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Phosphatase Activity (phosphatase + activity)
Kinds of Phosphatase Activity Selected AbstractsSmad3 Promotes Alkaline Phosphatase Activity and Mineralization of Osteoblastic MC3T3-E1 Cells,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2002Hideaki Sowa Abstract Transforming growth factor (TGF) , is abundantly stored in bone matrix and appears to regulate bone metabolism. Although the Smad family proteins are critical components of the TGF-, signaling pathways, the roles of Smad3 in the expression of osteoblastic phenotypes remain poorly understood. Therefore, this study was performed to clarify the roles of Smad3 in the regulation of proliferation, expression of bone matrix proteins, and mineralization in osteoblasts by using mouse osteoblastic cell line MC3T3-E1 cells stably transfected with Smad3. Smad3 significantly inhibited [3H]thymidine incorporation and fluorescent intensity of the MTT-dye assay, compared with empty vector. Moreover, Smad3 increased the levels of type I procollagen, osteopontin (OPN), and matrix Gla protein (MGP) mRNA in Northern blotting. These effects of Smad3 mimicked the effects of TGF-, on the same cells. On the other hand, Smad3 greatly enhanced ALP activity and mineralization of MC3T3-E1 cells compared with empty vector, although TGF-, inhibited ALP activity and mineralization of wild-type MC3T3-E1 cells. A type I collagen synthesis inhibitor L -azetidine-2-carboxylic acid, as well as osteocalcin (OCN), significantly antagonized Smad3-stimulated ALP activity and mineralization of MC3T3-E1 cells. In conclusion, this study showed that in mouse osteoblastic cells, Smad3 inhibited proliferation, but it also enhanced ALP activity, mineralization, and the levels of bone matrix proteins such as type I collagen (COLI), OPN, and MGP. We propose that Smad3 plays an important role in osteoblastic bone formation and might help to elucidate the transcriptional mechanism of bone formation and possibly lead to the development of bone-forming drugs. [source] Eyes Absent Proteins: Characterization of Substrate Specificity and Phosphatase Activity of Mutants Associated with Branchial, Otic and Renal AnomaliesCHEMBIOCHEM, Issue 14 2008Amna Musharraf Abstract The eyes absent (Eya) genes encode a family of proteins that combine the functions of transcriptional cofactors, signal transducers and enzymes, namely protein tyrosine phosphatases. The latter activity resides in the highly conserved C-terminal Eya domain (ED). Here, we investigated the substrate specificity of the Arabidopsis thaliana homologue (AtEya) by using low-molecular-weight compounds and synthetic phosphotyrosine (pY)-containing peptides that correspond either to phosphorylation sites in proteins or to peptides that were selected through the screening of a combinatorial peptide library. AtEya displayed modest peptide substrate specificity and was sensitive to charges adjacent to pY. In general, the presence of acidic residues on the N-terminal side of the phosphorylation site was critical for catalysis, whereas basic amino acids seemed to be preferred with respect to high-affinity binding. We also detected significant acyl phosphatase activity of AtEya; this suggests that Eya proteins might have further substrates in vivo. In addition, we analysed the phosphatase activity of a number of variants of the mouse Eya1 protein that harbours single point mutations that were associated with branchio,oto,renal syndrome (BOR), branchio,oto syndrome (BO) and ocular defects, respectively, in humans. While BOR mutations led to a significantly reduced phosphatase activity, BO mutants as well as those that are associated with ocular defects only displayed activity that was similar to wild-type levels. [source] Regulation of sperm flagellar motility activation and chemotaxis caused by egg-derived substance(s) in sea cucumberCYTOSKELETON, Issue 4 2009Masaya Morita Abstract The sea cucumber Holothuria atra is a broadcast spawner. Among broadcast spawners, fertilization occurs by means of an egg-derived substance(s) that induces sperm flagellar motility activation and chemotaxis. Holothuria atra sperm were quiescent in seawater, but exhibited flagellar motility activation near eggs with chorion (intact eggs). In addition, they moved in a helical motion toward intact eggs as well as a capillary filled with the water layer of the egg extracts, suggesting that an egg-derived compound(s) causes motility activation and chemotaxis. Furthermore, demembranated sperm flagella were reactivated in high pH (>7.8) solution without cAMP, and a phosphorylation assay using (,-32P)ATP showed that axonemal protein phosphorylation and dephosphorylation also occurred in a pH-dependent manner. These results suggest that the activation of sperm motility in holothurians is controlled by pH-sensitive changes in axonemal protein phosphorylation. Ca2+ concentration affected the swimming trajectory of demembranated sperm, indicating that Ca2+ -binding proteins present at the flagella may be associated with regulation of flagellar waveform. Moreover, the phosphorylation states of several axonemal proteins were Ca2+ -sensitive, indicating that Ca2+ impacts both kinase and phosphatase activities. In addition, in vivo sperm protein phosphorylation occurred after treatment with a water-soluble egg extract. Our results suggest that one or more egg-derived compounds activate motility and subsequent chemotactic behavior via Ca2+ -sensitive flagellar protein phosphorylation. