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Sample records for phosphatase shp-1 expression

  1. Expression, prognostic significance and mutational analysis of protein tyrosine phosphatase SHP-1 in chronic myeloid leukemia.

    Science.gov (United States)

    Papadopoulou, Vasiliki; Kontandreopoulou, Elina; Panayiotidis, Panayiotis; Roumelioti, Maria; Angelopoulou, Maria; Kyriazopoulou, Lydia; Diamantopoulos, Panagiotis T; Vaiopoulos, George; Variami, Eleni; Kotsianidis, Ioannis; Athina Viniou, Nora

    2016-05-01

    The protein tyrosine phosphatase SHP-1 dephosphorylates BCR-ABL1, thereby serving as a potential control mechanism of BCR-ABL1 kinase activity. Pathways regulating SHP-1 expression, which could be exploited in the therapeutics of TKI-resistant chronic myeloid leukemia (CML), remain unknown. Moreover, the questions of whether there is any kind of SHP-1 deregulation in CML, contributing to disease initiation or evolution, as well as the question of prognostic significance of SHP-1, have not been definitively answered. This study shows moderately lower SHP-1 mRNA expression in chronic phase CML patients in comparison to healthy individuals and no change in SHP-1 mRNA levels after successful TKI treatment. Mutational analysis of the aminoterminal and phosphatase domains of SHP-1 in patients did not reveal genetic lesions. This study also found no correlation of SHP-1 expression at diagnosis with response to treatment, although a trend for lower SHP-1 expression was noted in the very small non-responders' group of the 3-month therapeutic milestone.

  2. Study on expression of SH2 domain-containing protein tyrosine phosphatase SHP-1 and SHP-2 in γ-ray irradiation-induced thymus lymphoma in mice

    International Nuclear Information System (INIS)

    Huang Dingde; Chen Qi; Han Ling; Cai Jianming; Li Bailong; Huang Yuecheng; Gao Jianguo; Sun Suping

    2003-01-01

    Objective: To investigate the expression of SH2 domain containing-protein tyrosine phosphatase SHP-1 and SHP-2 in γ-ray irradiation-induced thymus lymphoma in mice. Methods: Altogether 338 BALB/c mice were randomly divided into irradiation groups and controls. Irradiation groups which were irradiated with γ-rays included canceration groups confirmed with histology and uncanceration groups. The controls were fed synchronistically with irradiation groups. The expression of SHP-1 and SHP-2 was detected with Western blot in thymus cells. Results: The expression of SHP-1 in canceration groups was much higher than that in uncanceration groups and controls significantly, while the expression of SHP-2 in canceration groups was higher than that in uncanceration groups and controls. When authors detected the expression of SHP-2 with Western blot, the authors found another protein with a molecular weight of 55x10 3 , which expression in canceration groups was higher than that in uncanceration groups and controls. Conclusion: The expression of SH2 domain-containing protein tyrosine phosphatase SHP-1 and SHP-2 is significantly increased in canceration groups, suggesting that SHP-1 and SHP-2 may be related with γ-ray induced thymus lymphoma in mice. Further research is expected on the relationship between development of cancer and SHP-1 and SHP-2

  3. Lack of Phosphotyrosine Phosphatase SHP-1 Expression in Malignant T-Cell Lymphoma Cells Results from Methylation of the SHP-1 Promoter

    DEFF Research Database (Denmark)

    Zhang, Q; Raghunath, P N; Vonderheid, E

    2000-01-01

    SHP-1 is an important negative regulator of signaling by several receptors including receptors for interleukin-2 (IL-2R) and other cytokines. SHP-1 acts by dephosphorylating the receptors and receptor-associated kinases such as IL-2R-associated Jak3 kinase. We found that SHP-1 protein was not det...

  4. Loss of SHP-1 tyrosine phosphatase expression correlates with the advanced stages of cutaneous T-cell lymphoma

    DEFF Research Database (Denmark)

    Witkiewicz, Agnieszka; Raghunath, Puthiyaveettil; Wasik, Agnieszka

    2007-01-01

    Cutaneous T-cell lymphoma (CTCL) comprises distinct and often progressive stages of skin involvement by patches, plaques, and tumors. We have previously demonstrated that CTCL-derived malignant T-cell lines display loss of a tumor suppressor SHP-1 tyrosine phosphatase because of epigenetic...

  5. Effect of NADPH oxidase inhibitor-apocynin on the expression of Src homology-2 domain-containing phosphatase-1 (SHP-1 exposed renal ischemia/reperfusion injury in rats

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    Zhiming Li

    2015-01-01

    Full Text Available This study was designed to evaluate whether NADPH oxidase inhibitor (apocynin preconditioning induces expression of Src homology-2 domain-containing phosphatase-1 (SHP-1 to protect against renal ischemia/reperfusion (I/R injury (RI/RI in rats. Rats were pretreated with 50 mg/kg apocynin, then subjected to 45 min ischemia and 24 h reperfusion. The results indicated that apocynin preconditioning improved the recovery of renal function and nitroso-redox balance, reduced oxidative stress injury and inflammation damage, and upregulated expression of SHP-1 as compared to RI/RI group. Therefore our study demonstrated that apocynin preconditioning provided a protection to the kidney against I/R injury in rats partially through inducing expression of SHP-1.

  6. Cell surface expression of channel catfish leukocyte immune-type receptors (IpLITRs) and recruitment of both Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1 and SHP-2.

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    Montgomery, Benjamin C S; Mewes, Jacqueline; Davidson, Chelsea; Burshtyn, Deborah N; Stafford, James L

    2009-04-01

    Channel catfish leukocyte immune-type receptors (IpLITRs) are immunoglobulin superfamily (IgSF) members believed to play a role in the control and coordination of cellular immune responses in teleost. Putative stimulatory and inhibitory IpLITRs are co-expressed by different types of catfish immune cells (e.g. NK cells, T cells, B cells, and macrophages) but their signaling potential has not been determined. Following cationic polymer-mediated transfections into human cell lines we examined the surface expression, tyrosine phosphorylation, and phosphatase recruitment potential of two types of putative inhibitory IpLITRs using 'chimeric' expression constructs and an epitope-tagged 'native' IpLITR. We also cloned and expressed the teleost Src homology 2 domain-containing protein tyrosine phosphatases (SHP)-1 and SHP-2 and examined their expression in adult tissues and developing zebrafish embryos. Co-immunoprecipitation experiments support the inhibitory signaling potential of distinct IpLITR-types that bound both SHP-1 and SHP-2 following the phosphorylation of tyrosine residues within their cytoplasmic tail (CYT) regions. Phosphatase recruitment by IpLITRs represents an important first step in understanding their influence on immune cell effector functions and suggests that certain inhibitory signaling pathways are conserved among vertebrates.

  7. Recruitment of SHP-1 protein tyrosine phosphatase and signalling by a chimeric T-cell receptor-killer inhibitory receptor

    DEFF Research Database (Denmark)

    Christensen, M D; Geisler, C

    2000-01-01

    Receptors expressing the immunoreceptor tyrosine-based inhibitory motif (ITIM) in their cytoplasmic tail play an important role in the negative regulation of natural killer and B-cell activation. A subpopulation of T cells expresses the ITIM containing killer cell inhibitory receptor (KIR), which...... recognize MHC class I molecules. Following coligation of KIR with an activating receptor, the tyrosine in the ITIM is phosphorylated and the cytoplasmic protein tyrosine phosphatase SHP-1 is recruited to the ITIM via its SH2 domains. It is still not clear how SHP-1 affects T-cell receptor (TCR) signalling...... regarding total protein tyrosine phosphorylation, TCR down-regulation, mobilization of intracellular free calcium, or induction of the activation markers CD69 and CD25....

  8. The tyrosine phosphatase SHP-1 regulates hypoxia inducible factor-1α (HIF-1α protein levels in endothelial cells under hypoxia.

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    Stefan K Alig

    Full Text Available The tyrosine phosphatase SHP-1 negatively influences endothelial function, such as VEGF signaling and reactive oxygen species (ROS formation, and has been shown to influence angiogenesis during tissue ischemia. In ischemic tissues, hypoxia induced angiogenesis is crucial for restoring oxygen supply. However, the exact mechanism how SHP-1 affects endothelial function during ischemia or hypoxia remains unclear. We performed in vitro endothelial cell culture experiments to characterize the role of SHP-1 during hypoxia.SHP-1 knock-down by specific antisense oligodesoxynucleotides (AS-Odn increased cell growth as well as VEGF synthesis and secretion during 24 hours of hypoxia compared to control AS-Odn. This was prevented by HIF-1α inhibition (echinomycin and apigenin. SHP-1 knock-down as well as overexpression of a catalytically inactive SHP-1 (SHP-1 CS further enhanced HIF-1α protein levels, whereas overexpression of a constitutively active SHP-1 (SHP-1 E74A resulted in decreased HIF-1α levels during hypoxia, compared to wildtype SHP-1. Proteasome inhibition (MG132 returned HIF-1α levels to control or wildtype levels respectively in these cells. SHP-1 silencing did not alter HIF-1α mRNA levels. Finally, under hypoxic conditions SHP-1 knock-down enhanced intracellular endothelial reactive oxygen species (ROS formation, as measured by oxidation of H2-DCF and DHE fluorescence.SHP-1 decreases half-life of HIF-1α under hypoxic conditions resulting in decreased cell growth due to diminished VEGF synthesis and secretion. The regulatory effect of SHP-1 on HIF-1α stability may be mediated by inhibition of endothelial ROS formation stabilizing HIF-1α protein. These findings highlight the importance of SHP-1 in hypoxic signaling and its potential as therapeutic target in ischemic diseases.

  9. Enhancing SHP-1 expression with 5-azacytidine may inhibit STAT3 activation and confer sensitivity in lestaurtinib (CEP-701)-resistant FLT3-ITD positive acute myeloid leukemia

    International Nuclear Information System (INIS)

    Al-Jamal, Hamid Ali Nagi; Mat Jusoh, Siti Asmaa; Hassan, Rosline; Johan, Muhammad Farid

    2015-01-01

    Tumor-suppressor genes are inactivated by methylation in several cancers including acute myeloid leukemia (AML). Src homology-2 (SH2)-containing protein-tyrosine phosphatase 1 (SHP-1) is a negative regulator of the JAK/STAT pathway. Transcriptional silencing of SHP-1 plays a critical role in the development and progression of cancers through STAT3 activation. 5-Azacytidine (5-Aza) is a DNA methyltransferase inhibitor that causes DNA demethylation resulting in re-expression of silenced SHP-1. Lestaurtinib (CEP-701) is a multi-targeted tyrosine kinase inhibitor that potently inhibits FLT3 tyrosine kinase and induces hematological remission in AML patients harboring the internal tandem duplication of the FLT3 gene (FLT3-ITD). However, the majority of patients in clinical trials developed resistance to CEP-701. Therefore, the aim of this study, was to assess the effect of re-expression of SHP-1 on sensitivity to CEP-701 in resistant AML cells. Resistant cells harboring the FLT3-ITD were developed by overexposure of MV4-11 to CEP-701, and the effects of 5-Aza treatment were investigated. Apoptosis and cytotoxicity of CEP-701 were determined using Annexin V and MTS assays, respectively. Gene expression was performed by quantitative real-time PCR. STATs activity was examined by western blotting and the methylation profile of SHP-1 was studied using MS-PCR and pyrosequencing analysis. Repeated-measures ANOVA and Kruskal–Wallis tests were used for statistical analysis. The cytotoxic dose of CEP-701 on resistant cells was significantly higher in comparison with parental and MV4-11R-cep + 5-Aza cells (p = 0.004). The resistant cells showed a significant higher viability and lower apoptosis compared with other cells (p < 0.001). Expression of SHP-1 was 7-fold higher in MV4-11R-cep + 5-Aza cells compared to parental and resistant cells (p = 0.011). STAT3 was activated in resistant cells. Methylation of SHP-1 was significantly decreased in MV4-11R-cep + 5-Aza cells (p = 0

  10. SHP-1 is a target of regorafenib in colorectal cancer

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    Lin, Hang; Hung, Man-Hsin; Chen, Yen-Lin; Huang, Jui-Wen; Tai, Wei-Tien; Yu, Hui-Chuan; Chen, Kuen-Feng

    2014-01-01

    Regorafenib is an inhibitor of multiple protein kinases which exerts antitumor and antimetastatic activities in metastatic colorectal cancer (CRC). SH2 domain-containing phosphatase 1 (SHP-1) is reported to have tumor suppressive potential because it acts as a negative regulator of p-STAT3Tyr705 signaling. However, little is known about the mechanism regarding regorafenib affects SHP-1 tyrosine phosphatase activity and leads to apoptosis and tumor suppression in CRC. Here, we found that regorafenib triggered apoptotic cell death and significantly enhanced SHP-1 activity, which dramatically decreased the phosphorylated form of STAT3 at Tyr705 (p-STAT3Tyr705). Importantly, regorafenib augmented SHP-1 activity by direct disruption of the association between N-SH2 and catalytic PTP domain of SHP-1. Deletion of the N-SH2 domain (dN1) or point mutation (D61A) of SHP-1 blocked the effect of regorafenib-induced SHP-1 activity, growth inhibition and a decrease of p-STAT3Tyr705 expression, suggesting that regorafenib triggers a conformational change in SHP-1 by relieving its autoinhibition. In vivo assay showed that regorafenib significantly inhibited xenograft growth and decreased p-STAT3Tyr705 expression but induced higher SHP-1 activity. Collectively, regorafenib is a novel SHP-1 agonist exerts superior anti-tumor effects by enhancing SHP-1 activity that directly targets p-STAT3Tyr705. Small molecule-enhancement of SHP-1 activity may be a promising therapeutic approach for CRC treatment. PMID:25071018

  11. 5-hydroxy-2-methyl-1,4-naphthoquinone, a vitamin K3 analogue, suppresses STAT3 activation pathway through induction of protein tyrosine phosphatase, SHP-1: potential role in chemosensitization.

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    Sandur, Santosh K; Pandey, Manoj K; Sung, Bokyung; Aggarwal, Bharat B

    2010-01-01

    The activation of signal transducers and activators of transcription 3 (STAT3) has been linked with carcinogenesis through survival, proliferation, and angiogenesis of tumor cells. Agents that can suppress STAT3 activation have potential not only for prevention but also for treatment of cancer. In the present report, we investigated whether 5-hydroxy-2-methyl-1,4-naphthoquinone (plumbagin), an analogue of vitamin K, and isolated from chitrak (Plumbago zeylanica), an Ayurvedic medicinal plant, can modulate the STAT3 pathway. We found that plumbagin inhibited both constitutive and interleukin 6-inducible STAT3 phosphorylation in multiple myeloma (MM) cells and this correlated with the inhibition of c-Src, Janus-activated kinase (JAK)1, and JAK2 activation. Vanadate, however, reversed the plumbagin-induced downregulation of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase. Indeed, we found that plumbagin induced the expression of the protein tyrosine phosphatase, SHP-1, and silencing of the SHP-1 abolished the effect of plumbagin. This agent also downregulated the expression of STAT3-regulated cyclin D1, Bcl-xL, and vascular endothelial growth factor; activated caspase-3; induced poly (ADP ribose) polymerase cleavage; and increased the sub-G(1) population of MM cells. Consistent with these results, overexpression of constitutive active STAT3 significantly reduced the plumbagin-induced apoptosis. When compared with AG490, a rationally designed STAT3/JAK2 inhibitor, plumbagin was found more potent in suppressing the proliferation of cells. Plumbagin also significantly potentiated the apoptotic effects of thalidomide and bortezomib in MM cells. Overall, these results suggest that the plumbagin inhibits STAT3 activation pathway through the induction of SHP-1 and this may mediate the sensitization of STAT3 overexpressing cancers to chemotherapeutic agents.

  12. Pharmacological Targeting SHP-1-STAT3 Signaling Is a Promising Therapeutic Approach for the Treatment of Colorectal Cancer

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    Li-Ching Fan

    2015-09-01

    Full Text Available STAT3 activation is associated with poor prognosis in human colorectal cancer (CRC. Our previous data demonstrated that regorafenib (Stivarga is a pharmacological agonist of SH2 domain-containing phosphatase 1 (SHP-1 that enhances SHP-1 activity and induces apoptosis by targeting STAT3 signals in CRC. This study aimed to find a therapeutic drug that is more effective than regorafenib for CRC treatment. Here, we showed that SC-43 was more effective than regorafenib at inducing apoptosis in vitro and suppressing tumorigenesis in vivo. SC-43 significantly increased SHP-1 activity, downregulated p-STAT3Tyr705 level, and induced apoptosis in CRC cells. An SHP-1 inhibitor or knockdown of SHP-1 by siRNA both significantly rescued the SC-43–induced apoptosis and decreased p-STAT3Tyr705 level. Conversely, SHP-1 overexpression increased the effects of SC-43 on apoptosis and p-STAT3Tyr705 level. These data suggest that SC-43–induced apoptosis mediated through the loss of p-STAT3Tyr705 was dependent on SHP-1 function. Importantly, SC-43–enhanced SHP-1 activity was because of the docking potential of SC-43, which relieved the autoinhibited N-SH2 domain of SHP-1 and inhibited p-STAT3Tyr705 signals. Importantly, we observed that a significant negative correlation existed between SHP-1 and p-STAT3Tyr705expression in CRC patients (P = .038. Patients with strong SHP-1 and weak p-STAT3Tyr705 expression had significantly higher overall survival compared with patients with weak SHP-1 and strong p-STAT3Tyr705 expression (P = .029. In conclusion, SHP-1 is suitable to be a useful prognostic marker and a pharmacological target for CRC treatment. Targeting SHP-1-STAT3 signaling by SC-43 may serve as a promising pharmacotherapy for CRC.

  13. Dovitinib Acts As a Novel Radiosensitizer in Hepatocellular Carcinoma by Targeting SHP-1/STAT3 Signaling

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    Huang, Chao-Yuan [Department of Oncology, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan (China); Department of Radiological Technology, Yuanpei University, Hsinchu, Taiwan (China); Tai, Wei-Tien [Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan (China); National Center of Excellence for Clinical Trial and Research, National Taiwan University Hospital, Taipei, Taiwan (China); Wu, Szu-Yuan [Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan (China); Department of Radiation Oncology, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan (China); Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan (China); Department of Biotechnology, Hungkuang University, Taichung, Taiwan (China); Shih, Chih-Ting; Chen, Min-Hsuan; Tsai, Ming-Hsien [Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan (China); Kuo, Chiung-Wen [Department of Medical Imaging and Radiological Technology, Yuanpei University of Medical Technology, Hsinchu, Taiwan (China); Shiau, Chung-Wai [Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei, Taiwan (China); Hung, Man-Hsin, E-mail: cindybeaty@gmail.com [Division of Medical Oncology, Department of Oncology, Taipei Veterans General Hospital, Taipei, Taiwan (China); Division of Hematology and Oncology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan (China); Program in Molecular Medicine, School of Life Science, National Yang-Ming University, Taipei, Taiwan (China); School of Medicine, National Yang-Ming University, Taipei, Taiwan (China); Chen, Kuen-Feng, E-mail: kfchen1970@ntu.edu.tw [Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan (China); National Center of Excellence for Clinical Trial and Research, National Taiwan University Hospital, Taipei, Taiwan (China)

    2016-06-01

    Purpose: Hepatocellular carcinoma (HCC) is among the most lethal human malignancies, and curative therapy is not an option for most patients. There is growing interest in the potential benefit of combining targeted therapies with radiation therapy (RT). This study aimed to characterize the efficacy and mechanism of an investigational drug, dovitinib, used in combination with RT. Methods and Materials: HCC cell lines (PLC5, Hep3B, SK-Hep1, HA59T, and Huh-7) were treated with dovitinib, RT, or both, and apoptosis and signal transduction were analyzed. Results: Dovitinib treatment resulted in Src homology region 2 (SH2) domain-containing phosphatase 1 (SHP-1)-mediated downregulation of p-STAT3 and promoted potent apoptosis of HCC cells. Ectopic expression of STAT3, or inhibition of SHP-1, diminished the effects of dovitinib on HCC cells. By ectopic expression and purified recombinant proteins of various mutant forms of SHP-1, the N-SH2 domain of SHP-1 was found to be required for dovitinib treatment. Overexpression of STAT3 or catalytic-dead mutant SHP-1 restored RT-induced reduction of HCC cell survival. Conversely, ectopic expression of SHP-1 or activation of SHP-1 by dovitinib enhanced the effects of RT against HCC in vitro and in vivo. Conclusions: SHP-1/STAT3 signaling is critically associated with the radiosensitivity of HCC cells. Combination therapy with RT and the SHP-1 agonist, such as dovitinib, resulted in enhanced in vitro and in vivo anti-HCC effects.

  14. Dovitinib Acts As a Novel Radiosensitizer in Hepatocellular Carcinoma by Targeting SHP-1/STAT3 Signaling

    International Nuclear Information System (INIS)

    Huang, Chao-Yuan; Tai, Wei-Tien; Wu, Szu-Yuan; Shih, Chih-Ting; Chen, Min-Hsuan; Tsai, Ming-Hsien; Kuo, Chiung-Wen; Shiau, Chung-Wai; Hung, Man-Hsin; Chen, Kuen-Feng

    2016-01-01

    Purpose: Hepatocellular carcinoma (HCC) is among the most lethal human malignancies, and curative therapy is not an option for most patients. There is growing interest in the potential benefit of combining targeted therapies with radiation therapy (RT). This study aimed to characterize the efficacy and mechanism of an investigational drug, dovitinib, used in combination with RT. Methods and Materials: HCC cell lines (PLC5, Hep3B, SK-Hep1, HA59T, and Huh-7) were treated with dovitinib, RT, or both, and apoptosis and signal transduction were analyzed. Results: Dovitinib treatment resulted in Src homology region 2 (SH2) domain-containing phosphatase 1 (SHP-1)-mediated downregulation of p-STAT3 and promoted potent apoptosis of HCC cells. Ectopic expression of STAT3, or inhibition of SHP-1, diminished the effects of dovitinib on HCC cells. By ectopic expression and purified recombinant proteins of various mutant forms of SHP-1, the N-SH2 domain of SHP-1 was found to be required for dovitinib treatment. Overexpression of STAT3 or catalytic-dead mutant SHP-1 restored RT-induced reduction of HCC cell survival. Conversely, ectopic expression of SHP-1 or activation of SHP-1 by dovitinib enhanced the effects of RT against HCC in vitro and in vivo. Conclusions: SHP-1/STAT3 signaling is critically associated with the radiosensitivity of HCC cells. Combination therapy with RT and the SHP-1 agonist, such as dovitinib, resulted in enhanced in vitro and in vivo anti-HCC effects.

  15. Shp-1 dephosphorylates TRPV1 in dorsal root ganglion neurons and alleviates CFA-induced inflammatory pain in rats.

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    Xiao, Xing; Zhao, Xiao-Tao; Xu, Ling-Chi; Yue, Lu-Peng; Liu, Feng-Yu; Cai, Jie; Liao, Fei-Fei; Kong, Jin-Ge; Xing, Guo-Gang; Yi, Ming; Wan, You

    2015-04-01

    Transient receptor potential vanilloid 1 (TRPV1) receptors are expressed in nociceptive neurons of rat dorsal root ganglions (DRGs) and mediate inflammatory pain. Nonspecific inhibition of protein-tyrosine phosphatases (PTPs) increases the tyrosine phosphorylation of TRPV1 and sensitizes TRPV1. However, less is known about tyrosine phosphorylation's implication in inflammatory pain, compared with that of serine/threonine phosphorylation. Src homology 2 domain-containing tyrosine phosphatase 1 (Shp-1) is a key phosphatase dephosphorylating TRPV1. In this study, we reported that Shp-1 colocalized with and bound to TRPV1 in nociceptive DRG neurons. Shp-1 inhibitors, including sodium stibogluconate and PTP inhibitor III, sensitized TRPV1 in cultured DRG neurons. In naive rats, intrathecal injection of Shp-1 inhibitors increased both TRPV1 and tyrosine-phosphorylated TRPV1 in DRGs and induced thermal hyperalgesia, which was abolished by pretreatment with TRPV1 antagonists capsazepine, BCTC, or AMG9810. Complete Freund's adjuvant (CFA)-induced inflammatory pain in rats significantly increased the expression of Shp-1, TRPV1, and tyrosine-phosphorylated TRPV1, as well as the colocalization of Shp-1 and TRPV1 in DRGs. Intrathecal injection of sodium stibogluconate aggravated CFA-induced inflammatory pain, whereas Shp-1 overexpression in DRG neurons alleviated it. These results suggested that Shp-1 dephosphorylated and inhibited TRPV1 in DRG neurons, contributing to maintain thermal nociceptive thresholds in normal rats, and as a compensatory mechanism, Shp-1 increased in DRGs of rats with CFA-induced inflammatory pain, which was involved in protecting against excessive thermal hyperalgesia.

  16. Association of 3BP2 with SHP-1 regulates SHP-1-mediated production of TNF-α in RBL-2H3 cells.

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    Chihara, Kazuyasu; Nakashima, Kenji; Takeuchi, Kenji; Sada, Kiyonao

    2011-12-01

    Adaptor protein 3BP2, a c-Abl Src homology 3 (SH3) domain-binding protein, is tyrosine phosphorylated and positively regulates mast cell signal transduction after the aggregation of the high affinity IgE receptor (FcεRI). Overexpression of the Src homology 2 (SH2) domain of 3BP2 results in the dramatic suppression of antigen-induced degranulation in rat basophilic leukemia RBL-2H3 cells. Previously, a linker for activation of T cells (LAT) was identified as one of the 3BP2 SH2 domain-binding protein. In this report, to further understand the functions of 3BP2 in FcεRI-mediated activation of mast cell, we explored the protein that associates with the SH2 domain of 3BP2 and found that SH2 domain-containing phosphatase-1 (SHP-1) inducibly interacts with the SH2 domain of 3BP2 after the aggregation of FcεRI. The phosphorylation of Tyr(564) in the carboxy (C)-terminal tail region of SHP-1 is required for the direct interaction of SHP-1 to the SH2 domain of 3BP2. The expression of the mutant form of SHP-1 which was unable to interact with 3BP2 resulted in the significant reduction in SHP-1-mediated tumor necrosis factor-α (TNF-α) production without any effects on the degranulation in antigen-stimulated RBL-2H3 cells. These findings suggest that 3BP2 directly interacts with Tyr(564) -phosphorylated form of SHP-1 and positively regulates the function of SHP-1 in FcεRI-mediated signaling in mast cells. © 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

  17. Advanced glycation end product Nε-carboxymethyllysine induces endothelial cell injury: the involvement of SHP-1-regulated VEGFR-2 dephosphorylation.

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    Liu, Shing Hwa; Sheu, Wayne Huey Herng; Lee, Maw Rong; Lee, Wen Jane; Yi, Yu Chiao; Yang, Tzung Jie; Jen, Jen Fon; Pan, Hung Chuan; Shen, Chin Chang; Chen, Wen Bao; Tien, Hsing Ru; Sheu, Meei Ling

    2013-06-01

    N(ε)-carboxymethyllysine (CML), a major advanced glycation end product, plays a crucial role in diabetes-induced vascular injury. The roles of protein tyrosine phosphatases and vascular endothelial growth factor (VEGF) receptors in CML-related endothelial cell injury are still unclear. Human umbilical vein endothelial cells (HUVECs) are a commonly used human EC type. Here, we tested the hypothesis that NADPH oxidase/reactive oxygen species (ROS)-mediated SH2 domain-containing tyrosine phosphatase-1 (SHP-1) activation by CML inhibits the VEGF receptor-2 (VEGFR-2, KDR/Flk-1) activation, resulting in HUVEC injury. CML significantly inhibited cell proliferation and induced apoptosis and reduced VEGFR-2 activation in parallel with the increased SHP-1 protein expression and activity in HUVECs. Adding recombinant VEGF increased forward biological effects, which were attenuated by CML. The effects of CML on HUVECs were abolished by SHP-1 siRNA transfection. Exposure of HUVECs to CML also remarkably escalated the integration of SHP-1 with VEGFR-2. Consistently, SHP-1 siRNA transfection and pharmacological inhibitors could block this interaction and elevating [(3)H]thymidine incorporation. CML also markedly activated the NADPH oxidase and ROS production. The CML-increased SHP-1 activity in HUVECs was effectively attenuated by antioxidants. Moreover, the immunohistochemical staining of SHP-1 and CML was increased, but phospho-VEGFR-2 staining was decreased in the aortic endothelium of streptozotocin-induced and high-fat diet-induced diabetic mice. We conclude that a pathway of tyrosine phosphatase SHP-1-regulated VEGFR-2 dephosphorylation through NADPH oxidase-derived ROS is involved in the CML-triggered endothelial cell dysfunction/injury. These findings suggest new insights into the development of therapeutic approaches to reduce diabetic vascular complications. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  18. SHP1-mediated cell cycle redistribution inhibits radiosensitivity of non-small cell lung cancer

    International Nuclear Information System (INIS)

    Cao, Rubo; Ding, Qian; Li, Pindong; Xue, Jun; Zou, Zhenwei; Huang, Jing; Peng, Gang

    2013-01-01

    Radioresistance is the common cause for radiotherapy failure in non-small cell lung cancer (NSCLC), and the degree of radiosensitivity of tumor cells is different during different cell cycle phases. The objective of the present study was to investigate the effects of cell cycle redistribution in the establishment of radioresistance in NSCLC, as well as the signaling pathway of SH2 containing Tyrosine Phosphatase (SHP1). A NSCLC subtype cell line, radioresistant A549 (A549S1), was induced by high-dose hypofractionated ionizing radiations. Radiosensitivity-related parameters, cell cycle distribution and expression of cell cycle-related proteins and SHP1 were investigated. siRNA was designed to down-regulate SHP1expression. Compared with native A549 cells, the proportion of cells in the S phase was increased, and cells in the G0/G1 phase were consequently decreased, however, the proportion of cells in the G2/M phase did not change in A549S1 cells. Moreover, the expression of SHP1, CDK4 and CylinD1 were significantly increased, while p16 was significantly down-regulated in A549S1 cells compared with native A549 cells. Furthermore, inhibition of SHP1 by siRNA increased the radiosensitivity of A549S1 cells, induced a G0/G1 phase arrest, down-regulated CDK4 and CylinD1expressions, and up-regulated p16 expression. SHP1 decreases the radiosensitivity of NSCLC cells through affecting cell cycle distribution. This finding could unravel the molecular mechanism involved in NSCLC radioresistance

  19. Src homology domain 2-containing protein-tyrosine phosphatase-1 (SHP-1) binds and dephosphorylates G(alpha)-interacting, vesicle-associated protein (GIV)/Girdin and attenuates the GIV-phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway.

    Science.gov (United States)

    Mittal, Yash; Pavlova, Yelena; Garcia-Marcos, Mikel; Ghosh, Pradipta

    2011-09-16

    GIV (Gα-interacting vesicle-associated protein, also known as Girdin) is a bona fide enhancer of PI3K-Akt signals during a diverse set of biological processes, e.g. wound healing, macrophage chemotaxis, tumor angiogenesis, and cancer invasion/metastasis. We recently demonstrated that tyrosine phosphorylation of GIV by receptor and non-receptor-tyrosine kinases is a key step that is required for GIV to directly bind and enhance PI3K activity. Here we report the discovery that Src homology 2-containing phosphatase-1 (SHP-1) is the major protein-tyrosine phosphatase that targets two critical phosphotyrosines within GIV and antagonizes phospho-GIV-dependent PI3K enhancement in mammalian cells. Using phosphorylation-dephosphorylation assays, we demonstrate that SHP-1 is the major and specific protein-tyrosine phosphatase that catalyzes the dephosphorylation of tyrosine-phosphorylated GIV in vitro and inhibits ligand-dependent tyrosine phosphorylation of GIV downstream of both growth factor receptors and GPCRs in cells. In vitro binding and co-immunoprecipitation assays demonstrate that SHP-1 and GIV interact directly and constitutively and that this interaction occurs between the SH2 domain of SHP-1 and the C terminus of GIV. Overexpression of SHP-1 inhibits tyrosine phosphorylation of GIV and formation of phospho-GIV-PI3K complexes, and specifically suppresses GIV-dependent activation of Akt. Consistently, depletion of SHP-1 enhances peak tyrosine phosphorylation of GIV, which coincides with an increase in peak Akt activity. We conclude that SHP-1 antagonizes the action of receptor and non-receptor-tyrosine kinases on GIV and down-regulates the phospho-GIV-PI3K-Akt axis of signaling.

  20. Effects of SOV-induced phosphatase inhibition and expression of protein tyrosine phosphatases in rat corneal endothelial cells.

    Science.gov (United States)

    Chen, Wei-Li; Harris, Deshea L; Joyce, Nancy C

    2005-11-01

    Contact inhibition is an important mechanism for maintaining corneal endothelium in a non-replicative state. Protein tyrosine phosphatases (PTPs) play a role in regulating the integrity of cell-cell contacts, differentiation, and growth. In this study, we aimed to evaluate whether phosphatases are involved in the maintenance of contact-dependent inhibition of proliferation in corneal endothelial cells and to identify candidate PTPs that are expressed in these cells and might be involved in regulation of contact inhibition. Confluent cultures of rat corneal endothelial cells or endothelium in ex vivo corneas were treated with the general phosphatase inhibitor, sodium orthovanadate (SOV). Immunocytochemistry (ICC) evaluated the effect of SOV on cell-cell contacts by staining for ZO-1, and on cell cycle progression by staining for Ki67. Transverse sections of rat cornea and cultured rat corneal endothelial cells were used to test for expression of the candidate PTPs: PTP-mu, PTP-LAR, PTP1B, SHP-1, SHP-2, and PTEN using ICC and either Western blots or RT-PCR. ZO-1 staining demonstrated that SOV induced a time-dependent release of cell-cell contacts in confluent cultures of corneal endothelial cells and in the endothelium of ex vivo corneas. Staining for Ki67 indicated that SOV promoted limited cell cycle progression in the absence of serum. PTP-mu, PTP1B, SHP-1, SHP-2, and PTEN, but not PTP-LAR, were expressed in rat corneal endothelial cells in situ and in culture. The subcellular location of PTP-mu and PTP1B differed in subconfluent and confluent cells, while that of SHP-1, SHP-2, and PTEN was similar, regardless of confluent status. Western blots confirmed the expression of PTP1B, SHP-1, SHP-2, and PTEN. RT-PCR confirmed expression of PTP-mu mRNA. Phosphatases are involved in regulation of junctional integrity and of cell proliferation in corneal endothelial cells. PTP-mu, PTP1B, SHP-1, SHP-2, and PTEN are expressed in rat corneal endothelium and may be involved in

  1. Andrographolide induces vascular smooth muscle cell apoptosis through a SHP-1-PP2A-p38MAPK-p53 cascade.

    Science.gov (United States)

    Chen, Yu-Ying; Hsieh, Cheng-Ying; Jayakumar, Thanasekaran; Lin, Kuan-Hung; Chou, Duen-Suey; Lu, Wan-Jung; Hsu, Ming-Jen; Sheu, Joen-Rong

    2014-07-10

    The abnormal growth of vascular smooth muscle cells (VSMCs) is considered a critical pathogenic process in inflammatory vascular diseases. We have previously demonstrated that protein phosphatase 2 A (PP2A)-mediated NF-κB dephosphorylation contributes to the anti-inflammatory properties of andrographolide, a novel NF-κB inhibitor. In this study, we investigated whether andrographolide causes apoptosis, and characterized its apoptotic mechanisms in rat VSMCs. Andrographolide activated the p38 mitogen-activated protein kinase (p38MAPK), leading to p53 phosphorylation. Phosphorylated p53 subsequently transactivated the expression of Bax, a pro-apoptotic protein. Transfection with pp2a small interfering RNA (siRNA) suppressed andrographolide-induced p38MAPK activation, p53 phosphorylation, and caspase 3 activation. Andrographolide also activated the Src homology 1 domain-containing protein tyrosine phosphatase (SHP-1), and induced PP2A dephosphorylation, both of which were inhibited by the SHP-1 inhibitor sodium stibogluconate (SSG) or shp-1 siRNA. SSG or shp-1 siRNA prevented andrographolide-induced apoptosis. These results suggest that andrographolide activates the PP2A-p38MAPK-p53-Bax cascade, causing mitochondrial dysfunction and VSMC death through an SHP-1-dependent mechanism.

  2. SHP-1 activation inhibits vascular smooth muscle cell proliferation and intimal hyperplasia in a rodent model of insulin resistance and diabetes

    DEFF Research Database (Denmark)

    Qi, Weier; Li, Qian; Liew, Chong Wee

    2017-01-01

    . However, the role of SHP-1 in intimal hyperplasia and restenosis has not been clarified in insulin resistance and diabetes. METHODS: We used a femoral artery wire injury mouse model, rodent models with insulin resistance and diabetes, and patients with type 2 diabetes. Further, we modulated SHP-1...... expression using a transgenic mouse that overexpresses SHP-1 in VSMCs (Shp-1-Tg). SHP-1 agonists were also employed to study the molecular mechanisms underlying the regulation of SHP-1 by oxidised lipids. RESULTS: Mice fed a high-fat diet (HFD) exhibited increased femoral artery intimal hyperplasia...... and decreased arterial SHP-1 expression compared with mice fed a regular diet. Arterial SHP-1 expression was also decreased in Zucker fatty rats, Zucker diabetic fatty rats and in patients with type 2 diabetes. In primary cultured VSMCs, oxidised LDL suppressed SHP-1 expression by activating Mek-1 (also known...

  3. Alteration of SHP-1/p-STAT3 Signaling: A Potential Target for Anticancer Therapy

    Directory of Open Access Journals (Sweden)

    Tzu-Ting Huang

    2017-06-01

    Full Text Available The Src homology 2 (SH2 domain-containing protein tyrosine phosphatase 1 (SHP-1, a non-receptor protein tyrosine phosphatase, has been reported as a negative regulator of phosphorylated signal transducer and activator of transcription 3 (STAT3 and linked to tumor development. In this present review, we will discuss the importance and function of SHP-1/p-STAT3 signaling in nonmalignant conditions as well as malignancies, its cross-talk with other pathways, the current clinical development and the potential role of inhibitors of this pathway in anticancer therapy and clinical relevance of SHP-1/p-STAT3 in cancers. Lastly, we will summarize and highlight work involving novel drugs/compounds targeting SHP-1/p-STAT3 signaling and combined strategies that were/are discovered in our and our colleagues’ laboratories.

  4. Calpain/SHP-1 interaction by honokiol dampening peritoneal dissemination of gastric cancer in nu/nu mice.

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    Shing Hwa Liu

    Full Text Available BACKGROUND: Honokiol, a small-molecular weight natural product, has previously been reported to activate apoptosis and inhibit gastric tumorigenesis. Whether honokiol inhibits the angiogenesis and metastasis of gastric cancer cells remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: We tested the effects of honokiol on angiogenic activity and peritoneal dissemination using in vivo, ex vivo and in vitro assay systems. The signaling responses in human gastric cancer cells, human umbilical vascular endothelial cells (HUVECs, and isolated tumors were detected and analyzed. In a xenograft gastric tumor mouse model, honokiol significantly inhibited the peritoneal dissemination detected by PET/CT technique. Honokiol also effectively attenuated the angiogenesis detected by chick chorioallantoic membrane assay, mouse matrigel plug assay, rat aortic ring endothelial cell sprouting assay, and endothelial cell tube formation assay. Furthermore, honokiol effectively enhanced signal transducer and activator of transcription (STAT-3 dephosphorylation and inhibited STAT-3 DNA binding activity in human gastric cancer cells and HUVECs, which was correlated with the up-regulation of the activity and protein expression of Src homology 2 (SH2-containing tyrosine phosphatase-1 (SHP-1. Calpain-II inhibitor and siRNA transfection significantly reversed the honokiol-induced SHP-1 activity. The decreased STAT-3 phosphorylation and increased SHP-1 expression were also shown in isolated peritoneal metastatic tumors. Honokiol was also capable of inhibiting VEGF generation, which could be reversed by SHP-1 siRNA transfection. CONCLUSIONS/SIGNIFICANCE: Honokiol increases expression and activity of SPH-1 that further deactivates STAT3 pathway. These findings also suggest that honokiol is a novel and potent inhibitor of angiogenesis and peritoneal dissemination of gastric cancer cells, providing support for the application potential of honokiol in gastric cancer therapy.

  5. Regulation of nasal airway homeostasis and inflammation in mice by SHP-1 and Th2/Th1 signaling pathways.

    Directory of Open Access Journals (Sweden)

    Seok Hyun Cho

    Full Text Available Allergic rhinitis is a chronic inflammatory disease orchestrated by Th2 lymphocytes. Src homology 2 domain-containing protein tyrosine phosphatase (SHP-1 is known to be a negative regulator in the IL-4α/STAT-6 signaling pathway of the lung. However, the role of SHP-1 enzyme and its functional relationship with Th2 and Th1 cytokines are not known in the nasal airway. In this study, we aimed to study the nasal inflammation as a result of SHP-1 deficiency in viable motheaten (mev mice and to investigate the molecular mechanisms involved. Cytology, histology, and expression of cytokines and chemokines were analyzed to define the nature of the nasal inflammation. Targeted gene depletion of Th1 (IFN-γ and Th2 (IL-4 and IL-13 cytokines was used to identify the critical pathways involved. Matrix metalloproteinases (MMPs were studied to demonstrate the clearance mechanism of recruited inflammatory cells into the nasal airway. We showed here that mev mice had a spontaneous allergic rhinitis-like inflammation with eosinophilia, mucus metaplasia, up-regulation of Th2 cytokines (IL-4 and IL-13, chemokines (eotaxin, and MMPs. All of these inflammatory mediators were clearly counter-regulated by Th2 and Th1 cytokines. Deletion of IFN-γ gene induced a strong Th2-skewed inflammation with transepithelial migration of the inflammatory cells. These findings suggest that SHP-1 enzyme and Th2/Th1 paradigm may play a critical role in the maintenance of nasal immune homeostasis and in the regulation of allergic rhinitis.

  6. SHP1 Regulates Bone Mass by Directing Mesenchymal Stem Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Menghui Jiang

    2016-07-01

    Full Text Available Osteoblasts and adipocytes are derived from a common precursor, mesenchymal stem cells (MSCs. Alterations in the normal fate of differentiating MSCs are involved in the development of obesity and osteoporosis. Here, we report that viable motheaten (mev mice, which are deficient in the SH2-domain-containing phosphatase-1 (SHP1, develop osteoporosis spontaneously. Consistently, MSCs from mev/mev mice exhibit significantly reduced osteogenic potential and greatly increased adipogenic potential. When MSCs were transplanted into nude mice, SHP1-deficient MSCs resulted in diminished bone formation compared with wild-type MSCs. SHP1 was found to bind to GSK3β and suppress its kinase activity by dephosphorylating pY216, thus resulting in β-catenin stabilization. Mice, in which SHP1 was deleted in MSCs using SHP1fl/flDermo1-cre, displayed significantly decreased bone mass and increased adipose tissue. Taken together, these results suggest a possible role for SHP1 in controlling tissue homeostasis through modulation of MSC differentiation via Wnt signaling regulation.

  7. SH2 domain-containing phosphatase 1 regulates pyruvate kinase M2 in hepatocellular carcinoma.

    Science.gov (United States)

    Tai, Wei-Tien; Hung, Man-Hsin; Chu, Pei-Yi; Chen, Yao-Li; Chen, Li-Ju; Tsai, Ming-Hsien; Chen, Min-Husan; Shiau, Chung-Wai; Boo, Yin-Pin; Chen, Kuen-Feng

    2016-04-19

    Pyruvate kinase M2 (PKM2) is known to promote tumourigenesis through dimer formation of p-PKM2Y105. Here, we investigated whether SH2-containing protein tyrosine phosphatase 1 (SHP-1) decreases p-PKM2Y105 expression and, thus, determines the sensitivity of sorafenib through inhibiting the nuclear-related function of PKM2. Immunoprecipitation and immunoblot confirmed the effect of SHP-1 on PKM2Y105 dephosphorylation. Lactate production was assayed in cells and tumor samples to determine whether sorafenib reversed the Warburg effect. Clinical hepatocellular carcinoma (HCC) tumor samples were assessed for PKM2 expression. SHP-1 directly dephosphorylated PKM2 at Y105 and further decreased the proliferative activity of PKM2; similar effects were found in sorafenib-treated HCC cells. PKM2 was also found to determine the sensitivity of targeted drugs, such as sorafenib, brivanib, and sunitinib, by SHP-1 activation. Significant sphere-forming activity was found in HCC cells stably expressing PKM2. Clinical findings suggest that PKM2 acts as a predicting factor of early recurrence in patients with HCC, particularly those without known risk factors (63.6%). SHP-1 dephosphorylates PKM2 at Y105 to inhibit nuclear function of PKM2 and determines the efficacy of targeted drugs. Targeting PKM2 by SHP-1 might provide new therapeutic insights for patients with HCC.

  8. SHP-1 is directly activated by the aryl hydrocarbon receptor and regulates BCL-6 in the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

    Energy Technology Data Exchange (ETDEWEB)

    Phadnis-Moghe, Ashwini S.; Li, Jinpeng [Genetics Program, Michigan State University, East Lansing, MI 48824 (United States); Institute for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Crawford, Robert B. [Institute for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Kaminski, Norbert E., E-mail: kamins11@msu.edu [Institute for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI 48824 (United States)

    2016-11-01

    The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which is a strong AHR agonist, causes significant suppression of human B cell activation and differentiation. The current studies describe the identification of Src homology phosphatase 1 (SHP-1) encoded by the gene PTPN6 as a putative regulator of TCDD-mediated suppression of B cell activation. Shp-1 was initially identified through a genome-wide analysis of AHR binding in mouse B cells in the presence of TCDD. The binding of AHR to the PTPN6 promoter was further confirmed using electrophoretic mobility shift assays in which, specific binding of AHR was detected at four putative DRE sites within PTPN6 promoter. Time-course measurements performed in human B cells highlighted a significant increase in SHP-1 mRNA and protein levels in the presence of TCDD. The changes in the protein levels of SHP-1 were also observed in a TCDD concentration-dependent manner. The increase in SHP-1 levels was also seen to occur due to a change in early signaling events in the presence of TCDD. We have shown that BCL-6 regulates B cell activation by repressing activation marker CD80 in the presence of TCDD. TCDD-treatment led to a significant increase in the double positive (SHP-1{sup hi} BCL-6{sup hi}) population. Interestingly, treatment of naïve human B cells with SHP-1 inhibitor decreased BCL-6 protein levels suggesting possible regulation of BCL-6 by SHP-1 for the first time. Collectively, these results suggest that SHP-1 is regulated by AHR in the presence of TCDD and may, in part through BCL-6, regulate TCDD-mediated suppression of human B cell activation. - Highlights: • SHP-1 encoded by the gene PTPN6 is directly activated by the AHR. • AHR binds to dioxin response elements within the SHP-1 promoter in a TCDD-inducible manner. • TCDD-mediated increase in SHP-1 levels is observed in primary human B cells. • Higher SHP-1 levels help in maintaining high BCL-6 levels in the presence of TCDD. • In

  9. Actin retrograde flow controls natural killer cell response by regulating the conformation state of SHP-1.

    Science.gov (United States)

    Matalon, Omri; Ben-Shmuel, Aviad; Kivelevitz, Jessica; Sabag, Batel; Fried, Sophia; Joseph, Noah; Noy, Elad; Biber, Guy; Barda-Saad, Mira

    2018-03-01

    Natural killer (NK) cells are a powerful weapon against viral infections and tumor growth. Although the actin-myosin (actomyosin) cytoskeleton is crucial for a variety of cellular processes, the role of mechanotransduction, the conversion of actomyosin mechanical forces into signaling cascades, was never explored in NK cells. Here, we demonstrate that actomyosin retrograde flow (ARF) controls the immune response of primary human NK cells through a novel interaction between β-actin and the SH2-domain-containing protein tyrosine phosphatase-1 (SHP-1), converting its conformation state, and thereby regulating NK cell cytotoxicity. Our results identify ARF as a master regulator of the NK cell immune response. Since actin dynamics occur in multiple cellular processes, this mechanism might also regulate the activity of SHP-1 in additional cellular systems. © 2018 The Authors.

  10. Detection of Intracellular Reduced (Catalytically Active) SHP-1 and Analyses of Catalytically Inactive SHP-1 after Oxidation by Pervanadate or H2O2.

    Science.gov (United States)

    Choi, Seeyoung; Love, Paul E

    2018-01-05

    Oxidative inactivation of cysteine-dependent Protein Tyrosine Phosphatases (PTPs) by cellular reactive oxygen species (ROS) plays a critical role in regulating signal transduction in multiple cell types. The phosphatase activity of most PTPs depends upon a 'signature' cysteine residue within the catalytic domain that is maintained in the de-protonated state at physiological pH rendering it susceptible to ROS-mediated oxidation. Direct and indirect techniques for detection of PTP oxidation have been developed (Karisch and Neel, 2013). To detect catalytically active PTPs, cell lysates are treated with iodoacetyl-polyethylene glycol-biotin (IAP-biotin), which irreversibly binds to reduced (S - ) cysteine thiols. Irreversible oxidation of SHP-1 after treatment of cells with pervanadate or H 2 O 2 is detected with antibodies specific for the sulfonic acid (SO 3 H) form of the conserved active site cysteine of PTPs. In this protocol, we describe a method for the detection of the reduced (S - ; active) or irreversibly oxidized (SO 3 H; inactive) form of the hematopoietic PTP SHP-1 in thymocytes, although this method is applicable to any cysteine-dependent PTP in any cell type.

  11. Inhibition of STAT3 signaling and induction of SHP1 mediate antiangiogenic and antitumor activities of ergosterol peroxide in U266 multiple myeloma cells

    International Nuclear Information System (INIS)

    Rhee, Yun-Hee; Jeong, Soo-Jin; Lee, Hyo-Jeong; Lee, Hyo-Jung; Koh, Wonil; Jung, Ji Hoon; Kim, Sun-Hee; Sung-Hoon, Kim

    2012-01-01

    Ergosterol peroxide (EP) derived from edible mushroom has been shown to exert anti-tumor activity in several cancer cells. In the present study, anti-angiogenic activity of EP was investigated with the underlying molecular mechanisms in human multiple myeloma U266 cells. Despite weak cytotoxicity against U266 cells, EP suppressed phosphorylation, DNA binding activity and nuclear translocalization of signal transducer and activator of transcription 3 (STAT3) in U266 cells at nontoxic concentrations. Also, EP inhibited phosphorylation of the upstream kinases Janus kinase 2 (JAK2) and Src in a time-dependent manner. Furthermore, EP increased the expression of protein tyrosine phosphatase SHP-1 at protein and mRNA levels, and conversely silencing of the SHP-1 gene clearly blocked EP-mediated STAT3 inactivation. In addition, EP significantly decreased vascular endothelial growth factor (VEGF), one of STAT3 target genes at cellular and protein levels as well as disrupted in vitro tube formation assay. Moreover, EP significantly suppressed the growth of U266 cells inoculated in female BALB/c athymic nude mice and immunohistochemistry revealed that EP effectively reduced the expression of STAT3 and CD34 in tumor sections compared to untreated control. These findings suggest that EP can exert antitumor activity in multiple myeloma U266 cells partly with antiangiogenic activity targeting JAK2/STAT3 signaling pathway as a potent cancer preventive agent for treatment of multiple myeloma cells

  12. Cloning and expression of a widely expressed receptor tyrosine phosphatase

    DEFF Research Database (Denmark)

    Sap, J; D'Eustachio, P; Givol, D

    1990-01-01

    We describe the identification of a widely expressed receptor-type (transmembrane) protein tyrosine phosphatase (PTPase; EC 3.1.3.48). Screening of a mouse brain cDNA library under low-stringency conditions with a probe encompassing the intracellular (phosphatase) domain of the CD45 lymphocyte...... antigen yielded cDNA clones coding for a 794-amino acid transmembrane protein [hereafter referred to as receptor protein tyrosine phosphatase alpha (R-PTP-alpha)] with an intracellular domain displaying clear homology to the catalytic domains of CD45 and LAR (45% and 53%, respectively). The 142-amino acid...

  13. SHP-1, a novel peptide isolated from seahorse inhibits collagen release through the suppression of collagenases 1 and 3, nitric oxide products regulated by NF-kappaB/p38 kinase.

    Science.gov (United States)

    Ryu, BoMi; Qian, Zhong-Ji; Kim, Se-Kwon

    2010-01-01

    Considerable efforts have been taken to identify natural peptides as potential bioactive substances. In this study, novel peptide (SHP-1) derived from seahorse (Hippocampus, Syngnathidae) hydrolysate was explored for its inhibitory effects on collagen release in arthritis with the investigation of its underlying mechanism of action. The efficacy of SHP-1 was determined on cartilage protective effects such as inhibition of collagen and GAG release. SHP-1 was able to suppress not only the expression of collagenases 1 and 3, but also the production of NO via down-regulation of iNOS. However, it presented an irrelevant effect on the level of GAG release in chondrocytic and osteoblastic cells. Inhibition of collagen release by SHP-1 is associated with restraining the phosphorylation of NF-kappaB and p38 kinase cascade. Therefore, it could be suggested that SHP-1 has a potential to be used in arthritis treatment.

  14. The MADS Box Genes ABS, SHP1, and SHP2 Are Essential for the Coordination of Cell Divisions in Ovule and Seed Coat Development and for Endosperm Formation in Arabidopsis thaliana.

    Science.gov (United States)

    Ehlers, Katrin; Bhide, Amey S; Tekleyohans, Dawit G; Wittkop, Benjamin; Snowdon, Rod J; Becker, Annette

    2016-01-01

    Seed formation is a pivotal process in plant reproduction and dispersal. It begins with megagametophyte development in the ovule, followed by fertilization and subsequently coordinated development of embryo, endosperm, and maternal seed coat. Two closely related MADS-box genes, SHATTERPROOF 1 and 2 (SHP1 and SHP2) are involved in specifying ovule integument identity in Arabidopsis thaliana. The MADS box gene ARABIDOPSIS BSISTER (ABS or TT16) is required, together with SEEDSTICK (STK) for the formation of endothelium, part of the seed coat and innermost tissue layer formed by the maternal plant. Little is known about the genetic interaction of SHP1 and SHP2 with ABS and the coordination of endosperm and seed coat development. In this work, mutant and expression analysis shed light on this aspect of concerted development. Triple tt16 shp1 shp2 mutants produce malformed seedlings, seed coat formation defects, fewer seeds, and mucilage reduction. While shp1 shp2 mutants fail to coordinate the timely development of ovules, tt16 mutants show less peripheral endosperm after fertilization. Failure in coordinated division of the innermost integument layer in early ovule stages leads to inner seed coat defects in tt16 and tt16 shp1 shp2 triple mutant seeds. An antagonistic action of ABS and SHP1/SHP2 is observed in inner seed coat layer formation. Expression analysis also indicates that ABS represses SHP1, SHP2, and FRUITFUL expression. Our work shows that the evolutionary conserved Bsister genes are required not only for endothelium but also for endosperm development and genetically interact with SHP1 and SHP2 in a partially antagonistic manner.

  15. The MADS Box Genes ABS, SHP1, and SHP2 Are Essential for the Coordination of Cell Divisions in Ovule and Seed Coat Development and for Endosperm Formation in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Katrin Ehlers

    Full Text Available Seed formation is a pivotal process in plant reproduction and dispersal. It begins with megagametophyte development in the ovule, followed by fertilization and subsequently coordinated development of embryo, endosperm, and maternal seed coat. Two closely related MADS-box genes, SHATTERPROOF 1 and 2 (SHP1 and SHP2 are involved in specifying ovule integument identity in Arabidopsis thaliana. The MADS box gene ARABIDOPSIS BSISTER (ABS or TT16 is required, together with SEEDSTICK (STK for the formation of endothelium, part of the seed coat and innermost tissue layer formed by the maternal plant. Little is known about the genetic interaction of SHP1 and SHP2 with ABS and the coordination of endosperm and seed coat development. In this work, mutant and expression analysis shed light on this aspect of concerted development. Triple tt16 shp1 shp2 mutants produce malformed seedlings, seed coat formation defects, fewer seeds, and mucilage reduction. While shp1 shp2 mutants fail to coordinate the timely development of ovules, tt16 mutants show less peripheral endosperm after fertilization. Failure in coordinated division of the innermost integument layer in early ovule stages leads to inner seed coat defects in tt16 and tt16 shp1 shp2 triple mutant seeds. An antagonistic action of ABS and SHP1/SHP2 is observed in inner seed coat layer formation. Expression analysis also indicates that ABS represses SHP1, SHP2, and FRUITFUL expression. Our work shows that the evolutionary conserved Bsister genes are required not only for endothelium but also for endosperm development and genetically interact with SHP1 and SHP2 in a partially antagonistic manner.

  16. Expression of Prostatic Acid Phosphatase in Rat Circumvallate Papillae.

    Directory of Open Access Journals (Sweden)

    Kentaro Nishida

    Full Text Available ATP and its metabolites are important for taste signaling in taste buds, and thus a clearance system for them would play critical roles in maintenance of gustatory function. A previous report revealed that mRNAs for ecto-5'-nucleotidase (NT5E and prostatic acid phosphatase (PAP were expressed by taste cells of taste buds, and NT5E-immunoreactivity was detected in taste cells. However, there was no information on PAP-immunoreactivity in taste buds. In this study, we examined the expression profile of PAP in rat taste buds. In the isolated rat taste buds, we detected expression of mRNA for PAP, but NT5E was not detected differing from the case of mouse ones (Dando et al., 2012, J Neuroscience. On immunohistochemical analysis, PAP-immunoreactivity was found predominantly in NTPDase2-positive type I and SNAP25-positive type III taste cells, while there were no apparent signals of it in PLC-β2-positive type II, α-gustducin-positive type II, AADC-positive type III and 5HT-positive type III ones. As for NT5E, we could not detect its immunoreactivity in rat taste buds, and co-localization of it with any taste cell markers, although mouse taste buds expressed NT5E as reported previously. These findings suggest that PAP expressed by type I and one of type III taste cells of rats may contribute to metabolic regulation of the extracellular levels of adenine nucleotides in the taste buds of circumvallate papillae, and the regulating mechanisms for adenine nucleotides in taste buds might be different between rats and mice.

  17. Phylogenetic characterization of phosphatase-expressing bacterial communities in Baltic Sea sediments

    NARCIS (Netherlands)

    Steenbergh, Anne; Bodelier, Paul; Hoogveld, H.L.; Slomp, C.P; Laanbroek, H.J.

    2015-01-01

    Phosphate release from sediments hampers the remediation of aquatic systems from a eutrophic state. Microbial phosphatases in sediments release phosphorus during organic matter degradation. Despite the important role of phosphatase-expressing bacteria, the identity of these bacteria in sediments is

  18. Cloning and characterization of rat density-enhanced phosphatase-1, a protein tyrosine phosphatase expressed by vascular cells.

    Science.gov (United States)

    Borges, L G; Seifert, R A; Grant, F J; Hart, C E; Disteche, C M; Edelhoff, S; Solca, F F; Lieberman, M A; Lindner, V; Fischer, E H; Lok, S; Bowen-Pope, D F

    1996-09-01

    We have cloned from cultured vascular smooth muscle cells a protein tyrosine phosphatase, rat density-enhanced phosphatase-1 (rDEP-1), which is a probable rat homologue of DEP-1/HPTP eta. rDEP-1 is encoded by an 8.7-kb transcript and is expressed as a 180- to 220-kD protein. The rDEP-1 gene is located on human chromosome 11 (region p11.2) and on mouse chromosome 2 (region 2E). The cDNA sequence predicts a transmembrane protein consisting of a single phosphatase catalytic domain in the intracellular region, a single transmembrane domain, and eight fibronectin type III repeats in the extracellular region (GenBank accession number U40790). In situ hybridization analysis demonstrates that rDEP-1 is widely expressed in vivo but that expression is highest in cells that form epithelioid monolayers. In cultured cells with epitheliod morphology, including endothelial cells and newborn smooth muscle cells, but not in fibroblast-like cells, rDEP-1 transcript levels are dramatically upregulated as population density increases. In vivo, quiescent endothelial cells in normal arteries express relatively high levels of rDEP-1. During repair of vascular injury, expression of rDEP-1 is downregulated in migrating and proliferating endothelial cells. In vivo, rDEP-1 transcript levels are present in very high levels in megakaryocytes, and circulating plates have high levels of the rDEP-1 protein. In vitro, initiation of differentiation of the human megakaryoblastic cell line CHRF-288-11 with phorbol 12-myristate 13-acetate leads to a very strong upregulation of rDEP-1 transcripts. The deduced structure and the regulation of expression of rDEP-1 suggest that it may play a role in adhesion and/or signaling events involving cell-cell and cell-matrix contact.

  19. Alkaline phosphatase expression during relapse after orthodontic tooth movement

    Directory of Open Access Journals (Sweden)

    Pinandi Sri Pudyani

    2014-03-01

    Full Text Available Background: The increasing of osteoblast activities during bone formation will be accompanied with the increasing expression of alkaline phosphatase enzyme (ALP. ALP can be obtained from clear fluid excreted by gingival crevicular fluid (GCF. Bone turnover, especially bone formation process, can be monitored through the expression of ALP secreted by GCF during orthodontic treatment. Thus, retention period is an important period that can be monitored through the level of bone metabolism around teeth. Purpose: This research were aimed to determine the relation of distance change caused by tooth relapse and ALP activities in gingival crevicular fluid after orthodontic; and to determine ALP as a potential biomarker of bone formation during retention period. Methods: Lower incisors of 25 guinea pigs were moved 3 mm to the distally by using open coil spring. Those relapse distance were measured and the gingival crevicular fluid was taken by using paper points to evaluate ALP levels on days 0, 3, 7, 14 and 21 respectivelly by using a spectrophotometer (405 nm. t-test and ANOVA test were conducted to determine the difference of ALP activities among the time intervals. The correlation regression analysis was conducted to determine the relation of distance change caused by the relapse tooth movement and ALP activities. Results: The greatest relapse movement was occurred on day 3 after open coil spring was removed. There was significant difference of the average of distance decrease among groups A1-A5 (p<0.05. It was also known that ALP level was increased on day 3, but there was no significant difference of the average level of ALP among groups A1-A5 (p>0.05. Finally, based on the results of correlation analysis between the ALP level decreasing and the relapse distance on both right and left of mesial and distal sides, it is known that there was no relation between those two variables (p>0.05. Conclusion: It can be concluded that relapse after orthodontic

  20. Methylation profiling of SOCS1, SOCS2, SOCS3, CISH and SHP1 in Philadelphia-negative myeloproliferative neoplasm.

    Science.gov (United States)

    Zhang, Min Yue; Fung, Tsz Kin; Chen, Fang Yuan; Chim, Chor Sang

    2013-10-01

    Janus kinase-signal transducer and activator of transcription (JAK/STAT) signalling, pivotal in Philadelphia-negative (Ph-ve) myeloproliferative neoplasm (MPN), is negatively regulated by molecules including SOCSs, CISH and SHP1. SOCS1, SOCS2 and SOCS3 methylation have been studied in MPN with discordant results. Herein, we studied the methylation status of SOCS1, SOCS2 and SOCS3, CISH and SHP1 by methylation-specific polymerase chain reaction (MSP) in cell lines and 45 diagnostic marrow samples of Ph-ve MPN. Moreover, we attempted to explain the discordance of methylation frequency by mapping the studied MSP primers to the respective genes. Methylation was detected in normal controls using SOCS2 MSP primers in the 3'translated exonic sequence, but not primers around the transcription start site in the 5' untranslated regions (5'UTR). SOCS1, SOCS2, SOCS3 and CISH were completely unmethylated in primary MPN samples and cell lines. In contrast, methylation of SHP1 was detected in 8.9% primary marrow samples. Moreover, SHP1 was completely methylated in K562 cell line, leading to reversible SHP1 silencing. A review of methylation studies of SOCS1 and SOCS3 showed that spuriously high rates of SOCS methylation had been reported using MSP primers targeting CpG sites in the 3'translated exonic sequence, which is also methylated in normal controls. However, using MSP primers localized to the 5'UTR, methylation of SOCS1, SOCS2 and SOCS3 is infrequent across all studies. In summary, methylation of SOCS1, SOCS2, SOCS3 and CISH is infrequent in Ph-ve MPN. Appropriate MSP primers are important for accurate estimation of the methylation frequency. The role of SHP1 methylation in the pathogenesis of MPN warrants further investigation. © 2013 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  1. Expression of protein-tyrosine phosphatases in the major insulin target tissues

    DEFF Research Database (Denmark)

    Norris, K; Norris, F; Kono, D H

    1997-01-01

    Protein-tyrosine phosphatases (PTPs) are key regulators of the insulin receptor signal transduction pathway. We have performed a detailed analysis of PTP expression in the major human insulin target tissues or cells (liver, adipose tissue, skeletal muscle and endothelial cells). To obtain a repre...

  2. Dihydrotestostenone increase the gene expression of androgen ...

    African Journals Online (AJOL)

    HNTEP cells were grown in basal medium and treated with DHT in different conditions. HNTEP cells under treatment with DHT (10-13 M) induced an increase in FHL-2 expression. In turn, high DHT concentrations (10-8 M) induced an increase in the expression SHP-1. The present data suggest that the SHP-1 and FHL-2 ...

  3. Expression, purification and crystallization of an atypical class C acid phosphatase from Mycoplasma bovis

    International Nuclear Information System (INIS)

    Singh, Harkewal; Reilly, Thomas J.; Calcutt, Michael J.; Tanner, John J.

    2011-01-01

    Methods for the expression, purification and crystallization of the class C acid phosphatase from M. bovis are reported. This enzyme is atypical in that it is nearly 20 kDa larger than other known class C acid phosphatases. Class C acid phosphatases (CCAPs) are 25–30 kDa bacterial surface proteins that are thought to function as broad-specificity 5′,3′-nucleotidases. Analysis of the newly published complete genome sequence of Mycoplasma bovis PG45 revealed a putative CCAP with a molecular weight of 49.9 kDa. The expression, purification and crystallization of this new family member are described here. Standard purification procedures involving immobilized metal-ion affinity chromatography and ion-exchange chromatography yielded highly pure and crystallizable protein. Crystals were grown in sitting drops at room temperature in the presence of PEG 3350 and HEPES buffer pH 7.5 and diffracted to 2.3 Å resolution. Analysis of diffraction data suggested a primitive monoclinic space group, with unit-cell parameters a = 78, b = 101, c = 180 Å, β = 92°. The asymmetric unit is predicted to contain six molecules, which are likely to be arranged as three dimers

  4. Receptor-like protein-tyrosine phosphatase alpha specifically inhibits insulin-increased prolactin gene expression

    DEFF Research Database (Denmark)

    Jacob, K K; Sap, J; Stanley, F M

    1998-01-01

    A physiologically relevant response to insulin, stimulation of prolactin promoter activity in GH4 pituitary cells, was used as an assay to study the specificity of protein-tyrosine phosphatase function. Receptor-like protein-tyrosine phosphatase alpha (RPTPalpha) blocks the effect of insulin...... is specific by two criteria. A number of potential RPTPalpha targets were ruled out by finding (a) that they are not affected or (b) that they are not on the pathway to insulin-increased prolactin-CAT activity. The negative effect of RPTPalpha on insulin activation of the prolactin promoter is not due...... to reduced phosphorylation or kinase activity of the insulin receptor or to reduced phosphorylation of insulin receptor substrate-1 or Shc. Inhibitor studies suggest that insulin-increased prolactin gene expression is mediated by a Ras-like GTPase but is not mitogen-activated protein kinase dependent...

  5. Deficient SOCS3 and SHP-1 Expression in Psoriatic T Cells

    DEFF Research Database (Denmark)

    Eriksen, Karsten W; Woetmann, Anders; Skov, Lone

    2010-01-01

    IFN-alpha and skin-infiltrating activated T lymphocytes have important roles in the pathogenesis of psoriasis. T cells from psoriatic patients display an increased sensitivity to IFN-alpha, but the pathological mechanisms behind the hyperresponsiveness to IFN-alpha remained unknown. In this study......-alpha signaling in psoriatic T cells. In conclusion, our data suggest that loss of regulatory control is involved in the aberrant hypersensitivity of psoriatic T cells to IFN-alpha.Journal of Investigative Dermatology advance online publication, 4 February 2010; doi:10.1038/jid.2010.6....

  6. Expression of yeast lipid phosphatase Sac1p is regulated by phosphatidylinositol-4-phosphate

    Directory of Open Access Journals (Sweden)

    Mayinger Peter

    2008-01-01

    Full Text Available Abstract Background Phosphoinositides play a central role in regulating processes at intracellular membranes. In yeast, a large number of phospholipid biosynthetic enzymes use a common mechanism for transcriptional regulation. Yet, how the expression of genes encoding lipid kinases and phosphatases is regulated remains unknown. Results Here we show that the expression of lipid phosphatase Sac1p in the yeast Saccharomyces cerevisiae is regulated in response to changes in phosphatidylinositol-4-phosphate (PI(4P concentrations. Unlike genes encoding enzymes involved in phospholipid biosynthesis, expression of the SAC1 gene is independent of inositol levels. We identified a novel 9-bp motif within the 5' untranslated region (5'-UTR of SAC1 that is responsible for PI(4P-mediated regulation. Upregulation of SAC1 promoter activity correlates with elevated levels of Sac1 protein levels. Conclusion Regulation of Sac1p expression via the concentration of its major substrate PI(4P ensures proper maintenance of compartment-specific pools of PI(4P.

  7. The structural insights of stem cell factor receptor (c-Kit interaction with tyrosine phosphatase-2 (Shp-2: An in silico analysis

    Directory of Open Access Journals (Sweden)

    Gurudutta Gangenahalli U

    2010-01-01

    Full Text Available Abstract Background Stem cell factor (SCF receptor c-Kit is recognized as a key signaling molecule, which transduces signals for the proliferation, differentiation and survival of stem cells. Binding of SCF to its receptor triggers transactivation, leading to the recruitment of kinases and phosphatases to the docking platforms of c-Kit catalytic domain. Tyrosine phosphatase-1 (Shp-1 deactivates/attenuates 'Kit' kinase activity. Whereas, Asp816Val mutation in the Kit activation loop transforms kinase domain to a constitutively activated state (switch off-to-on state, in a ligand-independent manner. This phenomenon completely abrogates negative regulation of Shp-1. To predict the possible molecular basis of interaction between c-Kit and Shp-1, we have performed an in silico protein-protein docking study between crystal structure of activated c-Kit (phosphorylated c-Kit and full length crystal structure of Shp-2, a close structural counterpart of Shp-1. Findings Study revealed a stretch of conserved amino acids (Lys818 to Ser821 in the Kit activation domain, which makes decisive H-bonds with N-sh2 and phosphotyrosine binding pocket residues of the phosphatase. These H-bonds may impose an inhibitory steric hindrance to the catalytic domain of c-Kit, there by blocking further interaction of the activation loop molecules with incoming kinases. We have also predicted a phosphotyrosine binding pocket in SH2 domains of Shp-1, which is found to be predominantly closer to a catalytic groove like structure in c-Kit kinase domain. Conclusions This study predicts that crucial hydrogen bonding between N-sh2 domain of Shp-1 and Kit activation loop can modulate the negative regulation of c-Kit kinase by Shp-1. Thus, this finding is expected to play a significant role in designing suitable gain-of-function c-Kit mutants for inducing conditional proliferation of hematopoietic stem cells.

  8. The Association of Endothelin-1 Signaling with Bone Alkaline Phosphatase Expression and Protumorigenic Activities in Canine Osteosarcoma.

    Science.gov (United States)

    Neumann, Z L; Pondenis, H C; Masyr, A; Byrum, M L; Wycislo, K L; Fan, T M

    2015-01-01

    Canine osteosarcoma (OS) is an aggressive sarcoma characterized by pathologic skeletal resorption and pulmonary metastases. A number of negative prognostic factors, including bone alkaline phosphatase, have been identified in dogs with OS, but the underlying biologic factors responsible for such observations have not been thoroughly investigated. Endothelin-1-mediated signaling is active during bone repair, and is responsible for osteoblast migration, survival, proliferation, and bone alkaline phosphatase expression. The endothelin-1 signaling axis is active in canine OS cells, and this pathway is utilized by malignant osteoblasts for promoting cellular migration, survival, proliferation, and bone alkaline phosphatase activities. 45 dogs with appendicular OS. The expressions of endothelin-1 and endothelin A receptor were studied in OS cell lines and in samples from spontaneously occurring tumors. Activities mediated by endothelin-1 signaling were investigated by characterizing responses in 3 OS cell lines. In 45 dogs with OS, bone alkaline phosphatase concentrations were correlated with primary tumor osteoproductivity. Canine OS cells express endothelin-1 and endothelin A receptor, and this signaling axis mediates OS migration, survival, proliferation, and bone alkaline phosphatase activities. In OS-bearing dogs, circulating bone alkaline phosphatase activities were positively correlated with primary tumor relative bone mineral densities. Canine OS cells express endothelin-1 and functional endothelin A receptors, with the potential for a protumorigenic signaling loop. Increases in bone alkaline phosphatase activity are associated with osteoblastic OS lesions, and might be an epiphenomenon of active endothelin-1 signaling or excessive osteoproduction within the localized bone microenvironment. Copyright © 2015 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

  9. Wnt5a attenuates Wnt3a-induced alkaline phosphatase expression in dental follicle cells

    International Nuclear Information System (INIS)

    Sakisaka, Yukihiko; Tsuchiya, Masahiro; Nakamura, Takashi; Tamura, Masato; Shimauchi, Hidetoshi; Nemoto, Eiji

    2015-01-01

    Wnt signaling regulates multiple cellular events such as cell proliferation, differentiation, and apoptosis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathways. Canonical Wnt/β-catenin signaling can promote the differentiation of dental follicle cells, putative progenitor cells for cementoblasts, osteoblasts, and periodontal ligament cells, toward a cementoblast/osteoblast phenotype during root formation, but little is known about the biological significance of noncanonical Wnt signaling in this process. We identified the expression of Wnt5a, a representative noncanonical Wnt ligand, in tooth root lining cells (i.e. precementoblasts/cementoblasts) and dental follicle cells during mouse tooth root development, as assessed by immunohistochemistry. Silencing expression of the Wnt5a gene in a dental follicle cell line resulted in enhancement of the Wnt3a (a representative canonical Wnt ligand)-mediated increase in alkaline phosphatase (ALP) expression. Conversely, treatment with recombinant Wnt5a inhibited the increase in ALP expression, suggesting that Wnt5a signaling functions as a negative regulator of canonical Wnt-mediated ALP expression of dental follicle cells. Wnt5a did not affect the nuclear translocation of β-catenin as well as β-catenin-mediated transcriptional activation of T-cell factor (Tcf) triggered by Wnt3a, suggesting that Wnt5a inhibits the downstream part of the β-catenin-Tcf pathway. These findings suggest the existence of a feedback mechanism between canonical and noncanonical Wnt signaling during the differentiation of dental follicle cells. - Highlights: • Dental follicle cells express Wnt5a during tooth root development. • Silencing of Wnt5a enhances Wnt3a-mediated ALP expression of dental follicle cells. • Conversely, treatment with rWnt5a inhibited the increase in ALP expression. • Wnt5a functions as a negative regulator of Wnt3a-mediated ALP expression

  10. Wnt5a attenuates Wnt3a-induced alkaline phosphatase expression in dental follicle cells

    Energy Technology Data Exchange (ETDEWEB)

    Sakisaka, Yukihiko [Department of Periodontology and Endodontology, Tohoku University Graduate School of Dentistry, Sendai 980-8575 (Japan); Tsuchiya, Masahiro [Department of Oral Diagnosis, Tohoku University Graduate School of Dentistry, Sendai 980-8575 (Japan); Tohoku Fukushi University, Sendai 989-3201 (Japan); Nakamura, Takashi [Department of Pediatric Dentistry, Tohoku University Graduate School of Dentistry, Sendai 980-8575 (Japan); Liason Center for Innovative Dentistry, Tohoku University Graduate School of Dentistry, Sendai 980-8575 (Japan); Tamura, Masato [Department of Biochemistry and Molecular Biology, Hokkaido University Graduate School of Dentistry, Sapporo 060-8586 (Japan); Shimauchi, Hidetoshi [Department of Periodontology and Endodontology, Tohoku University Graduate School of Dentistry, Sendai 980-8575 (Japan); Nemoto, Eiji, E-mail: e-nemoto@dent.tohoku.ac.jp [Department of Periodontology and Endodontology, Tohoku University Graduate School of Dentistry, Sendai 980-8575 (Japan)

    2015-08-01

    Wnt signaling regulates multiple cellular events such as cell proliferation, differentiation, and apoptosis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathways. Canonical Wnt/β-catenin signaling can promote the differentiation of dental follicle cells, putative progenitor cells for cementoblasts, osteoblasts, and periodontal ligament cells, toward a cementoblast/osteoblast phenotype during root formation, but little is known about the biological significance of noncanonical Wnt signaling in this process. We identified the expression of Wnt5a, a representative noncanonical Wnt ligand, in tooth root lining cells (i.e. precementoblasts/cementoblasts) and dental follicle cells during mouse tooth root development, as assessed by immunohistochemistry. Silencing expression of the Wnt5a gene in a dental follicle cell line resulted in enhancement of the Wnt3a (a representative canonical Wnt ligand)-mediated increase in alkaline phosphatase (ALP) expression. Conversely, treatment with recombinant Wnt5a inhibited the increase in ALP expression, suggesting that Wnt5a signaling functions as a negative regulator of canonical Wnt-mediated ALP expression of dental follicle cells. Wnt5a did not affect the nuclear translocation of β-catenin as well as β-catenin-mediated transcriptional activation of T-cell factor (Tcf) triggered by Wnt3a, suggesting that Wnt5a inhibits the downstream part of the β-catenin-Tcf pathway. These findings suggest the existence of a feedback mechanism between canonical and noncanonical Wnt signaling during the differentiation of dental follicle cells. - Highlights: • Dental follicle cells express Wnt5a during tooth root development. • Silencing of Wnt5a enhances Wnt3a-mediated ALP expression of dental follicle cells. • Conversely, treatment with rWnt5a inhibited the increase in ALP expression. • Wnt5a functions as a negative regulator of Wnt3a-mediated ALP expression.

  11. Expression of receptor-type protein tyrosine phosphatase in developing and adult renal vasculature.

    Directory of Open Access Journals (Sweden)

    Keiko Takahashi

    Full Text Available Renal vascular development is a coordinated process that requires ordered endothelial cell proliferation, migration, intercellular adhesion, and morphogenesis. In recent decades, studies have defined the pivotal role of endothelial receptor tyrosine kinases (RPTKs in the development and maintenance of renal vasculature. However, the expression and the role of receptor tyrosine phosphatases (RPTPs in renal endothelium are poorly understood, though coupled and counterbalancing roles of RPTKs and RPTPs are well defined in other systems. In this study, we evaluated the promoter activity and immunolocalization of two endothelial RPTPs, VE-PTP and PTPμ, in developing and adult renal vasculature using the heterozygous LacZ knock-in mice and specific antibodies. In adult kidneys, both VE-PTP and PTPμ were expressed in the endothelium of arterial, glomerular, and medullary vessels, while their expression was highly limited in peritubular capillaries and venous endothelium. VE-PTP and PTPμ promoter activity was also observed in medullary tubular segments in adult kidneys. In embryonic (E12.5, E13.5, E15.5, E17.5 and postnatal (P0, P3, P7 kidneys, these RPTPs were expressed in ingrowing renal arteries, developing glomerular microvasculature (as early as the S-shaped stage, and medullary vessels. Their expression became more evident as the vasculatures matured. Peritubular capillary expression of VE-PTP was also noted in embryonic and postnatal kidneys. Compared to VE-PTP, PTPμ immunoreactivity was relatively limited in embryonic and neonatal renal vasculature and evident immunoreactivity was observed from the P3 stage. These findings indicate 1 VE-PTP and PTPμ are expressed in endothelium of arterial, glomerular, and medullary renal vasculature, 2 their expression increases as renal vascular development proceeds, suggesting that these RPTPs play a role in maturation and maintenance of these vasculatures, and 3 peritubular capillary VE-PTP expression

  12. Diversity and Gene Expression of Phosphatase Genes Provide Insight into Soil Phosphorus Dynamics in a New Zealand Managed Grassland

    Science.gov (United States)

    Dunfield, K. E.; Gaiero, J. R.; Condron, L.

