Gene expression for glycosyltransferases in transfected neuroblastoma cells

Poster Sessions AP03: Lipids, Carbohydrates and Metabolism AP03-01 AP03-03 Iron, hydrogen peroxide and ascorbate as stimulants of lipid peroxidation in brain membranes A. C. Andorn, T. L. Bennett, D. Hogan and G. Taglialatella University of Texas Medical Branch, Galveston TX, USA c-Fos activates phospholipid metabolism in events associated to growth and differentiation B. L. Caputto, G. A. Gil, M. M. Portal, G. A. Borioli, M. L. Renner and M. E. Guido CIQUIBIC-Dpto. Qca Biológica, Fac. Cs. Quı́micas, Universidad Nacional de Córdoba, Córdoba, Argentina A standard way to assess lipid peroxidation (LP)in neurodegenerative diseases is to stimulate LP in membrane fragments (MF) from postmortem human brains both normal and diseased. Studies using 0.67 mM iron plus 67 mM hydrogen peroxide showed that stimulated LP is increased in Alzheimer’s diseased (AD) brain (Neurosci. Letts: 112, 333–337, 1990). In contrast, 0.1 mM ascorbate-stimulated LP is reduced in AD brain (Mol. Chem. Neuropath. 33, 15–26,1998). We have stimulated LP in MF from human prefrontal cortex with iron, iron plus hydrogen peroxide, hydrogen peroxide and ascorbate. Shown for brevity are two stimulants and the effect of some inhibitors of LP. The ingredients were incubated for 120 min with 0.1 mg protein and buffer. LP was determined spectrophotometrically by the formation of thiobarbituric acid positive reactants. The data shown are the mean ± SEM (N ¼ at least 3). Stimulants were FeCl2 + H2O2 (both 10 mM) or ascorbate (0.1 mM). Response was measured in nmols MDA/mg protein. Deferoxamine (10 lM), catalase (100 lg/mL) and alpha-tocopherol (Trolox) at 10 lM and are shown as percentage maximal. Stimulant Response Deferoxamine Catalase Trolox FeCl2 + H2O2 Ascorbate 270 ± 18 13.9 ± 2.0 97 ± 1.2 )0.67 ± 2.1 94 ± 6.0 190 ± 11 99 ± 0.0 )2.5 ± 5.9 These findings suggest that all methods of stimulating LP in MFP from human brain are not equivalent. Therefore caution is recommended in the interpretation of differences in LP between presumptive normal and neurodegenerative human brain. 32 P phospholipid labelling increases in quiescent fibroblasts stimulated with serum to re-enter growth. This stimulation of phospholipid synthesis shows two waves that depend on the transcription and translation of the immediate early gene c-fos. The time-course pattern of c-Fos protein expression resembles the activation pattern of phospholipid synthesis (The FASEB J, January 52001, 10.1096/fj.00-0446fje). NGF-treatment of PC12 cells promotes a strong increase in the expression of c-Fos and enhances phospholipid synthesis in a c-Fosconcentration-dependent way. Specifically blocking Fos expression with a c-Fos mRNA antisense oligonucleotide impairs activation of phospholipid synthesis and also greatly inhibits cell differentiation. By contrast, inhibition of Fos degradation in NGF-treated cells significantly increases c-Fos content, phospholipid synthesis and neurite elongation. Blocking nuclear import of c-Fos (AP-1) at the initiation of NGF treatment completely blocks neurite formation. However, if c-Fos nuclear import is blocked 16 h after addition of NGF to the cell cultures, neurite elongation continues almost normally. These results indicate that c-Fos has a dual function in developing cells: it first releases the nuclear program for differentiation and then sustains growth by activating key components required for membrane genesis, in the cytoplasm. The mechanisms underlying this novel regulatory activity of c-Fos on phospholipid synthesis, that is independent of its transcription factor activity, are being studied. Acknowledgements: Financed by Beca Carrillo-Oñativia, FONCyT, Agencia Córdoba. Ciencia (CONICOR), SeCyT-UNC, and CONICET. AP03-02 AP03-04 Regulation of ganglioside biosynthesis by enzyme complex formation of