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Biomedical subjects

David A Pearce

Publications and source records attributed to David A Pearce.

13 recordsLinked to original sources
PubMed2003

The yeast model for Batten disease: a role for Btn2p in the trafficking of the Golgi-associated vesicular targeting protein, Yif1p.

Btn2p is a novel coiled coil cytosolic protein in Saccharomyces cerevisiae. We report that Btn2p interacts with Yif1p, a component of a protein complex at the Golgi that functions in ER to Golgi transport. Deletion of Btn2p, btn2-delta, results in mis-localiztion of Yif1p to the vacuole. Therefore, Btn2p may have an apparent role in intracellular trafficking of proteins. Btn2p was originally identified as being up-regulated in a btn1-delta strain, which exhibits dysregulation of vacuolar pH, and this up-regulation of Btn2p was presumed to contribute to maintaining a stable vacuolar pH [Pearce et al. Nat. Genet. 22 (1999) 55]. We propose that up-regulation of Btn2p in btn1-delta is an indicator of altered trafficking within the cell, and as btn1-delta serves as a model for the lysosomal storage disorder Batten disease, that altered intracellular trafficking may contribute to some of the cellular pathological hallmarks of this disease.

Adaptor Proteins, Vesicular Transport↗
PubMed2003

Early changes in gene expression in two models of Batten disease.

Infantile and juvenile neuronal ceroid lipofuscinosis (NCLs) are progressive neurodegenerative disorders of childhood with distinct ages of clinical onset, but with a similar pathological outcome. Infantile and juvenile NCL are inherited in an autosomal recessive manner due to mutations in the CLN1 and CLN3 genes, respectively. Recently developed Cln1- and Cln3-knockout mouse models share similarities in pathology with the respective human disease. Using oligonucleotide arrays we identified reproducible changes in gene expression in the brains of both 10-week-old Cln1- and Cln3-knockout mice as compared to wild-type controls, and confirmed changes in levels of several of the cognate proteins by immunoblotting. Despite the similarities in pathology, the two mutations affect the expression of different, non-overlapping sets of genes. The possible significance of these changes and the pathological mechanisms underlying NCL diseases are discussed.

Animals↗
PubMed2003

Functional categorization of gene expression changes in the cerebellum of a Cln3-knockout mouse model for Batten disease.

Juvenile neuronal ceroid lipofuscinosis (JNCL or Batten Disease) is the most common progressive neurodegenerative disorder of childhood. The disease is inherited in an autosomal recessive manner and is the result of mutations in the CLN3 gene. One brain region severely affected in Batten disease is the cerebellum. Using a mouse model for Batten disease which shares pathological similarities to the disease in humans we have used oligonucleotide arrays to profile approximately 19000 mRNAs in the cerebellum. We have identified reproducible changes of twofold or more in the expression of 756 gene products in the cerebellum of 10-week-old Cln3-knockout mice as compared to wild-type controls. We have subsequently divided these genes with altered expression into 14 functional categories. We report a significant alteration in expression of genes associated with neurotransmission, neuronal cell structure and development, immune response and inflammation, and lipid metabolism. An apparent shift in metabolism toward gluconeogenesis is also evident in Cln3-knockout mice. Further experimentation will be necessary to understand the contribution of these changes in expression to a disease state. Detailed analysis of the functional consequences of altered expression of genes in the cerebellum of the Cln3-knockout mice may provide valuable clues in understanding the molecular basis of the pathological mechanisms underlying Batten disease.

Animals↗
PubMed2003

Prediction of biologically significant components from microarray data: Independently Consistent Expression Discriminator (ICED).

