ISOLATION AND CHARACTERIZATION OF A NOVEL ST3Gal V mRNA ISOFORM FROM HUMAN PLACENTA

INTRODUCTION: ST3Gal V (EC 2.4.99.9, GM3 synthase) is a key enzyme in the biosynthesis of gangliosides, a large and heterogeneous family of sialic acid-containing glycosphingolipids that, as mediator of cell-cell interactions and modulators of signalling transduction, play fundamental roles in many both physiological and pathological cellular processes (i.e., proliferation, differentiation, oncogenesis) (1). It catalyses the transfer of a sialic acid moiety from CMP-sialic acid onto LacCer producing GM3, a ganglioside ubiquitously distributed on the plasma membrane of all the eukaryotic cells, representing the common precursor of almost all ganglio-series gangliosides (2). However, the homologous rat protein purified from brain and liver can use other glycolipids as acceptor substrates, including GalCer, GlcCer, aGM2, and aGM1, although with a lower extent of specificity respect to LacCer (3). Contrasting results are also reported about the intracellular localization of the protein: GM3 synthase activity has been detected in both proximal and distal compartments of Golgi apparatus (4). Human ST3Gal V results expressed in a tissue-specific manner, especially in brain, muscle, testis, and placenta, and only one transcript, of about 2.4 kb, has been detected in all the tissues (5). Human ST3Gal V cDNAs have been already cloned from both TPA-differentiated HL60 cells and fetal and adult brain, and several mRNA variants have been identified (5). They differ in the 5\u2019-UTR sequences, but all of them seem to contain an identical coding region; the substrate activity of the encoded protein (362 aminoacids with a predicted molecular mass of 41.7 kDa) is highly restricted to LacCer. Studies on the structural organization and transcriptional regulation of the gene (6) also provided contrasting, but stimulating, results. MATERIALS AND METHODS: The complete ST3Gal V cDNA from human placenta was obtained by the 5\u2019- and 3\u2019-RACE technology (SMART RACE cDNA Amplification Kit, Clontech) using total RNA as template. The sequence of the PCR product was determined by M-Medical\u2019s DNA sequencing service. The transcription initiation site was evaluated by primer extension analysis. The translation initiation site was identified by in vitro experiments, using the TNT\uae T7 Quick coupled transcription/translation system (Promega), and it was confirmed by in vivo analyses. The isolated cDNA protein product, expressed as fusion protein with EGFP in CV1 cells using pEGFP-N3 as expression vector, was detected by western blot with specific anti-GFP antibodies. The enzymatic activity of the isolated cDNA protein product was assayed in rat mammary adenocarcinoma cells stably transfected with the isolated cDNA using the pRC/CMV (Promega) as expression vector; the enzymatic activity was determined by an in vitro radioactive assay using the microsomal enriched protein fraction as enzyme source, CMP-[14C]sialic acid as donor substrate, and different glycolipids as acceptor substrates. The genomic structure of the human ST3Gal V gene has been determined through a human genome BLAST homology search of the public database (GenBank) using the isolated cDNA as query sequence. RESULTS: A cDNA, consisting of 2149 bp (the poli-A tail is lacking) and showing high sequence identity with all the human ST3Gal V cDNAs until now registered in GeneBank, has been successfully isolated and cloned from human placenta. Primer extension experiments confirmed the transcription initiation site. Not surprisingly, it differs from all the other human cDNAs in the 5\u2019-end sequence, but, with respect to the other ones, it contains a new, possibly translation initiation, ATG codon; it is located upstream and in frame with the ATG indicated as translation initiation site in all the other human ST3Gal V cDNAs. In vitro and in vivo analyses on the placental cDNA product showed that it predominantly produces a protein with a larger molecular mass (about 44 kDa), having a higher substrate specifity for LacCer; however, GalCer, aGM1, and aGM2 could serve as substrates, although to a much lesser extent. Finally, a human genome BLAST homology search in the public database, using the isolated human placental cDNA as the query sequence, showed that the gene consists of seven exons which span over 28.5 kb, with exons ranging in size up to 1242 bp. This study, together with previously reported ones (3,5,6), suggests that the human ST3Gal V gene specifies at least two isozymes, having a tissue-specific expression, a different amino-terminal sequence, and a different substrate specificity (and why not different intracellular localization?). 1) Hakomori S.I. (2000) Glycoconj. J. 17, 143; Hakomori S.I. (2000) Glycoconj. J. 17, 627. 2) Kolter T. et al. (2002) J. Biol. Chem. 277, 25859. 3) Preuss U. et al. (1993) J. Biol. Chem. 268, 26273; Melkerson-Watson L.J. et al. (1991) J. Biol. Chem. 266, 4448. 4) Maccioni H.J.F. et al. (1999) Biochim. Biophys. Acta 1437, 101 5) Ishii A. et al. (1998) J. Biol. Chem. 273, 31652; Kapitonov D. et al.(1999) Glycoconj J. 16, 337. 6) Kim K.W. et al. (2001) Gene 273, 163; Zeng G. et al. (2003) Biochim. Biophys. Acta 1625, 30

