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Sample records for acid phosphatases paps

  1. Purification of prostatic acid phosphatase (PAP) for structural and functional studies.

    PubMed

    Herrala, Annakaisa M; Quintero, Ileana B; Vihko, Pirkko T

    2013-01-01

    High-scale purification methods are required for several protein studies such as crystallography, mass spectrometry, circular dichroism, and function. Here we describe a purification method for PAP based on anion exchange, L-(+)-tartrate affinity, and gel filtration chromatographies. Acid phosphatase activity and protein concentration were measured for each purification step, and to collect the fractions with the highest acid phosphatase activity the p-nitrophenyl phosphate method was used. The purified protein obtained by the procedure described here was used for the determination of the first reported three-dimensional structure of prostatic acid phosphatase.

  2. Cloning and characterization of a novel human phosphatidic acid phosphatase type 2, PAP2d, with two different transcripts PAP2d_v1 and PAP2d_v2.

    PubMed

    Sun, Liyun; Gu, Shaohua; Sun, Yaqiong; Zheng, Dan; Wu, Qihan; Li, Xin; Dai, Jianfeng; Dai, Jianliang; Ji, Chaoneng; Xie, Yi; Mao, Yumin

    2005-04-01

    This study reports the cloning and characterization of a novel human phosphatidic acid phosphatase type 2 isoform cDNAs (PAP2d) from the foetal brain cDNA library. The PAP2d gene is localized on chromosome 1p21.3. It contains six exons and spans 112 kb of the genomic DNA. By large-scale cDNA sequencing we found two splice variants of PAP2d, PAP2d_v1 and PAP2d_v2. The PAP2d_v1 cDNA is 1722 bp in length and spans an open reading frame from nucleotide 56 to 1021, encoding a 321aa protein. The PAP2d_v2 cDNA is 1707 bp in length encoding a 316aa protein from nucleotide 56-1006. The PAP2d_v1 cDNA is 15 bp longer than the PAP2d_v2 cDNA in the terminal of the fifth exon and it creates different ORF. Both of the proteins contain a well-conserved PAP2 motif. The PAP2d_v1 is mainly expressed in human brain, lung, kidney, testis and colon, while PAP2d_v2 is restricted to human placenta, skeletal muscle, and kidney. The two splice variants are co-expressed only in kidney. PMID:16010976

  3. Cloning and characterization of a novel human phosphatidic acid phosphatase type 2, PAP2d, with two different transcripts PAP2d_v1 and PAP2d_v2.

    PubMed

    Sun, Liyun; Gu, Shaohua; Sun, Yaqiong; Zheng, Dan; Wu, Qihan; Li, Xin; Dai, Jianfeng; Dai, Jianliang; Ji, Chaoneng; Xie, Yi; Mao, Yumin

    2005-04-01

    This study reports the cloning and characterization of a novel human phosphatidic acid phosphatase type 2 isoform cDNAs (PAP2d) from the foetal brain cDNA library. The PAP2d gene is localized on chromosome 1p21.3. It contains six exons and spans 112 kb of the genomic DNA. By large-scale cDNA sequencing we found two splice variants of PAP2d, PAP2d_v1 and PAP2d_v2. The PAP2d_v1 cDNA is 1722 bp in length and spans an open reading frame from nucleotide 56 to 1021, encoding a 321aa protein. The PAP2d_v2 cDNA is 1707 bp in length encoding a 316aa protein from nucleotide 56-1006. The PAP2d_v1 cDNA is 15 bp longer than the PAP2d_v2 cDNA in the terminal of the fifth exon and it creates different ORF. Both of the proteins contain a well-conserved PAP2 motif. The PAP2d_v1 is mainly expressed in human brain, lung, kidney, testis and colon, while PAP2d_v2 is restricted to human placenta, skeletal muscle, and kidney. The two splice variants are co-expressed only in kidney.

  4. An Approach to More Accurate Model Systems for Purple Acid Phosphatases (PAPs).

    PubMed

    Bernhardt, Paul V; Bosch, Simone; Comba, Peter; Gahan, Lawrence R; Hanson, Graeme R; Mereacre, Valeriu; Noble, Christopher J; Powell, Annie K; Schenk, Gerhard; Wadepohl, Hubert

    2015-08-01

    The active site of mammalian purple acid phosphatases (PAPs) have a dinuclear iron site in two accessible oxidation states (Fe(III)2 and Fe(III)Fe(II)), and the heterovalent is the active form, involved in the regulation of phosphate and phosphorylated metabolite levels in a wide range of organisms. Therefore, two sites with different coordination geometries to stabilize the heterovalent active form and, in addition, with hydrogen bond donors to enable the fixation of the substrate and release of the product, are believed to be required for catalytically competent model systems. Two ligands and their dinuclear iron complexes have been studied in detail. The solid-state structures and properties, studied by X-ray crystallography, magnetism, and Mössbauer spectroscopy, and the solution structural and electronic properties, investigated by mass spectrometry, electronic, nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR), and Mössbauer spectroscopies and electrochemistry, are discussed in detail in order to understand the structures and relative stabilities in solution. In particular, with one of the ligands, a heterovalent Fe(III)Fe(II) species has been produced by chemical oxidation of the Fe(II)2 precursor. The phosphatase reactivities of the complexes, in particular, also of the heterovalent complex, are reported. These studies include pH-dependent as well as substrate concentration dependent studies, leading to pH profiles, catalytic efficiencies and turnover numbers, and indicate that the heterovalent diiron complex discussed here is an accurate PAP model system. PMID:26196255

  5. The secreted purple acid phosphatase isozymes AtPAP12 and AtPAP26 play a pivotal role in extracellular phosphate-scavenging by Arabidopsis thaliana

    PubMed Central

    Plaxton, William C.

    2012-01-01

    Orthophosphate (Pi) is an essential but limiting macronutrient for plant growth. Extensive soil P reserves exist in the form of organic P (Po), which is unavailable for root uptake until hydrolysed by secretory acid phosphatases (APases). The predominant purple APase (PAP) isozymes secreted by roots of Pi-deficient (–Pi) Arabidopsis thaliana were recently identified as AtPAP12 (At2g27190) and AtPAP26 (At5g34850). The present study demonstrated that exogenous Po compounds such as glycerol-3-phosphate or herring sperm DNA: (i) effectively substituted for Pi in supporting the P nutrition of Arabidopsis seedlings, and (ii) caused upregulation and secretion of AtPAP12 and AtPAP26 into the growth medium. When cultivated under –Pi conditions or supplied with Po as its sole source of P nutrition, an atpap26/atpap12 T-DNA double insertion mutant exhibited impaired growth coupled with >60 and >30% decreases in root secretory APase activity and rosette total Pi concentration, respectively. Development of the atpap12/atpap26 mutant was unaffected during growth on Pi-replete medium but was completely arrested when 7-day-old Pi-sufficient seedlings were transplanted into a –Pi, Po-containing soil mix. Both PAPs were also strongly upregulated on root surfaces and in shoot cell-wall extracts of –Pi seedlings. It is hypothesized that secreted AtPAP12 and AtPAP26 facilitate the acclimation of Arabidopsis to nutritional Pi deficiency by: (i) functioning in the rhizosphere to scavenge Pi from the soil’s accessible Po pool, while (ii) recycling Pi from endogenous phosphomonoesters that have been leaked into cell walls from the cytoplasm. Thus, AtPAP12 and AtPAP26 are promising targets for improving crop P-use efficiency. PMID:23125358

  6. OsPAP10c, a novel secreted acid phosphatase in rice, plays an important role in the utilization of external organic phosphorus.

    PubMed

    Lu, Linghong; Qiu, Wenmin; Gao, Wenwen; Tyerman, Stephen D; Shou, Huixia; Wang, Chuang

    2016-10-01

    Under phosphate (Pi ) starvation, plants increase the secretion of purple acid phosphatases (PAPs) into the rhizosphere to scavenge organic phosphorus (P) for plant use. To date, only a few members of the PAP family have been characterized in crops. In this study, we identified a novel secreted PAP in rice, OsPAP10c, and investigated its role in the utilization of external organic P. OsPAP10c belongs to a monocotyledon-specific subclass of Ia group PAPs and is specifically expressed in the epidermis/exodermis cell layers of roots. Both the transcript and protein levels of OsPAP10c are strongly induced by Pi starvation. OsPAP10c overexpression increased acid phosphatase (APase) activity by more than 10-fold in the culture media and almost fivefold in both roots and leaves under Pi -sufficient and Pi -deficient conditions. This increase in APase activity further improved the plant utilization efficiency of external organic P. Moreover, several APase isoforms corresponding to OsPAP10c were identified using in-gel activity assays. Under field conditions with three different Pi supply levels, OsPAP10c-overexpressing plants had significantly higher tiller numbers and shorter plant heights. This study indicates that OsPAP10c encodes a novel secreted APase that plays an important role in the utilization of external organic P in rice. PMID:27411391

  7. A major root-associated acid phosphatase in Arabidopsis, AtPAP10, is regulated by both local and systemic signals under phosphate starvation

    PubMed Central

    Zhang, Ye; Wang, Xiaoyue; Lu, Shan; Liu, Dong

    2014-01-01

    The induction and secretion of acid phosphatases (APases) is a universal response of plants to phosphate (Pi) starvation. AtPAP10 (Arabidopsis purple acid phosphatase 10) is a major Pi starvation-induced APase that is associated with the root surface in Arabidopsis. So far, the roles of local and systemic signalling in regulating root-associated AtPAP10 activity remain largely unknown. In this work, we show that a decrease of local, external Pi availability is sufficient to induce AtPAP10 transcription in roots in the presence of sucrose, a systemic signal from shoots, whereas the magnitude of the induction is affected by the Pi status of the whole plant. Once the AtPAP10 mRNAs are synthesized in roots, subsequent accumulation of AtPAP10 proteins in root cells and increase in AtPAP10 activity on the root surface are mainly controlled by local signalling. Previously, ethylene has been demonstrated to be a positive regulator of AtPAP10 activity. In this study, we provide evidence that under Pi deficiency ethylene mainly modulates enzymatic activity of AtPAP10 on the root surface, but not AtPAP10 transcription and protein accumulation, suggesting that it functions as a local signal. Furthermore, our work indicates that the effect of ethylene on the induction of root-associated AtPAP10 activity depends on sucrose, but that the effect of sucrose does not depend on ethylene. These results reveal new insights into the distinct roles of local and systemic signalling in the regulation of root-associated AtPAP10 activity under Pi starvation. PMID:25246445

  8. Spatial structure of oligopeptide PAP(248-261), the N-terminal fragment of the HIV enhancer prostatic acid phosphatase peptide PAP(248-286), in aqueous and SDS micelle solutions

    NASA Astrophysics Data System (ADS)

    Blokhin, Dmitriy S.; Filippov, Andrei V.; Antzutkin, Oleg N.; Karataeva, Farida Kh.; Klochkov, Vladimir V.

    2014-07-01

    Prostatic acid phosphatase (PAP) is an enzyme that facilitates infection of cells by HIV. Its peptide fragment PAP(248-286) forms amyloid fibrils known as SEVI, which enhance attachment of the virus by viral adhesion to the host cell prior to receptor-specific binding via reducing the electrostatic repulsion between the membranes of the virus and the target cell. The secondary structure of PAP(248-286) in aqueous and SDS solutions can be divided into an N-terminal disordered region, an α-helical central part and an α/310-helical C-terminal region (Nanga et al., 2009). In this work, we used NMR spectroscopy to study the spatial structure of the isolated N-terminal fragment of PAP(248-286), PAP(248-261) (GIHKQKEKSRLQGG), in aqueous and SDS micelle solutions. Formation of a PAP(248-261)-SDS complex was confirmed by chemical shift alterations in the 1H NMR spectra of the peptide, as well as by the signs and values of Nuclear Overhauser Effect (NOE). In addition, the PAP(248-261) peptide does not form any specified secondary structure in either aqueous or SDS solutions.

  9. Real-Time Immune Monitoring to Guide Plasmid DNA Vaccination Schedule Targeting Prostatic Acid Phosphatase (PAP) in Patients with Castration-Resistant Prostate Cancer

    PubMed Central

    McNeel, Douglas G.; Becker, Jordan T.; Eickhoff, Jens C.; Johnson, Laura E.; Bradley, Eric; Pohlkamp, Isabel; Staab, Mary Jane; Liu, Glenn; Wilding, George; Olson, Brian M.

    2014-01-01

    BACKGROUND We have previously reported that a DNA vaccine encoding prostatic acid phosphatase (PAP) could elicit PAP-specific T cells in patients with early recurrent prostate cancer. In the current pilot trial we sought to evaluate whether prolonged immunization with regular booster immunizations, or “personalized” schedules of immunization determined using real-time immune monitoring, could elicit persistent, antigen-specific T cells, and whether treatment was associated with changes in PSA doubling time (PSA DT). METHODS 16 patients with castration-resistant, non-metastatic prostate cancer received six immunizations at two-week intervals, and then either quarterly (Arm 1) or as determined by multi-parameter immune monitoring (Arm 2). RESULTS Patients were on study a median of 16 months; four received 24 vaccinations. Only one event associated with treatment > grade 2 was observed. 6/16 (38%) remained metastasis-free at 2 years. PAP-specific T cells were elicited in 12/16 (75%), predominantly of a Th1 phenotype, which persisted in frequency and phenotype for at least one year. IFNγ-secreting T-cell responses measured by ELISPOT were detectable in 5/13 individuals at one year, and this was not statistically different between study arms. The overall median fold change in PSA DT from pre-treatment to post-treatment was 1.6 (range 0.6–7.0, p=0.036). CONCLUSIONS Repetitive immunization with a plasmid DNA vaccine was safe and elicited Th1-biased antigen-specific T cells that persisted over time. Modifications in the immunization schedule based on real-time immune monitoring did not increase the frequency of patients developing effector and memory T-cell responses with this DNA vaccine. PMID:24850844

  10. Penicillin inhibitors of purple acid phosphatase.

    PubMed

    Faridoon; Hussein, Waleed M; Ul Islam, Nazar; Guddat, Luke W; Schenk, Gerhard; McGeary, Ross P

    2012-04-01

    Purple acid phosphatases (PAPs) are binuclear metallohydrolases that have a multitude of biological functions and are found in fungi, bacteria, plants and animals. In mammals, PAP activity is linked with bone resorption and over-expression can lead to bone disorders such as osteoporosis. PAP is therefore an attractive target for the development of drugs to treat this disease. A series of penicillin conjugates, in which 6-aminopenicillanic acid was acylated with aromatic acid chlorides, has been prepared and assayed against pig PAP. The binding mode of most of these conjugates is purely competitive, and some members of this class have potencies comparable to the best PAP inhibitors yet reported. The structurally related penicillin G was shown to be neither an inhibitor nor a substrate for pig PAP. Molecular modelling has been used to examine the binding modes of these compounds in the active site of the enzyme and to rationalise their activities.

  11. Emerging Roles of Human Prostatic Acid Phosphatase

    PubMed Central

    Kong, Hoon Young; Byun, Jonghoe

    2013-01-01

    Prostate cancer is one of the most prevalent non-skin related cancers. It is the second leading cause of cancer deaths among males in most Western countries. If prostate cancer is diagnosed in its early stages, there is a higher probability that it will be completely cured. Prostatic acid phosphatase (PAP) is a non-specific phosphomonoesterase synthesized in prostate epithelial cells and its level proportionally increases with prostate cancer progression. PAP was the biochemical diagnostic mainstay for prostate cancer until the introduction of prostate-specific antigen (PSA) which improved the detection of early-stage prostate cancer and largely displaced PAP. Recently, however, there is a renewed interest in PAP because of its usefulness in prognosticating intermediate to high-risk prostate cancers and its success in the immunotherapy of prostate cancer. Although PAP is believed to be a key regulator of prostate cell growth, its exact role in normal prostate as well as detailed molecular mechanism of PAP regulation is still unclear. Here, many different aspects of PAP in prostate cancer are revisited and its emerging roles in other environment are discussed. PMID:24009853

  12. Radioimmunodetection of lymph node invasion in prostatic cancer. The use of iodine 123 (123I)-labeled monoclonal anti-prostatic acid phosphatase (PAP) 227 A F(ab')2 antibody fragments in vivo.

    PubMed

    Leroy, M; Teillac, P; Rain, J D; Saccavini, J C; Le Duc, A; Najean, Y

    1989-07-01

    The therapeutic indications in prostatic cancer depend on the regional and distant extension of the cancer and are difficult to assess before lymphadenectomy. Radioimmunodetection of lymph node involvement with monoclonal anti-prostatic acid phosphatase (PAP) antibodies can be proposed as a noninvasive alternative to lymphadenectomy. Fifteen patients with various stages of histologically proven prostatic cancer were examined by immunolymphoscintigraphy (ILS) before treatment to detect lymph node metastases. These patients had Stage A (n = 7), Stage B (n = 3), Stage C (n = 2), and Stage D (n = 3) tumors. They received between 100 and 400 micrograms of monoclonal antibody 227 A in the form of F(ab')2 fragments labeled with iodine 123 (123I). The antibody was injected directly into the periprostatic area. ILS images were obtained after 1, 3, 6, and 24 hours. Three days later, each patient underwent a lymphadenectomy for histologic examination. The results of the histologic examination and ILS were compared. In ten patients, the examination did not show any images capable of being interpreted as lymphadenopathy and histologic examination confirmed the integrity of the nodes examined. In five cases, scintigraphy suggested the presence of lymph node invasion by prostatic cancer and this was confirmed by histologic examination in three of the five cases. Overall, in terms of lymphadenopathy, this examination had a sensitivity of 100% and a specificity of 83%. Therefore, ILS appears to be capable of detecting lymph node metastases in prostatic cancer.

  13. Cloning and comparative protein modeling of two purple acid phosphatase isozymes from sweet potatoes (Ipomoea batatas).

    PubMed

    Durmus, A; Eicken, C; Spener, F; Krebs, B

    1999-09-14

    The sequence of cDNA fragments of two isozymes of the purple acid phosphatase from sweet potato (spPAP1 and spPAP2) has been determined by 5' and 3' rapid amplification of cDNA ends protocols using oligonucleotide primers based on amino acid information. The encoded amino acid sequences of these two isozymes show an equidistance of 72-77% not only to each other, but also to the primary structure of the purple acid phosphatase from red kidney bean (kbPAP). A three-dimensional model of the active site has been constructed for spPAP2 on the basis of the kbPAP crystallographic structure that helps to explain the reported differences in the visible and EPR spectra of spPAP2 and kbPAP.

  14. Cloning and comparative protein modeling of two purple acid phosphatase isozymes from sweet potatoes (Ipomoea batatas).

    PubMed

    Durmus, A; Eicken, C; Spener, F; Krebs, B

    1999-09-14

    The sequence of cDNA fragments of two isozymes of the purple acid phosphatase from sweet potato (spPAP1 and spPAP2) has been determined by 5' and 3' rapid amplification of cDNA ends protocols using oligonucleotide primers based on amino acid information. The encoded amino acid sequences of these two isozymes show an equidistance of 72-77% not only to each other, but also to the primary structure of the purple acid phosphatase from red kidney bean (kbPAP). A three-dimensional model of the active site has been constructed for spPAP2 on the basis of the kbPAP crystallographic structure that helps to explain the reported differences in the visible and EPR spectra of spPAP2 and kbPAP. PMID:10556574

  15. Molecular cloning of magnesium-independent type 2 phosphatidic acid phosphatases from airway smooth muscle.

    PubMed

    Tate, R J; Tolan, D; Pyne, S

    1999-07-01

    Members of the type 2 phosphatidic acid phosphatase (PAP2) family catalyse the dephosphorylation of phosphatidic acid (PA), lysophosphatidate and sphingosine 1-phosphate. Here, we demonstrate the presence of a Mg(2+)-independent and N-ethymaleimide-insensitive PAP2 activity in cultured guinea-pig airway smooth muscle (ASM) cells. Two PAP2 cDNAs of 923 and 926 base pairs were identified and subsequently cloned from these cells. The ORF of the 923 base pair cDNA encoded a protein of 285 amino acids (Mr = 32.1 kDa), which had 94% homology with human PAP2a (hPAP2a) and which probably represents a guinea-pig specific PAP2a (gpPAP2a1). The ORF of the 926 base pair cDNA encoded a protein of 286 amino acids (Mr = 32.1 kDa) which had 84% and 91% homology with hPAP2a and gpPAP2a1, respectively. This protein, termed gpPAP2a2, has two regions (aa 21-33 and 51-74) of marked divergence and altered hydrophobicity compared with hPAP2a and gpPAP2a1. This occurs in the predicted first and second transmembrane domains and at the extremes of the first outer loop. Other significant differences between gpPAP2a1/2 and hPAP2a, hPAP2b and hPAP2c occur at the cytoplasmic C-terminal. Transient expression of gpPAP2a2 in Cos-7 cells resulted in an approx. 4-fold increase in Mg(2+)-independent PAP activity, thereby confirming that gpPAP2a2 is another catalytically active member of an extended PAP2 family.

  16. Human prostatic acid phosphatase directly stimulates collagen synthesis and alkaline phosphatase content of isolated bone cells

    SciTech Connect

    Ishibe, M.; Rosier, R.N.; Puzas, J.E. )

    1991-10-01

    Human prostatic acid phosphatase (hPAP) directly enhances the differentiated characteristics of isolated bone cells in vitro. This enzyme, when added to cell cultures for 24 h in vitro stimulates collagen synthesis and the production of alkaline phosphatase. The effects are dose dependent, with statistically significant effects occurring from 0.1-100 nM hPAP. Concentrations higher than 100 nM do not evoke greater effects. The maximal effect of hPAP occurs between 12 and 24 h of exposure. The cells stimulated to the greatest degree are osteoprogenitor cells and osteoblasts. Fibroblasts isolated from the same tissue show a lesser sensitivity to hPAP. hPAP has no detectable effect on cell proliferation, as measured by radiolabeled thymidine incorporation or total DNA synthesis. None of the observations reported in this work can be attributed to contaminating proteins in the hPAP preparation. hPAP was radiolabeled with 125I and was used for affinity binding and cross-linking studies. Scatchard analysis of specific binding indicated the presence of 1.0 X 10(5) high affinity binding sites/cell, with a Kd of 6.5 nM. Cross-linking studies demonstrated the presence of one 320-kDa binding complex. The pH profile and kinetic determinations of Km and maximum velocity for hPAP were similar to those previously reported, except for the finding of positive cooperativity of the substrate with the enzyme under the conditions of our assay. We believe that the direct stimulation of bone-forming cells by hPAP may contribute to the sclerotic nature of skeletal bone around sites of neoplastic prostatic metastases and that the effect of the enzyme is probably mediated by a plasma membrane receptor.

  17. Synthesis, modelling and kinetic assays of potent inhibitors of purple acid phosphatase.

    PubMed

    Mohd-Pahmi, Siti Hajar; Hussein, Waleed M; Schenk, Gerhard; McGeary, Ross P

    2011-05-15

    Purple acid phosphatases (PAPs) are binuclear metallohydrolases that have been isolated from various mammals, plants, fungi and bacteria. In mammals PAP activity is associated with bone resorption and can lead to bone metabolic disorders such as osteoporosis; thus human PAP is an attractive target to develop anti-osteoporotic drugs. Based on a previous lead compound and rational drug design, acyl derivatives of α-aminonaphthylmethylphosphonic acid were synthesised and tested as PAP inhibitors. Kinetic analysis showed that they are good PAP inhibitors whose potencies improve with increasing acyl chain length. Maximum potency is reached when the number of carbons in the acyl chain is between 12 and 14. The most potent inhibitor of red kidney bean PAP is the dodecyl-derivative with K(ic)=5 μM, while the most potent pig PAP inhibitor is the tetradecyl-derivative with K(ic)=8 μM, the most potent inhibitor of a mammalian PAP yet reported.

  18. Prostatic acid phosphatase: structural aspects of inhibition by L-(+)-tartrate ions.

    PubMed

    Lovelace, L; Lewiński, K; Jakob, C G; Kuciel, R; Ostrowski, W; Lebioda, L

    1997-01-01

    The crystal structure of the complex between rat-prostatic acid phosphatase (PAP) and L-(+)-tartrate (Lindqvist et al., J. Biol. Chem., 1993, 268, 20744-20746) contains the model of the ligand with incorrect chirality. We report here the correct model and discuss the relation between this model and the model of the inhibitory complexes between PAP and oxy-anions.

  19. Molecular characterization and expression analysis of purple acid phosphatase gene from pearl oyster Pinctada martensii.

    PubMed

    Wang, Q H; Jiao, Y; Du, X D; Zhao, X X; Huang, R L; Deng, Y W; Yan, F

    2015-01-01

    Purple acid phosphatases (PAPs), also known as type 5 acid phosphatases, are widely present in animals, plants, and fungi. In mammal, PAP was reported to participate in immune defense and bone resorption. In this study, the characteristics and potential functions of a PAP gene from pearl oyster Pinctada martensii (pm-PAP) were examined. The Pm-PAP cDNA was found to be 2777 base pairs, containing a 1581-base pair open reading fragment encoding for 526 amino acids with an estimated molecular mass of 60.1 kDa and theoretical isoelectric point of 5.82. One signal peptide and five conserved motifs [GDXX/GDXXY/GNH(D/E)/XXXH/(A/G)HXH] were present in the entire sequence. Tissue expression profile analysis showed that pm-PAP mRNA was constitutively expressed in all tissues studied with abundant mRNA found in mollusk defense system, including hepatopancreas, gill, and hemocytes. After lipopolysaccharide stimulation, the expression of pm-PAP mRNA in hemocytes was dramatically upregulated at 2 h and achieved the highest level at 36 h. Additionally, pm-PAP mRNA expression was significantly increased and achieved the highest level at 2 days after the surgical implantation during pearl production. These results suggest that pm-PAP is a constitutive and inducible protein that may be involved in the immune defense of pearl oyster. PMID:25729991

  20. Insulin controls subcellular localization and multisite phosphorylation of the phosphatidic acid phosphatase, lipin 1.

    PubMed

    Harris, Thurl E; Huffman, Todd A; Chi, An; Shabanowitz, Jeffrey; Hunt, Donald F; Kumar, Anil; Lawrence, John C

    2007-01-01

    Brain, liver, kidney, heart, and skeletal muscle from fatty liver dystrophy (fld/fld) mice, which do not express lipin 1 (lipin), contained much less Mg(2+)-dependent phosphatidic acid phosphatase (PAP) activity than tissues from wild type mice. Lipin harboring the fld(2j) (Gly(84) --> Arg) mutation exhibited relatively little PAP activity. These results indicate that lipin is a major PAP in vivo and that the loss of PAP activity contributes to the fld phenotype. PAP activity was readily detected in immune complexes of lipin from 3T3-L1 adipocytes, where the protein was found both as a microsomal form and a soluble, more highly phosphorylated, form. Fifteen phosphorylation sites were identified by mass spectrometric analyses. Insulin increased the phosphorylation of multiple sites and promoted a gel shift that was due in part to phosphorylation of Ser(106). In contrast, epinephrine and oleic acid promoted dephosphorylation of lipin. The PAP-specific activity of lipin was not affected by the hormones or by dephosphorylation of lipin with protein phosphatase 1. However, the ratio of soluble to microsomal lipin was markedly increased in response to insulin and decreased in response to epinephrine and oleic acid. The results suggest that insulin and epinephrine control lipin primarily by changing localization rather than intrinsic PAP activity. PMID:17105729

  1. Insulin controls subcellular localization and multisite phosphorylation of the phosphatidic acid phosphatase, lipin 1.

    PubMed

    Harris, Thurl E; Huffman, Todd A; Chi, An; Shabanowitz, Jeffrey; Hunt, Donald F; Kumar, Anil; Lawrence, John C

    2007-01-01

    Brain, liver, kidney, heart, and skeletal muscle from fatty liver dystrophy (fld/fld) mice, which do not express lipin 1 (lipin), contained much less Mg(2+)-dependent phosphatidic acid phosphatase (PAP) activity than tissues from wild type mice. Lipin harboring the fld(2j) (Gly(84) --> Arg) mutation exhibited relatively little PAP activity. These results indicate that lipin is a major PAP in vivo and that the loss of PAP activity contributes to the fld phenotype. PAP activity was readily detected in immune complexes of lipin from 3T3-L1 adipocytes, where the protein was found both as a microsomal form and a soluble, more highly phosphorylated, form. Fifteen phosphorylation sites were identified by mass spectrometric analyses. Insulin increased the phosphorylation of multiple sites and promoted a gel shift that was due in part to phosphorylation of Ser(106). In contrast, epinephrine and oleic acid promoted dephosphorylation of lipin. The PAP-specific activity of lipin was not affected by the hormones or by dephosphorylation of lipin with protein phosphatase 1. However, the ratio of soluble to microsomal lipin was markedly increased in response to insulin and decreased in response to epinephrine and oleic acid. The results suggest that insulin and epinephrine control lipin primarily by changing localization rather than intrinsic PAP activity.

  2. Characterization of a soluble phosphatidic acid phosphatase in bitter melon (Momordica charantia).

    PubMed

    Cao, Heping; Sethumadhavan, Kandan; Grimm, Casey C; Ullah, Abul H J

    2014-01-01

    Momordica charantia is often called bitter melon, bitter gourd or bitter squash because its fruit has a bitter taste. The fruit has been widely used as vegetable and herbal medicine. Alpha-eleostearic acid is the major fatty acid in the seeds, but little is known about its biosynthesis. As an initial step towards understanding the biochemical mechanism of fatty acid accumulation in bitter melon seeds, this study focused on a soluble phosphatidic acid phosphatase (PAP, 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) that hydrolyzes the phosphomonoester bond in phosphatidate yielding diacylglycerol and P(i). PAPs are typically categorized into two subfamilies: Mg(2+)-dependent soluble PAP and Mg(2+)-independent membrane-associated PAP. We report here the partial purification and characterization of an Mg(2+)-independent PAP activity from developing cotyledons of bitter melon. PAP protein was partially purified by successive centrifugation and UNOsphere Q and S columns from the soluble extract. PAP activity was optimized at pH 6.5 and 53-60 °C and unaffected by up to 0.3 mM MgCl2. The K(m) and Vmax values for dioleoyl-phosphatidic acid were 595.4 µM and 104.9 ηkat/mg of protein, respectively. PAP activity was inhibited by NaF, Na(3)VO(4), Triton X-100, FeSO4 and CuSO4, but stimulated by MnSO4, ZnSO4 and Co(NO3)2. In-gel activity assay and mass spectrometry showed that PAP activity was copurified with a number of other proteins. This study suggests that PAP protein is probably associated with other proteins in bitter melon seeds and that a new class of PAP exists as a soluble and Mg(2+)-independent enzyme in plants. PMID:25203006

  3. Characterization of purple acid phosphatases involved in extracellular dNTP utilization in Stylosanthes.

    PubMed

    Liu, Pan-Dao; Xue, Ying-Bin; Chen, Zhi-Jian; Liu, Guo-Dao; Tian, Jiang

    2016-07-01

    Stylo (Stylosanthes spp.) is a pasture legume predominant in tropical and subtropical areas, where low phosphorus (P) availability is a major constraint for plant growth. Therefore, stylo might exhibit superior utilization of the P pool on acid soils, particularly organic P. However, little is known about mechanisms of inorganic phosphate (Pi) acquisition employed by stylo. In this study, the utilization of extracellular deoxy-ribonucleotide triphosphate (dNTP) and the underlying physiological and molecular mechanisms were examined for two stylo genotypes with contrasting P efficiency. Results showed that the P-efficient genotype, TPRC2001-1, was superior to the P-inefficient genotype, Fine-stem, when using dNTP as the sole P source. This was reflected by a higher dry weight and total P content for TPRC2001-1 than for Fine-stem, which was correlated with higher root-associated acid phosphatase (APase) activities in TPRC2001-1 under low P conditions. Subsequently, three PAP members were cloned from TPRC2001-1: SgPAP7, SgPAP10, and SgPAP26 Expression levels of these three SgPAPs were up-regulated by Pi starvation in stylo roots. Furthermore, there was a higher abundance of transcripts of SgPAP7 and SgPAP10 in TPRC2001-1 than in Fine-stem. Subcellular localization analysis demonstrated that these three SgPAPs were localized on the plasma membrane. Overexpression of these three SgPAPs could result in significantly increased root-associated APase activities, and thus extracellular dNTP utilization in bean hairy roots. Taken together, the results herein suggest that SgPAP7, SgPAP10, and SgPAP26 may differentially contribute to root-associated APase activities, and thus control extracellular dNTP utilization in stylo.

  4. Optimal level of purple acid phosphatase5 is required for maintaining complete resistance to Pseudomonas syringae

    PubMed Central

    Ravichandran, Sridhar; Stone, Sophia L.; Benkel, Bernhard; Zhang, Junzeng; Berrue, Fabrice; Prithiviraj, Balakrishnan

    2015-01-01

    Plants possess an exceedingly complex innate immune system to defend against most pathogens. However, a relative proportion of the pathogens overcome host's innate immunity and impair plant growth and productivity. We previously showed that mutation in purple acid phosphatase (PAP5) lead to enhanced susceptibility of Arabidopsis to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). Here, we report that an optimal level of PAP5 is crucial for mounting complete basal resistance. Overexpression of PAP5 impaired ICS1, PR1 expression and salicylic acid (SA) accumulation similar to pap5 knockout mutant plants. Moreover, plant overexpressing PAP5 was impaired in H2O2 accumulation in response to Pst DC3000. PAP5 is localized in to peroxisomes, a known site of generation of reactive oxygen species for activation of defense responses. Taken together, our results demonstrate that optimal levels of PAP5 is required for mounting resistance against Pst DC3000 as both knockout and overexpression of PAP5 lead to compromised basal resistance. PMID:26300891

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  6. The maize (Zea mays ssp. mays var. B73) genome encodes 33 members of the purple acid phosphatase family

    PubMed Central

    González-Muñoz, Eliécer; Avendaño-Vázquez, Aida-Odette; Montes, Ricardo A. Chávez; de Folter, Stefan; Andrés-Hernández, Liliana; Abreu-Goodger, Cei; Sawers, Ruairidh J. H.

    2015-01-01

    Purple acid phosphatases (PAPs) play an important role in plant phosphorus nutrition, both by liberating phosphorus from organic sources in the soil and by modulating distribution within the plant throughout growth and development. Furthermore, members of the PAP protein family have been implicated in a broader role in plant mineral homeostasis, stress responses and development. We have identified 33 candidate PAP encoding gene models in the maize (Zea mays ssp. mays var. B73) reference genome. The maize Pap family includes a clear single-copy ortholog of the Arabidopsis gene AtPAP26, shown previously to encode both major intracellular and secreted acid phosphatase activities. Certain groups of PAPs present in Arabidopsis, however, are absent in maize, while the maize family contains a number of expansions, including a distinct radiation not present in Arabidopsis. Analysis of RNA-sequencing based transcriptome data revealed accumulation of maize Pap transcripts in multiple plant tissues at multiple stages of development, and increased accumulation of specific transcripts under low phosphorus availability. These data suggest the maize PAP family as a whole to have broad significance throughout the plant life cycle, while highlighting potential functional specialization of individual family members. PMID:26042133

  7. The maize (Zea mays ssp. mays var. B73) genome encodes 33 members of the purple acid phosphatase family.

    PubMed

    González-Muñoz, Eliécer; Avendaño-Vázquez, Aida-Odette; Montes, Ricardo A Chávez; de Folter, Stefan; Andrés-Hernández, Liliana; Abreu-Goodger, Cei; Sawers, Ruairidh J H

    2015-01-01

    Purple acid phosphatases (PAPs) play an important role in plant phosphorus nutrition, both by liberating phosphorus from organic sources in the soil and by modulating distribution within the plant throughout growth and development. Furthermore, members of the PAP protein family have been implicated in a broader role in plant mineral homeostasis, stress responses and development. We have identified 33 candidate PAP encoding gene models in the maize (Zea mays ssp. mays var. B73) reference genome. The maize Pap family includes a clear single-copy ortholog of the Arabidopsis gene AtPAP26, shown previously to encode both major intracellular and secreted acid phosphatase activities. Certain groups of PAPs present in Arabidopsis, however, are absent in maize, while the maize family contains a number of expansions, including a distinct radiation not present in Arabidopsis. Analysis of RNA-sequencing based transcriptome data revealed accumulation of maize Pap transcripts in multiple plant tissues at multiple stages of development, and increased accumulation of specific transcripts under low phosphorus availability. These data suggest the maize PAP family as a whole to have broad significance throughout the plant life cycle, while highlighting potential functional specialization of individual family members. PMID:26042133

  8. A vaccine strategy with multiple prostatic acid phosphatase-fused cytokines for prostate cancer treatment

    PubMed Central

    FUJIO, KEI; WATANABE, MASAMI; UEKI, HIDEO; LI, SHUN-AI; KINOSHITA, RIE; OCHIAI, KAZUHIKO; FUTAMI, JUNICHIRO; WATANABE, TOYOHIKO; NASU, YASUTOMO; KUMON, HIROMI

    2015-01-01

    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 antigen

  9. Calcification in human osteoblasts cultured in medium conditioned by the prostatic cancer cell line PC-3 and prostatic acid phosphatase.

    PubMed

    Kimura, G; Sugisaki, Y; Masugi, Y; Nakazawa, N

    1992-01-01

    A medium that had been conditioned by PC-3 cells stimulated the calcification of a human osteoblastic cell line, Tak-10, in a nonmitogenic culture. The calcification of the osteoblasts was stimulated maximally at a 25% concentration of the conditioned medium. Calcification activity was markedly enhanced by the addition of both prostatic acid phosphatase (PAP) and its substrate, alpha-glycerophosphate, to the medium; however, PAP added alone did not enhance this activity. These results suggest that human prostatic carcinoma cells produce a factor that stimulates the calcification of the human osteoblasts. Results have also suggested that PAP is a requisite for osteogenesis provided that its substrates are abundant in the medium.

  10. Cloning and characterization of phosphorus starvation inducible Brassica napus PURPLE ACID PHOSPHATASE 12 gene family, and imprinting of a recently evolved MITE-minisatellite twin structure.

    PubMed

    Lu, Kun; Chai, You-Rong; Zhang, Kai; Wang, Rui; Chen, Li; Lei, Bo; Lu, Jun; Xu, Xin-Fu; Li, Jia-Na

    2008-10-01

    Purple acid phosphatase (PAP) is important for phosphorus assimilation and in planta redistribution. In this study, seven Brassica napus PAP12 (BnPAP12) genes orthologous to Arabidopsis thaliana PAP12 (AtPAP12) are isolated and characterized. NCBI BLASTs, multi-alignments, conserved domain prediction, and featured motif/residue characterization indicate that all BnPAP12 members encode dimeric high molecular weight plant PAPs. BnPAP12-1, BnPAP12-2, BnPAP12-3 and BnPAP12-7 (Group I) have six introns and encode 469-aa polypeptides structurally comparable to AtPAP12. BnPAP12-4 and BnPAP12-6 (Group II) have seven introns and encode 526-aa PAP12s. Encoding a 475-aa polypeptide, BnPAP12-5 (Group III) is evolved from a chimera of 5' part of Group I and 3' part of Group II. Sequence characterization and Southern detection suggest that there are about five BnPAP12 alleles. Homoeologous non-allelic fragment exchanges exist among BnPAP12 genes. BnPAP12-4 and BnPAP12-6 are imprinted with a Tourist-like miniature inverted-repeat transposable element (MITE) which is tightly associated with a novel minisatellite composed of four 36-bp tandem repeats. Existing solely in B. rapa/oleracea lineage, this recently evolved MITE-minisatellite twin structure does not impair transcription and coding capacity of the imprinted genes, and could be used to identify close relatives of B. rapa/oleracea lineage within Brassica. It is also useful for studying MITE activities especially possible involvement in minisatellite formation and gene structure evolution. BnPAP12-6 is silent in transcription. All other BnPAP12 genes basically imitate AtPAP12 in tissue specificity and Pi-starvation induced expression pattern, but divergence and complementation are distinct among them. Alternative polyadenylation and intron retention also exist in BnPAP12 mRNAs.

  11. Acid phosphatase/phosphotransferases from enteric bacteria.

    PubMed

    Mihara, Y; Utagawa, T; Yamada, H; Asano, Y

    2001-01-01

    We have investigated the enzymatic phosphorylation of nucleosides and found that Morganella morganii phoC acid phosphatase exhibits regioselective pyrophosphate (PP(i))-nucleoside phosphotransferase activity. In this study, we isolated genes encoding an acid phosphatase with regioselective phosphotransferase activity (AP/PTase) from Providencia stuartii, Enterobacter aerogenes, Escherichia blattae and Klebsiella planticola, and compared the primary structures and enzymatic characteristics of these enzymes with those of AP/PTase (PhoC acid phosphatase) from M. morganii. The enzymes were highly homologous in primary structure with M. morganii AP/PTase, and are classified as class A1 acid phosphatases. The synthesis of inosine-5'-monophosphate (5'-IMP) by E. coli overproducing each acid phosphatase was investigated. The P. stuartii enzyme, which is most closely related to the M. morganii enzyme, exhibited high 5'-IMP productivity, similar to the M. morganii enzyme. The 5'-IMP productivities of the E. aerogenes, E. blattae and K. planticola enzymes were inferior to those of the former two enzymes. This result underlines the importance of lower K(m) values for efficient nucleotide production. As these enzymes exhibited a very high degree of homology at the amino acid sequence level, it is likely that local sequence differences in the binding pocket are responsible for the differences in the nucleoside-PP(i) phosphotransferase reaction.

  12. Pap test

    MedlinePlus

    ... HSIL - Pap; Low-grade Pap; High-grade Pap; Carcinoma in situ - Pap; CIS - Pap; ASCUS - Pap; Atypical ... risk of cervical cancer is greater with HSIL CARCINOMA IN SITU (CIS): This result most often means ...

  13. The active site of purple acid phosphatase from sweet potatoes (Ipomoea batatas) metal content and spectroscopic characterization.

    PubMed

    Durmus, A; Eicken, C; Sift, B H; Kratel, A; Kappl, R; Hüttermann, J; Krebs, B

    1999-03-01

    Purple acid phosphatase from sweet potatoes Ipomoea batatas (spPAP) has been purified to homogeneity and characterized using spectroscopic investigations. Matrix-assisted laser desorption/ionization mass spectrometry analysis revealed a molecular mass of approximately 112 kDa. The metal content was determined by X-ray fluorescence using synchrotron radiation. In contrast to previous studies it is shown that spPAP contains a Fe(III)-Zn(II) center in the active site as previously determined for the purple acid phosphatase from red kidney bean (kbPAP). Moreover, an alignment of the amino acid sequences suggests that the residues involved in metal-binding are identical in both plant PAPs. Tyrosine functions as one of the ligands for the chromophoric Fe(III). Low temperature EPR spectra of spPAP show a signal near g = 4.3, characteristic for high-spin Fe(III) in a rhombic environment. The Tyr-Fe(III) charge transfer transition and the EPR signal are both very sensitive to changes in pH. The pH dependency strongly suggests the presence of an ionizable group with a pKa of 4.7, arising from an aquo ligand coordinated to Fe(III). EPR and UV/visible studies of spPAP in the presence of the inhibitors phosphate or arsenate suggest that both anions bind to Fe(III) in the binuclear center replacing the coordinated water or hydroxide ligand necessary for hydrolysis. The conserved histidine residues of spPAP corresponding to His202 and His296 in kbPAP probably interact in catalysis. PMID:10102999

  14. The active site of purple acid phosphatase from sweet potatoes (Ipomoea batatas) metal content and spectroscopic characterization.

    PubMed

    Durmus, A; Eicken, C; Sift, B H; Kratel, A; Kappl, R; Hüttermann, J; Krebs, B

    1999-03-01

    Purple acid phosphatase from sweet potatoes Ipomoea batatas (spPAP) has been purified to homogeneity and characterized using spectroscopic investigations. Matrix-assisted laser desorption/ionization mass spectrometry analysis revealed a molecular mass of approximately 112 kDa. The metal content was determined by X-ray fluorescence using synchrotron radiation. In contrast to previous studies it is shown that spPAP contains a Fe(III)-Zn(II) center in the active site as previously determined for the purple acid phosphatase from red kidney bean (kbPAP). Moreover, an alignment of the amino acid sequences suggests that the residues involved in metal-binding are identical in both plant PAPs. Tyrosine functions as one of the ligands for the chromophoric Fe(III). Low temperature EPR spectra of spPAP show a signal near g = 4.3, characteristic for high-spin Fe(III) in a rhombic environment. The Tyr-Fe(III) charge transfer transition and the EPR signal are both very sensitive to changes in pH. The pH dependency strongly suggests the presence of an ionizable group with a pKa of 4.7, arising from an aquo ligand coordinated to Fe(III). EPR and UV/visible studies of spPAP in the presence of the inhibitors phosphate or arsenate suggest that both anions bind to Fe(III) in the binuclear center replacing the coordinated water or hydroxide ligand necessary for hydrolysis. The conserved histidine residues of spPAP corresponding to His202 and His296 in kbPAP probably interact in catalysis.

  15. Acid phosphatase production by recombinant Arxula adeninivorans.

    PubMed

    Minocha, Neha; Kaur, Parvinder; Satyanarayana, T; Kunze, G

    2007-08-01

    Acid phosphatase production by recombinant Arxula adeninivorans was carried out in submerged fermentation. Using the Plackett-Burman design, three fermentation variables (pH, sucrose concentration, and peptone concentration) were identified to significantly affect acid phosphatase and biomass production, and these were optimized using response surface methodology of central composite design. The highest enzyme yields were attained in the medium with 3.9% sucrose and 1.6% peptone at pH 3.8. Because of optimization, 3.86- and 4.19-fold enhancement in enzyme production was achieved in shake flasks (17,054 U g(-1) DYB) and laboratory fermenter (18,465 U g(-1) DYB), respectively. PMID:17541580

  16. Acid Phosphatase Development during Ripening of Avocado.

    PubMed

    Sacher, J A

    1975-02-01

    The activity and subcellular distribution of acid phosphatase were assayed during ethylene-induced ripening of whole fruit or thick slices of avocado (Persea americana Mill. var. Fuerte and Hass). The activity increased up to 30-fold during ripening in both the supernatant fraction and the Triton X-100 extract of the precipitate of a 30,000g centrifugation of tissue homogenates from whole fruit or slices ripening in moist air. Enzyme activity in the residual precipitate after Triton extraction remained constant. The development of acid phosphatase in thick slices ripened in moist air was similar to that in intact fruit, except that enzyme development and ripening were accelerated about 24 hours in the slices. The increase in enzyme activity that occurs in slices ripening in moist air was inhibited when tissue sections were infiltrated with solutions, by aspiration for 2 minutes or by soaking for 2 hours, anytime 22 hours or more after addition of ethylene. This inhibition was independent of the presence or absence of cycloheximide or sucrose (0.3-0.5m). However, the large decline in enzyme activity in the presence of cycloheximide, as compared with the controls, indicated that synthesis of acid phosphatase was occurring at all stages of ripening.

  17. Potential Role for Purple Acid Phosphatase in the Dephosphorylation of Wall Proteins in Tobacco Cells1[W

    PubMed Central

    Kaida, Rumi; Serada, Satoshi; Norioka, Naoko; Norioka, Shigemi; Neumetzler, Lutz; Pauly, Markus; Sampedro, Javier; Zarra, Ignacio; Hayashi, Takahisa; Kaneko, Takako S.

    2010-01-01

    It is not yet known whether dephosphorylation of proteins catalyzed by phosphatases occurs in the apoplastic space. In this study, we found that tobacco (Nicotiana tabacum) purple acid phosphatase could dephosphorylate the phosphoryl residues of three apoplastic proteins, two of which were identified as α-xylosidase and β-glucosidase. The dephosphorylation and phosphorylation of recombinant α-xylosidase resulted in a decrease and an increase in its activity, respectively, when xyloglucan heptasaccharide was used as a substrate. Attempted overexpression of the tobacco purple acid phosphatase NtPAP12 in tobacco cells not only decreased the activity levels of the glycosidases but also increased levels of xyloglucan oligosaccharides and cello-oligosaccharides in the apoplast during the exponential phase. We suggest that purple acid phosphatase controls the activity of α-xylosidase and β-glucosidase, which are responsible for the degradation of xyloglucan oligosaccharides and cello-oligosaccharides in the cell walls. PMID:20357138

  18. Screening and Characterization of a Novel RNA Aptamer That Specifically Binds to Human Prostatic Acid Phosphatase and Human Prostate Cancer Cells

    PubMed Central

    Kong, Hoon Young; Byun, Jonghoe

    2015-01-01

    Prostatic acid phosphatase (PAP) expression increases proportionally with prostate cancer progression, making it useful in prognosticating intermediate to high-risk prostate cancers. A novel ligand that can specifically bind to PAP would be very helpful for guiding prostate cancer therapy. RNA aptamers bind to target molecules with high specificity and have key advantages such as low immunogenicity and easy synthesis. Here, human PAP-specific aptamers were screened from a 2′-fluoropyrimidine (FY)-modified RNA library by SELEX. The candidate aptamer families were identified within six rounds followed by analysis of their sequences and PAP-specific binding. A gel shift assay was used to identify PAP binding aptamers and the 6N aptamer specifically bound to PAP with a Kd value of 118 nM. RT-PCR and fluorescence labeling analyses revealed that the 6N aptamer bound to PAP-positive mammalian cells, such as PC-3 and LNCaP. IMR-90 negative control cells did not bind the 6N aptamer. Systematic minimization analyses revealed that 50 nucleotide sequences and their two hairpin structures in the 6N 2′-FY RNA aptamer were equally important for PAP binding. Renewed interest in PAP combined with the versatility of RNA aptamers, including conjugation of anti-cancer drugs and nano-imaging probes, could open up a new route for early theragnosis of prostate cancer. PMID:25591398

  19. Stabilization of human prostatic acid phosphatase by coupling with chondroitin sulfate.

    PubMed

    Luchter-Wasylewska, E; Dulińska, J; Ostrowski, W S; Torchilin, V P; Trubetskoy, V S

    1991-02-01

    Human prostatic acid phosphatase (PAP) (EC 3.1.3.2) was covalently linked to chondroitin sulfate A from whale cartilage. In order to bind the protein amino groups with the preactivated carboxyl groups of chondroitin sulfate, 1-ethyl-3-(3'-dimethylaminepropyl)carbodiimide and N-hydroxysulfosuccinimide were used as coupling agents. The product was soluble and enzymatically active. The activity was on average 25% higher than that of the free enzyme. The product was heterogeneous in respect to charge and Mr (50-1500) kDa, as determined by chromatography on Sephacryl S 300 and polyacrylamide gel electrophoresis. The resulting polymers contained covalently bound chondroitin sulfate, as shown by the biotin-avidin test. The modified enzyme is more resistant against various denaturing agents, e.g., urea, ethanol, and heat. Thus covalent modification of PAP by cross-linking to chondroitin sulfate could be the preferred method for stabilization of its biological activity.

  20. Digestion and the distribution of acid phosphatase in Blepharisma.

    PubMed

    Dembitzer, H M

    1968-05-01

    Suspensions of Blepharisma intermedium were fed latex particles for 5 min and then were separated from the particles by filtration. Samples were fixed at intervals after separation and incubated to demonstrate acid phosphatase activity. They were subsequently embedded and sectioned for electron microscopy. During formation of the food vacuole, the vacuolar membrane is acid phosphatase-negative. Within 5 min, dumbbell-shaped acid phosphatase-positive bodies, possibly derived from the the acid phosphatase-positive Golgi apparatus, apparently fuse with the food vacuole and render it acid phosphatase-positive. A larger type of acid phosphatase-positive, vacuolated body may also fuse with the food vacuole at later stages. At about 20 min after formation, acid phosphatase-positive secondary pinocytotic vesicles pinch off from the food vacuoles and approach a separate system of membrane-bounded spaces. By 1 hr after formation, the food vacuole becomes acid phosphatase-negative, and the undigested latex particles are voided into the membrane-bounded spaces. The membrane-bounded spaces are closely associated with the food vacuole at all stages of digestion and are generally acid phosphatase-negative. Within the membrane-bounded spaces, dense, pleomorphic, granular bodies are found, in which are embedded mitochondria, paraglycogen granules, membrane-limited acid phosphatase-containing structures, and Golgi apparatuses. The granular bodies may serve as vehicles for the transport of organelles through the extensive, ramifying membrane-bounded spaces.

  1. DIGESTION AND THE DISTRIBUTION OF ACID PHOSPHATASE IN BLEPHARISMA

    PubMed Central

    Dembitzer, Herbert M.

    1968-01-01

    Suspensions of Blepharisma intermedium were fed latex particles for 5 min and then were separated from the particles by filtration. Samples were fixed at intervals after separation and incubated to demonstrate acid phosphatase activity. They were subsequently embedded and sectioned for electron microscopy. During formation of the food vacuole, the vacuolar membrane is acid phosphatase-negative. Within 5 min, dumbbell-shaped acid phosphatase-positive bodies, possibly derived from the the acid phosphatase-positive Golgi apparatus, apparently fuse with the food vacuole and render it acid phosphatase-positive. A larger type of acid phosphatase-positive, vacuolated body may also fuse with the food vacuole at later stages. At about 20 min after formation, acid phosphatase-positive secondary pinocytotic vesicles pinch off from the food vacuoles and approach a separate system of membrane-bounded spaces. By 1 hr after formation, the food vacuole becomes acid phosphatase-negative, and the undigested latex particles are voided into the membrane-bounded spaces. The membrane-bounded spaces are closely associated with the food vacuole at all stages of digestion and are generally acid phosphatase-negative. Within the membrane-bounded spaces, dense, pleomorphic, granular bodies are found, in which are embedded mitochondria, paraglycogen granules, membrane-limited acid phosphatase-containing structures, and Golgi apparatuses. The granular bodies may serve as vehicles for the transport of organelles through the extensive, ramifying membrane-bounded spaces. PMID:4968524

  2. Phosphatidic acid phosphatase and diacylglycerol acyltransferase: potential targets for metabolic engineering of microorganism oil.

    PubMed

    Jin, Hong-Hao; Jiang, Jian-Guo

    2015-04-01

    Oleaginous microorganism is becoming one of the most promising oil feedstocks for biodiesel production due to its great advantages in triglyceride (TAG) accumulation. Previous studies have shown that de novo TAG biosynthesis can be divided into two parts: the fatty acid biosynthesis pathway (the upstream part which generates acyl-CoAs) and the glycerol-3-phosphate acylation pathway (the downstream part in which three acyl groups are sequentially added onto a glycerol backbone). This review mainly focuses on two enzymes in the G3P pathway, phosphatidic acid phosphatase (PAP) and diacylglycerol acyltransferase (DGAT). The former catalyzes a dephosphorylation reaction, and the latter catalyzes a subsequent acylation reaction. Genes, functional motifs, transmembrane domains, action mechanism, and new studies of the two enzymes are discussed in detail. Furthermore, this review also covers diacylglycerol kinase, an enzyme that catalyzes the reverse reaction of diacylglycerol formation. In addition, PAP and DGAT are the conjunction points of the G3P pathway, the Kennedy pathway, and the CDP-diacylglycerol pathway (CDP-DAG pathway), and the mutual transformation between TAGs and phospholipids is discussed as well. Given that both the Kennedy and CDP-diacylglycerol pathways are in metabolic interlock (MI) with the G3P pathway, it is suggested that, via metabolic engineering, TAG accumulation can be improved by the two pathways based on the pivotal function of PAP and DGAT.

  3. Unique structural features of red kidney bean purple acid phosphatase.

    PubMed

    Cashikar, A G; Rao, M N

    1995-06-01

    Purple acid phosphatase from red kidney beans (Phaseolus vulgaris) has been purified to homogeneity and characterized. The enzyme is a homodimer of 60 kDa subunits each containing one atom of zinc and iron in the active site. Circular dichroism spectral studies on the purified enzyme reveals that a large portion of the peptide backbone is in the unordered and beta-turn conformation. A unique feature of the red kidney bean acid phosphatase, which we have found, is that one of the two cysteines of each subunit is involved in the formation of an inter-subunit disulphide. The thiol group of the other cysteine is not necessary for the activity of the enzyme. Western blot analysis with antibodies raised against kidney bean acid phosphatase could not recognize acid phosphatases from other sources except from potato. This paper emphasizes the fact that acid phosphatases are functionally, but not structurally, conserved enzymes. PMID:7590853

  4. Acid phosphatase and protease activities in immobilized rat skeletal muscles

    NASA Technical Reports Server (NTRS)

    Witzmann, F. A.; Troup, J. P.; Fitts, R. H.

    1982-01-01

    The effect of hind-limb immobilization on selected Iysosomal enzyme activities was studied in rat hing-limb muscles composed primarily of type 1. 2A, or 2B fibers. Following immobilization, acid protease and acid phosphatase both exhibited signifcant increases in their activity per unit weight in all three fiber types. Acid phosphatase activity increased at day 14 of immobilization in the three muscles and returned to control levels by day 21. Acid protease activity also changed biphasically, displaying a higher and earlier rise than acid phosphatase. The pattern of change in acid protease, but not acid phosphatase, closely parallels observed muscle wasting. The present data therefore demonstrate enhanced proteolytic capacity of all three fiber types early during muscular atrophy. In addition, the data suggest a dependence of basal hydrolytic and proteolytic activities and their adaptive response to immobilization on muscle fiber composition.

  5. Structure-function relationships of purple acid phosphatase from red kidney beans based on heterologously expressed mutants.

    PubMed

    Truong, Ngoc Thanh; Naseri, Joseph Itor; Vogel, Andreas; Rompel, Annette; Krebs, B

    2005-08-01

    Purple acid phosphatases are binuclear metalloenzymes, which catalyze the conversion of orthophosphoric monoesters to alcohol and orthophosphate. The enzyme from red kidney beans is characterized with a Fe(III)-Zn(II) active center. So far, the reaction mechanisms postulated for PAPs assume the essentiality of two amino acids, residing near the bimetallic active site. Based on the amino acid sequence of kidney bean PAP (kbPAP), residues H296 and H202 are believed to be essential for catalytic function of the enzyme. In the present study, the role of residue H202 has been elucidated. Mutants H202A and H202R were prepared by site-directed mutagenesis and expressed in baculovirus-infected insect cells. Based on kinetic studies, residue H202 is assumed to play a role in stabilizing the transition state, particularly in charge compensation, steric positioning of the substrate, and facilitating the release of the product by protonating the substrate leaving groups. The study confirmed the essentiality and elucidates the functional role of H202 in the catalytic mechanism of kbPAP.

  6. Structure-function relationships of purple acid phosphatase from red kidney beans based on heterologously expressed mutants.

    PubMed

    Truong, Ngoc Thanh; Naseri, Joseph Itor; Vogel, Andreas; Rompel, Annette; Krebs, B

    2005-08-01

    Purple acid phosphatases are binuclear metalloenzymes, which catalyze the conversion of orthophosphoric monoesters to alcohol and orthophosphate. The enzyme from red kidney beans is characterized with a Fe(III)-Zn(II) active center. So far, the reaction mechanisms postulated for PAPs assume the essentiality of two amino acids, residing near the bimetallic active site. Based on the amino acid sequence of kidney bean PAP (kbPAP), residues H296 and H202 are believed to be essential for catalytic function of the enzyme. In the present study, the role of residue H202 has been elucidated. Mutants H202A and H202R were prepared by site-directed mutagenesis and expressed in baculovirus-infected insect cells. Based on kinetic studies, residue H202 is assumed to play a role in stabilizing the transition state, particularly in charge compensation, steric positioning of the substrate, and facilitating the release of the product by protonating the substrate leaving groups. The study confirmed the essentiality and elucidates the functional role of H202 in the catalytic mechanism of kbPAP. PMID:16009331

  7. Elevation of serum acid phosphatase in cancers with bone metastasis

    SciTech Connect

    Tavassoli, M.; Rizo, M.; Yam, L.T.

    1980-05-01

    In patients with nonprostatic cancer, serum acid phosphatase activity is usually elevated when bone metastasis is present but not when bone metastasis is absent. The fraction responsible for serum enzyme elevation is a normal component of serum; it appears in gel electrophoresis as band 5; and is tartrate-resistant. It is suggested that the origin of acid phosphatase elevation is bone osteoclasts rather than cancer tissue, as is the case with prostatic carcinoma. Determination of serum acid phosphatase activity may be useful in the detection of bone metastasis.

  8. Overexpression of a phosphatidic acid phosphatase type 2 leads to an increase in triacylglycerol production in oleaginous Rhodococcus strains.

    PubMed

    Hernández, Martín A; Comba, Santiago; Arabolaza, Ana; Gramajo, Hugo; Alvarez, Héctor M

    2015-03-01

    Oleaginous Rhodococcus strains are able to accumulate large amounts of triacylglycerol (TAG). Phosphatidic acid phosphatase (PAP) enzyme catalyzes the dephosphorylation of phosphatidic acid (PA) to yield diacylglycerol (DAG), a key precursor for TAG biosynthesis. Studies to establish its role in lipid metabolism have been mainly focused in eukaryotes but not in bacteria. In this work, we identified and characterized a putative PAP type 2 (PAP2) encoded by the ro00075 gene in Rhodococcus jostii RHA1. Heterologous expression of ro00075 in Escherichia coli resulted in a fourfold increase in PAP activity and twofold in DAG content. The conditional deletion of ro00075 in RHA1 led to a decrease in the content of DAG and TAG, whereas its overexpression in both RHA1 and Rhodococcus opacus PD630 promoted an increase up to 10 to 15 % by cellular dry weight in TAG content. On the other hand, expression of ro00075 in the non-oleaginous strain Rhodococcus fascians F7 promoted an increase in total fatty acid content up to 7 % at the expense of free fatty acid (FFA), DAG, and TAG fractions. Moreover, co-expression of ro00075/atf2 genes resulted in a fourfold increase in total fatty acid content by a further increase of the FFA and TAG fractions. The results of this study suggest that ro00075 encodes for a PAP2 enzyme actively involved in TAG biosynthesis. Overexpression of this gene, as single one or with an atf gene, provides an alternative approach to increase the biosynthesis and accumulation of bacterial oils as a potential source of raw material for biofuel production.

  9. Multiple forms of acid phosphatase activity in Gaucher's disease.

    PubMed

    Chambers, J P; Peters, S P; Glew, R H; Lee, R E; McCafferty, L R; Mercer, D W; Wenger, D A

    1978-07-01

    Although the primary genetic defect in all individuals with Gaucher's disease is a deficiency in glucocerebrosidase activity, the finding of marked elevations in splenic and serum acid phosphatase activity is almost as consistent a finding. Gaucher spleen and serum contain at least two forms of acid phosphatase that can be readily separated by chromatography on columns containing the cation exchange resin Sulphopropyl Sephadex. The major species of acid phosphatase (designated SP-I) contained in Triton X-100 (1% v/v) extracts of Gaucher spleen accounts for 65%--95% of the total activity and has the following properties: (1) it does not bind to the cation exchange column; (2) it exhibitis a pH optimum of 4.5--5.0; (3) it is inhibited by sodium fluoride (15 mM), L(+)-tartaric acid (20 mM), and beta-mercaptoethanol (2.1 M), and (4) it is resistant to inhibition by sodium dithionite (10 mM). The minor acid phosphatase activity (designated SP-II) present in extracts of Gaucher spleen has properties similar to those of the major species of acid phosphatase activity contained in serum from patients with Gaucher's disease: (1) it binds firmly to cation exchange columns (eluted by 0.5 M sodium chloride); (2) it exhibits a pH optimum of 5.0--6.0; (3) it is inhibited by sodium fluoride and sodium dithionite; and (4) it is resistant to inhibition by beta-mercaptoethanol (2.1 M) and L(+)-tartaric acid (20 mM). In addition, a second form of acid phosphatase that is tartrate resistant was found to be elevated in Gaucher serum. This form of serum acid phosphatase did not bind to Sulphopropyl Sephadex, was found to be significantly resistant to beta-mercaptoethanol (2.1 M), and was only partially inhibited by sodium dithionite (10 mM). The findings reported here indicate that at least three distinct forms of acid phosphatase activity are elevated in Gaucher's disease. Furthermore, the minor acid phosphatase activity contained in spleen homogenates has properties very similar to

  10. Structural and kinetic properties of a novel purple acid phosphatase from phosphate-starved tomato (Lycopersicon esculentum) cell cultures.

    PubMed Central

    Bozzo, Gale G; Raghothama, Kashchandra G; Plaxton, William C

    2004-01-01

    An intracellular acid phosphatase (IAP) from P(i)-starved (-P(i)) tomato ( Lycopersicon esculentum ) suspension cells has been purified to homogeneity. IAP is a purple acid phosphatase (PAP), as the purified protein was violet in colour (lambda(max)=546 nm) and was insensitive to L-tartrate. PAGE, periodic acid-Schiff staining and peptide mapping demonstrated that the enzyme exists as a 142 kDa heterodimer composed of an equivalent ratio of glycosylated and structurally dissimilar 63 (alpha-subunit) and 57 kDa (beta-subunit) polypeptides. However, the nine N-terminal amino acids of the alpha- and beta-subunits were identical, exhibiting similarity to the deduced N-terminal portions of several putative plant PAPs. Quantification of immunoblots probed with rabbit anti-(tomato acid phosphatase) immune serum revealed that the 4-fold increase in IAP activity due to P(i)-deprivation was correlated with similar increases in the amount of antigenic IAP alpha- and beta-subunits. IAP displayed optimal activity at pH 5.1, was activated 150% by 10 mM Mg(2+), but was potently inhibited by Zn(2+), Cu(2+), Fe(3+), molybdate, vanadate, fluoride and P(i). Although IAP demonstrated broad substrate selectivity, its specificity constant ( V (max)/ K (m)) with phosphoenolpyruvate was >250% greater than that obtained with any other substrate. IAP exhibited significant peroxidase activity, which was optimal at pH 9.0 and insensitive to Mg(2+) or molybdate. This IAP is proposed to scavenge P(i) from intracellular phosphate esters in -P(i) tomato. A possible secondary IAP role in the metabolism of reactive oxygen species is discussed. IAP properties are compared with those of two extracellular PAP isoenzymes that are secreted into the medium of -P(i) tomato cells [Bozzo, Raghothama and Plaxton (2002) Eur. J. Biochem. 269, 6278-6286]. PMID:14521509

  11. Prostate-specific acid phosphatase versus acid phosphatase in monitoring patients with prostate cancer.

    PubMed

    Killian, C S; Vargas, F P; Slack, N H; Murphy, G P; Chu, T M

    1982-01-01

    Serial levels of PAP and AcP activity were compared for their relative values in monitoring 57 early and 33 advanced prostate cancer patients. Several findings regarding the patients' disease status and the enzyme levels have been observed that may be beneficial to therapeutic management of these patients. They are: [1] an elevated PAP activity in disease recurrence and disease progression generally precedes an elevated AcP activity, and thus represents a more sensitive index for patients with early and advanced disease; [2] serial mean levels of PAP activity greater than the mean + 3 SD are more predictive for disease recurrence and progression than are those of AcP activity in both groups of patients; [3] PAP activity is a more sensitive monitor for changes in objective treatment response than is AcP activity; and [4] PAP is more specific than AcP for prostate, thus offering a more reliable marker to identify metastasis of unknown origin, or to confirm metastasis derived from a primary prostate tumor that may have been suggested by other non-prostate-specific marker[s]. In addition, data suggest a favorable prognosis for patients receiving therapy as inferred by a serial mean of PAP activity that is less than mean + 3 SD. PMID:6953924

  12. A cysteine-sulfinic acid in peroxiredoxin regulates H2O2-sensing by the antioxidant Pap1 pathway

    PubMed Central

    Vivancos, Ana P.; Castillo, Esther A.; Biteau, Benoît; Nicot, Carine; Ayté, José; Toledano, Michel B.; Hidalgo, Elena

    2005-01-01

    The Schizosaccharomyces pombe transcription factor Pap1 regulates antioxidant-gene transcription in response to H2O2. Pap1 activation occurs only at low, but not elevated, H2O2 concentrations that instead strongly trigger the mitogen-activated protein kinase Sty1 pathway. Here, we identify the peroxiredoxin Tpx1 as the upstream activator of Pap1. We show that, at low H2O2 concentrations, this oxidant scavenger can transfer a redox signal to Pap1, whereas higher concentrations of the oxidant inhibit the Tpx1-Pap1 redox relay through the temporal inactivation of Tpx1 by oxidation of its catalytic cysteine to a sulfinic acid. This cysteine modification can be reversed by the sulfiredoxin Srx1, its expression in response to high doses of H2O2 strictly depending on active Sty1. Thus, Tpx1 oxidation to the cysteine-sulfinic acid and its reversion by Srx1 constitutes a previously uncharacterized redox switch in H2O2 signaling, restricting Pap1 activation within a narrow range of H2O2 concentrations. PMID:15956211

  13. Comparing the results of Pap smear and Direct Visual Inspection (DVI) with 5% acetic acid in cervical cancer screening

    PubMed Central

    Pourasad-Shahrak, Shakiba; Salehi-Pourmehr, Hanieh; Mostafa-Garebaghi, Parvin; Asghari-Jafarabadi, Mohammad; Malakouti, Jamileh; Haghsay, Monireh

    2015-01-01

    Background: Cervical cancer is the most second common cancer among Iranian women. This study was carried out to compare the results of Pap smear method and Direct Visual Inspection (DVI) with 5% acetic acid in cervical cancer screening in Tabriz, Iran. Material and Methods: This cross-sectional study was carried out in Alzahra Therapeutic-Educational Centre, Tabriz, Iran in 2013 on 1000 women. First, Pap smear was done for all women, and then the cervix exposed with 5% acetic acid by cotton swab for 30 seconds and observed under adequate light. At the end, women with abnormal results in Pap smear or DVI method were referred to colposcopy and biopsy. Test's sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), LR+, LR- and confidence interval (CI) were determined (P < 0.05). Results: Nine-hundred and seventy-four (94.7%) cases were normal and had no abnormal findings and 26 (2.6%) participants had positive results in Pap smear or DVI test. Twelve women had abnormal Pap smear (nine women with atypical squamous cells of undetermined significance, ASCUS, three women with dysplasia, atypical endocervical, and low-grade squamous intraepithelial lesion, LSIL results) and 14 women had positive DVI (four women with human papillomavirus, HPV or koilocyte,) and one women with abnormality in both method had carcinoma in biopsy that referred to oncologist. In this study the sensitivity, specificity, PPV and NPV for DVI were 71.4%, 50%, 35.7%, and 81.8% respectively in comparison with 14.3%, 50%, 10%, and 60% for Pap smear. Conclusion: As the DVI method has higher sensitivity and positive predictive value than Pap smear, it could be used as a useful method beside the Pap smear. PMID:25657491

  14. Measuring phosphatidic acid phosphatase (EC 3.1.3.4) activity using two phosphomolybdate-based colorimetric methods

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phosphatidate phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4), which is also known as PAP, catalyzes the dephosphorylation of phosphatidate (PtdOH) to form diacylglycerol (DAG) and inorganic phosphate. In eukaryotes, PAP driven reaction is the committed step in the synthesis of triacyl...

  15. Ultrastructural localization of acid phosphatase in nonhuman primate vaginal epithelium.

    PubMed

    King, B F

    1985-01-01

    The vagina of the rhesus monkey is lined by a stratified squamous epithelium. However, little is known regarding the cytochemical composition of its cell organelles and the substances found in the intercellular spaces. In this study we have examined the ultrastructural distribution of acid phosphatase in the vaginal epithelium. In basal and parabasal cells reaction product was found in some Golgi cisternae and vesicles and in a variety of cytoplasmic granules. Reaction product was also found in some, but not all, membrane-coating granules. In the upper layers of the epithelium, the membrane-coating granules extruded their contents and acid phosphatase was localized in the intercellular spaces. The possible roles of acid phosphatase in keratinization, desquamation, or modification of substances in the intercellular compartment are discussed.

  16. Biocatalysis with Sol-Gel Encapsulated Acid Phosphatase

    ERIC Educational Resources Information Center

    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…

  17. Nucleotide and amino acid sequences of human intestinal alkaline phosphatase: close homology to placental alkaline phosphatase

    SciTech Connect

    Henthorn, P.S.; Raducha, M.; Edwards, Y.H.; Weiss, M.J.; Slaughter, C.; Lafferty, M.A.; Harris, H.

    1987-03-01

    A cDNA clone for human adult intestinal alkaline phosphatase (ALP) (orthophosphoric-monoester phosphohydrolase (alkaline optimum); EC 3.1.3.1) was isolated from a lambdagt11 expression library. The cDNA insert of this clone is 2513 base pairs in length and contains an open reading frame that encodes a 528-amino acid polypeptide. This deduced polypeptide contains the first 40 amino acids of human intestinal ALP, as determined by direct protein sequencing. Intestinal ALP shows 86.5% amino acid identity to placental (type 1) ALP and 56.6% amino acid identity to liver/bone/kidney ALP. In the 3'-untranslated regions, intestinal and placental ALP cDNAs are 73.5% identical (excluding gaps). The evolution of this multigene enzyme family is discussed.

  18. Spatial structure of heptapeptide Glu-Ile-Leu-Asn-His-Met-Lys, a fragment of the HIV enhancer prostatic acid phosphatase, in aqueous and SDS micelle solutions

    NASA Astrophysics Data System (ADS)

    Bloсhin, Dmitri S.; Aganova, Oksana V.; Yulmetov, Aidar R.; Filippov, Andrei V.; Gizatullin, Bulat I.; Afonin, Sergii; Antzutkin, Oleg N.; Klochkov, Vladimir V.

    2013-02-01

    Prostatic acid phosphatase (PAP) is a protein abundantly present in human seminal fluid. PAP plays important role in fertilization. Its 39-amino-acid fragment, PAP(248-286), is effective in enhancing infectivity of HIV virus. In this work, we determined the spatial structure in aqueous solution of a heptapeptide within the PAP fragment, containing amino acid residues 266-272 (Glu-Ile-Leu-Asn-His-Met-Lys). We also report the structure of the complex formed by this heptapeptide with sodium dodecyl sulfate micelles, a model of a biological membrane, as determined by 1H NMR spectroscopy and 2D NMR (TOCSY, HSQC-HECADE, NOESY) spectroscopy. Complex formation was confirmed by chemical shift alterations in the 1H NMR spectra of the heptapeptide, as well as by the signs and values of NOE effects. We also present a comparison of the spatial structure of Glu-Ile-Leu-Asn-His-Met-Lys in water and in complex with sodium dodecyl sulfate.

  19. Atomistic details of the Catalytic Mechanism of Fe(III)-Zn(II) Purple Acid Phosphatase.

    PubMed

    Alberto, Marta E; Marino, Tiziana; Ramos, Maria J; Russo, Nino

    2010-08-10

    In the present work, we performed a theoretical investigation of the reaction mechanism of the Fe(III)-Zn(II) purple acid phosphatase from red kidney beans (rkbPAP), using the hybrid density functional theory and employing different exchange-correlation potentials. Characterization of the transition states and intermediates involved and the potential energy profiles for the reaction in different environments (gas phase, protein environment, and water) are reported. Our results show that the Fe(III)-Zn(II)PAP catalyzes the hydrolysis of methylphosphate via direct attack by a bridging metals-coordinated hydroxide leading to the cleavage of the ester bond. From our study emerges that the rate-limiting step of the reaction is the nucleophilic attack followed by the less energetically demanding release of the leaving group. Furthermore, we provide insights into some important points of contention concerning the precatalytic complex and the substrate coordination mode into the active site prior to hydrolysis. In particular: (i) Two models of enzyme-substrate with different orientations of the substrate into the active site were tested to evaluate the possible roles played by the conserved histidine residues (His 202 and His 296); (ii) Different protonation states of the substrate were taken into account in order to reproduce different pH values and to verify its influence on the catalytic efficiency and on the substrate binding mode; (iii) The metals role in each step of the catalytic mechanism was elucidated. We were also able to ascertain that the activation of the leaving group by the protonated His 296 is decisive to reach an optimal catalytic efficiency, while the bond scission without activation requires higher energy to occur. PMID:26613496

  20. Crystallization of recombinant Haemophilus influenzaee (P4) acid phosphatase

    SciTech Connect

    Ou, Zhonghui; Felts, Richard L.; Reilly, Thomas J.; Nix, Jay C.; Tanner, John J.

    2006-05-01

    Lipoprotein e (P4) is a class C acid phosphatase and a potential vaccine candidate for nontypeable H. influenzae infections. This paper reports the crystallization of recombinant e (P4) and the acquisition of a 1.7 Å resolution native X-ray diffraction data set. Haemophilus influenzae infects the upper respiratory tract of humans and can cause infections of the middle ear, sinuses and bronchi. The virulence of the pathogen is thought to involve a group of surface-localized macromolecular components that mediate interactions at the host–pathogen interface. One of these components is lipoprotein e (P4), which is a class C acid phosphatase and a potential vaccine candidate for nontypeable H. influenzae infections. This paper reports the crystallization of recombinant e (P4) and the acquisition of a 1.7 Å resolution native X-ray diffraction data set. The space group is P4{sub 2}2{sub 1}2, with unit-cell parameters a = 65.6, c = 101.4 Å, one protein molecule per asymmetric unit and 37% solvent content. This is the first report of the crystallization of a class C acid phosphatase.

  1. Cytochemical characterization of yolk granule acid phosphatase during early development of the oyster Crassostrea gigas (Thunberg)

    NASA Astrophysics Data System (ADS)

    Wang, Yiyan; Sun, Hushan; Wang, Yanjie; Yan, Dongchun; Wang, Lei

    2015-03-01

    In this study, a cytochemical method and transmission electron microscopy was used to examine acid phosphatase activities of yolk granules throughout the early developmental stages of the Pacific oyster Crassostrea gigas. This study aimed to investigate the dynamic change of yolk granule acid phosphatase, and the mechanisms underlying its involvement in yolk degradation during the early developmental stages of molluscs. Three types of yolk granules (YGI, YGII, and YGIII) that differed in electron density and acid phosphatase reaction were identified in early cleavage, morula, blastula, gastrula, trochophore, and veliger stages. The morphological heterogeneities of the yolk granules were related to acid phosphatase activity and degrees of yolk degradation, indicating the association of acid phosphatase with yolk degradation in embryos and larvae of molluscs. Fusion of yolk granules was observed during embryogenesis and larval development of C. gigas. The fusion of YGI (free of acid phosphatase reaction) with YGII (rich in acid phosphatase reaction) could be the way by which yolk degradation is triggered.

  2. Enhancement of Chlorogenic Acid Production in Hairy Roots of Platycodon grandiflorum by Over-Expression of An Arabidopsis thaliana Transcription Factor AtPAP1

    PubMed Central

    Tuan, Pham Anh; Kwon, Do Yeon; Lee, Sanghyun; Arasu, Mariadhas Valan; Al-Dhabi, Naif Abdullah; Park, Nam Il; Park, Sang Un

    2014-01-01

    To improve the production of chlorogenic acid (CGA) in hairy roots of Platycodon grandiflorum, we induced over-expression of Arabidopsis thaliana transcription factor production of anthocyanin pigment (AtPAP1) using an Agrobacterium rhizogenes-mediated transformation system. Twelve hairy root lines showing over-expression of AtPAP1 were generated. In order to investigate the regulation of AtPAP1 on the activities of CGA biosynthetic genes, the expression levels of seven P. grandiflorum CGA biosynthetic genes were analyzed in the hairy root line that had the greatest accumulation of AtPAP1 transcript, OxPAP1-1. The introduction of AtPAP1 increased the mRNA levels of all examined CGA biosynthetic genes and resulted in a 900% up-regulation of CGA accumulation in OxPAP1-1 hairy roots relative to controls. This suggests that P. grandiflorum hairy roots that over-express the AtPAP1 gene are a potential alternative source of roots for the production of CGA. PMID:25153629

  3. [Tartrate-resistant acid phosphatase in free-living Amoeba proteus].

