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M P Fernández

Publications and source records attributed to M P Fernández.

At least 19 recordsLinked to original sources
PubMed2006

New contributions towards the understanding of the phylogenetic relationships among economically important fruit flies (Diptera: Tephritidae).

Fruit flies (Diptera: Tephritidae) are a species-rich and economically important group. The phylogenetic relationships among the many taxa are still to be fully resolved and the monophyly of several groups is still to be confirmed. This paper reports a study of the phylogenetic relationships among 23 economically important tephritid species (representing several major lineages of the family) which examines the sequence of a region of mitochondrial DNA encompassing the cytb, tRNA(Ser) and ND1 genes. Substitutions characteristic of particular taxa were found that could help classify members of the family at any developmental stage. The trees obtained by the maximum parsimony, neighbour joining and maximum likelihood methods were generally compatible with present morphological classification patterns. However, the data reveal some characteristics of the phylogenetic relationships of this family that do not agree with present classifications. The results support the probable non-monophyletic nature of the subfamily Trypetinae and suggest that Bactrocera cucurbitae (Coquillet) is more closely related to the genus Dacus than to other species of Bactrocera.

Animals↗
PubMed2001

Mesorhizobium chacoense sp. nov., a novel species that nodulates Prosopis alba in the Chaco Arido region (Argentina).

Low-molecular-weight RNA analysis was performed for the identification and classification of 20 Argentinian strains isolated from the root nodules of Prosopis alba. SDS-PAGE of total cellular proteins, determination of the DNA base composition, DNA-DNA reassociation experiments and physiological and biochemical tests were also carried out for these strains and the whole 16S rRNA gene was sequenced from one of the strains, strain LMG 19008T. Results of the genotypic and phenotypic characterization showed that the strains isolated in this study belong to a group that clustered in the genus Mesorhizobium. The results of DNA-DNA hybridizations showed that this group is a novel species of this genus. The name Mesorhizobium chacoense sp. nov. is proposed for this species. The type strain is LMG 19008T (= CECT 5336T).

Anti-Bacterial Agents↗
PubMed2000

Simultaneous determination of fat-soluble vitamins and provitamins in milk by microcolumn liquid chromatography.

The fat-soluble vitamins A, D2, D3, E and K1, retinyl acetate, retinyl palmitate, tocopherol acetate, ergosterol and 7-dehydrocholesterol in milk were separated on a packed reversed-phase fused-silica microcolumn, and determined with UV detection. Conventional liquid chromatographic equipment was adapted for such purposes, providing a highly efficient and sensitive analytical system. The vitamins could be separated in gradient mode with detection limits comprised between 0.02 ng/ml for retinol and 2 ng/ml for vitamin E. All vitamins were separated in less than 17 min. Recovery studies showed good results for all solutes (89-107%) and the intra-day coefficients of variations ranged from 2 to 8%. Application of the proposed method to the quantification of fat-soluble vitamins in milk is reported.

Animals↗
PubMed2000

Simultaneous determination of fat-soluble vitamins and provitamins in dairy products by liquid chromatography with a narrow-bore column.

A reversed-phase high-performance liquid chromatographic method is described for the simultaneous determination of vitamins A, D2, D3, E and K1, retinyl acetate, retinyl palmitate, tocopherol acetate, ergosterol and 7-dehydrocholesterol in milk and butter. Narrow-bore columns are recommended because this alternative provides a good separation and efficiency, plus greater economy and sensitivity. Detection limits for individual vitamins range from 0.14 to 6.9 ng. All vitamins are separated in less than 33 min. For the simultaneous determination of these vitamins and provitamins we use two sample pre-treatment methods, a liquid-liquid extraction with hexane or a solid-phase extraction with a C18 cartridge. Recovery studies show good results for all solutes (84-108% and 85-108% for milk and butter, respectively) and the intra-day coefficients of variations range from 1.6 to 4.5%. These methods permit the simple determination of fat-soluble vitamins using a small sample volume.

