Science.gov

Sample records for alkaline phosphate activity

  1. Lack of relationship between activity of intestinal alkaline phosphatase and calcium or phosphate absorption.

    PubMed

    Asteggiano, C; Tolosa, N; Pereira, R; Moreno, J; Cañas, F

    1981-01-01

    The effects of vitamin D3 and the aqueous extract of Solanum malacoxylon on intestinal alkaline phosphatase and tissue phosphate content were studied on rachitic chicks treated with large doses of ethane-1-hydroxy-1,1 diphosphonate (EHDP). The EHDP treatment blocks the increase of intestinal calcium or phosphate absorption induced by the vitamin D3, while it has no effects on the rise of intestinal alkaline phosphatase activity or the increment in tissue phosphate content. The lack of correlation between the increment of alkaline phosphatase and that of Ca or phosphate absorption in vitamin D3 plus EHDP treated chicks excludes a participation of the alkaline phosphatase in the mechanism of Ca or P intestinal absorption. The Ca or phosphorus absorption are elicited specifically by 1,25-(OH)2-D3, while alkaline phosphatase activity and phosphate tissue concentration respond to a broader spectrum of stimuli. PMID:6316731

  2. Relationship between Salivary Alkaline Phosphatase Enzyme Activity and The Concentrations of Salivary Calcium and Phosphate Ions

    PubMed Central

    Jazaeri, Mina; Malekzadeh, Hosein; Abdolsamadi, Hamidreza; Rezaei-Soufi, Loghman; Samami, Mohammad

    2015-01-01

    Although salivary alkaline phosphatase (ALP) can balance deand remineralization processes of enamel, there is no evidence regarding its effects on the concentrations of calcium and phosphate in saliva. The present study aims to determine the relationship between salivary ALP activity and the concentrations of calcium and phosphate in saliva. In this cross-sectional study, we evaluated salivary markers in 120 males, ages 19 to 44 years. All participants provided 5 mL of unstimulated whole saliva and the level of enzyme activity as well as calcium and phosphate concentrations were measured using a colorimetric method. Data were gathered and analyzed by statistical package for social sciences (SPSS) 13.00 using Pearson correlation test. A p value of <0.05 was considered statistically significant. The mean age of participants in the present study was 32.95 ± 8.09 years. The mean pH of saliva was 6.65 ± 0.62. Salivary parameters included average ALP activity (5.04 ± 1.866 U/dL), calcium (4.77 ± 0.877 mg/dL) and phosphate (10.38 ± 2.301 mg/dL). Pearson correlation test showed no significant relationship between ALP activity and calcium and phosphate concentrations in saliva (p>0.05). According to the results of the present study, there was no significant relation between salivary ALP activity and calcium and phosphate concentrations in saliva. However, further research is highly recommended. PMID:25870846

  3. Relationship between Salivary Alkaline Phosphatase Enzyme Activity and The Concentrations of Salivary Calcium and Phosphate Ions.

    PubMed

    Jazaeri, Mina; Malekzadeh, Hosein; Abdolsamadi, Hamidreza; Rezaei-Soufi, Loghman; Samami, Mohammad

    2015-01-01

    Although salivary alkaline phosphatase (ALP) can balance deand remineralization processes of enamel, there is no evidence regarding its effects on the concentrations of calcium and phosphate in saliva. The present study aims to determine the relationship between salivary ALP activity and the concentrations of calcium and phosphate in saliva. In this cross-sectional study, we evaluated salivary markers in 120 males, ages 19 to 44 years. All participants provided 5 mL of unstimulated whole saliva and the level of enzyme activity as well as calcium and phosphate concentrations were measured using a colorimetric method. Data were gathered and analyzed by statistical package for social sciences (SPSS) 13.00 using Pearson correlation test. A p value of <0.05 was considered statistically significant. The mean age of participants in the present study was 32.95 ± 8.09 years. The mean pH of saliva was 6.65 ± 0.62. Salivary parameters included average ALP activity (5.04 ± 1.866 U/dL), calcium (4.77 ± 0.877 mg/dL) and phosphate (10.38 ± 2.301 mg/dL). Pearson correlation test showed no significant relationship between ALP activity and calcium and phosphate concentrations in saliva (p>0.05). According to the results of the present study, there was no significant relation between salivary ALP activity and calcium and phosphate concentrations in saliva. However, further research is highly recommended. PMID:25870846

  4. Alkaline Phosphatase Activity : an overlooked player on the phosphate behavior in macrotidal estuaries

    NASA Astrophysics Data System (ADS)

    Delmas, Daniel; Labry, Claire; Youenou, Agnes; Quere, Julien; Auguet, Jean Christophe; Montanie, Helene

    2014-05-01

    The non-conservative behavior of phosphate within the estuarine salinity gradient is essentially assigned to physico-chemical processes, such as desorption at low salinity and to benthic exchanges. Microbial phosphatase activity (APA), generally related to phosphate deficiency, is seldom studied in phosphate rich estuarine waters. In order to address the impact of microbial activity (bacterial abundance, production BSP, APA) on phosphate behavior, we studied these activities on a seasonal basis within the salinity gradient of two macrotidal estuaries presenting different levels of suspended solids. Whatever the season the Charente estuary is characterized by high levels of Suspended Particulate Matter (SPM > 1g.L-1), particularly in the Maximum Turbidity Zone (MTZ) located at the 5-10 psu. In this area characterized by high BSP and APA there is a significant increase of PO4 levels especially during summer. In the Aulne estuary the particle load is significantly lower (1/10) but high BSP and APA are equally recorded. In the highly turbid waters of the Charente estuary, active phytoplankton is virtually absent as pheopigments constitute up to 80% of the total pigments, particularly in the MTZ, therefore APA may essentially have a bacterial origin. In the Aulne estuary attached bacteria are dominant, both in numbers and production, and their distribution along the haline gradient perfectly follows those of APA and phosphate levels. These observations, associated with the very close relationships observed between APA, SPM and BSP, suggest that APA derive mainly from bacterial (attached) origin and operate at the expense of particulate phosphorus and hence contribute to PO4 regeneration, especially in spring and summer. Finally, as APA increased as PO4, whereas the reverse is observed in both fresh and marine waters, an original scheme for APA regulation, related to the large dominance of attached bacteria can be described for the estuarine waters.

  5. Alkaline phosphatase activity in Zostera noltii hornem. and its contribution to the release of phosphate in the palmones river estuary

    NASA Astrophysics Data System (ADS)

    Hernández, I.; Pérez-Llorens, J. L.; Fernández, J. A.; Niell, F. X.

    Alkaline phosphatase activity (APA) was studied in Zostera noltii Hornem., a sea-grass collected in the Palmones river estuary (southern Spain). The higher activity was found in the leaves, with minor contributions in the stem and the underground parts of the plant. The enzymatic activity showed a two-phase kinetic versus substrate concentration between 5 μM and 25 mM. The influence of some environmental factors important in nature (temperature, pH, salinity, photon irradiance and external phosphate) on the enzymatic activity is discussed. Over an ecophysiological range of these factors, maximum APA was found at 30 0C (22·6 μmol pNP released g dry wt-1 h-1), pH 8·8 (35·6 μmol pNP g dry wt-1 h-1) and salinity 43·8 (27·8 pmol pNP g dry wt-1 h-1). With regard to light, APA and phosphate uptake in shoots were light-saturated and showed similar values for maximum velocity and half-saturation constant. In the range of phosphate concentration tested (0 20 μM), APA was independent of the external phosphate concentration. Finally, as Z. noltii incorporated only 16% of the phosphate hydrolysed from the model phosphomonoester used in the assay, the significance of Z. noltii population in the enzymatic release of phosphate to the estuary was estimated. A minimum of 8·4 nM Pi liberated per day and a maximum of 99·8 nM Pi day-1 was found.

  6. Alkaline Phosphatase Activity in Zostera noltii Hornem. and its Contribution to the Release of Phosphate in the Palmones River Estuary

    NASA Astrophysics Data System (ADS)

    Hernández, I.; Pérez-Llorens, J. L.; Fernández, J. A.; Niell, F. X.

    Alkaline phosphatase activity (APA) was studied in Zostera noltii Hornem., a sea-grass collected in the Palmones river estuary (southern Spain). The higher activity was found in the leaves, with minor contributions in the stem and the underground parts of the plant. The enzymatic activity showed a two-phase kinetic versus substrate concentration between 5 μM and 25 mM. The influence of some environmental factors important in nature (temperature, pH, salinity, photon irradiance and external phosphate) on the enzymatic activity is discussed. Over an ecophysiological range of these factors, maximum APA was found at 30 °C (22·6 μmol pNP released g dry wt -1 h -1), pH 8·8 (35·6 μmol pNP g dry wt -1 h -1) and salinity 43·8 (27·8 μmol pNP g dry wt -1 h -1). With regard to light, APA and phosphate uptake in shoots were light-saturated and showed similar values for maximum velocity and half-saturation constant. In the range of phosphate concentration tested (0-20 μM), APA was independent of the external phosphate concentration. Finally, as Z. noltii incorporated only 16% of the phosphate hydrolysed from the model phosphomonoester used in the assay, the significance of Z. noltii population in the enzymatic release of phosphate to the estuary was estimated. A minimum of 8·4 nM Pi liberated per day and a maximum of 99·8 nM Pi day -1 was found.

  7. Alkaline phosphatase activity in Zostera noltii hornem. and its contribution to the release of phosphate in the palmones river estuary

    NASA Astrophysics Data System (ADS)

    Hernández, I.; Pérez-Llorens, J. L.; Fernández, J. A.; Niell, F. X.

    Alkaline phosphatase activity (APA) was studied in Zostera noltii Hornem., a sea-grass collected in the Palmones river estuary (southern Spain). The higher activity was found in the leaves, with minor contributions in the stem and the underground parts of the plant. The enzymatic activity showed a two-phase kinetic versus substrate concentration between 5 μM and 25 mM. The influence of some environmental factors important in nature (temperature, pH, salinity, photon irradiance and external phosphate) on the enzymatic activity is discussed. Over an ecophysiological range of these factors, maximum APA was found at 30 0C (22·6 μmol pNP released g dry wt -1 h -1), pH 8·8 (35·6 μmol pNP g dry wt -1 h -1) and salinity 43·8 (27·8 pmol pNP g dry wt -1 h -1). With regard to light, APA and phosphate uptake in shoots were light-saturated and showed similar values for maximum velocity and half-saturation constant. In the range of phosphate concentration tested (0-20 μM), APA was independent of the external phosphate concentration. Finally, as Z. noltii incorporated only 16% of the phosphate hydrolysed from the model phosphomonoester used in the assay, the significance of Z. noltii population in the enzymatic release of phosphate to the estuary was estimated. A minimum of 8·4 nM Pi liberated per day and a maximum of 99·8 nM Pi day -1 was found.

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

    PubMed

    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

  9. Orbital fluid shear stress promotes osteoblast metabolism, proliferation and alkaline phosphates activity in vitro.

    PubMed

    Aisha, M D; Nor-Ashikin, M N K; Sharaniza, A B R; Nawawi, H; Froemming, G R A

    2015-09-10

    Prolonged disuse of the musculoskeletal system is associated with reduced mechanical loading and lack of anabolic stimulus. As a form of mechanical signal, the multidirectional orbital fluid shear stress transmits anabolic signal to bone forming cells in promoting cell differentiation, metabolism and proliferation. Signals are channeled through the cytoskeleton framework, directly modifying gene and protein expression. For that reason, we aimed to study the organization of Normal Human Osteoblast (NHOst) cytoskeleton with regards to orbital fluid shear (OFS) stress. Of special interest were the consequences of cytoskeletal reorganization on NHOst metabolism, proliferation, and osteogenic functional markers. Cells stimulated at 250 RPM in a shaking incubator resulted in the rearrangement of actin and tubulin fibers after 72 h. Orbital shear stress increased NHOst mitochondrial metabolism and proliferation, simultaneously preventing apoptosis. The ratio of RANKL/OPG was reduced, suggesting that orbital shear stress has the potential to inhibit osteoclastogenesis and osteoclast activity. Increase in ALP activity and OCN protein production suggests that stimulation retained osteoblast function. Shear stress possibly generated through actin seemed to hold an anabolic response as osteoblast metabolism and functional markers were enhanced. We hypothesize that by applying orbital shear stress with suitable magnitude and duration as a non-drug anabolic treatment can help improve bone regeneration in prolonged disuse cases. PMID:26163894

  10. Orbital fluid shear stress promotes osteoblast metabolism, proliferation and alkaline phosphates activity in vitro

    SciTech Connect

    Aisha, M.D.; Nor-Ashikin, M.N.K.; Sharaniza, A.B.R.; Nawawi, H.; Froemming, G.R.A.

    2015-09-10

    Prolonged disuse of the musculoskeletal system is associated with reduced mechanical loading and lack of anabolic stimulus. As a form of mechanical signal, the multidirectional orbital fluid shear stress transmits anabolic signal to bone forming cells in promoting cell differentiation, metabolism and proliferation. Signals are channeled through the cytoskeleton framework, directly modifying gene and protein expression. For that reason, we aimed to study the organization of Normal Human Osteoblast (NHOst) cytoskeleton with regards to orbital fluid shear (OFS) stress. Of special interest were the consequences of cytoskeletal reorganization on NHOst metabolism, proliferation, and osteogenic functional markers. Cells stimulated at 250 RPM in a shaking incubator resulted in the rearrangement of actin and tubulin fibers after 72 h. Orbital shear stress increased NHOst mitochondrial metabolism and proliferation, simultaneously preventing apoptosis. The ratio of RANKL/OPG was reduced, suggesting that orbital shear stress has the potential to inhibit osteoclastogenesis and osteoclast activity. Increase in ALP activity and OCN protein production suggests that stimulation retained osteoblast function. Shear stress possibly generated through actin seemed to hold an anabolic response as osteoblast metabolism and functional markers were enhanced. We hypothesize that by applying orbital shear stress with suitable magnitude and duration as a non-drug anabolic treatment can help improve bone regeneration in prolonged disuse cases. - Highlights: • OFS stress transmits anabolic signals to osteoblasts. • Actin and tubulin fibers are rearranged under OFS stress. • OFS stress increases mitochondrial metabolism and proliferation. • Reduced RANKL/OPG ratio in response to OFS inhibits osteoclastogenesis. • OFS stress prevents apoptosis and stimulates ALP and OCN.

  11. Enhanced osteogenic activity of a poly(butylene succinate)/calcium phosphate composite by simple alkaline hydrolysis.

    PubMed

    Arphavasin, Suphakit; Singhatanadgit, Weerachai; Ngamviriyavong, Patcharee; Janvikul, Wanida; Meesap, Preeyapan; Patntirapong, Somying

    2013-10-01

    Bone engineering offers the prospect of alternative therapies for clinically relevant skeletal defects. Poly(butylene succinate) (PBSu) is a biodegradable and biocompatible polyester which may possess some limitations in clinical use due to its hydrophobicity. In order to overcome these limitations and increase the bioactivity, a simple and convenient surface hydrolysis of PBSu, PBSu/hydroxyapatite and PBSu/β-tricalcium phosphate (TCP) films was performed. The resulting surfaces (i.e., HPBSu, HPBSu/HA and HPBSu/TCP) were tested for their physicochemical property, biocompatibility and osteogenic potency. The results showed that surface hydrolysis significantly increased surface roughness and hydrophilicity of the composites, with the HPBSu/TCP possessing the most pronounced results. All the materials appeared to be biocompatible and supported in vitro growth and osteoblast differentiation of hMSCs, and the alkaline hydrolysis significantly enhanced the hMSC cell proliferation and the osteogenic potency of PBSu/TCP compared with the non-hydrolyzed sample. In conclusion, the HPBSu/TCP possessed better hydrophilicity, biocompatibility and osteogenic potency in vitro, suggesting that this simple and convenient alkaline hydrolysis could be used to augment the biological property of PBSu-based composites for bone engineering in vivo. PMID:24057872

  12. A single electrochemical biosensor for detecting the activity and inhibition of both protein kinase and alkaline phosphatase based on phosphate ions induced deposition of redox precipitates.

    PubMed

    Shen, Congcong; Li, Xiangzhi; Rasooly, Avraham; Guo, Linyan; Zhang, Kaina; Yang, Minghui

    2016-11-15

    Protein kinase (PKA) and alkaline phosphatase (ALP) are clinically relevant enzymes for a number of diseases. In this work, we developed a new simple electrochemical biosensor for the detection of the activity and inhibition of both PKA and ALP. One common feature of the PKA and ALP catalyzing process is that PKA can hydrolysis adenosine-5'-triphosphate (ATP) and ALP can hydrolysis pyrophosphate, both reactions produce phosphate ions, and the amount of phosphate ion produced is proportional to enzyme activity. Our assay is based on the principle that phosphate ions react with molybdate to form redox molybdophosphate precipitates on the electrode surface, thus generating electrochemical current. The detection limit for PKA and ALP were much lower than existing assays. The biosensor has good specificity and was used to measure drug-stimulated PKA from lysates of HeLa cells. We also evaluated the use of the biosensor as a screening tool for enzyme inhibitors. To the best of our knowledge, this is the first report of a biosensor capable of detecting the activity of both PKA and ALP. This tool has the potential to simplify PKA and ALP clinical measurement, thereby improving diagnostics of relevant diseases. It also may serve as the basis for a simple screening method for new enzyme inhibitors for disease treatment. PMID:27179562

  13. ESTIMATION OF PHOSPHATE ESTER HYDROLYSIS RATE CONSTANTS. I. ALKALINE HYDROLYSIS

    EPA Science Inventory

    SPARC (SPARC Performs Automated Reasoning in Chemistry) chemical reactivity models were extended to allow the calculation of alkaline hydrolysis rate constants of phosphate esters in water. The rate is calculated from the energy difference between the initial and transition state...

  14. ESTIMATION OF PHOSPHATE ESTER HYDROLYSIS RATE CONSTANTS - ALKALINE HYDROLYSIS

    EPA Science Inventory

    SPARC (SPARC Performs Automated Reasoning in Chemistry) chemical reactivity models were extended to allow the calculation of alkaline hydrolysis rate constants of phosphate esters in water. The rate is calculated from the energy difference between the initial and transition state...

  15. Alkaline and alkaline earth metal phosphate halides and phosphors

    SciTech Connect

    Lyons, Robert Joseph; Setlur, Anant Achyut; Cleaver, Robert John

    2012-11-13

    Compounds, phosphor materials and apparatus related to nacaphite family of materials are presented. Potassium and rubidium based nacaphite family compounds and phosphors designed by doping divalent rare earth elements in the sites of alkaline earth metals in the nacaphite material families are descried. An apparatus comprising the phosphors based on the nacaphite family materials are presented herein. The compounds presented is of formula A.sub.2B.sub.1-yR.sub.yPO.sub.4X where the elements A, B, R, X and suffix y are defined such that A is potassium, rubidium, or a combination of potassium and rubidium and B is calcium, strontium, barium, or a combination of any of calcium, strontium and barium. X is fluorine, chlorine, or a combination of fluorine and chlorine, R is europium, samarium, ytterbium, or a combination of any of europium, samarium, and ytterbium, and y ranges from 0 to about 0.1.

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

    NASA Astrophysics Data System (ADS)

    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.

  17. Phosphate glass electrode with good selectivity for alkaline-earth cations

    USGS Publications Warehouse

    Truesdell, A.H.; Pommer, A.M.

    1963-01-01

    A phosphate glass has been found to have a significant electrode specificity toward alkaline-earth ions. The order of selectivity is 2H + > Ba++ > Sr++ > Ca++ > 2K+ > 2Na+ > Mg++. Exchange properties are discussed in relation to possible structure. Its use to determine activity of Ca++ in natural systems containing Mg++ is suggested.

  18. Cellular Performance Comparison of Biomimetic Calcium Phosphate Coating and Alkaline-Treated Titanium Surface

    PubMed Central

    Wei, Mei

    2013-01-01

    The influence of biomimetic calcium phosphate coating on osteoblasts behavior in vitro is not well established yet. In this study, we investigated the behavior of osteoblastic rat osteosarcoma 17/2.8 cells (ROS17/2.8) on two groups of biomaterial surfaces: alkaline-treated titanium surface (ATT) and biomimetic calcium phosphate coated ATT (CaP). The cell attachment, proliferation, differentiation, and morphology on these surfaces were extensively evaluated to reveal the impact of substrate surface on osteoblastic cell responses. It was found that the ROS17/2.8 cells cultured on the ATT surface had higher attachment and proliferation rates compared to those on the CaP surface. Our results also showed that the calcium phosphate coatings generated in this work have an inhibiting effect on osteoblast adhesion and further influenced the proliferation and differentiation of osteoblast compared to the ATT surface in vitro. Cells on the ATT surface also exhibited a higher alkaline phosphatase activity than on the CaP surface after two weeks of culture. Immunofluorescence staining and scanning electron microscopy results showed that the cells adhered and spread faster on the ATT surface than on the CaP surface. These results collectively suggested that substrate surface properties directly influence cell adhesion on different biomaterials, which would result in further influence on the cell proliferation and differentiation. PMID:24455730

  19. Responses of Phosphate Transporter Gene and Alkaline Phosphatase in Thalassiosira pseudonana to Phosphine

    PubMed Central

    Fu, Mei; Song, Xiuxian; Yu, Zhiming; Liu, Yun

    2013-01-01

    Phosphine, which is released continuously from sediment, can affect the eco-physiological strategies and molecular responses of phytoplankton. To examine the effects of phosphine on phosphorus uptake and utilization in Thalassiosira pseudonana, we examined the transcriptional level of the phosphate transporter gene (TpPHO) and the activity of alkaline phosphatase (AKP) in relation to supplement of various concentrations of phosphine. TpPHO expression was markedly promoted by phosphine in both the phosphate-deficient and phosphate-4 µM culture. However, high phosphine concentrations can inhibit TpPHO transcription in the declining growth phase. AKP activity was also higher in the phosphine treatment groups than that of the control. It increased with increasing phosphine concentration in the range of 0 to 0.056 µM but was inhibited by higher levels of phosphine. These responses revealed that phosphine can affect phosphate uptake and utilization in T. pseudonana. This result was consistent with the effect of phosphine on algal growth, while TpPHO expression and AKP were even more sensitive to phosphine than algal growth. This work provides a basic understanding for further research about how phosphine affects phytoplankton. PMID:23544096

  20. Chemostat Culture of Escherichia coli K-12 Limited by the Activity of Alkaline Phosphatase

    PubMed Central

    King, Stagg L.; Francis, J. C.

    1975-01-01

    The growth-limiting reaction of a chemostat culture of Escherichia coli K-12 was the hydrolysis of β-glycerophosphate by alkaline phosphatase. The culture was buffered at pH 5.2 where alkaline phosphatase was unable to supply phosphate to the cell at a rate sufficient to sustain the maximum rate of growth. Alkaline phosphatase activity in this system is discussed in terms of the so-called Flip-Flop mechanism. PMID:240310

  1. PAPSS2 Promotes Alkaline Phosphates Activity and Mineralization of Osteoblastic MC3T3-E1 Cells by Crosstalk and Smads Signal Pathways

    PubMed Central

    Wang, Weizhuo; Li, Fang; Wang, Kunzheng; Cheng, Bin; Guo, Xiong

    2012-01-01

    Several studies have indicated that PAPSS2 (3′-phosphoadenosine-5′-phosphosulfate synthetase 2) activity is important to normal skeletal development. Mouse PAPSS2 is predominantly expressed during the formation of the skeleton and cartilaginous elements of the mouse embryo and in newborn mice. However, the role and mechanism of PAPSS2 in bone formation remains largely unidentified. By analyzing the expression pattern of the PAPSS2 gene, we have found that PAPSS2 is expressed in bone tissue and bone formation. PAPSS2 transcripts increase during osteoblast differentiation and are in less level in RANKL-induced osteoclast like cells. By using lentivirus-mediated RNA interference (RNAi) technology, we knocked down PAPSS2 expression in MC3T3-E1 osteoblast. Silencing of PAPSS2 expression significantly decreases ALP activity and cell mineralization, inhibits expression of osteoblast marker osteopontin (OPN) and collagen I. Conversely, overexpression of PAPSS2 promotes the MC3T3-E1 to differentiate into osteoblast and mineralization. Moreover, compared to that in the control cells, the mRNA level and protein expression of phosphorylated Smad 2/3, which is a key transcriptional factor in the Smad osteoblast differentiation pathway, showed significant decreases in PAPSS2-silenced cells and increases in PAPSS2-overexpression cells. These results suggest that PAPSS2 might regulate osteoblast ALP activity and cell mineralization, probably through Smads signal pathways. PMID:22916269

  2. Phosphate Stress in Cultures and Field Populations of the Dinoflagellate Prorocentrum minimum Detected by a Single-Cell Alkaline Phosphatase Assay

    PubMed Central

    Dyhrman, Sonya T.; Palenik, Brian

    1999-01-01

    Alkaline phosphatase activity is a common marker of phosphate stress in many phytoplankton, but it has been difficult to attribute alkaline phosphatase activity to specific organisms or groups of phytoplankton in the field with traditional biochemical procedures. A new alkaline phosphatase substrate, ELF-97 (enzyme-labeled fluorescence), shows promise in this regard. When a phosphate group is cleaved from the ELF-97 reagent, the remaining molecule precipitates near the site of enzyme activity, thus fluorescently tagging cells with alkaline phosphatase activity. We characterized ELF-97 labeling in axenic cultures of a common dinoflagellate, Prorocentrum minimum, in order to understand ELF-97 labeling dynamics when phosphate nutrition varies. Enzyme activity, as detected by ELF-97 labeling, appears to be induced in late-log- or early-stationary-phase cultures if cells are grown in low-phosphate media and is lost when phosphate-stressed cells are refed with phosphate. ELF-97 appears to label an inducible intracellular alkaline phosphatase in P. minimum based on confocal microscopy studies. This may limit the use of this reagent to organisms that lack high levels of constitutive intracellular phosphatases. After laboratory cultures were characterized, ELF-97 was used to assay field populations of P. minimum in Narragansett Bay during two 1-week periods, and 12 to 100% of the P. minimum cells were labeled. The level of cell labeling was reduced by 3 days of incubation with added inorganic phosphate. Our results indicate that ELF-97 is an excellent new tool for monitoring phytoplankton phosphate stress in the environment when the data are supported by appropriate laboratory studies. PMID:10388722

  3. Alkaline resistant phosphate glasses and method of preparation and use thereof

    DOEpatents

    Brow, Richard K.; Reis, Signo T.; Velez, Mariano; Day, Delbert E.

    2010-01-26

    A substantially alkaline resistant calcium-iron-phosphate (CFP) glass and methods of making and using thereof. In one application, the CFP glass is drawn into a fiber and dispersed in cement to produce glass fiber reinforced concrete (GFRC) articles having the high compressive strength of concrete with the high impact, flexural and tensile strength associated with glass fibers.

  4. High-Strength / High Alkaline Resistant Fe-Phosphate Glass Fibers as Concrete Reinforcement

    SciTech Connect

    Mariano Velez

    2008-03-31

    Calcium-iron-phosphate glasses were developed whose chemical durabilities in alkaline solutions (pH 13) were comparable or superior to those of commercial alkaline-resistant (AR) silica-based glasses. However, the tensile strength of Ca-Fe-phosphate fibers, after being exposed to alkaline environments, including wet Portland cement pastes, is lower than that of current AR silicate fibers. Another series of Ca-Fe-phosphate glasses were developed with excellent chemical durability in strong acidic solutions (H2SO4, HF), indicating potential applications where silica-based fibers degrade very quickly, including E-glass. The new Ca-Fe-phosphate glasses can be melted and processed 300 to 500°C lower than silica-based glasses. This offers the possibility of manufacturing glass fibers with lower energy costs by 40-60% and the potential to reduce manufacturing waste and lower gas emissions. It was found that Ca-Fe-phosphate melts can be continuously pulled into fibers depending on the slope of the viscosity-temperature curve and with viscosity ~100 poise, using multi-hole Pt/Rh bushings.

  5. Kinetic Study of the Alkaline Degradation of Oseltamivir Phosphate and Valacyclovir Hydrochloride using Validated Stability Indicating HPLC.

    PubMed

    Al-Bagary, Ramzia I; El-Zaher, Asmaa A; Morsy, Fahima A; Fouad, Mai M

    2014-01-01

    Aqueous alkaline degradation was performed for oseltamivir phosphate (OP) and valacyclovir hydrochloride (VA). Isocratic stability indicating the use of high-performance liquid chromatography (HPLC) was presented for each drug in the presence of its degradation product. The separations were performed using the Nucleosil ODS column and a mobile phase consisting of phosphate buffer (pH = 7), acetonitrile, and methanol 50:25:25 (v/v/v) for OP. For VA separation, a Nucleosil CN column using phosphate buffer (pH = 7) and methanol 85:15 (v/v) was used as a mobile phase. Ultraviolet detection at 210 nm and 254 nm was used for OP and VA, respectively. The method showed high sensitivity concerning linearity, accuracy, and precision over the range 1-250 μg mL(-1) for both drugs. The proposed method was used to determine the drug in its pharmaceutical formulation and to investigate the degradation kinetics of each drug's alkaline-stressed samples. The reactions were found to follow a first-order reaction. The activation energy could also be estimated. International Conference on Harmonisation guidelines were adopted for method validation. PMID:24932100

  6. Alkaline phosphatase assay using a near-infrared fluorescent substrate merocyanine 700 phosphate.

    PubMed

    Gong, Haibiao; Little, Garrick; Cradduck, Mark; Draney, Daniel R; Padhye, Nisha; Olive, D Michael

    2011-05-15

    Alkaline phosphatase (ALP) is a phosphomonoester hydrolase that is commonly used as a conjugating enzyme in biological research. A wide variety of substrates have been developed to assay its activity. In this study, we developed an ALP assay method utilizing merocyanine 700 (MC700) based substrate MC700 phosphate (MC700p). MC700 is a near-infrared fluorescent merocyanine dye, and has excitation/emission maxima at 686 nm/722 nm in ALP assay buffer. Upon hydrolysis by ALP, MC700p is converted to MC700. The fluorescence of MC700 is dependent on the pH and detergent concentration in the buffer. The fluorescence signal produced by MC700p hydrolysis is linearly related to the ALP amount and substrate concentration. A stop solution containing EDTA could be used to stop the ALP/MC700p reaction. It was also demonstrated that MC700p could substitute pNpp as the ALP substrate in a commercial 17β-Estradiol enzyme immunoassay kit. PMID:21482307

  7. Phosphorane lifetime and stereo-electronic effects along the alkaline hydrolysis of phosphate esters.

    PubMed

    Pereira, Eufrásia S; Da Silva, Júlio C S; Brandão, Tiago A S; Rocha, Willian R

    2016-07-21

    Hybrid quantum mechanical/effective fragment potential (QM/EFP) calculations, in conjunction with the quantum theory of atoms in molecules (QTAIM) and energy decomposition analysis (EDA), were employed to investigate the reaction mechanism and stereo-electronic effects along the alkaline hydrolysis of the monoethyl phosphate dianion (MEP) and the diethylphosphate monoanion (DEP). Reactions proceed through a synchronous bimolecular ANDN mechanism for MEP and a stepwise (AN + DN) mechanism for DEP, with the formation of a phosphorane intermediate, having an overall reaction free energy and barrier of 11.5 and 43.0 kcal mol(-1), respectively. In addition, ab initio molecular dynamics simulations were performed to investigate the stability of the phosphorane pentacoordinate intermediate observed in the reaction of the phosphate diester. The phosphorane intermediate has a lifetime of ∼1 ps after which it decomposes into the corresponding alcohol and phosphate monoester dianion. Electrostatics governs the interaction between the nucleophile and the phosphate ester. However, some degree of covalence in the interaction starts to appear at distances shorter than 2.45 Å for MEP and 2.63 Å for DEP. For the monoester, the electrostatic repulsive terms are the dominant contributions for the formation of the transition state. On the other hand, for the phosphate diester, the formation of the P-OH bond is dominated by associative terms of electrostatic nature. PMID:27332044

  8. Effect of cobalt on synthesis and activation of Bacillus licheniformis alkaline phosphatase.

    PubMed Central

    Spencer, D B; Chen, C P; Hulett, F M

    1981-01-01

    The effect of CO2+ on the synthesis and activation of Bacillus licheniformis MC14 alkaline phosphatase has been shown by the development of a defined minimal salts medium in which this organism produces 35 times more (assayable) alkaline phosphatase than when grown in a low-phosphate complex medium or in the defined medium without cobalt. Stimulation of enzyme activity with cobalt is dependent on a low phosphate concentration in the medium (below 0.075 mM) and continued protein synthesis. Cobalt stimulation resulted in alkaline phosphate production being a major portion of total protein synthesized during late-logarithmic and early-stationary-phase culture growth. Cells cultured in the defined medium minus cobalt, or purified enzyme partially inactivated with a chelating agent, showed a 2.5-fold increase in activity when assayed in the presence of cobalt. Atomic spectral analysis indicated the presence of 3.65 +/- 0.45 g-atoms of cobalt associated with each mole of purified active alkaline phosphatase. A biochemical localization as a function of culture age in this medium showed that alkaline phosphatase was associated with the cytoplasmic membrane and was also found as a soluble enzyme in the periplasmic region and secreted into the growth medium. PMID:7462163

  9. Effect of cobalt on synthesis and activation of Bacillus licheniformis alkaline phosphatase.

    PubMed

    Spencer, D B; Chen, C P; Hulett, F M

    1981-02-01

    The effect of CO2+ on the synthesis and activation of Bacillus licheniformis MC14 alkaline phosphatase has been shown by the development of a defined minimal salts medium in which this organism produces 35 times more (assayable) alkaline phosphatase than when grown in a low-phosphate complex medium or in the defined medium without cobalt. Stimulation of enzyme activity with cobalt is dependent on a low phosphate concentration in the medium (below 0.075 mM) and continued protein synthesis. Cobalt stimulation resulted in alkaline phosphate production being a major portion of total protein synthesized during late-logarithmic and early-stationary-phase culture growth. Cells cultured in the defined medium minus cobalt, or purified enzyme partially inactivated with a chelating agent, showed a 2.5-fold increase in activity when assayed in the presence of cobalt. Atomic spectral analysis indicated the presence of 3.65 +/- 0.45 g-atoms of cobalt associated with each mole of purified active alkaline phosphatase. A biochemical localization as a function of culture age in this medium showed that alkaline phosphatase was associated with the cytoplasmic membrane and was also found as a soluble enzyme in the periplasmic region and secreted into the growth medium. PMID:7462163

  10. Recovery of phosphorus and nitrogen from alkaline hydrolysis supernatant of excess sludge by magnesium ammonium phosphate.

    PubMed

    Bi, Wei; Li, Yiyong; Hu, Yongyou

    2014-08-01

    Magnesium ammonium phosphate (MAP) method was used to recover orthophosphate (PO₄(3-)-P) and ammonium nitrogen (NH4(+)-N) from the alkaline hydrolysis supernatant of excess sludge. To reduce alkali consumption and decrease the pH of the supernatant, two-stage alkaline hydrolysis process (TSAHP) was designed. The results showed that the release efficiencies of PO₄(3-)-P and NH₄(+)-N were 41.96% and 7.78%, respectively, and the pH of the supernatant was below 10.5 under the running conditions with initial pH of 13, volume ratio (sludge dosage/water dosage) of 1.75 in second-stage alkaline hydrolysis reactor, 20 g/L of sludge concentration in first-stage alkaline hydrolysis reactor. The order of parameters influencing MAP reaction was analyzed and the optimized conditions of MAP reaction were predicted through the response surface methodology. The recovery rates of PO₄(3-)-P and NH₄(+)-N were 46.88% and 16.54%, respectively under the optimized conditions of Mg/P of 1.8, pH 9.7 and reaction time of 15 min. PMID:24880806

  11. Promiscuous sulfatase activity and thio-effects in a phosphodiesterase of the alkaline phosphatase superfamily†

    PubMed Central

    Lassila, Jonathan K.; Herschlag, Daniel

    2009-01-01

    The nucleotide phosphodiesterase/pyrophosphatase from Xanthomonas axonopodis (NPP) is a structural and evolutionary relative of alkaline phosphatase that preferentially hydrolyzes phosphate diesters. With the goal of understanding how these two enzymes with nearly identical Zn2+ bimetallo sites achieve high selectivity for hydrolysis of either phosphate monoesters or diesters, we have measured a promiscuous sulfatase activity in NPP. Sulfate esters are nearly isosteric with phosphate esters but carry less charge, offering a probe of electrostatic contributions to selectivity. NPP exhibits sulfatase activity with kcat/KM value of 2 × 10−5 M−1s−1, similar to the R166S mutant of alkaline phosphatase. We further report the effects of thio-substitution on phosphate monoester and diester reactions. Reactivities with these non-cognate substrates illustrate a reduced dependence of NPP reactivity on the charge of the nonbridging oxygen situated between the Zn2+ ions relative to that in alkaline phosphatase. This reduced charge dependence can explain about 102 of the 107-fold differential catalytic proficiency for the most similar monoester and diester substrates in the two enzymes. The results further suggest that active site contacts to substrate oxygen atoms that do not contact the Zn2+ ions may play an important role in defining the selectivity of the enzymes. PMID:18975918

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

    PubMed

    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

  13. Smart nanoprobes for the detection of alkaline phosphatase activity during osteoblast differentiation.

    PubMed

    Lim, Eun-Kyung; Keem, Joo Oak; Yun, Hui-suk; Jung, Jinyoung; Chung, Bong Hyun

    2015-02-21

    Gold nanoparticle-conjugated fluorescent hydroxyapatite (AuFHAp) was developed as a smart nanoprobe for measuring alkaline phosphatase (ALP) activity. AuFHAp showed NIR fluorescence due to the hydrolysis of its phosphate groups by ALP. In addition, gold nanoparticles help reduce the nonspecific signal by absorbing nonspecific fluorescence. Through in vitro tests, we confirmed that the AuFHAp probe was capable of detecting ALP levels related to osteoblast activity in living cells with high fluorescence intensity. PMID:25623488

  14. Alkaline Phosphatase Activity in San Francisco and Monterey Bays

    NASA Astrophysics Data System (ADS)

    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

  15. XANES Demonstrates the Release of Calcium Phosphates from Alkaline Vertisols to Moderately Acidified Solution.

    PubMed

    Andersson, Karl O; Tighe, Matthew K; Guppy, Christopher N; Milham, Paul J; McLaren, Timothy I; Schefe, Cassandra R; Lombi, Enzo

    2016-04-19

    Calcium phosphate (CaP) minerals may comprise the main phosphorus (P) reserve in alkaline soils, with solubility dependent on pH and the concentration of Ca and/or P in solution. Combining several techniques in a novel way, we studied these phenomena by progressively depleting P from suspensions of two soils (low P) using an anion-exchange membrane (AEM) and from a third soil (high P) with AEM together with a cation-exchange membrane. Depletions commenced on untreated soil, then continued as pH was manipulated and maintained at three constant pH levels: the initial pH (pHi) and pH 6.5 and 5.5. Bulk P K-edge X-ray absorption near-edge structure (XANES) spectroscopy revealed that the main forms of inorganic P in each soil were apatite, a second more soluble CaP mineral, and smectite-sorbed P. With moderate depletion of P at pHi or pH 6.5, CaP minerals became more prominent in the spectra compared to sorbed species. The more soluble CaP minerals were depleted at pH 6.5, and all CaP minerals were exhausted at pH 5.5, showing that the CaP species present in these alkaline soils are soluble with decreases of pH in the range achievable by rhizosphere acidification. PMID:26974327

  16. Enzyme activity in dialkyl phosphate ionic liquids.

    PubMed

    Thomas, Marie F; Li, Luen-Luen; Handley-Pendleton, Jocelyn M; van der Lelie, Daniel; Dunn, John J; Wishart, James F

    2011-12-01

    The activity of four metagenomic enzymes and an enzyme cloned from the straw mushroom, Volvariella volvacea were studied in the following ionic liquids, 1,3-dimethylimidazolium dimethyl phosphate, [mmim][dmp], 1-ethyl-3-methylimidazolium dimethyl phosphate, [emim][dmp], 1-ethyl-3-methylimidazolium diethyl phosphate, [emim][dep] and 1-ethyl-3-methylimidazolium acetate, [emim][OAc]. Activity was determined by analyzing the hydrolysis of para-nitrobenzene carbohydrate derivatives. In general, the enzymes were most active in the dimethyl phosphate ionic liquids, followed by acetate. Generally speaking, activity decreased sharply for concentrations of [emim][dep] above 10% v/v, while the other ionic liquids showed less impact on activity up to 20% v/v. PMID:22001053

  17. Enzyme activity in dialkyl phosphate ionic liquids

    SciTech Connect

    Thomas, M.F.; Dunn, J.; Li, L.-L.; Handley-Pendleton, J. M.; van der lelie, D.; Wishart, J. F.

    2011-12-01

    The activity of four metagenomic enzymes and an enzyme cloned from the straw mushroom, Volvariellavolvacea were studied in the following ionic liquids, 1,3-dimethylimidazolium dimethyl phosphate, [mmim][dmp], 1-ethyl-3-methylimidazolium dimethyl phosphate, [emim][dmp], 1-ethyl-3-methylimidazolium diethyl phosphate, [emim][dep] and 1-ethyl-3-methylimidazolium acetate, [emim][OAc]. Activity was determined by analyzing the hydrolysis of para-nitrobenzene carbohydrate derivatives. In general, the enzymes were most active in the dimethyl phosphate ionic liquids, followed by acetate. Generally speaking, activity decreased sharply for concentrations of [emim][dep] above 10% v/v, while the other ionic liquids showed less impact on activity up to 20% v/v.

  18. Phase Stability of Chromium(III) Oxide Hydroxide in Alkaline Sodium Phosphate Solutions

    SciTech Connect

    S.E. Ziemniak; E.P. Opalka

    2003-07-08

    Grimaldiite ({alpha}-CrOOH) is shown to transform to a sodium-chromium(III)-hydroxyphosphate compound (SCHP) in alkaline sodium phosphate solutions at elevated temperatures via CrOOH(s) + 4Na{sup +} + 2HPO{sub 4}{sup 2-} = Na{sub 4}Cr(OH)(PO{sub 4}){sub 2}(s) + H{sub 2}O. X-ray diffraction analyses indicate that SCHP possesses an orthorhombic lattice having the same space group symmetry (Ibam, No.72) as sodium ferric hydroxyphosphate. A structurally-consistent designation for SCHP is Na{sub 3}Cr(PO{sub 4}){sub 2} {center_dot} NaOH; the molar volume of SCHP is estimated to be 1552 cm{sup 3}. The thermodynamic equilibrium for the above reaction was defined in the system Na{sub 2}O-P{sub 2}O{sub 5}-Cr{sub 2}O{sub 3}-H{sub 2}O for Na/P molar ratios between 2.0 and 2.4. On the basis of observed reaction threshold values for sodium phosphate concentration and temperature, the standard molar entropy (S{sup o}), heat capacity (C{sub p}{sup o}) and free energy of formation ({Delta}G{sub f}{sup o}) for SCHP were calculated to be 690 J/(mol-K), 622 J/(mol-K) and -3509.97 kJ/mol, respectively.

  19. Oral antibodies to human intestinal alkaline phosphatase reduce dietary phytate phosphate bioavailability in the presence of dietary 1α-hydroxycholecalciferol.

    PubMed

    Bobeck, Elizabeth A; Hellestad, Erica M; Helvig, Christian F; Petkovich, P Martin; Cook, Mark E

    2016-03-01

    While it is well established that active vitamin D treatment increases dietary phytate phosphate utilization, the mechanism by which intestinal alkaline phosphatase (IAP) participates in phytate phosphate use is less clear. The ability of human IAP (hIAP) oral antibodies to prevent dietary phytate phosphate utilization in the presence of 1α-hydroxycholecalciferol (1α-(OH) D3) in a chick model was investigated. hIAP specific chicken immunoglobulin Y (IgY) antibodies were generated by inoculating laying hens with 17 synthetic peptides derived from the human IAP amino acid sequence and harvesting egg yolk. Western blot analysis showed all antibodies recognized hIAP and 6 of the 8 antibodies selected showed modest inhibition of hIAP activity in vitro (6 to 33% inhibition). In chicks where dietary phosphate was primarily in the form of phytate, 4 selected hIAP antibodies inhibited 1α-(OH) D3-induced increases in blood phosphate, one of which, generated against selected peptide (MFPMGTPD), was as effective as sevelamer hydrochloride in preventing the 1α-(OH) D3-induced increase in blood phosphate, but ineffective in preventing an increase in body weight gain and bone ash induced by 1α-(OH) D3. These studies demonstrated that orally-delivered antibodies to IAP limit dietary phytate-phosphate utilization in chicks treated with 1α-(OH) D3, and implicate IAP as an important host enzyme in increasing phytate phosphate bioavailability in 1α-(OH) D3 fed chicks. PMID:26666254

  20. Downscaling Alkaline Phosphatase Activity in a Subtropical Reservoir

    NASA Astrophysics Data System (ADS)

    Tseng, Y.

    2011-12-01

    This research was conducted by downscaling study to understand phosphorus (P)-deficient status of different plankton and the role of alkaline phosphatase activity (APA) in subtropical Feitsui Reservoir. Results from field survey showed that bulk APA (1.6~95.2 nM h-1) was widely observed in the epilimnion (0~20 m) with an apparent seasonal variations, suggesting that plankton in the system were subjected to P-deficient seasonally. Mixed layer depth (an index of phosphate availability) is the major factor influencing the variation of bulk APA and specific APA (124~1,253 nmol mg C-1 h-1), based on multiple linear regression analysis. Size-fractionated APA assays showed that picoplankton (size 0.2~3 um) contributed most of the bulk APA in the system. In addition, single-cell APA detected by enzyme-labeled fluorescence (ELF) assay indicated that heterotrophic bacteria are the major contributors of APA. Thus, we can infer that bacteria play an important role in accelerating P-cycle within P-deficient systems. Light/nutrient manipulation bioassays showed that bacterial growth was directly controlled by phosphate, while picocyanobacterial growth is controlled by light and can out-compete bacteria under P-limited condition with the aid of light. Further analysis revealed that the strength of summer typhoon is a factor responsible for the inter-annual variability of bulk and specific APA. APA study demonstrated the episodic events (e.g. strong typhoon and extreme precipitation) had significant influence on APA variability in sub-tropical to tropical aquatic ecosystems. Hence, the results herein will allow future studies on monitoring typhoon disturbance (intensity and frequency) as well as the APA of plankton during summer-to-autumn in subtropical systems.

  1. Probing the origins of catalytic discrimination between phosphate and sulfate monoester hydrolysis: comparative analysis of alkaline phosphatase and protein tyrosine phosphatases.

    PubMed

    Andrews, Logan D; Zalatan, Jesse G; Herschlag, Daniel

    2014-11-01

    Catalytic promiscuity, the ability of enzymes to catalyze multiple reactions, provides an opportunity to gain a deeper understanding of the origins of catalysis and substrate specificity. Alkaline phosphatase (AP) catalyzes both phosphate and sulfate monoester hydrolysis reactions with a ∼10(10)-fold preference for phosphate monoester hydrolysis, despite the similarity between these reactions. The preponderance of formal positive charge in the AP active site, particularly from three divalent metal ions, was proposed to be responsible for this preference by providing stronger electrostatic interactions with the more negatively charged phosphoryl group versus the sulfuryl group. To test whether positively charged metal ions are required to achieve a high preference for the phosphate monoester hydrolysis reaction, the catalytic preference of three protein tyrosine phosphatases (PTPs), which do not contain metal ions, were measured. Their preferences ranged from 5 × 10(6) to 7 × 10(7), lower than that for AP but still substantial, indicating that metal ions and a high preponderance of formal positive charge within the active site are not required to achieve a strong catalytic preference for phosphate monoester over sulfate monoester hydrolysis. The observed ionic strength dependences of kcat/KM values for phosphate and sulfate monoester hydrolysis are steeper for the more highly charged phosphate ester with both AP and the PTP Stp1, following the dependence expected based on the charge difference of these two substrates. However, the dependences for AP were not greater than those of Stp1 and were rather shallow for both enzymes. These results suggest that overall electrostatics from formal positive charge within the active site is not the major driving force in distinguishing between these reactions and that substantial discrimination can be attained without metal ions. Thus, local properties of the active site, presumably including multiple positioned dipolar

  2. Glucosamine: fructose-6-phosphate amidotransferase in the white shrimp Litopenaeus vannamei: characterization and regulation under alkaline and cadmium stress.

    PubMed

    Liu, Y; Cai, D X; Wang, L; Li, J Z; Wang, W N

    2015-10-01

    Heavy metal residues and chemical contaminators considered as relevant sources of aquatic environmental pollutants have a generally immunosuppressive effect on aquatic organisms, depressing metabolic activities and immune response. Glutamine: fructose-6-phosphate aminotransferase (GFAT, EC2.6.1.16) is the first, and rate-limiting, enzyme in the hexosamine biosynthetic pathway, and is involved in the regulation of chitin biosynthesis and glycosylation of proteins. We have isolated and characterized GFAT from the white shrimp Litopenaeus vannamei. Amino acid sequence similarity of the Lv-GFAT (L.vannamei-GFAT) was highest to GFATs isolated from insects and mammals (83 % similarity to that of Haemaphysalis longicornis). The open-reading frame of the Lv-GFAT codes for a protein of 41.6 kDa with a calculated isoelectric point of 5.03. RT-PCR assays showed that endogenous Lv-GFAT mRNA is most strongly expressed in the intestine. Further analysis of Lv-GFAT gene expression in hepatopancreas by quantitative real-time PCR demonstrated that Lv-GFAT transcript levels increased when the shrimp were exposed to alkaline pH (9.3) and cadmium stress, but the time when its mRNA expression level peaked differed under these stresses. We also first expressed the recombinant protein of GFAT from shrimps in Escherichia coli. Western blot analyses confirmed that the Lv-GFAT protein was strongly expressed in the hepatopancreas after exposure to the LC-Cd stress. These results suggest that Lv-GFAT expression is stimulated by alkaline pH and cadmium stress and that it may play important roles in resistance of shrimp to environmental stresses. PMID:25956985

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

    PubMed

    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

  4. Effects of Alkaline Phosphatase Activity on Nucleotide Measurements in Aquatic Microbial Communities †

    PubMed Central

    Karl, D. M.; Craven, D. B.

    1980-01-01

    Alkaline phosphatase (APase) activity was detected in aquatic microbial assemblages from the subtropics to Antarctica. The occurrence of APase in environmental nucleotide extracts was shown to significantly affect the measured concentrations of cellular nucleotides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, guanosine triphosphate, uridine triphosphate, and cytidine triphosphate), adenylate energy charge, and guanosine triphosphate/adenosine triphosphate ratios, when conventional methods of nucleotide extraction were employed. Under the reaction conditions specified in this report, the initial rate of hydrolysis of adenosine triphosphate was directly proportional to the activity of APase in the sample extracts and consequently can be used as a sensitive measure of APase activity. A method was devised for obtaining reliable nucleotide measurements in naturally occurring microbial populations containing elevated levels of APase activity. The metabolic significance of APase activity in microbial cells is discussed, and it is concluded that the occurrence and regulation of APase in nature is dependent upon microscale inorganic phosphate limitation of the autochthonous microbial communities. PMID:16345634

  5. Cholesterol modulates alkaline phosphatase activity of rat intestinal microvillus membranes.

    PubMed

    Brasitus, T A; Dahiya, R; Dudeja, P K; Bissonnette, B M

    1988-06-25

    Experiments were conducted, using a nonspecific lipid transfer protein, to vary the cholesterol/phospholipid molar ratio of rat proximal small intestinal microvillus membranes in order to assess the possible role of cholesterol in modulating enzymatic activities of this plasma membrane. Cholesterol/phospholipid molar ratios from 0.71 to 1.30 were produced from a normal value of 1.05 by incubation with the transfer protein and an excess of either phosphatidylcholine or cholesterol/phosphatidylcholine liposomes for 60 min at 37 degrees C. Cholesterol loading or depletion of the membranes was accompanied by a decrease or increase, respectively, in their lipid fluidity, as assessed by steady-state fluorescence polarization techniques using the lipid-soluble fluorophore 1,6-diphenyl-1,3,5-hexatriene. Increasing the cholesterol/phospholipid molar ratio also decreased alkaline phosphatase specific activity by approximately 20-30%, whereas decreasing this ratio increased this enzymatic activity by 20-30%. Sucrase, maltase, and lactase specific activities were not affected in these same preparations. Since the changes in alkaline phosphatase activity could be secondary to alterations in fluidity, cholesterol, or both, additional experiments were performed using benzyl alcohol, a known fluidizer. Benzyl alcohol (25 mM) restored the fluidity of cholesterol-enriched preparations to control levels, did not change the cholesterol/phospholipid molar ratio, and failed to alter alkaline phosphatase activity. These findings, therefore, indicate that alterations in the cholesterol content and cholesterol/phospholipid molar ratio of microvillus membranes can modulate alkaline phosphatase but not sucrase, maltase, or lactase activities. Moreover, membrane fluidity does not appear to be an important physiological regulator of these enzymatic activities. PMID:3379034

  6. Molecular and biochemical characterization of a new alkaline active multidomain xylanase from alkaline wastewater sludge.

    PubMed

    Zhao, Yanyu; Meng, Kun; Luo, Huiying; Huang, Huoqing; Yuan, Tiezheng; Yang, Peilong; Yao, Bin

    2013-02-01

    A xylanase gene, xyn-b39, coding for a multidomain glycoside hydrolase (GH) family 10 protein was cloned from the genomic DNA of the alkaline wastewater sludge of a paper mill. Its deduced amino acid sequence of 1,481 residues included two carbohydrate-binding modules (CBM) of family CBM_4_9, one catalytic domain of GH 10, one family 9 CBM and three S-layer homology (SLH) domains. xyn-b39 was expressed heterologously in Escherichia coli, and the recombinant enzyme was purified and characterized. Xyn-b39 exhibited maximum activity at pH 7.0 and 60 °C, and remained highly active under alkaline conditions (more than 80 % activity at pH 9.0 and 40 % activity at pH 10.0). The enzyme was thermostable at 55 °C, retaining more than 90 % of the initial activity after 2 h pre-incubation. Xyn-b39 had wide substrate specificity and hydrolyzed soluble substrates (birchwood xylan, beechwood xylan, oat spelt xylan, wheat arabinoxylan) and insoluble substrates (oat spelt xylan and wheat arabinoxylan). Hydrolysis product analysis indicated that Xyn-b39 was an endo-type xylanase. The K (m) and V (max) values of Xyn-b39 for birchwood xylan were 1.01 mg/mL and 73.53 U/min/mg, respectively. At the charge of 10 U/g reed pulp for 1 h, Xyn-b39 significantly reduced the Kappa number (P < 0.05) with low consumption of chlorine dioxide alone. PMID:23117673

  7. Identification of Active Radical Species in Alkaline Persulfate Oxidation.

    PubMed

    Liang, Chenju; Lei, Jung-Hsuan

    2015-07-01

    A proposed mechanism for alkaline activation of persulfate involves generation of sulfate (SO(4)(-)), hydroxyl (HO·), and superoxide radicals (O(2)(-)). The present study investigated the feasibility of chloroform (CF) degradation using alkaline activated persulfate and identified the active radical species using a radical inhibition technique. 2-propanol (PrOH) (preferentially reacted with HO·), phenol (preferentially reacted with both HO· and SO(4)(-)), and carbon tetrachloride (CT) (preferentially reacted with O(2)(-)) were used to inhibit the degradation of CF, and the extent of inhibited degradation was used to indicate the predominant radical species. Additions of PrOH and phenol appeared to significantly scavenge SO(4)(-) and HO· and resulted in inhibited CF degradation. Here, the authors demonstrated that SO(4)(-) and HO· were predominant radicals in the alkaline activated persulfate system. The presence of O(2)(-) scavengers (i.e., CT) resulted in a partial inhibition of CF degradation and, hence, one can speculate that O(2)(-) is a minor radical species. PMID:26163502

  8. Overproduction of acetate kinase activates the phosphate regulon in the absence of the phoR and phoM functions in Escherichia coli.

    PubMed Central

    Lee, T Y; Makino, K; Shinagawa, H; Nakata, A

    1990-01-01

    A DNA fragment of Escherichia coli cloned on pBR322 elevated the production of alkaline phosphatase and phosphate-binding protein in a phoR phoM strain. Nucleotide sequence analysis and enzyme assays revealed that the DNA fragment contained the ackA gene, which codes for acetate kinase. A high gene dosage of ackA was needed to induce the production of alkaline phosphatase and phosphate-binding protein in this strain. Overexpression of ackA elevated the intracellular ATP concentration, an effect that might be related to activation of the phosphate regulon in the phoR phoM strain. Images PMID:2158965

  9. Effectiveness of recovered magnesium phosphates as fertilizers in neutral and slightly alkaline soils

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Magnesium phosphates such as struvite (MgNH4PO4x6H2O) can be recovered from municipal, industrial and agricultural wastewaters. However, minimal research has been conducted on the beneficial reuse of these recovered products; conducted research has focused on low pH soils. This study determined wh...

  10. Positive Active Material For Alkaline Electrolyte Storage Battert Nickel Electrodes

    DOEpatents

    Bernard, Patrick; Baudry, Michelle

    2000-12-05

    A method of manufacturing a positive active material for nickel electrodes of alkaline storage batteries which consists of particles of hydroxide containing mainly nickel and covered with a layer of a hydroxide phase based on nickel and yttrium is disclosed. The proportion of the hydroxide phase is in the range 0.15% to 3% by weight of yttrium expressed as yttrium hydroxide relative to the total weight of particles.

  11. Preparation of alkaline earth phosphates with sol containing sodium alginate and sodium diphosphate.

    PubMed

    Sugiyama, Shigeru; Fujii, Minako; Fukuta, Kazuya; Seyama, Kazunori; Sotowa, Ken-Ichiro; Shigemoto, Naoya

    2006-03-01

    Magnesium hydrogen phosphate, calcium hydroxyapatite, and strontium hydroxyapatite were successfully prepared from sol consisting of sodium alginate and Na4P2O7 with Mg2+, Ca2+, and Sr2+ in the corresponding nitrates, respectively. It is revealed that the order of the addition of those substrates and the role of sodium alginate are important factors for the preparation of desired phosphate compounds. According to the previous paper on the preparation of calcium hydroxyapatite, sodium alginate was mixed with aqueous Na4P2O7, followed by the addition of the aqueous divalent cations, resulting in the poor formation of the target phosphates. However, as a revised sol-gel technique, sodium alginate was added to the mixture of Na4P2O7 and aqueous Mg2+ and Sr2+, resulting in a rather favorable formation of MgHPO4 and strontium hydroxyapatite, respectively, while the sol thus obtained was stable within a few days. However for aqueous Ca2+, calcium hydroxyapatite could not be obtained through the revised sol-gel technique. In the preparation of magnesium hydrogen phosphate, sodium alginate contributes mainly to the sol formation of the precursor. The ion exchange between Na+ in sodium alginate and aqueous Ca2+ was important for the preparation of calcium hydroxyapatite. In contrast, the reaction of sodium alginate with the mixture of Na4P2O7 and aqueous Sr2+ afforded strontium hydroxyapatite at the specific ratio of those three substrates. The structure of calcium and strontium phosphates prepared from the revised sol-gel process evidently depended on the amount of sodium alginate introduced into the mixture of Na4P2O7 and the corresponding divalent cations. PMID:16154579

  12. Early Events Induced by the Elicitor Cryptogein in Tobacco Cells: Involvement of a Plasma Membrane NADPH Oxidase and Activation of Glycolysis and the Pentose Phosphate Pathway.

    PubMed Central

    Pugin, A.; Frachisse, J. M.; Tavernier, E.; Bligny, R.; Gout, E.; Douce, R.; Guern, J.

    1997-01-01

    Application of the elicitor cryptogein to tobacco (cv Xanthi) is known to evoke external medium alkalinization, active oxygen species production, and phytoalexin synthesis. These are all dependent on an influx of calcium. We show here that cryptogein also induces calcium-dependent plasma membrane depolarization, chloride efflux, cytoplasm acidification, and NADPH oxidation without changes in NAD+ and ATP levels, indicating that the elicitor-activated redox system, responsible for active oxygen species production, uses NADPH in vivo. NADPH oxidation activates the functioning of the pentose phosphate pathway, leading to a decrease in glucose 6-phosphate and to the accumulation of glyceraldehyde 3-phosphate, 3- and 2-phosphoglyceric acid, and phosphoenolpyruvate. By inhibiting the pentose phosphate pathway, we demonstrate that the activation of the plasma membrane NADPH oxidase is responsible for active oxygen species production, external alkalinization, and acidification of the cytoplasm. A model is proposed for the organization of the cryptogein responses measured to date. PMID:12237354

  13. Interfacial activity in alkaline flooding enhanced oil recovery

    SciTech Connect

    Chan, M.K.

    1981-01-01

    The ionization of long-chained organic acids in the crude oil to form soaps was shown to be primarily responsible for the lowering of oil-water interfacial tension at alkaline pH. These active acids can be concentrated by silica gel chromatography into a minor polar fraction. An equilibrium chemical model was proposed based on 2 competing reactions: the ionization of acids to form active anions, and the formation of undissociated soap between acid anions and sodium ions. It correlates the interfacial activity with the interfacial concentration of active acid anions which is expressed in terms of the concentrations of the chemical species in the system. The model successfully predicts the observed oil-alkaline solution interfacial phenomenon, including its dependence on pH, alkali and salt concentrations, type of acid present and type of soap formed. Flooding at different alkali concentrations to activate different acid species present in the crude was shown to give better recovery than flooding at a single high alkali concentration. Treating the crude oil with a dilute solution of mineral acids liberates additional free active acids and yields better interfacial activity during subsequent alkali contact.

  14. Additive for activating iron electrodes in alkaline batteries

    SciTech Connect

    Berger, G.; Haschka, F.

    1981-02-10

    An additive is disclosed for the iron electrodes of alkaline batteries which prevents and counteracts the tendency of iron electrodes to become passive and ineffective. The additive consists of sulfide, selenide or telluride which is sparingly soluble in the electrolyte and has a decomposition potential more electronegative than the final charging potential of the iron electrode. The additive may be placed in the active electrode material during manufacture but may also be placed in the battery during or after manufacture, for example in tablet form. The additive may also be introduced in a manner permitting subsequent activation by electrochemical methods. A number of examples is presented.

  15. EF-G Activation by Phosphate Analogs.

    PubMed

    Salsi, Enea; Farah, Elie; Ermolenko, Dmitri N

    2016-05-22

    Elongation factor G (EF-G) is a universally conserved translational GTPase that promotes the translocation of tRNA and mRNA through the ribosome. EF-G binds to the ribosome in a GTP-bound form and subsequently catalyzes GTP hydrolysis. The contribution of the ribosome-stimulated GTP hydrolysis by EF-G to tRNA/mRNA translocation remains debated. Here, we show that while EF-G•GDP does not stably bind to the ribosome and induce translocation, EF-G•GDP in complex with phosphate group analogs BeF3(-) and AlF4(-) promotes the translocation of tRNA and mRNA. Furthermore, the rates of mRNA translocation induced by EF-G in the presence of GTP and a non-hydrolyzable analog of GTP, GDP•BeF3(-) are similar. Our results are consistent with the model suggesting that GTP hydrolysis is not directly coupled to mRNA/tRNA translocation. Hence, GTP binding is required to induce the activated, translocation-competent conformation of EF-G while GTP hydrolysis triggers EF-G release from the ribosome. PMID:27063503

  16. Structural investigation of Eu{sup 2+} emissions from alkaline earth zirconium phosphate

    SciTech Connect

    Hirayama, Masaaki; Sonoyama, Noriyuki; Yamada, Atsuo; Kanno, Ryoji

    2009-04-15

    Eu{sup 2+} doped A{sub 0.5}Zr{sub 2}(PO{sub 4}){sub 3} (A=Ca, Sr, Ba) phosphors with the NASICON structure were synthesized by a co-precipitation method. Their photoluminescent and structural properties were investigated by photoluminescent spectroscopy and powder X-ray Rietveld analysis, which determined two sites for Eu{sup 2+} ions in the host structure, 3a and 3b. The Eu-O bond lengths were increased by changing alkaline earth ions from Ca to Ba, causing Eu{sup 2+} emission bands to shift from blue-green to blue. A correlation was observed between the peak wavelength positions and the Eu-O bond length. The photoluminescent properties are discussed in terms of crystal field strength and nephelauxetic effect, and a schematic diagram of Eu{sup 2+} emissions is proposed for the Eu{sup 2+} doped NASICON phosphor. - Graphical abstract: Eu{sup 2+} doped NASICON structured A{sub 0.5}Zr{sub 2}(PO{sub 4}){sub 3} (A=Ca, Sr, Ba) showed the blue and blue-green colored emissions attributed to 4f{sup 6}5d{sup 1}-4f{sup 7} transitions. The photoluminescent properties are discussed in terms of crystal field strength and nephelauxetic effect using powder X-ray Rietveld analysis.

  17. Theoretical Studies of the Spin Hamiltonian Parameters and Local Distortions for Cu2+ in Alkaline Earth Lead Zinc Phosphate Glasses

    NASA Astrophysics Data System (ADS)

    Wang, Bo-Kun; Wu, Shao-Yi; Yuan, Zi-Yi; Liu, Zi-Xuan; Jiang, Shi-Xin; Liu, Zheng; Yao, Zi-Jian; Teng, Bao-Hua; Wu, Ming-He

    2016-08-01

    The spin Hamiltonian parameters and local structures are theoretically studied for Cu2+-doped alkaline earth lead zinc phosphate (RPPZ, R=Mg, Ca, Sr, and Ba) glasses based on the high-order perturbation calculations for a tetragonally elongated octahedral 3d9 cluster. The relative elongation ratios are found to be ρ≈3.2%, 4.4%, 4.6%, and 3.3% for R=Mg, Ca, Sr, and Ba, respectively, because of the Jahn-Teller effect. The whole decreasing crystal-field strength Dq and orbital reduction factor k from Mg to Sr are ascribed to the weakening electrostatic coulombic interactions and the increasing probability of productivity of nonbridge oxygen (and hence increasing Cu2+-O2- electron cloud admixtures) under PbO addition, respectively, with increasing alkali earth ionic radius. The anomalies (the largest Dq and the next highest k among the systems) for R=Ba are attributed to the cross linkage of this large cation in the network. The overall increasing order (Mg≤Ba

  18. Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Ca2+ level in osteoblasts (SaOS-2 cells) in vitro.

    PubMed

    Müller, Werner E G; Wang, Xiaohong; Diehl-Seifert, Bärbel; Kropf, Klaus; Schlossmacher, Ute; Lieberwirth, Ingo; Glasser, Gunnar; Wiens, Matthias; Schröder, Heinz C

    2011-06-01

    Inorganic polymeric phosphate is a physiological polymer that accumulates in bone cells. In the present study osteoblast-like SaOS-2 cells were exposed to this polymer, complexed in a 2:1 stoichiometric ratio with Ca(2+), polyP (Ca(2+) salt). At a concentration of 100 μM, polyP (Ca(2+) salt) caused a strong increase in the activity of the alkaline phosphatase and also an induction of the steady-state expression of the gene encoding this enzyme. Comparative experiments showed that polyP (Ca(2+) salt) can efficiently replace β-glycerophosphate in the in vitro hydroxyapatite (HA) biomineralization assay. In the presence of polyP (Ca(2+) salt) the cells extensively form HA crystallites, which remain intimately associated with or covered by the plasma membrane. Only the tips of the crystallites are directly exposed to the extracellular space. Element mapping by scanning electron microscopy/energy-dispersive X-ray spectroscopy coupled to a silicon drift detector supported the finding that organic material was dispersed within the crystallites. Finally, polyP (Ca(2+) salt) was found to cause an increase in the intracellular Ca(2+) level, while polyP, as well as inorganic phosphate (P(i)) or Ca(2+) alone, had no effect at the concentrations used. These findings are compatible with the assumption that polyP (Ca(2+) salt) is locally, on the surface of the SaOS-2 cells, hydrolyzed to P(i) and Ca(2+). We conclude that the inorganic polymer polyP (Ca(2+) salt) in concert with a second inorganic, and physiologically occurring, polymer, biosilica, activates osteoblasts and impairs the maturation of osteoclasts. PMID:21397057

  19. Alkaline phosphatase activity in normal and inflamed dental pulps.

    PubMed

    Spoto, G; Fioroni, M; Rubini, C; Tripodi, D; Di Stilio, M; Piattelli, A

    2001-03-01

    Alkaline phosphatase (ALP) seems to be important in the formation of mineralized tissues. High levels of ALP have been demonstrated in dental pulp cells. In the present study ALP activity was analyzed in normal healthy human dental pulps, in reversible pulpitis, and in irreversible pulpitis. Enzymatic ALP control values for the normal healthy pulps were 110.96+/-20.93. In the reversible pulpitis specimens the ALP activity increased almost eight times to 853.6+/-148.27. In the irreversible pulpitis specimens the values decreased sharply to 137.15+/-21.28 and were roughly equivalent to those seen in normal healthy pulps. The differences between the groups (control vs. reversible pulpitis and reversible pulpitis vs. irreversible pulpitis) were statistically significant. These results could point to a role of ALP in the initial pulp response after injury. PMID:11487147

  20. Regulation of alkaline ceramidase activity by the c-Src-mediated pathway.

    PubMed

    Sasaki, Hirotsune; Toyomura, Kaori; Matsuzaki, Wataru; Okamoto, Aya; Yamaguchi, Naoto; Nakamura, Hiroyuki; Murayama, Toshihiko

    2014-05-15

    Ceramidase hydrolyzes ceramide to fatty acids and sphingosine, and sphingosine is then converted to sphingosine-1-phosphate. Ceramide and sphingosine-1-phosphate act as signaling molecules. Although stimuli coupling to protein kinases-dependent systems have been shown to regulate ceramidase activity, the exact role of c-Src-mediated signal has not been elucidated. We examined the effects of the downregulation of c-Src activity and c-Src overexpression on ceramidase activity in cells. In A549, CHO, and HeLa cells labeled with a fluorescent ceramide, 4-nitrobenzo-2-oxa-1,3-diazole-labeled C6-ceramide (NBD-ceramide), the downregulation of c-Src by c-Src-shRNA and pharmacological inhibitors including SU6656 decreased levels of NBD-caproic acid. The overexpression of c-Src increased NBD-caproic acid levels in CHO and HeLa cells. Similar results were obtained in Na3VO4-treated cells having higher NBD-caproic acid levels. The downregulation and overexpression of c-Src decreased and increased ceramidase activity, respectively, in the lysates of A549 cells at pH 8.8. The ceramidase sensitivity to substrates, pH, and Ca(2+) suggest that the c-Src- and SU6656-sensitive ceramidase is alkaline ceramidase (ACER), possibly Ca(2+)-activated ACER2. Serum starvation increased both ceramidase activity at pH 8.8 and expression of ACER2. Our data suggest that c-Src-mediated signal positively regulates ACER activity in a Ca(2+)-independent manner. PMID:24708996

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

    PubMed

    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. PMID:25476378

  2. Relationship between Nitrite Reduction and Active Phosphate Uptake in the Phosphate-Accumulating Denitrifier Pseudomonas sp. Strain JR 12

    PubMed Central

    Barak, Yoram; van Rijn, Jaap

    2000-01-01

    Phosphate uptake by the phosphate-accumulating denitrifier Pseudomonas sp. JR12 was examined with different combinations of electron and carbon donors and electron acceptors. Phosphate uptake in acetate-supplemented cells took place with either oxygen or nitrate but did not take place when nitrite served as the final electron acceptor. Furthermore, nitrite reduction rates by this denitrifier were shown to be significantly reduced in the presence of phosphate. Phosphate uptake assays in the presence of the H+-ATPase inhibitor N,N′-dicyclohexylcarbodiimide (DCCD), in the presence of the uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP), or with osmotic shock-treated cells indicated that phosphate transport over the cytoplasmic membrane of this bacterium was mediated by primary and secondary transport systems. By examining the redox transitions of whole cells at 553 nm we found that phosphate addition caused a significant oxidation of a c-type cytochrome. Based on these findings, we propose that this c-type cytochrome serves as an intermediate in the electron transfer to both nitrite reductase and the site responsible for active phosphate transport. In previous studies with this bacterium we found that the oxidation state of this c-type cytochrome was significantly higher in acetate-supplemented, nitrite-respiring cells (incapable of phosphate uptake) than in phosphate-accumulating cells incubated with different combinations of electron donors and acceptors. Based on the latter finding and results obtained in the present study it is suggested that phosphate uptake in this bacterium is subjected to a redox control of the active phosphate transport site. By means of this mechanism an explanation is provided for the observed absence of phosphate uptake in the presence of nitrite and inhibition of nitrite reduction by phosphate in this organism. The implications of these findings regarding denitrifying, phosphate removal wastewater plants is discussed. PMID

  3. Microbial activities and phosphorus cycling: An application of oxygen isotope ratios in phosphate

    NASA Astrophysics Data System (ADS)

    Stout, Lisa M.; Joshi, Sunendra R.; Kana, Todd M.; Jaisi, Deb P.

    2014-08-01

    Microorganisms carry out biochemical transformations of nutrients that make up their cells. Therefore, understanding how these nutrients are transformed or cycled in natural environments requires knowledge of microbial activity. Commonly used indicators for microbial activity typically include determining microbial respiration by O2/CO2 measurements, cell counts, and measurement of enzyme activities. However, coupled studies on nutrient cycling and microbial activity are not given enough emphasis. Here we apply phosphate oxygen isotope ratios (δ18OP) as a tool for measurement of microbial activity and compare the rate of isotope exchange with methods of measuring microbial activities that are more commonly applied in environmental studies including respiration, dehydrogenase activity, alkaline phosphatase activity, and cell counts. Our results show that different bacteria may have different strategies for P uptake, storage and release, their respiration and consequently expression of DHA and APase activities, but in general the trend of their enzyme activities are comparable. Phosphate δ18OP values correlated well with these other parameters used to measure microbial activity with the strongest linear relationships between δ18OP and CO2 evolution (r = -0.99). Even though the rate of isotope exchange for each microorganism used in this study is different, the rate per unit CO2 respiration showed one general trend, where δ18OP values move towards equilibrium while CO2 is generated. While this suggests that P cycling among microorganisms used in this study can be generalized, further research is needed to determine whether the microorganism-specific isotope exchange trend may occur in natural environments. In summary, phosphate oxygen isotope measurements may offer an alternative for use as a tracer to measure microbial activity in soils, sediments, and many other natural environments.

  4. Structural basis for glucose-6-phosphate activation of glycogen synthase

    SciTech Connect

    Baskaran, Sulochanadevi; Roach, Peter J.; DePaoli-Roach, Anna A.; Hurley, Thomas D.

    2010-11-22

    Regulation of the storage of glycogen, one of the major energy reserves, is of utmost metabolic importance. In eukaryotes, this regulation is accomplished through glucose-6-phosphate levels and protein phosphorylation. Glycogen synthase homologs in bacteria and archaea lack regulation, while the eukaryotic enzymes are inhibited by protein kinase mediated phosphorylation and activated by protein phosphatases and glucose-6-phosphate binding. We determined the crystal structures corresponding to the basal activity state and glucose-6-phosphate activated state of yeast glycogen synthase-2. The enzyme is assembled into an unusual tetramer by an insertion unique to the eukaryotic enzymes, and this subunit interface is rearranged by the binding of glucose-6-phosphate, which frees the active site cleft and facilitates catalysis. Using both mutagenesis and intein-mediated phospho-peptide ligation experiments, we demonstrate that the enzyme's response to glucose-6-phosphate is controlled by Arg583 and Arg587, while four additional arginine residues present within the same regulatory helix regulate the response to phosphorylation.

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

    PubMed

    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. PMID:26002329

  6. Growth and alkaline phosphatase activity of Chattonella marina and Heterosigma akashiwo in response to phosphorus limitation.

