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P Herrero

Publications and source records attributed to P Herrero.

At least 19 recordsLinked to original sources
PubMed2006

Tpk3 and Snf1 protein kinases regulate Rgt1 association with Saccharomyces cerevisiae HXK2 promoter.

Hexokinase 2 is an essential factor for signalling repression through the Saccharomyces cerevisiae high-glucose sensing pathway. The main regulatory mechanism that controls the HXK2 gene expression in yeast is mediated by the Rgt1 and Med8 transcription factors, which repress HXK2 expression in low-glucose containing media. In this study, we show that the repression activity of Rgt1 is regulated by Snf1 and Tpk3 protein kinases. Binding of Rgt1 to the HXK2 promoter requires Rgt1 phosphorylation by Snf1 or by an Snf1-dependent protein kinase. Conversely, Rgt1 hyperphosphorylation by the Tpk3 or by a Tpk3-dependent protein kinase dissociates Rgt1 from the repressor complex. Two-hybrid and chromatin immunoprecipitation experiments indicate that an Snf1-dependent interaction between Rgt1 and Med8 in the repressor complex is also essential for Rgt1 repression. The repression of HXK2 transcription by Rgt1 likely occurs through the formation of a DNA loop in the HXK2 locus, spanning the promoter and coding regions. These results suggest that a novel silent-chromatin loop is responsible for Rgt1-dependent transcriptional regulation of the HXK2 gene.

Binding Sites↗
PubMed2006

[Renal function and cardiovascular risk in patients with essential hypertension. The "FRESHA" study].

BACKGROUND: In the past few years there has been a growing amount of information about renal dysfunction and cardiovascular risk. The objectives of this study were to assess the prevalence of renal dysfunction and evaluate the relation between renal function and cardiovascular risk in patients with essential hypertension. METHODS: A multicenter, cross-sectional survey of unselected patients with essential hypertension attending primary care settings in Spain was performed between june and november 2004. Renal function was evaluated with the abbreviated equation of the Modification of Diet in Renal Disease study. Renal insufficiency was defined as an estimated glomerular filtration rate <60 ml/min/1.73 m2. RESULTS: Eighty-eight investigators from 50 centers recruited 2130 patients being mean age 65.6 +/- 11 years and female 53%. Prevalence of diabetes, lipid abnormalities, and previous cardiovascular disease were 30.3%, 45.9%, and 42.1% respectively. Prevalence of renal insufficiency was 32.4% (95% CI 30.4-34.4). Patients suffering from renal insufficiency showed a higher prevalence of cardiovascular disease when comparing with those with an estimated glomerular filtration rate = or >60 ml/min/1.73 m2 (56.2% vs. 35.3%, OR 2.35, 95% CI 1.95-2.82, p < 0.001). A logistic regression analysis showed that the relation of renal dysfunction with cardiovascular disease was independent of other variables or classical cardiovascular risk factors as age, female sex, diabetes, smoking, hypercholesterolemia, and systolic blood pressure. CONCLUSIONS: Renal insufficiency was present in 32.4% of patients with essential hypertension attending primary care settings. Cases with renal dysfunction showed a higher cardiovascular risk. Hypertensive patients with renal insufficiency should be considered as candidates for an aggressive approach of cardiovascular risk management.

Aged↗
PubMed2005

Glucose sensing through the Hxk2-dependent signalling pathway.

In this work, we describe the hexokinase 2 (Hxk2) signalling pathway within the yeast cell. Hxk2 and Mig1 are the two major factors of glucose repression in Saccharomyces cerevisiae. The functions of both proteins have been extensively studied but there is no information about possible interactions among them in the repression pathway. Our results demonstrate that Hxk2 interacts directly with Mig1 in vivo and in vitro and that the ten amino acids motif between K6 and M15 is required for their interaction. This interaction has been detected at the DNA level both in vivo by chromatin immunoprecipitation experiments and in vitro using purified proteins and a DNA fragment containing the MIG1 site of the SUC2 promoter. This demonstrates that the interaction is of physiological relevance. Our findings show that the main role of Hxk2 in the glucose signalling pathway is the interaction with Mig1 to generate a repressor complex located in the nucleus of S. cerevisiae.

Cell Nucleus↗
PubMed2003

Textured hydroxyapatite interface onto biomedical titanium-based coatings.

