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Pilar Pérez

Publications and source records attributed to Pilar Pérez.

At least 19 recordsLinked to original sources
PubMed2006

Excited state proton transfer in 3-methyl-7-azaindole dimer. Symmetry control.

The concerted double proton transfer undergone by the C(2)(h) dimer of 7-azaindole upon electronic excitation has also been reported to occur in 3-methyl-7-azaindole monocrystals and in dimers of this compound under free-jet conditions. However, the results obtained in this work for the 3-methyl-7-azaindole dimer formed in a 10(-4) M solution of the compound in 2-methylbutane suggest that the dimer produces no fluorescent signal consistent with a double proton transfer in the liquid phase or in a matrix. In this paper, the spectroscopic behavior of the doubly hydrogen bonded dimer of 3-methyl-7-azaindole is shown to provide a prominent example of molecular symmetry control over the spectroscopy of a substance. This interpretation opens up a new, interesting research avenue for exploring the ability of molecular symmetry to switch between proton-transfer mechanisms. It should be noted that symmetry changes in the 3-methyl-7-azaindole dimer are caused by an out-of-phase internal rotation of the two methyl groups.

Journal Article↗
PubMed2006

Efficacy of selective 5-HT6 receptor ligands determined by monitoring 5-HT6 receptor-mediated cAMP signaling pathways.

1. Two novel selective 5-HT6 receptor ligands E-6801 (6-chloro-N-(3-(2-(dimethylamino)ethyl)-1H-indol-5-yl)imidazo[2,1-b]thiazole-5-sulfonamide) and E-6837 (5-chloro-N-(3-(2-(dimethylamino)ethyl)-1H-indol-5-yl)naphthalene-2-sulfonamide) were investigated and compared to the putative 5-HT6 receptor antagonists SB-271046 (5-chloro-N-(4-methoxy-3-(piperazin-1-yl)phenyl)-3-methylbenzo[b]thiophene-2-sulfonamide) and Ro 04-06790 (N-(2,6-bis(methylamino)pyrimidin-4-yl)-4-aminobenzenesulfonamide) using a cAMP-mediated pathway. 2. Forskolin stimulation, to increase the magnitude of agonist cAMP responses, and site-directed mutagenesis of the 5-HT6 receptor, in order to yield constitutively active receptor, were applied. 3. 5-HT (E(max), % over basal: 200), E-6801 (120) and E-6837 (23) induced cAMP formation at the rat 5-HT6 receptor. In the copresence of forskolin, cAMP responses were more potent and enhanced to 294 (5-HT, % over forskolin), 250 (E-6801) and 207 (E-6837), respectively. 5-HT-mediated cAMP formation was dose-dependently blocked by SB-271046 (pA(2): 8.76+/-0.22) and Ro 04-6790 (pA(2): 7.89+/-0.10) and not affected by the copresence of forskolin. Both E-6801 and E-6837 yielded partial antagonism of the 5-HT response in the absence of forskolin, whereas antagonism was either completely absent (E-6801) or attenuated (E-6837) in the copresence of forskolin. Intrinsic activity of these 5-HT6 receptor ligands at a constitutively active human S267K 5-HT6 receptor in Cos-7 cells indicated similar efficacy (E(max), % over basal) for 5-HT (97), E-6801 (91) and E-6837 (100), while Ro 04-6790 (-33) and SB-271046 (-39) were equi-efficacious inverse agonists. 4. The use of either forskolin or a constitutively active S267K 5-HT6 receptor enhances the resolution for monitoring the efficacy of 5-HT6 receptor ligands. E-6801 and E-6837 are potent partial agonists at the 5-HT6 receptor. Ro 04-6790 and SB-271046 appear to act as inverse agonists/antagonists.

Animals↗
PubMed2006

A new expert systems (SeDeM diagram) for control batch powder formulation and preformulation drug products.

