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  • Monoclonal antibodies to monoamine oxidase B and another mitochondrial protein from human liver. 3527152

    A monoclonal antibody has been generated to human liver monoamine oxidase (MAO) B by fusion of mouse myeloma cells with spleen cells from a mouse immunized with a mixture of semi-purified MAO A and MAO B. The antibody, 3F12/G10, an immunoglobulin G1, reacts with its antigen in cryostat sections of human liver, showing an intracellular particulate distribution as demonstrated by immunoperoxidase staining. The antibody indirectly precipitates [3H]pargyline-labelled human MAO B both from liver and platelet extracts but fails to precipitate MAO A from liver extracts. The antibody does not recognise rat liver MAO B, showing that the determinant is not universally expressed on MAO B. The antibody has no effect on the catalytic activity of MAO B. Other monoclonal antibodies were generated but they are directed to a protein with a subunit Mr of 54 000, a contaminant of the MAO preparation. One of these antibodies, A8/C2, an IgG2a, reacts with the same protein in both rat and human liver extracts.
    Document Type:
    Reference
    Product Catalog Number:
    MABN304
  • The p53 protein influences the sensitivity of testicular germ cells to mono-(2-ethylhexyl) phthalate-induced apoptosis by increasing the membrane levels of Fas and DR5 an ... 15371270

    The consequence of mono-(2-ethylhexyl) phthalate (MEHP)-induced injury of testicular Sertoli cells is the Fas-dependent apoptotic elimination of germ cells. In addition to the well-known ability of p53 to regulate the transcription of various apoptosis-associated proteins, p53 also has been implicated in mediating the localization of Fas to the plasma membrane of various cell types in a transcription-independent manner. To resolve the role of p53 in MEHP-mediated testicular toxicity, we used wild-type (p53(+/+)) and p53 knockout (p53(-/-)) mice. A significantly lower incidence of TUNEL-positive germ cells was observed in p53(-/-) mice compared to p53(+/+) mice at 1, 1.5, and 24 h after MEHP exposure. In these same mice, an induction of Fas and death receptor-5 (DR5) in testicular membrane preparations was observed only in p53(+/+) mice. Analyses of mRNA levels in testes of p53(+/+) and p53(-/-) mice by reverse transcription-polymerase chain reaction revealed that increases in membrane levels of Fas occurred in the absence of their transcriptional up-regulation. Processing of procaspase-8 was observed only in MEHP-treated p53(+/+) mice, and this correlated with the observed incidence of germ cell apoptosis. Interestingly, the p53 status of mice also influenced the stability of c-FLIP (L), a caspase-8 inhibitory protein, that was measured at levels approximately two- to fivefold higher in p53(-/-) mice after MEHP-exposure compared to those in p53(+/+) mice. Taken together, these data suggest that MEHP-induced germ cell apoptosis is dependent, in part, on the p53 protein and on its abilities to increase the localization of Fas and DR5 on the germ cell membrane as well as to decrease the cellular levels of c-FLIP (L).
    Document Type:
    Reference
    Product Catalog Number:
    06-697
  • Microtubule-severing ATPase spastin in glioblastoma: increased expression in human glioblastoma cell lines and inverse roles in cell motility and proliferation. 21865889

    We studied the expression and distribution of the microtubule-severing enzyme spastin in 3 human glioblastoma cell lines (U87MG, U138MG, and T98G) and in clinical tissue samples representative of all grades of diffuse astrocytic gliomas (n = 45). In adult human brains, spastin was distributed predominantly in neuronsand neuropil puncta and, to a lesser extent, in glia. Compared with normal mature brain tissues, spastin expression and cellular distribution were increased in neoplastic glial phenotypes, especiallyin glioblastoma (p < 0.05 vs low-grade diffuse astrocytomas). Overlapping punctate and diffuse patterns of localization wereidentified in tumor cells in tissues and in interphase and mitotic cells ofglioblastoma cell lines. There was enrichment of spastin in the leading edges of cells in T98G glioblastoma cell cultures and in neoplastic cell populations in tumor specimens. Real-time polymerase chain reaction and immunoblotting experiments revealed greater levels of spastin messenger RNA and protein expression in theglioblastoma cell lines versus normal human astrocytes. Functional experiments indicated that spastin depletion resulted in reduced cell motility and higher cell proliferation of T98G cells. Toour knowledge, this is the first report of spastin involvement incellmotility. Collectively, our results indicate that spastinexpression in glioblastomas might be linked to tumor cell motility, migration, and invasion.
    Document Type:
    Reference
    Product Catalog Number:
    MAB5634
  • A cellular gene encodes scrapie PrP 27-30 protein. 2859120