    PubMed

    Sopina, V A

    2002-01-01

    Tartrate-resistant acid phosphatase (TRAP) of Amoeba proteus (strain B) was represented by 3 of 6 bands (= electromorphs) revealed after disc-electrophoresis in polyacrylamide gels with the use of 2-naphthyl phosphate as a substrate at pH 4.0. The presence of MgCl2, CaCl2 or ZnCl2 (50 mM) in the incubation mixture used for gel staining stimulated activities of all 3 TRAP electromorphs or of two of them (in the case of ZnCl2). When gels were treated with MgCl2, CaCl2 or ZnCl2 (10 and 100 mM, 30 min) before their staining activity of TRAP electromorphs also increased. But unlike 1 M MgCl2 or 1 M CaCl2, 1 M ZnCl2 partly inactivated two of the three TRAP electromorphs. EDTA and EGTA (5 mM), and H2O2 (10 mM) completely inhibited TRAP electromorphs after gel treatment for 10, 20 and 30 min, resp. Of 5 tested ions (Mg2+, Ca2+, Fe2+, Fe3+ and Zn2+), only the latter reactivated the TRAP electromorphs previously inactivated by EDTA or EGTA treatment. In addition, after EDTA inactivation, TRAP electromorphs were reactivated better than after EGTA. The resistance of TRAP electromorphs to okadaic acid and phosphatase inhibitor cocktail 1 used in different concentrations is indicative of the absence of PP1 and PP2A among these electromorphs. Mg2+, Ca2+ and Zn2+ dependence of TRAP activity, and the resistance of its electromorphs to vanadate and phosphatase inhibitor cocktail 2 prevents these electromorphs from being classified as PTP. It is suggested that the active center of A. proteus TRAP contains zinc ion, which is essential for catalytic activity of the enzyme. Thus, TRAP of these amoebae is metallophosphatase showing phosphomonoesterase activity in acidic medium. This metalloenzyme differs from both mammalian tartrate-resistant PAPs and tartrate-resistant metallophosphatase of Rana esculenta.

  4. Phosphorus-containing fluorinated organics: polyfluoroalkyl phosphoric acid diesters (diPAPs), perfluorophosphonates (PFPAs), and perfluorophosphinates (PFPIAs) in residential indoor dust.

    PubMed

    De Silva, Amila O; Allard, Cody N; Spencer, Christine; Webster, Glenys M; Shoeib, Mahiba

    2012-11-20

    Indoor dust is thought to be a source of human exposure to perfluorocarboxylates (PFCAs) and perfluorosulfonates (PFSAs), but exposures to emerging organofluorine compounds, including precursors to PFCAs and PFSAs via indoor dust, remain unknown. We report an analytical method for measuring several groups of emerging phosphorus-containing fluorinated compounds, including polyfluoroalkyl phosphoric acid diesters (diPAP), perfluorophosphonates (PFPA), and perfluorophosphinates (PFPIA), as well as perfluoroethylcyclohexane sulfonate (PFECHS) in indoor dust. This method was used to analyze diPAP, PFPA, and PFPIA levels in 102 residential dust samples collected in 2007-2008 from Vancouver, Canada. The results indicated a predominant and ubiquitous presence of diPAPs (frequency of detection 100%, mean and median ΣdiPAPs 7637 and 2215 ng/g). Previously measured median concentrations of perfluorooctane sulfonate (PFOS), perfluorooctanoate (PFOA), and fluorotelomer alcohols (FTOHs) in the same samples were 14-74 times lower than ΣdiPAP levels, i.e. 71 ng/g PFOS, 30 ng/g PFOA, and 152 ng/g ΣFTOHs. PFPAs and PFPIAs were detected in 62% and 85% of samples, respectively, at concentrations nearly 3 orders of magnitude lower than diPAPs (median 2.3 ng/g ΣPFPAs and 2.3 ng/g ΣPFPIAs). PFECHS was detected in only 8% of dust samples. To the best of our knowledge, this is the first report of these compounds in indoor dust. In this study, diPAP concentrations represented 98% ± 7% of the total measured analytes in the dust samples. Detection of diPAPs at such high concentrations in indoor dust may represent an important and as-yet unrecognized indirect source of PFCA exposure in humans, given the identified biotransformation pathways. Identifying the sources of diPAPs to the indoor environment is a priority for future research to improve air quality in households.

  5. Inhibition of the Hematopoietic Protein Tyrosine Phosphatase by Phenoxyacetic Acids.

    PubMed

    Bobkova, Ekaterina V; Liu, Wallace H; Colayco, Sharon; Rascon, Justin; Vasile, Stefan; Gasior, Carlton; Critton, David A; Chan, Xochella; Dahl, Russell; Su, Ying; Sergienko, Eduard; Chung, Thomas D Y; Mustelin, Tomas; Page, Rebecca; Tautz, Lutz

    2011-02-01

    Protein tyrosine phosphatases (PTPs) have only recently become the focus of attention in the search for novel drug targets despite the fact that they play vital roles in numerous cellular processes and are implicated in many human diseases. The hematopoietic protein tyrosine phosphatase (HePTP) is often found dysregulated in preleukemic myelodysplastic syndrome (MDS), as well as in acute myelogenous leukemia (AML). Physiological substrates of HePTP include the mitogen-activated protein kinases (MAPKs) ERK1/2 and p38. Specific modulators of HePTP catalytic activity will be useful for elucidating mechanisms of MAPK regulation in hematopietic cells, and may also provide treatments for hematopoietic malignancies such as AML. Here we report the discovery of phenoxyacetic acids as inhibitors of HePTP. Structure-activity relationship (SAR) analysis and in silico docking studies reveal the molecular basis of HePTP inhibition by these compounds. We also show that these compounds are able to penetrate cell membranes and inhibit HePTP in human T lymphocytes.

  6. Senescence-inducible cell wall and intracellular purple acid phosphatases: implications for phosphorus remobilization in Hakea prostrata (Proteaceae) and Arabidopsis thaliana (Brassicaceae)

    PubMed Central

    Shane, Michael W.; Stigter, Kyla; Fedosejevs, Eric T.; Plaxton, William C.

    2014-01-01

    Despite its agronomic importance, the metabolic networks mediating phosphorus (P) remobilization during plant senescence are poorly understood. Highly efficient P remobilization (~85%) from senescing leaves and proteoid roots of harsh hakea (Hakea prostrata), a native ‘extremophile’ plant of south-western Australia, was linked with striking up-regulation of cell wall-localized and intracellular acid phosphatase (APase) and RNase activities. Non-denaturing PAGE followed by in-gel APase activity staining revealed senescence-inducible 120kDa and 60kDa intracellular APase isoforms, whereas only the 120kDa isoform was detected in corresponding cell wall fractions. Kinetic and immunological properties of the 120kDa and 60kDa APases partially purified from senescing leaves indicated that they are purple acid phosphatases (PAPs). Results obtained with cell wall-targeted hydrolases of harsh hakea were corroborated using Arabidopsis thaliana in which an ~200% increase in cell wall APase activity during leaf senescence was paralleled by accumulation of immunoreactive 55kDa AtPAP26 polypeptides. Senescing leaves of an atpap26 T-DNA insertion mutant displayed a >90% decrease in cell wall APase activity. Previous research established that senescing leaves of atpap26 plants exhibited a similar reduction in intracellular (vacuolar) APase activity, while displaying markedly impaired P remobilization efficiency and delayed senescence. It is hypothesized that up-regulation and dual targeting of PAPs and RNases to the cell wall and vacuolar compartments make a crucial contribution to highly efficient P remobilization that dominates the P metabolism of senescing tissues of harsh hakea and Arabidopsis. To the best of the authors’ knowledge, the apparent contribution of cell wall-targeted hydrolases to remobilizing key macronutrients such as P during senescence has not been previously suggested. PMID:25170100

  7. Phosphatidic acid phosphatase and phospholipdase A activities in plasma membranes from fusing muscle cells.

    PubMed

    Kent, C; Vagelos, P R

    1976-06-17

    Plasma membrane from fusing embryonic muscle cells were assayed for phospholipase A activity to determine if this enzyme plays a role in cell fusion. The membranes were assayed under a variety of conditions with phosphatidylcholine as the substrate and no phospholipase A activity was found. The plasma membranes did contain a phosphatidic acid phosphatase which was optimally active in the presence of Triton X-100 and glycerol. The enzyme activity was constant from pH 5.2 to 7.0, and did not require divalent cations. Over 97% of the phosphatidic acid phosphatase activity was in the particulate fraction. The subcellular distribution of the phosphatidic acid phosphatase was the same as the distributions of the plasma membrane markers, (Na+ + k+)-ATPase and the acetylcholine receptor, which indicates that this phosphatase is located exclusively in the plasma membranes. There was no detectable difference in the phosphatidic acid phosphatase activities of plasma membranes from fusing and non-fusing cells.

  8. Expression of acid phosphatase in the seminiferous epithelium of vertebrates.

    PubMed

    Peruquetti, R L; Taboga, S R; Azeredo-Oliveira, M T V

    2010-01-01

    Acid phosphatases (AcPs) are known to provide phosphate to tissues that have high energy requirements, especially during development, growth and maturation. During spermatogenesis AcP activity is manifested in heterophagous lysosomes of Sertoli cells. This phagocytic function appears to be hormone-independent. We examined the expression pattern of AcP during the reproductive period of four species belonging to different vertebrate groups: Tilapia rendalli (Teleostei, Cichlidae), Dendropsophus minutus (Amphibia, Anura), Meriones unguiculatus (Mammalia, Rodentia), and Oryctolagus cuniculus (Mammalia, Lagomorpha). To demonstrate AcP activity, cryosections were processed for enzyme histochemistry by a modification of the method of Gömöri. AcP activity was similar in the testes of these four species. Testes of T. rendalli, D. minutus and M. unguiculatus showed an intense reaction in the Sertoli cell region. AcP activity was detected in the testes of D. minutus and O. cuniculus in seminiferous epithelium regions, where cells are found in more advanced stages of development. The seminiferous epithelium of all four species exhibited AcP activity, mainly in the cytoplasm of either Sertoli cells or germ cells. These findings reinforce the importance of AcP activity during the spermatogenesis process in vertebrates. PMID:20391346

  9. Pregnancy-secreted Acid phosphatase, uteroferrin, enhances fetal erythropoiesis.

    PubMed

    Ying, Wei; Wang, Haiqing; Bazer, Fuller W; Zhou, Beiyan

    2014-11-01

    Uteroferrin (UF) is a progesterone-induced acid phosphatase produced by uterine glandular epithelia in mammals during pregnancy and targeted to sites of hematopoiesis throughout pregnancy. The expression pattern of UF is coordinated with early fetal hematopoietic development in the yolk sac and then liver, spleen, and bone to prevent anemia in fetuses. Our previous studies suggested that UF exerts stimulatory impacts on hematopoietic progenitor cells. However, the precise role and thereby the mechanism of action of UF on hematopoiesis have not been investigated previously. Here, we report that UF is a potent regulator that can greatly enhance fetal erythropoiesis. Using primary fetal liver hematopoietic cells, we observed a synergistic stimulatory effect of UF with erythropoietin and other growth factors on both burst-forming unit-erythroid and colony-forming unit-erythroid formation. Further, we demonstrated that UF enhanced erythropoiesis at terminal stages using an in vitro culture system. Surveying genes that are crucial for erythrocyte formation at various stages revealed that UF, along with erythropoietin, up-regulated transcription factors required for terminal erythrocyte differentiation and genes required for synthesis of hemoglobin. Collectively, our results demonstrate that UF is a cytokine secreted by uterine glands in response to progesterone that promotes fetal erythropoiesis at various stages of pregnancy, including burst-forming unit-erythroid and colony-forming unit-erythroid progenitor cells and terminal stages of differentiation of hematopoietic cells in the erythroid lineage. PMID:25093463

  10. Synthesis of functionalized fluorescent gold nanoclusters for acid phosphatase sensing

    NASA Astrophysics Data System (ADS)

    Sun, Jian; Yang, Fan; Yang, Xiurong

    2015-10-01

    A novel and convenient one-pot but two-step synthesis of fluorescent gold nanoclusters, incorporating glutathione (GSH) and 11-mercaptoundecanoic acid (MUA) as the functionalized ligands (i.e. AuNCs@GSH/MUA), is demonstrated. Herein, the mixing of HAuCl4 and GSH in aqueous solution results in the immediate formation of non-fluorescent GSH-Au+ complexes, and then a class of ~2.6 nm GSH-coated AuNCs (AuNCs@GSH) with mild orange-yellow fluorescence after several days. Interestingly, the intense orange-red emitting ~1.7 nm AuNCs@GSH/MUA can be synthesized within seconds by introducing an alkaline aqueous solution of MUA into the GSH-Au+ complexes or AuNC@GSH solution. Subsequently, a reliable AuNC@GSH/MUA-based real-time assay of acid phosphatase (ACP) is established for the first time, inspired by the selective coordination of Fe3+ with surface ligands of AuNCs, the higher binding affinity between the pyrophosphate ion (PPi) and Fe3+, and the hydrolysis of PPi into orthophosphate by ACP. Our fluorescent chemosensor can also be applied to assay ACP in a real biological sample and, furthermore, to screen the inhibitor of ACP. This report paves a new avenue for synthesizing AuNCs based on either the bottom-up reduction or top-down etching method, establishing real-time fluorescence assays for ACP by means of PPi as the substrate, and further exploring the sensing applications of fluorescent AuNCs.A novel and convenient one-pot but two-step synthesis of fluorescent gold nanoclusters, incorporating glutathione (GSH) and 11-mercaptoundecanoic acid (MUA) as the functionalized ligands (i.e. AuNCs@GSH/MUA), is demonstrated. Herein, the mixing of HAuCl4 and GSH in aqueous solution results in the immediate formation of non-fluorescent GSH-Au+ complexes, and then a class of ~2.6 nm GSH-coated AuNCs (AuNCs@GSH) with mild orange-yellow fluorescence after several days. Interestingly, the intense orange-red emitting ~1.7 nm AuNCs@GSH/MUA can be synthesized within seconds by

  11. Synthesis of functionalized fluorescent gold nanoclusters for acid phosphatase sensing.

    PubMed

    Sun, Jian; Yang, Fan; Yang, Xiurong

    2015-10-21

    A novel and convenient one-pot but two-step synthesis of fluorescent gold nanoclusters, incorporating glutathione (GSH) and 11-mercaptoundecanoic acid (MUA) as the functionalized ligands (i.e. AuNCs@GSH/MUA), is demonstrated. Herein, the mixing of HAuCl4 and GSH in aqueous solution results in the immediate formation of non-fluorescent GSH-Au(+) complexes, and then a class of ∼2.6 nm GSH-coated AuNCs (AuNCs@GSH) with mild orange-yellow fluorescence after several days. Interestingly, the intense orange-red emitting ∼1.7 nm AuNCs@GSH/MUA can be synthesized within seconds by introducing an alkaline aqueous solution of MUA into the GSH-Au(+) complexes or AuNC@GSH solution. Subsequently, a reliable AuNC@GSH/MUA-based real-time assay of acid phosphatase (ACP) is established for the first time, inspired by the selective coordination of Fe(3+) with surface ligands of AuNCs, the higher binding affinity between the pyrophosphate ion (PPi) and Fe(3+), and the hydrolysis of PPi into orthophosphate by ACP. Our fluorescent chemosensor can also be applied to assay ACP in a real biological sample and, furthermore, to screen the inhibitor of ACP. This report paves a new avenue for synthesizing AuNCs based on either the bottom-up reduction or top-down etching method, establishing real-time fluorescence assays for ACP by means of PPi as the substrate, and further exploring the sensing applications of fluorescent AuNCs. PMID:26391420

  12. Antitumor effects of methotrexate-monoclonal anti-prostatic acid phosphatase antibody conjugate on human prostate tumor

    SciTech Connect

    Deguchi, T.; Chu, T.M.; Leong, S.S.; Horoszewicz, J.S.; Lee, C.L.

    1986-03-01

    Methotrexate (MTX) was conjugated to an IgG/sub 1/ monoclonal antibody (MCA) specific for human prostatic acid phosphatase (PAP) by an active ester method, resulting in a molar ratio of MTX to IgG/sub 1/ of 14. MTX-MCA conjugate retained 94% of free antibody activity and preserved 90% of dihydrofolate reductase inhibitory activity of free MTX. MTX-MCA conjugate was shown to be accumulated in vitro by prostate tumor cells (LNCaP) 1.3 times higher than that of MTX conjugate to normal mouse IgG (NIgG) and 6.2 times higher than that of free MTX. Antitumor activity in vitro exhibited that MTX-MCA conjugate is more effective on inhibition (52%) of /sup 3/H-deoxyuridine incorporation into LNCaP cells than that of MTX-NIgG (39%), but both were less effective than free MTX (70%). The in vivo distribution of /sup 3/H-MTX-MCA conjugate in human prostate tumor xenograft (tumor: blood ratio 5.1) was higher than those of /sup 3/H-MTX-NIgG conjugate (1.1) and of free /sup 3/H-MTX (1.5). Anti-tumor activity in vivo demonstrated that MTX-MCA conjugate retarded the growth of xenografted human prostate tumor greatly and persistently, as compared with the control groups. These results suggested that MTX-monoclonal anti-PAP antibody conjugate represents a potential reagent for immunochemotherapy of human prostate tumor (NIH CA-34536, CA-15437 and ACS CH-269.

  13. The development of ribonuclease and acid phosphatase during germination of Pisum arvense.

    PubMed

    Barker, G R; Bray, C M; Walter, T J

    1974-08-01

    1. Development of ribonuclease activity in the cotyledons of germinating peas is biphasic, the time of appearance of the two phases depending on the conditions of growth. 2. Acid phosphatase exhibits a single phase of development. 3. Cycloheximide inhibits development of ribonuclease activity in phase II but not in phase I. 4. (14)C-labelled amino acids are not incorporated into ribonuclease isolated during phase I. 5. The buoyant density of ribonuclease isolated during phase I is not affected by imbibition of the seed in 80% deuterium oxide. 6. Acid phosphatase was isolated from the supernatant fraction of the cotyledons of germinating peas and partially purified. 7. Development of acid phosphatase activity during germination is inhibited by treatment of the seed with cycloheximide or actinomycin D. 8. Partial purification of acid phosphatase from peas germinated in the presence of (14)C-labelled amino acids suggests that the enzyme is radioactively labelled. 9. Germination of peas in the presence of 80% deuterium oxide results in an increase in the buoyant density of acid phosphatase. 10. The results suggest that increase in ribonuclease activity during the first 4 days of germination does not result from synthesis of protein de novo, but that the corresponding increase in acid phosphatase activity does result from synthesis de novo.

  14. Effects of multivalent cations on cell wall-associated acid phosphatase activity

    SciTech Connect

    Tu, S.I.; Brouillette, J.N.; Nagahashi, G.; Kumosinski, T.F.

    1988-09-01

    Primary cell walls, free from cytoplasmic contamination were prepared from corn (Zea mays L.) roots and potato (Solanum tuberosum) tubers. After EDTA treatment, the bound acid phosphatase activities were measured in the presence of various multivalent cations. Under the conditions of minimized Donnan effect and at pH 4.2, the bound enzyme activity of potato tuber cell walls (PCW) was stimulated by Cu/sup 2 +/, Mg/sup 2 +/, Za/sup 2 +/, and Mn/sup 2 +/; unaffected by Ba/sup 2 +/, Cd/sup 2 +/, and Pb/sup 2 +/; and inhibited by Al/sup 3 +/. The bound acid phosphatase of PCW was stimulated by a low concentration but inhibited by a higher concentration of Hg/sup 2 +/. On the other hand, in the case of corn root cells walls (CCW), only inhibition of the bound acid phosphatase by Al/sup 3 +/ and Hg/sup 2 +/ was observed. Kinetic analyses revealed that PCW acid phosphatase exhibited a negative cooperativity under all employed experimental conditions except in the presence of Mg/sup 2 +/. In contrast, CCW acid phosphatase showed no cooperative behavior. The presence of Ca/sup 2 +/ significantly reduced the effects of Hg/sup 2 +/ or Al/sup 3 +/, but not Mg/sup 2 +/, to the bound cell wall acid phosphatases. The salt solubilized (free) acid phosphatases from both PCW and CCW were not affected by the presence of tested cations except for Hg/sup 2 +/ or Al/sup 3 +/ which caused a Ca/sup 2 +/-insensitive inhibition of the enzymes. The induced stimulation or inhibition of bound acid phosphatases was quantitatively related to cation binding in the cell wall structure.

  15. Arabidopsis poly(A) polymerase PAPS1 limits founder-cell recruitment to organ primordia and suppresses the salicylic acid-independent immune response downstream of EDS1/PAD4.

    PubMed

    Trost, Gerda; Vi, Son Lang; Czesnick, Hjördis; Lange, Peggy; Holton, Nick; Giavalisco, Patrick; Zipfel, Cyril; Kappel, Christian; Lenhard, Michael

    2014-03-01

    Polyadenylation of pre-mRNAs by poly(A) polymerase (PAPS) is a critical process in eukaryotic gene expression. As found in vertebrates, plant genomes encode several isoforms of canonical nuclear PAPS enzymes. In Arabidopsis thaliana these isoforms are functionally specialized, with PAPS1 affecting both organ growth and immune response, at least in part by the preferential polyadenylation of subsets of pre-mRNAs. Here, we demonstrate that the opposite effects of PAPS1 on leaf and flower growth reflect the different identities of these organs, and identify a role for PAPS1 in the elusive connection between organ identity and growth patterns. The overgrowth of paps1 mutant petals is due to increased recruitment of founder cells into early organ primordia, and suggests that PAPS1 activity plays unique roles in influencing organ growth. By contrast, the leaf phenotype of paps1 mutants is dominated by a constitutive immune response that leads to increased resistance to the biotrophic oomycete Hyaloperonospora arabidopsidis and reflects activation of the salicylic acid-independent signalling pathway downstream of ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1)/PHYTOALEXIN DEFICIENT4 (PAD4). These findings provide an insight into the developmental and physiological basis of the functional specialization amongst plant PAPS isoforms.

  16. Alkaline, acid, and neutral phosphatase activities are induced during development in Myxococcus xanthus.

    PubMed Central

    Weinberg, R A; Zusman, D R

    1990-01-01

    One of the signals that has been reported to be important in stimulating fruiting body formation of Myxococcus xanthus is starvation for phosphate. We therefore chose to study phosphatase activity during M. xanthus development. Many phosphatases can cleave the substrate p-nitrophenol phosphate. Using this substrate in buffers at various pHs, we obtained a profile of phosphatase activities during development and germination of M. xanthus. These experiments indicated that there are five patterns of phosphatase activity in M. xanthus: two vegetative and three developmental. The two uniquely vegetative activities have pH optima at 7.2 and 8.5. Both require magnesium and both are inhibited by the reducing agent dithiothreitol. The developmental (spores) patterns of activity have pH optima of 5.2, 7.2, and 8.5. All three activities are Mg independent. Only the alkaline phosphatase activity is inhibited by dithiothreitol. The acid phosphatase activity is induced very early in development, within the first 2 to 4 h. Both the neutral and alkaline phosphatase Mg-independent activities are induced much later, about the time that myxospores become evident (24 to 30 h). The three activities are greatly diminished upon germination; however, the kinetics of loss differ for all three. The acid phosphatase activity declines very rapidly, the neutral activity begins to decline only after spores begin to convert to rods, and the alkaline phosphatase activity remains high until the time the cells begin to divide. All three developmental activities were measured in the developmental signalling mutants carrying asg, csg, and dsg. The pattern of expression obtained in the mutants was consistent with that of other developmentally regulated genes which exhibit similar patterns of expression during development. The ease with which phosphatases can be assayed should make the activities described in this report useful biochemical markers of stages of both fruiting body formation and

  17. Effects of precipitation on soil acid phosphatase activity in three successional forests in Southern China

    NASA Astrophysics Data System (ADS)

    Huang, W.; Liu, J.; Zhou, G.; Zhang, D.; Deng, Q.

    2011-01-01

    Phosphorus (P) is often a limiting nutrient for plant growth in tropical and subtropical forests. Global climate change has led to alterations in precipitation in the recent years, which inevitably influences P cycling. Soil acid phosphatase plays a vital role in controlling P mineralization, and its activity reflects the capacity of P supply to ecosystems. In order to study the effects of precipitation on soil acid phosphatase activity, an experiment of precipitation treatments (no precipitation, natural precipitation and doubled precipitation) in three forests of early-, mid- and advanced-successional stages in Southern China was carried out. Results showed that driven by seasonality of precipitation, changes in soil acid phosphatase activities coincided with the seasonal climate pattern, with significantly higher values in the wet season than in the dry season. Soil acid phosphatase activities were closely linked to forest successional stages, with enhanced values in the later stages of forest succession. In the dry season, soil acid phosphatase activities in the three forests showed a rising trend with increasing precipitation treatments. In the wet season, no precipitation treatment depressed soil acid phosphatase activity, while doubled precipitation treatment exerted no positive effects on it, and even significantly lowered it in the advanced forest. These indicate the potential transformation rate of organic P might be more dependent on water in the dry season than in the wet season. The negative responses of soil acid phosphatase activity to precipitation suggest that P supply in subtropical ecosystems might be reduced if there was a drought in a whole year or more rainfall in the wet season in the future. NP, no precipitation; Control, natural precipitation; DP, double precipitation.

  18. Vanadate inhibition of fungal phyA and bacterial appA2 histidine acid phosphatases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The fungal PhyA protein, which was first identified as an acid optimum phosphomonoesterase (EC 3.1.3.8), could also serve as a vanadate haloperoxidase (EC 1.11.1.10) provided the acid phosphatase activity is shutdown by vanadate. To understand how vanadate inhibits both phytate and pNPP degrading ac...

  19. Crystallization of a newly discovered histidine acid phosphatase from Francisella tularensis

    SciTech Connect

    Felts, Richard L.; Reilly, Thomas J.; Calcutt, Michael J.; Tanner, John J.

    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.

  20. Stabilization of human prostate acid phosphatase by cross-linking with diimidoesters.

    PubMed

    Wasylewska, E; Dulińska, J; Trubetskoy, V S; Torchilin, V P; Ostrowski, W S

    1987-01-01

    1. Modification of dimeric human prostate acid phosphatase (EC 3.1.3.2) by diimidoesters leads to the formation of water-soluble preparations of high enzymatic activity, resistant to denaturing agents. 2. Monomeric, dimeric, trimeric and tetrameric species were found in SDS-polyacrylamide gel electrophoresis of the phosphatase cross-linked with dimethyl-suberimidate, and dimeric, trimeric and tetrameric enzymatically active species on thin-layer Sephadex 200 gel filtration. This molecular pattern evidenced formation of the inter-subunit covalent linkages. All molecular forms are immunoreactive against the polyclonal rabbit anti-phosphatase antibodies. 3. The catalytic properties of the modified phosphatase are almost the same as those of the native enzyme. Differences in the optical properties between the modified and the native enzymes point to slight conformational transitions in the modified enzyme.

  1. The inhibitory effect of metals and other ions on acid phosphatase activity from Vigna aconitifolia seeds.

    PubMed

    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.

  2. Inhibition of lymphoid tyrosine phosphatase by benzofuran salicylic acids.

    PubMed

    Vang, Torkel; Xie, Yuli; Liu, Wallace H; Vidović, Dusica; Liu, Yidong; Wu, Shuangding; Smith, Deborah H; Rinderspacher, Alison; Chung, Caty; Gong, Gangli; Mustelin, Tomas; Landry, Donald W; Rickert, Robert C; Schürer, Stephan C; Deng, Shi-Xian; Tautz, Lutz

    2011-01-27

    The lymphoid tyrosine phosphatase (Lyp, PTPN22) is a critical negative regulator of T cell antigen receptor (TCR) signaling. A single-nucleotide polymorphism (SNP) in the ptpn22 gene correlates with the incidence of various autoimmune diseases, including type 1 diabetes, rheumatoid arthritis, and systemic lupus erythematosus. Since the disease-associated allele is a more potent inhibitor of TCR signaling, specific Lyp inhibitors may become valuable in treating autoimmunity. Using a structure-based approach, we synthesized a library of 34 compounds that inhibited Lyp with IC(50) values between 0.27 and 6.2 μM. A reporter assay was employed to screen for compounds that enhanced TCR signaling in cells, and several inhibitors displayed a dose-dependent, activating effect. Subsequent probing for Lyp's direct physiological targets by immunoblot analysis confirmed the ability of the compounds to inhibit Lyp in T cells. Selectivity profiling against closely related tyrosine phosphatases and in silico docking studies with the crystal structure of Lyp yielded valuable information for the design of Lyp-specific compounds. PMID:21190368

  3. Effects of precipitation on soil acid phosphatase activity in three successional forests in southern China

    NASA Astrophysics Data System (ADS)

    Huang, W.; Liu, J.; Zhou, G.; Zhang, D.; Deng, Q.

    2011-07-01

    Phosphorus (P) is often a limiting nutrient for plant growth in tropical and subtropical forests. Global climate change has led to alterations in precipitation in the recent years, which inevitably influences P cycling. Soil acid phosphatase plays a vital role in controlling P mineralization, and its activity reflects the capacity of organic P mineralization potential in soils. In order to study the effects of precipitation on soil acid phosphatase activity, an experiment with precipitation treatments (no precipitation, natural precipitation and doubled precipitation) in three successional forests in southern China was carried out. The three forests include Masson pine forest (MPF), coniferous and broad-leaved mixed forest (MF) and monsoon evergreen broad-leaved forest (MEBF). Results showed that driven by seasonality of precipitation, changes in soil acid phosphatase activities coincided with the seasonal climate pattern, with significantly higher values in the wet season than in the dry season. Soil acid phosphatase activities were closely linked to forest successional stages, with enhanced values in the later stages of forest succession. In the dry season, soil acid phosphatase activities in the three forests showed a rising trend with increasing precipitation treatments. In the wet season, soil acid phosphatase activity was depressed by no precipitation treatment in the three forests. However, doubled precipitation treatment exerted a significantly negative effect on it only in MEBF. These results indicate that the potential transformation rate of organic P might be more dependent on water in the dry season than in the wet season. A decrease in organic P turnover would occur in the three forests if there was a drought in a whole year in the future. More rainfall in the wet season would also be adverse to organic P turnover in MEBF due to its high soil moisture.

  4. The "manganese(III)-containing" purple acid phosphatase from sweet potatoes is an iron enzyme.

    PubMed

    Hefler, S K; Averill, B A

    1987-08-14

    An improved purification of the purple acid phosphatase from sweet potatoes has been developed, and the properties of the enzyme have been reexamined. Contrary to previous reports, (e.g., Y. Sugiura, et al., J. Biol. Chem., 256, 10664-10670 (1981) ), the enzyme contains two moles of iron and insignificant amounts of manganese. The specific activity of the iron-containing preparations is ca. 14 times higher than that reported previously for the purported "Mn(III)" enzyme. The sweet potato purple acid phosphatase does indeed bind manganese, but it can be removed by dialysis with no changes in specific activity or spectral properties.

  5. Autosomal dominant aniridia: probable linkage to acid phosphatase-1 locus on chromosome 2.

    PubMed Central

    Ferrell, R E; Chakravarti, A; Hittner, H M; Riccardi, V M

    1980-01-01

    Maximum likelihood analysis for linkage between autosomal dominant aniridia and 12 biochemical and serological markers in a single large family showed a probable linkage between autosomal dominant aniridia and the enzyme acid phosphatase-1. The presence of an autosomal dominant aniridia gene linked to acid phosphatase-1 on chromosome arm 2p and the existence of an aniridia syndrome resulting from deletion of band 13 of the short arm of chromosome 11 establishes a chromosome basis for genetic heterogeneity of aniridia phenotypes. PMID:6929510

  6. PURIFICATION AND PARTIAL CHARACTERIZATION OF AN ACID PHOSPHATASE FROM SPIRODELA OLIGORRHIZA AND ITS AFFINITY FOR SELECTED ORGANOPHOSPHATE PESTICIDES

    EPA Science Inventory

    An acid phosphatase from the aquatic plant Spirodela oligorrhiza (duckweed) was isolated by fast protein liquid chromatography (FPLC) and partially characterized. The enzyme was purified 1871-fold with a total yield of 40%. SDS-PAGE electrophoresis of the pure acid phosphatase ...

  7. [Biological profile of tartrate-resistant acid phosphatase as a marker of bone resorption].

    PubMed

    Rico, H; Iritia, M; Arribas, I; Revilla, M

    1990-12-01

    Tartrate-resistant serum acid phosphatase was measured in 123 subjects, 80 of which were normal and the rest pathologic, in order to define the profile and value of this parameter as a biological marker of osteoclastic activity. Normal subjects were divided into age groups based on the period where skeletal growth ends (under 20 years), at the age of menopause in women (50 years, between 20 and 50 years) and those over 50 years. There was an increase in tartrate-resistant serum acid phosphatase coinciding with puberty and no sex differences were observed after the 50 year mark, when women showed higher values than men (p less than 0.001). Such tartrate-resistant serum acid phosphatase increase, is reflected as higher values in the 50 year group than in the 20 to 50 year group (p less than 0.001), the only age limit where a negative significant correlation between tartrate-resistant serum acid phosphatase values and age could be observed (p less than 0.05). Values were higher up to the age of 20 years (p less than 0.001) than in any other older age group. Levels increased significantly (p less than 0.001 for both groups) in post-menopausal osteoporosis (n = 20) and in Paget's disease of bone (n = 15), and decreased significantly (p less than 0.05) in imperfect osteogenesis (n = 8), thus revealing its value as a biological marker of osteoclastic activity. PMID:2099535

  8. ISOLATION AND PARTIAL CHARACTERIZATION OF AN ACID PHOSPHATASE ACTIVITY FROM SPIRODELA OLIGORHIZA

    EPA Science Inventory

    An acid phosphatase activity from the aquatic plant Spirodela oligorhiza (duckweed) was isolated and partially characterized. S. oligorhiza was grown in a hydroponic growth medium, harvested, and ground up in liquid nitrogen. The ground plant material was added to a biological ...

  9. [Effect of phosphorus deficiency on activity of acid phosphatase exuded by wheat roots].

    PubMed

    Sun, Haiguo; Zhang, Fusuo

    2002-03-01

    The activity of acid phosphatase exuded by roots, the tissue location of the enzyme, and the relationship between the enzyme activity and phosphorus efficiency of wheat were studied. The results showed that the activity of acid phosphatase exuded by wheat 81(85)5-3-3-3 and NC37 under P-sufficiency treat were lower than those under P-deficiency, and the enzyme activity of the former variety was significantly higher than that of the latter. There was a significant difference in the enzyme activity among 12 wheat genotypes grown under P-deficiency treat. Acid phosphatase was exuded by epidermis cell of root, especially by epidermal cell of root apex. Thus, there was a linear relationship between the enzyme activity and the surface area of root or the number of root apexes. It implied that the enzyme activity was markedly related to the size of root system. The linear relationship between relative grain yield and acid phosphatase activity was significant. It indicates that the enzyme activity could be used as an early indicator to select P-efficient wheat genotypes.

  10. 21 CFR 862.1020 - Acid phosphatase (total or prostatic) test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Acid phosphatase (total or prostatic) test system. 862.1020 Section 862.1020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  11. 21 CFR 862.1020 - Acid phosphatase (total or prostatic) test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Acid phosphatase (total or prostatic) test system. 862.1020 Section 862.1020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  12. 21 CFR 862.1020 - Acid phosphatase (total or prostatic) test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Acid phosphatase (total or prostatic) test system. 862.1020 Section 862.1020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  13. 21 CFR 862.1020 - Acid phosphatase (total or prostatic) test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Acid phosphatase (total or prostatic) test system. 862.1020 Section 862.1020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  14. 21 CFR 862.1020 - Acid phosphatase (total or prostatic) test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Acid phosphatase (total or prostatic) test system. 862.1020 Section 862.1020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  15. Alterations in activities of acid phosphatase, alkaline phosphatase, ATPase and ATP content in response to seasonally varying Pi status in okra (Abelmoschus esculentus).

    PubMed

    Sen, Supatra; Mukherji, S

    2004-04-01

    Phosphorus (P) is the second most important macronutrient for plant growth. Plants exhibit numerous physiological and metabolic adaptations in response to seasonal variations in phosphorus content. Activities of acid and alkaline phosphatases, ATPase and ATP content were studied in summer, rainy and winter seasons at two different developmental stages (28 and 58 days after sowing) in Okra. Activities of both acid and alkaline phosphatases increased manifold in winter to cope up with low phosphorus content. ATP content and ATPase activity were high in summer signifying an active metabolic period. Phosphorus deficiency is characterized by low ATP content and ATPase activity (which are in turn partly responsible for a drastic reduction in growth and yield) and enhanced activities of acid and alkaline phosphatases which increase the availability of P in P-deficient seasons.

  16. Lipid phosphate phosphatases regulate lysophosphatidic acid production and signaling in platelets: studies using chemical inhibitors of lipid phosphate phosphatase activity.

    PubMed

    Smyth, Susan S; Sciorra, Vicki A; Sigal, Yury J; Pamuklar, Zehra; Wang, Zuncai; Xu, Yong; Prestwich, Glenn D; Morris, Andrew J

    2003-10-31

    Blood platelets play an essential role in ischemic heart disease and stroke contributing to acute thrombotic events by release of potent inflammatory agents within the vasculature. Lysophosphatidic acid (LPA) is a bioactive lipid mediator produced by platelets and found in the blood and atherosclerotic plaques. LPA receptors on platelets, leukocytes, endothelial cells, and smooth muscle cells regulate growth, differentiation, survival, motility, and contractile activity. Definition of the opposing pathways of synthesis and degradation that control extracellular LPA levels is critical to understanding how LPA bioactivity is regulated. We show that intact platelets and platelet membranes actively dephosphorylate LPA and identify the major enzyme responsible as lipid phosphate phosphatase 1 (LPP1). Localization of LPP1 to the platelet surface is increased by exposure to LPA. A novel receptor-inactive sn-3-substituted difluoromethylenephosphonate analog of phosphatidic acid that is a potent competitive inhibitor of LPP1 activity potentiates platelet aggregation and shape change responses to LPA and amplifies LPA production by agonist-stimulated platelets. Our results identify LPP1 as a pivotal regulator of LPA signaling in the cardiovascular system. These findings are consistent with genetic and cell biological evidence implicating LPPs as negative regulators of lysophospholipid signaling and suggest that the mechanisms involve both attenuation of lysophospholipid actions at cell surface receptors and opposition of lysophospholipid production. PMID:12909631

  17. ISOZYMES OF ACID PHOSPHATASE IN NORMAL AND CALMETTE-GUÉRIN BACILLUS-INDUCED RABBIT ALVEOLAR MACROPHAGES

    PubMed Central

    Axline, S. G.

    1968-01-01

    The acid phosphatase activity of normal alveolar and BCG-induced alveolar macrophages has been examined. Five electrophoretically distinct forms of acid phosphatase have been identified in both normal and BCG-induced macrophages. The acid phosphatases can be divided into two major categories. One category, containing four distinct forms, is readily solubilized after repeated freezing and thawing or mechanical disruption The second category, containing one form, is firmly bound to the lysosomal membrane and can be solubilized by treatment of the lysosomal fraction with Triton X-100. The Triton-extractable acid phosphatase and the predominant aqueous soluble acid phosphatase have been shown to differ in the degree of membrane binding, in solubility, in net charge, and in molecular weight. The two pre-dominant phosphatases possess identical pH optimum and do not differ in response to enzyme inhibitors. BCG stimulation has been shown to result in a nearly twofold increase in acid phosphatase activity. A nearly proportionate increase in the major acid phosphatase forms has been observed. PMID:4878908

  18. High affinity of acid phosphatase encoded by PHO3 gene in Saccharomyces cerevisiae for thiamin phosphates.

    PubMed

    Nosaka, K

    1990-02-01

    The enzymatic properties of acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) encoded by PHO3 gene in Saccharomyces cerevisiae, which is repressed by thiamin and has thiamin-binding activity at pH 5.0, were investigated to study physiological functions. The following results led to the conclusion that thiamin-repressible acid phosphatase physiologically catalyzes the hydrolysis of thiamin phosphates in the periplasmic space of S. cerevisiae, thus participating in utilization of the thiamin moiety of the phosphates by yeast cells: (a) thiamin-repressible acid phosphatase showed Km values of 1.6 and 1.7 microM at pH 5.0 for thiamin monophosphate and thiamin pyrophosphate, respectively. These Km values were 2-3 orders of magnitude lower than those (0.61 and 1.7 mM) for p-nitrophenyl phosphate; (b) thiamin exerted remarkable competitive inhibition in the hydrolysis of thiamin monophosphate (Ki 2.2 microM at pH 5.0), whereas the activity for p-nitrophenyl phosphate was slightly affected by thiamin; (c) the inhibitory effect of inorganic phosphate, which does not repress the thiamin-repressible enzyme, on the hydrolysis of thiamin monophosphate was much smaller than that of p-nitrophenyl phosphate. Moreover, the modification of thiamin-repressible acid phosphatase of S. cerevisiae with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide resulted in the complete loss of thiamin-binding activity and the Km value of the modified enzyme for thiamin monophosphate increased nearly to the value of the native enzyme for p-nitrophenyl phosphate. These results also indicate that the high affinity of the thiamin-repressible acid phosphatase for thiamin phosphates is due to the thiamin-binding properties of this enzyme.

  19. Comparative analysis of the 5'-end regions of two repressible acid phosphatase genes in Saccharomyces cerevisiae.

    PubMed Central

    Thill, G P; Kramer, R A; Turner, K J; Bostian, K A

    1983-01-01

    The nucleotide sequence of 5'-noncoding and N-terminal coding regions of two coordinately regulated, repressible acid phosphatase genes from Saccharomyces cerevisiae were determined. These unlinked genes encode different, but structurally related polypeptides of molecular weights 60,000 and 56,000. The DNA sequences of their 5'-flanking regions show stretches of extensive homology upstream of, and surrounding, a "TATA" sequence and in a region in which heterogeneous 5' ends of the p60 mRNA were mapped. The predicted amino acid sequences encoded by the N-terminal regions of both genes were confirmed by determination of the amino acid sequence of the native exocellular acid phosphatase and the partial sequence of the presecretory polypeptide synthesized in a cell-free protein synthesizing system. The N-terminal region of the p60 polypeptide was shown to be characterized by a hydrophobic 17-amino acid signal polypeptide which is absent in the native exocellular protein and thought to be necessary for acid phosphatase secretion. Images PMID:6343840

  20. LEPS2, a Phosphorus Starvation-Induced Novel Acid Phosphatase from Tomato1

    PubMed Central

    Baldwin, James C.; Karthikeyan, Athikkattuvalasu S.; Raghothama, Kashchandra G.

    2001-01-01

    Phosphate (Pi) is one of the least available plant nutrients found in the soil. A significant amount of phosphate is bound in organic forms in the rhizosphere. Phosphatases produced by plants and microbes are presumed to convert organic phosphorus into available Pi, which is absorbed by plants. In this study we describe the isolation and characterization of a novel tomato (Lycopersicon esculentum) phosphate starvation-induced gene (LePS2) representing an acid phosphatase. LePS2 is a member of a small gene family in tomato. The cDNA is 942 bp long and contains an open reading frame encoding a 269-amino acid polypeptide. The amino acid sequence of LePS2 has a significant similarity with a phosphatase from chicken. Distinct regions of the peptide also share significant identity with the members of HAD and DDDD super families of phosphohydrolases. Many plant homologs of LePS2 are found in the databases. The LePS2 transcripts are induced rapidly in tomato plant and cell culture in the absence of Pi. However, the induction is repressible in the presence of Pi. Divided root studies indicate that internal Pi levels regulate the expression of LePS2. The enhanced expression of LePS2 is a specific response to Pi starvation, and it is not affected by starvation of other nutrients or abiotic stresses. The bacterially (Escherichia coli) expressed protein exhibits phosphatase activity against the synthetic substrate p-nitrophenyl phosphate. The pH optimum of the enzyme activity suggests that LePS2 is an acid phosphatase. PMID:11161030

  1. LEPS2, a phosphorus starvation-induced novel acid phosphatase from tomato.

    PubMed

    Baldwin, J C; Karthikeyan, A S; Raghothama, K G

    2001-02-01

    Phosphate (Pi) is one of the least available plant nutrients found in the soil. A significant amount of phosphate is bound in organic forms in the rhizosphere. Phosphatases produced by plants and microbes are presumed to convert organic phosphorus into available Pi, which is absorbed by plants. In this study we describe the isolation and characterization of a novel tomato (Lycopersicon esculentum) phosphate starvation-induced gene (LePS2) representing an acid phosphatase. LePS2 is a member of a small gene family in tomato. The cDNA is 942 bp long and contains an open reading frame encoding a 269-amino acid polypeptide. The amino acid sequence of LePS2 has a significant similarity with a phosphatase from chicken. Distinct regions of the peptide also share significant identity with the members of HAD and DDDD super families of phosphohydrolases. Many plant homologs of LePS2 are found in the databases. The LePS2 transcripts are induced rapidly in tomato plant and cell culture in the absence of Pi. However, the induction is repressible in the presence of Pi. Divided root studies indicate that internal Pi levels regulate the expression of LePS2. The enhanced expression of LePS2 is a specific response to Pi starvation, and it is not affected by starvation of other nutrients or abiotic stresses. The bacterially (Escherichia coli) expressed protein exhibits phosphatase activity against the synthetic substrate p-nitrophenyl phosphate. The pH optimum of the enzyme activity suggests that LePS2 is an acid phosphatase.

  2. Recognition of Nucleoside Monophosphate Substrates by Haemophilus influenzae Class C Acid Phosphatase

    SciTech Connect

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

    2010-12-08

    The e (P4) phosphatase from Haemophilus influenzae functions in a vestigial NAD{sup +} utilization pathway by dephosphorylating nicotinamide mononucleotide to nicotinamide riboside. P4 is also the prototype of class C acid phosphatases (CCAPs), which are nonspecific 5{prime},3{prime}-nucleotidases localized to the bacterial outer membrane. To understand substrate recognition by P4 and other class C phosphatases, we have determined the crystal structures of a substrate-trapping mutant P4 enzyme complexed with nicotinamide mononucleotide, 5{prime}-AMP, 3{prime}-AMP, and 2{prime}-AMP. The structures reveal an anchor-shaped substrate-binding cavity comprising a conserved hydrophobic box that clamps the nucleotide base, a buried phosphoryl binding site, and three solvent-filled pockets that contact the ribose and the hydrogen-bonding edge of the base. The span between the hydrophobic box and the phosphoryl site is optimal for recognizing nucleoside monophosphates, explaining the general preference for this class of substrate. The base makes no hydrogen bonds with the enzyme, consistent with an observed lack of base specificity. Two solvent-filled pockets flanking the ribose are key to the dual recognition of 5{prime}-nucleotides and 3{prime}-nucleotides. These pockets minimize the enzyme's direct interactions with the ribose and provide sufficient space to accommodate 5{prime} substrates in an anti conformation and 3{prime} substrates in a syn conformation. Finally, the structures suggest that class B acid phosphatases and CCAPs share a common strategy for nucleotide recognition.

  3. Recognition of nucleoside monophosphate substrates by Haemophilus influenzae class C acid phosphatase.

    PubMed

    Singh, Harkewal; Schuermann, Jonathan P; Reilly, Thomas J; Calcutt, Michael J; Tanner, John J

    2010-12-10

    The e (P4) phosphatase from Haemophilus influenzae functions in a vestigial NAD(+) utilization pathway by dephosphorylating nicotinamide mononucleotide to nicotinamide riboside. P4 is also the prototype of class C acid phosphatases (CCAPs), which are nonspecific 5',3'-nucleotidases localized to the bacterial outer membrane. To understand substrate recognition by P4 and other class C phosphatases, we have determined the crystal structures of a substrate-trapping mutant P4 enzyme complexed with nicotinamide mononucleotide, 5'-AMP, 3'-AMP, and 2'-AMP. The structures reveal an anchor-shaped substrate-binding cavity comprising a conserved hydrophobic box that clamps the nucleotide base, a buried phosphoryl binding site, and three solvent-filled pockets that contact the ribose and the hydrogen-bonding edge of the base. The span between the hydrophobic box and the phosphoryl site is optimal for recognizing nucleoside monophosphates, explaining the general preference for this class of substrate. The base makes no hydrogen bonds with the enzyme, consistent with an observed lack of base specificity. Two solvent-filled pockets flanking the ribose are key to the dual recognition of 5'-nucleotides and 3'-nucleotides. These pockets minimize the enzyme's direct interactions with the ribose and provide sufficient space to accommodate 5' substrates in an anti conformation and 3' substrates in a syn conformation. Finally, the structures suggest that class B acid phosphatases and CCAPs share a common strategy for nucleotide recognition. PMID:20934434

  4. Structural basis of the inhibition of class C acid phosphatases by adenosine 5;#8242;-phosphorothioate

    SciTech Connect

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

    2012-01-20

    The inhibition of phosphatases by adenosine 5'-phosphorothioate (AMPS) was first reported in the late 1960s; however, the structural basis for the inhibition has remained unknown. Here, it is shown that AMPS is a submicromolar inhibitor of class C acid phosphatases, a group of bacterial outer membrane enzymes belonging to the haloacid dehalogenase structural superfamily. Furthermore, the 1.35-{angstrom} resolution crystal structure of the inhibited recombinant Haemophilus influenzae class C acid phosphatase was determined; this is the first structure of a phosphatase complexed with AMPS. The conformation of AMPS is identical to that of the substrate 5'-AMP, except that steric factors force a rotation of the thiophosphoryl out of the normal phosphoryl-binding pocket. This conformation is catalytically nonproductive, because the P atom is not positioned optimally for nucleophilic attack by Asp64, and the O atom of the scissile O-P bond is too far from the Asp (Asp66) that protonates the leaving group. The structure of 5'-AMP complexed with the Asp64 {yields} Asn mutant enzyme was also determined at 1.35-{angstrom} resolution. This mutation induces the substrate to adopt the same nonproductive binding mode that is observed in the AMPS complex. In this case, electrostatic considerations, rather than steric factors, underlie the movement of the phosphoryl. The structures not only provide an explanation for the inhibition by AMPS, but also highlight the precise steric and electrostatic requirements of phosphoryl recognition by class C acid phosphatases. Moreover, the structure of the Asp64 {yields} Asn mutant illustrates how a seemingly innocuous mutation can cause an unexpected structural change.

  5. Crystal structures and biochemical studies of human lysophosphatidic acid phosphatase type 6.

    PubMed

    Li, Jun; Dong, Yu; Lü, Xingru; Wang, Lu; Peng, Wei; Zhang, Xuejun C; Rao, Zihe

    2013-07-01

    Lysophosphatidic acid (LPA) is an important bioactive phospholipid involved in cell signaling through Gprotein-coupled receptors pathways. It is also involved in balancing the lipid composition inside the cell, and modulates the function of lipid rafts as an intermediate in phospholipid metabolism. Because of its involvement in these important processes, LPA degradation needs to be regulated as precisely as its production. Lysophosphatidic acid phosphatase type 6 (ACP6) is an LPA-specific acid phosphatase that hydrolyzes LPA to monoacylglycerol (MAG) and phosphate. Here, we report three crystal structures of human ACP6 in complex with malonate, L-(+)-tartrate and tris, respectively. Our analyses revealed that ACP6 possesses a highly conserved Rossmann-foldlike body domain as well as a less conserved cap domain. The vast hydrophobic substrate-binding pocket, which is located between those two domains, is suitable for accommodating LPA, and its shape is different from that of other histidine acid phosphatases, a fact that is consistent with the observed difference in substrate preferences. Our analysis of the binding of three molecules in the active site reveals the involvement of six conserved and crucial residues in binding of the LPA phosphate group and its catalysis. The structure also indicates a water-supplying channel for substrate hydrolysis. Our structural data are consistent with the fact that the enzyme is active as a monomer. In combination with additional mutagenesis and enzyme activity studies, our structural data provide important insights into substrate recognition and the mechanism for catalytic activity of ACP6.

  6. Phosphatidic acid phosphatase activity in subcellular fractions of normal and dystrophic human muscle.

    PubMed

    Kunze, D; Rüstow, B; Olthoff, D; Jung, K

    1985-03-15

    Biopsy samples from normal and dystrophic human muscle (Duchenne type) were fractionated by differential centrifugation and microsomes, mitochondria and cytosol were assayed for phosphatidic acid phosphatase (EC 3.1.3.4) and marker enzymes of mitochondria and cytosol. The activity of phosphatidic acid phosphatase was significantly lower in microsomes and higher in cytosol and mitochondria of dystrophic muscle than in the corresponding subcellular fractions of normal muscle. The results support an explanation of earlier findings that there is reduced G3P incorporation into diglycerides and phosphatidylcholine and a qualitative and quantitative change in the amount of phosphatidylcholine in dystrophic microsomes. The possible reasons for the reduction in the activity of only microsomal PA-P-ase were discussed.

  7. The prostatic acid phosphatase (ACPP) gene is localized to human chromosome 3q21-q23

    SciTech Connect

    Li, S.S.L.; Sharief, F.S. )

    1993-09-01

    Human prostatic acid phosphatase (ACPP) has been used as a diagnostic marker for prostate cancer. It is synthesized under androgen regulation and secreted by the epithelial cells of the prostate gland. The authors have confirmed the previous assignment of the ACPP gene to chromosome 3 by probing a panel of 25 human-Chinese hamster somatic cell hybrids, and they have further localized the ACPP gene to chromosome 3q21-q23 by fluorescence in situ hybridization. 10 refs., 1 fig.

  8. A COMPARATIVE ANALYSIS OF THREE CLASSES OF BACTERIAL NON-SPECIFIC ACID PHOSPHATASES AND ARCHAEAL PHOSPHOESTERASES: EVOLUTIONARY PERSPECTIVE

    PubMed Central

    U. Gandhi, Neha; B. Chandra, Sathees

    2012-01-01

    Introduction: Bacterial nonspecific acid phosphohydrolases (NSAPs) or phosphatases are group of enzymes secreted as soluble periplasmic proteins or retained as membrane bound lipoproteins that are usually able to dephosphorylate a broad array of structurally unrelated organic phosphoesters (nucleotides, sugar phosphates, phytic acid etc.) to acquire inorganic phosphate (Pi) and organic byproducts. They exhibit optimal catalytic activity at acidic to neutral pH values. On the basis of amino acid sequence relatedness, phosphatase are grouped into different molecular families namely Class A, Class B and Class C acid phosphatase respectively. Results and discussion: In this article out of thirty three sequences, twenty six belonging to each of the three classes of bacterial acid phosphatase and seven belonging to archaeal phosphoesterases were analyzed using various tools of bioinformatics. Phylogenetic analysis, dot plot comparisons and motif analysis were done to identify a number of similarities and differences between three classes of bacterial acid phosphatases and archaeal phosphoesterases. In this research we have attempted to decipher evolutionary relationship between three classes of bacterial acid phosphatase and archaeal phosphoesterases using bioinformatics approach. PMID:23322973

  9. Cloning, purification and crystallization of Bacillus anthracis class C acid phosphatase

    SciTech Connect

    Felts, Richard L.; Reilly, Thomas J.; Calcutt, Michael J.; Tanner, John J.

    2006-07-01

    Crystallization of a surface-localized acid phosphatase from Bacillus anthracis is reported. Flash annealing increased the high-resolution limit of usable data from 1.8 to 1.6 Å. Cloning, expression, purification and crystallization studies of a recombinant class C acid phosphatase from the Category A pathogen Bacillus anthracis are reported. Large diffraction-quality crystals were grown in the presence of HEPES and Jeffamine ED-2001 at pH 7.0. The crystals belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 53.4, b = 90.1, c = 104.2 Å. The asymmetric unit is predicted to contain two protein molecules with a solvent content of 38%. Two native data sets were collected from the same crystal before and after flash-annealing. The first data set had a mosaicity of 1.6° and a high-resolution limit of 1.8 Å. After flash-annealing, the apparent mosaicity decreased to 0.9° and the high-resolution limit of usable data increased to 1.6 Å. This crystal form is currently being used to determine the structure of B. anthracis class C acid phosphatase with experimental phasing techniques.

  10. Expression, purification and crystallization of class C acid phosphatases from Francisella tularensis and Pasteurella multocida

    PubMed Central

    Singh, Harkewal; Felts, Richard L.; Ma, Li; Malinski, Thomas J.; Calcutt, Michael J.; Reilly, Thomas J.; Tanner, John J.

    2009-01-01

    Class C nonspecific acid phosphatases are bacterial enzymes that are secreted across the cytoplasmic membrane and hydrolyze a variety of phosphomono­esters at acidic pH. These enzymes are of interest for the development of improved vaccines and clinical diagnostic methods. In one case, the category A pathogen Francisella tularensis, the class C phosphatase plays a role in bacterial fitness. Here, the cloning, expression, purification and crystallization methods for the class C acid phosphatases from F. tularensis and Pasteurella multocida are reported. Crystals of the F. tularensis enzyme diffracted to 2.0 Å resolution and belonged to space group C2221, with one enzyme molecule in the asymmetric unit. Crystals of the P. multocida enzyme diffracted to 1.85 Å resolution and belonged to space group C2, with three molecules in the asymmetric unit. Diffraction patterns from crystals of the P. multocida enzyme exhibited multiple interpenetrating reciprocal-space lattices, indicating epitaxial twinning. Despite this aberrance, autoindexing was robust and the data could be satisfactorily processed to 1.85 Å resolution using MOSFLM and SCALA. PMID:19255471

  11. Identification of purple acid phosphatase inhibitors by fragment-based screening: promising new leads for osteoporosis therapeutics.

    PubMed

    Feder, Daniel; Hussein, Waleed M; Clayton, Daniel J; Kan, Meng-Wei; Schenk, Gerhard; McGeary, Ross P; Guddat, Luke W

    2012-11-01

    Purple acid phosphatases are metalloenzymes found in animals, plants and fungi. They possess a binuclear metal centre to catalyse the hydrolysis of phosphate esters and anhydrides under acidic conditions. In humans, elevated purple acid phosphatases levels in sera are correlated with the progression of osteoporosis and metabolic bone malignancies, making this enzyme a target for the development of new chemotherapeutics to treat bone-related illnesses. To date, little progress has been achieved towards the design of specific and potent inhibitors of this enzyme that have drug-like properties. Here, we have undertaken a fragment-based screening approach using a 500-compound library identifying three inhibitors of purple acid phosphatases with K(i) values in the 30-60 μm range. Ligand efficiency values are 0.39-0.44 kcal/mol per heavy atom. X-ray crystal structures of these compounds in complex with a plant purple acid phosphatases (2.3-2.7 Å resolution) have been determined and show that all bind in the active site within contact of the binuclear centre. For one of these compounds, the phenyl ring is positioned within 3.5 Å of the binuclear centre. Docking simulations indicate that the three compounds fit into the active site of human purple acid phosphatases. These studies open the way to the design of more potent and selective inhibitors of purple acid phosphatases that can be tested as anti-osteoporotic drug leads.

  12. AN ANALYSIS OF CELLULAR AND SUBCELLULAR SYSTEMS WHICH TRANSFORM THE SPECIES CHARACTER OF ACID PHOSPHATASE IN ACETABULARIA.

    PubMed

    KECK, K; CHOULES, E A

    1963-08-01

    Several species-specific molecular forms of acid phosphatase are known to exist in the unicellular green alga Acetabularia. In graft combinations between cells of Acetabularia mediterranea (med) and Acicularia Schenckii (acic) the expression of the med phosphatase is dominant over acic phosphatase. There is good evidence that in such grafts the preexisting acic phosphatase is converted on the molecular level via an intermediate form to the med phosphatase. This conversion can be initiated by the transplantation of a med cell nucleus to an anucleate acic cell, but will also take place in grafts between anucleate med and anucleate acic cells, indicating that the direct participation of a cell nucleus is not required. An incomplete conversion of acic phosphatase, which terminates at the intermediate stage, is induced in acic cells by injection of a concentrated homogenate of med cytoplasm. A similar partial conversion occurs in vitro in a mixture of homogenates from med and acic cells. Subcellular particles, such as chloroplasts or mitochondria, can be removed from the homogenates by centrifugation without impairing the reactions leading to the intermediate phosphatase type. Experimental evidence suggests that the transformation of phosphatase types is enzymatically catalyzed and may involve the conjugation of small molecules with the phosphatase protein. It was shown, however, that sialic acid is not involved, since the incubation of med or acic homogenates with neuraminidase did not modify the electrophoretic mobility of either enzyme type. Another type of phosphatase, which occurs in Acetabularia erenulata (cren) and can be distinguished electrophoretically from the aforementioned types, is not subject to interaction. In various mono- and multi-nucleate graft combinations between cren cells on one hand, and med or acic cells on the other hand, the cren phosphatase is synthesized independently of the enzyme of the graft partner.

  13. Crystal structure and tartrate inhibition of Legionella pneumophila histidine acid phosphatase.

    PubMed

    Dhatwalia, Richa; Singh, Harkewal; Reilly, Thomas J; Tanner, John J

    2015-11-01

    Histidine acid phosphatases (HAPs) utilize a nucleophilic histidine residue to catalyze the transfer of a phosphoryl group from phosphomonoesters to water. HAPs function as protein phosphatases and pain suppressors in mammals, are essential for Giardia lamblia excystation, and contribute to virulence of the category A pathogen Francisella tularensis. Herein we report the first crystal structure and steady-state kinetics measurements of the HAP from Legionella pneumophila (LpHAP), also known as Legionella major acid phosphatase. The structure of LpHAP complexed with the inhibitor l(+)-tartrate was determined at 2.0 Å resolution. Kinetics assays show that l(+)-tartrate is a 50-fold more potent inhibitor of LpHAP than of other HAPs. Electrostatic potential calculations provide insight into the basis for the enhanced tartrate potency: the tartrate pocket of LpHAP is more positive than other HAPs because of the absence of an ion pair partner for the second Arg of the conserved RHGXRXP HAP signature sequence. The structure also reveals that LpHAP has an atypically expansive active site entrance and lacks the nucleotide substrate base clamp found in other HAPs. These features imply that nucleoside monophosphates may not be preferred substrates. Kinetics measurements confirm that AMP is a relatively inefficient in vitro substrate of LpHAP. PMID:26380880

  14. Biochemical Characterization and Subcellular Localization of the Red Kidney Bean Purple Acid Phosphatase.

    PubMed Central

    Cashikar, A. G.; Kumaresan, R.; Rao, N. M.

    1997-01-01

    Phosphatases are known to play a crucial role in phosphate turnover in plants. However, the exact role of acid phosphatases in plants has been elusive because of insufficient knowledge of their in vivo substrate and subcellular localization. We investigated the biochemical properties of a purple acid phosphatase isolated from red kidney bean (Phaseolus vulgaris) (KBPAP) with respect to its substrate and inhibitor profiles. The kinetic parameters were estimated for five substrates. We used 31P nuclear magnetic resonance to investigate the in vivo substrate of KBPAP. Chemical and enzymological estimation of polyphosphates and ATP, respectively, indicated the absence of polyphosphates and the presence of ATP in trace amounts in the seed extracts. Immunolocalization using antibodies raised against KBPAP was unsuccessful because of the non-specificity of the antiserum toward glycoproteins. Using histoenzymological methods with ATP as a substrate, we could localize KBPAP exclusively in the cell walls of the peripheral two to three rows of cells in the cotyledons. KBPAP activity was not detected in the embryo. In vitro experiments indicated that pectin, a major component of the cell wall, significantly altered the kinetic properties of KBPAP. The substrate profile and localization suggest that KBPAP may have a role in mobilizing organic phosphates in the soil during germination. PMID:12223752

  15. Acid phosphatase localization in the digestive glands of Dionaea muscipula Ellis flytraps.

    PubMed

    Henry, Y; Steer, M W

    1985-04-01

    The intracellular localization of acid phosphatases in stimulated digestive glands of Dionaea flytraps has been studied to provide evidence for the route taken by this enzyme during secretion. Previous studies have either included or excluded a role for the dictyosomes in this pathway. Both p-nitrophenyl phosphate and beta-glycerophosphate were used as substrates, and both gave similar localization patterns. Unstimulated glands contained little phosphatase activity in the endomembrane system, whereas 24 and 48 hr after stimulation, heavy deposits of lead were located in the endoplasmic reticulum cisternae, including the nuclear envelope, the dictyosome cisternae, and secretory vesicles. Since dictyosome activation, as judged by the presence of secretory vesicles in the cytoplasm, also coincides with gland stimulation, we conclude that secretion of the hydrolase enzymes occurs via this route and not, as suggested elsewhere, via direct endoplasmic reticulum to plasma membrane connections.

  16. Crystal Structures of the Histidine Acid Phosphatase from Francisella tularensis Provide Insight into Substrate Recognition

    SciTech Connect

    Singh, Harkewal; Felts, Richard L.; Schuermann, Jonathan P.; Reilly, Thomas J.; Tanner, John J.

    2009-12-01

    Histidine acid phosphatases catalyze the transfer of a phosphoryl group from phosphomonoesters to water at acidic pH using an active-site histidine. The histidine acid phosphatase from the category A pathogen Francisella tularensis (FtHAP) has been implicated in intramacrophage survival and virulence, motivating interest in understanding the structure and mechanism of this enzyme. Here, we report a structure-based study of ligand recognition by FtHAP. The 1.70-{angstrom}-resolution structure of FtHAP complexed with the competitive inhibitor L(+)-tartrate was solved using single-wavelength anomalous diffraction phasing. Structures of the ligand-free enzyme and the complex with inorganic phosphate were determined at resolutions of 1.85 and 1.70 {angstrom}, respectively. The structure of the Asp261Ala mutant enzyme complexed with the substrate 3'-AMP was determined at 1.50 {angstrom} resolution to gain insight into substrate recognition. FtHAP exhibits a two-domain fold similar to that of human prostatic acid phosphatase, consisting of an {alpha}/{beta} core domain and a smaller domain that caps the core domain. The structures show that the core domain supplies the phosphoryl binding site, catalytic histidine (His17), and an aspartic acid residue (Asp261) that protonates the leaving group, while the cap domain contributes residues that enforce substrate preference. FtHAP and human prostatic acid phosphatase differ in the orientation of the crucial first helix of the cap domain, implying differences in the substrate preferences of the two enzymes. 3'-AMP binds in one end of a 15-{angstrom}-long tunnel, with the adenine clamped between Phe23 and Tyr135, and the ribose 2'-hydroxyl interacting with Gln132. The importance of the clamp is confirmed with site-directed mutagenesis; mutation of Phe23 and Tyr135 individually to Ala increases K{sub m} by factors of 7 and 10, respectively. The structural data are consistent with a role for FtHAP in scavenging phosphate from small

  17. The first structure of a bacterial class B Acid phosphatase reveals further structural heterogeneity among phosphatases of the haloacid dehalogenase fold.

    PubMed

    Calderone, Vito; Forleo, Costantino; Benvenuti, Manuela; Cristina Thaller, Maria; Rossolini, Gian Maria; Mangani, Stefano

    2004-01-16

    AphA is a periplasmic acid phosphatase of Escherichia coli belonging to class B bacterial phosphatases, which is part of the DDDD superfamily of phosphohydrolases. The crystal structure of AphA has been determined at 2.2A and its resolution extended to 1.7A on an AuCl(3) derivative. This represents the first crystal structure of a class B bacterial phosphatase. Despite the lack of sequence homology, the AphA structure reveals a haloacid dehalogenase-like fold. This finding suggests that this fold could be conserved among members of the DDDD superfamily of phosphohydrolases. The active enzyme is a homotetramer built by using an extended N-terminal arm intertwining the four monomers. The active site of the native enzyme, as prepared, hosts a magnesium ion, which can be replaced by other metal ions. The structure explains the non-specific behaviour of AphA towards substrates, while a structure-based alignment with other phosphatases provides clues about the catalytic mechanism.

  18. Acid phosphatase complex from the freshwater snail Viviparus viviparus L. under standard conditions and intoxication by cadmium ions.

    PubMed

    Tsvetkov, I L; Popov, A P; Konichev, A S

    2003-12-01

    Acid phosphatases differing in both subcellular localization and substrate specificity were isolated for the first time from the liver of the freshwater snail Viviparus viviparus L. by preparative isoelectrofocusing. One of five characterized phosphatases is highly specific to ADP and the others can hydrolyze (at variable rate) a series of natural substrates. A scheme is proposed for the involvement of the studied phosphatases in carbohydrate metabolism. We have also studied some peculiarities of the effect of Cd2+ in vitro and in vivo on the activities of individual components of the acid phosphatase complex and corresponding changes in metabolism of the freshwater snail as a new test-object allowing the estimation of toxicity in water.

  19. Gibberellic acid controls specific acid-phosphatase isozymes in aleurone cells and protoplasts of Avena fatua L.

    PubMed

    Hooley, R

    1984-06-01

    In the presence of gibberellic acid (GA3) aleurone layers and isolated aleurone protoplasts of Avena fatua accumulate specific isozymes of acid phosphatase (EC 3.1.3.2). Some of these may be involved in mobilizing aleurone-grain phosphate reserves during germination. The hormone also controls secretion of other specific molecular forms of the enzyme that probably assist in endosperm hydrolysis. The accumulation and secretion of putative cell-wall-associated isozymes are stimulated by the action of GA3 in isolated protoplasts. This effect however, is apparently over-ridden in the intact tissue, possibly by a cell-wall-based feedback mechanism.

  20. In vitro effects of cinnamic acid derivatives on protein tyrosine phosphatase 1B.

    PubMed

    Adisakwattana, Sirichai; Pongsuwan, Jirawan; Wungcharoen, Chompunut; Yibchok-anun, Sirintorn

    2013-10-01

    Protein Tyrosine Phosphatase 1B (PTP1B) is a major negative regulator of insulin signaling pathways. Finding selective PTP1B inhibitors from natural sources has been widely recognized as a potential drug target for the treatment of diabetes mellitus and obesity. In the present study, we evaluated the inhibitory activity of cinnamic acid derivatives against PTP1B in vitro. Among 14 cinnamic acid derivatives and related compounds, the most potent inhibitor PTP1Bs were o-hydroxycinnamic acid and p-hydroxycinnamic acid, which had IC50 values of 137.67 ± 13.37 and 181.60 ± 9.34 µM, respectively. The kinetics analysis revealed that PTP1B was inhibited by o-hydroxycinnamic acid and p-hydroxycinnamic acid in a non-competitive manner. o-Hydroxycinnamic acid (25 μM) and p-hydroxycinnamic acid (25 μM), in combination with sodium orthovanadate (0.0125 μM), demonstrated a synergistic effect to inhibit PTP1B activity. In conclusion, the findings provide a new insight into naturally occurring PTP1B inhibitors that could be useful for treatment of diabetes and obesity.

  1. Inhibition kinetics of acid and alkaline phosphatases by atrazine and methomyl pesticides.

    PubMed

    El-Aswad, Ahmed F; Badawy, Mohamed E I

    2015-01-01

    The main objective of this work was to investigate the kinetic characteristics of acid and alkaline phosphatases isolated from different sources and to study the effects of the herbicide atrazine and insecticide methomyl on the activity and kinetic properties of the enzymes. Acid phosphatase (ACP) was isolated from the tomato plant (Solanum lycopersicum L. var. lycopersicum); alkaline phosphatase (ALP) was isolated from two sources, including mature earthworms (Aporrectodea caliginosa) and larvae of the Egyptian cotton leafworm (Spodoptera littoralis). The specific activities of the enzymes were 33.31, 5.56 and 0.72 mmol substrate hydrolyzed per minute per milligram protein for plant ACP, earthworms ALP and cotton leafworm ALP, respectively. The inhibition kinetics indicated that atrazine and methomyl caused competitive-non-competitive inhibition of the enzymes. The relationships between estimates of K(m) and V(max) calculated from the Michaelis-Menten equation have been explored. The extent of the inhibition was different, as estimated by the values of the inhibition constant Ki that were found to be 3.34 × 10(-3), 1.12 × 10(-2) and 1.07 × 10(-2) mM for plant ACP, earthworms ALP and cotton leafworm ALP, respectively, with methomyl. In the case of atrazine, K(i) were found to be 8.99 × 10(-3), 3.55 × 10(-2) and 1.36 × 10(-2) mM for plant ACP, earthworms ALP and cotton leafworm ALP, respectively. PMID:25996812

  2. Acid and Alkaline Phosphatase Levels in GCF during Orthodontic Tooth Movement

    PubMed Central

    Farahani, Mohammad; Safavi, Seyed Mohammadreza; Dianat, Omid; Khoramian Tusi, Somayeh; Younessian, Farnaz

    2015-01-01

    Statement of the Problem The present constituents of gingival crevicular fluid (GCF) can reflect the changes occurring in underlying tissues. Considering variety of biologic bone markers, alkaline phosphatase and acid phosphatase have been examined as bone turn over markers in orthodontic tooth movement. Purpose The current study designed in a longitudinal pattern to determine the changes of acid and alkaline phosphatase (ACP & ALP) in GCF during orthodontic tooth movement. Materials and Method An upper canines from twelve patients (mean age: 14±2 years) undergoing extraction orthodontic treatment for distal movement served as the test tooth (DC), and its contralateral (CC) and antagonist (AC) canines were used as controls. The CC was included in orthodontic appliance without orthodontic force; the AC was free from any orthodontic appliance. The GCF around the experimental teeth was harvested from mesial and distal tooth sites immediately before appliance placement (T0), and 14 (T2) and 28 days (T3) after it and ALP and ACP concentration were determined spectrophotometrically. Results ALP concentration was elevated significantly in DC and CC groups at days 14 and 28 compared with the AC. In DC group, the ALP was significantly greater in mesial sites than distal site, while no significant changes were found between both sites of CC. The peak level of ALP was observed in mesial sites of DC at T2. Regarding ACP, significant elevation of this enzyme was seen in DC group both in mesial and distal sites at T2 and T3. The peak level of this enzyme was seen at T2. Conclusion Monitoring simultaneous changes of ALP and ACP levels in GCF can reflect the tissue responses occur in periodontium during bone formation and bone resorption during orthodontic tooth movement, respectively. PMID:26535403

  3. Structure of thermotoga maritima stationary phase survival protein SurE : a novel acid phosphatase.

    SciTech Connect

    Zhang, R.-G; Skarina, T.; Katz, J. E.; Khachatryan, A; Vyas, S.; Arrowsmith, C. H.; Clarke, S.; Edwards, A.; Joachimiak, A.; Savchenko, A.; Biosciences Division; Univ. of Toronto; Univ. of California; Clinical Genomics Centre /Proteomics, Univ. Health Network

    2001-11-01

    Background: The rpoS, nlpD, pcm, and surE genes are among many whose expression is induced during the stationary phase of bacterial growth. rpoS codes for the stationary-phase RNA polymerase {sigma} subunit, and nlpD codes for a lipoprotein. The pcm gene product repairs damaged proteins by converting the atypical isoaspartyl residues back to L-aspartyls. The physiological and biochemical functions of surE are unknown, but its importance in stress is supported by the duplication of the surE gene in E. coli subjected to high-temperature growth. The pcm and surE genes are highly conserved in bacteria, archaea, and plants. Results: The structure of SurE from Thermotoga maritima was determined at 2.0 Angstroms. The SurE monomer is composed of two domains; a conserved N-terminal domain, a Rossman fold, and a C-terminal oligomerization domain, a new fold. Monomers form a dimer that assembles into a tetramer. Biochemical analysis suggests that SurE is an acid phosphatase, with an optimum pH of 5.5-6.2. The active site was identified in the N-terminal domain through analysis of conserved residues. Structure-based site-directed point mutations abolished phosphatase activity. T. maritima SurE intra- and intersubunit salt bridges were identified that may explain the SurE thermostability. Conclusions: The structure of SurE provided information about the protein's fold, oligomeric state, and active site. The protein possessed magnesium-dependent acid phosphatase activity, but the physiologically relevant substrate(s) remains to be identified. The importance of three of the assigned active site residues in catalysis was confirmed by site-directed mutagenesis.

  4. Trichoderma harzianum Produces a New Thermally Stable Acid Phosphatase, with Potential for Biotechnological Application

    PubMed Central

    Souza, Amanda Araújo; Leitão, Vanessa Oliveira; Ramada, Marcelo Henrique; Mehdad, Azadeh; Georg, Raphaela de Castro; Ulhôa, Cirano José; de Freitas, Sonia Maria

    2016-01-01

    Acid phosphatases (ACPases) are produced by a variety of fungi and have gained attention due their biotechnological potential in industrial, diagnosis and bioremediation processes. These enzymes play a specific role in scavenging, mobilization and acquisition of phosphate, enhancing soil fertility and plant growth. In this study, a new ACPase from Trichoderma harzianum, named ACPase II, was purified and characterized as a glycoprotein belonging to the acid phosphatase family. ACPase II presents an optimum pH and temperature of 3.8 and 65°C, respectively, and is stable at 55°C for 120 min, retaining 60% of its activity. The enzyme did not require metal divalent ions, but was inhibited by inorganic phosphate and tungstate. Affinity for several phosphate substrates was observed, including phytate, which is the major component of phosphorus in plant foods. The inhibition of ACPase II by tungstate and phosphate at different pH values is consistent with the inability of the substrate to occupy its active site due to electrostatic contacts that promote conformational changes, as indicated by fluorescence spectroscopy. A higher affinity for tungstate rather than phosphate at pH 4.0was observed, in accordance with its highest inhibitory effect. Results indicate considerable biotechnological potential of the ACPase II in soil environments. PMID:26938873

  5. A purple acid phosphatase plays a role in nodule formation and nitrogen fixation in Astragalus sinicus.

    PubMed

    Wang, Jianyun; Si, Zaiyong; Li, Fang; Xiong, Xiaobo; Lei, Lei; Xie, Fuli; Chen, Dasong; Li, Yixing; Li, Youguo

    2015-08-01

    The AsPPD1 gene from Astragalus sinicus encodes a purple acid phosphatase. To address the functions of AsPPD1 in legume-rhizobium symbiosis, its expression patterns, enzyme activity, subcellular localization, and phenotypes associated with its over-expression and RNA interference (RNAi) were investigated. The expression of AsPPD1 was up-regulated in roots and nodules after inoculation with rhizobia. Phosphate starvation reduced the levels of AsPPD1 transcripts in roots while increased those levels in nodules. We confirmed the acid phosphatase and phosphodiesterase activities of recombinant AsPPD1 purified from Pichia pastoris, and demonstrated its ability to hydrolyze ADP and ATP in vitro. Subcellular localization showed that AsPPD1 located on the plasma membranes in hairy roots and on the symbiosomes membranes in root nodules. Over-expression of AsPPD1 in hairy roots inhibited nodulation, while its silencing resulted in nodules early senescence and significantly decreased nitrogenase activity. Furthermore, HPLC measurement showed that AsPPD1 overexpression affects the ADP levels in the infected roots and nodules, AsPPD1 silencing affects the ratio of ATP/ADP and the energy charge in nodules, and quantitative observation demonstrated the changes of AsPPD1 transcripts level affected nodule primordia formation. Taken together, it is speculated that AsPPD1 contributes to symbiotic ADP levels and energy charge control, and this is required for effective nodule organogenesis and nitrogen fixation.