The new SeDeM Method is proposed for testing the batch-to-batch reproducibility of the same active pharmaceutical ingredient (API) in powder form. The procedure describes the study of the galenic properties of substances in powder form in terms of the applicability of direct compression technology. Through experimental determination of the SeDeM Method parameters, and their subsequent mathematical treatment and graphical expression (SeDeM Diagram), three batches of the same API were analysed to determine whether it was suitable for direct compression. Batch-to-batch reproducibility of the results was verified. It was concluded that the SeDeM Method is suitable for testing batch-to-batch reproducibility of characteristics in powdered APIs substances. The results obtained confirm that the SeDeM Method is a useful, effective tool for drug-preformulation studies providing the pharmacotechnical data required when formulating a drug in tablet form. In addition, the results were effective for defining the most appropriate manufacturing technology.

Chemistry, Pharmaceutical↗
PubMed2006

Whole spectrum analysis of ligand efficacy at constitutively active human wild-type and S267K 5-HT6 receptors in HEK-293F cells.

INTRODUCTION: Modulation of constitutive activity by the recombinant wild-type human 5-HT6 receptor was investigated with a series of 5-HT6 receptor ligands by monitoring the cAMP signalling pathway. The impact of the mutation S267K near the B(261)BXXB(265) CIII-loop motif was analyzed on the magnitude of constitutive receptor activity as previously conflicting results have been reported. METHODS: The wild-type 5-HT6 receptor plasmid was obtained by PCR and the mutant S267K5-HT6 receptor was constructed by site-directed mutagenesis and stably transfected in HEK-293F cells by electroporation. The cAMP signalling pathway was monitored as a functional read-out to investigate ligands' responses using homogeneous time resolved fluorescence. RESULTS: Constitutive activity was present both at wild-type and mutant S267K 5-HT6 receptors. Negative efficacy (E(max), % versus basal) as observed at nanomolar concentrations with SB-271046 was larger for mutant (-92+/-1%) than wild-type 5-HT6 receptor (-45+/-1%). Ro 04-6790 also demonstrated negative efficacy at the wild-type 5-HT6 receptor with a magnitude similar to SB-271046 but with a 36-fold lower potency. MS-245 demonstrated at nanomolar concentrations intermediate negative efficacy; -48+/-3% and -16+/-2% at mutant and wild-type 5-HT6 receptor, respectively. The 5-HT-mediated cAMP response was blocked by SB-271046, MS-245 and Ro 04-6790 to their respective level of negative efficacy with pKB values fitting with their binding pK(i) values. E-6801 was a highly potent (pEC50: 10.17 to 10.19) and efficacious agonist (+98 to +102% versus 5-HT) at both wild-type and mutant 5-HT6 receptors. DISCUSSION: The recombinant wild-type human 5-HT6 receptor is constitutively active in HEK-293F cells and displays a high resolution to monitor efficacy properties of 5-HT6 receptor ligands. The resolution capacity to differentiate between efficacy properties of 5-HT6 receptor ligands, in particular for negative efficacy, can be further enhanced by monitoring the mutant S267K 5-HT6 receptor.

Cyclic AMP↗
PubMed2006

Fission yeast Rho5p GTPase is a functional paralogue of Rho1p that plays a role in survival of spores and stationary-phase cells.

The Rho GTPase family and their effectors are key regulators involved in many eukaryotic cell functions related to actin organization and polarity establishment. Schizosaccharomyces pombe Rho1p is essential, directly activates the (1,3)-beta-d-glucan synthase, and participates in regulation of cell wall growth and morphogenesis. Here we describe the characterization of the fission yeast Rho5p GTPase, highly homologous to Rho1p, sharing 86% identity and 95% similarity. Overexpression of the hyperactive allele rho5-G15V causes a morphological effect similar to that of rho1-G15V, but the penetrance is significantly lower, and overexpression of the dominant-negative allele rho5-T20N causes lysis like that of rho1-T20N. Importantly, overexpression of rho5(+) but no other rho genes is able to rescue the lethality of rho1Delta cells. Shutoff experiments indicated that Rho5p can replace Rho1p, but it is not as effective in maintaining cell wall integrity or actin organization. rho5(+) expression is hardly detected during log-phase growth but is induced under nutritional starvation conditions. rho5Delta cells are viable and do not display any defects during logarithmic growth. However, when rho1(+) expression is repressed during stationary phase, rho5Delta cells display reduced viability. Ascospores lacking Rho5p are less resistant to heat or lytic enzymes than wild-type spores. Moreover, h(90) mutant strains carrying the hyperactive rho5-G15V or the dominant-negative rho5-T20N alleles display severe ascospore formation defects. These results suggest that Rho5p functions in a way similar to, but less efficient than, Rho1p, plays a nonessential role during stationary phase, and participates in the spore wall formation.