IDENTIFICATION OF A NOVEL GM3 SYNTHASE mRNA ISOFORM FROM HUMAN PLACENTA AND FUNCTIONAL ANALYSIS OF THE 5\u2019-FLANKING REGION OF THE TRANSCRIPT

GM3 synthase is a key enzyme in the biosynthesis of gangliosides that, as mediators of cell-cell interactions and modulators of signaling transduction, play fundamental roles in many both physiological and pathological cellular processes (i.e., proliferation, differentiation, oncogenesis) (1,2). GM3 synthase results expressed in a tissue-specific manner, especially in brain, muscle, testis, and placenta, and only one transcript, of about 2.4 kb, has been detected in all the tissues. Up to date GM3 synthase cDNA has been isolated and characterized from both TPA-differentiated HL-60 cells and human fetal and adult brain (3,4) and five mRNA variants have been identified. They differ in the 5\u2019-untranslated region, but all of them predict a 41.7 kDa protein having substrate activity highly restricted to lactosylceramide. On the contrary, our study, directed to the isolation of the full length GM3 synthase cDNA from human placenta, led to the identification of an unusual GM3 synthase mRNA variant that strongly differs from all the other GM3 synthase transcripts until now identified. In fact, its 5\u2019-terminal region introduces a new translation initiation site located upstream and in-frame with that usually considered as translation initiation codon in the GM3 synthase gene. Studies of in vitro translation and in vivo expression indicate that the human placental cDNA leads to the synthesis of two polypeptides with apparent molecular mass, in SDS-PAGE, of 42.1 and 38.3 kDa, respectively. Moreover, the stable transfection of the human placental cDNA into mammalian MG1361 cells resulted in a threefold increase of GM3 synthase activity, associated to a broader substrate specificity, and, more importantly, in significant alterations of cell ganglioside profile. Therefore, our data provide the first demonstration of the existence of two GM3 synthase isoforms and indicate that, at least in the human placenta, they apparently derive from a single transcript. In addition, to elucidate the mechanism that regulates the tissue-specific expression of the human GM3 synthase gene we isolated the genomic DNA region (~3 kb) flanking the placental GM3 synthase transcript. Sequence analysis with the MatInspector 2.2 program revealed that this region lacks canonical TATA e CAAT boxes, but contains several putative transcription factor binding sites including AP1, GATA1, NFAT, MZF1, IK2; unfortunately no placenta-specific transcription factor is reported in none of the available transcription factor databases. Up to date, functional analysis of the isolated genomic region has been assessed in human placental choriocarcinoma JEG3 cells by transient transfection, but preliminary data seem to indicate that it doesn\u2019t promote transcription of the reporter SEAP gene. Future plans are to analyze the functional activity of DNA fragments containing varying lengths of the 5\u2019-flanking genomic sequence in both JEG3 cells and other suitable cell lines so to evaluate the eventual presence of positive and negative regulatory elements and factors in both the DNA sequence and the cell line. 1) Hakomori S.I. (2000) Glycoconj. J. 17, 143. 2) Hakomori S.I. (2000) Glycoconj. J. 17, 627. 3) Ishii A. et al. (1998) J. Biol. Chem. 273, 31652. 4) Kapitonov D. et al.(1999) Glycoconj J. 16, 337