Hydroxyapatite (HAP) bioceramic coatings grown onto titanium-nitride (TiN) buffer layers by the aerosol-gel procedure present interfaces with a preferred growth orientation. These coatings were crystallized at 800 degrees C and subsequently etched to ease the study of the interface by Auger electron spectroscopy depth profiling. Ion beam milling was applied to cross-section samples to analyze the interface structures using transmission electron microscopy. At the interface, the HAP crystals showed a <002> orientation. It was shown by Auger electron spectroscopy depth profiling that O atoms diffuse into the nitride interlayer, indicating that the formation of O channels in the HAP structure is the driving force inducing the textured film. The outstanding biocompatible properties of both the materials and properties of their interface suggest that HAP/TiN structures are particularly well suited for endoprosthetic applications.

Coated Materials, Biocompatible↗
PubMed2002

Mediator factor Med8p interacts with the hexokinase 2: implication in the glucose signalling pathway of Saccharomyces cerevisiae.

In the presence of glucose the protein hexokinase 2 (Hxk2p), normally resident in the cytosol, is translocated to the nucleus where it impairs the activation of transcription of the glucose-repressed genes HXK1, GLK1 and SUC2, and promotes the activation of transcription of the glucose-induced genes HXK2 and HXT1. Here, we demonstrate the involvement of an heptameric motif, named the MED8 site, in the direct binding of the mediator protein Med8p, either as a monomer or as a homodimer. Because this site was previously involved in the Hxk2p-dependent glucose-induced regulation of gene transcription, we tested whether Hxk2p interacts with Med8p. Our results show that Hxk2 and Med8 proteins are physically associated and that this Hxk2p-Med8p interaction is of physiological significance because both proteins have been found interacting together in a cluster with DNA fragments containing the MED8 site. We conclude that Hxk2p operates through the MED8 site, by interacting with Med8p, in the glucose signal transduction pathway of Saccharomyces cerevisiae.

DNA-Binding Proteins↗
PubMed2001

The hexokinase 2 protein regulates the expression of the GLK1, HXK1 and HXK2 genes of Saccharomyces cerevisiae.

The key glycolytic HXK2 gene, coding for the enzyme hexokinase 2 (Hxk2p), is expressed when cells of the yeast Saccharomyces cerevisiae are grown on a fermentable medium using glucose, fructose or mannose as a carbon source. After shifting the cells to a non-fermentable carbon source, the HXK2 gene is repressed and the HXK1 and GLK1 genes are rapidly de-repressed, producing the enzymes hexokinase 1 (Hxk1p) and glucokinase (Glk1p) respectively. Because the in vivo functions of the Hxk1p and Glk1p enzymes have remained a mystery so far, we have investigated this glucose-induced regulatory process. Here we demonstrate the involvement of Hxk2p in the glucose-induced repression of the HXK1 and GLK1 genes and the glucose-induced expression of the HXK2 gene. We have also demonstrated the involvement of Hxk1p as a negative factor in the expression of the GLK1 and HXK2 genes. Further experimental evidence, using mutant cells expressing a truncated version of Hxk2p unable to enter the nucleus, shows that nuclear localization of Hxk2p is necessary for glucose-induced repression signalling of the HXK1 and GLK1 genes and for glucose-induced expression of the HXK2 gene. Gel mobility-shift analysis shows that Hxk2p-mediated regulation is exerted through ERA (ethanol repression autoregulation)-like regulatory sequences present in the HXK1 and GLK1 promoters and in two downstream repressing sequences of the HXK2 gene. These findings reveal a novel mechanism of gene regulation whereby the product of a glycolytic gene, normally resident in the cytosol, interacts directly with nuclear proteins to regulate the transcription of the HXK1 and GLK1 genes and to autoregulate its own transcription.

DNA↗
PubMed2001

Imaging and mapping protein-binding sites on DNA regulatory regions with atomic force microscopy.

Regulation of gene expression is fundamental in biological systems. A systematic search for protein binding sites in gene promoters has been done in recent years. Biochemical techniques are easy and reliable when analysing protein interactions with short pieces of DNA, but are difficult and tedious when long pieces of DNA have to be analysed. Here we propose AFM as a reliable and easy technique for identifying protein interaction sites in long DNA molecules like gene promoters. We support this idea using a well-known model: the interaction of the Pho4 protein with the PHO5 gene promoter. We have also applied the technique to demonstrate that Mig1 protein binds to two motifs in the promoter of HXK2 gene. Our results allow us to define Mig1p as a new factor probably contributing to the carbon source-dependent transcription regulation of HXK2 gene.