Amino Acid Sequence↗
PubMed2005

Stress-induced response, localization, and regulation of the Pmk1 cell integrity pathway in Schizosaccharomyces pombe.

Mitogen-activated protein kinase (MAPK) signaling pathways are critical for the sensing and response of eukaryotic cells to extracellular changes. In Schizosaccharomyces pombe, MAPK Pmk1/Spm1 has been involved in cell wall construction, morphogenesis, cytokinesis, and ion homeostasis, as part of the so-called cell integrity pathway together with MAPK kinase kinase Mkh1 and MAPK kinase Pek1. We show that Pmk1 is activated in multiple stress situations, including hyper- or hypotonic stress, glucose deprivation, presence of cell wall-damaging compounds, and oxidative stress induced by hydrogen peroxide or pro-oxidants. The stress-induced activation of Pmk1 was completely dependent on Mkh1 and Pek1 function, supporting a nonbranched pathway in the regulation of MAPK activation. Fluorescence microscopy revealed that Mkh1, Pek1, and Pmp1 (a protein phosphatase that inactivates Pmk1) are cytoplasmic proteins. Mkh1 and Pek1 were also found at the septum, whereas Pmk1 localized in both cytoplasm and nucleus as well as in the mitotic spindle and septum during cytokinesis. Interestingly, Pmk1 subcellular localization was unaffected by stress or the absence of Mkh1 and Pek1, suggesting that its activation by the Mkh1-Pek1 cascade takes place at the cytoplasm and/or septum and that the active and inactive forms of this kinase cross the nuclear membrane. Cdc42 GTPase and its effectors, p21-activated kinases Pak2 and Pak1, are not upstream elements controlling the basal level or the stress-induced activation of Pmk1. However, Sty1 MAPK was essential for proper Pmk1 deactivation after hypertonic stress in a process regulated by Atf1 transcription factor. These results provide the first evidence for the existence of cross-talk between two MAPK cascades during the stress response in fission yeast.

Cell Cycle Proteins↗
PubMed2005

Synthesis of new benzoxazinone derivatives as neuropeptide Y5 antagonists for the treatment of obesity.

Screening of our internal chemical collection against the neuropeptide Y5 (NPY Y5) receptor allowed the identification of a benzoxazine derivative 5f as a hit that showed moderate affinity (IC(50) = 300 nM). With the aim of improving the in vitro potency, a series of 2-benzoxazinone derivatives have been synthesized and tested for NPY Y5 activity. Most of the compounds were found to be potent and selective NPY Y5 antagonists having nanomolar binding affinities for the NPY Y5 receptor and showing functional antagonism in the forskolin-induced cyclic AMP test. Prelimminary studies in order to understand the structure-activity relationship were undertaken. Selected compounds were further evaluated for in vivo efficacy, affording the lead compound 2-[4-(8-methyl-2-oxo-4H-benzo[d][1,3]oxazin-1-yl)piperidin-1-yl]-N-(9-oxo-9H-fluoren-3-yl)acetamide 5p, which displayed in vivo activity reducing food intake in rodents.

Animals↗
PubMed2005

Endoureterotomy by intraluminal invagination for nonmalignant ureterointestinal anastomotic strictures: description of a new surgical technique and long-term followup.

