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Emilio Fernández

Publications and source records attributed to Emilio Fernández.

17 recordsLinked to original sources
PubMed2006

Differential regulation of the Chlamydomonas Nar1 gene family by carbon and nitrogen.

Six genes of the Nar1 multigene family from Chlamydomonas reinhardtii were identified and are located on chromosomes I, VI, VII, IX, and XII/XIII. The first known member Nar1.1 encodes a chloroplast nitrite transporter that regulates nitrate assimilation according to carbon availability, and data supporting the idea that NAR1 proteins may participate in adjusting both nitrite and carbon utilization by Chlamydomonas cells are presented herein. The protein sequences deduced from their corresponding cDNAs show the typical signature of the FNT family, but also have particular differences: (1) NAR1.1, NAR1.2, and NAR1.5 contain putative chloroplast transit peptides; and (2) NAR1.3 and NAR1.6 have long C-termini. The expression patterns for Nar1 transcripts showed differential responses to changes in nitrogen or carbon status, as well as a particular regulation by the nitrate assimilation regulatory gene Nit2. One gene, Nar1.2, was strongly carbon-regulated independently of Nit2; two genes, Nar1.1 and Nar1.6, were regulated by nitrogen and Nit2; and the other genes, Nar1.3, Nar1.4, and Nar1.5 were independent of Nit2 and responded to nitrogen or carbon treatments in a transient and not easily understandable way. We have used Xenopus oocytes as a heterologous system for functional expression of NAR1.2. The electrophysiological response to HCO3- and NO2- provides evidence that NAR1.2 is involved in both HCO3- and NO2- transport.

Algal Proteins↗
PubMed2006

Effect of copper on the photochemical efficiency, growth, and chlorophyll a biomass of natural phytoplankton assemblages.

In this investigation, we have tested the potential usefulness of variable fluorescence, i.e., the potential photochemical efficiency of photosystem II (F(v)/F(m)), determined with a fast repetition rate fluorometer (FRRF), as an indicator of the effect of metal pollution on natural phytoplankton assemblages. Phytoplankton populations were collected from an eutrophic embayment and exposed to different copper concentrations within the ppb range for 4 d. Enhanced photosynthesis was found for Cu concentrations of 10 and 20 microg/L, at very short exposure times (1 and 5 h). However, after 72 h even at 10 microg/L of Cu, F(v)/F(m)-values were significantly lower than those measured in the control. The highest Cu concentration tested (80 microg/L) caused a statistically significant decrease of F(v)/F(m) after 5 h. This response was contrasted with parallel determinations of chlorophyll a concentration, measurements of photosynthetic O2 production rates, and changes in the taxonomic composition and size structure of the microplankton communities. A reduction in overall particle size was found at Cu concentrations of 80 and 40 microg/L. Enhanced abundance of flagellates and the diatom Pseudonitzschia pungens was observed at 20 microg/L of copper. This study demonstrates the utility of this methodological approach as a rapid, nondestructive way for detecting trace-metal toxicity on natural phytoplankton.

Biomass↗
PubMed2005

Restriction enzyme site-directed amplification PCR: a tool to identify regions flanking a marker DNA.

An innovative combination of various recently described molecular methods was set up to efficiently identify regions flanking a marker DNA in insertional mutants of Chlamydomonas. The technique is named restriction enzyme site-directed amplification PCR (RESDA-PCR) and is based on the random distribution of frequent restriction sites in a genome and on a special design of primers. The primer design is based on the presence of a restriction site included in a low degenerated sequence at the 3' end and of a specific adapter sequence at the 5' end, with the two ends being linked by a polyinosine bridge. Specific primers of the marker DNA combined with the degenerated primers allow amplification of DNA fragments adjacent to the marker insertion by using two rounds of either short or long cycling procedures. Amplified fragments from 0.3 to 2 kb or more are routinely obtained at sufficient purity and quantity for direct sequencing. This method is fast, is reliable (87% success rate), and can be easily extrapolated to any organism and marker DNA by designing the appropriate primers. A procedure involving the PCR over enzyme digest fragments is also proposed for when, exceptionally, positive results are not obtained.

Animals↗
PubMed2005

Ammonium transporter genes in Chlamydomonas: the nitrate-specific regulatory gene Nit2 is involved in Amt1;1 expression.