    PubMed

    Wang, Zhao-Hui; Liang, Yu

    2015-02-01

    The growth and alkaline phosphatase activity (APA) of two raphidophyceae species Chattonella marina and Heterosigma akashiwo were investigated in response to P-limitation and subsequent addition of dissolved inorganic phosphorus (DIP, NaH2PO4) and two dissolved organic phosphorus (DOP) compounds: guanosine 5-monophosphate (GMP) and triethyl phosphate (TEP). APA levels increased greatly after P-starvation as the decrease of the cellular phosphorus quotes (Qp). C. marina responded to P-limitation quickly and strongly, with 10-fold increase in APA within 24 hr after P-starvation. The larger difference between maximal and minimal QP values in C. marina indicated its high capacity in P storage. APA of H. akashiwo was maximally enlarged about 2.5 times at 48 hr of P-starvation. After the addition of nutrients, cell numbers of C. marina increased in all treatments including the P-free culture, demonstrating the higher endurance of C. marina to P-limitation. However, those of H. akashiwo increased only in DIP and GMP cultures. APA increased only after the addition of the monophosphate ester GMP. The results suggest that quick responses of C. marina to P-limitation, high capacity in P storage as well as endurance for P-depletion provide this species an ecological advantage in phytoplankton community competition under DIP-limited conditions. PMID:25662231

  7. The dynamics of alkaline phosphatase activity during operculum regeneration in the polychaete Pomatoceros lamarckii.

    PubMed

    Szabó, Réka; Ferrier, David E K

    2014-01-01

    Alkaline phosphatase enzymes are found throughout the living world and fulfil a variety of functions. They have been linked to regeneration, stem cells and biomineralisation in a range of animals. Here we describe the pattern of alkaline phosphatase activity in a spiralian appendage, the operculum of the serpulid polychaete Pomatoceros lamarckii. The P. lamarckii operculum is reinforced by a calcified opercular plate and is capable of rapid regeneration, making it an ideal model system to study these key processes in annelids. Alkaline phosphatase activity is present in mesodermal tissues of both intact and regenerating opercular filaments, in a strongly regionalised pattern correlated with major morphological features. Based on the lack of epidermal activity and the broad distribution of staining in mesodermal tissues, calcification- or stem cell-specific roles are unlikely. Transcriptomic data reveal that at least four distinct genes contribute to the detected activity. Opercular alkaline phosphatase activity is sensitive to levamisole. Phylogenetic analysis of metazoan alkaline phosphatases indicates homology of the P. lamarckii sequences to other annelid alkaline phosphatases, and shows that metazoan alkaline phosphatase evolution was characterised by extensive lineage-specific duplications. PMID:25690977

  8. Effect of zinc phosphate chemical conversion coating on corrosion behaviour of mild steel in alkaline medium: protection of rebars in reinforced concrete

    NASA Astrophysics Data System (ADS)

    Simescu, Florica; Idrissi, Hassane

    2008-12-01

    We outline the ability of zinc phosphate coatings, obtained by chemical conversion, to protect mild steel rebars against localized corrosion, generated by chloride ions in alkaline media. The corrosion resistance of coated steel, in comparison with uncoated rebars and coated and uncoated steel rebars embedded in mortar, were evaluated by open-circuit potential, potentiodynamic polarization, cronoamperometry and electrochemical impedance spectroscopy. The coated surfaces were characterized by x-ray diffraction and scanning electron microscopy. First, coated mild steel rebars were studied in an alkaline solution with and without chloride simulating a concrete pore solution. The results showed that the slow dissolution of the coating generates hydroxyapatite Ca10(PO4)6(OH)2. After a long immersion, the coating became dense and provided an effective corrosion resistance compared with the mild steel rebar. Secondly, the coated and uncoated steel rebars embedded in mortar and immersed in chloride solution showed no corrosion or deterioration of the coated steel. Corrosion rate is considerably lowered by this phosphate coating.

  9. Use of solid phase extraction for the sequential injection determination of alkaline phosphatase activity in dynamic water systems.

    PubMed

    Santos, Inês C; Mesquita, Raquel B R; Bordalo, Adriano A; Rangel, António O S S

    2012-08-30

    In this work, a solid phase extraction sequential injection methodology for the determination of alkaline phosphatase activity in dynamic water systems was developed. The determination of the enzymatic activity was based on the spectrophotometric detection of a coloured product, p-nitrophenol, at 405 nm. The p-nitrophenol is the product of the catalytic decomposition of p-nitrophenyl phosphate, a non-coloured substrate. Considering the low levels expected in natural waters and exploiting the fact of alkaline phosphatase being a metalloprotein, the enzyme was pre-concentrated in-line using a NTA Superflow resin charged with Zn(2+) ions. The developed sequential injection method enabled a quantification range of 0.044-0.441 unit mL(-1) of enzyme activity with a detection limit of 0.0082 unit mL(-1) enzyme activity (1.9 μmol L(-1) of pNP) and a determination rate of 17 h(-1). Recovery tests confirmed the accuracy of the developed sequential injection method and it was effectively applied to different natural waters and to plant root extracts. PMID:22939148

  10. Alkaline phosphatase activity in salivary gland cells of Rhodnius neglectus and R. prolixus (Hemiptera, Triatominae).

    PubMed

    Lima-Oliveira, A P M; Alevi, K C C; Anhê, A C B; Azeredo-Oliveira, M T V

    2016-01-01

    Alkaline phosphatase activity was detected in salivary gland cells of the Rhodnius neglectus Lent, 1954, and R. prolixus Stal, 1859, vectors of Trypanosoma cruzi Chagas, 1909 (etiological agent of Chagas disease) and T. rangeli Tejera, 1920 (pathogenic to insect). The Gomori technique was used to demonstrate alkaline phosphatase activity. Alkaline phosphatase activity was observed throughout the entire gland, with an increased activity in the posterior region of the principal gland. In particular, phosphatase activity was found in the nucleolar corpuscles, suggesting a relationship with the rRNA transcription and ribosomal biogenesis. Alkaline phosphatase was also detected in the nuclear membrane and nuclear matrix, suggesting an association with the nucleo-cytoplasmic transport of ribonucleoproteins and the mechanisms of cell cycle and DNA replication, respectively. This study highlights the importance of alkaline phosphatase in the salivary gland of R. prolixus and R. neglectus and emphasizes its importance in secretory activity. Secretory activity is directly involved in hematophagy and, consequently, in development during metamorphosis. The observed presence of alkaline phosphatase suggests its involvement in the production of saliva allowing feeding of these insects that are important vectors of Chagas disease. PMID:27525888

  11. Glycerolipid biosynthesis in Saccharomyces cerevisiae: sn-glycerol-3-phosphate and dihydroxyacetone phosphate acyltransferase activities.

    PubMed Central

    Schlossman, D M; Bell, R M

    1978-01-01

    Yeast acyl-coenzyme A:dihydroxyacetone-phosphate O-acyltransferase (DHAP acyltransferase; EC 2.3.1.42) was investigated to (i) determine whether its activity and that of acyl-coenzyme A:sn-glycerol-3-phosphate O-acyltransferase (glycerol-P acyltransferase; EC 2.3.1.15) represent dual catalytic functions of a single membranous enzyme, (ii) estimate the relative contributions of the glycerol-P and DHAP pathways for yeast glycerolipid synthesis, and (iii) evaluate the suitability of yeast for future genetic investigations of the eucaryotic glycerol-P and DHAP acyltransferase activities. The membranous DHAP acyltransferase activity showed an apparent Km of 0.79 mM for DHAP, with a Vmax of 5.3 nmol/min per mg, whereas the glycerol-P acyltransferase activity showed an apparent Km of 0.05 mM for glycerol-P, with a Vmax of 3.4 nmol/min per mg. Glycerol-P was a competitive inhibitor (Ki, 0.07 mM) of the DHAP acyltransferase activity, and DHAP was a competitive inhibitor (Ki, 0.91 mM) of the glycerol-P acyltransferase activity. The two acyltransferase activities exhibited marked similarities in their pH dependence, acyl-coenzyme A chain length preference and substrate concentration dependencies, thermolability, and patterns of inactivation by N-ethylmaleimide, trypsin, and detergents. Thus, the data strongly suggest that yeast glycerol-P and DHAP acyltransferase activities represent dual catalytic functions of a single membrane-bound enzyme. Furthermore, since no acyl-DHAP oxidoreductase activity could be detected in yeast membranes, the DHAP pathway for glycerolipid synthesis may not operate in yeast. PMID:25265

  12. Biosynthesis of Dolichyl Phosphate

    PubMed Central

    Hopp, H. Esteban; Daleo, Gustavo R.; Romero, Pedro A.; Lezica, Rafael Pont

    1978-01-01

    This is the first report not only on the presence of polyprenyl phosphates and their site of synthesis in algae, but also on the formation of their sugar derivatives in this system. A glucose acceptor lipid was isolated from the nonphotosynthetic alga Prototheca zopfii. The lipid was acidic and resistant to mild acid and alkaline treatments. The glucosylated lipid was labile to mild acid hydrolysis and resistant to phenol treatment and catalytic hydrogenation, as dolichyl phosphate glucose is. These results are consistent with the properties of an α-saturated polyprenyl phosphate. The polyprenylic nature of the lipid was confirmed by biosynthesis from radioactive mevalonate. The [14C]lipid had the same chromatographic properties as dolichyl phosphate in DEAE-cellulose and Sephadex LH-20. Strong alkaline treatment and enzymic hydrolysis liberated free alcohols with chain lengths ranging from C90 to C105, C95 and C100 being the most abundant molecular forms. The glucose acceptor activity of the biosynthesized polyprenyl phosphate was confirmed. The ability of different subcellular fractions to synthesize dolichyl phosphate was studied. Mitochondria and the Golgi apparatus were the sites of dolichyl phosphate synthesis from mevalonate. PMID:16660269

  13. Pyridoxamine-5-phosphate enzyme-linked immune mass spectrometric assay substrate for linear absolute quantification of alkaline phosphatase to the yoctomole range applied to prostate specific antigen.

    PubMed

    Florentinus-Mefailoski, Angelique; Marshall, John G

    2014-11-01

    There is a need to measure proteins that are present in concentrations below the detection limits of existing colorimetric approaches with enzyme-linked immunoabsorbent assays (ELISA). The powerful enzyme alkaline phosphatase conjugated to the highly specific bacterial protein streptavidin binds to biotinylated macromolecules like proteins, antibodies, or other ligands and receptors with a high affinity. The binding of the biotinylated detection antibody, with resulting amplification of the signal by the catalytic production of reporter molecules, is key to the sensitivity of ELISA. The specificity and amplification of the signal by the enzyme alkaline phosphatase in ELISA together with the sensitivity of liquid chromatography electrospray ionization and mass spectrometry (LC-ESI-MS) to detect femtomole to picomole amounts of reporter molecules results in an ultrasensitive enzyme-linked immune mass spectrometric assay (ELIMSA). The novel ELIMSA substrate pyridoxamine-5-phosphate (PA5P) is cleaved by the enzyme alkaline phosphatase to yield the basic and hydrophilic product pyridoxamine (PA) that elutes rapidly with symmetrical peaks and a flat baseline. Pyridoxamine (PA) and (13)C PA were both observed to show a linear relationship between log ion intensity and quantity from picomole to femtomole amounts by liquid chromatography-electrospray ionization and mass spectrometry. Four independent methods, (i) internal (13)C isotope PA dilution curves, (ii) internal (13)C isotope one-point calibration, (iii) external PA standard curve, and (iv) external (13)C PA standard curve, all agreed within 1 digit in the same order of magnitude on the linear quantification of PA. Hence, a mass spectrometer can be used to robustly detect 526 ymol of the alkaline phosphatase streptavidin probe and accurately quantify zeptomole amounts of PSA against log linear absolute standard by micro electrospray on a simple ion trap. PMID:25259405

  14. Shp2 protein tyrosine phosphatase inhibitor activity of estramustine phosphate and its triterpenoid analogs

    PubMed Central

    Scott, Latanya M.; Chen, Liwei; Daniel, Kenyon G.; Brooks, Wesley H.; Guida, Wayne C.; Lawrence, Harshani R.; Sebti, Said M.; Lawrence, Nicholas J.; Wu, Jie

    2010-01-01

    Shp2 protein tyrosine phosphate (PTP) is a novel target for anticancer drug discovery. We identified estramustine phosphate as a Shp2 PTP inhibitor from the National Cancer Institute Approved Oncology Drug set. A focused structure-activity relationship study indicated that the 17- phosphate group is required for the Shp2 PTP inhibitor activity of estramustine phosphate. A search for estramustine phosphate analogs led to identification of two triperpenoids, enoxolone and celastrol, having Shp2 PTP inhibitor activity. With the previously reported PTP1B inhibitor trodusquemine, our study reveals steroids and triterpenoids with negatively charged phosphate, carboxylate, or sulfonate groups as novel pharmacophores of selective PTP inhibitors. PMID:21193311

  15. QM/MM analysis suggests that Alkaline Phosphatase (AP) and Nucleotide pyrophosphatase/phosphodiesterase slightly tighten the transition state for phosphate diester hydrolysis relative to solution: implication for catalytic promiscuity in the AP superfamily

    PubMed Central

    Hou, Guanhua

    2011-01-01

    Several members of the Alkaline Phosphatase (AP) superfamily exhibit a high level of catalytic proficiency and promiscuity in structurally similar active sites. A thorough characterization of the nature of transition state for different substrates in these enzymes is crucial for understanding the molecular mechanisms that govern those remarkable catalytic properties. In this work, we study the hydrolysis of a phosphate diester, MpNPP−, in solution, two experimentally well-characterized variants of AP (R166S AP, R166S/E322Y AP) and wild type Nucleotide pyrophosphatase/phosphodiesterase (NPP) by QM/MM calculations in which the QM method is an approximate density functional theory previously parameterized for phosphate hydrolysis (SCC-DFTBPR). The general agreements found between these calculations and available experimental data for both solution and enzymes support the use of SCC-DFTBPR/MM for a semi-quantitative analysis of the catalytic mechanism and nature of transition state in AP and NPP. Although phosphate diesters are cognate substrates for NPP but promiscuous substrates for AP, the calculations suggest that their hydrolysis reactions catalyzed by AP and NPP feature similar synchronous transition states that are slightly tighter in nature compared to that in solution, due in part to the geometry of the bimetallic zinc motif. Therefore, this study provides the first direct computational support to the hypothesis that enzymes in the AP superfamily catalyze cognate and promiscuous substrates via similar transition states to those in solution. Our calculations do not support the finding of recent QM/MM studies by López-Canut and coworkers, who suggested that the same diester substrate goes through a much looser transition state in NPP/AP than in solution, a result likely biased by the large structural distortion of the bimetallic zinc site in their simulations. Finally, our calculations for different phosphate diester orientations and phosphorothioate diesters

  16. Enhancing alkaline hydrogen evolution reaction activity through Ni-Mn3O4 nanocomposites.

    PubMed

    Li, Xu; Liu, Peng Fei; Zhang, Le; Zu, Meng Yang; Yang, Yun Xia; Yang, Hua Gui

    2016-08-18

    Developing efficient, stable and cost-effective electrocatalysts towards hydrogen production in alkaline environments is vital to improve energy efficiency for water splitting. In this work, we prepared Ni-Mn3O4 nanocomposites on Ni foam which exhibit an excellent hydrogen evolution reaction catalytic activity with a current density (j) of 10 mA cm(-2) at an overpotential (η) of 91 mV and show good stability in an alkaline medium. PMID:27500290

  17. Removal of lead from aqueous solutions by activated phosphate.

    PubMed

    Mouflih, M; Aklil, A; Sebti, S

    2005-03-17

    The potential of using activated phosphate as a new adsorbent for the removal of Pb from aqueous solutions was investigated. The kinetic of lead adsorption and the adsorption process were compared for natural phosphate (NP) and activated phosphate (AP). The results indicate that equilibrium was established in about 1h for NP and 3 h for AP. The effect of the pH was examined in the range 2-6. The maximum removal obtained is between two and three for NP and between three and four for AP. The maximum adsorption capacities at 25 degrees C are 155.04 and 115.34 mg/g for AP and NP, respectively. The effect of temperature has been carried out at 25, 35 and 45 degrees C. The data obtained from adsorption isotherms of lead at different temperatures fit to linear form of Langmuir adsorption equation. The thermodynamic parameters such as enthalpy (DeltaH), free energy (DeltaG) and entropy (DeltaS) were calculated. They show that adsorption of lead on NP and AP is an endothermic process more effective at high temperatures. These results show that AP is a good adsorbent for heavy metals from aqueous solutions and could be used as a purifier for water and wastewater. PMID:15752864

  18. Alkaline phosphatase activity and its relationship to inorganic phosphorus in the transition zone of the North-western African upwelling system

    NASA Astrophysics Data System (ADS)

    Sebastián, Marta; Arístegui, Javier; Montero, María F.; Escanez, Jose; Xavier Niell, F.

    2004-08-01

    The enzymatic activity of alkaline phosphatase (APA) was studied in the transition zone between the African upwelling system and the open ocean waters of the Canary Islands region. This region is recurrently dominated by the presence of upwelling filaments that may transport nutrient-enriched waters out into the open ocean before nutrients become exhausted by plankton. Turnover rates by APA were generally low in the whole region, but detectable in all the measurements carried out. On average, turnover rates were higher in the upwelling stations, and APA in those waters seemed to be mainly generated by heterotrophic bacteria to supply easily assimilable organic C. APA outside the upwelling area showed an inverse hyperbolic relationship with increasing phosphate, suggesting the presence of both constitutive and Pi-inducible APA. In these offshore waters, a threshold of 0.1 μM of phosphate could be defined for the regulatory function of Pi on APA. Thus, APA in nutrient-poor waters seemed to be induced to compensate for Pi-deficiency. Turnover rates in the filaments showed basal (probably constitutive) levels, whereas they increased in the surrounding waters, where phosphate concentration presumably did not satisfy plankton P-demands. The fertilising effect of the filaments and associated cyclonic eddies extended to at least 175 km offshore, where basal alkaline phosphatase activities were still found. The magnitude of this effect depends probably on the intensity of upwelling events and the degree of recirculation of filament water back to the coastal jet.

  19. DL-Buthionine-S,R-sulfoximine affects intestinal alkaline phosphatase activity.

    PubMed

    Marchionatti, A; Alisio, A; Díaz de Barboza, G; Baudino, V; Tolosa de Talamoni, N

    2001-06-01

    The susceptibility of intestinal alkaline phosphatase to DL-buthionine-S,R-sulfoximine was investigated in chicks fed a commercial diet. The results show that DL-buthionine-S,R-sulfoximine produced inhibition of intestinal alkaline phosphatase activity. This effect showed dose- and time-dependency and it was caused by either in vivo DL-buthionine-S,R- sulfoximine administration or in vitro DL-buthionine-S,R-sulfoximine incubation with villus tip enterocytes. DL-Buthionine-S,R-sulfoximine did not act directly on intestinal alkaline phosphatase but it provoked glutathione depletion which led to changes in the redox state of the enterocyte as shown by the production of free hydroxyl radicals and an incremental increase in the carbonyl content of proteins. The reversibility of the buthionine sulfoximine effect on intestinal alkaline phosphatase was proved by addition of glutathione monoester to the duodenal loop. PMID:11423381

  20. Alkaline pH activates the transport activity of GLUT1in L929 fibroblast cells

    PubMed Central

    Gunnink, Stephen M.; Kerk, Samuel A.; Kuiper, Benjamin D.; Alabi, Ola D.; Kuipers, David P.; Praamsma, Riemer C.; Wrobel, Kathryn E.; Louters, Larry L.

    2016-01-01

    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

  1. Alkaline pH activates the transport activity of GLUT1 in L929 fibroblast cells.

    PubMed

    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

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

    PubMed Central

    Villa-Bellosta, Ricardo; González-Parra, Emilio; Egido, Jesús

    2016-01-01

    Background 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. Methods and Findings 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. Conclusion 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

  3. Peroxymonosulfate activation by phosphate anion for organics degradation in water.

    PubMed

    Lou, Xiaoyi; Wu, Liuxi; Guo, Yaoguang; Chen, Chuncheng; Wang, Zhaohui; Xiao, Dongxue; Fang, Changling; Liu, Jianshe; Zhao, Jincai; Lu, Shuyu

    2014-12-01

    Activation of peroxygens is a critical method to generate oxidative species, but often consumes additional chemical reagents and/or energy. Here we report a novel and efficient activation reaction for peroxymonosulfate (PMS) by phosphate anions (PBS). The PBS/PMS coupled system, at neutral pH, is able to decompose efficiently even mineralize a variety of organic pollutants, such as Acid Orange 7, Rhodamine B and 2,4,6-trichlorophenol. In contrast, no measurable degradation was observed when the PMS was replaced by other peroxygens (i.e. hydrogen peroxide and peroxydisulfate). Both PMS and PBS are indispensable for the oxidative degradation of pollutants. Increasing pH and concentrations of PMS and PBS significantly accelerate the degradation of organics. It is proposed that OH would be the major radical for contamination degradation at pH 7.0 through the radical quenching experiments. This work provides a new way of PMS activation for decontamination at neutral pH, in particular for phosphate-rich wastewater treatment. PMID:25303463

  4. Dependence of the spectral properties of alkaline-free alumino-phosphate glasses on chemical changes during synthesis

    SciTech Connect

    Antonenko, N.I.; Abdrashitova, E.I.; Artomova, M.V.

    1986-07-01

    In order to obtain information about glass microstructure within the first coordination sphere, glasses are irradiated with ionizing radiation. Doses of gamma-radiation not exceeding 10/sup 4/-10/sup 5/ Gy generally produce paramagnetic (PO/sub 4/)/sup 2 -/ radicals and more rarely (PO/sub 3/)/sup 2 -/ in phosphate glasses. This paper shows that in glasses of the same composition structural units of the same composition can exist, but with different chemical bonding depending on the chemical changes occurring during synthesis. Glass samples and batches were irradiated with a Co 60 source at room temperature and the ses were 10/sup 4/ Gy.

  5. Treatment of Hypophosphatemic Rickets with Phosphate and Active Vitamin D in Japan: A Questionnaire-based Survey

    PubMed Central

    Fujiwara, Makoto; Namba, Noriyuki; Ozono, Keiichi; Arisaka, Osamu; Yokoya, Susumu

    2013-01-01

    Hereditary hypophosphatemic rickets represented by X-linked hypophosphatemic rickets (XLH) is a rare disorder characterized by hypophosphatemia, elevated alkaline phosphatase (ALP) and undermineralization of bone. Active vitamin D and phosphate are administered to correct hypophosphatemia and elevation of ALP. Overtreatment with phosphate leads to secondary hyperparathyroidism, and a large dose of active vitamin D has a risk of hypercalciuria. To understand the situation concerning treatment of patients with hereditary hypophosphatemic rickets in Japan, we conducted a questionnaire survey of pediatric endocrinologists. Answers were obtained from 53 out of 68 hospitals where the pediatric endocrinologists worked. One hundred and thirty-five patients were treated in 28 hospitals during November 2009 and May 2010; 126 patients suffered from hereditary hypophosphatemic rickets, and 9 had hypophosphatemia caused by other miscellaneous reasons. The distribution of patient age was as follows: 27 (21%) were between 6 mo and 6 yr of age, 39 (31%) were between 6 and 12 yr of age, and 60 (48%) were more than 12 yr of age. Active vitamin D was given to 123 patients, and phosphate was given to 106 patients. As for the dose of phosphorus, 37.2–58.1 mg/kg/d was given divided into 2 to 6 aliquots. There were various control targets of treatment, including serum phosphate, serum ALP, rachitic change, urinary Ca/Cr, parathyroid hormone and growth. It is very important to avoid side effects of these treatments. No evidence is available about the optimal dose of phosphate or number of administrations in the treatment of patients with hypophosphatemic rickets. Although there is a recommendation for clinical management of patients with hypophosphatemic rickets, we should set a clinical guideline for it in Japan. PMID:23966755

  6. Yam (Dioscorea batatas) Root and Bark Extracts Stimulate Osteoblast Mineralization by Increasing Ca and P Accumulation and Alkaline Phosphatase Activity

    PubMed Central

    Kim, Suji; Shin, Mee-Young; Son, Kun-Ho; Sohn, Ho-Yong; Lim, Jae-Hwan; Lee, Jong-Hwa; Kwun, In-Sook

    2014-01-01

    Yam (Dioscorea batatas) is widely consumed as functional food for health promotion mainly in East Asia countries. We assessed whether yam root (tuber) or bark (peel) extracts stimulated the activity of osteoblasts for osteogenesis. MC3T3-E1 cells (mouse osteoblasts) were treated with yam root extracts (water or methanol) (study I) or bark extracts (water or hexane) (study II) within 0~10 μg/mL during the periods of osteoblast proliferation (5~10 day), matrix maturation (11~15 day) and mineralization (16~20 day) as appropriate. In study I, both yam root water and methanol extracts increased cell proliferation as concentration-dependent manner. Cellular collagen synthesis and alkaline phosphatase (ALP) activity, both the indicators of bone matrix protein and inorganic phosphate production for calcification respectively, were also increased by yam root water and methanol extract. Osteoblast calcification as cell matrix Ca and P accumulation was also increased by the addition of yam root extracts. In study II, yam bark extracts (water and hexane) increased osteoblast proliferation and differentiation, as collagen synthesis and ALP activity and osteoblast matrix Ca and P deposition. The study results suggested that both yam root and bark extracts stimulate osteogenic function in osteoblasts by stimulating bone matrix maturation by increasing collagen synthesis, ALP activity, and matrix mineralization. PMID:25320717

  7. Bactericidal activity of alkaline salts of fatty acids towards bacteria associated with poultry processing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Antibacterial activity of alkaline salts of caproic, caprylic, capric, lauric, and myristic acids were determined using the agar diffusion assay. A 0.5M concentration of each fatty acid (FA) was dissolved in 1.0 M potassium hydroxide (KOH), and pH of the mixtures was adjusted to 10.5 with citric aci...

  8. Proton/Phosphate Stoichiometry in Uptake of Inorganic Phosphate by Cultured Cells of Catharanthus roseus (L.) G. Don

    PubMed Central

    Sakano, Katsuhiro

    1990-01-01

    Upon absorption of phosphate, cultured cells of Catharanthus roseus (L.) G. Don caused a rapid alkalinization of the medium in which they were suspended. The alkalinization continued until the added phosphate was completely exhausted from the medium, at which time the pH of the medium started to drop sharply toward the original pH value. Phosphate exposure caused the pH of the medium to increase from pH 3.5 to values as high as 5.8, while the rate of phosphate uptake was constant throughout (10-17 micromoles per hour per gram fresh weight). This indicates that no apparent pH optimum exists for the phosphate uptake by the cultured cells. The amount of protons cotransported with phosphate was calculated from the observed pH change up to the maximum alkalinization and the titration curve of the cell suspension. Proton/phosphate transport stoichiometry ranged from less than unity to 4 according to the amount of phosphate applied. At low phosphate doses, the stoichiometries were close to 4, while at high phosphate doses, smaller stoichiometries were observed. This suggests that, at high phosphate doses, activation of the proton pump is induced by the longer lasting proton influx acidifying the cytoplasm. The increased H+ efflux due to the proton pump could partially compensate protons taken up via the proton-phosphate cotransport system. Thus, the H+/H2PO4− stoichiometry of the cotransport is most likely to be 4. PMID:16667491

  9. Transketolase activity modulates glycerol-3-phosphate levels in Escherichia coli.

    PubMed

    Vimala, A; Harinarayanan, R

    2016-04-01

    Transketolase activity provides an important link between the metabolic pathways of glycolysis and pentose phosphate shunt and catalyzes inter-conversions between pentose phosphates and glycolytic intermediates. It is widely conserved in life forms. A genetic screen for suppression of the growth defect of Escherichia coli tktA tktB mutant in LB medium revealed two mutations, one that rendered the glpK expression constitutive and another that inactivated deoB. Characterizing these mutations aided in uncovering the role of ribose-5-P (a transketolase substrate) as an inhibitor of glycerol assimilation and de novo glycerol-3-P synthesis. Using lacZ fusions, we show that ribose-5-P enhances GlpR-mediated repression of the glpFKX operon and inhibits glycerol assimilation. Electrophoretic Mobility Shift Assay (EMSA) showed ribose-5-P made the DNA-GlpR complex less sensitive to the inducer glycerol-3-P. In addition to inhibition of glycerol assimilation, obstruction of ribose-5-P metabolism retards growth from glycerol-3-P limitation. Glucose helps to overcome this limitation through a mechanism involving catabolite repression. To our knowledge, this report is the first to show ribose-5-P can modulate glycerol-3-P concentration in the cell by regulation of glycerol assimilation as well as its de novo synthesis. This regulation could be prevalent in other organisms. PMID:26691989

  10. Osteogenic activity of cyclodextrin-encapsulated doxycycline in a calcium phosphate PCL and PLGA composite.

    PubMed

    Trajano, V C C; Costa, K J R; Lanza, C R M; Sinisterra, R D; Cortés, M E

    2016-07-01

    Composites of biodegradable polymers and calcium phosphate are bioactive and flexible, and have been proposed for use in tissue engineering and bone regeneration. When associated with the broad-spectrum antibiotic doxycycline (DOX), they could favor antimicrobial action and enhance the action of osteogenic composites. Composites of polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), and a bioceramic of biphasic calcium phosphate Osteosynt® (BCP) were loaded with DOX encapsulated in β-cyclodextrin (βCD) and were evaluated for effects on osteoblastic cell cultures. The DOX/βCD composite was prepared with a double mixing method. Osteoblast viability was assessed with methyl tetrazolium (MTT) assays after 1day, 7day, and 14days of composite exposure; alkaline phosphatase (AP) activity and collagen production were evaluated after 7days and 14days, and mineral nodule formation after 14days. Composite structures were evaluated by scanning electron microscopy (SEM). Osteoblasts exposed to the composite containing 25μg/mL DOX/βCD had increased cell proliferation (p<0.05) compared to control osteoblast cultures at all experimental time points, reaching a maximum in the second week. AP activity and collagen secretion levels were also elevated in osteoblasts exposed to the DOX/βCD composite (p<0.05 vs. controls) and reached a maximum after 14days. These results were corroborated by Von Kossa test results, which showed strong formation of mineralization nodules during the same time period. SEM of the composite material revealed a surface topography with pore sizes suitable for growing osteoblasts. Together, these results suggest that osteoblasts are viable, proliferative, and osteogenic in the presence of a DOX/βCD-containing BCP ceramic composite. PMID:27127066

  11. Cold-active alkaline phosphatase is irreversibly transformed into an inactive dimer by low urea concentrations.

    PubMed

    Hjörleifsson, Jens Guðmundur; Ásgeirsson, Bjarni

    2016-07-01

    Alkaline phosphatase is a homodimeric metallo-hydrolase where both Zn(2+) and Mg(2+) are important for catalysis and stability. Cold-adapted alkaline phosphatase variants have high activity at low temperatures and lower thermal stability compared with variants from mesophilic hosts. The instability, and thus inactivation, could be due to loose association of the dimers and/or loosely bound Mg(2)(+) in the active site, but this has not been studied in detail for the cold-adapted variants. Here, we focus on using the intrinsic fluorescence of Trp in alkaline phosphatase from the marine bacterium Vibrio splendidus (VAP) to probe for dimerization. Trp→Phe substitutions showed that two out of the five native Trp residues contributed mostly to the fluorescence emission. One residue, 15Å away from the active site (W460) and highly solvent excluded, was phosphorescent and had a distant role in substrate binding. An additional Trp residue was introduced to the dimer interface to act as a possible probe for dimerization. Urea denaturation curves indicated that an inactive dimer intermediate, structurally equivalent to the native state, was formed before dimer dissociation took place. This is the first example of the transition of a native dimer to an inactive dimer intermediate for alkaline phosphatase without using mutagenesis, ligands, or competitive inhibition. PMID:27043172

  12. Coordination sphere of the third metal site is essential to the activity and metal selectivity of alkaline phosphatases.

    PubMed

    Koutsioulis, Dimitris; Lyskowski, Andrzej; Mäki, Seija; Guthrie, Ellen; Feller, Georges; Bouriotis, Vassilis; Heikinheimo, Pirkko

    2010-01-01

    Alkaline phosphatases (APs) are commercially applied enzymes that catalyze the hydrolysis of phosphate monoesters by a reaction involving three active site metal ions. We have previously identified H135 as the key residue for controlling activity of the psychrophilic TAB5 AP (TAP). In this article, we describe three X-ray crystallographic structures on TAP variants H135E and H135D in complex with a variety of metal ions. The structural analysis is supported by thermodynamic and kinetic data. The AP catalysis essentially requires octahedral coordination in the M3 site, but stability is adjusted with the conformational freedom of the metal ion. Comparison with the mesophilic Escherichia coli, AP shows differences in the charge transfer network in providing the chemically optimal metal combination for catalysis. Our results provide explanation why the TAB5 and E. coli APs respond in an opposite way to mutagenesis in their active sites. They provide a lesson on chemical fine tuning and the importance of the second coordination sphere in defining metal specificity in enzymes. Understanding the framework of AP catalysis is essential in the efforts to design even more powerful tools for modern biotechnology. PMID:19916164

  13. Somatic cell count and alkaline phosphatase activity in milk for evaluation of mastitis in buffalo

    PubMed Central

    Patil, M. P.; Nagvekar, A. S.; Ingole, S. D.; Bharucha, S. V.; Palve, V. T.

    2015-01-01

    Background and Aim: Mastitis is a serious disease of dairy animals causing great economic losses due to a reduction in milk yield as well as lowering its nutritive value. The application of somatic cell count (SCC) and alkaline phosphatase activity in the milk for diagnosis of mastitis in buffalo is not well documented. Therefore, the present study was conducted to observe the SCC and alkaline phosphatase activity for evaluation of mastitis in buffalo. Materials and Methods: Milk samples of forty apparently healthy lactating buffaloes were selected and categorized into five different groups viz. normal buffaloes, buffaloes with subclinical mastitis with CMT positive milk samples (+1 Grade), (+2 Grade), (+3 Grade), and buffaloes with clinical mastitis with 8 animals in each group. The milk samples were analyzed for SCC and alkaline phosphatase activity. Results: The levels of SCC (×105 cells/ml) and alkaline phosphatase (U/L) in different groups were viz. normal (3.21±0.179, 16.48±1.432), subclinical mastitis with CMT positive milk samples with +1 Grade (4.21±0.138, 28.11±1.013), with +2 Grade (6.34±0.183, 34.50±1.034), with +3 Grade (7.96±0.213, 37.73±0.737) and buffaloes with clinical mastitis (10.21±0.220, 42.37±0.907) respectively, indicating an increasing trend in the values and the difference observed among various group was statistically significant. Conclusion: In conclusion, the results of the present study indicate that the concentration of milk SCC and alkaline phosphatase activity was higher in the milk of buffaloes with mastitis than in the milk of normal buffaloes. PMID:27047098

  14. Catalytically active alkaline molten globular enzyme: Effect of pH and temperature on the structural integrity of 5-aminolevulinate synthase.

    PubMed

    Stojanovski, Bosko M; Breydo, Leonid; Hunter, Gregory A; Uversky, Vladimir N; Ferreira, Gloria C

    2014-12-01

    5-Aminolevulinate synthase (ALAS), a pyridoxal-5'phosphate (PLP)-dependent enzyme, catalyzes the first step of heme biosynthesis in mammals. Circular dichroism (CD) and fluorescence spectroscopies were used to examine the effects of pH (1.0-3.0 and 7.5-10.5) and temperature (20 and 37°C) on the structural integrity of ALAS. The secondary structure, as deduced from far-UV CD, is mostly resilient to pH and temperature changes. Partial unfolding was observed at pH2.0, but further decreasing pH resulted in acid-induced refolding of the secondary structure to nearly native levels. The tertiary structure rigidity, monitored by near-UV CD, is lost under acidic and specific alkaline conditions (pH10.5 and pH9.5/37°C), where ALAS populates a molten globule state. As the enzyme becomes less structured with increased alkalinity, the chiral environment of the internal aldimine is also modified, with a shift from a 420nm to 330nm dichroic band. Under acidic conditions, the PLP cofactor dissociates from ALAS. Reaction with 8-anilino-1-naphthalenesulfonic acid corroborates increased exposure of hydrophobic clusters in the alkaline and acidic molten globules, although the reaction is more pronounced with the latter. Furthermore, quenching the intrinsic fluorescence of ALAS with acrylamide at pH1.0 and 9.5 yielded subtly different dynamic quenching constants. The alkaline molten globule state of ALAS is catalytically active (pH9.5/37°C), although the kcat value is significantly decreased. Finally, the binding of 5-aminolevulinate restricts conformational fluctuations in the alkaline molten globule. Overall, our findings prove how the structural plasticity of ALAS contributes to reaching a functional enzyme. PMID:25240868

  15. Development of New Cementitious Caterials by Alkaline Activating Industrial by-Products

    NASA Astrophysics Data System (ADS)

    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.

  16. The Potential of Soft Soil Improvement Through a Coupled Technique Between Electro Kinetic and Alkaline Activation of Soft Soil

    NASA Astrophysics Data System (ADS)

    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.

  17. Cycling of Dissolved Organic Phosphorus and Alkaline Phosphatase Activity in Euphotic Zone of the Western North Pacific

    NASA Astrophysics Data System (ADS)

    Suzumura, M.

    2010-12-01

    Phosphorus is an essential nutrient for marine organisms. In oligotrophic environments, concentrations of dissolved inorganic phosphate (SRP), the most bioavailable form of phosphorus, are low and have been hypothesized to constrain the primary productivity. Evidence has been found that dissolved organic phosphorus (DOP) supports a significant fraction of primary production through hydrolytic remineralization of DOP to SRP by alkaline phosphatase (APA). In this study, DOP biogeochemistry was investigated at three locations of the open-ocean environment in the Kuroshio region and at a semi-eutrophic coastal site of the western North Pacific. Concentrations of SRP, DOP and hydrolyzable ester-P were measured in the euphotic zone. Kinetic parameters of APA were determined using a fluorogenic substrate, including potential maximum velocity (Vmax), apparent Michaelis-Menten half-saturation constant (Km), and turnover time (TA) of APA hydrolyzable DOP. SRP concentrations were quite low (≤ 10 nM) in the surface seawater and rapidly increased below the chlorophyll a maximum layer (CML). DOP concentration ranged from 29 to 223 nM. Above the CML, DOP composed a major fraction accounting for 60-100% of dissolved total P. A significant linear relationship was found between the concentrations of SRP and hydrolyzable ester-P (R2 = 0.83, P < 0.01). This suggests active utilization of ester-P under phosphate-depleted conditions. In the Kuroshio region, Vmax of APA exhibited the highest value at the surface water (0 m) and decreased rapidly with depth, while at the coastal site the peak value was found at CML. TA of hydrolyzable DOP was quite variable among the locations and increased with depth especially below CML. The estimated values of in situ hydrolysis rate were much lower (2-34%) than the potential Vmax which was determined with the addition of an excess amount of the substrate. The results suggest that marine microbes can efficiently and rapidly utilize hydrolyzable DOP

  18. Competitive, uncompetitive, and mixed inhibitors of the alkaline phosphatase activity associated with the isolated brush border membrane of the tapeworm Hymenolepis diminuta.

    PubMed

    Pappas, P W; Leiby, D A

    1989-06-01

    Several compounds were tested as inhibitors of the alkaline phosphatase (AlkPase) activity associated with the isolated brush border membrane of the tapeworm, Hymenolepis diminuta. Molybdate, arsenate, arsenite and beta-glycerophosphate (BGP) were competitive inhibitors of the hydrolysis of p-nitrophenyl phosphate, while levamisole and clorsulon were uncompetitive and mixed inhibitors, respectively. Molybdate was also a competitive inhibitor of the hydrolysis of BGP and 5'-adenosine monophosphate, and levamisole was an uncompetitive inhibitor of BGP hydrolysis. The apparent inhibitor constants (Ki') for molybdate and levamisole were virtually identical regardless of the substrate, and these data support the hypothesis that the AlkPase activity is represented by a single membrane-bound enzyme with low substrate specificity. Quinacrine, Hg2+, and ethylenediaminetetraacetate were also potent inhibitors of the AlkPase activity, but the mechanisms by which these latter three inhibitors function were not clear. PMID:2768348

  19. Diversity and activity of methanotrophs in alkaline soil from a Chinese coal mine.

    PubMed

    Han, Bing; Chen, Yin; Abell, Guy; Jiang, Hao; Bodrossy, Levente; Zhao, Jiangang; Murrell, J Colin; Xing, Xin-Hui

    2009-11-01

    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 (13)CH(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. PMID:19515201

  20. Common Variants at Putative Regulatory Sites of the Tissue Nonspecific Alkaline Phosphatase Gene Influence Circulating Pyridoxal 5′-Phosphate Concentration in Healthy Adults123

    PubMed Central

    Carter, Tonia C; Pangilinan, Faith; Molloy, Anne M; Fan, Ruzong; Wang, Yifan; Shane, Barry; Gibney, Eileen R; Midttun, Øivind; Ueland, Per M; Cropp, Cheryl D; Kim, Yoonhee; Wilson, Alexander F; Bailey-Wilson, Joan E; Brody, Lawrence C; Mills, James L

    2015-01-01

    Background: Vitamin B-6 interconversion enzymes are important for supplying pyridoxal 5′-phosphate (PLP), the co-enzyme form, to tissues. Variants in the genes for these enzymes [tissue nonspecific alkaline phosphatase (ALPL), pyridoxamine 5′-phosphate oxidase, pyridoxal kinase, and pyridoxal phosphatase] could affect enzyme function and vitamin B-6 status. Objectives: We tested whether single-nucleotide polymorphisms (SNPs) in these genes influence vitamin B-6 status markers [plasma PLP, pyridoxal (PL), and 4-pyridoxic acid (PA)], and explored potential functional effects of the SNPs. Methods: Study subjects were young, healthy adults from Ireland (n = 2345). We measured plasma PLP, PL, and PA with liquid chromatography–tandem mass spectrometry and genotyped 66 tag SNPs in the 4 genes. We tested for associations with single SNPs in candidate genes and also performed genome-wide association study (GWAS) and gene-based analyses. Results: Seventeen SNPs in ALPL were associated with altered plasma PLP in candidate gene analyses (P < 1.89 × 10−4). In the GWAS, 5 additional ALPL SNPs were associated with altered plasma PLP (P < 5.0 × 10−8). Gene-based analyses that used the functional linear model β-spline (P = 4.04 × 10−15) and Fourier spline (P = 5.87 × 10−15) methods also showed associations between ALPL and altered plasma PLP. No SNPs in other genes were associated with plasma PLP. The association of the minor CC genotype of 1 ALPL SNP, rs1256341, with reduced ALPL expression in the HapMap Northern European ancestry population is consistent with the positive association between the CC genotype and plasma PLP in our study (P = 0.008). No SNP was associated with altered plasma PL or PA. Conclusions: In healthy adults, common variants in ALPL influence plasma PLP concentration, the most frequently used biomarker for vitamin B-6 status. Whether these associations are indicative of functional changes in vitamin B-6 status requires more investigation

  1. Evidence for alkaline igneous activity and associated metasomatism in the Reelfoot rift, south-central Midcontinent, U. S. A

    SciTech Connect

    Goldhaber, M.B.; Diehl, S.F.; Sutley, S.J. ); Flohr, M.J.K. )

    1993-03-01

    Alkaline igneous magmatism is commonly associated with intracontinental rifts such as the Reelfoot rift (RR). Direct evidence for alkaline magmatism in the area of the RR occurs as lamprophyre and syenite encountered in deep wells. The authors' new studies of lamprophyres and sedimentary rocks from wells in the region provide additional examples of alkaline magmatism and emphasize the effects of related metasomatism. Sedimentary rocks in the Dow Chemical No. 1 Garrigan well, which is not known to contain lamprophyre dikes, probably also were metasomatically altered, as they contain authigenic fluorapatite, Ce-phosphates, and other REE-rich minerals. Enrichments of incompatible and large ion lithophile elements commonly associated with alkaline magmatism occur in the New Madrid test well, near the crest of the Pascola Arch. The carbonate-free fraction of Paleozoic rocks in this well is highly enriched in Nb (500 ppm), Ba (> 5,000 ppm), La (500 ppm), Th (1,000 ppm), and F (2,400 ppm). Abundant inclusion-rich potassium-feldspar cement in a nearby well may also be the result of alkaline metasomatism. Fluorite and elevated F concentrations are found in several wells in the RR, and contrast with stratigraphically correlative platform carbonates of the Ozark uplift, which lack F enrichment. Well and spring water samples above the RR are enriched in fluorine (as much as 5,000 ppb) compared to samples away from the rift which typically have concentrations two orders of magnitude smaller. The data and observations are consistent with relatively widespread alkaline metasomatism, which was associated with the intrusion of alkaline magmas in the RR.

  2. Characterization of a novel low-temperature-active, alkaline and sucrose-tolerant invertase

    PubMed Central

    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

  3. Characterization of a novel low-temperature-active, alkaline and sucrose-tolerant invertase.

    PubMed

    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

  4. Conditional iron and pH-dependent activity of a non-enzymatic glycolysis and pentose phosphate pathway

    PubMed Central

    Keller, Markus A.; Zylstra, Andre; Castro, Cecilia; Turchyn, Alexandra V.; Griffin, Julian L.; Ralser, Markus

    2016-01-01

    Little is known about the evolutionary origins of metabolism. However, key biochemical reactions of glycolysis and the pentose phosphate pathway (PPP), ancient metabolic pathways central to the metabolic network, have non-enzymatic pendants that occur in a prebiotically plausible reaction milieu reconstituted to contain Archean sediment metal components. These non-enzymatic reactions could have given rise to the origin of glycolysis and the PPP during early evolution. Using nuclear magnetic resonance spectroscopy and high-content metabolomics that allowed us to measure several thousand reaction mixtures, we experimentally address the chemical logic of a metabolism-like network constituted from these non-enzymatic reactions. Fe(II), the dominant transition metal component of Archean oceanic sediments, has binding affinity toward metabolic sugar phosphates and drives metabolism-like reactivity acting as both catalyst and cosubstrate. Iron and pH dependencies determine a metabolism-like network topology and comediate reaction rates over several orders of magnitude so that the network adopts conditional activity. Alkaline pH triggered the activity of the non-enzymatic PPP pendant, whereas gentle acidic or neutral conditions favored non-enzymatic glycolytic reactions. Fe(II)-sensitive glycolytic and PPP-like reactions thus form a chemical network mimicking structural features of extant carbon metabolism, including topology, pH dependency, and conditional reactivity. Chemical networks that obtain structure and catalysis on the basis of transition metals found in Archean sediments are hence plausible direct precursors of cellular metabolic networks. PMID:26824074

  5. Phosphoinositide and Inositol Phosphate Analysis in Lymphocyte Activation

    PubMed Central

    Sauer, Karsten; Huang, Yina Hsing; Lin, Hongying; Sandberg, Mark; Mayr, Georg W.

    2015-01-01

    Lymphocyte antigen receptor engagement profoundly changes the cellular content of phosphoinositide lipids and soluble inositol phosphates. Among these, the phosphoinositides phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) play key signaling roles by acting as pleckstrin homology (PH) domain ligands that recruit signaling proteins to the plasma membrane. Moreover, PIP2 acts as a precursor for the second messenger molecules diacylglycerol and soluble inositol 1,4,5-trisphosphate (IP3), essential mediators of PKC, Ras/Erk, and Ca2+ signaling in lymphocytes. IP3 phosphorylation by IP3 3-kinases generates inositol 1,3,4,5-tetrakisphosphate (IP4), an essential soluble regulator of PH domain binding to PIP3 in developing T cells. Besides PIP2, PIP3, IP3, and IP4, lymphocytes produce multiple other phosphoinositides and soluble inositol phosphates that could have important physiological functions. To aid their analysis, detailed protocols that allow one to simultaneously measure the levels of multiple different phosphoinositide or inositol phosphate isomers in lymphocytes are provided here. They are based on thin layer, conventional and high-performance liquid chromatographic separation methods followed by radiolabeling or non-radioactive metal-dye detection. Finally, less broadly applicable nonchromatographic methods for detection of specific phosphoinositide or inositol phosphate isomers are discussed. Support protocols describe how to obtain pure unstimulated CD4+CD8+ thymocyte populations for analyses of inositol phosphate turnover during positive and negative selection, key steps in T cell development. PMID:19918943

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

    NASA Astrophysics Data System (ADS)

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

    2013-12-01

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

  7. Lithium Iron Phosphate Cell Performance Evaluations for Lunar Extravehicular Activities

    NASA Technical Reports Server (NTRS)

    Reid, Concha

    2007-01-01

    Lithium-ion battery cells are being evaluated for their ability to provide primary power and energy storage for NASA s future Exploration missions. These missions include the Orion Crew Exploration Vehicle, the Ares Crew Launch Vehicle Upper Stage, Extravehicular Activities (EVA, the advanced space suit), the Lunar Surface Ascent Module (LSAM), and the Lunar Precursor and Robotic Program (LPRP), among others. Each of these missions will have different battery requirements. Some missions may require high specific energy and high energy density, while others may require high specific power, wide operating temperature ranges, or a combination of several of these attributes. EVA is one type of mission that presents particular challenges for today s existing power sources. The Portable Life Support System (PLSS) for the advanced Lunar surface suit will be carried on an astronaut s back during eight hour long sorties, requiring a lightweight power source. Lunar sorties are also expected to occur during varying environmental conditions, requiring a power source that can operate over a wide range of temperatures. Concepts for Lunar EVAs include a primary power source for the PLSS that can recharge rapidly. A power source that can charge quickly could enable a lighter weight system that can be recharged while an astronaut is taking a short break. Preliminary results of Al23 Ml 26650 lithium iron phosphate cell performance evaluations for an advanced Lunar surface space suit application are discussed in this paper. These cells exhibit excellent recharge rate capability, however, their specific energy and energy density is lower than typical lithium-ion cell chemistries. The cells were evaluated for their ability to provide primary power in a lightweight battery system while operating at multiple temperatures.

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

    NASA Astrophysics Data System (ADS)

    Mamatha, S. S.; Malik, Ashish; Varik, Sandesh; Parvathi, V.; Jineesh, V. K.; Gauns, Mangesh U.; LokaBharathi, P. A.

    2015-01-01

    The realization of the potential importance of phosphorus (P) as a limiting nutrient in marine ecosystem is increasing globally. Hence, the contribution of biotic variables in mobilizing this nutrient would be relevant especially in productive coastal waters. As alkaline phosphatase activity (APA) indicates the status of P for primary production in aquatic environments, we asked the following question: is the level of APA indicative of P sufficiency or deficiency in coastal waters, especially, where upwelling is a regular phenomenon? Therefore, we have examined the total APA, chlorophyll a along with phosphatase producing bacteria (PPB) and related environmental parameters from nearshore to offshore in coastal waters off Trivandrum and Kochi regions differently affected by upwelling during the onset of monsoon. Off Trivandrum, APA in the offshore waters of 5-m layer at 2.23 μM P h- 1 was > 4 times higher than nearshore. Thus, low APA could be indicative of P sufficiency in coastal waters and higher activity suggestive of deficiency in offshore waters off Trivandrum. In contrast, there was less difference in APA between near and offshore surface waters off Kochi. Our results show that the regions differently affected by upwelling respond differently according to ambient P concentration, distance from shore or depth of water. These observations could apparently be applicable to other coastal systems as well, where gradients in upwelling and phosphate runoff have been noticed. Further studies on other transects would throw more light on the extent and direction of the relationship between APA and ambient P concentration. Such studies would help in understanding the level of control of this nutrient on the productivity of coastal waters.

  9. alpha-Tocopheryl phosphate – an active lipid mediator?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The vitamin E (alpha-tocopherol, alphaT) derivative, alpha-tocopheryl phosphate (alphaTP), is detectable in small amounts in plasma, tissues, and cultured cells. Studies done in vitro and in vivo suggest that alphaT can become phosphorylated and alphaTP dephosphorylated, suggesting the existence of ...

  10. Non-precious metal electrocatalysts with high activity for hydrogen oxidation reaction in alkaline electrolytes

    SciTech Connect

    Sheng, WC; Bivens, AP; Myint, M; Zhuang, ZB; Forest, RV; Fang, QR; Chen, JG; Yan, YS

    2014-05-01

    A ternary metallic CoNiMo catalyst is electrochemically deposited on a polycrystalline gold (Au) disk electrode using pulse voltammetry, and characterized for hydrogen oxidation reaction (HOR) activity by temperature-controlled rotating disk electrode measurements in 0.1 M potassium hydroxide (KOH). The catalyst exhibits the highest HOR activity among all non-precious metal catalysts (e.g., 20 fold higher than Ni). At a sufficient loading, the CoNiMo catalyst is expected to outperform Pt and thus provides a promising low cost pathway for alkaline or alkaline membrane fuel cells. Density functional theory (DFT) calculations and parallel H-2-temperature programmed desorption (TPD) experiments on structurally much simpler model alloy systems show a trend that CoNiMo has a hydrogen binding energy (HBE) similar to Pt and much lower than Ni, suggesting that the formation of multi-metallic bonds modifies the HBE of Ni and is likely a significant contributing factor for the enhanced HOR activity.

  11. Stabilization of Different Types of Transition States in a Single Enzyme Active Site: QM/MM Analysis of Enzymes in the Alkaline Phosphatase Superfamily

    PubMed Central

    Hou, Guanhua; Cui, Qiang

    2013-01-01

    The first step for the hydrolysis of a phosphate monoester (pNPP2−) in enzymes of the alkaline phosphatase (AP) superfamily, R166S AP and wild type NPP, is studied using QM/MM simulations based on an approximate density functional theory (SCC-DFTBPR) and a recently introduced QM/MM interaction Hamiltonian. The calculations suggest that similar loose transition states are involved in both enzymes, despite the fact that phosphate monoesters are the cognate substrates for AP but promiscuous substrates for NPP. The computed loose transition states are clearly different from the more synchronous ones previously calculated for diester reactions in the same AP enzymes. Therefore, our results explicitly support the proposal that AP enzymes are able to recognize and stabilize different types of transition states in a single active site. Analysis of the structural features of computed transition states indicates that the plastic nature of the bi-metallic site plays a minor role in accommodating multiple types of transition states, and that the high degree of solvent accessibility of the AP active site also contributes to its ability to stabilize diverse transition state structures without the need of causing large structural distortions of the bimetallic motif. The binding mode of the leaving group in the transition state highlights that vanadate may not always be an ideal transition state analog for loose phosphoryl transfer transition states. PMID:23786365

  12. Defective Multilayer Carbon Nanotubes Increase Alkaline Phosphatase Activity and Bone-Like Nodules in Osteoblast Cultures.

    PubMed

    Zancanela, Daniela Cervelle; Simaã, Ana Maria Sper; Matsubara, Elaine Yoshiko; Rosolen, José Maurício; Ciancaglini, Pietro

    2016-02-01

    Carbon nanotubes (CNT) is one of the most studied biomaterials, and issues about its cytotoxicity remain. The objective of our study was to investigate the in vitro influence of defective CNT on culture growth and on the formation of mineralized matrix nodules by primary osteoblastic cells grown in plastic or titanium (Ti) surfaces. Cellular viability, alkaline phosphatase activity and formation of mineral nodules were evaluated, besides the CNT characterization tests. The CNT studies showed better cell viability for osteoblasts incubated at stationary phase of culture in the presence of Ti (about 70%), but for the other phases, the cells suffered a significant reduction in viability. A peak of maximum alkaline phosphatase activity in the intermediate stage of growth (14 days of culture), which is characteristic for osteoblasts, was not affected, regardless of the presence of Ti or combination of CNT and Ti. Mineralized matrix nodules grew much more when the cells were incubated with CNT in the last 2 phases than when incubated in the first week, mainly when the cultures were grown on Ti discs. This study provides information for the application of CNT associated or not with Ti in processes of mineralization biostimulation. PMID:27433601

  13. Biological Activities of a Mixture of Biosurfactant from Bacillus subtilis and Alkaline Lipase from Fusarium oxysporum

    PubMed Central

    Pereira de Quadros, Cedenir; Cristina Teixeira Duarte, Marta; Maria Pastore, Gláucia

    2011-01-01

    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). PMID:24031642

  14. Correlating the hydrogen evolution reaction activity in alkaline electrolytes with the hydrogen binding energy on monometallic surfaces

    SciTech Connect

    Sheng, WC; Myint, M; Chen, JGG; Yan, YS

    2013-05-01

    The slow reaction kinetics of the hydrogen evolution and oxidation reactions (HER/HOR) on platinum in alkaline electrolytes hinders the development of alkaline electrolysers, solar hydrogen cells and alkaline fuel cells. A fundamental understanding of the exchange current density of the HER/HOR in alkaline media is critical for the search and design of highly active electrocatalysts. By studying the HER on a series of monometallic surfaces, we demonstrate that the HER exchange current density in alkaline solutions can be correlated with the calculated hydrogen binding energy (HBE) on the metal surfaces via a volcano type of relationship. The HER activity varies by several orders of magnitude from Pt at the peak of the plot to W and Au located on the bottom of each side of the plot, similar to the observation in acids. Such a correlation suggests that the HBE can be used as a descriptor for identifying electrocatalysts for HER/HOR in alkaline media, and that the HER exchange current density can be tuned by modifying the surface chemical properties.

  15. Insights into the activation mechanism of class I HDAC complexes by inositol phosphates

    PubMed Central

    Watson, Peter J.; Millard, Christopher J.; Riley, Andrew M.; Robertson, Naomi S.; Wright, Lyndsey C.; Godage, Himali Y.; Cowley, Shaun M.; Jamieson, Andrew G.; Potter, Barry V. L.; Schwabe, John W. R.

    2016-01-01

    Histone deacetylases (HDACs) 1, 2 and 3 form the catalytic subunit of several large transcriptional repression complexes. Unexpectedly, the enzymatic activity of HDACs in these complexes has been shown to be regulated by inositol phosphates, which bind in a pocket sandwiched between the HDAC and co-repressor proteins. However, the actual mechanism of activation remains poorly understood. Here we have elucidated the stereochemical requirements for binding and activation by inositol phosphates, demonstrating that activation requires three adjacent phosphate groups and that other positions on the inositol ring can tolerate bulky substituents. We also demonstrate that there is allosteric communication between the inositol-binding site and the active site. The crystal structure of the HDAC1:MTA1 complex bound to a novel peptide-based inhibitor and to inositol hexaphosphate suggests a molecular basis of substrate recognition, and an entropically driven allosteric mechanism of activation. PMID:27109927

  16. Endothelial alkaline phosphatase activity loss as an early stage in the development of radiation-induced heart disease in rats

    SciTech Connect

    Lauk, S.

    1987-04-01

    Alkaline phosphatase activity of capillary endothelial cells in the heart of Wistar and Sprague-Dawley rats was studied sequentially after single doses of 10, 15, 20, or 25 Gy. Following irradiation capillary density and alkaline phosphatase activity were focally lost before myocardial degeneration or clinical symptoms of heart disease developed. Recovery from both changes took place after doses of 10 or 15 Gy. The decrease in capillary density and enzyme activity showed the same strain difference in latency times and in the extent of the lesions as previously described for pathological and clinical signs of heart disease.

  17. A Relationship Between Microbial Activity in Soils and Phosphate Levels in Tributaries to Lake Champlain

    NASA Astrophysics Data System (ADS)

    Larose, R.; Lee, S.; Lane, T.

    2015-12-01

    Lake Champlain is a large natural freshwater lake. It forms the western boundary of Vermont and drains over half of the state. It is bordered by the state of New York on its western side and drains to the north into Quebec, Canada. Lake Champlain is the source of fresh drinking water for over quarter of a million people and provides for the livelihoods and recreational opportunities of many well beyond its borders. The health of this lake is important. During the summer month's algae blooms plague the lake. These unsightly growths, which affect other aquatic organisms, are the result of excess phosphate flowing into the lake from many sources. Examining whether there is a relationship between microbial activity in the soils bordering tributaries to Lake Champlain and phosphate levels in those tributaries sheds insight into the origins and paths by which phosphate moves into Lake Champlain. Understanding the how phosphate moves into the water system may assist in mitigation efforts.Total Phosphate levels and Total Suspended Solids were measured in second and third order streams in the Lake Champlain Basin over a three-year period. In addition microbial activity was measured within the toe, bank and upland riparian zone areas of these streams during the summer months. In general in areas showing greater microbial activity in the soil(s) there were increased levels of phosphate in the streams.

  18. Diarylsulfonamides and their bioisosteres as dual inhibitors of alkaline phosphatase and carbonic anhydrase: Structure activity relationship and molecular modelling studies.

    PubMed

    Al-Rashida, Mariya; Ejaz, Syeda Abida; Ali, Sharafat; Shaukat, Aisha; Hamayoun, Mehwish; Ahmed, Maqsood; Iqbal, Jamshed

    2015-05-15

    The effect of bioisosteric replacement of carboxamide linking group with sulfonamide linking group, on alkaline phosphatase (AP) and carbonic anhydrase (CA) inhibition activity of aromatic benzenesulfonamides was investigated. A series of carboxamide linked aromatic benzenesulfonamides 1a-1c, 2a-2d and their sulfonamide linked bioisosteres 3a-3d, 4a-4d was synthesized and evaluated for inhibitory activity against bovine tissue non-specific alkaline phosphatase (TNAP), intestinal alkaline phosphatase (IAP) and bCA II. A significant increase in CA inhibition activity was observed upon bioisosteric replacement of carboxamide linking group with a sulfonamide group. Some of these compounds were identified as highly potent and selective AP inhibitors. Compounds 1b, 2b, 3d, 4d 5b and 5c were found to be selective bTNAP inhibitors, whereas compounds 1a, 1c, 2a, 2c, 2d, 3a, 3c, 4a, 4b, 4c, 5a were found to be selective bIAP inhibitors. For most active AP inhibitor 3b, detailed kinetic studies indicated a competitive mode of inhibition against tissue non-specific alkaline phosphatase (TNAP) and non-competitive mode of inhibition against intestinal alkaline phosphatase (IAP). Molecular docking studies were carried out to rationalize important binding site interactions. PMID:25865133

  19. Sphingosine 1-phosphate receptor activation enhances BMP-2-induced osteoblast differentiation

    SciTech Connect

    Sato, Chieri; Iwasaki, Tsuyoshi; Kitano, Sachie; Tsunemi, Sachi; Sano, Hajime

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer We investigated the role of S1P signaling for osteoblast differentiation. Black-Right-Pointing-Pointer Both S1P and FTY enhanced BMP-2-stimulated osteoblast differentiation by C2C12 cells. Black-Right-Pointing-Pointer S1P signaling enhanced BMP-2-stimulated Smad and ERK phosphorylation by C2C12 cells. Black-Right-Pointing-Pointer MEK/ERK signaling is a pathway underlying S1P signaling for osteoblast differentiation. -- Abstract: We previously demonstrated that sphingosine 1-phosphate (S1P) receptor-mediated signaling induced proliferation and prostaglandin productions by synovial cells from rheumatoid arthritis (RA) patients. In the present study we investigated the role of S1P receptor-mediated signaling for osteoblast differentiation. We investigated osteoblast differentiation using C2C12 myoblasts, a cell line derived from murine satellite cells. Osteoblast differentiation was induced by the treatment of bone morphogenic protein (BMP)-2 in the presence or absence of either S1P or FTY720 (FTY), a high-affinity agonist of S1P receptors. Osteoblast differentiation was determined by osteoblast-specific transcription factor, Runx2 mRNA expression, alkaline phosphatase (ALP) activity and osteocalcin production by the cells. Smad1/5/8 and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was examined by Western blotting. Osteocalcin production by C2C12 cells were determined by ELISA. Runx2 expression and ALP activity by BMP-2-stimulated C2C12 cells were enhanced by addition of either S1P or FTY. Both S1P and FTY enhanced BMP-2-induced ERK1/2 and Smad1/5/8 phosphorylation. The effect of FTY was stronger than that of S1P. S1P receptor-mediated signaling on osteoblast differentiation was inhibited by addition of mitogen-activated protein kinase/ERK kinase (MEK) 1/2 inhibitor, indicating that the S1P receptor-mediated MEK1/2-ERK1/2 signaling pathway enhanced BMP-2-Smad signaling. These results indicate that S1P

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

    PubMed Central

    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

  1. A binary palladium-bismuth nanocatalyst with high activity and stability for alkaline glucose electrooxidation

    NASA Astrophysics Data System (ADS)

    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.

  2. An alkaline D-stereospecific endopeptidase with beta-lactamase activity from Bacillus cereus.

    PubMed

    Asano, Y; Ito, H; Dairi, T; Kato, Y

    1996-11-22

    We purified a novel extracellular D-stereospecific endopeptidase, alkaline D-peptidase (D-stereospecific peptide hydrolase, EC 3.4.11.-), to homogeneity from the culture broth of the soil bacterium Bacillus cereus strain DF4-B. The Mr of the enzyme was 37,952, and it was composed of a single polypeptide chain. The optimal pH for activity was approximately 10.3. The enzyme was strictly D-stereospecific toward oligopeptides composed of Dphenylalanine such as (D-Phe)3 and (D-Phe)4. The enzyme also acted to a lesser extent on (D-Phe)6, Boc-(D-Phe)4 (where Boc is tert-butoxycarbonyl), Boc-(D-Phe)4 methyl ester, Boc-(D-Phe)3 methyl ester, Boc-(D-Phe)2, (D-Phe)2, and others, but not upon their corresponding peptides composed of L-Phe, (D-Ala)n (n = 2-5), (D-Val)3, and (D-Leu)2. The mode of action of the enzyme was clarified with synthetic substrates ((D-Phe)2-D-Tyr and D-Tyr-(D-Phe)2) and eight stereoisomers of (Phe)3. The enzyme had beta-lactamase activity toward ampicillin and penicillin G, although carboxypeptidase DD and D-aminopeptidase activities were undetectable. The gene coding for alkaline D-peptidase (adp) was cloned into plasmid pUC118, and a 1164-base pair open reading frame consisting of 388 codons was identified as the adp gene. The predicted polypeptide was similar to carboxypeptidase DD from Streptomyces R61, penicillin-binding proteins from Streptomyces lactamdurans and Bacillus subtilis, and class C beta-lactamases. Thus, the enzyme was categorized as a new "penicillin-recognizing enzyme." PMID:8939979

  3. Segregated Pt on Pd nanotubes for enhanced oxygen reduction activity in alkaline electrolyte.

    PubMed

    St John, Samuel; Atkinson, Robert W; Dyck, Ondrej; Sun, Cheng-Jun; Zawodzinski, Thomas A; Papandrew, Alexander B

    2015-12-01

    Nanoscaled Pt domains were integrated with Pd nanotubes via vapor deposition to yield a highly active electrocatalyst for the oxygen reduction reaction (ORR) in alkaline media. The surface-area-normalized ORR activity of these bi-metallic Pt-on-Pd nanotubes (PtPdNTs) was nearly 6× the corresponding carbon-supported Pt nanoparticle (Pt/C) activity at 0.9 V vs. RHE (1.5 vs. 0.24 mA cmmetal(-2), respectively). Furthermore, the high specific activity of the PtPdNTs was achieved without sacrificing mass-normalized activity, which is more than twice that of Pt/C (0.333 A mgPtPdNT(-1)vs. 0.141 A mgPt/C(-1)) and also greater than that of Pd/C (0.221 A mgPd/C(-1)). We attribute the enhancements in specific and mass activity to modifications of the segregated Pt electronic structure and to nanoscale porosity, respectively. PMID:26553367

  4. Sphingomyelinase D Activity in Model Membranes: Structural Effects of in situ Generation of Ceramide-1-Phosphate

    PubMed Central

    Stock, Roberto P.; Brewer, Jonathan; Wagner, Kerstin; Ramos-Cerrillo, Blanca; Duelund, Lars; Jernshøj, Kit Drescher; Olsen, Lars Folke; Bagatolli, Luis A.

    2012-01-01

    The toxicity of Loxosceles spider venom has been attributed to a rare enzyme, sphingomyelinase D, which transforms sphingomyelin to ceramide-1-phosphate. The bases of its inflammatory and dermonecrotic activity, however, remain unclear. In this work the effects of ceramide-1-phosphate on model membranes were studied both by in situ generation of this lipid using a recombinant sphingomyelinase D from the spider Loxosceles laeta and by pre-mixing it with sphingomyelin and cholesterol. The systems of choice were large unilamellar vesicles for bulk studies (enzyme kinetics, fluorescence spectroscopy and dynamic light scattering) and giant unilamellar vesicles for fluorescence microscopy examination using a variety of fluorescent probes. The influence of membrane lateral structure on the kinetics of enzyme activity and the consequences of enzyme activity on the structure of target membranes containing sphingomyelin were examined. The findings indicate that: 1) ceramide-1-phosphate (particularly lauroyl ceramide-1-phosphate) can be incorporated into sphingomyelin bilayers in a concentration-dependent manner and generates coexistence of liquid disordered/solid ordered domains, 2) the activity of sphingomyelinase D is clearly influenced by the supramolecular organization of its substrate in membranes and, 3) in situ ceramide-1-phosphate generation by enzymatic activity profoundly alters the lateral structure and morphology of the target membranes. PMID:22558302

  5. A Mg(2+)-dependent ecto-phosphatase activity on the external surface of Trypanosoma rangeli modulated by exogenous inorganic phosphate.

    PubMed

    Fonseca-de-Souza, André L; Dick, Claudia Fernanda; Dos Santos, André Luiz Araújo; Meyer-Fernandes, José Roberto

    2008-08-01

    In this work, we characterized a Mg(2+)-dependent ecto-phosphatase activity present in live Trypanosoma rangeli epimastigotes. This enzyme showed capacity to hydrolyze the artificial substrate for phosphatases, p-nitrophenylphosphate (p-NPP). At saturating concentration of p-NPP, half-maximal p-NPP hydrolysis was obtained with 0.23mM Mg(2+). Ca(2+) had no effect on the basal phosphatase activity, could not substitute Mg(2+) as an activator and in contrast inhibited the p-NPP hydrolysis stimulated by Mg(2+). The dependence on p-NPP concentration showed a normal Michaelis-Menten kinetics for this phosphatase activity with values of V(max) of 8.94+/-0.36 nmol p-NP x h(-1) x 10(-7) cells and apparent K(m) of 1.04+/-0.16 mM p-NPP. Mg(2+)-dependent ecto-phosphatase activity was stimulated by the alkaline pH range. Experiments using inhibitors, such as, sodium fluoride, sodium orthovanadate and ammonium molybdate, inhibited the Mg(2+)-dependent ecto-phosphatase activity. Inorganic phosphate (Pi), a product of phosphatases, inhibited reversibly in 50% this activity. Okadaic acid and microcystin-LR, specific phosphoserine/threonine phosphatase inhibitors, inhibited significantly the Mg(2+)-dependent ecto-phosphatase activity. In addition, this phosphatase activity was able to recognize as substrates only o-phosphoserine and o-phosphothreonine, while o-phosphotyrosine was not a good substrate for this phosphatase. Epimastigote forms of T. rangeli exhibit a typical growth curve, achieving the stationary phase around fifth or sixth day and the Mg(2+)-dependent ecto-phosphatase activity decreased around 10-fold with the cell growth progression. Cells maintained at Pi-deprived medium (2 mM Pi) present Mg(2+)-dependent ecto-phosphatase activity approximately threefold higher than that maintained at Pi-supplemented medium (50 mM Pi). PMID:18599005

  6. Extensive site-directed mutagenesis reveals interconnected functional units in the alkaline phosphatase active site.

    PubMed

    Sunden, Fanny; Peck, Ariana; Salzman, Julia; Ressl, Susanne; Herschlag, Daniel

    2015-01-01

    Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called 'catalytic residues' are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modes of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes. PMID:25902402

  7. A smart fluorescence nanoprobe for the detection of cellular alkaline phosphatase activity and early osteogenic differentiation.

    PubMed

    Cao, Feng-Yi; Fan, Jin-Xuan; Long, Yue; Zeng, Xuan; Zhang, Xian-Zheng

    2016-07-01

    In the past decades, biomaterials were designed to induce stem cell toward osteogenic differentiation. However, conventional methods for evaluation osteogenic differentiation all required a process of cell fixation or lysis, which induce waste of a large number of cells. In this study, a fluorescence nanoprobe was synthesized by combining phosphorylated fluoresceinamine isomer I (FLA) on the surface of mesoporous silica-coated superparamagnetic iron oxide (Fe3O4@mSiO2) nanoparticles. In the presence of alkaline phosphatase (ALP), the phosphorylated FLA on the nanoprobe would be hydrolyzed, resulting in a fluorescence recovery of FLA. During early osteogenic differentiation, a high-level expression of cellular ALP was induced, which accelerated the hydrolysis of phosphorylated FLA, resulting in an enhancement of cellular fluorescence intensity. This fluorescence nanoprobe provides us a rapid and non-toxic method for the detection of cellular ALP activity and early osteogenic differentiation. PMID:26961462

  8. Spatial variability of dissolved phosphorous concentrations and alkaline phosphatase activity in the East China Sea

    NASA Astrophysics Data System (ADS)

    Liu, H.; Chang, J.; Ho, T.; Gong, G.

    2010-12-01

    The concentrations of dissolved inorganic phosphorus (DIP) and alkaline phosphatase activity (APA) have been determined at about 25 sampling stations in the East China Sea since 2003. The stations are mainly distributed from the Changjiang river mouth to northern Taiwan and east to the shelf break. In addition to the Changjiang discharge, we have found a specific nutrient source around a coastal site (122° 2’30’’ E, 28° 40’ N). Elevated DIP and nitrate concentrations have been constantly observed around the sampling station for 8 years, where the surface DIP concentrations are generally around 0.3 µM. The nutrient source may either originate from ground water discharge or coastal upwelling, where lower temperature has been observed in the water column around the station. In general, APA has been negatively correlated with DIP concentrations in the studies sites, with lowest APA around the high DIP station and the Changjiang river mouth.

  9. The Cryptococcus neoformans Alkaline Response Pathway: Identification of a Novel Rim Pathway Activator

    PubMed Central

    Ost, Kyla S.; O’Meara, Teresa R.; Huda, Naureen; Esher, Shannon K.; Alspaugh, J. Andrew

    2015-01-01

    The Rim101/PacC transcription factor acts in a fungal-specific signaling pathway responsible for sensing extracellular pH signals. First characterized in ascomycete fungi such as Aspergillus nidulans and Saccharomyces cerevisiae, the Rim/Pal pathway maintains conserved features among very distantly related fungi, where it coordinates cellular adaptation to alkaline pH signals and micronutrient deprivation. However, it also directs species-specific functions in fungal pathogens such as Cryptococcus neoformans, where it controls surface capsule expression. Moreover, disruption of the Rim pathway central transcription factor, Rim101, results in a strain that causes a hyper-inflammatory response in animal infection models. Using targeted gene deletions, we demonstrate that several genes encoding components of the classical Rim/Pal pathway are present in the C. neoformans genome. Many of these genes are in fact required for Rim101 activation, including members of the ESCRT complex (Vps23 and Snf7), ESCRT-interacting proteins (Rim20 and Rim23), and the predicted Rim13 protease. We demonstrate that in neutral/alkaline pH, Rim23 is recruited to punctate regions on the plasma membrane. This change in Rim23 localization requires upstream ESCRT complex components but does not require other Rim101 proteolysis components, such as Rim20 or Rim13. Using a forward genetics screen, we identified the RRA1 gene encoding a novel membrane protein that is also required for Rim101 protein activation and, like the ESCRT complex, is functionally upstream of Rim23-membrane localization. Homologs of RRA1 are present in other Cryptococcus species as well as other basidiomycetes, but closely related genes are not present in ascomycetes. These findings suggest that major branches of the fungal Kingdom developed different mechanisms to sense and respond to very elemental extracellular signals such as changing pH levels. PMID:25859664

  10. Alkaline phosphatase activity in the western English Channel: Elevations induced by high summertime rainfall

    NASA Astrophysics Data System (ADS)

    Rees, Andrew P.; Hope, Sam B.; Widdicombe, Claire E.; Dixon, Joanna L.; Woodward, E. Malcolm S.; Fitzsimons, Mark F.

    2009-03-01

    Alkaline phosphatase activity (APA) was determined in bulk particulate material and in a single-cell (ELF) assay at station L4 in the western English Channel during the summer of 2007. Throughout this period, the UK experienced its heaviest summertime rainfall since records began in 1914; with the result that riverine run-off into coastal waters was also elevated relative to long-term averages. Between May and August 2007, three distinct periods of elevated river run-off were observed which resulted in salinity minima at L4 on days 141, 190 and 232. An extended period of high river run-off between days 170 and 210 was responsible for decreases in near-surface salinity at L4 from 35.2068 to a minimum on day 190 of 34.7422. This contributed to the development of haline stratification which supported the development of an intense bloom of the centric diatom Chaetoceros debelis, with maximum observed chlorophyll a concentration of 8.69 μg l -1. Minima in salinity, and maxima in chlorophyll concentration on day 190 were coincident with a peak in river-derived dissolved inorganic nitrogen (DIN) of 1.9 μmol l -1 which was >5 times greater than the summertime mean and 24 times the concentrations experienced at L4 on weeks immediately before and after. There was no accompanying increase in dissolved inorganic phosphorus (DIP), and the DIN:DIP ratio increased to 49. With the inherent phosphorus stress that this caused, rates of APA increased from <4 to 42.4 nmolP l -1 h -1. ELF analysis on day 197 identified two taxa actively expressing alkaline phosphatase: the dinoflagellate Prorocentrum micans and ciliate Tiarana sp.

