Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 36
Filter
Add more filters











Publication year range
1.
Neuroscience ; 274: 229-41, 2014 Aug 22.
Article in English | MEDLINE | ID: mdl-24881576

ABSTRACT

Zellweger syndrome (ZS) is a severe peroxisomal disorder caused by mutations in peroxisome biogenesis, or PEX, genes. A central hallmark of ZS is abnormal neuronal migration and neurodegeneration, which manifests as widespread neurological dysfunction. The molecular basis of ZS neuropathology is not well understood. Here we present findings using a mouse model of ZS neuropathology with conditional brain inactivation of the PEX13 gene. We demonstrate that PEX13 brain mutants display changes that reflect an abnormal serotonergic system - decreased levels of tryptophan hydroxylase-2, the rate-limiting enzyme of serotonin (5-hydroxytryptamine, 5-HT) synthesis, dysmorphic 5-HT-positive neurons, abnormal distribution of 5-HT neurons, and dystrophic serotonergic axons. The raphe nuclei region of PEX13 brain mutants also display increased levels of apoptotic cells and reactive, inflammatory gliosis. Given the role of the serotonergic system in brain development and motor control, dysfunction of this system would account in part for the observed neurological changes of PEX13 brain mutants.


Subject(s)
Brain/pathology , Serotonergic Neurons/pathology , Zellweger Syndrome/pathology , Animals , Apoptosis , Axons/metabolism , Axons/pathology , Brain/metabolism , Cell Count , Disease Models, Animal , Fluorescent Antibody Technique , Gliosis/metabolism , Gliosis/pathology , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice, Transgenic , Mutation , Neuroimmunomodulation/physiology , Peroxisomes/metabolism , Serotonergic Neurons/metabolism , Tryptophan Hydroxylase/deficiency , Zellweger Syndrome/metabolism
2.
DNA Seq ; 12(1): 59-65, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11697145

ABSTRACT

A full length cDNA for the beta subunit of chick (Gallus gallus) eukaryotic translation initiation factor-2 is described. This cDNA was isolated by screening a chick cDNA library with a probe derived via differential display of developing chick heart tissue. Up-regulated expression of eIF-2 beta mRNA was confirmed by reverse Northern dot blot analysis. eIF-2 beta, together with eIF-2 alpha and eIF-2 gamma, comprise subunits of a complex that promotes the binding of methionyl-tRNA to ribosomes during the initiation of protein translation. The nucleotide sequence of the chick eIF-2 beta cDNA predicts a protein of 334 amino acids that has 95%, 93%, 56% and 37% sequence identity with rabbit, human, drosophila and yeast eIF-2 beta, respectively. The deduced eIF-2 beta protein contains a number of functional motifs and domains consistent with the putative function of this protein; these include a potential C2-C2 zinc-finger binding domain, three polylysine regions, and three acidic regions.


Subject(s)
DNA, Complementary/genetics , Eukaryotic Initiation Factor-2/genetics , Amino Acid Sequence , Animals , Base Sequence , Chick Embryo , Gene Expression Regulation, Developmental , Heart/embryology , Molecular Sequence Data , Sequence Homology, Amino Acid , Up-Regulation
3.
J Neurochem ; 78(3): 424-34, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11483645

ABSTRACT

Antibodies were raised against specific peptides from N-terminal regions of the alpha1 and alpha3 isoforms of the GABA(A) receptor, and used to assess the relative expression of these proteins in the superior frontal and primary motor cortices of 10 control, nine uncomplicated alcoholic and six cirrhotic alcoholic cases were matched for age and post-mortem delay. The regression of expression on post-mortem delay was not statistically significant for either isoform in either region. In both cortical areas, the regression of alpha1 expression on age differed significantly between alcoholic cases, which showed a decrease, and normal controls, which did not. Age had no effect on alpha3 expression. The alpha1 and alpha3 isoforms were found to be expressed differentially across cortical regions and showed a tendency to be expressed differentially across case groups. In cirrhotic alcoholics, alpha1 expression was greater in superior frontal than in motor cortex, whereas this regional difference was not significant in controls or uncomplicated alcoholics. In uncomplicated alcoholics, alpha3 expression was significantly lower in superior frontal than in motor cortex. Expression of alpha1 was significantly different from that of alpha3 in the superior frontal cortex of alcoholics, but not in controls. In motor cortex, there were no significant differences in expression between the isoforms in any case group.


