Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

From 1978 to 1982 at the Oncology Unit of the Rho Hospital, we followed 96 women who had been operated for breast cancer. In 22 cases (23%) the first signs of recurrence were changes in the following: physical examination (9), symptoms (7), ESR (3), bone scan (2), alkaline phosphatase (1), chest X-ray (1). An adequate follow-up schedule is based on the following: a) limited examinations causing little disturbance to the patient, easily feasible, sensitive, specific, and of limited cost; b) lead-intervals of various tests set according to the risk of relapse; c) critical periodic review of the series, with constant updating of information in the literature.
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PMID:Follow-up after mastectomy for breast cancer. Observations in 96 patients. 400 49

Human erythrocyte membranes were solubilized in sodium dodecyl sulfate at 100 degree C and subjected to polyacrylamide gel electrophoresis. The gels were sliced into segments and each segment was incubated with anti-Rho(D) IgG, washed, and then incubated with goat anti-human IgG covalently linked to alkaline phosphatase. Para-nitrophenyl phosphate was added to each slice and the absorbance of the solution surrounding each slice was measured at 405 nm. This technique demonstrated that the Rho (D) antigen is a protein with a mol, wt between 13, 000 and 30,000. This method should be applicable to the investigation of other membrane-bound antigens.
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PMID:Identification of Rho(D) antigen in polyacrylamide gels by an enzyme-linked immunoassay. 681 99

Specific [32P]ADP-ribosylation by Clostridium botulinum exoenzyme C3 was used to study the involvement of phosphorylation in the regulation of the low-molecular-mass GTP-binding protein Rho. Dephosphorylation of CHO cell extracts by alkaline phosphatase treatment resulted in a 80-90% reduction in the C3-catalysed [32P]ADP-ribosylation of Rho proteins in both cytosolic and membrane fractions. Similar results were obtained after dephosphorylation with protein phosphatase type-1 from bovine retina, whereas type-2B and type-2C phosphatases had no effect on the level of subsequent [32P]ADP-ribosylation of Rho by C3. Incubation of CHO cell lysate under phosphorylation conditions increased the subsequent C3-mediated [32P]ADP-ribosylation of Rho proteins. The protein kinase inhibitors H7 and H9 had no effect on [32P]ADP-ribosylation at concentrations which are specific for inhibition of protein kinase A or C. Recombinant glutathione S-transferase-RhoA fusion protein (GST-RhoA) was phosphorylated by protein kinase A; however, the phosphorylation had no stimulatory effect on the ADP-ribosylation of GST-RhoA by C3. An approx. 48 kDa phosphoprotein was identified which bound specifically to recombinant GST-RhoA fusion protein. By gel-permeation chromatography, Rho-containing complexes of approx. 50 kDa and 130-170 kDa were detected. The ADP-ribosylation of Rho in the 130-170 kDa complex was reduced by alkaline phosphatase pretreatment. The data suggest that Rho activity is influenced by phosphorylation of Rho-associated regulatory factors. Phosphorylation/dephosphorylation of these Rho-regulating factors appears to alter the ability of Rho to serve as a substrate for C3-induced [32P]ADP-ribosylation.
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PMID:ADP-ribosylation of Rho proteins by Clostridium botulinum exoenzyme C3 is influenced by phosphorylation of Rho-associated factors. 819 24

The GDP dissociation inhibitor Rho GDI from bovine neutrophil cytosol was purified in association with prenylated Rho A. Upon treatment of this complex with alkaline phosphatase, the Rho A and Rho GDI components were released to their free forms. Following migration in 2D-PAGE and specific immunodetection, the shape of the spot of Rho GDI was found to depend markedly on whether Rho GDI subjected to electrophoresis was present in a Rho A-Rho GDI complex or in a free form. In the first case Rho GDI focused as an elongated spot between pI 5.2 and pI 4.6 whereas in the later case it focused at a pI of 5.0-5.2 as a round spot. Activation of neutrophils by anaphylatoxin C5a in a [32Pi] supplemented medium resulted in radiolabeling of Rho GDI. In vitro incubation of Rho GDI with a neutrophil homogenate in the presence of [gamma 32P] ATP led also to radiolabeling of Rho GDI. Taken together these results suggest that Rho GDI in the Rho A-Rho GDI complex is phosphorylated and that the stability of the complex depends on the phosphorylation state of Rho GDI.
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PMID:Phosphorylation of Rho GDI stabilizes the Rho A-Rho GDI complex in neutrophil cytosol. 857 75

