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  • 1
    Electronic Resource
    Electronic Resource
    Immunological approach to identify Calmodulin-stimulated phosphatase isozymes from bovine brain (1994)
    Springer
    Molecular and cellular biochemistry 132 (1994), S. 101-108 
    Details
    ISSN: 1573-4919
    Keywords: CaM-stimulated phosphatase isozymes ; PP2B ; calcineurin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Molecular cloning of human, mouse and rat brain CaM-stimulated phosphatase has suggested the existence of two genes for the α subunit of the enzymes. Aα and Aβ fragments of Aα and Aβ from rat brain library have been expressed in bacteria to produce specific anti-calcineurin Aα and anti-calcineurin Aβ antibodies (Kunoet al., J Neurochem 58: 1643–1651, 1992). Alternative mRNA splicing gives rise to additional calcineurin isozymes with some containing an insertion sequence of ATVEAIEADE. Antibody against synthetic peptide of this insertion sequence has been raised in this study. Three CaM-stimulated phosphatase isozymes previously purified from bovine brain (BPI, BPII, BPIII) (Yokoyama & Wang, J Biol Chem 266: 14822–14829, 1991), along with the bacterially expressed rat Aα and Aβ fragments, were analyzed by two calcineurin α subunit monoclonal antibodies VJ6 and VD3, the rat anti-calcineurin Aα and anti-calcineurin Aβ specific polyclonal antibodies, and the insertion peptide antibody. The bovine brain CaM-stimulated phosphatase isozymes BPI and BPIII reacted with both anti-calcineurin Aα and anti-calcineurin Aβ antibodies. While BPII reacted with anti-calcineurin Aα but not anti-calcineurin Aβ antibody, it differed from the expressed Aα fragment in immunoreactivity towards the monoclonal antibodies. The results show that the bovine brain CaM-stimulated phosphatase isozymes cannot be simply categorized as derived from Aα or Aβ genes products. On the other hand, on the basis of immunoreactivity toward the insertion antibody, the isozymes can be readily classified into those containing the insertion sequence (BPI, LPI) and those without the inserting (BPII, BPIII, LPII, LPIII).
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00926918
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    Paper (German National Licenses)
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  • 2
    Electronic Resource
    Electronic Resource
    Activation of NFκB is necessary for IL-1β-induced cyclooxygenase-2 (COX-2) expression in human gingival fibroblasts (2000)
    Springer
    Molecular and cellular biochemistry 209 (2000), S. 113-118 
    Details
    ISSN: 1573-4919
    Keywords: COX-2 ; gene regulation ; IL-1β ; NFκB ; tyrosine phosphorylation ; human gingival fibroblasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The immediate-early cyclooxygenase-2 (COX-2) gene encodes an inducible prostaglandin synthase enzyme which is implicated in inflammatory and proliferative diseases. COX-2 is highly induced during cell activation by various factors, including mitogens, hormones and cytokines. Since pro-inflammatory cytokine IL-1β has been shown to induce prostaglandin E2 (PGE2) release in human gingival fibroblasts (HGF), here we analyzed the effect of IL-1β on the expression of COX-2 and the activation of NFκB in HGF. Northern hybridization analysis revealed that IL-1β (200 pg/ml) increased the expression of COX-2 mRNA in HGF. The effect of IL-1β was abrogated by herbimycin A, a protein tyrosine kinase inhibitor, and enhanced by orthovanadate, a protein tyrosine phosphatase inhibitor. IL-1β-induced PGE2 release was blocked by the tyrosine kinase inhibitor and increased by the tyrosine phosphatase inhibitor. The results of transient transfection assays using chimeric constructs of the human COX-2 promoter (nt -1432 ~ +59) ligated to a luciferase reporter gene indicated that IL-1β stimulated the transcriptional activity ~ 1.5-fold. Gel mobility shift assays with a radiolabelled COX-2-NFκB oligonucleotide (nts -223 to -214) revealed an increase in the binding of nuclear proteins from IL-1β-stimulated HGF. This increase of DNA-protein complex formation induced by IL-1β was blocked by herbimycin A and another tyrosine kinase inhibitor, genistein. These results suggest that NFκB is an important transcription factor for IL-1β-induced COX-2 gene expression, and is involved in inducing COX-2 gene transcription through tyrosine phosphorylation in HGF.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1007155525020
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  • 3
    Electronic Resource
    Electronic Resource
    Transforming growth factor-β1 regulation of bone sialoprotein gene transcription: Identification of a TGF-β activation element in the rat BSP gene promoter (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 65 (1997), S. 501-512 
    Details
    ISSN: 0730-2312
    Keywords: bone sialoprotein ; gene regulation ; mineralized tissues ; TGF-β1 ; transcription ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Transforming growth factor-β (TGF-β) increases steady-state mRNA levels of several extracellular matrix proteins in mineralized connective tissues. Bone sialoprotein (BSP) is a major constituent of the bone matrix, thought to initiate and regulate the formation of mineral crystals. To determine the molecular pathways of TGF-β1 regulation of bone proteins, we have analyzed the effects of the TGF-β1 on the expression of the BSP in the rat osteosarcoma cell line (ROS 17/2.8). TGF-β1 at 1 ng/ml, increased BSP mRNA levels in ROS 17/2.8 cells ∼8-fold; the stimulation was first evident at 3 hr, reached maximal levels at 12 hr and slowly declined thereafter. Since the stability of the BSP mRNA was not significantly affected by TGF-β1, and nuclear “run-on” transcription analyses revealed only a ∼2-fold increase in the transcription of the BSP gene, most of the increase in BSP mRNA appeared to involve a nuclear post-transcriptional mechanism. Moreover, the effects of TGF-β1 were indirect, since the increase in BSP mRNA was abrogated by cycloheximide (28 μg/ml). To identify the site of transcriptional regulation by TGF-β1, transient transfection analyses were performed using BSP gene promoter constructs linked to a luciferase reporter gene. Constructs that included nt -801 to -426 of the promoter sequence were found to enhance transcriptional activity ∼1.8-fold in cells treated with TGF-β1. Within this sequence, ∼500 nt upstream of the transcription start site, a putative TGF-β activation element (TAE) was identified that contained the 5′-portion of the nuclearfactor-1 (NF-1) canonical sequence (TTGGC) overlapping a consensus sequence for activator protein-2 (AP-2). The functionality of the TAE was shown by an increased binding of a nuclear protein from TGF-β1 stimulated cells in gel mobility shift assays and from the attenuation of TGF-β1-induced luciferase activity when cells were co-transfected with a double-stranded TAE oligonucleotide. Competition gel mobility shift analyses revealed that the nuclear protein that binds to the TAE has similar properties to, but is distinct from, NF-1 nuclear protein. These studies have therefore identified a TGF-β activation element (TAE) in the rat BSP gene promoter that mediates the stimulatory effects of TGF-β1 on BSP gene transcription. J. Cell. Biochem. 65:501-512. © 1997 Wiley-Liss Inc.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
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