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Sample records for alkaline phosphatase activity

  1. Low serum alkaline phosphatase activity in Wilson's disease.

    Science.gov (United States)

    Shaver, W A; Bhatt, H; Combes, B

    1986-01-01

    Low values for serum alkaline phosphatase activity were observed early in the course of two patients with Wilson's disease presenting with the combination of severe liver disease and Coombs' negative acute hemolytic anemia. A review of other cases of Wilson's disease revealed that 11 of 12 patients presenting with hemolytic anemia had values for serum alkaline phosphatase less than their respective sex- and age-adjusted mean values; in eight, serum alkaline phosphatase activity was less than the lower value for the normal range of the test. Low values for serum alkaline phosphatase were much less common in Wilson's disease patients with more chronic forms of presentation. Copper added in high concentration to serum in vitro did not have an important effect on serum alkaline phosphatase activity. The mechanism responsible for the decrease in serum alkaline phosphatase activity in patients is uncertain.

  2. Activation of Calf Intestinal Alkaline Phosphatase by Trifluoroethanol

    Institute of Scientific and Technical Information of China (English)

    曹志方; 徐真; 朴龙斗; 周海梦

    2001-01-01

    Alkaline phosphatase is a stable enzyme which is strongly resistant to urea, guanidine hydrochloride, acid pH, and heat. But there have been few studies on the effect of organic cosolvents on the activity and structure of alkaline phosphatase. The activity of calf intestinal alkaline phosphatase (CIAP) is markedly increased when incubated in solutions with elevated trifluoroethanol (TFE) concentrations. The activation is a time dependent course. There is a very fast phase in the activation kinetics in the mixing dead time (30 s) using convential methods. Further activation after the very fast phase follows biphasic kinetics. The structural basis of the activation has been monitored by intrinsic fluorescence and far ultraviolet circular dichroism. TFE (0 - 60%) did not lead to any significant change in the intrinsic fluorescence emission maximum, indicating no significant change in the tertiary structure of CIAP. But TFE did significantly change the secondary structure of CIAP, especially increasing α-helix content. We conclude that the activation of ClAP is due to its secondary structural change. The time for the secondary structure change induced by TFE preceds that of the activity increase. These results suggest that a rapid conformational change of ClAP induced by TFE results in the enhancement of ClAP activity, followed by further increase of this activity because of the further slightly slower rearrangements of the activated conformation. It is concluded that the higher catalytic activity of ClAP can be attained with various secondary structures.

  3. Alkaline phosphatase for immunocytochemical labelling: problems with endogenous enzyme activity.

    OpenAIRE

    Bulman, A. S.; Heyderman, E

    1981-01-01

    Alkaline phosphatase may be used as a label for immunocytochemistry and can be demonstrated in tissue sections using the single step naphthol phosphate method. Endogenous enzyme activity may not be destroyed by fixation in formalin, formol alcohol, Carnoy's or Baker's solutions and should be inhibited before results are assessed. Either Bouin's solution or periodic acid followed by potassium borohydride are satisfactory inhibitor and do not adversely affect immunocytochemical results.

  4. ALKALINE PHOSPHATASE ACTIVITY AS A MARKER OF DOG SEMEN FREEZABILITY

    Directory of Open Access Journals (Sweden)

    KOSINIAK-KAMYSZ K.

    2007-01-01

    Full Text Available The investigation was performed to evaluate the dog semen freezability and itsquality after thawing allowing its use for artificial insemination (AI. On the basis ofsperm motility, concentration and alkaline phosphatase (AP activity in semenplasma it was possible to establish that AP activity corresponds with the basic factorof semen examination. Significant statistical differences occurred between thequality of ejaculates which were qualified or disqualified to deep freezing and AI.These results show that AP activity in raw dog semen plasma can be used as amarker for the dog semen qualification for deep freezing and AI with 95%probability of the prognosis of the results.

  5. ALP (Alkaline Phosphatase) Test

    Science.gov (United States)

    ... Also 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 ...

  6. Alkaline Phosphatase in Stem Cells

    Directory of Open Access Journals (Sweden)

    Kateřina Štefková

    2015-01-01

    Full Text Available Alkaline phosphatase is an enzyme commonly expressed in almost all living organisms. In humans and other mammals, determinations of the expression and activity of alkaline phosphatase have frequently been used for cell determination in developmental studies and/or within clinical trials. Alkaline phosphatase also seems to be one of the key markers in the identification of pluripotent embryonic stem as well as related cells. However, alkaline phosphatases exist in some isoenzymes and isoforms, which have tissue specific expressions and functions. Here, the role of alkaline phosphatase as a stem cell marker is discussed in detail. First, we briefly summarize contemporary knowledge of mammalian alkaline phosphatases in general. Second, we focus on the known facts of its role in and potential significance for the identification of stem cells.

  7. Modulators of intestinal alkaline phosphatase.

    Science.gov (United States)

    Bobkova, Ekaterina V; Kiffer-Moreira, Tina; Sergienko, Eduard A

    2013-01-01

    Small molecule modulators of phosphatases can lead to clinically useful drugs and serve as invaluable tools to study functional roles of various phosphatases in vivo. Here, we describe lead discovery strategies for identification of inhibitors and activators of intestinal alkaline phosphatases. To identify isozyme-selective inhibitors and activators of the human and mouse intestinal alkaline phosphatases, ultrahigh throughput chemiluminescent assays, utilizing CDP-Star as a substrate, were developed for murine intestinal alkaline phosphatase (mIAP), human intestinal alkaline phosphatase (hIAP), human placental alkaline phosphatase (PLAP), and human tissue-nonspecific alkaline phosphatase (TNAP) isozymes. Using these 1,536-well assays, concurrent HTS screens of the MLSMR library of 323,000 compounds were conducted for human and mouse IAP isozymes monitoring both inhibition and activation. This parallel screening approach led to identification of a novel inhibitory scaffold selective for murine intestinal alkaline phosphatase. SAR efforts based on parallel testing of analogs against different AP isozymes generated a potent inhibitor of the murine IAP with IC50 of 540 nM, at least 65-fold selectivity against human TNAP, and >185 selectivity against human PLAP. PMID:23860652

  8. Sensitive optical detection of alkaline phosphatase activity with quantum dots

    Energy Technology Data Exchange (ETDEWEB)

    Ren, Xiangling [Laboratory of Controllable Preparation and Application of Nanomaterials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, No. 29, Zhongguancun East Road, Haidian District, Beijing 100190 (China); The State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096 (China); Chen, Zhenzhen; Chen, Xiaoying; Liu, Jing [Laboratory of Controllable Preparation and Application of Nanomaterials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, No. 29, Zhongguancun East Road, Haidian District, Beijing 100190 (China); Tang, Fangqiong, E-mail: tangfq@mail.ipc.ac.cn [Laboratory of Controllable Preparation and Application of Nanomaterials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, No. 29, Zhongguancun East Road, Haidian District, Beijing 100190 (China)

    2014-01-15

    A simple method has been developed to detect the activity of alkaline phosphatase (ALP) by the changing of fluorescence intensities of the quantum dots (QDs). In this system, the fluorescence intensities of the QDs were quenched by p-nitrophenol (pNP) which was produced in the process of ALP catalytic reaction. A series of linear calibration curves of the activity of ALP were obtained in different pH buffer solutions. The wide linear range was 3–1000 U L{sup −1} and the detection limit was 3 U L{sup −1} (S/N=3). Furthermore, the experimental conditions of biosensor were optimized, and anti-interference ability was presented. The activity of ALP was also detected in serum and the recovery of ALP in serum samples was more than 95%. The excellent performance of this biosensor indicates that it can be used in practice detection of ALP. -- Highlights: • A sensitive ALP biosensor is constructed based on QDs without complex processes. • The analysis processing is very convenient, simple and rapid. • The detection mechanism of the ALP biosensor is studied by XPS. • The paper proposes a feasible approach for some substrates or enzymes detecting.

  9. How Should an Increase in Alkaline Phosphatase Activity Be Interpreted?

    International Nuclear Information System (INIS)

    Low-level laser therapy, commonly known as LLLT, is the application of low power, monochromatic, and coherent light to injuries and lesions to stimulate healing and give pain relief. There are conflicting reports in the literature regarding the role of ALP. Objective: this study aimed to compare the cellular responses of wounded human skin fibroblasts exposed to doses of 0.5 J/cm2, 2.5 J/cm2, 5 J/cm2, or 16 J/cm2 using LLLT with a Helium-Neon laser (632.8 nm, 18.8 mW power output, 2.07 mW/cm2 power density, and 3.4 cm diameter spot size or area 9.1?cm2) to elucidate the role of alkaline phosphatase (ALP) in cell proliferation. Methods: cellular responses to laser irradiation were evaluated using ALP enzyme activity, LDH membrane integrity, neutral red for cell proliferation, optical density at 540?nm, and basic fibroblast growth factor (bFGF) expression. Results: results suggest that an increase in ALP is negatively correlated with cell growth depending on the concentration of growth factors in the medium. Results also indicate that an increase in ALP may be related to cellular damage. Conclusion: since the exact role of ALP is unknown, the ALP enzyme activity assay should be considered in conjunction with other cell proliferation assays such as neutral red, optical density, or more specifically bFGF expression.

  10. Alkaline Phosphatase Activity in San Francisco and Monterey Bays

    Science.gov (United States)

    Nicholson, D. P.

    2002-12-01

    Phosphorus (P) is an essential nutrient utilized by all living organisms, and has been recognized as a limiting nutrient in some oceanic systems (Cotner et al., 1997; Karl et al., 1995; Michaels et al., 1996; Wu et al., 2000). However, relatively little is known about the extent of P limitation in natural environments, how P limitation varies spatially and temporally, and what determines how and when P becomes limiting (Benitez-Nelson, 2000). A more direct estimate of the degree of P limitation in a variety of oceanic systems is needed to better understand P cycling and dynamics within the ocean and how these have and will change in response to global climate and environmental perturbation. Accordingly, the objective this study is to assess the P-status of marine planktonic communities in Monterey and San Francisco Bays using the activity of alkaline phosphatase in the water column. Alkaline phosphatase (AP) is the most widely used enzyme that marine organisms use to hydrolize organic P compounds to biologically available orthophosphate. Accordingly it is expected that in areas where P is a limiting nutrient organisms will produce and release more AP to seawater so they can utilize the dissolved and particulate organic P compounds. Indeed it has been suggested that the AP activity is a reliable indicator of P-availability to planktonic communities (Ammerman and Azam, 1985; Cotner and Wetzel, 1991; Hong et al., 1998). High enzyme activities indicate low dissolved inorganic phosphate (DIP) availability while low levels suggest that DIP supply satisfies the community P-demand. This study examines AP activity in San Francisco and Monterey Bays over a 12 month period, from November, 2001 through November, 2002 using two enzyme assays. The study encompasses data from a three-station transect in Monterey Bay, at depths ranging from 0-60 meters. The stations range from coastal waters to open ocean depths of several thousand meters. In San Francisco Bay, surface water from

  11. Application of Intracellular Alkaline Phosphatase Activity Measurement in Detection of Neutrophil Adherence In Vitro

    OpenAIRE

    Katarzyna Bednarska; Magdalena Klink; Zofia Sulowska

    2006-01-01

    We have proposed the use of the fluorimetric method with 4-methylumbelliferyl phosphate (4-MUP) specific substrate for the alkaline phosphatase determination in the neutrophil adhesion assay. We provide evidence that the endogenous neutrophil alkaline phosphatase (NAP) activity evaluation is reliable to quantify neutrophil adhesion at a wide range of cell numbers (104–106). The results obtained by fluorimetric NAP activity test correlate to the results of adherence evaluated using...

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

    Directory of Open Access Journals (Sweden)

    Lacramioara Oprica

    2011-12-01

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

  13. Application of intracellular alkaline phosphatase activity measurement in detection of neutrophil adherence in vitro.

    Science.gov (United States)

    Bednarska, Katarzyna; Klink, Magdalena; Sulowska, Zofia

    2006-01-01

    We have proposed the use of the fluorimetric method with 4-methylumbelliferyl phosphate (4-MUP) specific substrate for the alkaline phosphatase determination in the neutrophil adhesion assay. We provide evidence that the endogenous neutrophil alkaline phosphatase (NAP) activity evaluation is reliable to quantify neutrophil adhesion at a wide range of cell numbers (10(4)-10(6)). The results obtained by fluorimetric NAP activity test correlate to the results of adherence evaluated using the MTT reduction assay. The fluorimetric NAP activity test may be applied for resting as well as activated neutrophils without the risk of the activators interferences into the test. The alkaline phosphatase survey with the use of 4-MUP substrate is recommended herein as a sensitive, repeatable, simple, and reliable method of the neutrophil adherence determination in vitro. PMID:17047286

  14. Tubulin polymerization by paclitaxel (taxol) phosphate prodrugs after metabolic activation with alkaline phosphatase.

    Science.gov (United States)

    Mamber, S W; Mikkilineni, A B; Pack, E J; Rosser, M P; Wong, H; Ueda, Y; Forenza, S

    1995-08-01

    Paclitaxel (taxol) phosphate derivatives BMY46366, BMY-46489, BMS180661 and BMS180820 were used to determine the ability of alkaline phosphatase to convert these water-soluble potential prodrugs to tubulin-polymerizing metabolites (i.e., paclitaxel). Compounds were treated up to 180 min with an in vitro metabolic activation system composed of 10% bovine alkaline phosphatase in 0.2 M tris, pH 7.4, or in 0.2 M glycine, pH 8.8, plus 0.05 M MgCl2. Samples were tested (either by direct addition or after methylene chloride extraction/dimethyl-sulfoxide resuspension) in spectrophotometric tubulin polymerization assays utilizing bovine-derived microtubule protein. Pretreatment of 2'- and 7-phosphonoxyphenylpropionate prodrugs BMS180661 and BMS180820 with alkaline phosphatase for 30 to 120 min yielded relative initial slopes of about 20 to 100% at test concentrations equimolar to paclitaxel. High-performance liquid chromatography/mass spectrometry of BMS180661 treated with alkaline phosphatase confirmed the production of paclitaxel from the prodrug. In contrast, 2'- and 7-phosphate analogs BMY46366 and BMY46489 treated with alkaline phosphatase were not active in tubulin assays. None of the paclitaxel phosphate prodrugs polymerized tubulin in the absence of metabolic activation. The differences in tubulin polymerization with metabolic activation may be related both to accessibility of the phosphate group to the enzyme and to anionic charge effects. These results demonstrate that certain paclitaxel phosphate prodrugs can be metabolized by alkaline phosphatase to yield effective tubulin polymerization. PMID:7636751

  15. Intraspecific variation in alkaline phosphatase activity in Phaeodactylum tricornutum (Bacillariophyceae, Bohlin

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    Domênica Teixeira de Lima

    2016-01-01

    Full Text Available ABSTRACT To describe potential intraspecific variation in phosphorus incorporation in two strains of Phaeodactylum tricornutum (Bohlin, Ub3 and Ub7, alkaline phosphatase (AP activity was evaluated via enzyme-labeled fluorescence assay. Analysis using the probe ELF-97(r provides individual evaluation, and therefore can determine the nutritional status of inorganic phosphorus in phytoplanktonic cells. Bioassays compared the control treatment to both phosphate-enriched and phosphate-depleted treatments by varying only the phosphate concentration in the media. The P. tricornutum strains exhibited differences in their development when incubated in the phosphate-enriched media. The development of the Ub7 strain differed by exhibiting "luxury uptake" and utilization of organic phosphorus, and the alkaline phosphatase analysis indicated limitations of this clone under such conditions. The Ub7 strain showed higher AP activity, when compared to Ub3, in the P-enriched condition. P. tricornutum presented increases in AP activity and low variation in Surface/Volume ratio, by increasing biovolume and its maximum linear dimension, as strategies for phosphate incorporation. Our results highlight intraspecific differences in alkaline phosphatase activity, and hence differences in the incorporation of organic phosphorus, as the tested species regulated enzymatic activity under different external phosphate concentrations.

  16. The Effect of Titanium Dioxide Nanoparticles on Salivary Alkaline Phosphatase Activity

    OpenAIRE

    Eaman A. S. AL-Rubaee; Suha T. Abd; Nadia M. Kadim

    2015-01-01

    The structural and optical properties of the titanium dioxide nanoparticles [TiO2 NPs] have been investigated using [UV-Vis] spectrophotometer and SEM. The produced nanoparticles show small and about sharp round peaks around 220 nm. The produced nanoparticles have a spherical shape with an average particle size ˂50 nm. The effect of titanium dioxide NPs was studied on the activity of Alkaline Phosphatase [ALP] in the saliva of 25 patients with gingivitis in comparison to 20 healthy subjects w...

  17. Effects of sodium nitroprusside activity of acid and alkaline invertases and alkaline phosphatase in lemongrass (Cymbopogon flexuosus Steud Wats

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    Deepak Ganjewala

    2010-01-01

    Full Text Available Here we report the effects of SNP, a nitric oxide donor on sucrose metabolizing enzymes, acid and alkaline invertase (EC 3.2.1.26 and 3.2.1.153 and ubiquitous alkaline phosphatase (EC 3.1.3.1 in four lemongrass varieties viz., Krishna, Cauveri, Nima and Cheerharit. For the study, two 15 d lemongrass tillers were cut and immediately dipped into the test tubes containing SNP solution (5 mL of variable strength (1 to 5 mM and one without SNP (as control; kept for 4 h under mild sunlight. The results revealed that moderate SNP concentration (2 mM was most effective, caused drastic reduction (40% in protein content in var. Nima followed by Krishna (33%, Cauveri (17% and Cheerharit (12%. In contrast, SNP (1 mM has impressively enhanced protein content in all the lemongrass varieties. The SNP (2 mM markedly inhibited the activity of acid invertase by 38% in Cheerharit, 35% Nima and 28% Cauveri whereas and alkaline invertase by 21, 28 and 24% respectively in var. Cheerharit, Nima and Krishna. Similarly, SNP (5 mM severely inhibited (~ 63% the activity of the ALP in lemongrass var. Cauveri and Nima, 50% in Krishna and relatively less 23% in Cheerharit as compared to the control. However, in var. Nima, 50% loss in ALP activity had already been occurred after 2 mM SNP treatment. These results primarily suggests that NO interferes sucrose metabolism by anonymously hindering the activity of acid and alkaline invertase and ubiquitous alkaline phosphatase in lemongrasses.

  18. Persistently increased intestinal fraction of alkaline phosphatase

    DEFF Research Database (Denmark)

    Nathan, E; Baatrup, G; Berg, H;

    1984-01-01

    Persistent elevation of the intestinal fraction of the alkaline phosphatase (API) as an isolated finding has to our knowledge not been reported previously. It was found in a boy followed during a period of 5.5 years. The only symptom was transient periodic fatigue observed at home, but not apparent...... phosphatase activity could be demonstrated....

  19. Host plant effects on alkaline phosphatase activity in the whiteflies, Bemisia tabaci Biotype B and Trialeurodes vaporariorum.

    Science.gov (United States)

    Yan, Ying; Peng, Lu; Liu, Wan-Xue; Wan, Fang-Hao; Harris, Marvin K

    2011-01-01

    Bemisia tabaci (Gennadius) B-biotype and Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae) often coexist on greenhouse-grown vegetable crops in northern China. The recent spread of B. tabaci B-biotype has largely replaced T. vaporariorum, and B-biotype now overlaps with T. vaporariorum where common hosts occur in most invaded areas. The impact of the B-biotype on the agro eco system appears to be widespread, and involves the ability to compete with and perhaps replace other phytophages like T. vaporariorum. An emerging hypothesis is that the B-biotype is physiologically superior due at least in part to an improved ability to metabolically utilize the alkaline phosphatase pathway. To test this hypothesis, alkaline phosphatase activity was studied in the B-biotype and T. vaporariorum after feeding on a number of different hosts for a range of durations, with and without host switching. Alkaline phosphatase activity in T. vaporariorum was 1.45 to 2.53-fold higher than that of the B-biotype when fed on tomato for 4 and 24 h, or switched from tomato to cotton and cabbage for the same durations. However, alkaline phosphatase activity in the B-biotype was 1.40 to 3.35-fold higher than that of T. vaporariorum when the host switching time was ∼72 and ∼120 h on the same plant. Both short-term (4 h) and long-term (72 h) switching of plant hosts can significantly affect the alkaline phosphatase activity in the two species. After ∼120 h, feeding on tomato and cotton alkaline phosphatase activity in the B-biotype was significantly higher than that of T. vaporariorum. It was shown that alkaline phosphatase aids the species feeding on different plant species, and that the B-biotype is physiologically superior to T. vaporariorum in utilizing the enzyme compared to T. vaporariorum over longer periods of feeding.

  20. Alkaline phosphatase activity and the phosphorus mineralization rate of Lake Taihu

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The phosphorus fractions, the alkaline phosphatase activity (APA) and other water chemical parameters were concomitantly monitored from April 2003 to October 2004 in different ecotype sites of Lake Taihu. During the stages of algae growth, the phosphorus fractions and their relationships with APA in different ecotype sites were discussed and the phosphorus mineralization rate was calculated. In the water of Lake Taihu, most of the phosphorus (70.2%) could be attributed to the suspended particulate phosphorus, while the dissolved reactive phosphorus (DRP) seems to contribute less than 7%. About 58% of the total phosphorus, however, can be hydrolyzed as inorganic phosphate to compensate for phosphorus deficiency of algae and bacteria growth. During the different algae growth stages, the APA and its Kinetic parameters were varied significantly between different ecotype sites of Lake Taihu. This trend is also visible by comparing the phosphorus mineralization rate,and the most rapidly phosphorus turnover time is only several minutes. The fast recycle of phosphorus can, to some extent, be explained that the phosphorus source of algal blooms. The phytoplankton seems to compensate for phosphorus deficiency by using the alkaline phosphatase to hydrolyze phosphomonoesters.

  1. Chronic Cadmium Exposure Lead to Inhibition of Serum and Hepatic Alkaline Phosphatase Activity in Wistar Rats.

    Science.gov (United States)

    Treviño, Samuel; Andrade-García, Alejandra; Herrera Camacho, Irma; León-Chavez, Bertha Alicia; Aguilar-Alonso, Patricia; Flores, Gonzalo; Brambila, Eduardo

    2015-12-01

    Alkaline phosphatase (ALP) activity in the serum and liver from rats administered with cadmium (Cd) in drinking water was studied. After metal administration, Cd showed a time-dependent accumulation in the liver, meanwhile metallothionein had a maximum increase at 1 month, remaining in this level until the end of the study. On the other hand, serum and liver ALP activity was decreased after 3 months exposure. To determine if Cd produced an inhibition on enzyme, apo-ALP prepared from both nonexposed and exposed rats was reactivated with Zn, showing 60% more activity as compared with the enzyme isolated from nonexposed rats. In vitro assays showed that Cd-ALP was partially reactivated with Zn; however, in the presence of cadmium, Zn-ALP was completely inhibited. Kinetic studies indicate a noncompetitive inhibition by Cd; these results suggest that Cd can substitute Zn, and/or Cd can interact with nucleophilic ligands essential for the enzymatic activity.

  2. Ionizing Groups at the Active Site of the Alkaline Phosphatase from Ostrea cucullate

    Institute of Scientific and Technical Information of China (English)

    CHEN Qiao; WANG Qin; LIAO Jinhua; CHEN Qingxi

    2006-01-01

    The ionizing groups at the active site of alkaline phosphatase (ALP, EC 3.1.3.1) from Ostrea cucullate were studied using kinetic methods. The ionization constant, pKe, of the ionizing groups at the active site of the enzyme was found to be 10.11 at 37.0℃ and the organic solvent, dioxin, had no effect on the pKe. The standard dissociation enthalpy (△Ho) was determined to be 10.57 kcal/mol (1 cal=4.18 J). The results show that the dissociation group of the enzyme active center is the з-NH3+ of lysine. Chemical modification of the enzyme by acetic anhydride and succinic anhydride demonstrates that the amino group is one of the enzyme's functional groups.

  3. A Novel Bifunctional Hybrid with Marine Bacterium Alkaline Phosphatase and Far Eastern Holothurian Mannan-Binding Lectin Activities

    OpenAIRE

    Larissa Balabanova; Vasily Golotin; Svetlana Kovalchuk; Alexander Bulgakov; Galina Likhatskaya; Oksana Son; Valery Rasskazov

    2014-01-01

    A fusion between the genes encoding the marine bacterium Cobetia marina alkaline phosphatase (CmAP) and Far Eastern holothurian Apostichopus japonicus mannan-binding C-type lectin (MBL-AJ) was performed. Expression of the fusion gene in E. coli cells resulted in yield of soluble recombinant chimeric protein CmAP/MBL-AJ with the high alkaline phosphatase activity and specificity of the lectin MBL-AJ. The bifunctional hybrid CmAP/MBL-AJ was produced as a dimer with the molecular mass of 200 kDa...

  4. The Effects of Two Species of Daphne, Betulin and Betulinic Acid on Alkaline Phosphatase Activity in Two Human Cancer Cell lines, K562 and MCF-7

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    E Panahi Kokhdan

    2014-02-01

    Full Text Available Abstract Background & aim: Changes of alkaline phosphatase activity is one of the symptoms of many diseases. The aim of this study was to evaluate the effect of two types of Daphne, Betulin and Betulinic acid, on alkaline phosphatase activity in K562 and MCF-7 cell lines, respectively. Methods: In this study, 106 cancer cell lines of K562 and MCF-7 were cultured in presence of 5% carbon dioxide at 37 ° C. at doses near the IC50. The viability of cells, inside and outside alkaline phosphatase activity and the amount of total protein in each treatment were studied. The collected data was analyzed with a multivariate analysis of variance (Nested Design and Dunnett test. Results: The intracellular alkaline phosphatase activity of the cells showed different behavior compared to the extracellular alkaline phosphatase activity (p< 0.01. The highest increase of alkaline phosphatase activity in two cell lines (K562 and MCF-7 were 339% and 236% which was related to the treatment by macronata daphne. Conclusion: Unexpected increase in intracellular alkaline phosphatase activity in D. mucronata, D. oleides, Betulin, and Betulinic acid treatment may be due to changes in the composition of plasma membrane component and an increase the non-connected membrane of the protein which is due to the creation of more active proteins. Keywords: Daphne mucronata, Daphne oleoides, Alkaline Phosphatase, Betulinic Acid, Betulin

  5. Correlation of alkaline phosphatase activity to clinical parameters of inflammation in smokers suffering from chronic periodontitis

    Science.gov (United States)

    Grover, Vishakha; Malhotra, Ranjan; Kapoor, Anoop; Bither, Rupika; Sachdeva, Sonia

    2016-01-01

    Context: Current clinical periodontal diagnostic techniques emphasize the assessment of clinical and radiographic signs of periodontal diseases which can provide a measure of history of disease. Hence, new methodologies for early identification and determination of periodontal disease activity need to be explored which will eventually result in expedited treatment. Aim: To evaluate the correlation of alkaline phosphatase (ALP) activity in gingival crevicular fluid (GCF) to clinical parameters of periodontal inflammation in smokers with chronic periodontitis. Materials and Methods: Study population included 15 smoker male patients in the age group of 35–55 years suffering from moderate generalized chronic periodontitis with history of smoking present. Following parameters were evaluated at baseline, 1 month and 3 months after scaling and root planing: plaque index, bleeding index, probing pocket depth (PD), relative attachment level (RAL), and GCF ALP activity. Statistical Analysis Used: Independent variables for measurements over time were analyzed by using Wilcoxon signed rank test. Results: A statistically significant reduction in all the clinical parameters and GCF ALP activity was observed from baseline to 1 month and 3 months. A correlation was observed between change in GCF ALP activity and PD reduction as well as gain in RAL at 3 months. Conclusion: The present study emphasizes that total ALP activity could be used as a marker for periodontal disease activity in smokers. Estimation of changes in the levels of this enzyme has a potential to aid in the detection of progression of periodontal disease and monitoring the response to periodontal therapy. PMID:27563197

  6. Correlation of alkaline phosphatase activity to clinical parameters of inflammation in smokers suffering from chronic periodontitis

    Directory of Open Access Journals (Sweden)

    Vishakha Grover

    2016-01-01

    Full Text Available Context: Current clinical periodontal diagnostic techniques emphasize the assessment of clinical and radiographic signs of periodontal diseases which can provide a measure of history of disease. Hence, new methodologies for early identification and determination of periodontal disease activity need to be explored which will eventually result in expedited treatment. Aim: To evaluate the correlation of alkaline phosphatase (ALP activity in gingival crevicular fluid (GCF to clinical parameters of periodontal inflammation in smokers with chronic periodontitis. Materials and Methods: Study population included 15 smoker male patients in the age group of 35–55 years suffering from moderate generalized chronic periodontitis with history of smoking present. Following parameters were evaluated at baseline, 1 month and 3 months after scaling and root planing: plaque index, bleeding index, probing pocket depth (PD, relative attachment level (RAL, and GCF ALP activity. Statistical Analysis Used: Independent variables for measurements over time were analyzed by using Wilcoxon signed rank test. Results: A statistically significant reduction in all the clinical parameters and GCF ALP activity was observed from baseline to 1 month and 3 months. A correlation was observed between change in GCF ALP activity and PD reduction as well as gain in RAL at 3 months. Conclusion: The present study emphasizes that total ALP activity could be used as a marker for periodontal disease activity in smokers. Estimation of changes in the levels of this enzyme has a potential to aid in the detection of progression of periodontal disease and monitoring the response to periodontal therapy.

  7. Effects of overlying water aeration on phosphorus fractions and alkaline phosphatase activity in surface sediment

    Institute of Scientific and Technical Information of China (English)

    Jianjun Chen; Shaoyong Lu; Yikun Zhao; Wei Wang; Minsheng Huang

    2011-01-01

    Microbial activity may influence phosphorus (P) deposit and release at the water sediment interface.The properties of DO (dissolved oxygen), pH, P fractions (TP, Ca-P, Fe-P, OP, IP), and APA (alkaline phosphatase activity) at the water sediment interface were measured to investigate microbial activity variations in surface sediment under conditions of two-month intermittent aeration in overlying water.Results showed that DO and TP of overlying water increased rapidly in the first week and then decreased gradually after 15 day of intermittent aeration.Microorganism metabolism in surface sediment increased pH and decreased DO and TP in the overlying water.After two-month intermittent aeration, APA and OP from surface sediment (0-2 crm) were both significantly higher than those from bottom sediment (6-8 cm) (p < 0.05), and surface sediment Fe-P was transferred to OP during the course of microorganism reproduction on the surface sediment.These results suggest that microbial activity and microorganism biomass from the surface sediment were higher than those from bottom sediment afar two-month intermittent aeration in the overlying water.

  8. Interactive effects of temperature, ultraviolet radiation and food quality on zooplankton alkaline phosphatase activity.

    Science.gov (United States)

    Wolinski, Laura; Modenutti, Beatriz; Souza, Maria Sol; Balseiro, Esteban

    2016-06-01

    Ultraviolet Radiation (UVR) is a stressor for aquatic organisms affecting enzyme activities in planktonic populations because of the increase in reactive oxygen species. In addition, UVR exposure combined with other environmental factors (i.e. temperature and food quality) could have even higher detrimental effects. In this work, we aimed to determine the effect of UVR on somatic Alkaline Phosphatase Activity (APA) and Glutathione S-Transferase (GST) activity on the cladoceran Daphnia commutata under two different temperatures (10 °C and 20 °C) and under three food qualities (carbon:phosphorus ratios: 1150, 850 and 550). APA is a biomarker that is considered as a P deficiency indicator in zooplankton. Since recovery from UVR damage under dark conditions is an ATP depending reaction we also measured APA during recovery phases. We carried out a laboratory experiment combining different temperatures and food qualities with exposition to UVR followed by luminic and dark phases for recovery. In addition, we exposed organisms to H2O2, to establish if the response on APA to UVR was a consequence of the reactive oxygen species produced these short wavelengths. Our results showed that somatic APA was negatively affected by UVR exposure and this effect was enhanced under high temperature and low food quality. Consistently, GST activity was higher when exposed to UVR under both temperatures. The H2O2 experiments showed the same trend as UVR exposure, indicating that APA is affected mainly by oxidative stress than by direct effect of UVR on the enzyme. Finally, APA was affected in the dark phase of recovery confirming the P demands. These results enlighten the importance of food quality in the interacting effect of UVR and temperature, showing that C:P food ratio could determine the success or failure of zooplanktonic populations in a context of global change. PMID:26895537

  9. [Influences of uncommon isoenzymes on determination of alkaline phosphatase activity by dry-chemistry analyzers].

    Science.gov (United States)

    Tozawa, T; Hashimoto, M

    2001-04-01

    Dry-chemistry(DC) analysis may be influenced by some matrix effects for measuring uncommon isoenzyme forms. Placental and intestinal alkaline phosphatase(AP) are overestimated by the VITROS DC, compared with results obtained with the wet-chemistry(WC) method of Bretaudiere, et al. using 2-amino-2-methyl-1-propanol (AMP) buffer, however, no such discrepancy between AP results in any DC method and that with a routine WC method recommended by Japanese Society of Clinical Chemistry in that 2-ethylaminoethanol(EAE) buffer is used, has been demonstrated. The type of buffer used affects differently the rates of AP isoenzymes activities. We therefore examined whether the presence of uncommon AP isoenzyme forms in serum caused aberrant DC results for AP in comparison with a routine WC method using EAE buffer. Here, serum samples with only liver AP and bone AP(n : 32); high-molecular-mass AP(n : 11); placental AP(n : 12); intestinal AP(n : 13) and immunoglobulin (Ig) bound AP(n : 12) were analyzed for total AP activity on three different DC analyzers: VITROS 700XR, FUJIDRYCHEM 5000, SPOTCHEM 4410 and a WC analyzer: HITACHI 7350. Values obtained in all of the DCs for sera containing only liver/bone AP agreed with those with the WC method. For sera containing placental AP, the VITROS values were higher than those with the WC method, while the FUJIDRYCHEM values and the SPOTCHEM values were lower. The VITROS values and the FUJIDRYCHEM values for sera containing intestinal AP were lower than those with the WC method, while the SPOTCHEM values were higher. All of the DCs did not affect high-molecular-mass AP and Ig bound liver/bone AP types of macro AP, but underestimated Ig bound intestinal type. Ig bound intestinal AP may be sieved by DC multilayer elements. PMID:11391954

  10. Alkaline phosphatase activity related to phosphorus stress of microphytoplankton in different trophic conditions

    Science.gov (United States)

    Ivančić, Ingrid; Pfannkuchen, Martin; Godrijan, Jelena; Djakovac, Tamara; Marić Pfannkuchen, Daniela; Korlević, Marino; Gašparović, Blaženka; Najdek, Mirjana

    2016-08-01

    The northern Adriatic (NA) is a favorable basin for studying the adaptive strategies of plankton to a variety of conditions along the steep gradients of environmental parameters over the year. Earlier studies identified phosphorus (P)-limitation as one of the key stresses within the NA that shape the biological response in terms of biodiversity and metabolic adjustments. A wide range of reports supports the notion that P-limitation is a globally important phenomenon in aquatic ecosystems. In this study P stress of marine microphytoplankton was determined at species level along a trophic gradient in the NA. In P-limitation all species with considerable contributions to the diatom community expressed alkaline phosphatase activity (APA), compared to only a few marginal dinoflagellate species. Nevertheless, APA expressing species did not always dominate the phytoplankton community, suggesting that APA is also an important strategy for species to survive and maintain active metabolism outside of their mass abundances. A symbiotic relationship could be supposed for diatoms that did not express APA themselves and probably benefited from APA expressed by attached bacteria. APA was not expressed by any microphytoplankton species during the autumn when P was not limiting, while most of the species did express APA during the P-limitation. This suggests that APA expression is regulated by orthophosphate availability. The methods employed in this study allowed the microscopic detection of APA for each microphytoplankton cell with simultaneous morphologic/taxonomic analysis. This approach uncovered a set of strategies to compete in P-limited conditions within the marine microphytoplankton community. This study confirms the role of P-limitation as a shaping factor in marine ecosystems.

  11. A Novel Bifunctional Hybrid with Marine Bacterium Alkaline Phosphatase and Far Eastern Holothurian Mannan-Binding Lectin Activities

    Science.gov (United States)

    Balabanova, Larissa; Golotin, Vasily; Kovalchuk, Svetlana; Bulgakov, Alexander; Likhatskaya, Galina; Son, Oksana; Rasskazov, Valery

    2014-01-01

    A fusion between the genes encoding the marine bacterium Cobetia marina alkaline phosphatase (CmAP) and Far Eastern holothurian Apostichopus japonicus mannan-binding C-type lectin (MBL-AJ) was performed. Expression of the fusion gene in E. coli cells resulted in yield of soluble recombinant chimeric protein CmAP/MBL-AJ with the high alkaline phosphatase activity and specificity of the lectin MBL-AJ. The bifunctional hybrid CmAP/MBL-AJ was produced as a dimer with the molecular mass of 200 kDa. The CmAP/MBL-AJ dimer model showed the two-subunit lectin part that is associated with two molecules of alkaline phosphatase functioning independently from each other. The highly active CmAP label genetically linked to MBL-AJ has advantaged the lectin-binding assay in its sensitivity and time. The double substitution A156N/F159K in the lectin domain of CmAP/MBL-AJ has enhanced its lectin activity by 25±5%. The bifunctional hybrid holothurian's lectin could be promising tool for developing non-invasive methods for biological markers assessment, particularly for improving the MBL-AJ-based method for early detection of a malignant condition in cervical specimens. PMID:25397876

  12. A novel bifunctional hybrid with marine bacterium alkaline phosphatase and Far Eastern holothurian mannan-binding lectin activities.

    Directory of Open Access Journals (Sweden)

    Larissa Balabanova

    Full Text Available A fusion between the genes encoding the marine bacterium Cobetia marina alkaline phosphatase (CmAP and Far Eastern holothurian Apostichopus japonicus mannan-binding C-type lectin (MBL-AJ was performed. Expression of the fusion gene in E. coli cells resulted in yield of soluble recombinant chimeric protein CmAP/MBL-AJ with the high alkaline phosphatase activity and specificity of the lectin MBL-AJ. The bifunctional hybrid CmAP/MBL-AJ was produced as a dimer with the molecular mass of 200 kDa. The CmAP/MBL-AJ dimer model showed the two-subunit lectin part that is associated with two molecules of alkaline phosphatase functioning independently from each other. The highly active CmAP label genetically linked to MBL-AJ has advantaged the lectin-binding assay in its sensitivity and time. The double substitution A156N/F159K in the lectin domain of CmAP/MBL-AJ has enhanced its lectin activity by 25 ± 5%. The bifunctional hybrid holothurian's lectin could be promising tool for developing non-invasive methods for biological markers assessment, particularly for improving the MBL-AJ-based method for early detection of a malignant condition in cervical specimens.

  13. Alkaline phosphatase activity in the subtropical ocean: insights from nutrient, dust and trace metal addition experiments

    Directory of Open Access Journals (Sweden)

    Claire eMahaffey

    2014-12-01

    Full Text Available Phosphorus is an essential nutrient for all life on earth. In the ocean, the most bioavailable form of phosphorus is inorganic phosphate, but in the extensive subtropical gyres, phosphate concentrations can be chronically low and limit primary productivity and nitrogen fixation. In these regions, organisms produce hydrolytic enzymes, such as alkaline phosphatase (AP, that enable them to utilize the more replete dissolved organic phosphorus (DOP pool to meet their cellular phosphorus demands. In this study, we synthesized data from 14 published studies and present our own findings from two research cruises (D326 and D361 in the eastern subtropical Atlantic to explore the relationship between AP activity (APA and nutrients, Saharan dust and trace metals. We found that below a threshold phosphate concentration of ~ 30 nM, APA increased with an inverse hyperbolic relationship with phosphate concentration. Meanwhile, DOP concentrations decreased with enhanced APA, indicating utilization of the DOP pool. We found APA rates were significantly higher in the subtropical Atlantic compared to the subtropical Pacific Ocean, even over the same low phosphate concentration range (0 to 50 nM. While the phosphate concentration may have a first order control on the APA rates, we speculate that other factors influence this basin scale contrast. Using bioassay experiments, we show that the addition of Saharan dust and zinc significantly increased the rate of APA. To our knowledge, our results are the first direct field-based evidence that APA is limited by zinc in the subtropical ocean. Further work is required to explore the relationship between trace metals such as iron and zinc, which are co-factors of phosphohydrolytic enzymes, specifically PhoX and PhoA, respectively, and APA in the ocean.

  14. Differentiating Intracellular from Extracellular Alkaline Phosphatase Activity in Soil by Sonication

    NARCIS (Netherlands)

    Qin, S.P.; Hu, C.S.; Oenema, O.

    2013-01-01

    Differentiating intracellular from extracellular enzyme activity is important in soil enzymology, but not easy. Here, we report on an adjusted sonication method for the separation of intracellular from extracellular phosphatase activity in soil. Under optimal sonication conditions [soil:water ratio

  15. Phosphatidylinositol anchor of HeLa cell alkaline phosphatase

    International Nuclear Information System (INIS)

    Alkaline phosphatase from cancer cells, HeLa TCRC-1, was biosynthetically labeled with either 3H-fatty acids or [3H]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 3H-fatty acid label from immunoaffinity-purified alkaline phosphatase but had no effect on the radioactivity of [3H]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 3H-fatty acid labeled cells by PI-PLC was not radioactive. By contrast, treatment with bromelain removed both the 3H-fatty acid and the [3H]ethanolamine label from purified alkaline phosphatase. Subtilisin was also able to remove the [3H]ethanolamine label from the purified alkaline phosphatase. The 3H radioactivity in alkaline phosphatase purified from [3H]ethanolamine-labeled cells comigrated with authentic [3H]ethanolamine by anion-exchange chromatography after acid hydrolysis. The data suggest that the 3H-fatty acid and [3H]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

  16. Phosphatidylinositol anchor of HeLa cell alkaline phosphatase

    Energy Technology Data Exchange (ETDEWEB)

    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.

  17. 21 CFR 864.7660 - Leukocyte alkaline phosphatase test.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Leukocyte alkaline phosphatase test. 864.7660... Leukocyte alkaline phosphatase test. (a) Identification. A leukocyte alkaline phosphatase test is a device used to identify the enzyme leukocyte alkaline phosphatase in neutrophilic granulocytes...

  18. Valproic acid induces hair regeneration in murine model and activates alkaline phosphatase activity in human dermal papilla cells.

    Directory of Open Access Journals (Sweden)

    Soung-Hoon Lee

    Full Text Available BACKGROUND: Alopecia is the common hair loss problem that can affect many people. However, current therapies for treatment of alopecia are limited by low efficacy and potentially undesirable side effects. We have identified a new function for valproic acid (VPA, a GSK3β inhibitor that activates the Wnt/β-catenin pathway, to promote hair re-growth in vitro and in vivo. METHODOLOGY/ PRINCIPAL FINDINGS: Topical application of VPA to male C3H mice critically stimulated hair re-growth and induced terminally differentiated epidermal markers such as filaggrin and loricrin, and the dermal papilla marker alkaline phosphatase (ALP. VPA induced ALP in human dermal papilla cells by up-regulating the Wnt/β-catenin pathway, whereas minoxidil (MNX, a drug commonly used to treat alopecia, did not significantly affect the Wnt/β-catenin pathway. VPA analogs and other GSK3β inhibitors that activate the Wnt/β-catenin pathway such as 4-phenyl butyric acid, LiCl, and BeCl(2 also exhibited hair growth-promoting activities in vivo. Importantly, VPA, but not MNX, successfully stimulate hair growth in the wounds of C3H mice. CONCLUSIONS/ SIGNIFICANCE: Our findings indicate that small molecules that activate the Wnt/β-catenin pathway, such as VPA, can potentially be developed as drugs to stimulate hair re-growth.

  19. Characterization of alkaline phosphatase activity in seminal plasma and in fresh and frozen-thawed stallion spermatozoa.

    Science.gov (United States)

    Bucci, Diego; Giaretta, Elisa; Spinaci, Marcella; Rizzato, Giovanni; Isani, Gloria; Mislei, Beatrice; Mari, Gaetano; Tamanini, Carlo; Galeati, Giovanna

    2016-01-15

    Alkaline phosphatase (AP) has been studied in several situations to elucidate its role in reproductive biology of the male from different mammalian species; at present, its role in horse sperm physiology is not clear. The aim of the present work was to measure AP activity in seminal plasma and sperm extracts from freshly ejaculated as well as in frozen-thawed stallion spermatozoa and to verify whether relationship exists between AP activity and sperm quality parameters. Our data on 40 freshly ejaculated samples from 10 different stallions demonstrate that the main source of AP activity is seminal plasma, whereas sperm extracts contribution is very low. In addition, we found that AP activity at physiological pH (7.0) is significantly lower than that observed at pH 8.0, including the optimal AP pH (pH 10.0). Alkaline phosphatase did not exert any effect on sperm-oocyte interaction assessed by heterologous oocyte binding assay. Additionally, we observed a thermal stability of seminal plasma AP, concluding that it is similar to that of bone isoforms. Positive correlations were found between seminal plasma AP activity and sperm concentration, whereas a negative correlation was present between both spermatozoa extracts and seminal plasma AP activity and seminal plasma protein content. A significant decrease in sperm extract AP activity was found in frozen-thawed samples compared with freshly ejaculated ones (n = 21), concomitantly with the decrease in sperm quality parameters. The positive correlation between seminal plasma AP activity measured at pH 10 and viability of frozen-thawed spermatozoa suggests that seminal plasma AP activity could be used as an additional predictive parameter for stallion sperm freezability. In conclusion, we provide some insights into AP activity in both seminal plasma and sperm extracts and describe a decrease in AP after freezing and thawing.

  20. Copper sulfide nanoparticle-decorated graphene as a catalytic amplification platform for electrochemical detection of alkaline phosphatase activity.

    Science.gov (United States)

    Peng, Juan; Han, Xiao-Xia; Zhang, Qing-Chun; Yao, Hui-Qin; Gao, Zuo-Ning

    2015-06-01

    Copper sulfide nanoparticle-decorated graphene sheet (CuS/GR) was successfully synthesized and used as a signal amplification platform for electrochemical detection of alkaline phosphatase activity. First, CuS/GR was prepared through a microwave-assisted hydrothermal approach. The CuS/GR nanocomposites exhibited excellent electrocatalytic activity toward the oxidation of ALP hydrolyzed products such as 1-naphthol, which produced a current response. Thus, a catalytic amplification platform based on CuS/GR nanocomposite for electrochemical detection of ALP activity was designed using 1-naphthyl phosphate as a model substrate. The current response increased linearly with ALP concentration from 0.1 to 100 U L(-1) with a detection limit of 0.02 U L(-1). The assay was applied to estimate ALP activity in human serum samples with satisfactory results. This strategy may find widespread and promising applications in other sensing systems that involves ALP.

  1. Prophylactic treatment with alkaline phosphatase in cardiac surgery induces endogenous alkaline phosphatase release

    NARCIS (Netherlands)

    Kats, Suzanne; Brands, Ruud; Hamad, Mohamed A. Soliman; Seinen, Willem; Schamhorst, Volkher; Wulkan, Raymond W.; Schoenberger, Jacques P.; van Oeveren, Wim

    2012-01-01

    Introduction: Laboratory and clinical data have implicated endotoxin as an important factor in the inflammatory response to cardiopulmonary bypass. We assessed the effects of the administration of bovine intestinal alkaline phosphatase (bIAP), an endotoxin detoxifier, on alkaline phosphatase levels

  2. Comparison of the expression, activity, and fecal concentration of intestinal alkaline phosphatase between healthy dogs and dogs with chronic enteropathy.

    Science.gov (United States)

    Ide, Kaori; Kato, Kazuki; Sawa, Yuki; Hayashi, Akiko; Takizawa, Rei; Nishifuji, Koji

    2016-07-01

    OBJECTIVE To compare expression, activity, and fecal concentration of intestinal alkaline phosphatase (IAP) between healthy dogs and dogs with chronic enteropathy (CE). ANIMALS 9 healthy university-owned Beagles and 109 healthy client-owned dogs (controls) and 28 dogs with CE (cases). PROCEDURES Cases were defined as dogs with persistent (> 3 weeks) gastrointestinal signs that failed to respond to antimicrobials and anti-inflammatory doses of prednisolone or dietary trials, did not have mechanical gastrointestinal abnormalities as determined by abdominal radiography and ultrasonography, and had a diagnosis of lymphoplasmacytic enteritis or eosinophilic gastroenteritis on histologic examination of biopsy specimens. Duodenal and colonic mucosa biopsy specimens were obtained from the 9 university-owned Beagles and all cases for histologic examination and determination of IAP expression (by real-time quantitative PCR assay) and activity (by enzyme histochemical analysis). Fecal samples were obtained from all dogs for determination of fecal IAP concentration by a quantitative enzyme reaction assay. RESULTS For dogs evaluated, IAP expression and activity were localized at the luminal side of epithelial cells in the mucosa and intestinal crypts, although both were greater in the duodenum than in the colon. Active IAP was detected in the feces of all dogs. Intestinal alkaline phosphatase expression and activity were lower for cases than for controls, and fecal IAP concentration for dogs with moderate and severe CE was lower than that for dogs with mild CE. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that dogs with CE had impaired IAP expression and activity. Additional research is necessary to elucidate the role of IAP in the pathogenesis of CE. PMID:27347825

  3. Evaluating the levels of salivary alkaline and acid phosphatase activities as biochemical markers for periodontal disease: A case series

    Directory of Open Access Journals (Sweden)

    Sarita Dabra

    2012-01-01

    Full Text Available Background: The purpose of this study was to determine the salivary levels of alkaline phosphatase (ALP and acid phosphatase (ACP activities in patients with periodontal disease and to evaluate the use of these enzymes as biochemical markers for periodontal tissue damage. Materials and Methods: In this prospective analytical study, we examined the activities of salivary ALP and ACP in patients with periodontal disease, before and after periodontal treatment. The experimental groups consisted of 20 gingivitis patients and 20 periodontitis patients and the control group had healthy subjects (20 samples. The stimulated saliva of the patient was collected in a sterile test tube and analyzed using Hitachi′s Diagnostic Automatic Analyser. Periodontal disease was determined based on clinical parameters such as gingival index, probing depth and clinical attachment loss. Patients with periodontal disease were under conventional periodontal treatment. The statistical analysis applied was Student′s t-test. Probabilities less than 0.05 (P < 0.05 were considered significant. Results: The obtained results showed statistically significant increased activities of ALP and ACP in saliva from patients with periodontal disease in relation to control group. A significant reduction in the enzyme levels was seen after conventional periodontal therapy. Conclusions: Based on these results, salivary ALP and ACP can be considered to be the biomarkers for evaluating periodontal tissue damage.

  4. Temperature dependence of the absorbance of alkaline solutions of 4-nitrophenyl phosphate--a potential source of error in the measurement of alkaline phosphatase activity.

    Science.gov (United States)

    Burtis, C A; Seibert, L E; Baird, M A; Sampson, E J

    1977-09-01

    The absorbance of an alkaline solution of 4-nitrophenyl phosphate is a function of temperature. Quantitative evaluation of this phenomenon indicates that it (a) depends on the concentration of the compound and is independent of source, buffer concentration, and pH above 9.0; (b) is reversible; (c) is not a result of alkaline hydrolysis or 4-nitrophenol contamination; and (d) correlates with a temperature-induced shift of its absorbance spectrum. The phenomenon may represent a potential analytical problem in methods for alkaline phosphatase in which this compound is the substrate. If thermal equilibrium is not reached and maintained during an alkaline phosphatase assay, the thermochromic response will be included in the measured rate. The magnitude of this error depends on the thermal response and control characteristics of each particular instrument and the reaction conditions under which such an analysis is performed. PMID:19164

  5. Effect of noise exposure (85 dB ) on testicular adrenocortical steroidogenic key enzymes, acid and alkaline phosphatase activities of sex organs in mature albino rats

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Changes in the activities of △5-3β-hydroysteroid dehydrogenase (HSD) in testis and adrenal gland, 17β-hydroxysteroid dehydrogenase in testis, acid and alkaline phosphatase in testis, prostate and seminal vesicle were observed in noise exposed mature rats at the intensity of 85 dB for 8 h/day for 45 days. The results indicated that noise exposed group showed a significant diminution in the activities of androgenic key enzymes △5-3β and 17β-HSD, acid phosphatase in testis, prostate and seminal vesicle. There was a significant elevation in the activities of adrenal △5-3β-HSD, alkaline phosphatase in testis and other accessory sex organ in noise exposed group. Gonadosomatic, prostatosomatic and seminal vesiculo-somatic indexes were decreased significantly in noise exposed group. Therefore, it is evident that noise exposure at 85dB exerts a deleterious effect on testicular and adrenocortical activities.

  6. Endotoxin detoxification by alkaline phosphatase in cholestatic livers

    NARCIS (Netherlands)

    Poelstra, K; Bakker, WW; Hardonk, MJ; Meijer, DKF; Wisse, E; Knook, DL; Balabaud, C

    1997-01-01

    Increased expression of alkaline phosphatase (AP) in the liver is a hallmark of cholestasis but the pathophysiological role of this is not clear. We argue that deprotonation of carboxyl groups at the active site of the enzyme may be a prerequisite for optimal AP activity. Such a creation of negative

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

    DEFF Research Database (Denmark)

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

    2005-01-01

    The intestinal alkaline phosphatase gene (ALPI) encodes a digestive brush-border enzyme, which is highly upregulated during small intestinal epithelial cell differentiation. To identify new putative promoter motifs responsible for the regulation of ALPI expression during differentiation of the en......The intestinal alkaline phosphatase gene (ALPI) encodes a digestive brush-border enzyme, which is highly upregulated during small intestinal epithelial cell differentiation. To identify new putative promoter motifs responsible for the regulation of ALPI expression during differentiation...... of the enterocytes, we have conducted a computer-assisted cis-element search of the proximal human ALPI promoter sequence. A putative recognition site for the transcription factor hepatocyte nuclear factor (HNF)-4 was predicted at the positions from -94 to -82 in relation to the translational start site. The ability...

  8. Monitoring the activity and inhibition of alkaline phosphatase via quenching and restoration of the fluorescence of carbon dots

    International Nuclear Information System (INIS)

    We report that the fluorescence of carbon dots (C-dots) in water is quenched by the addition of Cu2+ ions, and that the subsequent addition of pyrophosphate (PPi) restores fluorescence. This is likely to be due to the coordination of Cu2+ by PPi. This effect forms the basis for a method to determine the activity and inhibition of the enzyme alkaline phosphatase (ALP). If ALP is added to a system composed of C-dots, Cu2+ and PPi, fluorescence will decrease over time because ALP catalyzes the hydrolysis of PPi to form orthophosphate (Pi). This results in a release of the quencher Cu2+. The decrease in fluorescence is related to the activity of ALP. The method is simple and displays good sensitivity (with a limit of detection of 1 units per L) and selectivity. The method was successfully applied to the determination of ALP in serum samples. We also have studied the inhibitory effect of Pi on the activity of ALP. We presume that this method holds a large potential in terms of diagnosis of ALP-related diseases, to evaluate the function of ALP in biological systems and in screening for potential inhibitors of ALP. (author)

  9. Assessment of lactate dehydrogenase, alkaline phosphatase and aspartate aminotransferase activities in cow's milk as an indicator of subclinical mastitis.

    Science.gov (United States)

    Babaei, H; Mansouri-Najand, L; Molaei, M M; Kheradmand, A; Sharifan, M

    2007-05-01

    This study examined the activities of lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in the milk of lactating Holstein cows in association with subclinical mastitis (SCM). A total of 94 milk samples were collected from 58 lactating dairy cows representing stages of lactation from the second to the tenth week after calving. Those which were classified as positive by California mastitis test (CMT) were deemed to have subclinical mastitis. All the milk samples were skimmed by centrifugation at 10 000g at 0 degrees C and were used for enzyme activities estimations. The mean activities of LDH and ALP were higher in the milk from udders with SCM than in the milk from healthy udders (p CMT results and LDH and ALP values were seen at thresholds of > 180 IU/L and > 40 IU/L respectively (kappa values 0.65 and 0.79, respectively). However, the sensitivity of the tests for identifying SCM at these thresholds was higher for ALP (96.4%) than for LDH (68.5%). In this study, LDH and ALP tests were standardized for cow's milk and results showed that only the ALP test was reliable in the early diagnosis of subclinical mastitis. PMID:17268916

  10. Low dietary copper increases fecal free radical production, fecal water alkaline phosphatase activity and cytotoxicity in healthy men.

    Science.gov (United States)

    Davis, Cindy D

    2003-02-01

    One possible dietary factor that may increase susceptibility to colon cancer is inadequate copper intake. The objective of this study was to investigate the effects of low and adequate copper intakes on copper nutriture and putative risk factors for colon cancer susceptibility in healthy men. Seventeen healthy free-living nonsmoking men aged 21-52 y completed a 13-wk controlled feeding study in a randomized crossover design. The basal diet contained 0.59 mg Cu/13.65 MJ. After a 1-wk equilibration period in which the men consumed the basal diet supplemented with 1.0 mg Cu/d, they were randomly assigned to receive either the basal diet or the basal diet supplemented with 2 mg Cu/d for 6 wk. After the first dietary period, the men immediately began to consume the other level of Cu for the last 6 wk. They collected their feces during the equilibration period and during the last 2 wk of the two dietary periods for free radical and fecal water analysis. Low dietary copper significantly (P copper significantly (P copper concentrations but did not affect fecal water volume, pH, iron or zinc concentrations. In contrast to the fecal analysis, hematological indicators of copper status were not significantly affected by the dietary treatments. These results suggest that low dietary copper adversely affects fecal free radical production and fecal water alkaline phosphatase activity, which are putative risk factors for colon cancer.

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

    Science.gov (United States)

    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.

  12. Evaluation of Milk Trace Elements, Lactate Dehydrogenase, Alkaline Phosphatase and Aspartate Aminotransferase Activity of Subclinical Mastitis as and Indicator of Subclinical Mastitis in Riverine Buffalo (Bubalus bubalis)

    OpenAIRE

    Guha, Anirban; Gera, Sandeep; Sharma, Anshu

    2012-01-01

    Mastitis is a highly morbid disease that requires detection at the subclinical stage. Tropical countries like India mainly depend on milch buffaloes for milk. The present study was conducted to investigate whether the trace minerals viz. copper (Cu), iron (Fe), zinc (Zn), cobalt (Co) and manganese (Mn) and enzyme activity of lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in riverine buffalo milk can be used as an indicator of subclinical mastitis ...

  13. Evaluation of alkaline phosphatase activity and availability of various P fractions for bean (Phaseolus vulgaris in some calcareous soils amended with municipal sewage sludge

    Directory of Open Access Journals (Sweden)

    T. Raeisi

    2014-07-01

    Full Text Available To evaluate the relationship of various P fractions and alkaline phosphatase activity with bean indices growing in 10 calcareous soils, amended with municipal sewage sludge from Chaharmahal-Va-Bakhtiari province, a greenhouse research was carried out. Soil samples were incubated for one month with sludge at a rate equivalent to 1% (w/w. Then, the P fractions, including P adsorbed by Fe and Al oxides (]NaOH+CB]-P, occluded P (CBD-P and P absorbed by Ca (HCl-P, were determined by Olsen and Summers' sequential fractionation procedure. Furthermore, total P, organic P and residual P were determined. Also, alkaline phosphatase activity was measured. A pot experiment in a completely randomized design with three replications in the ten soils was done to evaluate the bean plant indices. The results showed that the amount of P fractions decreased in the following order: HCl-P>residual-P>]NaOH+CB]-P > OP>CBD-P. The results also indicated that alkaline phosphatase activity was significantly correlated with CBD-P fraction, organic P and total P. In addition, significant correlations were found between ([NaOH+CB]-P and HCl-P and plant shoots. In general, the results of this research showed that P fractionation method appears to be a powerful tool to identify the P status and availability in the soils amended with sewage sludge.

  14. Purification and properties of alkaline phosphatase of silkworm Bombyx mori

    Institute of Scientific and Technical Information of China (English)

    TANG Yunming; CEN Liang; CHU Bo; LI Changchun; XU Min; LUO Ying; LU Cheng

    2006-01-01

    Alkaline phosphatase(AKP),from the succus entericus of silkworm,was purified using 10%-50% ammonium sulfate fractions,ion exchange chromatography Of DEAE-Sepharose,and size exclusion chromatography of Sephacryl S-200.The purification fold was 464 times and specified activity was 3936 U/mg.Optimum pH value of the phosphatase was 10.5,and was stable between pH 7.5 and 11.The optimum temperature of the phosphatase was 40℃ and it was unstable over 50℃.Km value of the phosphatase was 1.25 mmol/L.In a given condition,the phosphatase was selectively modified by PCMB,NBS,PMSE TNBS,SUAN,DTT,BrAc,and IAc,the results indicate that PMSF,SUA,BrAc,IAc,and TNBS could Obviously inhibit the activity of the phosphatase,and the degree of inhibition depended on the concentration of these reagents.There was little effect on the activity of phosphatase after treatment by PMSF,DTT,and NBT.We primarily conclude that mercapto and imidazole are essential for AKP from silkworm.Also,Lys residue and disulfide bands are necessary to protect the catalysis of the AKP.

  15. Effects of Dihydroartemisinin and Artemether on the Growth, Chlorophyll Fluorescence, and Extracellular Alkaline Phosphatase Activity of the Cyanobacterium Microcystis aeruginosa

    Science.gov (United States)

    Wang, Shoubing; Xu, Ziran

    2016-01-01

    Increased eutrophication in the recent years has resulted in considerable research focus on identification of methods for preventing cyanobacterial blooms that are rapid and efficient. The objectives of this study were to investigate the effects of dihydroartemisinin and artemether on the growth of Microcystis aeruginosa and to elucidate its mode of action. Variations in cell density, chlorophyll a, soluble protein, malondialdehyde, extracellular alkaline phosphatase activity (APA), and chlorophyll fluorescence parameters (Fv/Fm, ΦPSII, ETR, rapid light curves, fast chlorophyll fluorescence curves on fluorescence intensity, and relative variable fluorescence) were evaluated by lab-cultured experiments. Our results demonstrated that both dihydroartemisinin and artemether inhibited the growth of M.aeruginosa by impairing the photosynthetic center in photosystem II and reducing extracellular APA, with a higher sensitivity exhibited toward artemether. The inhibitory effects of dihydroartemisinin on M.aeruginosa increased with concentration, and the maximum growth inhibitory rate was 42.17% at 24 mg·L-1 after 120h exposure, whereas it was 55.72% at 6 mg·L-1 artemetherafter 120h exposure. Moreover, the chlorophyll fluorescence was significantly inhibited (p<0.05) after 120h exposure to 12 and 24 mg·L-1 dihydroartemisinin. Furthermore, after 120h exposure to 6 mg·L-1 artemether, Fv/Fm, ΦPSII, ETR and rETRmax showed a significant decrease (p<0.01) from initial values of 0.490, 0.516, 17.333, and 104.800, respectively, to 0. One-way analysis of variance showed that 6 mg·L-1 artemether and 24 mg·L-1 dihydroartemisinin had significant inhibitory effects on extracellular APA (p<0.01). The results of this study would be useful to further studies to validate the feasibility of dihydroartemisinin and artemether treatment to inhibit overall cyanobacterial growth in water bodies, before this can be put into practice. PMID:27755566

  16. Presence and patterns of alkaline phosphatase activity and phosphorus cycling in natural riparian zones under changing nutrient conditions

    Directory of Open Access Journals (Sweden)

    Peifang Wang

    2014-08-01

    Full Text Available Phosphorus (P is an important limiting nutrient in aquatic ecosystems and knowledge of P cycling is fundamental for reducing harmful algae blooms and other negative effects in water. Despite their importance, the characteristics of P cycling under changing nutrient conditions in shallow lakes were poorly investigated. In this study, in situ incubation experiments were conducted in a natural riparian zone in the main diversion channel used for water transfer into Lake Taihu (Wangyu River. Variations in microbial biomass, dissolved P fractions (organic and inorganic, and alkaline phosphatase activity (bulk APA and specific APA were determined after incubation with and without the addition of P and nitrogen (N (4 total water treatments: +P, +N, +NP, and control. Experiments were conducted during two seasons (late spring and early fall to account for natural differences in nutrient levels that may occur in situ. Our results demonstrated that low levels of DRP may not necessarily indicate P limitation. Phytoplankton exhibited “serial N limitation with P stress” in May, such that chlorophyll a (Chl a increased significantly with N addition, while the limiting nutrient shifted to P in October and phytoplankton biomass increased with P addition. Phytoplankton contributed greatly to APA production and was significantly influenced by P bioavailability, yet high levels of bulk APA were also not necessarily indicative of P limitation. In contrast to phytoplankton, bacteria were less P stressed. As a consequence of enhanced utilization of dissolved reactive P (DRP and dissolved organic P (DOP, +N treatment elevated APA significantly. By contrast, APA could be repressed to low values and phytoplankton converted a large portion of DRP to DOP with P addition. But this was not consistent with bacteria APA (bact-APA in the absence or presence of abundant phytoplankton biomass. The correlation between bulk APA and DRP was good at separate sites and discrepant

  17. Alkaline phosphatase activity: new assay for the Reflotron system. Results of the evaluation in eight clinical laboratories.

    Science.gov (United States)

    Schumann, G; Dominick, H C; Hellmann, D; Klauke, R; Möckesch, M; Stekel, H; von Schenck, H; Kraft, M; Nagel, R; Hänseler, E

    2001-01-01

    A new reagent carrier, Reflotron ALP, has been developed for the Reflotron system, allowing easy and rapid measurement (in less than 3 minutes) of alkaline phosphatase (ALP) activity in capillary blood, venous blood, heparinized plasma or serum. The evaluation of the analytical performance of the assay was carried out at eight clinical laboratories. The study of the imprecision using the measurements in human samples resulted in coefficients of variation ranging from 1.3% to 4.6% (within-run) and from 3.2% to 4.0% (day-to-day). The analytical specificity of the Reflotron ALP assay agrees well with ALP methods using a N-methyl-D-glucamine buffer solution. The calibration of the Reflotron ALP assay, however, is related to the reference intervals for ALP methods using a diethanolamine buffer solution. Method comparisons were performed with the ALP method on Hitachi instruments using diethanolamine buffer. Reflotron ALP measurements in blood and plasma in 157 randomly selected split samples showed excellent agreement (slope: 0.99; intercept: 0.7 U/l; median bias: 2.3%; median difference from the comparison method: -0.3%). Specimens from pregnant women and adolescents were excluded from this study. Differing values were obtained in a method comparison using 48 samples containing predominantly the ALP bone isoform (slope: 0.81; intercept: 31.5 U/l; median bias: 5.7%; median difference from the comparison method: -12.2%). Regression analysis of the results from 21 sera with prevailing placental ALP gave a slope of 1.51, and an intercept of -41.1 U/l (median bias: 8.6%; median difference from the comparison method: 35.6%). Reflotron ALP was compared with three different wet chemistry procedures using different buffer compounds: N-methyl-D-glucamine or diethanolamine or 2-amino-2-methyl-1-propanol. In samples containing predominantly ALP isoforms not of liver origin, the measurements with N-methyl-D-glucamine buffer gave the best fit with respect to Reflotron. In an

  18. Francisella DnaK Inhibits Tissue-nonspecific Alkaline Phosphatase*

    Science.gov (United States)

    Arulanandam, Bernard P.; Chetty, Senthilnath Lakshmana; Yu, Jieh-Juen; Leonard, Sean; Klose, Karl; Seshu, Janakiram; Cap, Andrew; Valdes, James J.; Chambers, James P.

    2012-01-01

    Following pulmonary infection with Francisella tularensis, we observed an unexpected but significant reduction of alkaline phosphatase, an enzyme normally up-regulated following inflammation. However, no reduction was observed in mice infected with a closely related Gram-negative pneumonic organism (Klebsiella pneumoniae) suggesting the inhibition may be Francisella-specific. In similar fashion to in vivo observations, addition of Francisella lysate to exogenous alkaline phosphatase (tissue-nonspecific isozyme) was inhibitory. Partial purification and subsequent proteomic analysis indicated the inhibitory factor to be the heat shock protein DnaK. Incubation with increasing amounts of anti-DnaK antibody reduced the inhibitory effect in a dose-dependent manner. Furthermore, DnaK contains an adenosine triphosphate binding domain at its N terminus, and addition of adenosine triphosphate enhances dissociation of DnaK with its target protein, e.g. alkaline phosphatase. Addition of adenosine triphosphate resulted in decreased DnaK co-immunoprecipitated with alkaline phosphatase as well as reduction of Francisella-mediated alkaline phosphatase inhibition further supporting the binding of Francisella DnaK to alkaline phosphatase. Release of DnaK via secretion and/or bacterial cell lysis into the extracellular milieu and inhibition of plasma alkaline phosphatase could promote an orchestrated, inflammatory response advantageous to Francisella. PMID:22923614

  19. Inhibition of Alkaline Phosphatase from Pearl Oyster Pinctada fucata by o-Phthalaldehyde: Involvement of Lysine and Histidine Residues at the Active Site

    Institute of Scientific and Technical Information of China (English)

    CHEN Hongtao; XIE Liping; YU Zhenyan; ZHANG Rongqing

    2005-01-01

    Alkaline phosphatase from Pinctada fucata was inactivated by o-phthalaldehyde (OPA). The inactivation followed pseudo first-order kinetics with a second rate constant of 0.167 (mmol/L)-1·min-1 at pH 7.5 and 25°C. A Tsou's plot analysis showed that inactivation occurred upon formation of one isoindole group. The OPA-modified enzyme lost the ability to bind with the specific affinity column and the presence of substrates or competitive inhibitors protected the enzyme from inactivation. The results revealed that the OPA-reaction site was at the enzyme substrate binding site. Prior modification of the enzyme by lysine or histidine specific reagent abolished formation of the isoindole derivatives, suggesting that lysine and histidine residues were involved in the OPA-induced inactivation. Taken together, OPA inactivated the alkaline phosphatase from Pinctada fucata by cross-linking lysine and histidine residues at the active site and formed an isoindole group at the substrate binding site of the enzyme.

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

    International Nuclear Information System (INIS)

    A cDNA clone for human adult intestinal alkaline phosphatase (ALP) [orthophosphoric-monoester phosphohydrolase (alkaline optimum); EC 3.1.3.1] was isolated from a λgt11 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

  1. Placental-type alkaline phosphatase in cervical neoplasia.

    OpenAIRE

    McLaughlin, P. J.; Warne, P H; Hutchinson, G. E.; Johnson, P. M.; Tucker, D. F.

    1987-01-01

    Monoclonal antibodies reactive with placental-type alkaline phosphatase have formed the basis of methods for detection of this oncodevelopmental antigen in patients with pre-invasive and invasive cervical neoplasia, with or without evidence of papilloma virus infection. Disease-related elevations of placental-type alkaline phosphatase were not observed in patients' sera. Solubilised cervical smears or biopsy material, and cervical mucus swabs, often contained substantial amounts of this isoen...

  2. Chromatographic separation of alkaline phosphatase from dental enamel

    DEFF Research Database (Denmark)

    Moe, D; Kirkeby, S; Salling, E

    1989-01-01

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

  3. 21 CFR 862.1050 - Alkaline phosphatase or isoenzymes test system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Alkaline phosphatase or isoenzymes test system... Test Systems § 862.1050 Alkaline phosphatase or isoenzymes test system. (a) Identification. An alkaline phosphatase or isoenzymes test system is a device intended to measure alkaline phosphatase or its...

  4. Optical Algal Biosensor using Alkaline Phosphatase for Determination of Heavy Metals

    OpenAIRE

    Durrieu, Claude; Tran-Minh, Canh

    2002-01-01

    International audience A biosensor is constructed to detect heavy metals from inhibition of alkaline phosphatase (AP) present on the external membrane of Chlorella vulgaris microalgae. The microalgal cells are immobilized on removable membranes placed in front of the tip of an optical fiber bundle inside a homemade microcell. C. vulgaris was cultivated in the laboratory and its alkaline phosphatase activity is strongly inhibited in the presence of heavy metals. This property has been used ...

  5. Variation of Alkaline Phosphatase Activity in Sediments of Shrimp Culture Ponds and Its Relationship with the Contents of C, N and P

    Institute of Scientific and Technical Information of China (English)

    SU Yuepeng; MA Shen; DONG Shuanglin

    2005-01-01

    Nine enclosures (5 m × 5 m) were built in a Fenneropenaeus chinensis culture pond of Rushan Gulf in April, 2001.The probiotics and BIO ENERGIZER solution were applied for disparate treatments. Variations of alkaline phosphatase activity (APA) and its relationship with the contents of C, N and P in sediments were studied. Results show that APA of sediments increases from 3.096 nmol g-1min-1 to 5.407 nmol g-1nin-1 in culture period; the bacteria biomass is not the only factor to determine APA; the contents of total P and total organic carbon have a significant positive correlation with APA, while that of total nitrogen has a negative correlation. In addition, the contents of inorganic P and organic P are not regular with APA. By comparison, TOC shows a more significant coherence with APA, meaning that organic pollution in sediments affects APA remarkably.

