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Vimentine and cytokeratines expression in LoVo cell clones intrinsically resistant to doxorubicin
No full textCLONING OF THE GM3 SYNTHASE cDNA FROM HUMAN PLACENTA AND GENOMIC ORGANISATION OF THE GENE
Full text linkINTRODUCTION: 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’- and 3’-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 (“Progetto Camilla”, 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’-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’s 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
Potential Applications of AFM (Atomic Force Microscopy) in Cosmetology
No full textThe Atomic Force Microscope (AFM) is able to image the surface properties of all types of materials (conductors, insulators, biological and vegetal samples) with a very high resolution and in the native state. In particular, AFM is used to study a number of biologic system such as DNA-replication and protein/ protein, DNA/protein interactions; furthermore, in cosmetology, it's suitable to study the fundamental characteristics of the skin, like elasticity and wrinkledness, and to compare for example morphological properties of human virgin hair and chemical or polymer treated hair. The most important characteristics of this instrument in cosmetological analyses are: • the samples can be directely examined without previus treatment, i.e. in native conditions, • data are collect in a digital form, • 3-D image rendering with variable magnification and shading, • view of the recorded image from any point of the space. In our laboratory we analyse numerous samples of cosmetic interest and in particular human hair and replica of human skin. In this short paper we present preliminary data on human vergin hairs
Xenopus laevis embryos : an animal model to study the effects of space stress and possible countermeasures (ASSC-MoMa)
No full textISOLATION AND CHARACTERIZATION OF THE GM3 SYNTHASE cDNA FROM HUMAN PLACENTA
Full text linkIt 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’- and 3’-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’-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’s 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
ISOLATION AND CHARACTERIZATION OF A NOVEL ST3Gal V mRNA ISOFORM FROM HUMAN PLACENTA
Full text linkINTRODUCTION: 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’-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’- and 3’-RACE technology (SMART RACE cDNA Amplification Kit, Clontech) using total RNA as template. The sequence of the PCR product was determined by M-Medical’s 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® 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’-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
EGFR AND ErbB2 PHOSPHORYLATION IS STRONGLY DEPENDENT ON GANGLIOSIDES
Full text linkErbB2 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°C, followed by stimulation of receptors with 10 nM EGF for 15 min at 37°C.
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
Proteolytic processing of the beta-amyloid precursor protein (APP) in membranes of the human neuroblastoma SH-SY5Y cells
Full text linkThe two human ST3Gal V isoforms, derived by alternative translation start site usage, are differently N-glycosylated but functionally active
Full text linkGanglioside depletion in mammary epithelial cells alters EGFR phosphorylation and reduces prolactin-induced beta-casein mRNA expression
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