Biomedical subjects

J A Navarro

Publications and source records attributed to J A Navarro.

At least 19 recordsLinked to original sources

PubMed2006

Characterization of a murine model of intranasal infection suitable for testing vaccines against C. abortus.

Mouse models have been widely used to test candidate vaccines against Chlamydophila abortus infection in mice. Although the induction of a systemic infection by endogenous or intraperitoneal inoculation is a useful tool for understanding the immune mechanism involved in the protection conferred by the vaccination, a different approach is necessary to understand other factors of the infection, such as mucosal immunity or the colonization of target organs. To test whether C. abortus intranasal model of infection in mice is a useful tool for testing vaccines in a first group of experiments mice, were infected intranasally with C. abortus to characterize the model of infection. When this model was used to test vaccines, two inactivated experimental vaccines, one of them adjuvated with QS-21 and another with aluminium hydroxide, and a live attenuated vaccine (strain 1B) were used. Non-vaccinated control mice died within the first 8 days, after displaying substantial loss of weight. Histologically, the mice showed lobar fibrinopurulent bronchointerstitial pneumonia. Prior immunization with QS-21 adjuvated vaccine or 1B vaccine presented mortality and the recipients showed a greater number of T cells in the lesions, especially CD8(+) T cells, than the control mice and mice immunized with vaccine adjuvated with aluminium hydroxide. The results confirm that the C. abortus intranasal model of infection in mice is a useful tool for testing vaccines.

Abortion, Veterinary↗

PubMed2006

RNA-binding properties and membrane insertion of Melon necrotic spot virus (MNSV) double gene block movement proteins.

Advances in structural and biochemical properties of carmovirus movement proteins (MPs) have only been obtained in p7 and p9 from Carnation mottle virus (CarMV). Alignment of carmovirus MPs revealed a low conservation of amino acid identity but interestingly, similarity was elevated in regions associated with the functional secondary structure elements reported for CarMV which were conserved in all studied proteins. Nevertheless, some differential features in relation with CarMV MPs were identified in those from Melon necrotic virus (MNSV) (p7A and p7B). p7A was a soluble non-sequence specific RNA-binding protein, but unlike CarMV p7, its central region alone could not account for the RNA-binding properties of the entire protein. In fact, a 22-amino acid synthetic peptide whose sequence corresponds to this central region rendered an apparent dissociation constant (K(d)) significantly higher than that of the corresponding entire protein (9 mM vs. 0.83-25.7 microM). This p7A-derived peptide could be induced to fold into an alpha-helical structure as demonstrated for other carmovirus p7-like proteins. Additionally, in vitro fractionation of p7B transcription/translation mixtures in the presence of ER-derived microsomal membranes strongly suggested that p7B is an integral membrane protein. Both characteristics of these two small MPs forming the double gene block (DGB) of MNSV are discussed in the context of the intra- and intercellular movement of carmovirus.

Amino Acid Sequence↗

PubMed2006

Relative importance of CD4+ and CD8+ T cells in the resolution of Chlamydophila abortus primary infection in mice.

The role of the specific cellular immune response is well established in Chlamydiaceae infections, but the importance of each T-cell subset seems to be species-dependent. This study was designed to clarify the role of T-cell subsets in the response to Chlamydophila abortus primary infection. C57BL/6 mice were depleted of CD4+ or CD8+, or both, by monoclonal antibody injections and subsequently infected with C. abortus. Mice were killed at intervals and samples were collected for bacteriological and histopathological analysis. Also carried out were spleen cell culture, cytokine quantification, immunolabelling for C. abortus antigen, and a TUNEL assay for apoptosis. CD8+ T cell-depleted mice all died within 12 days of C. abortus infection, while no mortality was observed in the other groups; surprisingly, CD4+ T cell-depleted mice showed lower morbidity (expressed as weight loss) than did a non-depleted (control) group. CD8+ T cell-depleted mice also differed from the other groups in showing a significantly higher chlamydial burden in the liver. CD8+ T cell-depleted mice also had a higher number of apoptotic cells in hepatic inflammatory foci and showed exacerbated IFN-gamma production by spleen cells after specific stimulation. Simultaneous depletion of both T-cell subpopulations led to a chronic infection, but not to early mortality. It is concluded that CD8+ T cells may play a role in the regulatory control of the CD4+ T-cell response and may have a direct cytotoxic or IFN-gamma-mediated effect on infected cells.

Animals↗

PubMed2006

Functional analysis of the five melon necrotic spot virus genome-encoded proteins.