PURPOSE: We describe a new surgical endoscopic technique for nonmalignant ureterointestinal anastomotic strictures. This procedure involving endoureterotomy by intraluminal invagination (the Lovaco technique) is performed by adopting a combined percutaneous antegrade and endoscopic retrograde approach. The results obtained by this technique are reviewed with long-term followup. MATERIALS AND METHODS: A total of 25 ureterointestinal anastomotic strictures were subjected to endoureterotomy by intraluminal invagination, including 12 left, 7 right and 3 bilateral cases. Surgical success was defined by radiological improvement and/or the ability to recover normal activity in the absence of flank pain, infection, or the need for ureteral stents or nephrostomy tubes. RESULTS: At a median followup of 51 months (range 2 to 145) the success rate for endoureterotomy by intraluminal invagination was 80% (20 of 25 ureterointestinal anastomotic strictures). No complications were recorded in the patients following endoureterotomy. CONCLUSIONS: This new endoureterotomy technique for ureterointestinal strictures following urinary diversion can be applied to any type of urinary diversion. It allows direct visualization of the stricture and stricture tissue biopsy. Intraluminal invagination makes it possible to increase the distance between the stricture, and the retroperitoneal vessels and bowels. The technique provides the control required to ensure full-thickness and full-length stricture incision. The success rate is high and it persists after long-term followup.

Adult↗
PubMed2005

Rho4 GTPase is involved in secretion of glucanases during fission yeast cytokinesis.

Rho GTPases are regulators of signaling pathways that control actin organization and cell polarity processes in all eukaryotic cells. In Schizosaccharomyces pombe, Rho4p is involved in the regulation of septum degradation during cytokinesis. Here we show that Rho4p participates in the secretion of the glucanases Eng1p and Agn1p, which are responsible for the septum degradation. First, eng1+ or agn1+ overexpression suppressed the rho4delta multiseptation phenotype, and simultaneous overproduction of Rho4p and Eng1p or of Rho4p and Agn1p caused a dramatic lysis. Second, Rho4p was not necessary for Eng1p-mediated glucanase activity as measured in cell extracts; however, rho4delta cells have a lower level of (1,3)-beta-D-glucanase activity in the culture medium. Additionally, Eng1- or Agn1-green fluorescent protein did not properly localize to the septum in rho4delta cells grown at 37 degrees C. There was a decreased amount of these enzymes in the cell wall and in the culture medium of rho4delta cells at 37 degrees C. These results provide evidence that Rho4p is involved in the regulation of Eng1p and Agn1p secretion during cytokinesis.

Cytokinesis↗
PubMed2004

The p75NTR-interacting protein SC1 inhibits cell cycle progression by transcriptional repression of cyclin E.

Schwann cell factor 1 (SC1), a p75 neurotrophin receptor-interacting protein, is a member of the positive regulatory/suppressor of variegation, enhancer of zeste, trithorax (PR/SET) domain-containing zinc finger protein family, and it has been shown to be regulated by serum and neurotrophins. SC1 shows a differential cytoplasmic and nuclear distribution, and its presence in the nucleus correlates strongly with the absence of bromodeoxyuridine (BrdU) in these nuclei. Here, we investigated potential transcriptional activities of SC1 and analyzed the function of its various domains. We show that SC1 acts as a transcriptional repressor when it is tethered to Gal4 DNA-binding domain. The repressive activity requires a trichostatin A-sensitive histone deacetylase (HDAC) activity, and SC1 is found in a complex with HDACs 1, 2, and 3. Transcriptional repression exerted by SC1 requires the presence of its zinc finger domains and the PR domain. Additionally, these two domains are involved in the efficient block of BrdU incorporation by SC1. The zinc finger domains are also necessary to direct SC1's nuclear localization. Lastly, SC1 represses the promoter of a promitotic gene, cyclin E, suggesting a mechanism for how growth arrest is regulated by SC1.

3T3 Cells↗
PubMed2004

Cell wall analysis.

The cell wall is a rigid structure essential for survival of the fungal cell. Because of its absence in mammalian cells, the cell wall is an attractive target for antifungal agents. Thus, for different reasons, it is important to know how the cell wall is synthesized and how different molecules regulate that synthesis. The Schizosaccharomyces pombe cell wall is mainly formed by glucose polysaccharides and some galactomannoproteins. Here, we describe a fast and reliable method to analyze changes in S. pombe cell wall composition by using specific enzymatic degradation and chemical treatment of purified cell walls. This approach provides a powerful means to analyze changes in (1,3)beta-glucan and (1,3)alpha-glucan, two main polysaccharides present in fungal cell walls. Analysis of cell wall polymers will be useful to search for new antifungal drugs that may inhibit cell wall biosynthesis and/or alter cell wall structure.