    2017-12-01

    Healthy and diverse communities of soil organisms influence key soil ecosystem services such as carbon sequestration, water quality protection, climate regulation and nutrient cycling. Microbially driven mineralization of organic phosphorus is an important contributor to plant available inorganic orthophosphates. In acidic soils, microbes produce non-specific acid phosphatases (NSAPs) which act on common forms of organic phosphorus (P). Our current understanding of P turnover in soils has been limited by lack of research tools capable of targeting these genes. Thus, we developed a set of oligonucleotide PCR primers that targeted bacteria with the genetic potential for acid phosphatase production. A long term randomized-block pasture trial was sampled following 22 years of continued aerial biomass removal and retention. Primers were used to target genes encoding alkaline phosphatase (phoD) and the three classes (CAAP, CBAP, CCAP) of non-specific acid phosphatases. PCR amplicons targeting total genes and gene transcripts were sequenced using Illumina MiSeq to understand the diversity of the bacterial phosphatase producing communities. In general, the majority of operational taxonomic units (OTUs) were shared across both treatments and across metagenomes and transcriptomes. However, analysis of DNA OTUs revealed significantly different communities driven by treatment differences (P reduced Olsen P levels (15 vs. 36 mg kg-1 in retained treatment). Acid phosphatase activity was measured in all samples, and found to be highest in the biomass retained treatment (16.8 vs. 11.4 µmol g-1 dry soil h-1), likely elevated due to plant-derived enzymes; however, was still correlated to bacterial gene abundances. Overall, the phosphatase producing microbial communities responded to the effect of consistent P limitation as expected, through alteration in the composition of the community structure and through increased levels of gene expression of the phosphatase genes.

  13. Expression of phosphatase of regenerating liver-3 is associated with prognosis of Wilms’ tumor

    Directory of Open Access Journals (Sweden)

    Sun F

    2017-01-01

    Full Text Available Fengyin Sun,1 Wenyi Li,2,3 Lie Wang,2 Changfeng Jiao3 1Department of Pediatric Surgery, Qilu Hospital, Shandong University, Jinan, Shandong Province, 2Department of General Surgery, Fuzhou General Hospital of Nanjing Command, PLA, Fuzhou, Fujian Province, 3Department of Vascular Surgery, Xinzhou City People’s Hospital, Xinzhou, Shanxi Province, People’s Republic of China Objective: The current study was undertaken to explore the clinical and prognostic value of phosphatase of regenerating liver-3 (PRL-3 expression in Wilms’ tumor. Methods: Seventy-six patients with Wilms’ tumor in Qilu Hospital from January 2003 to July 2009 were enrolled in the study. Protein expression level of PRL-3 was examined by immunohistochemical staining, and the correlation between PRL-3 expression and histopathological parameters, clinical variables, and outcome of patients with Wilms’ tumor were analyzed. Results: We found that 19% of patients with unfavorable histology had tumor recurrence and 16% of patients died following the operation. PRL-3 was expressed in 15 out of 76 tumors (19% and expressed highly in unfavorable histology Wilms’ tumor (P=0.04. PRL-3 protein expression level was correlated to 2.5-fold increase in recurrence rate of Wilms’ tumor (P=0.06 without any statistically significant difference. However, in favorable histology Wilms’ tumor, PRL-3 expression was correlated to an increase of 3.4-fold in recurrence rate (P=0.03. Conclusion: The expression of PRL-3 protein was correlated with an increased recurrence rate of favorable histology Wilms’ tumor. PRL-3 may serve as a promising biomarker for predicting patients with high risk of Wilms’ tumor. Further investigations are warranted to investigate the clinical function of PRL-3 in Wilms’ tumor. Keywords: Wilms’ tumor, prognosis, tumorigenesis, recurrence

  14. Evidence for an indirect transcriptional regulation of glucose-6-phosphatase gene expression by liver X receptors

    International Nuclear Information System (INIS)

    Grempler, Rolf; Guenther, Susanne; Steffensen, Knut R.; Nilsson, Maria; Barthel, Andreas; Schmoll, Dieter; Walther, Reinhard

    2005-01-01

    Liver X receptor (LXR) paralogues α and β (LXRα and LXRβ) are members of the nuclear hormone receptor family and have oxysterols as endogenous ligands. LXR activation reduces hepatic glucose production in vivo through the inhibition of transcription of the key gluconeogenic enzymes phosphoenolpyruvate carboxykinase and glucose-6-phosphatase (G6Pase). In the present study, we investigated the molecular mechanisms involved in the regulation of G6Pase gene expression by LXR. Both T0901317, a synthetic LXR agonist, and the adenoviral overexpression of either LXRα or LXRβ suppressed G6Pase gene expression in H4IIE hepatoma cells. However, compared to the suppression of G6Pase expression seen by insulin, the decrease of G6Pase mRNA by LXR activation was delayed and was blocked by cycloheximide, an inhibitor of protein synthesis. These observations, together with the absence of a conserved LXR-binding element within the G6Pase promoter, suggest an indirect inhibition of G6Pase gene expression by liver X receptors

  15. Obstructive sleep apnea and intermittent hypoxia increase expression of dual specificity phosphatase 1.

    Science.gov (United States)

    Hoffmann, Michal S; Singh, Prachi; Wolk, Robert; Narkiewicz, Krzysztof; Somers, Virend K

    2013-12-01

    Dual specificity phosphatase 1 (DUSP1) inhibits mitogen activated protein kinase activity, and is activated by several stimuli such as sustained hypoxia, oxidative stress, and hormones. However, the effect of intermittent hypoxia is not known. The aim of this study was to evaluate the role of intermittent hypoxia on DUSP1 expression, and to validate its role in a human model of intermittent hypoxia, as seen in obstructive sleep apnea (OSA). OSA is characterized by recurrent episodes of hypoxemia/reoxygenation and is a known risk factor for cardiovascular morbidity. In-vitro studies using human coronary artery endothelial cells (HCAEC) and ex-vivo studies using white blood cells isolated from healthy and OSA subjects. Intermittent hypoxia induced DUSP1 expression in human coronary artery endothelial cells (HCAEC), and in granulocytes isolated from healthy human subjects. Functionally, DUSP1 increased the expression and activity of manganese superoxide dismutase (MnSOD) in HCAEC. Further, significant increases in DUSP1 mRNA from total blood, and in DUSP1 protein in mononuclear cells and granulocytes isolated from OSA subjects, were observed in the early morning hours after one night of intermittent hypoxemia due to untreated OSA. This early-morning OSA-induced augmentation of DUSP1 gene expression was attenuated by continuous positive airway pressure (CPAP) treatment of OSA. Intermittent hypoxia increases MnSOD activity via increased DUSP1 expression in HCAEC. Similarly, overnight intermittent hypoxemia in patients with OSA induces expression of DUSP1, which may mediate increases of MnSOD expression and activity. This may contribute significantly to neutralizing the effects of reactive oxygen species, a consequence of the intermittent hypoxemia/reperfusion elicited by OSA. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  16. Expression and clinical significance of tyrosine phosphatase SHP-2 in colon cancer.

    Science.gov (United States)

    Cai, Peifen; Guo, Wenjie; Yuan, Huaqin; Li, Qian; Wang, Weicheng; Sun, Yang; Li, Xiaomin; Gu, Yanhong

    2014-04-01

    Protein-tyrosine phosphatase SHP-2, encoded by gene PTPN11, has been identified as a tumor-promoting factor in several types of leukemia and is hyper-activated by other mechanisms in some solid tumors including gastric cancer, breast cancer, non-small cell lung cancer (NSCLC), etc. But few were reported on the expression and significances of SHP-2 in colon cancer. Here, we detect SHP-2 expression in colon cancer cells, colon cancer-induced by AOM+DSS in mice and 232 human colon cancer specimens, including 58 groups of self-matched adjacent peritumor tissues and normal tissues. We found that compared to the normal colon tissues, SHP-2 significantly decreased in tumor tissues (Pcolon tumor cells as well as mice colon tumors. And in humans samples, low SHP-2 expression showed a significantly correlation with poor tumor differentiation (P<0.05), late TNM stage (P=0.1666) and lymph node metastasis (P<0.05). Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  17. Kinetics of gene expression of alkaline phosphatase during healing of alveolar bone in rats.

    Science.gov (United States)

    Rodrigues, Willian Caetano; Fabris, André Luís da Silva; Hassumi, Jaqueline Suemi; Gonçalves, Alaíde; Sonoda, Celso Koogi; Okamoto, Roberta

    2016-06-01

    Immunohistochemical studies and molecular biology have enabled us to identify numerous proteins that are involved in the metabolism of bone, and their encoding genes. Among these is alkaline phosphatase (ALP), an enzyme that is responsible for the initiation of mineralisation of the extracellular matrix during alveolar bone repair. To evaluate the gene expression of ALP during this process, we studied nine healthy adult male rats, which had their maxillary central incisors extracted from the right side and were randomly divided into three groups. During three experimental periods, 7 days, 14 days, and 28 days, the alveoli were curetted, the rats killed, and samples analysed by real-time reverse transcription polymerase chain reaction (qRT-PCR). The RNAm that encodes the gene for the synthesis of ALP was expressed during the three periods analysed, but its concentration was significantly increased at 14 and 28 days compared with at 7 days. There was no significant difference between 14 and 28 days (p=0.0005). We conclude that genes related to ALP are expressed throughout the healing process and more intensively during the later periods (14 and 28 days), which coincides with the increased formation of mineralised bone. Copyright © 2016 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

  18. AR-v7 protein expression is regulated by protein kinase and phosphatase

    Science.gov (United States)

    Li, Yinan; Xie, Ning; Gleave, Martin E.; Rennie, Paul S.; Dong, Xuesen

    2015-01-01

    Failure of androgen-targeted therapy and progression of castration-resistant prostate cancer (CRPC) are often attributed to sustained expression of the androgen receptor (AR) and its major splice variant, AR-v7. Although the new generation of anti-androgens such as enzalutamide effectively inhibits AR activity, accumulating pre-clinical and clinical evidence indicates that AR-v7 remains constitutively active in driving CRPC progression. However, molecular mechanisms which control AR-v7 protein expression remain unclear. We apply multiple prostate cancer cell models to demonstrate that enzalutamide induces differential activation of protein phosphatase-1 (PP-1) and Akt kinase depending on the gene context of cancer cells. The balance between PP-1 and Akt activation governs AR phosphorylation status and activation of the Mdm2 ubiquitin ligase. Mdm2 recognizes phosphorylated serine 213 of AR-v7, and induces AR-v7 ubiquitination and protein degradation. These findings highlight the decisive roles of PP-1 and Akt for AR-v7 protein expression and activities when AR is functionally blocked. PMID:26378044

  19. Cocoa Enriched Diets Enhance Expression of Phosphatases and Decrease Expression of Inflammatory Molecules in Trigeminal Ganglion Neurons

    Science.gov (United States)

    Cady, Ryan J.; Durham, Paul L.

    2010-01-01

    Activation of trigeminal nerves and release of neuropeptides that promote inflammation are implicated in the underlying pathology of migraine and temporomandibular joint (TMJ) disorders. The overall response of trigeminal nerves to peripheral inflammatory stimuli involves a balance between enzymes that promote inflammation, kinases, and those that restore homeostasis, phosphatases. The goal of this study was to determine the effects of a cocoa-enriched diet on the expression of key inflammatory proteins in trigeminal ganglion neurons under basal and inflammatory conditions. Rats were fed a control diet or an isocaloric diet enriched in cocoa for 14 days prior to an injection of noxious stimuli to cause acute or chronic excitation of trigeminal neurons. In animals fed a cocoa-enriched diet, basal levels of the mitogen-activated kinase (MAP) phosphatases MKP-1 and MKP-3 were elevated in neurons. Importantly, the stimulatory effects of acute or chronic peripheral inflammation on neuronal expression of the MAPK p38 and extracellular signal-regulated kinases (ERK) were significantly repressed in response to cocoa. Similarly, dietary cocoa significantly suppressed basal neuronal expression of calcitonin gene-related peptide (CGRP) as well as stimulated levels of the inducible form of nitric oxide synthase (iNOS), proteins implicated in the underlying pathology of migraine and TMJ disorders. To our knowledge, this is first evidence that a dietary supplement can cause upregulation of MKP, and that cocoa can prevent inflammatory responses in trigeminal ganglion neurons. Furthermore, our data provide evidence that cocoa contains biologically active compounds that would be beneficial in the treatment of migraine and TMJ disorders. PMID:20138852

  20. Expression, purification and preliminary crystallographic analysis of Mycobacterium tuberculosis CysQ, a phosphatase involved in sulfur metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Erickson, Anna I.; Sarsam, Reta D.; Fisher, Andrew J., E-mail: ajfisher@ucdavis.edu

    2014-05-10

    The cysQ gene from Mycobacterium tuberculosis was cloned and the expressed protein, a 3′-phosphoadenosine-5′’-phosphatase, was purified and crystallized. X-ray diffraction data were collected to 1.7 Å resolution.

  1. Transient expression of protein tyrosine phosphatases encoded in Cotesia plutellae bracovirus inhibits insect cellular immune responses

    Science.gov (United States)

    Ibrahim, Ahmed M. A.; Kim, Yonggyun

    2008-01-01

    Several immunosuppressive factors are associated with parasitism of an endoparasitoid wasp, Cotesia plutellae, on the diamondback moth, Plutella xylostella. C. plutellae bracovirus (CpBV) encodes a large number of putative protein tyrosine phosphatases (PTPs), which may play a role in inhibiting host cellular immunity. To address this inhibitory hypothesis of CpBV-PTPs, we performed transient expression of individual CpBV-PTPs in hemocytes of the beet armyworm, Spodoptera exigua, and analyzed their cellular immune responses. Two different forms of CpBV-PTPs were chosen and cloned into a eukaryotic expression vector under the control of the p10 promoter of baculovirus: one with the normal cysteine active site (CpBV-PTP1) and the other with a mutated active site (CpBV-PTP5). The hemocytes transfected with CpBV-PTP1 significantly increased in PTP activity compared to control hemocytes, but those with CpBV-PTP5 exhibited a significant decrease in the PTP activity. All transfected hemocytes exhibited a significant reduction in both cell spreading and encapsulation activities compared to control hemocytes. Co-transfection of CpBV-PTP1 together with its double-stranded RNA reduced the messenger RNA (mRNA) level of CpBV-PTP1 and resulted in recovery of both hemocyte behaviors. This is the first report demonstrating that the polydnaviral PTPs can manipulate PTP activity of the hemocytes to interrupt cellular immune responses.

  2. Protein tyrosine phosphatase, PTP1B, expression and activity in rat corneal endothelial cells

    Science.gov (United States)

    Harris, Deshea L.

    2007-01-01

    Purpose The current studies were conducted to determine whether the protein tyrosine phosphatase, PTP1B, plays a role in regulating epidermal growth factor receptor (EGFR) Tyr992 phosphorylation and cell cycle entry in rat corneal endothelial cells. Methods Corneas were obtained from male Sprague-Dawley rats. PTP1B mRNA and protein expression were compared in confluent and subconfluent cells by RT-PCR and western blots. Immunocytochemistry was used to determine the subcellular localization of both PTP1B and EGFR following epidermal growth factor (EGF) stimulation. Western blots were used to analyze the time-dependent effect of EGF on phosphorylation of EGFR Tyr992 plus or minus CinnGEL 2Me, an inhibitor of PTP1B activity. The effect of PTP1B inhibition on cell cycle entry was determined by calculating the percent of Ki67-positive cells following EGF treatment. Results PTP1B mRNA expression was similar in confluent and subconfluent cells, but PTP1B protein was expressed at 3 fold higher levels in subconfluent cells. Positive staining for PTP1B was localized in vesicular structures below the plasma membrane. EGFR staining was located at cell-cell borders in untreated endothelium, but was mainly cytoplasmic by 15 min after EGF treatment. In control cultures, phosphorylation of EGFR Tyr992 peaked by 5 min following EGF stimulation and rapidly decreased to basal levels by 30 min. In cultures pretreated with CinnGEL 2Me, Tyr992 phosphorylation peaked 2 min following EGF addition and was consistently sustained at a higher level than controls until 60 min after treatment. By 18 h following EGF treatment, cultures pretreated with CinnGEL 2Me exhibited a 1.7 fold increase in the number of Ki67-positive cells compared with control cultures. Conclusions Comparison of PTP1B mRNA and protein levels indicates that PTP1B expression is regulated mainly at the protein level and is higher in subconfluent cells. PTP1B was located in vesicles below the plasma membrane. The fact that

  3. Wnt/β-catenin expression does not correlate with serum alkaline phosphatase concentration in canine osteosarcoma patients.

    Directory of Open Access Journals (Sweden)

    Caroline M Piskun

    Full Text Available Osteosarcoma is an aggressive malignancy of the bone and an increase in serum alkaline phosphatase concentration has clinical prognostic value in both humans and canines. Increased serum alkaline phosphatase concentration at the time of diagnosis has been associated with poorer outcomes for osteosarcoma patients. The biology underlying this negative prognostic factor is poorly understood. Given that activation of the Wnt signaling pathway has been associated with alkaline phosphatase expression in osteoblasts, we hypothesized that the Wnt/β-catenin signaling pathway would be differentially activated in osteosarcoma tissue based on serum ALP status. Archived canine osteosarcoma samples and primary canine osteosarcoma cell lines were used to evaluate the status of Wnt/β-catenin signaling pathway activity through immunohistochemical staining, western immunoblot analyses, quantitative reverse-transcription polymerase chain reaction, and a Wnt-responsive promoter activity assay. We found no significant difference in β-catenin expression or activation between OSA populations differing in serum ALP concentration. Pathway activity was mildly increased in the primary OSA cell line generated from a patient with increased serum ALP compared to the normal serum ALP OSA cell line. Further investigation into the mechanisms underlying differences in serum ALP concentration is necessary to improve our understanding of the biological implications of this negative prognostic indicator.

  4. Identification of soybean purple acid phosphatase genes and their expression responses to phosphorus availability and symbiosis

    Science.gov (United States)

    Background and Aims Purple acid phosphatases (PAPs) are members of the metallo-phosphoesterase family and have been known to play important roles in phosphorus (P) acquisition and recycling in plants. Low P availability is a major constraint to growth and production of soybean, Glycine max. Comparat...

  5. Expression of the voltage-sensing phosphatase gene in the chick embryonic tissues and in the adult cerebellum.

    Science.gov (United States)

    Yamaguchi, Shinji; Aoki, Naoya; Kitajima, Takaaki; Okamura, Yasushi; Homma, Koichi J

    2014-10-01

    Voltage-sensing phosphatase (VSP) consists of a transmembrane voltage sensor domain (VSD) and the cytoplasmic domain with phosphoinositide-phosphatase activities. It operates as the voltage sensor and directly translates membrane potential into phosphoinositide turnover by coupling VSD to the cytoplasmic domain. VSPs are evolutionarily conserved from marine invertebrate up to humans. Recently, we demonstrated that ectopic expression of the chick ortholog of VSP, Gg-VSP, in a fibroblast cell line caused characteristic cell process outgrowths. Co-expression of chick PTEN suppressed such morphological change, suggesting that VSP regulates cell shape by increasing PI(3,4)P2. However, the in vivo function of Gg-VSP remains unclear. Here, we showed that in chick embryos Gg-VSP is expressed in the stomach, mesonephros, pharyngeal arch, limb bud, somites, floor plate of neural tube, and notochord. In addition, both Gg-VSP transcripts and the protein were found in the cerebellar Purkinje neurons. These findings provide an insight into the physiological functions of VSP.

  6. Control of Acid Phosphatases Expression from Aspergillus niger by Soil Characteristics

    Directory of Open Access Journals (Sweden)

    Ely Nahas

    2015-10-01

    Full Text Available ABSTRACTThis work studied the acid phosphatase (APase activity from culture medium (extracellular, eAPase and mycelial extract (intracellular, iAPase ofAspergillus niger F111. The influence of fungus growth and phosphate concentration of the media on the synthesis and secretion of phosphatase was demonstrated. The effects of pH, substrate concentration and inorganic and organic compounds added to the reaction mixture on APase activity were also studied. Both enzymes were repressed by high concentrations of phosphate. Overexpression of iAPase in relation to eAPase was detected; iAPase activity was 46.1 times higher than eAPase. The maximal activity of eAPase was after 24h of fungus growth and for iAPase was after 96h. Optimal pH and substrate concentrations were 4.5 and 8.0 mM, respectively. Michaelis-Menten constant (Km for the hydrolysis of p-nitrophenyl phosphate was 0.57 mM with Vmax = 14,285.71 U mg-1 mycelium for the iAPase and 0.31 mM with V max = 147.06 U mg-1 mycelium for eAPase. Organic substances had little effect on acid phosphatases when compared with the salts. Both the APases were inhibited by 10 mM KH 2PO4 and 5 mM (NH42MoO4; eAPase was also inhibited by 1 mM CoCl2.

  7. cAMP signalling of Bordetella adenylate cyclase toxin through the SHP-1 phosphatase activates the BimEL-Bax pro-apoptotic cascade in phagocytes

    Czech Academy of Sciences Publication Activity Database

    Ahmad, Jawid Nazir; Černý, Ondřej; Linhartová, Irena; Mašín, Jiří; Osička, Radim; Šebo, Peter

    2016-01-01

    Roč. 18, č. 3 (2016), s. 384-398 ISSN 1462-5814 R&D Projects: GA ČR GA13-14547S; GA ČR(CZ) GAP302/11/0580; GA ČR GAP302/12/0460 Institutional support: RVO:61388971 Keywords : FORKHEAD TRANSCRIPTION FACTOR * HUMAN T- CELL S * DENDRITIC CELL S Subject RIV: EE - Microbiology, Virology Impact factor: 4.554, year: 2016

  8. Bordetella pertussis Adenylate Cyclase Toxin Blocks Induction of Bactericidal Nitric Oxide in Macrophages through cAMP-Dependent Activation of the SHP-1 Phosphatase

    Czech Academy of Sciences Publication Activity Database

    Černý, Ondřej; Kamanová, Jana; Mašín, Jiří; Bíbová, Ilona; Škopová, Karolína; Šebo, Peter

    2015-01-01

    Roč. 194, č. 10 (2015), s. 4901-4913 ISSN 0022-1767 R&D Projects: GA ČR GAP302/12/0460; GA ČR GA13-14547S Institutional support: RVO:61388971 Keywords : CYCLIC-AMP * MURINE MACROPHAGE S * IFN-GAMMA Subject RIV: EE - Microbiology, Virology Impact factor: 4.985, year: 2015

  9. Dynamics of Protein Phosphatase Gene Expression in Corbicula fluminea Exposed to Microcystin-LR and to Toxic Microcystis aeruginosa Cells

    Directory of Open Access Journals (Sweden)

    Vitor Vasconcelos

    2011-12-01

    Full Text Available This study investigated the in vivo effects of microcystins on gene expression of several phosphoprotein phosphatases (PPP in the freshwater clam Corbicula fluminea with two different exposure scenarios. Clams were exposed for 96 h to 5 µg L−1 of dissolved microcystin-LR and the relative changes of gene expression of three different types of PPP (PPP1, 2 and 4 were analyzed by quantitative real-time PCR. The results showed a significant induction of PPP2 gene expression in the visceral mass. In contrast, the cyanotoxin did not cause any significant changes on PPP1 and PPP4 gene expression. Based on these results, we studied alterations in transcriptional patterns in parallel with enzymatic activity of C. fluminea for PPP2, induced by a Microcystis aeruginosa toxic strain (1 × 105 cells cm−3 during 96 h. The relative changes of gene expression and enzyme activity in visceral mass were analyzed by quantitative real-time PCR and colorimetric assays respectively. The clams exhibited a significant reduction of PPP2 activity with a concomitant enhancement of gene expression. Considering all the results we can conclude that the exposure to an ecologically relevant concentration of pure or intracellular microcystins (-LR promoted an in vivo effect on PPP2 gene expression in C. fluminea.

  10. The expression of a novel receptor-type tyrosine phosphatase suggests a role in morphogenesis and plasticity of the nervous system

    DEFF Research Database (Denmark)

    Canoll, P D; Barnea, G; Levy, J B

    1993-01-01

    Analysis of the localization of receptor-type protein tyrosine phosphatase-beta (RPTP-beta) by in situ hybridization and immunocytochemistry indicates that it is predominantly expressed in the developing central nervous system (CNS). RPTP-beta is highly expressed in radial glia and other forms....... In the adult, high levels of RPTP-beta are seen in regions of the brain where there is continued neurogenesis and neurite outgrowth. The spatial and temporal patterns of RPTP-beta expression suggest that this receptor phosphatase plays a role in morphogenesis and plasticity of the nervous system....

  11. Increased PTP1B expression and phosphatase activity in colorectal cancer results in a more invasive phenotype and worse patient outcome.

    Science.gov (United States)

    Hoekstra, Elmer; Das, Asha M; Swets, Marloes; Cao, Wanlu; van der Woude, C Janneke; Bruno, Marco J; Peppelenbosch, Maikel P; Kuppen, Peter J K; Ten Hagen, Timo L M; Fuhler, Gwenny M

    2016-04-19

    Cell signaling is dependent on the balance between phosphorylation of proteins by kinases and dephosphorylation by phosphatases. This balance if often disrupted in colorectal cancer (CRC), leading to increased cell proliferation and invasion. For many years research has focused on the role of kinases as potential oncogenes in cancer, while phosphatases were commonly assumed to be tumor suppressive. However, this dogma is currently changing as phosphatases have also been shown to induce cancer growth. One of these phosphatases is protein tyrosine phosphatase 1B (PTP1B). Here we report that the expression of PTP1B is increased in colorectal cancer as compared to normal tissue, and that the intrinsic enzymatic activity of the protein is also enhanced. This suggests a role for PTP1B phosphatase activity in CRC formation and progression. Furthermore, we found that increased PTP1B expression is correlated to a worse patient survival and is an independent prognostic marker for overall survival and disease free survival. Knocking down PTP1B in CRC cell lines results in a less invasive phenotype with lower adhesion, migration and proliferation capabilities. Together, these results suggest that inhibition of PTP1B activity is a promising new target in the treatment of colorectal cancer and the prevention of metastasis.

  12. Regulation of hemolysin expression and virulence of Staphylococcus aureus by a serine/threonine kinase and phosphatase.

    Directory of Open Access Journals (Sweden)

    Kellie Burnside

    2010-06-01

    Full Text Available Exotoxins, including the hemolysins known as the alpha (alpha and beta (beta toxins, play an important role in the pathogenesis of Staphylococcus aureus infections. A random transposon library was screened for S. aureus mutants exhibiting altered hemolysin expression compared to wild type. Transposon insertions in 72 genes resulting in increased or decreased hemolysin expression were identified. Mutations inactivating a putative cyclic di-GMP synthetase and a serine/threonine phosphatase (Stp1 were found to reduce hemolysin expression, and mutations in genes encoding a two component regulator PhoR, LysR family transcriptional regulator, purine biosynthetic enzymes and a serine/threonine kinase (Stk1 increased expression. Transcription of the hla gene encoding alpha toxin was decreased in a Deltastp1 mutant strain and increased in a Deltastk1 strain. Microarray analysis of a Deltastk1 mutant revealed increased transcription of additional exotoxins. A Deltastp1 strain is severely attenuated for virulence in mice and elicits less inflammation and IL-6 production than the Deltastk1 strain. In vivo phosphopeptide enrichment and mass spectrometric analysis revealed that threonine phosphorylated peptides corresponding to Stk1, DNA binding histone like protein (HU, serine-aspartate rich fibrinogen/bone sialoprotein binding protein (SdrE and a hypothetical protein (NWMN_1123 were present in the wild type and not in the Deltastk1 mutant. Collectively, these studies suggest that Stk1 mediated phosphorylation of HU, SrdE and NWMN_1123 affects S. aureus gene expression and virulence.

  13. Characterization of a human cell line stably over-expressing the candidate oncogene, dual specificity phosphatase 12.

    Directory of Open Access Journals (Sweden)

    Erica L Cain

    2011-04-01

    Full Text Available Analysis of chromosomal rearrangements within primary tumors has been influential in the identification of novel oncogenes. Identification of the "driver" gene(s within cancer-derived amplicons is, however, hampered by the fact that most amplicons contain many gene products. Amplification of 1q21-1q23 is strongly associated with liposarcomas and microarray-based comparative genomic hybridization narrowed down the likely candidate oncogenes to two: the activating transcription factor 6 (atf6 and the dual specificity phosphatase 12 (dusp12. While atf6 is an established transcriptional regulator of the unfolded protein response, the potential role of dusp12 in cancer remains uncharacterized.To evaluate the oncogenic potential of dusp12, we established stable cell lines that ectopically over-express dusp12 in isolation and determined whether this cell line acquired properties frequently associated with transformed cells. Here, we demonstrate that cells over-expressing dusp12 display increased cell motility and resistance to apoptosis. Additionally, over-expression of dusp12 promoted increased expression of the c-met proto-oncogene and the collagen and laminin receptor intergrin alpha 1 (itga1 which is implicated in metastasis.Collectively, these results suggest that dusp12 is oncologically relevant and exposes a potential association between dusp12 and established oncogenes that could be therapeutically targeted.

  14. Enteral intestinal alkaline phosphatase administration in newborns decreases iNOS expression in a neonatal necrotizing enterocolitis rat model.

    Science.gov (United States)

    Rentea, Rebecca M; Liedel, Jennifer L; Fredrich, Katherine; Pritchard, Kirkwood; Oldham, Keith T; Simpson, Pippa M; Gourlay, David M

    2013-01-01

    To determine if intestinal alkaline phosphatase (IAP) decreases intestinal injury resulting from experimentally induced necrotizing enterocolitis (NEC). We hypothesized that IAP administration prevents the initial development of NEC related intestinal inflammation. Pre- and full-term newborn Sprague-Dawley rat pups were sacrificed on day 1 of life. Pre-term pups were exposed to intermittent hypoxia and formula containing LPS to induce NEC. Select NEC pups were given 40, 4 or 0.4 units/kg of bovine IAP (NEC+IAP40u, IAP4u or IAP0.4u) enterally, once daily. Ileal sections were evaluated by real-time PCR (qRT-PCR) for IAP, iNOS, IL-1β, IL-6, and TNF-α mRNA and immunofluorescence for 3-nitrotyrosine (3-NT). Experimentally induced NEC decreased IAP mRNA expression by 66% (p ≤ 0.001). IAP supplementation increased IAP mRNA expression to control. Supplemental enteral IAP decreased nitrosative stress as measured by iNOS mRNA expression and 3-NT staining in the NEC stressed pups (p ≤ 0.01), as well as decreased intestinal TNF-α mRNA expression. In addition, IAP decreased LSP translocation into the serum in the treated pups. We conclude that enterally administered IAP prevents NEC-related intestinal injury and inflammation. Enteral IAP may prove a useful strategy in the prevention of NEC in preterm neonates. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Kinetic comparison of tissue non-specific and placental human alkaline phosphatases expressed in baculovirus infected cells: application to screening for Down's syndrome.

    Science.gov (United States)

    Denier, Colette C; Brisson-Lougarre, Andrée A; Biasini, Ghislaine G; Grozdea, Jean J; Fournier, Didier D

    2002-01-01

    In humans, there are four alkaline phosphatases, and each form exhibits a characteristic pattern of tissue distribution. The availability of an easy method to reveal their activity has resulted in large amount of data reporting correlations between variations in activity and illnesses. For example, alkaline phosphatase from neutrophils of mothers pregnant with a trisomy 21 fetus (Down's syndrome) displays significant differences both in its biochemical and immunological properties, and in its affinity for some specific inhibitors. To analyse these differences, the biochemical characteristics of two isozymes (non specific and placental alkaline phosphatases) were expressed in baculovirus infected cells. Comparative analysis of the two proteins allowed us to estimate the kinetic constants of denaturation and sensitivity to two inhibitors (L-p-bromotetramisole and thiophosphate), allowing better discrimination between the two enzymes. These parameters were then used to estimate the ratio of the two isoenzymes in neutrophils of pregnant mothers with or without a trisomy 21 fetus. It appeared that the placental isozyme represented 13% of the total activity of neutrophils of non pregnant women. This proportion did not significantly increase with normal pregnancy. By contrast, in pregnancies with trisomy 21 fetus, the proportion reached 60-80% of activity. Over-expression of the placental isozyme compared with the tissue-nonspecific form in neutrophils of mother with a trisomy 21 fetus may explain why the characteristics of the alkaline phosphatase in these cells is different from normal. Application of this knowledge could improve the potential of using alkaline phosphatase measurements to screen for Down's syndrome.

  16. Expression and function of the protein tyrosine phosphatase receptor J (PTPRJ in normal mammary epithelial cells and breast tumors.

    Directory of Open Access Journals (Sweden)

    Chanel E Smart

    Full Text Available The protein tyrosine phosphatase receptor J, PTPRJ, is a tumor suppressor gene that has been implicated in a range of cancers, including breast cancer, yet little is known about its role in normal breast physiology or in mammary gland tumorigenesis. In this paper we show that PTPRJ mRNA is expressed in normal breast tissue and reduced in corresponding tumors. Meta-analysis revealed that the gene encoding PTPRJ is frequently lost in breast tumors and that low expression of the transcript associated with poorer overall survival at 20 years. Immunohistochemistry of PTPRJ protein in normal human breast tissue revealed a distinctive apical localisation in the luminal cells of alveoli and ducts. Qualitative analysis of a cohort of invasive ductal carcinomas revealed retention of normal apical PTPRJ localization where tubule formation was maintained but that tumors mostly exhibited diffuse cytoplasmic staining, indicating that dysregulation of localisation associated with loss of tissue architecture in tumorigenesis. The murine ortholog, Ptprj, exhibited a similar localisation in normal mammary gland, and was differentially regulated throughout lactational development, and in an in vitro model of mammary epithelial differentiation. Furthermore, ectopic expression of human PTPRJ in HC11 murine mammary epithelial cells inhibited dome formation. These data indicate that PTPRJ may regulate differentiation of normal mammary epithelia and that dysregulation of protein localisation may be associated with tumorigenesis.

  17. Intestinal alkaline phosphatase administration in newborns decreases systemic inflammatory cytokine expression in a neonatal necrotizing enterocolitis rat model.

    Science.gov (United States)

    Rentea, Rebecca M; Liedel, Jennifer L; Fredrich, Katherine; Welak, Scott R; Pritchard, Kirkwood A; Oldham, Keith T; Simpson, Pippa M; Gourlay, David M

    2012-10-01

    Supplementation of intestinal alkaline phosphatase (IAP), an endogenous protein expressed in the intestines, decreases the severity of necrotizing enterocolitis (NEC)-associated intestinal injury and permeability. We hypothesized that IAP administration is protective in a dose-dependent manner of the inflammatory response in a neonatal rat model. Pre- and full-term newborn Sprague-Dawley rat pups were sacrificed on day of life 3. Control pups were vaginally delivered and dam fed. Preterm pups were delivered via cesarean section and exposed to intermittent hypoxia and formula feeds containing lipopolysaccharide (NEC) with and without IAP. Three different standardized doses were administered to a group of pups treated with 40, 4, and 0.4U/kg of bovine IAP (NEC+IAP40, IAP4, or IAP0.4U). Reverse transcription-real-time polymerase chain reaction (RT-PCR) for inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF)-α on liver and lung tissues and serum cytokine analysis for interleukin (IL)-1β, IL-6, IL-10, and TNF-α were performed. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests, expressed as mean±standard error of the mean and P≤0.05 considered significant. Levels of cytokines IL-1β, IL-6, and TNF-α increased significantly in NEC versus control, returning to control levels with increasing doses of supplemental enteral IAP. Hepatic and pulmonary TNF-α and iNOS messenger ribonucleic acid expressions increased in NEC, and the remaining elevated despite IAP supplementation. Proinflammatory cytokine expression is increased systemically with intestinal NEC injury. Administration of IAP significantly reduces systemic proinflammatory cytokine expression in a dose-dependent manner. Early supplemental enteral IAP may reduce NEC-related injury and be useful for reducing effects caused by a proinflammatory cascade. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. Mitogen-activated protein kinase phosphatase-1 expression in macrophages is controlled by lymphocytes during macrophage activation.

    Science.gov (United States)

    Luo, Chong; Yang, Xiqiang; Yao, Lan; Jiang, Liping; Liu, Wei; Li, Xin; Wang, Lijia

    2012-01-01

    The viewpoints on the control of innate immune cells by the adaptive immune system during sepsis remain controversial. Mitogen-activated protein kinase phosphatase-1 (MKP-1) is essential to the negative control of innate immunity and suppresses the activation of macrophages by inhibiting activated mitogen-activated protein kinase (MAPK). The purpose of the current study was to observe inflammatory response and macrophage activation in mice with severe combined immunodeficiency (SCID) with endotoxemia and to determine the role of MKP-1 in the control of macrophage activation by the adaptive immune system. Endotoxemia was induced in wild-type and SCID mice by an intraperitoneal injection of lipopolysaccharide (LPS), and all of the SCID mice died. SCID mice produced more inflammatory cytokines than BALB/c mice systemically and locally. TNF-α mRNA expression was higher and MKP-1 mRNA expression was lower in peritoneal macrophages (PMa) from SCID mice compared to PMa from wild-type mice after and even before LPS injection. Thioglycollate-stimulated PMa from wild-type mice were stimulated with LPS in vitro in the presence or absence of pan-T cells. The levels of TNF-α and IL-6 were higher in the supernatants from PMa cultured alone compared to PMa co-cultured with pan-T cells, and PMa MKP-1 mRNA and protein expression were higher when PMa were co-cultured with pan-T cells. Therefore, pan-T cells can up-regulate MKP-1 expression in macrophages and inhibit the secretion of inflammatory cytokines secretion by macrophages. In SCID mice, lymphocyte deficiency, especially T cell deficiency, causes insufficient MKP-1 expression in macrophages, which can be responsible for the severe inflammation and bad prognosis of septic SCID mice. MKP-1 plays an important role in the control of macrophage activation by the adaptive immune system.

  19. Yeast PAH1-encoded phosphatidate phosphatase controls the expression of CHO1-encoded phosphatidylserine synthase for membrane phospholipid synthesis.

    Science.gov (United States)

    Han, Gil-Soo; Carman, George M

    2017-08-11

    The PAH1 -encoded phosphatidate phosphatase (PAP), which catalyzes the committed step for the synthesis of triacylglycerol in Saccharomyces cerevisiae , exerts a negative regulatory effect on the level of phosphatidate used for the de novo synthesis of membrane phospholipids. This raises the question whether PAP thereby affects the expression and activity of enzymes involved in phospholipid synthesis. Here, we examined the PAP-mediated regulation of CHO1 -encoded phosphatidylserine synthase (PSS), which catalyzes the committed step for the synthesis of major phospholipids via the CDP-diacylglycerol pathway. The lack of PAP in the pah1 Δ mutant highly elevated PSS activity, exhibiting a growth-dependent up-regulation from the exponential to the stationary phase of growth. Immunoblot analysis showed that the elevation of PSS activity results from an increase in the level of the enzyme encoded by CHO1 Truncation analysis and site-directed mutagenesis of the CHO1 promoter indicated that Cho1 expression in the pah1 Δ mutant is induced through the inositol-sensitive upstream activation sequence (UAS INO ), a cis -acting element for the phosphatidate-controlled Henry (Ino2-Ino4/Opi1) regulatory circuit. The abrogation of Cho1 induction and PSS activity by a CHO1 UAS INO mutation suppressed pah1 Δ effects on lipid synthesis, nuclear/endoplasmic reticulum membrane morphology, and lipid droplet formation, but not on growth at elevated temperature. Loss of the DGK1 -encoded diacylglycerol kinase, which converts diacylglycerol to phosphatidate, partially suppressed the pah1 Δ-mediated induction of Cho1 and PSS activity. Collectively, these data showed that PAP activity controls the expression of PSS for membrane phospholipid synthesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Kinetic comparison of tissue non-specific and placental human alkaline phosphatases expressed in baculovirus infected cells: application to screening for Down's syndrome

    Science.gov (United States)

    Denier, Colette C; Brisson-Lougarre, Andrée A; Biasini, Ghislaine G; Grozdea, Jean J; Fournier, Didier D

    2002-01-01

    Background In humans, there are four alkaline phosphatases, and each form exibits a characteristic pattern of tissue distribution. The availability of an easy method to reveal their activity has resulted in large amount of data reporting correlations between variations in activity and illnesses. For example, alkaline phosphatase from neutrophils of mothers pregnent with a trisomy 21 fetus (Down's syndrome) displays significant differences both in its biochemical and immunological properties, and in its affinity for some specific inhibitors. Results To analyse these differences, the biochemical characteristics of two isozymes (non specific and placental alkaline phosphatases) were expressed in baculovirus infected cells. Comparative analysis of the two proteins allowed us to estimate the kinetic constants of denaturation and sensitivity to two inhibitors (L-p-bromotetramisole and thiophosphate), allowing better discrimination between the two enzymes. These parameters were then used to estimate the ratio of the two isoenzymes in neutrophils of pregnant mothers with or without a trisomy 21 fetus. It appeared that the placental isozyme represented 13% of the total activity of neutrophils of non pregnant women. This proportion did not significantly increase with normal pregnancy. By contrast, in pregnancies with trisomy 21 fetus, the proportion reached 60–80% of activity. Conclusion Over-expression of the placental isozyme compared with the tissue-nonspecific form in neutrophils of mother with a trisomy 21 fetus may explain why the characteristics of the alkaline phosphatase in these cells is different from normal. Application of this knowledge could improve the potential of using alkaline phosphatase measurements to screen for Down's syndrome. PMID:11818032

  1. Kinetic comparison of tissue non-specific and placental human alkaline phosphatases expressed in baculovirus infected cells: application to screening for Down's syndrome

    Directory of Open Access Journals (Sweden)

    Grozdea Jean J

    2002-01-01

    Full Text Available Abstract Background In humans, there are four alkaline phosphatases, and each form exibits a characteristic pattern of tissue distribution. The availability of an easy method to reveal their activity has resulted in large amount of data reporting correlations between variations in activity and illnesses. For example, alkaline phosphatase from neutrophils of mothers pregnent with a trisomy 21 fetus (Down's syndrome displays significant differences both in its biochemical and immunological properties, and in its affinity for some specific inhibitors. Results To analyse these differences, the biochemical characteristics of two isozymes (non specific and placental alkaline phosphatases were expressed in baculovirus infected cells. Comparative analysis of the two proteins allowed us to estimate the kinetic constants of denaturation and sensitivity to two inhibitors (L-p-bromotetramisole and thiophosphate, allowing better discrimination between the two enzymes. These parameters were then used to estimate the ratio of the two isoenzymes in neutrophils of pregnant mothers with or without a trisomy 21 fetus. It appeared that the placental isozyme represented 13% of the total activity of neutrophils of non pregnant women. This proportion did not significantly increase with normal pregnancy. By contrast, in pregnancies with trisomy 21 fetus, the proportion reached 60–80% of activity. Conclusion Over-expression of the placental isozyme compared with the tissue-nonspecific form in neutrophils of mother with a trisomy 21 fetus may explain why the characteristics of the alkaline phosphatase in these cells is different from normal. Application of this knowledge could improve the potential of using alkaline phosphatase measurements to screen for Down's syndrome.

  2. Changes in Expression of Connexin 32, Bile Canaliculus-Like Structures, and Localization of Alkaline Phosphatase in Primary Cultures of Fetal Rat Hepatocytes

    International Nuclear Information System (INIS)

    Fukazawa, Shoko; Chida, Kohsuke; Taguchi, Meiko; Takeuchi, Akihiro; Ikeda, Noriaki

    2013-01-01

    We devised an experimental design in primary cultures of fetal rat hepatocytes for studying hepatocyte differentiation over a short period. In the present study, hepatocytes were first cultured for 3 days in dexamethasone-supplemented medium and then for an additional 3 days in dexamethasone- or epidermal growth factor-supplemented medium. In hepatocytes cultured continuously in dexamethasone-supplemented medium, the expression of connexin 32 increased and bile canaliculus-like structures and localization of alkaline phosphatase in the plasma membrane around bile canaliculus-like structures were maintained. Few cells incorporated bromodeoxyuridine. On the other hand, in most of the hepatocytes cultured in epidermal growth factor-supplemented medium, the expression of connexin 32 was minimally recognized, bile canaliculus-like structures were shortened or eliminated, and alkaline phosphatase was localized as numerous fine spots throughout the cytoplasm. More than 20% of all hepatocytes incorporated bromodeoxyuridine. The present study suggests that in hepatocytes, there is a close relationship among connexin 32 expression, the maintenance of bile canaliculus-like structures, and the localization of alkaline phosphatase to the plasma membrane around the bile canaliculus-like structures, and this indicates that the present experimental model is useful for studying hepatocyte differentiation over a short period

  3. Up-regulation of phosphoinositide metabolism in tobacco cells constitutively expressing the human type I inositol polyphosphate 5-phosphatase

    Science.gov (United States)

    Perera, Imara Y.; Love, John; Heilmann, Ingo; Thompson, William F.; Boss, Wendy F.; Brown, C. S. (Principal Investigator)

    2002-01-01

    To evaluate the impact of suppressing inositol 1,4,5-trisphosphate (InsP(3)) in plants, tobacco (Nicotiana tabacum) cells were transformed with the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an enzyme which specifically hydrolyzes InsP(3). The transgenic cell lines showed a 12- to 25-fold increase in InsP 5-ptase activity in vitro and a 60% to 80% reduction in basal InsP(3) compared with wild-type cells. Stimulation with Mas-7, a synthetic analog of the wasp venom peptide mastoparan, resulted in an approximately 2-fold increase in InsP(3) in both wild-type and transgenic cells. However, even with stimulation, InsP(3) levels in the transgenic cells did not reach wild-type basal values, suggesting that InsP(3) signaling is compromised. Analysis of whole-cell lipids indicated that phosphatidylinositol 4,5-bisphosphate (PtdInsP(2)), the lipid precursor of InsP(3), was greatly reduced in the transgenic cells. In vitro assays of enzymes involved in PtdInsP(2) metabolism showed that the activity of the PtdInsP(2)-hydrolyzing enzyme phospholipase C was not significantly altered in the transgenic cells. In contrast, the activity of the plasma membrane PtdInsP 5 kinase was increased by approximately 3-fold in the transgenic cells. In vivo labeling studies revealed a greater incorporation of (32)P into PtdInsP(2) in the transgenic cells compared with the wild type, indicating that the rate of PtdInsP(2) synthesis was increased. These studies show that the constitutive expression of the human type I InsP 5-ptase in tobacco cells leads to an up-regulation of the phosphoinositide pathway and highlight the importance of PtdInsP(2) synthesis as a regulatory step in this system.

  4. Protein tyrosine phosphatases: regulatory mechanisms.

    NARCIS (Netherlands)

    den Hertog, J.; Ostman, A.; Bohmer, F.D.

    2008-01-01

    Protein-tyrosine phosphatases are tightly controlled by various mechanisms, ranging from differential expression in specific cell types to restricted subcellular localization, limited proteolysis, post-translational modifications affecting intrinsic catalytic activity, ligand binding and

  5. Down-regulated expression of the protein-tyrosine phosphatase 1B (PTP1B) is associated with aggressive clinicopathologic features and poor prognosis in hepatocellular carcinoma

    International Nuclear Information System (INIS)

    Zheng, Long-Yi; Zhou, Dong-Xun; Lu, Jin; Zhang, Wen-Jun; Zou, Da-Jin

    2012-01-01

    Highlights: ► PTP1B protein showed decreased expression in 67.79% of the HCC patients. ► Low PTP1B expression predicts poor prognosis of HCC. ► Low PTP1B expression is correlated with expansion of OV6 + tumor-initiating cells. ► Down-regulation of PTP1B is associated with activation of Wnt/β-Catenin signaling. -- Abstract: The protein-tyrosine phosphatase 1B (PTP1B) is a classical non-transmembrane protein tyrosine phosphatase that plays a key role in metabolic signaling and can exert both tumor suppressing and tumor promoting effects in different cancers depending on the substrate involved and the cellular context. However, the expression level and function of PTP1B in hepatocellular carcinoma (HCC) remain unclear. In this study, PTP1B expression was detected by immunohistochemistry in normal liver tissue (n = 16) and hepatocellular carcinoma (n = 169). The correlations between PTP1B expression level and clinicopathologic features and patient survival were also analyzed. One hundred and eleven of 169 HCC patients (65.7%) had negative or low PTP1B expression in tumorous tissues, whereas normal tissues always expressed strong PTP1B. Decreased PTP1B expression was significantly associated with aggressive clinicopathologic features and poor prognosis. Immunohistochemistry also showed that low PTP1B expression level was correlated with high percentage of OV6 + tumor-initiating cells (T-ICs) and high frequency of nuclear β-Catenin expression in HCC specimens. Our findings demonstrate for the first time that the loss of inhibitory effect of PTP1B may contribute to progression and invasion of HCC through activation of Wnt/β-Catenin signaling and expansion of liver T-ICs. PTP1B may serve as a valuable prognostic biomarker and potential therapeutic target in HCC.

  6. Down-regulated expression of the protein-tyrosine phosphatase 1B (PTP1B) is associated with aggressive clinicopathologic features and poor prognosis in hepatocellular carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Long-Yi [Department of Endocrinology, Changhai Hospital, 168 Changhai Road, Shanghai 200433 (China); Zhou, Dong-Xun [Department of Comprehensive Treatment II, Eastern Hepatobiliary Surgery Hospital, 225 Changhai Road, Shanghai 200438 (China); Lu, Jin [Department of Endocrinology, Changhai Hospital, 168 Changhai Road, Shanghai 200433 (China); Zhang, Wen-Jun [Department of Emergency, Changhai Hospital, 168 Changhai Road, Shanghai 200433 (China); Zou, Da-Jin, E-mail: dajinzou@hotmail.com [Department of Endocrinology, Changhai Hospital, 168 Changhai Road, Shanghai 200433 (China)

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer PTP1B protein showed decreased expression in 67.79% of the HCC patients. Black-Right-Pointing-Pointer Low PTP1B expression predicts poor prognosis of HCC. Black-Right-Pointing-Pointer Low PTP1B expression is correlated with expansion of OV6{sup +} tumor-initiating cells. Black-Right-Pointing-Pointer Down-regulation of PTP1B is associated with activation of Wnt/{beta}-Catenin signaling. -- Abstract: The protein-tyrosine phosphatase 1B (PTP1B) is a classical non-transmembrane protein tyrosine phosphatase that plays a key role in metabolic signaling and can exert both tumor suppressing and tumor promoting effects in different cancers depending on the substrate involved and the cellular context. However, the expression level and function of PTP1B in hepatocellular carcinoma (HCC) remain unclear. In this study, PTP1B expression was detected by immunohistochemistry in normal liver tissue (n = 16) and hepatocellular carcinoma (n = 169). The correlations between PTP1B expression level and clinicopathologic features and patient survival were also analyzed. One hundred and eleven of 169 HCC patients (65.7%) had negative or low PTP1B expression in tumorous tissues, whereas normal tissues always expressed strong PTP1B. Decreased PTP1B expression was significantly associated with aggressive clinicopathologic features and poor prognosis. Immunohistochemistry also showed that low PTP1B expression level was correlated with high percentage of OV6{sup +} tumor-initiating cells (T-ICs) and high frequency of nuclear {beta}-Catenin expression in HCC specimens. Our findings demonstrate for the first time that the loss of inhibitory effect of PTP1B may contribute to progression and invasion of HCC through activation of Wnt/{beta}-Catenin signaling and expansion of liver T-ICs. PTP1B may serve as a valuable prognostic biomarker and potential therapeutic target in HCC.

  7. A Role for Protein Phosphatase 2A in Regulating p38 Mitogen Activated Protein Kinase Activation and Tumor Necrosis Factor-Alpha Expression during Influenza Virus Infection

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    Anna H. Y. Law

    2013-04-01

    Full Text Available Influenza viruses of avian origin continue to pose pandemic threats to human health. Some of the H5N1 and H9N2 virus subtypes induce markedly elevated cytokine levels when compared with the seasonal H1N1 virus. We previously showed that H5N1/97 hyperinduces tumor necrosis factor (TNF-alpha through p38 mitogen activated protein kinase (MAPK. However, the detailed mechanisms of p38MAPK activation and TNF-alpha hyperinduction following influenza virus infections are not known. Negative feedback regulations of cytokine expression play important roles in avoiding overwhelming production of proinflammatory cytokines. Here we hypothesize that protein phosphatases are involved in the regulation of cytokine expressions during influenza virus infection. We investigated the roles of protein phosphatases including MAPK phosphatase-1 (MKP-1 and protein phosphatase type 2A (PP2A in modulating p38MAPK activation and downstream TNF-alpha expressions in primary human monocyte-derived macrophages (PBMac infected with H9N2/G1 or H1N1 influenza virus. We demonstrate that H9N2/G1 virus activated p38MAPK and hyperinduced TNF-alpha production in PBMac when compared with H1N1 virus. H9N2/G1 induced PP2A activity in PBMac and, with the treatment of a PP2A inhibitor, p38MAPK phosphorylation and TNF-alpha production were further increased in the virus-infected macrophages. However, H9N2/G1 did not induce the expression of PP2A indicating that the activation of PP2A is not mediated by p38MAPK in virus-infected PBMac. On the other hand, PP2A may not be the targets of H9N2/G1 in the upstream of p38MAPK signaling pathways since H1N1 also induced PP2A activation in primary macrophages. Our results may provide new insights into the control of cytokine dysregulation.

  8. Endoplasmic reticulum stress-induced apoptosis accompanies enhanced expression of multiple inositol polyphosphate phosphatase 1 (Minpp1): a possible role for Minpp1 in cellular stress response.

    Science.gov (United States)

    Kilaparty, Surya P; Agarwal, Rakhee; Singh, Pooja; Kannan, Krishnaswamy; Ali, Nawab

    2016-07-01

    Inositol polyphosphates represent a group of differentially phosphorylated inositol metabolites, many of which are implicated to regulate diverse cellular processes such as calcium mobilization, vesicular trafficking, differentiation, apoptosis, etc. The metabolic network of these compounds is complex and tightly regulated by various kinases and phosphatases present predominantly in the cytosol. Multiple inositol polyphosphate phosphatase 1 (Minpp1) is the only known endoplasmic reticulum (ER) luminal enzyme that hydrolyzes various inositol polyphosphates in vitro as well as in vivo conditions. However, access of the Minpp1 to cytosolic substrates has not yet been demonstrated clearly and hence its physiological function. In this study, we examined a potential role for Minpp1 in ER stress-induced apoptosis. We generated a custom antibody and characterized its specificity to study the expression of Minpp1 protein in multiple mammalian cells under experimentally induced cellular stress conditions. Our results demonstrate a significant increase in the expression of Minpp1 in response to a variety of cellular stress conditions. The protein expression was corroborated with the expression of its mRNA and enzymatic activity. Further, in an attempt to link the role of Minpp1 to apoptotic stress, we studied the effect of Minpp1 expression on apoptosis following silencing of the Minpp1 gene by its specific siRNA. Our results suggest an attenuation of apoptotic parameters following knockdown of Minpp1. Thus, in addition to its known role in inositol polyphosphate metabolism, we have identified a novel role for Minpp1 as a stress-responsive protein. In summary, our results provide, for the first time, a probable link between ER stress-induced apoptosis and Minpp1 expression.

  9. A Novel Phytase with Sequence Similarity to Purple Acid Phosphatases Is Expressed in Cotyledons of Germinating Soybean Seedlings 1

    Science.gov (United States)

    Hegeman, Carla E.; Grabau, Elizabeth A.

    2001-01-01

    Phytic acid (myo-inositol hexakisphosphate) is the major storage form of phosphorus in plant seeds. During germination, stored reserves are used as a source of nutrients by the plant seedling. Phytic acid is degraded by the activity of phytases to yield inositol and free phosphate. Due to the lack of phytases in the non-ruminant digestive tract, monogastric animals cannot utilize dietary phytic acid and it is excreted into manure. High phytic acid content in manure results in elevated phosphorus levels in soil and water and accompanying environmental concerns. The use of phytases to degrade seed phytic acid has potential for reducing the negative environmental impact of livestock production. A phytase was purified to electrophoretic homogeneity from cotyledons of germinated soybeans (Glycine max L. Merr.). Peptide sequence data generated from the purified enzyme facilitated the cloning of the phytase sequence (GmPhy) employing a polymerase chain reaction strategy. The introduction of GmPhy into soybean tissue culture resulted in increased phytase activity in transformed cells, which confirmed the identity of the phytase gene. It is surprising that the soybean phytase was unrelated to previously characterized microbial or maize (Zea mays) phytases, which were classified as histidine acid phosphatases. The soybean phytase sequence exhibited a high degree of similarity to purple acid phosphatases, a class of metallophosphoesterases. PMID:11500558

  10. Over-expression of the mycobacterial trehalose-phosphate phosphatase OtsB2 results in a defect in macrophage phagocytosis associated with increased mycobacterial-macrophage adhesion

    Directory of Open Access Journals (Sweden)

    Hao Li

    2016-11-01

    Full Text Available Trehalose-6-phosphate phosphatase (OtsB2 is involved in the OtsAB trehalose synthesis pathway to produce free trehalose and is strictly essential for mycobacterial growth. We wished to determine the effects of OtsB2 expression on mycobacterial phenotypes such as growth, phagocytosis and survival in macrophages. Mycobacterium bovis-BCG (BCG over-expressing OtsB2 were able to better survive in stationary phase. Over-expression of OtsB2 led to a decrease in phagocytosis but not survival in THP-1 macrophage-like cells, and this was not due to a decrease in general macrophage phagocytic activity. Surprisingly, when we investigated macrophage-mycobacterial interactions by flow cytometry and atomic force microscopy, we discovered that BCG over-expressing OtsB2 have stronger binding to THP-1 cells than wild-type BCG. These results suggest that altering OtsB2 expression has implications for mycobacterial host-pathogen interactions. Macrophage-mycobacteria phagocytic interactions are complex and merit further study.

  11. Disruption of a Guard Cell–Expressed Protein Phosphatase 2A Regulatory Subunit, RCN1, Confers Abscisic Acid Insensitivity in Arabidopsis

    Science.gov (United States)

    Kwak, June M.; Moon, Ji-Hye; Murata, Yoshiyuki; Kuchitsu, Kazuyuki; Leonhardt, Nathalie; DeLong, Alison; Schroeder, Julian I.

    2002-01-01

    Pharmacological studies have led to a model in which the phytohormone abscisic acid (ABA) may be positively transduced via protein phosphatases of the type 1 (PP1) or type 2A (PP2A) families. However, pharmacological evidence also exists that PP1s or PP2As may function as negative regulators of ABA signaling. Furthermore, recessive disruption mutants in protein phosphatases that function in ABA signal transduction have not yet been identified. A guard cell–expressed PP2A gene, RCN1, which had been characterized previously as a molecular component affecting auxin transport and gravity response, was isolated. A T-DNA disruption mutation in RCN1 confers recessive ABA insensitivity to Arabidopsis. The rcn1 mutation impairs ABA-induced stomatal closing and ABA activation of slow anion channels. Calcium imaging analyses show a reduced sensitivity of ABA-induced cytosolic calcium increases in rcn1, whereas mechanisms downstream of cytosolic calcium increases show wild-type responses, suggesting that RCN1 functions in ABA signal transduction upstream of cytosolic Ca2+ increases. Furthermore, rcn1 shows ABA insensitivity in ABA inhibition of seed germination and ABA-induced gene expression. The PP1 and PP2A inhibitor okadaic acid phenocopies the rcn1 phenotype in wild-type plants both in ABA-induced cytosolic calcium increases and in seed germination, and the wild-type RCN1 genomic DNA complements rcn1 phenotypes. These data show that RCN1 functions as a general positive transducer of early ABA signaling. PMID:12417706

  12. Effects of Src Kinase Inhibition on Expression of Protein Tyrosine Phosphatase 1B after Brain Hypoxia in a Piglet Animal Model

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    Dimitrios Angelis

    2017-01-01

    Full Text Available Background. Protein tyrosine phosphatases (PTPs in conjunction with protein tyrosine kinases (PTKs regulate cellular processes by posttranslational modifications of signal transduction proteins. PTP nonreceptor type 1B (PTP-1B is an enzyme of the PTP family. We have previously shown that hypoxia induces an increase in activation of a class of nonreceptor PTK, the Src kinases. In the present study, we investigated the changes that occur in the expression of PTP-1B in the cytosolic component of the brain of newborn piglets acutely after hypoxia as well as long term for up to 2 weeks. Methods. Newborn piglets were divided into groups: normoxia, hypoxia, hypoxia followed by 1 day and 15 days in FiO2 0.21, and hypoxia pretreated with Src kinase inhibitor PP2, prior to hypoxia followed by 1 day and 15 days. Hypoxia was achieved by providing 7% FiO2 for 1 hour and PTP-1B expression was measured via immunoblotting. Results. PTP-1B increased posthypoxia by about 30% and persisted for 2 weeks while Src kinase inhibition attenuated the expected PTP-1B-increased expression. Conclusions. Our study suggests that Src kinase mediates a hypoxia-induced increased PTP-1B expression.

  13. Hematopoietic cell phosphatase is recruited to CD22 following B cell antigen receptor ligation

    NARCIS (Netherlands)

    Lankester, A. C.; van Schijndel, G. M.; van Lier, R. A.

    1995-01-01

    Hematopoietic cell phosphatase is a nonreceptor protein tyrosine phosphatase that is preferentially expressed in hematopoietic cell lineages. Motheaten mice, which are devoid of (functional) hematopoietic cell phosphatase, have severe disturbances in the regulation of B cell activation and

  14. Distinct expression of alkaline phosphatase activity in epilimnetic bacteria: Implication for persistent DOC consumption in a P-limited reservoir

    Science.gov (United States)

    Tseng, Y.; Kao, S.; Shiah, F.

    2013-12-01

    In a P-deficient system, P availability usually controls the microbial activity and thus the ecosystem function. Thingstad et al. (1997) first addressed a 'Malfunctioning Microbial-loop' theory, which stated that low bacterial production (BP) caused by insufficient nutrient supply would result in DOC accumulation in an oligotrophic ecosystem. In this study we re-examined the theory by conducting seasonal patterns and correlations among soluble reactive phosphate (SRP) and DOC, microbial abundances (picocyanobacteria, bacteria, and heterotrophic nanoflagellate; HNF) and activities (primary production, bacterial production, and alkaline phosphatase activity; APA) coupled with enzyme-labeled fluorescence (ELF) assays on bacterioplankton in a subtropical reservoir sharing the common features, nitrate-replete and P-deficient, with most natural freshwater system during Oct 2007-Oct 2008. Persistently high APA was recorded during most of time, implying that the system was P-deficient. Size fractionated APA and ELF assay revealed that bacteria were the major APA contributor. However, significantly low epilimnion DOC was recorded during the stratified summer season accompanying with high BP and APA as well as high PP, implying that heterotrophic bacteria can well sustain in P-deficient system by utilizing DOP to rapidly lower down DOC under relatively high PP. Such findings oppose the 'Malfunctioning Microbial-loop' theory. On the other hand, strong epilimnetic DOC accumulation occurred in Oct 2007 under low light and low PP condition accompanying with high abundance of HNF, implying that HNF grazing may contribute to a certain degree of DOC accumulation. Correlation matrix supported our suggestions. This study testified the DOC dynamics in P-deficient ecosystem are tightly coupled with the source (PP and grazing) and sink (BP). We also suggested that in SRP-limited freshwater systems bacteria are capable of breaking down autochthonous DOC to reduce the chance of DOC

  15. Microarray Expression Analyses of Arabidopsis Guard Cells and Isolation of a Recessive Abscisic Acid Hypersensitive Protein Phosphatase 2C MutantW⃞

    Science.gov (United States)

    Leonhardt, Nathalie; Kwak, June M.; Robert, Nadia; Waner, David; Leonhardt, Guillaume; Schroeder, Julian I.

    2004-01-01

    Oligomer-based DNA Affymetrix GeneChips representing about one-third of Arabidopsis (Arabidopsis thaliana) genes were used to profile global gene expression in a single cell type, guard cells, identifying 1309 guard cell–expressed genes. Highly pure preparations of guard cells and mesophyll cells were isolated in the presence of transcription inhibitors that prevented induction of stress-inducible genes during cell isolation procedures. Guard cell expression profiles were compared with those of mesophyll cells, resulting in identification of 64 transcripts expressed preferentially in guard cells. Many large gene families and gene duplications are known to exist in the Arabidopsis genome, giving rise to redundancies that greatly hamper conventional genetic and functional genomic analyses. The presented genomic scale analysis identifies redundant expression of specific isoforms belonging to large gene families at the single cell level, which provides a powerful tool for functional genomic characterization of the many signaling pathways that function in guard cells. Reverse transcription–PCR of 29 genes confirmed the reliability of GeneChip results. Statistical analyses of promoter regions of abscisic acid (ABA)–regulated genes reveal an overrepresented ABA responsive motif, which is the known ABA response element. Interestingly, expression profiling reveals ABA modulation of many known guard cell ABA signaling components at the transcript level. We further identified a highly ABA-induced protein phosphatase 2C transcript, AtP2C-HA, in guard cells. A T-DNA disruption mutation in AtP2C-HA confers ABA-hypersensitive regulation of stomatal closing and seed germination. The presented data provide a basis for cell type–specific genomic scale analyses of gene function. PMID:14973164

  16. Expression of a truncated receptor protein tyrosine phosphatase kappa in the brain of an adult transgenic mouse

    DEFF Research Database (Denmark)

    Shen, P; Canoll, P D; Sap, J

    1999-01-01

    that goal, we have used this mouse model to map the distribution of the truncated RPTP-kappa/beta-geo fusion protein in the adult mouse brain using beta-galactosidase as a marker enzyme. Visualization of the beta-galactosidase activity revealed a non-random pattern of expression, and identified cells......-6596]. Nevertheless, since the transgene's expression is driven by the endogenous RPTP-kappa promoter, distribution of the truncated RPTP-kappa/beta-geo fusion protein should reflect the regional and cellular expression of wild-type RPTP-kappa, and thus may identify sites where RPTP-kappa is important. Towards...

  17. Expression of a truncated receptor protein tyrosine phosphatase kappa in the brain of an adult transgenic mouse

    DEFF Research Database (Denmark)

    Shen, P; Canoll, P D; Sap, J

    1999-01-01

    processes such as axonal growth and target recognition, as has been demonstrated for certain Drosophila RPTPs. The brain distribution of RPTP-kappa-expressing cells has not been determined, however. In a gene-trap mouse model with a beta-gal+neo (beta-geo) insertion in the endogenous RPTP-kappa gene......-6596]. Nevertheless, since the transgene's expression is driven by the endogenous RPTP-kappa promoter, distribution of the truncated RPTP-kappa/beta-geo fusion protein should reflect the regional and cellular expression of wild-type RPTP-kappa, and thus may identify sites where RPTP-kappa is important. Towards...... that goal, we have used this mouse model to map the distribution of the truncated RPTP-kappa/beta-geo fusion protein in the adult mouse brain using beta-galactosidase as a marker enzyme. Visualization of the beta-galactosidase activity revealed a non-random pattern of expression, and identified cells...

  18. Protein phosphatase magnesium-dependent 1δ (PPM1D mRNA expression is a prognosis marker for hepatocellular carcinoma.

    Directory of Open Access Journals (Sweden)

    Guang-Bing Li

    Full Text Available Protein phosphatase magnesium-dependent 1δ (PPM1D is an oncogene, overexpressed in many solid tumors, including ovarian cancer and breast cancer. The current study examined the expression and the prognostic value of PPM1D mRNA in human hepatocellular carcinoma (HCC.Total RNA was extracted from 86 HCC and paired non-cancerous liver tissues. PPM1D mRNA expression was determined by real-time quantitative reverse transcriptase-polymerase chain reaction (qPCR. Immunohistochemistry assay was used to verify the expression of ppm1d protein in the HCC and non-cancerous liver tissues. HCC patients were grouped according to PPM1D mRNA expression with the average PPM1D mRNA level in non-cancerous liver tissue samples as the cut-off. Correlations between clinicopathologic variables, overall survival and PPM1D mRNA expression were analyzed.PPM1D mRNA was significantly higher in HCC than in the paired non-cancerous tissue (p<0.01. This was confirmed by ppm1d staining. 56 patients were classified as high expression group and the other 30 patients were categorized as low expression group. There were significant differences between the two groups in term of alpha-fetoprotein (α-FP level (p<0.01, tumor size (p<0.01, TNM stage (p<0.01, recurrence incidence (p<0.01 and family history of liver cancer (p<0.01. The current study failed to find significant differences between the two groups in the following clinical characteristics: age, gender, portal vein invasion, lymphnode metastasis, hepatitis B virus (HBV infection and alcohol intake. Survival time of high expression group was significantly shorter than that of low expression group (median survival, 13 months and 32 months, respectively, p<0.01.Up-regulation of PPM1D mRNA was associated with progressive pathological feature and poor prognosis in HCC patients. PPM1D mRNA may serve as a prognostic marker in HCC.

  19. Expression of the MAP kinase phosphatase DUSP4 is associated with microsatellite instability in colorectal cancer (CRC) and causes increased cell proliferation.

    Science.gov (United States)

    Gröschl, Benedikt; Bettstetter, Marcus; Giedl, Christian; Woenckhaus, Matthias; Edmonston, Tina; Hofstädter, Ferdinand; Dietmaier, Wolfgang

    2013-04-01

    DUSP4 (MKP-2), a member of the mitogen-activated protein kinase phosphatase (MKP) family and potential tumor suppressor, negatively regulates the MAPKs (mitogen-activated protein kinases) ERK, p38 and JNK. MAPKs play a crucial role in cancer development and progression. Previously, using microarray analyses we found a conspicuously frequent overexpression of DUSP4 in colorectal cancer (CRC) with high frequent microsatellite instability (MSI-H) compared to microsatellite stable (MSS) CRC. Here we studied DUSP4 expression on mRNA level in 38 CRC (19 MSI-H and 19 MSS) compared to matched normal tissue as well as in CRC cell lines by RT-qPCR. DUSP4 was overexpressed in all 19 MSI-H tumors and in 14 MSS tumors. Median expression levels in MSI-H tumors were significantly higher than in MSS-tumors (p CRC cell lines showed 6.8-fold higher DUSP4 mRNA levels than MSS cell lines. DUSP4 expression was not regulated by promoter methylation since no methylation was found by quantitative methylation analysis of DUSP4 promoter in CRC cell lines neither in tumor samples. Furthermore, no DUSP4 mutation was found on genomic DNA level in four CRC cell lines. DUSP4 overexpression in CRC cell lines through DUSP4 transfection caused upregulated expression of MAPK targets CDC25A, CCND1, EGR1, FOS, MYC and CDKN1A in HCT116 as well as downregulation of mismatch repair gene MSH2 in SW480. Furthermore, DUSP4 overexpression led to increased proliferation in CRC cell lines. Our findings suggest that DUSP4 acts as an important regulator of cell growth within the MAPK pathway and causes enhanced cell growth in MSI-H CRC. Copyright © 2012 UICC.

  20. Ethylene signalling is involved in regulation of phosphate starvation-induced gene expression and production of acid phosphatases and anthocyanin in Arabidopsis

    KAUST Repository

    Lei, Mingguang

    2010-11-30

    With the exception of root hair development, the role of the phytohormone ethylene is not clear in other aspects of plant responses to inorganic phosphate (Pi) starvation. The induction of AtPT2 was used as a marker to find novel signalling components involved in plant responses to Pi starvation. Using genetic and chemical approaches, we examined the role of ethylene in the regulation of plant responses to Pi starvation. hps2, an Arabidopsis mutant with enhanced sensitivity to Pi starvation, was identified and found to be a new allele of CTR1 that is a key negative regulator of ethylene responses. 1-aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, increases plant sensitivity to Pi starvation, whereas the ethylene perception inhibitor Ag+ suppresses this response. The Pi starvation-induced gene expression and acid phosphatase activity are also enhanced in the hps2 mutant, but suppressed in the ethylene-insensitive mutant ein2-5. By contrast, we found that ethylene signalling plays a negative role in Pi starvation-induced anthocyanin production. These findings extend the roles of ethylene in the regulation of plant responses to Pi starvation and will help us to gain a better understanding of the molecular mechanism underlying these responses. © 2010 The Authors. New Phytologist © 2010 New Phytologist Trust.

  1. Expression of Mycobacterium tuberculosis Protein Tyrosine Phosphatase B in Escherichia coli and Its Recovery from Inclusion Body

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    Lalu Rudyat Telly Savalas

    2017-12-01

    Full Text Available The present study aims at expressing and partially purifying PtpB in active form. To achieve this, Mtb PtpB gene has been cloned into pET30a vector and overexpressed in Escherichia coli BL 21(DE3 under IPTG induction in the form of an inclusion body. Following resolubilization by urea and dialysis, the resulted PtpB has been shown to be active against para-Nitrophenyl phosphate.  It is concluded that the resulted PtpB has had been recovered from inclusion body to give the active form of the enzyme, and thus the success in overexpressing PtpB provides the required material to investigate the biochemical properties of the pathogen virulence factor further. 

  2. Deletion of Protein Tyrosine Phosphatase 1B (PTP1B Enhances Endothelial Cyclooxygenase 2 Expression and Protects Mice from Type 1 Diabetes-Induced Endothelial Dysfunction.

    Directory of Open Access Journals (Sweden)

    David J Herren

    Full Text Available Protein tyrosine phosphatase 1B (PTP1B dephosphorylates receptors tyrosine kinase and acts as a molecular brake on insulin signaling pathway. Conditions of metabolic dysfunction increase PTP1B, when deletion of PTP1B protects against metabolic disorders by increasing insulin signaling. Although vascular insulin signaling contributes to the control of glucose disposal, little is known regarding the direct role of PTP1B in the control of endothelial function. We hypothesized that metabolic dysfunctions increase PTP1B expression in endothelial cells and that PTP1B deletion prevents endothelial dysfunction in situation of diminished insulin secretion. Type I diabetes (T1DM was induced in wild-type (WT and PTP1B-deficient mice (KO with streptozotocin (STZ injection. After 28 days of T1DM, KO mice exhibited a similar reduction in body weight and plasma insulin levels and a comparable increase in glycemia (WT: 384 ± 20 vs. Ko: 432 ± 29 mg/dL, cholesterol and triglycerides, as WT mice. T1DM increased PTP1B expression and impaired endothelial NO-dependent relaxation, in mouse aorta. PTP1B deletion did not affect baseline endothelial function, but preserved endothelium-dependent relaxation, in T1DM mice. NO synthase inhibition with L-NAME abolished endothelial relaxation in control and T1DM WT mice, whereas L-NAME and the cyclooxygenases inhibitor indomethacin were required to abolish endothelium relaxation in T1DM KO mice. PTP1B deletion increased COX-2 expression and PGI2 levels, in mouse aorta and plasma respectively, in T1DM mice. In parallel, simulation of diabetic conditions increased PTP1B expression and knockdown of PTP1B increased COX-2 but not COX-1 expression, in primary human aortic endothelial cells. Taken together these data indicate that deletion of PTP1B protected endothelial function by compensating the reduction in NO bioavailability by increasing COX-2-mediated release of the vasodilator prostanoid PGI2, in T1DM mice.

  3. Overexpression of tissue-nonspecific alkaline phosphatase increases the expression of neurogenic differentiation markers in the human SH-SY5Y neuroblastoma cell line.

    Science.gov (United States)

    Graser, Stephanie; Mentrup, Birgit; Schneider, Doris; Klein-Hitpass, Ludger; Jakob, Franz; Hofmann, Christine

    2015-10-01

    Patients suffering from the rare hereditary disease hypophosphatasia (HPP), which is based on mutations in the ALPL gene, tend to develop central nervous system (CNS) related issues like epileptic seizures and neuropsychiatric illnesses such as anxiety and depression, in addition to well-known problems with the mineralization of bones and teeth. Analyses of the molecular role of tissue-nonspecific alkaline phosphatase (TNAP) in transgenic SH-SY5Y(TNAPhigh) neuroblastoma cells compared to SH-SY5Y(TNAPlow) cells indicate that the enzyme influences the expression levels of neuronal marker genes like RNA-binding protein, fox-1 homolog 3 (NEUN) and enolase 2, gamma neuronal (NSE) as well as microtubule-binding proteins like microtubule-associated protein 2 (MAP2) and microtubule-associated protein tau (TAU) during neurogenic differentiation. Fluorescence staining of SH-SY5Y(TNAPhigh) cells reveals TNAP localization throughout the whole length of the developed projection network and even synapsin Ι co-localization with strong TNAP signals at some spots at least at the early time points of differentiation. Additional immunocytochemical staining shows higher MAP2 expression in SH-SY5Y(TNAPhigh) cells and further a distinct up-regulation of tau and MAP2 in the course of neurogenic differentiation. Interestingly, transgenic SH-SY5Y(TNAPhigh) cells are able to develop longer cellular processes compared to control cells after stimulation with all-trans retinoic acid (RA). Current therapies for HPP prioritize improvement of the bone phenotype. Unraveling the molecular role of TNAP in extraosseous tissues, like in the CNS, will help to improve treatment strategies for HPP patients. Taking this rare disease as a model may also help to dissect TNAP's role in neurodegenerative diseases and even improve future treatment of common pathologies. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Kinetic comparison of tissue non-specific and placental human alkaline phosphatases expressed in baculovirus infected cells: application to screening for Down's syndrome

    OpenAIRE

    Grozdea Jean J; Fournier Didier D; Biasini Ghislaine G; Brisson-Lougarre Andrée A; Denier Colette C

    2002-01-01

    Abstract Background In humans, there are four alkaline phosphatases, and each form exibits a characteristic pattern of tissue distribution. The availability of an easy method to reveal their activity has resulted in large amount of data reporting correlations between variations in activity and illnesses. For example, alkaline phosphatase from neutrophils of mothers pregnent with a trisomy 21 fetus (Down's syndrome) displays significant differences both in its biochemical and immunological pro...