glycosyltransferases E. Bieberich,* S. MacKinnon,* J. Silva,* D. D. Li,* T. Tencomnao,* L. N. Irwin, D. Kapitonovà and R. K. Yu* *Institute of Molecular and Medical General, Medical College of Georgia, Augusta, GA 30912; Department of Biological Sciences, University of Texas, El Paso, TX 79968; àDepartment of Neurosurgery, University of Pennsylvania, Philadelphia, PA 19104, USA Effects of gangliosides on the distribution of a GPI-anchored protein in plasma membrane J. L. Daniotti, P. Crespo and A. Zurita CIQUIBIC, Facultad de Ciencias Quı́micas, Universidad Nacional de Córdoba, Argentina Two key regulatory enzymes in ganglioside biosynthesis, sialyltransferase II (ST2) and N-acetylgalactosaminyltransferase I (GalNAcT), have been expressed as fusion proteins with GFP, YFP, or RFP. Transient or stable transfection has been achieved in a substrain of murine neuroblastoma F-11 cells (F-11 A) that contains only low endogenous ST2 and GalNAcT activity. ST2-GFP (85 kDa) shows a distinct Golgi localization, whereas GalNAcT linked to YFP or RFP (115 kDa) is broadly distributed in ER and Golgi. Untransfected F-11 A cells contain mainly GM3, whereas stable transfection with ST2 or GalNAcT results in the predominant expression of b-series complex gangliosides (BCGs). This suggests that the expression of ST2 enhances the activity of endogenous GalNAcT and vice versa, which has been verified by in vitro activity assays with detergent-solubilized enzymes. A potential complex formation between ST2 and GalNAcT has been analyzed by transient transfection of ST2-GFP expressing cells with GalNAcT-RFP. Co-immunoprecipitation studies and fluorescence resonance energy transfer (FRET) show that the Golgi-localized fraction of GalNAcTRFP forms a complex with ST2-GFP. FRET-analysis also indicates that ST2-GFP forms a stable complex with pyrene-labeled GM3. We hypothesize that an ST2-GM3 complex associates with GalNAcT, resulting in the enhanced conversion of GM3 to GD3 and BCGs. Taken together, our results indicate that ganglioside biosynthesis is tightly regulated by the formation of glycosyltransferase complexes in the Golgi. GPI-anchored proteins are mainly clustered in sphingolipid-cholesterol microdomains of the plasma membrane (PM), which are characterized by a high insolubility in Triton X-100. We investigate here the distribution of a GPIanchored chimera, which contains an ER-import signal, and the total sequence of the yellow fluorescence protein fused to a GPI-attachment signal (GPI-YFP) in CHO-K1 cells with different glycolipid compositions. Cells depleted of glycosphingolipids by inhibiting glucosylceramide synthase activity, or cell lines expressing different gangliosides due to stable transfection of appropriate ganglioside glycosyltransferases, or treated with exogenous GM1 were used. The association of GPI-YFP chimera on the cell surface was investigated by using the membrane-impermeable cross-linking agent bis(sulfosuccinimidyl)suberate (BS3). Parental cell expressing GM3, or cells depleted of glycolipids, or transfected cells expressing mostly GM1 and GD1a or GD3 and GT3 subjected to chemical cross-linking with BS3 showed a major 80 kDa band (dimer) and a band of high molecular weight (400 kDa) detected by Western blots. However, minor changes in the relative proportion between these two bands were detected in the different cell clones. On the other hand, parental cells loaded with varying concentration (100–300 lM) of GM1 for 1 h before cross-linking with BS3 showed a dramatic reduction in the efficiency of cross-linking. Results indicate that loading with exogenous GM1 results in formation of particular species that alter the GPIYFP membrane distribution. These species would not form when the composition of gangliosides is changed keeping normal the process of synthesis and insertion into membranes. Acknowledgements: Supported by grant NS11853 (to R.K.Y.).
