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)

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

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

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)

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

Measurement of the cross-section and charge asymmetry of WW bosons produced in proton-proton collisions at s=8\sqrt{s}=8 TeV with the ATLAS detector

This paper presents measurements of the $W^+ \rightarrow \mu^+\nu$ and $W^- \rightarrow \mu^-\nu$ cross-sections and the associated charge asymmetry as a function of the absolute pseudorapidity of the decay muon. The data were collected in proton--proton collisions at a centre-of-mass energy of 8 TeV with the ATLAS experiment at the LHC and correspond to a total integrated luminosity of 20.2~\mbox{fb^{-1}}. The precision of the cross-section measurements varies between 0.8% to 1.5% as a function of the pseudorapidity, excluding the 1.9% uncertainty on the integrated luminosity. The charge asymmetry is measured with an uncertainty between 0.002 and 0.003. The results are compared with predictions based on next-to-next-to-leading-order calculations with various parton distribution functions and have the sensitivity to discriminate between them.Comment: 38 pages in total, author list starting page 22, 5 figures, 4 tables, submitted to EPJC. All figures including auxiliary figures are available at https://atlas.web.cern.ch/Atlas/GROUPS/PHYSICS/PAPERS/STDM-2017-13

Reduced ventricular proliferation in the foetal cortex following maternal inflammation in the mouse

It has been well established that maternal inflammation during pregnancy alters neurological function in the offspring, but its impact on cortical development and long-term consequences on the cytoarchitecture is largely unstudied. Here we report that lipopolysaccharide-induced systemic maternal inflammation in C57Bl/6 mice at embryonic Day 13.5 of pregnancy, as early as 8 h after challenge, caused a significant reduction in cell proliferation in the ventricular zone of the developing cerebral cortex, as revealed by quantification of anti-phospho-Histone H3 immunoreactivity and bromodeoxyuridine pulse labelling. The angle of mitotic cleavage, determined from analysis of haematoxylin and eosin staining, cyclin E1 gene expression and the pattern of β-catenin immunoreactivity were also altered by the challenge, which suggests a change from symmetric to asymmetric division in the radial progenitor cells. Modifications of cortical lamination and gene expression patterns were detected at post-natal Day 8 suggesting prolonged consequences of these alterations during embryonic development. Cellular uptake of proteins from the cerebrospinal fluid was observed in brains from lipopolysaccharide-treated animals in radial progenitor cells. However, the foetal blood–brain barrier to plasma proteins remained intact. Together, these results indicate that maternal inflammation can disrupt the ventricular surface and lead to decreased cellular proliferation. Changes in cell density in Layers IV and V at post-natal Day 8 show that these initial changes have prolonged effects on cortical organization. The possible shift in the fate of progeny and the resulting alterations in the relative cell numbers in the cerebral cortex following a maternal inflammatory response shown here will require further investigation to determine the long-term consequences of inflammation on the development of neuronal circuitry and behaviour