  11. Circadian Regulation of Sucrose Phosphate Synthase Activity in Tomato by Protein Phosphatase Activity.

    PubMed Central

    Jones, T. L.; Ort, D. R.

    1997-01-01

    Sucrose phosphate synthase (SPS), a key enzyme in sucrose biosynthesis, is regulated by protein phosphorylation and shows a circadian pattern of activity in tomato. SPS is most active in its dephosphorylated state, which normally coincides with daytime. Applying okadaic acid, a potent protein phosphatase inhibitor, prevents SPS activation. More interesting is that a brief treatment with cycloheximide, a cytoplasmic translation inhibitor, also prevents the light activation of SPS without any effect on the amount of SPS protein. Cordycepin, an inhibitor of transcript synthesis and processing, has the same effect. Both of these inhibitors also prevent the activation phase of the circadian rhythm in SPS activity. Conversely, cycloheximide and cordycepin do not prevent the decline in circadian SPS activity that normally occurs at night. These observations indicate that SPS phosphatase activity but not SPS kinase activity is controlled, directly or indirectly, at the level of gene expression. Taken together, these data imply that there is a circadian rhythm controlling the transcription of a protein phosphatase that subsequently dictates the circadian rhythm in SPS activity via effects on this enzyme's phosphorylation state. PMID:12223667

  12. Physicochemical characteristics and sorption capacities of heavy metal ions of activated carbons derived by activation with different alkyl phosphate triesters

    NASA Astrophysics Data System (ADS)

    Wang, Jing; Liu, Hai; Yang, Shaokun; Zhang, Jian; Zhang, Chenglu; Wu, Haiming

    2014-10-01

    Five alkyl phosphate triesters (APTEs), including trimethyl phosphate (TMP), triethyl phosphate (TEP), triisopropyl phosphate (TPP), tributyl phosphate (TBP) and trioctyl phosphate (TOP), were used as activating agents for preparing activated carbons (AC-APTEs) with high surface acidity and metal ion sorption capacity. N2 adsorption/desorption isotherms, surface morphologies, elemental compositions, results of Boehm's titration and sorption capacities of heavy metal ions of the carbons were investigated. AC-APTEs contained much more acidic groups and exhibited much less surface area (<500 m2/g) in comparison with activated carbon (AC-PPA, 1145 m2/g) obtained from phosphoric acid activation. For the AC-APTEs, AC-TOP had the highest surface area (488 m2/g), AC-TMP showed the highest yield (41.1%), and AC-TBP possessed the highest acidic groups (2.695 mmol/g), oxygen content (47.0%) and metal ion sorption capacities (40.1 mg/g for Ni(II) and 53.5 mg/g for Cd(II)). For the carbons, AC-APTEs showed much larger Ni(II) and Cd(II) sorption capacities than AC-PPA, except AC-TPP. The differences of the carbons in the physicochemical and sorption properties suggested surface chemistry of the carbons was the main factor influencing their sorption capacities whereas the pore structure played a secondary role.

  13. Role of phosphate on stability and catalase mimetic activity of cerium oxide nanoparticles.

    PubMed

    Singh, Ragini; Singh, Sanjay

    2015-08-01

    Cerium oxide nanoparticles (CeNPs) have been recently shown to scavenge reactive oxygen and nitrogen species (ROS and RNS) in different experimental model systems. CeNPs (3+) and CeNPs (4+) have been shown to exhibit superoxide dismutase (SOD) and catalase mimetic activity, respectively. Due to their nanoscale dimension, CeNPs are expected to interact with the components of biologically relevant buffers and medium, which could alter their catalytic properties. We have demonstrated earlier that CeNPs (3+) interact with phosphate and lose the SOD activity. However, very little is known about the interaction of CeNPs (4+) with the phosphate and other anions, predominantly present in biological buffers and their effects on the catalase mimetic-activity of these nanoparticles. In this study, we report that catalase mimetic-activity of CeNPs (4+) is resistant to the phosphate anions, pH changes and composition of cell culture media. Given the abundance of phosphate anions in the biological system, it is likely that internalized CeNPs would be influenced by cytoplasmic and nucleoplasmic concentration of phosphate. PMID:26011425

  14. High expression and biosilica encapsulation of alkaline-active carbonic anhydrase for CO2 sequestration system development.

    PubMed

    Min, Ki-Ha; Son, Ryeo Gang; Ki, Mi-Ran; Choi, Yoo Seong; Pack, Seung Pil

    2016-01-01

    Carbonic anhydrase (CA) is a biocatalyst for CO2 sequestration because of its distinctive ability to accelerate CO2 hydration. High production and efficient immobilization of alkaline-active CAs are required, because one potential application of CA is its use in the alkaline solvent-based CO2 absorption/desorption process. Here, we designed and applied an α-type CA from Hahella chejuensis (HCA), which was reported as highly active in alkaline conditions, but was mostly expressed as insoluble forms. We found that the signal peptide-removed form of HCA [HCA(SP-)] was successfully expressed in the soluble form [∼70mg of purified HCA(SP-) per L of culture]. HCA(SP-) also displayed high pH stability in alkaline conditions, with maximal activity at pH 10; at this pH, ∼90% activity was maintained for 2h. Then, we prepared HCA(SP-)-encapsulated silica particles [HCA(SP-)@silica] via a spermine-mediated bio-inspired silicification method. HCA(SP-)@silica exhibited high-loading and highly stable CA activity. In addition, HCA(SP-)@silica retained more than 90% of the CA activity even after 10 cycles of use in mild conditions, and ∼80% in pH 10 conditions. These results will be useful for the development of practical CO2 sequestration processes employing CA. PMID:26206748

  15. Electrochemical determination of activation energies for methanol oxidation on polycrystalline platinum in acidic and alkaline electrolytes.

    PubMed

    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

  16. Platinum and Palladium Overlayers Dramatically Enhance the Activity of Ruthenium Nanotubes for Alkaline Hydrogen Oxidation

    DOE PAGESBeta

    St. John, Samuel; Atkinson, Robert W.; Unocic, Kinga A.; Unocic, Raymond R.; Zawodzinski, Thomas A.; Papandrew, Alexander B.

    2015-10-18

    Templated vapor synthesis and thermal annealing were used to synthesize unsupported metallic Ru nanotubes with Pt or Pd overlayers. By controlling the elemental composition and thickness of these overlayers, we obtain nanostructures with very high alkaline hydrogen oxidation activity. For nanotubes with a nominal atomic composition of Ru0.90Pt0.10 display a surface-specific activity (2.4 mA/cm2) that is 35 times greater than that of pure Ru nanotubes at a 50 mV overpotential and 2.5 times greater than that of pure Pt nanotubes (0.98 mA/cm2). The surface-segregated structure also confers dramatically increased Pt utilization efficiency. We find a platinum-mass-specific activity of 1240 A/gPtmore » for the optimized nanotube versus 280 A/gPt for carbon-supported Pt nanoparticles and 109 A/gPt for monometallic Pt nanotubes. Here, we attribute the enhancement of both area- and platinum-mass-specific activity to the atomic-scale homeomorphism of the nanotube form factor with adlayer-modified polycrystals. Subsurface ligand and bifunctional effects previously observed on segregated, adlayer-modified polycrystals are translated to nanoscale catalysts.« less

  17. Platinum and Palladium Overlayers Dramatically Enhance the Activity of Ruthenium Nanotubes for Alkaline Hydrogen Oxidation

    SciTech Connect

    St. John, Samuel; Atkinson, Robert W.; Unocic, Kinga A.; Unocic, Raymond R.; Zawodzinski, Thomas A.; Papandrew, Alexander B.

    2015-10-18

    Templated vapor synthesis and thermal annealing were used to synthesize unsupported metallic Ru nanotubes with Pt or Pd overlayers. By controlling the elemental composition and thickness of these overlayers, we obtain nanostructures with very high alkaline hydrogen oxidation activity. For nanotubes with a nominal atomic composition of Ru0.90Pt0.10 display a surface-specific activity (2.4 mA/cm2) that is 35 times greater than that of pure Ru nanotubes at a 50 mV overpotential and 2.5 times greater than that of pure Pt nanotubes (0.98 mA/cm2). The surface-segregated structure also confers dramatically increased Pt utilization efficiency. We find a platinum-mass-specific activity of 1240 A/gPt for the optimized nanotube versus 280 A/gPt for carbon-supported Pt nanoparticles and 109 A/gPt for monometallic Pt nanotubes. Here, we attribute the enhancement of both area- and platinum-mass-specific activity to the atomic-scale homeomorphism of the nanotube form factor with adlayer-modified polycrystals. Subsurface ligand and bifunctional effects previously observed on segregated, adlayer-modified polycrystals are translated to nanoscale catalysts.

  18. A facile stable-isotope dilution method for determination of sphingosine phosphate lyase activity.

    PubMed

    Suh, Jung H; Eltanawy, Abeer; Rangan, Apoorva; Saba, Julie D

    2016-01-01

    A new technique for quantifying sphingosine phosphate lyase activity in biological samples is described. In this procedure, 2-hydrazinoquinoline is used to convert (2E)-hexadecenal into the corresponding hydrazone derivative to improve ionization efficiency and selectivity of detection. Combined utilization of liquid chromatographic separation and multiple reaction monitoring-mass spectrometry allows for simultaneous quantification of the substrate S1P and product (2E)-hexadecenal. Incorporation of (2E)- d5-hexadecenal as an internal standard improves detection accuracy and precision. A simple one-step derivatization procedure eliminates the need for further extractions. Limits of quantification for (2E)-hexadecenal and sphingosine-1-phosphate are 100 and 50fmol, respectively. The assay displays a wide dynamic detection range useful for detection of low basal sphingosine phosphate lyase activity in wild type cells, SPL-overexpressing cell lines, and wild type mouse tissues. Compared to current methods, the capacity for simultaneous detection of sphingosine-1-phosphate and (2E)-hexadecenal greatly improves the accuracy of results and shows excellent sensitivity and specificity for sphingosine phosphate lyase activity detection. PMID:26408264

  19. Mechanisms for solubilization of various insoluble phosphates and activation of immobilized phosphates in different soils by an efficient and salinity-tolerant Aspergillus niger strain An2.

    PubMed

    Li, Xiaolong; Luo, Lijin; Yang, Jinshui; Li, Baozhen; Yuan, Hongli

    2015-03-01

    Mechanisms for solubilization of different types of phosphates and activation of immobilized phosphates in different types of soils by an efficient fungal strain An2 were explored and evaluated in this study. An2 was isolated from a Chinese cabbage rhizosphere soil and identified as Aspergillus niger. It could fast release up to 1722, 2066, and 2356 mg L(-1) of soluble phosphorus (P) from 1 % Ca3(PO4)2, Mg3(PO4)2, and AlPO4 (Ca-P, Mg-P, and Al-P) and 215 and 179 mg L(-1) from 0.5 % FePO4 and rock phosphate (Fe-P and RP), respectively. HPLC assay demonstrated that An2 mainly secreted oxalic acid to solubilize Ca-P, Mg-P, Al-P, and Fe-P whereas secreted tartaric acid to solubilize RP. Furthermore, An2 could tolerate salinity up to 4 % NaCl without impairing its phosphate-solubilizing ability. The simulation experiments validated that An2 was able to effectively activate immobilized phosphates in general calcareous, acidic, as well as saline-alkali soils with high total P content. This study shows new insights into the mechanisms for microbial solubilization of different types of phosphates and supports the future application of strain An2 in different types of soils to effectively activate P for plants. PMID:25561059

  20. Histochemical localization of alkaline phosphatase activity in decalcified bone and cartilage.

    PubMed

    Miao, Dengshun; Scutt, Andrew

    2002-03-01

    We have developed methodology that enables alkaline phosphatase (ALP) to be histochemically stained reproducibly in decalcified paraffin-embedded bone and cartilage of rodents. Proximal tibiae and fourth lumbar vertebrae were fixed in periodate-lysine-paraformaldehyde (PLP) fixative, decalcified in an EDTA-G solution, and embedded in paraffin. In the articular cartilage of the proximal tibia, ALP activity was localized to the hypertrophic chondrocytes and cartilage matrix of the deep zone and the maturing chondrocytes of the intermediate zone. The cells and matrix in the superficial zone did not exhibit any enzyme activity. In tibial and vertebral growth plates, a progressive increase in ALP expression was seen in chondrocytes and cartilage matrix, with activity being weakest in the proliferative zone, higher in the maturing zone, and highest in the hypertrophic zone. In bone tissue, ALP activity was detected widely in pre-osteoblasts, osteoblasts, lining cells on the surface of trabeculae, some newly embedded osteocytes, endosteal cells, and subperiosteal cells. In areas of new bone formation, ALP activity was detected in osteoid. In the bone marrow, about 20% of bone marrow cells expressed ALP activity. In adult rats, the thickness of the growth plates was less and ALP activity was enhanced in maturing and hypertrophic chondrocytes, cartilage matrix in the hypertrophic zone, and primary spongiosa. This is the first time that ALP activity has been successfully visualized histochemically in decalcified, paraffin-embedded mineralized tissues. This technique should prove to be a very convenient adjunct for studying the behavior of osteoblasts during osteogenesis. PMID:11850436

  1. The Effect of Covalently Immobilized FGF-2 on Biphasic Calcium Phosphate Bone Substitute on Enhanced Biological Compatibility and Activity

    PubMed Central

    Moon, Kyung-Suk; Choi, Eun-Joo; Oh, Seunghan; Kim, Sungtae

    2015-01-01

    The purpose of this research was to covalently graft fibroblast growth factor 2 (FGF-2) onto biphasic calcium phosphate (BCP) via a bifunctional cross-linker technique and to estimate the optimal dose of FGF-2 resulting in the best osteogenic differentiation of human mesenchymal stem cells (hMSCs). SEM observation revealed that the surface of the 100 ng FGF-2 coated BCP was completely covered with the nanoparticles expected to be from the silane coupling agent. XRD, FT-IR, and XPS analysis showed that silane treatment, bifunctional cross-linker coating, and FGF-2 covalent grafts were conducted successfully without deforming the crystalline structure of BCP. An MTT assay demonstrated that FGF-2 coated BCP had good biocompatibility, regardless of the concentration of FGF-2, after 24 or 48 h of incubation. An alkaline phosphatase (ALP) activity assay (14 days of incubation) and the ALP gene expression level of real-time PCR analysis (7 days of incubation) revealed that 50, 100, and 200 ng FGF-2 coated BCP induced the highest activities among all experimental groups and control group (P < 0.05). Thus, low concentrations of FGF-2 facilitated excellent osteogenesis and were effective at enhancing osteogenic potential. Also, the bifunctional cross-linker technique is expected to be a more feasible way to induce osteogenic differentiation while minimizing the risk of FGF-2 overdose. PMID:26436096

  2. Bactericidal, virucidal, and mycobactericidal activities of reused alkaline glutaraldehyde in an endoscopy unit.

    PubMed Central

    Mbithi, J N; Springthorpe, V S; Sattar, S A; Pacquette, M

    1993-01-01

    Baths with 2% alkaline glutaraldehyde are often reused for 14 days to decontaminate flexible fiberoptic endoscopes (FFEs) between patients, but the effect of such reuse on the disinfectant's activity has not been known. Many busy endoscopy units also disinfect FFEs with contact times shorter than those recommended by the disinfectant manufacturer. We therefore collected samples of the disinfectant over the 14-day reuse period from two manual and one automatic bath used for bronchoscopes and gastroscopes at a local hospital. Control samples were also collected from a manual bath of 2% alkaline glutaraldehyde which did not receive any endoscopes. The germicidal activities of the samples were assessed in a carrier test against a mixture of hepatitis A virus, poliovirus 1 (Sabin), and Pseudomonas aeruginosa; the mixture also contained either Mycobacterium bovis or Mycobacterium gordonae. Bovine serum (5%) was the organic load. The criterion of efficacy was a minimum of a 3-log10-unit reduction in the infectivity titers of the organisms tested. The initial disinfectant concentration in all the baths was nearly 2.25%; it became about 1.8% in the control bath and fell to approximately 1% in the three test baths after 14 days. No protein was detected in the control bath, while its concentration rose gradually in the test baths to a maximum of 1,267 micrograms/ml after 14 days. With a contact time of 10 min at 20 +/- 2 degrees C, all the samples from the control bath were effective against all the test organisms and all the samples from all the test baths were also effective against P. aeruginosa. With a contact time of 10 or 20 min at 20+/-2 degrees C, the virucidal and mycobactericidal activities of the samples from the test baths showed broad-spectrum germicidal activity when the contact time was increased to 45 min and the temperature was raised to 25 degrees C. These findings emphasize the care needed in the disinfection of FFEs, especially in view of the increasing

  3. Pyrophosphate-regulated Zn(2+)-dependent DNAzyme activity: an amplified fluorescence sensing strategy for alkaline phosphatase.

    PubMed

    Kong, Rong-Mei; Fu, Ting; Sun, Ni-Na; Qu, Feng-Li; Zhang, Shu-Fang; Zhang, Xiao-Bing

    2013-12-15

    In this work, based on the fact that pyrophosphate (PPi) could regulate the activity of Zn(2+)-dependent DNAzyme, we for the first time report a fluorescence turn-on sensing system for alkaline phosphatase (ALP) with improved sensitivity via nonprotein-enzymatic signal amplification. A catalytic and molecular beacon (CAMB) design was employed to further improve its sensitivity. Taking advantage of the strong interactions between PPi and the Zn(2+), the cofactor Zn(2+) was caged, and the DNAzyme activity was effectively inhibited. The introduction of ALP, however, could catalyze the hydrolysis of PPi and release free Zn(2+), resulting in the activation of DNAzyme to catalyze the cleavage of the molecular beacon substrate with a remarkable increase of fluorescent signal. These optimized designs together allow a high sensitivity for ALP, with a detection limit of 20 pM observed, much lower than previously reported methods. It has also been used for detection of ALP in human serum with satisfactory results, demonstrating its potential applications in clinical diagnosis. PMID:23891797

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

    PubMed

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

    2003-10-31

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

  5. Structure characterization and antitumor activity of a polysaccharide from the alkaline extract of king oyster mushroom.

    PubMed

    Liu, Xinkui; Wang, Lin; Zhang, Chunmei; Wang, Hongmin; Zhang, Xiaohong; Li, Yuexia

    2015-03-15

    A water-soluble polysaccharide, designated as KOMAP, was isolated and purified from the alkaline extract of king oyster mushroom, which was composed of glucose (Glc), mannose (Man) and arabinose (Ara) in a molar ratio of 6.2:2.1:2.0. It had an average molecular weight of 2.1×10(4)Da. GC-MS analysis revealed that KOMAP was a linear structure of the polymer with a backbone composed of β-1,4-linked glucopyranosyl and β-1,3,6-linked mannopyranosyl units, which was terminated with α-1-linked arabinofuranosyl unit at C-6 position of β-1,3,6-linked mannopyranosyl residue along the main chain in the ratio of 3.1:1. The results in the animal experiment showed that 50, 100 and 200mg/mL of KOMAP not only inhibited the tumor growth, but also increased relative thymus and spleen indices, LPS- or ConA-induced lymphocytes proliferation, and serum cytokine IL-2, TNF-α, and IFN-γ levels, as well as the activities of NK cells and CTLs in spleen of Renca tumor-bearing mice. In summary, our data indicate that the KOMAP exerts effective immunoregulatory and anti-tumor activities in vivo. PMID:25542113

  6. Surface alkaline phosphatase activities of macroalgae on coral reefs of the central Great Barrier Reef, Australia

    NASA Astrophysics Data System (ADS)

    Schaffelke, B.

    2001-05-01

    Inshore reefs of the Great Barrier Reef (GBR) are subject to episodic nutrient supply, mainly by flood events, whereas midshelf reefs have a more consistent low nutrient availability. Alkaline phosphatase activity (APA) enables macroalgae to increase their phosphorus (P) supply by using organic P. APA was high (~4.0 to 15.5 µmol PO4 3- g DW-1 h-1) in species colonising predominantly inshore reefs and low (<2 µmol PO4 3- g DW-1 h-1) in species with a cross-shelf distribution. However, APA values of GBR algae in this study were much lower than data reported from other coral reef systems. In experiments with two Sargassum species tissue P levels were correlated negatively, and N:P ratios were positively correlated with APA. High APA can compensate for a relative P-limitation of macroalgae in coral reef systems that are subject to significant N-inputs, such as the GBR inshore reefs. APA and other mechanisms to acquire a range of nutrient species allow inshore species to thrive in habitats with episodic nutrient supply. These species also are likely to benefit from an increased nutrient supply caused by human activity, which currently is a global problem.

  7. Alkalinity of neutrophil phagocytic vacuoles is modulated by HVCN1 and has consequences for myeloperoxidase activity.

    PubMed

    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

  8. Luminescence properties of Eu-activated alkaline and alkaline-earth silicate Na{sub 2}Ca{sub 3}Si{sub 6}O{sub 16}

    SciTech Connect

    Wang, Jing; Huang, Yanlin; Wang, Xigang; Qin, Lin; Seo, Hyo Jin

    2014-07-01

    Highlights: • A novel yellow-emitting alkaline and alkaline-earth silicate Na{sub 2}Ca{sub 3}Si{sub 6}O{sub 16}:Eu{sup 2+} was first developed. • Under excitation with UV or near UV light the silicate presents broad emission band centered at 580 nm. - Abstract: Yellow-emitting phosphors of Na{sub 2}Ca{sub 3}Si{sub 6}O{sub 16}:Eu{sup 2+} was prepared by wet chemistry sol–gel method. X-ray powder diffraction and SEM measurements were applied to characterize the structure and morphology, respectively. The luminescence properties were investigated by the photoluminescence excitation and emission spectra, decay curve (lifetimes), CIE coordinates and the internal quantum efficiencies. The excitation spectra can match well with the emission light of near UV-LED chips (360–400 nm). Na{sub 2}Ca{sub 3}Si{sub 6}O{sub 16}:Eu{sup 2+} presents a symmetric emission band from 4f{sup 6}5d{sup 1} ⟶ 4f{sup 7}({sup 8}S{sub 7/2}) transitions of Eu{sup 2+} ions on doping below 3.0 mol%. On increasing Eu-doping levels, the sample contains two kinds of emission centers, i.e., Eu{sup 2+} and Eu{sup 3+} ions, which present the characteristic broad band (5d ⟶ 4f) and narrower (4f ⟶ 4f) luminescence lines, respectively. The energy transfer, the luminescence thermal stability (activation energy ΔE for thermal quenching) and luminescence mechanism of Na{sub 2}Ca{sub 3}Si{sub 6}O{sub 16}:Eu{sup 2+} phosphors were discussed by analyzing the relationship between the luminescence characteristics and the crystal structure.

  9. Microbial processes and factors controlling their activities in alkaline lakes of the Mongolian plateau

    NASA Astrophysics Data System (ADS)

    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.

  10. Combining phosphate and bacteria removal on chemically active filter membranes allows prolonged storage of drinking water.

    PubMed

    Rotzetter, A C C; Kellenberger, C R; Schumacher, C M; Mora, C; Grass, R N; Loepfe, M; Luechinger, N A; Stark, W J

    2013-11-13

    A chemically active filtration membrane with incorporated lanthanum oxide nanoparticles enables the removal of bacteria and phosphate at the same time and thus provides a simple device for preparation of drinking water and subsequent safe storage without using any kind of disinfectants. PMID:23913409

  11. Biochemical methods for quantifying sphingolipids: ceramide, sphingosine, sphingosine kinase-1 activity, and sphingosine-1-phosphate.

    PubMed

    Brizuela, Leyre; Cuvillier, Olivier

    2012-01-01

    Sphingolipids (ceramide, sphingosine, and sphingosine-1-phosphate) are bioactive lipids with important biological functions in proliferation, apoptosis, angiogenesis, and inflammation. Herein, we describe easy and rapid biochemical methods with the use of radiolabeled molecules ((3)H, (32)P) for their mass determination. Quantitation of sphingosine kinase-1 activity, the most studied isoform, is also included. PMID:22528435

  12. Measurement of the gain in a disk amplification stage with neodymium phosphate glass active elements

    SciTech Connect

    Voronich, Ivan N; Galakhov, I V; Garanin, Sergey G; Eroshenko, V A; Zaretskii, Aleksei I; Zimalin, B G; Ignat'ev, Ivan V; Kirdyashkin, M Yu; Kirillov, G A; Osin, Vladimir A; Rukavishnikov, N N; Sukharev, Stanislav A; Sharov, Oleg A; Charukhchev, Aleksandr V

    2003-06-30

    The measuring technique is described and time-resolved measurements of the small-signal gain as a function of the pump energy in a disk amplification stage with neodymium phosphate glass active elements in the 'Luch' facility are presented. The distribution of the gain over the amplifier aperture in the horizontal plane is measured. (lasers)

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

    PubMed

    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

  14. The effect of ultrasound on the catalytic activity of alkaline carbons: preparation of N-alkyl imidazoles

    NASA Astrophysics Data System (ADS)

    Durán-Valle, C. J.; Ferrera-Escudero, S.; Calvino-Casilda, V.; Díaz-Terán, J.; Martín-Aranda, R. M.

    2004-11-01

    N-Alkyl imidazoles have been prepared by sonochemical irradiation of imidazole and 1-bromobutane using alkaline promoted carbons. Under the experimental conditions, N-alkyl imidazoles can be prepared with a high activity and selectivity. It is observed that imidazole conversion increases in parallel with increasing basicity of the catalyst. For comparison, the alkylation of imidazole has also been performed in a batch reactor system under thermal activation.

  15. Fluorescent assay for alkaline phosphatase activity based on graphene oxide integrating with λ exonuclease.

    PubMed

    Liu, Xue-Guo; Xing, Xiao-Jing; Li, Bo; Guo, Yong-Ming; Zhang, Ye-Zhen; Yang, Yan; Zhang, Lian-Feng

    2016-07-15

    A novel fluorescence turn-on strategy for the alkaline phosphatase (ALP) assay is developed based on the preferential binding of graphene oxide (GO) to single-stranded DNA (ssDNA) over double-stranded DNA (dsDNA) coupled with λ exonuclease (λ exo) cleavage. Specifically, in the absence of ALP, the substrate-dsDNA constructed by one oligonucleotide with a fluorophore at the 3'-end (F-DNA) and its complementary sequence modified with a 5'-phosphoryl termini (p-DNA), is promptly cleaved by λ exo, and the resulting F-DNA is adsorbed on GO surface, allowing fluorescence quenching. Whereas the introduction of ALP leads to the hydrolysis of the P-DNA, and the yielding 5'-hydroxyl end product hampers the λ exo cleavage, inducing significant fluorescence enhancement due to the weak binding of dsDNA with GO. Under the optimized conditions, the approach exhibits high sensitivity and specificity to ALP with a detection limit of 0.19 U/L, and the determination of ALP in spiked human serum samples has also been realized. Notably, this new approach not only provides a novel and sensitive platform for the ALP activity detection but also promotes the exploitation of the GO-based biosensing for the detection of the protein with no specific binding element, and thus extending the GO-based sensing applications into a new field. PMID:27015149

  16. Electrocatalytic activities of alkyne-functionalized copper nanoparticles in oxygen reduction in alkaline media

    NASA Astrophysics Data System (ADS)

    Liu, Ke; Song, Yang; Chen, Shaowei

    2014-12-01

    Stable alkyne-capped copper nanoparticles were prepared by chemical reduction of copper acetate with sodium borohydride in the presence of alkyne ligands. Transmission electron microscopic measurements showed that nanoparticles were well dispersed with a diameter in the range of 4-6 nm. FTIR and photoluminescence spectroscopic measurements confirmed the successful attachment of the alkyne ligands onto the nanoparticle surface most likely forming Cu-Ctbnd interfacial bonds. XPS measurements indicated the formation of a small amount of CuO in the nanoparticles with a satellite peak where the binding energy red-shifted with increasing Cu(II) concentration. Cu2O was also detected in the nanoparticles. Similar results were observed with commercial CuO nanoparticles. Electrochemical studies showed that the as-prepared alkyne-capped copper nanoparticles exhibited apparent electrocatalytic activity in oxygen reduction in alkaline media, a performance that was markedly better than those reported earlier with poly- or single-crystalline copper electrodes; and the fraction of peroxides in the final products decreased with decreasing concentration of oxide components in the nanoparticles.

  17. Highly active nitrogen-doped nanocarbon electrocatalysts for alkaline direct methanol fuel cell

    NASA Astrophysics Data System (ADS)

    Kruusenberg, Ivar; Ratso, Sander; Vikkisk, Merilin; Kanninen, Petri; Kallio, Tanja; Kannan, Arunachala M.; Tammeveski, Kaido

    2015-05-01

    Direct methanol fuel cells are assembled and evaluated using Fumatech FAA3 alkaline anion exchange membrane. Two novel metal-free cathode catalysts are synthesised, investigated and compared with the commercial Pt-based catalyst. In this work nitrogen-doped few-layer graphene/multi-walled carbon nanotube (N-FLG/MWCNT) composite and nitrogen-doped MWCNT (N-MWCNT) catalyst are prepared by pyrolysing the mixture of dicyandiamide (DCDA) and carbon nanomaterials at 800 °C. The resulting cathode catalyst material shows a remarkable electrocatalytic activity for oxygen reduction reaction (ORR) in 0.1 M KOH solution employing the rotating disk electrode (RDE) method. Fuel cell tests are performed by using 1 M methanol as anode and pure oxygen gas cathode feed. The maximum power density obtained with the N-FLG/MWCNT material (0.72 mW cm-2) is similar to that of the Pt/C catalyst (0.72 mW cm-2), whereas the N-MWCNT material shows higher peak power density (0.92 mW cm-2) than the commercial Pt/C catalyst.

  18. Glucose-6-phosphate mediates activation of the carbohydrate responsive binding protein (ChREBP)

    SciTech Connect

    Li, Ming V.; Chen, Weiqin; Harmancey, Romain N.; Nuotio-Antar, Alli M.; Imamura, Minako; Saha, Pradip; Taegtmeyer, Heinrich; Chan, Lawrence

    2010-05-07

    Carbohydrate response element binding protein (ChREBP) is a Mondo family transcription factor that activates a number of glycolytic and lipogenic genes in response to glucose stimulation. We have previously reported that high glucose can activate the transcriptional activity of ChREBP independent of the protein phosphatase 2A (PP2A)-mediated increase in nuclear entry and DNA binding. Here, we found that formation of glucose-6-phosphate (G-6-P) is essential for glucose activation of ChREBP. The glucose response of GAL4-ChREBP is attenuated by D-mannoheptulose, a potent hexokinase inhibitor, as well as over-expression of glucose-6-phosphatase (G6Pase); kinetics of activation of GAL4-ChREBP can be modified by exogenously expressed GCK. Further metabolism of G-6-P through the two major glucose metabolic pathways, glycolysis and pentose-phosphate pathway, is not required for activation of ChREBP; over-expression of glucose-6-phosphate dehydrogenase (G6PD) diminishes, whereas RNAi knockdown of the enzyme enhances, the glucose response of GAL4-ChREBP, respectively. Moreover, the glucose analogue 2-deoxyglucose (2-DG), which is phosphorylated by hexokinase, but not further metabolized, effectively upregulates the transcription activity of ChREBP. In addition, over-expression of phosphofructokinase (PFK) 1 and 2, synergistically diminishes the glucose response of GAL4-ChREBP. These multiple lines of evidence support the conclusion that G-6-P mediates the activation of ChREBP.

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

    PubMed

    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

  20. Ureaplasma urealyticum-induced crystallization of magnesium ammonium phosphate and calcium phosphates in synthetic urine.

    PubMed

    Grenabo, L; Brorson, J E; Hedelin, H; Pettersson, S

    1984-10-01

    Crystallization of struvite and calcium phosphates was studied in vitro as encrustations on glass rods immersed in synthetic urine, to evaluate the crystallization capacity of Ureaplasma urealyticum and compare it with that of known urease and non-urease-producing bacteria. Inoculation of the synthetic urine with Ureaplasma urealyticum resulted in alkalinization of the synthetic urine and crystallization of struvite and brushite. Inoculation with Proteus mirabilis caused a faster and more pronounced alkalinization as well as crystallization of struvite and apatite. The alkalinization and crystallization caused by Ureaplasma urealyticum and Proteus mirabilis was completely prevented by acetohydroxamic acid, a potent urease inhibitor, linking the crystallization to the urease activity of the microorganisms. When the synthetic urine was inoculated with urease-negative Escherichia coli no alkalinization and no crystallization were seen. PMID:6381769

  1. Properties of Ce-activated alkali-lutetium double phosphate scintillators

    SciTech Connect

    Wiśniewski, D.; Wojtowicz, A. J.; Boatner, Lynn A

    2010-01-01

    The scintillation properties of Ce-activated alkali-lutetium double phosphate single crystals that vary with the alkali ion type and activation level are summarized and compared. The materials investigated here have been identified as fast and efficient scintillators for the detection of x-ray and radiation, and in case of Li3Lu(PO4)2:Ce, for thermal neutron detection as well.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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

  4. Hydrogen-rich water affected blood alkalinity in physically active men.

    PubMed

    Ostojic, Sergej M; Stojanovic, Marko D

    2014-01-01

    Possible appliance of effective and safe alkalizing agent in the treatment of metabolic acidosis could be of particular interest to humans experiencing an increase in plasma acidity, such as exercise-induced acidosis. In the present study we tested the hypothesis that the daily oral intake of 2L of hydrogen-rich water (HRW) for 14 days would increase arterial blood alkalinity at baseline and post-exercise as compared with the placebo. This study was a randomized, double blind, placebo-controlled trial involving 52 presumably healthy physically active male volunteers. Twenty-six participants received HRW and 26 a placebo (tap water) for 14 days. Arterial blood pH, partial pressure for carbon dioxide (pCO2), and bicarbonates were measured at baseline and postexercise at the start (day 0) and at the end of the intervention period (day 14). Intake of HRW significantly increased fasting arterial blood pH by 0.04 (95% confidence interval; 0.01 - 0.08; p < 0.001), and postexercise pH by 0.07 (95% confidence interval; 0.01 - 0.10; p = 0.03) after 14 days of intervention. Fasting bicarbonates were significantly higher in the HRW trial after the administration regimen as compared with the preadministration (30.5 ± 1.9 mEq/L vs. 28.3 ± 2.3 mEq/L; p < 0.0001). No volunteers withdrew before the end of the study, and no participant reported any vexatious side effects of supplementation. These results support the hypothesis that HRW administration is safe and may have an alkalizing effect in young physically active men. PMID:24392771

  5. Determination of phosphate in natural waters by activation analysis of tungstophosphoric acid

    USGS Publications Warehouse

    Allen, Herbert E.; Hahn, Richard B.

    1969-01-01

    Activation analysis may be used to determine quantitatively traces of phosphate in natural waters. Methods based on the reaction 31P(n,γ)32P are subject to interference by sulfur and chlorine which give rise to 32P through n,p and n,α reactions. If the ratio of phosphorus to sulfur or chlorine is small, as it is in most natural waters, accurate analyses by these methods are difficult to achieve. In the activation analysis method, molybdate and tungstate ions are added to samples containing phosphate ion to form tungstomolybdophosphoric acid. The complex is extracted with 2,6-dimethyl-4-heptanone. After activation of an aliquot of the organic phase for 1 hour at a flux of 1013 neutrons per cm2, per second, the gamma spectrum is essentially that of tungsten-187. The induced activity is proportional to the concentration of phosphate in the sample. A test of the method showed it to give accurate results at concentrations of 4 to at least 200 p.p.b. of phosphorus when an aliquot of 100 μl. was activated. By suitable reagent purification, counting for longer times, and activation of larger aliquots, the detection limit could be lowered several hundredfold.

  6. Regulation by sphingosine 1-phosphate of Bax and Bad activities during apoptosis in a MEK-dependent manner.

    PubMed

    Betito, Susan; Cuvillier, Olivier

    2006-02-24

    Herein we report that the prosurvival sphingolipid sphingosine 1-phosphate regulates the activities of both Bad and Bax during apoptosis of Jurkat cells. First, sphingosine 1-phosphate treatment results in Bad inactivation via the ERK/Rsk-1 pathway. Second, sphingosine 1-phosphate blocks the translocation of Bax to the mitochondria induced by Fas ligation. MEK inhibition by PD98059 or U0126 not only abrogates sphingosine 1-phosphate-induced Bad phosphorylation, but also its cytoprotective effect. Furthermore, inhibition of both mitochondrial cytochrome c efflux and Bax translocation to the mitochondria by sphingosine 1-phosphate could be overcome by PD98059 or U0126. Hence, the MEK/ERK pathway seems to be crucial for the survival effects initiated by sphingosine 1-phosphate. PMID:16414356

  7. Hollow raspberry-like PdAg alloy nanospheres: High electrocatalytic activity for ethanol oxidation in alkaline media

    NASA Astrophysics Data System (ADS)

    Peng, Cheng; Hu, Yongli; Liu, Mingrui; Zheng, Yixiong

    2015-03-01

    Palladium-silver (PdAg) alloy nanospheres with unique structure were prepared using a one-pot procedure based on the galvanic replacement reaction. Their electrocatalytic activity for ethanol oxidation in alkaline media was evaluated. The morphology and crystal structure of the samples were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and X-ray diffraction (XRD). Electrochemical characterization techniques, including cyclic voltammetry (CV) and chronoamperometry (CA) measurements were used to analyze the electrochemical performance of the PdAg alloy nanospheres. The SEM and TEM images showed that the PdAg alloy nanospheres exhibit a hierarchical nanostructure with hollow interiors and porous walls. Compared to the commercial Pd/C catalyst, the as-prepared PdAg alloy nanospheres exhibit superior electrocatalytic activity and stability towards ethanol electro-oxidation in alkaline media, showing its potential as a new non-Pt electro-catalyst for direct alcohol fuel cells (DAFCs).

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

    SciTech Connect

    Kato, Y.; Shimazu, A.; Nakashima, K.; Suzuki, F.; Jikko, A.; Iwamoto, M. )

    1990-07-01

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

  9. Toxicity mitigation and solidification of municipal solid waste incinerator fly ash using alkaline activated coal ash

    SciTech Connect

    Ivan Diaz-Loya, E.; Allouche, Erez N.; Eklund, Sven; Joshi, Anupam R.; Kupwade-Patil, Kunal

    2012-08-15

    Highlights: Black-Right-Pointing-Pointer Incinerator fly ash (IFA) is added to an alkali activated coal fly ash (CFA) matrix. Black-Right-Pointing-Pointer Means of stabilizing the incinerator ash for use in construction applications. Black-Right-Pointing-Pointer Concrete made from IFA, CFA and IFA-CFA mixes was chemically characterized. Black-Right-Pointing-Pointer Environmentally friendly solution to IFA disposal by reducing its toxicity levels. - Abstract: Municipal solid waste (MSW) incineration is a common and effective practice to reduce the volume of solid waste in urban areas. However, the byproduct of this process is a fly ash (IFA), which contains large quantities of toxic contaminants. The purpose of this research study was to analyze the chemical, physical and mechanical behaviors resulting from the gradual introduction of IFA to an alkaline activated coal fly ash (CFA) matrix, as a mean of stabilizing the incinerator ash for use in industrial construction applications, where human exposure potential is limited. IFA and CFA were analyzed via X-ray fluorescence (XRF), X-ray diffraction (XRD) and Inductive coupled plasma (ICP) to obtain a full chemical analysis of the samples, its crystallographic characteristics and a detailed count of the eight heavy metals contemplated in US Title 40 of the Code of Federal Regulations (40 CFR). The particle size distribution of IFA and CFA was also recorded. EPA's Toxicity Characteristic Leaching Procedure (TCLP) was followed to monitor the leachability of the contaminants before and after the activation. Also images obtained via Scanning Electron Microscopy (SEM), before and after the activation, are presented. Concrete made from IFA, CFA and IFA-CFA mixes was subjected to a full mechanical characterization; tests include compressive strength, flexural strength, elastic modulus, Poisson's ratio and setting time. The leachable heavy metal contents (except for Se) were below the maximum allowable limits and in many cases

  10. Measurements of metabolically active inorganic phosphate in plants growing in natural and agronomic settings and under water stress. [Stromal Phosphate

    SciTech Connect

    Sharkey, T.D.

    1988-01-01

    At high rates of photosynthesis, the conflicting requirements of adenosine triphosphate (ATP) synthesis for phosphate and starch and sucrose synthesis for low phosphate, may limit the overall rate of photosynthesis. This is called feedback limitation of photosynthesis. A nonaqueous fractionation technique was used to measure stromal phosphate levels without contamination from vacuolar phosphate. Under normal conditions the stromal phosphate level was found to be 7mM. Under feedback limited photosynthesis, this value dropped to <1mM. In a related study, the effect of water stress on photosynthesis was examined. Water stress was shown to cause a decrease in total leaf photosynthesis, due not to a total loss of photosynthetic ability, but rather due to photosynthesis only occurring in patches of the leaf. Water stress was shown to cause a reduction in starch and sucrose synthesis. Since this decline can be reversed by increasing the CO{sub 2} level around the plant, this is proposed to be due to closing of stomata due to the water stress. (MHB)

  11. Leucine aminopeptidase, beta-glucosidase and alkaline phosphatase activity rates and their significance in nutrient cycles in some coastal Mediterranean sites.

    PubMed

    Caruso, Gabriella

    2010-01-01

    In aquatic microbial ecology, knowledge of the processes involved in the turnover of organic matter is of utmost importance to understand ecosystem functioning. Microorganisms are major players in the cycling of nutrients (nitrogen, phosphorus) and carbon, thanks to their enzymatic activities (leucine aminopeptidase, LAP, alkaline phosphatase, AP, and beta-glucosidase, beta-GLU) on organic polymers (proteins, organic phosphates and polysaccharides, respectively). Estimates of the decomposition rates of organic polymers are performed using fluorogenic compounds, whose hydrolysis rate allow us to obtain information on the "potential" metabolic activity of the prokaryotic community. This paper refers the enzyme patterns measured during recent oceanographic cruises performed in some coastal Mediterranean sites, not yet fully investigated in terms of microbial biogeochemical processes. Mean enzyme activity rates ranged from 5.24 to 5558.1 nM/h, from 12.68 to 244.73 nM/h and from 0.006 to 9.51 nM/h for LAP, AP and beta-GLU, respectively. The highest LAP and AP activity rates were measured in the Gulf of Milazzo (Tyrrhenian Sea) and in the Straits of Messina, in association with the lowest bacterioplankton abundance; in contrast, the lowest ones were found in the northern Adriatic Sea. beta-GLU was more active in the Straits of Messina. Activity rates were analysed in relation to the main environmental variables. Along the northern Adriatic coastal side affected by the Po river, significant inverse relationships linked LAP and AP with salinity, pointing out that fluvial inputs provided organic substrates for microbial metabolism. Both in the Gulf of Manfredonia and in the Straits of Messina, LAP and AP levels were inversely related with the concentration of nitrate and inorganic phosphorus, respectively. In the Gulf of Milazzo, high cell-specific AP measured in spite of phosphorus availability suggested the role of this enzyme not only in phosphorus, but also in carbon

  12. Leucine Aminopeptidase, β-Glucosidase and Alkaline Phosphatase Activity Rates and Their Significance in Nutrient Cycles in Some Coastal Mediterranean Sites

    PubMed Central

    Caruso, Gabriella

    2010-01-01

    In aquatic microbial ecology, knowledge of the processes involved in the turnover of organic matter is of utmost importance to understand ecosystem functioning. Microorganisms are major players in the cycling of nutrients (nitrogen, phosphorus) and carbon, thanks to their enzymatic activities (leucine aminopeptidase, LAP, alkaline phosphatase, AP, and β-glucosidase, β-GLU) on organic polymers (proteins, organic phosphates and polysaccharides, respectively). Estimates of the decomposition rates of organic polymers are performed using fluorogenic compounds, whose hydrolysis rate allow us to obtain information on the “potential” metabolic activity of the prokaryotic community. This paper refers the enzyme patterns measured during recent oceanographic cruises performed in some coastal Mediterranean sites, not yet fully investigated in terms of microbial biogeochemical processes. Mean enzyme activity rates ranged from 5.24 to 5558.1 nM/h, from 12.68 to 244.73 nM/h and from 0.006 to 9.51 nM/h for LAP, AP and β-GLU, respectively. The highest LAP and AP activity rates were measured in the Gulf of Milazzo (Tyrrhenian Sea) and in the Straits of Messina, in association with the lowest bacterioplankton abundance; in contrast, the lowest ones were found in the northern Adriatic Sea. β-GLU was more active in the Straits of Messina. Activity rates were analysed in relation to the main environmental variables. Along the northern Adriatic coastal side affected by the Po river, significant inverse relationships linked LAP and AP with salinity, pointing out that fluvial inputs provided organic substrates for microbial metabolism. Both in the Gulf of Manfredonia and in the Straits of Messina, LAP and AP levels were inversely related with the concentration of nitrate and inorganic phosphorus, respectively. In the Gulf of Milazzo, high cell-specific AP measured in spite of phosphorus availability suggested the role of this enzyme not only in phosphorus, but also in carbon

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

    PubMed

    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

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

    PubMed Central

    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

  15. Toxicity mitigation and solidification of municipal solid waste incinerator fly ash using alkaline activated coal ash.

    PubMed

    Diaz-Loya, E Ivan; Allouche, Erez N; Eklund, Sven; Joshi, Anupam R; Kupwade-Patil, Kunal

    2012-08-01

    Municipal solid waste (MSW) incineration is a common and effective practice to reduce the volume of solid waste in urban areas. However, the byproduct of this process is a fly ash (IFA), which contains large quantities of toxic contaminants. The purpose of this research study was to analyze the chemical, physical and mechanical behaviors resulting from the gradual introduction of IFA to an alkaline activated coal fly ash (CFA) matrix, as a mean of stabilizing the incinerator ash for use in industrial construction applications, where human exposure potential is limited. IFA and CFA were analyzed via X-ray fluorescence (XRF), X-ray diffraction (XRD) and Inductive coupled plasma (ICP) to obtain a full chemical analysis of the samples, its crystallographic characteristics and a detailed count of the eight heavy metals contemplated in US Title 40 of the Code of Federal Regulations (40 CFR). The particle size distribution of IFA and CFA was also recorded. EPA's Toxicity Characteristic Leaching Procedure (TCLP) was followed to monitor the leachability of the contaminants before and after the activation. Also images obtained via Scanning Electron Microscopy (SEM), before and after the activation, are presented. Concrete made from IFA, CFA and IFA-CFA mixes was subjected to a full mechanical characterization; tests include compressive strength, flexural strength, elastic modulus, Poisson's ratio and setting time. The leachable heavy metal contents (except for Se) were below the maximum allowable limits and in many cases even below the reporting limit. The leachable Chromium was reduced from 0.153 down to 0.0045 mg/L, Arsenic from 0.256 down to 0.132 mg/L, Selenium from 1.05 down to 0.29 mg/L, Silver from 0.011 down to .001 mg/L, Barium from 2.06 down to 0.314 mg/L and Mercury from 0.007 down to 0.001 mg/L. Although the leachable Cd exhibited an increase from 0.49 up to 0.805 mg/L and Pd from 0.002 up to 0.029 mg/L, these were well below the maximum limits of 1.00 and 5

  16. Order of Activity of Nitrogen, Iron Oxide, and FeNx Complexes towards Oxygen Reduction in Alkaline Medium.

    PubMed

    Zhu, Yansong; Zhang, Bingsen; Wang, Da-Wei; Su, Dang Sheng

    2015-12-01

    In alkaline medium, it seems that both metal-free and iron-containing carbon-based catalysts, such as nitrogen-doped nanocarbon materials, FeOx -doped carbon, and Fe/N/C catalysts, are active for the oxygen reduction reaction (ORR). However, the order of activity of these different active compositions has not been clearly determined. Herein, we synthesized nitrogen-doped carbon black (NCB), Fe3 O4 /CB, Fe3 O4 /NCB, and FeN4 /CB. Through the systematic study of the ORR catalytic activity of these four catalysts in alkaline solution, we confirmed the difference in the catalytic activity and catalytic mechanism for nitrogen, iron oxides, and Fe-N complexes, respectively. In metal-free NCB, nitrogen can improve the ORR catalytic activity with a four-electron pathway. Fe3 O4 /CB catalyst did not exhibit improved activity over that of NCB owing to the poor conductivity and spinel structure of Fe3 O4 . However, FeN4 coordination compounds as the active sites showed excellent ORR catalytic activity. PMID:26609795

  17. Phosphate oxygen isotopic evidence for a temperate and biologically active Archaean ocean.

    PubMed

    Blake, Ruth E; Chang, Sae Jung; Lepland, Aivo

    2010-04-15

    Oxygen and silicon isotope compositions of cherts and studies of protein evolution have been interpreted to reflect ocean temperatures of 55-85 degrees C during the early Palaeoarchaean era ( approximately 3.5 billion years ago). A recent study combining oxygen and hydrogen isotope compositions of cherts, however, makes a case for Archaean ocean temperatures being no greater than 40 degrees C (ref. 5). Ocean temperature can also be assessed using the oxygen isotope composition of phosphate. Recent studies show that (18)O:(16)O ratios of dissolved inorganic phosphate (delta(18)O(P)) reflect ambient seawater temperature as well as biological processing that dominates marine phosphorus cycling at low temperature. All forms of life require and concentrate phosphorus, and as a result of biological processing, modern marine phosphates have delta(18)O(P) values typically between 19-26 per thousand (VSMOW), highly evolved from presumed source values of approximately 6-8 per thousand that are characteristic of apatite in igneous rocks and meteorites. Here we report oxygen isotope compositions of phosphates in sediments from the 3.2-3.5-billion-year-old Barberton Greenstone Belt in South Africa. We find that delta(18)O(P) values range from 9.3 per thousand to 19.9 per thousand and include the highest values reported for Archaean rocks. The temperatures calculated from our highest delta(18)O(P) values and assuming equilibrium with sea water with delta(18)O = 0 per thousand (ref. 12) range from 26 degrees C to 35 degrees C. The higher delta(18)O(P) values are similar to those of modern marine phosphate and suggest a well-developed phosphorus cycle and evolved biologic activity on the Archaean Earth. PMID:20393560

  18. Recombinant production and characterization of a highly active alkaline phosphatase from marine bacterium Cobetia marina.

    PubMed

    Golotin, Vasily; Balabanova, Larissa; Likhatskaya, Galina; Rasskazov, Valery

    2015-04-01

    The psychrophilic marine bacterium, Cobetia marina, recovered from the mantle tissue of the marine mussel, Crenomytilus grayanus, which contained a gene encoding alkaline phosphatase (AP) with apparent biotechnology advantages. The enzyme was found to be more efficient than its counterparts and showed k cat value 10- to 100-fold higher than those of all known commercial APs. The enzyme did not require the presence of exogenous divalent cations and dimeric state of its molecule for activity. The recombinant enzyme (CmAP) production and purification were optimized with a final recovery of 2 mg of the homogenous protein from 1 L of the transgenic Escherichia coli Rosetta(DE3)/Pho40 cells culture. CmAP displayed a half-life of 16 min at 45 °C and 27 min at 40 °C in the presence of 2 mM EDTA, thus suggesting its relative thermostability in comparison with the known cold-adapted analogues. A high concentration of EDTA in the incubation mixture did not appreciably inhibit CmAP. The enzyme was stable in a wide range of pH (6.0-11.0). CmAP exhibited its highest activity at the reaction temperature of 40-50 °C and pH 9.5-10.3. The structural features of CmAP could be the reason for the increase in its stability and catalytic turnover. We have modeled the CmAP 3D structure on the base of the high-quality experimental structure of the close homologue Vibrio sp. AP (VAP) and mutated essential residues predicted to break Mg(2+) bonds in CmAP. It seems probable that the intrinsically tight binding of catalytic and structural metal ions together with the flexibility of intermolecular and intramolecular links in CmAP could be attributed to the adapted mutualistic lifestyle in oceanic waters. PMID:25260971

  19. Reproducible quantification of osteoclastic activity: characterization of a biomimetic calcium phosphate assay.

    PubMed

    Maria, Salwa M; Prukner, Christiane; Sheikh, Zeeshan; Mueller, Frank; Barralet, Jake E; Komarova, Svetlana V

    2014-07-01

    Osteoclasts are responsible for bone and joint destruction in rheumatoid arthritis, periodontitis, and osteoporosis. Animal tusk slice assays are standard for evaluating the effect of therapeutics on these cells. However, in addition to batch-to-batch variability inherent to animal tusks, their use is clearly not sustainable. Our objective was to develop and characterize a biomimetic calcium phosphate assay based on the use of phase pure hydroxyapatite coated as a thin film on the surface of culture plates, to facilitate the reproducible quantification of osteoclast resorptive activity. Osteoclasts were formed from RAW 264.7 mouse monocyte cell line using a pro-resorptive cytokine RANKL (50 ng/mL). No change in substrate appearance was noted after culture with media without cells, or undifferentiated monocytes. Only in the presence of osteoclasts localized areas of calcium phosphate dissolution were observed. The total area resorbed positively correlated with the osteoclast numbers (R(2) = 0.99). The resorbed area was significantly increased by the addition of RANKL, and decreased after application of known inhibitors of osteoclast resorptive activity, calcitonin (10 μM), or alendronate (100 μM). Thus, calcium phosphate coated substrates allow reliable monitoring of osteoclast resorptive activity and offer an alternative to animal tusk slice assays. PMID:24259122

  20. Inactivation of highly activated spinach leaf sucrose-phosphate synthase by dephosphorylation. [Spinacia oleracea

    SciTech Connect

    Huber, J.L. ); Huber, S.C. North Carolina State Univ., Raleigh ); Hite, D.R.C.; Outlaw, W.H. Jr. )

    1991-01-01

    Spinach (Spinacia oleracea L.) leaf sucrose-phosphate synthase (SPS) can be phosphorylated and inactivated in vitro with ({gamma}-{sup 32}P)ATP. Thus, it was surprising to find that SPS, extracted from leaves fed mannose in the light to highly activate the enzyme, could be inactivated in an ATP-independent manner when desalted crude extracts were preincubated at 25{degrees}C before assay. The spontaneous inactivation involved a loss in activity measured with limiting substrate concentrations in the presence of the inhibitor, Pi, without affecting maximum catalytic activity. The spontaneous inactivation was unaffected by exogenous carrier proteins and protease inhibitors, but was inhibited by inorganic phosphate, fluoride, and molybdate, suggesting that a phosphatase may be involved. Okadaic acid, a potent inhibitor of mammalian type 1 and 2A protein phosphatases, had no effect up to 5 micromolar. Inactivation was stimulated about twofold by exogenous Mg{sup 2+} and was relatively insensitive to Ca{sup 2+} and to pH over the range pH 6.5 to 8.5. Radioactive phosphate incorporated into SPS during labeling of excised leaves with ({sup 32}P)Pi (initially in the dark and then in the light with mannose) was lost with time when desalted crude extracts were incubated at 25 C, and the loss in radiolabel was substantially reduced by fluoride. These results provide direct evidence for action of an endogenous phosphatase(s) using SPS as substrate.

  1. 2,3-diphosphoglycerate phosphatase activity of phosphoglycerate mutase: stimulation by vanadate and phosphate

    SciTech Connect

    Stankiewicz, P.J.; Gresser, M.J.; Tracey, A.S.; Hass, L.F.

    1987-03-10

    The binding of inorganic vanadate (V/sub i/) to rabbit muscle phosphoglycerate mutase (PGM), studied by using /sup 51/V nuclear magnetic resonance spectroscopy, shows a sigmoidal dependence on vanadate concentration with a stoichiometry of four vanadium atoms per PGM molecule at saturating (V/sub i/). The data are consistent with binding of one divanadate ion to each of the two subunits of PGM in a noncooperative manner with an intrinsic dissociation constant of 4 x 10/sup -6/ M. The relevance of this result to other studies which have shown that the V/sub i/-stimulated 2,3-diphosphoglycerate (2,3-DPG) phosphatase activity of PGM has a sigmoidal dependence on (V/sub i/) with a Hill coefficient of 2.0 is discussed. At pH 7.0, inorganic phosphate has little effect on the 2,3-DPG phosphatase activity of PGM, even at concentrations as high as 50 mM. Similarly, 25 ..mu..M V/sub i/ has little effect on the phosphatase activity. However, in the presence of 25 ..mu..M V/sub i/, a phosphate concentration of 20 mM increases the phosphatase activity by more than 3-fold. This behavior is rationalized in terms of activation of the phosphatase activity by a phosphate/vanadate mixed anhydride. This interpretation is supported by the observation of strong activation of the phosphatase activity by inorganic pyrophosphate. A molecular mechanism for the observed effects of vanadate is proposed, and the relevance of this study to the possible use of vanadate as a therapeutic agent for the treatment of sickle cell anemia is discussed.

  2. Glucose-6-phosphate mediates activation of the carbohydrate responsive binding protein (ChREBP)

    PubMed Central

    Li, Ming V.; Chen, Weiqin; Harmancey, Romain N; Nuotio-Antar, Alli M.; Imamura, Minako; Saha, Pradip; Taegtmeyer, Heinrich; Chan, Lawrence

    2010-01-01

    Carbohydrate Response Element Binding Protein (ChREBP) is a Mondo family transcription factor that activates a number of glycolytic and lipogenic genes in response to glucose stimulation. We have previously reported that high glucose can activate the transcriptional activity of ChREBP independent of the protein phosphatase 2A (PP2A)-mediated increase in nuclear entry and DNA binding. Here we found that formation of glucose-6-phosphate (G-6-P) is essential for glucose activation of ChREBP. The glucose response of GAL4-ChREBP is attenuated by D-mannoheptulose, a potent hexokinase inhibitor, as well as over-expression of glucose-6-phosphatase (G6Pase); kinetics of activation of GAL4-ChREBP can be modified by exogenously expressed GCK. Further metabolism of G-6-P through the two major glucose metabolic pathways, glycolysis and pentose phosphate pathway, is not required for activation of ChREBP; over-expression of glucose-6-phosphate dehydrogenase (G6PD) diminishes, whereas RNAi knockdown of the enzyme enhances, the glucose response of GAL4-ChREBP, respectively. Moreover, the glucose analogue 2-deoxyglucose (2-DG), which is phosphorylated by hexokinase, but not further metabolized, effectively upregulates the transcription activity of ChREBP. In addition, over-expression of phosphofructokinase (PFK) 1 and 2, synergistically diminishes the glucose response of GAL4-ChREBP. These multiple lines of evidence support the conclusion that G-6-P mediates the activation of ChREBP. PMID:20382127

  3. Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides. Binding of pyridoxal phosphate to 5-aminolaevulinate synthetase.

    PubMed Central

    Davies, R C; Neuberger, A

    1979-01-01

    1. Pyridoxal 5'-phosphate is a cofactor essential for the enzymic activity of aminolaevulinate synthetase from Rhodopseudomonas spheroides. It also aids activation of the low-activity enzyme by trisulphides such as cystine trisulphide, whereas inactivation of enzyme is facilitated by its absence. 2. The fluorescence spectrum of purified high-activity enzyme is that expected for a pyridoxal phosphate--Schiff base, but the firmly bound cofactor does not appear to be at the active centre. In dilute solutions of enzyme this grouping is inaccessible to nucleophiles such as glycine, hydroxylamine, borohydride and cyanide, at pH 7.4. 3. An active-centre Schiff base is formed between enzyne and added pyridoxal phosphate, which is accessible to nucleophiles. Concentrated solutions of this enzyme--Schiff base on treatment with glycine yield apo- and semi-apoenzyme, which can re-bind pyridoxal phosphate. 4. Two types of binding of pyridoxal phosphate are distinguishable in dilute solution of enzyme, but these become indistinguishable when concentrated solutions are treated with cofactor. A change occurs in the susceptibility towards borohydride of the fluorescence of the "structural" pyridoxal phosphate. 5. One or two molecules of cofactor are bound per subunit of mol. wt. 50 000 in semiapo- or holo-enzyme. The fluorescence of pyridoxamine phosphate covalently bound to enzyme also indicates one to two nmol of reducible Schiff base per 7000 units of activity in purified and partially purified samples of enzyme. 6. Cyanide does not convert high-activity into low-activity enzyme, but with the enzyme-pyridoxal phosphate complex it forms a yellow fluorescent derivative that is enzymically active. PMID:312102

  4. Mesophilic and thermophilic alkaline fermentation of waste activated sludge for hydrogen production: Focusing on homoacetogenesis.

    PubMed

    Wan, Jingjing; Jing, Yuhang; Zhang, Shicheng; Angelidaki, Irini; Luo, Gang

    2016-10-01

    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 from 19.2 mL H2/gVSS at 37 °C and pH 10-80.1 mL H2/gVSS at 55 °C and pH 10. However, the production of volatile fatty acids (mainly acetate) was higher at 37 °C and pH 10 by comparison with 55 °C and pH 10. Hydrogen consumption due to homoacetogenesis was observed at 37 °C and pH 10 but not 55 °C and pH 10. Higher expression levels of genes relating with homoacetogenesis and lower expression levels of genes relating with hydrogen production were found at 37 °C and pH 10 compared to 55 °C and pH 10. The continuous experiment demonstrated the steady-state hydrogen yield of WAS was comparable to that obtained from batch experiments at 55 °C and pH 10, and homoacetogenesis was still inhibited. However, the steady-state hydrogen yield of WAS (6.5 mL H2/gVSS) was much lower than that (19.2 mL H2/gVSS) obtained from batch experiments at 37 °C and pH 10 due to the gradual enrichment of homoacetogens as demonstrated by qPCR analysis. The high-throughput sequencing analysis of 16S rRNA genes showed that the abundance of genus Clostridium, containing several homoacetogens, was 5 times higher at 37 °C and pH 10 than 55 °C and pH 10. PMID:27420808

  5. Continuous cellularization of calcium phosphate hybrid scaffolds induced by plasma polymer activation.

    PubMed

    Bergemann, Claudia; Cornelsen, Matthias; Quade, Antje; Laube, Thorsten; Schnabelrauch, Matthias; Rebl, Henrike; Weißmann, Volker; Seitz, Hermann; Nebe, Barbara

    2016-02-01

    The generation of hybrid materials based on β-tricalcium phosphate (TCP) and various biodegradable polymers like poly(l-lactide-co-d,l-lactide) (PLA) represents a common approach to overcoming the disadvantages of pure TCP devices. These disadvantages lie in TCP's mechanical properties, such as brittleness. The positive characteristic of PLA - improvement of compressive strength of calcium phosphate scaffolds - is diametrically opposed to its cell attractiveness. Therefore, the objective of this work was to optimize osteoblast migration and cellularization inside a three-dimensionally (3D) printed, PLA polymer stabilized TCP hybrid scaffold by a plasma polymer process depositing amino groups via allylamine. MG-63 osteoblastic cells inside the 10mm hybrid scaffold were dynamically cultivated for 14days in a 3D model system integrated in a perfusion reactor. The whole TCP/PLA hybrid scaffold was continuously colonized due to plasma polymerized allylamine activation inducing the migration potential of osteoblasts. PMID:26652403

  6. MESOPOROUS IRON PHOSPHATE AS AN ACTIVE, SELECTIVE AND RECYCLABLE CATALYST FOR THE SYNTHESIS OF NOPOL BY PRINS CONDENSATION

    EPA Science Inventory


    Mesoporous iron phosphate is found to be a highly active and recyclable heterogeneous catalyst for the selective synthesis of nopol by Prins condensation of ?-pinene and paraformaldehyde in acetonitrile at 80 oC.



  7. Sewage sludge pretreatment by microwave irradiation combined with activated carbon fibre at alkaline pH for anaerobic digestion.

    PubMed

    Sun, Dedong; Guo, Sixiao; Ma, Nina; Wang, Guowen; Ma, Chun; Hao, Jun; Xue, Mang; Zhang, Xinxin

    2016-01-01

    This research focuses on the effects of microwave-assisted activated carbon fibre (ACF) (MW-ACF) treatment on sewage sludge at alkaline pH. The disintegration and biodegradability of sewage sludge were studied. It was found that the MW-ACF process at alkaline pH provided a rapid and efficient process to disrupt the microbial cells in the sludge. The results suggested that when irradiated at 800 W MW for 110 s with a dose of 1.0 g ACF/g solid concentration (SS) at pH 10.5, the MW-ACF pretreatment achieved 55% SS disintegration, 23% greater than the value of MW alone (32%). The concentration of total nitrogen, total phosphorus, supernatant soluble chemical oxygen demand, protein, and polysaccharide increased by 60%, 144%, 145%, 74%, and 77%, respectively. An increase in biogas production by 63.7% was achieved after 20 days of anaerobic digestion (AD), compared to the control. The results indicated that the MW-ACF pretreatment process at alkaline pH provides novel sludge management options in disintegration of sewage sludge for further AD. PMID:27332832

  8. The utilization of fructose by Escherichia coli. Properties of a mutant defective in fructose 1-phosphate kinase activity.

    PubMed

    Ferenci, T; Kornberg, H L

    1973-02-01

    1. The isolation and properties of a mutant of Escherichia coli devoid of fructose 1-phosphate kinase activity are described. 2. This mutant grew in media containing any one of a variety of substances, including hexoses, hexose 6-phosphates, sugar acids and glucogenic substrates, at rates not significantly different from those at which the parent organism grew on these substrates. However, only the parent grew on fructose or fructose 1-phosphate. 3. Fructose and fructose 1-phosphate inhibit the growth of the mutant, but not of its parent, on other carbon sources. 4. Even though not previously exposed to fructose, the mutant took up [(14)C]fructose rapidly but to only a small extent: [(14)C]fructose 1-phosphate was identified as the predominant labelled product. In contrast, the equally rapid but more extensive uptake of [(14)C]fructose by the parent organism required prior growth in the presence of fructose. PMID:4579702

  9. Bifunctional enhancement of oxygen reduction reaction activity on Ag catalysts due to water activation on LaMnO3 supports in alkaline media

    NASA Astrophysics Data System (ADS)

    Park, Shin-Ae; Lee, Eun-Kyung; Song, Hannah; Kim, Yong-Tae

    2015-08-01

    Ag is considered to be one of the best candidates for oxygen reduction reaction electrocatalysts in alkaline media for application in various electrochemical energy devices. In this study, we demonstrate that water activation is a key factor in enhancing the ORR activity in alkaline media, unlike in acid environments. Ag supported on LaMnO3 having a high oxophilicity showed a markedly higher ORR activity than that on carbon with inert surfaces. Through various electrochemical tests, it was revealed that the origin of the enhanced ORR activity of Ag/LaMnO3 is the bifunctional effect mainly due to the water activation at the interface between Ag and LaMnO3. Furthermore, the ligand effect due to the charge transfer from Mn to Ag leads to the enhancement of both oxygen activation on Ag and water activation on Mn sites, and hence, an improvement in the ORR activity of Ag/LaMnO3. On the other hand, the strain effect based on the fine structure variation in the lattice was negligible. We therefore suggest that the employment of a co-catalyst or support with highly oxophilic nature and the maximization of the interface between catalyst and support should be considered in the design of electrocatalysts for the ORR in alkaline media.

  10. Bifunctional enhancement of oxygen reduction reaction activity on Ag catalysts due to water activation on LaMnO3 supports in alkaline media.

    PubMed

    Park, Shin-Ae; Lee, Eun-Kyung; Song, Hannah; Kim, Yong-Tae

    2015-01-01

    Ag is considered to be one of the best candidates for oxygen reduction reaction electrocatalysts in alkaline media for application in various electrochemical energy devices. In this study, we demonstrate that water activation is a key factor in enhancing the ORR activity in alkaline media, unlike in acid environments. Ag supported on LaMnO3 having a high oxophilicity showed a markedly higher ORR activity than that on carbon with inert surfaces. Through various electrochemical tests, it was revealed that the origin of the enhanced ORR activity of Ag/LaMnO3 is the bifunctional effect mainly due to the water activation at the interface between Ag and LaMnO3. Furthermore, the ligand effect due to the charge transfer from Mn to Ag leads to the enhancement of both oxygen activation on Ag and water activation on Mn sites, and hence, an improvement in the ORR activity of Ag/LaMnO3. On the other hand, the strain effect based on the fine structure variation in the lattice was negligible. We therefore suggest that the employment of a co-catalyst or support with highly oxophilic nature and the maximization of the interface between catalyst and support should be considered in the design of electrocatalysts for the ORR in alkaline media. PMID:26310526

  11. Bifunctional enhancement of oxygen reduction reaction activity on Ag catalysts due to water activation on LaMnO3 supports in alkaline media

    PubMed Central

    Park, Shin-Ae; Lee, Eun-Kyung; Song, Hannah; Kim, Yong-Tae

    2015-01-01

    Ag is considered to be one of the best candidates for oxygen reduction reaction electrocatalysts in alkaline media for application in various electrochemical energy devices. In this study, we demonstrate that water activation is a key factor in enhancing the ORR activity in alkaline media, unlike in acid environments. Ag supported on LaMnO3 having a high oxophilicity showed a markedly higher ORR activity than that on carbon with inert surfaces. Through various electrochemical tests, it was revealed that the origin of the enhanced ORR activity of Ag/LaMnO3 is the bifunctional effect mainly due to the water activation at the interface between Ag and LaMnO3. Furthermore, the ligand effect due to the charge transfer from Mn to Ag leads to the enhancement of both oxygen activation on Ag and water activation on Mn sites, and hence, an improvement in the ORR activity of Ag/LaMnO3. On the other hand, the strain effect based on the fine structure variation in the lattice was negligible. We therefore suggest that the employment of a co-catalyst or support with highly oxophilic nature and the maximization of the interface between catalyst and support should be considered in the design of electrocatalysts for the ORR in alkaline media. PMID:26310526

  12. Biocontrol activity of an alkaline serine protease from Aureobasidium pullulans expressed in Pichia pastoris against four postharvest pathogens on apple.

    PubMed

    Banani, Houda; Spadaro, Davide; Zhang, Dianpeng; Matic, Slavica; Garibaldi, Angelo; Gullino, Maria Lodovica

    2014-07-16

    The yeast-like fungus Aureobasidium pullulans PL5 is a microbial antagonist against postharvest pathogens of fruits. The strain is able to produce hydrolases, including glucanases, chitinases and proteases. The alkaline serine protease gene ALP5 from A. pullulans was cloned, inserted into the vector pPIC9 to construct pPIC9/ALP5, and then expressed in Pichia pastoris strain KM71. ALP5 had a molecular mass of 42.9kDa after 5days growth with 1% methanol induction at 28°C. The recombinant protease expressed in P. pastoris showed its highest activity under alkaline conditions (at pH10) and a temperature of 50°C. The antifungal activity of the recombinant protease was investigated against Penicillium expansum, Botrytis cinerea, Monilinia fructicola and Alternaria alternata in vitro and on apple. The recombinant protease reduced significantly the spore germination and the germ tube length of the tested pathogens in PDB medium. The highest level of protease efficacy was observed against M. fructicola and B. cinerea, whereas a lower efficacy was observed against P. expansum and A. alternata indicating a possible effect of the pathogen cell wall composition on the proteolytic activity of the recombinant protease. The presence of protease was able to cause the swelling of the hyphae of B. cinerea, under an optical microscope. The recombinant protease expressed in P. pastoris was more active against the pathogens in vitro than the same enzyme expressed in E. coli in previous studies. The efficacy of ALP5 was also evaluated against the pathogens in vivo on cv Golden Delicious apples. The protease was more efficient in controlling M. fructicola, B. cinerea and P. expansum than A. alternata. However, the extent of the activity was dependent on the enzyme concentration and the length of fruit storage. This study demonstrated the capacity of the alkaline serine protease to keep its enzymatic activity for some days in the unfavorable environment of the fruit wounds. The alkaline

  13. Mutational analysis of the active site of indoleglycerol phosphate synthase from Escherichia coli.

    PubMed Central

    Darimont, B.; Stehlin, C.; Szadkowski, H.; Kirschner, K.

    1998-01-01

    Indoleglycerol phosphate synthase catalyzes the ring closure of 1-(2-carboxyphenylamino)-1-deoxyribulose 5'-phosphate to indoleglycerol phosphate, the fifth step in the pathway of tryptophan biosynthesis from chorismate. Because chemical synthesis of indole derivatives from arylamino ketones requires drastic solvent conditions, it is interesting by what mechanism the enzyme catalyzes the same condensation reaction. Seven invariant polar residues in the active site of the enzyme from Escherichia coli have been mutated directly or randomly, to identify the catalytically essential ones. A strain of E. coli suitable for selecting and classifying active mutants by functional complementation was constructed by precise deletion of the trpC gene from the genome. Judged by growth rates of transformants on selective media, mutants with either S58 or S60 replaced by alanine were indistinguishable from the wild-type, but R186 replaced by alanine was still partially active. Saturation random mutagenesis of individual codons showed that E53 was partially replaceable by aspartate and cysteine, whereas K114, E163, and N184 could not be replaced by any other residue. Partially active mutant proteins were purified and their steady-state kinetic and inhibitor binding constants determined. Their relative catalytic efficiencies paralleled their relative complementation efficiencies. These results are compatible with the location of the essential residues in the active site of the enzyme and support a chemically plausible catalytic mechanism. It involves two enzyme-bound intermediates and general acid-base catalysis by K114 and E163 with the support of E53 and N184. PMID:9605328

  14. Phosphate oxygen isotope ratio proxy for specific microbial activity in marine sediments (Peru Margin)

    NASA Astrophysics Data System (ADS)

    Liang, Y.; Blake, R. E.

    2005-12-01

    Oxygen (O) isotope ratios of biogenic apatites have been widely used as paleotemperature and environmental geochemical proxies. With improved knowledge of the phosphate O isotope effects of different P cycling pathways, the δ18O value of inorganic phosphate (δ18OP) has been proposed as a useful proxy and tracer of biological reactions and P cycling in natural environments[1,2,3,4]. Being the only way of removing P from oceanic water, sedimentary P burial is one of the most important processes during biogeochemical cycling of P. The high concentrations of organic matter and pronounced microbial activity at ODP Site 1230 along the Peru Margin result in unusually high interstitial water phosphate concentrations, which provides a unique opportunity to use δ18OP to investigate inorganic phosphate (Pi) regeneration and P cycling pathways in marine sediments. The isotopic measurements of both dissolved inorganic phosphate (DIP) and bulk sediment Pi show that DIP δ18OP values are affected by three different processes, which are all induced by specific microbial activities present in the sediments. In sediments at ~ 65 to 120 mbsf, porewater DIP is derived from dissolved organophosphorus compounds (DOP) through enzymatic degradation pathways, evidenced by both DIP δ18OP values and interstitial water chemistry. Measured porewater DIP δ18OP values also suggest that 4 to 8% of interstitial water DIP reflects regeneration of Pi from Porg by microbially-synthesized enzymes. Throughout the sediment column and especially at ~ 120 to 150 mbsf, DIP is released from the sediments by microbially-induced reductive dissolution of Fe-oxides, which contributes to the overall high DIP concentrations at Site 1230. The third and dominant process controlling measured DIP δ18OP values is microbial turnover of regenerated Pi. The presence of high microbial activities in organic-rich Site 1230 sediments promotes the remobilization of P and affects marine P cycling by potentially enhancing

  15. TWEAK favors phosphate-induced calcification of vascular smooth muscle cells through canonical and non-canonical activation of NFκB

    PubMed Central

    Hénaut, L; Sanz, A B; Martin-Sanchez, D; Carrasco, S; Villa-Bellosta, R; Aldamiz-Echevarria, G; Massy, Z A; Sanchez-Nino, M D; Ortiz, A

    2016-01-01

    Vascular calcification (VC) is associated with increased cardiovascular mortality in aging, chronic kidney disease (CKD), type 2 diabetes mellitus (T2DM) and atherosclerosis. TNF-like weak inducer of apoptosis (TWEAK) recently emerged as a new biomarker for the diagnosis and prognosis of cardiovascular diseases. TWEAK binding to its functional receptor Fn14 was reported to promote several steps of atherosclerotic plaque progression. However, no information is currently available on the role of TWEAK/Fn14 on the development of medial calcification, which is highly prevalent in aging, CKD and T2DM. This study explored the involvement of TWEAK in human vascular smooth muscle cells (h-VSMCs) calcification in vitro. We report that TWEAK binding to Fn14 promotes inorganic phosphate-induced h-VSMCs calcification, favors h-VSMCs osteogenic transition, decreasing acta2 and myh11 and increasing bmp2 mRNA and tissue non-specific alkaline phosphatase (TNAP), and increases MMP9 activity. Blockade of the canonical NFκB pathway reduced by 80% TWEAK pro-calcific properties and decreased osteogenic transition, TNAP and MMP9 activity. Blockade of non-canonical NFκB signaling by a siRNA targeting RelB reduced by 20% TWEAK pro-calcific effects and decreased TWEAK-induced loss of h-VSMCs contractile phenotype and MMP9 activity, without modulating bmp2 mRNA or TNAP activity. Inhibition of ERK1/2 activation by a MAPK kinase inhibitor did not influence TWEAK pro-calcific properties. Our results suggest that TWEAK/Fn14 directly favors inorganic phosphate-induced h-VSMCs calcification by activation of both canonical and non-canonical NFκB pathways. Given the availability of neutralizing anti-TWEAK strategies, our study sheds light on the TWEAK/Fn14 axis as a novel therapeutic target in the prevention of VC. PMID:27441657

  16. TWEAK favors phosphate-induced calcification of vascular smooth muscle cells through canonical and non-canonical activation of NFκB.

