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R Serrano

Publications and source records attributed to R Serrano.

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

Multiresidue determination of persistent organochlorine and organophosphorus compounds in whale tissues using automated liquid chromatographic clean up and gas chromatographic-mass spectrometric detection.

A multiresidue method based on normal-phase LC for the sample clean up of whale tissues extracts prior to GC-MS determination of residues of polychlorinated biphenyls, organochlorine pesticides and derivatives and lipophylic organophosphorus pesticides has been developed. Pesticides were extracted from blubber by fusing and dissolving the fat in n-hexane and from liver and kidney by reflux in n-hexane. Hexanic extracts were directly injected on the silicagel column of the automated LC clean up system, using n-hexane as mobile phase. Diode array detection allowed the on-line monitoring of lipids elution from the LC system. Purified extracts were analysed by GC using mass selective detection. The developed procedure was applied to different tissues from a whale specimen appeared in the Valencian coast, finding high concentrations of OCs (up to 7.3 micrograms g-1 pp'-DDE, and 7.2 micrograms g-1 PCBs). The method was validated by means of recovery tests for all the compounds detected in the whale and also for some other OCs and OPs studied in this paper. The method improves other current methods for the analysis of persistent organochlorines in marine mammals with regard to time of analysis, solvent expend and automation; solvent exchanges are not necessary before GC analysis, and it allows the simultaneous determination of organophosphorus pesticides.

Animals↗
PubMed1999

A novel mammalian lithium-sensitive enzyme with a dual enzymatic activity, 3'-phosphoadenosine 5'-phosphate phosphatase and inositol-polyphosphate 1-phosphatase.

We report the molecular cloning in Rattus norvegicus of a novel mammalian enzyme (RnPIP), which shows both 3'-phosphoadenosine 5'-phosphate (PAP) phosphatase and inositol-polyphosphate 1-phosphatase activities. This enzyme is the first PAP phosphatase characterized at the molecular level in mammals, and it represents the first member of a novel family of dual specificity enzymes. The phosphatase activity is strictly dependent on Mg2+, and it is inhibited by Ca2+ and Li+ ions. Lithium chloride inhibits the hydrolysis of both PAP and inositol-1,4-bisphosphate at submillimolar concentration; therefore, it is possible that the inhibition of the human homologue of RnPIP by lithium ions is related to the pharmacological action of lithium. We propose that the PAP phosphatase activity of RnPIP is crucial for the function of enzymes sensitive to inhibition by PAP, such as sulfotransferase and RNA processing enzymes. Finally, an unexpected connection between PAP and inositol-1,4-bisphosphate metabolism emerges from this work.

Amino Acid Sequence↗
PubMed1999

A proposal for nomenclature of aldehyde dehydrogenases in Saccharomyces cerevisiae and characterization of the stress-inducible ALD2 and ALD3 genes.

The complete sequencing of the genome of Saccharomyces cerevisiae indicated that this organism contains five genes encoding aldehyde dehydrogenases. YOR374w and YER073w correspond to the mitochondrial isoforms and we propose as gene names ALD4 and ALD5, respectively. YPL061w has been described as the cytoplasmic constitutive isoform and named ALD6. We characterize here the tandem-repeated ORFs YMR170c and YMR169c as the cytoplasmic stress-inducible isoforms, with gene names ALD2 and ALD3, respectively. The expression of ALD2 and ALD3 is dependent on the general-stress transcription factors Msn2,4 but independent of the HOG MAP kinase pathway. ALD3 is induced by a variety of stresses, including osmotic shock, heat shock, glucose exhaustion, oxidative stress and drugs. ALD2 is only induced by osmotic stress and glucose exhaustion. A double null mutant, ald2 ald3, exhibited unchanged sensitivity to any of the above stresses. The only phenotype detected in this mutant was a reduced growth rate in ethanol medium as compared to the wild type.

Aldehyde Dehydrogenase↗
PubMed1999

The yeast inositol monophosphatase is a lithium- and sodium-sensitive enzyme encoded by a non-essential gene pair.

