BackgroundThe use of molecular biology-based methods for species identification and establishing phylogenetic relationships has supplanted traditional methods relying on morphological characteristics. While PCR-based methods are now the commonly accepted gold standards for these types of analysis, relatively high costs, time-consuming assay development or the need for a priori information about species-specific sequences constitute major limitations. In the present study, we explored the possibility to differentiate between 13 different species from the genus Drosophila via a molecular proteomic approach.ResultsAfter establishing a simple protein extraction procedure and performing matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) with intact proteins and peptides, we could show that most of the species investigated reproducibly yielded mass spectra that were adequate for species classification. Furthermore, a dendrogram generated by cluster analysis of total protein patterns agrees reasonably well with established phylogenetic relationships.ConclusionConsidering the intra- and interspecies similarities and differences between spectra obtained for specimens of closely related Drosophila species, we estimate that species typing of insects and possibly other multicellular organisms by intact protein profiling (IPP) can be established successfully for species that diverged from a common ancestor about 3 million years ago.

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Neuronally enriched primary cerebrocortical cultures were exposed to glucose-free medium saturated with argon (in vitro ischemia) instead of oxygen (normoxia). Ischemia did not alter P2X 7 receptor mRNA, although serum deprivation clearly increased it. Accordingly, P2X 7 receptor immunoreactivity (IR) of microtubuline-associated protein 2 (MAP2)-IR neurons or of glial fibrillary acidic protein (GFAP)-IR astrocytes was not affected; serum deprivation augmented the P2X 7 receptor IR only in the astrocytic, but not the neuronal cell population. However, ischemia markedly increased the ATP-and 2¢-3¢-O-(4-benzoylbenzoyl)-adenosine 5¢-triphosphate (

ATP, ADPbetaS and UTP induced a comparable rise in the intracellular Ca2+ concentration ([Ca2+]i) in HEK-293 cells using fura-2 microfluorimetry. The responses persisted in Ca2+-free medium, but were abolished following depletion of intracellular Ca2+ stores by cyclopiazonic acid. Cross-desensitisation experiments demonstrated that exposure to ADPbetaS has no marked effect on UTP-induced [Ca2+]i transients and vice versa. Whereas the P2Y1 receptor-selective antagonist 2'-deoxy-N6-methyladenosine 3',5'-diphosphate (MRS 2179) abolished the responses to ADPbetaS, it decreased and did not alter the responses to ATP and UTP respectively. Although the P2Y1/P2Y4 receptor-preferential antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) abolished the responses to ADPbetaS, and decreased those to ATP, it also depressed the UTP-induced [Ca2+]i transients. Suramin, an antagonist with preference for P2Y2 receptors decreased both the ATP- and UTP-induced [Ca2+]i reactions. After numerous splittings, HEK-293 cells failed to react to ADPbetaS; however, repeated superfusion with this P2Y1 receptor agonist restored the [Ca2+]i signals. In agreement with the functional data, real-time polymerase chain reaction and immunocytochemical studies indicated the presence of P2Y1, P2Y2 and P2Y4 receptors. Our findings raise doubt with respect to the reliability of HEK-293 cells as expression systems for recombinant P2X receptors, because of a possible functional interaction with endogenous P2Y receptors.

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