    PubMed

    Hénaut, L; Sanz, A B; Martin-Sanchez, D; Carrasco, S; Villa-Bellosta, R; Aldamiz-Echevarria, G; Massy, Z A; Sanchez-Nino, M D; Ortiz, A

    2016-01-01

    Vascular calcification (VC) is associated with increased cardiovascular mortality in aging, chronic kidney disease (CKD), type 2 diabetes mellitus (T2DM) and atherosclerosis. TNF-like weak inducer of apoptosis (TWEAK) recently emerged as a new biomarker for the diagnosis and prognosis of cardiovascular diseases. TWEAK binding to its functional receptor Fn14 was reported to promote several steps of atherosclerotic plaque progression. However, no information is currently available on the role of TWEAK/Fn14 on the development of medial calcification, which is highly prevalent in aging, CKD and T2DM. This study explored the involvement of TWEAK in human vascular smooth muscle cells (h-VSMCs) calcification in vitro. We report that TWEAK binding to Fn14 promotes inorganic phosphate-induced h-VSMCs calcification, favors h-VSMCs osteogenic transition, decreasing acta2 and myh11 and increasing bmp2 mRNA and tissue non-specific alkaline phosphatase (TNAP), and increases MMP9 activity. Blockade of the canonical NFκB pathway reduced by 80% TWEAK pro-calcific properties and decreased osteogenic transition, TNAP and MMP9 activity. Blockade of non-canonical NFκB signaling by a siRNA targeting RelB reduced by 20% TWEAK pro-calcific effects and decreased TWEAK-induced loss of h-VSMCs contractile phenotype and MMP9 activity, without modulating bmp2 mRNA or TNAP activity. Inhibition of ERK1/2 activation by a MAPK kinase inhibitor did not influence TWEAK pro-calcific properties. Our results suggest that TWEAK/Fn14 directly favors inorganic phosphate-induced h-VSMCs calcification by activation of both canonical and non-canonical NFκB pathways. Given the availability of neutralizing anti-TWEAK strategies, our study sheds light on the TWEAK/Fn14 axis as a novel therapeutic target in the prevention of VC. PMID:27441657

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

    PubMed

    Sibirnyi, A A; Shavlovskii, G M

    1978-01-01

    The rate of p-nitrophenyl phosphate and flavin mononucleotide (FMN) hydrolysis by the partially purified preparation of alkaline phosphatase I of Pichia guilliermondii flavinogenic yeast was studied as affected by different substrates and inorganic ions. Their Km was established to be 2.0 X 10(-4) m and 2.5 X 10(-4) M, respectively. Dephosphorylation of p-nitrophenylphosphate and FMN was inhibited competitively by beta-glycerophosphate (Ki = 3.1 X 10(-3) M, respectively). The presence of inorganic phosphate ions in the reaction mixture decreases or removes inhibition of these compounds hydrolysis by other substrates of alkaline phosphatase I. The activity of alkaline phosphatase I increases in the presence of Mg2+ and was strongly inhibited in the presence of Be2+, Cu2+, Zn2+, Cd2+ and inorganic phosphate, the mixture of Be2+ and F- being the most effective. This mixture inhibited the phosphatase activity of the partially purified preparation of alkaline phosphatase I of the cell-free extract as well as of intact cells in both the alkaline and acid zones of pH (8.6 and 5.5, respectively). Incubation of the washed iron-deficient P. guilliermondii cells in the presence of Be2+ and F- did not result in accumulation of FMN in the yeast culture. A possible role of nonspecific phosphomonoesterases in hydrolysis of FMN in vivo is discussed. PMID:208203

  18. Type I phosphatidylinositol 4-phosphate 5-kinase homo- and heterodimerization determines its membrane localization and activity.

    PubMed

    Lacalle, Rosa Ana; de Karam, Juan C; Martínez-Muñoz, Laura; Artetxe, Ibai; Peregil, Rosa M; Sot, Jesús; Rojas, Ana M; Goñi, Félix M; Mellado, Mario; Mañes, Santos

    2015-06-01

    Type I phosphatidylinositol 4-phosphate 5-kinases (PIP5KIs; α, β, and γ) are a family of isoenzymes that produce phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] using phosphatidylinositol 4-phosphate as substrate. Their structural homology with the class II lipid kinases [type II phosphatidylinositol 5-phosphate 4-kinase (PIP4KII)] suggests that PIP5KI dimerizes, although this has not been formally demonstrated. Neither the hypothetical structural dimerization determinants nor the functional consequences of dimerization have been studied. Here, we used Förster resonance energy transfer, coprecipitation, and ELISA to show that PIP5KIβ forms homo- and heterodimers with PIP5KIγ_i2 in vitro and in live human cells. Dimerization appears to be a general phenomenon for PIP5KI isoenzymes because PIP5KIβ/PIP5KIα heterodimers were also detected by mass spectrometry. Dimerization was independent of actin cytoskeleton remodeling and was also observed using purified proteins. Mutagenesis studies of PIP5KIβ located the dimerization motif at the N terminus, in a region homologous to that implicated in PIP4KII dimerization. PIP5KIβ mutants whose dimerization was impaired showed a severe decrease in PI(4,5)P2 production and plasma membrane delocalization, although their association to lipid monolayers was unaltered. Our results identify dimerization as an integral feature of PIP5K proteins and a central determinant of their enzyme activity. PMID:25713054

  19. Active Site Detection by Spatial Conformity and Electrostatic Analysis—Unravelling a Proteolytic Function in Shrimp Alkaline Phosphatase

    PubMed Central

    Chakraborty, Sandeep; Minda, Renu; Salaye, Lipika; Bhattacharjee, Swapan K.; Rao, Basuthkar J.

    2011-01-01

    Computational methods are increasingly gaining importance as an aid in identifying active sites. Mostly these methods tend to have structural information that supplement sequence conservation based analyses. Development of tools that compute electrostatic potentials has further improved our ability to better characterize the active site residues in proteins. We have described a computational methodology for detecting active sites based on structural and electrostatic conformity - CataLytic Active Site Prediction (CLASP). In our pipelined model, physical 3D signature of any particular enzymatic function as defined by its active sites is used to obtain spatially congruent matches. While previous work has revealed that catalytic residues have large pKa deviations from standard values, we show that for a given enzymatic activity, electrostatic potential difference (PD) between analogous residue pairs in an active site taken from different proteins of the same family are similar. False positives in spatially congruent matches are further pruned by PD analysis where cognate pairs with large deviations are rejected. We first present the results of active site prediction by CLASP for two enzymatic activities - β-lactamases and serine proteases, two of the most extensively investigated enzymes. The results of CLASP analysis on motifs extracted from Catalytic Site Atlas (CSA) are also presented in order to demonstrate its ability to accurately classify any protein, putative or otherwise, with known structure. The source code and database is made available at www.sanchak.com/clasp/. Subsequently, we probed alkaline phosphatases (AP), one of the well known promiscuous enzymes, for additional activities. Such a search has led us to predict a hitherto unknown function of shrimp alkaline phosphatase (SAP), where the protein acts as a protease. Finally, we present experimental evidence of the prediction by CLASP by showing that SAP indeed has protease activity in vitro. PMID

  20. Endocytosis of Ligand-Activated Sphingosine 1-Phosphate Receptor 1 Mediated by the Clathrin-Pathway.

    PubMed

    Reeves, Patrick M; Kang, Yuan-Lin; Kirchhausen, Tom

    2016-01-01

    The sphingosine 1-phosphate receptor 1 (S1PR1) is one of five G protein-coupled receptors activated by the lipid sphingosine 1-phosphate (S1P). Stimulation of S1PR1 by binding S1P or the synthetic agonist FTY720P results in rapid desensitization, associated in part with depletion of receptor from the cell surface. We report here combining spinning disc confocal fluorescence microscopy and flow cytometry to show that rapid internalization of activated S1PR1 relies on a functional clathrin-mediated endocytic pathway. Uptake of activated S1PR1 was strongly inhibited in cells disrupted in their clathrin-mediated endocytosis by depleting clathrin or AP-2 or by treating cells with dynasore-OH. The uptake of activated S1P1R was strongly inhibited in cells lacking both β-arrestin 1 and β-arrestin 2, indicating that activated S1PR1 follows the canonical route of endocytosis for G-protein coupled receptor's (GPCR)'s. PMID:26481905

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

    PubMed

    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. PMID:26433714

  2. The influence of age on intestinal dipeptidyl peptidase IV (DPP IV/CD26), disaccharidases, and alkaline phosphatase enzyme activity in C57BL/6 mice.

    PubMed

    Detel, Dijana; Baticic, Lara; Varljen, Jadranka

    2008-01-01

    The objective of this study was to determine and describe the age-related changes in intestinal brush border membrane enzyme activities that occur in C57Bl/6 mice. Specifically, jejunal, duodenal, and ileal dipeptidyl peptidase IV/CD26, disaccharidase (lactase, sucrase, and maltase), and alkaline phosphatase activities were determined. A significant correlation between analyzed intestinal brush border membrane enzyme activities and animal age was found. Our study revealed that intestinal dipeptidyl peptidase IV/CD26, lactase, sucrase, maltase, and alkaline phosphatase activities decline significantly with age (p < .05). Nevertheless, the horizontal (duodenum to ileum) enzyme activity patterns are not affected by age. PMID:18189167

  3. Protein phosphorylation as a mechanism for regulation of spinach leaf sucrose-phosphate synthase activity

    SciTech Connect

    Huber, J.L.A.; Huber, S.C. )

    1989-04-01

    Protein phosphorylation has been identified as a mechanism for the light-dark regulation of spinach sucrose-phosphate synthase (SPS) activity, previously shown to involve some type of covalent modification of the enzyme. The 120 kD subunit of SPS in extracts of light-treated leaves was labeled with {sup 32}P in the presence of ({gamma}-{sup 32}P) ATP. In this in vitro system, {sup 32}P incorporation into light-activated SPS was dependent upon ATP and magnesium concentrations as well as time, and was closely paralleled by inactivation of the enzyme. The soluble protein kinase involved in the interconversion of SPS between activated and deactivated forms may be specific for SPS as it co-purifies with SPS during partial purification of the enzyme. The kinase appears not to be calcium activated and no evidence has been obtained for metabolite control of SPS phosphorylation/inactivation.

  4. Bacillus cereus Phosphopentomutase Is an Alkaline Phosphatase Family Member That Exhibits an Altered Entry Point into the Catalytic Cycle

    SciTech Connect

    Panosian, Timothy D.; Nannemann, David P.; Watkins, Guy R.; Phelan, Vanessa V.; McDonald, W. Hayes; Wadzinski, Brian E.; Bachmann, Brian O.; Iverson, Tina M.

    2011-09-15

    Bacterial phosphopentomutases (PPMs) are alkaline phosphatase superfamily members that interconvert {alpha}-D-ribose 5-phosphate (ribose 5-phosphate) and {alpha}-D-ribose 1-phosphate (ribose 1-phosphate). We investigated the reaction mechanism of Bacillus cereus PPM using a combination of structural and biochemical studies. Four high resolution crystal structures of B. cereus PPM revealed the active site architecture, identified binding sites for the substrate ribose 5-phosphate and the activator {alpha}-D-glucose 1,6-bisphosphate (glucose 1,6-bisphosphate), and demonstrated that glucose 1,6-bisphosphate increased phosphorylation of the active site residue Thr-85. The phosphorylation of Thr-85 was confirmed by Western and mass spectroscopic analyses. Biochemical assays identified Mn{sup 2+}-dependent enzyme turnover and demonstrated that glucose 1,6-bisphosphate treatment increases enzyme activity. These results suggest that protein phosphorylation activates the enzyme, which supports an intermolecular transferase mechanism. We confirmed intermolecular phosphoryl transfer using an isotope relay assay in which PPM reactions containing mixtures of ribose 5-[{sup 18}O{sub 3}]phosphate and [U-{sup 13}C{sub 5}]ribose 5-phosphate were analyzed by mass spectrometry. This intermolecular phosphoryl transfer is seemingly counter to what is anticipated from phosphomutases employing a general alkaline phosphatase reaction mechanism, which are reported to catalyze intramolecular phosphoryl transfer. However, the two mechanisms may be reconciled if substrate encounters the enzyme at a different point in the catalytic cycle.

  5. A segmental pattern of alkaline phosphatase activity within the notochord coincides with the initial formation of the vertebral bodies

    PubMed Central

    Sindre, Grotmol; Kari, Nordvik; Harald, Kryvi; Geir, K Totland

    2005-01-01

    This study shows that segmental expression of alkaline phosphatase (ALP) activity by the notochord of the Atlantic salmon (Salmo salar L.) coincides with the initial mineralization of the vertebral body (chordacentrum), and precedes ALP expression by presumed somite-derived cells external to the notochordal sheath. The early expression of ALP indicates that the notochord plays an instructive role in the segmental patterning of the vertebral column. The chordacentra form segmentally as mineralized rings within the notochordal sheath, and ALP activity spreads within the chordoblast layer from ventral to dorsal, displaying the same progression and spatial distribution as the mineralization process. No ALP activity was observed in sclerotomal mesenchyme surrounding the notochordal sheath during initial formation of the chordacentra. Our results support previous findings indicating that the chordoblasts initiate a segmental differentiation of the notochordal sheath into chordacentra and intervertebral regions. PMID:15857363

  6. Inorganic polymers from laterite using activation with phosphoric acid and alkaline sodium silicate solution: Mechanical and microstructural properties

    SciTech Connect

    Lassinantti Gualtieri, Magdalena

    2015-01-15

    Geopolymers from laterite, an iron-rich soil available in developing countries, have great potential as building materials. In this work, laterite from Togo (Africa) was used to prepare geopolymers using both phosphoric acid and alkaline sodium silicate solution. Microstructural properties were investigated by scanning electron microscopy, X-ray powder diffraction and mercury porosimetry, whereas thermal properties were evaluated by thermal analyses. The local environment of iron was studied by X-ray Absorption Spectroscopy (XANES region). The mechanical properties were determined. Modulus of Rupture and Young's modulus fell in the ranges 3.3–4.5 MPa and 12–33 GPa, respectively, rendering the materials good candidates for construction purposes. Heating above 900 °C results in weight-gain, presumably due to iron redox reactions. X-ray Absorption Spectroscopy data evidence changes in the chemical and structural environments of iron following thermal treatment of geopolymers. These changes indicate interaction between the geopolymer structure and iron during heating, possibly leading to redox properties. -- Highlights: •Geopolymerization of laterite is promising for fabrication of building materials. •Both phosphoric acid and alkaline sodium silicate solution can be used for activation. •Thermally activated redox properties of the inorganic polymers were observed.

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

    PubMed

    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

  8. Dosage Compensation in DROSOPHILA MELANOGASTER Triploids. II. Glucose-6-Phosphate Dehydrogenase Activity

    PubMed Central

    Maroni, Gustavo; Plaut, Walter

    1973-01-01

    The level of activity of the enzyme glucose-6-phosphate dehydrogenase was determinel in flies having seven different chromosomic constitutions. All those having an integral number of chromosomes [XAA, XXAA, XAAA, XXAAA, and XXXAAA (X=X chromosome, A=set of autosomes)] were found to have similar units of enzyme activity/mg live weight, while diploid females with a duplication and triploid females with a deficiency showed dosage effect. The amount of enzyme activity per cell, on the other hand, is also independent of the number of X's present but appears roughly proportional to the number of sets of autosomes.—It is proposed that dosage-compensated sex-linked genes are controlled by a positively acting regulatory factor(s) of autosomal origin. With this hypothesis it is possible to explain dosage compensation as a consequence of general regulatory mechanisms without invoking a special device which applies only to the X chromosomes. PMID:17248620

  9. MRI-based detection of alkaline phosphatase gene reporter activity using a porphyrin solubility switch

    PubMed Central

    Westmeyer, Gil G.; Emer, Elena G.; Lintelmann, Jutta; Jasanoff, Alan

    2014-01-01

    SUMMARY The ability to map patterns of gene expression noninvasively in living animals could have impact in many areas of biology. Reporter systems compatible with magnetic resonance imaging (MRI) could be particularly valuable, but existing strategies tend to lack sensitivity or specificity. Here we address the challenge of MRI-based gene mapping using the reporter enzyme secreted alkaline phosphatase (SEAP), in conjunction with a water soluble metalloporphyrin contrast agent. SEAP cleaves the porphyrin into an insoluble product that accumulates at sites of enzyme expression and can be visualized by MRI and optical absorbance. The contrast mechanism functions in vitro, in brain slices, and in animals. The system also provides the possibility of readout both in the living animal and by post mortem histology, and it notably does not require intracellular delivery of the contrast agent. The solubility switch mechanism used to detect SEAP could be adapted for imaging of additional reporter enzymes or endogenous targets. PMID:24613020

  10. Effect of CCK-8 on pentose phosphate shunt activity, pyridine nucleotides, and glucokinase of rat islets

    SciTech Connect

    Verspohl, E.J.; Breuning, I.; Ammon, H.P.

    1989-01-01

    In rat pancreatic islets the effects of cholecystokinin octapeptide (CCK-8) on pentose phosphate shunt (PPS) activity, glucokinase and hexokinase activity, and NADPH, NADP+, NADH, and NAD+ were studied. By elevating the glucose concentration from 3.0 to 8.3 and 16.7 mM the oxidation of (1-14C)- and (6-14C)glucose and the calculated PPS activity were increased in a concentration-dependent manner; 10 nM CCK-8 enhanced selectively the effect on (1-14C)glucose oxidation thereby increasing the PPS activity but only at an intermediate glucose concentration (8.3 mM). CCK-8 had no effect on glucokinase or hexokinase activity and CCK-8 did not influence glucose utilization. By elevating the glucose concentration, total NADPH and NADH were increased and total NADP+ and NAD+ were decreased. CCK-8 (10 nM) increased selectively NADPH and decreased NADP+ but did not change NADH or NAD+; the effect of CCK-8 on NADPH and NADH was only observed in the presence of an intermediate stimulatory glucose concentration (8.3 mM) but not at either a substimulatory glucose concentration or a maximally stimulatory glucose concentration for insulin release (3.0 or 16.7 mM). The data indicate first that CCK-8 does not act on glucose phosphorylation or glucose utilization and second that CCK-8 increases PPS activity and NADPH levels in rat pancreatic islets. Since the concentrations of glucose necessary for these CCK-8 effects are in the range of 8.3 mM and parallel with those necessary for insulin release as shown in earlier observations, glucose oxidation via pentose phosphate shunt and NADPH are suggested to be related to the CCK-8-modulated insulin release.

  11. Overexpression of SIRT5 confirms its involvement in deacetylation and activation of carbamoyl phosphate synthetase 1.

    PubMed

    Ogura, Masahito; Nakamura, Yasuhiko; Tanaka, Daisuke; Zhuang, Xiaotong; Fujita, Yoshihito; Obara, Akio; Hamasaki, Akihiro; Hosokawa, Masaya; Inagaki, Nobuya

    2010-02-26

    SIR2 protein, an NAD-dependent deacetylase, is localized to nucleus and is involved in life span extension by calorie restriction in yeast. In mammals, among the seven SIR2 homologues (SIRT1-7), SIRT3, 4, and 5 are localized to mitochondria. As SIRT5 mRNA levels in liver are increased by fasting, the physiological role of SIRT5 was investigated in liver of SIRT5-overexpressing transgenic (SIRT5 Tg) mice. We identified carbamoyl phosphate synthetase 1 (CPS1), a key enzyme of the urea cycle that catalyzes condensation of ammonia with bicarbonate to form carbamoyl phosphate, as a target of SIRT5 by two-dimensional electrophoresis comparing mitochondrial proteins in livers of SIRT5 Tg and wild-type mice. CPS1 protein was more deacetylated and activated in liver of SIRT5 Tg mice than in wild-type. In addition, urea production was upregulated in hepatocytes of SIRT5 Tg mice. These results agree with those of a previous study using SIRT5 knockout (KO) mice. Because ammonia generated during fasting is toxic, SIRT5 protein might play a protective role by converting ammonia to non-toxic urea through deacetylation and activation of CPS1. PMID:20097174

  12. The structural role of manganese ions in soil active silicate-phosphate glasses

    NASA Astrophysics Data System (ADS)

    Szumera, Magdalena

    2014-08-01

    Silicate-phosphate glasses of SiO2sbnd P2O5sbnd K2Osbnd MgOsbnd CaO system containing manganese ions were synthesized by the melt-quenching technique and were investigated to obtain information about the influence of Mn-cations on the glass structure and their chemical activity. Structural properties were studied using X-ray method, FTIR and Raman spectroscopies. The chemical activity of analyzed glasses in the 2 wt.% citric acid solution was measured by chemical analysis (ICP-AES, EDS) and SEM observations. It has been found that increasing amount of MnO2 in the structure of investigated glasses causes their gradual depolymerization. This process is more apparent in the case of the silico-oxygen subnetwork than phospho-oxygen one. This is related to increasing amounts of SiO4 tetrahedra containing two nonbridging oxygen atoms in silico-oxygen subnetwork. It has been also found that the presence of “weaker” chemical bonds of Sisbnd Osbnd Mn type in comparison to Sisbnd Osbnd Ca and Sisbnd Osbnd Mg bonds is responsible for the increase in solubility of the analyzed silicate-phosphate glasses in conditions simulating natural soil environment.

  13. [Effect of temperature acclimation on glucose-6-phosphate dehydrogenase activity in various tissues of the Mullus barbatus ponticus].

    PubMed

    Rusinova, O S

    1997-01-01

    Acclimation of the Mullus barbatus ponticus to the temperature fall (from 16 to 8 degrees C) induces an increase in the activity of glucose-6-phosphate dehydrogenase both in summer and in autumn. These changes are more expressed in the liver and red muscle than in the white ones. The values of Km of glucose-6-phosphate (only in autumn) and NADP are also higher at 8 than at 16 degrees C. Actinomycin D does not prevent from cold activation of this enzyme in liver only in autumn, when the activity of glucose-6-phosphate dehydrogenase is 8-10 times higher than in summer, and in the red muscle in the both seasons, although this injection decreases the level of enzyme activity in this muscle. This injection does not change the values of Km for glucose-6-phosphate and NADP. It is supposed that the activation of glucose-6-phosphate dehydrogenase in the liver and red muscle during cold adaptation may be a result of change of substrate-binding ability and enzyme interaction with NADP without changes in the enzyme biosynthesis. PMID:9606826

  14. Characterization of Streptococcus pneumoniae 5-enolpyruvylshikimate 3-phosphate synthase and its activation by univalent cations.

    PubMed

    Du, W; Wallis, N G; Mazzulla, M J; Chalker, A F; Zhang, L; Liu, W S; Kallender, H; Payne, D J

    2000-01-01

    The aroA gene (Escherichia coli nomenclature) encoding 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase from the gram-positive pathogen Streptococcus pneumoniae has been identified, cloned and overexpressed in E. coli, and the enzyme purified to homogeneity. It was shown to catalyze a reversible conversion of shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) to EPSP and inorganic phosphate. Activation by univalent cations was observed in the forward reaction, with NH+4, Rb+ and K+ exerting the greatest effects. Km(PEP) was lowered by increasing [NH+4] and [K+], whereas Km(S3P) rose with increasing [K+], but fell with increasing [NH+4]. Increasing [NH+4] and [K+] resulted in an overall increase in kcat. Glyphosate (GLP) was found to be a competitive inhibitor with PEP, but the potency of inhibition was profoundly affected by [NH+4] and [K+]. For example, increasing [NH+4] and [K+] reduced Ki(GLP versus PEP) up to 600-fold. In the reverse reaction, the enzyme catalysis was less sensitive to univalent cations. Our analysis included univalent cation concentrations comparable with those found in bacterial cells. Therefore, the observed effects of these metal ions are more likely to reflect the physiological behavior of EPSP synthase and also add to our understanding of how to inhibit this enzyme in the host organism. As there is a much evidence to suggest that EPSP synthase is essential for bacterial survival, its discovery in the serious gram-positive pathogen S. pneumoniae and its inhibition by GLP indicate its potential as a broad-spectrum antibacterial target. PMID:10601870

  15. Phosphatidylinositol-4-phosphate 5-Kinase Isoforms Exhibit Acyl Chain Selectivity for Both Substrate and Lipid Activator*

    PubMed Central

    Shulga, Yulia V.; Anderson, Richard A.; Topham, Matthew K.; Epand, Richard M.

    2012-01-01

    Phosphatidylinositol 4,5-bisphosphate is mostly produced in the cell by phosphatidylinositol-4-phosphate 5-kinases (PIP5K) and has a crucial role in numerous signaling events. Here we demonstrate that in vitro all three isoforms of PIP5K, α, β, and γ, discriminate among substrates with different acyl chains for both the substrates phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol (PtdIns) although to different extents, with isoform γ being the most selective. Fully saturated dipalmitoyl-PtdIns4P was a poor substrate for all three isoforms, but both the 1-stearoyl-2-arachidonoyl and the 1-stearoyl-2-oleoyl forms of PtdIns4P were good substrates. Vmax was greater for the 1-stearoyl-2-arachidonoyl form compared with the 1-stearoyl-2-oleoyl form, although for PIP5Kβ the difference was small. For the α and γ isoforms, Km was much lower for 1-stearoyl-2-oleoyl PtdIns4P, making this lipid the better substrate of the two under most conditions. Activation of PIP5K by phosphatidic acid is also acyl chain-dependent. Species of phosphatidic acid with two unsaturated acyl chains are much better activators of PIP5K than those containing one saturated and one unsaturated acyl chain. PtdIns is a poor substrate for PIP5K, but it also shows acyl chain selectivity. Curiously, there is no acyl chain discrimination among species of phosphatidic acid in the activation of the phosphorylation of PtdIns. Together, our findings indicate that PIP5K isoforms α, β, and γ act selectively on substrates and activators with different acyl chains. This could be a tightly regulated mechanism of producing physiologically active unsaturated phosphatidylinositol 4,5-bisphosphate species in the cell. PMID:22942276

  16. Improving the catalytic activity of isopentenyl phosphate kinase through protein coevolution analysis

    PubMed Central

    Liu, Ying; Yan, Zhihui; Lu, Xiaoyun; Xiao, Dongguang; Jiang, Huifeng

    2016-01-01

    Protein rational design has become more and more popular for protein engineering with the advantage of biological big-data. In this study, we described a method of rational design that is able to identify desired mutants by analyzing the coevolution of protein sequence. We employed this approach to evolve an archaeal isopentenyl phosphate kinase that can convert dimethylallyl alcohol (DMA) into precursor of isoprenoids. By designing 9 point mutations, we improved the catalytic activities of IPK about 8-fold in vitro. After introducing the optimal mutant of IPK into engineered E. coli strain for β-carotenoids production, we found that β-carotenoids production exhibited 97% increase over the starting strain. The process of enzyme optimization presented here could be used to improve the catalytic activities of other enzymes. PMID:27052337

  17. Improving the catalytic activity of isopentenyl phosphate kinase through protein coevolution analysis.

    PubMed

    Liu, Ying; Yan, Zhihui; Lu, Xiaoyun; Xiao, Dongguang; Jiang, Huifeng

    2016-01-01

    Protein rational design has become more and more popular for protein engineering with the advantage of biological big-data. In this study, we described a method of rational design that is able to identify desired mutants by analyzing the coevolution of protein sequence. We employed this approach to evolve an archaeal isopentenyl phosphate kinase that can convert dimethylallyl alcohol (DMA) into precursor of isoprenoids. By designing 9 point mutations, we improved the catalytic activities of IPK about 8-fold in vitro. After introducing the optimal mutant of IPK into engineered E. coli strain for β-carotenoids production, we found that β-carotenoids production exhibited 97% increase over the starting strain. The process of enzyme optimization presented here could be used to improve the catalytic activities of other enzymes. PMID:27052337

  18. Application of acid-activated Bauxsol for wastewater treatment with high phosphate concentration: Characterization, adsorption optimization, and desorption behaviors.

    PubMed

    Ye, Jie; Cong, Xiangna; Zhang, Panyue; Zeng, Guangming; Hoffmann, Erhard; Liu, Yang; Wu, Yan; Zhang, Haibo; Fang, Wei; Hahn, Hermann H

    2016-02-01

    Acid-activated Bauxsol was applied to treat wastewater with high phosphate concentration in a batch adsorption system in this paper. The effect of acid activation on the change of Bauxsol structure was systematically investigated. The mineralogical inhomogeneity and intensity of Bauxsol decreased after acid activation, and FeCl3·2H2O and Al(OH)3 became the dominant phases of acid-activated Bauxsol adsorption. Moreover, the BET surface area and total pore volume of Bauxsol increased after acid activation. Interaction of initial solution pH and adsorption temperature on phosphate adsorption onto acid-activated Bauxsol was investigated by using response surface methodology with central composite design. The maximum phosphate adsorption capacity of 192.94 mg g(-1) was achieved with an initial solution pH of 4.19 and an adsorption temperature of 52.18 °C, which increased by 7.61 times compared with that of Bauxsol (22.40 mg g(-1)), and was higher than other adsorbents. Furthermore, the desorption studies demonstrated that the acid-activated Bauxsol was successfully regenerated with 0.5 mol L(-1) HCl solution. The adsorption capacity and desorption efficiency of acid-activated Bauxsol maintained at 80.48% and 93.02% in the fifth adsorption-desorption cycle, respectively, suggesting that the acid-activated Bauxsol could be repeatedly used in wastewater treatment with high phosphate concentration. PMID:26606195

  19. Beta glucosidase from Bacillus polymyxa is activated by glucose-6-phosphate.

    PubMed

    Weiss, Paulo H E; Álvares, Alice C M; Gomes, Anderson A; Miletti, Luiz C; Skoronski, Everton; da Silva, Gustavo F; de Freitas, Sonia M; Magalhães, Maria L B

    2015-08-15

    Optimization of cellulose enzymatic hydrolysis is crucial for cost effective bioethanol production from lignocellulosic biomass. Enzymes involved in cellulose hydrolysis are often inhibited by their end-products, cellobiose and glucose. Efforts have been made to produce more efficient enzyme variants that are highly tolerant to product accumulation; however, further improvements are still necessary. Based on an alternative approach we initially investigated whether recently formed glucose could be phosphorylated into glucose-6-phosphate to circumvent glucose accumulation and avoid inhibition of beta-glucosidase from Bacillus polymyxa (BGLA). The kinetic properties and structural analysis of BGLA in the presence of glucose-6-phosphate (G6P) were investigated. Kinetic studies demonstrated that enzyme was not inhibited by G6P. In contrast, the presence of G6P activated the enzyme, prevented beta glucosidase feedback inhibition by glucose accumulation and improved protein stability. G6P binding was investigated by fluorescence quenching experiments and the respective association constant indicated high affinity binding of G6P to BGLA. Data reported here are of great impact for future design strategies for second-generation bioethanol production. PMID:26116788

  20. Chloroplast Activity and 3'phosphadenosine 5'phosphate Signaling Regulate Programmed Cell Death in Arabidopsis.

    PubMed

    Bruggeman, Quentin; Mazubert, Christelle; Prunier, Florence; Lugan, Raphaël; Chan, Kai Xun; Phua, Su Yin; Pogson, Barry James; Krieger-Liszkay, Anja; Delarue, Marianne; Benhamed, Moussa; Bergounioux, Catherine; Raynaud, Cécile

    2016-03-01

    Programmed cell death (PCD) is a crucial process both for plant development and responses to biotic and abiotic stress. There is accumulating evidence that chloroplasts may play a central role during plant PCD as for mitochondria in animal cells, but it is still unclear whether they participate in PCD onset, execution, or both. To tackle this question, we have analyzed the contribution of chloroplast function to the cell death phenotype of the myoinositol phosphate synthase1 (mips1) mutant that forms spontaneous lesions in a light-dependent manner. We show that photosynthetically active chloroplasts are required for PCD to occur in mips1, but this process is independent of the redox state of the chloroplast. Systematic genetic analyses with retrograde signaling mutants reveal that 3'-phosphoadenosine 5'-phosphate, a chloroplast retrograde signal that modulates nuclear gene expression in response to stress, can inhibit cell death and compromises plant innate immunity via inhibition of the RNA-processing 5'-3' exoribonucleases. Our results provide evidence for the role of chloroplast-derived signal and RNA metabolism in the control of cell death and biotic stress response. PMID:26747283

  1. Improved volatile fatty acids anaerobic production from waste activated sludge by pH regulation: Alkaline or neutral pH?

    PubMed

    Ma, Huijun; Chen, Xingchun; Liu, He; Liu, Hongbo; Fu, Bo

    2016-02-01

    In this study, the anaerobic fermentation was carried out for volatile fatty acids (VFAs) production at different pH (between 7.0 and 10.0) conditions with untreated sludge and heat-alkaline pretreated waste activated sludge. In the fermentation with untreated sludge, the extent of hydrolysis of organic matters and extent of acidification at alkaline pH are 54.37% and 30.37%, respectively, resulting in the highest VFAs yield at 235.46mg COD/gVS of three pH conditions. In the fermentation with heat-alkaline pretreated sludge, the acidification rate and VFAs yield at neutral pH are 30.98% and 240.14mg COD/gVS, respectively, which are higher than that at other pH conditions. With the glucose or bovine serum albumin as substrate for VFAs production, the neutral pH showed a higher VFAs concentration than the alkaline pH condition. The results of terminal restriction fragment length polymorphism (T-RFLP) analysis indicated that the alkaline pH caused low microbial richness. Based on the results in this study, we demonstrated that the alkaline pH is favor of hydrolysis of organic matter in sludge while neutral pH improved the acidogenesis for the VFAs production from sludge. Our finding is obvious different to the previous research and helpful for the understanding of how heat-alkaline pretreatment and alkaline fermentation influence the VFAs production, and beneficial to the development of VFAs production process. PMID:26652215

  2. Phosphate binding in the active centre of tomato multifunctional nuclease TBN1 and analysis of superhelix formation by the enzyme.

    PubMed

    Stránský, Jan; Koval', Tomáš; Podzimek, Tomáš; Týcová, Anna; Lipovová, Petra; Matoušek, Jaroslav; Kolenko, Petr; Fejfarová, Karla; Dušková, Jarmila; Skálová, Tereza; Hašek, Jindřich; Dohnálek, Jan

    2015-11-01

    Tomato multifunctional nuclease TBN1 belongs to the type I nuclease family, which plays an important role in apoptotic processes and cell senescence in plants. The newly solved structure of the N211D mutant is reported. Although the main crystal-packing motif (the formation of superhelices) is conserved, the details differ among the known structures. A phosphate ion was localized in the active site of the enzyme. The binding of the surface loop to the active centre is stabilized by the phosphate ion, which correlates with the observed aggregation of TBN1 in phosphate buffer. The conserved binding of the surface loop to the active centre suggests biological relevance of the contact in a regulatory function or in the formation of oligomers. PMID:26527269

  3. FTY720 Phosphate Activates Sphingosine-1-Phosphate Receptor 2 and Selectively Couples to Gα12/13/Rho/ROCK to Induce Myofibroblast Contraction.

    PubMed

    Sobel, Katrin; Monnier, Lucile; Menyhart, Katalin; Bolinger, Matthias; Studer, Rolf; Nayler, Oliver; Gatfield, John

    2015-06-01

    FTY720 phosphate (FTY720-P; 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol, monodihydrogen phosphate ester) is a nonselective sphingosine-1-phosphate (S1P) receptor agonist thought to be devoid of activity at the S1P2 receptor subtype. However, we have recently shown that FTY720-P displays significant S1P2 receptor agonist activity in recombinant cells and fibroblasts expressing endogenous S1P2 receptors. To elucidate the S1P2-dependent signaling pathways that were activated by FTY720-P, we employed second messenger assays and impedance-based assays in combination with pharmacological and small interfering RNA-based pathway inhibition in recombinant Chinese hamster ovary (CHO)-S1P2 cells as well as human lung myofibroblasts generated in vitro. In CHO-S1P2 cells, FTY720-P did not modulate cAMP or calcium levels. However, reporter-gene assays, impedance-based assays with a selective Rho-associated kinase (ROCK) inhibitor, Gα12/13 knockdown and activated Rho-pull-down assays demonstrated that FTY720-P potently activated Gα12/13/Rho/ROCK signaling. S1P similarly activated Gα12/13/Rho/ROCK signaling via S1P2 receptors, whereas the two selective S1P1 receptor agonists (Z,Z)-5-(3-chloro-4-[(2R)-2,3-dihydroxy-propoxy]-benzylidene)-2-propylimino-3-o-tolyl-thiazolidin-4-one (ponesimond) and 5-[4-phenyl-5-(trifluoromethyl)thiophen-2-yl]-3-[3-(trifluoromethyl)phenyl]1,2,4-oxadiazole (SEW2871) were inactive. In lung myofibroblasts, which mainly expressed the S1P2 receptor subtype, we showed that FTY720-P selectively activated the Gα12/13/Rho/ROCK pathway via the S1P2 receptor. Moreover, the activation of the Gα12/13/Rho/ROCK pathway in myofibroblasts by FTY720-P caused potent myofibroblast contraction similar to that induced by the natural ligand S1P. Thus, complementing second messenger assays with unbiased label-free assays or phenotypic assays in native expression systems can uncover activation of additional pathways, such as Gα12/13/Rho/ROCK signaling. PMID

  4. Synthesis and electrocatalytic activity of Au/Pt bimetallic nanodendrites for ethanol oxidation in alkaline medium.

    PubMed

    Han, Xinyi; Wang, Dawei; Liu, Dong; Huang, Jianshe; You, Tianyan

    2012-02-01

    Gold/Platinum (Au/Pt) bimetallic nanodendrites were successfully synthesized through seeded growth method using preformed Au nanodendrites as seeds and ascorbic acid as reductant. Cyclic voltammograms (CVs) of a series of Au/Pt nanodendrites modified electrodes in 1M KOH solution containing 1M ethanol showed that the electrocatalyst with a molar ratio (Au:Pt) of 3 exhibited the highest peak current density and the lowest onset potential. The peak current density of ethanol electro-oxidation on the Au(3)Pt(1) nanodendrites modified glassy carbon electrode (Au(3)Pt(1) electrode) is about 16, 12.5, and 4.5 times higher than those on the polycrystalline Pt electrode, polycrystalline Au electrode, and Au nanodendrites modified glassy carbon electrode (Au dendrites electrode), respectively. The oxidation peak potential of ethanol electro-oxidation on the Au(3)Pt(1) electrode is about 299 and 276 mV lower than those on the polycrystalline Au electrode and Au dendrites electrode, respectively. These results demonstrated that the Au/Pt bimetallic nanodendrites may find potential application in alkaline direct ethanol fuel cells (ADEFCs). PMID:22071516

  5. The effect of salinity on waste activated sludge alkaline fermentation and kinetic analysis.

    PubMed

    Jin, Baodan; Wang, Shuying; Xing, Liqun; Li, Baikun; Peng, Yongzhen

    2016-05-01

    The effect of salinity on sludge alkaline fermentation at low temperature (20°C) was investigated, and a kinetic analysis was performed. Different doses of sodium chloride (NaCl, 0-25g/L) were added into the fermentation system. The batch-mode results showed that the soluble chemical oxygen demand (SCOD) increased with salinity. The hydrolysate (soluble protein, polysaccharide) and the acidification products (short chain fatty acids (SCFAs), NH4(+)-N, and PO4(3-)-P) increased with salinity initially, but slightly declined respectively at higher level salinity (20g/L or 20-25g/L). However, the hydrolytic acidification performance increased in the presence of salt compared to that without salt. Furthermore, the results of Haldane inhibition kinetics analysis showed that the salt enhanced the hydrolysis rate of particulate organic matter from sludge particulate and the specific utilization of hydrolysate, and decreased the specific utilization of SCFAs. Pearson correlation coefficient analysis indicated that the importance of polysaccharide on the accumulation of SCFAs was reduced with salt addition, but the importance of protein and NH4(+)-N on SCFA accumulation was increased. PMID:27155412

  6. Escherichia coli N-Acetylglucosamine-1-Phosphate-Uridyltransferase/Glucosamine-1-Phosphate-Acetyltransferase (GlmU) Inhibitory Activity of Terreic Acid Isolated from Aspergillus terreus.

    PubMed

    Sharma, Rashmi; Lambu, Mallikharjuna Rao; Jamwal, Urmila; Rani, Chitra; Chib, Reena; Wazir, Priya; Mukherjee, Debaraj; Chaubey, Asha; Khan, Inshad Ali

    2016-04-01

    Secondary metabolite of Aspergillus terreus, terreic acid, is a reported potent antibacterial that was identified more than 60 years ago, but its cellular target(s) are still unknown. Here we screen its activity against the acetyltransferase domain of a bifunctional enzyme, Escherichia coli N-acetylglucosamine-1-phosphate-uridyltransferase/glucosamine-1-phosphate-acetyltransferase (GlmU). An absorbance-based assay was used to screen terreic acid against the acetyltransferase activity of E. coli GlmU. Terreic acid was found to inhibit the acetyltransferase domain of E. coli GlmU with an IC50 of 44.24 ± 1.85 µM. Mode of inhibition studies revealed that terreic acid was competitive with AcCoA and uncompetitive with GlcN-1-P. It also exhibited concentration-dependent killing of E. coli ATCC 25922 up to 4× minimum inhibitory concentration and inhibited the growth of biofilms generated by E. coli. Characterization of resistant mutants established mutation in the acetyltransferase domain of GlmU. Terreic acid was also found to be metabolically stable in the in vitro incubations with rat liver microsome in the presence of a NADPH regenerating system. The studies reported here suggest that terreic acid is a potent antimicrobial agent and support that E. coli GlmU acetyltransferase is a molecular target of terreic acid, resulting in its antibacterial activity. PMID:26762501

  7. In situ generated highly active copper oxide catalysts for the oxygen evolution reaction at low overpotential in alkaline solutions.

    PubMed

    Liu, Xiang; Cui, Shengsheng; Qian, Manman; Sun, Zijun; Du, Pingwu

    2016-04-25

    Developing efficient water oxidation catalysts made up of earth-abundant elements has attracted much attention as a step toward for future clean energy production. Herein we report a simple one-step method to generate a low cost copper oxide catalyst film in situ from a copper(ii) ethylenediamine complex. The resulting catalyst has excellent activity toward the oxygen evolution reaction in alkaline solutions. A catalytic current density of 1.0 mA cm(-2) and 10 mA cm(-2) for the catalyst film requires the overpotentials of only ∼370 mV and ∼475 mV in 1.0 M KOH, respectively. This catalytic performance shows that the new catalyst is one of the best Cu-based heterogeneous OER catalysts to date. PMID:27020763

  8. Molecular Basis of Reduced Pyridoxine 5′-Phosphate Oxidase Catalytic Activity in Neonatal Epileptic Encephalopathy Disorder*

    PubMed Central

    Musayev, Faik N.; Di Salvo, Martino L.; Saavedra, Mario A.; Contestabile, Roberto; Ghatge, Mohini S.; Haynes, Alexina; Schirch, Verne; Safo, Martin K.

    2009-01-01

    Mutations in pyridoxine 5′-phosphate oxidase are known to cause neonatal epileptic encephalopathy. This disorder has no cure or effective treatment and is often fatal. Pyridoxine 5′-phosphate oxidase catalyzes the oxidation of pyridoxine 5′-phosphate to pyridoxal 5′-phosphate, the active cofactor form of vitamin B6 required by more than 140 different catalytic activities, including enzymes involved in amino acid metabolism and biosynthesis of neurotransmitters. Our aim is to elucidate the mechanism by which a homozygous missense mutation (R229W) in the oxidase, linked to neonatal epileptic encephalopathy, leads to reduced oxidase activity. The R229W variant is ∼850-fold less efficient than the wild-type enzyme due to an ∼192-fold decrease in pyridoxine 5′-phosphate affinity and an ∼4.5-fold decrease in catalytic activity. There is also an ∼50-fold reduction in the affinity of the R229W variant for the FMN cofactor. A 2.5 Å crystal structure of the R229W variant shows that the substitution of Arg-229 at the FMN binding site has led to a loss of hydrogen-bond and/or salt-bridge interactions between FMN and Arg-229 and Ser-175. Additionally, the mutation has led to an alteration of the configuration of a β-strand-loop-β-strand structure at the active site, resulting in loss of two critical hydrogen-bond interactions involving residues His-227 and Arg-225, which are important for substrate binding and orientation for catalysis. These results provide a molecular basis for the phenotype associated with the R229W mutation, as well as providing a foundation for understanding the pathophysiological consequences of pyridoxine 5′-phosphate oxidase mutations. PMID:19759001

  9. Highly active cobalt phosphate and borate based oxygen evolving catalysts operating in neutral and natural waters

    SciTech Connect

    Esswein, AJ; Surendranath, Y; Reece, SY; Nocera, DG

    2011-02-01

    A high surface area electrode is functionalized with cobalt-based oxygen evolving catalysts (Co-OEC = electrodeposited from pH 7 phosphate, Pi, pH 8.5 methylphosphonate, MePi, and pH 9.2 borate electrolyte, Bi). Co-OEC prepared from MePi and operated in Pi and Bi achieves a current density of 100 mA cm(-2) for water oxidation at 442 and 363 mV overpotential, respectively. The catalyst retains activity in near-neutral pH buffered electrolyte in natural waters such as those from the Charles River (Cambridge, MA) and seawater (Woods Hole, MA). The efficacy and ease of operation of anodes functionalized with Co-OEC at appreciable current density together with its ability to operate in near neutral pH buffered natural water sources bodes well for the translation of this catalyst to a viable renewable energy storage technology.

  10. Potassium ion-activated hydrolysis of p-nitrophenyl phosphate in pancreatic islet-cell membranes.

    PubMed Central

    Lernmark, A; Parman, A; Täljedal, I B

    1977-01-01

    Hydrolysis of p-nitrophenyl phosphate was measured in a fraction enriched in plasma membranes from pancreatic islets of non-inbred ob/ob mice. Hydrolysis was stimulated by K+ (10mM) in the pH range 5--10; a small peak of K+-induced activation was observed between pH7.5 and 8. Both the K+-induced activation and the hydrolysis in the absence of K+ were Mg2+-dependent; maximum activation was obtained with 10mM-K+ plus 5 mM-Mg2+. Rb+ was as effective an activator as K+. Ouabain was inhibitory, the effect being inversely related to the K+ concentration; 0.1--0.2mM-ouabain caused about 50% inhibition in the presence of 1 mM-K+, but had no demonstrable effect in the presence of 4--5mM-K+. The K+-stimulated activity was markedly inhibited by 0.1mM-ATP, 35--140 MM-Na+, or 0.01 mM-p-chloromercuribenzenesulphonic acid. Similarities to Rb+ accumulation suggest that catalysis of univalent cation flow in pancreatic beta-cells may be coupled to a phosphoryl-transfer reaction with ATP as natural substrate or regulator. PMID:20876

  11. Immobilization and phytotoxicity of Pb in contaminated soil amended with γ-polyglutamic acid, phosphate rock, and γ-polyglutamic acid-activated phosphate rock.

    PubMed

    Zhu, Jun; Cai, Zhijian; Su, Xiaojuan; Fu, Qingling; Liu, Yonghong; Huang, Qiaoyun; Violante, Antonio; Hu, Hongqing

    2015-02-01

    Pot experiments were conducted to investigate the effects of γ-polyglutamic acid (γ-PGA), phosphate rock (PR), and γ-PGA-activated PR (γ-PGA-PR) on the immobilization and phytotoxicity of Pb in a contaminated soil. The proportion of residual Pb (Re-Pb) in soil was reduced by the addition of γ-PGA but was increased by the application of PR and γ-PGA-PR. The addition of γ-PGA in soil improved the accumulation of Pb in pak choi and decreased the growth of pak choi, suggesting the intensification of Pb phytotoxicity to pak choi. However, opposite effects of PR and γ-PGA-PR on the phytotoxicity of Pb to pak choi in soil were observed. Moreover, in the examined range, γ-PGA-PR activated by a higher amount of γ-PGA resulted in a greater proportion of Re-Pb in soil and weaker phytotoxicity of Pb to pak choi. The predominance of γ-PGA-PR in relieving the phytotoxicity of Pb was ascribed mainly to the increase of soil pH and available phosphate after the amendment, which could facilitate the precipitation of Pb in soil and provide pak choi with more phosphorus nutrient. PMID:25196962

  12. New SIRT1 activator from alkaline hydrolysate of total saponins in the stems-leaves of Panax ginseng.

    PubMed

    Ma, Li-Yuan; Zhou, Qi-Le; Yang, Xiu-Wei

    2015-11-15

    Two new dammarane-type triterpenes, namely ginsenoslaloside-I [3β,12β,24S-trihydroxy-dammara-20(22)E,25-diene-3-O-β-D-glucopyranoside, 1] and 20(S)-ginsenoside-Rh1-6'-acetate (2), together with twelve known compounds (3-14) were isolated from the alkaline hydrolysate of total saponins of the stems-leaves of Panax ginseng C.A. Meyer. Their chemical structures were elucidated by extensive spectroscopic analyses and comparison with the reported data. All 14 compounds were evaluated for their anti-proliferative activities against two human cancer cell lines (HL-60 and Hep-G2) and promotion activities of SIRT1. Compound 6 exhibited significant inhibitory activity in a concentration-dependent manner against HL-60 and Hep-G2 with the IC50 values of 10.32 and 24.33μM, respectively, and had comparable IC50 values with those of vinorelbine, a positive control agent. Meanwhile, compounds 1 and 6 were found to be a potential activator of SIRT1. The preliminary structure-activity relationship was also discussed based on the experimental data obtained. PMID:26420067

  13. Alkaline Phosphatase in Stem Cells

    PubMed Central

    Štefková, Kateřina; Procházková, Jiřina; Pacherník, Jiří

    2015-01-01

    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. PMID:25767512

  14. Acidic and alkaline pretreatments of activated carbon and their effects on the performance of air-cathodes in microbial fuel cells.

    PubMed

    Wang, Xin; Gao, Ningshengjie; Zhou, Qixing; Dong, Heng; Yu, Hongbing; Feng, Yujie

    2013-09-01

    Activated carbon (AC) is a high performing and cost effective catalyst for oxygen reduction reactions (ORRs) of air-cathodes in microbial fuel cells (MFCs). Acidic (HNO3) and alkaline (KOH) pretreatments on AC at low temperature (85°C) are conducted to enhance the performance of MFCs. The alkaline pretreatment increased the power density by 16% from 804±70 to 957±31 mW m(-2), possibly due to the decrease of ohmic resistance (from 20.58 to 19.20 Ω) and the increase of ORR activities provided by the adsorbed hydroxide ion and extra micropore area/volume after alkaline pretreatment. However, acidic pretreatment decreased the power output to 537±36 mW m(-2), which can be mainly attributed to the corrosion by adsorbed proton at the interface of AC powder and stainless steel mesh and the decreased pore area. PMID:23890977

  15. Temperature dependence of active tension in mammalian (rabbit psoas) muscle fibres: effect of inorganic phosphate.

    PubMed

    Coupland, M E; Puchert, E; Ranatunga, K W

    2001-11-01

    1. The effect of added inorganic phosphate (P(i), range 3-25 mM) on active tension was examined at a range of temperatures (5-30 degrees C) in chemically skinned (0.5 % Brij) rabbit psoas muscle fibres. Three types of experiments were carried out. 2. In one type of experiment, a muscle fibre was maximally activated at low temperature (5 degrees C) and its tension change was recorded during stepwise heating to high temperature in approximately 60 s. As found in previous studies, the tension increased with temperature and the normalised tension-(reciprocal) temperature relation was sigmoidal, with a half-maximal tension at 8 degrees C. In the presence of 25 mM added P(i), the temperature for half-maximal tension of the normalised curve was approximately 5 degrees C higher than in the control. The difference in the slope was small. 3. In a second type of experiment, the tension increment during a large temperature jump (from 5 to 30 degrees C) was examined during an active contraction. The relative increase of active tension on heating was significantly higher in the presence of 25 mM added P(i) (30/5 degrees C tension ratio of 6-7) than in the control with no added P(i) (tension ratio of approximately 3). 4. In a third type of experiment, the effect on the maximal Ca(2+)-activated tension of different levels of added P(i) (3-25 mM) (and P(i) mop adequate to reduce contaminating P(i) to micromolar levels) was examined at 5, 10, 20 and 30 degrees C. The tension was depressed with increased [P(i)] in a concentration-dependent manner at all temperatures, and the data could be fitted with a hyperbolic relation. The calculated maximal tension depression in excess [P(i)] was approximately 65 % of the control at 5-10 degrees C, in contrast to a maximal depression of 40 % at 20 degrees C and 30 % at 30 degrees C. 5. These experiments indicate that the active tension depression induced by P(i) in psoas fibres is temperature sensitive, the depression becoming less marked at

  16. Quantification of Galactose-1-Phosphate Uridyltransferase Enzyme Activity by Liquid Chromatography–Tandem Mass Spectrometry

    PubMed Central

    Li, Yijun; Ptolemy, Adam S.; Harmonay, Lauren; Kellogg, Mark; Berry, Gerard T.

    2013-01-01

    Background The diagnosis of galactosemia usually involves the measurement of galactose-1-phosphate uridyltransferase (GALT) activity. Traditional radioactive and fluorescent GALT assays are nonspecific, laborious, and/or lack sufficient analytical sensitivity. We developed a liquid chromatography–tandem mass spectrometry (LC-MS/MS)–based assay for GALT enzyme activity measurement. Method Our assay used stable isotope-labeled α-galactose-1-phosphate ([13C6]-Gal-1-P) as an enzyme substrate. Sample cleanup and separation were achieved by reversed-phase ion-pair chromatography, and the enzymatic product, isotope-labeled uridine diphosphate galactose ([13C6]-UDPGal), was detected by MS/MS at mass transition (571 > 323) and quantified by use of [13C6]-Glu-1-P (265 > 79) as an internal standard. Results The method yielded a mean (SD) GALT enzyme activity of 23.8 (3.8) µmol · (gHgb)−1 · h−1 in erythrocyte extracts from 71 controls. The limit of quantification was 0.04 µmol · (g Hgb)−1 · h−1 (0.2% of normal control value). Intraassay imprecision was determined at 4 different levels (100%, 25%, 5%, and 0.2% of the normal control values), and the CVs were calculated to be 2.1%, 2.5%, 4.6%, and 9.7%, respectively (n = 3). Interassay imprecision CVs were 4.5%, 6.7%, 8.2%, and 13.2% (n = 5), respectively. The assay recoveries at the 4 levels were higher than 90%. The apparent Km of the 2 substrates, Gal-1-P and UDPGlc, were determined to be 0.38 mmol/L and 0.071 mmol/L, respectively. The assay in erythrocytes of 33 patients with classical galactosemia revealed no detectable activity. Conclusions This LC-MS/MS–based assay for GALT enzyme activity will be useful for the diagnosis and study of biochemically heterogeneous patients with galactosemia, especially those with uncommon genotypes and detectable but low residual activities. PMID:20348403

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

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

  18. Serum placental-type alkaline phosphatase activity in women with squamous and glandular malignancies of the reproductive tract.

    PubMed Central

    Ind, T E; Iles, R K; Carter, P G; Lowe, D G; Shepherd, J H; Hudson, C N; Chard, T

    1994-01-01

    AIM--To investigate serum placental-type alkaline phosphatase (PLAP-type) activities in women with squamous and glandular malignancies of the reproductive tract using an immunoradiometric assay. METHODS--PLAP-type immunoreactivity was measured in 180 women with non-ovarian malignancies of the reproductive tract and the values were compared with those from 334 controls. The cases comprised 18 vulval, nine vaginal, 103 cervical, 46 endometrial, and five fallopian tube cancers. RESULTS--Serum PLAP-type activities were no different from controls in patients with squamous cell tumours. Women with adenocarcinoma of the cervix, endometrium, and fallopian tube had increased values: women with endometrial cancer had a median value nearly four times greater than that of controls. There was no direct correlation between PLAP-type activities and stage of disease in patients with endometrial cancer, but values reverted to normal after treatment. CONCLUSIONS--Serum PLAP-type measurements are of no value in the management of patients with squamous cell tumours of the female reproductive tract. Raised activities can, however, be found in glandular tumours, in particular endometrial cancer where serum PLAP-type measurements may be of value in predicting remission. PMID:7829680

  19. Dengue Virus NS1 Protein Modulates Cellular Energy Metabolism by Increasing Glyceraldehyde-3-Phosphate Dehydrogenase Activity

    PubMed Central

    Allonso, Diego; Andrade, Iamara S.; Conde, Jonas N.; Coelho, Diego R.; Rocha, Daniele C. P.; da Silva, Manuela L.; Ventura, Gustavo T.

    2015-01-01

    ABSTRACT Dengue is one of the main public health concerns worldwide. Recent estimates indicate that over 390 million people are infected annually with the dengue virus (DENV), resulting in thousands of deaths. Among the DENV nonstructural proteins, the NS1 protein is the only one whose function during replication is still unknown. NS1 is a 46- to 55-kDa glycoprotein commonly found as both a membrane-associated homodimer and a soluble hexameric barrel-shaped lipoprotein. Despite its role in the pathogenic process, NS1 is essential for proper RNA accumulation and virus production. In the present study, we identified that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with intracellular NS1. Molecular docking revealed that this interaction occurs through the hydrophobic protrusion of NS1 and the hydrophobic residues located at the opposite side of the catalytic site. Moreover, addition of purified recombinant NS1 enhanced the glycolytic activity of GAPDH in vitro. Interestingly, we observed that DENV infection promoted the relocalization of GAPDH to the perinuclear region, where NS1 is commonly found. Both DENV infection and expression of NS1 itself resulted in increased GAPDH activity. Our findings indicate that the NS1 protein acts to increase glycolytic flux and, consequently, energy production, which is consistent with the recent finding that DENV induces and requires glycolysis for proper replication. This is the first report to propose that NS1 is an important modulator of cellular energy metabolism. The data presented here provide new insights that may be useful for further drug design and the development of alternative antiviral therapies against DENV. IMPORTANCE Dengue represents a serious public health problem worldwide and is caused by infection with dengue virus (DENV). Estimates indicate that half of the global population is at risk of infection, with almost 400 million cases occurring per year. The NS1 glycoprotein is found in both the

  20. Array of amorphous calcium phosphate particles improves cellular activity on a hydrophobic surface.

    PubMed

    Kim, InAe; Kim, Hyun Jung; Kim, Hyun-Man

    2010-04-01

    Poor interaction between cells and surfaces, especially hydrophobic surfaces, results in delayed proliferation and increased apoptosis due to low cell adhesion signaling. To improve cell adhesion, hydrophilic array of amorphous calcium phosphate (ACP) was fabricated on a surface. A phosphate-buffered solution containing calcium ions was prepared at low temperature to prevent spontaneous precipitation. Then, the ion solution was heated to generate nuclei of ACP nanoparticles. The ACP nanoparticles adhered to the hydrophobic polystyrene surface forming an array composed of ACP particles. Multiple treatments of these nuclei with fresh CaP ion solutions increased the diameter and decreased the solubility of ACP particles enough to mediate cellular adhesion. The particle density in the array was dependent on the ion concentration of the CaP ion solutions. The ACP array improved a wide variety of activities when osteoblastic MC3T3-E1 cells were cultured on the ACP array fabricated on a hydrophobic bacteriological dish surface, compared to those cultured without the ACP array in vitro. The use of ACP array resulted in a lower apoptosis and also increased the spreading of cells to form stress fibers and focal contacts. Cells cultured on the ACP array proliferated more than cells cultured on a hydrophobic surface without the ACP array. The ACP array increased the expression of markers of differentiation in osteoblast. These results indicate that an array of ACP can be used as a coating material for enhancing biocompatibility in tissue engineering or biomaterials rather than modifying the surface with organic molecules. PMID:20119940

  1. Derivatives of phosphate Schiff base transition metal complexes: synthesis, studies and biological activity

    NASA Astrophysics Data System (ADS)

    El-Wahab, Z. H. Abd; El-Sarrag, M. R.

    2004-01-01

    We report the synthesis and structural characterization of series of tetra- and hexacoordinate metal chelate complexes of phosphate Schiff base ligands having the general composition LMX n·H 2O and L 2MX n (L=phosphate Schiff base ligand; M=Ag +, Mn 2+, Cu 2+, Zn 2+, Cd 2+, Hg 2+, or Fe 3+ and X=NO 3-, Br - or Cl -). The structure of the prepared compounds was investigated using elemental analysis, IR, 1H and 31P NMR, UV-vis, mass spectra, solid reflectance, magnetic susceptibility and conductance measurements as well as conductometric titration. In all the complexes studied, the ligands act as a chelate ligand with coordination involving the phosphateO-atom and the azomethineN-atom. IR, solid reflectance spectra and magnetic moment measurement are used to infer the structure and to illustrate the coordination capacity of ligand. IR spectra show the presence of coordinated nitrate and water molecule, the magnetic moments of all complexes show normal magnetic behavior and the electronic spectra of the metal complexes indicate a tetra- and octahedral structure for Mn 2+, octahedral structure of Fe 3+ and both square-planar and distorted octahedral structure for Cu 2+ complexes. Antimicrobial activity of the ligands and their complexes were tested using the disc diffusion method and the chosen strains include Staphylococcus aureus, Pseudomonas aereuguinosa, Klebsiella penumoniae, Escherichia coli, Microsporum canis, Trichophyton mentagrophyte and Trichophyton rubrum. Some known antibiotics are included for the sake of comparison and the chosen antibiotic are Amikacin, Doxycllin, Augmantin, Sulperazon, Unasyn, Septrin, Cefobid, Ampicillin, Nitrofurantion, Traivid and Erythromycin.

  2. Structural and chemical basis for glucosamine 6-phosphate binding and activation of the glmS ribozyme.

    PubMed

    Cochrane, Jesse C; Lipchock, Sarah V; Smith, Kathryn D; Strobel, Scott A

    2009-04-21

    The glmS ribozyme is the first naturally occurring catalytic RNA that relies on an exogenous, nonnucleotide cofactor for reactivity. From a biochemical perspective, the glmS ribozyme derived from Bacillus anthracis is the best characterized. However, much of the structural work to date has been done on a variant glmS ribozyme, derived from Thermoanaerobacter tengcongensis. Here we present structures of the B. anthracis glmS ribozyme in states before the activating sugar, glucosamine 6-phosphate (GlcN6P), has bound and after the reaction has occurred. These structures show an active site preorganized to bind GlcN6P that retains some affinity for the sugar even after cleavage of the RNA backbone. A structure of an inactive glmS ribozyme with a mutation distal from the ligand-binding pocket highlights a nucleotide critical to the reaction that does not affect GlcN6P binding. Structures of the glmS ribozyme bound to a naturally occurring inhibitor, glucose 6-phosphate (Glc6P), and a nonnatural activating sugar, mannosamine 6-phosphate (MaN6P), reveal a binding mode similar to that of GlcN6P. Kinetic analyses show a pH dependence of ligand binding that is consistent with titration of the cofactor's phosphate group and support a model in which the major determinant of activity is the sugar amine independent of its stereochemical presentation. PMID:19228039

  3. Structural and Chemical Basis for Glucosamine 6-Phosphate Binding and Activation of the glmS Ribozyme

    SciTech Connect

    Cochrane, J.; Lipchock, S; Smith, K; Strobel, S

    2009-01-01

    The glmS ribozyme is the first naturally occurring catalytic RNA that relies on an exogenous, nonnucleotide cofactor for reactivity. From a biochemical perspective, the glmS ribozyme derived from Bacillus anthracis is the best characterized. However, much of the structural work to date has been done on a variant glmS ribozyme, derived from Thermoanaerobacter tengcongensis. Here we present structures of the B. anthracis glmS ribozyme in states before the activating sugar, glucosamine 6-phosphate (GlcN6P), has bound and after the reaction has occurred. These structures show an active site preorganized to bind GlcN6P that retains some affinity for the sugar even after cleavage of the RNA backbone. A structure of an inactive glmS ribozyme with a mutation distal from the ligand-binding pocket highlights a nucleotide critical to the reaction that does not affect GlcN6P binding. Structures of the glmS ribozyme bound to a naturally occurring inhibitor, glucose 6-phosphate (Glc6P), and a nonnatural activating sugar, mannosamine 6-phosphate (MaN6P), reveal a binding mode similar to that of GlcN6P. Kinetic analyses show a pH dependence of ligand binding that is consistent with titration of the cofactor's phosphate group and support a model in which the major determinant of activity is the sugar amine independent of its stereochemical presentation.

  4. Electrocatalytic activity of alkyne-functionalized AgAu alloy nanoparticles for oxygen reduction in alkaline media

    NASA Astrophysics Data System (ADS)

    Hu, Peiguang; Song, Yang; Chen, Limei; Chen, Shaowei

    2015-05-01

    1-Dodecyne-functionalized AgAu alloy nanoparticles were synthesized by chemical reduction of metal salt precursors at varied initial feed ratios. Transmission electron microscopic measurements showed that the nanoparticles were all rather well dispersed with the average core diameter in the narrow range of 3 to 5 nm. X-ray photoelectron spectroscopic studies confirmed the formation of AgAu alloy nanoparticles with the gold concentration ranging from approximately 25 at% to 55 at%. Consistent results were obtained in UV-vis spectroscopic measurements where the nanoparticle surface plasmon resonance red-shifted almost linearly with increasing gold concentrations. The self-assembly of 1-dodecyne ligands on the nanoparticle surface was manifested in infrared spectroscopic measurements. Importantly, the resulting nanoparticles exhibited apparent electrocatalytic activity for oxygen reduction in alkaline media, and the performance was found to show a volcano variation in the Au content in the alloy nanoparticles, with the best performance observed for the samples with ca. 35.5 at% Au. The enhanced catalytic activity, as compared to pure Ag nanoparticles or even commercial Pt/C catalysts, was accounted for by the unique metal-ligand interfacial bonding interactions as well as alloying effects that increased metal-oxygen affinity.1-Dodecyne-functionalized AgAu alloy nanoparticles were synthesized by chemical reduction of metal salt precursors at varied initial feed ratios. Transmission electron microscopic measurements showed that the nanoparticles were all rather well dispersed with the average core diameter in the narrow range of 3 to 5 nm. X-ray photoelectron spectroscopic studies confirmed the formation of AgAu alloy nanoparticles with the gold concentration ranging from approximately 25 at% to 55 at%. Consistent results were obtained in UV-vis spectroscopic measurements where the nanoparticle surface plasmon resonance red-shifted almost linearly with increasing gold

  5. Interactions between organic additives and active powders in water-based lithium iron phosphate electrode slurries

    NASA Astrophysics Data System (ADS)

    Li, Chia-Chen; Lin, Yu-Sheng

    2012-12-01

    The interactions of organic additives with active powders are investigated and are found to have great influence on the determination of the mixing process for preparing electrode slurries with good dispersion and electrochemical properties of lithium iron phosphate (LiFePO4) electrodes. Based on the analyses of zeta potential, sedimentation, and rheology, it is shown that LiFePO4 prefers to interact with styrene-butadiene rubber (SBR) relative to other organic additives such as sodium carboxymethyl cellulose (SCMC), and thus shows preferential adsorption by SBR, whereas SBR has much lower efficiency than SCMC in dispersing LiFePO4. Therefore, for SCMC to interact with and disperse LiFePO4 before the interaction of LiFePO4 with SBR, it is suggested to mix SCMC with LiFePO4 prior to the addition of SBR during the slurry preparation process. For the electrode prepared via the suggested process, i.e., the sequenced adding process in which SCMC is mixed with active powders prior to the addition of SBR, a much better electrochemical performance is obtained than that of the one prepared via the process referred as the simultaneous adding process, in which mixing of SCMC and SBR with active powders in simultaneous.

  6. Aldosterone impairs vascular reactivity by decreasing glucose-6-phosphate dehydrogenase activity

    PubMed Central

    Leopold, Jane A.; Dam, Aamir; Maron, Bradley A.; Scribner, Anne W.; Liao, Ronglih; Handy, Diane E.; Stanton, Robert C.; Pitt, Bertram; Loscalzo, Joseph

    2013-01-01

    Hyperaldosteronism is associated with impaired vascular reactivity; however, the mechanism by which aldosterone promotes endothelial dysfunction remains unknown. Glucose-6-phosphate dehydrogenase (G6pd), the principal source of Nadph, modulates vascular function by limiting oxidant stress to preserve bioavailable nitric oxide (NO•). In these studies, we show that aldosterone (10−9-10−7 mol/l) decreases endothelial G6pd expression and activity in vitro resulting in increased oxidant stress and decreased cGMP levels similar to what is observed in G6pd-deficient cells. Aldosterone decreases G6pd expression by protein kinase A activation to increase expression of Crem, which interferes with Creb binding to the G6pd promoter. In vivo, infusion of aldosterone decreases vascular G6pd expression and impairs vascular reactivity. These effects are abrogated by spironolactone or vascular gene transfer of G6pd. These studies demonstrate that aldosterone induces a G6pd-deficient phenotype to impair endothelial function; aldosterone antagonism or gene transfer of G6pd improves vascular reactivity by restoring G6pd activity. PMID:17273168

  7. QSAR study and the hydrolysis activity prediction of three alkaline lipases from different lipase-producing microorganisms

    PubMed Central

    2012-01-01

    The hydrolysis activities of three alkaline lipases, L-A1, L-A2 and L-A3 secreted by different lipase-producing microorganisms isolated from the Bay of Bohai, P. R. China were characterized with 16 kinds of esters. It was found that all the lipases have the ability to catalyze the hydrolysis of the glycerides, methyl esters, ethyl esters, especially for triglycerides, which shows that they have broad substrate spectra, and this property is very important for them to be used in detergent industry. Three QSAR models were built for L-A1, L-A2 and L-A3 respectively with GFA using Discovery studio 2.1. The models equations 1, 2 and 3 can explain 95.80%, 97.45% and 97.09% of the variances (R2adj) respectively while they could predict 95.44%, 89.61% and 93.41% of the variances (R2cv) respectively. With these models the hydrolysis activities of these lipases to mixed esters were predicted and the result showed that the predicted values are in good agreement with the measured values, which indicates that this method can be used as a simple tool to predict the lipase activities for single or mixed esters. PMID:23016923

  8. QSAR study and the hydrolysis activity prediction of three alkaline lipases from different lipase-producing microorganisms.

    PubMed

    Wang, Haikuan; Wang, Xiaojie; Li, Xiaolu; Zhang, Yehong; Dai, Yujie; Guo, Changlu; Zheng, Heng

    2012-01-01

    The hydrolysis activities of three alkaline lipases, L-A1, L-A2 and L-A3 secreted by different lipase-producing microorganisms isolated from the Bay of Bohai, P. R. China were characterized with 16 kinds of esters. It was found that all the lipases have the ability to catalyze the hydrolysis of the glycerides, methyl esters, ethyl esters, especially for triglycerides, which shows that they have broad substrate spectra, and this property is very important for them to be used in detergent industry. Three QSAR models were built for L-A1, L-A2 and L-A3 respectively with GFA using Discovery studio 2.1. The models equations 1, 2 and 3 can explain 95.80%, 97.45% and 97.09% of the variances (R(2)(adj)) respectively while they could predict 95.44%, 89.61% and 93.41% of the variances (R(2)(cv)) respectively. With these models the hydrolysis activities of these lipases to mixed esters were predicted and the result showed that the predicted values are in good agreement with the measured values, which indicates that this method can be used as a simple tool to predict the lipase activities for single or mixed esters. PMID:23016923

  9. Catalytic Activity-d-Band Center Correlation for the O2 Reduction on Platinum in Alkaline Solutions

    SciTech Connect

    Lima,F.; Zhang, J.; Shao, M.; Sasaki, K.; Vukmirovic, M.; Ticianelli, E.; Adzic, R.

    2007-01-01

    We determined, by the rotating disk electrode technique, the kinetics of the oxygen-reduction reaction (ORR) on the surfaces of single crystals of Au(111), Ag(111), Pd(111), Rh(111), Ir(111), and Ru(0001), on Pt monolayers deposited on their surfaces, and also on nanoparticles of these metals dispersed on high-surface-area carbon. Plotting the correlation between the experimentally determined activities of these three types of electrocatalysts with the calculated metal d-band center energies,{var_epsilon}{sub d}, revealed a volcano-type dependence. In all cases, the electronic properties of the metal electrocatalysts, represented by the {var_epsilon}{sub d} value, were used for elucidating the metal-dependent catalytic activities, and establishing their electronic properties-the ORR kinetics relationship. Pt(111), Pt/C, and Pt/Pd(111) were found to top their corresponding volcano plots. Pd in alkaline solutions showed particularly high activity, suggesting it may offer potential replacement for Pt in fuel cells.

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

    PubMed

    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 < 0.05). There were no significant differences in AST values. The maximum agreement rates between the 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

  11. Sub-cellular localisation of alkaline phosphatase activity in the cytoplasm of tammar wallaby (Macropus eugenii) neutrophils and eosinophils.

    PubMed

    Hulme-Moir, K Lisa; Clark, Phillip

    2011-07-15

    Alkaline phosphatase (ALP) has been used in studies of neutrophil morphology and function as a marker for identifying different granule populations. In human neutrophils, ALP is found within secretory vesicles, a rapidly mobilisable vesicle population important for upregulating membrane receptors during early activation. Intra-cellular ALP activity in the heterophils of rabbits and guinea pigs, in contrast, is found only in secondary granules. The neutrophils and eosinophils of tammar wallabies (Macropus eugenii) have previously been reported to contain large amounts of ALP activity when stained using routine cytochemical techniques. To define the subcellular location of ALP in this species, cell suspensions were examined using cerium chloride cytochemistry and transmission electron microscopy (TEM). ALP was found in 2 distinct cytoplasmic compartments. One compartment displayed morphology consistent with a subpopulation of secondary granules while a second tubulo-vesicular population appeared similar to the secretory vesicles of human neutrophils. Thin tubular vesicles containing ALP were also identified within the cytoplasm of tammar wallaby eosinophils. Large numbers of ALP-containing vesicles have not been recognised previously in eosinophils and this may represent a novel cytoplasmic compartment. In both cell types, ALP-containing structures showed alteration in morphology following stimulation with N-formyl-Met-Leu-Phe (fMLP) and PMA. PMID:21596444

  12. Panax notoginseng stimulates alkaline phosphatase activity, collagen synthesis, and mineralization in osteoblastic MC3T3-E1 cells.

