Cloning and real-time PCR testing of 14 potential biomarkers in Eisenia fetida following cadmium exposure.

Important biological activities could be affected in metal exposed species, and amongthe main physiological functions, immunity may provide one (or more) effector(s) which expression can be directly affected by a metal exposure in various macroinvertebrates. As many proteinic effectors showed a high degree of homology between species, we have developed a PCR approach to characterize partial mRNA sequences of selected effectors in the laboratory model, Eisenia fetida. After cloning, levels of expression of each gene were analyzed following exposures (80 and 800 mg/kg) to cadmium spiked soils using real-time PCR. An implemented approach was allowed to test quickly potential biomarkers in Eisenia fetida. Selected effectors were calmodulin, heat shock proteins, superoxide dismutase, catalase, metallothionein, beta-adrenergic receptor kinase, pyruvate carboxylase, trancriptionally controlled tumor protein, protein kinase C, and ubiquitin. Most of the selected effectors did not show variations of expression level after exposure. Others expressed weak changes of expression as heat shock proteins. At lastfor catalase and metallothionein, early suitable variations of expression were observed.

Leech immunocytes contain proopiomelanocortin: nitric oxide mediates hemolymph proopiomelanocortin processing.

This report establishes the presence of mammalian-like proopiomelanocotropic hormone (POMC), and six of its peptides, including adrenocorticotropic hormone (ACTH) and melanocyte-stimulating hormone (MSH), in the immune tissues of the leech Theromyzon tessulatum. The 25.4-kDa protein was purified by high pressure gel permeation chromatography, anti-ACTH-affinity column, and reverse-phase HPLC. Its characterization was performed by Edman degradation, enzymatic treatments, and electrospray mass spectrometry. Leech POMC exhibits considerable amino acid sequence similarity to mammalian POMC. Of the six peptides, three showed high sequence similarity to their vertebrate counterparts met-enkephalin, alpha-MSH, and ACTH: 100, 84.6, and 70%, respectively; whereas gamma-MSH, beta-endorphin, and gamma-lipotropin hormone exhibited only 45, 20, and 10% sequence identity, respectively. No dibasic amino acid residues were found at the C terminus of the gamma- and beta-MSH peptides. In contrast, the leech alpha-MSH was flanked at its C-terminal by the Gly-Arg-Lys amidation signal. ACTH and corticotropin-like intermediary pituitary peptide were also C-terminally flanked by dibasic amino acid residues. The coding region of leech POMC was obtained by reverse transcription-PCR using degenerated oligonucleotide primers. Circulating levels of ACTH and MSH were 10 and 1 fmol/microl hemolymph, respectively. Morphine, in a dose-dependent manner, increased the levels of both peptides threefold; this effect was blocked by naloxone treatment. Similar results were found with the anandamide. Leech ACTH was processed to MSH by the enzymes neutral endopeptidase (24.11) and angiotensin-converting enzyme. Leech alpha-MSH had the same activity as authentic alpha-MSH in two bioasssay systems. Taken together, the study demonstrates that POMC is present in invertebrates and its immunoregulatory actions have been conserved during evolution.

Shift from fetal-type to Alzheimer-type phosphorylated Tau proteins in SKNSH-SY 5Y cells treated with okadaic acid.

Tau proteins are abnormally phosphorylated in Alzheimer's disease. Pathological Tau proteins named PHF-Tau 55, PHF-Tau 64, and PHF-Tau 69, are the main constituents of the paired helical filaments (PHF). When treating SKNSH-SY 5Y cells with okadaic acid (OA), Tau 55 protein was clearly induced whereas Tau 64 protein was only faintly induced. Here, we show that the absence of Tau 69 could be explained by the fact that adult isoforms containing N-terminal inserts are not detected. Phosphorylation is similar for untreated cellular Tau proteins and fetal Tau proteins, while OA cell treatment transformed fetal-type into Alzheimer-type phosphorylated proteins.

Mis-splicing yields circular RNA molecules.

We previously identified novel human ets-1 transcripts in which the normal order of exons is inverted, and demonstrated that although the order of exons is different than in the genomic DNA, splicing of these exons out of order occurs in pairs using genuine splice sites (1). Here we determine the structure of these novel transcripts, showing that they correspond to circular RNA molecules containing only exons in genomic order. These transcripts are stable molecules, localized in the cytoplasmic component of the cells. To our knowledge, this is the first case of circular transcripts being processed from nuclear pre-mRNA in eukaryotes. This new type of transcript might represent a novel aspect of gene expression and hold some interesting clues about the splicing mechanism.

Splicing with inverted order of exons occurs proximal to large introns.

Following our studies which showed that the alpha and beta exons of the chicken c-ets-1 gene are not conserved in the human homologue, we succeeded in identifying a novel human c-ets-1 transcript in which the normal order of exons is scrambled. By PCR and RNase protection assays, we demonstrated that while the order of exons is different from that in genomic DNA, splicing of these exons in aberrant order occurs in pairs and at the same conserved consensus splice sites used in the normally spliced transcript. The scrambled transcript is non-polyadenylated and is expressed at much lower levels than the normal transcript. It is not the consequence of genomic rearrangement at the ets-1 locus nor is it due to the transcription of any ets-1 pseudogene. These results confirm previous observations of scrambled splicing.