CLONING OF THE GM3 SYNTHASE cDNA FROM HUMAN PLACENTA AND GENOMIC ORGANISATION OF THE GENE

INTRODUCTION: Gangliosides are a large family of sialic acid-containing glycosphingolipids that play important roles in a large variety of biological processes. Both their functions and their biosynthetic pathways are currently clarified (1, 2). In vertebrates, almost all the ganglio-series gangliosides are synthesized from a common precursor, ganglioside GM3, which has the simplest structure among the major ganglioside. GM3 itself is known to participate in induction of differentiation, modulation of proliferation, signal transduction and integrin-mediated cell adhesion. GM3 synthase (EC 2.4.99.9, ST3Gal V) is the enzyme involved in the last step of GM3 biosynthesis: it catalyses the transfer of a sialic acid moiety from CMP-sialic acid onto lactosylceramide, forming an a2-3 linkage. Whereas GM3 is ubiquitously distributed in the plasma membranes of all eukaryotic cells, GM3 synthase results expressed in a tissue-specific manner, especially in brain, placenta, muscle and testis (3). Although its cDNA has been cloned from some mouse (4, 5) and human tissues (3, 6), studies on the genomic structure (7, 8) and on its transcriptional regulation (8, 9) provides contrasting results. MATERIALS AND METHODS: To isolate the complete coding sequence of the gene of GM3 synthase from human placenta we used the 5\u2019- and 3\u2019-Rapid Amplification of cDNA Ends technology (SMART RACE cDNA Amplification Kit, Clontech) using, as specific primers, oligonucleotides derived from the human GM3 synthase cDNA sequence from differentiated HL60 cells (3). The identity of some amplified DNA fragments was confirmed by Southern blot analysis (Gene ImagesTM AlkPhos DirectTM labelling and detection system, Amersham Pharmacia Biotech). The different PCR products were cloned into the pCR2.1 vector (TA Cloning Kit, InVitrogen) and the nucleotide sequence was determined (\u201cProgetto Camilla\u201d, M-Medical). The genomic structure of the human GM3 synthase gene has been determined through a human genome BLAST homology search of the public database (GenBank) using the GM3 synthase cDNA from human placenta as the query sequence. RESULTS: A cDNA, consisting of 2149 bp and showing high sequence homology with those encoding the human GM3 synthase from other human tissues (3, 6), has been successfully isolated and cloned from human placenta. Notwithstanding our approach, our cDNA has not the poli(A) tail. Between our cDNA and the other published ones, the major difference is in the 5\u2019-end, according to the existence of different promoter regions, responsible for tissue-specific expression of the gene. Furthermore, the cDNA from the human placenta contains, upstream and in frame with the ATG indicated as translation initiation site for the GM3 synthase of HL60 cells, another ATG codon inserted in a sequence compatible with Kozak\u2019s rule, suggesting that the protein of the human placenta could have an additional portion in NH2-terminus. The complete and partial coding regions of the human placenta cDNA are going to be cloned in an expression vector, under the control of the CMV promoter, in order to evaluate their GM3 synthase activity. The results of the human genome BLAST homology search of the public database using the GM3 synthase cDNA from human placenta as the query sequence showed that the gene consists of seven exons which span over 28.5 kb, with exons ranging in size up to 1242 bp. All exon-intron boundaries follows the GT-AG rule (10). 1) Hakomori S.I. (2000) Glycoconj. J. 17, 627-647 2) Kolter T. et al. (2002) J. Biol. Chem. 277, 25859-25862 3) Ishii A. et al. (1998) J. Biol. Chem. 273, 31652-31655 4) Kono M. et al. (1998) Biochem. Biophys. Res. Comm. 253, 170-175 5) Fukumoto S. et al. (1999) J. Biol. Chem. 274, 9271-9276 6) Kapitonov D. et al.(1999) Glycoconj J. 16, 337-350 7) Kim K.W. et al. (2001) Gene 273, 163-171 8) Kim S.W. et al. (2002) ) Biochim. Biophys. Acta 1578, 84-89 9) Zeng G. et al. (2003) Biochim. Biophys. Acta 1625, 30-35 10) Breathnach R. and Chambon P. (1981) Annu. Rev. Biochem. 50, 349-38