MOTIVATION: Class distinction is a supervised learning approach that has been successfully employed in the analysis of high-throughput gene expression data. Identification of a set of genes that predicts differential biological states allows for the development of basic and clinical scientific approaches to the diagnosis of disease. The Independent Consistent Expression Discriminator (ICED) was designed to provide a more biologically relevant search criterion during predictor selection by embracing the inherent variability of gene expression in any biological state. The four components of ICED include (i) normalization of raw data; (ii) assignment of weights to genes from both classes; (iii) counting of votes to determine optimal number of predictor genes for class distinction; (iv) calculation of prediction strengths for classification results. The search criteria employed by ICED is designed to identify not only genes that are consistently expressed at one level in one class and at a consistently different level in another class but identify genes that are variable in one class and consistent in another. The result is a novel approach to accurately select biologically relevant predictors of differential disease states from a small number of microarray samples. RESULTS: The data described herein utilized ICED to analyze the large AML/ALL training and test data set (Golub et al., 1999, Science, 286, 531-537) in addition to a smaller data set consisting of an animal model of the childhood neurodegenerative disorder, Batten disease, generated for this study. Both of the analyses presented herein have correctly predicted biologically relevant perturbations that can be used for disease classification, irrespective of sample size. Furthermore, the results have provided candidate proteins for future study in understanding the disease process and the identification of potential targets for therapeutic intervention.

Algorithms↗
PubMed2003

Optic nerve degeneration in a murine model of juvenile ceroid lipofuscinosis.

PURPOSE: To investigate optic nerve degeneration associated with CLN3 deficiency in a murine model of juvenile neuronal ceroid lipofuscinosis (Batten disease). METHODS: Using light and electron microscopy, the density and diameter of axons and the thickness of myelin in optic nerve were compared between age-matched cln3 knock-out (cln3-/-) and wild-type (129ev/TAC) mice. Western blot analysis was used to assay expression of Cln3 in mouse and primate retina and optic nerve. RESULTS: Morphologically identified mast cells were present in the meningeal sheaths surrounding the cln3-/- nerve and in the nerve itself. The cln3-/- optic nerve exhibited an overall loss of uniformity and integrity. Axon density in cln3-/- optic nerve was only 64% of that in wild-type optic nerve (P < 0.01). Accounting for differences in axon density, the diameter of axons in cln3-/- optic nerve was 1.2 times greater than in wild-type optic nerve (P < 0.01). Electron micrographs revealed large spaces between axons and 32% thinner myelin surrounding axons in cln3-/- mice than in wild type (P < 0.01). Western blot analysis demonstrated that Cln3 was expressed in retinas and optic nerves of mouse and primate. CONCLUSIONS: The presence of apparent mast cells in cln3-/- optic nerve suggests compromise of the blood-brain barrier. The absence of Cln3 causes loss of axons, axonal hypertrophy, and a reduction in myelination of retinal ganglion cells. Furthermore, expression of CLN3 in mouse and primate optic nerve links degeneration to loss of Cln3.

Animals↗
PubMed2002

An autoantibody inhibitory to glutamic acid decarboxylase in the neurodegenerative disorder Batten disease.

Mutations in the CLN3 gene are responsible for the neurodegenerative disorder Batten disease; however, the molecular basis of this disease remains unknown. In studying a mouse model for Batten disease, we report the presence of an autoantibody to glutamic acid decarboxylase (GAD65) in cln3-knockout mice serum that associates with brain tissue but is not present in sera or brain of normal mice. The autoantibody to GAD65 has the ability to inhibit the activity of glutamic acid decarboxylase. Furthermore, brains from cln3-knockout mice have decreased activity of glutamic acid decarboxylase as a result of the inhibition of this enzyme by the autoantibody, resulting in brain samples from cln3-knockout mice having elevated levels of glutamate as compared with normal. This elevated glutamate in the brain of cln3-knockout mice co-localizes with presynaptic markers. The decreased activity of GAD65 and increased levels of glutamate may have a causative role in astrocytic hypertrophy evident in cln3-knockout mice, and in altered expression of genes involved in the synthesis and utilization of glutamate that underlie a shift from synthesis to utilization of glutamate. An autoantibody to GAD65 is also present in sera of 20 out of 20 individuals tested who have Batten disease. Postmortem tissue shows decreased reactivity to an anti-GAD65 antibody that may be due to loss of GAD65-positive neurons or due to the reactive epitope being blocked by the presence of the autoantibody. We propose that an autoimmune response to GAD65 may contribute to a preferential loss of GABAergic neurons associated with Batten disease.

Adult↗
PubMed2002

Altered flurothyl seizure induction latency, phenotype, and subsequent mortality in a mouse model of juvenile neuronal ceroid lipofuscinosis/batten disease.