Ammonium transport is a key process in nitrogen metabolism. In the green alga Chlamydomonas, we have characterized molecularly the largest family of ammonium transporters (AMT1) so far described consisting of eight members. CrAmt1 genes have an interesting transcript structure with some very small exons. Differential expression patterns were found for each CrAmt1 gene in response to the nitrogen source by using Real Time PCR. These expression patterns were similar under high and low CO2 atmosphere. CrAmt1;1 expression was characterized in detail. It was repressed in both ammonium and nitrate medium, and strongly expressed in nitrogen-free media. Treatment with a Glutamine synthetase inhibitor released partially repression in ammonium and nitrate suggesting that ammonium and its derivatives participate in the observed repressing effects. By studying CrAmt1;1 expression in mutants deficient at different steps of the nitrate assimilation pathway, it has been shown that nitrate has a double negative effect on this gene expression; one related to its reduction to ammonium, and a second one by itself. This second effect of nitrate was dependent on the functionality of the regulatory gene Nit2, specific for nitrate assimilation. Thus, NIT2 would have a dual role on gene expression: the well-known positive one on nitrate assimilation and a novel negative one on Amt1;1 regulation.

Algal Proteins↗
PubMed2005

Functional genomics of the regulation of the nitrate assimilation pathway in Chlamydomonas.

The existence of mutants at specific steps in a pathway is a valuable tool of functional genomics in an organism. Heterologous integration occurring during transformation with a selectable marker in Chlamydomonas (Chlamydomonas reinhardtii) has been used to generate an ordered mutant library. A strain, having a chimeric construct (pNia1::arylsulfatase gene) as a sensor of the Nia1 gene promoter activity, was transformed with a plasmid bearing the paramomycin resistance AphVIII gene to generate insertional mutants defective at regulatory steps of the nitrate assimilation pathway. Twenty-two thousand transformants were obtained and maintained in pools of 96 for further use. The mutant library was screened for the following phenotypes: insensitivity to the negative signal of ammonium, insensitivity to the positive signal of nitrate, overexpression in nitrate, and inability to use nitrate. Analyses of mutants showed that (1) the number or integrated copies of the gene marker is close to 1; (2) the probability of cloning the DNA region at the marker insertion site is high (76%); (3) insertions occur randomly; and (4) integrations at different positions and orientations of the same genomic region appeared in at least three cases. Some of the mutants analyzed were found to be affected at putative new genes related to regulatory functions, such as guanylate cyclase, protein kinase, peptidyl-prolyl isomerase, or DNA binding. The Chlamydomonas mutant library constructed would also be valuable to identify any other gene with a screenable phenotype.

Ammonia↗
PubMed2005

Chlamydomonas reinhardtii strains expressing nitrate reductase under control of the cabII-1 promoter: isolation of chlorate resistant mutants and identification of new loci for nitrate assimilation.

The Chlamydomonas reinhardtii strain Tx11-8 is a transgenic alga that bears the nitrate reductase gene (Nia1) under control of the CabII-1 gene promoter (CabII-1-Nia1). Approximately nine copies of the chimeric CabII-1-Nia1 gene were found to be integrated in this strain and to confer a phenotype of chlorate sensitivity in the presence of ammonium. We have used this strain for the isolation of spontaneous chlorate resistant mutants in the presence of ammonium that were found to be defective at loci involved in MoCo metabolism and light-dependent growth in nitrate media. Of a total of 45 mutant strains analyzed first, 44 were affected in the MoCo activity (16 Nit(-), unable to grow in nitrate, and 28 Nit(+), able to grow in nitrate). All the Nit(-) strains lacked MoCo activity. Diploid complementation of Nit(-), MoCo(-) strains with C. reinhardtii MoCo mutants and genetic analysis indicated that some strains were defective at known loci for MoCo biosynthesis, while three strains were defective at two new loci, hereafter named Nit10 and Nit11. The other 28 Nit(+) strains showed almost undetectable MoCo activity or activity was below 20% of the parental strain. Second, only one strain (named 23c(+)) showed MoCo and NR activities comparable to those in the parental strain. Strain 23c(+) seems to be affected in a locus, Nit12, required for growth in nitrate under continuous light. It is proposed that this locus is required for nitrate/chlorate transport activity. In this work, mechanisms of chlorate toxicity are reviewed in the light of our results.

Animals↗
PubMed2005

REM1, a new type of long terminal repeat retrotransposon in Chlamydomonas reinhardtii.