Subject(s)
Alcoholism/metabolism , Frontal Lobe/metabolism , Motor Cortex/metabolism , Peptides/metabolism , Receptors, GABA-A/metabolism , Adult , Aged , Aged, 80 and over , Aging , Amino Acid Sequence , Animals , Antibodies/immunology , Antibodies/metabolism , Blotting, Western , CHO Cells , Cricetinae , Female , Humans , Liver Cirrhosis, Alcoholic/metabolism , Male , Middle Aged , Molecular Sequence Data , Neurons/metabolism , Peptides/chemistry , Peptides/immunology , Protein Isoforms , Protein Subunits , Rabbits , Receptors, GABA-A/chemistry , Receptors, GABA-A/genetics , Receptors, GABA-A/immunology , Regression Analysis , Sequence Alignment , Transfection
4.
DNA Seq ; 11(1-2): 155-62, 2000.
Article in English | MEDLINE | ID: mdl-10902924

ABSTRACT

The RPB6 cDNA of chicken, encoding one of the small subunits common to all three nuclear DNA-dependent RNA polymerases, has been isolated from an expression cDNA library by screening with a differential display derived probe, representing a gene shown to be highly up-regulated in early heart development. The nucleotide sequence of the cDNA isolated predicts a protein sequence of 127 amino acids. This sequence shares 124 amino acids (98% homology) with the human RNA polymerase II subunit 14.4 kDa (RPB6) and hamster hRPB6 and 123 amino acids (97% homology) with Rattus norvegicus RNA polymerase II subunit RPB6. Other conserved motifs in this protein and potential functions of RPB6 are discussed.


Subject(s)
DNA-Directed RNA Polymerases/genetics , Amino Acid Sequence , Animals , Base Sequence , Chick Embryo , Conserved Sequence , Cricetinae , DNA, Complementary , Humans , Molecular Sequence Data , Rats , Sequence Homology, Amino Acid
5.
J Clin Endocrinol Metab ; 85(3): 1143-50, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10720053

ABSTRACT

We previously described significant changes in GH-binding protein (GHBP) in pathological human pregnancy. There was a substantial elevation of GHBP in cases ofnoninsulin-dependent diabetes mellitus and a reduction in insulin-dependent diabetes mellitus. GHBP has the potential to modulate the proportion of free placental GH (PGH) and hence the impact on the maternal GH/insulin-like growth factor I (IGF-I) axis, fetal growth, and maternal glycemic status. The present study was undertaken to investigate the relationship among glycemia, GHBP, and PGH during pregnancy and to assess the impact of GHBP on the concentration of free PGH. We have extended the analysis of specimens to include measurements of GHBP, PGH, IGF-I, IGF-II, IGF-binding protein-1 (IGFBP-1), IGFBP-2, and IGFBP-3 and have related these to maternal characteristics, fetal growth, and glycemia. The simultaneous measurement of GHBP and PGH has for the first time allowed calculation of the free component of PGH and correlation of the free component to indexes of fetal growth and other endocrine markers. PGH, free PGH, IGF-I, and IGF-II were substantially decreased in IUGR at 28-30 weeks gestation (K28) and 36-38 weeks gestation (K36). The mean concentration (+/-SEM) of total PGH increased significantly from K28 to K36 (30.0 +/- 2.2 to 50.7 +/- 6.2 ng/mL; n = 40), as did the concentration of free PGH (23.4 +/- 2.3 to 43.7 +/- 6.0 ng/mL; n = 38). The mean percentage of free PGH was significantly less in IUGR than in normal subjects (67% vs. 79%; P < 0.01). Macrosomia was associated with an increase in these parameters that did not reach statistical significance. Multiple regression analysis revealed that PGH/IGF-I and IGFBP-3 account for 40% of the variance in birth weight. IGFBP-3 showed a significant correlation with IGF-I, IGF-II, and free and total PGH at K28 and K36. Noninsulin-dependent diabetes mellitus patients had a lower mean percentage of free PGH (65%; P < 0.01), and insulin-dependent diabetics had a higher mean percentage of free PGH (87%; P < 0.01) than normal subjects. Mean postprandial glucose at K28 correlated positively with PGH and free PGH (consistent with the hyperglycemic action of GH). GHBP correlated negatively with both postprandial and fasting glucose. Although GHBP correlated negatively with PGH (r = -0.52; P < .001), free PGH and total PGH correlated very closely (r = 0.98). The results are consistent with an inhibitory function for GHBP in vivo and support a critical role for placental GH and IGF-I in driving normal fetal growth.


