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Biomedical subjects

P Sheppard

Publications and source records attributed to P Sheppard.

12 recordsLinked to original sources
PubMed2001

Dishevelled regulates the metabolism of amyloid precursor protein via protein kinase C/mitogen-activated protein kinase and c-Jun terminal kinase.

Alzheimer's disease (AD) is a disorder of two pathologies: amyloid plaques, the core of which is a peptide derived from the amyloid precursor protein (APP), and neurofibrillary tangles composed of highly phosphorylated tau. Protein kinase C (PKC) is known to increase non-amyloidogenic alpha-secretase cleavage of APP, producing secreted APP (sAPPalpha), and glycogen synthase kinase (GSK)-3beta is known to increase tau phosphorylation. Both PKC and GSK-3beta are components of the wnt signaling cascade. Here we demonstrate that overexpression of another member of this pathway, dishevelled (dvl-1), increases sAPPalpha production. The dishevelled action on APP is mediated via both c-jun terminal kinase (JNK) and protein kinase C (PKC)/mitogen-activated protein (MAP) kinase but not via p38 MAP kinase. These data position dvl-1 upstream of both PKC and JNK, thereby explaining the previously observed dual signaling action of dvl-1. Furthermore, we show that human dvl-1 and wnt-1 also reduce the phosphorylation of tau by GSK-3beta. Therefore, both APP metabolism and tau phosphorylation are potentially linked through wnt signaling.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Glycogen synthase kinase-3beta immunoreactivity is reduced in the prefrontal cortex in schizophrenia.

Cytoarchitectural abnormalities have been reported in the cortex in schizophrenia. These suggest a developmental origin for this disorder. The Wnt signalling pathway is involved in the regulation of brain development; disruption of this pathway may lead to abnormal cortical development. In this study levels of three components of the Wnt signalling pathway; glycogen synthase kinase-3beta(GSK-3beta), beta-catenin and dishevelled-2 (Dvl-2) were determined in the prefrontal cortex of ten schizophrenic and ten control individuals using immunoblotting. GSK-3beta levels were significantly reduced in the schizophrenic group, while levels of beta-catenin and Dvl-2 did not differ between groups. This provides further evidence for an abnormality of the Wnt signalling pathway in schizophrenia.

Adaptor Proteins, Signal Transducing↗
PubMed1997

An islet-cell protein tyrosine phosphatase is a likely precursor to the 37-kDa autoantigen in type 1 diabetes: human and macaque sequences, tissue distribution, unique and shared epitopes, and predictive autoantibodies.

BACKGROUND: We sought to identify novel islet-cell autoantigens to better understand the pathogenesis, prediction, and immunotherapy of type 1 diabetes. MATERIALS AND METHODS: Macaque and human islet cDNA libraries expressed in mammalian cells were screened with human diabetes sera. A positive clone was sequenced directly and after 5' rapid amplification of cDNA ends (RACE). Northern blotting and in situ hybridization revealed the tissue distribution of the corresponding protein. Antigen, expressed by in vitro translation, and tryptic peptides were analyzed by SDS-PAGE. For the immunoprecipitations, 183 diabetic, 60 prediabetic, and 91 control sera were used. Truncated antigens were used in immunoprecipitations for epitope mapping. Recombinant antigen expressed in transfected fibroblasts was used in competition assays. RESULTS: Sequencing yielded an 111-kDa, 1,013 amino acid, transmembrane protein (M1851) containing consensus protein tyrosine phosphatase (PTPase) sequence. M1851 was 77% identical in the intracellular domain, but only 31% identical extracellularly, to the islet-cell autoantigen ICA512. mRNA localized to brain, prostate, pancreatic islets, and adrenal medulla. After limited trypsinization, the in vitro translated antigen was 37 kDa. M1851 was recognized by 47% of prediabetes sera, 31% of new diabetes sera, but only 1% of healthy controls. Only 1/73 sera binding M1851 failed to bind ICA512, whereas 42/114 binding ICA512 did not bind M1851. M1851 reactivity was not fully displaced by ICA512 in 24/49 sera. Removing the C-terminal 27, 80, or 160 amino acids of M1851 decreased reactivity by 70%, 90%, and 100%, respectively. CONCLUSIONS: This new islet-cell PTPase is likely to be the precursor to the 37-kDa tryptic fragment antigen. It is structurally related to ICA512 but has distinct diabetes autoantibody epitopes located at the C terminus.

Amino Acid Sequence↗
PubMed1995

Structure, expression, and extracellular targeting of PM27, a skeletal protein associated specifically with growth of the sea urchin larval spicule.

