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8. hExo70, a subunit of the exocyst complex – a possible link between vesicular transport, pre-mRNA splicing and aging?
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Abstracts / Experimental Gerontology 44 (2009) 126–134
Ames dwarf mice and their control siblings were used. The expression of clock genes: BMAL-1, CLOCK, PER1 and 2 and CRY 1 and 2 were studied in the hypothalamic pituitary axis. Gene expression was measured by real time PCR. No differences in CLOCK and BMAL-1 gene expression were observed in either control or dwarf mice with age at the hypothalamic level. However, aging reduced PER1 and 2 and CRY 1 and 2 in either dwarf and control mice. No differences between dwarf and control animals, in any of the clock genes analyzed, were observed. At the pituitary level, the expression of all clock genes was increased in dwarf as compared to control siblings. There was a decrease in the expression of all clock genes with age in both control and dwarf mice, with the exception of PER2 that was not modified. These data may suggest that the increased expression of all clock genes in the dwarf mice, at the pituitary level, could be related at least in part with the longer life-spam in this group. doi:10.1016/j.exger.2008.08.020
7. Comparative effects of curcumin on C6 rat glioma and T98G human glioblastoma cells T. Chang, H. Panchal, A.M. Gouw, T. Kuo, P.S. Timiras Department of Molecular and Cell Biology, University of California, Berkeley, CA, 94720, USA Curcumin from the dietary spice turmeric Curcuma longa, is the biologically active ingredient with antioxidant, anti-inflammatory, and anti-proliferative properties. These beneficial actions of curcumin may provide preventative treatment for various neurodegenerative diseases such as Alzheimer’s and Parkinson’s Diseases. Previous dose-effect studies have shown 5 lM of curcumin to be the effective dose for reducing cell proliferation with minimal toxicity on C6 rat glioma cells, which contains a mixture of astrocytes and oligodendrocytes. These neuroglia cells have been studied extensively for their close relationship with different neurologic conditions and their important roles in brain function such as neurotransmission (astrocytes) and neuronal myelination (oligodendrocytes). Furthermore, DNA microarrays show considerable plasticity of neuroglia cells with up- and down-regulation of several glial cell constituents. For example, a three fold increase in neurofilament M, a neuronal marker, in curcumin-treated cells after 48 h, suggests possible transdifferentiation of neuroglia cells into neuronal progenitor cells. Current study was undertaken to investigate whether similar effects of curcumin apply to human neuroglia. T98G human glioblastoma cells were chosen for this purpose due to their similarity in cell population composition when compared to C6 rat glioma. Growth curves were constructed which showed that 5 lM concentration of curcumin reduces proliferation after 4 days of treatment in T98G human glioblastoma when compared to 2 days in C6 rat glioma, suggesting delayed sensitivity of human neuroglia to curcumin. Both cell lines showed an increase in GFAP (an intermediate filament protein found in astrocytes) fluorescence through fluorescent microscopy after treatment with curcumin, confirming previous studies from our laboratory that suggest the ability of curcumin to activate glial cell maturation. This study is supported by NIH Grant AG 19145-05 and BioTime, Inc. doi:10.1016/j.exger.2008.08.021
8. hExo70, a subunit of the exocyst complex – a possible link between vesicular transport, pre-mRNA splicing and aging? H. Dellago a, J. Grillari a, Marlies Löscher a, P. Ajuh b, G. Ritter a, F. Eisenhaber c, A. Lamond b, H. Katinger a
a
Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria b University of Dundee, Scotland, UK c Institute of Molecular Pathology, Vienna, Austria The exocyst is a complex consisting of eight subunits and involved in protein targeting to plasma membrane domains. So far only cytoplasmic localization of the exocyst has been reported. We have previously shown that one of its subunits, hExo70, interacts directly with the nuclear splicing factor SNEV, a protein which is implicated in regulation of the replicative life-span of human endothelial cells. The interaction domain was mapped to the N-terminal 100 amino acids of hExo70, that presumably form a coiled coil (CC) domain. Addition of this domain alone to in vitro splicing reactions resulted in inhibition of pre-mRNA splicing, while equimolar amounts of hExo70 or a mutant lacking the CC domain (CC-hExo70) did not affect the reaction. In addition, in vivo splicing assays in Hela cells indicate that hExo70 changes the splicing pattern of the E1A minigene, verifying its role as splicing factor. During our investigations we found that hExo70 itself has differentially spliced isoforms and that the distribution of its splicing variants is cell type dependent. These results suggest a novel function of the exocyst in splicing, a hypothesis that is supported by the fact that we found at least one other exocyst subunit, Sec6, in the purified, assembled spliceosome. The influence of hExo70 on life span regulation in Saccharomyces cerevisiae and Drosophila melanogaster is currently being investigated. doi:10.1016/j.exger.2008.08.022
