PubMed Health⌕ Search

Biomedical subjects

David A Pearce

Publications and source records attributed to David A Pearce.

At least 37 records · Page 2Linked to original sources
PubMed2005

CLN3, the protein associated with batten disease: structure, function and localization.

Batten disease, an inherited neurodegenerative storage disease affecting children, results from the autosomal recessive inheritance of mutations in Cln3. The function of the CLN3 protein remains unknown. A key to understanding the pathology of this devastating disease will be to elucidate the function of CLN3 at the cellular level. CLN3 has proven difficult to study as it is predicted to be a membrane protein expressed at relatively low levels. This article is a critical review of various approaches used in examining the structure, trafficking, and localization of CLN3. We conclude that CLN3 is likely resident in the lysosomal/endosomal membrane. Different groups have postulated conflicting orientations for CLN3 within this membrane. In addition, CLN3 undergoes several posttranslational modifications and is trafficked through the endoplasmic reticulum and Golgi. Recent evidence also suggests that CLN3 traffics via the plasma membrane. Although the function of this protein remains elusive, it is apparent that genetic alterations in Cln3 may have a direct affect on lysosomal function.

Animals↗
PubMed2005

Characterization of lipid-linked oligosaccharide accumulation in mouse models of Batten disease.

The neuronal ceroid lipofuscinoses (NCLs, also known collectively as Batten disease) are a group of lysosomal storage disorders characterized by the accumulation of autofluorescent storage material in the brain and other tissues. A number of genes underlying various forms of NCL have been cloned, but the basis for the neurodegeneration in any of these is unknown. High levels of dolichol pyrophosphoryl oligosaccharides have previously been demonstrated in brain tissue from several NCL patients, but the specificity of the effect for the NCLs has been unclear. In the present study, we examine eight mouse models of lysosomal storage disorders by modern FACE and found striking lipid-linked oligosaccharide (LLO) accumulation in NCL mouse models (especially CLN1, CLN6, and CLN8 knockout or mutant mice) but not in several other lysosomal storage disorders affecting the brain. Using a mouse model of the most severe form of NCL (the PPT1 knockout mouse), we show that accumulated LLOs are not the result of a defect in LLO synthesis, extension, or transfer but rather are catabolic intermediates derived from LLO degradation. LLOs are enriched about 60-fold in the autofluorescent storage material purified from PPT1 knockoutmouse brain but comprise only 0.3% of the autofluorescent storage material by mass. The accumulation of LLOs is postulated to result from inhibition of late stages of lysosomal degradation of autophagosomes, which may be enriched in these metabolic precursors.

Animals↗
PubMed2005

Molecular detection and isolation from antarctica of methylotrophic bacteria able to grow with methylated sulfur compounds.

This study is the first demonstration that a diverse facultatively methylotrophic microbiota exists in some Antarctic locations. PCR amplification of genes diagnostic for methylotrophs was carried out with bacterial DNA isolated from 14 soil and sediment samples from ten locations on Signy Island, South Orkney Islands, Antarctica. Genes encoding the mxaF of methanol dehydrogenase, the fdxA for Afipia ferredoxin, the msmA of methanesulfonate monooxygenase, and the 16S rRNA gene of Methylobacterium were detected in all samples tested. The mxaF gene sequences corresponded to those of Hyphomicrobium, Methylobacterium, and Methylomonas. Over 30 pure cultures of methylotrophs were isolated on methanesulfonate, dimethylsulfone, or dimethylsulfide from ten Signy Island lakes. Some were identified from 16S rRNA gene sequences (and morphology) as Hyphomicrobium species, strains of Afipia felis, and a methylotrophic Flavobacterium strain. Antarctic environments thus contain diverse methylotrophic bacteria, growing on various C1-substrates, including C1-sulfur compounds.

Alphaproteobacteria↗
PubMed2005

Significant changes in the bacterioplankton community structure of a maritime Antarctic freshwater lake following nutrient enrichment.