  5. Cationized dextran nanoparticle-encapsulated CXCR4-siRNA enhanced correlation between CXCR4 expression and serum alkaline phosphatase in a mouse model of colorectal cancer

    Directory of Open Access Journals (Sweden)

    Abedini F

    2012-07-01

    Full Text Available Fatemeh Abedini,1 Hossein Hosseinkhani,2 Maznah Ismail,1,3 Abraham J Domb,4 Abdul Rahman Omar,1,5 Pei Pei Chong,1,2 Po-Da Hong,3 Dah-Shyong Yu,6 Ira-Yudovin Farber41Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia, Selangor, 2Graduate Institute of Biomedical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan, 3Faculty of Medicine & Health Sciences, Universiti Putra Malaysia, Selangor, Malaysia, 4Institute of Drug Research, The Center for Nanoscience and Nanotechnology, School of Pharmacy-Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel, 5Faculty of Veterinary Medicine, Universiti Putra Malaysia, Selangor, Malaysia, 6Nanomedicine Research Center, National Defense Medical Center, Taipei, TaiwanPurpose: The failure of colorectal cancer treatments is partly due to overexpression of CXCR4 by tumor cells, which plays a critical role in cell metastasis. Moreover, serum alkaline phosphatase (ALP levels are frequently elevated in patients with metastatic colorectal cancer. A polysaccharide, dextran, was chosen as the vector of siRNA. Spermine was conjugated to oxidized dextran by reductive amination process to obtain cationized dextran, so-called dextran-spermine, in order to prepare CXCR4-siRNAs/dextran-spermine nanoparticles. The fabricated nanoparticles were used in order to investigate whether downregulation of CXCR4 expression could affect serum ALP in mouse models of colorectal cancer.Methods: Colorectal cancer was established in BALB/C mice following injection of mouse colon carcinoma cells CT.26WT through the tail vein. CXCR4 siRNA for two sites of the target gene was administered following injection of naked siRNA or siRNA encapsulated into nanoparticles.Results: In vivo animal data revealed that CXCR4 silencing by dextran-spermine nanoparticles significantly downregulated CXCR4 expression compared with naked CXCR4 siRNA. Furthermore, there was

  6. Bacillus subtilis NhaC, an Na+/H+ antiporter, influences expression of the phoPR operon and production of alkaline phosphatases

    NARCIS (Netherlands)

    Pragai, Z; Eschevins, C; Bron, S; Harwood, CR

    When Bacillus subtilis is subjected to phosphate starvation, genes of the Pho regulon are either induced or repressed. Among those induced are genes encoding alkaline phosphatases (APases). A set of isogenic mutants, with a beta -galactosidase gene transcriptionally fused to the inactivated target

  7. Glucose-6-phosphatase deficiency

    Directory of Open Access Journals (Sweden)

    Labrune Philippe

    2011-05-01

    Full Text Available Abstract Glucose-6-phosphatase deficiency (G6P deficiency, or glycogen storage disease type I (GSDI, is a group of inherited metabolic diseases, including types Ia and Ib, characterized by poor tolerance to fasting, growth retardation and hepatomegaly resulting from accumulation of glycogen and fat in the liver. Prevalence is unknown and annual incidence is around 1/100,000 births. GSDIa is the more frequent type, representing about 80% of GSDI patients. The disease commonly manifests, between the ages of 3 to 4 months by symptoms of hypoglycemia (tremors, seizures, cyanosis, apnea. Patients have poor tolerance to fasting, marked hepatomegaly, growth retardation (small stature and delayed puberty, generally improved by an appropriate diet, osteopenia and sometimes osteoporosis, full-cheeked round face, enlarged kydneys and platelet dysfunctions leading to frequent epistaxis. In addition, in GSDIb, neutropenia and neutrophil dysfunction are responsible for tendency towards infections, relapsing aphtous gingivostomatitis, and inflammatory bowel disease. Late complications are hepatic (adenomas with rare but possible transformation into hepatocarcinoma and renal (glomerular hyperfiltration leading to proteinuria and sometimes to renal insufficiency. GSDI is caused by a dysfunction in the G6P system, a key step in the regulation of glycemia. The deficit concerns the catalytic subunit G6P-alpha (type Ia which is restricted to expression in the liver, kidney and intestine, or the ubiquitously expressed G6P transporter (type Ib. Mutations in the genes G6PC (17q21 and SLC37A4 (11q23 respectively cause GSDIa and Ib. Many mutations have been identified in both genes,. Transmission is autosomal recessive. Diagnosis is based on clinical presentation, on abnormal basal values and absence of hyperglycemic response to glucagon. It can be confirmed by demonstrating a deficient activity of a G6P system component in a liver biopsy. To date, the diagnosis is most

  8. Field-Evolved Mode 1 Resistance of the Fall Armyworm to Transgenic Cry1Fa-Expressing Corn Associated with Reduced Cry1Fa Toxin Binding and Midgut Alkaline Phosphatase Expression

    Science.gov (United States)

    Jakka, Siva R. K.; Gong, Liang; Hasler, James; Banerjee, Rahul; Sheets, Joel J.; Narva, Kenneth; Blanco, Carlos A.

    2015-01-01

    Insecticidal protein genes from the bacterium Bacillus thuringiensis (Bt) are expressed by transgenic Bt crops (Bt crops) for effective and environmentally safe pest control. The development of resistance to these insecticidal proteins is considered the most serious threat to the sustainability of Bt crops. Resistance in fall armyworm (Spodoptera frugiperda) populations from Puerto Rico to transgenic corn producing the Cry1Fa insecticidal protein resulted, for the first time in the United States, in practical resistance, and Bt corn was withdrawn from the local market. In this study, we used a field-collected Cry1Fa corn-resistant strain (456) of S. frugiperda to identify the mechanism responsible for field-evolved resistance. Binding assays detected reduced Cry1Fa, Cry1Ab, and Cry1Ac but not Cry1Ca toxin binding to midgut brush border membrane vesicles (BBMV) from the larvae of strain 456 compared to that from the larvae of a susceptible (Ben) strain. This binding phenotype is descriptive of the mode 1 type of resistance to Bt toxins. A comparison of the transcript levels for putative Cry1 toxin receptor genes identified a significant downregulation (>90%) of a membrane-bound alkaline phosphatase (ALP), which translated to reduced ALP protein levels and a 75% reduction in ALP activity in BBMV from 456 compared to that of Ben larvae. We cloned and heterologously expressed this ALP from susceptible S. frugiperda larvae and demonstrated that it specifically binds with Cry1Fa toxin. This study provides a thorough mechanistic description of field-evolved resistance to a transgenic Bt crop and supports an association between resistance and reduced Cry1Fa toxin binding and levels of a putative Cry1Fa toxin receptor, ALP, in the midguts of S. frugiperda larvae. PMID:26637593

  9. Effect of growth conditions on expression of the acid phosphatase (cyx-appA) operon and the appY gene, which encodes a transcriptional activator of Escherichia coli

    DEFF Research Database (Denmark)

    Brøndsted, Lone; Atlung, Tove

    1996-01-01

    The expression and transcriptional regulation of the Escherichia coli cyx-appA operon and the appY gene has been investigated during different environmental conditions using single copy transcriptional lacZ fusions. The cyx-appA operon encodes acid phosphatase and a putative cytochrome oxidase...... of the cyx-appA operon. The nitrate repression was partially dependent on NarL. A high expression of the operon was obtained in glucose medium supplemented with formate, where E.coli obtains energy by fermentation. The formate induction was independent of the fhlA gene product. The results presented...... in this paper indicate a clear difference in the regulation of the cyx-appA operon compared to the cyd operon, encoding the cytochrome d oxidase complex. The results suggest that cytochrome x oxidase has a function at even more oxygen limiting conditions than cytochrome d oxidase. The expression of the app...

  10. Characterisation and expression of a PP1 serine/threonine protein phosphatase (PfPP1 from the malaria parasite, Plasmodium falciparum: demonstration of its essential role using RNA interference

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    Musiyenko Alla

    2002-04-01

    Full Text Available Abstract Background Reversible protein phosphorylation is relatively unexplored in the intracellular protozoa of the Apicomplexa family that includes the genus Plasmodium, to which belong the causative agents of malaria. Members of the PP1 family represent the most highly conserved protein phosphatase sequences in phylogeny and play essential regulatory roles in various cellular pathways. Previous evidence suggested a PP1-like activity in Plasmodium falciparum, not yet identified at the molecular level. Results We have identified a PP1 catalytic subunit from P. falciparum and named it PfPP1. The predicted primary structure of the 304-amino acid long protein was highly similar to PP1 sequences of other species, and showed conservation of all the signature motifs. The purified recombinant protein exhibited potent phosphatase activity in vitro. Its sensitivity to specific phosphatase inhibitors was characteristic of the PP1 class. The authenticity of the PfPP1 cDNA was further confirmed by mutational analysis of strategic amino acid residues important in catalysis. The protein was expressed in all erythrocytic stages of the parasite. Abrogation of PP1 expression by synthetic short interfering RNA (siRNA led to inhibition of parasite DNA synthesis. Conclusions The high sequence similarity of PfPP1 with other PP1 members suggests conservation of function. Phenotypic gene knockdown studies using siRNA confirmed its essential role in the parasite. Detailed studies of PfPP1 and its regulation may unravel the role of reversible protein phosphorylation in the signalling pathways of the parasite, including glucose metabolism and parasitic cell division. The use of siRNA could be an important tool in the functional analysis of Apicomplexan genes.

  11. Cdc14 phosphatase

    DEFF Research Database (Denmark)

    Machín, Félix; Quevedo Rodriguez, Oliver; Ramos-Pérez, Cristina

    2016-01-01

    and cancer cells uncontrollably divide, much attention has been put into knocking down CDK activity. However, much less is known on the consequences of interfering with the phosphatases that put an end to the cell cycle. We have addressed in recent years the consequences of transiently inactivating the only...

  12. Analysis of the expression level and methylation of tumor protein p53, phosphatase and tensin homolog and mutS homolog 2 in N-methyl-N-nitrosourea-induced thymic lymphoma in C57BL/6 mice.

    Science.gov (United States)

    Huo, Xueyun; Li, Zhenkun; Zhang, Shuangyue; Li, Changlong; Guo, Meng; Lu, Jing; Lv, Jianyi; Du, Xiaoyan; Chen, Zhenwen

    2017-10-01

    Tumorigenesis is often caused by somatic mutation or epigenetic changes in genes that regulate aspects of cell death, proliferation and survival. Although the functions of multiple tumor suppressor genes have been well studied in isolation, how these genes cooperate during the progression of a single tumor remains unclear in numerous cases. The present study used N-methyl-N-nitrosourea (MNU), one of the most potent mutagenic nitrosourea compounds, to induce thymic lymphoma in C57BL/6J mice. Subsequently, the protein expression levels of phosphatase and tensin homolog (PTEN), transformation protein 53 and mutS homolog 2 (MSH2) were evaluated concomitantly in the thymus, liver, kidney and spleen of MNU-treated mice by western blotting. To determine whether changes in expression level were due to aberrant epigenetic regulation, the present study further examined the methylation status of each gene by MassARRAY analysis. During the tumorigenesis process of an MNU-induced single thymic lymphoma, the expression level of PTEN was revealed to be reduced in thymic lymphoma samples but not in normal or non-tumor thymus tissue samples. Furthermore, a marked reduction of P53 expression levels were demonstrated in thymic lymphomas and spleens with a metastatic tumor. Conversely, MSH2 upregulation was identified only in liver, kidney, and spleen samples that were infiltrated by thymic lymphoma cells. Furthermore, the present study revealed that a number of 5'-C-phosphate-G-3' sites located in the promoter of aberrantly expressed genes had significantly altered methylation statuses. These results improve the understanding of the course of mutagen-induced cancer, and highlight that epigenetic regulation may serve an important function in cancer.

  13. Direct determination of phosphatase activity from physiological substrates in cells.

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    Zhongyuan Ren

    Full Text Available A direct and continuous approach to determine simultaneously protein and phosphate concentrations in cells and kinetics of phosphate release from physiological substrates by cells without any labeling has been developed. Among the enzymes having a phosphatase activity, tissue non-specific alkaline phosphatase (TNAP performs indispensable, multiple functions in humans. It is expressed in numerous tissues with high levels detected in bones, liver and neurons. It is absolutely required for bone mineralization and also necessary for neurotransmitter synthesis. We provided the proof of concept that infrared spectroscopy is a reliable assay to determine a phosphatase activity in the osteoblasts. For the first time, an overall specific phosphatase activity in cells was determined in a single step by measuring simultaneously protein and substrate concentrations. We found specific activities in osteoblast like cells amounting to 116 ± 13 nmol min(-1 mg(-1 for PPi, to 56 ± 11 nmol min(-1 mg(-1 for AMP, to 79 ± 23 nmol min(-1 mg(-1 for beta-glycerophosphate and to 73 ± 15 nmol min(-1 mg(-1 for 1-alpha-D glucose phosphate. The assay was also effective to monitor phosphatase activity in primary osteoblasts and in matrix vesicles. The use of levamisole--a TNAP inhibitor--served to demonstrate that a part of the phosphatase activity originated from this enzyme. An IC50 value of 1.16 ± 0.03 mM was obtained for the inhibition of phosphatase activity of levamisole in osteoblast like cells. The infrared assay could be extended to determine any type of phosphatase activity in other cells. It may serve as a metabolomic tool to monitor an overall phosphatase activity including acid phosphatases or other related enzymes.

  14. HD-PTP is a catalytically inactive tyrosine phosphatase due to a conserved divergence in its phosphatase domain.

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    Marie-Claude Gingras

    Full Text Available The HD-PTP protein has been described as a tumor suppressor candidate and based on its amino acid sequence, categorized as a classical non-transmembrane protein tyrosine phosphatase (PTP. To date, no HD-PTP phosphorylated substrate has been identified and controversial results concerning its catalytic activity have been recently reported.Here we report a rigorous enzymatic analysis demonstrating that the HD-PTP protein does not harbor tyrosine phosphatase or lipid phosphatase activity using the highly sensitive DiFMUP substrate and a panel of different phosphatidylinositol phosphates. We found that HD-PTP tyrosine phosphatase inactivity is caused by an evolutionary conserved amino acid divergence of a key residue located in the HD-PTP phosphatase domain since its back mutation is sufficient to restore the HD-PTP tyrosine phosphatase activity. Moreover, in agreement with a tumor suppressor activity, HD-PTP expression leads to colony growth reduction in human cancer cell lines, independently of its catalytic PTP activity status.In summary, we demonstrate that HD-PTP is a catalytically inactive protein tyrosine phosphatase. As such, we identify one residue involved in its inactivation and show that its colony growth reduction activity is independent of its PTP activity status in human cancer cell lines.

  15. The protein histidine phosphatase LHPP is a tumour suppressor.

    Science.gov (United States)

    Hindupur, Sravanth K; Colombi, Marco; Fuhs, Stephen R; Matter, Matthias S; Guri, Yakir; Adam, Kevin; Cornu, Marion; Piscuoglio, Salvatore; Ng, Charlotte K Y; Betz, Charles; Liko, Dritan; Quagliata, Luca; Moes, Suzette; Jenoe, Paul; Terracciano, Luigi M; Heim, Markus H; Hunter, Tony; Hall, Michael N

    2018-03-29

    Histidine phosphorylation, the so-called hidden phosphoproteome, is a poorly characterized post-translational modification of proteins. Here we describe a role of histidine phosphorylation in tumorigenesis. Proteomic analysis of 12 tumours from an mTOR-driven hepatocellular carcinoma mouse model revealed that NME1 and NME2, the only known mammalian histidine kinases, were upregulated. Conversely, expression of the putative histidine phosphatase LHPP was downregulated specifically in the tumours. We demonstrate that LHPP is indeed a protein histidine phosphatase. Consistent with these observations, global histidine phosphorylation was significantly upregulated in the liver tumours. Sustained, hepatic expression of LHPP in the hepatocellular carcinoma mouse model reduced tumour burden and prevented the loss of liver function. Finally, in patients with hepatocellular carcinoma, low expression of LHPP correlated with increased tumour severity and reduced overall survival. Thus, LHPP is a protein histidine phosphatase and tumour suppressor, suggesting that deregulated histidine phosphorylation is oncogenic.

  16. Probing protein phosphatase substrate binding

    DEFF Research Database (Denmark)

    Højlys-Larsen, Kim B.; Sørensen, Kasper Kildegaard; Jensen, Knud Jørgen

    2012-01-01

    Proteomics and high throughput analysis for systems biology can benefit significantly from solid-phase chemical tools for affinity pull-down of proteins from complex mixtures. Here we report the application of solid-phase synthesis of phosphopeptides for pull-down and analysis of the affinity...... profile of the integrin-linked kinase associated phosphatase (ILKAP), a member of the protein phosphatase 2C (PP2C) family. Phosphatases can potentially dephosphorylate these phosphopeptide substrates but, interestingly, performing the binding studies at 4 °C allowed efficient binding to phosphopeptides......, without the need for phosphopeptide mimics or phosphatase inhibitors. As no proven ILKAP substrates were available, we selected phosphopeptide substrates among known PP2Cδ substrates including the protein kinases: p38, ATM, Chk1, Chk2 and RSK2 and synthesized directly on PEGA solid supports through a BAL...

  17. Overexpression of Human Bone Alkaline Phosphatase in Pichia Pastoris

    Science.gov (United States)

    Karr, Laurel; Malone, Christine, C.; Rose, M. Franklin (Technical Monitor)

    2000-01-01

    The Pichiapastoris expression system was utilized to produce functionally active human bone alkaline phosphatase in gram quantities. Bone alkaline phosphatase is a key enzyme in bone formation and biomineralization, yet important questions about its structural chemistry and interactions with other cellular enzymes in mineralizing tissues remain unanswered. A soluble form of human bone alkaline phosphatase was constructed by deletion of the 25 amino acid hydrophobic C-terminal region of the encoding cDNA and inserted into the X-33 Pichiapastoris strain. An overexpression system was developed in shake flasks and converted to large-scale fermentation. Alkaline phosphatase was secreted into the medium to a level of 32mgAL when cultured in shake flasks. Enzyme activity was 12U/mg measured by a spectrophotometric assay. Fermentation yielded 880mgAL with enzymatic activity of 968U/mg. Gel electrophoresis analysis indicates that greater than 50% of the total protein in the fermentation is alkaline phosphatase. A purification scheme has been developed using ammonium sulfate precipitation followed by hydrophobic interaction chromatography. We are currently screening crystallization conditions of the purified recombinant protein for subsequent X-ray diffraction analyses. Structural data should provide additional information on the role of alkaline phosphatase in normal bone mineralization and in certain bone mineralization anomalies.

  18. Detergent insolubility of alkaline phosphatase during biosynthetic transport and endocytosis. Role of cholesterol

    NARCIS (Netherlands)

    Cerneus, D. P.; Ueffing, E.; Posthuma, G.; Strous, G. J.; van der Ende, A.

    1993-01-01

    Alkaline phosphatase is anchored to the outer leaflet of the plasma membrane by a covalently attached glycosyl-phosphatidylinositol anchor. We have studied the biosynthetic transport and endocytosis of alkaline phosphatase in the choriocarcinoma cell line BeWo, which endogenously expresses this

  19. Influence of triethyl phosphate on phosphatase activity in shooting range soil: Isolation of a zinc-resistant bacterium with an acid phosphatase.

    Science.gov (United States)

    Story, Sandra; Brigmon, Robin L

    2017-03-01

    Phosphatase-mediated hydrolysis of organic phosphate may be a viable means of stabilizing heavy metals via precipitation as a metal phosphate in bioremediation applications. We investigated the effect of triethyl phosphate (TEP) on soil microbial-phosphatase activity in a heavy-metal contaminated soil. Gaseous TEP has been used at subsurface sites for bioremediation of organic contaminants but not applied in heavy-metal contaminated areas. Little is known about how TEP affects microbial activity in soils and it is postulated that TEP can serve as a phosphate source in nutrient-poor groundwater and soil/sediments. Over a 3-week period, TEP amendment to microcosms containing heavy-metal contaminated soil resulted in increased activity of soil acid-phosphatase and repression of alkaline phosphatase, indicating a stimulatory effect on the microbial population. A soil-free enrichment of microorganisms adapted to heavy-metal and acidic conditions was derived from the TEP-amended soil microcosms using TEP as the sole phosphate source and the selected microbial consortium maintained a high acid-phosphatase activity with repression of alkaline phosphatase. Addition of 5mM zinc to soil-free microcosms had little effect on acid phosphatase but inhibited alkaline phosphatase. One bacterial member from the consortium, identified as Burkholderia cepacia sp., expressed an acid-phosphatase activity uninhibited by high concentrations of zinc and produced a soluble, indigo pigment under phosphate limitation. The pigment was produced in a phosphate-free medium and was not produced in the presence of TEP or phosphate ion, indicative of purple acid-phosphatase types that are pressed by bioavailable phosphate. These results demonstrate that TEP amendment was bioavailable and increased overall phosphatase activity in both soil and soil-free microcosms supporting the possibility of positive outcomes in bioremediation applications. Copyright © 2016. Published by Elsevier Inc.

  20. Immunocytochemical detection of the microsomal glucose-6-phosphatase in human brain astrocytes.

    Science.gov (United States)

    Bell, J E; Hume, R; Busuttil, A; Burchell, A

    1993-10-01

    Using an antibody raised against the catalytic subunit of glucose-6-phosphatase, this enzyme was immunolocalized in many astrocytes in 20 normal human brains. Double immunofluorescence studies showed co-localization of glial fibrillary acidic protein (GFAP) with glucose-6-phosphatase in astrocytes. However, not all GFAP-positive cells were also glucose-6-phosphatase positive, indicating that some astrocytes do not contain demonstrable expression of this enzyme. Reactive astrocytes in a variety of abnormal brains were strongly glucose-6-phosphatase positive, but neoplastic astrocytes were often only weakly positive. Expression of the enzyme could not be demonstrated in radial glia, neurons or oligodendroglia. Astrocytes normally contain glycogen and the demonstration that some astrocytes also contain glucose-6-phosphatase indicates that they are competent for both glycogenolysis and gluconeogenesis, which may be critical for neuronal welfare.

  1. Characterization of Human Bone Alkaline Phosphatase in Pichia Pastoris

    Science.gov (United States)

    Malone, Christine C.; Ciszak, Eva; Karr, Laurel J.

    1999-01-01

    A soluble form of human bone alkaline phosphatase has been expressed in a recombinant strain of the methylotrophic yeast Pichia pastoris. We constructed a plasmid containing cDNA encoding for human bone alkaline phosphatase, with the hydrophobic carboxyl terminal portion deleted. Alkaline phosphatase was secreted into the medium to a level of 32mg/L when cultured in shake flasks, and enzyme activity was 12U/mg, as measured by a spectrophotometric assay. By conversion to a fermentation system, a yield of 880mg/L has been achieved with an enzyme activity of 968U/mg. By gel electrophoresis analysis, it appears that greater than 50% of the total protein in the fermentation media is alkaline phosphatase. Although purification procedures are not yet completely optimized, they are expected to include filtration, ion exchange and affinity chromatography. Our presentation will focus on the purification and crystallization results up to the time of the conference. Structural data should provide additional information on the role of alkaline phosphatase in normal bone mineralization and in certain bone mineralization anomalies.

  2. Phosphatases in Cancer : Shifting the balance

    NARCIS (Netherlands)

    E. Hoekstra (Elmer)

    2015-01-01

    markdownabstractAbstract The role of phosphatases in cancer is an ignored research field, mostly based on the dogma that phosphatases function as tumor suppressor genes. However, in our opinion dephosphorylation events by phosphatases can also enhance signaling in cancer. The current research

  3. Identification and characterization of a pyridoxal 5'-phosphate phosphatase in the silkworm (Bombyx mori).

    Science.gov (United States)

    Huang, ShuoHao; Han, CaiYun; Ma, ZhenQiao; Zhou, Jie; Zhang, JianYun; Huang, LongQuan

    2017-03-01

    Vitamin B 6 comprises six interconvertible pyridine compounds, among which pyridoxal 5'-phosphate (PLP) is a coenzyme for over 140 enzymes. PLP is also a very reactive aldehyde. The most well established mechanism for maintaining low levels of free PLP is its dephosphorylation by phosphatases. A human PLP-specific phosphatase has been identified and characterized. However, very little is known about the phosphatase in other living organisms. In this study, a cDNA clone of putative PLP phosphatase was identified from B. mori and characterized. The cDNA encodes a polypeptide of 343 amino acid residues, and the recombinant enzyme purified from E. coli exhibited properties similar to that of human PLP phosphatase. B. mori has a single copy of the PLPP gene, which is located on 11th chromosome, spans a 5.7kb region and contains five exons and four introns. PLP phosphatase transcript was detected in every larva tissue except hemolymph, and was most highly represented in Malpighian tube. We further down-regulated the gene expression of the PLP phosphatase in 5th instar larvae with the RNA interference. However, no significant changes in the gene expression of PLP biosynthetic enzymes and composition of B 6 vitamers were detected as compared with the control. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. FIG4 regulates lysosome membrane homeostasis independent of phosphatase function

    OpenAIRE

    Bharadwaj, Rajnish; Cunningham, Kathleen M.; Zhang, Ke; Lloyd, Thomas E.

    2015-01-01

    FIG4 is a phosphoinositide phosphatase that is mutated in several diseases including Charcot-Marie-Tooth Disease 4J (CMT4J) and Yunis-Varon syndrome (YVS). To investigate the mechanism of disease pathogenesis, we generated Drosophila models of FIG4-related diseases. Fig4 null mutant animals are viable but exhibit marked enlargement of the lysosomal compartment in muscle cells and neurons, accompanied by an age-related decline in flight ability. Transgenic animals expressing Drosophila Fig4 mi...

  5. Catalytic activity of a novel serine/threonine protein phosphatase PP5 from Leishmania major

    Directory of Open Access Journals (Sweden)

    Norris-Mullins Brianna

    2014-01-01

    Full Text Available Leishmaniasis is a vector-borne disease caused by protozoan parasites of the genus Leishmania. Our knowledge of protein phosphatases (PPs and their implication in signaling events is very limited. Here we report the expression, characterization and mutagenesis analysis of a novel protein phosphatase 5 (PP5 in Leishmania major. Recombinant PP5 is a bona fide phosphatase and is enzymatically active. Site-directed mutagenesis revealed auto-inhibitory roles of the N-terminal region. This is a rational first approach to understand the role of PP5 in the biology of the parasite better as well as its potential future applicability to anti-parasitic intervention.

  6. Protein phosphatases decrease their activity during capacitation: a new requirement for this event.

    Directory of Open Access Journals (Sweden)

    Janetti R Signorelli

    Full Text Available There are few reports on the role of protein phosphatases during capacitation. Here, we report on the role of PP2B, PP1, and PP2A during human sperm capacitation. Motile sperm were resuspended in non-capacitating medium (NCM, Tyrode's medium, albumin- and bicarbonate-free or in reconstituted medium (RCM, NCM plus 2.6% albumin/25 mM bicarbonate. The presence of the phosphatases was evaluated by western blotting and the subcellular localization by indirect immunofluorescence. The function of these phosphatases was analyzed by incubating the sperm with specific inhibitors: okadaic acid, I2, endothall, and deltamethrin. Different aliquots were incubated in the following media: 1 NCM; 2 NCM plus inhibitors; 3 RCM; and 4 RCM plus inhibitors. The percent capacitated sperm and phosphatase activities were evaluated using the chlortetracycline assay and a phosphatase assay kit, respectively. The results confirm the presence of PP2B and PP1 in human sperm. We also report the presence of PP2A, specifically, the catalytic subunit and the regulatory subunits PR65 and B. PP2B and PP2A were present in the tail, neck, and postacrosomal region, and PP1 was present in the postacrosomal region, neck, middle, and principal piece of human sperm. Treatment with phosphatase inhibitors rapidly (≤1 min increased the percent of sperm depicting the pattern B, reaching a maximum of ∼40% that was maintained throughout incubation; after 3 h, the percent of capacitated sperm was similar to that of the control. The enzymatic activity of the phosphatases decreased during capacitation without changes in their expression. The pattern of phosphorylation on threonine residues showed a sharp increase upon treatment with the inhibitors. In conclusion, human sperm express PP1, PP2B, and PP2A, and the activity of these phosphatases decreases during capacitation. This decline in phosphatase activities and the subsequent increase in threonine phosphorylation may be an important

  7. Increased liver alkaline phosphatase and aminotransferase ...

    African Journals Online (AJOL)

    The effect of daily, oral administration of ethanolic extract of Khaya senegalensis stem bark (2mg/kg body weight) for 18days on the alkaline phosphatase, aspartate and alanine aminotransferase activities of rat liver and serum were investigated. Compared with the control, the activities of liver alkaline phosphatase (ALP), ...

  8. Functional Analysis of Mouse G6pc1 Mutations Using a Novel In Situ Assay for Glucose-6-Phosphatase Activity and the Effect of Mutations in Conserved Human G6PC1/G6PC2 Amino Acids on G6PC2 Protein Expression.

    Directory of Open Access Journals (Sweden)

    Kayla A Boortz

    Full Text Available Elevated fasting blood glucose (FBG has been associated with increased risk for development of type 2 diabetes. Single nucleotide polymorphisms (SNPs in G6PC2 are the most important common determinants of variations in FBG in humans. Studies using G6pc2 knockout mice suggest that G6pc2 regulates the glucose sensitivity of insulin secretion. G6PC2 and the related G6PC1 and G6PC3 genes encode glucose-6-phosphatase catalytic subunits. This study describes a functional analysis of 22 non-synonymous G6PC2 SNPs, that alter amino acids that are conserved in human G6PC1, mouse G6pc1 and mouse G6pc2, with the goal of identifying variants that potentially affect G6PC2 activity/expression. Published data suggest strong conservation of catalytically important amino acids between all four proteins and the related G6PC3 isoform. Because human G6PC2 has very low glucose-6-phosphatase activity we used an indirect approach, examining the effect of these SNPs on mouse G6pc1 activity. Using a novel in situ functional assay for glucose-6-phosphatase activity we demonstrate that the amino acid changes associated with the human G6PC2 rs144254880 (Arg79Gln, rs149663725 (Gly114Arg and rs2232326 (Ser324Pro SNPs reduce mouse G6pc1 enzyme activity without affecting protein expression. The Arg79Gln variant alters an amino acid mutation of which, in G6PC1, has previously been shown to cause glycogen storage disease type 1a. We also demonstrate that the rs368382511 (Gly8Glu, rs138726309 (His177Tyr, rs2232323 (Tyr207Ser rs374055555 (Arg293Trp, rs2232326 (Ser324Pro, rs137857125 (Pro313Leu and rs2232327 (Pro340Leu SNPs confer decreased G6PC2 protein expression. In summary, these studies identify multiple G6PC2 variants that have the potential to be associated with altered FBG in humans.

  9. Characterization of protein phosphatase 5 from three lepidopteran insects: Helicoverpa armigera, Mythimna separata and Plutella xylostella.

    Directory of Open Access Journals (Sweden)

    Xi'en Chen

    Full Text Available Protein phosphatase 5 (PP5, a unique member of serine/threonine phosphatases, regulates a variety of biological processes. We obtained full-length PP5 cDNAs from three lepidopteran insects, Helicoverpa armigera, Mythimna separata and Plutella xylostella, encoding predicted proteins of 490 (55.98 kDa, 490 (55.82 kDa and 491 (56.07 kDa amino acids, respectively. These sequences shared a high identity with other insect PP5s and contained the TPR (tetratricopeptide repeat domains at N-terminal regions and highly conserved C-terminal catalytic domains. Tissue- and stage-specific expression pattern analyses revealed these three PP5 genes were constitutively expressed in all stages and in tested tissues with predominant transcription occurring at the egg and adult stages. Activities of Escherichia coli-produced recombinant PP5 proteins could be enhanced by almost 2-fold by a known PP5 activator: arachidonic acid. Kinetic parameters of three recombinant proteins against substrate pNPP were similar both in the absence or presence of arachidonic acid. Protein phosphatases inhibitors, okadaic acid, cantharidin, and endothall strongly impeded the activities of the three recombinant PP5 proteins, as well as exerted an inhibitory effect on crude protein phosphatases extractions from these three insects. In summary, lepidopteran PP5s share similar characteristics and are all sensitive to the protein phosphatases inhibitors. Our results also imply protein phosphatase inhibitors might be used in the management of lepidopteran pests.

  10. Displacement affinity chromatography of protein phosphatase one (PP1 complexes

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    Gourlay Robert

    2008-11-01

    Full Text Available Abstract Background Protein phosphatase one (PP1 is a ubiquitously expressed, highly conserved protein phosphatase that dephosphorylates target protein serine and threonine residues. PP1 is localized to its site of action by interacting with targeting or regulatory proteins, a majority of which contains a primary docking site referred to as the RVXF/W motif. Results We demonstrate that a peptide based on the RVXF/W motif can effectively displace PP1 bound proteins from PP1 retained on the phosphatase affinity matrix microcystin-Sepharose. Subsequent co-immunoprecipitation experiments confirmed that each identified binding protein was either a direct PP1 interactor or was in a complex that contains PP1. Our results have linked PP1 to numerous new nuclear functions and proteins, including Ki-67, Rif-1, topoisomerase IIα, several nuclear helicases, NUP153 and the TRRAP complex. Conclusion This modification of the microcystin-Sepharose technique offers an effective means of purifying novel PP1 regulatory subunits and associated proteins and provides a simple method to uncover a link between PP1 and additional cellular processes.

  11. Activation of the protein tyrosine phosphatase SHP2 via the interleukin-6 signal transducing receptor protein gp130 requires tyrosine kinase Jak1 and limits acute-phase protein expression.

    Science.gov (United States)

    Schaper, F; Gendo, C; Eck, M; Schmitz, J; Grimm, C; Anhuf, D; Kerr, I M; Heinrich, P C

    1998-11-01

    Stimulation of the interleukin-6 (IL-6) signalling pathway occurs via the IL-6 receptor-glycoprotein 130 (IL-6R-gp130) receptor complex and results in the regulation of acute-phase protein genes in liver cells. Ligand binding to the receptor complex leads to tyrosine phosphorylation and activation of Janus kinases (Jak), phosphorylation of the signal transducing subunit gp130, followed by recruitment and phosphorylation of the signal transducer and activator of transcription factors STAT3 and STAT1 and the src homology domain (SH2)-containing protein tyrosine phosphatase (SHP2). The tyrosine phosphorylated STAT factors dissociate from the receptor, dimerize and translocate to the nucleus where they bind to enhancer sequences of IL-6 target genes. Phosphorylated SHP2 is able to bind growth factor receptor bound protein (grb2) and thus might link the Jak/STAT pathway to the ras/raf/mitogen-activated protein kinase pathway. Here we present data on the dose-dependence, kinetics and kinase requirements for SHP2 phosphorylation after the activation of the signal transducer, gp130, of the IL-6-type family receptor complex. When human fibrosarcoma cell lines deficient in Jak1, Jak2 or tyrosine kinase 2 (Tyk2) were stimulated with IL-6-soluble IL-6R complexes it was found that only in Jak1-, but not in Jak 2- or Tyk2-deficient cells, SHP2 activation was greatly impaired. It is concluded that Jak1 is required for the tyrosine phosphorylation of SHP2. This phosphorylation depends on Tyr-759 in the cytoplasmatic domain of gp130, since a Tyr-759-->Phe exchange abrogates SHP2 activation and in turn leads to elevated and prolonged STAT3 and STAT1 activation as well as enhanced acute-phase protein gene induction. Therefore, SHP2 plays an important role in acute-phase gene regulation.

  12. Tea Contains Potent Inhibitors of Tyrosine Phosphatase PTP1B

    OpenAIRE

    Ma, Junfeng; Li, Zhe; Xing, Shu; Ho, Wanting Tina; Fu, Xueqi; Zhao, Zhizhuang Joe

    2011-01-01

    Tea is widely consumed all over the world. Studies have demonstrated the role of tea in prevention and treatment of various chronic diseases including diabetes and obesity, but the underlying mechanism is unclear. PTP1B is a widely expressed tyrosine phosphatase which has been defined as a target for therapeutic drug development to treat diabetes and obesity. In screening for inhibitors of PTP1B, we found that aqueous extracts of teas exhibited potent PTP1B inhibitory effects with an IC50 val...

  13. Autophagy Signaling in Prostate Cancer: Identification of a Novel Phosphatase

    Science.gov (United States)

    2013-01-01

    chloroquine treatment] and LC3 accumulation used as a measure of autophagic flux (Tanida et al., 2005). We found that both knockdown of PTPs and amino acid...6 Jeffrey P. MacKeigan,3 Kyle A. Furge,2 H. Eric Xu1,7†D ow nloaded The antimalaria drug chloroquine has been used as an anti-inflammatory agent for...verified and characterized for phosphatase activity in vitro (task 2). In addition to inserting these constructs into mammalian expression vectors, used

  14. Enzyme domain affects the movement of the voltage sensor in ascidian and zebrafish voltage-sensing phosphatases.

    Science.gov (United States)

    Hossain, Md Israil; Iwasaki, Hirohide; Okochi, Yoshifumi; Chahine, Mohamed; Higashijima, Shinichi; Nagayama, Kuniaki; Okamura, Yasushi

    2008-06-27

    The ascidian voltage-sensing phosphatase (Ci-VSP) consists of the voltage sensor domain (VSD) and a cytoplasmic phosphatase region that has significant homology to the phosphatase and tensin homolog deleted on chromosome TEN (PTEN). The phosphatase activity of Ci-VSP is modified by the conformational change of the VSD. In many proteins, two protein modules are bidirectionally coupled, but it is unknown whether the phosphatase domain could affect the movement of the VSD in VSP. We addressed this issue by whole-cell patch recording of gating currents from a teleost VSP (Dr-VSP) cloned from Danio rerio expressed in tsA201 cells. Replacement of a critical cysteine residue, in the phosphatase active center of Dr-VSP, by serine sharpened both ON- and OFF-gating currents. Similar changes were produced by treatment with phosphatase inhibitors, pervanadate and orthovanadate, that constitutively bind to cysteine in the active catalytic center of phosphatases. The distinct kinetics of gating currents dependent on enzyme activity were not because of altered phosphatidylinositol 4,5-bisphosphate levels, because the kinetics of gating current did not change by depletion of phosphatidylinositol 4,5-bisphosphate, as reported by coexpressed KCNQ2/3 channels. These results indicate that the movement of the VSD is influenced by the enzymatic state of the cytoplasmic domain, providing an important clue for understanding mechanisms of coupling between the VSD and its effector.

  15. Proteomic analysis of protein phosphatase Z1 from Candida albicans.

    Directory of Open Access Journals (Sweden)

    Bernadett Márkus

    Full Text Available Protein phosphatase Z is a "novel type" fungus specific serine/threonine protein phosphatase. Previously our research group identified the CaPPZ1 gene in the opportunistic pathogen Candida albicans and reported that the gene deletion had several important physiological consequences. In order to reveal the protein targets and the associated mechanisms behind the functions of the phosphatase a proteomic method was adopted for the comparison of the cappz1 deletion mutant and the genetically matching QMY23 control strain. Proteins extracted from the control and deletion mutant strains were separated by two-dimensional gel electrophoresis and the protein spots were stained with RuBPS and Pro-Q Diamond in order to visualize the total proteome and the phosphoproteome, respectively. The alterations in spot intensities were determined by densitometry and were analysed with the Delta2D (Decodon software. Spots showing significantly different intensities between the mutant and control strains were excised from the gels and were digested with trypsin. The resulting peptides were identified by LC-MS/MS mass spectrometry. As many as 15 protein spots were found that exhibited significant changes in their intensity upon the deletion of the phosphatase and 20 phosphoproteins were identified in which the level of phosphorylation was modified significantly in the mutant. In agreement with previous findings we found that the affected proteins function in protein synthesis, oxidative stress response, regulation of morphology and metabolism. Among these proteins we identified two potential CaPpz1 substrates (Eft2 and Rpp0 that may regulate the elongation step of translation. RT-qPCR experiments revealed that the expression of the genes coding for the affected proteins was not altered significantly. Thus, the absence of CaPpz1 exerted its effects via protein synthesis/degradation and phosphorylation/dephosphorylation. In addition, our proteomics data strongly

  16. Proteomic analysis of protein phosphatase Z1 from Candida albicans

    Science.gov (United States)

    Pfliegler, Walter P.; Petrényi, Katalin; Boros, Enikő; Pócsi, István; Tőzsér, József; Dombrádi, Viktor

    2017-01-01

    Protein phosphatase Z is a “novel type” fungus specific serine/threonine protein phosphatase. Previously our research group identified the CaPPZ1 gene in the opportunistic pathogen Candida albicans and reported that the gene deletion had several important physiological consequences. In order to reveal the protein targets and the associated mechanisms behind the functions of the phosphatase a proteomic method was adopted for the comparison of the cappz1 deletion mutant and the genetically matching QMY23 control strain. Proteins extracted from the control and deletion mutant strains were separated by two-dimensional gel electrophoresis and the protein spots were stained with RuBPS and Pro-Q Diamond in order to visualize the total proteome and the phosphoproteome, respectively. The alterations in spot intensities were determined by densitometry and were analysed with the Delta2D (Decodon) software. Spots showing significantly different intensities between the mutant and control strains were excised from the gels and were digested with trypsin. The resulting peptides were identified by LC-MS/MS mass spectrometry. As many as 15 protein spots were found that exhibited significant changes in their intensity upon the deletion of the phosphatase and 20 phosphoproteins were identified in which the level of phosphorylation was modified significantly in the mutant. In agreement with previous findings we found that the affected proteins function in protein synthesis, oxidative stress response, regulation of morphology and metabolism. Among these proteins we identified two potential CaPpz1 substrates (Eft2 and Rpp0) that may regulate the elongation step of translation. RT-qPCR experiments revealed that the expression of the genes coding for the affected proteins was not altered significantly. Thus, the absence of CaPpz1 exerted its effects via protein synthesis/degradation and phosphorylation/dephosphorylation. In addition, our proteomics data strongly suggested a role for

  17. Posttranslational heterogeneity of bone alkaline phosphatase in metabolic bone disease.

    Science.gov (United States)

    Langlois, M R; Delanghe, J R; Kaufman, J M; De Buyzere, M L; Van Hoecke, M J; Leroux-Roels, G G

    1994-09-01

    Bone alkaline phosphatase is a marker of osteoblast activity. In order to study the posttranscriptional modification (glycosylation) of bone alkaline phosphatase in bone disease, we investigated the relationship between mass and catalytic activity of bone alkaline phosphatase in patients with osteoporosis and hyperthyroidism. Serum bone alkaline phosphatase activity was measured after lectin precipitation using the Iso-ALP test kit. Mass concentration of bone alkaline phosphatase was determined with an immunoradiometric assay (Tandem-R Ostase). In general, serum bone alkaline phosphatase mass and activity concentration correlated well. The activity : mass ratio of bone alkaline phosphatase was low in hyperthyroidism. Activation energy of the reaction catalysed by bone alkaline phosphatase was high in osteoporosis and in hyperthyroidism. Experiments with neuraminidase digestion further demonstrated that the thermodynamic heterogeneity of bone alkaline phosphatase can be explained by a different glycosylation of the enzyme.

  18. Delivery of small interfering RNA for inhibition of endothelial cell apoptosis by hypoxia and serum deprivation

    International Nuclear Information System (INIS)

    Cho, Seung-Woo; Hartle, Lauren; Son, Sun Mi; Yang, Fan; Goldberg, Michael; Xu, Qiaobing; Langer, Robert; Anderson, Daniel G.

    2008-01-01

    RNA interference (RNAi) for anti-angiogenic or pro-apoptotic factors in endothelial cells (ECs) has great potential for the treatment of ischemic diseases by promoting angiogenesis or inhibiting apoptosis. Here, we report the utility of small interfering RNA (siRNA) in inhibiting EC apoptosis induced by tumor necrosis factor-α (TNF-α). siRNA was designed and synthesized targeting tumor necrosis factor-α receptor-1 (TNFR-1) and Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1). Human umbilical vein endothelial cells (HUVECs) were cultured under in vitro hypoxic and serum-deprived conditions to simulate in vivo ischemic conditions. Two days after liposomal delivery of siRNA targeting TNFR-1 and SHP-1, significant silencing of each target (TNFR-1; 76.5% and SHP-1; 97.2%) was detected. Under serum-deprived hypoxic (1% oxygen) conditions, TNF-α expression in HUVECs increased relative to normoxic (20% oxygen) and serum-containing conditions. Despite enhanced TNF-α expression, suppression of TNFR-1 or SHP-1 by siRNA delivery not only enhanced expression of angiogenic factors (KDR/Flk-1 and eNOS) and anti-apoptotic factor (Bcl-xL) but also reduced expression of a pro-apoptotic factor (Bax). Transfection of TNFR-1 or SHP-1 siRNA significantly decreased the HUVEC apoptosis while significantly enhancing HUVEC proliferation and capillary formation. The present study demonstrates that TNFR-1 and SHP-1 may be useful targets for the treatment of myocardial or hindlimb ischemia

  19. Defining Starch Binding by Glucan Phosphatases

    DEFF Research Database (Denmark)

    Auger, Kyle; Raththagala, Madushi; Wilkens, Casper

    2015-01-01

    Starch is a vital energy molecule in plants that has a wide variety of uses in industry, such as feedstock for biomaterial processing and biofuel production. Plants employ a three enzyme cyclic process utilizing kinases, amylases, and phosphatases to degrade starch in a diurnal manner. Starch...... is comprised of the branched glucan amylopectin and the more linear glucan amylose. Our lab has determined the first structures of these glucan phosphatases and we have defined their enzymatic action. Despite this progress, we lacked a means to quickly and efficiently quantify starch binding to glucan...

  20. Ligand-mediated negative regulation of a chimeric transmembrane receptor tyrosine phosphatase

    DEFF Research Database (Denmark)

    Desai, D M; Sap, J; Schlessinger, J

    1993-01-01

    CD45, a transmembrane protein tyrosine phosphatase (PTPase), is required for TCR signaling. Multiple CD45 isoforms, differing in the extracellular domain, are expressed in a tissue- and activation-specific manner, suggesting an important function for this domain. We report that a chimeric protein...... that ligand-mediated regulation of receptor-PTPases may have mechanistic similarities with receptor tyrosine kinases....

  1. Functional diversity of voltage-sensing phosphatases in two urodele amphibians.

    Science.gov (United States)

    Mutua, Joshua; Jinno, Yuka; Sakata, Souhei; Okochi, Yoshifumi; Ueno, Shuichi; Tsutsui, Hidekazu; Kawai, Takafumi; Iwao, Yasuhiro; Okamura, Yasushi

    2014-07-16

    Voltage-sensing phosphatases (VSPs) share the molecular architecture of the voltage sensor domain (VSD) with voltage-gated ion channels and the phosphoinositide phosphatase region with the phosphatase and tensin homolog (PTEN), respectively. VSPs enzymatic activities are regulated by the motions of VSD upon depolarization. The physiological role of these proteins has remained elusive, and insights may be gained by investigating biological variations in different animal species. Urodele amphibians are vertebrates with potent activities of regeneration and also show diverse mechanisms of polyspermy prevention. We cloned cDNAs of VSPs from the testes of two urodeles; Hynobius nebulosus and Cynops pyrrhogaster, and compared their expression and voltage-dependent activation. Their molecular architecture is highly conserved in both Hynobius VSP (Hn-VSP) and Cynops VSP (Cp-VSP), including the positively-charged arginine residues in the S4 segment of the VSD and the enzymatic active site for substrate binding, yet the C-terminal C2 domain of Hn-VSP is significantly shorter than that of Cp-VSP and other VSP orthologs. RT-PCR analysis showed that gene expression pattern was distinct between two VSPs. The voltage sensor motions and voltage-dependent phosphatase activities were investigated electrophysiologically by expression in Xenopus oocytes. Both VSPs showed "sensing" currents, indicating that their voltage sensor domains are functional. The phosphatase activity of Cp-VSP was found to be voltage dependent, as shown by its ability to regulate the conductance of coexpressed GIRK2 channels, but Hn-VSP lacked such phosphatase activity due to the truncation of its C2 domain. © 2014 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  2. Characterization of the Functional Domains of a Mammalian Voltage-Sensitive Phosphatase.

    Science.gov (United States)

    Rosasco, Mario G; Gordon, Sharona E; Bajjalieh, Sandra M

    2015-12-15

    Voltage-sensitive phosphatases (VSPs) are proteins that directly couple changes in membrane electrical potential to inositol lipid phosphatase activity. VSPs thus couple two signaling pathways that are critical for cellular functioning. Although a number of nonmammalian VSPs have been characterized biophysically, mammalian VSPs are less well understood at both the physiological and biophysical levels. In this study, we aimed to address this gap in knowledge by determining whether the VSP from mouse, Mm-VSP, is expressed in the brain and contains a functional voltage-sensing domain (VSD) and a phosphatase domain. We report that Mm-VSP is expressed in neurons and is developmentally regulated. To address whether the functions of the VSD and phosphatase domain are retained in Mm-VSP, we took advantage of the modular nature of these domains and expressed each independently as a chimeric protein in a heterologous expression system. We found that the Mm-VSP VSD, fused to a viral potassium channel, was able to drive voltage-dependent gating of the channel pore. The Mm-VSP phosphatase domain, fused to the VSD of a nonmammalian VSP, was also functional: activation resulted in PI(4,5)P2 depletion that was sufficient to inhibit the PI(4,5)P2-regulated KCNQ2/3 channels. While testing the functionality of the VSD and phosphatase domain, we observed slight differences between the activities of Mm-VSP-based chimeras and those of nonmammalian VSPs. Although the properties of VSP chimeras may not completely reflect the properties of native VSPs, the differences we observed in voltage-sensing and phosphatase activity provide a starting point for future experiments to investigate the function of Mm-VSP and other mammalian VSPs. In conclusion, our data reveal that both the VSD and the lipid phosphatase domain of Mm-VSP are functional, indicating that Mm-VSP likely plays an important role in mouse neurophysiology. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All

  3. Evidence for phosphoprotein phosphatase in Streptomyces granaticolor

    Czech Academy of Sciences Publication Activity Database

    Bobek, J.; Hercík, K.; Dobrová, Zuzana; Branny, Pavel; Nádvorník, Richard; Janeček, Jiří

    2000-01-01

    Roč. 45, č. 4 (2000), s. 310-312 ISSN 0015-5632 R&D Projects: GA ČR GA204/99/1534 Institutional research plan: CEZ:AV0Z5020903 Keywords : streptomycetes * phosphoprotein phosphatase Subject RIV: EE - Microbiology, Virology Impact factor: 0.752, year: 2000

  4. Enzyme kinetic characterization of protein tyrosine phosphatases

    DEFF Research Database (Denmark)

    Peters, Günther H.J.; Branner, S.; Møller, K. B.

    2003-01-01

    Protein tyrosine phosphatases (PTPs) play a central role in cellular signaling processes, resulting in an increased interest in modulating the activities of PTPs. We therefore decided to undertake a detailed enzyme kinetic evaluation of various transmembrane and cytosolic PTPs (PTPalpha, PTPbeta...

  5. FIG4 regulates lysosome membrane homeostasis independent of phosphatase function.

    Science.gov (United States)

    Bharadwaj, Rajnish; Cunningham, Kathleen M; Zhang, Ke; Lloyd, Thomas E

    2016-02-15

    FIG4 is a phosphoinositide phosphatase that is mutated in several diseases including Charcot-Marie-Tooth Disease 4J (CMT4J) and Yunis-Varon syndrome (YVS). To investigate the mechanism of disease pathogenesis, we generated Drosophila models of FIG4-related diseases. Fig4 null mutant animals are viable but exhibit marked enlargement of the lysosomal compartment in muscle cells and neurons, accompanied by an age-related decline in flight ability. Transgenic animals expressing Drosophila Fig4 missense mutations corresponding to human pathogenic mutations can partially rescue lysosomal expansion phenotypes, consistent with these mutations causing decreased FIG4 function. Interestingly, Fig4 mutations predicted to inactivate FIG4 phosphatase activity rescue lysosome expansion phenotypes, and mutations in the phosphoinositide (3) phosphate kinase Fab1 that performs the reverse enzymatic reaction also causes a lysosome expansion phenotype. Since FIG4 and FAB1 are present together in the same biochemical complex, these data are consistent with a model in which FIG4 serves a phosphatase-independent biosynthetic function that is essential for lysosomal membrane homeostasis. Lysosomal phenotypes are suppressed by genetic inhibition of Rab7 or the HOPS complex, demonstrating that FIG4 functions after endosome-to-lysosome fusion. Furthermore, disruption of the retromer complex, implicated in recycling from the lysosome to Golgi, does not lead to similar phenotypes as Fig4, suggesting that the lysosomal defects are not due to compromised retromer-mediated recycling of endolysosomal membranes. These data show that FIG4 plays a critical noncatalytic function in maintaining lysosomal membrane homeostasis, and that this function is disrupted by mutations that cause CMT4J and YVS. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  6. Osteocalcin and bone-specific alkaline phosphatase in Sickle cell ...

    African Journals Online (AJOL)

    specific alkaline phosphatase (b-AP) total protein levels were evaluated as indicators of bone turnover in twenty patients with sickle cell haemoglobinopathies and in twenty normal healthy individuals. The serum bonespecific alkaline phosphatase ...

  7. Elevated serum tartrate-resistant acid phosphatase isoform 5a levels in metabolic syndrome.

    Science.gov (United States)

    Huang, Yi-Jhih; Huang, Tsai-Wang; Chao, Tsu-Yi; Sun, Yu-Shan; Chen, Shyi-Jou; Chu, Der-Ming; Chen, Wei-Liang; Wu, Li-Wei

    2017-09-29

    Tartrate-resistant phosphatase isoform 5a is expressed in tumor-associated macrophages and is a biomarker of chronic inflammation. Herein, we correlated serum tartrate-resistant phosphatase isoform 5a levels with metabolic syndrome status and made comparisons with traditional markers of inflammation, including c-reactive protein and interleukin-6. One hundred healthy volunteers were randomly selected, and cut-off points for metabolic syndrome related inflammatory biomarkers were determined using receiver operating characteristic curves. Linear and logistic regression models were subsequently used to correlate inflammatory markers with the risk of metabolic syndrome. Twenty-two participants met the criteria for metabolic syndrome, and serum tartrate-resistant phosphatase isoform 5a levels of >5.8 μg/L were associated with metabolic syndrome (c-statistics, 0.730; p = 0.001; 95% confidence interval, 0.618-0.842). In addition, 1 μg/L increases in tartrate-resistant phosphatase isoform 5a levels were indicative of a 1.860 fold increase in the risk of metabolic syndrome (p = 0.012). Elevated serum tartrate-resistant phosphatase isoform 5a levels are associated with the risk of metabolic syndrome, with a cut-off level of 5.8 μg/L.

  8. [Spectroscopic analysis of the interaction of ethanol and acid phosphatase from wheat germ].

    Science.gov (United States)

    Xu, Dong-mei; Liu, Guang-shen; Wang, Li-ming; Liu, Wei-ping

    2004-11-01

    Conformational and activity changes of acid phosphatase from wheat germ in ethanol solutions of different concentrations were measured by fluorescence spectra and differential UV-absorption spectra. The effect of ethanol on kinetics of acid phosphatase was determined by using the double reciprocal plot. The results indicate the ethanol has a significant effect on the activity and conformation of acid phosphatase. The activity of acid phosphatase decreased linearly with increasing the concentration of ethanol. Differential UV-absorption spectra of the enzyme denatured in ethanol solutions showed two positive peaks at 213 and 234 nm, respectively. The peaks on the differential UV-absorption spectra suggested that the conformation of enzyme molecule changed from orderly structure to out-of-order crispation. The fluorescence emission peak intensity of the enzyme gradually strengthened with increasing ethanol concentration, which is in concordance with the conformational change of the microenvironments of tyrosine and tryptophan residues. The results indicate that the expression of the enzyme activity correlates with the stability and integrity of the enzyme conformation to a great degree. Ethanol is uncompetitive inhibitor of acid phosphatase.

  9. Src inhibitor herbimycin A prevents 132.7 kDa tyrosine phosphatase activity in Ramos Burkitt's lymphoma B cell line

    International Nuclear Information System (INIS)

    Hristov, K.; Mitev, V.; Knox, K.

    2006-01-01

    Reversible tyrosine phosphorylation, regulation of expression and proteolytic cleavage control tyrosine phosphatase contribution for the signalling pathways of B-cell antigen receptor (BCR), and CD40 during B cell selection. We used Ramos-BL B cell line to determine whether BCR and CD40 stimulation, or inhibition of the Src - tyrosine kinase, tyrosine phosphatase and caspase activity have an effect on the tyrosine phosphatase activities determined on in-gel phosphatase assay. The tyrosine phosphatase activities present in whole cell lysates of Ramos-BL B cells following treatment with 20 μg/ml anti-IgM, 1 μg/ml anti-CD40, 10 μM herbimycin A, 178 μM vanadate,100 μM phenylarsine oxide and 10 μM zVAD-fmk were detected with an in-gel phosphatase assay. Seven major tyrosine phosphatase activities with approximate molecular weight of 132.7, 63.9, 60.3, 54.2, 49.7, 44.6, and 39 kDa are present in whole cell lysates of Ramos-BL B cells. Treatment with Src-PTK inhibitor herbimycin A prevents 132.7 kDa tyrosine phosphatase activity. We conclude that the catalytic activity of Src-PTK in Ramos-BL B cells is critical for the presence of this 132.7 kDa tyrosine phosphatase activity. (authors)

  10. SH2-inositol phosphatase 1 negatively influences early megakaryocyte progenitors.

    Directory of Open Access Journals (Sweden)

    Lia E Perez

    Full Text Available The SH2-containing-5'inositol phosphatase-1 (SHIP influences signals downstream of cytokine/chemokine receptors that play a role in megakaryocytopoiesis, including thrombopoietin, stromal-cell-derived-Factor-1/CXCL-12 and interleukin-3. We hypothesize that SHIP might control megakaryocytopoiesis through effects on proliferation of megakaryocyte progenitors (MKP and megakaryocytes (MK.Herein, we report the megakaryocytic phenotype and MK functional assays of hematopoietic organs of two strains of SHIP deficient mice with deletion of the SHIP promoter/first exon or the inositol phosphatase domain. Both SHIP deficient strains exhibit a profound increase in MKP numbers in bone marrow (BM, spleen and blood as analyzed by flow cytometry (Lin(-c-Kit+CD41+ and functional assays (CFU-MK. SHIP deficient MKP display increased phosphorylation of Signal Transducers and Activators of Transcription 3 (STAT-3, protein kinase B (PKB/AKT and extracellular signal-regulated kinases (ERKs. Despite increased MKP content, total body number of mature MK (Lin(-c-kit(-CD41+ are not significantly changed as SHIP deficient BM contains reduced MK while spleen MK numbers are increased. Reduction of CXCR-4 expression in SHIP deficient MK may influence MK localization to the spleen instead of the BM. Endomitosis, process involved in MK maturation, was preserved in SHIP deficient MK. Circulating platelets and red blood cells are also reduced in SHIP deficient mice.SHIP may play an important role in regulation of essential signaling pathways that control early megakaryocytopoiesis in vivo.

  11. The function of Shp2 tyrosine phosphatase in the dispersal of acetylcholine receptor clusters

    Directory of Open Access Journals (Sweden)

    Madhavan Raghavan

    2008-07-01

    Full Text Available Abstract Background A crucial event in the development of the vertebrate neuromuscular junction (NMJ is the postsynaptic enrichment of muscle acetylcholine (ACh receptors (AChRs. This process involves two distinct steps: the local clustering of AChRs at synapses, which depends on the activation of the muscle-specific receptor tyrosine kinase MuSK by neural agrin, and the global dispersal of aneural or "pre-patterned" AChR aggregates, which is triggered by ACh or by synaptogenic stimuli. We and others have previously shown that tyrosine phosphatases, such as the SH2 domain-containing phosphatase Shp2, regulate AChR cluster formation in muscle cells, and that tyrosine phosphatases also mediate the dispersal of pre-patterned AChR clusters by synaptogenic stimuli, although the specific phosphatases involved in this latter step remain unknown. Results Using an assay system that allows AChR cluster assembly and disassembly to be studied separately and quantitatively, we describe a previously unrecognized role of the tyrosine phosphatase Shp2 in AChR cluster disassembly. Shp2 was robustly expressed in embryonic Xenopus muscle in vivo and in cultured myotomal muscle cells, and treatment of the muscle cultures with an inhibitor of Shp2 (NSC-87877 blocked the dispersal of pre-patterned AChR clusters by synaptogenic stimuli. In contrast, over-expression in muscle cells of either wild-type or constitutively active Shp2 accelerated cluster dispersal. Significantly, forced expression in muscle of the Shp2-activator SIRPα1 (signal regulatory protein α1 also enhanced the disassembly of AChR clusters, whereas the expression of a truncated SIRPα1 mutant that suppresses Shp2 signaling inhibited cluster disassembly. Conclusion Our results suggest that Shp2 activation by synaptogenic stimuli, through signaling intermediates such as SIRPα1, promotes the dispersal of pre-patterned AChR clusters to facilitate the selective accumulation of AChRs at developing NMJs.

  12. Human liver phosphatase 2A: cDNA and amino acid sequence of two catalytic subunit isotypes

    International Nuclear Information System (INIS)

    Arino, J.; Woon, Chee Wai; Brautigan, D.L.; Miller, T.B. Jr.; Johnson, G.L.

    1988-01-01

    Two cDNA clones were isolated from a human liver library that encode two phosphatase 2A catalytic subunits. The two cDNAs differed in eight amino acids (97% identity) with three nonconservative substitutions. All of the amino acid substitutions were clustered in the amino-terminal domain of the protein. Amino acid sequence of one human liver clone (HL-14) was identical to the rabbit skeletal muscle phosphatase 2A cDNA (with 97% nucleotide identity). The second human liver clone (HL-1) is encoded by a separate gene, and RNA gel blot analysis indicates that both mRNAs are expressed similarly in several human clonal cell lines. Sequence comparison with phosphatase 1 and 2A indicates highly divergent amino acid sequences at the amino and carboxyl termini of the proteins and identifies six highly conserved regions between the two proteins that are predicted to be important for phosphatase enzymatic activity

  13. Protein tyrosine phosphatase receptor type R deficient mice exhibit increased exploration in a new environment and impaired novel object recognition memory

    NARCIS (Netherlands)

    Erkens, M.; Bakker, B.; Duijn, L.M. van; Hendriks, W.J.A.J.; Zee, C.E.E.M. van der

    2014-01-01

    Mouse gene Ptprr encodes multiple protein tyrosine phosphatase receptor type R (PTPRR) isoforms that negatively regulate mitogen-activated protein kinase (MAPK) signaling pathways. In the mouse brain, PTPRR proteins are expressed in cerebellum, olfactory bulb, hippocampus, amygdala and perirhinal

  14. Monomeric tartrate resistant acid phosphatase induces insulin sensitive obesity.

    Directory of Open Access Journals (Sweden)

    Pernilla Lång

    2008-03-01

    Full Text Available Obesity is associated with macrophage infiltration of adipose tissue, which may link adipose inflammation to insulin resistance. However, the impact of inflammatory cells in the pathophysiology of obesity remains unclear. Tartrate resistant acid phosphatase (TRAP is an enzyme expressed by subsets of macrophages and osteoclasts that exists either as an enzymatically inactive monomer or as an active, proteolytically processed dimer.Using mice over expressing TRAP, we show that over-expression of monomeric, but not the dimeric form in adipose tissue leads to early onset spontaneous hyperplastic obesity i.e. many small fat cells. In vitro, recombinant monomeric, but not proteolytically processed TRAP induced proliferation and differentiation of mouse and human adipocyte precursor cells. In humans, monomeric TRAP was highly expressed in the adipose tissue of obese individuals. In both the mouse model and in the obese humans the source of TRAP in adipose tissue was macrophages. In addition, the obese TRAP over expressing mice exhibited signs of a low-grade inflammatory reaction in adipose tissue without evidence of abnormal adipocyte lipolysis, lipogenesis or insulin sensitivity.Monomeric TRAP, most likely secreted from adipose tissue macrophages, induces hyperplastic obesity with normal adipocyte lipid metabolism and insulin sensitivity.

  15. Identification of a variant form of tyrosine phosphatase LYP

    Directory of Open Access Journals (Sweden)

    Ho Wanting T

    2010-11-01

    Full Text Available Abstract Background Protein tyrosine phosphatases (PTPs are important cell signaling regulators with major pathological implications. LYP (also known as PTPN22 is an intracellular enzyme initially found to be predominately expressed in lymphocytes. Importantly, an allelic R620W variant of LYP is strongly associated with multiple autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, and autoimmune thyroid disease. Results In this study, we isolated a novel isoform of LYP designated LYP3. LYP3 differs from LYP1, the known isoform of LYP, in that it lacks a 28 amino acid segment right after the R620W site embedded in a proline-rich protein-protein interaction motif. Genomic sequence analysis revealed that LYP3 resulted from alternative splicing of the LYP gene located on chromosome 1p 13.3-13.1. Reverse transcription PCR analyses of 48 human tissues demonstrated that both LYP1 and LYP3 are predominantly expressed in primary and secondary lymphoid tissues but the relative expression levels of the two isoforms varies in different human tissues and individuals. Conclusions We thus identified a new variant form of LYP and conducted a comprehensive analysis of LYP tissue expressions. Considering the pathogenesis of LYP R620W, we believe that the expression of LYP3 may have an important role in regulating activity and function of LYP and may be implicated in autoimmune diseases.

  16. Voltage-sensing phosphatase: its molecular relationship with PTEN.

    Science.gov (United States)

    Okamura, Yasushi; Dixon, Jack E

    2011-02-01

    Voltage-sensing phosphoinositide phosphatase (VSP) contains voltage sensor and cytoplasmic phosphatase domains. A unique feature of this protein is that depolarization-induced motions of the voltage sensor activate PtdIns(3,4,5)P(3) and PtdIns(4,5)P(2) phosphatase activities. VSP exhibits remarkable structural similarities with PTEN, the phosphatase and tensin homolog deleted on chromosome 10. These similarities include the cytoplasmic phosphatase region, the phosphoinositide binding region, and the putative membrane interacting C2 domain.

  17. Purification of acidic phosphatase from mustard seedlings

    OpenAIRE

    sprotocols

    2014-01-01

    ### INTRODUCTION Phosphate esters are widely distributed in any organism. Nucleic acids, metabolic intermediates like glucose-6-phosphate, energy-rich substrates (AMP, creatine phosphate) are some obvious examples. While many metabolic intermediates are activated through the transfer of phosphate groups (e.g., by kinases) it is equally important that phosphate esters can also be rapidly broken down. The hydrolytic removal of phosphate groups from phosphoesters is catalyzed by phosphatases...

  18. Phosphatase activity of Poa pratensis seeds. I. Preliminary studies on acid phosphatase II

    Directory of Open Access Journals (Sweden)

    I. Lorenc-Kubis

    2015-01-01

    Full Text Available Acid phosphatase (EC 3.1.3.2 was extracted with 0.1 M sodium acetate buffer pH 5.1 from Poa pratensis seeds, and separated into three fractions by chromatography on DEAE cellulose. The highest activity was found in fraction Il-b (acid phosphatase II. The activity of the enzyme was optimal at pH 4.9. It hydrolyzed p-nitrophenyl phosphate most readily among the various phosphomonoesters examined. Acid phosphatase II showed also a high activity toward β-naphtyl phosphate and phenyl phosphate, very low activity towards β-glycero phosphate, 5'-GMP and no activity with glucose-1 phosphate. The enzyme was inhibited by Ca2+ and fluoride, but activated by Mg2+. EDTA had no influence on the activity of the enzyme.

  19. Phosphatase activity of Poa pratensis seeds. l. Preliminary studies on acid phosphatase II

    Energy Technology Data Exchange (ETDEWEB)

    Lorenc-Kubis, I.; Morawiecka, B.

    1973-01-01

    Acid phosphatase (EC 3.1.3.2) was extracted from 0.1 M sodium acetate buffer, pH 5.1 from Poa pratensis seeds, and separated into three fractions by chromatography on DEAE cellulose. The highest activity was found in fraction II-b (acid phosphatase II). The activity of the enzyme was optimal at pH 4.9. It hydrolyzed p-nitrophenyl phosphate most readily among the various phosphomonoesters examined. Acid phosphatase II showed also a high activity toward ..beta..-naphtyl phosphate and phenyl phosphate, very low activity towards ..beta..-glycero phosphate, 5'-GMP and no activity with glucose-1 phosphate. The enzyme was inhibited by Ca/sup 2 +/ and fluoride, but activated by Mg/sup 2 +/. EDTA had no influence on the activity of the enzyme. 12 references, 3 figures, 4 tables.

  20. STAT3 mediates regorafenib-induced apoptosis in hepatocellular carcinoma.

    Science.gov (United States)

    Tai, Wei-Tien; Chu, Pei-Yi; Shiau, Chung-Wai; Chen, Yao-Li; Li, Yong-Shi; Hung, Man-Hsin; Chen, Li-Ju; Chen, Pei-Lung; Su, Jung-Chen; Lin, Ping-Yi; Yu, Hui-Chuan; Chen, Kuen-Feng

    2014-11-15

    Here, we aim to investigate the molecular mechanism of regorafenib and verify the potential druggable target for the treatment of hepatocellular carcinoma (HCC). HCC cell lines (PLC5, HepG2, Hep3B, SK-Hep1, and HA59T) were used to investigate the in vitro effect of regorafenib. Phosphatase activity was analyzed in HCC cells and purified SHP-1 proteins. PLC5-bearing mice were used to test the therapeutic efficiency of 20 and 40 mg/kg/d treatment with regorafenib ([Formula: see text] mice). The clinical relevance of STAT3 signaling was investigated with 142 tumor samples from different patients with HCC. Descriptive statistical analysis was used to compare the baseline characteristics of patients and the expression of p-STAT3. Regorafenib inhibited STAT3-related signaling in a dose-dependent manner and was a more potent inhibitor of STAT3 than sorafenib. Regorafenib increased SHP-1 phosphatase activity in purified SHP-1 protein directly. N-SH2 domain deletion and D61A mutants mimicking open-form SHP-1 partially abolished regorafenib-induced STAT3 inhibition and apoptosis. Importantly, a higher level of expression of STAT3 was found in patients with advanced clinical stages (P = 0.009) and poorly differentiated tumors (P = 0.035). Regorafenib induced significant tumor inhibition by relieving the autoinhibited N-SH2 domain of SHP-1 directly and inhibiting p-STAT3 signals. STAT3 may be suitable as a prognostic marker of HCC development, and may be a druggable target for HCC-targeted therapy using regorafenib. ©2014 American Association for Cancer Research.

  1. Expression

    Directory of Open Access Journals (Sweden)

    Wang-Xia Wang

    2014-02-01

    Full Text Available The miR-15/107 family comprises a group of 10 paralogous microRNAs (miRNAs, sharing a 5′ AGCAGC sequence. These miRNAs have overlapping targets. In order to characterize the expression of miR-15/107 family miRNAs, we employed customized TaqMan Low-Density micro-fluid PCR-array to investigate the expression of miR-15/107 family members, and other selected miRNAs, in 11 human tissues obtained at autopsy including the cerebral cortex, frontal cortex, primary visual cortex, thalamus, heart, lung, liver, kidney, spleen, stomach and skeletal muscle. miR-103, miR-195 and miR-497 were expressed at similar levels across various tissues, whereas miR-107 is enriched in brain samples. We also examined the expression patterns of evolutionarily conserved miR-15/107 miRNAs in three distinct primary rat brain cell preparations (enriched for cortical neurons, astrocytes and microglia, respectively. In primary cultures of rat brain cells, several members of the miR-15/107 family are enriched in neurons compared to other cell types in the central nervous system (CNS. In addition to mature miRNAs, we also examined the expression of precursors (pri-miRNAs. Our data suggested a generally poor correlation between the expression of mature miRNAs and their precursors. In summary, we provide a detailed study of the tissue and cell type-specific expression profile of this highly expressed and phylogenetically conserved family of miRNA genes.

  2. Persistently increased intestinal fraction of alkaline phosphatase

    DEFF Research Database (Denmark)

    Nathan, E; Baatrup, G; Berg, H

    1984-01-01

    Persistent elevation of the intestinal fraction of the alkaline phosphatase (API) as an isolated finding has to our knowledge not been reported previously. It was found in a boy followed during a period of 5.5 years. The only symptom was transient periodic fatigue observed at home, but not apparent...... during hospitalization. His blood type was O, RH+, Le (a-, b+) and he was a secretor of H-substance, which may be associated with rising API activity after fat-loading. In this case API was unchanged after fat-loading. Neither intestinal nor liver diseases were found, and no other cause for the elevated...

  3. Regulation of tyrosine phosphatases in the adventitia during vascular remodelling

    International Nuclear Information System (INIS)

    Micke, Patrick; Hackbusch, Daniel; Mercan, Sibel; Stawowy, Philipp; Tsuprykov, Oleg; Unger, Thomas; Ostman, Arne; Kappert, Kai

    2009-01-01

    Protein tyrosine phosphatases (PTPs) are regulators of growth factor signalling in vascular remodelling. The aim of this study was to evaluate PTP expression in the context of PDGF-signalling in the adventitia after angioplasty. Utilising a rat carotid artery model, the adventitial layers of injured and non-injured vessels were laser microdissected. The mRNA expression of the PDGF β-receptor, the ligands PDGF-A/B/C/D and the receptor-antagonising PTPs (DEP-1, TC-PTP, SHP-2, PTP1B) were determined and correlated to vascular morphometrics, proliferation markers and PDGF β-receptor phosphorylation. The levels of the PDGF β-receptor, PDGF-C and PDGF-D were upregulated concurrently with the antagonising PTPs DEP-1 and TC-PTP at day 8, and normalised at day 14 after vessel injury. Although the proliferation parameters were time-dependently altered in the adventitial layer, the phosphorylation of the PDGF β-receptor remained unchanged. The expression dynamics of specific PTPs indicate a regulatory role of PDGF-signalling also in the adventitia during vascular remodelling.

  4. Protein phosphatase 2A dysfunction in Alzheimer’s disease

    Directory of Open Access Journals (Sweden)

    Jean-Marie eSontag

    2014-03-01

    Full Text Available Protein Phosphatase 2A (PP2A is a large family of enzymes that account for the majority of brain Ser/Thr phosphatase activity. While PP2A enzymes collectively modulate most cellular processes, sophisticated regulatory mechanisms are ultimately responsible for ensuring isoform-specific substrate specificity. Of particular interest to the Alzheimer’s disease (AD field, alterations in PP2A regulators and PP2A catalytic activity, subunit expression, methylation and/or phosphorylation, have been reported in AD-affected brain regions. PP2A dysfunction has been linked to Tau hyperphosphorylation, amyloidogenesis and synaptic deficits that are pathological hallmarks of this neurodegenerative disorder. Deregulation of PP2A enzymes also affects the activity of many Ser/Thr protein kinases implicated in AD. This review will more specifically discuss the role of the PP2A/B holoenzyme and PP2A methylation in AD pathogenesis. The PP2A/B isoform binds to tau and is the primary tau phosphatase. Its deregulation correlates with increased tau phosphorylation in vivo and in AD. Disruption of PP2A/B-Tau protein interactions likely contribute to Tau deregulation in AD. Significantly, alterations in one-carbon metabolism that impair PP2A methylation are associated with increased risk for sporadic AD, and enhanced AD-like pathology in animal models. Experimental studies have linked deregulation of PP2A methylation with down-regulation of PP2A/B, enhanced phosphorylation of Tau and amyloid precursor protein, Tau mislocalization, microtubule destabilization and neuritic defects. While it remains unclear what are the primary events that underlie PP2A dysfunction in AD, deregulation of PP2A enzymes definitely affects key players in the pathogenic process. As such, there is growing interest in developing PP2A-centric therapies for AD, but this may be a daunting task without a better understanding of the regulation and function of specific PP2A enzymes.

  5. Genome-wide functional analysis of plasmodium protein phosphatases reveals key regulators of parasite development and differentiation

    KAUST Repository

    Guttery, David S.

    2014-07-09

    Reversible protein phosphorylation regulated by kinases and phosphatases controls many cellular processes. Although essential functions for the malaria parasite kinome have been reported, the roles of most protein phosphatases (PPs) during Plasmodium development are unknown. We report a functional analysis of the Plasmodium berghei protein phosphatome, which exhibits high conservation with the P. falciparum phosphatome and comprises 30 predicted PPs with differential and distinct expression patterns during various stages of the life cycle. Gene disruption analysis of P. berghei PPs reveals that half of the genes are likely essential for asexual blood stage development, whereas six are required for sexual development/sporogony in mosquitoes. Phenotypic screening coupled with transcriptome sequencing unveiled morphological changes and altered gene expression in deletion mutants of two N-myristoylated PPs. These findings provide systematic functional analyses of PPs in Plasmodium, identify how phosphatases regulate parasite development and differentiation, and can inform the identification of drug targets for malaria. © 2014 The Authors.

  6. Genome-wide functional analysis of plasmodium protein phosphatases reveals key regulators of parasite development and differentiation

    KAUST Repository

    Guttery, David  S.; Poulin, Benoit; Ramaprasad, Abhinay; Wall, Richard  J.; Ferguson, David  J.P.; Brady, Declan; Patzewitz, Eva-Maria; Whipple, Sarah; Straschil, Ursula; Wright, Megan  H.; Mohamed, Alyaa  M.A.H.; Radhakrishnan, Anand; Arold, Stefan T.; Tate, Edward  W.; Holder, Anthony  A.; Wickstead, Bill; Pain, Arnab; Tewari, Rita

    2014-01-01

    Reversible protein phosphorylation regulated by kinases and phosphatases controls many cellular processes. Although essential functions for the malaria parasite kinome have been reported, the roles of most protein phosphatases (PPs) during Plasmodium development are unknown. We report a functional analysis of the Plasmodium berghei protein phosphatome, which exhibits high conservation with the P. falciparum phosphatome and comprises 30 predicted PPs with differential and distinct expression patterns during various stages of the life cycle. Gene disruption analysis of P. berghei PPs reveals that half of the genes are likely essential for asexual blood stage development, whereas six are required for sexual development/sporogony in mosquitoes. Phenotypic screening coupled with transcriptome sequencing unveiled morphological changes and altered gene expression in deletion mutants of two N-myristoylated PPs. These findings provide systematic functional analyses of PPs in Plasmodium, identify how phosphatases regulate parasite development and differentiation, and can inform the identification of drug targets for malaria. © 2014 The Authors.

  7. Low molecular weight protein tyrosine phosphatases control antibiotic production in Streptomyces coelicolor A3(2)

    DEFF Research Database (Denmark)

    Sohoni, Sujata Vijay; Lieder, Sarah; Bapat, Prashant Madhusudhan

    2014-01-01

    3700 was established usingpara-nitrophenyl phosphate and the tyrosine-phosphorylated protein PtkA from Bacillus subtilis as substrates. Theoptimum pH for the Sco3700 phosphatase activity was 6.8, and KM for pNPP was 14.3 mM compared to pH 6.0and KM0.75 mM for PtpA. The potential of Sco3700...... of ACT in the ptpA over expression strain. Furthermore, a significantly earlier onset of ACT productionwas observed when ptpA was over expressed. Sco3700 overexpression had a pleiotropic effect on the cell, and thestrain exhibited lower productivities and final concentrations of antibiotics. We conclude...... that Sco3700 is indeed atyrosine phosphatase, and it contributes to regulation of antibiotic production in S. coelicolor affecting the timing ofonset of the antibiotic production...

  8. Crystal structure and putative substrate identification for the Entamoeba histolytica low molecular weight tyrosine phosphatase.

    Science.gov (United States)

    Linford, Alicia S; Jiang, Nona M; Edwards, Thomas E; Sherman, Nicholas E; Van Voorhis, Wesley C; Stewart, Lance J; Myler, Peter J; Staker, Bart L; Petri, William A

    2014-01-01

    Entamoeba histolytica is a eukaryotic intestinal parasite of humans, and is endemic in developing countries. We have characterized the E. histolytica putative low molecular weight protein tyrosine phosphatase (LMW-PTP). The structure for this amebic tyrosine phosphatase was solved, showing the ligand-induced conformational changes necessary for binding of substrate. In amebae, it was expressed at low but detectable levels as detected by immunoprecipitation followed by immunoblotting. A mutant LMW-PTP protein in which the catalytic cysteine in the active site was replaced with a serine lacked phosphatase activity, and was used to identify a number of trapped putative substrate proteins via mass spectrometry analysis. Seven of these putative substrate protein genes were cloned with an epitope tag and overexpressed in amebae. Five of these seven putative substrate proteins were demonstrated to interact specifically with the mutant LMW-PTP. This is the first biochemical study of a small tyrosine phosphatase in Entamoeba, and sets the stage for understanding its role in amebic biology and pathogenesis. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Structural stability of human protein tyrosine phosphatase ρ catalytic domain: effect of point mutations.

    Directory of Open Access Journals (Sweden)

    Alessandra Pasquo

    Full Text Available Protein tyrosine phosphatase ρ (PTPρ belongs to the classical receptor type IIB family of protein tyrosine phosphatase, the most frequently mutated tyrosine phosphatase in human cancer. There are evidences to suggest that PTPρ may act as a tumor suppressor gene and dysregulation of Tyr phosphorylation can be observed in diverse diseases, such as diabetes, immune deficiencies and cancer. PTPρ variants in the catalytic domain have been identified in cancer tissues. These natural variants are nonsynonymous single nucleotide polymorphisms, variations of a single nucleotide occurring in the coding region and leading to amino acid substitutions. In this study we investigated the effect of amino acid substitution on the structural stability and on the activity of the membrane-proximal catalytic domain of PTPρ. We expressed and purified as soluble recombinant proteins some of the mutants of the membrane-proximal catalytic domain of PTPρ identified in colorectal cancer and in the single nucleotide polymorphisms database. The mutants show a decreased thermal and thermodynamic stability and decreased activation energy relative to phosphatase activity, when compared to wild- type. All the variants show three-state equilibrium unfolding transitions similar to that of the wild- type, with the accumulation of a folding intermediate populated at ~4.0 M urea.

