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1.
Neuron ; 31(6): 913-27, 2001 Sep 27.
Article in English | MEDLINE | ID: mdl-11580893

ABSTRACT

Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant disorder caused by a CAG repeat expansion. To determine the mechanism of neurotoxicity, we produced transgenic mice and observed a cone-rod dystrophy. Nuclear inclusions were present, suggesting that the disease pathway involves the nucleus. When yeast two-hybrid assays indicated that cone-rod homeobox protein (CRX) interacts with ataxin-7, we performed further studies to assess this interaction. We found that ataxin-7 and CRX colocalize and coimmunoprecipitate. We observed that polyglutamine-expanded ataxin-7 can dramatically suppress CRX transactivation. In SCA7 transgenic mice, electrophoretic mobility shift assays indicated reduced CRX binding activity, while RT-PCR analysis detected reductions in CRX-regulated genes. Our results suggest that CRX transcription interference accounts for the retinal degeneration in SCA7 and thus may provide an explanation for how cell-type specificity is achieved in this polyglutamine repeat disease.


Subject(s)
Cell Nucleus/metabolism , Homeodomain Proteins/antagonists & inhibitors , Nerve Tissue Proteins/physiology , Nuclear Proteins/physiology , Peptides/chemistry , Trans-Activators/antagonists & inhibitors , Trinucleotide Repeats , Age Factors , Animals , Ataxin-7 , Cell Line , Cell Nucleus/ultrastructure , Disease Models, Animal , Electroretinography , Eye Proteins/chemistry , Eye Proteins/genetics , Eye Proteins/physiology , Gene Expression Profiling , Genes, Synthetic , Homeodomain Proteins/physiology , Humans , Macromolecular Substances , Mice , Mice, Transgenic , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/deficiency , Nerve Tissue Proteins/genetics , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Photoreceptor Cells, Vertebrate/metabolism , Prions/genetics , Promoter Regions, Genetic , Protein Binding , Retinal Degeneration/genetics , Retinal Degeneration/metabolism , Spinocerebellar Ataxias/genetics , Spinocerebellar Ataxias/metabolism , Synaptic Transmission , Trans-Activators/physiology , Transcriptional Activation , Transfection , Transgenes , Two-Hybrid System Techniques
2.
Cytogenet Cell Genet ; 93(1-2): 117-23, 2001.
Article in English | MEDLINE | ID: mdl-11474193

ABSTRACT

The synuclein family of proteins is a group of primarily brain-expressed polypeptides that show a high degree of amino acid conservation. alpha-Synuclein is the best known of the synuclein family, as it is a major component of the Lewy body, a cytoplasmic inclusion characteristic of Parkinson's disease as well as a variety of related neurodegenerative disorders. With the discovery that mutations in alpha-synuclein can cause Parkinson's disease, a potential role for the other synuclein family members in neurodegenerative disease is being considered. beta-Synuclein in particular may deserve special attention, as it is co-expressed with alpha-synuclein at presynaptic nerve terminals, is subject to phosphorylation by Ca(2+) calmodulin protein kinase II, appears important for neural plasticity, and forms aggregates in the brains of patients with Parkinson's disease and a related disorder. To facilitate study of beta-synuclein, we have cloned the mouse beta-synuclein gene (Sncb) and determined its genomic organization, size, and intron-exon structure. Using an interspecific backcross mapping panel from The Jackson Laboratory, we were then able to localize Sncb to chromosome 13 at the MGD 35.0 cM position. Like the human beta-synuclein gene, Sncb appears to consist of six exons separated by five introns. Unlike the human beta-synuclein gene, the mouse ortholog possesses a variant GC 5' splice donor sequence at the exon 4 - intron 4 boundary in a highly conserved splice junction consensus. Northern blot analysis and Western blot analysis both indicate that Sncb is highly expressed in the brain. Knowledge of the genomic organization and expression pattern of Sncb will allow functional studies of its potential role in neurodegeneration to commence in the mouse.