  6. Serum alkaline phosphatase screening for vitamin D deficiency states

    International Nuclear Information System (INIS)

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

  7. Expression and Characterization of Recombinant Thermostable Alkaline Phosphatase from a Novel Thermophilic Bacterium Thermus thermophilus XM

    Institute of Scientific and Technical Information of China (English)

    Jianbo LI; Limei XU; Feng YANG

    2007-01-01

    A gene (tap) encoding a thermostable alkaline phosphatase from the thermophilic bacterium Thermus thermophilus XM was cloned and sequenced. It is 1506 bp long and encodes a protein of 501 amino acid residues with a calculated molecular mass of 54.7 kDa. Comparison of the deduced amino acid sequence with other alkaline phosphatases showed that the regions in the vicinity of the phosphorylation site and metal binding sites are highly conserved. The recombinant thermostable alkaline phosphatase was expressed as a His6-tagged fusion protein in Escherichia coli and its enzymatic properties were characterized after purification. The pH and temperature optima for the recombinant thermostable alkaline phosphatases activity were pH 12 and 75 ℃. As expected, the enzyme displayed high thermostability, retaining more than 50% activity after incubating for 6 h at 80 ℃. Its catalytic function was accelerated in the presence of 0.1 mM Co2+, Fe2+, Mg2+, or Mn2+ but was strongly inhibited by 2.0 mM Fe2+. Under optimal conditions, the Michaelis constant (Km) for cleavage of p-nitrophenyl-phosphate was 0.034 mM. Although it has much in common with other alkaline phosphatases, the recombinant thermostable alkaline phosphatase possesses some unique features, such as high optimal pH and good thermostability.

  8. CONTROL OF ALKALINE PHOSPHATASE ACTIVITY IN C3H10T1/2 CELLS: ROLE OF RETINOIC ACID AND CELL DENSITY

    Science.gov (United States)

    The enzyme alkaline phosphatase (AP) has been shown to be lost or inappropriately expressed during carcinogenesis in some tissues. ecause retinoic acid (RA) appears to play a role in the normal regulation of the enzyme (RA up-regulates AP in a variety of cell types) we have sugge...

  9. Dairy products and the French paradox: Could alkaline phosphatases play a role?

    Science.gov (United States)

    Lallès, Jean-Paul

    2016-07-01

    The French paradox - high saturated fat consumption but low incidence of cardiovascular disease (CVD) and mortality - is still unresolved and continues to be a matter of debate and controversy. Recently, it was hypothesised that the high consumption of dairy products, and especially cheese by the French population might contribute to the explanation of the French paradox, in addition to the "(red) wine" hypothesis. Most notably this would involve milk bioactive peptides and biomolecules from cheese moulds. Here, we support the "dairy products" hypothesis further by proposing the "alkaline phosphatase" hypothesis. First, intestinal alkaline phosphatase (IAP), a potent endogenous anti-inflammatory enzyme, is directly stimulated by various components of milk (e.g. casein, calcium, lactose and even fat). This enzyme dephosphorylates and thus detoxifies pro-inflammatory microbial components like lipopolysaccharide, making them unable to trigger inflammatory responses and generate chronic low-grade inflammation leading to insulin resistance, glucose intolerance, type-2 diabetes, metabolic syndrome and obesity, known risk factors for CVD. Various vitamins present in high amounts in dairy products (e.g. vitamins A and D; methyl-donors: folate and vitamin B12), and also fermentation products such as butyrate and propionate found e.g. in cheese, all stimulate intestinal alkaline phosphatase. Second, moulded cheeses like Roquefort contain fungi producing an alkaline phosphatase. Third, milk itself contains a tissue nonspecific isoform of alkaline phosphatase that may function as IAP. Milk alkaline phosphatase is present in raw milk and dairy products increasingly consumed in France. It is deactivated by pasteurization but it can partially reactivate after thermal treatment. Experimental consolidation of the "alkaline phosphatase" hypothesis will require further work including: systematic alkaline phosphatase activity measurements in dairy products, live dairy ferments and

  10. Effects of sediment and turbulence on alkaline phosphatase activity and photosynthetic activity of phytoplankton in the shallow hyper-eutrophic Lake Taihu, China.

    Science.gov (United States)

    Ding, Yanqing; Qin, Boqiang; Xu, Hai; Wang, Xiaodong

    2016-08-01

    Sediments play important roles, as nutrient reservoir, especially in shallow lake ecosystem. The water column of large shallow lakes is often stable but also disturbed by turbulence causing resuspension of sediments. While considerable research has been carried out to investigate the influence of sediment resuspension on nutrient release, fewer studies have been done to understand the contribution of alkaline phosphatase activity (APA) in water as a response to the two conditions (turbulence and stability). Also, effects of the two lake conditions on photosynthetic efficiency of phytoplankton are still poorly understood. This study will evaluate the effect of these two conditions on photosynthetic efficiency and APA. Sediments used in the indoor experiments were collected from Zhushan Bay in Lake Taihu. Turbulence was generated by rotors to simulate the strong wind-induced disturbance in Lake Taihu. Results of the experiments showed that TN and TP in the stable and episodically turbulent conditions were not significantly different, with TN ranging from 1.34 to 1.90 mg/L and TP from 0.08 to 0.18 mg/L. Whereas, the soluble reactive phosphorus in the episodically turbulent condition was significantly higher than in the stable condition. Episodic turbulence could enhance P cycling by resuspending sediment-associated P, which alleviated algal P limitation. In stable conditions, P deficiency induced the production of high APA, which enhanced the availability of P. Although episodic turbulence could also cause increased algal biomass, photosynthetic efficiency of the algae was also affected not only by the nutrients but also by many other factors, especially light availability. Our results suggest that episodic turbulence is an important driver of biogeochemical cycling in large shallow hypertrophic lake ecosystem. PMID:27151245

  11. Targeting the active site of the placental isozyme of alkaline phosphatase by phage-displayed scFv antibodies selected by a specific uncompetitive inhibitor

    Directory of Open Access Journals (Sweden)

    Kala Mrinalini

    2005-12-01

    Full Text Available Abstract Background The isozymes of alkaline phosphatase, the tissue non-specific, intestinal and placental, have similar properties and a high degree of identity. The placental isozyme (PLAP is an oncofetal antigen expressed in several malignancies including choriocarcinoma, seminoma and ovarian carcinoma. We had earlier attempted to isolate PLAP-specific scFv from a synthetic human immunoglobulin library but were unable to do so, presumably because of the similarity between the isozymes. In this work, we have employed a PLAP-specific uncompetitive inhibitor, L-Phe-Gly-Gly, to select isozyme specific scFvs. An uncompetitive inhibitor binds to the enzyme in the presence of substrate and stabilizes the enzyme-substrate complex. Several uncompetitive inhibitors have varying degrees of isozyme specificity for human alkaline phosphatase isozymes. A specific uncompetitive inhibitor would be able to unmask conformational differences between the otherwise very similar molecules. Also, such inhibitors would be directed to regions at/close to the active site of the enzyme. In this work, the library was first incubated with PLAP and the bound clones then eluted by incubation with L-Phe-Gly-Gly along with the substrate, para-nitro phenyl phosphate (pNPP. The scFvs were then studied with regard to the biochemical modulation of their binding, isozyme specificity and effect on enzyme activity. Results Of 13 clones studied initially, the binding of 9 was inhibited by L-Phe-Gly-Gly (with pNPP and 2 clones were inhibited by pNPP alone. Two clones had absolute and 2 clones had partial specificity to PLAP. Two clones were cross-reactive with only one other isozyme. Three scFv clones, having an accessible His6-tag, were purified and studied for their modulation of enzyme activity. All the three scFvs inhibited PLAP activity with the kinetics of competitive inhibition. Cell ELISA could demonstrate binding of the specific scFvs to the cell surface expressed PLAP

  12. Direct Promotion of Collagen Calcification by Alkaline Phosphatase

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Alkaline phosphatase promotes hydrolysis of phosphate containing substrates, causes a rise in inorganic phosphate and, therefore, enhances calcification of biological tissues. In this work, the calcification of collagen in a model serum was used as a model of collagenous tissue biomaterials to study the possible calcification promotion mechanism of alkaline phosphatase. In the enzyme concentration range of 0.10.5mg/mL, the enzyme shows a direct calcification promoting effect which is independent of the hydrolysis of its phosphate containing substrates but proportional to the enzyme concentration. Potassium pyrophosphate somewhat inhibits the calcification promotion.

  13. Arbuscular mycorrhizal fungal diversity, root colonization, and soil alkaline phosphatase activity in response to maize-wheat rotation and no-tillage in North China.

    Science.gov (United States)

    Hu, Junli; Yang, Anna; Zhu, Anning; Wang, Junhua; Dai, Jue; Wong, Ming Hung; Lin, Xiangui

    2015-07-01

    Monitoring the effects of no-tillage (NT) in comparison with conventional tillage (CT) on soil microbes could improve our understanding of soil biochemical processes and thus help us to develop sound management strategies. The objective of this study was to compare the species composition and ecological function of soil arbuscular mycorrhizal (AM) fungi during the growth and rotation of crops under NT and CT. From late June 2009 to early June 2010, 32 topsoil (0-15 cm) samples from four individual plots per treatment (CT and NT) were collected at both the jointing and maturation stages of maize (Zea mays L.) and wheat (Triticum aestivum L.) from a long-term experimental field that was established in an Aquic Inceptisol in North China in June 2006. The AM fungal spores were isolated and identified and then used to calculate species diversity indices, including the Shannon- Wiener index (H'), Evenness (E), and Simpson's index (D). The root mycorrhizal colonization and soil alkaline phosphatase activity were also determined. A total of 34 species of AM fungi within nine genera were recorded. Compared with NT, CT negatively affected the soil AM fungal community at the maize sowing stage, leading to decreases in the average diversity indices (from 2.12, 0.79, and 0.82 to 1.79, 0.72, and 0.74 for H', E, and D, respectively), root mycorrhizal colonization (from 28% to 20%), soil alkaline phosphatase activity (from 0.24 to 0.19 mg/g/24 h) and available phosphorus concentration (from 17.4 to 10.5 mg/kg) at the maize jointing stage. However, reductions in diversity indices of H', E, and D were restored to 2.20, 0.81, and 0.84, respectively, at the maize maturation stage. CT should affect the community again at the wheat sowing stage; however, a similar restoration in the species diversity of AM fungi was completed before the wheat jointing stage, and the highest Jaccard index (0.800) for similarity in the species composition of soil AM fungi between CT and NT was recorded at

  14. Bone alkaline phosphatase and mortality in dialysis patients

    NARCIS (Netherlands)

    C. Drechsler; M. Verduijn; S. Pilz; R.T. Krediet; F.W. Dekker; C. Wanner; M. Ketteler; E.W. Boeschoten; V. Brandenburg

    2011-01-01

    Serum alkaline phosphatase (AP) is associated with vascular calcification and mortality in hemodialysis patients, but AP derives from various tissues of origin. The aim of this study was to assess the effect of bone-specific AP (BAP) on morbidity and mortality in dialysis patients. From a prospectiv

  15. A physiologic function for alkaline phosphatase : Endotoxin detoxification

    NARCIS (Netherlands)

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

    1997-01-01

    Alkaline phosphatase (AP), a common enzyme present in many species including humans, has been studied extensively. Although the enzyme is routinely applied as a marker for liver function, its biologic relevance is poorly understood. The reason for this is obvious: the pH optimum of AP in vitro, as m

  16. Evaluation of Milk Trace Elements, Lactate Dehydrogenase, Alkaline Phosphatase and Aspartate Aminotransferase Activity of Subclinical Mastitis as and Indicator of Subclinical Mastitis in Riverine Buffalo (Bubalus bubalis).

    Science.gov (United States)

    Guha, Anirban; Gera, Sandeep; Sharma, Anshu

    2012-03-01

    Mastitis is a highly morbid disease that requires detection at the subclinical stage. Tropical countries like India mainly depend on milch buffaloes for milk. The present study was conducted to investigate whether the trace minerals viz. copper (Cu), iron (Fe), zinc (Zn), cobalt (Co) and manganese (Mn) and enzyme activity of lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in riverine buffalo milk can be used as an indicator of subclinical mastitis (SCM) with the aim of developing suitable diagnostic kit for SCM. Trace elements and enzyme activity in milk were estimated with Atomic absorption Spectrophotometer, GBC 932 plus and biochemical methods, respectively. Somatic cell count (SCC) was done microscopically. The cultural examination revealed Gram positive bacteria as the most prevalent etiological agent. A statistically significant (pbenchmark. Only ALP and Zn, the former being superior, were found to be suitable for diagnosis of SCM irrespective of etiological agents. LDH, Co and Fe can be introduced in the screening programs where Gram positive bacteria are omnipresent. It is recommended that both ALP and Zn be measured together in milk to diagnose buffalo SCM, irrespective of etiology. PMID:25049573

  17. A fluorometric assay for alkaline phosphatase activity based on β-cyclodextrin-modified carbon quantum dots through host-guest recognition.

    Science.gov (United States)

    Tang, Cong; Qian, Zhaosheng; Huang, Yuanyuan; Xu, Jiamin; Ao, Hang; Zhao, Meizhi; Zhou, Jin; Chen, Jianrong; Feng, Hui

    2016-09-15

    A convenient, reliable and highly sensitive assay for alkaline phosphatase (ALP) activity in the real-time manner is developed based on β-cyclodextrin-modified carbon quantum dots (β-CD-CQDs) nanoprobe through specific host-guest recognition. Carbon quantum dots were first functionalized with 3-aminophenyl boronic acid to produce boronic acid-functionalized CQDs, and then further modified with hydropropyl β-cyclodextrins (β-CD) through B-O bonds to form β-CD-CQDs nanoprobe. p-Nitrophenol phosphate disodium salt is used as the substrate of ALP, and can hydrolyze to p-nitrophenol under the catalysis of ALP. The resulting p-nitrophenol can enter the cavity of β-CD moiety in the nanoprobe due to their specific host-guest recognition, where photoinduced electron transfer process between p-nitrophenol and CQDs takes place to efficiently quench the fluorescence of the probe. The correlation between quenched fluorescence and ALP level can be used to establish quantitative evaluation of ALP activity in a broad range from 3.4 to 100.0U/L with the detection limit of 0.9U/L. This assay shows a high sensitivity to ALP even in the presence of a very high concentration of glucose. This study demonstrates a good electron donor/acceptor pair, which can be used to design general detection strategy through PET process, and also broadens the application of host-guest recognition for enzymes detection in clinical practice. PMID:27132001

  18. Copper(II) complexes with cyanoguanidine and o-phenanthroline: Theoretical studies, in vitro antimicrobial activity and alkaline phosphatase inhibitory effect

    Science.gov (United States)

    Martínez Medina, Juan J.; Islas, María S.; López Tévez, Libertad L.; Ferrer, Evelina G.; Okulik, Nora B.; Williams, Patricia A. M.

    2014-01-01

    Calculations based on density functional methods are carried out for two Cu(II) complexes with cyanoguanidine (cnge) and o-phenanthroline (o-phen): [Cu(o-phen)2(cnge)](NO3)2ṡ2H2O (1) and [Cu(o-phen)(cnge)(H2O)(NO3)2] (2). The calculated geometrical parameters are in agreement with the experimental values. The results of Atoms in Molecules (AIM) topological analysis of the electron density indicate that the Cu-N(phen) bonds in complex (1) have lower electron density, suggesting that those bonds are stronger in complex (2). Moreover, the ionic character of the Cu-N bond in the complex (1) is slightly stronger than that in the complex (2) and this situation would explain the fact that only complex (2) was stable in water solution. For this reason, the antimicrobial and enzymatic assays were performed using complex (2). It is well known that the increased use of antibiotics has resulted in the development of resistant bacterial and fungal strains. In this context, the study of novel antimicrobial agents has an enormous importance and metal complexes represent an interesting alternative for the treatment of infectious diseases. The aim of this work is to prove the modification of some biological properties like antimicrobial activity or alkaline phosphatase inhibitory activity upon copper complexation.

  19. [Inhibition of alkaline phosphatase I of Pichia guilliermondii yeast in vitro and in vivo].

    Science.gov (United States)

    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

  20. Comparative evaluation of Schistosoma mansoni, Schistosoma intercalatum, and Schistosoma haematobium alkaline phosphatase antigenicity by the alkaline phosphatase immunoassay (APIA).

    Science.gov (United States)

    Cesari, I M; Ballén, D E; Mendoza, L; Ferrer, A; Pointier, J-P; Kombila, M; Richard-Lenoble, D; Théron, A

    2014-04-01

    To know if alkaline phosphatase (AP) from schistosomes other than Schistosoma mansoni can be used as diagnostic marker for schistosomiasis in alkaline phosphatase immunocapture assay (APIA), we comparatively tested n-butanol extracts of adult worm membranes from a Venezuelan (JL) strain of S. mansoni (Ven/AWBE/Sm); a Cameroonian (EDEN) strain of Schistosoma intercalatum (Cam/AWBE/Si) and a Yemeni strain of Schistosoma haematobium (Yem/AWBE/Sh). APIA was evaluated with sera of patients from Venezuela, Senegal, and Gabon infected with S. mansoni, from Gabon infected with S. intercalatum or S. haematobium, from Chine infected with Schistosoma japonicum and from Cambodian patients infected with Schistosoma mekongi. Results indicate that 92.5% (37/40) of Venezuela sera, 75% (15/20) of Senegal sera, 39.5% (17/43) of S. haematobium sera, and 19.2% (5/26) S. intercalatum sera were APIA-positive with the Ven/AWBE/Sm preparation. APIA with the Cam/AWBE/Si preparation showed that 53.8% of S. intercalatum-positive sera had anti-AP antibodies, and 51.2% S. haematobium-positive sera cross-immunocapturing the S. intercalatum AP. APIA performed with Yem/AWBE/Sh showed that 55.8% S. haematobium sera were positive. Only two out of nine S. japonicum sera were APIA-positive with the Ven/AWBE/Sm and Cam/AWBE/Si, and no reaction was observed with Cambodian S. mekongi-positive sera. AP activity was shown to be present in all the schistosome species/strains studied. The use of APIA as a tool to explore the APs antigenicity and the presence of Schistosoma sp. infections through the detection of anti-Schistosoma sp. AP antibodies in a host, allowed us to demonstrate the antigenicity of APs of S. mansoni, S. intercalatum, and S. haematobium.

  1. Identification of a macro-alkaline phosphatase complex in a patient with inflammatory bowel disease.

    Science.gov (United States)

    McTaggart, Malcolm P; Rawson, Catherine; Lawrence, David; Raney, Barbara S; Jaundrill, Linnet; Miller, Lorna A; Murtinho-Braga, Joseph; Kearney, Edward M

    2012-07-01

    We report the rare finding of a macro-alkaline phosphatase (macroALP) complex in a patient with a previously unexplained raised alkaline phosphatase activity. The clinical symptoms were persistent, daily diarrhoea for two months with blood in the stool. The patient was subsequently diagnosed with inflammatory bowel disease, specifically ulcerative colitis, following a rectal biopsy and colonoscopy. Two cases of macroALP associated with ulcerative colitis have been reported before, suggesting there could be an increased prevalence of macroALP in these patients. PMID:22454544

  2. Alkaline phosphatase activity at the southwest coast of India: A comparison of locations differently affected by upwelling..

    Digital Repository Service at National Institute of Oceanography (India)

    Mamatha, S.S.; Malik, A.; Varik, S.; Parvathi, V.; Jineesh, V.K.; Gauns, M.; LokaBharathi, P.A.

    nearshore to offshore Trivandrum, the pattern is more patchy. However, we could discern an increase in activity at station 5 and beyond, synchronizing with the decrease in Chl a. Access to DOP data would have clarified these aspects better... for their comments and suggestions which significantly improved the content of the manuscript. This is NIO contribution no. is xxxx. References Ahmed, N., Shahab, S., 2011. Phosphate Solubilization: Their Mechanism Genetics and Application. The Internet. J...

  3. Gamma-glutamyltransferase, aspartate aminotransferase and alkaline phosphatase as markers of alcohol consumption in out-patient alcoholics

    DEFF Research Database (Denmark)

    Gluud, C; Andersen, I; Dietrichson, O;

    1981-01-01

    Serum activity of gamma-glutamyltransferase, aspartate aminotransferase and alkaline phosphatase were determined in 316 patients attending an out-patients clinic for treatment of alcoholism. The activity of gamma-glutamyltransferase was raised in 34% and that of aspartate aminotransferase...... and alkaline phosphatase in 18% and 7%. Neither the activity of gamma-glutamyltransferase, aspartate aminotransferase nor alkaline phosphatase showed any significant (P greater than 0.05) correlation with the history of alcohol consumption. The activities of gamma-glutamyltransferase and aspartate...... aminotransferase were raised significantly more often in patients with recent alcohol consumption than in patients who had abstained for more than 9 days. The concentration of alkaline phosphatase was not significantly (P greater than 0.05) different in these groups. The predictive value of raised and normal...

  4. Display of E. coli Alkaline Phosphatase pIII or pVIII Fusions on Phagemid Surfaces Reveals Monovalent Decoration with Active Molecules

    Science.gov (United States)

    Weichel, Michael; Jaussi, Rolf; Rhyner, Claudio; Crameri, Reto

    2008-01-01

    Active alkaline phosphatase of Escherichia coli (PhoA, EC 3.1.3.1) was displayed via the leucine zipper element of the Jun-Fos heterodimer on the surface of filamentous phage and the kinetic parameters Km and kcat were determined. The phoA gene was cloned downstream of fos while jun was inserted upstream of pIII or pVIII, alternatively, in the pJuFo phagemid vector. Both fusion genes are regulated by independent lacZ promoters. PhoA displayed on the phagemid pIII surface exhibited a Km of 11.2 µM with 4-nitrophenyl phosphate as substrate, which is consistent with data published for soluble PhoA. Based on these data we calculated the decoration of pJuFo phagemid with PhoA using the minor and major coat proteins pIII and pVIII as fusion partners under variable inducing conditions. We found that, even if the promoters are fully induced at a concentration of 1000 µM IPTG, the phagemids display maximally one copy of PhoA-Fos-Jun-coat protein fusion, irrespective of whether the protein is presented via pIII or pVIII. However, since PhoA is displayed in a native-like fashion, as deduced from the kinetic parameters of the enzymatic reaction, the pJuFo technology provides a versatile tool for the functional screening of complex cDNA libraries displayed on the phagemids' surface. PMID:18949073

  5. Crystallographic studies on shrimp alkaline phosphatase

    NARCIS (Netherlands)

    Backer, M.M.E. (Maaike Maria Eva)

    2003-01-01

    The earth has large cold areas, such as mountains, oceans and (ant)arctic regions, in which organisms have evolved to survive. This adaptation happens at a molecular level. The question is, how do proteins adjust such that they function at low temperatures? "Cold-active" or "cold-adapted" enzymes ha

  6. Interplay between intestinal alkaline phosphatase, diet, gut microbes and immunity.

    Science.gov (United States)

    Estaki, Mehrbod; DeCoffe, Daniella; Gibson, Deanna L

    2014-11-14

    Intestinal alkaline phosphatase (IAP) plays an essential role in intestinal homeostasis and health through interactions with the resident microbiota, diet and the gut. IAP's role in the intestine is to dephosphorylate toxic microbial ligands such as lipopolysaccharides, unmethylated cytosine-guanosine dinucleotides and flagellin as well as extracellular nucleotides such as uridine diphosphate. IAP's ability to detoxify these ligands is essential in protecting the host from sepsis during acute inflammation and chronic inflammatory conditions such as inflammatory bowel disease. Also important in these complications is IAP's ability to regulate the microbial ecosystem by forming a complex relationship between microbiota, diet and the intestinal mucosal surface. Evidence reveals that diet alters IAP expression and activity and this in turn can influence the gut microbiota and homeostasis. IAP's ability to maintain a healthy gastrointestinal tract has accelerated research on its potential use as a therapeutic agent against a multitude of diseases. Exogenous IAP has been shown to have beneficial effects when administered during ulcerative colitis, coronary bypass surgery and sepsis. There are currently a handful of human clinical trials underway investigating the effects of exogenous IAP during sepsis, rheumatoid arthritis and heart surgery. In light of these findings IAP has been marked as a novel agent to help treat a variety of other inflammatory and infectious diseases. The purpose of this review is to highlight the essential characteristics of IAP in protection and maintenance of intestinal homeostasis while addressing the intricate interplay between IAP, diet, microbiota and the intestinal epithelium.

  7. [ATPase and phosphatase activity of drone brood].

    Science.gov (United States)

    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

  8. Calcium-phosphate biomineralization induced by alkaline phosphatase activity in Escherichia coli: localization, kinetics and potential signatures in the fossil record

    Science.gov (United States)

    Cosmidis, Julie; Benzerara, Karim; Guyot, François; Skouri-Panet, Fériel; Duprat, Elodie; Férard, Céline; Guigner, Jean-Michel; Babonneau, Florence; Coelho, Cristina

    2015-12-01

    Bacteria are thought to play an important role in the formation of calcium-phosphate minerals composing marine phosphorites, as supported by the common occurrence of fossil microbes in these rocks. Phosphatase enzymes may play a key role in this process. Indeed, they may increase the supersaturation with respect to Ca-phosphates by releasing orthophosphate ions following hydrolysis of organic phosphorus. However, several questions remain unanswered about the cellular-level mechanisms involved in this model, and its potential signatures in the mineral products. We studied Ca-phosphate precipitation by different strains of Escherichia coli which were genetically modified to differ in the abundance and cellular localization of the alkaline phosphatase (PHO A) produced. The mineral precipitated by either E. coli or purified PHO A was invariably identified as a carbonate-free non-stoichiometric hydroxyapatite. However, the bacterial precipitates could be discriminated from the ones formed by purified PHO A at the nano-scale. PHO A localization was shown to influence the pattern of Ca-phosphate nucleation and growth. Finally, the rate of calcification was proved to be consistent with the PHO A enzyme kinetics. Overall, this study provides mechanistic keys to better understand phosphogenesis in the environment, and experimental references to better interpret the microbial fossil record in phosphorites.

  9. A description of alkaline phosphatases from marine organisms

    Science.gov (United States)

    Tian, Jiyuan; Jia, Hongbing; Yu, Juan

    2016-07-01

    Alkaline phosphatases (APs) are non-specific phosphohydrolases, and they are widely used in clinical diagnostics and biological studies. APs are widespread in nature and exhibit different structural formulations. Based on the diversity of biogenetic sources, APs exhibit temperature-propensity traits, and they are classified as psychrophilic, mesophilic, and thermophilic. In this article, the characteristics of psychrophilic APs from marine organisms were described, accompanied by a simple description of APs from other organisms. This review will facilitate better utilization of marine APs in the biotechnology field.

  10. Study on Temporal and Spatial Variation of Alkaline Phosphatase Activity in the Surface Sediments of Erhai Lake%洱海表层沉积物碱性磷酸酶活性时空变化

    Institute of Scientific and Technical Information of China (English)

    黄睿智; 王圣瑞; 赵海超; 朱乐辉

    2013-01-01

    The effects of physicochemical parameters in the overlying water and sediments on the alkaline phosphatase activity in Erhai Lake were discussed by studying the temporal and spatial variation of this activity from November 2010 to August 2011. The results indicated that; ①The activity of alkaline phosphatase of the surface sediments changed from 45.63 to 144.67 μmol/(g·h) , and the decreasing order of the activities in different regions were the north, the south and the central. The regions where the exogenous pollutant input had a significant impact had high activity levels of nitrogen, phosphorus and the alkaline phosphatase. ② The alkaline phosphatase activity in the surface sediment showed a obvious seasonal variation in general and its seasonal increasing order were autumn ( November) , winter (February), spring (May) and summer (August). Its seasonal variation showed significant differences in the western coastal region. ③ The alkaline phosphatase activity had significant positive correlations with concentrations of OM, TN, OP, TP and C/N in the sediments and with concentrations of OP, TN and CODMn in the overlying water. The physicochemical parameters in the sediments and overlying water were the internal factors and the exogenous pollutants input were the external factors that are important to affect alkaline phosphatase activity.%研究了2010-2011年洱海表层沉积物中碱性磷酸酶活性的时空变化,并探讨了沉积物及水体理化因子对碱性磷酸酶活性的影响.结果表明:①洱海表层沉积物中碱性磷酸酶活性为45.63 ~ 144.67 μmol/(g·h),空间分布总体呈北部>南部>中部,受外源污染物输入影响较大的区域,其沉积物中w(TN)、w(TP)较高,碱性磷酸酶活性也较高;②洱海表层沉积物中碱性磷酸酶活性呈明显的季节性变化特征,秋季(11月)<冬季(2月)<春季(5月)<夏季(8月),并且西部沿岸区域的季节性变化差异显著;③碱性磷酸酶

  11. 19416例健康成人碱性磷酸酶活性分析%19416 cases of serum alkaline phosphatase activity analysis of healthy adults

    Institute of Scientific and Technical Information of China (English)

    吉利; 连丽丽; 孙淑艳; 孙小慧

    2011-01-01

    目的 分析健康成人血清ALP活性,探讨ALP水平在成人性别和年龄的分布规律.方法 随机选择2008年来吉大一院健康体检各项指标正常的健康人19416例,对其血清ALP活性进行回顾性统计分析.结果 21-50岁男性ALP值显著高于同年龄组女性(P<0.01);51岁以上男性ALP值显著低于同年龄段女性(P<0.01).男性20-30岁ALP水平显著高于31岁以上各年龄组,31岁以上各年龄组间差异无统计学意义;女性20-40岁年龄段ALP水平差异无统计学意义,40岁以后,ALP水平随年龄增长而增高.结论 ALP在性别和年龄的分布规律,反映了健康成人骨代谢情况,雌激素可降低ALP活性,为研究雌激素对成骨细胞标志物ALP的影响提供科学的依据.%bjective To Analyze the activity of Alkaline phosphatase (ALP) in serum of healthy adults ,in terms of discussing the distribution rule of ALP levels in gender and age .Methods Making retrospective analysis of the ALP activity data of healthy people who has normal medical examination results from the physical examination center our hospital in 2008 .Results The ALP levels of 21-30,31-40 and 41-50 age group male were significantly higher than female of the same age group ( P<0.01 ) ;the ALP levels of 51-60 and older than 60 age groups male were significantly lower than those of the same age female ( P<0.01 ) .The variance analysis results of ALP levels between different age groups of male and female respectively showed that the ALP levds of 21-30 yearold male were obviously hgher than those of all age groups above 31 year-old and the male ALP results above 31 age groups has no different significance in statistics ;the ALP levels of 20-40 year-old female has no different significance in statistics and the other age groups has statistical significance the female ALP levels increased from 40 year-old as age grew .Conclusion Ths research obtained the distribution rule of adults serum ALP activity in gender and

  12. Altered alkaline phosphatase activity in obese Zucker rats liver respect to lean Zucker and Wistar rats discussed in terms of all putative roles ascribed to the enzyme

    Directory of Open Access Journals (Sweden)

    V. Bertone

    2011-02-01

    Full Text Available Biliary complications often lead to acute and chronic liver injury after orthotopic liver transplantation (OLT. Bile composition and secretion depend on the integrated action of all the components of the biliary tree, starting from hepatocytes. Fatty livers are often discarded as grafts for OLT, since they are extremely vulnerable to conventional cold storage (CS. However, the insufficiency of donors has stimulated research to improve the usage of such marginal organs as well as grafts. Our group has recently developed a machine perfusion system at subnormothermic temperature (20°C; MP20 that allows a marked improvement in preservation of fatty and even of normal rat livers as compared with CS. We sought to evaluate the response of the biliary tree of fatty liver to MP20, and a suitable marker was essential to this purpose. Alkaline phosphatase (AlkP, EC 3.1.3.1, frequently used as marker of membrane transport in hepatocytes and bile ducts, was our first choice. Since no histochemical data were available on AlkP distribution and activity in fatty liver, we have first settled to investigate AlkP activity in the steatotic liver of fatty Zucker rats (fa/fa, using as controls lean Zucker (fa/+ and normal Wistar rats. The AlkP reaction in Wistar rats was in accordance with the existing data and, in particular, was present in bile canaliculi of hepatocytes in the periportal region and midzone, in the canals of Hering and in small bile ducts but not in large bile ducts. In lean ZR liver the AlkP reaction in Hering canals and small bile ducts was similar to Wistar rat liver but hepatocytes had lower canalicular activity and besides presented moderate basolateral reaction. The difference between lean Zucker and Wistar rats, both phenotypically normal animals, could be related to the fact that lean Zucker rats are genotypically heterozygous for a recessive mutated allele. In fatty liver, the activity in ductules and small bile ducts was unchanged, but

  13. Improved double immunohistochemical staining method for cryostat and paraffin wax sections, combining alkaline phosphatase anti-alkaline phosphatase and indirect immunofluorescence

    OpenAIRE

    Tao, Q.; Srivastava, G; Loke, S L; Chan, E. Y.; Ho, F C

    1994-01-01

    Aims - To develop an immunohistochemical staining method for cryostat and paraffin wax sections so that two different antigens in the same section of tissues could be detected by combining immunoenzyme and immunofluorescence techniques. Methods - This double immunohistochemical staining method combines alkaline phosphatase-anti-alkaline phosphatase (APAAP) using New Fuchsin as a chromogen and indirect immunofluorescence. Results - APAAP staining for one antigen of this double immunohistochemi...

  14. Effects of Different Fertilizer on Soil Urease and Alkaline Phosphatase Activity%不同肥料对土壤脲酶和碱性磷酸酶活性的影响

    Institute of Scientific and Technical Information of China (English)

    周俊国; 杨鹏鸣

    2012-01-01

    Ureas and alkaline phosphatase activity of soil in different fertilizer was studied in this experiment. The results shewed urea, chicken manure, straw increased soil urease activity by 15. 38 % , 55.94 % , 27.97 % and soil alkaline phosphatase activity by 2.31 % , 16.20 % , 10.42 % respectively compared with the control in the Ⅰ fertilization levrl. Three fertiliters byd soil urease activity by 24. 48 % ,67.83 % ,46.15 % and alkaline phosphatase activity by 9.72% , 28.01 % , 37.04 % reapectively in the Ⅱ fertilisation level. Urea, chicken manure, straw increased soil urease activity by 37.76 % , 86. 71 % , 62.24 % , and soil alkaline phosphatase activity by 28.47 % , 65.74 % , 58.10 % respectively in the Ⅲ fertilization level. The affect order for soil urease aclivity by 3 fertilizers in different fertilizer levels was chicken manure, straw,urea.The affecting order for soil alkaline phosphstase activity was chicken manure, stravt, urea in Ⅱ fertilization level. The affecting order for soil alkaline phoaphatase activity was straw, chicken manure, urea in Ⅰ and Ⅲ fertilization level.%本实验对不同施肥条件下土壤脲酶活性、碱性磷酸酶活性进行了系统研究.结果表明,和对照相比,尿素、鸡粪、秸秆在Ⅰ施肥水平上,使土壤脲(酶)活性分别增加15.38%、55.94%、27.97%,土壤碱性磷酸酶活性分别增加2.31%、16.20%、10.42%;在Ⅱ施肥水平上使土壤脲酶活性分别增加24.48%,67.83%,46.15%,使土壤碱性磷酸酶活性分别增加9.72%、28.01%和37.04%.在Ⅲ施肥水平上使土壤脲酶分别增加37.76%、86.71%、62.24%,土壤碱性磷酸酶活性分别增加28.47%、65.74%和58.10%.3种不同施肥水平对土壤腺酶活性影响顺序依次是鸡粪、秸秆、尿素.在Ⅱ施肥水平上,对土壤碱性磷酸酶活性的影响大小依次是鸡粪、秸秆、尿素.在Ⅰ、Ⅲ施肥水平上是秸秆、鸡粪、尿素.

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

    Science.gov (United States)

    Chon, Jung-Whan; Kim, Hyunsook; Kim, Kwang-Yup

    2016-01-01

    The alkaline phosphatase (ALP) assay is a rapid and convenient method for verifying milk pasteurization. Since colorimetric ALP assays rely on subjective visual assessments, their results are especially unreliable near the detection limits. In this study, we attempted to establish quantitative criteria for residual ALP in milk by using a more objective method based on spectrophotometric measurements. Raw milk was heat-treated for 0, 10, 20, 30, and 40 min and then subjected to ALP assays. The quantitative criteria for residual ALP in the milk was determined as 2 μg phenol/mL of milk, which is just above the ALP value of milk samples heat-treated for 30 min. These newly proposed methodology and criteria could facilitate the microbiological quality control of milk. PMID:27194927

  16. Development of conductometric biosensors based on alkaline phosphatases for the water quality control

    CERN Document Server

    Berezhetskyy, A

    2008-01-01

    Researches are focused on the elaboration of enzymatic microconductometric device for heavy metal ions detection in water solutions. The manuscript includes a general introduction, the first chapter contains bibliographic review, the second chapter described the fundamentals of conductometric transducers, the third chapter examining the possibility to create and to optimize conductometric biosensor based on bovine alkaline phosphatase for heavy metals ions detection, the fourth chapter devoted to creation and optimization of conductometric biosensor based on alkaline phosphatase active microalgae and sol gel technology, the last chapter described application of the proposed algal biosensor for measurements of heavy metal ions toxicity of waste water, general conclusions stating the progresses achieved in the field of environmental monitoring

  17. Measurement of bone alkaline phosphatase and relative study with osteosarcoma

    Institute of Scientific and Technical Information of China (English)

    YANG Zhiping; HUO Yanqing; SUN Guangzhi; LI Jianmin; LI Xin

    2007-01-01

    The objective of this paper is to explore the value of bone alkaline phosphatase (BALP) for diagnosing osteosarcoma,evaluating the effect of the chemotherapy,judging the prognosis and supervising the relapse and metastasis.The immunoassay was used to check the BALP of the blood serum that was from 42 primary osteosarcoma patients.Alkaline phosphatase (ALP) in blood serum was checked with auto biochemistry equipment.The biopsy tissue and the lesion resected in operation were treated with pathology and histological response was counted.The patients were followed up from five months to 49 months with an average of 24.3 months.Eighteen cases relapsed and transferred,among which,16 of them were dead,and others were survival to the end of the follow-up.BALP was more sensitive than ALP in diagnosing osteosarcoma (P = 0.015).Fifteen cases decreased to normal value in ALP after preoperative chemotherapy,and 34 cases decreased in BALP.Both ALP and BALP in all cases decreased to normal value in postoperative.There was significant difference in positive correlation between the decrease of BALP and the increase of histological response (P = 0.001,r = 0.642).In the followup,there was significant difference in BALP between the group of relapse and transfer and the group of free disease survival (P=0.000).As a check marker in blood serum,BALP,reflecting the process of ossification,has a higher sensitivity than ALP.It has applied value in the diagnosis of osteosarcoma,reflection of the effect of chemotherapy and forecast the prognosis.

  18. Characterization of the alkaline phosphatase expressed on the surface of a Hodgkin's lymphoma cell line

    NARCIS (Netherlands)

    Belland, L; Visser, L; Poppema, S; Stinson, R A

    1993-01-01

    Alkaline phosphatase solubilized from a human Hodgkin's lymphoma cell line (L428) was compared with purified amphiphilic and hydrophilic forms of the enzyme from human liver, and with the enzyme solubilized from a cultured osteosarcoma cell line (Saos-2). Purified hydrophilic alkaline phosphatases f

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

    Directory of Open Access Journals (Sweden)

    Babić Olivera B.

    2013-01-01

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

  20. Purification and characterization of an alkaline phosphatase induced by phosphorus starvation in common bean (Phaseolus vulgaris L.) roots

    Energy Technology Data Exchange (ETDEWEB)

    Morales, L.; Gutierrez, N.; Maya, V.; Parra, C.; Martinez B, E.; Coello, P., E-mail: pcoello@servidor.unam.mx [UNAM, Facultad de Quimica, Departamento de Bioquimica, Ciudad Universitaria, 04510 Mexico D. F. (Mexico)

    2012-07-01

    Two phosphatase isoforms from roots of the common bean (Phaseolus vulgaris L.) showed an increase in activity in response to phosphate deficiency. One of them (APIII) was chosen for further purification through ionic exchange chromatography and preparative electrophoresis. The estimated molecular mass of APIII was 35 kDa by both SDS-Page and gel filtration analyses, suggesting a monomeric form of the active enzyme. The phosphatase was classified as an alkaline phosphatase based on the requirement of ph 8 for optimum catalysis. It not only exhibited broad substrate specificity, with the most activity against pyrophosphate, but also effectively catalyzed the hydrolysis of polyphosphate, glucose-1-phosphate and phospho enol-pyruvate. Activity was completely inhibited by molybdate, vanadate and phosphate but was only partially inhibited by fluoride. Although divalent cations were not essential for the pyro phosphatase activity of this enzyme, the hydrolysis of pyro phosphatase increased substantially in the presence of Mg{sup 2+}.

  1. Mechanical loading induced expression of bone morphogenetic protein-2,alkaline phosphatase activity,and collagen synthesis in osteoblastic MC3T3-E1 cells

    Institute of Scientific and Technical Information of China (English)

    LU Hong-fei; MAI Zhi-hui; XU Ye; WANG Wei; AI Hong

    2012-01-01

    Background Bone morphogenetic protein(BMP)-2,alkaline phosphatase(ALP),and collagen typeⅠ?are known to play a critical role in the process of bone remodeling.However,the relationship between mechanical strain and the expression of BMP-2,ALP,and COL-Ⅰ?in osteoblasts was still unknown.The purpose of this study was to investigate the effects of different magnitudes of mechanical strain on osteoblast morphology and on the expression of BMP-2,ALP,and COL-Ⅰ.Methods Osteoblast-like cells were flexed at four deformation rates(0,6%,12%,and 18% elongation).The expression of BMP-2 mRNA,ALP,and COL-Ⅰ?in osteoblast-like cells were determined by real-time quantitative reverse transcription polymerase chain reaction,respectively.The results were subjected to analysis of variance(ANOVA)using SPSS 13.0 statistical software.Results The cells changed to fusiform and grew in the direction of the applied strain after the mechanical strain was loaded.Expression level of the BMP-2,ALP,and COL-Ⅰ?increased magnitude-dependently with mechanical loading in the experimental groups,and the 12% elongation group had the highest expression(P<0.05).Conclusion Mechanical strain can induce morphological change and a magnitude-dependent increase in the expression of BMP-2,ALP,and COL-Ⅰ?mRNA in osteoblast-like cells,which might influence bone remodeling in orthodontic treatment.

  2. Expression of a human placental alkaline phosphatase gene in transfected cells: Use as a reporter for studies of gene expression

    International Nuclear Information System (INIS)

    The human placental alkaline phosphatase gene has been cloned and reintroduced into mammalian cells. When a plasmid carrying the gene under control of the simian virus 40 early promoter (pSV2Apap) is transfected into a variety of different cell types, placental alkaline phosphatase activity can readily be detected by using whole cell suspensions or cell lysates. Alkaline phosphatase activity can also be visualized directly in individual transfected cells by histochemical staining. The gene is appropriate for use as a reporter in studies of gene regulation since its expression is dependent on the presence of exogenous transcription control elements. The overall assay to detect the expression of the gene is quantitative, very rapid, and inexpensive. Cotransfections of cells with pSV2Apap and a related plasmid carrying the bacterial chloramphenicol acetyltransferase gene (pSV2Acat) indicate that transcription of these two genes is detected with roughly the same sensitivity

  3. Bone mineralisation in premature infants cannot be predicted from serum alkaline phosphatase or serum phosphate

    DEFF Research Database (Denmark)

    Faerk, J; Peitersen, Birgit; Petersen, S;

    2002-01-01

    BACKGROUND: The bone mineral content of premature infants at term is lower than in mature infants at the same postconceptional age. Serum alkaline phosphatase and serum phosphate are often used as indicators of bone mineralisation. OBJECTIVE: To analyse the association between bone mineral content...... and serum alkaline phosphatase and serum phosphate. METHODS: Serum alkaline phosphatase and phosphate were measured at weekly intervals during admission in 108 premature infants of gestational age below 32 weeks (mean (SD) gestational age 29 (2) weeks; mean (SD) birth weight 1129 (279) g). Bone mineral...... content was measured at term (mean gestational age 41 weeks) by dual energy x ray absorptiometry and corrected for body size. RESULTS: Serum alkaline phosphatase was significantly negatively associated with serum phosphate (p alkaline...

  4. SERUM VALUES OF ALKALINE PHOSPHATASE AND LACTATE DEHYDROGENASE IN OSTEOSARCOMA

    Science.gov (United States)

    ZUMÁRRAGA, JUAN PABLO; BAPTISTA, ANDRÉ MATHIAS; ROSA, LUIS PABLO DE LA; CAIERO, MARCELO TADEU; CAMARGO, OLAVO PIRES DE

    2016-01-01

    ABSTRACT Objective: To study the relationship between the pre and post chemotherapy (CT) serum levels of alkaline phosphatase (AP) and lactate dehydrogenase (LDH), and the percentage of tumor necrosis (TN) found in specimens after the pre surgical CT in patients with osteosarcoma. Methods: Series of cases with retrospective evaluation of patients diagnosed with osteosarcoma. Participants were divided into two groups according to serum values of both enzymes. The values of AP and LDH were obtained before and after preoperative CT. The percentage of tumor necrosis (TN) of surgical specimens of each patient was also included. Results: One hundred and thirty seven medical records were included from 1990 to 2013. Both the AP as LDH decreased in the patients studied, being the higher in pre CT than post CT. The average LHD decrease was 795.12U/L and AP decrease was 437.40 U/L. The average TN was 34.10 %. There was no statistically significant correlation between the serums values and the percentage of tumoral necrosis. Conclusion: The serum levels values of AP and LDH are not good predictors for the chemotherapy-induced necrosis in patients with osteosarcoma. Level of Evidence IV, Case Series. PMID:27217815

  5. Evolution of osteocalcin, alkaline phosphatase and parathyroid hormone after parathyroidectomy in patients receiving chronic maintenance hemodialysis.

    Science.gov (United States)

    Peretz, A M; Dhaene, M M; Drowart, A; Kinnaert, P; Vertongen, F M; Bourdoux, P P

    1992-03-01

    Parathyroid hormone (PTH), osteocalcin and alkaline phosphatase (AP) were investigated before and after parathyroidectomy in 12 patients receiving hemodialysis. Early post-parathyroidectomy, PTH decreased (p less than 0.001), AP increased (p less than 0.05), but osteocalcin remained unchanged. At 3 months, osteocalcin and AP declined. A negative correlation was observed between aluminum staining and post-parathyroidectomy osteocalcin. In conclusion, early post-parathyroidectomy, osteocalcin and AP reflect persistent osteoblastic activity, which declined after 3 months. In patients receiving hemodialysis both variables may represent different aspects of osteoblastic activity and osteocalcin allows mixed uremic osteodystrophy after parathyroidectomy. PMID:1578455

  6. Comparison of Inactivation and Unfolding of Calf Intestinal Alkaline Phosphatase in Guanidinium Chloride Solution

    Institute of Scientific and Technical Information of China (English)

    张英侠; 闫淑莲; 刘永利; 席宏伟; 周海梦

    2002-01-01

    The changes in activity and unfolding of calf intestinal alkaline phosphatase (CIP) during denaturation in guanidinium chloride solutions of different concentrations were investigated using ultraviolet difference absorption spectra and fluorescence emission spectra. Unfolding and inactivation rate constants were measured and compared. The inactivation course is much faster than that of unfolding, which suggests that the active site of CIP containing two zinc ions and one magnesium ion is situated in a limited and flexible region of the enzyme molecule, which is more fragile to the denaturant than the protein as a whole.

  7. Characterization of the phosphatidylinositol-glycan membrane anchor of human placental alkaline phosphatase

    International Nuclear Information System (INIS)

    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 [14C]ethanolamine, [14C]myristic acid, or myo[3H]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

  8. Bone mineralisation in premature infants cannot be predicted from serum alkaline phosphatase or serum phosphate

    OpenAIRE

    Faerk, J; Peitersen, B; Petersen, S; Michaelsen, K

    2002-01-01

    Background: The bone mineral content of premature infants at term is lower than in mature infants at the same postconceptional age. Serum alkaline phosphatase and serum phosphate are often used as indicators of bone mineralisation.

  9. Activation of p38 mitogen-activated protein kinase contribute to BMP4-induced alkaline phosphatase expression in MC3T3-E1 preosteoblast

    Institute of Scientific and Technical Information of China (English)

    YUAN Ye; Wu Zhi-jun; YAO Hui-yu; YU Xiao-dan; GUO Zi-kuan; CHEN Xiao-san; TANG Pei-xian; MAO Ning

    2006-01-01

    @@ Bone morphogenetic proteins (BMPs) induce ectopic bone formation and promote osteoblast differentiation.1 It has been documented that Smad transcriptional factors function as primary mediators of BMPs activity. Receptor-regulated Smad (Smad1, 5, 8) could be phosphorylated by activated BMPR-I and form complex with Smad4. The Smad complex translocates to the nucleus and regulate target gene transcription.2

  10. Associations between renal hyperfiltration and serum alkaline phosphatase.

    Directory of Open Access Journals (Sweden)

    Se Won Oh

    Full Text Available Renal hyperfiltration, which is associated with renal injury, occurs in diabetic or obese individuals. Serum alkaline phosphatase (ALP level is also elevated in patients with diabetes (DM or metabolic syndrome (MS, and increased urinary excretion of ALP has been demonstrated in patients who have hyperfiltration and tubular damage. However, little was investigated about the association between hyperfiltration and serum ALP level. A retrospective observational study of the 21,308 adults in the Korea National Health and Nutrition Examination Survey IV-V databases (2008-2011 was performed. Renal hyperfiltration was defined as exceeding the age- and sex-specific 97.5th percentile. We divided participants into 4 groups according to their estimated glomerular filtration rate (eGFR: >120, 90-119, 60-89, and 120 mL/min/1.73 m2 showed the highest risk for MS, in the highest ALP quartiles (3.848, 95% CI, 1.876-7.892, compared to the lowest quartile. Similarly, the highest risk for DM, in the highest ALP quartiles, was observed in participants with eGFR >120 ml/min/1.73 m2 (2.166, 95% CI, 1.084-4.329. ALP quartiles were significantly associated with albuminuria in participants with eGFR ≥ 60 ml/min/1.73m2. The highest ALP quartile had a 1.631-fold risk elevation for albuminuria with adjustment of age and sex. (95% CI, 1.158-2.297, P = 0.005. After adjustment, the highest ALP quartile had a 1.624-fold risk elevation, for renal hyperfiltration (95% CI, 1.204-2.192, P = 0.002. In addition, hyperfiltration was significantly associated with hemoglobin, triglyceride, white blood cell count, DM, smoking, and alcohol consumption (P<0.05. The relationship between serum ALP and metabolic disorders is stronger in participants with an upper-normal range of eGFR. Higher ALP levels are significantly associated with renal hyperfiltration in Korean general population.

  11. Localized cranial hyperostosis of meningiomas: a result of neoplastic enzymatic activity?

    DEFF Research Database (Denmark)

    Heick, A.; Mosdal, C.; Klinken, Leif

    1993-01-01

    Neuropathology, alkaline phosphatase, cranial hyperostosis, meningioma, ossifying enzymatic activity......Neuropathology, alkaline phosphatase, cranial hyperostosis, meningioma, ossifying enzymatic activity...

  12. Alkaline phosphatase in osteoblasts is down-regulated by pulsatile fluid flow

    Science.gov (United States)

    Hillsley, M. V.; Frangos, J. A.

    1997-01-01

    It is our hypothesis that interstitial fluid flow plays a role in the bone remodeling response to mechanical loading. The fluid flow-induced expression of three proteins (collagen, osteopontin, and alkaline phosphatase) involved in bone remodeling was investigated. Rat calvarial osteoblasts subjected to pulsatile fluid flow at an average shear stress of 5 dyne/cm2 showed decreased alkaline phosphatase (AP) mRNA expression after only 1 hour of flow. After 3 hours of flow, AP mRNA levels had decreased to 30% of stationary control levels and remained at this level for an additional 5 hours of flow. Steady flow (4 dyne/cm2 fluid shear stress), in contrast, resulted in a delayed and less dramatic decrease in AP mRNA expression to 63% of control levels after 8 hours of flow. The reduced AP mRNA expression under pulsatile flow conditions was followed by reduced AP enzyme activity after 24 hours. No changes in collagen or osteopontin mRNA expression were detected over 8 hours of pulsatile flow. This is the first time fluid flow has been shown to affect gene expression in osteoblasts.

  13. Electrochemical biosensor for detection of DNA hydroxymethylation based on glycosylation and alkaline phosphatase catalytic signal amplification

    International Nuclear Information System (INIS)

    Highlights: • DNA Hydroxymethylation was detected by electrochemical method. • 5-Hydroxymethylation cytosine in target DNA was chemically modified with glucose group. • Alkaline phosphatase catalytic signal amplification strategy was used. • The developed method also showed excellent reproducibility and stability. - Abstract: DNA hydroxymethylation (5-hydroxymethylcytosine, 5hmC) is a kind of new epigenetic modification, which plays key roles in nuclear reprogramming, regulates the gene activity, and initiates the DNA demethylation in mammals. For further understanding the functions of 5hmC and the correlation with tumour, it is essential to develop sensitive and selective methods for detecting and sequencing 5hmC. Herein, a kind of electrochemical biosensor was fabricated for 5hmC detection based on the glycosylation modification of 5hmC and enzymatic signal amplification. Under the catalytic effect of T4 β-glucosyltransferase, the 5hmC in target DNA was chemically modified with glucose. Then with the bridge connection of 1,4-phenyldiboronic acid, alkaline phosphatase was further captured on the electrode surface to catalyze the hydrolysis of p-nitrophenyl phosphate disodium salt to produce p-nitrophenol. Based on the relationship between the electrochemical oxidation signal of p-nitrophenol and the concentration of target DNA, the 5hmC level can be detected with high sensitivity and selectivity. The developed method also showed excellent reproducibility and stability

  14. X-ray structure reveals a new class and provides insight into evolution of alkaline phosphatases.

    Directory of Open Access Journals (Sweden)

    Subhash C Bihani

    Full Text Available The alkaline phosphatase (AP is a bi-metalloenzyme of potential applications in biotechnology and bioremediation, in which phosphate monoesters are nonspecifically hydrolysed under alkaline conditions to yield inorganic phosphate. The hydrolysis occurs through an enzyme intermediate in which the catalytic residue is phosphorylated. The reaction, which also requires a third metal ion, is proposed to proceed through a mechanism of in-line displacement involving a trigonal bipyramidal transition state. Stabilizing the transition state by bidentate hydrogen bonding has been suggested to be the reason for conservation of an arginine residue in the active site. We report here the first crystal structure of alkaline phosphatase purified from the bacterium Sphingomonas. sp. Strain BSAR-1 (SPAP. The crystal structure reveals many differences from other APs: 1 the catalytic residue is a threonine instead of serine, 2 there is no third metal ion binding pocket, and 3 the arginine residue forming bidentate hydrogen bonding is deleted in SPAP. A lysine and an aspargine residue, recruited together for the first time into the active site, bind the substrate phosphoryl group in a manner not observed before in any other AP. These and other structural features suggest that SPAP represents a new class of APs. Because of its direct contact with the substrate phosphoryl group, the lysine residue is proposed to play a significant role in catalysis. The structure is consistent with a mechanism of in-line displacement via a trigonal bipyramidal transition state. The structure provides important insights into evolutionary relationships between members of AP superfamily.

  15. A Toxin-Binding Alkaline Phosphatase Fragment Synergizes Bt Toxin Cry1Ac against Susceptible and Resistant Helicoverpa armigera

    OpenAIRE

    Wenbo Chen; Chenxi Liu; Yutao Xiao; Dandan Zhang; Yongdong Zhang; Xianchun Li; Bruce E Tabashnik; Kongming Wu

    2015-01-01

    Evolution of resistance by insects threatens the continued success of pest control using insecticidal crystal (Cry) proteins from the bacterium Bacillus thuringiensis (Bt) in sprays and transgenic plants. In this study, laboratory selection with Cry1Ac yielded five strains of cotton bollworm, Helicoverpa armigera, with resistance ratios at the median lethal concentration (LC50) of activated Cry1Ac ranging from 22 to 1700. Reduced activity and reduced transcription of an alkaline phosphatase p...

  16. Stimulus Response of Au-NPs@GMP-Tb Core-Shell Nanoparticles: Toward Colorimetric and Fluorescent Dual-Mode Sensing of Alkaline Phosphatase Activity in Algal Blooms of a Freshwater Lake.

    Science.gov (United States)

    Zhang, Xiaolei; Deng, Jingjing; Xue, Yumeng; Shi, Guoyue; Zhou, Tianshu

    2016-01-19

    In this study, we demonstrate a colorimetric and fluorescent dual-mode method for alkaline phosphatase activity (APA) sensing in freshwater lake with stimuli-responsive gold nanoparticles@terbium-guanosine monophosphate (Au-NPs@GMP-Tb) core-shell nanoparticles. Initially, the core-shell nanoparticles were fabricated based on Au-NPs decorated with a fluorescent GMP-Tb shell. Upon being excited at 290 nm, the as-formed Au-NPs@GMP-Tb core-shell nanoparticles emit green fluorescence, and the decorated GMP-Tb shell causes the aggregation of Au-NPs. However, the addition of ALP destroys GMP-Tb shell, resulting in the release of Au-NPs from the shell into the solvent. As a consequence, the aggregated Au-NPs solubilizes with the changes in the UV-vis spectrum of the dispersion, and in the meantime, the fluorescence of GMP-Tb shell turns off, which constitutes a new mechanism for colorimetric and fluorescent dual-mode sensing of APA. With the method developed here, we could monitor the dynamic change of APA during an algal bloom of a freshwater lake, both by the naked eye and further confirmed by fluorometric determination. This study not only offers a new method for on-site visible detection of APA but also provides a strategy for dual-mode sensing mechanisms by the rational design of the excellent optical properties of Au-NPs and the adaptive inclusion properties of the luminescent infinite coordination polymers.

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

    DEFF Research Database (Denmark)

    Joner, E.J.; Jakobsen, I.

    1995-01-01

    length density was twice as high in soil with added straw compared to the two other treatments. Mycorrhizal colonization resulted in lower activity of acid phosphatase in the HC for two out of three treatments. Alkaline phosphatase activity was only decreased by mycorrhiza in soil without organic matter...... additions. In soil with added clover alkaline phosphatase activity increased due to the presence of mycorrhizal hyphae. We suggest that mycorrhizas may influence the exudation of acid phosphatase by roots. Hyphae of G. invermaium did apparently not excrete extracellular phosphatases, but their presence may......Two experiments were set up to investigate the influence of soil organic matter on growth of arbuscular mycorrhizal (AM) hyphae and concurrent changes in soil inorganic P, organic P and phosphatase activity. A sandy loam soil was kept for 14 months under two regimes (outdoor where surplus...

  18. Inhibition of acid, alkaline, and tyrosine (PTP1B) phosphatases by novel vanadium complexes.

    Science.gov (United States)

    McLauchlan, Craig C; Hooker, Jaqueline D; Jones, Marjorie A; Dymon, Zaneta; Backhus, Emily A; Greiner, Bradley A; Dorner, Nicole A; Youkhana, Mary A; Manus, Lisa M

    2010-03-01

    In the course of our investigations of vanadium-containing complexes for use as insulin-enhancing agents, we have generated a series of novel vanadium coordination complexes with bidentate ligands. Specifically we have focused on two ligands: anthranilate (anc(-)), a natural metabolite of tryptophan, and imidizole-4-carboxylate (imc(-)), meant to mimic naturally occurring N-donor ligands. For each ligand, we have generated a series of complexes containing the V(III), V(IV), and V(V) oxidation states. Each complex was investigated using phosphatase inhibition studies of three different phosphatases (acid, alkaline, and tyrosine (PTP1B) phosphatase) as prima facia evidence for potential use as an insulin-enhancing agent. Using p-nitrophenyl phosphate as an artificial phosphatase substrate, the levels of inhibition were determined by measuring the absorbance of the product at 405nm using UV/vis spectroscopy. Under our experimental conditions, for instance, V(imc)(3) appears to be as potent an inhibitor of alkaline phosphatase as sodium orthovanadate when comparing the K(cat)/K(m) term. VO(anc)(2) is as potent an inhibitor of acid phosphatase and tyrosine phosphatase as the Na(3)VO(4). Thus, use of these complexes can increase our mechanistic understanding of the effects of vanadium in vivo. PMID:20071031

  19. X-Ray Structure Reveals a New Class and Provides Insight into Evolution of Alkaline Phosphatases

    OpenAIRE

    Bihani, Subhash C.; Das, Amit; Nilgiriwala, Kayzad S.; Prashar, Vishal; Pirocchi, Michel; Apte, Shree Kumar; Ferrer, Jean-Luc; Hosur, Madhusoodan V.

    2011-01-01

    The alkaline phosphatase (AP) is a bi-metalloenzyme of potential applications in biotechnology and bioremediation, in which phosphate monoesters are nonspecifically hydrolysed under alkaline conditions to yield inorganic phosphate. The hydrolysis occurs through an enzyme intermediate in which the catalytic residue is phosphorylated. The reaction, which also requires a third metal ion, is proposed to proceed through a mechanism of in-line displacement involving a trigonal bipyramidal transitio...

  20. Detection and analysis on bone alkaline phosphatase activity of 2540 infants%2540例婴幼儿骨源性碱性磷酸酶活性检测分析

    Institute of Scientific and Technical Information of China (English)

    盛巍

    2012-01-01

    目的:调查长兴地区0~5岁婴幼儿骨健康状况.方法:使用骨源性碱性磷酸酶(BAP)试剂盒对2009~2010年来长兴妇幼保健院门诊就诊的2 540例0~5岁婴幼儿进行检测.结果:在2 540例0~5岁婴幼儿中,BAP活性正常的1 215例,占总人数的47.83%;BAP活性处于预防水平的1 196例,占总人数的47.09%;BAP活性处于治疗水平的129例,占总人数的5.08%.结论:长兴地区约50.00%的婴幼儿骨骼处于亚健康状态,易患佝偻病,需及早预防.%Objective: To investigate the bone health status of 0 - 5 - year - old infants in Changxing county. Methods: Bone alkaline phosphatase ( BAP) activity of 2 540 infants aged 0-5 years who went to outpatient department of the hospital from 2009 to 2010 was detected by BAP kit. Results; Among2 540 infante aged 0 -5 years old, I 215 infants were found with normal BAP activity, accounting for 47. 83% ; BAP activity of 1 196 infants was at prevention level, accounting for 47.09% ; BAP activity of 129 infants was at treatment level, accounting for 5.08%. Conclusion: The bones of about 50.00% of the infants in Changxing county were subhealthy, the infants are susceptible to rickets, and prevention should be earned out early.

  1. A Disposable Alkaline Phosphatase-Based Biosensor for Vanadium Chronoamperometric Determination

    Directory of Open Access Journals (Sweden)

    Ana Lorena Alvarado-Gámez

    2014-02-01

    Full Text Available A chronoamperometric method for vanadium ion determination, based on the inhibition of the enzyme alkaline phosphatase, is reported. Screen-printed carbon electrodes modified with gold nanoparticles were used as transducers for the immobilization of the enzyme. The enzymatic activity over 4-nitrophenyl phosphate sodium salt is affected by vanadium ions, which results in a decrease in the chronoamperometric current registered. The developed method has a detection limit of 0.39 ± 0.06 µM, a repeatability of 7.7% (n = 4 and a reproducibility of 8% (n = 3. A study of the possible interferences shows that the presence of Mo(VI, Cr(III, Ca(II and W(VI, may affect vanadium determination at concentration higher than 1.0 mM. The method was successfully applied to the determination of vanadium in spiked tap water.

  2. A Disposable Alkaline Phosphatase-Based Biosensor for Vanadium Chronoamperometric Determination

    Science.gov (United States)

    Alvarado-Gámez, Ana Lorena; Alonso-Lomillo, María Asunción; Domínguez-Renedo, Olga; Arcos-Martínez, María Julia

    2014-01-01

    A chronoamperometric method for vanadium ion determination, based on the inhibition of the enzyme alkaline phosphatase, is reported. Screen-printed carbon electrodes modified with gold nanoparticles were used as transducers for the immobilization of the enzyme. The enzymatic activity over 4-nitrophenyl phosphate sodium salt is affected by vanadium ions, which results in a decrease in the chronoamperometric current registered. The developed method has a detection limit of 0.39 ± 0.06 μM, a repeatability of 7.7% (n = 4) and a reproducibility of 8% (n = 3). A study of the possible interferences shows that the presence of Mo(VI), Cr(III), Ca(II) and W(VI), may affect vanadium determination at concentration higher than 1.0 mM. The method was successfully applied to the determination of vanadium in spiked tap water. PMID:24569772

  3. Electrochemical Determination of Alkaline Phosphatase in Human Serum by Differential Pulse Voltammetry

    Institute of Scientific and Technical Information of China (English)

    SUN Wei; JIAO Kui; WANG Hai-yu; LU Lu-de

    2004-01-01

    Differential pulse voltammetry(DPV) was applied to the determination of alkaline phosphatase(ALP) activity in human serum with phenyl phosphate as the substrate. Phenyl phosphate can enzymatically be hydrolyzed to produce phenol which is quantified by DPV at +660 mV(vs.Ag/AgCl) in the concentration range of 2.0-100 μmol/L. The standard curve for ALP is linear over the range from 0.06 to 1000 U/L with a relative standard deviation of 3.0%. The conditions for the enzymatic reaction and voltammetric detection were optimized and the kinetic constants were also examined.The human serum samples were tested by this method and the results were in good agreement with those obtained by the routine p-nitrophenyl phosphate spectrophotometric method.

  4. Extreme Elevation of Alkaline Phosphatase in a Pregnancy Complicated by Gestational Diabetes and Infant with Neonatal Alloimmune Thrombocytopenia.

    Science.gov (United States)

    Lozo, Svjetlana; Atabeygi, Amir; Healey, Michael

    2016-01-01

    There have been few case reports of isolated elevation of alkaline phosphatase beyond the normal physiologic amount with subsequent return to baseline after delivery. Here we present a similar case of extreme elevation of alkaline phosphatase in a pregnancy complicated by gestational diabetes and subsequently by neonatal alloimmune thrombocytopenia (NAIT). PMID:27610256

  5. Distribution and impact factor of alkaline phosphatase activity in the intertidal surface sediments of the Yangtze Estuary%长江口潮滩表层沉积物中碱性磷酸酶活性及其影响因素

    Institute of Scientific and Technical Information of China (English)

    姜经梅; 赵慧; 沈铭能; 丁平兴; 尹国宇; 赵迪; 侯立军

    2011-01-01

    以长江口潮滩作为研究区域,对表层沉积物中碱性磷酸酶活性、解磷微生物量、磷组分及理化性质的时空分布特征进行了分析.结果表明,长江口潮滩湿地沉积物中碱性磷酸酶活性具有显著的季节性、区域性差异,冬季(1月)碱性磷酸酶活性较低,介于0.65~1.59μg.g-.1h-1之间;夏季(8、9月份)最高,变化范围为1.80~5.64μg.g-.1h-1.相关性分析结果显示,碱性磷酸酶活性与解磷细菌数量呈显著的正相关关系(rOPB=0.91,rIPB=0.89,p〈0.01),说明解磷细菌是影响碱性磷酸酶活性的主要因素之一.同时,碱性磷酸酶活性与磷组分、C/N、盐分等因素显著相关,而与粒度、有机碳氮含量的相关性不显著.%Spatial and temporal distributions of alkaline phosphatase activity(APA) in the intertidal sediments of the Yangtze Estuary were investigated,together with abundances of phosphate solubilizing bacteria and the phosphorus forms.It was shown that there were significantly seasonal and regional variations of alkaline phosphatase activity in the intertidal sediments.The alkaline phosphatase activities were relatively lower in winter(January),with values of 0.65 ~ 1.59 μg.g-.1h-1.In contrast,higher alkaline phosphatase activities were detected in summer(August and September),with values of 1.80 ~ 5.64 μg.g-1.h-1.Statistical analyses indicated that the alkaline phosphatase activities were related significantly to the numbers of phosphate solubilizing bacteria(rOPB = 0.91,rIPB = 0.89,p 0.01).This implied that the numbers of phosphate solubilizing bacteria were the primary factor controlling the changes of alkaline phosphatase activities.In addition,the alkaline phosphatase activities in the intertidal sediments were also affected by phosphorus forms,C:N molar ratios and salinity.However,there were no significant correlations of the alkaline phosphatase activities with the contents of sand

  6. A toxin-binding alkaline phosphatase fragment synergizes Bt toxin Cry1Ac against susceptible and resistant Helicoverpa armigera.

    Directory of Open Access Journals (Sweden)

    Wenbo Chen

    Full Text Available Evolution of resistance by insects threatens the continued success of pest control using insecticidal crystal (Cry proteins from the bacterium Bacillus thuringiensis (Bt in sprays and transgenic plants. In this study, laboratory selection with Cry1Ac yielded five strains of cotton bollworm, Helicoverpa armigera, with resistance ratios at the median lethal concentration (LC50 of activated Cry1Ac ranging from 22 to 1700. Reduced activity and reduced transcription of an alkaline phosphatase protein that binds Cry1Ac was associated with resistance to Cry1Ac in the four most resistant strains. A Cry1Ac-binding fragment of alkaline phosphatase from H. armigera (HaALP1f was not toxic by itself, but it increased mortality caused by Cry1Ac in a susceptible strain and in all five resistant strains. Although synergism of Bt toxins against susceptible insects by toxin-binding fragments of cadherin and aminopeptidase N has been reported previously, the results here provide the first evidence of synergism of a Bt toxin by a toxin-binding fragment of alkaline phosphatase. The results here also provide the first evidence of synergism of a Bt toxin by any toxin-binding peptide against resistant insects.

  7. A toxin-binding alkaline phosphatase fragment synergizes Bt toxin Cry1Ac against susceptible and resistant Helicoverpa armigera.

    Science.gov (United States)

    Chen, Wenbo; Liu, Chenxi; Xiao, Yutao; Zhang, Dandan; Zhang, Yongdong; Li, Xianchun; Tabashnik, Bruce E; Wu, Kongming

    2015-01-01

    Evolution of resistance by insects threatens the continued success of pest control using insecticidal crystal (Cry) proteins from the bacterium Bacillus thuringiensis (Bt) in sprays and transgenic plants. In this study, laboratory selection with Cry1Ac yielded five strains of cotton bollworm, Helicoverpa armigera, with resistance ratios at the median lethal concentration (LC50) of activated Cry1Ac ranging from 22 to 1700. Reduced activity and reduced transcription of an alkaline phosphatase protein that binds Cry1Ac was associated with resistance to Cry1Ac in the four most resistant strains. A Cry1Ac-binding fragment of alkaline phosphatase from H. armigera (HaALP1f) was not toxic by itself, but it increased mortality caused by Cry1Ac in a susceptible strain and in all five resistant strains. Although synergism of Bt toxins against susceptible insects by toxin-binding fragments of cadherin and aminopeptidase N has been reported previously, the results here provide the first evidence of synergism of a Bt toxin by a toxin-binding fragment of alkaline phosphatase. The results here also provide the first evidence of synergism of a Bt toxin by any toxin-binding peptide against resistant insects. PMID:25885820

  8. Acceleration of gelation and promotion of mineralization of chitosan hydrogels by alkaline phosphatase

    NARCIS (Netherlands)

    Douglas, T.E.L.; Skwarczynska, A.; Modrzejewska, Z.; Balcaen, L.; Schaubroeck, D.; Lycke, S.; Vanhaecke, F.; Vandenabeele, P.; Dubruel, P.; Jansen, J.A.; Leeuwenburgh, S.C.G.

    2013-01-01

    Thermosensitive chitosan hydrogels containing sodium beta-glycerophosphate (beta-GP), whose gelation is induced by increasing temperature to body temperature, were functionalized by incorporation of alkaline phosphatase (ALP), an enzyme involved in mineralization of bone. ALP incorporation led to ac

  9. Protection against an Escherichia Coli-induced sepsis by alkaline phosphatase in mice

    NARCIS (Netherlands)

    Verweij, Willem; Bentala, Hafida; Huizenga-Van der Vlag, A; van Loenen - Weemaes, Anne-miek; Kooi, K.; Meijer, Dick; Poelstra, Klaas

    2004-01-01

    Alkaline phosphatase (AP) is a phosphate transferase present in bacteria and eukaryotes. In previous studies, we have shown that AP is able to dephosphorylate lipopolysaccharide (LPS) at physiological pH levels. Because LPS is the causative agent of gram-negative sepsis, we hypothesize that AP might

  10. The effect of alkaline phosphatase coated onto titanium alloys on bone responses in rats.

    NARCIS (Netherlands)

    Schouten, C.; Beucken, J.J.J.P. van den; Jonge, L.T. de; Bronkhorst, E.M.; Meijer, G.J.; Spauwen, P.H.M.; Jansen, J.A.

    2009-01-01

    The enzyme alkaline phosphatase (ALP) was recently proposed as an implant coating material in order to improve the biological performance of orthopedic and dental implants. The present study evaluated the in vivo bone response to electrosprayed coatings, consisting of ALP, calcium phosphate (CaP) or

  11. 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.

