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Sample records for alkaline phosphatase isoenzyme

  1. Identification of human pulmonary alkaline phosphatase isoenzymes.

    Science.gov (United States)

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

    1997-04-01

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

  2. Changes of serum alkaline phosphatase isoenzymes in fasted rats.

    Science.gov (United States)

    Wada, H; Niwa, N; Hayakawa, T; Tsuge, H

    1996-10-01

    Changes of serum alkaline phosphatase (sALP) isoenzymes under fasting conditions were examined using polyacrylamide gel electrophoresis (PAGE), amino-acids (L-phenylalanine (L-Phe), L-homoarginine (L-HArg)) inhibition and wheat germ agglutinin (WGA) treatment. The sALP of non-fasted rats was separated into three bands (S1, S2, S3) by PAGE. The molecular weight (M.W.) of S1 corresponded to that of an isoenzyme found in the ileum. By the addition of L-Phe, the staining intensity of S1 was weakened, S2 and S3 remained unchanged and the total activity of the isoenzymes extracted from intestine decreased. On the other hand, the activity of isoenzymes extracted from kidney and bone decreased by the addition of L-HArg. Therefore, S1 was judged to be derived from intestine. The activities of total sALP and S1 decreased from 16 h of fasting. Total sALP activity and sALP activity of the supernatant prepared by WGA treatment decreased, whereas the ALP activity of the precipitate (difference between total sALP activity and supernatant sALP activity) did not change. The activity band of the precipitate corresponded to that of S3 by PAGE. Therefore, S3 was judged to be derived from bone. In conclusion, under fasting conditions, the activity of S1 decreased while the activities of S2 and S3 remained unchanged.

  3. Identification of rat serum alkaline phosphatase isoenzyme by means of wheat germ agglutinin.

    Science.gov (United States)

    Wada, H; Niwa, N; Hayakawa, T; Tsuge, H

    1997-01-01

    Wheat germ agglutinin (WGA) precipitates bone type serum alkaline phosphatase (sALP) isoenzyme specifically. The precipitates are composed of the macromolecules of WGA and "bone type sALP" (WGA-ALP complex). In order to use bone type sALP as a marker in polyacrylamide gel electrophoresis (PAGE), a method to separate "bone type sALP" from the "WGA-ALP complex" was established by using N-acetyl-D-glucosamine (GlcNAc)-Sepharose 6E column chromatography. It was concluded that this method is useful for clinical examination in the rat.

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

    DEFF Research Database (Denmark)

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

    1994-01-01

    Recent studies have demonstrated that growth hormone (GH)-deficient adults have a markedly decreased bone mineral content compared to healthy adults. However, there are conflicting results regarding the effects of GH treatment on bone mineral content in GH-deficient adults. Therefore, we evaluated...... the effect of GH treatment on a marker of bone formation (bone alkaline phosphatase), hepatic excretory function and distal forearm bone mineral content in GH-deficient adults. Growth hormone was administered subcutaneously in 21 adults (13 males and 8 females) with GH deficiency of childhood onset for 4...

  5. Increased liver alkaline phosphatase and aminotransferase ...

    African Journals Online (AJOL)

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

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

    DEFF Research Database (Denmark)

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

    1994-01-01

    Recent studies have demonstrated that growth hormone (GH)-deficient adults have a markedly decreased bone mineral content compared to healthy adults. However, there are conflicting results regarding the effects of GH treatment on bone mineral content in GH-deficient adults. Therefore, we evaluated...... the effect of GH treatment on a marker of bone formation (bone alkaline phosphatase), hepatic excretory function and distal forearm bone mineral content in GH-deficient adults. Growth hormone was administered subcutaneously in 21 adults (13 males and 8 females) with GH deficiency of childhood onset for 4...... months in a double-blind, placebo-controlled GH trial, while 13 of the patients then received further GH for an additional 14 months. Serum insulin-like growth factor I (IGF-I) increased significantly from 100 to 279 micrograms/l and IGF binding protein-3 (IGFBP-3) from 1930 to 3355 micrograms/l after 4...

  7. Isolation and characterization of a homogeneous isoenzyme of wheat germ acid phosphatase.

    Science.gov (United States)

    Waymack, P P; Van Etten, R L

    1991-08-01

    An acid phosphatase (orthophosphoric monoester phosphohydrolase, acid optimum; EC 3.1.3.2) isoenzyme from wheat germ was purified 7000-fold to homogeneity. The effect of wheat germ sources and their relationship to the isoenzyme content and purification behavior of acid phosphatases was investigated. Extensive information about the purification and stabilization of the enzyme is provided. The instability of isoenzymes in the latter stages of purification appeared to be the result of surface inactivation together with a sensitivity to dilution that could be partially offset by addition of Triton X-100 during chromatographic procedures. Added sulfhydryl protecting reagents had no effect on activity or stability, which was greatest in the pH range 4-7. The purified isoenzyme was homogeneous by polyacrylamide gel electrophoresis and exhibited the highest specific activity and turnover number reported for any acid phosphatase. The molecular weights of the pure isoenzyme and of related isoenzymes from wheat germ were found to be identical (58,000). The pure isoenzyme contained a single polypeptide chain and had a negligible carbohydrate content. The amino acid composition was determined. Of the various reasons that were considered to explain isoenzyme occurrence, a genetic basis was considered most likely. The enzyme was found to exhibit substrate inhibition with some substrates below pH 6, while above pH 8 it exhibited downwardly curving Lineweaver-Burk plots of the type that are generally described as "substrate activation". The observation of a phosphotransferase activity was consistent with the formation of a covalent phosphoenzyme intermediate, while inactivation by diethyl pyrocarbonate was consistent with the presence of an active site histidine.

  8. Further characterization of serum alkaline phosphatase from male and female beagle dogs.

    Science.gov (United States)

    Amacher, D E; Higgins, C V; Schomaker, S J; Clay, R J

    1989-01-01

    Alkaline phosphatase (AP) from the sera of both male and female beagle dogs was partially purified and then analyzed for the presence of AP isoenzymes having intestinal or osseous characteristics as detected by bromotetramisole inhibition or wheat germ lectin agarose electrophoresis, respectively. The sera from both sexes were similar in regard to the presence of AP isoenzymes with intestinal (16 vs. 20%) or osseous (19 vs. 23%) characteristics, but serum AP from the male had a greater sialic acid content and only the male serum contained a detectable constitutive acidic (pI = 3.4) AP isoenzyme. This was similar to a serum AP isoenzyme previously found elevated in the sera of dogs afflicted with hyperadrenocorticalism or of dogs treated with certain corticosteroids.

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

    Science.gov (United States)

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

    1994-09-01

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

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

    African Journals Online (AJOL)

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

  11. Osteomalacia with low alkaline phosphatase: a not so rare condition with important consequences.

    Science.gov (United States)

    Belkhouribchia, Jamal; Bravenboer, Bert; Meuwissen, Marije; Velkeniers, Brigitte

    2016-01-28

    Hypophosphatasia is a genetic disorder, characterised by a dysfunctional tissue-non-specific isoenzyme of alkaline phosphatase that impacts bone metabolism and predisposes to osteomalacia or rickets. The clinical presentation is very diverse, depending on the age of onset and the severity of the disease. Several forms of hypophosphatasia are recognised. We present a case of a 50-year-old woman with low impact fractures and loss of teeth at a young age. She also had a low alkaline phosphatase and was diagnosed with adult hypophosphatasia. Although the severe forms of hypophosphatasia are rather rare, the adult form is thought to occur quite frequently. As this condition is not well known by healthcare professionals, the time to diagnosis and initiation of adequate treatment is often postponed. When encountering a patient with low alkaline phosphatase, low bone density or a history of bone fractures, the possibility of hypophosphatasia should be considered. 2016 BMJ Publishing Group Ltd.

  12. Persistently increased intestinal fraction of alkaline phosphatase

    DEFF Research Database (Denmark)

    Nathan, E; Baatrup, G; Berg, H

    1984-01-01

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

  13. A generally applicable sequential alkaline phosphatase immunohistochemical double staining

    NARCIS (Netherlands)

    van der Loos, Chris M.; Teeling, Peter

    2008-01-01

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

  14. Relation of Serum Alkaline Phosphatase to liver scintigram in patients with hepatocellular carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Nishimura, H; Harada, T; Nawata, J; Hayakawa, M; Nishioka, M; Takemoto, T; Yokoyama, T; Takahashi, M

    1982-12-01

    Serum Alkaline Phosphatase (ALP) was studied in relation to liver scintigrams of 54 patients with hepatocellular carcinoma. The ALP activity was higher with larger tumors and in multiple tumors. Within the single tumor group, the activity was higher when the tumor was located in the hilum than in the periphery. The incidence of ALP-1 isoenzyme (bile ALP) roughly paralleled the total ALP activity. These results suggest that the variation of serum ALP seen in each individual patients with hepatocellular carcinoma reflects the volume of cholestatic liver tissue, which is changed by the number, size and localization of the tumor nodules in the liver.

  15. Tissue sources of serum alkaline phosphatase in 34 hyperthyroid cats: a qualitative and quantitative study.

    Science.gov (United States)

    Foster, D J; Thoday, K L

    2000-02-01

    The concentration of serum alkaline phosphatase (SALP) is commonly elevated in hyperthyroid cats. Agarose gel electrophoresis, in tris -barbital-sodium barbital buffer, with and without the separation enhancer neuraminidase, was used to investigate the sources of the constituent isoenzymes of SALP in serum samples from 34 hyperthyroid cats, comparing them to sera from five healthy cats and to tissue homogenates from liver, kidney, bone and duodenum. Contrary to previous reports, treatment of serum with neuraminidase made differentiation of the various isoenzymes more difficult to achieve. A single band corresponding to the liver isoenzyme (LALP) was found in 100 per cent of healthy cats. Eighty-eight per cent of the hyperthyroid cats showed two bands, corresponding to the liver and bone (BALP) isoenzymes while 12 per cent showed a LALP band alone. In hyperthyroid cats, there was a significant correlation between the serum L-thyroxine concentrations and the SALP concentrations. These findings suggest pathological changes in both bone and liver in most cases of feline thyrotoxicosis. Copyright 2000 Harcourt Publishers LtdCopyright 2000 Harcourt Publishers Ltd.

  16. Properties of Na+/K+ ATPase and alkaline phosphatase alter during spontaneous and radiation-induced leukemogenesis in mice

    International Nuclear Information System (INIS)

    Gonta-Grabiec, K.; Rossowski, W.

    1986-01-01

    Properties are characterized of Na + /K + ATPase and alkaline phosphatase in thymocytes or thymoblasts from mice of two strains: AKR in which thymoma developed spontaneously, and C57Bl in which the development was induced by X-irradiation (total dose: 5.4 Gy in 3 fractions). It was found that before thymoma could be discerned morphologically the properties of the two enzymes changed. There was a decrease in 86 Rb uptake and in the rate of ATP hydrolysis per cell (both strains) as well as an increase in alkaline phosphatase activity per cell (C57Bl mice). In both spontaneous and radiation-induced thymomas 86 Rb uptake, ATP hydrolysis and 3 H-ouabain binding per cell were higher than in normal thymuses. Likewise, alkaline phosphatase activity per cell was higher in the thymomas than in the thymuses; this increase was accompanied by the appearance of additional isoenzyme(s) (1 in AKR, 2 in C57Bl). These changes were compared with cAMP content and 3 H-thymidine incorporation, taken as indicators of the proliferative activity, and their high correlation in both AKR and C57Bl mice allowed to distinguish a pre-leukemic period. In that period thymoblasts clearly differed from the normal ones in Na + /K + ATPase and alkaline phosphatase properties as well as proliferation, although the morphology of the thymus was still unchanged. (author)

  17. Alkaline phosphatase activity of rumen bacteria.

    Science.gov (United States)

    Cheng, K J; Costerton, J W

    1977-11-01

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

  18. Alkaline Phosphatase, an Unconventional Immune Protein

    Directory of Open Access Journals (Sweden)

    Bethany A. Rader

    2017-08-01

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

  19. Overexpression of Human Bone Alkaline Phosphatase in Pichia Pastoris

    Science.gov (United States)

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

    2000-01-01

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

  20. Serum alkaline phosphatase screening for vitamin D deficiency states

    International Nuclear Information System (INIS)

    Shaheen, S.; Barrakzai, Q.

    2012-01-01

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

  1. Characterization of Human Bone Alkaline Phosphatase in Pichia Pastoris

    Science.gov (United States)

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

    1999-01-01

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

  2. Elevated Serum Level of Human Alkaline Phosphatase in Obesity

    International Nuclear Information System (INIS)

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

    2015-01-01

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

  3. Dephosphorylation of endotoxin by alkaline phosphatase in vivo

    NARCIS (Netherlands)

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

    1997-01-01

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

  4. 21 CFR 864.7660 - Leukocyte alkaline phosphatase test.

    Science.gov (United States)

    2010-04-01

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

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

    Science.gov (United States)

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

    2006-02-01

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

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

    Science.gov (United States)

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

    1985-01-01

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

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

    Science.gov (United States)

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

    2002-01-01

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

  8. Alkaline phosphatase as a screening test for osteomalacia.

    Science.gov (United States)

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

    2011-01-01

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

  9. A double antibody radioimmunoassay specific for placental alkaline phosphatase

    International Nuclear Information System (INIS)

    Dass, S.; Bagshawe, K.D.

    1984-01-01

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

  10. Chromatographic separation of alkaline phosphatase from dental enamel

    DEFF Research Database (Denmark)

    Moe, D; Kirkeby, S; Salling, E

    1989-01-01

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

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  13. The influence of complexing pharmaceutical compositions on alkaline phosphatase

    Science.gov (United States)

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

    2011-06-01

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

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

    NARCIS (Netherlands)

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

    1993-01-01

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

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

    Science.gov (United States)

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

    2015-12-01

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

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

    Science.gov (United States)

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

    2017-02-01

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

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

    DEFF Research Database (Denmark)

    Faerk, J; Peitersen, Birgit; Petersen, S

    2002-01-01

    BACKGROUND: The bone mineral content of premature infants at term is lower than in mature infants at the same postconceptional age. Serum alkaline phosphatase and serum phosphate are often used as indicators of bone mineralisation. OBJECTIVE: To analyse the association between bone mineral content...... content was measured at term (mean gestational age 41 weeks) by dual energy x ray absorptiometry and corrected for body size. RESULTS: Serum alkaline phosphatase was significantly negatively associated with serum phosphate (p mineral content was not associated with mean serum alkaline...... and serum alkaline phosphatase and serum phosphate. METHODS: Serum alkaline phosphatase and phosphate were measured at weekly intervals during admission in 108 premature infants of gestational age below 32 weeks (mean (SD) gestational age 29 (2) weeks; mean (SD) birth weight 1129 (279) g). Bone mineral...

  18. Genetic evaluations of Chinese patients with odontohypophosphatasia resulting from heterozygosity for mutations in the tissue-non-specific alkaline phosphatase gene.