GM3 IN THE REGULATION OF THE EXPRESSION AND ACTIVATION OF ErbB-2/EGFR HETERODIMERS

Gangliosides are a large and heterogeneous family of sialic acid containing glycosphingolipids, ubiquitous components of all eukaryotic cell membranes. They can partially segregate, together with cholesterol and specific signaling transduction proteins, such as receptor tyrosine-kinases, into unique more or less stable clusters or microdomains, indicated as \u201cglycolipid-enriched domains\u201d, \u201clipid rafts\u201d, \u201ccaveolae\u201d, contributing to the membrane structure, organization and function (1). Gangliosides are well-characterized modulators of receptor tyrosine-kinase (RTKs) phophorylation and dimerization (2). Our interest is particularly directed to study the relationship between gangliosides and two members of the tyrosine kinase ErbB receptor family: the epidermal growth factor receptor, EGFR or ErbB-1, and the structurally related protein ErbB-2. Unlike EGFR, ErbB-2 is a ligandless receptor: it can be activated by heterodimerization and cross-phosphorylation by other ligand-activated ErbB receptors (3,4). Our previous experimental evidence supports the functional relationship between ErbB-2 and gangliosides, in particular GM3 (5,6). In the present study, using the HC11 mouse mammary epithelial cell line, we investigated the ErbB-2 activation state and its tendency to form stable molecular complexes with EGFR and with ganglioside GM3, before and after EGF stimulation, by co-immunoprecipitation experiments with anti-ErbB-2 antibody and Western blot analyses. As HC11 cells express different ganglioside species, the exclusive association of GM3 with ErbB-2 and EGFR was ascertained by high performance-thin layer chromatography (HP-TLC) and TLC-immunostaining analyses of gangliosides extracted from the immunoprecipitates. Results display that in EGF-stimulated HC11 cells stable and tyrosine-phosphorylated ErbB2/EGFR dimers are formed and that GM3 is the unique ganglioside tightly associated to ErbB-2/EGFR dimers and to EGFR monomers, but not to ErbB2 monomers. In cells not stimulated with EGF a spontaneous but unproductive dimerization of ErbB2 and EGFR takes place and no ganglioside is found associated to the receptors. These observations indicate that the modulation of ErbB2 activation by GM3 may be mediated by EGFR, but that EGF stimulation is indispensable. After ganglioside depletion by [D]-PDMP, phosphorylated EGFR monomers are observed both before and after EGF stimulation, whereas ErbB-2 is present in the monomeric and unphosphorylated state even after EGF stimulation, suggesting that GM3 might have a bivalent key role in modulating the activation of ErbB-2 and EGFR. References 1. Fivaz, M., Abrami, L., Van Der Goot. F.G. Trends Cell Biol. 9(6), 212-213 (1999); 2. Bremer, E.G., Current topics in membranes 40, 387-411(1999); 3. Qian, X., LeVea, C.M., Freeman, J.K., Dougall, W.C., Greene, M.I., Proc. Natl. Acad. Sci. USA 91, 1500-1504 (1994); 4. Gulliford, T.J., Huang, G.C., Ouyang, X., Epstein, R.J., Oncogene 15, 2219-2223 (1997); 5. Sottocornola E., Berra B., and Colombo I., Biochim. Biophys. Acta-Molecolar and cell Biology of Lipids, 1635, 55-66 (2003); 6. Sottocornola E., Misasi R., Mattei V., Ciarlo L., Gradini R., Garofalo T., Berra B., Colombo I., and Sorice M., FEBS J. 273, 1821-1830 (2006)

