Expression levels of GM3 synthase is transcriptionally regulated in HL60 cells differentiated in monocytoid lineage by phorbol esters

INTRODUCTION: During bi-directional differentiation of human myelogenous leukemia cell line HL-60 into monocytoid and granulocytoid lineages, ganglioside GM3 and neolacto series gangliosides (NeuAc-nLCs) are expressed in differentiation direction-specific manner (1). That is, GM3 markedly increases during monocytic differentiation of HL-60 cells induced by 12-O-tetradecanoyilphorbol-13-acetate (TPA), whereas NeuAc-nLCs noticeably increase in granulocytic differentiation induced by all-trans retinoic acid (RA). These observations suggest that the accumulation of specific gangliosides on the cell membrane plays an important role as a trigger in differentiation induction and as determinant of the differentiation direction in human hematopoietic cell lines (1). It is known that two key upstream glycosyltransferases, Lc3Cer synthase and GM3 synthase, play a critical role regulating the glycosphingolipid biosynthesis in HL-60 cells during bi-directional differentiation (2), but the mechanisms controlling expression and activity levels of these enzymes have not yet been elucidated. In this study our attention is directed to investigate the regulation of GM3 synthase activity. MATERIALS AND METHODS: HL-60 cells were grown in RPMI 1640 complete medium at 37\ub0C in a humidified atmosphere enriched with 5% CO2. Granulocytoid differentiation of HL-60 cells was induced by treatment with 1 mM RA for 48 hours; macrophage-like cell differentiation was produced by 4 nM TPA addition to the culture medium for the same period of time. Acidic and neutral glycolipid profiles of control, RA- and TPA-treated cells were quali-quantitatively analysed by HP-TLC and digital scanning of the plates. GM3 synthase activities were determined in control, RA- and TPA-treated cells by an in vitro radioactive assay using 50 mg and 100 mg of the microsomal enriched protein fraction as enzyme source. mRNA expression levels of GM3 synthase gene was determined by RT-PCR. The house-keeping gene encoding for hypoxantine phospho-ribosyl transferase (HPRT) was used as internal standard for quantitative evaluation of the RT-PCR products. RESULTS: After 48 hours RA treatment, a 30% granulocytic differentiation degree of HL-60 cells was evaluated by conventional cytoplasmic/nuclear histochemical staining of the cells. On the contrary, quite 80% of TPA-treated cells showed evident macrophage-like adherent ability and prominent pseudopods, phenotipic markers of differentiation. Indeed, no modification in the glycosphingolipid profiles, in the enzyme activities and in mRNA expression levels of the crucial glycosyltransferases (Lc3Cer synthase and GM3 synthase) were observed in RA-treated cells. On the other hand, in TPA-treated cells there is a sensible increase in ganglioside GM3 content, accompanied by a consistent up-regulation of GM3 synthase activity with respect to undifferentiated and to RA-treated cells. Through quantitative RT-PCR experiments performed on total RNA from undifferentiated, RA- and TPA-treated HL-60 cells, we demonstrate the strict correlation between GM3 synthase activity and its mRNA level: the GM3 synthase transcript is present in equal amount in either undifferentiated and RA-treated cells, but it is dramatically increased (quite 3 times) in TPA-treated cells. These results first give support to a regulation mechanism at the transcriptional level for this enzyme. REFERENCES (1) H. Nojiri et al., Characteristic expression of glycosphingolipid profiles in the bipotential cell differentiation of human promyelocytic leukemia cell line HL-60. Blood 64, 2:534-541 (1984); (2) M. Nakamura et al., Total metabolic flow of glycosphingolipid biosynthesis is regulated by UDP-GlcNAc:Lactosylceramide beta-1,3-N-Acetylglucosaminyltransferase and CMP-NeuAc:Lactosylceramide alfa-2,3sialyltransferase in human hematopoietic cell line HL-60 during differentiation. J. Biol. Chem. 267, 33: 23507-23541 (1992)

Proteolytic processing of the beta-amyloid precursor protein (APP) in membranes of the human neuroblastoma SH-SY5Y cells

Bibliothèques et publics, bibliothèques publiques dans l’Europe d’Ancien Régime (XVIe-XVIIIe siècle) - Yann Sordet (Bibliothèque Mazarine) 18hLiège, ULg, Grand Physique (Bât. A1) Organisation : Cycle de conférences de Transitions En savoir plus. Source de l'information : http://web.philo.ulg.ac.be/transitions/agenda-general

Multimedia Object Modelling and Storage Allocation Strategies for Heterogeneous Parallel Access Storage Devices in Real Time Multimedia Computing Systems

The improvements in disk speeds have not kept up with improvements in processor and memory speeds. Conventional storage techniques, in the face of multimedia data, are inefficient and/or inadequate. Here, an efficient multimedia object allocation strategy is presented. We describe a multimedia object model, the object and storage device characteristics, and the fragmentation strategy. A bipartite graph approach is used for mapping fragments to storage devices and a cost function is used to determine an efficient allocation of an object and to balance the loads on the devices

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

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

An Architecture for distributed multimedia database systems

In the past few years considerable demand for user oriented multimedia information systems has developed. These systems must provide a rich set of functionality so that new, complex, and interesting applications can be addressed. This places considerable importance on the management of diverse data types including text, images, audio and video. These requirements generate the need for a new generation of distributed heterogeneous multimedia database systems. In this paper we identify a set of functional requirements for a multimedia server considering database management, object synchronization and integration, and multimedia query processing. A generalization of the requirements to a distributed system is presented, and some of our current research and developing activities are discussed