Animals↗
PubMed1998

Mouse annexin III cDNA, genetic mapping and evolution.

Mouse annexin III cDNA was characterized from I.M.A.G.E. Consortium (LLNL) expressed sequence tag clones by molecular sequencing, chromosomal mapping and systematic analysis. cDNA sequences extended the known 5' and 3' untranslated regions and confirmed the location of intron 7 with respect to the human gene. The Anx3 locus mapped to the middle of mouse chromosome 5 between Areg and Fgf5. Protein-coding regions were compared with homologous annexins to establish subfamily identity, structural conservation and divergence pattern. Annexin III exhibited low functional constraint against structural change and weak phylogenetic association with known annexins. The rapid, constant divergence of human and rodent annexins III from each other and from other annexin subfamilies was used to estimate gene separation times. Phylogenetic, phenetic and structural data suggested a possible direct or indirect separation of annexin III from XI approximately 317 million years ago.

Amino Acid Sequence↗
PubMed1998

The genetic origin of mouse annexin VIII.

Mouse annexin VIII cDNA was characterized by DNA sequencing of expressed sequence tag clones, molecular systematic analysis, and genetic linkage mapping to investigate its evolutionary origin. Its subfamily identity, divergence pattern, and nucleotide substitution rate were established by comparison with other annexin cDNA and deduced protein sequences. The known phylogenetic association of annexin VIII in an evolutionary clade with annexins XI, IV, V, and VIa identified these close homologs as potential progenitors or duplication products. Cladistic analysis confirmed the base position of annexin XI and its relationship to annexin IV as a direct duplication product. Although annexin VIII also derived from annexin XI, the evolutionary branching order, gene separation times, and mapping results indicated that it was probably a subsequent duplication product of annexin IV about 300 million years ago. Dates were calibrated against the assumed separation time of 75 Mya for rodents from other mammals, divergence rates were based on comparisons of all available annexin species, and relative rate tests implied individually stable gene clocks for most annexins. Linkage mapping of mouse Anx8 to the centromeric region of Chromosome (Chr) 14 placed it in a more distal homology group from previously mapped Anx7 and Anx11. Despite their synteny, the combined proximity and segregation of these three annexins diminished the likelihood that they were mutual gene duplication products.

Amino Acid Sequence↗
PubMed1997

Annexin gene structures and molecular evolutionary genetics.

Annexins provide an exemplary model for studying the pattern and process of molecular evolution in multigene families. Their related gene structures, broad dispersal in eukaryotic genomes and abundant coding sequences permit a phylogenetic reconstruction of their genetic history. The emerging picture is one of prolific expansion by gene duplication to more than 27 paralogous subfamilies that have undergone steady sequence divergence, speciation and differential selection. Homologous recombination via the common tetrad of internal repeats has, nevertheless, strictly preserved this core structure for over 1200 million years, implying a basic functional role. The existence of multiple annexins with unique 5' coding and regulatory regions has facilitated their adaptation to the varying ontogenetic and cell-specific needs of diverse organisms. Computational and cladistic sequence analyses have permitted the determination of original gene duplication dates and mutation rates for the ten known vertebrate annexins. Molecular genetic and evolutionary studies of annexins can help to define their structure-function relationships elucidate their individual physiological roles and ultimately link them to hereditary phenotypes.

Annexins↗
PubMed1997

Desmethyl tirilazad reduces brain nitric oxide synthase activity and cyclic guanosine monophosphate during cerebral global transient ischemia in rats.