Nutrient enrichment is known to increase bacterioplankton population density in a variety of Antarctic freshwater lakes. However, relatively little is known about the associated changes in species composition. In this study, the bacterioplankton community composition of one such lake was studied following natural nutrient enrichment to investigate the resistance of the system to environmental change. Heywood Lake is an enriched freshwater maritime Antarctic lake, with nitrogen and phosphorus concentrations significantly higher than its more oligotrophic neighbours (by at least an order of magnitude). This major change in lake chemistry has occurred following large increases in the fur seal population over the last 30 years. Using analysis of 16S rRNA gene fragments, fatty acid methyl ester analysis, denaturing gradient gel electrophoresis and fluorescence in situ hybridization, significant changes are reported in lake microbiology which have resulted in a distinct bacterioplankton community. In comparison to its more oligotrophic neighbours, nutrient-enriched Heywood Lake has a high bacterioplankton population density, reduced species richness and an increasing evenness among key groups. Only 42.3 % of the clones found with > or =97 % similarity to a named genus were also present in adjacent oligotrophic lakes, including three of the dominant groups. Critically, there was an apparent shift in dominance with trophic status (from the beta-Proteobacteria to the Actinobacteria). Other key observations included the absence of a dominant group of Cyanobacteria and the presence of marine bacteria. The significant impact of natural nutrient enrichment on the microbiology of Heywood Lake, therefore, suggests that low-temperature oligotrophic freshwater lake systems might have low resistance to environmental change.

Actinobacteria↗
PubMed2005

Isolation and properties of methanesulfonate-degrading Afipia felis from Antarctica and comparison with other strains of A. felis.

Three novel strains of methylotrophic Afipia felis were isolated from several locations on Signy Island, Antarctica, and a fourth from estuary sediment from the River Douro, Portugal. They were identified as strains of the alpha-2 proteobacterium A. felis by 16S rRNA gene sequence analysis. Two strains tested were shown to contain the fdxA gene, diagnostic for A. felis. All strains grew with methanesulfonate (and two strains with dimethylsulfone) as sole carbon substrate. Growth on methanesulfonate required methanesulfonate monooxygenase (MSAMO), using NADH as the reductant and stimulated by reduced flavin nucleotides and Fe(II). Polymerase chain reaction amplification of DNA from an Antarctic strain showed a typical msmA gene for the alpha-hydroxylase of MSAMO, and both Antarctic and Portuguese strains contained mxaF, the methanol dehydrogenase large subunit gene. This is the first report of methanesulfonate-degrading bacteria from the Antarctic and of methylotrophy in Afipia, and the first description of any bacterium able to use both methanesulfonate and dimethylsulfone. In contrast, the type strain of A. felis DSM 7326(T) was not methylotrophic, but grew in defined mineral medium with a wide range of single simple organic substrates. Free-living Afipia strains occurring widely in the natural environment may be significant as methylotrophs, degrading C(1)-sulfur compounds, including the recalcitrant organosulfur compound methanesulfonate.

Afipia↗
PubMed2005

Interaction among Btn1p, Btn2p, and Ist2p reveals potential interplay among the vacuole, amino acid levels, and ion homeostasis in the yeast Saccharomyces cerevisiae.