We have previously demonstrated that RGD peptides prevent tubular obstruction in ischemic acute renal failure (ARF) suggested that exposed unoccupied integrin receptors represent the target for such therapy. The present study investigated the topography of RGD binding sites and integrin receptors in ischemic rat kidneys. Two RGD peptides were synthesized: a cyclic biotinylated (Bt) RGD peptide and a linear RGD peptide (GRGDSP) labeled with rhodamine green (RhoG). Rats were subjected to 45 minutes of renal artery occlusion kidneys were harvested at different times post-ischemia, and stained with RGD peptides and a panel of antibodies to integrins. In control, Bt-RGD staining was undetectable in alkaline phosphatase histochemistry, whereas immunofluorescence detection with Rho-streptavidin conjugate as well as RhoG-GRGDSP staining faintly decorated the basolateral aspect of the proximal tubular cells in a punctate fashion. In contrast, ischemic kidneys showed binding to the basolateral and apical aspects of proximal tubules, peritubular capillaries, and desquamated cells within tubular lumen. The most conspicuous staining of ischemic kidneys was obtained with antibodies to the beta 1 (labeling of the apical aspect of proximal and distal tubules, as well as desquamated cells obstructing tubular lumen) and the alpha V (glomeruli, tubular epithelia, intima of blood vessels stained faintly, while the obstructing cellular conglomerates showed intense staining) subunits. Double staining with Bt-RGD and antibodies against the beta 1 and alpha V beta 3 integrins showed co-localization of staining within the tubules and vasculature, respectively. In vitro attachment of HL-60 leukocytes to the endothelial cells was inhibited by the cyclic RGD peptide. In conclusion, expression of RGD binding sites and beta 1 integrin subunits along the apical aspect of tubular epithelia and on the surface of desquamated cells is in concert with the hypothesis on the pathogenetic role of RGD-recognizing integrins in tubular obstruction. The expression of RGD binding sites along the intimal surface of blood vessels in ischemic kidneys suggests an additional target for RGD peptides in vascular endothelial cells.
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PMID:Two novel probes reveal tubular and vascular Arg-Gly-Asp (RGD) binding sites in the ischemic rat kidney. 921 51

The Rho GDP dissociation inhibitor (RhoGDI) regulates the activation-inactivation cycle of Rho small GTPases, such as Cdc42 and RhoA, by extracting them from the membrane. To study the roles of Mg(2+), phosphatidylinositol 4,5-bisphosphate (PIP(2)), ionic strength and phosphorylation on the interactions of RhoGDI with Cdc42 and RhoA, we developed a new, efficient and reliable method to produce prenylated Rho proteins using the yeast Saccharomyces cerevisiae. It has been previously reported that protein kinase A (PKA)-treatment of isolated membranes increased RhoA extraction from membranes by RhoGDI [Lang, Gesbert, Delespine-Carmagnat, Stancou, Pouchelet and Bertoglio (1996) EMBO J. 16, 510-519]. In the present study, we used an in vitro affinity chromatography system to show that phosphorylation of RhoA and Cdc42 significantly increased their interaction with RhoGDI under physiological conditions of ionic strength. This increase was independent of the nucleotide (GDP or guanosine 5'-[gamma-thio]triphosphate) loaded on to the Rho proteins, as well as of Mg(2+) and PIP(2). Moreover, dephosphorylation of rat brain membranes by alkaline phosphatase significantly decreased the extraction of RhoA and Cdc42 by RhoGDI. Subsequent re-phosphorylation by PKA restored the extraction levels, indicating the reversibility of this process. These results clearly demonstrate that the phosphorylation states of Cdc42 and RhoA regulate their interactions with RhoGDI and, consequently, their extraction from rat brain membranes. We therefore suggest that phosphorylation is a mechanism of regulation of Cdc42 and RhoA activity that is independent of GDP-GTP cycling.
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PMID:Phosphorylation states of Cdc42 and RhoA regulate their interactions with Rho GDP dissociation inhibitor and their extraction from biological membranes. 1177 96