Experiments were conducted to evaluate the effects of desmethyl tirilazad (10 mg/kg, i.p.), a 21-aminosteroid, on constitutive nitric oxide synthase (cNOS) activity and cyclic guanosine monophosphate (cGMP) levels in brain homogenates of rats subjected to cerebral global transient ischemia induced by bilateral clamping of the carotids for 30 minutes and reduction of arterial pressure (to 50-60 mmHg) by intravenous infusion of 1.5 ml of a solution of trimethaphan (5 mg/ml). Our results show that ischemia induces a rise in cNOS activity (from 62.0 +/- 6.1 to 133.3 +/- 13.3 pmol/min/mg protein) and cGMP levels (from 459.3 +/- 49.6 to 1074.1 +/- 132.1 fmol/mg protein). Pretreatment with desmethyl tirilazad abolishes these increases. These results are in agreement with the neuroprotective efficacy of desmethyl tirilazad in cerebral ischemia.

Animals↗
PubMed1996

Annexin V autoantibodies in rheumatoid arthritis.

OBJECTIVE: To investigate the occurrence of anti-annexin V autoantibodies in sera of patients with rheumatoid arthritis to assess involvement with the disease and any relation to glucocorticoid treatment. METHODS: Anti-annexin V antibodies were measured by an enzyme linked immunosorbent assay (ELISA) which used the purified human recombinant protein as antigen. RESULTS: Concentrations of anti-annexin V autoantibodies, predominantly of the IgG class, were significantly raised in sera from patients with rheumatoid arthritis compared to normal controls. This was not correlated with other indices of disease activity such as erythrocyte sedimentation rate or C reactive protein and was unrelated to glucocorticoid treatment. CONCLUSIONS: Extracellular annexin V provides an antigenic stimulus for autoantibody production and its in vivo expression is independent of glucocorticoid control. Such autoantibodies may have a detrimental role in the arthritic condition by interfering with putative functions of annexin V, including collagen type II binding, inhibition of phospholipase A2 activity, and Fc receptor activity.

Adult↗
PubMed1995

A BC200-derived element and Z-DNA as structural markers in annexin I genes: relevance to Alu evolution and annexin tetrad formation.

We have identified two types of structural elements in genomic DNA for annexin I that provide physical evidence of genetic events leading to conserved changes in gene structure. The sequence upstream of the transcribed region in human annexin I contained a rare, Alu-like repetitive element with flanking direct repeats, probably derived from the active BC200 gene via germline retroposition. Nucleotide substitutions in this BC200 insert relative to the 7SL gene and its absence in rodent annexins I identified it as a recent primate pseudogene. Phylogenetic analysis showed that the BC200 gene represents a new clade of primate Alu evolution that branched near the time of appearance of the progenitor to the free left Alu monomer, FLAM-C. Separate analysis identified a Z-DNA motif in pigeon annexin I intron 7 that may represent the vestigial recombination site involved in primordial assembly of the annexin tetrad. These distinct structural features in annexin I genes provide insight into the evolution of Alu repeats and the mechanism of annexin tetrad formation.

Animals↗
PubMed1995

Molecular phylogeny of annexins and identification of a primitive homologue in Giardia lamblia.

The homologous repeats of annexin tetrads are believed to have originated by successive duplication and fusion from a putative monomeric precursor, but neither the nature of their ancestor nor the events leading to the formation of different subfamilies have been elucidated. We have performed molecular phylogenetic analysis of aligned annexin nucleotide and amino acids sequences to characterize subfamily branching, to delineate the temporal order of appearance of individual repeat units, and to gain insight into the origin and nature of the primordial unit. All extant annexins appear to have a common tetrad precursor that may have originated from a progenitor unit resembling repeat 3, followed by the generation of repeats 4, 1, and 2 from a more evolved progenitor with subsequent fusion. Repeat sequences of the earliest human annexins VII and XIII were used to identify alpha-giardin genes as primitive homologues from the unicellular protozoan Giardia lamblia, which diverged from eukaryote lineage 1-1.5 billion yr ago. The significant homology between alpha-giardins and annexins suggested that the cell membrane adhesive role of these proteins may be a common, fundamental property of the annexin C-terminal core region. Purported annexin VII of Dictyostelium discoideum was reclassified as new annexin XIV, three Caenorhabditis elegans genes were assigned to new subfamilies XV, XVI, and XVII, and plant annexin XVIII from Medicago sativa was among the earliest diverging subfamilies. Annexins I and II were found to be closely related, but analysis of protein mutation rates confirmed that the former is evolving up to three times more rapidly. The inclusion of early phyla in annexin taxonomy provides a useful basis for assessing the structural and functional changes associated with annexin evolution.