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Extracellular phosphatase activity of natural plankton studied with ELF97 phosphate: fluorescence quantification and labelling kineticsENVIRONMENTAL MICROBIOLOGY, Issue 6 2003í Nedoma Summary ELF®97 phosphate (ELFP) is a phosphatase substrate which produces ELF®97 alcohol (ELFA), a fluorescent water-insoluble product, upon hydrolysis. We studied the kinetics of ELFA precipitation in freshwater samples at levels of total plankton and single phytoplankton cells, and tested the suitability of ELFP for measurement of surface-bound algal extracellular phosphatases. Samples from acidic Ple,né Lake (pH , 5; high phosphatase activity) and eutrophic ,ímov reservoir (pH ,7,10; moderate phosphatase activity) were incubated with ELFP for 5,300 min, fixed with HgCl2 and filtered through polycarbonate filters. Relative fluorescence of filter-retained ELFA precipitates was quantified with image analysis. Time-courses of ELFA formation exhibited lag periods followed by finite periods of linear increase. In Ple,né Lake, lag-times were shorter (1,18 min) and rates of increase in ELFA fluorescence higher (by ,2 orders of magnitude) than in ,ímov reservoir (lag-times 30,200 min). Similar patterns of ELFA formation kinetics were also observed in Ple,né Lake samples in cuvette spectrofluorometer measurements (which failed in ,ímov reservoir). Linear regression of seasonal data on rates of increase in ELFA fluorescence from image cytometry and spectrofluorometry (r2 = 0.65, n = 10) allowed for calibration of image cytometry in terms of amount of cell-associated ELFA. Preliminary measurements of extracellular phosphatase activities of several algae resulted in rates (10,2260 fmol cell,1 h,1) which are comparable to data reported in the literature for algal cultures. [source] Aminopeptidase and phosphatase activities in basins of Lake Hiidenvesi dominated by cyanobacteria and in laboratory grown AnabaenaFRESHWATER BIOLOGY, Issue 9 2002JAANA VAITOMAA 1.,Extracellular enzyme activities were examined in freshwater basins representing a transition from hypertrophy to mesotrophy and in axenic cyanobacterial cultures to evaluate the ecological role of extracellular enzyme activities of cyanobacteria. 2.,Aminopeptidase activity was related to the trophic status of the lake basins. The activity was highest in the most eutrophic basin and decreased in the less nutrient-rich basins. Cyanobacteria were the most important autotrophic organisms and aminopeptidase activity was positively associated with cyanobacterial biomass. 3.,In an axenic Anabaena batch culture, nitrogenase activity was several orders of magnitude higher than leucine aminopeptidase activity. Nitrate did not have an effect on aminopeptidase activity or growth, but significantly reduced the rate of nitrogen fixation. A high phosphorus concentration at the beginning of the Anabaena batch-culture experiment resulted in reduced phosphatase activity. 4.,In Lake Hiidenvesi, aminopeptidase activity probably originated mostly from attached bacteria and less so from cyanobacteria. [source] Rapid in vitro conversion of fosphenytoin into phenytoin in sera of patients with liver disease: Role of alkaline phosphatase ,JOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 5 2001Amitava Dasgupta Abstract Fosphenytoin, a phosphate ester pro drug of phenytoin, also cross-reacts with the fluorescence polarization immunoassay (FPIA) for phenytoin. We measured fosphenytoin concentrations using the FPIA kit and TDx analyzer. We prepared serum pools from normal volunteers and patients with liver disease. None of them received either fosphenytoin or phenytoin. Fosphenytoin standard solution (1 mg/ml) was prepared in water. We supplemented aliquots of normal and liver pools with known amounts of fosphenytoin and measured the concentrations at different time intervals. The conversion of fosphenytoin to phenytoin was slow in sera with normal alkaline phosphatase activities. The conversion was rapid in sera collected from patients with liver disease who also had high alkaline phosphatase activities. The observed concentrations were close to target concentrations within 0,2 min of supplementation with fosphenytoin. Surprisingly, the observed concentration then started to decline slightly but significantly with longer incubation time. In contrast, the observed concentration increased steadily in serum with normal alkaline phosphatase activity. For example, in the normal pool supplemented with 15.0 ,g/ml fosphenytoin (as the phenytoin equivalent), the observed concentrations were 6.9, 7.3, 7.7, 8.3, and 9.8 ,g/ml at 0,2, 10, 20, 30, and 60 min, respectively. However, in a serum pool prepared from patients with liver disease and supplemented with 15.0 ,g/ml of fosphenytoin (alkaline phosphatase: 2547 U/l), the observed phenytoin concentrations were 12.9, 12.1, 11.0, 10.7, and 10.7 ,g/ml at 0,2, 10, 20, 30, and 60 min, respectively. When we added alkaline phosphatase to the normal serum pool, we observed rapid conversion of fosphenytoin into phenytoin within 10 min, but the concentrations then declined with longer incubation time. However, when we repeated the experiment with protein-free ultrafiltrate, we observed rapid conversion of fosphenytoin to phenytoin, but the concentration did not decline with longer incubation time. J. Clin. Lab. Anal. 15:244,250, 2001. © 2001 Wiley-Liss, Inc. [source] Impaired long-term depression in P2X3 deficient mice is not associated with a spatial learning deficitJOURNAL OF NEUROCHEMISTRY, Issue 5 2006Yue Wang Abstract The hippocampus is a brain region critical for learning and memory processes believed to result from long-lasting changes in the function and structure of synapses. Recent findings suggest that ATP functions as a neurotransmitter or neuromodulator in the mammalian brain, where it activates several different types of ionotropic and G protein-coupled ATP receptors that transduce calcium signals. However, the roles of specific ATP receptors in synaptic plasticity have not been established. Here we show that mice lacking the P2X3 ATP receptor (P2X3KO mice) exhibit abnormalities in hippocampal synaptic plasticity that can be restored by pharmacological