Cell Wall↗
PubMed2003

Comparison of prognostic value of atrial fibrillation versus sinus rhythm in patients on long-term hemodialysis.

The influence of atrial fibrillation (AF) on the clinical pattern of patients with chronic renal insufficiency on hemodialysis remains unknown despite the cardiovascular pathology in these patients being well documented and being the primary cause of death in this patient population. The objective of this study was to compare the long-term outcome in those patients on our dialysis unit in sinus rhythm with those in AF.

Age Distribution↗
PubMed2003

Ought dialysis patients with atrial fibrillation be treated with oral anticoagulants?

BACKGROUND: Dialysis patients with atrial fibrillation have an increased thrombolic risk. Dicoumarin anticoagulant therapy is often considered contra-indicated in chronic renal insufficiency in which the risk of haemorrhage, though not defined, is perceived to be high. We assessed haemorrhage complications in dialysis patients receiving dicoumarin anticoagulant therapy to establish whether the haemorrhage risk justifies the contra-indication of anticoagulant therapy in patients with atrial fibrillation. PATIENTS AND METHODS: Over a period of a decade in our dialysis centre, 29 patients receiving anticoagulant therapy over a protracted period presented haemorrhage complications. These were classified with respect to severity and location and compared with 211 patients not receiving anticoagulant therapy. The relative risk of haemorrhage was calculated and was compared to risk of thrombo-embolism in dialysis patients with atrial fibrillation. RESULTS: Of the 29 patients, nine had 13 episodes of haemorrhage complications (26 episodes/100 patient-years). None was fatal, nor intra-cranial nor with serious clinical sequelae. In the group without anticoagulants, 29 patients had 39 haemorrhage complications (11 episodes/100 patient-years); four (10.2%) intra-cranial and all fatal. The relative risk of bleeding with anticoagulant therapy was 2.36 (95% confidence interval=1.19-4.27). CONCLUSIONS: (1) Dialysis patients with anticoagulant therapy presented with a higher risk of haemorrhage; (2) the relative risk of bleeding was double that of the dialysis population without anticoagulant therapy; (3) despite the high risk of haemorrhage that we observed, the high risk of thrombo-embolism and the attendant serious sequelae to which dialysis patients with atrial fibrillation are predisposed indicates that oral anticoagulation therapy ought not to be considered automatically contra-indicated in this patient group but that an exhaustive evaluation of the risk-benefit needs to be conducted on an individual patient basis.

Administration, Oral↗
PubMed2003

Novel Rho GTPase involved in cytokinesis and cell wall integrity in the fission yeast Schizosaccharomyces pombe.

The Rho family of GTPases is present in all eukaryotic cells from yeast to mammals; they are regulators in signaling pathways that control actin organization and morphogenetic processes. In yeast, Rho GTPases are implicated in cell polarity processes and cell wall biosynthesis. It is known that Rho1 and Rho2 are key proteins in the construction of the cell wall, an essential structure that in Schizosaccharomyces pombe is composed of beta-glucan, alpha-glucan, and mannoproteins. Rho1 regulates the synthesis of 1,3-beta-D-glucan by activation of the 1,3-beta-D-glucan synthase, and Rho2 regulates the synthesis of alpha-glucan by the 1,3-alpha-D-glucan synthase Mok1. Here we describe the characterization of another Rho GTPase in fission yeast, Rho4. rho4Delta cells are viable but display cell separation defects at high temperature. In agreement with this observation, Rho4 localizes to the septum. Overexpression of rho4(+) causes lysis and morphological defects. Several lines of evidence indicate that both rho4(+) deletion or rho4(+) overexpression result in a defective cell wall, suggesting an additional role for Rho4 in cell wall integrity. Rho4Delta cells also accumulate secretory vesicles around the septum and are defective in actin polarization. We propose that Rho4 could be involved in the regulation of the septum degradation during cytokinesis.

Alleles↗
PubMed2002

A comparison between two brine shrimp assays to detect in vitro cytotoxicity in marine natural products.