Coordinated proliferation and differentiation of growth plate chondrocytes is required for normal growth and development of the endochondral skeleton, but little is known about the intracellular signal transduction pathways regulating these processes. We have investigated the roles of the GTPase RhoA and its effector kinases ROCK1/2 in hypertrophic chondrocyte differentiation. RhoA, ROCK1, and ROCK2 are expressed throughout chondrogenic differentiation. RhoA overexpression in chondrogenic ATDC5 cells results in increased proliferation and a marked delay of hypertrophic differentiation, as shown by decreased induction of alkaline phosphatase activity, mineralization, and expression of the hypertrophic markers collagen X, bone sialoprotein, and matrix metalloproteinase 13. These effects are accompanied by activation of cyclin D1 transcription and repression of the collagen X promoter by RhoA. In contrast, inhibition of Rho/ROCK signaling by the pharmacological inhibitor Y27632 inhibits chondrocyte proliferation and accelerates hypertrophic differentiation. Dominant-negative RhoA also inhibits induction of the cyclin D1 promoter by parathyroid hormone-related peptide. Finally, Y27632 treatment partially rescues the effects of RhoA overexpression. In summary, we identify the RhoA/ROCK signaling pathway as a novel and important regulator of chondrocyte proliferation and differentiation.
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PMID:RhoA/ROCK signaling suppresses hypertrophic chondrocyte differentiation. 1472 36

Some years ago we showed that the Pasteurella multocida toxin (PMT) is a potent mitogen for cells in culture. It is an intracellularly acting toxin that stimulates several signal transduction pathways. The heterotrimeric G-protein, Gq, is stimulated, which in turn causes activation of protein kinase C and an increase in inositol trisphosphates. The Rho GTPase is also activated, leading via the Rho kinase, to activation of the focal adhesion kinase and to cytoskeletal rearrangements. Analysis of the PMT sequence suggested the presence of three domains that encode receptor binding, translocation and catalytic domains. The location of all three domains has been confirmed directly. Competitive binding assays confirmed that the N-terminus of PMT encoded the receptor-binding domain, while cytoplasmic microinjection of expressed PMT fragments identified the location of the C-terminal catalytic domain. Recently, we have demonstrated the presence of key amino acids that affect membrane insertion within the putative transmembrane domain. Several lines of evidence suggest that PMT activates Galphaq, and that this is one potential molecular target for the toxin. Galphaq is known to be tyrosine phosphorylated when activated normally via a G-protein-coupled receptor (GPCR), and it has been suggested that this is an essential part of the activation process. We have shown that PMT induces Galphaq tyrosine phosphorylation, but that this is not essential for activation of the G-protein. Furthermore, a totally inactive mutant of PMT stimulates Galpha phosphorylation without leading to its activation. Phosphorylation of Galphaq triggered by the inactive mutant potentiates activation of Gq via a GPCR, demonstrating that phosphorylation of Gq cannot lead to receptor uncoupling. Natural or experimental infection of animals with toxigenic P. multocida, or injection with purified recombinant PMT causes loss of nasal turbinate bone. The effects on bone have been analysed in vitro using cultures of osteoblasts--cells that lay down bone. PMT blocks the formation of mature calcified bone nodules and the expression of differentiation markers such as CBFA-1, alkaline phosphatase and osteocalcin. These effects can be partially prevented by inhibitors of Rho or Rho kinase function, implicating this pathway in osteoblast differentiation. Indeed, inhibitors of Rho stimulate the formation of bone nodules in vitro. In summary, PMT is a novel toxin that acts via signalling pathways to promote proliferation in many cells, while specifically inhibiting differentiation in osteoblast cells.
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PMID:The pasteurella multocida toxin interacts with signalling pathways to perturb cell growth and differentiation. 1514 25