modification of calcium-sensitive kinase and phosphatase activities. Calcium imaging studies revealed an attenuated calcium response to ATP in hippocampal neurons from P2X3KO mice. Basal synaptic transmission, paired-pulse facilitation and long-term potentiation are normal at synapses in hippocampal slices from P2X3KO. However, long-term depression is severely impaired at CA1, CA3 and dentate gyrus synapses. Long-term depression can be partially rescued in slices treated with a protein phosphatase 1,2 A activator or by postsynaptic inhibition of calcium/calmodulin-dependent protein kinase II. Despite the deficit in hippocampal long-term depression, P2X3KO mice performed normally in water maze tests of spatial learning, suggesting that long-term depression is not critical for this type of hippocampus-dependent learning and memory. [source] Arbuscular mycorrhizal fungi respond to the substrate pH of their extraradical mycelium by altered growth and root colonizationNEW PHYTOLOGIST, Issue 1 2002Ingrid M. Van Aarle Summary ,,To test the response of arbuscular mycorrhizal (AM) fungi to a difference in soil pH, the extraradical mycelium of Scutellospora calospora or Glomus intraradices, in association with Plantago lanceolata, was exposed to two different pH treatments, while the root substrate pH was left unchanged. ,,Seedlings of P. lanceolata, colonized by one or other of the fungal symbionts, and nonmycorrhizal controls, were grown in mesh bags placed in pots containing pH-buffered sand (pH around 5 or 6). The systems were harvested at approximately 2-wk intervals between 20 and 80 d. ,,Both fungi formed more extraradical mycelium at the higher pH. Glomus intraradices formed almost no detectable extraradical mycelium at lower pH. The extraradical mycelium of S. calospora had higher acid phosphatase activity than that of G. intraradices. Total AM root colonization decreased for both fungi at the higher pH, and high pH also reduced arbuscule and vesicle formation in G. intraradices. ,,In conclusion, soil pH influences AM root colonization as well as the growth and phosphatase activities of extraradical mycelium, although the two fungi responded differently. [source] Effects of dietary protein levels on the growth performance, digestive capacity and amino acid metabolism of juvenile Jian carp (Cyprinus carpio var. Jian)AQUACULTURE RESEARCH, Issue 9 2009Yong Liu Abstract This experiment was conducted to evaluate the effects of protein levels on the growth performance, digestive capacity and amino acid metabolism of juvenile Jian carp. Brown fish meal was used as the sole protein source in the present study. Six isoenergetic experimental diets containing 14.4 MJ kg,1 of digestible energy and 220,495 g crude protein kg,1 diets were fed to triplicate groups of 50 fish with a mean initial weight of 16.67 ± 0.01 g for 45 days. Per cent weight gain (PWG) and feed efficiency ratio (FER) improved with an increase in the dietary protein levels up to 330 g kg,1 diet. The condition factor, relative gut length, intestinal folds height, hepatopancreas and intestine protein content improved with an increase in the protein levels up to 330,385 g kg,1 diet. Trypsin, creatinkinase, Na+, K+ -ATPase and alkaline phosphatase activities generally followed the same tendency as that of growth parameters. Amylase and ,-glutamyl transpeptidase (,-GT) activities were negatively correlated with increasing protein levels from 220 to 330 g kg,1 diet, and no differences were found thereafter. Lipase activity was unaffected by protein levels. Lactobacillus amount was increased with protein levels up to 275 g kg,1 diet, while Aeromonas amount followed the opposite pattern. Escherichia coli amount was not influenced by dietary protein levels. Glutamate,oxaloacetate transaminase (GOT) activities in the hepatopancreas and plasma ammonia concentration (PAC) were not influenced by protein levels between 220 and 275 g kg,1 diet, but significantly increased with increasing protein levels from 275 to 440 g kg,1 diet, and remained similar thereafter. Glutamate,pyruvate transaminase (GPT) activities significantly increased with protein levels >275 g kg,1 diet. Based on the broken-line model, the dietary protein requirement for PWG of Jian carp (16.7,55.0 g) was estimated to be 341 g kg,1 diet with a digestible energy of 14.4 MJ kg,1 diet. [source] Comparative evaluation of human embryonic stem cell lines derived from zygotes with normal and abnormal pronucleiDEVELOPMENTAL DYNAMICS, Issue 2 2010Qing Huan Abstract Human embryonic stem (hES) cell lines have been derived from normally or abnormally fertilized zygotes. However, the similar and different properties of these two types of hES cell lines are not well-known. To address this question, we generated nine hES cell lines from zygotes containing normal (2PN) and abnormal (0PN, 1PN, 3PN) pronuclei. A side-by-side comparison showed that all cell lines exhibited distinct identity and karyotypical stability. They expressed similar "stemness" markers and alkaline phosphatase activity and differentiated into three embryonic germ lineages in embryoid bodies and teratomas. Under neural differentiation-promoting conditions, they were directed into neural progenitors and neurons. However, a variation in cell cycle and the relative abundance of gene expression of undifferentiated and differentiated markers were observed. These variations were also seen among individually derived normal hES cell lines. Thus, normal hES cell lines can be developed from fertilized zygotes with abnormal pronuclei usually excluded from clinical use. Developmental Dynamics 239:425,438, 2010. © 2009 Wiley-Liss, Inc. [source] Amperometric Algal Chlorella vulgaris Cell Biosensors Based on Alginate and Polypyrrole-Alginate GelsELECTROANALYSIS, Issue 11 2006Rodica Abstract The successful development and analytical performances of