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Biomedical subjects

J M Pardo

Publications and source records attributed to J M Pardo.

At least 19 recordsLinked to original sources
PubMed2004

Impact of the human immunodeficiency virus on the epidemiology of tuberculosis in area 15 of the Valencian community in Spain.

SETTING: Area 15 in Valencia. OBJECTIVES: To describe the epidemiology (1987-2001) of tuberculosis (TB) in human immunodeficiency virus (HIV) patients. METHODS: Study of annual incidence, age distribution, excess cases attributed to HIV, etiological risk fraction (ERF), population attributable fraction (PAF) and f factor. RESULTS: Of 476 cases diagnosed, 459 were TB, 16 environmental and one mixed; 76% of environmental cases were HIV-positive (P < 0.001). There was a mean annual TB incidence of 24.6/100000, with an annual reduction of 4%. Seventy-three patients were HIV coinfected (16%) (mean incidence 3834/100 000 seropositives). The principal risk factor was drug use (59%) for HIV+ and contact with TB for HIV-. We found no difference in pulmonary or extra-pulmonary location between groups, contrary to mixed cases (P < 0.001). In HIV+ there was a lower frequency of infiltrates (P < 0.001) and cavitation (P < 0.01), and a higher frequency of adenitis (P < 0.001), miliary or nodular pattern and normal X-ray (P < 0.001). Seropositives had a 174 times higher probability of developing TB. The mean ERF attributed to HIV was 99%, the PAF was 16% and the f factor was 1.19. Highly active antiretroviral therapy (HAART) reduced the risk of TB in HIV+ by 80%. CONCLUSIONS: TB has continued its decline, although HIV generated an excess of cases in the 1990s. HAART has reduced the TB risk in HIV+ and possibly the overall rate of TB.

Adolescent↗
PubMed2001

A plant defense response effector induces microbial apoptosis.

Osmotin is a tobacco PR-5 protein that has antifungal activity and is implicated in host-plant defense. We show here that osmotin induces apoptosis in Saccharomyces cerevisiae. Induction of apoptosis was correlated with intracellular accumulation of reactive oxygen species and was mediated by RAS2, but not RAS1. Osmotin treatment resulted in suppression of transcription of stress-responsive genes via the RAS2/cAMP pathway. It was therefore concluded that osmotin induced proapoptotic signaling in yeast. The results indicate that the ability of antimicrobial proteins to induce microbial apoptosis could be an important factor in determining a pathogen's virulence and could therefore be targeted for the design of new antifungal drugs.

Animals↗
PubMed2001

Tobacco and Arabidiopsis SLT1 mediate salt tolerance of yeast.

A tobacco cDNA (NtSLT1, for Nicotiana tabacum sodium- and lithium-tolerant) was isolated by functional complementation of the salt-sensitive phenotype of a calcineurin (CaN)-deficient yeast mutant (cnb delta, regulatory subunit null). CaN is a Ca2+/calmodulin-dependent type 2B protein phosphatase that regulates Na+ homeostasis in yeast. This phosphatase modulates plasma membrane K+/Na+ selectivity through the activation of high-affinity K+ transport, and increaseses extracellular Na+ efflux by activation and transcriptional induction of the Na+/Li+ translocating P-type ATPase encoded by ENA1. Expression of N-terminally truncated NtSLT1 (Met-304), but not full-length protein, suppressed salt sensitivity of cnb1. Truncated NtSLT1 also increased salt tolerance of wild-type yeast, indicating functional sufficiency. NtSLT1 encodes a protein of yet unknown function but experimentation in yeast confirms it as a salt tolerance determinant. The Arabidopsis thaliana orthologue, AtSLT1, also suppressed salt sensitivity of cnb delta but only when expressed without the N-terminus (Met-301), suggesting that this region of the proteins from these evolutionarily diverse plant species contains an autoinhibitory domain. NtSLT1 enhanced transcription of the CaN-dependent ENA1 gene promoter and compensated the salt sensitivity of a mutant deficient in TCN1--a transcription factor that is activated by CaN and then induces ENA1 expression. NtSLT1 partially suppressed the salt sensitivity of ena1-4 indicating that NtSLT1 has both ENA-dependent and independent functions. NtSLT1 suppressed spk1 hal4 (SPK1/HAL4 which encodes a serine-threonine kinase that regulates TRK1-2 transporters to have high K+/Na+ selectivity) but not ena1-4 trk1-2 implicating the ENA-independent function to be through TRK1-2. Together, these results implicate SLT1 as a signal regulatory molecule that mediates salt tolerance by modulating Na+ homeostasis.