  6. Trichoderma harzianum Produces a New Thermally Stable Acid Phosphatase, with Potential for Biotechnological Application.

    PubMed

    Souza, Amanda Araújo; Leitão, Vanessa Oliveira; Ramada, Marcelo Henrique; Mehdad, Azadeh; Georg, Raphaela de Castro; Ulhôa, Cirano José; de Freitas, Sonia Maria

    2016-01-01

    Acid phosphatases (ACPases) are produced by a variety of fungi and have gained attention due their biotechnological potential in industrial, diagnosis and bioremediation processes. These enzymes play a specific role in scavenging, mobilization and acquisition of phosphate, enhancing soil fertility and plant growth. In this study, a new ACPase from Trichoderma harzianum, named ACPase II, was purified and characterized as a glycoprotein belonging to the acid phosphatase family. ACPase II presents an optimum pH and temperature of 3.8 and 65 °C, respectively, and is stable at 55 °C for 120 min, retaining 60% of its activity. The enzyme did not require metal divalent ions, but was inhibited by inorganic phosphate and tungstate. Affinity for several phosphate substrates was observed, including phytate, which is the major component of phosphorus in plant foods. The inhibition of ACPase II by tungstate and phosphate at different pH values is consistent with the inability of the substrate to occupy its active site due to electrostatic contacts that promote conformational changes, as indicated by fluorescence spectroscopy. A higher affinity for tungstate rather than phosphate at pH 4.0 was observed, in accordance with its highest inhibitory effect. Results indicate considerable biotechnological potential of the ACPase II in soil environments. PMID:26938873

  7. Regulation of collagenase gene expression by okadaic acid, an inhibitor of protein phosphatases.

    PubMed Central

    Kim, S J; Lafyatis, R; Kim, K Y; Angel, P; Fujiki, H; Karin, M; Sporn, M B; Roberts, A B

    1990-01-01

    Human collagenase gene expression is regulated transcriptionally and is inducible by various mitogens in many cell types. To investigate the molecular mechanisms of this response, we examined the effects on collagenase gene expression of okadaic acid, a non-12-O-tetradecanoyl-phorbol-13-acetate (TPA)-type tumor promoter, which induces apparent "activation" of protein kinases by inhibition of protein phosphatases. Steady state levels of collagenase mRNA were markedly increased by okadaic acid treatment. We show that the AP-1 consensus sequence in the collagenase promoter is required for the induction of collagenase gene expression by okadaic acid, even though sequences upstream of the AP-1 consensus site have an additive effect. We also examined the regulation by okadaic acid of expression of the components of the AP-1 complex, c-fos and c-jun. c-fos expression is dramatically stimulated by okadaic acid, whereas c-jun expression is stimulated to a lesser extent. Induction of c-fos gene mRNA occurs through a region known to contain multiple regulatory elements. These results suggest that phosphorylation regulates collagenase gene expression mediated by an AP-1 binding site. Images PMID:1966042

  8. A possible role for acid phosphatase with thiamin-binding activity encoded by PHO3 in yeast.

    PubMed

    Nosaka, K; Kaneko, Y; Nishimura, H; Iwashima, A

    1989-07-01

    Periplasmic soluble thiamin-binding protein in Saccharomyces cerevisiae (Iwashima, A. et al. (1979) Biochim. Biophys. Acta 577, 217-220) was demonstrated to be encoded by PHO3 gene that codes for thiamin repressible acid phosphatase (Schweingruber, M.E. et al. (1986) J. Biol. Chem. 261, 15877-15882) by genetic analysis. The pho3 mutant cells of S. cerevisiae in contrast to the parent cells have markedly reduced activity of the uptake of [14C]thiamin phosphates, suggesting that thiamin repressible acid phosphatase plays a role in the hydrolysis of thiamin phosphates in the periplasmic space prior to the uptake of their thiamin moieties by S. cerevisiae.

  9. 4-Quinolone-3-carboxylic acids as cell-permeable inhibitors of protein tyrosine phosphatase 1B.

    PubMed

    Zhi, Ying; Gao, Li-Xin; Jin, Yi; Tang, Chun-Lan; Li, Jing-Ya; Li, Jia; Long, Ya-Qiu

    2014-07-15

    Protein tyrosine phosphatase 1B is a negative regulator in the insulin and leptin signaling pathways, and has emerged as an attractive target for the treatment of type 2 diabetes and obesity. However, the essential pharmacophore of charged phosphotyrosine or its mimetic confer low selectivity and poor cell permeability. Starting from our previously reported aryl diketoacid-based PTP1B inhibitors, a drug-like scaffold of 4-quinolone-3-carboxylic acid was introduced for the first time as a novel surrogate of phosphotyrosine. An optimal combination of hydrophobic groups installed at C-6, N-1 and C-3 positions of the quinolone motif afforded potent PTP1B inhibitors with low micromolar IC50 values. These 4-quinolone-3-carboxylate based PTP1B inhibitors displayed a 2-10 fold selectivity over a panel of PTP's. Furthermore, the bidentate inhibitors of 4-quinolone-3-carboxylic acids conjugated with aryl diketoacid or salicylic acid were cell permeable and enhanced insulin signaling in CHO/hIR cells. The kinetic studies and molecular modeling suggest that the 4-quinolone-3-carboxylates act as competitive inhibitors by binding to the PTP1B active site in the WPD loop closed conformation. Taken together, our study shows that the 4-quinolone-3-carboxylic acid derivatives exhibit improved pharmacological properties over previously described PTB1B inhibitors and warrant further preclinical studies.

  10. The internalization signal in the cytoplasmic tail of lysosomal acid phosphatase consists of the hexapeptide PGYRHV.

    PubMed Central

    Lehmann, L E; Eberle, W; Krull, S; Prill, V; Schmidt, B; Sander, C; von Figura, K; Peters, C

    1992-01-01

    Lysosomal acid phosphatase (LAP) is rapidly internalized from the cell surface due to a tyrosine-containing internalization signal in its 19 amino acid cytoplasmic tail. Measuring the internalization of a series of LAP cytoplasmic tail truncation and substitution mutants revealed that the N-terminal 12 amino acids of the cytoplasmic tail are sufficient for rapid endocytosis and that the hexapeptide 411-PGYRHV-416 is the tyrosine-containing internalization signal. Truncation and substitution mutants of amino acid residues following Val416 can prevent internalization even though these residues do not belong to the internalization signal. It was shown recently that part of the LAP cytoplasmic tail peptide corresponding to 410-PPGY-413 forms a well-ordered beta turn structure in solution. Two-dimensional NMR spectroscopy of two modified LAP tail peptides, in which the single tyrosine was substituted either by phenylalanine or by alanine, revealed that the tendency to form a beta turn is reduced by 25% in the phenylalanine-containing peptide and by approximately 50% in the alanine-containing mutant peptide. Our results suggest, that in the short cytoplasmic tail of LAP tyrosine is required for stabilization of the right turn and that the aromatic ring system of the tyrosine residue is a contact point to the putative cytoplasmic receptor. Images PMID:1425575

  11. The hppA gene of Helicobacter pylori encodes the class C acid phosphatase precursor.

    PubMed

    Godlewska, Renata; Bujnicki, Janusz M; Ostrowski, Jerzy; Jagusztyn-Krynicka, Elzbieta K

    2002-08-14

    Screening of the Helicobacter pylori genomic library with sera from infected humans and from immunized rabbits resulted in identification of the 25 kDa protein cell envelope (HppA) which exhibits acid phosphatase activity. Enzyme activity was demonstrated by specific enzymatic assays with whole-cell protein preparations of H. pylori strain N6 and from Escherichia coli carrying the hppA gene (pUWM192). HppA showed optimum activity at pH 5.6 and was resistant to inhibition by EDTA. Bioinformatics analysis and site-directed mutagenesis of two putative active site residues (D73 and D192) provide further insight into the sequence-structure-function relationships of HppA as a member of the DDDD phosphohydrolase superfamily.

  12. Lysosomal Acid Phosphatase Biosynthesis and Dysfunction: A Mini Review Focused on Lysosomal Enzyme Dysfunction in Brain.

    PubMed

    Ashtari, N; Jiao, X; Rahimi-Balaei, M; Amiri, S; Mehr, S E; Yeganeh, B; Marzban, H

    2016-01-01

    Lysosomes are membrane-bound organelles that are responsible for degrading and recycling macromolecules. Lysosomal dysfunction occurs in enzymatic and non-enzymatic deficiencies, which result in abnormal accumulation of materials. Although lysosomal storage disorders affect different organs, the central nervous system is the most vulnerable. Evidence shows the role of lysosomal dysfunction in different neurodegenerative diseases, such as Niemann-Pick Type C disease, juvenile neuronal ceroid lipofuscinosis, Alzheimer's disease and Parkinson's disease. Lysosomal enzymes such as lysosomal acid phosphatase 2 (Acp2) play a critical role in mannose-6-phosphate removal and Acp2 controls molecular and cellular functions in the brain during development and adulthood. Acp2 is essential in cerebellar development, and mutations in this gene cause severe cerebellar neurodevelopmental and neurodegenerative disorders. In this mini-review, we highlight lysosomal dysfunctions in the pathogenesis of neurodevelopmental and/or neurodegenerative diseases with special attention to Acp2 dysfunction. PMID:27132795

  13. Strigolactone Regulates Anthocyanin Accumulation, Acid Phosphatases Production and Plant Growth under Low Phosphate Condition in Arabidopsis

    PubMed Central

    Ito, Shinsaku; Nozoye, Tomoko; Sasaki, Eriko; Imai, Misaki; Shiwa, Yuh; Shibata-Hatta, Mari; Ishige, Taichiro; Fukui, Kosuke; Ito, Ken; Nakanishi, Hiromi; Nishizawa, Naoko K.; Yajima, Shunsuke; Asami, Tadao

    2015-01-01

    Phosphate is an essential macronutrient in plant growth and development; however, the concentration of inorganic phosphate (Pi) in soil is often suboptimal for crop performance. Accordingly, plants have developed physiological strategies to adapt to low Pi availability. Here, we report that typical Pi starvation responses in Arabidopsis are partially dependent on the strigolactone (SL) signaling pathway. SL treatment induced root hair elongation, anthocyanin accumulation, activation of acid phosphatase, and reduced plant weight, which are characteristic responses to phosphate starvation. Furthermore, the expression profile of SL-response genes correlated with the expression of genes induced by Pi starvation. These results suggest a potential overlap between SL signaling and Pi starvation signaling pathways in plants. PMID:25793732

  14. Sequential processing of lysosomal acid phosphatase by a cytoplasmic thiol proteinase and a lysosomal aspartyl proteinase.

    PubMed Central

    Gottschalk, S; Waheed, A; Schmidt, B; Laidler, P; von Figura, K

    1989-01-01

    BHK cells expressing human lysosomal acid phosphatase (LAP) transport LAP to lysosomes as an integral membrane protein. In lysosomes LAP is released from the membrane by proteolytic processing, which involves at least two cleavages at the C terminus of LAP. The first cleavage is catalysed by a thiol proteinase at the outside of the lysosomal membrane and removes the bulk of the cytoplasmic tail of LAP. The second cleavage is catalysed by an aspartyl proteinase inside the lysosomes and releases the luminal part of LAP from the membrane-spanning domain. The first cleavage at the cytoplasmic side of the lysosomal membrane depends on acidification of lysosomes and the second cleavage inside the lysosomes depends on prior processing of the cytoplasmic tail. These results suggest that the cytoplasmic tail controls the conformation of the luminal portion of LAP and vice versa. Images PMID:2684640

  15. Phosphatidic acid inhibits blue light-induced stomatal opening via inhibition of protein phosphatase 1 [corrected].

    PubMed

    Takemiya, Atsushi; Shimazaki, Ken-ichiro

    2010-08-01

    Stomata open in response to blue light under a background of red light. The plant hormone abscisic acid (ABA) inhibits blue light-dependent stomatal opening, an effect essential for promoting stomatal closure in the daytime to prevent water loss. However, the mechanisms and molecular targets of this inhibition in the blue light signaling pathway remain unknown. Here, we report that phosphatidic acid (PA), a phospholipid second messenger produced by ABA in guard cells, inhibits protein phosphatase 1 (PP1), a positive regulator of blue light signaling, and PA plays a role in stimulating stomatal closure in Vicia faba. Biochemical analysis revealed that PA directly inhibited the phosphatase activity of the catalytic subunit of V. faba PP1 (PP1c) in vitro. PA inhibited blue light-dependent stomatal opening but did not affect red light- or fusicoccin-induced stomatal opening. PA also inhibited blue light-dependent H(+) pumping and phosphorylation of the plasma membrane H(+)-ATPase. However, PA did not inhibit the autophosphorylation of phototropins, blue light receptors for stomatal opening. Furthermore, 1-butanol, a selective inhibitor of phospholipase D, which produces PA via hydrolysis of phospholipids, diminished the ABA-induced inhibition of blue light-dependent stomatal opening and H(+) pumping. We also show that hydrogen peroxide and nitric oxide, which are intermediates in ABA signaling, inhibited the blue light responses of stomata and that 1-butanol diminished these inhibitions. From these results, we conclude that PA inhibits blue light signaling in guard cells by PP1c inhibition, accelerating stomatal closure, and that PP1 is a cross talk point between blue light and ABA signaling pathways in guard cells.

  16. Comparative Evaluation of Efficacy of Three Different Herbal Toothpastes on Salivary Alkaline Phosphatase and Salivary Acid Phosphatase - A Randomized Controlled Trial

    PubMed Central

    Dodamani, Arun; Karibasappa, G. N.; Deshmukh, Manjiri; Naik, Rahul

    2016-01-01

    Introduction Very few researches in the past have tried to evaluate the effect of herbal toothpaste on saliva and salivary constituents like alkaline phosphatase and acid phosphatase which play an important role in maintaining oral health. Aim To evaluate and compare the effect of three different herbal toothpastes on Salivary Alkaline Phosphatase (ALP) and salivary Acid Phosphatase (ACP). Material and Methods The present study was a preliminary study conducted among 45 dental students (15 subjects in each group) in the age group of 19-21 years. Subjects in each group were randomly intervened with three different herbal toothpastes respectively (Group A – Patanjali Dant Kanti, Group B - Himalaya Complete Care and Group C – Vicco Vajradanti). Unstimulated saliva sample were collected before and after brushing and salivary ACP and salivary ALP levels were assessed at an interval of one week each for a period of four weeks starting from day one. Compiled data was analyzed using chi square test, paired t-test and ANOVA based on the nature of the obtained data. Results All the three toothpastes showed significant (p<0.001) reduction in ACP and ALP levels at each interval. For patanjali toothpaste, the mean reduction was in the range of 2.55 – 2.62 IU/L for ACP and 2.94 – 2.99 IU/L for ALP. For Himalaya toothpaste, the mean reduction was in the range of 1.39 – 1.47 IU/L for ACP and 1.55 – 1.61 IU/L for ALP. For Vicco toothpaste, the mean reduction was in the range of 2.46 – 2.50 IU/L for ACP and 2.64 – 2.77 IU/L for ALP. Patanjali and Vicco toothpaste were significantly effective in reducing the levels of salivary ACP and ALP more than Himalaya toothpaste (p<0.05). Conclusion Herbal toothpastes, especially Dant Kanti and Vicco Vajradanti, showed significant reduction in levels of ACP and ALP resulting in overall improvement towards the oral health. PMID:27790584

  17. A colorimetric and near-infrared fluorescent probe with high sensitivity and selectivity for acid phosphatase and inhibitor screening.

    PubMed

    Xu, Yongqian; Li, Benhao; Xiao, Liangliang; Ouyang, Jia; Sun, Shiguo; Pang, Yi

    2014-08-14

    A dual-channel including a colorimetric and fluorescent probe based on the aggregation-caused quenching (ACQ) and enzymolysis approach has been presented to screen acid phosphatase (ACP) and its inhibitor. Moreover, the ACP activity was determined by real time assay. PMID:24957006

  18. Effect of gingival application of melatonin on alkaline and acid phosphatase, osteopontin and osteocalcin in patients with diabetes and periodontal disease

    PubMed Central

    López-Valverde, Antonio; Gómez-de-Diego, Rafel; Arias-Santiago, Salvador; de Vicente-Jiménez, Joaquín

    2013-01-01

    Objectives: To assess the effect of topical application of melatonin to the gingiva on salivary fluid concentrations of acid phosphatase, alkaline phosphatase, osteopontin, and osteocalcin. Study Design: Cross-sectional study of 30 patients with diabetes and periodontal disease and 30 healthy subjects. Diabetic patients were treated with topical application of melatonin (1% orabase cream formula) once daily for 20 days and controls with a placebo formulation. Results: Before treatment with melatonin, diabetic patients showed significantly higher mean salivary levels of alkaline and acid phosphatase, osteopontin and osteocalcin than healthy subjects (P < 0.01). After treatment with melatonin, there was a statistically significant decrease of the gingival index (15.84± 10.3 vs 5.6 ± 5.1) and pocket depth (28.3 ± 19.5 vs 11.9 ± 9.0) (P < 0.001). Also, use of melatonin was associated with a significant reduction of the four biomarkers. Changes of salivary acid phosphatase and osteopontin correlated significantly with changes in the gingival index, whereas changes of alkaline phosphatase and osteopontin correlated significantly with changes in the pocket depth. Conclusions: Treatment with topical melatonin was associated with an improvement in the gingival index and pocket depth, a reduction in salivary concentrations of acid phosphatase, alkaline phosphatase, osteopontin and osteocalcin. Key words:Melatonin, diabetes mellitus, alkaline phosphatase, acid phosphatase, osteopontin, osteocalcin. PMID:23524437

  19. Association of the acid phosphatase (ACP1) gene with triglyceride levels in obese women.

    PubMed

    Bottini, Nunzio; MacMurray, James; Peters, Warren; Rostamkhani, Masoud; Comings, David E

    2002-11-01

    The acid phosphatase (ACP1) locus codes for a low molecular weight protein tyrosine phosphatase (LMPTP) that is found ubiquitously in human tissues. The *A allele of the ACP1 gene is associated with lower total enzymatic activity than the *B and *C alleles. An association between the *A allele and extreme values of body-mass-index (BMI) and dyslipidemia has previously been described in several samples of obese subjects from the Italian population. In the present study, we investigated the relationship between ACP1 *A allele genotypes (*A/*A, *A/*B, and *A/*C) and non-*A allele genotypes (*B/*B, *B/*C, and *C/*C) and metabolic variables in 277 Caucasian post-menopausal subjects consisting of 82 non-obese subjects (BMI/=35) subjects. ACP1 genotypes were found to be significantly associated with total cholesterol (p

  20. Activity of soil dehydrogenases, urease, and acid and alkaline phosphatases in soil polluted with petroleum.

    PubMed

    Wyszkowska, Jadwiga; Wyszkowski, Mirosław

    2010-01-01

    This study was undertaken to (1) determine the effects of petroleum pollution on changes in the biochemical properties of soil and (2) demonstrate whether the application of compost, bentonite, and calcium oxide is likely to restore biological balance. Petroleum soil pollution at a dose ranging from 2.5 to 10 cm(3)/kg disturbed the biochemical balance as evidenced by inhibition of the activities of soil dehydrogenases (SDH), urease (URE), and acid phosphatase (ACP). The greatest change was noted in the activity of SDH, whereas the least change occurred in URE. Petroleum significantly increased the activity of soil alkaline phosphatase (ALP) in soil used for spring rape, whereas in soil used for oat harvest there was decreased ALP activity. The application of compost, bentonite, and calcium oxide to soil proved effective in mitigating the adverse effects of petroleum on the activities of soil enzymes. Soil enrichment with compost, bentonite, and calcium oxide was found to stimulate the activities of URE and ALP and inhibit the activity of ACP. The influence of bentonite and calcium oxide was greater than that of compost. Calcium oxide and, to a lesser extent, compost were found to increase the activity of SDH, whereas bentonite exerted the opposite effect, especially in the case of the main crop, spring rape. The activities of SDH, URE, and ACP were higher in soil used for rape than that for oats. In contrast the activity of ALP was higher in soil used for oats. Data thus indicate that compost and especially bentonite and calcium oxide exerted a positive effect on activities of some enzymes in soil polluted with petroleum. Application of neutralizing additives to soil restored soil biological balance by counteracting the negative influence of petroleum on activities of URE and ALP. PMID:20706945

  1. A convenient and label-free fluorescence "turn off-on" nanosensor with high sensitivity and selectivity for acid phosphatase.

    PubMed

    Liu, Ziping; Lin, Zihan; Liu, Linlin; Su, Xingguang

    2015-05-30

    In this study, we reported a convenient label-free fluorescence nanosensor for rapid detection of acid phosphatase on the basis of aggregation-caused quenching (ACQ) and enzymolysis approach. The selectivity nanosensor was based on the fluorescence "turn off-on" mode, which possessed high sensitivity features. The original strong fluorescence intensity of CuInS2 QDs was quenched by sodium hexametaphosphate (NaPO3)6. The high efficiency of the quenching was caused by the non-covalent binding of positively charged CuInS2 QDs to the negatively charged (NaPO3)6 through electrostatic interactions, aggregating to form a CuInS2 QDs/(NaPO3)6 complex. Adding acid phosphatase caused intense fluorescence of CuInS2 QDs/(NaPO3)6 to be recovered, and this was because of enzymolysis. (NaPO3)6 was hydrolyzed into small fragments and the high negative charge density decreased, which would weaken the strong electrostatic interactions. As a result, the quenched fluorescence "turned on". Under the optimum conditions, there was a good linear relationship between I/I0 (I and I0 were the fluorescence intensity of CuInS2 QDs/(NaPO3)6 system in the presence and absence of acid phosphatase, respectively) and acid phosphatase concentration in the range of 75-1500 nU mL(-1) with the detection limit of 9.02 nU mL(-1). The proposed nanosensor had been utilized to detect and accurately quantify acid phosphatase in human serum samples with satisfactory results.

  2. Probing the interaction induced conformation transitions in acid phosphatase with cobalt ferrite nanoparticles: Relation to inhibition and bio-activity of Chlorella vulgaris acid phosphatase.

    PubMed

    Ahmad, Farooq; Zhou, Xing; Yao, Hongzhou; Zhou, Ying; Xu, Chao

    2016-09-01

    The present study explored the interaction and kinetics of cobalt ferrite nanoparticles (NPs) with acid phosphatase (ACP) by utilizing diverse range of spectroscopic techniques. The results corroborate, the CoFe2O4 NPs cause fluorescence quenching in ACP by static quenching mechanism. The negative values of van't Hoff thermodynamic expressions (ΔH=-0.3293Jmol(-1)K(-1) and ΔG=-3.960kJmol(-1)K(-1)) corroborate the spontaneity and exothermic nature of static quenching. The positive value of ΔS (13.2893Jmol(-1)K(-1)) corroborate that major contributors of higher and stronger binding affinity among CoFe2O4 NPs with ACP were electrostatic. In addition, FTIR, UV-CD, UV-vis spectroscopy and three dimensional fluorescence (3D) techniques confirmed that CoFe2O4 NPs binding induces microenvironment perturbations leading to secondary and tertiary conformation changes in ACP to a great extent. Furthermore, synchronous fluorescence spectroscopy (SFS) affirmed the comparatively significant changes in microenvironment around tryptophan (Trp) residue by CoFe2O4 NPs. The effect of CoFe2O4 NPs on the activation kinetics of ACP was further examined in Chlorella vulgaris. Apparent Michaelis constant (Km) values of 0.57 and 26.5mM with activation energy values of 0.538 and 3.428kJmol(-1) were determined without and with 200μM CoFe2O4 NPs. Apparent Vmax value of -7Umml(-1) corroborate that enzyme active sites were completely captured by the NPs leaving no space for the substrate. The results confirmed that CoFe2O4 NPs ceased the activity by unfolding of ACP enzyme. This suggests CoFe2O4 NPs perturbed the enzyme activity by transitions in conformation and hence the metabolic activity of ACP. This study provides the pavement for novel and simple approach of using sensitive biomarkers for sensing NPs in environment. PMID:27209386

  3. High throughput virtual screening and in silico ADMET analysis for rapid and efficient identification of potential PAP248-286 aggregation inhibitors as anti-HIV agents

    NASA Astrophysics Data System (ADS)

    Malik, Ruchi; Bunkar, Devendra; Choudhary, Bhanwar Singh; Srivastava, Shubham; Mehta, Pakhuri; Sharma, Manish

    2016-10-01

    Human semen is principal vehicle for transmission of HIV-1 and other enveloped viruses. Several endogenous peptides present in semen, including a 39-amino acid fragments of prostatic acid phosphatase (PAP248-286) assemble into amyloid fibrils named as semen-derived enhancer of viral infection (SEVI) that promote virion attachment to target cells which dramatically enhance HIV virus infection by up to 105-fold. Epigallocatechin-3-gallate (EGCG), a polyphenolic compound, is the major catechin found in green tea which disaggregates existing SEVI fibers, and inhibits the formation of SEVI fibers. The aim of this study was to screen a number of relevant polyphenols to develop a rational approach for designing PAP248-286 aggregation inhibitors as potential anti-HIV agents. The molecular docking based virtual screening results showed that polyphenolic compounds 2-6 possessed good docking score and interacted well with the active site residues of PAP248-286. Amino acid residues of binding site namely; Lys255, Ser256, Leu258 and Asn265 are involved in binding of these compounds. In silico ADMET prediction studies on these hits were also found to be promising. Polyphenolic compounds 2-6 identified as hits may act as novel leads for inhibiting aggregation of PAP248-286 into SEVI.

  4. The Jasper Ridge elevated CO{sub 2} experiment: Root acid phosphatase activity in Bromus hordeaceus and Avena barbata remains unchanged under elevated [CO{sub 2}

    SciTech Connect

    Cardon, Z.G.; Jackson, R.

    1995-06-01

    Root acid phosphatase activity increases phosphate available to plants by cleaving phosphate esters in soil organic matter. Because of increased plant growth potential under elevated [CO{sub 2}], we hypothesized that high [CO{sub 2}]-grown plants might exhibit higher phosphatase activity than low [CO{sub 2}]-grown plants. We assayed phosphatase activity in two species grown on two substrates (Bromus on serpentine soil and Bromus and Avena on sandstone soil) under high and low [CO{sub 2}] and under several nutrient treatments. Phosphatase activity was expressed per gram fresh weight of roots. Phosphatase activity of Bromus roots (on sandstone) was first assayed in treatments where only P and K, or only N, were added to soil. Bromus roots in this case showed strong induction of phosphatase activity when N only had been added to soil, indicating that Bromus regulated its phosphatase activity in response to phosphate availability. Both Bromus and Avena growing in sandstone, and Bromus growing in serpentine, showed enhanced phosphatase activity at high nutrient (N, P, and K) levels over that at low nutrient levels, but no differences between phosphatase activity were apparent between [CO{sub 2}] treatments. The increased phosphatase activity at high N, P, and K may indicate enhanced {open_quotes}growth demand{close_quotes} (reflected in higher biomass) in both Avena and Bromus. In contrast, though Bromus {open_quotes}growth demand{close_quotes} (biomass) increased under high [CO{sub 2}] on sandstone, phosphatase activity did not increase.

  5. Optimization of the tartrate-resistant acid phosphatase detection by histochemical method.

    PubMed

    Galvão, M J; Santos, A; Ribeiro, M D; Ferreira, A; Nolasco, F

    2011-01-01

    According to the new KDIGO (Kidney Disease Improving Global Outcomes) guidelines, the term of renal osteodystrophy, should be used exclusively in reference to the invasive diagnosis of bone abnormalities. Due to the low sensitivity and specificity of biochemical serum markers of bone remodelling,the performance of bone biopsies is highly stimulated in dialysis patients and after kidney transplantation. The tartrate-resistant acid phosphatase (TRACP) is an iso-enzyme of the group of acid phosphatases, which is highly expressed by activated osteoclasts and macrophages. TRACP in osteoclasts is in intracytoplasmic vesicles that transport the products of bone matrix degradation. Being present in activated osteoclasts, the identification of this enzyme by histochemistry in undecalcified bone biopsies is an excellent method to quantify the resorption of bone. Since it is an enzymatic histochemical method for a thermolabile enzyme, the temperature at which it is performed is particularly relevant. This study aimed to determine the optimal temperature for identification of TRACP in activated osteoclasts in undecalcified bone biopsies embedded in methylmethacrylate. We selected 10 cases of undecalcified bone biopsies from hemodialysis patients with the diagnosis of secondary hyperparathyroidism. Sections of 5 μm were stained to identify TRACP at different incubation temperatures (37º, 45º, 60º, 70º and 80ºC) for 30 minutes. Activated osteoclasts stained red and trabecular bone (mineralized bone) was contrasted with toluidine blue. This approach also increased the visibility of the trabecular bone resorption areas (Howship lacunae). Unlike what is suggested in the literature and in several international protocols, we found that the best results were obtained with temperatures between 60ºC and 70ºC. For technical reasons and according to the results of the present study, we recommended that, for an incubation time of 30 minutes, the reaction should be carried out at 60

  6. Optimization of the tartrate-resistant acid phosphatase detection by histochemical method

    PubMed Central

    Galvão, M.J.; Santos, A. R.; Ribeiro, M.D.; Ferreira, A.; Nolasco, F.

    2011-01-01

    According to the new kidney disease improving global outcomes (KDIGO) guidelines, the term of renal osteodystrophy, should be used exclusively in reference to the invasive diagnosis of bone abnormalities. Due to the low sensitivity and specificity of biochemical serum markers of bone remodelling, the performance of bone biopsies is highly stimulated in dialysis patients and after kidney transplantation. The tartrate-resistant acid phosphatase (TRACP) is an iso-enzyme of the group of acid phosphatases, which is highly expressed by activated osteoclasts and macrophages. TRACP in osteoclasts is in intracytoplasmic vesicles that transport the products of bone matrix degradation. Being present in activated osteoclasts, the identification of this enzyme by histochemistry in undecalcified bone biopsies is an excellent method to quantify the resorption of bone. Since it is an enzymatic histochemical method for a thermolabile enzyme, the temperature at which it is performed is particularly relevant. This study aimed to determine the optimal temperature for identification of TRACP in activated osteoclasts in undecalcified bone biopsies embedded in methylmethacrylate. We selected 10 cases of undecalcified bone biopsies from hemodialysis patients with the diagnosis of secondary hyperparathyroidism. Sections of 5 µm were stained to identify TRACP at different incubation temperatures (37°, 45°, 60°, 70° and 80°C) for 30 minutes. Activated osteoclasts stained red and trabecular bone (mineralized bone) was contrasted with toluidine blue. This approach also increased the visibility of the trabecular bone resorption areas (Howship lacunae). Unlike what is suggested in the literature and in several international protocols, we found that the best results were obtained with temperatures between 60°C and 70°C. For technical reasons and according to the results of the present study, we recommended that, for an incubation time of 30 min, the reaction should be carried out at 60

  7. A novel antimicrobial protein isolated from potato (Solanum tuberosum) shares homology with an acid phosphatase.

    PubMed

    Feng, Jie; Yuan, Fenghua; Gao, Yin; Liang, Chenggang; Xu, Jin; Zhang, Changling; He, Liyuan

    2003-12-01

    The nucleotide and amino acids sequences for AP(1) will appear in the GenBank(R) and NCBI databases under accession number AY297449. A novel antimicrobial protein (AP(1)) was purified from leaves of the potato ( Solanum tuberosum, variety MS-42.3) with a procedure involving ammonium sulphate fractionation, molecular sieve chromatography with Sephacryl S-200 and hydrophobic chromatography with Butyl-Sepharose using a FPLC system. The inhibition spectrum investigation showed that AP(1) had good inhibition activity against five different strains of Ralstonia solanacearum from potato or other crops, and two fungal pathogens, Rhizoctonia solani and Alternaria solani from potato. The full-length cDNA encoding AP(1) has been successfully cloned by screening a cDNA expression library of potato with an anti-AP(1) antibody and RACE (rapid amplification of cDNA ends) PCR. Determination of the nucleotide sequences revealed the presence of an open reading frame encoding 343 amino acids. At the C-terminus of AP(1) there is an ATP-binding domain, and the N-terminus exhibits 58% identity with an/the acid phosphatase from Mesorhizobium loti. SDS/PAGE and Western blotting analysis suggested that the AP(1) gene can be successfully expressed in Escherichia coli and recognized by an antibody against AP(1). Also the expressed protein showed an inhibition activity the same as original AP(1) protein isolated from potato. We suggest that AP(1) most likely belongs to a new group of proteins with antimicrobial characteristics in vitro and functions in relation to phosphorylation and energy metabolism of plants.

  8. Blood groups and red cell acid phosphatase types in a Mixteca population resident in Mexico City.

    PubMed

    Buentello, L.; García, P.; Lisker, R.; Salamanca, F.; Peñaloza, R.

    1999-01-01

    Several blood groups, ABO, Rh, Ss, Fy, Jk, and red cell acid phosphatase (ACP) types were studied in a native Mixteca population that has resided in Mexico City since 1950. Gene frequencies were obtained and used to establish admixture estimates with blacks and whites. The subjects came from three different geographical areas: High Mixteca, Low Mixteca, and Coast Mixteca. All frequencies were in Hardy-Weinberg equilibrium. The difference in the ABO frequencies was statistically significant when subjects from the three areas were compared simultaneously. Rh frequencies differed only between the High and the Low Mixteca populations. The ACP frequencies were similar between the Low Mixteca population and a previously reported Mestizo population. However, there were significant differences between the High Mixteca group and a Mestizo population, all the subjects being from Oaxaca. This is the first report of Ss, Fy, Jk, and ACP frequencies in a Mixteca population. Am. J. Hum. Biol. 11:525-529, 1999. Copyright 1999 Wiley-Liss, Inc.

  9. Macrophage expression of tartrate-resistant acid phosphatase as a prognostic indicator in colon cancer

    PubMed Central

    How, Joan; Brown, Jason R.; Saylor, Sasha; Rimm, David L.

    2014-01-01

    Recent research has indicated that separate populations of macrophages are associated with differing outcomes in cancer survival. In our study, we examine macrophage expression of tartrate resistant acid phosphatase (TRAP) and its effect on survival in colon cancer. Immunohistochemical analysis on colorectal adenocarcinomas confirmed macrophage expression of TRAP. Co-localization of TRAP with CD68, a pan-macrophage marker, revealed that TRAP is present in some but not all subpopulations of macrophages. Further co-localization of TRAP with CD163, an M2 marker, revealed that TRAP is expressed by both M2 and non-M2 macrophages. TRAP expression was then measured using the AQUA method of quantitative immunofluorescence in a tissue microarray consisting of 233 colorectal cancer patients seen at Yale-New Haven Hospital. Survival analysis revealed that patients with high TRAP expression have a 22% increase in 5-year survival (uncorrected log rank p=0.025) and a 47% risk reduction for disease specific death (p=0.02). This finding was validated in a second cohort of older cases consisting of 505 colorectal cancer patients. Patients with high TRAP expression in the validation set had a 19% increase in 5-year survival (log rank p=0.0041) and a 52% risk reduction of death (p=0.0019). These results provide evidence that macrophage expression of TRAP is associated with improved outcome, and implicates TRAP as a potential biomarker in colon cancer. PMID:24429833

  10. Macrophage expression of tartrate-resistant acid phosphatase as a prognostic indicator in colon cancer.

    PubMed

    How, Joan; Brown, Jason R; Saylor, Sasha; Rimm, David L

    2014-08-01

    Recent research has indicated that separate populations of macrophages are associated with differing outcomes in cancer survival. In our study, we examine macrophage expression of tartrate-resistant acid phosphatase (TRAP) and its effect on survival in colon cancer. Immunohistochemical analysis on colorectal adenocarcinomas confirmed macrophage expression of TRAP. Co-localization of TRAP with CD68, a pan-macrophage marker, revealed that TRAP is present in some but not all sub-populations of macrophages. Further co-localization of TRAP with CD163, an M2 marker, revealed that TRAP is expressed by both M2 and non-M2 macrophages. TRAP expression was then measured using the AQUA method of quantitative immunofluorescence in a tissue microarray consisting of 233 colorectal cancer patients seen at Yale-New Haven Hospital. Survival analysis revealed that patients with high TRAP expression have a 22 % increase in 5-year survival (uncorrected log-rank p = 0.025) and a 47 % risk reduction in disease-specific death (p = 0.02). This finding was validated in a second cohort of older cases consisting of 505 colorectal cancer patients. Patients with high TRAP expression in the validation set had a 19 % increase in 5-year survival (log-rank p = 0.0041) and a 52 % risk reduction in death (p = 0.0019). These results provide evidence that macrophage expression of TRAP is associated with improved outcome and implicates TRAP as a potential biomarker in colon cancer.

  11. Biochemical characterization of the class B acid phosphatase (AphA) of Escherichia coli MG1655.

    PubMed

    Passariello, Claudio; Forleo, Costantino; Micheli, Vanna; Schippa, Serena; Leone, Rosalida; Mangani, Stefano; Thaller, Maria Cristina; Rossolini, Gian Maria

    2006-01-01

    The AphA enzyme of Escherichia coli, a molecular class B periplasmic phosphatase that belongs to the DDDD superfamily of phosphohydrolases, was purified and subjected to biochemical characterization. Kinetic analysis with several substrates revealed that the enzyme essentially behaves as a broad-spectrum nucleotidase highly active on 3'- and 5'-mononucleotides and monodeoxynucleotides, but not active on cyclic nucleotides, or nucleotides di- and triphosphate. Mononucleotides are degraded to nucleosides, and AphA apparently does not exhibit any nucleotide phosphomutase activity. However, it can transphosphorylate nucleosides in the presence of phosphate donors. Kinetic properties of AphA are consistent with structural data, and suggest a role for the hydrophobic pocket present in the active site crevice, made by residues Phe 56, Leu71, Trp77 and Tyr193, in conferring preferential substrate specificity by accommodating compounds with aromatic rings. AphA was inhibited by several chelating agents, including EDTA, EGTA, 1,10-phenanthroline and dipicolinic acid, with EDTA being apparently the most powerful inhibitor.

  12. Purification and characterization of an acid phosphatase from the commercial mushroom Agaricus bisporus.

    PubMed

    Wannet, W J; Wassenaar, R W; Jorissen, H J; van der Drift, C; Op den Camp, H J

    2000-04-01

    Acid phosphatase [AP; EC 3.1.3.2], a key enzyme involved in the synthesis of mannitol in Agaricus bisporus, was purified to homogeneity and characterized. The native enzyme appeared to be a high molecular weight type glycoprotein. It has a molecular weight of 145 kDa and consists of four identical 39-kDa subunits. The isoelectric point of the enzyme was found at 4.7. Maximum activity occurred at 65 degrees C. The optimum pH range was between 3.5 and 5.5, with maximum activity at pH 4.75. The enzyme was unaffected by EDTA, and inhibited by tartrate and inorganic phosphate. The enzyme exhibits a Km for p-nitrophenylphosphate and fructose-6-phosphate of 370 microM and 3.1 mM, respectively. A broad substrate specificity was observed with significant activities for fructose-6-phosphate, glucose-6-phosphate, mannitol-1-phosphate, AMP and beta-glycerol phosphate. Only phosphomonoesters were dephosphorylated. Antibodies raised against the purified enzyme could precipitate AP activity from a cell-free extract in an anticatalytic immunoprecipitation test.

  13. Avian prostatic acid phosphatase: estrogen regulation in the oviduct and epithelial cell-derived ovarian carcinomas.

    PubMed

    Bae, Hyocheol; Lim, Whasun; Bae, Seung-Min; Bazer, Fuller W; Choi, Youngsok; Song, Gwonhwa

    2014-07-01

    Prostatic acid phosphatase (ACPP) is a glycoprotein that is mainly synthesized and secreted by glandular epithelial cells (GE) of the prostate, and it is well known as a biomarker for prostate cancer. Although ACPP was used as prognostic/diagnostic indicator and studied to elucidate regulatory mechanism(s) during several decades in humans, its role is not clearly understood. Gene profiling data using a chicken DNA microarray revealed that ACPP increased significantly during remodeling and recrudescence of the oviduct in response to estrogen. Thus, in this study, we investigated the expression and hormonal regulation of ACPP gene in the reproductive tracts of chickens. ACPP was specifically detected in the luminal cells (LE) and GE of chicken oviduct, and diethylstilbestrol (a synthetic nonsteroidal estrogen) stimulated its expression during development of the oviduct. In addition, ACPP mRNA and protein were localized to LE and GE during the regeneration phase of the oviduct of laying hens during induced molting. Furthermore, ACPP mRNA and protein were abundant in GE of ovarian carcinoma, but not in normal ovaries. Moreover, strong expression of ACPP protein was detected in epithelial cells of cancerous ovaries from women. Collectively, results of the present study are the first to show that ACPP is a novel estrogen-stimulated gene in the oviductal epithelial cells of the chicken and that its expression increases significantly in epithelial cells of ovarian carcinoma, which indicates that it may be a candidate biomarker for diagnosis of epithelia-derived ovarian cancer in women. PMID:24829029

  14. Effect of endosulfan on acid and alkaline phosphatase activity in liver, kidney, and muscles of Channa gachua

    SciTech Connect

    Sharma, R.M. )

    1990-03-01

    The widespread use of a great many toxic chemicals to eliminate unwanted plant or animal species has resulted in the contamination of most aquatic habitats with these substances on a regular basis. Endosulfan, a polycyclic chlorinated hydrocarbon of cyclodien group, is a well known organochlorine insecticide on the activity of acid and alkaline phosphatase in liver, kidney and muscles of a freshwater teleost, Channa gachua.

  15. Dehydrogenases, Acid and Alkaline Phosphatases, and Esterases for Chemotaxonomy of Selected Meloidogyne, Ditylenchus, Heterodera and Aphelenchus spp.

    PubMed Central

    Dickson, D. W.; Huisingh, D.; Sasser, J. N.

    1971-01-01

    Various taxonomically useful profiles of four dehydrogenases (lactate, malate, glucose-6-phosphate, and a-glycerophosphate) and three hydrolases (acid and alkaline phosphatase and esterase) were detected in whole nematode homogenates of Meloidogyne javanica, M. hapla, M. incognita, M. arenaria, Ditylenchus dipsaci, D. triformis, Heterodera glycines, and Aphelenchus avenae. The enzyme profiles were stable in populations cultured on several different hosts. A tentative enzymically-determined phylogeny of Meloidogyne is given. PMID:19322334

  16. Unsymmetrical Fe(III)Co(II) and Ga(III)Co(II) complexes as chemical hydrolases: biomimetic models for purple acid phosphatases (PAPs).

    PubMed

    Xavier, Fernando R; Neves, Ademir; Casellato, Annelise; Peralta, Rosely A; Bortoluzzi, Adailton J; Szpoganicz, Bruno; Severino, Patricia C; Terenzi, Hernán; Tomkowicz, Zbigniew; Ostrovsky, Sergei; Haase, Wolfgang; Ozarowski, Andrew; Krzystek, Jerzy; Telser, Joshua; Schenk, Gerhard; Gahan, Lawrence R

    2009-08-17

    The design and development of suitable biomimetic catalytic systems capable of mimicking the functional properties of enzymes continues to be a challenge for bioinorganic chemists. In this study, we report on the synthesis, X-ray structures, and physicochemical characterization of the novel isostructural [Fe(III)Co(II)(BPBPMP)(mu-OAc)(2)]ClO(4) (1) and [Ga(III)Co(II)(BPBPMP)(mu-OAc)(2)]ClO(4) (2) complexes with the unsymmetrical dinucleating ligand H(2)BPBPMP (2-bis[{(2-pyridyl-methyl)-aminomethyl}-6-{(2-hydroxy-benzyl)-(2-pyridyl-methyl)}-aminomethyl]-4-methylphenol). The previously reported complex [Fe(III)Zn(II)(BPBPMP)(mu-OAc)(2)]ClO(4) (3) was investigated here by electron paramagnetic resonance for comparison with such studies on 1 and 2. A magneto-structural correlation between the exchange parameter J (cm(-1)) and the average bond lengh d (A) of the [Fe(III)-O-M(II)] structural unit for 1 and for related isostructural Fe(III)M(II) complexes using the correlation J = -10(7) exp(-6.8d) reveals that this parameter is the major factor that determines the degree of antiferromagnetic coupling in the series [(BPBPMP)Fe(III)(mu-OAc)(2)M(II)](+) (M(II) = Mn, Fe, Co, Ni) of complexes. Potentiometric and spectrophotometric titrations along with electronic absorption studies show that, in aqueous solution, complexes 1 and 2 generate the [(HO)M(III)(mu-OH)Co(II)(H(2)O)] complex as the catalytically active species in diester hydrolysis reactions. Kinetic studies on the hydrolysis of the model substrate bis(2,4-dinitrophenyl)phosphate by 1 and 2 show Michaelis-Menten behavior, with 2 being 35% more active than 1. In combination with k(H)/k(D) isotope effects, the kinetic studies suggest a mechanism in which a terminal M(III)-bound hydroxide is the hydrolysis-initiating nucleophilic catalyst. In addition, the complexes show maximum catalytic activity in DNA hydrolysis near physiological pH. The modest reactivity difference between 1 and 2 is consistent with the slightly increased nucleophilic character of the Ga(III)-OH terminal group in comparison to Fe(III)-OH in the dinuclear M(III)Co(II) species. PMID:19603814

  17. Cellular prostatic acid phosphatase, a PTEN-functional homologue in prostate epithelia, functions as a prostate-specific tumor suppressor

    PubMed Central

    Muniyan, Sakthivel; Ingersoll, Matthew A.; Batra, Surinder K.; Lin, Ming-Fong

    2014-01-01

    The inactivation of tumor suppressor genes (TSGs) plays a vital role in the progression of human cancers. Nevertheless, those ubiquitous TSGs have been shown with limited roles in various stages of diverse carcinogenesis. Investigation on identifying unique TSG, especially for early stage of carcinogenesis, is imperative. As such, the search for organ-specific TSGs has emerged as a major strategy in cancer research. Prostate cancer (PCa) has the highest incidence in solid tumors in US males. Cellular prostatic acid phosphatase (cPAcP) is a prostate-specific differentiation antigen. Despite intensive studies over the past several decades on PAcP as a PCa biomarker, the role of cPAcP as a PCa-specific tumor suppressor has only recently been emerged and validated. The mechanism underlying the pivotal role of cPAcP as a prostate-specific TSG is, in part, due to its function as a protein tyrosine phosphatase (PTP) as well as a phosphoinositide phosphatase (PIP), an apparent functional homologue to Phosphatase and tensin homolog (PTEN) in PCa cells. This review is focused on discussing the function of this authentic prostate-specific tumor suppressor and the mechanism behind the loss of cPAcP expression leading to prostate carcinogenesis. We review other phosphatases’ roles as TSGs which regulate oncogenic PI3K signaling in PCa and discuss the functional similarity between cPAcP and PTEN in prostate carcinogenesis. PMID:24747769

  18. A novel phytase with sequence similarity to purple acid phosphatases is expressed in cotyledons of germinating soybean seedlings.

    PubMed

    Hegeman, C E; Grabau, E A

    2001-08-01

    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.

  19. Mouse monoclonal antibodies against Phytolacca americana antiviral protein PAP I.

    PubMed

    Kaloyanova, D; Kyurkchiev, S; Xu, J; Abouhaidar, M; Ivanov, I

    1999-08-01

    Four hybridoma lines are constructed producing monoclonal antibodies against the pokeweed (Phytolacca americana) antiviral protein PAP I. Two of the antibodies, 4E8 and 5D3, are characterized in more detail. They recognize amino acid sequences rather than conformational changes and their epitopes are 65% distinct. One of these antibodies (5D3) is used to study localization of recombinant PAP I in Escherichia coli cells by immuno-gold electron microscopy.

  20. Separation of acid phosphatases in the rat ventral prostate by gel filtration, isoelectric focusing, and chromatofocusing.

    PubMed

    Jauhiainen, A; Rytöluoto-Kärkkäinen, R; Vanha-Perttula, T

    1983-01-01

    Acid phosphatases of the rat ventral prostate were fractionated by gel filtration (GF) on Sepharose 6B, isoelectric focusing (IEF), and chromatofocusing (CF). In GF three activity peaks (GF-1, GF-2, GF-3) were disclosed. They showed some differences in substrate preference when six substrates (p-nitrophenyl phosphate; p-NPP; phenolphthalein phosphate, Phe-P; thymolphthalein phosphate, Tym-P; alpha-naphthyl phosphate, alpha-NP; beta-naphthyl phosphate, beta-NP; naphthol ASBI phosphate, N-ASBI-P) were tested. Differences were also encountered in their sensitivity to tartrate and fluoride. IEF gave seven bands at different pI values (8.3, 8.1, 7.9, 7.1, 6.4, 5.5, and 5.0) with alpha-NP and beta-NP but only four with N-ASBI-P. Four of the bands (8.3, 8.1, 7.9, 5.5) were sensitive to tartrate. In CF eight activity peaks (CF-1 to CF-8) were resolved with the six substrates. They differed from each other in pI values, pH optima, substrate preference, and modifier characteristics. Peaks CF-1 (pI 8.3, pH 5.5), CF-2 (pI 8.1, pH 4.2) and CF-3 (pI 7.9, pH 4.2) had a large substrate spectrum and high sensitivity to tartrate and fluoride. CF-4 (pI 7.1, pH 6.0) and CF-7 (pI 5.5, pH 4.2) were low in activity, preferred alpha-NP as substrate, and were moderately sensitive to tartrate. CF-5 (pI 6.4, pH 5.5) and CF-8 (pI 5.0, pH 5.0) were able to hydrolyse all substrates tested with moderate inhibition by tartrate. CF-6 (pI 6.0, pH 5.0) showed a relative preference for p-NPP and Phe-P with no hydrolysis of N-ASBI-P and Tym-P. Of these activities CF-6 and CF-7 were also clearly activated by Co2+. Peaks CF-6 and CF-7 appeared the most sensitive to p-chloromercuribenzoate. It is concluded that activities CF-1, CF-2, and CF-3 are lysosomal isoenzymes with minor structural differences. The others are possibly all nonlysosomal with greater biochemical differences. Some of them apparently represent the secretory form(s) of acid phosphatase in the rat ventral prostate.

  1. Regulation of alkaline phosphatase expression in a neonatal rat clonal calvarial cell strain by retinoic acid.

    PubMed

    Ng, K W; Gummer, P R; Michelangeli, V P; Bateman, J F; Mascara, T; Cole, W G; Martin, T J

    1988-02-01

    A clonal cell strain, UMR 201, was established from a culture of rat calvarial cells by the process of limiting dilution on a collagen substratum. One-day-old neonatal rat calvaria stripped of periosteum were placed on collagen in alpha-MEM with 10% fetal bovine serum (FBS). Cells that grew out from the calvaria were passaged eight times to select cells with the ability to proliferate in culture before cloning was attempted. Cells from the clonal strain were homogeneous in appearance with a doubling time in culture of about 24 hours. The UMR 201 cells formed predominantly type 1 collagen. When treated with retinoic acid (RA), all cells showed an intense staining for alkaline phosphatase (ALP). This effect of RA on the expression of ALP activity was reversible and was time and dose dependent. The earliest change was observed within 6 hours. In contrast, single and isolated clumps of untreated cells stained positively for ALP only when they were confluent. Coincubation with dactinomycin up to 3 hours after the addition of RA completely prevented the expression of ALP, whereas dactinomycin became progressively less effective when added at later times. This is interpreted as indicating a regulatory role of RA on the gene expression of ALP. Other hormones acting on bone, such as 1,25(OH)2 vitamin D3 and dexamethasone, also modulate ALP activity. The cells showed morphologic evidence of senescence after passage 12. Our preliminary studies showed that the UMR 201 cells had the characteristics of relatively undifferentiated mesenchymal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Root surface acid phosphatases and their role in phosphorus assimilation by Eriophorum vaginatum

    SciTech Connect

    Kroehler, C.J.; Linkins, A.E.

    1988-01-01

    Eriophorum vaginatum is a dominant plant in much of the arctic tundra ecosystem where phosphorus is frequently a limiting nutrient. The mineralization of this organic phosphorus was thought to be principally controlled by microbial respiration, however, more recent work shows that extracellular soil phosphatases are the principal regulators. The existence of plant root and mycorrhizal surface phosphatases which are capable of hydrolyzing organic phosphorus compounds, suggests that soil organic phosphorus may be directly utilized by plants. Since E. vaginatum is a tussock forming sedge with a very dense annually produced rooting system which can exploit most of the tussock soil volume, its surface phosphatases may play a dominant role in organic phosphorus hydrolysis into inorganic phosphorus. Of equal significance would be the potential for this activity to contribute to the phosphorus nutrition through the coupling of phosphorus hydrolysis on the root and root uptake of the resultant inorganic phosphorus. Phosphatase activity was investigated and found to be uniformly distributed along the surface of the root. Kinetic analysis of the enzyme gave estimates of 9.23 mM for the apparent Km and 1.61 * 10/sup -3/ ..mu..moles mm-2 hr/sup -1/ for the apparent Vmax. Saturation values for E. vaginatum phosphatases are about 3 times higher than average soil solution organic phosphorus concentrations. 12 refs., 4 figs.

  3. Heterologous expression of AtPAP2 in transgenic potato influences carbon metabolism and tuber development.

    PubMed

    Zhang, Youjun; Sun, Feng; Fettke, Joerg; Schöttler, Mark Aurel; Ramsden, Lawrence; Fernie, Alisdair R; Lim, Boon Leong

    2014-10-16

    Changes in carbon flow and sink/source activities can affect floral, architectural, and reproductive traits of plants. In potato, overexpression (OE) of the purple acid phosphatase 2 of Arabidopsis (AtPAP2) resulted in earlier flowering, faster growth rate, increased tubers and tuber starch content, and higher photosynthesis rate. There was a significant change in sucrose, glucose and fructose levels in leaves, phloem and sink biomass of the OE lines, consistent with an increased expression of sucrose transporter 1 (StSUT1). Furthermore, the expression levels and enzyme activity of sucrose-phosphate synthase (SPS) were also significantly increased in the OE lines. These findings strongly suggest that higher carbon supply from the source and improved sink strength can improve potato tuber yield.

  4. The IBO germination quantitative trait locus encodes a phosphatase 2C-related variant with a nonsynonymous amino acid change that interferes with abscisic acid signaling.

    PubMed

    Amiguet-Vercher, Amélia; Santuari, Luca; Gonzalez-Guzman, Miguel; Depuydt, Stephen; Rodriguez, Pedro L; Hardtke, Christian S

    2015-02-01

    Natural genetic variation is crucial for adaptability of plants to different environments. Seed dormancy prevents precocious germination in unsuitable conditions and is an adaptation to a major macro-environmental parameter, the seasonal variation in temperature and day length. Here we report the isolation of IBO, a quantitative trait locus (QTL) that governs c. 30% of germination rate variance in an Arabidopsis recombinant inbred line (RIL) population derived from the parental accessions Eilenburg-0 (Eil-0) and Loch Ness-0 (Lc-0). IBO encodes an uncharacterized phosphatase 2C-related protein, but neither the Eil-0 nor the Lc-0 variant, which differ in a single amino acid, have any appreciable phosphatase activity in in vitro assays. However, we found that the amino acid change in the Lc-0 variant of the IBO protein confers reduced germination rate. Moreover, unlike the Eil-0 variant of the protein, the Lc-0 variant can interfere with the activity of the phosphatase 2C ABSCISIC ACID INSENSITIVE 1 in vitro. This suggests that the Lc-0 variant possibly interferes with abscisic acid signaling, a notion that is supported by physiological assays. Thus, we isolated an example of a QTL allele with a nonsynonymous amino acid change that might mediate local adaptation of seed germination timing. PMID:25490966

  5. Acid phosphatases of the rat epididymis. II. Biochemical characteristics, subcellular distribution and histochemical localization.

    PubMed

    Nikkanen, V; Vanha-Perttula, T

    1977-01-01

    After separation of three epididymal acid phosphatases their biochemical properties were differently studied. With appropriate substrate and inhibitor selection the distribution of the enzymes in different segments as well as the subcellular fractions of the rat epididymis was also demonstrated. The same biochemical differences were also utilized in the histochemical localization of the enzymes. It was found that Enzyme I had a pH-optimum at 5.0, a molecular weight of 97 000 and Km-constant of 0.901 mM. It was highly sensitive to tartrate and fluoride and it was localized in lysosomes as well as in the epididymal spermatozoa. Enzyme II had an optimum at pH 5.7, a molecular weight of 67 000 and Km-constant of 0.806 mM. It was also inhibited by fluoride but more resistant to tartrate. Its subcellular site was also particulate, but it was also found in the epididymal fluid. Enzyme III had an optimum at pH 5.2, a molecular weight of 135 000 and Km-constant of 0.685 m. It was resistant to low concentrations of fluoride and tartrate but sensitive to heavy metal ions. The enzyme was soluble and it behaved incoherently in thermal inactivation. All enzymes revealed the highest activity in the thin middle segments of the epididymis. Histochemical naphthol substrates gave a diffuse reaction in the epididymal epithelial cells. With the lead salt methods glycerophosphates and p-nitrophenylphosphate gave somewhat different results depending on their specificity as substrates for the epididymal enzymes. Both substrates gave a strong reaction supranuclearly in the Golgi area of the chief cells. This activity was inhibited by tartrate and was most probably due to Enzyme I. The epididymal corpus and cauda showed additionally a very strong apical activity in the chief cells with p-nitrophenylphosphate. This activity was resitant to tartrate but sensitive to fluoride. It was concluded that this enzyme represents Enzyme II activity. Similar activity was also found in the dissolving

  6. Identification of Genes Required for Secretion of the Francisella Oxidative Burst-Inhibiting Acid Phosphatase AcpA.

    PubMed

    Hoang, Ky Van; Chen, Carolyn G; Koopman, Jacob; Moshiri, Jasmine; Adcox, Haley E; Gunn, John S

    2016-01-01

    Francisella tularensis is a Tier 1 bioterror threat and the intracellular pathogen responsible for tularemia in humans and animals. Upon entry into the host, Francisella uses multiple mechanisms to evade killing. Our previous studies have shown that after entering its primary cellular host, the macrophage, Francisella immediately suppresses the oxidative burst by secreting a series of acid phosphatases including AcpA-B-C and HapA, thereby evading the innate immune response of the macrophage and enhancing survival and further infection. However, the mechanism of acid phosphatase secretion by Francisella is still unknown. In this study, we screened for genes required for AcpA secretion in Francisella. We initially demonstrated that the known secretion systems, the putative Francisella-pathogenicity island (FPI)-encoded Type VI secretion system and the Type IV pili, do not secrete AcpA. Using random transposon mutagenesis in conjunction with ELISA, Western blotting and acid phosphatase enzymatic assays, a transposon library of 5450 mutants was screened for strains with a minimum 1.5-fold decrease in secreted (culture supernatant) AcpA, but no defect in cytosolic AcpA. Three mutants with decreased supernatant AcpA were identified. The transposon insertion sites of these mutants were revealed by direct genomic sequencing or inverse-PCR and sequencing. One of these mutants has a severe defect in AcpA secretion (at least 85% decrease) and is a predicted hypothetical inner membrane protein. Interestingly, this mutant also affected the secretion of the FPI-encoded protein, VgrG. Thus, this screen identified novel protein secretion factors involved in the subversion of host defenses. PMID:27199935

  7. Identification of Genes Required for Secretion of the Francisella Oxidative Burst-Inhibiting Acid Phosphatase AcpA

    PubMed Central

    Hoang, Ky Van; Chen, Carolyn G.; Koopman, Jacob; Moshiri, Jasmine; Adcox, Haley E.; Gunn, John S.

    2016-01-01

    Francisella tularensis is a Tier 1 bioterror threat and the intracellular pathogen responsible for tularemia in humans and animals. Upon entry into the host, Francisella uses multiple mechanisms to evade killing. Our previous studies have shown that after entering its primary cellular host, the macrophage, Francisella immediately suppresses the oxidative burst by secreting a series of acid phosphatases including AcpA-B-C and HapA, thereby evading the innate immune response of the macrophage and enhancing survival and further infection. However, the mechanism of acid phosphatase secretion by Francisella is still unknown. In this study, we screened for genes required for AcpA secretion in Francisella. We initially demonstrated that the known secretion systems, the putative Francisella-pathogenicity island (FPI)-encoded Type VI secretion system and the Type IV pili, do not secrete AcpA. Using random transposon mutagenesis in conjunction with ELISA, Western blotting and acid phosphatase enzymatic assays, a transposon library of 5450 mutants was screened for strains with a minimum 1.5-fold decrease in secreted (culture supernatant) AcpA, but no defect in cytosolic AcpA. Three mutants with decreased supernatant AcpA were identified. The transposon insertion sites of these mutants were revealed by direct genomic sequencing or inverse-PCR and sequencing. One of these mutants has a severe defect in AcpA secretion (at least 85% decrease) and is a predicted hypothetical inner membrane protein. Interestingly, this mutant also affected the secretion of the FPI-encoded protein, VgrG. Thus, this screen identified novel protein secretion factors involved in the subversion of host defenses. PMID:27199935

  8. Toxic impact of aldrin on acid and alkaline phosphatase activity of penaeid prawn, Metapenaeus monoceros: In vitro study

    SciTech Connect

    Reddy, M.S.; Jayaprada, P.; Rao, K.V.R. )

    1991-03-01

    The increasing contamination of the aquatic environment by the indiscriminate and widespread use of different kinds of pesticides is a serious problem for environmental biologists. Organochlorine insecticides are more hazardous since they are not only more toxic but also leave residues in nature. The deleterious effects of aldrin on several crustaceans have been studied. But studies concerning the impact of aldrin on biochemical aspects of crustaceans are very much limited. The present study is aimed at probing the in vitro effects of aldrin on the acid and alkaline phosphatase activity levels in selected tissues of penaeid prawn, Metapenaeus monoceros (Fabricius).

  9. Comparative acid phosphatase distribution in the suprarenal gland of Discoglossus pictus, Xenopus laevis and Bufo bufo (Anurans, Amphibia).

    PubMed

    Manelli, H; Mastrolia, L; Arizzi, M

    1981-01-01

    Acid phosphatase activity was found to have a similar distribution in the suprarenal glands of Discoglossus pictus, Xenopus laevis and Bufo Bufo (Anurans, Amphibia) as determined by light and electron histochemical localization. The enzymatic activity is localized in the lysosomes of both the interrenal cells and the chromaffin cells. It is, moreover, positive on the granule membranes of the adrenaline cells whereas it appears only occasionally on the granule membranes of the noradrenaline cells. Some precipitates can also be seen occasionally at the level of the Golgi membranes.

  10. Human lysosomal acid phosphatase is transported as a transmembrane protein to lysosomes in transfected baby hamster kidney cells.

    PubMed Central

    Waheed, A; Gottschalk, S; Hille, A; Krentler, C; Pohlmann, R; Braulke, T; Hauser, H; Geuze, H; von Figura, K

    1988-01-01

    BHK cells transfected with human lysosomal acid phosphatase (LAP) cDNA (CT29) expressed 70-fold higher enzyme activities of acid phosphatase than non-transfected BHK cells. The CT29-LAP was synthesized in BHK cells as a heterogeneously glycosylated precursor that was tightly membrane associated. Transfer to the trans-Golgi was associated with a small increase in size (approximately 7 kd) and partial processing of the oligosaccharides to complex type structures. CT29-LAP was transferred into lysosomes as shown by subcellular fractionation, immunofluorescence and immunoelectron microscopy. Lack of mannose-6-phosphate residues suggested that transport does not involve mannose-6-phosphate receptors. Part of the membrane-associated CT29-LAP was processed to a soluble form. The mechanism that converts CT29-LAP into a soluble form was sensitive to NH4Cl, and reduced the size of the polypeptide by 7 kd. In vitro translation of CT29-derived cRNA in the presence of microsomal membranes yielded a CT29-LAP precursor that is protected from proteinase K except for a small peptide of approximately 2 kd. In combination with the sequence data available for LAP, these observations suggest that CT29-LAP is synthesized and transported to lysosomes as a transmembrane protein. In the lysosomes, CT29-LAP is released from the membrane by proteolytic cleavage, which removes a C-terminal peptide including the transmembrane domain and the cytosolic tail of 18 amino acids. Images PMID:3056714

  11. High Uric Acid (UA) Negatively Affects Serum Tartrate-Resistant Acid Phosphatase 5b (TRACP 5b) Immunoassay

    PubMed Central

    Wu, Zhi-Qi; Zhang, Yan; Xie, Erfu; Song, Wei-Juan; Yang, Rui-Xia; Yan, Cheng-Jing; Zhang, Bing-Feng; Xu, Hua-Guo

    2016-01-01

    Background Bone metastases often occur in the majority of patients with advanced cancer, such as prostate cancer, lung cancer and breast cancer. Serum tartrate-resistant acid phosphatase 5b (TRACP 5b), a novel bone resorption marker, has been used gradually in the clinics as a specific and sensitive marker of bone resorption for the early diagnosis of cancer patients with bone metastasis. Here, we reported that high concentrations of uric acid (UA) lead to decrease of TRACP 5b levels and determined whether TRACP 5b level was associated with UA in interference experiment. Methods A total of 77 patients with high concentrations of UA and 77 healthy subjects were tested to evaluate the differences in their TRACP 5b levels. Serial dilutions of UA were respectively spiked with a known concentration of TRACP 5b standard sample, then Serum TRACP 5b was detected by using bone TRAP® Assay. A correction equation was set to eliminate UA-derived TRACP 5b false-decrease. The effect of this correction was evaluated in high-UA individuals. Results The average TRACP level of the high-UA individuals (1.47± 0.62 U/L) was significantly lower than that of the healthy subjects (2.62 ± 0.63 U/L) (t-test, p<0.0001). The UA correction equation derived: ΔTRACP 5b = -1.9751lgΔUA + 3.7365 with an R2 = 0.98899. Application of the UA correction equation resulted in a statistically non-significant difference in TRACP 5b values between the healthy subjects and high-UA individuals (p = 0.24). Conclusions High UA concentrations can falsely decrease TRACP 5b levels due to a method-related systematic error. To avoid misdiagnoses or inappropriate therapeutic decisions, increased attention should be paid to UA interference, when TRACP 5b is used for early diagnosis of cancer patients with bone metastasis, evaluation of the aggressiveness of osteosarcoma or prediction of survival in prostate cancer and breast cancer with bone metastases. PMID:26800211

  12. The catalytic role of aspartic acid-92 in a human dual-specific protein-tyrosine-phosphatase.

    PubMed

    Denu, J M; Zhou, G; Guo, Y; Dixon, J E

    1995-03-14

    The mechanism of catalysis for the human dual-specific (vaccinia H1-related) protein-tyrosine-phosphatase was investigated. The pH dependence of the kcat value is bell-shaped when p-nitrophenyl phosphate was employed as a model substrate. The kcat/Km pH profile rises with a slope of 2 and decreases with a slope of -1, indicating that two groups must be unprotonated and one group must be protonated for activity. An amino acid residue with an apparent pKa value of 5.5 +/- 0.2 must be unprotonated and a residue with a pKa value of 5.7 must be unprotonated for activity. The pKa value of the catalytic cysteine-124 (C124) was 5.6 +/- 0.1. The aspartic acid-92-asparagine (D92N) mutant enzyme was 100-fold less active than the native enzyme and exhibited the loss of the basic limb in the pH profiles, suggesting that in the native enzyme D92 must be protonated for activity. The D92 residue is conserved throughout the entire family of dual-specific phosphatases. Mutants glutamic acid-6-glutamine, glutamic acid-32-glutamine, aspartic acid-14-asparagine, and aspartic acid-110-asparagine had less than a 2-fold effect on the kinetic parameters when compared to native enzyme. Based upon the lack of a "burst" in rapid reaction kinetics, formation of the intermediate is rate-limiting with both native and D92N mutant enzymes. In agreement with rate-limiting formation of the intermediate, the pKa value of 5.5 for the group which must be unprotonated for activity was assigned to C124.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Relation of fatty acid composition in lead-exposed mallards to fat mobilization, lipid peroxidation and alkaline phosphatase activity

    USGS Publications Warehouse

    Mateo, R.; Beyer, W.N.; Spann, J.W.; Hoffman, D.J.

    2003-01-01

    The increase of n-6 polyunsaturated fatty acids (PUFA) in animal tissues has been proposed as a mechanism of Pb poisoning through lipid peroxidation or altered eicosanoids metabolism. We have studied fatty acid (FA) composition in liver and brain of mallards (Anas platyrhynchos) feeding for three weeks on diets containing combinations of low or high levels of vitamin E (20 or 200 UI/kg) and Pb (0 or 2 g/kg). Saturated FA, n-6 PUFA and total concentrations of FA were higher in livers of Pb-exposed mallards, but not in their brains. The percentage of n-6 PUFA in liver and brain was slightly higher in Pb-exposed mallards. The increase of n-6 PUFA in liver was associated with increased triglycerides and cholesterol in plasma, thus could be in part attributed to feed refusal and fat mobilization. The hepatic ratios between adrenic acid (22:4 n-6) and arachidonic acid (20:4 n-6) or between adrenic acid and linoleic acid (18:2 n-6) were higher in Pb exposed birds, supporting the existing hypothesis of increased fatty acid elongation by Pb. Among the possible consequences of increased n-6 PUFA concentration in tissues, we found increased lipid peroxidation in liver without important histopathological changes, and decreased plasma alkaline phosphatase activity that may reflect altered bone metabolism in birds.

  14. X-ray absorption studies of the purple acid phosphatase from red kidney beans (native enzyme, metal exchanged form)

    NASA Astrophysics Data System (ADS)

    Ahlers, F.; Zippel, F.; Klabunde, T.; Krebs, B.; Löcke, R.; Witzel, H.; Nolting, H.-F.

    1995-02-01

    Purple acid phosphatase from red kidney beans (KBP) catalyzes the hydrolysis of activated phosphoric acid monoesters and contains a heterodinuclear Fe(III)Zn(II) core in its active site. Iron K-edge X-ray absorption data have been obtained for the native enzyme and for a metal exchanged derivative, where Zn(II) was substituted by Fe(III). The environment of the native enzyme consists of 2.5 O/N at 1.91 Å, 3 O/N at 2.09 Å, and 1 Zn at 4.05 Å. For the metal exchanged form we obtained 2.5 O/N at 1.94 Å, 2.5 O/N at 2.09 Å, and 1 Fe at 3.79 Å.

  15. A structure-based proposal for the catalytic mechanism of the bacterial acid phosphatase AphA belonging to the DDDD superfamily of phosphohydrolases.

    PubMed

    Calderone, Vito; Forleo, Costantino; Benvenuti, Manuela; Thaller, Maria Cristina; Rossolini, Gian Maria; Mangani, Stefano

    2006-01-27

    The Escherichia coli gene aphA codes for a periplasmic acid phosphatase called AphA, belonging to class B bacterial phosphatases, which is part of the DDDD superfamily of phosphohydrolases. After our first report about its crystal structure, we have started a series of crystallographic studies aimed at understanding of the catalytic mechanism of the enzyme. Here, we report three crystal structures of the AphA enzyme in complex with the hydrolysis products of nucleoside monophosphate substrates and a fourth with a proposed intermediate analogue that appears to be covalently bound to the enzyme. Comparison with the native enzyme structure and with the available X-ray structures of different phosphatases provides clues about the enzyme chemistry and allows us to propose a catalytic mechanism for AphA, and to discuss it with respect to the mechanism of other bacterial and human phosphatases.

  16. Variation of Photosynthesis, Fatty Acid Composition, ATPase and Acid Phosphatase Activities, and Anatomical Structure of Two Tea (Camellia sinensis (L.) O. Kuntze) Cultivars in Response to Fluoride

    PubMed Central

    Wang, L. X.; Tang, J. H.; Xiao, B.; Yang, Y. J.; Liu, J.

    2013-01-01

    The changes of photosynthetic parameters, water use efficiency (WUE), fatty acid composition, chlorophyll (Chl) content, malondialdehyde (MDA) content, ATPase and acid phosphatase activities, fluoride (F) content, and leaf anatomical structure of two tea cultivars, “Pingyangtezao” (PY) and “Fudingdabai” (FD), after F treatments were investigated. The results show that net photosynthetic rate (Pn), stomatal conductance (gs), and transpiration rate (E) significantly decreased in both cultivars after 0.3 mM F treatment, but FD had higher Pn, gs, and WUE and lower E than PY. Chl content in PY significantly decreased after 0.2 and 0.3 mM F treatments, while no significant changes were observed in FD. The proportions of shorter chain and saturated fatty acids increased and those of longer chain and unsaturated fatty acids decreased in both cultivars under F treatments. The contents of MDA increased after F treatments but were higher in PY than in FD. In addition, F treatments decreased the activities of ATPase and acid phosphatase and increased F content in both cultivars; however, compared with PY, FD showed higher enzymatic activities and lower F content in roots and leaves. Leaf anatomical structure in FD indicated that cells in leaf midrib region were less injured by F than in PY. PMID:24023526

  17. Variation of photosynthesis, fatty acid composition, ATPase and acid phosphatase activities, and anatomical structure of two tea (Camellia sinensis (L.) O. Kuntze) cultivars in response to fluoride.

    PubMed

    Wang, L X; Tang, J H; Xiao, B; Yang, Y J; Liu, J

    2013-01-01

    The changes of photosynthetic parameters, water use efficiency (WUE), fatty acid composition, chlorophyll (Chl) content, malondialdehyde (MDA) content, ATPase and acid phosphatase activities, fluoride (F) content, and leaf anatomical structure of two tea cultivars, "Pingyangtezao" (PY) and "Fudingdabai" (FD), after F treatments were investigated. The results show that net photosynthetic rate (P(n)), stomatal conductance (g(s)), and transpiration rate (E) significantly decreased in both cultivars after 0.3 mM F treatment, but FD had higher P(n), g(s), and WUE and lower E than PY. Chl content in PY significantly decreased after 0.2 and 0.3 mM F treatments, while no significant changes were observed in FD. The proportions of shorter chain and saturated fatty acids increased and those of longer chain and unsaturated fatty acids decreased in both cultivars under F treatments. The contents of MDA increased after F treatments but were higher in PY than in FD. In addition, F treatments decreased the activities of ATPase and acid phosphatase and increased F content in both cultivars; however, compared with PY, FD showed higher enzymatic activities and lower F content in roots and leaves. Leaf anatomical structure in FD indicated that cells in leaf midrib region were less injured by F than in PY.

  18. World-Wide Indoor Exposure to Polyfluoroalkyl Phosphate Esters (PAPs) and other PFASs in Household Dust.

    PubMed

    Eriksson, Ulrika; Kärrman, Anna

    2015-12-15

    Human exposure to perfluorooctanoic acid (PFOA) and other per- and polyfluoroalkyl substances (PFASs) is ongoing and in some cases increasing, despite efforts made to reduce emissions. The role of precursor compounds such as polyfluorinated phosphate esters (PAPs) has received increasing attention, but there are knowledge gaps regarding their occurrence and impact on human exposure. In this study, mono-, di-, and triPAPs, perfluorinated alkyl acids (PFAAs), saturated, and unsaturated fluorotelomer carboxylic acids (FTCA/FTUCAs), perfluoroalkane sulfonamides, and sulfonamidethanols (FOSA/FOSEs), and one fluorotelomer sulfonic acid (FTSA)) were compared in household dust samples from Canada, the Faroe Islands, Sweden, Greece, Spain, Nepal, Japan, and Australia. Mono-, di-, and triPAPs, including several diPAP homologues, were frequently detected in dust from all countries, revealing an ubiquitous spread in private households from diverse geographic areas, with significant differences between countries. The median levels of monoPAPs and diPAPs ranged from 3.7 ng/g to 1 023 ng/g and 3.6 ng/g to 692 ng/g, respectively, with the lowest levels found in Nepal and the highest in Japan. The levels of PAPs exceeded those of the other PFAS classes. These findings reveal the importance of PAPs as a source of PFAS exposure worldwide.

  19. Molecular cloning and catalytic activity of a membrane-bound prenyl diphosphate phosphatase from Croton stellatopilosus Ohba.

    PubMed

    Nualkaew, Natsajee; Guennewich, Nils; Springob, Karin; Klamrak, Anuwatchakit; De-Eknamkul, Wanchai; Kutchan, Toni M

    2013-07-01

    Geranylgeraniol (GGOH), a bioactive acyclic diterpene with apoptotic induction activity, is the immediate precursor of the commercial anti-peptic, plaunotol (18-hydroxy geranylgeraniol), which is found in Croton stellatopilosus (Ohba). From this plant, a cDNA encoding a prenyl diphosphate phosphatase (CsPDP), which catalyses the dephosphorylation of geranylgeranyl diphosphate (GGPP) to GGOH, was isolated using a PCR approach. The full-length cDNA contained 888bp and encoded a 33.6 kDa protein (295 amino acids) that was phylogenetically grouped into the phosphatidic acid phosphatase (PAP) enzyme family. The deduced amino acid sequence showed 6 hydrophobic transmembrane regions with 57-85% homology to the sequences of other plant PAPs. The recombinant CsPDP and its 4 truncated constructs exhibited decreasing dephosphorylation activities relative to the lengths of the N-terminal deletions. While the full-length CsPDP successfully performed the two sequential monodephosphorylation steps on GGPP to form GGOH, the larger N-terminal deletion in the truncated enzymes appeared to specifically decrease the catalytic efficiency of the second monodephosphorylation step. The information presented here on the CsPDP cDNA and factors affecting the dephosphorylation activity of its recombinant protein may eventually lead to the discovery of the specific GGPP phosphatase gene and enzyme that are involved in the formation of GGOH in the biosynthetic pathway of plaunotol in C. stellatopilosus.

  20. Enhanced degradation of five organophosphorus pesticides in skimmed milk by lactic acid bacteria and its potential relationship with phosphatase production.

    PubMed

    Zhang, Ying-Hua; Xu, Di; Liu, Jia-Qi; Zhao, Xin-Huai

    2014-12-01

    Skimmed milk spiked with five organophosphorus pesticides (OPPs), chlorpyrifos, diazinon, fenitrothion, malathion and methyl parathion, was fermented by ten lactic acid bacteria (LAB) and four strain combinations at 42°C for 24h. OPPs left in the samples at different times were extracted, purified, detected by gas chromatography and calculated for degradation rate constants, based on a first-order reaction model. OPPs degradation was enhanced by the inoculated LAB, resulting in 0.8-225.4% increase in the rate constants. Diazinon and methyl parathion were more stable whereas chlorpyrifos, fenitrothion and malathion were more labile. Lactobacillus brevis 1.0209 showed the strongest acceleration on OPPs degradation while strain combination could bring about a synergy between the strains of lower ability. Phosphatase production of the strains might be one of the key factors responsible for the enhanced OPPs degradation, as the detected phosphatase activities were positively correlated to the measured degradation rate constants of OPPs (r=0.636-0.970, P<0.05). PMID:24996321

  1. Enhanced degradation of five organophosphorus pesticides in skimmed milk by lactic acid bacteria and its potential relationship with phosphatase production.