A new long terminal repeat (LTR) retrotransposon, named REM1, has been identified in the green alga Chlamydomonas reinhardtii. It was found in low copy number, highly methylated, and with an inducible transpositional activity. This retrotransposon is phylogenetically related to Ty3-gypsy LTR retrotransposons and possesses new and unusual structural features. A regulatory module, ORF3p, is present in an inverse transcriptional orientation to that of the polyprotein and contains PHD-finger and chromodomains, which might confer specificity of the target site and are highly conserved in proteins involved in transcriptional regulation by chromatin remodeling. By using different wild-type and mutant strains, we show that CrREM1 was active with a strong transcriptional activity and amplified its copy number in strains that underwent foreign DNA integration and/or genetic crosses. However, integration of CrREM1 was restricted to these events even though the expression of its full-length transcripts remained highly activated. A regulatory mechanism of CrREM1 retrotransposition which would help to minimize its deleterious effects in the host genome is proposed.

Algal Proteins↗
PubMed2003

Mcp1 encodes the molybdenum cofactor carrier protein in Chlamydomonas reinhardtii and participates in protection, binding, and storage functions of the cofactor.

The molybdenum cofactor (Moco) is essential for the activity of all molybdoenzymes except nitrogenase. The cDNA for the Moco carrier protein (MocoCP) of Chlamydomonas reinhardtii has been cloned by reverse transcription PCR approaches with primers designed from microsequenced peptides of this protein. The C. reinhardtii MocoCP has been expressed in Escherichia coli. The recombinant protein has been purified to electrophoretic homogeneity and is found assembled into a homotetramer when Moco is not present under native conditions. Recombinant MocoCP has the same biochemical characteristics as MocoCP from C. reinhardtii, as it bound Moco from milk xanthine oxidase with high affinity, prevented Moco inactivation by oxygen, and transferred Moco efficiently to aponitrate reductase from the Neurospora crassa nit1 mutant. The genomic DNA sequence corresponding to the Chlamydomonas MocoCP gene, CrMcp1, also was isolated. This gene contained three introns in the coding region. The deduced amino acid sequence of CrMcp1 did not show a significant identity to functionally known proteins in the GenBank data base, although a significant conservation was found with bacterial proteins of unknown function. The results suggest that proteins having a Moco binding function probably exist in other organisms.

Amino Acid Sequence↗
PubMed2003

The cesarean delivery scar pouch: clinical implications and diagnostic correlation between transvaginal sonography and hysteroscopy.

OBJECTIVE: We sought (1) to describe an anatomic defect of the uterine cavity in the anterior isthmus diagnosed by transvaginal sonography in a group of premenopausal women with previous cesarean deliveries, (2) to establish whether there is an association between the presence of the pouch and a bleeding disturbance, and (3) to compare the diagnostic efficacy of transvaginal sonography versus hysteroscopy for the detection of this defect. METHODS: We performed a retrospective study of 92 premenopausal women with histories of at least 1 cesarean delivery. Age, number of previous cesarean deliveries, time elapsed between last cesarean delivery and first consultation, and total area of the pouch were recorded in groups of women with and without abnormal bleeding. Hysteroscopy was also performed in 43.8% of the patients who had abnormal bleeding. RESULTS: In all women, transvaginal sonography revealed the presence of a pouch on the anterior uterine segment at the site of the expected previous cesarean delivery scar. Hysteroscopy showed 100% correlation with transvaginal sonography in detection of this pouch. CONCLUSIONS: The high correlation between bleeding disturbances and the presence of a pouch, in the absence of other pathologic entities, suggests this anatomic defect as the possible cause, especially in view of the fact that women who had heavier and longer bleeding episodes tended to have a larger pouch. Transvaginal sonography is a very simple, noninvasive, low-cost examination that should be considered as the first choice for screening, because it highly correlates (100%) with hysteroscopy in the diagnosis of this defect and may help rule out other causes.

Adult↗
PubMed2002

The activity of the high-affinity nitrate transport system I (NRT2;1, NAR2) is responsible for the efficient signalling of nitrate assimilation genes in Chlamydomonas reinhardtii.