two biosensor configurations based on the entrapment of algal cells of Chlorella vulgaris into either a regular alginate gel or a newly synthesized pyrrole-alginate matrix are reported. These biosensors were compared in terms of their amperometric current measurements to p -nitrophenyl phosphate when used as substrate for the detection of an algal alkaline phosphatase activity. The high stability of the pyrrole-alginate gel when compared to that of the alginate coating is herein demonstrated. [source] Molecular analysis of the phosphorus starvation response in Trichodesmium spp.ENVIRONMENTAL MICROBIOLOGY, Issue 9 2009Elizabeth D. Orchard Summary The marine diazotroph Trichodesmium is a major contributor to primary production and nitrogen fixation in the tropical and subtropical oceans. These regions are often characterized by low phosphorus (P) concentrations, and P starvation of Trichodesmium could limit growth, and potentially constrain nitrogen fixation. To better understand how this genus responds to P starvation we examined four genes involved in P acquisition: two copies of a high-affinity phosphate binding protein (pstS and sphX) and two putative alkaline phosphatases (phoA and phoX). Sequence analysis of these genes among cultured species of Trichodesmium (T. tenue, T. erythraeum, T. thiebautii and T. spiralis) showed that they all are present and conserved within the genus. In T. erythraeum IMS101, the expression of sphX, phoA and phoX were sensitive to P supply whereas pstS was not. The induction of alkaline phosphatase activity corresponded with phoA and phoX expression, but enzyme activity persisted after the expression of these genes returned to basal levels. Additionally, nifH (nitrogenase reductase; involved in nitrogen fixation) expression was downregulated under P starvation conditions. These data highlight molecular level responses to low P and lay a foundation for better understanding the dynamics of Trichodesmium P physiology in low-P environments. [source] Extracellular phosphatase activity of natural plankton studied with ELF97 phosphate: fluorescence quantification and labelling kineticsENVIRONMENTAL MICROBIOLOGY, Issue 6 2003í Nedoma Summary ELF®97 phosphate (ELFP) is a phosphatase substrate which produces ELF®97 alcohol (ELFA), a fluorescent water-insoluble product, upon hydrolysis. We studied the kinetics of ELFA precipitation in freshwater samples at levels of total plankton and single phytoplankton cells, and tested the suitability of ELFP for measurement of surface-bound algal extracellular phosphatases. Samples from acidic Ple,né Lake (pH , 5; high phosphatase activity) and eutrophic ,ímov reservoir (pH ,7,10; moderate phosphatase activity) were incubated with ELFP for 5,300 min, fixed with HgCl2 and filtered through polycarbonate filters. Relative fluorescence of filter-retained ELFA precipitates was quantified with image analysis. Time-courses of ELFA formation exhibited lag periods followed by finite periods of linear increase. In Ple,né Lake, lag-times were shorter (1,18 min) and rates of increase in ELFA fluorescence higher (by ,2 orders of magnitude) than in ,ímov reservoir (lag-times 30,200 min). Similar patterns of ELFA formation kinetics were also observed in Ple,né Lake samples in cuvette spectrofluorometer measurements (which failed in ,ímov reservoir). Linear regression of seasonal data on rates of increase in ELFA fluorescence from image cytometry and spectrofluorometry (r2 = 0.65, n = 10) allowed for calibration of image cytometry in terms of amount of cell-associated ELFA. Preliminary measurements of extracellular phosphatase activities of several algae resulted in rates (10,2260 fmol cell,1 h,1) which are comparable to data reported in the literature for algal cultures. [source] Chronic toxicity and responses of several important enzymes in Daphnia magna on exposure to sublethal microcystin-LRENVIRONMENTAL TOXICOLOGY, Issue 3 2005Wei Chen Abstract In the current study, the toxicological mechanisms of microcystin-LR and its disadvantageous effects on Daphnia magna were examined. Survival rate, number of newborn, activity of several important enzymes [glutathione S-transferase (GST), lactate dehydrogenase (LDH), phosphatases, and glutathione], accumulated microcystins, and ultrastructural changes in different organs of Daphnia were monitored over the course of 21-day chronic tests. The results indicated that low concentrations of dissolved microcystin had no harmful effect on Daphnia. On the contrary, stimulatory effects were detected. In the presence of toxin at high dosage and for long-term exposure, GST and glutathione levels decreased significantly. The decreased enzyme activity in the antioxidant system probably was caused by detoxification reactions with toxins. And these processes of detoxification at the beginning of chronic tests may enable phosphatases in Daphnia magna to withstand inhibition by the toxins. At the same time, we also found that the LDH activity in test animals increased with exposure to microcystin-LR, indicating that adverse effects occurred in Daphnia. With microcystin given at a higher dosage or for a longer exposure, the effect on Daphnia magna was fatal. In the meantime, microcystin began to accumulate in Daphnia magna, and phosphatase activity started to be inhibited. From the ultrastructure results of cells in D. magna, we obtained new information: the alimentary canal may be the target organ affected by exposure of microcystins to D. magna. The results of the current study also suggested that the oxidative damage and PPI (protein phosphatase inhibition) mechanisms of vertebrates also are adapted to Daphnia. © 2005 Wiley Periodicals, Inc. Environ Toxicol 20: 323,330, 2005. [source] Evaluation of the ishikawa cell line bioassay for the detection of estrogenic substances from sediment extractsENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 