  10. Trehalose 6-phosphate phosphatases of Pseudomonas aeruginosa.

    Science.gov (United States)

    Cross, Megan; Biberacher, Sonja; Park, Suk-Youl; Rajan, Siji; Korhonen, Pasi; Gasser, Robin B; Kim, Jeong-Sun; Coster, Mark J; Hofmann, Andreas

    2018-04-24

    The opportunistic bacterium Pseudomonas aeruginosa has been recognized as an important pathogen of clinical relevance and is a leading cause of hospital-acquired infections. The presence of a glycolytic enzyme in Pseudomonas, which is known to be inhibited by trehalose 6-phosphate (T6P) in other organisms, suggests that these bacteria may be vulnerable to the detrimental effects of intracellular T6P accumulation. In the present study, we explored the structural and functional properties of trehalose 6-phosphate phosphatase (TPP) in P. aeruginosa in support of future target-based drug discovery. A survey of genomes revealed the existence of 2 TPP genes with either chromosomal or extrachromosomal location. Both TPPs were produced as recombinant proteins, and characterization of their enzymatic properties confirmed specific, magnesium-dependent catalytic hydrolysis of T6P. The 3-dimensional crystal structure of the chromosomal TPP revealed a protein dimer arising through β-sheet expansion of the individual monomers, which possess the overall fold of halo-acid dehydrogenases.-Cross, M., Biberacher, S., Park, S.-Y., Rajan, S., Korhonen, P., Gasser, R. B., Kim, J.-S., Coster, M. J., Hofmann, A. Trehalose 6-phosphate phosphatases of Pseudomonas aeruginosa.

  11. Increased ethanol accumulation from glucose via reduction of ATP level in a recombinant strain of Saccharomyces cerevisiae overexpressing alkaline phosphatase.

    Science.gov (United States)

    Semkiv, Marta V; Dmytruk, Kostyantyn V; Abbas, Charles A; Sibirny, Andriy A

    2014-05-15

    The production of ethyl alcohol by fermentation represents the largest scale application of Saccharomyces cerevisiae in industrial biotechnology. Increased worldwide demand for fuel bioethanol is anticipated over the next decade and will exceed 200 billion liters from further expansions. Our working hypothesis was that the drop in ATP level in S. cerevisiae cells during alcoholic fermentation should lead to an increase in ethanol production (yield and productivity) with a greater amount of the utilized glucose converted to ethanol. Our approach to achieve this goal is to decrease the intracellular ATP level via increasing the unspecific alkaline phosphatase activity. Intact and truncated versions of the S. cerevisiae PHO8 gene coding for vacuolar or cytosolic forms of alkaline phosphatase were fused with the alcohol dehydrogenase gene (ADH1) promoter. The constructed expression cassettes used for transformation vectors also contained the dominant selective marker kanMX4 and S. cerevisiae δ-sequence to facilitate multicopy integration to the genome. Laboratory and industrial ethanol producing strains BY4742 and AS400 overexpressing vacuolar form of alkaline phosphatase were characterized by a slightly lowered intracellular ATP level and biomass accumulation and by an increase in ethanol productivity (13% and 7%) when compared to the parental strains. The strains expressing truncated cytosolic form of alkaline phosphatase showed a prolonged lag-phase, reduced biomass accumulation and a strong defect in ethanol production. Overexpression of vacuolar alkaline phosphatase leads to an increased ethanol yield in S. cerevisiae.

  12. Differentiation-dependent activation of the human intestinal alkaline phosphatase promoter by HNF-4 in intestinal cells

    DEFF Research Database (Denmark)

    Olsen, Line; Bressendorff, Simon; Troelsen, Jesper T

    2005-01-01

    The intestinal alkaline phosphatase gene (ALPI) encodes a digestive brush-border enzyme, which is highly upregulated during small intestinal epithelial cell differentiation. To identify new putative promoter motifs responsible for the regulation of ALPI expression during differentiation of the en...

  13. A generally applicable sequential alkaline phosphatase immunohistochemical double staining

    NARCIS (Netherlands)

    van der Loos, Chris M.; Teeling, Peter

    2008-01-01

    A universal type of sequential double alkaline phosphatase immunohistochemical staining is described that can be used for formalin-fixed, paraffin-embedded and cryostat tissue sections from human and mouse origin. It consists of two alkaline phosphatase detection systems including enzymatic

  14. Voltage sensitive phosphatases: emerging kinship to protein tyrosine phosphatases from structure-function research

    Directory of Open Access Journals (Sweden)

    Kirstin eHobiger

    2015-02-01

    Full Text Available The transmembrane protein Ci-VSP from the ascidian Ciona intestinalis was described as first member of a fascinating family of enzymes, the voltage sensitive phosphatases (VSPs. Ci-VSP and its voltage-activated homologs from other species are stimulated by positive membrane potentials and dephosphorylate the head groups of negatively charged phosphoinositide phosphates (PIPs. In doing so, VSPs act as control centers at the cytosolic membrane surface, because they intervene in signaling cascades that are mediated by PIP lipids. The characteristic motif CX5RT/S in the active site classifies VSPs as members of the huge family of cysteine-based protein tyrosine phosphatases (PTPs. Although PTPs have already been well characterized regarding both, structure and function, their relationship to VSPs has drawn only limited attention so far. Therefore, the intention of this review is to give a short overview about the extensive knowledge about PTPs in relation to the facts known about VSPs. Here, we concentrate on the structural features of the catalytic domain which are similar between both classes of phosphatases and their consequences for the enzymatic function. By discussing results obtained from crystal structures, molecular dynamics simulations, and mutagenesis studies, a possible mechanism for the catalytic cycle of VSPs is presented based on that one proposed for PTPs. In this way, we want to link the knowledge about the catalytic activity of VSPs and PTPs.

  15. EXPRESS

    International Nuclear Information System (INIS)

    Ancelin, C.; Le, P.; DeSaint-Quentin, S.; Villatte, N.

    1987-01-01

    This paper presents EXPRESS, an expert system developed for the automation of reliability studies. The first part consists in the description of the method for static thermohydraulic systems. In this step, the authors define the knowledge representation based on the two inference engines - ALOUETTE and LCR developed by EDF. They explain all the process to construct a fault tree from a topological and functional description of the system. Numerous examples are exhibited in illustration of the method. This is followed by the lessons derived from the studies performed on some safety systems of the PALUEL nuclear plant. The development of the same approach for electric power systems is described, insisting on the difference resulting from the sequential nature of these systems. Finally, they show the main advantages identified during the studies

  16. Phosphatase activity of Poa pratensis seeds. II. Purification and characterization of acid phosphatase Ia2 and Ia3

    Directory of Open Access Journals (Sweden)

    I. Lorenc-Kubis

    2015-01-01

    Full Text Available Two acid phosphatases (Ia2, Ia3 have been isolated from Poa pratensis seeds and partially purified. Both enzymes showed maximal activity at pH 4,9. They exhibited high activity towards p-nitrophenyl phosphate, inorganic pyrophosphate and phenyl phosphate, much less activity towards glucose-6 phosphate, and mononucleotides. Phosphatases a2 and a3 differed in their activity towards ADP. Orthophosphate, fluoride and Zn2+ were effective inhibitors. EDTA, β-mercaptoethanol and Mg2+ activated phophatase a2 but had no effect on phosphatase a3. Zn2+ inhibited the activity of phosphatase a2 noncompetitively, whereas phosphatase a3 showed inhibition of mixed type. Trypsin, chymotrypsin and pronase had no effect on the enzyme activities of both molecular forms.

  17. Uranium Biomineralization By Natural Microbial Phosphatase Activities in the Subsurface

    Energy Technology Data Exchange (ETDEWEB)

    Taillefert, Martial [Georgia Tech Research Corporation, Atlanta, GA (United States)

    2015-04-01

    This project investigated the geochemical and microbial processes associated with the biomineralization of radionuclides in subsurface soils. During this study, it was determined that microbial communities from the Oak Ridge Field Research subsurface are able to express phosphatase activities that hydrolyze exogenous organophosphate compounds and result in the non-reductive bioimmobilization of U(VI) phosphate minerals in both aerobic and anaerobic conditions. The changes of the microbial community structure associated with the biomineralization of U(VI) was determined to identify the main organisms involved in the biomineralization process, and the complete genome of two isolates was sequenced. In addition, it was determined that both phytate, the main source of natural organophosphate compounds in natural environments, and polyphosphate accumulated in cells could also be hydrolyzed by native microbial population to liberate enough orthophosphate and precipitate uranium phosphate minerals. Finally, the minerals produced during this process are stable in low pH conditions or environments where the production of dissolved inorganic carbon is moderate. These findings suggest that the biomineralization of U(VI) phosphate minerals is an attractive bioremediation strategy to uranium bioreduction in low pH uranium-contaminated environments. These efforts support the goals of the SBR long-term performance measure by providing key information on "biological processes influencing the form and mobility of DOE contaminants in the subsurface".

  18. Alkaline phosphatase activity of rumen bacteria.

    Science.gov (United States)

    Cheng, K J; Costerton, J W

    1977-11-01

    Of the 54 strains of rumen bacteria examined for alkaline phosphatase (APase) production, 9 of 33 gram-negative strains and none of 21 gram-positive strains produced the enzyme. The APase of the cells of the three strains of Bacteroides ruminicola that produced significant amounts of the enzyme was located in the periplasmic area of the cell envelope, whereas the enzyme was located in the strains of Selenomonas ruminantium and Succinivibrio dextrinosolvens was associated with the outer membrane. The localization of APase production in the cells of natural populations of rumen bacteria from hay-fed sheep was accomplished by reaction product deposition, and both the proportion of APase-producing bacteria and the location of the enzyme in the cell envelope of the producing cells could be determined. We suggest that this procedure is useful in detecting shifts in the bacterial population and the release of cell-bound APase that accompany feedlot bloat and other sequelae of dietary manipulation in ruminants.

  19. Identification of human pulmonary alkaline phosphatase isoenzymes.

    Science.gov (United States)

    Capelli, A; Cerutti, C G; Lusuardi, M; Donner, C F

    1997-04-01

    An increase of alkaline phosphatase (ALP) activity has been observed in the bronchoalveolar lavage fluid (BALF) of patients affected by pulmonary fibrosis in chronic interstitial lung disorders. To characterize the ALP isoenzymes in such cases, we used gel filtration, agarose gel electrophoresis, heat and amino acid inhibition assays, wheat-germ agglutinin (WGA) precipitation, and an immunoassay specific for the bone-isoform of ALP. Only one anodic band representing a high-molecular-weight isoform of ALP (Mr approximately 2,000 kDa) was observed on electrophoresis of BALF. The inhibition assay results were consistent for a tissue-nonspecific isoenzyme sensitive to a temperature of 56 degrees C (71.9 +/- 2.5% inhibition) and to homoarginine (65.7 +/- 1.9%), and resistant to L-phenylalanine and L-leucine. Less than 13% of ALP activity was heat-stable. After incubation of BALF specimens with glycosyl-phosphatidylinositol-phospholipase D plus Nonidet P-40, or with phosphatidylinositol-phospholipase C alone, an electrophoretic cathodic band (Mr approximately 220 kDa) appeared near the bone band of a standard serum. With the WGA assay, 84.4 +/- 3.3% of ALP precipitated and the band disappeared. After immunoassay for the bone isoform, a mean of less than 5% enzyme activity was measured. We conclude that the ALP found in BALF is a pulmonary isoform of a tissue nonspecific isoenzyme.

  20. Alkaline Phosphatase, an Unconventional Immune Protein

    Directory of Open Access Journals (Sweden)

    Bethany A. Rader

    2017-08-01

    Full Text Available Recent years have seen an increase in the number of studies focusing on alkaline phosphatases (APs, revealing an expanding complexity of function of these enzymes. Of the four human AP (hAP proteins, most is known about tissue non-specific AP (TNAP and intestinal AP (IAP. This review highlights current understanding of TNAP and IAP in relation to human health and disease. TNAP plays a role in multiple processes, including bone mineralization, vitamin B6 metabolism, and neurogenesis, is the genetic cause of hypophosphatasia, influences inflammation through regulation of purinergic signaling, and has been implicated in Alzheimer’s disease. IAP regulates fatty acid absorption and has been implicated in the regulation of diet-induced obesity and metabolic syndrome. IAP and TNAP can dephosphorylate bacterial-derived lipopolysaccharide, and IAP has been identified as a potential regulator of the composition of the intestinal microbiome, an evolutionarily conserved function. Endogenous and recombinant bovine APs and recombinant hAPs are currently being explored for their potential as pharmacological agents to treat AP-associated diseases and mitigate multiple sources of inflammation. Continued research on these versatile proteins will undoubtedly provide insight into human pathophysiology, biochemistry, and the human holobiont.

  1. Defective interleukin-4/Stat6 activity correlates with increased constitutive expression of negative regulators SOCS-3, SOCS-7, and CISH in colon cancer cells.

    Science.gov (United States)

    Liu, Xiao Hong; Xu, Shuang Bing; Yuan, Jia; Li, Ben Hui; Zhang, Yan; Yuan, Qin; Li, Pin Dong; Li, Feng; Zhang, Wen Jie

    2009-12-01

    Interleukin-4 (IL-4)-induced Stat6 activities (phenotypes) vary among human cancer cells, of which the HT-29 cell line carries an active Stat6(high) phenotype, while Caco-2 carries a defective Stat6(null) phenotype, respectively. Cancer cells with Stat6(high) show resistance to apoptosis and exaggerated metastasis, suggesting the clinical significance of Stat6 phenotypes. We previously showed that Stat6(high) HT-29 cells exhibited low constitutive expression of Stat6-negative regulators SOCS-1 and SHP-1 because of gene hypermethylation. This study further examined the constitutive expression of other closely related SOCS family numbers including SOCS-3, SOCS-5, SOCS-7, and CISH using RT-PCR. Similar to SOCS-1 and SHP-1, Stat6(high) HT-29 cells expressed low constitutive mRNA of SOCS-3, SOCS-7, and CISH than Stat6(null) Caco-2 cells. Interestingly, DNA demethylation using 5-aza-2'-deoxycytidine in HT-29 cells up-regulated mRNA expression of the above genes, indicating a hypermethylation status, which was confirmed by methylation-specific sequencing in selected SOCS-3 gene. Furthermore, defective Stat6(null) Caco-2 exhibited impaired phosphorylation of Stat6 after IL-4 stimulation by flow cytometry, in keeping with the notion of an over-performed negative regulation. The findings that IL-4/Stat6 phenotypes show differential expression of multiple negative regulators suggest a model that a collective force of powerful negative regulators, directly and indirectly, acts on Stat6 activation, which may result in differential Stat6 phenotypes.

  2. Pst I restriction fragment length polymorphism of the human placental alkaline phosphatase gene in normal placentae and tumors

    International Nuclear Information System (INIS)

    Tsavaler, L.; Penhallow, R.C.; Kam, W.; Sussman, H.H.

    1987-01-01

    The structure of the human placental alkaline phosphatase gene from normal term placentae was studied by restriction enzyme digestion and Southern blot analysis using a cDNA probe to the gene for the placental enzyme. The DNA digests fall into three distinct patterns based on the presence and intensity of an extra 1.1-kilobase Pst I Band. The extra 1.1-kilobase band is present in 9 of 27 placenta samples, and in 1 of these samples the extra band is present at double intensity. No polymorphism was revealed by digestion with restriction enzymes EcoRI, Sma I, BamHI, or Sac I. The extra Pst I-digestion site may lie in a noncoding region of the gene because no correlation was observed between the restriction fragment length polymorphism and the common placental alkaline phosphatase alleles identified by starch gel electrophoresis. In addition, because placental alkaline phosphatase is frequently re-expressed in neoplasms, the authors examined tissue from ovarian, testicular, and endometrial tumors and from BeWo choriocarcinoma cells in culture. The Pst I-DNA digestion patterns from these cells and tissues were identical to those seen in the normal ovary and term placentae. The consistent reproducible digestion patterns seen in DNA from normal and tumor tissue indicate that a major gene rearrangement is not the basis for the ectopic expression of placental alkaline phosphatase in neoplasia

  3. Redox Regulation of Receptor Protein-Tyrosine Phosphatases

    NARCIS (Netherlands)

    Groen, A.J.

    2006-01-01

    Phosphorylation is of major importance in cell signalling processes like cell migration, cell proliferation and cell differentiation within higher eukaryotic organisms. Therefore, the balance between phosphorylation, mediated by kinases, and dephosphorylation, mediated by phosphatases, must be

  4. Synthesis and phosphatase activity of a Cobalt(II) phenanthroline ...

    Indian Academy of Sciences (India)

    MAMONI GARAI

    2017-09-19

    Sep 19, 2017 ... Synthesis and phosphatase activity of a Cobalt(II) phenanthroline complex. MAMONI GARAIa ... tion, cobalt complexes have gained importance because of their application as ... 2.3 Physical measurements. Infrared spectrum ...

  5. Novel Combinatorial Chemistry-Derived Inhibitors of Oncogenic Phosphatases

    National Research Council Canada - National Science Library

    Lazo, John

    1999-01-01

    Our overall goal of this US Army Breast Cancer Grant entitled "Novel Combinatorial Chemistry-Derived Inhibitors of Oncogenic Phosphatases" is to identity and develop novel therapeutic agents for human breast cancer...

  6. Research on Phosphatases of Belladona Leaves and Their Purification

    Directory of Open Access Journals (Sweden)

    M. Khorsand

    1957-01-01

    Full Text Available Through experimentation with several leaves it has been possible for us to point out the existance of two different acid phosphatases. We have studied in more detail the phosphatases of belldon a leaves (Atropa Belladona L. Solanacees. The great part of the phosphatase activity is water extractable. We have compared the activity of the soluble fraction with that not directly extractable by means of water. The insoluble fraction could not be solubilized in a satisfaetC'fY m.anner.The digestion by papaine produced a slight solubilizing effect; on the other hand salt solutions, neutral or alkaline, or water glycerol mixtures had no solubilizing effect on the enzyme, It has been possible to demonstrate the existence of two different phosphatases in the insoluble fraction: the first of the type II,

  7. Crystallization and preliminary X-ray diffraction analysis of rat protein tyrosine phosphatase η

    Energy Technology Data Exchange (ETDEWEB)

    Matozo, Huita C.; Nascimento, Alessandro S.; Santos, Maria A. M. [Instituto de Física de São Carlos, Departamento de Física e Informática, Universidade de São Paulo, Avenida Trabalhador São Carlense 400, CEP 13566-590 São Carlos, SP (Brazil); Iuliano, Rodolfo [Dipartimento di Medicina Sperimentale e Clinica, Facoltà di Medicina e Chirurgia, Università di Catanzaro, 88100 Catanzaro (Italy); Fusco, Alfredo [Dipartimento di Biologia e Patologia Cellulare e Molecolare, c/o Instituto di Endocrinologia ed Oncologia Sperimentale del CNR, Facolta di Medicina e Chirurgia, Università degli Studi di Napoli ‘Federico II’, Via Pansini 5, 80131 Naples (Italy); NOGEC (Naples Oncogenomocs Center)-CEINGE, Biotecnologie Avanzate, Via Comunale Margherita 482, 80145 Naples (Italy); Polikarpov, Igor, E-mail: ipolikarpov@if.sc.usp.br [Instituto de Física de São Carlos, Departamento de Física e Informática, Universidade de São Paulo, Avenida Trabalhador São Carlense 400, CEP 13566-590 São Carlos, SP (Brazil); Laboratório Nacional de Luz Síncrotron, Campinas, SP (Brazil)

    2006-09-01

    In this study, the catalytic domain of rat protein tyrosine phosphatase η was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals were obtained by the hanging-drop vapour-diffusion method. The rat protein tyrosine phosphatase η (rPTPη) is a cysteine-dependent phosphatase which hydrolyzes phosphoester bonds in proteins and other molecules. rPTPη and its human homologue DEP-1 are involved in neoplastic transformations. Thus, expression of the protein is reduced in all oncogene-transformed thyroid cell lines and is absent in highly malignant thyroid cells. Moreover, consistent with the suggested tumour suppression role of PTPη, inhibition of the tumorigenic process occurs after its exogenous reconstitution, suggesting that PTPη might be important for gene therapy of cancers. In this study, the catalytic domain of rPTPη was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals were obtained by the hanging-drop vapour-diffusion method. Diffraction data were collected to 1.87 Å resolution. The crystal belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 46.46, b = 63.07, c = 111.64 Å, and contains one molecule per asymmetric unit.

  8. Crystallization and preliminary X-ray diffraction analysis of rat protein tyrosine phosphatase η

    International Nuclear Information System (INIS)

    Matozo, Huita C.; Nascimento, Alessandro S.; Santos, Maria A. M.; Iuliano, Rodolfo; Fusco, Alfredo; Polikarpov, Igor

    2006-01-01

    In this study, the catalytic domain of rat protein tyrosine phosphatase η was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals were obtained by the hanging-drop vapour-diffusion method. The rat protein tyrosine phosphatase η (rPTPη) is a cysteine-dependent phosphatase which hydrolyzes phosphoester bonds in proteins and other molecules. rPTPη and its human homologue DEP-1 are involved in neoplastic transformations. Thus, expression of the protein is reduced in all oncogene-transformed thyroid cell lines and is absent in highly malignant thyroid cells. Moreover, consistent with the suggested tumour suppression role of PTPη, inhibition of the tumorigenic process occurs after its exogenous reconstitution, suggesting that PTPη might be important for gene therapy of cancers. In this study, the catalytic domain of rPTPη was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals were obtained by the hanging-drop vapour-diffusion method. Diffraction data were collected to 1.87 Å resolution. The crystal belongs to space group P2 1 2 1 2 1 , with unit-cell parameters a = 46.46, b = 63.07, c = 111.64 Å, and contains one molecule per asymmetric unit

  9. Identification and analysis of OsttaDSP, a phosphoglucan phosphatase from Ostreococcus tauri.

    Directory of Open Access Journals (Sweden)

    Julieta B Carrillo

    Full Text Available Ostreococcus tauri, the smallest free-living (non-symbiotic eukaryote yet described, is a unicellular green alga of the Prasinophyceae family. It has a very simple cellular organization and presents a unique starch granule and chloroplast. However, its starch metabolism exhibits a complexity comparable to higher plants, with multiple enzyme forms for each metabolic reaction. Glucan phosphatases, a family of enzymes functionally conserved in animals and plants, are essential for normal starch or glycogen degradation in plants and mammals, respectively. Despite the importance of O. tauri microalgae in evolution, there is no information available concerning the enzymes involved in reversible phosphorylation of starch. Here, we report the molecular cloning and heterologous expression of the gene coding for a dual specific phosphatase from O. tauri (OsttaDSP, homologous to Arabidopsis thaliana LSF2. The recombinant enzyme was purified to electrophoretic homogeneity to characterize its oligomeric and kinetic properties accurately. OsttaDSP is a homodimer of 54.5 kDa that binds and dephosphorylates amylopectin. Also, we also determined that residue C162 is involved in catalysis and possibly also in structural stability of the enzyme. Our results could contribute to better understand the role of glucan phosphatases in the metabolism of starch in green algae.

  10. Detection of phosphatase activity in aquatic and terrestrial cyanobacterial strains

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    Babić Olivera B.

    2013-01-01

    Full Text Available Cyanobacteria, as highly adaptable microorganisms, are characterized by an ability to survive in different environmental conditions, in which a significant role belongs to their enzymes. Phosphatases are enzymes produced by algae in relatively large quantities in response to a low orthophosphate concentration and their activity is significantly correlated with their primary production. The activity of these enzymes was investigated in 11 cyanobacterial strains in order to determine enzyme synthesis depending on taxonomic and ecological group of cyanobacteria. The study was conducted with 4 terrestrial cyanobacterial strains, which belong to Nostoc and Anabaena genera, and 7 filamentous water cyanobacteria of Nostoc, Oscillatoria, Phormidium and Microcystis genera. The obtained results showed that the activity of acid and alkaline phosphatases strongly depended on cyanobacterial strain and the environment from which the strain originated. Higher activity of alkaline phosphatases, ranging from 3.64 to 85.14 μmolpNP/s/dm3, was recorded in terrestrial strains compared to the studied water strains (1.11-5.96 μmolpNP/s/dm3. The activity of acid phosphatases was higher in most tested water strains (1.67-6.28 μmolpNP/s/dm3 compared to the activity of alkaline phosphatases (1.11-5.96 μmolpNP/s/dm3. Comparing enzyme activity of nitrogen fixing and non-nitrogen fixing cyanobacteria, it was found that most nitrogen fixing strains had a higher activity of alkaline phosphatases. The data obtained in this work indicate that activity of phosphatases is a strain specific property. The results further suggest that synthesis and activity of phosphatases depended on eco-physiological characteristics of the examined cyanobacterial strains. This can be of great importance for the further study of enzymes and mechanisms of their activity as a part of cyanobacterial survival strategy in environments with extreme conditions. [Projekat Ministarstva nauke Republike

  11. Effect of vanadium compounds on acid phosphatase activity

    OpenAIRE

    Vescina, Cecilia M.; Sálice, Viviana C.; Cortizo, Ana María; Etcheverry, Susana B.

    1996-01-01

    The direct effect of different vanadium compounds on acid phosphatase (ACP) activity was investigated. Vanadate and vanadyl but not pervanadate inhibited the wheat germ ACP activity. These vanadium derivatives did not alter the fibroblast Swiss 3T3 soluble fraction ACP activity. Using inhibitors of tyrosine phosphatases (PTPases), the wheat germ ACP was partially characterized as a PTPase. This study suggests that the inhibitory ability of different vanadium derivatives to modulate ACP activi...

  12. Dual-specificity phosphatase 3 deficiency or inhibition limits platelet activation and arterial thrombosis.

    Science.gov (United States)

    Musumeci, Lucia; Kuijpers, Marijke J; Gilio, Karen; Hego, Alexandre; Théâtre, Emilie; Maurissen, Lisbeth; Vandereyken, Maud; Diogo, Catia V; Lecut, Christelle; Guilmain, William; Bobkova, Ekaterina V; Eble, Johannes A; Dahl, Russell; Drion, Pierre; Rascon, Justin; Mostofi, Yalda; Yuan, Hongbin; Sergienko, Eduard; Chung, Thomas D Y; Thiry, Marc; Senis, Yotis; Moutschen, Michel; Mustelin, Tomas; Lancellotti, Patrizio; Heemskerk, Johan W M; Tautz, Lutz; Oury, Cécile; Rahmouni, Souad

    2015-02-17

    A limitation of current antiplatelet therapies is their inability to separate thrombotic events from bleeding occurrences. A better understanding of the molecular mechanisms leading to platelet activation is important for the development of improved therapies. Recently, protein tyrosine phosphatases have emerged as critical regulators of platelet function. This is the first report implicating the dual-specificity phosphatase 3 (DUSP3) in platelet signaling and thrombosis. This phosphatase is highly expressed in human and mouse platelets. Platelets from DUSP3-deficient mice displayed a selective impairment of aggregation and granule secretion mediated by the collagen receptor glycoprotein VI and the C-type lectin-like receptor 2. DUSP3-deficient mice were more resistant to collagen- and epinephrine-induced thromboembolism compared with wild-type mice and showed severely impaired thrombus formation on ferric chloride-induced carotid artery injury. Intriguingly, bleeding times were not altered in DUSP3-deficient mice. At the molecular level, DUSP3 deficiency impaired Syk tyrosine phosphorylation, subsequently reducing phosphorylation of phospholipase Cγ2 and calcium fluxes. To investigate DUSP3 function in human platelets, a novel small-molecule inhibitor of DUSP3 was developed. This compound specifically inhibited collagen- and C-type lectin-like receptor 2-induced human platelet aggregation, thereby phenocopying the effect of DUSP3 deficiency in murine cells. DUSP3 plays a selective and essential role in collagen- and C-type lectin-like receptor 2-mediated platelet activation and thrombus formation in vivo. Inhibition of DUSP3 may prove therapeutic for arterial thrombosis. This is the first time a protein tyrosine phosphatase, implicated in platelet signaling, has been targeted with a small-molecule drug. © 2014 American Heart Association, Inc.

  13. Knockout mice reveal a role for protein tyrosine phosphatase H1 in cognition

    Directory of Open Access Journals (Sweden)

    Ardizzone Michele

    2008-08-01

    Full Text Available Abstract Background The present study has investigated the protein tyrosine phosphatase H1 (PTPH1 expression pattern in mouse brain and its impact on CNS functions. Methods We have previously described a PTPH1-KO mouse, generated by replacing the PTP catalytic and the PDZ domain with a LacZ neomycin cassette. PTPH1 expression pattern was evaluated by LacZ staining in the brain and PTPH1-KO and WT mice (n = 10 per gender per genotype were also behaviorally tested for CNS functions. Results In CNS, PTPH1 is expressed during development and in adulthood and mainly localized in hippocampus, thalamus, cortex and cerebellum neurons. The behavioral tests performed on the PTPH1-KO mice showed an impact on working memory in male mice and an impaired learning performance at rotarod in females. Conclusion These results demonstrate for the first time a neuronal expression of PTPH1 and its functionality at the level of cognition.

  14. Interaction of Uranium with Bacterial Cell Surfaces: Inferences from Phosphatase-Mediated Uranium Precipitation

    Science.gov (United States)

    Kulkarni, Sayali; Misra, Chitra Seetharam; Gupta, Alka; Ballal, Anand

    2016-01-01

    ABSTRACT Deinococcus radiodurans and Escherichia coli expressing either PhoN, a periplasmic acid phosphatase, or PhoK, an extracellular alkaline phosphatase, were evaluated for uranium (U) bioprecipitation under two specific geochemical conditions (GCs): (i) a carbonate-deficient condition at near-neutral pH (GC1), and (ii) a carbonate-abundant condition at alkaline pH (GC2). Transmission electron microscopy revealed that recombinant cells expressing PhoN/PhoK formed cell-associated uranyl phosphate precipitate under GC1, whereas the same cells displayed extracellular precipitation under GC2. These results implied that the cell-bound or extracellular location of the precipitate was governed by the uranyl species prevalent at that particular GC, rather than the location of phosphatase. MINTEQ modeling predicted the formation of predominantly positively charged uranium hydroxide ions under GC1 and negatively charged uranyl carbonate-hydroxide complexes under GC2. Both microbes adsorbed 6- to 10-fold more U under GC1 than under GC2, suggesting that higher biosorption of U to the bacterial cell surface under GC1 may lead to cell-associated U precipitation. In contrast, at alkaline pH and in the presence of excess carbonate under GC2, poor biosorption of negatively charged uranyl carbonate complexes on the cell surface might have resulted in extracellular precipitation. The toxicity of U observed under GC1 being higher than that under GC2 could also be attributed to the preferential adsorption of U on cell surfaces under GC1. This work provides a vivid description of the interaction of U complexes with bacterial cells. The findings have implications for the toxicity of various U species and for developing biological aqueous effluent waste treatment strategies. IMPORTANCE The present study provides illustrative insights into the interaction of uranium (U) complexes with recombinant bacterial cells overexpressing phosphatases. This work demonstrates the effects of aqueous

  15. Role of tyrosine phosphatase inhibitors in cancer treatment with emphasis on SH2 domain-containing tyrosine phosphatases (SHPs)

    NARCIS (Netherlands)

    Irandoust, Mahban; van den Berg, Timo K.; Kaspers, Gertjan J. L.; Cloos, Jacqueline

    2009-01-01

    Protein tyrosine phosphorylation is one of the key mechanisms involved in signal transduction pathways. This modification is regulated by concerted action of protein tyrosine phosphatases and protein tyrosine kinases. Deregulation of either of these key regulators lead to abnormal cellular

  16. Target of rapamycin complex 1 and Tap42-associated phosphatases are required for sensing changes in nitrogen conditions in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Li, Jinmei; Yan, Gonghong; Liu, Sichi; Jiang, Tong; Zhong, Mingming; Yuan, Wenjie; Chen, Shaoxian; Zheng, Yin; Jiang, Yong; Jiang, Yu

    2017-12-01

    In yeast target of rapamycin complex 1 (TORC1) and Tap42-associated phosphatases regulate expression of genes involved in nitrogen limitation response and the nitrogen discrimination pathway. However, it remains unclear whether TORC1 and the phosphatases are required for sensing nitrogen conditions. Utilizing temperature sensitive mutants of tor2 and tap42, we examined the role of TORC1 and Tap42 in nuclear entry of Gln3, a key transcription factor in yeast nitrogen metabolism, in response to changes in nitrogen conditions. Our data show that TORC1 is essential for Gln3 nuclear entry upon nitrogen limitation and downshift in nitrogen quality. However, Tap42-associated phosphatases are required only under nitrogen limitation condition. In cells grown in poor nitrogen medium, the nitrogen permease reactivator kinase (Npr1) inhibits TORC1 activity and alters its association with Tap42, rendering Tap42-associated phosphatases unresponsive to nitrogen limitation. These findings demonstrate a direct role for TORC1 and Tap42-associated phosphatases in sensing nitrogen conditions and unveil an Npr1-dependent mechanism that controls TORC1 and the phosphatases in response to changes in nitrogen quality. © 2017 John Wiley & Sons Ltd.

  17. Serum alkaline phosphatase screening for vitamin D deficiency states

    International Nuclear Information System (INIS)

    Shaheen, S.; Barrakzai, Q.

    2012-01-01

    Objective: To determine whether serum vitamin D levels are correlated with serum levels of alkaline phosphatase or not. Study Design: Cross-sectional, observational study. Place and Duration of Study: Multi-centre study, conducted at Liaquat National Hospital and Medical College, National Medical Centre and Medicare Hospital, Karachi, from January to October 2009. Methodology: Patients attending the Orthopaedic OPDs with complaints of pain in different body regions and serum vitamin D/sub 3/ levels of greater or equal to 30 ng/ml were included in the study. Patients with vitamin D deficiency were further categorized into mild deficiency or insufficiency (vit. D/sub 3/ = 20-29 ng/ml), moderate deficiency (vit. D/sub 3/ = 5 - 19 ng/ml) and severe deficiency forms (vit. D/sub 3/ < 5 ng/ml). Pearson correlation was applied to test the correlation of serum alkaline phosphatase levels with serum vitamin D/sub 3/ levels. P-value < 0.05 was considered to be significant. Results: Out of 110 samples, 26 had mild (23%), 61 had moderate (55%) and 21 had severe (19.1%) vitamin D deficiencies. All of the patients in the three groups had alkaline phosphatase with in normal limits and the total mean value of the enzyme was 135.97 +- 68.14I U/L. The inter group comparison showed highest values of alkaline phosphatase in the moderate vitamin D deficiency group. The correlation coefficient of alkaline phosphatase and serum vitamin D/sub 3/ levels was r =0.05 (p =0.593). Conclusion: Serum vitamin D/sub 3/ levels may not be correlated with increased serum alkaline phosphatase levels. Therefore, alkaline phosphatase may not be used as a screening test to rule out vitamin D deficiency. (author)

  18. Molecular characterization and functional analysis of serine/threonine protein phosphatase of Toxocara canis.

    Science.gov (United States)

    Ma, Guang Xu; Zhou, Rong Qiong; Hu, Shi Jun; Huang, Han Cheng; Zhu, Tao; Xia, Qing You

    2014-06-01

    Toxocara canis (T. canis) is a widely prevalent zoonotic parasite that infects a wide range of mammalian hosts, including humans. We generated the full-length complementary DNA (cDNA) of the serine/threonine phosphatase gene of T. canis (Tc stp) using 5' rapid amplification of the cDNA ends. The 1192-bp sequence contained a continuous 942-nucleotide open reading frame, encoding a 313-amino-acid polypeptide. The Tc STP polypeptide shares a high level of amino-acid sequence identity with the predicted STPs of Loa loa (89%), Brugia malayi (86%), Oesophagostomum columbianum (76%), and Oesophagostomumdentatum (76%). The Tc STP contains GDXHG, GDXVDRG, GNHE motifs, which are characteristic of members of the phosphoprotein phosphatase family. Our quantitative real-time polymerase chain reaction analysis showed that the Tc STP was expressed in six different tissues in the adult male, with high-level expression in the spermary, vas deferens, and musculature, but was not expressed in the adult female, suggesting that Tc STP might be involved in spermatogenesis and mating behavior. Thus, STP might represent a potential molecular target for controlling T. canis reproduction. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Phosphatase of Regenerating Liver-3 Promotes Motility and Metastasis of Mouse Melanoma Cells

    Science.gov (United States)

    Wu, Xiaopeng; Zeng, Hu; Zhang, Xianming; Zhao, Ying; Sha, Haibo; Ge, Xiaomei; Zhang, Minyue; Gao, Xiang; Xu, Qiang

    2004-01-01

    Recent reports suggested that phosphatase of regenerating liver (PRL)-3 might be involved in colorectal carcinoma metastasis with an unknown mechanism. Here we demonstrated that PRL-3 expression was up-regulated in human liver carcinoma compared with normal liver. PRL-3 was also highly expressed in metastatic melanoma B16-BL6 cells but not in its lowly metastatic parental cell line, B16 cells. B16 cells transfected with PRL-3 cDNA displayed morphological transformation from epithelial-like shape to fibroblast-like shape. PRL-3-overexpressed cells showed much higher migratory ability, which could be reversed by specific anti-sense oligodeoxynucleotide and the phosphatase inhibitors sodium orthovanadate or potassium bisperoxo oxovanadate V. Meanwhile, the expression of the catalytically inactive PRL-3 mutations (D72A or C104S) significantly reduced the cell migratory capability. In addition, PRL-3 transfectants demonstrated altered extracellular matrix adhesive property and up-regulated integrin-mediated cell spreading efficiency. Furthermore, we confirmed that PRL-3 could facilitate lung and liver metastasis of B16 cells in an experimental metastasis model in mice, consistent with accelerated proliferation and growth rate both in vitro and in vivo. Together, these observations provide convincing evidence that PRL-3 truly plays a causal role in tumor metastasis. PMID:15161639

  20. A novel tetratricopeptide repeat (TPR containing PP5 serine/threonine protein phosphatase in the malaria parasite, Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Adams Brian

    2001-11-01

    Full Text Available Abstract Background The malarial parasite, Plasmodium falciparum (Pf, is responsible for nearly 2 million deaths worldwide. However, the mechanisms of cellular signaling in the parasite remain largely unknown. Recent discovery of a few protein kinases and phosphatases point to a thriving reversible phosphorylation system in the parasite, although their function and regulation need to be determined. Results We provide biochemical and sequence evidence for a protein serine/threonine phosphatase type PP5 in Plasmodium falciparum, and named it PfPP5. The 594-amino acid polypeptide was encoded by a 1785 nucleotide long intronless gene in the parasite. The recombinant protein, expressed in bacteria, was indistinguishable from native PfPP5. Sequencing comparison indicated that the extra-long N-terminus of PfPP5 outside the catalytic core contained four tetratricopeptide repeats (TPRs, compared to three such repeats in other PP5 phosphatases. The PfPP5 N-terminus was required for stimulation of the phosphatase activity by polyunsaturated fatty acids. Co-immunoprecipitation demonstrated an interaction between native PfPP5 and Pf heat shock protein 90 (hsp90. PfPP5 was expressed in all the asexual erythrocytic stages of the parasite, and was moderately sensitive to okadaic acid. Conclusions This is the first example of a TPR-domain protein in the Apicomplexa family of parasites. Since TPR domains play important roles in protein-protein interaction, especially relevant to the regulation of PP5 phosphatases, PfPP5 is destined to have a definitive role in parasitic growth and signaling pathways. This is exemplified by the interaction between PfPP5 and the cognate chaperone hsp90.

  1. Phosphorylcholine Phosphatase: A Peculiar Enzyme of Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Carlos Eduardo Domenech

    2011-01-01

    Full Text Available Pseudomonas aeruginosa synthesizes phosphorylcholine phosphatase (PchP when grown on choline, betaine, dimethylglycine or carnitine. In the presence of Mg2+ or Zn2+, PchP catalyzes the hydrolysis of p-nitrophenylphosphate (p-NPP or phosphorylcholine (Pcho. The regulation of pchP gene expression is under the control of GbdR and NtrC; dimethylglycine is likely the metabolite directly involved in the induction of PchP. Therefore, the regulation of choline metabolism and consequently PchP synthesis may reflect an adaptive response of P. aeruginosa to environmental conditions. Bioinformatic and biochemistry studies shown that PchP contains two sites for alkylammonium compounds (AACs: one in the catalytic site near the metal ion-phosphoester pocket, and another in an inhibitory site responsible for the binding of the alkylammonium moiety. Both sites could be close to each other and interact through the residues 42E, 43E and 82YYY84. Zn2+ is better activator than Mg2+ at pH 5.0 and it is more effective at alleviating the inhibition produced by the entry of Pcho or different AACs in the inhibitory site. We postulate that Zn2+ induces at pH 5.0 a conformational change in the active center that is communicated to the inhibitory site, producing a compact or closed structure. However, at pH 7.4, this effect is not observed because to the hydrolysis of the [Zn2+L2−1L20(H2O2] complex, which causes a change from octahedral to tetrahedral in the metal coordination geometry. This enzyme is also present in P. fluorescens, P. putida, P. syringae, and other organisms. We have recently crystallized PchP and solved its structure.

  2. Regulation of Brain-Derived Neurotrophic Factor and Growth Factor Signaling Pathways by Tyrosine Phosphatase Shp2 in the Retina: A Brief Review

    Directory of Open Access Journals (Sweden)

    Mojdeh Abbasi

    2018-03-01

    Full Text Available SH2 domain-containing tyrosine phosphatase-2 (PTPN11 or Shp2 is a ubiquitously expressed protein that plays a key regulatory role in cell proliferation, differentiation and growth factor (GF signaling. This enzyme is well expressed in various retinal neurons and has emerged as an important player in regulating survival signaling networks in the neuronal tissues. The non-receptor phosphatase can translocate to lipid rafts in the membrane and has been implicated to regulate several signaling modules including PI3K/Akt, JAK-STAT and Mitogen Activated Protein Kinase (MAPK pathways in a wide range of biochemical processes in healthy and diseased states. This review focuses on the roles of Shp2 phosphatase in regulating brain-derived neurotrophic factor (BDNF neurotrophin signaling pathways and discusses its cross-talk with various GF and downstream signaling pathways in the retina.

  3. Effects of parathyroid hormone and calcitonin on alkaline phosphatase activity and matrix calcification in rabbit growth-plate chondrocyte cultures

    Energy Technology Data Exchange (ETDEWEB)

    Kato, Y.; Shimazu, A.; Nakashima, K.; Suzuki, F.; Jikko, A.; Iwamoto, M. (Osaka Univ. (Japan))

    1990-07-01

    The effects of PTH and calcitonin (CT) on the expression of mineralization-related phenotypes by chondrocytes were examined. In cultures of pelleted growth-plate chondrocytes. PTH caused 60-90% decreases in alkaline phosphatase activity, the incorporation of {sup 45}Ca into insoluble material, and the calcium content during the post-mitotic stage. These effects of PTH were dose-dependent and reversible. In contrast, CT increased alkaline phosphatase activity, {sup 45}Ca incorporation into insoluble material, and the calcium content by 1.4- to 1.8-fold. These observations suggest that PTH directly inhibits the expression of the mineralization-related phenotypes by growth-plate chondrocytes, and that CT has the opposite effects.

  4. Cloning and Characterization of Purple Acid Phosphatase Phytases from Wheat, Barley, Maize and Rice

    DEFF Research Database (Denmark)

    Dionisio, Giuseppe; Madsen, Claus Krogh; Holm, Preben Bach

    2011-01-01

    development and germination. In wheat, it was demonstrated that a and b isogene expression is driven by different promoters (approximately 31% identity). TaPAPhy_a/b promoter reporter gene expression in transgenic grains and peptide mapping of TaPAPhy purified from wheat bran and germinating grains confirmed......Barley (Hordeum vulgare) and wheat (Triticum aestivum) possess significant phytase activity in the mature grains. Maize (Zea mays) and rice (Oryza sativa) possess little or virtually no preformed phytase activity in the mature grain and depend fully on de novo synthesis during germination. Here......, it is demonstrated that wheat, barley, maize, and rice all possess purple acid phosphatase (PAP) genes that, expressed in Pichia pastoris, give fully functional phytases (PAPhys) with very similar enzyme kinetics. Preformed wheat PAPhy was localized to the protein crystalloid of the aleurone vacuole. Phylogenetic...

  5. New procedures to measure synthase and phosphatase activities of bis-phosphoglycerate mutase. Interest for development of therapeutic drugs

    International Nuclear Information System (INIS)

    Ravel, P.; Garel, M.C.; Toullec, D.

    1997-01-01

    In red blood cells, a modulation of the level of the allosteric effector of hemoglobin, 2,3-diphosphoglycerate (2,3-DPG) would have implications in the treatment of ischemia and sickle cell anemia. Its concentrations is determined by the relative activities of the synthase and phosphatase reactions of the multifunctional bis-phosphoglycerate mutase (BPGM). In this report we develop first a more direct synthase assay which uses glyceraldehyde phosphate to suppress the aldolase and triose phosphate isomerase reactions. Secondly we propose a radioactive phosphatase assay coupled to chromatographic separation and identification of the reaction products by paper electrophoresis. Such identification of these products allows us to show that the multifunctional BPGM expresses its mutase instead of its phosphatase activity in conditions of competition between the 3-phosphoglycerate and the 2-phospho-glycolate activator in the phosphatase reaction. These two more precise procedures could be used to study the effects of substrate and cofactor analogues regarding potential therapeutic approaches and could be used for clinical analyses to detect deficiency of BPGM. (author)

  6. Acid phosphatase and lipid peroxidation in human cataractous lens epithelium

    Directory of Open Access Journals (Sweden)

    Vasavada Abhay

    1993-01-01

    Full Text Available The anterior lens epithelial cells undergo a variety of degenerative and proliferative changes during cataract formation. Acid phosphatase is primarily responsible for tissue regeneration and tissue repair. The lipid hydroperoxides that are obtained by lipid peroxidation of polysaturated or unsaturated fatty acids bring about deterioration of biological membranes at cellular and tissue levels. Acid phosphatase and lipid peroxidation activities were studied on the lens epithelial cells of nuclear cataract, posterior subcapsular cataract, mature cataract, and mixed cataract. Of these, mature cataractous lens epithelium showed maximum activity for acid phosphatase (516.83 moles of p-nitrophenol released/g lens epithelium and maximum levels of lipid peroxidation (86.29 O.D./min/g lens epithelium. In contrast, mixed cataractous lens epithelium showed minimum activity of acid phosphatase (222.61 moles of p-nitrophenol released/g lens epithelium and minimum levels of lipid peroxidation (54.23 O.D./min/g lens epithelium. From our study, we correlated the maximum activity of acid phosphatase in mature cataractous lens epithelium with the increased areas of superimposed cells associated with the formation of mature cataract. Likewise, the maximum levels of lipid peroxidation in mature cataractous lens epithelium was correlated with increased permeability of the plasma membrane. Conversely, the minimum levels of lipid peroxidation in mixed cataractous lens epithelium makes us presume that factors other than lipid peroxidation may also account for the formation of mixed type of cataract.

  7. Elevated Serum Level of Human Alkaline Phosphatase in Obesity

    International Nuclear Information System (INIS)

    Khan, A. R.; Awan, F. R.; Najam, S. S.; Islam, M.; Siddique, T.; Zain, M.

    2015-01-01

    Objective: To investigate a correlation between serum alkaline phosphatase level and body mass index in human subjects. Methods: The comparative cross-sectional study was carried out at the National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan, from April 2012 to June 2013. Blood serum alkaline phosphatase levels were estimated and the subjects were divided into three sub-groups on the basis of their body mass index: normal weight (<25kg/m2), overweight (25-27kg/m2) and obese (>27kg/m2) subjects. The serum samples were used for the estimation of clinically important biochemical parameters, using commercial kits on clinical chemistry analyser. Results: Of the 197 subjects, 97(49 percent) were obese and 100(51 percent) were non-obese. The serum alkaline phosphatase level increased in obese (214±6.4 IU/L) compared to the non-obese subjects (184.5±5 IU/L). Furthermore, a significant linear relationship (r=0.3;p-0.0001) was found between serum alkaline phosphatase and body mass index. Other biochemical variables were not correlated to the body mass index. Conclusion: Over activity and higher amounts of alkaline phosphatase were linked to the development of obesity. (author)

  8. Domain-to-domain coupling in voltage-sensing phosphatase.

    Science.gov (United States)

    Sakata, Souhei; Matsuda, Makoto; Kawanabe, Akira; Okamura, Yasushi

    2017-01-01

    Voltage-sensing phosphatase (VSP) consists of a transmembrane voltage sensor and a cytoplasmic enzyme region. The enzyme region contains the phosphatase and C2 domains, is structurally similar to the tumor suppressor phosphatase PTEN, and catalyzes the dephosphorylation of phosphoinositides. The transmembrane voltage sensor is connected to the phosphatase through a short linker region, and phosphatase activity is induced upon membrane depolarization. Although the detailed molecular characteristics of the voltage sensor domain and the enzyme region have been revealed, little is known how these two regions are coupled. In addition, it is important to know whether mechanism for coupling between the voltage sensor domain and downstream effector function is shared among other voltage sensor domain-containing proteins. Recent studies in which specific amino acid sites were genetically labeled using a fluorescent unnatural amino acid have enabled detection of the local structural changes in the cytoplasmic region of Ciona intestinalis VSP that occur with a change in membrane potential. The results of those studies provide novel insight into how the enzyme activity of the cytoplasmic region of VSP is regulated by the voltage sensor domain.

  9. TCTEX1D4, a novel protein phosphatase 1 interactor: connecting the phosphatase to the microtubule network

    Science.gov (United States)

    Korrodi-Gregório, Luís; Vieira, Sandra I.; Esteves, Sara L. C.; Silva, Joana V.; Freitas, Maria João; Brauns, Ann-Kristin; Luers, Georg; Abrantes, Joana; Esteves, Pedro J.; da Cruz e Silva, Odete A. B.; Fardilha, Margarida; da Cruz e Silva, Edgar F.

    2013-01-01

    Summary Reversible phosphorylation plays an important role as a mechanism of intracellular control in eukaryotes. PPP1, a major eukaryotic Ser/Thr-protein phosphatase, acquires its specificity by interacting with different protein regulators, also known as PPP1 interacting proteins (PIPs). In the present work we characterized a physiologically relevant PIP in testis. Using a yeast two-hybrid screen with a human testis cDNA library, we identified a novel PIP of PPP1CC2 isoform, the T-complex testis expressed protein 1 domain containing 4 (TCTEX1D4) that has recently been described as a Tctex1 dynein light chain family member. The overlay assays confirm that TCTEX1D4 interacts with the different spliced isoforms of PPP1CC. Also, the binding domain occurs in the N-terminus, where a consensus PPP1 binding motif (PPP1BM) RVSF is present. The distribution of TCTEX1D4 in testis suggests its involvement in distinct functions, such as TGFβ signaling at the blood–testis barrier and acrosome cap formation. Immunofluorescence in human ejaculated sperm shows that TCTEX1D4 is present in the flagellum and in the acrosome region of the head. Moreover, TCTEX1D4 and PPP1 co-localize in the microtubule organizing center (MTOC) and microtubules in cell cultures. Importantly, the TCTEX1D4 PPP1BM seems to be relevant for complex formation, for PPP1 retention in the MTOC and movement along microtubules. These novel results open new avenues to possible roles of this dynein, together with PPP1. In essence TCTEX1D4/PPP1C complex appears to be involved in microtubule dynamics, sperm motility, acrosome reaction and in the regulation of the blood–testis barrier. PMID:23789093

  10. MAPK phosphatase AP2C3 induces ectopic proliferation of epidermal cells leading to stomata development in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Julija Umbrasaite

    2010-12-01

    Full Text Available In plant post-embryonic epidermis mitogen-activated protein kinase (MAPK signaling promotes differentiation of pavement cells and inhibits initiation of stomata. Stomata are cells specialized to modulate gas exchange and water loss. Arabidopsis MAPKs MPK3 and MPK6 are at the core of the signaling cascade; however, it is not well understood how the activity of these pleiotropic MAPKs is constrained spatially so that pavement cell differentiation is promoted only outside the stomata lineage. Here we identified a PP2C-type phosphatase termed AP2C3 (Arabidopsis protein phosphatase 2C that is expressed distinctively during stomata development as well as interacts and inactivates MPK3, MPK4 and MPK6. AP2C3 co-localizes with MAPKs within the nucleus and this localization depends on its N-terminal extension. We show that other closely related phosphatases AP2C2 and AP2C4 are also MAPK phosphatases acting on MPK6, but have a distinct expression pattern from AP2C3. In accordance with this, only AP2C3 ectopic expression is able to stimulate cell proliferation leading to excess stomata development. This function of AP2C3 relies on the domains required for MAPK docking and intracellular localization. Concomitantly, the constitutive and inducible AP2C3 expression deregulates E2F-RB pathway, promotes the abundance and activity of CDKA, as well as changes of CDKB1;1 forms. We suggest that AP2C3 downregulates the MAPK signaling activity to help maintain the balance between differentiation of stomata and pavement cells.

  11. A Global Protein Kinase and Phosphatase Interaction Network in Yeast

    Science.gov (United States)

    Breitkreutz, Ashton; Choi, Hyungwon; Sharom, Jeffrey R.; Boucher, Lorrie; Neduva, Victor; Larsen, Brett; Lin, Zhen-Yuan; Breitkreutz, Bobby-Joe; Stark, Chris; Liu, Guomin; Ahn, Jessica; Dewar-Darch, Danielle; Reguly, Teresa; Tang, Xiaojing; Almeida, Ricardo; Qin, Zhaohui Steve; Pawson, Tony; Gingras, Anne-Claude; Nesvizhskii, Alexey I.; Tyers, Mike

    2011-01-01

    The interactions of protein kinases and phosphatases with their regulatory subunits and substrates underpin cellular regulation. We identified a kinase and phosphatase interaction (KPI) network of 1844 interactions in budding yeast by mass spectrometric analysis of protein complexes. The KPI network contained many dense local regions of interactions that suggested new functions. Notably, the cell cycle phosphatase Cdc14 associated with multiple kinases that revealed roles for Cdc14 in mitogen-activated protein kinase signaling, the DNA damage response, and metabolism, whereas interactions of the target of rapamycin complex 1 (TORC1) uncovered new effector kinases in nitrogen and carbon metabolism. An extensive backbone of kinase-kinase interactions cross-connects the proteome and may serve to coordinate diverse cellular responses. PMID:20489023

  12. cAMP response element binding protein (CREB activates transcription via two distinct genetic elements of the human glucose-6-phosphatase gene

    Directory of Open Access Journals (Sweden)

    Stefano Luisa

    2005-01-01

    Full Text Available Abstract Background The enzyme glucose-6-phosphatase catalyzes the dephosphorylation of glucose-6-phosphatase to glucose, the final step in the gluconeogenic and glycogenolytic pathways. Expression of the glucose-6-phosphatase gene is induced by glucocorticoids and elevated levels of intracellular cAMP. The effect of cAMP in regulating glucose-6-phosphatase gene transcription was corroborated by the identification of two genetic motifs CRE1 and CRE2 in the human and murine glucose-6-phosphatase gene promoter that resemble cAMP response elements (CRE. Results The cAMP response element is a point of convergence for many extracellular and intracellular signals, including cAMP, calcium, and neurotrophins. The major CRE binding protein CREB, a member of the basic region leucine zipper (bZIP family of transcription factors, requires phosphorylation to become a biologically active transcriptional activator. Since unphosphorylated CREB is transcriptionally silent simple overexpression studies cannot be performed to test the biological role of CRE-like sequences of the glucose-6-phosphatase gene. The use of a constitutively active CREB2/CREB fusion protein allowed us to uncouple the investigation of target genes of CREB from the variety of signaling pathways that lead to an activation of CREB. Here, we show that this constitutively active CREB2/CREB fusion protein strikingly enhanced reporter gene transcription mediated by either CRE1 or CRE2 derived from the glucose-6-phosphatase gene. Likewise, reporter gene transcription was enhanced following expression of the catalytic subunit of cAMP-dependent protein kinase (PKA in the nucleus of transfected cells. In contrast, activating transcription factor 2 (ATF2, known to compete with CREB for binding to the canonical CRE sequence 5'-TGACGTCA-3', did not transactivate reporter genes containing CRE1, CRE2, or both CREs derived from the glucose-6-phosphatase gene. Conclusions Using a constitutively active CREB2

  13. Effect of vanadium compounds on acid phosphatase activity.

    Science.gov (United States)

    Vescina, C M; Sálice, V C; Cortizo, A M; Etcheverry, S B

    1996-01-01

    The direct effect of different vanadium compounds on acid phosphatase (ACP) activity was investigated. Vanadate and vanadyl but not pervanadate inhibited the wheat germ ACP activity. These vanadium derivatives did not alter the fibroblast Swiss 3T3 soluble fraction ACP activity. Using inhibitors of tyrosine phosphatases (PTPases), the wheat germ ACP was partially characterized as a PTPase. This study suggests that the inhibitory ability of different vanadium derivatives to modulate ACP activity seems to depend on the geometry around the vanadium atom more than on the oxidation state. Our results indicate a correlation between the PTPase activity and the sensitivity to vanadate and vanadyl cation.

  14. Phosphotyrosine as a substrate of acid and alkaline phosphatases.

    Science.gov (United States)

    Apostoł, I; Kuciel, R; Wasylewska, E; Ostrowski, W S

    1985-01-01

    A new spectrophotometric method for following dephosphorylation of phosphotyrosine has been described. The absorption spectra of phosphotyrosine and tyrosine were plotted over the pH range from 3 to 9. The change in absorbance accompanying the conversion of phosphotyrosine to tyrosine was the greatest at 286 nm. The difference absorption coefficients were calculated for several pH values. Dephosphorylation of phosphotyrosine by acid phosphatases from human prostate gland, from wheat germ and potatoes obeys the Michaelis-Menten equation, whereas alkaline phosphatases calf intestine and E. coli are inhibited by excess of substrate.

  15. A Dictyostelium secreted factor requires a PTEN-like phosphatase to slow proliferation and induce chemorepulsion.

    Directory of Open Access Journals (Sweden)

    Sarah E Herlihy

    Full Text Available In Dictyostelium discoideum, AprA and CfaD are secreted proteins that inhibit cell proliferation. We found that the proliferation of cells lacking CnrN, a phosphatase and tensin homolog (PTEN-like phosphatase, is not inhibited by exogenous AprA and is increased by exogenous CfaD. The expression of CnrN in cnrN cells partially rescues these altered sensitivities, suggesting that CnrN is necessary for the ability of AprA and CfaD to inhibit proliferation. Cells lacking CnrN accumulate normal levels of AprA and CfaD. Like cells lacking AprA and CfaD, cnrN cells proliferate faster and reach a higher maximum cell density than wild type cells, tend to be multinucleate, accumulate normal levels of mass and protein per nucleus, and form less viable spores. When cnrN cells expressing myc-tagged CnrN are stimulated with a mixture of rAprA and rCfaD, levels of membrane-associated myc-CnrN increase. AprA also causes chemorepulsion of Dictyostelium cells, and CnrN is required for this process. Combined, these results suggest that CnrN functions in a signal transduction pathway downstream of AprA and CfaD mediating some, but not all, of the effects of AprA and CfaD.

  16. A Dictyostelium secreted factor requires a PTEN-like phosphatase to slow proliferation and induce chemorepulsion.

    Science.gov (United States)

    Herlihy, Sarah E; Tang, Yitai; Gomer, Richard H

    2013-01-01

    In Dictyostelium discoideum, AprA and CfaD are secreted proteins that inhibit cell proliferation. We found that the proliferation of cells lacking CnrN, a phosphatase and tensin homolog (PTEN)-like phosphatase, is not inhibited by exogenous AprA and is increased by exogenous CfaD. The expression of CnrN in cnrN cells partially rescues these altered sensitivities, suggesting that CnrN is necessary for the ability of AprA and CfaD to inhibit proliferation. Cells lacking CnrN accumulate normal levels of AprA and CfaD. Like cells lacking AprA and CfaD, cnrN cells proliferate faster and reach a higher maximum cell density than wild type cells, tend to be multinucleate, accumulate normal levels of mass and protein per nucleus, and form less viable spores. When cnrN cells expressing myc-tagged CnrN are stimulated with a mixture of rAprA and rCfaD, levels of membrane-associated myc-CnrN increase. AprA also causes chemorepulsion of Dictyostelium cells, and CnrN is required for this process. Combined, these results suggest that CnrN functions in a signal transduction pathway downstream of AprA and CfaD mediating some, but not all, of the effects of AprA and CfaD.

  17. Fabrication of hydrogels with elasticity changed by alkaline phosphatase for stem cell culture.

    Science.gov (United States)

    Toda, Hiroyuki; Yamamoto, Masaya; Uyama, Hiroshi; Tabata, Yasuhiko

    2016-01-01

    The objective of this study is to design hydrogels whose elasticity can be changed by alkaline phosphatase (ALP) in cell culture and evaluate the effect of hydrogel elasticity on an osteogenic gene expression of cells. Hydrogels were prepared by the radical polymerization of acrylamide (AAm), N,N'-methylenebisacrylamide (BIS), and Phosmer™M containing phosphate groups (PE-PAAm hydrogels). The storage modulus of PE-PAAm hydrogels prepared was changed by the preparation conditions. When human mesenchymal stem cells (hMSC) were cultured on the ALP-responsive PE-PAAm hydrogels in the presence or absence of ALP, the morphology of hMSC was observed and one of the osteogenic differentiation markers, Runx2, was evaluated. By ALP addition into the culture medium, the morphology of hMSC was changed into an elongated shape without cell damage. ALP addition modified the level of Runx2 gene expression, which was influenced by the modulus of PE-PAAm hydrogels. It is concluded that the elasticity change of hydrogel substrates in cell culture had an influence on the Runx2 gene expression of hMSC. Stem cells sense the surface elasticity of culture substrates, and their differentiation fate is biologically modified by substrate properties. Most of experiments have been performed in static conditions during cell culture, while the in vivo microenvironment is dynamically changed. In this study, we established to design an enzyme-responsive hydrogel whose elasticity can be changed by alkaline phosphatase (ALP) in cell culture to mimic in vivo conditions. As a result, the cells were deformed and the gene expression level of an osteogenic maker, Runx2, was modified by ALP treatment. This is the novel report describing to demonstrate that the dynamic alteration of hydrogel substrate elasticity could modulate the osteoblastic gene expression of human MSC in vitro. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  18. Inhibition of DNA methyltransferase induces G2 cell cycle arrest and apoptosis in human colorectal cancer cells via inhibition of JAK2/STAT3/STAT5 signalling.

    Science.gov (United States)

    Xiong, Hua; Chen, Zhao-Fei; Liang, Qin-Chuan; Du, Wan; Chen, Hui-Min; Su, Wen-Yu; Chen, Guo-Qiang; Han, Ze-Guang; Fang, Jing-Yuan

    2009-09-01

    DNA methyltransferase inhibitors (MTIs) have recently emerged as promising chemotherapeutic or preventive agents for cancer, despite their poorly characterized mechanisms of action. The present study shows that DNA methylation is integral to the regulation of SH2-containing protein tyrosine phosphatase 1 (SHP1) expression, but not for regulation of suppressors of cytokine signalling (SOCS)1 or SOCS3 in colorectal cancer (CRC) cells. SHP1 expression correlates with down-regulation of Janus kinase/signal transducers and activators of transcription (JAK2/STAT3/STAT5) signalling, which is mediated in part by tyrosine dephosphorylation events and modulation of the proteasome pathway. Up-regulation of SHP1 expression was achieved using a DNA MTI, 5-aza-2'-deoxycytidine (5-aza-dc), which also generated significant down-regulation of JAK2/STAT3/STAT5 signalling. We demonstrate that 5-aza-dc suppresses growth of CRC cells, and induces G2 cell cycle arrest and apoptosis through regulation of downstream targets of JAK2/STAT3/STAT5 signalling including Bcl-2, p16(ink4a), p21(waf1/cip1) and p27(kip1). Although 5-aza-dc did not significantly inhibit cell invasion, 5-aza-dc did down-regulate expression of focal adhesion kinase and vascular endothelial growth factor in CRC cells. Our results demonstrate that 5-aza-dc can induce SHP1 expression and inhibit JAK2/STAT3/STAT5 signalling. This study represents the first evidence towards establishing a mechanistic link between inhibition of JAK2/STAT3/STAT5 signalling and the anticancer action of 5-aza-dc in CRC cells that may lead to the use of MTIs as a therapeutic intervention for human colorectal cancer.

  19. Phosphatase and tensin homologue deleted on chromosome 10 ...

    African Journals Online (AJOL)

    Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a tumor suppressor gene deleted or mutated in many human cancers such as glioblastoma, spinal tumors, prostate, bladder, adrenals, thyroid, breast, endometrium, and colon cancers. They result from loss of heterozygosity (LOH) for the PTEN ...

  20. Phosphatases as an index of biotic contamination of dust

    NARCIS (Netherlands)

    Kniest, F.M.; Bronswijk, van J.E.M.H.; Schober, G.; Bouma, C.

    1990-01-01

    Enzymatic (phosphatase) activity (naphthol-release made visible with diazonium salt) of 10 mattress dust samples was correlated with number of counted arthropods, fungal spores and bacteria. This method can be helpful in the evaluation of large number of dust samples e.g. from riskful areas or from

  1. Biocatalysis with Sol-Gel Encapsulated Acid Phosphatase

    Science.gov (United States)

    Kulkarni, Suhasini; Tran, Vu; Ho, Maggie K.-M.; Phan, Chieu; Chin, Elizabeth; Wemmer, Zeke; Sommerhalter, Monika

    2010-01-01

    This experiment was performed in an upper-level undergraduate biochemistry laboratory course. Students learned how to immobilize an enzyme in a sol-gel matrix and how to perform and evaluate enzyme-activity measurements. The enzyme acid phosphatase (APase) from wheat germ was encapsulated in sol-gel beads that were prepared from the precursor…

  2. Anxious moments for the protein tyrosine phosphatase PTP1B

    OpenAIRE

    Krishnan, Navasona; Tonks, Nicholas K.

    2015-01-01

    Chronic stress can lead to the development of anxiety and mood disorders. Thus, novel therapies for preventing adverse effects of stress are vitally important. Recently, the protein tyrosine phosphatase PTP1B was identified as a novel regulator of stress-induced anxiety. This opens up exciting opportunities to exploit PTP1B inhibitors as anxiolytics.

  3. WIP1 phosphatase as pharmacological target in cancer therapy

    Czech Academy of Sciences Publication Activity Database

    Pecháčková, Soňa; Burdová, Kamila; Macůrek, Libor

    2017-01-01

    Roč. 95, č. 6 (2017), s. 589-599 ISSN 0946-2716 R&D Projects: GA MŠk 7F14061; GA MŠk LO1220 Institutional support: RVO:68378050 Keywords : Cancer * Phosphatase * Checkpoint * DNA damage response * Inhibitor * p53 Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Medicinal chemistry Impact factor: 4.686, year: 2016

  4. Defining carbohydrate binding of glucan phosphatases via Affinity gel electrophoresis

    DEFF Research Database (Denmark)

    Auger, Kyle; Raththagala, Madushi; Wilkens, Casper

    2016-01-01

    was to determine a technique to measure carbohydrate binding quickly and efficiently. We established a protocol to reproducibly and quantitatively measure the binding of the enzymes to glucans utilizing Affinity Gel Electrophoresis (AGE). The results show that the various glucan phosphatases possess differing...

  5. Dephosphorylation of endotoxin by alkaline phosphatase in vivo

    NARCIS (Netherlands)

    Poelstra, Klaas; Bakker, W.W; Klok, P.A; Kamps, J.AAM; Hardonk, M.J; Meijer, D.K F

    1997-01-01

    Natural substrates for alkaline phosphatase (AP) are at present not identified despite extensive investigations. Difficulties in imagining a possible physiological function involve its extremely high pH optimum for the usual exogenous substrates and its localization as an ecto-enzyme. As endotoxin

  6. Targeted deletion of kidney glucose-6 phosphatase leads to nephropathy

    NARCIS (Netherlands)

    Clar, Julie; Gri, Blandine; Calderaro, Julien; Birling, Marie-Christine; Herault, Yann; Smit, G. Peter A.; Mithieux, Gilles; Rajas, Fabienne

    2014-01-01

    Renal failure is a major complication that arises with aging in glycogen storage disease type 1a and type 1b patients. In the kidneys, glucose-6 phosphatase catalytic subunit (encoded by G6pc) deficiency leads to the accumulation of glycogen, an effect resulting in marked nephromegaly and

  7. Impact of ionic aluminium on extracellular phosphatases in acidified lakes

    Czech Academy of Sciences Publication Activity Database

    Bittl, T.; Vrba, Jaroslav; Nedoma, Jiří; Kopáček, Jiří

    2001-01-01

    Roč. 3, č. 9 (2001), s. 578-587 ISSN 1462-2912 R&D Projects: GA ČR GA206/97/0072; GA ČR GA206/00/0063 Keywords : acid phosphatases * pH effect * inhibition Subject RIV: DA - Hydrology ; Limnology Impact factor: 3.276, year: 2001

  8. 21 CFR 864.7660 - Leukocyte alkaline phosphatase test.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Leukocyte alkaline phosphatase test. 864.7660 Section 864.7660 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7660...

  9. In vitro production of growth regulators and phosphatase activity by ...

    African Journals Online (AJOL)

    The result showed that the population levels of phosphobacteria were higher in the rhizosphere soil of groundnut plant. Further, all the strains of phosphobacteria were able to produce phytohormones and phosphatase enzyme under in vitro conditions. Keywords: In vitro, phosphobacteria, growth regulators ...

  10. Alkaline phosphatase activity in gingival crevicular fluid during canine retraction.

    Science.gov (United States)

    Batra, P; Kharbanda, Op; Duggal, R; Singh, N; Parkash, H

    2006-02-01

    The aim of the study was to investigate alkaline phosphatase activity in the gingival crevicular fluid (GCF) during orthodontic tooth movement in humans. Postgraduate orthodontic clinic. Ten female patients requiring all first premolar extractions were selected and treated with standard edgewise mechanotherapy. Canine retraction was done using 100 g sentalloy springs. Maxillary canine on one side acted as experimental site while the contralateral canine acted as control. Gingival crevicular fluid was collected from mesial and distal of canines before initiation of canine retraction (baseline), immediately after initiation of retraction, and on 1st, 7th, 14th and 21st day and the alkaline phosphatase activity was estimated. The results show significant (p < 0.05) changes in alkaline phosphatase activity on the 7th, 14th and 21st day on both mesial and distal aspects of the compared experimental and control sides. The peak in enzyme activity occurred on the 14th day of initiation of retraction followed by a significant fall in activity especially on the mesial aspect. The study showed that alkaline phosphatase activity could be successfully estimated in the GCF using calorimetric estimation assay kits. The enzyme activity showed variation according to the amount of tooth movement.

  11. Yeast Acid Phosphatases and Phytases: Production, Characterization and Commercial Prospects

    Science.gov (United States)

    Kaur, Parvinder; Satyanarayana, T.

    The element phosphorus is critical to all life forms as it forms the basic component of nucleic acids and ATP and has a number of indispensable biochemical roles. Unlike C or N, the biogeochemical cycling of phosphorus is very slow, and thus making it the growth-limiting element in most soils and aquatic systems. Phosphohydrolases (e.g. acid phosphatases and phytases) are enzymes that break the C-O-P ester bonds and provide available inorganic phosphorus from various inassimilable organic forms of phosphorus like phytates. These enzymes are of significant value in effectively combating phosphorus pollution. Although phytases and acid phosphatases are produced by various plants, animals and micro organisms, microbial sources are more promising for the production on a commercial scale. Yeasts being the simplest eukaryotes are ideal candidates for phytase and phos-phatase research due to their mostly non-pathogenic and GRAS status. They have not, however, been utilized to their full potential. This chapter focuses attention on the present state of knowledge on the production, characterization and potential commercial prospects of yeast phytases and acid phosphatases.

  12. Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Ying-Nan P.; LaMarche, Matthew J.; Chan, Ho Man; Fekkes, Peter; Garcia-Fortanet, Jorge; Acker, Michael G.; Antonakos, Brandon; Chen, Christine Hiu-Tung; Chen, Zhouliang; Cooke, Vesselina G.; Dobson, Jason R.; Deng, Zhan; Fei, Feng; Firestone, Brant; Fodor, Michelle; Fridrich, Cary; Gao, Hui; Grunenfelder, Denise; Hao, Huai-Xiang; Jacob, Jaison; Ho, Samuel; Hsiao, Kathy; Kang, Zhao B.; Karki, Rajesh; Kato, Mitsunori; Larrow, Jay; La Bonte, Laura R.; Lenoir, Francois; Liu, Gang; Liu, Shumei; Majumdar, Dyuti; Meyer, Matthew J.; Palermo, Mark; Perez, Lawrence; Pu, Minying; Price, Edmund; Quinn, Christopher; Shakya, Subarna; Shultz, Michael D.; Slisz, Joanna; Venkatesan, Kavitha; Wang, Ping; Warmuth, Markus; Williams, Sarah; Yang, Guizhi; Yuan, Jing; Zhang, Ji-Hu; Zhu, Ping; Ramsey, Timothy; Keen, Nicholas J.; Sellers, William R.; Stams, Travis; Fortin , Pascal D. (Novartis)

    2016-06-29

    The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase1. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma1, 2, 3, 4, 5. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS–ERK signalling pathway2, 3. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways6, 7. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy8, 9. Here we report the discovery of a highly potent (IC50 = 0.071 μM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RAS–ERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers.

  13. Nuclear protein phosphatase-1: an epigenetic regulator of fear memory and amygdala long-term potentiation.

    Science.gov (United States)

    Koshibu, K; Gräff, J; Mansuy, I M

    2011-01-26

    Complex brain diseases and neurological disorders in human generally result from the disturbance of multiple genes and signaling pathways. These disturbances may derive from mutations, deletions, translocations or rearrangements of specific gene(s). However, over the past years, it has become clear that such disturbances may also derive from alterations in the epigenome affecting several genes simultaneously. Our work recently demonstrated that epigenetic mechanisms in the adult brain are in part regulated by protein phosphatase 1 (PP1), a protein Ser/Thr phosphatase that negatively regulates hippocampus-dependent long-term memory (LTM) and synaptic plasticity. PP1 is abundant in brain structures involved in emotional processing like the amygdala, it may therefore be involved in the regulation of fear memory, a form of memory related to post-traumatic stress disorder (PTSD) in human. Here, we demonstrate that PP1 is a molecular suppressor of fear memory and synaptic plasticity in the amygdala that can control chromatin remodeling in neurons. We show that the selective inhibition of the nuclear pool of PP1 in amygdala neurons significantly alters posttranslational modifications (PTMs) of histones and the expression of several memory-associated genes. These alterations correlate with enhanced fear memory, and with an increase in long-term potentiation (LTP) that is transcription-dependent. Our results underscore the importance of nuclear PP1 in the amygdala as an epigenetic regulator of emotional memory, and the relevance of protein phosphatases as potential targets for therapeutic treatment of brain disorders like PTSD. © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

  14. PTEN phosphatase-independent maintenance of glandular morphology in a predictive colorectal cancer model system.

    Science.gov (United States)

    Jagan, Ishaan C; Deevi, Ravi K; Fatehullah, Aliya; Topley, Rebecca; Eves, Joshua; Stevenson, Michael; Loughrey, Maurice; Arthur, Kenneth; Campbell, Frederick Charles

    2013-11-01

    Organotypic models may provide mechanistic insight into colorectal cancer (CRC) morphology. Three-dimensional (3D) colorectal gland formation is regulated by phosphatase and tensin homologue deleted on chromosome 10 (PTEN) coupling of cell division cycle 42 (cdc42) to atypical protein kinase C (aPKC). This study investigated PTEN phosphatase-dependent and phosphatase-independent morphogenic functions in 3D models and assessed translational relevance in human studies. Isogenic PTEN-expressing or PTEN-deficient 3D colorectal cultures were used. In translational studies, apical aPKC activity readout was assessed against apical membrane (AM) orientation and gland morphology in 3D models and human CRC. We found that catalytically active or inactive PTEN constructs containing an intact C2 domain enhanced cdc42 activity, whereas mutants of the C2 domain calcium binding region 3 membrane-binding loop (M-CBR3) were ineffective. The isolated PTEN C2 domain (C2) accumulated in membrane fractions, but C2 M-CBR3 remained in cytosol. Transfection of C2 but not C2 M-CBR3 rescued defective AM orientation and 3D morphogenesis of PTEN-deficient Caco-2 cultures. The signal intensity of apical phospho-aPKC correlated with that of Na(+)/H(+) exchanger regulatory factor-1 (NHERF-1) in the 3D model. Apical NHERF-1 intensity thus provided readout of apical aPKC activity and associated with glandular morphology in the model system and human colon. Low apical NHERF-1 intensity in CRC associated with disruption of glandular architecture, high cancer grade, and metastatic dissemination. We conclude that the membrane-binding function of the catalytically inert PTEN C2 domain influences cdc42/aPKC-dependent AM dynamics and gland formation in a highly relevant 3D CRC morphogenesis model system.

  15. PTEN Phosphatase-Independent Maintenance of Glandular Morphology in a Predictive Colorectal Cancer Model System

    Directory of Open Access Journals (Sweden)

    Ishaan C. Jagan

    2013-11-01

    Full Text Available Organotypic models may provide mechanistic insight into colorectal cancer (CRC morphology. Three-dimensional (3D colorectal gland formation is regulated by phosphatase and tensin homologue deleted on chromosome 10 (PTEN coupling of cell division cycle 42 (cdc42 to atypical protein kinase C (aPKC. This study investigated PTEN phosphatase-dependent and phosphatase-independent morphogenic functions in 3D models and assessed translational relevance in human studies. Isogenic PTEN-expressing or PTEN-deficient 3D colorectal cultures were used. In translational studies, apical aPKC activity readout was assessed against apical membrane (AM orientation and gland morphology in 3D models and human CRC. We found that catalytically active or inactive PTEN constructs containing an intact C2 domain enhanced cdc42 activity, whereas mutants of the C2 domain calcium binding region 3 membrane-binding loop (M-CBR3 were ineffective. The isolated PTEN C2 domain (C2 accumulated in membrane fractions, but C2 M-CBR3 remained in cytosol. Transfection of C2 but not C2 M-CBR3 rescued defective AM orientation and 3D morphogenesis of PTEN-deficient Caco-2 cultures. The signal intensity of apical phospho-aPKC correlated with that of Na+/H+ exchanger regulatory factor-1 (NHERF-1 in the 3D model. Apical NHERF-1 intensity thus provided readout of apical aPKC activity and associated with glandular morphology in the model system and human colon. Low apical NHERF-1 intensity in CRC associated with disruption of glandular architecture, high cancer grade, and metastatic dissemination. We conclude that the membrane-binding function of the catalytically inert PTEN C2 domain influences cdc42/aPKC-dependent AM dynamics and gland formation in a highly relevant 3D CRC morphogenesis model system.

  16. Decryptification of Acid Phosphatase in Arthrospores of Geotrichum Species Treated with Dimethyl Sulfoxide and Acetone

    Science.gov (United States)

    Cotter, David A.; Martel, Anita J.; MacDonald, Paul

    1975-01-01

    Decryptification of acid phosphatase in Geotrichum sp. arthrospores was accomplished using acetone or dimethyl sulfoxide treatment. Both dimethyl sulfoxide and acetone irreversibly destroyed the integrity of the spore membranes without solubilizing acid phosphatase. PMID:1167386

  17. Receptor protein tyrosine phosphatase alpha is essential for hippocampal neuronal migration and long-term potentiation

    DEFF Research Database (Denmark)

    Petrone, Angiola; Battaglia, Fortunato; Wang, Cheng

    2003-01-01

    Despite clear indications of their importance in lower organisms, the contributions of protein tyrosine phosphatases (PTPs) to development or function of the mammalian nervous system have been poorly explored. In vitro studies have indicated that receptor protein tyrosine phosphatase alpha...