9. Aging and environmental enrichment modify plasma prolactin and corticosterone levels as well as the immune response of submaxillary lymph nodes M.P. Fernández-Mateos a, G. Segovia b, V. Jimenez c, A. Del Arco b, J. Ríos-Lugos c, F. Mora b, A.I. Esquifino c a
Departamento de Biología Celular, Facultad de Medicina, Universidad Complutense, Madrid, Spain b Departamento de Fisiologaí y Facultad de Medicina, Universidad Complutense, Madrid, Spain c Departamento de Bioquímica y Biología Molecular III, Facultad de Medicina, Universidad Complutense, Madrid, Spain The effects of environmental enrichment on age changes in plasma prolactin, corticosterone and distribution and activity of T and B lymphocytes in submaxillary lymph nodes were determined. Three months old male rats were housed in control (1 animal per cage) or enriched (10–12 animals in large cages containing running wheels, plastic tunnels and toys) conditions up to the age of 6, 15 and 24 months. Hormones were measured by specific RIAs. Lymphocyte subsets were measured by FACSCAN and lymphocyte proliferation by tymidine incorporation in culture. Plasma prolactin and corticosterone levels that increased with age in controls were significantly decreased by environmental enrichment. Percentage of CD4+ and T lymphocytes did not change with age and were not modified by environmental enrichment as happened with CD8+ lymphocytes that decreased with age. However, B lymphocytes increased with age and were not modified by environmental enrichment. Proliferative capacity of T lymphocytes was not modified by age in the control group nor in environmental enrichmed rats. However, proliferative capacity of B lymphocytes was normalized in the oldest group by environmental enrichment. These data suggest that environmental
Abstracts / Experimental Gerontology 44 (2009) 126–134
Ames dwarf mice and their control siblings were used. The expression of clock genes: BMAL-1, CLOCK, PER1 and 2 and CRY 1 and 2 were studied in the hypothalamic pituitary axis. Gene expression was measured by real time PCR. No differences in CLOCK and BMAL-1 gene expression were observed in either control or dwarf mice with age at the hypothalamic level. However, aging reduced PER1 and 2 and CRY 1 and 2 in either dwarf and control mice. No differences between dwarf and control animals, in any of the clock genes analyzed, were observed. At the pituitary level, the expression of all clock genes was increased in dwarf as compared to control siblings. There was a decrease in the expression of all clock genes with age in both control and dwarf mice, with the exception of PER2 that was not modified. These data may suggest that the increased expression of all clock genes in the dwarf mice, at the pituitary level, could be related at least in part with the longer life-spam in this group. doi:10.1016/j.exger.2008.08.020
7. Comparative effects of curcumin on C6 rat glioma and T98G human glioblastoma cells T. Chang, H. Panchal, A.M. Gouw, T. Kuo, P.S. Timiras Department of Molecular and Cell Biology, University of California, Berkeley, CA, 94720, USA Curcumin from the dietary spice turmeric Curcuma longa, is the biologically active ingredient with antioxidant, anti-inflammatory, and anti-proliferative properties. These beneficial actions of curcumin may provide preventative treatment for various neurodegenerative diseases such as Alzheimer’s and Parkinson’s Diseases. Previous dose-effect studies have shown 5 lM of curcumin to be the effective dose for reducing cell proliferation with minimal toxicity on C6 rat glioma cells, which contains a mixture of astrocytes and oligodendrocytes. These neuroglia cells have been studied extensively for their close relationship with different neurologic conditions and their important roles in brain function such as neurotransmission (astrocytes) and neuronal myelination (oligodendrocytes). Furthermore, DNA microarrays show considerable plasticity of neuroglia cells with up- and down-regulation of several glial cell constituents. For