Subject(s)
Carrier Proteins/metabolism , Embryonic and Fetal Development/physiology , Fetal Growth Retardation/metabolism , Human Growth Hormone/metabolism , Placenta/metabolism , Pregnancy in Diabetics/metabolism , Somatomedins/metabolism , Adult , Birth Weight/physiology , Blood Glucose/metabolism , Diabetes Mellitus, Type 1/metabolism , Diabetes Mellitus, Type 2/metabolism , Female , Humans , Insulin-Like Growth Factor Binding Proteins/metabolism , Predictive Value of Tests , Pregnancy , Reference Values
6.
J Biol Chem ; 275(6): 4127-36, 2000 Feb 11.
Article in English | MEDLINE | ID: mdl-10660573

ABSTRACT

Pex5p, a receptor for peroxisomal matrix proteins with a type 1 peroxisome targeting signal (PTS1), has been proposed to cycle from the cytoplasm to the peroxisomal membrane where it docks with Pex14p and Pex13p, the latter an SH3 domain-containing protein. Using in vitro binding assays we have demonstrated that binding of Pex5p to Pex14p is enhanced when Pex5p is loaded with a PTS1-containing peptide. In contrast, Pex5p binding to Pex13p, which involves only the SH3 domain, occurs at 20-40-fold lower levels and is reduced when Pex5p is preloaded with a PTS1 peptide. Pex14p was also shown to bind weakly to the Pex13p SH3 domain. Site-directed mutagenesis of the Pex13p SH3 domain attenuated binding to Pex5p and Pex14p, consistent with both of these proteins being binding partners for this domain. The SH3 binding site in Pex5p was determined to lie within a 114-residue peptide (Trp(100)-Glu(213)) in the amino-terminal region of the protein. The interaction between this peptide and the SH3 domain was competitively inhibited by Pex14p. We interpret these data as suggesting that docking of the Pex5p-PTS1 protein complex at the peroxisome membrane occurs at Pex14p and that the Pex13p SH3 domain functions as an associated component possibly involved in sequestering Pex5p after relinquishment of the PTS1 protein cargo to components of the translocation machinery.


Subject(s)
Carrier Proteins , Fungal Proteins/metabolism , Membrane Proteins/metabolism , Peroxisomes/metabolism , Protein Sorting Signals/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Repressor Proteins , Amino Acid Sequence , Binding Sites , Membrane Transport Proteins , Models, Molecular , Molecular Sequence Data , Mutation , Peroxins , Peroxisome-Targeting Signal 1 Receptor , Pichia , Protein Binding , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae Proteins , src Homology Domains/genetics
7.
J Biol Chem ; 274(48): 34277-82, 1999 Nov 26.
Article in English | MEDLINE | ID: mdl-10567403

ABSTRACT

The gene mutated in the human genetic disorder ataxia-telangiectasia codes for a protein, ATM, the known functions of which include response to DNA damage, cell cycle control, and meiotic recombination. Consistent with these functions, ATM is predominantly present in the nucleus of proliferating cells; however, a significant proportion of the protein has also been detected outside the nucleus in cytoplasmic vesicles. To understand the possible role of extra-nuclear ATM, we initially investigated the nature of these vesicles. In this report we demonstrate that a portion of ATM co-localizes with catalase, that ATM is present in purified mouse peroxisomes, and that there are reduced levels of ATM in the post-mitochondrial membrane fraction of cells from a patient with a peroxisome biogenesis disorder. Furthermore the use of the yeast two-hybrid system demonstrated that ATM interacts directly with a protein involved in the import of proteins into the peroxisome matrix. Because peroxisomes are major sites of oxidative metabolism, we investigated catalase activity and lipid hydroperoxide levels in normal and A-T fibroblasts. Significantly decreased catalase activity and increased lipid peroxidation was observed in several A-T cell lines. The localization of ATM to peroxisomes may contribute to the pleiotropic nature of A-T.