    PubMed

    Ji, Zhe; Cheng, Yizhao; Yuan, Puwei; Dang, Xiaoqian; Guo, Xiong; Wang, Weizhuo

    2015-10-01

    Total Panax notoginseng saponin (PNS) has been extensively used to treat a variety of diseases, such as bone fractures, soft tissue injuries, etc. In this study, mouse calvaria-original osteoblastic MC3T3-E1 cells were cultured in various concentrations of PNS (0.005-5 mg/mL) during the period (1, 5, 14, and 23 d). At the endpoint, the osteogenic capacity of MC3T3-E1 cells was investigated by measuring the alkaline phosphatase (ALP) activity, the deposited calcium, and the expression of osteogenic-related markers, including bone collagen type 1 (Col1) and osteocalcin (OCN). Compared with all groups in each period, the most pronounced effect was observed at the concentration range between 0.05 and 0.5 mg/mL (P < 0.05) and the cell proliferation with PNS treatment was found during the whole osteogenic period. Moreover, cellular ALP activity with PNS was increased during 7, 14, and 21 d and cell mineralization with PNS was enhanced in 14 and 21 d. Furthermore, the differentiation markers Col1 and OCN increased in the PNS-treated cells. Our work suggests that PNS may stimulate the osteogenesis process which contains osteoblastic proliferation, differentiation, and mineralization by increasing cellular ALP activity, extracellular matrix mineralization, and osteoblast-associated molecules in the osteoblasts. PMID:25904074

  13. Equilibrium and kinetic studies of adsorption of phosphate onto ZnCl2 activated coir pith carbon.

    PubMed

    Namasivayam, C; Sangeetha, D

    2004-12-15

    Phosphate removal from aqueous solution was investigated using ZnCl(2)-activated carbon developed from coir pith, an agricultural solid waste. Studies were conducted to delineate the effect of contact time, adsorbent dose, phosphate concentration, pH, and temperature. The adsorption equilibrium data followed both Langmuir and Freundlich isotherms. Langmuir adsorption capacity was found to be 5.1 mg/g. Adsorption followed second-order kinetics. The removal was maximum in the pH range 3-10. pH effect and desorption studies showed that adsorption occurred by both ion exchange and chemisorption mechanisms. Adsorption was found to be spontaneous and endothermic. Effect of foreign ions on adsorption shows that perchlorate, sulfate, and selenite decreased the percent removal of phosphate. PMID:15533408

  14. Sphingosine-1-phosphate mediates epidermal growth factor-induced muscle satellite cell activation

    SciTech Connect

    Nagata, Yosuke Ohashi, Kazuya; Wada, Eiji; Yuasa, Yuki; Shiozuka, Masataka; Nonomura, Yoshiaki; Matsuda, Ryoichi

    2014-08-01

    Skeletal muscle can regenerate repeatedly due to the presence of resident stem cells, called satellite cells. Because satellite cells are usually quiescent, they must be activated before participating in muscle regeneration in response to stimuli such as injury, overloading, and stretch. Although satellite cell activation is a crucial step in muscle regeneration, little is known of the molecular mechanisms controlling this process. Recent work showed that the bioactive lipid sphingosine-1-phosphate (S1P) plays crucial roles in the activation, proliferation, and differentiation of muscle satellite cells. We investigated the role of growth factors in S1P-mediated satellite cell activation. We found that epidermal growth factor (EGF) in combination with insulin induced proliferation of quiescent undifferentiated mouse myoblast C2C12 cells, which are also known as reserve cells, in serum-free conditions. Sphingosine kinase activity increased when reserve cells were stimulated with EGF. Treatment of reserve cells with the D-erythro-N,N-dimethylsphingosine, Sphingosine Kinase Inhibitor, or siRNA duplexes specific for sphingosine kinase 1, suppressed EGF-induced C2C12 activation. We also present the evidence showing the S1P receptor S1P2 is involved in EGF-induced reserve cell activation. Moreover, we demonstrated a combination of insulin and EGF promoted activation of satellite cells on single myofibers in a manner dependent on SPHK and S1P2. Taken together, our observations show that EGF-induced satellite cell activation is mediated by S1P and its receptor. - Highlights: • EGF in combination with insulin induces proliferation of quiescent C2C12 cells. • Sphingosine kinase activity increases when reserve cells are stimulated with EGF. • EGF-induced activation of reserve cells is dependent on sphingosine kinase and ERK. • The S1P receptor S1P2 is involved in EGF-induced reserve cell activation. • EGF-induced reserve cell activation is mediated by S1P and its

  15. Synthesis and comparison of the biological activity of monocyclic phosphonate, difluorophosphonate and phosphate analogs of the natural AChE inhibitor cyclophostin.

    PubMed

    Martin, Benjamin P; Vasilieva, Elena; Dupureur, Cynthia M; Spilling, Christopher D

    2015-12-15

    New monocyclic phosphate, phosphonate and difluorophosphonate analogs of the natural AChE inhibitor cyclophostin were synthesized and their activity toward human AChE examined. Surprisingly, the phosphate, phosphonate, and difluorophosphonate analogs all showed diminished activity when compared with the natural product. PMID:26585276

  16. Use of agar diffusion assay to measure bactericidal activity of alkaline salts of fatty acids against bacteria associated with poultry processing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The agar diffusion assay was used to examine antibacterial activity of alkaline salts of caproic, caprylic, capric, lauric, and myristic acids. A 0.5M concentration of each fatty acid was dissolved in 1.0 M potassium hydroxide (KOH), and pH of the mixtures was adjusted to 10.5 with citric acid. Solu...

  17. Use of agar diffusion assay to evaluate bactericidal activity of formulations of alkaline salts of fatty acids against bacteria associated with poultry processing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The agar diffusion assay was used to examine antibacterial activity of alkaline salts of fatty acids (FA). Wells in agar media seeded with bacteria were filled with FA-potassium hydroxide (KOH) solutions, plates were incubated, and zones of inhibition were measured. The relationship between bacteric...

  18. Sphingosine-1-Phosphate Is a Novel Regulator of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Activity

    PubMed Central

    Semenkov, Illya; Molinski, Steven; Pasyk, Stan; Ahmadi, Saumel; Bui, Hai H.; Bear, Christine E.; Lidington, Darcy; Bolz, Steffen-Sebastian

    2015-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) attenuates sphingosine-1-phosphate (S1P) signaling in resistance arteries and has emerged as a prominent regulator of myogenic vasoconstriction. This investigation demonstrates that S1P inhibits CFTR activity via adenosine monophosphate-activated kinase (AMPK), establishing a potential feedback link. In Baby Hamster Kidney (BHK) cells expressing wild-type human CFTR, S1P (1μmol/L) attenuates forskolin-stimulated, CFTR-dependent iodide efflux. S1P’s inhibitory effect is rapid (within 30 seconds), transient and correlates with CFTR serine residue 737 (S737) phosphorylation. Both S1P receptor antagonism (4μmol/L VPC 23019) and AMPK inhibition (80μmol/L Compound C or AMPK siRNA) attenuate S1P-stimluated (i) AMPK phosphorylation, (ii) CFTR S737 phosphorylation and (iii) CFTR activity inhibition. In BHK cells expressing the ΔF508 CFTR mutant (CFTRΔF508), the most common mutation causing cystic fibrosis, both S1P receptor antagonism and AMPK inhibition enhance CFTR activity, without instigating discernable correction. In summary, we demonstrate that S1P/AMPK signaling transiently attenuates CFTR activity. Since our previous work positions CFTR as a negative S1P signaling regulator, this signaling link may positively reinforce S1P signals. This discovery has clinical ramifications for the treatment of disease states associated with enhanced S1P signaling and/or deficient CFTR activity (e.g. cystic fibrosis, heart failure). S1P receptor/AMPK inhibition could synergistically enhance the efficacy of therapeutic strategies aiming to correct aberrant CFTR trafficking. PMID:26079370

  19. Sphingosine-1-Phosphate Is a Novel Regulator of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Activity.

    PubMed

    Malik, Firhan A; Meissner, Anja; Semenkov, Illya; Molinski, Steven; Pasyk, Stan; Ahmadi, Saumel; Bui, Hai H; Bear, Christine E; Lidington, Darcy; Bolz, Steffen-Sebastian

    2015-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) attenuates sphingosine-1-phosphate (S1P) signaling in resistance arteries and has emerged as a prominent regulator of myogenic vasoconstriction. This investigation demonstrates that S1P inhibits CFTR activity via adenosine monophosphate-activated kinase (AMPK), establishing a potential feedback link. In Baby Hamster Kidney (BHK) cells expressing wild-type human CFTR, S1P (1μmol/L) attenuates forskolin-stimulated, CFTR-dependent iodide efflux. S1P's inhibitory effect is rapid (within 30 seconds), transient and correlates with CFTR serine residue 737 (S737) phosphorylation. Both S1P receptor antagonism (4μmol/L VPC 23019) and AMPK inhibition (80μmol/L Compound C or AMPK siRNA) attenuate S1P-stimluated (i) AMPK phosphorylation, (ii) CFTR S737 phosphorylation and (iii) CFTR activity inhibition. In BHK cells expressing the ΔF508 CFTR mutant (CFTRΔF508), the most common mutation causing cystic fibrosis, both S1P receptor antagonism and AMPK inhibition enhance CFTR activity, without instigating discernable correction. In summary, we demonstrate that S1P/AMPK signaling transiently attenuates CFTR activity. Since our previous work positions CFTR as a negative S1P signaling regulator, this signaling link may positively reinforce S1P signals. This discovery has clinical ramifications for the treatment of disease states associated with enhanced S1P signaling and/or deficient CFTR activity (e.g. cystic fibrosis, heart failure). S1P receptor/AMPK inhibition could synergistically enhance the efficacy of therapeutic strategies aiming to correct aberrant CFTR trafficking. PMID:26079370

  20. Activated platelets release sphingosine 1-phosphate and induce hypersensitivity to noxious heat stimuli in vivo

    PubMed Central

    Weth, Daniela; Benetti, Camilla; Rauch, Caroline; Gstraunthaler, Gerhard; Schmidt, Helmut; Geisslinger, Gerd; Sabbadini, Roger; Proia, Richard L.; Kress, Michaela

    2015-01-01

    At the site of injury activated platelets release various mediators, one of which is sphingosine 1-phosphate (S1P). It was the aim of this study to explore whether activated human platelets had a pronociceptive effect in an in vivo mouse model and whether this effect was based on the release of S1P and subsequent activation of neuronal S1P receptors 1 or 3. Human platelets were prepared in different concentrations (105/μl, 106/μl, 107/μl) and assessed in mice with different genetic backgrounds (WT, S1P1fl/fl, SNS-S1P1−/−, S1P3−/−). Intracutaneous injections of activated human platelets induced a significant, dose-dependent hypersensitivity to noxious thermal stimulation. The degree of heat hypersensitivity correlated with the platelet concentration as well as the platelet S1P content and the amount of S1P released upon platelet activation as measured with LC MS/MS. Despite the significant correlations between S1P and platelet count, no difference in paw withdrawal latency (PWL) was observed in mice with a global null mutation of the S1P3 receptor or a conditional deletion of the S1P1 receptor in nociceptive primary afferents. Furthermore, neutralization of S1P with a selective anti-S1P antibody did not abolish platelet induced heat hypersensitivity. Our results suggest that activated platelets release S1P and induce heat hypersensitivity in vivo. However, the platelet induced heat hypersensitivity was caused by mediators other than S1P. PMID:25954148

  1. Cellular recovery of glyceraldehyde-3-phosphate dehydrogenase activity and thiol status after exposure to hydroperoxides

    SciTech Connect

    Brodie, A.E.; Reed, D.J. )

    1990-01-01

    The activity of the thiol-dependent enzyme glyceraldehyde-3-phosphate dehydrogenase (GPD), in vertebrate cells, was modulated by a change in the intracellular thiol:disulfide redox status. Human lung carcinoma cells (A549) were incubated with 1-120 mM H2O2, 1-120 mM t-butyl hydroperoxide, 1-6 mM ethacrynic acid, or 0.1-10 mM N-ethylmaleimide for 5 min. Loss of reduced protein thiols, as measured by binding of the thiol reagent iodoacetic acid to GPD, and loss of GPD enzymatic activity occurred in a dose-dependent manner. Incubation of the cells, following oxidative treatment, in saline for 30 min or with 20 mM dithiothreitol (DTT) partially reversed both changes in GPD. The enzymatic recovery of GPD activity was observed either without addition of thiols to the medium or by incubation of a sonicated cell mixture with 2 mM cysteine, cystine, cysteamine, or glutathione (GSH); GSSG had no effect. Treatment of cells with buthionine sulfoximine (BSO) to decrease cellular GSH by varying amounts caused a dose-related increase in sensitivity of GPD activity to inactivation by H2O2 and decreased cellular ability for subsequent recovery. GPD responded in a similar fashion with oxidative treatment of another lung carcinoma cell line (A427) as well as normal lung tissue from human and rat. These findings indicate that the cellular thiol redox status can be important in determining GPD enzymatic activity.

  2. In vitro activity of Tedizolid phosphate against multidrug-resistant Streptococcus pneumoniae isolates from Asian countries.

    PubMed

    Baek, Jin Yang; Kang, Cheol-In; Kim, So Hyun; Ko, Kwan Soo; Chung, Doo Ryeon; Peck, Kyong Ran; Hsueh, Po-Ren; Thamlikitkul, Visanu; So, Thomas Man-Kit; Lee, Nam Yong; Song, Jae-Hoon

    2016-06-01

    Tedizolid phosphate is a second-generation oxazolidinone prodrug that is potential activity against a wide range of Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus, penicillin-resistant streptococci, and vancomycin-resistant enterococci. The in vitro activity of tedizolid and other comparator agents against multidrug-resistant (MDR) pneumococci from various Asian countries were evaluated. Of the S. pneumoniae clinical pneumonia isolates collected during 2008 and 2009 from 8 Asian countries (Korea, Taiwan, Thailand, Hong Kong, Vietnam, Malaysia, Philippines, and Sri Lanka), 104 isolates of MDR pneumococci were included in this study. Antimicrobial susceptibility testing for 18 antimicrobial agents was performed by broth microdilution method. Tedizolid was highly active against pneumococci. All isolates tested were inhibited at a tedizolid minimum inhibitory concentration (MIC) value of ≤0.25μg/ml (ranged from ≤0.03μg/ml to 0.25μg/ml). The MIC50 and MIC90 of tedizolid against MDR pneumococci were both 0.12μg/ml, while MIC50 and MIC90 of linezolid were 0.5μg/ml and 1μg/ml, respectively. In addition, tedizolid maintained the activity against S. pneumoniae regardless of the extensively drug-resistant (XDR) phenotype of the isolates. The activity of tedizolid was excellent against all types of MDR pneumococci, exhibiting and maintaining at least 4-fold-greater potency compared to linezolid, regardless of resistance phenotypes to other commonly utilized agents. Tedizolid has the potential to be an agent to treat infections caused by MDR pneumococci in the Asia. PMID:27083121

  3. Dual mode antibacterial activity of ion substituted calcium phosphate nanocarriers for bone infections.

    PubMed

    Sampath Kumar, T S; Madhumathi, K; Rubaiya, Y; Doble, Mukesh

    2015-01-01

    Nanotechnology has tremendous potential for the management of infectious diseases caused by multi-drug resistant bacteria, through the development of newer antibacterial materials and efficient modes of antibiotic delivery. Calcium phosphate (CaP) bioceramics are commonly used as bone substitutes due to their similarity to bone mineral and are widely researched upon for the treatment of bone infections associated with bone loss. CaPs can be used as local antibiotic delivery agents for bone infections and can be substituted with antibacterial ions in their crystal structure to have a wide spectrum, sustained antibacterial activity even against drug resistant bacteria. In the present work, a dual mode antibiotic delivery system with antibacterial ion substituted calcium deficient hydroxyapatite (CDHA) nanoparticles has been developed. Antibacterial ions such as zinc, silver, and strontium have been incorporated into CDHA at concentrations of 6, 0.25-0.75, and 2.5-7.5 at. %, respectively. The samples were found to be phase pure, acicular nanoparticles of length 40-50 nm and width 5-6 nm approximately. The loading and release profile of doxycycline, a commonly used antibiotic, was studied from the nanocarriers. The drug release was studied for 5 days and the release profile was influenced by the ion concentrations. The release of antibacterial ions was studied over a period of 21 days. The ion substituted CDHA samples were tested for antibacterial efficacy on Staphylococcus aureus and Escherichia coli by MIC/MBC studies and time-kill assay. AgCDHA and ZnCDHA showed high antibacterial activity against both bacteria, while SrCDHA was weakly active against S. aureus. Present study shows that the antibiotic release can provide the initial high antibacterial activity, and the sustained ion release can provide a long-term antibacterial activity. Such dual mode antibiotic and antibacterial ion release offers an efficient and potent way to treat an incumbent drug

  4. Subcellular Characterization of Porcine Oocytes with Different Glucose-6-phosphate Dehydrogenase Activities

    PubMed Central

    Fu, Bo; Ren, Liang; Liu, Di; Ma, Jian-Zhang; An, Tie-Zhu; Yang, Xiu-Qin; Ma, Hong; Zhang, Dong-Jie; Guo, Zhen-Hua; Guo, Yun-Yun; Zhu, Meng; Bai, Jing

    2015-01-01

    The in vitro maturation (IVM) efficiency of porcine embryos is still low because of poor oocyte quality. Although brilliant cresyl blue positive (BCB+) oocytes with low glucose-6-phosphate dehydrogenase (G6PDH) activity have shown superior quality than BCB negative (−) oocytes with high G6PDH activity, the use of a BCB staining test before IVM is still controversial. This study aimed to shed more light on the subcellular characteristics of porcine oocytes after selection using BCB staining. We assessed germinal vesicle chromatin configuration, cortical granule (CG) migration, mitochondrial distribution, the levels of acetylated lysine 9 of histone H3 (AcH3K9) and nuclear apoptosis features to investigate the correlation between G6PDH activity and these developmentally related features. A pattern of chromatin surrounding the nucleoli was seen in 53.0% of BCB+ oocytes and 77.6% of BCB+ oocytes showed peripherally distributed CGs. After IVM, 48.7% of BCB+ oocytes had a diffused mitochondrial distribution pattern. However, there were no significant differences in the levels of AcH3K9 in the nuclei of blastocysts derived from BCB+ and BCB− oocytes; at the same time, we observed a similar incidence of apoptosis in the BCB+ and control groups. Although this study indicated that G6PDH activity in porcine oocytes was correlated with several subcellular characteristics such as germinal vesicle chromatin configuration, CG migration and mitochondrial distribution, other features such as AcH3K9 level and nuclear apoptotic features were not associated with G6PDH activity and did not validate the BCB staining test. In using this test for selecting porcine oocytes, subcellular characteristics such as the AcH3K9 level and apoptotic nuclear features should also be considered. Adding histone deacetylase inhibitors or apoptosis inhibitors into the culture medium used might improve the efficiency of IVM of BCB+ oocytes. PMID:26580437

  5. Modulation of glyceraldehyde-3-phosphate dehydrogenase activity by surface functionalized quantum dots.

    PubMed

    Ghosh, Srabanti; Ray, Manju; Das, Mahua Rani; Chakrabarti, Adrita; Khan, Ali Hossain; Sarma, D D; Acharya, Somobrata

    2014-03-21

    Enzymatic regulation is a fast and reliable diagnosis tool via identification and design of inhibitors for modulation of enzyme function. Previous reports on quantum dots (QDs)-enzyme interactions reveal a protein-surface recognition ability leading to promising applications in protein stabilization, protein delivery, bio-sensing and detection. However, the direct use of QDs to control enzyme inhibition has never been revealed to date. Here we show that a series of biocompatible surface-functionalized metal-chalcogenide QDs can be used as potent inhibitors for malignant cells through the modulation of enzyme activity, while normal cells remain unaffected. The in vitro activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an enzyme involved critically in the glycolysis of cancer cells, is inactivated selectively in a controlled way by the QDs at a significantly low concentration (nM). Cumulative kinetic studies delineate that the QDs undergo both reversible and irreversible inhibition mechanisms owing to the site-specific interactions, enabling control over the inhibition kinetics. These complementary loss-of-function probes may offer a novel route for rapid clinical diagnosis of malignant cells and biomedical applications. PMID:24496476

  6. Enhanced Catalytic Activities of NiPt Truncated Octahedral Nanoparticles toward Ethylene Glycol Oxidation and Oxygen Reduction in Alkaline Electrolyte.

    PubMed

    Xia, Tianyu; Liu, Jialong; Wang, Shouguo; Wang, Chao; Sun, Young; Gu, Lin; Wang, Rongming

    2016-05-01

    The high cost and poor durability of Pt nanoparticles (NPs) are great limits for the proton exchange membrane fuel cells (PEMFCs) from being scaled-up for commercial applications. Pt-based bimetallic NPs together with a uniform distribution can effectively reduce the usage of expensive Pt while increasing poison resistance of intermediates. In this work, a simple one-pot method was used to successfully synthesize ultrafine (about 7.5 nm) uniform NiPt truncated octahedral nanoparticles (TONPs) in dimethylformamid (DMF) without any seeds or templates. The as-prepared NiPt TONPs with Pt-rich surfaces exhibit greatly improved catalytic activities together with good tolerance and better stability for ethylene glycol oxidation reaction (EGOR) and oxygen reduction reaction (ORR) in comparison with NiPt NPs and commercial Pt/C catalysts in alkaline electrolyte. For example, the value of mass and specific activities for EGOR are 23.2 and 17.6 times higher comparing with those of commercial Pt/C, respectively. Our results demonstrate that the dramatic enhancement is mainly attributed to Pt-rich surface, larger specific surface area, together with coupling between Ni and Pt atoms. This developed method provides a promising pathway for simple preparation of highly efficient electrocatalysts for PEMFCs in the near future. PMID:27093304

  7. Dissolved organic phosphorus utilization and alkaline phosphatase activity of the dinoflagellate Gymnodinium impudicum isolated from the South Sea of Korea

    NASA Astrophysics Data System (ADS)

    Oh, Seok Jin; Kwon, Hyeong Kyu; Noh, Il Hyeon; Yang, Han-Soeb

    2010-09-01

    This study investigated alkaline phosphatase (APase) activity and dissolved organic and inorganic phosphorus utilization by the harmful dinoflagellate Gymnodinium impudicum (Fraga et Bravo) Hansen et Moestrup isolated from the South Sea of Korea. Under conditions of limited phosphorus, observation of growth kinetics in batch culture yielded a maximum growth rate (μmax) of 0.41 /day and a half saturation constant (Ks) of 0.71 μM. In time-course experiments, APase was induced as dissolved inorganic phosphorus (DIP) concentrations fell below 0.83 μM, a threshold near the estimated Ks; APase activity increased with further DIP depletion to a maximum of 0.70 pmol/cell/h in the senescent phase. Thus, Ks may be an important index of the threshold DIP concentration for APase induction. G. impudicum utilizes a wide variety of dissolved organic phosphorus compounds in addition to DIP. These results suggest that DIP limitation in the Southern Sea of Korea may have led to the spread of G. impudicum along with the harmful dinoflagellate Cochlodinium polykrikoides in recent years.

  8. Electrocatalytic Activity of Transition Metal Oxide-Carbon Composites for Oxygen Reduction in Alkaline Batteries and Fuel Cells

    SciTech Connect

    Malkhandi, S; Trinh, P; Manohar, AK; Jayachandrababu, KC; Kindler, A; Prakash, GKS; Narayanan, SR

    2013-06-07

    Conductive transition metal oxides (perovskites, spinels and pyrochlores) are attractive as catalysts for the air electrode in alkaline rechargeable metal-air batteries and fuel cells. We have found that conductive carbon materials when added to transition metal oxides such as calcium-doped lanthanum cobalt oxide, nickel cobalt oxide and calcium-doped lanthanum manganese cobalt oxide increase the electrocatalytic activity of the oxide for oxygen reduction by a factor of five to ten. We have studied rotating ring-disk electrodes coated with (a) various mass ratios of carbon and transition metal oxide, (b) different types of carbon additives and (c) different types of transition metal oxides. Our experiments and analysis establish that in such composite catalysts, carbon is the primary electro- catalyst for the two-electron electro-reduction of oxygen to hydroperoxide while the transition metal oxide decomposes the hydroperoxide to generate additional oxygen that enhances the observed current resulting in an apparent four-electron process. These findings are significant in that they change the way we interpret previous reports in the scientific literature on the electrocatalytic activity of various transition metal oxide- carbon composites for oxygen reduction, especially where carbon is assumed to be an additive that just enhances the electronic conductivity of the oxide catalyst. (C) 2013 The Electrochemical Society. All rights reserved.

  9. Mononuclear and dinuclear peroxotungsten complexes with co-ordinated dipeptides as potent inhibitors of alkaline phosphatase activity.

    PubMed

    Hazarika, Pankaj; Kalita, Diganta; Islam, Nashreen S

    2008-08-01

    New molecular peroxotungstate(VI) complexes with dipeptides as ancillary ligands of the type, [WO(O(2))(2)(dipeptide)(H(2)O)].3H(2)O, dipeptide = glycyl-glycine or glycyl-leucine, have been synthesized and characterized by elemental analysis, spectral and physico-chemical methods including thermal analysis. The complexes contain side-on bound peroxo groups and a peptide zwitterion bonded to the metal centre unidentately through an O(carboxylate) atom. Investigations on certain biologically important key properties of these compounds and a set of dimeric compounds in analogous co-ligand environment, Na(2)[W(2)O(3)(O(2))(4)(dipeptide)(2)].3H(2)O, dipeptide = glycyl-glycine and glycyl-leucine, reported previously by us revealed interesting features of the compounds. Each of the compounds despite having a 7 co-ordinated metal centre exerts a strong inhibitory effect on alkaline phosphatase activity with a potency higher than that of the free dipeptide, tungstate or peroxotungstate. The compounds exhibit remarkable stability in solutions of acidic as well as physiological pH and are weaker as substrate to the enzyme catalase, compared to H(2)O(2). The mononuclear and dinuclear peroxotungsten compounds are efficient oxidants of reduced glutathione (GSH), a reaction in which only one of the peroxo groups of a diperoxotungsten moiety of the complexes was found to be active. PMID:18665997

  10. Investigation on the near-infrared-emitting thermal stability of Bi activated alkaline-earth aluminoborosilicate glasses

    NASA Astrophysics Data System (ADS)

    Wan, Ronghua; Song, Zhiguo; Li, Yongjin; Zhou, Yuting; Liu, Qun; Qiu, Jianbei; Yang, Zhengwen; Yin, Zhaoyi

    2015-02-01

    Stability of near-infrared (NIR) emission form Bi doped 42.5SiO2-12.5B2O3-25Al2O3-20RO (SBAR, R = Ca, Sr, Ba) glasses under treatment between annealing and softening temperature were studied. Results show that the thermal stability of Bi-NIR-emitting centers in SBAR glasses generally decreases with the increase of the radius of modifier cations but is greatly higher that in similar alkali glasses. Comparative experiments indicate these phenomena can be understood by the tendency that the smaller and higher charged alkaline earth ions as higher field strength modifier cations will increase the concentration of negative charge on non-bridging oxygens and also help to stabilize the non-bridging oxygens, which can restrain the thermally activated diffusion and valence change of Bi-activated centers, respectively. The results can provide an improved understanding for the NIR-emitting thermal stability of Bi doped glasses and a scientific reference for composition design of Bi-doped optical fiber.

  11. Replacement of a phenylalanine by a tyrosine in the active site confers fructose-6-phosphate aldolase activity to the transaldolase of Escherichia coli and human origin.

    PubMed

    Schneider, Sarah; Sandalova, Tatyana; Schneider, Gunter; Sprenger, Georg A; Samland, Anne K

    2008-10-31

    Based on a structure-assisted sequence alignment we designed 11 focused libraries at residues in the active site of transaldolase B from Escherichia coli and screened them for their ability to synthesize fructose 6-phosphate from dihydroxyacetone and glyceraldehyde 3-phosphate using a newly developed color assay. We found one positive variant exhibiting a replacement of Phe(178) to Tyr. This mutant variant is able not only to transfer a dihydroxyacetone moiety from a ketose donor, fructose 6-phosphate, onto an aldehyde acceptor, erythrose 4-phosphate (14 units/mg), but to use it as a substrate directly in an aldolase reaction (7 units/mg). With a single amino acid replacement the fructose-6-phosphate aldolase activity was increased considerably (>70-fold compared with wild-type). Structural studies of the wild-type and mutant protein suggest that this is due to a different H-bond pattern in the active site leading to a destabilization of the Schiff base intermediate. Furthermore, we show that a homologous replacement has a similar effect in the human transaldolase Taldo1 (aldolase activity, 14 units/mg). We also demonstrate that both enzymes TalB and Taldo1 are recognized by the same polyclonal antibody. PMID:18687684

  12. Replacement of a Phenylalanine by a Tyrosine in the Active Site Confers Fructose-6-phosphate Aldolase Activity to the Transaldolase of Escherichia coli and Human Origin*S⃞

    PubMed Central

    Schneider, Sarah; Sandalova, Tatyana; Schneider, Gunter; Sprenger, Georg A.; Samland, Anne K.

    2008-01-01

    Based on a structure-assisted sequence alignment we designed 11 focused libraries at residues in the active site of transaldolase B from Escherichia coli and screened them for their ability to synthesize fructose 6-phosphate from dihydroxyacetone and glyceraldehyde 3-phosphate using a newly developed color assay. We found one positive variant exhibiting a replacement of Phe178 to Tyr. This mutant variant is able not only to transfer a dihydroxyacetone moiety from a ketose donor, fructose 6-phosphate, onto an aldehyde acceptor, erythrose 4-phosphate (14 units/mg), but to use it as a substrate directly in an aldolase reaction (7 units/mg). With a single amino acid replacement the fructose-6-phosphate aldolase activity was increased considerably (>70-fold compared with wild-type). Structural studies of the wild-type and mutant protein suggest that this is due to a different H-bond pattern in the active site leading to a destabilization of the Schiff base intermediate. Furthermore, we show that a homologous replacement has a similar effect in the human transaldolase Taldo1 (aldolase activity, 14 units/mg). We also demonstrate that both enzymes TalB and Taldo1 are recognized by the same polyclonal antibody. PMID:18687684

  13. Mechanical properties of materials obtained via alkaline activation of illite-based clays of Latvia

    NASA Astrophysics Data System (ADS)

    Sperberga, I.; Rundans, M.; Cimmers, A.; Krage, L.; Sidraba, I.

    2015-04-01

    Materials has been synthesized in the temperature range from 60-100 °C from two illite based clays of Latvia under activation of KOH and NaOH solutions (4-6 M). Compressive strength and apparent porosity were measured. The effect of concentration of KOH and NaOH solutions on the material mechanical properties was investigated by means of infrared spectroscopy (IR). Compressive strength data of the materials showed that via such activation could obtain building materials with good quality.

  14. Conjugated bile acids activate the sphingosine-1-phosphate receptor 2 in primary rodent hepatocytes.

    PubMed

    Studer, Elaine; Zhou, Xiqiao; Zhao, Renping; Wang, Yun; Takabe, Kazuaki; Nagahashi, Masayuki; Pandak, William M; Dent, Paul; Spiegel, Sarah; Shi, Ruihua; Xu, Weiren; Liu, Xuyuan; Bohdan, Pat; Zhang, Luyong; Zhou, Huiping; Hylemon, Phillip B

    2012-01-01

    Bile acids have been shown to be important regulatory molecules for cells in the liver and gastrointestinal tract. They can activate various cell signaling pathways including extracellular regulated kinase (ERK)1/2 and protein kinase B (AKT) as well as the G-protein-coupled receptor (GPCR) membrane-type bile acid receptor (TGR5/M-BAR). Activation of the ERK1/2 and AKT signaling pathways by conjugated bile acids has been reported to be sensitive to pertussis toxin (PTX) and dominant-negative Gα(i) in primary rodent hepatocytes. However, the GPCRs responsible for activation of these pathways have not been identified. Screening GPCRs in the lipid-activated phylogenetic family (expressed in HEK293 cells) identified sphingosine-1-phosphate receptor 2 (S1P(2) ) as being activated by taurocholate (TCA). TCA, taurodeoxycholic acid (TDCA), tauroursodeoxycholic acid (TUDCA), glycocholic acid (GCA), glycodeoxycholic acid (GDCA), and S1P-induced activation of ERK1/2 and AKT were significantly inhibited by JTE-013, a S1P(2) antagonist, in primary rat hepatocytes. JTE-013 significantly inhibited hepatic ERK1/2 and AKT activation as well as short heterodimeric partner (SHP) mRNA induction by TCA in the chronic bile fistula rat. Knockdown of the expression of S1P(2) by a recombinant lentivirus encoding S1P(2) shRNA markedly inhibited the activation of ERK1/2 and AKT by TCA and S1P in rat primary hepatocytes. Primary hepatocytes prepared from S1P(2) knock out (S1P(2) (-/-) ) mice were significantly blunted in the activation of the ERK1/2 and AKT pathways by TCA. Structural modeling of the S1P receptors indicated that only S1P(2) can accommodate TCA binding. In summary, all these data support the hypothesis that conjugated bile acids activate the ERK1/2 and AKT signaling pathways primarily through S1P(2) in primary rodent hepatocytes. PMID:21932398

  15. Structural definition of the active site and catalytic mechanism of 3,4-dihydroxy-2-butanone-4-phosphate synthase.

    PubMed

    Liao, Der-Ing; Zheng, Ya-Jun; Viitanen, Paul V; Jordan, Douglas B

    2002-02-12

    X-ray crystal structures of L-3,4-dihydroxy-2-butanone-4-phosphate synthase from Magnaporthe grisea are reported for the E-SO(4)(2-), E-SO(4)(2-)-Mg(2+), E-SO(4)(2)(-)-Mn(2+), E-SO(4)(2)(-)-Mn(2+)-glycerol, and E-SO(4)(2)(-)-Zn(2+) complexes with resolutions that extend to 1.55, 0.98, 1.60, 1.16, and 1.00 A, respectively. Active-site residues of the homodimer are fully defined. The structures were used to model the substrate ribulose 5-phosphate in the active site with the phosphate group anchored at the sulfate site and the placement of the ribulose group guided by the glycerol site. The model includes two Mg(2+) cations that bind to the oxygen substituents of the C2, C3, C4, and phosphate groups of the substrate, the side chains of Glu37 and His153, and water molecules. The position of the metal cofactors and the substrate's phosphate group are further stabilized by an extensive hydrogen-bond and salt-bridge network. On the basis of their proximity to the substrate's reaction participants, the imidazole of an Asp99-His136 dyad from one subunit, the side chains of the Asp41, Cys66, and Glu174 residues from the other subunit, and Mg(2+)-activated water molecules are proposed to serve specific roles in the catalytic cycle as general acid-base functionalities. The model suggests that during the 1,2-shift step of the reaction, the substrate's C3 and C4 hydroxyl groups are cis to each other. A cis transition state is calculated to have an activation barrier that is 2 kcal/mol greater than that of the trans transition state in the absence of the enzyme. PMID:11827524

  16. Structural definition of the active site and catalytic mechanism of 3,4-dihydroxy-2-butanone 4-phosphate synthase

    SciTech Connect

    Liao, D.-I.; Zheng, Y.-J.; Viitanen, P.V.; Jordan, D.B.

    2010-03-08

    X-ray crystal structures of L-3,4-dihydroxy-2-butanone-4-phosphate synthase from Magnaporthe grisea are reported for the E-SO{sub 4}{sup 2-}, E-{sub 4}{sup 2-}-Mg{sup 2+}, E-SO{sub 4}{sup 2-}-Mn{sup 2+}, E-SO{sub 4}{sup 2-}-Mn{sup 2+}-glycerol, and E-SO{sub 4}{sup 2-}-Zn{sup 2+} complexes with resolutions that extend to 1.55, 0.98, 1.60, 1.16, and 1.00 {angstrom}, respectively. Active-site residues of the homodimer are fully defined. The structures were used to model the substrate ribulose 5-phosphate in the active site with the phosphate group anchored at the sulfate site and the placement of the ribulose group guided by the glycerol site. The model includes two Mg{sup 2+} cations that bind to the oxygen substituents of the C2, C3, C4, and phosphate groups of the substrate, the side chains of Glu37 and His153, and water molecules. The position of the metal cofactors and the substrate's phosphate group are further stabilized by an extensive hydrogen-bond and salt-bridge network. On the basis of their proximity to the substrate's reaction participants, the imidazole of an Asp99-His136 dyad from one subunit, the side chains of the Asp41, Cys66, and Glu174 residues from the other subunit, and Mg{sup 2+}-activated water molecules are proposed to serve specific roles in the catalytic cycle as general acid-base functionalities. The model suggests that during the 1,2-shift step of the reaction, the substrate's C3 and C4 hydroxyl groups are cis to each other. A cis transition state is calculated to have an activation barrier that is 2 kcal/mol greater than that of the trans transition state in the absence of the enzyme.

  17. Adsorption between TC-stabilized AuNPs and the phosphate group: application of the PTP1B activity assay.

    PubMed

    Lv, Jun; Wang, Xiaonan; Zhang, Yuanyuan; Li, Defeng; Zhang, Juan; Sun, Lizhou

    2015-12-01

    Based on the adsorption between tetracycline (TC) and phosphate groups, a general colorimetric method is explored in this work by using TC-stabilized gold nanoparticles (TC/AuNPs) and 4-aminophenyl phosphate-functionalized Fe3O4 magnetic nanoparticles (APP/MNPs). Taking protein tyrosine phosphatase 1B (PTP1B) as an example, 4-aminophenyl phosphate (APP) can be hydrolyzed into 4-aminophenol (AP) by PTP1B, resulting in the disappearance of the phosphate group on the outer layer of MNPs and the loss of corresponding adsorptive ability. Upon addition of TC/AuNP solution, TC/AuNPs will remain in the supernatant solution after magnetic separation and a high absorbance value can be observed. So PTP1B activity is related to the concentrations of TC/AuNPs in the supernatant solution. In this work, the enzyme activity can be determined at levels as low as 0.0885 U mL(-1) and over a linear detection range as wide as 0.1 U mL(-1) to 0.9 U mL(-1). Moreover, using the proposed method, the inhibition effect of betulinic acid (BA) and sodium orthovanadate (Na3VO4) on PTP1B activity can be tested with IC50 values of 30 μM and 4 μM, respectively. Therefore, a universal platform for the accurate colorimetric analysis of kinase and phosphatase activities can be established through the adsorption between TC and phosphate groups. PMID:26523458

  18. Molecular modeling of human alkaline sphingomyelinase.

    PubMed

    Suresh, Panneer Selvam; Olubiyi, Olujide; Thirunavukkarasu, Chinnasamy; Strodel, Birgit; Kumar, Muthuvel Suresh

    2011-01-01

    Alkaline sphingomyelinase, which is expressed in the human intestine and hydrolyses sphingomyelin, is a component of the plasma and the lysosomal membranes. Hydrolase of sphingomyelin generates ceramide, sphingosine, and sphingosine 1-phosphate that have regulatory effects on vital cellular functions such as proliferation, differentiation, and apoptosis. The enzyme belongs to the Nucleotide Pyrophosphatase/Phosphodiesterase family and it differs in structural similarity with acidic and neutral sphingomyelinase. In the present study we modeled alkaline sphingomyelinase using homology modeling based on the structure of Nucleotide Pyrophosphatase/Phosphodiesterase from Xanthomonas axonopodis with which it shares 34% identity. Homology modeling was performed using Modeller9v7. We found that Cys78 and Cys394 form a disulphide bond. Further analysis shows that Ser76 may be important for the function of this enzyme, which is supported by the findings of Wu et al. (2005), that S76F abolishes the activity completely. We found that the residues bound to Zn(2+) are conserved and geometrically similar with the template. Molecular Dynamics simulations were carried out for the modeled protein to observe the effect of Zinc metal ions. It was observed that the metal ion has little effect with regard to the stability but induces increased fluctuations in the protein. These analyses showed that Zinc ions play an important role in stabilizing the secondary structure and in maintaining the compactness of the active site. PMID:21544170

  19. Determination of Phosphate-activated Glutaminase Activity and Its Kinetics in Mouse Tissues using Metabolic Mapping (Quantitative Enzyme Histochemistry)

    PubMed Central

    Botman, Dennis; Tigchelaar, Wikky

    2014-01-01

    Phosphate-activated glutaminase (PAG) converts glutamine to glutamate as part of the glutaminolysis pathway in mitochondria. Two genes, GLS1 and GLS2, which encode for kidney-type PAG and liver-type PAG, respectively, differ in their tissue-specific activities and kinetics. Tissue-specific PAG activity and its kinetics were determined by metabolic mapping using a tetrazolium salt and glutamate dehydrogenase as an auxiliary enzyme in the presence of various glutamine concentrations. In kidney and brain, PAG showed Michaelis-Menten kinetics with a Km of 0.6 mM glutamine and a Vmax of 1.1 µmol/mL/min when using 5 mM glutamine. PAG activity was high in the kidney cortex and inner medulla but low in the outer medulla, papillary tip, glomeruli and the lis of Henle. In brain tissue sections, PAG was active in the grey matter and inactive in myelin-rich regions. Liver PAG showed allosteric regulation with a Km of 11.6 mM glutamine and a Vmax of 0.5 µmol/mL/min when using 20 mM glutamine. The specificity of the method was shown after incubation of brain tissue sections with the PAG inhibitor 6-diazo-5-oxo-L-norleucine. PAG activity was decreased to 22% in the presence of 2 mM 6-diazo-5-oxo-L-norleucine. At low glutamine concentrations, PAG activity was higher in periportal regions, indicating a lower Km for periportal PAG. When compared with liver, kidney and brain, other tissues showed 3-fold to 6-fold less PAG activity. In conclusion, PAG is mainly active in mouse kidney, brain and liver, and shows different kinetics depending on which type of PAG is expressed. PMID:25163927

  20. Sphingosine-1-phosphate lyase downregulation promotes colon carcinogenesis through STAT3-activated microRNAs

    PubMed Central

    Degagné, Emilie; Pandurangan, Ashok; Bandhuvula, Padmavathi; Kumar, Ashok; Eltanawy, Abeer; Zhang, Meng; Yoshinaga, Yuko; Nefedov, Mikhail; de Jong, Pieter J.; Fong, Loren G.; Young, Stephen G.; Bittman, Robert; Ahmedi, Yasmin; Saba, Julie D.

    2014-01-01

    Growing evidence supports a link between inflammation and cancer; however, mediators of the transition between inflammation and carcinogenesis remain incompletely understood. Sphingosine-1-phosphate (S1P) lyase (SPL) irreversibly degrades the bioactive sphingolipid S1P and is highly expressed in enterocytes but downregulated in colon cancer. Here, we investigated the role of SPL in colitis-associated cancer (CAC). We generated mice with intestinal epithelium-specific Sgpl1 deletion and chemically induced colitis and tumor formation in these animals. Compared with control animals, mice lacking intestinal SPL exhibited greater disease activity, colon shortening, cytokine levels, S1P accumulation, tumors, STAT3 activation, STAT3-activated microRNAs (miRNAs), and suppression of miR-targeted anti-oncogene products. This phenotype was attenuated by STAT3 inhibition. In fibroblasts, silencing SPL promoted tumorigenic transformation through a pathway involving extracellular transport of S1P through S1P transporter spinster homolog 2 (SPNS2), S1P receptor activation, JAK2/STAT3-dependent miR-181b-1 induction, and silencing of miR-181b-1 target cylindromatosis (CYLD). Colon biopsies from patients with inflammatory bowel disease revealed enhanced S1P and STAT3 signaling. In mice with chemical-induced CAC, oral administration of plant-type sphingolipids called sphingadienes increased colonic SPL levels and reduced S1P levels, STAT3 signaling, cytokine levels, and tumorigenesis, indicating that SPL prevents transformation and carcinogenesis. Together, our results suggest that dietary sphingolipids can augment or prevent colon cancer, depending upon whether they are metabolized to S1P or promote S1P metabolism through the actions of SPL. PMID:25347472

  1. Redox balance and mitochondrial glycerol phosphate dehydrogenase activity in trained rats.

    PubMed

    Casimiro-Lopes, Gustavo; Ramos, Dionizio; Sorenson, Martha M; Salerno, Verônica P

    2012-11-01

    Free radical production is increased in many disease states and during exercise, but in the latter the concurrent stimulation of the antioxidant defense system seems to protect the organism from excessive production of reactive oxygen species. Chronic exercise can exert negative effects on the activity of mitochondrial glycerol phosphate dehydrogenase (mGPdH), which may offer some explanation for the antioxidant effects of training, since this enzyme is a relevant producer of free radicals. To test this correlation, we compared mGPdH activity, two antioxidant defense markers and two markers of oxidative stress in sedentary and trained (Tr) rats. Training was through a swimming exercise 3 days a week. After 8 weeks, Tr rats lasted twice as long as controls in an acute swimming test with a 5% load. Forty-eight hours after the last exercise, the animals were killed to collect blood and tissues. Tr animals presented lower body weight and visceral fat mass with lower triglyceride content in visceral fat and plasma (p < 0.05). The specific activity of mGPdH in muscle mitochondria was reduced in Tr rats by 88% (p < 0.05). Total antioxidant capacity, lipid peroxidation and reduced glutathione (GSH) in liver and muscle were unaltered, while plasma GSH increased by 21% (p < 0.05). These data suggest a profile of successful redox equilibrium maintenance in Tr rats, with a potentially significant contribution from the lower level of mGPdH activity in muscle. This training protocol appears to be suitable for use in detailed studies of biochemical adaptations to oxidative stress. PMID:22391683

  2. Sphingosine-1-phosphate lyase downregulation promotes colon carcinogenesis through STAT3-activated microRNAs.

    PubMed

    Degagné, Emilie; Pandurangan, Ashok; Bandhuvula, Padmavathi; Kumar, Ashok; Eltanawy, Abeer; Zhang, Meng; Yoshinaga, Yuko; Nefedov, Mikhail; de Jong, Pieter J; Fong, Loren G; Young, Stephen G; Bittman, Robert; Ahmedi, Yasmin; Saba, Julie D

    2014-12-01

    Growing evidence supports a link between inflammation and cancer; however, mediators of the transition between inflammation and carcinogenesis remain incompletely understood. Sphingosine-1-phosphate (S1P) lyase (SPL) irreversibly degrades the bioactive sphingolipid S1P and is highly expressed in enterocytes but downregulated in colon cancer. Here, we investigated the role of SPL in colitis-associated cancer (CAC). We generated mice with intestinal epithelium-specific Sgpl1 deletion and chemically induced colitis and tumor formation in these animals. Compared with control animals, mice lacking intestinal SPL exhibited greater disease activity, colon shortening, cytokine levels, S1P accumulation, tumors, STAT3 activation, STAT3-activated microRNAs (miRNAs), and suppression of miR-targeted anti-oncogene products. This phenotype was attenuated by STAT3 inhibition. In fibroblasts, silencing SPL promoted tumorigenic transformation through a pathway involving extracellular transport of S1P through S1P transporter spinster homolog 2 (SPNS2), S1P receptor activation, JAK2/STAT3-dependent miR-181b-1 induction, and silencing of miR-181b-1 target cylindromatosis (CYLD). Colon biopsies from patients with inflammatory bowel disease revealed enhanced S1P and STAT3 signaling. In mice with chemical-induced CAC, oral administration of plant-type sphingolipids called sphingadienes increased colonic SPL levels and reduced S1P levels, STAT3 signaling, cytokine levels, and tumorigenesis, indicating that SPL prevents transformation and carcinogenesis. Together, our results suggest that dietary sphingolipids can augment or prevent colon cancer, depending upon whether they are metabolized to S1P or promote S1P metabolism through the actions of SPL. PMID:25347472

  3. Glucose-6-phosphate transport activity in liver microsomes exposed to stilbene disulfonate derivatives

    SciTech Connect

    Countaway, J.L.; Arion, W.J.

    1986-05-01

    Glucose-6-P (G6P) hydrolysis by hepatic microsomes (MS) is mediated by a coupled system composed of the G6P transporter (T1), the enzyme (E) and a phosphate transporter (T2). Zoccoli et al. concluded that T1 is a 54 kDa protein based on a linear correlation of labeling by /sup 3/H-4,4'diisothiocyano-1,2-diphenylethane-2,2'-disulfonate (/sup 3/H-H/sub 2/DIDS) and inhibition of system activity. The authors cannot support this conclusion: (1) in their hands the reaction of /sup 3/H-H/sub 2/DIDS with MS proteins is extremely nonspecific, and (2) the linear correlation must be between labeling and inhibition of T1 activity, because transport per se is not the absolute rate limiting step in hydrolysis by the system. Point 2 is readily demonstrated by examining the influence of the enzyme inhibitor, D-glucose, on the sensitivity of the system to inhibition by H/sub 2/DIDS. Studies of H/sub 2/DIDS inhibition of the system in MS from fasted and diabetic rats revealed that the observed inhibition constant for the system, K/sub i(S)/, is inversely proportional to the fraction of latent G6Pase activity (LF) seen before exposure to H/sub 2/DIDS, and K/sub i(S)/ x LF - K/sub i(T1)/, the inhibition constant for T1 activity. This relationship is derived from the equation 1/V/sub (S)/ - 1/V/sub (E)/ = 1/V/sub (T1)/, where V denotes the initial rates of S, E and T1, respectively. The latter equation can be used to calculate V/sub (T1)/ for any preparation of intact MS, and it predicts that labeling and inhibition of T1 will be linearly correlated with V/sub (T1)/ but not V/sub (S)/.

  4. Catalysis of hydrolysis and nucleophilic substitution at the P-N bond of phosphoimidazolide-activated nucleotides in phosphate buffers

    NASA Technical Reports Server (NTRS)

    Kanavarioti, A.; Rosenbach, M. T.

    1991-01-01

    Phosphoimidazolide-activated derivatives of guanosine and cytidine 5'-monophosphates, henceforth called ImpN's, exhibit enhanced rates of degradation in the presence of aqueous inorganic phosphate in the range 4.0 < or = pH < or = 8.6. This degradation is been attributed to (i) nucleophilic substitution of the imidazolide and (ii) catalysis of the P-N bond hydrolysis by phosphate. The first reaction results in the formation of nucleoside 5'-diphosphate and the second in nucleoside 5'-monophosphate. Analysis of the observed rates as well as the product ratios as a function of pH and phosphate concentration allow distinction between various mechanistic possibilities. The results show that both H2PO4- and HPO4(2-) participate in both hydrolysis and nucleophilic substitution. Statistically corrected biomolecular rate constants indicate that the dianion is 4 times more effective as a general base than the monoanion, and 8 times more effective as nucleophile. The low Bronsted value beta = 0.15 calculated for these phosphate species, presumed to act as general bases in facilitating water attack, is consistent with the fact that catalysis of the hydrolysis of the P-N bond in ImpN's has not been detected before. The beta nuc = 0.35 calculated for water, H2PO4-, HPO4(2-), and hydroxide acting as nucleophiles indicates a more associative transition state for nucleotidyl (O2POR- with R = nucleoside) transfers than that observed for phosphoryl (PO3(2-)) transfers (beta nuc = 0.25). With respect to the stability/reactivity of ImpN's under prebiotic conditions, our study shows that these materials would not suffer additional degradation due to inorganic phosphate, assuming the concentrations of phosphate, Pi, on prebiotic Earth were similar to those in the present oceans ([Pi] approximately 2.25 micromoles).

  5. Purification and characterization of an extracellular alkaline phosphatase from Penicillium chrysogenum.

    PubMed

    Politino, M; Brown, J; Usher, J J

    1996-01-01

    An extracellular alkaline phosphatase from Penicillium chrysogenum was purified to homogeneity using DEAE ion-exchange chromatography and size exclusion chromatography. SDS-PAGE of the purified enzyme indicated a molecular weight of 58,000. The mobility of the native enzyme on a Superose 12 column suggests that the active form of the enzyme is a monomer. The enzyme catalyzes the hydrolysis of phosphate from a variety of substrates including p-nitrophenyl phosphate, alpha-naphthyl phosphate and the anti-tumor compound etoposide phosphate. The apparent K(m) for the substrate p-nitrophenyl phosphate is 1.3 mM and the enzyme is inhibited by inorganic phosphate. The pH optimum of the enzyme is 9.0 with a broad optimal temperature range between 40 and 50 degrees C. The isoelectric point of the enzyme is approximately 5.5. The enzyme is a glycoprotein; digestion with endoglycosidase H indicates that the protein consists primarily of N-linked carbohydrates. Enzymatic activity is enhanced by the addition of divalent cations such as Mg+2 and Mn+2 and inhibited by addition of a chelator such as EDTA suggesting a metal ion requirement. The enzyme was found to be an inexpensive catalyst for the conversion of etoposide phosphate to etoposide in the manufacture of this anti-tumor compound. PMID:8958566

  6. Joint effect of phosphorus limitation and temperature on alkaline phosphatase activity and somatic growth in Daphnia magna.

    PubMed

    Wojewodzic, Marcin W; Kyle, Marcia; Elser, James J; Hessen, Dag O; Andersen, Tom

    2011-04-01

    Alkaline phosphatase (AP) is a potential biomarker for phosphorus (P) limitation in zooplankton. However, knowledge about regulation of AP in this group is limited. In a laboratory acclimation experiment, we investigated changes in body AP concentration for Daphnia magna kept for 6 days at 10, 15, 20 and 25 °C and fed algae with 10 different molar C:P ratios (95-660). In the same experiment, we also assessed somatic growth of the animals since phosphorus acquisition is linked to growth processes. Overall, non-linear but significant relationships of AP activity with C:P ratio were observed, but there was a stronger impact of temperature on AP activity than of P limitation. Animals from the lowest temperature treatment had higher normalized AP activity, which suggests the operation of biochemical temperature compensation mechanisms. Body AP activity increased by a factor of 1.67 for every 10 °C decrease in temperature. These results demonstrate that temperature strongly influences AP expression. Therefore, using AP as a P limitation marker in zooplankton needs to consider possible confounding effects of temperature. Both temperature and diet affected somatic growth. The temperature effect on somatic growth, expressed as the Q (10) value, responded non-linearly with C:P, with Q(10) ranging between 1.9 for lowest food C:P ratio and 1.4 for the most P-deficient food. The significant interaction between those two variables highlights the importance of studying temperature-dependent changes of growth responses to food quality. PMID:21153741

  7. Porous Mn2 O3 : A Low-Cost Electrocatalyst for Oxygen Reduction Reaction in Alkaline Media with Comparable Activity to Pt/C.

    PubMed

    Wang, Wenhai; Geng, Jing; Kuai, Long; Li, Min; Geng, Baoyou

    2016-07-11

    Preparing nonprecious metal catalysts with high activity in the oxygen reduction reaction (ORR) can promote the development of energy conversion devices. Support-free porous Mn2 O3 was synthesized by a facile aerosol-spray-assisted approach (ASAA) and subsequent thermal treatment, and exhibited ORR activity that is comparable to commercial Pt/C The catalyst also exhibits notably higher activity than other Mn-based oxides, such as Mn3 O4 and MnO2 . The rotating ring disk electrode (RRDE) study indicates a typical 4-electron ORR pathway on Mn2 O3 . Furthermore, the porous Mn2 O3 demonstrates considerable stability and a good methanol tolerance in alkaline media. In light of the low cost and high earth abundance of Mn, the highly active Mn2 O3 is a promising candidate to be used as a cathode material in metal-air batteries and alkaline fuel cells. PMID:27258474

  8. Fabrications of zinc-releasing biocement combining zinc calcium phosphate to calcium phosphate cement.

    PubMed

    Horiuchi, Shinya; Hiasa, Masahiro; Yasue, Akihiro; Sekine, Kazumitsu; Hamada, Kenichi; Asaoka, Kenzo; Tanaka, Eiji

    2014-01-01

    Recently, zinc-releasing bioceramics have been the focus of much attention owing to their bone-forming ability. Thus, some types of zinc-containing calcium phosphate (e.g., zinc-doped tricalcium phosphate and zinc-substituted hydroxyapatite) are examined and their osteoblastic cell responses determined. In this investigation, we studied the effects of zinc calcium phosphate (ZCP) derived from zinc phosphate incorporated into calcium phosphate cement (CPC) in terms of its setting reaction and MC3T3-E1 osteoblast-like cell responses. Compositional analysis by powder X-ray diffraction analysis revealed that HAP crystals were precipitated in the CPC containing 10 or 30wt% ZCP after successfully hardening. However, the crystal growth observed by scanning electron microscopy was delayed in the presence of additional ZCP. These findings indicate that the additional zinc inhibits crystal growth and the conversion of CPC to the HAP crystals. The proliferation of the cells and alkaline phosphatase (ALP) activity were enhanced when 10wt% ZCP was added to CPC. Taken together, ZCP added CPC at an appropriate fraction has a potent promotional effect on bone substitute biomaterials. PMID:24090874

  9. Nucleotide sequences encoding a thermostable alkaline protease

    DOEpatents

    Wilson, David B.; Lao, Guifang

    1998-01-01

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium.

  10. Nucleotide sequences encoding a thermostable alkaline protease

    DOEpatents

    Wilson, D.B.; Lao, G.

    1998-01-06

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium. 3 figs.

  11. Alkaline-earth oxide network modifier on optical properties of Ce3+-activated borogermanate glasses

    NASA Astrophysics Data System (ADS)

    Sun, Xin-Yuan; Xiao, Zhuo-Hao; Zhong, Jiu-Ping

    2015-12-01

    Transparent and colorless CeO2-activated borogermanate glasses, with the nominal composition of 25B2O3-40GeO2-14Gd2O3-1CeO2-20MO (M = Ba, Sr, Ca and Mg), were synthesized by a melt-quenching method in air. Their optical properties including the transmittance, photoluminescence (excitation and emission spectra), the luminescence decay curves, as well as the temperature-dependent emission spectra were studied in detail. The room temperature photoluminescence spectra reveal that the emission intensity of the MgO glass is about two times stronger than that of the BaO glass. The blue shift of the cut-off edge, excitation and emission spectra of Ce3+-activated borogermanate glass were clearly observed in the order of BaO, SrO, CaO and MgO. And the emission intensity of Ce3+ ions as a function of temperature range in 325-475 K was also investigated.

  12. Calc-alkaline Magmatic Activities and Related Mineralization in the Northwest of the Lut Block (Eastern Iran)

    NASA Astrophysics Data System (ADS)

    Saadat, S.; Stern, C.; Hadizadeh, H.; Ghoorchi, M.

    2008-12-01

    The Lut Block of Eastern Iran has significant mineral potential based on its tectonic setting, geologic evolution, Tertiary igneous rock cover and old mining records. In the Ahang prospecting area (~ 40Km2), located in northwest of the Lut block, magmatic activities started in Eocene with the eruption of mafic to intermediate extrusives, mainly andesites. These rocks are intruded by Oligocene-Miocene monzodiorite and quartz monzonite stocks and cut by felsic dikes. Intrusive rocks are meta-aluminous and belong to the high-K calc-alkaline series. The results of REE analysis show that these rocks are enriched in La relative to Yb. Their ratios of Ta+Yb versus Rb suggest the VAG (volcanic arc granites) geotectonic regime for their origin. Propylitic, sericitic, argillic and silicification alteration zones are well developed in this area. Based on heavy minerals studies of stream sediments, Wulfenite, Stolzite, Galena, Sphalerite, Diaboleite, Dechenite, Mimetite, Pyrite, Celestine, Barite, Flourite, Malachite and Sapphire are present in this area. Geochemical analysis of stream sediment samples indicates considerable anomalous zones of W, Pb, Zn, Cu, Au, As, Sb and also Ba and F. Mineralization as veins, veinlet and minor stockwork are mainly controlled by structural features and brecciated zones. Mineralization occurs in three types: Quartz- Hematite- Galena (high grade of Pb, Zn, Au, As, W); Quarts- Pyrite- Chalcopyrite and Galena (high grade of Cu, Mo, Ag, Zn, Pb), and Quartz- Barite- Celestine- Galena and flurine (high grade of F, Sr, Ba, Pb). The range of value for these elements in mineralized rock samples are: Cu (5-1500ppm), Mo (0.5-109ppm), Pb (20ppm-14%), Zn (8ppm-7.8%), W (5-1350ppm), As (1-723ppm), Ag (0.5-56.5ppm), Au (7ppb-7.8ppm). According to the field observations and data presented here, this area has a high potential for base-metal, W and Au mineral exploration. Keywords: Calc-alkaline magmatism, Lut Block, mineralization, Iran

  13. Nicotinic acid-adenine dinucleotide phosphate activates the skeletal muscle ryanodine receptor.

    PubMed Central

    Hohenegger, Martin; Suko, Josef; Gscheidlinger, Regina; Drobny, Helmut; Zidar, Andreas

    2002-01-01

    Calcium is a universal second messenger. The temporal and spatial information that is encoded in Ca(2+)-transients drives processes as diverse as neurotransmitter secretion, axonal outgrowth, immune responses and muscle contraction. Ca(2+)-release from intracellular Ca(2+) stores can be triggered by diffusible second messengers like Ins P (3), cyclic ADP-ribose or nicotinic acid-adenine dinucleotide phosphate (NAADP). A target has not yet been identified for the latter messenger. In the present study we show that nanomolar concentrations of NAADP trigger Ca(2+)-release from skeletal muscle sarcoplasmic reticulum. This was due to a direct action on the Ca(2+)-release channel/ryanodine receptor type-1, since in single channel recordings, NAADP increased the open probability of the purified channel protein. The effects of NAADP on Ca(2+)-release and open probability of the ryanodine receptor occurred over a similar concentration range (EC(50) approximately 30 nM) and were specific because (i) they were blocked by Ruthenium Red and ryanodine, (ii) the precursor of NAADP, NADP, was ineffective at equimolar concentrations, (iii) NAADP did not affect the conductance and reversal potential of the ryanodine receptor. Finally, we also detected an ADP-ribosyl cyclase activity in the sarcoplasmic reticulum fraction of skeletal muscle. This enzyme was not only capable of synthesizing cyclic GDP-ribose but also NAADP, with an activity of 0.25 nmol/mg/min. Thus, we conclude that NAADP is generated in the vicinity of type 1 ryanodine receptor and leads to activation of this ion channel. PMID:12102654

  14. Effect of phosphate activating group on oligonucleotide formation on montmorillonite: the regioselective formation of 3',5'-linked oligoadenylates

    NASA Technical Reports Server (NTRS)

    Prabahar, K. J.; Cole, T. D.; Ferris, J. P.

    1994-01-01

    The effects of amine structure on the montmorillonite-catalyzed oligomerization of the 5'-phosphoramidates of adenosine are investigated. 4-Aminopyridine derivatives yielded oligoadenylates as long as dodecamers with a regioselectivity for 3',5'-phosphodiester bond formation averaging 88%. Linear and cyclic oligomers are obtained and no A5'ppA-containing products are detected. Oligomers as long as the hexanucleotide are obtained using 2-aminobenzimidazole as the activating group. A predominance of pA2'pA is detected in the dimer fraction along with cyclic 3',5'-trimer; no A5'ppA-containing oligomers were detected. Little or no oligomer formation was observed when morpholine, piperidine, pyrazole, 1,2,4-triazole, and 2-pyridone are used as phosphate-activating groups. The effects of the structure of the phosphate activating group on the oligomer structure and chain lengths are discussed.

  15. Antibacterial activity and ion release of bonding agent containing amorphous calcium phosphate nanoparticles

    PubMed Central

    Chen, Chen; Weir, Michael D.; Cheng, Lei; Lin, Nancy; Lin-Gibson, Sheng; Chow, Laurence C.; Zhou, Xuedong; Xu, Hockin H. K.

    2015-01-01

    Objectives Recurrent caries at the margins is a primary reason for restoration failure. The objectives of this study were to develop bonding agent with the double benefits of antibacterial and remineralizing capabilities, to investigate the effects of NACP filler level and solution pH on Ca and P ion release from adhesive, and to examine the antibacterial and dentin bond properties. Methods Nanoparticles of amorphous calcium phosphate (NACP) and a quaternary ammonium monomer (dimethylaminododecyl methacrylate, DMADDM) were synthesized. Scotchbond Multi-Purpose (SBMP) primer and adhesive served as control. DMADDM was incorporated into primer and adhesive at 5% by mass. NACP was incorporated into adhesive at filler mass fractions of 10%, 20%, 30% and 40%. A dental plaque microcosm biofilm model was used to test the antibacterial bonding agents. Calcium (Ca) and phosphate (P) ion releases from the cured adhesive samples were measured vs. filler level and solution pH of 7, 5.5 and 4. Results Adding 5% DMADDM and 10–40% NACP into bonding agent, and water-aging for 28 days, did not affect dentin bond strength, compared to SBMP control at 1 day (p > 0.1). Adding DMADDM into bonding agent substantially decreased the biofilm metabolic activity and lactic acid production. Total microorganisms, total streptococci, and mutans streptococci were greatly reduced for bonding agents containing DMADDM. Increasing NACP filler level from 10% to 40% in adhesive increased the Ca and P ion release by an order of magnitude. Decreasing solution pH from 7 to 4 increased the ion release from adhesive by 6–10 folds. Significance Bonding agents containing antibacterial DMADDM and remineralizer NACP were formulated to have Ca and P ion release, which increased with NACP filler level from 10% to 40% in adhesive. NACP adhesive was “smart” and dramatically increased the ion release at cariogenic pH 4, when these ions would be most-needed to inhibit caries. Therefore, bonding agent

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

    USGS Publications Warehouse

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

    2003-01-01

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

  17. New oxo-bridged peroxotungsten complexes containing biogenic co-ligand as potent inhibitors of alkaline phosphatase activity.

    PubMed

    Hazarika, Pankaj; Kalita, Diganta; Sarmah, Swapnalee; Islam, Nashreen S

    2006-03-01

    Novel dinuclear peroxo complexes of tungsten with coordinated cystine of the type A(2)[W(2)O(3)(O(2))(4)(cystine)].4H(2)O, A = Na (1) or K (2) have been synthesized from the reaction of A(2)WO(4,)cysteine and 30% H(2)O(2)at pH 2.5. The synthesized compounds were characterized by elemental analysis, spectral and physico-chemical methods. The two W(VI) centres with side-on bound peroxo groups of the dinuclear complex species are bridged by an oxo group and a cystine ligand, formed from the oxidation of cysteine. Cystine occurring as zwitterion binds the metal centers of the complex ion through O(carboxylate) atoms leading to hepta co-ordination around each W(VI). The compounds exhibit high stability toward decomposition in solution of acidic as well as physiological pH and serve as weak substrates to catalase, undergoing degradation in presence of the enzyme at a rate much slower relative to H(2)O(2). The compounds efficiently oxidized GSH to GSSG, a reaction in which only two of the peroxide groups of the complex species were found to participate. The compounds induce strong inhibitory effect on alkaline phosphatase activity with a potency higher than that of the free cystine, tungstate, or peroxotungstate. PMID:16477386

  18. Enhancement of Alkaline Protease Activity and Stability via Covalent Immobilization onto Hollow Core-Mesoporous Shell Silica Nanospheres.

    PubMed

    Ibrahim, Abdelnasser Salah Shebl; Al-Salamah, Ali A; El-Toni, Ahmed M; Almaary, Khalid S; El-Tayeb, Mohamed A; Elbadawi, Yahya B; Antranikian, Garabed

    2016-01-01

    The stability and reusability of soluble enzymes are of major concerns, which limit their industrial applications. Herein, alkaline protease from Bacillus sp. NPST-AK15 was immobilized onto hollow core-mesoporous shell silica (HCMSS) nanospheres. Subsequently, the properties of immobilized proteases were evaluated. Non-, ethane- and amino-functionalized HCMSS nanospheres were synthesized and characterized. NPST-AK15 was immobilized onto the synthesized nano-supports by physical and covalent immobilization approaches. However, protease immobilization by covalent attachment onto the activated HCMSS-NH₂ nanospheres showed highest immobilization yield (75.6%) and loading capacity (88.1 μg protein/mg carrier) and was applied in the further studies. In comparison to free enzyme, the covalently immobilized protease exhibited a slight shift in the optimal pH from 10.5 to 11.0, respectively. The optimum temperature for catalytic activity of both free and immobilized enzyme was seen at 60 °C. However, while the free enzyme was completely inactivated when treated at 60 °C for 1 h the immobilized enzyme still retained 63.6% of its initial activity. The immobilized protease showed higher V(max), k(cat) and k(cat)/K(m), than soluble enzyme by 1.6-, 1.6- and 2.4-fold, respectively. In addition, the immobilized protease affinity to the substrate increased by about 1.5-fold. Furthermore, the enzyme stability in various organic solvents was significantly enhanced upon immobilization. Interestingly, the immobilized enzyme exhibited much higher stability in several commercial detergents including OMO, Tide, Ariel, Bonux and Xra by up to 5.2-fold. Finally, the immobilized protease maintained significant catalytic efficiency for twelve consecutive reaction cycles. These results suggest the effectiveness of the developed nanobiocatalyst as a candidate for detergent formulation and peptide synthesis in non-aqueous media. PMID:26840303

  19. Enhancement of Alkaline Protease Activity and Stability via Covalent Immobilization onto Hollow Core-Mesoporous Shell Silica Nanospheres

    PubMed Central

    Ibrahim, Abdelnasser Salah Shebl; Al-Salamah, Ali A.; El-Toni, Ahmed M.; Almaary, Khalid S.; El-Tayeb, Mohamed A.; Elbadawi, Yahya B.; Antranikian, Garabed

    2016-01-01

    The stability and reusability of soluble enzymes are of major concerns, which limit their industrial applications. Herein, alkaline protease from Bacillus sp. NPST-AK15 was immobilized onto hollow core-mesoporous shell silica (HCMSS) nanospheres. Subsequently, the properties of immobilized proteases were evaluated. Non-, ethane- and amino-functionalized HCMSS nanospheres were synthesized and characterized. NPST-AK15 was immobilized onto the synthesized nano-supports by physical and covalent immobilization approaches. However, protease immobilization by covalent attachment onto the activated HCMSS–NH2 nanospheres showed highest immobilization yield (75.6%) and loading capacity (88.1 μg protein/mg carrier) and was applied in the further studies. In comparison to free enzyme, the covalently immobilized protease exhibited a slight shift in the optimal pH from 10.5 to 11.0, respectively. The optimum temperature for catalytic activity of both free and immobilized enzyme was seen at 60 °C. However, while the free enzyme was completely inactivated when treated at 60 °C for 1 h the immobilized enzyme still retained 63.6% of its initial activity. The immobilized protease showed higher Vmax, kcat and kcat/Km, than soluble enzyme by 1.6-, 1.6- and 2.4-fold, respectively. In addition, the immobilized protease affinity to the substrate increased by about 1.5-fold. Furthermore, the enzyme stability in various organic solvents was significantly enhanced upon immobilization. Interestingly, the immobilized enzyme exhibited much higher stability in several commercial detergents including OMO, Tide, Ariel, Bonux and Xra by up to 5.2-fold. Finally, the immobilized protease maintained significant catalytic efficiency for twelve consecutive reaction cycles. These results suggest the effectiveness of the developed nanobiocatalyst as a candidate for detergent formulation and peptide synthesis in non-aqueous media. PMID:26840303

  20. A low-temperature-active alkaline pectate lyase from Xanthomonas campestris ACCC 10048 with high activity over a wide pH range.

    PubMed

    Yuan, Peng; Meng, Kun; Wang, Yaru; Luo, Huiying; Shi, Pengjun; Huang, Huoqing; Tu, Tao; Yang, Peilong; Yao, Bin

    2012-11-01

    Alkaline pectate lyases are favorable for the textile industry. Here, we report the gene cloning and expression of a low-temperature-active alkaline pectate lyase (PL D) from Xanthomonas campestris ACCC 10048. Deduced PL D consists of a putative 27-residue signal peptide and a catalytic domain of 320 residues belonging to family PF09492. Recombinant PL D (r-PL D) produced in Escherichia coli was purified to electrophoretic homogeneity with a single step of Ni(2+)-NTA affinity chromatography and showed an apparent molecular weight of ~38 kDa. The pH and temperature optima of r-PL D were found to be 9.0 °C and 30 °C, respectively. Compared with its microbial counterparts, r-PL D had higher activity over a wide pH range (>45 % of the maximum activity at pH 3.0-12.0) and at lower temperatures (>35 % of activity even at 0 °C). The K(m) and V(max) values of r-PL D for polygalacturonic acid were 4.9 gl(-1) and 30.1 μmolmin(-1) mg(-1), respectively. Compared with the commercial compound pectinase from Novozymes, r-PL D showed similar efficacy in reducing the intrinsic viscosity of polygalacturonic acid (35.1 % vs. 36.5 %) and in bioscouring of jute (10.25 % vs. 10.82 %). Thus, r-PL D is a valuable additive candidate for the textile industry. PMID:22983714

  1. Modulators of intestinal alkaline phosphatase.

    PubMed

    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

  2. Antioxidative activity of protein hydrolysates prepared from alkaline-aided channel catfish protein isolates.

    PubMed

    Theodore, Ann E; Raghavan, Sivakumar; Kristinsson, Hordur G

    2008-08-27

    Antioxidative activity of hydrolyzed protein prepared from alkali-solubilized catfish protein isolates was studied. The isolates were hydrolyzed to 5, 15, and 30% degree of hydrolysis using the protease enzyme, Protamex. Hydrolyzed protein was separated into hydrolysates and soluble supernatants, and both of these fractions were studied for their metal chelating ability, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging ability, ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC), and their ability to inhibit the formation of thiobarbituric acid reactive substances (TBARS) in washed tilapia muscle containing tilapia hemolysate. Both hydrolysates and supernatants were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results showed that DPPH radical scavenging ability and reducing power of catfish protein hydrolysates decreased, whereas the ORAC value, metal chelating ability, and ability to inhibit TBARS increased, with an increase in the degree of hydrolysis. Hydrolysate samples showed higher DPPH radical scavenging ability and Fe(3+) reducing ability, and supernatant samples had higher metal chelating ability. In general, low molecular weight (MW) peptides had high ORAC values and high metal chelating ability, and high MW peptides had a higher reducing power (FRAP) and were more effective in scavenging DPPH radicals. In a washed muscle model system, the ability of catfish protein hydrolysates and their corresponding supernatants to inhibit the formation of TBARS increased with an increase in the degree of hydrolysis. PMID:18662014

  3. The effects of alkaline earth metal ions and halogen ions on the chromium oxide activities in alkaline earth metal oxide-halide-Cr2O3 system fluxes

    NASA Astrophysics Data System (ADS)

    Li, Lian-Fu; Jiang, Mao-Fa; Wang, Wen-Zhong; Chen, Zhao-Ping

    2000-06-01

    The solid electrolyte cell — Mo|Cr + Cr2O3‖ZrO2(MgO)‖{Cu-Cr}alloy + (Cr2O3)fluxes|Mo+ is used at 1673 K to determine Cr2O3 activities in MO-MX 2-Cr2O3 (M = Ca2+, Ba2-, X = F- or Cl-) ternary fluxes, which are in equilibrium with the copper-chromium binary alloy. The ternary isothermal phase diagrams of CaO-CaF2-Cr2O3 and BaO-BaCl2-Cr2O3 system fluxes are inferred on the basis of the experimental results and binary phase diagrams. The results indicate that Cr2O3 activities in all fluxes always decrease with the increase of the X MO /X MX2 ratio. Partial replacement of BaO in BaO-BaF2-Cr2O3 fluxes by CaO is acceptable for economy and efficiency considerations. At the same time, partial substitution of BaO for CaO in CaO-CaF2-Cr2O3 fluxes is advantageous for phosphorus removal and chromium retention as a result of the increased Cr2O3 activities, increased basicities, and widening of the liquid zones. Compared to those in BaO-BaF2-Cr2O3 fluxes, Cr2O3 activities in CaO-CaF2-Cr2O3 fluxes approximately follow the same curve as the former, although the position and the width of the liquid zones are considerably different, and activities in BaO-BaCl2-Cr2O3 fluxes are higher at the lower Cr2O3 content, or vice versa. The activity coefficients of Cr2O3 in the fluxes decrease with the increase of the X MO /X MX 2 ratios.

  4. Comparison of the adjuvant activity of aluminum hydroxide and calcium phosphate on the antibody response towards Bothrops asper snake venom.

    PubMed

    Olmedo, Hidekel; Herrera, María; Rojas, Leonardo; Villalta, Mauren; Vargas, Mariángela; Leiguez, Elbio; Teixeira, Catarina; Estrada, Ricardo; Gutiérrez, José María; León, Guillermo; Montero, Mavis L

    2014-01-01

    The adjuvanticity of aluminum hydroxide and calcium phosphate on the antibody response in mice towards the venom of the snake Bothrops asper was studied. It was found that, in vitro, most of the venom proteins are similarly adsorbed by both mineral salts, with the exception of some basic phospholipases A2, which are better adsorbed by calcium phosphate. After injection, the adjuvants promoted a slow release of the venom, as judged by the lack of acute toxicity when lethal doses of venom were administered to mice. Leukocyte recruitment induced by the venom was enhanced when it was adsorbed on both mineral salts; however, venom adsorbed on calcium phosphate induced a higher antibody response towards all tested HPLC fractions of the venom. On the other hand, co-precipitation of venom with calcium phosphate was the best strategy for increasing: (1) the capacity of the salt to couple venom proteins in vitro; (2) the venom ability to induce leukocyte recruitment; (3) phagocytosis by macrophages; and (4) a host antibody response. These findings suggest that the chemical nature is not the only one determining factor of the adjuvant activity of mineral salts. PMID:23506358

  5. Phosphate salts

    MedlinePlus

    ... taken by mouth or used as enemas. Indigestion. Aluminum phosphate and calcium phosphate are FDA-permitted ingredients ... Phosphate salts containing sodium, potassium, aluminum, or calcium are LIKELY SAFE for most people when taken by mouth short-term, when sodium phosphate is inserted into the ...

