ognizant Communication Corporation

GENE EXPRESSION

ABSTRACTS
VOLUME 12, NUMBER 2

Gene Expression, Vol. 12, pp. 61-67
1052-2166/05 $20.00 + .00
Copyright © 2005 Cognizant Comm. Corp.
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The Transcriptional Response to Hypoxic Insult Controlled by FRA-2

Tanya L. Butler and Keith R. Pennypacker

Department of Pharmacology and Therapeutics, University of South Florida, Tampa, FL 33612

FRA-2 is involved in cellular differentiation and is also upregulated in response to ischemic injury to the brain. To shed light on the function of this transcription factor, a novel microarray analysis was utilized to identify FRA-2-dependent gene expression increased in the hypoxic response. Genes were identified that were upregulated by exposure of neuronally differentiated PC12 cells to hypoxia. Using a dominant negative construct to block FRA-2, a second subset of genes that were FRA-2 dependent was found. Cross comparison then allowed isolation of a list of genes that were induced in response to hypoxia in a FRA-2-dependent manner. These data suggest that FRA-2 is involved in the transcriptional control of neuroprotective genes and in the switch from aerobic to anaerobic metabolism.

Key words: Rat pheochromocytoma; PC12; AP-1; Neuroprotection

Address correspondence to Keith R. Pennypacker, College of Medicine, Department of Pharmacology and Therapeutics, University of South Florida, 12901 Bruce B Downs Boulevard, MDC 9, Tampa, FL 33612. Tel: (813) 974-9913; Fax: (813) 974-2565; E-mail: kpennypa@hsc.usf.edu




Gene Expression, Vol. 12, pp. 69-81
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Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Quantification of G Protein Gas Subunit Splice Variants in Different Human Tissues and Cells Using Pyrosequencing

Ulrich H. Frey,1 Holger Nückel,1,2 Dobromir Dobrev,3 Iris Manthey,1 I. E. Sandalcioglu,4 Andreas Eisenhardt,5 Karl Worm,6 Hans Hauner,7 and Winfried Siffert1

1Institut für Pharmakologie, Universitätsklinikum, D-45122 Essen, Germany
2Klinik für Hämatologie, Universitätsklinikum, D-45122 Essen, Germany
3Institut für Pharmakologie und Toxikologie, D-01307 Dresden, Germany
4Klinik für Neurochirurgie, Universitätsklinikum, D-45122 Essen, Germany
5Klinik für Urologie, Universitätsklinikum, D-45122 Essen, Germany
6Institut für Pathologie, Universitätsklinikum, D-45122 Essen, Germany
7Else-Kröner-Fresenius-Zentrum für Ernährungsmedizin, Technische Universität München, D-81675 München, Germany

The G protein Gas is derived from four alternatively spliced transcripts, two long variants (GasL+CAG and GasL-CAG), which include an extra 45-bp segment, and two short variants (GasS+CAG and GasS-CAG). The long and short forms differ in each case by splicing in or out of a serine residue encoded at the 3´ end of the variable exon 3. The relative expression of all four variants in human tissues is poorly investigated due to experimental limitations. We therefore established a method for reliable relative mRNA quantification of these splice variants based on the Pyrosequencing technology, and determined Gas transcript ratios in various human tissues and cells. GasS/Gas ratio was highest in blood mononuclear cells (0.84 ± 0.02, n = 16) and lowest in the brain (0.51 ± 0.14, n = 3). The different ranges resulted from differences in GvsS+CAG ratios, which ranged from a total Gas ratio of 0.32 ± 0.07 (n = 12) in heart tissue to 0.57 ± 0.03 (n = 16) in blood mononuclear cells (p < 0.0001), whereas the GasS-CAG ratio was rather constant and ranged from 0.22 ± 0.04 (n = 7) in retinoblastoma cells to 0.27 ± 0.04 in lymphocytes (p = 0.19). The GasL+CAG ratio ranged from 0.02 ± 0.02 in heart tissue to 0.05 ± 0.01 in retinoblastoma cells, with a varying proportion of GasL-CAG, which ranged from 0.14 ± 0.02 in blood mononuclear cells to 0.41 ± 0.08 in heart tissue. Stimulation of immortalized B lymphoblasts with isoproterenol resulted in significant changes of splice variant ratios. Our data indicate that changes of long and short ratios of Gas in different tissues affected GasL-CAG and GasS+CAG rather than GasL+CAG and GasS-CAG. Furthermore, stimulation of cells seemed to affect splice variant ratios. These results are, therefore, suggestive of different biological functions of these variants.