Subject(s)
Peroxisomes/chemistry , Protein Serine-Threonine Kinases/analysis , Amino Acid Sequence , Animals , Ataxia Telangiectasia/enzymology , Ataxia Telangiectasia/metabolism , Ataxia Telangiectasia/pathology , Ataxia Telangiectasia Mutated Proteins , Catalase/analysis , Catalase/genetics , Catalase/metabolism , Cell Cycle Proteins , Cell Line , Cell Nucleus/chemistry , DNA-Binding Proteins , Fibroblasts/chemistry , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Immunohistochemistry , Lipid Peroxides/metabolism , Male , Mice , Molecular Sequence Data , Peroxisomal Disorders/metabolism , Peroxisomal Disorders/pathology , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Tumor Suppressor Proteins , Two-Hybrid System Techniques
8.
Hum Genet ; 105(1-2): 38-44, 1999.
Article in English | MEDLINE | ID: mdl-10480353

ABSTRACT

Peroxisome biogenesis disorders are a heterogeneous group of human neurodegenerative diseases caused by peroxisomal metabolic dysfunction. At the molecular level, these disorders arise from mutations in PEX genes that encode proteins required for the import of proteins into the peroxisomal lumen. The Zellweger syndrome spectrum of diseases is a major sub-set of these disorders and represents a clinical continuum from Zellweger syndrome (the most severe) through neonatal adrenoleukodystrophy to infantile Refsum disease. The PEX1 gene, which encodes a cytoplasmic AAA ATPase, is the responsible gene in more than half of the Zellweger syndrome spectrum patients, and mutations in PEX1 can account for the full spectrum of phenotypes seen in these patients. In these studies, we have undertaken mutation analysis of PEX1 in skin fibroblast cell lines from Australasian Zellweger syndrome spectrum patients. A previously reported common PEX1 mutation that gives rise to a G843D substitution and correlates with the less severe disease phenotypes has been found to be present at high frequency in our patient cohort. We also report a novel PEX1 mutation that occurs at high frequency in Zellweger syndrome spectrum patients. This mutation produces a frameshift in exon 13, a change that leads to the premature truncation of the PEX1 protein. A Zellweger syndrome patient who was homozygous for this mutation and who survived for less than two months from birth had undetectable levels of PEX1 mRNA. This new common mutation therefore correlates with a severe disease phenotype. We have adopted procedures for the detection of this mutation for successful prenatal diagnosis.


Subject(s)
Frameshift Mutation , Glycoproteins/genetics , Membrane Proteins , Peroxisomal Disorders/genetics , Zellweger Syndrome/genetics , ATPases Associated with Diverse Cellular Activities , Acyltransferases/analysis , Acyltransferases/genetics , Alleles , Amino Acid Sequence , Base Sequence , Blotting, Northern , Cell Line , Child , DNA Mutational Analysis , Exons , Genetic Testing , Genotype , Glycoproteins/metabolism , Humans , Infant , Infant, Newborn , Microbodies/metabolism , Molecular Sequence Data , Phenotype , Prenatal Diagnosis , RNA, Messenger/analysis
9.
Am J Hum Genet ; 65(3): 621-34, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10441568

ABSTRACT

The peroxisome-biogenesis disorders (PBDs) are a genetically and phenotypically diverse group of diseases caused by defects in peroxisome assembly. One of the milder clinical variants within the PBDs is neonatal adrenoleukodystrophy (NALD), a disease that is usually associated with partial defects in the import of peroxisomal matrix proteins that carry the type 1 or type 2 peroxisomal targeting signals. Here, we characterize the sole representative of complementation group 13 of the PBDs, a patient with NALD (patient PBD222). Skin fibroblasts from patient PBD222 display defects in the import of multiple peroxisomal matrix proteins. However, residual matrix-protein import can be detected in cells from patient PBD222, consistent with the relatively mild phenotypes of the patient. PEX13 encodes a peroxisomal membrane protein with a cytoplasmically exposed SH3 domain, and we find that expression of human PEX13 restores peroxisomal matrix-protein import in cells from patient PBD222. Furthermore, these cells are homozygous for a missense mutation at a conserved position in the PEX13 SH3 domain. This mutation attenuated the activity of human PEX13, and an analogous mutation in yeast PEX13 also reduced its activity. The mutation was absent in >100 control alleles, indicating that it is not a common polymorphism. Previous studies have demonstrated extragenic suppression in the PBDs, but the phenotypes of patient PBD222 cells could not be rescued by expression of any other human PEX genes. Taken together, these results provide strong evidence that mutations in PEX13 are responsible for disease in patient PBD222 and, by extension, in complementation group 13 of the PBDs.