    Science.gov (United States)

    Wan, Jia; Zhang, Li; Liu, Tang; Wang, Yewei

    2017-08-01

    Hypophosphatasia is a rare heritable metabolic disorder characterized by defective bone and tooth mineralization accompanied by a deficiency of tissue-non-specific (liver/bone/kidney) isoenzyme of alkaline phosphatase activity, caused by a number of loss-of-function mutations in the alkaline phosphatase liver type gene. We seek to explore the clinical manifestations and identify the mutations associated with the disease in a Chinese odonto- hypophosphatasia family. The proband and his younger brother affected with premature loss of primary teeth at their 2-year-old. They have mild abnormal serum alkaline phosphatase and 25-hydroxy vitamin D values, but the serum alkaline phosphatase activity of their father, mother and grandmother, who showed no clinical symptoms of hypophosphatasia, was exhibited significant decreased. In addition to premature loss of primary teeth, the proband and his younger brother showed low bone mineral density, X-rays showed that they had slight metaphyseal osteoporosis changes, but no additional skeletal abnormalities. Deoxyribonucleic acid sequencing and analysis revealed a single nucleotide polymorphism c.787T>C (p.Y263H) in exon 7 and/or a novel mutation c.-92C>T located at 5'UTR were found in the affected individuals. We examined all individuals of an odonto- hypophosphatasia family by clinical and radiographic examinations as well as laboratory assays. Furthermore, all 12 exons and the exon-intron boundaries of the alkaline phosphatase liver type gene were amplified and directly sequenced for further analysis and screened for mutations. Our present findings suggest the single nucleotide polymorphism c.787T>C and c.-92C>T should be responsible for the odonto- hypophosphatasia disorders in this family.

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

    Science.gov (United States)

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

    2002-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Grozdea Jean J

    2002-01-01

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

  1. COMPARISON OF METHODS FOR ALKALINE PHOSPHATASE AND PEROXIDASE DETECTION IN MILK

    Directory of Open Access Journals (Sweden)

    felipe Nael Seixas

    2014-02-01

    Full Text Available This study evaluated the performance of strips for colorimetric detection of alkaline phosphatase and peroxidase in milk, comparing them with a kit of reagents for alkaline phosphatase and the official methodology for peroxidase. The samples were analyzed at the Laboratory Inspection of Products of Animal Origin, State University of Londrina. For the comparison tests for the detection of alkaline phosphatase four treatments were made by adding different percentages of raw milk (1%, 2%, 5% and 10% in the pasteurized milk, plus two control treatments. Thirty-eight samples triplicate for each treatment were analyzed. To compare the performance of tests for peroxidase 80 pasteurized milk samples were evaluated simultaneously by official methodology and by colorimetric strips. The performance of the alkaline phosphatase were different for the treatments with 1% and 2% of raw milk which had all the strips change color as the reagent kit showed the presence of phosphatase in just 2.63% and 5.26% the cases, respectively for each treatment. The colorimetric strips for alkaline phosphatase are more sensitive for the identification of small quantities compared to the reagent kit. The performance of tests for peroxidase showed no difference. The strips for the detection of peroxidase or alkaline phosphatase were effective and can replace traditional methods.

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

    NARCIS (Netherlands)

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

    2013-01-01

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

  3. Subcellular localization of alkaline phosphatase in Bacillus licheniformis 749/C by immunoelectron microscopy with colloidal gold

    International Nuclear Information System (INIS)

    Tinglu, G.; Ghosh, A.; Ghosh, B.K.

    1984-01-01

    Subcellular distribution of the alkaline phosphatase of Bacillus licheniformis 749/C was determined by an immunoelectron microscopy method. Anti-alkaline phosphatase antibody labeled with 15- to 18-nm colloidal gold particles (gold-immunoglobulin G [IgG] complex) were used for the study. Both the plasma membrane and cytoplasmic material were labeled with the gold-IgG particles. These particles formed clusters in association with the plasma membrane; in contrast, in the cytoplasm the particles were largely dispersed, and only a few clusters were found. The gold-IgG binding was quantitatively estimated by stereological analysis of labeled, frozen thin sections. This estimation of a variety of control samples showed that the labeling was specific for the alkaline phosphatase. Cluster formation of the gold -IgG particles in association with the plasma membrane suggests that existence of specific alkaline phosphatase binding sites (receptors) in the plasma membrane of B. licheniformis 749/C. 27 references, 6 figures, 1 table

  4. Imaging of alkaline phosphatase activity in bone tissue.

    Directory of Open Access Journals (Sweden)

    Terence P Gade

    Full Text Available The purpose of this study was to develop a paradigm for quantitative molecular imaging of bone cell activity. We hypothesized the feasibility of non-invasive imaging of the osteoblast enzyme alkaline phosphatase (ALP using a small imaging molecule in combination with (19Flourine magnetic resonance spectroscopic imaging ((19FMRSI. 6, 8-difluoro-4-methylumbelliferyl phosphate (DiFMUP, a fluorinated ALP substrate that is activatable to a fluorescent hydrolysis product was utilized as a prototype small imaging molecule. The molecular structure of DiFMUP includes two Fluorine atoms adjacent to a phosphate group allowing it and its hydrolysis product to be distinguished using (19Fluorine magnetic resonance spectroscopy ((19FMRS and (19FMRSI. ALP-mediated hydrolysis of DiFMUP was tested on osteoblastic cells and bone tissue, using serial measurements of fluorescence activity. Extracellular activation of DiFMUP on ALP-positive mouse bone precursor cells was observed. Concurringly, DiFMUP was also activated on bone derived from rat tibia. Marked inhibition of the cell and tissue activation of DiFMUP was detected after the addition of the ALP inhibitor levamisole. (19FMRS and (19FMRSI were applied for the non-invasive measurement of DiFMUP hydrolysis. (19FMRS revealed a two-peak spectrum representing DiFMUP with an associated chemical shift for the hydrolysis product. Activation of DiFMUP by ALP yielded a characteristic pharmacokinetic profile, which was quantifiable using non-localized (19FMRS and enabled the development of a pharmacokinetic model of ALP activity. Application of (19FMRSI facilitated anatomically accurate, non-invasive imaging of ALP concentration and activity in rat bone. Thus, (19FMRSI represents a promising approach for the quantitative imaging of bone cell activity during bone formation with potential for both preclinical and clinical applications.

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

    Directory of Open Access Journals (Sweden)

    Lacramioara Oprica

    2011-12-01

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

  6. Alkaline phosphatase expression during relapse after orthodontic tooth movement

    Directory of Open Access Journals (Sweden)

    Pinandi Sri Pudyani

    2014-03-01

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

  7. Associations between renal hyperfiltration and serum alkaline phosphatase.

    Directory of Open Access Journals (Sweden)

    Se Won Oh

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

  8.  Alkaline phosphatase normalization is a biomarker of improved survival in primary sclerosing cholangitis.

    Science.gov (United States)

    Hilscher, Moira; Enders, Felicity B; Carey, Elizabeth J; Lindor, Keith D; Tabibian, James H

    2016-01-01

     Introduction. Recent studies suggest that serum alkaline phosphatase may represent a prognostic biomarker in patients with primary sclerosing cholangitis. However, this association remains poorly understood. Therefore, the aim of this study was to investigate the prognostic significance and clinical correlates of alkaline phosphatase normalization in primary sclerosing cholangitis. This was a retrospective cohort study of patients with a new diagnosis of primary sclerosing cholangitis made at an academic medical center. The primary endpoint was time to hepatobiliaryneoplasia, liver transplantation, or liver-related death. Secondary endpoints included occurrence of and time to alkaline phosphatase normalization. Patients who did and did not achieve normalization were compared with respect to clinical characteristics and endpoint-free survival, and the association between normalization and the primary endpoint was assessed with univariate and multivariate Cox proportional-hazards analyses. Eighty six patients were included in the study, with a total of 755 patient-years of follow-up. Thirty-eight patients (44%) experienced alkaline phosphatase normalization within 12 months of diagnosis. Alkaline phosphatase normalization was associated with longer primary endpoint-free survival (p = 0.0032) and decreased risk of requiring liver transplantation (p = 0.033). Persistent normalization was associated with even fewer adverse endpoints as well as longer survival. In multivariate analyses, alkaline phosphatase normalization (adjusted hazard ratio 0.21, p = 0.012) and baseline bilirubin (adjusted hazard ratio 4.87, p = 0.029) were the only significant predictors of primary endpoint-free survival. Alkaline phosphatase normalization, particularly if persistent, represents a robust biomarker of improved long-term survival and decreased risk of requiring liver transplantation in patients with primary sclerosing cholangitis.

  9. A study of the alkaline and acid phosphatase activities in acute uranium intoxication

    International Nuclear Information System (INIS)

    Bokova, N.; Pavlova, V.; Stancheva, Yu.; Khadzhirusev, S.; Kiradzhiev, G.

    1975-01-01

    Comparative study of the ability of the sodium salt of diethylbarbituric acid and acetazolamide to protect the kidneys is conducted under conditions of acute uranium intoxication in rats. The parameters studied are alkaline and acid phosphatase activities in the serum and urine and phosphatase activity in the kidneys (histochemically as described by Gomori) followed up until the 30th day after the total uranyl acetate dose was reached (2 or 7 mg per kg bodyweight). Either compound exerted only minor effect on serum alkaline phosphatase activity. Sodium diethylbarbiturate induced distinct fluctuations in urinary alkaline phosphatase activity throughout the entire study period, but the differences never reached statistical significance. Acetazolamide caused essential decrease in urinary alkaline phosphatase activity. In either case renal tissue protection from the action of the uranyl ion may be suggested. This assumption is supported by the histochemical analysis. The compounds appeared to have no effect on serum acid phosphatase activity which showed high variability both in control and in treated rats. (Ch.K.)

  10. Sensitive detection of alkaline phosphatase by switching on gold nanoclusters fluorescence quenched by pyridoxal phosphate.

    Science.gov (United States)

    Halawa, Mohamed Ibrahim; Gao, Wenyue; Saqib, Muhammad; Kitte, Shimeles Addisu; Wu, Fengxia; Xu, Guobao

    2017-09-15

    In this work, we designed highly sensitive and selective luminescent detection method for alkaline phosphatase using bovine serum albumin functionalized gold nanoclusters (BSA-AuNCs) as the nanosensor probe and pyridoxal phosphate as the substrate of alkaline phosphatase. We found that pyridoxal phosphate can quench the fluorescence of BSA-AuNCs and pyridoxal has little effect on the fluorescence of BSA-AuNCs. The proposed mechanism of fluorescence quenching by PLP was explored on the basis of data obtained from high-resolution transmission electron microscopy (HRTEM), dynamic light scattering (DLS), UV-vis spectrophotometry, fluorescence spectroscopy, fluorescence decay time measurements and circular dichroism (CD) spectroscopy. Alkaline phosphatase catalyzes the hydrolysis of pyridoxal phosphate to generate pyridoxal, restoring the fluorescence of BSA-AuNCs. Therefore, a recovery type approach has been developed for the sensitive detection of alkaline phosphatase in the range of 1.0-200.0U/L (R 2 =0.995) with a detection limit of 0.05U/L. The proposed sensor exhibit excellent selectivity among various enzymes, such as glucose oxidase, lysozyme, trypsin, papain, and pepsin. The present switch-on fluorescence sensing strategy for alkaline phosphatase was successfully applied in human serum plasma with good recoveries (100.60-104.46%), revealing that this nanosensor probe is a promising tool for ALP detection. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Relationship of serum and saliva calcium, phosphorus and alkaline phosphatase with dry mouth feeling in menopause.

    Science.gov (United States)

    Agha-Hosseini, Farzaneh; Mirzaii-Dizgah, Iraj; Moosavi, Mahdieh-Sadat

    2012-06-01

    The aim of this study was to compare serum and saliva calcium, phosphorus and alkaline phosphatase of menopausal women with/without dry mouth (DM) feeling. The composition of saliva in menopause women with/without DM feeling is different. Some of these differences are in hormones that are related to bone turnover. A case-control study was carried out on 60 selected menopausal women aged 45-79 years with or without DM feeling (30 as case, 30 as control), conducted at the Clinic of Oral Medicine, Tehran University of Medical Sciences. The phosphorus concentration was measured by photometrical measurement of the blue colour formed after the addition of ammonium molybdate and stannous chloride; calcium was measured by Arsenazo reaction; and alkaline phosphatase by the pNPP-AMP method. Statistical analysis of Student's t-test was used. The mean serum phosphorus and alkaline phosphatase, stimulated and unstimulated saliva calcium and alkaline phosphatase levels were significantly higher in the menopausal women suffering from DM. There were no significant differences between groups regarding saliva phosphorus and serum calcium concentration. Calcium, phosphorus and alkaline phosphatase appear associated with DM feeling in menopause. © 2012 The Gerodontology Society and John Wiley & Sons A/S.

  12. Water Quality Interaction with Alkaline Phosphatase in the Ganga River: Implications for River Health.

    Science.gov (United States)

    Yadav, Amita; Pandey, Jitendra

    2017-07-01

    Carbon, nitrogen and phosphorus inputs through atmospheric deposition, surface runoff and point sources were measured in the Ganga River along a gradient of increasing human pressure. Productivity variables (chlorophyll a, gross primary productivity, biogenic silica and autotrophic index) and heterotrophy (respiration, substrate induced respiration, biological oxygen demand and fluorescein diacetate hydrolysis) showed positive relationships with these inputs. Alkaline phosphatase (AP), however, showed an opposite trend. Because AP is negatively influenced by available P, and eutrophy generates a feedback on P fertilization, the study implies that the alkaline phosphatase can be used as a high quality criterion for assessing river health.

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

    DEFF Research Database (Denmark)

    Gluud, C; Andersen, I; Dietrichson, O

    1981-01-01

    and alkaline phosphatase in 18% and 7%. Neither the activity of gamma-glutamyltransferase, aspartate aminotransferase nor alkaline phosphatase showed any significant (P greater than 0.05) correlation with the history of alcohol consumption. The activities of gamma-glutamyltransferase and aspartate...

  14. Alkaline phosphatase immobilization onto Bio-Gide(R) and Bio-Oss(R) for periodontal and bone regeneration.

    NARCIS (Netherlands)

    Oortgiesen, D.A.W.; Plachokova, A.S.; Geenen, C.; Meijer, G.J.; Walboomers, X.F.; Beucken, J.J.J.P van den; Jansen, J.B.M.J.

    2012-01-01

    AIM: To evaluate the effect of alkaline phosphatase (ALP) immobilization onto Bio-Gide((R)) in vitro, and to study the in vivo performance of ALP-enriched Bio-Gide((R)) and/or Bio-Oss((R)) with the purpose to enhance periodontal regeneration. MATERIALS AND METHODS: Alkaline phosphatase ALP was

  15. Enzymatic mineralization of hydrogels for bone tissue engineering by incorporation of alkaline phosphatase.

    NARCIS (Netherlands)

    Douglas, T.E.L.; Messersmith, P.B.; Chasan, S.; Mikos, A.G.; Mulder, E.L.W. de; Dickson, G.; Schaubroeck, D.; Balcaen, L.; Vanhaecke, F.; Dubruel, P.; Jansen, J.A.; Leeuwenburgh, S.C.G.

