed by an extracellular N terminus of four b-strands, seven transmembrane helices (TM1 to TM7) connected by alternating intracellular (I1 to I3) and extracellular (E1 to E3) hydrophilic loops, a disulfide bridge between E2 and TM3, and a cytoplasmic C terminus containing an a-helix (Hx8) parallel to the cell membrane. Statistical analysis of the residues forming the TM helices of the rhodopsin family of GPCRs shows a large number of conserved sequence patterns; this suggests a common TM structure. Thus, the availability of the rhodopsin structure allows the use of homology modeling techniques to build three-dimensional models of other homologous GPCRs. The putative structural homology between rhodopsin and other GPCRs probably does not extend to the extracellular domain, since the extracellular N terminus and loop fragments are highly variable in length and amino acid content. The class A family of GPCRs contains highly conserved Pro residues in the middle of TMs 5 (P5.50, conserved in 77% of the sequences), 6 (P6.50, 100%), and 7 (P7.50, 96%; residues are identified by the generic numbering scheme of Ballesteros and Weinstein, which allows easy comparison among residues in the 7TM segments of different receptors). Pro residues are normally observed in the TM helices of membrane proteins where they usually induce a significant distortion named a “Pro-kink”. This break arises in order to avoid a steric clash between the pyrrolidine ring of the Pro side chain (at position i) and the carbonyl oxygen of the residue in the preceding turn (position i 4) and leads to a distortion of the helical structure. However, TM segments of rhodopsin, either with or without Pro residues in their sequence are far from being standard Pro-kinked or ideal helices, respectively. Their distortions are energetically stabilized through complementary intraand interhelical interactions involving polar side chains, backbone carbonyls, and, in some cases, specific structural and functional water molecules embedded in the TM bundle. Here we review the role of these water molecules in the structure and function of GPCRs and in building computergenerated homology models of class A GPCRs. We propose that water molecules present in the vicinity of highly conserved motifs are most likely present in the rhodopsin family of GPCRs, being another conserved structural element in the family.

Ser and Thr Residues Modulate the Conformation of Pro-Kinked Transmembrane α-Helices

Functionally required conformational plasticity of transmembrane proteins implies that specific structural motifs have been integrated in transmembrane helices. Surveying a database of transmembrane helices and the large family of G-protein coupled receptors we identified a series of overrepresented motifs associating Pro with either Ser or Thr. Thus, we have studied the conformation of Pro-kinked transmembrane helices containing Ser or Thr residues, in both g+ and g- rotamers, by molecular dynamics simulations in a hydrophobic environment. Analysis of the simulations shows that Ser or Thr can significantly modulate the deformation of the Pro. A series of motifs, such as (S/T)P and (S/T)AP in the g+ rotamer and the TAP and PAA(S/T) motifs in the g- rotamer, induce an increase in bending angle of the helix compared to a standard Pro-kink, apparently due to the additional hydrogen bond formed between the side chain of Ser/Thr and the backbone carbonyl oxygen. In contrast, (S/T)AAP and PA(S/T) motifs, in both g+ and g-, and PAA(S/T) in g+ rotamers decrease the bending angle of the helix by either reducing the steric clash between the pyrrolidine ring of Pro and the helical backbone, or by adding a constrain in the form of a hydrogen bond in the curved-in face of the helix. Together with a number of available experimental data, our results strongly suggest that association of Ser and Thr with Pro is commonly used in transmembrane helices to accommodate the structural needs of specific functions.

Analysis of nine chromosome probes in first polar bodies and metaphase II oocytes for the detection of aneuploidies.