Subject(s)
Membrane Proteins/genetics , Mutation , Peroxisomal Disorders/genetics , Peroxisomal Disorders/metabolism , Amino Acid Substitution , Biological Transport , Biomarkers/analysis , Cell Line , Fibroblasts/metabolism , Fibroblasts/pathology , Fungal Proteins/genetics , Fungal Proteins/metabolism , Genetic Complementation Test , Humans , Infant, Newborn , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Microbodies/enzymology , Microbodies/metabolism , Peroxisomal Disorders/enzymology , Phenotype , Pichia/genetics , Polymorphism, Genetic/genetics , Skin , Transfection , src Homology Domains
10.
Genomics ; 54(3): 521-8, 1998 Dec 15.
Article in English | MEDLINE | ID: mdl-9878256

ABSTRACT

The peroxisome biogenesis disorders (PBDs) are a set of lethal genetic diseases characterized by peroxisomal metabolic deficiencies, multisystem abnormalities, mental retardation, and premature death. These disorders are genetically heterogeneous and are caused by mutations in genes, termed PEX genes, required for import of proteins into the peroxisomal matrix. We have previously reported the identification of human PEX13, the gene encoding the docking factor for the PTS1 receptor, or PEX5 protein. As such, mutations in PEX13 would be expected to abrogate peroxisomal protein import and result in PBD phenotypes. We report here the structure of the human PEX13 gene. PEX13 spans approximately 11 kb on chromosome 2 and contains four exons, one more than previously thought. The corrected PEX13 cDNA is predicted to encode a protein product with a molecular mass of 44,312 Da. We examined the ability of PEX13 expression to rescue the peroxisomal protein import defects of fibroblast cells representing all known PBD complementation groups. No complementation was observed, suggesting that this gene is not mutated in any set of existing patients. However, given that complementation group assignments have been determined for only a subset of PBD patients, it is possible that PEX13-deficient patients may exist at a low frequency within our existing PBD patient population or within ethnic groups underrepresented in our patient pool.


Subject(s)
Chromosomes, Human, Pair 2 , Membrane Proteins/genetics , Peroxisomal Disorders/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Genetic Complementation Test , Humans , Membrane Proteins/metabolism , Mice , Molecular Sequence Data
11.
Brain Res Brain Res Protoc ; 1(4): 347-56, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9384815

ABSTRACT

The GABAA receptor is the site of action of the inhibitory neurotransmitter GABA, as well as a number of pharmacologically important drugs such as benzodiazepines, barbiturates, and ethanol. The GABAA receptor is a pentameric complex composed of distinct polypeptides, which have been divided into five subunit classes on the basis of sequence homology. To date, 17 isoforms of the receptor have been identified and cloned in mammalian brain, and designated alpha 1-6, beta 1-4, gamma 1-4, delta and rho 1-2. In addition, several isoforms exist in alternatively spliced forms (for review see ref.). Studies on recombinant receptors have revealed that receptors constituted from different isoforms exhibit distinct pharmacological properties. For example, the alpha subunit class appears to be responsible for GABA enhancement of benzodiazepine binding. GABAA receptor function is modulated by benzodiazepine agonists such as flunitrazepam and diazepam, barbiturates, anaesthetics, neurosteroids, and ethanol. Chronic treatment of animals with many of these compounds can bring about profound changes in receptor expression and pharmacology. The RT/PCR assay described here was developed to quantify the alpha 1, alpha 2 and alpha 3 isoforms in the same assay. The amount of each isoform was quantified on the basis of a standard curve generated under identical PCR conditions to the target sequences. In this way it is possible to quantify multiple samples in each RT/PCR assay, thereby reducing inter-assay variability. The assay can be applied to quantify the expression of these isoforms in response to acute and chronic drug administration, or in particular disease states. Altered expression may reflect a corresponding change in protein synthesis, or an alteration of the subtype composition of GABAA receptor.


Subject(s)
Brain/metabolism , Polymerase Chain Reaction/methods , Receptors, GABA-A/metabolism , Humans , Isomerism , Transcription, Genetic
12.
Eur J Pharmacol ; 326(2-3): 265-72, 1997 May 20.
Article in English | MEDLINE | ID: mdl-9196280

ABSTRACT

The displacement of [3H]flunitrazepam by unlabelled flunitrazepam or zolpidem was used to assess the affinity and density of sub-types of GABA(A) receptors in the superior frontal and primary motor cortices of ten alcoholic, seven alcoholic-cirrhotic and ten matched control cases. The binding was best fitted by a model with a single site for flunitrazepam, but two sites for zolpidem. Neither the patients' age nor the post-mortem interval were significantly correlated with the affinity or density of any of the binding sites. The affinity of all ligands did not differ either between cortical regions or across case groups. Hence, the density of each binding site was analyzed at constant affinity. The densities of flunitrazepam and high-affinity zolpidem binding sites were invariant across cortical regions and case groups. Low-affinity zolpidem binding sites were significantly more dense in the frontal than in the motor cortex of alcoholic cases irrespective of cirrhosis, whereas this regional difference was not significant in control cases.