Function of the melon necrotic spot virus (MNSV) genome-encoded proteins (p29, p89, p7A, p7B and p42) has been studied. Protein-expression mutants of an infectious, full-length cDNA clone of a Spanish MNSV-Al isolate and a recombinant green fluorescent protein (GFP)-expressing virus were used in infection bioassays on melon plants. Results revealed that p29 and p89 are both essential for virus replication, whereas small proteins p7A and p7B are sufficient to support viral movement between adjacent cells operating in trans. It is also demonstrated that, in addition to its structural role as coat protein, p42 is an important factor controlling symptoms and is required for systemic transport. Moreover, both p42 and p7B, among all of the MNSV-encoded proteins, were able to delay RNA silencing in transient-expression assays on GFP-transgenic Nicotiana benthamiana plants. Finally, the presence of p42 also produced an enhancing effect on local spread similar to that of potyviral helper component proteinase (HC-Pro), probably due to its RNA silencing-suppression ability.

Carmovirus↗

PubMed2005

Immunophenotypical characterization of lymphocyte subpopulations of the uterus of non-pregnant and pregnant goats.

The increased susceptibility during pregnancy to certain pathogens that cause abortions may be related to changes in the distribution and phenotype of lymphocyte subpopulations in the uterus. Histological, electron microscopic and immunocytochemical techniques were used in this study to examine whether such variations occur in different stages of the reproductive cycle of goats. The study of non-pregnant goats showed that most uterine lymphocytes were T cells and displayed both an intraepithelial and stromal distribution. CD8+ T lymphocytes were more numerous than CD4+ T lymphocytes. In the endometrial epithelium two lymphocyte subpopulations were observed: non-granulated CD2+ CD8+ T lymphocytes and granulated CD2+ CD8- T lymphocytes. During gestation, no lymphocytes were observed in the placentomal area, while a decreased number of T lymphocyte subpopulations were found in the inter-placentomal area. In the inter-caruncular epithelium, non-granulated CD2+ CD8+ T lymphocytes disappeared, whereas the granulated CD2+ CD8- T lymphocyte subpopulations increased their number and changed their morphology.

Animals↗

PubMed2005

Canine carcinosarcomas in the head.

Four cases of neoplasms in the heads of old dogs were studied. All the dogs showed both carcinomatous and sarcomatous malignant components in an admixed growing pattern. Histologic analysis of the tumors showed that the carcinomatous cells resembled squamous cell carcinoma in all dogs except one, where an adenoid arrangement of the neoplastic cells was also observed. The sarcomatous component showed osteoid matrix produced by pleomorphic poorly differentiated cells, which is regarded as a typical feature of osteosarcomas. Immunohistochemical analysis demonstrated that the components were positive only for cytokeratin (carcinomatous component) or vimentin (sarcomatous component). This observation led us to classify the neoplasms as true carcinosarcomas, thus providing evidence of a new preferential location for this unusual tumor in dog.

Animals↗

PubMed2004

Genetic variability in the coat protein genes of lettuce big-vein associated virus and Mirafiori lettuce big-vein virus.

Available data suggests that lettuce big-vein disease is caused by the ophiovirus Mirafiori lettuce big-vein virus (MLBVV) but not by the varicosavirus Lettuce big-vein-associated virus (LBVaV), although the latter is frequently associated with the disease. Since the disease occurs worldwide, the putative coat protein (CP) open reading frames of geographically distinct isolates of MLBVV and LBVaV were sequenced. Comparison of both nucleotide and amino acid sequences showed a high level of sequence similarity among LBVaV isolates. Phylogenetic analysis of LBVaV CP nucleotide sequences showed that most of the Spanish isolates clustered in a phylogenetic group whereas English isolates were more similar to the USA isolate. An Australian isolate was closely related to the Dutch isolate. Genetic diversity among MLBVV CP nucleotide sequences was higher ranging from 0.2% to 12%. Phylogenetic analysis of MLBVV CP nucleotide sequences revealed two distinct subgroups. However, this grouping was not correlated with symptom development on lettuce or the geographic origin of the MLBVV isolates. Finally, a quick method based on RFLP analysis of RT-PCR amplicons was developed for assigning MLBVV isolates to the two subgroups.

Amino Acid Sequence↗

PubMed2004

Natural killer (NK) cells play a critical role in the early innate immune response to Chlamydophila abortus infection in mice.