The PM27 gene encodes a sea urchin skeletal protein. Both the transcript and encoded protein appear at the mesenchyme blastula stage and are restricted to the primary mesenchyme cell (PMC) lineage throughout development. Transgenic expression of PM27 promoter constructs demonstrates that this cell specificity is regulated at the level of transcription. The PM27 sequence predicts a nonglycosylated secretory product of 27 kDa in mature form. The N-terminal "repeat" domain of the deduced amino acid sequence consists of a series of tandem repeats and shares both sequence and predicted structural similarities with several fiber-forming proteins. The C-terminal "lectin" domain is similar to the C-class lectins. Antisera against either domain of PM27 detect two major proteins in embryo extracts, with apparent molecular weights of 27 and 30 kDa. Immunolocalization in whole embryos demonstrates that PM27 antigen is produced uniformly and exclusively by PMCs through the early prism stage and that this specificity is further restricted during skeletogenesis to a subpopulation of PMCs associated with the growing tips of the spicules. It is secreted to the skeletal compartment and accumulates predominantly at the advancing mineralizing surface of the spicule tips. As the spicules elongate PM27 protein disappears from the more mature mid-shaft regions. The observed characteristics of PM27 are consistent with a role in the regulation or execution of skeletal growth, as opposed to maintenance or structural integrity of the spicules.

Amino Acid Sequence↗
PubMed1993

Substitution of valine-865 by methionine or leucine in the human androgen receptor causes complete or partial androgen insensitivity, respectively with distinct androgen receptor phenotypes.

We have identified two different single nucleotide missense substitutions at valine-865 in exon 7 of the human androgen receptor (AR) gene in two families with androgen resistance. Val-->methionine is associated with the complete syndrome; Val-->leucine is associated with the partial form. In genital skin fibroblasts, both alterations yield a normal maximum binding capacity, but an increased apparent equilibrium dissociation constant for all androgens tested. In genital skin fibroblasts, Val865-Met A-R complexes have increased rate constants of dissociation with 5 alpha-dihydrotestosterone, and the nonmetabolized ligands methyltrienolone or mibolerone (MB); their Val865-Leu counterparts have increased rates with methyltrienolone and MB, but not with 5 alpha-dihydrotestosterone. In transiently transfected COS-1 or PC-3 cells, Met865 AR is more severely impaired than Leu865 AR in transactivating two different androgen-responsive reporter constructs, thereby correlating with clinical phenotype. In COS-1 cells exposed to MB for 74 h, this relative impairment correlates with the relative instability of the MB-binding activity of each mutant AR, suggesting that their respective intrinsic transcriptional regulatory competence is normal. Notably, these mutant ARs lose significantly more MB-binding activity than immunoreactivity, suggesting that prolonged MB exposure induces them to adopt a nonbinding state. The position homologous to Val865 in the AR is occupied by Leu or Met in the three steroid receptors closely related to the AR. This indicates the structural subtlety that underlies the steroid-binding activity of different steroid receptors.

Amino Acid Sequence↗
PubMed1987

Sequence variation of bovine elastin mRNA due to alternative splicing.

Poly A+ RNA, isolated from a single 210 day fetal bovine nuchal ligament, was used to synthesize cDNA by the RNase H method, using AMV reverse transcriptase for first strand synthesis and DNA polymerase I for the second strand. The cDNA was inserted into lambda gt10 using EcoRI linkers, and recombinant phage containing elastin sequences were identified by hybridization with a 1.3 kb sheep elastin cDNA clone, pcSELI (Yoon, K. et al., Biochem. Biophys. Res. Comm. 118: 261-265, 1984). Three clones containing the largest inserts of 2.9, 2.8, and 2.6 kb were selected for further study. The complete sequence analysis of the 3 clones was correlated with the sequence of 10.2 kb of the bovine elastin gene. The analyses: (i) showed that the cDNA encompassed the great majority of the translated sequence, (ii) ordered the tryptic peptides of porcine tropoelastin, (iii) determined new amino acid sequences not previously found in the porcine peptides and (iv) demonstrated that alternative splicing of the primary transcript leads to significant variation in the sequence of the translated portion of the mRNA.

Amino Acid Sequence↗
PubMed1987

Alternative splicing of human elastin mRNA indicated by sequence analysis of cloned genomic and complementary DNA.

Poly(A)+ RNA, isolated from a single 7-mo fetal human aorta, was used to synthesize cDNA by the RNase H method, and the cDNA was inserted into lambda gt10. Recombinant phage containing elastin sequences were identified by hybridization with cloned, exon-containing fragments of the human elastin gene. Three clones containing inserts of 3.3, 2.7, and 2.3 kilobases were selected for further analysis. Three overlapping clones containing 17.8 kilobases of the human elastin gene were also isolated from genomic libraries. Complete sequence analysis of the six clones demonstrated that: the cDNA encompassed the entire translated portion of the mRNA encoding 786 amino acids, including several unusual hydrophilic amino acid sequences not previously identified in porcine tropoelastin, exons encoding either hydrophobic or crosslinking domains in the protein alternated in the gene, and a great abundance of Alu repetitive sequences occurred throughout the introns. The data also indicated substantial alternative splicing of the mRNA. These results suggest the potential for significant variation in the precise molecular structure of the elastic fiber in the human population.

Amino Acid Sequence↗