Although lipid-lowering therapy with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) decreases the progression of coronary artery and aortic valve calcification, the mechanism of action of these drugs to inhibit the calcification process remains unclear. In this study, we investigated the effect of statins such as cerivastatin and atorvastatin on vascular calcification by utilizing an in vitro model of inflammatory vascular calcification. Cerivastatin and atorvastatin dose-dependently inhibited in vitro calcification of human vascular smooth muscle cells (HVSMCs) induced by the following inflammatory mediators (IM): interferon-gamma, 1alpha,25-dihydroxyvitamin D3, tumor necrosis factor-alpha, and oncostatin M. These statins also depressed expression of alkaline phosphatase (ALP) in HVSMCs induced by these factors. Mevalonate and geranylgeranylpyrophosphate reversed the inhibitory effect of cerivastatin on ALP expression in HVSMCs, while farnesylpyrophosphate showed no effect on the ALP activities inhibited by this drug, suggesting that inhibition of Rho and its downstream target, Rho kinase may mediate the inhibitory effect of cerivastatin. Cerivastatin prevented RhoA activation in HVSMCs induced by the IM. A specific inhibitor of Rho kinase (Y-27632) inhibited in vitro calcification and induction of ALP in HVSMCs. These findings provide a possible mechanism of statins to prevent the progression of calcification in inflammatory vascular diseases such as atherosclerosis and cardiac valvular calcification.
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PMID:Statins inhibit in vitro calcification of human vascular smooth muscle cells induced by inflammatory mediators. 1538 84

Rho family proteins can coordinate multiple signaling pathways through their ability to regulate both gene transcription and the actin cytoskeleton. With respect to the neuronal Nogo receptor (NgR), recent data assign a key role for the GTPase Rho in the control of cellular responses leading to actin cytoskeletal rearrangements and finally resulting in axonal outgrowth inhibition and growth cone collapse in the adult human central nervous system. In order to evaluate potential NgR antagonists, human embryonic kidney 293 cells stably overexpressing RhoA in the absence or presence of NgR have been generated. RhoA activation induced by stimulation with the alkaline phosphatase-tagged NgR ligand Nogo66 (AP-Nogo66) was confirmed by affinity-precipitation of the GTPase with the Rho-binding domain from Rhotekin. As this pull-down assay is not applicable to a higher-throughput format, a cellular Rho GTPase activation assay strategy based on the ability of Rho to regulate the actin cytoskeleton was developed. Stimulation with L-alpha-lysophosphatidic acid (LPA), a Rho activator acting through the ubiquitiously expressed LPA receptors, induced significant cytoskeletal rearrangement resulting in cell contraction in all RhoA-overexpressing cell lines. In contrast, stimulation with AP-Nogo66 resulted in Rho-dependent cell contraction with a similar time course only in the NgR-expressing cell line. Moreover, the NgR-induced Rho-dependent morphological changes could be analyzed and quantified with customary image analysis software. In conclusion, the cytoskeletal rearrangement assay is amenable to automated high-content screening and has the potential to eliminate major technical bottlenecks of the pull-down assay. The increased throughput of the cellular GTPase activation assay compared with the biochemical method should facilitate the evaluation of compounds that modulate the actin cytoskeleton through Rho.
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PMID:A high-content screening assay for the Nogo receptor based on cellular Rho activation. 1671 17


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