  6. Conjugated bile acids promote cholangiocarcinoma cell invasive growth through activation of sphingosine 1-phosphate receptor 2

    PubMed Central

    Liu, Runping; Zhao, Renping; Zhou, Xiqiao; Liang, Xiuyin; Campbell, Deanna JW; Zhang, Xiaoxuan; Zhang, Luyong; Shi, Ruihua; Wang, Guangji; Pandak, William M; Sirica, Alphonse E; Hylemon, Phillip B; Zhou, Huiping

    2014-01-01

    Cholangiocarcinoma (CCA) is an often fatal primary malignancy of the intra- and extrahepatic biliary tract that is commonly associated with chronic cholestasis and significantly elevated levels of primary and conjugated bile acids (CBAs), which are correlated with bile duct obstruction (BDO). BDO has also recently been shown to promote CCA progression. However, whereas there is increasing evidence linking chronic cholestasis and abnormal bile acid profiles to CCA development and progression, the specific mechanisms by which bile acids may be acting to promote cholangiocarcinogenesis and invasive biliary tumor growth have not been fully established. Recent studies have shown that CBAs, but not free bile acids, stimulate CCA cell growth, and that an imbalance in the ratio of free to CBAs may play an important role in the tumorigenesis of CCA. Also, CBAs are able to activate extracellular signal-regulated kinase (ERK)1/2- and phosphatidylinositol-3-kinase/protein kinase B (AKT)-signaling pathways through sphingosine 1-phosphate receptor 2 (S1PR2) in rodent hepatocytes. In the current study, we demonstrate S1PR2 to be highly expressed in rat and human CCA cells, as well as in human CCA tissues. We further show that CBAs activate the ERK1/2- and AKT-signaling pathways and significantly stimulate CCA cell growth and invasion in vitro. Taurocholate (TCA)-mediated CCA cell proliferation, migration, and invasion were significantly inhibited by JTE-013, a chemical antagonist of S1PR2, or by lentiviral short hairpin RNA silencing of S1PR2. In a novel organotypic rat CCA coculture model, TCA was further found to significantly increase the growth of CCA cell spheroidal/“duct-like” structures, which was blocked by treatment with JTE-013. Conclusion: Our collective data support the hypothesis that CBAs promote CCA cell-invasive growth through S1PR2. PMID:24700501

  7. Community Structure and Activity Dynamics of Nitrifying Bacteria in a Phosphate-Removing Biofilm

    PubMed Central

    Gieseke, Armin; Purkhold, Ulrike; Wagner, Michael; Amann, Rudolf; Schramm, Andreas

    2001-01-01

    The microbial community structure and activity dynamics of a phosphate-removing biofilm from a sequencing batch biofilm reactor were investigated with special focus on the nitrifying community. O2, NO2−, and NO3− profiles in the biofilm were measured with microsensors at various times during the nonaerated-aerated reactor cycle. In the aeration period, nitrification was oxygen limited and restricted to the first 200 μm at the biofilm surface. Additionally, a delayed onset of nitrification after the start of the aeration was observed. Nitrate accumulating in the biofilm in this period was denitrified during the nonaeration period of the next reactor cycle. Fluorescence in situ hybridization (FISH) revealed three distinct ammonia-oxidizing populations, related to the Nitrosomonas europaea, Nitrosomonas oligotropha, and Nitrosomonas communis lineages. This was confirmed by analysis of the genes coding for 16S rRNA and for ammonia monooxygenase (amoA). Based upon these results, a new 16S rRNA-targeted oligonucleotide probe specific for the Nitrosomonas oligotropha lineage was designed. FISH analysis revealed that the first 100 μm at the biofilm surface was dominated by members of the N. europaea and the N. oligotropha lineages, with a minor fraction related to N. communis. In deeper biofilm layers, exclusively members of the N. oligotropha lineage were found. This separation in space and a potential separation of activities in time are suggested as mechanisms that allow coexistence of the different ammonia-oxidizing populations. Nitrite-oxidizing bacteria belonged exclusively to the genus Nitrospira and could be assigned to a 16S rRNA sequence cluster also found in other sequencing batch systems. PMID:11229931

  8. Glutamyl Phosphate Is an Activated Intermediate in Actin Crosslinking by Actin Crosslinking Domain (ACD) Toxin

    PubMed Central

    Kudryashova, Elena; Kalda, Caitlin; Kudryashov, Dmitri S.

    2012-01-01

    Actin Crosslinking Domain (ACD) is produced by several life-threatening Gram-negative pathogenic bacteria as part of larger toxins and delivered into the cytoplasm of eukaryotic host cells via Type I or Type VI secretion systems. Upon delivery, ACD disrupts the actin cytoskeleton by catalyzing intermolecular amide bond formation between E270 and K50 residues of actin, leading to the formation of polymerization-deficient actin oligomers. Ultimately, accumulation of the crosslinked oligomers results in structural and functional failure of the actin cytoskeleton in affected cells. In the present work, we advanced in our understanding of the ACD catalytic mechanism by discovering that the enzyme transfers the gamma-phosphoryl group of ATP to the E270 actin residue, resulting in the formation of an activated acyl phosphate intermediate. This intermediate is further hydrolyzed and the energy of hydrolysis is utilized for the formation of the amide bond between actin subunits. We also determined the pH optimum for the reaction and the kinetic parameters of ACD catalysis for its substrates, ATP and actin. ACD showed sigmoidal, non-Michaelis-Menten kinetics for actin (K0.5 = 30 µM) reflecting involvement of two actin molecules in a single crosslinking event. We established that ACD can also utilize Mg2+-GTP to support crosslinking, but the kinetic parameters (KM = 8 µM and 50 µM for ATP and GTP, respectively) suggest that ATP is the primary substrate of ACD in vivo. The optimal pH for ACD activity was in the range of 7.0–9.0. The elucidated kinetic mechanism of ACD toxicity adds to understanding of complex network of host-pathogen interactions. PMID:23029200

  9. Structural Diversity Within the Mononuclear and Binuclear Active Sites of N-Acetyl-D-Glucosamine-6-Phosphate Deacetylase

    SciTech Connect

    Hall,R.; Brown, S.; Fedorov, A.; Fedorov, E.; Xu, C.; Babbitt, P.; Almo, S.; Raushel, F.

    2007-01-01

    NagA catalyzes the hydrolysis of N-acetyl-D-glucosamine-6-phosphate to D-glucosamine-6-phosphate and acetate. X-ray crystal structures of NagA from Escherichia coli were determined to establish the number and ligation scheme for the binding of zinc to the active site and to elucidate the molecular interactions between the protein and substrate. The three-dimensional structures of the apo-NagA, Zn-NagA, and the D273N mutant enzyme in the presence of a tight-binding N-methylhydroxyphosphinyl-D-glucosamine-6-phosphate inhibitor were determined. The structure of the Zn-NagA confirms that this enzyme binds a single divalent cation at the beta-position in the active site via ligation to Glu-131, His-195, and His-216. A water molecule completes the ligation shell, which is also in position to be hydrogen bonded to Asp-273. In the structure of NagA bound to the tight binding inhibitor that mimics the tetrahedral intermediate, the methyl phosphonate moiety has displaced the hydrolytic water molecule and is directly coordinated to the zinc within the active site. The side chain of Asp-273 is positioned to activate the hydrolytic water molecule via general base catalysis and to deliver this proton to the amino group upon cleavage of the amide bond of the substrate. His-143 is positioned to help polarize the carbonyl group of the substrate in conjunction with Lewis acid catalysis by the bound zinc. The inhibitor is bound in the {alpha}-configuration at the anomeric carbon through a hydrogen bonding interaction of the hydroxyl group at C-1 with the side chain of His-251. The phosphate group of the inhibitor attached to the hydroxyl at C-6 is ion paired with Arg-227 from the adjacent subunit. NagA from Thermotoga maritima was shown to require a single divalent cation for full catalytic activity.

  10. Structural diversity within the mononuclear and binuclear active sites of N-acetyl-D-glucosamine-6-phosphate deacetylase.

    PubMed

    Hall, Richard S; Brown, Shoshana; Fedorov, Alexander A; Fedorov, Elena V; Xu, Chengfu; Babbitt, Patricia C; Almo, Steven C; Raushel, Frank M

    2007-07-10

    NagA catalyzes the hydrolysis of N-acetyl-d-glucosamine-6-phosphate to d-glucosamine-6-phosphate and acetate. X-ray crystal structures of NagA from Escherichia coli were determined to establish the number and ligation scheme for the binding of zinc to the active site and to elucidate the molecular interactions between the protein and substrate. The three-dimensional structures of the apo-NagA, Zn-NagA, and the D273N mutant enzyme in the presence of a tight-binding N-methylhydroxyphosphinyl-d-glucosamine-6-phosphate inhibitor were determined. The structure of the Zn-NagA confirms that this enzyme binds a single divalent cation at the beta-position in the active site via ligation to Glu-131, His-195, and His-216. A water molecule completes the ligation shell, which is also in position to be hydrogen bonded to Asp-273. In the structure of NagA bound to the tight binding inhibitor that mimics the tetrahedral intermediate, the methyl phosphonate moiety has displaced the hydrolytic water molecule and is directly coordinated to the zinc within the active site. The side chain of Asp-273 is positioned to activate the hydrolytic water molecule via general base catalysis and to deliver this proton to the amino group upon cleavage of the amide bond of the substrate. His-143 is positioned to help polarize the carbonyl group of the substrate in conjunction with Lewis acid catalysis by the bound zinc. The inhibitor is bound in the alpha-configuration at the anomeric carbon through a hydrogen bonding interaction of the hydroxyl group at C-1 with the side chain of His-251. The phosphate group of the inhibitor attached to the hydroxyl at C-6 is ion paired with Arg-227 from the adjacent subunit. NagA from Thermotoga maritima was shown to require a single divalent cation for full catalytic activity. PMID:17567048

  11. Bismuth citrate in the quantification of inorganic phosphate and its utility in the determination of membrane-bound phosphatases.

    PubMed

    Cariani, L; Thomas, L; Brito, J; del Castillo, J R

    2004-01-01

    This paper describes a rapid and sensitive method to determine inorganic phosphate, even in the presence of labile organic phosphate compounds and large quantities of proteins. The method eliminates the use of sodium arsenite, a highly toxic compound, substituting bismuth citrate for it to stabilize the phosphomolybdic acid complex formed during the interaction of inorganic phosphate and molybdate reduced by ascorbic acid. This method has also been adapted to microplates and has been used to determine the activities of Na/K ATPase and alkaline phosphatase of intestinal basolateral and luminal plasma membranes. PMID:14654048

  12. A limitation of the continuous spectrophotometric assay for the measurement of myo-inositol-1-phosphate synthase activity.

    PubMed

    Huang, Xinyi; Hernick, Marcy

    2011-10-15

    Myo-inositol-1-phosphate synthase (MIPS) catalyzes the conversion of glucose-6-phosphate to myo-inositol-1-phosphate. The reaction catalyzed by MIPS is the first step in the biosynthesis of inositol and inositol-containing molecules that serve important roles in both eukaryotes and prokaryotes. Consequently, MIPS is a target for the development of therapeutic agents for the treatment of infectious diseases and bipolar disorder. We recently reported a continuous spectrophotometric method for measuring MIPS activity using a coupled assay that allows the rapid characterization of MIPS in a multiwell plate format. Here we validate the continuous assay as a high-throughput alternative for measuring MIPS activity and report on one limitation of this assay-the inability to examine the effect of divalent metal ions (at high concentrations) on MIPS activity. In addition, we demonstrate that the activity of MIPS from Arabidopsis thaliana is moderately enhanced by the addition Mg(2+) and is not enhanced by other divalent metal ions (Zn(2+) and Mn(2+)), consistent with what has been observed for other eukaryotic MIPS enzymes. Our findings suggest that the continuous assay is better suited for characterizing eukaryotic MIPS enzymes that require monovalent cations as cofactors than for characterizing bacterial or archeal MIPS enzymes that require divalent metal ions as cofactors. PMID:21729692

  13. Dual Mode Antibacterial Activity of Ion Substituted Calcium Phosphate Nanocarriers for Bone Infections

    PubMed Central

    Sampath Kumar, T. S.; Madhumathi, K.; Rubaiya, Y.; Doble, Mukesh

    2015-01-01

    Nanotechnology has tremendous potential for the management of infectious diseases caused by multi-drug resistant bacteria, through the development of newer antibacterial materials and efficient modes of antibiotic delivery. Calcium phosphate (CaP) bioceramics are commonly used as bone substitutes due to their similarity to bone mineral and are widely researched upon for the treatment of bone infections associated with bone loss. CaPs can be used as local antibiotic delivery agents for bone infections and can be substituted with antibacterial ions in their crystal structure to have a wide spectrum, sustained antibacterial activity even against drug resistant bacteria. In the present work, a dual mode antibiotic delivery system with antibacterial ion substituted calcium deficient hydroxyapatite (CDHA) nanoparticles has been developed. Antibacterial ions such as zinc, silver, and strontium have been incorporated into CDHA at concentrations of 6, 0.25–0.75, and 2.5–7.5 at. %, respectively. The samples were found to be phase pure, acicular nanoparticles of length 40–50 nm and width 5–6 nm approximately. The loading and release profile of doxycycline, a commonly used antibiotic, was studied from the nanocarriers. The drug release was studied for 5 days and the release profile was influenced by the ion concentrations. The release of antibacterial ions was studied over a period of 21 days. The ion substituted CDHA samples were tested for antibacterial efficacy on Staphylococcus aureus and Escherichia coli by MIC/MBC studies and time-kill assay. AgCDHA and ZnCDHA showed high antibacterial activity against both bacteria, while SrCDHA was weakly active against S. aureus. Present study shows that the antibiotic release can provide the initial high antibacterial activity, and the sustained ion release can provide a long-term antibacterial activity. Such dual mode antibiotic and antibacterial ion release offers an efficient and potent way to treat an incumbent

  14. Alkaline Phosphatase-Mimicking Peptide Nanofibers for Osteogenic Differentiation.

    PubMed

    Gulseren, Gulcihan; Yasa, I Ceren; Ustahuseyin, Oya; Tekin, E Deniz; Tekinay, Ayse B; Guler, Mustafa O

    2015-07-13

    Recognition of molecules and regulation of extracellular matrix synthesis are some of the functions of enzymes in addition to their catalytic activity. While a diverse array of enzyme-like materials have been developed, these efforts have largely been confined to the imitation of the chemical structure and catalytic activity of the enzymes, and it is unclear whether enzyme-mimetic molecules can also be used to replicate the matrix-regulatory roles ordinarily performed by natural enzymes. Self-assembled peptide nanofibers can provide multifunctional enzyme-mimetic properties, as the active sequences of the target enzymes can be directly incorporated into the peptides. Here, we report enhanced bone regeneration efficiency through peptide nanofibers carrying both catalytic and matrix-regulatory functions of alkaline phosphatase, a versatile enzyme that plays a critical role in bone formation by regulating phosphate homeostasis and calcifiable bone matrix formation. Histidine presenting peptide nanostructures were developed to function as phosphatases. These molecules are able to catalyze phosphate hydrolysis and serve as bone-like nodule inducing scaffolds. Alkaline phosphatase-like peptide nanofibers enabled osteogenesis for both osteoblast-like and mesenchymal cell lines. PMID:26039144

  15. Alkaline quinone flow battery.

    PubMed

    Lin, Kaixiang; Chen, Qing; Gerhardt, Michael R; Tong, Liuchuan; Kim, Sang Bok; Eisenach, Louise; Valle, Alvaro W; Hardee, David; Gordon, Roy G; Aziz, Michael J; Marshak, Michael P

    2015-09-25

    Storage of photovoltaic and wind electricity in batteries could solve the mismatch problem between the intermittent supply of these renewable resources and variable demand. Flow batteries permit more economical long-duration discharge than solid-electrode batteries by using liquid electrolytes stored outside of the battery. We report an alkaline flow battery based on redox-active organic molecules that are composed entirely of Earth-abundant elements and are nontoxic, nonflammable, and safe for use in residential and commercial environments. The battery operates efficiently with high power density near room temperature. These results demonstrate the stability and performance of redox-active organic molecules in alkaline flow batteries, potentially enabling cost-effective stationary storage of renewable energy. PMID:26404834

  16. Activator anion binding site in pyridoxal phosphorylase b: the binding of phosphite, phosphate, and fluorophosphate in the crystal.

    PubMed

    Oikonomakos, N G; Zographos, S E; Tsitsanou, K E; Johnson, L N; Acharya, K R

    1996-12-01

    It has been established that phosphate analogues can activate glycogen phosphorylase reconstituted with pyridoxal in place of the natural cofactor pyridoxal 5'-phosphate (Change YC. McCalmont T, Graves DJ. 1983. Biochemistry 22:4987-4993). Pyridoxal phosphorylase b has been studied by kinetic, ultracentrifugation, and X-ray crystallographic experiments. In solution, the catalytically active species of pyridoxal phosphorylase b adopts a conformation that is more R-state-like than that of native phosphorylase b, but an inactive dimeric species of the enzyme can be stabilized by activator phosphite in combination with the T-state inhibitor glucose. Co-crystals of pyridoxal phosphorylase b complexed with either phosphite, phosphate, or fluorophosphate, the inhibitor glucose, and the weak activator IMP were grown in space group P4(3)2(1)2, with native-like unit cell dimensions, and the structures of the complexes have been refined to give crystallographic R factors of 18.5-19.2%, for data between 8 and 2.4 A resolution. The anions bind tightly at the catalytic site in a similar but not identical position to that occupied by the cofactor 5'-phosphate group in the native enzyme (phosphorus to phosphorus atoms distance = 1.2 A). The structural results show that the structures of the pyridoxal phosphorylase b-anion-glucose-IMP complexes are overall similar to the glucose complex of native T-state phosphorylase b. Structural comparisons suggest that the bound anions, in the position observed in the crystal, might have a structural role for effective catalysis. PMID:8976550

  17. Activator anion binding site in pyridoxal phosphorylase b: the binding of phosphite, phosphate, and fluorophosphate in the crystal.

    PubMed Central

    Oikonomakos, N. G.; Zographos, S. E.; Tsitsanou, K. E.; Johnson, L. N.; Acharya, K. R.

    1996-01-01

    It has been established that phosphate analogues can activate glycogen phosphorylase reconstituted with pyridoxal in place of the natural cofactor pyridoxal 5'-phosphate (Change YC. McCalmont T, Graves DJ. 1983. Biochemistry 22:4987-4993). Pyridoxal phosphorylase b has been studied by kinetic, ultracentrifugation, and X-ray crystallographic experiments. In solution, the catalytically active species of pyridoxal phosphorylase b adopts a conformation that is more R-state-like than that of native phosphorylase b, but an inactive dimeric species of the enzyme can be stabilized by activator phosphite in combination with the T-state inhibitor glucose. Co-crystals of pyridoxal phosphorylase b complexed with either phosphite, phosphate, or fluorophosphate, the inhibitor glucose, and the weak activator IMP were grown in space group P4(3)2(1)2, with native-like unit cell dimensions, and the structures of the complexes have been refined to give crystallographic R factors of 18.5-19.2%, for data between 8 and 2.4 A resolution. The anions bind tightly at the catalytic site in a similar but not identical position to that occupied by the cofactor 5'-phosphate group in the native enzyme (phosphorus to phosphorus atoms distance = 1.2 A). The structural results show that the structures of the pyridoxal phosphorylase b-anion-glucose-IMP complexes are overall similar to the glucose complex of native T-state phosphorylase b. Structural comparisons suggest that the bound anions, in the position observed in the crystal, might have a structural role for effective catalysis. PMID:8976550

  18. Fructose-1,6-Bisphosphate Is an Allosteric Activator of Pyrophosphate:Fructose-6-Phosphate 1-Phosphotransferase.

    PubMed

    Nielsen, T. H.

    1995-05-01

    The activity of highly purified pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP) from barley (Hordeum vulgare) leaves was studied under conditions where the catalyzed reaction was allowed to approach equilibrium. The activity of PFP was monitored by determining the changes in the levels of fructose-6-phosphate, orthophosphate, and fructose-1,6-bisphosphate (Fru-1,6-bisP). Under these conditions PFP activity was not dependent on activation by fructose-2,6-bisphosphate (Fru-2,6-bisP). Inclusion of aldolase in the reaction mixture temporarily restored the dependence of PFP on Fru-2,6-bisP. Alternatively, PFP was activated by Fru-1,6-bisP in the presence of aldolase. It is concluded that Fru-1,6-bisP is an allosteric activator of barley PFP, which can substitute for Fru-2,6-bisP as an activator. A significant activation was observed at a concentration of 5 to 25 [mu]M Fru-1,6-bisP, which demonstrates that the allosteric site of barley PFP has a very high affinity for Fru-1,6-bisP. The high affinity for Fru-1,6-bisP at the allosteric site suggests that the observed activation of PFP by Fru-1,6-bisP constitutes a previously unrecognized in vivo regulation mechanism. PMID:12228454

  19. Facile and Sensitive Fluorescence Sensing of Alkaline Phosphatase Activity with Photoluminescent Carbon Dots Based on Inner Filter Effect.

    PubMed

    Li, Guoliang; Fu, Huili; Chen, Xuejie; Gong, Peiwei; Chen, Guang; Xia, Lian; Wang, Hua; You, Jinmao; Wu, Yongning

    2016-03-01

    A simple and sensitive fluorescent assay for detecting alkaline phosphatase (ALP) based on the inner filter effect (IFE) has been proven, which is conceptually different from the previously reported ALP fluorescent assays. In this sensing platform, N-doped carbon dots (CDs) with a high quantum yield of 49% were prepared by one-pot synthesis and were directly used as a fluorophore in IFE. p-Nitrophenylphosphate (PNPP) was employed to act as an ALP substrate, and its enzyme catalytic product (p-nitrophenol (PNP)) was capable of functioning as a powerful absorber in IFE to influence the excitation of fluorophore (CDs). When in the presence of ALP, PNPP was transformed into PNP and induced the absorption band transition from 310 to 405 nm, which resulted in the complementary overlap between the absorption of PNP and the excitation of CDs. Because of the competitive absorption, the excitation of CDs was significantly weakened, resulting in the quenching of CDs. The present IFE-based sensing strategy showed a good linear relationship from 0.01 to 25 U/L (R(2) = 0.996) and provided an exciting detection limit of 0.001 U/L (signal-to-noise ratio of 3). The proposed sensing approach was successfully applied to ALP sensing in serum samples, ALP inhibitor investigation and phosphatase cell imaging. The presented IFE-based CDs fluorescence sensing strategy gives new insight on the development of the facile and sensitive optical probe for enzyme activity assay because the surface modification or the linking between the receptor and the fluorophore is no longer required. PMID:26820049

  20. Complete genome sequence of a low-temperature active and alkaline-stable endoglucanase-producing Paenibacillus sp. strain IHB B 3084 from the Indian Trans-Himalayas.

    PubMed

    Dhar, Hena; Swarnkar, Mohit Kumar; Rana, Aditi; Kaushal, Kanishak; Singh, Anil Kumar; Kasana, Ramesh Chand; Gulati, Arvind

    2016-07-20

    A genome of 5.88Mb with 46.83% G+C content is reported for an endoglucanase-producing bacterium Paenibacillus sp. strain IHB B 3084 isolated from the cold environments of the Indian Trans-Himalayas. The psychrotrophic bacterium produces low-temperature active and alkaline-stable endoglucanases of industrial importance. The genomic data has provided insight into genomic basis of cellulase production and survival of the bacterium in the cold environments. PMID:27114323

  1. Cryopreservation of glucose-6-phosphate dehydrogenase activity inside red blood cells: developing a specimen repository in support of development and evaluation of glucose-6-phosphate dehydrogenase deficiency tests

    PubMed Central

    2013-01-01

    Background Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common human enzyme deficiency. It is characterized by abnormally low levels of G6PD activity. Individuals with G6PD deficiency are at risk of undergoing acute haemolysis when exposed to 8‒aminoquinoline-based drugs, such as primaquine. For this reason it is imperative to identify individuals with G6PD deficiency prior to administering these anti-malarial drugs. There is a need for the development and evaluation of point-of-care G6PD deficiency screening tests suitable for areas of the developing world where malarial treatments are frequently administered. The development and evaluation of new G6PD tests will be greatly assisted with the availability of specimen repositories. Methods Cryopreservation of erythrocytes was evaluated as a means to preserve G6PD activity. Blood specimens from 31 patients including ten specimens with normal G6PD activity, three with intermediate activity, and 18 with deficient activity were cryopreserved for up to six months. Results Good correlation in G6PD activity between fresh and cryopreserved specimens (R2 = 0.95). The cryopreserved specimens show an overall small drop in mean G6PD activity of 0.23 U/g Hb (P=0.23). Cytochemical staining showed that intracellular G6PD activity distribution within the red blood cell populations is preserved during cryopreservation. Furthermore, the mosaic composition of red blood cells in heterozygous women is also preserved for six months or more. The fluorescent spot and the BinaxNOW qualitative tests for G6PD deficiency also showed high concordance in G6PD status determination between cryopreserved specimens and fresh specimens. Conclusions A methodology for establishing a specimen panel for evaluation of G6PD tests is described. The approach is similar to that used in several malaria research facilities for the cryopreservation of parasites in clinical specimens and axenic cultures. Specimens stored in this manner will aid

  2. Metabolic activation of herbicide products by Vicia faba detected in human peripheral lymphocytes using alkaline single cell gel electrophoresis.

    PubMed

    Calderón-Segura, María Elena; Gómez-Arroyo, Sandra; Molina-Alvarez, Bertha; Villalobos-Pietrini, Rafael; Calderón-Ezquerro, Carmen; Cortés-Eslava, Josefina; Valencia-Quintana, Pedro Rafael; López-González, Lucina; Zúñiga-Reyes, Rubén; Sánchez-Rincón, José

    2007-09-01

    activated by the V. faba root metabolism to produce DNA damage in human peripheral lymphocyte. The alkaline comet technique is a rapid and sensitive assay, to quickly evaluate DNA damage the metabolic activation of herbicide products by V. faba root in human cells in vitro. PMID:17482794

  3. Comparison of inhibitory activity on calcium phosphate precipitation by acidic proline-rich proteins, statherin, and histatin-1.

    PubMed

    Tamaki, N; Tada, T; Morita, M; Watanabe, T

    2002-07-01

    This study quantitatively compares the inhibition of calcium phosphate (CaP) precipitation by the salivary acidic proline-rich proteins (PRPs) statherin and histatin-1. Saliva and CaCl2 in 125 mM imidazole buffer (pH 7.0) were incubated with potassium phosphate and a hydroxyapatite (HAP) suspension, for 30 min at 25 degrees C, then filtered through nitrocellulose. The calcium (Ca) concentration in the filtrate was measured by atomic absorption spectrophotometry, then deducted from that in the initial solution to determine the amount of CaP precipitation after 30 min. The values of the inhibitory activities on CaP precipitation relative to crude parotid saliva were 4.7, 4.9, 6.9, and 65.8 for histatin-1, large PRPs, small PRPs, and statherin, respectively. PMID:12060866

  4. Fructose 6-phosphate phosphoketolase activity in wild-type strains of Lactobacillus, isolated from the intestinal tract of pigs.

    PubMed

    Bolado-Martínez, E; Acedo-Félix, E; Peregrino-Uriarte, A B; Yepiz-Plascencia, G

    2012-01-01

    Phosphoketolases are key enzymes of the phosphoketolase pathway of heterofermentative lactic acid bacteria, which include lactobacilli. In heterofermentative lactobacilli xylulose 5-phosphate phosphoketolase (X5PPK) is the main enzyme of the phosphoketolase pathway. However, activity of fructose 6-phosphate phosphoketolase (F6PPK) has always been considered absent in lactic acid bacteria. In this study, the F6PPK activity was detected in 24 porcine wild-type strains of Lactobacillus reuteri and Lactobacillus mucosae, but not in the Lactobacillus salivarius or in L. reuteri ATCC strains. The activity of F6PPK increased after treatment of the culture at low-pH and diminished after porcine bile-salts stress conditions in wild-type strains of L. reuteri. Colorimetric quantification at 505 nm allowed to differentiate between microbial strains with low activity and without the activity of F6PPK. Additionally, activity of F6PPK and the X5PPK gene expression levels were evaluated by real time PCR, under stress and nonstress conditions, in 3 L. reuteri strains. Although an exact correlation, between enzyme activity and gene expression was not obtained, it remains possible that the xpk gene codes for a phosphoketolase with dual substrate, at least in the analyzed strains of L. reuteri. PMID:23101386

  5. Synthesis of 3,3'-carbonyl-bis(chromones) and their activity as mammalian alkaline phosphatase inhibitors.

    PubMed

    Miliutina, Mariia; Ejaz, Syeda Abida; Iaroshenko, Viktor O; Villinger, Alexander; Iqbal, Jamshed; Langer, Peter

    2016-01-14

    Hitherto unknown 3,3'-carbonyl-bis(chromones) 8, dimeric chromones bridged by a carbonyl group, were prepared by reaction of chromone-3-carboxylic acid chloride with 3-(dimethylamino)-1- (2-hydroxyphenyl)-2-propen-1-ones 9. The method is generally applicable for the synthesis of novel symmetrical or non-symmetrical products which were found to inhibit mammalian alkaline phosphatases. PMID:26490672

  6. Specificity of hydrolysis of phytic acid by alkaline phytase from lily pollen.

    PubMed Central

    Barrientos, L; Scott, J J; Murthy, P P

    1994-01-01

    Phytases are the primary enzymes responsible for the hydrolysis of phytic acid, myo-inositol-1,2,3,4,5,6-hexakisphosphate (I-1,2,3,4,5,6-P6). A number of phytases with varying specificities, properties, and localizations hydrolyze phytic acid present in cells. The specificity of hydrolysis of phytic acid by alkaline phytase from lily (Lilium longiflorum L.) pollen is described. Structures of the intermediate inositol phosphates and the final product were established by a variety of nuclear magnetic resonance techniques (1H-, 31P-, and 31P-1H-detected multiple quantum coherence spectroscopy, and total correlation spectroscopy). On the basis of the structures identified we have proposed a scheme of hydrolysis of phytic acid. Initial hydrolysis of the phosphate ester occurs at the D-5 position of phytic acid to yield the symmetrical I-1,2,3,4,6-P5. The two subsequent dephosphorylations occur adjacent to the D-5 hydroxyl group to yield I-1,2,3-P3 as the final product. Alkaline phytase differs from other phytases in the specificity of hydrolysis of phosphate esters on the inositol ring, its high substrate specificity for phytic acid, and biochemical properties such as susceptibility to activation by calcium and inhibition by fluoride. The physiological significance of alkaline phytase and the biological role of I-1,2,3-P3 remain to be identified. PMID:7846160

  7. An amorphous CoSe film behaves as an active and stable full water-splitting electrocatalyst under strongly alkaline conditions.

    PubMed

    Liu, Tingting; Liu, Qian; Asiri, Abdullah M; Luo, Yonglan; Sun, Xuping

    2015-12-01

    It is attractive but still remains a big challenge to develop non-noble metal bifunctional electrocatalysts efficient for both oxygen evolution reaction (OER) and hydrogen evolution reaction (HER) under alkaline conditions. Herein, an amorphous CoSe film electrodeposited on a Ti mesh (a-CoSe/Ti) is demonstrated to exhibit high electrocatalytic activity and stability for both reactions in 1.0 M KOH. It needs overpotentials of 292 and 121 mV to drive 10 mA cm(-2) for OER and HER, respectively. The two-electrode alkaline water electrolyzer affords a water-splitting current of 10 mA cm(-2) at a cell voltage of 1.65 V. This work offers an attractive cost-effective catalytic material toward full water splitting applications. PMID:26431349

  8. Cerium-activated rare-earth orthophosphate and double-phosphate scintillators for x-and gamma-ray detection

    SciTech Connect

    Boatner, Lynn A; Keefer, Lara A; Farmer, James Matthew; Wisniewski, D.; Wojtowicz, A. J.

    2004-01-01

    When activated with an appropriate rare-earth ion (e.g., Ce or Nd), rare-earth orthophosphates of the form REPO4 (where RE = a rare-earth cation) and alkali rare-earth double phosphates of the form A{sub 3}RE(PO{sub 4}){sub 2} (where A = K, Rb, or Cs) are characterized by light yields and decay times that make these materials of interest for radiation-detection applications. Crystals of the compound Rb{sub 3}Lu(PO{sub 4}){sub 2} when activated with {approx}0.1 mol % Ce exhibit a light yield that is {approx}250% that of BGO with a decay time on the order of {approx}40 nsec. The cerium-activated rare-earth orthophosphate LuPO{sub 4}:Ce is also characterized by a high light yield and a relatively fast decay time of {approx}25 nsec. Additionally, the rare-earth orthophosphates are extremely chemically, physically, and thermally durable hosts that recover easily from radiation damage effects. The properties of the rare-earth orthophosphates and double phosphates that pertain to their use as X- and gamma-ray detectors are reviewed. This review includes information related to the use of Nd-doped LuPO{sub 4} as a scintillator with a sufficiently energetic, short-wavelength output ({lambda} = 90 nm) so that it can be used in conjunction with appropriately activated proportional counters. Information is presented on the details of the synthesis, structure, and luminescence properties of lanthanide double phosphates that, when activated with cerium, are efficient scintillators with output wavelengths that are sufficiently long to be well matched to the response of silicon photodiode detectors.

  9. Phosphate-activated glutaminase activity is enhanced in brain, intestine and kidneys of rats following portacaval anastomosis

    PubMed Central

    Romero-Gómez, Manuel; Jover, María; Díaz-Gómez, Daniel; de Terán, Laura Collantes; Rodrigo, Regina; Camacho, Inés; Echevarría, Miriam; Felipo, Vicente; Bautista, Juan D

    2006-01-01

    AIM: To assess whether portacaval anastomosis (PCA) in rats affects the protein expression and/or activity of glutaminase in kidneys, intestines and in three brain areas of cortex, basal ganglia and cerebellum and to explain the neurological alterations found in hepatic encephalopathy (HE). METHODS: Sixteen male Wistar rats weighing 250-350 g were grouped into sham-operation control (n = 8) or portacaval shunt (n = 8). Twenty-eight days after the procedure, the animals were sacrificed. The duodenum, kidney and brain were removed, homogenised and mitochondria were isolated. Ammonia was measured in brain and blood. Phosphate-activated glutaminase (PAG) activity was determined by measuring ammonia production following incubation for one hour at 37 °C with O-phthalaldehyde (OPA) and specific activity expressed in units per gram of protein (µkat/g of protein). Protein expression was measured by immunoblotting. RESULTS: Duodenal and kidney PAG activities together with protein content were significantly higher in PCA group than in control or sham-operated rats (duodenum PAG activity was 976.95±268.87 µkat/g of protein in PCA rats vs 429.19±126.92 µkat/g of protein in sham-operated rats; kidneys PAG activity was 1259.18 ± 228.79 µkat/g protein in PCA rats vs 669.67± 400.8 µkat/g of protein in controls, P < 0.05; duodenal protein content: 173% in PCA vs sham-operated rats; in kidneys the content of protein was 152% in PCA vs sham-operated rats). PAG activity and protein expression in PCA rats were higher in cortex and basal ganglia than those in sham-operated rats (cortex: 6646.6 ± 1870.4 µkat/g of protein vs 3573.8 ± 2037.4 µkat/g of protein in control rats, P < 0.01; basal ganglia, PAG activity was 3657.3 ± 1469.6 μkat/g of protein in PCA rats vs 2271.2 ± 384 μkat/g of protein in sham operated rats, P < 0.05; In the cerebellum, the PAG activity was 2471.6 ± 701.4 μkat/g of protein vs 1452.9 ± 567.8

  10. Phosphate salts

    MedlinePlus

    ... as a laxative to clean the bowels before surgery or intestinal tests. Healthcare providers sometimes give potassium phosphate intravenously (by IV) for treating low phosphate and high calcium levels in the blood, and for preventing low phosphate in patients who are being tube-fed.

  11. Similar healthy osteoclast and osteoblast activity on nanocrystalline hydroxyapatite and nanoparticles of tri-calcium phosphate compared to natural bone

    PubMed Central

    MacMillan, Adam K; Lamberti, Francis V; Moulton, Julia N; Geilich, Benjamin M; Webster, Thomas J

    2014-01-01

    While there have been numerous studies to determine osteoblast (bone forming cell) functions on nanocrystalline compared to micron crystalline ceramics, there have been few studies which have examined osteoclast activity (including tartrate-resistant acid phosphatase, formation of resorption pits, size of resorption pits, and receptor activator of nuclear factor κB [RANK]). This is despite the fact that osteoclasts are an important part of maintaining healthy bone since they resorb bone during the bone remodeling process. Moreover, while it is now well documented that bone formation is enhanced on nanoceramics compared to micron ceramics, some have pondered whether osteoblast functions (such as osteoprotegerin and RANK ligand [RANKL]) are normal (ie, non-diseased) on such materials compared to natural bone. For these reasons, the objective of the present in vitro study was to determine various functions of osteoclasts and osteoblasts on nanocrystalline and micron crystalline hydroxyapatite as well as tri-calcium phosphate materials and compare such results to cortical and cancellous bone. Results showed for the first time similar osteoclast activity (including tartrate-resistant acid phosphatase, formation of resorption pits, size of resorption pits, and RANK) and osteoblast activity (osteoprotegerin and RANKL) on nanocrystalline hydroxyapatite compared to natural bone, whereas osteoclast and osteoblast functions on micron crystalline versions of these ceramics were much different than natural bone. In this manner, this study provides additional evidence that nanocrystalline calcium phosphates can serve as suitable synthetic analogs to natural bone to improve numerous orthopedic applications. It also provides the first data of healthy osteoclast and osteoblast functions on nanocrystalline calcium phosphates compared to natural bone. PMID:25506216

  12. Aspirin inhibits glucose-6-phosphate dehydrogenase activity in HCT 116 cells through acetylation: Identification of aspirin-acetylated sites

    PubMed Central

    Ai, Guoqiang; Dachineni, Rakesh; Kumar, D. Ramesh; Alfonso, Lloyd F.; Marimuthu, Srinivasan; Bhat, G. Jayarama

    2016-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the first reaction in the pentose phosphate pathway, and generates ribose sugars, which are required for nucleic acid synthesis, and nicotinamide adenine dinucleotide phosphate (NADPH), which is important for neutralization of oxidative stress. The expression of G6PD is elevated in several types of tumor, including colon, breast and lung cancer, and has been implicated in cancer cell growth. Our previous study demonstrated that exposure of HCT 116 human colorectal cancer cells to aspirin caused acetylation of G6PD, and this was associated with a decrease in its enzyme activity. In the present study, this observation was expanded to HT-29 colorectal cancer cells, in order to compare aspirin-mediated acetylation of G6PD and its activity between HCT 116 and HT-29 cells. In addition, the present study aimed to determine the acetylation targets of aspirin on recombinant G6PD to provide an insight into the mechanisms of inhibition. The results demonstrated that the extent of G6PD acetylation was significantly higher in HCT 116 cells compared with in HT-29 cells; accordingly, a greater reduction in G6PD enzyme activity was observed in the HCT 116 cells. Mass spectrometry analysis of aspirin-acetylated G6PD (isoform a) revealed that aspirin acetylated a total of 14 lysine residues, which were dispersed throughout the length of the G6PD protein. One of the important amino acid targets of aspirin included lysine 235 (K235, in isoform a) and this corresponds to K205 in isoform b, which has previously been identified as being important for catalysis. Acetylation of G6PD at several sites, including K235 (K205 in isoform b), may mediate inhibition of G6PD activity, which may contribute to the ability of aspirin to exert anticancer effects through decreased synthesis of ribose sugars and NADPH. PMID:27356773

  13. Characterization of the mineral phosphate-solubilizing activity of Pantoea agglomerans MMB051 isolated from an iron-rich soil in southeastern Venezuela (Bolívar State).

    PubMed

    Sulbarán, Miguel; Pérez, Elizabeth; Ball, María M; Bahsas, Alí; Yarzábal, Luis Andrés

    2009-04-01

    The mineral phosphate-solubilizing (MPS) activity of a Pantoea agglomerans strain, namely MMB051, isolated from an iron-rich, acidic soil near Ciudad Piar (Bolívar State, Venezuela), was characterized on a chemically defined medium (NBRIP). Various insoluble inorganic phosphates, including tri-calcium phosphate [Ca(3)(PO(4))(2)], iron phosphate (FePO(4)), aluminum phosphate (AlPO(4)), and Rock Phosphate (RP) were tested as sole sources of P for bacterial growth. Solubilization of Ca(3)(PO(4))(2) was very efficient and depended on acidification of the external milieu when MMB051 cells were grown in the presence of glucose. This was also the case when RP was used as the sole P source. On the other hand, the solubilization efficiency toward more insoluble mineral phosphates (FePO(4) and AlPO(4)) was shown to be very low. Even though gluconic acid (GA) was detected on culture supernatants of strain MMB051, a consequence of the direct oxidation pathway of glucose, inorganic-P solubilization seemed also to be related to other processes dependent on active cell growth. Among these, proton release by ammonium (NH(4)(+) ) fixation appeared to be of paramount importance to explain inorganic-P solubilization mediated by strain MMB051. On the contrary, the presence of nitrate (NO(3)(-) ) salts as the sole N source affected negatively the ability of MMB051 cells to solubilize inorganic P. PMID:19067045

  14. Complete Genome Sequence of the Rhizobacterium Pseudomonas trivialis Strain IHBB745 with Multiple Plant Growth-Promoting Activities and Tolerance to Desiccation and Alkalinity.

    PubMed

    Gulati, Arvind; Swarnkar, Mohit Kumar; Vyas, Pratibha; Rahi, Praveen; Thakur, Rishu; Thakur, Namika; Singh, Anil Kumar

    2015-01-01

    The complete genome sequence of 6.45 Mb is reported here for Pseudomonas trivialis strain IHBB745 (MTCC 5336), which is an efficient, stress-tolerant, and broad-spectrum plant growth-promoting rhizobacterium. The gene-coding clusters predicted the genes for phosphate solubilization, siderophore production, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, indole-3-acetic acid (IAA) production, and stress response. PMID:26337878

  15. Transmembrane signal transduction in bacterial chemotaxis involves ligand-dependent activation of phosphate group transfer.

    PubMed Central

    Borkovich, K A; Kaplan, N; Hess, J F; Simon, M I

    1989-01-01

    Signal transduction in Escherichia coli involves the interaction of transmembrane receptor proteins such as the aspartate receptor, Tar, and the products of four chemotaxis genes, cheA, cheY, cheW, and cheZ. It was previously shown that the cheA gene product is an autophosphorylating protein kinase that transfers phosphate to CheY, whereas the cheZ gene product acts as a specific CheY phosphatase. Here we report that the system can be reconstituted in vitro and receptor function can be coupled to CheY phosphorylation. Coupling requires the presence of the CheW protein, the appropriate form of the receptor, and the CheA and CheY proteins. Under these conditions the accumulation of CheY-phosphate is enhanced approximately 300-fold. This rate enhancement is seen in reactions using wild-type and "tumble" mutant receptors but not "smooth" mutant receptors. The increased accumulation of phosphoprotein was inhibited by micromolar concentrations of aspartate, using wild-type, but not tumble, receptors. These results provide evidence that the signal transduction pathway in bacterial chemotaxis involves receptor-mediated alteration of the levels of phosphorylated proteins. They suggest that CheW acts as the coupling factor between receptor and phosphorylation. The results also support the suggestion that CheY-phosphate is the tumble signal. Images PMID:2645576

  16. Role of chemical composition in the enhanced catalytic activity of Pt-based alloyed ultrathin nanowires for the hydrogen oxidation reaction under alkaline conditions

    DOE PAGESBeta

    Megan E. Scofield; Wong, Stanislaus S.; Zhou, Yuchen; Yue, Shiyu; Wang, Lei; Su, Dong; Tong, Xiao; Vukmirovic, Miomir B.; Adzic, Radoslav R.

    2016-05-19

    With the increased interest in the development of hydrogen fuel cells as a plausible alternative to internal combustion engines, recent work has focused on creating alkaline fuel cells (AFC), which employ an alkaline environment. Working in alkaline as opposed to acidic media yields a number of tangible benefits, including (i) the ability to use cheaper and plentiful precious-metal-free catalysts, due to their increased stability, (ii) a reduction in the amount of degradation and corrosion of Pt-based catalysts, and (iii) a longer operational lifetime for the overall fuel cell configuration. However, in the absence of Pt, no catalyst has achieved activitiesmore » similar to those of Pt. Herein, we have synthesized a number of crystalline ultrathin PtM alloy nanowires (NWs) (M = Fe, Co, Ru, Cu, Au) in order to replace a portion of the costly Pt metal without compromising on activity while simultaneously adding in metals known to exhibit favorable synergistic ligand and strain effects with respect to the host lattice. In fact, our experiments confirm theoretical insights about a clear and correlative dependence between measured activity and chemical composition. We have conclusively demonstrated that our as-synthesized alloy NW catalysts yield improved hydrogen oxidation reaction (HOR) activities as compared with a commercial Pt standard as well as with our as-synthesized Pt NWs. The Pt7Ru3 NW system, in particular, quantitatively achieved an exchange current density of 0.493 mA/cm2, which is higher than the corresponding data for Pt NWs alone. In addition, the HOR activities follow the same expected trend as their calculated hydrogen binding energy (HBE) values, thereby confirming the critical importance and correlation of HBE with the observed activities.« less

  17. [Activity of NADP-dependent glyceraldehyde-phosphate dehydrogenase and phosphoenolpyruvate carboxylase in wheat leaves under water stress].

    PubMed

    Cherniad'ev, I I; Monakhova, O F

    2006-01-01

    The activities of NADP: glyceraldehyde-phosphate dehydrogenase (GAPDH), an enzyme complex comprising of phosphoglycerate kinase (EC 2.7.2.3) and glyceraldehyde-phosphate dehydrogenase (EC 1.2.1.13), and phosphoenolpyruvate carboxylase (PEPK; EC 4.1.1.31) in seedlings and leaves of wheat (Triticum aestivum L.) plants of the cultivars Mironovskaya 808 and Lutescens 758 have been compared under conditions of normal water supply, water deficiency, and subsequent rehydration. GAPDH activity, which determines the carbohydrate route of photosynthetic metabolism at the initial stages, is decreased by water stress to a greater extent than that of PEPK, on the activity of which non-carbohydrate metabolic pathways depend. Pretreatment of seedlings and mature plants with natural (6-benzylaminopurine) and synthetic (tidiazuron, kartolin-2, and kartolin-4) cytokinins attenuates the loss of enzyme activities during drought and facilitates their recovery within the period of rehydration; both effects are underlain by augmentation of reparation processes. The relative intensification of non-carbohydrate pathways of photosynthetic metabolism, observed under conditions of water deficiency, is accompanied by an increase in the osmotic pressure of cell sap. Possible mechanisms of this protector effect of cytokinin preparations are discussed. PMID:16878554

  18. 5'-(E)-Vinylphosphonate: A Stable Phosphate Mimic Can Improve the RNAi Activity of siRNA-GalNAc Conjugates.

    PubMed

    Parmar, Rubina; Willoughby, Jennifer L S; Liu, Jingxuan; Foster, Donald J; Brigham, Benjamin; Theile, Christopher S; Charisse, Klaus; Akinc, Akin; Guidry, Erin; Pei, Yi; Strapps, Walter; Cancilla, Mark; Stanton, Matthew G; Rajeev, Kallanthottathil G; Sepp-Lorenzino, Laura; Manoharan, Muthiah; Meyers, Rachel; Maier, Martin A; Jadhav, Vasant

    2016-06-01

    Small interfering RNA (siRNA)-mediated silencing requires siRNA loading into the RNA-induced silencing complex (RISC). Presence of 5'-phosphate (5'-P) is reported to be critical for efficient RISC loading of the antisense strand (AS) by anchoring it to the mid-domain of the Argonaute2 (Ago2) protein. Phosphorylation of exogenous duplex siRNAs is thought to be accomplished by cytosolic Clp1 kinase. However, although extensive chemical modifications are essential for siRNA-GalNAc conjugate activity, they can significantly impair Clp1 kinase activity. Here, we further elucidated the effect of 5'-P on the activity of siRNA-GalNAc conjugates. Our results demonstrate that a subset of sequences benefit from the presence of exogenous 5'-P. For those that do, incorporation of 5'-(E)-vinylphosphonate (5'-VP), a metabolically stable phosphate mimic, results in up to 20-fold improved in vitro potency and up to a threefold benefit in in vivo activity by promoting Ago2 loading and enhancing metabolic stability. PMID:27121751

  19. Role of Acetyl-Phosphate in Activation of the Rrp2-RpoN-RpoS Pathway in Borrelia burgdorferi

    PubMed Central

    Xu, Haijun; Caimano, Melissa J.; Lin, Tao; He, Ming; Radolf, Justin D.; Norris, Steven J.; Gheradini, Frank; Wolfe, Alan J.; Yang, X. Frank

    2010-01-01

    Borrelia burgdorferi, the Lyme disease spirochete, dramatically alters its transcriptome and proteome as it cycles between the arthropod vector and mammalian host. During this enzootic cycle, a novel regulatory network, the Rrp2-RpoN-RpoS pathway (also known as the σ54–σS sigma factor cascade), plays a central role in modulating the differential expression of more than 10% of all B. burgdorferi genes, including the major virulence genes ospA and ospC. However, the mechanism(s) by which the upstream activator and response regulator Rrp2 is activated remains unclear. Here, we show that none of the histidine kinases present in the B. burgdorferi genome are required for the activation of Rrp2. Instead, we present biochemical and genetic evidence that supports the hypothesis that activation of the Rrp2-RpoN-RpoS pathway occurs via the small, high-energy, phosphoryl-donor acetyl phosphate (acetyl∼P), the intermediate of the Ack-Pta (acetate kinase-phosphate acetyltransferase) pathway that converts acetate to acetyl-CoA. Supplementation of the growth medium with acetate induced activation of the Rrp2-RpoN-RpoS pathway in a dose-dependent manner. Conversely, the overexpression of Pta virtually abolished acetate-induced activation of this pathway, suggesting that acetate works through acetyl∼P. Overexpression of Pta also greatly inhibited temperature and cell density-induced activation of RpoS and OspC, suggesting that these environmental cues affect the Rrp2-RpoN-RpoS pathway by influencing acetyl∼P. Finally, overexpression of Pta partially reduced infectivity of B. burgdorferi in mice. Taken together, these findings suggest that acetyl∼P is one of the key activating molecule for the activation of the Rrp2-RpoN-RpoS pathway and support the emerging concept that acetyl∼P can serve as a global signal in bacterial pathogenesis. PMID:20862323

  20. International symposium on cellular and molecular biology of phosphate and phosphorylated compounds in microorganisms: Proceedings

    SciTech Connect

    1993-12-31

    This report contains the abstracts of papers presented at the conference. Attention is focused on the following topics: regulation of phosphate metabolism in bacteria; structure-function of alkaline phosphatase; regulation of phosphate metabolism in yeast; transport of phosphate and phosphorylated compounds; and phosphate regulation in pathogenesis and secondary metabolism.

  1. 2-Aminoethoxydiphenyl borate (2-APB) reduces alkaline phosphatase release, CD63 expression, F-actin polymerization and chemotaxis without affecting the phagocytosis activity in bovine neutrophils.

    PubMed

    Conejeros, I; Velásquez, Z D; Carretta, M D; Alarcón, P; Hidalgo, M A; Burgos, R A

    2012-01-15

    2-Aminoethoxydiphenyl borate (2-APB) interferes with the Ca(2+) influx and reduces the ROS production, gelatinase secretion and CD11b expression in bovine neutrophils. Moreover, it has been suggested that inhibition of the Ca(2+) channel involved in the store operated Ca(2+) entry (SOCE) is a potential target for the development of new anti-inflammatory drugs in cattle, however it is unknown whether 2-APB affects neutrophil functions associated with the innate immune response. This study describes the effect of 2-APB, a putative SOCE inhibitor, on alkaline phosphatase activity a marker of secretory vesicles, CD63 a marker for azurophil granules, F-actin polymerization and in vitro chemotaxis in bovine neutrophils stimulated with platelet-activating factor (PAF). Also, we evaluated the effect of 2-APB in the phagocytic activity against Escherichia coli and Staphylococcus aureus bioparticles. We observed that doses of 2-APB ≥10 μM significantly reduced alkaline phosphatase activity and in vitro chemotaxis, whereas concentrations of 2-APB ≥50 μM reduced CD63 expression and F-actin polymerization. Finally, we observed that 2-APB did not affect the phagocytic activity in neutrophils incubated with E. coli and S. aureus bioparticles. We concluded that inhibition of Ca(2+) influx could be a useful strategy to reduce inflammatory process in cattle. PMID:22226550

  2. Truncation of the unique N-terminal domain improved the thermos-stability and specific activity of alkaline α-amylase Amy703.

    PubMed

    Lu, Zhenghui; Wang, Qinhong; Jiang, Sijing; Zhang, Guimin; Ma, Yanhe

    2016-01-01

    High pH condition is of special interest for the potential applications of alkaline α-amylase in textile and detergent industries. Thus, there is a continuous demand to improve the amylase's properties to meet the requirements set by specific applications. Here we reported the systematic study of modular domain engineering to improve the specific activity and stability of the alkaline α-amylase from Bacillus pseudofirmus 703. The specific activity of the N-terminal domain truncated mutant (N-Amy) increased by ~35-fold with a significantly improved thermo-stability. Kinetic analysis demonstrated that the Kcat and Kcat/Kmof N-Amy were enhanced by 1300-fold and 425.7-fold, respectively, representing the largest catalytic activity improvement of the engineered α-amylases through the methods of domain deletion, fusion or swapping. In addition, different from the wild-type Amy703, no exogenous Ca(2+) were required for N-Amy to maintain its full catalytic activity, implying its superior potential for many industrial processes. Circular dichroism analysis and structure modeling revealed that the increased compactness and α-helical content were the main contributors for the improved thermo-stability of N-Amy, while the improved catalytic efficiency was mainly attributed by the increased conformational flexibility around the active center. PMID:26926401

  3. Truncation of the unique N-terminal domain improved the thermos-stability and specific activity of alkaline α-amylase Amy703

    PubMed Central

    Lu, Zhenghui; Wang, Qinhong; Jiang, Sijing; Zhang, Guimin; Ma, Yanhe

    2016-01-01

    High pH condition is of special interest for the potential applications of alkaline α-amylase in textile and detergent industries. Thus, there is a continuous demand to improve the amylase’s properties to meet the requirements set by specific applications. Here we reported the systematic study of modular domain engineering to improve the specific activity and stability of the alkaline α-amylase from Bacillus pseudofirmus 703. The specific activity of the N-terminal domain truncated mutant (N-Amy) increased by ~35-fold with a significantly improved thermo-stability. Kinetic analysis demonstrated that the Kcat and Kcat/Kmof N-Amy were enhanced by 1300-fold and 425.7-fold, respectively, representing the largest catalytic activity improvement of the engineered α-amylases through the methods of domain deletion, fusion or swapping. In addition, different from the wild-type Amy703, no exogenous Ca2+ were required for N-Amy to maintain its full catalytic activity, implying its superior potential for many industrial processes. Circular dichroism analysis and structure modeling revealed that the increased compactness and α-helical content were the main contributors for the improved thermo-stability of N-Amy, while the improved catalytic efficiency was mainly attributed by the increased conformational flexibility around the active center. PMID:26926401

  4. Control of Pyrophosphated-Fructose-6-Phosphate 1-Phosphotransferase Activity in the Cotyledons of Citrullus lanatus1

    PubMed Central

    Botha, Anna-Maria; Botha, Frederik C.

    1990-01-01

    After initiation of radicle elongation, the pyrophosphate:d-fructose-6-phosphate 1-phosphotransferase (PFP) activity sharply increases in the cotyledons of Citrullus lanatus. Removal of the radicle early during incubation prevents the increase in PFP activity in the cotyledons evident in the control. Removal of the radicle at any stage after germination results in a decrease in PFP activity in the cotyledons. Application of kinetin (0.5 micromolar) or 2-chlorophosphonic acid (0.1 micromolar) to isolated cotyledons replaces the effect of the radicle. Gibberellic acid (0.09 micromolar GA3) also partially mimics the presence of the radicle. Anaerobic conditions, as well as cycloheximide application (0.18 micromolar) to intact embryos or to kinetin and ethrel treated isolated cotyledons prevent the increase in PFP activity evident in the control. PMID:16667523

  5. Exposure to the Proton Scavenger Glycine under Alkaline Conditions Induces Escherichia coli Viability Loss

    PubMed Central

    Vanhauteghem, Donna; Janssens, Geert Paul Jules; Lauwaerts, Angelo; Sys, Stanislas; Boyen, Filip; Cox, Eric; Meyer, Evelyne

    2013-01-01

    Our previous work described a clear loss of Escherichia coli (E. coli) membrane integrity after incubation with glycine or its N-methylated derivatives N-methylglycine (sarcosine) and N,N-dimethylglycine (DMG), but not N,N,N-trimethylglycine (betaine), under alkaline stress conditions. The current study offers a thorough viability analysis, based on a combination of real-time physiological techniques, of E. coli exposed to glycine and its N-methylated derivatives at alkaline pH. Flow cytometry was applied to assess various physiological parameters such as membrane permeability, esterase activity, respiratory activity and membrane potential. ATP and inorganic phosphate concentrations were also determined. Membrane damage was confirmed through the measurement of nucleic acid leakage. Results further showed no loss of esterase or respiratory activity, while an instant and significant decrease in the ATP concentration occurred upon exposure to either glycine, sarcosine or DMG, but not betaine. There was a clear membrane hyperpolarization as well as a significant increase in cellular inorganic phosphate concentration. Based on these results, we suggest that the inability to sustain an adequate level of ATP combined with a decrease in membrane functionality leads to the loss of bacterial viability when exposed to the proton scavengers glycine, sarcosine and DMG at alkaline pH. PMID:23544135

  6. Exposure to the proton scavenger glycine under alkaline conditions induces Escherichia coli viability loss.

    PubMed

    Vanhauteghem, Donna; Janssens, Geert Paul Jules; Lauwaerts, Angelo; Sys, Stanislas; Boyen, Filip; Cox, Eric; Meyer, Evelyne

    2013-01-01

    Our previous work described a clear loss of Escherichia coli (E. coli) membrane integrity after incubation with glycine or its N-methylated derivatives N-methylglycine (sarcosine) and N,N-dimethylglycine (DMG), but not N,N,N-trimethylglycine (betaine), under alkaline stress conditions. The current study offers a thorough viability analysis, based on a combination of real-time physiological techniques, of E. coli exposed to glycine and its N-methylated derivatives at alkaline pH. Flow cytometry was applied to assess various physiological parameters such as membrane permeability, esterase activity, respiratory activity and membrane potential. ATP and inorganic phosphate concentrations were also determined. Membrane damage was confirmed through the measurement of nucleic acid leakage. Results further showed no loss of esterase or respiratory activity, while an instant and significant decrease in the ATP concentration occurred upon exposure to either glycine, sarcosine or DMG, but not betaine. There was a clear membrane hyperpolarization as well as a significant increase in cellular inorganic phosphate concentration. Based on these results, we suggest that the inability to sustain an adequate level of ATP combined with a decrease in membrane functionality leads to the loss of bacterial viability when exposed to the proton scavengers glycine, sarcosine and DMG at alkaline pH. PMID:23544135

  7. Amorphous calcium phosphate and its application in dentistry

    PubMed Central

    2011-01-01

    Amorphous Calcium Phosphate (ACP) is an essential mineral phase formed in mineralized tissues and the first commercial product as artificial hydroxyapatite. ACP is unique among all forms of calcium phosphates in that it lacks long-range, periodic atomic scale order of crystalline calcium phosphates. The X-ray diffraction pattern is broad and diffuse with a maximum at 25 degree 2 theta, and no other different features compared with well-crystallized hydroxyapatite. Under electron microscopy, its morphological form is shown as small spheroidal particles in the scale of tenths nanometer. In aqueous media, ACP is easily transformed into crystalline phases such as octacalcium phosphate and apatite due to the growing of microcrystalline. It has been demonstrated that ACP has better osteoconductivity and biodegradability than tricalcium phosphate and hydroxyapatite in vivo. Moreover, it can increase alkaline phosphatase activities of mesoblasts, enhance cell proliferation and promote cell adhesion. The unique role of ACP during the formation of mineralized tissues makes it a promising candidate material for tissue repair and regeneration. ACP may also be a potential remineralizing agent in dental applications. Recently developed ACP-filled bioactive composites are believed to be effective anti-demineralizing/remineralizing agents for the preservation and repair of tooth structures. This review provides an overview of the development, structure, chemical composition, morphological characterization, phase transformation and biomedical application of ACP in dentistry. PMID:21740535

  8. Sensitive and selective determining ascorbic acid and activity of alkaline phosphatase based on electrochemiluminescence of dual-stabilizers-capped CdSe quantum dots in carbon nanotube-nafion composite.

    PubMed

    Ma, Xiaolong; Zhang, Xin; Guo, Xinli; Kang, Qi; Shen, Dazhong; Zou, Guizheng

    2016-07-01

    Sensitive and selective determining bio-related molecule and enzyme play an important role in designing novel procedure for biological sensing and clinical diagnosis. Herein, we found that dual-stabilizers-capped CdSe quantum dots (QDs) in composite film of multi-walled carbon nanotubes (CNTs) and Nafion, displaying eye-visible monochromatic electrochemiluminescence (ECL) with fwhm of 37nm, which offers promising ECL signal for detecting ascorbic acid (AA) as well as the activity of alkaline phosphatase (ALP) in biological samples. It was also shown that the dual-stabilizers-capped CdSe QDs can preserve their highly passivated surface states with prolonged lifetime of excited states in Nafion mixtures, and facilitate electron-transfer ability of Nafion film along with CNTs. Compared with the QDs/GCE, the ECL intensity is enhanced 1.8 times and triggering potential shifted to lower energy by 0.12V on the CdSe-CNTs-Nafion/GCE. The ECL quenching degree increases with increasing concentration of AA in the range of 0.01-30nM with a limit of detection (LOD) of 5pM. The activity of ALP was determined indirectly according to the concentration of AA, generated in the hydrolysis reaction of l-ascorbic acid 2-phosphate sesquimagnesium (AA-P) in the presence of ALP as a catalyst, with an LOD of 1μU/L. The proposed strategy is favorable for developing simple ECL sensor or device with high sensitivity, spectral resolution and less electrochemical interference. PMID:27154663

  9. Artefactual nanoparticle activation of the inflammasome platform: in vitro evidence with a nano-formed calcium phosphate

    PubMed Central

    Pele, Laetitia; Haas, Carolin T; Hewitt, Rachel; Faria, Nuno; Brown, Andy; Powell, Jonathan

    2015-01-01

    Aim To determine whether in vitro experimental conditions dictate cellular activation of the inflammasome by apatitic calcium phosphate nanoparticles. Material & methods The responses of blood-derived primary human cells to in situ-formed apatite were investigated under different experimental conditions to assess the effect of aseptic culture, cell rest and duration of particle exposure. Cell death and particle uptake were assessed, while IL-1β and caspase 1 responses, with and without lipopolysaccharide prestimulation, were evaluated as markers of inflammasome activation. Results Under carefully addressed experimental conditions, apatitic nanoparticles did not induce cell death or engage the inflammasome platform, although both could be triggered through artefacts of experimentation. Conclusion In vitro studies often predict that engineered nanoparticles, such as synthetic apatite, are candidates for inflammasome activation and, hence, are toxic. However, the experimental setting must be very carefully considered as it may promote false-positive outcomes. PMID:24991724

  10. Monte Carlo simulations of phosphate polyhedron connectivity in glasses

    SciTech Connect

    ALAM,TODD M.

    2000-01-01

    Monte Carlo simulations of phosphate tetrahedron connectivity distributions in alkali and alkaline earth phosphate glasses are reported. By utilizing a discrete bond model, the distribution of next-nearest neighbor connectivities between phosphate polyhedron for random, alternating and clustering bonding scenarios was evaluated as a function of the relative bond energy difference. The simulated distributions are compared to experimentally observed connectivities reported for solid-state two-dimensional exchange and double-quantum NMR experiments of phosphate glasses. These Monte Carlo simulations demonstrate that the polyhedron connectivity is best described by a random distribution in lithium phosphate and calcium phosphate glasses.

  11. Monte Carlo Simulations of Phosphate Polyhedron Connectivity in Glasses

    SciTech Connect

    ALAM,TODD M.

    1999-12-21

    Monte Carlo simulations of phosphate tetrahedron connectivity distributions in alkali and alkaline earth phosphate glasses are reported. By utilizing a discrete bond model, the distribution of next-nearest neighbor connectivities between phosphate polyhedron for random, alternating and clustering bonding scenarios was evaluated as a function of the relative bond energy difference. The simulated distributions are compared to experimentally observed connectivities reported for solid-state two-dimensional exchange and double-quantum NMR experiments of phosphate glasses. These Monte Carlo simulations demonstrate that the polyhedron connectivity is best described by a random distribution in lithium phosphate and calcium phosphate glasses.

  12. Reprogramming metabolism by histone methyltransferase NSD2 drives endocrine resistance via coordinated activation of pentose phosphate pathway enzymes.

    PubMed

    Wang, Junjian; Duan, Zhijian; Nugent, Zoann; Zou, June X; Borowsky, Alexander D; Zhang, Yanhong; Tepper, Clifford G; Li, Jian Jian; Fiehn, Oliver; Xu, Jianzhen; Kung, Hsing-Jien; Murphy, Leigh C; Chen, Hong-Wu

    2016-08-10

    Metabolic reprogramming such as the aerobic glycolysis or Warburg effect is well recognized as a common feature of tumorigenesis. However, molecular mechanisms underlying metabolic alterations for tumor therapeutic resistance are poorly understood. Through gene expression profiling analysis we found that histone H3K36 methyltransferase NSD2/MMSET/WHSC1 expression was highly elevated in tamoxifen-resistant breast cancer cell lines and clinical tumors. IHC analysis indicated that NSD2 protein overexpression was associated with the disease recurrence and poor survival. Ectopic expression of NSD2 wild type, but not the methylase-defective mutant, drove endocrine resistance in multiple cell models and xenograft tumors. Mechanistically, NSD2 was recruited to and methylated H3K36me2 at the promoters of key glucose metabolic enzyme genes. Its overexpression coordinately up-regulated hexokinase 2 (HK2) and glucose-6-phosphate dehydrogenase (G6PD), two key enzymes of glycolysis and the pentose phosphate pathway (PPP), as well as TP53-induced glycolysis regulatory phosphatase TIGAR. Consequently, NSD2-driven tamoxifen-resistant cells and tumors displayed heightened PPP activity, elevated NADPH production, and reduced ROS level, without significantly altered glycolysis. These results illustrate a coordinated, epigenetic activation of key glucose metabolic enzymes in therapeutic resistance and nominate methyltransferase NSD2 as a potential therapeutic target for endocrine resistant breast cancer. PMID:27164560

  13. ALP (Alkaline Phosphatase) Test

    MedlinePlus

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

  14. Electrocatalytic activity and operational stability of electrodeposited Pd-Co films towards ethanol oxidation in alkaline electrolytes

    NASA Astrophysics Data System (ADS)

    Tsui, Lok-kun; Zafferoni, Claudio; Lavacchi, Alessandro; Innocenti, Massimo; Vizza, Francesco; Zangari, Giovanni

    2015-10-01

    Direct alkaline ethanol fuel cells (DEFCs) are usually run with Pd anodic catalysts, but their performance can be improved by utilizing alloys of Pd and Co. The oxyphilic Co serves to supply ample -OH to the ethanol oxidation reaction, accelerating the rate limiting step at low overpotential under alkaline conditions. Pd-Co films with compositions between 20 and 80 at% Co can be prepared by electrodeposition from a NH3 complexing electrolyte. Cyclic voltammetry studies show that the ethanol oxidation peak exhibits increasing current density with increasing Co content, reaching a maximum at 77% Co. In contrast, potentiostatic measurements under conditions closer to fuel cell operating conditions show that a 50 at% Co alloy has the highest performance. Importantly, the Co-Pd film is also found to undergo phase and morphological transformations during ethanol oxidation, resulting in a change from a compact film to high surface area flake-like structures containing Co3O4 and CoOOH; such a transformation instead is not observed when operating at a constant potential of 0.7 VRHE.

  15. Sphingosine-1-Phosphate Enhances Satellite Cell Activation in Dystrophic Muscles through a S1PR2/STAT3 Signaling Pathway

    PubMed Central

    Carlson, Morgan E.; Oskouian, Babak; Kumar, Ashok; Fyrst, Henrik; Zhang, Meng; Proia, Richard L.; Hoffman, Eric P.; Saba, Julie D.

    2012-01-01

    Sphingosine-1-phosphate (S1P) activates a widely expressed family of G protein-coupled receptors, serves as a muscle trophic factor and activates muscle stem cells called satellite cells (SCs) through unknown mechanisms. Here we show that muscle injury induces dynamic changes in S1P signaling and metabolism in vivo. These changes include early and profound induction of the gene encoding the S1P biosynthetic enzyme SphK1, followed by induction of the catabolic enzyme sphingosine phosphate lyase (SPL) 3 days later. These changes correlate with a transient increase in circulating S1P levels after muscle injury. We show a specific requirement for SphK1 to support efficient muscle regeneration and SC proliferation and differentiation. Mdx mice, which serve as a model for muscular dystrophy (MD), were found to be S1P-deficient and exhibited muscle SPL upregulation, suggesting that S1P catabolism is enhanced in dystrophic muscle. Pharmacological SPL inhibition increased muscle S1P levels, improved mdx muscle regeneration and enhanced SC proliferation via S1P receptor 2 (S1PR2)-dependent inhibition of Rac1, thereby activating Signal Transducer and Activator of Transcription 3 (STAT3), a central player in inflammatory signaling. STAT3 activation resulted in p21 and p27 downregulation in a S1PR2-dependent fashion in myoblasts. Our findings suggest that S1P promotes SC progression through the cell cycle by repression of cell cycle inhibitors via S1PR2/STAT3-dependent signaling and that SPL inhibition may provide a therapeutic strategy for MD. PMID:22606352

  16. Sphingosine-1-phosphate enhances satellite cell activation in dystrophic muscles through a S1PR2/STAT3 signaling pathway.

    PubMed

    Loh, Kenneth C; Leong, Weng-In; Carlson, Morgan E; Oskouian, Babak; Kumar, Ashok; Fyrst, Henrik; Zhang, Meng; Proia, Richard L; Hoffman, Eric P; Saba, Julie D

    2012-01-01

    Sphingosine-1-phosphate (S1P) activates a widely expressed family of G protein-coupled receptors, serves as a muscle trophic factor and activates muscle stem cells called satellite cells (SCs) through unknown mechanisms. Here we show that muscle injury induces dynamic changes in S1P signaling and metabolism in vivo. These changes include early and profound induction of the gene encoding the S1P biosynthetic enzyme SphK1, followed by induction of the catabolic enzyme sphingosine phosphate lyase (SPL) 3 days later. These changes correlate with a transient increase in circulating S1P levels after muscle injury. We show a specific requirement for SphK1 to support efficient muscle regeneration and SC proliferation and differentiation. Mdx mice, which serve as a model for muscular dystrophy (MD), were found to be S1P-deficient and exhibited muscle SPL upregulation, suggesting that S1P catabolism is enhanced in dystrophic muscle. Pharmacological SPL inhibition increased muscle S1P levels, improved mdx muscle regeneration and enhanced SC proliferation via S1P receptor 2 (S1PR2)-dependent inhibition of Rac1, thereby activating Signal Transducer and Activator of Transcription 3 (STAT3), a central player in inflammatory signaling. STAT3 activation resulted in p21 and p27 downregulation in a S1PR2-dependent fashion in myoblasts. Our findings suggest that S1P promotes SC progression through the cell cycle by repression of cell cycle inhibitors via S1PR2/STAT3-dependent signaling and that SPL inhibition may provide a therapeutic strategy for MD. PMID:22606352

  17. Alkaline-resistance model of subtilisin ALP I, a novel alkaline subtilisin.

    PubMed

    Maeda, H; Mizutani, O; Yamagata, Y; Ichishima, E; Nakajima, T

    2001-05-01

    The alkaline-resistance mechanism of the alkaline-stable enzymes is not yet known. To clarify the mechanism of alkaline-resistance of alkaline subtilisin, structural changes of two typical subtilisins, subtilisin ALP I (ALP I) and subtilisin Sendai (Sendai), were studied by means of physicochemical methods. Subtilisin NAT (NAT), which exhibits no alkaline resistance, was examined as a control. ALP I gradually lost its activity, accompanied by protein degradation, but, on the contrary, Sendai was stable under alkaline conditions. CD spectral measurements at neutral and alkaline pH indicated no apparent differences between ALP I and Sendai. A significant difference was observed on measurement of fluorescence emission spectra of the tryptophan residues of ALP I that were exposed on the enzyme surface. The fluorescence intensity of ALP I was greatly reduced under alkaline conditions; moreover, the reduction was reversed when alkaline-treated ALP I was neutralized. The fluorescence spectrum of Sendai remained unchanged. The enzymatic and optical activities of NAT were lost at high pH, indicating a lack of functional and structural stability in an alkaline environment. Judging from these results, the alkaline resistance is closely related to the surface structure of the enzyme molecule. PMID:11328588

  18. Site-directed mutagenesis maps interactions that enhance cognate and limit promiscuous catalysis by an alkaline phosphatase superfamily phosphodiesterase.

    PubMed

    Wiersma-Koch, Helen; Sunden, Fanny; Herschlag, Daniel

    2013-12-23

    Catalytic promiscuity, an evolutionary concept, also provides a powerful tool for gaining mechanistic insights into enzymatic reactions. Members of the alkaline phosphatase (AP) superfamily are highly amenable to such investigation, with several members having been shown to exhibit promiscuous activity for the cognate reactions of other superfamily members. Previous work has shown that nucleotide pyrophosphatase/phosphodiesterase (NPP) exhibits a >10⁶-fold preference for the hydrolysis of phosphate diesters over phosphate monoesters, and that the reaction specificity is reduced 10³-fold when the size of the substituent on the transferred phosphoryl group of phosphate diester substrates is reduced to a methyl group. Here we show additional specificity contributions from the binding pocket for this substituent (herein termed the R' substituent) that account for an additional ~250-fold differential specificity with the minimal methyl substituent. Removal of four hydrophobic side chains suggested on the basis of structural inspection to interact favorably with R' substituents decreases phosphate diester reactivity 10⁴-fold with an optimal diester substrate (R' = 5'-deoxythymidine) and 50-fold with a minimal diester substrate (R' = CH₃). These mutations also enhance the enzyme's promiscuous phosphate monoesterase activity by nearly an order of magnitude, an effect that is traced by mutation to the reduction of unfavorable interactions with the two residues closest to the nonbridging phosphoryl oxygen atoms. The quadruple R' pocket mutant exhibits the same activity toward phosphate diester and phosphate monoester substrates that have identical leaving groups, with substantial rate enhancements of ~10¹¹-fold. This observation suggests that the Zn²⁺ bimetallo core of AP superfamily enzymes, which is equipotent in phosphate monoester and diester catalysis, has the potential to become specialized for the hydrolysis of each class of phosphate esters via addition

  19. Calcium phosphate: an alternative calcium compound for dietary prevention of colon cancer? A study on intestinal and faecal parameters in healthy volunteers.