Key words: G proteins; Splice variants; Pyrosequencing; Signal transduction; Quantification

Address correspondence to Ulrich H. Frey, Department of Pharmacology, University Hospital Essen, D-45122 Essen, Germany. Tel: +49 201 723 3459; Fax: +49 201 723 5968; E-mail: ulrich.frey@uni-essen.de




Gene Expression, Vol. 12, pp. 83-98
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Copyright © 2005 Cognizant Comm. Corp.
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Expression of Progenitor Cell Markers During Expansion of Sorted Human Pancreatic Beta Cells

Thomas Bouckenooghe,1 Brigitte Vandewalle,1 Ericka Moerman,1 Pierre-Marie Danzé,2 Bruno Lukowiak,1 Ghaffar Muharram,1 Julie Kerr-Conte,1 Valery Gmyr,1 Bernard Laine,2 and François Pattou1

1INSERM ERIT-M 0106 and 2INSERM U459, Faculty of Medicine, Place de Verdun 59045 Lille, France

Functional pancreatic beta cell mass is dynamic and although fully differentiated, beta cells are capable of reentering the cell cycle upon appropriate stimuli. Stimulating regeneration-competent cells in situ is clearly the most desirable way to restore damaged tissue. Regeneration by dedifferentiation and transdifferentiation is a potential source of cells exhibiting a more developmentally immature phenotype and a wide differentiation potential. In this context and to gain a better understanding of the transformation induced in human beta cells during forced in vitro expansion, we focused on identifying differences in gene expression along with phenotypical transformation between proliferating and quiescent human beta cells. FACS-purified beta cells from three different human pancreata were cultured during 3-4 months (8-10 subcultures) on HTB-9 cell matrix with hepatocyte growth factor. Gene expression profiling was performed on cells from each subculture on "in-house" pancreas-specific microarrays consisting of 218 genes and concomitant morphological transformations were studied by immunocytochemistry. Immunocytochemical studies indicated a shift from epithelial to neuroepithelial cell phenotype, including progenitor cell features such as protein gene product 9.5 (PGP 9.5), Reg, vimentin, and neurogenin 3 protein expression. The expression of 49 genes was downregulated, including several markers of endocrine differentiation while 76 were induced by cell expansion including several markers of progenitor cells. Their pattern also argues for the transdifferentiation of beta cells into progenitor cells, demonstrating neuroepithelial features and overexpressing both PBX1, a homeodomain protein that can bind as a heterodimer with PDX1 and could switch the nature of its transcriptional activity, and neurogenin 3, a key factor for the generation of endocrine islet cells. Our study of the machinery that regulates human beta cell expansion and dedifferentiation may help elucidate some of the critical genes that control the formation of adult pancreatic progenitor cells and hence design targets to modify their expression in view of the production of insulin-secreting cells.

Key words: DNA microarray; Beta cell expansion; Gene expression; Human purified beta cell

Address correspondence to B. Vandewalle, Thérapie Cellulaire du Diabète, INSERM, ERIT-M 0106, Faculté de Médecine, Place de Verdun, 59045 Lille, France. Tel/Fax: (33) 3 20 62 68 77; E-mail: bvandewalle@univ-lille2.fr




Gene Expression, Vol. 12, pp. 99-106
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Copyright © 2005 Cognizant Comm. Corp.
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Translational Downregulation of the Noncatalytic Growth Factor Receptor TrkB.T1 by Ischemic Preconditioning of Primary Neurons

Julius A. Steinbeck and Axel Methner

Research Group Protective Signaling, Zentrum für Molekulare Neurobiologie and Klinik und Poliklinik für Neurologie, Universitätsklinikum Hamburg-Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany

Short episodes of ischemia can protect neuronal cells and tissue against a subsequent lethal ischemia by a phenomenon called ischemic preconditioning. The development of this tolerance depends on protein synthesis and takes at least 1 day. It therefore seems reasonable that preconditioning leads to upregulation and translation of protective genes or posttranslational modification of pro- or antiapoptotic proteins. We recently used suppression subtractive hybridization to identify transcripts upregulated in rat primary neuronal cultures preconditioned by oxygen glucose deprivation. In this contribution, we describe the previously unknown 7-kb full-length sequence of an upregulated expressed sequence tag and show that it constitutes the 3&prime; end of the large untranslated region of the noncatalytic "truncated" growth factor receptor TrkB.T1. TrkB.T1 is expressed most prominently in the adult brain and its mRNA was found to be 2.1-fold upregulated by ischemic preconditioning. At the protein level, however, TrkB.T1 was clearly downregulated, possibly by increased degradation in preconditioned cultures. TrKB.T1 can act as a dominant-negative inhibitor of its catalytic counterpart TrkB, which is the receptor for brain-derived neurotrophic factor (BDNF), a factor induced by ischemia that can protect from ischemia-induced neuron loss. We hypothesize that the downregulation of TrkB.T1 at the protein level can prolong BDNF-mediated protective signaling via the catalytic receptor and thus participate in the development of ischemic preconditioning.