Subject(s)
Alcoholism/metabolism , Brain/metabolism , Liver Cirrhosis, Alcoholic/metabolism , Pyridines/metabolism , Receptors, GABA-A/metabolism , Adult , Aged , Aged, 80 and over , Binding Sites , Female , Flunitrazepam/metabolism , Frontal Lobe/metabolism , Humans , Linear Models , Male , Middle Aged , Motor Cortex/metabolism , Postmortem Changes , RNA, Messenger/biosynthesis , Up-Regulation , Zolpidem
13.
Brain Res ; 751(1): 102-12, 1997 Mar 14.
Article in English | MEDLINE | ID: mdl-9098573

ABSTRACT

The expression of the alpha 1, alpha 2 and alpha 3 isoforms of the GABAA receptor was studied in the superior frontal and motor cortices of 10 control, 10 uncomplicated alcoholic and 7 cirrhotic alcoholic cases matched for age and post-mortem delay. The assay was based on competitive RT/PCR using a single set of primers specific to the alpha class of isoform mRNA species, and was normalized against a synthetic cRNA internal standard. The assay was shown to be quantitative for all three isoform mRNA species. Neither the patient's age nor the post-mortem interval significantly affected the expression of any isoform in either cortical area. The profile of expression was shown to be significantly different between the case groups, particularly because alpha 1 expression was raised in both groups of alcoholics of controls. The two groups of alcoholics could be differentiated on the basis of regional variations in alpha 1 expression. In frontal cortex, alpha 1 mRNA expression was significantly increased when uncomplicated alcoholics were compared with control cases whereas alcoholic-cirrhotic cases were not significantly different from either controls or uncomplicated alcoholic cases. In the motor cortex, alpha 1 expression was elevated only when alcoholic-cirrhotic cases were compared with control cases. There was no significant difference between case groups or areas for any other isoform.


Subject(s)
Alcoholism/metabolism , Cerebral Cortex/chemistry , Receptors, GABA-A/chemistry , Receptors, GABA-A/genetics , Adult , Age Factors , Aged , Aged, 80 and over , Blotting, Southern , Cerebral Cortex/metabolism , DNA Primers , Female , Gene Expression/physiology , Humans , Isomerism , Male , Middle Aged , Oligonucleotide Probes , Polymerase Chain Reaction/methods , Polymerase Chain Reaction/standards , Postmortem Changes , RNA, Messenger/analysis , Reference Standards
14.
Neurochem Int ; 29(6): 677-84, 1996 Dec.
Article in English | MEDLINE | ID: mdl-9113136

ABSTRACT

Selective variations in cerebral GABAA receptor pharmacology and function are observed in experimental animals subjected to a number of alcohol-treatment and -withdrawal paradigms, and where human alcoholics with and without a range of concomitant diseases are compared with non-alcoholic cases. Recombination studies have shown that variations in GABAA receptor pharmacology and function can result from altering its subunit isoform composition. This commentary examines the rôle of subunit isoform expression in the response to long-term alcohol administration in animals, and in the pathogenesis of alcoholism-related brain damage in human cases.


Subject(s)
Alcoholism/metabolism , Brain Damage, Chronic/metabolism , Ethanol/adverse effects , Gene Expression Regulation/drug effects , Receptors, GABA-A/drug effects , Alcoholism/complications , Animals , Brain Damage, Chronic/etiology , Disease Models, Animal , Humans , Receptors, GABA-A/genetics
16.
J Cell Biol ; 135(1): 85-95, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8858165

ABSTRACT

Import of newly synthesized PTS1 proteins into the peroxisome requires the PTS1 receptor (Pex5p), a predominantly cytoplasmic protein that cycles between the cytoplasm and peroxisome. We have identified Pex13p, a novel integral peroxisomal membrane from both yeast and humans that binds the PTS1 receptor via a cytoplasmically oriented SH3 domain. Although only a small amount of Pex5p is bound to peroxisomes at steady state (< 5%), loss of Pex13p further reduces the amount of peroxisome-associated Pex5p by approximately 40-fold. Furthermore, loss of Pex13p eliminates import of peroxisomal matrix proteins that contain either the type-1 or type-2 peroxisomal targeting signal but does not affect targeting and insertion of integral peroxisomal membrane proteins. We conclude that Pex13p functions as a docking factor for the predominantly cytoplasmic PTS1 receptor.