Amino Acid Sequence↗
PubMed2001

Characterization of altered myocardial fatty acid metabolism in patients with inherited cardiomyopathy.

Inherited defects in myocardial long-chain fatty acid metabolism are increasingly recognized as a cause of cardiomyopathy and sudden death in children. To evaluate whether the phenotypic expression of these genetic diseases could be delineated using positron emission tomography (PET), 11 patients with inherited defects in fatty acid metabolism were evaluated and results were compared with those of 6 nonaffected siblings. Myocardial perfusion, myocardial oxygen consumption (MVO2), and long-chain fatty acid metabolism were determined noninvasively with PET using quantitative mathematical models. There were no differences in haemodynamics, perfusion, MVO2 or plasma substrate levels between groups. Patients with defects in enzymes of fatty acid beta-oxidation (acyl-CoA dehydrogenase and 3-hydroxyacyl-CoA dehydrogenase deficiencies) (n = 5) had diminished myocardial palmitate oxidation compared with healthy siblings (3.2 +/- 3.0 vs. 13.0 +/- 5.6 nmol/g per min, p < 0.03) and a decrease in the percentage of MVO2 accounted for by palmitate (2% +/- 3% vs. 9% +/- 5%, p < 0.04). In these patients, extracted palmitate was shunted into a slow-turnover compartment (predominantly reflecting esterification to triglycerides) with expansion of palmitate in that pool (185 +/- 246 compared with 27 +/- 67 nmol/g in healthy siblings,p < 0.02). In contrast, myocardium of patients with carnitine deficiency (n = 6) (all on oral carnitine therapy) had normal palmitate extraction but expansion of the interstitial/cytosolic fatty acid pool (617 +/- 399 vs. 261 +/- 73 nmol/g in healthy siblings, p < 0.04), suggesting different mechanisms for handling upstream fatty acyl intermediates. Thus, PET can be used to noninvasively assess abnormal myocardial handling of fatty acids in patients with inherited defects of metabolism. This approach should be useful in the assessment of altered myocardial fatty acid metabolism associated with cardiomyopathy as well as for evaluating the efficacy of therapeutic interventions in affected patients.

Acetates↗
PubMed2001

Short-term oral estrogen replacement therapy does not augment endothelium-independent myocardial perfusion in postmenopausal women.

OBJECTIVES: The purpose of this study was to determine the effect of usual-dose estrogen replacement therapy (ERT) on myocardial perfusion and myocardial perfusion reserve (MPR) (evoked by an endothelium-independent vasodilator) in healthy postmenopausal women. Postmenopausal women have a decreased myocardial perfusion reserve compared with younger women. Estrogen infusions are known to enhance endothelium-dependent vasodilation of the epicardial coronary arteries in postmenopausal women, but whether ERT also enhances endothelium-independent myocardial perfusion and perfusion reserve is unclear. METHODS: In 24 healthy postmenopausal women who were not taking ERT, myocardial perfusion at rest, perfusion during the infusion of adenosine (a primarily endothelium-independent vasodilator), and MPR were determined by positron-emission tomography (PET) and oxygen 15-labeled water. The women were then randomly assigned in a double-blind fashion to receive either 0.625 mg of oral conjugated estrogens (Premarin) or placebo per day for 4 to 6 weeks, after which they underwent a repeat cardiac PET study. RESULTS: There was no statistical difference between those assigned to ERT and those assigned to placebo in the measurement of myocardial perfusion at rest (1.21 +/- 0.31 vs 1.16 +/- 0.18 mL/g/min, respectively) in response to adenosine (2.66 +/- 0.96 vs 3.3 +/- 0.45 mL/g/min) or MPR (2.24 +/- 0.83 vs 2.88 +/- 0.64 mL/g/min) after 4 to 6 weeks of oral ERT. There was also no difference between the groups in any of the myocardial perfusion measurements after correction for the rate-pressure product. CONCLUSIONS: Short-term oral ERT does not affect myocardial perfusion at rest in response to adenosine or MPR in healthy postmenopausal women. Thus potential beneficial effects of ERT on vasomotor function may be limited to enhancement of endothelium-dependent vasodilative mechanisms affecting conduit vessels.

Adenosine↗
PubMed2001

A novel mouse model of lipotoxic cardiomyopathy.