The expression of nitrite uptake activity and the induction of transcripts from several nitrate assimilation genes (Nii1, Nrt2;1, Nrt2;3, and Nar1) have been analysed in Chlamydomonas reinhardtii Dang. strains bearing several sets of genes encoding nitrate transport systems. Different nitrate concentrations resulted in a differential induction pattern of nitrite uptake activity depending on which particular nitrate transport system was present in the cells, and that was directly related to their relative efficiency for nitrate transport. The presence of the high-affinity nitrate transport system I (NRT2;1, NAR2) made cells able to sense the very low concentrations of nitrate present in culture medium with no added nitrate and to express optimally Nii1, Nrt2;1, Nrt2;3, and Nar1 genes involved in nitrate/nitrite assimilation. In addition, strains lacking nitrate reductase activity overexpressed these gene transcripts as a result of continuous signalling by nitrate, but only those bearing an active system I. This study supports the hypothesis that signalling of nitrate assimilation genes occurs intracellularly in a process dependent on the nitrate uptake activity. The bispecific nitrate/nitrite transport system I with the highest affinity for nitrate is the most efficient one for signalling the expression of nitrate/nitrite assimilation genes in C. reinhardtii.

Algal Proteins↗
PubMed2002

NADP-malate dehydrogenase from Chlamydomonas: prediction of new structural determinants for redox regulation by homology modelling.

The function of a gene closely linked to nitrate assimilation loci from Chlamydomonas reinhardtii has been investigated. Gene expression analysis shows that its mRNA accumulation is modulated by light, carbon source and adaptation to light/dark cyclic conditions of growth. A full-length cDNA was isolated for the light-regulated transcript, and sequence characterization indicates that it encodes the NADP-malate dehydrogenase from C. reinhardtii (NADP-MDH;Cr). The primary structure of NADP-MDH;Cr is closely related to plant, mossfern and algal NADP-malate dehydrogenases, and shares structural determinants for chloroplast targeting, cofactor binding and catalysis. Sequence conservation extends to the carboxy end of the protein, where plant and mossfern enzymes have two cysteines and an acidic C-terminus with a critical role for regulation of NADP-MDH activity by the thioredoxin/ferredoxin system. Accordingly, incubation with DTT activates NADP-MDH enzyme in cell-free extracts from C. reinhardtii. Like NADP-malate dehydrogenases from two other green algae, the N-terminal extension of NADP-MDH;Cr lacks two thiol residues whose reduction constitutes the rate-limiting step in the activation reaction of plant enzymes. Homology-based 3D modelling of NADP-MDH;Cr, the first structure predicted for NADP-malate dehydrogenase from a lower eukaryote, evidences close positioning of two new cysteines in an accessible region of the protein surface. These results suggest that the algal enzyme has a different arrangement of regulatory disulfide bridges, which might involve the existence of new mechanisms that control functioning of the malate valve, the main system to export reducing power from the chloroplast of plant cells.

Algal Proteins↗
PubMed2002

Nitrate signalling on the nitrate reductase gene promoter depends directly on the activity of the nitrate transport systems in Chlamydomonas.

Nitrate signalling on the nitrate reductase (Nia1) gene promoter from Chlamydomonas reinhardtii has been studied by using a construct of the Nia1 promoter transcriptionally fused to the Chlamydomonas arylsulphatase gene as a reporter in strains bearing different sets of nitrate/nitrite transport genes. The high-affinity nitrate transport (HANT) system I is required for efficient signalling by nitrate, even at submicromolar concentrations of the anion. In addition, the autogenous regulation of nitrate reductase has been found to depend on the presence of system I. The low-affinity nitrate transport system III promoted signalling optimally on the promoter at millimolar nitrate concentrations. The HANT system IV, which is insensitive to ammonium and active at low CO2, allowed nitrate signalling at micromolar concentrations even in the presence of ammonium, suggesting that the balance of these two effectors controls Nia1 transcription. Our data indicate that nitrate signalling on the Nia1 gene promoter occurs intracellularly and depends on the activity of nitrate transporters.

Animals↗
PubMed2002

Nitrite transport to the chloroplast in Chlamydomonas reinhardtii: molecular evidence for a regulated process.

Nitrite transport to the chloroplast is not a well documented process in spite of being a central step in the nitrate assimilation pathway. The lack of molecular evidence, as well as the easy diffusion of nitrite through biological membranes, have made this physiological process difficult to understand in plant nutrition. The aim of this review is to illustrate that nitrite transport to the chloroplast is a regulated step, intimately related to the efficiency of nitrate utilization. In Chlamydomonas reinhardtii, the Nar1;1 gene has been shown to have this role in nitrate assimilation. NAR1;1 corresponds to a plastidic membrane transporter protein related to the bacterial formate/nitrite transporters. At least four Nar1 genes might exist in Chlamydomonas. The existence of orthologous Nar1 genes in plants is discussed.