Chlamydophila abortus, the aetiological agent of ovine enzootic abortion, induces a strong inflammatory reaction that leads to the T helper cell (Th1) specific immune response necessary for the clearance of infection. Because the role of natural killer (NK) cells during the first stages of this response has received little attention, this study focused on determining the function of these cells in a mouse model of infection. The location of NK cells in the liver and spleen of infected mice was examined immunohistochemically with an anti-Ly49G monoclonal antibody. The number of NK cells increased during the infection both in spleen and liver. In subsequent experiments, an anti-asialo GM1 polyclonal antibody was injected to deplete the NK cells. NK-depleted mice showed a substantial increase in their susceptibility to C. abortus infection, with high mortality rates and an increased burden of bacteria in the liver. Histopathological studies showed that inflammatory foci, composed mainly of neutrophils, were greater in size and number in depleted mice, while numerous chlamydial inclusions were associated with the foci. Serum concentrations of IFN-gamma, a key cytokine in the control of C. abortus infection, were substantially reduced in the NK-depleted mice. To establish the relationship between NK cells and other components of the innate immune response, neutrophils were depleted with the RB6-8C5 antibody. These cells were shown to be crucial in the recruitment of NK cells to the inflammatory foci.

Animals↗

PubMed2004

Kinetics of infection and effects on the placenta of Chlamydophila abortus in experimentally infected pregnant ewes.

A Chlamydophila abortus-induced abortion model was carried out on the basis of the experimental infection of ewes at day 75 of gestation. The infection induced abortions and the birth of weak lambs during the last 3 weeks of pregnancy. To study the kinetics of the infection in the placenta and in other organs, infected ewes were killed at 105, 120, and 130 days of gestation and also several days after abortion or parturition. Infected ewes developed a systemic infection that caused a mild and transient pneumonia and focal hepatitis. Pathologic changes were observed in placentas at 120 day of gestation, although the lesions varied between animals and even between placentomes of the same placenta. The first placental area infected was the maternal stroma and epithelium next to the intercaruncular areas, where neutrophilic response seemed to control the infection. A substantial degree of multiplication of C. abortus was then observed in the trophoblast cells of the placentome, periplacentomal choriallantoic membranes, and hilius, with an inflammatory exudate composed mainly of neutrophils, some macrophages, and very scarce lymphocytes. After abortion, the lesions affected the intercotyledonary areas of the aborted placentas, whereas in the uterus significant lymphocyte infiltration was observed, together with a rapid decrease of the C. abortus antigen in the degenerated caruncular tissues.

Abortion, Veterinary↗

PubMed2003

Detection of melon necrotic spot virus in water samples and melon plants by molecular methods.

Melon necrotic spot virus (MNSV) is a water and soil-borne pathogen affecting species of the Cucurbitaceae family both in hydroponic and soil crops. Molecular methods for detecting MNSV in water samples, nutrient solutions and melon plants were developed. For this purpose, water samples from a water source pool of a hydroponic culture or from the recirculating nutrient solution were concentrated by ultracentrifugation or PEG precipitation followed by RT-PCR analysis. Both concentration methods were suitable to allow the detection of MNSV and represent, as far as we know, the first time that this virus has been detected in water samples. A non-isotopic riboprobe specific for MNSV was obtained and used to detect the virus in plant tissue. Different parts of mechanically infected plants were examined including the roots, stems, inoculated cotyledons and young leaves. Excluding the inoculated cotyledons, the tissues showing the highest accumulation levels of the virus were the roots. The potential inclusion of such tools in management programs is discussed.

Antibodies, Viral↗

PubMed2001

The unique proline of the Prochlorothrix hollandica plastocyanin hydrophobic patch impairs electron transfer to photosystem I.

A number of surface residues of plastocyanin from Prochlorothrix hollandica have been modified by site-directed mutagenesis. Changes have been made in amino acids located in the amino-terminal hydrophobic patch of the copper protein, which presents a variant structure as compared with other plastocyanins. The single mutants Y12G, Y12F, Y12W, P14L, and double mutant Y12G/P14L have been produced. Their reactivity toward photosystem I has been analyzed by laser flash absorption spectroscopy. Plots of the observed rate constant with all mutants versus plastocyanin concentration show a saturation profile similar to that with wild-type plastocyanin, thus suggesting the formation of a plastocyanin-photosystem I transient complex. The mutations do not induce relevant changes in the equilibrium constant for complex formation but induce significant variations in the electron transfer rate constant, mainly with the two mutants at proline 14. Additionally, molecular dynamics calculations indicate that mutations at position 14 yield small changes in the geometry of the copper center. The comparative kinetic analysis of the reactivity of plastocyanin mutants toward photosystem I from different organisms (plants and cyanobacteria) reveals that reversion of the unique proline of Prochlorothrix plastocyanin to the conserved leucine of all other plastocyanins at this position enhances the reactivity of the Prochlorothrix protein.