2002 International Society for Neurochemistry, Journal of Neurochemistry, 81 (Suppl. 1) 9 AP03-05 AP03-07 High brain glycogen levels: lability during tissue sampling & extraction N. F. Cruz and G. Dienel University AR Medical Sci, Little Rock, USA IGF-1 and PI3-kinase regulate folate-dependent methylation in an ethanol-sensitive manner R. C. Deth and M. Waly North-eastern University, Boston, USA High brain glycogen levels suggest an unrecognized role for rapid mobilization of astrocytic energy metabolism during brain activation. Brain glycogen is usually reported to be 2–3 lmol/g, and occasionally up to 5–6 lmol/g. In two recent studies, we found 5–6 and 8–14 lmol glycogen/g in brains of nonfasted rats that were sequestered to prevent stress prior to assessment of metabolic changes induced by sensory stimulation. Because neurotransmitters stimulate glycogenolysis, stress or sensory activation during tissue sampling (e.g. handling, restraint, freeze-blowing) should reduce brain glycogen content, perhaps explaining, in part, the lower values. Lability of glycogen during extraction might also be a critical factor. In our first study, frozen brain powders were thawed in 0.03 M HCl at )12C, homogenized, heated (100C, 45 min), and assayed for glucose (glc) released by amyloglucosidase (using glc oxidase); in our second study the powders were first extracted with buffered aqueous ethanol and the insoluble pellet was assayed for glycogen by exactly the same procedure. To determine if all glc released by amyloglucosidase is metabolically active, powders were incubated at 37C for 0–30 min, boiled in HCl, and assayed; 95% of the glycogen was degraded and its Ôcarbon equivalentsÕ recovered mainly as glc, glc-p, and lactate. Comparison of the HCl vs. ethanol procedures in parallel assays of the same brain powders revealed that glycogen was about 50% lower even when carefully thawed in weak acid, suggesting incomplete phosphorylase inactivation. Glycogen levels in perchloric acid extracts (insoluble + soluble fractions) of the same powders were also lower than in ethanol extracts. Thus, elimination of stressful procedures and extreme care during tissue denaturation are essential for detection of high levels of brain glycogen. Ethanol is known to inhibit the folate-dependent methylation of homocysteine, and is also known to inhibit the signalling activity of insulin-like growth factor-1 (IGF-1). In cultured SH-SY5Y human neuroblastoma cells we find that ethanol potently inhibits basal folate-dependent phospholipid methylation (PLM), with an IC50 of approximately 5 mM (0.025%), but has no effect on PLM measured using [3H-methyl]methionine. IGF-1 stimulates folate-dependent PLM, with an EC50 of 0.4 nM, but has no effect on PLM measured with [3H-methyl]methionine. Thus both ethanol and IGF-1 appear to affect methionine synthase-dependent remethylation of homocysteine. Selective inhibitors of PI3-kinase (wortmannin and LY294004) each inhibited folate-dependent PLM, and eliminated the effect of ethanol, suggesting that PI3-kinase activity mediates the stimulatory effect of IGF-1 and that ethanol interferes with this mechanism. In contrast to its inhibition of basal PLM, ethanol augments folate-dependent PLM measured in the presence of dopamine, resulting in a shift of folate-derived methyl groups toward the D4R-mediated PLM cycle, away from the nonreceptor-mediated cycle. Among a series of alkanols, all of which inhibited basal PLM, ethanol was the only one that augmented D4R-mediated PLM. Our studies reveal a previously unrecognized action of IGF-1 to regulate folate-dependent homocysteine remethylation at the level of methionine synthase. This mechanism appears to be mediated by activation of PI3-kinase and is potently inhibited by ethanol. AP03-06 AP03-08 Cearmide generated during apoptosis is localized to detergent-insoluble fractions of the cells (Rafts) G. Dawson, J. K. Kilkus and R. Goswami Deaprtments of Pediatrics, Biochemsitry and Molecualr Biology, University of Chicago, Chicago, USA Peculiarities of lipid composition disorders in rat brain and myocardium at unilateral right ganglion sympathectomy E. P. Gasparyan, G. A. Hoveyan, H. M. Amirkhanyan, M. A. Simonyan, A. R. Zakharyan, L. T. Amirkhanyan and K. G. Karageuzyan Institute of Molecular Biology of NAS RA, Yerevan, Armenia The detergent-insoluble fraction (Raft), isolated from human oligodendroglioma cells by density-gradient ultracentifugation was shown to be greatly enriched in sphingolipids, especially ceramide. Treatment of HOG cells with staurosporine (to induce apoptosis) or exogenous sphingomyelinase increased the ceramide content of the Raft fraction, whereas b-methylcyclodextran reduced it by preventing Raft formation. Assay of endogenous sphingomyelinase in unstimulated HOG cells revealed lysosomal (acid) activity to be 10-times higher than neutral (Mg-dependent) activity but concentrations of staurosporine necessary to induce apoptosis only activated neutral sphingomyelinase. A similar raft fraction was isolated from neonatal rat oligodendrocyte primary cultures and was shown to be similarly enriched in ceramide. As the oligodendrocytes differentiated and lipid content increased it was more difficult to isolate a discrete Raft fraction by this procedure. Proteins associated with the Raft fraction included caveolin-1 and the palmitoylated tyrosine kinase Fyn. Rafts isolated from a human neuroblastoma cell line (LAN-5) engineered to overexpress palmitoyl: protein thioesterase (PPT1) showed a 50% reduction in ceramide content and an increased resistance to apoptosis induced by either staurosporine or ceramide. We therefore propose that the formation of ceramide in Rafts in cells is an essential initial step in pro-apoptotic signalling. 10 Unilateral gangliosympathectomy (UGS) is accompanied by a decrease in the brain and myocardial tissues of the levels of ATP, cAMP, ATP/ADP ratio, quantity of DNA and histones. The simultaneous decrease of intranuclear sphingomyelins and phosphatidylcholines concentrations was observed too. The latter was accompanied by the parallel increase of lysophosphatidylcholines content, which is the result of a significant activation of phospholipase A2. On the seventh day of UGS the pronounced changes were noticed in glycolipids qualitative and quantitative composition, especially in cerebrosides and sulfatides content from the operated side. The detected changes are similar to those described under conditions of demyelinization. It was also established a development of pronounced changes in the quantitative composition of new categories of metal-containing proteins recently discovered by the Armenian biochemists. Those proteins are presented as a series of B558 (I–IV) cytochrome and as so-called suprol. The latter is a new phospholipid-containing lipoprotein with a very high physiological activity. As a reflection of UGS, the oxidative stress is characterized by disorders in ratio of interrelations between different representatives of cytochromes mentioned both in the whole blood, and in the erythrocyte membranes. These abnormalities play a great role in disorders of membrane-bound fatty acids composition of phospholipids, and have a definite significance in conditioning the pathological changes of physiological state in the whole organism.