Subject(s)
Exons/genetics , Gene Expression Profiling , Introns/genetics , Nerve Tissue Proteins/genetics , Neurodegenerative Diseases/genetics , Physical Chromosome Mapping , Amino Acid Sequence , Animals , Base Sequence , Blotting, Western , Central Nervous System/metabolism , Crosses, Genetic , Female , Male , Mice , Molecular Sequence Data , Nerve Tissue Proteins/metabolism , Organ Specificity , RNA, Messenger/genetics , RNA, Messenger/metabolism , Restriction Mapping , Sequence Alignment , Synucleins , alpha-Synuclein , beta-Synuclein
3.
Anesthesiology ; 92(1): 156-63, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10638912

ABSTRACT

BACKGROUND: High-dose intravenously administered methylprednisolone has been shown to improve outcome after spinal cord injury. The resultant glucocorticoid-induced immunosuppression, however, results in multiple complications including sepsis, pneumonia, and wound infection. These complications could be reduced by techniques that increase the spinal bioavailability of intravenously administered methylprednisolone while simultaneously decreasing plasma bioavailability. This study aimed to characterize the spinal and plasma bioavailability of methylprednisolone after intravenous and intrathecal administration and to identify barriers to the distribution of methylprednisolone from plasma into spinal cord. METHODS: The spinal and plasma pharmacokinetics of intravenous (30-mg/kg bolus dose plus 5.4 mg x kg(-1) x h(-1)) and intrathecal (1-mg/kg bolus dose plus 1 mg x kg(-1) x h(-1)) methylprednisolone infusions were compared in pigs. In addition, wild-type mice and P-glycoprotein knockout mice were used to determine the role of P-glycoprotein in limiting spinal bioavailability of methylprednisolone. RESULTS: Despite the greater intravenous dose, concentrations of methylprednisolone in pig spinal cord were far higher and plasma concentrations much lower after intrathecal administration. After intraperitoneal administration in the mouse, the concentrations of methylprednisolone in muscle were not different between mice expressing P-glycoprotein (2.39 +/- 1.79 microg/g) and those lacking P-glycoprotein (2.83 +/- 0.46 microg/g). In contrast, methylprednisolone was undetectable in spinal cords of wild-type mice, whereas concentrations in spinal cords of P-glycoprotein-deficient mice were similar to those in skeletal muscle (2.83 +/- 0.27 microg/g). CONCLUSIONS: These pig studies demonstrate that the spinal cord bioavailability of methylprednisolone is poor after intravenous administration. The studies in knockout mice suggest that this poor bioavailability results from P-glycoprotein-mediated exclusion of methylprednisolone from the spinal cord.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/physiology , Glucocorticoids/administration & dosage , Glucocorticoids/pharmacokinetics , Methylprednisolone/administration & dosage , Methylprednisolone/pharmacokinetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Animals , Biological Availability , Chromatography, High Pressure Liquid , Female , Glucocorticoids/blood , Infusions, Intravenous , Injections, Spinal , Male , Methylprednisolone/blood , Mice , Mice, Knockout , Microdialysis , Species Specificity , Swine , Tissue Distribution
4.
Biochem Biophys Res Commun ; 187(1): 152-7, 1992 Aug 31.
Article in English | MEDLINE | ID: mdl-1520296

ABSTRACT

The polymerase chain reaction was used to amplify a novel fucosyltransferase cDNA (FucT-VI) from A431 and from HL60 cells. The amplified cDNA has a high degree of sequence identity to FucT-V and to FucT-III, and a much lower level of similarity to FucT-IV. Transfection of the FucT-VI gene into mammalian cells confers alpha-1,3 fucosyltransferase activity to the cells, resulting in cell surface expression of Lewis x and sialyl-Lewis x carbohydrates. In contrast to FucT-IV activity, FucT-VI catalyzes the transfer of fucose from GDP-beta-fucose to alpha-2,3 sialylated substrates. The substrate specificity of the FucT-VI gene product suggests that FucT-VI may be an enzyme involved in the biosynthesis of the E-Selectin ligand, sialyl-Lewis x, in myeloid cells.


Subject(s)
Cloning, Molecular , Fucosyltransferases/genetics , Gene Expression , Isoenzymes/genetics , Amino Acid Sequence , Base Sequence , Cell Line , DNA/chemistry , DNA/genetics , Fucosyltransferases/chemistry , Fucosyltransferases/metabolism , Humans , Isoenzymes/chemistry , Isoenzymes/metabolism , Molecular Sequence Data , Polymerase Chain Reaction , Transfection
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