Subject(s)
Cell Membrane/chemistry , Membrane Proteins/metabolism , Microbodies/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , src Homology Domains , Acetyl-CoA C-Acetyltransferase/metabolism , Amino Acid Sequence , Biological Transport , Catalase/metabolism , Cell Adhesion Molecules/metabolism , Cell Line , Cloning, Molecular , Cytoplasm/metabolism , Fibroblasts , Genes/genetics , Genes, Fungal/genetics , Humans , Intracellular Membranes/chemistry , Membrane Proteins/analysis , Membrane Proteins/genetics , Membrane Proteins/physiology , Microbodies/chemistry , Molecular Sequence Data , Peroxisomal Targeting Signal 2 Receptor , Peroxisome-Targeting Signal 1 Receptor , Pichia/genetics , Sequence Analysis, DNA
17.
Metab Brain Dis ; 11(1): 39-54, 1996 Mar.
Article in English | MEDLINE | ID: mdl-8815389

ABSTRACT

Synaptic plasma membranes were prepared from four cerebrocortical areas from six male Angora goats made chronically thiamine deficient (TD) by the administration of AmproliumTM (600-900 mg/kg daily for 38-44 d). Four male controls were matched for age (27-30 mo). Four different radioligands were used to characterise GABAA and Glu-RNMDA receptor binding sites. There were marked, localised and contrasting changes in motor cortex, with an increase in GABAA and a decrease in Glu-RNMDA binding site densities. Less clearcut changes of a similar nature were seen in visual cortex. There was no variation in the parameters of GABA-activated [3H]diazepam binding between cortical areas in control goats, but there was a reduction in the maximal response to GABA in all areas in TD goats. There were regional variations in glutamate-activated [3H]MK-801 binding in control goat brain, and a non-selectively reduced maximal response in TD. Alterations in these indices of GABA- and glutamate-mediated neurotransmission may underlie the neurological signs of acute thiamine deficiency in these animals.


Subject(s)
Brain Chemistry/physiology , Receptors, GABA-A/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Thiamine Deficiency/metabolism , Alcoholism/metabolism , Alcoholism/pathology , Amprolium , Animals , Brain Chemistry/drug effects , Diazepam/pharmacokinetics , Disease Models, Animal , Dizocilpine Maleate/pharmacokinetics , Excitatory Amino Acid Antagonists/pharmacokinetics , Female , Flunitrazepam/pharmacokinetics , GABA Agonists/pharmacology , GABA Modulators/pharmacokinetics , Goats , In Vitro Techniques , Male , Muscimol/pharmacology , Synaptic Membranes/metabolism , Synaptosomes/metabolism , Thiamine Deficiency/chemically induced , Thiamine Deficiency/pathology , gamma-Aminobutyric Acid/physiology
18.
J Clin Invest ; 97(3): 681-8, 1996 Feb 01.
Article in English | MEDLINE | ID: mdl-8609223

ABSTRACT

Pristanic acid oxidation measurements proved a reliable tool for assessing complementation in fused heterokaryons from patients with peroxisomal biogenesis defects. We, therefore, used this method to determine the complementation groups of patients with isolated defects in peroxisomal beta-oxidation. The rate of oxidation of pristanic acid was reduced in affected cell lines from all of the families with inherited defects in peroxisomal beta-oxidation, thus excluding the possibility of a defective acyl CoA oxidase. Complementation analyses indicated that all of the patients belonged to the same complementation group, which corresponded to cell lines with bifunctional protein defects. Phytanic acid oxidation was reduced in fibroblasts from some, but not all, of the patients. Plasma samples were still available from six of the patients. The ratio of pristanic acid to phytanic acid was elevated in all of these samples, as were the levels of saturated very long chain fatty acids (VLCFA). However, the levels of bile acid intermediates, polyenoic VLCFA, and docosahexaenoic acid were abnormal in only some of the samples. Pristanic acid oxidation measurements were helpful in a prenatal assessment for one of the families where previous experience had shown that cellular VLCFA levels were not consistently elevated in affected individuals.