7 2005Shinya Hashimoto Abstract This study examines the application of Ishikawa human endometrial adenocarcinoma cells to measure the estrogenic activity of fractionated extracts of sediments from Tokyo Bay, Japan. Estrogen stimulates alkaline phosphatase activity in this cell line. The results of these assays were compared with those of a yeast estrogen screen (YES) assay. The Ishikawa cell line bioassay showed higher sensitivity to 17,-estradiol (median effective concentration [EC50], 10.7 pM) than did the YES assay (EC50, 480 pM). Fractionation of sediment extracts (all samples collected from 5 sites) showed that the nonpolar fraction was poisonous to yeast cells; the estrogenic activity of this fraction, therefore, could not be measured by YES. However, the nonpolar fraction did not kill the Ishikawa cells. The 17,-estradiol-equivalent values of 15 extracts (3 fractions from each of 5 sediment samples) ranged from 5.7 to 697 pg/g dry weight according to the Ishikawa cell line bioassay. Chemical analysis using gas chromatography-mass spectrometry revealed that the highest concentrations of endocrine-disrupting chemicals were observed at the sampling station near the sewage treatment plant. The results support that the Ishikawa cell line bioassay is suitable for measuring the estrogenic activity of sediment samples. [source] Association of non-alcoholic steatohepatitis (NASH) with chronic neutrophilic leukemiaEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 3 2004Chikashi Yoshida Abstract: A 54-yr-old female having chronic neutrophilic leukemia (CNL) associated with severe liver injury is presented. Physical examination on admission showed severe jaundice, hepatosplenomegaly, massive ascites, and pretibial edema. Complete blood count showed a hemoglobin level of 9.1 g/dL, platelet count of 25.8 × 104/,L, and white blood cell count of 36.6 × 103/,L with 89.7% neutrophils. Blood chemistry showed hyperbilirubinemia (21.9 mg/dL) with normal transaminase levels. There was no abnormality in serum cholesterol, triglyceride, or glucose levels. Neutrophil alkaline phosphatase activity was significantly elevated. Bone marrow aspiration showed myeloid hyperplasia with normal karyotype. Rearrangement of the bcr/abl was not detected by either polymerase chain reaction or fluorescence in situ hybridization. Human androgen receptor gene assay (HUMARA) of the bone marrow cells showed clonal proliferation of neutrophils. The patient was diagnosed as having CNL. To evaluate the pathogenesis of the liver injury, a needle biopsy was performed, which showed steatohepatitis with infiltration of neutrophils. As the patient had no history of alcohol abuse, a diagnosis of non-alcoholic steatohepatitis (NASH) was made. Assuming that the infiltration of abnormal neutrophils into the liver contributed to the development of NASH, she was treated with cytoreductive chemotherapy (cytosine arabinoside: 100 mg/d, 1,3 doses/wk). With decreases in white blood cell counts, serum bilirubin levels decreased gradually to 1.5 mg/mL. A postchemotherapy liver biopsy specimen showed marked improvement of the fatty degenerative change. To our knowledge, this is the first report describing the development of NASH in a myeloproliferative disorder. We believe that the infiltration of leukemic cells contributed to the development of NASH in this patient. [source] Molecular cloning and expression regulation of PRG-3, a new member of the plasticity-related gene familyEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2004Nicolai E. Savaskan Abstract Phospholipid-mediated signalling on neurons provokes diverse responses such as neurogenesis, pattern formation and neurite remodelling. We have recently uncovered a novel set of molecules in the mammalian brain, named plasticity-related genes (PRGs), which mediate lipid phosphate phosphatase activity and provide evidence for their involvement in mechanisms of neuronal plasticity. Here, we report on a new member of the vertebrate-specific PRG family, which we have named plasticity-related gene-3 (PRG-3). PRG-3 is heavily expressed in the brain and shows a specific expression pattern during brain development where PRG-3 expression is found predominantly in neuronal cell layers and is already expressed at embryonic day 16. In the mature brain, strongest PRG-3 expression occurs in the hippocampus and cerebellum. Overexcitation of neurons induced by kainic acid leads to a transient down-regulation of PRG-3. Furthermore, PRG-3 is expressed on neurite extensions and promotes neurite growth and a spreading-like cell body in neuronal cells and COS-7 cells. In contrast to previously described members of the PRG family, PRG-3 does not perform its function through enzymatic phospholipid degradation. In summary, our findings feature a new member of the PRG family which shows dynamic expression regulation during brain development and neuronal excitation. [source] Lipopolysaccharide alters decorin and biglycan synthesis in rat alveolar bone osteoblasts: consequences for bone repair during periodontal diseaseEUROPEAN JOURNAL OF ORAL SCIENCES, Issue 3 2008Helen C. Roberts A prime pathogenic agent associated with periodontitis is lipopolysaccharide (LPS) derived from Porphyromonas gingivalis. This study investigated the effects of P. gingivalis LPS on osteoblasts, which are responsible for alveolar bone repair. Bone cells were obtained from explants of rat alveolar bone chips and cultured with 0,200 ng ml,1 of P. gingivalis LPS. Porphyromonas gingivalis LPS significantly increased cell proliferation and inhibited osteoblast differentiation, as judged by reduced alkaline phosphatase activity. Analysis of biglycan mRNA and protein levels indicated that P. gingivalis LPS significantly delayed the normally high expression of biglycan during the early stages of culture, which are associated with