Key words: Ischemic preconditioning; Oxygen-glucose deprivation; SMART cDNA synthesis; Subtractive suppression hybridization; TrkB.T1

Address correspondence to Axel Methner, Klinik und Poliklinik für Neurologie, Universitätsklinikum Hamburg-Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany. E-mail: methner@uke.uni-hamburg.de




Gene Expression, Vol. 12, pp. 107-121
1052-2166/05 $20.00 + .00
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Exercise-Induced Gene Expression Changes in the Rat Spinal Cord

Victoria M. Perreau,1 Paul A. Adlard,1 Aileen J. Anderson,2 and Carl W. Cotman1

1Institute for Brain Aging and Dementia, 1113 Gillespie N.R.F., University of California Irvine, Irvine, CA
2Department of Physical Medicine and Rehabilitation, 1107 Gillespie N.R.F., University of California Irvine, Irvine, CA

There is growing evidence that exercise benefits recovery of neuromuscular function from spinal cord injury (SCI). However, the effect of exercise on gene expression in the spinal cord is poorly understood. We used oligonucleotide microarrays to compare thoracic and lumbar regions of spinal cord of either exercising (voluntary wheel running for 21 days) or sedentary rats. The expression data were filtered using statistical tests for significance, and K-means clustering was then used to segregate lists of significantly changed genes into sets based upon expression patterns across all experimental groups. Levels of brain-derived neurotrophic factor (BDNF) protein were also measured after voluntary exercise, across different regions of the spinal cord. BDNF mRNA increased with voluntary exercise, as has been previously shown for other forms of exercise, contributed to by increases in both exon I and exon III. The exercise-induced gene expression changes identified by microarray analysis are consistent with increases in pathways promoting neuronal health, signaling, remodeling, cellular transport, and development of oligodendrocytes. Taken together these data suggest cellular pathways through which exercise may promote recovery in the SCI population.

Key words: Exercise; Gene expression; Brain-derived neurotrophic factor (BDNF); Microarray; Rat; Spinal cord

Address correspondence to Victoria Perreau, Ph.D., Institute for Brain Aging and Dementia, 1113 Gillespie N.R.F., University of California, Irvine, Irvine, CA 92697-4540. Tel: (949) 824-6071; Fax: (949) 824-2071; E-mail: vperreau@uci.edu




Gene Expression, Vol. 12, pp. 123-136
1052-2166/05 $20.00 + .00
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Transcriptional Profile of NeuroD1 Expression in a Human Fetal Astroglial Cell Line

Siddharth G. Kamath,1 Ning Chen,2 Steve A. Enkemann,3 and Juan Sanchez-Ramos1,2,4

Departments of 1Neurology, 2Neurosurgery, 3Moffitt Cancer Center, and 4James Haley VA Hospital, University of South Florida College of Medicine, Tampa, FL 33612

NeuroD1, a member of the basic helix-loop-helix (bHLH) protein family, is a transcription factor that plays a pivotal role in terminal differentiation of neural progenitors. The primary objective was to generate an early transcriptional profile triggered by NeuroD1 to guide future studies on mechanisms of neuronal differentiation. The human NeuroD1 coding region was amplified from human fetal brain RNA using specific primers and cloned into a CMV expression vector (CT-GFP-TOPO/pcDNA3.1). Transfection of a fetal glial cell line with this construct resulted in expression of NeuroD1 in 13-15% of the cells. Markers typical of early neuronal development were observed by immunocytochemical staining in a small proportion of transfected cells. To enrich the population of NeuroD1-expressing cells, fluorescence-activated cell sorting (FACS) was used to purify and collect the NeuroD1/GFP+ cells. Total RNA was extracted from the pair of cultures (NeuroD1/GFP vs. control plasmid/GFP) and processed for gene expression studies. A final gene list was composed from those probe sets that were either increased or decreased in the NeuroD1-expressing cells in three independent experiments (p < 0.001). Each gene was investigated further for possible roles in neurogenesis and a subset of 177 genes was chosen based on the following characteristics: a) genes that are potential NeuroD1 dimerization partners, b) genes that modulate other bHLH transcription factors, c) genes related to development, and d) genes associated with neural induction, outgrowth, and terminal differentiation. DNA microarray analysis of NeuroD1 expression in an astroglial cell line produced a "snapshot" transcriptional profile that will be useful in deciphering the complex molecular code that specifies a neuronal fate.

Key words: Transcription factors; Neuronal differentiation; Gene expression

Address correspondence to J. R. Sanchez-Ramos, Ph.D., M.D., Department of Neurology MDC 55, University of South Florida, 12901 Bruce B. Downs Blvd., Tampa, FL 33620. Tel: (813) 974-5841; E-mail: jsramos@hsc.usf.edu