Btn2p, a novel cytosolic coiled-coil protein in Saccharomyces cerevisiae, was previously shown to interact with and to be necessary for the correct localization of Rhb1p, a regulator of arginine uptake, and Yif1p, a Golgi protein. We now report the biochemical and physical interactions of Btn2p with Ist2p, a plasma membrane protein that is thought to have a function in salt tolerance. A deletion in Btn2p (btn2Delta strains) results in a failure to correctly localize Ist2p, and strains lacking Btn2p and Ist2p (btn2Delta ist2Delta strains) are unable to grow in the presence of 0.5 or 1.0 M NaCl. Btn2p was originally identified as being up-regulated in a btn1Delta strain, which lacks the vacuolar-lysosomal membrane protein, Btn1p, and serves as a model for Batten disease. This up-regulation of Btn2p was shown to contribute to the maintenance of a stable vacuolar pH in the btn1Delta strain. Btn1p was subsequently shown to be required for the optimal transport of arginine into the vacuole. Interestingly, btn1Delta ist2Delta strains are also unable to grow in the presence of 0.5 or 1.0 M NaCl, and ist2Delta suppresses the vacuolar arginine transport defect in btn1Delta strains. Although further investigation is required, we speculate that altered vacuolar arginine transport in btn1Delta strains represents a mechanism for maintaining or balancing cellular ion homeostasis. Btn2p interacts with at least three proteins that are seemingly involved in different biological functions in different subcellular locations. Due to these multiple interactions, we conclude that Btn2p may play a regulatory role across the cell in response to alterations in the intracellular environment that may be caused by changes in amino acid levels or pH, a disruption in protein trafficking, or imbalances in ion homeostasis resulting from either genetic or environmental manipulation.

Amino Acid Transport Systems↗
PubMed2004

Enhanced mitochondrial degradation of yeast cytochrome c with amphipathic structures.

The dispensable N-terminus of iso-1-cytochrome c (iso-1) in the yeast Saccharomyces cerevisiae was replaced by 11 different amphipathic structures. Rapid degradation of the corresponding iso-1 occurred, with the degree of degradation increasing with the amphipathic moments; and this amphipathic-dependent degradation was designated ADD. ADD occurred with the holo-forms in the mitochondria but not as the apo-forms in the cytosol. The extreme mutant type degraded with a half-life of approximately 12 min, whereas the normal iso-1 was stable over hours. ADD was influenced by the rho+/rho- state and by numerous chromosomal genes. Most importantly, ADD appeared to be specifically suppressed to various extents by deletions of any of the YME1, AFG3, or RCA1 genes encoding membrane-associated mitochondrial proteases, probably because the amphipathic structures caused a stronger association with the mitochondrial inner membrane and its associated proteases. The use of ADD assisted in the differentiation of substrates of different mitochondrial degradation pathways.

Amino Acid Sequence↗
PubMed2004

Glutamic acid decarboxylase autoimmunity in Batten disease and other disorders.

Degenerative diseases of the CNS, such as stiff-person syndrome (SPS), progressive cerebellar ataxia, and Rasmussen encephalitis, have been characterized by the presence of autoantibodies. Recent findings in individuals with Batten disease and in animal models for the disorder indicate that this condition may be associated with autoantibodies against glutamic acid decarboxylase (GAD), an enzyme that converts the excitatory neurotransmitter glutamate to the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Anti-GAD autoantibodies could result in excess excitatory neurotransmitters, leading to the seizures and other symptoms observed in patients with Batten disease. The pathogenic potential of GAD autoantibodies is examined in light of what is known for other autoimmune disorders, such as multiple sclerosis, SPS, Rasmussen encephalitis, and type 1 diabetes, and may have radical implications for diagnosis and management of Batten disease.

Animals↗
PubMed2004

Late onset neurodegeneration in the Cln3-/- mouse model of juvenile neuronal ceroid lipofuscinosis is preceded by low level glial activation.

Mouse models of neuronal ceroid lipofuscinosis (NCL) exhibit many features of the human disorder, with widespread regional atrophy and significant loss of GABAergic interneurons in the hippocampus and neocortex. Reactive gliosis is a characteristic of all forms of NCL, but it is unclear whether glial activation precedes or is triggered by neuronal loss. To explore this issue we undertook detailed morphological characterization of the Cln3 null mutant (Cln3(-/-)) mouse model of juvenile NCL (JNCL) that revealed a delayed onset neurodegenerative phenotype with no significant regional atrophy, but with widespread loss of hippocampal interneurons that was first evident at 14 months of age. Quantitative image analysis demonstrated upregulation of markers of astrocytic and microglial activation in presymptomatic Cln3(-/-) mice at 5 months of age, many months before significant neuronal loss occurs. These data provide evidence for subtle glial responses early in JNCL pathogenesis.