example, a three fold increase in neurofilament M, a neuronal marker, in curcumin-treated cells after 48 h, suggests possible transdifferentiation of neuroglia cells into neuronal progenitor cells. Current study was undertaken to investigate whether similar effects of curcumin apply to human neuroglia. T98G human glioblastoma cells were chosen for this purpose due to their similarity in cell population composition when compared to C6 rat glioma. Growth curves were constructed which showed that 5 lM concentration of curcumin reduces proliferation after 4 days of treatment in T98G human glioblastoma when compared to 2 days in C6 rat glioma, suggesting delayed sensitivity of human neuroglia to curcumin. Both cell lines showed an increase in GFAP (an intermediate filament protein found in astrocytes) fluorescence through fluorescent microscopy after treatment with curcumin, confirming previous studies from our laboratory that suggest the ability of curcumin to activate glial cell maturation. This study is supported by NIH Grant AG 19145-05 and BioTime, Inc. doi:10.1016/j.exger.2008.08.021
8. hExo70, a subunit of the exocyst complex – a possible link between vesicular transport, pre-mRNA splicing and aging? H. Dellago a, J. Grillari a, Marlies Löscher a, P. Ajuh b, G. Ritter a, F. Eisenhaber c, A. Lamond b, H. Katinger a
a
Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria b University of Dundee, Scotland, UK c Institute of Molecular Pathology, Vienna, Austria The exocyst is a complex consisting of eight subunits and involved in protein targeting to plasma membrane domains. So far only cytoplasmic localization of the exocyst has been reported. We have previously shown that one of its subunits, hExo70, interacts directly with the nuclear splicing factor SNEV, a protein which is implicated in regulation of the replicative life-span of human endothelial cells. The interaction domain was mapped to the N-terminal 100 amino acids of hExo70, that presumably form a coiled coil (CC) domain. Addition of this domain alone to in vitro splicing reactions resulted in inhibition of pre-mRNA splicing, while equimolar amounts of hExo70 or a mutant lacking the CC domain (CC-hExo70) did not affect the reaction. In addition, in vivo splicing assays in Hela cells indicate that hExo70 changes the splicing pattern of the E1A minigene, verifying its role as splicing factor. During our investigations we found that hExo70 itself has differentially spliced isoforms and that the distribution of its splicing variants is cell type dependent. These results suggest a novel function of the exocyst in splicing, a hypothesis that is supported by the fact that we found at least one other exocyst subunit, Sec6, in the purified, assembled spliceosome. The influence of hExo70 on life span regulation in Saccharomyces cerevisiae and Drosophila melanogaster is currently being investigated. doi:10.1016/j.exger.2008.08.022
9. Aging and environmental enrichment modify plasma prolactin and corticosterone levels as well as the immune response of submaxillary lymph nodes M.P. Fernández-Mateos a, G. Segovia b, V. Jimenez c, A. Del Arco b, J. Ríos-Lugos c, F. Mora b, A.I. Esquifino c a
Departamento de Biología Celular, Facultad de Medicina, Universidad Complutense, Madrid, Spain b Departamento de Fisiologaí y Facultad de Medicina, Universidad Complutense, Madrid, Spain c Departamento de Bioquímica y Biología Molecular III, Facultad de Medicina, Universidad Complutense, Madrid, Spain The effects of environmental enrichment on age changes in plasma prolactin, corticosterone and distribution and activity of T and B lymphocytes in submaxillary lymph nodes were determined. Three months old male rats were housed in control (1 animal per cage) or enriched (10–12 animals in large cages containing running wheels, plastic tunnels and toys) conditions up to the age of 6, 15 and 24 months. Hormones were measured by specific RIAs. Lymphocyte subsets were measured by FACSCAN and lymphocyte proliferation by tymidine incorporation in culture. Plasma prolactin and corticosterone levels that increased with age in controls were significantly decreased by environmental enrichment. Percentage of CD4+ and T lymphocytes did not change with age and were not modified by environmental enrichment as happened with CD8+ lymphocytes that decreased with age. However, B lymphocytes increased with age and were not modified by environmental enrichment. Proliferative capacity of T lymphocytes was not modified by age in the control group nor in environmental enrichmed rats. However, proliferative capacity of B lymphocytes was normalized in the oldest group by environmental enrichment. These data suggest that environmental