  18. An Ancient Protein Phosphatase, SHLP1, Is Critical to Microneme Development in Plasmodium Ookinetes and Parasite Transmission

    Directory of Open Access Journals (Sweden)

    Eva-Maria Patzewitz

    2013-03-01

    Full Text Available Signaling pathways controlled by reversible protein phosphorylation (catalyzed by kinases and phosphatases in the malaria parasite Plasmodium are of great interest, for both increased understanding of parasite biology and identification of novel drug targets. Here, we report a functional analysis in Plasmodium of an ancient bacterial Shewanella-like protein phosphatase (SHLP1 found only in bacteria, fungi, protists, and plants. SHLP1 is abundant in asexual blood stages and expressed at all stages of the parasite life cycle. shlp1 deletion results in a reduction in ookinete (zygote development, microneme formation, and complete ablation of oocyst formation, thereby blocking parasite transmission. This defect is carried by the female gamete and can be rescued by direct injection of mutant ookinetes into the mosquito hemocoel, where oocysts develop. This study emphasizes the varied functions of SHLP1 in Plasmodium ookinete biology and suggests that it could be a novel drug target for blocking parasite transmission.

  19. Recent insights into Protein Phosphatase 2A structure and regulation: the reasons why PP2A is no longer considered as a lazy passive housekeeping enzyme

    Directory of Open Access Journals (Sweden)

    Martin, M.

    2010-01-01

    Full Text Available Although intracellular signal transduction is often portrayed as a protein kinase "domino effect", the counterbalancing function of phosphatases, and thus the control of phosphatase activity, is equally relevant to proper regulation of cellular function. Protein Phosphatase 2A (PP2A is a widely expressed family of protein phosphatases made of a core dimer, composed of a catalytic (C subunit and a structural (A subunit, in association with a third variable regulatory (B subunit. Although viewed as a constitutive housekeeping enzyme in the past, PP2A is a highly regulated phosphatase and is emerging as an important regulator of multiple cellular processes involving protein phosphorylation. The regulation of PP2A is mainly accomplished by the identity of the regulatory B-type subunit, which determines substrate specificity, subcellular localization and catalytic activity of the PP2A holoenzyme. In agreement with this, recent findings on the structure and post-translational modifications of PP2A emphasize the importance of PP2A holoenzyme composition in its regulation and pleiotropic activities.

  20. Correlations between calcineurin phosphatase inhibition and cyclosporine metabolites concentrations in kidney transplant recipients: Implications for immunoassays

    DEFF Research Database (Denmark)

    Jørgensen, Kaj Anker; Karamperis, Nikolaos; Koefoed-Nielsen, Pernille Bundgaard

    2006-01-01

    by inhibiting the enzyme calcineurin phosphatase. Determination of the enzyme's activity is one of the most promising pharmacodynamic markers. It is unknown how calcineurin phosphatase inhibition correlates with various cyclosporine monitoring assays and what is the potential impact of metabolites...... by the enzyme multiplied immunoassay technique (EMIT) and by the polyclonal fluorescence polarization immunoassay (pFPIA). Calcineurin phosphatase activity was measured by its ability to dephosphorylate a previously phosphorylated 19-amino acid peptide. We found that calcineurin phosphatase inhibition...

  1. Correlations between calcineurin phosphatase inhibition and cyclosporine metabolites concentrations in kidney transplant recipients: implications for immunoassays

    DEFF Research Database (Denmark)

    Karamperis, N; Koefoed-Nielsen, PB; Brahe, P

    2006-01-01

    by inhibiting the enzyme calcineurin phosphatase. Determination of the enzyme's activity is one of the most promising pharmacodynamic markers. It is unknown how calcineurin phosphatase inhibition correlates with various cyclosporine monitoring assays and what is the potential impact of metabolites...... by the enzyme multiplied immunoassay technique (EMIT) and by the polyclonal fluorescence polarization immunoassay (pFPIA). Calcineurin phosphatase activity was measured by its ability to dephosphorylate a previously phosphorylated 19-amino acid peptide. We found that calcineurin phosphatase inhibition...

  2. An acid phosphatase in the plasma membranes of human astrocytoma showing marked specificity toward phosphotyrosine protein.

    OpenAIRE

    Leis, J F; Kaplan, N O

    1982-01-01

    The plasma membrane from the human tumor astrocytoma contains an active acid phosphatase activity based on hydrolysis of p-nitrophenyl phosphate. Other acid phosphatase substrates--beta-glycerophosphate, O-phosphorylcholine, and 5'-AMP--are not hydrolyzed significantly. The phosphatase activity is tartrate insensitive and is stimulated by Triton X-100 and EDTA. Of the three known phosphoamino acids, only free O-phosphotyrosine is hydrolyzed by the membrane phosphatase activity. Other acid pho...

  3. Functional requirements for inhibitory signal transmission by the immunomodulatory receptor CD300a.

    Science.gov (United States)

    DeBell, Karen E; Simhadri, Venkateswara R; Mariano, John L; Borrego, Francisco

    2012-04-26

    Activation signals can be negatively regulated by cell surface receptors bearing immunoreceptor tyrosine-based inhibitory motifs (ITIMs). CD300a, an ITIM bearing type I transmembrane protein, is expressed on many hematopoietic cells, including subsets of lymphocytes. We have taken two approaches to further define the mechanism by which CD300a acts as an inhibitor of immune cell receptor signaling. First, we have expressed in Jurkat T cells a chimeric receptor consisting of the extracellular domains of killer-cell immunoglobulin-like receptor (KIR)2DL2 fused to the transmembrane and cytoplasmic segments of CD300a (KIR-CD300a) to explore surrogate ligand-stimulated inhibition of superantigen stimulated T cell receptor (TCR) mediated cell signaling. We found that intact CD300a ITIMs were essential for inhibition and that the tyrosine phosphorylation of these ITIMs required the src tyrosine kinase Lck. Tyrosine phosphorylation of the CD300a ITIMs created docking sites for both src homology 2 domain containing protein tyrosine phosphatase (SHP)-1 and SHP-2. Suppression of SHP-1 and SHP-2 expression in KIR-CD300a Jurkat T cells with siRNA and the use of DT40 chicken B cell lines expressing CD300a and deficient in several phosphatases revealed that SHP-1, but not SHP-2 or the src homology 2 domain containing inositol 5' phosphatase SHIP, was utilized by CD300a for its inhibitory activity. These studies provide new insights into the function of CD300a in tuning T and B cell responses.

  4. Protein phosphatase 2A regulates central sensitization in the spinal cord of rats following intradermal injection of capsaicin

    Directory of Open Access Journals (Sweden)

    Fang Li

    2006-03-01

    Full Text Available Abstract Background Intradermal injection of capsaicin into the hind paw of rats induces spinal cord central sensititzation, a process in which the responsiveness of central nociceptive neurons is amplified. In central sensitization, many signal transduction pathways composed of several cascades of intracellular enzymes are involved. As the phosphorylation state of neuronal proteins is strictly controlled and balanced by the opposing activities of protein kinases and phosphatases, the involvement of phosphatases in these events needs to be investigated. This study is designed to determine the influence of serine/threonine protein phosphatase type 2A (PP2A on the central nociceptive amplification process, which is induced by intradermal injection of capsaicin in rats. Results In experiment 1, the expression of PP2A protein in rat spinal cord at different time points following capsaicin or vehicle injection was examined using the Western blot method. In experiment 2, an inhibitor of PP2A (okadaic acid, 20 nM or fostriecin, 30 nM was injected into the subarachnoid space of the spinal cord, and the spontaneous exploratory activity of the rats before and after capsaicin injection was recorded with an automated photobeam activity system. The results showed that PP2A protein expression in the spinal cord was significantly upregulated following intradermal injection of capsaicin in rats. Capsaicin injection caused a significant decrease in exploratory activity of the rats. Thirty minutes after the injection, this decrease in activity had partly recovered. Infusion of a phosphatase inhibitor into the spinal cord intrathecal space enhanced the central sensitization induced by capsaicin by making the decrease in movement last longer. Conclusion These findings indicate that PP2A plays an important role in the cellular mechanisms of spinal cord central sensitization induced by intradermal injection of capsaicin in rats, which may have implications in

  5. Detection of endogenous alkaline phosphatase activity in intact cells by flow cytometry using the fluorogenic ELF-97 phosphatase substrate

    Science.gov (United States)

    Telford, W. G.; Cox, W. G.; Stiner, D.; Singer, V. L.; Doty, S. B.

    1999-01-01

    BACKGROUND: The alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF((R))-97 for enzyme-labeled fluorescence) has been found useful for the histochemical detection of endogenous AP activity and AP-tagged proteins and oligonucleotide probes. In this study, we evaluated its effectiveness at detecting endogenous AP activity by flow cytometry. METHODS: The ELF-97 phosphatase substrate was used to detect endogenous AP activity in UMR-106 rat osteosarcoma cells and primary cultures of chick chondrocytes. Cells were labeled with the ELF-97 reagent and analyzed by flow cytometry using an argon ultraviolet (UV) laser. For comparison purposes, cells were also assayed for AP using a Fast Red Violet LB azo dye assay previously described for use in detecting AP activity by flow cytometry. RESULTS: The ELF-97 phosphatase substrate effectively detected endogenous AP activity in UMR-106 cells, with over 95% of the resulting fluorescent signal resulting from AP-specific activity (as determined by levamisole inhibition of AP activity). In contrast, less than 70% of the fluorescent signal from the Fast Red Violet LB (FRV) assay was AP-dependent, reflecting the high intrinsic fluorescence of the unreacted components. The ELF-97 phosphatase assay was also able to detect very low AP activity in chick chondrocytes that was undetectable by the azo dye method. CONCLUSIONS: The ELF-97 phosphatase assay was able to detect endogenous AP activity in fixed mammalian and avian cells by flow cytometry with superior sensitivity to previously described assays. This work also shows the applicability of ELF-97 to flow cytometry, supplementing its previously demonstrated histochemical applications. Copyright 1999 Wiley-Liss, Inc.

  6. Zinc-ion-dependent acid phosphatase exhibits magnesium-ion-dependent myo-inositol-1-phosphatase activity.

    Science.gov (United States)

    Fujimoto, S; Okano, I; Tanaka, Y; Sumida, Y; Tsuda, J; Kawakami, N; Shimohama, S

    1996-06-01

    We have purified bovine brain Zn(2+)-dependent acid phosphatase (Zn(2+)-APase), which requires Zn2+ ions to hydrolyze the substrate p-nitrophenyl phosphate (pNPP) in an acidic environment. The substrate specificity and metal requirement of Zn(2+)-APase at a physiological pH was also studied. The enzyme exhibited hydrolytic activity on myo-inositol-1- and -2-monophosphates, 2'-adenosine monophosphate, 2'-guanosine monophosphate, and the alpha- and beta-glycerophosphates, glucose-1-phosphate, and fructose-6-phosphate in 50 mM Tris-HCl buffer (pH 7.4) in the presence of Mg2+ ions, but not on pNPP and phosphotyrosine. Zn2+, Mn2+ and Co2+ ions were less effective for activation. Among the above substrates, myo-inositol-1-phosphate was the most susceptible to hydrolysis by the enzyme in the presence of 3 mM Mg2+ ions. The enzyme exhibited an optimum pH at around 8 for myo-inositol-1-phosphate in the presence of 3 mM Mg2+ ions. The Mg(2+)-dependent myo-inositol-1-phosphatase activity of the enzyme was significantly inhibited by Li+ ions. The Zn(2+)-dependent p-nitrophenyl phosphatase activity and Mg(2+)-dependent myo-inositol-1-phosphatase activity of the purified enzyme fraction exhibited similar behavior on Sephadex G-100 and Mono Q colomns. These findings suggest that Zn(2+)-APase also exhibits Mg(2+)-dependent myo-inositol-1-phosphatase activity under physiological conditions.

  7. Phosphatase activity of Poa pratensis seeds. III. Effect of fluoride, citrate, urea and other substances on the activity of acid phosphatase Ia2 and Ia3

    Directory of Open Access Journals (Sweden)

    Irena Lorenc-Kubis

    2015-01-01

    Full Text Available Effects of fluoride, citrate, urea and other substances on the activity of acid phosphatase a2 and a3 toward p-nitrophenylphosphate and phenylphosphate were investigated. Both enzymes were inhibited by fluoride, p-chloromercuribenzoate and oxalate. Fluoride inhibited acid phosphatase a2 noncorapetitively with p-mitrophenylphosphate, whereas acid phosphatase a3 showed inhibition of mixed type. Hydrolysis of phenylphosphate by both acid phosphatases was activated by citrate. Cytosine and uridine inhibited the activity of phosphatase a2 toward p-nitrophenylphosphate and phenylphosphate, but no effect was observed in case of acid phosphatase a3. After 30 min. incubation with 4 M urea both enzymes lost about 30% of activity.

  8. Alkaline phosphatase binds tenaciously to titanium; implications for biological surface evaluation following bone implant retrieval.

    Science.gov (United States)

    Mansell, J P; Shiel, A I; Harwood, C; Stephens, D

    2017-07-01

    Enhancing the performance and longevity of titanium (Ti) implants continues to be a significant developmental theme in contemporary biomaterials design. Our specific focus pertains to the surface functionalisation of Ti using the bioactive lipid, lysophosphatidic acid (LPA) and certain phosphatase-resistant analogues of LPA. Coating survivorship to a plethora of testing regimens is required to align with due regulatory process before novel biomaterials can enter clinical trials. One of the key acceptance criteria is coating retention to the physical stresses experienced during implantation. In assessing coating stability to insertion into porcine bone we found that a subsequent in vitro assessment to confirm coating persistence was masked by abundant alkaline phosphatase (ALP) contamination adsorbed to the metal surface. Herein we report that ALP can bind to Ti in a matter of minutes by simply immersing Ti samples in aqueous solutions of the enzyme. We strongly discourage the in vitro monitoring of osteoblast and stromal cell ALP expression when assessing bioactive coating survivorship following Ti implant retrieval form native bone tissue. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Protection against gamma-radiation injury by protein tyrosine phosphatase 1B

    Directory of Open Access Journals (Sweden)

    Marina Mojena

    2018-07-01

    Full Text Available Protein tyrosine phosphatase 1B (PTP1B is widely expressed in mammalian tissues, in particular in immune cells, and plays a pleiotropic role in dephosphorylating many substrates. Moreover, PTP1B expression is enhanced in response to pro-inflammatory stimuli and to different cell stressors. Taking advantage of the use of mice deficient in PTP1B we have investigated the effect of γ-radiation in these animals and found enhanced lethality and decreased respiratory exchange ratio vs. the corresponding wild type animals. Using bone-marrow derived macrophages and mouse embryonic fibroblasts (MEFs from wild-type and PTP1B-deficient mice, we observed a differential response to various cell stressors. PTP1B-deficient macrophages exhibited an enhanced response to γ-radiation, UV-light, LPS and S-nitroso-glutathione. Macrophages exposed to γ-radiation show DNA damage and fragmentation, increased ROS production, a lack in GSH elevation and enhanced acidic β-galactosidase activity. Interestingly, these differences were not observed in MEFs. Differential gene expression analysis of WT and KO macrophages revealed that the main pathways affected after irradiation were an up-regulation of protein secretion, TGF-β signaling and angiogenesis among other, and downregulation of Myc targets and Hedgehog signaling. These results demonstrate a key role for PTP1B in the protection against the cytotoxicity of irradiation in intact animal and in macrophages, which might be therapeutically relevant. Keywords: Protein tyrosine phosphatase, Cell viability, Irradiation sensitivity, Lethality, p53

  10. Structure determination of T-cell protein-tyrosine phosphatase

    DEFF Research Database (Denmark)

    Iversen, L.F.; Møller, K. B.; Pedersen, A.K.

    2002-01-01

    Protein-tyrosine phosphatase 1B (PTP1B) has recently received much attention as a potential drug target in type 2 diabetes. This has in particular been spurred by the finding that PTP1B knockout mice show increased insulin sensitivity and resistance to diet-induced obesity. Surprisingly, the highly...... homologous T cell protein-tyrosine phosphatase (TC-PTP) has received much less attention, and no x-ray structure has been provided. We have previously co-crystallized PTP1B with a number of low molecular weight inhibitors that inhibit TC-PTP with similar efficiency. Unexpectedly, we were not able to co...... the high degree of functional and structural similarity between TC-PTP and PTP1B, we have been able to identify areas close to the active site that might be addressed to develop selective inhibitors of each enzyme....

  11. Myosin phosphatase Fine-tunes Zebrafish Motoneuron Position during Axonogenesis.

    Directory of Open Access Journals (Sweden)

    Juliane Bremer

    2016-11-01

    Full Text Available During embryogenesis the spinal cord shifts position along the anterior-posterior axis relative to adjacent tissues. How motor neurons whose cell bodies are located in the spinal cord while their axons reside in adjacent tissues compensate for such tissue shift is not well understood. Using live cell imaging in zebrafish, we show that as motor axons exit from the spinal cord and extend through extracellular matrix produced by adjacent notochord cells, these cells shift several cell diameters caudally. Despite this pronounced shift, individual motoneuron cell bodies stay aligned with their extending axons. We find that this alignment requires myosin phosphatase activity within motoneurons, and that mutations in the myosin phosphatase subunit mypt1 increase myosin phosphorylation causing a displacement between motoneuron cell bodies and their axons. Thus, we demonstrate that spinal motoneurons fine-tune their position during axonogenesis and we identify the myosin II regulatory network as a key regulator.

  12. Phosphatase activity tunes two-component system sensor detection threshold.

    Science.gov (United States)

    Landry, Brian P; Palanki, Rohan; Dyulgyarov, Nikola; Hartsough, Lucas A; Tabor, Jeffrey J

    2018-04-12

    Two-component systems (TCSs) are the largest family of multi-step signal transduction pathways in biology, and a major source of sensors for biotechnology. However, the input concentrations to which biosensors respond are often mismatched with application requirements. Here, we utilize a mathematical model to show that TCS detection thresholds increase with the phosphatase activity of the sensor histidine kinase. We experimentally validate this result in engineered Bacillus subtilis nitrate and E. coli aspartate TCS sensors by tuning their detection threshold up to two orders of magnitude. We go on to apply our TCS tuning method to recently described tetrathionate and thiosulfate sensors by mutating a widely conserved residue previously shown to impact phosphatase activity. Finally, we apply TCS tuning to engineer B. subtilis to sense and report a wide range of fertilizer concentrations in soil. This work will enable the engineering of tailor-made biosensors for diverse synthetic biology applications.

  13. Central regulation of metabolism by protein tyrosine phosphatases

    Directory of Open Access Journals (Sweden)

    Ryan eTsou

    2013-01-01

    Full Text Available Protein tyrosine phosphatases (PTPs are important regulators of intracellular signaling pathways via the dephosphorylation of phosphotyrosyl residues on various receptor and non-receptor substrates. The phosphorylation state of central nervous system (CNS signaling components underlies the molecular mechanisms of a variety of physiological functions including the control of energy balance and glucose homeostasis. In this review, we summarize the current evidence implicating PTPs as central regulators of metabolism, specifically highlighting their interactions with the neuronal leptin and insulin signaling pathways. We discuss the role of a number of PTPs (PTP1B, SHP2, TCPTP, RPTPe, and PTEN, reviewing the findings from genetic mouse models and in vitro studies which highlight these phosphatases as key central regulators of energy homeostasis.

  14. Light availability may control extracellular phosphatase production in turbid environments

    Czech Academy of Sciences Publication Activity Database

    Rychtecký, Pavel; Řeháková, Klára; Kozlíková, Eliška; Vrba, Jaroslav

    2015-01-01

    Roč. 69, č. 1 (2015), s. 37-44 ISSN 0095-3628 R&D Projects: GA ČR(CZ) GA206/09/0309; GA ČR(CZ) GAP504/11/2177; GA ČR(CZ) GAP504/11/2182 Institutional support: RVO:60077344 Keywords : phytoplankton * phosphatase activity * ELF97 phosphate Subject RIV: DA - Hydrology ; Limnology Impact factor: 3.232, year: 2015

  15. Membrane interaction and functional plasticity of inositol polyphosphate 5-phosphatases.

    Science.gov (United States)

    Braun, Werner; Schein, Catherine H

    2014-05-06

    In this issue of Structure, Trésaugues and colleagues determined the interaction of membrane-bound phosphoinositides with three clinically significant human inositol polyphosphate 5-phosphatases (I5Ps). A comparison to the structures determined with soluble substrates revealed differences in the binding mode and suggested how the I5Ps and apurinic endonuclease (APE1) activities evolved from the same metal-binding active center. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Acid phosphatases in seeds and developing of squash (Cucurbita ficifolia

    Directory of Open Access Journals (Sweden)

    Irena Lorenc-Kubis

    2014-01-01

    Full Text Available Changes in protein content and acid phosphatase activity were followed during germination (imbition through seedlings development in extracts from cotyledons of squash (Cucurbita ficifolia. It has been shown that the activity of acid phosphatase was initially low and than increased to a maximum after 6 days of imbition. Acid phosphates were isolated from cotyledons of seeds and from 6-, 10- and 22-days old seedlings by extraction the proteins with 0.1 M acetate buffer pH 5.1, precipitation with ethanol and by affinity chromatography on con A-Sepharose. Two glycoprotein enzymes AcPase Ba and AcPase Bb which differ in their affinity to immobilized con A were obtained. Both acid phosphatates retained the enzyme activity after binding to free con A. Rocket affinity electrophoresis of AcPase Ba and AcPase Bb, isolated from cotyledons of seeds and seedlings, revealed differences in their ability to bind to con A during seeds germination and seedling develop-ment indicating changes in their sugar component. Con A was found to activate both enzymes. The enzymes cross-reacted with monospecific antibodies raised against grass seed acid phosphatate Ba indicating an antigenic relationship between squash and grass acid phosphatases.

  17. A histochemical study of rat salivary gland acid phosphatase.

    Science.gov (United States)

    Isacsson, G

    1986-01-01

    Male Sprague-Dawley rats received 4 mg pilocarpine/100 g body wt intraperitoneally or physiological saline as control and were killed at various intervals. Acid phosphatase was reacted on frozen sections from soft palate, parotid and submandibular glands using sodium-alpha-naphthyl acid phosphate as substrate. Various inhibitors were added to the incubation medium. The strongest acid phosphatase activity was in the parotid gland acinar and proximal secretory duct cells; the mucous minor glands of the palate were completely negative. Activity was found in the acinar cells, proximal secretory duct cells, granular and striated duct and excretory duct cells. Pilocarpine injection slightly reduced the activity up to 6 h after injection. Cupric chloride added to the incubation medium lowered the overall activity. Fluoride and molybdate inhibited the acid phosphatase reaction in all structures. Tartrate inhibited the reaction in all structures except the submandibular striated duct cells. The tartrate-resistant activity may be a Na+K+-dependent ATPase involved in re-absorbing water and electrolytes from the primary saliva.

  18. Comparative studies on the carbohydrate, protein and acid phosphatase contents in seeds of some rye (Secale cereale varieties

    Directory of Open Access Journals (Sweden)

    B. Morawiecka

    2015-01-01

    Full Text Available The contents of sugars. proteins and acid phosphatase extracted with 0.1 M acetate buffer, pH 5.1, from some rye varieties were determined. The total sugar level amounted to 3.25-9.70 g per 100 g of seeds; the estimates for pentoses were 1.7-2.9 g and those for proteins 0.91-1.60 g per 100 g of seeds. Acid phosphatase showed and activity level between 0.18 and 1.26 units/mg protein. After disc electrophoresis proteins were separated into 10 to 11 bands: at pH 9.4 or into 4-7 bands at pH 3.8. Essential variety differences were expressed in protein patterns after electrophoresis at pH 3.8. Acid phosphatase was separated into 5 and 4 activity bands at pH 9.4 and 3.3,. respectively. No variations in zymogram patterns were observed in respect to variety differences or cultivation in various climate and soil conditions.

  19. Phospholipid metabolism and nuclear function: roles of the lipin family of phosphatidic acid phosphatases.

    Science.gov (United States)

    Siniossoglou, Symeon

    2013-03-01

    Phospholipids play important roles in nuclear function as dynamic building blocks for the biogenesis of the nuclear membrane, as well as signals by which the nucleus communicates with other organelles, and regulate a variety of nuclear events. The mechanisms underlying the nuclear roles of phospholipids remain poorly understood. Lipins represent a family of phosphatidic acid (PA) phosphatases that are conserved from yeasts to humans and perform essential functions in lipid metabolism. Several studies have identified key roles for lipins and their regulators in nuclear envelope organization, gene expression and the maintenance of lipid homeostasis in yeast and metazoans. This review discusses recent advances in understanding the roles of lipins in nuclear structure and function. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Regulation of protein phosphatase 2A during embryonic diapause process in the silkworm, Bombyx mori.

    Science.gov (United States)

    Gu, Shi-Hong; Hsieh, Hsiao-Yen; Lin, Pei-Ling

    2017-11-01

    Regulation of protein phosphorylation requires coordinated interactions between protein kinases and protein phosphatases. In the present study, we investigated regulation of protein phosphatase 2A (PP2A) during the embryonic diapause process of B. mori. An immunoblotting analysis showed that Bombyx eggs contained a catalytic C subunit, a major regulatory B subunit (B55/PR55 subunit), and a structural A subunit, with the A and B subunits undergoing differential changes between diapause and non-diapause eggs during embryonic process. In non-diapause eggs, eggs whose diapause initiation was prevented by HCl, and eggs in which diapause had been terminated by chilling of diapausing eggs at 5°C for 70days and then were transferred to 25°C, protein levels of the A and B subunits of PP2A gradually increased toward embryonic development. However, protein levels of the A and B subunits in diapause eggs remained at low levels during the first 8days after oviposition. The direct determination of PP2A enzymatic activity showed that the activity remained at low levels in diapause eggs during the first 8days after oviposition. However, in non-diapause eggs, eggs whose diapause initiation was prevented by HCl, and eggs in which diapause had been terminated by chilling, PP2A enzymatic activity sharply increased during the first several days, reached a peak during the middle embryonic development, and then greatly decreased 3 or 4days before hatching. Examination of temporal changes in mRNA expression levels of the catalytic β subunit and regulatory subunit of PP2A showed high levels in eggs whose diapause initiation was prevented by HCl compared to those in diapause eggs. These results demonstrate that the higher PP2A gene expression and PP2A A and B subunit protein levels and increased enzymatic activity are related to embryonic development of B. mori. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Identification and bioinformatics comparison of two novel phosphatases in monoecious and gynoecious cucumber lines

    Science.gov (United States)

    Pawełkowicz, Magdalena E.; Wojcieszek, Michał; Osipowski, Paweł; Krzywkowski, Tomasz; PlÄ der, Wojciech; Przybecki, Zbigniew

    2016-09-01

    Two Arabidopsis thaliana genes from the PP2C family of protein phosphatases (AtABI1 and AtABI2) were used to find orthologous genes in the Cucumis sativus L. cv. Borszczagowski (cucumber) genome. Cucumber has been used as a model plant for sex expression studies because although it has been defined as a monoecious species, numerous genotypes are known to produce only female, only male, or hermaphroditic flowers. We identified two new orthologous genes of AtABI1 and AtABI2 in the cucumber genome and named them CsABI1 and CsABI2. To determine the relationships between the regulation of CsABI1 and CsABI2 and flower morphogenesis in cucumber, we performed various computational analyses to define the structure of the genes, and to predict regulatory elements and protein motifs in their sequences. We also performed an expression analysis to identify differences in the expression levels of CsABI1 and CsABI2 in vegetative and generative tissues (leaf, shoot apex, and flower buds) of monoecious (B10) and gynoecious (2gg) cucumber lines. We found that the expressions of CsABI1 and CsABI2 differed in male and female floral buds, and correlated these findings with the abscisic acid signaling pathways in male and female flowers.

  2. The Phosphatase Inhibitor Calyculin-A Impairs Clot Retraction, Platelet Activation, and Thrombin Generation

    Directory of Open Access Journals (Sweden)

    Renáta Hudák

    2017-01-01

    Full Text Available The aim of this study was to investigate the effect of the serine/threonine protein phosphatase inhibitor, calyculin-A (CLA, on clot formation and on the procoagulant activity of human platelets. Platelet-rich plasma (PRP samples were preincubated with buffer or CLA and subsequently platelets were activated by the protease-activated receptor 1 (PAR-1 activator, thrombin receptor activating peptide (TRAP. Clot retraction was detected by observing clot morphology up to 1 hour, phosphatidylserine- (PS- expression was studied by flow cytometry, and thrombin generation was measured by a fluorimetric assay. For the intracellular Ca2+ assay, platelets were loaded with calcium-indicator dyes and the measurements were carried out using a ratiometric method with real-time confocal microscopy. CLA preincubation inhibited clot retraction, PS-expression, and thrombin formation. TRAP activation elicited Ca2+ response and PS-expression in a subset of platelets. The activated PRP displayed significantly faster and enhanced thrombin generation compared to nonactivated samples. CLA pretreatment abrogated PS-exposure and clot retraction also in TRAP-activated samples. As a consequence of the inhibitory effect on calcium elevation and PS-expression, CLA significantly downregulated thrombin generation in PRP. Our results show that CLA pretreatment may be a useful tool to investigate platelet activation mechanisms that contribute to clot formation and thrombin generation.

  3. Dephosphorylation of chicken cardiac myofibril C-protein by protein phosphatases 1 and 2A

    International Nuclear Information System (INIS)

    Thysseril, T.J.; Hegazy, M.G.; Schlender, K.K.

    1987-01-01

    C-Protein, which is a regulatory component of cardiac muscle myofibrils, is phosphorylated in response to β-adrenergic agonists by a cAMP-dependent mechanism and dephosphorylated in response to cholinergic agonists. It is believed that the cAMP-dependent phosphorylation is due to cAMP-dependent protein kinase. The protein phosphatase(s) involved in the dephosphorylation of C-protein has not been determined. In this study, chicken cardiac C-protein was phosphorylated with the cAMP-dependent protein kinase to about 3 mol phosphate/mol C-protein. Incubation of [ 32 P]C-protein with the catalytic subunit of protein phosphatase 1 or 2A rapidly removed 30-40% of 32 [P]. Phosphopeptide maps and phosphoamino acid analysis revealed that the major site(s) dephosphorylated by either phosphatase was a phosphothreonine residue(s) located on the same tryptic peptide and on the same CNBr fragment. Increasing the incubation period or the phosphatase concentration did not result in any further dephosphorylation of C-protein by phosphatase 1, but phosphatase 2A completely dephosphorylated C-protein. Preliminary studies showed that the major protein phosphatase associated with the myofibril was phosphatase 2A. These results indicate the phosphatase 2A may be important in the regulation of the phosphorylation state of C-protein

  4. Pattern-Recognition Receptor Signaling Regulator mRNA Expression in Humans and Mice, and in Transient Inflammation or Progressive Fibrosis

    Science.gov (United States)

    Günthner, Roman; Kumar, Vankayala Ramaiah Santhosh; Lorenz, Georg; Anders, Hans-Joachim; Lech, Maciej

    2013-01-01

    The cell type-, organ-, and species-specific expression of the pattern-recognition receptors (PRRs) are well described but little is known about the respective expression profiles of their negative regulators. We therefore determined the mRNA expression levels of A20, CYLD, DUBA, ST2, CD180, SIGIRR, TANK, SOCS1, SOCS3, SHIP, IRAK-M, DOK1, DOK2, SHP1, SHP2, TOLLIP, IRF4, SIKE, NLRX1, ERBIN, CENTB1, and Clec4a2 in human and mouse solid organs. Humans and mice displayed significant differences between their respective mRNA expression patterns of these factors. Additionally, we characterized their expression profiles in mononuclear blood cells upon bacterial endotoxin, which showed a consistent induction of A20, SOCS3, IRAK-M, and Clec4a2 in human and murine cells. Furthermore, we studied the expression pattern in transient kidney ischemia-reperfusion injury versus post-ischemic atrophy and fibrosis in mice. A20, CD180, ST2, SOCS1, SOCS3, SHIP, IRAK-M, DOK1, DOK2, IRF4, CENTB1, and Clec4a2 were all induced, albeit at different times of injury and repair. Progressive fibrosis was associated with a persistent induction of these factors. Thus, the organ- and species-specific expression patterns need to be considered in the design and interpretation of studies related to PRR-mediated innate immunity, which seems to be involved in tissue injury, tissue regeneration and in progressive tissue scarring. PMID:24009023

  5. Regulation of tumor cell migration by protein tyrosine phosphatase (PTP)-proline-, glutamate-, serine-, and threonine-rich sequence (PEST)

    Science.gov (United States)

    Zheng, Yanhua; Lu, Zhimin

    2013-01-01

    Protein tyrosine phosphatase (PTP)–proline-, glutamate-, serine-, and threonine-rich sequence (PEST) is ubiquitously expressed and is a critical regulator of cell adhesion and migration. PTP-PEST activity can be regulated transcriptionally via gene deletion or mutation in several types of human cancers or via post-translational modifications, including phosphorylation, oxidation, and caspase-dependent cleavage. PTP-PEST interacts with and dephosphorylates cytoskeletal and focal adhesion-associated proteins. Dephosphorylation of PTP-PEST substrates regulates their enzymatic activities and/or their interaction with other proteins and plays an essential role in the tumor cell migration process. PMID:23237212

  6. c-Jun controls the efficiency of MAP kinase signaling by transcriptional repression of MAP kinase phosphatases

    International Nuclear Information System (INIS)

    Sprowles, Amy; Robinson, Dan; Wu Yimi; Kung, H.-J.; Wisdom, Ron

    2005-01-01

    The mammalian JNK signaling pathway regulates the transcriptional response of cells to environmental stress, including UV irradiation. This signaling pathway is composed of a classical MAP kinase cascade; activation results in phosphorylation of the transcription factor substrates c-Jun and ATF2, and leads to changes in gene expression. The defining components of this pathway are conserved in the fission yeast S. pombe, where the genetic studies have shown that the ability of the JNK homolog Spc1 to be activated in response to UV irradiation is dependent on the presence of the transcription factor substrate Atf1. We have used genetic analysis to define the role of c-Jun in activation of the mammalian JNK signaling pathway. Our results show that optimal activation of JNK requires the presence of its transcription factor substrate c-Jun. Mutational analysis shows that the ability of c-Jun to support efficient activation of JNK requires the ability of Jun to bind DNA, suggesting a transcriptional mechanism. Consistent with this, we show that c-Jun represses the expression of several MAP kinase phosphatases. In the absence of c-Jun, the increased expression of MAP kinase phosphatases leads to impaired activation of the ERK, JNK, and p38 MAP kinases after pathway activation. The results show that one function of c-Jun is to regulate the efficiency of signaling by the ERK, p38, and JNK MAP kinases, a function that is likely to affect cellular responses to many different stimuli

  7. Phosphoprotein phosphatase of bovine spleen cell nuclei: physicochemical properties

    International Nuclear Information System (INIS)

    Rezyapkin, V.I.; Leonova, L.E.; Komkova, A.I.

    1986-01-01

    The physicochemical properties of phosphoprotein phosphatase (EC 1.3.1.16) from bovine spleen cell nuclei were studied. The enzyme possesses broad substrate specificity and catalyzes the dephosphorylation of phosphocasein, ATP, ADP, and p-nitrophenyl phosphate (pNPP). K/sub m/ for ATP, ADP, and pNPP are equal to 0.44, 0.43, and 1.25 mM, respectively. M/sub r/ of the enzyme, according to the data of gel filtraction of Sephadex G-75 and electrophoresis in polyacrylamide gel of various concentrations is ∼ 33,000. In electrophoresis in the presence of SDS, two protein bands with M/sub r/ 12,000 and 18,000 are detected. In the enzyme molecule, acid amino acid residues predominate; two free SH groups and two disulfide bridges are detected. Phosphoprotein phosphatase is a glycoprotein, containing ∼ 22% carbonhydrates. The protein possesses a supplementary absorption maximum at 560 nm. Ammonium molybdate is a competitive inhibitor with K/sub i/ 0.37 μM, while sodium fluoride is a noncompetitive inhibitor with K/sub i/ 1.3 mM. Incubation in the presence of 2 mM phenylmethylsulfonyl fluoride for 25 h leads to a loss of ∼ 46% of the enzymatic activity. Ammonium molybdate, sodium fluoride, and PMSF are reversible inhibitors. Modifications of the SH groups, NH 2 groups, and histidine leads to a decrease in the enzymatic activity. Incubation of phosphoprotein phosphatase with [γ- 32 P]ATP leads to the incorporation of 0.33 mole 33 P per mole of the enzyme. The mechanism of hydrolysis of the phosphodiester bond, catalyzed by the enzyme, is discussed

  8. A double antibody radioimmunoassay specific for placental alkaline phosphatase

    International Nuclear Information System (INIS)

    Dass, S.; Bagshawe, K.D.

    1984-01-01

    Placental alkaline phosphatase (PLAP) is normally found in enzymically measurable amounts in second and third trimester pregnancy serum. Its occurrence in sera and tumours from patients with malignant disease has led to the development of methods to specifically identify and quantitate the enzyme. Recently immunological techniques have been used, employing antibodies raised to purified PLAP; these include solid phase radioimmunoassays and enzyme-immunoassay. The development of a sensitive, specific, automated double-antibody radioimmunoassay for the measurement of PLAP in serum is reported. (Auth.)

  9. Radioimmunoassay for prostatic acid phosphatase in human serum. Methodologic aspects

    Energy Technology Data Exchange (ETDEWEB)

    Pradalier, N; Canal, P; Pujol, A; Fregevu, Y [Groupe de Recherches du Centre Claudius-Regaud, Toulouse (France); Soula, G [Faculte des Sciences Pharmaceutiques, Toulouse (France)

    1982-01-01

    We propose a double antibody radioimmunoassay for human prostatic acid phosphatase (PAP) in serum for diagnosis and management of prostatic adenocarcinoma under treatment. The antigen is purified from human prostatic fluid by a gel-filtration on Sephadex G 100 followed by affinity chromatography on Con A Sepharose. A specific antibody is raised in rabbits and purified by immunoadsorption with a female serum. The described technique offers both radioisotopic sensibility and immunologic specificity. Physiological values determined in the serum of 125 healthy males are below 2 ng/ml. No significative differences are observed with age. The proposed technique also shows significant differences between values evaluated for benign prostatic hyperplasia and prostatic adenocarcinoma.

  10. Promoting Uranium Immobilization by the Activities of Microbial Phosphatases

    Energy Technology Data Exchange (ETDEWEB)

    Martinez, Robert J.; Beazley, Melanie J.; Wilson, Jarad J.; Taillefert, Martial; Sobecky, Patricia A.

    2005-04-05

    The overall goal of this project is to examine the role of nonspecific phosphohydrolases present in naturally occurring subsurface microorganisms for the purpose of promoting the immobilization of radionuclides through the production of uranium [U(VI)] phosphate precipitates. Specifically, we hypothesize that the precipitation of U(VI) phosphate minerals may be promoted through the microbial release and/or accumulation of PO{sub 4}{sup 3-}. During this phase of the project we have been conducting assays to determine the effects of pH, inorganic anions and organic ligands on U(VI) mineral formation and precipitation when FRC bacterial isolates were grown in simulated groundwater medium. The molecular characterization of FRC isolates has also been undertaken during this phase of the project. Analysis of a subset of gram-positive FRC isolates cultured from FRC soils (Areas 1, 2 and 3) and background sediments have indicated a higher percentage of isolates exhibiting phosphatase phenotypes (i.e., in particular those surmised to be PO{sub 4}{sup 3-}-irrepressible) relative to isolates from the reference site. A high percentage of strains that exhibited such putatively PO{sub 4}{sup 3-}-irrepressible phosphatase phenotypes were also resistant to the heavy metals lead and cadmium. Previous work on FRC strains, including Arthrobacter, Bacillus and Rahnella spp., has demonstrated differences in tolerance to U(VI) toxicity (200 {micro}M) in the absence of organophosphate substrates. For example, Arthrobacter spp. exhibited the greatest tolerance to U(VI) while the Rahnella spp. have been shown to facilitate the precipitation of U(VI) from solution and the Bacillus spp. demonstrate the greatest sensitivity to acidic conditions and high concentrations of U(VI). PCR-based detection of FRC strains are being conducted to determine if non-specific acid phosphatases of the known molecular classes [i.e., classes A, B and C] are present in these FRC isolates. Additionally, these

  11. Chromatographic separation of alkaline phosphatase from dental enamel

    DEFF Research Database (Denmark)

    Moe, D; Kirkeby, S; Salling, E

    1989-01-01

    Alkaline phosphatase (AP) was prepared from partly mineralized bovine enamel by extraction in phosphate buffer, centrifugation and various chromatographic techniques. Chromatofocusing showed that the enamel enzyme possessed five isoelectric points at the acid pH level ranging from pH 5.7 to pH 4.......4. Three enzyme peaks were eluted using low pressure chromatography with a Bio-gel column. With a HPLC gel filtration column the separation of the enamel extract resulted in only one peak with AP activity. The fractions of this peak were used to produce an antibody against bovine AP....

  12. Receptor tyrosine phosphatase R-PTP-kappa mediates homophilic binding

    DEFF Research Database (Denmark)

    Sap, J; Jiang, Y P; Friedlander, D

    1994-01-01

    Receptor tyrosine phosphatases (R-PTPases) feature PTPase domains in the context of a receptor-like transmembrane topology. The R-PTPase R-PTP-kappa displays an extracellular domain composed of fibronectin type III motifs, a single immunoglobulin domain, as well as a recently defined MAM domain (Y...... not require PTPase activity or posttranslational proteolytic cleavage of the R-PTP-kappa protein and is calcium independent. The results suggest that R-PTPases may provide a link between cell-cell contact and cellular signaling events involving tyrosine phosphorylation....

  13. Phosphoglycolate phosphatase and 2,3-diphosphoglycerate in red cells of normal and anemic subjects.

    Science.gov (United States)

    Somoza, R; Beutler, E

    1983-10-01

    Red cell phosphoglycolate phosphatase (PGP) and 2,3-diphosphoglycerate (2,3-DPG) were investigated in normal and anemic patients and rabbits. In hemolytic anemia and blood-loss anemia, characterized by a young red cell population, there was an increase in both phosphoglycolate phosphatase activity and 2,3-diphosphoglycerate levels. In aplastic anemia, the phosphoglycolate phosphatase activity was normal, but the 2,3-diphosphoglycerate values were nonetheless increased. Thus, no relationship was found between phosphoglycolate phosphatase activity and 2,3-diphosphoglycerate levels. The lack of correlation between the activity of phosphoglycolate phosphatase and 2,3-DPG levels suggests that modulation of phosphoglycolate phosphatase activity does not control the level of 2,3-DPG in erythrocytes.

  14. A Novel Molecular Diagnostic of Glioblastomas: Detection of an Extracellular Fragment of Protein Tyrosine Phosphatase μ

    Directory of Open Access Journals (Sweden)

    Susan M. Burden-Gulley

    2010-04-01

    Full Text Available We recently found that normal human brain and low-grade astrocytomas express the receptor protein tyrosine phosphatase mu (PTPμ and that the more invasive astrocytomas, glioblastoma multiforme (GBM, downregulate full-length PTPμ expression. Loss of PTPμ expression in GBMs is due to proteolytic cleavage that generates an intracellular and potentially a cleaved and released extracellular fragment of PTPμ. Here, we identify that a cleaved extracellular fragment containing the domains required for PTPμ-mediated adhesion remains associated with GBM tumor tissue. We hypothesized that detection of this fragment would make an excellent diagnostic tool for the localization of tumor tissue within the brain. To this end, we generated a series of fluorescently tagged peptide probes that bind the PTPμ fragment. The peptide probes specifically recognize GBM cells in tissue sections of surgically resected human tumors. To test whether the peptide probes are able to detect GBM tumors in vivo, the PTPμ peptide probes were tested in both mouse flank and intracranial xenograft human glioblastoma tumor model systems. The glial tumors were molecularly labeled with the PTPμ peptide probes within minutes of tail vein injection using the Maestro FLEX In Vivo Imaging System. The label was stable for at least 3 hours. Together, these results indicate that peptide recognition of the PTPμ extracellular fragment provides a novel molecular diagnostic tool for detection of human glioblastomas. Such a tool has clear translational applications and may lead to improved surgical resections and prognosis for patients with this devastating disease.

  15. Striatal-enriched Tyrosine Protein Phosphatase (STEP) in the Mechanisms of Depressive Disorders.

    Science.gov (United States)

    Kulikova, Elizabeth; Kulikov, Alexander

    2017-08-30

    Striatal-enriched tyrosine protein phosphatase (STEP) is expressed mainly in the brain. Its dysregulation is associated with Alzheimer's and Huntington's diseases, schizophrenia, fragile X syndrome, drug abuse and stroke/ischemia. However, an association between STEP and depressive disorders is still obscure. The review discusses the theoretical foundations and experimental facts concerning possible relationship between STEP dysregulation and depression risk. STEP dephosphorylates and inactivates several key neuronal signaling proteins such as extracellular signal-regulating kinase 1 and 2 (ERK1/2), stress activated protein kinases p38, the Src family tyrosine kinases Fyn, Pyk2, NMDA and AMPA glutamate receptors. The inactivation of these proteins decreases the expression of brain derived neurotrophic factor (BDNF) necessary for neurogenesis and neuronal survival. The deficit of BDNF results in progressive degeneration of neurons in the hippocampus and cortex and increases depression risk. At the same time, a STEP inhibitor, 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6-amine hydrochloride (TC-2153), increases BDNF expression in the hippocampus and attenuated the depressivelike behavior in mice. Thus, STEP is involved in the mechanism of depressive disorders and it is a promising molecular target for atypical antidepressant drugs of new generation. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  16. Sp1 transcriptional activity is up-regulated by phosphatase 2A in dividing T lymphocytes.

    Science.gov (United States)

    Lacroix, Isabelle; Lipcey, Carol; Imbert, Jean; Kahn-Perlès, Brigitte

    2002-03-15

    We have followed Sp1 expression in primary human T lymphocytes induced, via CD2 plus CD28 costimulation, to sustained proliferation and subsequent return to quiescence. Binding of Sp1 to wheat germ agglutinin lectin was not modified following activation, indicating that the overall glycosylation of the protein was unchanged. Sp1 underwent, instead, a major dephosphorylation that correlated with cyclin A expression and, thus, with cell cycle progression. A similar change was observed in T cells that re-entered cell cycle following secondary interleukin-2 stimulation, as well as in serum-induced proliferating NIH/3T3 fibroblasts. Phosphatase 2A (PP2A) appears involved because 1) treatment of dividing cells with okadaic acid or cantharidin inhibited Sp1 dephosphorylation and 2) PP2A dephosphorylated Sp1 in vitro and strongly interacted with Sp1 in vivo. Sp1 dephosphorylation is likely to increase its transcriptional activity because PP2A overexpression potentiated Sp1 site-driven chloramphenicol acetyltransferase expression in dividing Kit225 T cells and okadaic acid reversed this effect. This increase might be mediated by a stronger affinity of dephosphorylated Sp1 for DNA, as illustrated by the reduced DNA occupancy by hyperphosphorylated Sp factors from cantharidin- or nocodazole-treated cells. Finally, Sp1 dephosphorylation appears to occur throughout cell cycle except for mitosis, a likely common feature to all cycling cells.

  17. MAPK Phosphatase-1 Deficiency Exacerbates the Severity of Imiquimod-Induced Psoriasiform Skin Disease

    Directory of Open Access Journals (Sweden)

    Weiheng Zhao

    2018-03-01

    Full Text Available Persistent activation of mitogen-activated protein kinase (MAPK is believed to be involved in psoriasis pathogenesis. MAPK phosphatase-1 (MKP-1 is an important negative regulator of MAPK activity, but the cellular and molecular mechanisms of MKP-1 in psoriasis development are largely unknown. In this study, we found that the expression of MKP-1 was decreased in the imiquimod (IMQ-induced psoriasiform mouse skin. MKP-1-deficient (MKP-1−/− mice were highly susceptible to IMQ-induced skin inflammation, which was associated with increased production of inflammatory cytokines and chemokines. MKP-1 acted on both hematopoietic and non-hematopoietic cells to regulate psoriasis pathogenesis. MKP-1 deficiency in macrophages led to enhanced p38 activation and higher expression of interleukin (IL-1β, CXCL2, and S100a8 upon R848 stimulation. Moreover, MKP-1 deficiency in the non-hematopoietic compartments led to an enhanced IL-22 receptor signaling and higher expression of CXCL1 and CXCL2 upon IMQ treatment. Collectively, our data suggest a critical role for MKP-1 in the regulation of skin inflammation.

  18. Tissue non-specific alkaline phosphatase production by human dental pulp stromal cells is enhanced by high density cell culture.

    Science.gov (United States)

    Tomlinson, Matthew J; Dennis, Caitriona; Yang, Xuebin B; Kirkham, Jennifer

    2015-08-01

    The cell surface hydrolase tissue non-specific alkaline phosphatase (TNAP) (also known as MSCA-1) is used to identify a sub-population of bone marrow stromal cells (BMSCs) with high mineralising potential and is found on subsets of cells within the dental pulp. We aim to determine whether TNAP is co-expressed by human dental pulp stromal cells (hDPSCs) alongside a range of BMSC markers, whether this is an active form of the enzyme and the effects of culture duration and cell density on its expression. Cells from primary dental pulp and culture expanded hDPSCs expressed TNAP. Subsequent analyses revealed persistent TNAP expression and co-expression with BMSC markers such as CD73 and CD90. Flow cytometry and biochemical assays showed that increased culture durations and cell densities enhanced TNAP expression by hDPSCs. Arresting the hDPSC cell cycle also increased TNAP expression. These data confirm that TNAP is co-expressed by hDPSCs together with other BMSC markers and show that cell density affects TNAP expression levels. We conclude that TNAP is a potentially useful marker for hDPSC selection especially for uses in mineralised tissue regenerative therapies.

  19. Simplified preparation of a phosphatase inhibitor and further studies of its action.

    Science.gov (United States)

    Coburn, S P; Schaltenbrand, W E

    1978-05-01

    1-Pyrrolidinecarbothioic acid (2-pyridylmethylene) hydrazide chelates Zn2+ but not Mg2+. This compound is about twice as effective as EDTA for inhibiting alkaline phosphatase from calf mucosa, and approx. 1000-fold more effective than EDTA for inhibiting acid phosphatase from wheat germ. The compound did not inhibit pyridoxine kinase activity in human leucocytes at the highest concentration tested (33 micron). Therefore it may be a useful tool for either examining or eliminating the effects of phosphatases in complex enzyme systems.

  20. Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase[S

    OpenAIRE

    Oguro, Ami; Imaoka, Susumu

    2012-01-01

    Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hyd...

  1. The Role of DmCatD, a Cathepsin D-Like Peptidase, and Acid Phosphatase in the Process of Follicular Atresia in Dipetalogaster maxima (Hemiptera: Reduviidae), a Vector of Chagas' Disease

    Science.gov (United States)

    Leyria, Jimena; Fruttero, Leonardo L.; Nazar, Magalí; Canavoso, Lilián E.

    2015-01-01

    In this work, we have investigated the involvement of DmCatD, a cathepsin D-like peptidase, and acid phosphatase in the process of follicular atresia of Dipetalogaster maxima, a hematophagous insect vector of Chagas’ disease. For the studies, fat bodies, ovaries and hemolymph were sampled from anautogenous females at representative days of the reproductive cycle: pre-vitellogenesis, vitellogenesis as well as early and late atresia. Real time PCR (qPCR) and western blot assays showed that DmCatD was expressed in fat bodies and ovaries at all reproductive stages, being the expression of its active form significantly higher at the atretic stages. In hemolymph samples, only the immunoreactive band compatible with pro-DmCatD was observed by western blot. Acid phosphatase activity in ovarian tissues significantly increased during follicular atresia in comparison to pre-vitellogenesis and vitellogenesis. A further enzyme characterization with inhibitors showed that the high levels of acid phosphatase activity in atretic ovaries corresponded mainly to a tyrosine phosphatase. Immunofluorescence assays demonstrated that DmCatD and tyrosine phosphatase were associated with yolk bodies in vitellogenic follicles, while in atretic stages they displayed a different cellular distribution. DmCatD and tyrosine phosphatase partially co-localized with vitellin. Moreover, their interaction was supported by FRET analysis. In vitro assays using homogenates of atretic ovaries as the enzyme source and enzyme inhibitors demonstrated that DmCatD, together with a tyrosine phosphatase, were necessary to promote the degradation of vitellin. Taken together, the results strongly suggested that both acid hydrolases play a central role in early vitellin proteolysis during the process of follicular atresia. PMID:26091289

  2. Characterization and site-directed mutagenesis of Wzb, an O-phosphatase from Lactobacillus rhamnosus

    Directory of Open Access Journals (Sweden)

    Gilbert Christophe

    2008-04-01

    Full Text Available Abstract Background Reversible phosphorylation events within a polymerisation complex have been proposed to modulate capsular polysaccharide synthesis in Streptococcus pneumoniae. Similar phosphatase and kinase genes are present in the exopolysaccharide (EPS biosynthesis loci of numerous lactic acid bacteria genomes. Results The protein sequence deduced from the wzb gene in Lactobacillus rhamnosus ATCC 9595 reveals four motifs of the polymerase and histidinol phosphatase (PHP superfamily of prokaryotic O-phosphatases. Native and modified His-tag fusion Wzb proteins were purified from Escherichia coli cultures. Extracts showed phosphatase activity towards tyrosine-containing peptides. The purified fusion protein Wzb was active on p-nitrophenyl-phosphate (pNPP, with an optimal activity in presence of bovine serum albumin (BSA 1% at pH 7.3 and a temperature of 75°C. At 50°C, residual activity decreased to 10 %. Copper ions were essential for phosphatase activity, which was significantly increased by addition of cobalt. Mutated fusion Wzb proteins exhibited reduced phosphatase activity on p-nitrophenyl-phosphate. However, one variant (C6S showed close to 20% increase in phosphatase activity. Conclusion These characteristics reveal significant differences with the manganese-dependent CpsB protein tyrosine phosphatase described for Streptococcus pneumoniae as well as with the polysaccharide-related phosphatases of Gram negative bacteria.

  3. Growth and extracellular phosphatase activity of arbuscular mycorrhizal hyphae as influenced by soil organic matter

    DEFF Research Database (Denmark)

    Joner, E.J.; Jakobsen, I.

    1995-01-01

    Two experiments were set up to investigate the influence of soil organic matter on growth of arbuscular mycorrhizal (AM) hyphae and concurrent changes in soil inorganic P, organic P and phosphatase activity. A sandy loam soil was kept for 14 months under two regimes (outdoor where surplus...... additions. In soil with added clover alkaline phosphatase activity increased due to the presence of mycorrhizal hyphae. We suggest that mycorrhizas may influence the exudation of acid phosphatase by roots. Hyphae of G. invermaium did apparently not excrete extracellular phosphatases, but their presence may...

  4. ATP catabolism by tissue nonspecific alkaline phosphatase contributes to development of ARDS in influenza-infected mice.

    Science.gov (United States)

    Woods, Parker S; Doolittle, Lauren M; Hickman-Davis, Judy M; Davis, Ian C

    2018-01-01

    Influenza A viruses are highly contagious respiratory pathogens that are responsible for significant morbidity and mortality worldwide on an annual basis. We have shown previously that influenza infection of mice leads to increased ATP and adenosine accumulation in the airway lumen. Moreover, we demonstrated that A 1 -adenosine receptor activation contributes significantly to influenza-induced acute respiratory distress syndrome (ARDS). However, we found that development of ARDS in influenza-infected mice does not require catabolism of ATP to adenosine by ecto-5'-nucleotidase (CD73). Hence, we hypothesized that increased adenosine generation in response to infection is mediated by tissue nonspecific alkaline phosphatase (TNAP), which is a low-affinity, high-capacity enzyme that catabolizes nucleotides in a nonspecific manner. In the current study, we found that whole lung and BALF TNAP expression and alkaline phosphatase enzymatic activity increased as early as 2 days postinfection (dpi) of C57BL/6 mice with 10,000 pfu/mouse of influenza A/WSN/33 (H1N1). Treatment at 2 and 4 dpi with a highly specific quinolinyl-benzenesulfonamide TNAP inhibitor (TNAPi) significantly reduced whole lung alkaline phosphatase activity at 6 dpi but did not alter TNAP gene or protein expression. TNAPi treatment attenuated hypoxemia, lung dysfunction, histopathology, and pulmonary edema at 6 dpi without impacting viral replication or BALF adenosine. Treatment also improved epithelial barrier function and attenuated cellular and humoral immune responses to influenza infection. These data indicate that TNAP inhibition can attenuate influenza-induced ARDS by reducing inflammation and fluid accumulation within the lung. They also further emphasize the importance of adenosine generation for development of ARDS in influenza-infected mice.

  5. A new monoclonal antibody detects downregulation of protein tyrosine phosphatase receptor type γ in chronic myeloid leukemia patients

    Directory of Open Access Journals (Sweden)

    Marzia Vezzalini

    2017-06-01

    Full Text Available Abstract Background Protein tyrosine phosphatase receptor gamma (PTPRG is a ubiquitously expressed member of the protein tyrosine phosphatase family known to act as a tumor suppressor gene in many different neoplasms with mechanisms of inactivation including mutations and methylation of CpG islands in the promoter region. Although a critical role in human hematopoiesis and an oncosuppressor role in chronic myeloid leukemia (CML have been reported, only one polyclonal antibody (named chPTPRG has been described as capable of recognizing the native antigen of this phosphatase by flow cytometry. Protein biomarkers of CML have not yet found applications in the clinic, and in this study, we have analyzed a group of newly diagnosed CML patients before and after treatment. The aim of this work was to characterize and exploit a newly developed murine monoclonal antibody specific for the PTPRG extracellular domain (named TPγ B9-2 to better define PTPRG protein downregulation in CML patients. Methods TPγ B9-2 specifically recognizes PTPRG (both human and murine by flow cytometry, western blotting, immunoprecipitation, and immunohistochemistry. Results Co-localization experiments performed with both anti-PTPRG antibodies identified the presence of isoforms and confirmed protein downregulation at diagnosis in the Philadelphia-positive myeloid lineage (including CD34+/CD38bright/dim cells. After effective tyrosine kinase inhibitor (TKI treatment, its expression recovered in tandem with the return of Philadelphia-negative hematopoiesis. Of note, PTPRG mRNA levels remain unchanged in tyrosine kinase inhibitors (TKI non-responder patients, confirming that downregulation selectively occurs in primary CML cells. Conclusions The availability of this unique antibody permits its evaluation for clinical application including the support for diagnosis and follow-up of these disorders. Evaluation of PTPRG as a potential therapeutic target is also facilitated by the

  6. Cloning of soluble alkaline phosphatase cDNA and molecular basis of the polymorphic nature in alkaline phosphatase isozymes of Bombyx mori midgut.

    Science.gov (United States)

    Itoh, M; Kanamori, Y; Takao, M; Eguchi, M

    1999-02-01

    A cDNA coding for soluble type alkaline phosphatase (sALP) of Bombyx mori was isolated. Deduced amino acid sequence showed high identities to various ALPs and partial similarities to ATPase of Manduca sexta. Using this cDNA sequence as a probe, the molecular basis of electrophoretic polymorphism in sALP and membrane-bound type ALP (mALP) was studied. As for mALP, the result suggested that post-translational modification was important for the proteins to express activity and to represent their extensive polymorphic nature, whereas the magnitude of activities was mainly regulated by transcription. On the other hand, sALP zymogram showed poor polymorphism, but one exception was the null mutant, in which the sALP gene was largely lost. Interestingly, the sALP gene was shown to be transcribed into two mRNAs of different sizes, 2.0 and 2.4 Kb. In addition to the null mutant of sALP, we found a null mutant for mALP. Both of these mutants seem phenotypically silent, suggesting that the functional differentiation between these isozymes is not perfect, so that they can still work mutually and complement each other as an indispensable enzyme for B. mori.

  7. Protein kinase and phosphatase activities of thylakoid membranes

    International Nuclear Information System (INIS)

    Michel, H.; Shaw, E.K.; Bennett, J.

    1987-01-01

    Dephosphorylation of the 25 and 27 kDa light-harvesting Chl a/b proteins (LHCII) of the thylakoid membranes is catalyzed by a phosphatase which differs from previously reported thylakoid-bound phosphatases in having an alkaline pH optimum (9.0) and a requirement for Mg 2+ ions. Dephosphorylation of the 8.3 kDa psb H gene product requires a Mg 2+ ion concentration more than 200 fold higher than that for dephosphorylation of LHC II. The 8.3 kDa and 27 kDa proteins appear to be phosphorylated by two distinct kinases, which differ in substrate specificity and sensitivity to inhibitors. The plastoquinone antagonist 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone (DBMIB) inhibits phosphorylation of the 27 kDa LHC II much more readily than phosphorylation of the 8.3 kDa protein. A similar pattern of inhibition is seen for two synthetic oligopeptides (MRKSATTKKAVC and ATQTLESSSRC) which are analogs of the phosphorylation sites of the two proteins. Possible modes of action of DBMIB are discussed. 45 refs., 7 figs., 3 tabs

  8. Semi-Automatic Rating Method for Neutrophil Alkaline Phosphatase Activity.

    Science.gov (United States)

    Sugano, Kanae; Hashi, Kotomi; Goto, Misaki; Nishi, Kiyotaka; Maeda, Rie; Kono, Keigo; Yamamoto, Mai; Okada, Kazunori; Kaga, Sanae; Miwa, Keiko; Mikami, Taisei; Masauzi, Nobuo

    2017-01-01

    The neutrophil alkaline phosphatase (NAP) score is a valuable test for the diagnosis of myeloproliferative neoplasms, but it has still manually rated. Therefore, we developed a semi-automatic rating method using Photoshop ® and Image-J, called NAP-PS-IJ. Neutrophil alkaline phosphatase staining was conducted with Tomonaga's method to films of peripheral blood taken from three healthy volunteers. At least 30 neutrophils with NAP scores from 0 to 5+ were observed and taken their images. From which the outer part of neutrophil was removed away with Image-J. These were binarized with two different procedures (P1 and P2) using Photoshop ® . NAP-positive area (NAP-PA) and granule (NAP-PGC) were measured and counted with Image-J. The NAP-PA in images binarized with P1 significantly (P < 0.05) differed between images with NAP scores from 0 to 3+ (group 1) and those from 4+ to 5+ (group 2). The original images in group 1 were binarized with P2. NAP-PGC of them significantly (P < 0.05) differed among all four NAP score groups. The mean NAP-PGC with NAP-PS-IJ indicated a good correlation (r = 0.92, P < 0.001) to results by human examiners. The sensitivity and specificity of NAP-PS-IJ were 60% and 92%, which might be considered as a prototypic method for the full-automatic rating NAP score. © 2016 Wiley Periodicals, Inc.

  9. Alkaline phosphatase as a screening test for osteomalacia.

    Science.gov (United States)

    Chinoy, Muhammad Amin; Javed, Muhammad Imran; Khan, Alamzeb; Sadruddin, Nooruddin

    2011-01-01

    Vitamin D deficiency remains common in children and adults in Pakistan despite adequate sunlight exposure. Diagnosis in adults is usually delayed and is made following pathological fractures that result in significant morbidity. The objective of this study was to see whether Serum Alkaline Phosphatase levels could be used as a screening test for osteomalacia. The Study was conducted at Fatima Hospital, Baqai Medical University, Gadap, Karachi, between July 2002 and June 2005. Serum calcium levels are commonly used to screen patients suspected of osteomalacia, and raised serum alkaline phosphatase (SALP) is considered a diagnostic finding. We used SALP to screen patients who presented with back or non-specific aches and pain of more than six months duration. Three hundred thirty-four (334) patients were screened of which 116 (35%) had raised SALP. Osteomalacia was diagnosed in 92 (79.3%) of these 116 either by plain radiographs, bone biopsy or isotope bone scan. Fifty-four (53.4%) of the 101 cases had a normal level of serum calcium. Osteomalacia is likely to be missed if only serum calcium is used to screen patients. Serum Alkaline Phosphate should be used as the preferred method for screening these patients.

  10. Spatial control of protein phosphatase 2A (de)methylation

    International Nuclear Information System (INIS)

    Longin, Sari; Zwaenepoel, Karen; Martens, Ellen; Louis, Justin V.; Rondelez, Evelien; Goris, Jozef; Janssens, Veerle

    2008-01-01

    Reversible methylation of the protein phosphatase 2A catalytic subunit (PP2A C ) is an important regulatory mechanism playing a crucial role in the selective recruitment of regulatory B subunits. Here, we investigated the subcellular localization of leucine carboxyl methyltransferase (LCMT1) and protein phosphatase methylesterase (PME-1), the two enzymes catalyzing this process. The results show that PME-1 is predominantly localized in the nucleus and harbors a functional nuclear localization signal, whereas LCMT1 is underrepresented in the nucleus and mainly localizes to the cytoplasm, Golgi region and late endosomes. Indirect immunofluorescence with methylation-sensitive anti-PP2A C antibodies revealed a good correlation with the methylation status of PP2A C , demethylated PP2A C being substantially nuclear. Throughout mitosis, demethylated PP2A C is associated with the mitotic spindle and during cytokinesis with the cleavage furrow. Overexpression of PME-1, but not of an inactive mutant, results in increased demethylation of PP2A C in the nucleus, whereas overexpression of a cytoplasmic PME-1 mutant lacking the NLS results in increased demethylation in the cytoplasm-in all cases, however, without any obvious functional consequences. PME-1 associates with an inactive PP2A population, regardless of its esterase activity or localization. We propose that stabilization of this inactive, nuclear PP2A pool is a major in vivo function of PME-1

  11. Src-homology 2 domain-containing tyrosine phosphatase 2 promotes oral cancer invasion and metastasis

    Science.gov (United States)

    2014-01-01

    Background Tumor invasion and metastasis represent a major unsolved problem in cancer pathogenesis. Recent studies have indicated the involvement of Src-homology 2 domain-containing tyrosine phosphatase 2 (SHP2) in multiple malignancies; however, the role of SHP2 in oral cancer progression has yet to be elucidated. We propose that SHP2 is involved in the progression of oral cancer toward metastasis. Methods SHP2 expression was evaluated in paired oral cancer tissues by using immunohistochemical staining and real-time reverse transcription polymerase chain reaction. Isogenic highly invasive oral cancer cell lines from their respective low invasive parental lines were established using a Boyden chamber assay, and changes in the hallmarks of the epithelial-mesenchymal transition (EMT) were assessed to evaluate SHP2 function. SHP2 activity in oral cancer cells was reduced using si-RNA knockdown or enforced expression of a catalytically deficient mutant to analyze migratory and invasive ability in vitro and metastasis toward the lung in mice in vivo. Results We observed the significant upregulation of SHP2 in oral cancer tissues and cell lines. Following SHP2 knockdown, the oral cancer cells markedly attenuated migratory and invasion ability. We observed similar results in phosphatase-dead SHP2 C459S mutant expressing cells. Enhanced invasiveness was associated with significant upregulation of E-cadherin, vimentin, Snail/Twist1, and matrix metalloproteinase-2 in the highly invasive clones. In addition, we determined that SHP2 activity is required for the downregulation of phosphorylated ERK1/2, which modulates the downstream effectors, Snail and Twist1 at a transcript level. In lung tissue sections of mice, we observed that HSC3 tumors with SHP2 deletion exhibited significantly reduced metastatic capacity, compared with tumors administered control si-RNA. Conclusions Our data suggest that SHP2 promotes the invasion and metastasis of oral cancer cells. These results

  12. TORC1 regulates Pah1 phosphatidate phosphatase activity via the Nem1/Spo7 protein phosphatase complex.

    Directory of Open Access Journals (Sweden)

    Emmanuelle Dubots

    Full Text Available The evolutionarily conserved target of rapamycin complex 1 (TORC1 controls growth-related processes such as protein, nucleotide, and lipid metabolism in response to growth hormones, energy/ATP levels, and amino acids. Its deregulation is associated with cancer, type 2 diabetes, and obesity. Among other substrates, mammalian TORC1 directly phosphorylates and inhibits the phosphatidate phosphatase lipin-1, a central enzyme in lipid metabolism that provides diacylglycerol for the synthesis of membrane phospholipids and/or triacylglycerol as neutral lipid reserve. Here, we show that yeast TORC1 inhibits the function of the respective lipin, Pah1, to prevent the accumulation of triacylglycerol. Surprisingly, TORC1 regulates Pah1 in part indirectly by controlling the phosphorylation status of Nem1 within the Pah1-activating, heterodimeric Nem1-Spo7 protein phosphatase module. Our results delineate a hitherto unknown TORC1 effector branch that controls lipin function in yeast, which, given the recent discovery of Nem1-Spo7 orthologous proteins in humans, may be conserved.

  13. Compensatory Role of Inositol 5-Phosphatase INPP5B to OCRL in Primary Cilia Formation in Oculocerebrorenal Syndrome of Lowe.

    Directory of Open Access Journals (Sweden)

    Na Luo

    Full Text Available Inositol phosphatases are important regulators of cell signaling, polarity, and vesicular trafficking. Mutations in OCRL, an inositol polyphosphate 5-phosphatase, result in Oculocerebrorenal syndrome of Lowe, an X-linked recessive disorder that presents with congenital cataracts, glaucoma, renal dysfunction and mental retardation. INPP5B is a paralog of OCRL and shares similar structural domains. The roles of OCRL and INPP5B in the development of cataracts and glaucoma are not understood. Using ocular tissues, this study finds low levels of INPP5B present in human trabecular meshwork but high levels in murine trabecular meshwork. In contrast, OCRL is localized in the trabecular meshwork and Schlemm's canal endothelial cells in both human and murine eyes. In cultured human retinal pigmented epithelial cells, INPP5B was observed in the primary cilia. A functional role for INPP5B is revealed by defects in cilia formation in cells with silenced expression of INPP5B. This is further supported by the defective cilia formation in zebrafish Kupffer's vesicles and in cilia-dependent melanosome transport assays in inpp5b morphants. Taken together, this study indicates that OCRL and INPP5B are differentially expressed in the human and murine eyes, and play compensatory roles in cilia development.

  14. Reduced levels of membrane-bound alkaline phosphatase are common to lepidopteran strains resistant to Cry toxins from Bacillus thuringiensis.

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    Juan Luis Jurat-Fuentes

    Full Text Available Development of insect resistance is one of the main concerns with the use of transgenic crops expressing Cry toxins from the bacterium Bacillus thuringiensis. Identification of biomarkers would assist in the development of sensitive DNA-based methods to monitor evolution of resistance to Bt toxins in natural populations. We report on the proteomic and genomic detection of reduced levels of midgut membrane-bound alkaline phosphatase (mALP as a common feature in strains of Cry-resistant Heliothis virescens, Helicoverpa armigera and Spodoptera frugiperda when compared to susceptible larvae. Reduced levels of H. virescens mALP protein (HvmALP were detected by two dimensional differential in-gel electrophoresis (2D-DIGE analysis in Cry-resistant compared to susceptible larvae, further supported by alkaline phosphatase activity assays and Western blotting. Through quantitative real-time polymerase chain reaction (qRT-PCR we demonstrate that the reduction in HvmALP protein levels in resistant larvae are the result of reduced transcript amounts. Similar reductions in ALP activity and mALP transcript levels were also detected for a Cry1Ac-resistant strain of H. armigera and field-derived strains of S. frugiperda resistant to Cry1Fa. Considering the unique resistance and cross-resistance phenotypes of the insect strains used in this work, our data suggest that reduced mALP expression should be targeted for development of effective biomarkers for resistance to Cry toxins in lepidopteran pests.

  15. Characterizing the interactions between prolyl isomerase pin1 and phosphatase inhibitor-2 in living cells with FRET and FCS

    Science.gov (United States)

    Sun, Yuansheng; Wang, Lifu; Jyothikumar, Vinod; Brautigan, David L.; Periasamy, Ammasi

    2012-03-01

    Phosphatase inhibitor-2 (I2) was discovered as a regulator of protein Ser/Thr phosphatase-1 and is conserved from yeast to human. Binding between purified recombinant I2 from different species and the prolyl isomerase Pin1 has been demonstrated with pull-down assays, size exclusion chromatography and nuclear magnetic resonance spectroscopy. Despite this, questions persist as to whether these proteins associate together in living cells. In this study, we prepared fluorescent protein (FP) fusions of I2 and Pin1 and employed both Förster Resonance Energy Transfer (FRET) and Fluorescence Correlation Spectroscopy (FCS) imaging techniques to characterize their interactions in living cells. In both intensity-based and time-resolved FRET studies, we observed FRET uniformly across whole cells co-expressing I2-Cerulean and Pin1-Venus that was significantly higher than in negative controls expressing Cerulean FP (without fusing to I2) as the FRET donor and Pin1-Venus, showing a specific interaction between I2-Cerulean and Pin1-Venus in living cells. We also observed the co-diffusion of I2-Cerulean and Pin1-mCherry in Fluorescence Cross Correlation Spectroscopy (FCCS) measurements. We further showed that I2 itself as well as I2-Pin1 formed complexes in living cells (predicted from in vitro studies) via a quantitative FRET assay, and demonstrated from FCS measurements that both I2 and Pin1 (fused to Cerulean) are highly mobile in living cells.

  16. The dual-specificity phosphatase MKP-1 limits the cardiac hypertrophic response in vitro and in vivo.

    Science.gov (United States)

    Bueno, O F; De Windt, L J; Lim, H W; Tymitz, K M; Witt, S A; Kimball, T R; Molkentin, J D

    2001-01-19

    Mitogen-activated protein kinase (MAPK) signaling pathways are important regulators of cell growth, proliferation, and stress responsiveness. A family of dual-specificity MAP kinase phosphatases (MKPs) act as critical counteracting factors that directly regulate the magnitude and duration of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) activation. Here we show that constitutive expression of MKP-1 in cultured primary cardiomyocytes using adenovirus-mediated gene transfer blocked the activation of p38, JNK1/2, and ERK1/2 and prevented agonist-induced hypertrophy. Transgenic mice expressing physiological levels of MKP-1 in the heart showed (1) no activation of p38, JNK1/2, or ERK1/2; (2) diminished developmental myocardial growth; and (3) attenuated hypertrophy in response to aortic banding and catecholamine infusion. These results provide further evidence implicating MAPK signaling factors as obligate regulators of cardiac growth and hypertrophy and demonstrate the importance of dual-specificity phosphatases as counterbalancing regulatory factors in the heart.

  17. Protein tyrosine phosphatase receptor delta acts as a neuroblastoma tumor suppressor by destabilizing the aurora kinase a oncogene

    LENUS (Irish Health Repository)

    Meehan, Maria

    2012-02-05

    Abstract Background Protein tyrosine phosphatase receptor delta (PTPRD) is a member of a large family of protein tyrosine phosphatases which negatively regulate tyrosine phosphorylation. Neuroblastoma is a major childhood cancer arising from precursor cells of the sympathetic nervous system which is known to acquire deletions and alterations in the expression patterns of PTPRD, indicating a potential tumor suppressor function for this gene. The molecular mechanism, however, by which PTPRD renders a tumor suppressor effect in neuroblastoma is unknown. Results As a molecular mechanism, we demonstrate that PTPRD interacts with aurora kinase A (AURKA), an oncogenic protein that is over-expressed in multiple forms of cancer, including neuroblastoma. Ectopic up-regulation of PTPRD in neuroblastoma dephosphorylates tyrosine residues in AURKA resulting in a destabilization of this protein culminating in interfering with one of AURKA\\'s primary functions in neuroblastoma, the stabilization of MYCN protein, the gene of which is amplified in approximately 25 to 30% of high risk neuroblastoma. Conclusions PTPRD has a tumor suppressor function in neuroblastoma through AURKA dephosphorylation and destabilization and a downstream destabilization of MYCN protein, representing a novel mechanism for the function of PTPRD in neuroblastoma.

  18. Co-ordinate regulation of growth factor receptors and lipid phosphate phosphatase-1 controls cell activation by exogenous lysophosphatidate.

    Science.gov (United States)

    Pilquil, C; Ling, Z C; Singh, I; Buri, K; Zhang, Q X; Brindley, D N

    2001-11-01

    The serum-derived lipid growth factors, lysophosphatidate (LPA) and sphingosine 1-phosphate (S1P), activate cells selectively through different members of a family of endothelial differentiation gene (EDG) receptors. Activation of EDG receptors by LPA and S1P provides a variety of signalling cascades depending upon the G-protein coupling of the different EDG receptors. This leads to chemotactic and mitogenic responses, which are important in wound healing. For example, LPA stimulates fibroblast division and S1P stimulates the chemotaxis and division of endothelial cells leading to angiogenesis. Counteracting these effects of LPA and S1P, are the actions of lipid phosphate phosphatases (LPP, or phosphatidate phosphohydrolases, Type 2). The isoform LPP-1 is expressed in the plasma membrane with its active site outside the cell. This enzyme is responsible for 'ecto-phosphatase' activity leading to the degradation of exogenous lipid phosphate mediators, particularly LPA. Expression of LPP-1 decreases cell activation by exogenous LPA. The mechanism for this is controversial and several mechanisms have been proposed. Evidence will be presented that the LPPs cross-talk with EDG and other growth factor receptors, thus, regulating the responses of the cells to lipid phosphate mediators of signal transduction.

  19. Crystallization and preliminary X-ray analysis of the stress-response PPM phosphatase RsbX from Bacillus subtilis

    International Nuclear Information System (INIS)

    Suganuma, Masatoshi; Teh, Aik Hong; Makino, Masatomo; Shimizu, Nobutaka; Kaneko, Tomonori; Hirata, Kunio; Yamamoto, Masaki; Kumasaka, Takashi

    2009-01-01

    The bacterial PPM phosphatase RsbX from B. subtilis was expressed in E. coli, purified and crystallized. The crystal belonged to space group P1 and diffracted to 1.06 Å resolution. RsbX from Bacillus subtilis is a manganese-dependent PPM phosphatase and negatively regulates the signal transduction of the general stress response by the dephosphorylation of RsbS and RsbR, which are activators of the alternative RNA polymerase σ factor SigB. In order to elucidate the structural–functional relationship of its Ser/Thr protein-phosphorylation mechanism, an X-ray crystallographic diffraction study of RsbX was performed. Recombinant RsbX was expressed in Escherichia coli, purified and crystallized. Crystals were obtained using the sitting-drop vapour-diffusion method and X-ray diffraction data were collected to 1.06 Å resolution with an R merge of 8.1%. The crystals belonged to the triclinic space group P1, with unit-cell parameters a = 33.3, b = 41.7, c = 68.6 Å, α = 98.8, β = 90.0, γ = 108.4°

  20. Dynamic Changes in Yeast Phosphatase Families Allow for Specialization in Phosphate and Thiamine Starvation.

    Science.gov (United States)

    Nahas, John V; Iosue, Christine L; Shaik, Noor F; Selhorst, Kathleen; He, Bin Z; Wykoff, Dennis D

    2018-05-10

    Convergent evolution is often due to selective pressures generating a similar phenotype. We observe relatively recent duplications in a spectrum of Saccharomycetaceae yeast species resulting in multiple phosphatases that are regulated by different nutrient conditions - thiamine and phosphate starvation. This specialization is both transcriptional and at the level of phosphatase substrate specificity. In Candida glabrata , loss of the ancestral phosphatase family was compensated by the co-option of a different histidine phosphatase family with three paralogs. Using RNA-seq and functional assays, we identify one of these paralogs, CgPMU3 , as a thiamine phosphatase. We further determine that the 81% identical paralog CgPMU2 does not encode thiamine phosphatase activity; however, both are capable of cleaving the phosphatase substrate, 1-napthyl-phosphate. We functionally demonstrate that members of this family evolved novel enzymatic functions for phosphate and thiamine starvation, and are regulated transcriptionally by either nutrient condition, and observe similar trends in other yeast species. This independent, parallel evolution involving two different families of histidine phosphatases suggests that there were likely similar selective pressures on multiple yeast species to recycle thiamine and phosphate. In this work, we focused on duplication and specialization, but there is also repeated loss of phosphatases, indicating that the expansion and contraction of the phosphatase family is dynamic in many Ascomycetes. The dynamic evolution of the phosphatase gene families is perhaps just one example of how gene duplication, co-option, and transcriptional and functional specialization together allow species to adapt to their environment with existing genetic resources. Copyright © 2018, G3: Genes, Genomes, Genetics.

