PubMed Health⌕ Search

Biomedical subjects

R Serrano

Publications and source records attributed to R Serrano.

At least 73 records · Page 4Linked to original sources
PubMed1997

Automated sample clean-up and fractionation of chlorpyrifos, chlorpyrifos-methyl and metabolites in mussels using normal-phase liquid chromatography.

An automated method based on normal-phase LC has been developed for the sample clean-up of mussel extracts prior to gas chromatographic analysis of residues of chlorpyrifos, chlorpyrifos-methyl and their metabolites chlorpyrifos-methyl-oxon and 3,5,6-trichloro-2-pyridinol. Pesticides were extracted by means of a high speed blender using acetonitrile-acetone (90:10, v/v). The extract obtained was filtered and concentrated using rotavapor and the residue was dissolved in hexane. One ml of the hexanoic extract was injected on the silica-gel column, using hexane as mobile phase. Pesticides and metabolites were eluted in fat-free fractions with different mixtures of hexane-ethyl acetate. Diode array detection allowed monitoring on-line the elution of lipids. Purified extracts were analyzed by GC using nitrogen-phosphorus detection for quantitation and MS for confirmatory purposes. The method is fully automated from the injection of the extract to the collection of fractions, which are directly injected into the GC system. In this way, neither further clean-up nor solvent exchange were necessary prior to GC analysis. Recoveries obtained from fortified mussel samples at two concentration levels-100 and 20 ng g-1 for parent pesticides and 200 and 40 ng g-1 for metabolites-were higher than 90%. Limits of detection of the whole procedure of analysis were lower than 1 ng g-1 for parent pesticides and than 10 ng g-1 for metabolites. This method has been successfully applied to bioconcentration studies with mussels exposed to chlorpyrifos. Chlorpyrifos and its metabolic derivative 3,5,6-trichloro-2-pyridinol were detected and confirmed by MS in analyzed samples.

Animals↗
PubMed1997

An assessment of heavy metals and boron contamination in workplace atmospheres from ceramic factories.

The occupational exposure in workplace atmospheres of ceramic factories to some metals (Ba, Cd, Co, Cr, Cu, Ni, Zn) and boron has been studied. During 1994 samples were collected at several ceramic factories from the Comunitat Valenciana (Spain) in a random fashion to avoid correlation problems regarding data analysis; factories were sampled to provide a representative cross-section. Powdered samples were treated with concentrated nitric acid according to the National Institute for Occupational Safety and Health (NIOSH) digestion procedure for the analysis of metals in air samples. Heavy metals were analyzed by AAS and boron by UV/Visible spectrophotometry using the Azomethine-H method. Concentrations found for Cd, Cr, Cu, Ni and Zn were considered very low in the environment of this industrial sector. However, Co, Ba and B were detected in several samples with concentrations above the threshold limit value (TLV), referred to airborne concentrations of substances in workplace atmospheres. Principal component analysis (PCA) has also been carried out in order to classify the workplaces studied and their potential hazard to the workers.

Air Pollutants, Occupational↗
PubMed1997

Mechanisms of salt tolerance conferred by overexpression of the HAL1 gene in Saccharomyces cerevisiae.

Overexpression of the HAL1 gene improves the tolerance of Saccharomyces cerevisiae to NaCl by increasing intracellular K+ and decreasing intracellular Na+. The effect of HAL1 on intracellular Na+ was mediated by the PMR2/ENA1 gene, corresponding to a major Na+ efflux system. The expression level of ENA1 was dependent on the gene dosage of HAL1 and overexpression of HAL1 suppressed the salt sensitivity of null mutants in calcineurin and Hal3p, other known regulators of ENA1 expression. The effect of HAL1 on intracellular K+ was independent of the TRK1 and TOK1 genes, corresponding to a major K+ uptake system and to a K+ efflux system activated by depolarization, respectively. Overexpression of HAL1 reduces K+ loss from the cells upon salt stress, a phenomenon mediated by an unidentified K+ efflux system. Overexpression of HAL1 did not increase NaCl tolerance in galactose medium. NaCl poses two types of stress, osmotic and ionic, counteracted by glycerol synthesis and sodium extrusion, respectively. As compared to glucose, with galactose as carbon source glycerol synthesis is reduced and the expression of ENA1 is increased. As a consequence, osmotic adjustment through glycerolsynthesis, a process not affected by HAL1, is the limiting factor for growth on galactose under NaCl stressed.

Adaptation, Physiological↗
PubMed1997

Transfer of the yeast salt tolerance gene HAL1 to Cucumis melo L. cultivars and in vitro evaluation of salt tolerance.