Inositol monophosphatases (IMPases) are lithium-sensitive enzymes that participate in the inositol cycle of calcium signalling and in inositol biosynthesis. Two open reading frames (YHR046c and YDR287w) with homology to animal and plant IMPases are present in the yeast genome. The two recombinant purified proteins were shown to catalyse inositol-1-phosphate hydrolysis sensitive to lithium and sodium. A double gene disruption had no apparent growth defect and was not auxotroph for inositol. Therefore, lithium effects in yeast cannot be explained by inhibition of IMPases and inositol depletion, as suggested for animal systems. Overexpression of yeast IMPases increased lithium and sodium tolerance and reduced the intracellular accumulation of lithium. This phenotype was blocked by a null mutation in the cation-extrusion ATPase encoded by the ENA1/PMR2A gene, but it was not affected by inositol supplementation. As overexpression of IMPases increased intracellular free Ca2+, it is suggested that yeast IMPases are limiting for the optimal operation of the inositol cycle of calcium signalling, which modulates the Ena1 cation-extrusion ATPase.

Amino Acid Sequence↗
PubMed1999

The Arabidopsis HAL2-like gene family includes a novel sodium-sensitive phosphatase.

The yeast HAL2 gene encodes a lithium- and sodium-sensitive phosphatase that hydrolyses 3'-phosphoadenosine-5'-phosphate (PAP). Salt toxicity in yeast results from Hal2 inhibition and accumulation of PAP, which inhibits sulphate assimilation and RNA processing. We have investigated whether the model plant Arabidopsis thaliana contains sodium-sensitive PAP phosphatases. The Arabidopsis HAL2-like gene family is composed of three members: AtAHL and AtSAL2, characterized in the present work, and the previously identified AtSAL1. The AtAHL and AtSAL2 cDNAs complement the auxotrophy for methionine of the yeast hal2 mutant and the recombinant proteins catalyse the conversion of PAP to AMP in a Mg(2+)-dependent reaction sensitive to inhibition by Ca2+ and Li+. The PAP phosphatase activity of AtAHL is sensitive to physiological concentrations of Na+, whereas the activities of AtSAL1 and AtSAL2 are not. Another important difference is that AtAHL is very specific for PAP while AtSAL1 and AtSAL2 also act as inositol polyphosphate 1-phosphatases. AtAHL constitutes a novel type of sodium-sensitive PAP phosphatase which could act co-ordinately with plant sulphotransferases and serve as target of salt toxicity in plants.

Amino Acid Sequence↗
PubMed1999

Arabidopsis thaliana AtHAL3: a flavoprotein related to salt and osmotic tolerance and plant growth.

We have isolated two Arabidopsis thaliana genes, AtHAL3a and AtHAL3b, showing homology with HAL3, a yeast protein which regulates the cell cycle and tolerance to salt stress through inhibition of the PPZ1 type-1 protein phosphatase. Expression of AtHAL3a in yeast hal3 mutants partially complements their LiCl sensitivity, suggesting possible conserved functions between both proteins. AtHAL3a and AtHAL3b are induced by salt stress and AtHAL3a is the most expressed in non-stressed plants, particularly in seeds. In situ hybridization demonstrates enrichment of AtHAL3a mRNA in seed embryos and in the vascular phloem of different plant tissues. AtHAL3 proteins show striking homology with a group of proteins found in fungi, plants and animals and some homology with a large family of prokaryotic flavoproteins. Recombinant AtHAL3a protein purified from Escherichia coli was yellow because it contained a non-covalently bound chromophore revealed as flavin mononucleotide. Trans- genic Arabidopsis plants, with gain of AtHAL3a function, show altered growth rates and improved tolerance to salt and osmotic stress.

Amino Acid Sequence↗
PubMed1999

Repressors and upstream repressing sequences of the stress-regulated ENA1 gene in Saccharomyces cerevisiae: bZIP protein Sko1p confers HOG-dependent osmotic regulation.