  1. The TriTryp Phosphatome: analysis of the protein phosphatase catalytic domains

    Directory of Open Access Journals (Sweden)

    Huxley-Jones Julie

    2007-11-01

    Full Text Available Abstract Background The genomes of the three parasitic protozoa Trypanosoma cruzi, Trypanosoma brucei and Leishmania major are the main subject of this study. These parasites are responsible for devastating human diseases known as Chagas disease, African sleeping sickness and cutaneous Leishmaniasis, respectively, that affect millions of people in the developing world. The prevalence of these neglected diseases results from a combination of poverty, inadequate prevention and difficult treatment. Protein phosphorylation is an important mechanism of controlling the development of these kinetoplastids. With the aim to further our knowledge of the biology of these organisms we present a characterisation of the phosphatase complement (phosphatome of the three parasites. Results An ontology-based scan of the three genomes was used to identify 86 phosphatase catalytic domains in T. cruzi, 78 in T. brucei, and 88 in L. major. We found interesting differences with other eukaryotic genomes, such as the low proportion of tyrosine phosphatases and the expansion of the serine/threonine phosphatase family. Additionally, a large number of atypical protein phosphatases were identified in these species, representing more than one third of the total phosphatase complement. Most of the atypical phosphatases belong to the dual-specificity phosphatase (DSP family and show considerable divergence from classic DSPs in both the domain organisation and sequence features. Conclusion The analysis of the phosphatome of the three kinetoplastids indicates that they possess orthologues to many of the phosphatases reported in other eukaryotes, including humans. However, novel domain architectures and unusual combinations of accessory domains, suggest distinct functional roles for several of the kinetoplastid phosphatases, which await further experimental exploration. These distinct traits may be exploited in the selection of suitable new targets for drug development to prevent

  2. Functional analysis of the glycogen binding subunit CG9238/Gbs-70E of protein phosphatase 1 in Drosophila melanogaster.

    Science.gov (United States)

    Kerekes, Éva; Kókai, Endre; Páldy, Ferenc Sándor; Dombrádi, Viktor

    2014-06-01

    The product of the CG9238 gene that we termed glycogen binding subunit 70E (Gbs-70E) was characterized by biochemical and molecular genetics methods. The interaction between Gbs-70E and all catalytic subunits of protein phosphatase 1 (Pp1-87B, Pp1-9C, Pp1-96A and Pp1-13C) of Drosophila melanogaster was confirmed by pairwise yeast two-hybrid tests, co-immunoprecipitation and pull down experiments. The binding of Gbs-70E to glycogen was demonstrated by sedimentation analysis. With RT-PCR we found that the mRNAs coding for the longer Gbs-70E PB/PC protein were expressed in all developmental stages of the fruit flies while the mRNA for the shorter Gbs-70E PA was restricted to the eggs and the ovaries of the adult females. The development specific expression of the shorter splice variant was not conserved in different Drosophila species. The expression level of the gene was manipulated by P-element insertions and gene deletion to analyze the functions of the gene product. A small or moderate reduction in the gene expression resulted in no significant changes, however, a deletion mutant expressing very low level of the transcript lived shorter and exhibited reduced glycogen content in the imagos. In addition, the gene deletion decreased the fertility of the fruit flies. Our results prove that Gbs-70E functions as the glycogen binding subunit of protein phosphatase 1 that regulates glycogen content and plays a role in the development of eggs in D. melanogaster. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Curcumin and Chronic Kidney Disease (CKD: Major Mode of Action through Stimulating Endogenous Intestinal Alkaline Phosphatase

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    Siddhartha S. Ghosh

    2014-12-01

    Full Text Available Curcumin, an active ingredient in the traditional herbal remedy and dietary spice turmeric (Curcuma longa, has significant anti-inflammatory properties. Chronic kidney disease (CKD, an inflammatory disease, can lead to end stage renal disease resulting in dialysis and transplant. Furthermore, it is frequently associated with other inflammatory disease such as diabetes and cardiovascular disorders. This review will focus on the clinically relevant inflammatory molecules that play a role in CKD and associated diseases. Various enzymes, transcription factors, growth factors modulate production and action of inflammatory molecules; curcumin can blunt the generation and action of these inflammatory molecules and ameliorate CKD as well as associated inflammatory disorders. Recent studies have shown that increased intestinal permeability results in the leakage of pro-inflammatory molecules (cytokines and lipopolysaccharides from gut into the circulation in diseases such as CKD, diabetes and atherosclerosis. This change in intestinal permeability is due to decreased expression of tight junction proteins and intestinal alkaline phosphatase (IAP. Curcumin increases the expression of IAP and tight junction proteins and corrects gut permeability. This action reduces the levels of circulatory inflammatory biomolecules. This effect of curcumin on intestine can explain why, despite poor bioavailability, curcumin has potential anti-inflammatory effects in vivo and beneficial effects on CKD.

  4. Mice deficient in transmembrane prostatic acid phosphatase display increased GABAergic transmission and neurological alterations.

    Directory of Open Access Journals (Sweden)

    Heidi O Nousiainen

    Full Text Available Prostatic acid phosphatase (PAP, the first diagnostic marker and present therapeutic target for prostate cancer, modulates nociception at the dorsal root ganglia (DRG, but its function in the central nervous system has remained unknown. We studied expression and function of TMPAP (the transmembrane isoform of PAP in the brain by utilizing mice deficient in TMPAP (PAP-/- mice. Here we report that TMPAP is expressed in a subpopulation of cerebral GABAergic neurons, and mice deficient in TMPAP show multiple behavioral and neurochemical features linked to hyperdopaminergic dysregulation and altered GABAergic transmission. In addition to increased anxiety, disturbed prepulse inhibition, increased synthesis of striatal dopamine, and augmented response to amphetamine, PAP-deficient mice have enlarged lateral ventricles, reduced diazepam-induced loss of righting reflex, and increased GABAergic tone in the hippocampus. TMPAP in the mouse brain is localized presynaptically, and colocalized with SNARE-associated protein snapin, a protein involved in synaptic vesicle docking and fusion, and PAP-deficient mice display altered subcellular distribution of snapin. We have previously shown TMPAP to reside in prostatic exosomes and we propose that TMPAP is involved in the control of GABAergic tone in the brain also through exocytosis, and that PAP deficiency produces a distinct neurological phenotype.

  5. 3' Phosphatase activity toward phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] by voltage-sensing phosphatase (VSP).

    Science.gov (United States)

    Kurokawa, Tatsuki; Takasuga, Shunsuke; Sakata, Souhei; Yamaguchi, Shinji; Horie, Shigeo; Homma, Koichi J; Sasaki, Takehiko; Okamura, Yasushi

    2012-06-19

    Voltage-sensing phosphatases (VSPs) consist of a voltage-sensor domain and a cytoplasmic region with remarkable sequence similarity to phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor phosphatase. VSPs dephosphorylate the 5' position of the inositol ring of both phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] upon voltage depolarization. However, it is unclear whether VSPs also have 3' phosphatase activity. To gain insights into this question, we performed in vitro assays of phosphatase activities of Ciona intestinalis VSP (Ci-VSP) and transmembrane phosphatase with tensin homology (TPTE) and PTEN homologous inositol lipid phosphatase (TPIP; one human ortholog of VSP) with radiolabeled PI(3,4,5)P(3). TLC assay showed that the 3' phosphate of PI(3,4,5)P(3) was not dephosphorylated, whereas that of phosphatidylinositol 3,4-bisphosphate [PI(3,4)P(2)] was removed by VSPs. Monitoring of PI(3,4)P(2) levels with the pleckstrin homology (PH) domain from tandem PH domain-containing protein (TAPP1) fused with GFP (PH(TAPP1)-GFP) by confocal microscopy in amphibian oocytes showed an increase of fluorescence intensity during depolarization to 0 mV, consistent with 5' phosphatase activity of VSP toward PI(3,4,5)P(3). However, depolarization to 60 mV showed a transient increase of GFP fluorescence followed by a decrease, indicating that, after PI(3,4,5)P(3) is dephosphorylated at the 5' position, PI(3,4)P(2) is then dephosphorylated at the 3' position. These results suggest that substrate specificity of the VSP changes with membrane potential.

  6. Structural basis for inhibition of the protein tyrosine phosphatase 1B by phosphotyrosine peptide mimetics

    NARCIS (Netherlands)

    Groves, M R; Yao, Z J; Roller, P P; Burke, T R; Barford, D

    1998-01-01

    Protein tyrosine phosphatases regulate diverse cellular processes and represent important targets for therapeutic intervention in a number of diseases. The crystal structures of protein tyrosine phosphatase 1B (PTP1B) in complex with small molecule inhibitors based upon two classes of

  7. Emerging issues in receptor protein tyrosine phosphatase function: lifting fog or simply shifting?

    DEFF Research Database (Denmark)

    Petrone, A; Sap, J

    2000-01-01

    Transmembrane (receptor) tyrosine phosphatases are intimately involved in responses to cell-cell and cell-matrix contact. Several important issues regarding the targets and regulation of this protein family are now emerging. For example, these phosphatases exhibit complex interactions with signal...

  8. Preparative resolution of D,L-threonine catalyzed by immobilized phosphatase.

    Science.gov (United States)

    Scollar, M P; Sigal, G; Klibanov, A M

    1985-03-01

    Hydrolysis of L- and D-O-phosphothreonines catalyzed by four different phosphatases, alkaline phosphatases from calf intestine and E. coli and acid phosphatases from wheat germ and potato, has been kinetically studied. Alkaline phosphatases were found to have comparable reactivities towards the optical isomers. On the other hand, both acid phosphatases displayed a marked stereoselectivity, hydrolyzing the L-ester much faster than its D counterpart. Wheat germ acid phosphatase was the most stereoselective enzyme: V(L)/V(D) = 24 and K(m,L)/K(m,D) = 0.17. This enzyme was immobilized (in k-carrageenan gel, followed by crosslinking with glutaraldehyde) and used for the preparative resolution of D,L-threonine: the latter was first chemically O-phosphorylated and then asymmetrically hydrolyzed by the immobilized phosphatase. As a result, gram quantities of L-threonine of high optical purity and O-phospho-D-threonine were prepared. Immobilized wheat germ phosphatase has been tested for the resolution of other racemic alcohols: serine, 2-amino-1-butanol, 1-amino-2-propanol, 2-octanol, and menthol. In all those cases, the enzyme was either not sufficiently stereoselective or too slow for preparative resolutions.

  9. Coupling between the voltage-sensing and phosphatase domains of Ci-VSP.

    Science.gov (United States)

    Villalba-Galea, Carlos A; Miceli, Francesco; Taglialatela, Maurizio; Bezanilla, Francisco

    2009-07-01

    The Ciona intestinalis voltage sensor-containing phosphatase (Ci-VSP) shares high homology with the phosphatidylinositol phosphatase enzyme known as PTEN (phosphatase and tensin homologue deleted on chromosome 10). We have taken advantage of the similarity between these proteins to inquire about the coupling between the voltage sensing and the phosphatase domains in Ci-VSP. Recently, it was shown that four basic residues (R11, K13, R14, and R15) in PTEN are critical for its binding onto the membrane, required for its catalytic activity. Ci-VSP has three of the basic residues of PTEN. Here, we show that when R253 and R254 (which are the homologues of R14 and R15 in PTEN) are mutated to alanines in Ci-VSP, phosphatase activity is disrupted, as revealed by a lack of effect on the ionic currents of KCNQ2/3, where current decrease is a measure of phosphatase activity. The enzymatic activity was not rescued by the introduction of lysines, indicating that the binding is an arginine-specific interaction between the phosphatase binding domain and the membrane, presumably through the phosphate groups of the phospholipids. We also found that the kinetics and steady-state voltage dependence of the S4 segment movement are affected when the arginines are not present, indicating that the interaction of R253 and R254 with the membrane, required for the catalytic action of the phosphatase, restricts the movement of the voltage sensor.

  10. The tillage effect on the soil acid and alkaline phosphatase activity

    Directory of Open Access Journals (Sweden)

    Lacramioara Oprica

    2011-12-01

    Full Text Available Phosphatases (acid and alkaline are important in soils because these extracellular enzymes catalyze the hydrolysis of organic phosphate esters to orthophosphate; thus they form an important link between biologically unavailable and mineral phosphorous. Phosphatase activity is sensitive to environmental perturbations such as organic amendments, tillage, waterlogging, compaction, fertilizer additions and thus it is often used as an environmental indicator of soil quality in riparian ecosystems. The aim of the study was to assess the effect of tillage systems on phosphatases activity in a field experiment carried out in Ezăreni farm. The phosphatase activitiy were determined at two depths (7-10 cm and 15-25cm layers of a chernozem soil submitted to conventional tillage (CT in a fertilised and unfertilised experiment. Monitoring soil alkaline phosphatase activity showed, generally, the same in fertilized soil profiles collected from both depths; the values being extremely close. In unfertilized soils, alkaline phosphatase activity is different only in soils that were exposed to unconventional work using disc harrows and 30cm tillage. Both works type (no tillage and conventional tillage cause an intense alkaline phosphatase activity in 7-10 cm soil profile. Acid phosphatase activity is highly fluctuating in both fertilized as well unfertilized soil, this enzyme being influenced by the performed works.

  11. Establishing Quantitative Standards for Residual Alkaline Phosphatase in Pasteurized Milk.

    Science.gov (United States)

    Kim, Dong-Hyeon; Chon, Jung-Whan; Lim, Jong-Soo; Kim, Hong-Seok; Kang, Il-Byeong; Jeong, Dana; Song, Kwang-Young; Kim, Hyunsook; Kim, Kwang-Yup; Seo, Kun-Ho

    2016-01-01

    The alkaline phosphatase (ALP) assay is a rapid and convenient method for verifying milk pasteurization. Since colorimetric ALP assays rely on subjective visual assessments, their results are especially unreliable near the detection limits. In this study, we attempted to establish quantitative criteria for residual ALP in milk by using a more objective method based on spectrophotometric measurements. Raw milk was heat-treated for 0, 10, 20, 30, and 40 min and then subjected to ALP assays. The quantitative criteria for residual ALP in the milk was determined as 2 μg phenol/mL of milk, which is just above the ALP value of milk samples heat-treated for 30 min. These newly proposed methodology and criteria could facilitate the microbiological quality control of milk.

  12. Radioimmunoassay for prostatic acid phosphatase in human serum. Methodologic aspects

    International Nuclear Information System (INIS)

    Pradalier, N.; Canal, P.; Pujol, A.; Fregevu, Y.; Soula, G.

    1982-01-01

    We propose a double antibody radioimmunoassay for human prostatic acid phosphatase (PAP) in serum for diagnosis and management of prostatic adenocarcinoma under treatment. The antigen is purified from human prostatic fluid by a gel-filtration on Sephadex G 100 followed by affinity chromatography on Con A Sepharose. A specific antibody is raised in rabbits and purified by immunoadsorption with a female serum. The described technique offers both radioisotopic sensibility and immunologic specificity. Physiological values determined in the serum of 125 healthy males are below 2 ng/ml. No significative differences are observed with age. The proposed technique also shows significant differences between values evaluated for benign prostatic hyperplasia and prostatic adenocarcinoma [fr

  13. The influence of complexing pharmaceutical compositions on alkaline phosphatase

    Science.gov (United States)

    Atyaksheva, L. F.; Chukhrai, E. S.; Stepina, N. D.; Novikova, N. N.; Yur'eva, E. A.

    2011-06-01

    It is established that the pharmaceutical compositions xydiphon, medifon, succimer, and EDTA, which are used as complexing agents for accelerating the excretion of heavy metals from human organism, at certain concentrations inhibit enzyme alkaline phosphatase (AP). It is concluded that xydiphon and EDTA have a noticeable effect on AP activity at concentrations over 0.01 mM; medifon and succimer, at concentrations of over 0.3-0.5 mM. The enzyme's inhibition constants and type of inhibition are determined. Xydiphon is found to manifest the highest affinity to AP ( K I = 0.35 mM). It is shown by kinetic analysis that dissociative chemoinactivation of the enzyme takes place under the action of complexing agents. The corresponding kinetic parameters are calculated.

  14. Suppressing Type 2C Protein Phosphatases Alters Fruit Ripening and the Stress Response in Tomato.

    Science.gov (United States)

    Zhang, Yushu; Li, Qian; Jiang, Li; Kai, Wenbin; Liang, Bin; Wang, Juan; Du, Yangwei; Zhai, Xiawan; Wang, Jieling; Zhang, Yingqi; Sun, Yufei; Zhang, Lusheng; Leng, Ping

    2018-01-01

    Although ABA signaling has been widely studied in Arabidopsis, the roles of core ABA signaling components in fruit remain poorly understood. Herein, we characterize SlPP2C1, a group A type 2C protein phosphatase that negatively regulates ABA signaling and fruit ripening in tomato. The SlPP2C1 protein was localized in the cytoplasm close to AtAHG3/AtPP2CA. The SlPP2C1 gene was expressed in all tomato tissues throughout development, particularly in flowers and fruits, and it was up-regulated by dehydration and ABA treatment. SlPP2C1 expression in fruits was increased at 30 d after full bloom and peaked at the B + 1 stage. Suppression of SlPP2C1 expression significantly accelerated fruit ripening which was associated with higher levels of ABA signaling genes that are reported to alter the expression of fruit ripening genes involved in ethylene release and cell wall catabolism. SlPP2C1-RNAi (RNA interference) led to increased endogenous ABA accumulation and advanced release of ethylene in transgenic fruits compared with wild-type (WT) fruits. SlPP2C1-RNAi also resulted in abnormal flowers and obstructed the normal abscission of pedicels. SlPP2C1-RNAi plants were hypersensitized to ABA, and displayed delayed seed germination and primary root growth, and increased resistance to drought stress compared with WT plants. These results demonstrated that SlPP2C1 is a functional component in the ABA signaling pathway which participates in fruit ripening, ABA responses and drought tolerance. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  15. Association between absolute tumor burden and serum bone-specific alkaline phosphatase in canine appendicular osteosarcoma.

    Science.gov (United States)

    Sternberg, R A; Pondenis, H C; Yang, X; Mitchell, M A; O'Brien, R T; Garrett, L D; Helferich, W G; Hoffmann, W E; Fan, T M

    2013-01-01

    In dogs with appendicular osteosarcoma (OSA), increased pretreatment serum bone-specific alkaline phosphatase (BALP) activity is a negative prognostic factor, associated with shorter disease-free intervals and survival times, but a biologic basis for observed differential serum BALP activities in canine OSA patients remains incompletely defined. Serum BALP activity will correlate with absolute tumor burden in dogs with OSA. This study included 96 client-owned dogs with appendicular OSA. In canine OSA cell lines, the expression and membranous release of BALP was evaluated in vitro. The correlation between serum BALP activity and radiographic primary tumor size was evaluated in OSA-bearing dogs. In dogs developing visceral OSA metastases, serial changes in serum BALP activities were evaluated in relation to progression of macroscopic metastases, and visceral metastatic OSA cells were evaluated for BALP expression. In vitro, BALP expression was not associated with either tumorigenic or metastatic phenotype, rather the quantity of membranous BALP released was proportional with cell density. In dogs devoid of macroscopic metastases, there was a positive correlation between serum BALP activity and absolute primary tumor size. In dogs with progressive OSA metastases, serum BALP activity increased and coincided with the development of macroscopic metastases. OSA cells derived from visceral metastatic lesions retained BALP expression. Tumor burden is a determinant of serum BALP activity in dogs with appendicular OSA. The association between increased pretreatment BALP activity and negative clinical prognosis may simply be attributed to greater initial tumor burden, and consequently more advanced tumor stage. Copyright © 2013 by the American College of Veterinary Internal Medicine.

  16. Serum creatinine and alkaline phosphatase levels are associated with severe chronic periodontitis.

    Science.gov (United States)

    Caúla, A L; Lira-Junior, R; Tinoco, E M B; Fischer, R G

    2015-12-01

    Periodontitis may alter systemic homeostasis and influence creatinine and alkaline phosphatase levels. Therefore, the aim of this study was to evaluate the relationship between severe chronic periodontitis and serum creatinine and alkaline phosphatase levels. One hundred patients were evaluated, 66 with severe chronic periodontitis (test group) and 34 periodontally healthy controls (control group). Medical, demographic and periodontal parameters were registered. Blood sample was collected after an overnight fast and serum creatinine and alkaline phosphatase levels were determined. There were significant differences between test and control groups in ethnicity, gender and educational level (p creatinine level (p creatinine and alkaline phosphatase levels. Severe chronic periodontitis was associated to lower creatinine and higher alkaline phosphatase levels. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. The involvement of glucose-6-phosphatase in mucilage secretion by root cap cells of Zea mays

    Science.gov (United States)

    Moore, R.; McClelen, C. E.

    1985-01-01

    In order to determine the involvement of glucose-6-phosphatase in mucilage secretion by root cap cells, we have cytochemically localized the enzyme in columella and peripheral cells of root caps of Zea mays. Glucose-6-phosphatase is associated with the plasmalemma and cell wall of columella cells. As columella cells differentiate into peripheral cells and begin to produce and secrete mucilage, glucose-6-phosphatase staining intensifies and becomes associated with the mucilage and, to a lesser extent, the cell wall. Cells being sloughed from the cap are characterized by glucose-6-phosphatase staining being associated with the vacuole and plasmalemma. These changes in enzyme localization during cellular differentiation in root caps suggest that glucose-6-phosphatase is involved in the production and/or secretion of mucilage by peripheral cells of Z. mays.

  18. Vanadate monomers and dimers both inhibit the human prostatic acid phosphatase.

    Science.gov (United States)

    Crans, D C; Simone, C M; Saha, A K; Glew, R H

    1989-11-30

    A combination of enzyme kinetics and 51V NMR spectroscopy was used to identify the species of vanadate that inhibits acid phosphatases. Monomeric vanadate was shown to inhibit wheat germ and potato acid phosphatases. At pH 5.5, the vanadate dimer inhibits the human prostatic acid phosphatase whereas at pH 7.0 it is the vanadate monomer that inhibits this enzyme. The pH-dependent shift in the affinity of the prostatic phosphatase for vanadate is presumably due to deprotonation of an amino acid side chain in or near the binding site resulting in a conformational change in the protein. pH may be a subtle effector of the insulin-like vanadate activity in biological systems and may explain some of the differences in selectivity observed with the protein phosphatases.

  19. Alkaline phosphatase levels in patients with coronary heart disease saliva and its relation with periodontal status

    Science.gov (United States)

    Yunita, Dina Suci; Masulili, Sri Lelyati C.; Tadjoedin, Fatimah M.; Radi, Basuni

    2017-02-01

    Coronary heart disease (CHD) is a disease that causes narrowing of the coronary arteries. Currently, there is a hypothesis regarding periodontal infection that increases risk for heart disease. Alkaline phosphatase (ALP) as a marker of inflammation will increase in atherosclerosis and periodontal disease. The objective of this research is analyzing the relationship between the levels of alkaline phosphatase in saliva with periodontal status in patients with CHD and non CHD. Here, saliva of 104 subjects were taken, each 1 ml, and levels of Alkaline Phosphatase was analyzed using Abbott ci4100 architect. We found that no significant difference of Alkaline Phosphatase levels in saliva between CHD patients and non CHD. Therefore, it can be concluded that Alkaline Phosphatase levels in patients with CHD saliva was higher than non CHD and no association between ALP levels with periodontal status.

  20. A new family of phosphoinositide phosphatases in microorganisms: identification and biochemical analysis

    Directory of Open Access Journals (Sweden)

    Bennett Hayley J

    2010-08-01

    Full Text Available Abstract Background Phosphoinositide metabolism is essential to membrane dynamics and impinges on many cellular processes, including phagocytosis. Modulation of phosphoinositide metabolism is important for pathogenicity and virulence of many human pathogens, allowing them to survive and replicate in the host cells. Phosphoinositide phosphatases from bacterial pathogens are therefore key players in this modulation and constitute attractive targets for chemotherapy. MptpB, a virulence factor from Mycobacterium tuberculosis, has phosphoinositide phosphatase activity and a distinct active site P-loop signature HCXXGKDR that shares characteristics with eukaryotic lipid phosphatases and protein tyrosine phosphatases. We used this P-loop signature as a "diagnostic motif" to identify related putative phosphatases with phosphoinositide activity in other organisms. Results We found more than 200 uncharacterised putative phosphatase sequences with the conserved signature in bacteria, with some related examples in fungi and protozoa. Many of the sequences identified belong to recognised human pathogens. Interestingly, no homologues were found in any other organisms including Archaea, plants, or animals. Phylogenetic analysis revealed that these proteins are unrelated to classic eukaryotic lipid phosphatases. However, biochemical characterisation of those from Listeria monocytogenes and Leishmania major, demonstrated that, like MptpB, they have phosphatase activity towards phosphoinositides. Mutagenesis studies established that the conserved Asp and Lys in the P-loop signature (HCXXGKDR are important in catalysis and substrate binding respectively. Furthermore, we provide experimental evidence that the number of basic residues in the P-loop is critical in determining activity towards poly-phosphoinositides. Conclusion This new family of enzymes in microorganisms shows distinct sequence and biochemical characteristics to classic eukaryotic lipid phosphatases

  1. A new family of phosphoinositide phosphatases in microorganisms: identification and biochemical analysis.

    Science.gov (United States)

    Beresford, Nicola J; Saville, Charis; Bennett, Hayley J; Roberts, Ian S; Tabernero, Lydia

    2010-08-02

    Phosphoinositide metabolism is essential to membrane dynamics and impinges on many cellular processes, including phagocytosis. Modulation of phosphoinositide metabolism is important for pathogenicity and virulence of many human pathogens, allowing them to survive and replicate in the host cells. Phosphoinositide phosphatases from bacterial pathogens are therefore key players in this modulation and constitute attractive targets for chemotherapy. MptpB, a virulence factor from Mycobacterium tuberculosis, has phosphoinositide phosphatase activity and a distinct active site P-loop signature HCXXGKDR that shares characteristics with eukaryotic lipid phosphatases and protein tyrosine phosphatases. We used this P-loop signature as a "diagnostic motif" to identify related putative phosphatases with phosphoinositide activity in other organisms. We found more than 200 uncharacterised putative phosphatase sequences with the conserved signature in bacteria, with some related examples in fungi and protozoa. Many of the sequences identified belong to recognised human pathogens. Interestingly, no homologues were found in any other organisms including Archaea, plants, or animals. Phylogenetic analysis revealed that these proteins are unrelated to classic eukaryotic lipid phosphatases. However, biochemical characterisation of those from Listeria monocytogenes and Leishmania major, demonstrated that, like MptpB, they have phosphatase activity towards phosphoinositides. Mutagenesis studies established that the conserved Asp and Lys in the P-loop signature (HCXXGKDR) are important in catalysis and substrate binding respectively. Furthermore, we provide experimental evidence that the number of basic residues in the P-loop is critical in determining activity towards poly-phosphoinositides. This new family of enzymes in microorganisms shows distinct sequence and biochemical characteristics to classic eukaryotic lipid phosphatases and they have no homologues in humans. This study provides

  2. Combining affinity proteomics and network context to identify new phosphatase substrates and adapters in growth pathways.

    Directory of Open Access Journals (Sweden)

    Francesca eSacco

    2014-05-01

    Full Text Available Protein phosphorylation homoeostasis is tightly controlled and pathological conditions are caused by subtle alterations of the cell phosphorylation profile. Altered levels of kinase activities have already been associated to specific diseases. Less is known about the impact of phosphatases, the enzymes that down-regulate phosphorylation by removing the phosphate groups. This is partly due to our poor understanding of the phosphatase-substrate network. Much of phosphatase substrate specificity is not based on intrinsic enzyme specificity with the catalytic pocket recognizing the sequence/structure context of the phosphorylated residue. In addition many phosphatase catalytic subunits do not form a stable complex with their substrates. This makes the inference and validation of phosphatase substrates a non-trivial task. Here, we present a novel approach that builds on the observation that much of phosphatase substrate selection is based on the network of physical interactions linking the phosphatase to the substrate. We first used affinity proteomics coupled to quantitative mass spectrometry to saturate the interactome of eight phosphatases whose down regulations was shown to affect the activation of the RAS-PI#K pathway. By integrating information from functional siRNA with protein interaction information, we develop a strategy that aims at inferring phosphatase physiological substrates. Graph analysis is used to identify protein scaffolds that may link the catalytic subunits to their substrates. By this approach we rediscover several previously described phosphatase substrate interactions and characterize two new protein scaffolds that promote the dephosphorylation of PTPN11 and ERK by DUSP18 and DUSP26 respectively.

  3. 2-D Difference in gel electrophoresis combined with Pro-Q Diamond staining: a successful approach for the identification of kinase/phosphatase targets.

    Science.gov (United States)

    Orsatti, Laura; Forte, Eleonora; Tomei, Licia; Caterino, Marianna; Pessi, Antonello; Talamo, Fabio

    2009-07-01

    The protein tyrosine phosphatase PRL-3 is an appealing therapeutic cancer target for its well described involvement in the metastasis progression. Nevertheless, very little is known about PRL-3 role in tumorigenesis. In the attempt to identify the protein target of this phosphatase we have devised a model system based on the use of highly invasive HCT116 colon cancer cells over-expressing PRL-3. We used 2-D difference gel electrophoresis combined with the fluorescence staining Pro-Q Diamond selective for phosphorylated proteins to monitor changes in the phosphorylation status of possible substrates. Proteins whose phosphorylation level was negatively affected by PRL-3 over-expression were identified by MS. Two proteins were found to be significantly dephosphorylated in this condition, the cytoskeletal protein ezrin and elongation factor 2. Ezrin has already been described as having a proactive role in cancer metastasis through control of its phosphorylation status, and the PRL-3-induced modulation of ezrin phosphorylation in HCT116 and human umblical vascular endothelial cells is the subject of a separate paper by Forte et al. [Biochim. Biophys. Acta 2008, 1783, 334-344]. The combination of 2-D difference in gel electrophoresis and Pro-Q Diamond was hence confirmed successful in analyzing changes of protein phosphorylation which enable the identification of kinase/phosphatase targets.

  4. A phosphatase-independent gain-of-function mutation in PTEN triggers aberrant cell growth in astrocytes through an autocrine IGF-1 loop.

    Science.gov (United States)

    Fernández, S; Genis, L; Torres-Alemán, I

    2014-08-07

    Loss-of-function mutations in the phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome10) contribute to aberrant cell growth in part through upregulation of the mitogenic IGF-1/PI3K/Akt pathway. In turn, this pathway exerts a homeostatic feedback over PTEN. Using mutagenesis analysis to explore a possible impact of this mutual control on astrocyte growth, we found that truncation of the C-terminal region of PTEN (Δ51) associates with a marked increase in NFκB activity, a transcription factor overactivated in astrocyte tumors. Whereas mutations of PTEN are considered to lead to a loss-of-function, PTENΔ51, a truncation that comprises a region frequently mutated in human gliomas, displayed a neomorphic (gain-of-function) activity that was independent of its phosphatase activity. This gain-of-function of PTENΔ51 includes stimulation of IGF-1 synthesis through protein kinase A activation of the IGF-1 promoter. Increased IGF-1 originates an autocrine loop that activates Akt and NFκB. Constitutive activation of NFκB in PTENΔ51-expressing astrocytes leads to aberrant cell growth; astrocytes expressing this mutant PTEN generate colonies in vitro and tumors in vivo. Mutations converting a tumor suppressor such as PTEN into a tumor promoter through a gain-of-function involving IGF-1 production may further our understanding of the role played by this growth factor in glioma growth and help us define druggable targets for personalized therapy.

  5. Regulation of Akt/Protein Kinase B Signaling by a Novel Protein Phosphatase in Breast Cancer Cells

    National Research Council Canada - National Science Library

    Brognard, John; Newton, Alexandra

    2008-01-01

    ...: cell proliferation, growth, and apoptosis. Finally, since this phosphatase resides in a location of frequent loss of heterozygosity in breast cancer, we sought to determine if this phosphatase played a role in breast tumorigenesis...

  6. Protein tyrosine phosphatase PTP1B is involved in hippocampal synapse formation and learning.

    Directory of Open Access Journals (Sweden)

    Federico Fuentes

    Full Text Available ER-bound PTP1B is expressed in hippocampal neurons, and accumulates among neurite contacts. PTP1B dephosphorylates ß-catenin in N-cadherin complexes ensuring cell-cell adhesion. Here we show that endogenous PTP1B, as well as expressed GFP-PTP1B, are present in dendritic spines of hippocampal neurons in culture. GFP-PTP1B overexpression does not affect filopodial density or length. In contrast, impairment of PTP1B function or genetic PTP1B-deficiency leads to increased filopodia-like dendritic spines and a reduction in mushroom-like spines, while spine density is unaffected. These morphological alterations are accompanied by a disorganization of pre- and post-synapses, as judged by decreased clustering of synapsin-1 and PSD-95, and suggest a dynamic synaptic phenotype. Notably, levels of ß-catenin-Tyr-654 phosphorylation increased ∼5-fold in the hippocampus of adult PTP1B(-/- (KO mice compared to wild type (WT mice and this was accompanied by a reduction in the amount of ß-catenin associated with N-cadherin. To determine whether PTP1B-deficiency alters learning and memory, we generated mice lacking PTP1B in the hippocampus and cortex (PTP1B(fl/fl-Emx1-Cre. PTP1B(fl/fl-Emx1-Cre mice displayed improved performance in the Barnes maze (decreased time to find and enter target hole, utilized a more efficient strategy (cued, and had better recall compared to WT controls. Our results implicate PTP1B in structural plasticity within the hippocampus, likely through modulation of N-cadherin function by ensuring dephosphorylation of ß-catenin on Tyr-654. Disruption of hippocampal PTP1B function or expression leads to elongation of dendritic filopodia and improved learning and memory, demonstrating an exciting novel role for this phosphatase.

  7. Differential regulation of protein phosphatase 1 (PP1) isoforms in human heart failure and atrial fibrillation.

    Science.gov (United States)

    Meyer-Roxlau, Stefanie; Lämmle, Simon; Opitz, Annett; Künzel, Stephan; Joos, Julius P; Neef, Stefan; Sekeres, Karolina; Sossalla, Samuel; Schöndube, Friedrich; Alexiou, Konstantin; Maier, Lars S; Dobrev, Dobromir; Guan, Kaomei; Weber, Silvio; El-Armouche, Ali

    2017-07-01

    Protein phosphatase 1 (PP1) is a key regulator of important cardiac signaling pathways. Dysregulation of PP1 has been heavily implicated in cardiac dysfunctions. Accordingly, pharmacological targeting of PP1 activity is considered for therapeutic intervention in human cardiomyopathies. Recent evidence from animal models implicated previously unrecognized, isoform-specific activities of PP1 in the healthy and diseased heart. Therefore, this study examined the expression of the distinct PP1 isoforms PP1α, β, and γ in human heart failure (HF) and atrial fibrillation (AF) and addressed the consequences of β-adrenoceptor blocker (beta-blocker) therapy for HF patients with reduced ejection fraction on PP1 isoform expression. Using western blot analysis, we found greater abundance of PP1 isoforms α and γ but unaltered PP1β levels in left ventricular myocardial tissues from HF patients as compared to non-failing controls. However, expression of all three PP1 isoforms was higher in atrial appendages from patients with AF compared to patients with sinus rhythm. Moreover, we found that in human failing ventricles, beta-blocker therapy was associated with lower PP1α abundance and activity, as indicated by higher phosphorylation of the PP1α-specific substrate eIF2α. Greater eIF2α phosphorylation is a known repressor of protein translation, and accordingly, we found lower levels of the endoplasmic reticulum (ER) stress marker Grp78 in the very same samples. We propose that isoform-specific targeting of PP1α activity may be a novel and innovative therapeutic strategy for the treatment of human cardiac diseases by reducing ER stress conditions.

  8. Implication of protein tyrosine phosphatase 1B in MCF-7 cell proliferation and resistance to 4-OH tamoxifen

    International Nuclear Information System (INIS)

    Blanquart, Christophe; Karouri, Salah-Eddine; Issad, Tarik

    2009-01-01

    The protein tyrosine phosphatase 1B (PTP1B) and the T-cell protein tyrosine phosphatase (TC-PTP) were initially thought to be mainly anti-oncogenic. However, overexpression of PTP1B and TC-PTP has been observed in human tumors, and recent studies have demonstrated that PTP1B contributes to the appearance of breast tumors by modulating ERK pathway. In the present work, we observed that decreasing the expression of TC-PTP or PTP1B in MCF-7 cells using siRNA reduced cell proliferation without affecting cell death. This reduction in proliferation was associated with decreased ERK phosphorylation. Moreover, selection of tamoxifen-resistant MCF-7 cells, by long-term culture in presence of 4-OH tamoxifen, resulted in cells that display overexpression of PTP1B and TC-PTP, and concomitant increase in ERK and STAT3 phosphorylation. siRNA experiments showed that PTP1B, but not TC-PTP, is necessary for resistance to 4-OH tamoxifen. Therefore, our work indicates that PTP1B could be a relevant therapeutic target for treatment of tamoxifen-resistant breast cancers.

  9. Arctigenin inhibits triple-negative breast cancers by targeting CIP2A to reactivate protein phosphatase 2A.

    Science.gov (United States)

    Huang, Qiuyue; Qin, Shanshan; Yuan, Xiaoning; Zhang, Liang; Ji, Juanli; Liu, Xuewen; Ma, Wenjing; Zhang, Yunfei; Liu, Pengfei; Sun, Zhiting; Zhang, Jingxuan; Liu, Ying

    2017-07-01

    We have shown that a novel STAT3 inhibitor arctigenin (Atn) induces significant cytotoxicity in triple-negative breast cancer (TNBC) cells. This study further delineated molecular mechanisms where by Atn triggered cytotoxicity in TNBC cells. We found Atn can also inhibit metastasis in TNBC cells through cancerous inhibitor of protein phosphatase 2A (CIP2A) pathway. CIP2A is an endogenous inhibitor of protein phosphatase 2A (PP2A), which can increase the migration and invasion of various cancer cells. PP2A is a tumor suppressor, which is functionally defective in various cancers. Atn-induced metastasis inhibition was associated with reactivation of PP2A, downregulation of CIP2A and Akt phosphorylation. Silencing CIP2A enhanced Atn-induced metastasis inhibition and apoptosis in TNBCs. Furthermore, ectopic expression of CIP2A or inhibition of PP2A in TNBC cells abolished the effects of Atn. In conclusion, we found that enhancement of PP2A activity by inhibition of CIP2A, at least in part, promotes the anti-metastasis effect induced by Atn. Our findings disclose the novel therapeutic mechanism of this targeted agent, and suggest the therapeutic potential and feasibility of developing PP2A enhancers as a novel anticancer strategy.

  10. Microvillus-Specific Protein Tyrosine Phosphatase SAP-1 Plays a Role in Regulating the Intestinal Paracellular Transport of Macromolecules.

    Science.gov (United States)

    Mori, Shingo; Kamei, Noriyasu; Murata, Yoji; Takayama, Kozo; Matozaki, Takashi; Takeda-Morishita, Mariko

    2017-09-01

    The stomach cancer-associated protein tyrosine phosphatase 1 (SAP-1) is a receptor-type protein tyrosine phosphatase that is specifically expressed on the apical membrane of the intestinal epithelium. SAP-1 is known to maintain the balance of phosphorylation of proteins together with protein kinases; however, its biological function and impact on pharmacokinetics in the intestine remain unclear. The present study, therefore, aimed at clarifying the relationship between SAP-1 and the intestinal absorption behaviors of typical transporter substrates and macromolecules. The endogenous levels of glucose and total cholesterol in the blood were similar between wild-type and SAP-1-deficient mice (Sap1 -/- ), suggesting no contribution of SAP-1 to biogenic influx. Moreover, in vitro transport study with everted ileal sacs demonstrated that there was no difference in the absorption of breast cancer resistance protein, P-glycoprotein, and peptide transporter substrates between both mice. However, absorptive clearance of macromolecular model dextrans (FD-4 and FD-10) in Sap1 -/- mice was significantly higher than that in wild-type mice, and this was confirmed by the trend of increased FD-4 absorption from colonic loops of Sap1 -/- mice. Therefore, the results of this study suggest the partial contribution of SAP-1 to the regulated transport of hydrophilic macromolecules through paracellular tight junctions. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  11. An acid phosphatase in the plasma membranes of human astrocytoma showing marked specificity toward phosphotyrosine protein.

    Science.gov (United States)

    Leis, J F; Kaplan, N O

    1982-11-01

    The plasma membrane from the human tumor astrocytoma contains an active acid phosphatase activity based on hydrolysis of p-nitrophenyl phosphate. Other acid phosphatase substrates--beta-glycerophosphate, O-phosphorylcholine, and 5'-AMP--are not hydrolyzed significantly. The phosphatase activity is tartrate insensitive and is stimulated by Triton X-100 and EDTA. Of the three known phosphoamino acids, only free O-phosphotyrosine is hydrolyzed by the membrane phosphatase activity. Other acid phosphatases tested from potato, wheat germ, milk, and bovine prostate did not show this degree of specificity. The plasma membrane activity also dephosphorylated phosphotyrosine histone at a much greater rate than did the other acid phosphatases. pH profiles for free O-phosphotyrosine and phosphotyrosine histone showed a shift toward physiological pH, indicating possible physiological significance. Phosphotyrosine histone dephosphorylation activity was nearly 10 times greater than that seen for phosphoserine histone dephosphorylation, and Km values were much lower for phosphotyrosine histone dephosphorylation (0.5 microM vs. 10 microM). Fluoride and zinc significantly inhibited phosphoserine histone dephosphorylation. Vanadate, on the other hand, was a potent inhibitor of phosphotyrosine histone dephosphorylation (50% inhibition at 0.5 microM) but not of phosphoserine histone. ATP stimulated phosphotyrosine histone dephosphorylation (160-250%) but inhibited phosphoserine histone dephosphorylation (95%). These results suggest the existence of a highly specific phosphotyrosine protein phosphatase activity associated with the plasma membrane of human astrocytoma.

  12. COMPARISON OF METHODS FOR ALKALINE PHOSPHATASE AND PEROXIDASE DETECTION IN MILK

    Directory of Open Access Journals (Sweden)

    felipe Nael Seixas

    2014-02-01

    Full Text Available This study evaluated the performance of strips for colorimetric detection of alkaline phosphatase and peroxidase in milk, comparing them with a kit of reagents for alkaline phosphatase and the official methodology for peroxidase. The samples were analyzed at the Laboratory Inspection of Products of Animal Origin, State University of Londrina. For the comparison tests for the detection of alkaline phosphatase four treatments were made by adding different percentages of raw milk (1%, 2%, 5% and 10% in the pasteurized milk, plus two control treatments. Thirty-eight samples triplicate for each treatment were analyzed. To compare the performance of tests for peroxidase 80 pasteurized milk samples were evaluated simultaneously by official methodology and by colorimetric strips. The performance of the alkaline phosphatase were different for the treatments with 1% and 2% of raw milk which had all the strips change color as the reagent kit showed the presence of phosphatase in just 2.63% and 5.26% the cases, respectively for each treatment. The colorimetric strips for alkaline phosphatase are more sensitive for the identification of small quantities compared to the reagent kit. The performance of tests for peroxidase showed no difference. The strips for the detection of peroxidase or alkaline phosphatase were effective and can replace traditional methods.

  13. Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase[S

    Science.gov (United States)

    Oguro, Ami; Imaoka, Susumu

    2012-01-01

    Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hydrolyzed products. Although lecithin itself did not inhibit the phosphatase activity, the hydrolyzed lecithin significantly inhibited it, suggesting that lysophospholipid or fatty acid can inhibit it. Next, we investigated the inhibition of phosphatase activity by lysophosphatidyl choline, palmitoyl lysophosphatidic acid, monopalmitoyl glycerol, and palmitic acid. Palmitoyl lysophosphatidic acid and fatty acid efficiently inhibited phosphatase activity, suggesting that lysophosphatidic acids (LPAs) are substrates for the phosphatase activity of sEH. As expected, palmitoyl, stearoyl, oleoyl, and arachidonoyl LPAs were efficiently dephosphorylated by sEH (Km, 3–7 μM; Vmax, 150–193 nmol/min/mg). These results suggest that LPAs are substrates of sEH, which may regulate physiological functions of cells via their metabolism. PMID:22217705

  14. Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase.

    Science.gov (United States)

    Oguro, Ami; Imaoka, Susumu

    2012-03-01

    Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hydrolyzed products. Although lecithin itself did not inhibit the phosphatase activity, the hydrolyzed lecithin significantly inhibited it, suggesting that lysophospholipid or fatty acid can inhibit it. Next, we investigated the inhibition of phosphatase activity by lysophosphatidyl choline, palmitoyl lysophosphatidic acid, monopalmitoyl glycerol, and palmitic acid. Palmitoyl lysophosphatidic acid and fatty acid efficiently inhibited phosphatase activity, suggesting that lysophosphatidic acids (LPAs) are substrates for the phosphatase activity of sEH. As expected, palmitoyl, stearoyl, oleoyl, and arachidonoyl LPAs were efficiently dephosphorylated by sEH (Km, 3-7 μM; Vmax, 150-193 nmol/min/mg). These results suggest that LPAs are substrates of sEH, which may regulate physiological functions of cells via their metabolism.

  15. Regulatory dephosphorylation of CDK at G₂/M in plants: yeast mitotic phosphatase cdc25 induces cytokinin-like effects in transgenic tobacco morphogenesis.

    Science.gov (United States)

    Lipavská, Helena; Masková, Petra; Vojvodová, Petra

    2011-05-01

    During the last three decades, the cell cycle and its control by cyclin-dependent kinases (CDKs) have been extensively studied in eukaryotes. This endeavour has produced an overall picture that basic mechanisms seem to be largely conserved among all eukaryotes. The intricate regulation of CDK activities includes, among others, CDK activation by CDC25 phosphatase at G₂/M. In plants, however, studies of this regulation have lagged behind as a plant Cdc25 homologue or other unrelated phosphatase active at G₂/M have not yet been identified. Failure to identify a plant mitotic CDK activatory phosphatase led to characterization of the effects of alien cdc25 gene expression in plants. Tobacco, expressing the Schizosaccharomyces pombe mitotic activator gene, Spcdc25, exhibited morphological, developmental and biochemical changes when compared with wild type (WT) and, importantly, increased CDK dephosphorylation at G₂/M. Besides changes in leaf shape, internode length and root development, in day-neutral tobacco there was dramatically earlier onset of flowering with a disturbed acropetal floral capacity gradient typical of WT. In vitro, de novo organ formation revealed substantially earlier and more abundant formation of shoot primordia on Spcdc25 tobacco stem segments grown on shoot-inducing media when compared with WT. Moreover, in contrast to WT, stem segments from transgenic plants formed shoots even without application of exogenous growth regulator. Spcdc25-expressing BY-2 cells exhibited a reduced mitotic cell size due to a shortening of the G₂ phase together with high activity of cyclin-dependent kinase, NtCDKB1, in early S-phase, S/G₂ and early M-phase. Spcdc25-expressing tobacco ('Samsun') cell suspension cultures showed a clustered, more circular, cell phenotype compared with chains of elongated WT cells, and increased content of starch and soluble sugars. Taken together, Spcdc25 expression had cytokinin-like effects on the characteristics studied

  16. Purification and characterization of a phosphotyrosyl-protein phosphatase from wheat seedlings.

    Science.gov (United States)

    Cheng, H F; Tao, M

    1989-10-19

    A neutral phosphatase which catalyzes the hydrolysis of p-nitrophenylphosphate has been purified to homogeneity from wheat seedlings. The enzyme is a monomeric glycoprotein exhibiting a molecular weight of 35,000, frictional ratio of 1.22, Stokes' radius of 260 nm, and sedimentation coefficient of 3.2 S. That the enzyme is a glycoprotein is surmised from its chromatographic property on Concanavalin A-Sepharose column. An examination of the substrate specificity indicates that the enzyme exhibits a preference for phosphotyrosine over a number of phosphocompounds, including p-nitrophenylphosphate and several glycolytic intermediates. Both phosphoserine and phosphothreonine are not hydrolyzed by the enzyme. The phosphatase activity is not affected by high concentrations of chelating agents and does not require metal ions. Molybdate, orthovanadate, Zn2+, and Hg2+ are all potent inhibitors of the phosphatase activity. The ability of the phosphatase to dephosphorylate protein phosphotyrosine has been investigated. [32P-Tyr]poly(Glu,Tyr)n, [32P-Tyr]alkylated bovine serum albumin, [32P-Tyr]angiotensin-I, and [32P-Tyr]band 3 (from human erythrocyte) are all substrates of the phosphatase. On the other hand, the enzyme has no activity toward protein phosphoserine and phosphothreonine. Our result further indicates that the neutral phosphatase is distinct from the wheat germ acid phosphatase. The latter enzyme is found to dephosphorylate phosphotyrosyl as well as phosphoseryl and phosphothreonyl groups in proteins. In light of the many similarities in properties to phosphotyrosyl protein phosphatases isolated from several sources, it is suggested that the wheat seedling phosphatase may participate in cellular regulation involving protein tyrosine phosphorylation.

  17. Antitumor effects of metformin via indirect inhibition of protein phosphatase 2A in patients with endometrial cancer.

    Directory of Open Access Journals (Sweden)

    Shinsuke Hanawa

    Full Text Available Metformin, an antidiabetic drug, inhibits the endometrial cancer cell growth in vivo by improving the insulin resistance; however, its mechanism of action is not completely understood. Protein phosphatase 2A (PP2A is a serine/threonine phosphatase associated with insulin resistance and type 2 diabetes, and its inhibition restores the insulin resistance. This study investigated the antitumor effect of metformin on endometrial cancer with a focus on PP2A.Metformin (1,500-2,250 mg/day was preoperatively administered to patients with endometrial cancer for 4 to 6 weeks. Expression of the PP2A regulatory subunits, 4 (PPP2R4 and B (PP2A-B, was evaluated using real-time polymerase chain reaction (RT-PCR and immunohistochemistry (IHC using paired specimens obtained before and after metformin treatment. The effect of PPP2R4 inhibition with small interfering RNA was evaluated in the endometrial cancer cell lines HEC265 and HEC1B. P values of < .05 were considered statistically significant.Preoperative metformin treatment significantly reduced the expression of PP2A-B, as determined using IHC, and the mRNA expression of PPP2R4, as determined using RT-PCR, in the patients with endometrial cancer. However, metformin could not directly alter the PPP2R4 mRNA levels in the endometrial cancer cell lines in vitro. PPP2R4 knockdown reduced the proliferation and induced the apoptosis by activating caspases 3/7 in HEC265 and HEC1B cells.Downregulation of the PP2A-B subunit, including PPP2R4, is an important indirect target of metformin. Inhibition of PP2A may be an option for the treatment of endometrial cancer patients with insulin resistance.This trial is registered with UMIN-CTR (number UMIN000004852.

  18. Inhibition of receptor tyrosine kinase signalling by small molecule agonist of T-cell protein tyrosine phosphatase

    International Nuclear Information System (INIS)

    Mattila, Elina; Marttila, Heidi; Sahlberg, Niko; Kohonen, Pekka; Tähtinen, Siri; Halonen, Pasi; Perälä, Merja; Ivaska, Johanna

    2010-01-01

    T-cell protein tyrosine phosphatase (TCPTP/TC45) is a ubiquitously expressed intra-cellular non-receptor protein tyrosine phosphatase involved in the negative regulation of several cancer relevant cellular signalling pathways. We have previously shown that interaction between the α-cytoplasmic tail of α1β1 integrin and TCPTP activates TCPTP by disrupting an inhibitory intra-molecular bond in TCPTP. Thus, inhibition of the regulatory interaction in TCPTP is a desirable strategy for TCPTP activation and attenuation of oncogenic RTK signalling. However, this is challenging with low molecular weight compounds. We developed a high-throughput compatible assay to analyse activity of recombinant TCPTP in vitro. Using this assay we have screened 64280 small molecules to identify novel agonists for TCPTP. Dose-dependent response to TCPTP agonist was performed using the in vitro assay. Inhibition effects and specificity of TCPTP agonists were evaluated using TCPTP expressing and null mouse embryonic fibroblasts. Western blot analysis was used to evaluate attenuation of PDGFRβ and EGFR phosphorylation. Inhibition of VEGF signalling was analysed with VEGF-induced endothelial cell sprouting assays. From the screen we identified six TCPTP agonists. Two compounds competed with α1-cytoplasmic domain for binding to TCPTP, suggesting that they activate TCPTP similar to α1-cyt by disrupting the intra-molecular bond in TCPTP. Importantly, one of the compounds (spermidine) displayed specificity towards TCPTP in cells, since TCPTP -/- cells were 43-fold more resistant to the compound than TCPTP expressing cells. This compound attenuates PDGFRβ and VEGFR2 signalling in cells in a TCPTP-dependent manner and functions as a negative regulator of EGFR phosphorylation in cancer cells. In this study we showed that small molecules mimicking TCPTP-α1 interaction can be used as TCPTP agonists. These data provide the first proof-of-concept description of the use of high-throughput screening

  19. Repression of class I transcription by cadmium is mediated by the protein phosphatase 2A

    Science.gov (United States)

    Zhou, Lei; Le Roux, Gwenaëlle; Ducrot, Cécile; Chédin, Stéphane; Labarre, Jean; Riva, Michel; Carles, Christophe

    2013-01-01

    Toxic metals are part of our environment, and undue exposure to them leads to a variety of pathologies. In response, most organisms adapt their metabolism and have evolved systems to limit this toxicity and to acquire tolerance. Ribosome biosynthesis being central for protein synthesis, we analyzed in yeast the effects of a moderate concentration of cadmium (Cd2+) on Pol I transcription that represents >60% of the transcriptional activity of the cells. We show that Cd2+ rapidly and drastically shuts down the expression of the 35S rRNA. Repression does not result from a poisoning of any of the components of the class I transcriptional machinery by Cd2+, but rather involves a protein phosphatase 2A (PP2A)-dependent cellular signaling pathway that targets the formation/dissociation of the Pol I–Rrn3 complex. We also show that Pol I transcription is repressed by other toxic metals, such as Ag+ and Hg2+, which likewise perturb the Pol I–Rrn3 complex, but through PP2A-independent mechanisms. Taken together, our results point to a central role for the Pol I–Rrn3 complex as molecular switch for regulating Pol I transcription in response to toxic metals. PMID:23640330

  20. HONSU, a protein phosphatase 2C, regulates seed dormancy by inhibiting ABA signaling in Arabidopsis.

    Science.gov (United States)

    Kim, Woohyun; Lee, Yeon; Park, Jeongmoo; Lee, Nayoung; Choi, Giltsu

    2013-04-01

    Seed dormancy, a seed status that prohibits germination even in the presence of inductive germination signals, is a poorly understood process. To identify molecular components that regulate seed dormancy, we screened T-DNA insertion lines and identified a mutant designated honsu (hon). HON loss-of-function mutants display deep seed dormancy, whereas HON-overexpressing lines display shallow seed dormancy. HON encodes a seed-specific group A phosphatase 2C (PP2C) and is one of the major negative regulators of seed dormancy among group A PP2Cs. Like other PP2C family members, HON interacts with PYR1/RCAR11 in the presence of ABA. Our analysis indicates that HON inhibits ABA signaling and activates gibberellic acid signaling, and both of these conditions must be satisfied to promote the release of seed dormancy. However, HON mRNA levels are increased in mutants displaying deep seed dormancy or under conditions that deepen seed dormancy, and decreased in mutants displaying shallow seed dormancy or under conditions that promote the release of seed dormancy. Taken together, our results indicate that the expression of HON mRNA is homeostatically regulated by seed dormancy.

  1. Transmembrane prostatic acid phosphatase (TMPAP interacts with snapin and deficient mice develop prostate adenocarcinoma.

    Directory of Open Access Journals (Sweden)

    Ileana B Quintero

    Full Text Available The molecular mechanisms underlying prostate carcinogenesis are poorly understood. Prostatic acid phosphatase (PAP, a prostatic epithelial secretion marker, has been linked to prostate cancer since the 1930's. However, the contribution of PAP to the disease remains controversial. We have previously cloned and described two isoforms of this protein, a secretory (sPAP and a transmembrane type-I (TMPAP. The goal in this work was to understand the physiological function of TMPAP in the prostate. We conducted histological, ultra-structural and genome-wide analyses of the prostate of our PAP-deficient mouse model (PAP(-/- with C57BL/6J background. The PAP(-/- mouse prostate showed the development of slow-growing non-metastatic prostate adenocarcinoma. In order to find out the mechanism behind, we identified PAP-interacting proteins byyeast two-hybrid assays and a clear result was obtained for the interaction of PAP with snapin, a SNARE-associated protein which binds Snap25 facilitating the vesicular membrane fusion process. We confirmed this interaction by co-localization studies in TMPAP-transfected LNCaP cells (TMPAP/LNCaP cells and in vivo FRET analyses in transient transfected LNCaP cells. The differential gene expression analyses revealed the dysregulation of the same genes known to be related to synaptic vesicular traffic. Both TMPAP and snapin were detected in isolated exosomes. Our results suggest that TMPAP is involved in endo-/exocytosis and disturbed vesicular traffic is a hallmark of prostate adenocarcinoma.

  2. Involvement of Sac1 phosphoinositide phosphatase in the metabolism of phosphatidylserine in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Tani, Motohiro; Kuge, Osamu

    2014-04-01

    Sac1 is a phosphoinositide phosphatase that preferentially dephosphorylates phosphatidylinositol 4-phosphate. Mutation of SAC1 causes not only the accumulation of phosphoinositides but also reduction of the phosphatidylserine (PS) level in the yeast Saccharomyces cerevisiae. In this study, we characterized the mechanism underlying the PS reduction in SAC1-deleted cells. Incorporation of (32) P into PS was significantly delayed in sac1∆ cells. Such a delay was also observed in SAC1- and PS decarboxylase gene-deleted cells, suggesting that the reduction in the PS level is caused by a reduction in the rate of biosynthesis of PS. A reduction in the PS level was also observed with repression of STT4 encoding phosphatidylinositol 4-kinase or deletion of VPS34 encoding phophatidylinositol 3-kinase. However, the combination of mutations of SAC1 and STT4 or VPS34 did not restore the reduced PS level, suggesting that both the synthesis and degradation of phosphoinositides are important for maintenance of the PS level. Finally, we observed an abnormal PS distribution in sac1∆ cells when a specific probe for PS was expressed. Collectively, these results suggested that Sac1 is involved in the maintenance of a normal rate of biosynthesis and distribution of PS. Copyright © 2014 John Wiley & Sons, Ltd.

  3. Interaction of Protein Phosphatase 1δ with Nucleophosmin in Human Osteoblastic Cells

    International Nuclear Information System (INIS)

    Haneji, Tatsuji; Teramachi, Jumpei; Hirashima, Kanji; Kimura, Koji; Morimoto, Hiroyuki

    2012-01-01

    Protein phosphorylation and dephosphorylation has been recognized as an essential mechanism in the regulation of cellular metabolism and function in various tissues. Serine and threonine protein phosphatases (PP) are divided into four categories: PP1, PP2A, PP2B, and PP2C. At least four isoforms of PP1 catalytic subunit in rat, PP1α, PP1γ1, PP1γ2, and PP1δ, were isolated. In the present study, we examined the localization and expression of PP1δ in human osteoblastic Saos-2 cells. Anti-PP1δ antibody recognized a protein present in the nucleolar regions in Saos-2 cells. Cellular fractionation revealed that PP1δ is a 37 kDa protein localized in the nucleolus. Nucleophosmin is a nucleolar phosphoprotein and located mainly in the nucleolus. Staining pattern of nucleophosmin in Saos-2 cells was similar to that of PP1δ. PP1δ and nucleophosmin were specifically stained as dots in the nucleus. Dual fluorescence images revealed that PP1δ and nucleophosmin were localized in the same regions in the nucleolus. Similar distribution patterns of PP1δ and nucleophosmin were observed in osteoblastic MG63 cells. The interaction of PP1δ and nucleophosmin was also shown by immunoprecipitation and Western analysis. These results indicated that PP1δ associate with nucleophosmin directly in the nucleolus and suggested that nucleophosmin is one of the candidate substrate for PP1δ

  4. Research on Phosphatases of Belladona Leaves and Their Purification (Part 1

    Directory of Open Access Journals (Sweden)

    M. Khorsand

    1956-12-01

    Full Text Available Belladona leaves as well as all other studied leaves contains two distinct phosphatase fractions belonging respectively to types II and IIIi the major parts of these enzymes is extraetible by water. It was not possible to extract the non soluble fraction which is solidly retained by the cellular constituents. Phosphatase II does not differ from other phosphatnses of the same type. Whereas phosphatase III is distinetely different from enzymes of the same type of vegetal or animal origins. It is activated by bivalent metallic ions which are specific activators of the alkaline phcspbatnses: Mg-Zn-Ni and Co.

  5. Uranium Biomineralization by Natural Microbial Phosphatase Activities in the Subsurface

    Energy Technology Data Exchange (ETDEWEB)

    Sobecky, Patricia A. [Univ. of Alabama, Tuscaloosa, AL (United States)

    2015-04-06

    In this project, inter-disciplinary research activities were conducted in collaboration among investigators at The University of Alabama (UA), Georgia Institute of Technology (GT), Lawrence Berkeley National Laboratory (LBNL), Brookhaven National Laboratory (BNL), the DOE Joint Genome Institute (JGI), and the Stanford Synchrotron Radiation Light source (SSRL) to: (i) confirm that phosphatase activities of subsurface bacteria in Area 2 and 3 from the Oak Ridge Field Research Center result in solid U-phosphate precipitation in aerobic and anaerobic conditions; (ii) investigate the eventual competition between uranium biomineralization via U-phosphate precipitation and uranium bioreduction; (iii) determine subsurface microbial community structure changes of Area 2 soils following organophosphate amendments; (iv) obtain the complete genome sequences of the Rahnella sp. Y9-602 and the type-strain Rahnella aquatilis ATCC 33071 isolated from these soils; (v) determine if polyphosphate accumulation and phytate hydrolysis can be used to promote U(VI) biomineralization in subsurface sediments; (vi) characterize the effect of uranium on phytate hydrolysis by a new microorganism isolated from uranium-contaminated sediments; (vii) utilize positron-emission tomography to label and track metabolically-active bacteria in soil columns, and (viii) study the stability of the uranium phosphate mineral product. Microarray analyses and mineral precipitation characterizations were conducted in collaboration with DOE SBR-funded investigators at LBNL. Thus, microbial phosphorus metabolism has been shown to have a contributing role to uranium immobilization in the subsurface.

  6. Sensing charges of the Ciona intestinalis voltage-sensing phosphatase.

    Science.gov (United States)

    Villalba-Galea, Carlos A; Frezza, Ludivine; Sandtner, Walter; Bezanilla, Francisco

    2013-11-01

    Voltage control over enzymatic activity in voltage-sensitive phosphatases (VSPs) is conferred by a voltage-sensing domain (VSD) located in the N terminus. These VSDs are constituted by four putative transmembrane segments (S1 to S4) resembling those found in voltage-gated ion channels. The putative fourth segment (S4) of the VSD contains positive residues that likely function as voltage-sensing elements. To study in detail how these residues sense the plasma membrane potential, we have focused on five arginines in the S4 segment of the Ciona intestinalis VSP (Ci-VSP). After implementing a histidine scan, here we show that four arginine-to-histidine mutants, namely R223H to R232H, mediate voltage-dependent proton translocation across the membrane, indicating that these residues transit through the hydrophobic core of Ci-VSP as a function of the membrane potential. These observations indicate that the charges carried by these residues are sensing charges. Furthermore, our results also show that the electrical field in VSPs is focused in a narrow hydrophobic region that separates the extracellular and intracellular space and constitutes the energy barrier for charge crossing.

  7. Allosteric substrate switching in a voltage-sensing lipid phosphatase.

    Science.gov (United States)

    Grimm, Sasha S; Isacoff, Ehud Y

    2016-04-01

    Allostery provides a critical control over enzyme activity, biasing the catalytic site between inactive and active states. We found that the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP), which modifies phosphoinositide signaling lipids (PIPs), has not one but two sequential active states with distinct substrate specificities, whose occupancy is allosterically controlled by sequential conformations of the voltage-sensing domain (VSD). Using fast fluorescence resonance energy transfer (FRET) reporters of PIPs to monitor enzyme activity and voltage-clamp fluorometry to monitor conformational changes in the VSD, we found that Ci-VSP switches from inactive to a PIP3-preferring active state when the VSD undergoes an initial voltage-sensing motion and then into a second PIP2-preferring active state when the VSD activates fully. This two-step allosteric control over a dual-specificity enzyme enables voltage to shape PIP concentrations in time, and provides a mechanism for the complex modulation of PIP-regulated ion channels, transporters, cell motility, endocytosis and exocytosis.

  8. Allosteric substrate switching in a voltage sensing lipid phosphatase

    Science.gov (United States)

    Grimm, Sasha S.; Isacoff, Ehud Y.

    2016-01-01

    Allostery provides a critical control over enzyme activity, biasing the catalytic site between inactive and active states. We find the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP), which modifies phosphoinositide signaling lipids (PIPs), to have not one but two sequential active states with distinct substrate specificities, whose occupancy is allosterically controlled by sequential conformations of the voltage sensing domain (VSD). Using fast FRET reporters of PIPs to monitor enzyme activity and voltage clamp fluorometry to monitor conformational changes in the VSD, we find that Ci-VSP switches from inactive to a PIP3-preferring active state when the VSD undergoes an initial voltage sensing motion and then into a second PIP2-preferring active state when the VSD activates fully. This novel 2-step allosteric control over a dual specificity enzyme enables voltage to shape PIP concentrations in time, and provides a mechanism for the complex modulation of PIP-regulated ion channels, transporters, cell motility and endo/exocytosis. PMID:26878552

  9. Changes of serum alkaline phosphatase isoenzymes in fasted rats.

    Science.gov (United States)

    Wada, H; Niwa, N; Hayakawa, T; Tsuge, H

    1996-10-01

    Changes of serum alkaline phosphatase (sALP) isoenzymes under fasting conditions were examined using polyacrylamide gel electrophoresis (PAGE), amino-acids (L-phenylalanine (L-Phe), L-homoarginine (L-HArg)) inhibition and wheat germ agglutinin (WGA) treatment. The sALP of non-fasted rats was separated into three bands (S1, S2, S3) by PAGE. The molecular weight (M.W.) of S1 corresponded to that of an isoenzyme found in the ileum. By the addition of L-Phe, the staining intensity of S1 was weakened, S2 and S3 remained unchanged and the total activity of the isoenzymes extracted from intestine decreased. On the other hand, the activity of isoenzymes extracted from kidney and bone decreased by the addition of L-HArg. Therefore, S1 was judged to be derived from intestine. The activities of total sALP and S1 decreased from 16 h of fasting. Total sALP activity and sALP activity of the supernatant prepared by WGA treatment decreased, whereas the ALP activity of the precipitate (difference between total sALP activity and supernatant sALP activity) did not change. The activity band of the precipitate corresponded to that of S3 by PAGE. Therefore, S3 was judged to be derived from bone. In conclusion, under fasting conditions, the activity of S1 decreased while the activities of S2 and S3 remained unchanged.

  10. Uranium Biomineralization by Natural Microbial Phosphatase Activities in the Subsurface

    International Nuclear Information System (INIS)

    Sobecky, Patricia A.

    2015-01-01

    In this project, inter-disciplinary research activities were conducted in collaboration among investigators at The University of Alabama (UA), Georgia Institute of Technology (GT), Lawrence Berkeley National Laboratory (LBNL), Brookhaven National Laboratory (BNL), the DOE Joint Genome Institute (JGI), and the Stanford Synchrotron Radiation Light source (SSRL) to: (i) confirm that phosphatase activities of subsurface bacteria in Area 2 and 3 from the Oak Ridge Field Research Center result in solid U-phosphate precipitation in aerobic and anaerobic conditions; (ii) investigate the eventual competition between uranium biomineralization via U-phosphate precipitation and uranium bioreduction; (iii) determine subsurface microbial community structure changes of Area 2 soils following organophosphate amendments; (iv) obtain the complete genome sequences of the Rahnella sp. Y9-602 and the type-strain Rahnella aquatilis ATCC 33071 isolated from these soils; (v) determine if polyphosphate accumulation and phytate hydrolysis can be used to promote U(VI) biomineralization in subsurface sediments; (vi) characterize the effect of uranium on phytate hydrolysis by a new microorganism isolated from uranium-contaminated sediments; (vii) utilize positron-emission tomography to label and track metabolically-active bacteria in soil columns, and (viii) study the stability of the uranium phosphate mineral product. Microarray analyses and mineral precipitation characterizations were conducted in collaboration with DOE SBR-funded investigators at LBNL. Thus, microbial phosphorus metabolism has been shown to have a contributing role to uranium immobilization in the subsurface.

  11. Ly49Q, a member of the Ly49 family that is selectively expressed on myeloid lineage cells and involved in regulation of cytoskeletal architecture

    Science.gov (United States)

    Toyama-Sorimachi, Noriko; Tsujimura, Yusuke; Maruya, Mikako; Onoda, Atsuko; Kubota, Toshiyuki; Koyasu, Shigeo; Inaba, Kayo; Karasuyama, Hajime

    2004-01-01

    Here, we identified and characterized a Ly49 family member, designated as Ly49Q. The Ly49q gene encodes a 273-aa protein with an immunoreceptor tyrosine-based inhibitory motif (ITIM) at the N terminus of its cytoplasmic domain. We show that the ITIM of Ly49Q can recruit SHP-2 and SHP-1 in a tyrosine phosphorylation-dependent manner. In contrast to other known members of the Ly49 family, Ly49Q was found not to be expressed on NK1.1+ cells, but instead was detectable on virtually all Gr-1+ cells, such as myeloid precursors in bone marrow. Monocytes/macrophages also expressed low levels of Ly49Q, and the expression was enhanced by the treatment of cells with IFN-γ. Treatment of activated macrophages with anti-Ly49Q mAb induced rapid formation of polarized actin structures, showing filopodia-like structure on one side and lamellipodial-like structure on the other side. A panel of proteins became tyrosine-phosphorylated in myeloid cells when treated with the mAb. Induction of the phosphorylation depends on the ITIM of Ly49Q. Thus, Ly49Q has unique features different from other known Ly49 family members and appears to be involved in regulation of cytoskeletal architecture of macrophages through ITIM-mediated signaling. PMID:14732700

  12. Phosphatases modulate the bistable sporulation gene expression pattern in Bacillus subtilis

    NARCIS (Netherlands)

    Veening, JW; Hamoen, LW; Kuipers, OP

    Spore formation in the Gram- positive bacterium Bacillus subtilis is a last resort adaptive response to starvation. To initiate sporulation, the key regulator in this process, Spo0A, needs to be activated by the so-called phosphorelay. Within a sporulating culture of B. subtilis, some cells initiate

  13. Interaction of Myosin Phosphatase Target Subunit (MYPT1) with Myosin Phosphatase-RhoA Interacting Protein (MRIP): A Role of Glutamic Acids in the Interaction.

    Science.gov (United States)

    Lee, Eunhee; Stafford, Walter F

    2015-01-01

    Scaffold proteins bind to and functionally link protein members of signaling pathways. Interaction of the scaffold proteins, myosin phosphatase target subunit (MYPT1) and myosin phosphatase-RhoA interacting protein (MRIP), causes co-localization of myosin phosphatase and RhoA to actomyosin. To examine biophysical properties of interaction of MYPT1 with MRIP, we employed analytical ultracentrifugation and surface plasmon resonance. In regard to MRIP, its residues 724-837 are sufficient for the MYPT1/MRIP interaction. Moreover, MRIP binds to MYPT1 as either a monomer or a dimer. With respect to MYPT1, its leucine repeat region, LR (residues 991-1030) is sufficient to account for the MYPT1/MRIP interaction. Furthermore, point mutations that replace glutamic acids 998-1000 within LR reduced the binding affinity toward MRIP. This suggests that the glutamic acids of MYPT1 play an important role in the interaction.

  14. Regulation of brown fat adipogenesis by protein tyrosine phosphatase 1B.

    Directory of Open Access Journals (Sweden)

    Kosuke Matsuo

    2011-01-01

    Full Text Available Protein-tyrosine phosphatase 1B (PTP1B is a physiological regulator of insulin signaling and energy balance, but its role in brown fat adipogenesis requires additional investigation.To precisely determine the role of PTP1B in adipogenesis, we established preadipocyte cell lines from wild type and PTP1B knockout (KO mice. In addition, we reconstituted KO cells with wild type, substrate-trapping (D/A and sumoylation-resistant (K/R PTP1B mutants, then characterized differentiation and signaling in these cells. KO, D/A- and WT-reconstituted cells fully differentiated into mature adipocytes with KO and D/A cells exhibiting a trend for enhanced differentiation. In contrast, K/R cells exhibited marked attenuation in differentiation and lipid accumulation compared with WT cells. Expression of adipogenic markers PPARγ, C/EBPα, C/EBPδ, and PGC1α mirrored the differentiation pattern. In addition, the differentiation deficit in K/R cells could be reversed completely by the PPARγ activator troglitazone. PTP1B deficiency enhanced insulin receptor (IR and insulin receptor substrate 1 (IRS1 tyrosyl phosphorylation, while K/R cells exhibited attenuated insulin-induced IR and IRS1 phosphorylation and glucose uptake compared with WT cells. In addition, substrate-trapping studies revealed that IRS1 is a substrate for PTP1B in brown adipocytes. Moreover, KO, D/A and K/R cells exhibited elevated AMPK and ACC phosphorylation compared with WT cells.These data indicate that PTP1B is a modulator of brown fat adipogenesis and suggest that adipocyte differentiation requires regulated expression of PTP1B.

  15. Influence of acid phosphatase activity on the saccharification of potato maltodextrins by Aspergillus niger glucoamylase

    Energy Technology Data Exchange (ETDEWEB)

    Zyla, K. (Akademia Rolnicza, Cracow (Poland). Dept. of Biotechnology)

    1990-01-01

    A preparation of Aspergillus niger acid phosphatase, which had the temperature optimum 60deg C, pH optimum 1.8-3.0; good stability at pH 4-5, the ability to hydrolyze glucose-6-phosphate at a high rate, and substantial lack of glucogenic activities, was used simultaneously with a glucoamylase in order to learn its influence on the saccharification of potato maltodextrins. The addition of the acid phosphatase activity in amounts that gave the 50 fold increase, as compared to phosphatase activity which naturally occurs in the gluocoamylase (GA) preparation 'AMG-200', was found to influence on the DE level, mainly at the high substrate concentration (40% d.s.) and low glucoamylase dosage (60-100 GAU/kg d.s.). It may also be possible, when using the acid phosphatase addition, to shorten the saccharification time. (orig.).

  16. Acceleration of gelation and promotion of mineralization of chitosan hydrogels by alkaline phosphatase

    NARCIS (Netherlands)

    Douglas, T.E.L.; Skwarczynska, A.; Modrzejewska, Z.; Balcaen, L.; Schaubroeck, D.; Lycke, S.; Vanhaecke, F.; Vandenabeele, P.; Dubruel, P.; Jansen, J.A.; Leeuwenburgh, S.C.G.

    2013-01-01

    Thermosensitive chitosan hydrogels containing sodium beta-glycerophosphate (beta-GP), whose gelation is induced by increasing temperature to body temperature, were functionalized by incorporation of alkaline phosphatase (ALP), an enzyme involved in mineralization of bone. ALP incorporation led to

  17. Diagnosis of prostate cancer using a radioimmunoassay for prostatic acid phosphatase in serum

    International Nuclear Information System (INIS)

    Lea, O.A.; Hoeisaeter, P.Aa.

    1981-01-01

    The paper describes the development and evaluation of a specific radioimmunoassay for the determination of prostatic acid phosphatase in serum as a useful aid in the detection of prostatic cancer. (Auth.)

  18. Molecular cloning and chromosome mapping of the human gene encoding protein phosphotyrosyl phosphatase 1B

    International Nuclear Information System (INIS)

    Brown-Shimer, S.; Johnson, K.A.; Bruskin, A.; Green, N.R.; Hill, D.E.; Lawrence, J.B.; Johnson, C.

    1990-01-01

    The inactivation of growth suppressor genes appears to play a major role in the malignant process. To assess whether protein phosphotyrosyl phosphatases function as growth suppressors, the authors have isolated a cDNA clone encoding human protein phosphotyrosyl phosphatase 1B for structural and functional characterization. The translation product deduced from the 1,305-nucleotide open reading frame predicts a protein containing 435 amino acids and having a molecular mass of 49,966 Da. The amino-terminal 321 amino acids deduced from the cDNA sequence are identical to the empirically determined sequence of protein phosphotyrosyl phosphatase 1B. A genomic clone has been isolated and used in an in situ hybridization to banded metaphase chromosomes to determine that the gene encoding protein phosphotyrosyl phosphatase 1B maps as a single-copy gene to the long arm of chromosome 20 in the region q13.1-q13.2

  19. Subcellular localization of alkaline phosphatase in Bacillus licheniformis 749/C by immunoelectron microscopy with colloidal gold

    International Nuclear Information System (INIS)

    Tinglu, G.; Ghosh, A.; Ghosh, B.K.

    1984-01-01

    Subcellular distribution of the alkaline phosphatase of Bacillus licheniformis 749/C was determined by an immunoelectron microscopy method. Anti-alkaline phosphatase antibody labeled with 15- to 18-nm colloidal gold particles (gold-immunoglobulin G [IgG] complex) were used for the study. Both the plasma membrane and cytoplasmic material were labeled with the gold-IgG particles. These particles formed clusters in association with the plasma membrane; in contrast, in the cytoplasm the particles were largely dispersed, and only a few clusters were found. The gold-IgG binding was quantitatively estimated by stereological analysis of labeled, frozen thin sections. This estimation of a variety of control samples showed that the labeling was specific for the alkaline phosphatase. Cluster formation of the gold -IgG particles in association with the plasma membrane suggests that existence of specific alkaline phosphatase binding sites (receptors) in the plasma membrane of B. licheniformis 749/C. 27 references, 6 figures, 1 table

  20. Acid phosphatase from stored Poa pratensis caryopses and its ability for binding to lectins

    Directory of Open Access Journals (Sweden)

    Irena Lorenc-Kubis

    2014-01-01

    Full Text Available The effect of the storage period of Poa pratensis caryopses on acid phosphatase activity and on the ability of this enzyme to interact with lectins has been studied. It has been shown that after ten years of caryopses storage, the activity of acid phosphatase decreased about 50 per cent, while the content of proteins and carbohydrates did not change. The decrease of enzyme activity during the long period of storage was found only in seeds, but not in chaffs. Acid phosphatase was isolated from caryopses stored one, two, three, five and ten years. The enzyme showed the ability to bind to immoblized as well as to free conA during the whole period of storage, hut did not react with Wheat Germen Agglutinin (WGA. The activation of acid phosphatase by binding to conA decreased with the length of storage period.

  1. Bone mineralisation in premature infants cannot be predicted from serum alkaline phosphatase or serum phosphate

    DEFF Research Database (Denmark)

    Faerk, J; Peitersen, Birgit; Petersen, S

    2002-01-01

    BACKGROUND: The bone mineral content of premature infants at term is lower than in mature infants at the same postconceptional age. Serum alkaline phosphatase and serum phosphate are often used as indicators of bone mineralisation. OBJECTIVE: To analyse the association between bone mineral content...... content was measured at term (mean gestational age 41 weeks) by dual energy x ray absorptiometry and corrected for body size. RESULTS: Serum alkaline phosphatase was significantly negatively associated with serum phosphate (p mineral content was not associated with mean serum alkaline...... and serum alkaline phosphatase and serum phosphate. METHODS: Serum alkaline phosphatase and phosphate were measured at weekly intervals during admission in 108 premature infants of gestational age below 32 weeks (mean (SD) gestational age 29 (2) weeks; mean (SD) birth weight 1129 (279) g). Bone mineral...

  2. Membrane-bound 2,3-diphosphoglycerate phosphatase of human erythrocytes.

    Science.gov (United States)

    Schröter, W; Neuvians, M

    1970-12-01

    Gradual osmotic hemolysis of human erythrocytes reduces the cell content of whole protein, hemoglobin, 2,3-diphosphoglycerate and triosephosphate isomerase extensively, but not that of membrane protein and 2,3-diphosphoglycerate phosphatase. After the refilling of the ghosts with 2,3-diphosphoglycerate and reconstitution of the membrane, the 2,3-diphosphoglycerate phosphatase activity equals that of intact red cells. The membrane-bound 2,3-diphosphoglycerate phosphatase can be activated by sodium hyposulfite. The enzyme system of ghosts seems to differ from that of intact red cells with regard to the optima of pH and temperature. It remains to be elucidated if the membrane binding of the 2,3-diphosphoglycerate phosphatase is related to the transfer of inorganic phosphate across the red cell membrane.