Adaptation, Physiological↗
PubMed2001

Resistance to the plant PR-5 protein osmotin in the model fungus Saccharomyces cerevisiae is mediated by the regulatory effects of SSD1 on cell wall composition.

The capacity of plants to counter the challenge of pathogenic fungal attack depends in part on the ability of plant defense proteins to overcome fungal resistance by being able to recognize and eradicate the invading fungi. Fungal genes that control resistance to plant defense proteins are therefore important determinants that define the range of fungi from which an induced defense protein can protect the plant. Resistance of the model fungus Saccharomyces cerevisiae to osmotin, a plant defense PR-5 protein, is strongly dependent on the natural polymorphism of the SSD1 gene. Expression of the SSD1-v allele afforded resistance to the antifungal protein. Conversely, yeast strains carrying the SSD1-d allele or a null ssd1Delta mutation displayed high sensitivity to osmotin. The SSD1-v protein mediates osmotin resistance in a cell wall-dependent manner. Deletion of SSD1-v or SSD1-d impeded sorting of the PIR proteins (osmotin-resistance factors) to the cell wall without affecting mRNA levels, indicating that SSD1 functions in post-transcriptional regulation of gene expression. The sensitivity of ssd1Delta cells to osmotin was only partially suppressed by over-accumulation of PIR proteins in the cell wall, suggesting an additional function for SSD1 in cell wall-mediated resistance. Accordingly, cells carrying a null ssd1 mutation also displayed aberrant cell-wall morphology and lower levels of alkali-insoluble cell-wall glucans. Therefore SSD1 is an important regulator of fungal cell-wall biogenesis and composition, including the deposition of PIR proteins which block the action of plant antifungal PR-5 proteins.

Alleles↗
PubMed2000

Functional conservation between yeast and plant endosomal Na(+)/H(+) antiporters.

Vacuolar compartmentation of Na(+) is an essential mechanism for salinity tolerance since it lowers cytosolic Na(+) levels while contributing to osmotic adjustment for cell turgor and expansion. The AtNHX1 protein of Arabidopsis thaliana substituted functionally for ScNHX1, the endosomal Na(+)/H(+) antiporter of yeast. Ion tolerance conferred by AtNHX1 and ScNHX1 correlated with ion uptake into an intracellular pool that was energetically dependent on the vacuolar (H(+))ATPase. AtNHX1 localized to vacuolar membrane fractions of yeast. Hence, both transporters share an evolutionarily conserved function in Na(+) compartmentation. AtNHX1 mRNA levels were upregulated by ABA and NaCl treatment in leaf but not in root tissue.

Abscisic Acid↗
PubMed2000

Fungal cell wall phosphomannans facilitate the toxic activity of a plant PR-5 protein.