2002 International Society for Neurochemistry, Journal of Neurochemistry, 81 (Suppl. 1) AP03-09 AP03-11 Inheritance of brain beta-galactosidase activities and gangliosides in crosses of DBA/2 and knockout mice E. C. Hauser,* A. d’Azzo and T. N. Seyfried* *Biology Department, Boston College, Chestnut Hill, MA, USA; Genetics Department, St Jude Children’s Res. Hosp., Memphis, TN, USA Brain phospholipid levels and composition are altered in alpha-synuclen gene-abated mice E. J. Murphy,* N. Cole, A. Glynn* and R. L. Nussbaum *University of North Dakota, Grand Forks, ND 58201; NHGRI, NIH, Bethesda, MD 20892, USA GM1-gangliosidosis is a progressive neurological disease caused by deficiencies in lysosomal acid beta-galactosidase (b-gal) and involves CNS accumulation and storage of ganglioside GM1 and its asialo form (GA1). In humans, this disease is characterized as infantile, juvenile, or adult onset. Similar to the infantile/juvenile human diseases, b-gal knockout (ko) mice express residual brain b-gal activity and elevations of GM1/GA1 and total brain gangliosides. Previous studies suggested that inbred DBA/2 (D2) mice may model the adult human disease since total brain gangliosides and GM1 were higher and b-gal activity was lower (by 70–80%) in D2 mice than in inbred C57BL (B6) mice. An analysis was conducted to determine if the b-gal genes in the D2 and the ko mice were functionally allelic and if the reduced brain b-gal activity in D2 mice could account for elevations in GM1 and brain gangliosides. D2 mice (bgald/bgald, d/d) were crossed with B6/ 129sv mice (bgal+/bgal–, +/–) to generate d/+ and d/– offspring. The relative specific activity (nmol/mg protein/h) of whole brain b-gal in 21 day-old+/ +,+/d, d/d, d/–, and –/– mice was 65 + 4, 45 + 4, 18 + 1, 14 + 2, and 2 + 0.2, respectively (n ¼ 4). These results show that the D2 and ko genes are allelic. The average brain ganglioside sialic acid concentration (ug/100 mg dry wt) in the 21 day-old+/+, d/d, d/–, and –/– mice was 425, 468, 443, and 597, respectively. The ratio of GM1 to GD1a in these mice was 0.2, 0.4, 0.3, and 1.1, respectively. Furthermore, GA1 was present only in ko mice. These results suggest that the reduced brain b-gal activity in D2 mice cannot alone account for the elevation of total gangliosides and GM1 in these mice. Elucidating the physiological function of alpha-synuclein is important because mutations in this protein are implicated as a causitive factor in familial Parkinson disease. Further, alpha-synuclein is also associated with a number of other neurodegenerative diseases. To determine the effects of alpha-synuclein on brain lipid metabolism, phospholipid acyl chain composition, phospholipid and cholesterol mass were assessed in brains and synaptosomes isolated from control and gene-abated mice. Lipids were separated using standard techniques. There was a significant rearrangement of phospholipid metabolism in the gene-abated mice as indicated by a 75% reduction in phosphatidic acid. Phosphatidylinositol and cardiolipin mass were reduced 30 and 55%, respectively. Choline glycerophospholipid and sphingomyelin mass were reduced 8 and 40%, respectively, while ethanolamine glycerophospholipid mass was increased 1.4-fold. This included an increase in ethanolamine plasmalogen mass. Despite these changes, total phospholipid levels were not significantly altered. Surprisingly, cholesterol mass was decreased 16% as well as a 16% decrease in the cholesterol to phospholipid ratio. The major acyl chain changes were a 13–18% decrease in 22:6 n-3 proportions in the major phospholipid classes and a specific 10% reduction in 20:4 n-6 in phosphatidylinositol. Changes in synaptosomes were limited, but there was a substantial 65% reduction in choline plasmalogen. This is important as choline plasmalogen appears to be involved in signal transduction and synaptic function. These changes observed in the geneabated