Amino Acid Sequence↗
PubMed1994

The gene encoding human annexin V has a TATA-less promoter with a high G+C content.

Annexin V is a phospholipase A2 and protein kinase C inhibitory protein with calcium channel activity and an undefined role in cellular signal transduction, inflammation, growth and differentiation. Three genomic clones for human annexin V (ANX5) were characterized by restriction analysis, Southern blotting and sequencing. ANX5 spans at least 29 kb of the human genome and contains 13 exons ranging in length from 44 to 513 bp and 12 introns from 232 bp to 8 kb. The absence of a typical TATA box and the presence of high G+C content and Sp1-binding sites in its promoter characterize it as a 'housekeeping' gene and account for its broad pattern of expression. Potential binding sites for cis-regulatory elements identified in the 5'-upstream region of annexin V are consistent with its known regulation by oncogenic and growth-related stimuli. ANX5, like its chick homologue, differs from the genes encoding annexins I, II and III in features of its promoter and in the size of its exons 1, 2 and 3 in ways that may impart individuality to its regulation and function.

Amino Acid Sequence↗
PubMed1994

Structure of the gene encoding anchorin CII (chick annexin V).

Anchorin CII (annexin V) was first characterized as a collagen-binding protein and later identified as the chick homologue of human endonexin II, a member of the annexin gene family. Its gene (anx5) structure and sequence have been investigated to provide insight into the evolution and regulation of this important protein, and to elucidate its putative role in signal transduction and cellular differentiation. Four chick genomic clones encoding anchorin CII were isolated and characterized by restriction analysis, Southern blotting and sequencing. The anchorin CII-encoding gene spans about 24 kb and consists of 13 exons ranging in length from 50 to 561 bp, interrupted by 12 introns of 94 bp to 7 kb. Its promoter sequence contained no TATA box, but did display a high G+C content and multiple Sp1-binding sites typical of 'housekeeping' genes. Potential binding sites for transcription factors in the 5'-upstream region are consistent with regulation of anx5 expression by mitogens, oncoproteins, steroids and possibly metals. Genomic Southern blotting confirmed that chick anx5 is present as a single-copy gene.

Amino Acid Sequence↗
PubMed1991

[ABO materno-fetal incompatibility in the newborn infant: clinical usefulness of Coombs test and elution for the study of hemolytic disease].

A decision to establish early treatment for hemolytic disease in the newborn is usually based upon results of the Coombs test. In order to evaluate a possible advantage of using the Elution test to help in the diagnosis of the hemolytic state, samples of cord blood from 197 consecutive newborns with ABO incompatibility were studied. Hemolysis occurred in 41. Each test (Coombs and Elution) was positive in 26 patients, for a total of 35 positive results and a combined sensitivity of 85%. False positives occurred in 24 of 156 patients with Coombs test and in 30 for the Elution test. Thus, combined results of both test would have suggested a need for phototherapy in 72 of 197 patients when in fact it was needed for only 41. Conversely, 13 patients with hemolytic disease were missed by both tests. The negative predictive value of both tests combined was 95% which is useful for clinical practice.

Coombs Test↗
PubMed1990

Anchorin CII, a collagen-binding chondrocyte surface protein of the calpactin family.