    A clone encoding PrP 27-30, the major protein in purified preparations of scrapie agent, was selected from a scrapie-infected hamster brain cDNA library by oligonucleotide probes corresponding to the N terminus of the protein. Southern blotting with PrP cDNA revealed a single gene with the same restriction patterns in normal and scrapie-infected brain DNA. A single PrP-related gene was also detected in murine and human DNA. PrP-related mRNA was found at similar levels in normal and scrapie-infected hamster brain, as well as in many other normal tissues. Using antisera against PrP 27-30, a PrP-related protein was detected in crude extracts of infected brain and to a lesser extent in extracts of normal brain. Proteinase K digestion yielded PrP 27-30 in infected brain extract, but completely degraded the PrP-related protein in normal brain extract. No PrP-related nucleic acids were found in purified preparations of scrapie prions, indicating that PrP 27-30 is not encoded by a nucleic acid carried within the infectious particles.
    Document Type:
    Reference
    Product Catalog Number:
    AG210
    Product Catalog Name:
    Prion Protein, recombinant
  • Preparation of a novel monoclonal antibody specific for myelin basic protein phosphorylated on Thr98. 7759601

    Phosphorylation is one of a number of post-translational modifications resulting in charge microheterogeneity of myelin basic protein (MBP). This phosphorylation is claimed to destabilise the compact myelin sheath by decreasing the interaction of membrane bilayers, thereby creating or maintaining pockets of cytoplasm. To further investigate and localise MBP phosphorylation to discrete regions of the myelin sheath we raised a monoclonal antibody with specificity for a known phosphorylation site in MBP. A synthetic peptide was made by Fmoc peptide chemistry and phosphorylation of Thr98 was achieved on the resin by the global phosphorylation methodology, utilising dibenzyl-N,N-diethylphosphoramidite phosphitylation and t-butylhydroperoxide oxidation. The peptide coupled to tuberculin was used to immunise mice for monoclonal antibody production. The selected hybridoma (Clone P12) secreted an IgG2a antibody which reacted strongly with the phosphorylated immunogen and with phosphorylated fractions of bovine MBP obtained by ion exchange chromatography. The antibody had minimal reactivity with the unphosphorylated peptide; the same peptide phosphorylated at another site Ser102; a preparation of unphosphorylated MBP obtained by ion exchange chromatography; and with an irrelevant phosphorylated protein (histone). Similar phosphorylation state-specific monoclonal antibodies could be made to recognise other specific phosphorylation sites in MBP or other proteins. It is planned to use these antibodies to quantify and locate the extent of MBP phosphorylation in normal and multiple sclerosis myelin.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
  • Partitioning the proteome: phase separation for targeted analysis of membrane proteins in human post-mortem brain. 22745773

    Neuroproteomics is a powerful platform for targeted and hypothesis driven research, providing comprehensive insights into cellular and sub-cellular disease states, Gene × Environmental effects, and cellular response to medication effects in human, animal, and cell culture models. Analysis of sub-proteomes is becoming increasingly important in clinical proteomics, enriching for otherwise undetectable proteins that are possible markers for disease. Membrane proteins are one such sub-proteome class that merit in-depth targeted analysis, particularly in psychiatric disorders. As membrane proteins are notoriously difficult to analyse using traditional proteomics methods, we evaluate a paradigm to enrich for and study membrane proteins from human post-mortem brain tissue. This is the first study to extensively characterise the integral trans-membrane spanning proteins present in human brain. Using Triton X-114 phase separation and LC-MS/MS analysis, we enriched for and identified 494 membrane proteins, with 194 trans-membrane helices present, ranging from 1 to 21 helices per protein. Isolated proteins included glutamate receptors, G proteins, voltage gated and calcium channels, synaptic proteins, and myelin proteins, all of which warrant quantitative proteomic investigation in psychiatric and neurological disorders. Overall, our sub-proteome analysis reduced sample complexity and enriched for integral membrane proteins by 2.3 fold, thus allowing for more manageable, reproducible, and targeted proteomics in case vs. control biomarker studies. This study provides a valuable reference for future neuroproteomic investigations of membrane proteins, and validates the use Triton X-114 detergent phase extraction on human post mortem brain.
    Document Type:
    Reference
    Product Catalog Number:
    MAB386
    Product Catalog Name:
    Anti-Myelin Basic Protein Antibody, a.a. 82-87
  • Scrapie and cellular PrP isoforms are encoded by the same chromosomal gene. 2873895

    PrP 27-30 is the major protein in purified preparations of scrapie agent. An almost complete PrP cDNA was used to select PrP-related genomic clones from normal hamster DNA. The gene contains a noncoding exon of 56 to 82 bp and a 2 kb coding exon, separated by a 10 kb intron. Transcription initiates at the same multiple sites in vivo and in vitro. The promoter lacks a TATA box and contains three repeats of the sequence GCCCCGCCC, which resembles the Sp1 binding site found in "housekeeping" genes. The PrP coding sequence encodes a presumptive amino-terminal signal peptide. The primary structure of PrP encoded by the gene of a healthy animal does not differ from that encoded by a cDNA from a scrapie-infected animal, suggesting that the different properties of PrP from normal and scrapie-infected brains are due to post-translational events.
    Document Type:
    Reference
    Product Catalog Number:
    AG210
    Product Catalog Name:
    Prion Protein, recombinant
  • Na+/K+-ATPase is present in scrapie-associated fibrils, modulates PrP misfolding in vitro and links PrP function and dysfunction. 22073199