    PubMed

    Zhang, Ying-Hua; Xu, Di; Liu, Jia-Qi; Zhao, Xin-Huai

    2014-12-01

    Skimmed milk spiked with five organophosphorus pesticides (OPPs), chlorpyrifos, diazinon, fenitrothion, malathion and methyl parathion, was fermented by ten lactic acid bacteria (LAB) and four strain combinations at 42°C for 24h. OPPs left in the samples at different times were extracted, purified, detected by gas chromatography and calculated for degradation rate constants, based on a first-order reaction model. OPPs degradation was enhanced by the inoculated LAB, resulting in 0.8-225.4% increase in the rate constants. Diazinon and methyl parathion were more stable whereas chlorpyrifos, fenitrothion and malathion were more labile. Lactobacillus brevis 1.0209 showed the strongest acceleration on OPPs degradation while strain combination could bring about a synergy between the strains of lower ability. Phosphatase production of the strains might be one of the key factors responsible for the enhanced OPPs degradation, as the detected phosphatase activities were positively correlated to the measured degradation rate constants of OPPs (r=0.636-0.970, P<0.05).

  2. Effects of synthetic retinoids and retinoic acid isomers on the expression of alkaline phosphatase in F9 teratocarcinoma cells.

    PubMed

    Gianni, M; Zanotta, S; Terao, M; Garattini, S; Garattini, E

    1993-10-15

    Expression of ALP in F9 teratocarcinoma cells is induced by all-trans retinoic acid (ATRA) (Gianni' et al., Biochem. J. 274: 673-678, 1991). The specific ligand for retinoic acid related receptors (RXRs), 9-cis retinoic acid (9-cis RA), and three synthetic analogs binding to the alpha, beta and gamma forms of the retinoic acid receptors (RARs), AM580, CD2019, and CD437, were used to study their effects on alkaline phosphatase (ALP) enzymatic activity and mRNA levels. At concentrations close to the Kd for their respective receptors, 9-cis RA, AM580 (the RAR alpha agonist) and CD437 (the RAR gamma agonist) clearly upregulate the expression of the ALP gene, whereas the effect of CD2019 (the RAR beta agonist) is very modest. A specific inhibitor of the RAR alpha, Ro 41-5253, completely blocks the induction of ALP triggered by AM580, while it has minor effects on the upregulation caused by ATRA, 9-cis RA, CD437 and CD2019. The induction of ALP observed with the various retinoids is inhibited by the contemporaneous treatment with dibutyryl cAMP. The levels of the RAR alpha and gamma transcripts are unaltered, while RAR beta mRNAs are induced by ATRA, AM580, CD437 and to a lower extent by 9-cis RA and CD2019.

  3. A study of acid phosphatase locus 1 in women with high fat content and normal body mass index.

    PubMed

    De Lorenzo, Antonino; Di Renzo, Laura; Puja, Alberto; Saccucci, Patrizia; Gloria-Bottini, Fulvia; Bottini, Egidio

    2009-03-01

    De Lorenzo and coworkers have recently described a class of women with normal body mass index (BMI) and high fat content (normal weight obese syndrome [NWO]). This observation prompted us to study the possible role of acid phosphatase locus 1 (ACP(1)) in the differentiation of this special class of obese subjects. Acid phosphatase locus 1 is a polymorphic gene associated with severe obesity and with total cholesterol and triglycerides levels. The enzyme is composed by 2 isoforms--F and S--that have different biochemical properties and probably different functions. The sample study was composed of 130 white women from the population of Rome. Total fat mass and percentage of fat mass were measured by dual-energy x-ray absorptiometry. Thirty-six women had a BMI less than 25 and percentage of fat mass greater than 30 (high fat, normal BMI [HFHB]), and 94 women showed a BMI greater than 25 and a percentage of fat mass greater than 30 (high fat, high BMI [HFHB]). In the whole sample, the proportion of low-activity ACP(1) genotypes (*A/*A and *B/*A) was higher than in controls. However, whereas HFNB showed a very high frequency of ACP(1) *A/*A genotype, high-fat, high-BMI women showed an increase of *B/*A genotype. These 2 genotypes differ in the concentration of F isoform and the F/S ratio, which are lower in ACP(1)*A/*A genotype than in ACP(1)*B/*A genotype. The genetic differentiation of the class of women with normal BMI and high fat content from the class showing a concordant level of the 2 parameters supports the hypothesis that HFNB class represents a special cluster of obese subjects not revealed by BMI evaluation. Because ACP(1) is present in adipocytes, the present observation suggests that F isoform may have a specific role in the regulation of quantity of adipose tissue.

  4. Induction of a Major Leaf Acid Phosphatase Does Not Confer Adaptation to Low Phosphorus Availability in Common Bean1

    PubMed Central

    Yan, Xiaolong; Liao, Hong; Trull, Melanie C.; Beebe, Steve E.; Lynch, Jonathan P.

    2001-01-01

    Acid phosphatase is believed to be important for phosphorus scavenging and remobilization in plants, but its role in plant adaptation to low phosphorus availability has not been critically evaluated. To address this issue, we compared acid phosphatase activity (APA) in leaves of common bean (Phaseolus vulgaris) in a phosphorus-inefficient genotype (DOR364), a phosphorus-efficient genotype (G19833), and their F5.10 recombinant inbred lines (RILs). Phosphorus deficiency substantially increased leaf APA, but APA was much higher and more responsive to phosphorus availability in DOR364 than in G19833. Leaf APA segregated in the RILs, with two discrete groups having either high (mean = 1.71 μmol/mg protein/min) or low (0.36 μmol/mg protein/min) activity. A chi-square test indicated that the observed difference might be controlled by a single gene. Non-denaturing protein electrophoresis revealed that there are four visible isoforms responsible for total APA in common bean, and that the difference in APA between contrasting genotypes could be attributed to the existence of a single major isoform. Qualitative mapping of the APA trait and quantitative trait loci analysis with molecular markers indicated that a major gene contributing to APA is located on linkage group B03 of the unified common bean map. This locus was not associated with loci conferring phosphorus acquisition efficiency or phosphorus use efficiency. RILs contrasting for APA had similar phosphorus pools in old and young leaves under phosphorus stress, arguing against a role for APA in phosphorus remobilization. Our results do not support a major role for leaf APA induction in regulating plant adaptation to phosphorus deficiency. PMID:11299369

  5. TsPAP1 encodes a novel plant prolyl aminopeptidase whose expression is induced in response to suboptimal growth conditions

    SciTech Connect

    Szawlowska, Urszula; Grabowska, Agnieszka; Zdunek-Zastocka, Edyta; Bielawski, Wieslaw

    2012-03-02

    Highlights: Black-Right-Pointing-Pointer A cDNA encoding a novel plant prolyl aminopeptidase, TsPAP1, was obtained from triticale. Black-Right-Pointing-Pointer The cloned TsPAP1 cDNA is 1387 bp long and encodes a protein of 390 amino acids. Black-Right-Pointing-Pointer The deduced TsPAP1 protein revealed characteristics of the monomeric bacterial PAPs. Black-Right-Pointing-Pointer The TsPAP1 mRNA level increased under drought, salinity and in the presence of metal ions. -- Abstract: A triticale cDNA encoding a prolyl aminopeptidase (PAP) was obtained by RT-PCR and has been designated as TsPAP1. The cloned cDNA is 1387 bp long and encodes a protein of 390 amino acids with a calculated molecular mass of 43.9 kDa. The deduced TsPAP1 protein exhibits a considerable sequence identity with the biochemically characterized bacterial and fungal PAP proteins of small molecular masses ({approx}35 kDa). Moreover, the presence of conserved regions that are characteristic for bacterial monomeric PAP enzymes (the GGSWG motif, the localization of the catalytic triad residues and the segment involved in substrate binding) has also been noted. Primary structure analysis and phylogenetic analysis revealed that TsPAP1 encodes a novel plant PAP protein that is distinct from the multimeric proteins that have thus far been characterized in plants and whose counterparts have been recognized only in bacteria and fungi. A significant increase in the TsPAP1 transcript level in the shoots of triticale plants was observed under drought and saline conditions as well as in the presence of cadmium and aluminium ions in the nutrient medium. This paper is the first report describing changes in the transcript levels of any plant PAP in response to suboptimal growth conditions.

  6. Characterization of a soluble phosphatidic acid phosphatase in bitter melon (Momordica charantia)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Momordica charantia is often called bitter melon, bitter gourd or bitter squash because its fruit has a bitter taste. The fruit has been widely used as vegetable and herbal medicine. Alpha-eleostearic acid is the major fatty acid in the seeds, but little is known about its biosynthesis. As an initia...

  7. Time-dependent effect of p-Aminophenol (PAP) toxicity in renal slices and development of oxidative stress

    SciTech Connect

    Harmon, R. Christopher; Terneus, Marcus V.; Kiningham, Kinsley K.; Valentovic, Monica . E-mail: Valentov@marshall.edu

    2005-11-15

    p-Aminophenol (PAP), a metabolite of acetaminophen, is nephrotoxic. This study investigated PAP-mediated changes as a function of time that occur prior to loss of membrane integrity. Experiments further evaluated the development of oxidative stress by PAP. Renal slices from male Fischer 344 (F344) rats (N = 4-6) were exposed to 0.1, 0.25, and 0.5 mM PAP for 15-120 min under oxygen and constant shaking at 37 {sup o}C. Pyruvate-stimulated gluconeogenesis, adenine nucleotide levels, and total glutathione (GSH) levels were diminished in a concentration- and time-dependent manner prior to detection of a rise in lactate dehydrogenase (LDH) leakage. Glutathione disulfide (GSSG) levels were increased by PAP suggesting the induction of oxidative stress. Western blot analysis confirmed a rise in 4-hydroxynonenal (4-HNE)-adducted proteins in tissues exposed to 0.1 and 0.25 mM PAP for 90 min. The appearance of 4-HNE-adducted proteins at the 0.1 mM concentration of PAP occurred prior to development of increased LDH leakage. Pretreatment with 1 mM glutathione (GSH) for 30 min only partially reduced PAP toxicity as LDH values were less severely depleted relative to tissues not pretreated with GSH. In contrast, pretreatment for 15 min with 2 mM ascorbic acid completely protected against PAP toxicity. Further studies showed that ascorbic acid pretreatment prevented PAP-mediated depletion of GSH. In summary, PAP rapidly depletes GSH and adenine nucleotides and inhibits gluconeogenesis prior to a rise in LDH leakage. PAP induces oxidative stress as indicated by an increase in GSSG and 4-HNE-adducted proteins. Ascorbic acid pretreatment prevents PAP toxicity by maintaining GSH status.

  8. Structure of recombinant Haemophilus influenzae e (P4) acid phosphatase reveals a new member of the haloacid dehalogenase superfamily.

    PubMed

    Felts, Richard L; Ou, Zhonghui; Reilly, Thomas J; Tanner, John J

    2007-10-01

    Lipoprotein e (P4) from Haemophilus influenzae belongs to the "DDDD" superfamily of phosphohydrolases and is the prototype of class C nonspecific acid phosphatases. P4 is also a component of a H. influenzae vaccine. We report the crystal structures of recombinant P4 in the ligand-free and tungstate-inhibited forms, which are the first structures of a class C phosphatase. P4 has a two-domain architecture consisting of a core alpha/beta domain and a smaller alpha domain. The core domain features a five-stranded beta-sheet flanked by helices on both sides that is reminiscent of the haloacid dehalogenase superfamily. The alpha domain appears to be unique and plays roles in substrate binding and dimerization. The active site is solvent accessible and located in a cleft between the two domains. The structure shows that P4 is a metalloenzyme and that magnesium is the most likely metal ion in the crystalline recombinant enzyme. The ligands of the metal ion are the carboxyl groups of the first and third Asp residues of the DDDD motif, the backbone carbonyl of the second Asp of the DDDD motif, and two water molecules. The structure of the tungstate-bound enzyme suggests that Asp64 is the nucleophile that attacks the substrate P atom. Dimerization appears to be important for catalysis because intersubunit contacts stabilize the active site. Analysis of the structural context of mutations engineered for vaccine studies shows that the most promising mutations are located in the dimer interface. This observation suggests a structure-based vaccine design strategy in which the dimer interface is disrupted in order to expose epitopes that are buried in dimeric P4.

  9. Clinical usefulness of serum tartrate-resistant fluoride-sensitive acid phosphatase activity in evaluating bone turnover.

    PubMed

    Nakanishi, M; Yoh, K; Uchida, K; Maruo, S; Rai, S K; Matsuoka, A

    1999-01-01

    This study was carried out to evaluate the clinical validity and usefulness of serum tartrate-resistant fluoride-sensitive acid phosphatase (TrFsACP) activity using 2,6-dichloro-4-acetylphenyl phosphate as substrate at pH 6.2 in metabolic bone diseases. The mean Z-scores of TrFsACP activity in patients on hemodialysis were higher than in healthy subjects (male: 2.04+/-1.98, n = 49, P < .05; female: 1.49+/-2.43, n = 39, P < .05) and increased with duration of hemodialysis (r = .516, P < .01). Bone alkaline phosphatase also was found to be significantly higher in hemodialysis patients (male: 0.93+/-1.49, P < .05; female: 1.66+/-2.42, P < .05) compared with normal subjects: but had lower correlation with duration of hemodialysis than TrFsACP (r = .277, P < .05). Ulcerative colitis (1.37+/-2.21, n = 15) in males showed a significantly higher Z-score of TrFsACP compared with control subjects (P < .05). The relationship of TrFsACP activity and ultrasound findings (stiffness; speed of sound [SOS]; broadband ultra sound attenuation [BUA]) in healthy women aged 30-75 years (n = 95) were inversely and significantly correlated with stiffness (r = -.465, P < .01 ), SOS (r = -.484, P < .01), and BUA (r = -.366, P < .01), but were age dependent. TrFsACP activity significantly correlated with stiffness (r = -.521, P < .05) and SOS (r = -.527, P < .05) only in the age group of 46-55 years. BUA (r = -.313, P > .05) did not correlate significantly in any subject in the present study. We conclude that serum TrFsACP activity is useful in the diagnosis and monitoring of bone turnover.

  10. Boswellic Acid Blocks STAT3 Signaling, Proliferation, and Survival of Multiple Myeloma via the Protein Tyrosine Phosphatase SHP-1

    PubMed Central

    Kunnumakkara, Ajaikumar B.; Nair, Asha S.; Sung, Bokyung; Pandey, Manoj K.; Aggarwal, Bharat B.

    2009-01-01

    Activation of signal transducers and activators of transcription (STAT)-3 factors has been linked with survival, proliferation, chemoresistance and angiogenesis of tumor cells, including human multiple myeloma (MM). Thus agents that can suppress STAT3 activation have potential as cancer therapeutics. In our search for such agents, we identified acetyl-11-keto-β-boswellic acid (AKBA), originally isolated from Boswellia serrata. Our results show that AKBA inhibited constitutive STAT3 activation in human MM cells. AKBA suppressed IL-6-induced STAT3 activation, and the inhibition was reversible. The phosphorylation of both Jak 2 and Src, constituents of the STAT3 pathway, was inhibited by AKBA. Interestingly, treatment of cells with pervanadate suppressed AKBA’s effect to inhibit the phosphorylation of STAT3, thus suggesting the involvement of a protein tyrosine phosphatase. We found that AKBA induced Src homology region 2 domain-containing phosphatase 1 (SHP-1), which may account for its role in dephosphorylation of STAT3. Moreover, deletion of SHP-1 gene by SiRNA abolished the ability of AKBA to inhibit STAT3 activation. The inhibition of STAT3 activation by AKBA led to the suppression of gene products involved in proliferation (cyclin D1), survival (Bcl-2, Bcl-xL and Mcl-1), and angiogenesis (VEGF). This affect correlated with the inhibition of proliferation and apoptosis in MM cells. Consistent with these results, overexpression of constitutive active STAT3 significantly reduced the AKBA induced apoptosis. Overall, our results suggest that AKBA is a novel inhibitor of STAT3 activation and has potential in the treatment of cancer. PMID:19147543

  11. Okadaic acid indicates a major function for protein phosphatases in stimulus-response coupling of RINm5F rat insulinoma cells.

    PubMed

    Mayer, P; Jochum, C; Schatz, H; Pfeiffer, A

    1994-01-01

    Stimulus-induced insulin secretion involves the activation of several protein kinases within the beta cell. Most prominent are protein kinase A, protein kinase C and calcium/calmodulin-dependent protein kinases. Protein kinase action is functionally antagonized by protein phosphatases. The four ubiquious serine/threonine protein phosphatases are termed PP-1, PP-2A, -2B and -2C. PP-1 and PP-2A are in vivo parts of major protein complexes. These complexes presumably regulate the phosphatase activity and direct the enzyme to its site of action. Therefore, PP-1 and -2A could play an important role in controlling intracellular signal transmission. Two different toxins, okadaic acid and calyculin A, both from marine invertebrates, were recently discovered and identified as potent and highly specific inhibitors of PP-1 and PP-2A. Both compounds emerged as very useful tools for studying intracellular phosphorylation events. We took advantage of these substances to investigate the significance of protein phosphatase action in stimulus-induced insulin secretion. To avoid major complexity, we confined our study to the cAMP and the phosphoinositide signal pathway. Okadaic acid alone evoked virtually no secretory response. cAMP-dependent secretion was markedly enhanced by 1 microM okadaic acid. The stimulatory effect of okadaic acid was strongly dependent on the concentration of cAMP analoga. In contrast, insulin release caused by the cholinergic agonist carbachol was not influenced by okadaic acid. Calyculin A (10 nM) slightly increased cAMP-induced secretion, but its high toxicity prohibited accurate interpretation of the data. Our findings support the idea that serine/threonine phosphatases act as important regulators in stimulus response coupling.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Frequency and organization of papA homologous DNA sequences among uropathogenic digalactoside-binding Escherichia coli strains.

    PubMed

    Denich, K; Craiu, A; Rugo, H; Muralidhar, G; O'Hanley, P

    1991-06-01

    The frequency of selected papA DNA sequences among 89 digalactoside-binding, uropathogenic Escherichia coli strains was evaluated with 12 different synthetic 15-base probes corresponding to papA genes from four digalactoside-binding piliated recombinant strains (HU849, 201B, and 200A). The papA probes encode amino acids which are common at the carboxy terminus of all strains, adjacent to the proximal portion of the intramolecular disulfide loop of strain 210B, or predicted to constitute the type-specific epitope for each of the four recombinant strains or other epitopes of strain HU849. The presence among the strains of DNA sequence homology to the papA probes was determined by in situ colony hybridization. Hybridization data suggest that there is a high frequency of homologous papA DNA sequences corresponding to selected regions of the papA gene from strain HU849 among the clinical strains. The following nucleotide locations which encode portions of the mature HU849 PapA are detected in a high percentage (42 to 70%) of clinical isolates: 208 to 222, 310 to 324, 478 to 492, 517 to 531, 553 to 567, and 679 to 693. These sequences encode portions of the predicted protective, immunogenic, and/or antigenic epitopes of this PapA. The data also indicate considerable heterogeneity of papA sequences among the strains, especially in the region of nucleotide bases corresponding to positions 391 to 418. These oligonucleotides encode the predicted PapA type-specific immunogenic dominant epitope. Determination of the extent of genetic variability in the papA gene among digalactoside-binding strains will require more extensive DNA sequencing of prototypic papA genes, additional hybridization studies employing other papA gene oligonucleotide probes, and assessment of the different pap operons and their copy number in each strain.

  13. Voltage-dependent motion of the catalytic region of voltage-sensing phosphatase monitored by a fluorescent amino acid

    PubMed Central

    Sakata, Souhei; Jinno, Yuka; Kawanabe, Akira; Okamura, Yasushi

    2016-01-01

    The cytoplasmic region of voltage-sensing phosphatase (VSP) derives the voltage dependence of its catalytic activity from coupling to a voltage sensor homologous to that of voltage-gated ion channels. To assess the conformational changes in the cytoplasmic region upon activation of the voltage sensor, we genetically incorporated a fluorescent unnatural amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), into the catalytic region of Ciona intestinalis VSP (Ci-VSP). Measurements of Anap fluorescence under voltage clamp in Xenopus oocytes revealed that the catalytic region assumes distinct conformations dependent on the degree of voltage-sensor activation. FRET analysis showed that the catalytic region remains situated beneath the plasma membrane, irrespective of the voltage level. Moreover, Anap fluorescence from a membrane-facing loop in the C2 domain showed a pattern reflecting substrate turnover. These results indicate that the voltage sensor regulates Ci-VSP catalytic activity by causing conformational changes in the entire catalytic region, without changing their distance from the plasma membrane. PMID:27330112

  14. Over-expression of AtPAP2 in Camelina sativa leads to faster plant growth and higher seed yield

    PubMed Central

    2012-01-01

    Background Lipids extracted from seeds of Camelina sativa have been successfully used as a reliable source of aviation biofuels. This biofuel is environmentally friendly because the drought resistance, frost tolerance and low fertilizer requirement of Camelina sativa allow it to grow on marginal lands. Improving the species growth and seed yield by genetic engineering is therefore a target for the biofuels industry. In Arabidopsis, overexpression of purple acid phosphatase 2 encoded by Arabidopsis (AtPAP2) promotes plant growth by modulating carbon metabolism. Overexpression lines bolt earlier and produce 50% more seeds per plant than wild type. In this study, we explored the effects of overexpressing AtPAP2 in Camelina sativa. Results Under controlled environmental conditions, overexpression of AtPAP2 in Camelina sativa resulted in longer hypocotyls, earlier flowering, faster growth rate, higher photosynthetic rate and stomatal conductance, increased seed yield and seed size in comparison with the wild-type line and null-lines. Similar to transgenic Arabidopsis, activity of sucrose phosphate synthase in leaves of transgenic Camelina was also significantly up-regulated. Sucrose produced in photosynthetic tissues supplies the building blocks for cellulose, starch and lipids for growth and fuel for anabolic metabolism. Changes in carbon flow and sink/source activities in transgenic lines may affect floral, architectural, and reproductive traits of plants. Conclusions Lipids extracted from the seeds of Camelina sativa have been used as a major constituent of aviation biofuels. The improved growth rate and seed yield of transgenic Camelina under controlled environmental conditions have the potential to boost oil yield on an area basis in field conditions and thus make Camelina-based biofuels more environmentally friendly and economically attractive. PMID:22472516

  15. Roles of arachidonic acid, lipoxygenases and phosphatases in calcium-dependent modulation of M-current in bullfrog sympathetic neurons.

    PubMed Central

    Yu, S P

    1995-01-01

    1. M-current (IM) is regulated by intracellular free Ca2+ ([Ca2+]i). Suppression and overrecovery of IM induced by muscarine and luteinizing-hormone releasing hormone (LHRH) are also regulated by [Ca2+]i. The role of the arachidonic acid (AA) pathway in the Ca(2+)-dependent modulation of IM was investigated using whole-cell voltage clamp and intracellular perfusion in dissociated bullfrog sympathetic B neurons. 2. Quinacrine (10-20 microM) and 4-bromophenacyl bromide (4-BPB; 4-10 microM), the inhibitors of phospholipase A2, blocked the enhancement of IM evoked by raising [Ca2+]i. 3. AA (6-120 microM) increased IM by about 50% of the control current in a Ca(2+)-dependent manner. 4. Enhancements of IM by Ca2+ and AA were blocked by the lipoxygenase (LO) inhibitors nordihydroguaiaretic acid (NDGA; 1-5 microM) and 5,8,11-eicosatrynoic acid (ETI; 10 microM). The cyclo-oxygenase inhibitor indomethacin (10 microM) had no effect. 5. Enhancement of IM by Ca2+ was abolished by the selective 12-LO inhibitors baicalein (1-2 microM) and 15(S)-hydroxy-5-cis-8-cis-11-cis-13-trans-eicosatetraenoic acid (15-HETE; 6.5 microM). A 12-LO product, 2(S)-hydroxy-5-cis-8-cis-10-trans-14-cis- eicosatetraenoic acid (12-HETE; 13-20 microM), increased IM without Ca2+ requirement. 6. Enhancement of IM by Ca2+ was not affected by the selective 5-LO inhibitors AA-861 (10 microM), 5,6-dehydroarachidonic acid (5,6-DAA, 10 microM) and L-651,896 (10 microM). The 5-LO metabolites leukotriene C4 (1.5-8 microM) and leukotriene B4 (1.5-5 microM) showed no obvious effect on IM. 7. NDGA alone inhibited IM with an IC50 of 0.73 microM at 120 nM Cai(2+). 8. NDGA did not affect suppression of IM by muscarine or LHRH; however, overrecovery of IM upon removing these agonists was totally eliminated by 1 microM NDGA. 9. Inhibitors of phosphatases, calyculin A (0.1 microM) and okadaic acid (1 microM), completely abolished overrecovery of IM. Calyculin A also blocked the Ca(2+)-induced IM enhancement. 10. It is

  16. Phosphatase Under-Producer Mutants Have Altered Phosphorus Relations1

    PubMed Central

    Tomscha, Jennifer L.; Trull, Melanie C.; Deikman, Jill; Lynch, Jonathan P.; Guiltinan, Mark J.

    2004-01-01

    Phosphorus (P) acquisition and partitioning are essential for plant homeostasis. P is available for plant uptake when in its inorganic form (H2PO4−, or Pi), but Pi is often limiting in soils. Plants secrete acid phosphatases (APases) into the apoplastic space, which may be important for obtaining Pi from organic P sources; however, the relative importance of these enzymes for plant P nutrition has yet to be determined. We demonstrate that the root-associated APase pool is increased in Arabidopsis when Pi is limiting and document five APase isoforms secreted from Arabidopsis roots. Previously, we presented the identification of the phosphatase under-producer (pup) mutants, which have decreased in vivo root APase staining when grown under low P conditions. Here, we present the characterization of one of these, pup3, and further studies with pup1. pup3 has 49%, 38%, and 37% less specific APase activity in exudates, roots, and shoots, respectively. Root-associated APase activity is decreased by 16% in pup1 and 25% in pup3, regardless of P treatment. Two APase activity isoforms are reduced in pup3 exudates, and root and shoot isoforms are also affected. One of the two exudate isoforms is recognized by a polyclonal antibody raised to an Arabidopsis purple APase recombinant protein (AtPAP12); however, AtPAP12 transcript levels are unaffected in the mutant. The pup3 mutation was mapped to 68.4 ± 6.0 centimorgans on chromosome 5. Although P concentrations were not altered in pup1 and pup3 tissues when grown in nutrient solution in which Pi was the sole source of P, the mutants had 10% (pup1) and 17% (pup3) lower shoot P concentrations when grown in a peat-vermiculite mix in which the majority of the total P was present as organic P. Therefore, the pup defects, which include secreted APases, are functionally important for plant P nutrition. PMID:15122033

  17. Membrane Topology and Biochemical Characterization of the Escherichia coli BacA Undecaprenyl-Pyrophosphate Phosphatase

    PubMed Central

    Manat, Guillaume; El Ghachi, Meriem; Auger, Rodolphe; Baouche, Karima; Olatunji, Samir; Kerff, Frédéric; Touzé, Thierry; Mengin-Lecreulx, Dominique; Bouhss, Ahmed

    2015-01-01

    Several integral membrane proteins exhibiting undecaprenyl-pyrophosphate (C55-PP) phosphatase activity were previously identified in Escherichia coli that belonged to two distinct protein families: the BacA protein, which accounts for 75% of the C55-PP phosphatase activity detected in E. coli cell membranes, and three members of the PAP2 phosphatidic acid phosphatase family, namely PgpB, YbjG and LpxT. This dephosphorylation step is required to provide the C55-P carrier lipid which plays a central role in the biosynthesis of various cell wall polymers. We here report detailed investigations of the biochemical properties and membrane topology of the BacA protein. Optimal activity conditions were determined and a narrow-range substrate specificity with a clear preference for C55-PP was observed for this enzyme. Alignments of BacA protein sequences revealed two particularly well-conserved regions and several invariant residues whose role in enzyme activity was questioned by using a site-directed mutagenesis approach and complementary in vitro and in vivo activity assays. Three essential residues Glu21, Ser27, and Arg174 were identified, allowing us to propose a catalytic mechanism for this enzyme. The membrane topology of the BacA protein determined here experimentally did not validate previous program-based predicted models. It comprises seven transmembrane segments and contains in particular two large periplasmic loops carrying the highly-conserved active site residues. Our data thus provide evidence that all the different E. coli C55-PP phosphatases identified to date (BacA and PAP2) catalyze the dephosphorylation of C55-PP molecules on the same (outer) side of the plasma membrane. PMID:26560897

  18. [Acid phosphatase of leukocytes in patients with diffuse toxic goiter in evaluating the stage of the disease].

    PubMed

    Vaiuta, N P; Livshits, A Kh; Mendeleev, I M

    1987-01-01

    Acid phosphatase (AP) of lymphocytes and neutrophils was examined cytochemically in 23 patients with associated diffuse toxic goiter (DTG) and thyrotoxicosis during thiamazol treatment, in 20 persons with a DTG remission, and in 5 patients with postradiation and postoperative hypothyroses. AP activity with multiple-granule distribution of the enzyme was high in the majority of the patients with thyrotoxicosis, decreasing with thiamazol treatment. Thyrotoxicosis recurred in 4 out of 11 patients whose lymphocytes had an increased amount of AP during a DTG remission. In 9 patients with normal AP content, no relapses were noted during a DTG remission. Two patients with iatrogenic hypothyroses associated with high lymphocytic AP activity manifested elevated titres of antithyroglobulin immunoglobulins (ATI). In 3 patients with normal content of cellular AP, the ATI titre was low. Cellular AP, a non-specific marker of immunogenesis activity, makes it possible to presumably differentiate the stages of DTG and to evaluate to a definite degree the character of a remission. The preserved high activity of lymphocytic AP in patients with a DTG remission is a prognostically unfavourable factor as regards thyrotoxicosis relapses. The high titre of ATI and activity of lymphocytic AP attest to the predominant autoimmune component of such hypothyroses in part of DTG patients. The authors stress that such patients should receive combined thyroid and glucocorticoid therapy.

  19. A spheroid-based 3-D culture model for pancreatic cancer drug testing, using the acid phosphatase assay.

    PubMed

    Wen, Z; Liao, Q; Hu, Y; You, L; Zhou, L; Zhao, Y

    2013-07-01

    Current therapy for pancreatic cancer is multimodal, involving surgery and chemotherapy. However, development of pancreatic cancer therapies requires a thorough evaluation of drug efficacy in vitro before animal testing and subsequent clinical trials. Compared to two-dimensional culture of cell monolayer, three-dimensional (3-D) models more closely mimic native tissues, since the tumor microenvironment established in 3-D models often plays a significant role in cancer progression and cellular responses to the drugs. Accumulating evidence has highlighted the benefits of 3-D in vitro models of various cancers. In the present study, we have developed a spheroid-based, 3-D culture of pancreatic cancer cell lines MIAPaCa-2 and PANC-1 for pancreatic drug testing, using the acid phosphatase assay. Drug efficacy testing showed that spheroids had much higher drug resistance than monolayers. This model, which is characteristically reproducible and easy and offers rapid handling, is the preferred choice for filling the gap between monolayer cell cultures and in vivo models in the process of drug development and testing for pancreatic cancer.

  20. Salicylic Acid Based Small Molecule Inhibitor for the Oncogenic Src Homology-2 Domain Containing Protein Tyrosine Phosphatase-2 (SHP2)

    SciTech Connect

    Zhang, Xian; He, Yantao; Liu, Sijiu; Yu, Zhihong; Jiang, Zhong-Xing; Yang, Zhenyun; Dong, Yuanshu; Nabinger, Sarah C.; Wu, Li; Gunawan, Andrea M.; Wang, Lina; Chan, Rebecca J.; Zhang, Zhong-Yin

    2010-08-13

    The Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) plays a pivotal role in growth factor and cytokine signaling. Gain-of-function SHP2 mutations are associated with Noonan syndrome, various kinds of leukemias, and solid tumors. Thus, there is considerable interest in SHP2 as a potential target for anticancer and antileukemia therapy. We report a salicylic acid based combinatorial library approach aimed at binding both active site and unique nearby subpockets for enhanced affinity and selectivity. Screening of the library led to the identification of a SHP2 inhibitor II-B08 (compound 9) with highly efficacious cellular activity. Compound 9 blocks growth factor stimulated ERK1/2 activation and hematopoietic progenitor proliferation, providing supporting evidence that chemical inhibition of SHP2 may be therapeutically useful for anticancer and antileukemia treatment. X-ray crystallographic analysis of the structure of SHP2 in complex with 9 reveals molecular determinants that can be exploited for the acquisition of more potent and selective SHP2 inhibitors.

  1. Influence of swine manure on growth, P uptake and activities of acid phosphatase and phytase of Polygonum hydropiper.

    PubMed

    Ye, Daihua; Li, Tingxuan; Chen, Guangdeng; Zheng, Zicheng; Yu, Haiying; Zhang, Xizhou

    2014-06-01

    Excessive application of animal manure to the farmland results in enrichment of P in the soil. Phytoremediation is a promising strategy for extracting excess P from manure impacted soil. P uptake characteristics of a mining ecotype (ME) and a non-mining ecotype (NME) of Polygonum hydropiper were investigated in this study by adopting soil culture containing various concentrations of swine manure (0-200 g swine manure kg(-1) soil). A peak value in the biomass of P. hydropiper was determined in 100 g kg(-1) soil. Significant increase of P content in tissues of two ecotypes was noticed with an increase in swine manure concentration. Maximum P accumulation in shoots and roots was observed at the concentration of 100 g kg(-1) soil, however, the ME accumulated more P as compared to the NME. The ME showed a lower plant effective number and a higher P extraction ratio compared to the NME. Both acid phosphatase and phytase activities of P. hydropiper were obviously enhanced under swine manure impacted soil compared with control, while those of ME higher than the NMEs. Therefore, the two ecotypes of P. hydropiper can accumulate P from soil amended with swine manure and establishes the foundation for phytoremediation.

  2. Active inclusion bodies of acid phosphatase PhoC: aggregation induced by GFP fusion and activities modulated by linker flexibility

    PubMed Central

    2013-01-01

    Background Biologically active inclusion bodies (IBs) have gained much attention in recent years. Fusion with IB-inducing partner has been shown to be an efficient strategy for generating active IBs. To make full use of the advantages of active IBs, one of the key issues will be to improve the activity yield of IBs when expressed in cells, which would need more choices on IB-inducing fusion partners and approaches for engineering IBs. Green fluorescent protein (GFP) has been reported to aggregate when overexpressed, but GFP fusion has not been considered as an IB-inducing approach for these fusion proteins so far. In addition, the role of linker in fusion proteins has been shown to be important for protein characteristics, yet impact of linker on active IBs has never been reported. Results Here we report that by fusing GFP and acid phosphatase PhoC via a linker region, the resultant PhoC-GFPs were expressed largely as IBs. These IBs show high levels of specific fluorescence and specific PhoC activities (phosphatase and phosphotransferase), and can account for up to over 80% of the total PhoC activities in the cells. We further demonstrated that the aggregation of GFP moiety in the fusion protein plays an essential role in the formation of PhoC-GFP IBs. In addition, PhoC-GFP IBs with linkers of different flexibility were found to exhibit different levels of activities and ratios in the cells, suggesting that the linker region can be utilized to manipulate the characteristics of active IBs. Conclusions Our results show that active IBs of PhoC can be generated by GFP fusion, demonstrating for the first time the potential of GFP fusion to induce active IB formation of another soluble protein. We also show that the linker sequence in PhoC-GFP fusion proteins plays an important role on the regulation of IB characteristics, providing an alternative and important approach for engineering of active IBs with the goal of obtaining high activity yield of IBs. PMID:23497261

  3. Phosphatase inhibitors remove the run-down of γ-aminobutyric acid type A receptors in the human epileptic brain

    PubMed Central

    Palma, E.; Ragozzino, D. A.; Di Angelantonio, S.; Spinelli, G.; Trettel, F.; Martinez-Torres, A.; Torchia, G.; Arcella, A.; Di Gennaro, G.; Quarato, P. P.; Esposito, V.; Cantore, G.; Miledi, R.; Eusebi, F.

    2004-01-01

    The properties of γ-aminobutyric acid (GABA) type A receptors (GABAA receptors) microtransplanted from the human epileptic brain to the plasma membrane of Xenopus oocytes were compared with those recorded directly from neurons, or glial cells, in human brains slices. Cell membranes isolated from brain specimens, surgically obtained from six patients afflicted with drug-resistant temporal lobe epilepsy (TLE) were injected into frog oocytes. Within a few hours, these oocytes acquired GABAA receptors that generated GABA currents with an unusual run-down, which was inhibited by orthovanadate and okadaic acid. In contrast, receptors derived from membranes of a nonepileptic hippocampal uncus, membranes from mouse brain, or recombinant rat α1β2γ2-GABA receptors exhibited a much less pronounced GABA-current run-down. Moreover, the GABAA receptors of pyramidal neurons in temporal neocortex slices from the same six epileptic patients exhibited a stronger run-down than the receptors of rat pyramidal neurons. Interestingly, the GABAA receptors of neighboring glial cells remained substantially stable after repetitive activation. Therefore, the excessive GABA-current run-down observed in the membrane-injected oocytes recapitulates essentially what occurs in neurons, rather than in glial cells. Quantitative RT-PCR analyses from the same TLE neocortex specimens revealed that GABAA-receptor β1, β2, β3, and γ2 subunit mRNAs were significantly overexpressed (8- to 33-fold) compared with control autopsy tissues. Our results suggest that an abnormal GABA-receptor subunit transcription in the TLE brain leads to the expression of run-down-enhanced GABAA receptors. Blockage of phosphatases stabilizes the TLE GABAA receptors and strengthens GABAergic inhibition. It may be that this process can be targeted to develop new treatments for intractable epilepsy. PMID:15218107

  4. Estimation of the rate constants associated with the inhibitory effect of okadaic acid on type 2A protein phosphatase by time-course analysis.

    PubMed Central

    Takai, A; Ohno, Y; Yasumoto, T; Mieskes, G

    1992-01-01

    As is often the case with tightly binding inhibitors, okadaic acid produces its inhibitory effect on type 2A protein phosphatase (PP2A) in a time-dependent manner. We measured the rate constants associated with the binding of okadaic acid to PP2A by analysing the time-course of the reduction of the p-nitrophenyl phosphate (pNPP) phosphatase activity of the enzyme after application of okadaic acid. The rate constants for dissociation of okadaic acid from PP2A were also estimated from the time-course of the recovery of the activity from inhibition by okadaic acid after addition of a mouse IgG1 monoclonal antibody raised against the inhibitor. Our results show that the rate constants for the binding of okadaic acid and PP2A are of the order of 10(7) M-1.s-1, a typical value for reactions involving relatively large molecules, whereas those for their dissociation are in the range 10(-4)-10(-3) s-1. The very low values of the latter seems to be the determining factor for the exceedingly high affinity of okadaic acid for PP2A. The dissociation constants for the interaction of okadaic acid with the free enzyme and the enzyme-substrate complex, estimated as the ratio of the rate constants, are both in the range 30-40 pM, in agreement with the results of previous dose-inhibition analyses. PMID:1329723

  5. Activation of protein phosphatase 2A is responsible for increased content and inactivation of respiratory chain complex i induced by all-trans retinoic acid in human keratinocytes.

    PubMed

    Papa, F; Sardaro, N; Lippolis, R; Panelli, D; Scacco, S

    2016-01-01

    This study presents the effect of all-trans retinoic acid (ATRA) on cell growth and respiratory chain complex I in human keratinocyte cultures. Keratinocyte treatment results in increased level of GRIM-19 and other subunits of complex I, in particular of their carbonylated forms, associated with inhibition of its enzymatic activity. The results show that in keratinocytes ATRA-promoted phosphatase activity controls the proteostasis and activity of complex I. PMID:27358125

  6. Effect of Induced Oxidative Stress and Herbal Extracts on Acid Phosphatase Activity in Lysosomal and Microsomal Fractions of Midgut Tissue of the Silkworm, Bombyx mori

    PubMed Central

    Gaikwad, Y. B.; Gaikwad, S. M.; Bhawane, G. P.

    2010-01-01

    Lysosomal and microsomal acid phosphatase activity was estimated in midgut tissue of silkworm larvae, Bombyx mori L. (Lepidoptera: Bombycidae), after induced oxidative stress by D-galactose. The larvae were simultaneously were treated with ethanolic extracts of Bacopa monniera and Lactuca sativa to study their antioxidant properties. Lipid peroxidation and fluorescence was measured to analyze extent of oxidative stress. The ethanolic extract of Lactuca sativa was found to be more effective in protecting membranes against oxidative stress than Bacopa monniera. PMID:20874583

  7. Detection of Ca2+-dependent acid phosphatase activity identifies neuronal integrity in damaged rat central nervous system after application of bacterial melanin

    PubMed Central

    Petrosyan, Tigran R.; Ter-Markosyan, Anna S.; Hovsepyan, Anna S.

    2016-01-01

    The study aims to confirm the neuroregenerative effects of bacterial melanin (BM) on central nervous system injury using a special staining method based on the detection of Ca2+-dependent acid phosphatase activity. Twenty-four rats were randomly assigned to undergo either unilateral destruction of sensorimotor cortex (group I; n = 12) or unilateral rubrospinal tract transection at the cervical level (C3–4) (group II; n = 12). In each group, six rats were randomly selected after surgery to undergo intramuscular injection of BM solution (BM subgroup) and the remaining six rats were intramuscularly injected with saline (saline subgroup). Neurological testing confirmed that BM accelerated the recovery of motor function in rats from both BM and saline subgroups. Two months after surgery, Ca2+-dependent acid phosphatase activity detection in combination with Chilingarian’s calcium adenoside triphosphate method revealed that BM stimulated the sprouting of fibers and dilated the capillaries in the brain and spinal cord. These results suggest that BM can promote the recovery of motor function of rats with central nervous system injury; and detection of Ca2+-dependent acid phosphatase activity is a fast and easy method used to study the regeneration-promoting effects of BM on the injured central nervous system.

  8. Detection of Ca2+-dependent acid phosphatase activity identifies neuronal integrity in damaged rat central nervous system after application of bacterial melanin

    PubMed Central

    Petrosyan, Tigran R.; Ter-Markosyan, Anna S.; Hovsepyan, Anna S.

    2016-01-01

    The study aims to confirm the neuroregenerative effects of bacterial melanin (BM) on central nervous system injury using a special staining method based on the detection of Ca2+-dependent acid phosphatase activity. Twenty-four rats were randomly assigned to undergo either unilateral destruction of sensorimotor cortex (group I; n = 12) or unilateral rubrospinal tract transection at the cervical level (C3–4) (group II; n = 12). In each group, six rats were randomly selected after surgery to undergo intramuscular injection of BM solution (BM subgroup) and the remaining six rats were intramuscularly injected with saline (saline subgroup). Neurological testing confirmed that BM accelerated the recovery of motor function in rats from both BM and saline subgroups. Two months after surgery, Ca2+-dependent acid phosphatase activity detection in combination with Chilingarian’s calcium adenoside triphosphate method revealed that BM stimulated the sprouting of fibers and dilated the capillaries in the brain and spinal cord. These results suggest that BM can promote the recovery of motor function of rats with central nervous system injury; and detection of Ca2+-dependent acid phosphatase activity is a fast and easy method used to study the regeneration-promoting effects of BM on the injured central nervous system. PMID:27630700

  9. Detection of Ca(2+)-dependent acid phosphatase activity identifies neuronal integrity in damaged rat central nervous system after application of bacterial melanin.

    PubMed

    Petrosyan, Tigran R; Ter-Markosyan, Anna S; Hovsepyan, Anna S

    2016-07-01

    The study aims to confirm the neuroregenerative effects of bacterial melanin (BM) on central nervous system injury using a special staining method based on the detection of Ca(2+)-dependent acid phosphatase activity. Twenty-four rats were randomly assigned to undergo either unilateral destruction of sensorimotor cortex (group I; n = 12) or unilateral rubrospinal tract transection at the cervical level (C3-4) (group II; n = 12). In each group, six rats were randomly selected after surgery to undergo intramuscular injection of BM solution (BM subgroup) and the remaining six rats were intramuscularly injected with saline (saline subgroup). Neurological testing confirmed that BM accelerated the recovery of motor function in rats from both BM and saline subgroups. Two months after surgery, Ca(2+)-dependent acid phosphatase activity detection in combination with Chilingarian's calcium adenoside triphosphate method revealed that BM stimulated the sprouting of fibers and dilated the capillaries in the brain and spinal cord. These results suggest that BM can promote the recovery of motor function of rats with central nervous system injury; and detection of Ca(2+)-dependent acid phosphatase activity is a fast and easy method used to study the regeneration-promoting effects of BM on the injured central nervous system. PMID:27630700

  10. A simple-potentiometric method for determination of acid and alkaline phosphatase enzymes in biological fluids and dairy products using a nitrophenylphosphate plastic membrane sensor.

    PubMed

    Hassan, Saad S M; Sayour, Hossam E M; Kamel, Ayman H

    2009-04-27

    A novel poly(vinyl chloride) matrix membrane sensor responsive to 4-nitrophenylphosphate (4-NPP) substrate is described, characterized and used for the potentiometric assay of acid (ACP) and alkaline (ALP) phosphatase enzymes. The sensor is based on the use of the ion-association complex of 4-NPP anion with nickel(II)-bathophenanthroline cation as an electroactive material and nitrophenyloctyl ether (NPOE) as a solvent mediator. The sensor displays good selectivity and stability and demonstrates a near-Nernstian response for 4-NPP over the concentration range 9.6x10(-6) to 1.0x10(-2) M with an anionic slope of 28.6+/-0.3 mV decade(-1) and a detection limit of 6.3x10(-6) M over the pH range 4.5-10. The sensor is used to measure the decrease of a fixed concentration of 4-NPP substrate as a function of acid and alkaline phosphatase enzyme activities at optimized conditions of pH and temperature. A linear relationship between the initial rate of 4-NPP substrate hydrolysis and enzyme activity holds over 0.05-3.0 and 0.03-3.4 IU L(-1) of ACP and ALP enzymes, respectively. Validation of the method by measuring the lower detection limit, range, accuracy, precision, within-day repeatability and between-day-variability reveals good performance characteristics of the proposed sensor. The sensor is used for the determination of acid and alkaline phosphatase enzyme activities in biological fluids of some patients suffering from alcoholic cirrhosis, acute myelocytic leukemia, pre-eclampsia and prostatic cancer. The sensor is also utilized for assessment of alkaline phosphatase enzyme in milk and dairy products. The results obtained agree fairly well with data obtained by the standard spectrophotometric methods.

  11. Release of an acid phosphatase activity during lily pollen tube growth involves components of the secretory pathway.

    PubMed

    Ibrahim, Hala; Pertl, Heidi; Pittertschatscher, Klaus; Fadl-Allah, Ezzat; el-Shahed, Ahmed; Bentrup, Friedrich-Wilhelm; Obermeyer, Gerhard

    2002-05-01

    An acid phosphatase (acPAse) activity was released during germination and tube growth of pollen of Lilium longiflorum Thunb. By inhibiting components of the secretory pathway, the export of the acPase activity was affected and tube growth stopped. Brefeldin A (1 microM) and cytochalasin D (1 microM), which block the production and transport of secretory vesicles, respectively, inhibited the acPase secretion. The Ca2+ channel blocker gadolinium (100 microM Gd3+) also inhibited acPase secretion and tube growth, whereas 3 mM caffeine, another Ca2+ uptake inhibitor, stimulated the acPase release, while tube growth was inhibited. The Yariv reagent (beta-D-glucosyl)3 Yariv phenylglycoside stopped tube growth by binding to arabinogalactan proteins of the tube tip cell wall but did not affect acPase secretion. A strong correlation between tube growth and acPase release was detected. The secreted acPase activity had a pH optimum at pH 5.5, a KM of 0.4 mM for p-nitrophenyl phosphate, and was inhibited by zinc, molybdate, phosphate, and fluoride ions, but not by tartrate. In electrophoresis gels the main acPase activity was detected at 32 kDa. The conspicuous correlation between activity of the secretory pathway and acPase secretion during tube elongation strongly indicates an important role of the acPase during pollen tube growth and the secreted acPase activity may serve as a useful marker enzyme assay for secretory activity in pollen tubes.

  12. Gambogic Acid Inhibits STAT3 Phosphorylation Through Activation of Protein Tyrosine Phosphatase SHP-1: Potential Role in Proliferation and Apoptosis

    PubMed Central

    Prasad, Sahdeo; Pandey, Manoj K.; Yadav, Vivek R.; Aggarwal, Bharat B.

    2011-01-01

    The transcription factor, signal transducer and activator of transcription 3 (STAT3), is associated with proliferation, survival, and metastasis of cancer cells. We investigated whether gambogic acid (GA), a xanthone derived from the resin of traditional Chinese medicine, Gamboge hanburyi (mangosteen), can regulate the STAT3 pathway, leading to suppression of growth and sensitization of cancer cells. We found that GA induced apoptosis in human multiple myeloma cells that correlated with the inhibition of both constitutive and inducible STAT3 activation. STAT3 phosphorylation at both tyrosine residue 705 and serine residue 727 was inhibited by GA. STAT3 suppression was mediated through the inhibition of activation of the protein tyrosine kinases Janus-activated kinase (JAK) 1, and JAK2. Treatment with the protein tyrosine phosphatase (PTP) inhibitor pervanadate reversed the GA-induced down-regulation of STAT3, suggesting the involvement of a PTP. We also found that GA induced the expression of the PTP SHP-1. Deletion of the SHP-1 gene by small interfering RNA suppressed the ability of GA to inhibit STAT3 activation and to induce apoptosis, suggesting the critical role of SHP-1 in its action. Moreover, GA down-regulated the expression of STAT3-regulated antiapoptotic (Bcl-2, Bcl-xL, and Mcl-1), proliferative (cyclin D1), and angiogenic (VEGF) proteins, and this correlated with suppression of proliferation and induction of apoptosis. Overall, these results suggest that GA blocks STAT3 activation, leading to suppression of tumor cell proliferation and induction of apoptosis. PMID:21490133

  13. Involvement of lysophosphatidic acid, sphingosine 1-phosphate and ceramide 1-phosphate in the metabolization of phosphatidic acid by lipid phosphate phosphatases in bovine rod outer segments.

    PubMed

    Pasquaré, Susana J; Salvador, Gabriela A; Giusto, Norma Maria

    2008-07-01

    The aim of the present research was to evaluate the generation of [2-3H]diacylglycerol ([2-3H]DAG) from [2-3H]-Phosphatidic acid ([2-3H]PA) by lipid phosphate phosphatases (LPPs) at different concentrations of lysophosphatidic acid (LPA), sphingosine 1-phosphate (S1P), and ceramide 1-phosphate (C1P) in purified ROS obtained from dark-adapted retinas (DROS) or light-adapted retinas (BLROS) as well as in ROS membrane preparations depleted of soluble and peripheral proteins. Western blot analysis revealed the presence of LPP3 exclusively in all membrane preparations. Immunoblots of entire ROS and depleted ROS did not show dark-light differences in LPP3 levels. LPPs activities were diminished by 53% in BLROS with respect to DROS. The major competitive effect on PA hydrolysis was exerted by LPA and S1P in DROS and by C1P in BLROS. LPPs activities in depleted ROS were similar to the activity observed in entire DROS and BLROS, respectively. LPA, S1P and C1P competed at different extent in depleted DROS and BLROS. Sphingosine and ceramide inhibited LPPs activities in entire and depleted DROS. Ceramide also inhibited LPPs activities in entire and in depleted BLROS. Our findings are indicative of a different degree of competition between PA and LPA, S1P and C1P by LPPs depending on the illumination state of the retina. PMID:18288612

  14. Protein tyrosine phosphatases regulate arachidonic acid release, StAR induction and steroidogenesis acting on a hormone-dependent arachidonic acid-preferring acyl-CoA synthetase.

    PubMed

    Cano, Florencia; Poderoso, Cecilia; Cornejo Maciel, Fabiana; Castilla, Rocío; Maloberti, Paula; Castillo, Fernanda; Neuman, Isabel; Paz, Cristina; Podestá, Ernesto J

    2006-06-01

    The activation of the rate-limiting step in steroid biosynthesis, that is the transport of cholesterol into the mitochondria, is dependent on PKA-mediated events triggered by hormones like ACTH and LH. Two of such events are the protein tyrosine dephosphorylation mediated by protein tyrosine phosphatases (PTPs) and the release of arachidonic acid (AA) mediated by two enzymes, ACS4 (acyl-CoA synthetase 4) and Acot2 (mitochondrial thioesterase). ACTH and LH regulate the activity of PTPs and Acot2 and promote the induction of ACS4. Here we analyzed the involvement of PTPs on the expression of ACS4. We found that two PTP inhibitors, acting through different mechanisms, are both able to abrogate the hormonal effect on ACS4 induction. PTP inhibitors also reduce the effect of cAMP on steroidogenesis and on the level of StAR protein, which facilitates the access of cholesterol into the mitochondria. Moreover, our results indicate that exogenous AA is able to overcome the inhibition produced by PTP inhibitors on StAR protein level and steroidogenesis. Then, here we describe a link between PTP activity and AA release, since ACS4 induction is under the control of PTP activity, being a key event for AA release, StAR induction and steroidogenesis.

  15. Activity of hydrolytic enzymes in fungi isolated from diabetic pregnant women: is there any relationship between fungal alkaline and acid phosphatase activity and glycemic control?

    PubMed

    Nowakowska, Dorota; Kurnatowska, Alicja; Stray-Pedersen, Babill; Wilczyński, Jan

    2004-06-01

    Ability to respond to environmental changes and secretion of hydrolases are considered to be important for Candida virulence. In this study we determined and compared the activities of 19 different hydrolases of the fungal strains isolated from diabetic and non-diabetic pregnant women. We also looked for the presence of a relationship between hydrolase activities and glycemic control, and, furthermore, evaluated the influence of gestational age on the activity of hydrolases. Mycological examinations were performed for 119 diabetic pregnant women: 47 with diabetes mellitus type I (DM), 72 with gestational diabetes (GDM), and for 132 healthy women (CON). Samples were collected from the vagina, rectum and oral cavity and cultured on Sabouraud media. The fungal hydrolase activities were evaluated using the API ZYM test (bioMerieux). For the 19 different fungal hydrolases tested, 13 activities were present in the isolated fungal strains. The activity of alkaline phosphatase (ALP) in vaginal strains (p=0.028) and acid phosphatase (ACP) in strains from the vagina (p=0.006) and rectum (p=0.049) was significantly lower in DM than in GDM and CON women. In conclusion, we describe for the first time that fungi isolated from pregnant diabetic women have lower activity of both phosphatases compared to fungi isolated from healthy women. Furthermore, similar differences of mean ALP and ACP activities were observed in the course of pregnancy in strains from the vagina and rectum of DM and CON women. However, strains from DM had lower activity at each stage of pregnancy. The highest activity of ALP and ACP was detected at the beginning, then declined, and had the lowest values between the 24(th) and 33(rd) week of gestation. After that period the activity of both phosphatases increased.

  16. Genetic studies of water buffalo blood markers. I. Red cell acid phosphatase, albumin, catalase, red cell alpha-esterase-3, group-specific component, and protease inhibitor.

    PubMed

    Tan, S G; Barker, J S; Selvaraj, O S; Mukherjee, T K; Wong, Y F

    1993-06-01

    We have developed the methodologies for typing and family studies to establish the modes of inheritance of water buffalo red cell acid phosphatase (Acp), protease inhibitor (Pi), and group-specific component (Gc) on isoelectric focusing and albumin (Alb), red cell alpha-esterase-3 (Est-3), and catalase (Cat) on polyacrylamide gel electrophoresis. Family studies showed that Pi, Gc, Alb, and Cat are coded by autosomal genes with two codominant alleles, while Est-3 is autosomal with two codominant alleles and a recessive null allele and Acp exhibits three codominant alleles.

  17. Okadaic acid, a protein phosphatase inhibitor, blocks calcium changes, gene expression, and cell death induced by gibberellin in wheat aleurone cells.

    PubMed Central

    Kuo, A; Cappelluti, S; Cervantes-Cervantes, M; Rodriguez, M; Bush, D S

    1996-01-01

    The cereal aleurone functions during germination by secreting hydrolases, mainly alpha-amylase, into the starchy endosperm. Multiple signal transduction pathways exist in cereal aleurone cells that enable them to modulate hydrolase production in response to both hormonal and environmental stimuli. Gibberellic acid (GA) promotes hydrolase production, whereas abscisic acid (ABA), hypoxia, and osmotic stress reduce amylase production. In an effort to identify the components of transduction pathways in aleurone cells, we have investigated the effect of okadaic acid (OA), a protein phosphatase inhibitor, on stimulus-response coupling for GA, ABA, and hypoxia. We found that OA (100 nM) completely inhibited all the GA responses that we measured, from rapid changes in cytosolic Ca2+ through changes in gene expression and accelerated cell death. OA (100 nM) partially inhibited ABA responses, as measured by changes in the level of PHAV1, a cDNA for an ABA-induced mRNA in barley. In contrast, OA had no effect on the response to hypoxia, as measured by changes in cytosolic Ca2+ and by changes in enzyme activity and RNA levels of alcohol dehydrogenase. Our data indicate that OA-sensitive protein phosphatases act early in the transduction pathway of GA but are not involved in the response to hypoxia. These data provide a basis for a model of multiple transduction pathways in which the level of cytosolic Ca2+ is a key point of convergence controlling changes in stimulus-response coupling. PMID:8742711

  18. Aspartic acid-484 of nascent placental alkaline phosphatase condenses with a phosphatidylinositol glycan to become the carboxyl terminus of the mature enzyme.

    PubMed Central

    Micanovic, R; Bailey, C A; Brink, L; Gerber, L; Pan, Y C; Hulmes, J D; Udenfriend, S

    1988-01-01

    A carboxyl-terminal chymotryptic peptide from mature human placental alkaline phosphatase was purified by HPLC and monitored by a specific RIA. Sequencing and amino acid assay showed that the carboxyl terminus of the peptide was aspartic acid, representing residue 484 of the proenzyme as deduced from the corresponding cDNA. Further analysis of the peptide showed it to be a peptidoglycan containing one residue of ethanolamine, one residue of glucosamine, and two residues of neutral hexose. The inositol glycan is apparently linked to the alpha carboxyl group of the aspartic acid through the ethanolamine. Location of the inositol glycan on Asp-484 of the proenzyme indicates that a 29-residue peptide is cleaved from the nascent protein during the post-translational condensation with the phosphatidylinositol-glycan. PMID:3422741

  19. Nature of immobilization surface affects antibody specificity to placental alkaline phosphatase.

    PubMed

    Kumar, Mukesh; Khan, Imran; Sinha, Subrata

    2015-01-01

    Retention of native conformation of immobilized protein is essential for various applications including selection and detection of specific recombinant antibodies (scFvs). Placental alkaline phosphatase (PAP), an onco-fetal antigen expressed on the surface of several tumors, was immobilized on supermagnetic particles for selection of recombinant antibodies from a human phage display antibody library. The isolated antibodies were found to be cross-reactive to either of the isozymes of alkaline phosphatase, i.e., bone alkaline phosphatase (BAP) or intestinal alkaline phosphatase (IAP) and could not be used for tumor targeting. A specific anti-PAP monoclonal antibody H17E2 was tested for retention of specificity under these conditions. Binding of the antibody to magnetic beads conjugated IAP and BAP along with PAP and the ability of the two isozymes to inhibit its binding to PAP depicted the loss of isozyme specificity of the antibody. However, the antibody retained its specificity to PAP immobilized on polyvinyl chloride (PVC) surface. Enzyme activity was observed on both surfaces. This demonstrates that nature of immobilization may affect antigen-antibody binding in subtle ways, resulting in alteration of conformation of the epitopes. This may have consequences for determining the specificity of antibody binding for proteins that share a high degree of homology.

  20. Regulation of anthocyanin biosynthesis in Arabidopsis thaliana red pap1-D cells metabolically programmed by auxins.

    PubMed

    Liu, Zhong; Shi, Ming-Zhu; Xie, De-Yu

    2014-04-01

    Red pap1-D cells of Arabidopsis thaliana have been cloned from production of anthocyanin pigmentation 1-Dominant (pap1-D) plants. The red cells are metabolically programmed to produce high levels of anthocyanins by a WD40-bHLH-MYB complex that is composed of the TTG1, TT8/GL3 and PAP1 transcription factors. Here, we report that indole 3-acetic acid (IAA), naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) regulate anthocyanin biosynthesis in these red cells. Seven concentrations (0, 0.2, 0.4, 2.2, 9, 18 and 27 μM) were tested for the three auxins. IAA and 2,4-D at 2.2-27 μM reduced anthocyanin levels. NAA at 0-0.2 μM or above 9 μM also decreased anthocyanin levels, but from 0.4 to 9 μM, it increased them. HPLC-ESI-MS analysis identified seven cyanin molecules that were produced in red pap1-D cells, and their levels were affected by auxins. The expression levels of ten genes, including six transcription factors (TTG1, EGL3, MYBL2, TT8, GL3 and PAP1) and four pathway genes (PAL1, CHS, DFR and ANS) involved in anthocyanin biosynthesis were analyzed upon various auxin treatments. The resulting data showed that 2,4-D, NAA and IAA control anthocyanin biosynthesis by regulating the expression of TT8, GL3 and PAP1 as well as genes in the anthocyanin biosynthetic pathway, such as DFR and ANS. In addition, the expression of MYBL2, PAL1 and CHS in red pap1-D and wild-type cells differentially respond to the three auxins. Our data demonstrate that the three auxins regulate anthocyanin biosynthesis in metabolically programmed red cells via altering the expression of transcription factor genes and pathway genes. PMID:24370633

  1. Discovery and Evaluation of Novel Inhibitors of Mycobacterium Protein Tyrosine Phosphatase B from the 6-Hydroxy-Benzofuran-5-Carboxylic Acid Scaffold

    PubMed Central

    He, Yantao; Xu, Jie; Yu, Zhi-hong; Gunawan, Andrea M.; Wu, Li; Wang, Lina; Zhang, Zhong-Yin

    2013-01-01

    Mycobacterium tuberculosis (Mtb) protein tyrosine phosphatase B (mPTPB) is a virulence factor secreted by the pathogen and mediates mycobacterial survival in macrophages by targeting host cell immune responses. Consequently, mPTPB represents an exciting new target to combat TB infection. We describe a medicinal chemistry-oriented approach that transforms a benzofuran salicylic acid scaffold into a highly potent (IC50 = 38 nM) and selective mPTPB inhibitor (>50 fold against a large panel of PTPs). Importantly, the inhibitor is capable of reversing the altered host immune responses induced by the bacterial phosphatase and restoring the macrophage’s full capacity to secrete IL-6 and undergo apoptosis in response to IFN-γ stimulation, validating the concept that chemical inhibition of mPTPB may be therapeutically useful for novel TB treatment. The study further demonstrates that bicyclic salicylic acid pharmacophores can be used to deliver PTP inhibitors with high potency, selectivity, and cellular efficacy. PMID:23305444

  2. Differential Expression of 1-Aminocyclopropane-1-Carboxylate Synthase Genes during Orchid Flower Senescence Induced by the Protein Phosphatase Inhibitor Okadaic Acid1

    PubMed Central

    Wang, Ning Ning; Yang, Shang Fa; Charng, Yee-yung

    2001-01-01

    Applying 10 pmol of okadaic acid (OA), a specific inhibitor of type 1 or type 2A serine/threonine protein phosphatases, to the orchid (Phalaenopsis species) stigma induced a dramatic increase in ethylene production and an accelerated senescence of the whole flower. Aminoethoxyvinylglycine or silver thiosulfate, inhibitors of ethylene biosynthesis or action, respectively, effectively inhibited the OA-induced ethylene production and retarded flower senescence, suggesting that the protein phosphatase inhibitor induced orchid flower senescence through an ethylene-mediated signaling pathway. OA treatment induced a differential expression pattern for the 1-aminocyclopropane-1-carboxylic acid synthase multigene family. Accumulation of Phal-ACS1 transcript in the stigma, labelum, and ovary induced by OA were higher than those induced by pollination as determined by “semiquantitative” reverse transcriptase-polymerase chain reaction. In contrast, the transcript levels of Phal-ACS2 and Phal-ACS3 induced by OA were much lower than those induced by pollination. Staurosporine, a protein kinase inhibitor, on the other hand, inhibited the OA-induced Phal-ACS1 expression in the stigma and delayed flower senescence. Our results suggest that a hyper-phosphorylation status of an unidentified protein(s) is involved in up-regulating the expression of Phal-ACS1 gene resulting in increased ethylene production and accelerated the senescence process of orchid flower. PMID:11351088

  3. Lipin 2 binds phosphatidic acid by the electrostatic hydrogen bond switch mechanism independent of phosphorylation.

    PubMed

    Eaton, James M; Takkellapati, Sankeerth; Lawrence, Robert T; McQueeney, Kelley E; Boroda, Salome; Mullins, Garrett R; Sherwood, Samantha G; Finck, Brian N; Villén, Judit; Harris, Thurl E

    2014-06-27

    Lipin 2 is a phosphatidic acid phosphatase (PAP) responsible for the penultimate step of triglyceride synthesis and dephosphorylation of phosphatidic acid (PA) to generate diacylglycerol. The lipin family of PA phosphatases is composed of lipins 1-3, which are members of the conserved haloacid dehalogenase superfamily. Although genetic alteration of LPIN2 in humans is known to cause Majeed syndrome, little is known about the biochemical regulation of its PAP activity. Here, in an attempt to gain a better general understanding of the biochemical nature of lipin 2, we have performed kinetic and phosphorylation analyses. We provide evidence that lipin 2, like lipin 1, binds PA via the electrostatic hydrogen bond switch mechanism but has a lower rate of catalysis. Like lipin 1, lipin 2 is highly phosphorylated, and we identified 15 phosphosites. However, unlike lipin 1, the phosphorylation of lipin 2 is not induced by insulin signaling nor is it sensitive to inhibition of the mammalian target of rapamycin. Importantly, phosphorylation of lipin 2 does not negatively regulate either membrane binding or PAP activity. This suggests that lipin 2 functions as a constitutively active PA phosphatase in stark contrast to the high degree of phosphorylation-mediated regulation of lipin 1. This knowledge of lipin 2 regulation is important for a deeper understanding of how the lipin family functions with respect to lipid synthesis and, more generally, as an example of how the membrane environment around PA can influence its effector proteins. PMID:24811178

  4. Fine structural and histochemical studies on salivary glands of Peripatoides novae-zealandiae (Onychophora) with special reference to acid phosphatase distribution.

    PubMed

    Nelson, L; van der Lande, V; Robson, E A

    1980-01-01

    The Onychophora feed on small arthropods and produce saliva when ingesting prey. Although saliva undoubtedly helps to liquefy the food its constituents have not yet been fully described. The salivary glands, two long tubes of glandularepithelium, are known to secrete a powerful protease, however, besides other enzymes and mucus. In Peripatoides novae-zealandiae there are protein-secreting cells of three types, referred to here as columnar, cuboidal and modified cells, and mucus cells. The anterior two-thirds of the gland show most cell diversity, while the posterior regionconsists mainly of columnar cells. These are the most numerous elements overall and they probably secrete salivary protease. In thick resin sections the granules of all protein-secreting cells stain strongly with methylene blue. Those of columnar cells are markedly uneven in size and accumulate distally, eventually filling the cytoplasm. More proximal Golgi regions may be discernible. Mucus cells are all of one type and their secretion droplets are stained lightly by methylene blue. The electron microscope shows that distal microvilli, desmosomes and septate junctions are common to all gland cells. In columnar cells, secretory material is contributed by Golgi complexes and by rough endoplasmic reticulum. Early secretory vacuoles containing dense material are seen in the concavity of Golgi regions. They are precursors to larger condensing vacuoles whose contents have a more flocculent appearance, and which may attain 3--4 micrometers in diameter. These evolve into secretory granules, usually of uneven texture, which are up to 2.5 micrometers in diameter. Histochemical tests for acid phosphatase show moderate amounts of enzyme throughout the gland. In whole mounts and sections the strongest reaction is in a band of cuboidal cells along the anterior median border. Columnar cells show a diffuse cytoplasmic reaction towards the base and sometimes distal to the nucleus, and mucus cells may also react

  5. Purple acid phosphatase of the human macrophage and osteoclast. Characterization, molecular properties, and crystallization of the recombinant di-iron-oxo protein secreted by baculovirus-infected insect cells.

    PubMed

    Hayman, A R; Cox, T M

    1994-01-14

    The purple phosphatases catalyze hydrolysis of phosphate esters (optimum pH approximately 5) and are resistant to inhibition by dextro-rotatory tartrate; their distinctive color is due to Fe(III)-phenolate charge-transfer transitions at their active site. Expression of human purple phosphatase, designated type 5 acid phosphatase, is restricted to osteoclasts and other activated cells of monohistiocytic lineage, but its biological rôle in relation to bone resorption and phagocytosis is unknown. To characterize this enzyme further, we have engineered the human type 5 acid phosphatase into a baculovirus vector expression system that enabled milligram quantities of purple protein to be purified from medium containing Sf9 host cells. The phosphatase cDNA was transcribed as a single RNA species of 1.5 kilobases as in human tissues. Tartrate-resistant acid phosphatase activity reacting with uteroferrin antisera appeared in the culture medium, from which up to 8 mg/liter was purified by two-step cation-exchange chromatography at pH 8.0. Two isoforms of approximately 36 kDa were identified by SDS-polyacrylamide electrophoresis and were converted to a single species of apparent molecular size 34 kDa upon treatment with N-glycosidase F, indicating secreted glycoforms of a single polypeptide. Mass spectroscopy showed that the mean molecular mass of the active, secreted glycoprotein was 35849 Da. The recombinant enzyme (specific activity, 190 mumol p-nitrophenol/min/mg at 37 degrees C) contained 2 iron atoms/molecule and formed purple, monoclinic crystals. Exposure to the ferric chelator, 1,2-dimethyl-3-hydroxypyrid-4-one, rapidly inactivated the enzyme, which was not inhibited by alpha, alpha'-bipyridyl, a ferrous chelator. That ferric iron is essential for enzymatic catalysis, was further indicated by the synergistic effects of the reductant, dithiothreitol, and bipyridyl on phosphatase activity. The recombinant purple phosphatase catalyzed the peroxidation of 5

  6. 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

    PubMed Central

    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

  7. Differential induction of apoptosis in human breast tumor cells by okadaic acid and related inhibitors of protein phosphatases 1 and 2A

    SciTech Connect

    Kiguchi, Kaoru; Chubb, C.H.; Glesne, D.; Huberman, E. |; Fujiki, Hirota

    1994-09-01

    To investigate a possible relationship between apoptosis induction and protein phosphorylation in human breast carcinoma cells, the authors treated three such cell types, MB-231, MCF-7, and AU-565, wit okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A, or phorbol 12 myristate 13-acetate, an activator of protein kinase C. They then examined these cells of the appearance of apoptosis markers. While OA caused multiplication arrest and cytotoxicity in all three cell lines, apoptosis was induced in MB-231 and MCF-7 cells but not in AU-565 cells. A similar cell-specific apoptosis induction was also observed after treatment with dinophysistoxin-1 (an active OA analogue) and with calyculin A (a structurally unrelated protein phosphatase inhibitor) but not with analogues that either ar inactive or penetrate epithelial cells poorly. Phorbol 12-myristate 13-acetate also inhibited cell multiplication but was without effect in inducing apoptosis in these cells. Levels of the apoptosis-inhibitory protein BCL2 were examined in these cells, but they did to correlate with this differential susceptibility. They additionally treated the three cell types with 1-{beta}-D-arabinofuranosylcytosine and genistein to determine whether the AU-565 cell line would also be resistant to apoptosis induction by other chemical stimuli. Both of these agents led to the induction of apoptosis in all three cell lines. These results indicate that the AU-565 cells are specifically resistant to apoptosis induction by inhibitors of protein phosphatases 1 and 2A. This cell-specific resistance may thus allow one to identify cellular mediators of apoptosis by comparing protein phosphorylation patterns in these cells before and after treatment with OA or related inhibitors.

  8. FERONIA interacts with ABI2-type phosphatases to facilitate signaling cross-talk between abscisic acid and RALF peptide in Arabidopsis.

    PubMed

    Chen, Jia; Yu, Feng; Liu, Ying; Du, Changqing; Li, Xiushan; Zhu, Sirui; Wang, Xianchun; Lan, Wenzhi; Rodriguez, Pedro L; Liu, Xuanming; Li, Dongping; Chen, Liangbi; Luan, Sheng

    2016-09-13

    Receptor-like kinase FERONIA (FER) plays a crucial role in plant response to small molecule hormones [e.g., auxin and abscisic acid (ABA)] and peptide signals [e.g., rapid alkalinization factor (RALF)]. It remains unknown how FER integrates these different signaling events in the control of cell growth and stress responses. Under stress conditions, increased levels of ABA will inhibit cell elongation in the roots. In our previous work, we have shown that FER, through activation of the guanine nucleotide exchange factor 1 (GEF1)/4/10-Rho of Plant 11 (ROP11) pathway, enhances the activity of the phosphatase ABA Insensitive 2 (ABI2), a negative regulator of ABA signaling, thereby inhibiting ABA response. In this study, we found that both RALF and ABA activated FER by increasing the phosphorylation level of FER. The FER loss-of-function mutant displayed strong hypersensitivity to both ABA and abiotic stresses such as salt and cold conditions, indicating that FER plays a key role in ABA and stress responses. We further showed that ABI2 directly interacted with and dephosphorylated FER, leading to inhibition of FER activity. Several other ABI2-like phosphatases also function in this pathway, and ABA-dependent FER activation required PYRABACTIN RESISTANCE (PYR)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR)-A-type protein phosphatase type 2C (PP2CA) modules. Furthermore, suppression of RALF1 gene expression, similar to disruption of the FER gene, rendered plants hypersensitive to ABA. These results formulated a mechanism for ABA activation of FER and for cross-talk between ABA and peptide hormone RALF in the control of plant growth and responses to stress signals. PMID:27566404

  9. /sup 18/O isotope effect in /sup 13/C nuclear magnetic resonance spectroscopy. Part 9. Hydrolysis of benzyl phosphate by phosphatase enzymes and in acidic aqueous solutions

    SciTech Connect

    Parente, J.E.; Risley, J.M.; Van Etten, R.L.

    1984-12-26

    The /sup 18/O isotope-induced shifts in /sup 13/C and /sup 31/P nuclear magnetic resonance (NMR) spectroscopy were used to establish the position of bond cleavage in the phosphatase-catalyzed and acid-catalyzed hydrolysis reactions of benzyl phosphate. The application of the /sup 18/O-isotope effect in NMR spectroscopy affords a continuous, nondestructive assay method for following the kinetics and position of bond cleavage in the hydrolytic process. The technique provides advantages over most discontinuous methods in which the reaction components must be isolated and converted to volatile derivatives prior to analysis. In the present study, (..cap alpha..-/sup 13/C,ester-/sup 18/O)benzyl phosphate and (ester-/sup 18/O)benzyl phosphate were synthesized for use in enzymatic and nonenzymatic studies. Hydrolysis reactions catalyzed by the alkaline phosphatase from E. coli and by the acid phosphatases isolated from human prostate and human liver were all accompanied by cleavage of the substrate phosphorus-oxygen bond consistent with previously postulated mechanisms involving covalent phosphoenzyme intermediates. An extensive study of the acid-catalyzed hydrolysis of benzyl phosphate at 75/sup 0/C revealed that the site of bond cleavage is dependent on pH. At pH less than or equal to 1.3, the hydrolysis proceeds with C-O bond cleavage; at 1.3 < pH < 2.0, there is a mixture of C-O and P-O bond scission, the latter progressively predominating as the pH is raised; at pH greater than or equal to 2.0, the hydrolysis proceeds with exclusive P-O bond scission. (S)-(+)-(..cap alpha..-/sup 2/H)Benzyl phosphate was also synthesized. Hydrolysis of this chiral benzyl derivative demonstrated that the acid-catalyzed C-O bond scission of benzyl phosphate proceeds by an A-1 (S/sub N/1) mechanism with 70% racemization and 30% inversion at carbon. 37 references, 4 figures, 2 tables.

  10. A Second Two-Component Regulatory System of Bordetella bronchiseptica Required for Bacterial Resistance to Oxidative Stress, Production of Acid Phosphatase, and In Vivo Persistence

    PubMed Central

    Jungnitz, Heidrun; West, Nicholas P.; Walker, Mark J.; Chhatwal, Gursharan S.; Guzmán, Carlos A.

    1998-01-01

    Random minitransposon mutagenesis was used to identify genes involved in the survival of Bordetella bronchiseptica within eukaryotic cells. One of the mutants which exhibited a reduced ability to survive intracellularly harbored a minitransposon insertion in a locus (ris) which displays a high degree of homology to two-component regulatory systems. This system exhibited less than 25% amino acid sequence homology to the only other two-component regulatory system described in Bordetella spp., the bvg locus. A risA′-′lacZ translational fusion was constructed and integrated into the chromosome of B. bronchiseptica. Determination of β-galactosidase activity under different environmental conditions suggested that ris is regulated independently of bvg and is optimally expressed at 37°C, in the absence of Mg2+, and when bacteria are in the intracellular niche. This novel regulatory locus, present in all Bordetella spp., is required for the expression of acid phosphatase by B. bronchiseptica. Although catalase and superoxide dismutase production were unaffected, the ris mutant was more sensitive to oxidative stress than the wild-type strain. Complementation of bvg-positive and bvg-negative ris mutants with the intact ris operon incorporated as a single copy into the chromosome resulted in the reestablishment of the ability of the bacterium to produce acid phosphatase and to resist oxidative stress. Mouse colonization studies demonstrated that the ris mutant is cleared by the host much earlier than the wild-type strain, suggesting that ris-regulated products play a significant role in natural infections. The identification of a second two-component system in B. bronchiseptica highlights the complexity of the regulatory network needed for organisms with a life cycle requiring adaptation to both the external environment and a mammalian host. PMID:9746560

  11. A STRESS-RESPONSIVE NAC1-Regulated Protein Phosphatase Gene Rice Protein Phosphatase18 Modulates Drought and Oxidative Stress Tolerance through Abscisic Acid-Independent Reactive Oxygen Species Scavenging in Rice1[W][OPEN

    PubMed Central

    You, Jun; Zong, Wei; Hu, Honghong; Li, Xianghua; Xiao, Jinghua; Xiong, Lizhong

    2014-01-01

    Plants respond to abiotic stresses through a complexity of signaling pathways, and the dephosphorylation mediated by protein phosphatase (PP) is an important event in this process. We identified a rice (Oryza sativa) PP2C gene, OsPP18, as a STRESS-RESPONSIVE NAC1 (SNAC1)-regulated downstream gene. The ospp18 mutant was more sensitive than wild-type plants to drought stress at both the seedling and panicle development stages. Rice plants with OsPP18 suppressed through artificial microRNA were also hypersensitive to drought stress. Microarray analysis of the mutant revealed that genes encoding reactive oxygen species (ROS) scavenging enzymes were down-regulated in the ospp18 mutant, and the mutant exhibited reduced activities of ROS scavenging enzymes and increased sensitivity to oxidative stresses. Overexpression of OsPP18 in rice led to enhanced osmotic and oxidative stress tolerance. The expression of OsPP18 was induced by drought stress but not induced by abscisic acid (ABA). Although OsPP18 is a typical PP2C with enzymatic activity, it did not interact with SNF1-RELATED PROTEIN KINASE2 protein kinases, which function in ABA signaling. Meanwhile, the expression of ABA-responsive genes was not affected in the ospp18 mutant, and the ABA sensitivities of the ospp18 mutant and OsPP18-overexpressing plants were also not altered. Together, these findings suggest that OsPP18 is a unique PP2C gene that is regulated by SNAC1 and confers drought and oxidative stress tolerance by regulating ROS homeostasis through ABA-independent pathways. PMID:25318938

  12. A STRESS-RESPONSIVE NAC1-regulated protein phosphatase gene rice protein phosphatase18 modulates drought and oxidative stress tolerance through abscisic acid-independent reactive oxygen species scavenging in rice.