An Agrobacterium-mediated gene transfer method for production of transgenic melon plants has been optimized. The HAL1 gene, an halotolerance gene isolated from yeast, was inserted in a chimaeric construct and joined to two marker genes: a selectable-neomycin phosphotransferase-II (nptII)-, and a reporter-beta-glucuronidase (gus)-. The entire construct was introduced into commercial cultivars of melon. Transformants were selected for their ability to grow on media containing kanamycin. Transformation was confirmed by GUS assays, PCR analysis and Southern hybridization. Transformation efficiency depended on the cultivar, selection scheme used and the induction of vir-genes by the addition of acetosyringone during the cocultivation period. The highest transformation frequency, 3% of the total number of explants cocultivated, was obtained with cotyledonary explants of cv. 'Pharo'. Although at a lower frequency (1.3%), we have also succeeded in the transformation of leaf explants. A loss of genetic material was detected in some plants, and results are in accordance with the directional model of T-DNA transfer. In vitro cultured shoots from transgenic populations carrying the HAL1 gene were evaluated for salt tolerance on shoot growth medium containing 10 gl-1 NaCl. Although root and vegetative growth were reduced, transgenic HAL1-positive plants consistently showed a higher level of tolerance than control HAL1-negative plants.

Acetophenones↗
PubMed1996

The yeast HAL2 nucleotidase is an in vivo target of salt toxicity.

The yeast halotolerance gene HAL2 encodes a nucleotidase that dephosphorylates 3'-phosphoadenosine 5'-phosphate (PAP) and 3'-phosphoadenosine 5'-phosphosulfate (PAPS), intermediates of the sulfate assimilation pathway. This nucleotidase is inhibited by Na+ and Li+ but not by K+. Incubation of wild-type yeast cells with NaCl and LiCl, but not with KCl, increased intracellular PAP to millimolar concentrations. No depletion of the pool of adenine nucleotides (AMP, ADP, ATP) was observed. Other stresses such as heat shock or oxidative stress did not result in PAP accumulation. PAPS concentrations also increased during salt stress but remained lower than 0.5 microM. S-Adenosylmethionine concentrations decreased by 50%, reflecting inhibition of sulfate assimilation during salt stress. Salt-induced PAP accumulation was attenuated in a yeast strain overexpressing HAL2. This strain grew better than the wild type under salt stress. These results suggest that the cation sensitivity of the HAL2 nucleotidase is an important determinant of the inhibition of yeast growth by sodium and lithium salts. In addition to blocking sulfate assimilation by product inhibition of PAPS reductase, PAP accumulation may have other unidentified toxic effects.

Fungal Proteins↗
PubMed1996

Kinetics and energetics of trehalose transport in Saccharomyces cerevisiae.

Cells of Saccharomyces cerevisiae are able to transport trehalose against a concentration gradient, without efflux or counterflow of the labeled substrate. Uptake was inhibited by uncouplers, acetic acid, and organic mercury compounds. The addition of trehalose resulted in alkalinization of the medium. The ratio of H+ depletion to trehalose uptake by yeast cells was approximately 1:1, which indicates the existence of a trehalose-H+ symporter in these cells. The optimum pH for this active H+-trehalose symport was 5.0, and both the Km and the Vmax were negatively affected by increasing or decreasing the extracellular pH from its optimum value. Kinetic studies showed the existence of at least two different trehalose transport activities in yeast cells: a high-affinity H+-trehalose symporter (Km = 4 mM), and a low-affinity transport activity (Km > 100 mM) that could be a facilitated diffusion process. The high-affinity H+-trehalose symporter was repressed by glucose, whereas the low-affinity uptake was constitutively expressed in S. cerevisiae.

Biological Transport, Active↗
PubMed1996

Multiple transduction pathways regulate the sodium-extrusion gene PMR2/ENA1 during salt stress in yeast.

The yeast PMR2/ENA1 gene encodes an ATPase involved in sodium extrusion and induced by NaCl. At low salt concentrations (0.3 M) induction is mediated by the HOG-MAP kinase pathway, a system activated by non-specific osmotic stress. At high salt concentrations (0.8 M) induction is mediated by the protein phosphatase calcineurin and is specific for sodium. Protein kinase A and Sis2p/Hal3p modulate PMR2/ENA1 expression as negative and positive factors, respectively but Sis2p/Hal3p does not participate in the transduction of the salt signal. Salt stress decreases the level of cAMP and the resulting decrease in protein kinase A activity may contribute to HOG-mediated induction.

Adenosine Triphosphatases↗
PubMed1996

CtCdc55p and CtHa13p: two putative regulatory proteins from Candida tropicalis with long acidic domains.

The salt-tolerance gene HAL3 from Saccharomyces cerevisiae encodes a novel regulatory protein (Hal3p) which modulates the expression of the ENA1 sodium-extrusion ATPase (Ferrando et al., Mol. Cell. Biol. vol. 15, 1995, pp. 5470-5481). Hal3p contains an essential acidic domain rich in aspartates at its carboxyl terminus. We have isolated two cross-hybridizing genes from a genomic library of Candida tropicalis. One of the genes (CtHAL3) is a true homolog of HAL3 and it partially complements the salt sensitivity of a S. cerevisiae hal3 mutant. The activity of CtHAL3 was equivalent to that of an open reading frame (YKL088w) identified by genome sequencing of S. cerevisiae and with homology to HAL3. The other cross-hybridizing gene (CtCDC55) is a CDC55 homolog, encoding a protein with an internal acidic domain not present in the S. cerevisiae CDC55 product. Cdc55p is a regulatory subunit of protein phosphatase 2A and CtCDC55 complements the cold sensitivity of a S. cerevisiae cdc55 mutant. The presence of acidic domains in different putative regulatory proteins may suggest a role for this type of domain in molecular interactions.