cell proliferation and early differentiation of progenitor cells. In the presence of P. gingivalis LPS, decorin expression by the alveolar bone cells was reduced during periods of culture relating to collagen fibrillogenesis and mineral deposition. Analysis of glycosaminoglycan chains conjugated to these proteoglycans suggested that in the presence of P. gingivalis LPS, dermatan sulfate persisted within the matrix. This study suggests that P. gingivalis LPS influences the expression and processing of decorin and biglycan in the matrix, altering alveolar bone cell activity and osteoblast phenotype development. The consequences of this altered expression in relation to hindering bone repair as part of the cycle of events during periodontal disease are discussed. [source] Chronic Hypoxia Induces Prolonged Angiogenesis in Skeletal Muscles of RatEXPERIMENTAL PHYSIOLOGY, Issue 3 2002D. Deveci Skeletal muscle capillarity and fibre cross-sectional area were investigated within and between diaphragm (Diaph), extensor digitorum longus (EDL), soleus (SOL) and tibialis anterior (TA) muscles of control and chronic hypoxic (12% O2 for 6 weeks) adult male Wistar rats (final body mass ,355 g). Cryostat sections were stained for alkaline phosphatase activity to depict all capillaries, and for succinic dehydrogenase to demonstrate regional differences in oxidative capacity within the muscles. Hypoxia-induced angiogenesis occurred in all muscles (P < 0.01), with capillary-to-fibre ratio (C:F) being higher in the more active and oxidative muscles, Diaph (27%) and SOL (26%), than phasically active and glycolytic muscles, TA (21%) and EDL (15%). Diaph, SOL and EDL maintained fibre size, and hence showed an increased capillary density (CD) and reduced intramuscular diffusion distance (DD), whereas TA showed fibre hypertrophy and maintained CD and DD compared to control muscles. The extent of angiogenesis among different regions of muscle varied so as to suggest that muscle fibre size has an additional influence on capillary growth during chronic systemic hypoxia, which is progressive over an extended period of systemic hypoxia. [source] Molecular basis of perinatal hypophosphatasia with tissue-nonspecific alkaline phosphatase bearing a conservative replacement of valine by alanine at position 406FEBS JOURNAL, Issue 11 2008Structural importance of the crown domain Hypophosphatasia, a congenital metabolic disease related to the tissue-nonspecific alkaline phosphatase gene (TNSALP), is characterized by reduced serum alkaline phosphatase levels and defective mineralization of hard tissues. A replacement of valine with alanine at position 406, located in the crown domain of TNSALP, was reported in a perinatal form of hypophosphatasia. To understand the molecular defect of the TNSALP (V406A) molecule, we examined this missense mutant protein in transiently transfected COS-1 cells and in stable CHO-K1 Tet-On cells. Compared with the wild-type enzyme, the mutant protein showed a markedly reduced alkaline phosphatase activity. This was not the result of defective transport and resultant degradation of TNSALP (V406A) in the endoplasmic reticulum, as the majority of newly synthesized TNSALP (V406A) was conveyed to the Golgi apparatus and incorporated into a cold detergent insoluble fraction (raft) at a rate similar to that of the wild-type TNSALP. TNSALP (V406A) consisted of a dimer, as judged by sucrose gradient centrifugation, suggestive of its proper folding and correct assembly, although this mutant showed increased susceptibility to digestion by trypsin or proteinase K. When purified as a glycosylphosphatidylinositol-anchorless soluble form, the mutant protein exhibited a remarkably lower Kcat/Km value compared with that of the wild-type TNSALP. Interestingly, leucine and isoleucine, but not phenylalanine, were able to substitute for valine, pointing to the indispensable role of residues with a longer aliphatic side chain at position 406 of TNSALP. Taken together, this particular mutation highlights the structural importance of the crown domain with respect to the catalytic function of TNSALP. [source] Aldehydes release zinc from proteins.FEBS JOURNAL, Issue 18 2006A pathway from oxidative stress/lipid peroxidation to cellular functions of zinc Oxidative stress, lipid peroxidation, hyperglycemia-induced glycations and environmental exposures increase the cellular concentrations of aldehydes. A novel aspect of the molecular actions of aldehydes, e.g. acetaldehyde and acrolein, is their reaction with the cysteine ligands of zinc sites in proteins and concomitant zinc release. Stoichiometric amounts of acrolein release zinc from zinc,thiolate coordination sites in proteins such as metallothionein and alcohol dehydrogenase. Aldehydes also release zinc intracellularly in cultured human hepatoma (HepG2) cells and interfere with zinc-dependent signaling processes such as gene expression and phosphorylation. Thus both acetaldehyde and acrolein induce the expression of metallothionein and modulate protein tyrosine phosphatase activity in a zinc-dependent way. Since minute changes in the availability of cellular zinc have potent effects, zinc release is a mechanism of amplification that may account for many of the biological effects of aldehydes. The zinc-releasing activity of aldehydes establishes relationships among cellular zinc, the functions of endogenous and xenobiotic aldehydes, and redox stress, with implications for pathobiochemical and toxicologic mechanisms. [source] Characterization of a eukaryotic type serine/threonine protein kinase and protein phosphatase of Streptococcus pneumoniae and identification of kinase substratesFEBS JOURNAL, Issue 5 2005Linda Nováková Searching the genome sequence of Streptococcus pneumoniae revealed the presence of a single Ser/Thr protein kinase gene stkP linked to protein phosphatase phpP. Biochemical