    PubMed

    You, Jun; Zong, Wei; Hu, Honghong; Li, Xianghua; Xiao, Jinghua; Xiong, Lizhong

    2014-12-01

    Plants respond to abiotic stresses through a complexity of signaling pathways, and the dephosphorylation mediated by protein phosphatase (PP) is an important event in this process. We identified a rice (Oryza sativa) PP2C gene, OsPP18, as a STRESS-RESPONSIVE NAC1 (SNAC1)-regulated downstream gene. The ospp18 mutant was more sensitive than wild-type plants to drought stress at both the seedling and panicle development stages. Rice plants with OsPP18 suppressed through artificial microRNA were also hypersensitive to drought stress. Microarray analysis of the mutant revealed that genes encoding reactive oxygen species (ROS) scavenging enzymes were down-regulated in the ospp18 mutant, and the mutant exhibited reduced activities of ROS scavenging enzymes and increased sensitivity to oxidative stresses. Overexpression of OsPP18 in rice led to enhanced osmotic and oxidative stress tolerance. The expression of OsPP18 was induced by drought stress but not induced by abscisic acid (ABA). Although OsPP18 is a typical PP2C with enzymatic activity, it did not interact with SNF1-RELATED PROTEIN KINASE2 protein kinases, which function in ABA signaling. Meanwhile, the expression of ABA-responsive genes was not affected in the ospp18 mutant, and the ABA sensitivities of the ospp18 mutant and OsPP18-overexpressing plants were also not altered. Together, these findings suggest that OsPP18 is a unique PP2C gene that is regulated by SNAC1 and confers drought and oxidative stress tolerance by regulating ROS homeostasis through ABA-independent pathways.

  13. A Novel Phosphatidic Acid-Protein-tyrosine Phosphatase D2 Axis Is Essential for ERBB2 Signaling in Mammary Epithelial Cells*

    PubMed Central

    Ramesh, Mathangi; Krishnan, Navasona; Muthuswamy, Senthil K.; Tonks, Nicholas K.

    2015-01-01

    We used a loss-of-function screen to investigate the role of classical protein-tyrosine phosphatases (PTPs) in three-dimensional mammary epithelial cell morphogenesis and ERBB2 signaling. The study revealed a novel role for PTPD2 as a positive regulator of ERBB2 signaling. Suppression of PTPD2 attenuated the ERBB2-induced multiacinar phenotype in three-dimensional cultures specifically by inhibiting ERBB2-mediated loss of polarity and lumen filling. In contrast, overexpression of PTPD2 enhanced the ERBB2 phenotype. We also found that a lipid second messenger, phosphatidic acid, bound PTPD2 in vitro and enhanced its catalytic activity. Small molecule inhibitors of phospholipase D (PLD), an enzyme that produces phosphatidic acid in cells, also attenuated the ERBB2 phenotype. Exogenously added phosphatidic acid rescued the PLD-inhibition phenotype, but only when PTPD2 was present. These findings illustrate a novel pathway involving PTPD2 and the lipid second messenger phosphatidic acid that promotes ERBB2 function. PMID:25681440

  14. Characterization of Yersinia enterocolitica, Y. intermedia, Y. aldovae, Y. frederiksenii, Y. kristensenii and Y. pseudotuberculosis by electrophoretic polymorphism of acid phosphatase, esterases, and glutamate and malate dehydrogenases.

    PubMed

    Goullet, P; Picard, B

    1988-02-01

    Acid phosphatase, esterases, and glutamate and malate dehydrogenases of 192 strains of Yersinia enterocolitica, Y. intermedia, Y. aldovae, Y. frederiksenii, Y. kristensenii and Y. pseudotuberculosis were analysed by horizontal polyacrylamide agarose gel electrophoresis and by isoelectrofocusing in thin-layer polyacrylamide gels. The six species were clearly separated from each other by their distinct enzyme electrophoretic polymorphism. For Y. enterocolitica, the strains of biotype 5 were differentiated from the other biotypes by the mobility of glutamate dehydrogenase. For Y. frederiksenii, six zymotypes were delineated by pI and by the mobility of the enzymes. Variation in number or mobility of esterases within each species could represent a marker for epidemiological and ecological analyses. A linear relationship was obtained between the mean genetic diversity coefficient of enzymes and the mean percentage DNA-DNA relatedness of Y. intermedia, Y. aldovae, Y. enterocolitica and Y. frederiksenii.

  15. Complex coordinated extracellular metabolism: Acid phosphatases activate diluted human leukocyte proteins to generate energy flow as NADPH from purine nucleotide ribose

    PubMed Central

    Hibbs, John B.; Vavrin, Zdenek; Cox, James E.

    2016-01-01

    Complex metabolism is thought to occur exclusively in the crowded intracellular environment. Here we report that diluted enzymes from lysed human leukocytes produce extracellular energy. Our findings involve two pathways: the purine nucleotide catabolic pathway and the pentose phosphate pathway, which function together to generate energy as NADPH. Glucose6P fuel for NADPH production is generated from structural ribose of purine ribonucleoside monophosphates, ADP, and ADP-ribose. NADPH drives glutathione reductase to reduce an oxidized glutathione disulfide-glutathione redox couple. Acid phosphatases initiate ribose5P salvage from purine ribonucleoside monophosphates, and transaldolase controls the direction of carbon chain flow through the nonoxidative branch of the pentose phosphate pathway. These metabolic control points are regulated by pH. Biologically, this energy conserving metabolism could function in perturbed extracellular spaces. PMID:26895212

  16. Stimulation of a Gs-like G protein in the osteoclast inhibits bone resorption but enhances tartrate-resistant acid phosphatase secretion.

    PubMed

    Moonga, B S; Pazianas, M; Alam, A S; Shankar, V S; Huang, C L; Zaidi, M

    1993-01-29

    Previous studies have demonstrated that G-protein agonists induce quiescence (Q effect) or retraction (R effect) in isolated osteoclasts. We now report the functional effects of such agonists on osteoclastic bone resorption and enzyme release. Exposure of osteoclasts to tetrafluoro-aluminate anions (AlF4-), a universal G protein stimulator, resulted in a marked concentration-dependent inhibition of bone resorption. This was associated with a dramatic increase in the secretion of the osteoclast-specific enzyme, tartrate-resistant acid phosphatase (TRAP). Cholera toxin, a Gs stimulator and a selective Q effect agonist, similarly abolished bone resorption and enhanced TRAP secretion. In contrast, pertussis toxin, a Gi inhibitor and a selective R effect agonist, inhibited bone resorption significantly, but slightly reduced enzyme release. The results suggest an involvement of a Gs-like G protein in TRAP secretion from the osteoclast, possibly through a cyclic AMP-dependent mechanism.

  17. Complex coordinated extracellular metabolism: Acid phosphatases activate diluted human leukocyte proteins to generate energy flow as NADPH from purine nucleotide ribose.

    PubMed

    Hibbs, John B; Vavrin, Zdenek; Cox, James E

    2016-08-01

    Complex metabolism is thought to occur exclusively in the crowded intracellular environment. Here we report that diluted enzymes from lysed human leukocytes produce extracellular energy. Our findings involve two pathways: the purine nucleotide catabolic pathway and the pentose phosphate pathway, which function together to generate energy as NADPH. Glucose6P fuel for NADPH production is generated from structural ribose of purine ribonucleoside monophosphates, ADP, and ADP-ribose. NADPH drives glutathione reductase to reduce an oxidized glutathione disulfide-glutathione redox couple. Acid phosphatases initiate ribose5P salvage from purine ribonucleoside monophosphates, and transaldolase controls the direction of carbon chain flow through the nonoxidative branch of the pentose phosphate pathway. These metabolic control points are regulated by pH. Biologically, this energy conserving metabolism could function in perturbed extracellular spaces.

  18. Coordinated Regulation of the Neutral Amino Acid Transporter SNAT2 and the Protein Phosphatase Subunit GADD34 Promotes Adaptation to Increased Extracellular Osmolarity*

    PubMed Central

    Krokowski, Dawid; Jobava, Raul; Guan, Bo-Jhih; Farabaugh, Kenneth; Wu, Jing; Majumder, Mithu; Bianchi, Massimiliano G.; Snider, Martin D.; Bussolati, Ovidio; Hatzoglou, Maria

    2015-01-01

    Cells respond to shrinkage induced by increased extracellular osmolarity via programmed changes in gene transcription and mRNA translation. The immediate response to this stress includes the induction of expression of the neutral amino acid transporter SNAT2. Increased SNAT2-mediated uptake of neutral amino acids is an essential adaptive mechanism for restoring cell volume. In contrast, stress-induced phosphorylation of the α subunit of the translation initiation factor eIF2 (eIF2α) can promote apoptosis. Here we show that the response to mild hyperosmotic stress involves regulation of the phosphorylation of eIF2α by increased levels of GADD34, a regulatory subunit of protein phosphatase 1 (PP1). The induction of GADD34 was dependent on transcriptional control by the c-Jun-binding cAMP response element in the GADD34 gene promoter and posttranscriptional stabilization of its mRNA. This mechanism differs from the regulation of GADD34 expression by other stresses that involve activating transcription factor 4 (ATF4). ATF4 was not translated during hyperosmotic stress despite an increase in eIF2α phosphorylation. The SNAT2-mediated increase in amino acid uptake was enhanced by increased GADD34 levels in a manner involving decreased eIF2α phosphorylation. It is proposed that the induction of the SNAT2/GADD34 axis enhances cell survival by promoting the immediate adaptive response to stress. PMID:26041779

  19. Ecto-alkaline phosphatase activity identified at physiological pH range on intact P19 and HL-60 cells is induced by retinoic acid.

    PubMed

    Scheibe, R J; Kuehl, H; Krautwald, S; Meissner, J D; Mueller, W H

    2000-01-01

    The activity of membrane-bound alkaline phosphatase (ALP) expressed on the external surface of cultured murine P19 teratocarcinoma and human HL-60 myeloblastic leukemia cells was studied at physiological pH using p-nitrophenylphosphate (pNPP) as substrate. The rate of substrate hydrolysis catalyzed by intact viable cells remained constant for eight successive incubations of 30 min and was optimal at micromolar substrate concentrations over the pH range 7.4-8.5. The value of apparent K(m) for pNPP in P19 and HL-60 cells was 120 microM. Hydrolytic activity of the ecto-enzyme at physiological pH decreased by the addition of levamisole, a specific and noncompetitive inhibitor of ALP (K(i) P19 = 57 microM; K(i) HL-60 = 50 microM). Inhibition of hydrolysis was reversed by removal of levamisole within 30 min. Retinoic acid (RA), which promotes the differentiation of P19 and HL-60 cells, induced levamisole-sensitive ecto-phosphohydrolase activity at pH 7.4. After its autophosphorylation by ecto-kinase activity, a 98-kDa membrane protein in P19 cells was found to be sensitive to ecto-ALP, and protein dephosphorylation increased after incubation of cells with RA for 24 h and 48 h. Orthovanadate, an inhibitor of all phosphatase activities, blocked the levamisole-sensitive dephosphorylation of the membrane phosphoproteins, while (R)-(-)-epinephrine reversed the effect by complexation of the inhibitor. The results demonstrate that the levamisole-sensitive phosphohydrolase activity on the cell surface is consistent with ecto-ALP activity degrading both physiological concentrations of exogenously added substrate and endogenous surface phosphoproteins under physiological pH conditions. The dephosphorylating properties of ecto-ALP are induced by RA, suggesting a specific function in differentiating P19 teratocarcinoma and HL-60 myeloblastic leukemia cells. PMID:10649440

  20. Structure and function of an archaeal homolog of survival protein E (SurEalpha): an acid phosphatase with purine nucleotide specificity.

    PubMed

    Mura, Cameron; Katz, Jonathan E; Clarke, Steven G; Eisenberg, David

    2003-03-01

    The survival protein E (SurE) family was discovered by its correlation to stationary phase survival of Escherichia coli and various repair proteins involved in sustaining this and other stress-response phenotypes. In order to better understand this ancient and well-conserved protein family, we have determined the 2.0A resolution crystal structure of SurEalpha from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum (Pae). This first structure of an archaeal SurE reveals significant similarities to and differences from the only other known SurE structure, that from the eubacterium Thermatoga maritima (Tma). Both SurE monomers adopt similar folds; however, unlike the Tma SurE dimer, crystalline Pae SurEalpha is predominantly non-domain swapped. Comparative structural analyses of Tma and Pae SurE suggest conformationally variant regions, such as a hinge loop that may be involved in domain swapping. The putative SurE active site is highly conserved, and implies a model for SurE bound to a potential substrate, guanosine-5'-monophosphate (GMP). Pae SurEalpha has optimal acid phosphatase activity at temperatures above 90 degrees C, and is less specific than Tma SurE in terms of metal ion requirements. Substrate specificity also differs between Pae and Tma SurE, with a more specific recognition of purine nucleotides by the archaeal enzyme. Analyses of the sequences, phylogenetic distribution, and genomic organization of the SurE family reveal examples of genomes encoding multiple surE genes, and suggest that SurE homologs constitute a broad family of enzymes with phosphatase-like activities.

  1. Multiplexing Spheroid Volume, Resazurin and Acid Phosphatase Viability Assays for High-Throughput Screening of Tumour Spheroids and Stem Cell Neurospheres

    PubMed Central

    Ivanov, Delyan P.; Parker, Terry L.; Walker, David A.; Alexander, Cameron; Ashford, Marianne B.; Gellert, Paul R.; Garnett, Martin C.

    2014-01-01

    Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity) were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money. PMID:25119185

  2. Lipid phosphate phosphatase-1 regulates lysophosphatidic acid-induced calcium release, NF-κB activation and interleukin-8 secretion in human bronchial epithelial cells

    PubMed Central

    2004-01-01

    LPA (lysophosphatidic acid), a potent bioactive phospholipid, elicits diverse cellular responses through activation of the G-protein-coupled receptors LPA1–LPA4. LPA-mediated signalling is partially regulated by LPPs (lipid phosphate phosphatases; LPP-1, -2 and -3) that belong to the phosphatase superfamily. This study addresses the role of LPPs in regulating LPA-mediated cell signalling and IL-8 (interleukin-8) secretion in HBEpCs (human bronchial epithelial cells). Reverse transcription–PCR and Western blotting revealed the presence and expression of LPP-1–3 in HBEpCs. Exogenous [3H]oleoyl LPA was hydrolysed to [3H]-mono-oleoylglycerol. Infection of HBEpCs with an adenoviral construct of human LPP-1 for 48 h enhanced the dephosphorylation of exogenous LPA by 2–3-fold compared with vector controls. Furthermore, overexpression of LPP-1 partially attenuated LPA-induced increases in the intracellular Ca2+ concentration, phosphorylation of IκB (inhibitory κB) and translocation of NF-κB (nuclear factor-κB) to the nucleus, and almost completely prevented IL-8 secretion. Infection of cells with an adenoviral construct of the mouse LPP-1 (R217K) mutant partially attenuated LPA-induced IL-8 secretion without altering LPA-induced changes in intracellular Ca2+ concentration, phosphorylation of IκB, NF-κB activation or IL-8 gene expression. Our results identify LPP-1 as a key regulator of LPA signalling and IL-8 secretion in HBEpCs. Thus LPPs could represent potential targets in regulating leucocyte infiltration and airway inflammation. PMID:15461590

  3. Abscisic Acid Promotion of Arbuscular Mycorrhizal Colonization Requires a Component of the PROTEIN PHOSPHATASE 2A Complex1[W][OPEN

    PubMed Central

    Charpentier, Myriam; Sun, Jongho; Wen, Jiangqi; Mysore, Kirankumar S.; Oldroyd, Giles E.D.

    2014-01-01

    Legumes can establish intracellular interactions with symbiotic microbes to enhance their fitness, including the interaction with arbuscular mycorrhizal (AM) fungi. AM fungi colonize root epidermal cells to gain access to the root cortex, and this requires the recognition by the host plant of fungus-made mycorrhizal factors. Genetic dissection has revealed the symbiosis signaling pathway that allows the recognition of AM fungi, but the downstream processes that are required to promote fungal infection are poorly understood. Abscisic acid (ABA) has been shown to promote arbuscule formation in tomato (Solanum lycopersicum). Here, we show that ABA modulates the establishment of the AM symbiosis in Medicago truncatula by promoting fungal colonization at low concentrations and impairing it at high concentrations. We show that the positive regulation of AM colonization via ABA requires a PROTEIN PHOSPHATASE 2A (PP2A) holoenzyme subunit, PP2AB′1. Mutations in PP2AB′1 cause reduced levels of AM colonization that cannot be rescued with permissive ABA application. The action of PP2AB′1 in response to ABA is unlinked to the generation of calcium oscillations, as the pp2aB′1 mutant displays a normal calcium response. This contrasts with the application of high concentrations of ABA that impairs mycorrhizal factor-induced calcium oscillations, suggesting different modes of action of ABA on the AM symbiosis. Our work reveals that ABA functions at multiple levels to regulate the AM symbiosis and that a PP2A phosphatase is required for the ABA promotion of AM colonization. PMID:25293963

  4. ALP (Alkaline Phosphatase) Test

    MedlinePlus

    ... known as: ALK PHOS; Alkp Formal name: Alkaline Phosphatase Related tests: AST ; ALT ; GGT ; Bilirubin ; Liver Panel ; Bone Markers ; Alkaline Phosphatase Isoenzymes; Bone Specific ALP All content on Lab ...

  5. Enzyme kinetics of conjugating enzymes: PAPS sulfotransferase.

    PubMed

    James, Margaret O

    2014-01-01

    The sulfotransferase (SULT) enzymes catalyze the formation of sulfate esters or sulfamates from substrates that contain hydroxy or amine groups, utilizing 3'-phosphoadenosyl-5'-phosphosulfate (PAPS) as the donor of the sulfonic group. The rate of product formation depends on the concentrations of PAPS and substrate as well as the sulfotransferase enzyme; thus, if PAPS is held constant while varying substrate concentration (or vice versa), the kinetic constants derived are apparent constants. When studied over a narrow range of substrate concentrations, classic Michaelis-Menten kinetics can be observed with many SULT enzymes and most substrates. Some SULT enzymes exhibit positive or negative cooperativity during conversion of substrate to product, and the kinetics fit the Hill plot. A characteristic feature of most sulfotransferase-catalyzed reactions is that, when studied over a wide range of substrate concentrations, the rate of product formation initially increases as substrate concentration increases, then decreases at high substrate concentrations, i.e., they exhibit substrate inhibition or partial substrate inhibition. This chapter gives an introduction to sulfotransferases, including a historical note, the nomenclature, a description of the function of SULTs with different types of substrates, presentation of examples of enzyme kinetics with SULTs, and a discussion of what is known about mechanisms of substrate inhibition in the sulfotransferases.

  6. Biological activities of Zn(II)-S-methyl-cysteine complex as antiradical, inhibitor of acid phosphatase enzyme and in vivo antidepressant effects.

    PubMed

    Escudero, Graciela E; Martini, Nancy; Jori, Khalil; Jori, Nadir; Maresca, Nahuel R; Laino, Carlos H; Naso, Luciana G; Williams, Patricia A M; Ferrer, Evelina G

    2016-12-01

    The antidepressant effect of simple Zn(II) salts has been proved in several animal models of depression. In this study, a coordination metal complex of Zn(II) having a sulfur containing ligand is tested as antidepressant for the first time. Forced swimming test method on male Wistar rats shows a decrease in the immobility and an increase in the swimming behavior after treatment with [Zn(S-Met)2] (S-Met=S-methyl-l-cysteine) being more effective and remarkable than ZnCl2. The thiobarbituric acid and the pyranine consumption (hydroxyl and peroxyl radicals, respectively) methods were applied to evaluate the antioxidant activity of S-Met and [Zn(S-Met)2] showing evidence of attenuation of hydroxyl but not peroxyl radicals activities. UV-vis studies on the inhibition of acid phosphatase enzyme (AcP) demonstrated that S-methyl-l-cysteine did not produce any effect but, in contrast, [Zn(S-Met)2] complex behaved as a moderate inhibitor. Finally, bioavailability studies were performed by fluorescence spectroscopy denoting the ability of the albumin to transport the complex.

  7. Cell- and ligand-specific dephosphorylation of acid hydrolases: Evidence that the mannose 6-phosphatase is controlled by compartmentalization

    SciTech Connect

    Einstein, R.; Gabel, C.A. )

    1991-01-01

    Mouse L cells that possess the cation-independent mannose 6-phosphate (Man 6-P)/insulin-like growth factor (IGF) II receptor change the extent to which they dephosphorylate endocytosed acid hydrolases in response to serum. To investigate the mechanism by which dephosphorylation competence is regulated, the dephosphorylation of individual acid hydrolases was studied in Man 6-P/IGF II receptor-positive and -deficient cell lines. 125I-labeled Man 6-P-containing acid hydrolases were proteolytically processed but remained phosphorylated when endocytosed by receptor-positive L cells maintained in the absence of serum; after the addition of serum, however, the cell-associated hydrolases were dephosphorylated. Individual hydrolases were dephosphorylated at distinct rates and to different extents. In contrast, the same hydrolases were dephosphorylated equally and completely after entry into Man 6-P/IGF II receptor-positive Chinese hamster ovary (CHO) cells. The dephosphorylation competence of Man 6-P/IGF II receptor-deficient mouse J774 cells was more limited. beta-Glucuronidase produced by these cells underwent a limited dephosphorylation in transit to lysosomes such that diphosphorylated oligosaccharides were converted to monophosphorylated species. The overall quantity of phosphorylated oligosaccharides associated with the enzyme, however, did not decrease within the lysosomal compartment. Likewise, beta-glucuronidase was not dephosphorylated when introduced into J774 cells via Fc receptor-mediated endocytosis. The CHO and J774 cell lysosomes, therefore, display opposite extremes with respect to their capacity to dephosphorylate acid hydrolases; within CHO cell lysosomes acid hydrolases are rapidly and efficiently dephosphorylated, but within J774 cell lysosomes the same acid hydrolases remain phosphorylated.

  8. Improvement of Student Understanding of How Kinetic Data Facilitates the Determination of Amino Acid Catalytic Function through an Alkaline Phosphatase Structure/Mechanism Bioinformatics Exercise

    ERIC Educational Resources Information Center

    Grunwald, Sandra K.; Krueger, Katherine J.

    2008-01-01

    Laboratory exercises, which utilize alkaline phosphatase as a model enzyme, have been developed and used extensively in undergraduate biochemistry courses to illustrate enzyme steady-state kinetics. A bioinformatics laboratory exercise for the biochemistry laboratory, which complements the traditional alkaline phosphatase kinetics exercise, was…

  9. Terpenes and sterols from the fruits of Prunus mume and their inhibitory effects on osteoclast differentiation by suppressing tartrate-resistant acid phosphatase activity.

    PubMed

    Yan, Xi-Tao; Lee, Sang-Hyun; Li, Wei; Jang, Hae-Dong; Kim, Young-Ho

    2015-02-01

    The fruits of Prunus mume are a common commercial product and a valuable source of food and medicinal material in Eastern Asian countries. Our phytochemical investigation of the P. mume fruit led to the isolation of nine terpenes, including three ursane-type triterpenes (1-3), two cycloartane-type triterpenes (4 and 5), and four tocopherols (10-13), as well as four sterols (6-9). Their structures were elucidated based on extensive spectroscopic analysis, including 1D and 2D NMR and ESI-MS, and the majority of these compounds were isolated from this plant for the first time. The anti-osteoporosis activities of 1-13 were evaluated by measuring their inhibitory effects on tartrate-resistant acid phosphatase (TRAP) activity in receptor activator of nuclear factor κB ligand-induced osteoclastic RAW 264.7 macrophage cells. Compounds 2-7 and 9-12 significantly suppressed TRAP activity down to 47.96 ± 2.45-86.45 ± 3.07 % relative to the control at a concentration of 1 μM. These results suggest that the fruits of P. mume could be an excellent source of anti-osteoporosis phytochemicals that may be developed as natural nutraceuticals and functional foods.

  10. [The importance of studying the acid phosphatase of the blood serum and bone marrow lymphoblasts and polymorphonuclear neutrophils in the prognosis of the course of acute lymphoblastic leukemia].

    PubMed

    Vaiuta, N P; Khaĭfets, L M; Mendeleev, I M

    1988-01-01

    The activity of serum acid phosphatase (AP), bone marrow lymphoblasts and polymorphonuclear neutrophils was studied in 45 ALL patients. Cytochemical coefficients (CCC) and the percentage of positively reacting bone marrow cells were determined. All the patients received programmed polychemotherapy. They were investigated before the start of therapy, during recurrence and at different time of remission (from 1 to 60 mos) during each reinduction cycle. At the climax of ALL the activity of serum AP was increased 2.8-fold, a CCC value for lymphoblastic AP--10-fold, for polymorphonuclear neutrophils--3-fold as compared with normal values. A tendency toward the reduction of indices was noted at different time of remission, the approximation to normal values was noted on the 40th-46th months of remission only. In recurrence development the level of the serum and cellular enzyme as well as the percentage of positively reacting cells significantly exceeded normal values and were close to indices at the climax of disease. The above tendency permitted the use of these tests to evaluate the completeness of remission and to predict recurrences during a follow-up of ALL patients.

  11. A comparative study on phosphotransferase activity of acid phosphatases from Raoultella planticola and Enterobacter aerogenes on nucleosides, sugars, and related compounds.

    PubMed

    Médici, Rosario; Garaycoechea, Juan I; Valino, Ana L; Pereira, Claudio A; Lewkowicz, Elizabeth S; Iribarren, Adolfo M

    2014-04-01

    Natural and modified nucleoside-5'-monophosphates and their precursors are valuable compounds widely used in biochemical studies. Bacterial nonspecific acid phosphatases (NSAPs) are a group of enzymes involved in the hydrolysis of phosphoester bonds, and some of them exhibit phosphotransferase activity. NSAP containing Enterobacter aerogenes and Raoultella planticola whole cells were evaluated in the phosphorylation of a wide range of nucleosides and nucleoside precursors using pyrophosphate as phosphate donor. To increase the productivity of the process, we developed two genetically modified strains of Escherichia coli which overexpressed NSAPs of E. aerogenes and R. planticola. These new recombinant microorganisms (E. coli BL21 pET22b-phoEa and E. coli BL21 pET22b-phoRp) showed higher activity than the corresponding wild-type strains. Reductions in the reaction times from 21 h to 60 min, from 4 h to 15 min, and from 24 h to 40 min in cases of dihydroxyacetone, inosine, and fludarabine, respectively, were obtained.

  12. Extending the Mannose 6-Phosphate Glycoproteome by High Resolution/Accuracy Mass Spectrometry Analysis of Control and Acid Phosphatase 5-Deficient Mice*

    PubMed Central

    Sleat, David E.; Sun, Pengling; Wiseman, Jennifer A.; Huang, Ling; El-Banna, Mukarram; Zheng, Haiyan; Moore, Dirk F.; Lobel, Peter

    2013-01-01

    In mammals, most newly synthesized lumenal lysosomal proteins are delivered to the lysosome by the mannose 6-phosphate (Man6P) targeting pathway. Man6P -containing proteins can be affinity-purified and characterized using proteomic approaches, and such studies have led to the discovery of new lysosomal proteins and associated human disease genes. One limitation to this approach is that in most cell types the Man6P modification is rapidly removed by acid phosphatase 5 (ACP5) after proteins are targeted to the lysosome, and thus, some lysosomal proteins may escape detection. In this study, we have extended the analysis of the lysosomal proteome using high resolution/accuracy mass spectrometry to identify and quantify proteins in a combined analysis of control and ACP5-deficient mice. To identify Man6P glycoproteins with limited tissue distribution, we analyzed multiple tissues and used statistical approaches to identify proteins that are purified with high specificity. In addition to 68 known Man6P glycoproteins, 165 other murine proteins were identified that may contain Man6P and may thus represent novel lysosomal residents. For four of these lysosomal candidates, (lactoperoxidase, phospholipase D family member 3, ribonuclease 6, and serum amyloid P component), we demonstrate lysosomal residence based on the colocalization of fluorescent fusion proteins with a lysosomal marker. PMID:23478313

  13. Acid phosphatase activity in liver macrophage aggregates as a marker for pollution-induced immunomodulation of the non-specific immune response in fish

    NASA Astrophysics Data System (ADS)

    Broeg, Katja

    2003-10-01

    The activity of acid phosphatase in liver macrophage aggregates (MA-AP) of different fish species was used as a marker for a pollution-induced modulation of the digestive capacity of phagocytes, since functions of the non-specific immune response play a central role in the maintenance of animals' health. Based upon the investigation of more than 900 individual flounders (Platichthys flesus) and mullets (Liza aurata), natural variations, gender-specific differences and pollution-induced alterations in AP activity are demonstrated in this study. MA-AP activity was dependent on temperature and season but, nevertheless, distinctions between differently polluted areas were visible in all sampling campaigns with lowest MA-AP activity in fish from the polluted areas of the German Bight and the Israeli coast of the Mediterranean Sea. For organochlorine contaminants, as well as for mercury and copper, a significant correlation could be observed between residue concentrations in fish tissues and MA-AP activity. In all cases, except mercury which showed a positive correlation, AP activity was suppressed in animals with a high contaminant burden. MA-AP activity turned out to give reliable and consistent results for a quantification of immunomodulation in both fish species.

  14. Comparison of the effects of eldecalcitol with either raloxifene or bisphosphonate on serum tartrate resistant acid phosphatase-5b, a bone resorption marker, in postmenopausal osteoporosis

    PubMed Central

    Takada, Junichi; Ikeda, Satoshi; Kusanagi, Tetsuya; Mizuno, Satoshi; Wada, Hiroshi; Iba, Kousuke; Yoshizaki, Takashi; Yamashita, Toshihiko

    2016-01-01

    Summary Objective This study analyzes whether concomitant raloxifene (RLX) or bisphosphonates (BP) plus eldecalcitol (ELD) has excessive suppressive effects on a bone resorption marker during the first 6 months of treatment in postmenopausal women in real-world setting. Methods 285 postmenopausal osteoporotic patients who had been treated with RLX or BP plus ELD were evaluated the bone resorption marker, serum tartrate resistant acid phosphatase-5b (TRACP-5b), during the first 6 months of treatment. Results In drug-naïve group (not received osteoporosis medications before the administration, n=70), the concomitant RLX or BP with ELD significantly decreased levels of TRACP-5b without severe suppression. In vitamin D switch group [RLX or BP plus alfacalcidol (ALF) and then switched to RLX or BP plus ELD, n=215], the replacing ALF with ELD further and significantly decreased TRACP-5b and tertile analyses based on baseline values were significantly decreased far more in the highest, compared with the lowest tertile in the ELD+RLX and ELD+BP groups. Conclusion ELD combined with RLX or BP administered for 6 months to postmenopausal women with osteoporosis who were drug-naïve or who had switched medications significantly reduced and maintained TRACP-5b values within the reference range. PMID:27252739

  15. Biochemical effect of a histidine phosphatase acid (phytase) of Aspergillus japonicus var. Saito on performance and bony characteristics of broiler.

    PubMed

    Maller, Alexandre; de Quadros, Thays Cristina Oliveira; Junqueira, Otto M; Graña, Alfredo Lora; de Lima Montaldi, Ana Paula; Alarcon, Ricardo Fernandes; Jorge, João Atílio; de Lourdes T M Polizeli, Maria

    2016-01-01

    Phytases are enzymes that hydrolyze the ester linkage of phytic acid, releasing inositol and inorganic phosphate. The phytic acid (phytate) is a major form of phosphorus in plant foods. Knowing that diet for animal of production has the cereal base (corn and soybean), primarily, broilers need for an alternative to use of the phosphate present in these ingredients, since it does not naturally produce the enzyme phytase, which makes it available. The aims of this work was studding the safe supplementation of Aspergillus japonicus var. Saito crude phytase in feeding broilers and check the biochemical effect on performance and bones of these animals. The enzymatic extract did not have aflatoxins B1, B2, G2 and G1 and zearalenone and ochratoxin, and low concentrations of this extract did not have cytotoxic effects on cells derived from lung tissue. The in vivo experiments showed that the phytase supplied the available phosphate reduction in the broiler feed formulation, with a live weight, weight gain, feed intake, feed conversion, viability, productive efficiency index and carcass yield similar to the control test. Furthermore, the phytase supplementation favored the formation of bone structure and performance of the broilers. The results show the high biotechnological potential of A. japonicus phytase on broiler food supplementation to reduce phosphorus addition in the food formulation. So, this enzyme could be used as a commercial alternative to animal diet supplementation.

  16. Biochemical effect of a histidine phosphatase acid (phytase) of Aspergillus japonicus var. Saito on performance and bony characteristics of broiler.

    PubMed

    Maller, Alexandre; de Quadros, Thays Cristina Oliveira; Junqueira, Otto M; Graña, Alfredo Lora; de Lima Montaldi, Ana Paula; Alarcon, Ricardo Fernandes; Jorge, João Atílio; de Lourdes T M Polizeli, Maria

    2016-01-01

    Phytases are enzymes that hydrolyze the ester linkage of phytic acid, releasing inositol and inorganic phosphate. The phytic acid (phytate) is a major form of phosphorus in plant foods. Knowing that diet for animal of production has the cereal base (corn and soybean), primarily, broilers need for an alternative to use of the phosphate present in these ingredients, since it does not naturally produce the enzyme phytase, which makes it available. The aims of this work was studding the safe supplementation of Aspergillus japonicus var. Saito crude phytase in feeding broilers and check the biochemical effect on performance and bones of these animals. The enzymatic extract did not have aflatoxins B1, B2, G2 and G1 and zearalenone and ochratoxin, and low concentrations of this extract did not have cytotoxic effects on cells derived from lung tissue. The in vivo experiments showed that the phytase supplied the available phosphate reduction in the broiler feed formulation, with a live weight, weight gain, feed intake, feed conversion, viability, productive efficiency index and carcass yield similar to the control test. Furthermore, the phytase supplementation favored the formation of bone structure and performance of the broilers. The results show the high biotechnological potential of A. japonicus phytase on broiler food supplementation to reduce phosphorus addition in the food formulation. So, this enzyme could be used as a commercial alternative to animal diet supplementation. PMID:27625972

  17. AtPP2CG1, a protein phosphatase 2C, positively regulates salt tolerance of Arabidopsis in abscisic acid-dependent manner

    SciTech Connect

    Liu, Xin; Zhu, Yanming; Zhai, Hong; Cai, Hua; Ji, Wei; Luo, Xiao; Li, Jing; Bai, Xi

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer AtPP2CG1 positively regulates salt tolerance in ABA-dependent manner. Black-Right-Pointing-Pointer AtPP2CG1 up-regulates the expression of marker genes in different pathways. Black-Right-Pointing-Pointer 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.

  18. Involvement of phosphatidate phosphatase in the biosynthesis of triacylglycerols in Chlamydomonas reinhardtii * #

    PubMed Central

    Deng, Xiao-dong; Cai, Jia-jia; Fei, Xiao-wen

    2013-01-01

    Lipid biosynthesis is essential for eukaryotic cells, but the mechanisms of the process in microalgae remain poorly understood. Phosphatidic acid phosphohydrolase or 3-sn-phosphatidate phosphohydrolase (PAP) catalyzes the dephosphorylation of phosphatidic acid to form diacylglycerols and inorganic orthophosphates. This reaction is integral in the synthesis of triacylglycerols. In this study, the mRNA level of the PAP isoform CrPAP2 in a species of Chlamydomonas was found to increase in nitrogen-free conditions. Silencing of the CrPAP2 gene using RNA interference resulted in the decline of lipid content by 2.4%–17.4%. By contrast, over-expression of the CrPAP2 gene resulted in an increase in lipid content by 7.5%–21.8%. These observations indicate that regulation of the CrPAP2 gene can control the lipid content of the algal cells. In vitro CrPAP2 enzyme activity assay indicated that the cloned CrPAP2 gene exhibited biological activities. PMID:24302712

  19. Global Transcriptomic Analysis of Interactions between Pseudomonas aeruginosa and Bacteriophage PaP3

    PubMed Central

    Zhao, Xia; Chen, Canhuang; Shen, Wei; Huang, Guangtao; Le, Shuai; Lu, Shuguang; Li, Ming; Zhao, Yan; Wang, Jing; Rao, Xiancai; Li, Gang; Shen, Mengyu; Guo, Keke; Yang, Yuhui; Tan, Yinling; Hu, Fuquan

    2016-01-01

    The interactions between Bacteriophage (phage) and host bacteria are widespread in nature and influences of phage replication on the host cells are complex and extensive. Here, we investigate genome-wide interactions of Pseudomonas aeruginosa (P. aeruginosa) and its temperate phage PaP3 at five time points during phage infection. Compared to the uninfected host, 38% (2160/5633) genes of phage-infected host were identified as differentially expressed genes (DEGs). Functional analysis of the repressed DEGs revealed infection-stage-dependent pathway communications. Based on gene co-expression analysis, most PaP3 middle genes were predicted to have negative impact on host transcriptional regulators. Sub-network enrichment analysis revealed that adjacent genes of PaP3 interacted with the same host genes and might possess similar functions. Finally, our results suggested that during the whole infection stage, the early genes of PaP3 had stronger regulatory role in host gene expression than middle and late genes, while the host genes involved amino acid metabolism were the most “vulnerable” targets of these phage genes. This work provides the basis for understanding survival mechanisms of parasites and host, and seeking phage gene products that could potentially be used in anti-bacterial infection. PMID:26750429

  20. Comparison of alkaline phosphatase activity of MC3T3-E1 cells cultured on different Ti surfaces: modified sandblasted with large grit and acid-etched (MSLA), laser-treated, and laser and acid-treated Ti surfaces

    PubMed Central

    Li, Lin-Jie; Kim, So-Nam

    2016-01-01

    PURPOSE In this study, the aim of this study was to evaluate the effect of implant surface treatment on cell differentiation of osteoblast cells. For this purpose, three surfaces were compared: (1) a modified SLA (MSLA: sand-blasted with large grit, acid-etched, and immersed in 0.9% NaCl), (2) a laser treatment (LT: laser treatment) titanium surface and (3) a laser and acid-treated (LAT: laser treatment, acid-etched) titanium surface. MATERIALS AND METHODS The MSLA surfaces were considered as the control group, and LT and LAT surfaces as test groups. Alkaline phosphatase expression (ALP) was used to quantify osteoblastic differentiation of MC3T3-E1 cell. Surface roughness was evaluated by a contact profilometer (URFPAK-SV; Mitutoyo, Kawasaki, Japan) and characterized by two parameters: mean roughness (Ra) and maximum peak-to-valley height (Rt). RESULTS Scanning electron microscope revealed that MSLA (control group) surface was not as rough as LT, LAT surface (test groups). Alkaline phosphatase expression, the measure of osteoblastic differentiation, and total ALP expression by surface-adherent cells were found to be highest at 21 days for all three surfaces tested (P<.05). Furthermore, ALP expression levels of MSLA and LAT surfaces were significantly higher than expression levels of LT surface-adherent cells at 7, 14, and 21 days, respectively (P<.05). However, ALP expression levels between MSLA and LAT surface were equal at 7, 14, and 21 days (P>.05). CONCLUSION This study suggested that MSLA and LAT surfaces exhibited more favorable environment for osteoblast differentiation when compared with LT surface, the results that are important for implant surface modification studies. PMID:27350860

  1. Histochemical and electrophoretic studies on phosphatases of some Indian trematodes.

    PubMed

    Haque, M; Siddiqi, A H

    1982-06-01

    The isoenzymes of acid and alkaline phosphatases and their histochemical localization were studied by polyacrylamide disc gel electrophoresis in four species of trematodes: Gigantocotyle explanatum from the liver and Gastrothylax crumenifer from the rumen of water buffalo (Bubalus bubalis) and Echinostoma malayanum and Fasciolopsis buski from the small intestine of the pig (Sus scrofa). Both acid and alkaline phosphatases were present in the tegument, gastrodermis, suckers, testes, ovary, eggs, vitellaria and uterus but alkaline phosphatase activity was demonstrated only in the parenchyma and excretory ducts. Polyacrylamide gel electrophoresis revealed two to four isoenzymes for both acid and alkaline phosphatase.

  2. PAP/HIP protein is an obesogenic factor.

    PubMed

    Secq, Veronique; Mallmann, Cecilia; Gironella, Meritxell; Lopez, Belen; Closa, Daniel; Garcia, Stéphane; Christa, Laurence; Montalto, Giuseppe; Dusetti, Nelson; Iovanna, Juan L

    2014-02-01

    In this article we report the obesogenic role of the acute phase protein PAP/HIP. We found that the transgenic TgPAP/HIP mice develop spontaneous obesity under standard nutritional conditions, with high levels of glucose, leptin, and LDL and low levels of triglycerides and HDL in blood. Accordingly, PAP/HIP-deficient mice are skinny under standard nutritional conditions. We also found that expression of PAP/HIP is induced in intestinal epithelial cells in response to gavage with olive oil and this induction is AG490 sensitive. We demonstrated that incubation of 3T3-L1 preadipocytes with a low concentration as 1 ng/ml of recombinant PAP/HIP results in accelerated BrdU incorporation in vitro. PAP/HIP-dependent adipocytes growth is sensitive to the MEK inhibitor U0126. Finally, patients with severe obesity present higher blood levels of PAP/HIP than non-obese control individuals. Altogether our data suggest that PAP/HIP could be a mediator of fat tissue development, released by the intestine and induced by the presence of food into the gut.

  3. Who is getting Pap smears in urban Peru?

    PubMed Central

    Paz Soldan, Valerie A; Lee, Frank H; Carcamo, Cesar; Holmes, King K; Garnett, Geoff P; Garcia, Patricia

    2008-01-01

    Background Cervical cancer, although usually preventable by Pap smear screening, remains the leading cause of cancer-related deaths among women in Peru. The percentages and characteristics of women in Peru who have or have not had a Pap smear have not been defined. Methods In an urban community randomized trial of sexually transmitted infection (STI)/HIV prevention in Peru, 6712 randomly selected women between the ages of 18 and 29 from 20 cities were interviewed regarding having had cervical Pap smears. Results Among women sampled, only 30.9% had had a Pap smear. By multivariate analysis, the main predictors of having a Pap smear were having had sex, having had children, completion of secondary education and increasing age. Regional variations were also found: women from the highlands and rainforest were less likely to have had Pap smears than women from the coast. Conclusion A norm of seeking and receiving Pap smears has not been established among sexually active young Peruvian women. To improve Pap smear coverage in Peru, promotion efforts should target underserved women and regions with less coverage. PMID:18653515

  4. Effect of Bacteria and Amoebae on Rhizosphere Phosphatase Activity

    PubMed Central

    Gould, W. Douglas; Coleman, David C.; Rubink, Amy J.

    1979-01-01

    The contributions of various components of soil microflora and microfauna to rhizosphere phosphatase activity were determined with hydroponic cultures. Three treatments were employed: (i) plants alone (Bouteloua gracilis (H.B.K.) Lag. ex Steud.) (ii) plants plus bacteria (Pseudomonas sp.), and (iii) plants plus bacteria plus amoebae (Acanthamoeba sp.). No alkaline phosphatase was detected, but an appreciable amount of acid phosphatase activity (120 to 500 nmol of p-nitrophenylphosphate hydrolyzed per h per plant) was found in the root culture solutions. The presence of bacteria or bacteria and amoebae increased the amount of acid phosphatase in solution, and properties of additional activity were identical to properties of plant acid phosphatase. The presence of bacteria or bacteria and amoebae increased both solution and root phosphatase activities at most initial phosphate concentrations. PMID:16345390

  5. CIR, a corepressor of CBF1, binds to PAP-1 and effects alternative splicing

    SciTech Connect

    Maita, Hiroshi; Kitaura, Hirotake; Ariga, Hiroyoshi . E-mail: hiro@pharm.hokudai.ac.jp; Iguchi-Ariga, Sanae M.M.

    2005-02-15

    We have reported that PAP-1, a product of a causative gene for autosomal retinitis pigmentosa, plays a role in splicing. In this study, CIR, a protein originally identified as a CBF1-interacting protein and reported to act as a transcriptional corepressor, was identified as a PAP-1 binding protein and its function as a splicing factor was investigated. In addition to a basic lysine and acidic serine-rich (BA) domain and a zinc knuckle-like motif, CIR has an arginine/serine dipeptide repeat (RS) domain in its C terminal region. The RS domain has been reported to be present in the superfamily of SR proteins, which are involved in splicing reactions. We generated CIR mutants with deletions of each BA and RS domain and studied their subcellular localizations and interactions with PAP-1 and other SR proteins, including SC35, SF2/ASF, and U2AF{sup 35}. CIR was found to interact with U2AF{sup 35} through the BA domain, with SC35 and SF2/ASF through the RS domain, and with PAP-1 outside the BA domain in vivo and in vitro. CIR was found to be colocalized with SC35 and PAP-1 in nuclear speckles. Then the effect of CIR on splicing was investigated using the E1a minigene as a reporter in HeLa cells. Ectopic expression of CIR with the E1a minigene changed the ratio of spliced isoforms of E1a that were produced by alternative selection of 5'-splice sites. These results indicate that CIR is a member of the family of SR-related proteins and that CIR plays a role in splicing regulation.

  6. Extracellular phosphatases of Chlamydomonas reinhardi and their regulation.

    PubMed

    Patni, N J; Dhawale, S W; Aaronson, S

    1977-04-01

    Chlamydomonas reinhardi, cultured under normal growth conditions, secreted significant amounts of protein and carbohydrates but not lipids or nucleic acids. A fivefold increase in light intensity led to a tenfold increase in secreted protein and carbohydrate. Among the proteins secreted was acid phosphatase with a pH optimum at 4.8 like the enzyme in the cells. Phosphorus depleted algae grown on minimal orthophosphate contained and secreted both acid and alkaline phosphatase. The pH optimum of the intracellular alkaline phosphatase was 9.2. When phosphorus-depleted cells were grown with increasing orthophosphate, intra- and extracellular alkaline phosphatase was almost completely repressed and intra- and extracellular acid phosphatase was partially repressed. Extracellular acid and alkaline phosphatase increased with the age of the culture. Electrophoresis indicated only one acid and one alkaline phosphatase in phosphorus-satisfied and phosphorus-depleted cells. Chlamydomonas cells suspended in an inorganic salt solution secreted only acid phosphatase; the absence of any extr-cellular cytoplasmic marker enzyme indicated that there was little, if any, autolysis to account for the extracellular acid enzyme. Phosphorus-depleted cells were able to grow on organic phosphates as the sole source of orthophosphate. Ribose-5-phosphate was the best for cell multiplication, and its utility was shown to be due to the cell's ability to use the ribose as well as the orthophosphatase for cell multiplication.

  7. High mature grain phytase activity in the Triticeae has evolved by duplication followed by neofunctionalization of the purple acid phosphatase phytase (PAPhy) gene

    PubMed Central

    Brinch-Pedersen, Henrik

    2013-01-01

    The phytase activity in food and feedstuffs is an important nutritional parameter. Members of the Triticeae tribe accumulate purple acid phosphatase phytases (PAPhy) during grain filling. This accumulation elevates mature grain phytase activities (MGPA) up to levels between ~650 FTU/kg for barley and 6000 FTU/kg for rye. This is notably more than other cereals. For instance, rice, maize, and oat have MGPAs below 100 FTU/kg. The cloning and characterization of the PAPhy gene complement from wheat, barley, rye, einkorn, and Aegilops tauschii is reported here. The Triticeae PAPhy genes generally consist of a set of paralogues, PAPhy_a and PAPhy_b, and have been mapped to Triticeae chromosomes 5 and 3, respectively. The promoters share a conserved core but the PAPhy_a promoter have acquired a novel cis-acting regulatory element for expression during grain filling while the PAPhy_b promoter has maintained the archaic function and drives expression during germination. Brachypodium is the only sequenced Poaceae sharing the PAPhy duplication. As for the Triticeae, the duplication is reflected in a high MGPA of ~4200 FTU/kg in Brachypodium. The sequence conservation of the paralogous loci on Brachypodium chromosomes 1 and 2 does not extend beyond the PAPhy gene. The results indicate that a single-gene segmental duplication may have enabled the evolution of high MGPA by creating functional redundancy of the parent PAPhy gene. This implies that similar MGPA levels may be out of reach in breeding programs for some Poaceae, e.g. maize and rice, whereas Triticeae breeders should focus on PAPhy_a. PMID:23918958

  8. The THO/TREX Complex Active in miRNA Biogenesis Negatively Regulates Root-Associated Acid Phosphatase Activity Induced by Phosphate Starvation1[OPEN

    PubMed Central

    Tao, Sibo; Zhang, Ye; Wang, Xiaoyue; Xu, Le; Fang, Xiaofeng; Lu, Zhi John

    2016-01-01

    Induction and secretion of acid phosphatases (APases) is an adaptive response that plants use to cope with P (Pi) deficiency in their environment. The molecular mechanism that regulates this response, however, is poorly understood. In this work, we identified an Arabidopsis (Arabidopsis thaliana) mutant, hps8, which exhibits enhanced APase activity on its root surface (also called root-associated APase activity). Our molecular and genetic analyses indicate that this altered Pi response results from a mutation in the AtTHO1 gene that encodes a subunit of the THO/TREX protein complex. The mutation in another subunit of this complex, AtTHO3, also enhances root-associated APase activity under Pi starvation. In Arabidopsis, the THO/TREX complex functions in mRNA export and miRNA biogenesis. When treated with Ag+, an inhibitor of ethylene perception, the enhanced root-associated APase activity in hps8 is largely reversed. hpr1-5 is another mutant allele of AtTHO1 and shows similar phenotypes as hps8. ein2 is completely insensitive to ethylene. In the hpr1-5ein2 double mutant, the enhanced root-associated APase activity is also greatly suppressed. These results indicate that the THO/TREX complex in Arabidopsis negatively regulates root-associated APase activity induced by Pi starvation by inhibiting ethylene signaling. In addition, we found that the miRNA399-PHO2 pathway is also involved in the regulation of root-associated APase activity induced by Pi starvation. These results provide insight into the molecular mechanism underlying the adaptive response of plants to Pi starvation. PMID:27329222

  9. Caveolae-mediated endocytosis of the glucosaminoglycan-interacting adipokine tartrate resistant acid phosphatase 5a in adipocyte progenitor lineage cells.

    PubMed

    Patlaka, Christina; Norgård, Maria; Paulie, Staffan; Nordvall-Bodell, Annica; Lång, Pernilla; Andersson, Göran

    2014-03-01

    Adipogenesis depends on growth factors controlling proliferation/differentiation of mesenchymal stem cells (MSCs). Membrane binding and endocytosis of growth factors are often coupled to receptor activation and downstream signaling leading to specific cellular responses. The novel adipokine tartrate-resistant acid phosphatase (TRAP) 5a exhibits a growth factor-like effect on MSCs and pre-adipocytes and induces hyperplastic obesity in vivo. However its molecular interaction with pre-adipocytes remains unknown. Therefore, this study aimed to investigate membrane interaction of TRAP and its endocytosis routes in pre-adipocytes. Confocal and/or electron microscopy were used to detect TRAP in untreated or TRAP 5a/b treated pre-adipocytes under conditions that allow or inhibit endocytosis in combination with co-staining of endocytotic vesicles. TRAP interaction with heparin/heparan sulfate was verified by gel filtration. It could be shown that TRAP 5a, but not 5b, binds to the membrane of pre-adipocytes where it co-localizes with heparin-sulfate proteoglycan glypican-4. Also in vitro, TRAP 5a exhibited affinity for both heparin and heparan sulfate with heparin inhibiting its enzyme activity. Upon caveolae-mediated endocytosis of saturating levels of TRAP 5a, TRAP 5a co-localized intracellularly with glypican-4 and late endosomal marker Rab-7 positive vesicles. The protein was also located in multivesicular bodies (MVBs) but did not co-localize with lysosomal marker LAMP-1. TRAP 5a endocytosis was also detectable in pre-osteoblasts, but not fibroblasts, embryonic MSCs or mature adipocytes. These results indicate that TRAP 5a exhibits binding to cell surface, endocytosis and affinity to glucosaminoglycans (GAGs) in pre-adipocyte and pre-osteoblast lineage cells in a manner similar to other heparin-binding growth factors.

  10. Effects of cadmium alone and in combination with low molecular weight chitosan on metallothionein, glutathione-S-transferase, acid phosphatase, and ATPase of freshwater crab Sinopotamon yangtsekiense.

    PubMed

    Li, Ruijin; Zhou, Yanying; Wang, Lan; Ren, Guorui; Zou, Enmin

    2014-03-01

    Cadmium (Cd) is an environmental contaminant showing a variety of deleterious effects, including the potential threat for the ecological environment and human health via food chains. Low molecular weight chitosan (LMWC) has been demonstrated to be an effective antioxidant. Metallothionein (MT) mRNA levels and activities of glutathione-S-transferase (GST), superoxide dismutase (SOD), acid phosphatase (ACP), Na(+),K(+)-ATPase, and Ca(2+)-ATPase as well as malondialdehyde (MDA) contents in the gills of the freshwater crab Sinopotamon yangtsekiense were analyzed in vivo in order to determine the injury of Cd exposure on the gill tissues as well as the protective effect of LMWC against this injury. The results showed that there was an apparent accumulation of Cd in the gills, which was lessened by the presence of LMWC. Moreover, Cd(2+) significantly increased the gill MT mRNA levels, ACP activity and MDA content while decreasing the activities of SOD, GST, Na(+),K(+)-ATPase, and Ca(2+)-ATPase in the crabs relative to the control. Cotreatment with LMWC reduced the levels of MT mRNA and ACP but raised the activities of GST, Na(+),K(+)-ATPase, and Ca(2+)-ATPase in gill tissues compared with the crabs exposed to Cd(2+) alone. These results suggest that LMWC may exert its protective effect through chelating Cd(2+) to form LMWC-Cd(2+) complex, elevating the antioxidative activities of GST, Na(+),K(+)-ATPase, and Ca(2+)-ATPase as well as alleviating the stress pressure on MT and ACP, consequently protecting the cell from the adverse effects of Cd.

  11. Secreted Acid Phosphatase (SapM) of Mycobacterium tuberculosis Is Indispensable for Arresting Phagosomal Maturation and Growth of the Pathogen in Guinea Pig Tissues

    PubMed Central

    Puri, Rupangi Verma; Reddy, P. Vineel; Tyagi, Anil K.

    2013-01-01

    Tuberculosis (TB) is responsible for nearly 1.4 million deaths globally every year and continues to remain a serious threat to human health. The problem is further complicated by the growing incidence of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB), emphasizing the need for the development of new drugs against this disease. Phagosomal maturation arrest is an important strategy employed by Mycobacterium tuberculosis to evade the host immune system. Secretory acid phosphatase (SapM) of M.tuberculosis is known to dephosphorylate phosphotidylinositol 3-phosphate (PI3P) present on phagosomes. However, there have been divergent reports on the involvement of SapM in phagosomal maturation arrest in mycobacteria. This study was aimed at reascertaining the involvement of SapM in phagosomal maturation arrest in M.tuberculosis. Further, for the first time, we have also studied whether SapM is essential for the pathogenesis of M.tuberculosis. By deleting the sapM gene of M.tuberculosis, we demonstrate that MtbΔsapM is defective in the arrest of phagosomal maturation as well as for growth in human THP-1 macrophages. We further show that MtbΔsapM is severely attenuated for growth in the lungs and spleen of guinea pigs and has a significantly reduced ability to cause pathological damage in the host when compared with the parental strain. Also, the guinea pigs infected with MtbΔsapM exhibited a significantly enhanced survival when compared with M.tuberculosis infected animals. The importance of SapM in phagosomal maturation arrest as well as in the pathogenesis of M.tuberculosis establishes it as an attractive target for the development of new therapeutic molecules against tuberculosis. PMID:23923000

  12. Polarized osteoclasts put marks of tartrate-resistant acid phosphatase on dentin slices--a simple method for identifying polarized osteoclasts.

    PubMed

    Nakayama, Takahiro; Mizoguchi, Toshihide; Uehara, Shunsuke; Yamashita, Teruhito; Kawahara, Ichiro; Kobayashi, Yasuhiro; Moriyama, Yoshinori; Kurihara, Saburo; Sahara, Noriyuki; Ozawa, Hidehiro; Udagawa, Nobuyuki; Takahashi, Naoyuki

    2011-12-01

    Osteoclasts form ruffled borders and sealing zones toward bone surfaces to resorb bone. Sealing zones are defined as ringed structures of F-actin dots (actin rings). Polarized osteoclasts secrete protons to bone surfaces via vacuolar proton ATPase through ruffled borders. Catabolic enzymes such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K are also secreted to bone surfaces. Here we show a simple method of identifying functional vestiges of polarized osteoclasts. Osteoclasts obtained from cocultures of mouse osteoblasts and bone marrow cells were cultured for 48 h on dentin slices. Cultures were then fixed and stained for TRAP to identify osteoclasts on the slices. Cells were removed from the slices with cotton swabs, and the slices subjected to TRAP and Mayer's hematoxylin staining. Small TRAP-positive spots (TRAP-marks) were detected in the resorption pits stained with Mayer's hematoxylin. Pitted areas were not always located in the places of osteoclasts, but osteoclasts existed on all TRAP-marks. A time course experiment showed that the number of TRAP-marks was maintained, while the number of resorption pits increased with the culture period. The position of actin rings formed in osteoclasts corresponded to that of TRAP-marks on dentin slices. Immunostaining of dentin slices showed that both cathepsin K and vacuolar proton ATPase were colocalized with the TRAP-marks. Treatment of osteoclast cultures with alendronate, a bisphosphonate, suppressed the formation of TRAP-marks and resorption pits without affecting the cell viability. Calcitonin induced the disappearance of both actin rings and TRAP-marks in osteoclast cultures. These results suggest that TRAP-marks are vestiges of proteins secreted by polarized osteoclasts. PMID:21983021

  13. 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 Responses.

    PubMed

    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

    2015-09-01

    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.

  14. 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 Responses.

    PubMed

    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

    2015-09-01

    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

  15. 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

    PubMed Central

    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

  16. Identification of Biomarkers of Exposure to FTOHs and PAPs in Humans Using a Targeted and Nontargeted Analysis Approach.

    PubMed

    Dagnino, Sonia; Strynar, Mark J; McMahen, Rebecca L; Lau, Christopher S; Ball, Carol; Garantziotis, Stavros; Webster, Thomas F; McClean, Michael D; Lindstrom, Andrew B

    2016-09-20

    Although historic perfluorinated compounds are currently under scrutiny and growing regulatory control in the world, little is known about human exposure to other polyfluorinated compounds presently in use. Fluorotelomer alcohols (FTOHs) and polyfluoroalkyl phosphate esters (PAPs) are known to degrade to terminal perfluorinated acids and toxic reactive intermediates through metabolic pathways. Therefore, it is important to characterize their human exposure by the identification of unique biomarkers. With the use of liquid chromatography-mass spectrometry-time-of-flight analysis (LC-MS-TOF), we developed a workflow for the identification of metabolites for the 8:2 FTOH and 8:2 diPAP. Analysis of serum and urine of dosed rats indicated the 8:2 FTOH-sulfate and the 8:2 diPAP as potential biomarkers. These compounds, as well as 25 other fluorinated compounds and metabolites, were analyzed in human serum and urine samples from the general population (n = 100) and office workers (n = 30). The 8:2 FTOH-sulfate was measured for the first time in human samples in 5 to 10% of the serum samples, ranging from 50 to 80 pg/mL. The 8:2 diPAP was measured in 58% of the samples, ranging from 100 to 800 pg/mL. This study indicates the FTOH-sulfate conjugate as a biomarker of exposure to FTOHs and PAPs in humans.

  17. Identification of Biomarkers of Exposure to FTOHs and PAPs in Humans Using a Targeted and Nontargeted Analysis Approach.

    PubMed

    Dagnino, Sonia; Strynar, Mark J; McMahen, Rebecca L; Lau, Christopher S; Ball, Carol; Garantziotis, Stavros; Webster, Thomas F; McClean, Michael D; Lindstrom, Andrew B

    2016-09-20

    Although historic perfluorinated compounds are currently under scrutiny and growing regulatory control in the world, little is known about human exposure to other polyfluorinated compounds presently in use. Fluorotelomer alcohols (FTOHs) and polyfluoroalkyl phosphate esters (PAPs) are known to degrade to terminal perfluorinated acids and toxic reactive intermediates through metabolic pathways. Therefore, it is important to characterize their human exposure by the identification of unique biomarkers. With the use of liquid chromatography-mass spectrometry-time-of-flight analysis (LC-MS-TOF), we developed a workflow for the identification of metabolites for the 8:2 FTOH and 8:2 diPAP. Analysis of serum and urine of dosed rats indicated the 8:2 FTOH-sulfate and the 8:2 diPAP as potential biomarkers. These compounds, as well as 25 other fluorinated compounds and metabolites, were analyzed in human serum and urine samples from the general population (n = 100) and office workers (n = 30). The 8:2 FTOH-sulfate was measured for the first time in human samples in 5 to 10% of the serum samples, ranging from 50 to 80 pg/mL. The 8:2 diPAP was measured in 58% of the samples, ranging from 100 to 800 pg/mL. This study indicates the FTOH-sulfate conjugate as a biomarker of exposure to FTOHs and PAPs in humans. PMID:27477586

  18. Ternary oxovanadium(IV) complexes with amino acid-Schiff base and polypyridyl derivatives: synthesis, characterization, and protein tyrosine phosphatase 1B inhibition.

    PubMed

    Lu, Liping; Yue, Jinjun; Yuan, Caixia; Zhu, Miaoli; Han, Hong; Liu, Zhiwei; Guo, Maolin

    2011-10-01

    To investigate the structure-activity relationship of vanadium complexes in inhibiting protein tyrosine phosphatase1B (PTP1B), eight mixed-ligand oxovanadium(IV) complexes, [V(IV)O(SalAla)(NN)] (H(2)SalAla for salicylidene alanine, NN for N,N'-donor heterocyclic base, namely, 2,2'-bipyridine (bpy, 1), 1,10-phenanthroline (phen, 2), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq, 3), dipyrido[3,2-a:2',3'-c]phenazine (dppz, 4)), [V(IV)O(SalLys)(dpq)] (5), [V(IV)O(SalLys)(dppz)] (6), [V(IV)O(SalAsp)(dppz)], (7) and [V(IV)O(SalTrp)(dppz)] (8)), of which 3-8 are new, have been prepared and characterized by elemental analysis, infrared, UV-visible, electrospray ionization mass spectrometry and conductivity. The molar conductance data confirmed the non-electrolytic nature of the complexes in DMSO solution. The coordination in [V(IV)O (SalAla)(phen)] (2) was confirmed by X-ray crystal structure analysis. The oxidation state of V(IV) with d(1) configuration in 2 was confirmed by EPR. The speciation of VO-SalAla-phen in aqueous solution was investigated by potentiometric pH titrations. The results indicate that the main species are two ternary complexes at the pH range 7.0-7.4. Biochemical assays demonstrate that the mixed-ligand oxovanadium(IV) complexes are potent inhibitors of PTP1B with IC(50) values in the range of 62-597nM, approximately 3-10 fold weaker in potency than those of similar mixed-ligand oxovanadium(IV) complexes of salicylidene anthranilic acid (SAA) derivative with polypyridyl ligands, except complex 8, which exhibits comparable or better inhibition activity than those of the mixed-ligand oxovanadium(IV) complexes of SAA derivative with polypyridyl ligands. The results demonstrate that the structures of vanadium complexes influence the PTP1B inhibition activity. Kinetics assays reveal that complex 2 inhibits PTP1B in a competitive manner.

  19. Relationship of spermatoscopy, prostatic acid phosphatase activity and prostate-specific antigen (p30) assays with further DNA typing in forensic samples from rape cases.

    PubMed

    Romero-Montoya, Lydia; Martínez-Rodríguez, Hugo; Pérez, Miguel Antonio; Argüello-García, Raúl

    2011-03-20

    In the forensic laboratory the biological analyses for rape investigation commonly include vaginal swabs as sample material combined to biochemical tests including sperm cytology (SC) and detection of acid phosphatase activity (AP) and prostate-specific antigen (PSA, p30) for the conclusive identification of semen components. Most reports comparing these tests relied on analysis of semen samples or donor swabs taken under controlled conditions; however their individual or combined efficacy under real live sampling conditions in different laboratories is largely unknown. We carried out SC, APA and PSA analyses in vaginal swabs collected from casework rapes submitted to Mexican Forensic Laboratories at Texcoco and Toluca. On the basis of positive and negative results from each assay and sample, data were classified into eight categories (I-VIII) and compared with those obtained in the two only similar studies reported in Toronto, Canada and Hong Kong, China. SC and APA assays had the higher overall positivity in Toluca and Texcoco samples respectively and otherwise PSA had a lower but very similar positivity between these two laboratories. When compared to the previous studies some similarities were found, namely similar frequencies (at a ratio of approximately 1 out of 3) of samples being positive or negative by all techniques (Categories I and VI respectively) and a comparable overall positivity of APA and SC but higher than that of PSA. Indeed the combined results of using SC, APA and PSA tests was considered as conclusive for semen detection from approximately 1 out of 3 cases (Category I) to approximately 1 out of 2 cases in a scenario where at least SC is positive, strongly presumptive in 2 out of 3 cases (with at least one test positive) and the remainder 1 out of 3 cases (Category VI) suggested absence of semen. By determining Y-STR polymorphisms (12-loci) in additional samples obtained at Toluca laboratory, complete DNA profiles were determined from all

  20. Oligoribonuclease is a common downstream target of lithium-induced pAp accumulation in Escherichia coli and human cells

    PubMed Central

    Mechold, Undine; Ogryzko, Vasily; Ngo, Saravuth; Danchin, Antoine

    2006-01-01

    We identified Oligoribonuclease (Orn), an essential Escherichia coli protein and the only exonuclease degrading small ribonucleotides (5mer to 2mer) and its human homologue, small fragment nuclease (Sfn), in a screen for proteins that are potentially regulated by 3′-phosphoadenosine 5′-phosphate (pAp). We show that both enzymes are sensitive to micromolar amounts of pAp in vitro. We also demonstrate that Orn can degrade short DNA oligos in addition to its activity on RNA oligos, similar to what was documented for Sfn. pAp was shown to accumulate as a result of inhibition of the pAp-degrading enzyme by lithium, widely used to treat bipolar disorder, thus its regulatory targets are of significant medical interest. CysQ, the E.coli pAp-phosphatase is strongly inhibited by lithium and calcium in vitro and is a main target of lithium toxicity in vivo. Our findings point to remarkable conservation of the connection between sulfur- and RNA metabolism between E.coli and humans. PMID:16682444

  1. Biogeochemical drivers of phosphatase activity in salt marsh sediments

    NASA Astrophysics Data System (ADS)

    Freitas, Joana; Duarte, Bernardo; Caçador, Isabel

    2014-10-01

    Although nitrogen has become a major concern for wetlands scientists dealing with eutrophication problems, phosphorous represents another key element, and consequently its biogeochemical cycling has a crucial role in eutrophication processes. Microbial communities are a central component in trophic dynamics and biogeochemical processes on coastal systems, since most of the processes in sediments are microbial-mediated due to enzymatic action, including the mineralization of organic phosphorus carried out by acid phosphatase activity. In the present work, the authors investigate the biogeochemical sediment drivers that control phosphatase activities. Authors also aim to assess biogeochemical factors' influence on the enzyme-mediated phosphorous cycling processes in salt marshes. Plant rhizosediments and bare sediments were collected and biogeochemical features, including phosphatase activities, inorganic and organic phosphorus contents, humic acids content and pH, were assessed. Acid phosphatase was found to give the highest contribution for total phosphatase activity among the three pH-isoforms present in salt marsh sediments, favored by acid pH in colonized sediments. Humic acids also appear to have an important role inhibiting phosphatase activity. A clear relation of phosphatase activity and inorganic phosphorous was also found. The data presented reinforces the role of phosphatase in phosphorous cycling.

  2. Engineering of the PapMV vaccine platform with a shortened M2e peptide leads to an effective one dose influenza vaccine.

    PubMed

    Carignan, Damien; Thérien, Ariane; Rioux, Gervais; Paquet, Geneviève; Gagné, Marie-Ève Laliberté; Bolduc, Marilène; Savard, Pierre; Leclerc, Denis

    2015-12-16

    The emergence of highly virulent influenza strains and the risks of pandemics as well as the limited efficiency of the current seasonal vaccines are important public health concerns. There is a major need for new influenza vaccines that would be broadly cross-protective. The ectodomain of matrix protein 2 (M2e) is highly conserved amongst different influenza strains and could be used as a broad spectrum antigen. To overcome its low immunogenicity we have fused a short peptide epitope derived from the human consensus sequence of M2e (amino acids 6-14, EVETPIRNE) to the N-terminus of papaya mosaic virus coat protein. The fusion harboring coat proteins were assembled around a single stranded RNA into virus-like particles (PapMV-sM2e). The resulting PapMV-sM2e rod-shaped particle was stable and indistinguishable from regular PapMV particles. A single intramuscular immunization with PapMV-sM2e was sufficient to mount appreciable levels of CD4 dependent M2e specific total IgG and IgG2a antibody in mice sera. PapMV-sM2e proved to be self-adjuvanting since the addition of PapMV as an exogenous adjuvant did not result in significantly improved antibody titers. In addition, we confirmed the adjuvant property of PapMV-sM2e using the trivalent inactivated flu vaccine as antigen and demonstrated that the newly engineered nanoparticles areas efficacious as an adjuvant than the original PapMV nanoparticles. Upon infection with a sub-lethal dose of influenza, PapMV-sM2e vaccinated animals were completely protected from virus induced morbidity and mortality. Mice immunized with decreasing amounts of PapMV-sM2e and challenged with a more stringent dose of influenza virus displayed dose-dependent levels of protection. Seventy percent of the mice immunized once with the highest dose of PapMV-sM2e survived the challenged. The survival of the mice correlated mainly with the levels of anti-M2e IgG2a antibodies obtained before the infection. These results demonstrate that PapMV-sM2e can

  3. Structure-Function Analysis of the 3' Phosphatase Component of T4 Polynucleotide Kinase/phosphatase

    SciTech Connect

    Zhu,H.; Smith, P.; Wang, L.; Shuman, S.

    2007-01-01

    T4 polynucleotide kinase/phosphatase (Pnkp) exemplifies a family of bifunctional enzymes with 5'-kinase and 3' phosphatase activities that function in nucleic acid repair. T4 Pnkp is a homotetramer of a 301-aa polypeptide, which consists of an N-terminal kinase domain of the P-loop phosphotransferase superfamily and a C-terminal phosphatase domain of the DxD acylphosphatase superfamily. The homotetramer is formed via pairs of phosphatase-phosphatase and kinase-kinase homodimer interfaces. Here we identify four side chains-Asp187, Ser211, Lys258, and Asp277-that are required for 3' phosphatase activity. Alanine mutations at these positions abolished phosphatase activity without affecting kinase function or tetramerization. Conservative substitutions of asparagine or glutamate for Asp187 did not revive the 3' phosphatase, nor did arginine or glutamine substitutions for Lys258. Threonine in lieu of Ser211 and glutamate in lieu of Asp277 restored full activity, whereas asparagine at position 277 had no salutary effect. We report a 3.0 A crystal structure of the Pnkp tetramer, in which a sulfate ion is coordinated between Arg246 and Arg279 in a position that we propose mimics one of the penultimate phosphodiesters (5'NpNpNp-3') of the polynucleotide 3'-PO(4) substrate. The amalgam of mutational and structural data engenders a plausible catalytic mechanism for the phosphatase that includes covalent catalysis (via Asp165), general acid-base catalysis (via Asp167), metal coordination (by Asp165, Asp277 and Asp278), and transition state stabilization (via Lys258, Ser211, backbone amides, and the divalent cation). Other critical side chains play architectural roles (Arg176, Asp187, Arg213, Asp254). To probe the role of oligomerization in phosphatase function, we introduced six double-alanine cluster mutations at the phosphatase-phosphatase domain interface, two of which (R297A-Q295A and E292A-D300A) converted Pnkp from a tetramer to a dimer and ablated phosphatase activity.

  4. Inhibition of specific binding of okadaic acid to protein phosphatase 2A by microcystin-LR, calyculin-A and tautomycin: method of analysis of interactions of tight-binding ligands with target protein.

    PubMed Central

    Takai, A; Sasaki, K; Nagai, H; Mieskes, G; Isobe, M; Isono, K; Yasumoto, T

    1995-01-01

    Several groups have reported that okadaic acid (OA) and some other tight-binding protein phosphatase inhibitors including microcystin-LR (MCLR), calyculin-A and tautomycin prevent each other from binding to protein phosphatase 2A (PP2A). In this paper, we have introduced an improved procedure for examining to what extent the affinity of an enzyme for a labelled tight-binding ligand is reduced by binding of an unlabelled tight-binding, ligand to the enzyme. Using this procedure, we have analysed the dose-dependent reduction of PP2A binding of [24-3H]OA by addition of OA, MCLR, calyculin-A and tautomycin. The results indicate that the binding of the unlabelled inhibitors to the PP2A molecule causes a dramatic (10(6)-10(8)-fold) increase in the dissociation constant associated with the interaction of [24-3H]OA and PP2A. This suggests that OA and the other inhibitors bind to PP2A in a mutually exclusive manner. The protein phosphatase inhibitors may share the same binding site on the PP2A molecule. We have also measured values of the dissociation constant (Ki) for the interaction of these toxins with protein phosphatase 1 (PP1). For MCLR and calyculin-A, the ratio of the Ki value obtained for PP1 to that for PP2A was in the range 4-9, whereas it was 0.01-0.02 for tautomycin. The value of tautomycin is considerably smaller than that (0.4) calculated from previously reported Ki values. PMID:7702557

  5. Molecular cloning, genomic organization, chromosomal mapping and subcellular localization of mouse PAP7: a PBR and PKA-RIalpha associated protein.

    PubMed

    Liu, Jun; Cavalli, Luciane R; Haddad, Bassem R; Papadopoulos, Vassilios

    2003-04-10

    A mouse protein that interacts with the peripheral-type benzodiazepine receptor (PBR) and the cAMP-dependent protein kinase A (PKA) regulatory subunit RIalpha (PKA-RIalpha), named PBR and PKA associated protein 7 (PAP7) was identified and shown to be involved in hormone-induced steroid biosynthesis in testicular Leydig cells. In the present study, mouse PAP7 cDNA was extended by 5'-rapid amplification of cDNA ends; and a 3432 bp sequence, encoding a 525-amino-acid protein with a calculated molecular weight of 60 kDa, was re-assembled. Mouse and human PAP7 share an 85% amino acid identity and contain a conserved acyl-CoA-binding protein/diazepam binding inhibitor (ACBP/DBI) motif. ACBP/DBI has been identified as the endogenous PBR ligand able to stimulate mitochondrial steroid formation in all steroidogenic cells. The full-length mouse PAP7 gene was cloned and assembled by screening a BAC clone, polymerase chain reaction and searching the mouse genome database. The gene is approximately 29 kb in length and includes eight exons and seven introns. Although it is shorter than the human PAP7 gene, all exons are conserved between the mouse and human. The mouse PAP7 gene was mapped to chromosome 1H3-5 by fluorescence in situ hybridization in agreement with in silico search of the mouse genome database that mapped the PAP7 cDNA sequence to the 1H4 area. Immunofluorescence confocal microscopy demonstrated that PAP7 is mainly localized in the trans-Golgi apparatus and mitochondria in mouse tumor Leydig cells, in agreement with its proposed function in targeting the PKA isoenzyme to organelles rich in PBR, i.e. mitochondria, where phosphorylation of specific protein substrates mediates the hormone-induced steroid formation.

  6. N-Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites.

    PubMed

    Niu, Canfang; Luo, Huiying; Shi, Pengjun; Huang, Huoqing; Wang, Yaru; Yang, Peilong; Yao, Bin

    2015-12-04

    N-Glycosylation can modulate enzyme structure and function. In this study, we identified two pepsin-resistant histidine acid phosphatase (HAP) phytases from Yersinia kristensenii (YkAPPA) and Yersinia rohdei (YrAPPA), each having an N-glycosylation motif, and one pepsin-sensitive HAP phytase from Yersinia enterocolitica (YeAPPA) that lacked an N-glycosylation site. Site-directed mutagenesis was employed to construct mutants by altering the N-glycosylation status of each enzyme, and the mutant and wild-type enzymes were expressed in Pichia pastoris for biochemical characterization. Compared with those of the N-glycosylation site deletion mutants and N-deglycosylated enzymes, all N-glycosylated counterparts exhibited enhanced pepsin resistance. Introduction of the N-glycosylation site into YeAPPA as YkAPPA and YrAPPA conferred pepsin resistance, shifted the pH optimum (0.5 and 1.5 pH units downward, respectively) and improved stability at acidic pH (83.2 and 98.8% residual activities at pH 2.0 for 1 h). Replacing the pepsin cleavage sites L197 and L396 in the immediate vicinity of the N-glycosylation motifs of YkAPPA and YrAPPA with V promoted their resistance to pepsin digestion when produced in Escherichia coli but had no effect on the pepsin resistance of N-glycosylated enzymes produced in P. pastoris. Thus, N-glycosylation may improve pepsin resistance by enhancing the stability at acidic pH and reducing pepsin's accessibility to peptic cleavage sites. This study provides a strategy, namely, the manipulation of N-glycosylation, for improvement of phytase properties for use in animal feed.

