Phosphatase 2A Beta Research Articles

A Combined Proteomics and Bioinformatics Approach Reveals Novel Signaling Pathways and Molecular Targets After Intracerebral Hemorrhage

Intracerebral hemorrhage (ICH) is a non-traumatic cerebrovascular disorder with very high morbidity and mortality and regarded as one of the deadliest stroke subtypes. Notably, there is no effective treatment for ICH. Despite an overall increase in preclinical studies, the pathophysiology of ICH is complex and remains enigmatic. To this end, ICH was induced in male CD-1 mice and the ipsilateral brain tissue was characterized in an unbiased manner using a combination of proteomics and bioinformatics approaches. A total of 4833 proteins were revealed by quantitative proteomic analysis. Of those, 207 proteins exhibited significantly altered expression after ICH in comparison to sham. It was found that 46 proteins were significantly upregulated and 161 proteins were significantly downregulated after ICH compared to sham. The quantitative proteomics approach combined with bioinformatics revealed several novel molecular targets (cyclin-dependent-like kinase 5, E3 ubiquitin-protein ligase, protein phosphatase 2A-alpha, protein phosphatase 2A-beta, serine/threonine-protein kinase PAK1, alpha-actinin-4, calpain-8, axin-1, NCK1, and septin-4), and related signaling pathways, which could play roles in secondary brain injury and long-term neurobehavioral outcomes after ICH warranting further investigation.

Gene expression profile following stable expression of the cellular prion protein.

1. The cellular prion protein (PrPC) is expressed widely in neural and nonneural tissues at the highest level in neurons in the central nervous system (CNS). 2. Recent studies indicated that transgenic mice with the cytoplasmic accumulation of PrPC exhibited extensive neurodegeneration in the cerebellum, although the underlying mechanism remains unknown. To identify the genes whose expression is controlled by over-expression of PrPC in human cells, we have established a stable PrPC-expressing HEK293 cell line designated P1 by the site-specific recombination technique. 3. Microarray analysis identified 33 genes expressed differentially between P1 and the parent PrPC-non-expressing cell line among 12,814 genes examined. They included 18 genes involved in neuronal and glial functions, 5 related to production of extracellular matrix proteins, and 2 located in the complement cascade. 4. Northern blot analysis verified marked upregulation in P1 of the brain-specific protein phosphatase 2A beta subunit (PPP2R2B), a causative gene of spinocerebellar ataxia 12, and the cerebellar degeneration-related autoantigen (CDR34) gene associated with development of paraneoplastic cerebellar degeneration. 5. These results indicate that accumulation of PrPC in the cell caused aberrant regulation of a battery of the genes important for specific neuronal function. This represents a possible mechanism underlying PrPC-mediated selective neurodegeneration.

Physical map of the human chromosome 8p12-p21 encompassing tumor suppressor and Werner's syndrome gene loci.

Detailed physical maps of the human genome are important resources for identification and isolation of genes responsible for diseases and for the study of their structure and function. We constructed a 2.0-Mb high-resolution physical map within the human chromosome 8p12-p21 region extending from marker D8S131 to D8S283. The map comprises a series of contigs mostly P1/PAC clones, which span the loci of potential tumor suppressor genes and the Werner's syndrome gene. Each P1/PAC DNA was defined by its size, restriction sites, terminal sequences, intermarker distances and location relative to major genes and markers. The genes on these P1/PAC DNAs were analyzed by an exon amplification method to determine their locations. The genes newly found by the exon amplification method together with other known genes, including those of glutathion reductase, a general transcription factor, protein phosphatase 2A beta subunit and Werner's syndrome, were precisely mapped within the contigs. These P1/PAC DNAs are useful reagents for the generation of new microsatellite markers to narrow the candidate region of the tumor suppressor gene(s) and/or genes responsible for other diseases, which are believed to exist in this region by linkage analysis.