Amino Acid Sequence↗
PubMed1996

Localization of plasma membrane H+-ATPase in nodules of Phaseolus vulgaris L.

Legume nodules have specialized transport functions for the exchange of carbon and nitrogen compounds between bacteroids and root cells. Plasma membrane-type (vanadate-sensitive) H+-ATPase energizes secondary active transporters in plant cells and it could drive exchanges across peribacteroidal and plasmatic membranes. A nodule cDNA corresponding to a major isoform of Phaseolus vulgaris H+-ATPase (designated BHA1) has been cloned. BHA1 is a functional proton pump because after removal of its inhibitory domain and can complement a yeast mutant unable to synthesize a H+-ATPase. BHA1 is not nodule-specific, since it is also expressed in roots of uninfected plants. It belongs to the subfamily of plasma membrane H+-ATPases defined by the Arabidopsis AHA1, AHA2 and AHA3 genes and the tobacco PMA4 and corn MHA2 genes. In situ hybridization in nodule sections indicates high expression of BHA1 limited to uninfected cells. These results were confirmed by immunocytochemistry. The relatively low expression of plasma membrane-type H+-ATPase in Rhizobium-infected cells put a note of caution on the origin of the vanadate-sensitive ATPase described in preparations of peribacteroidal membranes. Also, our results indicate that active transport in symbiotic nodules is most intense at the plasma membrane of uninfected cells and support a specialized role of uninfected tissue for nitrogen transport.

Amino Acid Sequence↗
PubMed1996

Salt tolerance in plants and microorganisms: toxicity targets and defense responses.

Salt tolerance of crops could be improved by genetic engineering if basic questions on mechanisms of salt toxicity and defense responses could be solved at the molecular level. Mutant plants accumulating proline and transgenic plants engineered to accumulate mannitol or fructans exhibit improved salt tolerance. A target of salt toxicity has been identified in Saccharomyces cerevisiae: it is a sodium-sensitive nucleotidase involved in sulfate activation and encoded by the HAL2 gene. The major sodium-extrusion system of S. cerevisiae is a P-ATPase encoded by the ENA1 gene. The regulatory system of ENA1 expression includes the protein phosphatase calcineurin and the product of the HAL3 gene. In Escherichia coli, the Na(+)-H+ antiporter encoded by the nhaA gene is essential for salt tolerance. No sodium transport system has been identified at the molecular level in plants. Ion transport at the vacuole is of crucial importance for salt accumulation in this compartment, a conspicuous feature of halophytic plants. The primary sensors of osmotic stress have been identified only in E. coli. In S. cerevisiae, a protein kinase cascade (the HOG pathway) mediates the osmotic induction of many, but not all, stress-responsive genes. In plants, the hormone abscisic acid mediates many stress responses and both a protein phosphatase and a transcription factor (encoded by the ABI1 and ABI3 genes, respectively) participate in its action.

Drug Tolerance↗
PubMed1996

A major isoform of the maize plasma membrane H(+)-ATPase: characterization and induction by auxin in coleoptiles.

The plasma membrane (PM) H(+)-ATPase has been proposed to play important transport and regulatory roles in plant physiology, including its participation in auxin-induced acidification in coleoptile segments. This enzyme is encoded by a family of genes differing in tissue distribution, regulation, and expression level. A major expressed isoform of the maize PM H(+)-ATPase (MHA2) has been characterized. RNA gel blot analysis indicated that MHA2 is expressed in all maize organs, with highest levels being in the roots. In situ hybridization of sections from maize seedlings indicated enriched expression of MHA2 in stomatal guard cells, phloem cells, and root epidermal cells. MHA2 mRNA was induced threefold when nonvascular parts of the coleoptile segments were treated with auxin. This induction correlates with auxin-triggered proton extrusion by the same part of the segments. The PM H(+)-ATPase in the vascular bundies does not contribute significantly to auxin-induced acidification, is not regulated by auxin, and masks the auxin effect in extracts of whole coleoptile segments. We conclude that auxin-induced acidification in coleoptile segments most often occurs in the nonvascular tissue and is mediated, at least in part, by increased levels of MHA2.

Amino Acid Sequence↗
PubMed1995

A salt-sensitive 3'(2'),5'-bisphosphate nucleotidase involved in sulfate activation.

Overexpression of a yeast gene, HAL2, allows the cells to tolerate higher than normal extracellular salt concentrations. HAL2 encodes a 3'(2')5'-bisphosphate nucleotidase that serves to remove the end products of sulfate transfer during cellular metabolism. The enzyme is inhibited by lithium and sodium and is activated by potassium. Metabolic systems that are sensitive to salt, as well as those governing osmolyte synthesis and ion transport, offer routes by which genetic engineering can be used to improve the tolerance of various organisms to salt.

Adenosine Diphosphate↗