PURPOSE: Juvenile neuronal ceroid lipofuscinosis (JNCL), or Batten disease, is a pediatric neurodegenerative disease characterized by vision loss, seizure activity, cognitive decline, and premature death. Discovery of the Batten disease-related gene, CLN3, led to creation of a Cln3 protein-deficient mouse model (Cln3-/-), which recapitulates some of the histopathologic characteristics of the human condition. We hypothesized that lack of Cln3 would alter seizure-related behavioral parameters. METHODS: Using flurothyl gas inhalation, we examined seizure-induction latencies in Cln3-/- mice and wildtype (wt) controls at time points that represent late neonatal, immature, mature, and aged time points. We examined latency to first myoclonic jerk (LMJ), latency to loss of posture (LOP), and subsequent mortality. RESULTS: Our results demonstrate an age-dependent alteration of seizure-induction latencies in Cln3-/-. Immature Cln-/- mice aged 35-42 days had an increased latency to both LMJ and LOP compared with age-matched wt controls. There were no significant latency differences between Cln3-/- and wt at other time points examined. Mortality after generalized seizure was high in both Cln3-/- and wt animals at late neonatal and immature developmental stages. No mortality was seen in wt mice past maturity at 6 weeks. Mature and aged Cln3-/- animals retained a vulnerability to death after seizure activity. CONCLUSIONS: These results suggest that a deficiency of Cln3 protein in the Batten model mice may result in age-dependent alteration of the neuroanatomic and biochemical substrates involved in seizure propagation and recovery. This may be important in understanding seizures, neurodegeneration, and premature death in human Batten disease.

Administration, Inhalation↗
PubMed2002

Interaction with Btn2p is required for localization of Rsglp: Btn2p-mediated changes in arginine uptake in Saccharomyces cerevisiae.

Btn2p, a novel coiled-coil protein, is up-regulated in btn1delta yeast strains, and this up-regulation is thought to contribute to maintaining a stable vacuolar pH in btn1delta strains (D. A. Pearce, T. Ferea, S. A. Nosel, B. Das, and F. Sherman, Nat. Genet. 22:55-58, 1999). We now report that Btn2p interacts biochemically and functionally with Rsglp, a down-regulator of the Can1p arginine and lysine permease. Rsglp localizes to a distinct structure toward the cell periphery, and strains lacking Btn2p (btn2delta strains) fail to correctly localize Rsg1p. btn2delta strains, like rsg1delta strains, are sensitive for growth in the presence of the arginine analog canavanine. Furthermore, btn2delta strains, like rsg1delta strains, demonstrate an elevated rate of uptake of [14C]arginine, which leads to increased intracellular levels of arginine. Overexpression of BTN2 results in a decreased rate of arginine uptake. Collectively, these results indicate that altered levels of Btn2p can modulate arginine uptake through localization of the Can1p-arginine permease regulatory protein, Rsglp. Our original identification of Btn2p was that it is up-regulated in the btn1delta strain which serves as a model for the lysosomal storage disorder Batten disease. Btnlp is a vacuolar/lysosomal membrane protein, and btn1delta suppresses both the canavanine sensitivity and the elevated rate of uptake of arginine displayed by btn2delta rsg1delta strains. We conclude that Btn2p interacts with Rsglp and modulates arginine uptake. Up-regulation of BTN2 expression in btn1delta strains may facilitate either a direct or indirect effect on intracellular arginine levels.

Arginine↗
PubMed2002

The neuronal ceroid lipofuscinoses: mutations in different proteins result in similar disease.