studies performed with recombinant StkP suggest that this protein is a functional eukaryotic-type Ser/Thr protein kinase. In vitro kinase assays and Western blots of S. pneumoniae subcellular fractions revealed that StkP is a membrane protein. PhpP is a soluble protein with manganese-dependent phosphatase activity in vitro against a synthetic substrate RRA(pT)VA. Mutations in the invariant aspartate residues implicated in the metal binding completely abolished PhpP activity. Autophosphorylated form of StkP was shown to be a substrate for PhpP. These results suggest that StkP and PhpP could operate as a functional pair in vivo. Analysis of phosphoproteome maps of both wild-type and stkP null mutant strains labeled in vivo and subsequent phosphoprotein identification by peptide mass fingerprinting revealed two possible substrates for StkP. The evidence is presented that StkP can phosphorylate in vitro phosphoglucosamine mutase GlmM which catalyzes the first step in the biosynthetic pathway leading to the formation of UDP- N -acetylglucosamine, an essential common precursor to cell envelope components. [source] Study of the subunit interactions in myosin phosphatase by surface plasmon resonanceFEBS JOURNAL, Issue 6 2000Attila Tóth The interactions of the catalytic subunit of type 1 protein phosphatase (PP1c) and the N-terminal half (residues 1,511) of myosin phosphatase target subunit 1 (MYPT1) were studied. Biotinylated MYPT1 derivatives were immobilized on streptavidin-biosensor chips, and binding parameters with PP1c were determined by surface plasmon resonance (SPR). The affinity of binding of PP1c was: MYPT11,296 > MYPT11,38 > MYPT123,38. No binding was detected with MYPT11,34, suggesting a critical role for residues 35,38, i.e. the PP1c binding motif. Binding of residues 1,22 was inferred from: a higher affinity binding to PP1c for MYPT11,38 compared to MYPT123,38, as deduced from SPR kinetic data and ligand competition assays; and an activation of the myosin light chain phosphatase activity of PP1c by MYPT11,38, but not by MYPT123,38. Residues 40,296 (ankyrin repeats) in MYPT11,296 inhibited the phosphorylase phosphatase activity of PP1c (IC50 = 0.2 nm), whereas MYPT11,38, MYPT123,38 or MYPT11,34 were without effect. MYPT140,511, which alone did not bind to PP1c, showed facilitated binding to the complexes of PP1c,MYPT11,38 and PP1c,MYPT123,38. The inhibitory effect of MYPT140,511 on the phosphorylase phosphatase activity of PP1c also was increased in the presence of MYPT11,38. The binding of MYPT1304,511 to complexes of PP1c and MYPT11,38, or MYPT11,296, was detected by SPR. These results suggest that within the N-terminal half of MYPT1 there are at least four binding sites for PP1c. The essential interaction is with the PP1c-binding motif and the other interactions are facilitated in an ordered and cooperative manner. [source] Distribution of ecto 5,-nucleotidase on Mycoplasma species associated with arthritisFEMS MICROBIOLOGY LETTERS, Issue 1 2000Sheena Johnson Abstract The enzyme ecto 5,-nucleotidase (5,N) was found to be active on 8/14 strains of Mycoplasma fermentans, Km (±S.D.) 3.8±2.8 ,M 5,-AMP, and on the type strain of Mycoplasma pulmonis, Km 0.63 ,M 5,-AMP. The six M. fermentans strains lacking 5,N activity were related by restriction fragment length polymorphism typing. At pH 8.5, the type strains of Mycoplasma arthritidis, Mycoplasma buccale and Ureaplasma urealyticum showed a relatively non-specific phosphatase activity against 5,-AMP but no activity was shown by the type strains of Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma orale, Mycoplasma penetrans, Mycoplasma pneumoniae and Mycoplasma salivarium at this pH. M. fermentans has been reported from rheumatoid joints, which show a raised 5,N activity on their synovial cells and in their fluid which may be associated directly or indirectly with the mycoplasma. [source] Aminopeptidase and phosphatase activities in basins of Lake Hiidenvesi dominated by cyanobacteria and in laboratory grown AnabaenaFRESHWATER BIOLOGY, Issue 9 2002JAANA VAITOMAA 1.,Extracellular enzyme activities were examined in freshwater basins representing a transition from hypertrophy to mesotrophy and in axenic cyanobacterial cultures to evaluate the ecological role of extracellular enzyme activities of cyanobacteria. 2.,Aminopeptidase activity was related to the trophic status of the lake basins. The activity was highest in the most eutrophic basin and decreased in the less nutrient-rich basins. Cyanobacteria were the most important autotrophic organisms and aminopeptidase activity was positively associated with cyanobacterial biomass. 3.,In an axenic Anabaena batch culture, nitrogenase activity was several orders of magnitude higher than leucine aminopeptidase activity. Nitrate did not have an effect on aminopeptidase activity or growth, but significantly reduced the rate of nitrogen fixation. A high phosphorus concentration at the beginning of the Anabaena batch-culture experiment resulted in reduced phosphatase activity. 