    Transmissible spongiform encephalopathies are characterised by widespread deposition of fibrillar and/or plaque-like forms of the prion protein. These aggregated forms are produced by misfolding of the normal prion protein, PrP(C), to the disease-associated form, PrP(Sc), through mechanisms that remain elusive but which require either direct or indirect interaction between PrP(C) and PrP(Sc) isoforms. A wealth of evidence implicates other non-PrP molecules as active participants in the misfolding process, to catalyse and direct the conformational conversion of PrP(C) or to provide a scaffold ensuring correct alignment of PrP(C) and PrP(Sc) during conversion. Such molecules may be specific to different scrapie strains to facilitate differential prion protein misfolding. Since molecular cofactors may become integrated into the growing protein fibril during prion conversion, we have investigated the proteins contained in prion disease-specific deposits by shotgun proteomics of scrapie-associated fibrils (SAF) from mice infected with 3 different strains of mouse-passaged scrapie. Concomitant use of negative control preparations allowed us to identify and discount proteins that are enriched non-specifically by the SAF isolation protocol. We found several proteins that co-purified specifically with SAF from infected brains but none of these were reproducibly and demonstrably specific for particular scrapie strains. The α-chain of Na(+)/K(+)-ATPase was common to SAF from all 3 strains and we tested the ability of this protein to modulate in vitro misfolding of recombinant PrP. Na(+)/K(+)-ATPase enhanced the efficiency of disease-specific conversion of recombinant PrP suggesting that it may act as a molecular cofactor. Consistent with previous results, the same protein inhibited fibrillisation kinetics of recombinant PrP. Since functional interactions between PrP(C) and Na(+)/K(+)-ATPase have previously been reported in astrocytes, our data highlight this molecule as a key link between PrP function, dysfunction and misfolding.
    Document Type:
    Reference
    Product Catalog Number:
    AB9094
    Product Catalog Name:
    Anti-Sodium Pump (Na+ pump (Na+-K+-ATPase)) α 2 Isoform Antibody
  • Two distinct monokines, interleukin 1 and tumor necrosis factor, each independently induce biosynthesis and transient expression of the same antigen on the surface of cul ... 3485132

    A murine monoclonal antibody (H4/18) raised against cultured human endothelial cells (HEC) prestimulated by the monokine interleukin 1 (IL 1) recognizes a cell surface molecule inducible by IL 1 or by the distinct monokine tumor necrosis factor (TNF) in primary or serially passaged HEC. H4/18 binding is not basally expressed or inducible by IL 1 in an SV-40 transformed HEC line, in human dermal fibroblasts, or in blood leukocytes. Expression of this molecule by HEC in response to IL 1 can be blocked by protein and RNA synthesis inhibitors but not by cyclooxygenase inhibitors. In addition, H4/18 can immunoprecipitate two biosynthetically labeled polypeptides (Mr 100,000 and 120,000) from HEC stimulated with IL 1 but not from control HEC. Thus, the H4/18 binding site appears to be an inducible surface protein specific for HEC. The majority of HEC in a culture can be induced to express the H4/18 binding protein, but expression is transient (peak 4 to 6 hr) and over the next 24 hr declines to near basal levels either in the continued presence of or upon removal of IL 1. The magnitude of the peak response depends upon IL 1 concentration (peak 5 to 10 U/ml), and the response is optimized by the continued presence of IL 1 during the initial 4- to 6-hr induction period. The time of peak H4/18 binding does not appear to be a function of IL 1 concentration. The decline of H4/18 binding from peak levels is prevented by cycloheximide, a protein synthesis inhibitor. HEC maintained in the presence of IL 1 for 24 hr become refractory to restimulation by IL 1; however, IL 1-stimulated cells rested in the absence of IL 1 for 20 hr can be stimulated by fresh IL 1. HEC expression of the H4/18 binding protein is not induced by interleukin 2 or by interferon-alpha, -beta, or -gamma. Induction of H4/18 binding by TNF is also concentration dependent, transient, and dependent upon protein and RNA synthesis. Several observations suggest that IL1 and TNF act independently on HEC. Our TNF is a recombinant protein, expressed from a cloned cDNA and thus free of IL 1 contamination; it also has no activity in a highly sensitive IL 1 assay. Our standard IL 1 preparation is affinity purified and lacks TNF activity on L929 cells. Thus, our monokine preparations are not cross-contaminated. Most interestingly, HEC incubated with IL 1 and refractory to IL1 restimulation can be restimulated by TNF to express H4/18 binding and vice versa.(ABSTRACT TRUNCATED AT 400 WORDS)
    Document Type:
    Reference
    Product Catalog Number:
    ECM645