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  • 1
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Under typical culture conditions, cerebellar granule cells die abruptly after 17 days in vitro. This burst of neuronal death involves ultrastructural changes and internucleosomal DNA fragmentations characteristic of apoptosis and is effectively arrested by pretreatment with actinomycin-D and cycloheximide. The level of a 38-kDa protein in the particulate fraction is markedly increased during age-induced cell death and by pretreatment with NMDA, which potentiates this cell death. Conversely, the age-induced increment of the 38-kDa particulate protein is suppressed by actinomycin-D and cycloheximide. N-terminal microsequencing of the 38-kDa protein revealed sequence identity with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A GAPDH antisense oligodeoxyribonucleotide blocks age-induced expression of the particulate 38-kDa protein and effectively inhibits neuronal apoptosis. In contrast, the corresponding sense oligonucleotide of GAPDH was completely ineffective in preventing the age-induced neuronal death and the 38-kDa protein overexpression. Moreover, the age-induced expression of the 38-kDa protein is preceded by a pronounced increase in the GAPDH mRNA level, which is abolished by actinomycin-D, cycloheximide, or the GAPDH antisense, but not sense, oligonucleotide. Thus, our results suggest that overexpression of GAPDH in the particulate fraction has a direct role in age-induced apoptosis of cerebellar neurons.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1442-2042
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Background:Prostatic acid phosphatase (PAP) is an important protein which should be studied further as a tumor marker or as a biologically functional molecule. The purpose of the study was to establish a simple and reliable method to obtain highly pure PAP. Methods: Spleen cells from mice immunized with prostatic epithelial cells prepared from benign prostatic hyperplasia tissue were fused with myeloma cells X63Ag8–653. Hybrid cells of interest were selected using the indirect immunofluorescence method with unfixed frozen tissue sections. One clone of the hybrid cell lines was established which secreted the monoclonal antibody specifically reactive to prostatic acid phosphatase. Using this monoclonal antibody, we purified the antigen from human prostatic tissue by means of single-step immunoaffinity chromatography.Results:SDS-PAGE profiling under reducing conditions indicated that the protein recognized by this antibody consisted of several components of molecular weight 41,000–45,000. Partial amino acid sequence analysis of this protein indicated that these components involved a heterogeneously modified single polypeptide, and that this antigen is identical to human prostatic acid phosphatase. Conclusions: This single-step method saves the time needed to purify prostatic acid phosphatase and requires only half a day for the whole procedure. Moreover, the purity of the isolated protein was extremely high. This method seems to be useful not only for purifying prostatic acid phosphatase but also for purifying other proteins from the prostate gland and for analysis of antigenic macromolecules.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1436-2813
    Keywords: Key Words: obstructive jaundice ; afferent loop syndrome ; biliary drainage
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1617-4623
    Keywords: Binding protein-dependent transport system ; Cyanobacterium ; Nitrate reductase ; Nitrate transport ; Nitrate assimilation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The nrtA gene, which has been proposed to be involved in nitrate transport of Synechococcus sp. PCC7942 (Anacystis nidulans R2), was mapped at 3.9 kb upstream of the nitrate reductase gene, narB. Three closely linked genes (designated nrtB, nrtC, and nrtD), which encode proteins of 279, 659, and 274 amino acids, respectively, were found between the nrtA and narB genes. NrtB is a hydrophobic protein having structural similarity to the integral membrane components of bacterial transport systems that are dependent on periplasmic substrate-binding proteins. The N-terminal portion of NrtC (amino acid residues 1–254) and NrtD are 58% identical to each other in their amino acid sequences, and resemble the ATP-binding components of binding protein-dependent transport systems. The C-terminal portion of NrtC is 30% identical to NrtA. Mutants constructed by interrupting each of nrtB and nrtC were unable to grow on nitrate, and the nrtD mutant required high concentration of nitrate for growth. The rate of nitrate-dependent O2 evolution (photosynthetic O2 evolution coupled to nitrate reduction) in wild-type cells measured in the presence of l-methionine d,l-sulfoximine and glycolaldehyde showed a dual-phase relationship with nitrate concentration. It followed saturation kinetics up to 10 mM nitrate (the concentration required for half-saturation = 1 μM), and the reaction rate then increased above the saturation level of the first phase as the nitrate concentration increased. The high-affinity phase of nitrate-dependent O2 evolution was absent in the nrtD mutant. The results suggest that there are two independent mechanisms of nitrate uptake and that the nrtB-nrtC-nrtD cluster encodes a high-affinity nitrate transport system.
    Type of Medium: Electronic Resource
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