animals suggest that alpha-synuclein has a substantial role in brain lipid metabolism. AP03-10 AP03-12 Cyclooxygenase-2 protein is cleaved following activation by arachidonic acid and endocannabinoids J. A. Hewett, A. S. Vidwans and S. J. Hewett Department of Neuroscience, The University of Connecticut Health Center, Farmington, CT, USA Effect of gangliosides on adenylyl cyclase activity in the cortex and striatum of rats N. N. Nalivaeva, S. A. Plesneva, N. M. Dubrovskaya and I. A. Zhuravin University of Leeds, Leeds, UK; Institute Evol. Physiol. BIochem., St Petersburg, Russia Cyclooxygenase-2 (COX-2) is an 70 kDa protein of the endoplasmic reticulum that catalyzes the initial step in the metabolism of arachidonic acid (AA) to bioactive lipid mediators, including prostaglandins. It is constitutively expressed in certain neurons of the central nervous system and its expression and activity are up-regulated during pathological conditions, including cerebral ischemia and epilepsy. In COS-7 cells overexpressing murine COX-2, a 33–35 kDa COX-2 fragment doublet was observed after exposure to AA. COX-2 cleavage occurred rapidly, as early as 1 min after substrate administration, and correlated temporally with catalytic activity. Pretreatment of cells with NS-398, a selective inhibitor of COX-2 activity, blocked AA-induced cleavage. Results with NS-398 were verified using an inactive COX-2 mutant protein, confirming the notion that cleavage is dependent on enzyme activity. COX-2 cleavage was also induced by the endocannabinoid, 2-arachidonoyl glycerol, which was recently described as a COX-2 substrate. Activity-dependent COX-2 cleavage was not unique to transfected COS-7 cells, since it was observed in primary murine astrocytes expressing endogenous COX-2. Finally, neither the proteosome inhibitor, MG-132, nor the golgi toxin, brefeldin A, blocked COX-2 cleavage. These results suggest that activity-dependent COX-2 cleavage occurs locally within the endoplasmic reticulum, perhaps by an endogenous protease, raising the intriguing possibility that local proteolysis may modulate the level of COX-2 in cells Acknowledgements: Supported by grants from the NINDS (NS36812) and The Patrick and Catherine Weldon Medical Research Foundation. By studying the effects of gangliosides (G) on learning and memory we have found that i.p. administration of G led to a decrease in AC activity in the cortex (Cx) and striatum (Str) as well as in the threshold of the sensitivity of striatal neurons to the effect of cholinergic agonists. G also modified the sensitivity of AC from the Cx and Str to such agents as Gpp[NH]p and forskolin. The aim of this work was to analyze the correlation between the changes in the activity of AC in the Cx and Str, concentration of G in these brain structures as well as formation of motor reactions of newborn rats during the first month of postnatal ontogenesis. It was found that new born rats develop normal body rotation by sixth day, locomotion by fifteenth day and stabilization of locomotor activity and supporting body balance by 20–22nd day. As shown previously, the concentration of gangliosides in the Cx and Str is gradually increasing during the first month of animal development. The activity of AC (pmol cAMP/min/mg of protein) in the Cx was found to decrease from 34.75 to 4.09 and in the Str from 46.00 to 11.67 during the first week after birth. However in the periods of formation of general behavioural reactions we observed a statistically significant increase in AC activity: in the Str on 10th and 26th days (p < 0.01) and in the Cx from 10th to 19th days (p < 0.05) compared to the AC activity on 5th and 30th days. Thus, formation of locomotor activity and posture-tonic reactions during development of rats in early postnatal ontogenesis correlates with increasing concentration of G and basal activity of AC. Supported by RFBR (99-04-49751) and RAS (99-06-287).