    PubMed

    Cats, A; Mulder, N H; de Vries, E G; Oremus, E T; Kreumer, W M; Kleibeuker, J H

    1993-09-01

    In an effort to reduce the risk of colorectal cancer development, oral calcium carbonate supplementation has been used in previous studies for the precipitation of cytotoxic bile acids and fatty acids. In human intervention trials its effect on mucosal hyperproliferation in the colorectum has not always been satisfactory. Because the complexation of calcium and bile acids requires the formation of calcium phosphate, we performed an intervention study in 14 healthy volunteers, giving them 1,500 mg calcium as Ca3(PO4)2 for 1 week. The effects of tricalcium phosphate on luminal and faecal parameters of cytolytic activity were evaluated before, during, and after calcium phosphate supplementation. The cytolytic activity of faecal water and intestinal alkaline phosphatase activity in faecal water were not affected by supplemental calcium phosphate. In duodenal bile, the proportion of cholic acid tended to increase, whereas that of chenodeoxycholic acid tended to decrease during calcium phosphate supplementation. Neither concentrations of total and individual faecal bile acids, nor that of faecal fat were affected during calcium phosphate supplementation. It is suggested that, although phosphate is involved in bile acid precipitation, phosphate competes for calcium in the binding of fatty acids. This might possibly explain the unchanged cytolytic potency of faecal water, and therefore does not make tricalcium phosphate a suitable calcium compound for dietary intervention. PMID:8401176

  20. A unique alkaline pH-regulated and fatty acid-activated tandem pore domain potassium channel (K₂P) from a marine sponge.

    PubMed

    Wells, Gregory D; Tang, Qiong-Yao; Heler, Robert; Tompkins-MacDonald, Gabrielle J; Pritchard, Erica N; Leys, Sally P; Logothetis, Diomedes E; Boland, Linda M

    2012-07-15

    A cDNA encoding a potassium channel of the two-pore domain family (K(2P), KCNK) of leak channels was cloned from the marine sponge Amphimedon queenslandica. Phylogenetic analysis indicated that AquK(2P) cannot be placed into any of the established functional groups of mammalian K(2P) channels. We used the Xenopus oocyte expression system, a two-electrode voltage clamp and inside-out patch clamp electrophysiology to determine the physiological properties of AquK(2P). In whole cells, non-inactivating, voltage-independent, outwardly rectifying K(+) currents were generated by external application of micromolar concentrations of arachidonic acid (AA; EC(50) ∼30 μmol l(-1)), when applied in an alkaline solution (≥pH 8.0). Prior activation of channels facilitated the pH-regulated, AA-dependent activation of AquK(2P) but external pH changes alone did not activate the channels. Unlike certain mammalian fatty-acid-activated K(2P) channels, the sponge K(2P) channel was not activated by temperature and was insensitive to osmotically induced membrane distortion. In inside-out patch recordings, alkalinization of the internal pH (pK(a) 8.18) activated the AquK(2P) channels independently of AA and also facilitated activation by internally applied AA. The gating of the sponge K(2P) channel suggests that voltage-independent outward rectification and sensitivity to pH and AA are ancient and fundamental properties of animal K(2P) channels. In addition, the membrane potential of some poriferan cells may be dynamically regulated by pH and AA. PMID:22723483

  1. Preparation, characterization, biological activity, and transport study of polystyrene based calcium-barium phosphate composite membrane.

    PubMed

    Khan, Mohammad Mujahid Ali; Rafiuddin

    2013-10-01

    Calcium-barium phosphate (CBP) composite membrane with 25% polystyrene was prepared by co-precipitation method. Scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transformed infrared (FTIR), and Thermogravimetric analysis (TGA) were used to characterize the membrane. The membrane was found to be crystalline in nature with consistent arrangement of particles and no indication of visible cracks. The electrical potentials measured across the composite membrane in contact with univalent electrolytes (KCl, NaCl and LiCl), have been found to increase with decrease in concentrations. Thus the membrane was found to be cation-selective. Transport properties of developed membranes may be utilized for the efficient desalination of saline water and more importantly demineralization process. The antibacterial study of this composite membrane shows good results for killing the disease causing bacteria along with waste water treatment. PMID:23910337

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

    PubMed

    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

  3. Separating and Stabilizing Phosphate from High-Level Radioactive Waste: Process Development and Spectroscopic Monitoring

    SciTech Connect

    Lumetta, Gregg J.; Braley, Jenifer C.; Peterson, James M.; Bryan, Samuel A.; Levitskaia, Tatiana G.

    2012-05-09

    Removing phosphate from alkaline high-level waste sludges at the Department of Energy's Hanford Site in Washington State is necessary to increase the waste loading in the borosilicate glass waste form that will be used to immobilize the highly radioactive fraction of these wastes. We are developing a process which first leaches phosphate from the high-level waste solids with aqueous sodium hydroxide, and then isolates the phosphate by precipitation with calcium oxide. Tests with actual tank waste confirmed that this process is an effective method of phosphate removal from the sludge and offers an additional option for managing the phosphorus in the Hanford tank waste solids. The presence of vibrationally active species, such as nitrate and phosphate ions, in the tank waste processing streams makes the phosphate removal process an ideal candidate for monitoring by Raman or infrared spectroscopic means. As a proof-of-principle demonstration, Raman and Fourier transform infrared (FTIR) spectra were acquired for all phases during a test of the process with actual tank waste. Quantitative determination of phosphate, nitrate, and sulfate in the liquid phases was achieved by Raman spectroscopy, demonstrating the applicability of Raman spectroscopy for the monitoring of these species in the tank waste process streams.

  4. Separating and stabilizing phosphate from high-level radioactive waste: process development and spectroscopic monitoring.

    PubMed

    Lumetta, Gregg J; Braley, Jenifer C; Peterson, James M; Bryan, Samuel A; Levitskaia, Tatiana G

    2012-06-01

    Removing phosphate from alkaline high-level waste sludges at the Department of Energy's Hanford Site in Washington State is necessary to increase the waste loading in the borosilicate glass waste form that will be used to immobilize the highly radioactive fraction of these wastes. We are developing a process which first leaches phosphate from the high-level waste solids with aqueous sodium hydroxide, and then isolates the phosphate by precipitation with calcium oxide. Tests with actual tank waste confirmed that this process is an effective method of phosphate removal from the sludge and offers an additional option for managing the phosphorus in the Hanford tank waste solids. The presence of vibrationally active species, such as nitrate and phosphate ions, in the tank waste processing streams makes the phosphate removal process an ideal candidate for monitoring by Raman or infrared spectroscopic means. As a proof-of-principle demonstration, Raman and Fourier transform infrared (FTIR) spectra were acquired for all phases during a test of the process with actual tank waste. Quantitative determination of phosphate, nitrate, and sulfate in the liquid phases was achieved by Raman spectroscopy, demonstrating the applicability of Raman spectroscopy for the monitoring of these species in the tank waste process streams. PMID:22571620

  5. Biochemical characteristics of an alkaline pectate lyase PelA from Volvariella volvacea: roles of the highly conserved N-glycosylation site in its secretion and activity.

    PubMed

    Shi, Aiqin; Hu, Hang; Zheng, Fei; Long, Liangkun; Ding, Shaojun

    2015-04-01

    Alkaline pectate lyases have great application potential in the bioscouring of textiles. They are isolated predominantly from bacteria and a few fungi. Here, we report the biochemical characteristics of a novel alkaline pectate lyase PelA from the basidiomycete Volvariella volvacea. The full-length pelA encodes a 321-amino-acid polypeptide containing a putative 18-residue signal peptide and a pectate lyase family 1 catalytic domain. It contains one conserved and one non-conserved potential N-glycosylation site (N-X-S/T) at the residues N95 and N198, respectively. The enzyme showed optimal activity at 60 °C and pH 10, although it was stable between pH 4 and pH 11. Additional Ca(2+) was not required to measure PelA activity in vitro, but it could significantly enhance its activity and thermal stability. The V max values using polygalacturonic acid as substrate were increased from 50.71 to 89.96 IU mg(-1) by the addition of 0.1 mM Ca(2+), whereas the K m values were decreased from 0.681 to 0.514 mg ml(-1). Site-directed mutagenesis revealed PelA has only one N-glycan attached to the residue N95. This N-glycan is crucial to its efficient secretion and activity possibly due to its role in maintaining the secondary structure of PelA. Amino acid substitution at the residue N198 had no effect on PelA secretion, but resulted in a slight (5.16 %) to modest (27.37 %) decrease in specific activity and less thermal stability, indicating the amino acid itself is also important for activity due to it being highly conserved and because of its proximity to the catalytic site. PMID:25341402

  6. A method for making an alkaline battery electrode plate

    NASA Technical Reports Server (NTRS)

    Chida, K.; Ezaki, T.

    1983-01-01

    A method is described for making an alkaline battery electrode plate where the desired active substances are filled into a nickel foam substrate. In this substrate an electrolytic oxidation reduction occurs in an alkaline solution containing lithium hydroxide.

  7. Comparative Enzymology in the Alkaline Phosphatase Superfamily to Determine the Catalytic Role of an Active Site Metal Ion

    PubMed Central

    Zalatan, Jesse G.; Fenn, Timothy D.; Herschlag, Daniel

    2009-01-01

    Mechanistic models for biochemical systems are frequently proposed from structural data. Site-directed mutagenesis can be used to test the importance of proposed functional sites, but these data do not necessarily indicate how these sites contribute to function. Herein we apply an alternative approach to the catalytic mechanism of alkaline phosphatase (AP), a widely-studied, prototypical bimetallo enzyme. A third metal ion site in AP has been suggested to provide general base catalysis, but comparison with an evolutionarily-related enzyme casts doubt on this model. Removal of this metal site from AP has large differential effects on reactions of cognate and promiscuous substrates, and the results are inconsistent with general base catalysis. Instead, these and additional results suggest that the third metal ion stabilizes the transferred phosphoryl group in the transition state. These results establish a new mechanistic model for this prototypical bimetallo enzyme and demonstrate the power of a comparative approach for probing biochemical function. PMID:18851975

  8. Adenine derivatives as phosphate-activating groups for the regioselective formation of 3',5'-linked oligoadenylates on montmorillonite: possible phosphate-activating groups for the prebiotic synthesis of RNA

    NASA Technical Reports Server (NTRS)

    Prabahar, K. J.; Ferris, J. P.

    1997-01-01

    Methyladenine and adenine N-phosphoryl derivatives of adenosine 5'-monophosphate (5'-AMP) and uridine 5'-monophosphate (5'-UMP) are synthesized, and their structures are elucidated. The oligomerization reactions of the adenine derivatives of 5'-phosphoramidates of adenosine on montmorillonite are investigated. 1-Methyladenine and 3-methyladenine derivatives on montmorillonite yielded oligoadenylates as long as undecamer, and the 2-methyladenine and adenine derivatives on montmorillonite yielded oligomers up to hexamers and pentamers, respectively. The 1-methyladenine derivative yielded linear, cyclic, and A5'ppA-derived oligonucleotides with a regioselectivity for the 3',5'-phosphodiester linkages averaging 84%. The effect of pKa and amine structure of phosphate-activating groups on the montmorillonite-catalyzed oligomerization of the 5'-phosphoramidate of adenosine are discussed. The binding and reaction of methyladenine and adenine N-phosphoryl derivatives of adenosine are described.

  9. Nicotine inhibits collagen synthesis and alkaline phosphatase activity, but stimulates DNA synthesis in osteoblast-like cells

    SciTech Connect

    Ramp, W.K.; Lenz, L.G.; Galvin, R.J. )

    1991-05-01

    Use of smokeless tobacco is associated with various oral lesions including periodontal damage and alveolar bone loss. This study was performed to test the effects of nicotine on bone-forming cells at concentrations that occur in the saliva of smokeless tobacco users. Confluent cultures of osteoblast-like cells isolated from chick embryo calvariae were incubated for 2 days with nicotine added to the culture medium (25-600 micrograms/ml). Nicotine inhibited alkaline phosphatase in the cell layer and released to the medium, whereas glycolysis (as indexed by lactate production) was unaffected or slightly elevated. The effects on medium and cell layer alkaline phosphatase were concentration dependent with maximal inhibition occurring at 600 micrograms nicotine/ml. Nicotine essentially did not affect the noncollagenous protein content of the cell layer, but did inhibit collagen synthesis (hydroxylation of ({sup 3}H)proline and collagenase-digestible protein) at 100, 300, and 600 micrograms/ml. Release of ({sup 3}H)hydroxyproline to the medium was also decreased in a dose-dependent manner, as was the collagenase-digestible protein for both the medium and cell layer. In contrast, DNA synthesis (incorporation of ({sup 3}H)thymidine) was more than doubled by the alkaloid, whereas total DNA content was slightly inhibited at 600 micrograms/ml, suggesting stimulated cell turnover. Morphologic changes occurred in nicotine-treated cells including rounding up, detachment, and the occurrence of numerous large vacuoles. These results suggest that steps to reduce the salivary concentration of nicotine in smokeless tobacco users might diminish damaging effects of this product on alveolar bone.

  10. Calc-alkaline mafic rocks of the Black Dyke Formation: Remnants of the final activity of a submerged Permian volcano

    SciTech Connect

    Blein, O.; Lapierre, H.; Pecher, A. ); Schweickert, R.A. . Dept. of Geological Science)

    1993-04-01

    The Permian Black Dyke Fm., which occurs as large tectonic slices within the Luning allochthon in the Excelsior Mountains, NV, forms an E-W trending anticline at Black Dyke Mountain. The 800-m thick stratigraphic succession consists of volcanic and pyroclastic rocks overlain conformably by volcaniclastic sediments. Along the northern limb of the anticline, the rocks consist of mafic porphyritic lavas, breccias, and graded and ungraded pyroclastic beds. The sedimentary unit consists of thick volcaniclastic turbidites overlain by conglomerates, sandstones, and mudstones. Along the southern limb of the anticline, the sequence is replaced by reworked breccia, tuffs, and sandstones. Mafic plutonic rocks occur as xenoliths in the lavas and breccias, and as coeval plugs intruding the section. Gabbros show cumulate or porphyritic textures and are composed of amph, cpx, and zoned plag. Their Ti/V (14.5--15) and Nb/Y (0.25--0.3) ratios fall in the range commonly found in calc-alkaline rocks. Diorite porphyry shows high Al[sub 2]O[sub 3], ZrO[sub 2], and REE abundances indicating that this rock is more fractionated. Basalts and andesites are plag-cpx-opx phyric. They often include glomeroporphyritic clots of cpx with amph coronas. Some rocks exhibit fluidal textures. Both volcanic and plutonic rocks show homogeneous geochemical features and similar crystallization sequences: Fe-Ti oxides---->plag---->opx + cpx----> brown zoned hbl, suggesting that they are cogenetic. Thus, the lower part of the Black Dyke Fm. likely represents the final products formed in a calc-alkaline magma chamber because pyroclastic rocks prevail over lava flows and abundant early crystal cumulates occur as plugs or as inclusions in the lavas and breccias.

  11. Flaxseed Protects Against Diabetes-Induced Glucotoxicity by Modulating Pentose Phosphate Pathway and Glutathione-Dependent Enzyme Activities in Rats.

    PubMed

    Gök, Müslüm; Ulusu, Nuray N; Tarhan, Nilay; Tufan, Can; Ozansoy, Gülgün; Arı, Nuray; Karasu, Çimen

    2016-01-01

    This study investigated the effects of flaxseed (Linum usitatissimum L.) intake on general metabolism, pentose phosphate pathway (PPP) and glutathione-dependent enzymes in diabetic rats. Diabetes was induced by streptozotocin injection (40 mg/kg, i.p.) and the enzyme activities were determined spectrophotometrically. Diabetic and control rats were divided in two subgroups, one untreated, and one treated with flaxseed (0.714 g/kg body weight/day; orally) for 12 weeks. Flaxseed ameliorated decreased body weight (p < .05) and increased blood glucose (p < .001), triglyceride (p < .001), ALT (p < .001) and AST (p < .001) in diabetic rats. Diabetes resulted in increased glucose-6-phosphate dehydrogenase (G6PD) (p < .05) and decreased glutathione-S-transferase (GST) (p < .01), but unchanged 6-phosphogluconate dehydrogenase (6PGD) and glutathione reductase (GR) in the brain of rats. These alterations were partially improved by flaxseed in comparison to diabetic untreated group (p < .05). G6PD, 6PGD, GR were elevated (p < .001), while GST unchanged in the lung of diabetic untreated group compared to control. Flaxseed partially prevented the increase in 6PGD (p < .05) and GR (p < .01), but unaffected G6PD in the lung of diabetic rats. G6PD (p < .001), 6PGD (p < .05), GR (p < .001) were augmented, while GST showed a significant (p < .001) depletion in the pancreas of diabetic untreated rats compared to control. Diabetic alterations observed in pancreatic enzyme activities were significantly prevented by flaxseed. Furthermore, a remarkable decrease in 6PGD (p < .001) and an increase in G6PD (threefold of control) were found in the lens of diabetic untreated group that were completely prevented by flaxseed (p < .001). Flaxseed has beneficial effects against diabetes-induced glucotoxicity by modulating G6PD, 6PGD, GR and GST activities in tissues. PMID:26317558

  12. Synthesis of unsaturated phosphatidylinositol 4-phosphates and the effects of substrate unsaturation on SopB phosphatase activity.

    PubMed

    Furse, Samuel; Mak, LokHang; Tate, Edward W; Templer, Richard H; Ces, Oscar; Woscholski, Rüdiger; Gaffney, Piers R J

    2015-02-21

    In this paper evidence is presented that the fatty acid component of an inositide substrate affects the kinetic parameters of the lipid phosphatase Salmonella Outer Protein B (SopB). A succinct route was used to prepare the naturally occurring enantiomer of phosphatidylinositol 4-phosphate (PI-4-P) with saturated, as well as singly, triply and quadruply unsaturated, fatty acid esters, in four stages: (1) The enantiomers of 2,3:5,6-O-dicyclohexylidene-myo-inositol were resolved by crystallisation of their di(acetylmandelate) diastereoisomers. (2) The resulting diol was phosphorylated regio-selectively exclusively on the 1-O using the new reagent tri(2-cyanoethyl)phosphite. (3) With the 4-OH still unprotected, the glyceride was coupled using phosphate tri-ester methodology. (4) A final phosphorylation of the 4-O, followed by global deprotection under basic then acidic conditions, provided PI-4-P bearing a range of sn-1-stearoyl, sn-2-stearoyl, -oleoyl, -γ-linolenoyl and arachidonoyl, glycerides. Enzymological studies showed that the introduction of cis-unsaturated bonds has a measurable influence on the activity (relative Vmax) of SopB. Mono-unsaturated PI-4-P exhibited a five-fold higher activity, with a two-fold higher KM, over the saturated substrate, when presented in DOPC vesicles. Poly-unsaturated PI-4-P showed little further change with respect to the singly unsaturated species. This result, coupled with our previous report that saturated PI-4-P has much higher stored curvature elastic stress than PI, supports the hypothesis that the activity of inositide phosphatase SopB has a physical role in vivo. PMID:25515724

  13. Cloning, characterization, expression and antifungal activity of an alkaline serine protease of Aureobasidium pullulans PL5 involved in the biological control of postharvest pathogens.

    PubMed

    Zhang, Dianpeng; Spadaro, Davide; Valente, Silvia; Garibaldi, Angelo; Gullino, Maria Lodovica

    2012-02-15

    An alkaline protease gene was amplified from genomic DNA and cDNA of the antagonistic yeast-like fungus Aureobasidium pullulans PL5, a biocontrol agent effective against Monilinia laxa on stone fruit and Botrytis cinerea and Penicillium expansum on pome fruits. An open reading frame of 1248 bp encoding a 415-amino acid (aa) protein with a calculated molecular weight (M(r)) of 42.9 kDa and an isoelectric point (pI) of 4.5 was characterized. The cDNAALP5 gene had an 18-amino acid signal peptide, one N-gylcosylation, one histidine active site, and one serine active site. The ALP5 gene with a M(r) of 1351 bp contained two introns. One intron was of 54 bp, while the other was of 50 bp. Protein BLAST and phylogenetic tree analysis of the deduced amino sequences from the cDNAALP5 gene showed that the encoded protein had 100% homology to a protease enzyme (ALP2) of a sea strain of A. pullulans, suggesting that the protein ALP5 was an alkaline serine protease. Expression of ALP5 in Escherichia coli BL21 (DE3), followed by identification with Western-blotting, purification with Ni-NTA and analysis of enzymatic activity, yielded an homogeneous recombinant ALP5 which hydrolysed the substrate casein and inhibited the mycelial growth of the pathogens. At its optimal pH of 10.0 and reaction temperature of 50°C, the recombinant protease exhibited the highest activity towards the substrate casein, though the highest stability was at lower temperatures and pH between 7.0 and 9.0. This study provided the direct evidence that extracellular proteases secreted by the antagonist A. pullulans PL5 played a role in the biocontrol activities against some postharvest pathogens of apple and peach. PMID:22225984

  14. Identification and characterization of a thermostable bifunctional enzyme with phosphomannose isomerase and sugar-1-phosphate nucleotidylyltransferase activities from a hyperthermophilic archaeon, Pyrococcus horikoshii OT3.

    PubMed

    Akutsu, Jun-ichi; Zhang, Zilian; Morita, Rihito; Kawarabayasi, Yutaka

    2015-11-01

    Mannosylglycerate is known as a compatible solute, and plays important roles for salinity adaptation and high temperature stability of microorganisms. In the gene cluster for the mannosylglycerate biosynthetic pathway predicted from the genomic data of Pyrococcus horikoshii OT3, the PH0925 protein was found as a putative bifunctional enzyme with phosphomannose isomerase (PMI) and mannose-1-phosphate guanylyltransferase (Man-1-P GTase) activities, which can synthesize GDP-mannose when accompanied by a phosphomannomutase/phosphoglucomutase (PMM/PGM) enzyme (PH0923). The recombinant PH0925 protein, expressed in E. coli, exhibited both expected PMI and Man-1-P GTase activities, as well as absolute thermostability; 95 °C was the optimum reaction temperature. According to the guanylyltransferase activity (GTase) of the PH0925 protein, it was found that the protein can catalyze glucose-1-phosphate (Glc-1-P) and glucosamine-1-phosphate (GlcN-1-P) in addition to Man-1-P. The analyses of C-terminus-truncated forms of the PH0925 protein indicated that sugar-1-phosphate nucleotidylyltransferase (Sugar-1-P NTase) activity was located in the region from the N-terminus to the 345th residue, and that the C-terminal 114 residue region of the PH0925 protein inhibited the Man-1-P GTase activity. Conversely, the PMI activity was abolished by deletion of the C-terminal 14 residues. This is the first report of a thermostable enzyme with both PMI and multiple Sugar-1-P NTase activities. PMID:26290359

  15. Sex and depot differences in ex vivo adipose tissue fatty acid storage and glycerol-3-phosphate acyltransferase activity

    PubMed Central

    Morgan-Bathke, Maria; Chen, Liang; Oberschneider, Elisabeth; Harteneck, Debra

    2015-01-01

    Adipose tissue fatty acid storage varies according to sex, adipose tissue depot, and degree of fat gain. However, the mechanism(s) for these variations is not completely understood. We examined whether differences in adipose tissue glycerol-3-phosphate acyltransferase (GPAT) might play a role in these variations. We optimized an enzyme activity assay for total GPAT and GPAT1 activity in human adipose tissue and measured GPAT activity. Omental and subcutaneous adipose tissue was collected from obese and nonobese adults for measures of GPAT and GPAT1 activities, ex vivo palmitate storage, acyl-CoA synthetase (ACS) and diacylglycerol-acyltransferase (DGAT) activities, and CD36 protein. Total GPAT and GPAT1 activities decreased as a function of adipocyte size in both omental (r = −0.71, P = 0.003) and subcutaneous (r = −0.58, P = 0.04) fat. The relative contribution of GPAT1 to total GPAT activity increased as a function of adipocyte size, accounting for up to 60% of GPAT activity in those with the largest adipocytes. We found strong, positive correlations between ACS, GPAT, and DGAT activities for both sexes and depots (r values 0.58–0.91) and between these storage factors and palmitate storage rates into TAG (r values 0.55–0.90). We conclude that: 1) total GPAT activity decreases as a function of adipocyte size; 2) GPAT1 can account for over half of adipose GPAT activity in hypertrophic obesity; and 3) ACS, GPAT, and DGAT are coordinately regulated. PMID:25738782

  16. Novel As-doped, As and N-codoped carbon nanotubes as highly active and durable electrocatalysts for O2 reduction in alkaline medium

    NASA Astrophysics Data System (ADS)

    Liu, Ziwu; Li, Meng; Wang, Fang; Wang, Quan-De

    2016-02-01

    To develop more efficient metal-free cathode electrocatalysts for fuel cells, novel arsenic (As)-doped, As and N-codoped carbon nanotubes are synthesized by chemical vapor deposition in this work. The as-prepared As-containing carbon nanotubes exhibit significantly enhanced activity and long-term durability for the oxygen reduction reaction (ORR) in alkaline medium, indicating that the doping of As or codoping As with other heteroatoms into carbon matrix could improve the ORR activity of carbon materials due to the changes in electronic and physical properties of carbon nanotubes evidenced by density functional theory calculations. Moreover, As-containing carbon nanotubes also display much better methanol tolerance, showing a good potential application for future fuel cells.

  17. Sorption of water alkalinity and hardness from high-strength wastewater on bifunctional activated carbon: process optimization, kinetics and equilibrium studies.

    PubMed

    Amosa, Mutiu K

    2016-08-01

    Sorption optimization and mechanism of hardness and alkalinity on bifunctional empty fruit bunch-based powdered activation carbon (PAC) were studied. The PAC possessed both high surface area and ion-exchange properties, and it was utilized in the treatment of biotreated palm oil mill effluent. Batch adsorption experiments designed with Design Expert(®) were conducted in correlating the singular and interactive effects of the three adsorption parameters: PAC dosage, agitation speed and contact time. The sorption trends of the two contaminants were sequentially assessed through a full factorial design with three factor interaction models and a central composite design with polynomial models of quadratic order. Analysis of variance revealed the significant factors on each design response with very high R(2) values indicating good agreement between model and experimental values. The optimum operating conditions of the two contaminants differed due to their different regions of operating interests, thus necessitating the utility of desirability factor to get consolidated optimum operation conditions. The equilibrium data for alkalinity and hardness sorption were better represented by the Langmuir isotherm, while the pseudo-second-order kinetic model described the adsorption rates and behavior better. It was concluded that chemisorption contributed majorly to the adsorption process. PMID:26752149

  18. Characterization of a thermostable alkaline phosphatase from a novel species Thermus yunnanensis sp. nov. and investigation of its cobalt activation at high temperature.

    PubMed

    Gong, Ningping; Chen, Chaoyin; Xie, Liping; Chen, Hongtao; Lin, Xianzhi; Zhang, Rongqing

    2005-06-30

    A thermostable alkaline phosphatase with high specific activity and thermal resistance was purified from a novel species of Thermus sp. named as Thermus yunnanensis sp. nov. The enzyme contains a single peptide with a molecular mass of about 52 kDa on SDS-PAGE analysis and appears to be a homodimer in solution with the molecular mass of 104 kDa. The optimal pH and temperature for its activities are pH 8.0-10.0 and 70-80 degrees C, respectively. The catalytic activities of the enzyme are metal ion dependent, and Mg2+, Zn2+ and Co2+ are the main activators. Among these, Co2+ is the most active stimulator and has unique activation effect at high temperature. Metal binding analysis showed the binding of Mg2+ at the metal binding site was easy to loss in the thermoinactivation, and Co2+ was apt to bind at that site and kept the favorable configuration of catalysis, which would result high activation in the incubation with Co2+ at high temperature. According to this study, a model was proposed for the explanation of the activation and the results of actual experiments demonstrated the validity of the model. PMID:15955749

  19. Characterization of the highly active fragment of glyceraldehyde-3-phosphate dehydrogenase gene promoter for recombinant protein expression in Pleurotus ostreatus.

    PubMed

    Yin, Chaomin; Zheng, Liesheng; Zhu, Jihong; Chen, Liguo; Ma, Aimin

    2015-03-01

    Developing efficient native promoters is important for improving recombinant protein expression by fungal genetic engineering. The promoter region of glyceraldehyde-3-phosphate dehydrogenase gene in Pleurotus ostreatus (Pogpd) was isolated and optimized by upstream truncation. The activities of these promoters with different lengths were further confirmed by fluorescence, quantitative real-time PCR and Western blot analysis. A truncated Pogpd-P2 fragment (795 bp) drove enhanced green fluorescence protein (egfp) gene expression in P. ostreatus much more efficiently than full-length Pogpd-P1. Further truncating Pogpd-P2 to 603, 403 and 231 bp reduced the eGFP expression significantly. However, the 403-bp fragment between -356 bp and the start codon was the minimal but sufficient promoter element for eGFP expression. Compact native promoters for genetic engineering of P. ostreatus were successfully developed and validated in this study. This will broaden the preexisting repertoire of fungal promoters for biotechnology application. PMID:25743073

  20. Synthesis, characterization and antimicrobial activity of the micro/nano structured biogenic silver doped calcium phosphate

    NASA Astrophysics Data System (ADS)

    Supraja, N.; Prasad, T. N. V. K. V.; David, Ernest

    2016-01-01

    Scale formation in PVC pipelines reduces the water flow efficiency and enhances microbial contamination. A bio-based composite material comprising of silver doped calcium phosphate (Cp-Ag) was synthesized using a simple technique (photo catalysis) and herein, we report for the first time on preparation and evaluation of the antimicrobial efficacy of silver doped calcite extracted from the scale in drinking water pipe lines. Five concentrations of silver doped calcite materials viz,5, 10, 15, 20 and 25 ppm were prepared using chemical ammonia mediated synthetic method. The material Cp-Ag was characterized by using the techniques UV-Visible spectroscopy, X-ray diffraction, Fourier transform infrared spectroscopy, Scanning electron microscopy, Raman spectroscopy, Thermo gravimetric analysis, X-ray photo electron spectroscopy (XPS), Nuclear magnetic resonance spectrometer and X-ray flouresence microscopy (XRF). Typical rhombohedral structure of the silver doped calcite was observed. XRF and XPS studies confirmed the presence of both calcium and silver in the composite material (Cp-Ag). The silver doped calcite material exhibited enhanced inhibition against Escherichia coli and staphylococcus aureus (Kirby-Bauer discs diffusion assay) which is also dependent on the concentration of the Cp-Ag material.

  1. Timing of inorganic phosphate release modulates the catalytic activity of ATP-driven rotary motor protein

    NASA Astrophysics Data System (ADS)

    Watanabe, Rikiya; Noji, Hiroyuki

    2014-04-01

    F1-ATPase is a rotary motor protein driven by ATP hydrolysis. The rotary motion of F1-ATPase is tightly coupled to catalysis, in which the catalytic sites strictly obey the reaction sequences at the resolution of elementary reaction steps. This fine coordination of the reaction scheme is thought to be important to achieve extremely high chemomechanical coupling efficiency and reversibility, which is the prominent feature of F1-ATPase among molecular motor proteins. In this study, we intentionally change the reaction scheme by using single-molecule manipulation, and we examine the resulting effect on the rotary motion of F1-ATPase. When the sequence of the products released, that is, ADP and inorganic phosphate, is switched, we find that F1 frequently stops rotating for a long time, which corresponds to inactivation of catalysis. This inactive state presents MgADP inhibition, and thus, we find that an improper reaction sequence of F1-ATPase catalysis induces MgADP inhibition.

  2. Inhibition of lipid phosphate phosphatase activity by VPC32183 suppresses the ability of diacylglycerol pyrophosphate to activate ERK(1/2) MAP kinases.

    PubMed

    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. PMID:22820196

  3. Elevation of Glucose 6-Phosphate Dehydrogenase Activity Induced by Amplified Insulin Response in Low Glutathione Levels in Rat Liver.

    PubMed

    Taniguchi, Misako; Mori, Nobuko; Iramina, Chizuru; Yasutake, Akira

    2016-01-01

    Weanling male Wistar rats were fed on a 10% soybean protein isolate (SPI) diet for 3 weeks with or without supplementing 0.3% sulfur-containing amino acids (SAA; methionine or cystine) to examine relationship between glutathione (GSH) levels and activities of NADPH-producing enzymes, glucose 6-phosphate dehydrogenase (G6PD) and malic enzyme (ME), in the liver. Of rats on the 10% SPI diet, GSH levels were lower and the enzyme activities were higher than of those fed on an SAA-supplemented diet. Despite the lower GSH level, γ-glutamylcysteine synthetase (γ-GCS) activity was higher in the 10% SPI group than other groups. Examination of mRNAs of G6PD and ME suggested that the GSH-suppressing effect on enzyme induction occurred prior to and/or at transcriptional levels. Gel electrophoresis of G6PD indicated that low GSH status caused a decrease in reduced form and an increase in oxidized form of the enzyme, suggesting an accelerated turnover rate of the enzyme. In primary cultured hepatocytes, insulin response to induce G6PD activity was augmented in low GSH levels manipulated in the presence of buthionine sulfoximine. These findings indicated that elevation of the G6PD activity in low GSH levels was caused by amplified insulin response for expression of the enzyme and accelerated turnover rate of the enzyme molecule. PMID:27597985

  4. Elevation of Glucose 6-Phosphate Dehydrogenase Activity Induced by Amplified Insulin Response in Low Glutathione Levels in Rat Liver

    PubMed Central

    Taniguchi, Misako; Mori, Nobuko; Iramina, Chizuru

    2016-01-01

    Weanling male Wistar rats were fed on a 10% soybean protein isolate (SPI) diet for 3 weeks with or without supplementing 0.3% sulfur-containing amino acids (SAA; methionine or cystine) to examine relationship between glutathione (GSH) levels and activities of NADPH-producing enzymes, glucose 6-phosphate dehydrogenase (G6PD) and malic enzyme (ME), in the liver. Of rats on the 10% SPI diet, GSH levels were lower and the enzyme activities were higher than of those fed on an SAA-supplemented diet. Despite the lower GSH level, γ-glutamylcysteine synthetase (γ-GCS) activity was higher in the 10% SPI group than other groups. Examination of mRNAs of G6PD and ME suggested that the GSH-suppressing effect on enzyme induction occurred prior to and/or at transcriptional levels. Gel electrophoresis of G6PD indicated that low GSH status caused a decrease in reduced form and an increase in oxidized form of the enzyme, suggesting an accelerated turnover rate of the enzyme. In primary cultured hepatocytes, insulin response to induce G6PD activity was augmented in low GSH levels manipulated in the presence of buthionine sulfoximine. These findings indicated that elevation of the G6PD activity in low GSH levels was caused by amplified insulin response for expression of the enzyme and accelerated turnover rate of the enzyme molecule. PMID:27597985

  5. Rheb Protein Binds CAD (Carbamoyl-phosphate Synthetase 2, Aspartate Transcarbamoylase, and Dihydroorotase) Protein in a GTP- and Effector Domain-dependent Manner and Influences Its Cellular Localization and Carbamoyl-phosphate Synthetase (CPSase) Activity*

    PubMed Central

    Sato, Tatsuhiro; Akasu, Hitomi; Shimono, Wataru; Matsu, Chisa; Fujiwara, Yuki; Shibagaki, Yoshio; Heard, Jeffrey J.; Tamanoi, Fuyuhiko; Hattori, Seisuke

    2015-01-01

    Rheb small GTPases, which consist of Rheb1 and Rheb2 (also known as RhebL1) in mammalian cells, are unique members of the Ras superfamily and play central roles in regulating protein synthesis and cell growth by activating mTOR. To gain further insight into the function of Rheb, we carried out a search for Rheb-binding proteins and found that Rheb binds to CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), a multifunctional enzyme required for the de novo synthesis of pyrimidine nucleotides. CAD binding is more pronounced with Rheb2 than with Rheb1. Rheb binds CAD in a GTP- and effector domain-dependent manner. The region of CAD where Rheb binds is located at the C-terminal region of the carbamoyl-phosphate synthetase domain and not in the dihydroorotase and aspartate transcarbamoylase domains. Rheb stimulated carbamoyl-phosphate synthetase activity of CAD in vitro. In addition, an elevated level of intracellular UTP pyrimidine nucleotide was observed in Tsc2-deficient cells, which was attenuated by knocking down of Rheb. Immunostaining analysis showed that expression of Rheb leads to increased accumulation of CAD on lysosomes. Both a farnesyltransferase inhibitor that blocks membrane association of Rheb and knockdown of Rheb mislocalized CAD. These results establish CAD as a downstream effector of Rheb and suggest a possible role of Rheb in regulating de novo pyrimidine nucleotide synthesis. PMID:25422319

  6. Production of alkaline protease from Cellulosimicrobium cellulans

    PubMed Central

    Ferracini-Santos, Luciana; Sato, Hélia H

    2009-01-01

    Cellulosimicrobium cellulans is one of the microorganisms that produces a wide variety of yeast cell wall-degrading enzymes, β-1,3-glucanase, protease and chitinase. Dried cells of Saccharomyces cerevisiae were used as carbon and nitrogen source for cell growth and protease production. The medium components KH2PO4, KOH and dried yeast cells showed a significant effect (p<0.05) on the factorial fractional design. A second design was prepared using two factors: pH and percentage of dried yeast cells. The results showed that the culture medium for the maximum production of protease was 0.2 g/l of MgSO4.7H2O, 2.0 g/l of (NH4)2SO4 and 8% of dried yeast cells in 0.15M phosphate buffer at pH 8.0. The maximum alkaline protease production was 7.0 ± 0.27 U/ml over the center point. Crude protease showed best activity at 50ºC and pH 7.0-8.0, and was stable at 50ºC. PMID:24031317

  7. Structural and biochemical characterization of a halophilic archaeal alkaline phosphatase.

    PubMed

    Wende, Andy; Johansson, Patrik; Vollrath, Ronnald; Dyall-Smith, Mike; Oesterhelt, Dieter; Grininger, Martin

    2010-07-01

    Phosphate is an essential component of all cells that must be taken up from the environment. Prokaryotes commonly secrete alkaline phosphatases (APs) to recruit phosphate from organic compounds by hydrolysis. In this study, the AP from Halobacterium salinarum, an archaeon that lives in a saturated salt environment, has been functionally and structurally characterized. The core fold and the active-site architecture of the H. salinarum enzyme are similar to other AP structures. These generally form dimers composed of dominant beta-sheet structures sandwiched by alpha-helices and have well-accessible active sites. The surface of the enzyme is predicted to be highly negatively charged, like other proteins of extreme halophiles. In addition to the conserved core, most APs contain a crown domain that strongly varies within species. In the H. salinarum AP, the crown domain is made of an acyl-carrier-protein-like fold. Different from other APs, it is not involved in dimer formation. We compare the archaeal AP with its bacterial and eukaryotic counterparts, and we focus on the role of crown domains in enhancing protein stability, regulating enzyme function, and guiding phosphoesters into the active-site funnel. PMID:20438737

  8. Effects of phosphate and ADP on shortening velocity during maximal and submaximal calcium activation of the thin filament in skeletal muscle fibers.

    PubMed Central

    Metzger, J M

    1996-01-01

    The effects of added phosphate and MgADP on unloaded shortening velocity during maximal and submaximal Ca2+ activation of the thin filament were examined in skinned single skeletal fibers from rabbit psoas muscle. During maximal Ca2+ activation, added phosphate (10-30 mM) had no effect on unloaded shortening velocity as determined by the slack-test technique. In fibers activated at submaximal concentrations of Ca2+ in the absence of added phosphate, plots of slack length versus duration of unloaded shortening were biphasic, consisting of an initial high velocity phase of shortening and a subsequent low velocity phase of shortening. Interestingly, in the presence of added phosphate, biphasic slack-test plots were no longer apparent. This result was obtained in control fibers over a range of submaximal Ca2+ concentrations and in maximally Ca2+ activated fibers, which were first treated to partially extract troponin C. Thus, under conditions that favor the appearance of biphasic shortening (i.e., low [Ca2+], troponin C extraction), added phosphate eliminated the low velocity component. In contrast, in fibers activated in the presence of 5 mM added MgADP, biphasic slack-test plots were apparent even during maximal Ca2+ activation. The basis of biphasic shortening is not known but it may be due to the formation of axially compressed cross-bridges that become strained to bear a tension that opposes the relative sliding of the myofilaments. The present findings could be explained if added phosphate and MgADP bind to cross-bridges in a strain-dependent manner. In this case, the results suggest that phosphate inhibits the formation of cross-bridges that bear a compressive strain. Added MgADP, on the other hand, may be expected to detain cross-bridges in strong binding states, thus promoting an increase in the population of cross-bridges bearing a compressive strain. Alterations in the population of strained cross-bridges by added phosphate and MgADP would alter the internal

  9. Ribose 5-Phosphate Glycation Reduces Cytochrome c Respiratory Activity and Membrane Affinity‡

    PubMed Central

    Hildick-Smith, Gordon J.; Downey, Michael C.; Gretebeck, Lisa M.; Gersten, Rebecca A.; Sandwick, Roger K.

    2011-01-01

    Spontaneous glycation of bovine heart cytochrome c (cyt c) by the sugar ribose 5-phosphate (R5P) decreases the ability of the heme protein to transfer electrons in the respiratory pathway and to bind to membranes. Trypsin fragmentation studies suggest the preferential sites of glycation include Lys72 and Lys87/88 of a cationic patch involved in the association of the protein with its respiratory chain partners and with cardiolipin-containing membranes. Reaction of bovine cyt c with R5P (50 mM) for 8 h modified the protein in a manner that decreased its ability to transfer electrons to cytochrome oxidase by 60%. An 18 hour treatment with R5P decreased bovine cyt c’s binding affinity with cardiolipin-containing liposomes by an estimated eightfold. A similar lower binding of glycated cyt c was observed with mitoplasts. The reversal of the effects of R5P on membrane binding by ATP further supports an A-site modification. A significant decrease in the rate of spin state change for ferro-cyt c, thought to be due to cardiolipin insertion disrupting the Met coordination to heme, was found for the R5P-treated cyt c. This change occurred to a greater extent than explained by the permanent attachment of the protein onto the liposome. Turbidity changes resulting from the multi-lamellar liposome fusion that is readily promoted by cyt c binding were not seen for the R5P-glycated cyt c samples. Collectively, these results demonstrate the negative impact that R5P glycation can have on critical electron transfer and membrane association functions of cyt c. PMID:22091532

  10. Red light generation through the lead boro-telluro-phosphate glasses activated by Eu3+ ions

    NASA Astrophysics Data System (ADS)

    Selvi, S.; Marimuthu, K.; Suriya Murthy, N.; Muralidharan, G.

    2016-09-01

    Lead boro-telluro-phosphate glasses containing 0.05 to 2.0 wt% of Eu3+ ions were prepared through melt quenching technique. Structural characteristics of title glasses were identified through XRD, FTIR and Raman studies. The optical properties of the prepared glasses were studied using UV-Vis-NIR absorption and photoluminescence spectra. From the resultant spectra, we have obtained the bonding parameters (δ), nephelauxetic ratio (β), direct and indirect band gaps and Urbach energy (ΔE) values. A deep red luminescence due to 5D0 → 7F2 transition of Eu3+ ions could be observed for the title glasses. The local site symmetry around the Eu3+ ions and the degree of Eu3+-O2- covalence were assessed from the luminescence intensity ratio of 5D0 → 7F2/5D0 → 7F1 transitions. Judd-Ofelt intensity parameters, calculated from the luminescence spectra, were used to estimate the radiative parameters like transition probability (A), branching ratio (βexp, βcal) and stimulated emission cross-section (σPE) concerning the 5D0 → 7FJ (J = 0, 1, 2, 3 and 4) transitions. The important laser parameters, gain bandwidth and optical gain are also estimated. The decay curves associated with the transition from 5D0 state was found to be single-exponential at all Eu3+ ion concentrations. CIE colour coordinates and colour purity of the prepared glasses were estimated from the CIE chromaticity diagram.

  11. Enhancing Fluoride Mediated Dentine Sensitivity Relief through Functionalised Tricalcium Phosphate Activity

    PubMed Central

    Naoum, Steven J.; Lenard, Andrea; Martin, F. Elizabeth

    2015-01-01

    Background. To assess the clinical efficacy of a dentifrice containing fluoride and functionalised tricalcium phosphate (fTCP) in reducing dentine sensitivity. Methods. A 10-week parallel blind randomised control trial was conducted. Subjects were assigned to one of four groups and instructed to brush twice daily: A: Colgate Cavity Protection (1000 ppmF-MFP); B: Sensodyne Total Care (1000 ppmF-NaF + 19300 ppmK+-KNO3); C: Clinpro Tooth Crème (950 ppmF-NaF + fTCP); and D: Clinpro Tooth Crème (brushing + additional topical application). Seventy-one patients were assessed at baseline, 6 weeks, and 10 weeks for cold, tactile, and hypertonic sensitivity using the NRS-11 pain rating scale. A combined modalities sensitivity score (CMS) was calculated. Results. At 6 weeks, patients reported the following reduction in CMS: A (20%); B (30%); C (42%); D (52%). At 10 weeks, patients reported the following reduction in CMS: A (18%), B (40%), C (24%), and D (54%). The only CMS comparisons to show a significant difference (P < 0.05) were between Groups A and D (6 and 10 weeks). Conclusions. Addition of fTCP to a dentifrice enhances the ability of dentifrice fluoride in reducing dentine sensitivity. Using Clinpro Tooth Crème twice daily for brushing can be as effective to reduce dentine sensitivity as twice daily brushing using Sensodyne Total Care. However, additional nightly topical application of fTCP, in addition to twice daily brushing, showed an enhanced reduction in dentine sensitivity. PMID:27347553

  12. Matrix Domain Modulates HIV-1 Gag's Nucleic Acid Chaperone Activity via Inositol Phosphate Binding ▿

    PubMed Central

    Jones, Christopher P.; Datta, Siddhartha A. K.; Rein, Alan; Rouzina, Ioulia; Musier-Forsyth, Karin

    2011-01-01

    Retroviruses replicate by reverse transcribing their single-stranded RNA genomes into double-stranded DNA using specific cellular tRNAs to prime cDNA synthesis. In HIV-1, human tRNA3Lys serves as the primer and is packaged into virions during assembly. The viral Gag protein is believed to chaperone tRNA3Lys placement onto the genomic RNA primer binding site; however, the timing and possible regulation of this event are currently unknown. Composed of the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 domains, the multifunctional HIV-1 Gag polyprotein orchestrates the highly coordinated process of virion assembly, but the contribution of these domains to tRNA3Lys annealing is unclear. Here, we show that NC is absolutely essential for annealing and that the MA domain inhibits Gag's tRNA annealing capability. During assembly, MA specifically interacts with inositol phosphate (IP)-containing lipids in the plasma membrane (PM). Surprisingly, we find that IPs stimulate Gag-facilitated tRNA annealing but do not stimulate annealing in Gag variants lacking the MA domain or containing point mutations involved in PM binding. Moreover, we find that IPs prevent MA from binding to nucleic acids but have little effect on NC or Gag. We propose that Gag binds to RNA either with both NC and MA domains or with NC alone and that MA-IP interactions alter Gag's binding mode. We propose that MA's interactions with the PM trigger the switch between these two binding modes and stimulate Gag's chaperone function, which may be important for the regulation of events such as tRNA primer annealing. PMID:21123373

  13. 2C-Methyl-d-erythritol 4-phosphate enhances and sustains cyclodiphosphate synthase IspF activity

    PubMed Central

    Bitok, J. Kipchirchir; Freel Meyers, Caren

    2012-01-01

    There is significant progress toward understanding catalysis throughout the essential MEP pathway to isoprenoids in human pathogens; however, little is known about pathway regulation. The present study begins by testing the hypothesis that isoprenoid biosynthesis is regulated via feedback inhibition of the fifth enzyme cyclodiphosphate IspF by downstream isoprenoid diphosphates. Here, we demonstrate recombinant E. coli IspF is not inhibited by downstream metabolites and isopentenyl diphosphate (IDP), dimethylallyl diphosphate (DMADP), geranyl diphosphate (GDP) and farnesyl diphosphate (FDP) under standard assay conditions. However, 2C-methyl-d-erythritol 4-phosphate (MEP), the product of reductoisomerase IspC and first committed MEP pathway intermediate, activates and sustains this enhanced IspF activity, and the IspF-MEP complex is inhibited by FDP. We further show that the methylerythritol scaffold itself, which is unique to this pathway, drives the activation and stabilization of active IspF. Our results suggest a novel feed-forward regulatory mechanism for 2Cmethyl-d-erythritol 2,4-cyclodiphosphate (MEcDP) production and support an isoprenoid biosynthesis regulatory mechanism via feedback inhibition of the IspF-MEP complex by FDP. The results have important implications for development of inhibitors against the IspF-MEP complex, which may be the physiologically relevant form of the enzyme. PMID:22839733

  14. Effect of feeding and of DDT on the activity of hepatic glucose 6- phosphate dehydrogenase in two salmonids

    USGS Publications Warehouse

    Buhler, Donald R.; Benville, P.

    1969-01-01

    The specific activity of liver glucose 6-phosphate dehydrogenase in yearling rainbow trout remained unchanged when the fish were starved for periods as long as 8 weeks and when starved animals were fed diets of various compositions. Injection of insulin concurrently with refeeding also failed to alter the specific activity of the enzyme in trout. The absence of a dietary or insulin influence on the teleost enzyme system is to be contrasted with studies in mammals in which the activity of hepatic glucose 6-P dehydrogenase was markedly stimulated after refeeding starved animals or injection of insulin.Ingestion of the pesticide DDT by juvenile coho salmon or adult rainbow trout also had no effect on the specific activity of liver glucose 6-P dehydrogenase and DDT failed to inhibit the rainbow trout enzyme in vitro. These results also differ considerably from those found in higher animals.These results suggest that the glucose 6-P dehydrogenase enzyme in teleosts may be under a different type of regulatory control from that found in mammals.

  15. 2C-Methyl-d-erythritol 4-phosphate enhances and sustains cyclodiphosphate synthase IspF activity.

    PubMed

    Bitok, J Kipchirchir; Meyers, Caren Freel

    2012-10-19

    There is significant progress toward understanding catalysis throughout the essential MEP pathway to isoprenoids in human pathogens; however, little is known about pathway regulation. The present study begins by testing the hypothesis that isoprenoid biosynthesis is regulated via feedback inhibition of the fifth enzyme cyclodiphosphate synthase IspF by downstream isoprenoid diphosphates. Here, we demonstrate recombinant E. coli IspF is not inhibited by downstream metabolites isopentenyl diphosphate (IDP), dimethylallyl diphosphate (DMADP), geranyl diphosphate (GDP), and farnesyl diphosphate (FDP) under standard assay conditions. However, 2C-methyl-d-erythritol 4-phosphate (MEP), the product of reductoisomerase IspC and first committed MEP pathway intermediate, activates and sustains this enhanced IspF activity, and the IspF-MEP complex is inhibited by FDP. We further show that the methylerythritol scaffold itself, which is unique to this pathway, drives the activation and stabilization of active IspF. Our results suggest a novel feed-forward regulatory mechanism for 2C-methyl-d-erythritol 2,4-cyclodiphosphate (MEcDP) production and support an isoprenoid biosynthesis regulatory mechanism via feedback inhibition of the IspF-MEP complex by FDP. The results have important implications for development of inhibitors against the IspF-MEP complex, which may be the physiologically relevant form of the enzyme. PMID:22839733

  16. The oxygen isotope composition of phosphate released from phytic acid by the activity of wheat and Aspergillus niger phytase

    NASA Astrophysics Data System (ADS)

    Sperber, C. v.; Tamburini, F.; Brunner, B.; Bernasconi, S. M.; Frossard, E.

    2015-03-01

    Phosphorus (P) is an essential nutrient for living organisms. Under P-limiting conditions plants and microorganisms can exude extracellular phosphatases that release inorganic phosphate (Pi) from organic phosphorus compounds (Porg). Phytic acid (IP6) is an important form of Porg in many soils. The enzymatic hydrolysis of IP6 by phytase yields plant available inorganic phosphate (Pi) and less phosphorylated inositol derivates as products. The hydrolysis of organic P-compounds by phosphatases leaves an isotopic imprint on the oxygen isotope composition (δ18O) of released Pi, which might be used to trace P in the environment. This study aims at determining the effect of phytase on the oxygen isotope composition of released Pi. For this purpose, enzymatic assays with histidine acid phytases from wheat and Aspergillus niger were prepared using IP6, adenosine 5'monophosphate (AMP) and glycerophosphate (GPO4) as substrates. For a comparison to the δ18O of Pi released by other extracellular enzymes, enzymatic assays with acid phosphatases from potato and wheat germ with IP6 as substrate were prepared. During the hydrolysis of IP6 by phytase, four Pi are released, and one oxygen atom from water is incorporated into each Pi. This incorporation of oxygen from water into Pi is subject to an apparent inverse isotopic fractionation (ϵ ∼ 6 to 10‰), which is similar to that imparted by acid phosphatase from potato during the hydrolysis of IP6 (ϵ ∼ 7‰) where less than three Pi are released. The incorporation of oxygen from water into Pi during the hydrolysis of AMP and GPO4 by phytase yielded a normal isotopic fractionation (ϵ ∼ -12‰), again similar to values reported for acid phosphatases from potato and wheat germ. We attribute this similarity in ɛ to the same amino acid sequence motif (RHGXRXP) at the active site of these enzymes, which leads to similar reaction mechanisms. We suggest that the striking substrate-dependency of

  17. BIOLOGICAL ELECTRODES SPECIFIC FOR PHOSPHATE AND NITRITE IONS

    EPA Science Inventory

    Biological electrodes have been developed for the selective analysis of phosphate and nitrate ions at micro-levels. A phosphate electrode was constructed by using two enzymes, alkaline phosphatase and glucose oxidase which were immobilized and mounted on the surface of the Platin...

  18. Novel mode of inhibition by D-tagatose 6-phosphate through a Heyns rearrangement in the active site of transaldolase B variants.

    PubMed

    Stellmacher, Lena; Sandalova, Tatyana; Schneider, Sarah; Schneider, Gunter; Sprenger, Georg A; Samland, Anne K

    2016-04-01

    Transaldolase B (TalB) and D-fructose-6-phosphate aldolase A (FSAA) from Escherichia coli are C-C bond-forming enzymes. Using kinetic inhibition studies and mass spectrometry, it is shown that enzyme variants of FSAA and TalB that exhibit D-fructose-6-phosphate aldolase activity are inhibited covalently and irreversibly by D-tagatose 6-phosphate (D-T6P), whereas no inhibition was observed for wild-type transaldolase B from E. coli. The crystal structure of the variant TalB(F178Y) with bound sugar phosphate was solved to a resolution of 1.46 Å and revealed a novel mode of covalent inhibition. The sugar is bound covalently via its C2 atom to the ℇ-NH2 group of the active-site residue Lys132. It is neither bound in the open-chain form nor as the closed-ring form of D-T6P, but has been converted to β-D-galactofuranose 6-phosphate (D-G6P), a five-membered ring structure. The furanose ring of the covalent adduct is formed via a Heyns rearrangement and subsequent hemiacetal formation. This reaction is facilitated by Tyr178, which is proposed to act as acid-base catalyst. The crystal structure of the inhibitor complex is compared with the structure of the Schiff-base intermediate of TalB(E96Q) formed with the substrate D-fructose 6-phosphate determined to a resolution of 2.20 Å. This comparison highlights the differences in stereochemistry at the C4 atom of the ligand as an essential determinant for the formation of the inhibitor adduct in the active site of the enzyme. PMID:27050126

  19. Variation of alkaline phosphatase activity in sediments of shrimp culture ponds and its relationship with the contents of C, N and P

    NASA Astrophysics Data System (ADS)

    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 5.407nmol g-1min-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.

  20. Improvement of anaerobic digestion of waste-activated sludge by using H₂O₂ oxidation, electrolysis, electro-oxidation and thermo-alkaline pretreatments.

    PubMed

    Feki, Emna; Khoufi, Sonia; Loukil, Slim; Sayadi, Sami

    2015-10-01

    Disintegration of municipal waste-activated sludge (WAS) is regarded as a prerequisite of the anaerobic digestion process to reduce sludge volume and improve biogas yield. Pretreatment of WAS using thermo-alkaline (TA), H2O2 oxidation, electrolysis and electro-oxidation (EO) processes were investigated and compared in term of COD solubilization and biogas production. For each pretreatment, the influences of different operational variables were studied in detail. At optimum conditions, EO gave the maximum COD solubilization (28 %). The effects of pretreatments under the optimum conditions on anaerobic digestion were experienced with biochemical methane potential assay. Significant increases in biogas yield up to 78 and 40 % were observed respectively in the EO and TA pretreated samples compared to raw sludge. Results clearly revealed that the application of EO is a significant alternative method for the improvement of WAS anaerobic digestion. PMID:25982985

  1. Stimulatory effect of 1,25-dihydroxycholecalciferol-like substances from Solanum malacoxylon and Cestrum diurnum on phosphate transport in chick jejunum.

    PubMed

    Peterlik, M; Wasserman, R H

    1978-10-01

    Extracts of the calcinogenic plants Solanum malocoxylon and Cestrum diurnum stimulate phosphate absorption by the jejunum of vitamin D-deficient chicks, as determined by everted gut sac technique. Their action on cellular pathways of transepithelial phosphate transport is indistinguishable thereby from that of cholecalciferol. Increased net absorption from the lumen was due to enhanced uptake of phosphate from the luminal side, while leakage of tissue phosphate in the opposite direction was apparently unaffected. Steep serosa/mucosa concentration gradients were observed as consequence of enhanced levels of transepithelial phosphate flux in the mucosa-to-serosa direction. With respect to their stimulatory action on phosphate absorption, the calcinogenic plant factors retained their biological activity when phosphate transport was depressed by a high strontium diet. Their action in overcoming the strontium inhibition of phosphate absorption, calcium-binding protein synthesis, and alkaline phosphatase activity, was comparable to the effect of 1,25-dihydroxycholecalciferol. On the basis of these biological responses, the action of the plant factors from Solanum malacoxylon and Cestrum diurnum provides further evidence for their close resemblance to the hormonally active sterol. PMID:702209

  2. Structure-guided redesign of D-fructose-6-phosphate aldolase from E. coli: remarkable activity and selectivity towards acceptor substrates by two-point mutation.

    PubMed

    Gutierrez, Mariana; Parella, Teodor; Joglar, Jesús; Bujons, Jordi; Clapés, Pere

    2011-05-28

    Structure-guided re-design of the acceptor binding site of D-fructose-6-phosphate aldolase from E. coli leads to the construction of FSA A129S/A165G double mutant with an activity between 5- to >900-fold higher than that of wild-type towards N-Cbz-aminoaldehyde derivatives. PMID:21499643

  3. Polyphenolic compounds from flowers of Hibiscus rosa-sinensis Linn. and their inhibitory effect on alkaline phosphatase enzyme activity in vitro.

    PubMed

    Salib, Josline Y; Daniel, Enas N; Hifnawy, Mohamed S; Azzam, Shadia M; Shaheed, Iman B; Abdel-Latif, Sally M

    2011-01-01

    Graded concentrations (0.1-100 mg/mL reaction mixture) of the methanolic extract of the flowers of Hibiscus rosa-sinensis Linn., its water-soluble fraction as well as compounds isolated from this fraction were tested for their inhibitory effect on alkaline phosphatase enzyme activity in vitro. Both the methanolic extract and its water-soluble fraction showed significant inhibitory effects on the enzyme activity in vitro. On screening the activity of the compounds isolated from the water-soluble fraction, its high inhibitory activity was attributed to the presence of quercetin-7-O-galactoside which showed a high potent inhibition of the enzyme activity reaching 100% at 100 mg/mL reaction mixture. Phytochemical investigations of the water-soluble fraction were also carried out and afforded ten polyphenolic compounds including two new natural compounds, namely kaempferol-7-O-[6'''-O-p-hydroxybenzoyl-beta-D-glucosyl-(1-->6)-beta-D-glucopyranoside] and scutellarein-6-O-alpha-L-rhamnopyranoside-8-C-beta-D-glucopyranoside). The chemical structure of the isolated compounds was elucidated on the basis of chemical and spectral data. PMID:22191209

  4. Proteomics demonstration that histone H4 is a colchicine-induced retro-modulator of growth and alkaline phosphatase activity in hair follicle dermal papilla culture.

    PubMed

    Hsia, Ching-Wu; Shui, Hao-Ai; Wang, Chih-Yuan; Yu, Hui-Ming; Ho, Ming-Yi; Cheng, Kur-Ta; Tseng, Min-Jen

    2011-05-16

    Dermal papilla cells (DPCs) control the development of hair follicles via cell-cell interactions and extracellular molecules. Colchicine affected active anagen DPCs to result in hair loss in the clinical setting. The purpose of this study was to identify the retro-modulator released by DPCs exposed to sub-toxic dose of colchicine and elucidate its effect on dermal papilla culture. The molecular-weight cutoff ultrafiltration and HPLC were used to purify the components of colchicine-treated DPC secretomes and examined their ability to down-regulate the growth and alkaline phosphatase (ALP) activity of DPCs. The active product was identified by in-gel trypsin digestion, nano-LC-ESI-MS/MS and validated by Western blot to be histone H4 (P62804), which inhibited the proliferation and diminished the ALP activity of cultured DPCs. Treating DPCs with recombinant histone H4 reproduced the growth inhibition effect whereas adding antibody to immunoneutralize histone H4 abolished this growth inhibitory consequence. DPCs with high ALP activity can induce the neogenesis of hair follicles and support the hair fiber growth in vivo. Our results indicated that sub-lethal colchicine can inactivate DPCs through releasing histone H4. Through the investigation of the retro-modulation of histone H4 on dermal papillae may give implications for understanding the mechanism of colchicine-induced hair disorder. PMID:21362507

  5. Effects of dietary vitamin E on mucosal maltase and alkaline phosphatase enzyme activities and on the amount of mucosal malonyldialdehyde in broiler chickens

    PubMed Central

    Farrokhifar, Seyed Hamid; Ali Jafari, Ramezan; Erfani Majd, Naeem; Fatemi Tabatabaee, Seyed Reza; Mayahi, Mansour

    2013-01-01

    The effects of dietary vitamin E levels on mucosal maltase and alkaline phosphatase (ALP) enzyme activities and on the amount of mucosal malonyldialdehyde (MDA) in broiler chickens were studied in the present study. One hundred and eighty of male day old broiler chicks (Ross 308 strain) were randomly assigned into five groups, each with three replicates and 12 chicks in each replicate. Chickens in group A were fed corn-soy- based diet, while those in groups B, C, D and E were fed the same diet with 20, 60, 180, and 540 mg kg-1 vitamin E supplement (d-alpha tocopherol), respectively. Six birds were randomly chosen from each group, and were euthanized on days 10, 21, 32, and 42 of age. One segment of small intestine outset was homogenized and mucosal ALP and maltase activity were measured. Moreover, mucosal lipid peroxidate amount was measured to reveal the impact of vitamin E on oxidative stress. Maltase activity was increased with the increase of vitamin E up to 60 mg kg-1 of diet while with further levels, it was decreased. Addition of 60 mg kg-1 of vitamin E to the diet significantly increased ALP enzyme activity (p ≤ 0.001). Addition of 540 mg kg-1 of vitamin E supplement to the diet led to the minimum amount of MDA at 32 days of age. It may be concluded that supplementation of broiler's diet with 60 mg kg-1 of vitamin E can increase mucosal maltase and ALP enzyme activity. PMID:25568675

  6. Phosphate control in dialysis

    PubMed Central

    Cupisti, Adamasco; Gallieni, Maurizio; Rizzo, Maria Antonietta; Caria, Stefania; Meola, Mario; Bolasco, Piergiorgio

    2013-01-01

    Prevention and correction of hyperphosphatemia is a major goal of chronic kidney disease–mineral and bone disorder (CKD–MBD) management, achievable through avoidance of a positive phosphate balance. To this aim, optimal dialysis removal, careful use of phosphate binders, and dietary phosphate control are needed to optimize the control of phosphate balance in well-nourished patients on a standard three-times-a-week hemodialysis schedule. Using a mixed diffusive–convective hemodialysis tecniques, and increasing the number and/or the duration of dialysis tecniques are all measures able to enhance phosphorus (P) mass removal through dialysis. However, dialytic removal does not equal the high P intake linked to the high dietary protein requirement of dialysis patients; hence, the use of intestinal P binders is mandatory to reduce P net intestinal absorption. Unfortunately, even a large dose of P binders is able to bind approximately 200–300 mg of P on a daily basis, so it is evident that their efficacy is limited in the case of an uncontrolled dietary P load. Hence, limitation of dietary P intake is needed to reach the goal of neutral phosphate balance in dialysis, coupled to an adequate protein intake. To this aim, patients should be informed and educated to avoid foods that are naturally rich in phosphate and also processed food with P-containing preservatives. In addition, patients should preferentially choose food with a low P-to-protein ratio. For example, patients could choose egg white or protein from a vegetable source. Finally, boiling should be the preferred cooking procedure, because it induces food demineralization, including phosphate loss. The integrated approach outlined in this article should be actively adapted as a therapeutic alliance by clinicians, dieticians, and patients for an effective control of phosphate balance in dialysis patients. PMID:24133374

  7. Activity of the response regulator CiaR in mutants of Streptococcus pneumoniae R6 altered in acetyl phosphate production

    PubMed Central

    Marx, Patrick; Meiers, Marina; Brückner, Reinhold

    2015-01-01

    The two-component regulatory system (TCS) CiaRH of Streptococcus pneumoniae is implicated in competence, ß-lactam resistance, maintenance of cell integrity, bacteriocin production, host colonization, and virulence. Depending on the growth conditions, CiaR can be highly active in the absence of its cognate kinase CiaH, although phosphorylation of CiaR is required for DNA binding and gene regulation. To test the possibility that acetyl phosphate (AcP) could be the alternative phosphodonor, genes involved in pyruvate metabolism were disrupted to alter cellular levels of acetyl phosphate. Inactivating the genes of pyruvate oxidase SpxB, phosphotransacetylase Pta, and acetate kinase AckA, resulted in very low AcP levels and in strongly reduced CiaR-mediated gene expression in CiaH-deficient strains. Therefore, alternative phosphorylation of CiaR appears to proceed via AcP. The AcP effect on CiaR is not detected in strains with CiaH. Attempts to obtain elevated AcP by preventing its degradation by acetate kinase AckA, were not successful in CiaH-deficient strains with a functional SpxB, the most important enzyme for AcP production in S. pneumoniae. The ciaH-spxB-ackA mutant producing intermediate amounts of AcP could be constructed and showed a promoter activation, which was much higher than expected. Since activation was dependent on AcP, it can apparently be used more efficiently for CiaR phosphorylation in the absence of AckA. Therefore, high AcP levels in the absence of CiaH and AckA may cause extreme overexpression of the CiaR regulon leading to synthetic lethality. AckA is also involved in a regulatory response, which is mediated by CiaH. Addition of acetate to the growth medium switch CiaH from kinase to phosphatase. This switch is lost in the absence of AckA indicating metabolism of acetate is required, which starts with the production of AcP by AckA. Therefore, AckA plays a special regulatory role in the control of the CiaRH TCS. PMID:25642214

  8. Glucose-6-phosphate isomerase.

    PubMed

    Achari, A; Marshall, S E; Muirhead, H; Palmieri, R H; Noltmann, E A

    1981-06-26

    Glucose-6-phosphate isomerase (EC 5.3.1.9) is a dimeric enzyme of molecular mass 132000 which catalyses the interconversion of D-glucose-6-phosphate and D-fructose-6-phosphate. The crystal structure of the enzyme from pig muscle has been determined at a nominal resolution of 2.6 A. The structure is of the alpha/beta type. Each subunit consists of two domains and the active site is in both the domain interface and the subunit interface (P.J. Shaw & H. Muirhead (1976), FEBS Lett. 65, 50-55). Each subunit contains 13 methionine residues so that cyanogen bromide cleavage will produce 14 fragments, most of which have been identified and at least partly purified. Sequence information is given for about one-third of the molecule from 5 cyanogen bromide fragments. One of the sequences includes a modified lysine residue. Modification of this residue leads to a parallel loss of enzymatic activity. A tentative fit of two of the peptides to the electron density map has been made. It seems possible that glucose-6-phosphate isomerase, triose phosphate isomerase and pyruvate kinase all contain a histidine and a glutamate residue at the active site. PMID:6115414

  9. Active lithium replenishment to extend the life of a cell employing carbon and iron phosphate electrodes

    NASA Astrophysics Data System (ADS)

    Wang, John; Soukiazian, Souren; Verbrugge, Mark; Tataria, Harshad; Coates, Dwaine; Hall, David; Liu, Ping

    We describe and implement a method of extending the life of a LiFePO 4/graphite lithium ion battery by replenishing the lost active lithium during cell operation and concomitant capacity fade. The approach may prove helpful in terms of increasing lithium ion cell life. After the cell had lost 30% of its capacity, analysis showed that the cell had not experienced significant impedance increase or cathode capacity loss, and the anode had lost about 5% of its storage capacity. The analysis confirmed that the loss of active lithium greatly outpaced the loss of capacity for either electrode and is responsible for cell capacity decay. The cathode was then discharged against an external lithium electrode to increase the amount of active lithium within the cell. About half of the lost capacity was recovered, and the cell cycled for 1500 more cycles. Active lithium replenishment from a reserve electrode may be an effective method of extending the life of lithium ion batteries.

  10. Alkaline "Permanent" Paper.

    ERIC Educational Resources Information Center

    Pacey, Antony

    1991-01-01

    Discussion of paper manufacturing processes and their effects on library materials focuses on the promotion of alkaline "permanent" paper, with less acid, by Canadian library preservation specialists. Standards for paper acidity are explained; advantages of alkaline paper are described, including decreased manufacturing costs; and recyclability is…

  11. Anodes for alkaline electrolysis

    DOEpatents

    Soloveichik, Grigorii Lev

    2011-02-01

    A method of making an anode for alkaline electrolysis cells includes adsorption of precursor material on a carbonaceous material, conversion of the precursor material to hydroxide form and conversion of precursor material from hydroxide form to oxy-hydroxide form within the alkaline electrolysis cell.

  12. Sucrose phosphate synthase activity rises in correlation with high-rate cellulose synthesis in three heterotrophic systems.

    PubMed

    Babb, V M; Haigler, C H

    2001-11-01

    Based on work with cotton fibers, a particulate form of sucrose (Suc) synthase was proposed to support secondary wall cellulose synthesis by degrading Suc to fructose and UDP-glucose. The model proposed that UDP-glucose was then channeled to cellulose synthase in the plasma membrane, and it implies that Suc availability in cellulose sink cells would affect the rate of cellulose synthesis. Therefore, if cellulose sink cells could synthesize Suc and/or had the capacity to recycle the fructose released by Suc synthase back to Suc, cellulose synthesis might be supported. The capacity of cellulose sink cells to synthesize Suc was tested by analyzing the Suc phosphate synthase (SPS) activity of three heterotrophic systems with cellulose-rich secondary walls. SPS is a primary regulator of the Suc synthesis rate in leaves and some Suc-storing, heterotrophic organs, but its activity has not been previously correlated with cellulose synthesis. Two systems analyzed, cultured mesophyll cells of Zinnia elegans L. var. Envy and etiolated hypocotyls of kidney beans (Phaseolus vulgaris), contained differentiating tracheary elements. Cotton (Gossypium hirsutum L. cv Acala SJ-1) fibers were also analyzed during primary and secondary wall synthesis. SPS activity rose in all three systems during periods of maximum cellulose deposition within secondary walls. The Z. elegans culture system was manipulated to establish a tight linkage between the timing of tracheary element differentiation and rising SPS activity and to show that SPS activity did not depend on the availability of starch for degradation. The significance of these findings in regard to directing metabolic flux toward cellulose will be discussed. PMID:11706202

  13. The level of glucose-6-phosphate dehydrogenase activity strongly influences xylose fermentation and inhibitor sensitivity in recombinant Saccharomyces cerevisiae strains.

    PubMed

    Jeppsson, Marie; Johansson, Björn; Jensen, Peter Ruhdal; Hahn-Hägerdal, Bärbel; Gorwa-Grauslund, Marie F

    2003-11-01

    Disruption of the ZWF1 gene encoding glucose-6-phosphate dehydrogenase (G6PDH) has been shown to reduce the xylitol yield and the xylose consumption in the xylose-utilizing recombinant Saccharomyces cerevisiae strain TMB3255. In the present investigation we have studied the influence of different production levels of G6PDH on xylose fermentation. We used a synthetic promoter library and the copper-regulated CUP1 promoter to generate G6PDH-activities between 0% and 179% of the wild-type level. G6PDH-activities of 1% and 6% of the wild-type level resulted in 2.8- and 5.1-fold increase in specific xylose consumption, respectively, compared with the ZWF1-disrupted strain. Both strains exhibited decreased xylitol yields (0.13 and 0.19 g/g xylose) and enhanced ethanol yields (0.36 and 0.34 g/g xylose) compared with the control strain TMB3001 (0.29 g xylitol/g xylose, 0.31 g ethanol/g xylose). Cytoplasmic transhydrogenase (TH) from Azotobacter vinelandii has previously been shown to transfer NADPH and NAD(+) into NADP(+) and NADH, and TH-overproduction resulted in lower xylitol yield and enhanced glycerol yield during xylose utilization. Strains with low G6PDH-activity grew slower in a lignocellulose hydrolysate than the strain with wild-type G6PDH-activity, which suggested that the availability of intracellular NADPH correlated with tolerance towards lignocellulose-derived inhibitors. Low G6PDH-activity strains were also more sensitive to H(2)O(2) than the control strain TMB3001. PMID:14618564

  14. Electrocatalysis of carbon black- or poly(diallyldimethylammonium chloride)-functionalized activated carbon nanotubes-supported Pd-Tb towards methanol oxidation in alkaline media

    NASA Astrophysics Data System (ADS)

    Wang, Li; Wang, Yi; Li, An; Yang, Yunshang; Tang, Qinghu; Cao, Hongbin; Qi, Tao; Li, Changming

    2014-07-01

    The Pd-Tb/C catalysts with different Pd/Tb ratios were synthesized by a simple simultaneous reduction reaction with sodium borohydride in aqueous solution. The structure and morphology of those catalysts had been characterized by X-ray diffraction (XRD) and transmission electron microscopy (TEM). The electrocatalytic performance of those catalysts for methanol oxidation in alkaline media was investigated using cyclic voltammetry (CV), linear sweep voltammetry (LSV) and CO stripping experiments. It is found that the 20%Pd-1%Tb/C catalyst has a higher catalytic activity than the 20%Pd/C catalyst, but the effect of Tb cannot be explained by a bi-functional mechanism. According to the X-Ray photoelectron spectroscopy (XPS) analyses, it is suggested that the higher content of metallic Pd caused by the addition of Tb contributes to the better catalytic activity of 20%Pd-1%Tb/C. Based on the good electrocatalytic performance of 20%Pd-1%Tb/C, the 20%Pd-1%Tb catalyst supported on poly(diallyldimethylammonium chloride) (PDDA)-functionalized activated carbon nanotubes was prepared, and it exhibits a better catalytic activity. The improvement mainly results from the further increase of metallic Pd due to the presence of PDDA.

  15. Effect of calcination temperature on the activity of solid Ca/Al composite oxide-based alkaline catalyst for biodiesel production.

    PubMed

    Meng, Yong-Lu; Wang, Bo-Yang; Li, Shu-Fen; Tian, Song-Jiang; Zhang, Min-Hua

    2013-01-01

    A solid Ca/Al composite oxide-based alkaline catalyst containing Ca(12)Al(14)O(33) and CaO was prepared by chemical synthesis and thermal activation from sodium aluminate solution and calcium hydroxide emulsion. The effect of calcination temperatures ranging from 120 °C to 1000 °C on activity of the catalyst was investigated. The catalyst calcined at 600 °C showed the highest activity with >94% yield of fatty acid methyl esters (i.e. biodiesel) when applied to the transesterification of rapeseed oil at a methanol:oil molar ratio of 15:1 at 65 °C for 3h. Structure and properties of the catalyst were studied and the characterizations with XRD, TGA, FTIR, BET, and SEM demonstrated that the performance of the catalyst was closely related to its specific surface area and crystalline structure. In particular, the generation of crystalline Ca(12)Al(14)O(33) improved the catalytic activity due its synergistic effect with CaO. PMID:23196252

  16. Novel one-step synthesis of wool-ball-like Ni-carbon nanotubes composite cathodes with favorable electrocatalytic activity for hydrogen evolution reaction in alkaline solution

    NASA Astrophysics Data System (ADS)

    Chen, Zhouhao; Ma, Zhipeng; Song, Jianjun; Wang, Lixin; Shao, Guangjie

    2016-08-01

    In this work, supergravity fields are performed to prepare Ni-CNTs composite cathodes with wool-ball-like morphology from the Watts bath containing well-distributed functionalized CNTs. The prepared Ni-CNTs composite cathodes are used as noble metal-free electrocatalyst with favorable electrocatalytic activity for hydrogen evolution reaction (HER) in alkaline solutions. The crystal structure and morphology of the composite cathodes are characterized by XRD and SEM measurements. The electrochemical activities of the cathodes are characterized through Tafel polarization measurement, electrochemical impedance spectroscopy and cyclic voltammetric study in 1.0 M NaOH solution. The results indicate that catalytic activities of the Ni-CNTs cathodes prepared under supergravity fields are enhanced significantly, and the sample prepared at rotating speed 3000 rpm from the bath containing 1 g dm-3 CNTs exhibits the highest HER activity with smallest Tafel slope and largest exchange current density of 823.9 μA cm-2. Furthermore, the effects of both the CNTs concentrations and the intensities of supergravity fields on the properties of the Ni-CNTs cathodes are investigated.