    Science.gov (United States)

    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

  12. Enslavement in the water body by toxic Aphanizomenon ovalisporum, inducing alkaline phosphatase in phytoplanktons.

    Science.gov (United States)

    Bar-Yosef, Yehonathan; Sukenik, Assaf; Hadas, Ora; Viner-Mozzini, Yehudit; Kaplan, Aaron

    2010-09-14

    The hepatotoxin cylindrospermopsin (CYN) produced by certain cyanobacteria, including Aphanizomenon ovalisporum (hereafter Aphanizomenon) [1], seriously affects lake water quality [2], but its biological role is not known. Strong correlation between Aphanizomenon abundance in Lake Kinneret, Israel, and alkaline phosphatase (APase) activity suggests that inorganic phosphate (Pi) limitation induces the PHO regulon and APase secretion [3]. Staining lake samples with DAPI [4] revealed a high level of polyphosphate bodies (PPB) in Aphanizomenon. Application of enzyme-labeled fluorescence (ELF-APase) [5] showed APase in various organisms, but not in Aphanizomenon. ELF-APase signals and extracellular APase activity in Aphanizomenon were detected only after exploiting PPB under prolonged Pi deprivation in cultures or toward the end of its autumn bloom. Pi deprivation of Aphanizomenon induces CYN production, high-affinity Pi uptake, and an internal, not external, APase. Addition of Aphanizomenon spent media or CYN to various phytoplanktons, including Chlamydomonas reinhardtii, induced genes typically upregulated under Pi limitation and a rise in extracellular APase activity, despite ample surrounding Pi. Coculturing Aphanizomenon with Chlamydomonas or with Debarya sp. showed positive ELF-APase signals, but not in Aphanizomenon. CYN producers promote Pi supply by inducing APase secretion by other phytoplanktons, possibly explaining their increased abundance despite reduced Pi supply from watersheds.

  13. Correlation Between Down Syndrome and the Level of Placental Alkaline Phosphatase in Amniotic Fluid

    OpenAIRE

    UÇAR, Canan; Balci, Sevim; TUNÇBİLEK, Ergül; ÜNSAL, İbrahim

    2005-01-01

    The aim of this study was to investigate the correlation between Down syndrome and the level of placental alkaline phosphatase (PLAP) in amniotic fluid. A total of 279 amniotic fluid samples taken between 14 and 24 gestational weeks were investigated in this study. Karyotype analysis was made in all samples. In 10 samples trisomy 21 was determined, in one sample trisomy 18, in two samples 47,XXY, in six samples various structural chromosomal anomalies and in 260 samples normal karyotype. PLA...

  14. Analytical validation of serum bone alkaline phosphatase (BAP OSTASE) on Liaison

    OpenAIRE

    Cavalier, Etienne; Rozet, Eric; Carlisi, Agnès; Bekaert, Anne-Catherine; ROUSSELLE, Olivier; Hubert, Philippe; Chapelle, Jean-Paul; Delanaye, Pierre

    2009-01-01

    Background: The goal of this study was to validate the DiaSorin Liaison BAP OSTASE, a new method for measurement of bone alkaline phosphatase (BAP), and to compare this method with the Beckman-Coulter Access Ostase. We also wanted to establish the reference range for BAP in adults and children. Methods: We determined the precision, functional sensitivity, recovery, linearity and measurement uncertainty, accuracy profile and β-expectation limits. We defined an adult reference interval using i...

  15. Structural characteristics of alkaline phosphatase from the moderately halophilic bacterium Halomonas sp. 593

    Energy Technology Data Exchange (ETDEWEB)

    Arai, Shigeki; Yonezawa, Yasushi [Japan Atomic Energy Agency, 2-4 Shirakata-shirane, Tokai, Ibaraki 319-1195 (Japan); Ishibashi, Matsujiro [Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065 (Japan); Matsumoto, Fumiko; Adachi, Motoyasu; Tamada, Taro [Japan Atomic Energy Agency, 2-4 Shirakata-shirane, Tokai, Ibaraki 319-1195 (Japan); Tokunaga, Hiroko [Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065 (Japan); Blaber, Michael [Florida State University, 1115 West Call Street, Tallahassee, FL 32306-4300 (United States); Tokunaga, Masao [Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065 (Japan); Kuroki, Ryota, E-mail: kuroki.ryota@jaea.go.jp [Japan Atomic Energy Agency, 2-4 Shirakata-shirane, Tokai, Ibaraki 319-1195 (Japan)

    2014-03-01

    In order to clarify the structural basis of the halophilic characteristics of an alkaline phosphatase derived from the moderate halophile Halomonas sp. 593 (HaAP), the tertiary structure of HaAP was determined to 2.1 Å resolution by X-ray crystallography. The structural properties of surface negative charge and core hydrophobicity were shown to be intermediate between those characteristic of halophiles and non-halophiles, and may explain the unique functional adaptation to a wide range of salt concentrations. Alkaline phosphatase (AP) from the moderate halophilic bacterium Halomonas sp. 593 (HaAP) catalyzes the hydrolysis of phosphomonoesters over a wide salt-concentration range (1–4 M NaCl). In order to clarify the structural basis of its halophilic characteristics and its wide-range adaptation to salt concentration, the tertiary structure of HaAP was determined by X-ray crystallography to 2.1 Å resolution. The unit cell of HaAP contained one dimer unit corresponding to the biological unit. The monomer structure of HaAP contains a domain comprised of an 11-stranded β-sheet core with 19 surrounding α-helices similar to those of APs from other species, and a unique ‘crown’ domain containing an extended ‘arm’ structure that participates in formation of a hydrophobic cluster at the entrance to the substrate-binding site. The HaAP structure also displays a unique distribution of negatively charged residues and hydrophobic residues in comparison to other known AP structures. AP from Vibrio sp. G15-21 (VAP; a slight halophile) has the highest similarity in sequence (70.0% identity) and structure (C{sup α} r.m.s.d. of 0.82 Å for the monomer) to HaAP. The surface of the HaAP dimer is substantially more acidic than that of the VAP dimer (144 exposed Asp/Glu residues versus 114, respectively), and thus may enable the solubility of HaAP under high-salt conditions. Conversely, the monomer unit of HaAP formed a substantially larger hydrophobic interior

  16. Structural studies of human alkaline phosphatase in complex with strontium: Implication for its secondary effect in bones

    OpenAIRE

    Llinas, Paola; Masella, Michel; Stigbrand, Torgny; Ménez, André; Stura, Enrico A.; Le Du, Marie Hélène

    2006-01-01

    Strontium is used in the treatment of osteoporosis as a ranelate compound, and in the treatment of painful scattered bone metastases as isotope. At very high doses and in certain conditions, it can lead to osteomalacia characterized by impairment of bone mineralization. The osteomalacia symptoms resemble those of hypophosphatasia, a rare inherited disorder associated with mutations in the gene encoding for tissue-nonspecific alkaline phosphatase (TNAP). Human alkaline phosphatases have four m...

  17. Salivary alkaline phosphatase levels speak about association of smoking, diabetes and potentially malignant diseases???

    Directory of Open Access Journals (Sweden)

    A Ravi Prakash

    2016-01-01

    Full Text Available Background: Alkaline phosphatase (ALP is a hydrolase intracellular enzyme participating in the metabolic processes of cells. Rise in salivary ALP (S-ALP levels reflects inflammation and destruction of healthy tissues suggesting it as a clinical biomarker. S-ALP is used in analyzing the severity of the disease occurrence in smokers and nonsmokers who are diabetic and nondiabetic. S-ALP levels are analyzed using autoanalyzer in 40 patients who visited our department. Aims and Objectives: To determine the levels of S-ALP in diagnosing potentially malignant conditions and debilitating diseases in early stages of inflammation and altered cellular metabolism. Materials and Methods: The study groups include: (1 Group A - 10 smokers who are diabetic. (2 Group B - 10 smokers who are nondiabetic. (3 Group C - 10 nonsmokers who are diabetic. (4 Group D - 10 nonsmokers and nondiabetic as control. Unstimulated saliva samples are collected and run in auto-analyzer with ALP enzyme reagent to analyze ALP levels. Comparison is made between all the four groups. Results: Results were statistically significant with increased activity of ALP levels in saliva from Group A when compared to Group D. The results are Group A > Group B > Group C > Group D. The results also revealed significant raise in levels of ALP levels in saliva from smokers when compared to diabetes. Thus explaining adverse effects of smoking. Conclusion: S-ALP can be considered to be the biomarker for evaluating adverse effects of smoking, diabetes and other debilitating diseases in early stages.

  18. Invited review: The application of alkaline phosphatase assays for the validation of milk product pasteurization.

    Science.gov (United States)

    Rankin, S A; Christiansen, A; Lee, W; Banavara, D S; Lopez-Hernandez, A

    2010-12-01

    Standard practices for indirectly assessing the pasteurization status of milk products are primarily based on the thermal inactivation kinetics of the endogenous milk enzyme, alkaline phosphatase (ALP). This assessment provides an invaluable, if not required, tool for both regulatory and in-house process control and validation. Endogenous milk ALP manifests a slightly higher heat resistance than the pathogenic microflora upon which pasteurization time and temperature requirements are based. Hence, ALP activity is recognized and accepted as the method of choice for the rapid validation of milk product pasteurization. However, ALP assays have notable limitations that must be understood if they are to be administered and interpreted correctly and the results are to be applied judiciously. Issues such as the reactivation of heat-denatured ALP and the presence of both heat-stable and -labile microbial ALP are addressed. A discussion of ALP in the milk of nonbovine species is presented based on the limited literature available. Some discussion of research involving alternative pasteurization indicators also is presented. This article is intended to summarize the pertinent details of the ALP assay for dairy products (noting the basis and limitations of various methods) and the processing, handling, and known compositional factors that influence the assay results.

  19. EXPRESSION OF ALKALINE PHOSPHATASE DURING OSTEOGENIC DIFFERENTIATION OF RAT BONE MARROW STROMAL CELLS

    Directory of Open Access Journals (Sweden)

    AKBARI M

    2001-01-01

    Full Text Available Introduction: Bone marrow contains a population of stem cells capable of differentiating to osteoblast and forming the bone nodule by dexamethasone. Material and Methods: The stromal cells of bone marrow obtained from 4 to 6 weeks old Spruge-Dawely male rats were grown in primary culture for 7 days and subcultured for 18 days. The cells were cultured in either DMEM medium containing 15% fetal calf serum and antibiotics as the controls or the above medium supplemented with osteogenic supplements (OS: include 10 mM Na-beta glycerophosphate (Na-betaGp, 10 nM dexamethasone (Dex and 50 g/ml ascordic acid (AsA as the examined cultures. After 6, 12 and 18 days of grow up in subculture, the cultures were examined for mineralization and alkaline phosphatase (Apase expression. Results: Mesenchymal stem cells (MSCs in examined cultures underwent a dramatic change in cellular morphology and a significat increase in Apase activity by day 12. The deposition of a calcified matrix on the surface of the culture flasks became evident between days 12 and 18. Conclusion: The addition of osteogenic supplements (OS to MSCs cultures induced Apase expression that contributes to cellular differentiation and mineralization of extracellular matrix.

  20. Kidney bone disease and mortality in CKD: revisiting the role of vitamin D, calcimimetics, alkaline phosphatase, and minerals.

    Science.gov (United States)

    Kalantar-Zadeh, Kamyar; Shah, Anuja; Duong, Uyen; Hechter, Rulin C; Dukkipati, Ramanath; Kovesdy, Csaba P

    2010-08-01

    Recent evidence suggests that the traditional syndromes known as renal osteodystrophy, secondary hyperparathyroidism, and vitamin D deficiency are related to mortality in persons with moderate to advanced chronic kidney disease (CKD). The so-called 'kidney bone disease', also known as 'mineral and bone disorders', is defined to include bone disorders, mineral disarrays, and vascular calcification. We have identified 14 common and clinically relevant conditions of contemporary nature that are related to the kidney bone disease, including calcitriol (active vitamin D) deficiency, 25(OH)-vitamin D deficiency, biochemical hyperparathyroidism, relatively low parathyroid hormone (PTH) level, increased serum alkaline phosphatase (hyperphosphatasemia), elevated fibroblast growth factor (FGF)-23, high turnover bone disease, adynamic bone disease, uremic osteoporosis, vascular calcification, hyper- and hypophosphatemia, and hyper- and hypocalcemia. We present a critical review of these 14 conditions with emphasis on patient survival and other pertinent clinical outcomes. We also review unresolved controversies surrounding the management of these conditions by administration of nutritional vitamin D (ergocalciferol and cholecalciferol), vitamin D receptor activators (calcitriol, alphacalcidiol, doxercalciferol), D-mimetics (paricalcitol, maxacalcitol), calcimimetics (cinacalcet), recombinant PTH (teriparatide), and receptor activator of nuclear factor-kappaB ligand modulators (denosumab); compare mortality predictability of PTH and alkaline phosphatase; and examine potential risks of bone disorders and mineral disarrays in CKD patients. PMID:20671739

  1. Enzymatic methods for the determination of pollution in seawater using salt resistant alkaline phosphatase from eggs of the sea urchin Strongylocentrotus intermedius

    International Nuclear Information System (INIS)

    Highlights: • Alkaline phosphatase from eggs of the sea urchin (StAP) proved to be active in seawater. • Activity of StAP is inhibited by very low concentrations of heavy metal. • A test to assess sea and fresh water quality has been developed basing on StAP. • For the first time a salt resistant alkaline phosphatase has been found in eukaryote. - Abstract: A new salt resistant alkaline phosphatase from eggs of the sea urchin Strongylocentrotus intermedius (StAP) has been shown to have a unique property to hydrolyze substrate in seawater without loss of enzymatic activity. The enzyme has pH optimum at 8.0–8.5. Model experiments showed various concentrations of copper, zinc, cadmium and lead added to seawater or a standard buffer mixture to inhibit completely the enzyme activity at the concentrations of 15–150 μg/l. StAP sensitivity to the presence in seawater of metals, pesticides, detergents and oil products appears to be considerably less. Samples of seawater taken from aquatic areas of the Troitsy Bay of the Peter the Great Bay, Japan Sea have been shown to inhibit the enzyme activity; the same was shown for the samples of fresh waters. The phosphatase inhibition assay developed proved to be highly sensitive, technically easy-to use allowing to test a great number of samples

  2. Reduced L/B/K alkaline phosphatase gene expression in renal cell carcinoma: plausible role in tumorigenesis.

    Science.gov (United States)

    Sharma, Ujjawal; Pal, Deeksha; Singh, Shrawan Kumar; Kakkar, Nandita; Prasad, Rajendra

    2014-09-01

    Renal cell carcinoma (RCC) is the most common kidney cancer in adults. Although several genes have been found to be involved in carcinogenesis of RCC, more great efforts are needed to identify new genes which are responsible for the process. Clear cell RCC, originates from proximal tubule cells, is the most common pathological type of RCC. Alkaline phosphatase (ALP) is a marker enzyme of brush border membrane of proximal tubular cells. Our previous studies showed a significant decreased activity of Liver/Bone/Kidney (L/B/K) alkaline phosphatase in RCC. In the present study, we explored the molecular basis of the decreased activity of ALP in RCC. Immunohistochemistry, immunofluorescence and flow cytometry analysis showed decreased ALP protein in RCC. Additionally, real time PCR documented significantly reduced ALP gene expression (P = 0.009). Moreover, RCC cell lines (ACHN and A498) transfected with full length L/B/K cDNA showed decreased migratory property as well as viability of these cells as compared with controls (P = 0.000). Further, L/B/K ALP cDNA transfected cells (ACHN and A498) showed significant increased apoptosis as compared to control (P = 0.000). These findings suggest the new role of ALP in cell viability and apoptosis and involvement in RCC tumorigenesis. However, further studies are needed to explore the exact molecular mechanism. PMID:24909115

  3. Assessing the Biological Activity of the Glucan Phosphatase Laforin.

    Science.gov (United States)

    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

  4. Diagnostic Utility of Heat Stable Alkaline Phosphatase in Hypertensive Disorders of Pregnancy

    Science.gov (United States)

    Abu Raghavan, Srinivasan; Ghosh, Seethesh; Basu, Sharbari; Ramasamy, Ramesh; Murugaiyan, Sathish Babu

    2014-01-01

    Background: Hypertensive disorders in pregnancy (HDP) complicate 3-10% of all pregnancies. Though there are several biochemical parameters which aid in predicting hypertension of pregnancy, human placental alkaline phosphatase (PLAP), synthesized in placenta during pregnancy by placental syncytiotrophoblast, assumes diagnostic relevance. The purpose of this study was to compare the total alkaline phosphatase (ALP) and heat stable placental alkaline phosphatase (PLAP) levels in the serum of normotensive and hypertensive disorders of pregnancy and to evaluate the clinical utility of ALP and PLAP as a reliable, sensitive, specific and economical biochemical marker of HDP. Materials and Methods: This was a case control study, carried out on pregnant women with hypertension, of south Indian population. Study included pregnant women, 60 patients with hypertension and 60 controls. Biochemical assays were carried out by the IFCC approved procedures based on spectrophotometric method and using fully automated random access chemistry analyser. Data was compared by using student t-test. ROC was drawn to find out optimum cut off for ALP, PLAP and PLAP/ALP ratio in HDP. Pearson’s correlation was performed to ascertain the association among markers. Results: Serum total ALP, PLAP and PLAP/ALP ratio levels were significantly higher in hypertensive pregnant women when compared to controls (p<0.05). There was significant correlation among ALP, PLAP and DBP. ROC analysis of ALP (169.5), PLAP (69) and PLAP/ALP (0.44) ratios showed optimum cut-offs in diagnosis of hypertension in pregnancy. Conclusion: Serum heat stable ALP isoenzyme and PLAP/ALP ratio could be useful adjuvant markers in diagnosis of HDP in association with other relevant and economically viable biochemical tests. PMID:25584211

  5. The Detection of Alkaline Phosphatase Using an Electrochemical Biosensor in a Single-Step Approach

    OpenAIRE

    Chung-Chiun Liu; Kevin Wang; Wang, Joanne H.; Brandon Bartling

    2009-01-01

    A one-step, single use, disposable Alkaline Phosphatase (ALP) biosensor has been developed. It is based on the detection of phenol produced by an ALP enzymatic reaction. It can operate at 25 °C in a pH 10 medium. It measures ALP of 0–300 IU/L. The permissible concentrations of glucose, ascorbic acid and urea without interference are 10 mM/L, 5 mg/L and 400 mg/L, respectively. Experimental results are compared to those obtained by spectrophotometric measurements in bovine serum. Excellent line...

  6. Intestinal alkaline phosphatase: a summary of its role in clinical disease.

    Science.gov (United States)

    Fawley, Jason; Gourlay, David M

    2016-05-01

    Over the past few years, there is increasing evidence implicating a novel role for Intestinal Alkaline Phosphatase (IAP) in mitigating inflammatory mediated disorders. IAP is an endogenous protein expressed by the intestinal epithelium that is believed to play a vital role in maintaining gut homeostasis. Loss of IAP expression or function is associated with increased intestinal inflammation, dysbiosis, bacterial translocation and subsequently systemic inflammation. As these events are a cornerstone of the pathophysiology of many diseases relevant to surgeons, we sought to review recent research in both animal and humans on IAP's physiologic function, mechanisms of action and current research in specific surgical diseases.

  7. Serial serum alkaline phosphatase as an early biomarker for osteopenia of prematurity.

    Science.gov (United States)

    Abdallah, Enas A A; Said, Reem N; Mosallam, Dalia S; Moawad, Eman M I; Kamal, Naglaa M; Fathallah, Mohammed G E-D

    2016-09-01

    Metabolic bone disease of prematurity is a condition characterized by reduction in bone mineral content (osteopenia). It is a problem faced by very low birth weight (VLBW) infants because of lack of fetal mineralization during the last trimester. Our aim was to assess serum alkaline phosphatase (ALP) level as an early biomarker for osteopenia in premature infants and to estimate an optimal cutoff value of serum ALP at which osteopenia is detected radiologically in premature newborns.This prospective study was conducted on a cohort of 120 newborn infants of both sex of ≤34 weeks' gestational age and premature infants particularly those <1000-g birth weight and <32 weeks' gestation. PMID:27631238

  8. Properties of a constitutive alkaline phosphatase from strain 74A of the mold Neurospora crassa

    OpenAIRE

    Morales A.C.; Nozawa S.R.; Thedei Jr. G.; Maccheroni Jr. W.; De Rossi A.

    2000-01-01

    A constitutive alkaline phosphatase was purified to apparent homogeneity as determined by polyacrylamide gel electrophoresis from mycelia of the wild strain 74A of the mold Neurospora crassa, after growth on acetate and in the presence of saturating amounts of inorganic phosphate (Pi) for 72 h at 30ºC. The molecular mass was 58 kDa and 56 kDa as determined by exclusion chromatography and SDS-PAGE, respectively. This monomeric enzyme shows an apparent optimum pH ranging from 9.5 to 10.5 and Mi...

  9. A New Voltammetric Enzyme Immunoassay System for the Detection of Alkaline Phosphatase

    Institute of Scientific and Technical Information of China (English)

    KuiJIAO; WeiSUN; 等

    2002-01-01

    A new voltammetric enzyme immunoassay system was invesigated based on p-nitrophenyl phosphate (PNPP) as the subsrate for alkaline phosphatase (ALP). PNPP is enzymatically hydrolyzed and the product p-nitrophenol (PNP) is detected by differential pulse voltammetry (DPV), which can be oxidized at +1.02 V(vs.Ag/AgCl) on bare glass carbon electrode (GCE). The conditions for enzymatic reaction and electrochemical detection were studied. According to this method,ALP can be detected with a detection limit of 2.8×102 mU/L and a linear range of 4.0×102-1.0×106mU/L.

  10. A New Voltammetric Enzyme Immunoassay System for the Detection of Alkaline Phosphatase

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A new voltammetric enzyme immunoassay system was investigated based on p-nitrophenyl phosphate (PNPP) as the substrate for alkaline phosphatase (ALP). PNPP is enzymatically hydrolyzed and the product p-nitrophenol (PNP) is detected by differential pulse voltammetry (DPV), which can be oxidized at +1.02 V (vs. Ag/AgCl) on bare glass carbon electrode (GCE). The conditions for enzymatic reaction and electrochemical detection were studied. According to this method, ALP can be detected with a detection limit of 2.8′102 mU/L and a linear range of 4.0′102 ~ 1.0′106 mU/L.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-08-01

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

  12. The mechanism of mineralization and the role of alkaline phosphatase in health and disease.

    Science.gov (United States)

    Orimo, Hideo

    2010-02-01

    Biomineralization is the process by which hydroxyapatite is deposited in the extracellular matrix. Physiological mineralization occurs in hard tissues, whereas pathological calcification occurs in soft tissues. The first step of mineralization is the formation of hydroxyapatite crystals within matrix vesicles that bud from the surface membrane of hypertrophic chondrocytes, osteoblasts, and odontoblasts. This is followed by propagation of hydroxyapatite into the extracellular matrix and its deposition between collagen fibrils. Extracellular inorganic pyrophosphate, provided by NPP1 and ANKH, inhibits hydroxyapatite formation. Tissue-nonspecific alkaline phosphatase (TNAP) hydrolyzes pyrophosphate and provides inorganic phosphate to promote mineralization. Inorganic pyrophosphate, pyridoxal phosphate, and phosphoethanolamine are thought to be the physiologic substrates of TNAP. These accumulate in the event of TNAP deficiency, e.g., in cases of hypophosphatasia. The gene encoding TNAP is mapped to chromosome 1, consists of 12 exons, and possesses regulatory motifs in the 5'-untranslated region. Inhibition of TNAP enzymatic activity suppresses TNAP mRNA expression and mineralization in vitro. Hypophosphatasia is an inherited systemic bone disease characterized by hypomineralization of hard tissues. The phenotype of hypophosphatasia is varied. To date, more than 200 mutations in the TNAP gene have been reported. Knockout mice mimic the phenotypes of severe hypophosphatasia. Among the mutations in the TNAP gene, c.1559delT is frequent in the Japanese population. This frameshift mutation results in the expression of an abnormally long protein that is degraded in cells. DNA-based prenatal diagnosis using chorionic villus sampling has been developed, but requires thorough genetic counseling. Although hypophosphatasia is untreatable at present, the recent success of enzyme replacement therapy offers promise. The problems presented by impaired mineralization in age

  13. Genetic optimization of a bacteriophage-delivered alkaline phosphatase reporter to detect Escherichia coli.

    Science.gov (United States)

    Jackson, Angelyca A; Hinkley, Troy C; Talbert, Joey N; Nugen, Sam R; Sela, David A

    2016-10-01

    A large fraction of foodborne illnesses are linked to (∼46%) leafy green vegetables contaminated by pathogens harbored in agricultural water. To prevent this, accurate point-of-production detection tools are required to identify and quantify bacterial contaminants in produce before consumers are impacted. In this study, a proof-of-concept model was engineered for a phage-based Escherichia coli detection system. We engineered the coliphage T7 to express alkaline phosphatase (ALP) to serve as the signal for E. coli detection. Wild type phoA (T7ALP) and a dominant-active allele, phoA D153G D330N (T7ALP*) was inserted into the T7 genome, with engineered constructs selected by CRISPR-mediated cleavage of unaltered chromosomes and confirmed by PCR. Engineered phages and E. coli target cells were co-incubated for 16 hours to produce lysates with liberated ALP correlated with input cell concentrations. A colorimetric assay used p-nitrophenyl phosphate (pNPP) to demonstrate significant ALP production by T7ALP and T7ALP* compared to the vector control (T7EV) (p≤ 0.05). Furthermore, T7ALP* produced 2.5-fold more signal than T7ALP (p≤ 0.05) at pH 10. Due to the increase in signal for the modified ALP* allele, we assessed T7ALP* sensitivity in a dose-responsive manner. We observed 3-fold higher signal for target cell populations as low as ∼2 × 10(5) CFU mL(-1) (p≤ 0.05 vs. no-phage control). PMID:27412402

  14. Alkaline phosphatase inhibition based conductometric biosensor for phosphate estimation in biological fluids.

    Science.gov (United States)

    Upadhyay, Lata Sheo Bachan; Verma, Nishant

    2015-06-15

    Determination of phosphate ions concentration is very important from both, environmental and clinical point of view. In this study, a simple and novel conductometric biosensor for indirect determination of the phosphate ions in aqueous solution has been developed. The developed biosensor is based on the inhibition of immobilized alkaline phosphatase activity, in the presence of the phosphate ions. This is the first time we developed a mono-enzymatic biosensor for indirect estimation of phosphate ions. The developed biosensor showed a broad linear response (as compared to other reported biosensors) for phosphate ions in the range of 0.5-5.0 mM (correlation coefficient=0.995), with a detection limit of 50 µM. Different optimized parameters were obtained as the buffer concentration of 30 mM, substrate concentration of 1.0mM, and a pH of 9.0. All the optimized parameters were analyzed by analysis of variance, and were found to be statistically significant at a level of α=0.05. The developed biosensor is also suitable to determine the serum phosphate concentration, with a recovery of 86-104%, while a recovery of 102% was obtained from the water samples that were spiked with 500 µM phosphate. A relative standard deviation in the conductance response for five successive measurements (n=5) did not exceed 7%, with a shelf life of 30 days. With a lower detection limit and a higher recovery, the biosensor provides a facile approach for phosphate estimation in biological fluids.

  15. Properties of a constitutive alkaline phosphatase from strain 74A of the mold Neurospora crassa

    Directory of Open Access Journals (Sweden)

    Morales A.C.

    2000-01-01

    Full Text Available A constitutive alkaline phosphatase was purified to apparent homogeneity as determined by polyacrylamide gel electrophoresis from mycelia of the wild strain 74A of the mold Neurospora crassa, after growth on acetate and in the presence of saturating amounts of inorganic phosphate (Pi for 72 h at 30ºC. The molecular mass was 58 kDa and 56 kDa as determined by exclusion chromatography and SDS-PAGE, respectively. This monomeric enzyme shows an apparent optimum pH ranging from 9.5 to 10.5 and Michaelis kinetics for the hydrolysis of p-nitrophenyl phosphate (the Km and Hill coefficient values were 0.35 mM and 1.01, respectively, alpha-naphthyl phosphate (the Km and Hill coefficient values were 0.44 mM and 0.97, respectively, ß-glycerol phosphate (the Km and Hill coefficient values were 2.46 mM and 1.01, respectively and L-histidinol phosphate (the Km and Hill coefficient values were 0.47 mM and 0.94, respectively at pH 8.9. The purified enzyme is activated by Mg2+, Zn2+ and Tris-HCl buffer, and is inhibited by Be2+, histidine and EDTA. Also, 0.3 M Tris-HCl buffer protected the purified enzyme against heat inactivation at 70ºC(half-life of 19.0 min, k = 0.036 min-1 as compared to 0.3 M CHES (half-life of 2.3 min, k = 0.392 min-1 in the same experiment.

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

    International Nuclear Information System (INIS)

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

  17. Phosphatase Activity of Microbial Populations in Different Milk Samples in Relation to Protein and Carbohydrate Content

    Directory of Open Access Journals (Sweden)

    Sosanka Protim SANDILYA

    2014-12-01

    Full Text Available Cattle milk is a rich source of protein, carbohydrate, vitamins, minerals and all other major and micro nutrients. At a moderate pH, milk is an excellent media for the growth of microbes and thus, intake of raw milk is precarious. In this study, attempt was made for a qualitative study of eight raw milk samples of different varieties of cow and goat milk, collected from Jorhat district of Assam, India, on the basis of nutritional value and microbial population. The highest microbial population was found in the milk collected from cross hybrid variety of cow, whereas microbial contamination was the least in Jersey cow milk. Samples of C1 (Jersey cow variety showed presence of the highest amount of protein and carbohydrate content as compared to the others. Almost all the milk samples showed positive acid and alkaline phosphatase activity. Maximum acid phosphatase activity was observed in cross hybrid cow milk, whereas local cow milk exhibited the highest alkaline phosphatase activity. Phosphatase activity did not show any co-relationship with microbial population of the milk samples. Similarly, the protein and carbohydrate content of the samples did not have any significant impact on both acid and alkaline phosphatase activity.

  18. Carbon and Nitrogen Sources Influence Tricalcium Phosphate Solubilization and Extracellular Phosphatase Activity by Talaromyces flavus.

    Science.gov (United States)

    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.

  19. Tissue-nonspecific Alkaline Phosphatase Regulates Purinergic Transmission in the Central Nervous System During Development and Disease

    Directory of Open Access Journals (Sweden)

    Álvaro Sebastián-Serrano

    2015-01-01

    Full Text Available Tissue-nonspecific alkaline phosphatase (TNAP is one of the four isozymes in humans and mice that have the capacity to hydrolyze phosphate groups from a wide spectrum of physiological substrates. Among these, TNAP degrades substrates implicated in neurotransmission. Transgenic mice lacking TNAP activity display the characteristic skeletal and dental phenotype of infantile hypophosphatasia, as well as spontaneous epileptic seizures and die around 10 days after birth. This physiopathology, linked to the expression pattern of TNAP in the central nervous system (CNS during embryonic stages, suggests an important role for TNAP in neuronal development and synaptic function, situating it as a good target to be explored for the treatment of neurological diseases. In this review, we will focus mainly on the role that TNAP plays as an ectonucleotidase in CNS regulating the levels of extracellular ATP and consequently purinergic signaling.

  20. RELATION BETWEEN ALKALINE PHOSPHATASE IN GINGIGVAL CREVICULAR FLUID OF IMPLANT TEETH AND THE CURING RESULT

    Institute of Scientific and Technical Information of China (English)

    周正; 周坚; 邹石莹; 吴效民

    2001-01-01

    Objective. To discover the relation between alkaline phosphatase (ALP) in gingival crevicular fluid (GCF)of implant teeth aad the curing results.Methods. We measured the ALP level in GCF among 56 cases of implant teeth which included 2 failed cas-es, 5 cases with bad oral hygiene and gingivitis, and compared it with that in the normal group composed of 10persons.Results. The ALP levels in normal group and success implant group showed no difference. The ALP levelsin normal group and success with gingivitis group showed obvious difference. The ALP levels of the 2 failed cas-es are the highest of all.Conclusions. The ALP level in GCF is an important index in evaluating the curing result of the implantteeth.``

  1. Dietary free fatty acids form alkaline phosphatase-enriched microdomains in the intestinal brush border membrane

    DEFF Research Database (Denmark)

    Hansen, Gert H; Rasmussen, Karina; Niels-Christiansen, Lise-Lotte;

    2011-01-01

    mimicking a physiological solution of dietary mixed micelles, rearranged the lipid raft microdomain organization of the membranes. Thus, the fat mixture generated a low-density subpopulation of microvillar detergent resistant membranes (DRMs) highly enriched in alkaline phosphatase (AP). Since this GPI......-linked enzyme is the membrane protein in the brush border with the highest affinity for lipid rafts, this implies that free fatty acids selectively insert stably into these membrane microdomains. We have previously shown that absorption of dietary lipids transiently induce a selective endocytosis of AP from...... the brush border, and from work by others it is known that fat absorption is accompanied by a rise in serum AP and secretion of surfactant-like particles from enterocytes. We propose that these physiological processes may be triggered by the sequestering of dietary free fatty acids in lipid raft...

  2. Fluoride stimulates [3H]thymidine incorporation and alkaline phosphatase production by human osteoblasts

    International Nuclear Information System (INIS)

    The effect of sodium fluoride on alkaline phosphatase (ALP) release and [3H]thymidine uptake by human osteoblasts in culture was investigated. Sodium fluoride stimulated both ALP release and [3H]thymidine uptake at concentrations of sodium fluoride greater than 250 mumol/L. This stimulation was similar in magnitude to that induced by 1,25-dihydroxycholecalciferol. The fluoride-induced increase in ALP was inhibited by verapamil, a calcium channel blocker. We conclude that sodium fluoride stimulates osteoblasts to proliferate and to release ALP. This stimulation by fluoride is dependent on calcium influx. Fluoride-induced stimulation of human osteoblasts may be relevant to its effect in enhancing bone formation in patients with osteoporosis

  3. Diagnostic evaluation of canine serum alkaline phosphatase by immunochemical means and interpretation of results.

    Science.gov (United States)

    Saini, P K; Peavy, G M; Hauser, D E; Saini, S K

    1978-09-01

    Sera of several canine patients contained an isoenzyme of alkaline phosphatase (ALP) that resembled intestinal ALP with respect to heat inactivation, L-phenylalanine inhibition, and sensitivity to anti-canine intestinal ALP antibody, but differed with regard to the electrophoretic migration. The electrophoretic mobility of the isoenzyme was slightly cathodal than that of hepatic ALP, and its migration was reduced, similar to that of hepatic isoenzyme after neuraminidase treatment. This isoenzyme, which could be corticosteroid induced, was in the sera of numerous dogs with hepatobiliary disorders and was different from the hepatic isoenzyme that appeared in the sera of dogs with acute hepatitis, based on anti-canine intestinal ALP antibody interaction, heat inactivation, and electrophoretic migration. PMID:358873

  4. The Detection of Alkaline Phosphatase Using an Electrochemical Biosensor in a Single-Step Approach

    Directory of Open Access Journals (Sweden)

    Chung-Chiun Liu

    2009-10-01

    Full Text Available A one-step, single use, disposable Alkaline Phosphatase (ALP biosensor has been developed. It is based on the detection of phenol produced by an ALP enzymatic reaction. It can operate at 25 °C in a pH 10 medium. It measures ALP of 0–300 IU/L. The permissible concentrations of glucose, ascorbic acid and urea without interference are 10 mM/L, 5 mg/L and 400 mg/L, respectively. Experimental results are compared to those obtained by spectrophotometric measurements in bovine serum. Excellent linearity between the biosensor outputs and the ALP concentrations exists. The agreement between the measurements of this biosensor and the spectrophotometer is also outstanding.

  5. Relationship between serum heat—stable alkaline phosphatase level and p[regnancy

    Institute of Scientific and Technical Information of China (English)

    CaoGuo-Xian; DingMei-Juan; 等

    1998-01-01

    Serum heat-stable alkaline phosphatase(HSAP) level in 649 cases of normal pregnancy and 164 cases of high-risk pregnancy is measured by raioimmunoassay (RIA).The results indicate that the HSAP level in normal prenancy increased proportionally with gestation weeks(r=0.9843).In 33 cases of pregnancy induced hypertension and 21 cases of intrauterine fetal growth retardation,the HSAP level is significantly low.In 7 cases of neonatal asphyxia and 26 cases of ftal distress,the HSAP level in the mother's serum is also low.In 53 cases of intrahepatic cholestasis of pregnancy,the HSAP level in similar to those of normal pregnancy,This study illustrates that HSAP RIA can play an important role in the evaluation of placental function and fetal prognosis for cases of high-risk pregancy.

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

    Science.gov (United States)

    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…

  7. Electrochemical Detection of Alkaline Phosphatase in BALB/c Mouse Fetal Liver Stromal Cells with Capillary Electrophoresis

    Institute of Scientific and Technical Information of China (English)

    Xue Mei SUN; Dong LI; Zeng Liang BAI; Wen Rui JIN

    2004-01-01

    A method for determination of alkaline phosphatase (ALP) in BALB/c mouse fetal liver stromal cells has been described based on the catalytic reaction. After the cell extract is incubated with the substrate disodium phenyl phosphate, the reaction product phenol generated by ALP is determined by capillary electrophoresis with electrochemical detection.

  8. Changes in Bone Alkaline Phosphatase and Procollagen Type-1 C-Peptide after Static and Dynamic Exercises

    Science.gov (United States)

    Kubo, Keitaro; Yuki, Kazuhito; Ikebukuro, Toshihiro

    2012-01-01

    We investigated the effects of two types of nonweight-bearing exercise on changes in bone-specific alkaline phosphatase (BAP) and pro-collagen type 1 C-peptide (P1P). BAP is a specific marker of bone synthesis, whereas P1P reflects synthesis of type 1 collagen in other organs as well as bone. Eight participants performed static and dynamic…

  9. Treatment with bortezomib in multiple myeloma is associated with only a transient and brief increase of bone specific alkaline phosphatase

    DEFF Research Database (Denmark)

    Haidl, Felix; Plesner, Torben; Lund, Thomas

    2012-01-01

    There are indications of a bone anabolic effect associated with bortezomib treatment. We present a study with long follow up, measuring bone specific alkaline phosphatase (bALP) for a year during and after treatment in an unselected cohort of myeloma patients treated with bortezomib, and assess...

  10. The activity of some phosphatases in tissues of adult Hymenolepis nana Siebold (Csetoda).

    Science.gov (United States)

    Humiczewska, M

    1989-01-01

    Histochemical methods were used to study the localization and activity of acid and alkaline phosphatases, ATP-ase, 5-nucleotidase, and glucose-6-phosphatase in tissues of the mature form of Hymenolepis nana. Considerable differences in activity and localization of particular enzymes were observed in the organs of the parasite. The results obtained permit the statement that the integument is the most active enzymatically; in connection with the literature data, this gives grounds for the thesis that the integument of the cestodes functions as an absorbent-digestive organ. PMID:2558920

  11. Biological Apatite Formed from Polyphosphate and Alkaline Phosphatase May Exchange Oxygen Isotopes from Water through Carbonate

    Science.gov (United States)

    Omelon, S. J.; Stanley, S. Y.; Gorelikov, I.; Matsuura, N.

    2011-12-01

    The oxygen isotopic composition in bone mineral phosphate is known to reflect the local water composition, environmental humidity, and diet1. Once ingested, biochemical processes presumably equilibrate PO43- with "body water" by the many biochemical reactions involving PO43- 2. Blake et al. demonstrated that enzymatic release of PO43- from organophosphorus compounds, and microbial metabolism of dissolved orthophosphate, significantly exchange the oxygen in precipitated apatite within environmental water3,4, which otherwise does not exchange with water at low temperatures. One of the enzymes that can cleave phosphates from organic substrates is alkaline phosphastase5, the enzyme also associated with bone mineralization. The literature often states that the mineral in bone in hydroxylapatite, however the mineral in bone is carbonated apatite that also contains some fluoride6. Deprotonation of HPO32- occurs at pH 12, which is impossibly high for biological system, and the predominate carbonate species in solution at neutral pH is HCO3-. To produce an apatite mineral without a significant hydroxyl content, it is possible that apatite biomineralization occurs through a polyphosphate pathway, where the oxygen atom required to transform polyphosphate into individual phosphate ions is from carbonate: [PO3-]n + CO32- -> [PO3-]n-1 + PO43- + CO2. Alkaline phosphatase can depolymerise polyphosphate into orthophosphate5. If alkaline phosphatase cleaves an oxygen atom from a calcium-carbonate complex, then there is no requirement for removing a hydrogen atom from the HCO3- or HPO43- ions of body water to form bioapatite. A mix of 1 mL of 1 M calcium polyphosphate hydogel, or nano-particles of calcium polyphosphate, and amorphous calcium carbonate were reacted with alkaline phosphatase, and maintained at neutral to basic pH. After two weeks, carbonated apatite and other calcium phosphate minerals were identified by powder x-ray diffraction. Orthophosphate and unreacted

  12. Estimation and Comparison of Salivary Calcium, Phosphorous, Alkaline Phosphatase and pH Levels in Periodontal Health and Disease: A Cross-sectional Biochemical Study

    Science.gov (United States)

    Patel, Rufi Murad; Suragimath, Girish; Zope, Sameer

    2016-01-01

    Introduction In oral diagnostics there is a great challenge to determine biomarkers for screening and evaluating the disease activity. Biomarkers can also serve as a useful tool to measure the efficacy of the therapy. Aim To evaluate and compare the levels of salivary calcium, phosphorous, alkaline phosphatase and pH levels in periodontally healthy subjects and patients with gingivitis and periodontitis. Materials and Methods The present study consisted of 150 subjects aged between 20-45 years who were divided into three groups; periodontally healthy, gingivitis and chronic periodontitis. Prior to the clinical examination the demographic details, relevant information of the subject, gingival index, plaque index, Oral Hygiene Index (OHI) and pH were recorded. Biochemical assay of saliva i.e., inorganic calcium, phosphorous and alkaline phosphatase were estimated by colorimetric method. ANOVA and Tukey’s test were applied for statistical analysis. Results The mean levels of biomarkers studied were; inorganic calcium (12.55μg/dl), phosphorous (14.50μg/dl), alkaline phosphatase (49.62μg/dl) and pH (11.65). There was a gradual increase in these levels as the condition progressed from health to gingivitis or periodontitis which was statistically significant at pperiodontal tissues in disease and health. PMID:27630955

  13. Intestinal Alkaline Phosphatase: Potential Roles in Promoting Gut Health in Weanling Piglets and Its Modulation by Feed Additives - A Review.

    Science.gov (United States)

    Melo, A D B; Silveira, H; Luciano, F B; Andrade, C; Costa, L B; Rostagno, M H

    2016-01-01

    The intestinal environment plays a critical role in maintaining swine health. Many factors such as diet, microbiota, and host intestinal immune response influence the intestinal environment. Intestinal alkaline phosphatase (IAP) is an important apical brush border enzyme that is influenced by these factors. IAP dephosphorylates bacterial lipopolysaccharides (LPS), unmethylated cytosine-guanosine dinucleotides, and flagellin, reducing bacterial toxicity and consequently regulating toll-like receptors (TLRs) activation and inflammation. It also desphosphorylates extracellular nucleotides such as uridine diphosphate and adenosine triphosphate, consequently reducing inflammation, modulating, and preserving the homeostasis of the intestinal microbiota. The apical localization of IAP on the epithelial surface reveals its role on LPS (from luminal bacteria) detoxification. As the expression of IAP is reported to be downregulated in piglets at weaning, LPS from commensal and pathogenic gram-negative bacteria could increase inflammatory processes by TLR-4 activation, increasing diarrhea events during this phase. Although some studies had reported potential IAP roles to promote gut health, investigations about exogenous IAP effects or feed additives modulating IAP expression and activity yet are necessary. However, we discussed in this paper that the critical assessment reported can suggest that exogenous IAP or feed additives that could increase its expression could show beneficial effects to reduce diarrhea events during the post weaning phase. Therefore, the main goals of this review are to discuss IAP's role in intestinal inflammatory processes and present feed additives used as growth promoters that may modulate IAP expression and activity to promote gut health in piglets. PMID:26732323

  14. Prognostic Significance of Serum Alkaline Phosphatase Level in Osteosarcoma: A Meta-Analysis of Published Data

    Directory of Open Access Journals (Sweden)

    Hai-Yong Ren

    2015-01-01

    Full Text Available Background. Serum alkaline phosphatase (SALP is commonly elevated in osteosarcoma patients. A number of studies have investigated the prognostic role of SALP level in patients with osteosarcoma but yielded inconsistent results. Method. Systematic computerized searches were performed in PubMed, Embase, and Web of Science databases for relevant original articles. The pooled hazard ratios (HRs and relative risks (RRs with corresponding confidence intervals (CIs were calculated to assess the prognostic value of SALP level. Results. Finally, 21 studies comprising 3228 patients were included. Overall, the pooled HRs of SALP suggested that elevated level had an unfavorable impact on osteosarcoma patients’ overall survival (OS (HR = 1.82; 95% CI: 1.61–2.06; p<0.001 and event-free survival (EFS (HR = 1.97; 95% CI: 1.61–2.42; p<0.001. Combined RRs of SALP indicated that elevated level was associated with presence of metastasis at diagnosis (RR = 5.55; 95% CI: 1.61–9.49; p=0.006. No significantly different results were obtained after stratified by variables of age range, cancer stage, sample size, and geographic region. Conclusion. This meta-analysis demonstrated that high SALP level is significantly associated with poor OS or EFS rate and presence of metastasis at diagnosis. SALP level is a convenient and effective biomarker of prognosis for osteosarcoma.

  15. Dynamic evolution of the LPS-detoxifying enzyme intestinal alkaline phosphatase in zebrafish and other vertebrates

    Directory of Open Access Journals (Sweden)

    Ye eYang

    2012-10-01

    Full Text Available Alkaline phosphatases (Alps are well-studied enzymes that remove phosphates from a variety of substrates. Alps function in diverse biological processes, including modulating host-bacterial interactions by dephosphorylating the Gram-negative bacterial cell wall component lipopolysaccharide (LPS. In animals, Alps are encoded by multiple genes characterized by either ubiquitous expression (named Alpls, for their liver expression, or their tissue-specific expression, for example in the intestine (Alpi. We previously characterized a zebrafish alpi gene (renamed here alpi.1 that is regulated by Myd88-dependent innate immune signaling and that is required to prevent a host’s excessive inflammatory reactions to its resident microbiota. Here we report the characterization of two new alp genes in zebrafish, alpi.2 and alp3. To understand their origins, we investigated the phylogenetic history of Alp genes in animals. We find that vertebrate Alp genes are organized in three clades with one of these clades missing from the mammals. We present evidence that these three clades originated during the two vertebrate genome duplications. We show that in zebrafish alpl is ubiquitously expressed, as it is in mammals, whereas the other three alps are specific to the intestine. Our phylogenetic analysis reveals that in contrast to Alpl, which has been stably maintained as a single gene throughout the vertebrates, the Alpis have been lost and duplicated multiple times independently in vertebrate lineages, likely reflecting the rapid and dynamic evolution of vertebrate gut morphologies, driven by changes in bacterial associations and diet.

  16. Alkaline phosphatase levels in diagnostic peritoneal lavage fluid as a predictor of hollow visceral injury.

    Science.gov (United States)

    Jaffin, J H; Ochsner, M G; Cole, F J; Rozycki, G S; Kass, M; Champion, H R

    1993-06-01

    Isolated injuries to hollow viscera may result in equivocal diagnostic peritoneal lavage (DPL) findings. Small bowel injuries cause alkaline phosphatase (AP) levels to increase in DPL effluent. The goal of this study was to better define the role of AP levels in the evaluation of the injured abdomen. We prospectively measured AP levels in 672 patients undergoing DPL. These were retrospectively compared with the clinical findings. All 12 patients with small bowel injuries and three of four with large bowel injuries had an AP level > 10 IU/L. There was one patient with an AP level > 10 IU/L without clinically significant intra-abdominal injury. An AP level > 10 IU/L in the DPL effluent predicted injury requiring laparotomy with a specificity of 99.8% and a sensitivity of 94.7%. We recommend using AP levels only in the management of patients with equivocal findings on DPL who would otherwise not undergo laparotomy. This selective use of AP levels will improve the probability of early diagnosis of bowel injury without increasing the cost of care.

  17. Biodistribution and translational pharmacokinetic modeling of a human recombinant alkaline phosphatase.

    Science.gov (United States)

    Peters, Esther; Stevens, Jasper; Arend, Jacques; Guan, Zheng; Raaben, Willem; Laverman, Peter; van Elsas, Andrea; Masereeuw, Rosalinde; Pickkers, Peter

    2015-11-10

    Clinical trials showed renal protective effects of bovine intestinal alkaline phosphatase (AP) in patients with sepsis-associated acute kidney injury (AKI). Subsequently, a human recombinant chimeric AP (recAP) was developed as a pharmaceutically acceptable alternative. Here, we investigated the biodistribution and pharmacokinetics (PK) of recAP and developed a translational population PK model. Biodistribution was studied during LPS-induced AKI in rats. Iodine-125-labeled recAP was primarily taken up by liver, spleen, adrenals, heart, lungs and kidneys followed by the gastro-intestinal tract and thyroid. Tissue distribution was not critically affected by endotoxemia. PK parameters were determined in rats and minipigs during IV bolus injections of recAP, administered once, or once daily during seven consecutive days. Plasma concentrations of recAP increased with increasing dose and disappeared in a biphasic manner. Exposure to recAP, estimated by AUC and Cmax, was similar on days 1 and 7. Subsequently, population approach nonlinear mixed effects modeling was performed with recAP rat and minipig and biAP phase I PK data. Concentration versus time data was accurately described in all species by a two-compartmental model with allometric scaling based on body weight. This model provides a solid foundation for determining the optimal dose and duration of first-in-man recAP studies. PMID:26325308

  18. Titanium dioxide nanotube films: Preparation, characterization and electrochemical biosensitivity towards alkaline phosphatase.

    Science.gov (United States)

    Roman, Ioan; Trusca, Roxana Doina; Soare, Maria-Laura; Fratila, Corneliu; Krasicka-Cydzik, Elzbieta; Stan, Miruna-Silvia; Dinischiotu, Anca

    2014-04-01

    Titania nanotubes (TNTs) were prepared by anodization on different substrates (titanium, Ti6Al4V and Ti6Al7Nb alloys) in ethylene glycol and glycerol. The influence of the applied potential and processing time on the nanotube diameter and length is analyzed. The as-formed nanotube layers are amorphous but they become crystalline when subjected to subsequent thermal treatment in air at 550°C; TNT layers grown on titanium and Ti6Al4V alloy substrates consist of anatase and rutile, while those grown on Ti6Al7Nb alloy consist only of anatase. The nanotube layers grown on Ti6Al7Nb alloy are less homogeneous, with supplementary islands of smaller diameter nanotubes, spread across the surface. Better adhesion and proliferation of osteoblasts was found for the nanotubes grown on all three substrates by comparison to an unprocessed titanium plate. The sensitivity towards bovine alkaline phosphatase was investigated mainly by electrochemical impedance spectroscopy in relation to the crystallinity, the diameter and the nature of the anodization electrolyte of the TNT/Ti samples. The measuring capacity of the annealed nanotubes of 50nm diameter grown in glycerol was demonstrated and the corresponding calibration curve was built for the concentration range of 0.005-0.1mg/mL.

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

    Science.gov (United States)

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

    2016-06-01

    Immunohistochemical studies and molecular biology have enabled us to identify numerous proteins that are involved in the metabolism of bone, and their encoding genes. Among these is alkaline phosphatase (ALP), an enzyme that is responsible for the initiation of mineralisation of the extracellular matrix during alveolar bone repair. To evaluate the gene expression of ALP during this process, we studied nine healthy adult male rats, which had their maxillary central incisors extracted from the right side and were randomly divided into three groups. During three experimental periods, 7 days, 14 days, and 28 days, the alveoli were curetted, the rats killed, and samples analysed by real-time reverse transcription polymerase chain reaction (qRT-PCR). The RNAm that encodes the gene for the synthesis of ALP was expressed during the three periods analysed, but its concentration was significantly increased at 14 and 28 days compared with at 7 days. There was no significant difference between 14 and 28 days (p=0.0005). We conclude that genes related to ALP are expressed throughout the healing process and more intensively during the later periods (14 and 28 days), which coincides with the increased formation of mineralised bone. PMID:26935214

  20. Evaluation of biodegradation and biocompatibility of collagen/chitosan/alkaline phosphatase biopolymeric membranes

    Indian Academy of Sciences (India)

    E BERTEANU; D IONITA; M SIMOIU; M PARASCHIV; R TATIA; A APATEAN; M SIDOROFF; L TCACENCO

    2016-04-01

    The aim of this study was to develop a new variant of membranes based on collagen (COL), chitosan (CHI) and alkaline phosphatase (ALP) immobilized and cross-linking with glutaraldehyde (GA) at different concentrations. The biodegradation in the presence of collagenase was investigated. Biocompatibility was evaluated by MTT assay using a mouse fibroblast cell culture type NCTC (clone 929). Non-cross-linked samples were biocompatible and membranes cross-linked with low concentrations of GA (0.04, 0.08%) were also iocompatible. However, high concentrations of GA lead to a decreased biocompatibility. The adsorption behaviour of Ca$^{2+}$ ions to all membraneswere evaluated using the Freundlich isotherms. Haemolytic studies were performed in order to consider their applications in biomineralization process. By the addition of collagen and ALP to chitosan, the haemolytic indexdecreases, the COL–CHI–ALP membrane being in the non-haemolytic domain, while the COL–CHI–ALP–GA membrane has a haemolytic index greater than 2, and is slightly haemolytic.

  1. Alkaline phosphatase, cytokeratin 7, cytokeratin 8 in the diagnosis of primary lung adenocarcinoma from 148 pleura fluids specimens.

    OpenAIRE

    Temelli Ozlem; Ozhavzali MĂźzeyyen; Atay Meral; OÄuztĂźzĂźn Serpil; Yirtici Umit; TĂźrk Mustafa; Atay Ziya

    2009-01-01

    Adenocarcinomas are the most common cause of malignancy in pleura fluids. Usual primary sites include the lung, breast, gastrointestinal tract, and genitourinary tracts. Predicting the site of origin of an adenocarcinoma can be difficult due to overlapping morphologic characteristics. We investigated the use of alkaline phosphatase (AP), Cytokeratin7 (CK7) Cytokeratin8 (CK8) to distinguish adenocarcinomas of lung in 148 body cavity fluid samples. Overall results for primary lung adenocarcinom...

  2. A High Level of Intestinal Alkaline Phosphatase Is Protective Against Type 2 Diabetes Mellitus Irrespective of Obesity ☆

    OpenAIRE

    Malo, Madhu S.

    2015-01-01

    Highlights • Diabetes patients have very low levels of intestinal alkaline phosphatase (IAP) in their stool. • Obese people with high IAP do not develop diabetes. • A low level of IAP in a healthy person is indicative of having the incipient (latent) metabolic syndrome (diabetes). • Approximately 65% of the healthy population have the incipient metabolic syndrome including incipient diabetes. • ‘Temporal IAP profiling’ should diagnose the incipient metabolic syndrome (diabetes). Currently, di...

  3. Effects of Intercropping with Potato Onion on the Growth of Tomato and Rhizosphere Alkaline Phosphatase Genes Diversity

    OpenAIRE

    Wu, Xia; Wu, Fengzhi; Zhou, Xingang; Fu, Xuepeng; Tao, Yue; Xu, Weihui; Pan, Kai; Liu, Shouwei

    2016-01-01

    Background and Aims: In China, excessive fertilization has resulted in phosphorus (P) accumulation in most greenhouse soils. Intercropping can improve the efficiency of nutrient utilization in crop production. In this study, pot experiments were performed to investigate the effects of intercropping with potato onion (Allium cepa L. var. aggregatum G. Don) on tomato (Solanum lycopersicum L.) seedlings growth and P uptake, the diversity of rhizosphere phosphobacteria and alkaline phosphatase (A...

  4. Structural studies of human alkaline phosphatase in complex with strontium: Implication for its secondary effect in bones

    Science.gov (United States)

    Llinas, Paola; Masella, Michel; Stigbrand, Torgny; Ménez, André; Stura, Enrico A.; Le Du, Marie Hélène

    2006-01-01

    Strontium is used in the treatment of osteoporosis as a ranelate compound, and in the treatment of painful scattered bone metastases as isotope. At very high doses and in certain conditions, it can lead to osteomalacia characterized by impairment of bone mineralization. The osteomalacia symptoms resemble those of hypophosphatasia, a rare inherited disorder associated with mutations in the gene encoding for tissue-nonspecific alkaline phosphatase (TNAP). Human alkaline phosphatases have four metal binding sites—two for zinc, one for magnesium, and one for calcium ion—that can be substituted by strontium. Here we present the crystal structure of strontium-substituted human placental alkaline phosphatase (PLAP), a related isozyme of TNAP, in which such replacement can have important physiological implications. The structure shows that strontium substitutes the calcium ion with concomitant modification of the metal coordination. The use of the flexible and polarizable force-field TCPEp (topological and classical polarization effects for proteins) predicts that calcium or strontium has similar interaction energies at the calcium-binding site of PLAP. Since calcium helps stabilize a large area that includes loops 210–228 and 250–297, its substitution by strontium could affect the stability of this region. Energy calculations suggest that only at high doses of strontium, comparable to those found for calcium, can strontium substitute for calcium. Since osteomalacia is observed after ingestion of high doses of strontium, alkaline phosphatase is likely to be one of the targets of strontium, and thus this enzyme might be involved in this disease. PMID:16815919

  5. Alkaline phosphatase and OmpA protein can be translocated posttranslationally into membrane vesicles of Escherichia coli.

    OpenAIRE

    Chen, L.; Rhoads, D; Tai, P C

    1985-01-01

    We previously described a system for translocating the periplasmic enzyme alkaline phosphatase and the outer membrane protein OmpA into inverted membrane vesicles of Escherichia coli. We have now optimized and substantially improved the translocation system by including polyamines and by reducing the amount of membrane used. Under these conditions, efficient translocation was seen even posttranslationally, i.e., when vesicles were not added until after protein synthesis was stopped. This was ...

  6. Energy-requiring translocation of the OmpA protein and alkaline phosphatase of Escherichia coli into inner membrane vesicles.

    OpenAIRE

    Rhoads, D B; Tai, P C; Davis, B D

    1984-01-01

    In developing a reliable in vitro system for translocating bacterial proteins, we found that the least dense subfraction of the membrane of Escherichia coli was superior to the total inner membrane, both for a secreted protein (alkaline phosphatase) and for an outer membrane protein (OmpA). Compounds that eliminated the proton motive force inhibited translocation, as already observed in cells; since protein synthesis continued, the energy for translocation appears to be derived from the energ...

  7. A single-molecule digital enzyme assay using alkaline phosphatase with a cumarin-based fluorogenic substrate.

    Science.gov (United States)

    Obayashi, Yusuke; Iino, Ryota; Noji, Hiroyuki

    2015-08-01

    Digitalization of fluorogenic enzymatic assays through the use of femtoliter chamber array technology is an emerging approach to realizing highly quantitative bioassays with single-molecule sensitivity. However, only a few digital fluorogenic enzyme assays have been reported, and the variations of the digital enzyme assays are basically limited to fluorescein- and resorufin-based fluorogenic assays. This limitation hampers the realization of a multiplex digital enzyme assay such as a digital enzyme-linked immunosorbent assay (ELISA). In this study, after optimization of buffer conditions, we achieved a single-molecule digital enzyme alkaline phosphatase (ALP) assay with a cumarin-based fluorogenic substrate, 4-methylunbelliferyl phosphate (4-MUP). When ALP molecules were encapsulated in a 44-femtoliter chamber array at a low ratio of less than 1 molecule per chamber, each chamber showed a discrete fluorescence signal in an all-or-none manner, allowing the digital counting of the number of active enzyme molecules. The fraction of fluorescent chambers linearly decreased with the enzyme concentration, obeying the Poisson distribution as expected. We also demonstrated a dual-color digital enzyme assay with a ALP/4-MUP and β-galactosidase (β-gal)/resorufin-β-d-galactopyranoside combination. The activities of single ALP and β-gal molecules were clearly detected simultaneously. The method developed in this study will enable us to carry out a parallelized, multiplex digital ELISA. PMID:26101788

  8. PrognosticValue of PINP,BoneAlkaline Phosphatase, CTX-I, andYKL-40 in Patients With Metastatic Prostate Carcinoma

    DEFF Research Database (Denmark)

    Brasso, Klaus; Christensen, Ib Jarle; Johansen, Julia S;

    2006-01-01

    Prognostic value of PINP, bone alkaline phosphatase, CTX-I, and YKL-40 in patients with metastatic prostate carcinoma. Prostate. 2006 Apr 1;66(5):503-13. PMID: 16372331 [PubMed - indexed for MEDLINE]......Prognostic value of PINP, bone alkaline phosphatase, CTX-I, and YKL-40 in patients with metastatic prostate carcinoma. Prostate. 2006 Apr 1;66(5):503-13. PMID: 16372331 [PubMed - indexed for MEDLINE]...

  9. Synthesis of benzofuran derivatives as selective inhibitors of tissue-nonspecific alkaline phosphatase: effects on cell toxicity and osteoblast-induced mineralization.

    Science.gov (United States)

    Marquès, Stéphanie; Buchet, René; Popowycz, Florence; Lemaire, Marc; Mebarek, Saïda

    2016-03-01

    Tissue-nonspecific alkaline phosphatase (TNAP) by hydrolyzing pyrophosphate, an inhibitor of apatite formation, promotes extracellular matrix calcification during bone formation and growth, as well as during ectopic calcification under pathological conditions. TNAP is a target for the treatment of soft tissue pathological ossification. We synthesized a series of benzofuran derivatives. Among these, SMA14, displayed TNAP activity better than levamisole. SMA14 was found to be not toxic at doses of up to 40μM in osteoblast-like Saos-2 cells and primary osteoblasts. As probed by Alizarin Red staining, this compound inhibited mineral formation in murine primary osteoblast and in osteoblast-like Saos-2 cells.

  10. Contribution of matrix vesicles and alkaline phosphatase to ectopic bone formation

    Directory of Open Access Journals (Sweden)

    Ciancaglini P.

    2006-01-01

    Full Text Available Endochondral calcification involves the participation of matrix vesicles (MVs, but it remains unclear whether calcification ectopically induced by implants of demineralized bone matrix also proceeds via MVs. Ectopic bone formation was induced by implanting rat demineralized diaphyseal bone matrix into the dorsal subcutaneous tissue of Wistar rats and was examined histologically and biochemically. Budding of MVs from chondrocytes was observed to serve as nucleation sites for mineralization during induced ectopic osteogenesis, presenting a diameter with Gaussian distribution with a median of 306 ± 103 nm. While the role of tissue-nonspecific alkaline phosphatase (TNAP during mineralization involves hydrolysis of inorganic pyrophosphate (PPi, it is unclear how the microenvironment of MV may affect the ability of TNAP to hydrolyze the variety of substrates present at sites of mineralization. We show that the implants contain high levels of TNAP capable of hydrolyzing p-nitrophenylphosphate (pNPP, ATP and PPi. The catalytic properties of glycosyl phosphatidylinositol-anchored, polidocanol-solubilized and phosphatidylinositol-specific phospholipase C-released TNAP were compared using pNPP, ATP and PPi as substrates. While the enzymatic efficiency (k cat/Km remained comparable between polidocanol-solubilized and membrane-bound TNAP for all three substrates, the k cat/Km for the phosphatidylinositol-specific phospholipase C-solubilized enzyme increased approximately 108-, 56-, and 556-fold for pNPP, ATP and PPi, respectively, compared to the membrane-bound enzyme. Our data are consistent with the involvement of MVs during ectopic calcification and also suggest that the location of TNAP on the membrane of MVs may play a role in determining substrate selectivity in this micro-compartment.

  11. Effects of vasectomy on seminal plasma alkaline phosphatase in male alpacas (Vicugña pacos).

    Science.gov (United States)

    Pearson, L K; Campbell, A J; Sandoval, S; Tibary, A

    2013-12-01

    Azoospermia is a common finding in male alpacas which present for infertility. The challenge is to differentiate azoospermia of testicular origin from non-testicular origin. In several species, alkaline phosphatase (AP) concentrations in seminal plasma have been used as a diagnostic marker of contributions of the testis and epididymis to the ejaculate. The purpose of this study was to determine whether AP assay could differentiate testicular from non-testicular azoospermia in male alpacas. An experimental model of bilateral outflow obstruction (pre-scrotal vasectomy) was used in 22 male alpacas, aged 2-9 years. No reproductive history was available. Animals were submitted for electroejaculation (EE) under general anaesthesia and vasectomy performed. Five weeks later, animals were submitted for EE. Vasectomy was not successful in one animal, which was removed from analysis. AP levels were compared in seminal plasma in the pre- and post-vasectomy samples. The mean ± SEM concentration of AP in pre-vasectomy seminal plasma was 504.29 ± 166.45 U/l (range 10-2910); the post-vasectomy levels were 252.48 ± 81.77 U/l (range 0-1640; p = 0.06). In 71.4% of animals, AP levels decreased, varying from 18% to 100% reduction. Results of this study suggest that AP is not produced exclusively by the testis and epididymis in alpacas and that AP assay is not a valid diagnostic test for determination of origin of azoospermia; the gold standard for diagnosis of origin of azoospermia remains testicular biopsy. PMID:23790090

  12. Preoperative serum alkaline phosphatase: a predictive factor for early hypocalcaemia following parathyroidectomy of primary hyperparathyroidism

    Institute of Scientific and Technical Information of China (English)

    Sun Longhao; He Xianghui; Liu Tong

    2014-01-01

    Background Postoperative hypocalcemia is one of the most common complications following parathyroidectomy for primary hyperparathyroidism (PHPT).The aim of this study was to analyze the predictive value of biochemical parameters as indicators for episodes of hypocalcemia in patients undergoing parathyroidectomy for PHPT.Methods The patients with PHPT who underwent parathyroidectomy between February 2004 and February 2014 were studied retrospectively at a single medical center.The patients were divided into biochemical,clinical,and no postoperative hypocalcemia groups,based on different clinical manifestations.Potential risk factors for postoperative hypocalcemia were identified and investigated by univariate and multivariate Logistic regression analysis.Results Of the 139 cases,25 patients (18.0%) were diagnosed with postoperative hypocalcemia according to the traditional criterion.Univariate analysis revealed only alkaline phosphatase (ALP) and the small area under the curve (AUC) of receiver operating characteristics (ROC) curve for ALP demonstrates low accuracy in predicting the occurrence of postoperative hypocalcemia.Based on new criteria,22 patients were added to the postoperative hypocalcemia group and similar biochemical parameters were compared.The serum ALP was a significant independent risk factor for postoperative hypocalcemia (P=0.000) and its AUC of ROC curve was 0.783.The optimal cutoff point was 269 U/L and the sensitivity and specificity for prediction were 89.2% and 64.3%,respectively.Conclusions The risk of postoperative hypocalcemia after parathyroidectomy should be emphasized for patients with typical symptoms of hypocalcemia despite their serum calcium level is in normal or a little higher range.Serum ALP is a predictive factor for the occurrence of postoperative hypocalcemia.

  13. Spatial-temporal Variations of Alkaline Phosphatase Activity in the Kuilei Lake and Their Influencing Factors%傀儡湖水体中碱性磷酸酶活性的时空变化及其影响因素

    Institute of Scientific and Technical Information of China (English)

    张成艳; 成小英; 王建军; 张路; 杜应旸

    2013-01-01

    Alkaline phosphatase activity (APA) plays an important role in the biogeochemical cycling of phosphorus.The spatial-temporal distribution of APA and related environmental variables were studied in the Kuilei Lake.The research found significant seasonal and regional variations of APA in the water body,with lower APAs in outlet waters and higher in the estuarine.APAs were relatively low in winter (January),ranging from 2.06 to 17.33 nmol mL-1 h-1,and high in summer (July),ranging from 5.78 to 13.52 nmol mL-1 h-1.Correlation analyses indicated that APAs were significantly related to particulate organic nitrogen,total nitrogen and NO2--N.Other nitrogen forms (NO3--N and NH4+-N) were also related,though not significantly.The results implied that the factors related to nitrogen are the primary factors affecting APAs.%碱性磷酸酶在磷的生物地球化学循环过程中发挥着重要作用.以傀儡湖为研究区域,对其水体中碱性磷酸酶活性(Alkaline phosphatase activity,APA)及其理化性质的时空分布进行分析.结果表明,傀儡湖水体APA呈时空异质性:河道较低,河口则易出现较高的APA;全湖的APA存在较强的季节性变化,冬季(1月)最低,变化范围为2.06-17.33nmol mL-1h-1;夏季(7月)最高,变化范围为5.78-13.52 nmol mL-1 h-1.APA与环境因子相关性分析显示,APA与颗粒态有机氮浓度关系最为密切,与总氮含量的相关性其次,随后显著相关的因子为NO2--N,此外是NO3--N和NH4+-N.这一结果表明与氮元素有关的环境因子可能是影响APA的主要因素.

  14. In vitro induction of alkaline phosphatase levels predicts in vivo bone forming capacity of human bone marrow stromal cells

    Directory of Open Access Journals (Sweden)

    Henk-Jan Prins

    2014-03-01

    Full Text Available One of the applications of bone marrow stromal cells (BMSCs that are produced by ex vivo expansion is for use in in vivo bone tissue engineering. Cultured stromal cells are a mixture of cells at different stages of commitment and expansion capability, leading to a heterogeneous cell population that each time can differ in the potential to form in vivo bone. A parameter that predicts for in vivo bone forming capacity is thus far lacking. We employed single colony-derived BMSC cultures to identify such predictive parameters. Using limiting dilution, we have produced sixteen single CFU-F derived BMSC cultures from human bone marrow and found that only five of these formed bone in vivo. The single colony-derived BMSC strains were tested for proliferation, osteogenic-, adipogenic- and chondrogenic differentiation capacity and the expression of a variety of associated markers. The only robust predictors of in vivo bone forming capacity were the induction of alkaline phosphatase, (ALP mRNA levels and ALP activity during in vitro osteogenic differentiation. The predictive value of in vitro ALP induction was confirmed by analyzing “bulk-cultured” BMSCs from various bone marrow biopsies. Our findings show that in BMSCs, the additional increase in ALP levels over basal levels during in vitro osteogenic differentiation is predictive of in vivo performance.

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

    Science.gov (United States)

    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.

  16. High-throughput screening of tissue-nonspecific alkaline phosphatase for identification of effectors with diverse modes of action.

    Science.gov (United States)

    Sergienko, Eduard A; Millán, José Luis

    2010-08-01

    Here we describe a protocol for the identification of effectors of tissue-nonspecific alkaline phosphatase (TNAP). It is based on a highly sensitive method for detecting TNAP activity. After dephosphorylation by TNAP, a dioxetane-based substrate undergoes a series of chemical transformations resulting in light production. Light intensity serves as a quantitative measure of the velocity of the TNAP-catalyzed reaction in the steady state. This protocol includes guidelines for optimizing the assay and for high-throughput screening in multiwell plates. The assay is sensitive to the influence of diverse effectors of TNAP as long as the assay optimization steps are repeated for each new batch of the enzyme; full optimization is accomplished in under 2 d. Depending on the available equipment, 10,000-100,000 compounds can be screened in an 8-h period. This protocol provides a method of screening TNAP that is 1,000-fold more sensitive and 10-fold faster than a conventional colorimetric assay with p-nitrophenyl phosphate. PMID:20671726

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

    Science.gov (United States)

    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.

  18. Association between Serum Alkaline Phosphatase Level and Cerebral Small Vessel Disease.

    Directory of Open Access Journals (Sweden)

    Han-Bin Lee

    Full Text Available Serum alkaline phosphatase (ALP is a marker of vascular calcification. A high serum ALP level is associated with an increase in cardiovascular events, and predicts poor functional outcome in patients with stroke. We investigated whether serum ALP was associated with cerebral small vessel disease (cSVD and large cerebral artery stenosis (LCAS.We evaluated vascular risk factors, brain magnetic resonance images (MRIs, and MR angiograms from 1,011 neurologically healthy participants. The presence of silent lacunar infarction (SLI and moderate-to-severe cerebral white matter hyperintensities (MS-cWMH were evaluated as indices of cSVD on brain MRIs. Findings of extracranial arterial stenosis (ECAS or intracranial arterial stenosis (ICAS were considered to be indices of LCAS on MR angiograms.Subjects with SLI (odds ratio [OR]: 2.09; 95% confidence interval [CI]: 1.27-3.42; p = 0.004 and MS-cWMH (OR: 1.48; 95% CI; 1.03-2.13, p = 0.036 were significantly more likely to have ALP levels in the third tertile (ALP ≥ 195 IU/L than the first tertile (ALP ≤ 155 IU/L, after adjusting for cardiovascular risk factors. The mean serum ALP level was significantly higher in patients with SLI or MS-cWMH compared to patients without those findings. After adjustment for confounding factors, the multivariate model found that the statistical significance of serum ALP remained when the presence of SLI (OR: 1.05 per 10 IU/L increase in ALP; 95% CI: 1.02-1.08; p = 0.003 or MS-cWMH (OR: 1.03 per 10 IU/L increase in ALP; 95% CI: 1.00-1.06; p = 0.025 were added to the model. There were no differences in the proportions of patients with LCAS, ICAS, and ECAS across the serum ALP tertiles.Our study of neurologically healthy participants found a positive association between serum ALP level and indicators of cSVD, but no association between serum ALP level and the indicators of LCAS.

  19. Ecto-phosphatase activity on the external surface of Rhodnius prolixus salivary glands: modulation by carbohydrates and Trypanosoma rangeli.