2002 International Society for Neurochemistry, Journal of Neurochemistry, 81 (Suppl. 1) 11 AP03-15 AP03-13 Gene expression for glycosyltransferases in transfected neuroblastoma cells 1 L. N. Irwin,* E. Bieberich, D. D. Li, J. Silvaà and R. K. Yu *Department Biol. Sci., University of Texas at El Paso, TX Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA, USA To study regulatory mechanisms of ganglioside biosynthesis, genes for sialyltransferase II (ST2) or acetylgalactosaminyltransferase I (GalNAcT) were transfected into different cell lines of a substrain of murine neuroblastoma F-11 cells (F-11A). While complex b-series ganglioside synthesis is negligible in the parental line, cells transfected with genes for either enzyme express complex gangliosides requiring both enzyme activities. We have used primers specific for each of the two genes to determine the level of gene expression (1) in the parental cells where endogenous ST2 and GalNAcT activities are very low, and (2) in each of the transfected cell lines where both enzyme activities are present and complex gangliosides are synthesized. Transcripts for both ST2 and GalNAcT were amplified from cDNA that was reverse-transcribed from the parental as well as both transfected cell lines. While some differences in transcript number and size were noted between parental, ST2-transfected, and GalNAcT-transfected cells, suggesting some degree of transcriptional control, all the cell lines clearly expressed transcripts for both genes. These results support the view that ST2 and GalNAcT act in a coordinated fashion, perhaps as a multienzyme complex, to catalyze the synthesis of complex gangliosides, and that synthesis of complex b-series gangliosides in these cells is regulated primarily post-transcriptionally. Acknowledgements: Supported by NIH R15-DC05179 to L.N.I, NIH NS-11853 to R.K.Y, and NIH-NCRR(RCMI) G12-RRO8124 to the Border Biomedical Research Center at the University of Texas at El Paso. Transcription factors NF-jB and Sp1 are major determinants of the basal promoter activity of the rat GD3-synthase gene G.Zeng, L. Gao, T. Xia, T. Tencomao and R. K. Yu Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia, USA GD3-synthase is one of the key sialyltransferases responsible for synthesis of ganglioside GD3, the substrate for initiation of the ÔbÕ and ÔcÕ series ganglioside synthesis. We have previously cloned the rat GD3-synthase gene promoter, and preliminary characterization has identified a minimal 0.5-kb region that has a strong basal promoter activity, and is GC-rich and has no CAAT or TATA boxes. In this study, we showed that the Sp1 and NF-jB sites in this region significantly contributed to basal GD3-synthase promoter activity. When either the Sp1 or NF-jB sites were deleted, a 50% decrease in promoter activity was observed. The same results were obtained by a decoy strategy using oligonucleotides containing the Sp1 or NF-jB sites. The binding to the Sp1 and NF-jB sites was confirmed by electrophoretic mobility shift assay (EMSA), competition and supershift EMSA. In addition, cell-type specific activation of the promoter was also determined. The promoter was highly activated in the GD3-expressing F11 cells while repressed in NG-108 cells in which GD3 is almost undetectable. An additional band of NF-jB family was identified only in the F-11 nuclear extract using the NF-jB consensus probe by EMSA. DNA pull-down assays were further carried out to screen proteins that bound to the promoter including the basal region and the potential negative-regulatory region between )526 and )769. More than 10 major binding proteins were pulled down, some of which were present only in the F-11 or NG-108 nuclear extracts. Our data demonstrate that NF-jB and Sp1 are the major determinants for the basal promoter activity and some factors such as NF-jB may be involved in cell type-specific expression of the gene. AP03-14 AP03-16 Analysis of glycosphingolipid composition of a neural progenitor cell line K. Suetake, S. S. Liour, T. Tencomnao, Stacy Kraemer and Robert K. Yu Program in Neurobiology, Institute of Molecular Medicine and Genetics, Medical College of Georgia, GA, USA Interleukin-1 increases expression of cytosolic and membrane PGE synthase in mouse astrocytes and brain M. K. O’Banion, M. E. Maida and A. H. Moore University of Rochester Medical Center, Rochester, New York, USA Continuous neurogenesis from neural stem cells in adult brain suggests the possibility that these newly generated neural tissue may be capable of repair of the neurodegeneration associated with genetic disorders, trauma, and aging. Glycosphingolipids (GSLs) play important roles in cellular growth, differentiation and cell signalling; however, little is known about the GSL composition in neural progenitor and/or stem cells. In order to characterize the GSL composition in neural stem cells, we studied the multipotential C17.2 cell line that was generated after myc-transformation of isolated from neonatal mouse cerebellar neural progenitor cells. The developmental potential