We used fluorescent in situ hybridisation (FISH) to detect nine chromosomes (1, 13, 15, 16, 17, 18, 21, 22 and X) in 89 first Polar Bodies (1PBs), from in vitro matured oocytes discarded from IVF cycles. In 54 1PBs, we also analysed the corresponding oocyte in metaphase II (MII) to confirm the results; the other 35 1PBs were analysed alone as when preimplantation genetic diagnosis using 1PB (PGD-1PB) is performed. The frequency of aneuploid oocytes found was 47.5%; if the risk of aneuploidy for 23 chromosomes is estimated, the percentage rises to 57.2%. Missing chromosomes or chromatids found in 1PBs of 1PB/MII doublets were confirmed by MII results in 74.2%, indicating that only 25.8% of them were artefactual. Abnormalities observed in 1PBs were 55.8% whole-chromosome alterations and 44.2% chromatid anomalies. We observed a balanced predivision of chromatids for all chromosomes analysed. Differences between balanced predivision in 1PB and MII were statistically significant (P<0.0001, χ2 test); the 1PB was most affected. The mean abnormal segregation frequency for each chromosome was 0.89% (range 0.52–1.70%); so, each of the 23 chromosomes of an oocyte has a risk of 0.89% to be involved in aneuploidy. No significant differences were observed regarding age, type of abnormality (chromosome or chromatid alterations) or frequency of aneuploidy. Nine of the 35 patients (25.7%) whose 1PB and MII were studied presented abnormalities (extra chromosomes) that probably originated in early oogenesis. Analysis of 1PBs to select euploid oocytes could help patients of advanced age undergoing in vitro fertilization (IVF) treatment.

Risk, Predictors, and Clinical Characteristics of Lymphoma Development in Primary Sjögren's Syndrome

OBJECTIVE To assess the risk and predictors of lymphoma development in a large cohort of patients with primary Sjögrens syndrome (pSS). METHODS Cox-regression analyses were used to study the predictive value of clinical and laboratory findings at pSS diagnosis, and Kaplan-Meier survival curves to compare survival probability between patients who developed lymphoma and the total cohort. Expected risk for lymphoma was calculated by comparison with the background population. RESULTS Eleven (4.5%) from 244 patients developed a non-Hodgkin lymphoma (NHL). Diffuse large B-cell and mucosa-associated lymphoid tissue lymphomas occurred at a similar frequency. Three (27.3%) patients died: 2 due to transformation from mucosa-associated lymphoid tissue to diffuse large B-cell. Purpura (HR 8.04, 95% confidence interval [CI] 2.33-27.67), parotidomegaly (HR 6.75, 95%CI 1.89-23.99), anemia (HR 3.43, 95%CI 1.04-11.35), leukopenia (HR 8.70, 95%CI 2.38-31.82), lymphocytopenia (HR 16.47, 95%CI 3.45-78.67), hypergammaglobulinemia (HR 4.06, 95%CI 1.06-15.58), low C3 (HR 36.65, 95%CI 10.65-126.18), and low C4 (HR 39.70, 95%CI 8.85-126.18) levels at pSS diagnosis were significant predictors of NHL development, but only hypocomplementemia and lymphocytopenia were independent risk factors. Hypocomplementemia was related to earlier development of NHL and higher mortality. The cumulative risk of developing lymphoma ranged from 3.4% in the first 5 years to 9.8% at 15 years. Standardized incidence ratio (95%CI) for NHL development was 15.6 (95%CI 8.7-28.2). CONCLUSIONS Patients with pSS have a 16-fold increased risk of developing lymphoma. This risk increases with time. Hypocomplementemia and lymphocytopenia at pSS diagnosis are the strongest predictors. Survival is clearly reduced in patients with hypocomplementemia. Indolent lymphomas tend to evolve over time toward a more aggressive histologic type.

Conformational Toggle Switches Implicated in Basal Constitutive and Agonist-Induced Activated States of 5-Hydroxytryptamine-4 Receptors