    Science.gov (United States)

    Gomes, Suzete A O; Fonseca de Souza, André L; Kiffer-Moreira, Tina; Dick, Claudia F; dos Santos, André L A; Meyer-Fernandes, José R

    2008-05-01

    The salivary glands of insect's vectors are target organs to study the vectors-pathogens interactions. Rhodnius prolixus an important vector of Trypanosoma cruzi can also transmit Trypanosoma rangeli by bite. In the present study we have investigated ecto-phosphatase activity on the surface of R. prolixus salivary glands. Ecto-phosphatases are able to hydrolyze phosphorylated substrates in the extracellular medium. We characterized these ecto-enzyme activities on the salivary glands external surface and employed it to investigate R. prolixus-T. rangeli interaction. Salivary glands present a low level of hydrolytic activity (4.30+/-0.35 nmol p-nitrophenol (p-NP)xh(-1)xgland pair(-1)). The salivary glands ecto-phosphatase activity was not affected by pH variation; and it was insensitive to alkaline inhibitor levamisole and inhibited approximately 50% by inorganic phosphate (Pi). MgCl2, CaCl2 and SrCl2 enhanced significantly the ecto-phosphatase activity detected on the surface of salivary glands. The ecto-phosphatase from salivary glands surface efficiently releases phosphate groups from different phosphorylated amino acids, giving a higher rate of phosphate release when phospho-tyrosine is used as a substrate. This ecto-phosphatase activity was inhibited by carbohydrates as d-galactose and d-mannose. Living short epimastigotes of T. rangeli inhibited salivary glands ecto-phosphatase activity at 75%, while boiled parasites did not. Living long epimastigote forms induced a lower, but significant inhibitory effect on the salivary glands phosphatase activity. Interestingly, boiled long epimastigote forms did not loose the ability to modulate salivary glands phosphatase activity. Taken together, these data suggest a possible role for ecto-phosphatase on the R. prolixus salivary glands-T. rangeli interaction. PMID:18407240

  20. Effects of synthetic detergents on in vivo activity of tissue phosphatases and succinic dehydrogenase from Mystus vittatus

    Energy Technology Data Exchange (ETDEWEB)

    Mohan, D.; Verma, S.R.

    1981-05-01

    African catfish (Mystus vittatus) were exposed to three sub-lethal concentrations of Swascofix E45 (13.8, 9.2 and 4.6 mg/l) and Swascol 3L (69.3, 46.2 and 23.1 mg/l) for 15 and 30 days, and their effects on alkaline and acid phosphatase, and succinic dehydrogenase in liver, kidney and intestine were measured. The enzymes were found to be inhibited in all the tissues. Maximum inhibition (38.44%) was observed in liver alkaline phosphatase activity after 30 days with the highest concentration of Swascofix E45 and the lowest inhibition (0.118%) was found in kidney acid phosphatase activity with the lowest concentration of Swascol 3L after 15 days. Insignificant enzyme stimulation in some cases was also observed.

  1. Effect of andrographolide on phosphatases activity and cytotoxicity against Spodoptera litura

    Directory of Open Access Journals (Sweden)

    E Edwin

    2016-05-01

    Full Text Available Andrographolide was isolated from ethanol extraction of Andrographis paniculata by column chromatography. Evaluation of larvicidal efficacy, enzymatic changes and cytotoxic activities against Spodoptera litura were conducted across a range of concentrations. The compound showed significant larvicidal activity between 5 - 25 ppm, post ingestion. Morphological deformities observed in larval-pupal stages. Enzymatic profiles were altered by reduction in acid phosphatase, ACP activity by 69.18 %, alkaline phosphatase, ALP activity 75.3 % and 74.9 % reduction in ATPase. Binding affinity to midgut epithelium cells suggests disintegration of cellular organelles observed was directly associated with ingestion of the compound. The results suggest that andrographolide has potential for development as a significant inhibitor of development against the pest Spodoptera litura.

  2. Unraveling the Alkaline Phosphatase Inhibition, Anticancer, and Antileishmanial Potential of Coumarin-Triazolothiadiazine Hybrids: Design, Synthesis, and Molecular Docking Analysis.

    Science.gov (United States)

    Ibrar, Aliya; Zaib, Sumera; Jabeen, Farukh; Iqbal, Jamshed; Saeed, Aamer

    2016-07-01

    A series of new coumarin-triazolothiadiazine hybrid compounds (5a-j) was designed and synthesized by using the molecular hybridization concept. The cyclocondensation reaction involves the coumarinyl 4-amino-1,2,4-triazole and a range of bromo-acetophenones, delivering the desired products in good yields. The structures of the synthesized compounds were established on the basis of spectro-analytical data. The prepared compounds were evaluated against alkaline phosphatase (ALP) where compound 5j incorporating bis-coumarinyl motifs at the 3- and 6-positions of the heteroaromatic core turned out to be a potent inhibitor with an IC50 value of 1.15 ± 1.0 µM. The synthesized compounds were also tested against Leishmania major and 5h was the lead member with an IC50 value of 0.89 ± 0.08 μM. Anticancer activity was also determined using kidney fibroblast (BHK-21) and lung carcinoma (H-157) cancer cell lines. Compound 5i showed highest cytotoxic potential against H-157 cells with an IC50 value of 1.01 ± 0.12 μM, which is an improved inhibition compared to the standards (vincristine and cisplatin) used in this assay. Molecular docking studies were carried out on the synthesized library of coumarin-triazolothiadiazine hybrids against ALP. Almost all of the compounds showed strong interactions with the key residues of the active site of the receptor. In case of compounds 5a-c, 5h, and 5j, docking results positively complemented the experimental screening. These results provided substantial evidence for the further development of these compounds as potent inhibitors of ALP.

  3. 泥鳅仔稚鱼发育期间消化酶及碱性磷酸酶比活力的变化%Changes of digestive enzymes and alkaline phosphatase specific activities in Misgurnus anguillicaudatus during the development of early larval ontogeny

    Institute of Scientific and Technical Information of China (English)

    张云龙; 樊启学; 彭聪; 胡培培; 宗克金; 宋林; 沈凡

    2013-01-01

    The specific activities of several digestive enzymes (pensin, trypsin, lipase, amylase) and alkaline phosphatase of Misgurnus anguillicaudatus were determined from hatching to 30 DAH ( days after hatching). Pepsin activity could not be detected during the entire period of the experiment. In contrast, trypsin performed a high level activity after hatching , and its specific activity increased significantly after first feeding, peaking at 6 DAH, and decreased abruptly after that. The variation of lipase and amylase shared a similar pattern, their specific activities had two obvious peaks around the time of the transition of endogenous to exdogenous feeding and the beginning of the transportation of larval stage to juvenile stage. From 2 to 6 DAH, the specific activity of alkaline phosphatase significantly increased and then declined to a roughly stable level. This study demonstrated that M. anguillicaudatus has only a structural not functional stomach during the larval stage. The period of 2 to 6 DAH was the stage of the maturation of digestive gut in M. anguillicaudatus larvae and the crucial step forward the digestion mode of adult M. anguillicaudatus. The steadiness of these specific activities suggested that M. anguillicaudatus was efficient to utilize carbohydrate and lipid in food.%研究了泥鳅(Misgurnus anguillicaudatus)从孵化至30 DAH(日龄,Days after hatching)几种消化酶及碱性磷酸酶比活力的变化情况.胃蛋白酶直至30 DAH仍未检出活性.而胰蛋白酶表现出较高的比活力,其比活力在初次摄食之后显著上升,6 DAH达到最大值之后开始显著降低(P<0.05).脂肪酶与淀粉酶的变化模式相似,在内源性营养向外源性营养转变及仔鱼向稚鱼转变这两个时间段出现两个高峰值.碱性磷酸酶比活力在2-6 DAH显著上升(P<0.05),之后开始下降并趋于平稳.研究表明,泥鳅在仔稚鱼阶段只具有结构性的胃而缺乏分泌细胞的分化.2-6 DAH是泥鳅仔鱼肠

  4. A GPI-anchored alkaline phosphatase is a functional midgut receptor of Cry11Aa toxin in Aedes aegypti larvae.

    Science.gov (United States)

    Fernandez, Luisa E; Aimanova, Karlygash G; Gill, Sarjeet S; Bravo, Alejandra; Soberón, Mario

    2006-02-15

    A 65 kDa GPI (glycosylphosphatidyl-inositol)-anchored ALP (alkaline phosphatase) was characterized as a functional receptor of the Bacillus thuringiensis subsp. israelensis Cry11Aa toxin in Aedes aegypti midgut cells. Two (a 100 kDa and a 65 kDa) GPI-anchored proteins that bound Cry11Aa toxin were preferentially extracted after treatment of BBMV (brush boder membrane vesicles) from Ae. aegypti midgut epithelia with phospholipase C. The 65 kDa protein was further purified by toxin affinity chromatography. The 65 kDa protein showed ALP activity. The peptide-displaying phages (P1.BBMV and P8.BBMV) that bound to the 65 kDa GPI-ALP (GPI-anchored ALP) and competed with the Cry11Aa toxin to bind to BBMV were isolated by selecting BBMV-binding peptide-phages by biopanning. GPI-ALP was shown to be preferentially distributed in Ae. aegypti in the posterior part of the midgut and in the caeca, by using P1.BBMV binding to fixed midgut tissue sections to determine the location of GPI-ALP. Cry11Aa binds to the same regions of the midgut and competed with P1.BBMV and P8.BBMV to bind to BBMV. The importance of this interaction was demonstrated by the in vivo attenuation of Cry11Aa toxicity in the presence of these phages. Our results shows that GPI-ALP is an important receptor molecule involved in Cry11Aa interaction with midgut cells and toxicity to Ae. aegypti larvae.

  5. One-step immunoassay for tetrabromobisphenol a using a camelid single domain antibody-alkaline phosphatase fusion protein.

    Science.gov (United States)

    Wang, Jia; Majkova, Zuzana; Bever, Candace R S; Yang, Jun; Gee, Shirley J; Li, Ji; Xu, Ting; Hammock, Bruce D

    2015-01-01

    Tetrabromobisphenol A (TBBPA) is a ubiquitous brominated flame retardant, showing widespread environmental and human exposures. A variable domain of the heavy chain antibody (VHH), naturally occurring in camelids, approaches the lower size limit of functional antigen-binding entities. The ease of genetic manipulation makes such VHHs a superior choice to use as an immunoreagent. In this study, a highly selective anti-TBBPA VHH T3-15 fused with alkaline phosphatase (AP) from E. coli was expressed, showing both an integrated TBBPA-binding capacity and enzymatic activity. A one-step immunoassay based on the fusion protein T3-15-AP was developed for TBBPA in 5% dimethyl sulfoxide (DMSO)/phosphate buffered saline (PBS, pH 7.4), with a half-maximum signal inhibition concentration (IC50) of 0.20 ng mL(-1). Compared to the parental VHH T3-15, T3-15-AP was able to bind to a wider variety of coating antigens and the assay sensitivity was slightly improved. Cross-reactivity of T3-15-AP with a set of important brominated analogues was negligible (<0.1%). Although T3-15-AP was susceptible to extreme heat (90 °C), much higher binding stability at ambient temperature was observed in the T3-15-AP-based assay for at least 70 days. A simple pretreatment method of diluting urine samples with DMSO was developed for a one-step assay. The recoveries of TBBPA from urine samples via this one-step assay ranged from 96.7% to 109.9% and correlated well with a high-performance liquid chromatography-tandem mass spectroscopy (HPLC-MS/MS) method. It is expected that the dimerized fusion protein, VHH-AP, will show promising applications in human exposure and environmental monitoring.

  6. Characterization of Six Missense Mutations in the Tissue-Nonspecific Alkaline Phosphatase (TNSALP Gene in Chinese Children with Hypophosphatasia

    Directory of Open Access Journals (Sweden)

    Haiou Yang

    2013-09-01

    Full Text Available Aims: Hypophosphatasia, a rare inherited disease characterized by defective mineralization of bone and teeth, is caused by various mutations in the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP gene. Our aim was to determine the mutations on TNSALP gene in three Chinese children diagnosed as having hypophosphatasia. Methods: Genomic DNA was extracted from whole blood samples of patients and their parents. The TNSALP coding regions were then sequenced. Plasmids expressing wild-type or various mutants were built and in vitro studies were performed in order to determine whether these amino acid replacements could affect the TNSALP enzymatic activity. Results: Six missense mutations were identified from three independent pedigrees. Of the six missense mutations, four were novel and two had been previously reported. The Y28D, A111T and T389N mutants displayed only negligible ALP activity in vitro compared to the wild-type (WT TNSALP. The defect was mainly due to the significantly decreased protein expression in the 66 KD immature forms and the nearly undetectable protein expression in the 80 KD mature forms. Moreover, all three mutants had a dominant negative effect on the WT protein when co-transfected with TNSALP (WT. M219V and R136L mutants both exhibited partial enzymatic activities which were consistent with reduced protein expression in both forms of TNSALP which further exhibited moderate dominant-negative effect. In addition, Y388H mutant showed weak ALP activity. Western blot analysis indicated that the extreme reduction in signal from the mature forms of TNSALP could be the main cause of decreased enzymatic activity, since a strong signal was observed in the immature forms. Conclusion: Six missense mutations were identified in three Chinese hypophosphatasia pedigrees with subnormal serum ALP activity. Our results show that the low activity of serum ALP in the three patients is due mainly to a defect in the protein expression

  7. Biomineralization of Uranium by PhoY Phosphatase Activity Aids Cell Survival in Caulobacter crescentus

    Energy Technology Data Exchange (ETDEWEB)

    Yung, M C [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Jiao, Y [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2014-07-22

    Caulobacter crescentus is known to tolerate high levels of uranium [U(VI)], but its detoxification mechanism is poorly understood. Here we show that C. crescentus is able to facilitate U(VI) biomineralization through the formation of U-Pi precipitates via its native alkaline phosphatase activity. The U-Pi precipitates, deposited on the cell surface in the form of meta-autunite structures, have a lower U/Pi ratio than do chemically produced precipitates. The enzyme that is responsible for the phosphatase activity and thus the biomineralization process is identified as PhoY, a periplasmic alkaline phosphatase with broad substrate specificity. Furthermore, PhoY is shown to confer a survival advantage on C. crescentus toward U(VI) under both growth and nongrowth conditions. Results obtained in this study thus highlight U(VI) biomineralization as a resistance mechanism in microbes, which not only improves our understanding of bacterium-mineral interactions but also aids in defining potential ecological niches for metal-resistant bacteria.

  8. Biomineralization of uranium by PhoY phosphatase activity aids cell survival in Caulobacter crescentus.

    Science.gov (United States)

    Yung, Mimi C; Jiao, Yongqin

    2014-08-01

    Caulobacter crescentus is known to tolerate high levels of uranium [U(VI)], but its detoxification mechanism is poorly understood. Here we show that C. crescentus is able to facilitate U(VI) biomineralization through the formation of U-Pi precipitates via its native alkaline phosphatase activity. The U-Pi precipitates, deposited on the cell surface in the form of meta-autunite structures, have a lower U/Pi ratio than do chemically produced precipitates. The enzyme that is responsible for the phosphatase activity and thus the biomineralization process is identified as PhoY, a periplasmic alkaline phosphatase with broad substrate specificity. Furthermore, PhoY is shown to confer a survival advantage on C. crescentus toward U(VI) under both growth and nongrowth conditions. Results obtained in this study thus highlight U(VI) biomineralization as a resistance mechanism in microbes, which not only improves our understanding of bacterium-mineral interactions but also aids in defining potential ecological niches for metal-resistant bacteria.

  9. Study on oxidative stress and activity of alkaline phosphatase of rats exposed to different period of fluoride%投氟不同时间大鼠血清氧化应激和碱性磷酸酶活性的变化研究

    Institute of Scientific and Technical Information of China (English)

    徐辉; 范海清; 张金铭; 李广生

    2010-01-01

    Objective To observe the status of oxidative stress and activity of alkaline phosphatase(ALP) in rats exposed to high fluoride for the different periods and to analyze the effect of fluoride on the activity of ALP and oxidative stress in fluorosis rats. Methods Twenty-four Wistar rats were divided into control and high-fluoride groups according to their body mass, 12 rats in each group. The control group drank tap water(sodium fluoride concentrations 0.05); the level of uric acid in serum was significantly higher in high-fluoride group for 1,4 week[ (89.53 ± 13.21 ), (88.47 ± 19.78 )μmol/L] compared to the control [ (77.79 ± 11.43 ), (65.42 ± i 3.42) μ mol/L, all P 0.05);投氟1、4周大鼠血清尿酸[(89.53±13.21)、(88.47±19.78)μmol/L]较对照组[(77.79±11.43)、(65.42±13.42)μmol/L]明显增高(P均<0.05),而投氟8、12周大鼠尿酸[(67.21±9.44)、(73.95±9.52)μmol/L]低于对照组[(77.79±11.43)、(65.42±13.42)μmol/L,P均<0.05].结论 投与过量氟大鼠的ALP活性变化具有一定的时间依赖性;而投氟刺激大鼠氧化应激水平增强,这种刺激与投氟时间没有明显关联.

  10. Ultrasensitive electrochemical DNAzyme sensor for lead ion based on cleavage-induced template-independent polymerization and alkaline phosphatase amplification.

    Science.gov (United States)

    Liu, Shufeng; Wei, Wenji; Sun, Xinya; Wang, Li

    2016-09-15

    In this article, a simple, highly sensitive and selective electrochemical DNAzyme sensor for Pb(2+) was developed on the basis of a 8-17 DNAzyme cleavage-induced template-independent polymerization and alkaline phosphatase amplification strategy. The hairpin-like substrate strand (HP DNA) of 8-17 DNAzyme was firstly immobilized onto the electrode. In the presence of Pb(2+) and the catalytic strand of 8-17 DNAzyme, the HP DNA could be cleaved to expose the free 3'-OH terminal, which could be then utilized for the cascade operation by terminal deoxynucleotidyl transferase (TdTase) for the base extension to incorporate biotinylated dUTP (dUTP-biotin). The further conjugated streptavidin-labeled alkaline phosphatase (SA-ALP) then catalyzed conversion of electrochemically inactive 1-naphthyl phosphate (1-NP) for the generation of electrochemical response signal. The currently fabricated Pb(2+) sensor effectively combines triply cascade amplification effects including cyclic Pb(2+)-dependent DNAzyme cleavage, TdTase-mediated base extension and enzymatic catalysis of ALP. An impressive detection limit of 0.043nM toward Pb(2+) with an excellent selectivity could be ultimately obtained, which was superior than most of the electrochemical methods. Thus, the developed amplification strategy opens a promising avenue for the detection of metal ions and may extend for the detection of other nucleic acid-related analytes. PMID:27093488

  11. Ultrasensitive detection of amifostine and alkaline phosphatase based on the growth of CdS quantum dots.

    Science.gov (United States)

    Na, Weidan; Liu, Siyu; Liu, Xiaotong; Su, Xingguang

    2015-11-01

    In this study, we reported a simple and sensitive fluorescence nanosensor for rapid detection of amifostine and alkaline phosphatase (ALP). The novel nanosensor was based on the fluorescence "turn on-off" of CdS quantum dots (QDs). Firstly, Cd(2+) cation could react with S(2-) anion to generate fluorescent CdS QDs in the presence of amifostine. The fluorescence (FL) intensity of amifostine-capped CdS QDs (Amifostine-CdS QDs) was increased with the increasing amounts of amifostine, and could be used for amifostine detection. However, amifostine could be converted to 2-(3-aminopropylamino) ethanethiol (WR1065) in the presence of ALP based on the dephosphorylation of ALP. Under the optimum conditions, the affinity of WR1065 to CdS QDs was weaker than that of amifostine. Therefore the new generation of WR1065-CdS QDs would reduce the FL intensity with the increase of ALP concentration, and the fluorescence of CdS QDs was turn off. The metabolic process of amifostine in the presence of alkaline phosphatase could be also studied via the change of FL intensity of CdS QDs. The present method was cost-effective, convenient, and does not require any complicated synthetic procedures. PMID:26452927

  12. 秦皇岛近海褐潮高发区浮游植物的碱性磷酸酶活性分析%Alkaline phosphatase activity of phytoplankton in Qin-huangdao coastal waters with frequent brown tide occurrences

    Institute of Scientific and Technical Information of China (English)

    刘晓红; 覃仙玲; 蔡阳扬; 欧林坚; 吕颂辉

    2016-01-01

    Alkaline phosphatase (AP) is a type of ectoenzyme that is expressed by phytoplankton under phosphorus (P) stress states and can hydrolyze dissolved organic P (DOP). AP has been widely used to indicate the P stress status of phytoplankton in the sea. In this study, the AP activity (APA) of phytoplankton during the brown tide of Aureococcus anophagefferens that occurred near the coastal waters of Qinghuangdao was investigated in July 2013. The P status of phytoplankton and the capability of phytoplankton in using DOP during the bloom were analyzed. The results show that the density of A. anophagefferens was as high as 108 cells/L when the bloom occurred in the coastal area of Qinhuangdao. DOP was the main P source for phytoplankton growth. The densities of A. ano-phagefferens showed positive correlations with NO3−, DOP, dissolved inorganic P, and so forth. Phytoplankton ex-pressed abundant AP, and the average APA in water reached as high as 217.72 nmol/(µg·h)chla ± 90.86 nmol/(µg·h)chla (350.44 nmol/(L·h) ± 130.57 nmol/(L·h)). APA increased significantly with phytoplankton biomass. Our results suggest that A. anophagefferens experienced severe P stress or even P limitation when the brown tide occurred. The bioavailability of P, particularly DOP, may play a key role in the occurrence and persistence of the brown tide of A. anophagefferens in the coastal waters of Qinhuangdao.%碱性磷酸酶(Alkaline phosphatase, AP)是浮游植物在磷胁迫状态下表达的一种水解有机磷源的胞外酶,可用于指示海区浮游植物的磷胁迫状态。本研究于2013年7月,对秦皇岛近海抑食金球藻(Aureococcus anophagefferens)褐潮发生期间浮游植物的碱性磷酸酶活性(AP activity, APA)进行研究,结合其他理化参数,分析藻华发生时浮游植物的磷营养状态及其对海水中磷源的水解与利用情况。结果表明,褐潮发生时,抑食金球藻细胞密度高达108个/L,溶解有机磷(Dissolved organic phosphorus

  13. Versatile and Amplified Biosensing through Enzymatic Cascade Reaction by Coupling Alkaline Phosphatase in Situ Generation of Photoresponsive Nanozyme.

    Science.gov (United States)

    Jin, Lu-Yi; Dong, Yu-Ming; Wu, Xiu-Ming; Cao, Gen-Xia; Wang, Guang-Li

    2015-10-20

    The alkaline phosphatase (ALP) biocatalysis followed by the in situ enzymatic generation of a visible light responsive nanozyme is coupled to elucidate a novel amplification strategy by enzymatic cascade reaction for versatile biosensing. The enzymatic hydrolysis of o-phosphonoxyphenol (OPP) to catechol (CA) by ALP is allowed to coordinate on the surface of TiO2 nanoparticles (NPs) due to the specificity and high affinity of enediol ligands to Ti(IV). Upon the stimuli by CA generated from ALP, the inert TiO2 NPs is activated, which demonstrates highly efficient oxidase mimicking activity for catalyzing the oxidation of the typical substrate of 3,3',5,5'-tetramethylbenzidine (TMB) under visible light (λ ≥ 400 nm) irradiation utilizing dissolved oxygen as an electron acceptor. On the basis of the cascade reaction of ALP and the nanozyme of CA coordinated TiO2 (TiO2-CA) NPs, we design exquisitely colorimetric biosensors for probing ALP activity and its inhibitor of 2, 4-dichlorophenoxyacetic acid (2,4-DA). Quantitative probing of ALP activity in a wide linear range from 0.01 to 150 U/L with the detection limit of 0.002 U/L is realized, which endows the methodology with sufficiently high sensitivity for potentially practical applications in real samples of human serum (ALP level of 40-190 U/L for adults). In addition, a novel immunoassay protocol by taking mouse IgG as an example is validated using the ALP/nanozyme cascade amplification reaction as the signal transducer. A low detection limit of 2.0 pg/mL is attained for mouse IgG, which is 4500-fold lower than that of the standard enzyme-linked immuno-sorbent assay (ELISA) kit. Although only mouse IgG is used as a proof-of-concept in our experiment, we believe that this approach is generalizable to be readily extended to other ELISA systems. This methodology opens a new horizon for amplified and versatile biosensing including probing ALP activity and following ALP-based ELISA immunoassays. PMID:26419907

  14. Versatile and Amplified Biosensing through Enzymatic Cascade Reaction by Coupling Alkaline Phosphatase in Situ Generation of Photoresponsive Nanozyme.

    Science.gov (United States)

    Jin, Lu-Yi; Dong, Yu-Ming; Wu, Xiu-Ming; Cao, Gen-Xia; Wang, Guang-Li

    2015-10-20

    The alkaline phosphatase (ALP) biocatalysis followed by the in situ enzymatic generation of a visible light responsive nanozyme is coupled to elucidate a novel amplification strategy by enzymatic cascade reaction for versatile biosensing. The enzymatic hydrolysis of o-phosphonoxyphenol (OPP) to catechol (CA) by ALP is allowed to coordinate on the surface of TiO2 nanoparticles (NPs) due to the specificity and high affinity of enediol ligands to Ti(IV). Upon the stimuli by CA generated from ALP, the inert TiO2 NPs is activated, which demonstrates highly efficient oxidase mimicking activity for catalyzing the oxidation of the typical substrate of 3,3',5,5'-tetramethylbenzidine (TMB) under visible light (λ ≥ 400 nm) irradiation utilizing dissolved oxygen as an electron acceptor. On the basis of the cascade reaction of ALP and the nanozyme of CA coordinated TiO2 (TiO2-CA) NPs, we design exquisitely colorimetric biosensors for probing ALP activity and its inhibitor of 2, 4-dichlorophenoxyacetic acid (2,4-DA). Quantitative probing of ALP activity in a wide linear range from 0.01 to 150 U/L with the detection limit of 0.002 U/L is realized, which endows the methodology with sufficiently high sensitivity for potentially practical applications in real samples of human serum (ALP level of 40-190 U/L for adults). In addition, a novel immunoassay protocol by taking mouse IgG as an example is validated using the ALP/nanozyme cascade amplification reaction as the signal transducer. A low detection limit of 2.0 pg/mL is attained for mouse IgG, which is 4500-fold lower than that of the standard enzyme-linked immuno-sorbent assay (ELISA) kit. Although only mouse IgG is used as a proof-of-concept in our experiment, we believe that this approach is generalizable to be readily extended to other ELISA systems. This methodology opens a new horizon for amplified and versatile biosensing including probing ALP activity and following ALP-based ELISA immunoassays.

  15. Crystallization and preliminary X-ray crystallographic analysis of PhoK, an extracellular alkaline phosphatase from Sphingomonas sp. BSAR-1

    International Nuclear Information System (INIS)

    A new alkaline phosphatase enzyme from Sphingomonas sp. strain BSAR-1, termed PhoK, has been shown to be useful in uranium bioprecipitation. PhoK has been expressed, purified and crystallized. Alkaline phosphatases (APs) are widely distributed from microbes to humans and are involved in several important biological processes such as phosphate nutrition, signal transduction and pathogenesis. Alkaline phosphatases are also useful in various industrial applications and in recombinant DNA technology. A new AP enzyme from Sphingomonas sp. strain BSAR-1, termed PhoK, has been shown to be useful in uranium bioprecipitation. PhoK was expressed, purified and crystallized. The crystals belonged to space group P43212 or P41212, with unit-cell parameters a = b = 87.37, c = 168.16 Å, and contained one enzyme molecule in the asymmetric unit. Native diffraction data have been collected to 1.95 Å resolution at the ESRF

  16. Phosphatase activity in sandy soil influenced by mycorrhizal and non-mycorrhizal cover crops

    Directory of Open Access Journals (Sweden)

    Alceu Kunze

    2011-06-01

    Full Text Available Cover crops may difffer in the way they affect rhizosphere microbiota nutrient dynamics. The purpose of this study was to evaluate the effect of mycorrhizal and non-mycorrhizal cover crops on soil phosphatase activity and its persistence in subsequent crops. A three-year experiment was carried out with a Typic Quartzipsamment. Treatments were winter species, either mycorrhizal black oat (Avena strigosa Schreb or the non-mycorrhizal species oilseed radish (Raphanus sativus L. var. oleiferus Metzg and corn spurry (Spergula arvensis L.. The control treatment consisted of resident vegetation (fallow in the winter season. In the summer, a mixture of pearl millet (Pennisetum americanum L. with sunnhemp (Crotalaria juncea L. or with soybean (Glycine max L. was sown in all plots. Soil cores (0-10 cm and root samples were collected in six growing seasons (winter and summer of each year. Microbial biomass P was determined by the fumigation-extraction method and phosphatase activity using p-nitrophenyl-phosphate as enzyme substrate. During the flowering stage of the winter cover crops, acid phosphatase activity was 30-35 % higher in soils with the non-mycorrhizal species oilseed radish, than in the control plots, regardless of the amount of P immobilized in microbial biomass. The values of enzyme activity were intermediate in the plots with corn spurry and black oat. Alkaline phosphatase activity was 10-fold lower and less sensitive to the treatments, despite the significant relationship between the two phosphatase activities. The effect of plant species on the soil enzyme profile continued in the subsequent periods, during the growth of mycorrhizal summer crops, after completion of the life cycle of the cover crops.

  17. Using a Personal Glucose Meter and Alkaline Phosphatase for Point-of-Care Quantification of Galactose-1-Phosphate Uridyltransferase in Clinical Galactosemia Diagnosis.

    Science.gov (United States)

    Zhang, Jingjing; Xiang, Yu; Novak, Donna E; Hoganson, George E; Zhu, Junjie; Lu, Yi

    2015-10-01

    The personal glucose meter (PGM) was recently shown to be a general meter to detect many targets. Most studies, however, focus on transforming either target binding or enzymatic activity that cleaves an artificial substrate into the production of glucose. More importantly, almost all reports exhibit their methods by using artificial samples, such as buffers or serum samples spiked with the targets. To expand the technology to even broader targets and to validate its potential in authentic, more complex clinical samples, we herein report expansion of the PGM method by using alkaline phosphatase (ALP) that links the enzymatic activity of galactose-1-phosphate uridyltransferase to the production of glucose, which allows point-of-care galactosemia diagnosis in authentic human clinical samples. Given the presence of ALP in numerous enzymatic assays for clinical diagnostics, the methods demonstrated herein advance the field closer to point-of-care detection of a wide range of targets in real clinical samples. PMID:26350570

  18. Labelling of T cell subsets under field conditions in tropical countries. Adaptation of the immuno-alkaline phosphatase staining method for blood smears

    DEFF Research Database (Denmark)

    Lisse, I M; Whittle, H; Aaby, P;

    1990-01-01

    Immuno-alkaline phosphatase (AP) staining for T cell subsets (CD4 and CD8) of smears from fingerprick blood functioned well under tropical climatic conditions when smears were stored frozen with silica gel before being labelled. Unlabelled smears were stored for up to 12 months and could be trans......Immuno-alkaline phosphatase (AP) staining for T cell subsets (CD4 and CD8) of smears from fingerprick blood functioned well under tropical climatic conditions when smears were stored frozen with silica gel before being labelled. Unlabelled smears were stored for up to 12 months and could...

  19. Enzymatic methods for the determination of pollution in seawater using salt resistant alkaline phosphatase from eggs of the sea urchin Strongylocentrotus intermedius.

    Science.gov (United States)

    Menzorova, Natalie I; Seitkalieva, Alexandra V; Rasskazov, Valerу A

    2014-02-15

    A new salt resistant alkaline phosphatase from eggs of the sea urchin Strongylocentrotus intermedius (StAP) has been shown to have a unique property to hydrolyze substrate in seawater without loss of enzymatic activity. The enzyme has pH optimum at 8.0-8.5. Model experiments showed various concentrations of copper, zinc, cadmium and lead added to seawater or a standard buffer mixture to inhibit completely the enzyme activity at the concentrations of 15-150 μg/l. StAP sensitivity to the presence in seawater of metals, pesticides, detergents and oil products appears to be considerably less. Samples of seawater taken from aquatic areas of the Troitsy Bay of the Peter the Great Bay, Japan Sea have been shown to inhibit the enzyme activity; the same was shown for the samples of fresh waters. The phosphatase inhibition assay developed proved to be highly sensitive, technically easy-to use allowing to test a great number of samples.

  20. Immunocytochemical Phenotyping of Disseminated Tumor Cells in Bone Marrow by uPA Receptor and CK18: Investigation of Sensitivity and Specificity of an Immunogold/Alkaline Phosphatase Double Staining Protocol

    OpenAIRE

    Allgayer, Heike; Heiss, Markus Maria; Riesenberg, Rainer; Babic, Rudolf; Jauch, Karl Walter; Schildberg, Friedrich Wilhelm

    1997-01-01

    Phenotyping of cytokeratin (CK) 18-positive cells in bone marrow is gaining increasing importance for future prognostic screening of carcinoma patients. Urokinase-type plasminogen activator receptor (uPA-R) is one example of a potential aggressive marker for those cells. However, a valid and reliable double staining method is needed. Using monoclonal antibodies against uPA-R and CK18, we modified an immunogold/alkaline phosphatase double staining protocol. UPA-R/CK18-positive tumor cell contr...

  1. The cellular prion protein interacts with the tissue non-specific alkaline phosphatase in membrane microdomains of bioaminergic neuronal cells.

    Directory of Open Access Journals (Sweden)

    Myriam Ermonval

    Full Text Available BACKGROUND: The cellular prion protein, PrP(C, is GPI anchored and abundant in lipid rafts. The absolute requirement of PrP(C in neurodegeneration associated to prion diseases is well established. However, the function of this ubiquitous protein is still puzzling. Our previous work using the 1C11 neuronal model, provided evidence that PrP(C acts as a cell surface receptor. Besides a ubiquitous signaling function of PrP(C, we have described a neuronal specificity pointing to a role of PrP(C in neuronal homeostasis. 1C11 cells, upon appropriate induction, engage into neuronal differentiation programs, giving rise either to serotonergic (1C11(5-HT or noradrenergic (1C11(NE derivatives. METHODOLOGY/PRINCIPAL FINDINGS: The neuronal specificity of PrP(C signaling prompted us to search for PrP(C partners in 1C11-derived bioaminergic neuronal cells. We show here by immunoprecipitation an association of PrP(C with an 80 kDa protein identified by mass spectrometry as the tissue non-specific alkaline phosphatase (TNAP. This interaction occurs in lipid rafts and is restricted to 1C11-derived neuronal progenies. Our data indicate that TNAP is implemented during the differentiation programs of 1C11(5-HT and 1C11(NE cells and is active at their cell surface. Noteworthy, TNAP may contribute to the regulation of serotonin or catecholamine synthesis in 1C11(5-HT and 1C11(NE bioaminergic cells by controlling pyridoxal phosphate levels. Finally, TNAP activity is shown to modulate the phosphorylation status of laminin and thereby its interaction with PrP. CONCLUSION/SIGNIFICANCE: The identification of a novel PrP(C partner in lipid rafts of neuronal cells favors the idea of a role of PrP in multiple functions. Because PrP(C and laminin functionally interact to support neuronal differentiation and memory consolidation, our findings introduce TNAP as a functional protagonist in the PrP(C-laminin interplay. The partnership between TNAP and PrP(C in neuronal cells may

  2. P depletion and activity of phosphatases in the rhizosphere of mycorrhizal and non-mycorrhizal cucumber (Cucumis Sativus L.)

    DEFF Research Database (Denmark)

    Joner, E.J.; Magid, J.; Gahoonia, T.S.;

    1995-01-01

    was sectioned in a freezing microtome and analyzed for extracellular acid (pH 5.2) and alkaline (pH 8.5) phosphatase activity as well as depletion of NaHCO-3-extractable inorganic P (P-i) and P-o. Roots and mycorrhizal hyphae depleted the soil of P-i but did not influence the concentration of P-o in spite......An experiment was set up to test the ability of arbuscular mycorrhizal (AM) roots and hyphae to produce extracellular phosphatases and to study the relationship between phosphatase activity and soil organic P (P-o). Non-mycorrhizal cucumber and cucumber in symbiosis with either of two mycorrhizal...... of increased phosphatase activity in soil influenced by roots. Phosphatase activity at both pH values was highest in soil influenced by uncolonized roots, but this was attributed to higher root length densities as compared to mycorrhizal roots. Mycorrhizal hyphae showed no influence on soil phosphatase...

  3. A phage-displayed chicken single-chain antibody fused to alkaline phosphatase detects Fusarium pathogens and their presence in cereal grains

    International Nuclear Information System (INIS)

    Graphical abstract: A phage-displayed chicken scFv antibody, FvSG7, binds on the surface antigen of conidiospores and the mycelia of F. verticillioides. Its fusion with alkaline phosphatase (AP) through a 218 linker displayed a 4-fold higher affinity compared with the parent scFv antibody and efficiently detected toxigenic Fusarium pathogens in cereal grains. Highlights: ► Generation of a highly reactive scFv antibody against F. verticillioides. ► Localization of the antibody binding to the surface target of F. verticillioides. ► Expression of the antibody–alkaline phosphatase (AP) fusion linked by a 218 linker. ► The antibody–AP fusion has a higher affinity than the parental antibody. ► The antibody–AP fusion detects toxigenic Fusarium pathogens in cereal grains. -- Abstract: Fusarium and its poisonous mycotoxins are distributed worldwide and are of particular interest in agriculture and food safety. A simple analytical method to detect pathogens is essential for forecasting diseases and controlling mycotoxins. This article describes a proposed method for convenient and sensitive detection of Fusarium pathogens that uses the fusion of single-chain variable fragment (scFv) and alkaline phosphatase (AP). A highly reactive scFv antibody specific to soluble cell wall-bound proteins (SCWPs) of F. verticillioides was selected from an immunized chicken phagemid library by phage display. The antibody was verified to bind on the surface of ungerminated conidiospores and mycelia of F. verticillioides. The scFv–AP fusion was constructed, and soluble expression in bacteria was confirmed. Both the antibody properties and enzymatic activity were retained, and the antigen-binding capacity of the fusion was enhanced by the addition of a linker. Surface plasmon resonance measurements confirmed that the fusion displayed 4-fold higher affinity compared with the fusion's parental scFv antibody. Immunoblot analyses showed that the fusion had good binding capacity to

  4. A phage-displayed chicken single-chain antibody fused to alkaline phosphatase detects Fusarium pathogens and their presence in cereal grains

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Zu-Quan [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); Li, He-Ping [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Zhang, Jing-Bo [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Huang, Tao [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Liu, Jin-Long; Xue, Sheng [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); Wu, Ai-Bo [Institute for Agri-food Standards and Testing Technology, Laboratory of Quality and Safety Risk Assessment for Agro-products, Ministry of Agriculture, Shanghai Academy of Agricultural Sciences, 1000 Jinqi Road, Shanghai 201403 (China); Liao, Yu-Cai, E-mail: ycliao06@yahoo.com.cn [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); National Center of Plant Gene Research, Wuhan 430070 (China)

    2013-02-18

    Graphical abstract: A phage-displayed chicken scFv antibody, FvSG7, binds on the surface antigen of conidiospores and the mycelia of F. verticillioides. Its fusion with alkaline phosphatase (AP) through a 218 linker displayed a 4-fold higher affinity compared with the parent scFv antibody and efficiently detected toxigenic Fusarium pathogens in cereal grains. Highlights: ► Generation of a highly reactive scFv antibody against F. verticillioides. ► Localization of the antibody binding to the surface target of F. verticillioides. ► Expression of the antibody–alkaline phosphatase (AP) fusion linked by a 218 linker. ► The antibody–AP fusion has a higher affinity than the parental antibody. ► The antibody–AP fusion detects toxigenic Fusarium pathogens in cereal grains. -- Abstract: Fusarium and its poisonous mycotoxins are distributed worldwide and are of particular interest in agriculture and food safety. A simple analytical method to detect pathogens is essential for forecasting diseases and controlling mycotoxins. This article describes a proposed method for convenient and sensitive detection of Fusarium pathogens that uses the fusion of single-chain variable fragment (scFv) and alkaline phosphatase (AP). A highly reactive scFv antibody specific to soluble cell wall-bound proteins (SCWPs) of F. verticillioides was selected from an immunized chicken phagemid library by phage display. The antibody was verified to bind on the surface of ungerminated conidiospores and mycelia of F. verticillioides. The scFv–AP fusion was constructed, and soluble expression in bacteria was confirmed. Both the antibody properties and enzymatic activity were retained, and the antigen-binding capacity of the fusion was enhanced by the addition of a linker. Surface plasmon resonance measurements confirmed that the fusion displayed 4-fold higher affinity compared with the fusion's parental scFv antibody. Immunoblot analyses showed that the fusion had good binding

  5. A fluorescence turn on assay for alkaline phosphatase based on the Cu(2+) catalyzed Fenton-like reaction.

    Science.gov (United States)

    Zhang, Qingfeng; Zhang, Cuiyun; Shahzad, Sohail Anjum; Yu, Cong

    2016-09-01

    A fluorescence turn-on assay was established for ALP (alkaline phosphatase) based on Cu(2+) catalyzed Fenton-like reaction and Graphene Oxide (GO). GO was utilized to quench the fluorescence of fluorescein (FAM) labeled single strand DNA (F-DNA). ALP can remove the phosphate group in sodium ascorbyl phosphate (SAP), and convert it into reducing ascorbate. Highly reactive hydroxyl radicals (·OH) were generated in the presence of ascorbate and Cu(2+) through the Fenton-like reaction. The reactive radicals generated in situ caused the cleavage of F-DNA into small fragments. When GO was added, the fluorescence emission of the sample without ALP was quenched and fluorescence emission recovered in the presence of ALP. The intensity of the recovered fluorescence was directly related to the concentration of ALP in the assay solution, and a sensitive and selective facile ALP assay is therefore established. PMID:27343614

  6. A Further Investigation of the Effects of Extremely Low Frequency Magnetic Fields on Alkaline Phosphatase and Acetylcholinesterase.

    Directory of Open Access Journals (Sweden)

    Gary Silkstone

    Full Text Available Using a custom build spectrophotometer equipped with Helmholtz coils and designed to study the effects of magnetic fields on enzyme reactions in real-time we have investigated the influence of fields, from 100 μT to 10 mT and at a variety of field frequencies, on the membrane bound enzymes alkaline phosphatase and acetylcholinesterase. We have also employed other methods to apply a magnetic field, e.g. Biostim. In contrast to earlier reports we have been unable to detect any field effects on these enzymes under any field/frequency regime. We discuss possible reasons for the discrepancy between this and earlier work and note the particularly complex influence of small temperature changes that may confound analysis.

  7. Significant Association Between Bone-Specific Alkaline Phosphatase and Vascular Calcification of the Hand Arteries in Male Hemodialysis Patients

    Directory of Open Access Journals (Sweden)

    Eiji Ishimura

    2014-09-01

    Full Text Available Background/Aims: Bone-specific alkaline phosphatase (BAP hydrolyzes pyrophosphate, which inhibits vascular calcification. We examined association between serum BAP and vascular calcification of male hemodialysis patients. Methods: Hand roentgenography of 167 male maintenance hemodialysis patients was conducted, and visible vascular calcification of the hand arteries was evaluated. Serum levels of 3 bone formation markers (BAP, osteocalcin, and N-terminal propeptide of type I collagen and 2 bone resorption markers (C-terminal telopeptide of type I collagen, and cross-linked N-telopeptide of type I collagen were measured, along with serum intact parathyroid hormone (PTH. Results: Of 167 patients, visible vascular calcification was seen in 37 patients. Among the bone formation and resorption markers, serum BAP was significantly higher in patients with vascular calcification than in those without (pConclusions: Higher serum BAP, but not other bone markers, is significantly associated with the presence of vascular calcification in male hemodialysis patients.

  8. Sensitive and direct electrochemical detection of double-stranded DNA utilizing alkaline phosphatase-labelled zinc finger proteins.

    Science.gov (United States)

    Noh, Soodong; Ha, Dat Thinh; Yang, Haesik; Kim, Moon-Soo

    2015-06-21

    Direct detection of double-stranded DNA (dsDNA) using zinc finger proteins (ZFPs) is of great importance in biomedical applications such as identifying pathogens and circulating DNAs. However, its sensitivity is still not sufficiently high because limited signalling labels can be conjugated or fused. Herein, we report sensitive and direct detection of dsDNA using (i) alkaline phosphatase (ALP) as a fast catalytic label conjugated to ZFPs along with (ii) electrochemical measurement of an ALP product (l-ascorbic acid) at the indium-tin oxide electrode with a high signal-to-background ratio. ALP is simply conjugated to a ZFP through lysine residues in a ZFP purification tag, a maltose binding protein (MBP). Sandwich-type electrochemical detection of dsDNA allows a detection limit of ca. 100 fM without using DNA amplification. PMID:25969923

  9. Activity of Follicular Fluid Phosphatases and Their Correlation with Levels of Serum Esteroidal Hormones and Gonadotropins

    Directory of Open Access Journals (Sweden)

    Sh Byranvand

    2006-10-01

    Full Text Available Introduction: The aim of this study was to evaluate the correlation between serum levels of ovarian and gonadotropin hormones, age and number of follicles with follicular alkaline and acid phosphatase activity in infertile women under controlled ovarian hyperstimulation. Methods: After collection of follicular fluid and calculation of the number of follicles, the specific activity of alkaline (ALP and acid phosphatase (ACP was determined according to the total protein in 19 women at the time of puncture. Also at that time, the levels of progesterone, estradiol, and follicle stimulating hormone (FSH and leuteinizing hormone (LH of their sera were measured. The correlation of follicular ALP and ACP with each serum hormone levels, women age and number of follicles was calculated using non-parametric analysis. Results: The ALP has a correlation with progesterone (P=0.01 levels but doesn’t have any correlation with the other factors. However, the ACP activity has a correlation not only with follicular number but also with estradiol and progesterone levels (P=0.05. Conclusion: Thus ACP activity is more affected by ovarian hormone than ALP and it can affect the ovarian microenvironment and oocyte development.

  10. 僵蚕提取物对棉铃虫的胃毒作用和碱性磷酸酶活性的影响%Toxicity and Alkaline Phosphatase Activity Inhibition of the Extracts from Bombyx batryticatus Against Helicoverpa armigera

    Institute of Scientific and Technical Information of China (English)

    曹军; 于欢; 张瑞凤; 王沛; 张雅林; 王敦

    2012-01-01

    僵蚕提取物对棉铃虫有胃毒作用,对饲毒后不同时间棉铃虫3龄幼虫体内碱性磷酸酶的活性进行了研究.结果表明,在3龄棉铃虫人工饲料中添加僵蚕提取物浓度为3 mg·g-1时,饲毒第3天试虫的校正死亡率为66.73%;5 mg·g-1的剂量导致试虫死亡率可以高达83.33%,表明僵蚕提取物对棉铃虫具有很强的胃毒作用.试虫碱性磷酸酶活性分析表明,1~5 mg·g-1浓度处理组的试虫碱性磷酸酶活性与对照组相比均显著下降,并有明显的剂量相关性.推测侵蚕对棉铃虫幼虫的毒杀作用机制之一与其对碱性磷酸酶活性的抑制有关.%This paper reported the study results on feeding toxicity and inhibition effects on alkaline phos-phatase (ALP) of Helicoverpa armigera treated with the extracts of Bombyx batryticatus. The corrected mortality of H. armigera treated with the extracts of B. batryticatus at 3 mg · g-1 concentration in insect diet was 66. 7% after feeding on the toxic diet three days, while the corrected mortality was up to 83% to the larvae treated with a concentration of 5 mg · g-1, indicating that the B. batryticatus extracts was high toxic to the third instar larvae of H. armigera. The enzyme assay showed that the ALP activities decreased significantly while treated with the B. batryticatus extracts concentration ranged from 1 mg · g-1 to 5 mg · g-1, and the correlationship was responsible to the dosage of B. batryticatus extracts. Thus, the inhibition on ALP activity is a possible mechanism for the pesticidal activity of B. batryticatus extracts.

  11. Metals in the active site of native protein phosphatase-1.

    Science.gov (United States)

    Heroes, Ewald; Rip, Jens; Beullens, Monique; Van Meervelt, Luc; De Gendt, Stefan; Bollen, Mathieu

    2015-08-01

    Protein phosphatase-1 (PP1) is a major protein Ser/Thr phosphatase in eukaryotic cells. Its activity depends on two metal ions in the catalytic site, which were identified as manganese in the bacterially expressed phosphatase. However, the identity of the metal ions in native PP1 is unknown. In this study, total reflection X-ray fluorescence (TXRF) was used to detect iron and zinc in PP1 that was purified from rabbit skeletal muscle. Metal exchange experiments confirmed that the distinct substrate specificity of recombinant and native PP1 is determined by the nature of their associated metals. We also found that the iron level associated with native PP1 is decreased by incubation with inhibitor-2, consistent with a function of inhibitor-2 as a PP1 chaperone. PMID:25890482

  12. Investigations into the stabilisation of drugs by sugar glasses : II: Delivery of an inulin-stabilised alkaline phosphatase in the intestinal lumen via the oral route

    NARCIS (Netherlands)

    Eriksson, H.J.C.; Verweij, W.R.; Poelstra, K.; Hinrichs, W.L.J.; de Jong, G.J.; Somsen, G.W.; Frijlink, H.W.

    2003-01-01

    In this study the possibility to deliver the acid-sensitive enzyme alkaline phosphatase (AP) from calf intestine (CIAP) to the intestinal system by oral administration was investigated. Tablets were prepared and in vitro evaluated. Final proof of concept studies were performed in rats. This acid lab

  13. Growth hormone (GH) treatment increases serum insulin-like growth factor binding protein-3, bone isoenzyme alkaline phosphatase and forearm bone mineral content in young adults with GH deficiency of childhood onset

    DEFF Research Database (Denmark)

    Juul, A; Pedersen, S A; Sørensen, S;

    1994-01-01

    the effect of GH treatment on a marker of bone formation (bone alkaline phosphatase), hepatic excretory function and distal forearm bone mineral content in GH-deficient adults. Growth hormone was administered subcutaneously in 21 adults (13 males and 8 females) with GH deficiency of childhood onset for 4...... months of GH treatment (p alkaline phosphatase increased significantly from 38.6 to 92.9 U/l during GH therapy in male patients (p ...-derived alkaline phosphatase was unaltered by GH. In the females, the increase in bone alkaline phosphatase did not reach statistical significance (19.1 vs 40.0 U/l, p = 0.06). The GH-induced increase in bone alkaline phosphatase correlated significantly with the increase in serum IGFBP-3 (r = 0.46, p = 0...

  14. Cloning of the gene and characterization of the enzymatic properties of the monomeric alkaline phosphatase (PhoX) from Pasteurella multocida strain X-73.

    Science.gov (United States)

    Wu, Jin-Ru; Shien, Jui-Hung; Shieh, Happy K; Hu, Chung-Chi; Gong, Shuen-Rong; Chen, Ling-Yun; Chang, Poa-Chun

    2007-02-01

    We have identified a new phoX gene encoding the monomeric alkaline phosphatase from Pasteurella multocida X-73. This gene was not found in the published genome sequence of Pasteurella multocida pm70. Characterization of the recombinant PhoX of Pasteurella multocida X-73 showed that it is a monomeric enzyme, activated by Ca(2+) and possibly secreted by the Tat pathway. These features distinguish phosphatases of the PhoX family from those of the PhoA family. All proteins of the PhoX family were found to contain a conserved motif that shares significant sequence homology with the calcium-binding site of a phosphotriesterase known as diisopropylfluorophosphatase. Site-directed mutagenesis revealed that D527 of PhoX might be the ligand bound to the catalytic calcium. This is the first report on identification of homologous sequences between PhoX and the phosphotriesterase and on the potential calcium-binding site of PhoX. PMID:17156125

  15. Redox Regulation of Protein Tyrosine Phosphatase Activity by Hydroxyl Radical

    OpenAIRE

    Meng, Fan-Guo; Zhang, Zhong-Yin

    2012-01-01

    Substantial evidence suggests that transient production of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) is an important signaling event triggered by the activation of various cell surface receptors. Major targets of H2O2 include protein tyrosine phosphatases (PTPs). Oxidation of the active site Cys by H2O2 abrogates PTP catalytic activity, thereby potentially furnishing a mechanism to ensure optimal tyrosine phosphorylation in response to a variety of physiological stimuli. ...

  16. Protein phosphatase 2A regulatory subunit B56α limits phosphatase activity in the heart.

    Science.gov (United States)

    Little, Sean C; Curran, Jerry; Makara, Michael A; Kline, Crystal F; Ho, Hsiang-Ting; Xu, Zhaobin; Wu, Xiangqiong; Polina, Iuliia; Musa, Hassan; Meadows, Allison M; Carnes, Cynthia A; Biesiadecki, Brandon J; Davis, Jonathan P; Weisleder, Noah; Györke, Sandor; Wehrens, Xander H; Hund, Thomas J; Mohler, Peter J

    2015-07-21

    Protein phosphatase 2A (PP2A) is a serine/threonine-selective holoenzyme composed of a catalytic, scaffolding, and regulatory subunit. In the heart, PP2A activity is requisite for cardiac excitation-contraction coupling and central in adrenergic signaling. We found that mice deficient in the PP2A regulatory subunit B56α (1 of 13 regulatory subunits) had altered PP2A signaling in the heart that was associated with changes in cardiac physiology, suggesting that the B56α regulatory subunit had an autoinhibitory role that suppressed excess PP2A activity. The increase in PP2A activity in the mice with reduced B56α expression resulted in slower heart rates and increased heart rate variability, conduction defects, and increased sensitivity of heart rate to parasympathetic agonists. Increased PP2A activity in B56α(+/-) myocytes resulted in reduced Ca(2+) waves and sparks, which was associated with decreased phosphorylation (and thus decreased activation) of the ryanodine receptor RyR2, an ion channel on intracellular membranes that is involved in Ca(2+) regulation in cardiomyocytes. In line with an autoinhibitory role for B56α, in vivo expression of B56α in the absence of altered abundance of other PP2A subunits decreased basal phosphatase activity. Consequently, in vivo expression of B56α suppressed parasympathetic regulation of heart rate and increased RyR2 phosphorylation in cardiomyocytes. These data show that an integral component of the PP2A holoenzyme has an important inhibitory role in controlling PP2A enzyme activity in the heart.

  17. 中性粒细胞碱性磷酸酶活性在红细胞增多症鉴别诊断中的临床意义%The significance of neutrophil alkaline phosphatase activity in the differential diagnosis of polycythemia

    Institute of Scientific and Technical Information of China (English)

    汤俊峰; 叶龙飞

    2016-01-01

    Objective To discuss clinical significance of the neutrophil alkaline phosphatase(NAP) activity in polycythemia pa‐tients .Methods 35 cases of patients with secondary polycythemia(secondary group) and 22 patients with polycythemia vera (poly‐cythemia vera group) were enrolled in the study ,whose peripheral blood smears were stained by using chemical method and the pos‐itive rate and scores of NAP were assessed .In addition to that ,30 cases of healthy people who had underwent physical examination were recruited as control group .Results NAP positive rate and scores of the polycythemia vera group were significantly higher than those of secondary and control group(P0 .05) .Conclusion NAP score is a good methord to diagnose polycythemia ,which could facilitate rapid ,accurate diagno‐sis and be used as auxiliary indicators in the differential diagnosis of polycythemia .%目的:探讨中性粒细胞碱性磷酸酶(NAP)活性在红细胞增多症鉴别诊断中的临床意义。方法对35例继发性红细胞增多症患者(继红组)、22例真性红细胞增多症患者(真红组)外周血涂片用化学法染色并对NAP阳性率和积分进行判断并以30例健康体检者作为对照组。结果真性组患者NAP阳性率及积分明显高于继红组及对照组,差异均有统计学意义(P<0.05);继红组NAP积分及阳性率与健康组比较差异均无统计学意义(P>0.05)。结论NAP积分应用于诊断红细胞增多症是一种有效的方法,有助于快速、准确的鉴别诊断,可作为红细胞增多症鉴别诊断的重要辅助指标。

  18. Mechanisms of Action for Total Flavones of Hippophae Rhamnoides and Quercetin on Alkaline Phosphatase Activity in Chicken Osteoblasts%沙棘总黄酮、槲皮素对鸡成骨细胞碱性磷酸酶活性作用机制的体外研究

    Institute of Scientific and Technical Information of China (English)

    刘洪柱; 李垚; 陈鑫; 赵伟; 张丽欣; 欧阳文文; 刘红南

    2011-01-01

    The experiment was conducted to study the effects of total flavones of Hippophae rhamnoides (TFH) and quercetin on alkaline phosphatase (ALP) activity in osteoblasts of arbor acres (AA) broilers' embryo in vitro and its mechanisms of action. Cells were cultured in basal culture medium, basal culture medium supplemented with β-estradiol (E2) , TFH and quercetin for 24 h, respectively; after treated by estrogen receptor (ER) antagonist for 2 h, the cells were cultured for another 24 h in basal culture medium, basal culture medium supplemented with TFH and quercetin, respectively; after treated with extracellular signal-regulated ki-nase (ERK) inhibitor for 2 h, the cells were cultured for another 24 h in basal culture medium, basal culture medium supplemented with TFH and quercetin, respectively; the cells were collected for the analysis of ALP activity. Cells were cultured in basal culture medium supplemented with TFH or quercetin, and collected at six time points (0, 2, 5, 15, 30, 60 min) for the determination of phosphorylated extracellular-regulated kinase (p-ERK) concentration by ELASE. Cells were cultured in basal culture medium, basal culture medium supplemented with E2, TFH or quercetin for 30 min, respectively; after treated by ER antagonist for 15 min, the cells were cultured for 30 min in basal culture medium, basal culture medium supplemented with E2, TFH and quercetin, respectively; cells were collected for the determination of p-ERK concentration by ELASE. The results showed as follows:1) culture medium supplemented with TFH or quercetin significantly increased the ALP activity {P <0.05), and the effects of which were totally prevented by ER antagonist and MEK inhibitor (P<0.05);2)the phosphorylation level of ERK were significantly increased in culture medium supplemented with TFH after 5, 15 min incubation and with quercetin after 2, 5, 15, 30, 60 min incubation (P < 0.05); 3) culture medium supplemented with TFH or quercetin significantly increased

  19. Protein phosphatase 1α is a Ras-activated Bad phosphatase that regulates interleukin-2 deprivation-induced apoptosis

    Science.gov (United States)

    Ayllón, Verónica; Martínez-A, Carlos; García, Alphonse; Cayla, Xavier; Rebollo, Angelita

    2000-01-01

    Growth factor deprivation is a physiological mechanism to regulate cell death. We utilize an interleukin-2 (IL-2)-dependent murine T-cell line to identify proteins that interact with Bad upon IL-2 stimulation or deprivation. Using the yeast two-hybrid system, glutathione S-transferase (GST) fusion proteins and co-immunoprecipitation techniques, we found that Bad interacts with protein phosphatase 1α (PP1α). Serine phosphorylation of Bad is induced by IL-2 and its dephosphorylation correlates with appearance of apoptosis. IL-2 deprivation induces Bad dephosphorylation, suggesting the involvement of a serine phosphatase. A serine/threonine phosphatase activity, sensitive to the phosphatase inhibitor okadaic acid, was detected in Bad immunoprecipitates from IL-2-stimulated cells, increasing after IL-2 deprivation. This enzymatic activity also dephosphorylates in vivo 32P-labeled Bad. Treatment of cells with okadaic acid blocks Bad dephosphorylation and prevents cell death. Finally, Ras activation controls the catalytic activity of PP1α. These results strongly suggest that Bad is an in vitro and in vivo substrate for PP1α phosphatase and that IL-2 deprivation-induced apoptosis may operate by regulating Bad phosphorylation through PP1α phosphatase, whose enzymatic activity is regulated by Ras. PMID:10811615

  20. An in vitro study of alkaline phosphatase sensitivity to mixture of aflatoxin B1 and fumonisin B1 in the hepatopancreas of coastal lagoon wild and farmed shrimp Litopenaeus vannamei.

    Science.gov (United States)

    Pérez-Acosta, Jesús A; Burgos-Hernandez, Armando; Velázquez-Contreras, Carlos A; Márquez-Ríos, Enrique; Torres-Arreola, Wilfrido; Arvizu-Flores, Aldo A; Ezquerra-Brauer, J Marina

    2016-08-01

    This study aimed to establish the combined effect of aflatoxin B1 (AFB1) and fumonisin B1 (FB1) on wild Litopenaeus vannamei hepatopancreas alkaline phosphatase (AP) activity compared with that of farmed shrimp. AP activity in hepatopancreas extract was confirmed by several specific inhibitor assays. AP activity of wild shrimp was higher than that of farmed shrimp (p mycotoxins concentrations used in the study, AP from farmed L. vannamei was sensitive to the presence of both mycotoxins; however, AP is more sensitive to the combination of AFB1 + FB1 suggesting a possible synergistic or potentiating inhibitory effect. PMID:27040818

  1. Structural elucidation of the NADP(H) phosphatase activity of staphylococcal dual-specific IMPase/NADP(H) phosphatase.

    Science.gov (United States)

    Bhattacharyya, Sudipta; Dutta, Anirudha; Dutta, Debajyoti; Ghosh, Ananta Kumar; Das, Amit Kumar

    2016-02-01

    NADP(H)/NAD(H) homeostasis has long been identified to play a pivotal role in the mitigation of reactive oxygen stress (ROS) in the intracellular milieu and is therefore critical for the progression and pathogenesis of many diseases. NAD(H) kinases and NADP(H) phosphatases are two key players in this pathway. Despite structural evidence demonstrating the existence and mode of action of NAD(H) kinases, the specific annotation and the mode of action of NADP(H) phosphatases remains obscure. Here, structural evidence supporting the alternative role of inositol monophosphatase (IMPase) as an NADP(H) phosphatase is reported. Crystal structures of staphylococcal dual-specific IMPase/NADP(H) phosphatase (SaIMPase-I) in complex with the substrates D-myo-inositol-1-phosphate and NADP(+) have been solved. The structure of the SaIMPase-I-Ca(2+)-NADP(+) ternary complex reveals the catalytic mode of action of NADP(H) phosphatase. Moreover, structures of SaIMPase-I-Ca(2+)-substrate complexes have reinforced the earlier proposal that the length of the active-site-distant helix α4 and its preceding loop are the predisposing factors for the promiscuous substrate specificity of SaIMPase-I. Altogether, the evidence presented suggests that IMPase-family enzymes with a shorter α4 helix could be potential candidates for previously unreported NADP(H) phosphatase activity.

  2. Metavanadate at the active site of the phosphatase VHZ.

    Science.gov (United States)

    Kuznetsov, Vyacheslav I; Alexandrova, Anastassia N; Hengge, Alvan C

    2012-09-01

    Vanadate is a potent modulator of a number of biological processes and has been shown by crystal structures and NMR spectroscopy to interact with numerous enzymes. Although these effects often occur under conditions where oligomeric forms dominate, the crystal structures and NMR data suggest that the inhibitory form is usually monomeric orthovanadate, a particularly good inhibitor of phosphatases because of its ability to form stable trigonal-bipyramidal complexes. We performed a computational analysis of a 1.14 Å structure of the phosphatase VHZ in complex with an unusual metavanadate species and compared it with two classical trigonal-bipyramidal vanadate-phosphatase complexes. The results support extensive delocalized bonding to the apical ligands in the classical structures. In contrast, in the VHZ metavanadate complex, the central, planar VO(3)(-) moiety has only one apical ligand, the nucleophilic Cys95, and a gap in electron density between V and S. A computational analysis showed that the V-S interaction is primarily ionic. A mechanism is proposed to explain the formation of metavanadate in the active site from a dimeric vanadate species that previous crystallographic evidence has shown to be able to bind to the active sites of phosphatases related to VHZ. Together, the results show that the interaction of vanadate with biological systems is not solely reliant upon the prior formation of a particular inhibitory form in solution. The catalytic properties of an enzyme may act upon the oligomeric forms primarily present in solution to generate species such as the metavanadate ion observed in the VHZ structure. PMID:22876963

  3. PROTEN TYROSINE PHOSPHATASE ACTIVITY IN RAT ASCITES HEPATOMA CELLS

    Directory of Open Access Journals (Sweden)

    M.Saadat

    1998-10-01

    Full Text Available Protein tyrosine phosphatases (PTPases regulate tyrosine phosphorylation of target proteins involved in several aspects of cellular functions. Enzyme activities of the PTPases in cytosolic and particulate fractions of rat ascites hepatoma cell lines were determined and compared with those of normal rat liver. Our present data revealed that although there was no neoplatic-specific alteration of the PTPase activity in examined hepatomas, the activity in particulate fractions of island type of hepatomas was remarkably decreased compared with either rat liver or free type hepatomas.

  4. Alkaline phosphatase, cytokeratin 7, cytokeratin 8 in the diagnosis of primary lung adenocarcinoma from 148 pleura fluids specimens.

    Directory of Open Access Journals (Sweden)

    Temelli Ozlem

    2009-05-01

    Full Text Available Adenocarcinomas are the most common cause of malignancy in pleura fluids. Usual primary sites include the lung, breast, gastrointestinal tract, and genitourinary tracts. Predicting the site of origin of an adenocarcinoma can be difficult due to overlapping morphologic characteristics. We investigated the use of alkaline phosphatase (AP, Cytokeratin7 (CK7 Cytokeratin8 (CK8 to distinguish adenocarcinomas of lung in 148 body cavity fluid samples. Overall results for primary lung adenocarcinomas, demonstrated CK8 reactivity in 106 (72% of 148 cases. 95 primary lung carcinoma samples (65% were positive for CK7. AP was expressed in 81% of primary lung adenocarcinomas. Positive immunoreactivity for AP was characterized by a red, diffusely apical cytoplasmic staining in tumor cells that ocurred singly or in groups. There was a significant difference between AP, CK 7 and CK 8 expressions in primary lung adenocarcinomas (P=0.02; Chi-squared test. The sensitivity of AP, CK8, CK7 as a marker for primary lung adenocarcinomas were 82%, 72%, 64%, respectively. Thus the AP positive staining largely confirmed the cytologic diagnosis of lung adenocarcinoma.

  5. The relationship between the alkaline phosphatase network and the haematopoiesis in mice subjected to whole-body irradiation

    Directory of Open Access Journals (Sweden)

    Almohamad Khaled M.

    2014-08-01

    Full Text Available Purpose: To investigate the relationship between the alkaline phosphatase (ALP network of the marrow stroma and the haematopoietic regeneration after mice whole-body irradiation. Materials and methods: Three groups of mice were irradiated with a non-lethal ionising radiation dose: the fi rst one received an intraperitoneal injection of Levamisole, ALP inhibitor, 24 h before irradiation; the second one received an intraperitoneal injection of Lisinopril, haematopoiesis inhibitor, 24 h before irradiation; the third was left untreated, but irradiated. The fourth group, untreated and not irradiated, was the control. The total surface occupied by ALP positive processes, revealed by means of ALP cytochemistry in the marrow area, was evaluated semi-quantitively. Nucleated bone marrow cells were also counted. Results: ALP network began to increase 24 h after irradiation to reach a maximum after 72 h, when the bone marrow was almost become completely empty of the haematopoietic cells. This increase advances the haematopoietic recovery. This process was substantially delayed when the mice were injected with Levamisole 24 h before irradiation. On the contrary, ALP network increased strongly since the fi rst day after irradiation when the mice were injected with Lisinopril 24 h before irradiation. Conclusions: These data have indicated that the haematopoietic recovery and repopulation of the bone marrow were advanced by the ALP network recovery.

  6. A High Level of Intestinal Alkaline Phosphatase Is Protective Against Type 2 Diabetes Mellitus Irrespective of Obesity.

    Science.gov (United States)

    Malo, Madhu S

    2015-12-01

    Mice deficient in intestinal alkaline phosphatase (IAP) develop type 2 diabetes mellitus (T2DM). We hypothesized that a high level of IAP might be protective against T2DM in humans. We determined IAP levels in the stools of 202 diabetic patients and 445 healthy non-diabetic control people. We found that compared to controls, T2DM patients have approx. 50% less IAP (mean +/- SEM: 67.4 +/- 3.2 vs 35.3 +/- 2.5 U/g stool, respectively; p diabetes status. With each 25 U/g decrease in stool IAP, there is a 35% increased risk of diabetes. The study revealed that obese people with high IAP (approx. 65 U/g stool) do not develop T2DM. Approx. 65% of the healthy population have diabetes', and might develop T2DM and other metabolic disorders in the near future. In conclusion, high IAP levels appear to be protective against diabetes irrespective of obesity, and a 'temporal IAP profile' might be a valuable tool for predicting 'the incipient metabolic syndrome', including 'incipient diabetes'.

  7. Steric hindrance regulated supramolecular assembly between β-cyclodextrin polymer and pyrene for alkaline phosphatase fluorescent sensing

    Science.gov (United States)

    Song, Chunxia; Yang, Xiaohai; Wang, Kemin; Wang, Qing; Liu, Jianbo; Huang, Jin; Zhou, Maogui; Guo, Xiaochen

    2016-03-01

    We herein report a strategy for sensitive alkaline phosphatase (ALP) fluorescent sensing based on steric hindrance regulated supramolecular assembly between β-cyclodextrin polymer (polyβ-CD) and pyrene. The fluorescence of pyrene was enhanced more than 10 times through supramolecular assembly with polyβ-CD. The 5‧-phosphorylated dsDNA probe with pyrene attached on the 3‧-terminal could be cleaved by λ exonuclease (λ exo), yielding pyrene attached on mononucleotides. Pyrene attached on mononucleotides could easily enter the cavity of polyβ-CD, resulting in fluorescence enhancement. When ALP was introduced, it could remove 5‧-phosphate groups from dsDNA and then prevented the cleavage of dsDNA. Pyrene attached on dsDNA was difficult to enter the cavity of polyβ-CD because of steric hindrance, resulting in an inconspicuous fluorescence enhancement. Owing to the excellent fluorescence enhancement during steric hindrance regulated supramolecular assembly, excellent performance of the assay method was achieved for ALP with a detection limit of 0.04 U mL- 1. The detection limit was superior or comparable with the reported methods. Besides, this method was simple in design, avoiding double-labeling of probe.

  8. Can Serum Levels of Alkaline Phosphatase and Phosphate Predict Cardiovascular Diseases and Total Mortality in Individuals with Preserved Renal Function? A Systemic Review and Meta-Analysis

    OpenAIRE

    Jing-Wei Li; Cui Xu; Ye Fan; Yong Wang; Ying-Bin Xiao

    2014-01-01

    Background It is demonstrated that elevated serum levels of alkaline phosphatase (ALP) and phosphate indicate a higher risks of cardiovascular disease (CVD) and total mortality in population with chronic kidney disease (CKD), but it remains unclear whether this association exists in people with normal or preserved renal function. Method Clinical trials were searched from Embase and PubMed from inception to 2013 December using the keywords “ALP”, “phosphate”, “CVD”, “mortality” and so on, and ...

  9. The Characteristic Absorption Peak Analysis of Alkaline Phosphatase Kit%对碱性磷酸酶试剂盒特征吸收峰的分析

    Institute of Scientific and Technical Information of China (English)

    何乐春

    2012-01-01

      The paper analysis the reasonableness of enterprise standards about detection wavelength according to the test result of reagent blank absorbance value of alkaline phosphatase ( ALP ) kit.%  从检测一厂家生产的碱性磷酸酶(ALP)试剂盒的试剂空白吸光度所得的值,分析企业标准中所涉及的检测波长是否合理。

  10. Improvement of the skeletal and dental hypophosphatasia phenotype in Alpl−/− mice by administration of soluble (non-targeted) chimeric alkaline phosphatase

    OpenAIRE

    Gasque, Kellen; Foster, Brian L.; Kuss, Pia; Yadav, Manisha C; Jin LIU; Kiffer-Moreira, Tina; van Elsas, Andrea; Hatch, Nan; Somerman, Martha J.; Millán, JoséLuis

    2014-01-01

    Hypophosphatasia (HPP) results from ALPL gene mutations, which lead to a deficiency of tissue-nonspecific alkaline phosphatase (TNAP), and accumulation of inorganic pyrophosphate, a potent inhibitor of mineralization that is also a natural substrate of TNAP, in the extracellular space. HPP causes mineralization disorders including soft bones (rickets or osteomalacia) and defects in teeth and periodontal tissues. Enzyme replacement therapy using mineral-targeting recombinant TNAP has proven ef...

  11. Assessment of the alkaline phosphatase level in gingival crevicular fluid, as a biomarker to evaluate the effect of scaling and root planing on chronic periodontitis: An in vivostudy

    Directory of Open Access Journals (Sweden)

    Jimly James Kunjappu

    2012-01-01

    Full Text Available Context: Clinical evaluation of gingivitis and/or periodontitis does not predict the progression or remission of the disease. Due to this diagnostic constraint, clinicians assume that the pathology has an increased risk of progression and plan treatments, despite the knowledge that all inflamed sites are not necessarily progressing. Extensive research has been carried out on gingival crevicular fluid (GCF components that might serve as potential diagnostic markers for periodontitis. Among them alkaline phosphatase (ALP levels in GCF has shown promise as a diagnostic marker. Aim: This study compares the levels of GCF alkaline phosphatase in patients with chronic periodontitis before and after scaling and root planing. Materials and Methods: This study is an in vivo longitudinal study conducted on twenty patients with localized periodontitis. The GCF was collected from the affected site prior to scaling and root planing and ALP level estimated. The probing depth and plaque index at the site were also measured for correlation. Patients were recalled after 7, 30, and 60 days for reassessment. Results: The GCF ALP values showed a sustained, statistically significant decrease after treatment. There was a positive correlation with probing depth but not with plaque index measured at each interval. Conclusion: The assessment of level of periodontal disease and effect of mechanical plaque control on the progression and regression of the disease can be evaluated precisely by the corresponding GCF ALP levels. Thus, alkaline phosphatase level is not only a biomarker for the pathology but also an indicator of prognosis of periodontitis.

