Development and validation of a specific real-time PCR assay for the detection of the parasite Perkinsus olseni.

Perkinsus olseni is a protozoan parasite that infects a wide variety of molluscs worldwide, causing economic losses in the aquaculture sector. In the present study, a quantitative PCR (qPCR) assay was developed for the detection and quantification of P. olseni in clam gill tissue and hemolymph (Ruditapes philippinarum and R. decussatus), and the results were compared with those of the standard diagnostic methods recommended by the O.I.E. (World Organisation for Animal Health): Ray's fluid thioglycollate culture method (RFTM), a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay and histopathology. The efficiency, sensitivity and reproducibility of the newly described qPCR assay were also determined. The highest prevalence was detected using the qPCR assay, and the strongest linear correlation was obtained between the RFTM infection levels and the threshold cycle (Ct) number from the gill tissue. Although better results were obtained from gill than from the hemolymph in the qPCR assays, especially with lower infection levels of the parasite, a significant linear correlation was observed between Ct values from the gill and hemolymph. The qPCR assay that was developed in this study showed high sensitivity, specificity and reproducibility for the detection and quantification of P. olseni.

Pigment conformation and pigment-protein interactions in the reconstituted Lhcb4 antenna protein.

Resonance Raman spectra of the native Lhcb4 antenna protein are compared with those of a recombinant protein prepared by in vitro refolding of its polypeptide, over-expressed in Escherichia coli, with added pigments [Giuffra et al. (1996) Eur. J. Biochem. 238, 112-120]. The results indicate that the native pigment conformation is reproduced almost perfectly in the reconstituted protein, with only small differences which are attributed to a slight shift in the Soret absorption peak of two or more chlorophylls. This procedure therefore represents a model system for the investigation of site-directed mutant LHC proteins, which are otherwise very difficult to obtain.

Suborganellar localisation and effect of light on Helianthus tuberosus chloroplast transglutaminases and their substrates.

The light stimulation of transglutaminase (TGase EC 2.3.2.13) activity was verified by incubating isolated chloroplasts of Helianthus tuberosus L. continuously or for alternate periods of light or dark (light/dark and dark/light). The first 10 min of incubation always represented the critical period. Light-harvesting complexes of photosystem II (LHCII) were more intensely labelled by (14)C-polyamines under light and light/dark than under dark and dark/light conditions. Chloroplasts were fractionated into thylakoid- and stroma-enriched fractions in which multiple TGase forms and substrates were found. Antibodies against TGase recognised 58- and 24-kDa bands in thylakoids and a 150-kDa band in the stroma. The latter, and its 150-kDa fraction, catalysed the conjugation of 14C-polyamines to Rubisco. In both fractions (thylakoid-pre and stroma-pre) the analysis of polyamine glutamyl derivatives showed a significant light-affected conjugation of polyamines to endogenous proteins. Alternatively, entire chloroplasts were incubated and afterwards their sub-fractions were isolated (thylakoid-post and stroma-post). The PSII and LHCII complexes were more intensely immunodetected in thylakoid-post than in thylakoid-pre, especially under dark conditions. Conversely, the conjugation of polyamines to thylakoid proteins was clearly light-stimulated in thylakoid-post, and much less in thylakoid-pre. Stroma-pre proteins were poorly polyamine-conjugated and not light-affected; on the contrary, stroma-post proteins were much more polyamine-modified and strongly light-stimulated. Thus, the light-activated conjugation depends mainly on the presence of the thylakoid fraction during the assay. The protective effect on chloroplasts under photo-damage, stress or senescence conditions attributed in the literature to free polyamines is discussed with regard to the occurrence of polyamine conjugates catalysed by TGases.