In an attempt to identify collagen-binding proteins on the chondrocyte surface, a protein of Mr 34KD, called Anchorin CII was isolated from chondrocyte membranes by affinity chromatography on type II collagen sepharose (Mollenhauer & von der Mark, 1983). The protein was localized on the chondrocyte surface by immunofluorescence labeling using a specific rabbit antibody (Mollenhauer et al., 1984), by immunogold labeling and by cell surface iodination (Pfäffle et al., 1988). Fab'fragments of anti anchorin CII reduced the binding of chondrocytes to type II collagen substrates (Mollenhauer et al., 1984). Analysis of the complete primary structure of anchorin CII revealed 4 repetitive domains of each 70-80 amino acid residues, and the absence of hydrophobic transmembrane sequences or signal peptides (Fernández et al., 1988). Thus, anchorin CII is another member of the calpactin/lipocortin/annexin family, although most other members of this family are located strictly intracellularly. Similar to lipocortin I, however, anchorin CII can be identified extracellularly, e.g. in the culture medium of chondrocytes and fibroblasts (Pfäffle et al., 1988). Here we report on further studies on sequence homologies to other annexins, and on the Ca(++)- and phospholipide binding of this protein.

Amino Acid Sequence↗
PubMed1988

Biosynthesis, secretion and extracellular localization of anchorin CII, a collagen-binding protein of the calpactin family.

The amino acid sequence of anchorin CII, a collagen-binding protein isolated originally from chondrocyte membranes, was previously determined by sequencing of cDNA and proteolytic fragments of the protein. Computer analysis of the protein sequence revealed four internal repeats of approximately 70-80 residues, each containing a highly conserved consensus sequence of 17 residues. These repeats show considerable homology with sequences in human and bovine calpactin, lipocortin, endonexin and protein II, which are members of a family of Ca2+- and phospholipid-binding proteins, as well as major substrates of tyrosine kinases. While these proteins have been located at the inner side of the plasma membrane of fibroblasts and epithelial cells, here we present experimental evidence that anchorin CII is at least partially released from cells and binds to the outer cell surface. Biosynthesis studies in cell-free systems and in cell culture indicate that anchorin CII is not processed, which is consistent with the absence of signal sequences from the protein. Yet, pulse-chase experiments show that anchorin is released into the culture medium of fibroblasts after 30 min, and in chondrocyte cultures after 20 h. Anchorin CII was located to the outer cell surface of chondrocytes by lactoperoxidase-catalyzed cell surface iodination as well as by antibody labeling both at light- and electron-microscopical level. The pericellular localization of anchorin CII is consistent with the notion that this protein is involved in the interaction of chondrocytes and fibroblasts with extracellular collagen.

Amino Acid Sequence↗
PubMed1985

Methylation of type II and type I collagen genes in differentiated and dedifferentiated chondrocytes.

The methyl-sensitive restriction endonucleases HpaII and HhaI as well as the methyl-insensitive enzyme MspI were used to examine the methylation status of the pro-alpha 1(II) collagen gene of cartilage. Five different cell types with varying abilities to express type II collagen were studied. Chick embryo chondrocytes express type II collagen, while 5-bromodeoxyuridine-treated chondrocytes, retinoic acid-treated chondrocytes, chick embryo fibroblasts, and erythrocytes do not synthesize type II collagen. Both cDNA and genomic probes for the pro-alpha 1(II) collagen gene were used, covering the complete 3' end of the gene and its flanking sequences. The pro-alpha 1(II) collagen DNA was undermethylated in chondrocytes, compared to either fibroblasts or erythrocytes. However, the methylation of the 5-bromodeoxyuridine-treated and retinoic acid-treated chondrocytes was identical to that of control chondrocytes. The methylation pattern of two regions of the gene of the pro-alpha 2(I) collagen chain was identical in all cell types tested, whether or not the gene was expressed. Our results indicate that genes for these collagen chains differ in their methylation pattern. The type II collagen gene shows reduced methylation in expressing cartilage, but does not acquire an increase in methylation in "dedifferentiated" chondrocytes. The changes in DNA methylation that occur during cell differentiation do not appear to be sufficient to explain gene activation and deactivation.

Animals↗