The neuronal ceroid-lipofuscinoses (NCL) are the most common group of progressive neurodegenerative diseases in children, with an incidence as high as one in 12,500 live births. The main features of this disease are failure of psychomotor development, impaired vision, seizures, and premature death. Many biochemical and physiological studies have been initiated to determine the cellular defect underlying the disease, although only a few traits have been truly associated with the disorders. One of the paradox's of the NCL-diseases is the characteristic accumulation of autofluorescent hydrophobic material in the lysosomes of neurons and other cell types. However, the accumulation of this lysosomal storage material, which no doubt contributes to the neurologic disease, does not apparently lead to disease outside the CNS, and how these cellular alterations relate to the neurodegeneration in NCLs is unknown. Mutations have been identified in six distinct genes/proteins, namely CLN1, which encodes PPT1, a protein thiolesterase; CLN2, which encodes TPP1, a serine protease; and CLN3, CLN5, CLN6, and CLN8, which encode novel transmembrane proteins. Mutation in any one of these CLN-proteins results in a distinct type of NCL-disease. However, there are many shared similarities in the pathology of these diseases. The most obvious connection between PPT1, TPP1, CLN3, CLN5, CLN6, and CLN8 is their subcellular localization. To date, three of the four proteins whose subcellular localization has been confirmed, namely PPT1, TPP1, and CLN3, reside in the lysosome. We review the function of the CLN-proteins and discuss the possibility that a disruption in a common biological process leads to an NCL-disease.

Child↗
PubMed1998

Identification of the contiguous Paracoccus denitrificans ccmF and ccmH genes: disruption of ccmF, encoding a putative transporter, results in formation of an unstable apocytochrome c and deficiency in siderophore production.

Apocytochrome C550 was detected in the periplasm of a new mutant of Paracoccus denitrificans, HN48, that is pleiotropically lacking c-type cytochromes, produces reduced levels of siderophores and carries a Tn5 insertion in the ccmF gene for which sequence data, along with that for the contiguous ccmH, are reported. A counterpart to the ccmF gene was found in an archaebacterium but could not be located in the yeast genome, whereas mitochondrial haem lyases in the latter were not present in an archaeobacterial or in eubacterial genomes. A topological analysis for CcmF is presented which indicates at least eleven transmembrane helices, suggesting a role as a transporter; evidence against the substrate being haem is presented but sequence similarity with Escherichia coli gamma-aminobutyric acid transporter was identified. Analysis by pulse-chase methodology has shown that, in this and another cytochrome-c-deficient mutant, the apo form of P. denitrificans cytochrome C550 is much less stable than the holo form, directly demonstrating the presence of a periplasmic degradation system in P. denitrificans that removes non-functional proteins. A variety of phenotypes are observed for P. denitrificans mutated in different ccm genes, thus indicating that the stability of the ccm gene products does not require assembly of a complex of all the Ccm proteins.

Amino Acid Sequence↗
PubMed1997

The Paracoccus denitrificans ccmA, B and C genes: cloning and sequencing, and analysis of the potential of their products to form a haem or apo- c-type cytochrome transporter.

Two c-type cytochrome deficient mutants of Paracoccus denitrificans, HN49 and HN53, were isolated by Tn5 mutagenesis and screening for failure to oxidize dimethylphenylenediamine (the Nadi test). Both were completely deficient in c-type cytochromes. Genomic DNA flanking the site of Tn5 insertion in HN53 was cloned by marked rescue and a 3.1 kb region sequenced. Three of the genes, designated ccmA, ccmB and ccmC, present in this region are proposed to encode the components of a membrane transporter of the ABC-(ATP-binding cassette) superfamily, which is similar to a group of transporters postulated to translocate either haem or apocytochromes c. The Tn5 elements in HN49 and HN53 shown to be inserted in ccmB and ccmA, respectively. Sequence analysis suggested that both CcmB and CcmC have the potential to interact with CcmA and thus that the three gene products probably associate to form a complex with (CcmA)2-CcmB-CcmC stoichiometry; it is also indicated a lack of similarity between CcmB and CcmC and the membrane-integral components of transporters mediating uptake of haem or other iron complexes. Supplementation of growth media with haem did not stimulate c-type cytochrome formation in HN49 or HN53, although it elevated levels of soluble haemoproteins and membrane-bound cytochromes b, suggesting that exogenous haem can traverse both outer and inner membranes of P. denitrificans. HN49 and HN53 accumulated apocytochrome C550 to much lower levels than other c-type cytochrome deficient mutants of P. denitrificans but expression and translocation of an apocytochrome C550-alkaline phosphatase fusion protein and apocytochrome cd1 were unaffected in HN53. The results suggest that the substrate for the putative CcmABC-transporter is probably neither haem nor c-type apocytochromes.

ATP-Binding Cassette Transporters↗