  3. The tyrosine phosphatase Shp2 interacts with NPM-ALK and regulates anaplastic lymphoma cell growth and migration

    DEFF Research Database (Denmark)

    Voena, Claudia; Conte, Chiara; Ambrogio, Chiara

    2007-01-01

    Anaplastic large cell lymphomas (ALCL) are mainly characterized by the reciprocal translocation t(2;5)(p23;q35) that involves the anaplastic lymphoma kinase (ALK) gene and generates the fusion protein NPM-ALK with intrinsic tyrosine kinase activity. NPM-ALK triggers several signaling cascades......, leading to increased cell growth, resistance to apoptosis, and changes in morphology and migration of transformed cells. To search for new NPM-ALK interacting molecules, we developed a mass spectrometry-based proteomic approach in HEK293 cells expressing an inducible NPM-ALK and identified the tyrosine...... phosphatase Shp2 as a candidate substrate. We found that NPM-ALK was able to bind Shp2 in coprecipitation experiments and to induce its phosphorylation in the tyrosine residues Y542 and Y580 both in HEK293 cells and ALCL cell lines. In primary lymphomas, antibodies against the phosphorylated tyrosine Y542...

  4. Purification and properties of acid phosphatase from Avena elatior L. seeds

    Directory of Open Access Journals (Sweden)

    E. Wieczorek

    2015-01-01

    Full Text Available Acid phosphatase F1 from Avena elatior seeds was isolated and partially purified by means of alcohol precepitation, DEAE-, CM-column chromatography, Sephadex G-150, Sephadex G-200 and Sepharose 4B - gel filtration. The enzyme was stable at 50°C, pH 5.1. The pH optimum for phosphatase activity was 4.2. Fluoride, Zn2+, molybdate were effective inhibitors. EDTA and l, 10-phenanthroline activated the enzyme.

  5. Ivermectin resistant and susceptible third-stage larvae of Haemonchus contortus: cholinesterase and phosphatase activities

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    Consuelo Giménez-Pardo

    2004-03-01

    Full Text Available Cholinesterase and acid phosphatase (AP, but not alkaline phosphatase activities, were detected in cytosolic and membrane-bound fractions of ivermectin resistant and susceptible Haemonchus contortus infective-stage larvae. Some differences in acetylcholinesterase activity of cytosolic fractions and in the AP activity of these fractions as well as in the response to AP inhibitors by membrane-bound fractions were detected. Data are discussed.

  6. Tetranucleotide repeat polymorphism at the human prostatic acid phosphatase (ACPP) gene

    Energy Technology Data Exchange (ETDEWEB)

    Polymeropoulos, M H; Xiao, Hong; Rath, D S; Merril, C R [National Inst. of Mental Health Neuroscience Center, Washington, DC (United States)

    1991-09-11

    The polymorphic (AAAT){sub n} repeat begins at base pair 2342 of the human prostatic acid phosphatase gene on chromosome 3q21-qter. The polymorphism can be typed using the polymerase chain reaction (PCR) as described previously. The predicted length of the amplified sequence was 275 bp. Co-dominant segregation was observed in two informative families. The human prostatic acid phosphatase gene has been assigned to chromosome 3q21-qter.

  7. Tyrosine phosphorylation in T cells is regulated by phosphatase activity: studies with phenylarsine oxide.

    OpenAIRE

    Garcia-Morales, P; Minami, Y; Luong, E; Klausner, R D; Samelson, L E

    1990-01-01

    Activation of T cells induces rapid tyrosine phosphorylation on the T-cell receptor zeta chain and other substrates. These phosphorylations can be regulated by a number of protein-tyrosine kinases (ATP: protein-tyrosine O-phosphotransferase, EC 2.7.1.112) and protein-tyrosine-phosphatases (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48). In this study, we demonstrate that phenylarsine oxide can inhibit tyrosine phosphatases while leaving tyrosine kinase function intact. We use this ...

  8. Imaging of alkaline phosphatase activity in bone tissue.

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    Terence P Gade

    Full Text Available The purpose of this study was to develop a paradigm for quantitative molecular imaging of bone cell activity. We hypothesized the feasibility of non-invasive imaging of the osteoblast enzyme alkaline phosphatase (ALP using a small imaging molecule in combination with (19Flourine magnetic resonance spectroscopic imaging ((19FMRSI. 6, 8-difluoro-4-methylumbelliferyl phosphate (DiFMUP, a fluorinated ALP substrate that is activatable to a fluorescent hydrolysis product was utilized as a prototype small imaging molecule. The molecular structure of DiFMUP includes two Fluorine atoms adjacent to a phosphate group allowing it and its hydrolysis product to be distinguished using (19Fluorine magnetic resonance spectroscopy ((19FMRS and (19FMRSI. ALP-mediated hydrolysis of DiFMUP was tested on osteoblastic cells and bone tissue, using serial measurements of fluorescence activity. Extracellular activation of DiFMUP on ALP-positive mouse bone precursor cells was observed. Concurringly, DiFMUP was also activated on bone derived from rat tibia. Marked inhibition of the cell and tissue activation of DiFMUP was detected after the addition of the ALP inhibitor levamisole. (19FMRS and (19FMRSI were applied for the non-invasive measurement of DiFMUP hydrolysis. (19FMRS revealed a two-peak spectrum representing DiFMUP with an associated chemical shift for the hydrolysis product. Activation of DiFMUP by ALP yielded a characteristic pharmacokinetic profile, which was quantifiable using non-localized (19FMRS and enabled the development of a pharmacokinetic model of ALP activity. Application of (19FMRSI facilitated anatomically accurate, non-invasive imaging of ALP concentration and activity in rat bone. Thus, (19FMRSI represents a promising approach for the quantitative imaging of bone cell activity during bone formation with potential for both preclinical and clinical applications.

  9. [Glucose-6-phosphatase from nuclear envelope in rat liver].

    Science.gov (United States)

    González-Mujica, Freddy

    2008-06-01

    Nuclear envelope (NE) and microsomal glucosa-6-phosphatase (G-6-Pase) activities were compared. Intact microsomes were unable to hydrolyze mannose-6-phosphate (M-6-P), on the other hand, intact NE hydrolyzes this substrate. Galactose-6-phosphate showed to be a good substrate for both NE and microsomal enzymes, with similar latency to that obtained with M-6-P using microsomes. In consequence, this substrate was used to measure the NE integrity. The kinetic parameters (Kii and Kis) of the intact NE G-6-Pase for the phlorizin inhibition using glucose-6-phosphate (G-6-P) and M-6-P as substrates, were very similar. The NE T1 transporter was more sensitive to amiloride than the microsomal T1. The microsomal system was more sensitive to N-ethylmalemide (NEM) than the NE and the latter was insensitive to anion transport inhibitors DIDS and SITS, which strongly affect the microsomal enzyme. The above results allowed to postulate the presence of a hexose-6-phosphate transporter in the NE which is able to carry G-6-P and M-6-P, and perhaps other hexose-6-phosphate which could be different from that present in microsomes or, if it is the same, its activity could by modified by the membrane system where it is included. The higher PPi hydrolysis activity of the intact NE G-6-Pase in comparison to the intact microsomal, suggests differences between the Pi/PPi transport (T2) of both systems. The lower sensitivity of the NE G-6-Pase to NEM suggests that the catalytic subunit of this system has some differences with the microsomal isoform.

  10. Response to DNA damage: why do we need to focus on protein phosphatases?

    Directory of Open Access Journals (Sweden)

    Midori eShimada

    2013-01-01

    Full Text Available Eukaryotic cells are continuously threatened by unavoidable errors during normal DNA replication or various sources of genotoxic stresses that cause DNA damage or stalled replication. To maintain genomic integrity, cells have developed a coordinated signaling network, known as the DNA damage response (DDR. Following DNA damage, sensor molecules detect the presence of DNA damage and transmit signals to downstream transducer molecules. This in turn conveys the signals to numerous effectors, which initiate a large number of specific biological responses, including transient cell cycle arrest mediated by checkpoints, DNA repair, and apoptosis. It is recently becoming clear that dephosphorylation events are involved in keeping DDR factors inactive during normal cell growth. Moreover, dephosphorylation is required to shut off checkpoint arrest following DNA damage and has been implicated in the activation of the DDR. Spatial and temporal regulation of phosphorylation events is essential for the DDR, and fine-tuning of phosphorylation is partly mediated by protein phosphatases. While the role of kinases in the DDR has been well documented, the complex roles of protein dephosphorylation have only recently begun to be investigated. Therefore, it is important to focus on the role of phosphatases and to determine how their activity is regulated upon DNA damage. In this work, we summarize current knowledge on the involvement of serine/threonine phosphatases, especially the protein phosphatase 1, protein phosphatase 2A, and protein phosphatase Mg2+/Mn2+-dependent families, in the DDR.

  11. A study of the alkaline and acid phosphatase activities in acute uranium intoxication

    International Nuclear Information System (INIS)

    Bokova, N.; Pavlova, V.; Stancheva, Yu.; Khadzhirusev, S.; Kiradzhiev, G.

    1975-01-01

    Comparative study of the ability of the sodium salt of diethylbarbituric acid and acetazolamide to protect the kidneys is conducted under conditions of acute uranium intoxication in rats. The parameters studied are alkaline and acid phosphatase activities in the serum and urine and phosphatase activity in the kidneys (histochemically as described by Gomori) followed up until the 30th day after the total uranyl acetate dose was reached (2 or 7 mg per kg bodyweight). Either compound exerted only minor effect on serum alkaline phosphatase activity. Sodium diethylbarbiturate induced distinct fluctuations in urinary alkaline phosphatase activity throughout the entire study period, but the differences never reached statistical significance. Acetazolamide caused essential decrease in urinary alkaline phosphatase activity. In either case renal tissue protection from the action of the uranyl ion may be suggested. This assumption is supported by the histochemical analysis. The compounds appeared to have no effect on serum acid phosphatase activity which showed high variability both in control and in treated rats. (Ch.K.)

  12. The impact of phosphatases on proliferative and survival signaling in cancer.

    Science.gov (United States)

    Narla, Goutham; Sangodkar, Jaya; Ryder, Christopher B

    2018-05-03

    The dynamic and stringent coordination of kinase and phosphatase activity controls a myriad of physiologic processes. Aberrations that disrupt the balance of this interplay represent the basis of numerous diseases. For a variety of reasons, early work in this area portrayed kinases as the dominant actors in these signaling events with phosphatases playing a secondary role. In oncology, these efforts led to breakthroughs that have dramatically altered the course of certain diseases and directed vast resources toward the development of additional kinase-targeted therapies. Yet, more recent scientific efforts have demonstrated a prominent and sometimes driving role for phosphatases across numerous malignancies. This maturation of the phosphatase field has brought with it the promise of further therapeutic advances in the field of oncology. In this review, we discuss the role of phosphatases in the regulation of cellular proliferation and survival signaling using the examples of the MAPK and PI3K/AKT pathways, c-Myc and the apoptosis machinery. Emphasis is placed on instances where these signaling networks are perturbed by dysregulation of specific phosphatases to favor growth and persistence of human cancer.

  13. The Arabidopsis thiamin-deficient mutant pale green1 lacks thiamin monophosphate phosphatase of the vitamin B1 biosynthesis pathway.

    Science.gov (United States)

    Hsieh, Wei-Yu; Liao, Jo-Chien; Wang, Hsin-Tzu; Hung, Tzu-Huan; Tseng, Ching-Chih; Chung, Tsui-Yun; Hsieh, Ming-Hsiun

    2017-07-01

    Thiamin diphosphate (TPP, vitamin B 1 ) is an essential coenzyme present in all organisms. Animals obtain TPP from their diets, but plants synthesize TPPde novo. We isolated and characterized an Arabidopsis pale green1 (pale1) mutant that contained higher concentrations of thiamin monophosphate (TMP) and less thiamin and TPP than the wild type. Supplementation with thiamin, but not the thiazole and pyrimidine precursors, rescued the mutant phenotype, indicating that the pale1 mutant is a thiamin-deficient mutant. Map-based cloning and whole-genome sequencing revealed that the pale1 mutant has a mutation in At5g32470 encoding a TMP phosphatase of the TPP biosynthesis pathway. We further confirmed that the mutation of At5g32470 is responsible for the mutant phenotypes by complementing the pale1 mutant with constructs overexpressing full-length At5g32470. Most plant TPP biosynthetic enzymes are located in the chloroplasts and cytosol, but At5g32470-GFP localized to the mitochondrion of the root, hypocotyl, mesophyll and guard cells of the 35S:At5g32470-GFP complemented plants. The subcellular localization of a functional TMP phosphatase suggests that the complete vitamin B1 biosynthesis pathway may involve the chloroplasts, mitochondria and cytosol in plants. Analysis of PALE1 promoter-uidA activity revealed that PALE1 is mainly expressed in vascular tissues of Arabidopsis seedlings. Quantitative RT-PCR analysis of TPP biosynthesis genes and genes encoding the TPP-dependent enzymes pyruvate dehydrogenase, α-ketoglutarate dehydrogenase and transketolase revealed that the transcript levels of these genes were upregulated in the pale1 mutant. These results suggest that endogenous levels of TPP may affect the expression of genes involved in TPP biosynthesis and TPP-dependent enzymes. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  14. Adaptor protein GRB2 promotes Src tyrosine kinase activation and podosomal organization by protein-tyrosine phosphatase ϵ in osteoclasts.

    Science.gov (United States)

    Levy-Apter, Einat; Finkelshtein, Eynat; Vemulapalli, Vidyasiri; Li, Shawn S-C; Bedford, Mark T; Elson, Ari

    2014-12-26

    The non-receptor isoform of protein-tyrosine phosphatase ϵ (cyt-PTPe) supports adhesion of bone-resorbing osteoclasts by activating Src downstream of integrins. Loss of cyt-PTPe reduces Src activity in osteoclasts, reduces resorption of mineralized matrix both in vivo and in cell culture, and induces mild osteopetrosis in young female PTPe KO mice. Activation of Src by cyt-PTPe is dependent upon this phosphatase undergoing phosphorylation at its C-terminal Tyr-638 by partially active Src. To understand how cyt-PTPe activates Src, we screened 73 Src homology 2 (SH2) domains for binding to Tyr(P)-638 of cyt-PTPe. The SH2 domain of GRB2 bound Tyr(P)-638 of cyt-PTPe most prominently, whereas the Src SH2 domain did not bind at all, suggesting that GRB2 may link PTPe with downstream molecules. Further studies indicated that GRB2 is required for activation of Src by cyt-PTPe in osteoclast-like cells (OCLs) in culture. Overexpression of GRB2 in OCLs increased activating phosphorylation of Src at Tyr-416 and of cyt-PTPe at Tyr-638; opposite results were obtained when GRB2 expression was reduced by shRNA or by gene inactivation. Phosphorylation of cyt-PTPe at Tyr-683 and its association with GRB2 are integrin-driven processes in OCLs, and cyt-PTPe undergoes autodephosphorylation at Tyr-683, thus limiting Src activation by integrins. Reduced GRB2 expression also reduced the ability of bone marrow precursors to differentiate into OCLs and reduced the fraction of OCLs in which podosomal adhesion structures assume organization typical of active, resorbing cells. We conclude that GRB2 physically links cyt-PTPe with Src and enables cyt-PTPe to activate Src downstream of activated integrins in OCLs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Intestinal Alkaline Phosphatase: Potential Roles in Promoting Gut Health in Weanling Piglets and Its Modulation by Feed Additives - A Review.

    Science.gov (United States)

    Melo, A D B; Silveira, H; Luciano, F B; Andrade, C; Costa, L B; Rostagno, M H

    2016-01-01

    The intestinal environment plays a critical role in maintaining swine health. Many factors such as diet, microbiota, and host intestinal immune response influence the intestinal environment. Intestinal alkaline phosphatase (IAP) is an important apical brush border enzyme that is influenced by these factors. IAP dephosphorylates bacterial lipopolysaccharides (LPS), unmethylated cytosine-guanosine dinucleotides, and flagellin, reducing bacterial toxicity and consequently regulating toll-like receptors (TLRs) activation and inflammation. It also desphosphorylates extracellular nucleotides such as uridine diphosphate and adenosine triphosphate, consequently reducing inflammation, modulating, and preserving the homeostasis of the intestinal microbiota. The apical localization of IAP on the epithelial surface reveals its role on LPS (from luminal bacteria) detoxification. As the expression of IAP is reported to be downregulated in piglets at weaning, LPS from commensal and pathogenic gram-negative bacteria could increase inflammatory processes by TLR-4 activation, increasing diarrhea events during this phase. Although some studies had reported potential IAP roles to promote gut health, investigations about exogenous IAP effects or feed additives modulating IAP expression and activity yet are necessary. However, we discussed in this paper that the critical assessment reported can suggest that exogenous IAP or feed additives that could increase its expression could show beneficial effects to reduce diarrhea events during the post weaning phase. Therefore, the main goals of this review are to discuss IAP's role in intestinal inflammatory processes and present feed additives used as growth promoters that may modulate IAP expression and activity to promote gut health in piglets.

  16. A vaccine strategy with multiple prostatic acid phosphatase-fused cytokines for prostate cancer treatment.

    Science.gov (United States)

    Fujio, Kei; Watanabe, Masami; Ueki, Hideo; Li, Shun-Ai; Kinoshita, Rie; Ochiai, Kazuhiko; Futami, Junichiro; Watanabe, Toyohiko; Nasu, Yasutomo; Kumon, Hiromi

    2015-04-01

    Immunotherapy is one of the attractive treatment strategies for advanced prostate cancer. The US Food and Drug Administration (FDA) previously approved the therapeutic vaccine, sipuleucel-T, which is composed of autologous antigen-presenting cells cultured with a fusion protein [prostatic acid phosphatase (PAP) and granulocyte-macrophage colony-stimulating factor (GMCSF)]. Although sipuleucel-T has been shown to prolong the median survival of patients for 4.1 months, more robust therapeutic effects may be expected by modifying the vaccination protocol. In the present study, we aimed to develop and validate a novel vaccination strategy using multiple PAP-fused cytokines for prostate cancer treatment. Using a super gene expression (SGE) system that we previously established to amplify the production of a recombinant protein, significant amounts of PAP-fused cytokines [human GMCSF, interleukin-2 (IL2), IL4, IL7 and mouse GMCSF and IL4] were obtained. We examined the activity of the fusion proteins in vitro to validate their cytokine functions. A significant upregulation of dendritic cell differentiation from monocytes was achieved by PAP-GMCSF when used with the other PAP-fused cytokines. The PAP-fused human IL2 significantly increased the proliferation of lymphocytes, as determined by flow cytometry. We also investigated the in vivo therapeutic effects of multiple PAP-fused cytokines in a mouse prostate cancer model bearing prostate-specific antigen (PSA)- and PAP-expressing tumors. The simultaneous intraperitoneal administration of PAP-GMCSF, -IL2, -IL4 and -IL7 significantly prevented tumor induction and inhibited the tumor growth in the PAP-expressing tumors, yet not in the PSA-expressing tumors. The in vivo therapeutic effects with the multiple PAP-fused cytokines were superior to the effects of PAP-GMCSF alone. We thus demonstrated the advantages of the combined use of multiple PAP-fused cytokines including PAP-GMCSF, and propose a promising prostatic

  17. EFEITO DE FATORES AMBIENTAIS DA FOSFATASE ÁCIDA NO FEIJOEIRO EFFECTS OF ENVIRONMENTAL FACTORS ON THE ACTIVITY OF ACID PHOSPHATASE IN COMMON BEAN

    Directory of Open Access Journals (Sweden)

    José Renato de Freitas

    2007-09-01

    Full Text Available

    Plantas com 15 dias após a germinação foram colhidas em experimentos de campo com a finalidade de conhecer o pH, temperatura e tempo necessários para melhor expressar a atividade da fosfatase ácida em três variedades do feijoeiro (Phaseolus vulgaris L., Carioca, EMP-84 e CNF-l0, na presença e na ausência de fósforo. Os maiores valores de atividade da fosfatase ácida foram observadas quando as plantas foram colocadas em solução em pH 5,5 durante 120 minutos à temperatura de 30°C. A utilização de substâncias tamponantes como PNPP + Triton X-100 expressaram melhor a atividade da fosfatase ácida. As condições de vácuo constituíram um fator positivo para a atividade da fosfatase ácida. As plantas desenvolvidas sob estresse hídrico apresentaram menor atividade da fosfatase ácida. A relação folha-raiz da atividade da fosfatase ácida atingiu 5,72 para a variedade Carioca, 4,91 para a variedade EMP-84 e 4,36 para a variedade CNF-10.

    PALAVRAS-CHAVE: pH; temperatura; solução tamponada; tempo de reação; Phaseolus vulgaris.

    Plants with 15 days after the germination were picked in field experiments with the purpose of knowing the best pH, temperature and the necessary time to express the activity of the phosphatase acid in three bean varieties (Phaseolus vulgaris L., Carioca, EMP-84 and CNF-10, in the presence and in the phosphorus absence. The largest values of activity of the phosphatase acid were observed when the plants were tested in pH 5.5 solution during 120 minutes at the temperature of 30°C. The use of buffer substances as PNPP + Triton X-100 expressed better the activity of the phosphatase acid. The vacuum condition constituted a positive factor to express the activity of the phosphatase acid. The plants

  18. Genome wide identification of wheat and Brachypodium type one protein phosphatases and functional characterization of durum wheat TdPP1a.

    Directory of Open Access Journals (Sweden)

    Mariem Bradai

    Full Text Available Reversible phosphorylation is an essential mechanism regulating signal transduction during development and environmental stress responses. An important number of dephosphorylation events in the cell are catalyzed by type one protein phosphatases (PP1, which catalytic activity is driven by the binding of regulatory proteins that control their substrate specificity or subcellular localization. Plants harbor several PP1 isoforms accounting for large functional redundancies. While animal PP1s were reported to play relevant roles in controlling multiple cellular processes, plant orthologs remain poorly studied. To decipher the role of plant PP1s, we compared PP1 genes from three monocot species, Brachypodium, common wheat and rice at the genomic and transcriptomic levels. To gain more insight into the wheat PP1 proteins, we identified and characterized TdPP1a, the first wheat type one protein phosphatase from a Tunisian durum wheat variety Oum Rabiaa3. TdPP1a is highly conserved in sequence and structure when compared to mammalian, yeast and other plant PP1s. We demonstrate that TdPP1a is an active, metallo-dependent phosphatase in vitro and is able to interact with AtI2, a typical regulator of PP1 functions. Also, TdPP1a is capable to complement the heat stress sensitivity of the yeast mutant indicating that TdPP1a is functional also in vivo. Moreover, transient expression of TdPP1a::GFP in tobacco leaves revealed that it is ubiquitously distributed within the cell, with a strong accumulation in the nucleus. Finally, transcriptional analyses showed similar expression levels in roots and leaves of durum wheat seedlings. Interestingly, the expression in leaves is significantly induced following salinity stress, suggesting a potential role of TdPP1a in wheat salt stress response.

  19. Purkinje Cell Compartmentation in the Cerebellum of the Lysosomal Acid Phosphatase 2 Mutant Mouse (Nax - Naked-Ataxia Mutant Mouse)

    Science.gov (United States)

    Bailey, Karen; Rahimi Balaei, Maryam; Mannan, Ashraf; Del Bigio, Marc R.; Marzban, Hassan

    2014-01-01

    The Acp2 gene encodes the beta subunit of lysosomal acid phosphatase, which is an isoenzyme that hydrolyzes orthophosphoric monoesters. In mice, a spontaneous mutation in Acp2 results in severe cerebellar defects. These include a reduced size, abnormal lobulation, and an apparent anterior cerebellar disorder with an absent or hypoplastic vermis. Based on differential gene expression in the cerebellum, the mouse cerebellar cortex can normally be compartmentalized anteroposteriorly into four transverse zones and mediolaterally into parasagittal stripes. In this study, immunohistochemistry was performed using various Purkinje cell compartmentation markers to examine their expression patterns in the Acp2 mutant. Despite the abnormal lobulation and anterior cerebellar defects, zebrin II and PLCβ4 showed similar expression patterns in the nax mutant and wild type cerebellum. However, fewer stripes were found in the anterior zone of the nax mutant, which could be due to a lack of Purkinje cells or altered expression of the stripe markers. HSP25 expression was uniform in the central zone of the nax mutant cerebellum at around postnatal day (P) 18–19, suggesting that HSP25 immunonegative Purkinje cells are absent or delayed in stripe pattern expression compared to the wild type. HSP25 expression became heterogeneous around P22–23, with twice the number of parasagittal stripes in the nax mutant compared to the wild type. Aside from reduced size and cortical disorganization, both the posterior zone and nodular zone in the nax mutant appeared less abnormal than the rest of the cerebellum. From these results, it is evident that the anterior zone of the nax mutant cerebellum is the most severely affected, and this extends beyond the primary fissure into the rostral central zone/vermis. This suggests that ACP2 has critical roles in the development of the anterior cerebellum and it may regulate anterior and central zone compartmentation. PMID:24722417

  20. Overexpression of protein tyrosine phosphatase-alpha (PTP-alpha) but not PTP-kappa inhibits translocation of GLUT4 in rat adipose cells

    DEFF Research Database (Denmark)

    Cong, L N; Chen, H; Li, Y

    1999-01-01

    Protein tyrosine phosphatases (PTPases) are likely to play important roles in insulin action. We recently demonstrated that the nontransmembrane PTPase PTP1B can act as a negative modulator of insulin-stimulated translocation of GLUT4. We now examine the role of PTP-alpha and PTP-kappa (two...... of cell surface GLUT4 in response to insulin and a threefold decrease in insulin sensitivity when compared with control cells expressing only tagged GLUT4. Co-overexpression of PTP-alpha and PTP1B did not have additive effects, suggesting that these PTPases share common substrates. Cells overexpressing...

  1. Associations between renal hyperfiltration and serum alkaline phosphatase.

    Directory of Open Access Journals (Sweden)

    Se Won Oh

    Full Text Available Renal hyperfiltration, which is associated with renal injury, occurs in diabetic or obese individuals. Serum alkaline phosphatase (ALP level is also elevated in patients with diabetes (DM or metabolic syndrome (MS, and increased urinary excretion of ALP has been demonstrated in patients who have hyperfiltration and tubular damage. However, little was investigated about the association between hyperfiltration and serum ALP level. A retrospective observational study of the 21,308 adults in the Korea National Health and Nutrition Examination Survey IV-V databases (2008-2011 was performed. Renal hyperfiltration was defined as exceeding the age- and sex-specific 97.5th percentile. We divided participants into 4 groups according to their estimated glomerular filtration rate (eGFR: >120, 90-119, 60-89, and 120 mL/min/1.73 m2 showed the highest risk for MS, in the highest ALP quartiles (3.848, 95% CI, 1.876-7.892, compared to the lowest quartile. Similarly, the highest risk for DM, in the highest ALP quartiles, was observed in participants with eGFR >120 ml/min/1.73 m2 (2.166, 95% CI, 1.084-4.329. ALP quartiles were significantly associated with albuminuria in participants with eGFR ≥ 60 ml/min/1.73m2. The highest ALP quartile had a 1.631-fold risk elevation for albuminuria with adjustment of age and sex. (95% CI, 1.158-2.297, P = 0.005. After adjustment, the highest ALP quartile had a 1.624-fold risk elevation, for renal hyperfiltration (95% CI, 1.204-2.192, P = 0.002. In addition, hyperfiltration was significantly associated with hemoglobin, triglyceride, white blood cell count, DM, smoking, and alcohol consumption (P<0.05. The relationship between serum ALP and metabolic disorders is stronger in participants with an upper-normal range of eGFR. Higher ALP levels are significantly associated with renal hyperfiltration in Korean general population.

  2. Voltage-sensing phosphatase modulation by a C2 domain

    Directory of Open Access Journals (Sweden)

    Paul M. Castle

    2015-04-01

    Full Text Available The voltage-sensing phosphatase (VSP is the first example of an enzyme controlled by changes in membrane potential. VSP has four distinct regions: the transmembrane voltage-sensing domain (VSD, the inter-domain linker, the cytosolic catalytic domain and the C2 domain. The VSD transmits the changes in membrane potential through the inter-domain linker activating the catalytic domain which then dephosphorylates phosphatidylinositol phosphate lipids. The role of the C2, however, has not been established. In this study, we explore two possible roles for the C2: catalysis and membrane-binding. The Ci-VSP crystal structures show that the C2 residue Y522 lines the active site suggesting a contribution to catalysis. When we mutated Y522 to phenylalanine, we found a shift in the voltage dependence of activity. This suggests hydrogen bonding as a mechanism of action. Going one step further, when we deleted the entire C2 domain, we found voltage-dependent enzyme activity was no longer detectable. This result clearly indicates the entire C2 is necessary for catalysis as well as for modulating activity. As C2s are known membrane-binding domains, we tested whether the VSP C2 interacts with the membrane. We probed a cluster of four positively charged residues lining the top of the C2 and suggested by previous studies to interact with phosphatidylinositol 4,5-bisphosphate (PI(4,5P2 (Kalli et al., 2014. Neutralizing those positive charges significantly shifted the voltage dependence of activity to higher voltages. We tested membrane binding by depleting PI(4,5P2 from the membrane using the 5HT2C receptor and found that the VSD motions as measured by voltage clamp fluorometry were not changed. These results suggest that if the C2 domain interacts with the membrane to influence VSP function it may not occur exclusively through PI(4,5P2. Together, this data advances our understanding of the VSP C2 by demonstrating a necessary and critical role for the C2 domain in

  3. Voltage-sensing phosphatase modulation by a C2 domain.

    Science.gov (United States)

    Castle, Paul M; Zolman, Kevin D; Kohout, Susy C

    2015-01-01

    The voltage-sensing phosphatase (VSP) is the first example of an enzyme controlled by changes in membrane potential. VSP has four distinct regions: the transmembrane voltage-sensing domain (VSD), the inter-domain linker, the cytosolic catalytic domain, and the C2 domain. The VSD transmits the changes in membrane potential through the inter-domain linker activating the catalytic domain which then dephosphorylates phosphatidylinositol phosphate (PIP) lipids. The role of the C2, however, has not been established. In this study, we explore two possible roles for the C2: catalysis and membrane-binding. The Ci-VSP crystal structures show that the C2 residue Y522 lines the active site suggesting a contribution to catalysis. When we mutated Y522 to phenylalanine, we found a shift in the voltage dependence of activity. This suggests hydrogen bonding as a mechanism of action. Going one step further, when we deleted the entire C2 domain, we found voltage-dependent enzyme activity was no longer detectable. This result clearly indicates the entire C2 is necessary for catalysis as well as for modulating activity. As C2s are known membrane-binding domains, we tested whether the VSP C2 interacts with the membrane. We probed a cluster of four positively charged residues lining the top of the C2 and suggested by previous studies to interact with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] (Kalli et al., 2014). Neutralizing those positive charges significantly shifted the voltage dependence of activity to higher voltages. We tested membrane binding by depleting PI(4,5)P2 from the membrane using the 5HT2C receptor and found that the VSD motions as measured by voltage clamp fluorometry (VCF) were not changed. These results suggest that if the C2 domain interacts with the membrane to influence VSP function it may not occur exclusively through PI(4,5)P2. Together, this data advances our understanding of the VSP C2 by demonstrating a necessary and critical role for the C2 domain in

  4.  Alkaline phosphatase normalization is a biomarker of improved survival in primary sclerosing cholangitis.

    Science.gov (United States)

    Hilscher, Moira; Enders, Felicity B; Carey, Elizabeth J; Lindor, Keith D; Tabibian, James H

    2016-01-01

     Introduction. Recent studies suggest that serum alkaline phosphatase may represent a prognostic biomarker in patients with primary sclerosing cholangitis. However, this association remains poorly understood. Therefore, the aim of this study was to investigate the prognostic significance and clinical correlates of alkaline phosphatase normalization in primary sclerosing cholangitis. This was a retrospective cohort study of patients with a new diagnosis of primary sclerosing cholangitis made at an academic medical center. The primary endpoint was time to hepatobiliaryneoplasia, liver transplantation, or liver-related death. Secondary endpoints included occurrence of and time to alkaline phosphatase normalization. Patients who did and did not achieve normalization were compared with respect to clinical characteristics and endpoint-free survival, and the association between normalization and the primary endpoint was assessed with univariate and multivariate Cox proportional-hazards analyses. Eighty six patients were included in the study, with a total of 755 patient-years of follow-up. Thirty-eight patients (44%) experienced alkaline phosphatase normalization within 12 months of diagnosis. Alkaline phosphatase normalization was associated with longer primary endpoint-free survival (p = 0.0032) and decreased risk of requiring liver transplantation (p = 0.033). Persistent normalization was associated with even fewer adverse endpoints as well as longer survival. In multivariate analyses, alkaline phosphatase normalization (adjusted hazard ratio 0.21, p = 0.012) and baseline bilirubin (adjusted hazard ratio 4.87, p = 0.029) were the only significant predictors of primary endpoint-free survival. Alkaline phosphatase normalization, particularly if persistent, represents a robust biomarker of improved long-term survival and decreased risk of requiring liver transplantation in patients with primary sclerosing cholangitis.

  5. AtPP2CG1, a protein phosphatase 2C, positively regulates salt tolerance of Arabidopsis in abscisic acid-dependent manner

    International Nuclear Information System (INIS)

    Liu, Xin; Zhu, Yanming; Zhai, Hong; Cai, Hua; Ji, Wei; Luo, Xiao; Li, Jing; Bai, Xi

    2012-01-01

    Highlights: ► AtPP2CG1 positively regulates salt tolerance in ABA-dependent manner. ► AtPP2CG1 up-regulates the expression of marker genes in different pathways. ► AtPP2CG1 expresses in the vascular system and trichomes of Arabidopsis. -- Abstract: AtPP2CG1 (Arabidopsis thaliana protein phosphatase 2C G Group 1) was predicted as an abiotic stress candidate gene by bioinformatic analysis in our previous study. The gene encodes a putative protein phosphatase 2C that belongs to Group G of PP2C. There is no report of Group G genes involved in abiotic stress so far. Real-time RT-PCR analysis showed that AtPP2CG1 expression was induced by salt, drought, and abscisic acid (ABA) treatment. The expression levels of AtPP2CG1 in the ABA synthesis-deficient mutant abi2–3 were much lower than that in WT plants under salt stress suggesting that the expression of AtPP2CG1 acts in an ABA-dependent manner. Over-expression of AtPP2CG1 led to enhanced salt tolerance, whereas its loss of function caused decreased salt tolerance. These results indicate that AtPP2CG1 positively regulates salt stress in an ABA-dependent manner. Under salt treatment, AtPP2CG1 up-regulated the expression levels of stress-responsive genes, including RD29A, RD29B, DREB2A and KIN1. GUS activity was detected in roots, leaves, stems, flower, and trichomes of AtPP2CG1 promoter–GUS transgenic plants. AtPP2CG1 protein was localized in nucleus and cytoplasm via AtPP2CG1:eGFP and YFP:AtPP2CG1 fusion approaches.

  6. The wip1 phosphatase (PPM1D) antagonizes activation of the CHK2 tumor suppressor kinase

    Energy Technology Data Exchange (ETDEWEB)

    Manet, Oliva-Trastoy; Berthonaud, V.; Chevalier, A.; Ducrot, C.; Marsolier-Kergoat, M.C.; Mann, C.; Leteurtre, F. [CEA Saclay, DSV, DBJC, SBGM, Lab. du Controle du Cycle Cellulaire, 91 - Gif-sur-Yvette (France)

    2006-07-01

    adaptation). Our group previously demonstrated that type 2C protein phosphatases (PP2C) Ptc2 and Ptc3 are required for DNA checkpoint inactivation after DNA double-strand break repair or adaptation in S. cerevisiae. Here we show the conservation of this pathway in mammalian cells. In response to DNA damage, ATM (ataxia telangiectasia mutated) phosphorylates the Chk2 tumor suppressor kinase at threonine 68 (Thr68), allowing Chk2 kinase dimerization and activation by auto-phosphorylations in the T-loop. The oncogenic protein Wip1, a PP2C phosphatase, binds Chk2 and de-phosphorylates phospho-Thr68. Consequently, Wip1 opposes Chk2 activation by ATM after ionizing irradiation of cells. The recombinant Chk2 protein is fully phosphorylated and activated, due to the high protein concentrations obtained during production. In vitro, Wip 1 de-phosphorylates the phospho-T68 of Chk2, but does not reduce Chk2 kinase activity on its usual GST-CDC25C substrate. These observations suggest that Wip1 phosphatase controls Chk2 activation rather than its enzymatic activity that relies on phosphorylations in the T-loop. The physiological consequences of Wip1 overexpression were tested in human adenocarcinoma cells: the HCT15 cell line. The specificities of this cell line are (i ) the absence of functional p53 proteins, leading to a G2 delay in response to a genotoxic stress, and (ii) the absence of functional Chk2 proteins, because of one CHK2 allele being unexpressed and because the second allele codes for a mutated protein that is unstable and inactive. The HCT15 cell line was complemented by a functional form of HA-Chk2 and the selected clone expresses the protein to a level similar to that observed in other cell lines. In HCT15 colorectal cancer cells corrected for functional Chk2 activity, Wip 1 modest overexpression suppressed the contribution of Chk2 to the G2/M DNA damage checkpoint. These results indicate that Wip1 is one of the phosphatases regulating the activity of Chk2 in response to

  7. The wip1 phosphatase (PPM1D) antagonizes activation of the CHK2 tumor suppressor kinase

    International Nuclear Information System (INIS)

    Manet, Oliva-Trastoy; Berthonaud, V.; Chevalier, A.; Ducrot, C.; Marsolier-Kergoat, M.C.; Mann, C.; Leteurtre, F.

    2006-01-01

    ). Our group previously demonstrated that type 2C protein phosphatases (PP2C) Ptc2 and Ptc3 are required for DNA checkpoint inactivation after DNA double-strand break repair or adaptation in S. cerevisiae. Here we show the conservation of this pathway in mammalian cells. In response to DNA damage, ATM (ataxia telangiectasia mutated) phosphorylates the Chk2 tumor suppressor kinase at threonine 68 (Thr68), allowing Chk2 kinase dimerization and activation by auto-phosphorylations in the T-loop. The oncogenic protein Wip1, a PP2C phosphatase, binds Chk2 and de-phosphorylates phospho-Thr68. Consequently, Wip1 opposes Chk2 activation by ATM after ionizing irradiation of cells. The recombinant Chk2 protein is fully phosphorylated and activated, due to the high protein concentrations obtained during production. In vitro, Wip 1 de-phosphorylates the phospho-T68 of Chk2, but does not reduce Chk2 kinase activity on its usual GST-CDC25C substrate. These observations suggest that Wip1 phosphatase controls Chk2 activation rather than its enzymatic activity that relies on phosphorylations in the T-loop. The physiological consequences of Wip1 overexpression were tested in human adenocarcinoma cells: the HCT15 cell line. The specificities of this cell line are (i ) the absence of functional p53 proteins, leading to a G2 delay in response to a genotoxic stress, and (ii) the absence of functional Chk2 proteins, because of one CHK2 allele being unexpressed and because the second allele codes for a mutated protein that is unstable and inactive. The HCT15 cell line was complemented by a functional form of HA-Chk2 and the selected clone expresses the protein to a level similar to that observed in other cell lines. In HCT15 colorectal cancer cells corrected for functional Chk2 activity, Wip 1 modest overexpression suppressed the contribution of Chk2 to the G2/M DNA damage checkpoint. These results indicate that Wip1 is one of the phosphatases regulating the activity of Chk2 in response to DNA

  8. Intestinal alkaline phosphatase in the colonic mucosa of children with inflammatory bowel disease

    Science.gov (United States)

    Molnár, Kriszta; Vannay, Ádám; Szebeni, Beáta; Bánki, Nóra Fanni; Sziksz, Erna; Cseh, Áron; Győrffy, Hajnalka; Lakatos, Péter László; Papp, Mária; Arató, András; Veres, Gábor

    2012-01-01

    AIM: To investigate intestinal alkaline phosphatase (iAP) in the intestinal mucosa of children with inflammatory bowel disease (IBD). METHODS: Colonic biopsy samples were taken from 15 newly diagnosed IBD patients and from 10 healthy controls. In IBD patients, specimens were obtained both from inflamed and non-inflamed areas. The iAP mRNA and protein expression was determined by reverse transcription-polymerase chain reaction and Western blotting analysis, respectively. Tissue localization of iAP and Toll-like receptor (TLR) 4 was investigated by immunofluorescent staining. RESULTS: The iAP protein level in the inflamed mucosa of children with Crohn’s disease (CD) and ulcerative colitis (UC) was significantly decreased when compared with controls (both P < 0.05). Similarly, we found a significantly decreased level of iAP protein in the inflamed mucosa in CD compared with non-inflamed mucosa in CD (P < 0.05). In addition, the iAP protein level in inflamed colonic mucosa in patients with UC was decreased compared with non-inflamed mucosa in patients with CD (P < 0.05). iAP protein levels in the non-inflamed mucosa of patients with CD were similar to controls. iAP mRNA expression in inflamed colonic mucosa of children with CD and UC was not significantly different from that in non-inflamed colonic mucosa with CD. Expression of iAP mRNA in patients with non-inflamed mucosa and in controls were similar. Co-localization of iAP with TLR4 showed intense staining with a dotted-like pattern. iAP was present in the inflamed and non-inflamed mucosa of patients with CD, UC, and in control biopsy specimens, irrespective of whether it was present in the terminal ileum or in the colon. However, the fluorescent signal of TLR4 was more pronounced in the colon compared with the terminal ileum in all groups studied. CONCLUSION: Lower than normal iAP protein levels in inflamed mucosa of IBD patients may indicate a role for iAP in inflammatory lesions in IBD. Based on our results

  9. Macrophage fusion is controlled by the cytoplasmic protein tyrosine phosphatase PTP-PEST/PTPN12.

    Science.gov (United States)

    Rhee, Inmoo; Davidson, Dominique; Souza, Cleiton Martins; Vacher, Jean; Veillette, André

    2013-06-01

    Macrophages can undergo cell-cell fusion, leading to the formation of multinucleated giant cells and osteoclasts. This process is believed to promote the proteolytic activity of macrophages toward pathogens, foreign bodies, and extracellular matrices. Here, we examined the role of PTP-PEST (PTPN12), a cytoplasmic protein tyrosine phosphatase, in macrophage fusion. Using a macrophage-targeted PTP-PEST-deficient mouse, we determined that PTP-PEST was not needed for macrophage differentiation or cytokine production. However, it was necessary for interleukin-4-induced macrophage fusion into multinucleated giant cells in vitro. It was also needed for macrophage fusion following implantation of a foreign body in vivo. Moreover, in the RAW264.7 macrophage cell line, PTP-PEST was required for receptor activator of nuclear factor kappa-B ligand (RANKL)-triggered macrophage fusion into osteoclasts. PTP-PEST had no impact on expression of fusion mediators such as β-integrins, E-cadherin, and CD47, which enable macrophages to become fusion competent. However, it was needed for polarization of macrophages, migration induced by the chemokine CC chemokine ligand 2 (CCL2), and integrin-induced spreading, three key events in the fusion process. PTP-PEST deficiency resulted in specific hyperphosphorylation of the protein tyrosine kinase Pyk2 and the adaptor paxillin. Moreover, a fusion defect was induced upon treatment of normal macrophages with a Pyk2 inhibitor. Together, these data argue that macrophage fusion is critically dependent on PTP-PEST. This function is seemingly due to the ability of PTP-PEST to control phosphorylation of Pyk2 and paxillin, thereby regulating cell polarization, migration, and spreading.

  10. Transmembrane Prostatic Acid Phosphatase (TMPAP) Interacts with Snapin and Deficient Mice Develop Prostate Adenocarcinoma

    Science.gov (United States)

    Quintero, Ileana B.; Herrala, Annakaisa M.; Araujo, César L.; Pulkka, Anitta E.; Hautaniemi, Sampsa; Ovaska, Kristian; Pryazhnikov, Evgeny; Kulesskiy, Evgeny; Ruuth, Maija K.; Soini, Ylermi; Sormunen, Raija T.; Khirug, Leonard; Vihko, Pirkko T.

    2013-01-01

    The molecular mechanisms underlying prostate carcinogenesis are poorly understood. Prostatic acid phosphatase (PAP), a prostatic epithelial secretion marker, has been linked to prostate cancer since the 1930's. However, the contribution of PAP to the disease remains controversial. We have previously cloned and described two isoforms of this protein, a secretory (sPAP) and a transmembrane type-I (TMPAP). The goal in this work was to understand the physiological function of TMPAP in the prostate. We conducted histological, ultra-structural and genome-wide analyses of the prostate of our PAP-deficient mouse model (PAP−/−) with C57BL/6J background. The PAP−/− mouse prostate showed the development of slow-growing non-metastatic prostate adenocarcinoma. In order to find out the mechanism behind, we identified PAP-interacting proteins byyeast two-hybrid assays and a clear result was obtained for the interaction of PAP with snapin, a SNARE-associated protein which binds Snap25 facilitating the vesicular membrane fusion process. We confirmed this interaction by co-localization studies in TMPAP-transfected LNCaP cells (TMPAP/LNCaP cells) and in vivo FRET analyses in transient transfected LNCaP cells. The differential gene expression analyses revealed the dysregulation of the same genes known to be related to synaptic vesicular traffic. Both TMPAP and snapin were detected in isolated exosomes. Our results suggest that TMPAP is involved in endo-/exocytosis and disturbed vesicular traffic is a hallmark of prostate adenocarcinoma. PMID:24039861

  11. Glycation Contributes to Interaction Between Human Bone Alkaline Phosphatase and Collagen Type I.

    Science.gov (United States)

    Halling Linder, Cecilia; Enander, Karin; Magnusson, Per

    2016-03-01

    Bone is a biological composite material comprised primarily of collagen type I and mineral crystals of calcium and phosphate in the form of hydroxyapatite (HA), which together provide its mechanical properties. Bone alkaline phosphatase (ALP), produced by osteoblasts, plays a pivotal role in the mineralization process. Affinity contacts between collagen, mainly type II, and the crown domain of various ALP isozymes were reported in a few in vitro studies in the 1980s and 1990s, but have not attracted much attention since, although such interactions may have important implications for the bone mineralization process. The objective of this study was to investigate the binding properties of human collagen type I to human bone ALP, including the two bone ALP isoforms B1 and B2. ALP from human liver, human placenta and E. coli were also studied. A surface plasmon resonance-based analysis, supported by electrophoresis and blotting, showed that bone ALP binds stronger to collagen type I in comparison with ALPs expressed in non-mineralizing tissues. Further, the B2 isoform binds significantly stronger to collagen type I in comparison with the B1 isoform. Human bone and liver ALP (with identical amino acid composition) displayed pronounced differences in binding, revealing that post-translational glycosylation properties govern these interactions to a large extent. In conclusion, this study presents the first evidence that glycosylation differences in human ALPs are of crucial importance for protein-protein interactions with collagen type I, although the presence of the ALP crown domain may also be necessary. Different binding affinities among the bone ALP isoforms may influence the mineral-collagen interface, mineralization kinetics, and degree of bone matrix mineralization, which are important factors determining the material properties of bone.

  12. Isolation and partial characterization of an acid phosphatase from Artemisia vulgaris pollen extract

    Directory of Open Access Journals (Sweden)

    RATKO M. JANKOV

    2002-09-01

    Full Text Available An acid phosphatase from an extract of mugwort (Artemisia vulgaris pollen was purified by a factor of 48 by a combination of ion exchange and gel-chromatography. The molecular weights of the enzyme were 76 kDa and 73 kDa, determined by gel filtration on a Sephadex G-100 sf column and by SDS PAGE (under reducing and non-reducing conditions, respectively. In analytical isoelectrofocusing, the enzyme appears as two very close bands, pI at about 4.2. The optimum pH for the enzyme is 5.4. The apparent Km for p-nitrophenyl phosphate was estimated to be 0.16 mM. The purified enzyme has broad specificity, and hydrolyses p-nitrophenyl phosphate and a-naphthyl phosphate. Pyrophosphate and O-phospho-L-tyrosine were estimated to be the best substrates for this enzyme as potential in vivo substrates. The enzyme is inhibited competitively by phosphate (Ki = 1.25 mM, molybdate (Ki = 0.055 mM and pyrophosphate (Ki = 6.7 mM and non-competitively by fluoride (Ki = 9.8 mM. Metal ions such as Hg2+, Cu2+ and Zn2+ express an inhibitory effect on the enzyme, while the enzyme is slightly activated by non-ionic detergents, Tween 20 and Triton X-100. There is no change in the enzyme activity in the presence of tartrate, citrate, EDTA, 1,10-phenanthroline and sulfhydryl-group modifiers such as p-chloromercuribenzoate and N-ethylmaleimide.

  13. Downregulation of Wip1 phosphatase modulates the cellular threshold of DNA damage signaling in mitosis

    Science.gov (United States)

    Macurek, Libor; Benada, Jan; Müllers, Erik; Halim, Vincentius A.; Krejčíková, Kateřina; Burdová, Kamila; Pecháčková, Sona; Hodný, Zdeněk; Lindqvist, Arne; Medema, René H.; Bartek, Jiri

    2013-01-01

    Cells are constantly challenged by DNA damage and protect their genome integrity by activation of an evolutionary conserved DNA damage response pathway (DDR). A central core of DDR is composed of a spatiotemporally ordered net of post-translational modifications, among which protein phosphorylation plays a major role. Activation of checkpoint kinases ATM/ATR and Chk1/2 leads to a temporal arrest in cell cycle progression (checkpoint) and allows time for DNA repair. Following DNA repair, cells re-enter the cell cycle by checkpoint recovery. Wip1 phosphatase (also called PPM1D) dephosphorylates multiple proteins involved in DDR and is essential for timely termination of the DDR. Here we have investigated how Wip1 is regulated in the context of the cell cycle. We found that Wip1 activity is downregulated by several mechanisms during mitosis. Wip1 protein abundance increases from G1 phase to G2 and declines in mitosis. Decreased abundance of Wip1 during mitosis is caused by proteasomal degradation. In addition, Wip1 is phosphorylated at multiple residues during mitosis, and this leads to inhibition of its enzymatic activity. Importantly, ectopic expression of Wip1 reduced γH2AX staining in mitotic cells and decreased the number of 53BP1 nuclear bodies in G1 cells. We propose that the combined decrease and inhibition of Wip1 in mitosis decreases the threshold necessary for DDR activation and enables cells to react adequately even to modest levels of DNA damage encountered during unperturbed mitotic progression. PMID:23255129

  14. Control of cytoplasmic and nuclear protein kinase A by phosphodiesterases and phosphatases in cardiac myocytes

    Science.gov (United States)

    Haj Slimane, Zeineb; Bedioune, Ibrahim; Lechêne, Patrick; Varin, Audrey; Lefebvre, Florence; Mateo, Philippe; Domergue-Dupont, Valérie; Dewenter, Matthias; Richter, Wito; Conti, Marco; El-Armouche, Ali; Zhang, Jin; Fischmeister, Rodolphe; Vandecasteele, Grégoire

    2014-01-01

    Aims The cAMP-dependent protein kinase (PKA) mediates β-adrenoceptor (β-AR) regulation of cardiac contraction and gene expression. Whereas PKA activity is well characterized in various subcellular compartments of adult cardiomyocytes, its regulation in the nucleus remains largely unknown. The aim of the present study was to compare the modalities of PKA regulation in the cytoplasm and nucleus of cardiomyocytes. Methods and results Cytoplasmic and nuclear cAMP and PKA activity were measured with targeted fluorescence resonance energy transfer probes in adult rat ventricular myocytes. β-AR stimulation with isoprenaline (Iso) led to fast cAMP elevation in both compartments, whereas PKA activity was fast in the cytoplasm but markedly slower in the nucleus. Iso was also more potent and efficient in activating cytoplasmic than nuclear PKA. Similar slow kinetics of nuclear PKA activation was observed upon adenylyl cyclase activation with L-858051 or phosphodiesterase (PDE) inhibition with 3-isobutyl-1-methylxantine. Consistently, pulse stimulation with Iso (15 s) maximally induced PKA and myosin-binding protein C phosphorylation in the cytoplasm, but marginally activated PKA and cAMP response element-binding protein phosphorylation in the nucleus. Inhibition of PDE4 or ablation of the Pde4d gene in mice prolonged cytoplasmic PKA activation and enhanced nuclear PKA responses. In the cytoplasm, phosphatase 1 (PP1) and 2A (PP2A) contributed to the termination of PKA responses, whereas only PP1 played a role in the nucleus. Conclusion Our study reveals a differential integration of cytoplasmic and nuclear PKA responses to β-AR stimulation in cardiac myocytes. This may have important implications in the physiological and pathological hypertrophic response to β-AR stimulation. PMID:24550350

  15. Identification of protein tyrosine phosphatase 1B and casein as substrates for 124-v-Mos

    Directory of Open Access Journals (Sweden)

    Stabel Silvia

    2002-04-01

    Full Text Available Abstract Background The mos proto-oncogene encodes a cytoplasmic serine/threonine-specific protein kinase with crucial function during meiotic cell division in vertebrates. Based on oncogenic amino acid substitutions the viral derivative, 124-v-Mos, displays constitutive protein kinase activity and functions independent of unknown upstream effectors of mos protein kinase. We have utilized this property of 124-v-Mos and screened for novel mos substrates in immunocomplex kinase assays in vitro. Results We generated recombinant 124-v-Mos using the baculovirus expression system in Spodoptera frugiperda cells and demonstrated constitutive kinase activity by the ability of 124-v-Mos to auto-phosphorylate and to phosphorylate vimentin, a known substrate of c-Mos. Using this approach we analyzed a panel of acidic and basic substrates in immunocomplex protein kinase assays and identified novel in vitro substrates for 124-v-Mos, the protein tyrosine phosphatase 1B (PTP1B, alpha-casein and beta-casein. We controlled mos-specific phosphorylation of PTP1B and casein in comparative assays using a synthetic kinase-inactive 124-v-Mos mutant and further, tryptic digests of mos-phosphorylated beta-casein identified a phosphopeptide specifically targeted by wild-type 124-v-Mos. Two-dimensional phosphoamino acid analyses showed that 124-v-mos targets serine and threonine residues for phosphorylation in casein at a 1:1 ratio but auto-phosphorylation occurs predominantly on serine residues. Conclusion The mos substrates identified in this study represent a basis to approach the identification of the mos-consensus phosphorylation motif, important for the development of specific inhibitors of the Mos protein kinase.

  16. Trichostatin A, a histone deacetylase inhibitor, suppresses JAK2/STAT3 signaling via inducing the promoter-associated histone acetylation of SOCS1 and SOCS3 in human colorectal cancer cells.

    Science.gov (United States)

    Xiong, Hua; Du, Wan; Zhang, Yan-Jie; Hong, Jie; Su, Wen-Yu; Tang, Jie-Ting; Wang, Ying-Chao; Lu, Rong; Fang, Jing-Yuan

    2012-02-01

    Aberrant janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling is involved in the oncogenesis of several cancers. Suppressors of cytokine signaling (SOCS) genes and SH2-containing protein tyrosine phosphatase 1 (SHP1) proteins, which are negative regulators of JAK/STAT signaling, have been reported to have tumor suppressor functions. However, in colorectal cancer (CRC) cells, the mechanisms that regulate SOCS and SHP1 genes, and the cause of abnormalities in the JAK/STAT signaling pathway, remain largely unknown. The present study shows that trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, leads to the hyperacetylation of histones associated with the SOCS1 and SOCS3 promoters, but not the SHP1 promoter in CRC cells. This indicates that histone modifications are involved in the regulation of SOCS1 and SOCS3. Moreover, upregulation of SOCS1 and SOCS3 expression was achieved using TSA, which also significantly downregulated JAK2/STAT3 signaling in CRC cells. We also demonstrate that TSA suppresses the growth of CRC cells, and induces G1 cell cycle arrest and apoptosis through the regulation of downstream targets of JAK2/STAT3 signaling, including Bcl-2, survivin and p16(ink4a) . Therefore, our data demonstrate that TSA may induce SOCS1 and SOCS3 expression by inducing histone modifications and consequently inhibits JAK2/STAT3 signaling in CRC cells. These results also establish a mechanistic link between the inhibition of JAK2/STAT3 signaling and the anticancer action of TSA in CRC cells. Copyright © 2011 Wiley Periodicals, Inc.

  17. Crystal structure of the cytoplasmic phosphatase and tensin homolog (PTEN)-like region of Ciona intestinalis voltage-sensing phosphatase provides insight into substrate specificity and redox regulation of the phosphoinositide phosphatase activity.

    Science.gov (United States)

    Matsuda, Makoto; Takeshita, Kohei; Kurokawa, Tatsuki; Sakata, Souhei; Suzuki, Mamoru; Yamashita, Eiki; Okamura, Yasushi; Nakagawa, Atsushi

    2011-07-01

    Ciona intestinalis voltage-sensing phosphatase (Ci-VSP) has a transmembrane voltage sensor domain and a cytoplasmic region sharing similarity to the phosphatase and tensin homolog (PTEN). It dephosphorylates phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate upon membrane depolarization. The cytoplasmic region is composed of a phosphatase domain and a putative membrane interaction domain, C2. Here we determined the crystal structures of the Ci-VSP cytoplasmic region in three distinct constructs, wild-type (248-576), wild-type (236-576), and G365A mutant (248-576). The crystal structure of WT-236 and G365A-248 had the disulfide bond between the catalytic residue Cys-363 and the adjacent residue Cys-310. On the other hand, the disulfide bond was not present in the crystal structure of WT-248. These suggest the possibility that Ci-VSP is regulated by reactive oxygen species as found in PTEN. These structures also revealed that the conformation of the TI loop in the active site of the Ci-VSP cytoplasmic region was distinct from the corresponding region of PTEN; Ci-VSP has glutamic acid (Glu-411) in the TI loop, orienting toward the center of active site pocket. Mutation of Glu-411 led to acquirement of increased activity toward phosphatidylinositol 3,5-bisphosphate, suggesting that this site is required for determining substrate specificity. Our results provide the basic information of the enzymatic mechanism of Ci-VSP.

  18. Genetic variation of an acid phosphatase (Acp-2) in the laboratory rat: possible homology with mouse AP-1 and human ACP2.

    Science.gov (United States)

    Bender, K; Bissbort, S; Kuhn, A; Nagel, M; Günther, E

    1986-02-01

    A genetic locus controlling the electrophoretic mobility of an acid phosphatase in the rat (Rattus norvegicus) is described. The locus, designed Acp-2, is not expressed in erythrocytes but is expressed in all other tissues studied. The product of Acp-2 hydrolyzes a wide variety of phosphate monoesters and is inhibited by L(+)-tartaric acid. Inbred rat strains have fixed either allele Acp-2a or allele Acp-2b. Codominant expression is observed in the respective F1 hybrids. Backcross progenies revealed the expected 1:1 segregation ratio. Possible loose linkage was found between the Acp-2 and the Pep-3 gene loci at a recombination frequency of 0.36 +/- 0.06.

  19. Acid phosphatase turnover during repressed and derepressed cultivation of Aspergillus niger

    International Nuclear Information System (INIS)

    Komano, Teruya

    1975-01-01

    Enhancement of the activity of acid phosphatase (EC 3.1.3.2) by phosphate starvation in growing Aspergillus niger mycelia was prevented by cycloheximide. This indicates that the enhancement was due to de novo protein synthesis caused by derepression. Radioactive acid phosphatase extracted from mycelia labeled with 14 C-amino acid was separated into at least four fractions. Experiments on pulse labeling and the chasing of the four acid phosphatases revealed the synthesis and degradation of each fraction occurred at different rates; showing a different rate of turnover of the enzyme molecules. The results of similar experiments performed during culture in the presence of phosphate (partially repressed condition) suggested that the marked change in the activity ratios of the four acid phosphatases during cultivation was the result of the active turnover of enzyme molecules. In contrast, the slight changes in the ratios observed during derepressed cultivation seemed to be the result of similar of synthesis and degradation of each phosphatase fraction. (auth.)

  20. Sensitive detection of alkaline phosphatase by switching on gold nanoclusters fluorescence quenched by pyridoxal phosphate.

    Science.gov (United States)

    Halawa, Mohamed Ibrahim; Gao, Wenyue; Saqib, Muhammad; Kitte, Shimeles Addisu; Wu, Fengxia; Xu, Guobao

    2017-09-15

    In this work, we designed highly sensitive and selective luminescent detection method for alkaline phosphatase using bovine serum albumin functionalized gold nanoclusters (BSA-AuNCs) as the nanosensor probe and pyridoxal phosphate as the substrate of alkaline phosphatase. We found that pyridoxal phosphate can quench the fluorescence of BSA-AuNCs and pyridoxal has little effect on the fluorescence of BSA-AuNCs. The proposed mechanism of fluorescence quenching by PLP was explored on the basis of data obtained from high-resolution transmission electron microscopy (HRTEM), dynamic light scattering (DLS), UV-vis spectrophotometry, fluorescence spectroscopy, fluorescence decay time measurements and circular dichroism (CD) spectroscopy. Alkaline phosphatase catalyzes the hydrolysis of pyridoxal phosphate to generate pyridoxal, restoring the fluorescence of BSA-AuNCs. Therefore, a recovery type approach has been developed for the sensitive detection of alkaline phosphatase in the range of 1.0-200.0U/L (R 2 =0.995) with a detection limit of 0.05U/L. The proposed sensor exhibit excellent selectivity among various enzymes, such as glucose oxidase, lysozyme, trypsin, papain, and pepsin. The present switch-on fluorescence sensing strategy for alkaline phosphatase was successfully applied in human serum plasma with good recoveries (100.60-104.46%), revealing that this nanosensor probe is a promising tool for ALP detection. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Integrative proteomics and biochemical analyses define Ptc6p as the Saccharomyces cerevisiae pyruvate dehydrogenase phosphatase.

    Science.gov (United States)

    Guo, Xiao; Niemi, Natalie M; Coon, Joshua J; Pagliarini, David J

    2017-07-14

    The pyruvate dehydrogenase complex (PDC) is the primary metabolic checkpoint connecting glycolysis and mitochondrial oxidative phosphorylation and is important for maintaining cellular and organismal glucose homeostasis. Phosphorylation of the PDC E1 subunit was identified as a key inhibitory modification in bovine tissue ∼50 years ago, and this regulatory process is now known to be conserved throughout evolution. Although Saccharomyces cerevisiae is a pervasive model organism for investigating cellular metabolism and its regulation by signaling processes, the phosphatase(s) responsible for activating the PDC in S. cerevisiae has not been conclusively defined. Here, using comparative mitochondrial phosphoproteomics, analyses of protein-protein interactions by affinity enrichment-mass spectrometry, and in vitro biochemistry, we define Ptc6p as the primary PDC phosphatase in S. cerevisiae Our analyses further suggest additional substrates for related S. cerevisiae phosphatases and describe the overall phosphoproteomic changes that accompany mitochondrial respiratory dysfunction. In summary, our quantitative proteomics and biochemical analyses have identified Ptc6p as the primary-and likely sole- S. cerevisiae PDC phosphatase, closing a key knowledge gap about the regulation of yeast mitochondrial metabolism. Our findings highlight the power of integrative omics and biochemical analyses for annotating the functions of poorly characterized signaling proteins. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Relationship of serum and saliva calcium, phosphorus and alkaline phosphatase with dry mouth feeling in menopause.

    Science.gov (United States)

    Agha-Hosseini, Farzaneh; Mirzaii-Dizgah, Iraj; Moosavi, Mahdieh-Sadat

    2012-06-01

    The aim of this study was to compare serum and saliva calcium, phosphorus and alkaline phosphatase of menopausal women with/without dry mouth (DM) feeling. The composition of saliva in menopause women with/without DM feeling is different. Some of these differences are in hormones that are related to bone turnover. A case-control study was carried out on 60 selected menopausal women aged 45-79 years with or without DM feeling (30 as case, 30 as control), conducted at the Clinic of Oral Medicine, Tehran University of Medical Sciences. The phosphorus concentration was measured by photometrical measurement of the blue colour formed after the addition of ammonium molybdate and stannous chloride; calcium was measured by Arsenazo reaction; and alkaline phosphatase by the pNPP-AMP method. Statistical analysis of Student's t-test was used. The mean serum phosphorus and alkaline phosphatase, stimulated and unstimulated saliva calcium and alkaline phosphatase levels were significantly higher in the menopausal women suffering from DM. There were no significant differences between groups regarding saliva phosphorus and serum calcium concentration. Calcium, phosphorus and alkaline phosphatase appear associated with DM feeling in menopause. © 2012 The Gerodontology Society and John Wiley & Sons A/S.

  3. Phosphatase Activity of Microbial Populations in Different Milk Samples in Relation to Protein and Carbohydrate Content

    Directory of Open Access Journals (Sweden)

    Sosanka Protim SANDILYA

    2014-12-01

    Full Text Available Cattle milk is a rich source of protein, carbohydrate, vitamins, minerals and all other major and micro nutrients. At a moderate pH, milk is an excellent media for the growth of microbes and thus, intake of raw milk is precarious. In this study, attempt was made for a qualitative study of eight raw milk samples of different varieties of cow and goat milk, collected from Jorhat district of Assam, India, on the basis of nutritional value and microbial population. The highest microbial population was found in the milk collected from cross hybrid variety of cow, whereas microbial contamination was the least in Jersey cow milk. Samples of C1 (Jersey cow variety showed presence of the highest amount of protein and carbohydrate content as compared to the others. Almost all the milk samples showed positive acid and alkaline phosphatase activity. Maximum acid phosphatase activity was observed in cross hybrid cow milk, whereas local cow milk exhibited the highest alkaline phosphatase activity. Phosphatase activity did not show any co-relationship with microbial population of the milk samples. Similarly, the protein and carbohydrate content of the samples did not have any significant impact on both acid and alkaline phosphatase activity.

  4. Phosphate-solubility and phosphatase activity in Gangetic alluvial soil as influenced by organophosphate insecticide residues.

    Science.gov (United States)

    Majumder, Shyam Prasad; Das, Amal Chandra

    2016-04-01

    An experiment was conducted under laboratory conditions to investigate the effect of four organophosphate insecticides, viz. monocrotophos, profenophos, quinalphos and triazophos at their field application rates (0.75, 1.0, 0.5 and 0.6 kg a.i.ha(-1), respectively), on the growth and activities of phosphate solubilizing microorganisms in relation to availability of insoluble phosphates in the Gangetic alluvial soil of West Bengal, India. The proliferation of phosphate solubilizing microorganisms was highly induced with profenophos (38.3%), while monocrotophos exerted maximum stimulation (20.8%) towards the solubility of insoluble phosphates in soil. The phosphatase activities of the soil (both acid phosphatase and alkaline phosphatase) were significantly increased due to the incorporation of the insecticides in general, and the augmentation was more pronounced with quinalphos (43.1%) followed by profenophos (27.6%) for acid phosphatase, and with monocrotophos (25.2%) followed by profenophos (16.1%) for alkaline phosphatase activity in soil. The total phosphorus was highly retained by triazophos (19.9%) followed by monocrotophos (16.5%), while incorporation of triazophos and quinalphos manifested greater availability of water soluble phosphorus in soil. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Deficiency in Protein Tyrosine Phosphatase PTP1B Shortens Lifespan and Leads to Development of Acute Leukemia.

    Science.gov (United States)

    Le Sommer, Samantha; Morrice, Nicola; Pesaresi, Martina; Thompson, Dawn; Vickers, Mark A; Murray, Graeme I; Mody, Nimesh; Neel, Benjamin G; Bence, Kendra K; Wilson, Heather M; Delibegović, Mirela

    2018-01-01

    Protein tyrosine phosphatase PTP1B is a critical regulator of signaling pathways controlling metabolic homeostasis, cell proliferation, and immunity. In this study, we report that global or myeloid-specific deficiency of PTP1B in mice decreases lifespan. We demonstrate that myeloid-specific deficiency of PTP1B is sufficient to promote the development of acute myeloid leukemia. LysM-PTP1B -/- mice lacking PTP1B in the innate myeloid cell lineage displayed a dysregulation of bone marrow cells with a rapid decline in population at midlife and a concomitant increase in peripheral blood blast cells. This phenotype manifested further with extramedullary tumors, hepatic macrophage infiltration, and metabolic reprogramming, suggesting increased hepatic lipid metabolism prior to overt tumor development. Mechanistic investigations revealed an increase in anti-inflammatory M2 macrophage responses in liver and spleen, as associated with increased expression of arginase I and the cytokines IL10 and IL4. We also documented STAT3 hypersphosphorylation and signaling along with JAK-dependent upregulation of antiapoptotic proteins Bcl2 and BclXL. Our results establish a tumor suppressor role for PTP1B in the myeloid lineage cells, with evidence that its genetic inactivation in mice is sufficient to drive acute myeloid leukemia. Significance: This study defines a tumor suppressor function for the protein tyrosine phosphatase PTP1B in myeloid lineage cells, with evidence that its genetic inactivation in mice is sufficient to drive acute myeloid leukemia. Cancer Res; 78(1); 75-87. ©2017 AACR . ©2017 American Association for Cancer Research.

  6. Neurotrophin-3 Enhances the Synaptic Organizing Function of TrkC-Protein Tyrosine Phosphatase σ in Rat Hippocampal Neurons.

    Science.gov (United States)

    Ammendrup-Johnsen, Ina; Naito, Yusuke; Craig, Ann Marie; Takahashi, Hideto

    2015-09-09

    Neurotrophin-3 (NT-3) and its high-affinity receptor TrkC play crucial trophic roles in neuronal differentiation, axon outgrowth, and synapse development and plasticity in the nervous system. We demonstrated previously that postsynaptic TrkC functions as a glutamatergic synapse-inducing (synaptogenic) cell adhesion molecule trans-interacting with presynaptic protein tyrosine phosphatase σ (PTPσ). Given that NT-3 and PTPσ bind distinct domains of the TrkC extracellular region, here we tested the hypothesis that NT-3 modulates TrkC/PTPσ binding and synaptogenic activity. NT-3 enhanced PTPσ binding to cell surface-expressed TrkC and facilitated the presynapse-inducing activity of TrkC in rat hippocampal neurons. Imaging of recycling presynaptic vesicles combined with TrkC knockdown and rescue approaches demonstrated that NT-3 rapidly potentiates presynaptic function via binding endogenous postsynaptic TrkC in a tyrosine kinase-independent manner. Thus, NT-3 positively modulates the TrkC-PTPσ complex for glutamatergic presynaptic assembly and function independently from TrkC kinase activation. Our findings provide new insight into synaptic roles of neurotrophin signaling and mechanisms controlling synaptic organizing complexes. Significance statement: Although many synaptogenic adhesion complexes have been identified in recent years, little is known about modulatory mechanisms. Here, we demonstrate a novel role of neurotrophin-3 in synaptic assembly and function as a positive modulator of the TrkC-protein tyrosine phosphatase σ complex. This study provides new insight into the involvement of neurotrophin signaling in synapse development and plasticity, presenting a molecular mechanism that may underlie previous observations of short- and long-term enhancement of presynaptic function by neurotrophin. Given the links of synaptogenic adhesion molecules to autism and schizophrenia, this study might also contribute to a better understanding of the pathogenesis of

  7. Protein phosphatase 2ACα gene knock-out results in cortical atrophy through activating hippo cascade in neuronal progenitor cells.

    Science.gov (United States)

    Liu, Bo; Sun, Li-Hua; Huang, Yan-Fei; Guo, Li-Jun; Luo, Li-Shu

    2018-02-01

    Protein phosphatase 2ACα (PP2ACα), a vital member of the protein phosphatase family, has been studied primarily as a regulator for the development, growth and protein synthesis of a lot of cell types. Dysfunction of PP2ACα protein results in neurodegenerative disease; however, this finding has not been directly confirmed in the mouse model with PP2ACα gene knock-out. Therefore, in this study presented here, we generated the PP2ACα gene knock-out mouse model by the Cre-loxP targeting gene system, with the purpose to directly observe the regulatory role of PP2ACα gene in the development of mouse's cerebral cortex. We observe that knocking-out PP2ACα gene in the central nervous system (CNS) results in cortical neuronal shrinkage, synaptic plasticity impairments, and learning/memory deficits. Further study reveals that PP2ACα gene knock-out initiates Hippo cascade in cortical neuroprogenitor cells (NPCs), which blocks YAP translocation into the nuclei of NPCs. Notably, p73, directly targeted by Hippo cascade, can bind to the promoter of glutaminase2 (GLS2) that plays a dominant role in the enzymatic regulation of glutamate/glutamine cycle. Finally, we find that PP2ACα gene knock-out inhibits the glutamine synthesis through up-regulating the activity of phosphorylated-p73 in cortical NPCs. Taken together, it concludes that PP2ACα critically supports cortical neuronal growth and cognitive function via regulating the signaling transduction of Hippo-p73 cascade. And PP2ACα indirectly modulates the glutamine synthesis of cortical NPCs through targeting p73 that plays a direct transcriptional regulatory role in the gene expression of GLS2. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Tumor suppressor PTEN affects tau phosphorylation: deficiency in the phosphatase activity of PTEN increases aggregation of an FTDP-17 mutant Tau

    Directory of Open Access Journals (Sweden)

    Zhang Xue

    2006-07-01

    Full Text Available Abstract Background Aberrant hyperphosphorylation of tau protein has been implicated in a variety of neurodegenerative disorders. Although a number of protein kinases have been shown to phosphorylate tau in vitro and in vivo, the molecular mechanisms by which tau phosphorylation is regulated pathophysiologically are largely unknown. Recently, a growing body of evidence suggests a link between tau phosphorylation and PI3K signaling. In this study, phosphorylation, aggregation and binding to the microtubule of a mutant frontal temporal dementia and parkinsonism linked to chromosome 17 (FTDP-17 tau in the presence of tumor suppressor PTEN, a major regulatory component in PI3K signaling, were investigated. Results Phosphorylation of the human mutant FTDP-17 tau, T40RW, was evaluated using different phospho-tau specific antibodies in the presence of human wild-type or phosphatase activity null mutant PTEN. Among the evaluated phosphorylation sites, the levels of Ser214 and Thr212 phospho-tau proteins were significantly decreased in the presence of wild-type PTEN, and significantly increased when the phosphatase activity null mutant PTEN was ectopically expressed. Fractionation of the mutant tau transfected cells revealed a significantly increased level of soluble tau in cytosol when wild-type PTEN was expressed, and an elevated level of SDS-soluble tau aggregates in the presence of the mutant PTEN. In addition, the filter/trap assays detected more SDS-insoluble mutant tau aggregates in the cells overexpressing the mutant PTEN compared to those in the cells overexpressing wild-type PTEN and control DNA. This notion was confirmed by the immunocytochemical experiment which demonstrated that the overexpression of the phosphatase activity null mutant PTEN caused the mutant tau to form aggregates in the COS-7 cells. Conclusion Tumor suppressor PTEN can alleviate the phosporylation of the mutant FTDP-17 tau at specific sites, and the phosphatase activity

  9. Potential role of voltage-sensing phosphatases in regulation of cell structure through the production of PI(3,4)P2.

    Science.gov (United States)

    Yamaguchi, Shinji; Kurokawa, Tatsuki; Taira, Ikuko; Aoki, Naoya; Sakata, Souhei; Okamura, Yasushi; Homma, Koichi J

    2014-04-01

    Voltage-sensing phosphatase, VSP, consists of the transmembrane domain, operating as the voltage sensor, and the cytoplasmic domain with phosphoinositide-phosphatase activities. The voltage sensor tightly couples with the cytoplasmic phosphatase and membrane depolarization induces dephosphorylation of several species of phosphoinositides. VSP gene is conserved from urochordate to human. There are some diversities among VSP ortholog proteins; range of voltage of voltage sensor motions as well as substrate selectivity. In contrast with recent understandings of biophysical mechanisms of VSPs, little is known about its physiological roles. Here we report that chick ortholog of VSP (designated as Gg-VSP) induces morphological feature of cell process outgrowths with round cell body in DF-1 fibroblasts upon its forced expression. Expression of the voltage sensor mutant, Gg-VSPR153Q with shifted voltage dependence to a lower voltage led to more frequent changes of cell morphology than the wild-type protein. Coexpression of PTEN that dephosphorylates PI(3,4)P2 suppressed this effect by Gg-VSP, indicating that the increase of PI(3,4)P2 leads to changes of cell shape. In addition, visualization of PI(3,4)P2 with the fluorescent protein fused with the TAPP1-derived pleckstrin homology (PH) domain suggested that Gg-VSP influenced the distribution of PI(3,4)P2 . These findings raise a possibility that one of the VSP's functions could be to regulate cell morphology through voltage-sensitive tuning of phosphoinositide profile. © 2013 Wiley Periodicals, Inc.

  10. Signaling Cascades: Consequences of Varying Substrate and Phosphatase Levels

    DEFF Research Database (Denmark)

    Feliu, Elisenda; Knudsen, Michael; Wiuf, Carsten Henrik

    2012-01-01

    We study signaling cascades with an arbitrary number of layers of one-site phosphorylation cycles. Such cascades are abundant in nature and integrated parts of many pathways. Based on the Michaelis-Menten model of enzyme kinetics and the law of mass-action, we derive explicit analytic expressions...

  11. Evidence That Speciation of Oxovanadium Complexes Does Not Solely Account for Inhibition of Leishmania Acid Phosphatases

    Science.gov (United States)

    Dorsey, Benjamin M.; McLauchlan, Craig C.; Jones, Marjorie A.

    2018-01-01

    Leishmaniasis is an endemic disease affecting a diverse spectra of populations, with 1.6 million new cases reported each year. Current treatment options are costly and have harsh side effects. New therapeutic options that have been previously identified, but still underappreciated as potential pharmaceutical targets, are Leishmania secreted acid phosphatases (SAP). These acid phosphatases, which are reported to play a role in the survival of the parasite in the sand fly vector, and in homing to the host macrophage, are inhibited by orthovanadate and decavanadate. Here, we use L. tarentolae to further evaluate these inhibitors. Using enzyme assays, and UV-visible spectroscopy, we investigate which oxovanadium starting material (orthovanadate or decavanadate) is a better inhibitor of L. tarentolae secreted acid phosphatase activity in vitro at the same total moles of vanadium. Considering speciation and total vanadium concentration, decavanadate is a consistently better inhibitor of SAP in our conditions, especially at low substrate:inhibitor ratios. PMID:29707535

  12. Evidence that Speciation of Oxovanadium Complexes does not Solely Account for Inhibition of Leishmania Acid Phosphatases

    Science.gov (United States)

    Dorsey, Benjamin M.; McLauchlan, Craig C.; Jones, Marjorie A.

    2018-04-01

    Leishmaniasis is an endemic disease affecting a diverse spectra of populations, with 1.6 million new cases reported each year. Current treatment options are costly and have harsh side effects. New therapeutic options that have been previously identified, but still underappreciated as potential pharmaceutical targets, are Leishmania secreted acid phosphatases (SAP). These acid phosphatases, which are reported to play a role in the survival of the parasite in the sand fly vector, and in homing to the host macrophage, are inhibited by orthovanadate and decavanadate. Here, we use L. tarentolae to further evaluate these inhibitors. Using enzyme assays, and UV-visible spectroscopy, we investigate which oxovanadium starting material (orthovanadate or decavanadate) is a better inhibitor of L. tarentolae secreted acid phosphatase activity in vitro at the same total moles of vanadium. Considering speciation and total vanadium concentration, decavanadate is a consistently better inhibitor of SAP in our conditions, especially at low substrate:inhibitor ratios.