Osmotin is a plant PR-5 protein. It has a broad spectrum of antifungal activity, yet also exhibits specificity for certain fungal targets. The structural bases for this specificity remain unknown. We show here that full sensitivity of Saccharomyces cerevisiae cells to the PR-5 protein osmotin is dependent on the function of MNN2, MNN4 and MNN6. MNN2 is an alpha-1, 2-mannosyltransferase catalyzing the addition of the first mannose to the branches on the poly l,6-mannose backbone of the outer chain of cell wall N-linked mannans. MNN4 and MNN6 are required for the transfer of mannosylphosphate to cell wall mannans. Null mnn2, mnn4 or mnn6 mutants lack phosphomannans and are defective in binding osmotin to the fungal cell wall. Both antimannoprotein antibody and the cationic dye alcian blue protect cells against osmotin cytotoxicity. MNN1 is an alpha-1,3-mannosyltransferase that adds the terminal mannose to the outer chain branches of N-linked mannan, masking mannosylphosphate. Null mnn1 cells exhibit enhanced osmotin binding and sensitivity. Several cell wall mannoproteins can bind to immobilized osmotin, suggesting that their polysaccharide constituent determines osmotin binding. Our results demonstrating a causal relationship between cell surface phosphomannan and the susceptibility of a yeast strain to osmotin suggest that cell surface polysaccharides of invading pathogens control target specificity of plant PR-5 proteins.

Carbohydrate Conformation↗
PubMed1998

Stress signaling through Ca2+/calmodulin-dependent protein phosphatase calcineurin mediates salt adaptation in plants.

Calcineurin (CaN) is a Ca2+- and calmodulin-dependent protein phosphatase (PP2B) that, in yeast, is an integral intermediate of a salt-stress signal transduction pathway that effects NaCl tolerance through the regulation of Na+ influx and efflux. A truncated form of the catalytic subunit and the regulatory subunit of yeast CaN were coexpressed in transgenic tobacco plants to reconstitute a constitutively activated phosphatase in vivo. Several different transgenic lines that expressed activated CaN also exhibited substantial NaCl tolerance, and this trait was linked to the genetic inheritance of the CaN transgenes. Enhanced capacity of plants expressing CaN to survive NaCl shock was similar when evaluation was conducted on seedlings in tissue culture raft vessels or plants in hydroponic culture that were transpiring actively. Root growth was less perturbed than shoot growth by NaCl in plants expressing CaN. Also, NaCl stress survival of control shoots was enhanced substantially when grafted onto roots of plants expressing CaN, further implicating a significant function of the phosphatase in the preservation of root integrity during salt shock. Together, these results indicate that in plants, like in yeast, a Ca2+- and calmodulin-dependent CaN signal pathway regulates determinants of salt tolerance required for stress adaptation. Furthermore, modulation of this pathway by expression of an activated regulatory intermediate substantially enhanced salt tolerance.

Adaptation, Physiological↗
PubMed1998

Osmotin, a plant antifungal protein, subverts signal transduction to enhance fungal cell susceptibility.

The plant pathogenesis-related protein osmotin is an antifungal cytotoxic agent that causes rapid cell death in the yeast S. cerevisiae. We show here that osmotin uses a signal transduction pathway to weaken defensive cell wall barriers and increase its cytotoxic efficacy. The pathway activated by osmotin includes the regulatory elements of the mating pheromone response STE4, STE18, STE20, STE5, STE11, STE7, FUS3, KSS1, and STE12. Neither the pheromone receptor nor its associated G protein alpha subunit GPA1 are required for osmotin action. However, mutation of SST2, a negative regulator of G alpha proteins, resulted in supersensitivity to osmotin. Phosphorylation of STE7 was rapidly stimulated by osmotin preceding any changes in cell vitality or morphology. These results demonstrate that osmotin subverts target cell signal transduction as part of its mechanism of action.

Antifungal Agents↗
PubMed1997

Stress proteins on the yeast cell surface determine resistance to osmotin, a plant antifungal protein.

Strains of the yeast Saccharomyces cerevisiae differ in their sensitivities to tobacco osmotin, an antifungal protein of the PR-5 family. However, cells sensitive to tobacco osmotin showed resistance to osmotin-like proteins purified from the plant Atriplex nummularia, indicating a strict specificity between the antifungal protein and its target cell. A member of a gene family encoding stress proteins induced by heat and nitrogen limitation, collectively called Pir proteins, was isolated among the genes that conveyed resistance to tobacco osmotin to a susceptible strain. We show that overexpression of Pir proteins increased resistance to osmotin, whereas simultaneous deletion of all PIR genes in a tolerant strain resulted in sensitivity. Pir proteins have been immunolocalized to the cell wall. The enzymatic digestion of the cell wall of sensitive and resistant cells rendered spheroplasts equally susceptible to the cytotoxic action of tobacco osmotin but not to other osmotin-like proteins, indicating that the cell membrane interacts specifically with osmotin and facilitates its action. Our results demonstrate that fungal cell wall proteins are determinants of resistance to antifungal PR-5 proteins.