    2012-01-01

    Alkaline phosphatase (ALP), an enzyme involved in mineralization of bone, is incorporated into three hydrogel biomaterials to induce their mineralization with calcium phosphate (CaP). These are collagen type I, a mussel-protein-inspired adhesive consisting of PEG substituted with catechol groups,

  16. A Ten-Week Biochemistry Lab Project Studying Wild-Type and Mutant Bacterial Alkaline Phosphatase

    Science.gov (United States)

    Witherow, D. Scott

    2016-01-01

    This work describes a 10-week laboratory project studying wild-type and mutant bacterial alkaline phosphatase, in which students purify, quantitate, and perform kinetic assays on wild-type and selected mutants of the enzyme. Students also perform plasmid DNA purification, digestion, and gel analysis. In addition to simply learning important…

  17. Investigations into the stabilisation of drugs by sugar glasses : I. Tablets prepared from stabilised alkaline phosphatase

    NARCIS (Netherlands)

    Eriksson, H.J.C.; Hinrichs, W.L.J.; van Veen, B.; Somsen, G.W.; de Jong, G.J.; Frijlink, H.W.

    2002-01-01

    The aim of this study was to investigate the formulation of sugar glass stabilised alkaline phosphatase from bovine intestine (BIAP) into tablets. Two major subjects of tablet formulation were investigated. First, the compaction behaviour of the inulin sugar glass was investigated. Secondly, the

  18. Benomyl inhibits phosphorus transport but not fungal alkaline phosphatase activity in a Glomus-cucumber symbiosis

    DEFF Research Database (Denmark)

    Larsen, J.; Thingstrup, I.; Jakobsen, I.

    1996-01-01

    when benomyl was applied to the HC at 10 µg g-1 soil, whereas the uptake of 32P from RHC I roots + hyphae) was reduced only at the highest dose of application to the RHC (100 µ g g-1 soil). In contrast to the marked reduction of benomyl on fungal P transport, the activity of fungal alkaline phosphatase...

  19. [Serum calcium and phosphorus concentration and alkaline phosphatase activity in healthy children during growth and development].

    Science.gov (United States)

    Savić, Ljiljana; Savić, Dejan

    2008-01-01

    Many changes happen during growth and development in an organism as a result of important hormon changes, especially biohumoral ones. These changes make a problem when interpreting biochemical results in pediatric population. The most important changes are intensive calcium and phosphorus metabolic turnover in bone tissue with changes in alkaline phosphatase activity as a result of osteoblast activity. The aim of this study was to follow the serum calcium and phosphorus concentration and alkaline phosphatase activity in children 1-15 years old in different growth and development period and of different sexes and to fortify the influence of growth and development dynamics on biohumoral status in healthy male and female children. We evaluated 117 healthy children of both sexes from 1-15 years of age and divided them into three age groups: 1-5, 6-10 and 11-15 years. We followed the serum calcium and phosphorus concentration and alkaline phosphatase activity in different groups and in different sexes. Our investigation found significantly higher values of serum calcium in boys than in girls with no important changes between the age groups and significantly higher values of serum phosphorus in the youngest age group in all children and in different sexes with no important sex differences. Alkaline phosphatase activity followed the growth spurt and was the biggest in 6-10 years group in girls and in 11-15 years group in boys.

  20. Quantification of the histochemical reaction for alkaline phosphatase activity using the indoxyl-tetranitro BT method

    NARCIS (Netherlands)

    van Noorden, C. J.; Jonges, G. N.

    1987-01-01

    The indoxyl-tetranitro BT method for the demonstration of alkaline phosphatase activity has been optimized and its validity for quantitative histochemistry tested. The study has been performed with model films of polyacrylamide gel incorporating homogenate of rat liver and with cryostat sections

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    M. P. Patil

    2015-03-01

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

  3. [Kinetic study on inhibition effects of dansyl-L-phenylalanine and L-phenylalanine on calf intestinal alkaline phosphatase].

    Science.gov (United States)

    Li, Li-Na; Wu, Yu-Qing; Buchet, René

    2009-10-01

    To evaluate the inhibition effect of dansyl-L-phenylalanine on calf intestinal alkaline phosphatase (CIAP), UV-Vis spectrophotometric method was employed. It was found that dansyl-L-phenylalanine can selectively inhibit CIAP. The kinetic inhibition processes of dansyl-L-phenylalanine and L-phenylalanine were comparatively studied. The authors' finding elucidates that at the optimized alkaline pH of alkaline phosphatase (pH 10.4) and 37 degrees C, dansyl-L-phenylalanine can inhibit alkaline phosphatase activity of CIAP efficiently and specifically, similar as L-phenylalanine. Both inhibition types were uncompetitive inhibition resulting from the double reciprocal curve fitting of upsilon versus substrate concentrations, and the inhibition constants Ki of both inhibitors were determined to be 2.3 and 1.1 mmol L(-1) respectively, both of which were at millimolar level. The investigation of the inhibition effect of dansyl modified L-phenylalanine on calf intestinal alkaline phosphatase not only helped get insight into the detailed inhibition mechanism of L-phenylalanine on tissue specific alkaline phosphatase, such as in the case of intestinal alkaline phosphatase, but also provided the possibility to employ fluorescence spectroscopy by labeling the specific inhibitors of alkaline phosphatase with chromophoric groups.

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

    Science.gov (United States)

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

    1999-01-01

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

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

    Science.gov (United States)

    Berezhetskyy, A.

    2008-09-01

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

  6. Significance of bone specific alkaline phosphatase as a tumor marker in malignant bone tumor

    International Nuclear Information System (INIS)

    Kim, Sug Jun; Jeon, Dae Geun; Huh, Kwang

    1998-01-01

    The relationship between total alkaline phosphatase activity and bone forming lesion is a well known fact. But alkaline phosphatase consist mainly of two portion (liver, bone). To clarify the exact activity of bone forming tissue, quantitative measurement of BALP is essential. Two finds of tests were performed for their feasibility as a laboratory test (wheat germ lectin vs electrophoresis). We analyzed 40 bony lesion and got 58 samples. Lectin method was simple, economic, with reliable resproducability. Owing to the small number of test sample, we could not identify the relationship between the disease activity and measured BALP level. Further collection of clinical sample and analysis the pattern of BALP on each clinical settings. (author). 8 refs

  7. Assessment of corticosteroid-induced alkaline phosphatase as a prognostic indicator in canine lymphoma.

    Science.gov (United States)

    Wiedemann, A L; Charney, S C; Barger, A M; Schaeffer, D J; Kitchell, B E

    2005-04-01

    To examine the incidence of elevated corticosteroid-induced alkaline phosphatase (sALP) in dogs with lymphoma and to determine if sALP is a reliable prognostic indicator in canine lymphoma. The medical records of 62 canine lymphoma patients treated with a combination chemotherapy protocol from 1994 to 2003 at the University of Illinois Veterinary Teaching Hospital were examined. Variables assessed with respect to response rate and remission duration included age, bodyweight, sex, breed, World Health Organization stage (I to V), substage (a or b), pretreatment administration of corticosteroid, and serum levels of alkaline phosphatase, sALP and alanine aminotransferase. sALP was not statistically significant with respect to response rate or duration of remission, nor was preinduction glucocorticoid administration. Stage was significant with respect to achieving remission. It was found that sALP is not a useful prognostic indicator for response rate and remission duration in dogs with lymphoma.

  8. and alanine (EC. 2.6.1.2) transaminases, and alkaline phosphatase

    African Journals Online (AJOL)

    The activities of aspartate (E.C. 2.6.1.1) and alanine (E.C. 2.6.1.2) transaminases (AST and ALT, respectively), and alkaline phosphatase (ALP) (E.C. 3.1.3.1) were determined in erythrocytes obtained from 20 HbAA, 15 HbAS and 12 HbSS human subjects. The results showed that the three enzymes had different levels of ...

  9. The effects of Zinc supplementation on serum zinc, alkaline phosphatase activity and fracture healing of bones

    International Nuclear Information System (INIS)

    Sadighi, A.; Moradi, A.; Roshan, Marjan M.; Ostadrahimi, A.

    2009-01-01

    Objective was to determine the effect of zinc supplementation on callus information, serum zinc and alkaline phosphatase activity in humans. This randomized, double-blind, placebo controlled clinical trial was conducted on 60 patients with traumatic bone fracture referred to Shohada Hospital of Tabriz, Iran from August to December 2007. Subjects were randomly divided into 2 groups: cases (n=30), receiving one capsule of zinc sulfate consists of 50 mg zinc each day and the controls (n=30), receiving placebo for 60 days. Individual and clinical information was determined by a questionnaire: nutritional intake by 3 days food records at the beginning and the end of trial. Serum zinc and alkaline phosphatase was measured by atomic absorption spectroscopy and by enzymatic method. Callus information during fracture healing was evaluated by radiography of the bone. There was no significant difference in physical activity, gender, age, type of fractures and nutrient intake, between the 2 groups. The administration of zinc caused a significant elevation of serum zinc and alkaline phosphatase activity. Assessment of bone x-rays showed a significant progress in callus formation in cases compared to the controls. This study shows that zinc supplementation can stimulate fracture healing, however, it needs further study. (author)

  10. Application of Scharer's quantitative method for the determination of residual alkaline phosphatase activity in standard Minas

    Directory of Open Access Journals (Sweden)

    C.F. Soares

    2013-08-01

    Full Text Available Milk pasteurization is a critical issue in the dairy industry, and failures in this process can affect final product safety. Scharer's enzymatic method is still traditionally used to verify pasteurization efficiency compliance, and it is based on screening for residual alkaline phosphatase in milk. Although several methods are used to quantify enzymatic activity to assess milk pasteurization efficiency, there is a small amount of published data regarding the use of these methods to quantify alkaline phosphatase in cheese. In this study, the Scharer's modified method was used to determine the levels of residual alkaline phosphatase in standard minas cheese, before and after 20 days of ripening. The cheeses were made using raw or pasteurized milk with the addition of different concentrations of raw milk (0; 0.05%; 0.10%; 0.20%; and 0.50%. In the fresh cheese samples, the method showed a sensitivity of only 0.50% with the addition of raw milk to the pasteurized milk used to make cheese. In addition, levels of up 0.20% of raw milk in pasteurized milk, the concentrations of phenol was inferior to 1μg phenol/g of dairy product which is the preconized indicator value for adequate pasteurization.

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

    Digital Repository Service at National Institute of Oceanography (India)

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

    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...

  12. Dephosphorylation of microtubule-binding sites at the neurofilament-H tail domain by alkaline, acid, and protein phosphatases.

    Science.gov (United States)

    Hisanaga, S; Yasugawa, S; Yamakawa, T; Miyamoto, E; Ikebe, M; Uchiyama, M; Kishimoto, T

    1993-06-01

    The dephosphorylation-induced interaction of neurofilaments (NFs) with microtubules (MTs) was investigated by using several phosphatases. Escherichia coli alkaline and wheat germ acid phosphatases increased the electrophoretic mobility of NF-H and NF-M by dephosphorylation, and induced the binding of NF-H to MTs. The binding of NFs to MTs was observed only after the electrophoretic mobility of NF-H approached the exhaustively dephosphorylated level when alkaline phosphatase was used. The number of phosphate remaining when NF-H began to bind to MTs was estimated by measuring phosphate bound to NF-H. NF-H did not bind to MTs even when about 40 phosphates from the total of 51 had been removed by alkaline phosphatase. The removal of 6 further phosphates finally resulted in the association of NF-H with MTs. A similar finding, that the restricted phosphorylation sites in the NF-H tail domain, but not the total amount of phosphates, were important for binding to MTs, was also obtained with acid phosphatases. In contrast to alkaline and acid phosphatases, four classes of protein phosphatases (protein phosphatases 1, 2A, 2B, and 2C) were ineffective for shifting the electrophoretic mobility of NF proteins and for inducing the association of NFs to MTs.

  13. Alkaline and Acid Phosphatase Activity in Blood Plasma of Chickens Irradiated by Low dose Gamma Radiation

    International Nuclear Information System (INIS)

    Petar, K.; Marinko, V.; Saveta, M.; Miljenko, S.

    2004-01-01

    In our previous paper (Kraljevic et, al, 2000; Kraljevic et al 2002) we showed that the growth of the chickens hatched from eggs irradiated with 0.15 Gy gamma-rays before incubation was significantly higher than in controls during the fattening period (1-42 days). The concentration of total protein, glucose and cholesterol in the blood plasma of the same chickens was also significantly changed. In this paper an attempt was made to determine the effect of irradiation of eggs by low dose ionizing radiation before incubation upon activity of alkaline and acid phosphatase in the blood plasma of chickens hatched from irradiated eggs. The eggs of heavy breeding chickens were irradiated by dose of 0.15 Gy gamma radiation (60 Co) before incubation. Along with the chickens which were hatched from irradiated eggs, there was a control group of chickens hatched from nonirradiated eggs. All other conditions were the same for both groups. After hatching, blood samples were taken from the wing vein on days 1, 3, 5, 6, 10, 20, 30 and 42. The activity of both enzymes was determined spectrophotometrically by using Boehring Mannheim GmbH optimized kits. the activity of alkaline phosphatase in blood plasma was decreased on days 42, and the activity of acid phosphatase in the blood plasma of the same chickens was increased on day 42. Obtained results confirm our early obtained results that low dose of gamma radiation has effects upon metabolic processes in the chickens hatched from eggs irradiated before incubation. (Author)

  14. Study on alkaline and acid phosphatase activity in acute uranium intoxication

    International Nuclear Information System (INIS)

    Bokova, N.V.; Pavlova, V.B.; Stancheva, Yu.A.; Khadzhirusev, S.B.; Kiradzhiev, G.D.

    1975-01-01

    The protective potential of diethyl barbituric acid sodium salt is studied, in comparison with that of acetazolamide, on kidneys under acute uranium intoxication. Experiments involved rats given intraperitoneal injections with uranyl acetate on 12 successive days up to a total dose of 0.5, 2.0 or 7.0 mg/kg. The resulting effects are measured by chemical assays of serum and urine for alkaline and acid phosphatase and histochemical assays for phosphatase activities in kidneys, kinetics being followed over a 30-day period after total dose administration. Protection of kidneys from toxic uranium effects was found to be of about the same degree with sodium diethyl barbiturate as with acetazolamide. (A.B.)