BACKGROUND: The brine shrimp lethality assay is considered a useful tool for preliminary assessment of toxicity. It has also been suggested for screening pharmacological activities in plant extracts. However, we think that it is necessary to evaluate the suitability of the brine shrimp methods before they are used as a general bio-assay to test natural marine products for pharmacological activity. MATERIAL AND METHODS: The bioactivity of the isopropanolic (2-PrOH) extracts of 14 species of marine invertebrates and 6 species of macroalgae was evaluated with the shrimp lethality assay (lethality assay), as well as with another assay based on the inhibition of hatching of the cyst (hatchability assay). The extracts were also assayed for cytotoxicity against two human cell lines, lung carcinoma A-549 and colon carcinoma HT-29, in order to assess the sensitivity of the shrimp assays to detect cytotoxic activity. RESULTS: Two sponges (Hyatella sp, Dysidea sp.), two gorgonians (Pacifigorgia adamsii, Muricea sp.), one tunicate (Polyclinum laxum), and three echinoderms (Holothuria impatiens, Pseudoconus californica and Pharia pyramidata) showed a strong cytostatic (growth inhibition) and cytotoxic effect. The hatchability assay showed a strong activity in 4 of the species active against the two human cell lines tested (Hyatella sp, Dysidea sp., Pacifigorgia adamsii and Muricea sp.), and the lethality assay also showed a high lethality in 4 of them (Pacifigorgia adamsii, Muricea sp., Polyclinum laxum, and Pharia pyramidata). Each bioassay detected activity in 50% of the species that were considered active against the two human cell lines tested. However, the simultaneous use of both bioassays increased the percentage to 75%. CONCLUSIONS: Our results seem consistent with the correlation previously established between cytotoxicity and brine shrimp lethality in plant extracts. We suggest using both bioassays simultaneously to test natural marine products for pharmacological activity.

Animals↗
PubMed2002

Use of the pseudophase model in the interpretation of reactivity under restricted geometry conditions. An application to the study of the [Ru(NH(3))(5)pz](2+) + S(2)O(8)(2-) electron-transfer reaction in different microheterogeneous systems.

The title reaction has been studied under restricted geometry conditions, comprising aqueous solutions of a polymer (DNA), cyclodextrins, and dendrimers as well as (CTA)Cl micellar solutions and sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/water/oil microemulsions. The results are interpreted by taking as a general basis the pseudophase model, modified in some cases in order to take into account the specificity of the reaction medium. These modifications describe the anti-cooperative character of the binding of the substrates to the surfaces and the changes of the electric potential at the surfaces. The conclusion is that the pseudophase model is a powerful tool for the interpretation of kinetic data in the reaction media considered in this work, provided that the specificity of these media were incorporated into the model.

Cyclodextrins↗
PubMed2002

The concerted mechanism of photo-induced biprotonic transfer in 7-azaindole dimers: structure, quantum-theoretical analysis, and simultaneity principles.

Six stable dimer models for 7-azaindole (including the classic C(2h) doubly hydrogen-bonded, coplanar, centrosymmetric dimer) are considered to be observable in adiabatic nozzle jet molecular beams. They are analyzed by hybrid density functional theory (DFT), the MP2 ab initio method for the ground electronic state, and the single-excitation configuration interaction (CIS) (over frozen ground state optimized geometries obtained from DFT) excited state calculations, for global potential minima and proton-transfer potential energy curves. Three simultaneity principles are stated: (i) intermolecular coherent excitation molecular exciton simultaneity, (ii) intramolecular acid-base change simultaneity at the pyrrolo-N-H and aza-N proton-donor, proton-acceptor sites, and (iii) intermolecular simultaneity of catalytic proton-donor, proton-acceptor action. It is suggested that the formation of the classic C(2h) dimer of 7-azaindole, which is considered exclusively by previous researchers, can be formed from at least one of the several card-pack hydrogen-bonded dimers in a secondary slower step approaching a microsecond scale, instead of the picosecond events at the supersonic nozzle. It is proposed that the complexity of dimerization modes is the basis of the postexcitation, postionization diverse kinetic isotope results.

Catalysis↗