Bacterial Proteins↗

PubMed2001

A single arginyl residue in plastocyanin and in cytochrome c(6) from the cyanobacterium Anabaena sp. PCC 7119 is required for efficient reduction of photosystem I.

Positively charged plastocyanin from Anabaena sp. PCC 7119 was investigated by site-directed mutagenesis. The reactivity of its mutants toward photosystem I was analyzed by laser flash spectroscopy. Replacement of arginine at position 88, which is adjacent to the copper ligand His-87, by glutamine and, in particular, by glutamate makes plastocyanin reduce its availability for transferring electrons to photosystem I. Such a residue in the copper protein thus appears to be isofunctional with Arg-64 (which is close to the heme group) in cytochrome c(6) from Anabaena (Molina-Heredia, F. P., Diaz-Quintana, A., Hervás, M., Navarro, J. A., and De la Rosa, M. A. (1999) J. Biol. Chem. 274, 33565-33570) and Synechocystis (De la Cerda, B., Diaz-Quintana, A., Navarro, J. A. , Hervás, M., and De la Rosa, M. A. (1999) J. Biol. Chem. 274, 13292-13297). Other mutations concern specific residues of plastocyanin either at its positively charged east face (D49K, H57A, H57E, K58A, K58E, Y83A, and Y83F) or at its north hydrophobic pole (L12A, K33A, and K33E). Mutations altering the surface electrostatic potential distribution allow the copper protein to modulate its kinetic efficiency: the more positively charged the interaction site, the higher the rate constant. Whereas replacement of Tyr-83 by either alanine or phenylalanine has no effect on the kinetics of photosystem I reduction, Leu-12 and Lys-33 are essential for the reactivity of plastocyanin.

Amino Acid Substitution↗

PubMed2001

Crystal structure of low-potential cytochrome c549 from Synechocystis sp. PCC 6803 at 1.21 A resolution.

The crystal structure of low-potential cytochrome c549, an extrinsic component of the photosystem II (PS II) from Synechocystis sp. PCC 6803, was obtained directly from single-wavelength 1.21 A resolution diffraction data. This is the first monodomain bis-histidinyl monoheme cytochrome c to be structurally characterized. The extended N-terminal region of c549 builds up a two-strand antiparallel beta-sheet in a hairpin motif, which extends through two molecules owing to crystal packing. Both peptide termini are involved in crystal contacts, which may explain their protrusion out of the globular fold. The C-terminus is preceded by a 9 A-long hydrophobic finger extending from a positively charged base and could be involved in PSII interactions, as well as a protruding negative patch built by a set of conserved acidic residues among c549 sequences.

Amino Acid Sequence↗

PubMed2001

A comparative study of the thermal stability of plastocyanin, cytochrome c(6) and Photosystem I in thermophilic and mesophilic cyanobacteria.

Cytochrome c(6) (Cyt) from the thermophilic cyanobacterium Phormidium laminosum has been purified and characterized. It is a mildly acidic protein, with physicochemical properties very similar to those of plastocyanin (Pc). This is in agreement with the functional interchangeability of the two metalloproteins as electron donors to Photosystem I (PS I). The kinetic analyses of the interaction of Pc and Cyt with Photosystem I show that both metalloproteins reduce PS I with similar efficiencies, according to an oriented collisional kinetic model involving repulsive electrostatic interactions. The thermostability study of the Phormidium Pc/PS I system compared with those from mesophilic cyanobacteria (Synechocystis, Anabaena and Pseudanabaena) reveals that Pc is the partner limiting the thermostability of the Phormidium couple. The cross-reactions between Pc and PS I from different organisms demonstrate not only that Phormidium Pc enhances the stability of the Pc/PS I system using PS I from mesophilic cyanobacteria, but also that Phormidium PS I possesses a higher thermostability than the other photosystems.

Journal Article↗

PubMed2001

Differences in lymphocyte subpopulations from peripheral blood and lymphoid organs in natural caprine tuberculosis infection.