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

    Science.gov (United States)

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

    2015-01-01

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

  16. Production, secretion, and stability of human secreted alkaline phosphatase in tobacco NT1 cell suspension cultures.

    Science.gov (United States)

    Becerra-Arteaga, Alejandro; Mason, Hugh S; Shuler, Michael L

    2006-01-01

    Tobacco NT1 cell suspension cultures secreting active human secreted alkaline phosphatase (SEAP) were generated for the first time as a model system to study recombinant protein production, secretion, and stability in plant cell cultures. The SEAP gene encodes a secreted form of the human placental alkaline phosphatase (PLAP). During batch culture, the highest level of active SEAP in the culture medium (0.4 U/mL, corresponding to approximately 27 mg/L) was observed at the end of the exponential growth phase. Although the level of active SEAP decreased during the stationary phase, the activity loss did not appear to be due to SEAP degradation (based on Western blots) but due to SEAP denaturation. The protein-stabilizing agents polyvinylpirrolidone (PVP) and bacitracin were added extracellularly to test for their ability to reduce the loss of SEAP activity during the stationary phase. Bacitracin (100 mg/L) was the most effective treatment at sustaining activity levels for up to 17 days post-subculture. Commercially available human placental alkaline phosphatase (PLAP) was used to probe the mechanism of SEAP deactivation. Experiments with PLAP in sterile and conditioned medium corroborated the denaturation of SEAP by factors generated by cell growth and not due to simple proteolysis. We also show for the first time that the factors promoting activity loss are heat labile at 95 degrees C but not at 70 degrees C, and they are not inactivated after a 5 day incubation period under normal culture conditions (27 degrees C). In addition, there were no significant changes in pH or redox potential when comparing sterile and cell-free conditioned medium during PLAP incubation, indicating that these factors were unimportant.

  17. Alkaline phosphatase-fused repebody as a new format of immuno-reagent for an immunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Hyo-Deok; Lee, Joong-jae [Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701 (Korea, Republic of); Kim, Yu Jung [Industrial Biotechnology and Bioenergy Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon (Korea, Republic of); Hantschel, Oliver [School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne (Switzerland); Lee, Seung-Goo [Industrial Biotechnology and Bioenergy Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon (Korea, Republic of); Kim, Hak-Sung, E-mail: hskim76@kaist.ac.kr [Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701 (Korea, Republic of)

    2017-01-15

    Enzyme-linked immunoassays based on an antibody-antigen interaction are widely used in biological and medical sciences. However, the conjugation of an enzyme to antibodies needs an additional chemical process, usually resulting in randomly cross-linked molecules and a loss of the binding affinity and enzyme activity. Herein, we present the development of an alkaline phosphatase-fused repebody as a new format of immuno-reagent for immunoassays. A repebody specifically binding to human TNF-α (hTNF-α) was selected through a phage display, and its binding affinity was increased up to 49 nM using a modular engineering approach. A monomeric alkaline phosphatase (mAP), which was previously isolated from a metagenome library, was genetically fused to the repebody as a signal generator, and the resulting repebody-mAP fusion protein was used for direct and sandwich immunoassays of hTNF-α. We demonstrate the utility and potential of the repebody-mAP fusion protein as an immuno-reagent by showing the sensitivity of 216 pg mL{sup −1} for hTNF-α in a sandwich immunoassay. Furthermore, this repebody-mAP fusion protein enabled the detection of hTNF-α spiked in a serum-supplemented medium with high accuracy and reproducibility. It is thus expected that a mAP-fused repebody can be broadly used as an immuno-reagent in immunoassays. - Highlights: • A human TNF-α (hTNF-α)-specific repebody was selected using a phage display. • A monomeric alkaline phosphatase (mAP) was genetically fused to the repebody. • mAP-fused repebody enabled detection of hTNF-α with high sensitivity and accuracy. • mAP-fused repebody can be widely used as a new immuno-reagent in immunoassays.

  18. Study of serum levels of calcium, phosphorus and alkaline phosphatase in chronic kidney disease

    Directory of Open Access Journals (Sweden)

    R. Freethi

    2016-03-01

    Full Text Available Chronic kidney disease (CKD is a worldwide public health problem, with increasing prevalence and lethal adverse outcomes like progressive loss of kidney function, cardiovascular disease and premature death. Disturbances in mineral metabolism and bone disease are common complications of CKD and an important cause of morbidity and decreased quality of life in patients with CKD. Patients with renal failure have an increased risk of cardiovascular mortality that may be due in part to vascular calcification. To measure serum levels of calcium, phosphorus and alkaline phosphatase in patients in various stages of CKD and to correlate the same with creatinine and estimated Glomerular Filtration Rate (eGFRvalues. This is a cross sectional study done at Thanjavur Medical College Hospital. 60 CKD patients and 50 healthy controls were enrolled in this study. Serum levels of creatinine, calcium, phosphorus and alkaline phosphatase were measured and eGFR values correlated with the serum creatinine. The mean values of creatinine (4.9 ± 2.23 mg/dl, cal cium (9.8 ± 0.456 mg/dl , phosphorus (4.19 ± 0.404 mg/dl and alkaline phosphatase (94.01 ± 15.10 U/L in the stu dy group are significantly higher than the control group in which the mean levels are 0.89 ± 0.102 mg/dl, 10.17 ± 0.37 mg/dl, 4.02 ± 0.16 mg/dl and 25.16 ± 4.65U/ L respectively. We have found that there is a significant difference in the above said parameters among patients in different stages of CKD (stage 3-5 indicating the progression of mineral bone disease with advancing stage of CKD.

  19. Structural comparisons of two allelic variants of human placental alkaline phosphatase.

    Science.gov (United States)

    Millán, J L; Stigbrand, T; Jörnvall, H

    1985-01-01

    A simple immunosorbent purification scheme based on monoclonal antibodies has been devised for human placental alkaline phosphatase. The two most common allelic variants, S and F, have similar amino acid compositions with identical N-terminal amino acid sequences through the first 13 residues. Both variants have identical lectin binding properties towards concanavalin A, lentil-lectin, wheat germ agglutinin, phytohemagglutinin and soybean agglutinin, and identical carbohydrate contents as revealed by methylation analysis. CNBr fragments of the variants demonstrate identical high performance liquid chromatography patterns. The carbohydrate containing fragment is different from the 32P-labeled active site fragment and the N-terminal fragment.

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

    Science.gov (United States)

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

    2014-05-15

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

  1. Lectin histochemistry and alkaline phosphatase activity in the pia mater vessels of spontaneously hypertensive rats (SHR).

    Science.gov (United States)

    Szumańska, G; Gadamski, R

    1992-01-01

    Some lectins were used to study the localization of sugar residues on the endothelial cell surface in the pia mater blood vessels of control (WKY) and hypertensive rats (SHR). The lectins tested recognized the following residues: beta-D-galactosyl (Ricinus communis agglutinin 120, RCA-1), alpha-L-fucosyl (Ulex europaeus agglutinin, UEA-1), N-acetylglucosaminyl and sialyl (Wheat germ agglutinin, WGA), N-glycolyl-neuraminic acid (Limax flavus agglutinin, LFA), and N-acetyl-D-galactosaminyl (Helix pomatia agglutinin, HPA). Several differences were revealed in the presence of sugar receptors on the surface of endothelial cells between the control and the hypertensive rats. Our studies showed also differences in the localization of the tested glycoconjugates between pial capillaries, small, medium-size and large pial arteries. The histochemical evaluation of alkaline phosphatase revealed an increased activity of the enzyme in the pial vessels of SHRs as compared with control rats with a similar localization of the enzyme activity. Some differences in the distribution of lectin binding sites and alkaline phosphatase activity could be associated with the different functions of particular segments of the pial vascular network.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    1999-02-01

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

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

    Directory of Open Access Journals (Sweden)

    Subhash C Bihani

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-07-01

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

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

    International Nuclear Information System (INIS)

    Morales, L.; Gutierrez, N.; Maya, V.; Parra, C.; Martinez B, E.; Coello, P.

    2012-01-01

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

  7. A ten-week biochemistry lab project studying wild-type and mutant bacterial alkaline phosphatase.

    Science.gov (United States)

    Witherow, D Scott

    2016-11-12

    This work describes a 10-week laboratory project studying wild-type and mutant bacterial alkaline phosphatase, in which students purify, quantitate, and perform kinetic assays on wild-type and selected mutants of the enzyme. Students also perform plasmid DNA purification, digestion, and gel analysis. In addition to simply learning important techniques, students acquire novel biochemical data in their kinetic analysis of mutant enzymes. The experiments are designed to build on students' work from week to week in a way that requires them to apply quantitative analysis and reasoning skills, reinforcing traditional textbook biochemical concepts. Students are assessed through lab reports focused on journal style writing, quantitative and conceptual question sheets, and traditional exams. © 2016 by The International Union of Biochemistry and Molecular Biology, 44(6):555-564, 2016. © 2016 The International Union of Biochemistry and Molecular Biology.

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

    Directory of Open Access Journals (Sweden)

    Ana Lorena Alvarado-Gámez

    2014-02-01

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

  9. Relationship between serum heat-stable alkaline phosphatase level and pregnancy

    International Nuclear Information System (INIS)

    Cao Guoxian; Xiao Weihong; Yu Huixin; Li Weiyi; Huang Xuquan

    1998-01-01

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

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    this membrane passage in organ cultured intestinal mucosal explants. We found that in addition to a rapid uptake into the cytoplasm, a fraction of the fatty acid analogs were inserted directly into the brush border membrane. Furthermore, a brief exposure of microvillar membrane vesicles to a fat mixture...... mimicking a physiological solution of dietary mixed micelles, rearranged the lipid raft microdomain organization of the membranes. Thus, the fat mixture generated a low-density subpopulation of microvillar detergent resistant membranes (DRMs) highly enriched in alkaline phosphatase (AP). Since this GPI-linked...... enzyme is the membrane protein in the brush border with the highest affinity for lipid rafts, this implies that free fatty acids selectively insert stably into these membrane microdomains. We have previously shown that absorption of dietary lipids transiently induce a selective endocytosis of AP from...

  11. A sandwich-type optical immunosensor based on the alkaline phosphatase enzyme for Salmonella thypimurium detection

    Science.gov (United States)

    Widyastuti, E.; Puspitasari Schonherr, M. F.; Masruroh, A.; Anggraeni, R. A.; Nisak, Y. K.; Mursidah, S.

    2018-03-01

    Salmonella is pathogenic bacteria that caused foodborne diseases which being called Salmonellosis. Prevalence of Salmonellosis that being caused by Salmonella thypimurium in Indonesia is quite high. However, detection of Salmonella bacteria in food still limited, complicated, and required a lot time. Sensitive optical assay for Salmonella thypimurium paper based detection has been developed by integrating sandwich assay between antibody-antigen complex and alkaline phosphatase enzyme that produce visible bluish-purple colour with presence of NBT-BCIP substrate. The results showed that Limit of Quantitation of detection is 105 CFU mL-1 with detection time 15 minutes. Linearity test between Colour intensity that produced from Salmonella concentration presence on samples showed that detection has good linearity. Selectivity test exhibited excellent sensitivity with good discrimination against Escherichia coli.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

    Grunwald, Sandra K.; Krueger, Katherine J.

    2008-01-01

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

  14. Robotic implementation of assays: tissue-nonspecific alkaline phosphatase (TNAP) case study.

    Science.gov (United States)

    Chung, Thomas D Y

    2013-01-01

    Laboratory automation and robotics have "industrialized" the execution and completion of large-scale, enabling high-capacity and high-throughput (100 K-1 MM/day) screening (HTS) campaigns of large "libraries" of compounds (>200 K-2 MM) to complete in a few days or weeks. Critical to the success these HTS campaigns is the ability of a competent assay development team to convert a validated research-grade laboratory "benchtop" assay suitable for manual or semi-automated operations on a few hundreds of compounds into a robust miniaturized (384- or 1,536-well format), well-engineered, scalable, industrialized assay that can be seamlessly implemented on a fully automated, fully integrated robotic screening platform for cost-effective screening of hundreds of thousands of compounds. Here, we provide a review of the theoretical guiding principles and practical considerations necessary to reduce often complex research biology into a "lean manufacturing" engineering endeavor comprising adaption, automation, and implementation of HTS. Furthermore we provide a detailed example specifically for a cell-free in vitro biochemical, enzymatic phosphatase assay for tissue-nonspecific alkaline phosphatase that illustrates these principles and considerations.

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

    Science.gov (United States)

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

    2017-07-01

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

  16. Simulated bioavailability of phosphorus from aquatic macrophytes and hytoplankton by aqueous suspension and incubation with alkaline phosphatase

    Science.gov (United States)

    Bioavailability of phosphorus (P) in aquatic macrophytes and algae on lake eutrophication was studied by evaluation their P forms and quantities in their water suspensions and impact by alkaline phosphatase hydrolysis. using solution 31P-nuclear magnetic resonance (NMR). The laboratory suspension an...

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

    DEFF Research Database (Denmark)

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

    2005-01-01

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

  18. Inactivation of peroxidase, pectinesterase and alkaline phosphatase in polymers as a model for irradiation of dried foodstuffs

    NARCIS (Netherlands)

    Roozen, J.P.

    1972-01-01

    This thesis consists of a summary only of 6 refereed scientific papers published in Lebensmittel -Wissenschaft und -Technologie 3, 37-40 (1970); 4, 24-26, 93-96, 158-162, 196-200 (1971); 5, 128-131 (1972).