  12. AN INTRA - ABDOMINAL FOREI GN BOCORRELATION OF SERUM LIPID PROFILE, SERUM CALCIUM, ALKALINE PHOSPHATASE AND SERUM PROTEIN WITH HISTOPATHOLOGICAL GRADING AND STAGING IN HEAD AND NECK CANCER

    Directory of Open Access Journals (Sweden)

    Neha

    2014-02-01

    Full Text Available Lipids have a key role in the maintenance of cell integrity. Hypolipidemia is related with increased risk of cancer and mortality. Elevated calcium and alkaline phosphatase has also been seen in cancer patients having m etastasis. Alteration in serum prote ins also has been reported. Hence present study was undertaken in 100 histopathologically confirmed head and neck cancer patients, in Department of Pathology Gandhi Medical College Bhopal, after taking informed written c onsent and ethical clearance. Lipid Profile, Serum Calcium, Alkaline Phosphatase, Serum Protein with Albumin were estimated and compared with age and sex matched healthy controls. Statistical analysis was done using chi square test. Total cholesterol (TC, high density lipoprotein - cholesterol (HDLC, low density lipoprotein - cholesterol (LDLC, Very low density lipoprotein - cholesterol (VLDLC all decreased in head & neck cancer patients. Hypercalcemia was seen in 35% of patients. Raised Alkaline phosphatase (ALP was seen in 8 % cancer patients. Serum calcium was found to be increasing with advancing stage. Further studies should be carried out in large number of patients to confirm the role of these parameters with special attention to modifiable ones and their relation with staging and grading of cancer, which can be used as prognostic markers.

  13. Serum proteins, trace metals and phosphatases in psoriasis

    Directory of Open Access Journals (Sweden)

    Bhatnagar M

    1994-01-01

    Full Text Available Serum proteins, zinc, copper, acid phosphatase (AcPase and alkaline phosphatase (AlPase were studied in both active and remission phases of psoriasis. Data were compared with healthy controls, ?1, ? and ? globulins were high in active phase while ?1 and ? globulins were at par in remission phase. Serum copper was low but zinc and alkaline phosphatase were significantly high in both active and remission phases of the disease. Acid phosphatase level was at par in all the experimental groups. Study suggests a positive correlation of globulin, zinc and Alpase in active and remission phase of psoriasis.

  14. Changes in Phosphorus Fractions, pH, and phosphatase Activity in rhizosphere of Two Rice Genotypes

    Institute of Scientific and Technical Information of China (English)

    LI Yong-Fu; LUO An-Cheng; WEI Xing-Hua; YAO Xu-Guo

    2008-01-01

    A rhizobox experiment with two phosphorus (P) treatments, zero-P (0 mg P kg-1) and plus-P (100 mg P kg-1) as Ca(H2PO4)2·H2O, was conducted to study the chemical and biochemical properties in the rhizosphere of two rice genotypes (cv. Zhongbu 51 and Pembe) different in P uptake ability and their relationship with the depletion of soil P fractions. Plant P uptake, pH, phosphatase activity, and soil P fractions in the rhizcephere were measured. Both total dry weight and total P uptake of Pembe were significantly (P < 0.05) higher than those of Zhongbu 51 in the zero-P and plns-P treatments. Significant depletions of resin-Pi, NaHCO3-Pi, NaHCO3-Po, and NaOH-Pi, where Pi stands for inorganic P and Po for organic P, were observed in the rhizosphere of both Zhongbu 51 and Pembe under both P treatments. Pembe showed a greater ability than Zhongbu 51 in depleting resin-Pi, NaHCO2-Pi, NaHCO3-Po, NaOH-Pi, and NaOH-Po in the rhizosphere. HCl-Pi and residual-P were not depleted in the rhizosphere of both genotypes, regardless of P treatments despite significant acidification in the rhizosphere of Pembe under zero-P treatment. Higher acid phosphatase (AcPME) activity and alkaline phosphatase (A1PME) activity were observed in the rhizosphere of both Zhongbu 51 and Pembe compared to the corresponding controls without plant. AcPME activity was negatively (P < 0.01) correlated to NaHCO3-Po concentration in the rhizosphere of both Zhongbu 51 and Pembe, suggesting that AcPME was associated with the mineralization of soil organic P.

  15. Alkaline phosphatases are involved in the response of Aedes aegypti larvae to intoxication with Bacillus thuringiensis subsp. israelensis Cry toxins.

    Science.gov (United States)

    Stalinski, Renaud; Laporte, Frédéric; Després, Laurence; Tetreau, Guillaume

    2016-03-01

    Bacillus thuringiensis subsp. israelensis (Bti) is a natural pathogen of dipterans widely used as a biological insecticide for mosquito control. To characterize the response of mosquitoes to intoxication with Bti, the transcriptome profile of Bti-exposed susceptible Aedes aegypti larvae was analysed using Illumina RNA-seq. Gene expression of 11 alkaline phosphatases (ALPs) was further investigated by real time quantitative polymerase chain reaction and ALP activity was measured in the susceptible strain and in four strains resistant to a single Bti Cry toxin or to Bti. These strains were unexposed or exposed to their toxin of selection. Although all resistant strains constitutively exhibited a higher level of transcription of ALP genes than the susceptible strain, they showed a lower total ALP activity. The intoxication with different individual Cry toxins triggered a global pattern of ALP gene under-transcription in all the one-toxin-resistant strains but involving different specific sets of ALPs in each resistant phenotype. Most of the ALPs involved are not known Cry-binding proteins. RNA interference experiment demonstrated that reducing ALP expression conferred increased the survival of larvae exposed to Cry4Aa, confirming the involvement of ALP in Cry4Aa toxicity.

  16. Alkaline phosphatases are involved in the response of Aedes aegypti larvae to intoxication with Bacillus thuringiensis subsp. israelensis Cry toxins.

    Science.gov (United States)

    Stalinski, Renaud; Laporte, Frédéric; Després, Laurence; Tetreau, Guillaume

    2016-03-01

    Bacillus thuringiensis subsp. israelensis (Bti) is a natural pathogen of dipterans widely used as a biological insecticide for mosquito control. To characterize the response of mosquitoes to intoxication with Bti, the transcriptome profile of Bti-exposed susceptible Aedes aegypti larvae was analysed using Illumina RNA-seq. Gene expression of 11 alkaline phosphatases (ALPs) was further investigated by real time quantitative polymerase chain reaction and ALP activity was measured in the susceptible strain and in four strains resistant to a single Bti Cry toxin or to Bti. These strains were unexposed or exposed to their toxin of selection. Although all resistant strains constitutively exhibited a higher level of transcription of ALP genes than the susceptible strain, they showed a lower total ALP activity. The intoxication with different individual Cry toxins triggered a global pattern of ALP gene under-transcription in all the one-toxin-resistant strains but involving different specific sets of ALPs in each resistant phenotype. Most of the ALPs involved are not known Cry-binding proteins. RNA interference experiment demonstrated that reducing ALP expression conferred increased the survival of larvae exposed to Cry4Aa, confirming the involvement of ALP in Cry4Aa toxicity. PMID:26663676

  17. Promoting Uranium Immobilization by the Activities of Microbial Phosphatases

    International Nuclear Information System (INIS)

    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 PO43- 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 (micro)M dissolved uranium and that Rahnella is much more efficient in precipitating U(VI) than Bacillus sp. The

  18. Promoting Uranium Immobilization by the Activities of Microbial Phosphatases

    Energy Technology Data Exchange (ETDEWEB)

    Robert J. Martinez; Melanie J. Beazley; Samuel M. Webb; Martial Taillefert (co-PI); 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

  19. Ultrasensitive detection of cancer cells and glycan expression profiling based on a multivalent recognition and alkaline phosphatase-responsive electrogenerated chemiluminescence biosensor

    Science.gov (United States)

    Chen, Xiaojiao; He, Yao; Zhang, Youyu; Liu, Meiling; Liu, Yang; Li, Jinghong

    2014-09-01

    A multivalent recognition and alkaline phosphatase (ALP)-responsive electrogenerated chemiluminescence (ECL) biosensor for cancer cell detection and in situ evaluation of cell surface glycan expression was developed on a poly(amidoamine) (PAMAM) dendrimer-conjugated, chemically reduced graphene oxide (rGO) electrode interface. In this strategy, the multivalency and high affinity of the cell-targeted aptamers on rGO provided a highly efficient cell recognition platform on the electrode. The ALP and concanavalin A (Con A) coated gold nanoparticles (Au NPs) nanoprobes allowed the ALP enzyme-catalyzed production of phenols that inhibited the ECL reaction of Ru(bpy)32+ on the rGO electrode interface, affording fast and highly sensitive ECL cytosensing and cell surface glycan evaluation. Combining the multivalent aptamer interface and ALP nanoprobes, the ECL cytosensor showed a detection limit of 38 CCRF-CEM cells per mL in human serum samples, broad dynamic range and excellent selectivity. In addition, the proposed biosensor provided a valuable insight into dynamic profiling of the expression of different glycans on cell surfaces, based on the carbohydrates recognized by lectins applied to the nanoprobes. This biosensor exhibits great promise in clinical diagnosis and drug screening.A multivalent recognition and alkaline phosphatase (ALP)-responsive electrogenerated chemiluminescence (ECL) biosensor for cancer cell detection and in situ evaluation of cell surface glycan expression was developed on a poly(amidoamine) (PAMAM) dendrimer-conjugated, chemically reduced graphene oxide (rGO) electrode interface. In this strategy, the multivalency and high affinity of the cell-targeted aptamers on rGO provided a highly efficient cell recognition platform on the electrode. The ALP and concanavalin A (Con A) coated gold nanoparticles (Au NPs) nanoprobes allowed the ALP enzyme-catalyzed production of phenols that inhibited the ECL reaction of Ru(bpy)32+ on the rGO electrode

  20. The significance of amperometric detection of alkaline phosphatase in colorectal cancer diagnostics

    Science.gov (United States)

    Belkin, Anton; Freynd, Genrietta; Katsnelson, Mikhail

    2016-08-01

    Colorectal cancer (CRC) is one of the most common cancers in the world; it takes the second place in oncological morbidity. The ALP activity of intestinal epithelial differentiation marker (ALP) was investigated in surgical material and colon biopsies of 47 patients with the electrochemical method using biosensors (biochips). The average current obtained in the studies of colorectal cancer tissues was lower than in the studies of intact colon mucosa. Histology of tumors matched the differentiation types/stages of adenocarcinomas. The study of ALP activity in the surgical material and biopsies of colon tumors can become one of the most useful methods for evaluating the functional atypia in tumor tissue.

  1. Origin and production of phosphatases in the acid Lake Gardsjoen

    Energy Technology Data Exchange (ETDEWEB)

    Olsson, H.

    1983-01-01

    The activity of acid phosphatases was followed for one year in Lake Gardsjoen as well as in the inlet and the outlet of the lake. A budget of the phosphatases was calculated, including an estimation of the production of phosphatases. The phosphatase activity was also measured in two basins upstream of L. Gardsjoen: the north basin and the south basin of L. Stora Haestevatten. The acid phosphatase activity was very high compared with reported alkaline phosphatase activities in other lakes. About 95% of the phosphatases in L. Gardsjoen was produced in the lake, and the production was highest in early summer. Small Chrysophyceae (< 10 ..mu..m) probably produced the majority of the acid phosphatases in the investigated lakes, and accordingly could be favoured in environments with low phosphorus supply due to their ability to produce large amounts of phosphatases. 10 references, 8 figures, 2 tables.

  2. Orientation of mineral crystallites and mineral density during skeletal development in mice deficient in tissue nonspecific alkaline phosphatase.

    Science.gov (United States)

    Tesch, W; Vandenbos, T; Roschgr, P; Fratzl-Zelman, N; Klaushofer, K; Beertsen, W; Fratzl, P

    2003-01-01

    Tissue nonspecific alkaline phosphatase (TNALP) is thought to play an important role in mineralization processes, although its exact working mechanism is not known. In the present investigation we have studied mineral crystal characteristics in the developing skeleton of TNALP-deficient mice. Null mutants (n = 7) and their wild-type littermates (n = 7) were bred and killed between 8 and 22 days after birth. Skeletal tissues were processed to assess mineral characteristics (small angle X-ray scattering, quantitative backscattered electron imaging), and to analyze bone by light microscopy and immunolabeling. The results showed a reduced longitudinal growth and a strongly delayed epiphyseal ossification in the null mutants. This was accompanied by disturbances in mineralization pattern, in that crystallites were not orderly aligned with respect to the longitudinal axis of the cortical bone. Among the null mutants, a great variability in the mineralization parameters was noticed. Also, immunolabeling of osteopontin (OPN) revealed an abnormal distribution pattern of the protein within the bone matrix. Whereas in the wild-type animals OPN was predominantly observed in cement and reversal lines, in the null mutants, OPN was also randomly dispersed throughout the nonmineralized matrix, with focal densities. In contrast, the distribution pattern of osteocalcin (OC) was comparable in both types of animals. It is concluded that ablation of TNALP results not only in hypomineralization of the skeleton, but also in a severe disorder of the mineral crystal alignment pattern in the corticalis of growing long bone in association with a disordered matrix architecture, presumably as a result of impaired bone remodeling and maturation. PMID:12510812

  3. Construction of single chain Fv antibody against transferrin receptor and its protein fusion with alkaline phosphatase

    Institute of Scientific and Technical Information of China (English)

    Dao-Feng Yang; Hui-Fen Zhu; Zhi-Hua Wang; Guan-Xin Shen; De-Ying Tian

    2005-01-01

    AIM: To construct fusion protein of a single-chain antibody(scFv) against transferrin receptor (TfR) with alkalinephosphatase (AP).METHODS: The VH-linker-VL, namely scFv gene, wasprepared by amplifying the VH and VL genes from plasmid pGEM-T-VH and pGEM-T-VL with splicing overlap extension polymerase chain reaction (SOE PCR). After the ScFv gene was modified by SfiⅠ and NotⅠ, it was subcloned into the secretory expression vector pUC19/119, and then was transformed into E. coli TG1. The positive colonies were screened by colony PCR and their expressions were induced by IPTG. ScFv gene was gained by digesting ScFv expression vector pUC19/119 with Sfi I and NotⅠ restriction enzymes, then subcloned into expression vector pDAP2, followed by transformation in E. coli TG1. The positive colonies were selected by bacterial colony PCR. The expression of fusion protein (scFv-AP) was induced by IPTG. Its activity was detected by enzyme immunoassay. The molecular weights of scFv and scFv-AP were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).RESULTS: The product of SOE PCR formed a band of 700 bp in agarose gel electrophoresis. SDS-PAGE demonstrated the molecular weight of scFv was 27 ku. Immunofluorescent assay (IFA) demonstrated its reactivity with TfR. The molecular weight of scFv-AP was 75 ku. Enzyme immunoassay showed that scFv-AP could specifically bind to human TfR and play AP activity.CONCLUSION: We have successfully prepared the antihuman TfR scFv and constructed the fusion protein of scFv and AP. It is promising for immunological experiments.

  4. Bifunctional coating based on carboxymethyl chitosan with stable conjugated alkaline phosphatase for inhibiting bacterial adhesion and promoting osteogenic differentiation on titanium

    Science.gov (United States)

    Zheng, Dong; Neoh, Koon Gee; Kang, En-Tang

    2016-01-01

    In this work, alkaline phosphatase (ALP) was covalently immobilized on carboxymethyl chitosan (CMCS)-coated polydopamine (PDA)-functionalized Ti to achieve a bifunctional surface. Our results showed ∼89% reduction in Staphylococcus epidermidis adhesion on this surface compared to that on pristine Ti. The ALP-modified Ti supported cell proliferation, and significantly enhanced cellular ALP activity and calcium deposition of osteoblasts, human mesenchymal stem cells (hMSCs) and human adipose-derived stem cells (hADSCs). The extent of enhancement in the functions of these cells is dependent on the surface density of immobilized ALP. The substrate prepared using an ALP solution of 50 μg/cm2 resulted in 44%, 54% and 129% increase in calcium deposited by osteoblasts, hMSCs and hADSCs, respectively, compared to those cultured on pristine Ti. The ALP-modified substrates also promoted the osteogenic differentiation of hMSCs and hADSCs by up-regulating gene expressions of runt-related transcription factor 2 (RUNX2), osterix (OSX), and osteocalcin (OC) in the two types of stem cells. The surface-immobilized ALP was stable after being subjected to 1 h immersion in 70% ethanol and autoclaving at 121 °C for 20 min. However, the enzymatic bioactivity of the surface-immobilized ALP was reduced by about 50% after these substrates were immersed in phosphate buffered saline (PBS) or PBS containing lysozyme for 14 days.

  5. Development of a nanobody-alkaline phosphatase fusion protein and its application in a highly sensitive direct competitive fluorescence enzyme immunoassay for detection of ochratoxin A in cereal.

    Science.gov (United States)

    Liu, Xing; Xu, Yang; Wan, De-bin; Xiong, Yong-hua; He, Zhen-yun; Wang, Xian-xian; Gee, Shirley J; Ryu, Dojin; Hammock, Bruce D

    2015-01-20

    A rapid and sensitive direct competitive fluorescence enzyme immunoassay (dc-FEIA) for ochratoxin A (OTA) based on a nanobody (Nb)-alkaline phosphatase (AP) fusion protein was developed. The VHH (variable domain of heavy chain antibody) gene of Nb28 was subcloned into the expression vector pecan45 containing the AP double-mutant gene. The Nb28-AP construct was transformed into Escherichia coli BL21(DE3)plysS, and soluble expression in bacteria was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot. Both the Nb properties and AP enzymatic activity were validated by colorimetric and fluorometric analysis. The 50% inhibitory concentration and the detection limit of the dc-FEIA were 0.13 and 0.04 ng/mL, respectively, with a linear range of 0.06-0.43 ng/mL. This assay was compared with LC-MS/MS, and the results indicated the reliability of Nb-AP fusion protein-based dc-FEIA for monitoring OTA contamination in cereal.

  6. Second generation of pseudotype-based serum neutralization assay for Nipah virus antibodies: sensitive and high-throughput analysis utilizing secreted alkaline phosphatase.

    Science.gov (United States)

    Kaku, Yoshihiro; Noguchi, Akira; Marsh, Glenn A; Barr, Jennifer A; Okutani, Akiko; Hotta, Kozue; Bazartseren, Boldbaatar; Fukushi, Shuetsu; Broder, Christopher C; Yamada, Akio; Inoue, Satoshi; Wang, Lin-Fa

    2012-01-01

    Nipah virus (NiV), Paramyxoviridae, Henipavirus, is classified as a biosafety level (BSL) 4 pathogen, along with the closely related Hendra virus (HeV). A novel serum neutralization test was developed for measuring NiV neutralizing antibodies under BSL2 conditions using a recombinant vesicular stomatitis virus (VSV) expressing secreted alkaline phosphatase (SEAP) and pseudotyped with NiV F/G proteins (VSV-NiV-SEAP). A unique characteristic of this novel assay is the ability to obtain neutralization titers by measuring SEAP activity in supernatant using a common ELISA plate reader. This confers a remarkable advantage over the first generation of NiV-pseudotypes expressing green fluorescent protein or luciferase, which require expensive and specific measuring equipment. Using panels of NiV- and HeV-specific sera from various species, the VSV-NiV-SEAP assay demonstrated neutralizing antibody status (positive/negative) consistent with that obtained by conventional live NiV test, and gave higher antibody titers than the latter. Additionally, when screening sixty-six fruit bat sera at one dilution, the VSV-NiV-SEAP assay produced identical results to the live NiV test and only required a very small amount (2μl) of sera. The results suggest that this novel VSV-NiV-SEAP assay is safe, useful for high-throughput screening of sera using an ELISA plate reader, and has high sensitivity and specificity. PMID:22115786

  7. Differential Requirement for Pten Lipid and Protein Phosphatase Activity during Zebrafish Embryonic Development

    Science.gov (United States)

    Stumpf, Miriam; den Hertog, Jeroen

    2016-01-01

    The lipid- and protein phosphatase PTEN is one of the most frequently mutated tumor suppressor genes in human cancers and many mutations found in tumor samples directly affect PTEN phosphatase activity. In order to understand the functional consequences of these mutations in vivo, the aim of our study was to dissect the role of Pten phosphatase activities during zebrafish embryonic development. As in other model organisms, zebrafish mutants lacking functional Pten are embryonically lethal. Zebrafish have two pten genes and pten double homozygous zebrafish embryos develop a severe pleiotropic phenotype around 4 days post fertilization, which can be largely rescued by re-introduction of pten mRNA at the one-cell stage. We used this assay to characterize the rescue-capacity of Pten and variants with mutations that disrupt lipid, protein or both phosphatase activities. The pleiotropic phenotype at 4dpf could only be rescued by wild type Pten, indicating that both phosphatase activities are required for normal zebrafish embryonic development. An earlier aspect of the phenotype, hyperbranching of intersegmental vessels, however, was rescued by Pten that retained lipid phosphatase activity, independent of protein phosphatase activity. Lipid phosphatase activity was also required for moderating pAkt levels at 4 dpf. We propose that the role of Pten during angiogenesis mainly consists of suppressing PI3K signaling via its lipid phosphatase activity, whereas the complex process of embryonic development requires lipid and protein phosphatase of Pten. PMID:26848951

  8. Alkaline phosphatases and aminopeptidases are altered in a Cry11Aa resistant strain of Aedes aegypti.

    Science.gov (United States)

    Lee, Su-Bum; Aimanova, Karlygash G; Gill, Sarjeet S

    2014-11-01

    Bacillus thuringiensis subsp. israelensis (Bti) is widely used for the biological control of mosquito populations. However, the mechanism of Bti toxins is still not fully understood. To further elucidate the mechanism of Bti toxins, we developed an Aedes aegypti resistant strain that shows high-level resistance to Cry11Aa toxin. After 27 selections with Cry11Aa toxin, the larvae showed a 124-fold resistance ratio for Cry11Aa (strain G30). G30 larvae showed cross-resistance to Cry4Aa (66-fold resistance), less to Cry4Ba (13-fold), but not to Cry11Ba (2-fold). Midguts from these resistant larvae did not show detectable difference in the processing of the Cry11Aa toxin compared to that in susceptible larvae (WT). Brush border membrane vesicles (BBMV) from resistant larvae bound slightly less Cry11Aa compared to WT BBMV. To identify potential proteins associated with Cry11A resistance, not only transcript changes in the larval midgut were analyzed using Illumina sequencing and qPCR, but alterations of previously identified receptor proteins were investigated using immunoblots. The transcripts of 375 genes were significantly increased and those of 208 genes were down regulated in the resistant larvae midgut compared to the WT. None of the transcripts for previously identified receptors of Cry11Aa (Aedes cadherin, ALP1, APN1, and APN2) were altered in these analyses. The genes for the identified functional receptors in resistant larvae midgut did not contain any mutation in their sequences nor was there any change in their transcript expression levels compared to WT. However, ALP proteins were expressed at reduced levels (∼ 40%) in the resistant strain BBMV. APN proteins and their activity were also slightly reduced in resistance strain. The transcript levels of ALPs (AAEL013330 and AAEL015070) and APNs (AAEL008158, AAEL008162) were significantly reduced. These results strongly suggest that ALPs and APNs could be associated with Cry11Aa resistance in Ae. aegypti. PMID

  9. A bacterial tyrosine phosphatase inhibits plant pattern recognition receptor activation.

    Science.gov (United States)

    Macho, Alberto P; Schwessinger, Benjamin; Ntoukakis, Vardis; Brutus, Alexandre; Segonzac, Cécile; Roy, Sonali; Kadota, Yasuhiro; Oh, Man-Ho; Sklenar, Jan; Derbyshire, Paul; Lozano-Durán, Rosa; Malinovsky, Frederikke Gro; Monaghan, Jacqueline; Menke, Frank L; Huber, Steven C; He, Sheng Yang; Zipfel, Cyril

    2014-03-28

    Innate immunity relies on the perception of pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs) located on the host cell's surface. Many plant PRRs are kinases. Here, we report that the Arabidopsis receptor kinase EF-TU RECEPTOR (EFR), which perceives the elf18 peptide derived from bacterial elongation factor Tu, is activated upon ligand binding by phosphorylation on its tyrosine residues. Phosphorylation of a single tyrosine residue, Y836, is required for activation of EFR and downstream immunity to the phytopathogenic bacterium Pseudomonas syringae. A tyrosine phosphatase, HopAO1, secreted by P. syringae, reduces EFR phosphorylation and prevents subsequent immune responses. Thus, host and pathogen compete to take control of PRR tyrosine phosphorylation used to initiate antibacterial immunity.

  10. Observation on the activities of alpha-L-fucosidase,alkaline phosphatase and adenosine deaminase in pregnant women%妊娠女性α-L-岩藻糖苷酶、碱性磷酸酶和腺苷脱氨酶活性观察

    Institute of Scientific and Technical Information of China (English)

    王玮玮; 孙伟才; 周惠玉

    2014-01-01

    目的:妊娠期女性观察血清α-L-岩藻糖苷酶(AFU)、碱性磷酸酶(ALP)及腺苷脱氨酶(ADA)活性的变化及其与妊娠周期的相关性。方法分别检测352例体检正常的不同妊娠期女性(妊娠组,其中早期妊娠210例、晚期妊娠142例)及322例因不孕就诊且尚未妊娠的体检正常的女性(非妊娠组)血清 AFU、ALP、ADA 活性。对所有对象均进行动态监测(早期妊娠组于妊娠第14、22、30及38周分别检测,晚期妊娠组于妊娠第38及40周检测,非妊娠组于就诊时、就诊后第8及24周检测)并做比较。结果妊娠组血清 ALP、AFU 活性明显高于非妊娠组(P =0.000),ADA 活性则低于非妊娠组(P =0.000)。晚期妊娠组血清 ALP、AFU 活性明显高于早期妊娠组(P =0.000),但 ADA 活性两组间差异无统计学意义(P >0.05)。对早期妊娠组的动态监测显示,随着妊娠周期的增加,AFU 及 ALP 活性逐渐升高,但 ADA 基本无变化。晚期妊娠组第38周及第40周血清 AFU、ALP、ADA 活性差异均无统计学意义(P >0.05)。对非妊娠组的动态监测显示,与就诊时比较,就诊后第8周 ALP、ADA 活性差异有统计学意义(P =0.000),就诊后第24周 ADA 活性差异有统计学意义;与就诊后第8周比较,就诊后第24周 ALP、AFU、ADA 活性差异均有统计学意义(P <0.05)。结论妊娠期女性血清 AFU 及 ALP 活性明显升高,且随孕周的增加,升高趋势越明显。%Objective To observe the activity changes of alpha-L-fucosidase (AFU),alkaline phosphatase (ALP) and adenosine deaminase (ADA)in pregnant women and their correlations with pregnant period.Methods The activities of AFU,ALP and ADA were determined in 352 pregnant women (210 cases with early-stage pregnancy and 142 cases with late-stage pregnancy)and 322 unpregnant women.Dynamic monitoring was performed (early-stage pregnancy

  11. Advances on Characteristic and Application of Alkaline Phosphatase%碱性磷酸酶特性及其应用的研究进展

    Institute of Scientific and Technical Information of China (English)

    王秋颖

    2011-01-01

    Alkaline phosphatase(AP) is a kind of hydrolase,which catalyzes the nonspecific hydrolysis of phosphate monoester to produce phosphate or transfer phosphate. AP exists widely in microbial and animal kingdom. AP plays a vital important role in phosphate cycle of world creatures, and has been used widely in the fields of diagnostics, biochemistry and molecular biology. In this paper,the history, the reaction mechanism and application of alkaline phosphatase was reviewed.%碱性磷酸酶(alkaline pllosplaatase,AP)是一种非特异性磷酸单酯酶,能催化磷酸单酯的水解反应,产生无机磷酸和相应的醇、酚或糖,也能催化磷酸基团的转移反应.AP广泛存在于微生物和动物体内,在磷生物地球化学循环过程中有重要作用,并广泛应用于诊断学、生物化学及分子生物学等领域.作者对碱性磷酸酶的研究历史、催化作用机制及其应用进行了综述.

  12. Effects of Intercropping with Potato Onion on the Growth of Tomato and Rhizosphere Alkaline Phosphatase Genes Diversity

    Science.gov (United States)

    Wu, Xia; Wu, Fengzhi; Zhou, Xingang; Fu, Xuepeng; Tao, Yue; Xu, Weihui; Pan, Kai; Liu, Shouwei

    2016-01-01

    Background and Aims: In China, excessive fertilization has resulted in phosphorus (P) accumulation in most greenhouse soils. Intercropping can improve the efficiency of nutrient utilization in crop production. In this study, pot experiments were performed to investigate the effects of intercropping with potato onion (Allium cepa L. var. aggregatum G. Don) on tomato (Solanum lycopersicum L.) seedlings growth and P uptake, the diversity of rhizosphere phosphobacteria and alkaline phosphatase (ALP) genes in phosphorus-rich soil. Methods: The experiment included three treatments, namely tomato monoculture (TM), potato onion monoculture (OM), and tomato/potato onion intercropping (TI-tomato intercropping and OI-potato onion intercropping). The growth and P uptake of tomato and potato onion seedlings were evaluated. The dilution plating method was used to determine the population of phosphate-solubilizing bacteria (PSB) and phosphate-mineralizing bacteria (PMB). The genomic DNAs of PSB and PMB in the rhizosphere of tomato and potato onions were extracted and purified, and then, with the primer set of 338f /518r, the PCR amplification of partial bacterial 16S rDNA sequence was performed and sequenced to determine the diversities of PSB and PMB. After extracting the total genomic DNAs from the rhizosphere, the copy numbers and diversities of ALP genes were investigated using real-time PCR and PCR-DGGE, respectively. Results: Intercropping with potato onion promoted the growth and P uptake of tomato seedlings, but inhibited those of potato onion. After 37 days of transplanting, compared to the rhizosphere of TM, the soil pH increased, while the electrolytic conductivity and Olsen P content decreased (p < 0.05) in the rhizosphere of TI. The populations and diversities of PSB, PMB, and ALP genes increased significantly in the rhizosphere of TI, compared to the rhizosphere of TM. Conclusion: The results indicated that intercropping with potato onion promoted the growth and P

  13. INFLUENCE OF LIMING AND WASTE ORGANIC MATERIALS ON THE ACTIVITY OF PHOSPHATASE IN SOIL CONTAMINATED WITH NICKEL

    Directory of Open Access Journals (Sweden)

    Beata Kuziemska

    2014-10-01

    Full Text Available A study was carried out on soil following a two-year pot experiment that was conducted in 2009–2010, in three repetitions in Siedlce. The experiment included the following factors: 1 – amount of Ni in soil (0, 75, 150 and 225 mg·kg-1 soil by applying an aqueous NiSO4·7H2O solution; 2 – liming (0 and Ca according to 1 Hh as CaCO3; 3 – organic waste products (rye straw at a dose of 4 t·ha-1 and brown coal at a dose of 40 t·ha-1. In each experimental year, orchard grass was the test plant and four swaths were harvested. The activities of acidic and alkaline phosphatase, pH and the content of carbon in organic compounds were determined in the soil samples collected after each grass swath and in each experimental year. It was found that Ni at 75 mg·kg-1 soil activated the enzymes under study, whereas higher doses caused their statistically-confirmed inactivation. The lowest activity of the investigated enzymes was detected in soil supplemented with 225 Ni·kg-1 soil. Liming caused an increase in the activity of alkaline phosphatase and a reduction in the activity of acidic phosphatase. Straw and brown coal induced a substantial increase in the activity of both enzymes in the tested soil samples. Both liming and straw and carbon eliminated the negative effect of higher nickel doses on the activity of the enzymes under study.

  14. Patient pools and the use of "patient means" are valuable tools in quality control illustrated by a bone-specific alkaline phosphatase assay

    DEFF Research Database (Denmark)

    Hinge, Maja; Lund, Erik D.; Brandslund, Ivan;

    2016-01-01

    BACKGROUND: Quality control (QC) is an essential part of clinical biochemistry to ensure that laboratory test results are reliable and correct. Those tests without a defined reference method constitute a special challenge, as is the case with bone-specific alkaline phosphatase (BAP). METHODS...... AND RESULTS: The present study reports an example where a shift in a BAP assay was detected by use of a patient pool and supported by a retrospective calculation of "patient mean", while the external QC and specific assay control material were unaffected by the shift. CONCLUSIONS: Patient pools and the use...

  15. Down regulation of a gene for cadherin, but not alkaline phosphatase, associated with Cry1Ab resistance in the sugarcane borer Diatraea saccharalis.

    Directory of Open Access Journals (Sweden)

    Yunlong Yang

    Full Text Available The sugarcane borer, Diatraea saccharalis, is a major target pest of transgenic corn expressing Bacillus thuringiensis (Bt proteins (i.e., Cry1Ab in South America and the mid-southern region of the United States. Evolution of insecticide resistance in such target pests is a major threat to the durability of transgenic Bt crops. Understanding the pests' resistance mechanisms will facilitate development of effective strategies for delaying or countering resistance. Alterations in expression of cadherin- and alkaline phosphatase (ALP have been associated with Bt resistance in several species of pest insects. In this study, neither the activity nor gene regulation of ALP was associated with Cry1Ab resistance in D. saccharalis. Total ALP enzymatic activity was similar between Cry1Ab-susceptible (Cry1Ab-SS and -resistant (Cry1Ab-RR strains of D. saccharalis. In addition, expression levels of three ALP genes were also similar between Cry1Ab-SS and -RR, and cDNA sequences did not differ between susceptible and resistant larvae. In contrast, altered expression of a midgut cadherin (DsCAD1 was associated with the Cry1Ab resistance. Whereas cDNA sequences of DsCAD1 were identical between the two strains, the transcript abundance of DsCAD1 was significantly lower in Cry1Ab-RR. To verify the involvement of DsCAD1 in susceptibility to Cry1Ab, RNA interference (RNAi was employed to knock-down DsCAD1 expression in the susceptible larvae. Down-regulation of DsCAD1 expression by RNAi was functionally correlated with a decrease in Cry1Ab susceptibility. These results suggest that down-regulation of DsCAD1 is associated with resistance to Cry1Ab in D. saccharalis.

  16. 骨碱性磷酸酶与碱性磷酸酶在佝偻病诊断中的比较%Bone alkaline phosphatase versus serum alkaline phosphatase in the diagnosis of rickets

    Institute of Scientific and Technical Information of China (English)

    林岚; 陈力; 张春玲; 杨延萍; 肖玉联

    2011-01-01

    Objective To explore the significance of quantitative and qualitative measurements of bone alkaline phosphatase ( BAP-E and BAP-CIT ) and detection of serum AP in the diagnosis of rickets. Methods Serum AP level was measured, quantitative BAP was detected by ELISA, and qualitative BAP was determined by CIT in 55 children with rickets and 26 control subjects. Results AP level and BAP-E were ( 164.38 ± 23.87 ) U/L and ( 57.99 ± 7.67 ) U/L in controls, and were ( 209.50 ± 44.28 ) U/L, ( 80.38 ± 15.75 ) U/L, ( 206.06 ± 30.80 ) U/L, ( 78.05± 11.69) U/L, ( 1000.47 ± 464.65) U/L, and (283.22 ± 96.42) U/L in mild or severe rickets ,respectively. AP and BAP-E were elevated significantly in children with rickets ( P< 0.05 ), and there were significant differences between the severe groups and the mild groups ( P < 0.05 ). AP positively related with BAP-E ( r= 0.921, P< 0.01 ) and BAP-CIT ( r= 0.49, P< 0.01 ). In the control group,BAP-CIT within the normal range was only in one patient and that beyond the normal range was in the remaining patients. Conclusions AP and BAP-E are positively correlated with the severity of rickets.AP may be used in severe patients. BAP-CIT should be used carefully in the diagnosis of rickets.%目的 了解定量骨碱性磷酸酶( BAP-E)、定性骨碱性磷酸酶(BAP-CIT)、血清碱性磷酸酶(AP)三个指标在佝偻病诊断中的意义。方法 检测55例佝偻病患儿、26例对照婴幼儿血清AP、BAP-E、BAP-CIT,BAP-E采用ELISA法,BAP-CIT用全血干化学和免疫浓缩技术。结果 佝偻病患儿的AP和BAP-E较对照组(164.38 U/L±23.87 U/L,57.99 U/L±7.67 U/L)明显增高(P<0.05),中、重度组佝偻病患儿的AP和BAP-E( 1000.47 U/L±464.65U/L,283.22 U/L±96.42 U/L)较轻度组(209.50 U/L±44.28 U/L,80.38 U/L±15.75 U/L;206.06 U/L±30.80 U/L,78.05 U/L±11.69 U/L)明显增高(P<0.05),对照组BAP-CIT仅1例在试剂盒的正常范围≤200 U/L,余皆在异常范围(200-300) U/L,AP

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

    Science.gov (United States)

    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.

  18. Ultrasensitive electroanalysis of low-level free microRNAs in blood by maximum signal amplification of catalytic silver deposition using alkaline phosphatase-incorporated gold nanoclusters.

    Science.gov (United States)

    Si, Yanmei; Sun, Zongzhao; Zhang, Ning; Qi, Wei; Li, Shuying; Chen, Lijun; Wang, Hua

    2014-10-21

    An ultrasensitive sandwich-type analysis method has been initially developed for probing low-level free microRNAs (miRNAs) in blood by a maximal signal amplification protocol of catalytic silver deposition. Gold nanoclusters (AuNCs) were first synthesized and in-site incorporated into alkaline phosphatase (ALP) to form the ALP-AuNCs. Unexpectedly, the so incorporated AuNCs could dramatically enhance the catalysis activities of ALP-AuNCs versus native ALP. A sandwiched hybridization protocol was then proposed using ALP-AuNCs as the catalytic labels of the DNA detection probes for targeting miRNAs that were magnetically caught from blood samples by DNA capture probes, followed by the catalytic ligation of two DNA probes complementary to the targets. Herein, the ALP-AuNC labels could act as the bicatalysts separately in the ALP-catalyzed substrate dephosphorylation reaction and the AuNCs-accelerated silver deposition reaction. The signal amplification of ALP-AuNCs-catalyzed silver deposition was thereby maximized to be measured by the electrochemical outputs. The developed electroanalysis strategy could allow for the ultrasensitive detection of free miRNAs in blood with the detection limit as low as 21.5 aM, including the accurate identification of single-base mutant levels in miRNAs. Such a sandwich-type analysis method may circumvent the bottlenecks of the current detection techniques in probing short-chain miRNAs. It would be tailored as an ultrasensitive detection candidate for low-level free miRNAs in blood toward the diagnosis of cancer and the warning or monitoring of cancer metastasis in the clinical laboratory.

  19. Uranium Biomineralization by Natural Microbial Phosphatase Activities in the Subsurface

    Energy Technology Data Exchange (ETDEWEB)

    Sobecky, Patricia A. [Univ. of Alabama, Tuscaloosa, AL (United States)

    2015-04-06

    In this project, inter-disciplinary research activities were conducted in collaboration among investigators at The University of Alabama (UA), Georgia Institute of Technology (GT), Lawrence Berkeley National Laboratory (LBNL), Brookhaven National Laboratory (BNL), the DOE Joint Genome Institute (JGI), and the Stanford Synchrotron Radiation Light source (SSRL) to: (i) confirm that phosphatase activities of subsurface bacteria in Area 2 and 3 from the Oak Ridge Field Research Center result in solid U-phosphate precipitation in aerobic and anaerobic conditions; (ii) investigate the eventual competition between uranium biomineralization via U-phosphate precipitation and uranium bioreduction; (iii) determine subsurface microbial community structure changes of Area 2 soils following organophosphate amendments; (iv) obtain the complete genome sequences of the Rahnella sp. Y9-602 and the type-strain Rahnella aquatilis ATCC 33071 isolated from these soils; (v) determine if polyphosphate accumulation and phytate hydrolysis can be used to promote U(VI) biomineralization in subsurface sediments; (vi) characterize the effect of uranium on phytate hydrolysis by a new microorganism isolated from uranium-contaminated sediments; (vii) utilize positron-emission tomography to label and track metabolically-active bacteria in soil columns, and (viii) study the stability of the uranium phosphate mineral product. Microarray analyses and mineral precipitation characterizations were conducted in collaboration with DOE SBR-funded investigators at LBNL. Thus, microbial phosphorus metabolism has been shown to have a contributing role to uranium immobilization in the subsurface.

  20. Protein phosphatase-1 activates CDK9 by dephosphorylating Ser175.

    Directory of Open Access Journals (Sweden)

    Tatiana Ammosova

    Full Text Available The cyclin-dependent kinase CDK9/cyclin T1 induces HIV-1 transcription by phosphorylating the carboxyterminal domain (CTD of RNA polymerase II (RNAPII. CDK9 activity is regulated by protein phosphatase-1 (PP1 which was previously shown to dephosphorylate CDK9 Thr186. Here, we analyzed the effect of PP1 on RNAPII phosphorylation and CDK9 activity. The selective inhibition of PP1 by okadaic acid and by NIPP1 inhibited phosphorylation of RNAPII CTD in vitro and in vivo. Expression of the central domain of NIPP1 in cultured cells inhibited the enzymatic activity of CDK9 suggesting its activation by PP1. Comparison of dephosphorylation of CDK9 phosphorylated by ((32P in vivo and dephosphorylation of CDK9's Thr186 analyzed by Thr186 phospho-specific antibodies, indicated that a residue other than Thr186 might be dephosphorylated by PP1. Analysis of dephosphorylation of phosphorylated peptides derived from CDK9's T-loop suggested that PP1 dephosphorylates CDK9 Ser175. In cultured cells, CDK9 was found to be phosphorylated on Ser175 as determined by combination of Hunter 2D peptide mapping and LC-MS analysis. CDK9 S175A mutant was active and S175D--inactive, and dephosphorylation of CDK9's Ser175 upregulated HIV-1 transcription in PP1-dependent manner. Collectively, our results point to CDK9 Ser175 as novel PP1-regulatory site which dephosphorylation upregulates CDK9 activity and contribute to the activation of HIV-1 transcription.

  1. Treatment of PCR products with exonuclease I and heat-labile alkaline phosphatase improves the visibility of combined bisulfite restriction analysis

    International Nuclear Information System (INIS)

    Research highlights: → Incubating PCR products at a high temperature causes smears in gel electrophoresis. → Smears interfere with the interpretation of methylation analysis using COBRA. → Treatment with exonuclease I and heat-labile alkaline phosphatase eliminates smears. → The elimination of smears improves the visibility of COBRA. -- Abstract: DNA methylation plays a vital role in the regulation of gene expression. Abnormal promoter hypermethylation is an important mechanism of inactivating tumor suppressor genes in human cancers. Combined bisulfite restriction analysis (COBRA) is a widely used method for identifying the DNA methylation of specific CpG sites. Here, we report that exonuclease I and heat-labile alkaline phosphatase can be used for PCR purification for COBRA, improving the visibility of gel electrophoresis after restriction digestion. This improvement is observed when restriction digestion is performed at a high temperature, such as 60 oC or 65 oC, with BstUI and TaqI, respectively. This simple method can be applied instead of DNA purification using spin columns or phenol/chloroform extraction. It can also be applied to other situations when PCR products are digested by thermophile-derived restriction enzymes, such as PCR restriction fragment length polymorphism (RFLP) analysis.

  2. Treatment of PCR products with exonuclease I and heat-labile alkaline phosphatase improves the visibility of combined bisulfite restriction analysis

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, Kousuke; Emoto, Noriko; Sunohara, Mitsuhiro; Kawakami, Masanori; Kage, Hidenori; Nagase, Takahide; Ohishi, Nobuya [Department of Respiratory Medicine, The University of Tokyo Hospital, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan); Takai, Daiya, E-mail: dtakai-ind@umin.ac.jp [Department of Clinical Laboratory, The University of Tokyo Hospital, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan)

    2010-08-27

    Research highlights: {yields} Incubating PCR products at a high temperature causes smears in gel electrophoresis. {yields} Smears interfere with the interpretation of methylation analysis using COBRA. {yields} Treatment with exonuclease I and heat-labile alkaline phosphatase eliminates smears. {yields} The elimination of smears improves the visibility of COBRA. -- Abstract: DNA methylation plays a vital role in the regulation of gene expression. Abnormal promoter hypermethylation is an important mechanism of inactivating tumor suppressor genes in human cancers. Combined bisulfite restriction analysis (COBRA) is a widely used method for identifying the DNA methylation of specific CpG sites. Here, we report that exonuclease I and heat-labile alkaline phosphatase can be used for PCR purification for COBRA, improving the visibility of gel electrophoresis after restriction digestion. This improvement is observed when restriction digestion is performed at a high temperature, such as 60 {sup o}C or 65 {sup o}C, with BstUI and TaqI, respectively. This simple method can be applied instead of DNA purification using spin columns or phenol/chloroform extraction. It can also be applied to other situations when PCR products are digested by thermophile-derived restriction enzymes, such as PCR restriction fragment length polymorphism (RFLP) analysis.

  3. The synthesis of Phosphate-repressible alkaline phosphatase do not appear to be regulated by ambient pH in the filamentous mould Neurospora crassa

    Directory of Open Access Journals (Sweden)

    Nozawa Sérgio R.

    2002-01-01

    Full Text Available In order to investigate further the adaptive response of moulds to ambient pH, we have measured by ELISA the pho-2-encoded Pi-repressible alkaline phosphatase synthesised by Neurospora crassa. We showed that the 74A and pho-2A strains of this mould secrete similar amounts of the pho-2-encoded enzyme irrespective of ambient pH, when both the preg and pgov genes are not functional, i.e., in strains nuc-2+ growing under Pi-starvation. This suggests that pho-2, which is responsive to Pi starvation via the action of genes nuc-2, preg, pgov and nuc-1, is not a gene responsive to ambient pH and that the differential glycosylation observed for the Pi-repressible alkaline phosphatase retained by the mycelium at pH 5.6 or secreted into the growth medium at pH 8.0 is the genetic response to ambient pH sensing in N. crassa.

  4. Effects of TNF-α on the proliferation and alkaline phosphatase of the human dental follicle cells%TNF-α对体外培养的人牙囊细胞增殖特性及碱性磷酸酶的影响

    Institute of Scientific and Technical Information of China (English)

    毕迎春; 李芸; 金作林

    2012-01-01

    Objective To study the effects of different concentration TNF-a on the activity of proliferation and the activity of alkaline phosphatase of human dental follicle cells. Methods The 5th passage human dental follicle cells were seeded on 96-well plates and co-cultured with different concentration of TNF-a, the activity of proliferation and the activity of alkaline phosphatase was assayed. Results The 10~50ng/ml TNF-a enhanced the activity of proliferation profoundly and the effects were significant at 3 days after co-culture. 10~200ng/ml TNF-a decreased the activity of alkaline phosphatase. Conclusions TNF-a enhance the activity of proliferation and decreased the activity of alkaline phosphatase of human dental follicle cells at special concentration and time.%目的:检测不同浓度TNF-对体外培养人牙囊细胞增殖活性和碱性磷酸酶活性的影响.方法:第 5代人牙囊细胞接种于96孔培养板上,分别与不同浓度的TNF-共同孵育,检测TNF-对人牙囊细胞的增殖活性和碱性磷酸酶活性的影响. 结果:TNF-在浓度为10~ 50ng/ml,时间为3天时促进人牙囊细胞的增殖,浓度为10~ 200ng/ml时抑制碱性磷酸酶活性.结论:TNF-在特定的浓度和时间促进人牙囊细胞的增殖,抑制牙囊细胞的成骨特性.

  5. Fluorescence labelling of phosphatase activity in digestive glands of carnivorous plants.

    Science.gov (United States)

    Płachno, B J; Adamec, L; Lichtscheidl, I K; Peroutka, M; Adlassnig, W; Vrba, J

    2006-11-01

    A new ELF (enzyme labelled fluorescence) assay was applied to detect phosphatase activity in glandular structures of 47 carnivorous plant species, especially Lentibulariaceae, in order to understand their digestive activities. We address the following questions: (1) Are phosphatases produced by the plants and/or by inhabitants of the traps? (2) Which type of hairs/glands is involved in the production of phosphatases? (3) Is this phosphatase production a common feature among carnivorous plants or is it restricted to evolutionarily advanced species? Our results showed activity of the phosphatases in glandular structures of the majority of the plants tested, both from the greenhouse and from sterile culture. In addition, extracellular phosphatases can also be produced by trap inhabitants. In Utricularia, activity of phosphatase was detected in internal glands of 27 species from both primitive and advanced sections and different ecological groups. Further positive reactions were found in Genlisea, Pinguicula, Aldrovanda, Dionaea, Drosera, Drosophyllum, Nepenthes, and Cephalotus. In Utricularia and Genlisea, enzymatic secretion was independent of stimulation by prey. Byblis and Roridula are usually considered as "proto-carnivores", lacking digestive enzymes. However, we found high activity of phosphatases in both species. Thus, they should be classified as true carnivores. We suggest that the inflorescence of Byblis and some Pinguicula species might also be an additional "carnivorous organ", which can trap a prey, digest it, and finally absorb available nutrients. PMID:16865659

  6. Effects of Lanthanum and Cerium on Acid Phosphatase Activities in Two Soils

    Institute of Scientific and Technical Information of China (English)

    徐冬梅; 刘广深; 徐杰; 刘维屏

    2004-01-01

    To evaluate the security of using thulium,comparision between effects of La and those of Ce on acidic phosphatase activities in red soil and yellow soil in Zhejiang district was studied under conditions of ambient temperature and humidity. Results show that the acid phosphatase from different soil respondes to La and Ce differently. The activity of acid phosphatase in soil 1 declines with the increase of the concentration of La and Ce. The maximum inhibitory ratio of La and Ce reaches 69.8% and 71.0% respectively. But La and Ce have stimulative effect on the activity of acid phosphatase in soil 2. Under the effect of same concentration of the thulium,the acid phosphatase in two soils increases with the extending of culture time.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-04-01

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

  8. Extracellular Matrix Proteins, Alkaline Phosphatase and Pyrophosphate as Molecular Determinants of Bone, Tooth, Kidney and Vascular Calcification

    Science.gov (United States)

    McKee, Marc D.

    2008-09-01

    Progress in biomineralization research in recent years has identified, characterized and described functions for key noncollagenous extracellular matrix proteins regulating crystal growth in the skeleton and dentition. Some of these same proteins expressed in soft tissues undergoing pathologic calcification also inhibit ectopic crystal growth. In addition to extracellular matrix proteins regulating matrix mineralization, the enzyme tissue-nonspecific alkaline phosphatase—which is highly expressed by cells in mineralized tissues—cleaves pyrophosphate, an anionic small-molecule inhibitor of mineralization. Together with the required mineral ion availability necessary for crystal growth, these molecular determinants appear to function in limiting the spread of pathologic calcification seen in soft tissues such as blood vessels and kidneys. Osteopontin, in particular, is a potent calcification inhibitor that accumulates in mineralized tissues and in calcified deposits during vascular calcification and nephrolithiasis/urolithiasis. Additional research is required to establish the exact temporal sequence in which the molecular determinants of pathologic calcification appear relative to mineral crystal growth in different tissues, and to establish their relationship (if any) to the activation of osteogenic differentiation programs.

  9. The content of macro- and microelements and the phosphatase activity of soils under a varied plant cultivation technology

    Science.gov (United States)

    Bartkowiak, A.; Lemanowicz, J.; Kobierski, M.

    2015-12-01

    The paper presents the results of the analyses of selected physicochemical properties and the activity of alkaline and acid phosphatase in the soils which differed in terms of plant cultivation technology. Profile sI represented arable land in the crop rotation with cereals dominating (medium intensive technology), without irrigation, while profile sII—represented arable land with vegetable crops cultivation (intensive technology), intensively fertilized and irrigated. The content of available phosphorus in the two soil profiles investigated ranged from 6.6 to 69.1 mg/kg. The highest contents of phosphorus available to plants were reported in the plough horizon of both soils, while the abundance of potassium and magnesium was highest in the illuvial horizon of both soils. The soil profiles investigated showed a significant variation in terms of the cultivation technologies applied. The contents of plant-available Cu and Zn in soil were low and they resulted in the inhibition of neither alkaline nor acid phosphatase. The intensive vegetable crops cultivation technology decreased the content of organic matter and increased the content of the nutrients in soil. Using the Ward method, it was found that relatively similar physicochemical and chemical properties were reported for the genetic horizons of both soil profiles, especially Ap horizon of the soil representing arable land with intensive cultivation of vegetable crops.

  10. Calcineurin phosphatase activity and immunosuppression. A review on the role of calcineurin phosphatase activity and the immunosuppressive effect of cyclosporin A and tacrolimus

    DEFF Research Database (Denmark)

    Jørgensen, Kaj Anker; Koefoed-Nielsen, P.B.; Karamperis, N.

    2003-01-01

    The mode of immunosuppressive action of tacrolimus (FK506) and cyclosporin A has been elucidated. Both drugs bind to proteins in the cytoplasm to form complexes, which in turn inhibit the phosphatase activity of calcineurin, an important limiting step in the activation of T cells. The association...... between drug uptake (pharmacokinetics) and enzyme inhibition (pharmacodynamics) is under current investigation. Great variations in the correlation between blood drug levels and enzyme inhibition could indicate that monitoring calcineurin phosphatase activity for treatment might be superior to monitoring...... blood drug levels Udgivelsesdato: 2003/2...

  11. Microchannel conductivity measurements in microchip for on line monitoring of dephosphorylation rates of organic phosphates using paramagnetic-beads linked alkaline phosphatase.

    Science.gov (United States)

    Kechadi, Mohammed; Sotta, Bruno; Gamby, Jean

    2015-01-01

    This paper presents the use of polymer coated microelectrodes for the realtime conductivity monitoring in a microchannel photoablated through the polymer without contact. Based on this strategy, a small conductometry sensor has been developed to record in time conductivity variation when an enzymatic reaction occurs through the channel. The rate constant determination, k2, for the dephosphorylation of organic phosphate-alkaline phosphatase-superparamagnetic beads complex using chemically different substrates such as adenosine monoesterphosphate, adenosine diphosphate and adenosine triphosphate was taken as an example to demonstrate selectivity and sensivity of the detection scheme. The k2 value measured for each adenosine phosphate decreases from 39 to 30 s(-1) in proportion with the number (3, 2 and 1) of attached phosphate moiety, thus emphasizing the steric hindrance effect on kinetics.

  12. Comparison of measurements of canine plasma creatinine, glucose, proteins, urea, alanine aminotransferase, and alkaline phosphatase obtained with Spotchem SP 4430 and Vitros 250 analyzers.

    Science.gov (United States)

    Trumel, C; Diquélou, A; Germain, C; Palanché, F; Braun, J P

    2005-12-01

    The suitability of the Spotchem 4430 benchtop biochemistry analyzer for canine blood samples was tested for creatinine, glucose, proteins, urea, alkaline phosphatases and alanine aminotransferase. Results obtained from whole blood and corresponding heparin plasma were identical except for proteins which were higher in plasma (n=10). Between series imprecision (n=10) was 0.93). The slopes of the Passing-Bablock's regression ranged from 0.90 to 1.20 and intercepts were low. The mean biases were low, except for creatinine for which the results obtained by Spotchem (Jaffe reaction) were about 20 micromol/L higher than with the Vitros (enzymatic reaction). The results of this study show that the Spotchem analyzer is suitable for use in canine whole blood or plasma when small numbers of tests are to be performed and large analyzers are not available. PMID:16054888

  13. Multiwalled carbon nanotube modified screen-printed electrodes for the detection of p-aminophenol: Optimisation and application in alkaline phosphatase-based assays

    Energy Technology Data Exchange (ETDEWEB)

    Lamas-Ardisana, Pedro Jose [Departamento de Quimica Fisica y Analitica, Universidad de Oviedo, 33006 Oviedo, Asturias (Spain); Queipo, Paula [Departamento de Fisica, Universidad de Oviedo, 33007 Oviedo, Asturias (Spain); Fanjul-Bolado, Pablo [Departamento de Quimica Fisica y Analitica, Universidad de Oviedo, 33006 Oviedo, Asturias (Spain); Costa-Garcia, Agustin [Departamento de Quimica Fisica y Analitica, Universidad de Oviedo, 33006 Oviedo, Asturias (Spain)], E-mail: costa@fq.uniovi.es

    2008-05-12

    Carboxylated multiwalled carbon nanotubes (MWCNT-COOH) were used to modify the working electrode surface of different screen-printed electrodes. The effect of this modification on the electrodic characteristics (double layer capacitance, electroactive area and heterogeneous rate constants for the electron transfer) was evaluated and optimized for the cyclic voltammetric determination of p-aminophenol. The enzymatic hydrolysis of p-aminophenylphosphate was employed for the quantification of alkaline phosphatase, one of the most important label enzymes in immunoassays. Finally, ELISA assays were carried out to quantify pneumolysin using this enzymatic system. Results obtained indicated that low superficial densities of MWCNT-COOH (0.03-0.06 {mu}g mm{sup -2}) yielded the same electrodic improvements but with better analytical properties.

  14. ZZ亲和肽-碱性磷酸酶融合蛋白的制备与初步应用%Preparation of fusion protein between ZZ affinity peptide and alkaline phosphatase and its primary application

    Institute of Scientific and Technical Information of China (English)

    鲍如梦; 赵爽佳; 薛敏; 杨洪鸣; 唐金宝

    2015-01-01

    目的:构建、表达ZZ亲和肽与碱性磷酸酶( AP)融合蛋白,并研究其生物学活性。方法:将ZZ亲和肽基因与AP基因重组,构建原核表达载体pEZZ-AP并利用大肠杆菌表达,金属离子亲和层析纯化目的蛋白,Western blot鉴定ZZ-AP生物学活性并初步应用于免疫细胞化学。结果:所构建重组质粒pEZZ-AP 经E.coli DH5α表达,SDS-PAGE结果显示目的蛋白相对分子量为62 kD,与理论值相近。 Western blot结果表明ZZ-AP融合蛋白既具有兔IgG抗体结合活性,又具有碱性磷酸酶活性,在细胞免疫组化应用中与AP标记的羊抗兔二抗显色模式与效果相似。结论:ZZ-AP融合蛋白构建成功,该融合蛋白具有兔IgG 抗体结合活性和碱性磷酸酶活性,细胞免疫组化应用结果表明该融合蛋白在免疫分析中具有潜在的应用价值。%Objective:To construct and express the fusion protein between ZZ affinity peptide and alkaline phosphatase and examine its biological activities.Methods:The alkaline phosphatase gene was cloned into pEZZ 18 vector containing ZZ peptide gene resulted in the pEZZ-AP recombinant vector.Then the vector was transformed and expressed in E.coli DH5α.And HisTrap affinity chromatography was employed to separate and purify the target protein.After analyzed by Western blot , ZZ-AP fusion protein was applied to immunocytochemistry as an alterative second antibody.Results:The result of SDS-PAGE showed that the fusion protein with a relative molecular weight was consistent with the theoretical values (62 kD).The fusion protein has rabbit IgG-binding ability and en-zymatic activity of alkaline phosphatase ,those were validated in Western blot;and it produced a good signal that was comparable in its staining pattern to that generated with goat anti-rabbit IgG-AP in immunocytochemistry.Conclusion: The ZZ-AP fusion protein was constructed successfully ,it has rabbit IgG-binding ability and enzymatic

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

    Directory of Open Access Journals (Sweden)

    Janetti R Signorelli

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

  16. Loss of skeletal mineralization by the simultaneous ablation of PHOSPHO1 and alkaline phosphatase function: a unified model of the mechanisms of initiation of skeletal calcification.

    Science.gov (United States)

    Yadav, Manisha C; Simão, Ana Maria Sper; Narisawa, Sonoko; Huesa, Carmen; McKee, Marc D; Farquharson, Colin; Millán, José Luis

    2011-02-01

    Endochondral ossification is a carefully orchestrated process mediated by promoters and inhibitors of mineralization. Phosphatases are implicated, but their identities and functions remain unclear. Alkaline phosphatase (TNAP) plays a crucial role promoting mineralization of the extracellular matrix by restricting the concentration of the calcification inhibitor inorganic pyrophosphate (PP(i)). Mutations in the TNAP gene cause hypophosphatasia, a heritable form of rickets and osteomalacia. Here we show that PHOSPHO1, a phosphatase with specificity for phosphoethanolamine and phosphocholine, plays a functional role in the initiation of calcification and that ablation of PHOSPHO1 and TNAP function prevents skeletal mineralization. Phospho1(-/-) mice display growth plate abnormalities, spontaneous fractures, bowed long bones, osteomalacia, and scoliosis in early life. Primary cultures of Phospho1(-/-) tibial growth plate chondrocytes and chondrocyte-derived matrix vesicles (MVs) show reduced mineralizing ability, and plasma samples from Phospho1(-/-) mice show reduced levels of TNAP and elevated plasma PP(i) concentrations. However, transgenic overexpression of TNAP does not correct the bone phenotype in Phospho1(-/-) mice despite normalization of their plasma PP(i) levels. In contrast, double ablation of PHOSPHO1 and TNAP function leads to the complete absence of skeletal mineralization and perinatal lethality. We conclude that PHOSPHO1 has a nonredundant functional role during endochondral ossification, and based on these data and a review of the current literature, we propose an inclusive model of skeletal calcification that involves intravesicular PHOSPHO1 function and P(i) influx into MVs in the initiation of mineralization and the functions of TNAP, nucleotide pyrophosphatase phosphodiesterase-1, and collagen in the extravesicular progression of mineralization.

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

    OpenAIRE

    W. Huang; 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 organic P mineralization potential in soils. In order to study the effects of precipitation on soil acid phosphatase activity, an experiment with precipitation t...

  18. Multiple myeloma: Changes in serum C-terminal telopeptide of collagen type I and bone-specific alkaline phosphatase can be used in daily practice to detect imminent osteolysis

    DEFF Research Database (Denmark)

    Lund, Thomas; Abildgaard, Niels; Andersen, Thomas L;

    2010-01-01

    of collagen type-I (CTX-I), C-terminal crosslinked telopeptide of type-I collagen generated by MMPs (ICTP), N-terminal crosslinked telopeptide of type-I collagen (NTX-I), and the bone formation marker bone-specific alkaline phosphatase (bALP) monthly for two years. Retrospectively, we identified 40 cases...

  19. Production and characterization of a single-chain variable fragment linked alkaline phosphatase fusion protein for detection of O,O-diethyl organophosphorus pesticides in a one-step enzyme-linked immunosorbent assay

    Science.gov (United States)

    A single-chain variable fragment (scFv) and alkaline phosphatase (AP) fusion protein for detection of O, O-diethyl organophosphorus pesticides (OPs) was produced and characterized. The scFv gene was prepared by cloning VL and VH genes from a hybridoma cell secreting monoclonal antibody with broad-s...

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

    Directory of Open Access Journals (Sweden)

    Norris-Mullins Brianna

    2014-01-01

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

  1. Analysis of detection results of bone alkaline phosphatase in 2 184 children in Baoji area%宝鸡地区2184例儿童骨碱性磷酸酶结果的分析

    Institute of Scientific and Technical Information of China (English)

    乔正梅; 葛君琍; 赵秋剑; 张利强

    2014-01-01

    目的:评价骨碱性磷酸酶对儿童佝偻病早期诊断及疗效评价的临床价值,通过骨碱性磷酸酶的检测了解该地区儿童佝偻病的发病率。方法选择2013年1~12月来宝鸡市中心医院体检的2184例儿童,按年龄分成婴儿、幼儿、学龄前、学龄期4组,采集指血,采用北京中生金域诊断技术有限公司提供的骨源性碱性磷酸酶试剂盒检测末梢血中骨碱性磷酸酶水平。结果2184例受检儿童中,临床佝偻病(NBAP>250 U/L)的检出率为10.1%,且年龄越小检出率越高;亚临床佝偻病(NBAP 200~250 U/L)的检出率为65.5%,说明宝鸡地区大部分儿童处于亚健康状态;春冬季检出率高于夏秋季;NBAP 检出率无性别差异。结论儿童为生长发育较快时期,易引起维生素 D 缺乏,儿童骨碱性磷酸酶活性检测对维生素 D 缺乏引起的佝偻病的早期诊断有重要的临床价值。预防应从围产期开始,及时检测儿童骨碱性磷酸酶水平,以便早诊断、早治疗。%Objective To evaluate the clinical value of bone alkaline phosphatase in early diagnosis and treatment effect of chil-dren′rickets and to understand the incidence rate of rickets in children in the local region by the detection of neonatal bone alkaline phosphatase(NBAP).Methods 2 184 children with physical examination in our hospital from January to December 2013 were se-lected and divided into the infants,young children,preschool and school-age children groups according to age.The finger blood was collected for detecting the bone alkaline phosphatase in the peripheral blood by the bone alkaline phosphatase reagent kit provided by Beijing Jinyu diagnosis technology Co.,Ltd.Results Among detected 2 184 children,the detection rate of clinical rickets(NBAP>250 U/L)was 10.1 %,moreover the smaller the age,the higher the detection rate;the detection rate of subclinical rickets(NBAP 200~250 U/L)was 65

  2. Alkaline activation of ceramic waste materials

    OpenAIRE

    REIG CERDÁ, LUCÍA; Tashima, M. M.; Soriano, L.; Borrachero, M. V.; Monzó, J.; Payá, J.

    2013-01-01

    Ceramic materials represent around 45 % of construction and demolition waste, and originate not only from the building process, but also as rejected bricks and tiles from industry. Despite the fact that these wastes are mostly used as road sub-base or construction backfill materials, they can also be employed as supplementary cementitious materials, or even as raw material for alkali-activated binders This research aimed to investigate the properties and microstructure of alkali-activated cem...

  3. Single and Combined Effects of As (III) and Acetochlor on Phosphatase Activity in Soil

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yun; ZHANG Feng; ZHANG Guan-cai; GUAN Lian-zhu

    2013-01-01

    The actions and interactions of acetochlor and As on the soil phosphatase activity were investigated after 1, 3, 6, 10, 15, 30 and 60 d of exposure under control conditions. The soils were exposed to various concentrations of acetochlor and As individually and simultaneously. The results showed that acetochlor, As only, and combined pollution all clearly inhibited soil phosphatase activity. The maximum inhibition ratios of soil phosphatase activity by acetochlor, As only and combined pollution were 36.44, 74.12 and 61.29%, respectively. Two kinetic models,ν=c/(1+bi) (model 1) andν=c(1+ai)/(l+bi) (model 2), were used to describe the relationship between the concentrations of As and acetochlor and the activity of soil phosphatase. The semi-effect dose (ED50) values induced by As and acetochlor stress based on the inhibition of soil phosphatase were 18.1 and 33.11 mg kg-1, respectively, according to calculation by model 1. The interactive effect of acetochlor with As on soil phosphatase primarily consisted of significant antagonism effects at the higher concentrations tested. The step regression results show that the toxicity order was As (III)>acetochlor>As (III)×acetochlor throughout the incubation period.

  4. Application of Alcohol Soluble Fixing Solution in Alkaline Phosphatase Staining%醇溶性固定液在碱性磷酸酶染色中的应用

    Institute of Scientific and Technical Information of China (English)

    陈敬文; 张伟; 崔华娟; 陈晓东; 赖续文

    2011-01-01

    目的 探讨醇溶性固定液在碱性磷酸酶 (alkaline phosphatase,ALP)染色中的应用.方法 将60例新鲜标本各取2块组织设为A、B两组,A组冷丙酮固定,B组醇溶性固定液固定,两组均常规石蜡切片,同时设立对照组,进行碱性磷酸酶钙-钴法染色,并对染色结果 进行观察.结果 两组切片均可见黑色碱性磷酸酶存在,但B组的阳性强度明显高于A组,对照组呈阴性反应.结论 醇溶性固定液在对碱性磷酸酶的固定效果和显示方面,均优于冷丙酮固定液.%Objective To explore the application of alcohol soluble fixing solution in alkaline phosphatase staining.Methods Two tissues in each of 60 fresh samples were taken and assigned as group A and group B fixed by cold acetone and alcohol soluble fixing solution respectively.Conventional paraffin slice were obtained for the two groups and the corresponding control group was set.All groups underwent alkaline phosphatase Ca-Co staining.Results Black alkaline phosphatase were observed in groups A and B, the positive intensity in group B was higher, and the control group was negative.Conclusion Alcohol soluble fixing solution is better than cold acetone in fixing and staining alkaline phosphatase.

  5. Generic phosphatase activity detection using zinc mediated aggregation modulation of polypeptide-modified gold nanoparticles

    Science.gov (United States)

    Selegård, Robert; Enander, Karin; Aili, Daniel

    2014-11-01

    A challenge in the design of plasmonic nanoparticle-based colorimetric assays is that the change in colloidal stability, which generates the colorimetric response, is often directly linked to the biomolecular recognition event. New assay strategies are hence required for every type of substrate and enzyme of interest. Here, a generic strategy for monitoring of phosphatase activity is presented where substrate recognition is completely decoupled from the nanoparticle stability modulation mechanism, which enables detection of a wide range of enzymes using different natural substrates with a single simple detection scheme. Phosphatase activity generates inorganic phosphate that forms an insoluble complex with Zn2+. In a sample containing a preset concentration of Zn2+, phosphatase activity will markedly reduce the concentration of dissolved Zn2+ from the original value, which in turn affects the aggregation of gold nanoparticles functionalized with a designed Zn2+ responsive polypeptide. The change in nanoparticle stability thus provides a rapid and sensitive readout of the phosphatase activity. The assay is not limited to a particular enzyme or enzyme substrate, which is demonstrated using three completely different phosphatases and five different substrates, and thus constitutes a highly interesting system for drug screening and diagnostics.A challenge in the design of plasmonic nanoparticle-based colorimetric assays is that the change in colloidal stability, which generates the colorimetric response, is often directly linked to the biomolecular recognition event. New assay strategies are hence required for every type of substrate and enzyme of interest. Here, a generic strategy for monitoring of phosphatase activity is presented where substrate recognition is completely decoupled from the nanoparticle stability modulation mechanism, which enables detection of a wide range of enzymes using different natural substrates with a single simple detection scheme

  6. Activation of ERK induces phosphorylation of MAPK phosphatase-7, a JNK specific phosphatase, at Ser-446.

    Science.gov (United States)

    Masuda, Kouhei; Shima, Hiroshi; Katagiri, Chiaki; Kikuchi, Kunimi

    2003-08-22

    We previously showed that MKP-7 suppresses MAPK activation in COS-7 cells in the order of selectivity, JNK > p38 > ERK, but interacts with ERK as well as JNK and p38. In this study we found that, when expressed in COS-7 cells with HA-ERK2, the mobility of FLAG-MKP-7 was decreased on SDS-PAGE gels depending on several stimuli, including phorbol 12-myristate 13-acetate, fetal bovine serum, epidermal growth factor, H2O2, and ionomycin. By using U0126, a MEK inhibitor, and introducing several point mutations, we demonstrated that this upward mobility shift is because of phosphorylation and identified Ser-446 of MKP-7 as the phosphorylation site targeted by ERK activation. To determine how MKP-7 interacts with MAPKs, we identified three domains in MKP-7 required for interaction with MAPKs, namely, putative MAP kinase docking domains (D-domain) I and II and a long COOH-terminal stretch unique to MKP-7. The D-domain I is required for interaction with ERK and p38, whereas the D-domain II is required for interaction with JNK and p38, which is likely to be important for MKP-7 to suppress JNK and p38 activations. The COOH-terminal stretch of MKP-7 was shown to determine JNK preference for MKP-7 by masking MKP-7 activity toward p38 and is a domain bound by ERK. These data strongly suggested that Ser-446 of MKP-7 is phosphorylated by ERK. PMID:12794087

  7. Promoting Uranium Immobilization by the Activities of Microbial Phosphatases

    Energy Technology Data Exchange (ETDEWEB)

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

    2005-04-05

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

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  9. Alkalosis and Dialytic Clearance of Phosphate Increases Phosphatase Activity: A Hidden Consequence of Hemodialysis.

    Directory of Open Access Journals (Sweden)

    Ricardo Villa-Bellosta

    Full Text Available Extracellular pyrophosphate is a potent endogenous inhibitor of vascular calcification, which is degraded by alkaline phosphatase (ALP and generated by hydrolysis of ATP via ectonucleotide pyrophosphatase/phosphodiesterase 1 (eNPP1. ALP activity (as routinely measured in clinical practice represents the maximal activity (in ideal conditions, but not the real activity (in normal or physiological conditions. For the first time, the present study investigated extracellular pyrophosphate metabolism during hemodialysis sessions (including its synthesis via eNPP1 and its degradation via ALP in physiological conditions.45 patients in hemodialysis were studied. Physiological ALP activity represents only 4-6% of clinical activity. ALP activity increased post-hemodialysis by 2% under ideal conditions (87.4 ± 3.3 IU/L vs. 89.3 ± 3.6 IU/L and 48% under physiological conditions (3.5 ± 0.2 IU/L vs. 5.2 ± 0.2 IU/L. Pyrophosphate synthesis by ATP hydrolysis remained unaltered post-hemodialysis. Post-hemodialysis plasma pH (7.45 ± 0.02 significantly increased compared with the pre-dialysis pH (7.26 ± 0.02. The slight variation in pH (~0.2 units induced a significant increase in ALP activity (9%. Addition of phosphate in post-hemodialysis plasma significantly decreased ALP activity, although this effect was not observed with the addition of urea. Reduction in phosphate levels and increment in pH were significantly associated with an increase in physiological ALP activity post-hemodialysis. A decrease in plasma pyrophosphate levels (3.3 ± 0.3 μmol/L vs. 1.9 ± 0.1 μmol/L and pyrophosphate/ATP ratio (1.9 ± 0.2 vs. 1.4 ± 0.1 post-hemodialysis was also observed.Extraction of uremic toxins, primarily phosphate and hydrogen ions, dramatically increases the ALP activity under physiological conditions. This hitherto unknown consequence of hemodialysis suggests a reinterpretation of the clinical value of this parameter.