The yeast ENA1/PMR2A gene encodes a cation extrusion ATPase in Saccharomyces cerevisiae which is essential for survival under salt stress conditions. One important mechanism of ENA1 transcriptional regulation is based on repression under normal growth conditions, which is relieved by either osmotic induction or glucose starvation. Analysis of the ENA1 promoter revealed a Mig1p-binding motif (-533 to -544) which was characterized as an upstream repressing sequence (URSMIG-ENA1) regulated by carbon source. Its function was abolished in a mig1 mig2 double-deletion strain as well as in either ssn6 or tup1 single mutants. A second URS at -502 to -513 is responsible for transcriptional repression regulated by osmotic stress and is similar to mammalian cyclic AMP response elements (CREs) that are recognized by CREB proteins. This URSCRE-ENA1 element requires for its repression function the yeast CREB homolog Sko1p (Acr1p) as well as the integrity of the Ssn6p-Tup1p corepressor complex. When targeted to the GAL1 promoter by fusing with the Gal4p DNA-binding domain, Sko1p acts as an Ssn6/Tup1p-dependent repressor regulated by osmotic stress. A glutathione S-transferase-Sko1 fusion protein binds specifically to the URSCRE-ENA1 element. Furthermore, a hog1 mitogen-activated protein kinase deletion strain could not counteract repression on URSCRE-ENA1 during osmotic shock. The loss of SKO1 completely restored ENA1 expression in a hog1 mutant and partially suppressed the osmotic stress sensitivity, qualifying Sko1p as a downstream effector of the HOG pathway. Our results indicate that different signalling pathways (HOG osmotic pathway and glucose repression pathway) use distinct promoter elements of ENA1 (URSCRE-ENA1 and URSMIG-ENA1) via specific transcriptional repressors (Sko1p and Mig1/2p) and via the general Ssn6p-Tup1p complex. The physiological importance of the relief from repression during salt stress was also demonstrated by the increased tolerance of sko1 or ssn6 mutants to Na+ or Li+ stress.

Adenosine Triphosphatases↗
PubMed1999

A novel mechanism of ion homeostasis and salt tolerance in yeast: the Hal4 and Hal5 protein kinases modulate the Trk1-Trk2 potassium transporter.

The regulation of intracellular ion concentrations is a fundamental property of living cells. Although many ion transporters have been identified, the systems that modulate their activity remain largely unknown. We have characterized two partially redundant genes from Saccharomyces cerevisiae, HAL4/SAT4 and HAL5, that encode homologous protein kinases implicated in the regulation of cation uptake. Overexpression of these genes increases the tolerance of yeast cells to sodium and lithium, whereas gene disruptions result in greater cation sensitivity. These phenotypic effects of the mutations correlate with changes in cation uptake and are dependent on a functional Trk1-Trk2 potassium transport system. In addition, hal4 hal5 and trk1 trk2 mutants exhibit similar phenotypes: (i) they are deficient in potassium uptake; (ii) their growth is sensitive to a variety of toxic cations, including lithium, sodium, calcium, tetramethylammonium, hygromycin B, and low pH; and (iii) they exhibit increased uptake of methylammonium, an indicator of membrane potential. These results suggest that the Hal4 and Hal5 protein kinases activate the Trk1-Trk2 potassium transporter, increasing the influx of potassium and decreasing the membrane potential. The resulting loss in electrical driving force reduces the uptake of toxic cations and improves salt tolerance. Our data support a role for regulation of membrane potential in adaptation to salt stress that is mediated by the Hal4 and Hal5 kinases.

Biological Transport↗
PubMed1998

The yeast halotolerance determinant Hal3p is an inhibitory subunit of the Ppz1p Ser/Thr protein phosphatase.