    Aqueous solutions containing an enzyme (peroxidase, alkaline phosphatase,

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

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

    Science.gov (United States)

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

    2011-12-01

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

  1. Catalytic efficiency is a better predictor of arsenic toxicity to soil alkaline phosphatase.

    Science.gov (United States)

    Wang, Ziquan; Tian, Haixia; Lu, Guannan; Zhao, Yiming; Yang, Rui; Megharaj, Mallavarapu; He, Wenxiang

    2018-02-01

    Arsenic (As) is an inhibitor of phosphatase, however, in the complex soil system, the substrate concentration effect and the mechanism of As inhibition of soil alkaline phosphatase (ALP) and its kinetics has not been adequately studied. In this work, we investigated soil ALP activity in response to As pollution at different substrate concentrations in various types of soils and explored the inhibition mechanism using the enzyme kinetics. The results showed that As inhibition of soil ALP activity was substrate concentration-dependent. Increasing substrate concentration decreased inhibition rate, suggesting reduced toxicity. This dependency was due to the competitive inhibition mechanism of As to soil ALP. The kinetic parameters, maximum reaction velocity (V max ) and Michaelis constant (K m ) in unpolluted soils were 0.012-0.267mMh -1 and 1.34-3.79mM respectively. The competitive inhibition constant (K ic ) was 0.17-0.70mM, which was lower than K m , suggesting higher enzyme affinity for As than for substrate. The ecological doses, ED 10 and ED 50 (concentration of As that results in 10% and 50% inhibition on enzyme parameter) for inhibition of catalytic efficiency (V max /K m ) were lower than those for inhibition of enzyme activity at different substrate concentrations. This suggests that the integrated kinetic parameter, catalytic efficiency is substrate concentration independent and more sensitive to As than ALP activity. Thus, catalytic efficiency was proposed as a more reliable indicator than ALP activity for risk assessment of As pollution. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    International Nuclear Information System (INIS)

    Arai, Shigeki; Yonezawa, Yasushi; Ishibashi, Matsujiro; Matsumoto, Fumiko; Adachi, Motoyasu; Tamada, Taro; Tokunaga, Hiroko; Blaber, Michael; Tokunaga, Masao; Kuroki, Ryota

    2014-01-01

    In order to clarify the structural basis of the halophilic characteristics of an alkaline phosphatase derived from the moderate halophile Halomonas sp. 593 (HaAP), the tertiary structure of HaAP was determined to 2.1 Å resolution by X-ray crystallography. The structural properties of surface negative charge and core hydrophobicity were shown to be intermediate between those characteristic of halophiles and non-halophiles, and may explain the unique functional adaptation to a wide range of salt concentrations. Alkaline phosphatase (AP) from the moderate halophilic bacterium Halomonas sp. 593 (HaAP) catalyzes the hydrolysis of phosphomonoesters over a wide salt-concentration range (1–4 M NaCl). In order to clarify the structural basis of its halophilic characteristics and its wide-range adaptation to salt concentration, the tertiary structure of HaAP was determined by X-ray crystallography to 2.1 Å resolution. The unit cell of HaAP contained one dimer unit corresponding to the biological unit. The monomer structure of HaAP contains a domain comprised of an 11-stranded β-sheet core with 19 surrounding α-helices similar to those of APs from other species, and a unique ‘crown’ domain containing an extended ‘arm’ structure that participates in formation of a hydrophobic cluster at the entrance to the substrate-binding site. The HaAP structure also displays a unique distribution of negatively charged residues and hydrophobic residues in comparison to other known AP structures. AP from Vibrio sp. G15-21 (VAP; a slight halophile) has the highest similarity in sequence (70.0% identity) and structure (C α r.m.s.d. of 0.82 Å for the monomer) to HaAP. The surface of the HaAP dimer is substantially more acidic than that of the VAP dimer (144 exposed Asp/Glu residues versus 114, respectively), and thus may enable the solubility of HaAP under high-salt conditions. Conversely, the monomer unit of HaAP formed a substantially larger hydrophobic interior comprising 329

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-03-01

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

  4. Effect of Hypodynamy on Structure and Alkaline Phosphatase Activity of Kidney in Japanese Quails

    Directory of Open Access Journals (Sweden)

    V. Almášiová

    2008-01-01

    Full Text Available The objective of the study was to observe the effect of experimental hypodynamy simulating weightlessness in space on the structure, ultrastructure and alkaline phosphatase activity of kidney in Japanese quail (Coturnix coturnix japonica. Two days after hatching, the quails were suspended in special shirts below the cage ceiling so their feet did not touch the floor. They could consume food and water ad libitum. Experimental animals were sacrificed after 14, 21, 28, 35, 42, 49 and 56 days of hypodynamy. Birds of the same age, hatched at the same time, and fed the same diet were used as a control. Samples of kidney were processed for light (LM and transmission electron microscopy (TEM, and alkaline phosphatase (AP analysis. Short-term (14–28 days hypodynamy caused no marked damage to the structure and ultrastructure of kidneys. However, after long-term (35–59 days hypodynamy, morphological changes were observed in some cells of the proximal and distal tubules. The dying cells in proximal tubules, observed in semi-thin sections by LM, were dark and contained a nucleus of irregular shape. Observation by TEM showed that their nucleus was dark and shrivelled and the electron-dense cytoplasm contained long, dense, rod-shaped mitochondria with thin mitochondrial cristae. Microvilli were present on the apical surface of cells and formed a brush border. Sporadic dying cells were also observed in distal tubules. Large, light vacuoles were found in the cytoplasm of cells of collecting tubules, however, the structure of renal corpuscles and medullary loops remained undisturbed. Microscopical analysis by means of a direct TUNEL reaction on days 35 to 59 of hypodynamy showed a moderate occurrence of cellular apoptosis in the proximal and distal tubules of experimental Japanese quail. The activity of AP in the brush border of the proximal tubules on days 14–29 of hypodynamy was normal in experimental animals and showed no significant differences in

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

    Directory of Open Access Journals (Sweden)

    Siddhartha S. Ghosh

    2014-12-01

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

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

    Science.gov (United States)

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

    2016-06-01

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

  7. Colorimetric determination of alkaline phosphatase as indicator of mammalian feces in corn meal: collaborative study.

    Science.gov (United States)

    Gerber, H

    1986-01-01

    In the official method for rodent filth in corn meal, filth and corn meal are separated in organic solvents, and particles are identified by the presence of hair and a mucous coating. The solvents are toxic, poor separation yields low recoveries, and fecal characteristics are rarely present on all fragments, especially on small particles. The official AOAC alkaline phosphatase test for mammalian feces, 44.181-44.184, has therefore been adapted to determine the presence of mammalian feces in corn meal. The enzyme cleaves phosphate radicals from a test indicator/substrate, phenolphthalein diphosphate. As free phenolphthalein accumulates, a pink-to-red color develops in the gelled test agar medium. In a collaborative study conducted to compare the proposed method with the official method for corn meal, 44.049, the proposed method yielded 45.5% higher recoveries than the official method. Repeatability and reproducibility for the official method were roughly 1.8 times more variable than for the proposed method. The method has been adopted official first action.

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

    Directory of Open Access Journals (Sweden)

    Hai-Yong Ren

    2015-01-01

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

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

    Science.gov (United States)

    Ren, Hai-Yong; Sun, Ling-Ling; Li, Heng-Yuan; Ye, Zhao-Ming

    2015-01-01

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

  10. A case of polycythemia with low neutrophilic alkaline phosphatase and chromosome abnormalities in atomic bomb survivor

    International Nuclear Information System (INIS)

    Chiyoda, Shin; Toyoda, Shigeki; Shikaya, Takaaki; Tagawa, Masuko; Matsunaga, Masako.

    1978-01-01

    A case of mild polycythemia with low neutophilic alkaline phosphatase in a short-distance group was reported. The patient was exposed 1.4 km from the center of explosion (estimated exposure dose, 330 rad). He suffered from acute symptoms such as vomiting, diarrhea, increase in temperature, loss of hair, poor appetite, and hemorrhage. In an examination of a-bomb survivors in 1969, his erythrocyte count was 622 x 10 4 /mm 3 and his hemoglobin level was 18.3 gm/dl. Later his erythrocyte count was sometimes over 550 x 10 4 /mm 3 . Upon admission to a hospital for a detailed examination, a slight increase in erythrocyte count and hemoglobin level and low NAP values were observed. Bone marrow findings revealed a slight increase in erythroblasts. Chromosomal analysis of bone marrow cells and peripheral lymphocytes revealed various abnormalities, seemingly related to exposure to radiation. Low NAPS values continued for a long time, and the patient remained healthy. (Tsunoda, M.)

  11. Alkaline phosphatase labeled SERS active sandwich immunoassay for detection of Escherichia coli

    Science.gov (United States)

    Bozkurt, Akif Goktug; Buyukgoz, Guluzar Gorkem; Soforoglu, Mehmet; Tamer, Ugur; Suludere, Zekiye; Boyaci, Ismail Hakki

    2018-04-01

    In this study, a sandwich immunoassay method utilizing enzymatic activity of alkaline phosphatase (ALP) on 5-bromo-4-chloro-3-indolyl phosphate (BCIP) for Escherichia coli (E. coli) detection was developed using surface enhanced Raman spectroscopy (SERS). For this purpose, spherical magnetic gold coated core-shell nanoparticles (MNPs-Au) and rod shape gold nanoparticles (Au-NRs) were synthesized and modified for immunomagnetic separation (IMS) of E. coli from the solution. In order to specify the developed method to ALP activity, Au-NRs were labeled with this enzyme. After successful construction of the immunoassay, BCIP substrate was added to produce the SERS-active product; 5-bromo-4-chloro-3-indole (BCI). A good linearity (R2 = 0.992) was established between the specific SERS intensity of BCI at 600 cm- 1 and logarithmic E. coli concentration in the range of 1.7 × 101-1.7 × 106 cfu mL- 1. LOD and LOQ values were also calculated and found to be 10 cfu mL- 1 and 30 cfu mL- 1, respectively.

  12. Genetics of alkaline phosphatase of the small intestine of the house mouser (Mus musculus).

    Science.gov (United States)

    Wilcox, F H

    1983-08-01

    Four inbred strains of mice exhibited either slow (PL/J), intermediate (DBA/2J, LP/J), or fast (SWR/J) rates of migration of duodenal alkaline phosphatase on cellulose acetate electrophoresis. Hybrids of these strains also had intermediate rates of migration regardless of the combination of strains used as parents. Strain differences were present in all regions of the small but not the large intestine. Crosses of the PL/J strain to hybrids between this strain and the other three strains gave a 1:1 segregation of the slow and intermediate patterns. The symbol Akp-3 is proposed for the locus responsible for the slower migration of the enzyme in this strain. Data from the LP/J X PL/J hybrid crossed with the PL/J strain showed linkage with two loci on chromosome 1 as follows: centromere--Idh-1--13.8 cM--Akp-3--8.9 +/- 2.6 cM--Pep-3. The available data do not reveal the genetic basis for the faster migration rate of the enzyme from the SWR/J strain, but a different response to neuraminidase and apparent nonlinkage to the Pep-3 locus suggest that a locus other than Akp-3 is responsible.

  13. Fluorescent Biosensor for Phosphate Determination Based on Immobilized Polyfluorene-Liposomal Nanoparticles Coupled with Alkaline Phosphatase.

    Science.gov (United States)

    Kahveci, Zehra; Martínez-Tomé, Maria José; Mallavia, Ricardo; Mateo, C Reyes

    2017-01-11

    This work describes the development of a novel fluorescent biosensor based on the inhibition of alkaline phosphatase (ALP). The biosensor is composed of the enzyme ALP and the conjugated cationic polyfluorene HTMA-PFP. The working principle of the biosensor is based on the fluorescence quenching of this polyelectrolyte by p-nitrophenol (PNP), a product of the hydrolysis reaction of p-nitrophenyl phosphate (PNPP) catalyzed by ALP. Because HTMA-PFP forms unstable aggregates in buffer, with low fluorescence efficiency, previous stabilization of the polyelectrolyte was required before the development of the biosensor. HTMA-PFP was stabilized through its interaction with lipid vesicles to obtain stable blue-emitting nanoparticles (NPs). Fluorescent NPs were characterized, and the ability to be quenched by PNP was evaluated. These nanoparticles were coupled to ALP and entrapped in a sol-gel matrix to produce a biosensor that can serve as a screening platform to identify ALP inhibitors. The components of the biosensor were examined before and after sol-gel entrapment, and the biosensor was optimized to allow the determination of phosphate ion in aqueous medium.

  14. Risk Factors for Elevated Preoperative Alkaline Phosphatase in Patients with Refractory Secondary Hyperparathyroidism.

    Science.gov (United States)

    Yang, Meng; Zhang, Ling; Huang, Linping; Sun, Xiaoliang; Ji, Haoyang; Lu, Yao

    2017-12-01

    Elevated preoperative levels of alkaline phosphatase (ALP) in patients with refractory secondary hyperparathyroidism are correlated with postoperative hypocalcemia and mortality. The aim of this study was to identify the predictors of preoperative ALP in patients with secondary hyperparathyroidism. From April 2012 to December 2015, 220 patients with refractory secondary hyperparathyroidism undergoing total parathyroidectomy without autotransplantation were reviewed. A total of 164 patients presented with elevated preoperative ALP. Univariate analysis showed that patients with elevated ALP were significantly younger. The elevated ALP group had significantly higher levels of preoperative parathyroid hormone (PTH), lower preoperative serum calcium, higher preoperative phosphorus, lower postoperative hypocalcemia, and a longer hospital stay. Logistic regression analysis showed that elevated preoperative PTH was a significant independent risk factor for elevated preoperative ALP (P = 0.000), and its value of 1624 pg/mL was the optimal cutoff point. Factors predictive of elevated preoperative ALP in patients with secondary hyperparathyroidism include preoperative PTH. Earlier surgery, aggressive calcium supplementation, and more careful or aggressive postoperative care for high-risk patients are needed.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1990-07-01

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

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

    Directory of Open Access Journals (Sweden)

    Caroline M Piskun

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

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

    Directory of Open Access Journals (Sweden)

    Wenbo Chen

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

  18. Studies on alkaline and acid phosphatase activity of neutrophil leukicytes, 2

    International Nuclear Information System (INIS)

    Niki, Yoko

    1983-01-01

    With a view to analyzing the inhibiting effect of anticancer drugs and irradiation on hematopoiesis in rabbits neutrophil (pseudoeosinophil) counts and the neutrophilic activities of alkaline phosphatase (AP) and acid phosphatase (SP) were serially followed up after drug administration or irradiation. The enzym activity was estimated histochemically, using azo-dye staining. Each rabbit was given cyclophosphamid (CP) (25mg/kg x 10, at intervals of 5 - 7 days ; 50mg/kg x 5, every day; or 100mg/kg x 1, i.m.), Thio-TEPA (4mg/kg x 1, i.m.), Vinblastin (VBT) (1mg/kg x 1, i.v.), 6MP (25mg/kg x 1, p.o.), or Mitomycin C (MMC) (1.5mg/kg x 1, i.v.). The results obtained were as follows : 1) The neutrophil counts became slightly elevated at 24 hrs, reached their nadir at 48 to 72 hrs, and recovered to normal in 5 to 6 days thereafter, except with 6 MP which produced no significant change but for a temporary elevation after dosages. 2) Except in the group administrated 6MP, which caused no significant hematorogical changes, the AP changes were similar in all of the animal groups : after temporary depression, it became elevated for 5 to 6 days, and recovered to normal about 9 days thereafter. 3) SP showed no changes in the 25mg/kg x 10 CP and the 6MP groups, it became elevated in 2 or 3 days after the administration of MMC, VBT, or Thio-TEPA to recover to normal in 5 to 10 days thereafter. 4) 60 Co irradiation (1,000 rad/whole body x 1) led to a temporary ascent in phil count followed by a descent from the 6th day on, and then a slow recovery to normal. AP was elevated from the third to the sixth days, and, after a depression on the tenth day, it returned to normal 24 days after irradiation, while SP showed a continued elevation from the 2nd to the 13th day. (author)

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

    DEFF Research Database (Denmark)

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

    2006-01-01

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

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

    OpenAIRE

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

    2002-01-01

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

  1. Facile colorimetric assay of alkaline phosphatase activity using Fe(II)-phenanthroline reporter.

    Science.gov (United States)

    Hu, Qiong; Zhou, Baojing; Dang, Pengyun; Li, Lianzhi; Kong, Jinming; Zhang, Xueji