ISOLATION AND CHARACTERIZATION OF THE GM3 SYNTHASE cDNA FROM HUMAN PLACENTA

It is known that gangliosides have various important biological functions, and their functions as well as their biosynthesis are currently clarified (1, 2). In vertebrates, almost all the ganglio-series gangliosides are synthesized from a common precursor, ganglioside GM3, which has the simplest structure among the major gangliosides. GM3 itself is known to participate in induction of differentiation, modulation of proliferation, signal transduction and integrin-mediated cell adhesion. GM3 synthase (EC 2.4.99.9, ST3Gal V) is the enzyme involved in the last step of GM3 biosynthesis: it catalyses the transfer of a sialic acid moiety from CMP-sialic acid onto lactosylceramide, forming an a2-3 linkage. Whereas GM3 is ubiquitously distributed in the plasma membranes of all eukaryotic cells, GM3 synthase results expressed in a tissue specific manner, especially in brain, placenta, muscle and testis (3). Many important issues, such as human cDNA identification and characterization, genomic structure and regulation of gene expression, are still open. To isolate the coding sequence of the gene of GM3 synthase from human placenta we used the 5\u2019- and 3\u2019-Rapid Amplification of cDNA Ends technology (SMART RACE cDNA Amplification Kit, Clontech) using, as specific primers, oligonucleotides derived from the human GM3 synthase cDNA sequence from differentiated HL60 cells (3). The different PCR products were cloned into the pCR2.1 vector (TA Cloning Kit, InVitrogen) and the nucleotide sequence was determined. A cDNA, showing high sequence homology with that encoding the human GM3 synthase from TPA-differentiated HL60 cells (3), has been successfully isolated and cloned from human placenta. The major difference between these two cDNAs is in the 5\u2019-end, according to the existence of different promoter regions, responsible for tissue-specific expression of the gene. Furthermore, the cDNA from the human placenta contains, upstream and in frame with the ATG indicated as translation initiation site for the GM3 synthase of HL60 cells, another ATG codon inserted in a sequence compatible with Kozak\u2019s rule, suggesting that the protein of the human placenta has an additional portion in NH2-terminus. The complete coding region of the human placenta cDNA is going to be cloned in an expression vector, under the control of the CMV promoter, in order to evaluate its activity. On the other hand, in vitro translation experiments are going to be carried out to define the first start codon. 1) Hakomori S.I. (2000): Glycoconj. J. 17, 627-647 2) Kolter T. et al. (2002): J.Biol.Chem. 277, 25859-25862 3) Ishii A. et al. (1998): J.B.C. 273, 31652-3165

Intramolecular Pd(II)-Catalyzed Cyclization of Propargylamides: Straightforward Synthesis of 5-Oxazole-carbaldehydes

(Chemical Equation Presented) Direct synthesis of 2-substituted 5-oxazolecarbaldehydes was performed by intramolecular reaction of propargylamides through treatment with a catalytic amount of Pd(II) salts in the presence of a stoichiometric amount of reoxidant agent. The heterocyclization process was well-tolerated by a wide range of aryl, heteroaryl, and alkyl propargylamides. This protocol constitutes a valuable synthetic pathway to 5-oxazolecarbaldehydes, alternative to the formylation on oxazole rings, often unsatisfactory in term of regioselectivity and yields

Transient currents in HfO2 and their impact on circuit and memory applications

We investigate transient currents in HfO2 dielectrics, considering their dependence on electric field, temperature and gate stack composition. We show that transient currents remain an issue even at very low temperatures and irrespective of the HfO2/SiO2 bilayer properties. Finally, we assess their impact on the reliability of precision circuit and memory applications Transient currents in HfO2 and their impact on circuit and memory applications (PDF Download Available). Available from: http://www.researchgate.net/publication/224672970_Transient_currents_in_HfO2_and_their_impact_on_circuit_and_memory_applications [accessed Oct 22, 2015]

Palladium-catalyzed domino carbopalladation/5-exo-allylic amination of \u3b1-amino allenamides: an efficient entry to enantiopure imidazolidinones