Inherited and acquired cardiomyopathies are associated with marked intracellular lipid accumulation in the heart. To test the hypothesis that mismatch between myocardial fatty acid uptake and utilization leads to the accumulation of cardiotoxic lipid species, and to establish a mouse model of metabolic cardiomyopathy, we generated transgenic mouse lines that overexpress long-chain acyl-CoA synthetase in the heart (MHC-ACS). This protein plays an important role in vectorial fatty acid transport across the plasma membrane. MHC-ACS mice demonstrate cardiac-restricted expression of the transgene and marked cardiac myocyte triglyceride accumulation. Lipid accumulation is associated with initial cardiac hypertrophy, followed by the development of left-ventricular dysfunction and premature death. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining and cytochrome c release in transgenic hearts suggest that cardiac myocyte death occurs, in part, by lipid-induced programmed cell death. Taken together, our data demonstrate that fatty acid uptake/utilization mismatch in the heart leads to accumulation of lipid species toxic to cardiac myocytes. This novel mouse model will provide insight into the role of perturbations in myocardial lipid metabolism in the pathogenesis of inherited and acquired forms of heart failure.

Animals↗
PubMed2000

Absence of dc-conductivity in lambda-DNA.

The electrical conductivity of biomaterials on a molecular scale is of fundamental interest in the life sciences. We perform first principles electronic structure calculations, which clearly indicate that lambda-DNA chains should present large resistance values. We also present two direct procedures to measure electrical currents through DNA molecules adsorbed on mica. The lower limit for the resistivity is 10(6) Omega . cm, in agreement with our calculations. We also show that low energy electron bombardment induces a rapid contamination and dramatically affects the measured conductivity, thus providing an explanation to recent reports of high DNA conductivity.

Animals↗
PubMed2000

Electrodeposition of hydroxyapatite coatings in basic conditions.

Hydroxyapatite films have been grown in this work by an electrodeposition method involving both physical and chemical processes and presenting several differences with respect to other reported works. Description of the coating formation is based on the evolution of current through the sample placed as positive electrode in the basic electrolyte. The characterisation of hydroxyapatite films is of special importance since the bioactive properties related to HAP have been directly identified with its specific composition (Ca/P ratio) and crystalline structure. This characterisation has been traditionally fulfilled by the use of XRD, FTIR and SEM. Results of a further characterisation of the coatings by TEM and SFM, additional to the analysis by XRD, FTIR and SEM, are presented. Interpretation and comparison of our results with those obtained by other electrodeposition methods lead to arguments in favour of a deposition produced directly from ionic species.

Electrochemistry↗
PubMed2000

Identification of weak interfaces in composites using transmission electron microscopy

A new experimental technique was developed to identify crack paths with a resolution of nanometres in fibre-reinforced composites. Cracks were introduced through Vickers indentations on one side of the sample prior to starting the thinning process. Indentations were placed close to the fibres in order to get enough cracks at the fibre/matrix interface in the electron-transparent region of the thinned sample. The technique was used in a Nicalon-fibre Al2O3 matrix composite prior to and after a heat treatment at 1200 degrees C for 1 h. The analysis of the crack paths allowed the identification of the weakest interface in each condition.

Journal Article↗
PubMed1999

Analysis by atomic force microscopy of Med8 binding to cis-acting regulatory elements of the SUC2 and HXK2 genes of saccharomyces cerevisiae.

Med8 protein is a regulator that specifically binds to upstream activating sequences (UASs) of SUC2 promoter, to downstream repressing sequences (DRSs) of the HXK2 gene and to the carboxy-terminal domain of the RNA polymerase II. Atomic force microscopy has allowed for direct visualization of Med8 interactions with a 305 bp fragment of SUC2 promoter and with a 676 bp fragment of HXK2 gene, containing respectively the UASs and DRSs regulatory regions. This approach has provided complementary information about the position and the structure of the DNA-protein complexes. Med8 binding to DNA results in total covering of one of the two existing 7 bp motives (consensus, (A/C)(A/G)GAAAT) in the studied DNA fragments. No preference for binding either of the two UASs of SUC2 promoter as well as for the two DRSs of HXK2 gene has been found. We also discuss whether this protein works as dimer or as a monomer.

Carrier Proteins↗
PubMed1999

Functional characterization of transcriptional regulatory elements in the upstream region of the yeast GLK1 gene.