  7. N-Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

    PubMed Central

    Niu, Canfang; Luo, Huiying; Shi, Pengjun; Huang, Huoqing; Wang, Yaru; Yang, Peilong

    2015-01-01

    N-Glycosylation can modulate enzyme structure and function. In this study, we identified two pepsin-resistant histidine acid phosphatase (HAP) phytases from Yersinia kristensenii (YkAPPA) and Yersinia rohdei (YrAPPA), each having an N-glycosylation motif, and one pepsin-sensitive HAP phytase from Yersinia enterocolitica (YeAPPA) that lacked an N-glycosylation site. Site-directed mutagenesis was employed to construct mutants by altering the N-glycosylation status of each enzyme, and the mutant and wild-type enzymes were expressed in Pichia pastoris for biochemical characterization. Compared with those of the N-glycosylation site deletion mutants and N-deglycosylated enzymes, all N-glycosylated counterparts exhibited enhanced pepsin resistance. Introduction of the N-glycosylation site into YeAPPA as YkAPPA and YrAPPA conferred pepsin resistance, shifted the pH optimum (0.5 and 1.5 pH units downward, respectively) and improved stability at acidic pH (83.2 and 98.8% residual activities at pH 2.0 for 1 h). Replacing the pepsin cleavage sites L197 and L396 in the immediate vicinity of the N-glycosylation motifs of YkAPPA and YrAPPA with V promoted their resistance to pepsin digestion when produced in Escherichia coli but had no effect on the pepsin resistance of N-glycosylated enzymes produced in P. pastoris. Thus, N-glycosylation may improve pepsin resistance by enhancing the stability at acidic pH and reducing pepsin's accessibility to peptic cleavage sites. This study provides a strategy, namely, the manipulation of N-glycosylation, for improvement of phytase properties for use in animal feed. PMID:26637601

  8. CoPAP: Coevolution of presence-absence patterns.

    PubMed

    Cohen, Ofir; Ashkenazy, Haim; Levy Karin, Eli; Burstein, David; Pupko, Tal

    2013-07-01

    Evolutionary analysis of phyletic patterns (phylogenetic profiles) is widely used in biology, representing presence or absence of characters such as genes, restriction sites, introns, indels and methylation sites. The phyletic pattern observed in extant genomes is the result of ancestral gain and loss events along the phylogenetic tree. Here we present CoPAP (coevolution of presence-absence patterns), a user-friendly web server, which performs accurate inference of coevolving characters as manifested by co-occurring gains and losses. CoPAP uses state-of-the-art probabilistic methodologies to infer coevolution and allows for advanced network analysis and visualization. We developed a platform for comparing different algorithms that detect coevolution, which includes simulated data with pairs of coevolving sites and independent sites. Using these simulated data we demonstrate that CoPAP performance is higher than alternative methods. We exemplify CoPAP utility by analyzing coevolution among thousands of bacterial genes across 681 genomes. Clusters of coevolving genes that were detected using our method largely coincide with known biosynthesis pathways and cellular modules, thus exhibiting the capability of CoPAP to infer biologically meaningful interactions. CoPAP is freely available for use at http://copap.tau.ac.il/.

  9. Ga(III) complexes as models for the M(III) site of purple acid phosphatase: ligand effects on the hydrolytic reactivity toward bis(2,4-dinitrophenyl) phosphate.

    PubMed

    Coleman, Fergal; Hynes, Michael J; Erxleben, Andrea

    2010-07-19

    The effects of a series of Ga(III) complexes with tripodal ligands on the hydrolysis rate of the activated phosphate diester bis(2,4-dinitrophenyl)phosphate (BDNPP) have been investigated. In particular, the influence of the nature of the ligand donor sites on the reactivity of Ga(III) which represents a mimic of the Fe(III) ion in purple acid phosphatase has been evaluated. It has been shown that replacing neutral nitrogen donor atoms and carboxylate groups by phenolate groups enhanced the reactivity of the Ga complexes. Bell-shaped pH-rate profiles and the measured solvent deuterium isotope effects are indicative of a mechanism that involves nucleophilic attack on the coordinated substrate by Ga-OH. The trend in reactivity found for the different Ga complexes reveals that of the two effects of the metal, Lewis acid activation of the substrate and nucleophile activation, the latter one is more important in determining the intrinsic reactivity of the metal catalyst. The relevance of the present findings for the modulation of the activity of the M(III) ion in purple acid phosphatase whose active site contains a phenolate (tyrosine side chain) is discussed. PMID:20565083

  10. Phosphatidylinositol anchor of HeLa cell alkaline phosphatase

    SciTech Connect

    Jemmerson, R.; Low, M.G.

    1987-09-08

    Alkaline phosphatase from cancer cells, HeLa TCRC-1, was biosynthetically labeled with either /sup 3/H-fatty acids or (/sup 3/H)ethanolamine as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitated material. Phosphatidylinositol-specific phospholipase C (PI-PLC) released a substantial proportion of the /sup 3/H-fatty acid label from immunoaffinity-purified alkaline phosphatase but had no effect on the radioactivity of (/sup 3/H)ethanolamine-labeled material. PI-PLC also liberated catalytically active alkaline phosphatase from viable cells, and this could be selectively blocked by monoclonal antibodies to alkaline phosphatase. However, the alkaline phosphatase released from /sup 3/H-fatty acid labeled cells by PI-PLC was not radioactive. By contrast, treatment with bromelain removed both the /sup 3/H-fatty acid and the (/sup 3/H)ethanolamine label from purified alkaline phosphatase. Subtilisin was also able to remove the (/sup 3/H)ethanolamine label from the purified alkaline phosphatase. The /sup 3/H radioactivity in alkaline phosphatase purified from (/sup 3/H)ethanolamine-labeled cells comigrated with authentic (/sup 3/H)ethanolamine by anion-exchange chromatography after acid hydrolysis. The data suggest that the /sup 3/H-fatty acid and (/sup 3/H)ethanolamine are covalently attached to the carboxyl-terminal segment since bromelain and subtilisin both release alkaline phosphatase from the membrane by cleavage at that end of the polypeptide chain. The data are consistent with findings for other proteins recently shown to be anchored in the membrane through a glycosylphosphatidylinositol structure and indicate that a similar structure contributes to the membrane anchoring of alkaline phosphatase.

  11. Biochemical Characterization of Mycobacterium smegmatis RnhC (MSMEG_4305), a Bifunctional Enzyme Composed of Autonomous N-Terminal Type I RNase H and C-Terminal Acid Phosphatase Domains

    PubMed Central

    Jacewicz, Agata

    2015-01-01

    ABSTRACT Mycobacterium smegmatis encodes several DNA repair polymerases that are adept at incorporating ribonucleotides, which raises questions about how ribonucleotides in DNA are sensed and removed. RNase H enzymes, of which M. smegmatis encodes four, are strong candidates for a surveillance role. Here, we interrogate the biochemical activity and nucleic acid substrate specificity of M. smegmatis RnhC, a bifunctional RNase H and acid phosphatase. We report that (i) the RnhC nuclease is stringently specific for RNA:DNA hybrid duplexes; (ii) RnhC does not selectively recognize and cleave DNA-RNA or RNA-DNA junctions in duplex nucleic acid; (iii) RnhC cannot incise an embedded monoribonucleotide or diribonucleotide in duplex DNA; (iv) RnhC can incise tracts of 4 or more ribonucleotides embedded in duplex DNA, leaving two or more residual ribonucleotides at the cleaved 3′-OH end and at least one or two ribonucleotides on the 5′-PO4 end; (v) the RNase H activity is inherent in an autonomous 140-amino-acid (aa) N-terminal domain of RnhC; and (vi) the C-terminal 211-aa domain of RnhC is an autonomous acid phosphatase. The cleavage specificity of RnhC is clearly distinct from that of Escherichia coli RNase H2, which selectively incises at an RNA-DNA junction. Thus, we classify RnhC as a type I RNase H. The properties of RnhC are consistent with a role in Okazaki fragment RNA primer removal or in surveillance of oligoribonucleotide tracts embedded in DNA but not in excision repair of single misincorporated ribonucleotides. IMPORTANCE RNase H enzymes help cleanse the genome of ribonucleotides that are present either as ribotracts (e.g., RNA primers) or as single ribonucleotides embedded in duplex DNA. Mycobacterium smegmatis encodes four RNase H proteins, including RnhC, which is characterized in this study. The nucleic acid substrate and cleavage site specificities of RnhC are consistent with a role in initiating the removal of ribotracts but not in single

  12. Diacyltransferase Activity and Chain Length Specificity of Mycobacterium tuberculosis PapA5 in the Synthesis of Alkyl β-Diol Lipids

    SciTech Connect

    Touchette, Megan H.; Bommineni, Gopal R.; Delle Bovi, Richard J.; Gadbery, John; Nicora, Carrie D.; Shukla, Anil K.; Kyle, Jennifer E.; Metz, Thomas O.; Martin, Dwight W.; Sampson, Nicole S.; Miller, W. T.; Tonge, Peter J.; Seeliger, Jessica C.

    2015-09-08

    Although classified as Gram-positive bacteria, Corynebacterineae possess an asymmetric outer membrane that imparts structural and thereby physiological similarity to more distantly related Gram-negative bacteria. Like lipopolysaccharide in Gram-negative bacteria, lipids in the outer membrane of Corynebacterineae have been associated with the virulence of pathogenic species such as Mycobacterium tuberculosis (Mtb). For example, Mtb strains that lack long, branched-chain alkyl esters known as dimycocerosates (DIMs) are significantly attenuated in model infections. The resultant interest in the biosynthetic pathway of these unusual virulence factors has led to the elucidation of many of the steps leading to the final esterification of the alkyl beta-diol, phthiocerol, with branched-chain fatty acids know as mycocerosates. PapA5 is an acyltransferase implicated in these final reactions. We here show that PapA5 is indeed the terminal enzyme in DIM biosynthesis by demonstrating its dual esterification activity and chain-length preference using synthetic alkyl beta-diol substrate analogues. Applying these analogues to a series of PapA5 mutants, we also revise a model for the substrate binding within PapA5. Finally, we demonstrate that the Mtb Ser/Thr kinase PknB modifies PapA5 on three Thr residues, including two (T196, T198) located on an unresolved loop. These results clarify the DIM biosynthetic pathway and suggest possible mechanisms by which DIM biosynthesis may be regulated by the post-translational modification of PapA5.

  13. Zn-exchange and Mössbauer studies on the [Fe-Fe] derivatives of the purple acid Fe(III)-Zn(II)-phosphatase from kidney beans.

    PubMed

    Suerbaum, H; Körner, M; Witzel, H; Althaus, E; Mosel, B D; Müller-Warmuth, W

    1993-05-15

    In order to perform Mössbauer studies, Zn(II) in the Fe(III)-Zn(II) purple acid phosphatase of the red kidney bean has been exchanged by incubating the semiapoenzyme with 57Fe(II). The resulting Fe(III)-57Fe(II) enzyme has 125% activity, compared with that of the Zn(II) enzyme. It can be oxidized by H2O2 or peroxydisulfate to the Fe(III)-57Fe(III) species with a 30-times lower activity. Incubation of the metal-free apoenzyme with 57Fe(II) in the presence of O2 leads to the 57Fe(III)-57Fe(II) species which is stable in dilute solutions, but partially oxidized during the concentration procedure to the 57Fe(III)-57Fe(III) enzyme. Limited reduction of the oxidized enzyme with ascorbate delivers a mixture of the Fe(II)-Fe(II)/Fe(III)-Fe(III) species, but not the mixed valent Fe(III)-Fe(II) species, indicating that after the transfer of the first electron the second electron of the ascorbate radical is immediately transferred to the second Fe(III). The Mössbauer spectra of the oxidized species show at 4.2 K two quadrupole doublets with delta of 0.51 mm/s and 0.53 mm/s and delta E of 1.46 and 0.96 mm/s indicating high spin Fe(III) in two different binding sites, obviously with a higher asymmetry in the chromophoric Fe(III) site. The values are too low for a mu-oxo bridge. The mixed-valent Fe(III)-Fe(II) species shows two quadrupole doublets with delta values of 0.55 mm/s and 1.14 mm/s and delta E values of 1.43 mm/s and 3.01 mm/s at 70 K for high spin Fe(II) and Fe(III), but the signal of the Fe(II) component shows magnetic patterns at 4.2 K indicating a half-integer spin system with antiferromagnetic coupling. The Fe(II)-Fe(II) system exhibits two quadrupole doublets with delta values of 1.18 mm/s and 1.22 mm/s and with delta E values of 3.69 mm/s and 2.68 mm/s again indicating a higher asymmetry in the originally chromophoric Fe(III)-binding site. Addition of phosphate shows only minor differences in the oxidized enzyme and in the mixed valent Fe(III)-Fe(II) system

  14. Zn-exchange and Mössbauer studies on the [Fe-Fe] derivatives of the purple acid Fe(III)-Zn(II)-phosphatase from kidney beans.

    PubMed

    Suerbaum, H; Körner, M; Witzel, H; Althaus, E; Mosel, B D; Müller-Warmuth, W

    1993-05-15

    In order to perform Mössbauer studies, Zn(II) in the Fe(III)-Zn(II) purple acid phosphatase of the red kidney bean has been exchanged by incubating the semiapoenzyme with 57Fe(II). The resulting Fe(III)-57Fe(II) enzyme has 125% activity, compared with that of the Zn(II) enzyme. It can be oxidized by H2O2 or peroxydisulfate to the Fe(III)-57Fe(III) species with a 30-times lower activity. Incubation of the metal-free apoenzyme with 57Fe(II) in the presence of O2 leads to the 57Fe(III)-57Fe(II) species which is stable in dilute solutions, but partially oxidized during the concentration procedure to the 57Fe(III)-57Fe(III) enzyme. Limited reduction of the oxidized enzyme with ascorbate delivers a mixture of the Fe(II)-Fe(II)/Fe(III)-Fe(III) species, but not the mixed valent Fe(III)-Fe(II) species, indicating that after the transfer of the first electron the second electron of the ascorbate radical is immediately transferred to the second Fe(III). The Mössbauer spectra of the oxidized species show at 4.2 K two quadrupole doublets with delta of 0.51 mm/s and 0.53 mm/s and delta E of 1.46 and 0.96 mm/s indicating high spin Fe(III) in two different binding sites, obviously with a higher asymmetry in the chromophoric Fe(III) site. The values are too low for a mu-oxo bridge. The mixed-valent Fe(III)-Fe(II) species shows two quadrupole doublets with delta values of 0.55 mm/s and 1.14 mm/s and delta E values of 1.43 mm/s and 3.01 mm/s at 70 K for high spin Fe(II) and Fe(III), but the signal of the Fe(II) component shows magnetic patterns at 4.2 K indicating a half-integer spin system with antiferromagnetic coupling. The Fe(II)-Fe(II) system exhibits two quadrupole doublets with delta values of 1.18 mm/s and 1.22 mm/s and with delta E values of 3.69 mm/s and 2.68 mm/s again indicating a higher asymmetry in the originally chromophoric Fe(III)-binding site. Addition of phosphate shows only minor differences in the oxidized enzyme and in the mixed valent Fe(III)-Fe(II) system

  15. Differentiation, early response gene expression, and apoptosis induction in human breast tumor cells by Okadaic Acid and related inhibitors of protein phosphatases 1 and 2A. Okadaic acid effects on human breast tumor cells

    SciTech Connect

    Kiguchi, K.; Giometti, C.; Chubb, C.H.; Huberman, E.; Fujiki, H.

    1992-08-20

    Okadaic acid (OA), a tumor promoter and an inhibitor of protein phosphatases (PPH) 1 and 2A, was tested for its ability to induce events associated with differentiation and apoptosis induction in the human MCF-7, AU-565, and MB-231 breast tumor cells. Differentiation in these cells was characterized by inhibition of cell multiplication, reactivity with monoclonal antibodies to {alpha}-lactalbumin and {beta}-casein, and the appearance of large lipid droplets; apoptosis was characterized by the appearance of cells with segmented and fragmented nuclei. In the MCF-7 cell line, OA at nanomolar concentrations elicited within 5 min an increase in the phosphorylation of a set of cellular proteins, within hours expression of the early response genes, junB, c-jun, and c-fos and within days manifestation of differentiation and apoptosis markers. Differentiation and apoptosis were also induced by dinophysistoxin-1 and calyculin A, two other tumor promoters and inhibitors of PPH 1 and 2A, but not by OA tetramethyl ether, an inactive OA derivative, or microcystin LR, a PPH 1 and 2A inhibitor that penetrates epithelial cells poorly. OA induced both differentiation and apoptosis in MB-231 cells and MCF-7, but only differentiation in AU-565 cells. Phorbol 12-myristate 13-acetate (PMA), a tumor promoter that is not an inhibitor of PPH 1 and 2A but rather an activator of protein kinase C, also induced within minutes the phosphorylation of proteins, within hours the expression of early response genes, and within days differentiation, but not apoptosis; yet PMA was able to attenuate apoptosis induced by the okadaic acid class of tumor promoters. These results indicate that OA and related agents can induce processes that result in tumor breast cell differentiation and apoptosis, and this induction is associated with their ability to inhibit PPH 1 and 2A. Yet apoptosis is not necessarily required for differentiation induction by these agents.

  16. Resistance of leishmanial phosphatases to inactivation by oxygen metabolites.

    PubMed

    Saha, A K; Das, S; Glew, R H; Gottlieb, M

    1985-09-01

    Leishmania donovani promastigotes produce large quantities of two distinct acid phosphatases; a tartrate-resistant enzyme is localized to the external surface of the plasma membrane, and a tartrate-sensitive enzyme is secreted into the growth medium. It was shown previously that preincubation of human neutrophils and macrophages with the tartrate-resistant phosphatase markedly reduced the ability of these host cells to produce superoxide anions in response to stimulation with the activator formyl-methionyl-leucyl-phenylalanine. The possibility that the cell surface acid phosphatase or the phosphatase that is secreted into the extracellular fluid might compromise other host cell functions, especially intracellular ones, depends on the ability of the enzyme to resist exposure to toxic oxygen metabolites (e.g., superoxide anion, hydrogen peroxide, hypochlorite) generated by phagocytic cells. In the present report, we show that both leishmanial acid phosphatases were relatively resistant to inactivation by oxygen metabolites. At pH 5.5, the activity of the tartrate-resistant phosphatase was reduced 50% by incubation for 1 h with each of the following: 30 mM O2-, 500 mM hydrogen peroxide, and 6 mM hypochlorite ion. These concentrations are many fold greater than the concentrations of these substances that are generated by stimulated polymorphonuclear phagocytes. The tartrate-sensitive acid phosphatase differed markedly from the tartrate-resistant phosphatase in that the former was essentially insensitive to even very high concentrations of superoxide anion and hydrogen peroxide. Furthermore, 50% inactivation of the tartrate-sensitive leishmanial phosphatase required exposure to 35 mM hypochlorite for 30 min. These results indicate that the catalytic potential of these two leishmanial acid phosphatases probably survives exposure to toxic oxygen metabolites generated by neutrophils and macrophages.

  17. Socioeconomic and regional inequalities of pap smear coverage.

    PubMed

    Manica, Silvia Troyahn; Drachler, Maria de Lourdes; Teixeira, Luciana Barcellos; Ferla, Alcindo Antônio; Gouveia, Helga Geremias; Anschau, Fernando; Oliveira, Dora Lúcia Leidens Correa de

    2016-03-01

    Objectives To identify socioeconomic and regional inequalities of pap smear coverage in the state of Rio Grande do Sul. Methods An ecological study based on data of the 2011-2012 national health information system to estimate the annual coverage of pap smears for the overall female population of the state and for women without private health insurance. We estimated annual pap smear coverage according to the Municipal Social Vulnerability Index and health macro-regions and regions of the state. Results The percentage of women without private health insurance ranged from 38.1% to 94.2% in the health regions. Pap smear coverage was 17.3% for the overall female population and 23.8% for women without private health insurance. Pap smear coverage was higher in more socially vulnerable municipalities and regions with a higher percentage of women with private health insurance. Conclusions The prevalence of private health insurance should be considered in studies that address the coverage of the Brazilian Unified Health System (SUS). PMID:26982680

  18. Redox regulation of protein tyrosine phosphatase 1B (PTP1B): Importance of steric and electronic effects on the unusual cyclization of the sulfenic acid intermediate to a sulfenyl amide

    NASA Astrophysics Data System (ADS)

    Sarma, Bani Kanta

    2013-09-01

    The redox regulation of protein tyrosine phosphatase 1B (PTP1B) via the unusual transformation of its sulfenic acid (PTP1B-SOH) to a cyclic sulfenyl amide intermediate is studied by using small molecule chemical models. These studies suggest that the sulfenic acids derived from the H2O2-mediated reactions o-amido thiophenols do not efficiently cyclize to sulfenyl amides and the sulfenic acids produced in situ can be trapped by using methyl iodide. Theoretical calculations suggest that the most stable conformer of such sulfenic acids are stabilized by nO → σ*S-OH orbital interactions, which force the -OH group to adopt a position trans to the S⋯O interaction, leading to an almost linear arrangement of the O⋯S-O moiety and this may be the reason for the slow cyclization of such sulfenic acids to their corresponding sulfenyl amides. On the other hand, additional substituents at the 6-position of o-amido phenylsulfenic acids that can induce steric environment and alter the electronic properties around the sulfenic acid moiety by S⋯N or S⋯O nonbonded interactions destabilize the sulfenic acids by inducing strain in the molecule. This may lead to efficient the cyclization of such sulfenic acids. This model study suggests that the amino acid residues in the close proximity of the sulfenic acid moiety in PTP1B may play an important role in the cyclization of PTP1B-SOH to produce the corresponding sulfenyl amide.

  19. The annual Pap test: a dubious policy success.

    PubMed

    Foltz, A M; Kelsey, J L

    1978-01-01

    The annual Pap test became a recommended standard for American women without ever having been subjected to controlled trials to estimate its efficacy and effectiveness. After more than 30 years of routine use, the Pap test fails to meet most of the generally accepted criteria for a mass screening program. The policy persists, however, because the nation's ideology supports the maximum utilization of new technologies; and special interest groups have promoted the test as the major weapon in the "War on Cancer." With some exceptions, this is a questionable allocation of public and private health resources.

  20. Phosphatase activities as biosignatures of extant life

    NASA Astrophysics Data System (ADS)

    Kobayashi, K.; Itoh, Y.; Edazawa, Y.; Moroi, A.; Takano, Y.

    It has been recognized that terrestrial biosphere expands to such extreme environments as deep subsurface lithosphere high temperature hot springs and stratosphere Possible extraterrestrial biospheres in Mars Europa and Titan are being discussed Many biosignatures or biomarkers have been proposed to detect microbial activities in such extreme environments Phosphate esters are essential for the terrestrial life since they are constituents of nucleic acids and cell mebranes Thus all the terrestrial organisms have phosphatases that are enzymes catalyzing hydrolysis of phosphate esters We analyzed phosphatase activities in the samples obtained in extreme environments such as submarine hydrothermal systems and discussed whether they can be used as biosignatures for extant life Core samples and chimney samples were collected at the Suiyo Seamount Izu-Bonin Arc the Pacific Ocean in 2001 and 2002 and in South Mariana hydrothermal systems the Pacific Oceanas in 2003 both in a part of the Archaean Park Project Phosphatase activity in solid rock samples was measured spectrometrically by using 25 mM p-nitrophenyl phosphate pH 8 0 or pH 6 5 as a substrate as follows Pulverized samples were incuvated with substrate solution for an hour and then production rate of p-nitrophenol was calculated with absorbance at 410 nm Phosphatase activity in extracts was measured fluorometrically by using 4-methylumberyferryl phosphate as a substrate Concentration of amino acids and their enantiomeric ratio were determined by HPLC after HF digestion of the

  1. Pap, papG and prsG DNA sequences in Escherichia coli from the fecal flora and the urinary tract.

    PubMed

    Johanson, I M; Plos, K; Marklund, B I; Svanborg, C

    1993-08-01

    The pap gene clusters encode P fimbriae and fimbriae-associated G adhesins. DNA sequence analysis has resolved three G adhesin variants (papGJ96, papGIA2 and prsGJ96) that differ in receptor specificity and therefore in binding to epithelial cells. In this study, DNA probes specific for the pap gene cluster or the papGJ96, papGIA2 and prsGJ96 adhesin sequences were used to examine 74 fecal and 204 urinary Escherichia coli isolates (67 from acute pyelonephritis, 71 from acute cystitis and 66 from asymptomatic bacteriuria). In accordance with previous studies, a higher frequency of pap+ strains was found in the urinary strains (71%) than in the fecal (20%) E. coli isolates. The papGIA2, and prsGJ96 sequences were more frequent among urinary (42% papG+IA2, 23% prsG+J96) than among fecal (18% papG+IA2, 5% prsG+J96) isolates. None of the isolates hybridized with the papGJ96 probe. Pap+ strains accounted for 82% of the pyelonephritis, 69% of the cystitis and 61% of the asymptomatic bacteriuria strains. The papGIA2 genotype dominated in acute pyelonephritis strains (72% papG+IA2, 16% prsG+J96). The prsGJ96 genotype was most frequent in cystitis strains (25% papG+IA2, 37% prsG+J96). The asymptomatic bacteriuria strains formed an intermediate group (30% papG+IA2, 14% prsG+J96). Most of the papG+IA2 strains expressed P fimbriae which agglutinated human erythrocytes, sheep erythrocytes and Gal alpha 1-4Gal beta latex beads. The prsG+J96 strains varied in agglutination of human and sheep erythrocytes and Gal alpha 1-4Gal beta-latex beads. The results demonstrated that the papGIA2 and prsGJ96 adhesin DNA sequences differ in disease association.

  2. Molecular basis for TPR domain-mediated regulation of protein phosphatase 5.

    PubMed

    Yang, Jing; Roe, S Mark; Cliff, Matthew J; Williams, Mark A; Ladbury, John E; Cohen, Patricia T W; Barford, David

    2005-01-12

    Protein phosphatase 5 (Ppp5) is a serine/threonine protein phosphatase comprising a regulatory tetratricopeptide repeat (TPR) domain N-terminal to its phosphatase domain. Ppp5 functions in signalling pathways that control cellular responses to stress, glucocorticoids and DNA damage. Its phosphatase activity is suppressed by an autoinhibited conformation maintained by the TPR domain and a C-terminal subdomain. By interacting with the TPR domain, heat shock protein 90 (Hsp90) and fatty acids including arachidonic acid stimulate phosphatase activity. Here, we describe the structure of the autoinhibited state of Ppp5, revealing mechanisms of TPR-mediated phosphatase inhibition and Hsp90- and arachidonic acid-induced stimulation of phosphatase activity. The TPR domain engages with the catalytic channel of the phosphatase domain, restricting access to the catalytic site. This autoinhibited conformation of Ppp5 is stabilised by the C-terminal alphaJ helix that contacts a region of the Hsp90-binding groove on the TPR domain. Hsp90 activates Ppp5 by disrupting TPR-phosphatase domain interactions, permitting substrate access to the constitutively active phosphatase domain, whereas arachidonic acid prompts an alternate conformation of the TPR domain, destabilising the TPR-phosphatase domain interface.

  3. Regulation of synthase phosphatase and phosphorylase phosphatase in rat liver.

    PubMed

    Tan, A W; Nuttall, F Q

    1976-08-12

    Using substrates purified from liver, the apparent Km values of synthase phosphatase ([UDPglucose--glycogen glucosyltransferase-D]phosphohydrolase, EC 3.1.3.42) and phosphorylase phosphatase (phosphorylase a phosphohydrolase, EC 3.1.3.17) were found to be 0.7 and 60 units/ml respectively. The maximal velocity of phosphorylase phosphatase was more than a 100 times that of synthase phosphatase. In adrenalectomized, fasted animals there was a complete loss of synthase phosphatase but only a slight decrease in phosphorylase phosphatase when activity was measured using endogenous substrates in a concentrated liver extract. When assayed under optimal conditions with purified substrates, both activities were present but had decreased to very low levels. Mixing experiments indicated that synthase D present in the extract of adrenalectomized fasted animals was altered such that it was no longer a substrate for synthase phosphatase from normal rats. Phosphorylase a substrate on the other hand was unaltered and readily converted. When glucose was given in vivo, no change in percent of synthase in the I form was seen in adrenalectomized rats but the percent of phosphorylase in the a form was reduced. Precipitation of protein from an extract of normal fed rats with ethanol produced a large activation of phosphorylase phosphatase activity with no corresponding increase in synthase phosphatase activity. Despite the low phosphorylase phosphatase present in extracts of adrenalectomized fasted animals, ethanol precipitation increased activity to the same high level as obtained in the normal fed rats. Synthase phosphatase and phosphorylase phosphatase activities were also decreased in normal fasted, diabetic fed and fasted, and adrenalectomized fed rats. Both enzymes recovered in the same manner temporally after oral glucose administration to adrenalectomized, fasted rats. These results suggest an integrated regulatory mechanism for the two phosphatase.

  4. Continuous stress-induced dopamine dysregulation augments PAP-I and PAP-II expression in melanotrophs of the pituitary gland

    SciTech Connect

    Konishi, Hiroyuki; Ogawa, Tokiko; Kawahara, Shinichi; Matsumoto, Sakiko; Kiyama, Hiroshi

    2011-04-01

    Research highlights: {yields} We focused on the rat pituitary intermediate lobe (IL) under continuous stress (CS). {yields} CS induced PAP-I and PAP-II expression in melanotrophs of the IL. {yields} This gene induction was triggered by CS-related dopamine dysregulation. {yields} PAP-I and PAP-II may sustain homeostasis of the IL under CS. -- Abstract: Under continuous stress (CS) in rats, melanotrophs, the predominant cell-type in the intermediate lobe (IL) of the pituitary, are hyperactivated to secrete {alpha}-melanocyte-stimulating hormone and thereafter degenerate. Although these phenomena are drastic, the molecular mechanisms underlying the cellular changes are mostly unknown. In this study, we focused on the pancreatitis-associated protein (PAP) family members of the secretory lectins and characterized their expression in the IL of CS model rats because we had identified two members of this family as up-regulated genes in our previous microarray analysis. RT-PCR and histological studies demonstrated that prominent PAP-I and PAP-II expression was induced in melanotrophs in the early stages of CS, while another family member, PAP-III, was not expressed. We further examined the regulatory mechanisms of PAP-I and PAP-II expression and revealed that both were induced by the decreased dopamine levels in the IL under CS. Because the PAP family members are implicated in cell survival and proliferation, PAP-I and PAP-II secreted from melanotrophs may function to sustain homeostasis of the IL under CS conditions in an autocrine or a paracrine manner.

  5. [Is the PAP smear era coming to an end?].

    PubMed

    Nowak-Markwitz, Ewa; Spaczyński, Marek

    2015-12-01

    After the discovery of the role human papilloma virus (HPV) plays in the development of cervical cancer we are witnesses to a change in the conception and interpretation of cervical cancer prevention processes. Primary prevention gained a new tool in the form of HPV vaccines. Secondary prevention, i.e. detection of cervical intraepithelial neoplasia (GIN), acquired a new diagnostic method--the HPV test. Studies were initiated in order to determine the usefulness of HPV tests in cervical cancer prevention and screening. They revealed that the DNA HPV test used in screening has higher sensitivity in CIN detection than PAP smear and that HPV-negative patients are better and longer protected against developing cervical cancer in comparison to women with normal PAP smear results. HPV tests also possess a predictive value, which detects women more susceptible to developing cervical cancer in the future. PAP smear does not have a predictive value. Instead, it only detects a presence or an absence of neoplasia at that particular time. These results clearly indicate that the era of classic PAP smear is indeed coming to an end, replaced by a new primary CIN screening tool--HPV test. The entire cervical cancer screening system must therefore be redefined and reorganized. PMID:26995946

  6. Missed Opportunities for Health Education on Pap Smears in Peru

    ERIC Educational Resources Information Center

    Bayer, Angela M.; Nussbaum, Lauren; Cabrera, Lilia; Paz-Soldan, Valerie A.

    2011-01-01

    Despite cervical cancer being one of the leading causes of cancer-related deaths among women in Peru, cervical Pap smear coverage is low. This article uses findings from 185 direct clinician observations in four cities of Peru (representing the capital and each of the three main geographic regions of the country) to assess missed opportunities for…

  7. Factors Associated with Pap Testing among Hmong Women

    PubMed Central

    Fang, Dao Moua; Lee, Serge; Stewart, Susan; Ly, May Ying; Chen, Moon S.

    2014-01-01

    This paper reports the baseline data on the proportion of Hmong women (n=402), ages 18–65, in Sacramento, California who reported that they had a Pap test for the early detection of cervical cancer. We assessed the association between socio-demographic characteristics and Pap test receipt using chi-squared and multiple logistic regression analyses. Only 74% had ever had a Pap test, with 61% tested in the previous three years, compared with 91% and 86%, respectively, of California women overall. Women were more likely to have had a recent Pap test if they were age 21–30 (OR=3.0 vs. age 51–65, 95% CI 1.4–6.7) or 31–40 (OR=3.0, 95% CI 1.4–6.4), and less likely if they were single (OR=0.4 vs. married/partnered, 95% CI 0.2–1.0) or born in the U.S. (OR=0.3 vs. ,10 years in U.S., 95% CI 0.1–0.8). PMID:20693730

  8. [ATPase and phosphatase activity of drone brood].

    PubMed

    Bodnarchuk, L I; Stakhman, O S

    2004-01-01

    Most researches on insect enzymes concern carbohydrate and nitrogenous exchange. Data on ATPase activity for larval material of drone brood are absent in the available literature. The drone brood is one of the least investigated apiproducts. Allowing for the important role of ATPase in the vital functions of the insect cells our work was aimed at the study of ATPase of the drone blood activity and that of alkaline and acid phosphatases. When studying liophylised preparations of the drone brood homogenate we have found out high activity of Mg2+, Na+, K+-, Ca2+- and Mg2+-ATPase and of alkaline and acid phosphatase, that is the possible explanation of the high-intensity power and plastic processes proceeding during growth and development of larvae.

  9. [ATPase and phosphatase activity of drone brood].

    PubMed

    Bodnarchuk, L I; Stakhman, O S

    2004-01-01

    Most researches on insect enzymes concern carbohydrate and nitrogenous exchange. Data on ATPase activity for larval material of drone brood are absent in the available literature. The drone brood is one of the least investigated apiproducts. Allowing for the important role of ATPase in the vital functions of the insect cells our work was aimed at the study of ATPase of the drone blood activity and that of alkaline and acid phosphatases. When studying liophylised preparations of the drone brood homogenate we have found out high activity of Mg2+, Na+, K+-, Ca2+- and Mg2+-ATPase and of alkaline and acid phosphatase, that is the possible explanation of the high-intensity power and plastic processes proceeding during growth and development of larvae. PMID:16350755

  10. Manduca sexta proprophenoloxidase activating proteinase-3 (PAP3) stimulates melanization by activating proPAP3, proSPHs, and proPOs

    PubMed Central

    Wang, Yang; Lu, Zhiqiang; Jiang, Haobo

    2014-01-01

    Melanization participates in various insect physiological processes including antimicrobial immune responses. Phenoloxidase (PO), a critical component of the enzyme system catalyzing melanin formation, is produced as an inactive precursor prophenoloxidase (proPO) and becomes active via specific proteolytic cleavage by proPO activating proteinase (PAP). In Manduca sexta, three PAPs can activate proPOs in the presence of two serine proteinase homologs (SPH1 and SPH2). While the hemolymph proteinases (HPs) that generate the active PAPs are known, it is unclear how the proSPHs (especially proSPH1) are activated. In this study, we isolated from plasma of bar-stage M. sexta larvae an Ile-Glu-Ala-Arg-p-nitroanilide hydrolyzing enzyme that cleaved the proSPHs. This proteinase, PAP3, generated active SPH1 and SPH2, which function as cofactors for PAP3 in proPO activation. Cleavage of the purified recombinant proSPHs by PAP3 yielded 38 kDa bands similar in mobility to the SPHs formed in vivo. Surprisingly, PAP3 also can activate proPAP3 to stimulate melanization in a direct positive feedback loop. The enhanced proPO activation concurred with the cleavage activation of proHP6, proHP8, proPAP1, proPAP3, proSPH1, proSPH2, proPOs, but not proHP14 or proHP21. These results indicate that PAP3, like PAP1, is a key factor of the self-reinforcing mechanism in the proPO activation system, which is linked to other immune responses in M. sexta. PMID:24768974

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

    PubMed

    Ren, Zhongyuan; Do, Le Duy; Bechkoff, Géraldine; Mebarek, Saida; Keloglu, Nermin; Ahamada, Saandia; Meena, Saurabh; Magne, David; Pikula, Slawomir; Wu, Yuqing; Buchet, René

    2015-01-01

    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. PMID:25785438

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

    PubMed

    Ren, Zhongyuan; Do, Le Duy; Bechkoff, Géraldine; Mebarek, Saida; Keloglu, Nermin; Ahamada, Saandia; Meena, Saurabh; Magne, David; Pikula, Slawomir; Wu, Yuqing; Buchet, René

    2015-01-01

    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.

  13. Biomarkers for the activation of calcium metabolism in dairy cows: elevation of tartrate-resistant acid phosphatase activity by lowering dietary cation-anion difference is associated with the prevention of milk fever.

    PubMed

    Kurosaki, Naotoshi; Yamato, Osamu; Sato, Jun; Naito, Yoshihisa; Mori, Fuminobu; Imoto, Seiichi; Maede, Yoshimitsu

    2007-03-01

    In our previous study, it was demonstrated that the administration of anion salts, which slightly lower the dietary cation-anion difference (DCAD), in the prepartum period is safe and effective for preventing milk fever in multiparous cows. In the present study, several biomarkers, which might show activation of Ca metabolism, were analyzed using stored samples in the previous study to investigate the mechanism of the preventive effect on milk fever by lowering DCAD. Changes in bone-specific alkaline phosphatase activity, osteocalcin and insulin-like growth factor I concentrations in serum were almost the same among the three groups of multiparous cows with or without the oral administration of anion salts, while the levels of these serum biomarkers in the group of primiparous cows (heifer group) were much higher compared with those in the three multiparous groups throughout the experimental period. Urinary deoxypyridinoline excretion was not a useful biomarker for dairy cows because it hardly changed during the peripartum period in all groups. However, serum tartrate-resistant acid phosphatase (TRAP) activity, which is known as a biomarker of osteoclast activity, was well associated with the administration of anion salts lowering DCAD because among the three multiparous groups, only the group of multiparous cows fed the anion salts (anion group) showed an increased level, which rose to the level in the heifer group, and was markedly higher than those in the other control groups of multiparous cows. The increased activity of serum TRAP in the anion group suggested that Ca in the plasma pool was mobilized smoothly from bone-bound Ca via mature osteoclasts at parturition, which might be due to prior activation under mild acidosis induced by slightly lowering DCAD. Therefore, TRAP was the best biomarker to monitor the activation of Ca metabolism in dairy cows fed anion salts.

  14. Cytomorphology of unusual infectious entities in the Pap test

    PubMed Central

    Khalbuss, Walid E.; Michelow, Pam; Benedict, Cynthia; Monaco, Sara E.; Pantanowitz, Liron

    2012-01-01

    Rare entities in the Pap test, including neoplastic and non-neoplastic conditions, pose challenges due to their infrequent occurrence in the daily practice of cytology. Furthermore, these conditions give rise to important diagnostic pitfalls. Infections such as tuberculosis cervicitis may be erroneously diagnosed as carcinoma, whereas others, such as schistosomiasis, are associated with squamous cell carcinoma. These cases include granuloma inguinale (donovanosis), tuberculosis, coccidioidomycosis, schistosomiasis, taeniasis, and molluscum contagiosum diagnosed in Pap tests. Granuloma inguinale shows histiocytes that contain intracytoplasmic bacteria (Donovan bodies). Tuberculosis is characterized by necrotizing granulomatous inflammation with Langhans-multinucleated giant cells. Coccidioidomycosis may show large intact or ruptured fungal spherules associated with endospores. Schistosoma haematobium is diagnosed by finding characteristic ova with a terminal spine. Molluscum contagiosum is characterized by the appearance of squamous cells with molluscum bodies. This article reviews the cytomorphology of selected rare infections and focuses on their cytomorphology, differential diagnosis, and role of ancillary diagnostic studies. PMID:22919422

  15. Missed Opportunities for Health Education on Pap Smears in Peru

    PubMed Central

    Bayer, Angela M.; Nussbaum, Lauren; Cabrera, Lilia; Paz-Soldan, Valerie A.

    2013-01-01

    Despite cervical cancer being one of the leading causes of cancer-related deaths among women in Peru, cervical Pap smear coverage is low. This article uses findings from 185 direct clinician observations in four cities of Peru (representing the capital and each of the three main geographic regions of the country) to assess missed opportunities for health education on Pap smears and other preventive women’s health behaviors during women’s visits to a health care provider. Various types of health establishments, provider settings, and provider types were observed. Opportunities for patient education on the importance of prevention were rarely exploited. In fact, health education provided was minimal. Policy and programmatic implications are discussed. PMID:21464205

  16. Immunocytochemical localization of a tartrate-resistant and vanadate-sensitive acid nucleotide tri- and diphosphatase.

    PubMed

    Andersson, G N; Ek-Rylander, B; Hammarström, L E; Lindskog, S; Toverud, S U

    1986-03-01

    Purified rabbit antiserum to a tartrate-resistant and vanadate-sensitive acid phosphatase (nucleotide tri- and diphosphatase) prepared from rat bone was used in immunocytochemical studies. The antigen was localized in sections of fixed, decalcified tissue (head from rat) using the peroxidase-antiperoxidase bridge (PAP) or the avidin-biotin-peroxidase complex (ABC) technique. Both techniques resulted in similar and specific immunostaining in the following cells and tissues: osteoclasts situated in resorption lacunae, epithelium overlying enamel-free areas of tips of cusps of unerupted molars, cilia of respiratory epithelium, and tissue macrophages. This distribution corresponds to the cellular sites of tartrate-resistant acid phosphatase activity, as revealed by enzyme histochemistry. With the ABC method, staining in osteoclasts was obtained with antiserum dilutions of up to 1:10,000. Biochemical studies revealed that vanadate-sensitive acid ATPase activity in liver subcellular fractions was almost exclusively confined to lysosomes. Thus, the immunostaining has revealed the presence of the tartrate-resistant and vanadate-sensitive nucleotide phosphatase in many cells associated with tissue resorption and phagocytosis.

  17. Enhancing Potato System Sustainability: Crop Rotation Impacts on Soil Phosphatase Activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Potato is a species with a low efficiency of acquiring soil P. Rotation crops may potentially influence P uptake by potato by increasing soil organic acids, phosphatase activity, and microbial biomass. However, this kind of information is very limited. We measured the activities of acid phosphatase,...

  18. A HPLC-fluorescence detection method for determination of phosphatidic acid phosphohydrolase activity: application in human myocardium.

    PubMed

    Burgdorf, Christof; Prey, Antje; Richardt, Gert; Kurz, Thomas

    2008-03-15

    Phosphatidic acid phosphohydrolase (PAP) catalyzes the dephosphorylation of phosphatidic acid (PA) to diacylglycerol, the second messenger responsible for activation of protein kinase C. Despite the crucial role of PAP lipid signaling, there are no data on PAP signaling function in the human heart. Here we present a nonradioactive assay for the investigation of PAP activity in human myocardium using a fluorescent derivative of PA, 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphate (BODIPY-PA), as substrate in an in vitro PAP-catalyzed reaction. Unreacted BODIPY-PA was resolved from the PAP products by a binary gradient HPLC system and BODIPY-diacylglycerol was detected by fluorimetry. The reaction proceeded at a linear rate for up to 60 min and increased linearly with increasing amounts of cardiac protein in a range of 0.25 to 8.0 microg. This assay proved to be sensitive for accurate quantitation of total PAP activity, PAP-1 activity, and PAP-2 activity in human atrial tissue and right ventricular endomyocardial biopsies. Total PAP activity was approximately fourfold higher in ventricular myocardium than in atrial tissue. There was negligible PAP-1 activity in atrial myocardium compared with ventricular myocardium, indicating regional differences in activities and distribution pattern of PAP-1 and PAP-2 in the human heart. PMID:18023403

  19. A HPLC-fluorescence detection method for determination of phosphatidic acid phosphohydrolase activity: application in human myocardium.

    PubMed

    Burgdorf, Christof; Prey, Antje; Richardt, Gert; Kurz, Thomas

    2008-03-15

    Phosphatidic acid phosphohydrolase (PAP) catalyzes the dephosphorylation of phosphatidic acid (PA) to diacylglycerol, the second messenger responsible for activation of protein kinase C. Despite the crucial role of PAP lipid signaling, there are no data on PAP signaling function in the human heart. Here we present a nonradioactive assay for the investigation of PAP activity in human myocardium using a fluorescent derivative of PA, 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphate (BODIPY-PA), as substrate in an in vitro PAP-catalyzed reaction. Unreacted BODIPY-PA was resolved from the PAP products by a binary gradient HPLC system and BODIPY-diacylglycerol was detected by fluorimetry. The reaction proceeded at a linear rate for up to 60 min and increased linearly with increasing amounts of cardiac protein in a range of 0.25 to 8.0 microg. This assay proved to be sensitive for accurate quantitation of total PAP activity, PAP-1 activity, and PAP-2 activity in human atrial tissue and right ventricular endomyocardial biopsies. Total PAP activity was approximately fourfold higher in ventricular myocardium than in atrial tissue. There was negligible PAP-1 activity in atrial myocardium compared with ventricular myocardium, indicating regional differences in activities and distribution pattern of PAP-1 and PAP-2 in the human heart.

  20. Electrochemical studies of the mono-Fe, Fe-Zn, and Fe-Fe metalloisoforms of bacteriophage lambda protein phosphatase.

    PubMed

    Reiter, Tiffany A; Rusnak, Frank

    2004-01-27

    Bacteriophage lambda protein phosphatase (lambdaPP) is a member of a large superfamily of metallophosphoesterases, including serine/threonine protein phosphatases, purple acid phosphatases, 5'-nucleotidase, and DNA repair enzymes such as Mre11. Members of this family share several common characteristics, including a common phosphoesterase motif, secondary structural fold (betaalphabetaalphabeta), and metal ligand environment, and often accommodate a dinuclear metal center. The identity of the active site metals often differs between family members. Despite the extensive spectroscopic studies of several family members, only the standard redox potential of porcine purple acid phosphate (PAP) has been measured. In this report, we investigate the redox properties of another member of this protein family. The standard redox potentials of the mono-Fe, Fe-Zn, and Fe-Fe metalloisoforms of lambdaPP were determined from anaerobic redox titration experiments. Two different S = 5/2, mono-Fe3+ lambdaPP species were identified: the first with an E/D approximately 0.17, g = 8.9 and 4.8, and an Eo' approximately +130 mV; the second with E/D approximately 0.05, g = 6.7, 5.9, and 4.4, and an Eo' approximately +120 mV. The first and second mono-Fe3+ species are thought to represent Fe present in the M2 and M1 sites, respectively. The addition of Zn2+ to mono-Fe3+ lambdaPP results in a decrease in both mono-Fe3+ species and the appearance of a new S = 5/2, Fe(3+)-Zn2+ species (E/D approximately 0.02, g = 5.9, and an Eo' > +175 mV). The Fe-Fe lambdaPP titration revealed an S = 1/2, Fe(3+)-Fe2+ (g < 2) species with an Eo' > +128 mV. These results suggest that the active site of lambdaPP supports a high oxidation potential for both metal sites and may indicate an equally oxidizing active site for other member metallophosphoesterases.

  1. PapA1 and PapA2 are acyltransferases essential for the biosynthesis of the Mycobacterium tuberculosis virulence factor Sulfolipid-1

    PubMed Central

    Kumar, Pawan; Schelle, Michael W.; Jain, Madhulika; Lin, Fiona L.; Petzold, Christopher J.; Leavell, Michael D.; Leary, Julie A.; Cox, Jeffery S.; Bertozzi, Carolyn R.

    2007-01-01

    Mycobacterium tuberculosis produces numerous exotic lipids that have been implicated as virulence determinants. One such glycolipid, Sulfolipid-1 (SL-1), consists of a trehalose-2-sulfate (T2S) core acylated with four lipid moieties. A diacylated intermediate in SL-1 biosynthesis, SL1278, has been shown to activate the adaptive immune response in human patients. Although several proteins involved in SL-1 biosynthesis have been identified, the enzymes that acylate the T2S core to form SL1278 and SL-1, and the biosynthetic order of these acylation reactions, are unknown. Here we demonstrate that PapA2 and PapA1 are responsible for the sequential acylation of T2S to form SL1278 and are essential for SL-1 biosynthesis. In vitro, recombinant PapA2 converts T2S to 2′-palmitoyl T2S, and PapA1 further elaborates this newly identified SL-1 intermediate to an analog of SL1278. Disruption of papA2 and papA1 in M. tuberculosis confirmed their essential role in SL-1 biosynthesis and their order of action. Finally, the ΔpapA2 and ΔpapA1 mutants were screened for virulence defects in a mouse model of infection. The loss of SL-1 (and SL1278) did not appear to affect bacterial replication or trafficking, suggesting that the functions of SL-1 are specific to human infection. PMID:17592143

  2. Inhibition of pokeweed antiviral protein (PAP) by turnip mosaic virus genome-linked protein (VPg).

    PubMed

    Domashevskiy, Artem V; Miyoshi, Hiroshi; Goss, Dixie J

    2012-08-24

    Pokeweed antiviral protein (PAP) from Phytolacca americana is a ribosome-inactivating protein (RIP) and an RNA N-glycosidase that removes specific purine residues from the sarcin/ricin loop of large rRNA, arresting protein synthesis at the translocation step. PAP is also a cap-binding protein and is a potent antiviral agent against many plant, animal, and human viruses. To elucidate the mechanism of RNA depurination, and to understand how PAP recognizes and targets various RNAs, the interactions between PAP and turnip mosaic virus genome-linked protein (VPg) were investigated. VPg can function as a cap analog in cap-independent translation and potentially target PAP to uncapped IRES-containing RNA. In this work, fluorescence spectroscopy and HPLC techniques were used to quantitatively describe PAP depurination activity and PAP-VPg interactions. PAP binds to VPg with high affinity (29.5 nm); the reaction is enthalpically driven and entropically favored. Further, VPg is a potent inhibitor of PAP depurination of RNA in wheat germ lysate and competes with structured RNA derived from tobacco etch virus for PAP binding. VPg may confer an evolutionary advantage by suppressing one of the plant defense mechanisms and also suggests the possible use of this protein against the cytotoxic activity of ribosome-inactivating proteins.

  3. Phosphorylation regulates the Star-PAP-PIPKIα interaction and directs specificity toward mRNA targets.

    PubMed

    Mohan, Nimmy; Sudheesh, A P; Francis, Nimmy; Anderson, Richard; Laishram, Rakesh S

    2015-08-18

    Star-PAP is a nuclear non-canonical poly(A) polymerase (PAP) that shows specificity toward mRNA targets. Star-PAP activity is stimulated by lipid messenger phosphatidyl inositol 4,5 bisphoshate (PI4,5P2) and is regulated by the associated Type I phosphatidylinositol-4-phosphate 5-kinase that synthesizes PI4,5P2 as well as protein kinases. These associated kinases act as coactivators of Star-PAP that regulates its activity and specificity toward mRNAs, yet the mechanism of control of these interactions are not defined. We identified a phosphorylated residue (serine 6, S6) on Star-PAP in the zinc finger region, the domain required for PIPKIα interaction. We show that S6 is phosphorylated by CKIα within the nucleus which is required for Star-PAP nuclear retention and interaction with PIPKIα. Unlike the CKIα mediated phosphorylation at the catalytic domain, Star-PAP S6 phosphorylation is insensitive to oxidative stress suggesting a signal mediated regulation of CKIα activity. S6 phosphorylation together with coactivator PIPKIα controlled select subset of Star-PAP target messages by regulating Star-PAP-mRNA association. Our results establish a novel role for phosphorylation in determining Star-PAP target mRNA specificity and regulation of 3'-end processing.

  4. Phosphorylation regulates the Star-PAP-PIPKIα interaction and directs specificity toward mRNA targets

    PubMed Central

    Mohan, Nimmy; AP, Sudheesh; Francis, Nimmy; Anderson, Richard; Laishram, Rakesh S.

    2015-01-01

    Star-PAP is a nuclear non-canonical poly(A) polymerase (PAP) that shows specificity toward mRNA targets. Star-PAP activity is stimulated by lipid messenger phosphatidyl inositol 4,5 bisphoshate (PI4,5P2) and is regulated by the associated Type I phosphatidylinositol-4-phosphate 5-kinase that synthesizes PI4,5P2 as well as protein kinases. These associated kinases act as coactivators of Star-PAP that regulates its activity and specificity toward mRNAs, yet the mechanism of control of these interactions are not defined. We identified a phosphorylated residue (serine 6, S6) on Star-PAP in the zinc finger region, the domain required for PIPKIα interaction. We show that S6 is phosphorylated by CKIα within the nucleus which is required for Star-PAP nuclear retention and interaction with PIPKIα. Unlike the CKIα mediated phosphorylation at the catalytic domain, Star-PAP S6 phosphorylation is insensitive to oxidative stress suggesting a signal mediated regulation of CKIα activity. S6 phosphorylation together with coactivator PIPKIα controlled select subset of Star-PAP target messages by regulating Star-PAP-mRNA association. Our results establish a novel role for phosphorylation in determining Star-PAP target mRNA specificity and regulation of 3′-end processing. PMID:26138484

  5. Adolescents' intention and self-efficacy to follow Pap testing recommendations after receiving the HPV vaccine.

    PubMed

    Higgins, Lisa M; Dirksing, Kelsie N; Ding, Lili; Morrow, Charlene D; Widdice, Lea A; Kahn, Jessica A

    2016-06-01

    Human papillomavirus (HPV) vaccines are recommended in the US for girls and women 11-26 y of age. Because these vaccines do not prevent all cervical cancers, Papanicolaou (Pap) screening is still recommended after vaccination. Young women who have been vaccinated may perceive themselves at lower risk for HPV infection and cervical cancer, which could lead to lower intention and self-efficacy to follow cervical cancer screening guidelines, and subsequent nonadherence to Pap testing. The aim of this study was to examine whether perceived risk of human papillomavirus (HPV) after vaccination and other factors are associated with adolescents' intention and self-efficacy to get Pap testing after HPV vaccination. Women 13-21 y of age (N = 339) receiving their first HPV vaccine dose completed a survey. Multivariable logistic regression examined associations between perceived risk of HPV and intention/self-efficacy to get a Pap test while adjusting for other factors. Approximately half of participants reported high intention and half reported high self-efficacy to get a Pap test. Factors significantly associated with high intention were Pap testing history and knowledge about HPV/HPV vaccines; factors significantly associated with high self-efficacy included insurance plan, Pap testing history, communication with clinician about needing a Pap test after vaccination, lifetime number of male sexual partners, and recent smoking. In conclusion, educating adolescents about HPV/HPV vaccines and the need for Pap testing may increase self-efficacy/intention to get a Pap test after vaccination. PMID:26934107

  6. Differences in phosphatidic acid signalling and metabolism between ABA and GA treatments of barley aleurone cells.

    PubMed

    Villasuso, Ana Laura; Di Palma, Maria A; Aveldaño, Marta; Pasquaré, Susana J; Racagni, Graciela; Giusto, Norma M; Machado, Estela E

    2013-04-01

    Phosphatidic acid (PA) is the common lipid product in abscisic acid (ABA) and gibberellic acid (GA) response. In this work we investigated the lipid metabolism in response to both hormones. We could detect an in vivo phospholipase D activity (PLD, EC 3.1.4.4). This PLD produced [(32)P]PA (phosphatidic acid) rapidly (minutes) in the presence of ABA, confirming PA involvement in signal transduction, and transiently, indicating rapid PA removal after generation. The presence of PA removal by phosphatidate phosphatase 1 and 2 isoforms (E.C. 3.1.3.4) was verified in isolated aleurone membranes in vitro, the former but not the latter being specifically responsive to the presence of GA or ABA. The in vitro DGPP phosphatase activity was not modified by short time incubation with GA or ABA while the in vitro PA kinase - that allows the production of 18:2-DGPP from 18:2-PA - is stimulated by ABA. The long term effects (24 h) of ABA or GA on lipid and fatty acid composition of aleurone layer cells were then investigated. An increase in PC and, to a lesser extent, in PE levels is the consequence of both hormone treatments. ABA, in aleurone layer cells, specifically activates a PLD whose product, PA, could be the substrate of PAP1 and/or PAK activities. Neither PLD nor PAK activation can be monitored by GA treatment. The increase in PAP1 activity monitored after ABA or GA treatment might participate in the increase in PC level observed after 24 h hormone incubation.

  7. Phosphatase production and activity in Citrobacter freundii and a naturally occurring, heavy-metal-accumulating Citrobacter sp.

    PubMed

    Montgomery, D M; Dean, A C; Wiffen, P; Macaskie, L E

    1995-10-01

    The ability of a naturally occurring Citrobacter sp. to accumulate cadmium has been attributed to cellular precipitation of CdHPO4, utilizing HPO4(2-) liberated via the activity of an overproduced, Cd-resistant acid-type phosphatase. Phosphatase production and heavy metal accumulation by batch cultures of this strain (N14) and a phosphatase-deficient mutant were compared with two reference strains of Citrobacter freundii. Only strain N14 expressed a high level of acid phosphatase and accumulated lanthanum and uranyl ion enzymically. Acid phosphatase is regulated via carbon-starvation; although the C. freundii strains overexpressed phosphatase activity in carbon-limiting continuous culture, this was approximately 20-fold less than the activity of strain N14 grown similarly. Citrobacter strain N14 was originally isolated from a metal-contaminated soil environment; phosphatase overproduction and metal accumulation were postulated as a detoxification mechanism. However, application of Cd-stress, and enrichment for Cd-resistant C. freundii ('training'), reduced the phosphatase activity of this organism by about 50% as compared to Cd-unstressed cultures. The acid phosphatase of C. freundii and Citrobacter N14 had a similar pattern of resistance to some diagnostic reagents. The enzyme of the latter is similar to the PhoN acid phosphatase of Salmonella typhimurium described by other workers; the results are discussed with respect to the known phosphatases of the enterobacteria.

  8. The Extended Family of Protein Tyrosine Phosphatases.

    PubMed

    Alonso, Andrés; Nunes-Xavier, Caroline E; Bayón, Yolanda; Pulido, Rafael

    2016-01-01

    In higher eukaryotes, the Tyr phosphorylation status of cellular proteins results from the coordinated action of Protein Tyrosine Kinases (PTKs) and Protein Tyrosine Phosphatases (PTPs). PTPs have emerged as highly regulated enzymes with diverse substrate specificity, and proteins with Tyr-dephosphorylation or Tyr-dephosphorylation-like properties can be clustered as the PTPome. This includes proteins from the PTP superfamily, which display a Cys-based catalytic mechanism, as well as enzymes from other gene families (Asp-based phosphatases, His-based phosphatases) that have converged in protein Tyr-dephosphorylation-related functions by using non-Cys-based catalytic mechanisms. Within the Cys-based members of the PTPome, classical PTPs dephosphorylate specific phosphoTyr (pTyr) residues from protein substrates, whereas VH1-like dual-specificity PTPs dephosphorylate pTyr, pSer, and pThr residues, as well as nonproteinaceous substrates, including phosphoinositides and phosphorylated carbohydrates. In addition, several PTPs have impaired catalytic activity as a result of amino acid substitutions at their active sites, but retain regulatory functions related with pTyr signaling. As a result of their relevant biological activity, many PTPs are linked to human disease, including cancer, neurodevelopmental, and metabolic diseases, making these proteins important drug targets and molecular markers in the clinic. Here, a brief overview on the biochemistry and physiology of the different groups of proteins that belong to the mammalian PTPome is presented. PMID:27514797

  9. Two potential fish glycerol-3-phosphate phosphatases.

    PubMed

    Raymond, James A

    2015-06-01

    Winter-acclimated rainbow smelt (Osmerus mordax Mitchill) produce high levels of glycerol as an antifreeze. A common pathway to glycerol involves the enzyme glycerol-3-phosphate phosphatase (GPP), but no GPP has yet been identified in fish or any other animal. Here, two phosphatases assembled from existing EST libraries (from winter-acclimated smelt and cold-acclimated smelt hepatocytes) were found to resemble a glycerol-associated phosphatase from a glycerol-producing alga, Dunaliella salina, and a recently discovered GPP from a bacterium, Mycobacterium tuberculosis. Recombinant proteins were generated and were found to have GPP activity on the order of a few μMol Pi/mg enzyme/min. The two enzymes have acidic pH optima (~5.5) similar to that previously determined for GPP activity in liver tissue, with about 1/3 of their peak activities at neutral pH. The two enzymes appear to account for the GPP activity of smelt liver, but due to their reduced activities at neutral pH, their contributions to glycerol production in vivo remain unclear. Similar enzymes may be active in a glycerol-producing insect, Dendroctonus ponderosae.

  10. A chronoamperometric screen printed carbon biosensor based on alkaline phosphatase inhibition for W(IV) determination in water, using 2-phospho-L-ascorbic acid trisodium salt as a substrate.

    PubMed

    Alvarado-Gámez, Ana Lorena; Alonso-Lomillo, María Asunción; Domínguez-Renedo, Olga; Arcos-Martínez, María Julia

    2015-01-22

    This paper presents a chronoamperometric method to determine tungsten in water using screen-printed carbon electrodes modified with gold nanoparticles and cross linked alkaline phosphatase immobilized in the working electrode. Enzymatic activity over 2-phospho-l-ascorbic acid trisodium salt, used as substrate, was affected by tungsten ions, which resulted in a decrease of chronoamperometric current, when a potential of 200 mV was applied on 10 mM of substrate in a Tris HCl buffer pH 8.00 and 0.36 M of KCl. Calibration curves for the electrochemical method validation, give a reproducibility of 5.2% (n = 3), a repeatability of 9.4% (n = 3) and a detection limit of 0.29 ± 0.01 µM. Enriched tap water, purified laboratory water and bottled drinking water, with a certified tungsten reference solution traceable to NIST, gave a recovery of 97.1%, 99.1% and 99.1% respectively (n = 4 in each case) and a dynamic range from 0.6 to 30 µM. This study was performed by means of a Lineweaver-Burk plot, showing a mixed kinetic inhibition.

  11. A Chronoamperometric Screen Printed Carbon Biosensor Based on Alkaline Phosphatase Inhibition for W(VI) Determination in Water, Using 2-Phospho-l-Ascorbic Acid Trisodium Salt as a Substrate

    PubMed Central

    Alvarado-Gámez, Ana Lorena; Alonso-Lomillo, María Asunción; Domínguez-Renedo, Olga; Arcos-Martínez, María Julia

    2015-01-01

    This paper presents a chronoamperometric method to determine tungsten in water using screen-printed carbon electrodes modified with gold nanoparticles and cross linked alkaline phosphatase immobilized in the working electrode. Enzymatic activity over 2-phospho-l-ascorbic acid trisodium salt, used as substrate, was affected by tungsten ions, which resulted in a decrease of chronoamperometric current, when a potential of 200 mV was applied on 10 mM of substrate in a Tris HCl buffer pH 8.00 and 0.36 M of KCl. Calibration curves for the electrochemical method validation, give a reproducibility of 5.2% (n = 3), a repeatability of 9.4% (n = 3) and a detection limit of 0.29 ± 0.01 μM. Enriched tap water, purified laboratory water and bottled drinking water, with a certified tungsten reference solution traceable to NIST, gave a recovery of 97.1%, 99.1% and 99.1% respectively (n = 4 in each case) and a dynamic range from 0.6 to 30 μM. This study was performed by means of a Lineweaver–Burk plot, showing a mixed kinetic inhibition. PMID:25621602

  12. A rapid and efficient method for the screening of acid phosphatase 1 in young tomato seedlings, and for the identification of root-knot nematode species using miniaturized polyacrylamide gel electrophoresis.

    PubMed

    Cap, G B; Roberts, P A

    1992-05-01

    A relatively rapid and highly sensitive miniaturized polyacrylamide gel electrophoresis technique is described for the analysis of certain isozymes from single cotyledons of tomato seedlings and from single females of the root-know nematode (Meloidogyne spp.). Homogenates from single tomato cotyledons (7, 14, 21, and 28 days old) were electrophoresed and stained for acid phosphatase 1 (Aps 1) activity. Cotyledons from plants of all the above age groups showed good Aps 1 activity. Nondestructive screening for tomato Aps 1 is therefore feasible, using very small samples, from as young as 7-day-old tomato seedlings. This could be of important use in expediting root-knot nematode resistance (based on the Aps 1-linked resistance gene Mi) screening for breeding programs, or F1 testing for seed production purposes. In addition, the mini-polyacrylamide gel electrophoresis technique was useful for determination of the Aps 1 allelic contribution to the total enzyme activity. The system was also used to detect malate dehydrogenase and esterase isozyme activity from single adult females of the four common root-knot nematodes, Meloidogyne arenaria, M. hapla, M. incognita, and M. javanica, with equally good results, enabling species discrimination.

  13. Fatty acid composition of muscle fat and enzymes of storage lipid synthesis in whole muscle from beef cattle.

    PubMed

    Kazala, E Chris; Lozeman, Fred J; Mir, Priya S; Aalhus, Jennifer L; Schmutz, Sheila M; Weselake, Randall J

    2006-11-01

    Enhanced intramuscular fat content (i.e., marbling) in beef is a desirable trait, which can result in increased product value. This study was undertaken with the aim of revealing biochemical factors associated with the marbling trait in beef cattle. Samples of longissimus lumborum (LL) and pars costalis diaphragmatis (PCD) were taken from a group of intact crossbred males and females at slaughter, lipids extracted, and the resulting FAME examined for relationships with marbling fat deposition. For LL, significant associations were found between degree of marbling and myristic (14:0, r = 0.55, P < 0.01), palmitic (16:0, r = 0.80, P < 0.001), stearic (18:0, r = -0.58, P < 0.01), and oleic (18:1c-9, r = 0.79, P < 0.001) acids. For PCD, significant relationships were found between marbling and palmitic (r = 0.71, P < 0.001) and oleic (r = 0.74, P < 0.001) acids. Microsomal fractions prepared from PCD muscle were assayed for diacylglycerol acyltransferase (DGAT), lysophosphatidic acid acyltransferase (LPAAT), and phosphatidic acid phosphatase-1 (PAP-1) activity, and the results examined for relationships with degree of intramuscular fat deposition. None of the enzyme activities from PCD displayed an association with marbling fat content, but DGAT specific activity showed significant positive associations with LPAAT (r = 0.54, P < 0.01), total PAP (r = 0.66, P < 0.001), and PAP-1 (r = 0.63, P < 0.01) specific activities. The results on FA compositions of whole muscle tissues provide insight into possible enzyme action associated with the production of specific FA. The increased proportion of oleic acid associated with enhanced lipid content of whole muscle is noteworthy given the known health benefits of this FA. PMID:17263304

  14. Fatty acid composition of muscle fat and enzymes of storage lipid synthesis in whole muscle from beef cattle.

    PubMed

    Kazala, E Chris; Lozeman, Fred J; Mir, Priya S; Aalhus, Jennifer L; Schmutz, Sheila M; Weselake, Randall J

    2006-11-01

    Enhanced intramuscular fat content (i.e., marbling) in beef is a desirable trait, which can result in increased product value. This study was undertaken with the aim of revealing biochemical factors associated with the marbling trait in beef cattle. Samples of longissimus lumborum (LL) and pars costalis diaphragmatis (PCD) were taken from a group of intact crossbred males and females at slaughter, lipids extracted, and the resulting FAME examined for relationships with marbling fat deposition. For LL, significant associations were found between degree of marbling and myristic (14:0, r = 0.55, P < 0.01), palmitic (16:0, r = 0.80, P < 0.001), stearic (18:0, r = -0.58, P < 0.01), and oleic (18:1c-9, r = 0.79, P < 0.001) acids. For PCD, significant relationships were found between marbling and palmitic (r = 0.71, P < 0.001) and oleic (r = 0.74, P < 0.001) acids. Microsomal fractions prepared from PCD muscle were assayed for diacylglycerol acyltransferase (DGAT), lysophosphatidic acid acyltransferase (LPAAT), and phosphatidic acid phosphatase-1 (PAP-1) activity, and the results examined for relationships with degree of intramuscular fat deposition. None of the enzyme activities from PCD displayed an association with marbling fat content, but DGAT specific activity showed significant positive associations with LPAAT (r = 0.54, P < 0.01), total PAP (r = 0.66, P < 0.001), and PAP-1 (r = 0.63, P < 0.01) specific activities. The results on FA compositions of whole muscle tissues provide insight into possible enzyme action associated with the production of specific FA. The increased proportion of oleic acid associated with enhanced lipid content of whole muscle is noteworthy given the known health benefits of this FA.

  15. Structural Genomics of Protein Phosphatases

    SciTech Connect

    Almo,S.; Bonanno, J.; Sauder, J.; Emtage, S.; Dilorenzo, T.; Malashkevich, V.; Wasserman, S.; Swaminathan, S.; Eswaramoorthy, S.; et al

    2007-01-01

    The New York SGX Research Center for Structural Genomics (NYSGXRC) of the NIGMS Protein Structure Initiative (PSI) has applied its high-throughput X-ray crystallographic structure determination platform to systematic studies of all human protein phosphatases and protein phosphatases from biomedically-relevant pathogens. To date, the NYSGXRC has determined structures of 21 distinct protein phosphatases: 14 from human, 2 from mouse, 2 from the pathogen Toxoplasma gondii, 1 from Trypanosoma brucei, the parasite responsible for African sleeping sickness, and 2 from the principal mosquito vector of malaria in Africa, Anopheles gambiae. These structures provide insights into both normal and pathophysiologic processes, including transcriptional regulation, regulation of major signaling pathways, neural development, and type 1 diabetes. In conjunction with the contributions of other international structural genomics consortia, these efforts promise to provide an unprecedented database and materials repository for structure-guided experimental and computational discovery of inhibitors for all classes of protein phosphatases.

  16. Abscisic acid affects transcription of chloroplast genes via protein phosphatase 2C-dependent activation of nuclear genes: repression by guanosine-3'-5'-bisdiphosphate and activation by sigma factor 5.

    PubMed

    Yamburenko, Maria V; Zubo, Yan O; Börner, Thomas

    2015-06-01

    Abscisic acid (ABA) represses the transcriptional activity of chloroplast genes (determined by run-on assays), with the exception of psbD and a few other genes in wild-type Arabidopsis seedlings and mature rosette leaves. Abscisic acid does not influence chloroplast transcription in the mutant lines abi1-1 and abi2-1 with constitutive protein phosphatase 2C (PP2C) activity, suggesting that ABA affects chloroplast gene activity by binding to the pyrabactin resistance (PYR)/PYR1-like or regulatory component of ABA receptor protein family (PYR/PYL/RCAR) and signaling via PP2Cs and sucrose non-fermenting protein-related kinases 2 (SnRK2s). Further we show by quantitative PCR that ABA enhances the transcript levels of RSH2, RSH3, PTF1 and SIG5. RelA/SpoT homolog 2 (RSH2) and RSH3 are known to synthesize guanosine-3'-5'-bisdiphosphate (ppGpp), an inhibitor of the plastid-gene-encoded chloroplast RNA polymerase. We propose, therefore, that ABA leads to an inhibition of chloroplast gene expression via stimulation of ppGpp synthesis. On the other hand, sigma factor 5 (SIG5) and plastid transcription factor 1 (PTF1) are known to be necessary for the transcription of psbD from a specific light- and stress-induced promoter (the blue light responsive promoter, BLRP). We demonstrate that ABA activates the psbD gene by stimulation of transcription initiation at BLRP. Taken together, our data suggest that ABA affects the transcription of chloroplast genes by a PP2C-dependent activation of nuclear genes encoding proteins involved in chloroplast transcription. PMID:25976841

  17. Sensitive and selective determining ascorbic acid and activity of alkaline phosphatase based on electrochemiluminescence of dual-stabilizers-capped CdSe quantum dots in carbon nanotube-nafion composite.

    PubMed

    Ma, Xiaolong; Zhang, Xin; Guo, Xinli; Kang, Qi; Shen, Dazhong; Zou, Guizheng

    2016-07-01

    Sensitive and selective determining bio-related molecule and enzyme play an important role in designing novel procedure for biological sensing and clinical diagnosis. Herein, we found that dual-stabilizers-capped CdSe quantum dots (QDs) in composite film of multi-walled carbon nanotubes (CNTs) and Nafion, displaying eye-visible monochromatic electrochemiluminescence (ECL) with fwhm of 37nm, which offers promising ECL signal for detecting ascorbic acid (AA) as well as the activity of alkaline phosphatase (ALP) in biological samples. It was also shown that the dual-stabilizers-capped CdSe QDs can preserve their highly passivated surface states with prolonged lifetime of excited states in Nafion mixtures, and facilitate electron-transfer ability of Nafion film along with CNTs. Compared with the QDs/GCE, the ECL intensity is enhanced 1.8 times and triggering potential shifted to lower energy by 0.12V on the CdSe-CNTs-Nafion/GCE. The ECL quenching degree increases with increasing concentration of AA in the range of 0.01-30nM with a limit of detection (LOD) of 5pM. The activity of ALP was determined indirectly according to the concentration of AA, generated in the hydrolysis reaction of l-ascorbic acid 2-phosphate sesquimagnesium (AA-P) in the presence of ALP as a catalyst, with an LOD of 1μU/L. The proposed strategy is favorable for developing simple ECL sensor or device with high sensitivity, spectral resolution and less electrochemical interference. PMID:27154663

  18. Distinction in vitro between rat liver phosphatidate phosphatase and phospholipase C

    SciTech Connect

    Lamb, R.; Foster, K.; McGuffin, M.

    1986-03-05

    Hepatocellular membranes (1000 x g) were incubated with sn-(1,3-/sup 14/C) glycerol-3-P, ATP, Ca/sup 2 +/, NaF and palmitate to form labeled, membrane-associated phosphatidate(PA). Membranes incubated with 2mM oleate or 5mM bromobenzene showed rapid (5-10 min) and significant (2-6 fold) increases in the dephosphorylation of PA. However, oleate and bromobenzene activated the dephosphorylation of PA by phosphatidate phosphatase (PAP) and phospholipase C (PLC), respectively. This conclusion is supported by the observation that the PAP stimulated by oleate is: 1) Mg/sup 2 +/-dependent; 2) inhibited by Ca/sup 2 +/ and NaF; 3) specific for PA; 4) associated with a rise in liver cell triacylglycerol (TG) formation. Bromobenzene, however, activated a PLC that is: 1) stimulated by various metals; 2) enhanced by NaF; 3) is associated with a rise in the degradation of membrane phospholipids and liver cell injury. These results suggest that under the appropriate conditions in vitro the dephosphorylation of PA can be used to assess chemical-dependent changes in PAP and/or PLC activity.

  19. Glucose-6-phosphatase deficiency

    PubMed Central

    2011-01-01

    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 commonly confirmed

  20. Overexpression of Human Bone Alkaline Phosphatase in Pichia Pastoris

    NASA Technical Reports Server (NTRS)

    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.

  1. Protein tyrosine and serine–threonine phosphatases in the sea urchin, Strongylocentrotus purpuratus: Identification and potential functions

    PubMed Central

    Byrum, C.A.; Walton, K.D.; Robertson, A.J.; Carbonneau, S.; Thomason, R.T.; Coffman, J.A.; McClay, D.R.

    2011-01-01

    Protein phosphatases, in coordination with protein kinases, play crucial roles in regulation of signaling pathways. To identify protein tyrosine phosphatases (PTPs) and serine–threonine (ser–thr) phosphatases in the Strongylocentrotus purpuratus genome, 179 annotated sequences were studied (122 PTPs, 57 ser–thr phosphatases). Sequence analysis identified 91 phosphatases (33 conventional PTPs, 31 dual specificity phosphatases, 1 Class III Cysteine-based PTP, 1 Asp-based PTP, and 25 ser–thr phosphatases). Using catalytic sites, levels of conservation and constraint in amino acid sequence were examined. Nine of 25 receptor PTPs (RPTPs) corresponded to human, nematode, or fly homologues. Domain structure revealed that sea urchin-specific RPTPs including two, PTPRLec and PTPRscav, may act in immune defense. Embryonic transcription of each phosphatase was recorded from a high-density oligonucleotide tiling microarray experiment. Most RPTPs are expressed at very low levels, whereas nonreceptor PTPs (NRPTPs) are generally expressed at moderate levels. High expression was detected in MAP kinase phosphatases (MKPs) and numerous ser–thr phosphatases. For several expressed NRPTPs, MKPs, and ser–thr phosphatases, morpholino antisense-mediated knockdowns were performed and phenotypes obtained. Finally, to assess roles of annotated phosphatases in endomesoderm formation, a literature review of phosphatase functions in model organisms was superimposed on sea urchin developmental pathways to predict areas of functional activity. PMID:17087928

  2. RNA-seq analysis of antibiotic-producing Bacillus subtilis SC-8 in response to signal peptide PapR of Bacillus cereus.

    PubMed

    Yeo, In-Cheol; Lee, Nam Keun; Yang, Byung Wook; Hahm, Young Tae

    2014-01-01

    Bacillus subtilis SC-8 produces an antibiotic that has narrow antagonistic activity against bacteria in the Bacillus cereus group. In B. cereus group bacteria, peptide-activating PlcR (PapR) plays a significant role in regulating the transcription of virulence factors. When B. subtilis SC-8 and B. cereus are co-cultured, PapR is assumed to stimulate antibiotic production by B. subtilis SC-8. To better understand the effect of PapR on this interspecies interaction, the global transcriptome profile of B. subtilis SC-8 was analyzed in the presence of PapR. Significant changes were detected in 12.8 % of the total transcripts. Genes related to amino acid transport and metabolism (16.5 %) and transcription (15 %) were mainly upregulated, whereas genes involved in carbohydrate transport and metabolism (12.7 %) were markedly downregulated. The expression of genes related to transcription, including several transcriptional regulators and proteins involved in tRNA biosynthesis, was increased. The expression levels of genes associated with several transport systems, such as antibiotic, cobalt, and iron complex transporters, was also significantly altered. Among the downregulated genes were transcripts associated with spore formation, the subtilosin A gene cluster, and nitrogen metabolism.

  3. The five glucose-6-phosphatase paralogous genes are differentially regulated by insulin alone or combined with high level of amino acids and/or glucose in trout hepatocytes.

    PubMed

    Lucie, Marandel; Weiwei, Dai; Stéphane, Panserat; Sandrine, Skiba-Cassy

    2016-04-01

    A recent analysis of the newly sequenced rainbow trout (Oncorhynchus mykiss) genome suggested that duplicated gluconeogenic g6pc paralogues, fixed in this genome after the salmonid-specific 4th whole genome duplication, may have a role in the setting up of the glucose-intolerant phenotype in this carnivorous species. This should be due to the sub- or neo-functionalization of their regulation. In the present short communication we thus addressed the question of the regulation of these genes by insulin, hormone involved in the glucose homeostasis, and its interaction with glucose and amino acids in vitro. The stimulation of trout hepatocytes with insulin revealed an atypical up-regulation of g6pcb2 ohnologues and confirmed the sub- or neo-functionalization of the five g6pc genes at least at the regulatory level. Intriguingly, when hepatocytes were cultured with high levels of glucose and/or AAs in presence of insulin, most of the g6pc paralogues were up-regulated. It strongly suggested a cross-talk between insulin and nutrients for the regulation of these genes. Moreover these results strengthened the idea that g6pc duplicated genes may significantly contribute to the setting up of the glucose-intolerant phenotype in trout via their atypical regulation by insulin alone or in interaction with nutrients. These findings open new perspectives to better understand in vivo glucose-intolerant phenotype in trout fed a high carbohydrate diet.