Identification of vestibular compensation-associated molecules by means of differential display

The differential display method was used to identify gene expression which is altered in the cerebellar flocculus after unilateral labyrinthectomy (UL). Total RNA from flocculi of sham-operated and labyrinthectomized rats was isolated, amplified by PCR using arbitrary primer sets and separated by electrophoresis on a polyacrylamide gel. PCR products, whose amounts were significantly different in samples from labyrinthectomized animals and those from controls, were cut out of the gel and sequenced. One of the up-regulated products was the rat protein phosphatase 2A beta catalytic subunit mRNA and one of the down-regulated products was the rat glutamate receptor delta-2 subunit mRNA. Histochemical examination of in situ hybridization showed that those molecules were intensively localized in the Purkinje cell layer. In labyrinthectomized rats, UL-induced nystagmus gradually disappeared within 3 days after UL. These findings suggest that changes in expression of those molecules in the floccular Purkinje cells after UL is involved in vestibular compensation. So far various kinds of neural plasticity-associated molecules have been investigated, mainly by slice-in vitro studies. This study indicates that differential display is a feasible molecular biological in vivo method for investigation of the mechanism of neural plasticity.

Cloning and Characterization of Rep-8 (D8S2298E) in the Human Chromosome 8p11.2–p12

A novel human gene referred to as the Rep-8 gene (D8S2298E) was cloned by a combination of exon trapping, thermal asymmetric interlaced-PCR, and screening of a cDNA library. It is located in human chromosome 8p11.2–p12. The gene consists of eight exons and spans about 20 kb between the glutathioneS-reductase and the protein phosphatase 2A beta subunit genes. The full-length Rep-8 gene contains 1483 nucleotides and codes for a protein of 270 amino acids. Southern blot experiments showed that the Rep-8 gene exists as a single copy per haploid. With a zoo blot analysis, human Rep-8 DNA hybridized strongly with the monkey DNA, but only weakly with the DNAs of species other thanHomo sapiens.Northern blot analysis showed that it is expressed abundantly in the testis and ovary, suggesting that the Rep-8 gene product may play a role in reproduction.

1408 Role of the rat floccular protein phosphatase 2a beta subunit in lesion-induced vestibular plasticity:detected by means of defferential display

1408 Role of the rat floccular protein phosphatase 2a beta subunit in lesion-induced vestibular plasticity:detected by means of defferential display

Comparative Analysis of Mouse NotI Linking Clones with Mouse and Human Genomic Sequences and Transcripts

NotI cleavage sites are frequently associated with CpG islands that identify the 5' regulatory sites of functional genes in the genome. Therefore we analyzed a sample of 22 NotI linking clones prepared from mouse brain DNA, to determine whether these mouse NotI site associated clones could be used for comparative analysis of mouse and human genomes by cross-reaction with both mouse and human genomic DNA and RNA in Southern and Northern hybridization. We further examined whether we could establish the identity of these clones with known genes by comparing the nucleotide sequences surrounding the NotI site with the GenBank database. We observed that 70% of the clones cross-hybridized with human DNA and that 4 of 11 tested clones (36%) detected a transcript in human HeLa cells RNA whereas 73% clones (8/11) detected transcripts in mouse RNAs from one or more organs. Single pass sequence analysis was successful on 16 of 19 clones. The GC content in these sequence was very high (48.8% to 73.8%) suggesting that 12 of 16 sequenced clones contained a CpG island. Three out of 19 clones showed significant similarity with previously analyzed mouse gene sequences in GenBank, including the mouse rRNA gene family, cathepsin and the scip POU-domain genes. In addition, two sequences showed significant similarity to the human and rabbit protein phosphatase 2A-beta subunit and the human transforming growth factor-beta. Thus, 5 of 16 clones showed homology with identified genes. These results and the recent work of using RLGS methods for genetic mapping indicate that NotI linking clones can be used to efficiently cross reference a comparative analysis of the mouse and human genomic maps.

Molecular cloning of rat phosphoprotein phosphatase 2Aβ cDNA and increased expressions of phosphatase 2Aα and 2Aβ in rat liver tumors

A cDNA clone coding for an isotype of the catalytic subunit of rat phosphoprotein phosphatase 2A was isolated. The deduced amino acid sequence of the clone was different at 8 positions from that of rat phosphatase 2A alpha determined in a previous study. The deduced amino acid sequence of the clone was, however, identical to that of human phosphatase 2A beta and differed only at one position from that of rabbit 2A beta. Thus, the isolated cDNA was identified as a clone coding for rat phosphatase 2A beta. Using a 2A beta specific probe, two kinds of transcripts were detected in rat liver: a major 2.0 kb mRNA transcript and a minor 1.4 kb mRNA transcript. These transcripts were both greatly increased in rat liver tumors induced by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) regardless of the carcinoma or hyperplastic nodule.