  13. The inhibitory effect of metals and other ions on acid phosphatase activity from Vigna aconitifolia seeds.

    Science.gov (United States)

    Srivastava, Pramod Kumar; Anand, Asha

    2015-01-01

    Sensitivity of acid phosphatase from Vigna aconitifolia seeds to metal ions, fluoride, and phosphate was examined. All the effectors had different degree of inhibitory effect on the enzyme. Among metal ions, molybdate and ferric ion were observed to be most potent inhibitors and both exhibited mixed type of inhibition. Acid phosphatase activity was inhibited by Cu2+ in a noncompetitive manner. Zn and Mn showed mild inhibition on the enzyme activity. Inhibition kinetics analysis explored molybdate as a potent inhibitor for acid phosphatase in comparison with other effectors used in this study. Fluoride was the next most strong inhibitor for the enzyme activity, and caused a mixed type of inhibition. Phosphate inhibited the enzyme competitively, which demonstrates that inhibition due to phosphate is one of the regulatory factors for enzyme activity.

  14. Requirement for tyrosine phosphatase during serotonergic neuromodulation by protein kinase C.

    Science.gov (United States)

    Catarsi, S; Drapeau, P

    1997-08-01

    Tyrosine kinases and phosphatases are abundant in the nervous system, where they signal cellular differentiation, mediate the responses to growth factors, and direct neurite outgrowth during development. Tyrosine phosphorylation can also alter ion channel activity, but its physiological significance remains unclear. In an identified leech mechanosensory neuron, the ubiquitous neuromodulator serotonin increases the activity of a cation channel by activating protein kinase C (PKC), resulting in membrane depolarization and modulation of the receptive field properties. We observed that the effects on isolated neurons and channels were blocked by inhibiting tyrosine phosphatases. Serotonergic stimulation of PKC thus activates a tyrosine phosphatase activity associated with the channels, which reverses their constitutive inhibition by tyrosine phosphorylation, representing a novel form of neuromodulation.

  15. Osteomalacia with low alkaline phosphatase: a not so rare condition with important consequences.

    Science.gov (United States)

    Belkhouribchia, Jamal; Bravenboer, Bert; Meuwissen, Marije; Velkeniers, Brigitte

    2016-01-28

    Hypophosphatasia is a genetic disorder, characterised by a dysfunctional tissue-non-specific isoenzyme of alkaline phosphatase that impacts bone metabolism and predisposes to osteomalacia or rickets. The clinical presentation is very diverse, depending on the age of onset and the severity of the disease. Several forms of hypophosphatasia are recognised. We present a case of a 50-year-old woman with low impact fractures and loss of teeth at a young age. She also had a low alkaline phosphatase and was diagnosed with adult hypophosphatasia. Although the severe forms of hypophosphatasia are rather rare, the adult form is thought to occur quite frequently. As this condition is not well known by healthcare professionals, the time to diagnosis and initiation of adequate treatment is often postponed. When encountering a patient with low alkaline phosphatase, low bone density or a history of bone fractures, the possibility of hypophosphatasia should be considered. 2016 BMJ Publishing Group Ltd.

  16. Serine/threonine phosphatase tapp2cs might be served as an early signal molecule for water stress in wheat

    International Nuclear Information System (INIS)

    Song, K. H.; Tian, W. L.; Hou, B. Z.; Guo, J. X.; Mei, X. R.; Li, Y. Z.

    2015-01-01

    Much progress has been made towards understanding the role of serine/threonine phosphatases type 2C (PP2Cs) in abscisic acid (ABA) signaling transduction. However, how the negative regulator, PP2Cs, responds to plant water loss remains unclear. Here, we used a series of relative soil moisture (RSM: 85 percentage (well watered), 65 percentage (moderate stress), 45 percentage (severe stress) potted winter wheat (Triticum aestivum L.) and the detached leaves to detect ABA levels and transcripts of PP2Cs, including PP2C40, PP2C45, PP2C59 and PP2C6 as well as the core downstream signals of ABA, including ABF, SnRK2.4 and SnRK2.5. The results showed that the continual loss of water led to a consistent increase in ABA levels, and that the mRNA expression levels of PP2Cs were dependent on plant water condition. PP2Cs expression could be induced by a slight loss of water, and inhibited under severe loss of water. These results were further confirmed by the transcripts of ABF, SnRK2.4 and SnRK2.5. Furthermore, in slight loss of water, 100 μM exogenous ABA could promote PP2Cs expression; in severe loss of water, it inhibited PP2Cs expression. In conclusion, ABA accumulation is controlled by water condition and the PP2C expression is dependent on plant water condition, suggesting that PP2Cs might be served as an early signal molecule for water stress in wheat. (author)

  17. Crystallization of a newly discovered histidine acid phosphatase from Francisella tularensis

    Energy Technology Data Exchange (ETDEWEB)

    Felts, Richard L. [Department of Chemistry, University of Missouri-Columbia, Columbia, Missouri 65211 (United States); Reilly, Thomas J. [Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri-Columbia, Columbia, Missouri 65212 (United States); Veterinary Medical Diagnostic Laboratory, College of Veterinary Medicine, University of Missouri-Columbia, Columbia, Missouri 65212 (United States); Calcutt, Michael J. [Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri-Columbia, Columbia, Missouri 65212 (United States); Tanner, John J., E-mail: tannerjj@missouri.edu [Department of Chemistry, University of Missouri-Columbia, Columbia, Missouri 65211 (United States); Department of Biochemistry, University of Missouri-Columbia, Columbia, Missouri 65211 (United States)

    2006-01-01

    A histidine acid phosphatase from the CDC Category A pathogen F. tularensis has been crystallized in space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = 61.96, c = 210.78 Å. A 1.75 Å resolution data set was collected at Advanced Light Source beamline 4.2.2. Francisella tularensis is a highly infectious bacterial pathogen that is considered by the Centers for Disease Control and Prevention to be a potential bioterrorism weapon. Here, the crystallization of a 37.2 kDa phosphatase encoded by the genome of F. tularensis subsp. holarctica live vaccine strain is reported. This enzyme shares 41% amino-acid sequence identity with Legionella pneumophila major acid phosphatase and contains the RHGXRXP motif that is characteristic of the histidine acid phosphatase family. Large diffraction-quality crystals were grown in the presence of Tacsimate, HEPES and PEG 3350. The crystals belong to space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = 61.96, c = 210.78 Å. The asymmetric unit is predicted to contain one protein molecule, with a solvent content of 53%. A 1.75 Å resolution native data set was recorded at beamline 4.2.2 of the Lawrence Berkeley National Laboratory Advanced Light Source. Molecular-replacement trials using the human prostatic acid phosphatase structure as the search model (28% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of F. tularensis histidine acid phosphatase will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative.

  18. Crystallization of a newly discovered histidine acid phosphatase from Francisella tularensis

    International Nuclear Information System (INIS)

    Felts, Richard L.; Reilly, Thomas J.; Calcutt, Michael J.; Tanner, John J.

    2005-01-01

    A histidine acid phosphatase from the CDC Category A pathogen F. tularensis has been crystallized in space group P4 1 2 1 2, with unit-cell parameters a = 61.96, c = 210.78 Å. A 1.75 Å resolution data set was collected at Advanced Light Source beamline 4.2.2. Francisella tularensis is a highly infectious bacterial pathogen that is considered by the Centers for Disease Control and Prevention to be a potential bioterrorism weapon. Here, the crystallization of a 37.2 kDa phosphatase encoded by the genome of F. tularensis subsp. holarctica live vaccine strain is reported. This enzyme shares 41% amino-acid sequence identity with Legionella pneumophila major acid phosphatase and contains the RHGXRXP motif that is characteristic of the histidine acid phosphatase family. Large diffraction-quality crystals were grown in the presence of Tacsimate, HEPES and PEG 3350. The crystals belong to space group P4 1 2 1 2, with unit-cell parameters a = 61.96, c = 210.78 Å. The asymmetric unit is predicted to contain one protein molecule, with a solvent content of 53%. A 1.75 Å resolution native data set was recorded at beamline 4.2.2 of the Lawrence Berkeley National Laboratory Advanced Light Source. Molecular-replacement trials using the human prostatic acid phosphatase structure as the search model (28% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of F. tularensis histidine acid phosphatase will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative

  19. 2,3-diphosphoglycerate phosphatase activity of phosphoglycerate mutase: stimulation by vanadate and phosphate

    International Nuclear Information System (INIS)

    Stankiewicz, P.J.; Gresser, M.J.; Tracey, A.S.; Hass, L.F.

    1987-01-01

    The binding of inorganic vanadate (V/sub i/) to rabbit muscle phosphoglycerate mutase (PGM), studied by using 51 V nuclear magnetic resonance spectroscopy, shows a sigmoidal dependence on vanadate concentration with a stoichiometry of four vanadium atoms per PGM molecule at saturating [V/sub i/]. The data are consistent with binding of one divanadate ion to each of the two subunits of PGM in a noncooperative manner with an intrinsic dissociation constant of 4 x 10 -6 M. The relevance of this result to other studies which have shown that the V/sub i/-stimulated 2,3-diphosphoglycerate (2,3-DPG) phosphatase activity of PGM has a sigmoidal dependence on [V/sub i/] with a Hill coefficient of 2.0 is discussed. At pH 7.0, inorganic phosphate has little effect on the 2,3-DPG phosphatase activity of PGM, even at concentrations as high as 50 mM. Similarly, 25 μM V/sub i/ has little effect on the phosphatase activity. However, in the presence of 25 μM V/sub i/, a phosphate concentration of 20 mM increases the phosphatase activity by more than 3-fold. This behavior is rationalized in terms of activation of the phosphatase activity by a phosphate/vanadate mixed anhydride. This interpretation is supported by the observation of strong activation of the phosphatase activity by inorganic pyrophosphate. A molecular mechanism for the observed effects of vanadate is proposed, and the relevance of this study to the possible use of vanadate as a therapeutic agent for the treatment of sickle cell anemia is discussed

  20. Subcellular localisation and properties of histone phosphate phosphatase in human polymorphonuclear leukocytes: alterations in pregnancy and chronic granulocytic leukaemia and relationship to alkaline phosphatase

    International Nuclear Information System (INIS)

    Smith, G.P.; Peters, T.J.

    1981-01-01

    Using [ 32 P]histone as substrate, an assay for histone phosphate phosphatase was optimised for human polymorphonuclear leukocytes. Kinetic studies showed that the activity was optimal at pH 6.8, was stimulated by Mn 2+ and Co 2+ , and inhibited by sodium sulphite and zinc chloride. The apparent Ksub(m) of the enzyme for histone phosphate was 0.89 μmol/l. (Auth.)

  1. [Effect of elevated atmospheric CO2 on soil urease and phosphatase activities].

    Science.gov (United States)

    Chen, Lijun; Wu, Zhijie; Huang, Guohong; Zhou, Likai

    2002-10-01

    The response of soil urease and phosphatase activities at different rice growth stages to free air CO2 enrichment (FACE) was studied. The results showed that comparing with the ambient atmospheric CO2 concentration (370 mumol.mol-1), FACE (570 mumol.mol-1) significantly increased the urease activity of 0-5 cm soil layer at the vigorous growth stage of rice, whole that of 5-10 cm layer had no significant change during the whole growing season. Phosphatase activity of 0-5 cm and 5-10 cm soil layers significantly increased, and the peak increment was at the vigorous growth stage of rice.

  2. Water Quality Interaction with Alkaline Phosphatase in the Ganga River: Implications for River Health.

    Science.gov (United States)

    Yadav, Amita; Pandey, Jitendra

    2017-07-01

    Carbon, nitrogen and phosphorus inputs through atmospheric deposition, surface runoff and point sources were measured in the Ganga River along a gradient of increasing human pressure. Productivity variables (chlorophyll a, gross primary productivity, biogenic silica and autotrophic index) and heterotrophy (respiration, substrate induced respiration, biological oxygen demand and fluorescein diacetate hydrolysis) showed positive relationships with these inputs. Alkaline phosphatase (AP), however, showed an opposite trend. Because AP is negatively influenced by available P, and eutrophy generates a feedback on P fertilization, the study implies that the alkaline phosphatase can be used as a high quality criterion for assessing river health.

  3. Hydrolytic and alcoholytic dephosphorylation of nucleotides by acid phosphatase in the presence of ethanol.

    Science.gov (United States)

    Tomaszewski, M; Buchowicz, J

    1971-08-01

    The effect of ethanol on the activity of acid phosphatase from wheat germ was studied, by using ribonucleoside monophosphates as the enzyme substrates. The nucleotides were effectively degraded to the corresponding nucleosides in the presence of ethanol at all concentrations tested, including a 96% (v/v) solution. However, the nucleotide dephosphorylation was accompanied by the liberation of orthophosphate only when the concentration of ethanol in the assay mixture did not exceed 15%. No inorganic phosphate was liberated when ethanol was present at higher concentrations. Instead, monoethyl phosphate was formed in quantities expected for orthophosphate. The results are explained in terms of phosphatase-catalysed alcoholysis.

  4. A new fluorescence-based method identifies protein phosphatases regulating lipid droplet metabolism.

    Directory of Open Access Journals (Sweden)

    Bruno L Bozaquel-Morais

    Full Text Available In virtually every cell, neutral lipids are stored in cytoplasmic structures called lipid droplets (LDs and also referred to as lipid bodies or lipid particles. We developed a rapid high-throughput assay based on the recovery of quenched BODIPY-fluorescence that allows to quantify lipid droplets. The method was validated by monitoring lipid droplet turnover during growth of a yeast culture and by screening a group of strains deleted in genes known to be involved in lipid metabolism. In both tests, the fluorimetric assay showed high sensitivity and good agreement with previously reported data using microscopy. We used this method for high-throughput identification of protein phosphatases involved in lipid droplet metabolism. From 65 yeast knockout strains encoding protein phosphatases and its regulatory subunits, 13 strains revealed to have abnormal levels of lipid droplets, 10 of them having high lipid droplet content. Strains deleted for type I protein phosphatases and related regulators (ppz2, gac1, bni4, type 2A phosphatase and its related regulator (pph21 and sap185, type 2C protein phosphatases (ptc1, ptc4, ptc7 and dual phosphatases (pps1, msg5 were catalogued as high-lipid droplet content strains. Only reg1, a targeting subunit of the type 1 phosphatase Glc7p, and members of the nutrient-sensitive TOR pathway (sit4 and the regulatory subunit sap190 were catalogued as low-lipid droplet content strains, which were studied further. We show that Snf1, the homologue of the mammalian AMP-activated kinase, is constitutively phosphorylated (hyperactive in sit4 and sap190 strains leading to a reduction of acetyl-CoA carboxylase activity. In conclusion, our fast and highly sensitive method permitted us to catalogue protein phosphatases involved in the regulation of LD metabolism and present evidence indicating that the TOR pathway and the SNF1/AMPK pathway are connected through the Sit4p-Sap190p pair in the control of lipid droplet biogenesis.

  5. Sugar signalling and gene expression in relation to carbohydrate ...

    Indian Academy of Sciences (India)

    Sucrose is required for plant growth and development. The sugar status of plant cells is sensed by sensor proteins. The signal generated by signal transduction cascades, which could involve mitogen-activated protein kinases, protein phosphatases, Ca2+ and calmodulins, results in appropriate gene expression. A variety of ...

  6. Downregulation of PTP1B and TC-PTP phosphatases potentiate dendritic cell-based immunotherapy through IL-12/IFNγ signaling.

    Science.gov (United States)

    Penafuerte, Claudia; Feldhammer, Matthew; Mills, John R; Vinette, Valerie; Pike, Kelly A; Hall, Anita; Migon, Eva; Karsenty, Gerard; Pelletier, Jerry; Zogopoulos, George; Tremblay, Michel L

    2017-01-01

    PTP1B and TC-PTP are highly related protein-tyrosine phosphatases (PTPs) that regulate the JAK/STAT signaling cascade essential for cytokine-receptor activation in immune cells. Here, we describe a novel immunotherapy approach whereby monocyte-derived dendritic cell (moDC) function is enhanced by modulating the enzymatic activities of PTP1B and TC-PTP. To downregulate or delete the activity/expression of these PTPs, we generated mice with PTP-specific deletions in the dendritic cell compartment or used PTP1B and TC-PTP specific inhibitor. While total ablation of PTP1B or TC-PTP expression leads to tolerogenic DCs via STAT3 hyperactivation, downregulation of either phosphatase remarkably shifts the balance toward an immunogenic DC phenotype due to hyperactivation of STAT4, STAT1 and Src kinase. The resulting increase in IL-12 and IFNγ production subsequently amplifies the IL-12/STAT4/IFNγ/STAT1/IL-12 positive autocrine loop and enhances the therapeutic potential of mature moDCs in tumor-bearing mice. Furthermore, pharmacological inhibition of both PTPs improves the maturation of defective moDCs derived from pancreatic cancer (PaC) patients. Our study provides a new advance in the use of DC-based cancer immunotherapy that is complementary to current cancer therapeutics.

  7. A preliminary X-ray study of d,d-heptose-1,7-bisphosphate phosphatase from Burkholderia thailandensis E264

    International Nuclear Information System (INIS)

    Kim, Mi-Sun; Shin, Dong Hae

    2010-01-01

    In this study, d,d-heptose-1,7-bisphosphate phosphatase has been cloned, expressed, purified and crystallized. d,d-Heptose-1,7-bisphosphate phosphatase (GmhB), which is involved in the third step of the NDP-heptose biosynthesis pathway, converts d,d-heptose-1,7-bisphosphate to d,d-heptose-1-phosphate. This biosynthesis pathway is a target for new antibiotics or antibiotic adjuvants for Gram-negative pathogens. Burkholderia thailandensis is a useful surrogate organism for studying the pathogenicity of melioidosis owing to its extensive genomic similarity to B. pseudomallei. Melioidosis caused by B. pseudomallei is a serious invasive disease of animals and humans in tropical and subtropical areas. In this study, GmhB has been cloned, expressed, purified and crystallized. X-ray data have also been collected to 2.50 Å resolution using synchrotron radiation. The crystal belonged to space group P6, with unit-cell parameters a = 243.2, b = 243.2, c = 41.1 Å

  8. Resolving Early Signaling Events in T-Cell Activation Leading to IL-2 and FOXP3 Transcription

    Directory of Open Access Journals (Sweden)

    Jeffrey P. Perley

    2014-11-01

    Full Text Available Signal intensity and feedback regulation are known to be major factors in the signaling events stemming from the T-cell receptor (TCR and its various coreceptors, but the exact nature of these relationships remains in question. We present a mathematical model of the complex signaling network involved in T-cell activation with cross-talk between the Erk, calcium, PKC and mTOR signaling pathways. The model parameters are adjusted to fit new and published data on TCR trafficking, Zap70, calcium, Erk and Isignaling. The regulation of the early signaling events by phosphatases, CD45 and SHP1, and the TCR dynamics are critical to determining the behavior of the model. Additional model corroboration is provided through quantitative and qualitative agreement with experimental data collected under different stimulating and knockout conditions. The resulting model is analyzed to investigate how signal intensity and feedback regulation affect TCR- and coreceptor-mediated signal transduction and their downstream transcriptional profiles to predict the outcome for a variety of stimulatory and knockdown experiments. Analysis of the model shows that: (1 SHP1 negative feedback is necessary for preventing hyperactivity in TCR signaling; (2 CD45 is required for TCR signaling, but also partially suppresses it at high expression levels; and (3 elevated FOXP3 and reduced IL-2 signaling, an expression profile often associated with T regulatory cells (Tregs, is observed when the system is subjected to weak TCR and CD28 costimulation or a severe reduction in CD45 activity.

  9. Regorafenib (Stivarga) pharmacologically targets epithelial-mesenchymal transition in colorectal cancer.

    Science.gov (United States)

    Fan, Li-Ching; Teng, Hao-Wei; Shiau, Chung-Wai; Tai, Wei-Tien; Hung, Man-Hsin; Yang, Shung-Haur; Jiang, Jeng-Kai; Chen, Kuen-Feng

    2016-09-27

    Epithelial-to-mesenchymal transition (EMT) is well-known to evoke cancer invasion/metastasis, leading to a high frequency of mortality in patients with metastatic colorectal cancer (mCRC). Protein tyrosine phosphatase (PTPase)-targeted therapy has been identified as a novel cancer therapeutic. Previously, we proved that sorafenib with anti-EMT potency prevents TGF-β1-induced EMT/invasion by directly activating SH2-domain-containing phosphatase 1 (SHP-1)-dependent p-STAT3Tyr705 suppression in hepatocellular carcinoma. Regorafenib has a closely related chemical structure as sorafenib and is approved for the pharmacotherapy of mCRC. Herein, we evaluate whether regorafenib activates PTPase SHP-1 in the same way as sorafenib to abolish EMT-related invasion/metastasis in CRC. Notably, regorafenib exerted potent anti-EMT activity to curb TGF-β1-induced EMT/invasion in vitro as well inhibited lung metastatic outgrowth of SW480 mesenchymal cells in vivo. Mechanistically, regorafenib-enhanced SHP-1 activity significantly impeded TGF-β1-induced EMT/invasion via low p-STAT3Tyr705 level as proved by a SHP-1 inhibitor or siRNA-mediated SHP-1 depletion. Conversely, overexpression of SHP-1 further enhanced the inhibitory effects of regorafenib on TGF-β1-induced p-STAT3Tyr705 and EMT/invasion. Regorafenib directly activates SHP-1 by potently relieving the autoinhibited N-SH2 domain of SHP-1 to inhibit TGF-β1-induced p-STAT3Tyr705 and EMT/invasion. Importantly, the clinical evidence indicated that SHP-1 was positively correlated with E-cadherin and that significantly determined the overall survival of CRC patients. This result further confirms our in vitro data that SHP-1 is a negative regulatory PTPase in EMT regulation and serves as a pharmacological target for mCRC therapy. Collectively, activating PTPase SHP-1 by regorafenib focusing on its anti-EMT activity might be a useful pharmacotherapy for mCRC.

  10. Identification of Open Stomata1-Interacting Proteins Reveals Interactions with Sucrose Non-fermenting1-Related Protein Kinases2 and with Type 2A Protein Phosphatases That Function in Abscisic Acid Responses1[OPEN

    Science.gov (United States)

    Waadt, Rainer; Manalansan, Bianca; Rauniyar, Navin; Munemasa, Shintaro; Booker, Matthew A.; Brandt, Benjamin; Waadt, Christian; Nusinow, Dmitri A.; Kay, Steve A.; Kunz, Hans-Henning; Schumacher, Karin; DeLong, Alison; Yates, John R.; Schroeder, Julian I.

    2015-01-01

    The plant hormone abscisic acid (ABA) controls growth and development and regulates plant water status through an established signaling pathway. In the presence of ABA, pyrabactin resistance/regulatory component of ABA receptor proteins inhibit type 2C protein phosphatases (PP2Cs). This, in turn, enables the activation of Sucrose Nonfermenting1-Related Protein Kinases2 (SnRK2). Open Stomata1 (OST1)/SnRK2.6/SRK2E is a major SnRK2-type protein kinase responsible for mediating ABA responses. Arabidopsis (Arabidopsis thaliana) expressing an epitope-tagged OST1 in the recessive ost1-3 mutant background was used for the copurification and identification of OST1-interacting proteins after osmotic stress and ABA treatments. These analyses, which were confirmed using bimolecular fluorescence complementation and coimmunoprecipitation, unexpectedly revealed homo- and heteromerization of OST1 with SnRK2.2, SnRK2.3, OST1, and SnRK2.8. Furthermore, several OST1-complexed proteins were identified as type 2A protein phosphatase (PP2A) subunits and as proteins involved in lipid and galactolipid metabolism. More detailed analyses suggested an interaction network between ABA-activated SnRK2-type protein kinases and several PP2A-type protein phosphatase regulatory subunits. pp2a double mutants exhibited a reduced sensitivity to ABA during seed germination and stomatal closure and an enhanced ABA sensitivity in root growth regulation. These analyses add PP2A-type protein phosphatases as another class of protein phosphatases to the interaction network of SnRK2-type protein kinases. PMID:26175513

  11. A missense mutation in the human liver/bone/kidney alkaline phosphatase gene causing a lethal form of hypophosphatasia

    International Nuclear Information System (INIS)

    Weiss, M.J.; Cole, D.E.C.; Ray, K.; Whyte, M.P.; Lafferty, M.A.; Mulivor, R.A.; Harris, H.

    1988-01-01

    Hypophosphatasia is an inherited disorder characterized by defective bone mineralization and a deficiency of serum and tissue liver/bone/kidney alkaline phosphatase (L/B/K ALP) activity. Clinical severity is variable, ranging from death in utero (due to severe rickets) to pathologic fractures first presenting in adult life. Affected siblings, however, are phenotypically similar. Severe forms of the disease are inherited in an autosomal recessive fashion; heterozygotes often show reduced serum ALP activity. The specific gene defects in hypophosphatasia are unknown but are thought to occur either at the L/B/K ALP locus or within another gene that regulates L/B/K ALP expression. The authors used the polymerase chain reaction to examine L/B/K ALP cDNA from a patient with a perinatal (lethal) form of the disease. They observed a guanine-to-adenine transition in nucleotide 711 of the cDNA that converts alanine-162 of the mature enzyme to threonine. The affected individual, whose parents are second cousins, is homozygous for the mutant allele. Introduction of this mutation into an otherwise normal cDNA by site-directed mutagenesis abolished the expression of active enzyme, demonstrating that a defect in the L/B/K ALP gene results in hypophosphatasia and that the enzyme is, therefore, essential for normal skeletal mineralization

  12. A complex between contactin-1 and the protein tyrosine phosphatase PTPRZ controls the development of oligodendrocyte precursor cells

    Energy Technology Data Exchange (ETDEWEB)

    Lamprianou, Smaragda; Chatzopoulou, Elli; Thomas, Jean-Léon; Bouyain, Samuel; Harroch, Sheila (IP-Korea); (UPMC); (UMKC)

    2013-09-23

    The six members of the contactin (CNTN) family of neural cell adhesion molecules are involved in the formation and maintenance of the central nervous system (CNS) and have been linked to mental retardation and neuropsychiatric disorders such as autism. Five of the six CNTNs bind to the homologous receptor protein tyrosine phosphatases gamma (PTPRG) and zeta (PTPRZ), but the biological roles of these interactions remain unclear. We report here the cocrystal structure of the carbonic anhydrase-like domain of PTPRZ bound to tandem Ig repeats of CNTN1 and combine these structural data with binding assays to show that PTPRZ binds specifically to CNTN1 expressed at the surface of oligodendrocyte precursor cells. Furthermore, analyses of glial cell populations in wild-type and PTPRZ-deficient mice show that the binding of PTPRZ to CNTN1 expressed at the surface of oligodendrocyte precursor cells inhibits their proliferation and promotes their development into mature oligodendrocytes. Overall, these results implicate the PTPRZ/CNTN1 complex as a previously unknown modulator of oligodendrogenesis.

  13. Curcumin Inhibits NTHi-Induced MUC5AC Mucin Overproduction in Otitis Media via Upregulation of MAPK Phosphatase MKP-1

    Directory of Open Access Journals (Sweden)

    Anuhya Sharma Konduru

    2017-01-01

    Full Text Available Otitis media (OM, characterized by the presence of mucus overproduction and excess inflammation in the middle ear, is the most common childhood infection. Nontypeable Haemophilus influenzae (NTHi pathogen is responsible for approximately one-third of episodes of bacteria-caused OM. Current treatments for bacterial OM rely on the systemic use of antibiotics, which often leads to the emergence of multidrug resistant bacterial strains. Therefore there is an urgent need for developing alternative therapies strategies for controlling mucus overproduction in OM. MUC5AC mucin has been shown to play a critical role in the pathogenesis of OM. Here we show that curcumin derived from Curcuma longa plant is a potent inhibitor of NTHi-induced MUC5AC mucin expression in middle ear epithelial cells. Curcumin inhibited MUC5AC expression by suppressing activation of p38 MAPK by upregulating MAPK phosphatase MKP-1. Thus, our study identified curcumin as a potential therapeutic for inhibiting mucin overproduction in OM by upregulating MKP-1, a known negative regulator of inflammation.

  14. Receptor-type Protein Tyrosine Phosphatase β Regulates Met Phosphorylation and Function in Head and Neck Squamous Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Yiru Xu

    2012-11-01

    Full Text Available Head and neck squamous cell carcinoma (HNSCC is the sixth most common cancer and has a high rate of mortality. Emerging evidence indicates that hepatocyte growth factor receptor (or Met pathway plays a pivotal role in HNSCC metastasis and resistance to chemotherapy. Met function is dependent on tyrosine phosphorylation that is under direct control by receptor-type protein tyrosine phosphatase β (RPTP-β. We report here that RPTP-β expression is significantly downregulated in HNSCC cells derived from metastatic tumors compared to subject-matched cells from primary tumors. Knockdown of endogenous RPTP-β in HNSCC cells from primary tumor potentiated Met tyrosine phosphorylation, downstream mitogen-activated protein (MAP kinase pathway activation, cell migration, and invasion. Conversely, restoration of RPTP-β expression in cells from matched metastatic tumor decreased Met tyrosine phosphorylation and downstream functions. Furthermore, we observed that six of eight HNSCC tumors had reduced levels of RPTP-β protein in comparison with normal oral tissues. Collectively, the results demonstrate the importance of RPTP-β in tumor biology of HNSCC through direct dephosphorylation of Met and regulation of downstream signal transduction pathways. Reduced RPTP-β levels, with or without Met overexpression, could promote Met activation in HNSCC tumors.

  15. Protein tyrosine phosphatase 1B (PTP1B) is required for cardiac lineage differentiation of mouse embryonic stem cells.

    Science.gov (United States)

    Eshkiki, Zahra Shokati; Ghahremani, Mohammad Hossein; Shabani, Parisa; Firuzjaee, Sattar Gorgani; Sadeghi, Asie; Ghanbarian, Hossein; Meshkani, Reza

    2017-01-01

    Protein tyrosine phosphatase 1B (PTP1B) has been shown to regulate multiple cellular events such as differentiation, cell growth, and proliferation; however, the role of PTP1B in differentiation of embryonic stem (ES) cells into cardiomyocytes remains unexplored. In the present study, we investigated the effects of PTP1B inhibition on differentiation of ES cells into cardiomyocytes. PTP1B mRNA and protein levels were increased during the differentiation of ES cells into cardiomyocytes. Accordingly, a stable ES cell line expressing PTP1B shRNA was established. In vitro, the number and size of spontaneously beating embryoid bodies were significantly decreased in PTP1B-knockdown cells, compared with the control cells. Decreased expression of cardiac-specific markers Nkx2-5, MHC-α, cTnT, and CX43, as assessed by real-time PCR analysis, was further confirmed by immunocytochemistry of the markers. The results also showed that PTP1B inhibition induced apoptosis in both differentiated and undifferentiated ES cells, as presented by increasing the level of cleaved caspase-3, cytochrome C, and cleaved PARP. Further analyses revealed that PTP1B inhibition did not change proliferation and pluripotency of undifferentiated ES cells. Taken together, the data presented here suggest that PTP1B is essential for proper differentiation of ES cells into cardiomyocytes.

  16. The human tartrate-resistant acid phosphatase (TRAP): involvement of the hemin responsive elements (HRE) in transcriptional regulation.

    Science.gov (United States)

    Fleckenstein, E C; Dirks, W G; Drexler, H G

    2000-02-01

    The biochemical properties and protein structure of the tartrate-resistant acid phosphatase (TRAP), an iron-containing lysosomal glycoprotein in cells of the mononuclear phagocyte system, are well known. In contrast, little is known about the physiology and genic structure of this unique enzyme. In some diseases, like hairy cell leukemia, Gaucher's disease and osteoclastoma, cytochemically detected TRAP expression is used as a disease-associated marker. In order to begin to elucidate the regulation of this gene we generated different deletion constructs of the TRAP 5'-flanking region, placed them upstream of the luciferase reporter gene and assayed them for their ability to direct luciferase expression in human 293 cells. Treatment of these cells with the iron-modulating reagents transferrin and hemin causes opposite effects on the TRAP promoter activity. Two regulatory GAGGC tandem repeat sequences (the hemin responsive elements, HRE) within the 5'-flanking region of the human TRAP gene were identified. Studies with specific HRE-deletion constructs of the human TRAP 5'-flanking region upstream of the luciferase reporter gene document the functionality of these HRE-sequences which are apparently responsible for mediating transcriptional inhibition upon exposure to hemin. In addition to the previously published functional characterization of the murine TRAP HRE motifs, these results provide the first description of a new iron/hemin-responsive transcriptional regulation in the human TRAP gene.

  17. Squalene Inhibits ATM-Dependent Signaling in γIR-Induced DNA Damage Response through Induction of Wip1 Phosphatase.

    Directory of Open Access Journals (Sweden)

    Naoto Tatewaki

    Full Text Available Ataxia telangiectasia mutated (ATM kinase plays a crucial role as a master controller in the cellular DNA damage response. Inhibition of ATM leads to inhibition of the checkpoint signaling pathway. Hence, addition of checkpoint inhibitors to anticancer therapies may be an effective targeting strategy. A recent study reported that Wip1, a protein phosphatase, de-phosphorylates serine 1981 of ATM during the DNA damage response. Squalene has been proposed to complement anticancer therapies such as chemotherapy and radiotherapy; however, there is little mechanistic information supporting this idea. Here, we report the inhibitory effect of squalene on ATM-dependent DNA damage signals. Squalene itself did not affect cell viability and the cell cycle of A549 cells, but it enhanced the cytotoxicity of gamma-irradiation (γIR. The in vitro kinase activity of ATM was not altered by squalene. However, squalene increased Wip1 expression in cells and suppressed ATM activation in γIR-treated cells. Consistent with the potential inhibition of ATM by squalene, IR-induced phosphorylation of ATM effectors such as p53 (Ser15 and Chk1 (Ser317 was inhibited by cell treatment with squalene. Thus, squalene inhibits the ATM-dependent signaling pathway following DNA damage through intracellular induction of Wip1 expression.

  18. Detection of alkaline phosphatase in canine cells previously stained with Wright-Giemsa and its utility in differentiating osteosarcoma from other mesenchymal tumors.

    Science.gov (United States)

    Ryseff, Julia K; Bohn, Andrea A

    2012-09-01

    Osteosarcoma (OSA) is a common primary bone tumor in dogs. Demonstration of alkaline phosphatase (ALP) reactivity by tumor cells on unstained slides is useful in differentiating osteosarcoma from other types of sarcoma. However, unstained slides are not always available. The objectives of this study were to evaluate the diagnostic utility of detecting ALP expression in differentiating osteosarcoma from other sarcomas in dogs using cytologic material previously stained with Wright-Giemsa stain and to assess the sensitivity and specificity of ALP expression for diagnosing osteosarcoma using a specific protocol. Archived aspirates of histologically confirmed sarcomas in dogs that had been previously stained with Wright-Giemsa stain were treated with 5-bromo, 4-chloro, 3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) as a substrate for ALP. Cells were evaluated for expression of ALP after incubation with BCIP/NBT for 1 hour. Sensitivity and specificity of ALP expression for diagnosis of OSA were calculated. In samples from 83 dogs, cells from 15/17 OSAs and from 4/66 tumors other than OSA (amelanotic melanoma, gastrointestinal stromal tumor, collision tumor, and anaplastic sarcoma) expressed ALP. Sensitivity and specificity of ALP expression detected using BCIP/NBT substrate applied to cells previously stained with Wright-Giemsa stain for OSA were 88 and 94%, respectively. ALP expression detected using BCIP/NBT substrate applied to previously stained cells is useful in differentiating canine OSA from other mesenchymal neoplasms. © 2012 American Society for Veterinary Clinical Pathology.

  19. Dephosphorylation of microtubule-binding sites at the neurofilament-H tail domain by alkaline, acid, and protein phosphatases.

    Science.gov (United States)

    Hisanaga, S; Yasugawa, S; Yamakawa, T; Miyamoto, E; Ikebe, M; Uchiyama, M; Kishimoto, T

    1993-06-01

    The dephosphorylation-induced interaction of neurofilaments (NFs) with microtubules (MTs) was investigated by using several phosphatases. Escherichia coli alkaline and wheat germ acid phosphatases increased the electrophoretic mobility of NF-H and NF-M by dephosphorylation, and induced the binding of NF-H to MTs. The binding of NFs to MTs was observed only after the electrophoretic mobility of NF-H approached the exhaustively dephosphorylated level when alkaline phosphatase was used. The number of phosphate remaining when NF-H began to bind to MTs was estimated by measuring phosphate bound to NF-H. NF-H did not bind to MTs even when about 40 phosphates from the total of 51 had been removed by alkaline phosphatase. The removal of 6 further phosphates finally resulted in the association of NF-H with MTs. A similar finding, that the restricted phosphorylation sites in the NF-H tail domain, but not the total amount of phosphates, were important for binding to MTs, was also obtained with acid phosphatases. In contrast to alkaline and acid phosphatases, four classes of protein phosphatases (protein phosphatases 1, 2A, 2B, and 2C) were ineffective for shifting the electrophoretic mobility of NF proteins and for inducing the association of NFs to MTs.

  20. DMPD: DUSP meet immunology: dual specificity MAPK phosphatases in control of theinflammatory response. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17114416 DUSP meet immunology: dual specificity MAPK phosphatases in control of the...ml) (.csml) Show DUSP meet immunology: dual specificity MAPK phosphatases in control of theinflammatory resp...onse. PubmedID 17114416 Title DUSP meet immunology: dual specificity MAPK phospha

  1. Gamma-glutamyltransferase, aspartate aminotransferase and alkaline phosphatase as markers of alcohol consumption in out-patient alcoholics

    DEFF Research Database (Denmark)

    Gluud, C; Andersen, I; Dietrichson, O

    1981-01-01

    and alkaline phosphatase in 18% and 7%. Neither the activity of gamma-glutamyltransferase, aspartate aminotransferase nor alkaline phosphatase showed any significant (P greater than 0.05) correlation with the history of alcohol consumption. The activities of gamma-glutamyltransferase and aspartate...

  2. Identification of protein phosphatase involvement in the AT-receptor induced activation of endothelial nitric oxide synthase

    DEFF Research Database (Denmark)

    Peluso, A Augusto; Bertelsen, Jesper Bork; Andersen, Kenneth

    2018-01-01

    -antagonist), L-NAME (10µM; eNOS inhibitor), MK-2206 (100nM; Akt-inhibitor) sodium fluoride (1nM; serine/threonine-phosphatase inhibitor) or sodium orthovanadate (10nM; tyrosine-phosphatase inhibitor). NO release was estimated by quantifying DAF-FM fluorescence. The phosphorylation status of activating (e...

  3. Effects of sub-lethal dose of gamma-irradiation on levels of acid phosphatase in cerebellum of pigeons

    International Nuclear Information System (INIS)

    Shah, V.C.; Gadhia, P.K.

    1980-01-01

    The changes in the activities of acid phosphatase in the sham-irradiated and γ-irradiated cerebellum of pigeons have been studied both biochemically as well as histochemically after 400 rads. The specific activity of acid phosphatase decreased significantly after 48h and 72h of irradiation. The histochemical observations following total body irradiation confirmed the results obtained by quantitative biochemical studies. (author)

  4. Alkaline phosphatase immobilization onto Bio-Gide(R) and Bio-Oss(R) for periodontal and bone regeneration.

    NARCIS (Netherlands)

    Oortgiesen, D.A.W.; Plachokova, A.S.; Geenen, C.; Meijer, G.J.; Walboomers, X.F.; Beucken, J.J.J.P van den; Jansen, J.B.M.J.

    2012-01-01

    AIM: To evaluate the effect of alkaline phosphatase (ALP) immobilization onto Bio-Gide((R)) in vitro, and to study the in vivo performance of ALP-enriched Bio-Gide((R)) and/or Bio-Oss((R)) with the purpose to enhance periodontal regeneration. MATERIALS AND METHODS: Alkaline phosphatase ALP was

  5. Hepatic glucose-6-phosphatase-α deficiency leads to metabolic reprogramming in glycogen storage disease type Ia.

    Science.gov (United States)

    Cho, Jun-Ho; Kim, Goo-Young; Mansfield, Brian C; Chou, Janice Y

    2018-04-15

    Glycogen storage disease type Ia (GSD-Ia) is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC), a key enzyme in endogenous glucose production. This autosomal recessive disorder is characterized by impaired glucose homeostasis and long-term complications of hepatocellular adenoma/carcinoma (HCA/HCC). We have shown that hepatic G6Pase-α deficiency-mediated steatosis leads to defective autophagy that is frequently associated with carcinogenesis. We now show that hepatic G6Pase-α deficiency also leads to enhancement of hepatic glycolysis and hexose monophosphate shunt (HMS) that can contribute to hepatocarcinogenesis. The enhanced hepatic glycolysis is reflected by increased lactate accumulation, increased expression of many glycolytic enzymes, and elevated expression of c-Myc that stimulates glycolysis. The increased HMS is reflected by increased glucose-6-phosphate dehydrogenase activity and elevated production of NADPH and the reduced glutathione. We have previously shown that restoration of hepatic G6Pase-α expression in G6Pase-α-deficient liver corrects metabolic abnormalities, normalizes autophagy, and prevents HCA/HCC development in GSD-Ia. We now show that restoration of hepatic G6Pase-α expression normalizes both glycolysis and HMS in GSD-Ia. Moreover, the HCA/HCC lesions in L-G6pc-/- mice exhibit elevated levels of hexokinase 2 (HK2) and the M2 isoform of pyruvate kinase (PKM2) which play an important role in aerobic glycolysis and cancer cell proliferation. Taken together, hepatic G6Pase-α deficiency causes metabolic reprogramming, leading to enhanced glycolysis and elevated HMS that along with impaired autophagy can contribute to HCA/HCC development in GSD-Ia. Published by Elsevier Inc.

  6. Real-time fluorescence assay of alkaline phosphatase in living cells using boron-doped graphene quantum dots as fluorophores.

    Science.gov (United States)

    Chen, Li; Yang, Guancao; Wu, Ping; Cai, Chenxin

    2017-10-15

    This work reports a convenient and real-time assay of alkaline phosphatase (ALP) in living cells based on a fluorescence quench-recovery process at a physiological pH using the boron-doped graphene quantum dots (BGQDs) as fluorophore. The fluorescence of BGQDs is found to be effectively quenched by Ce 3+ ions because of the coordination of Ce 3+ ions with the carboxyl group of BGQDs. Upon addition of adenosine triphosphate (ATP) into the system, the quenched fluorescence can be recovered by the ALP-positive expressed cells (such as MCF-7 cells) due to the removal of Ce 3+ ions from BGQDs surface by phosphate ions, which are generated from ATP under catalytic hydrolysis of ALP that expressed in cells. The extent of fluorescence signal recovery depends on the level of ALP in cells, which establishes the basis of ALP assay in living cells. This approach can also be used for specific discrimination of the ALP expression levels in different type of cells and thus sensitive detection of those ALP-positive expressed cells (for example MCF-7 cells) at a very low abundance (10±5 cells mL -1 ). The advantages of this approach are that it has high sensitivity because of the significant suppression of the background due to the Ce 3+ ion quenching the fluorescence of BGQDs, and has the ability of avoiding false signals arising from the nonspecific adsorption of non-target proteins because it operates via a fluorescence quench-recovery process. In addition, it can be extended to other enzyme systems, such as ATP-related kinases. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Purification and characterization of an alkaline phosphatase induced by phosphorus starvation in common bean (Phaseolus vulgaris L.) roots

    Energy Technology Data Exchange (ETDEWEB)

    Morales, L.; Gutierrez, N.; Maya, V.; Parra, C.; Martinez B, E.; Coello, P., E-mail: pcoello@servidor.unam.mx [UNAM, Facultad de Quimica, Departamento de Bioquimica, Ciudad Universitaria, 04510 Mexico D. F. (Mexico)

    2012-07-01

    Two phosphatase isoforms from roots of the common bean (Phaseolus vulgaris L.) showed an increase in activity in response to phosphate deficiency. One of them (APIII) was chosen for further purification through ionic exchange chromatography and preparative electrophoresis. The estimated molecular mass of APIII was 35 kDa by both SDS-Page and gel filtration analyses, suggesting a monomeric form of the active enzyme. The phosphatase was classified as an alkaline phosphatase based on the requirement of ph 8 for optimum catalysis. It not only exhibited broad substrate specificity, with the most activity against pyrophosphate, but also effectively catalyzed the hydrolysis of polyphosphate, glucose-1-phosphate and phospho enol-pyruvate. Activity was completely inhibited by molybdate, vanadate and phosphate but was only partially inhibited by fluoride. Although divalent cations were not essential for the pyro phosphatase activity of this enzyme, the hydrolysis of pyro phosphatase increased substantially in the presence of Mg{sup 2+}.

  8. Purification and characterization of an alkaline phosphatase induced by phosphorus starvation in common bean (Phaseolus vulgaris L.) roots

    International Nuclear Information System (INIS)

    Morales, L.; Gutierrez, N.; Maya, V.; Parra, C.; Martinez B, E.; Coello, P.

    2012-01-01

    Two phosphatase isoforms from roots of the common bean (Phaseolus vulgaris L.) showed an increase in activity in response to phosphate deficiency. One of them (APIII) was chosen for further purification through ionic exchange chromatography and preparative electrophoresis. The estimated molecular mass of APIII was 35 kDa by both SDS-Page and gel filtration analyses, suggesting a monomeric form of the active enzyme. The phosphatase was classified as an alkaline phosphatase based on the requirement of ph 8 for optimum catalysis. It not only exhibited broad substrate specificity, with the most activity against pyrophosphate, but also effectively catalyzed the hydrolysis of polyphosphate, glucose-1-phosphate and phospho enol-pyruvate. Activity was completely inhibited by molybdate, vanadate and phosphate but was only partially inhibited by fluoride. Although divalent cations were not essential for the pyro phosphatase activity of this enzyme, the hydrolysis of pyro phosphatase increased substantially in the presence of Mg 2+ .

  9. Identification and characterization of an ATP.Mg-dependent protein phosphatase from pig brain

    International Nuclear Information System (INIS)

    Yang, S.D.; Fong, Y.L.

    1985-01-01

    Substantial amounts of ATP.Mg-dependent phosphorylase phosphatase (Fc. M) and its activator (kinase FA) were identified and extensively purified from pig brain, in spite of the fact that glycogen metabolism in the brain is of little importance. The brain Fc.M was completely inactive and could only be activated by ATP.Mg and FA, isolated either from rabbit muscle or pig brain. Kinetical analysis of the dephosphorylation of endogenous brain protein indicates that Fc.M could dephosphorylate 32 P-labeled myelin basic protein (MBP) and [ 32 P]phosphorylase alpha at a comparable rate and moreover, this associated MBP phosphatase activity was also strictly kinase FA/ATP.Mg-dependent, demonstrating that MBP is a potential substrate for Fc.M in the brain. By manipulating MBP and inhibitor-2 as specific potent phosphorylase phosphatase inhibitors, we further demonstrate that 1) Fc.M contains two distinct catalytic sites to dephosphorylate different substrates, and 2) brain MBP may be a physiological trigger involved in the regulation of protein phosphatase substrate specificity in mammalian nervous tissues

  10. The manometric determination of thiamine pyrophosphate and the inhibition of the acid yeast phosphatase

    NARCIS (Netherlands)

    Steyn-Parvé, Elizabeth P.

    1962-01-01

    Sodium molybdate is a powerful inhibitor of the acid yeast phosphatase in both fresh baker's yeast and dried brewer's yeast, provided that the yeast is suspended in a suitable buffer. It displays no action in citrate or phosphate buffers, but is active in acetate or maleate buffers, both at the

  11. Enzymatic mineralization of hydrogels for bone tissue engineering by incorporation of alkaline phosphatase.

    NARCIS (Netherlands)

    Douglas, T.E.L.; Messersmith, P.B.; Chasan, S.; Mikos, A.G.; Mulder, E.L.W. de; Dickson, G.; Schaubroeck, D.; Balcaen, L.; Vanhaecke, F.; Dubruel, P.; Jansen, J.A.; Leeuwenburgh, S.C.G.

    2012-01-01

    Alkaline phosphatase (ALP), an enzyme involved in mineralization of bone, is incorporated into three hydrogel biomaterials to induce their mineralization with calcium phosphate (CaP). These are collagen type I, a mussel-protein-inspired adhesive consisting of PEG substituted with catechol groups,

  12. Advances in lanthanide-based luminescent peptide probes for monitoring the activity of kinase and phosphatase.

    Science.gov (United States)

    Pazos, Elena; Vázquez, M Eugenio

    2014-02-01

    Signaling pathways based on protein phosphorylation and dephosphorylation play critical roles in the orchestration of complex biochemical events and form the core of most signaling pathways in cells (i.e. cell cycle regulation, cell motility, apoptosis, etc.). The understanding of these complex signaling networks is based largely on the biochemical study of their components, i.e. kinases and phosphatases. The development of luminescent sensors for monitoring kinase and phosphatase activity is therefore an active field of research. Examples in the literature usually rely on the modulation of the fluorescence emission of organic fluorophores. However, given the exceptional photophysical properties of lanthanide ions, there is an increased interest in their application as emissive species for monitoring kinase and phosphatase activity. This review summarizes the advances in the development of lanthanide-based luminescent peptide sensors as tools for the study of kinases and phosphatases and provides a critical description of current examples and synthetic approaches to understand these lanthanide-based luminescent peptide sensors. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. A Ten-Week Biochemistry Lab Project Studying Wild-Type and Mutant Bacterial Alkaline Phosphatase

    Science.gov (United States)

    Witherow, D. Scott

    2016-01-01

    This work describes a 10-week laboratory project studying wild-type and mutant bacterial alkaline phosphatase, in which students purify, quantitate, and perform kinetic assays on wild-type and selected mutants of the enzyme. Students also perform plasmid DNA purification, digestion, and gel analysis. In addition to simply learning important…

  14. Soil phosphatase and urease activities impacted by cropping systems and water management

    Science.gov (United States)

    Soil enzymes can play an important role in nutrient availability to plants. Consequently, soil enzyme measurements can provide useful information on soil fertility for crop production. We examined the impact of cropping system and water management on phosphatase, urease, and microbial biomass C in s...

  15. Investigations into the stabilisation of drugs by sugar glasses : I. Tablets prepared from stabilised alkaline phosphatase

    NARCIS (Netherlands)

    Eriksson, H.J.C.; Hinrichs, W.L.J.; van Veen, B.; Somsen, G.W.; de Jong, G.J.; Frijlink, H.W.

    2002-01-01

    The aim of this study was to investigate the formulation of sugar glass stabilised alkaline phosphatase from bovine intestine (BIAP) into tablets. Two major subjects of tablet formulation were investigated. First, the compaction behaviour of the inulin sugar glass was investigated. Secondly, the

  16. The effect of potassium iodide on the production of acid phosphatase by Sporothrix schenckii

    Directory of Open Access Journals (Sweden)

    P. S. Grover

    2003-06-01

    Full Text Available The present study was undertaken to find out the in vitro effect of potassium iodide (KI on the production of acid phosphatase by fully characterized strain of S.schenckii isolated from a patient of Cutaneous Sporotrichosis. The enzyme acid phosphatase was estimated during the 3 phases of growth of S.schenckii, without and with three concentrations of KI incorporated in the culture medium. In the control and in the test proper, with various concentrations of KI, no adverse effect of KI was observed on the production of acid phosphatase in early and mid log phase of fungal growth. Whereas in the exponential phase in test proper, there was a statistical significant decrease in the enzyme production with 0.8% and 3.2% of KI. The low activity at 0.8% and 3.2% KI indicates that KI has inhibitory effect on the growth of S.schenckii and has led to decrease in the activity of the enzyme. (Med J Indones 2003; 12: 65-8 Keywords: S.schenckii, acid phosphatase, potassium iodide

  17. The effects of drought stress on the activity of acid phosphatase and ...

    African Journals Online (AJOL)

    A model of drought was created on pigweed and the effects of drought stress on the activity of acid phosphatase and its protective enzymes were examined. The pot-cultured pigweeds were divided into 4 groups (ten plants per group) when they reached 6 leaves. (1) In the control group, the culture media contained 70 ...

  18. Functional Diversity of Haloacid Dehalogenase Superfamily Phosphatases from Saccharomyces cerevisiae: BIOCHEMICAL, STRUCTURAL, AND EVOLUTIONARY INSIGHTS.

    Science.gov (United States)

    Kuznetsova, Ekaterina; Nocek, Boguslaw; Brown, Greg; Makarova, Kira S; Flick, Robert; Wolf, Yuri I; Khusnutdinova, Anna; Evdokimova, Elena; Jin, Ke; Tan, Kemin; Hanson, Andrew D; Hasnain, Ghulam; Zallot, Rémi; de Crécy-Lagard, Valérie; Babu, Mohan; Savchenko, Alexei; Joachimiak, Andrzej; Edwards, Aled M; Koonin, Eugene V; Yakunin, Alexander F

    2015-07-24

    The haloacid dehalogenase (HAD)-like enzymes comprise a large superfamily of phosphohydrolases present in all organisms. The Saccharomyces cerevisiae genome encodes at least 19 soluble HADs, including 10 uncharacterized proteins. Here, we biochemically characterized 13 yeast phosphatases from the HAD superfamily, which includes both specific and promiscuous enzymes active against various phosphorylated metabolites and peptides with several HADs implicated in detoxification of phosphorylated compounds and pseudouridine. The crystal structures of four yeast HADs provided insight into their active sites, whereas the structure of the YKR070W dimer in complex with substrate revealed a composite substrate-binding site. Although the S. cerevisiae and Escherichia coli HADs share low sequence similarities, the comparison of their substrate profiles revealed seven phosphatases with common preferred substrates. The cluster of secondary substrates supporting significant activity of both S. cerevisiae and E. coli HADs includes 28 common metabolites that appear to represent the pool of potential activities for the evolution of novel HAD phosphatases. Evolution of novel substrate specificities of HAD phosphatases shows no strict correlation with sequence divergence. Thus, evolution of the HAD superfamily combines the conservation of the overall substrate pool and the substrate profiles of some enzymes with remarkable biochemical and structural flexibility of other superfamily members. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Dragmacidins: new protein phosphatase inhibitors from a southern australian deep-water marine sponge, spongosorites sp

    Science.gov (United States)

    Capon; Rooney; Murray; Collins; Sim; Rostas; Butler; Carroll

    1998-05-01

    A Spongosorites sp. collected during trawling operations off the southern coast of Australia returned the new alkaloid dragmacidin E (3), the structure of which was secured by detailed spectroscopic analysis. Dragmacidin E (3), and its co-metabolite dragmacidin D (1) have been identified as potent inhibitors of serine-threonine protein phosphatases.

  20. Protein phosphatase 5 promotes hepatocarcinogenesis through interaction with AMP-activated protein kinase.

    Science.gov (United States)

    Chen, Yao-Li; Hung, Man-Hsin; Chu, Pei-Yi; Chao, Tzu-I; Tsai, Ming-Hsien; Chen, Li-Ju; Hsiao, Yung-Jen; Shih, Chih-Ting; Hsieh, Feng-Shu; Chen, Kuen-Feng

    2017-08-15

    The serine-threonine protein phosphatase family members are known as critical regulators of various cellular functions, such as survival and transformation. Growing evidence suggests that pharmacological manipulation of phosphatase activity exhibits therapeutic benefits. Ser/Thr protein phosphatase 5 (PP5) is known to participate in glucocorticoid receptor (GR) and stress-induced signaling cascades that regulate cell growth and apoptosis, and has been shown to be overexpressed in various human malignant diseases. However, the role of PP5 in hepatocellular carcinoma (HCC) and whether PP5 may be a viable therapeutic target for HCC treatment are unknown. Here, by analyzing HCC clinical samples obtained from 215 patients, we found that overexpression of PP5 is tumor specific and associated with worse clinical outcomes. We further characterized the oncogenic properties of PP5 in HCC cells. Importantly, both silencing of PP5 with lentiviral-mediated short hairpin RNA (shRNA) and chemical inhibition of PP5 phosphatase activity using the natural compound cantharidin/norcantharidin markedly suppressed the growth of HCC cells and tumors in vitro and in vivo. Moreover, we identified AMP-activated protein kinase (AMPK) as a novel downstream target of oncogenic PP5 and demonstrated that the antitumor mechanisms underlying PP5 inhibition involve activation of AMPK signaling. Overall, our results establish a pathological function of PP5 in hepatocarcinogenesis via affecting AMPK signaling and suggest that PP5 inhibition is an attractive therapeutic approach for HCC. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Benomyl inhibits phosphorus transport but not fungal alkaline phosphatase activity in a Glomus-cucumber symbiosis

    DEFF Research Database (Denmark)

    Larsen, J.; Thingstrup, I.; Jakobsen, I.

    1996-01-01

    when benomyl was applied to the HC at 10 µg g-1 soil, whereas the uptake of 32P from RHC I roots + hyphae) was reduced only at the highest dose of application to the RHC (100 µ g g-1 soil). In contrast to the marked reduction of benomyl on fungal P transport, the activity of fungal alkaline phosphatase...

  2. A peptide export-import control circuit modulating bacterial development regulates protein phosphatases of the phosphorelay.

    Science.gov (United States)

    Perego, M

    1997-08-05

    The phosphorelay signal transduction system activates developmental transcription in sporulation of Bacillus subtilis by phosphorylation of aspartyl residues of the Spo0F and Spo0A response regulators. The phosphorylation level of these response regulators is determined by the opposing activities of protein kinases and protein aspartate phosphatases that interpret positive and negative signals for development in a signal integration circuit. The RapA protein aspartate phosphatase of the phosphorelay is regulated by a peptide that directly inhibits its activity. This peptide is proteolytically processed from an inactive pre-inhibitor protein encoded in the phrA gene. The pre-inhibitor is cleaved by the protein export apparatus to a putative pro-inhibitor that is further processed to the active inhibitor peptide and internalized by the oligopeptide permease. This export-import circuit is postulated to be a mechanism for timing phosphatase activity where the processing enzymes regulate the rate of formation of the active inhibitor. The processing events may, in turn, be controlled by a regulatory hierarchy. Chromosome sequencing has revealed several other phosphatase-prepeptide gene pairs in B. subtilis, suggesting that the use of this mechanism may be widespread in signal transduction.

  3. Phosphatase-regulated recruitment of the spindle- and kinetochore-associated (Ska complex to kinetochores

    Directory of Open Access Journals (Sweden)

    Sushama Sivakumar

    2017-11-01

    Full Text Available Kinetochores move chromosomes on dynamic spindle microtubules and regulate signaling of the spindle checkpoint. The spindle- and kinetochore-associated (Ska complex, a hexamer composed of two copies of Ska1, Ska2 and Ska3, has been implicated in both roles. Phosphorylation of kinetochore components by the well-studied mitotic kinases Cdk1, Aurora B, Plk1, Mps1, and Bub1 regulate chromosome movement and checkpoint signaling. Roles for the opposing phosphatases are more poorly defined. Recently, we showed that the C terminus of Ska1 recruits protein phosphatase 1 (PP1 to kinetochores. Here we show that PP1 and protein phosphatase 2A (PP2A both promote accumulation of Ska at kinetochores. Depletion of PP1 or PP2A by siRNA reduces Ska binding at kinetochores, impairs alignment of chromosomes to the spindle midplane, and causes metaphase delay or arrest, phenotypes that are also seen after depletion of Ska. Artificial tethering of PP1 to the outer kinetochore protein Nuf2 promotes Ska recruitment to kinetochores, and it reduces but does not fully rescue chromosome alignment and metaphase arrest defects seen after Ska depletion. We propose that Ska has multiple functions in promoting mitotic progression and that kinetochore-associated phosphatases function in a positive feedback cycle to reinforce Ska complex accumulation at kinetochores.

  4. Intercropping Acacia mangium stimulates AMF colonization and soil phosphatase activity in Eucalyptus grandis

    Directory of Open Access Journals (Sweden)

    Daniel Bini

    Full Text Available ABSTRACT: Arbuscular mycorrhizal fungi (AMF are very important to plant nutrition, mostly in terms of acquisition of P and micronutrients. While Acacia mangium is closely associated with AMF throughout the whole cycle, Eucalyptus grandis presents this symbiosis primarily at the seedling stage. The aim of this study was to evaluate the dynamics of AMF in these two tree species in both pure and mixed plantations during the first 20 months after planting. We evaluated the abundance, richness and diversity of AMF spores, the rate of AMF mycorrhizal root colonization, enzymatic activity and soil and litter C, N and P. There was an increase in AMF root colonization of E. grandis when intercropped with A. mangium as well as an increase in the activity of acid and alkaline phosphatase in the presence of leguminous trees. AMF colonization and phosphatase activities were both involved in improvements in P cycling and P nutrition in soil. In addition, P cycling was favored in the intercropped plantation, which showed negative correlation with litter C/N and C/P ratios and positive correlation with soil acid phosphatase activity and soil N and P concentrations. Intercropping A. mangium and E. grandis maximized AMF root colonization of E. grandis and phosphatase activity in the soil, both of which accelerate P cycling and forest performance.

  5. [Serum calcium and phosphorus concentration and alkaline phosphatase activity in healthy children during growth and development].

    Science.gov (United States)

    Savić, Ljiljana; Savić, Dejan

    2008-01-01

    Many changes happen during growth and development in an organism as a result of important hormon changes, especially biohumoral ones. These changes make a problem when interpreting biochemical results in pediatric population. The most important changes are intensive calcium and phosphorus metabolic turnover in bone tissue with changes in alkaline phosphatase activity as a result of osteoblast activity. The aim of this study was to follow the serum calcium and phosphorus concentration and alkaline phosphatase activity in children 1-15 years old in different growth and development period and of different sexes and to fortify the influence of growth and development dynamics on biohumoral status in healthy male and female children. We evaluated 117 healthy children of both sexes from 1-15 years of age and divided them into three age groups: 1-5, 6-10 and 11-15 years. We followed the serum calcium and phosphorus concentration and alkaline phosphatase activity in different groups and in different sexes. Our investigation found significantly higher values of serum calcium in boys than in girls with no important changes between the age groups and significantly higher values of serum phosphorus in the youngest age group in all children and in different sexes with no important sex differences. Alkaline phosphatase activity followed the growth spurt and was the biggest in 6-10 years group in girls and in 11-15 years group in boys.

  6. In vitro characterization of the Bacillus subtilis protein tyrosine phosphatase YwqE

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Musumeci, Lucia; Tautz, Lutz

    2005-01-01

    Both gram-negative and gram-positive bacteria possess protein tyrosine phosphatases (PTPs) with a catalytic Cys residue. In addition, many gram-positive bacteria have acquired a new family of PTPs, whose first characterized member was CpsB from Streptococcus pneumoniae. Bacillus subtilis contains...

  7. Essential domain of receptor tyrosine phosphatase beta (RPTPbeta) for interaction with Helicobacter pylori vacuolating cytotoxin

    DEFF Research Database (Denmark)

    Yahiro, Kinnosuke; Wada, Akihiro; Yamasaki, Eiki

    2004-01-01

    Helicobacter pylori produces a potent exotoxin, VacA, which causes progressive vacuolation as well as gastric injury. Although VacA was able to interact with two receptor-like protein tyrosine phosphatases, RPTPbeta and RPTPalpha, RPTPbeta was found to be responsible for gastric damage caused...

  8. MECHANISM OF PROTEIN TYROSINE PHOSPHATASE INHIBITION IN HUMAN AIRWAY EPITHELIAL CELLS (HAEC) EXPOSED TO ZN2+

    Science.gov (United States)

    A number of studies have implicated zinc in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to Zn2+ inhibits protein tyrosine phosphatase (PTP) activity and leads to activation of epidermal growth factor receptor (EGFR) signaling in ...

  9. Water molecule network and active site flexibility of apo protein tyrosine phosphatase 1B

    DEFF Research Database (Denmark)

    Pedersen, A.K.; Peters, Günther H.J.; Møller, K.B.

    2004-01-01

    Protein tyrosine phosphatase 1B (PTP1B) plays a key role as a negative regulator of insulin and leptin signalling and is therefore considered to be an important molecular target for the treatment of type 2 diabetes and obesity. Detailed structural information about the structure of PTP1B, including...

  10. Mechanism of protein tyrosine phosphatase 1B-mediated inhibition of leptin signalling

    DEFF Research Database (Denmark)

    Lund, I K; Hansen, J A; Andersen, H S

    2005-01-01

    Upon leptin binding, the leptin receptor is activated, leading to stimulation of the JAK/STAT signal transduction cascade. The transient character of the tyrosine phosphorylation of JAK2 and STAT3 suggests the involvement of protein tyrosine phosphatases (PTPs) as negative regulators...

  11. Pterocarpans with inhibitory effects on protein tyrosine phosphatase 1B from Erythrina lysistemon Hutch

    DEFF Research Database (Denmark)

    Dao, Trong Tuan; Nguyen, Phi Hung; Thuong, Phuong Thien

    2009-01-01

    ',5':3,4]-2'',2''-dimethyldihydropyrano[6'',5'':9,10]pterocarpan (1), furano[5',4':3,4]-9-hydroxy-10-prenylpterocarpan (2), and 8-formyl-3,9-dihydroxy-4,10-diprenylpterocarpan (3), based on spectroscopic analyses. All the isolates, with the exception of 3, 6, and 11, strongly inhibited protein tyrosine phosphatase 1B (PTP1B) activity...

  12. Multiple forms of the human tyrosine phosphatase RPTP alpha. Isozymes and differences in glycosylation

    DEFF Research Database (Denmark)

    Daum, G; Regenass, S; Sap, J

    1994-01-01

    Among all the receptor-linked protein-tyrosine-phosphatase RPTP alpha clones described from mammalian tissues, one differed in that it encoded a 9-amino-acid insert 3 residues upstream from the transmembrane segment (Kaplan, R., Morse, B., Huebner, K., Croce, C., Howk, R. Ravera, M., Ricca, G...

  13. Structural and biochemical analysis of atypically low dephosphorylating activity of human dual-specificity phosphatase 28.

    Directory of Open Access Journals (Sweden)

    Bonsu Ku

    Full Text Available Dual-specificity phosphatases (DUSPs constitute a subfamily of protein tyrosine phosphatases, and are intimately involved in the regulation of diverse parameters of cellular signaling and essential biological processes. DUSP28 is one of the DUSP subfamily members that is known to be implicated in the progression of hepatocellular and pancreatic cancers, and its biological functions and enzymatic characteristics are mostly unknown. Herein, we present the crystal structure of human DUSP28 determined to 2.1 Å resolution. DUSP28 adopts a typical DUSP fold, which is composed of a central β-sheet covered by α-helices on both sides and contains a well-ordered activation loop, as do other enzymatically active DUSP proteins. The catalytic pocket of DUSP28, however, appears hardly accessible to a substrate because of the presence of nonconserved bulky residues in the protein tyrosine phosphatase signature motif. Accordingly, DUSP28 showed an atypically low phosphatase activity in the biochemical assay, which was remarkably improved by mutations of two nonconserved residues in the activation loop. Overall, this work reports the structural and biochemical basis for understanding a putative oncological therapeutic target, DUSP28, and also provides a unique mechanism for the regulation of enzymatic activity in the DUSP subfamily proteins.

  14. Quantification of the histochemical reaction for alkaline phosphatase activity using the indoxyl-tetranitro BT method

    NARCIS (Netherlands)

    van Noorden, C. J.; Jonges, G. N.

    1987-01-01

    The indoxyl-tetranitro BT method for the demonstration of alkaline phosphatase activity has been optimized and its validity for quantitative histochemistry tested. The study has been performed with model films of polyacrylamide gel incorporating homogenate of rat liver and with cryostat sections

  15. ETS1 mediates MEK1/2-dependent overexpression of cancerous inhibitor of protein phosphatase 2A (CIP2A in human cancer cells.

    Directory of Open Access Journals (Sweden)

    Anchit Khanna

    2011-03-01

    Full Text Available EGFR-MEK-ERK signaling pathway has an established role in promoting malignant growth and disease progression in human cancers. Therefore identification of transcriptional targets mediating the oncogenic effects of the EGFR-MEK-ERK pathway would be highly relevant. Cancerous inhibitor of protein phosphatase 2A (CIP2A is a recently characterized human oncoprotein. CIP2A promotes malignant cell growth and is over expressed at high frequency (40-80% in most of the human cancer types. However, the mechanisms inducing its expression in cancer still remain largely unexplored. Here we present systematic analysis of contribution of potential gene regulatory mechanisms for high CIP2A expression in cancer. Our data shows that evolutionary conserved CpG islands at the proximal CIP2A promoter are not methylated both in normal and cancer cells. Furthermore, sequencing of the active CIP2A promoter region from altogether seven normal and malignant cell types did not reveal any sequence alterations that would increase CIP2A expression specifically in cancer cells. However, treatment of cancer cells with various signaling pathway inhibitors revealed that CIP2A mRNA expression was sensitive to inhibition of EGFR activity as well as inhibition or activation of MEK-ERK pathway. Moreover, MEK1/2-specific siRNAs decreased CIP2A protein expression. Series of CIP2A promoter-luciferase constructs were created to identify proximal -27 to -107 promoter region responsible for MEK-dependent stimulation of CIP2A expression. Additional mutagenesis and chromatin immunoprecipitation experiments revealed ETS1 as the transcription factor mediating stimulation of CIP2A expression through EGFR-MEK pathway. Thus, ETS1 is probably mediating high CIP2A expression in human cancers with increased EGFR-MEK1/2-ERK pathway activity. These results also suggest that in addition to its established role in invasion and angiogenesis, ETS1 may support malignant cellular growth via regulation of

  16. Optimization of extraction parameters of PTP1β (protein tyrosine phosphatase 1β), inhibitory polyphenols, and anthocyanins from Zea mays L. using response surface methodology (RSM).

    Science.gov (United States)

    Hwang, Seung Hwan; Kwon, Shin Hwa; Wang, Zhiqiang; Kim, Tae Hyun; Kang, Young-Hee; Lee, Jae-Yong; Lim, Soon Sung

    2016-08-26

    Protein tyrosine phosphatase expressed in insulin-sensitive tissues (such as liver, muscle, and adipose tissue) has a key role in the regulation of insulin signaling and pathway activation, making protein tyrosine phosphatase a promising target for the treatment of type 2 diabetes mellitus and obesity and response surface methodology (RSM) is an effective statistical technique for optimizing complex processes using a multi-variant approach. In this study, Zea mays L. (Purple corn kernel, PCK) and its constituents were investigated for protein tyrosine phosphatase 1β (PTP1β) inhibitory activity including enzyme kinetic study and to improve total yields of anthocyanins and polyphenols, four extraction parameters, including temperature, time, solid-liquid ratio, and solvent volume, were optimized by RSM. Isolation of seven polyphenols and five anthocyanins was achieved by PTP1β assay. Among them, cyanidin-3-(6"malonylglucoside) and 3'-methoxyhirsutrin showed the highest PTP1β inhibition with IC50 values of 54.06 and 64.04 μM, respectively and 4.52 mg gallic acid equivalent/g (GAE/g) of total polyphenol content (TPC) and 43.02 mg cyanidin-3-glucoside equivalent/100 g (C3GE/100g) of total anthocyanin content (TAC) were extracted at 40 °C for 8 h with a 33 % solid-liquid ratio and a 1:15 solvent volume. Yields were similar to predictions of 4.58 mg GAE/g of TPC and 42.28 mg C3GE/100 g of TAC. These results indicated that PCK and 3'-methoxyhirsutrin and cyanidin-3-(6"malonylglucoside) might be active natural compounds and could be apply by optimizing of extraction process using response surface methodology.

  17. Phosphatase and tensin homolog-β-catenin signaling modulates regulatory T cells and inflammatory responses in mouse liver ischemia/reperfusion injury.

    Science.gov (United States)

    Zhu, Qiang; Li, Changyong; Wang, Kunpeng; Yue, Shi; Jiang, Longfeng; Ke, Michael; Busuttil, Ronald W; Kupiec-Weglinski, Jerzy W; Zhang, Feng; Lu, Ling; Ke, Bibo

    2017-06-01

    The phosphatase and tensin homolog (PTEN) deleted on chromosome 10 plays an important role in regulating T cell activation during inflammatory response. Activation of β-catenin is crucial for maintaining immune homeostasis. This study investigates the functional roles and molecular mechanisms by which PTEN-β-catenin signaling promotes regulatory T cell (Treg) induction in a mouse model of liver ischemia/reperfusion injury (IRI). We found that mice with myeloid-specific phosphatase and tensin homolog knockout (PTEN M-KO ) exhibited reduced liver damage as evidenced by decreased levels of serum alanine aminotransferase, intrahepatic macrophage trafficking, and proinflammatory mediators compared with the PTEN-proficient (floxed phosphatase and tensin homolog [PTEN FL/FL ]) controls. Disruption of myeloid PTEN-activated b-catenin promoted peroxisome proliferator-activated receptor gamma (PPARγ)-mediated Jagged-1/Notch signaling and induced forkhead box P3 (FOXP3)1 Tregs while inhibiting T helper 17 cells. However, blocking of Notch signaling by inhibiting γ-secretase reversed myeloid PTEN deficiency-mediated protection in ischemia/reperfusion-triggered liver inflammation with reduced FOXP3 + and increased retinoid A receptor-related orphan receptor gamma t-mediated interleukin 17A expression in ischemic livers. Moreover, knockdown of β-catenin or PPARγ in PTEN-deficient macrophages inhibited Jagged-1/Notch activation and reduced FOXP3 + Treg induction, leading to increased proinflammatory mediators in macrophage/T cell cocultures. In conclusion, our findings demonstrate that PTEN-β-catenin signaling is a novel regulator involved in modulating Treg development and provides a potential therapeutic target in liver IRI. Liver Transplantation 23 813-825 2017 AASLD. © 2017 by the American Association for the Study of Liver Diseases.

  18. Somatic cell count and alkaline phosphatase activity in milk for evaluation of mastitis in buffalo

    Science.gov (United States)

    Patil, M. P.; Nagvekar, A. S.; Ingole, S. D.; Bharucha, S. V.; Palve, V. T.

    2015-01-01

    Background and Aim: Mastitis is a serious disease of dairy animals causing great economic losses due to a reduction in milk yield as well as lowering its nutritive value. The application of somatic cell count (SCC) and alkaline phosphatase activity in the milk for diagnosis of mastitis in buffalo is not well documented. Therefore, the present study was conducted to observe the SCC and alkaline phosphatase activity for evaluation of mastitis in buffalo. Materials and Methods: Milk samples of forty apparently healthy lactating buffaloes were selected and categorized into five different groups viz. normal buffaloes, buffaloes with subclinical mastitis with CMT positive milk samples (+1 Grade), (+2 Grade), (+3 Grade), and buffaloes with clinical mastitis with 8 animals in each group. The milk samples were analyzed for SCC and alkaline phosphatase activity. Results: The levels of SCC (×105 cells/ml) and alkaline phosphatase (U/L) in different groups were viz. normal (3.21±0.179, 16.48±1.432), subclinical mastitis with CMT positive milk samples with +1 Grade (4.21±0.138, 28.11±1.013), with +2 Grade (6.34±0.183, 34.50±1.034), with +3 Grade (7.96±0.213, 37.73±0.737) and buffaloes with clinical mastitis (10.21±0.220, 42.37±0.907) respectively, indicating an increasing trend in the values and the difference observed among various group was statistically significant. Conclusion: In conclusion, the results of the present study indicate that the concentration of milk SCC and alkaline phosphatase activity was higher in the milk of buffaloes with mastitis than in the milk of normal buffaloes. PMID:27047098

  19. The phosphatase inhibitor menadione (vitamin K3) protects cells from EGFR inhibition by erlotinib and cetuximab.

    Science.gov (United States)

    Perez-Soler, Roman; Zou, Yiyu; Li, Tianhong; Ling, Yi He

    2011-11-01

    Skin toxicity is the main side effect of epidermal growth factor receptor (EGFR) inhibitors, often leading to dose reduction or discontinuation. We hypothesized that phosphatase inhibition in the skin keratinocytes may prevent receptor dephosphorylation caused by EGFR inhibitors and be used as a new potential strategy for the prevention or treatment of this side effect. Menadione (Vitamin K3) was used as the prototype compound to test our hypothesis. HaCat human skin keratinocyte cells and A431 human squamous carcinoma cells were used. EGFR inhibition was measured by Western blotting and immunofluorescence. Phosphatase inhibition and reactive oxygen species (ROS) generation were measured by standard ELISA and fluorescence assays. Menadione caused significant and reversible EGFR activation in a dose-dependent manner starting at nontoxic concentrations. EGFR activation by menadione was associated with reversible protein tyrosine phosphatase inhibition, which seemed to be mediated by ROS generation as exposure to antioxidants prevented both menadione-induced ROS generation and phosphatase inhibition. Short-term coincubation of cells with nontoxic concentrations of menadione and the EGFR inhibitors erlotinib or cetuximab prevented EGFR dephosphorylation. Seventy-two-hour coincubation of cells with the highest nontoxic concentration of menadione and erlotinib provided for a fourfold cell growth inhibitory protection in HaCat human keratinocyte cells. Menadione at nontoxic concentrations causes EGFR activation and prevents EGFR dephosphorylation by erlotinib and cetuximab. This effect seems to be mediated by ROS generation and secondary phosphatase inhibition. Mild oxidative stress in skin keratinocytes by topical menadione may protect the skin from the toxicity secondary to EGFR inhibitors without causing cytotoxicity. ©2011 AACR

  20. Somatic cell count and alkaline phosphatase activity in milk for evaluation of mastitis in buffalo

    Directory of Open Access Journals (Sweden)

    M. P. Patil

    2015-03-01

    Full Text Available Background and Aim: Mastitis is a serious disease of dairy animals causing great economic losses due to a reduction in milk yield as well as lowering its nutritive value. The application of somatic cell count (SCC and alkaline phosphatase activity in the milk for diagnosis of mastitis in buffalo is not well documented. Therefore, the present study was conducted to observe the SCC and alkaline phosphatase activity for evaluation of mastitis in buffalo. Materials and Methods: Milk samples of forty apparently healthy lactating buffaloes were selected and categorized into five different groups viz. normal buffaloes, buffaloes with subclinical mastitis with CMT positive milk samples (+1 Grade, (+2 Grade, (+3 Grade, and buffaloes with clinical mastitis with 8 animals in each group. The milk samples were analyzed for SCC and alkaline phosphatase activity. Results: The levels of SCC (×105 cells/ml and alkaline phosphatase (U/L in different groups were viz. normal (3.21±0.179, 16.48±1.432, subclinical mastitis with CMT positive milk samples with +1 Grade (4.21±0.138, 28.11±1.013, with +2 Grade (6.34±0.183, 34.50±1.034, with +3 Grade (7.96±0.213, 37.73±0.737 and buffaloes with clinical mastitis (10.21±0.220, 42.37±0.907 respectively, indicating an increasing trend in the values and the difference observed among various group was statistically significant. Conclusion: In conclusion, the results of the present study indicate that the concentration of milk SCC and alkaline phosphatase activity was higher in the milk of buffaloes with mastitis than in the milk of normal buffaloes.

  1. Phosphatase activity in sandy soil influenced by mycorrhizal and non-mycorrhizal cover crops

    Directory of Open Access Journals (Sweden)

    Alceu Kunze

    2011-06-01

    Full Text Available Cover crops may difffer in the way they affect rhizosphere microbiota nutrient dynamics. The purpose of this study was to evaluate the effect of mycorrhizal and non-mycorrhizal cover crops on soil phosphatase activity and its persistence in subsequent crops. A three-year experiment was carried out with a Typic Quartzipsamment. Treatments were winter species, either mycorrhizal black oat (Avena strigosa Schreb or the non-mycorrhizal species oilseed radish (Raphanus sativus L. var. oleiferus Metzg and corn spurry (Spergula arvensis L.. The control treatment consisted of resident vegetation (fallow in the winter season. In the summer, a mixture of pearl millet (Pennisetum americanum L. with sunnhemp (Crotalaria juncea L. or with soybean (Glycine max L. was sown in all plots. Soil cores (0-10 cm and root samples were collected in six growing seasons (winter and summer of each year. Microbial biomass P was determined by the fumigation-extraction method and phosphatase activity using p-nitrophenyl-phosphate as enzyme substrate. During the flowering stage of the winter cover crops, acid phosphatase activity was 30-35 % higher in soils with the non-mycorrhizal species oilseed radish, than in the control plots, regardless of the amount of P immobilized in microbial biomass. The values of enzyme activity were intermediate in the plots with corn spurry and black oat. Alkaline phosphatase activity was 10-fold lower and less sensitive to the treatments, despite the significant relationship between the two phosphatase activities. The effect of plant species on the soil enzyme profile continued in the subsequent periods, during the growth of mycorrhizal summer crop