Cloning, Molecular↗
PubMed1996

Activated calcineurin confers high tolerance to ion stress and alters the budding pattern and cell morphology of yeast cells.

The PP2B protein phosphatase, also known as calcineurin, is a regulator of ion homeostasis in yeast cells. We have investigated the physiological consequences of constitutive expression of a recombinant form of calcineurin in which the Ca2+/calmodulin-binding and autoinhibitory domains of the catalytic subunit were deleted. The concomitant expression of the regulatory subunit along with the truncated catalytic subunit resulted in high tolerance to toxic levels of Na+ and Li+. This activated form of calcineurin substituted for the Na+ stress signal to promote the expression of the ENA1 gene, encoding a P-ATPase pump, and to induce the transition of the K+ uptake system to the high affinity mode that restricts influx of Na+ and Li+. In addition, the transcriptional responsiveness of ENA1 to Na+ stress was enhanced. These results demonstrate that calcineurin has a pivotal role in a signaling cascade activated by ion stress in yeast. Moreover, we found that changes in the level of calcineurin activity affected budding pattern and cell morphology. Cells expressing the truncated calcineurin were elongated and budded in an unipolar pattern, whereas calcineurin-deficient mutants budded randomly. These results suggest that calcineurin may also act in the establishment of cell polarity.

Biological Transport↗
PubMed1996

Phosphate transporters from the higher plant Arabidopsis thaliana.

Two cDNAs (AtPT1 and AtPT2) encoding plant phosphate transporters have been isolated from a library prepared with mRNA extracted from phosphate-starved Arabidopsis thaliana roots, The encoded polypeptides are 78% identical to each other and show high degree of amino acid sequence similarity with high-affinity phosphate transporters of Saccharomyces cerevisiae, Neurospora crassa, and the mycorrhizal fungus Glomus versiforme. The AtPT1 and AtPT2 polypeptides are integral membrane proteins predicted to contain 12 membrane-spanning domains separated into two groups of six by a large charged hydrophilic region. Upon expression, both AtPT1 and AtPT2 were able to complement the pho84 mutant phenotype of yeast strain NS219 lacking the high-affinity phosphate transport activity. AtPT1 and AtPT2 are representatives of two distinct, small gene families in A. thaliana. The transcripts of both genes are expressed in roots and are not detectable in leaves. The steady-state level of their mRNAs increases in response to phosphate starvation.

Amino Acid Sequence↗
PubMed1995

Molecular characterization of glyoxalase-I from a higher plant; upregulation by stress.

A cDNA, GLX1, encoding glyoxalase-I was isolated by differential screening of salt-induced genes in tomato. Glyoxalases-I and -II are ubiquitous enzymes whose functions are not clearly understood. They may serve to detoxify methylglyoxal produced from triosephosphates in all cells. The protein encoded by GLX1 shared 49.4% and 58.5% identity with glyoxalase-I isolated from bacteria and human, respectively. Furthermore, yeast cells expressing GLX1 showed a glyoxalase-I specific activity 20-fold higher than non-transformed cells. Both GLX1 mRNA and glyoxalase-I polypeptide levels increased 2- to 3-fold in roots, stems and leaves of plants treated with either NaCl, mannitol, or abscisic acid. Immunohistochemical localization indicated that glyoxalase-I was expressed in all cell types, with preferential accumulation in phloem sieve elements. This expression pattern was not appreciably altered by salt-stress. We suggest that the increased expression of glyoxalase-I may be linked to a higher demand for ATP generation and to enhanced glycolysis in salt-stressed plants.

Amino Acid Sequence↗
PubMed1994

The protein phosphatase calcineurin is essential for NaCl tolerance of Saccharomyces cerevisiae.