  10. Acidic-phosphoprotein phosphatase activity of rat ventral prostate nuclei: apparent lack of effect of androgens.

    Science.gov (United States)

    Wilson, M J; Ahmed, K; Fischbach, T J

    1978-08-01

    A protein phosphatase activity has been demonstrated in nuclei of rat ventral prostate utilizing 32P-labelled phosvitin as a model acidic phosphoprotein substrate. This phosphoprotein phosphatase has a pH optimum of 6.7, is unaffected by the sulphydryl protecting agent 2-mercaptoethanol, and requires a divalent cation for maximal activity. Of the various divalent cations tested, Mg2+ is the most effective in reactivating the EDTA-inhibited enzyme. The phosphatase is inhibited by sodium flouride, sodium oxalate, N-ethylmaleimide, ATP and ADP but is relatively insensitive to ammonium molybdate. Increased ionic strength of the reaction medium also causes a reduction in the enzyme activity, e.g., by 48% at 200 mM sodium chloride. The activity of the acidic phosphoprotein phosphatase did not change significantly at 48 h or 96 h post-orchiectomy when expressed per unit of nuclear protein. However, it is reduced by approx. 30% at these times after castration if based on DNA content. The decline in activity per nucleus reflects the decrease in the realtive nuclear protein content observed at 48 h or 96 h post-orchiectomy. This suggests that the decline in the phosphorylation of prostatic nuclear acidic proteins which occurs upon androgen withdrawal is not due to increased nuclear phosphatase activity.

  11. Phosphatase production and activity in Citrobacter freundii and a naturally occurring, heavy-metal-accumulating Citrobacter sp.

    Science.gov (United States)

    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.

  12. Eyes absent tyrosine phosphatase activity is not required for Drosophila development or survival.

    Directory of Open Access Journals (Sweden)

    Meng Jin

    Full Text Available Eyes absent (Eya is an evolutionarily conserved transcriptional coactivator and protein phosphatase that regulates multiple developmental processes throughout the metazoans. Drosophila eya is necessary for survival as well as for the formation of the adult eye. Eya contains a tyrosine phosphatase domain, and mutations altering presumptive active-site residues lead to strongly reduced activities in ectopic eye induction, in vivo genetic rescue using the Gal4-UAS system, and in vitro phosphatase assays. However, these mutations have not been analyzed during normal development with the correct levels, timing, and patterns of endogenous eya expression. To investigate whether the tyrosine phosphatase activity of Eya plays a role in Drosophila survival or normal eye formation, we generated three eya genomic rescue (eyaGR constructs that alter key active-site residues and tested them in vivo. In striking contrast to previous studies, all eyaGR constructs fully restore eye formation as well as viability in an eya null mutant background. We conclude that the tyrosine phosphatase activity of Eya is not required for normal eye development or survival in Drosophila. Our study suggests the need for a re-evaluation of the mechanism of Eya action and underscores the importance of studying genes in their native context.

  13. Effect of andrographolide on phosphatases activity and cytotoxicity against Spodoptera litura

    OpenAIRE

    Edwin, E.; P Vasantha-Srinivasan; A Thanigaivel; A Ponsankar; S Selin-Rani; K. Kalaivani; WB Hunter; Duraipandiyan, V; NA AlDhabi

    2016-01-01

    Andrographolide was isolated from ethanol extraction of Andrographis paniculata by column chromatography. Evaluation of larvicidal efficacy, enzymatic changes and cytotoxic activities against Spodoptera litura were conducted across a range of concentrations. The compound showed significant larvicidal activity between 5 - 25 ppm, post ingestion. Morphological deformities observed in larval-pupal stages. Enzymatic profiles were altered by reduction in acid phosphatase, ACP activity ...

  14. HYDROGEN SULFIDE ADSORPTION BY ALKALINE IMPREGNATED COCONUT SHELL ACTIVATED CARBON

    Directory of Open Access Journals (Sweden)

    HUI SUN CHOO

    2013-12-01

    Full Text Available Biogas is one type of renewable energy which can be burnt to produce heat and electricity. However, it cannot be burnt directly due to the presence of hydrogen sulfide (H2S which is highly corrosive to gas engine. In this study, coconut shell activated carbon (CSAC was applied as a porous adsorbent for H2S removal. The effect of amount of activated carbon and flow rate of gas stream toward adsorption capacity were investigated. Then, the activated carbons were impregnated by three types of alkaline (NaOH, KOH and K2CO3 with various ratios. The effects of various types of alkaline and their impregnation ratio towards adsorption capacity were analysed. In addition, H2S influent concentration and the reaction temperature on H2S adsorption were also investigated. The result indicated that adsorption capacity increases with the amount of activated carbon and decreases with flow rate of gas stream. Alkaline impregnated activated carbons had better performance than unimpregnated activated carbon. Among all impregnated activated carbons, activated carbon impregnated by K2CO3 with ratio 2.0 gave the highest adsorption capacity. Its adsorption capacity was 25 times higher than unimpregnated activated carbon. The result also indicated that the adsorption capacity of impregnated activated carbon decreased with the increment of H2S influent concentration. Optimum temperature for H2S adsorption was found to be 50˚C. In this study, the adsorption of H2S on K2CO3 impregnated activated carbon was fitted to the Langmuir isotherm. The fresh and spent K2CO3 impregnated activated carbon were characterized to study the adsorption process.

  15. Effect of phosphoric fertilizer and starter rates of nitrogen fertilizers on the phosphatase activity in the rhizosphere soil and nonlignified soybean roots under drought conditions

    Science.gov (United States)

    Emnova, E. E.; Daraban, O. V.; Bizgan, I. V.; Toma, S. I.

    2014-02-01

    In a small-plot field experiment, two soybean ( Glycine max L.) cultivars were grown on a calcareous chernozem under the drought conditions of 2012 with the preplanting application of simple superphosphate (Ps) at 60 kg/ha, urea (Nu) at 10 and 20 kg/ha, and ammonium nitrate (Nan) at 20 kg/ha. The phosphatase activity was measured in the rhizosphere soil (0- to 20-cm layer) and the fine nonlignified roots of soybean plants at the blossoming and pod-formation stages (the soil water content was 19 and 33% of the total water capacity, respectively). The maximum content of available phosphorus in the rhizosphere of both soybean cultivars (4.3-4.8 mg/100 g dry soil) was found at the simultaneous application of Ps and Nu20. Higher activities of the predominant phosphatases (alkaline phosphatase in the rhizosphere and acid phosphatase in the roots) were observed in the root-inhabited zone of the soil under the Indra cultivar compared to the Aura cultivar, which correlated with the lower content of available phosphorus in the rhizosphere soil (especially at the simultaneous application of Ps and Nu20) and the higher productivity of this cultivar in this treatment.

  16. Elevated Nitrogen Deposition from Alberta Oil Sands Development Stimulates Phosphatase Activity in Dominant Sphagnum Moss Species

    Science.gov (United States)

    Kashi, N. N.; Wieder, R.; Vile, M. A.

    2013-12-01

    Emissions of NOx associated with Alberta oil sands (AOS) development are leading to locally elevated atmospheric N deposition, in a region where background N deposition has been historically quite low (acid phosphatase activities in living plant capitulum of Sphagnum angustifolium, Sphagnum fuscum, and Sphagnum magellanicum were quantified in June and July using 4-methyumbelliferylphosphate and fluorescence detection of the enzymatically released methylumbelliferone (MUF). Phosphatase activities did not differ with N treatment for S. angustifolium in the bog (p=0.3409) or the poor fen (p=0.0629), or for S. fuscum in the bog (p=0.1950), averaging 35.0 × 0.7, 61.6 × 1.2, and 41.6 × 0.9 μmol MUF/g DWT/hr, respectively. For S. fuscum in the poor fen, phosphatase activities differed between N treatments (p=0.0275), ranging 40.6 × 1.1 μmol MUF/g DWT/hr in the control plots to 73.7 × 2.0 μmol MUF/g DWT/hr in the 5 kg/ha/yr N treatment plots; increasing N deposition did not result in a gradual change in enzyme activity. On the other hand, S. magellanicum phosphatase activities differed between N treatments (p=0.0189) and showed a pattern of generally increasing activity with increasing N deposition (37.4 × 0.5 μmol MUF/g DWT/hr in control plots; 97.9 × 4.5 μmol MUF/g DWT/hr in the 25 kg/ha/yr N treatment plots). The differing phosphatase responses between these dominant Sphagnum species suggest unique differences in nutrient balance and/or microbial activity. Combining the three moss species and weighting by their abundances within each plot (percent cover), phosphatase activities differed between N treatments in the bog (p=0.0388) and the poor fen (p=0.0005), with the latter exhibiting a clear increase in enzyme activity with increasing N deposition, and a doubling of phosphatase activity between the control plots and the 25 kg/kg/yr N deposition treatment. Although the three moss species responded differently, at the plot scale, increasing N deposition

  17. 骨源性碱性磷酸酶与血钙测定结果比较及临床探讨%Bone alkaline phosphatase compared with the serum calcium measurement results and clinical

    Institute of Scientific and Technical Information of China (English)

    黎智彪

    2013-01-01

      Objective: To investigate the to study bone alkaline phosphatase and serum calcium measurement results, analysis of bone alkaline phosphatase test value, in order to improve the clinical diagnosis and treatment of calcium deficiency patients. Methods: August 2009 to October 2012 infants undergo a medical examination of 150 patients for the study, given the determination of bone alkaline phosphatase and serum calcium al samples caught. Comparative observation test results, analysis of the significance of the detection of the two indicators of lack of calcium. Results: 150 cases of infants and young children, 37 cases of abnormalities of bone alkaline phosphatase, calcium abnormalities in 15 cases, two methods difference was significantly (p <0.05), the difference was statisticaly significant. Conclusion: bone alkaline phosphatase and serum calcium response to a lack of calcium infants, abnormal bone alkaline phosphatase detection rate higher, but also help the reaction of calcium nutrition of patients, doctors are required to pay close attention.%  目的探讨研究骨源性碱性磷酸酶与血钙的测定结果,分析骨源性碱性磷酸酶的检验价值,以提高临床对缺钙患者的诊治效果。方法选择2009年8月~2012年10月我院进行体检的婴幼儿150例为研究对象,对所有被检者均给予骨源性碱性磷酸酶和血钙的测定。对比观察检查结果,分析两种指标对钙离子缺乏的检测意义。结果150例婴幼儿中,骨源性碱性磷酸酶异常者37例,血钙异常15例,两种方法比较差异明显,p <0.05,差异有统计学意义。结论骨源性碱性磷酸酶与血钙均可以反应婴幼儿的钙缺乏,其中骨源性碱性磷酸酶异常的检出率更高,更有助于反应患者的钙营养情况,需要医生引起高度重视。

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

    Science.gov (United States)

    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

  19. Effects of Myriophyllum spicatum on Kinetic Characteristics of Alkaline Phosphatase in Water and Sediments%狐尾藻对水体和沉积物中碱性磷酸酶动力学特征的影响

    Institute of Scientific and Technical Information of China (English)

    赵海超; 王圣瑞; 金相灿; 焦立新

    2011-01-01

    The enzyme mechanism of an inactivating eutrophic lake by submerged plants was determined by analyzing the kinetic parameter variations of alkaline phosphataae in the overlying water, interstitial water and sediments under indoor simulating conditions with the plant Myriophyllum spicatum. The results indicated that under the experimental condition Vmax of the alkaline phosphatase, the overlying water, the interstitial water and the sediments were all reduced by planting Myriophyllum spicatum. Myriophyllum spicatum inactivated the alkaline phosphatase in the overlying water and sediments obviously, and mainly inactivated the dissolved alkaline phosphatase Vmax in the interstitial water. The effect of Myriophyllum spicatum on alkaline phosphatase in the soil was higher than that in the sediment of same nutrition. The Vmax and Km of the alkaline phosphatase in the overlying water of the soil substrate were higher than those of the sediment substrate, and the Vmax and Km of the alkaline phosphatase in the soil were lower than those in the sediment. The Vmax of the alkaline phosphatase in the sediments ascended opposite to that in the overlying water. The alkaline phosphatase in the interstitial water had obvious seasonal variation and characteristics, and Vmax attained the highest value from July to August.%在室内模拟条件下栽培狐尾藻,通过对上覆水、间隙水和沉积物中碱性磷酸酶动力学参数变化的分析,揭示了沉水植物对湖泊富营养化影响的酶学机制.结果表明:在试验条件下,栽培狐尾藻使上覆水、间隙水和沉积物中的碱性磷酸酶的最大反应速率(Vmax)均有所降低;狐尾藻对上覆水和底质中碱性磷酸酶反应速率及亲和力的抑制作用比较明显,对间隙水主要是抑制溶解性碱性磷酸酶的Vmax;狐尾藻对土壤中碱性磷酸酶的影响比同一营养水平的沉积物大,与沉积物相比,土壤作底质时上覆水中碱性磷酸酶的Vmax和K(ms)(米氏

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    W. Huang

    2011-07-01

    Full Text Available 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.

  2. An in vitro study of alkaline phosphatase sensitivity to mixture of aflatoxin B1 and fumonisin B1 in the hepatopancreas of coastal lagoon wild and farmed shrimp Litopenaeus vannamei.

    Science.gov (United States)

    Pérez-Acosta, Jesús A; Burgos-Hernandez, Armando; Velázquez-Contreras, Carlos A; Márquez-Ríos, Enrique; Torres-Arreola, Wilfrido; Arvizu-Flores, Aldo A; Ezquerra-Brauer, J Marina

    2016-08-01

    This study aimed to establish the combined effect of aflatoxin B1 (AFB1) and fumonisin B1 (FB1) on wild Litopenaeus vannamei hepatopancreas alkaline phosphatase (AP) activity compared with that of farmed shrimp. AP activity in hepatopancreas extract was confirmed by several specific inhibitor assays. AP activity of wild shrimp was higher than that of farmed shrimp (p shrimp was inhibited when incubated with AFB1 and FB1. The greatest inhibition occurred when AP was incubated with a mixture of AFB1 and FB1. The IC50 for AFB1 on AP activity of wild and farmed shrimp hepatopancreases was 0.790 and 0.398 μg/mL, respectively. The IC50 of FB1 was 0.87 μg/mL for wild shrimp and 0.69 μg/mL for farmed shrimp. These results suggest that, at the mycotoxins concentrations used in the study, AP from farmed L. vannamei was sensitive to the presence of both mycotoxins; however, AP is more sensitive to the combination of AFB1 + FB1 suggesting a possible synergistic or potentiating inhibitory effect.

  3. High serum alkaline phosphatase cooperating with MMP-9 predicts metastasis and poor prognosis in patients with primary osteosarcoma in Southern China

    Directory of Open Access Journals (Sweden)

    Han Ju

    2012-02-01

    Full Text Available Abstract Background Osteosarcoma is a malignant tumor with high ability to form invasion and metastasis. Identifying prognostic factor in osteosarcoma is helpful to select those patients for more aggressive management. Our study evaluated serum alkaline phosphatase (ALP cooperating with matrix metalloproteinase-9 (MMP-9 as an important prognostic predictor for local recurrence and distant metastasis of osteosarcoma. Methods 177 cases were included from the osteosarcoma patients treated at 1st Affiliated Hospital of Sun Yat-sen University (1999-2008. Pre-chemotherapy serum ALP (pre-ALP were studied and correlated with tumor recurrence, lung metastasis and patient survival. MMP-9 protein in tumor tissues was detected by immunohistochemistry and correlated with pre-ALP level. Results Pre-ALP were partitioned into normal, high, and very high groups, in each group the incidence of metastases was 12.2%, 21.2% and 34.6%, respectively (p = 0.007. In the three groups the mean disease-free survival (DFS was 57 ± 3.15, 28 ± 3.57 and 14 ± 3.35 months, respectively (p Conclusions Pre-ALP was an independent prognostic factor for the survival of osteosarcoma patients in south China, and correlated with MMP-9 expression and lung metastasis. ALP can also serve as a prognostic marker for treatment, and merit large-scale validation studies.

  4. The utility of alkaline phosphatase measurement as a screening test for rickets in breast-fed infants and toddlers: a study from the puget sound pediatric research network.

    Science.gov (United States)

    Taylor, James A; Richter, Monica; Done, Stephen; Feldman, Kenneth W

    2010-12-01

    To determine if alkaline phosphatase (AP) levels are a useful screening test for rickets, the authors measured serum AP levels in children 6 to 15 months old who were predominantly breast-fed for > 6 months without vitamin D supplementation. Radiographs were obtained on children with elevated AP levels to determine the presence of rickets. AP levels were obtained on 246 children; levels were elevated in 33 (13.4%). Rickets was present in 4 of 18 children with elevated levels on whom radiographs were obtained. The sensitivity and specificity of AP levels as a test for rickets was maximal at a cutoff value of 552 U/L. Using this cutoff value, the specificity of AP levels as a test for rickets was 97.4%, and the positive predictive value (PPV) was 40.0%. These results suggest that AP levels may be a useful screening test for rickets in children who are breast-fed for prolonged periods without vitamin D supplementation.

  5. Fluorescence detection of adenosine-5'-triphosphate and alkaline phosphatase based on the generation of CdS quantum dots

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Siyu; Wang, Xinyan; Pang, Shu; Na, Weidan; Yan, Xu; Su, Xingguang, E-mail: suxg@jlu.edu.cn

    2014-05-01

    Highlights: • Cd²⁺ reacts with S²⁻ to generate fluorescent CdS QDs with ATP. • ATP can be hydrolyzed by the enzymatic dephosphorylation of ALP. • Fluorescent CdS QDs could not be generated in the presence of ALP. • The analysis system was successfully applied to assay ATP and ALP. Abstract: We have developed an analytical method to detect adenosine-5'-triphosphate (ATP) and alkaline phosphatase (ALP) based on the generation of CdS quantum dots (QDs). We demonstrated that Cd²⁺ cation reacts with S²⁻anion to generate fluorescent CdS QDs in the presence of some certain amount of ATP. With increase in the ATP concentration, the fluorescence intensity of CdS QDs was also enhanced. ATP can be converted into adenosine by the dephosphorylation of ALP, so that the generation of CdS QDs would be inhibited in the presence of ALP. Therefore, this novel analysis system could be applied to assay ATP and ALP based on the growth of fluorescent CdS QDs.

  6. Alkaline Phosphatase Tagged Antibodies on Gold Nanoparticles/TiO2 Nanotubes Electrode: A Plasmonic Strategy for Label-Free and Amplified Photoelectrochemical Immunoassay.

    Science.gov (United States)

    Zhu, Yuan-Cheng; Zhang, Nan; Ruan, Yi-Fan; Zhao, Wei-Wei; Xu, Jing-Juan; Chen, Hong-Yuan

    2016-06-01

    This work reports a plasmonic strategy capable of label-free yet amplified photoelectrochemical (PEC) immunoassay for the sensitive and specific detection of model protein p53, an important transcription factor that regulates the cell cycle and functions as a tumor suppressor. Specifically, on the basis of Au nanoparticles (NPs) deposited on hierarchically ordered TiO2 nanotubes (NTs), a protein G molecular membrane was used for immobilization of alkaline phosphatase (ALP) conjugated anti-p53 (ALP-a-p53). Due to the immunological recognition between the receptor and target, the plasmonic charge separation from Au NPs to the conduction band of TiO2 NTs could be influenced greatly that originated from multiple factors. The degree of signal suppression is directly associated with the target concentration, so by monitoring the changes of the plasmonic photocurrent responding after the specific binding, a new plasmonic PEC immunoassay could be tailored for label-free and amplified detection. The operating principle of this study could be extended as a general protocol for numerous other targets of interest. PMID:27150939

  7. 髁突软骨细胞碱性磷酸酶活性及体外矿化能力的检测%Study on the activity of alkaline phosphatase and the calcified ability of mandibular condylar chondrocyte in vitro

    Institute of Scientific and Technical Information of China (English)

    毛勇; 段小红; 张玉梅

    2001-01-01

    @@0 引言髁突软骨细胞(mandibular condylar chondro cyte, MCC)体外培养时存在细胞形态变化、Ⅰ型和Ⅱ型胶原表达的更迭等表型变化特征[1],但还不清楚MCC碱性磷酸酶(alkaline phosphatase,ALP)活性的变化特点. 本实验采用ALP活性测定和Von Kossa染色法对兔MCC进行检测,以补充对MCC这方面生物学特征的认识.

  8. Lowering of phytic acid content by enhancement of phytase and acid phosphatase activities during sunflower germination

    OpenAIRE

    Juliana da Silva Agostini; Rosicler Balduíno Nogueira; Elza Iouko Ida

    2010-01-01

    The objective of this work was to investigate the germination of hybrid sunflowers BRS191 and C11 as a means of lowering phytic acid (PA) content by enhancing the activity of endogenous phytase and acid phosphatase. The concentration of PA in hybrid sunflower achenes varied from 2.16 to 2.83g/100g of sample (p < 0.05). The phytase and acid phosphatase activities of sunflowers BRS191 and C11 were the highest on the 4th and 5th days of germination, respectively, with the release of the phosphor...

  9. Rapid assessment of acid phosphatase activity in the mycorrhizosphere and in arbuscular mycorrhizal fungal hyphae

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A pot experiment has been carried out under controlled conditions to study the possibility of applying the technique of in vivo staining for acid phosphatase activity on the roots of mycorrhizal plants and arbuscular mycorrhizal hyphae. The pots had 5 compartments. The central root compartment was separated from the two adjacent hyphal compartments using nylon nets of 30 m m mesh, and the two hyphal compartments were separated from the two outermost compartments with 0.45 m m membranes. Red clover was grown in the root compartment and was either inoculated with the arbuscular mycorrhizal fungus (AMF) Glomus mosseae or uninoculated. Sodium phytate was applied to all compartments. The results show that AMF can increase acid phosphatase activity of clover roots. The plant roots acquired deep red "mycorrhizal prints". The external hyphae also had obvious "hyphal prints" on the test papers, indicating the ability of mycorrhizal hyphae to release acid phosphatase.

  10. Dexamethasone Causes Sustained Expression of Mitogen-Activated Protein Kinase (MAPK) Phosphatase 1 and Phosphatase-Mediated Inhibition of MAPK p38

    OpenAIRE

    Lasa, Marina; Abraham, Sonya M.; Boucheron, Christine; Saklatvala, Jeremy; Clark, Andrew R.

    2002-01-01

    The stress-activated protein kinase p38 stabilizes a number of mRNAs encoding inflammatory mediators, such as cyclooxygenase 2 (Cox-2). In HeLa cells the anti-inflammatory glucocorticoid dexamethasone destabilizes Cox-2 mRNA by inhibiting p38 function. Here we demonstrate that this effect is phosphatase dependent. Furthermore, in HeLa cells dexamethasone induced the sustained expression of mitogen-activated protein kinase phosphatase 1 (MKP-1), a potent inhibitor of p38 function. The inhibiti...

  11. The evaluation of 25-hydroxy vitamin D, calcium, phosphate and alkaline phosphatase levels in epileptic children under antiepileptic medication

    Directory of Open Access Journals (Sweden)

    Keyhani doost Z

    2011-01-01

    Full Text Available "n 800x600 Normal 0 false false false EN-US X-NONE AR-SA MicrosoftInternetExplorer4 st1":*{behavior:url(#ieooui } /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman","serif";} Background: Epilepsy is a common disease in the pediatric neurology. There are frequent anti-epileptic drugs which are used in management of epilepsy. Anti-epileptic drugs may have some complications on bone and vitamin-D metabolism. In this study we aimed to evaluate vitamin-D metabolism in epileptic children."n"nMethods: The study was a prospective and cross sectional one. A total 89 epileptic children who were taking anti-epileptic drugs for longer than six months with no underlying disorder in Imam Khomeini and Bahrami Hospitals in Tehran, Iran were enrolled in our study"n"nResults: Forty nine boys and 40 girls were enrolled in this study; mean age of the patients was 7.8±2.1 years. Mean duration of anti-epileptic drug therapy was 2.3 years (SD=0.4, 70 of patients were under monotherapy and 19 were under polytherapy. None of the patients had signs of rickets. Serum calcium and phosphor levels were within normal ranges. Serum alkaline phosphates levels were increased more than two times in 43%. 42% had vitamin-D deficiency (25-OH Vit D<10 ng/ml and another 33% had vitamin-D insufficiency (10<25-oh Vit D<20 ng/ml. 29 patients (32% were taking prophylactic supplemental Vit D (200-400 IU/day. There was significant difference between patients taking supplemental vitamin-D as prophylaxis and patients who did not (p=0.04. There was no significant difference in vitamin-D levels between patients according to age, gender or different drugs."n"nConclusion: Periodic

  12. Control of Sty1 MAPK activity through stabilisation of the Pyp2 MAPK phosphatase.

    Science.gov (United States)

    Kowalczyk, Katarzyna M; Hartmuth, Sonya; Perera, David; Stansfield, Peter; Petersen, Janni

    2013-08-01

    In all eukaryotes tight control of mitogen-activated protein kinase (MAPK) activity plays an important role in modulating intracellular signalling in response to changing environments. The fission yeast MAPK Sty1 (also known as Spc1 or Phh1) is highly activated in response to a variety of external stresses. To avoid segregation of damaged organelles or chromosomes, strong Sty1 activation transiently blocks mitosis and cell division until such stresses have been dealt with. MAPK phosphatases dephosphorylate Sty1 to reduce kinase activity. Therefore, tight control of MAPK phosphatases is central for stress adaptation and for cell division to resume. In contrast to Pyp1, the fission yeast Pyp2 MAPK phosphatase is under environmental control. Pyp2 has a unique sequence (the linker region) between the catalytic domain and the N-terminal MAPK-binding site. Here we show that the Pyp2 linker region is a destabilisation domain. Furthermore, the linker region is highly phosphorylated to increase Pyp2 protein stability and this phosphorylation is Sty1 dependent. Our data suggests that Sty1 activation promotes Pyp2 phosphorylation to increase the stability of the phosphatase. This MAPK-dependent Pyp2 stabilisation allows cells to attenuate MAPK signalling and resume cell division, once stresses have been dealt with. PMID:23690545

  13. Effects icariin of on the alkline phosphatase activity of human periodontal ligament cells inhibited by LPS%淫羊藿苷对内毒素抑制牙周膜细胞ALP表达的影响

    Institute of Scientific and Technical Information of China (English)

    姜瑛; 王祥; 许华; 刘英群

    2013-01-01

    Objective: To investigate the effects of icariin (ICA) on proliferation of huaman periodontal ligament cells (human periodontal ligament cells, hPDLC) and the alkaline phosphatase (alkaline phosphatase, ALP) activity inhibited by LPS. Method: HPDLC were cultured in virto and stimulated with icariin with different concentrations (0, 10-5、 10-6、 10-7、 10-8、 10-9 mol / L) for 96 hours.The ability of proliferation of HPDLC was detected by MTT method.The activity of alkaline phosphatase and the expression of alkaline phosphatase mRNA was seperately determined by p-Nitrophnyl phosphate (pNPP) method and RT-PCR after being treated with icariin at the concentration of 10-6 mol / L inhibited by LPS. Result: icariin had a dose-dependent effect on the proliferation the hPDLC in a suitable concentration range from l0-6-10-8 mol / L. The alkaline phosphatase activity was remarkably inhibited by incubation of PDLC with 10 μg / mL LPS and was improved in the presence of icariin at the concentration of 10-6 mol / L. Conclusion: Icariin have the effect on the proliferation of PDLC and improve the ALP activity inhibited by LPS, which could help for Peri—apical tissue regeneration.%目的:探讨淫羊藿苷对人牙周膜细胞(periodontal ligament cells,PDLC)增殖及对内毒素(Lipopolysaccharide,LPS)干扰下碱性磷酸酶(alkaline phosphatase,ALP)的影响.方法:原代培养PDLC,MTT法检测不同浓度(0、10-5~10-9 mol/L)淫羊藿苷对PDLC增殖的影响;RT-PCR、对硝基苯酚法测定淫羊藿苷对LPS抑制PDLC的ALP mRNA表达和分泌的影响.结果:淫羊藿苷在一定浓度下(10-6~10-7 mol/L)可促进PDLC增殖;10 μg/mL LPS可抑制PDLC的ALP活性;加入10-6 mol/L淫羊藿苷干扰后,对LPS抑制PDLC的ALP有拮抗作用,可提高其mRNA表达和活性.结论:淫羊藿苷在一定浓度时可促进PDLC增殖;可能通过拮抗LPS抑制其ALP的活性.

  14. Reversible oxidation controls the activity and oligomeric state of the mammalian phosphoglycolate phosphatase AUM.

    Science.gov (United States)

    Seifried, Annegrit; Bergeron, Alexandre; Boivin, Benoit; Gohla, Antje

    2016-08-01

    Redox-dependent switches of enzyme activity are emerging as important fine-tuning mechanisms in cell signaling. For example, protein tyrosine phosphatases employ a conserved cysteine residue for catalysis, which also renders them highly susceptible to reversible inactivation by oxidation. In contrast, haloacid dehalogenase (HAD)-type phosphatases perform catalysis via a phosphoaspartyltransferase reaction. The potential regulation of HAD phosphatases by reversible oxidation has not yet been explored. Here, we investigate the redox-sensitivity of the HAD-type phosphoglycolate phosphatase PGP, also known as AUM or glycerol-3-phosphate phosphatase. We show that recombinant, purified murine PGP is inhibited by oxidation and re-activated by reduction. We identify three reactive cysteine residues in the catalytic core domain of PGP (Cys35, Cys104 and Cys243) that mediate the reversible inhibition of PGP activity and the associated, redox-dependent conformational changes. Structural analysis suggests that Cys35 oxidation weakens van-der-Waals interactions with Thr67, a conserved catalytic residue required for substrate coordination. Cys104 and Cys243 form a redox-dependent disulfide bridge between the PGP catalytic core and cap domains, which may impair the open/close-dynamics of the catalytic cycle. In addition, we demonstrate that Cys297 in the PGP cap domain is essential for redox-dependent PGP oligomerization, and that PGP oxidation/oligomerization occurs in response to stimulation of cells with EGF. Finally, employing a modified cysteinyl-labeling assay, we show that cysteines of cellular PGP are transiently oxidized to sulfenic acids. Taken together, our findings establish that PGP, an aspartate-dependent HAD phosphatase, is transiently inactivated by reversible oxidation in cells. PMID:27179418

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

    Science.gov (United States)

    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 ...

  16. A protein tyrosine phosphatase-like protein from baculovirus has RNA 5'-triphosphatase and diphosphatase activities.

    Science.gov (United States)

    Takagi, T; Taylor, G S; Kusakabe, T; Charbonneau, H; Buratowski, S

    1998-08-18

    The superfamily of protein tyrosine phosphatases (PTPs) includes at least one enzyme with an RNA substrate. We recently showed that the RNA triphosphatase domain of the Caenorhabditis elegans mRNA capping enzyme is related to the PTP enzyme family by sequence similarity and mechanism. The PTP most similar in sequence to the capping enzyme triphosphatase is BVP, a dual-specificity PTP encoded by the Autographa californica nuclear polyhedrosis virus. Although BVP previously has been shown to have modest tyrosine and serine/threonine phosphatase activity, we find that it is much more potent as an RNA 5'-phosphatase. BVP sequentially removes gamma and beta phosphates from the 5' end of triphosphate-terminated RNA, leaving a 5'-monophosphate end. The activity was specific for polynucleotides; nucleotide triphosphates were not hydrolyzed. A mutant protein in which the active site cysteine was replaced with serine was inactive. Three other dual-specificity PTPs (VH1, VHR, and Cdc14) did not exhibit detectable RNA phosphatase activity. Therefore, capping enzyme and BVP are members of a distinct PTP-like subfamily that can remove phosphates from RNA. PMID:9707557

  17. The two neutrophil plasma membrane markers alkaline phosphatase and HLA class I antigen localize differently in granule-deficient cytoplasts. An ideal plasma membrane marker in human neutrophils is still lacking.

    Science.gov (United States)

    Pellmé, Sara; Dahlgren, Claes; Karlsson, Anna

    2007-08-31

    Neutrophil function relies largely on the ability of the cell to mobilize its different granules and vesicles to the cell surface and thereby expose and/or release effector molecules to the surrounding tissue. To properly identify these subcellular compartments is thus a prerequisite for studies of neutrophil physiology. A range of specific markers for the classical granules is available, but finding optimal markers for the secretory vesicles and plasma membrane has historically been more challenging. Latent and non-latent alkaline phosphatase activities are often used to distinguish these two light membrane structures, but the outcome using this technique depends on the level of cellular activation. Therefore, HLA-I was introduced some years ago as a specific, stimulation-independent marker for the plasma membrane. In this study we however report that detailed fractionation studies of neutrophil cytoplasts, lacking secretory vesicles, granules and other dense organelles, reveal that the HLA-I antigen is not only co-localizing with the plasma membrane marker ALP, but is also present in other, more dense organelles. Further, we found the mixed enzyme-linked immunosorbent assay (MELISA), detecting the beta(2)-microglobulin/HLA-I complex, to be negatively influenced by uncomplexed beta(2)-microglobulin present in the specific granules and secretory vesicles, making it difficult to use HLA-I as a plasma membrane marker during maturation of for example phagolysosomes. PMID:17673253

  18. 饲料锌含量对方格星虫稚虫生长性能、体成分、体腔液中锌含量及碱性磷酸酶活性的影响%Effects of Dietary Zinc Content on Growth Performance, Body Composition, Coelomic Fluid Zinc Content and Alkaline Phosphatase Activity of Juvenile Peanut Worm, Sipunculus nudus Linnaeus

    Institute of Scientific and Technical Information of China (English)

    许明珠; 张琴; 童潼; 董兰芳; 杨家林; 蒋艳; 黄国强

    2016-01-01

    This trial was conducted to investigate the effects of dietary zinc content on growth performance, body composition, coelomic fluid zinc content and alkaline phosphatase ( AKP ) activity of juvenile peanut worm, Sipunculus nudus Linnaeus ( S. nudus ) . Juvenile peanut worm with the average body weight of (14.54±0.10) mg were fed six isonitrogenous and isoenergetic microparticle diets with different supplemental levels of zinc ( zinc methionine as zinc source) for 8 weeks, and the measured zinc contents in diets were 9.3, 31.7, 49.9, 90.1, 168.6 and 326.5 mg/kg, respectively. Each diet had three replicates and each replicate had 400 juvenile peanut worm. The results showed that the dietary zinc content had significant effects on weight gain rate ( WGR) , specific growth rate ( SGR) and survival ratio ( SR) of S. nudus ( P0.05). Dietary zinc content significantly affected coelomic fluid zinc content and AKP activity of S. nudus ( P<0.05) . With the increase of dietary zinc content, the coelomic fluid zinc content of S. nudus was sustained rise, when the dietary zinc content was 326.5 mg/kg, it produced the maximum value. Moreover, the coelomic fluid AKP activity was firstly increased and then tended to be stable, when the dietary zinc content was 49.9 mg/kg, it produced the maximum value. With WGR as the evaluating index, the suitable dietary zinc content of juvenile peanut worm is 41.93 mg/kg by regression analysis.%以蛋氨酸锌为锌源,在等氮等能的基础饲料中添加不同水平的锌,配制实际锌含量分别为9.3、31.7、49.9、90.1、168.6和326.5 mg/kg 的6种试验饲料,饲喂平均体重为(14.54±0.10) mg的方格星虫稚虫8周,用以研究饲料锌含量对方格星虫稚虫生长、体成分、体腔液中锌含量及碱性磷酸酶活性的影响。每种试验饲料设3个重复,每个重复饲喂400尾方格星虫稚虫。结果表明:饲料锌含量对方格星虫的成活率、增重率和特定

  19. Reduced expression of PNUTS leads to activation of Rb-phosphatase and caspase-mediated apoptosis.

    Science.gov (United States)

    De Leon, Gabriel; Sherry, Tara C; Krucher, Nancy A

    2008-06-01

    There is abundant evidence that Retinoblastoma (Rb) activity is important in the control of cell proliferation and apoptosis. Reversible phosphorylation of the Rb protein that is carried out by cyclin dependent kinases and Protein phosphatase 1 (PP1) regulates its functions. A PP1 interacting protein, PNUTS (Phosphatase Nuclear Targeting Subunit) is proposed to be a regulator of Rb phosphorylation. In this study, PNUTS knockdown in MCF7, SKA and HCT116 cancer cells causes a reduction in viability due to increased apoptosis. However, normal cells (MCF10A breast and CCD-18Co colon) do not exhibit reduced viability when PNUTS expression is diminished. PNUTS knockdown has no effect in Rb-null Saos-2 cells. However, when Rb is stably expressed in Saos-2 cells, PNUTS knockdown reduces cell number. Knockdown of PNUTS in p53-/- HCT116 cells indicates that p53 is dispensable for the induction of apoptosis. Loss of PNUTS expression results in increased Rb-phosphatase activity and Rb dephosphorylation. E2F1 dissociates from Rb in cells depleted of PNUTS and the resulting apoptosis is dependent on caspase-8. These results indicate that Rb phosphorylation state can be manipulated by targeting Rb phosphatase activity and suggest that PNUTS may be a potential target for therapeutic pro-apoptotic strategies. PMID:18360108

  20. Contributions of phosphatase and microbial activity to internal phosphorus loading and their relation to lake eutrophication

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Phosphatase may accelerate the process of lake eutrophication through improving phosphorus bioavailability. This mechanism was studied in three Chinese eutrophic shallow lakes (Lake Taihu, Lake Longyang and Lake Lianhua). Phosphatase activity was related to the concentration of soluble reactive phosphorus (SRP) and chlorophyll a. Stability of dissolved phosphatase in reverse micelles may be attributed to molecular size, conformation and active residues of the enzyme.At the site with Microcystis bloomed in Lake Taihu, dissolved phosphatase activity was higher and more stable in micelles, SRP concentrations were lower in interstitial water, the contents of different forms of phosphorus and the amounts of aerobic bacteria were lower while respiration efficiency was higher in sediments. Phosphobacteria, both inorganic and organic and other microorganisms were abundant in surface water but rare in sediments. Therefore, internal phosphorus may substantially flux into water column by enzymatic hydrolysis and anaerobic release, together with mobility of bacteria,thereby initiating the bloom. In short, biological mechanism may act in concert with physical and chemical factors to drive the internal phosphorus release and accelerate lake eutrophication.

  1. Free Fatty Acids Inhibit Protein Tyrosine Phosphatase 1B and Activate Akt

    Directory of Open Access Journals (Sweden)

    Eisuke Shibata

    2013-09-01

    Full Text Available Background/Aims: Accumulating evidence has suggested that free fatty acids (FFAs interact with protein kinases and protein phosphatases. The present study examined the effect of FFAs on protein phosphatases and Akt. Methods: Activities of protein phosphatase 1 (PP1, protein phosphatase 2A (PP2A, and protein tyrosine phosphatase 1B (PTP1B were assayed under the cell-free conditions. Phosphorylation of Akt was monitored in MSTO-211H human malignant pleural mesothelioma cells without and with knocking-down phosphatidylinositol 3 kinase (PI3K or 3-phosphoinositide-dependent protein kinase-1 (PDK1. Results: In the cell-free assay, unsaturated FFAs (uFFAs such as oleic, linoleic and linolenic acid and saturated FFAs (sFFAs such as stearic, palmitic, myristic, and behenic acid markedly reduced PTP1B activity, with the potential for uFFAs greater than that for sFFAs. All the investigated sFFAs inhibited PP2A activity, but otherwise no inhibition was obtained with uFFAs. Both uFFAs and sFFAs had no effect on PP1 activity. Oleic acid phosphorylated Akt both on Thr308 and Ser473, while stearic acid phosphorylated Akt on Thr308 alone. The effects of oleic and stearic acid on Akt phosphorylation were abrogated by the PI3K inhibitor wortmannin or the PDK1 inhibitor BX912 and also by knocking-down PI3K or PDK1. Conclusion: The results of the present study indicate that uFFAs and sFFAs could activate Akt through a pathway along a PI3K/PDK1/Akt axis in association with PTP1B inhibition.

  2. Effects of Rice Straw Returning on Activities of Soil Phosphatase and Available P Values in Soil%水稻秸秆还田对土壤磷酸酶活性及速效磷含量的影响

    Institute of Scientific and Technical Information of China (English)

    战厚强; 颜双双; 王家睿; 马春梅; 龚振平; 董守坤; 张钦文

    2015-01-01

    The experiment focused on the cold rice,which used plot experiment and laboratory simulation experiment,and studied the effects of rice straw returning on activities of soil phosphatase and available P values in soil so as to provide evidence and reference for the rice straw returning.The result showed that activities of soil acid phosphatase were the highest in the soil,followed by activities of soil neutral phosphatase,activities of soil alkaline phosphatase.The activities of acid phosphatase with straw returning was significantly higher than that of no straw returning,the neutral phosphatase and alkaline phosphatase activities with straw returning were higher than that of no straw returning.Both the activities of acid phosphatase and available P values in soil increased with the amount of straw returned,the activities of alkaline phosphatase decreased with the amount of straw returned but the neutral phosphatase activities was relatively stable.There were a positive correlation between the activities of acid phosphatase and available P values in soil,therefore the activities of acid phosphatase could be used to report to the available P in soil,and the activities of acid phosphatase could be used as sensitive indicators of soil phosphorus status.%本试验针对中国东北稻作区,利用室内模拟试验和小区试验,研究了水稻秸秆还田对土壤磷酸酶活性及土壤速效磷含量的影响,为实现寒地水稻秸秆还田生产提供依据和参考.结果表明,土壤中酸性磷酸酶活性最大,碱性磷酸酶活性次之,中性磷酸酶活性最低;秸秆还田处理的酸性磷酸酶活性极显著高于不还田处理,秸秆还田处理的中性磷酸酶活性和碱性磷酸酶活性显著高于不还田处理;在一定范围内,酸性磷酸酶活性和土壤速效磷含量均随秸秆施入量增加而升高;中性磷酸酶活性相对稳定;碱性磷酸酶活性随秸秆施入量增加而降低.土壤酸性磷酸酶活性与土壤速效

  3. 碱性磷酸酶试剂开瓶的稳定性研究%Study on stability of alkaline phosphatase reagent after the packaging was opened

    Institute of Scientific and Technical Information of China (English)

    段爱军; 段爱华

    2015-01-01

    Objective:To study the stability of alkaline phosphatase(ALP) after the packaging was opened and to provide the data for purchasing right amount reageng. Methods:The method of synchronous measurement was used and five kits(S0, S1, S2, S3, S4) were chosen. ALP routine test was performed using the kit S4 at eight a.m. in first day. The remaining reagent was covered tightly and preserved at 2-8 ℃. Then S3, S2, S1 and S0 kits were opened at the same time in second, third, fourth and fifth day using the same method, respectively. Five of the same parameters for ALP detection channel (ALP0, ALP1, ALP2, ALP3, ALP4, corresponding to S0, S1, S2, S3, S4) were also set in the instrument according to the reagent instruction. The detection channels were calibrated using the calibration materials(BioSino Bio-technology and Science Inc) in Selectra-E fully automatic biochemical analyzer. Results: Compared with the results obtained by S0, the results from S3 and S4 showed statistically significance(P0.05). Conclusion: The ALP reagent is stable in 48 hours after the packaging was opened and the laboratory should choose suitable packing size of reagent kit according to the dosage to use.%目的:研究碱性磷酸酶(alkaline phosphatase, ALP)试剂开瓶后的稳定性,为实验室合理购买试剂包规格提供依据。方法:采用同步测量设计方案,首先选择5个试剂盒,分别记为S0、S1、S2、S3、S4,第1天上午8点启用试剂S4进行ALP常规检测,未用完试剂盖紧盖,置2~8℃保存备用;第2天、第3天、第4天上午相同时间、相同方式启用S3、S2、S1;第5天启用试剂S0;在仪器上按试剂说明书设置5个相同参数的ALP检测通道,记为ALP0、ALP1、ALP2、ALP3、ALP4,对应试剂为S0、S1、S2、S3、S4;在荷兰威图Selectra-E全自动生化仪上用校准品(中生北控生物科技有限公司)校准各个通道;测量样品并进行结果比较分析。结果:试剂 S3、S4与试剂 S0

  4. 妊娠妇女不同孕期血清碱性磷酸酶变化%Variation of serum alkaline phosphatase in pregnant in different stage

    Institute of Scientific and Technical Information of China (English)

    周洁

    2016-01-01

    Objective To explore the variation of serum alkaline phosphatase in Pregnancy in dif‐ferent stage .Methods Unicel Dxc800 fully automatic biochemical anaylzer was used to detect the con‐centrations of serum ALP in 522 pregnant and 157 control .Results The concentrations of serum ALP in control group ,early pregnancy ,midtrimester and late pregnancy group were (66 .58 ± 44 .09) U /L , (61 .64 ± 17 .86)U /L ,(79 .98 ± 26 .38)U /L ,and (202 .89 ± 68 .94 )U /L respectively .Statistic analysis dem‐onstrated that there was no difference between early pregnancy group and control one( P > 0 .05) .The concentrations of serum ALP in midtrimester pregnancy and late pregnancy groups were hiher than those in early pregnancy and control groups significantly ,and the concentration of serum ALP in late pregnancy group was hiher than that in midtrimester pregnancy group significantly ( P < 0 .05) .Condu‐sion The serum level of alkaline phosphatase in pregnant increased with gestational prolongation .%①目的探讨不同孕周妊娠期妇女血清碱性磷酸酶的变化。②方法采用贝克曼(Unicel Dxc800)全自动生化分析仪检测522例妊娠早、中、晚期妇女血清碱性磷酸酶值,同时选取同期157例健康育龄非妊娠妇女作为对照,方差分析不同组间血清碱性磷酸酶值差异。③结果健康育龄非妊娠妇女及妊娠期妇女孕早期、孕中期和孕晚期血清碱性磷酸酶浓度分别为(66.58±44.09)U /L 、(61.64±17.86)U /L 、(79.98±26.38)U /L 、(202.89±68.94)U /L 。统计学分析结果表明:孕早期血清碱性磷酸酶水平与对照组比较差异无统计学意义( P >0.05);孕中期、孕晚期高于孕早期及对照组,孕晚期高于孕中期,差异均具有统计学意义( P 均<0.05)。④结论妇女孕期血清碱性磷酸酶水平随孕周数增加呈逐渐上升趋势。

  5. Nitrate reductase and acid phosphatase activities as affected by inorganic phosphate in corn roots

    OpenAIRE

    Marie Kummerova; Józef Buczek

    2014-01-01

    The deficieny of inorganic phosphate in nutrient solution reduces by about 50 per cent NO3- absorption in corn seedlings, it decreases both in vitro and in vivo nitrate reductase (NR) activity, as well the potential and actual NR level and has a very weak effect on NR induction. Acid phosphatases activities increase in corn roots when the plants are grown in nutrient solution without phosphorus. We suggest that inorganic phosphate is required mainly for maintenance of NR activity rather, than...

  6. A New 4.0-Generation Dendrimer Phosphorescence Labeling Reagent and Its Application to Determination of Trace Alkaline Phosphatase by Affinity Adsorption Solid Substrate-room Temperature Phosphorimetry

    Institute of Scientific and Technical Information of China (English)

    LI, Zhi-Ming; LIU, Jia-Ming; CHEN, Xiao-Hua; YANG, Min-Lan; CHEN, Xin-Hua; SHI, Xiu-Mei

    2007-01-01

    A Triton X-100-4.0G-D (4.0G-D refers to a 4.0-generation dendrimer) was brought forward as a new phosphorescence labeling reagent. Two types of specific affinity adsorption (AA) reactions (direct method and sandwich method) were carried out between the labeling product of Triton X-100-4.0G-D-Wheat germ agglutinin (WGA) and alkaline phosphatase (ALP), the product of AA reaction preserved the good characteristics of room temperature phosphorescence (RTP) of 4.0G-D and △Ip of the product was proportional to the content of ALP. According to the fact stated above, a new method for the determination of trace ALP by affinity adsorption solid substrate-room temperature phosphorimetry (AA-SS-RTP) was established on the basis of WGA labeled with the Triton X-100-4.0G-D. The detection limits were 0.20 ag·spot-1 (corresponding concentration: 5.0 × 10-16 g·mL-1, namely 5.0× 10-18 mol·L-1) for a direct method and 0.14 ag·spot-1 (corresponding concentration: 3.5× 10-16 g·mL-1,namely 3.5 × 10-18 mol·L-1) for a sandwich method, respectively. For their high sensitivity, good repeatability and high accuracy, the direct method and sandwich method have been successfully applied to determine the content of ALP in human serum, and the results were coincided with the clinical detection results of the enzyme-linked immunosorbent assay method by the Zhangzhou Hospital of Traditional Chinese Medicine. Meanwhile, the mechanism for the determination of trace ALP by AA-SS-RTP was discussed.

  7. Identification of an alkaline phosphatase as a putative Cry1Ac binding protein in Ostrinia furnacalis (Guenée).

    Science.gov (United States)

    Jin, Tingting; Duan, Xiaoli; Bravo, Alejandra; Soberón, Mario; Wang, Zhenying; He, Kanglai

    2016-07-01

    Asian corn borer (ACB), Ostrinia furnacalis, is an important insect pest of maize susceptible to different Cry1A toxins. Based on amino acid sequence alignment of ALP sequences from lepidopteran larvae an alp gene was cloned from ACB, named ofalp. Pull dawn assays using biotinylated Cry1Ac and brush border membrane vesicles isolated from second instar ACB larvae showed that four proteins of 50, 65, 68 and 70kDa precipitated with the Cry1Ac. The 65kDa band cross-reacted with the anti-OfALP monoclonal antibody. GalNac was able to release the binding of Cry1Ac to the 65kDa OfALP in pull down assays. A 37kDa fragment from residues D173 to D473 of OfALP was cloned and expressed in Escherichia coli cells. We show that this ALP-fragment was able to bind Cry1Ac in ligand blot analysis. Our data also indicate that different ALP isoforms or variants may be also Cry1Ac binding proteins since more ALP enzymatic activity was pull down with Cry1Ac than with anti-OfALP antibody. We also analyzed the expression levels of ALP throughout the larval development by qPCR and ALP enzymatic activity. Our data indicated that ALP expression in ACB was observed preferentially in young instar larvae. Finally, we show that resistance in O. furnacalis ACB-AcR strain resistant to Cry1Ac did not correlate with changes in expression of this ALP protein since it shows similar gene expression of ofalp than the susceptible insect strain. Identification of Cry1Ac receptors will help to understand mechanism of action of Cry1Ac in O. furnacalis and to understand mechanism of Cry toxin resistance. Our data indicate that at least one ALP protein is involved in the binding interaction with Cry1Ac in O. furnacalis. PMID:27265829

  8. 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

    Science.gov (United States)

    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

  9. 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

    Directory of Open Access Journals (Sweden)

    Ana Lorena Alvarado-Gámez

    2015-01-01

    Full Text Available 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.

  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.

    Science.gov (United States)

    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. Phosphatase activity of Bombyx mori nucleopolyhedrovirus PTP is dispensable for enhanced locomotory activity in B. mori larvae.

    Science.gov (United States)

    Katsuma, Susumu

    2015-11-01

    Baculovirus-induced enhanced locomotory activity (ELA) is not induced in caterpillars infected with a mutant Bombyx mori nucleopolyhedrovirus (BmNPV) or Autographa californica multiple nucleopolyhedrovirus (AcMNPV) lacking a functional protein tyrosine phosphatase gene (ptp). Previous studies suggest that the PTP proteins from BmNPV and AcMNPV act in different ways to induce ELA, i.e., BmNPV PTP is utilized as a virion structural component, whereas AcMNPV PTP requires its phosphatase activity. Here, I generated and characterized two new BmNPV mutants expressing enzymatically inactive PTP proteins and confirmed that the phosphatase activity of PTP is not required for ELA induction in BmNPV-infected B. mori larvae.

  12. Phosphatase inhibitors activate normal and defective CFTR chloride channels.

    OpenAIRE

    Becq, F; Jensen, T J; Chang, X B; Savoia, A.; Rommens, J M; Tsui, L C; Buchwald, M; Riordan, J R; Hanrahan, J W

    1994-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is regulated by phosphorylation and dephosphorylation at multiple sites. Although activation by protein kinases has been studied in some detail, the dephosphorylation step has received little attention. This report examines the mechanisms responsible for the dephosphorylation and spontaneous deactivation ("rundown") of CFTR chloride channels excised from transfected Chinese hamster ovary (CHO) and human airway epi...

  13. Changes of Activities in NAD Kinase and NADP Phosphatase During Ripening and Senescence of Tomato and Strawberry Fruits

    Institute of Scientific and Technical Information of China (English)

    GU Cai-qin; GUAN Jun-feng; XI Yu-fang; LI Guang-min

    2002-01-01

    Activities of NAD kinase(NADK)and NADP phosphatase and relationship between the two enzymes and temperature, respiration, ethylene production and trifluoperazine(TFP) were studied during ripening and senescence of strawberry and tomato frnits after harvest at 4℃and 20℃. The activity of NAD kinase in strawberry decreased slowly during first four days, then increased gradually. The NADP phosphatase activity increased at the second day, decreased the next day,then increased again. In tomato fruit, the activities of NAD kinase and NADP phosphatase increased at the second day, decreased with the ripening and senescence of the fruit. The change trend of NAD kinase and respiration in the two fruits were similar, the same were NADP phosphatase and ethylene production. TFP enhanced the activity of NAD kinase and had little effect on NADP phosphatase. Low temperature(4℃ ) activated the NAD kinase and reduced the activity of NADP phosphatase. These results indicated that the NAD kinase and NADP phosphatase were related to the ripening and senescence of strawberry and tomato fruits. The activation of NAD kinase probably postponed the ripening and senescence of the fruits.

  14. IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 °C. Part 9: reference procedure for the measurement of catalytic concentration of alkaline phosphatase International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Scientific Division, Committee on Reference Systems of Enzymes (C-RSE) (1)).

    Science.gov (United States)

    Schumann, Gerhard; Klauke, Rainer; Canalias, Francesca; Bossert-Reuther, Steffen; Franck, Paul F H; Gella, F-Javier; Jørgensen, Poul J; Kang, Dongchon; Lessinger, Jean-Marc; Panteghini, Mauro; Ceriotti, Ferruccio

    2011-09-01

    Abstract This paper is the ninth in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 °C and the certification of reference preparations. Other parts deal with: Part 1. The concept of reference procedures for the measurement of catalytic activity concentrations of enzymes; Part 2. Reference procedure for the measurement of catalytic concentration of creatine kinase; Part 3. Reference procedure for the measurement of catalytic concentration of lactate dehydrogenase; Part 4. Reference procedure for the measurement of catalytic concentration of alanine aminotransferase; Part 5. Reference procedure for the measurement of catalytic concentration of aspartate aminotransferase; Part 6. Reference procedure for the measurement of catalytic concentration of γ-glutamyltransferase; Part 7. Certification of four reference materials for the determination of enzymatic activity of γ-glutamyltransferase, lactate dehydrogenase, alanine aminotransferase and creatine kinase at 37 °C; Part 8. Reference procedure for the measurement of catalytic concentration of α-amylase. The procedure described here is derived from the previously described 30 °C IFCC reference method. Differences are tabulated and commented on in Appendix 1.

  15. Association of Protein Phosphatase 1γ1 with Spinophilin Suppresses Phosphatase Activity in a Parkinson Disease Model*

    OpenAIRE

    Brown, Abigail M.; Baucum, Anthony J.; Bass, Martha A.; Roger J Colbran

    2008-01-01

    Sustained nigrostriatal dopamine depletion increases the serine/threonine phosphorylation of multiple striatal proteins that play a role in corticostriatal synaptic plasticity, including Thr286 phosphorylation of calcium/calmodulin-dependent protein kinase IIα (CaMKIIα). Mechanisms underlying these changes are unclear, but protein phosphatases play a critical role in the acute modulation of striatal protein phosphorylation. Here we show that dopamine depletion for periods ranging from 3 weeks...

  16. Protein phosphatases 1 and 2A promote Raf-1 activation by regulating 14-3-3 interactions.

    Science.gov (United States)

    Jaumot, M; Hancock, J F

    2001-07-01

    Raf-1 activation is a complex process which involves plasma membrane recruitment, phosphorylation, protein-protein and lipid-protein interactions. We now show that PP1 and PP2A serine-threonine phosphatases also have a positive role in Ras dependent Raf-1 activation. General serine-threonine phosphatase inhibitors such sodium fluoride, or ss-glycerophosphate and sodium pyrophosphate, or specific PP1 and PP2A inhibitors including microcystin-LR, protein phosphatase 2A inhibitor I(1) or protein phosphatase inhibitor 2 all abrogate H-Ras and K-Ras dependent Raf-1 activation in vitro. A critical Raf-1 target residue for PP1 and PP2A is S259. Serine phosphatase inhibitors block the dephosphorylation of S259, which accompanies Raf-1 activation, and Ras dependent activation of mutant Raf259A is relatively resistant to serine phosphatase inhibitors. Sucrose gradient analysis demonstrates that serine phosphatase inhibition increases the total amount of 14-3-3 and Raf-1 associated with the plasma membrane and significantly alters the distribution of 14-3-3 and Raf-1 across different plasma membrane microdomains. These observations suggest that dephosphorylation of S259 is a critical early step in Ras dependent Raf-1 activation which facilitates 14-3-3 displacement. Inhibition of PP1 and PP2A therefore causes plasma membrane accumulation of Raf-1/14-3-3 complexes which cannot be activated.

  17. Functional analysis of TPM domain containing Rv2345 of Mycobacterium tuberculosis identifies its phosphatase activity.

    Science.gov (United States)

    Sinha, Avni; Eniyan, Kandasamy; Sinha, Swati; Lynn, Andrew Michael; Bajpai, Urmi

    2015-07-01

    Mycobacterium tuberculosis (Mtb) is the causal agent of tuberculosis, the second largest infectious disease. With the rise of multi-drug resistant strains of M. tuberculosis, serious challenge lies ahead of us in treating the disease. The availability of complete genome sequence of Mtb has improved the scope for identifying new proteins that would not only further our understanding of biology of the organism but could also serve to discover new drug targets. In this study, Rv2345, a hypothetical membrane protein of M. tuberculosis H37Rv, which is reported to be a putative ortholog of ZipA cell division protein has been assigned function through functional annotation using bioinformatics tools followed by experimental validation. Sequence analysis showed Rv2345 to have a TPM domain at its N-terminal region and predicted it to have phosphatase activity. The TPM domain containing region of Rv2345 was cloned and expressed using pET28a vector in Escherichia coli and purified by Nickel affinity chromatography. The purified TPM domain was tested in vitro and our results confirmed it to have phosphatase activity. The enzyme activity was first checked and optimized with pNPP as substrate, followed by using ATP, which was also found to be used as substrate by the purified protein. Hence sequence analysis followed by in vitro studies characterizes TPM domain of Rv2345 to contain phosphatase activity.

  18. Comparative phytohormone profiles, lipid kinase and lipid phosphatase activities in barley aleurone, coleoptile, and root tissues.

    Science.gov (United States)

    Meringer, Maria V; Villasuso, Ana L; Pasquaré, Susana J; Giusto, Norma M; Machado, Estela E; Racagni, Graciela E

    2012-09-01

    We analyzed lipid kinase and lipid phosphatase activities and determined endogenous phytohormone levels by liquid chromatography-tandem mass spectrometry in root and coleoptile tissues following germination of barley (Hordeum vulgare) seeds. The enzymes showing highest activity in aleurone cells were diacylglycerol kinase (DAG-k, EC 2.7.1.107) and phosphatidate kinase (PA-k). The ratio of gibberellins (GAs) to abscisic acid (ABA) was 2-fold higher in aleurone than in coleoptile or root tissues. In coleoptiles, phosphatidylinositol 4-kinase (PI4-k, EC 2.7.1.67) showed the highest enzyme activity, and jasmonic acid (JA) level was higher than in aleurone. In roots, activities of PI4-k, DAG-k, and PA-k were similar, and salicylic acid (SA) showed the highest concentration. In the assays to evaluate the hydrolysis of DGPP (diacylglycerol pyrophosphate) and PA (phosphatidic acid) we observed that PA hydrolysis by LPPs (lipid phosphate phosphatases) was not modified; however, the diacylglycerol pyrophosphate phosphatase (DGPPase) was strikingly higher in coleoptile and root tissues than to aleurone. Relevance of these findings in terms of signaling responses and seedling growth is discussed.

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

    Science.gov (United States)

    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.

  20. Estudio de la fosfatasa ácida y alcalina en suelos de la Región Pampeana Norte del área sojera argentina Study of acid and alkaline phosphatase in soils of the Pampean North Region from argentine soybean area

    Directory of Open Access Journals (Sweden)

    Leticia Andrea Fernández

    2008-07-01

    ón de ambos métodos, es posible estudiar la fosfatasa ácida y alcalina de un suelo y obtener información sobre el potencial del mismo para movilizar Po.Transformation of organic phosphorus (Po into soluble inorganic phosphorus (Pi is called mineralization and is carried out by phosphatase enzymes. The present research focuses on the study of the phosphatase activity of five soils from the soybean area of the Northern Pampean region, by evaluating the phosphatase activity in soil samples and the number of bacteria and fungi with that activity. Soil samples were collected and the total number and phosphatase activity of cultivated heterotrophic aerobic bacteria (CHAB and cultivated fungi (CF was assessed. No significant differences were observed in the numbers of CHAB and CH between the studied soils. The number of bacteria with acid phosphatase activity was 6.85 10(5 CFU g-1 soil, while alkaline activity was 5.80 10(5 CFU g-1 soil. In contrast, the number of fungi with acid phosphatase activity was 1.78 10³ CFU g-1 soil and with alkaline activity was 1.77 10³ CFU g-1 soil. No significant differences were observed in the number of bacteria and fungi with both enzymes. However, acid activity was higher than alkaline activity in soil samples. Alkaline phosphatase activity ranged from 5.72 to 15.5 mg p- nitrofenol kg-1 soil h-1 while acid activity varied from 27.4 to 10(5 mg p-nitrofenol kg-1 soil h-1. There were significant differences in phosphatase activity between the soybean soils. Our results show that the mineralization activities of Po sources are in agreement with other cultivated soils. On the other hand, the number of bacteria and fungi complements the information on soil phosphatase activity. Clearly, both methods allow the study of alkaline and acid phosphatase activity in soil and give information about the soil potential to mobilize Po.

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

    Directory of Open Access Journals (Sweden)

    Abedini F

    2012-07-01

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

  2. Development of a single-chain variable fragment-alkaline phosphatase fusion protein and a sensitive direct competitive chemiluminescent enzyme immunoassay for detection of ractopamine in pork

    Energy Technology Data Exchange (ETDEWEB)

    Dong Jiexian; Li Zhenfeng; Lei Hongtao; Sun Yuanming [Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou 510642 (China); Ducancel, Frederic [CEA, iBiTec-S, Service de Pharmacologie et d' Immnoanalyse (SPI), CEA Saclay, F-91191 Gif sur Yvette (France); Xu Zhenlin [Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou 510642 (China); Boulain, Jean-Claude [CEA, iBiTec-S, Service de Pharmacologie et d' Immnoanalyse (SPI), CEA Saclay, F-91191 Gif sur Yvette (France); Yang Jinyi; Shen Yudong [Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou 510642 (China); Wang Hong, E-mail: gzwhongd@63.com [Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou 510642 (China)

    2012-07-29

    Graphical abstract: Detection model of dc-CLEIA based on anti-RAC scFv-AP fusion protein. Highlights: Black-Right-Pointing-Pointer The scFv-AP fusion protein against ractopamine (RAC) was produced. Black-Right-Pointing-Pointer A dc-CLEIA for RAC was developed based on the purified scFv-AP fusion protein. Black-Right-Pointing-Pointer The sensitivity of dc-CLEIA was 10 times as sensitive as dc-ELISA for RAC. Black-Right-Pointing-Pointer Recovery tests from pork samples were studied. Black-Right-Pointing-Pointer Good accuracy was obtained. - Abstract: A rapid, sensitive chemiluminescent enzyme immunoassay (CLEIA) for ractopamine (RAC) based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein was developed. The scFv gene was prepared by cloning the heavy- and light-chain variable region genes (V{sub H} and V{sub L}) from hybridoma cell line AC2, which secretes antibodies against RAC, and assembling V{sub H} and V{sub L} genes with a linker by means of splicing overlap extension polymerase chain reaction. The resulting scFv gene was inserted into the expression vector pLIP6/GN containing AP to produce the fusion protein in Escherichia coli strain BL21. The purified scFv-AP fusion protein was used to develop a direct competitive CLEIA (dcCLEIA) protocol for detection of RAC. The average concentration required for 50% inhibition of binding and the limit of detection of the assay were 0.25 {+-} 0.03 and 0.02 {+-} 0.004 ng mL{sup -1}, respectively, and the linear response range extended from 0.05 to 1.45 ng mL{sup -1}. The assay was 10 times as sensitive as the corresponding enzyme-linked immunosorbent assay based on the same fusion protein. Cross-reactivity studies showed that the fusion protein did not cross react with RAC analogs. DcCLEIA was used to analyze RAC spiked pork samples, and the validation was confirmed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS). The results showed a good correlation between

  3. Phosphatase activity and organic phosphorus turnover on a high Arctic glacier

    Directory of Open Access Journals (Sweden)

    M. Stibal

    2009-05-01

    Full Text Available Arctic glacier surfaces harbour abundant microbial communities consisting mainly of heterotrophic and photoautotrophic bacteria. The microbes must cope with low concentrations of nutrients and with the fact that both the dissolved and debris-bound nutrient pools are dominated by organic phases. Here we provide evidence that phosphorus (P is deficient in the supraglacial environment on a Svalbard glacier, we quantify the enzymatic activity of phosphatases in the system and we estimate the contribution of the microbes to the cycling of the dominant organic P in the supraglacial environment. Incubation of cryoconite debris revealed significant phosphatase activity in the samples (19–67 nmol MUP g−1 h−1. It was inhibited by inorganic P during incubations and had its optimum at around 30°C. The phosphatase activity measured at near-in situ temperature and substrate concentration suggests that the available dissolved organic P can be turned over by microbes within ~3–11 h on the glacier surface. By contrast, the amount of potentially bioavailable debris-bound organic P is sufficient for a whole ablation season. However, it is apparent that some of this potentially bioavailable debris-bound P is not accessible to the microbes.

  4. Root phosphatase activity, plant growth and phosphorus accumulation of maize genotypes

    Directory of Open Access Journals (Sweden)

    Machado Cynthia Torres de Toledo

    2004-01-01

    Full Text Available The activity of the enzyme phosphatase (P-ase is a physiological characteristic related to plant efficiency in relation to P acquisition and utilization, and is genetically variable. As part of a study on maize genotype characterization in relation to phosphorus (P uptake and utilization efficiency, two experiments were set up to measure phosphatase (P-ase activity in intact roots of six local and improved maize varieties and two sub-populations. Plants were grown at one P level in nutrient solution (4 mg L-1 and the P-ase activity assay was run using 17-day-old plants for varieties and 24-day-old plants for subpopulations. Shoot and root dry matter yields and P concentrations and contents in plant parts were determined, as well as P-efficiency indexes. Root P-ase activity differed among varieties, and highest enzimatic activities were observed in two local varieties -'Catetão' and 'Caiano' -and three improved varieties -'Sol da Manhã', 'Nitrodente' and 'BR 106'. 'Carioca', a local variety, had the lowest activity. Between subpopulations, 'ND2', with low yielding and poorly P-efficient plants, presented higher root P-ase activity as compared to 'ND10', high yielding and highly P-efficient plants. In general, subpopulations presented lower P-ase activities as compared to varieties. Positive and/or negative correlations were obtained between P-ase activity and P-efficiency characteristics, specific for the genotypes, not allowing inference on a general and clear association between root-secreted phosphatase and dry matter production or P acquisition. Genotypic variability must be known and considered before using P-ase activity as an indicator of P nutritional status, or P tolerance, adaptation and efficiency under low P conditions.

  5. Phosphate solubilizing bacteria and alkaline phosphatase activity in coastal waters off Trivandrum

    Digital Repository Service at National Institute of Oceanography (India)

    Mamatha, S.S.; Gobika, A.; Janani, P.

    nutrient to all life, being a structural and functional component of organisms. Large quantities of elemental phosphorus are immobilized in living organism, and are locked up in the sediments as insoluble organic and inorganic compounds (De et. al.,2011... of phenol released. Suitable controls were maintained in all analyses and the results represent the average of replicates. Biological data was log transformed for statistical analysis. Inter- relationships between biological and environmental parameters...

  6. Amino acid sequence of the cold-active alkaline phosphatase from Atlantic cod (Gadus morhua)

    DEFF Research Database (Denmark)

    Asgeirsson, Bjarni; Nielsen, Berit Noesgaard; Højrup, Peter

    2003-01-01

    -linked glycosylation sites were found. The glycan structure was determined as complex biantennary in type with fucose and sialic acid attached, although a trace of complex tri-antennary structure was also observed. A three-dimensional model was obtained by homology modelling using the human placental AP scaffold. Cod...

  7. Benomyl inhibits phosphorus transport but not fungal alkaline phosphatase activity in a Glomus–cucumber symbiosis

    DEFF Research Database (Denmark)

    Larsen, John; Thingstrup, Ida; Jakobsen, Iver;

    1996-01-01

    compartment systems where nylon mesh was used to separate n root-free hyphal compartment (HC) and a root + hyphal compartment(RHC) from The main root compartment (RC). Non-mycorrhizal control plants were grown in similar growth units. After 6 wk benomyl was applied to the plants in three ways: as soil...

  8. Bone-specific alkaline phosphatase and tartrate-resistant acid phosphatase levels in patients with cleidocranial dysplasia: one case report%颅骨锁骨发育不良患者骨特异性碱性磷酸酶和抗酒石酸酸性磷酸酶的水平:1例报告

    Institute of Scientific and Technical Information of China (English)

    李阳飞; 秦晗; 徐宏志

    2011-01-01

    BACKGROUND: Bone-specific alkaline phosphatase and tartrate-resistant acid phosphatase levels are closely related to skeletal development. OBJECTIVE: To observe bone-specific alkaline phosphatase and tartrate-resistant acid phosphatase levels in patients with cleidocranial dysplasia (CCD). METHODS: Clinical data of one case of CCD were collected. The osseous malformation over the entire body was examined by panoramic radiography. A CCD phenotype was defined by the presence of hypoplastic clavicles and delayed closure of the anterior fontanels in addition to the presence of classic eraniofaeial features. Laboratory examination was used to detect the change of bone-specific alkaline phosphatase and tartrate-resistant acid phosphatase levels. RESULTS AND CONCLUSION: The patients had obvious clinical characteristics of CCD, such as clavicle rudiment or absence, delayed closure of cranial fontanels and sutures, supernumerary and late erupting teeth, wide pubic symphysis, and other skeletal anomalies. The skeletal abnormalities as well as oral manifestations of the syndrome were variable within the affected patient. But abnormal chromosome was not found in this patient. Bone-specific alkaline phosphatase and tartrate-resistant acid phosphatase levels were increased. These findings suggest that CCD patients may accompany with the genetic variation of tartrate-resistant acid phosphatase.%背景:骨特异性碱性磷酸酶和抗酒石酸酸性磷酸酶与骨骼的发育密切相关.目的:观察颅骨锁骨发育不良患者骨特异性碱性磷酸酶和抗酒石酸酸性磷酸酶的变化.方法:收集1 例颅骨锁骨发育不良患者临床资料,利用全颌曲面断层片检查全面了解患者的颌骨发育情况;详细进行口腔检查,记录恒牙萌出及乳牙迟萌以及埋伏牙、多生牙等;综合临床资料和实验室检查确定临床诊断,并检测患者骨特异性碱性磷酸酶和抗酒石酸酸性磷酸酶的变化.结果与结论:该患者有

  9. Fine-Tuning of Pten Localization and Phosphatase Activity Is Essential for Zebrafish Angiogenesis

    Science.gov (United States)

    Stumpf, Miriam; Blokzijl-Franke, Sasja; den Hertog, Jeroen

    2016-01-01

    The lipid- and protein phosphatase PTEN is an essential tumor suppressor that is highly conserved among all higher eukaryotes. As an antagonist of the PI3K/Akt cell survival and proliferation pathway, it exerts its most prominent function at the cell membrane, but (PIP3-independent) functions of nuclear PTEN have been discovered as well. PTEN subcellular localization is tightly controlled by its protein conformation. In the closed conformation, PTEN localizes predominantly to the cytoplasm. Opening up of the conformation of PTEN exposes N-terminal and C-terminal regions of the protein that are required for both interaction with the cell membrane and translocation to the nucleus. Lack of Pten leads to hyperbranching of the intersegmental vessels during zebrafish embryogenesis, which is rescued by expression of exogenous Pten. Here, we observed that expression of mutant PTEN with an open conformation rescued the hyperbranching phenotype in pten double homozygous embryos and suppressed the increased p-Akt levels that are characteristic for embryos lacking Pten. In addition, in pten mutant and wild type embryos alike, open conformation PTEN induced stalled intersegmental vessels, which fail to connect with the dorsal longitudinal anastomotic vessel. Functional hyperactivity of open conformation PTEN in comparison to wild type PTEN seems to result predominantly from its enhanced recruitment to the cell membrane. Enhanced recruitment of phosphatase inactive mutants to the membrane did not induce the stalled vessel phenotype nor did it rescue the hyperbranching phenotype in pten double homozygous embryos, indicating that PTEN phosphatase activity is indispensable for its regulatory function during angiogenesis. Taken together, our data suggest that PTEN phosphatase activity needs to be carefully fine-tuned for normal embryogenesis and that the control of its subcellular localization is a key mechanism in this process. PMID:27138341

  10. 热灭活法与干化学法检测骨碱性磷酸酶技术的对照分析%Comparison between heat-inactivity method and dry chemistry method on assaying bone alkaline phosphatase

    Institute of Scientific and Technical Information of China (English)

    李常林; 张艳; 马胜; 林虹梅; 卢秀芬

    2009-01-01

    Objective Assaying the content of bone alkaline phosphatase in childen's large batch samples by the method of heat-inactivity in compensation for the deficiency of dry chemistry.Methods Collecting finger blood and examinging alkaline phosphatase by biochemistry analyzer before and after heat-inactivity of the samples,then calculating the ratio of results between after and before heat-inactivity to filtrate suspect positive cases.Detecting the suspect positive cases by dry chemistry method,comparing the results of the two methods.Results The positive results of this method are in accord with the results of dry chemistry.Conclusion In contrast with dry chemistry method, the method of heat-inactivity is in possession of the characteristics of lower cost,quick analysis,quantity,fitting for large batch samples asssaying,etc.%目的 应用热灭活法检测大批量儿童骨碱性磷酸酶(bone alkaline phosphatase,BALP)项目以弥补干化学法测定BALP之不足.方法 采末梢血于生化分析仪上测定灭活前后的碱性磷酸酶(alkaline phosphatase,ALP)值,计算灭活后与灭活前ALP的百分比以筛出疑似阳性病例.热灭活法筛查出的BALP阳性病例,再用干化学法测定其BALP,之后将二种方法的测定结果进行对照.结果 热灭活法筛查出的BALP阳性结果同干化学法测定的BALP结果基本一致.结论 热灭活法检测骨碱性磷酸酶技术具有成本低、检测速度快、可定量分析、适合大批量BALP检测的优点.