Components of cellular stress responses can be identified by correlating changes in stress tolerance with gain or loss of function of defined genes. Previous work has shown that yeast cells deficient in Ppz1 protein phosphatase or overexpressing Hal3p, a novel regulatory protein of unknown function, exhibit increased resistance to sodium and lithium, whereas cells lacking Hal3p display increased sensitivity. These effects are largely a result of changes in expression of ENA1, encoding the major cation extrusion pump of yeast cells. Disruption or overexpression of HAL3 (also known as SIS2) has no effect on salt tolerance in the absence of PPZ1, suggesting that Hal3p might function upstream of Ppz1p in a novel signal transduction pathway. Hal3p is recovered from crude yeast homogenates by using immobilized, bacterially expressed Ppz1p fused to glutathione S-transferase, and it also copurifies with affinity-purified glutathione S-transferase-Ppz1p from yeast extracts. In both cases, the interaction is stronger when only the carboxyl-terminal catalytic phosphatase domain of Ppz1p is expressed. In vitro experiments reveal that the protein phosphatase activity of Ppz1p is inhibited by Hal3p. Overexpression of Hal3p suppresses the reduced growth rate because of the overexpression of Ppz1p and aggravates the lytic phenotype of a slt2/mpk1 mitogen-activated protein kinase mutant (thus mimicking the deletion of PPZ1). Therefore, Hal3p might modulate diverse physiological functions of the Ppz1 phosphatase, such as salt stress tolerance and cell cycle progression, by acting as a inhibitory subunit.

Binding Sites↗
PubMed1998

The Ssn6-Tup1 repressor complex of Saccharomyces cerevisiae is involved in the osmotic induction of HOG-dependent and -independent genes.

The response of yeast to osmotic stress has been proposed to rely on the HOG-MAP kinase signalling pathway and on transcriptional activation mediated by STRE promoter elements. However, the osmotic induction of HAL1, an important determinant of salt tolerance, is HOG independent and occurs through the release of transcriptional repression. We have identified an upstream repressing sequence in HAL1 promoter (URSHAL1) located between -231 and -156. This promoter region was able to repress transcription from a heterologous promoter and to bind proteins in non-stressed cells, but not in salt-treated cells. The repression conferred by URSHAL1 is mediated through the Ssn6-Tup1 protein complex and is abolished in the presence of osmotic stress. The Ssn6-Tup1 co-repressor is also involved in the regulation of HOG-dependent genes such as GPD1, CTT1, ALD2, ENA1 and SIP18, and its deletion can suppress the osmotic sensitivity of hog1 mutants. We propose that the Ssn6-Tup1 repressor complex might be a general component in the regulation of osmostress responses at the transcriptional level of both HOG-dependent and -independent genes.

Base Sequence↗
PubMed1998

Yeast putative transcription factors involved in salt tolerance.

Four putative yeast transcription factors (Hal6-9p) have been identified which upon overexpression in multicopy plasmids increase sodium and lithium tolerance. This effect is mediated, at least in part, by increased expression of the Enalp Na+/Li+ extrusion pump. Hal6p and Hal7p are bZIP proteins and their gene disruptions affected neither salt tolerance nor ENA1 expression. Hal8p and Hal9p are putative zinc fingers and their gene disruptions decreased both salt tolerance and ENA1 expression. Therefore, Hal8p and Hal9p, but not Hal6p and Hal7p, qualify as transcriptional activators of ENA1 under physiological conditions. Hal8p seems to mediate the calcineurin-dependent part of ENA1 expression.

Adaptation, Physiological↗
PubMed1998

Familial mediterranean fever and acute myocardial infarction secondary to coronary vasculitis.

AIMS: We report a case study to elucidate the pathogenesis of polyarteritis nodosa (PAN) type vasculitis, a rare complication of familial mediterranean fever (FMF). METHODS AND RESULTS: A woman with amyloidosis complicating FMF underwent a cadaveric renal transplantation and 5 years later suffered an acute myocardial infarction secondary to an isolated coronary vasculitis. CONCLUSIONS: The histopathological findings of the vasculitis were not in keeping with PAN. We postulated that the pathogenesis of vasculitis in FMF is different from that of the classic PAN and might be similar to the mechanism of the serosal inflammation.

Adult↗
PubMed1998

Evaluation of an immunohistochemical test with polyclonal antibodies raised against mycobacteria used in formalin-fixed tissue compared with mycobacterial specific culture.