4.,In Lake Hiidenvesi, aminopeptidase activity probably originated mostly from attached bacteria and less so from cyanobacteria. [source] Schizosaccharomyces pombe cell division cycle under limited glucose requires Ssp1 kinase, the putative CaMKK, and Sds23, a PP2A-related phosphatase inhibitorGENES TO CELLS, Issue 5 2009Yuichiro Hanyu Calcium/calmodulin-dependent protein kinase (CaMK) is required for diverse cellular functions, and similar kinases exist in fungi. Although mammalian CaMK kinase (CaMKK) activates CaMK and also evolutionarily-conserved AMP-activated protein kinase (AMPK), CaMKK is yet to be established in yeast. We here report that the fission yeast Schizosaccharomyces pombe Ssp1 kinase, which controls G2/M transition and response to stress, is the putative CaMKK. Ssp1 has a CaM binding domain (CBD) and associates with 14-3-3 proteins as mammalian CaMKK does. Temperature-sensitive ssp1 mutants isolated are defective in the tolerance to limited glucose, and this tolerance requires the conserved stretch present between the kinase domain and CBD. Sds23, multi-copy suppressor for mutants defective in type 1 phosphatase and APC/cyclosome, also suppresses the ssp1 phenotype, and is required for the tolerance to limited glucose. We demonstrate that Sds23 binds to type 2A protein phosphatases (PP2A) and PP2A-related phosphatase Ppe1, and that Sds23 inhibits Ppe1 phosphatase activity. Ssp1 and Ppe1 thus seem to antagonize in utilizing limited glucose. We also show that Ppk9 and Ssp2 are the catalytic subunits of AMPK and AMPK-related kinases, respectively, which bind to common ,-(Amk2) and ,-(Cbs2) subunits. [source] Pigment epithelium-derived factor induces the production of chemokines by rat microgliaGLIA, Issue 4 2005Asako Takanohashi Abstract Many studies have shown that pigment epithelium-derived factor (PEDF) has neurotrophic effects on retinal cells and hippocampal, spinal cord, and cerebellar granule cell neurons, but much less work has examined the effects of PEDF on glia. In this study, we show that PEDF changes microglial morphology within 1 h of exposure, to a more deactivated form, while having no effect on the expression of such activation markers as OX-42 and ED-1. In contrast, urea activates acid phosphatase, and PEDF blocks that activation. PEDF also activates NF,B, accompanied by the induction of mRNAs and proteins for the chemokines macrophage inflammatory protein-1, (MIP-1,, MIP-2, and MIP-3,. All the chemokines stimulate acid phosphatase activity, and high doses of MIP-2 and MIP-3,), alter the morphology of the microglia at 1 h after treatment. These results suggest that the use of PEDF for clinical treatments, such as for retinal neovascularization, brain injury, or ischemia, should be undertaken with caution because of the possibility of induction of inflammation caused by microglial or other immune cell migration in response to the chemokines induced by PEDF. © 2005 Wiley-Liss, Inc. [source] Primary sclerosing cholangitis in children: A long-term follow-up studyHEPATOLOGY, Issue 1 2003Ariel E. Feldstein Primary sclerosing cholangitis (PSC) is increasingly diagnosed in children and adolescents, but its long-term prognosis remains uncertain. The aim of this longitudinal, cohort study was to determine the long-term outcome of children with PSC. Fifty-two children with cholangiography-proven PSC (34 boys and 18 girls; mean age 13.8 ± 4.2 years; range, 1.5-19.6 years) who were seen at our institution over a 20-year period were followed-up for up to 16.7 years. Two thirds presented with symptoms and/or signs of PSC and 81% had concomitant inflammatory bowel disease (IBD). Twenty-five percent had total alkaline phosphatase activity within the normal range for the age group, but all of them had elevated ,-glutamyl transpeptidase levels. Autoimmune hepatitis overlapping with PSC was present in 35% of children. A positive but transient clinical and/or biochemical response occurred under therapy with ursodeoxycholic acid, alone or in combination with immunosuppressive medications. During follow-up, 11 children underwent liver transplantation for end-stage PSC and 1 child died. The median (50%) survival free of liver transplantation was 12.7 years. Compared with an age- and gender-matched U.S. population, survival was significantly shorter in children with PSC (P < .001). In a Cox regression model, lower platelet count, splenomegaly, and older age were associated with shorter survival. Presence of autoimmune hepatitis overlapping with PSC (P = .2) or medical therapy (P = .2) did not affect survival. In conclusion, PSC significantly decreases survival in this child population. Although pharmacologic therapy may improve symptoms and liver test results initially, it does not seem to impact the long-term outcome. [source] Does l -carnitine have any effect on cold preservation injury of non-fatty liver in the University of Wisconsin solution?HEPATOLOGY RESEARCH, Issue 8 2007Abdurrahman Coskun Aim:, To evaluate the protective effect of l -carnitine on liver tissue preserved in University of Wisconsin (UW) solution. Methods:, Twenty Wistar Albino rats were divided into two groups, a control (UW) group and a UW plus l -carnitine group. Retrieved liver grafts were preserved in UW and UW plus l -carnitine solutions at +4°C. Preservation solution samples were assessed at 2, 24, 36, and 48 h to measure alanine aminotransferase and acid phosphatase activity. Tissue injury was scored on paraffin sections. Results:, No micro or macrovacuolar fat droplets were observed in the tissue slices. l -Carnitine effectively decreased enzyme release when added to UW solution (P < 0.05). Conclusion:, In addition to fatty liver, l -carnitine might be a metabolic adjunct in preservation solutions for non-fatty liver within UW solution. [source] Alkaline phosphatase activity in pasteurized milk: A quantitative comparison of Fluorophos and colourimetric proceduresINTERNATIONAL JOURNAL OF DAIRY TECHNOLOGY, Issue 3 2009CLARE PAYNE Fluorophos and colourimetric procedures for alkaline phosphatase (ALP) testing were compared using milk with raw milk additions, purified bovine ALP additions and heat treatments. Repeatability was between 0.9% and 10.1% for Fluorophos, 3.5% and 46.1% for the Aschaffenburg and Mullen (A&M) procedure and 4.4% and 8.8% for the Scharer rapid test. Linearity (,R2) using raw milk addition was 0.96 between Fluorophos and the Scharer procedure. Between the Fluorophos and the A&M procedures, R2 values were 0.98, 0.99 and 0.98 for raw milk additions, bovine ALP additions and heat treatments respectively. Fluorophos showed greater sensitivity and was both faster and simpler to perform. [source] | ||||||||||||||||||||||