NaCl-sensitive yeast mutants were isolated to identify genes essential for NaCl tolerance. Complementation of a mutant highly sensitive to Na+ and Li+ led to the isolation of the CNB1 gene. This gene encodes the regulatory subunit (CNB) of the Ca2+/calmodulin-dependent protein phosphatase calcineurin. Cells deficient in CNB accumulated Li+ due to reduced expression of ENA1, a gene encoding a P-type ATPase involved in Na+ and Li+ efflux. In addition, the K+ transport system of cnb1 delta cells was not converted to the high affinity state that facilitates better discrimination of K+ over Na+. Thus the cnb1 delta strain resembled a trk1 mutant. These results indicate that adaptation to NaCl stress in Saccharomyces cerevisiae requires a signal transduction pathway involving Ca2+ and protein phosphorylation-dephosphorylation. In this pathway, calcineurin would coordinate gene expression and activity of ion transporters to facilitate ion homeostasis.

Adenosine Triphosphatases↗
PubMed1994

Differential accumulation of S-adenosylmethionine synthetase transcripts in response to salt stress.

NaCl stress causes the accumulation of several mRNAs in tomato seedlings. An upregulated cDNA clone, SAM1, was found to encode a S-adenosyl-L-methionine synthetase enzyme (AdoMet synthetase). Expression of the cDNA SAM1 in a yeast mutant lacking functional SAM genes resulted in high AdoMet synthetase activity and AdoMet accumulation. We show that tomato plants contain at least four SAM isogenes. Clones corresponding to isogenes SAM2 and SAM3 have also been isolated and sequenced. They encode predicted polypeptides 95% and 92% identical, respectively, to the SAM1-encoded AdoMet Synthetase. RNA hybridization analysis showed a differential response of SAM genes to salt and other stress treatments. SAM1 and SAM3 mRNAs accumulated in the root in response to NaCl, mannitol or ABA treatments. SAM1 mRNA accumulated also in leaf tissue. These increases of mRNA level were apparent as soon as 8 h after the initiation of the salt treatment and were maintained for at least 3 days. A possible role for AdoMet synthetases in the adaptation to salt stress is discussed.

Abscisic Acid↗
PubMed1990

A tomato cDNA inducible by salt stress and abscisic acid: nucleotide sequence and expression pattern.

We have characterized a new tomato cDNA, TAS14, inducible by salt stress and abscisic acid (ABA). Its nucleotide sequence predicts an open reading frame coding for a highly hydrophilic and glycine-rich (23.8%) protein of 130 amino acids. Southern blot analysis of tomato DNA suggests that there is one TAS14 structural gene per haploid genome. TAS14 mRNA accumulates in tomato seedlings upon treatment with NaCl, ABA or mannitol. It is also induced in roots, stems and leaves of hydroponically grown tomato plants treated with NaCl or ABA. TAS14 mRNA is not induced by other stress conditions such as cold and wounding. The sequence of the predicted TAS14 protein shows four structural domains similar to the rice RAB21, cotton LEA D11 and barley and maize dehydrin genes.

Abscisic Acid↗
PubMed1990

Immunocytolocalization of Plasma Membrane H-ATPase.

The localization of plasma membrane H(+)-ATPase has been studied at the optical microscope level utilizing frozen and paraffin sections of Avena sativa and Pisum sativum, specific anti-ATPase polyclonal antibody, and second antibody coupled to alkaline phosphatase. In leaves and stems the ATPase is concentrated at the phloem, supporting the notion that it generates the driving force for phloem loading. In roots the ATPase is concentrated at both the periphery (rootcap and epidermis) and at the central cylinder, including endodermis and vascular cells. This supports a ;two-pump' mechanism for ion absorption, involving active uptake at the epidermis, symplast transport across the cortex, and active efflux at the xylem. The low ATPase content of root meristem and elongation zone may explain the observed transorgan H(+) currents, which leave nongrowing parts and enter growing tips.

Journal Article↗