  11. Glucose-induced posttranslational activation of protein phosphatases PP2A and PP1 in yeast

    Institute of Scientific and Technical Information of China (English)

    Dries Castermans; Ils Somers; Johan Kriel; Wendy Louwet; Stefaan Wera; Matthias Versele; Veerle Janssens; Johan M Thevelein

    2012-01-01

    The protein phosphatases PP2A and PP1 are major regulators of a variety of cellular processes in yeast and other eukaryotes.Here,we reveal that both enzymes are direct targets of glucose sensing.Addition of glucose to glucosedeprived yeast cells triggered rapid posttranslational activation of both PP2A and PP1.Glucose activation of PP2A is controlled by regulatory subunits Rts1,Cdc55,Rrd1 and Rrd2.It is associated with rapid carboxymethylation of the catalytic subnnits,which is necessary but not sufficient for activation.Glucose activation of PP1 was fully dependent on regulatory subunits Reg1 and Shp1.Absence of Gac1,GIc8,Reg2 or Red1 partially reduced activation while Pig1 and Pig2 inhibited activation.Full activation of PP2A and PP1 was also dependent on subunits classically considered to belong to the other phosphatase.PP2A activation was dependent on PP1 subunits Reg1 and Shpl while PP1 activation was dependent on PP2A subunit Rts1.Rts1 interacted with both Pph21 and Glc7 under different conditions and these interactions were Regl dependent.Regl-GIc7 interaction is responsible for PP1 involvement in the main glucose repression pathway and we show that deletion of Shpl also causes strong derepression of the invertase gene SUC2.Deletion of the PP2A subunits Pph21 and Pph22,Rrd1 and Rrd2,specifically enhanced the derepression level of SUC2,indicating that PP2A counteracts SUC2 derepression.Interestingly,the effect of the regulatory subunit Rtsl was consistent with its role as a subunit of both PP2A and PP1,affecting derepression and repression of SUC2,respectively.We also show that abolished phosphatase activation,except by reg1A,does not completely block Snf1 dephosphorylation after addition of glucose.Finally,we show that glucose activation of the cAMP-PKA (protein kinase A)pathway is required for glucose activation of both PP2A and PP1.Our results provide novel insight into the complex regulatory role of these two major protein phosphatases in glucose

  12. Alkaline activated slag cements. Determination of reaction degree

    Directory of Open Access Journals (Sweden)

    Fernández-Jiménez, A.

    2001-03-01

    Full Text Available The aim of the present work was to evaluate the validity of non-calorimetric different methods, used in the determination of reaction degree of alkaline activated slag pastes. The methods used were: (a chemical separation by methanol-salicylic acid; (b determination of the weight loss mass between 100-600°C in TG curves, associated to chemically combined water; (c quantification of the -74 ppm signal in 29Si MAS-NMR spectra. The parameters considered in the process were: nature of the alkaline activator (Waterglass, Na2CO3 and NaOH, activator concentration (4% and 3% Na2O in mass with respect to the slag, curing temperature (25 and 45°C, slag specific surface (460 and 900 m2/kg and time of reaction (from 7 days to 18 months. The results obtained indicate that none of the three methods is definitive but complementary and they provide to follow the reactive evolution of the alkaline activated slag cements. The method based on the quantification of the -74 ppm signal in the 29Si MAS NMR is the most suitable method.

    El objetivo del presente trabajo fue evaluar la validez de diferentes métodos, no calorimétricos, utilizados en la determinación del grado de reacción de pastas de escoria activada alcalinamente. Los métodos utilizados fueron: (a método de separación química por disolución en metanol ácido-salicílico; (b determinación de las pérdidas de masa entre 100-600°C en las curvas de TG, pérdidas asociadas a la cantidad de agua químicamente combinada: (c cuantificación de la señal de -74 ppm de los espectros de 29Si RMN MAS. Las variables consideradas en el proceso fueron: naturaleza del activador alcalino (Waterglass, Na2CO3 y NaOH, concentración del activador (4% y 3% de Na2O en masa respecto a la escoria, temperatura de curado (25 y 45°C, superficie específica de la escoria (460 y 900 m2/kg y

  13. Effect of gamma-irradiation on nucleic acids, proteins, respiration and phosphatase activity of carrot callus cultures

    International Nuclear Information System (INIS)

    Callus tissue cultures were subjected to 60Co qamma irradiation at 0.5 Krad and analysed for nucleic acids, proteins, respiration rate and phosphatase activity on 0, 10, 20 and 30 days. The RNA contents and respiratory rates were enhanced as a result of irradiation. The RNA contents were reduced than their non-irradiated counterparts. The acid phosphatase activity was enhanced immediately after irradiation, declined on 10th and 20th day and more thereafter. (author)

  14. Label-free electrochemical impedance detection of kinase and phosphatase activities using carbon nanofiber nanoelectrode arrays

    Science.gov (United States)

    Li, Yifen; Syed, Lateef; Liu, Jianwei; Hua, Duy H.; Li, Jun

    2012-01-01

    We demonstrate the feasibility of a label-free electrochemical method to detect the kinetics of phosphorylation and dephosphorylation of surface-attached peptides catalyzed by kinase and phosphatase, respectively. The peptides with a sequence specific to c-Src tyrosine kinase and protein tyrosine phosphatase 1B (PTP1B) were first validated with ELISA-based protein tyrosine kinase assay and then functionalized on vertically aligned carbon nanofiber (VACNF) nanoelectrode arrays (NEAs). Real-time electrochemical impedance spectroscopy (REIS) measurements showed reversible impedance changes upon the addition of c-Src kinase and PTP1B phosphatase. Only a small and unreliable impedance variation was observed during the peptide phosphorylation, but a large and fast impedance decrease was observed during the peptide dephosphorylation at different PTP1B concentrations. The REIS data of dephosphorylation displayed a well-defined exponential decay following the Michaelis-Menten heterogeneous enzymatic model with a specific constant, kcat/Km, of (2.1 ± 0.1) × 107 M−1 s−1. Consistent values of the specific constant was measured at PTP1B concentration varying from 1.2 to 2.4 nM with the corresponding electrochemical signal decay constant varying from 38.5 to 19.1 s. This electrochemical method can be potentially used as a label-free method for profiling enzyme activities in fast reactions. PMID:22935373

  15. PTEN蛋白磷酸酶活性的作用%The Function of PTEN's Protein Phosphatase Activity

    Institute of Scientific and Technical Information of China (English)

    郝礼森; 刘小娟; 张晓岚

    2012-01-01

    PTEN一个具有磷酸酶活性的肿瘤抑制基因,是编码具有脂质磷酸酶活性和蛋白磷酸酶活性的双重特异性磷酸酶,其缺失或功能异常与人类恶性肿瘤的发生发展密切相关.PTEN的脂质磷酸酶活性和蛋白磷酸酶活性在调控肿瘤细胞的生物学行为、维持细胞正常的生理活动中均发挥了重要作用.但二者的作用重点及机制仍有不同,其蛋白磷酸酶活性主要侧重于调控细胞的黏附迁移及侵袭.为更好地认识PTEN蛋白磷酸酶活性的作用,该文对PTEN蛋白磷酸酶活性的作用及其机制作一简要综述.%Phosphatase and tensin homolog deleted on chromosome ten (PTEN), a tumor suppressorgene found to exhibit a dual specificity protein and lipid phosphatase activity, occurs deletion or dysfunction in a wide range of advanced cancers. Both lipid phosphatase activity and protein phosphatase activity of PTEN play an important role in regulating the biological behavior of tumor cell and maintaining normal cellular physiologic function. However, there are differences between lipid phosphatase activity and protein phosphatase activity in the focal point and mechanism of the function. And the function of its protein phosphatase activity is mainly to regulate the adhesion, migration and invasion of cell. In this article, the funcion of PTEN's protein phosphatase activity and its mechanisms were briefly summarized for a better understanding of its role.

  16. Allosterically Regulated Phosphatase Activity from Peptide-PNA Conjugates Folded Through Hybridization.

    Science.gov (United States)

    Machida, Takuya; Dutt, Som; Winssinger, Nicolas

    2016-07-18

    The importance of spatial organization in short peptide catalysts is well recognized. We synthesized and screened a library of peptides flanked by peptide nucleic acids (PNAs) such that the peptide would be constrained in a hairpin loop upon hybridization. A screen for phosphatase activity led to the discovery of a catalyst with >25-fold rate acceleration over the linear peptide. We demonstrated that the hybridization-enforced folding of the peptide is necessary for activity, and designed a catalyst that is allosterically controlled using a complementary PNA sequence. PMID:27320214

  17. Phosphatase activities in rice-planting meadow brown soil and their responses to fertilization%草甸棕壤水稻田磷酸酶活性及对施肥措施的响应

    Institute of Scientific and Technical Information of China (English)

    沈菊培; 陈振华; 陈利军

    2005-01-01

    This study is aimed to investigate the activities of phosphomonoesterase (acid-, neutral-, and alkaline-), phosphodiesterase and phosphotriesterase in a rice-planting meadow brown soil at the lower reach of Liao River, and their responses to different fertilization treatments. The results showed that there was no significant difference in soil total P and organic P contents among all treatments, but soil available P content was significantly higher in treatment OM than in other treatments. Soil acid-and neutral phosphomonoesterase had a higher activity than alkaline phosphomonoesterase and phosphodiesterase, while phosphotriesterase had the lowest activity. No significant difference was found in phosphatase activities between different fertilization treatments. Soil acid phosphomonoesterase activity had a significant correlation with soil total P and available P contents, while soil phosphodiesterase activity significantly correlated with soil organic P content.

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

    Science.gov (United States)

    Jakka, Siva R K; Gong, Liang; Hasler, James; Banerjee, Rahul; Sheets, Joel J; Narva, Kenneth; Blanco, Carlos A; Jurat-Fuentes, Juan L

    2015-12-04

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

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

    Science.gov (United States)

    Jakka, Siva R K; Gong, Liang; Hasler, James; Banerjee, Rahul; Sheets, Joel J; Narva, Kenneth; Blanco, Carlos A; Jurat-Fuentes, Juan L

    2016-02-01

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

  20. Astragaloside Ⅱ triggers T cell activation through regulation of CD45 protein tyrosine phosphatase activity

    Institute of Scientific and Technical Information of China (English)

    Chun-ping WAN; Li-xin GAO; Li-fei HOU; Xiao-qian YANG; Pei-lan HE; Yi-fu YANG; Wei TANG

    2013-01-01

    Aim:To investigate the immunomodulating activity of astragalosides,the active compounds from a traditional tonic herb Astragalus membranaceus Bge,and to explore the molecular mechanisms underlying the actions,focusing on CD45 protein tyrosine phosphatase (CD45 PTPase),which plays a critical role in T lymphocyte activation.Methods:Primary splenocytes and T cells were prepared from mice.CD45 PTPase activity was assessed using a colorimetric assay.Cell proliferation was measured using a [3H]-thymidine incorporation assay.Cytokine proteins and mRNAs were examined with ELISA and RT-PCR,respectively.Activation markers,including CD25 and CD69,were analyzed using flow cytometry.Activation of LCK (Tyr505) was detected using Western blot analysis.Mice were injected with the immunosuppressant cyclophosphamide (CTX,80 mg/kg),and administered astragaloside Ⅱ (50 mg/kg).Results:Astragaloside Ⅰ,Ⅱ,Ⅲ,and Ⅳ concentration-dependently increased the CD45-mediated of pNPP/OMFP hydrolysis with the EC50 values ranged from 3.33 to 10.42 μg/mL.Astragaloside Ⅱ (10 and 30 μg/mL) significantly enhanced the proliferation of primary splenocytes induced by ConA,alloantigen or anti-CD3.Astragaloside Ⅱ (30 μg/mL) significantly increased IL-2 and IFN-y secretion,upregulated the mRNA levels of IFN-y and T-bet in primary splenocytes,and promoted CD25 and CD69 expression on primary CD4+T cells upon TCR stimulation.Furthermore,astragaloside Ⅱ (100 ng/mL) promoted CD45-mediated dephosphorylation of LCK (Tyr505) in primary T cells,which could be blocked by a specific CD45 PTPase inhibitor.In CTX-induced immunosuppressed mice,oral administration of astragaloside Ⅱ restored the proliferation of splenic T cells and the production of IFN-Y and IL-2.However,astragaloside Ⅱ had no apparent effects on B cell proliferation.Conclusion:Astragaloside Ⅱ enhances T cell activation by regulating the activity of CD45 PTPase,which may explain why Astragalus membranaceus Bge is used as a tonic

  1. Effect of Combined Heavy Metal Pollution on Nitrogen Mineralization Potential,Urease and Phosphatase Activities in a Typic Udic Ferrisol

    Institute of Scientific and Technical Information of China (English)

    ZHENGCHUNRONG; TUCONG; 等

    1999-01-01

    Individual and combined effects of Cu,Pb,Zn and Cd on N mineralization,urease and phosphatase were examined in a Typic Udic Ferrisol in laboratory by employing and uniform design and a single factor design,Soil pollution caused by heavy metals inhibited N mineralization (N0 value)and urease and phosphatase activities.The combined pollution of metals alleviated their toxicity to N mineralization to some extent whereas aggravated the toxicity to urease and phosphatase.Phosphorous application could mitigat the toxic effect of heavy metals on phosphatase activities,while alleviating effect of N application on the toxicity of heavy metals to urease was inconsistent.However,the mitigating effect of the fertilizers was limited in heavily polluted soils.

  2. Water molecule network and active site flexibility of apo protein tyrosine phosphatase 1B

    DEFF Research Database (Denmark)

    Pedersen, A.K.; Peters, Günther H.J.; Møller, K.B.;

    2004-01-01

    Protein tyrosine phosphatase 1B (PTP1B) plays a key role as a negative regulator of insulin and leptin signalling and is therefore considered to be an important molecular target for the treatment of type 2 diabetes and obesity. Detailed structural information about the structure of PTP1B, including...... the conformation and flexibility of active-site residues as well as the water-molecule network, is a key issue in understanding ligand binding and enzyme kinetics and in structure-based drug design. A 1.95 Angstrom apo PTP1B structure has been obtained, showing four highly coordinated water molecules...

  3. Negative regulation of caspase 3-cleaved PAK2 activity by protein phosphatase 1

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The p21-activated kinase 2 (PAK2) is activated by binding of small G proteins, Cdc42 and Rac, or through proteolytic cleavage by caspases or caspase-like proteases. Activation by both small G protein and caspase requires autophosphorylation at Thr-402 of PAK2. Although activation of PAK2 has been investigated for nearly a decade, the mechanism of PAK2 downregulation is unclear. In this study, we have applied the kinetic theory of substrate reaction during modification of enzyme activity to study the regulation mechanism of PAK2 activity by the catalytic subunit of protein phosphatase 1 (PP1α). On the basis of the kinetic equation of the substrate reaction during the reversible phosphorylation of PAK2, all microscopic kinetic constants for the free enzyme and enzyme-substrate(s) complexes have been determined. The results indicate that (1) PP1α can act directly on phosphorylated Thr-402 in the activation loop of PAK2 and down-regulate its kinase activity; (2) binding of the exogenous protein/peptide substrates at the active site of PAK2 decreases both the rates of PAK2 autoactivation and inactivation. The present method provides a novel approach for studying reversible phosphorylation reactions. The advantage of this method is not only its usefulness in study of substrate effects on enzyme modification but also its convenience in study of modification reaction directly involved in regulation of enzyme activity. This initial study should provide a foundation for future structural and mechanistic work of protein kinases and phosphatases.

  4. Negative regulation of caspase 3-cleaved PAK2 activity by protein phosphatase 1

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The p21-activated kinase 2 (PAK2) is activated by binding of small G proteins, Cdc42 and Rac, or through proteolytic cleavage by caspases or caspase-like proteases. Activation by both small G protein and caspase requires autophosphorylation at Thr-402 of PAK2. Although activation of PAK2 has been investigated for nearly a decade, the mechanism of PAK2 downregulation is unclear. In this study, we have applied the kinetic theory of substrate reaction during modification of enzyme activity to study the regulation mechanism of PAK2 activity by the catalytic subunit of protein phosphatase 1 (PP1α). On the basis of the kinetic equation of the substrate reaction during the reversible phosphorylation of PAK2, all microscopic kinetic constants for the free enzyme and enzyme-substrate(s) complexes have been determined. The results indicate that (1) PP1α can act directly on phosphorylated Thr-402 in the acti-vation loop of PAK2 and down-regulate its kinase activity; (2) binding of the exogenous protein/peptide substrates at the active site of PAK2 decreases both the rates of PAK2 autoactivation and inactivation. The present method provides a novel approach for studying reversible phosphorylation reactions. The advantage of this method is not only its usefulness in study of substrate effects on enzyme modifica-tion but also its convenience in study of modification reaction directly involved in regulation of enzyme activity. This initial study should provide a foundation for future structural and mechanistic work of protein kinases and phosphatases.

  5. Using "On/Off" (19)F NMR/Magnetic Resonance Imaging Signals to Sense Tyrosine Kinase/Phosphatase Activity in Vitro and in Cell Lysates.

    Science.gov (United States)

    Zheng, Zhen; Sun, Hongbin; Hu, Chen; Li, Gongyu; Liu, Xiaomei; Chen, Peiyao; Cui, Yusi; Liu, Jing; Wang, Junfeng; Liang, Gaolin

    2016-03-15

    Tyrosine kinase and phosphatase are two important, antagonistic enzymes in organisms. Development of noninvasive approach for sensing their activity with high spatial and temporal resolution remains challenging. Herein, we rationally designed a hydrogelator Nap-Phe-Phe(CF3)-Glu-Tyr-Ile-OH (1a) whose supramolecular hydrogel (i.e., Gel 1a) can be subjected to tyrosine kinase-directed disassembly, and its phosphate precursor Nap-Phe-Phe(CF3)-Glu-Tyr(H2PO3)-Ile-OH (1b), which can be subjected to alkaline phosphatase (ALP)-instructed self-assembly to form supramolecular hydrogel Gel 1b, respectively. Mechanic properties and internal fibrous networks of the hydrogels were characterized with rheology and cryo transmission electron microscopy (cryo-TEM). Disassembly/self-assembly of their corresponding supramolecular hydrogels conferring respective "On/Off" (19)F NMR/MRI signals were employed to sense the activity of these two important enzymes in vitro and in cell lysates for the first time. We anticipate that our new (19)F NMR/magnetic resonance imaging (MRI) method would facilitate pharmaceutical researchers to screen new inhibitors for these two enzymes without steric hindrance. PMID:26901415

  6. Dynamic recruitment of protein tyrosine phosphatase PTPD1 to EGF stimulation sites potentiates EGFR activation.

    Directory of Open Access Journals (Sweden)

    Pedro Roda-Navarro

    Full Text Available Balanced activity of protein tyrosine kinases and phosphatases (PTPs controls tyrosine phosphorylation levels and, consequently, is needed to prevent pathologies like cancer. Phosphatase activity is tightly regulated in space and time. Thus, in order to understand how phospho-tyrosine signalling is regulated, the intracellular dynamics of PTPs should be investigated. Here, we have studied the intracellular dynamics of PTPD1, a FERM (four-point-one, ezrin, radixin, moesin domain-containing PTP that is over expressed in cancer cells and potentiates EGFR signalling. Whereas PTPD1 was excluded from E-cadherin rich cell-cell adhesions in epithelial cell monolayers, it diffused from the cytoplasm to those membranes in contact with the extracellular medium. Localisation of PTPD1 at the plasma membrane was mediated by its FERM domain and enabled the formation of EGFR/PTPD1-containing signalling complexes that pre-existed at the plasma membrane before EGF stimulation. PTPD1 and EGFR transiently co-localised at EGF stimulation sites until the formation of macropinosomes containing active species of EGFR. Interference of PTPD1 expression caused a decrease in EGFR phosphorylated species at the periphery of the cell. Presented data suggest that the transient formation of dynamic PTPD1/EGFR signalling complexes strengthens EGF signalling by promoting the spatial propagation of EGFR phosphorylated species.

  7. Effects of Betaine Aldehyde Dehydrogenase-Transgenic Soybean on Phosphatase Activities and Rhizospheric Bacterial Community of the Saline-Alkali Soil

    Directory of Open Access Journals (Sweden)

    Ying Nie

    2016-01-01

    Full Text Available The development of transgenic soybean has produced numerous economic benefits; however the potential impact of root exudates upon soil ecological systems and rhizospheric soil microbial diversity has also received intensive attention. In the present study, the influence of saline-alkali tolerant transgenic soybean of betaine aldehyde dehydrogenase on bacterial community structure and soil phosphatase during growth stages was investigated. The results showed that, compared with nontransgenic soybean as a control, the rhizospheric soil pH of transgenic soybean significantly decreased at the seedling stage. Compared to HN35, organic P content was 13.5% and 25.4% greater at the pod-filling stage and maturity, respectively. The acid phosphatase activity of SRTS was significantly better than HN35 by 12.74% at seedling, 14.03% at flowering, and 59.29% at podding, while alkaline phosphatase achieved maximum activity in the flowering stage and was markedly lower than HN35 by 13.25% at pod-filling. The 454 pyrosequencing technique was employed to investigate bacterial diversity, with a total of 25,499 operational taxonomic units (OTUs obtained from the 10 samples. Notably, the effect of SRTS on microbial richness and diversity of rhizospheric soil was marked at the stage of podding and pod-filling. Proteobacteria, Acidobacteria, and Actinobacteria were the dominant phyla among all samples. Compared with HN35, the relative abundance of Proteobacteria was lower by 2.01%, 2.06%, and 5.28% at the stage of seedling, at pod-bearing, and at maturity. In genus level, the relative abundance of Gp6, Sphingomonas sp., and GP4 was significantly inhibited by SRTS at the stage of pod-bearing and pod-filling.

  8. Research on Phosphatases of Belladona Leaves and Their Purification

    Directory of Open Access Journals (Sweden)

    M. Khorsand

    1957-01-01

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

  9. Phosphatase activity and organic phosphorus turnover on a high Arctic glacier

    Directory of Open Access Journals (Sweden)

    M. Stibal

    2009-02-01

    Full Text Available Arctic glacier surfaces harbor abundant microbial communities consisting mainly of heterotrophic and photoautotrophic bacteria. The microbes must cope with very low concentrations of nutrients and with the fact that both the dissolved and debris-bound nutrient pools are dominated by organic phases. Here we provide evidence that phosphorus (P is deficient and limiting in the supraglacial environment on a Svalbard glacier, we show how the microbial community responds to the P stress and we quantify the contribution of the microbes to the cycling of the dominant organic P in the supraglacial environment. Incubation of cryoconite debris revealed significant phosphatase activity in the samples (19–67 nmol MUP g−1 h−1, which was controlled by the concentration of inorganic P during incubations and had its optimum at around 30°C. The phosphatase activity rates measured at near-in situ temperature and substrate concentration imply that the available dissolved organic P can be turned over by microbes within ~3–11 h on the glacier surface. By contrast, the amount of potentially bioavailable debris-bound organic P is sufficient for a whole ablation season. However, it is apparent that some of this potentially bioavailable debris-bound P is not accessible to the microbes.

  10. Histochemical demonstration of activity of acid phosphatase and beta-glucuronidase in bovine incisor tooth germs

    DEFF Research Database (Denmark)

    Kirkeby, S; Salling, E; Moe, D

    1983-01-01

    Activity of acid phosphatase and beta-glucuronidase was shown in bovine preodontoblasts and preameloblasts prior to the onset of secretion. In the preameloblasts the rather weak reaction consisted of small discrete granules dispersed in the cytoplasm apical, lateral, and proximal to the nucleus....... After initiation of enamel formation, a change in localization and intensity of the colored reaction product was observed in the ameloblasts. The activity appeared stronger and was restricted to a narrow zone just apical to the nucleus. It is proposed that the acid hydrolases in the tooth forming cells...... are located to the Golgi complex. The differences in activity of acid hydrolases between bone and tooth forming cells are expounded....

  11. 966 cases of infants with bone alkaline phosphatase detection analysis%婴幼儿骨源性碱性磷酸酶966例检测分析

    Institute of Scientific and Technical Information of China (English)

    张炬光

    2013-01-01

    Objective to understand the incidence of infant rickets in Yulin city. Methods 966 cases of infants with bone alkaline phosphatase detection results of a retrospective analysis. Results the detection of bone alkaline phosphatase in 966 cases, 132 cases were rickets, a 13.66%detection rate, Among them, the boy was detected in 617 patients, the detection of rickets in 70 cases, the detection rate of 11.35%; the girl was detected in 349 patients, 62 cases were rickets, a 17.77%detection rate. Conclusion The detection of bone alkaline phosphatase in early diagnosis of rickets in infants and young children with sensitivity and specificity, the detection method is simple, to provide basis for prevention and treatment of rickets, is worth popularizing.%  目的评价骨源性碱性磷酸酶检测对婴幼儿佝偻病的早期诊断价值。方法对966例婴幼儿行骨源性碱性磷酸酶检测。结果966例婴幼儿中检出佝偻病治疗水平132例,阳性检出率为13.66%。其中,男孩检测617例,检出佝偻病治疗水平70例,阳性检出率为11.35%;女孩检测349例,检出佝偻病治疗水平62例,阳性检出率为17.77%。结论骨源性碱性磷酸酶检测对婴幼儿佝偻病的早期诊断具有一定的灵敏性和特异性,检测方法简单,能够为佝偻病的防治提供参考依据,值得推广应用。

  12. Timely Degradation of Wip1 Phosphatase by APC/C Activator Protein Cdh1 is Necessary for Normal Mitotic Progression.

    Science.gov (United States)

    Jeong, Ho-Chang; Gil, Na-Yeon; Lee, Ho-Soo; Cho, Seung-Ju; Kim, Kyungtae; Chun, Kwang-Hoon; Cho, Hyeseong; Cha, Hyuk-Jin

    2015-08-01

    Wip1 belongs to the protein phosphatase C (PP2C) family, of which expression is up-regulated by a number of external stresses, and serves as a stress modulator in normal physiological conditions. When overexpressed, premature dephosphorylation of stress-mediators by Wip1 results in abrogation of tumor surveillance, thus Wip1 acts as an oncogene. Previously, the functional regulation of Wip1 in cell-cycle progression by counteracting cellular G1 and G2/M checkpoint activity in response to DNA damage was reported. However, other than in stress conditions, the function and regulatory mechanism of Wip1 has not been fully determined. Herein, we demonstrated that protein regulation of Wip1 occurs in a cell cycle-dependent manner, which is directly governed by APC/C(Cdh1) at the end of mitosis. In particular, we also showed evidence that Wip1 phosphatase activity is closely associated with its own protein stability, suggesting that reduced phosphatase activity of Wip1 during mitosis could trigger its degradation. Furthermore, to verify the physiological role of its phosphatase activity during mitosis, we established doxycycline-inducible cell models, including a Wip1 wild type (WT) and phosphatase dead mutant (Wip1 DA). When ectopically expressing Wip1 WT, we observed a delay in the transition from metaphase to anaphase. In conclusion, these studies show that mitotic degradation of Wip1 by APC/C(Cdh1) is important for normal mitotic progression.

  13. Procyanidins Negatively Affect the Activity of the Phosphatases of Regenerating Liver.

    Directory of Open Access Journals (Sweden)

    Sven Stadlbauer

    Full Text Available Natural polyphenols like oligomeric catechins (procyanidins derived from green tea and herbal medicines are interesting compounds for pharmaceutical research due to their ability to protect against carcinogenesis in animal models. It is nevertheless still unclear how intracellular pathways are modulated by polyphenols. Monomeric polyphenols were shown to affect the activity of some protein phosphatases (PPs. The three phosphatases of regenerating liver (PRLs are close relatives and promising therapeutic targets in cancer. In the present study we show that several procyanidins inhibit the activity of all three members of the PRL family in the low micromolar range, whereas monomeric epicatechins show weak inhibitory activity. Increasing the number of catechin units in procyanidins to more than three does not further enhance the potency. Remarkably, the tested procyanidins showed selectivity in vitro when compared to other PPs, and over 10-fold selectivity toward PRL-1 over PRL-2 and PRL-3. As PRL overexpression induces cell migration compared to control cells, the effect of procyanidins on this phenotype was studied. Treatment with procyanidin C2 led to a decrease in cell migration of PRL-1- and PRL-3-overexpressing cells, suggesting the compound-dependent inhibition of PRL-promoted cell migration. Treatment with procyanidin B3 led to selective suppression of PRL-1 overexpressing cells, thereby corroborating the selectivity toward PRL-1- over PRL-3 in vitro. Together, our results show that procyanidins negatively affect PRL activity, suggesting that PRLs could be targets in the polypharmacology of natural polyphenols. Furthermore, they are interesting candidates for the development of PRL-1 inhibitors due to their low cellular toxicity and the selectivity within the PRL family.

  14. Redox Modulation of PTEN Phosphatase Activity by Hydrogen Peroxide and Bisperoxidovanadium Complexes.

    Science.gov (United States)

    Lee, Chang-Uk; Hahne, Gernot; Hanske, Jonas; Bange, Tanja; Bier, David; Rademacher, Christoph; Hennig, Sven; Grossmann, Tom N

    2015-11-01

    PTEN is a dual-specificity protein tyrosine phosphatase. As one of the central tumor suppressors, a thorough regulation of its activity is essential for proper cellular homeostasis. The precise implications of PTEN inhibition by reactive oxygen species (e.g. H2 O2 ) and the subsequent structural consequences remain elusive. To study the effects of PTEN inhibition, bisperoxidovanadium (bpV) complexes serve as important tools with the potential for the treatment of nerve injury or cardiac ischemia. However, their mode of action is unknown, hampering further optimization and preventing therapeutic applications. Based on protein crystallography, mass spectrometry, and NMR spectroscopy, we elucidate the molecular basis of PTEN inhibition by H2O2 and bpV complexes. We show that both molecules inhibit PTEN via oxidative mechanisms resulting in the formation of the same intramolecular disulfide, therefore enabling the reactivation of PTEN under reductive conditions. PMID:26418532

  15. Research on Phosphatases of Belladona Leaves and Their Purification (Part 1

    Directory of Open Access Journals (Sweden)

    M. Khorsand

    1956-12-01

    Full Text Available Belladona leaves as well as all other studied leaves contains two distinct phosphatase fractions belonging respectively to types II and IIIi the major parts of these enzymes is extraetible by water. It was not possible to extract the non soluble fraction which is solidly retained by the cellular constituents. Phosphatase II does not differ from other phosphatnses of the same type. Whereas phosphatase III is distinetely different from enzymes of the same type of vegetal or animal origins. It is activated by bivalent metallic ions which are specific activators of the alkaline phcspbatnses: Mg-Zn-Ni and Co.

  16. Research on Phosphatases of Belladona Leaves and Their Purification (Part 1

    Directory of Open Access Journals (Sweden)

    M. Khorsand

    1956-07-01

    Full Text Available Belladona leaves as well as all other studied leaves contains two distinct phosphatase fractions belonging respectively to types II and IIIi the major parts of these enzymes is extraetible by water. It was not possible to extract the non soluble fraction which is solidly retained by the cellular constituents. Phosphatase II does not differ from other phosphatnses of the same type. Whereas phosphatase III is distinetely different from enzymes of the same type of vegetal or animal origins. It is activated by bivalent metallic ions which are specific activators of the alkaline phcspbatnses: Mg-Zn-Ni and Co.

  17. Iron content and acid phosphatase activity in hepatic parenchymal lysosomes of patients with hemochromatosis before and after phlebotomy treatment

    Energy Technology Data Exchange (ETDEWEB)

    Cleton, M.I.; de Bruijn, W.C.; van Blokland, W.T.; Marx, J.J.; Roelofs, J.M.; Rademakers, L.H.

    1988-03-01

    Lysosomal structures in liver parenchymal cells of 3 patients with iron overload and of 3 subjects without iron-storage disorders were investigated. A combination of enzyme cytochemistry--with cerium as a captive ion to demonstrate lysosomal acid phosphatase activity--and electron probe X-ray microanalysis (EPMA) was used. We were able (1) to define and quantify lysosomal structures as lysosomes, siderosomes, or residual bodies, (2) to quantify the amount of iron and cerium simultaneously in these structures, and (3) to evaluate a possible relation between iron storage and enzyme activity. With histopathologically increased iron storage, the number of siderosomes had increased at the cost of lysosomes, with a corresponding increase in acid phosphatase activity in both organelles. In histopahtologically severe iron overload, however, acid phosphatase activity was low or not detectable and most of the iron was stored in residual bodies. After phlebotomy treatment, the number of siderosomes had decreased in favor of the lysosomes, approaching values obtained in control subjects, and acid phosphatase activity was present in all iron-containing structures. In this way a relationship between iron storage and enzyme activity was established. The iron content of the individual lysosomal structures per unit area had increased with histopathologically increased iron storage and had decreased after phlebotomy treatment. From this observation, it is concluded that the iron status of the patient is not only reflected by the amount of iron-containing hepatocytes but, as well, by the iron content lysosomal unit area.

  18. Radioprotective effect of Panax ginseng on the phosphatases and lipid peroxidation level in testes of Swiss albino mice

    Energy Technology Data Exchange (ETDEWEB)

    Kumar M.; Sharma M.K.; Saxena P.S.; Kumar A. [Rajasthan Univ., Jaipur (India)

    2003-03-01

    The Panax ginseng has been used as traditional medicine for past several years among oriental people. The present investigation has been made to assess the radioprotective efficacy of ginseng root extract in the testicular enzymes of Swiss albino mice. The Swiss albino mice were divided into different groups. Ginseng treated group: The animals were administered 10 mg/kg body weight ginseng root extract intraperitoneal (i.p.). Radiation treated group: The animals were exposed to 8 Gy gamma radiation at the dose rate of 1.69 Gy/min at the distance of 80 cm. Combination group: Animals were administered ginseng extract continuously for 4 d and on 4th day they were irradiated to 8 Gy gamma radiation after 30 min of extract administration. The animals from above groups were autopsied on day 1, 3, 7, 14 and 30. Biochemical estimations of acid and alkaline phosphatases and Lipid peroxidation (LPO) in testes were done. In ginseng treated group acid and alkaline phosphatases activity and LPO level did not show any significant alteration. In irradiated animals there was a significant increase in acid phosphatase activity and LPO level. However, significant decline in alkaline phosphatase activity was observed. The treatment of ginseng before irradiation causes significant decrease in acid phosphatase and LPO level and significant increase in alkaline phosphatase activity. One of the cause of radiation damage is lipid peroxidation. Due to lipid peroxidation, lysosomal membrane permeability alters and thus results in release of hydrolytic enzymes. So, an increase in acid phosphatase was noticed after radiation treatment. The alkaline phosphatase activity is associated with membrane permeability and different stages of spermatogenesis. Due to membrane damage and depletion of germ cells of testes after irradiation the enzyme activity was decreased. Ginseng markedly inhibits lipid peroxidation. It acts in indirect fashion to protect radical processes by inhibition of initiation of

  19. The relationship between human skeletal muscle pyruvate dehydrogenase phosphatase activity and muscle aerobic capacity.

    Science.gov (United States)

    Love, Lorenzo K; LeBlanc, Paul J; Inglis, J Greig; Bradley, Nicolette S; Choptiany, Jon; Heigenhauser, George J F; Peters, Sandra J

    2011-08-01

    Pyruvate dehydrogenase (PDH) is a mitochondrial enzyme responsible for regulating the conversion of pyruvate to acetyl-CoA for use in the tricarboxylic acid cycle. PDH is regulated through phosphorylation and inactivation by PDH kinase (PDK) and dephosphorylation and activation by PDH phosphatase (PDP). The effect of endurance training on PDK in humans has been investigated; however, to date no study has examined the effect of endurance training on PDP in humans. Therefore, the purpose of this study was to examine differences in PDP activity and PDP1 protein content in human skeletal muscle across a range of muscle aerobic capacities. This association is important as higher PDP activity and protein content will allow for increased activation of PDH, and carbohydrate oxidation. The main findings of this study were that 1) PDP activity (r(2) = 0.399, P = 0.001) and PDP1 protein expression (r(2) = 0.153, P = 0.039) were positively correlated with citrate synthase (CS) activity as a marker for muscle aerobic capacity; 2) E1α (r(2) = 0.310, P = 0.002) and PDK2 protein (r(2) = 0.229, P =0.012) are positively correlated with muscle CS activity; and 3) although it is the most abundant isoform, PDP1 protein content only explained ∼ 18% of the variance in PDP activity (r(2) = 0.184, P = 0.033). In addition, PDP1 in combination with E1α explained ∼ 38% of the variance in PDP activity (r(2) = 0.383, P = 0.005), suggesting that there may be alternative regulatory mechanisms of this enzyme other than protein content. These data suggest that with higher muscle aerobic capacity (CS activity) there is a greater capacity for carbohydrate oxidation (E1α), in concert with higher potential for PDH activation (PDP activity). PMID:21596918

  20. Stimulation of receptor protein-tyrosine phosphatase alpha activity and phosphorylation by phorbol ester

    DEFF Research Database (Denmark)

    den Hertog, J; Sap, J; Pals, C E;

    1995-01-01

    Receptor Protein-Tyrosine Phosphatase alpha (RPTP alpha) is a transmembrane protein with two cytoplasmic catalytic protein-tyrosine phosphatase (PTP) domains and a relatively short (123 amino acids) extracellular domain. Here we report that treatment of transfected cells that express RPTP alpha...

  1. Synthesis of Zeolites by Alkaline Activation of Fly Ash

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    In terms of mineral transformation, and chemical composition of acid-soluble component as a function of reaction time, the effect of alkaline solution on zeolite-like fly ash was studied by employing fly ash and NaOH solution as starting materials. When fly ash and 1€? 0mol/L NaOH solution were processed at 100℃ for 24h with 1:10 W/S rat io in a relatively closed system, powder XRD patterns of resulting pro ducts indicated the formation of various zeolites. Zeolite P crystalli zed early at low alkaline concentration, which was replaced then by ze olites X and A. At high concentration, hydroxy sodalite was the only n ew phase. Quartz, in fly ash and NaOH solution system, gradually disso lved, and mullite, however, remained stable. It was concluded that, wi th Al/Si and Na/Si finally reaching equilibrium in molar ratio, compos ition of starting mixtures affects the crystallization of zeolite from fly ash.

  2. Key factors governing alkaline pretreatment of waste activated sludge

    Institute of Scientific and Technical Information of China (English)

    Xianli Shi; Li Deng; Fangfang Sun; Jieyu Liang; Xu Deng

    2015-01-01

    Alkaline pretreatment is an effective technology to disintegrate sewage sludge, where alkali dosage and sludge concentration are two important factors. pH value or alkali concentration is usually adjusted in order to deter-mine a proper dosage of alkali. Our work has found that this is not a good strategy. A new parameter, the ratio of alkali to sludge (Ra/s), is more sensitive in controlling the alkali dosage. The sludge concentration Cs and reten-tion time t are two other important factors to consider. The validity of these arguments is confirmed with model-ing and experiments. The individual effect of Ra/s, Cs and t was studied separately. Then the combined effect of these three factors was evaluated. The sludge disintegration degree of 44.7%was achieved with the optimized factors. Furthermore, an alkaline-microwave combined pretreatment process was carried out under these optimized conditions. A high disintegration degree of 62.3%was achieved while the energy consumption of microwave was much lower than previously reported.

  3. The molecular chaperone Hsp70 activates protein phosphatase 5 (PP5) by binding the tetratricopeptide repeat (TPR) domain.

    Science.gov (United States)

    Connarn, Jamie N; Assimon, Victoria A; Reed, Rebecca A; Tse, Eric; Southworth, Daniel R; Zuiderweg, Erik R P; Gestwicki, Jason E; Sun, Duxin

    2014-01-31

    Protein phosphatase 5 (PP5) is auto-inhibited by intramolecular interactions with its tetratricopeptide repeat (TPR) domain. Hsp90 has been shown to bind PP5 to activate its phosphatase activity. However, the functional implications of binding Hsp70 to PP5 are not yet clear. In this study, we find that both Hsp90 and Hsp70 bind to PP5 using a luciferase fragment complementation assay. A fluorescence polarization assay shows that Hsp90 (MEEVD motif) binds to the TPR domain of PP5 almost 3-fold higher affinity than Hsp70 (IEEVD motif). However, Hsp70 binding to PP5 stimulates higher phosphatase activity of PP5 than the binding of Hsp90. We find that PP5 forms a stable 1:1 complex with Hsp70, but the interaction appears asymmetric with Hsp90, with one PP5 binding the dimer. Solution NMR studies reveal that Hsc70 and PP5 proteins are dynamically independent in complex, tethered by a disordered region that connects the Hsc70 core and the IEEVD-TPR contact area. This tethered binding is expected to allow PP5 to carry out multi-site dephosphorylation of Hsp70-bound clients with a range of sizes and shapes. Together, these results demonstrate that Hsp70 recruits PP5 and activates its phosphatase activity which suggests dual roles for PP5 that might link chaperone systems with signaling pathways in cancer and development.

  4. Activation of the low molecular weight protein tyrosine phosphatase in keratinocytes exposed to hyperosmotic stress.

    Directory of Open Access Journals (Sweden)

    Rodrigo A Silva

    Full Text Available Herein, we provide new contribution to the mechanisms involved in keratinocytes response to hyperosmotic shock showing, for the first time, the participation of Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP activity in this event. We reported that sorbitol-induced osmotic stress mediates alterations in the phosphorylation of pivotal cytoskeletal proteins, particularly Src and cofilin. Furthermore, an increase in the expression of the phosphorylated form of LMWPTP, which was followed by an augment in its catalytic activity, was observed. Of particular importance, these responses occurred in an intracellular milieu characterized by elevated levels of reduced glutathione (GSH and increased expression of the antioxidant enzymes glutathione peroxidase and glutathione reductase. Altogether, our results suggest that hyperosmostic stress provides a favorable cellular environment to the activation of LMWPTP, which is associated with increased expression of antioxidant enzymes, high levels of GSH and inhibition of Src kinase. Finally, the real contribution of LMWPTP in the hyperosmotic stress response of keratinocytes was demonstrated through analysis of the effects of ACP1 gene knockdown in stressed and non-stressed cells. LMWPTP knockdown attenuates the effects of sorbitol induced-stress in HaCaT cells, mainly in the status of Src kinase, Rac and STAT5 phosphorylation and activity. These results describe for the first time the participation of LMWPTP in the dynamics of cytoskeleton rearrangement during exposure of human keratinocytes to hyperosmotic shock, which may contribute to cell death.

  5. Effect of Phosphatases Activity in the Hepatopancreas and Muscle of the Fresh Water Female Field Crab, Spiralothelphusa hydrodroma (Herbst Treated with Cypermethrin

    Directory of Open Access Journals (Sweden)

    R. S. Sreenivasan

    2011-04-01

    Full Text Available The fresh water field crab, Spiralothelphusa hydrodroma is an important human food source in parts of South India and the crab is constantly exposed to pesticides, which are used extensively to control agricultural pests. Evaluation of the toxic effect of cypermethrin on the experimental crab for the LC₅₀ value was carried out. Effect of cypermethrin on the biochemical changes in the hepatopancreas and muscle was observed. Quantitative study of biochemical changes of acid phosphatase and alkaline phosphatase were undertaken.

  6. Effects of Hg and Cu on the activities of soil acid phosphatase

    Institute of Scientific and Technical Information of China (English)

    XU Dong-mei; CHEN Bo; LIU Wen-li; LIU Guang-shen; LIU Wei-ping

    2007-01-01

    Comparative study on the activity and kinectic properties of acid phosphatase (ACPase) of three soils amended with Hg and Cu at constant temperature and humidity was carried out. The results indicated that the inhibition on ACPase of the three sample soils by Hg and Cu varied with the content of soil organic matter and pH, where, Soil 1 was the most seriously contaminated due to its lowest content of organic matter and the lowest pH among three samples, Soil 2 took the second place, and Soil 3was the least contaminated. Except Soil 3, the activity of soil ACPase tended to increase along with the contact time under the same type and the same concentration of heavy metal. In particular the Vmax values of ACPase in all three samples decreased with increasing Hg and Cu concentration, whereas the Km values were affected weakly. According to the change of Vmax and Km values,Cu and Hg had the same inhibition effect on soil ACPase. Both of them may be a type of compound of non-competitive and anti-competitive inhibition. Statistic analyses indicated that activities of soil ACPase and Vmax values could serve as bioindicator to partially denote the heavy metal Hg and Cu contamination degree.

  7. The wheat MAP kinase phosphatase 1 alleviates salt stress and increases antioxidant activities in Arabidopsis.

    Science.gov (United States)

    Zaidi, Ikram; Ebel, Chantal; Belgaroui, Nibras; Ghorbel, Mouna; Amara, Imène; Hanin, Moez

    2016-04-01

    Mitogen-activated protein kinase phosphatases (MKPs) are important negative regulators in the MAPK signaling pathways, which play crucial roles in plant growth, development and stress responses. We have previously shown that the heterologous expression of a durum wheat MKP, TMKP1, results in increased tolerance to salt stress in yeast but its particular contribution in salt stress tolerance in plants was not investigated. Here, TMKP1 was overexpressed in Arabidopsis thaliana and physiological changes were assessed in transgenic plants exposed to stress conditions. Under salt stress and especially LiCl, the TMKP1 overexpressors displayed higher germination rates in comparison to wild type plants. The enhancement of salt stress tolerance was accompanied by increased antioxidant enzyme activities, namely superoxide dismutase, catalase and peroxydases. Such increases in antioxidant activities were concomitant with lower malondialdehyde, superoxide anion O2(-) and hydrogen peroxide levels in the TMKP1 transgenic seedlings. Moreover, we provide evidence that, in contrast to the Arabidopsis ortholog AtMKP1, TMKP1 acts as a positive regulator of salt stress tolerance via its ectopic expression in the Arabidopsis mkp1 mutant. PMID:26927025

  8. Alkaline pH activates the transport activity of GLUT1 in L929 fibroblast cells.

    Science.gov (United States)

    Gunnink, Stephen M; Kerk, Samuel A; Kuiper, Benjamin D; Alabi, Ola D; Kuipers, David P; Praamsma, Riemer C; Wrobel, Kathryn E; Louters, Larry L

    2014-04-01

    The widely expressed mammalian glucose transporter, GLUT1, can be acutely activated in L929 fibroblast cells by a variety of conditions, including glucose deprivation, or treatment with various respiration inhibitors. Known thiol reactive compounds including phenylarsine oxide and nitroxyl are the fastest acting stimulators of glucose uptake, implicating cysteine biochemistry as critical to the acute activation of GLUT1. In this study, we report that in L929 cells glucose uptake increases 6-fold as the pH of the uptake solution is increased from 6 to 9 with the half-maximal activation at pH 7.5; consistent with the pKa of cysteine residues. This pH effect is essentially blocked by the pretreatment of the cells with either iodoacetamide or cinnamaldehyde, compounds that form covalent adducts with reduced cysteine residues. In addition, the activation by alkaline pH is not additive at pH 8 with known thiol reactive activators such as phenylarsine oxide or hydroxylamine. Kinetic analysis in L929 cells at pH 7 and 8 indicate that alkaline conditions both increases the Vmax and decreases the Km of transport. This is consistent with the observation that pH activation is additive to methylene blue, which activates uptake by increasing the Vmax, as well as to berberine, which activates uptake by decreasing the Km. This suggests that cysteine biochemistry is utilized in both methylene blue and berberine activation of glucose uptake. In contrast a pH increase from 7 to 8 in HCLE cells does not further activate glucose uptake. HCLE cells have a 25-fold higher basal glucose uptake rate than L929 cells and the lack of a pH effect suggests that the cysteine biochemistry has already occurred in HCLE cells. The data are consistent with pH having a complex mechanism of action, but one likely mediated by cysteine biochemistry. PMID:24333987

  9. Insulin resistance is associated with reduced fasting and insulin-stimulated glycogen synthase phosphatase activity in human skeletal muscle.

    OpenAIRE

    Kida, Y; Esposito-Del Puente, A; Bogardus, C; Mott, D M

    1990-01-01

    Insulin-stimulated glycogen synthase activity in human skeletal muscle correlates with insulin-mediated glucose disposal rate (M) and is reduced in insulin-resistant subjects. We have previously reported reduced insulin-stimulated glycogen synthase activity associated with reduced fasting glycogen synthase phosphatase activity in skeletal muscle of insulin-resistant Pima Indians. In this study we investigated the time course for insulin stimulation of glycogen synthase and synthase phosphatas...

  10. Activation of WIP1 phosphatase by HTLV-1 Tax mitigates the cellular response to DNA damage.

    Directory of Open Access Journals (Sweden)

    Tajhal Dayaram

    Full Text Available Genomic instability stemming from dysregulation of cell cycle checkpoints and DNA damage response (DDR is a common feature of many cancers. The cancer adult T cell leukemia (ATL can occur in individuals infected with human T cell leukemia virus type 1 (HTLV-1, and ATL cells contain extensive chromosomal abnormalities, suggesting that they have defects in the recognition or repair of DNA damage. Since Tax is the transforming protein encoded by HTLV-1, we asked whether Tax can affect cell cycle checkpoints and the DDR. Using a combination of flow cytometry and DNA repair assays we showed that Tax-expressing cells exit G(1 phase and initiate DNA replication prematurely following damage. Reduced phosphorylation of H2AX (γH2AX and RPA2, phosphoproteins that are essential to properly initiate the DDR, was also observed in Tax-expressing cells. To determine the cause of decreased DDR protein phosphorylation in Tax-expressing cells, we examined the cellular phosphatase, WIP1, which is known to dephosphorylate γH2AX. We found that Tax can interact with Wip1 in vivo and in vitro, and that Tax-expressing cells display elevated levels of Wip1 mRNA. In vitro phosphatase assays showed that Tax can enhance Wip1 activity on a γH2AX peptide target by 2-fold. Thus, loss of γH2AX in vivo could be due, in part, to increased expression and activity of WIP1 in the presence of Tax. siRNA knockdown of WIP1 in Tax-expressing cells rescued γH2AX in response to damage, confirming the role of WIP1 in the DDR. These studies demonstrate that Tax can disengage the G(1/S checkpoint by enhancing WIP1 activity, resulting in reduced DDR. Premature G(1 exit of Tax-expressing cells in the presence of DNA lesions creates an environment that tolerates incorporation of random mutations into the host genome.

  11. Phosphatase Activities of Endolithic Communities in Rocks of the Antarctic Dry Valleys.

    Science.gov (United States)

    Banerjee; Whitton; Wynn-Williams

    2000-01-01

    Phosphorus is scarce in Beacon Sandstone of the McMurdo Dry Valleys, Antarctica, and any input from precipitation is minimal. In endolithic microbial communities recycling of P by the action of phosphatases may therefore be important. The phosphatase activities of three different types of endolithic communities in the McMurdo Dry Valley, Antarctica, were studied in the laboratory. The dominant phototrophs were Chroococcidiopsis, mixed Gloeocapsa and Trebouxia, and Trebouxia. Bacteria were also visually conspicuous in the latter two communities, and the Trebouxia in both cases formed a lichenized association with fungal hyphae. In each case marked phosphomonoesterase (PMEase) activity was found in assays with 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate as substrate, and phosphodiesterase activity with bis-p-nitrophenyl phosphate as substrate. The pH optimum of PMEase (assayed at 0.5 pH intervals) of the Chroococcidiopsis, Gloeocapsa-Trebouxia, and Trebouxia communities was 9.5, 5.5, and 8.0, respectively. These values are similar for aqueous extracts of the respective rocks (pH 9.2, 6.2, 7.5). All three communities showed significantly higher PMEase activity at 5 degrees than 1 degrees C, and the first two also showed much higher activity at 5 degrees than 10 degrees C. All three communities also showed slightly lower activity in the light (7 µmol photon m(-2) s(-1)) than the dark; this was found with all substrates and substrate concentrations. Prior exposure of a moistened sample to light for 2 h led to a reduction in activity even when the subsequent assay was done in the dark. The rate of PMEase activity (using 100 µM MUP) in the Gloeocapsa-Trebouxia and Trebouxia communities was approximately linear with time up to 24 h, whereas the Chroococcidiopsis community showed a marked decrease after 6 h. At least part of this was due to retention of the 4-methylumbelliferone (MU) hydrolysis product. In spite of the assays being conducted on a whole

  12. Activation of protein phosphatase 1 by a small molecule designed to bind to the enzyme's regulatory site.

    Science.gov (United States)

    Tappan, Erin; Chamberlin, A Richard

    2008-02-01

    The activity of protein phosphatase 1 (PP1), a serine-threonine phosphatase that participates ubiquitously in cellular signaling, is controlled by a wide variety of regulatory proteins that interact with PP1 at an allosteric regulatory site that recognizes a "loose" consensus sequence (usually designated as RVXF) found in all such regulatory proteins. Peptides containing the regulatory consensus sequence have been found to recapitulate the binding and PP1 activity modulation of the regulatory proteins, suggesting that it might be possible to design small-molecule surrogates that activate PP1 rather than inhibiting it. This prospect constitutes a largely unexplored way of controlling signaling pathways that could be functionally complementary to the much more extensively explored stratagem of kinase inhibition. Based on these principles, we have designed a microcystin analog that activates PP1. PMID:18291321

  13. Study of possible changes brought about by plutonium oxide in the acid phosphatase activity of alveolar macrophages of the rabbit

    International Nuclear Information System (INIS)

    This report describes the various techniques used for determining the acid phosphatase activity of alveolar rabbit macrophages after inhalation of radioactive plutonium oxide particles, exposure of the animals, removal and sampling of the alveolar cells, and technical dosage. The results obtained are presented; they do not make it possible, in this particular case, to affirm that an important change in the enzymatic activity studied occurs. (author)

  14. Alkaline-sulphate activation processes of a Spanish blast furnace slag

    OpenAIRE

    Fernández Jiménez, A.; Puertas, F.; Fernández-Carrasco, L.

    1996-01-01

    Alkaline-sulphate activation processes of a Spanish granulated blast furnace slag (Avilés, Ensidesa) have been studied. Activator solutions used were: deionized water (as reference solution), Ca(OH)2 (3,5∙10-3N), NaOH (1N), Na2CO3 (2N), CaSO4∙2H2O (3,0∙10-3N) at 25ºC. The influence of the nature of alkaline or sulphate solution cation...

  15. Lowering of phytic acid content by enhancement of phytase and acid phosphatase activities during sunflower germination

    Directory of Open Access Journals (Sweden)

    Juliana da Silva Agostini

    2010-08-01

    Full Text Available The objective of this work was to investigate the germination of hybrid sunflowers BRS191 and C11 as a means of lowering phytic acid (PA content by enhancing the activity of endogenous phytase and acid phosphatase. The concentration of PA in hybrid sunflower achenes varied from 2.16 to 2.83g/100g of sample (p O objetivo deste trabalho foi investigar a germinação de girassóis híbridos BRS 191 e C11 com finalidade de reduzir o teor de AF e aumentar as atividades de phytases e fosfatases endógenas. A concentração do AF nos aquênios de girassóis híbridos variou de 2,16 a 2,83 g /100g de amostra (p< 0,005. As atividades de fitases e fosfatases de girassóis BRS191 e C11 foram elevadas no 4º e 5º dia de germinação, respectivamente, com liberação do fósforo necessário para o desenvolvimento da semente. Estes resultados indicam que o AF do girassol hibrido reduz e a atividade de phytase aumenta em períodos distintos da germinação, possibilitando assim a aplicação desta enzima no controle do teor de AF em cereais, melhorando o seu valor nutricional.

  16. Synthesis, characterization and antimicrobial activity of alkaline ion-exchanged ZnO/bentonite nanocomposites

    Institute of Scientific and Technical Information of China (English)

    Hamideh Pouraboulghasem; Mohammad Ghorbanpour; Razieh Shayegh; Samaneh Lotfiman

    2016-01-01

    Nanocomposites of zinc/bentonite clay were synthesized for use as an antibacterial material by a quick and simple alkaline ion exchange method. The synthesis of zinc doped bentonite nanocomposite was accomplished by placing bentonite in a melting bath of ZnSO4 for 10, 20, 40, 60 and 90 min. The complexes were characterized by XRD, SEM and DRS. XRD analyses and SEM observations confirmed the diffusion of zinc to the clay surfaces. Antibacterial activity tests againstEscherichia coli showed that bentonite did not present any antibacterial properties, but after alkaline ion exchange treatment, inhibition was noted. The highest antibacterial activity was observed with ZnO/bentonite composite alkaline ion exchange for 60 and 90 min. Interestingly, the leaching test indicated that ZnO/bentonite did not present any risk for drinking water treatment.

  17. Asp1 from Schizosaccharomyces pombe binds a [2Fe-2S](2+) cluster which inhibits inositol pyrophosphate 1-phosphatase activity.

    Science.gov (United States)

    Wang, Huanchen; Nair, Vasudha S; Holland, Ashley A; Capolicchio, Samanta; Jessen, Henning J; Johnson, Michael K; Shears, Stephen B

    2015-10-27

    Iron-sulfur (Fe-S) clusters are widely distributed protein cofactors that are vital to cellular biochemistry and the maintenance of bioenergetic homeostasis, but to our knowledge, they have never been identified in any phosphatase. Here, we describe an iron-sulfur cluster in Asp1, a dual-function kinase/phosphatase that regulates cell morphogenesis in Schizosaccharomyces pombe. Full-length Asp1, and its phosphatase domain (Asp1(371-920)), were each heterologously expressed in Escherichia coli. The phosphatase activity is exquisitely specific: it hydrolyzes the 1-diphosphate from just two members of the inositol pyrophosphate (PP-InsP) signaling family, namely, 1-InsP7 and 1,5-InsP8. We demonstrate that Asp1 does not hydrolyze either InsP6, 2-InsP7, 3-InsP7, 4-InsP7, 5-InsP7, 6-InsP7, or 3,5-InsP8. We also recorded 1-phosphatase activity in a human homologue of Asp1, hPPIP5K1, which was heterologously expressed in Drosophila S3 cells with a biotinylated N-terminal tag, and then isolated from cell lysates with avidin beads. Purified, recombinant Asp1(371-920) contained iron and acid-labile sulfide, but the stoichiometry (0.8 atoms of each per protein molecule) indicates incomplete iron-sulfur cluster assembly. We reconstituted the Fe-S cluster in vitro under anaerobic conditions, which increased the stoichiometry to approximately 2 atoms of iron and acid-labile sulfide per Asp1 molecule. The presence of a [2Fe-2S](2+) cluster in Asp1(371-920) was demonstrated by UV-visible absorption, resonance Raman spectroscopy, and electron paramagnetic resonance spectroscopy. We determined that this [2Fe-2S](2+) cluster is unlikely to participate in redox chemistry, since it rapidly degraded upon reduction by dithionite. Biochemical and mutagenic studies demonstrated that the [2Fe-2S](2+) cluster substantially inhibits the phosphatase activity of Asp1, thereby increasing its net kinase activity. PMID:26422458

  18. Influence of the activator concentration on the kinetics of the alkaline activation process of a blast furnace slag

    OpenAIRE

    Fernández-Jiménez, A.; Puertas, F.

    1997-01-01

    The influence of activator solution concentration on hydration kinetics of an alkaline activated blast furnace slag has been studied. The alkaline activator used was a mix of waterglass (Na2SiO3∙nH2O) and NaOH solution (of variable concentration). Final activator concentrations were 3,4 and 5 % Na2O wt. with respect to the slag total weight. Degree of reaction (α) was determined from hydration hea...

  19. Direct and indirect effects of ammonia, ammonium and nitrate on phosphatase activity and carbon fluxes from decomposing litter in peatland.

    Science.gov (United States)

    Johnson, David; Moore, Lucy; Green, Samuel; Leith, Ian D; Sheppard, Lucy J

    2010-10-01

    Here we investigate the response of soils and litter to 5 years of experimental additions of ammonium (NH4), nitrate (NO3), and ammonia (NH3) to an ombrotrophic peatland. We test the importance of direct (via soil) and indirect (via litter) effects on phosphatase activity and efflux of CO2. We also determined how species representing different functional types responded to the nitrogen treatments. Our results demonstrate that additions of NO3, NH4 and NH3 all stimulated phosphatase activity but the effects were dependent on species of litter and mechanism (direct or indirect). Deposition of NH3 had no effect on efflux of CO2 from Calluna vulgaris litter, despite it showing signs of stress in the field, whereas both NO3 and NH4 reduced CO2 fluxes. Our results show that the collective impacts on peatlands of the three principal forms of nitrogen in atmospheric deposition are a result of differential effects and mechanisms on individual components.

  20. Mesophilic and thermophilic alkaline fermentation of waste activated sludge for hydrogen production: Focusing on homoacetogenesis

    DEFF Research Database (Denmark)

    Wan, Jingjing; Jing, Yuhang; Zhang, Shicheng;

    2016-01-01

    The present study compared the mesophilic and thermophilic alkaline fermentation of waste activated sludge (WAS) for hydrogen production with focus on homoacetogenesis, which mediated the consumption of H2 and CO2 for acetate production. Batch experiments showed that hydrogen yield of WAS increased...

  1. The Correlation Analysis of Alkaline Phosphatase and Parathyroid Hormone in Chronic Kidney Disease%慢性肾脏病碱性磷酸酶与甲状旁腺素相关性分析

    Institute of Scientific and Technical Information of China (English)

    余娜

    2012-01-01

      Objective To explore the correlation analysis of alkaline phosphatase(ALP) and parathyroid hormone(PTH) in chronic kidney disease(CKD), and evaluate their significance in diagnosis and treatment of renal osteodystrophy(ROD). Methods 60 CKD3-5 stage patients were extracted of fasting venous blood, to determinate ALP and PTH and analysis their correlation. Results ALP and PHT showed a significant positive correlation when PTH>300pg/ml(r=0.865, P300pg/ml时,ALP与PTH相关性良好,且呈正相关,有统计学意义(r=0.865,P<0.05);当PTH≤300pm/dl时, ALP与PTH无相关性.结论 ALP与PTH相关性良好,随着PTH的增高,ALP增高越明显.两者结合,对诊断和治疗高转运骨病(HTO)有重要的临床价值.且ALP检测方法简单,花费少.

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

    Science.gov (United States)

    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.

  3. Activity and Tissue Expression of Tyrosine Phosphatase PTP-MEG2

    Institute of Scientific and Technical Information of China (English)

    DONG Hong-bo; LI Guo-dong; WANG Shao-feng; FU Xue-qi; ZHAO Zhi-zhuang Joe

    2011-01-01

    Protein tyrosine phosphatases(PTPs) are crucial regulators of signal transduction. Among them,PTP-MEG2 is an intracellular enzyme of 593 amino acid residues with a putative lipid-binding domain at the N-terminus. In the present study, we cloned the full-length form of the enzyme and expressed it in E. coli cells as a 6xHis-tagged protein. The majority of the expressed enzyme was found in the inclusion body of E. coli cell extracts.Upon extraction with a buffer containing urea, the recombinant enzyme was purified to near homogeneity on a single Ni-NTA-agarose column. This procedure resulted in the production of over 100 mg of purified recombinant PTP-MEG2 from 1 L E. coli cell culture. The purified protein displayed a single polypeptide band with expected molecular size on SDS-polyacrylamide gel electrophoresis under reducing conditions. Isolated under denatured conditions in urea, the purified enzyme was re-natured by dialyzing against a refolding buffer. The re-natured enzyme effectively dephosphorylated the common PTP substrate para-nitrophenylphosphate with a specific activity of 2000 units/mg. Meanwhile, the denatured enzyme was used to immunize a rabbit to produce antibodies. The resulting antiserum had extremely high sensitivity and specificity. When used for Western blot analysis, the anti-serum revealed a wide expression of PTP-MEG2 in many tissues of mice. Together, we developed a highly effective way to purify a large amount of PTP-MEG2 and generated highly sensitive antibodies that can specifically detect endogenous expression of the enzyme in tissues.

  4. Performance of alkaline activated slag at high temperatures

    OpenAIRE

    Mejía de Gutiérrez, R.; Maldonado, J.; C. Gutiérrez

    2004-01-01

    This paper presents an investigation into the performance of alkali-activated slag (AAS) mortar exposed to elevated temperatures. Sodium silicate, sodium hydroxide and a mix (waterglass) with a modulus (SiO2/Na20) of 1.5 were used as activators. The specimens were heated in an electric furnace up to 1000 ºC in steps of 200 ºC for a constant period of 2 hours. The weight loss, residual compressive strength, resistance to chloride ion penetration, p...

  5. Effecf of pH and some cations on activity of acid phosphatase secreted from Ustilago sp. isolated from acid sulphate soil

    Directory of Open Access Journals (Sweden)

    Chairatana Nilnond

    2007-03-01

    Full Text Available Acid phosphatase secreted from Ustilago sp. is able to hydrolyze organic phosphorus. These soil yeast microorganisms were isolated from rice roots grown in acid sulphate soil that generally contains highamount of aluminum (Al, iron (Fe and manganese (Mn ions. Therefore, the objectives of this study were to examine the effect of pH and some cations on acid phosphatase activity. Two isolates of Ustilago sp., AR101and AR102, were cultured in 100 mL of modified Pikovskaya's broth containing Na-phytate, pH 4, and acid phosphatase activity was determined at pH 2.0-7.0. Effect of Al, Fe, and Mn, including calcium (Ca ions,on growth of AR101 and AR102, secreted acid phosphatase activity, and the ability of acid phosphatase on the phosphorus release from Na-phytate by Ustilago sp. were investigated. It was found that the optimum pH for acid phosphatase activity was 3.5-4.5. The activity of acid phosphatase secreted from AR101 (3,690nmol min-1 mL-1 was remarkably higher than that from AR102 (956 nmol min-1 mL-1. Aluminum, iron, manganese and calcium ions in the medium did not affect the growth of either isolate. The activity of secretedacid phosphatase of AR101 was inhibited by Al and Ca ion, and synthesis of acid phosphatase of Ustilago sp. AR102 was possibly stimulated by Fe ion. Both AR101 and AR102 solubilized Na-phytate, resulting in therelease of P. However, some amount of released P was then precipitated with Al and Fe ions as the highly insoluble Fe- or Al- phosphate.

  6. 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.

    Science.gov (United States)

    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.

  7. Screening the active constituents of Chinese medicinal herbs as potent inhibitors of Cdc25 tyrosine phosphatase, an activator of the mitosis-inducing p34cdc2 kinase

    Institute of Scientific and Technical Information of China (English)

    YANG Hua; ZHENG Shu; MEIJER Laurent; LI Shi-min; LECLERC Sophie; YU Lin-lin; CHENG Jin-quan; ZHANG Su-zhan

    2005-01-01

    Objective: To screen and evaluate the active constituents of Chinese medicinal herbs as potent inhibitors of Cdc25phosphatase. Methods: The affinity chromatography purified glutashione-S-transferase/Cdc25A phosphatase fusion protein and Cdc2/cyclin B from the extracts of starfish M phase oocytes are used as the cell cycle-specific targets for screening the antimitotic constituents. We tested 9 extracts isolated from the Chinese medicinal herbs and vegetables including the agents currently used in cancer treatment by measuring the inhibition of Cdc25A phosphatase and Cdc2 kinase activity. The antitumor activity of the extracts was also evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and flow cytometry.Results: Cdc25A inhibitory activity and antitumor activity are detected in the extracts isolated from three Chinese medicinal herbs Agrimonapilosa; Herba solani lyrati; Galla chinesis. Conclusion: We found three extracts isolated from Chinese medicinal herbs have potential inhibitory activity of Cdc25 phosphatase using a highly specific mechanism-based screen assay for antimitotic drug discovery.

  8. Temporal profile of calcineurin phosphatase activity during acute allograft rejection in the heterotopic rat heart transplantation model

    DEFF Research Database (Denmark)

    Karamperis, N; Koefoed-Nielsen, P B; Marcussen, N;

    2008-01-01

    BACKGROUND: Regardless of the extensive worldwide use of calcineurin inhibitors, little is known about the behavior of calcineurin phosphatase (CaN) during acute allograft rejection. The aim of this study was to investigate the temporal profile of CaN during acute allograft rejection and reveal...... postoperative time points. CaN activity was measured in isolated peripheral blood and spleen mononuclear cells and in graft heart homogenates. CaN activity was measured as the release of radiolabeled phosphate from a previously phosphorylated 19 amino acid peptide. RESULTS: We have shown that CaN's activity...

  9. Serum total and bone alkaline phosphatase and tartrate-resistant acid phosphatase activities for the assessment of bone fracture healing in dogs

    Directory of Open Access Journals (Sweden)

    C. Sousa

    2011-08-01

    Full Text Available O objetivo deste trabalho foi estudar o padrão de variação da atividade sérica da fosfatase alcalina total (tALP, da isoenzima óssea da fosfatase alcalina (BALP e da fosfatase ácida resistente ao tartarato (TRAP, assim como a variação da concentração dos minerais séricos durante o processo de cicatrização de fraturas ósseas no cão. A variação sérica destes marcadores do metabolismo ósseo foi avaliada em nove cães com fraturas diafisárias fechadas de ossos longos, submetidas a tratamento cirúrgico para osteosíntese. Durante o período pós-operatório, sete animais evoluíram no sentido de uma normal união óssea, sendo que dois deles desenvolveram um processo de não união óssea. Foram observados, relativamente à BALP, valores de actividade sérica mais elevados e com diferença estatística (P<0,05 no grupo de animais que evoluiu no sentido de uma normal união óssea, comparativamente ao grupo de animais que evoluiu no sentido do processo de não união. No grupo de animais que evoluiu para a completa união óssea foram, adicionalmente, observados valores diminuidos (P<0,05 da atividade sérica da TRAP, até ao dia 60 do período pós-operatório seguido de uma elevação estatisticamente significativa após este período. Em conclusão, os biomarcadores do metabolismo ósseo poderão vir a constituir um método auxiliar de diagnóstico na monitorização do processo de cicatrização de fracturas ósseas, possibilitando, a detecção precoce de complicações pós-operatórias.

  10. The effect of pH and natural microbial phosphatase activity on the speciation of uranium in subsurface soils

    Science.gov (United States)

    Beazley, Melanie J.; Martinez, Robert J.; Webb, Samuel M.; Sobecky, Patricia A.; Taillefert, Martial

    2011-10-01

    The biomineralization of U(VI) phosphate as a result of microbial phosphatase activity is a promising new bioremediation approach to immobilize uranium in both aerobic and anaerobic conditions. In contrast to reduced uranium minerals such as uraninite, uranium phosphate precipitates are not susceptible to changes in oxidation conditions and may represent a long-term sink for uranium in contaminated environments. So far, the biomineralization of U(VI) phosphate has been demonstrated with pure cultures only. In this study, two uranium contaminated soils from the Department of Energy Oak Ridge Field Research Center (ORFRC) were amended with glycerol phosphate as model organophosphate source in small flow-through columns under aerobic conditions to determine whether natural phosphatase activity of indigenous soil bacteria was able to promote the precipitation of uranium(VI) at pH 5.5 and 7.0. High concentrations of phosphate (1-3 mM) were detected in the effluent of these columns at both pH compared to control columns amended with U(VI) only, suggesting that phosphatase-liberating microorganisms were readily stimulated by the organophosphate substrate. Net phosphate production rates were higher in the low pH soil (0.73 ± 0.17 mM d -1) compared to the circumneutral pH soil (0.43 ± 0.31 mM d -1), suggesting that non-specific acid phosphatase activity was expressed constitutively in these soils. A sequential solid-phase extraction scheme and X-ray absorption spectroscopy measurements were combined to demonstrate that U(VI) was primarily precipitated as uranyl phosphate minerals at low pH, whereas it was mainly adsorbed to iron oxides and partially precipitated as uranyl phosphate at circumneutral pH. These findings suggest that, in the presence of organophosphates, microbial phosphatase activity can contribute to uranium immobilization in both low and circumneutral pH soils through the formation of stable uranyl phosphate minerals.