Age Factors↗
PubMed2004

Homogeneous polymerase chain reaction nucleobase quenching assay to detect the 1-kbp deletion in CLN3 that causes Batten disease.

Batten disease is an autosomal recessive disorder also known as juvenile neuronal ceroid lipofuscinosis. The most common mutation for this disease is an approximately 1-kbp deletion in the CLN3 gene, which accounts for about 80 to 85% of the mutation load. We developed a rapid assay for this mutation using the PCR to produce amplicons that are detected by nucleobase quenching of the fluorescent signal from a probe labeled with a fluorescent dye. The probe overlaps the deletion breakpoint and is completely base paired to the mutant amplicon. However, three bases at the 5' end of the probe do not base pair with the wild-type amplicon. The alleles are distinguished by the different melting temperatures of the probe amplicon hybrids. Comparison of this new method with an allele-specific PCR and gel electrophoresis-based method showed 100% concordance in determination of the genotype for 30 specimens (11 homozygous mutant, 8 heterozygotes, and 11 homozygous normal). PCR followed by allele-specific melting curve analysis using nucleobase quenching has utility as a rapid method for detection of the most common mutation that causes Batten disease.

Female↗
PubMed2004

Altered gene expression in the eye of a mouse model for batten disease.

PURPOSE: Juvenile neuronal ceroid lipofuscinosis (JNCL or Batten Disease) is one of the most common progressive neurodegenerative disorders of childhood, resulting from autosomal recessive inheritance of mutations in the CLN3 gene. Pathologically, Batten disease is characterized by lysosomal storage of autofluorescent material in all tissue types. Although characterized by seizures, mental retardation, and loss of motor skills, the first presenting symptom of Batten disease is vision loss. METHODS: High-density oligonucleotide arrays were used to profile approximately 19,000 mRNAs in the eye of 10-week-old Cln3-knockout and normal mice, and the data were compared with that for the cerebellum in the same model as a means to identify gene expression changes that are specific to the eye. RESULTS: A detailed list was compiled of 285 functionally categorized genes that have altered expression in the eye of Cln3-knockout mice before the appearance of the characteristic lysosomal storage material. Furthermore, 18 genes were identified and 6 validated by semiquantitative RT-PCR that have altered expression in the eye, but not in the cerebellum of Cln3-knockout mice. The genes that have altered expression specific to the eye of the Cln3-knockout mouse may be of importance in understanding the function of CLN3 in different tissues. CONCLUSIONS: Downregulation of genes associated with energy production in the mitochondria appears to be specific to the eye. The CLN3 defect may result in altered mitochondrial function in eye but not other tissue. More detailed experimentation is needed to understand the contribution of these changes in expression to disease state, and whether these changes are specific for certain cell types within the eye.

Animals↗
PubMed2003

A role in vacuolar arginine transport for yeast Btn1p and for human CLN3, the protein defective in Batten disease.

In Saccharomyces cerevisiae, transport of arginine into the vacuole has previously been shown to be facilitated by a putative H+/arginine antiport. We confirm that transport of arginine into isolated yeast vacuoles requires ATP and we demonstrate a requirement for a functional vacuolar H+-ATPase. We previously reported that deletion of BTN1 (btn1-delta), an ortholog of the human Batten disease gene CLN3, resulted in a decrease in vacuolar pH during early growth. We report that this altered vacuolar pH in btn1-delta strains underlies a lack of arginine transport into the vacuole, which results in a depletion of endogenous vacuolar arginine levels. This arginine transport defect in btn1-delta is complemented by expression of either BTN1 or the human CLN3 gene and strongly suggests a function for transport of, or regulation of the transport of, basic amino acids into the vacuole or lysosome for yeast Btn1p, and human CLN3 protein, respectively. We propose that defective transport at the lysosomal membrane caused by an absence of functional CLN3 is the primary biochemical defect that results in Batten disease.

Arginine↗
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↗