An immunohistochemical (IH) test (commercially available polyclonal antiserum rabbit anti-Myco-bacterium bovis; DAKO A/S) was used to detect the presence of mycobacteria in 65 formalin-fixed, paraffin-embedded tissue blocks from different organs, showing necrotizing caseous granuloma lesions on hematoxylin and eosin sections from 65 patients. These 65 samples were dyed using an acid-fast fluorescent technique and compared using the immunohistochemical method. Both results were also compared with the mycobacterial cultures. The IH test, compared with the culture, showed a sensitivity (S) of 52%, a specificity (Sp) of 76%, a positive predictive value (PV pos) of 61% and a negative predictive value (PV neg) of 69%. We analyze these data and discuss the possible causes of false-positive and -negative results of the IH test. This rapid test on paraffin embedded tissue seems valuable in the period when waiting for the culture results.

Animals↗
PubMed1998

Altered Na+ and Li+ homeostasis in Saccharomyces cerevisiae cells expressing the bacterial cation antiporter NhaA.

The bacterial Na+ (Li+)/H+ antiporter NhaA has been expressed in the yeast Saccharomyces cerevisiae. NhaA was present in both the plasma membrane and internal membranes, and it conferred lithium but not sodium tolerance. In cells containing the yeast Ena1-4 (Na+, Li+) extrusion ATPase, the extra lithium tolerance conferred by NhaA was dependent on a functional vacuolar H+ ATPase and correlated with an increase of lithium in an intracellular pool which exhibited slow efflux of cations. In yeast mutants without (Na+, Li+) ATPase, lithium tolerance conferred by NhaA was not dependent on a functional vacuolar H+ ATPase and correlated with a decrease of intracellular lithium. NhaA was able to confer sodium tolerance and to decrease intracellular sodium accumulation in a double mutant devoid of both plasma membrane (Na+, Li+) ATPase and vacuolar H+ ATPase. These results indicate that the bacterial antiporter NhaA expressed in yeast is functional at both the plasma membrane and the vacuolar membrane. The phenotypes conferred by its expression depend on the functionally of plasma membrane (Na+, Li+) ATPase and vacuolar H+ ATPase.

Bacterial Proteins↗
PubMed1998

Toluene alters brainstem enkephalinergic system in rats.

Acute exposure to high doses of toluene can generate respiratory depression. However, neurotoxic mechanism of its action in the brainstem is not completely clear. In this work, acute, but not subchronic, exposure of rats to toluene increased leu-enkephalin immunostaining in several myelencephalic nuclei implicated in cardiorespiratory control. Due to the physiological role of enkephalins in the central regulation of breathing, it is suggested that the enkephalinergic system could play a role in neurotoxic respiratory depression induced by high dose acute toluene exposure.

Animals↗
PubMed1998

Effects of acute lidocaine administration on the rat prosencephalic enkephalinergic system.

Lidocaine is a local anesthetic widely used in therapeutics and as antiarrhythmic agent. However, information concerning possible alterations in the enkephalinergic system after acute treatment with this substance is not complete. This being the case, we focused on analyzing enkephalin immunostaining in several regions of the rat brain after lidocaine administration. We could not find significative changes in rat prosencephalon. These data might suggest that the enkephalinergic system is not altered by lidocaine.

Anesthetics, Local↗
PubMed1997

Single point dilution method for the quantitative analysis of antibodies to the gag24 protein of HIV-1.

In the present work a concept proposed in 1992 by Dopotka and Giesendorf was applied to the quantitative analysis of antibodies to the p24 protein of HIV-1 in infected asymptomatic individuals and AIDS patients. Two approaches were analyzed, a linear model OD = b0 + b1.log(titer) and a nonlinear log(titer) = alpha.OD beta, similar to the Dopotka-Giesendorf's model. The above two proposed models adequately fit the dependence of the optical density values at a single point dilution, and titers achieved by the end point dilution method (EPDM). Nevertheless, the nonlinear model better fits the experimental data, according to residuals analysis. Classical EPDM was compared with the new single point dilution method (SPDM) using both models. The best correlation between titers calculated using both models and titers achieved by EPDM was obtained with the nonlinear model. The correlation coefficients for the nonlinear and linear models were r = 0.85 and r = 0.77, respectively. A new correction factor was introduced into the nonlinear model and this reduced the day-to-day variation of titer values. In general, SPDM saves time, reagents and is more precise and sensitive to changes in antibody levels, and therefore has a higher resolution than EPDM.

Enzyme-Linked Immunosorbent Assay↗