Subject(s)
3-Hydroxyacyl CoA Dehydrogenases/deficiency , Enoyl-CoA Hydratase/deficiency , Fatty Acids/metabolism , Isomerases , Multienzyme Complexes/deficiency , Peroxisomal Disorders/diagnosis , Australia/epidemiology , Bile Acids and Salts/blood , Cells, Cultured , Fatty Acids/blood , Fibroblasts/metabolism , Genetic Complementation Test , Humans , Hybrid Cells , Microbodies/chemistry , Oxidation-Reduction , Peroxisomal Bifunctional Enzyme , Peroxisomal Disorders/epidemiology , Phytanic Acid/metabolism , Prenatal Diagnosis , Zellweger Syndrome/diagnosis
19.
Arch Biochem Biophys ; 321(2): 526-30, 1995 Aug 20.
Article in English | MEDLINE | ID: mdl-7544098

ABSTRACT

We have isolated and sequenced a cDNA which encodes 376 amino acids toward the carboxy-terminus, and encompassing the putative nucleotide binding fold, of PMP68 (mouse liver peroxisomal integral membrane protein of 68 kDa) the major integral membrane protein of mouse liver peroxisomes. The protein sequence predicted from this cDNA shows 97.9% amino acid identity to this same region of rat liver PMP70, a member of the ATP-binding cassette protein superfamily (K. Kamijo, S. Taketani, S. Yokota, T. Osumi, and T. Hashimoto, 1990, J. Biol. Chem. 265, 4534-4540). The section of the cDNA encoding the hydrophilic and putative cytoplasmic domain of PMP68 was expressed as a recombinant fusion protein in bacteria. Two monoclonal antibodies raised against this protein have been epitope-mapped to peptides generated by cyanogen bromide cleavage of the fusion protein. Antibody 1A4 recognizes a peptide whose sequence contains the first motif of the putative nucleotide binding fold of PMP68, and antibody 8F11 recognizes a carboxy-terminal peptide which includes the second motif of this nucleotide binding fold. These antibodies are expected to be useful in the elucidation of the biological function of this putative membrane transporter.


Subject(s)
ATP-Binding Cassette Transporters , Epitopes/analysis , Liver/metabolism , Membrane Proteins/biosynthesis , Membrane Proteins/chemistry , Amino Acid Sequence , Animals , Antibodies, Monoclonal , Base Sequence , Binding Sites , Chromatography, Gel , Chromatography, High Pressure Liquid , Cloning, Molecular , DNA, Complementary , Electrophoresis, Polyacrylamide Gel , Female , Immunoblotting , Membrane Proteins/metabolism , Mice , Mice, Mutant Strains , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/isolation & purification , Protein Folding , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism
20.
Mech Ageing Dev ; 82(1): 1-17, 1995 Jul 28.
Article in English | MEDLINE | ID: mdl-7475353

ABSTRACT

Actively transcribed genes are more susceptible to nuclease digestion, an observation suggested to reflect an altered state of chromatin organization. It has been hypothesized that exposure or sequestration of chromatin domains is a higher order gene regulatory mechanism. In order to test whether tissue lineage is organized by mechanisms at the level or chromatin structure, three cardiac phenotype-conferring genes (atrial natriuretic factor, myosin light chain-1-ventricular and alpha-tropomyosin) have been assessed for DNase 1 sensitivity in nuclei prepared from tissues of the developing guinea pig. These data have been related to the level of tissue mRNA expression of these genes to ascertain whether the exposed state of genes can occur when transcription is low or undetectable. Although this phenomenon was evident in some cases, the data were not consistent with mechanisms at the level of chromatin structure directing tissue type.


Subject(s)
Chromatin/genetics , Chromatin/metabolism , Gene Expression , Heart/physiology , Aging/metabolism , Animals , Animals, Newborn , Atrial Natriuretic Factor/genetics , Deoxyribonuclease I/metabolism , Fetus/metabolism , Guinea Pigs , Heart/embryology , Heart Atria , Heart Ventricles , Liver/metabolism , Muscle, Skeletal/embryology , Muscle, Skeletal/metabolism , Myocardium/metabolism , Myosin Light Chains/genetics , Protein Precursors/genetics , RNA, Messenger/metabolism , Tropomyosin/genetics
SELECTION OF CITATIONS
SEARCH DETAIL