Optimization of intracellular microcystin-LR extraction for its analysis by protein phosphatase inhibition assay.

Microcystins are toxins produced by some strains of cyanobacteria. Several methods have been developed to allow the quantification of microcystins, which are mainly endotoxins. Among those methods, the protein phosphatase inhibition assay is a good candidate as a screening method because of its sensitivity, simplicity and specificity. In this work a method for intracellular microcystin extraction in field water samples and lab cyanobacterial cultures prior to their analysis by protein phosphatase inhibition assay has been optimized. Microcystin-LR and Microcystis aeruginosa PCC 7806 were used as reference microcystin and strain, respectively, in order to optimize the protocol. The protocol consists on filtering the sample through a nylon filter of 0.8 microm, filter extraction with methanol 80% 0.1% trifluoroacetic acid (TFA) 0.1% tween 20, extract centrifugation and supernatant dilution (1/20). The establishment of an extraction protocol was carried out determining the extraction volume, time of extraction and number of extractions. The advantages of the method developed in this work are basically its simplicity and avoiding the use of specific and expensive equipment.

Development of a simple enzyme immunoassay for the determination of ovine luteinizing hormone.

The present study describes the development and validation of a simple sensitive and specific sandwich enzyme immunoassay (EIA) for the quantification of ovine luteinizing hormone (LH) in plasma. Microtitre plates were coated with the capture antibody 518b7 anti-bovine LH. A second peroxidase-labelled anti-ovine LH antibody was used as tracer. A simple 3-step procedure was used for the sample analysis; (1) incubation of standards and samples with the pre-coated antibody plates for 2 h at 37 degrees C; (2) incubation with the peroxidase-labelled antibody for 1 h at room temperature; and (3) colour development with TMB substrate. A linear dose-response curve was obtained in the range 0-10 ng/ml (r2 > 0.99). The detection limit was 0.05 ng/ml, and the intra-assay and inter-assay coefficients of variation were 7% and 11.7%, respectively. The theoretical stability of microplates and reagents was calculated, this being greater than one year. Low or undetectable cross-reactivities were recorded for follicle-stimulating hormone, bovine thyroid-stimulating hormone, equine chorionic gonadotrophin and a gonadotrophin-releasing hormone (GnRH) analogue. The EIA was biologically validated by the determination of plasma LH concentrations of nine Rasa Aragonesa ovariectomized and estradiol-implanted ewes after a double GnRH challenge. In conclusion, this enzyme immunoassay provides an efficient, simple and sensitive method for the routine analysis of ovine LH.

Expression of ferredoxin-NADP+ reductase in heterocysts from Anabaena sp.

The expression of ferredoxin-NADP+ reductase (FNR) from Anabaena sp. PCC 7119 in heterocysts and vegetative cells has been quantified. Specific reductase activity in heterocysts was approximately 10 times higher than in vegetative cells, corresponding to the increased FNR protein content. This was confirmed by immunoquantification of the FNR protein from whole filaments of Anabaena sp. PCC 7120 grown in media with and without combined nitrogen. Transcription of the petH gene was markedly enhanced in the absence of combined nitrogen. This suggests that the increased RNA level is mainly responsible for the up-regulation of FNR in heterocysts. As has been observed for nif genes, iron deficiency also increased transcription of petH. Characterization of the FNR purified from isolated heterocysts showed no detectable differences from the enzyme from vegetative cells. Although nitrogen stress was a key regulatory factor, localization of the petH gene in the genomic map of Anabaena PCC 7120 showed that this gene is not physically associated with the nif cluster.

Interference of nucleases in cyanobacterium ferredoxin purification.

Isolation of cyanobacterial ferredoxin is normally carried out using nucleases in order to degrade the nucleic acids that accompany this protein during the purification procedure. However, this practice presents the inconvenience that these proteins remain in trace amounts in the purified ferredoxin preparations, although they are not visible by electrophoretical techniques. Evidence of that fact is shown in this report and an alternative procedure is described for the rapid preparation of ferredoxin from crude extracts of Anabaena PCC 7119. The method involves a treatment of the crude extract with streptomycin sulphate, a high molecular weight polication that precipitates the nucleic acids in the beginning of the purification.

Differential activities of heterocyst ferredoxin, vegetative cell ferredoxin, and flavodoxin as electron carriers in nitrogen fixation and photosynthesis in Anabaena sp.

In cyanobacteria an increasing number of low potential electron carriers is found, but in most cases their contribution to metabolic pathways remains unclear. In this work, we compare recombinant plant-type ferredoxins from Anabaena sp. PCC 7120, encoded by the genes petF and fdxH, respectively, and flavodoxin from Anabaena sp. PCC 7119 as electron carriers in reconstituted in vitro assays with nitrogenase, Photosystem I, ferredoxin-NADP(+) reductase and pyruvate-ferredoxin oxidoreductase. In every experimental system only the heterocyst ferredoxin catalyzed an efficient electron transfer to nitrogenase while vegetative cell ferredoxin and flavodoxin were much less active. This implies that flavodoxin is not able to functionally replace heterocyst ferredoxin. When PFO-activity in heterocyst extracts was reconstituted under anaerobic conditions, both ferredoxins were more efficient than flavodoxin, which suggested that this PFO was of the ferredoxin dependent type. Flavodoxin, synthesized under iron limiting conditions, replaces PetF very efficiently in the electron transport from Photosystem I to NADP(+), using thylakoids from vegetative cells.

Transcriptional and translational analysis of ferredoxin and flavodoxin under iron and nitrogen stress in Anabaena sp. strain PCC 7120.

In Anabaena sp. strain PCC 7120, vegetative cell ferredoxin synthesis under iron starvation was repressed 25-fold, whereas heterocyst ferredoxin synthesis decreased only 2.8-fold. Induction of flavodoxin under iron depletion was independent of the availability of combined nitrogen. Under iron stress but in the presence of combined nitrogen, fdxH and nifH genes were transcriptionally active; although excision of the 11-kb element seemed to be completed, nitrogenase activity and the fdxH gene product were not detectable.