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GALIG gene expression induces apoptosis in cultured cells through a pathway still under investigation. It is highly expressed in leukocytes but weakly detectable in bone marrow, suggesting a role in the myeloid lineage homeostasis. We show here that GALIG-induced cell death is counteracted by the overexpression of MCL-1, a pro-survival member of the Bcl2 family. Moreover, during spontaneous neutrophil apoptosis, a substantial increase in GALIG gene expression is observed : GALIG still opposes MCL-1. Finally, in bone marrow and peripheral blood cells from patients with Acute Myeloid Leukemia type 2, the level of GALIG transcripts is massively down-regulated when compared to their normal counterparts, while MCL-1 is expressed to the same extent. These data suggest that GALIG could be a key player in the cell death pathway involved in leukocytes homeostasis and myeloid malignancies.
Galig, a gene embedded within the galectin-3 gene, induces cell death when transfected in human cells. This death is associated with cell shrinkage, nuclei condensation, and aggregation of mitochondria. Galig contains two different overlapping open reading frames encoding two unrelated proteins. Previous observations have shown that one of these proteins, named mitogaligin, binds to mitochondria and promotes the release of cytochrome c. However, the mechanism of action of this cytotoxic protein remains still obscure.
Galectin-3 internal gene (Galig) was recently identified as an internal gene transcribed from the second intron of the human galectin-3 gene that is implicated in cell growth, cell differentiation, and cancer development. In this study, we show that galig expression causes morphological alterations in human cells, such as cell shrinkage, cytoplasm vacuolization, nuclei condensation, and ultimately cell death. These alterations were associated with extramitochondrial release of cytochrome c, a known cell death effector.
We previously reported that alternative transcripts were initiated within the second intron of the human Galectin-3 gene (LGALS3), We now demonstrate that these transcripts arise from an internal gene embedded within LGALS3 and named galig (Galectin-3 internal gene). Tissue-specific expression of galig was assayed by screening of several human tissues. Contrary to LGALS3, galig appears to be tightly regulated and principally activated in leukocytes from peripheral blood. Cloning and characterization of galig transcripts revealed that they contain two out-of-frame overlapping open-reading frames (ORFs). Transfection of expression vectors encoding enhanced green fluorescent protein (EGFP) chimeras indicated that both ORFs could be translated in proteins unrelated to Galectin-3. The ORF1 polypeptide targets EGFP to cytosol and nucleus whereas ORF2 targets EGFP to mitochondria. These results revealed the exceptional genetic organization of the LGALS3 locus.
Analysis of antimicrobial activities that are present in the skin secretions of the South American frog Phyllomedusa bicolor revealed six polycationic (lysine-rich) and amphipathic alpha-helical peptides, 24-33 residues long, termed dermaseptins B1 to B6, respectively, Prepro-dermaseptins B all contain an almost identical signal peptide, which is followed by a conserved acidic propiece, a processing signal Lys-Arg, and a dermaseptin progenitor sequence. The 22-residue signal peptide plus the first 3 residues of the acidic propiece are encoded by conserved nucleotides encompassed by the first coding exon of the dermaseptin genes. The 25-residue amino-terminal region of prepro-dermaseptins B shares 50% identity with the corresponding region of precursors for D-amino acid containing opioid peptides or for antimicrobial peptides originating from the skin of distantly related frog species. The remarkable similarity found between prepro-proteins that encode end products with strikingly different sequences, conformations, biological activities and modes of action suggests that the corresponding genes have evolved through dissemination of a conserved "secretory cassette" exon.
Recently, we have shown that the dopamine D1B/D5 receptor displays binding and coupling properties that are reminiscent of those of the constitutively activated G protein-coupled receptors when compared with the related D1A/D1 receptor subtype (Tiberi, M., and Caron, M. G. (1994) J. Biol. Chem. 269, 27925-27931). The carboxyl-terminal region of the third cytoplasmic loop of several G protein coupled receptors has been demonstrated to be important for the regulation of the equilibrium between inactive and active receptor conformations. In this cytoplasmic region, the primary structure of dopamine D1A and D1B receptors differs by only two residues : Phe(264)/Arg(266) present in D1A receptor compared with Ile(288)/Lys(290) in the D1B receptor.
Dermenkephalin (Tyr-D-Met-Phe-His-Leu-Met-AspNH(2)) is a highly potent and selective d-opioid peptide isolated from frog skin. It was recently recognized that the C-terminus His(4)-Leu(5)-Met(6)-Asp(7)NH(2) of dermenkephalin was responsible for the addressing of the peptide towards the d-opioid receptor. In order to investigate the role played by residues 4, 5 and 6 in this ’d address’, we synthesized and evaluated 20 new analogues for their ability to displace tritiated ligands from mu- and d-opioid sites. Results showed that position 4 of dermenkephalin contributes to 6 selectivity independently of d-opioid receptor binding by preventing a high affinity mu binding. Position 5 requires a hydrophobic side chain to enhance d affinity. A high d affinity was obtained with any amino acids introduced in position 6 suggesting that residue 6 serves as a neutral spacer. Thus, the main features responsible for the high d-opioid selectivity of dermenkephalin are electrostatic repulsions with the mu-opioid receptor, additional hydrophobic interactions with the d-opioid receptor and folding of the C-terminal domain.
Dermenkephalin, H-Tyr-(D)Met-Phe-His-Leu-Met-Asp-NH2, a highly potent and selective d-opioid peptide isolated from frog skin, was studied in DMSO-d(6) solution by two-dimensional nmr spectroscopy, including the determination of NH temperature coefficients, the evaluation of (3)J coupling constants from phase-sensitive correlated spectroscopy (COSY) and the volumes of nuclear Overhauser effect (NOE) correlations. The two-dimensional NOE spectroscopy (NOESY) spectrum of dermenkephalin revealed sequential, medium-, and long-range effects. To put this information on a quantitative basis, special attention was devoted to J cross-peak suppression, quantification of the NOE volumes and analysis of the overlaps, normalization of the NOEs against diagonal peaks and H beta beta, geminal interactions.
A video-tracking technique has been used for the evaluation of climbing behavior in mice. An automated image analysis system, the Videotrack 512 (Electronique Lyonnaise), was adapted for this specific application. This allowed distinguishing between spontaneous climbing and stereotyped climbing. The activity duration of mice was simultaneously measured. In order to validate this method, in the present study the ability of apomorphine to induce climbing in mice, and the effects of the D1-dopamine receptor agonist SKF-38393 and the D2-dopamine agonist RU-24926 and their association were investigated.
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