The glucokinase gene GLK1 of the yeast Saccharomyces cerevisiae is transcriptionally regulated in response to the carbon source of the growth medium. Northern-blot analysis shows that the GLK1 gene is expressed at a basal level in the presence of glucose, de-repressed more than 6-fold under conditions of sugar limitation and more than 25-fold under conditions of ethanol induction. lacZ fusions of the GLK1 gene promoter were constructed and a deletion analysis was performed in order to identify the cis-acting regulatory elements of the promoter that controls GLK1 gene expression. First, the expression seemed to be mediated mainly by one GCR1 and three stress-responsive element (STRE) activating elements. Secondly, an ethanol repression autoregulation (ERA)/twelve-fold TA repeat (TAB) repressor element was identified within the promoter region of the GLK1 gene. A specific and differential protein binding to the STRE was observed with extracts from de-repressed and repressed cells. No differential binding to the GCR1 or ERA/TAB elements was observed with extracts from de-repressed and repressed cells, but, in both cases, the binding was competed for by an excess of the unlabelled GLK1(GCR1) and GLK1(ERA) sequence. The transcription factors Msn2 and Msn4, which bind to the GLK1 upstream region through the STRE, contribute to inductive activation. The transcription factor Gcr1, which binds through the GCR1 element, contributes to constitutive activation. In order to achieve the severe glucose repression of GLK1, constitutive repressor factors acting through the ERA/TAB element must counteract constitutive activation generated by Gcr1 binding to the GCR1 element. Full expression of the GLK1 gene is produced by inductive activation of three STRE when Msn2 and Msn4 proteins are translocated to the nucleus by covalent modification. The combinatorial effect of the entire region leads to the regulated transcription of GLK1, i.e., silent in media with glucose and other preferred carbon sources, such as fructose or mannose, and increased levels of expression upon glucose depletion.

Base Sequence↗
PubMed1999

Med8, a subunit of the mediator CTD complex of RNA polymerase II, directly binds to regulatory elements of SUC2 and HXK2 genes.

In a search to identify new factors required for expression of SUC2 gene in Saccharomyces cerevisiae, we have partially purified a 27 kDa protein (p27) that bound both the DRSs of the HXK2 gene and the UASs of SUC2 gene. The amino terminal sequence of p27 identified the MED8 gene (open reading frame YBR193C), located in chromosome II of S. cerevisiae, as the gene coding for the protein. Disruption of this gene has demonstrated that is an essential gene for yeast growth. To determine whether the p27 protein represents the Med8 product, we expressed MED8 gene in E. coli and demonstrated that the heterologous synthesized protein specifically binds to both UASSUC2 and DRS2HXK2. This observation suggests that Med8 may be important for the coupling of the glucose repression pathway of SUC2 gene to the HXK2 gene expression. Med8 has been described as a mediator protein interacting with the CTD of the RNA polymerase II. Thus, the role of Med8 could be to act as coupling factor by linking activating and repressing transcription complexes to the RNA polymerase II holoenzyme transcriptional machinery.

Chromosome Mapping↗
PubMed1999

Evaluation of gallium-68 tris(2-mercaptobenzyl)amine: a complex with brain and myocardial uptake.

Previous research into development of a gallium-radiolabeled agent that crosses the blood-brain barrier has met with limited success. In this study, we focused our attention on a Ga(III) complex of a 4-coordinate amine trithiolate tripod ligand, tris(2-mercaptobenzyl) amine (S3N). The Ga(III) S3N complex is small, neutral, and lipophilic, meeting the requirements for a potential brain imaging agent. The Ga-68 complex was easily formed with a radiochemical purity of >95%. In vitro stability of the Ga-S3N complex, determined in rat serum incubated at 37 degrees C, was greater than 95% intact at 2 h by silica gel and reversed-phase radio-thin layer chromatography. Biodistribution studies conducted in female Sprague-Dawley rats showed the complex cleared rapidly from the blood with initial high liver uptake followed by rapid washout. Significant uptake was observed in the brain, with brain:blood ratios increasing from 0.11 at 2 min postinjection to 3.8 at 60 min postinjection. Uptake was also observed in the heart going from a heart:blood ratio of 2.3 at 2 min postinjection to 11 at 60 min postinjection. Molecular mechanics were used to determine the coordination number, and demonstrated that the Ga(III) complex prefers to be 4-coordinate. Imaging studies with 68Ga-S3N in a Nemestrina macaque showed significant brain uptake, similar to other lipophilic agents. The extraction of 68Ga-S3N into the brains of both rodents and primates, higher than any 68Ga agent reported in the literature, suggests that this compound may have potential as a brain imaging agent for positron emission tomography.

Animals↗