  17. Mild water stress of Phaseolus vulgaris plants leads to reduced starch synthesis and extractable sucrose phosphate synthase activity

    SciTech Connect

    Vassey, T.L.; Sharkey, T.D. )

    1989-04-01

    Mild water stress, on the order of {minus}1.0 megapascals xylem water potential, can reduce the rate of photosynthesis and eliminate the inhibition of photosynthesis caused by O{sub 2} in water-stress-sensitive plants such as Phaseolus vulgaris. To investigate the lack of O{sub 2} inhibition of photosynthesis, we measured stromal and cytosolic fructose-1,6-bisphosphatase, sucrose phosphate synthase, and partitioning of newly fixed carbon between starch and sucrose before, during, and after mild water stress. The extractable activity of the fructose bisphosphatases was unaffected by mild water stress. The extractable activity of SPS was inhibited by more than 60% in plants stressed to water potentials of {minus}0.9 megapascals. Water stress caused a decline in the starch/sucrose partitioning ratio indicating that starch synthesis was inhibited more than sucrose synthesis. We conclude that the reduced rate of photosynthesis during water stress is caused by stomatal closure, and that the restriction of CO{sub 2} supply caused by stomatal closure leads to a reduction in the capacity for both starch and sucrose synthesis. This causes the reduced O{sub 2} inhibition and abrupt CO{sub 2} saturation of photosynthesis.

  18. Prediction of Muscle Energy States at Low Metabolic Rates Requires Feedback Control of Mitochondrial Respiratory Chain Activity by Inorganic Phosphate

    PubMed Central

    Schmitz, Joep P. J.; Jeneson, Jeroen A. L.; van Oorschot, Joep W. M.; Prompers, Jeanine J.; Nicolay, Klaas; Hilbers, Peter A. J.; van Riel, Natal A. W.

    2012-01-01

    The regulation of the 100-fold dynamic range of mitochondrial ATP synthesis flux in skeletal muscle was investigated. Hypotheses of key control mechanisms were included in a biophysical model of oxidative phosphorylation and tested against metabolite dynamics recorded by 31P nuclear magnetic resonance spectroscopy (31P MRS). Simulations of the initial model featuring only ADP and Pi feedback control of flux failed in reproducing the experimentally sampled relation between myoplasmic free energy of ATP hydrolysis (ΔGp = ΔGpo′+RT ln ([ADP][Pi]/[ATP]) and the rate of mitochondrial ATP synthesis at low fluxes (<0.2 mM/s). Model analyses including Monte Carlo simulation approaches and metabolic control analysis (MCA) showed that this problem could not be amended by model re-parameterization, but instead required reformulation of ADP and Pi feedback control or introduction of additional control mechanisms (feed forward activation), specifically at respiratory Complex III. Both hypotheses were implemented and tested against time course data of phosphocreatine (PCr), Pi and ATP dynamics during post-exercise recovery and validation data obtained by 31P MRS of sedentary subjects and track athletes. The results rejected the hypothesis of regulation by feed forward activation. Instead, it was concluded that feedback control of respiratory chain complexes by inorganic phosphate is essential to explain the regulation of mitochondrial ATP synthesis flux in skeletal muscle throughout its full dynamic range. PMID:22470528

  19. Carbon Monoxide Signaling in Human Red Blood Cells: Evidence for Pentose Phosphate Pathway Activation and Protein Deglutathionylation

    PubMed Central

    Metere, Alessio; Iorio, Egidio; Scorza, Giuseppe; Camerini, Serena; Casella, Marialuisa; Crescenzi, Marco; Minetti, Maurizio

    2014-01-01

    Abstract Aims: The biochemistry underlying the physiological, adaptive, and toxic effects of carbon monoxide (CO) is linked to its affinity for reduced transition metals. We investigated CO signaling in the vasculature, where hemoglobin (Hb), the CO most important metal-containing carrier is highly concentrated inside red blood cells (RBCs). Results: By combining NMR, MS, and spectrophotometric techniques, we found that CO treatment of whole blood increases the concentration of reduced glutathione (GSH) in RBC cytosol, which is linked to a significant Hb deglutathionylation. In addition, this process (i) does not activate glycolytic metabolism, (ii) boosts the pentose phosphate pathway (PPP), (iii) increases glutathione reductase activity, and (iv) decreases oxidized glutathione concentration. Moreover, GSH concentration was partially decreased in the presence of 2-deoxyglucose and the PPP antagonist dehydroepiandrosterone. Our MS results show for the first time that, besides Cys93, Hb glutathionylation occurs also at Cys112 of the β-chain, providing a new potential GSH source hitherto unknown. Innovation: This work provides new insights on the signaling and antioxidant-boosting properties of CO in human blood, identifying Hb as a major source of GSH release and the PPP as a metabolic mechanism supporting Hb deglutathionylation. Conclusions: CO-dependent GSH increase is a new RBC process linking a redox-inactive molecule, CO, to GSH redox signaling. This mechanism may be involved in the adaptive responses aimed to counteract stress conditions in mammalian tissues. Antioxid. Redox Signal. 20, 403–416. PMID:23815439

  20. Alkaline igneous rocks

    SciTech Connect

    Fitton, J.G.; Upton, B.G.J.

    1987-01-01

    In this volume, an international team of scientists provides an up-to-date overview of the nature, origin, and evolution of alkaline magmas. Particular attention is paid to carbonatites, lamprophyres, and lamproites which are rock suites of current interest not recently reviewed elsewhere. Recent work on the classical alkaline provinces of East Africa, South Greenland, and the Kola Peninsula is included together with reviews of other areas of alkaline magmatism in North and South America, East Greenland, Europe, West Africa, and the ocean basins. Other papers discuss the impact of experimental isotopic and geochemical studies of the petrogenesis of alkaline rocks. This book will be of interest to petrologists and geochemists studying alkaline igneous rocks, and to other earth scientists as a reference on the rapidly expanding field of igneous petrology.

  1. Phosphatidylinositol 4-Phosphate 5-Kinases in the Regulation of T Cell Activation.

    PubMed

    Porciello, Nicla; Kunkl, Martina; Viola, Antonella; Tuosto, Loretta

    2016-01-01

    Phosphatidylinositol 4,5-biphosphate kinases (PIP5Ks) are critical regulators of T cell activation being the main enzymes involved in the synthesis of phosphatidylinositol 4,5-biphosphate (PIP2). PIP2 is indeed a pivotal regulator of the actin cytoskeleton, thus controlling T cell polarization and migration, stable adhesion to antigen-presenting cells, spatial organization of the immunological synapse, and co-stimulation. Moreover, PIP2 also serves as a precursor for the second messengers inositol triphosphate, diacylglycerol, and phosphatidylinositol 3,4,5-triphosphate, which are essential for the activation of signaling pathways regulating cytokine production, cell cycle progression, survival, metabolism, and differentiation. Here, we discuss the impact of PIP5Ks on several T lymphocyte functions with a specific focus on the role of CD28 co-stimulation in PIP5K compartimentalization and activation. PMID:27242793

  2. Phosphatidylinositol 4-Phosphate 5-Kinases in the Regulation of T Cell Activation

    PubMed Central

    Porciello, Nicla; Kunkl, Martina; Viola, Antonella; Tuosto, Loretta

    2016-01-01

    Phosphatidylinositol 4,5-biphosphate kinases (PIP5Ks) are critical regulators of T cell activation being the main enzymes involved in the synthesis of phosphatidylinositol 4,5-biphosphate (PIP2). PIP2 is indeed a pivotal regulator of the actin cytoskeleton, thus controlling T cell polarization and migration, stable adhesion to antigen-presenting cells, spatial organization of the immunological synapse, and co-stimulation. Moreover, PIP2 also serves as a precursor for the second messengers inositol triphosphate, diacylglycerol, and phosphatidylinositol 3,4,5-triphosphate, which are essential for the activation of signaling pathways regulating cytokine production, cell cycle progression, survival, metabolism, and differentiation. Here, we discuss the impact of PIP5Ks on several T lymphocyte functions with a specific focus on the role of CD28 co-stimulation in PIP5K compartimentalization and activation. PMID:27242793

  3. Reductive dechlorination of carbon tetrachloride using buffered alkaline ascorbic acid.

    PubMed

    Lin, Ya-Ting; Liang, Chenju

    2015-10-01

    Alkaline ascorbic acid (AA) was recently discovered as a novel in-situ chemical reduction (ISCR) reagent for remediating chlorinated solvents in the subsurface. For this ISCR process, the maintenance of an alkaline pH is essential. This study investigated the possibility of the reduction of carbon tetrachloride (CT) using alkaline AA solution buffered by phosphate and by NaOH. The results indicated that CT was reduced by AA, and chloroform (CF) was a major byproduct at a phosphate buffered pH of 12. However, CT was completely reduced by AA in 2M NaOH without CF formation. In the presence of iron/soil minerals, iron could be reduced by AA and Fe(2+) tends to precipitate on the mineral surface to accelerate CT degradation. A simultaneous transfer of hydrogenolysis and dichloroelimination would occur under phosphate buffered pH 12. This implies that a high alkaline environment is a crucial factor for maintaining the dominant pathway of two electron transfer from dianionic AA to dehydroascorbic acid, and to undergo dichloroelimination of CT. Moreover, threonic acid and oxalic acid were identified to be the major AA decomposition products in alkaline solutions. PMID:25912910

  4. Human ASIC3 channel dynamically adapts its activity to sense the extracellular pH in both acidic and alkaline directions

    PubMed Central

    Delaunay, Anne; Gasull, Xavier; Salinas, Miguel; Noël, Jacques; Friend, Valérie; Lingueglia, Eric; Deval, Emmanuel

    2012-01-01

    In rodent sensory neurons, acid-sensing ion channel 3 (ASIC3) has recently emerged as a particularly important sensor of nonadaptive pain associated with tissue acidosis. However, little is known about the human ASIC3 channel, which includes three splice variants differing in their C-terminal domain (hASIC3a, hASIC3b, and hASIC3c). hASIC3a transcripts represent the main mRNAs expressed in both peripheral and central neuronal tissues (dorsal root ganglia [DRG], spinal cord, and brain), where a small proportion of hASIC3c transcripts is also detected. We show that hASIC3 channels (hASIC3a, hASIC3b, or hASIC3c) are able to directly sense extracellular pH changes not only during acidification (up to pH 5.0), but also during alkalization (up to pH 8.0), an original and inducible property yet unknown. When the external pH decreases, hASIC3 display a transient acid mode with brief activation that is relevant to the classical ASIC currents, as previously described. On the other hand, an external pH increase activates a sustained alkaline mode leading to a constitutive activity at resting pH. Both modes are inhibited by the APETx2 toxin, an ASIC3-type channel inhibitor. The alkaline sensitivity of hASIC3 is an intrinsic property of the channel, which is supported by the extracellular loop and involves two arginines (R68 and R83) only present in the human clone. hASIC3 is thus able to sense the extracellular pH in both directions and therefore to dynamically adapt its activity between pH 5.0 and 8.0, a property likely to participate in the fine tuning of neuronal membrane potential and to neuron sensitization in various pH environments. PMID:22829666

  5. Mineral induced formation of sugar phosphates

    NASA Astrophysics Data System (ADS)

    Pitsch, S.; Eschenmoser, A.; Gedulin, B.; Hui, S.; Arrhenius, G.

    1995-08-01

    Glycolaldehyde phosphate, sorbed from highly dilute, weakly alkaline solution into the interlayer of common expanding sheet structure metal hydroxide minerals, condenses extensively to racemic aldotetrose-2,4-diphosphates and aldohexose-2,4,6-triphosphates. The reaction proceeds mainly through racemic erythrose-2,4-phosphate, and terminates with a large fraction of racemic altrose-2,4,6-phosphate. In the absence of an inductive mineral phase, no detectable homogeneous reaction takes place in the concentration- and pH range used. The reactant glycolaldehyde phosphate is practically completely sorbed within an hour from solutions with concentrations as low as 50 µm; the half-time for conversion to hexose phosphates is of the order of two days at room temperature and pH 9.5. Total production of sugar phosphates in the mineral interlayer is largely independent of the glycolaldehyde phosphate concentration in the external solution, but is determined by the total amount of GAP offered for sorption up to the capacity of the mineral. In the presence of equimolar amounts of rac-glyceraldehyde-2-phosphate, but under otherwise similar conditions, aldopentose-2,4,-diphosphates also form, but only as a small fraction of the hexose-2,4,6-phosphates.

  6. Mineral induced formation of sugar phosphates

    NASA Technical Reports Server (NTRS)

    Pitsch, S.; Eschenmoser, A.; Gedulin, B.; Hui, S.; Arrhenius, G.

    1995-01-01

    Glycolaldehyde phosphate, sorbed from highly dilute, weakly alkaline solution into the interlayer of common expanding sheet structure metal hydroxide minerals, condenses extensively to racemic aldotetrose-2, 4-diphophates, and aldohexose-2, 4, 6-triphosphates. The reaction proceeds mainly through racemic erythrose-2, 4-phosphate, and terminates with a large fraction of racemic altrose-2, 4, 6-phosphate. In the absence of an inductive mineral phase, no detectable homogeneous reaction takes place in the concentration- and pH range used. The reactant glycolaldehyde phosphate is practically completely sorbed within an hour from solutions with concentrations as low as 50 micron; the half-time for conversion to hexose phosphates is of the order of two days at room temperature and pH 9.5. Total production of sugar phosphates in the mineral interlayer is largely independent of the glycolaldehyde phosphate concentration in the external solution, but is determined by the total amount of GAP offered for sorption up to the capacity of the mineral. In the presence of equimolar amounts of rac-glyceraldehyde-2-phosphate, but under otherwise similar conditions, aldopentose-2, 4, -diphosphates also form, but only as a small fraction of the hexose-2, 4, 6-phosphates.

  7. Electron microscope histochemical localization of alkaline phosphatase(s) in Bacillus licheniformis.

    PubMed Central

    McNicholas, J M; Hulett, F M

    1977-01-01

    Sites of alkaline phosphatase (APase) activity in a facultative thermophilic strain of Bacillus licheniformis MC14 have been localized by electron microscope histochemistry, using a lead capture method. The effects of 3% glutaraldehyde and 3.0 mM lead on APase activity were investigated, and these compounds were found to significantly inhibit enzyme activity, 68 and 18%, respectively. A number of parameters were varied in studies to localize APase activity, including: growth temperature (55 and 37 degrees C); substrate concentration in the histochemical mixture (0.06, 0.15, 0.30, 1.00 mM); fixatives; protoplast preparations and whole cells; phosphate-repressed and -derepressed cells; and age of vegetative cells (mid-log and late log). These variations affected the number but not the location of lead phosphate deposits, which appeared at discrete sites along the inner side of the cytoplasmic membrane. Control cells incubated in histochemical mixtures lacking substrate, lead, or both exhibited no lead phosphate depositis. The histochemical localization at membrane sites correlated well with biochemical localization data, which indicated that greater than 80% of the APase activity was associated with the membrane fraction in logarithmically growing cells. Images PMID:401501

  8. Electron microscope histochemical localization of alkaline phosphatase(s) in Bacillus licheniformis.

    PubMed

    McNicholas, J M; Hulett, F M

    1977-01-01

    Sites of alkaline phosphatase (APase) activity in a facultative thermophilic strain of Bacillus licheniformis MC14 have been localized by electron microscope histochemistry, using a lead capture method. The effects of 3% glutaraldehyde and 3.0 mM lead on APase activity were investigated, and these compounds were found to significantly inhibit enzyme activity, 68 and 18%, respectively. A number of parameters were varied in studies to localize APase activity, including: growth temperature (55 and 37 degrees C); substrate concentration in the histochemical mixture (0.06, 0.15, 0.30, 1.00 mM); fixatives; protoplast preparations and whole cells; phosphate-repressed and -derepressed cells; and age of vegetative cells (mid-log and late log). These variations affected the number but not the location of lead phosphate deposits, which appeared at discrete sites along the inner side of the cytoplasmic membrane. Control cells incubated in histochemical mixtures lacking substrate, lead, or both exhibited no lead phosphate depositis. The histochemical localization at membrane sites correlated well with biochemical localization data, which indicated that greater than 80% of the APase activity was associated with the membrane fraction in logarithmically growing cells. PMID:401501

  9. A comparative investigation of metal-support interactions on the catalytic activity of Pt nanoparticles for ethanol oxidation in alkaline medium

    NASA Astrophysics Data System (ADS)

    Godoi, Denis R. M.; Villullas, Hebe M.; Zhu, Fu-Chun; Jiang, Yan-Xia; Sun, Shi-Gang; Guo, Junsong; Sun, Lili; Chen, Rongrong

    2016-04-01

    The effects of interactions of Pt nanoparticles with hybrid supports on reactivity towards ethanol oxidation in alkaline solution are investigated. Studies involve catalysts with identical Pt nanoparticles on six hybrid supports containing carbon powder and transition metal oxides (TiO2, ZrO2, SnO2, CeO2, MoO3 and WO3). In situ X-ray absorption spectroscopy (XAS) results evidence that metal-support interactions produce changes in the Pt 5d band vacancy, which appears to determine the catalytic activity. The highest and lowest activities are observed for Pt nanoparticles on hybrid supports containing TiO2 and CeO2, respectively. Further studies are presented for these two catalysts. In situ FTIR reflection spectroscopy measurements, taken using both multi-stepped FTIR spectroscopy (MS-FTIR) and single potential alteration FTIR spectroscopy (SPA-FTIR), evidence that the main product of ethanol oxidation is acetate, although signals attributed to carbonate and CO2 indicate some differences in CO2 production. Fuel cell performances of these catalysts, tested in a 4.5 cm2 single cell at different temperatures (40-90 °C) show good agreement with data obtained by electrochemical techniques. Results of this comprehensive study point out the possibility of compensating a reduction of noble metal load with an increase in activity promoted by interactions between metallic nanoparticles and a support.

  10. Calcium phosphate surfaces promote osteogenic differentiation of mesenchymal stem cells

    PubMed Central

    Müller, Petra; Bulnheim, Ulrike; Diener, Annette; Lüthen, Frank; Teller, Marianne; Klinkenberg, Ernst-Dieter; Neumann, Hans-Georg; Nebe, Barbara; Liebold, Andreas; Steinhoff, Gustav; Rychly, Joachim

    2008-01-01

    Abstract Although studies in vivo revealed promising results in bone regeneration after implantation of scaffolds together with osteogenic progenitor cells, basic questions remain how material surfaces control the biology of mesenchymal stem cells (MSC). We used human MSC derived from bone marrow and studied the osteogenic differentiation on calcium phosphate surfaces. In osteogenic differentiation medium MSC differentiated to osteoblasts on hydroxyapatite and BONITmatrix®, a degradable xerogel composite, within 14 days. Cells revealed a higher alkaline phosphatase (ALP) activity and increased RNA expression of collagen I and osteocalcin using real-time RTPCR compared with cells on tissue culture plastic. To test whether material surface characteristics alone are able to stimulate osteogenic differentiation, MSC were cultured on the materials in expansion medium without soluble additives for osteogenic differentiation. Indeed, cells on calcium phosphate without osteogenic differentiation additives developed to osteoblasts as shown by increased ALP activity and expression of osteogenic genes, which was not the case on tissue culture plastic. Because we reasoned that the stimulating effect on osteogenesis by calcium phosphate surfaces depends on an altered cell–extracellular matrix interaction we studied the dynamic behaviour of focal adhesions using cells transfected with GFP labelled vinculin. On BONITmatrix®, an increased mobility of focal adhesions was observed compared with cells on tissue culture plastic. In conclusion, calcium phosphate surfaces are able to drive MSC to osteoblasts in the absence of osteogenic differentiation supplements in the medium. An altered dynamic behaviour of focal adhesions on calcium phosphate surfaces might be involved in the molecular mechanisms which promote osteogenic differentiation. PMID:18366455

  11. Active removal of inorganic phosphate from cerebrospinal fluid by the choroid plexus.

    PubMed

    Guerreiro, Pedro M; Bataille, Amy M; Parker, Sonda L; Renfro, J Larry

    2014-06-01

    The P(i) concentration of mammalian cerebrospinal fluid (CSF) is about one-half that of plasma, a phenomenon also shown here in the spiny dogfish, Squalus acanthias. The objective of the present study was to characterize the possible role of the choroid plexus (CP) in determining CSF P(i) concentration. The large sheet-like fourth CP of the shark was mounted in Ussing chambers where unidirectional (33)P(i) fluxes revealed potent active transport from CSF to the blood side under short-circuited conditions. The flux ratio was 8:1 with an average transepithelial resistance of 87 ± 17.9 Ω·cm(2) and electrical potential difference of +0.9 ± 0.17 mV (CSF side positive). Active P(i) absorption from CSF was inhibited by 10 mM arsenate, 0.2 mM ouabain, Na(+)-free medium, and increasing the K(+) concentration from 5 to 100 mM. Li(+) stimulated transport twofold compared with Na(+)-free medium. Phosphonoformic acid (1 mM) had no effect on active P(i) transport. RT-PCR revealed both P(i) transporter (PiT)1 and PiT2 (SLC20 family) gene expression, but no Na(+)-P(i) cotransporter II (SLC34 family) expression, in the shark CP. PiT2 immunoreactivity was shown by immunoblot analysis and localized by immunohistochemistry in (or near) the CP apical microvillar membranes of both the shark and rat. PiT1 appeared to be localized primarily to vascular endothelial cells. Taken together, these data indicate that the CP actively removes P(i) from CSF. This process has transport properties consistent with a PiT2, Na(+)-dependent transporter that is located in the apical region of the CP epithelium. PMID:24740787

  12. Alkaline phosphatase from venom of the endoparasitoid wasp, Pteromalus puparum.

    PubMed

    Zhu, Jia-Ying; Yin Ye, Gong; Fang, Qi; Hu, Cui

    2010-01-01

    Using chromogenic substrates 5-bromo-4-chloro-3'-indolyl phosphate and nitro blue tetrazolium, alkaline phosphatase (ALPase) was histochemically detected in the venom apparatus of an endoparasitoid wasp, Pteromalus puparum L. (Hymenoptera: Pteromalidae). Ultrastructural observations demonstrated its presence in the secretory vesicles and nuclei of the venom gland secretory cells. Using p-nitrophenyl phosphate as substrate to measure enzyme activity, the venom ALPase was found to be temperature dependent with bivalent cation effects. The full-length cDNA sequence of ALPase was amplified from the cDNA library of the venom apparatus of P. puparum, providing the first molecular characterization of ALPase in the venom of a parasitoid wasp. The cDNA consisted of 2645 bp with a 1623 bp open reading frame coding for 541 deduced amino acids with a predicted molecular mass of 59.83 kDa and pI of 6.98. Using multiple sequence alignment, the deduced amino acid sequence shared high identity to its counterparts from other insects. A signal peptide and a long conserved ALPase gene family signature sequence were observed. The amino acid sequence of this venom protein was characterized with different potential glycosylation, myristoylation, phosphorylation sites and metal ligand sites. The transcript of the ALPase gene was detected by RT-PCR in the venom apparatus with development related expression after adult wasp emergence, suggesting a possible correlation with the oviposition process. PMID:20575745

  13. Inorganic-organic separators for alkaline batteries

    NASA Technical Reports Server (NTRS)

    Sheibley, D. W. (Inventor)

    1978-01-01

    A flexible separator is reported for use between the electrodes of Ni-Cd and Ni-Zn batteries using alkaline electrolytes. The separator was made by coating a porous substrate with a battery separator composition. The coating material included a rubber-based resin copolymer, a plasticizer and inorganic and organic fillers which comprised 55% by volume or less of the coating as finally dried. One or more of the filler materials, whether organic or inorganic, is preferably active with the alkaline electrolyte to produce pores in the separator coating. The plasticizer was an organic material which is hydrolyzed by the alkaline electrolyte to improve conductivity of the separator coating.

  14. AMP-activated protein kinase inhibits alkaline pH- and PKA-induced apical vacuolar H+-ATPase accumulation in epididymal clear cells.

    PubMed

    Hallows, Kenneth R; Alzamora, Rodrigo; Li, Hui; Gong, Fan; Smolak, Christy; Neumann, Dietbert; Pastor-Soler, Núria M

    2009-04-01

    Acidic luminal pH and low [HCO(3)(-)] maintain sperm quiescent during maturation in the epididymis. The vacuolar H(+)-ATPase (V-ATPase) in clear cells is a major contributor to epididymal luminal acidification. We have shown previously that protein kinase A (PKA), acting downstream of soluble adenylyl cyclase stimulation by alkaline luminal pH or HCO(3)(-), induces V-ATPase apical membrane accumulation in clear cells. Here we examined whether the metabolic sensor AMP-activated protein kinase (AMPK) regulates this PKA-induced V-ATPase apical membrane accumulation. Immunofluorescence labeling of rat and non-human primate epididymides revealed specific AMPK expression in epithelial cells. Immunofluorescence labeling of rat epididymis showed that perfusion in vivo with the AMPK activators 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) or A-769662 induced a redistribution of the V-ATPase into subapical vesicles, even in the presence of a luminal alkaline (pH 7.8) buffer compared with that of controls perfused without drug. Moreover, preperfusion with AICAR blocked the PKA-mediated V-ATPase translocation to clear cell apical membranes induced by N(6)-monobutyryl-cAMP (6-MB-cAMP). Purified PKA and AMPK both phosphorylated V-ATPase A subunit in vitro. In HEK-293 cells [(32)P]orthophosphate in vivo labeling of the A subunit increased following PKA stimulation and decreased following RNA interference-mediated knockdown of AMPK. Finally, the extent of PKA-dependent in vivo phosphorylation of the A subunit increased with AMPK knockdown. In summary, our findings suggest that AMPK inhibits PKA-mediated V-ATPase apical accumulation in epididymal clear cells, that both kinases directly phosphorylate the V-ATPase A subunit in vitro and in vivo, and that AMPK inhibits PKA-dependent phosphorylation of this subunit. V-ATPase activity may be coupled to the sensing of acid-base status via PKA and to metabolic status via AMPK. PMID:19211918

  15. Oxysterol-binding Protein Activation at Endoplasmic Reticulum-Golgi Contact Sites Reorganizes Phosphatidylinositol 4-Phosphate Pools.

    PubMed

    Goto, Asako; Charman, Mark; Ridgway, Neale D

    2016-01-15

    Oxysterol-binding protein (OSBP) exchanges cholesterol and phosphatidylinositol 4-phosphate (PI-4P) at contact sites between the endoplasmic reticulum (ER) and the trans-Golgi/trans-Golgi network. 25-Hydroxycholesterol (25OH) competitively inhibits this exchange reaction in vitro and causes the constitutive localization of OSBP at the ER/Golgi interface and PI-4P-dependent recruitment of ceramide transfer protein (CERT) for sphingomyelin synthesis. We used PI-4P probes and mass analysis to determine how OSBP controls the availability of PI-4P for this metabolic pathway. Treatment of fibroblasts or Chinese hamster ovary (CHO) cells with 25OH caused a 50-70% reduction in Golgi-associated immunoreactive PI-4P that correlated with Golgi localization of OSBP. In contrast, 25OH caused an OSBP-dependent enrichment in Golgi PI-4P that was detected with a pleckstrin homology domain probe. The cellular mass of phosphatidylinositol monophosphates and Golgi PI-4P measured with an unbiased PI-4P probe (P4M) was unaffected by 25OH and OSBP silencing, indicating that OSBP shifts the distribution of PI-4P upon localization to ER-Golgi contact sites. The PI-4P and sterol binding activities of OSBP were both required for 25OH activation of sphingomyelin synthesis, suggesting that 25OH must be exchanged for PI-4P to be concentrated at contact sites. We propose a model wherein 25OH activation of OSBP promotes the binding and retention of PI-4P at ER-Golgi contact sites. This pool of PI-4P specifically recruits pleckstrin homology domain-containing proteins involved in lipid transfer and metabolism, such as CERT. PMID:26601944

  16. Activation of sphingosine 1-phosphate receptor-1 by SEW2871 improves cognitive function in Alzheimer's disease model rats

    PubMed Central

    Asle-Rousta, Masoumeh; Oryan, Shahrbanoo; Ahmadiani, Abolhassan; Rahnema, Mehdi

    2013-01-01

    Sphingosine-1 phosphate (S1P) is involved in a variety of cellular processes via activation of S1P receptors (S1PRs; S1PR1 to S1PR5) that are highly expressed in the brain. It has been shown that the level of S1P is reduced in the brain of Alzheimer's disease (AD) patients. However, there is no study designed to evaluate the expression of S1PRs in AD brains. The objectives of the present work are (1) to examine the expression of S1PR1-3 in the hippocampus of beta amyloid (Aβ) 1-42 injected rats and (2) to clarify the effects of chronic S1PR1 activation on S1PR1-3 levels, spatial memory deficit and hippocampal damage in AD rats. SEW2871, the S1PR1 selective agonist, repeatedly was injected intraperitoneally during a period of two weeks. Upon Western Blot data bilateral intrahippocampal injection of Aβ1-42 decreased the expression of S1PR1 while increased S1PR2 level and did not affect that of S1PR3. We found that chronic administration of SEW2871 inhibited the reduction of S1PR1 expression and ameliorated spatial memory impairment in the Morris water maze task in rats. In addition, SEW2871 attenuated the Aβ1-42-induced hippocampal neuronal loss according to Nissl staining findings. Data in the current study highlights the importance of S1PR1 signaling pathway deregulation in AD development and suggests that activation of S1PR1 may represent a potential approach for developing new therapeutics to manage memory deficit and apoptosis associated with neurodegenerative disorders such as AD. PMID:26417237

  17. Elevated nuclear sphingoid base-1-phosphates and decreased histone deacetylase activity after fumonisin B1 treatment in mouse embryonic fibroblasts.

    PubMed

    Gardner, Nicole M; Riley, Ronald T; Showker, Jency L; Voss, Kenneth A; Sachs, Andrew J; Maddox, Joyce R; Gelineau-van Waes, Janee B

    2016-05-01

    Fumonisin B1 (FB1) is a mycotoxin produced by a common fungal contaminant of corn. Administration of FB1 to pregnant LM/Bc mice induces exencephaly in embryos, and ingestion of FB1-contaminated food during early pregnancy is associated with increased risk for neural tube defects (NTDs) in humans. FB1 inhibits ceramide synthase enzymes in sphingolipid biosynthesis, causing sphinganine (Sa) and bioactive sphinganine-1-phosphate (Sa1P) accumulation in blood, cells, and tissues. Sphingosine kinases (Sphk) phosphorylate Sa to form Sa1P. Upon activation, Sphk1 associates primarily with the plasma membrane, while Sphk2 is found predominantly in the nucleus. In cells over-expressing Sphk2, accumulation of Sa1P in the nuclear compartment inhibits histone deacetylase (HDAC) activity, causing increased acetylation of histone lysine residues. In this study, FB1 treatment in LM/Bc mouse embryonic fibroblasts (MEFs) resulted in significant accumulation of Sa1P in nuclear extracts relative to cytoplasmic extracts. Elevated nuclear Sa1P corresponded to decreased histone deacetylase (HDAC) activity and increased histone acetylation at H2BK12, H3K9, H3K18, and H3K23. Treatment of LM/Bc MEFs with a selective Sphk1 inhibitor, PF-543, or with ABC294640, a selective Sphk2 inhibitor, significantly reduced nuclear Sa1P accumulation after FB1, although Sa1P levels remained significantly increased relative to basal levels. Concurrent treatment with both PF-543 and ABC294640 prevented nuclear accumulation of Sa1P in response to FB1. Other HDAC inhibitors are known to cause NTDs, so these results suggest that FB1-induced disruption of sphingolipid metabolism leading to nuclear Sa1P accumulation, HDAC inhibition, and histone hyperacetylation is a potential mechanism for FB1-induced NTDs. PMID:26905748

  18. The vascular disrupting activity of OXi8006 in endothelial cells and its phosphate prodrug OXi8007 in breast tumor xenografts.

    PubMed

    Strecker, Tracy E; Odutola, Samuel O; Lopez, Ramona; Cooper, Morgan S; Tidmore, Justin K; Charlton-Sevcik, Amanda K; Li, Li; MacDonough, Matthew T; Hadimani, Mallinath B; Ghatak, Anjan; Liu, Li; Chaplin, David J; Mason, Ralph P; Pinney, Kevin G; Trawick, Mary Lynn

    2015-12-01

    This study describes the vascular disrupting ability and the mechanism of action of the indole-based tubulin-binding compound, OXi8006, and its water-soluble phosphate prodrug OXi8007. Treatment of rapidly proliferating human umbilical vein endothelial cells (HUVECs), used as a model for the tumor vasculature, with OXi8006 or OXi8007, caused potent microtubule disruption followed by extensive reorganization of the cytoskeletal network. The mechanism of action involved an increase in focal adhesion formation associated with an increase in phosphorylation of both non-muscle myosin light chain and focal adhesion kinase. These effects were dramatically diminished by an inhibitor of RhoA kinase, a downstream effector of RhoA. Cell cycle blockade at G2/M and cytotoxicity toward rapidly proliferating HUVECs were also observed. Capillary-like networks of HUVECs were disrupted by the action of both OXi8006 and OXi8007. The prodrug OXi8007 exhibited potent and rapid dose-dependent antivascular activity assessed by dynamic bioluminescence imaging (BLI) in an MDA-MB-231-luc breast cancer xenograft mouse model. By 6 hours post treatment, over 93% of the BLI signal was abolished with only a slight recovery at 24 hours. These findings were confirmed by histology. The results from this study demonstrate that OXi8007 is a potent vascular disrupting agent acting through an anti-microtubule mechanism involving RhoA. PMID:26325604

  19. A Ternary Polyaniline/Active Carbon/Lithium Iron Phosphate Composite as Cathode Material for Lithium Ion Battery.

    PubMed

    Wang, Xiaohong; Zhang, Wuxing; Huang, Yunhui; Xia, Tian; Lian, Yongfu

    2016-06-01

    Lithium iron phosphate (LiFePO4) has been evaluated as the most promising cathode material for the next generation lithium-ion batteries because of its high operating voltage, good cycle performance, low cost, and environmentally friendly safety. However, pure LiFePO4 shows poor reversible capacity and charge/discharge performance at high current density. Many methods including optimization of particle size, introduction of coating carbon and conductive polymer, and the doping of metal and halogen ions have been developed to improve its electrochemical performance. In this study, conductive polymer polyaniline (PANI), active carbon and LiFePO4 (C-LFP/PANI) composite cathodes were successfully prepared by chemical oxidation method. Electrochemical performance shows that a remarkable improvement in capacity and rate performance can be achieved in the C-LFP/PANI composite cathodes with an addition of HCI. In comparison with C-LFP cathode, the C-LFP/PANI doped with HCl composite exhibits ca. 15% and 26% capacity enhancement at 0.2 C and 10 C, respectively. PMID:27427742

  20. Autophagy and endosomal trafficking inhibition by Vibrio cholerae MARTX toxin phosphatidylinositol-3-phosphate-specific phospholipase A1 activity

    PubMed Central

    Agarwal, Shivani; Kim, Hyunjin; Chan, Robin B.; Agarwal, Shivangi; Williamson, Rebecca; Cho, Wonhwa; Paolo, Gilbert D.; Satchell, Karla J. F.

    2015-01-01

    Vibrio cholerae, responsible for acute gastroenteritis secretes a large multifunctional-autoprocessing repeat-in-toxin (MARTX) toxin linked to evasion of host immune system, facilitating colonization of small intestine. Unlike other effector domains of the multifunctional toxin that target cytoskeleton, the function of alpha-beta hydrolase (ABH) remained elusive. This study demonstrates that ABH is an esterase/lipase with catalytic Ser–His–Asp triad. ABH binds with high affinity to phosphatidylinositol-3-phosphate (PtdIns3P) and cleaves the fatty acid in PtdIns3P at the sn1 position in vitro making it the first PtdIns3P-specific phospholipase A1 (PLA1). Expression of ABH in vivo reduces intracellular PtdIns3P levels and its PtdIns3P-specific PLA1 activity blocks endosomal and autophagic pathways. In accordance with recent studies acknowledging the potential of extracellular pathogens to evade or exploit autophagy to prevent their clearance and facilitate survival, this is the first report highlighting the role of ABH in inhibiting autophagy and endosomal trafficking induced by extracellular V. cholerae. PMID:26498860

  1. Altered chloroplast structure and function in a mutant of Arabidopsis deficient in plastid glycerol-3-phosphate acyltransferase activity

    SciTech Connect

    Kunst, L.; Somerville, C. ); Browse, J. )

    1989-07-01

    Mutants of Arabidopsis thaliana deficient in plastid glycerol-3-phosphate acyltransferase activity have altered chloroplast membrane lipid composition. This caused an increase in the number of regions of appressed membrane per chloroplast and a decrease in the average number of thylakoid membranes in the appressed regions. The net effect was a significant decrease in the ratio of appressed to nonappressed membranes. A comparison of 77 K fluorescence emission spectra of thylakoid membranes from the mutant and wild type indicated that the ultrastructural changes were associated with an altered distribution of excitation energy transfer from antenna chlorophyll to photosystem II and photosystem I in the mutant. The changes in leaf lipid composition did not significantly affect growth or development of the mutant under standard conditions. However, at temperatures above 28{degree}C the mutant grew slightly more rapidly than the wild type, and measurements of temperature-induced fluorescence yield enhancement suggested an increased thermal stability of the photosynthetic apparatus of the mutant. These effects are consistent with other evidence suggesting that membrane lipid composition is an important determinant of chloroplast structure but has relatively minor direct effects on the function of the membrane proteins associated with photosynthetic electron transport.

  2. The oxygen isotope composition of phosphate released from phytic acid by the activity of wheat and Aspergillus niger phytase

    NASA Astrophysics Data System (ADS)

    von Sperber, C.; Tamburini, F.; Brunner, B.; Bernasconi, S. M.; Frossard, E.

    2015-07-01

    Phosphorus (P) is an essential nutrient for living organisms. Under P-limiting conditions plants and microorganisms can exude extracellular phosphatases that release inorganic phosphate (Pi) from organic phosphorus compounds (Porg). Phytic acid (myo-inositol hexakisphosphate, IP6) is an important form of Porg in many soils. The enzymatic hydrolysis of IP6 by phytase yields available Pi and less phosphorylated inositol derivates as products. The hydrolysis of organic P compounds by phosphatases leaves an isotopic imprint on the oxygen isotope composition (δ18O) of released Pi, which might be used to trace P in the environment. This study aims at determining the effect of phytase on the oxygen isotope composition of released Pi. For this purpose, enzymatic assays with histidine acid phytases from wheat and Aspergillus niger were prepared using IP6, adenosine 5'-monophosphate (AMP) and glycerophosphate (GPO4) as substrates. For a comparison to the δ18O of Pi released by other extracellular enzymes, enzymatic assays with acid phosphatases from potato and wheat germ with IP6 as a substrate were prepared. During the hydrolysis of IP6 by phytase, four of the six Pi were released, and one oxygen atom from water was incorporated into each Pi. This incorporation of oxygen from water into Pi was subject to an apparent inverse isotopic fractionation (ϵ ~ 6 to 10 ‰), which was similar to that imparted by acid phosphatase from potato during the hydrolysis of IP6 (ϵ ~ 7 ‰), where less than three Pi were released. The incorporation of oxygen from water into Pi during the hydrolysis of AMP and GPO4 by phytase yielded a normal isotopic fractionation (ϵ ~ -12 ‰), similar to values reported for acid phosphatases from potato and wheat germ. We attribute this similarity in ϵ to the same amino acid sequence motif (RHGXRXP) at the active site of these enzymes, which leads to similar reaction mechanisms. We suggest that the striking

  3. Phosphonate analogues of cyclopropavir phosphates and their E-isomers. Synthesis and antiviral activity.

    PubMed

    Mhaske, Santosh B; Ksebati, Bashar; Prichard, Mark N; Drach, John C; Zemlicka, Jiri

    2009-06-01

    Z- and E-Phosphonate analogues 12 and 13 derived from cyclopropavir and the corresponding cyclic phosphonates 14 and 15 were synthesized and their antiviral activity was investigated. The 2,2-bis(hydroxymethylmethylenecyclopropane acetate (17) was transformed to tetrahydropyranyl acetate 18. Deacetylation gave intermediate 19 which was converted to bromide 20. Alkylation with diisopropyl methylphosphonate afforded after protecting group exchange (21 to 22) acetylated phosphonate intermediate 22. Addition of bromine gave the dibromo derivative 16 which was used in the alkylation-elimination procedure with 2-amino-6-chloropurine to give Z- and E-isomers 23 and 24. Hydrolytic dechlorination coupled with removal of all protecting groups gave the guanine phosphonates 12 and 13. Cyclization afforded the cyclic phosphonates 14 and 15. Z-Phosphonate 12 was a potent and non-cytotoxic inhibitor of human and murine cytomegalovirus (HCMV and MCMV) with EC(50) 2.2-2.7 and 0.13 microM, respectively. It was also an effective agent against Epstein-Barr virus (EBV, EC(50) 3.1 microM). The cyclic phosphonate 14 inhibited HCMV (EC(50) 2.4-11.5 microM) and MCMV (EC(50) 0.4 microM) but it was ineffective against EBV. Both phosphonates 12 and 14 were as active against two HCMV Towne strains with mutations in UL97 as they were against wild-type HCMV thereby circumventing resistance due to such mutations. Z-Phosphonate 12 was a moderate inhibitor of replication of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) but it was a potent agent against varicella zoster virus (VZV, EC(50) 2.9 microM). The cyclic phosphonate 14 lacked significant potency against these viruses. E-isomers 13 and 15 were devoid of antiviral activity. PMID:19410465

  4. Phosphonate Analogues of Cyclopropavir Phosphates and Their E-isomers. Synthesis and Antiviral Activity

    PubMed Central

    Mhaske, Santosh B.; Ksebati, Bashar; Prichard, Mark N.; Drach, John C.; Zemlicka, Jiri

    2009-01-01

    Z- and E-phosphonate analogues 12 and 13 derived from cyclopropavir and the corresponding cyclic phosphonates 14 and 15 were synthesized and their antiviral activity was investigated. The 2,2-bis(hydroxymethylmethylenecyclopropane acetate (17) was transformed to tetrahydropyranyl acetate 18. Deacetylation gave intermediate 19 which was converted to bromide 20. Alkylation with diisopropyl methylphosphonate afforded after protecting group exchange (21 to 22) acetylated phosphonate intermediate 22. Addition of bromine gave the dibromo derivative 16 which was used in the alkylation-elimination procedure with 2-amino-6-chloropurine to give Z- and E-isomers 23 and 24. Hydrolytic dechlorination coupled with removal of all protecting groups gave the guanine phosphonates 12 and 13. Cyclization afforded the cyclic phosphonates 14 and 15. Z-Phosphonate 12 was a potent and non-cytotoxic inhibitor of human and murine cytomegalovirus (HCMV and MCMV) with EC50 2.2-2.7 and 0.13 μM, respectively. It was also an effective agent against Epstein-Barr virus (EBV, EC50 3.1 μM). The cyclic phosphonate 14 inhibited HCMV (EC50 2.4-11.5 μM) and MCMV (EC50 0.4 μM) but it was ineffective against EBV. Both phosphonates 12 and 14 were as active against two HCMV Towne strains with mutations in UL97 as they were against wild-type HCMV thereby circumventing resistance due to such mutations. Z-Phosphonate 12 was a moderate inhibitor of replication of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) but it was a potent agent against varicella zoster virus (VZV, EC50 2.9 μM). The cyclic phosphonate 14 lacked significant potency against these viruses. E-isomers 13 and 15 were devoid of antiviral activity. PMID:19410465

  5. Alkaline battery operational methodology

    DOEpatents

    Sholklapper, Tal; Gallaway, Joshua; Steingart, Daniel; Ingale, Nilesh; Nyce, Michael

    2016-08-16

    Methods of using specific operational charge and discharge parameters to extend the life of alkaline batteries are disclosed. The methods can be used with any commercial primary or secondary alkaline battery, as well as with newer alkaline battery designs, including batteries with flowing electrolyte. The methods include cycling batteries within a narrow operating voltage window, with minimum and maximum cut-off voltages that are set based on battery characteristics and environmental conditions. The narrow voltage window decreases available capacity but allows the batteries to be cycled for hundreds or thousands of times.

  6. Characterization of biomimetic calcium phosphate labeled with fluorescent dextran for quantification of osteoclastic activity.

    PubMed

    Maria, Salwa M; Prukner, Christiane; Sheikh, Zeeshan; Müller, Frank A; Komarova, Svetlana V; Barralet, Jake E

    2015-07-01

    Bone resorbing osteoclasts represent an important therapeutic target for diseases associated with bone and joint destruction, such as rheumatoid arthritis, periodontitis, and osteoporosis. The quantification of osteoclast resorptive activity in vitro is widely used for screening new anti-resorptive medications. The aim of this paper was to develop a simplified semi-automated method for the quantification of osteoclastic resorption using fluorescently labeled biomimetic mineral layers which can replace time intensive, often subjective and clearly non-sustainable use of translucent slices of tusks from vulnerable or endangered species such as the elephant. Osteoclasts were formed from RAW 264.7 mouse monocyte cell line using the pro-resorptive cytokine receptor activator of nuclear factor kappa-B ligand (RANKL). We confirmed that fluorescent labeling did not interfere with the biomimetic features of hydroxyapatite, and developed an automated method for quantifying osteoclastic resorption. Correlation between our assay and traditional manual measurement techniques was found to be very strong (R(2)=0.99). In addition, we modified the technique to provide depth and volume data of the resorption pits by confocal imaging at defined depths. Thus, our method allows automatic quantification of total osteoclastic resorption as well as additional data not obtainable by the current tusk slice technique offering a better alternative for high throughput screening of potential antiresorptives. PMID:25829107

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

    NASA Astrophysics Data System (ADS)

    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.

  8. Carbon nanotubes/heteroatom-doped carbon core-sheath nanostructures as highly active, metal-free oxygen reduction electrocatalysts for alkaline fuel cells.

    PubMed

    Sa, Young Jin; Park, Chiyoung; Jeong, Hu Young; Park, Seok-Hee; Lee, Zonghoon; Kim, Kyoung Taek; Park, Gu-Gon; Joo, Sang Hoon

    2014-04-14

    A facile, scalable route to new nanocomposites that are based on carbon nanotubes/heteroatom-doped carbon (CNT/HDC) core-sheath nanostructures is reported. These nanostructures were prepared by the adsorption of heteroatom-containing ionic liquids on the walls of CNTs, followed by carbonization. The design of the CNT/HDC composite allows for combining the electrical conductivity of the CNTs with the catalytic activity of the heteroatom-containing HDC sheath layers. The CNT/HDC nanostructures are highly active electrocatalysts for the oxygen reduction reaction and displayed one of the best performances among heteroatom-doped nanocarbon catalysts in terms of half-wave potential and kinetic current density. The four-electron selectivity and the exchange current density of the CNT/HDC nanostructures are comparable with those of a Pt/C catalyst, and the CNT/HDC composites were superior to Pt/C in terms of long-term durability and poison tolerance. Furthermore, an alkaline fuel cell that employs a CNT/HDC nanostructure as the cathode catalyst shows very high current and power densities, which sheds light on the practical applicability of these new nanocomposites. PMID:24554521

  9. Chemical equilibrium model for interfacial activity of crude oil in aqueous alkaline solution: the effects of pH, alkali and salt

    SciTech Connect

    Chan, M.; Yen, T.F.

    1980-11-01

    A chemical equilibrium model for interfacial activity of crude in aqueous alkaline solution is proposed. The model predicts the observed effects of pH and concentrations of alkali and salt on the interfacial tension (IFT). The model proposed was shown to describe the observed effects of acid content, pH, and sodium ions on the interfacial activity of crude oil in water. Once the pH of the interface reaches the pKa of the acids, sometimes with the help of addition of some salt, the IFT experiences a sudden steep drop to the range of 10/sup -2/ dynes/cm. After that, further addition of sodium either in the form of NaOH or NaCl is going to increase the IFT due to a shift of equilibriumn to the formation of undissociated soap. This was confirmed by the difference in the observed effect of sodium on the IFT of the extracted soap molecules which are dissociated easily and those which are associated highly and precipitated easily. These soap molecules have dissociation constant values ranging from below 10/sup -2/ to above one. 13 references.

  10. 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)

    PubMed Central

    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 (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 (p<0.01) increase in SCC, Fe, Zn, Co and LDH occurred in SCM milk containing gram positive bacterial agents only. ALP was found to be elevated in milk infected by both gram positive and negative bacteria. The percent sensitivity, specificity and accuracy, predictive values and likelihood ratios were calculated taking bacterial culture examination and SCC≥2×105 cells/ml of milk as the benchmark. 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

  11. An alkaline-active and alkali-stable pectate lyase from Streptomyces sp. S27 with potential in textile industry.

    PubMed

    Yuan, Peng; Meng, Kun; Shi, Pengjun; Luo, Huiying; Huang, Huoqing; Tu, Tao; Yang, Peilong; Yao, Bin

    2012-06-01

    A pectate lyase gene (pl-str) was cloned from Streptomyces sp. S27 and expressed in Escherichia coli Rosetta. The full-length pl-str consists of 972 bp and encodes for a protein of 323 amino acids without signal peptide that belongs to family PF00544. The recombinant enzyme (r-PL-STR) was purified to electrophoretic homogeneity using Ni²⁺-NTA chromatography and showed apparent molecular mass of ~35 kDa. The pH optimum of r-PL-STR was found to be 10.0, and it exhibited >70% of the maximal activity at pH 12.0. After incubation at 37°C for 1 h without substrate, the enzyme retained more than 55% activity at pH 7.0-12.0. Compared with the commercial complex enzyme Scourzyme(@)301L from Novozymes, purified r-PL-STR showed similar efficacy in reducing the intrinsic viscosity of polygalacturonic acid (49.0 vs. 49.7%). When combined with cellulase and α-amylase, r-PL-STR had comparable performance in bioscouring of jute fabric (22.39 vs. 22.99%). Thus, r-PL-STR might represent a good candidate for use in alkaline industries such as textile. PMID:22278674

  12. Evaluation of the catalytic activity of Pd-Ag alloys on ethanol oxidation and oxygen reduction reactions in alkaline medium

    NASA Astrophysics Data System (ADS)

    Oliveira, M. C.; Rego, R.; Fernandes, L. S.; Tavares, P. B.

    2011-08-01

    Pd-Ag alloys containing different amounts of Ag (8, 21 and 34 at.%) were prepared in order to evaluate their catalytic activity towards the ethanol oxidation (EOR) and oxygen reduction (ORR) reactions. A sequential electroless deposition of Ag and Pd on a stainless steel disc, followed by annealing at 650 °C under Ar stream, was used as the alloy electrode deposition process. From half-cell measurements in a 1.0 M NaOH electrolyte at ≅20 °C, it was found that alloying Pd with Ag leads to an increases of the ORR and EOR kinetics, relative to Pd. Among the alloys under study, the 21 at.% Ag content alloy presents the highest catalytic activity for the EOR and the lowest Ag content alloy (8 at.% Ag) shows the highest ORR activity. Moreover, it was found that the selectivity of Pd-Ag alloys towards ORR is sustained when ethanol is present in the electrolyte.

  13. Effect of water soluble extract of nacre (Pinctada maxima) on alkaline phosphatase activity and Bcl-2 expression in primary cultured osteoblasts from neonatal rat calvaria.

    PubMed

    Moutahir-Belqasmi, F; Balmain, N; Lieberrher, M; Borzeix, S; Berland, S; Barthelemy, M; Peduzzi, J; Milet, C; Lopez, E

    2001-01-01

    The nacre (mother of pearl) layer of the oyster Pinctada maxima shell can initiate bone formation by human osteoblasts in vivo and in vitro and is a new biomaterial that induces osteogenesis. This activity of nacre could be due to its water-soluble matrix. We examined the action of a water-soluble extract of nacre on the osteoblast phenotype of cells isolated from rat neonatal calvaria by measuring alkaline phosphatase (ALP) activity and by localization of the anti-apoptotic protein Bcl-2 by immunocytochemistry. ALP activity was increased 7% (p<0.001) by 100 microg proteins/ml extract and 20% (p<0.001) by 50 microg proteins/ml extract, but a low concentration of extract decreased the ALP activity by 8%. Cells treated with a high aspartic acid content fraction of the extract had increased ALP activity (23%, p<0.0001). Nacre extract and the fraction have no effect on the proliferation of mature osteoblasts. Immunoreactive Bcl-2 was overproduced in the cytoplasm and nuclei of osteoblasts at all stages of culture. Bcl-2 was found over the whole chromatin in quiescent and mitotic cells at the end of mitosis in the two nuclei in one cell, before cytodieresis. Bcl-2 was also found over chromosomes. Thus, nacre extract stimulates Bcl-2 production in osteoblasts, that is correlated with the cell cycle. Bcl-2 was also abundant in the nucleoli of extract-treated cells. Thus, the concentration and subcellular distribution of Bcl-2 in osteoblasts in primary cultures is influenced by nacre extract, and related to the cell cycle and the regulation of gene expression. Hence, knowledge of how water-soluble extracts of Pinctada maxima nacre act on osteoblasts in vitro may reveal the mechanisms involved in its action in vivo on bone cells and bone regeneration. PMID:15348370

  14. Evaluation of pink-pigmented facultative methylotrophic bacteria for phosphate solubilization.

    PubMed

    Jayashree, Shanmugam; Vadivukkarasi, Ponnusamy; Anand, Kirupanithi; Kato, Yuko; Seshadri, Sundaram

    2011-08-01

    Thirteen pink-pigmented facultative methylotrophic (PPFM) strains isolated from Adyar and Cooum rivers in Chennai and forest soil samples in Tamil Nadu, India, along with Methylobacterium extorquens, M. organophilum, M. gregans, and M. komagatae were screened for phosphate solubilization in plates. P-solubilization index of the PPFMs grown on NBRIP-BPB plates for 7 days ranged from 1.1 to 2.7. The growth of PPFMs in tricalcium phosphate amended media was found directly proportional to the glucose concentration. Higher phosphate solubilization was observed in four strains MSF 32 (415 mg l(-l)), MDW 80 (301 mg l(-l)), M. komagatae (279 mg l(-l)), and MSF 34 (202 mg l(-l)), after 7 days of incubation. A drop in the media pH from 6.6 to 3.4 was associated with an increase in titratable acidity. Acid phosphatase activity was more pronounced in the culture filtrate than alkaline phosphatase activity. Adherence of phosphate to densely grown bacterial surface was observed under scanning electron microscope after 7-day-old cultures. Biochemical characterization and screening for methanol dehydrogenase gene (mxaF) confirmed the strains as methylotrophs. The mxaF gene sequence from MSF 32 clustered towards M. lusitanum sp. with 99% similarity. This study forms the first detailed report on phosphate solubilization by the PPFMs. PMID:21445558

  15. Digital database of mining-related features at selected historic and active phosphate mines, Bannock, Bear Lake, Bingham, and Caribou counties, Idaho

    USGS Publications Warehouse

    Causey, J. Douglas; Moyle, Phillip R.

    2001-01-01

    This report provides a description of data and processes used to produce a spatial database that delineates mining-related features in areas of historic and active phosphate mining in the core of the southeastern Idaho phosphate resource area. The data have varying degrees of accuracy and attribution detail. Classification of areas by type of mining-related activity at active mines is generally detailed; however, the spatial coverage does not differentiate mining-related surface disturbance features at many of the closed or inactive mines. Nineteen phosphate mine sites are included in the study. A total of 5,728 hc (14,154 ac), or more than 57 km2 (22 mi2), of phosphate mining-related surface disturbance are documented in the spatial coverage of the core of the southeast Idaho phosphate resource area. The study includes 4 active phosphate mines—Dry Valley, Enoch Valley, Rasmussen Ridge, and Smoky Canyon—and 15 historic phosphate mines—Ballard, Champ, Conda, Diamond Gulch, Gay, Georgetown Canyon, Henry, Home Canyon, Lanes Creek, Maybe Canyon, Mountain Fuel, Trail Canyon, Rattlesnake Canyon, Waterloo, and Wooley Valley. Spatial data on the inactive historic mines is relatively up-to-date; however, spatially described areas for active mines are based on digital maps prepared in early 1999. The inactive Gay mine has the largest total area of disturbance: 1,917 hc (4,736 ac) or about 19 km2 (7.4 mi2). It encompasses over three times the disturbance area of the next largest mine, the Conda mine with 607 hc (1,504 ac), and it is nearly four times the area of the Smoky Canyon mine, the largest of the active mines with 497 hc (1,228 ac). The wide range of phosphate mining-related surface disturbance features (approximately 80) were reduced to 13 types or features used in this study—adit and pit, backfilled mine pit, facilities, mine pit, ore stockpile, railroad, road, sediment catchment, tailings or tailings pond, topsoil stockpile, water reservoir, and disturbed

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

    PubMed

    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

  17. Phosphate Mines, Jordan

    NASA Technical Reports Server (NTRS)

    2008-01-01

    Jordan's leading industry and export commodities are phosphate and potash, ranked in the top three in the world. These are used to make fertilizer. The Jordan Phosphate Mines Company is the sole producer, having started operations in 1935. In addition to mining activities, the company produces phosphoric acid (for fertilizers, detergents, pharmaceuticals), diammonium phosphate (for fertilizer), sulphuric acid (many uses), and aluminum fluoride (a catalyst to make aluminum and magnesium).

    The image covers an area of 27.5 x 49.4 km, was acquired on September 17, 2005, and is located near 30.8 degrees north latitude, 36.1 degrees east longitude.

    The U.S. science team is located at NASA's Jet Propulsion Laboratory, Pasadena, Calif. The Terra mission is part of NASA's Science Mission Directorate.

  18. A distinct type of glycerol-3-phosphate acyltransferase with sn-2 preference and phosphatase activity producing 2-monoacylglycerol.

    PubMed

    Yang, Weili; Pollard, Mike; Li-Beisson, Yonghua; Beisson, Fred; Feig, Michael; Ohlrogge, John

    2010-06-29

    The first step in assembly of membrane and storage glycerolipids is acylation of glycerol-3-phosphate (G3P). All previously characterized membrane-bound, eukaryotic G3P acyltransferases (GPATs) acylate the sn-1 position to produce lysophosphatidic acid (1-acyl-LPA). Cutin is a glycerolipid with omega-oxidized fatty acids and glycerol as integral components. It occurs as an extracellular polyester on the aerial surface of all plants, provides a barrier to pathogens and resistance to stress, and maintains organ identity. We have determined that Arabidopsis acyltransferases GPAT4 and GPAT6 required for cutin biosynthesis esterify acyl groups predominantly to the sn-2 position of G3P. In addition, these acyltransferases possess a phosphatase domain that results in sn-2 monoacylglycerol (2-MAG) rather than LPA as the major product. Such bifunctional activity has not been previously described in any organism. The possible roles of 2-MAGs as intermediates in cutin synthesis are discussed. GPAT5, which is essential for the accumulation of suberin aliphatics, also exhibits a strong preference for sn-2 acylation. However, phosphatase activity is absent and 2-acyl-LPA is the major product. Clearly, plant GPATs can catalyze more reactions than the sn-1 acylation by which they are currently categorized. Close homologs of GPAT4-6 are present in all land plants, but not in animals, fungi or microorganisms (including algae). Thus, these distinctive acyltransferases may have been important for evolution of extracellular glycerolipid polymers and adaptation of plants to a terrestrial environment. These results provide insight into the biosynthetic assembly of cutin and suberin, the two most abundant glycerolipid polymers in nature. PMID:20551224

  19. Biofunctionalization of α-zirconium phosphate nanosheets: toward rational control of enzyme loading, affinities, activities and structure retention.

    PubMed

    Deshapriya, Inoka K; Kim, Christina S; Novak, Marc J; Kumar, Challa V

    2014-06-25

    Controlling the properties of enzymes bound to solid surfaces in a rational manner is a grand challenge. Here we show that preadsorption of cationized bovine serum albumin (cBSA) to α-Zr(IV) phosphate (α-ZrP) nanosheets promotes enzyme binding in a predictable manner, and surprisingly, the enzyme binding is linearly proportional to the number of residues present in the enzyme or its volume, providing a powerful, new predictable tool. The cBSA loaded α-ZrP (denoted as bZrP) was tested for the binding of pepsin, glucose oxidase (GOX), tyrosinase, catalase, myoglobin and laccase where the number of residues increased from the lowest value of ∼153 to the highest value of 2024. Loading depended linearly on the number of residues, rather than enzyme charge or its isoelectric point. No such correlation was seen for the binding of these enzymes to α-ZrP nanosheets without the preadsorption of cBSA, under similar conditions of pH and buffer. Enzyme binding to bZrP was supported by centrifugation studies, powder X-ray diffraction and scanning electron microscopy/energy-dispersive X-ray spectroscopy. All the bound enzymes retained their secondary structure and the extent of structure retention depended directly on the amount of cBSA preadsorbed on α-ZrP, prior to enzyme loading. Except for tyrosinase, all enzyme/bZrP biocatalysts retained their enzymatic activities nearly 90-100%, and biofunctionalization enhanced the loading, improved structure retention and supported higher enzymatic activities. This approach of using a chemically modified protein to serve as a glue, with a predictable affinity/loading of the enzymes, could be useful to rationally control enzyme binding for applications in advanced biocatalysis and biomedical applications. PMID:24853777

  20. Structural transformations in the a-tricalcium phosphate powders after mechanical activation and subsequent heat treatment

    NASA Astrophysics Data System (ADS)

    Shamray, V. F.; Karpihin, A. E.; Fedotov, A. Y.; Komlev, V. S.; Barinov, S. M.; Sirotinkin, V. P.

    2016-04-01

    X-ray diffraction analysis of the structural phase evolution in the a-TCP powder subjected to milling in the planetary mill in butanol, and followed by annealing at 1300 °C was performed. After milling, there was a systematic smoothing of the X-ray diffraction pattern of α-TCP. Significant changes in lattice constants and atomic parameters were not observed. The average particle size was reduced from 10 to 2 micrometers. The decrease of the size of coherently scattering domains CSD (250 to 80 nm) makes the main contribution to the X-ray lines broadening. The value of microstrain changes from 1 *10-6 to 2*10-4. After annealing at 1300 °C for 2 hours the phase with apatite structure was found. The content of the apatite phase increases with increasing of time of mechanical activation. Possible mechanisms of deformation of α-TCP particles and structural transformations α-TCP → HA are considered.

  1. Luminescent properties of orange emissive Sm3+-activated thermally stable phosphate phosphor for optical devices.

    PubMed

    Ratnam, B V; Jayasimhadri, M; Jang, Kiwan

    2014-11-11

    Rare earth ion activated orthophosphates have a great deal of interest due to their thermal stability for white light emitting diodes. In this regard, thermally stable Sm3+ doped NaCaPO4 (NCP) phosphor was synthesized by conventional solid state reaction technique. The phase and the structure of the as prepared powders were characterized by X-ray diffraction (XRD), field emission scanning electron microscope (FE-SEM), FT-IR, emission and excitation properties were extensively investigated for NCP phosphors. X-ray diffraction analysis confirmed the formation of NaCaPO4 with orthorhombic structure. The excitation spectra indicate that this phosphor can be effectively excited by UV light from 350 to 500 nm. All the transitions in the excitation spectrum of Sm3+ start from the ground state 6H5/2 to various excited states. The emission spectra indicated that the emitted radiation was dominated by the emission peak wavelength at 599 nm originated from the transition of 4G5/2→6H7/2. The optimum concentration of Sm3+ is determined as 1.0 mol% based on the concentration dependent emission spectra. These results suggest that the NaCaPO4:Sm3+ phosphor is a promising orange emitting phosphor under 404 nm excitation with CIE coordinates of x=0.545, y=0.41, which might be used in the development of materials for LED's and other optical devices in the visible region. PMID:24892535

  2. Calcium phosphate nanoparticles are associated with inorganic phosphate-induced osteogenic differentiation of rat bone marrow stromal cells.

    PubMed

    Chen, Xiao-rong; Bai, Jing; Yuan, Shuai-jun; Yu, Cai-xia; Huang, Jian; Zhang, Tian-lan; Wang, Kui

    2015-08-01

    In the present study, we demonstrated that calcium phosphate (CaP) nanoparticles formed in cell culture media were implicated in the process of high inorganic phosphate (Pi) mediated osteogenic differentiation of rat bone marrow stromal cells (BMSCs). Exposure of BMSCs in vitro to high Pi-containing media reduced alkaline phosphatase (ALP) activity and the expressions of osteoblast-specific genes. The sediments of CaP nanoparticles were observed at the cell surface and some of them were concomitantly found inside cells at high Pi concentration. In addition, treatment the cells with pyrophosphate (PPi), an inhibitor of calcium crystal formation, abrogated the ALP activity induced by high Pi, suggesting the contribution of CaP nanoparticles. Moreover, for isolated CaP nanoparticles, there was a trend of conversion from amorphous calcium phosphate to hydroxyapatite with elevated Pi. The particle size of CaP increased and the surface morphology changed from spherical to irregular due to increased concentrations of serum proteins incorporated into CaP nanoparticles. The study demonstrated that those physicochemical properties of CaP nanoparticles played an important role in modulating BMSCs differentiation. Furthermore, the addition of Pi in the osteogenic media resulted in a dose-dependent increase in matrix mineralization, while treatment of the cells with PPi suppressed Pi-induced calcium deposition. The findings indicated that calcium deposition in the matrix partly came from the spontaneous precipitation of CaP nanoparticles. PMID:26111760

  3. Cordysobin, a novel alkaline serine protease with HIV-1 reverse transcriptase inhibitory activity from the medicinal mushroom Cordyceps sobolifera.

    PubMed

    Wang, Shou-Xian; Liu, Yu; Zhang, Guo-Qing; Zhao, Shuang; Xu, Feng; Geng, Xiao-Li; Wang, He-Xiang

    2012-01-01

    A novel serine protease, designated as cordysobin, was purified from dried fruiting bodies of the mushroom Cordyceps sobolifera. The isolation procedure utilized ion exchange chromatography on DEAE-cellulose and SP-Sepharose followed by gel filtration on Superdex 75. The protease did not adsorb on DEAE-cellulose but bound to SP-Sepharose. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the protease resolved as a single band with an apparent molecular mass of 31 kDa. Its optimal pH was 10.0, and the optimal temperature was 65°C. The protease displayed a K(m) value of 0.41 μM and 13.44 μM·min⁻¹ using Suc-Leu-Leu-Val-Tyr-MCA as substrate at pH 10.0 and 37°C. Protease activity was enhanced by the Fe²⁺ ion at low concentration range of 1.25-10 mM and was strongly inhibited by Hg²⁺ up to 1.25 mM. The protease was strongly inhibited by chymostatin and phenylmethylsulfonyl fluoride (PMSF), suggesting that it is a serine protease. It manifested significant inhibitory activity toward HIV-1 reverse transcriptase (RT) with an IC₅₀ value of 8.2×10⁻³ μM, which is the highest anti-HIV-1 RT activity of reported mushroom proteins. PMID:22014786

  4. ATPase kinetics on activation of rabbit and frog permeabilized isometric muscle fibres: a real time phosphate assay.

    PubMed Central

    He, Z H; Chillingworth, R K; Brune, M; Corrie, J E; Trentham, D R; Webb, M R; Ferenczi, M A

    1997-01-01

    1. The rate of appearance of inorganic phosphate (Pi) and hence the ATPase activity of rabbit psoas muscle in single permeabilized muscle fibres initially in rigor was measured following laser flash photolysis of the P3-1-(2-nitrophenyl)ethyl ester of ATP (NPE-caged ATP) in the presence and absence of Ca2+. Pi appearance was monitored from the fluorescence signal of a Pi-sensitive probe, MDCC-PBP, a coumarin-labelled A197C mutant of the phosphate-binding protein from Escherichia coli. Fibres were immersed in oil to optimize the fluorescence signal and to obviate diffusion problems. The ATPase activity was also measured under similar conditions from the rate of NADH disappearance using an NADH-linked coupled enzyme assay. 2. On photolysis of NPE-caged ATP in the presence of Ca2+ at 20 degrees C, the fluorescence increase of MDCC-PBP was non-linear with time. ATPase activity was 41 s-1 in the first turnover based on a myosin subfragment 1 concentration of 150 microM. This was calculated from a linear regression of the fluorescence signal reporting 20-150 microM of Pi release. Tension was at 67% of its isometric level by the time 150 microM Pi was released. ATPase activities were 36 and 31 s-1 for Pi released in the ranges of 150-300 microM and 300-450 microM, respectively. The ATPase activity had a Q10 value of 2.9 based on measurements at 5, 12 and 20 degrees C. 3. An NADH-linked assay showed the ATPase activity had a lower limit of 12.7 s-1 at 20 degrees C. The response to photolytic release of ADP showed that the rate of NADH disappearance was partially limited by the flux through the coupled reactions. Simulations indicated that the linked assay data were consistent with an initial ATPase activity of 40 s-1. 4. On photolysis of NPE-caged ATP in the absence of Ca2+ the ATPase activity was 0.11 s-1 at 20 degrees C with no discernible rapid transient phase of Pi release during the first turnover of the ATPase. 5. To avoid the rigor state, the ATPase rate in the

  5. Comparison of phosphate materials for immobilizing cadmium in soil.

    PubMed

    Hong, Chang Oh; Chung, Doug Young; Lee, Do Kyoung; Kim, Pil Joo

    2010-02-01

    A study was conducted to compare the effects of phosphate (P) materials in reducing cadmium extractability. Seven P materials (commercial P fertilizers--fused phosphate (FP), 'fused and superphosphate' [FSP], and rock phosphate [RP]; P chemicals--Ca[H(2)PO(4)](2).H(2)O, [NH(4)](2)HPO(4), KH(2)PO(4), and K(2)HPO(4)) were selected for the test. The selected P source was mixed with Cd-contaminated soil at the rate of 0, 200, 400, 800, and 1,600 mg P kg(-1) under controlled moisture conditions at 70% of water holding capacity, then incubated for 8 weeks. FP, Ca(H(2)PO(4))(2) H(2)O, KH(2)PO(4), and K(2)HPO(4) significantly decreased NH(4)OAc-extractable Cd (plant-available form) concentrations with increasing application rates. Compared to other phosphate materials used, K(2)HPO(4) was found to be the most effective in reducing the plant-available Cd concentration in soil, mainly due to the negative charge increase caused by soil pH and phosphate adsorption. Contrary to the general information, FSP and (NH(4))(2)HPO(4) increased Cd extractability at low levels of P application (<400 mg kg(-1)), and thereafter Cd extractability decreased significantly with increasing application rate. RP scarcely had an effect on reducing Cd extractability. Ion activity products of CdHPO(4), Cd(OH)(2), and CdCO(3) analyzed by the MINTEQ program were significantly increased by K(2)HPO(4) addition, but the effect of Cd-P compound formation on reducing Cd extractability was negligible. Conclusively, the P-induced alleviation of Cd extractability can be attributed primarily to Cd immobilization due to the increase in soil pH and negative charge rather than Cd-P precipitation, and therefore, alkaline P materials such as K(2)HPO(4) are effective for immobilizing soil Cd. PMID:19633979

  6. Phosphate removal from domestic wastewater using thermally modified steel slag.

    PubMed

    Yu, Jian; Liang, Wenyan; Wang, Li; Li, Feizhen; Zou, Yuanlong; Wang, Haidong

    2015-05-01

    This study was performed to investigate the removal of phosphate from domestic wastewater using a modified steel slag as the adsorbent. The adsorption effects of alkalinity, salt, water, and thermal modification were investigated. The results showed that thermal activation at 800°C for 1 hr was the optimum operation to improve the adsorption capacity. The adsorption process of the thermally modified slag was well described by the Elovich kinetic model and the Langmuir isotherm model. The maximum adsorption capacity calculated from the Langmuir model reached 13.62 mg/g. Scanning electron microscopy indicated that the surface of the modified slag was cracked and that the texture became loose after heating. The surface area and pore volume did not change after thermal modification. In the treatment of domestic wastewater, the modified slag bed (35.5 kg) removed phosphate effectively and operated for 158 days until the effluent P rose above the limit concentration of 0.5 mg/L. The phosphate fractionation method, which is often applied in soil research, was used to analyze the phosphate adsorption behavior in the slag bed. The analysis revealed that the total contents of various Ca-P forms accounted for 81.4%-91.1%, i.e., Ca10-P 50.6%-65.1%, Ca8-P 17.8%-25.0%, and Ca2-P 4.66%-9.20%. The forms of Al-P, Fe-P, and O-P accounted for only 8.9%-18.6%. The formation of Ca10-P precipitates was considered to be the main mechanism of phosphate removal in the thermally modified slag bed. PMID:25968262

  7. Influence of phosphate and pH on myofibrillar ATPase activity and force in skinned cardiac trabeculae from rat.

    PubMed Central

    Ebus, J P; Stienen, G J; Elzinga, G

    1994-01-01

    1. The effects of inorganic phosphate (Pi) and pH on maximal calcium-activated isometric force and MgATPase activity were studied in chemically skinned cardiac trabeculae from rat. ATP hydrolysis was coupled enzymatically to the breakdown of NADH, and its concentration was determined photometrically. Measurements were performed at 2.1 microns sarcomere length and 20 degrees C. ATPase activity and force were also determined when square-wave-shaped length changes were applied, with a frequency of 23 Hz and an amplitude of 2.5%. 2. At pH 7.0 without added Pi, the average isometric force (+/- S.E.M.) was 51 +/- 3 kN m-2 (n = 23). The average isometric ATPase activity was 0.43 +/- 0.02 mM s-1 (n = 23). During the changes in length ATPase activity increased to 152 +/- 3% of the isometric value, while the average force level decreased to 48 +/- 2%. 3. Isometric force gradually decreased to 31 +/- 2% of the control value when the Pi concentration was increased to 30 mM. Isometric ATPase activity, however, remained constant for Pi concentrations up to 5 mM and decreased to 87 +/- 3% at 30 mM Pi. When Pi accumulation inside the preparation due to ATP hydrolysis was taken into account, a linear relationship was found between isometric force and log [Pi]. The decrease in relative force was found to be 44 +/- 4% per decade. 4. During the length changes, ATPase activity and average force showed, apart from the increase in ATPase activity and decrease in average force, the same dependence on Pi as the isometric values. Stiffness, estimated from the amplitude of the force responses during the length changes, decreased in proportion to isometric force when the Pi concentration was increased. The changes in the shape of the force responses due to the repetitive changes in length as a function of the Pi concentration were relatively small. These results suggest that the effect of Pi on the transitions which influence ATP turnover is rather insensitive to changes in cross

  8. Anaerobic phosphate release from activated sludge with enhanced biological phosphorus removal. A possible mechanism of intracellular pH control

    SciTech Connect

    Bond, P.L.; Keller, J.; Blackall, L.L.

    1999-06-05

    The biochemical mechanisms of the wastewater treatment process known as enhanced biological phosphorus removal (EBPR) are presently described in a metabolic model. The authors investigated details of the EBPR model to determine the nature of the anaerobic phosphate release and how this may be metabolically associated with polyhydroxyalkanoate (PHA) formation. Iodoacetate, an inhibitor of glycolysis, was found to inhibit the anaerobic formation of PHA and phosphate release, supporting the pathways proposed in the EBPR metabolic model. In the metabolic model, it is proposed that polyphosphate degradation provides energy for the microorganisms in anaerobic regions of these treatment systems. Other investigations have shown that anaerobic phosphate release depends on the extracellular pH. The authors observed that when the intracellular pH of EBPR sludge was raised, substantial anaerobic phosphate release was caused without volatile fatty