Allenamides of alpha-amino acids were converted into enantiopure 2-vinylimidazolidin-4-ones by a carbopalladation/exo-cyclization process. The products were obtained in 2.5:1-5.5:1 dr, with 94-99% ee. The palladium-catalyzed carbonylative cyclization of the same substrates afforded enone structures. Starting from properly substituted allenamides, an intramolecular carbopalladation followed by intramolecular amination gave rise to tricyclic fused-ring imidazolidinones

EGFR AND ErbB2 PHOSPHORYLATION IS STRONGLY DEPENDENT ON GANGLIOSIDES

ErbB2 and EGF receptors are two members of the ErbB receptor superfamily of tyrosine-kinases. Among the different tissues, they are of particular importance in the mammary gland during growth, differentiation, and suckling. Moreover the overexpression of ErbB2 and EGFR is main feature of pour prognosis mammary adenocarcinomas. Gangliosides are important glycosphingolipids involved in many physio-pathological cell events, because of their ability to mediate cell function through modulation of growth factor receptors, like EGFR and ErbB2. To point out the role of gangliosides in modulating ErbB2 and EGFR activation in HC11 mammary epithelial cell line, we analysed receptor activation in control and ganglioside-depleted cells. Ganglioside depletion was obtained by treatment with 30 mM 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) for 5 days at 37\ub0C, followed by stimulation of receptors with 10 nM EGF for 15 min at 37\ub0C. Western blot analyses of total cell lysates revealed that phosphorylated EGFR was visible as three bands, having an Mr range of 170-190 kDa, contrarily to EGFR in control cells that was identified as a single 170 kDa band. Moreover, EGFR was phosphorylated in ganglioside-depleted cells also in the absence of the proper stimuli. ErbB2 maintained the same phosphorylation profile in control and in PDMP-treated cells. Analyses of the immunoprecipitates, obtained with specific antibodies, indicated that only the band of EGFR with higher molecular mass immunoprecipitates with anti-ErbB2 antibody. To further investigate EGFR activation, we evaluated it by digestion with calf intestine alkaline phosphatase (CIAP) by adding 5 or 10 units CIAP for 30 min or 1 hour. Results clearly indicated that EGFR underwent sensitive variations after CIAP treatment. Indeed, EGFR displayed two co-migrating bands in PDMP-treated control and EGF-stimulated cells, further confirming the main role of gangliosides in EGFR activation and stimulation. 1) Milani S. et al. 2007 Ganglioside GM3 is stably associated to tyrosine-phosphorylated ErbB2/EGFR receptor complexes and EGFR monomers, but not to ErbB2. Biochim Biophys Acta, 1771, 873-8

Motor-based bodily self is selectively impaired in eating disorders

BACKGROUND: Body representation disturbances in body schema (i.e. unconscious sensorimotor body representations for action) have been frequently reported in eating disorders. Recently, it has been proposed that body schema relies on adequate functioning of the motor system, which is strongly implicated in discriminating between one’s own and someone else’s body. The present study aimed to investigate the motor-based bodily self in eating disorders and controls, in order to examine the role of the motor system in body representation disturbances at the body schema level. METHODS: Female outpatients diagnosed with eating disorders (N = 15), and healthy controls (N = 18) underwent a hand laterality task, in which their own (self-stimuli) and someone else’s hands (other-stimuli) were displayed at different orientations. Participants had to mentally rotate their own hand in order to provide a laterality judgement. Group differences in motor-based bodily self-recognition—i.e. whether a general advantage occurred when implicitly processing self- vs. other-stimuli − were evaluated, by analyzing response times and accuracy by means of mixed ANOVAs. RESULTS: Patients with eating disorders did not show a temporal advantage when mentally rotating self-stimuli compared to other-stimuli, as opposed to controls (F(1, 31) = 5.6, p = 0.02; eating disorders-other = 1092 ±256 msec, eating disorders-self = 1097±254 msec; healthy controls-other = 1239±233 msec, healthy controls -self = 1192±232 msec). CONCLUSIONS: This study provides initial indication that high-level motor functions might be compromised as part of body schema disturbances in eating disorders. Further larger investigations are required to test motor system abnormalities in the context of body schema disturbance in eating disorders