Although the cell-mediated immune response is known to be a critical factor in host defence against intracellular mycobacterial infection, the different components of the T-cell response are unclear, particularly in caprine infection. In this study we examine the differences in the lymphocyte population of peripheral blood, spleen and mediastinal and superficial lymph nodes in 11 naturally infected goats showing positive reactions in the comparative tuberculine intradermal test. According to the different types of lesion showing, the goats were classified into proliferative or exudative tuberculosis. The results obtained by fflow cytometry analysis indicated that the main differences in peripheral blood were in the CD4 T-cell population, which decreased markedly in goats with exudative tuberculosis, while the CD8 and B cells increased in number. The gamma/delta T cells did not show significant differences in either type of tuberculosis, while interleukin-2 receptor cells decreased slightly in the exudative tuberculosis. The CD4:CD8 ratio was higher than 1 in goats with proliferative tuberculosis and lower than 1 in goats with exudative tuberculosis. In general, the lymphoid organs of the goats with exudative tuberculosis showed a significant increase in the number of CD8 T cells (CD4:CD8 ratio of less than 1) whereas no significant differences were observed in the CD4 T population between either type of tuberculosis.

Animals↗

PubMed2001

Light and ultrastructural immunocytochemical study of prolactin cells in ovine adenohypophysis. Influence of lactation and weaning.

The influence of lactation and weaning on the number, distribution, and structure of mammotroph cells (PRL cells) in ewes was studied using immunocytochemical procedures for light and electron microscopy, as well as morphometric and stereological techniques. The adenohypophyseal gland of 12 ewes of the Segureña breed at different stages of milk production and weaning were analysed, while the same organ of 3 ewes in anestrus served as control. During lactation, PRL cells increased in number and decreased in size. Ultrastructurally, these cells showed a well-developed rough endoplasmic reticulum (RER) and Golgi complex (GC), and large secretory granules. These findings point to increased synthesis and secretion activities during lactation, and a decrease after weaning. One day after weaning 'storage' cells increased in number, and crinophagy became evident 7 days after weaning, when the first 'inactive' cells were seen (10% of total cells). In anestrus these last cells represented 40% of the total, and showed scarce granules, signs of inactivity, lysosomes and lipid droplets.

Anestrus↗

PubMed2000

dfh is a Drosophila homolog of the Friedreich's ataxia disease gene.

A putative Drosophila homolog of the Friedreich's ataxia disease gene (FRDA) has been cloned and characterized; it has been named Drosophila frataxin homolog (dfh). It is located at 8C/D position on X chromosome and is spread over 1kb, a much smaller genomic region than the human gene. Its genomic organization is simple, with a single intron dividing the coding region into two exons. The predicted encoded product has 190 amino acids, being considered a frataxin-like protein on the basis of the sequence and secondary structure conservation when compared with human frataxin and related proteins from other eukaryotes. The closest match between the Drosophila and the human proteins involved a stretch of 38 amino acids at C-terminus, encoded by dfh exon 2, and exons 4 and 5a of the FRDA gene, respectively. This highly conserved region is very likely to form a functional domain with a beta sheet structure flanked by alpha-helices where the sequence is less conserved. A signal peptide for mitochondrial import has also been predicted in the Drosophila frataxin-like protein, suggesting its mitochondrial localization, as occurs for human frataxin and other frataxin-like proteins described in eukaryotes. The Drosophila gene is expressed throughout the development of this organism, with a peak of expression in 6-12h embryos, and showing a spatial ubiquitous pattern from 4h embryos to the last embryonic stage examined. The isolation of dfh will soon make available specific dfh mutants that help in understanding the pathogenesis of FRDA.

Amino Acid Sequence↗

PubMed2000

Characterization of the initiation sites of both polarity strands of a viroid RNA reveals a motif conserved in sequence and structure.

Viroids replicate through a rolling-circle mechanism in which the infecting circular RNA and its complementary (-) strand are transcribed. The precise site at which transcription starts was investigated for the avocado sunblotch viroid (ASBVd), the type species of the family of viroids with hammerhead ribozymes. Linear ASBVd (+) and (-) RNAs begin with a UAAAA sequence that maps to similar A+U-rich terminal loops in their predicted quasi-rod-like secondary structures. The sequences around the initiation sites of ASBVd, which replicates and accumulates in the chloroplast, are similar to the promoters of a nuclear-encoded chloroplastic RNA polymerase (NEP), supporting the involvement of an NEP-like activity in ASBVd replication. Since RNA folding appears to be kinetically determined, the specific location of both ASBVd initiation sites provides a mechanistic insight into how the nascent ASBVd strands may fold in vivo. The approach used here, in vitro capping and RNase protection assays, may be useful for investigating the initiation sites of other small circular RNA replicons.

Base Sequence↗