  4. The pap Operon of Avian Pathogenic Escherichia coli Strain O1:K1 Is Located on a Novel Pathogenicity Island

    PubMed Central

    Kariyawasam, Subhashinie; Johnson, Timothy J.; Nolan, Lisa K.

    2006-01-01

    We have identified a 56-kb pathogenicity island (PAI) in avian pathogenic Escherichia coli strain O1:K1 (APEC-O1). This PAI, termed PAI IAPEC-O1, is integrated adjacent to the 3′ end of the pheV tRNA gene. It carries putative virulence genes of APEC (pap operon), other E. coli genes (tia and ireA), and a 1.5-kb region unique to APEC-O1. The kps gene cluster required for the biosynthesis of polysialic acid capsule was mapped to a location immediately downstream of this PAI. PMID:16369033

  5. Perfusion from angiogram and a priori (PAP) with temporal regularization

    NASA Astrophysics Data System (ADS)

    Taguchi, Katsuyuki; Geschwind, Jean-Francois H.

    2009-02-01

    Perfusion imaging is often used for diagnosis and for assessment of the response to the treatment. If perfusion can be measured during interventional procedures, it could lead to quantitative, more efficient and accurate treatment; however, imaging modalities that allow continuous dynamic scanning are not available in most of procedure rooms. Thus, we developed a method to measure the perfusion-time attenuation curves (TACs)-of regions-of-interest (ROIs) using xray C-arm angiography system with no gantry rotation but with a priori. The previous study revealed a problem of large oscillations in the estimated TACs and the lack of comparison with CT-based approaches. Thus the purposes of this study were (1) to reduce the variance of TDCs; and (2) to compare the performance of the improved PAP with that of the CT-based perfusion method. Our computer simulation study showed that the standard deviation of PAP method was decreased by 10.7-59.0% and that it outperformed (20× or 200× times) higher dose CT methods in terms of the accuracy, variance, and the temporal resolution.

  6. Predictors of Cervical Pap Smear Screening Awareness, Intention, and Receipt Among Vietnamese-American Women

    PubMed Central

    Nguyen, Tung T.; McPhee, Stephen J.; Nguyen, Thoa; Lam, Tram; Mock, Jeremiah

    2006-01-01

    Background Compared with white women, Vietnamese women in the United States have a higher rate of cervical cancer and lower Papanicolau (Pap) test utilization. We evaluated factors associated with awareness of the Pap test, intention to obtain it, and its receipt in Vietnamese-American women. Methods In 2000, we conducted a telephone survey of Vietnamese-American women aged ≥18 years living in Santa Clara County, California, and Harris County, Texas. We collected data on sociodemographics, healthcare system access and attitudes, as well as Pap test awareness, attitudes, intentions, and practices. Results Of 1566 subjects, 74% had heard of the Pap test, and 76% had had at least one. Only 42% of those who never had a Pap test had considered obtaining one. There were no significant differences between the two sites. Women aged ≥65 had the lowest rates for all three outcomes. For all women, younger age, being married, having requested a Pap test, physician recommendation, and preferring a female standby if the doctor was male were associated with Pap test intention. Being married, higher level of education, having a female doctor, having a respectful doctor, having requested the test, and physician recommendation were associated with Pap test receipt. Conclusion Vietnamese-American women have low rates of Pap test awareness, intention, and receipt. The patient–doctor interaction is an important determinant. Efforts to increase Pap test utilization in this population need to be directed at encouraging physicians to offer the Pap test and empowering women to ask for the test. PMID:12350454

  7. [Phosphatase activity in Amoeba proteus at low pH].

    PubMed

    Sopina, V A

    2009-01-01

    In free-living Amoeba proteus (strain B), three forms of tartrate-sensitive phosphatase were revealed using PAGE of the supernatant of ameba homogenates obtained with 1% Triton X-100 or distilled water and subsequent staining of gels with 2-naphthyl phosphate as substrate (pH 4.0). The form with the highest mobility in the ameba supernatant was sensitive to all tested phosphatase activity modulators. Two other forms with the lower mobilities were completely or significantly inactivated not only by sodium L-(+)-tartrate, but also by L-(+)-tartaric acid, sodium orthovanadate, ammonium molybdate, EDTA, EGTA, o-phospho-L-tyrosine, DL-dithiotreitol, H2O2, 2-mercaptoethanol, and ions of heavy metals - Fe2+, Fe3+, and Cu2+. Based on results of inhibitory analysis, lysosome location in the ameba cell, and wide substrate specificity of these two forms, it has been concluded that they belong to nonspecific acid phosphomonoesterases (AcP, EC 3.1.3.2). This AcP is suggested to have both phosphomonoesterase and phosphotyrosyl-protein phosphatase activitis. Two ecto-phosphatases were revealed in the culture medium, in which amebas were cultivated. One of them was inhibited by the same reagents as the ameba tartrate-sensitive AcP and seems to be the AcP released into the culture medium in the process of exocytosis of the content of food vacuoles. In the culture medium, apart from this AcP, another phosphatase was revealed, which was not inhibited by any tested inhibitors of AcP and alkaline phosphatase. It cannot be ruled out that this phosphatase belong to the ecto-ATPases found in many protists; however, its ability to hydrolyze ATP has not yet been proven.

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

    SciTech Connect

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

    1987-05-01

    C-Protein, which is a regulatory component of cardiac muscle myofibrils, is phosphorylated in response to US -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 (TSP)C-protein with the catalytic subunit of protein phosphatase 1 or 2A rapidly removed 30-40% of TS(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.

  9. Genomic and Proteomic Analyses of the Terminally Redundant Genome of the Pseudomonas aeruginosa Phage PaP1: Establishment of Genus PaP1-Like Phages

    PubMed Central

    Lu, Shuguang; Le, Shuai; Tan, Yinling; Zhu, Junmin; Li, Ming; Rao, Xiancai; Zou, Lingyun; Li, Shu; Wang, Jing; Jin, Xiaolin; Huang, Guangtao; Zhang, Lin; Zhao, Xia; Hu, Fuquan

    2013-01-01

    We isolated and characterized a new Pseudomonas aeruginosa myovirus named PaP1. The morphology of this phage was visualized by electron microscopy and its genome sequence and ends were determined. Finally, genomic and proteomic analyses were performed. PaP1 has an icosahedral head with an apex diameter of 68–70 nm and a contractile tail with a length of 138–140 nm. The PaP1 genome is a linear dsDNA molecule containing 91,715 base pairs (bp) with a G+C content of 49.36% and 12 tRNA genes. A strategy to identify the genome ends of PaP1 was designed. The genome has a 1190 bp terminal redundancy. PaP1 has 157 open reading frames (ORFs). Of these, 143 proteins are homologs of known proteins, but only 38 could be functionally identified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography-mass spectrometry allowed identification of 12 ORFs as structural protein coding genes within the PaP1 genome. Comparative genomic analysis indicated that the Pseudomonas aeruginosa phage PaP1, JG004, PAK_P1 and vB_PaeM_C2-10_Ab1 share great similarity. Besides their similar biological characteristics, the phages contain 123 core genes and have very close phylogenetic relationships, which distinguish them from other known phage genera. We therefore propose that these four phages be classified as PaP1-like phages, a new phage genus of Myoviridae that infects Pseudomonas aeruginosa. PMID:23675441

  10. Pap Smear Test Prevalence within Three Years (Age 18+) - Small Area Estimates

    Cancer.gov

    For Pap smear test, a woman 18 years of age or older must have reported having at least one Pap smear test in her life. Furthermore, she should have had one within the last three years by the time of interview.

  11. Roles of phagocytosis activating protein (PAP) in Aeromonas hydrophila infected Cyprinus carpio.

    PubMed

    Wonglapsuwan, Monwadee; Kongmee, Pataraporn; Suanyuk, Naraid; Chotigeat, Wilaiwan

    2016-06-01

    Cyprinus carpio (koi) is one of the most popular ornamental fish. A major problem for C. carpio farming is bacterial infections especially by Aeromonas hydrophila. Previously studies had shown that the Phagocytosis Activating Protein (PAP) gene was involved in the innate immune response of animals. Therefore, we attempted to identify a role for the PAP gene in the immunology of C. carpio. The expression of the PAP was found in C. carpio whole blood and increased when the fish were stimulated by inactivated A. hydrophila. In addition, PAP-phMGFP DNA was injected as an immunostimulant. The survival rate and the phagocytic index were significantly increased in the A. hydrophila infected fish that received the PAP-phMGFP DNA immunostimulant. A chitosan-PAP-phMGFP nanoparticle was then developed and feeded into fish which infected with A. hydrophila. These fish had a significantly lower mortality rate than the control. Therefore, this research confirmed a key role for PAP in protection fish from bacterial infection and the chitosan-PAP-phMGFP nanoparticle could be a good prototype for fish immunostimulant in the future. PMID:26748248

  12. Diacyltransferase Activity and Chain Length Specificity of Mycobacterium tuberculosis PapA5 in the Synthesis of Alkyl Beta-Diol Lipids

    PubMed Central

    Touchette, Megan H.; Bommineni, Gopal R.; Delle Bovi, Richard J.; Gadbery, John E.; Nicora, Carrie D.; Shukla, Anil K.; Kyle, Jennifer E.; Metz, Thomas O.; Martin, Dwight W.; Sampson, Nicole S.; Miller, W. Todd; Tonge, Peter J.; Seeliger, Jessica C.

    2015-01-01

    Although classified as Gram-positive bacteria, Corynebacterineae possess an asymmetric outer membrane that imparts structural and thereby physiological similarity to more distantly related Gram-negative bacteria. Like lipopolysaccharide in Gram-negative bacteria, lipids in the outer membrane of Corynebacterineae have been associated with the virulence of pathogenic species such as Mycobacterium tuberculosis (Mtb). For example, Mtb strains that lack long, branched-chain alkyl esters known as dimycocerosates (DIMs) are significantly attenuated in model infections. The resultant interest in the biosynthetic pathway of these unusual virulence factors has led to the elucidation of many of the steps leading to the final esterification of the alkyl beta-diol, phthiocerol, with branched-chain fatty acids known as mycocerosates. PapA5 is an acyltransferase implicated in these final reactions. We here show that PapA5 is indeed the terminal enzyme in DIM biosynthesis by demonstrating its dual esterification activity and chain-length preference using synthetic alkyl beta-diol substrate analogues. Applying these analogues to a series of PapA5 mutants, we also revise a model for the substrate binding within PapA5. Finally, we demonstrate that the Mtb Ser/Thr kinases PknB and PknE modify PapA5 on three overlapping Thr residues and a fourth Thr is unique to PknE phosphorylation. These results clarify the DIM biosynthetic pathway and indicate post-translational modifications that warrant further elucidation for their roles in regulation DIM biosynthesis. PMID:26271001

  13. Identification of Phase-I Metabolites of the Kv1.3 Blocker PAP-1 (5-(4-Phenoxybutoxy)psoralen) in the Rat

    PubMed Central

    Hao, Bin; Chen, Zhong-Wei; Zhou, Xiang-Jun; Zimin, Pavel I.; Miljanich, George P.; Wulff, Heike; Wang, Yong-Xiang

    2013-01-01

    PAP-1 (5-(4-phenoxybutoxy)psoralen), a potent small-molecule blocker of the voltage-gated potassium Kv1.3 channel, is currently in pre-clinical development for psoriasis. The present study was undertaken to identify the major phase-I metabolites of PAP-1 in rats. Following oral administration at 50 mg/kg, bile, plasma, urine and feces were collected, and separated by reversed-phase HPLC after sample preparation by solid-phase extraction. Five phase-I metabolites, i.e., 5-(oxybutyric-acid)psoralen (M1), 5-[4-(4-hydroxybutoxy)]psoralen (M2), 5-[4-(4-hydroxyphenoxy)]psoralen (M3), 5-[4-(3-hydroxyphenoxy)]psoralen (M4), 8-hydroxyl-5-(4-phenoxybutoxy)psoralen (M5), were isolated and identified by mass spectrometry and NMR spectroscopy. M3, M4 and M5 were hydroxylated products, while M1 and M2 were O-dealkylation products. Incubation of PAP-1 with rat liver microsomes rendered the same five major metabolites in a NADPH-dependent manner suggesting that cytochrome P450 (CYP) enzymes are involved in PAP-1 metabolism. Inhibitors of rat CYP1A1/2 (alpha-naphthoflavone) and CYP3A (ketoconazole) but not CYP2D6, CYP2E or CYP2C9 blocked the metabolism of PAP-1 in rat microsomes. Of the five metabolites M3, M4 and M5 were found to inhibit Kv1.3 currents with nanomolar IC50s, while M1 and M2 were inactive. Our results identified the Kv1.3-inactive M1 as the major phase-I metabolite, and suggest that hydroxylation and O-dealkylation are the major pathways of PAP-1 metabolism principally via CYP1A1/2 and CYP3A. We further conducted a 6-months repeat-dose toxicity study with PAP-1 at 50 mg/kg in both male and female rats and did not observe any changes in hematology, blood chemistry, body weight, histology of any major organ, or speeding up of metabolism suggesting induction of its metabolizing enzymes (presumably CYPs). PMID:21070145

  14. Species identification of processed animal proteins (PAPs) in animal feed containing feed materials from animal origin.

    PubMed

    Axmann, Sonja; Adler, Andreas; Brandstettner, Agnes Josephine; Spadinger, Gabriela; Weiss, Roland; Strnad, Irmengard

    2015-01-01

    Since June 2013 the total feed ban of processed animal proteins (PAPs) was partially lifted. Now it is possible to mix fish feed with PAPs from non-ruminants (pig and poultry). To guarantee that fish feed, which contains non-ruminant PAPs, is free of ruminant PAPs, it has to be analysed with a ruminant PCR assay to comply with the total ban of feeding PAPs from ruminants. However, PCR analysis cannot distinguish between ruminant DNA, which originates from proteins such as muscle and bones, and ruminant DNA, which comes from feed materials of animal origin such as milk products or fat. Thus, there is the risk of obtaining positive ruminant PCR signals based on these materials. The paper describes the development of the combination of two analysis methods, micro-dissection and PCR, to eliminate the problem of 'false-positive' PCR signals. With micro-dissection, single particles can be isolated and subsequently analysed with PCR.

  15. Molecular cloning, genomic organization, and chromosomal localization of the human pancreatitis-associated protein (PAP) gene

    SciTech Connect

    Dusetti, N.J.; Frigerio, J.M.; Dagorn, J.C.; Iovanna, J.L. ); Fox, M.F.; Swallow, D.M. )

    1994-01-01

    Pancreatitis-associated protein (PAP) is a secretory pancreatic protein present in small amounts in normal pancreas and overexpressed during the acute phase of pancreatitis. In this paper, the authors describe the cloning, characterization, and chromosomal mapping of the human PAP gene. The gene spans 2748 bp and contains six exons interrupted by five introns. The gene has a typical promoter containing the sequences TATAAA and CCAAT 28 and 52 bp upstream of the cap site, respectively. They found striking similarities in genomic organization as well as in the promoter sequences between the human and rat PAP genes. The human PAP gene was mapped to chromosome 2p12 using rodent-human hybrid cells and in situ chromosomal hybridization. This localization coincides with that of the reg/lithostathine gene, which encodes a pancreatic secretory protein structurally related to PAP, suggesting that both genes derived from the same ancestral gene by duplication. 35 refs., 4 figs., 1 tab.

  16. Phosphatase activity on the cell wall of Fonsecaea pedrosoi.

    PubMed

    Kneipp, L F; Palmeira, V F; Pinheiro, A A S; Alviano, C S; Rozental, S; Travassos, L R; Meyer-Fernandes, J R

    2003-12-01

    The activity of a phosphatase was characterized in intact mycelial forms of Fonsecaea pedrosoi, a pathogenic fungus that causes chromoblastomycosis. At pH 5.5, this fungus hydrolyzed p-nitrophenylphosphate (p-NPP) to p-nitrophenol (p-NP) at a rate of 12.78 +/- 0.53 nmol p-NP per h per mg hyphal dry weight. The values of Vmax and apparent Km for p-NPP hydrolyses were measured as 17.89 +/- 0.92 nmol p-NP per h per mg hyphal dry weight and 1.57 +/- 0.26 mmol/l, respectively. This activity was inhibited at increased pH, a finding compatible with an acid phosphatase. The enzymatic activity was strongly inhibited by classical inhibitors of acid phosphatases such as sodium orthovanadate (Ki = 4.23 micromol/l), sodium molybdate (Ki = 7.53 micromol/l) and sodium fluoride (Ki = 126.78 micromol/l) in a dose-dependent manner. Levamizole (1 mmol/l) and sodium tartrate (10 mmol/l), had no effect on the enzyme activity. Cytochemical localization of the acid phosphatase showed electrondense cerium phosphate deposits on the cell wall, as visualized by transmission electron microscopy. Phosphatase activity in F. pedrosoi seems to be associated with parasitism, as sclerotic cells, which are the fungal forms mainly detected in chromoblastomycosis lesions, showed much higher activities than conidia and mycelia did. A strain of F. pedrosoi recently isolated from a human case of chromoblastomycosis also showed increased enzyme activity, suggesting that the expression of surface phosphatases may be stimulated by interaction with the host.

  17. Does knowledge influence pap test screening among young African-American women?

    PubMed

    Bynum, Shalanda A; Guillaume, Daphnee A; Brandt, Heather M; Fletcher, Faith E

    2014-09-01

    Pap test screening among African-American women has substantially increased. However, African-American women continue to bear the burden of cervical cancer as compared to White women. The objective of this study was to assess the influence of Pap test knowledge on cervical screening history among young African-American women. Between January and April 2009, 320 women from historically black colleges and universities located in the southeastern United States who met study inclusion criteria completed an anonymous self-report questionnaire to assess their awareness, knowledge, and behaviors related to human papillomavirus and cervical cancer prevention and control. Seventy-six percent of women reported ever having a Pap test, 54 % reported having a Pap test less than 1 year ago, and 25 % reported ever having an abnormal Pap test result. The overall mean score on the six-point Pap test knowledge scale was 4.46 ± 1.02. Women who reported having an abnormal Pap test (4.96 ± 0.82) had significantly higher Pap test knowledge compared to those never having an abnormal result (4.49 ± 1.04), p < 0.01. No other differences were found. Efforts to improve Pap test knowledge among all women, including those with no prior abnormal Pap test history, are critical to cervical cancer prevention and control over the life course. Such efforts should include creating information that is relevant to the population and enables informed decision making about cervical health.

  18. Tyrosine phosphatases as key regulators of StAR induction and cholesterol transport: SHP2 as a potential tyrosine phosphatase involved in steroid synthesis.

    PubMed

    Cooke, Mariana; Mele, Pablo; Maloberti, Paula; Duarte, Alejandra; Poderoso, Cecilia; Orlando, Ulises; Paz, Cristina; Cornejo Maciel, Fabiana; Podestá, Ernesto J

    2011-04-10

    The phospho-dephosphorylation of intermediate proteins is a key event in the regulation of steroid biosynthesis. In this regard, it is well accepted that steroidogenic hormones act through the activation of serine/threonine (Ser/Thr) protein kinases. Although many cellular processes can be regulated by a crosstalk between different kinases and phosphatases, the relationship of Ser/Thr phosphorylation and tyrosine (Tyr)-dephosphorylation is a recently explored field in the regulation of steroid synthesis. Indeed in steroidogenic cells, one of the targets of hormone-induced Ser/Thr phosphorylation is a protein tyrosine phosphatase. Whereas protein tyrosine phosphatases were initially regarded as household enzymes with constitutive activity, dephosphorylating all the substrates they encountered, evidence is now accumulating that protein tyrosine phosphatases are tightly regulated by various mechanisms. Here, we will describe the role of protein tyrosine phosphatases in the regulation of steroid biosynthesis, relating them to steroidogenic acute regulatory protein, arachidonic acid metabolism and mitochondrial rearrangement.

  19. Determination of trace alkaline phosphatase by affinity adsorption solid substrate room temperature phosphorimetry based on wheat germ agglutinin labeled with 8-quinolineboronic acid phosphorescent molecular switch and prediction of diseases

    NASA Astrophysics Data System (ADS)

    Liu, Jia-Ming; Gao, Hui; Li, Fei-Ming; Shi, Xiu-Mei; Lin, Chang-Qing; Lin, Li-Ping; Wang, Xin-Xing; Li, Zhi-Ming

    2010-09-01

    The 8-quinolineboronic acid phosphorescent molecular switch (abbreviated as PMS-8-QBA. Thereinto, 8-QBA is 8-quinolineboronic acid, and PMS is phosphorescent molecular switch) was found for the first time. PMS-8-QBA, which was in the "off" state, could only emit weak room temperature phosphorescence (RTP) on the acetyl cellulose membrane (ACM). However, PMS-8-QBA turned "on" automatically for its changed structure, causing that the RTP of 8-QBA in the system increased, after PMS-8-QBA-WGA (WGA is wheat germ agglutinin) was formed by reaction between -OH of PMS-8-QBA and -COOH of WGA. More interesting is that the -NH 2 of PMS-8-QBA-WGA could react with the -COOH of alkaline phosphatase (AP) to form the affinity adsorption (AA) product WGA-AP-WGA-8-QBA-PMS (containing -NH-CO- bond), which caused RTP of the system to greatly increase. Thus, affinity adsorption solid substrate room temperature phosphorimetry using PMS-8-QBA as labelling reagent (PMS-8-QBA-AA-SSRTP) for the determination of trace AP was established. The method had many advantages, such as high sensitivity (the detection limit (LD) was 2.5 zg spot -1. For sample volume of 0.40 μl spot -1, corresponding concentration was 6.2 × 10 -18 g ml -1), good selectivity (the allowed concentration of coexisting material was higher, when the relative error was ±5%), high accuracy (applied to detection of AP content in serum samples, the result was coincided with those obtained by enzyme-linked immunoassay), which was suitable for the detection of trace AP content in serum samples and the forecast of human diseases. Meanwhile, the mechanism of PMS-8-QBA-AASSRTP was discussed. The new field of analytical application and clinic diagnosis technique of molecule switch are exploited, based on the phosphorescence characteristic of PMS-8-QBA, the AA reaction between WGA and AP, as well as the relation between AP content and human diseases. The research results promote the development and interpenetrate among molecule

  20. Yeast Acid Phosphatase in a Student Laboratory.

    ERIC Educational Resources Information Center

    Barbaric, Sloeodan; Ries, Blanka

    1988-01-01

    Examines the influence of enzyme and substrate concentrations, pH, temperature, and inhibitors on catalytic activity. Follows the influence of different phosphate concentrations in the growth medium on enzyme activity. Studies regulation of enzyme synthesis by repression. Includes methodology for six experiments. (MVL)

  1. Flavonoid production in transgenic hop (Humulus lupulus L.) altered by PAP1/MYB75 from Arabidopsis thaliana L.

    PubMed

    Gatica-Arias, A; Farag, M A; Stanke, M; Matoušek, J; Wessjohann, L; Weber, G

    2012-01-01

    Hop is an important source of secondary metabolites, such as flavonoids. Some of these are pharmacologically active. Nevertheless, the concentration of some classes as flavonoids in wild-type plants is rather low. To enhance the production in hop, it would be interesting to modify the regulation of genes in the flavonoid biosynthetic pathway. For this purpose, the regulatory factor PAP1/AtMYB75 from Arabidopsis thaliana L. was introduced into hop plants cv. Tettnanger by Agrobacterium-mediated genetic transformation. Twenty kanamycin-resistant transgenic plants were obtained. It was shown that PAP1/AtMYB75 was stably incorporated and expressed in the hop genome. In comparison to the wild-type plants, the color of female flowers and cones of transgenic plants was reddish to pink. Chemical analysis revealed higher levels of anthocyanins, rutin, isoquercitin, kaempferol-glucoside, kaempferol-glucoside-malonate, desmethylxanthohumol, xanthohumol, α-acids and β-acids in transgenic plants compared to wild-type plants. PMID:21912858

  2. Phosphatase-mediated heavy metal accumulation by a Citrobacter sp. and related enterobacteria.

    PubMed

    Macaskie, L E; Bonthrone, K M; Rouch, D A

    1994-08-15

    A Citrobacter sp. was reported previously to accumulate heavy metals as cell-bound heavy metal phosphates. Metal uptake is mediated by the activity of a periplasmic acid-type phosphatase that liberates inorganic phosphate to provide the precipitant ligand for heavy metals presented to the cells. Amino acid sequencing of peptide fragments of the purified enzyme revealed significant homology to the phoN product (acid phosphatase) of some other enterobacteria. These organisms, together with Klebsiella pneumoniae, previously reported to produce acid phosphatase, were tested for their ability to remove uranium and lanthanum from challenge solutions supplemented with phosphatase substrate. The coupling of phosphate liberation to metal bioaccumulation was limited to the metal accumulating Citrobacter sp.; therefore the participation of species-specific additional factors in metal bioaccumulation was suggested.

  3. Biochemical characterization of the extracellular phosphatases produced by phosphorus-deprived Chlamydomonas reinhardtii.

    PubMed Central

    Quisel, J D; Wykoff, D D; Grossman, A R

    1996-01-01

    We have examined the extracellular phosphatases produced by the terrestrial green alga Chlamydomonas reinhardtii in response to phosphorus deprivation. Phosphorus-deprived cells increase extra-cellular alkaline phosphatase activity 300-fold relative to unstarved cells. The alkaline phosphatases are released into the medium by cell-wall-deficient strains and by wild-type cells after treatment with autolysin, indicating that they are localized to the periplasm. Anion-exchange chromatography and analysis by nondenaturing polyacrylamide gel electrophoresis revealed that there are two major inducible alkaline phosphatases. A calcium-dependent enzyme composed of 190-kD glycoprotein subunits accounts for 85 to 95% of the Alkaline phosphatase activity. This phosphatase has optimal activity at pH 9.5 and a Km of 120 to 262 microns for all physiological substrates tested, with the exception of phytic acid, which it cleaved with a 50-fold lower efficiency. An enzyme with optimal activity at pH 9 and no requirement for divalent cations accounts for 2 to 10% of the alkaline phosphatase activity. This phosphatase was only able to efficiently hydrolyze arylphosphates. The information reported here, in conjunction with the results of previous studies, defines the complement of extracellular phosphatases produced by phosphorus-deprived Chlamydomonas cells. PMID:8754684

  4. Pap Test and HIV testing behaviors of South Carolina women 18–64 years old

    PubMed Central

    Wigfall, Lisa; Duffus, Wayne A.; Annang, Lucy; Richter, Donna L.; Torres, Myriam E.; Williams, Edith M.; Glover, Saundra

    2010-01-01

    Background Pap tests are used to detect abnormal cell growth in the cervix. Early detection of precancerous cells increases the likelihood of treatment success. In fact, the Pap test is one of only a few cancer screening procedures that can prevent cancer by virtue of identifying and intervening upon abnormal precancerous cells in the early stages. There has been a growing prevalence of cervical cancer among HIV-positive women. Early diagnosis is essential to improving survival outcomes of women living with HIV/AIDS. The purpose of this study was to describe Pap test behaviors among women in South Carolina and examine its relationship with HIV testing. Methods Behavioral Risk Factor Surveillance System (BRFSS) data from 2008 were analyzed. Chi-square tests and logistic regression analyses were performed to describe Pap test behaviors among our sample (n=3,404) of non-Hispanic White and non-Hispanic Black women (18–64 years old). Results The majority of participants (97%) reported ever having a Pap test. Participants who reported never having a Pap test were 50% less likely to have ever been tested for HIV. Conclusions The participants in our study met the Healthy People 2010 target of ever having a Pap test. However, less than half of participants (42%) had ever been tested for HIV. A larger proportion of women who have had a Pap test had also been tested for HIV. Practice Implications Our findings suggest that offering women an HIV test during routine Pap tests may present an opportunity to increase the number of women in South Carolina who know their HIV serostatus. PMID:20108718

  5. Relevance of HPV Screening for Triaging Equivocal Cytology Findings in the Pap II-p, Pap III and Pap IIID Groups – Results of Two Long-Term Studies

    PubMed Central

    Luyten, A.; Petry, K. U.

    2015-01-01

    Introduction: The use of HPV screening for the triage of ASC-US (atypical squamous cells of undetermined significance) cytology results has been established as a sound standard by international trials whereas the data for other cytology findings are in part contradictory. There is a lack of long-term studies on the use of HPV triage in Germany. Materials and Methods: For the present study data from a primary HPV screening project involving women aged over 30 years, ongoing since 2006, and an epidemiological study on women aged between 20 and 27 years, ongoing since 2009, were used. Upon recruitment, all women underwent a smear test for cytology and screening for “high-risk” HPV using Hybrid Capture 2 (HC2). If both tests were positive or if there were persisting remarkable cytology findings or a positive HPV test, then clarification by colposcopy was performed. Results: Altogether, among 282 women with Pap II-p (ASC-US), Pap III (ASC-H) or Pap IIID (LSIL + CIN2) and negative HPV test there was no case of CIN3+. Among the women under 30 years of age, however, 69 % (ASC-US) to 85 % (LSIL + CIN2) of the remarkable findings were HPV positive, also among the older women with Pap IIID, the 71 % prevalence of HPV was too high for a triage and even without triage there was a 23 % risk for CIN3+. On the other hand, of the women over 30 years old with ASC-US (Pap II-p) findings, only 21 % were positive for HPV and the risk for CIN3+ in this group was high at 29 %. Also for ASC-H (Pap III) findings in the age group of over 30 years with an HPV prevalence of 56 % there was an efficient triage for CIN3+. Discussion: In summary, the HPV triage of ASC-US (Pap II-p) findings in women aged over 30 years was found to be efficient; in contrast, LSIL + CIN2 (Pap IIID) findings in this age group justified an immediate referral to colposcopy whereas cytology control appeared to be sufficient for younger women. PMID:26556908

  6. Conserved sequence motifs among bacterial, eukaryotic, and archaeal phosphatases that define a new phosphohydrolase superfamily.

    PubMed

    Thaller, M C; Schippa, S; Rossolini, G M

    1998-07-01

    Members of a new molecular family of bacterial nonspecific acid phosphatases (NSAPs), indicated as class C, were found to share significant sequence similarities to bacterial class B NSAPs and to some plant acid phosphatases, representing the first example of a family of bacterial NSAPs that has a relatively close eukaryotic counterpart. Despite the lack of an overall similarity, conserved sequence motifs were also identified among the above enzyme families (class B and class C bacterial NSAPs, and related plant phosphatases) and several other families of phosphohydrolases, including bacterial phosphoglycolate phosphatases, histidinol-phosphatase domains of the bacterial bifunctional enzymes imidazole-glycerolphosphate dehydratases, and bacterial, eukaryotic, and archaeal phosphoserine phosphatases and threalose-6-phosphatases. These conserved motifs are clustered within two domains, separated by a variable spacer region, according to the pattern [FILMAVT]-D-[ILFRMVY]-D-[GSNDE]-[TV]-[ILVAM]-[AT S VILMC]-X-¿YFWHKR)-X-¿YFWHNQ¿-X( 102,191)-¿KRHNQ¿-G-D-¿FYWHILVMC¿-¿QNH¿-¿FWYGP¿-D -¿PSNQYW¿. The dephosphorylating activity common to all these proteins supports the definition of this phosphatase motif and the inclusion of these enzymes into a superfamily of phosphohydrolases that we propose to indicate as "DDDD" after the presence of the four invariant aspartate residues. Database searches retrieved various hypothetical proteins of unknown function containing this or similar motifs, for which a phosphohydrolase activity could be hypothesized.

  7. Two ancient bacterial-like PPP family phosphatases from Arabidopsis are highly conserved plant proteins that possess unique properties.

    PubMed

    Uhrig, R Glen; Moorhead, Greg B

    2011-12-01

    Protein phosphorylation, catalyzed by the opposing actions of protein kinases and phosphatases, is a cornerstone of cellular signaling and regulation. Since their discovery, protein phosphatases have emerged as highly regulated enzymes with specificity that rivals their counteracting kinase partners. However, despite years of focused characterization in mammalian and yeast systems, many protein phosphatases in plants remain poorly or incompletely characterized. Here, we describe a bioinformatic, biochemical, and cellular examination of an ancient, Bacterial-like subclass of the phosphoprotein phosphatase (PPP) family designated the Shewanella-like protein phosphatases (SLP phosphatases). The SLP phosphatase subcluster is highly conserved in all plants, mosses, and green algae, with members also found in select fungi, protists, and bacteria. As in other plant species, the nucleus-encoded Arabidopsis (Arabidopsis thaliana) SLP phosphatases (AtSLP1 and AtSLP2) lack genetic redundancy and phylogenetically cluster into two distinct groups that maintain different subcellular localizations, with SLP1 being chloroplastic and SLP2 being cytosolic. Using heterologously expressed and purified protein, the enzymatic properties of both AtSLP1 and AtSLP2 were examined, revealing unique metal cation preferences in addition to a complete insensitivity to the classic serine/threonine PPP protein phosphatase inhibitors okadaic acid and microcystin. The unique properties and high conservation of the plant SLP phosphatases, coupled to their exclusion from animals, red algae, cyanobacteria, archaea, and most bacteria, render understanding the function(s) of this new subclass of PPP family protein phosphatases of particular interest.

  8. Phosphatase acitivity as biosignatures in terrestrial extreme environments

    NASA Astrophysics Data System (ADS)

    Kawai, Jun; Nakamoto, Saki; Hara, Masashi; Obayashi, Yumiko; Kaneko, Takeo; Mita, Hajime; Yoshimura, Yoshitaka; Takano, Yoshinori; Kobayashi, Kensei

    Since phosphate esters are essential for the terrestrial life, phosphatase activity can be a can-didate for biosignatures of biological activity. It has been recognized that terrestrial biosphere expands to such extreme environments as deep subsurface lithosphere, high temperature hot springs and stratosphere. We analyzed phosphatase activities in the samples obtained in ex-treme environments such as submarine hydrothermal systems and Antarctica , and discussed whether they can be used as biosignatures for extant life. Core samples and chimney samples were collected at Tarama Knoll in Okinawa Trough in 2009, both in a part of the Archaean Park Project. Surface soil samples are obtained at the Sites 1-8 near Showa Base in Antarctica during the 47th Japan Antarctic exploration mission in 2005-6. Alkaline Phosphatase activ-ity in sea water and in soil was measured spectrometrically by using 25 mM p-nitrophenyl phosphate (pH 8.0) as a substrate. Phosphatase activities in extracts were measured fluoro-metrically by using 4-methylumberyferryl phosphate as a substrate. Concentration of amino acids and their enantiomeric ratios were also determined by HPLC . Significant enzymatic ac-tivities were revealed in both some of the hydrothermal sub-vent systems and Antarctica soils, which is crucial evidence of vigorous microbial oasis. It is consistent with the fact that large enantiomeric excess of L-form amino acids were found in the same core sequences. Optimum temperatures of ALP in the chimney, Antarctica soil and YNU campus soil were 353 K, 313 K, and 333 K, respectively. The present results suggested that phosphatase activities,, together with amino acids, can be used as possible biosignatures for extant life.

  9. Structure and copy number of gene clusters related to the pap P-adhesin operon of uropathogenic Escherichia coli.

    PubMed

    Arthur, M; Campanelli, C; Arbeit, R D; Kim, C; Steinbach, S; Johnson, C E; Rubin, R H; Goldstein, R

    1989-02-01

    The structurally related pap and prs operons of the uropathogenic Escherichia coli isolate J96 encode a P and an F adhesin that mediate bacterial attachment to the human P blood group antigen and the Forssman antigen, respectively. Using probes prepared from different segments of the pap operon, Southern blot hybridizations were performed to characterize pap-related sequences of 30 E. coli clinical isolates expressing different adhesin phenotypes. Gene clusters encoding P and F adhesins displayed no restriction site polymorphism in sequences homologous to the papH, papC, and papD genes that encode proteins essential to the transport and polymerization of the subunits of the P-pilus adhesin. In contrast, pap-related genetic elements associated with a null phenotype either lacked homology to the papH, papC, and papD genes or displayed a restriction site polymorphism in this region. Sequences within and surrounding the J96 papG and prsG adhesin genes that determine the binding specificities to the P and F antigens, respectively, were not conserved. However, gene clusters encoding different binding specificities could not be distinguished based on such restriction site polymorphisms. The majority of clinical isolates had more than one copy of pap-related sequences that involved gene clusters similar to the J96 pap operon, as well as genetic elements that were related only to a part of this operon. The implications of this unexpected copy number polymorphism with respect to possible recombination events involving pap-related sequences are discussed.

  10. Determination of liver microsomal glucose-6-phosphatase.

    PubMed

    Zak, B; Epstein, E; Baginski, E S

    1977-01-01

    A procedure for the determination of liver microsomal glucose-6-phosphatase is described. Homogenization and ultracentrifrigation were used to prepare a precipitate whose character was defined by monitoring the desire enzyme activity which serves as a marker. Activity of the enzyme was determined by means of a sensitive colorimetric reaction for the product, inorganic phosphate. Non-enzymatic hydrolysis problems with the substrate are minimized in this procedure by the masking action of citrate. The final heteropoly blue color appears to be considerably sensitized by interaction of phosphomolybdous ion with arsenite. The stability of the relatively labile enzyme was ensured by chelating any metals present with ethylene diamine tetraacetic acid. The overall results obtained by the procedure appear to be useful as an aid in the diagnosis of Type I glycogenosis, a glycogen storage disease called Von Gierke's disease. PMID:192125

  11. Pap, gruel, and panada: early approaches to artificial infant feeding.

    PubMed

    Obladen, Michael

    2014-01-01

    This paper collects information on artificial infant feeding published before 1860, the year when commercial formula became available. We have extensive artifactual evidence of thousands of feeding vessels since the Bronze Age. Special museum collections can be found in London, Paris, Cologne, Fécamp, Toronto, New Mexico, and elsewhere. The literature on the use of animal milk for infant feeding begins with Soranus in the 2nd century CE. Literature evidence from the very first printed books in the 15th century proves that physicians, surgeons, midwives, and the laity were aware of the opportunities and risks of artificial infant feeding. Most 17th to 19th century books on infant care contained detailed recipes for one or several of the following infant foods: pap, a semisolid food made of flour or bread crumbs cooked in water with or without milk; gruel, a thin porridge resulting from boiling cereal in water or milk, and panada, a preparation of various cereals or bread cooked in broth. During the 18th century, the published opinion on artificial feeding evolved from health concerns to a moral ideology. This view ignored the social and economic pressures which forced many mothers to forego or shorten breast-feeding. Bottle-feeding has been common practice throughout history.

  12. Negative HPV screening test predicts low cervical cancer risk better than negative Pap test

    Cancer.gov

    Based on a study that included more than 1 million women, investigators at NCI have determined that a negative test for HPV infection compared to a negative Pap test provides greater safety, or assurance, against future risk of cervical cancer.

  13. Cortisol modification of HeLa 65 alkaline phosphatase. Decreased phosphate content of the induced enzyme.

    PubMed

    Bazzell, K L; Price, G; Tu, S; Griffin, M

    1976-01-15

    Alkaline phosphatase activity of HeLa cells is increased 5-20-fold during growth in medium with cortisol. The increase in enzyme activity is due to an enhanced catalytic efficiency rather than an increase in alkaline phosphatase protein in induced cells. In the present study the chemical composition of control and induced forms of alkaline phosphatase were investigated to determine the enzyme modification that may be responsible for the increased catalytic activity. HeLa alkaline phosphatase is a phosphoprotein and the induced form of the enzyme has approximately one-half of the phosphate residues associated with control enzyme. The decrease in phosphate residues of the enzyme apparently alters its catalytic activity. Other chemical components of purified alkaline phosphatase from control and induced cells are similar; these include sialic acid, hexosamine and sulfhydryl residues. PMID:1248469

  14. Inhibitory effects of a peptide-fusion protein (Latarcin-PAP1-Thanatin) against chikungunya virus.

    PubMed

    Rothan, Hussin A; Bahrani, Hirbod; Shankar, Esaki M; Rahman, Noorsaadah Abd; Yusof, Rohana

    2014-08-01

    Chikungunya virus (CHIKV) outbreaks have led to a serious economic burden, as the available treatment strategies can only alleviate disease symptoms, and no effective therapeutics or vaccines are currently available for human use. Here, we report the use of a new cost-effective approach involving production of a recombinant antiviral peptide-fusion protein that is scalable for the treatment of CHIKV infection. A peptide-fusion recombinant protein LATA-PAP1-THAN that was generated by joining Latarcin (LATA) peptide with the N-terminus of the PAP1 antiviral protein, and the Thanatin (THAN) peptide to the C-terminus, was produced in Escherichia coli as inclusion bodies. The antiviral LATA-PAP1-THAN protein showed 89.0% reduction of viral plaque formation compared with PAP1 (46.0%), LATA (67.0%) or THAN (79.3%) peptides alone. The LATA-PAP1-THAN protein reduced the viral RNA load that was 0.89-fold compared with the untreated control cells. We also showed that PAP1 resulted in 0.44-fold reduction, and THAN and LATA resulting in 0.78-fold and 0.73-fold reductions, respectively. The LATA-PAP1-THAN protein inhibited CHIKV replication in the Vero cells at an EC50 of 11.2μg/ml, which is approximately half of the EC50 of PAP1 (23.7μg/ml) and protected the CHIKV-infected mice at the dose of 0.75mg/ml. We concluded that production of antiviral peptide-fusion protein in E. coli as inclusion bodies could accentuate antiviral activities, enhance cellular internalisation, and could reduce product toxicity to host cells and is scalable to epidemic response quantities.

  15. Selective indication for positive airway pressure (PAP) in sleep-related breathing disorders with obstruction

    PubMed Central

    Stasche, Norbert

    2006-01-01

    Positive airway pressure (PAP) is the therapy of choice for most sleep-related breathing disorders (SRBD). A variety of PAP devices using positive airway pressure (CPAP, BiPAP, APAP, ASV) must be carefully considered before application. This overview aims to provide criteria for choosing the optimal PAP device according to severity and type of sleep-related breathing disorder. In addition, the range of therapeutic applications, constraints and side effects as well as alternative methods to PAP will be discussed. This review is based on an analysis of current literature and clinical experience. The data is presented from an ENT-sleep-laboratory perspective and is designed to help the ENT practitioner initiate treatment and provide support. Different titration methods, current devices and possible applications will be described. In addition to constant pressure devices (CPAP), most commonly used for symptomatic obstructive sleep apnoea (OSA) without complicating conditions, BiPAP models will be introduced. These allow two different positive pressure settings and are thus especially suitable for patients with cardiopulmonary diseases or patients with pressure intolerance, increasing compliance in this subgroup considerably. Compliance can also be increased in patients during first night of therapy, patients with highly variable pressure demands or position-dependent OSA, by using self-regulating Auto-adjust PAP devices (Automatic positive airway pressure, APAP). Patients with Cheyne-Stokes breathing, a subtype of central sleep apnoea, benefit from adaptive servo-ventilation (ASV), which analyzes breathing patterns continually and adjusts the actual ventilation pressure accordingly. This not only reduces daytime sleepiness, but can also influence heart disease positively. Therapy with positive airway pressure is very effective in eliminating obstruction-related sleep diseases and symptoms. However, because therapy is generally applied for life, the optimal PAP device

  16. Light and the E3 ubiquitin ligase COP1/SPA control the protein stability of the MYB transcription factors PAP1 and PAP2 involved in anthocyanin accumulation in Arabidopsis.

    PubMed

    Maier, Alexander; Schrader, Andrea; Kokkelink, Leonie; Falke, Christian; Welter, Bastian; Iniesto, Elisa; Rubio, Vicente; Uhrig, Joachim F; Hülskamp, Martin; Hoecker, Ute

    2013-05-01

    Anthocyanins are natural pigments that accumulate only in light-grown and not in dark-grown Arabidopsis plants. Repression of anthocyanin accumulation in darkness requires the CONSTITUTIVELY PHOTOMORPHOGENIC1/SUPPRESSOR OF PHYA-105 (COP1/SPA) ubiquitin ligase, as cop1 and spa mutants produce anthocyanins also in the dark. Here, we show that COP1 and SPA proteins interact with the myeloblastosis (MYB) transcription factors PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP)1 and PAP2, two members of a small protein family that is required for anthocyanin accumulation and for the expression of structural genes in the anthocyanin biosynthesis pathway. The increased anthocyanin levels in cop1 mutants requires the PAP1 gene family, indicating that COP1 functions upstream of the PAP1 gene family. PAP1 and PAP2 proteins are degraded in the dark and this degradation is dependent on the proteasome and on COP1. Hence, the light requirement for anthocyanin biosynthesis results, at least in part, from the light-mediated stabilization of PAP1 and PAP2. Consistent with this conclusion, moderate overexpression of PAP1 leads to an increase in anthocyanin levels only in the light and not in darkness. Here we show that SPA genes are also required for reducing PAP1 and PAP2 transcript levels in dark-grown seedlings. Taken together, these results indicate that the COP1/SPA complex affects PAP1 and PAP2 both transcriptionally and post-translationally. Thus, our findings have identified mechanisms via which the COP1/SPA complex controls anthocyanin levels in Arabidopsis that may be useful for applications in biotechnology directed towards increasing anthocyanin content in plants.

  17. Control of placental alkaline phosphatase gene expression in HeLa cells: induction of synthesis by prednisolone and sodium butyrate

    SciTech Connect

    Chou, J.Y.; Takahashi, S.

    1987-06-16

    HeLa S/sub 3/ cells produce an alkaline phosphatase indistinguishable from the enzyme from human term placenta. The phosphatase activity in these cells was induced by both prednisolone and sodium butyrate. Both agents stimulated de novo synthesis of the enzyme. The increase in phosphatase activity paralleled the increase in immunoactivity and biosynthesis of placental alkaline phosphatase. The fully processed phosphatase monomer in control, prednisolone-treated or butyrate-treated cells was a 64.5 K polypeptide, measured by both incorporation of L-(/sup 35/S)methionine into enzyme protein and active-site labeling. The 64.5K polypeptide was formed by the incorporation of additional N-acetylneuraminic acid moieties to a precursor polypeptide of 61.5K. However, this biosynthetic pathway was identified only in butyrate-treated cells. In prednisolone-treated cells, the processing of 61.5K to 64.5K monomer was accelerated, and the presence of the 61.5 precursor could only be detected by either neuraminidase or monensin treatment. Phosphatase mRNA which comigrated with the term placental alkaline phosphatase mRNA of 2.7 kilobases was induced in the presence of either prednisolone or butyrate. Alkaline phosphatase mRNA is untreated HeLa S/sub 3/ cells migrated slightly faster than the term placental alkaline phosphatase mRNA. Butyrate also induced a second still faster migrating alkaline phosphatase mRNA. Both prednisolone and butyrate increased the steady-state levels of placental alkaline phosphatase mRNA. The data indicate that the increase in phosphatase mRNA by prednisolone and butyrate resulted in the induction of alkaline phosphatase activity and biosynthesis in HeLa S/sub 3/ cells. Furthermore, both agents induced the expression of different alkaline phosphatase gene transcripts without altering its protein product.

  18. The relationship between the MMP system, adrenoceptors and phosphoprotein phosphatases

    PubMed Central

    Rietz, A; Spiers, JP

    2012-01-01

    The MMPs and their inhibitors [tissue inhibitor of MMPs (TIMPs) ] form the mainstay of extracellular matrix homeostasis. They are expressed in response to numerous stimuli including cytokines and GPCR activation. This review highlights the importance of adrenoceptors and phosphoprotein phosphatases (PPP) in regulating MMPs in the cardiovascular system, which may help explain some of the beneficial effects of targeting the adrenoceptor system in tissue remodelling and will establish emerging crosstalk between these three systems. Although α- and β-adrenoceptor activation increases MMP but decreases TIMP expression, MMPs are implicated in the growth stimulatory effects of adrenoceptor activation through transactivation of epidermal growth factor receptor. Furthermore, they have recently been found to catalyse the proteolysis of β-adrenoceptors and modulate vascular tone. While the mechanisms underpinning these effects are not well defined, reversible protein phosphorylation by kinases and phosphatases may be key. In particular, PPP (Ser/Thr phosphatases) are not only critical in resensitization and internalization of adrenoceptors but also modulate MMP expression. The interrelationship is complex as isoprenaline (ISO) inhibits okadaic acid [phosphoprotein phosphatase type 1/phosphoprotein phosphatase type 2A (PP2A) inhibitor]-mediated MMP expression. While this may be simply due to its ability to transiently increase PP2A activity, there is evidence for MMP-9 that ISO prevents okadaic acid-mediated expression of MMP-9 through a β-arrestin, NF-κB-dependent pathway, which is abolished by knock-down of PP2A. It is essential that crosstalk between MMPs, adrenoceptors and PPP are investigated further as it will provide important insight into how adrenoceptors modulate cardiovascular remodelling, and may identify new targets for pharmacological manipulation of the MMP system. PMID:22364165

  19. Awareness of Pap testing and factors associated with intent to undergo Pap testing by level of sexual experience in unmarried university students in Korea: results from an online survey

    PubMed Central

    2014-01-01

    Background Young and unmarried women have not been a target group for cervical cancer prevention in Korea. No previous studies have investigated the awareness of Pap testing, the intention to undergo Pap testing, or the factors associated with that intention, in this group of women. This information would be useful for an expansion in the focus of primary cervical cancer prevention. This study aimed to compare the awareness of Pap testing between groups of unmarried university students in Korea, and to investigate the factors associated with the intention to undergo Pap testing, by level of sexual experience. Methods A total of 475 unmarried university students who had never undergone a Pap test completed a web-based survey. Differences in awareness of the importance of the Pap test, confidence in Pap testing, intention to undergo the test, attitudes, subjective norms, perceived control, stigma, and shame by level of sexual experience were analysed using independent t-tests. Associations between measurement variables and intention to undergo Pap testing were analysed using correlation analysis. Variables yielding significant associations (p < 0.05) were included in a stepwise multiple regression model of intention to undergo Pap testing. Results Most participants perceived that the need for regular Pap testing was less important (score, 77.76) than other methods of cervical cancer prevention. They were not confident that is was an effective method of cervical cancer prevention for themselves (score, 59.56). There were differences in confidence in Pap testing and in the factors associated with intention to undergo Pap testing between sexually experienced and sexually inexperienced students. Regardless of level of sexual experience, the subjective norm was the most important predictor of intention to undergo Pap testing. Conclusions There was a low level of Pap screening awareness among the students. The factors associated with intention to undergo Pap testing

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

    PubMed

    Chen, Xi'en; Lü, Shumin; Zhang, Yalin

    2014-01-01

    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. PMID:24823652

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

    PubMed

    Chen, Xi'en; Lü, Shumin; Zhang, Yalin

    2014-01-01

    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.

  2. Methods to distinguish various types of protein phosphatase activity

    SciTech Connect

    Brautigan, D.L.; Shriner, C.L.

    1988-01-01

    To distinguish the action of protein Tyr(P) and protein Ser(P)/Thr(P) phosphatases on /sup 32/P-labeled phosphoproteins in subcellular fractions different inhibitors and activators are utilized. Comparison of the effects of added compounds provides a convenient, indirect method to characterize dephosphorylation reactions. Protein Tyr(P) phosphatases are specifically inhibited by micromolar Zn2+ or vanadate, and show maximal activity in the presence of EDTA. The other class of cellular phosphatases, specific for protein Ser(P) and Thr(P) residues, are inhibited by fluoride and EDTA. In this class of enzymes two major functional types can be distinguished: those sensitive to inhibition by the heat-stable protein inhibitor-2 and not stimulated by polycations, and those not sensitive to inhibition and stimulated by polycations. Preparation of /sup 32/P-labeled Tyr(P) and Ser(P) phosphoproteins also is presented for the direct measurement of phosphatase activities in preparations by the release of acid-soluble (/sup 32/P)phosphate.

  3. The RCN1-encoded A subunit of protein phosphatase 2A increases phosphatase activity in vivo

    NASA Technical Reports Server (NTRS)

    Deruere, J.; Jackson, K.; Garbers, C.; Soll, D.; Delong, A.; Evans, M. L. (Principal Investigator)

    1999-01-01

    Protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine-specific protein phosphatase, comprises a catalytic C subunit and two distinct regulatory subunits, A and B. The RCN1 gene encodes one of three A regulatory subunits in Arabidopsis thaliana. A T-DNA insertion mutation at this locus impairs root curling, seedling organ elongation and apical hypocotyl hook formation. We have used in vivo and in vitro assays to gauge the impact of the rcn1 mutation on PP2A activity in seedlings. PP2A activity is decreased in extracts from rcn1 mutant seedlings, and this decrease is not due to a reduction in catalytic subunit expression. Roots of mutant seedlings exhibit increased sensitivity to the phosphatase inhibitors okadaic acid and cantharidin in organ elongation assays. Shoots of dark-grown, but not light-grown seedlings also show increased inhibitor sensitivity. Furthermore, cantharidin treatment of wild-type seedlings mimics the rcn1 defect in root curling, root waving and hypocotyl hook formation assays. In roots of wild-type seedlings, RCN1 mRNA is expressed at high levels in root tips, and accumulates to lower levels in the pericycle and lateral root primordia. In shoots, RCN1 is expressed in the apical hook and the basal, rapidly elongating cells in etiolated hypocotyls, and in the shoot meristem and leaf primordia of light-grown seedlings. Our results show that the wild-type RCN1-encoded A subunit functions as a positive regulator of the PP2A holoenzyme, increasing activity towards substrates involved in organ elongation and differential cell elongation responses such as root curling.

  4. Psychosocial burden of abnormal pap smears among HIV-infected women at Chon Buri hospital, Thailand.

    PubMed

    Jayathunge, Mangala P H; Bowanwatanuwong, Chureeratana; Maek-A-nantawat, Wirach; Pitisuttithum, Benjaluck Phonratland Punnee

    2010-01-01

    This retrospective case-control study assessed the psychological burden of abnormal Pap smears, and their prevalence and characteristics among HIV-infected women attending an HIV clinic. Women with positive (n = 73) and negative Pap-smear results (n = 317) were assessed for psychosocial burden using 4 questionnaires: Psycho-Social Impact of Abnormal Pap Smears (PEAPS-Q), Hospital Anxiety and Depression Scale (HADS), Work Productivity and Impairment (WPAI) and the EURO-Qol Thermometer. The prevalence of pre-cervical cancer lesions in HIV infected woman was 17.5% (ASCUS 2.9%, LSIL 3.8%, HSIL 7.4%, SCC 1.7%, and atypical glandular cells including adenocarcinoma 1.7%). HIV infected women with abnormal Pap smears showed higher anxiety levels on the HADS questionnaire (p = 0.015); this had a significant effect on regular daily activities (p = 0.009) per the WPAI questionnaire compared to HIV positive women with normal Pap smear. Ever married HIV infected woman with an abnormal Pap smear had a significantly lower psychosocial burden using the PEAPS-Q questionnaire (p < 0.001). After adjusting for age and duration since last Pap smear, the education level of the patient was a strong predictor for anxiety. Patients, with a college education had significantly lower anxiety (p = 0.001, 95% CI -5.74 to -1.37) than those with lower or higher education. Women with HSIL were more anxious (p = 0.014, 95% CI 0.49 -4.39) than those with low grade or normal lesions.

  5. Comparison of PapType to Digene Hybrid Capture 2, Roche linear array, and Amplicor for detection of high-risk human papillomavirus genotypes in women with previous abnormal pap smears.

    PubMed

    Tabrizi, Sepehr N; Stevens, Matthew P; Khan, Zaheer A; Chow, Conan; Devitt, Martin A; Garland, Suzanne M

    2012-08-01

    PapType human papillomavirus (HPV) assay was compared to Hybrid Capture 2 (HC2), Amplicor (Amp), and Linear Array (LA) HPV tests in 894 women undergoing management for a high-grade Pap smear abnormality. The sensitivity in detection of underlying high-grade histological diagnosis by PapType was 90.3% and by HC2 was 79.8%, while by Amp and LA it was 92.4% and 91.6%, respectively. The specificities were 52.5%, 55.3%, 49.4%, and 51.7% for PapType, HC2, Amp, and LA, respectively.

  6. [Phosphoprotein phosphatase nonspecifically hydrolyzes CoA].

    PubMed

    Reziapkin, V I; Moiseenok, A G

    1988-01-01

    CoA hydrolysis was studied by a homogenous phosphoprotein phosphatase (EC 3.1 3.16) preparation from bovine spleen nuclei at pH 5.8. Phosphoprotein phosphatase catalyzed hydrolysis of the CoA 3'-phosphoester bond to form dephospho-CoA and Pi. The Km value for phosphoprotein phosphatase with CoA as substrate was 3.7 mM, the specific activity - 0.26 mmol Pi.min-1.mg-1. Phosphoprotein phosphatase did not essentially catalyze the calcium pantothenate hydrolysis (not more than 2% as compared with the CoA hydrolysis rate). PMID:2849829

  7. Protein phosphatases in pancreatic islets

    PubMed Central

    Ortsäter, Henrik; Grankvist, Nina; Honkanen, Richard E.; Sjöholm1, Åke

    2014-01-01

    The prevalence of diabetes is increasing rapidly world-wide. A cardinal feature of most forms of diabetes is the lack of insulin-producing capability, due to the loss of insulin-producing β-cells, impaired glucose-sensitive insulin secretion from the β-cell, or a combination thereof, the reasons for which largely remain elusive. Reversible phosphorylation is an important and versatile mechanism for regulating the biological activity of many intracellular proteins, which, in turn, controls a variety of cellular functions. For instance, significant changes in protein kinase activities and in protein phosphorylation patterns occur subsequent to stimulation of insulin release by glucose. Therefore, the molecular mechanisms regulating phosphorylation of proteins involved in the insulin secretory process by the β-cell have been extensively investigated. However, far less is known about the role and regulation of protein dephosphorylation by various protein phosphatases. Herein we review extant data implicating serine/threonine and tyrosine phosphatases in various aspects of healthy and diabetic islet biology, ranging from control of hormonal stimulus-secretion coupling to mitogenesis and apoptosis. PMID:24681827

  8. Automatic cervical cell segmentation and classification in Pap smears.

    PubMed

    Chankong, Thanatip; Theera-Umpon, Nipon; Auephanwiriyakul, Sansanee

    2014-02-01

    Cervical cancer is one of the leading causes of cancer death in females worldwide. The disease can be cured if the patient is diagnosed in the pre-cancerous lesion stage or earlier. A common physical examination technique widely used in the screening is Papanicolaou test or Pap test. In this research, a method for automatic cervical cancer cell segmentation and classification is proposed. A single-cell image is segmented into nucleus, cytoplasm, and background, using the fuzzy C-means (FCM) clustering technique. Four cell classes in the ERUDIT and LCH datasets, i.e., normal, low grade squamous intraepithelial lesion (LSIL), high grade squamous intraepithelial lesion (HSIL), and squamous cell carcinoma (SCC), are considered. The 2-class problem can be achieved by grouping the last 3 classes as one abnormal class. Whereas, the Herlev dataset consists of 7 cell classes, i.e., superficial squamous, intermediate squamous, columnar, mild dysplasia, moderate dysplasia, severe dysplasia, and carcinoma in situ. These 7 classes can also be grouped to form a 2-class problem. These 3 datasets were tested on 5 classifiers including Bayesian classifier, linear discriminant analysis (LDA), K-nearest neighbor (KNN), artificial neural networks (ANN), and support vector machine (SVM). For the ERUDIT dataset, ANN with 5 nucleus-based features yielded the accuracies of 96.20% and 97.83% on the 4-class and 2-class problems, respectively. For the Herlev dataset, ANN with 9 cell-based features yielded the accuracies of 93.78% and 99.27% for the 7-class and 2-class problems, respectively. For the LCH dataset, ANN with 9 cell-based features yielded the accuracies of 95.00% and 97.00% for the 4-class and 2-class problems, respectively. The segmentation and classification performances of the proposed method were compared with that of the hard C-means clustering and watershed technique. The results show that the proposed automatic approach yields very good performance and is better than its

  9. 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

    PubMed Central

    Boortz, Kayla A.; Syring, Kristen E.; Pound, Lynley D.; Wang, Yingda; Oeser, James K.; O’Brien, Richard M.

    2016-01-01

    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. PMID:27611587

  10. 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.

    PubMed

    Boortz, Kayla A; Syring, Kristen E; Pound, Lynley D; Wang, Yingda; Oeser, James K; O'Brien, Richard M

    2016-01-01

    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. PMID:27611587

  11. Carbon and Nitrogen Sources Influence Tricalcium Phosphate Solubilization and Extracellular Phosphatase Activity by Talaromyces flavus.

    PubMed

    Stefanoni Rubio, P J; Godoy, M S; Della Mónica, I F; Pettinari, M J; Godeas, A M; Scervino, J M

    2016-01-01

    The aim of this work was to study phosphate (P) solubilization (and the processes involved in this event) by Talaromyces flavus (BAFC 3125) as a function of carbon and/or nitrogen sources. P solubilization was evaluated in NBRIP media supplemented with different carbon (glucose, sorbitol, sucrose, and fructose) and nitrogen (L-asparagine, urea, ammonium sulfate (AS), and ammonium nitrate (AN) combinations. The highest P solubilization was related to the highest organic acid production (especially gluconic acid) and pH drop for those treatments where glucose was present. Also P solubilization was higher when an inorganic nitrogen source was supplemented to the media when compared to an organic one. Although not being present an organic P source, phosphatase activity was observed. This shows that P mineralization and P solubilization can occur simultaneously, and that P mineralization is not induced by the enzyme substrate. The combination that showed highest P solubilization was for AN-glucose. The highest acid phosphatase activity was for AS-fructose, while for alkaline phosphatase were for AS-fructose and AN-fructose. Acid phosphatase activity was higher than alkaline. P solubilization and phosphatase activity (acid and alkaline) were influenced by the different carbon-nitrogen combinations. A better understanding of phosphate-solubilizing fungi could bring a better use of soil P.

  12. Phosphorylated TandeMBP: A unique protein substrate for protein phosphatase assay.

    PubMed

    Sugiyama, Yasunori; Yamashita, Sho; Uezato, Yuuki; Senga, Yukako; Katayama, Syouichi; Goshima, Naoki; Shigeri, Yasushi; Sueyoshi, Noriyuki; Kameshita, Isamu

    2016-11-15

    To analyze a variety of protein phosphatases, we developed phosphorylated TandeMBP (P-TandeMBP), in which two different mouse myelin basic protein isoforms were fused in tandem, as a protein phosphatase substrate. P-TandeMBP was prepared efficiently in four steps: (1) phosphorylation of TandeMBP by a protein kinase mixture (Ca(2+)/calmodulin-dependent protein kinase Iδ, casein kinase 1δ, and extracellular signal-regulated kinase 2); (2) precipitation of both P-TandeMBP and protein kinases to remove ATP, Pi, and ADP; (3) acid extraction of P-TandeMBP with HCl to remove protein kinases; and (4) neutralization of the solution that contains P-TandeMBP with Tris. In combination with the malachite green assay, P-TandeMBP can be used to detect protein phosphatase activity without using radioactive materials. Moreover, P-TandeMBP served as an efficient substrate for PPM family phosphatases (PPM1A, PPM1B, PPM1D, PPM1F, PPM1G, PPM1H, PPM1K, and PPM1M) and PPP family phosphatase PP5. Various phosphatase activities were also detected with high sensitivity in gel filtration fractions from mouse brain using P-TandeMBP. These results indicate that P-TandeMBP might be a powerful tool for the detection of protein phosphatase activities. PMID:27565380

  13. Purification, characterization, and bioinformatics studies of phosphatidic acid phosphohydrolase from Lagenaria siceraria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phosphatidic acid phosphohydrolase (PAP), EC 3.1.3.4, is the penultimate step in the Kennedy pathway of triacyl glycerol (TAG) synthesis leading to the formation of diacyl glycerol (DAG), which is a key intermediate in TAG synthesis. We partially purified a soluble PAP from mid maturing seeds of bot...

  14. Phosphoinositide Phosphatases in Cell Biology and Disease

    PubMed Central

    Liu, Yang; Bankaitis, Vytas A.

    2010-01-01

    Phosphoinositides are essential signaling molecules linked to a diverse array of cellular processes in eukaryotic cells. The metabolic interconversions of these phospholipids are subject to exquisite spatial and temporal regulation executed by arrays of phosphatidylinositol (PtdIns) and phosphoinositide-metabolizing enzymes. These include PtdIns- and phosphoinositide-kinases that drive phosphoinositide synthesis, and phospholipases and phosphatases that regulate phosphoinositide degradation. In the past decade, phosphoinositide phosphatases have emerged as topics of particular interest. This interest is driven by the recent appreciation that these enzymes represent primary mechanisms for phosphoinositide degradation, and because of their ever-increasing connections with human diseases. Herein, we review the biochemical properties of six major phosphoinositide phosphatases, the functional involvements of these enzymes in regulating phosphoinositide metabolism, the pathologies that arise from functional derangements of individual phosphatases, and recent ideas concerning the involvements of phosphoinositide phosphatases in membrane traffic control. PMID:20043944

  15. Protein binding associated with exposure to fluorotelomer alcohols (FTOHs) and polyfluoroalkyl phosphate esters (PAPs) in rats.

    PubMed

    Rand, Amy A; Mabury, Scott A

    2014-02-18

    The biotransformation of fluorotelomer-based compounds such as fluorotelomer alcohols (FTOHs) and polyfluoroalkyl phosphate esters (PAPs) are sources of exposure to perfluorinated carboxylates (PFCAs), leading in part to the observation of significant concentrations of PFCAs in human blood. The biotransformation of FTOHs and PAPs yield intermediate metabolites that have been observed to covalently modify proteins. In the current investigation, the extent of covalent protein binding in Sprague-Dawley rats upon exposure to 8:2 FTOH and the 6:2 polyfluoroalkyl phosphate diester (6:2 diPAP) was quantified. The animals were administered a single dose of 8:2 FTOH or 6:2 diPAP at 100 mg/kg by oral gavage to monitor biotransformation and extent of protein binding within the liver, kidney, and plasma. In the 8:2 FTOH-dosed animals, perfluorooctanoate (PFOA) was produced as the primary PFCA, at 623.13 ± 59.3, 459.5 ± 171.8, and 397.3 ± 133.0 ng/g in the plasma, liver, and kidney, respectively. For the animals exposed to 6:2 diPAPs, perfluorohexanoate (PFHxA) was the primary PFCA produced, with maximum concentrations of 57.4 ± 6.5, 9.0 ± 1.2, and 25.3 ± 1.2 ng/g in the plasma, liver, and kidney, respectively. Protein binding was observed in the plasma, liver, and kidney after 8:2 FTOH and 6:2 diPAP exposure, with the most significant binding occurring in the liver (>100 nmol/g protein). This is the first study to link the exposure and in vivo biotransformation of fluorotelomer-based compounds to covalent protein binding.

  16. 3'-Phosphoadenosine 5'-phosphosulfate (PAPS) synthases, naturally fragile enzymes specifically stabilized by nucleotide binding.

    PubMed

    van den Boom, Johannes; Heider, Dominik; Martin, Stephen R; Pastore, Annalisa; Mueller, Jonathan W

    2012-05-18

    Activated sulfate in the form of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is needed for all sulfation reactions in eukaryotes with implications for the build-up of extracellular matrices, retroviral infection, protein modification, and steroid metabolism. In metazoans, PAPS is produced by bifunctional PAPS synthases (PAPSS). A major question in the field is why two human protein isoforms, PAPSS1 and -S2, are required that cannot complement for each other. We provide evidence that these two proteins differ markedly in their stability as observed by unfolding monitored by intrinsic tryptophan fluorescence as well as circular dichroism spectroscopy. At 37 °C, the half-life for unfolding of PAPSS2 is in the range of minutes, whereas PAPSS1 remains structurally intact. In the presence of their natural ligand, the nucleotide adenosine 5'-phosphosulfate (APS), PAPS synthase proteins are stabilized. Invertebrates only possess one PAPS synthase enzyme that we classified as PAPSS2-type by sequence-based machine learning techniques. To test this prediction, we cloned and expressed the PPS-1 protein from the roundworm Caenorhabditis elegans and also subjected this protein to thermal unfolding. With respect to thermal unfolding and the stabilization by APS, PPS-1 behaved like the unstable human PAPSS2 protein suggesting that the less stable protein is evolutionarily older. Finally, APS binding more than doubled the half-life for unfolding of PAPSS2 at physiological temperatures and effectively prevented its aggregation on a time scale of days. We propose that protein stability is a major contributing factor for PAPS availability that has not as yet been considered. Moreover, naturally occurring changes in APS concentrations may be sensed by changes in the conformation of PAPSS2.

  17. Phosphonate monoesters on a thiacalix[4]arene framework as potential inhibitors of protein tyrosine phosphatase 1B.

    PubMed

    Trush, Viacheslav V; Kharchenko, Sergiy G; Tanchuk, Vsevolod Yu; Kalchenko, Vitaly I; Vovk, Andriy I

    2015-09-01

    Monoester derivatives of thiacalix[4]arene tetrakis(methylphosphonic) acid were found to be capable of inhibiting protein tyrosine phosphatase 1B. In addition, these compounds can strongly bind to human serum albumin. PMID:26205135

  18. Specificity of a protein phosphatase inhibitor from rabbit skeletal muscle.

    PubMed Central

    Cohen, P; Nimmo, G A; Antoniw, J F

    1977-01-01

    A hear-stable protein, which is a specific inhibitor of protein phosphatase-III, was purified 700-fold from skeletal muscle by a procedure that involved heat-treatment at 95 degrees C, chromatography on DEAE-cellulose and gel filtration on Sephadex G-100. The final step completely resolved the protein phosphatase inhibitor from the protein inhibitor of cyclic AMP-dependent protein kinase. The phosphorylase phosphatase, beta-phosphorylase kinase phosphatase, glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities of protein phosphatase-III [Antoniw, J. F., Nimmo, H. G., Yeaman, S. J. & Cohen, P.(1977) Biochem.J. 162, 423-433] were inhibited in a very similar manner by the protein phosphatase inhibitor and at least 95% inhibition was observed at high concentrations of inhibitor. The two forms of protein phosphatase-III, termed IIIA and IIIB, were equally susceptible to the protein phosphatase inhibitor. The protein phosphatase inhibitor was at least 200 times less effective in inhibiting the activity of protein phosphatase-I and protein phosphatase-II. The high degree of specificity of the inhibitor for protein phosphatase-III was used to show that 90% of the phosphorylase phosphatase and glycogen synthase phosphatase activities measured in muscle extracts are catalysed by protein phosphatase-III. Protein phosphatase-III was tightly associated with the protein-glycogen complex that can be isolated from skeletal muscle, whereas the protein phosphatase inhibitor and protein phosphatase-II were not. The results provide further evidence that the enzyme that catalyses the dephosphorylation of the alpha-subunit of phosphorylase kinase (protein phosphatase-II) and the enzyme that catalyses the dephosphorylation of the beta-subunit of phosphorylase kinase (protein phosphatase-III) are distinct. The results suggest that the protein phosphatase inhibitor may be a useful probe for differentiating different classes of protein phosphatases in mammalian

  19. Characterization of the phosphatidylinositol-glycan membrane anchor of human placental alkaline phosphatase

    SciTech Connect

    Howard, A.D.; Berger, J.; Gerber, L.; Familletti, P.; Udenfriend, S.

    1987-09-01

    Placental alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) is a member of a diverse group of membrane proteins whose attachment to the lipid bilayer is mediated by a phosphatidylinositol-glycan. To investigate structural aspects of the glycolipid anchor, cultured WISH cells were used because, they produce the enzyme in abundant quantities. When cell suspensions were incubated with purified phosphatidylinositol-specific phospholipase C, most of the placental alkaline phosphatase was released from membranes in a hydrophilic form. On incubation of the cells with (/sup 14/C)ethanolamine, (/sup 14/C)myristic acid, or myo(/sup 3/H)inositol, each was incorporated into the phosphatase near the carboxyl terminus, showing that these components, which are found in other phosphatidylinositol membrane-linked proteins, are also present in placental alkaline phosphatase.

  20. Protein phosphatase 2A in stretch-induced endothelial cell proliferation

    NASA Technical Reports Server (NTRS)

    Murata, K.; Mills, I.; Sumpio, B. E.

    1996-01-01

    We previously proposed that activation of protein kinase C is a key mechanism for control of cell growth enhanced by cyclic strain [Rosales and Sumpio (1992): Surgery 112:459-466]. Here we examined protein phosphatase 1 and 2A activity in bovine aortic endothelial cells exposed to cyclic stain. Protein phosphatase 2A activity in the cytosol was decreased by 36.1% in response to cyclic strain for 60 min, whereas the activity in the membrane did not change. Treatment with low concentration (0.1 nM) of okadaic acid enhanced proliferation of both static and stretched endothelial cells in 10% fetal bovine serum. These data suggest that protein phosphatase 2A acts as a growth suppressor and cyclic strain may enhance cellular proliferation by inhibiting protein phosphatase 2A as well as stimulating protein kinase C.

  1. Partial purification and characterization of an enzyme from pea nuclei with protein tyrosine phosphatase activity.

    PubMed

    Guo, Y L; Roux, S J

    1995-01-01

    A pea (Pisum sativum L.) nuclear enzyme with protein tyrosine phosphatase activity has been partially purified and characterized. The enzyme has a molecular mass of 90 kD as judged by molecular sieve column chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Like animal protein tyrosine phosphatases it can be inhibited by low concentrations of molybdate and vanadate. It is also inhibited by heparin and spermine but not by either the acid phosphatase inhibitors citrate and tartrate or the protein serine/threonine phosphatase inhibitor okadaic acid. The enzyme does not require Ca2+, Mg2+, or Mn2+ for its activity but is stimulated by ethylenediaminetetraacetate and by ethyleneglycolbis(beta-aminoethyl ether)-N,N'-tetraacetic acid. It dephosphorylates phosphotyrosine residues on the four different 32P-tyrosine-labeled peptides tested but not the phosphoserine/threonine residues on casein and histone. Like some animal protein tyrosine phosphatases, it has a variable pH optimum depending on the substrate used: the optimum is 5.5 when the substrate is [32P]tyrosine-labeled lysozyme, but it is 7.0 when the substrate is [32P]tyrosine-labeled poly(glutamic acid, tyrosine). It has a Km of 4 microM when the lysozyme protein is used as a substrate.

  2. The characteristics and relevant factors of Pap smear test use for women with intellectual disabilities in Taiwan

    PubMed Central

    2014-01-01

    Background This study examines the Pap smear usage conditions and relevant influential factors for 18,204 women aged 30 years and above with intellectual disabilities, using nationwide data from 2008. Methods The research method of this study is secondary data analysis. The data was obtained from three nationwide databases from 2006 to 2008. This study employed descriptive statistics to analyze the use and rate of Pap smear testing by women with intellectual disabilities. Chi-square test was used to assess the correlation between Pap smear test usage and several variables. Logistic regression analysis was employed to explore the factors that influence Pap smear test usage. Results The results show that 4.83% (n =880) of women with intellectual disabilities underwent Pap smear tests. Pap smear test usage rates exhibit a declining trend with increases in age. Factors that significantly influence Pap smear test use include age, urbanization level of resident area, monthly salary, aboriginal status, marital status, existence of DM, severity of disability. Conclusions The women with intellectual disabilities had a low use rate of Pap smear test, which is significantly less than the 28.8% usage rate for the general population of women aged 30 years and above. PMID:24890828

  3. Characterization of the major phosphofructokinase-dephosphorylating protein phosphatases from Ascaris suum muscle.

    PubMed

    Daum, G; Schmid, B; MacKintosh, C; Cohen, P; Hofer, H W

    1992-07-13

    In contrast to the mammalian enzyme, PFK from the nematode Ascaris suum is activated following phosphorylation (Daum et al. (1986) Biochem. Biophys. Res. Commun. 139, 215-221) catalyzed by a cAMP-dependent protein kinase (Thalhofer et al. (1988) J. Biol. Chem. 263, 952-957). In the present report, we describe the characterization of the major PFK dephosphorylating phosphatases from Ascaris muscle. Two of these phosphatases exhibit apparent M(r) values of 174,000 and 126,000, respectively, and are dissociated to active 33 kDa proteins by ethanol precipitation. Denaturing electrophoresis of each of the enzyme preparations showed two bands of M(r) 33,000 and 63,000. The enzymes are classified as type 2A phosphatases according to their inhibition by subnanomolar concentrations of okadaic acid, the lack of inhibition by heat-stable phosphatase inhibitors 1 and 2, and their preference for the alpha- rather than for the beta-subunit of phosphorylase kinase. Like other type 2A phosphatases, they exhibit broad substrate specificities, are activated by divalent cations and polycations, and inhibited by fluoride, inorganic phosphate and adenine nucleotides. In addition, we have found that PFK is also dephosphorylated by an unusual protein phosphatase. This exhibits kinetic properties similar to type 2A protein phosphatases, but has a distinctly lower sensitivity towards inhibition by okadaic acid (IC50 approx. 20 nM). Partial purification of the enzyme provided evidence that it is composed of a 30 kDa catalytic subunit and probably two other subunits (molecular masses 66 and 72 kDa). The dephosphorylation of PFK by protein phosphatases is strongly inhibited by heparin. This effect, however, is substrate-specific and does not occur with Ascaris phosphorylase a. PMID:1321672

  4. Functional characterization of two members of histidine phosphatase superfamily in Mycobacterium tuberculosis

    PubMed Central

    2013-01-01

    Background Functional characterization of genes in important pathogenic bacteria such as Mycobacterium tuberculosis is imperative. Rv2135c, which was originally annotated as conserved hypothetical, has been found to be associated with membrane protein fractions of H37Rv strain. The gene appears to contain histidine phosphatase motif common to both cofactor-dependent phosphoglycerate mutases and acid phosphatases in the histidine phosphatase superfamily. The functions of many of the members of this superfamily are annotated based only on similarity to known proteins using automatic annotation systems, which can be erroneous. In addition, the motif at the N-terminal of Rv2135c is ‘RHA’ unlike ‘RHG’ found in most members of histidine phosphatase superfamily. These necessitate the need for its experimental characterization. The crystal structure of Rv0489, another member of the histidine phosphatase superfamily in M. tuberculosis, has been previously reported. However, its biochemical characteristics remain unknown. In this study, Rv2135c and Rv0489 from M. tuberculosis were cloned and expressed in Escherichia coli with 6 histidine residues tagged at the C terminal. Results Characterization of the purified recombinant proteins revealed that Rv0489 possesses phosphoglycerate mutase activity while Rv2135c does not. However Rv2135c has an acid phosphatase activity with optimal pH of 5.8. Kinetic parameters of Rv2135c and Rv0489 are studied, confirming that Rv0489 is a cofactor dependent phosphoglycerate mutase of M. tuberculosis. Additional characterization showed that Rv2135c exists as a tetramer while Rv0489 as a dimer in solution. Conclusion Most of the proteins orthologous to Rv2135c in other bacteria are annotated as phosphoglycerate mutases or hypothetical proteins. It is possible that they are actually phosphatases. Experimental characterization of a sufficiently large number of bacterial histidine phosphatases will increase the accuracy of the automatic

  5. Pap smear screening among Asian Pacific Islander women in a multisite community-based cancer screening program.

    PubMed

    Fernandez, Maria E; Lin, Jennifer; Leong-Wu, Cindy; Aday, Luann

    2009-04-01

    This study assessed screening completion rates (SCR) and sociodemographic factors associated with Pap test screening among previously nonadherent, foreign-born Asian Pacific Islander (API) women across four sites participating in a community-based cancer screening program called ENCOREplus. At intake, 926 out of 1,140 women were nonadherent to recommended Pap test screening guidelines. Most participants were age 51 and older, had a high school education or higher, had been in the U.S. less than a decade, had annual household incomes less than $10,000, and were uninsured. Women with limited resources were more likely to get a Pap test after participating in ENCOREplus. Women from the Glendale site were almost 18 times more likely to get a Pap test than API women in other sites. Over half of the women in Glendale reported that help getting low cost Pap tests and having translators available were instrumental in completing screening. PMID:19372282

  6. TPIP: a novel phosphoinositide 3-phosphatase.

    PubMed Central

    Walker, S M; Downes, C P; Leslie, N R

    2001-01-01

    The PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumour suppressor is a phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] 3-phosphatase that plays a critical role in regulating many cellular processes by antagonizing the phosphoinositide 3-kinase signalling pathway. We have identified and characterized two human homologues of PTEN, which differ with respect to their subcellular localization and lipid phosphatase activities. The previously cloned, but uncharacterized, TPTE (transmembrane phosphatase with tensin homology) is localized to the plasma membrane, but lacks detectable phosphoinositide 3-phosphatase activity. TPIP (TPTE and PTEN homologous inositol lipid phosphatase) is a novel phosphatase that occurs in several differentially spliced forms of which two, TPIP alpha and TPIP beta, appear to be functionally distinct. TPIP alpha displays similar phosphoinositide 3-phosphatase activity compared with PTEN against PtdIns(3,4,5)P(3), PtdIns(3,5)P(2), PtdIns(3,4)P(2) and PtdIns(3)P, has N-terminal transmembrane domains and appears to be localized on the endoplasmic reticulum. This is unusual as most signalling-lipid-metabolizing enzymes are not integral membrane proteins. TPIP beta, however, lacks detectable phosphatase activity and is cytosolic. TPIP has a wider tissue distribution than the testis-specific TPTE, with specific splice variants being expressed in testis, brain and stomach. TPTE and TPIP do not appear to be functional orthologues of the Golgi-localized and more distantly related murine PTEN2. We suggest that TPIP alpha plays a role in regulating phosphoinositide signalling on the endoplasmic reticulum, and might also represent a tumour suppressor and functional homologue of PTEN in some tissues. PMID:11716755

  7. LOW KNOWLEDGE OF CERVICAL CANCER AND CERVICAL PAP SMEARS AMONG WOMEN IN PERU, AND THEIR IDEAS OF HOW THIS COULD BE IMPROVED

    PubMed Central

    PAZ-SOLDÁN, VALERIE A.; NUSSBAUM, LAUREN; BAYER, ANGELA M.; CABRERA, LILIA

    2013-01-01

    Estimates of the percentage of women who have had Pap smears in Peru vary between 7% and 43%. This study explores what women know about cervical cancer and Pap smears, as well as their barriers to obtaining Pap smears. Focus group discussions (FGD) were conducted with a total of 177 women in four Peruvian cities. Discussions reveal that most women did not know what causes cervical cancer. Most women did not know the purpose of Pap smears, although knowledge about Pap smears was higher than knowledge about cervical cancer. Fear, embarrassment, and lack of knowledge were the main barriers identified for not getting Pap smears. Programs and policies aiming to increase Pap smear coverage must start by educating women on cervical cancer and its prevention in order to improve women's perceptions about the screening test and increase Pap smear seeking behaviors in the long term. PMID:21988870

  8. [Technology and disciplinary fields: cytotechnicians and implementation of the Pap test in Brazil].