of C17.2 cells is similar to the endogenous neural stem cells, i.e. they are capable of self-renewal and differentiation into neurons and glia (Snyder et al. Cell 68, 33–51, 1992). HPTLC analysis of the total lipid fractions isolated undifferentiated C17.2 cells revealed that the major GSL expressed in these cells were glucosylceramide, GM3, GM2, GM1, and GD1a. Histochemical staining for GM1 using cholera toxin B subunit revealed punctate staining on the plasma membrane, suggesting GM1 at the plasma membrane, maybe clustered into microdomain-like structures. Our results indicate that the undifferentiated C17.2 neural stem cell line is unique in expression of a-series gangliosides. These gangliosides may prove to be useful markers for these cells. Acknowledgements: Supported by an NIH grant NS11853 and a grant from the Children’s Medical Research Foundation. 12 Increased production of prostaglandin E2 (PGE2) accompanies inflammatory responses, both peripherally and in the brain. Previous studies have demonstrated marked up-regulation of cyclooxygenase-2 (COX-2) expression by IL-1b in mouse astrocytes and brain. However, selective increases in PGE2 production are dependent on expression of downstream PGE synthases. Therefore, we have investigated regulation of two PGE synthase isoforms in cultured murine astrocytes and in mouse brain treated with IL-1b. In astrocytes exposed to 10 ng/mL IL-1b we see an early and dramatic increase in cytosolic PGE synthase mRNA and protein expression at 4 and 8 h that largely subsides by 24 h. This pattern is similar to that observed for COX-2 expression. Interestingly, increases in membrane PGE synthase levels also occur, but with a delayed time course. Similar increases in both isoforms are observed following intraparenchymal injection of 5 ng recombinant IL-1b. More specifically, the in vivo increase in COX-2 mRNA was accompanied by an increase in membrane PGE synthase mRNA levels 6 h following IL-1b injection. Subsequently, protein expression of both the cytosolic and membrane PGE synthases was elevated at 24 h. Interestingly, administration of NS-398, a COX-2 specific inhibitor, attenuated IL-1b-induced changes in PGE synthase expression, suggesting regulation of these isoforms by a COX-2-dependent mechanism. Together these data suggest that up-regulation of both PGE synthase isoforms contributes to cerebral inflammatory responses to IL-1b. Acknowledgements: Supported by RO1 NS33553 to MKO.
2002 International Society for Neurochemistry, Journal of Neurochemistry, 81 (Suppl. 1) AP03-17 AP03-19 Disruption of PPARb results in distinct gender differences in mouse brain phospholipid T. A. Rosenberger, J. T. Hovda and J. M. Peters Brain Physiology and Metabolism Section NIA/NIH, Bethesda, MD and Department of Veterinary Science and Center for Molecular Toxicology and Carcinogenesis, Pennsylvania State University, University Park, Pennsylvania, USA Prediction, isolation, and sequencing of the promoter for human aminophospholipid translocase (APTL) II T. Sobocki,* M. B. Sobocka and P. Banerjee *Biochem., SUNY Downstate Medical Cntr, Brooklyn, NY 10203; Neurosci. Prog. and Chem. Department, College of Staten Island (CUNY), NY 10314, USA A homozygous PPARb-null mouse has been developed in which the ligand-binding domain of the PPARb receptor is disrupted. Analysis of brains from these animals show that female null mice have a 24 and 17% increase of plasmenylethanolamine and phosphatidylserine and a 9% decrease in the level of phosphatidylinositol when compared to controls. The phospholipid changes found in female null mice were associated with significant increases in esterified 18:1n-9, 20:1n-9, 20:4n-6, and 22:5n-3 in plasmenylethanolamine, 20:1n-9 in phosphatidylinositol, and 18:0, 18:1n-9, 18:3n-6, 20:1n-9, and 20:4n-6 in phosphatidylserine. Increased esterified 18:1n-9, 18:2n-6, 18:3n-6, and 20:1n-9 were also found in the phosphatidylethanolamine fraction despite its cellular content remaining unchanged. Brain phospholipid content in male PPARb-null mice was not different than controls, but increased levels of 20:1n-9 in the phosphatidylinositol and 18:1n-9 in the phosphatidylserine fractions were observed. No changes were found in the content of brain cholesterol, triglycerides, and free fatty acid in either female or male PPARb-null mice. These data suggests that the peroxisomal proliferator-activated receptor beta is involved in maintaining fatty acid and phospholipid levels in adult female mouse brain and provides strong evidence suggesting a role for PPARb in peroxisomal acylCoA utilization. p-Type ATPases drive translocation of phosphatidylserine (PS) and phosphatidyl-ethanolamine (PE) from the outer to inner leaflet of the plasma membrane. This results in their inner-leaflet sequestration, which is typical for healthy cells. The externalization of PS (and PE) during apoptosis labels apoptotic cells for phagocytosis by scavenger cells (e.g. microglia). Recently, coding sequences (partial cDNA and coding sequence based on a partial gene sequence) of the type