Algal Proteins↗
PubMedDate not supplied

Inhibition of mitochondrial respiration by nitric oxide: its role in glucose metabolism and neuroprotection.

There is an increasing body of evidence demonstrating that inhibition of cytochrome c oxidase by nitric oxide (NO) may be one more step in a signaling cascade involved in the physiologic regulation of cell functions. For example, in both astrocytes and neurons the inhibition of mitochondrial respiration by endogenously produced NO induces transient and modest decreases in cellular ATP concentrations. This mitochondrial impairment may serve as a cellular sensor of energy charges, hence modulating metabolic pathways, such as glycolysis, through AMP-activated protein kinase (AMPK) in astrocytes. In neurons, the NO derivative peroxynitrite anion triggers signaling pathways leading to glucose oxidation through the pentose-phosphate pathway to form reducing equivalents in the form of NADPH. The modulation of these metabolic pathways by nitric oxide or its derivatives may be important for understanding the mechanisms by which this free radical affects neuronal death or survival.

Animals↗
PubMedDate not supplied

Surgical treatment and follow-up of women with intermenstrual bleeding due to cesarean section scar defect.

STUDY OBJECTIVE: Previous cesarean delivery scar (PCDS) defect has been described as a cause of intermenstrual bleeding in women with no other uterine pathology except for a pouch on the anterior uterine segment at the site of the cesarean scar. The objective of this study was to assess the effectiveness of hysteroscopic surgery to correct this anatomic defect and eliminate the bleeding disturbance in a group of women with this symptom. DESIGN: Retrospective study (Canadian Task Force classification XX). SETTING: Private hospital, department of obstetrics and gynecology. PATIENTS: Twenty-four women, age 29-41 years, who reported intermenstrual bleeding, especially postmenstrual spotting, with no other gynecologic pathology except for the presence of a PCDS defect. Diagnosis was established with transvaginal ultrasound, when a fluid-filled, triangular defect was seen in the anterior uterine isthmus, in relation to the cesarean section scar. INTERVENTION: Hysteroscopic resection of fibrotic tissue that overhangs underneath the triangular pouch, facilitating blood drainage through the cervix and fulguration of endometrial glands and/or dilated blood vessels. MEASUREMENTS AND MAIN RESULTS: The mean number of previous cesarean-section deliveries was 2.75. Postoperative follow-up was 24 months in 21 patients and at least 14 months in the other 3 patients. Eleven of these patients with the desire to become pregnant were unable to conceive after trying for a period of at least 2 years before hysteroscopy. Infertility work-up in the 11 patients revealed 9 with unknown infertility, 1 with male infertility, and 1 with failed tubal reversal surgery. Nine of them became pregnant between 14- and 24-months of follow-up. Eighty-four percent of patients (20/24) remained asymptomatic (without bleeding disturbances) after surgery. CONCLUSION: Previous cesarean delivery scar defect may be the cause of intermenstrual bleeding, and it is possible that it also may impair fertility, but it can be successfully treated by hysteroscopic surgery.

Adult↗
PubMedDate not supplied

Regulation of glucose metabolism by nitrosative stress in neural cells.

Following brain inflammatory stimuli, astrocytes actively synthesize nitric oxide and peroxynitrite. These nitrogen-derived species trigger a repertoire of biochemical effects, including alteration of mitochondrial function and redox status both in astrocytes and neighboring neurons. Furthermore, under such nitrosative stress astrocytes show remarkable resistance in spite of having their mitochondria impaired, whereas the neighboring neurons show vulnerability. In this review, we discuss recent evidence strongly suggesting that nitrogen-derived species modulate key regulatory steps of glucose metabolism. These involve up-regulation of high-affinity glucose transporter, stimulation of glycolysis at 6-phosphofructo-1-kinase, and activation of pentose-phosphate pathway at glucose-6-phosphate dehydrogenase. We conclude that the orchestrated stimulation of glucose-metabolising pathways by nitric oxide would be a transient attempt of certain neural cells to compensate for the impaired energy status and oxidised glutathione and thus emerge from an otherwise neuropathological outcome.

Animals↗