The extended classic ternary complex model predicts that a G protein-coupled receptor (GPCR) exists in only two interconvertible states: an inactive R, and an active R*. However, different structural active R* complexes may exist in addition to a silent inactive R ground state (Rg). Here we demonstrate, in a cellular context, that several R* states of 5-hydroxytryptamine-4 (5-HT4) receptors involve different side-chain conformational toggle switches. Using site-directed mutagenesis and molecular modeling approaches, we show that the basal constitutive receptor (R*basal) results from stabilization of an obligatory double toggle switch (Thr3.36 from inactive g- to active g+ and Trp6.48 from inactive g+ to active t). Mutation of either threonine or tryptophan to alanine resulted in a lowering of the activity of the R*basal similar to the Rg. The T3.36A mutation shows that the Thr3.36 toggle switch plays a minor role in the stabilization of R* induced by 5-HT (R*-5-HT) and BIMU8 (R*-BIMU8) and is fully required in the stabilization of R* induced by (S)-zacopride, cisapride, and 1-(4-amino-5-chloro-2-methoxyphenyl)-3-(1-butyl-4-piperidinyl)-1-propanone (RS 67333) (R*-benzamides). Thus, benzamides stabilize R*-benzamides by forming a specific hydrogen bond with Thr3.36 in the active g+ conformation. Conversely, R*-BIMU8 was probably the result of a direct conformational transition of Trp6.48 from inactive g+ to active t by hydrogen bonding of this residue to a carboxyl group of BIMU8. We were surprised that the Trp6.48 toggle switch was not necessary for receptor activation by the natural agonist 5-HT. R*-5-HT is probably attained through other routes of activation. Thus, different conformational arrangements occur during stabilization of R*basal, R*-5-HT, R*-benzamides, and R*-BIMU8.

Benzimidazole Derivatives as New Serotonin 5-HT6 Receptor Antagonists. Molecular Mechanisms of Receptor Inactivation

On the basis of our previously described pharmacophore model for serotonin 5-HT(6) receptor (5-HT(6)R) antagonists, we have designed, synthesized, and pharmacologically characterized a series of benzimidazole derivatives 1-20 that represent a new family of potent antagonists at the human 5-HT(6)R. Site-directed mutagenesis and a beta(2)-adrenoceptor-based homology model of the 5-HT(6)R were used to predict the mode of binding of antagonist SB-258585 and the new synthesized ligands. Substitution of W6.48, F6.52, or N6.55 by Ala fully impedes compound 4 to block 5-HT-induced activation. Thus, we propose that D3.32 in TM 3 anchors the protonated piperazine ring, the benzimidazole ring expands parallel to EL 2 to hydrogen bond N6.55 in TM 6, and the aromatic ring is placed between TMs 3 and 5 in CH(2)-containing compounds and between TMs 3 and 6 in CO-containing compounds. This combined experimental and computational study has permitted to propose the molecular mechanisms by which the new benzimidazole derivatives act as 5-HT(6)R antagonists.

The specificity of binding of glycoprotein hormones to their receptors.

Abstract.The glycoprotein hormone receptor family is peculiar because, in contrast to other G protein-coupled receptors, a large N-terminal extracellular ectodomain is responsible for hormone recognition. Hormone-receptor pairs have evolved in such a manner that a limited number of positions both at the ‘seat-belt’ domain of the hormone and the leucine-rich repeats of the receptor, play attractive and repulsive interactions for binding and specificity, respectively. Surprisingly, the constitutive activity of the receptor, mostly modulated by highly conserved amino acids within the heptahelical domain of the receptor (i.e., outside the hormone binding region), also regulates effectiveness of hormone recognition by the extracellular part. In this review we analyze, at the molecular level, these important discriminating determinants for selective binding of glycoprotein hormones to their receptors, as well as natural mutations, observed in patients with gestational hyperthyroidism or ovarian hyperstimulation syndrome, that modify the selectivity of binding.

Synthesis of new serotonin 5-HT7 receptor ligands. Determinants of 5-HT7/5-HT1A receptor selectivity.

We report the synthesis of a new set of compounds of general structure I (1-20) with structural modifications in the pharmacophoric elements of the previously reported lead UCM-5600. The new derivatives have been evaluated for binding affinity at 5-HT(7) and 5-HT(1A) receptors. The influence of the different structural features in terms of 5-HT(7)/5-HT(1A) receptor affinity and selectivity was analyzed by computational simulations of the complexes between compounds I and beta(2)-based 3-D models of these receptors. Compound 18 (HYD(1) = 1,3-dihydro-2H-indol-2-one; spacer = -(CH(2))(4)-; HYD(2) + HYD(3) = 3,4-dihydroisoquinolin-2(1H)-yl) exhibits high 5-HT(7)R affinity (K(i) = 7 nM) and selectivity over the 5-HT(1A)R (31-fold), and has been characterized as a partial agonist of the human 5-HT(7)R.