    2017-01-15

    We report a versatile approach for the colorimetric assay of alkaline phosphatase (ALP) activity based on the distinctive metal-to-ligand charge-transfer (MLCT) absorption properties of Fe(II)-phenanthroline reporter. In the presence of ALP, the applied substrate ascorbic acid 2-phosphate is enzymatically hydrolyzed to produce ascorbic acid, which then reduces Fe 3+ to Fe 2+ . The complexation of Fe 2+ with the bathophenanthroline disulfonate (BPS) ligand generates a blood-red Fe(BPS) 3 4- reporter, which is characterized by an intense MLCT absorption band at 535 nm in the visible range. Under optimal conditions, the spectral output exhibits a good quantitative relationship with ALP activity over the range of 0-220 mU mL -1 with a detection limit of 0.94 mU mL -1 . Moreover, the activity of ALP can also be conveniently judged through naked-eye observations. Results indicate that it is highly selective and can be applied to the screening of ALP inhibitors. In addition, it has been successfully employed to detect the endogenous ALP level of undiluted human serum samples, with a detection limit of 1.05 mU mL -1 being achieved. This approach avoids any elaborately designed substrates and holds considerable simplicity and flexibility for reporter design. This study broadens the horizon of the applications of phenanthroline-based transition metal complexes. Furthermore, an efficient and practical method like this has the potential to be widely used in clinical applications and in the point-of-care testing. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    2015-01-01

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

  3. Serum bone alkaline phosphatase and calcaneus bone density predict fractures: a prospective study.

    Science.gov (United States)

    Ross, P D; Kress, B C; Parson, R E; Wasnich, R D; Armour, K A; Mizrahi, I A

    2000-01-01

    The aim of this study was to assess the ability of serum bone-specific alkaline phosphatase (bone ALP), creatinine-corrected urinary collagen crosslinks (CTx) and calcaneus bone mineral density (BMD) to identify postmenopausal women who have an increased risk of osteoporotic fractures. Calcaneus BMD and biochemical markers of bone turnover (serum bone ALP and urinary CTx) were measured in 512 community-dwelling postmenopausal women (mean age at baseline 69 years) participating in the Hawaii Osteoporosis Study. New spine and nonspine fractures subsequent to the BMD and biochemical bone markers measurements were recorded over an average of 2.7 years. Lateral spinal radiographs were used to identify spine fractures. Nonspine fractures were identified by self-report at the time of each examination. During the 2.7-year follow-up, at least one osteoporotic fracture occurred in 55 (10.7%) of the 512 women. Mean baseline serum bone ALP and urinary CTx were significantly higher among women who experienced an osteoporotic fracture compared with those women who did not fracture. In separate age-adjusted logistic regression models, serum bone ALP, urinary CTx and calcaneus BMD were each significantly associated with new fractures (odds ratios of 1.53, 1.54 and 1.61 per SD, respectively). Multiple variable logistic regression analysis identified BMD and serum bone ALP as significant predictors of fracture (p = 0.002 and 0.017, respectively). The results from this investigation indicate that increased bone turnover is significantly associated with an increased risk of osteoporotic fracture in postmenopausal women. This association is similar in magnitude and independent of that observed for BMD.

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

    Directory of Open Access Journals (Sweden)

    Claire eMahaffey

    2014-12-01

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

  5. Glycation Contributes to Interaction Between Human Bone Alkaline Phosphatase and Collagen Type I.

    Science.gov (United States)

    Halling Linder, Cecilia; Enander, Karin; Magnusson, Per

    2016-03-01

    Bone is a biological composite material comprised primarily of collagen type I and mineral crystals of calcium and phosphate in the form of hydroxyapatite (HA), which together provide its mechanical properties. Bone alkaline phosphatase (ALP), produced by osteoblasts, plays a pivotal role in the mineralization process. Affinity contacts between collagen, mainly type II, and the crown domain of various ALP isozymes were reported in a few in vitro studies in the 1980s and 1990s, but have not attracted much attention since, although such interactions may have important implications for the bone mineralization process. The objective of this study was to investigate the binding properties of human collagen type I to human bone ALP, including the two bone ALP isoforms B1 and B2. ALP from human liver, human placenta and E. coli were also studied. A surface plasmon resonance-based analysis, supported by electrophoresis and blotting, showed that bone ALP binds stronger to collagen type I in comparison with ALPs expressed in non-mineralizing tissues. Further, the B2 isoform binds significantly stronger to collagen type I in comparison with the B1 isoform. Human bone and liver ALP (with identical amino acid composition) displayed pronounced differences in binding, revealing that post-translational glycosylation properties govern these interactions to a large extent. In conclusion, this study presents the first evidence that glycosylation differences in human ALPs are of crucial importance for protein-protein interactions with collagen type I, although the presence of the ALP crown domain may also be necessary. Different binding affinities among the bone ALP isoforms may influence the mineral-collagen interface, mineralization kinetics, and degree of bone matrix mineralization, which are important factors determining the material properties of bone.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-08-01

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

  7. Differences in sialic acid residues among bone alkaline phosphatase isoforms: a physical, biochemical, and immunological characterization.

    Science.gov (United States)

    Magnusson, P; Farley, J R

    2002-12-01

    High-performance liquid chromatography (HPLC) separates three human bone alkaline phosphatase (BALP) isoforms in serum; two major BALP isoforms, B1 and B2, and a minor fraction, B/I, which is composed on average of 70% bone and 30% intestinal ALP. The current studies were intended to identify an in vitro source of the BALP isoforms for physical, biochemical, and immunological characterizations. The three BALP isoforms were identified in extracts of human osteosarcoma (SaOS-2) cells, by HPLC, after separation by anion-exchange chromatography. All three BALP isoforms were similar with respect to freeze-thaw stability, solubility, heat inactivation, and inhibition by L-phenylalanine, L-homoarginine, and levamisole. The isoforms were also kinetically similar (i.e., maximal velocity and KM at pH 8.8 and pH 10.0). The isoforms differed, however, with respect to sensitivity to precipitation with wheat germ agglutinin (WGA), P acid residues was estimated to be 29 and 45, for each B1 and B2 homodimer, respectively. Apparent discrepancies between these estimates of molecular weight and estimates based on gel filtration chromatography were attributed to nonspecific interactions between carbohydrate residues and the gel filtration beads. All three BALP isoforms showed similar dose-dependent linearity in the commercial Alkphase-B and Tandem-MP Ostase immunoassays, r = 0.944 and r = 0.985, respectively (P acid residues compared with B/I, which mainly explains the apparent differences in molecular weight. Future investigations will focus on the clinical and functional significance of the revealed differences in sialic acid residues.

  8. Thermal and Carbon Dioxide Inactivation of Alkaline Phosphatase in Buffer and Milk

    Directory of Open Access Journals (Sweden)

    Osman Erkmen

    2004-01-01

    Full Text Available The effects of temperature and CO2 treatment on the inactivation of alkaline phosphatase (ALP were studied. The thermal stability of ALP was found to be significantly (P< 0.05 different in glycine/NaOH buffer, pasteurized milk and raw milk. ALP was completely inactivated in the buffer at 60, 70 and 80 °C but approximately 12 % of activity was present at 50 °C after 55 min of treatment. The time required for complete inactivation of the enzyme in the buffer was reduced from 50 to 4 min as temperature increased from 60 to 80 °C. Complete inactivation of the enzyme in pasteurized milk was achieved at 70 and 80 °C but 28 and 15 % of ALP activity was still present at 50 and 60 °C after 120 min of treatment. Inactivation time for raw milk was reduced nearly 18-fold by increasing temperature from 50 to 70 °C. ALP in the buffer exposed to CO2 (under atmospheric pressure treatment at different temperatures showed a decrease in enzyme activity. Inactivation was found to be higher as the temperature increased from 20 to 50 °C. At the end of a 30-min treatment, residual ALP activity was found to be 84 and 19 % at 20 and 50 °C, respectively. Faster drop in pH and enzyme activity occurred within 5 min. The change in pH and enzyme activity dependant on CO2 treatment was not observed in raw milk mainly due to strong buffering capacity of milk.

  9. Alkaline phosphatase in nasal secretion of cattle: biochemical and molecular characterisation.

    Science.gov (United States)

    Ghazali, M Faizal; Koh-Tan, H H Caline; McLaughlin, Mark; Montague, Paul; Jonsson, Nicholas N; Eckersall, P David

    2014-09-05

    Nasal secretion (NS) was investigated as a source of information regarding the mucosal and systemic immune status of cattle challenged by respiratory disease. A method for the collection of substantial volumes (~12 ml) of NS from cattle was developed to establish a reference range of analytes that are present in the NS of healthy cattle. Biochemical profiles of NS from a group of 38 healthy Holstein-Friesian cows revealed high alkaline phosphatase (AP) activity of up to 2392 IU/L. The character and source of the high activity of AP in bovine NS was investigated. Histochemical analysis confirmed the localization of the AP enzyme activity to epithelial cells and serous glands of the nasal respiratory mucosa. Analysis of mRNA levels from nasal mucosa by end point RT-PCR and PCR product sequencing confirmed that the AP was locally produced and is identical at the nucleotide level to the non-specific AP splice variant found in bovine liver, bone and kidney. Analysis by isoelectric focussing confirmed that AP was produced locally at a high level in nasal epithelium demonstrating that AP from nasal secretion and nasal mucosa had similar pI bands, though differing from those of the liver, kidney, bone and intestine, suggesting different post-translational modification (PTM) of AP in these tissues. A nasal isozyme of AP has been identified that is present at a high activity in NS, resulting from local production and showing distinctive PTM and may be active in NS as an anti-endotoxin mediator.

  10. Responses of alkaline phosphatase activity to phosphorus stress in Daphnia magna.

    Science.gov (United States)

    McCarthy, S D S; Rafferty, S P; Frost, P C

    2010-01-15

    We examined how alkaline phosphatase (AP) activity within the bodies and in the materials released by the crustacean Daphnia magna responds to variable algal food phosphorus (P)-content. We found that Daphnia eating P-poor food (C:P approximately 700) had significantly higher AP activity in their bodies on a mass-specific basis compared with individuals eating P-rich food (C:P approximately 100). This dietary P effect on AP activity was not altered by Daphnia starvation but was partially related to differences in the P concentration of animal body homogenates. By contrast, poor P-nutrition of Daphnia lowered AP activity in released materials compared with that measured from their P-sufficient conspecifics. Moreover, AP activity in Daphnia release was lowest in animals consuming P-poor food for longer time periods. Our results support the hypothesis that AP activity increases inside P-limited Daphnia as a mechanism to increase P-acquisition and retention from ingested algae in these nutritionally stressed animals. The lower level of AP activity present in the water of P-deprived animals could reflect a change from largely free to membrane-bound AP isotypes in the digestive tracts of P-starved animals or a decrease in the shedding of membrane-anchored AP from their intestinal lining. These results supplement accumulating evidence that P-poor algal food reduces the dietary mineral P available to Daphnia. In addition, animal body AP activity measurements, with some refinement, may prove useful as an in situ indicator of P-stress in aquatic consumers.

  11. The fate of purified radio-labelled alkaline phosphatase from the liver in the organism

    International Nuclear Information System (INIS)

    Herbert, V.

    1981-01-01

    Alkaline phosphatase (AP) from dog liver was enriched by a factor of 5.444 in various steps. Rabbit antiserum to the purified AP was produced; 125-I was used then to radiolabel the highly purified AP. Four dogs were cholecystectomized and subsequently received an extracorporal drainage of the bile ducts. Decrease rate of total radio-activity and of PBI in the serum was determined in one dog; likewise in three other dogs before and one week after occlusion of their main bile ducts. In addition, radioactivity above the organs was measured in some animals at short intervals. In the dogs with main bile duct drainage, bile was collected continuously for up to 70 h, samples were taken, and residual bile plus native dog bile were re-infused into the distal choledochus catheter. Total radioactivity, PBI and immunoprecipitability with antibodies were determined in the bile and serum samples. AP, GOT, CPT and bilirubin were determined in some serum samples. In addition, total radioactivity excreted by urine was established. Results show injected 125-I-AP to be rapidly stored in the liver and not to be excreted via bile to a decisive extent. The fact that 125-I-AP is not excreted via bile is further indicated by the identical decrease rate of injected 125-I-AP in the serum in dogs with and without main bile duct occlusion. Injected 125-I-AP appears to be metabolized very rapidly in the liver as is indicated by the rapid decrease of immuno precipitability of 125-I-AP in the serum. (orig./MG) [de

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

    Science.gov (United States)

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

    2013-01-01

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

  13. Apoptosis may determine the release of skeletal alkaline phosphatase activity from human osteoblast-line cells.

    Science.gov (United States)

    Farley, J R; Stilt-Coffing, B

    2001-01-01

    Although quantitative measurement of skeletal alkaline phosphatase (sALP) activity in serum can provide an index of the rate of bone formation, the metabolic process that determines the release of sALP - from the surface of osteoblasts, into circulation-is unknown. The current studies were intended to examine the hypothesis that the release of sALP from human osteoblasts is a consequence of apoptotic cell death. We measured the release of sALP activity from human osteosarcoma (SaOS-2) cells and normal human bone cells, under basal conditions and in response to agents that increased apoptosis (TNF-a, okadiac acid) and agents that inhibit apoptosis (IGF-I, calpain, and caspase inhibitors). Apoptosis was determined by the presence of nucleosomes (histone-associated DNA) in the cytoplasm of the cells by using a commercial kit. The results of these studies showed that TNF-a and okadiac acid caused dose- and time-dependent increases in apoptosis in the SaOS-2 cells (r = 0.78 for doses of TNF-a and r = 0.93 for doses of okadiac acid, P sALP activity (e.g., r = 0.89 for TNF-a and r = 0.75 for okadiac acid, P sALP activity (P sALP activity (P sALP release. The associations between apoptosis and sALP release were not unique to osteosarcoma (i.e., SaOS-2) cells, but also seen with osteoblast-line cells derived from normal human bone. Together, these data demonstrate that the release of sALP activity from human osteoblast-line cells in vitro is associated with, and may be a consequence of, apoptotic cell death. These findings are consistent with the general hypothesis that the appearance of sALP activity in serum may reflect the turnover of osteoblast-line cells.

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

    Science.gov (United States)

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

    2016-06-01

    Ultraviolet Radiation (UVR) is a stressor for aquatic organisms affecting enzyme activities in planktonic populations because of the increase in reactive oxygen species. In addition, UVR exposure combined with other environmental factors (i.e. temperature and food quality) could have even higher detrimental effects. In this work, we aimed to determine the effect of UVR on somatic Alkaline Phosphatase Activity (APA) and Glutathione S-Transferase (GST) activity on the cladoceran Daphnia commutata under two different temperatures (10 °C and 20 °C) and under three food qualities (carbon:phosphorus ratios: 1150, 850 and 550). APA is a biomarker that is considered as a P deficiency indicator in zooplankton. Since recovery from UVR damage under dark conditions is an ATP depending reaction we also measured APA during recovery phases. We carried out a laboratory experiment combining different temperatures and food qualities with exposition to UVR followed by luminic and dark phases for recovery. In addition, we exposed organisms to H2O2, to establish if the response on APA to UVR was a consequence of the reactive oxygen species produced these short wavelengths. Our results showed that somatic APA was negatively affected by UVR exposure and this effect was enhanced under high temperature and low food quality. Consistently, GST activity was higher when exposed to UVR under both temperatures. The H2O2 experiments showed the same trend as UVR exposure, indicating that APA is affected mainly by oxidative stress than by direct effect of UVR on the enzyme. Finally, APA was affected in the dark phase of recovery confirming the P demands. These results enlighten the importance of food quality in the interacting effect of UVR and temperature, showing that C:P food ratio could determine the success or failure of zooplanktonic populations in a context of global change. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Ontogeny and distribution of alkaline and acid phosphatases in the digestive system of California halibut larvae (Paralichthys californicus).