    PubMed

    Teixeira, Luiz Antonio; Pumar, Leticia

    2014-01-01

    The implementation of the Pap test as a primary technology in the control of cervical cancer in Brazil was the result of choices, agreements and disputes among certain professional groups, including physicians from various specialisations, pharmacists, biologists, biomedical scientists and cytotechnologists. The first part of the paper describes the process of formulating Brazil's first screening campaigns using the Pap smear, and the subsequent emergence of the profession of cytotechnology, whose practitioners interpret this test. Second, based on questions raised by international historiography in the field of science and technology, we explore in detail how the adoption of the Pap smear transpired within the Brazilian context, focussing on the debates among the various professional groups with an interest in the suitability of the test and on the relationships between the public and private healthcare sectors. We show that the professional career of cytotechnologists and the way in which the Pap smear has been implemented as a central technology for cervical cancer screening in Brazil have been shaped by the conflicting views of this technology held by different disciplines as well as by the relationship between these disciplines and labour market dynamics.

  9. Process improvement of pap smear tracking in a women's medicine center clinic in residency training.

    PubMed

    Calhoun, Byron C; Goode, Jeff; Simmons, Kathy

    2011-11-01

    Application of Six-Sigma methodology and Change Acceleration Process (CAP)/Work Out (WO) tools to track pap smear results in an outpatient clinic in a hospital-based residency-training program. Observational study of impact of changes obtained through application of Six-Sigma principles in clinic process with particular attention to prevention of sentinel events. Using cohort analysis and applying Six-Sigma principles to an interactive electronic medical record Soarian workflow engine, we designed a system of timely accession and reporting of pap smear and pathology results. We compared manual processes from January 1, 2007 to February 28, 2008 to automated processes from March 1, 2008 to December 31, 2009. Using the Six-Sigma principles, CAP/WO tools, including "voice of the customer" and team focused approach, no outlier events went untracked. Applying the Soarian workflow engine to track prescribed 7 day turnaround time for completion, we identified 148 pap results in 3,936, 3 non-gynecological results in 15, and 41 surgical results in 246. We applied Six-Sigma principles to an outpatient clinic facilitating an interdisciplinary team approach to improve the clinic's reporting system. Through focused problem assessment, verification of process, and validation of outcomes, we improved patient care for pap smears and critical pathology.

  10. Anal Pap Screening for HIV-infected Men Who Have Sex With Men: Practice Improvement.

    PubMed

    Welbeck, Monique

    2016-01-01

    HIV-infected men who have sex with men (MSM) have the highest rates of anal dysplasia and anal cancer when compared to HIV-uninfected MSM and when compared to HIV-infected heterosexual men and women. Despite significantly increasing rates of anal dysplasia and anal cancer in HIV-infected MSM, in many settings, no standard protocol is in place to screen for anal dysplasia in this high-risk group. A practice improvement project was conducted at a primary care health center to educate the HIV health care team about anal Pap screening in an effort to increase provider knowledge and rates of anal Pap screening performed as part of primary comprehensive care for HIV-infected MSM. Increased health care provider knowledge of anal Pap screening within this setting resulted in increased anal Pap screening for HIV-infected MSM. Routine screening leads to improved surveillance and treatment of precancerous lesions, decreasing morbidity and mortality in HIV-infected MSM.

  11. Frequency of pap and pil operons in Escherichia coli strains associated with urinary infections.

    PubMed

    Perugini, M R; Vidotto, M C

    1996-03-01

    Strains of E. coli isolated from patients with urinary tract infection were examined for P and type 1 adhesin production by colony hybridization with pap and pil operons. The P pili probe detected 45 (46.4%) of the total of 97 strains studied and the type 1 pili probe detected 83 (85.6%). The pap operon was detected in 39 (53.4%) of 73 strains isolated from urine of patients with urinary disease and in 6 (25.0%) of 24 strains isolated from feces of healthy individuals employed as controls (P = 0.029), and the pil operon was detected in 67 (91.8%) of the urinary strains and in 16 (66.6%) of the fecal strains (P = 0.007). Our data did not show significant differences in frequency of P pili among isolates from pyelonephritis (78.5%), cystitis (45.8%) and asymptomatic bacteriuria (54.5%). Type 1 pili were not associated with the different types of infection; the frequency of these pili was 100% in pyelonephritis and in asymptomatic bacteriuria, and 87.5% in cystitis. The incidence of pap operon in strains isolated from pyelonephritis and from asymptomatic bacteriuria was higher in 11- to 40-year old women. These data show a high frequency of pap and pil operons among uropathogenic strains of E. coli, which seems to be an important factor in the development of urinary infection.

  12. Mammography and Pap test screening among low-income foreign-born Hispanic women in USA.

    PubMed

    Fernández, M A; Tortolero-Luna, G; Gold, R S

    1998-01-01

    Little is known about the factors influencing screening among low-income Hispanic women particularly among recent immigrants. A sample of 148 low-income, low-literate, foreign-born Hispanic women residing in the Washington DC metropolitan area participated in the study. The mean age of the sample was 46.2 (SD=11.5), 84% reported annual household incomes ($15,000. All women were Spanish speakers and had low acculturation levels. Ninety six percent had reported having a Pap smear, but 24% were not in compliance with recommended screening (Pap test within the last 3 years). Among women 40 and older, 62% had received a mammogram, but only 33% were compliant with age appropriate recommended mammography screening guidelines. Women in this study had more misconceptions about cancer than Hispanics in other studies. Multivariate logistic models for correlates of Pap test and mammography screening behavior indicate that factors such as fear of the screening test, embarrassment, and lack of knowledge influenced screening behavior. In conclusion, women in this study had lower rates of mammography screening than non-Hispanic women and lower rates of compliance with recommended Mammography and Pap test screening guidelines.

  13. Pap Tests Every Three Years: Cost-Effective in the Long Run?

    ERIC Educational Resources Information Center

    Amschler, Denise Hope

    1983-01-01

    The American Cancer Society's guidelines, recommending that having Pap tests at three-year intervals is safe for many women, are questioned. Dangers to women with a high risk of cervical cancer, problems with faulty test results, and other gynecological problems that may be detected during pelvic examinations are discussed. (PP)

  14. Expression of Pokeweed Antiviral Protein in Transgenic Plants Induces Virus Resistance in Grafted Wild-Type Plants Independently of Salicylic Acid Accumulation and Pathogenesis-Related Protein Synthesis.

    PubMed Central

    Smirnov, S.; Shulaev, V.; Tumer, N. E.

    1997-01-01

    Pokeweed antiviral protein (PAP), a 29-kD protein isolated from Phytolacca americana, inhibits translation by catalytically removing a specific adenine residue from the large rRNA of the 60S subunit of eukaryotic ribosomes. Transgenic tobacco (Nicotiana tabacum) plants expressing PAP or a variant (PAP-v) were shown to be resistant to a broad spectrum of plant viruses. Expression of PAP-v in transgenic plants induces synthesis of pathogenesis-related proteins and a very weak (<2-fold) increase in salicylic acid levels. Using reciprocal grafting experiments, we demonstrate here that transgenic tobacco rootstocks expressing PAP-v induce resistance to tobacco mosaic virus infection in both N. tabacum NN and nn scions. Increased resistance to potato virus X was also observed in N. tabacum nn scions grafted on transgenic rootstocks. PAP expression was not detected in the wild-type scions or rootstocks that showed virus resistance, nor was there any increase in salicylic acid levels or pathogenesis-related protein synthesis. Grafting experiments with transgenic plants expressing an inactive PAP mutant demonstrated that an intact active site of PAP is necessary for induction of virus resistance in wild-type scions. These results indicate that enzymatic activity of PAP is responsible for generating a signal that renders wild-type scions resistant to virus infection in the absence of increased salicylic acid levels and pathogenesis-related protein synthesis. PMID:12223762

  15. CstF-64 and 3'-UTR cis-element determine Star-PAP specificity for target mRNA selection by excluding PAPα.

    PubMed

    Kandala, Divya T; Mohan, Nimmy; A, Vivekanand; A P, Sudheesh; G, Reshmi; Laishram, Rakesh S

    2016-01-29

    Almost all eukaryotic mRNAs have a poly (A) tail at the 3'-end. Canonical PAPs (PAPα/γ) polyadenylate nuclear pre-mRNAs. The recent identification of the non-canonical Star-PAP revealed specificity of nuclear PAPs for pre-mRNAs, yet the mechanism how Star-PAP selects mRNA targets is still elusive. Moreover, how Star-PAP target mRNAs having canonical AAUAAA signal are not regulated by PAPα is unclear. We investigate specificity mechanisms of Star-PAP that selects pre-mRNA targets for polyadenylation. Star-PAP assembles distinct 3'-end processing complex and controls pre-mRNAs independent of PAPα. We identified a Star-PAP recognition nucleotide motif and showed that suboptimal DSE on Star-PAP target pre-mRNA 3'-UTRs inhibit CstF-64 binding, thus preventing PAPα recruitment onto it. Altering 3'-UTR cis-elements on a Star-PAP target pre-mRNA can switch the regulatory PAP from Star-PAP to PAPα. Our results suggest a mechanism of poly (A) site selection that has potential implication on the regulation of alternative polyadenylation.

  16. CstF-64 and 3′-UTR cis-element determine Star-PAP specificity for target mRNA selection by excluding PAPα

    PubMed Central

    Kandala, Divya T.; Mohan, Nimmy; A, Vivekanand; AP, Sudheesh; G, Reshmi; Laishram, Rakesh S.

    2016-01-01

    Almost all eukaryotic mRNAs have a poly (A) tail at the 3′-end. Canonical PAPs (PAPα/γ) polyadenylate nuclear pre-mRNAs. The recent identification of the non-canonical Star-PAP revealed specificity of nuclear PAPs for pre-mRNAs, yet the mechanism how Star-PAP selects mRNA targets is still elusive. Moreover, how Star-PAP target mRNAs having canonical AAUAAA signal are not regulated by PAPα is unclear. We investigate specificity mechanisms of Star-PAP that selects pre-mRNA targets for polyadenylation. Star-PAP assembles distinct 3′-end processing complex and controls pre-mRNAs independent of PAPα. We identified a Star-PAP recognition nucleotide motif and showed that suboptimal DSE on Star-PAP target pre-mRNA 3′-UTRs inhibit CstF-64 binding, thus preventing PAPα recruitment onto it. Altering 3′-UTR cis-elements on a Star-PAP target pre-mRNA can switch the regulatory PAP from Star-PAP to PAPα. Our results suggest a mechanism of poly (A) site selection that has potential implication on the regulation of alternative polyadenylation. PMID:26496945

  17. Comparison of PCR, culturing and Pap smear microscopy for accurate diagnosis of genital Actinomyces.

    PubMed

    Kaya, Dilek; Demirezen, Şayeste; Hasçelik, Gülşen; Gülmez Kivanç, Dolunay; Beksaç, Mehmet Sinan

    2013-05-01

    Members of the genus Actinomyces, Gram-positive, non-spore-forming anaerobic bacteria, are normal inhabitants of the mucosal surfaces of the oral, gastrointestinal and genital tracts. Identification of these bacteria using conventional methods is generally difficult because of their complex transport and growth requirements and their fastidious and slow-growing nature. However, in recent years, the advancement of molecular techniques has provided much improved identification and differentiation of closely related Actinomyces species. The aim of the present study was to evaluate the efficacy of the PCR technique in the diagnosis of genital Actinomyces in comparison with culturing and Papanicolaou (Pap) smear microscopy. Multiple sampling was conducted from 200 women using smear microscopy, culturing and PCR. Cyto-brushes were smeared on glass slides and stained using the routine Pap technique. Culturing was performed from a sterile swab, and Actinomyces were determined using the BBL Crystal ANR ID kit. PCR was performed from a second swab, and the Actinomyces type was determined using type-specific primers designed in our laboratory. Only one vaginal fluid sample (0.5%) revealed Actinomyces-like organisms on Pap smear examination. Actinomyces were detected in nine samples (4.5%) using the BBL Crystal ANR ID kit. Using PCR, eight samples (4%) were found positive for Actinomyces. No specimens that gave positive results by Pap smear microscopy and culturing could be confirmed by PCR. Pap smear microscopy and culturing were both found to have zero sensitivity for Actinomyces. PCR appears to be a sensitive and reliable diagnostic method for the detection of Actinomyces, which are difficult to cultivate from genital samples. PCR can be used for diagnostic confirmation in cases diagnosed by conventional methods, to prevent false-positive results.

  18. Abnormal Pap Smear and Diagnosis of High-Grade Vaginal Intraepithelial Neoplasia

    PubMed Central

    Sopracordevole, Francesco; Mancioli, Francesca; Clemente, Nicolò; De Piero, Giovanni; Buttignol, Monica; Giorda, Giorgio; Ciavattini, Andrea

    2015-01-01

    Abstract The aim of this study was to analyze the correlation between the first diagnosis of high-grade Vaginal Intraepithelial Neoplasia (HG-VaIN: VaIN 2–VaIN 3) and the cytological abnormalities on the referral pap smear. All the women with histological diagnosis of HG-VaIN consecutively referred to the Gynecological Oncology Unit of the Aviano National Cancer Institute (Aviano, Italy) from January 1991 to April 2014 and with a pap smear performed in the 3 months before the diagnosis were considered, and an observational cohort study was performed. A total of 87 women with diagnosis of HG-VaIN were identified. Major cytological abnormalities (HSIL and ASC-H) on the referral pap smear were significantly more frequent than lesser abnormalities (ASC-US and LSIL) in postmenopausal women (64.9% vs 36.7%, P = 0.02) and in women with a previous diagnosis of HPV-related cervical preinvasive or invasive lesions (70.5% vs 39.5%, P = 0.01). Diagnosis of VaIN 3 was preceded by major cytological abnormalities in most of the cases (72.7% vs 27.3%, P < 0.001). The diagnosis of HG-VaIN can be preceded by different abnormalities on referral pap smear. Major abnormalities are usually reported in postmenopausal women and in women with previous cervical HPV-related disease. However, ASC-US or LSIL do not exclude HG-VaIN, especially VaIN2. An accurate examination of the whole vaginal walls (or vaginal vault) must be performed in all the women who underwent colposcopy for an abnormal pap smear, and a biopsy of all suspicious areas is mandatory. PMID:26496321

  19. Abnormal Pap Smear and Diagnosis of High-Grade Vaginal Intraepithelial Neoplasia: A Retrospective Cohort Study.

    PubMed

    Sopracordevole, Francesco; Mancioli, Francesca; Clemente, Nicolò; De Piero, Giovanni; Buttignol, Monica; Giorda, Giorgio; Ciavattini, Andrea

    2015-10-01

    The aim of this study was to analyze the correlation between the first diagnosis of high-grade Vaginal Intraepithelial Neoplasia (HG-VaIN: VaIN 2-VaIN 3) and the cytological abnormalities on the referral pap smear.All the women with histological diagnosis of HG-VaIN consecutively referred to the Gynecological Oncology Unit of the Aviano National Cancer Institute (Aviano, Italy) from January 1991 to April 2014 and with a pap smear performed in the 3 months before the diagnosis were considered, and an observational cohort study was performed.A total of 87 women with diagnosis of HG-VaIN were identified. Major cytological abnormalities (HSIL and ASC-H) on the referral pap smear were significantly more frequent than lesser abnormalities (ASC-US and LSIL) in postmenopausal women (64.9% vs 36.7%, P = 0.02) and in women with a previous diagnosis of HPV-related cervical preinvasive or invasive lesions (70.5% vs 39.5%, P = 0.01). Diagnosis of VaIN 3 was preceded by major cytological abnormalities in most of the cases (72.7% vs 27.3%, P < 0.001).The diagnosis of HG-VaIN can be preceded by different abnormalities on referral pap smear. Major abnormalities are usually reported in postmenopausal women and in women with previous cervical HPV-related disease. However, ASC-US or LSIL do not exclude HG-VaIN, especially VaIN2. An accurate examination of the whole vaginal walls (or vaginal vault) must be performed in all the women who underwent colposcopy for an abnormal pap smear, and a biopsy of all suspicious areas is mandatory. PMID:26496321

  20. Factors associated with Mexican women's familiarity with the purpose of the Pap test.

    PubMed

    Nájera Aguilar, P; Lazcano Ponce, E C; de Ruíz, P A; Ramírez Sánchez, T; Cantoral Uriza, L; Hernández Avila, M

    1996-12-01

    Use of health services is usually associated with a variety of factors, including the socioeconomic characteristics of the users, their familiarity with the usefulness of the services provided, and the acceptability and accessibility of those services. To study the factors associated with women's familiarity with the Pap test, a population-based study was carried out in Mexico City and two rural areas in the state of Oaxaca by means of household interviews. The sample consisted of 4208 women 15 to 49 years of age. Univariate, bivariate, and multivariate analyses were done using unconditional logistic regression; the independent variables were access to social security health services, age, education, housing quality, and place of residence (urban or rural); the dependent variable was the interview subject's familiarity with the purpose of the Pap test. The results were expressed as odds ratios with 95% confidence intervals. It was found that 41.5% of the women surveyed did not know the purpose of the Pap test, and that within this latter group, 97% had never had one. Factors found to be associated with not knowing the test's purpose were lack of access to the social security health services (OR = 1.9; 95% CI: 1.5-2.3); illiteracy (OR = 36.1; 95% CI: 17.9-72.7); and low socioeconomic level (OR = 2.9; 95% CI: 2.3-3.7). Also, rural dwellers had less familiarity with the Pap test than urban dwellers (OR = 0.5; 95% CI: 0.4-0.7). These results highlight the need to develop strategies for making the benefits of the Pap test known, bearing in mind the socioeconomic and cultural diversity of the populations involved.

  1. Elucidation of the active conformation of the APS-kinase domain of human PAPS synthetase 1.

    PubMed

    Sekulic, Nikolina; Dietrich, Kristen; Paarmann, Ingo; Ort, Stephan; Konrad, Manfred; Lavie, Arnon

    2007-03-23

    Bifunctional human PAPS synthetase (PAPSS) catalyzes, in a two-step process, the formation of the activated sulfate carrier 3'-phosphoadenosine 5'-phosphosulfate (PAPS). The first reaction involves the formation of the 5'-adenosine phosphosulfate (APS) intermediate from ATP and inorganic sulfate. APS is then further phosphorylated on its 3'-hydroxyl group by an additional ATP molecule to generate PAPS. The former reaction is catalyzed by the ATP-sulfurylase domain and the latter by the APS-kinase domain. Here, we report the structure of the APS-kinase domain of PAPSS isoform 1 (PAPSS1) representing the Michaelis complex with the products ADP-Mg and PAPS. This structure provides a rare glimpse of the active conformation of an enzyme catalyzing phosphoryl transfer without resorting to substrate analogs, inactivating mutations, or catalytically non-competent conditions. Our structure shows the interactions involved in the binding of the magnesium ion and PAPS, thereby revealing residues critical for catalysis. The essential magnesium ion is observed bridging the phosphate groups of the products. This function of the metal ion is made possible by the DGDN-loop changing its conformation from that previously reported, and identifies these loop residues unambiguously as a Walker B motif. Furthermore, the second aspartate residue of this motif is the likely candidate for initiating nucleophilic attack on the ATP gamma-phosphate group by abstracting the proton from the 3'-hydroxyl group of the substrate APS. We report the structure of the APS-kinase domain of human PAPSS1 in complex with two APS molecules, demonstrating the ability of the ATP/ADP-binding site to bind APS. Both structures reveal extended N termini that approach the active site of the neighboring monomer. Together, these results significantly increase our understandings of how catalysis is achieved by APS-kinase.

  2. Ratiometric electrochemical detection of alkaline phosphatase.

    PubMed

    Goggins, Sean; Naz, Christophe; Marsh, Barrie J; Frost, Christopher G

    2015-01-11

    A novel ferrocene-derived substrate for the ratiometric electrochemical detection of alkaline phosphatase (ALP) was designed and synthesised. It was demonstrated to be an excellent electrochemical substrate for the ALP-labelled enzyme-linked immunosorbent assay (ELISA).

  3. Uncoupling of 3'-phosphatase and 5'-kinase functions in budding yeast. Characterization of Saccharomyces cerevisiae DNA 3'-phosphatase (TPP1).

    PubMed

    Vance, J R; Wilson, T E

    2001-05-01

    Polynucleotide kinase is a bifunctional enzyme containing both DNA 3'-phosphatase and 5'-kinase activities seemingly suited to the coupled repair of single-strand nicks in which the phosphate has remained with the 3'-base. We show that the yeast Saccharomyces cerevisiae is able to repair transformed dephosphorylated linear plasmids by non-homologous end joining with considerable efficiency independently of the end-processing polymerase Pol4p. Homology searches and biochemical assays did not reveal a 5'-kinase that would account for this repair, however. Instead, open reading frame YMR156C (here named TPP1) is shown to encode only a polynucleotide kinase-type 3'-phosphatase. Tpp1p bears extensive similarity to the ancient L-2-halo-acid dehalogenase and DDDD phosphohydrolase superfamilies, but is specific for double-stranded DNA. It is present at high levels in cell extracts in a functional form and so does not represent a pseudogene. Moreover, the phosphatase-only nature of this gene is shared by Saccharomyces mikatae YMR156C and Arabidopsis thaliana K15M2.3. Repair of 3'-phosphate and 5'-hydroxyl lesions is thus uncoupled in budding yeast as compared with metazoans. Repair of transformed dephosphorylated plasmids, and 5'-hydroxyl blocking lesions more generally, likely proceeds by a cycle of base removal and resynthesis.

  4. Multiple Functions of the Eya Phosphotyrosine Phosphatase

    PubMed Central

    2015-01-01

    Eyes absent (Eya), a protein conserved from plants to humans and best characterized as a transcriptional coactivator, is also the prototype for a novel class of eukaryotic aspartyl protein tyrosine phosphatases. This minireview discusses recent breakthroughs in elucidating the substrates and cellular events regulated by Eya's tyrosine phosphatase function and highlights some of the complexities, new questions, and surprises that have emerged from efforts to understand how Eya's unusual multifunctionality influences developmental regulation and signaling. PMID:26667035

  5. Protein-tyrosine phosphatases: biological function, structural characteristics, and mechanism of catalysis.

    PubMed

    Zhang, Z Y

    1998-01-01

    The protein-tyrosine phosphatases (PTPases) superfamily consists of tyrosine-specific phosphatases, dual specificity phosphatases, and the low-molecular-weight phosphatases. They are modulators of signal transduction pathways that regulate numerous cell functions. Malfunction of PTPases have been linked to a number of oncogenic and metabolic disease states, and PTPases are also employed by microbes and viruses for pathogenicity. There is little sequence similarity among the three subfamilies of phosphatases. Yet, three-dimensional structural data show that they share similar conserved structural elements, namely, the phosphate-binding loop encompassing the PTPase signature motif (H/V)C(X)5R(S/T) and an essential general acid/base Asp residue on a surface loop. Biochemical experiments demonstrate that phosphatases in the PTPase superfamily utilize a common mechanism for catalysis going through a covalent thiophosphate intermediate that involves the nucleophilic Cys residue in the PTPase signature motif. The transition states for phosphoenzyme intermediate formation and hydrolysis are dissociative in nature and are similar to those of the solution phosphate monoester reactions. One strategy used by these phosphatases for transition state stabilization is to neutralize the developing negative charge in the leaving group. A conformational change that is restricted to the movement of a flexible loop occurs during the catalytic cycle of the PTPases. However, the relationship between loop dynamics and enzyme catalysis remains to be established. The nature and identity of the rate-limiting step in the PTPase catalyzed reaction requires further investigation and may be dependent on the specific experimental conditions such as temperature, pH, buffer, and substrate used. In-depth kinetic and structural analysis of a representative number of phosphatases from each group of the PTPase superfamily will most likely continue to yield insightful mechanistic information that may be

  6. Assessing the Biological Activity of the Glucan Phosphatase Laforin.

    PubMed

    Romá-Mateo, Carlos; Raththagala, Madushi; Gentry, Mathew S; Sanz, Pascual

    2016-01-01

    Glucan phosphatases are a recently discovered family of enzymes that dephosphorylate either starch or glycogen and are essential for proper starch metabolism in plants and glycogen metabolism in humans. Mutations in the gene encoding the only human glucan phosphatase, laforin, result in the fatal, neurodegenerative, epilepsy known as Lafora disease. Here, we describe phosphatase assays to assess both generic laforin phosphatase activity and laforin's unique glycogen phosphatase activity. PMID:27514803

  7. Saccharomyces cerevisiae protein phosphatase 2A performs an essential cellular function and is encoded by two genes.

    PubMed Central

    Sneddon, A A; Cohen, P T; Stark, M J

    1990-01-01

    Two genes (PPH21 and PPH22) encoding the yeast homologues of protein serine-threonine phosphatase 2A have been cloned from a Saccharomyces cerevisiae genomic library using a rabbit protein phosphatase 2A cDNA as a hybridization probe. The PPH genes are genetically linked on chromosome IV and are predicted to encode polypeptides each with 74% amino acid sequence identity to rabbit type 2A protein phosphatase, indicating once again the extraordinarily high degree of sequence conservation shown by protein-phosphatases from different species. The two PPH genes show less than 10% amino acid sequence divergence from each other and while disruption of either PPH gene alone is without any major effect, the double disruption is lethal. This indicates that protein phosphatase 2A activity is an essential cellular function in yeast. Measurement of type 2A protein phosphatase activity in yeast strains lacking one or other of the genes indicates that they account for most, if not all, protein phosphatase 2A activity in the cell. Images Fig. 5. PMID:2176150

  8. Phosphate solubilization potential and phosphatase activity of rhizospheric trichoderma spp.

    PubMed

    Anil, Kapri; Lakshmi, Tewari

    2010-07-01

    Trichoderma sp., a well known biological control agent against several phytopathogens, was tested for its phosphate (P) solubilizing potential. Fourteen strains of Trichoderma sp. were isolated from the forest tree rhizospheres of pinus, deodar, bamboo, guava and oak on Trichoderma selective medium. The isolates were tested for their in-vitro P-solubilizing potential using National Botanical Research Institute Phosphate (NBRIP) broth containing tricalcium phosphate (TCP) as the sole P source, and compared with a standard culture of T. harzianum. All the cultures were found to solubilize TCP but with varying potential. The isolate DRT-1 showed maximum amount of soluble phosphate (404.07 εg.ml(-1)), followed by the standard culture of T. harzianum (386.42 εg.ml(-1)) after 96 h of incubation at 30±1(0)C. Extra-cellular acid and alkaline phosphatases of the fungus were induced only in the presence of insoluble phosphorus source (TCP). High extra-cellular alkaline phosphatase activity was recorded for the isolate DRT-1 (14.50 U.ml(-1)) followed by the standard culture (13.41 U.ml(-1)) at 72h. The cultures showed much lesser acid phosphatase activities. Under glasshouse conditions, Trichoderma sp. inoculation increased chickpea (Cicer arietinum) growth parameters including shoot length, root length, fresh and dry weight of shoot as well as roots, in P-deficient soil containing only bound phosphate (TCP). Shoot weight was increased by 23% and 33% by inoculation with the isolate DRT-1 in the soil amended with 100 and 200 mg TCP kg(-1) soil, respectively, after 60 d of sowing. The study explores high P-solubilizing potential of Trichoderma sp., which can be exploited for the solubilization of fixed phosphates present in the soil, thereby enhancing soil fertility and plant growth.

  9. Phosphate-solubility and phosphatase activity in Gangetic alluvial soil as influenced by organophosphate insecticide residues.

    PubMed

    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.

  10. Phosphate-solubility and phosphatase activity in Gangetic alluvial soil as influenced by organophosphate insecticide residues.

    PubMed

    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. PMID:26720809

  11. A conserved serine residue is required for the phosphatidate phosphatase activity but not the transcriptional coactivator functions of lipin-1 and lipin-2.

    PubMed

    Donkor, Jimmy; Zhang, Peixiang; Wong, Samantha; O'Loughlin, Lauren; Dewald, Jay; Kok, Bernard P C; Brindley, David N; Reue, Karen

    2009-10-23

    Mammalian lipins (lipin-1, lipin-2, and lipin-3) are Mg2+-dependent phosphatidate phosphatase (PAP) enzymes, which catalyze a key reaction in glycerolipid biosynthesis. Lipin-1 also functions as a transcriptional coactivator in conjunction with members of the peroxisome proliferator-activated receptor family. An S734L mutation in LPIN2 causes Majeed syndrome, a human inflammatory disorder characterized by recurrent osteomyelitis, fever, dyserythropoietic anemia, and cutaneous inflammation. Here we demonstrate that mutation of the equivalent serine in mouse lipin-1 and lipin-2 to leucine or aspartate abolishes PAP activity but does not impair lipin association with microsomal membranes, the major site of glycerolipid synthesis. We also determined that lipin-2 has transcriptional coactivator activity for peroxisome proliferator-activated receptor-response elements similar to lipin-1 and that this activity is not affected by mutating the conserved serine. Therefore, our results indicate that the symptoms of the Majeed syndrome result from a loss of lipin-2 PAP activity. To characterize sites of lipin-2 action, we detected lipin-2 expression by in situ hybridization on whole mouse sections and by quantitative PCR of tissues relevant to Majeed syndrome. Lipin-2 was most prominently expressed in liver, where levels were much higher than lipin-1, and also in kidney, lung, gastrointestinal tract, and specific regions of the brain. Lipin-2 was also expressed in circulating red blood cells and sites of lymphopoiesis (bone marrow, thymus, and spleen). These results raise the possibility that the loss of lipin-2 PAP activity in erythrocytes and lymphocytes may contribute to the anemia and inflammation phenotypes observed in Majeed syndrome patients.

  12. Immunochemical Assays and Nucleic-Acid Detection Techniques for Clinical Diagnosis of Prostate Cancer

    PubMed Central

    Kanyong, Prosper; Rawlinson, Sean; Davis, James

    2016-01-01

    Prostate cancer (PCa) is a significant cause of morbidity and mortality and the most common cancer in men in Europe, North America, and some parts of Africa. The established methods for detecting PCa are normally based on tests using Prostate Specific Antigen (PSA) in blood, Prostate cancer antigen 3 (PCA3) in urine and tissue Alpha-methylacyl-CoA racemase (AMACR) as tumour markers in patient samples. Prior to the introduction of PSA in clinics, prostatic acid phosphatase (PAP) was the most widely used biomarker. An early diagnosis of PCa through the detection of these biomarkers requires the availability of simple, reliable, cost-effective and robust techniques. Immunoassays and nucleic acid detection techniques have experienced unprecedented growth in recent years and seem to be the most promising analytical tools. This growth has been driven in part by the surge in demand for near-patient-testing systems in clinical diagnosis. This article reviews immunochemical assays, and nucleic-acid detection techniques that have been used to clinically diagnose PCa. PMID:26958088

  13. Bacterial-like PPP protein phosphatases

    PubMed Central

    Kerk, David; Uhrig, R Glen; Moorhead, Greg B

    2013-01-01

    Reversible phosphorylation is a widespread modification affecting the great majority of eukaryotic cellular proteins, and whose effects influence nearly every cellular function. Protein phosphatases are increasingly recognized as exquisitely regulated contributors to these changes. The PPP (phosphoprotein phosphatase) family comprises enzymes, which catalyze dephosphorylation at serine and threonine residues. Nearly a decade ago, “bacterial-like” enzymes were recognized with similarity to proteins from various bacterial sources: SLPs (Shewanella-like phosphatases), RLPHs (Rhizobiales-like phosphatases), and ALPHs (ApaH-like phosphatases). A recent article from our laboratory appearing in Plant Physiology characterizes their extensive organismal distribution, abundance in plant species, predicted subcellular localization, motif organization, and sequence evolution. One salient observation is the distinct evolutionary trajectory followed by SLP genes and proteins in photosynthetic eukaryotes vs. animal and plant pathogens derived from photosynthetic ancestors. We present here a closer look at sequence data that emphasizes the distinctiveness of pathogen SLP proteins and that suggests that they might represent novel drug targets. A second observation in our original report was the high degree of similarity between the bacterial-like PPPs of eukaryotes and closely related proteins of the “eukaryotic-like” phyla Myxococcales and Planctomycetes. We here reflect on the possible implications of these observations and their importance for future research. PMID:24675170

  14. HvPap-1 C1A Protease and HvCPI-2 Cystatin Contribute to Barley Grain Filling and Germination.

    PubMed

    Diaz-Mendoza, Mercedes; Dominguez-Figueroa, Jose D; Velasco-Arroyo, Blanca; Cambra, Ines; Gonzalez-Melendi, Pablo; Lopez-Gonzalvez, Angeles; Garcia, Antonia; Hensel, Goetz; Kumlehn, Jochen; Diaz, Isabel; Martinez, Manuel

    2016-04-01

    Proteolysis is an essential process throughout the mobilization of storage proteins in barley (Hordeum vulgare) grains during germination. It involves numerous types of enzymes, with C1A Cys proteases the most abundant key players. Manipulation of the proteolytic machinery is a potential way to enhance grain yield and quality, and it could influence the mobilization of storage compounds along germination. Transgenic barley plants silencing or over-expressing the cathepsin F-like HvPap-1 Cys protease show differential accumulation of storage molecules such as starch, proteins, and free amino acids in the grain. It is particularly striking that the HvPap-1 artificial microRNA lines phenotype show a drastic delay in the grain germination process. Alterations to the proteolytic activities in the over-expressing and knock-down grains associated with changes in the level of expression of several C1A peptidases were also detected. Similarly, down-regulating cystatin Icy-2, one of the proteinaceous inhibitors of the cathepsin F-like protease, also has important effects on grain filling. However, the ultimate physiological influence of manipulating a peptidase or an inhibitor cannot be always predicted, since the plant tries to compensate the modified proteolytic effects by modulating the expression of some other peptidases or their inhibitors.

  15. Living with uncertainty: equivocal Pap test results and the evolution of ASC terminology.

    PubMed

    Howell, Lydia Pleotis; Wilton, Maaya; Bishop, John; Afify, Alaa

    2010-03-01

    Communication of equivocal findings and their significance has been a significant challenge related to Pap testing throughout its history. Terminology to report these findings has changed considerably to accommodate the changes in understanding of cervical neoplasia, and to accommodate new management strategies, tests, and technologies. This article reviews the evolution of terminology for equivocal Pap test findings from the original Papanicolaou classification to the current the Bethesda System 2001 atypical squamous cells terminology, the implication and use of these terms, and the changing landscape of cervical neoplasia screening, which prompted these terminology changes. Emerging issues related to improving risk stratification through the introduction of additional terms and the impact of human papillomavirus testing may alter terminology of equivocal findings in the future.

  16. Seleninate in Place of Phosphate: Irreversible Inhibition of Protein Tyrosine Phosphatases

    SciTech Connect

    Abdo, Mohannad; Liu, Sijiu; Zhou, Bo; Walls, Chad D.; Wu, Li; Knapp, Spencer; Zhang, Zhong-Yin

    2009-02-16

    A homotyrosine based seleninic acid irreversibly inhibits protein tyrosine phosphatases by forming a covalent selenosulfide linkage with the active site cysteine sulfhydryl specifically. The details of the event are revealed by model synthetic studies and by kinetic, mass spectrometric, and crystallographic characterization.

  17. Highly sensitive detection of alkaline phosphatase using molecular beacon probes based on enzymatic polymerization.

    PubMed

    Ma, Changbei

    2012-06-01

    We have developed a new methodology for highly sensitive alkaline phosphatase assay using molecular beacon probes. No incubation step is needed to obtain a limit of detection for ALP of 2×10(-16) M. Furthermore, ALP inhibition by the inhibitor okadaic acid is shown, demonstrating the potential for high-throughput screening for inhibitors.

  18. A Community–Academic Partnership to Increase Pap Testing in Appalachian Kentucky

    PubMed Central

    Collins, Tom; Stradtman, Lindsay R.; Vanderpool, Robin C.; Neace, Deborah R.; Cooper, Karen D.

    2016-01-01

    Introduction Appalachian Kentucky is recognized for elevated rates of cervical cancer, which exerts an undue burden in this medically underserved region. The purpose of this study was to examine the impact of an academic–community partnership, specifically a regional health department and a CDC Prevention Research Center, in conducting outreach aimed at improving Pap testing rates and examining barriers among under-screened women in Appalachian Kentucky. Differences between women with abnormal and negative results were also examined. Methods The Prevention Research Center provided technical assistance to the district health department that, in turn, hosted “Women’s Health Day” events at county health departments, providing incentives to women who had never had a Pap test or those who had not received one in at least 3 years to receive guideline-recommended screening. Results From 2011 to 2014, 317 women were screened for cervical cancer; data were analyzed in 2014. The mean age was 42.1 (SD=13.6) years. More than half (54.5%) of the sample reported high school as their highest level of education, and 57.7% had an annual household income of <$25,000. The most commonly reported barriers to Pap testing were cost (28.4%) and lack of a perceived need for screening (25.6%). Approximately one in five (21.7%) women received abnormal Pap results. Conclusions As a result of this community–academic public health partnership and its shared resources, Appalachian Kentucky women received needed cervical cancer screening and appropriate follow-up for abnormal results, thereby increasing this population’s compliance with guideline-recommended screening. PMID:26190807

  19. Cuando uno de tus papás tiene cáncer

    Cancer.gov

    Sugerencias para saber qué decir a los amigos, cómo superar la tensión y en dónde encontrar apoyo; también, información sobre el cáncer y sobre los tratamientos de cáncer, para jóvenes que tienen a uno de sus papás con cáncer.

  20. Pap testing among Vietnamese women: health care system and physician factors.

    PubMed

    Taylor, Victoria M; Schwartz, Stephen M; Yasui, Yutaka; Burke, Nancy; Shu, Jianfen; Lam, D Hien; Jackson, J Carey

    2004-12-01

    Cervical cancer occurs more frequently among Vietnamese Americans than women of any other race/ethnicity. In addition, previous studies in California have documented low Papanicolaou (Pap) testing rates in Vietnamese communities. This study focused on health care system factors and physician characteristics associated with recent cervical cancer screening among Vietnamese women. A population-based survey was conducted in Seattle during 2002. In-person interviews were conducted by bilingual, bicultural female survey workers. The survey response rate was 82% and 518 women were included in the analysis. Seventy-four percent of the respondents reported having been screened for cervical cancer on at least one occasion, and 64% reported a Pap smear within the previous 2 years. Women with a regular doctor were more likely to have been recently screened than those without a regular doctor (OR = 2.33, 95% CI = 1.45-3.74). Among those with a regular doctor, having a male physician, receiving care at a private doctor's office (rather than a community, hospital, or multi-specialty clinic), and concern about the cost of health care were independently associated with lower screening rates. Physician ethnicity was not associated with recent Pap smear receipt. The findings support targeted interventions for Vietnamese women without a regular physician and private doctors' offices that serve Vietnamese Americans. The availability of low cost screening services should be publicized in Vietnamese communities.

  1. Allosteric signalling in the outer membrane translocation domain of PapC usher

    PubMed Central

    Farabella, Irene; Pham, Thieng; Henderson, Nadine S; Geibel, Sebastian; Phan, Gilles; Thanassi, David G; Delcour, Anne H; Waksman, Gabriel; Topf, Maya

    2014-01-01

    PapC ushers are outer-membrane proteins enabling assembly and secretion of P pili in uropathogenic E. coli. Their translocation domain is a large β-barrel occluded by a plug domain, which is displaced to allow the translocation of pilus subunits across the membrane. Previous studies suggested that this gating mechanism is controlled by a β-hairpin and an α-helix. To investigate the role of these elements in allosteric signal communication, we developed a method combining evolutionary and molecular dynamics studies of the native translocation domain and mutants lacking the β-hairpin and/or the α-helix. Analysis of a hybrid residue interaction network suggests distinct regions (residue ‘communities’) within the translocation domain (especially around β12–β14) linking these elements, thereby modulating PapC gating. Antibiotic sensitivity and electrophysiology experiments on a set of alanine-substitution mutants confirmed functional roles for four of these communities. This study illuminates the gating mechanism of PapC ushers and its importance in maintaining outer-membrane permeability. DOI: http://dx.doi.org/10.7554/eLife.03532.001 PMID:25271373

  2. [Factors associated with failure to take a Pap smear test among Quilombola women].

    PubMed

    Oliveira, Márcio Vasconcelos; Guimarães, Mark Drew Crosland; França, Elisabeth Barboza

    2014-11-01

    The objective of this study was to analyze factors associated with failure to take a Pap smear test among quilombola women living in Vitória da Conquista in the state of Bahia. A cross-sectional, population-based study was conducted with women aged 18 to 64. Descriptive analysis and univariate and multivariate analysis using a multinomial logistic model was applied. Women who had never performed the test or had not had one for over three years were compared separately from those who were examined within the preceding three years. Of the 348 women included in the analysis, 27.3%, reported never having a Pap smear test. The following factors were independently associated with never having taken the test: age of 18 to 29 years and 50 to 59 years; lack of education; not having a partner; seeking care in health-related units/establishments other than their place of residence; and having a clinical breast examination three or more years ago or never having one. The findings indicate a need for reflection in order to combat factors that are associated with not having a Pap smear test among quilombola women, since it is important to implement actions for the prevention of cervical cancer.

  3. Allosteric signalling in the outer membrane translocation domain of PapC usher.

    PubMed

    Farabella, Irene; Pham, Thieng; Henderson, Nadine S; Geibel, Sebastian; Phan, Gilles; Thanassi, David G; Delcour, Anne H; Waksman, Gabriel; Topf, Maya

    2014-01-01

    PapC ushers are outer-membrane proteins enabling assembly and secretion of P pili in uropathogenic E. coli. Their translocation domain is a large β-barrel occluded by a plug domain, which is displaced to allow the translocation of pilus subunits across the membrane. Previous studies suggested that this gating mechanism is controlled by a β-hairpin and an α-helix. To investigate the role of these elements in allosteric signal communication, we developed a method combining evolutionary and molecular dynamics studies of the native translocation domain and mutants lacking the β-hairpin and/or the α-helix. Analysis of a hybrid residue interaction network suggests distinct regions (residue 'communities') within the translocation domain (especially around β12-β14) linking these elements, thereby modulating PapC gating. Antibiotic sensitivity and electrophysiology experiments on a set of alanine-substitution mutants confirmed functional roles for four of these communities. This study illuminates the gating mechanism of PapC ushers and its importance in maintaining outer-membrane permeability.

  4. A ubiquitous plant housekeeping gene, PAP, encodes a major protein component of bell pepper chromoplasts.

    PubMed

    Pozueta-Romero, J; Rafia, F; Houlné, G; Cheniclet, C; Carde, J P; Schantz, M L; Schantz, R

    1997-11-01

    We have isolated a cDNA (PAP) corresponding to a single nuclear gene that encodes an approximately 30-kD major protein of bell pepper (Capsicum annuum L.) fruit chromoplasts. RNA and protein analyses revealed that, although at a low level, this gene is also expressed in every organ of the plant, the amount of the corresponding transcript and protein dramatically increasing in the latter stages of fruit development. Western-blot and immunocytochemical analyses of purified chloroplasts from leaves and fruits and of chromoplasts from red fruits showed that the encoded protein is the major component of plastoglobules and fibrils and is localized on the outer surface of these lipid structures. Analyses of PAP in plants belonging to different taxa revealed that it is expressed and highly conserved in both monocotyledonous and dicotyledonous plants. The presence of the protein in plastids not differentiating into chromoplasts indicates that PAP is expressed irrespective of the ontogeny of various plastid lines. In light of our results and since the encoded protein, identical to that previously named ChrB or fibrillin, is present in plastoglobules from several species and accumulates in the fibrils of bell pepper chromoplast, we propose to designate it as a plastid-lipid-associated protein.

  5. Frequency and patterns of abnormal Pap smears in Sudanese women with infertility: What are the perspectives?

    PubMed Central

    Almobarak, Ahmed O; Elhoweris, Mohammed H; Nour, Hilmi M; Ahmed, Mohammed Abd Allah M; Omer, Al-Fadhil Alobeed; Ahmed, Mohamed H

    2013-01-01

    Background: Pelvic inflammatory disease is one of the major causes of infertility in females. Also, women with infertility are at high-risk of developing cervical intraepithelial lesions or even carcinoma. Aim: To assess the prevalence and patterns of epithelial cell abnormalities in cervical smears in Sudanese infertile and fertile women. Materials and Methods: The present study included 200 cases (women with infertility) of age group 20-60 years who had routine pap smear. A total of 700 fertile women constituted the control group. Results: Among the 200 infertile women, 93.5% reported as negative for intraepithelial lesion or malignancy (NILM), and 6.5% were epithelial cell abnormalities (atypical squamous cells of undetermined significance and above). In the control group 96.6% of the smears reported as NILM and 3.4% as epithelial cell abnormalities. The percentage of abnormal pap smears in the infertile women was significantly higher when compared with the control group. Conclusions: Epithelial cell abnormalities are significantly higher in women with infertility as compared with fertile women. Importantly, inflammatory smears were reported two times more than in the controls. We recommend pap smear as a routine practice for all women assessed for infertility problems. Further studies are necessary to evaluate the incidence of human papilloma virus infections in infertile women with abnormal cervical cytology. PMID:23833398

  6. Inhibition of sucrose phosphatase by sucrose

    PubMed Central

    Hawker, J. S.

    1967-01-01

    1. Partially purified sucrose phosphatase from immature stem tissue of sugarcane is inhibited by sucrose. The enzyme was also inhibited by maltose, melezitose and 6-kestose but not by eight other sugars, including glucose and fructose. 2. The relative effectiveness of sucrose, maltose and melezitose as inhibitors is different for sucrose phosphatase from different plants. 3. The inhibition of the sugar-cane enzyme by sucrose was shown to be partially competitive. The Ki for sucrose is about 10mm. 4. Melezitose is also a partially competitive inhibitor of the enzyme but the inhibition by maltose is probably mixed. 5. The possibility that sucrose controls both the rate of accumulation of sucrose in stems of sugar-cane and sucrose synthesis in leaves by inhibiting sucrose phosphatase is discussed. PMID:4291490

  7. A specific sucrose phosphatase from plant tissues

    PubMed Central

    Hawker, J. S.; Hatch, M. D.

    1966-01-01

    1. A phosphatase that hydrolyses sucrose phosphate (phosphorylated at the 6-position of fructose) was isolated from sugar-cane stem and carrot roots. With partially purified preparations fructose 6-phosphate, glucose 6-phosphate, fructose 1-phosphate, glucose 1-phosphate and fructose 1,6-diphosphate are hydrolysed at between 0 and 2% of the rate for sucrose phosphate. 2. The activity of the enzyme is increased fourfold by the addition of Mg2+ ions and inhibited by EDTA, fluoride, inorganic phosphate, pyrophosphate, Ca2+ and Mn2+ ions. Sucrose (50mm) reduces activity by 60%. 3. The enzyme exhibits maximum activity between pH6·4 and 6·7. The Michaelis constant for sucrose phosphate is between 0·13 and 0·17mm. 4. At least some of the specific phosphatase is associated with particles having the sedimentation properties of mitochondria. 5. A similar phosphatase appears to be present in several other plant species. PMID:4290548

  8. Promoting Uranium Immobilization by the Activities of Microbial Phosphatases

    SciTech Connect

    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

  9. The Oxidative Stress Responsive Transcription Factor Pap1 Confers DNA Damage Resistance on Checkpoint-Deficient Fission Yeast Cells

    PubMed Central

    Belfield, Carrie; Queenan, Craig; Rao, Hui; Kitamura, Kenji; Walworth, Nancy C.

    2014-01-01

    Eukaryotic cells invoke mechanisms to promote survival when confronted with cellular stress or damage to the genome. The protein kinase Chk1 is an integral and conserved component of the DNA damage response pathway. Mutation or inhibition of Chk1 results in mitotic death when cells are exposed to DNA damage. Oxidative stress activates a pathway that results in nuclear accumulation of the bZIP transcription factor Pap1. We report the novel finding that fission yeast Pap1 confers resistance to drug- and non-drug-induced DNA damage even when the DNA damage checkpoint is compromised. Multi-copy expression of Pap1 restores growth to chk1-deficient cells exposed to camptothecin or hydroxyurea. Unexpectedly, increased Pap1 expression also promotes survival of chk1-deficient cells with mutations in genes encoding DNA ligase (cdc17) or DNA polymerase δ (cdc6), but not DNA replication initiation mutants. The ability of Pap1 to confer resistance to DNA damage was not specific to chk1 mutants, as it also improved survival of rad1- and rad9-deficient cells in the presence of CPT. To confer resistance to DNA damage Pap1 must localize to the nucleus and be transcriptionally active. PMID:24587136

  10. Protein kinase A-mediated phosphorylation of Pah1p phosphatidate phosphatase functions in conjunction with the Pho85p-Pho80p and Cdc28p-cyclin B kinases to regulate lipid synthesis in yeast.

    PubMed

    Su, Wen-Min; Han, Gil-Soo; Casciano, Jessica; Carman, George M

    2012-09-28

    Pah1p, which functions as phosphatidate phosphatase (PAP) in the yeast Saccharomyces cerevisiae, plays a crucial role in lipid homeostasis by controlling the relative proportions of its substrate phosphatidate and its product diacylglycerol. The diacylglycerol produced by PAP is used for the synthesis of triacylglycerol as well as for the synthesis of phospholipids via the Kennedy pathway. Pah1p is a highly phosphorylated protein in vivo and has been previously shown to be phosphorylated by the protein kinases Pho85p-Pho80p and Cdc28p-cyclin B. In this work, we showed that Pah1p was a bona fide substrate for protein kinase A, and we identified by mass spectrometry and mutagenesis that Ser-10, Ser-677, Ser-773, Ser-774, and Ser-788 were the target sites of phosphorylation. Protein kinase A-mediated phosphorylation of Pah1p inhibited its PAP activity by decreasing catalytic efficiency, and the inhibitory effect was primarily conferred by phosphorylation at Ser-10. Analysis of the S10A and S10D mutations (mimicking dephosphorylation and phosphorylation, respectively), alone or in combination with the seven alanine (7A) mutations of the sites phosphorylated by Pho85p-Pho80p and Cdc28p-cyclin B, indicated that phosphorylation at Ser-10 stabilized Pah1p abundance and inhibited its association with membranes, PAP activity, and triacylglycerol synthesis. The S10A mutation enhanced the physiological effects imparted by the 7A mutations, whereas the S10D mutations attenuated the effects of the 7A mutations. These data indicated that the protein kinase A-mediated phosphorylation of Ser-10 functions in conjunction with the phosphorylations mediated by Pho85p-Pho80p and Cdc28p-cyclin B and that phospho-Ser-10 should be dephosphorylated for proper PAP function.

  11. Evaluation and significance of hyperchromatic crowded groups (HCG) in liquid-based paps

    PubMed Central

    Chivukula, Mamatha; Austin, R Marshall; Shidham, Vinod B

    2007-01-01

    Objective Hyperchromatic crowded groups (HCG), a term first introduced into the cytology literature by DeMay in 1995, are commonly observed in Pap tests and may rarely be associated with serious but difficult to interpret lesions. In this study, we specifically defined HCG as dark crowded cell groups with more than 15 cells which can be identified at 10× screening magnification. Methods We evaluated consecutive liquid-based (Surepath) Pap tests from 601 women (age 17–74 years, mean age 29.4 yrs) and observed HCG in 477 cases. In all 477 HCG cases, Pap tests were found to be satisfactory and to contain an endocervical sample. HCG were easily detectible at 10× screening magnification (size up to 400 um, mean 239.5 um) and ranged from 1 to 50 (mean 19.5) per Pap slide. Results HCG predominantly represented 3-Dimensional groups of endocervical cells with some nuclear overlap (379/477 – 79%), reactive endocervical cells with relatively prominent nucleoli and some nuclear crowding (29/477 – 6%), clusters of inflammatory cells (25/477 – 5.2%), parabasal cells (22/477 – 4.6%), endometrial cells (1/477 – 0.2%). Epithelial cell abnormalities (ECA) were present in only 21 of 477 cases (4.6%). 18 of 21 women with HCG-associated ECA were less than 40 years old; only 3 were =/> 40 years. HCG-associated final abnormal Pap test interpretations were as follows: ASCUS (6/21 – 28%), LSIL (12/21 – 57%), ASC-H (2/21 – 9.5%), and HSIL/CIN2-3 (3/21 – 14%). The association of HCG with ECA was statistically significant (p = 0.0174. chi-square test). In patients with ECA, biopsy results were available in 10 cases, and 4 cases of biopsy-proven CIN2/3 were detected. Among these four cases, HCG in the Pap tests, in retrospect represented the lesional high grade cells in three cases (one HSIL case and two ASC-H cases). Interestingly, none of the 124 cases without HCG were found to have an epithelial cell abnormality. Conclusion We conclude: a. HCG are observed in a high

  12. Genome-Wide Analysis of PAPS1-Dependent Polyadenylation Identifies Novel Roles for Functionally Specialized Poly(A) Polymerases in Arabidopsis thaliana

    PubMed Central

    Ramming, Anna; Kolbe, Benjamin; Vi, Son Lang; Bispo, Cláudia; Becker, Jörg D.; de Moor, Cornelia; Lenhard, Michael

    2015-01-01

    The poly(A) tail at 3’ ends of eukaryotic mRNAs promotes their nuclear export, stability and translational efficiency, and changes in its length can strongly impact gene expression. The Arabidopsis thaliana genome encodes three canonical nuclear poly(A) polymerases, PAPS1, PAPS2 and PAPS4. As shown by their different mutant phenotypes, these three isoforms are functionally specialized, with PAPS1 modifying organ growth and suppressing a constitutive immune response. However, the molecular basis of this specialization is largely unknown. Here, we have estimated poly(A)-tail lengths on a transcriptome-wide scale in wild-type and paps1 mutants. This identified categories of genes as particularly strongly affected in paps1 mutants, including genes encoding ribosomal proteins, cell-division factors and major carbohydrate-metabolic proteins. We experimentally verified two novel functions of PAPS1 in ribosome biogenesis and redox homoeostasis that were predicted based on the analysis of poly(A)-tail length changes in paps1 mutants. When overlaying the PAPS1-dependent effects observed here with coexpression analysis based on independent microarray data, the two clusters of transcripts that are most closely coexpressed with PAPS1 show the strongest change in poly(A)-tail length and transcript abundance in paps1 mutants in our analysis. This suggests that their coexpression reflects at least partly the preferential polyadenylation of these transcripts by PAPS1 versus the other two poly(A)-polymerase isoforms. Thus, transcriptome-wide analysis of poly(A)-tail lengths identifies novel biological functions and likely target transcripts for polyadenylation by PAPS1. Data integration with large-scale co-expression data suggests that changes in the relative activities of the isoforms are used as an endogenous mechanism to co-ordinately modulate plant gene expression. PMID:26305463

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

    PubMed

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

    2015-01-01

    Extracellular phosphatase production by phytoplankton was investigated in the moderately eutrophic Lipno reservoir, Czech Republic during 2009 and 2010. We hypothesized that production of extracellular phosphatases is an additional mechanism of phosphorus acquisition enabling producers to survive rather than to dominate the phytoplankton. Hence, we examined the relationship between light availability and phosphatase production, as light plays an important role in polymictic environments. Bulk phosphatase activity was measured using a common fluorometric assay, and the production of phosphatases was studied using the Fluorescently Labelled Enzyme Activity technique, which enabled direct microscopic detection of phosphatase-positive cells. In total, 29 taxa of phytoplankton were identified during both years. Only 17 taxa from the total number of 29 showed production of extracellular phosphatases. Species dominating the phytoplankton rarely produced extracellular phosphatases. In contrast, taxa exhibiting phosphatase activity were present in low biomass in the phytoplankton assemblage. Moreover, there was a significant relationship between the proportion of phosphatase positive species in samples and the Z(eu):Z(mix) ratio (a proxy of light availability). A laboratory experiment with different light intensities confirmed the influence of light on production of phosphatases. Our seasonal study confirmed that extracellular phosphatase production is common in low-abundance populations but not in dominant taxa of the phytoplankton. It also suggested the importance of sufficient light conditions for the production of extracellular phosphatases.

  14. [Appearance of phosphatase reticular cells in the spleen parenchyme of normal and tumor-bearing mice].

    PubMed

    Catayée, G; Chalet, M

    1976-01-01

    The splenic mouse parenchyma presents 3 reticular cells types revealed by histo-enzymatic technics: the phagocytic cells show a strong phosphatasic acid activity, the endothelial cells possess a phosphatasic alcalin or adenosine-triphosphatasic reaction, the perithelial cells are 5' nucleotidasic. These different cells are distributed by forming specific topographic structures in the splenic tissue. The phosphatasic alcalin reticular cells seem to be, with their distribution, characteristic elements of mouse spleen. Indeed the modifications in tumoral animal interest chiefly this cell category. In this case, the reticular cells form a deep and large membrane between marginal zone and perivascular lymphoid sheath of the white pulp. These different reticular cells probably react for the defense system of the animal.

  15. Phosphatase activity against neurofilament proteins from bovine spinal cord: effect of aluminium and neuropsychoactive drugs.

    PubMed

    Shetty, K T; Veeranna; Guru, S C

    1992-03-16

    Protein phosphatase activity associated with neurofilament (NF) rich (Triton X-100 insoluble) fraction was extracted and partially characterised by using known inhibitors of protein phosphatases such as vanadate and fluoride. Protein phosphatase activity was demonstrated with reference to the dephosphorylation of endogenous substrate, NF protein and exogenous protein substrates, casein and phosvitin. Phosphoamino acids and beta-glycerophosphate were found to be poor substrates. Further, new observations have been made regarding the in vitro inhibitory effect of aluminium and the differential effects of some of the neuropsychoactive drugs. The findings could possibly lead to studies explaining the biochemical basis of aluminium induced neurotoxicity as well as the side effects associated with the long term medication of neuropsychoactive drugs. PMID:1320755

  16. [Inhibition of alkaline phosphatase I of Pichia guilliermondii yeast in vitro and in vivo].

    PubMed

    Sibirnyi, A A; Shavlovskii, G M

    1978-01-01

    The rate of p-nitrophenyl phosphate and flavin mononucleotide (FMN) hydrolysis by the partially purified preparation of alkaline phosphatase I of Pichia guilliermondii flavinogenic yeast was studied as affected by different substrates and inorganic ions. Their Km was established to be 2.0 X 10(-4) m and 2.5 X 10(-4) M, respectively. Dephosphorylation of p-nitrophenylphosphate and FMN was inhibited competitively by beta-glycerophosphate (Ki = 3.1 X 10(-3) M, respectively). The presence of inorganic phosphate ions in the reaction mixture decreases or removes inhibition of these compounds hydrolysis by other substrates of alkaline phosphatase I. The activity of alkaline phosphatase I increases in the presence of Mg2+ and was strongly inhibited in the presence of Be2+, Cu2+, Zn2+, Cd2+ and inorganic phosphate, the mixture of Be2+ and F- being the most effective. This mixture inhibited the phosphatase activity of the partially purified preparation of alkaline phosphatase I of the cell-free extract as well as of intact cells in both the alkaline and acid zones of pH (8.6 and 5.5, respectively). Incubation of the washed iron-deficient P. guilliermondii cells in the presence of Be2+ and F- did not result in accumulation of FMN in the yeast culture. A possible role of nonspecific phosphomonoesterases in hydrolysis of FMN in vivo is discussed. PMID:208203

  17. Dephosphorylation of CDK9 by protein phosphatase 2A and protein phosphatase-1 in Tat-activated HIV-1 transcription

    PubMed Central

    Ammosova, Tatyana; Washington, Kareem; Debebe, Zufan; Brady, John; Nekhai, Sergei

    2005-01-01

    Background HIV-1 Tat protein recruits human positive transcription elongation factor P-TEFb, consisting of CDK9 and cyclin T1, to HIV-1 transactivation response (TAR) RNA. CDK9 is maintained in dephosphorylated state by TFIIH and undergo phosphorylation upon the dissociation of TFIIH. Thus, dephosphorylation of CDK9 prior to its association with HIV-1 preinitiation complex might be important for HIV-1 transcription. Others and we previously showed that protein phosphatase-2A and protein phosphatase-1 regulates HIV-1 transcription. In the present study we analyze relative contribution of PP2A and PP1 to dephosphorylation of CDK9 and to HIV-1 transcription in vitro and in vivo. Results In vitro, PP2A but not PP1 dephosphorylated autophosphorylated CDK9 and reduced complex formation between P-TEFb, Tat and TAR RNA. Inhibition of PP2A by okadaic acid inhibited basal as well as Tat-induced HIV-1 transcription whereas inhibition of PP1 by recombinant nuclear inhibitor of PP1 (NIPP1) inhibited only Tat-induced transcription in vitro. In cultured cells, low concentration of okadaic acid, inhibitory for PP2A, only mildly inhibited Tat-induced HIV-1 transcription. In contrast Tat-mediated HIV-1 transcription was strongly inhibited by expression of NIPP1. Okadaic acid induced phosphorylation of endogenous as well transiently expressed CDK9, but this induction was not seen in the cells expressing NIPP1. Also the okadaic acid did not induce phosphorylation of CDK9 with mutation of Thr 186 or with mutations in Ser-329, Thr-330, Thr-333, Ser-334, Ser-347, Thr-350, Ser-353, and Thr-354 residues involved in autophosphorylation of CDK9. Conclusion Our results indicate that although PP2A dephosphorylates autophosphorylated CDK9 in vitro, in cultured cells PP1 is likely to dephosphorylate CDK9 and contribute to the regulation of activated HIV-1 transcription. PMID:16048649

  18. Phosphatase hydrolysis of organic phosphorus compounds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phosphatases are diverse groups of enzymes that deserve special attention because of the significant roles they play in mineralizing organic phosphorus (P) into inorganic available form. For getting more insight on the enzymatically hydrolysis of organic P, in this work, we compared the catalytic pa...

  19. Probing Mechanistic Similarities between Response Regulator Signaling Proteins and Haloacid Dehalogenase Phosphatases.

    PubMed

    Immormino, Robert M; Starbird, Chrystal A; Silversmith, Ruth E; Bourret, Robert B

    2015-06-01

    Response regulator signaling proteins and phosphatases of the haloacid dehalogenase (HAD) superfamily share strikingly similar folds, active site geometries, and reaction chemistry. Proteins from both families catalyze the transfer of a phosphoryl group from a substrate to one of their own aspartyl residues, and subsequent hydrolysis of the phosphoprotein. Notable differences include an additional Asp that functions as an acid/base catalyst and an active site well-structured prior to phosphorylation in HAD phosphatases. Both features contribute to reactions substantially faster than those for response regulators. To investigate mechanisms underlying the functional differences between response regulators and HAD phosphatases, we characterized five double mutants of the response regulator CheY designed to mimic HAD phosphatases. Each mutant contained the extra Asp paired with a phosphatase-inspired substitution to potentially position the Asp properly. Only CheY DR (Arg as the anchor) exhibited enhanced rates of both autophosphorylation with phosphoramidate and autodephosphorylation compared to those of wild-type CheY. Crystal structures of CheY DR complexed with MoO4(2-) or WO4(2-) revealed active site hydrogen bonding networks similar to those in HAD·substrate complexes, with the extra Asp positioned for direct interaction with the leaving group (phosphorylation) or nucleophile (dephosphorylation). However, CheY DR reaction kinetics did not exhibit the pH sensitivities expected for acid/base catalysis. Biochemical analysis indicated CheY DR had an enhanced propensity to adopt the active conformation without phosphorylation, but a crystal structure revealed unphosphorylated CheY DR was not locked in the active conformation. Thus, the enhanced reactivity of CheY DR reflected partial acquisition of catalytic and structural features of HAD phosphatases. PMID:25928369

  20. Atomic structure of dual-specificity phosphatase 26, a novel p53 phosphatase.

    PubMed

    Lokareddy, Ravi Kumar; Bhardwaj, Anshul; Cingolani, Gino

    2013-02-01

    Regulation of p53 phosphorylation is critical to control its stability and biological activity. Dual-specificity phosphatase 26 (DUSP26) is a brain phosphatase highly overexpressed in neuroblastoma, which has been implicated in dephosphorylating phospho-Ser20 and phospho-Ser37 in the p53 transactivation domain. In this paper, we report the 1.68 Å crystal structure of a catalytically inactive mutant (Cys152Ser) of DUSP26 lacking the first 60 N-terminal residues (ΔN60-C/S-DUSP26). This structure reveals the architecture of a dual-specificity phosphatase domain related in structure to Vaccinia virus VH1. DUSP26 adopts a closed conformation of the protein tyrosine phosphatase (PTP)-binding loop, which results in an unusually shallow active site pocket and buried catalytic cysteine. A water molecule trapped inside the PTP-binding loop makes close contacts both with main chain and with side chain atoms. The hydrodynamic radius (R(H)) of ΔN60-C/S-DUSP26 measured from velocity sedimentation analysis (R(H) ∼ 22.7 Å) and gel filtration chromatography (R(H) ∼ 21.0 Å) is consistent with an ∼18 kDa globular monomeric protein. Instead in crystal, ΔN60-C/S-DUSP26 is more elongated (R(H) ∼ 37.9 Å), likely because of the extended conformation of C-terminal helix α9, which swings away from the phosphatase core to generate a highly basic surface. As in the case of phosphatase MKP-4, we propose that a substrate-induced conformational change, possibly involving rearrangement of helix α9 with respect to the phosphatase core, allows DUSP26 to adopt a catalytically active conformation. The structural characterization of DUSP26 presented in this paper provides the first atomic insight into this disease-associated phosphatase.

  1. Promoting Uranium Immobilization by the Activities of Microbial Phosphatases

    SciTech Connect

    Robert J. Martinez; Melanie J. Beazley; Samuel M. Webb; Martial Taillefert; and Patricia A. Sobecky

    2007-04-19

    The overall objective of this project is to examine the activity 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 PO4 3- as a means to detoxify radionuclides and heavy metals. An experimental approach was designed to determine the extent of phosphatase activity in bacteria previously isolated from contaminated subsurface soils collected at the ERSP Field Research Center (FRC) in Oak Ridge, TN. Screening of 135 metal resistant isolates for phosphatase activity indicated the majority (75 of 135) exhibited a phosphatase-positive phenotype. During this phase of the project, a PCR based approach has also been designed to assay FRC isolates for the presence of one or more classes of the characterized non-specific acid phophastase (NSAP) genes likely to be involved in promoting U(VI) precipitation. Testing of a subset of Pb resistant (Pbr) Arthrobacter, Bacillus and Rahnella strains indicated 4 of the 9 Pbr isolates exhibited phosphatase phenotypes suggestive of the ability to bioprecipitate U(VI). Two FRC strains, a Rahnella sp. strain Y9602 and a Bacillus sp. strain Y9-2, were further characterized. The Rahnella sp. exhibited enhanced phosphatase activity relative to the Bacillus sp. Whole-cell enzyme assays identified a pH optimum of 5.5, and inorganic phosphate accumulated in pH 5.5 synthetic groundwater (designed to mimic FRC conditions) incubations of both strains in the presence of a model organophosphorus substrate provided as the sole C and P source. Kinetic experiments showed that these two organisms can grow in the presence of 200 μM dissolved uranium and that Rahnella is much more efficient in precipitating U(VI) than Bacillus sp. The

  2. The microsomal glucose-6-phosphatase enzyme of pancreatic islets.

    PubMed Central

    Waddell, I D; Burchell, A

    1988-01-01

    Microsomal fractions isolated from pancreatic islet cells were shown to contain high specific glucose-6-phosphatase activity. The islet-cell glucose-6-phosphatase enzyme has the same Mr (36,500), similar immunological properties and kinetic characteristics to the hepatic microsomal glucose-6-phosphatase enzyme. Images Fig. 1. Fig. 2. PMID:2849415

  3. Protein phosphatase and kinase activities possibly involved in exocytosis regulation in Paramecium tetraurelia.

    PubMed Central

    Kissmehl, R; Treptau, T; Hofer, H W; Plattner, H

    1996-01-01

    In Paramecium tetraurelia cells synchronous exocytosis induced by aminoethyldextran (AED) is accompanied by an equally rapid dephosphorylation of a 63 kDa phosphoprotein (PP63) within 80 ms. In vivo, rephosphorylation occurs within a few seconds after AED triggering. In homogenates (P)P63 can be solubilized in all three phosphorylation states (phosphorylated, dephosphorylated and rephosphorylated) and thus tested in vitro. By using chelators of different divalent cations, de- and rephosphorylation of PP63 and P63 respectively can be achieved by an endogenous protein phosphatase/kinase system. Dephosphorylation occurs in the presence of EDTA, whereas in the presence of EGTA this was concealed by phosphorylation by endogenous kinase(s), thus indicating that phosphorylation of P63 is calcium-independent. Results obtained with protein phosphatase inhibitors (okadaic acid, calyculin A) allowed us to exclude a protein serine/threonine phosphatase of type I (with selective sensitivity in Paramecium). Protein phosphatase 2C is also less likely to be a candidate because of its requirement for high Mg2+ concentrations. According to previous evidence a protein serine/threonine phosphatase of type 2B (calcineurin; CaN) is possibly involved. We have now found that bovine brain CaN dephosphorylates PP63 in vitro. Taking into account the specific requirements of this phosphatase in vitro, with p-nitrophenyl phosphate as a substrate, we have isolated a cytosolic phosphatase of similar characteristics by combined preparative gel electrophoresis and affinity-column chromatography. In Paramecium this phosphatase also dephosphorylates PP63 in vitro (after 32P labelling in vivo). Using various combinations of ion exchange, affinity and hydrophobic interaction chromatography we have also isolated three different protein kinases from the soluble fraction, i.e. a cAMP-dependent protein kinase (PKA), a cGMP-dependent protein kinase (PKG) and a casein kinase. Among the kinases tested, PKA

  4. Evaluation of the management of Hr-HPV+/PapTest- women: results at 1-year recall

    PubMed Central

    Chiappetta, Caterina; Puggioni, Chiara; Lendaro, Eugenio; Cacciotti, Jessica; Zaralli, Roberto; Migliore, Giovanna; Bellardini, Paola; Petrozza, Vincenzo; Della Rocca, Carlo; Di Cristofano, Claudio

    2015-01-01

    With cervical cancer screening the choice of 1-year as a period of follow-up in positive high-risk HPV women without cytological lesions is still under discussion. We evaluated the management of these women and the role of HPV genotyping test. We did a cervical cancer screening study of women aged 35-64 with primary high-risk HPV test. Women positive for high-risk HPV with negative cytology were followed-up after 1 year. In this study we selected women with high-risk HPV+/PapTest- resulted high-risk HPV+ at recall and performed the PapTest and HPV genotyping test. The detection rate of squamous high grade (CIN2+) relative to the total screened cohort was 2.1‰, and it was 0.2‰ at the 1-year recall. The colposcopy performed in women referred at the 1-year recall accounted for 48.8% of the total (baseline + 1-year recall), and 84.3% of these women had no cytological lesions. The most frequent hr-HPV genotype detected was HPV16 and 66.7% of co-infections were due to HPV16 and HPV18. 54.5% of women presented a persistent infection at 1-year recall with the same HPV subtype, 50% of persistent infections was due to HPV16 and 16.7% of these were determined to be CIN2+ histological lesions. Our data show that it may be useful to extend the period of follow-up for women hr-HPV+/PapTest- so as to reduce the number of unnecessary colposcopies due to the transitory infections and that the genotyping test could help to identify the persistent infections in which HPV16 is involved. PMID:26884886

  5. Pap Smear

    MedlinePlus

    ... Formal name: Papanicolaou Test Related tests: HPV Test ; Trichomonas Testing All content on Lab Tests Online has ... to detect vaginal or uterine infections, such as trichomonas infections . Abnormal cells and infections can be present ...

  6. Bacterial vaginosis, aerobic vaginitis, vaginal inflammation and major Pap smear abnormalities.

    PubMed

    Vieira-Baptista, P; Lima-Silva, J; Pinto, C; Saldanha, C; Beires, J; Martinez-de-Oliveira, J; Donders, G

    2016-04-01

    The purpose of this investigation was to evaluate the impact of the vaginal milieu on the presence of abnormal Pap smears and a positive human papilloma virus (HPV) test. A cross-sectional study was conducted between June 2014 and May 2015, evaluating the vaginal discharge by fresh wet mount microscopy and comparing these data with Pap smear findings. Wet mount slides were scored for bacterial vaginosis (BV), aerobic vaginitis (AV), presence of Candida and Trichomonas vaginalis. Cytologic evaluation was done on all Pap smears according to the Bethesda criteria. The cobas© HPV Test (Roche) was performed for HPV detection. A total of 622 cases were evaluated. The mean age of the patients was 41.6 ± 10.65 years (range 21-75). Eighty-three women (13.3 %) had a cytology result worse than low-grade squamous intraepithelial lesion (LSIL). When comparing this group with the one with normal or minor [atypical squamous cells of undetermined significance (ASC-US) or LSIL] Pap smear abnormalities, there were no differences in the presence of Candida (32.5 % vs. 33.2 %, p = 1.0), absence of lactobacilli (38.6 % vs. 32.5 %, p = 0.32) or BV (20.5 % vs. 13.2 %, p = 0.09). On the other hand, moderate or severe inflammation (msI) (41.0 % vs. 28.8 %, p = 0,04), moderate or severe AV (msAV) (16.9 % vs. 7.2 %, p = 0.009) and msAV/BV (37.3 % vs. 20.0 %, p = 0.001) were more common in women with such major cervical abnormalities. No significant association was found between deviations of the vaginal milieu and high-risk HPV infection. The presence of msI or msAV, but not BV, is independently associated with an increased risk of major cervical cytological abnormalities, but not with HPV infection. PMID:26810061

  7. Does Cytological Laboratory Holds the Responsibility for the Low Sensitivity of the PAP Test in Detecting Endometrial Cancer?

    PubMed

    Milicić, Valerija; Matić, Tereza Solocki; Martinek, Vjenceslav; Tomasković, Igor; Ramljak, Vesna

    2015-09-01

    Endometrial cancer is the most common gynecological cancer but there is no economically justified screening method. Although we can detect endometrial cells in the sample using PAP test, many studies show low sensitivity and positive predictive value of PAP test for the diagnosis of endometrial cancer. The goal of this research was to determine significance of PAP test for the diagnostics of endometrial carcinoma. Sensitivity and specificity were analyzed with statistical parameters. VCE (vaginal, cervical, endocervical) smears of patients with histologically proven endometrial carcinoma were re-examined in order to determine the proportion of false negative results for endometrial cancer cells in the VCE samples. Study group consisted of all consecutive patients with PAP test performed at the Department of Clinical Cytology of the University Hospital Center Osijek from 2002 until the end of 2014. There was one inclusion criteria: subsequent hysterectomy or curettage within the six month after the PAP test, regardless of histological finding. From a total of 263 patients with previous PAP test and histologically proven endometrial cancer, endometrial cancer was cytologicaly diagnosed in 24.7% (including suspicious and positive findings), while 66.2% patients had normal cytological findings. The diagnostic value of PAP test in detection of endometrial cancer was statistically revealed with 25% sensitivity and 99% specificity. To determine false negative rate VCE samples were reviewed for patients with histologically proven endometrial cancer and negative VCE findings. There were a total of five negative results. In one case revision did not changed the original negative diagnosis, but benign endometrial cells, a lot of blood and inadequate cytohormonal status were found. In three out of four reviewed samples there were missed cells of endometrial adenocarcinoma. Review of remaining VCE sample upgraded the diagnosis from negative to suspicious for endometrial cancer