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Appl Environ Microbiol ; 77(5): 1651-9, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21193668


The frequency and intensity of Pseudo-nitzschia spp. blooms along the coast of Catalonia have been increasing over the past 20 years. As species from this genus that are documented as toxigenic have been found in local waters, with both toxic and nontoxic species cooccurring in the same bloom, there is a need to develop management tools for discriminating the difference. Currently, differentiation of toxic and nontoxic species requires time-consuming electron microscopy to distinguish taxonomic features that would allow identification as to species, and cryptic species can still remain misidentified. In this study, cells of Pseudo-nitzschia from clonal cultures isolated from seawater were characterized to their species identity using scanning electron microscopy, and subsamples of each culture were used to create an internal transcribed spacer 1 (ITS-1), 5.8S, and ITS-2 ribosomal DNA database for development of species-specific quantitative PCR (qPCR) assays. Once developed, these qPCR assays were applied to field samples collected over a 2-year period in Alfaques Bay in the northwestern Mediterranean Sea to evaluate the possibility of a comprehensive surveillance for all Pseudo-nitzschia spp. using molecular methods to supplement optical microscopy, which can discern taxonomy only to the genus level within this taxon. Total Pseudo-nitzschia cell density was determined by optical microscopy from water samples collected weekly and compared to results obtained from the sum of eight Pseudo-nitzschia species-specific qPCR assays using duplicate samples. Species-specific qPCR followed by melt curve analysis allowed differentiation of amplicons and identification of false positives, and results correlated well with the total Pseudo-nitzschia cell counts from optical microscopy.

Diatomáceas/classificação , Diatomáceas/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Água do Mar/microbiologia , Análise por Conglomerados , DNA Ribossômico/química , DNA Ribossômico/genética , DNA Espaçador Ribossômico/genética , Diatomáceas/genética , Diatomáceas/ultraestrutura , Genes de RNAr , Mar Mediterrâneo , Microscopia Eletrônica de Varredura , Filogenia , RNA Ribossômico 5,8S/genética , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico
J Invertebr Pathol ; 100(1): 50-3, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18823999


We present the first record in Spanish Mediterranean waters of the protozoan parasite Perkinsus olseni infecting the clam Ruditapes decussatus. Perkinsus infection was detected all year around albeit at a low level of infection intensity. Histological analysis, induction of zoospores and in situ hybridisation assay confirmed the presence of Perkinsus sp. The identity of the parasite was determined by species-specific PCR assay in DNA samples obtained from infected clams. Sequencing of amplified fragments showed 100% identity to the ITS region of P. olseni. We confirmed for the first time the presence of P. olseni in Spanish Mediterranean waters.

Bivalves/parasitologia , Parasitos/isolamento & purificação , Animais , Bivalves/citologia , Mar Mediterrâneo , Parasitos/classificação , Parasitos/genética , RNA Ribossômico/química , Análise de Sequência de RNA , Espanha
Parasitol Res ; 99(6): 631-7, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16710674


A P-glycoprotein homologue (Pgh1) is believed to play a role in modulating levels of chloroquine resistance in Plasmodium falciparum. To study the role of Pgh1 in the mechanism of chloroquine (CQ) resistance, antisera were raised against this protein. There was no direct association between the level of Pgh1 expression and chloroquine sensitivity. We also failed to detect phosphorylation of Pgh1 in the food vacuole (FV), suggesting that other mechanisms regulate the chloroquine-resistant (CQR) phenotype. Therefore, high levels of expression of Pgh1 or phosphorylation of this protein in the FV could not account for CQ sensitivity. In addition, the lack of inhibition of CQ accumulation by anti-Pgh1 antibodies suggests that Pgh1 is not involved as a CQ transporter in the plasma membrane of P. falciparum. Furthermore, resistance reversers do not appear to act at the plasma membrane level.

Transportadores de Cassetes de Ligação de ATP/metabolismo , Antimaláricos/farmacologia , Cloroquina/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Vacúolos/química , Animais , Membrana Celular/metabolismo , Cloroquina/metabolismo , Resistência a Medicamentos , Soros Imunes/metabolismo , Fosforilação , Plasmodium falciparum/citologia
Chemotherapy ; 52(1): 50-2, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16340201


Resistance of Plasmodium falciparum to chloroquine (CQ) has been associated with a decrease in CQ accumulation by parasitized erythrocytes. This study aimed at investigating the role of parasite plasma membranes (PPM) in the mechanism of CQ accumulation. CQ accumulation capabilities of membranes were determined using tritiated CQ. PPM isolated from chloroquine-sensitive parasites were found to accumulate less CQ than those isolated from chloroquine-resistant parasites. However, CQ accumulation was found to be ATP-independent suggesting that this accumulation results from binding rather than transport.

Membrana Celular/metabolismo , Cloroquina/metabolismo , Plasmodium falciparum/citologia , Plasmodium falciparum/metabolismo , Animais