    Science.gov (United States)

    Zacarias-Soto, Magali; Barón-Sevilla, Benjamín; Lazo, Juan P

    2013-10-01

    Studies aimed to assess the digestive physiology of marine fish larvae under culture conditions are important to further understand the functional characteristics and digestive capacities of the developing larvae. Most studies to date concentrate on intestinal lumen digestion and little attention to the absorption process. Thus, the objectives of this study were to histochemically detect and quantify some of the enzymes responsible for absorption and intracellular digestion of nutrients in the anterior and posterior intestine of California halibut larvae. Alkaline and acid phosphatases were detected from the first days post-hatch (dph). Alkaline phosphatase maintained a high level of activity during the first 20 dph in both intestinal regions. Thereafter, a clear intestinal regionalization of the activity was observed with the highest levels occurring in the anterior intestine. Acid phosphatase activity gradually increased in both intestinal regions during development, and a regionalization of the activity was not observed until late in development, once the ocular migration began. Highest levels were observed in the anterior intestine at the end of metamorphosis concomitant with the stomach development. The results from this study show some morphological and physiological changes are occurring during larval development and a clear regionalization of the absorption process as the larvae develops. These ontological changes must be considered in the elaboration of diets according to the digestive capacity of the larvae.

  16. A sensitive fluorescence biosensor for alkaline phosphatase activity based on the Cu(II)-dependent DNAzyme

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Mengmeng; Guo, Yajuan; Wang, Lixu [College of Chemistry, Fuzhou University, Fuzhou, Fujian 350116 (China); Luo, Fang, E-mail: luofang0812@163.com [College of Biological Science and Technology, Fuzhou University, Fuzhou, Fujian 350116 (China); Lin, Cuiying, E-mail: lcuiying@fzu.edu.cn [College of Chemistry, Fuzhou University, Fuzhou, Fujian 350116 (China); Lin, Zhenyu; Chen, Guonan [College of Chemistry, Fuzhou University, Fuzhou, Fujian 350116 (China)

    2016-12-15

    Alkaline phosphatase (ALP) plays an important role in phosphate metabolism processes; deviation from its normal level may indicate different kinds of diseases, so it is highly necessary to develop some simple and sensitive methods to monitor the ALP level. In this study, a simple, high selective, and sensitive fluorescent biosensor has been proposed for ALP activity determination. The Cu(II)-dependent DNAzyme (Cu-Enzyme) are divided into two parts: Cu-Enzyme 1 and Cu-Enzyme 2, and labelled with alkyne and azido groups, respectively. The Cu-substrate (Cu-Sub) is labelled with a FAM fluorophore (6-carboxyfluorescein) at the 3′-end and an additional quencher (BHQ1) at the 5′-end. The 5′-end of Cu-Enzyme 1 is labelled with BHQ1 as well. The hybridization of the Cu-Enzyme 1 and Cu-Enzyme 2 with Cu-Sub strand results in the low background fluorescence signal because the fluorescence from FAM is quenched. The addition of ALP can hydrolyze AA-P into AA, which can reduce Cu(II) into Cu(I) and in turn catalyze the cycloaddition of Cu-Enzyme 1 and Cu-Enzyme 2 to form a modified Cu-Enzyme. Then the modified Cu-Enzyme catalyzes the cleavage of the Cu-Sub strands into two pieces. One piece containing FAM fluorophore can easily diffuse into solution and give off a strong fluorescence signal. The enhanced fluorescent intensity has a linear relationship with the ALP concentration in the range of 0.36–54.55 U L{sup −1} with the detection limit of 0.14 U L{sup −1} (S/N = 3). The proposed biosensor has been successfully applied to detect ALP in serum samples with satisfied results. - Highlights: • We have proposed a simple, high selective and sensitive fluorescent biosensor for ALP. • The biosensor combines the high selectivity and the click reaction and the high sensitivity of the fluorescence detection. • The biosensor has been successfully applied to detect ALP in serum samples with satisfied results.

  17. Glycosylation differences contribute to distinct catalytic properties among bone alkaline phosphatase isoforms.

    Science.gov (United States)

    Halling Linder, Cecilia; Narisawa, Sonoko; Millán, José Luis; Magnusson, Per

    2009-11-01

    Three circulating human bone alkaline phosphatase (BALP) isoforms (B1, B2, and B/I) can be distinguished in healthy individuals and a fourth isoform (B1x) has been discovered in patients with chronic kidney disease and in bone tissue. The present study was designed to correlate differing glycosylation patterns of each BALP isoform with their catalytic activity towards presumptive physiological substrates and to compare those properties with two recombinant isoforms of the tissue-nonspecific ALP (TNALP) isozyme, i.e., TNALP-flag, used extensively for mutation analysis of hypophosphatasia mutations and sALP-FcD(10), a chimeric enzyme recently used as therapeutic drug in a mouse model of infantile hypophosphatasia. The BALP isoforms were prepared from human osteosarcoma (SaOS-2) cells and the kinetic properties were evaluated using the synthetic substrate p-nitrophenylphosphate (pNPP) at pH 7.4 and 9.8, and the three suggested endogenous physiological substrates, i.e., inorganic pyrophosphate (PP(i)), pyridoxal 5'-phosphate (PLP), and phosphoethanolamine (PEA) at pH 7.4. Qualitative glycosylation differences were also assessed by lectin binding and precipitation. The k(cat)/K(M) was higher for B2 for all the investigated substrates. The catalytic activity towards PEA was essentially undetectable. The kinetic activity for TNALP-flag and sALP-FcD(10) was similar to the activity of the human BALP isoforms. The BALP isoforms differed in their lectin binding properties and dose-dependent lectin precipitation, which also demonstrated differences between native and denatured BALP isoforms. The observed differences in lectin specificity were attributed to N-linked carbohydrates. In conclusion, we demonstrate significantly different catalytic properties among the BALP isoforms due to structural differences in posttranslational glycosylation. Our data also suggests that PEA is not an endogenous substrate for the BALP isoforms or for the recombinant TNALP isoforms. The TNALP

  18. Intestinal alkaline phosphatase in the colonic mucosa of children with inflammatory bowel disease

    Science.gov (United States)

    Molnár, Kriszta; Vannay, Ádám; Szebeni, Beáta; Bánki, Nóra Fanni; Sziksz, Erna; Cseh, Áron; Győrffy, Hajnalka; Lakatos, Péter László; Papp, Mária; Arató, András; Veres, Gábor

    2012-01-01

    AIM: To investigate intestinal alkaline phosphatase (iAP) in the intestinal mucosa of children with inflammatory bowel disease (IBD). METHODS: Colonic biopsy samples were taken from 15 newly diagnosed IBD patients and from 10 healthy controls. In IBD patients, specimens were obtained both from inflamed and non-inflamed areas. The iAP mRNA and protein expression was determined by reverse transcription-polymerase chain reaction and Western blotting analysis, respectively. Tissue localization of iAP and Toll-like receptor (TLR) 4 was investigated by immunofluorescent staining. RESULTS: The iAP protein level in the inflamed mucosa of children with Crohn’s disease (CD) and ulcerative colitis (UC) was significantly decreased when compared with controls (both P < 0.05). Similarly, we found a significantly decreased level of iAP protein in the inflamed mucosa in CD compared with non-inflamed mucosa in CD (P < 0.05). In addition, the iAP protein level in inflamed colonic mucosa in patients with UC was decreased compared with non-inflamed mucosa in patients with CD (P < 0.05). iAP protein levels in the non-inflamed mucosa of patients with CD were similar to controls. iAP mRNA expression in inflamed colonic mucosa of children with CD and UC was not significantly different from that in non-inflamed colonic mucosa with CD. Expression of iAP mRNA in patients with non-inflamed mucosa and in controls were similar. Co-localization of iAP with TLR4 showed intense staining with a dotted-like pattern. iAP was present in the inflamed and non-inflamed mucosa of patients with CD, UC, and in control biopsy specimens, irrespective of whether it was present in the terminal ileum or in the colon. However, the fluorescent signal of TLR4 was more pronounced in the colon compared with the terminal ileum in all groups studied. CONCLUSION: Lower than normal iAP protein levels in inflamed mucosa of IBD patients may indicate a role for iAP in inflammatory lesions in IBD. Based on our results

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

    Science.gov (United States)

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

    2018-01-01

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

  20. Método fosfatasa alcalina anti-fosfatasa alcalina para el diagnóstico de inmunodeficiencias celulares Alkaline phosphatase-anti-alkaline phosphatase method for the diagnosis of cell immunodeficiencies

    Directory of Open Access Journals (Sweden)

    Berta B Socarrás Ferrer

    2001-12-01

    Full Text Available Para el estudio de 25 pacientes con infecciones recurrentes y un grupo control de 25 individuos supuestamente sanos, se aplicó, en nuestro laboratorio, el método inmunocitoquímico de fosfatasa alcalina anti-fosfatasa alcalina, para la cuantificación de las principales subpoblaciones de linfocitos T identificados con los anticuerpos monoclonales: anti-CD3, anti-CD4 y anti-CD8. Se obtuvieron diferencias significativas para las subpoblaciones TCD3 y CD4 positivos (p For the study of 25 patients with recurrent infections and a control group of 25 supposedly healthy individuals, our Laboratory applied the immunocytochemical method of alkaline phosphatase - anti-alkaline phosphatase for the quantitation of the main T-lymphocyte subgroups identified with monoclonal antibodies:antiCD3, anti-CD4 and anti-CD8. There were significant differences in positive TCD3 and CD4 subsets (p<0,05. Because this is a low cost and quick method, it may be applied by other immunodiagnosis labs throughout the country

  1. LDH isoenzyme blood test

    Science.gov (United States)

    ... medlineplus.gov/ency/article/003499.htm LDH isoenzyme blood test To use the sharing features on this page, ... Names LD; LDH; Lactic (lactate) dehydrogenase isoenzymes Images Blood test References Carty RP, Pincus MR, Sarafraz-Yazdi E. ...

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

    Directory of Open Access Journals (Sweden)

    Juan Luis Jurat-Fuentes

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

  3. Variations of alkaline phosphatase activity and P fractions in sediments of a shallow Chinese eutrophic lake (Lake Taihu)

    International Nuclear Information System (INIS)

    Zhang Tingxi; Wang Xiaorong; Jin Xiangcan

    2007-01-01

    The distribution of alkaline phosphatase activity (APA) and P fractions in sediment cores and the relationship between them were studied in a shallow Chinese freshwater lake (Lake Taihu). Sediment cores were collected from four sites, characterized by different degrees of eutrophication in June 2004. Sediment P was fractionated into Fe/Al-P, Ca-P, organic P (OP), inorganic P (IP) and total P (TP). The former two species made the largest contribution to the sediment P pool. Results show that trophic status and hydrological conditions have great impact on the APA of the sediments. The order of the APA in sediments was conjectured to be: macrophyte dominated lake > transitional lake > algal dominated lake. APA profiles follow a similar downcore decreasing trend. There was a positive relationship between the APA and the TP, IP. The multiple linear regression equation of the APA and P fractions is: APA = -97 + 0.768TP - 0.985Fe/Al-P. - Characteristics of the alkaline phosphatase activity and P fractions in sediments of different trophic status lake were studied in Lake Taihu

  4. [Alkaline phosphatase activity in blood group B or O secretors is fluctuated by the dinner intake of previous night].

    Science.gov (United States)

    Matsushita, Makoto; Harajiri, Sanae; Tabata, Shiori; Yukimasa, Nobuyasu; Muramoto, Yoshimi; Komoda, Tsugikazu

    2013-04-01

    We previously reported that two intestinal alkaline phosphatase (IAP) isoforms, high molecular mass IAP (HIAP) and normal molecular mass IAP (NIAP), appear in healthy serum with our Triton-PAGE method for determination of ALP isozymes. In addition, HIAP is chiefly present in blood group B or O secretors, and a large amount of NIAP is secreted into the circulation after high-fat meal in blood group B or O secretors. In the present paper, we investigated the relationship between alkaline phosphatase (ALP) activity in early morning with the patient in a fasted state and the dinner intake of previous night. Two types of dinner were prepared; a low-fat meal (520 kcal), and a high-fat meal (1,040 kcal). Subjects ate the 2 types of dinner on different days. The mean ALP activities at 14 h after high-fat meal ingestion in blood group B or O secretors (n=14) from JSCC and IFCC methods were 8.8% and 5.2% higher than those at 14 h after low-fat meal ingestion in blood group B or O secretors, respectively. The increases in ALP activity between after high-fat meal and low-fat meal were nearly identical to the increases in NIAP activity. These results suggest that a high-fat meal is more likely to affect ALP activity at the early morning with the patient in a fasted state in blood group B or O secretors.

  5. Temporal and spatial variations in kinetics of alkaline phosphatase in sediments of a shallow Chinese eutrophic lake (Lake Donghu).

    Science.gov (United States)

    Yiyong, Zhou; Jianqiu, Li; Min, Zhang

    2002-04-01

    Monthly sediment and interstitial water samples were collected in a shallow Chinese freshwater lake (Lake Donghu) from three areas to determine if alkaline phosphatase activity (APA) plays an important role, in phosphorus cycling in sediment. The seasonal variability in the kinetics of APA and other relevant parameters were investigated from 1995-1996. The phosphatase hydrolyzable phosphorus (PHP) fluctuated seasonally in interstitial water, peaking in the spring. A synchronous pattern was observed in chlorophyll a contents in surface water in general. The orthophosphate (o-P) concentrations in the interstitial water increased during the spring. An expected negative relationship between PHP and Vmax of APA is not evident in interstitial water. The most striking feature of the two variables is their co-occurring, which can be explained in terms of an induction mechanism. It is argued that phosphatase activity mainly contributes to the driving force of o-P regeneration from PHP in interstitial water, supporting the development of phytoplankton biomass in spring. The Vmax values in sediment increased during the summer, in conjunction with lower Km values in interstitial water that suggest a higher affinity for the substrate. The accumulation of organic matter in the sediment could be traced back to the breakdown of the algal spring bloom, which may stimulate APA with higher kinetic efficiency, by a combination of the higher Vmax in sediments plus lower Km values in interstitial water, in summer. In summary, a focus on phosphatase and its substrate in annual scale may provide a useful framework for the development of novel P cycling, possible explanations for the absence of a clear relationship between PHP and APA were PHP released from the sediment which induced APA, and the presence of kinetically higher APA both in sediment and interstitial water which permitted summer mineralization of organic matter derived from the spring bloom to occur. The study highlighted the

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

    Science.gov (United States)

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

    2014-11-04

    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

  7. Canine osteosarcoma cell lines from patients with differing serum alkaline phosphatase concentrations display no behavioural differences in vitro.