  11. Performance of alkaline activated slag at high temperatures

    Directory of Open Access Journals (Sweden)

    Mejía de Gutiérrez, R.

    2004-12-01

    Full Text Available This paper presents an investigation into the performance of alkali-activated slag (AAS mortar exposed to elevated temperatures. Sodium silicate, sodium hydroxide and a mix (waterglass with a modulus (SiO2/Na20 of 1.5 were used as activators. The specimens were heated in an electric furnace up to 1000 ºC in steps of 200 ºC for a constant period of 2 hours. The weight loss, residual compressive strength, resistance to chloride ion penetration, porosity and capillary sorption were evaluated and the results were compared with those of ordinary and blended Portland cement mortar

    En el presente traba jo se estudió el comportamiento frente a ¡a temperatura de morteros producidos a partir de escorias siderúrgicas activadas alcalinamente (EAA, utilizando diferentes activantes tales como silicato sódico, hidróxido de sodio y sus correspondientes mezclas. Cada espécimen se expuso por dos horas a temperaturas hasta de 1.000 ºC, en intervalos de 200 °C y en cada caso se determinaron los cambios de color peso, resistencia mecánica y durabilidad. Esta última propiedad se evaluó determinando las modificaciones de porosidad y permeabilidad a cloruros. Los resultados se comparan con los obtenidos en morteros de cemento Portland con y sin adición, específicamente con aquéllos que incorporan humo de sílice.

  12. DevS/DosS sensor is bifunctional and its phosphatase activity precludes aerobic DevR/DosR regulon expression in Mycobacterium tuberculosis.

    Science.gov (United States)

    Kaur, Kohinoor; Kumari, Priyanka; Sharma, Saurabh; Sehgal, Snigdha; Tyagi, Jaya Sivaswami

    2016-08-01

    Two-component systems, comprising histidine kinases and response regulators, empower bacteria to sense and adapt to diverse environmental stresses. Some histidine kinases are bifunctional; their phosphorylation (kinase) and dephosphorylation (phosphatase) activities toward their cognate response regulators permit the rapid reversal of genetic responses to an environmental stimulus. DevR-DevS/DosR-DosS is one of the best-characterized two-component systems of Mycobacterium tuberculosis. The kinase function of DevS is activated by gaseous stress signals, including hypoxia, resulting in the induction of ~ 48-genes DevR dormancy regulon. Regulon expression is tightly controlled and lack of expression in aerobic Mtb cultures is ascribed to the absence of phosphorylated DevR. Here we show that DevS is a bifunctional sensor and possesses a robust phosphatase activity toward DevR. We used site-specific mutagenesis to generate substitutions in conserved residues in the dimerization and histidine phosphotransfer domain of DevS and determined their role in kinase/phosphatase functions. In vitro and in vivo experiments, including a novel in vivo phosphatase assay, collectively establish that these conserved residues are critical for regulating kinase/phosphatase functions. Our findings establish DevS phosphatase function as an effective control mechanism to block aerobic expression of the DevR dormancy regulon. Asp-396 is essential for both kinase and phosphatase functions, whereas Gln-400 is critical for phosphatase function. The positive and negative functions perform opposing roles in DevS: the kinase function triggers regulon induction under hypoxia, whereas its phosphatase function prevents expression under aerobic conditions. A finely tuned balance in these opposing activities calibrates the dormancy regulon response output. PMID:27327040

  13. New procedures to measure synthase and phosphatase activities of bis-phosphoglycerate mutase. Interest for development of therapeutic drugs; Nouveaux procedes pour mesurer les activites synthase et phosphatase de la bisphosphoglycerate mutase. Interet pour le developpement de drogues therapeutiques

    Energy Technology Data Exchange (ETDEWEB)

    Ravel, P.; Garel, M.C. [Hopital Henri-Mondor, 94 - Creteil (France); Toullec, D. [Laboratoire Glaxo Wellcome, 91- Les Ulis (France)

    1997-12-31

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

  14. Development of New Cementitious Caterials by Alkaline Activating Industrial by-Products

    Science.gov (United States)

    Fernández-Jimenez, A.; García-Lodeiro, I.; Palomo, A.

    2015-11-01

    The alkaline activation of aluminosiliceous industrial by-products such as blast furnace slag and fly ash is widely known to yield binders whose properties make them comparable to or even stronger and more durable than ordinary Portland cement. The present paper discusses activation fundamentals (such as the type and concentration of alkaline activator and curing conditions) as well as the structure of the cementitious gels formed (C-A-S-H, N-A-S-H). The durability and strength of these systems make these materials apt for use in many industrial applications, such as precast concrete elements (masonery blocks, railroad sleepers), protective coatings for materials with low fire ratings and lightweight elements.

  15. Low Soil Phosphorus Availability Increases Acid Phosphatases Activities and Affects P Partitioning in Nodules, Seeds and Rhizosphere of Phaseolus vulgaris

    Directory of Open Access Journals (Sweden)

    Jean-Jacques Drevon

    2012-06-01

    Full Text Available The effect of phosphorus (P deficiency on phosphatases activities in N2-fixing legumes has been widely studied in hydroponic culture. However, the response of acid phosphatase (APase and phytase in rhizosphere, nodules and seeds of Phaseolus vulgaris to low soil’s P-availability is not yet fully understood. In this study, six genotypes of N2-fixing P. vulgaris were grown under contrasting soil P-availabilities; i.e., low  (4.3 mg P kg−1 and sufficient (16.7 mg P kg−1 in the Haouz region of Morocco. At flowering and maturity stages, plants were harvested and analyzed for their phosphatases activities, growth and P content. Results show that, low P decreased nodulation, growth, P uptake and N accumulation in all the genotypes, but to a greater extent in the sensitive recombinant inbreed line 147. In addition, while seed P content was slightly reduced under low P soil; a higher P was noticed in the Flamingo and Contender large seeded-beans (6.15 to 7.11 mg g−1. In these latter genotypes, high APase and phytase activities in seeds and nodules were associated with a significant decline in rhizosphere’s available P. APase activity was mainly stimulated in nodules, whereas phytase activity was highly induced in seeds (77%. In conclusion, the variations of APase and phytase activities in nodules and seeds depend on genotype and can greatly influence the internal utilization of P, which might result in low P soil tolerance in N2-fixing legumes.

  16. The effect of water and salt stresses on the phosphorus content and acid phosphatase activity in oilseed rape

    Directory of Open Access Journals (Sweden)

    Stanisław Flasiński

    2014-02-01

    Full Text Available Oilseed rape plants responded to water and salt stresses (-0.5 MPa, PEG 6000 and NaCI by reduction of the fresh and dry weights of shoots and roots. When PEG was used, the ratio of dry weights of roots:shoots surpassed that of controls. The leaf protein content increased considerably. The phosphorus content decreased only in the roots, most significantly after three days of stress. Immediately after the stresses were induced, an increase in the acid phosphatase (AP activity was noted. Water and salt stresses caused four- and two-fold increases in AP activity in leaves, respectively. Changes in the enzyme activity were negligible in stems and roots. There are nine forms of AP in young leaves of oilseed rape. In the stressed plants, from No. 5 revealed lower activity and forms Nos 8 and 9, higher activities than in the control. The increase in AP activity was directly accompanied by the decrease in the water potential of the tissues. Oilseed rape is considerably less sensitive to salt stress than to water stress, which is manifested as the lower inhibition of plant growth and also by a smaller increase in acid phosphatase activity.

  17. Activation of fly ashes by the high temperature and high alkalinity in ASR tests

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    High temperature and high alkalinity are typical testing conditions to accelerate the appraisal process of the suppressing effect of fly ashes on alkali silica reaction(ASR),but the reaction mechanism of fly ashes would be quite different under such conditions compared to the normal condition of temperature and alkalinity.To make a reasonable analysis of the suppressing effect of fly ashes,13 types of fly ashes were tested in this paper by both the accelerated mortar bar test method and the 60°C accelerated concrete prism test method.The results showed that the effect of fly ashes would be magnified under the condition of high temperature and high alkalinity.The XRD analysis showed that all the phases of fly ash could react with the hot alkaline solution except for mullite and a small amount of quartz.Fly ash could be significantly activated by the 80°C 1 mol/L NaOH solution,and form mainly C-S-H phase and P type zeolite,but its effect on inhibiting ASR was exaggerated then.According to the mortar strength test and the ASR suppressing test results,C-S-H phase contributed to mortar strength,but its amount did not decide the ASR suppressing effect of fly ash.

  18. Methane production and microbial community structure for alkaline pretreated waste activated sludge.

    Science.gov (United States)

    Sun, Rui; Xing, Defeng; Jia, Jianna; Zhou, Aijuan; Zhang, Lu; Ren, Nanqi

    2014-10-01

    Alkaline pretreatment was studied to analyze the influence on waste activated sludge (WAS) reduction, methane production and microbial community structure during anaerobic digestion. Methane production from alkaline pretreated sludge (A-WAS) (pH = 12) increased from 251.2 mL/Ld to 362.2 mL/Ld with the methane content of 68.7% compared to raw sludge (R-WAS). Sludge reduction had been improved, and volatile suspended solids (VSS) removal rate and protein reduction had increased by ∼ 10% and ∼ 35%, respectively. The bacterial and methanogenic communities were analyzed using 454 pyrosequencing and clone libraries of 16S rRNA gene. Remarkable shifts were observed in microbial community structures after alkaline pretreatment, especially for Archaea. The dominant methanogenic population changed from Methanosaeta for R-WAS to Methanosarcina for A-WAS. In addition to the enhancement of solubilization and hydrolysis of anaerobic digestion of WAS, alkaline pretreatment showed significant impacts on the enrichment and syntrophic interactions between microbial communities.

  19. The Potential of Soft Soil Improvement Through a Coupled Technique Between Electro Kinetic and Alkaline Activation of Soft Soil

    Science.gov (United States)

    Ahmed, G. E.; Ismail, H. B.; Huat, B. K.; Afshin, A.; Azhar, A. T. S.

    2016-07-01

    Soil stabilization techniques have been in development for decades with different rates of success. Alkaline activation of soft soil is one of those techniques that has proved to deliver some of the best shear strength values with minor drawbacks in comparison with conventional soil stabilization methods. However, environmental considerations have not been taken into account, as major mineral glassy phase activators are poisoning alkaline solutions, such as sodium-, potassium-hydroxide, and sodium-, potassium-silicate, which poses serious hazards to man and environment. This paper addresses the ways of discarding the involvement of the aforementioned alkaline solutions in soft soil stabilization by investigating the potential of a coupled electro kinetic alkaline activation technique for soft soil strengthening, through which the provision of alkaline pH is governed by electro kinetic potential. Uncertainties in regard to the dissolution of aluminosilicate as well as the dominance of acidic front are challenges that need to be overcome.

  20. Quantitative Analysis of Protein Concentration by Absorption Peak Integration Method of UV Spectroscopy-Taking Alkaline Phosphatase as an Example%紫外吸收光谱积分法分析蛋白质浓度-以碱性磷酸酶为例

    Institute of Scientific and Technical Information of China (English)

    朱元荣; 吴丰昌; 林樱

    2013-01-01

    利用矿物(针铁矿,蒙脱石)和太湖沉积物吸附碱性磷酸酶(alkaline phosphatase,APase),测定吸附后上清液中剩余碱性磷酸酶浓度时发现其紫外吸收光谱发生了变化,利用传统280 nm处紫外吸收法无法直接准确测定其浓度值.基于对碱性磷酸酶252~305 nm处吸收峰面积积分方法可以消除影响,并准确分析测定碱性磷酸酶浓度.其测定结果与考马斯亮蓝法测定结果进行比较,表明了该方法可以方便,快速和准确地测定此类实验中碱性磷酸酶浓度.同时,该方法还可以扩展至其他蛋白质的定量分析,甚至其他类似实验中,一定程度上克服传统方法应用单波长进行定量分析中存在的易受干扰的缺点.%Adsorption of alkaline phosphatase (APase) on minerals (goethite,montmorillonite) and sediments from Taihu Lake was studied.However,the concentration of alkaline phosphatase in the supernatant cannot be analyzed by the adsorption at 280 nm due to that the UV spectroscopy was changed.Quantitative analysis of alkaline phosphatase in the supernatant by the absorption peak (252~305 nm) integration method of UV spectroscopy was developed.This method determined the concentrations of alkaline phosphatase well.Compared to the results of Bradford,the absorption peak integration method can determine the concentrations of supernatant fast,conveniently,and accurately.This method can also be applied to other protein solution analysis and similar experiments.The drawbacks of traditional single wavelength method (280nm) were overcome to some extent based on the method of absorption peak integration.

  1. Maternal antibiotic-induced early changes in microbial colonization selectively modulate colonic permeability and inducible heat shock proteins, and digesta concentrations of alkaline phosphatase and TLR-stimulants in swine offspring.

    Directory of Open Access Journals (Sweden)

    Marie-Edith Arnal

    Full Text Available Elevated intake of high energy diets is a risk factor for the development of metabolic diseases and obesity. High fat diets cause alterations in colonic microbiota composition and increase gut permeability to bacterial lipopolysaccharide, and subsequent low-grade chronic inflammation in mice. Chronic inflammatory bowel diseases are increasing worldwide and may involve alterations in microbiota-host dialog. Metabolic disorders appearing in later life are also suspected to reflect changes in early programming. However, how the latter affects the colon remains poorly studied. Here, we hypothesized that various components of colonic physiology, including permeability, ion exchange and protective inducible heat shock proteins (HSP are influenced in the short- and long-terms by early disturbances in microbial colonization. The hypothesis was tested in a swine model. Offspring were born to control mothers (n = 12 or mothers treated with the antibiotic (ATB amoxicillin around parturition (n = 11. Offspring were slaughtered between 14 and 42 days of age to study short-term effects. For long-term effects, young adult offspring from the same litters consumed a normal or a palm oil-enriched diet for 4 weeks between 140 and 169 days of age. ATB treatment transiently modified maternal fecal microbiota although the minor differences observed for offspring colonic microbiota were nonsignificant. In the short-term, consistently higher HSP27 and HSP70 levels and transiently increased horseradish peroxidase permeability in ATB offspring colon were observed. Importantly, long-term consequences included reduced colonic horseradish peroxidase permeability, and increased colonic digesta alkaline phosphatase (AP and TLR2- and TLR4-stimulant concentrations in rectal digesta in adult ATB offspring. Inducible HSP27 and HSP70 did not change. Interactions between early ATB treatment and later diet were noted for paracellular permeability and concentrations of colonic

  2. A Novel Interaction of the Catalytic Subunit of Protein Phosphatase 2A with the Adaptor Protein CIN85 Suppresses Phosphatase Activity and Facilitates Platelet Outside-in αIIbβ3 Integrin Signaling.

    Science.gov (United States)

    Khatlani, Tanvir; Pradhan, Subhashree; Da, Qi; Shaw, Tanner; Buchman, Vladimir L; Cruz, Miguel A; Vijayan, K Vinod

    2016-08-12

    The transduction of signals generated by protein kinases and phosphatases are critical for the ability of integrin αIIbβ3 to support stable platelet adhesion and thrombus formation. Unlike kinases, it remains unclear how serine/threonine phosphatases engage the signaling networks that are initiated following integrin ligation. Because protein-protein interactions form the backbone of signal transduction, we searched for proteins that interact with the catalytic subunit of protein phosphatase 2A (PP2Ac). In a yeast two-hybrid study, we identified a novel interaction between PP2Ac and an adaptor protein CIN85 (Cbl-interacting protein of 85 kDa). Truncation and alanine mutagenesis studies revealed that PP2Ac binds to the P3 block ((396)PAIPPKKPRP(405)) of the proline-rich region in CIN85. The interaction of purified PP2Ac with CIN85 suppressed phosphatase activity. Human embryonal kidney 293 αIIbβ3 cells overexpressing a CIN85 P3 mutant, which cannot support PP2Ac binding, displayed decreased adhesion to immobilized fibrinogen. Platelets contain the ∼85 kDa CIN85 protein along with the PP2Ac-CIN85 complex. A myristylated cell-permeable peptide derived from residues 395-407 of CIN85 protein (P3 peptide) disrupted the platelet PP2Ac-CIN85 complex and decreased αIIbβ3 signaling dependent functions such as platelet spreading on fibrinogen and thrombin-mediated fibrin clot retraction. In a phospho-profiling study P3 peptide treated platelets also displayed decreased phosphorylation of several signaling proteins including Src and GSK3β. Taken together, these data support a role for the novel PP2Ac-CIN85 complex in supporting integrin-dependent platelet function by dampening the phosphatase activity. PMID:27334924

  3. Diversity and activity of methanotrophs in alkaline soil from a Chinese coal mine

    Energy Technology Data Exchange (ETDEWEB)

    Han, Bing; Chen, Yin; Abell, Guy; Jiang, Hao; Bodrossy, Levente; Zhao, Jiangang; Murrell, J. Colin; Xing, Xin-Hui [Tsinghua University, Beijing (China). Dept. of Chemical Engineering

    2009-11-15

    Culture-independent molecular biological techniques, including 16S rRNA gene and functional gene clone libraries and microarray analyses using pmoA (encoding a key subunit of particulate methane monooxygenase), were applied to investigate the methanotroph community structure in alkaline soil from a Chinese coal mine. This environment contained a high diversity of methanotrophs, including the type II methanotrophs Methylosinus/Methylocystis, type I methanotrophs related to Methylobacter/Methylosoma and Methylococcus, and a number of as yet uncultivated methanotrophs. In order to identify the metabolically active methane-oxidizing bacteria from this alkaline environment, DNA stable isotope probing (DNA-SIP) experiments using {sup 13}CH{sub 4} were carried out. This showed that both type I and type II methanotrophs were active, together with methanotrophs related to Methylocella, which had previously been found only in acidic environments. Methylotrophs, including Methylopila and Hyphomicrobium, were also detected in soil DNA and after DNA-SIP experiments. DNA sequence information on the most abundant, active methanotrophs in this alkaline soil will facilitate the design of oligonucleotide probes to monitor enrichment cultures when isolating key alkaliphilic methanotrophs from such environments.

  4. Characterization of an adapter subunit to a phosphatidylinositol (3)P 3-phosphatase: Identification of a myotubularin-related protein lacking catalytic activity

    OpenAIRE

    Nandurkar, H. H.; Caldwell, K K; Whisstock, J C; Layton, M. J.; Gaudet, E. A.; Norris, F. A.; Majerus, P W; Mitchell, C. A.

    2001-01-01

    The D3-phosphoinositides act as second messengers by recruiting, and thereby activating, diverse signaling proteins. We have previously described the purification of a rat phosphatidylinositol 3-phosphate [PtdIns(3)P] 3-phosphatase, comprising a heterodimer of a 78-kDa adapter subunit in complex with a 65-kDa catalytic subunit. Here, we have cloned and characterized the cDNA encoding the human 3-phosphatase adapter subunit (3-PAP). Sequence alignment showed that 3-PAP ...

  5. Castor oil increases intestinal formation of platelet-activating factor and acid phosphatase release in the rat.

    OpenAIRE

    Pinto, A; Calignano, A; Mascolo, N; Autore, G; Capasso, F

    1989-01-01

    1. When castor oil was administered by gavage to rats, the duodenum and jejunum but not ileum and colon produced large amounts (5-6 fold greater than control) of platelet activating factor (Paf). 2. Intraluminal release of acid phosphatase (AP) was also markedly increased (5-6 fold greater than control) in the duodenum and jejunum of castor oil-treated rats and there was a correlation between the elevated release of AP and intestinal hyperaemia. 3. These findings support a role for Paf as a m...

  6. Inhibition of lipid phosphate phosphatase activity by VPC32183 suppresses the ability of diacylglycerol pyrophosphate to activate ERK(1/2) MAP kinases.

    Science.gov (United States)

    Violet, Pierre-Christian; Billon-Denis, Emmanuelle; Robin, Philippe

    2012-11-01

    The lipidic metabolite, diacylglycerol pyrophosphate (DGPP), in its dioctanoyl form (DGPP 8:0), has been described as an antagonist for mammalian lysophosphatidic acid (LPA) receptors LPA1 and LPA3. In this study we show that DGPP 8:0 does not antagonize LPA dependent activation of ERK(1/2) MAP kinases but strongly stimulated them in various mammalian cell lines. LPA and DGPP 8:0 stimulation of ERK(1/2) occurred through different pathways. The DGPP 8:0 effect appeared to be dependent on PKC, Raf and MEK but was insensitive to pertussis toxin and did not involve G protein activation. Finally we showed that DGPP 8:0 effect on ERK(1/2) was dependent on its dephosphorylation by a phosphatase activity sharing lipid phosphate phosphatase properties. The inhibition of this phosphatase activity by VPC32183, a previously characterized LPA receptor antagonist, blocked the DGPP 8:0 effect on ERK(1/2) activation. Moreover, down-regulation of lipid phosphate phosphatase 1 (LPP1) expression by RNA interference technique also reduced DGPP 8:0-induced ERK(1/2) activation. Consistently, over expression of LPP1 in HEK293 cells increases DGPP 8:0 hydrolysis and this increased activity was inhibited by VPC32183. In conclusion, DGPP 8:0 does not exert its effect by acting on a G protein coupled receptor, but through its dephosphorylation by LPP1, generating dioctanoyl phosphatidic acid which in turn activates PKC. These results suggest that LPP1 could have a positive regulatory function on cellular signaling processes such as ERK(1/2) activation.

  7. Changes of the Biomass and Acid Phosphatase Activity in Maize (Zea mays L.) Lines Under Low-P Stress

    Institute of Scientific and Technical Information of China (English)

    YAO Qilun

    2008-01-01

    A pot culture trial was conducted to investigate the changes of the biomass and acid phosphatase (APase) activity in 10 maize lines under low-P stress. P-deficiency significantly decreased the biomass, but induced the significant enhancement of the APase activity. Since P-deficiency had smaller effects on the low-P tolerant maize lines compared with P-sensitive lines, it was demonstrated that differences of tolerance to P-deficiency existed among 10 different maize lines. In addition, the relative biomass and APase activity changed during the vegetative stage of development, and there existed a significant correlation between the biomass and APase activity under low-P stress. These results suggest that the biomass and APase activity can be regarded as indicative traits of maize lines for tolerance to low-P stress at seedling stage.

  8. High frequencies of elevated alkaline phosphatase activity and rickets exist in extremely low birth weight infants despite current nutritional support

    Directory of Open Access Journals (Sweden)

    Parker Bruce R

    2009-07-01

    Full Text Available Abstract Background Osteopenia and rickets are common among extremely low birth weight infants (ELBW, Methods We evaluated all ELBW infants admitted to Texas Children's Hospital NICU in 2006 and 2007. Of 211 admissions, we excluded 98 patients who were admitted at >30 days of age or did not survive/stay for >6 weeks. Bone radiographs obtained in 32 infants were reviewed by a radiologist masked to laboratory values. Results In this cohort of 113 infants, P-APA was found to have a significant inverse relationship with BW, gestational age and serum phosphorus. In paired comparisons, P-APA of infants Conclusion Elevation of P-APA >600 IU/L was very common in ELBW infants. BW was significantly inversely related to both P-APA and radiologic rickets. No single value of P-APA was related to radiological findings of rickets. Given the very high risk of osteopenia and rickets among ELBW infants, we recommend consideration of early screening and early mineral supplementation, especially among infants

  9. Characterization of a novel low-temperature-active, alkaline and sucrose-tolerant invertase

    Science.gov (United States)

    Zhou, Junpei; He, Limei; Gao, Yajie; Han, Nanyu; Zhang, Rui; Wu, Qian; Li, Junjun; Tang, Xianghua; Xu, Bo; Ding, Junmei; Huang, Zunxi

    2016-01-01

    A glycoside hydrolase family 32 invertase from Bacillus sp. HJ14 was expressed in Escherichia coli. The purified recombinant enzyme (rInvHJ14) showed typical biochemical properties of low-temperature-active and alkaline enzymes: (i) rInvHJ14 was active and stable in the range of pH 7.0–9.5 with an apparent pH optimum of 8.0; (ii) rInvHJ14 was most active but not stable at 30–32.5 °C, with 19.7, 48.2 and 82.1% of its maximum activity when assayed at 0, 10 and 20 °C, respectively, and the Ea, ΔG* (30 °C), Km (30 °C) and kcat (30 °C) values for hydrolysis of sucrose by rInvHJ14 was 47.6 kJ mol−1, 57.6 kJ mol−1, 62.9 mM and 746.2 s−1, respectively. The enzyme also showed strong sucrose tolerance. rInvHJ14 preserved approximately 50% of its highest activity in the presence of 2045.0 mM sucrose. Furthermore, potential factors for low-temperature-active and alkaline adaptations of rInvHJ14 were presumed. Compared with more thermostable homologs, rInvHJ14 has a higher frequency of glycine residues and a longer loop but a lower frequency of proline residues (especially in a loop) in the catalytic domain. The catalytic pockets of acid invertases were almost negatively charged while that of alkaline rInvHJ14 was mostly positively charged. PMID:27553125

  10. Characterization of a novel low-temperature-active, alkaline and sucrose-tolerant invertase.

    Science.gov (United States)

    Zhou, Junpei; He, Limei; Gao, Yajie; Han, Nanyu; Zhang, Rui; Wu, Qian; Li, Junjun; Tang, Xianghua; Xu, Bo; Ding, Junmei; Huang, Zunxi

    2016-01-01

    A glycoside hydrolase family 32 invertase from Bacillus sp. HJ14 was expressed in Escherichia coli. The purified recombinant enzyme (rInvHJ14) showed typical biochemical properties of low-temperature-active and alkaline enzymes: (i) rInvHJ14 was active and stable in the range of pH 7.0-9.5 with an apparent pH optimum of 8.0; (ii) rInvHJ14 was most active but not stable at 30-32.5 °C, with 19.7, 48.2 and 82.1% of its maximum activity when assayed at 0, 10 and 20 °C, respectively, and the Ea, ΔG(*) (30 °C), Km (30 °C) and kcat (30 °C) values for hydrolysis of sucrose by rInvHJ14 was 47.6 kJ mol(-1), 57.6 kJ mol(-1), 62.9 mM and 746.2 s(-1), respectively. The enzyme also showed strong sucrose tolerance. rInvHJ14 preserved approximately 50% of its highest activity in the presence of 2045.0 mM sucrose. Furthermore, potential factors for low-temperature-active and alkaline adaptations of rInvHJ14 were presumed. Compared with more thermostable homologs, rInvHJ14 has a higher frequency of glycine residues and a longer loop but a lower frequency of proline residues (especially in a loop) in the catalytic domain. The catalytic pockets of acid invertases were almost negatively charged while that of alkaline rInvHJ14 was mostly positively charged. PMID:27553125

  11. Use of an Anaerobic Chamber Environment for the Assay of Endogenous Cellular Protein-Tyrosine Phosphatase Activities

    Directory of Open Access Journals (Sweden)

    Zhu Li

    2002-01-01

    Full Text Available Protein-tyrosine phosphatases (PTPases have a catalytic cysteine residue whose reduced state is integral to the reaction mechanism. Since exposure to air can artifactually oxidize this highly reactive thiol, PTPase assays have typically used potent reducing agents to reactivate the enzymes present; however, this approach does not allow for the measurement of the endogenous PTPase activity directly isolated from the in vivo cellular environment. Here we provide a method for using an anaerobic chamber to preserve the activity of the total PTPase complement in a tissue lysate or of an immunoprecipitated PTPase homolog to characterize their endogenous activation state. Comparison with a sample treated with biochemical reducing agents allows the determination of the activatable (reducible fraction of the endogenous PTPase pool.

  12. Nitrate reducing bacterial activity under alkaline conditions found in intermediate-level nuclear waste repositories

    International Nuclear Information System (INIS)

    This study focuses on the bituminous waste, a mixture of bitumen and insoluble salts, mainly of nitrate and sulphate carrying low and intermediate level radioactivity. The re-saturation of the disposal cells after closure will likely lead to the development of microbial activity. One of the most critical aspects limiting bacterial activity is the high alkalinity prevailing in the barrier concrete system and at the concrete-clay rock interface (pH = 11-13.5). The objective of this work is to evaluate the possible activity of nitrate reducing bacteria under these alkaline conditions, building on knowledge for alkaliphilic bacteria gained from similar conditions. Alkaliphilic bacteria develop optimally at pH values above 9, often between 10 and 12, in some cases above 12. They are likely to be predominant under alkaline conditions, but they will be replaced by neutrophilic bacteria as soon as local zones of near-neutral pH develop. In the case of a waste cell this could be at the interface to the clay rock or in the vicinity of organic matter releasing bituminous waste. Two sets of experiments have been carried out, bringing nitrate in contact with a solution containing organic matter (acetate) and a culture of alkaliphilic microbes, one focuses on the influence of nitrate concentrations, the other on the impact of changes in pH (10.0 and 12.4). The results indicate a low microbial activity at pH 12.4 whereas a significant nitrate reduction is obtained at pH 10.0. Furthermore, nitrite is the main product of nitrate reduction; traces of ammonium have also been measured. (authors)

  13. Protein phosphatase PP1-NIPP1 activates mesenchymal genes in HeLa cells.

    Science.gov (United States)

    Van Dessel, Nele; Boens, Shannah; Lesage, Bart; Winkler, Claudia; Görnemann, Janina; Van Eynde, Aleyde; Bollen, Mathieu

    2015-05-22

    The deletion of the protein phosphatase-1 (PP1) regulator known as Nuclear Inhibitor of PP1 (NIPP1) is embryonic lethal during gastrulation, hinting at a key role of PP1-NIPP1 in lineage specification. Consistent with this notion we show here that a mild, stable overexpression of NIPP1 in HeLa cells caused a massive induction of genes of the mesenchymal lineage, in particular smooth/cardiac-muscle and matrix markers. This reprogramming was associated with the formation of actin-based stress fibers and retracting filopodia, and a reduced proliferation potential. The NIPP1-induced mesenchymal transition required functional substrate and PP1-binding domains, suggesting that it involves the selective dephosphorylation of substrates of PP1-NIPP1. PMID:25907536

  14. 基于不同方法测定土壤酸性磷酸酶活性的比较%Comparison of soil acid phosphatase activity determined by different methods

    Institute of Scientific and Technical Information of China (English)

    李莹飞; 耿玉清; 周红娟; 杨英

    2016-01-01

    Method, edited by Songyin Guan, for measurement method of soil acid phosphatase activity based on phenyl phosphate disodium salt substrate. In contrast, researchers outside China mainly cite the book entitled Methods of Soil Enzymology, edited by Dick, that was based on disodium p-Nitrophenyl phosphate tetrahydrate (PNPP) substrate. However, non-conspicuous coloration has existed for the measurement of products based on phenyl phosphate disodium salt substrate. Furthermore, it has been difficult for researchers to select an optimal method for determining acid phosphatase activity since these methods use different substrates. To determine the optimal method for measuring soil acid phosphatase activity, three different methods were used to measure the acid phosphatase activity of 10 soil samples of acid, neutral and alkaline soils, respectively. The three selected methods were 1) based on phenyl phosphate disodium salt substrate and colored using pH 5.0 acetate buffer (DPP 1);2) based on phenyl phosphate disodium salt substrate and colored using pH 9.4 borate buffer (DPP 2) during chromogenic process;or 3) the PNPP method. Furthermore, the study analyzed the effects of different pH buffers and phenol concentrations on product absorbance. The results showed that chromogenic reaction of phenol with 2,6-dibromchinone-chlorimide was colorless within pH≤6 buffer solution with phenyl phosphate disodium salt as the substrate. In contrast, the above chromogenic reaction was observed under alkaline buffer (pH≥ 8) in all the samples. And there were significant differences in the Pearson correlation coefficient (R2) between phenol concentration and product absorbance at 0.01 level. Therefore, pH was a significant factor in determining the coloration between phenol and 2,6-dibromchinone-chlorimide. Furthermore, when acid phosphatase activity was determined using the PNPP method, the coefficient of variation of acid phosphatase activities in the 10 soil samples increased by 70

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

    Directory of Open Access Journals (Sweden)

    Stefano Luisa

    2005-01-01

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

  16. 2143例0~12岁儿童血清碱性磷酸酶结果探讨%Clinical value of serum alkaline phosphatase in children aged 0 to 12 years

    Institute of Scientific and Technical Information of China (English)

    穆迎娟; 李忠信; 郑小玲

    2012-01-01

    目的 探讨2143例0~12岁儿童血清碱性磷酸酶(ALP)的结果.方法 应用SIEMENS DimensionRxL Max全自动生化分析仪检测2 143例来本院体检的0~12岁儿童血清ALP,对所得的数据进行统计学处理.结果 儿童血清ALP值在3~岁、9~岁和10~岁组男女性别间差异有统计学意义(P<0.05),其余组男女性别间差异均无统计学意义.儿童血清ALP值在1~岁、10~岁、11~12岁年龄组间差异有统计学意义(P<0.05),而其他年龄组之间差异无统计学意义(P>0.05).结论 0~12岁儿童血清ALP水平显著高于成人.儿童时期血清ALP水平与其年龄和性别相关,应根据不同性别、年龄的儿童相应的发育情况给予不同的参考范围.%Objective To analyze the clinical significance of serum alkaline phosphatase (ALP) in children. Methods Serum level of ALP from 2 143 children with the age of 0 to 12 years old children in our hospital attending for the physical examination were measured by SIEMENS Dimension RxL Max AUTOLAB, and the results were analyzed statistically. Results There was a significant difference in the level of serum ALP in children aged 3 to 4 years old, 9 to 10 years old and 10 to 11 years old. The level of ALP in children aged 0 to 1 year old and 2 to 10 years was similar in both sex. Conclusion The serum level of ALP in children was higher than the adult. The level of ALP is related to their age and sex. so children serum ALP values should be based on the development situation of children of different gender, age, given the different reference ranges.

  17. Rapamycin induces mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) expression through activation of protein kinase B and mitogen-activated protein kinase kinase pathways.

    Science.gov (United States)

    Rastogi, Ruchi; Jiang, Zhongliang; Ahmad, Nisar; Rosati, Rita; Liu, Yusen; Beuret, Laurent; Monks, Robert; Charron, Jean; Birnbaum, Morris J; Samavati, Lobelia

    2013-11-22

    Mitogen-activated protein kinase phosphatase-1 (MKP-1), also known as dual specificity phosphatase-1 (DUSP-1), plays a crucial role in the deactivation of MAPKs. Several drugs with immune-suppressive properties modulate MKP-1 expression as part of their mechanism of action. We investigated the effect of mTOR inhibition through rapamycin and a dual mTOR inhibitor (AZD2014) on MKP-1 expression. Low dose rapamycin led to a rapid activation of both AKT and ERK pathways with a subsequent increase in MKP-1 expression. Rapamycin treatment led to phosphorylation of CREB, transcription factor 1 (ATF1), and ATF2, three transcription factors that bind to the cyclic AMP-responsive elements on the Mkp-1 promoter. Inhibition of either the MEK/ERK or the AKT pathway attenuated rapamycin-mediated MKP-1 induction. AZD2014 did not activate AKT but activated the ERK pathway, leading to a moderate MKP-1 induction. Using bone marrow-derived macrophages (BMDMs) derived from wild-type (WT) mice or mice deficient in AKT1 and AKT2 isoforms or BMDM from targeted deficiency in MEK1 and MEK2, we show that rapamycin treatment led to an increased MKP1 expression in BMDM from WT but failed to do so in BMDMs lacking the AKT1 isoform or MEK1 and MEK2. Importantly, rapamycin pretreatment inhibited LPS-mediated p38 activation and decreased nitric oxide and IL-6 production. Our work provides a conceptual framework for the observed immune modulatory effect of mTOR inhibition.

  18. Biological activities of a mixture of biosurfactant from Bacillus subtilis and alkaline lipase from Fusarium oxysporum

    Directory of Open Access Journals (Sweden)

    Cedenir Pereira de Quadros

    2011-03-01

    Full Text Available In this study, we investigate the antimicrobial effects of a mixture of a biosurfactant from Bacillus subtilis and an alkaline lipase from Fusarium oxysporum (AL/BS mix on several types of microorganisms, as well as their abilities to remove Listeria innocua ATCC 33093 biofilm from stainless steel coupons. The AL/BS mix had a surface tension of around 30 mN.m-1, indicating that the presence of alkaline lipase did not interfere in the surface activity properties of the tensoactive component. The antimicrobial activity of the AL/BS mix was determined by minimum inhibitory concentration (MIC micro-assays. Among all the tested organisms, the presence of the mixture only affected the growth of B. subtilis CCT 2576, B. cereus ATCC 10876 and L. innocua. The most sensitive microorganism was B. cereus (MIC 0.013 mg.mL-1. In addition, the effect of the sanitizer against L. innocua attached to stainless steel coupons was determined by plate count after vortexing. The results showed that the presence of the AL/BS mix improved the removal of adhered cells relative to treatment done without the sanitizer, reducing the count of viable cells by 1.72 log CFU.cm-2. However, there was no significant difference between the sanitizers tested and an SDS detergent standard (p<0.05.

  19. Effects of nitrogen fertilization on soil nutrient concentration and phosphatase activity and forage nutrient uptake from a grazed pasture system.

    Science.gov (United States)

    Dillard, Sandra Leanne; Wood, Charles Wesley; Wood, Brenda Hall; Feng, Yucheng; Owsley, Walter Frank; Muntifering, Russell Brian

    2015-05-01

    Over a 3-year period, the effect of differing N-application regimes on soil extractable-P concentration, soil phosphatase activity, and forage P uptake in a P-enriched grazed-pasture system was investigated. In the fall of each year, six 0.28-ha plots were overseeded with triticale ( × Triticosecale rimpaui Wittm.) and crimson clover (Trifolium incarnatum) into a tall fescue (Lolium arundinacea)/bermudagrass (Cynodon dactylon) sod and assigned to 1 of 3 N-fertilizer treatments (n = 2): 100% of N recommendation in a split application (100N), 50% in a single application (50N), and 0% of N recommendation (0N) for triticale. Cattle commenced grazing the following spring and grazed until May. In the summer, plots were overseeded with cowpea (Vigna unguiculata), fertilized at the same rates by reference to N recommendations for bermudagrass, and grazed by cattle until September. There were no effects of N fertilization on soil phosphatase activity, electrical conductivity, or concentrations of water-soluble P. Concentrations of extractable P decreased in plots receiving 50N, but increasing N fertilization to 100N resulted in no further reduction in extractable P. Forage biomass, foliar P concentrations, and forage P mass were not affected by N fertilization rates at the plant-community level, but responses were observed within individual forage species. Results are interpreted to mean that N fertilization at 50% of the agronomic recommendation for the grass component can increase forage P mass of specific forages and decrease soil extractable P, thus providing opportunity for decreasing P losses from grazed pasture. PMID:25728918

  20. Nutrient addition modifies phosphatase activities along an altitudinal gradient in a tropical montane forest in Southern Ecuador

    Directory of Open Access Journals (Sweden)

    Karla eDietrich

    2016-02-01

    Full Text Available Atmospheric nutrient deposition and climate change are expected to endanger the diversity of tropical forest ecosystems. Nitrogen (N deposition might influence nutrient fluxes beyond the N cycle by a concomitant increased demand for other nutritional elements such as phosphorus (P. Organisms might respond to the increased P demand by enhanced activity of enzymes involved in releasing inorganic P from organic matter (OM. Our aims were to assess the effect of i climate shifts (approximated by an altitudinal gradient, and ii nutrient addition (N, P, N+P on phosphatase activity (PA in organic layer and mineral soil of a tropical montane rainforest in Southern Ecuador. A nutrient manipulation experiment (NUMEX was set up along an altitudinal gradient (1000, 2000, and 3000 m a.s.l.. We determined PA and inorganic and total P concentrations. PA at 1000 m was significantly lower (mean ± standard error: 48 ± 20 µmol p-NP g-1 dm h-1 as compared to 2000 m and 3000 m (119 ± 11 and 137 ± 19, respectively. One explanation might be that very rapid decomposition of OM at 1000 m results in very thin organic layers reducing the stabilization of enzymes and thus, resulting in leaching loss of enzymes under the humid tropical climate. We found no effect of N addition on PA neither in the organic layer nor in mineral soil, probably because of the low nutrient addition rates that showed ambiguous results so far on productivity measures as a proxy for P demand. In the organic layers of P and N+P treatments, we found decreased PA and increased concentrations of inorganic P. This indicates that the surplus of inorganic P reduced the biosynthesis of phosphatase enzymes. PA in megadiverse montane rainforests is likely to be unaffected by increased atmospheric N deposition but reduced upon atmospheric P deposition.

  1. The catalytic activity of the CD45 membrane-proximal phosphatase domain is required for TCR signaling and regulation

    DEFF Research Database (Denmark)

    Desai, D M; Sap, J; Silvennoinen, O;

    1994-01-01

    Cell surface expression of CD45, a receptor-like protein tyrosine phosphatase (PTPase), is required for T cell antigen receptor (TCR)-mediated signal transduction. Like the majority of transmembrane PTPases, CD45 contains two cytoplasmic phosphatase domains, whose relative in vivo function is not...

  2. Electrocatalytic activity of porous nanostructured Fe/Pt-Fe electrode for methanol electrooxidation in alkaline media

    Institute of Scientific and Technical Information of China (English)

    Javad Hosseini; Mehdi Abdolmaleki; Hamid Reza Pouretedal; Mohammad Hossein Keshavarz

    2015-01-01

    An electrochemical approach to fabricate a nanostructured Fe/Pt-Fe catalyst through electrodepo-sition followed by galvanic replacement is presented. An Fe/Pt-Fe nanostructured electrode was prepared by deposition of Fe-Zn onto a Fe electrode surface, followed by replacement of the Zn by Pt at open-circuit potential in a Pt-containing alkaline solution. Scanning electron microscopy and energy-dispersive X-ray techniques reveal that the Fe/Pt-Fe electrode is porous and contains Pt. The electrocatalytic activity of the Fe/Pt-Fe electrode for oxidation of methanol was examined by cyclic voltammetry and chronoamperometry. The electrooxidation current on the Fe/Pt-Fe catalyst is much higher than that on flat Pt and smooth Fe catalysts. The onset potential and peak potential on the Fe/Pt-Fe catalyst are more negative than those on flat Pt and smooth Fe electrodes for methanol electrooxidation. All results show that this nanostructured Fe/Pt-Fe electrode is very attractive for integrated fuel cell applications in alkaline media.

  3. Value of detection of calcium in whole blood and bone alkaline phosphatase in the diagnosis of rickets%全血钙和骨碱性磷酸酶检测在佝偻病诊断中的应用

    Institute of Scientific and Technical Information of China (English)

    韦孟兰

    2013-01-01

    目的 探讨全血钙和骨碱性磷酸酶(BALP)在儿童维生素D缺乏性佝偻病中的诊断价值.方法 对755例体检儿童采末梢血,用全血干化学法和免疫浓缩技术检测BALP,全血钙检测采用原子吸收光谱分析法.结果 755例体检儿童中BALP≤200 U/L 138例,200~250 U/L 386例,≥250U/L231例.血钙<1.55 mmol/L 79例,≥1.55mmol/L676例.BALP异常检出率为81.7%(617/755),血钙异常检出率为10.5%(79/755).血钙在各年龄组和男女性别间差异均无统计学意义.BALP在男女性别间差异无统计学意义,在不同年龄组间差异有统计学意义,1岁以下组BALP异常检出率最高,3~5岁组最低.大部分儿童处于维生素D轻度缺乏或缺乏状态,且随着年龄增长发病率有逐渐降低的趋势.结论 BALP的检测在维生素D缺乏性佝偻病诊断中较血钙测定敏感,更能反映人体钙营养水平.为更好地预防、控制和治疗佝偻病,婴幼儿应定期做相关项目的健康体检.%Objective To investigate the diagnostic value of calcium in whole blood and bone alkaline phosphatase (BALP) in vitamin D deficiency rickets in children. Methods Peripheral blood was collected from 755 children. Levels of BALP were detected based on dry chemical and immune concentrated technique, and calcium was detected by atomic absorption spectroscopy. Results BALP levels were showed ≤200 U/L in 138 children, 200-250 U/L in 386 children, and ≥ 250 U/L in 231 children. Calcium was found <1.55 mmo1/L in 79 children and ≥ 1.55 mmol/L in 676 children. The detection rate of abnormal BALP was 81.7% (617/755), and that of abnormal calcium was 0.5% (79/755). Calcium showed no statistically significant difference between different age groups and gender groups. The BALP showed no statistically significant difference between males and females, but significant differences between different age groups. The detection rate of abnormal BALP was highest in age group that smaller than one

  4. Effect of insecticides alone and in combination with fungicides on nitrification and phosphatase activity in two groundnut (Arachis hypogeae L.) soils.

    Science.gov (United States)

    Srinivasulu, M; Jaffer Mohiddin, G; Subramanyam, K; Rangaswamy, V

    2012-06-01

    The effect of selected pesticides, monocrotophos, chlorpyrifos alone and in combination with mancozeb and carbendazim, respectively, was tested on nitrification and phosphatase activity in two groundnut (Arachis hypogeae L.) soils. The oxidation of ammonical nitrogen was significantly enhanced under the impact of selected pesticides alone and in combinations at 2.5 kg ha(-1) in black soil, and furthermore, increase in concentration of pesticides decreased the rate of nitrification, whereas in the case of red soil, the nitrification was increased up to 5.0 kg ha(-1) after 4 weeks, and then decline phase was started gradually from 6 to 8 weeks of incubation. The activity of phosphatase was increased in soils, which received the monocrotophos alone and in combination with mancozeb up to 2.5 and 5.0 kg ha(-1), whereas the application of chlorpyrifos singly and in combination with carbendazim at 2.5 kg ha(-1) profoundly increased the phosphatase activity after 20 days of incubation, in both soils. But higher concentrations of pesticides were either innocuous or inhibitory to the phosphatase activity.

  5. A binary palladium-bismuth nanocatalyst with high activity and stability for alkaline glucose electrooxidation

    Science.gov (United States)

    Chen, Cheng-Chuan; Lin, Cheng-Lan; Chen, Lin-Chi

    2015-08-01

    Binary palladium-bismuth nanocatalysts supported on functionalized multi-walled carbon nanotubes (Pd-Bi/C) are synthesized using a one-pot polyol method. The prepared Pd-Bi/C catalysts have a metal particle range from 5.25 to 12.98 nm and are investigated for alkaline electrocatalytic glucose oxidation reaction (GOR). The physical properties of the catalysts are characterized by X-ray diffraction (XRD), scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDS), transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS). The electrochemical activities are determined by cyclic voltammetry (CV), linear sweep voltammetry (LSV), Tafel analysis and chronoamperomtry (CA) for comparing the electrochemical active surface area (ECSA), GOR onset potential, GOR peak current density, Tafel slope, poisoning rate and cycling stability of the Pd-Bi/C catalysts. It is found that Pd-Bi/C (1:0.14) can significantly enhance the electrocatalytic activity on GOR about 40% times higher than Pd/C and as well as has a 3.7-fold lower poisoning rate. The in-use stability of Pd-Bi/C (1:0.14) is also remarkably improved, according to the results of the 200 cycling CV test. The effects of the operating temperature and the concentration of glucose and NaOH electrolyte on Pd-Bi/C (1:0.14) are further studied in this work. The highest Pd-Bi/C catalyzed GOR current density of 29.5 mA cm-2 is attained in alkaline medium.

  6. Synthesis and protein tyrosine phosphatase 1B inhibition activities of two new synthetic bromophenols and their methoxy derivatives

    Science.gov (United States)

    Cui, Yongchao; Shi, Dayong; Hu, Zhiqiang

    2011-11-01

    3-bromo-4,5-bis(2,3-dibromo-4,5-dihydroxybenzyl)-1,2-benzenediol ( 1) is a natural bromophenol isolated from the red algae Rhodomela confervoides that exhibits significant inhibition against protein tyrosine phosphatase 1B (PTP1B). Based on its activity, we synthesized two new synthetic bromophenols and their methoxy derivatives from vanillin using the structure of natural bromophenol 1 as a scaffold. The structures of these bromophenols were elucidated from 1H NMR, 13C NMR, and high resolution electron ionization mass spectrometry as 2,3-dibromo-1-(2'-bromo-6'-(3″,4″-dimethoxybenzyl)-3',4'-dimethoxybenzyl)-4,5-dimethoxybenzene ( 2), 2,3-dibromo-1-(2'-bromo-6'-(2″-bromo-4″,5″-dimethoxybenzyl)-3',4'-dimethoxybenzyl)-4,5-dimethoxybenzene ( 3), 3,4-dibromo-5-(2'-bromo-6'-(2″-bromo-4″,5″-dihydroxybenzyl)-3',4'-dihydroxybenzyl)pyrocatechol ( 4) and 3,4-dibromo-5-(2'-bromo-6'-(3″,4″-dihydroxybenzyl)-3',4'-dihydroxybenzyl)pyrocatechol ( 5). PTP1B inhibition activities of these compounds were evaluated using a colorimetric assay, and compounds 3 and 4 demonstrated interesting activity against PTP1B.

  7. Ubiquitination of the bacterial inositol phosphatase, SopB, regulates its biological activity at the plasma membrane.

    LENUS (Irish Health Repository)

    Knodler, Leigh A

    2009-11-01

    The Salmonella type III effector, SopB, is an inositol polyphosphate phosphatase that modulates host cell phospholipids at the plasma membrane and the nascent Salmonella-containing vacuole (SCV). Translocated SopB persists for many hours after infection and is ubiquitinated but the significance of this covalent modification has not been investigated. Here we identify by mass spectrometry six lysine residues of SopB that are mono-ubiquitinated. Substitution of these six lysine residues with arginine, SopB-K(6)R, almost completely eliminated SopB ubiquitination. We found that ubiquitination does not affect SopB stability or membrane association, or SopB-dependent events in SCV biogenesis. However, two spatially and temporally distinct events are dependent on ubiquitination, downregulation of SopB activity at the plasma membrane and prolonged retention of SopB on the SCV. Activation of the mammalian pro-survival kinase Akt\\/PKB, a downstream target of SopB, was intensified and prolonged after infection with the SopB-K(6)R mutant. At later times, fewer SCV were decorated with SopB-K(6)R compared with SopB. Instead SopB-K(6)R was present as discrete vesicles spread diffusely throughout the cell. Altogether, our data show that ubiquitination of SopB is not related to its intracellular stability but rather regulates its enzymatic activity at the plasma membrane and intracellular localization.

  8. The mitogen-activated protein kinase (MAPK) cascade controls phosphatase and tensin homolog (PTEN) expression through multiple mechanisms.

    Science.gov (United States)

    Ciuffreda, Ludovica; Di Sanza, Cristina; Cesta Incani, Ursula; Eramo, Adriana; Desideri, Marianna; Biagioni, Francesca; Passeri, Daniela; Falcone, Italia; Sette, Giovanni; Bergamo, Paola; Anichini, Andrea; Sabapathy, Kanaga; McCubrey, James A; Ricciardi, Maria Rosaria; Tafuri, Agostino; Blandino, Giovanni; Orlandi, Augusto; De Maria, Ruggero; Cognetti, Francesco; Del Bufalo, Donatella; Milella, Michele

    2012-06-01

    The mitogen-activated protein kinase (MAPK) and PI3K pathways are regulated by extensive crosstalk, occurring at different levels. In tumors, transactivation of the alternate pathway is a frequent "escape" mechanism, suggesting that combined inhibition of both pathways may achieve synergistic antitumor activity. Here we show that, in the M14 melanoma model, simultaneous inhibition of both MEK and mammalian target of rapamycin (mTOR) achieves synergistic effects at suboptimal concentrations, but becomes frankly antagonistic in the presence of relatively high concentrations of MEK inhibitors. This observation led to the identification of a novel crosstalk mechanism, by which either pharmacologic or genetic inhibition of constitutive MEK signaling restores phosphatase and tensin homolog (PTEN) expression, both in vitro and in vivo, and inhibits downstream signaling through AKT and mTOR, thus bypassing the need for double pathway blockade. This appears to be a general regulatory mechanism and is mediated by multiple mechanisms, such as MAPK-dependent c-Jun and miR-25 regulation. Finally, PTEN upregulation appears to be a major effector of MEK inhibitors' antitumor activity, as cancer cells in which PTEN is inactivated are consistently more resistant to the growth inhibitory and anti-angiogenic effects of MEK blockade. PMID:22215152

  9. Surface-bound phosphatase activity in living hyphae of ectomycorrhizal fungi of Nothofagus obliqua.

    Science.gov (United States)

    Alvarez, Maricel; Godoy, Roberto; Heyser, Wolfgang; Härtel, Steffen

    2004-01-01

    We determined the location and the activity of surface-bound phosphomonoesterase (SBP) of five ectomycorrhizal (EM) fungi of Nothofagus oblique. EM fungal mycelium of Paxillus involutus, Austropaxillus boletinoides, Descolea antartica, Cenococcum geophilum and Pisolithus tinctorius was grown in media with varying concentrations of dissolved phosphorus. SBP activity was detected at different pH values (3-7) under each growth regimen. SBP activity was assessed using a colorimetric method based on the hydrolysis of p-nitrophenyl phosphate (pNPP) to p-nitrophenol phosphate (pNP) + P. A new technique involving confocal laser-scanning microscopy (LSM) was used to locate and quantify SBP activity on the hyphal surface. EM fungi showed two fundamentally different patterns of SBP activity in relation to varying environmental conditions (P-concentrations and pH). In the cases of D. antartica, A. boletinoides and C. geophilum, changes in SBP activity were induced primarily by changes in the number of SBP-active centers on the hyphae. In the cases of P. tinctorius and P. involutus, the number of SBP-active centers per μm hyphal length changed much less than the intensity of the SBP-active centers on the hyphae. Our findings not only contribute to the discussion about the role of SBP-active centers in EM fungi but also introduce LSM as a valuable method for studying EM fungi.

  10. Electrochemical determination of activation energies for methanol oxidation on polycrystalline platinum in acidic and alkaline electrolytes.

    Science.gov (United States)

    Cohen, Jamie L; Volpe, David J; Abruña, Héctor D

    2007-01-01

    The oxidation pathways of methanol (MeOH) have been the subject of intense research due to its possible application as a liquid fuel in polyelectrolyte membrane (PEM) fuel cells. The design of improved catalysts for MeOH oxidation requires a deep understanding of these complex oxidation pathways. This paper will provide a discussion of the literature concerning the extensive research carried out in acidic and alkaline electrolytes. It will highlight techniques that have proven useful in the determination of product ratios, analysis of surface poisoning, anion adsorption, and oxide formation processes, in addition to the effects of temperature on the MeOH oxidation pathways at bulk polycrystalline platinum (Pt(poly)) electrodes. This discussion will provide a framework with which to begin the analysis of activation energy (E(a)) values. This kinetic parameter may prove useful in characterizing the rate-limiting step of the MeOH oxidation at an electrode surface. This paper will present a procedure for the determination of E(a) values for MeOH oxidation at a Pt(poly) electrode in acidic and alkaline media. Values from 24-76 kJ mol(-1) in acidic media and from 36-86 kJ mol(-1) in alkaline media were calculated and found to be a function of applied potential and direction of the potential sweep in a voltammetric experiment. Factors that influence the magnitude of the calculated E(a) include surface poisoning from MeOH oxidation intermediates, anion adsorption from the electrolyte, pH effects, and oxide formation processes. These factors are all potential, and temperature, dependent and must clearly be addressed when citing E(a) values in the literature. Comparison of E(a) values must be between systems of comparable electrochemical environment and at the same potential. E(a) values obtained on bulk Pt(poly), compared with other catalysts, may give insight into the superiority of other Pt-based catalysts for MeOH oxidation and lead to the development of new catalysts

  11. Adiponectin haploinsufficiency promotes mammary tumor development in MMTV-PyVT mice by modulation of phosphatase and tensin homolog activities.

    Directory of Open Access Journals (Sweden)

    Janice B B Lam

    Full Text Available BACKGROUND: Adiponectin is an adipokine possessing beneficial effects on obesity-related medical complications. A negative association of adiponectin levels with breast cancer development has been demonstrated. However, the precise role of adiponectin deficiency in mammary carcinogenesis remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, MMTV-polyomavirus middle T antigen (MMTV-PyVT transgenic mice with reduced adiponectin expressions were established and the stromal effects of adiponectin haploinsufficiency on mammary tumor development evaluated. In mice from both FVB/N and C57BL/6J backgrounds, insufficient adiponectin production promoted mammary tumor onset and development. A distinctive basal-like subtype of tumors, with a more aggressive phenotype, was derived from adiponectin haplodeficient MMTV-PyVT mice. Comparing with those from control MMTV-PyVT mice, the isolated mammary tumor cells showed enhanced tumor progression in re-implanted nude mice, accelerated proliferation in primary cultures, and hyperactivated phosphatidylinositol-3-kinase (PI3K/Akt/beta-catenin signaling, which at least partly attributed to the decreased phosphatase and tensin homolog (PTEN activities. Further analysis revealed that PTEN was inactivated by a redox-regulated mechanism. Increased association of PTEN-thioredoxin complexes was detected in tumors derived from mice with reduced adiponectin levels. The activities of thioredoxin (Trx1 and thioredoxin reductase (TrxR1 were significantly elevated, whereas treatment with either curcumin, an irreversible inhibitor of TrxR1, or adiponectin largely attenuated their activities and resulted in the re-activation of PTEN in these tumor cells. Moreover, adiponectin could inhibit TrxR1 promoter-mediated transcription and restore the mRNA expressions of TrxR1. CONCLUSION: Adiponectin haploinsufficiency facilitated mammary tumorigenesis by down-regulation of PTEN activity and activation of PI3K

  12. Enhancement of thermophilic anaerobic digestion of thickened waste activated sludge by combined microwave and alkaline pretreatment

    Institute of Scientific and Technical Information of China (English)

    Yongzhi Chi; Yuyou Li; Xuening Fei; Shaopo Wang; Hongying Yun

    2011-01-01

    Pretreatment of thickened waste activated sludge (TWAS) by combined microwave and alkaline pretreatment (MAP) was studied to improve thermophilic anaerobic digestion efficiency.Uniform design was applied to determine the combination of target temperature (110-210℃),microwave holding time (1-51 min),and NaOH dose (0-2.5 g NaOH/g suspended solids (SS)) in terms of their effect on volatile suspended solids (VSS) solubilization.Maximum solubilization ratio (85.1%) of VSS was observed at 210℃ with 0.2 g-NaOH/g-SS and 35 min holding time.The effects of 12 different pretreatment methods were investigated in 28 thermophilic batch reactors by monitoring cumulative methane production (CMP).Improvements in methane production in the TWAS were directly related to the microwave and alkaline pretreatment of the sludge.The highest CMP was a 27% improvement over the control.In spite of the increase in soluble chemical oxygen demand concentration and the decrease in dewaterability of digested sludge,a semi-continuous thennophilic reactor fed with pretreated TWAS without neutralization (at 170℃ with 1 rain holding time and 0.05 g NaOH/g SS) was stable and functioned well,with volatile solid (VS) and total chemical oxygen demand (TCOD) reductions of 28% and 18%,respectively,which were higher than those of the control system.Additionally,methane yields (L@STP/g-CODadded,at standard temperature and pressure (STP) conditions of 0℃ and 101.325 kPa) and (L@STP/g VSadded) increased by 17% and 13%,respectively,compared to the control reactor.

  13. Avicin D: a protein reactive plant isoprenoid dephosphorylates Stat 3 by regulating both kinase and phosphatase activities.

    Directory of Open Access Journals (Sweden)

    Valsala Haridas

    Full Text Available Avicins, a class of electrophilic triterpenoids with pro-apoptotic, anti-inflammatory and antioxidant properties, have been shown to induce redox-dependant post-translational modification of cysteine residues to regulate protein function. Based on (a the cross-talk that occurs between redox and phosphorylation processes, and (b the role of Stat3 in the process of apoptosis and carcinogenesis, we chose to study the effects of avicins on the processes of phosphorylation/dephosphorylation in Stat3. Avicins dephosphorylate Stat3 in a variety of human tumor cell lines, leading to a decrease in the transcriptional activity of Stat3. The expression of Stat3-regulated proteins such as c-myc, cyclin D1, Bcl2, survivin and VEGF were reduced in response to avicin treatment. Underlying avicin-induced dephosphorylation of Stat3 was dephosphorylation of JAKs, as well as activation of protein phosphatase-1. Downregulation of both Stat3 activity and expression of Stat 3-controlled pro-survival proteins, contributes to the induction of apoptosis in avicin treated tumor cells. Based on the role of Stat3 in inflammation and wounding, and the in vivo inhibition of VEGF by avicins in a mouse skin carcinogenesis model, it is likely that avicin-induced inhibition of Stat3 activity results in the suppression of the pro-inflammatory and pro-oxidant stromal environment of tumors. Activation of PP-1, which also acts as a cellular economizer, combined with the redox regulation by avicins, can aid in redirecting metabolism from growth promoting anabolic to energy sparing pathways.

  14. Adiponectin Increases Skeletal Muscle Mitochondrial Biogenesis by Suppressing Mitogen-Activated Protein Kinase Phosphatase-1

    OpenAIRE

    Qiao, Liping; Kinney, Brice; Yoo, Hyung sun; Lee, Bonggi; Schaack, Jerome; Shao, Jianhua

    2012-01-01

    Adiponectin enhances mitochondrial biogenesis and oxidative metabolism in skeletal muscle. This study aimed to investigate the underlying mechanisms through which adiponectin induces mitochondrial biogenesis in skeletal muscle. Mitochondrial contents, expression, and activation status of p38 mitogen-activated protein kinase (MAPK) and PPARγ coactivator 1α (PGC-1α) were compared between skeletal muscle samples from adiponectin gene knockout, adiponectin-reconstituted, and control mice. Adenovi...

  15. Alkaline-sulphate activation processes of a Spanish blast furnace slag

    Directory of Open Access Journals (Sweden)

    Fernández Jiménez, A.

    1996-03-01

    Full Text Available Alkaline-sulphate activation processes of a Spanish granulated blast furnace slag (Avilés, Ensidesa have been studied. Activator solutions used were: deionized water (as reference solution, Ca(OH2 (3,5∙10-3N, NaOH (1N, Na2CO3 (2N, CaSO4∙2H2O (3,0∙10-3N at 25ºC. The influence of the nature of alkaline or sulphate solution cation on slag activation process was verified. Sodium solutions decrease the induction period while calcium solutions increase it. Slag reaction degree was also determined, likewise the nature of the different reaction products formed as a function of the activator solution nature.

    Se han estudiado los procesos de activación alcalinosulfáticos de una escoria granulada de alto horno española (Avilés, Ensidesa. Las disoluciones activantes utilizadas fueron: H2O desionizada (como disolución de referencia, Ca(OH2 (3,5∙10-3N, NaOH (1N, Na2CO3 (2N, CaSO4∙2H2O (3,0∙10-3N a 25ºC. Se ha comprobado la influencia de la naturaleza del catión de la disolución alcalina o sulfática sobre el proceso de activación de la escoria. Las disoluciones sódicas disminuyen el período de inducción, mientras que las disoluciones cálcicas lo incrementan. También se determinó el grado de reacción de la escoria, así como la naturaleza de los distintos productos de reacción formados, en función de la naturaleza de la disolución activante.

  16. Phage & phosphatase: a novel phage-based probe for rapid, multi-platform detection of bacteria.

    Science.gov (United States)

    Alcaine, S D; Pacitto, D; Sela, D A; Nugen, S R

    2015-11-21

    Genetic engineering of bacteriophages allows for the development of rapid, highly specific, and easily manufactured probes for the detection of bacterial pathogens. A challenge for novel probes is the ease of their adoption in real world laboratories. We have engineered the bacteriophage T7, which targets Escherichia coli, to carry the alkaline phosphatase gene, phoA. This inclusion results in phoA overexpression following phage infection of E. coli. Alkaline phosphatase is commonly used in a wide range of diagnostics, and thus a signal produced by our phage-based probe could be detected using common laboratory equipment. Our work demonstrates the successful: (i) modification of T7 phage to carry phoA; (ii) overexpression of alkaline phosphatase in E. coli; and (iii) detection of this T7-induced alkaline phosphatase activity using commercially available colorimetric and chemilumiscent methods. Furthermore, we demonstrate the application of our phage-based probe to rapidly detect low levels of bacteria and discern the antibiotic resistance of E. coli isolates. Using our bioengineered phage-based probe we were able to detect 10(3) CFU per mL of E. coli in 6 hours using a chemiluminescent substrate and 10(4) CFU per mL within 7.5 hours using a colorimetric substrate. We also show the application of this phage-based probe for antibiotic resistance testing. We were able to determine whether an E. coli isolate was resistant to ampicillin within 4.5 hours using chemiluminescent substrate and within 6 hours using a colorimetric substrate. This phage-based scheme could be readily adopted in labs without significant capital investments and can be translated to other phage-bacteria pairs for further detection.

  17. Alkalinity of neutrophil phagocytic vacuoles is modulated by HVCN1 and has consequences for myeloperoxidase activity.

    Science.gov (United States)

    Levine, Adam P; Duchen, Michael R; de Villiers, Simon; Rich, Peter R; Segal, Anthony W

    2015-01-01

    The NADPH oxidase of neutrophils, essential for innate immunity, passes electrons across the phagocytic membrane to form superoxide in the phagocytic vacuole. Activity of the oxidase requires that charge movements across the vacuolar membrane are balanced. Using the pH indicator SNARF, we measured changes in pH in the phagocytic vacuole and cytosol of neutrophils. In human cells, the vacuolar pH rose to ~9, and the cytosol acidified slightly. By contrast, in Hvcn1 knock out mouse neutrophils, the vacuolar pH rose above 11, vacuoles swelled, and the cytosol acidified excessively, demonstrating that ordinarily this channel plays an important role in charge compensation. Proton extrusion was not diminished in Hvcn1-/- mouse neutrophils arguing against its role in maintaining pH homeostasis across the plasma membrane. Conditions in the vacuole are optimal for bacterial killing by the neutral proteases, cathepsin G and elastase, and not by myeloperoxidase, activity of which was unphysiologically low at alkaline pH. PMID:25885273

  18. Microbial processes and factors controlling their activities in alkaline lakes of the Mongolian plateau

    Science.gov (United States)

    Namsaraev, Zorigto B.; Zaitseva, Svetlana V.; Gorlenko, Vladimir M.; Kozyreva, Ludmila P.; Namsaraev, Bair B.

    2015-11-01

    A striking feature of the Mongolian plateau is the wide range of air temperatures during a year, -30 to 30°C. High summer temperatures, atmospheric weathering and the arid climate lead to formation of numerous alkaline soda lakes that are covered by ice during 6-7 months per year. During the study period, the lakes had pH values between 8.1 to 10.4 and salinity between 1.8 and 360 g/L. According to chemical composition, the lakes belong to sodium carbonate, sodium chloride-carbonate and sodium sulfate-carbonate types. This paper presents the data on the water chemical composition, results of the determination of the rates of microbial processes in microbial mats and sediments in the lakes studied, and the results of a Principal Component Analysis of environmental variables and microbial activity data. Temperature was the most important factor that influenced both chemical composition and microbial activity. pH and salinity are also important factors for the microbial processes. Dark CO2 fixation is impacted mostly by salinity and the chemical composition of the lake water. Total photosynthesis and sulfate-reduction are impacted mostly by pH. Photosynthesis is the dominant process of primary production, but the highest rate (386 mg C/(L•d)) determined in the lakes studied were 2-3 times lower than in microbial mats of lakes located in tropical zones. This can be explained by the relatively short warm period that lasts only 3-4 months per year. The highest measured rate of dark CO2 assimilation (59.8 mg C/(L•d)) was much lower than photosynthesis. The highest rate of sulfate reduction was 60 mg S/(L•d), while that of methanogenesis was 75.6 μL CN4/(L•d) in the alkaline lakes of Mongolian plateau. The rate of organic matter consumption during sulfate reduction was 3-4 orders of magnitude higher than that associated with methanogenesis.

  19. Phosphatase activity in commercial spleen exonuclease decreases the recovery of benzo[a]pyrene and N-hydroxy-2-naphthylamine DNA adducts by 32P-postlabeling.

    Science.gov (United States)

    Adams, S P; Laws, G M; Selden, J R; Nichols, W W

    1994-05-15

    Spleen exonuclease, which degrades nucleic acids into single 3'-nucleotides, is used in the detection of DNA adducts by 32P-postlabeling. Contamination of the exonuclease with phosphatase activity can reduce the recovery of benzo[a]pyrene and N-hydroxy-2-naphthylamine DNA adducts by 32P-postlabeling. Four preparations of spleen exonuclease containing varying levels of phosphatase activity (2-naphthylamine DNA adducts. Surprisingly, recovery of these DNA adducts was nearly 10 times greater using nuclease P1 than when using 1-butanol extraction for adduct enrichment, since arylamine DNA adducts have previously been reported to be poorly detected by 32P-postlabeling after nuclease P1 treatment. Our data indicate that the hydrolysis of DNA by spleen exonuclease may be an important source of variability in both qualitative and quantitative analysis of adducts by 32P-postlabeling. PMID:8059938

  20. The change of serum alkaline phosphatase and fracture healing about the fracture with type 2 diabetes%骨折合并Ⅱ型糖尿病患者骨转换生化指标及骨折愈合的变化

    Institute of Scientific and Technical Information of China (English)

    杨东辉

    2012-01-01

    [ Objective ] To observe the change of serum alkaline phosphatase and fracture-healing about the fracture with type 2 diabetes. [ Method ] The patients with fracture were divided into two groups; fracture-patient with type 2 diabetes, and fracture-patient with normal blood glucose. The change of serum alkaline phosphatase( AKP)and fracture-healing were observed in different groups. [Result] AKP in the group of the fracture-patient with type 2 diabetes changed significantly as compared with the group of the fracture-patient with normal blood glucose. Type 2 diabetes would slow down the speed of fracture-healing (P<0.01). [ Conclusion ] Type 2 diabetes could influence the metabolism-speed of fracture-healing.%[目的]观察Ⅱ型糖尿病骨折患者骨转换生化指标-碱性磷酸酶及骨折愈合的变化.[方法]将在我科住院行手术治疗的骨折患者分为非糖尿病骨折组72例及Ⅱ型糖尿病骨折组79例,观察术前、术后两组患者血浆碱性磷酸酶(AKP)及骨折愈合情况.[结果]非糖尿病骨折组与Ⅱ型糖尿病骨折组比较,血AKP检测指标及骨折愈合情况差异具有显著性意义(P<0.01,P<0.05).Ⅱ型糖尿病可影响骨折后血AKP浓度的变化及骨折愈合速度.[结论]Ⅱ型糖尿病可影响骨质代谢,并易造成骨折延缓愈合.

  1. 骨碱性磷酸酶检测技术诊治小儿佝偻病在浙江农村推广的适宜性评估%Evaluation of diagnosis of infantile vitamin D deficiency rickets by bone alkaline phosphatase detection in township-level health organizations of Zhejiang rural areas

    Institute of Scientific and Technical Information of China (English)

    朱雯; 朱炜; 杨再峰; 王红妹; 张扬; 沈毅; 李鲁; 姒健敏; 蒋健敏; 夏志俊; 蒋海瑛

    2009-01-01

    Objective: To evaluate the health technology of diagnosis of infantile vitamin D deficiency rickets by bone alkaline phosphatase detection in township-level health organizations of Zhejiang rural areas. Methods: The health technology was evaluated by the assessment model for selection and assessment of rural appropriate health technology in Zhejiang province. Results: The weighted score of first-level indicators were 8.05, 8.06, 6.32, 18.13, 18.88, 4.84 and 4.36 respectively. The total score was 68.64. Maturity: good; need: fair; convenience: good; safety: good; validity: very good; economic attributes: fair; sociality and ethicality: good. Conclusion: Diagnosis of infantile vitamin D deficiency rickets by bone alkaline phosphatase detection is suitable for dissemination and application in township-level health organizations in Zhejiang rural areas.%目的:对浙江省农村乡级卫生机构在小儿佝偻病诊治中应用骨碱性磷酸酶检测技术进行系统全面的评估.方法:使用"浙江省农村卫生适宜技术筛选评估指标体系"进行评估.结果:7个一级指标的加权分分别为8.05、8.06、6.32、18.13、18.88、4.84、4.36,总分为68.64;该技术成熟性较好;需要性一般;简便性较好;安全性较好;有效性很好;经济性一般;社会和伦理适应性较好.结论:该技术适宜在浙江省乡级农村卫生机构推广应用.

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

    Directory of Open Access Journals (Sweden)

    Tigran R Petrosyan

    2016-01-01

    Full Text Available 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 underg