II ATPase have been reported. Initial analysis of these sequences revealed an apparent lack of the 5¢UTRs. In our attempts to elucidate the promoter, the transcription start site and the 5¢ UTR of the APTL II gene, we compared these partial coding sequences to the EST’s and genomic databases. This allowed us to elaborate exon/intron organization of ATPase II gene and revealed the possibility of existence of alternative splicing. We then analyzed the region in genomic DNA immediately upstream of the 5¢end of the longest 5¢EST clone, which revealed the presence of a CpG island. A TATA-less promoter was predicted. We then amplified the part of the gene containing the promoter by PCR using genomic DNA as template. Cloning and sequencing revealed minor, though significant, differences between our sequence and its counterpart reported by Human Genome Project. Our studies will be followed up by 5¢ RACE and analysis of the putative promoter region by its ability to drive an expression of a reporter gene. This will allow us to understand tissue specificity and the functional role of the APTLII enzyme. AP03-18 AP03-20 Effects of ethanol on gangliosides in the plasma, liver, and brain of inbred mouse strains M. I. Saito, M. A. Saito, I. Szakall and C. Vadasz The Nathan S. Kline Institute for Psychiatric Research and New York University, Orangeburg, USA NMR study of 13C-glucose and 13C-acetate metabolism in the brain of rats with acute liver failure C. Zwingmann, N. Chatauret, D. Leibfritz and R. F. Butterworth Neuroscience Research Unit, CHUM Hôpital Saint-Luc, Montreal, Canada; Department of Organic Chemistry, University of Bremen, Germany The mouse inbred strains C57BL/6ByJ and BALB/cJ show genetically different alcohol-related behaviors. Using these strains, we examined ganglioside contents in the plasma, liver, and brain with or without acute ethanol treatment. The quantification of GM1 was performed with a TLCimmunostaining procedure using choleragenoid, and the contents of other gangliosides were measured after staining with resorcinol reagents. It is known that there are polymorphisms in ganglioside compositions among inbred mouse strains. We found that the plasma GM1 level in BALB/cJ mice (1.6 ± 0.6 ng/lL) was 12 times higher than the level found in C57BL/6ByJ mice (0.13 ± 0.03 ng/lL) although the major ganglioside in both strains was GM2. The ganglioside profiles in the liver were similar to those of the plasma, and the GM1 level in BALB/cJ was 25 times higher than that of C57BL/6ByJ. The liver probably synthesizes the plasma gangliosides as suggested in other studies. The total brain ganglioside compositions were also different between BALB/cJ and C57BL/6ByJ. The levels of GD1b and GQ1b were higher in BALB/cJ although the GM1 contents were similar. These animals were injected with 20% ethanol intraperitoneally in a single dose of 3 g/kg and the ganglioside contents were measured after 4 h. The GM1 levels in the liver and plasma were lower in ethanol-treated animals while the GM1 levels were higher in erythrocytes and brains. Since it has been shown that the administration of GM1 attenuates or modifies the effects of ethanol in several animal models, the difference in GM1 contents in the plasma between BALB/cJ and C57BL/6ByJ may contribute to some of the differences in ethanol-related behaviors or toxicity between these strains. Acute liver failure (ALF) is characterized by hepatic encephalopathy (HE) with brain edema as the main cause of death. The role of brain energy failure in ALF is still controversial. In the present study we investigated the association of encephalopathy with brain energy metabolism in rats with ALF induced by liver devascularization. Using [1–13]glucose and [2–13C]acetate combined with [1H, 13C]NMR spectroscopy anaerobic and aerobic carbon fluxes were studied in brain extracts from ALF rats. The anaplerotic flux of 13 C-label from glucose was found to be stimulated resulting in a 10-fold increase of [13C]glutamine (Gln) starting at precoma stages of encephalopathy. In contrast, de novo synthesis of alanine and lactate increased to 320 and 210% in precoma, followed by a further elevation at coma stages to 615 and 425% of sham-operated control values. Using [2–13C]acetate, the higher 13 C-enrichment of C-2 in Gln, glutamate (Glu) and the corresponding C-4 in GABA compared to C-3 of either compound suggests cleavage of [2–13C]citrate and formation of [3–13C]oxaloacetate, which enters the glial TCA cycle. On the other hand, decreased synthesis of [4–13C]Glu and [2–13C]GABA was observed. This may result from decreased neuronal oxidative metabolism, since the ratio of [4–13C]Glu/[2–13C]GABA is unchanged and increased [2–13C]Glu and corresponding [4–13C]GABA suggests a stimulated flux of astrocytic label to neurons. Impaired neuronal energy metabolism and selective stimulation of lactate synthesis may contribute to the pathogenesis of HE in ALF. Acknowledgements: Funded by CIHR Canada.
2002 International Society for Neurochemistry, Journal of Neurochemistry, 81 (Suppl. 1) 13