    Science.gov (United States)

    Holmes, K E; Thompson, V; Piskun, C M; Kohnken, R A; Huelsmeyer, M K; Fan, T M; Stein, T J

    2015-09-01

    Osteosarcoma is an aggressive malignancy and represents the most frequent primary bone malignancy of dogs and humans. Prognostic factors reported for osteosarcoma include tumour size, presence of metastatic disease and serum alkaline phosphatase (ALP) concentration at the time of diagnosis. To date, there have been no studies to determine whether the behaviour of osteosarcoma cells differ based on serum ALP concentration. Here, we report on the generation of six canine osteosarcoma cell lines from osteosarcoma-bearing dogs with differences in serum ALP concentration. To determine whether in vitro behaviour differs between primary osteosarcoma cell lines generated from patients with normal or increased serum ALP, assays were performed to evaluate proliferation, migration, invasion and chemosensitivity. There were no significant differences in cell proliferation, migration, invasion or chemosensitivity between cell lines associated with normal or increased serum ALP concentration. © 2013 Blackwell Publishing Ltd.

  8. Canine osteosarcoma cell lines from patients with differing serum alkaline phosphatase concentrations display no behavioral differences in vitro

    Science.gov (United States)

    Holmes, Katie E.; Thompson, Victoria; Piskun, Caroline M.; Kohnken, Rebecca A.; Huelsmeyer, Michael K.; Fan, Timothy M.; Stein, Timothy J.

    2013-01-01

    Osteosarcoma is an aggressive malignancy and represents the most frequent primary bone malignancy of dogs and humans. Prognostic factors reported for osteosarcoma include tumor size, presence of metastatic disease, and serum alkaline phosphatase (ALP) concentration at the time of diagnosis. To date, there have been no studies to determine whether the behavior of osteosarcoma cells differ based on serum ALP concentration. Here we report on the generation of six canine osteosarcoma cell lines from osteosarcoma-bearing dogs with differences in serum ALP concentration. To determine whether in vitro behavior differs between primary osteosarcoma cell lines generated from patients with normal or increased serum ALP assays were performed to evaluate proliferation, migration, invasion, and chemosensitivity. There were no significant differences in cell proliferation, migration, invasion, or chemosensitivity between cell lines associated normal or increased serum ALP concentration. PMID:23489774

  9. Localization of Alkaline Phosphatase and Cathepsin D during Cell Restoration after Colchicine Treatment in Primary Cultures of Fetal Rat Hepatocytes

    International Nuclear Information System (INIS)

    Chida, Kohsuke; Taguchi, Meiko

    2011-01-01

    Localization of alkaline phosphatase (ALP) and cathepsin D (CAPD) in primary cultures of fetal rat hepatocytes was examined using double immunofluorescent staining in order to investigate the relationship between lysosome movement and the fate of ALP during cell restoration after microtubule disruption by colchicine. At 3 hr and 24 hr after colchicine treatment, numerous coarse dots containing ALP were observed throughout the cytoplasm, and some of these showed colocalization with CAPD. At 48 hr and 72 hr after colchicine treatment, although most of the dots containing ALP in the cytoplasm disappeared, dots containing CAPD remained. The present results suggest that the denatured ALP proteins remaining in the cytoplasm of hepatocytes during cell restoration after colchicine treatment are digested by lysosomes

  10. Ratiometric detection of copper ions and alkaline phosphatase activity based on semiconducting polymer dots assembled with rhodamine B hydrazide.

    Science.gov (United States)

    Sun, Junyong; Mei, Han; Gao, Feng

    2017-05-15

    The rational surface functionalization of semiconducting polymer dots (Pdots) has attracted much attention to extend their applications in fabricating chemo/biosensing platform. In this study, a novel ratiometric fluorescent sensing platform using functionalized Pdots as probes for fluorescence signal transmission has been designed for sensing Cu(Ⅱ) and activity of alkaline phosphatase (ALP) with high selectivity and enhanced sensitivity. The highly fluorescent Pdots were firstly prepared with Poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1',3}-thiadiazole)] (PFBT) via nanoprecipitation method, and then assembled with non-fluorescent rhodamine B hydrazide (RB-hy), which shows special binding activity to Cu(Ⅱ), through adsorption process to obtain functionalized nanohybrids, Pdots@RB-hy. As thus, a FRET donors/acceptors pair, in which PFBT Pdots act as energy donors while RB-hy-Cu(II) complexes act as energy acceptors were constructed. On the basis of the varies in fluorescence intensities of donors/acceptors in the presence of different amounts of Cu(II), a ratiometric method for sensing Cu(II) has been proposed. The proposed ratiometric Cu(II) sensor shows a good linear detection range from 0.05 to 5μM with a detection limit of 15nM. Furthermore, using the Pdots@RB-hy-Cu(II) system as signal transducer, a ratiometric sensing for alkaline phosphatase (ALP) activity has also been established with pyrophosphate (PPi) as substrates. The constructed ratiometric sensor of ALP activity displays a linear detection range from 0.005 to 15UL -1 with a detection limit of 0.0018UL -1 . The sensor was further successfully used for ALP activity detection in human serum with satisfactory results. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Nicotine inhibits collagen synthesis and alkaline phosphatase activity, but stimulates DNA synthesis in osteoblast-like cells

    International Nuclear Information System (INIS)

    Ramp, W.K.; Lenz, L.G.; Galvin, R.J.

    1991-01-01

    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 [ 3 H]proline and collagenase-digestible protein) at 100, 300, and 600 micrograms/ml. Release of [ 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 [ 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

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

    Science.gov (United States)

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

    2016-07-01

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

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

    NARCIS (Netherlands)

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

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

  14. Oxidative Stress as Estimated by Gamma-Glutamyl Transferase Levels Amplifies the Alkaline Phosphatase-Dependent Risk for Mortality in ESKD Patients on Dialysis

    NARCIS (Netherlands)

    C. Torino (Claudia); F.U.S. Mattace Raso (Francesco); J.L.C.M. van Saase (Jan); M. Postorino (Maurizio); G.L. Tripepi (Giovanni); F. Mallamaci (Francesca); C. Zoccali (Carmine)

    2016-01-01

    textabstractAlkaline phosphatase (Alk-Phos) is a powerful predictor of death in patients with end-stage kidney disease (ESKD) and oxidative stress is a strong inducer of Alk-Phos in various tissues. We tested the hypothesis that oxidative stress, as estimated by a robust marker of systemic oxidative

  15. The flare in alkaline phosphatase activity post-orchidectomy predicts which patient may benefit from early chemotherapy in metastatic prostate cancer

    NARCIS (Netherlands)

    Pelger, Rob C. M.; Lycklama A Nijeholt, Guus A. B.; Zwinderman, Aielko H.; Hamdy, Neveen A. T.

    2002-01-01

    BACKGROUND: A flare in serum alkaline phosphatase (ALP) activity post-orchidectomy has been shown to be of negative prognostic value for progression-free survival (PFS) in patients with prostate cancer. The aim of this study was to investigate whether a flare in ALP may help identify patients in

  16. Activity of alkaline phosphatase and its fractions in blood serum of rats with experimental hyperthyroidism, kept in a radionuclide contaminated area

    International Nuclear Information System (INIS)

    Markova, A.G.; Bagel', I.M.

    2002-01-01

    In studies on male rats, with experimentally induced thyrotoxicosis a modifying effect of the stay in radioactively contaminated zone on the total alkaline phosphatase activity was shown. The data indicate disturbances in calcium-phosphoric metabolism, resulting in pronounced deviations from the normal range in the activity of thermolabile (bone) fraction (authors)

  17. Application of ascorbic acid 2-phosphate as a new voltammetric substrate for alkaline phosphatase determination in human serum

    Directory of Open Access Journals (Sweden)

    Wei Sun

    2005-12-01

    Full Text Available An electrochemical assay of the enzyme alkaline phosphatase (ALP using ascorbic acid 2-phosphate (AAP as a new voltammetric substrate has been described in this paper. In the alkaline buffer solution the ALP enzymatic hydrolysis product of AAP was ascorbic acid (AA, which was an electro-active substance and had a sensitive differential pulse voltammetric (DPV oxidative response on glassy carbon electrode (GCE at +380 mV (versus Ag/AgCl, so the activity of ALP could be monitored voltammetrically of the oxidative peak current of AA. The electrochemical behaviours of AA were carefully studied and the AA standard solution could be measured by DPV method in the linear range from 10.0 to 1000.0 μmol/L with the detection limit of 8.0 μmol/L. The optimal conditions for ALP enzymatic reaction and the voltammetric detection were optimized. Under the optimal conditions the calibration curve for ALP assay exhibited a linear range from 0.4 to 2000.0 U/L with a detection limit of 0.3 U/L. This proposed method was further applied to determine the ALP content in healthy human serum and the results were in good agreement with the traditional p-nitrophenyl phosphate spectrophotometric method. The kinetic constants of enzymatic reaction were also investigated with the apparent kinetic constant Km as 2.77 mmol/L and the maximum velocity Vmax as 0.33 mol/min.

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

    International Nuclear Information System (INIS)

    Menzorova, Natalie I.; Seitkalieva, Alexandra V.; Rasskazov, Valery A.

    2014-01-01

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

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

    Science.gov (United States)

    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.

  20. Assessment of the colorimetric and fluorometric assays for alkaline phosphatase activity in cow's, goat's, and sheep's milk.

    Science.gov (United States)

    Klotz, V; Hill, Art; Warriner, K; Griffiths, M; Odumeru, J

    2008-09-01

    Raw milk is a well-established vehicle for the carriage of human pathogens, and many regulatory bodies have consequently mandated compulsory pasteurization as a food safety intervention. The residual activity of alkaline phosphatase (ALP) has historically been used to verify the adequacy of pasteurization of cow's milk. However, there is uncertainty on how the current ALP standards and methods of analysis can be applied to sheep's and goat's milk, which naturally contain different levels of the enzyme than that found in cow's milk. The official ALP methods applied in Canada (colorimetric assay; MFO-3) and in the United States (Fluorophos) were assessed for their ability to detect enzyme activity in raw and pasteurized milk derived from cows, sheep, and goats. The detection limit and the limit of quantitation were 0.8 and 2.02 microg/ml phenol, respectively, for the MFO-3 method and 43 and 85 mU/liter, respectively, for the Fluorophos method. The average ALP levels in raw goat's, cow's, and sheep's milk were 165, 1,562, and 3,512 microg/ml phenol, respectively. Raw milk detection limits, which correspond to raw milk phosphatase levels, were 0.051, 0.485, and 0.023% in cow's, goat's, and sheep's milk, respectively, for the MFO-3 method and 0.007, 0.070, and 0.004%, respectively, for the Fluorophos method. Although both methods can be used for ALP determination in cow's, goat's, and sheep's milk, the Fluorophos assay was superior to the colorimetric MFO-3 method based on sensitivity and time required to complete the analysis.

  1. Effects of a human recombinant alkaline phosphatase on renal hemodynamics, oxygenation and inflammation in two models of acute kidney injury

    Energy Technology Data Exchange (ETDEWEB)

    Peters, Esther, E-mail: esther.peters@radboudumc.nl [Department of Intensive Care Medicine, Radboud university medical center, PO Box 9101, Internal Mailbox 710, 6500 HB, Nijmegen (Netherlands); Department of Pharmacology and Toxicology, Radboud university medical center, PO Box 9101, Internal Mailbox 149, 6500 HB, Nijmegen (Netherlands); Ergin, Bülent, E-mail: b.ergin@amc.uva.nl [Department of Translational Physiology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam (Netherlands); Kandil, Asli, E-mail: aslikandil@istanbul.edu.tr [Department of Biology, Faculty of Science, Istanbul University, PK 34134, Vezneciler, Istanbul (Turkey); Gurel-Gurevin, Ebru, E-mail: egurelgurevin@gmail.com [Department of Biology, Faculty of Science, Istanbul University, PK 34134, Vezneciler, Istanbul (Turkey); Elsas, Andrea van, E-mail: a.vanelsas@am-pharma.com [AM-Pharma, Rumpsterweg 6, 3981 AK, Bunnik (Netherlands); Masereeuw, Rosalinde, E-mail: r.masereeuw@uu.nl [Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, PO Box 80082, 3508 TB Utrecht (Netherlands); Pickkers, Peter, E-mail: peter.pickkers@radboudumc.nl [Department of Intensive Care Medicine, Radboud university medical center, PO Box 9101, Internal Mailbox 710, 6500 HB, Nijmegen (Netherlands); Ince, Can, E-mail: c.ince@amc.uva.nl [Department of Translational Physiology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam (Netherlands)

    2016-12-15

    Two small clinical trials indicated that administration of bovine intestinal alkaline phosphatase (AP) improves renal function in critically ill patients with sepsis-associated acute kidney injury (AKI), for which the mechanism of action is not completely understood. Here, we investigated the effects of a newly developed human recombinant AP (recAP) on renal oxygenation and hemodynamics and prevention of kidney damage and inflammation in two in vivo AKI models. To induce AKI, male Wistar rats (n = 18) were subjected to renal ischemia (30 min) and reperfusion (I/R), or sham-operated. In a second model, rats (n = 18) received a 30 min infusion of lipopolysaccharide (LPS; 2.5 mg/kg), or saline, and fluid resuscitation. In both models, recAP (1000 U/kg) was administered intravenously (15 min before reperfusion, or 90 min after LPS). Following recAP treatment, I/R-induced changes in renal blood flow, renal vascular resistance and oxygen delivery at early, and cortical microvascular oxygen tension at late reperfusion were no longer significantly affected. RecAP did not influence I/R-induced effects on mean arterial pressure. During endotoxemia, recAP treatment did not modulate the LPS-induced changes in systemic hemodynamics and renal oxygenation. In both models, recAP did exert a clear renal protective anti-inflammatory effect, demonstrated by attenuated immunostaining of inflammatory, tubular injury and pro-apoptosis markers. Whether this renal protective effect is sufficient to improve outcome of patients suffering from sepsis-associated AKI is being investigated in a large clinical trial. - Highlights: • Human recombinant alkaline phosphatase (recAP) is a potential new therapy for sepsis-associated acute kidney injury (AKI). • RecAP can modulate renal oxygenation and hemodynamics immediately following I/R-induced AKI. • RecAP did not modulate endotoxemia-induced changes in systemic hemodynamics and renal oxygenation. • RecAP did exert a clear renal protective

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

    Science.gov (United States)

    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.

  3. Effects of garlic and diallyl trisulfide on the growth, photosynthesis, and alkaline phosphatase activity of the toxic cyanobacterium Microcystis aeruginosa.

    Science.gov (United States)

    Wang, Shoubing; Wang, Yuanan; Ma, Xiaoxue; Xu, Ziran

    2016-03-01

    To identify a botanical algicide and elucidate the response of cyanobacteria to the extract from higher plants, the effects of garlic and garlic-derived diallyl trisulfide on Microcystis aeruginosa were studied. Effects were evaluated by changes in cell density, chlorophyll a, maximum effective quantum yield (Fv/Fm), effective quantum yield (YII), non-photochemical quenching (NPQ), and rapid light curves of M. aeruginosa. In addition, alkaline phosphatase activity (APA) was measured when M. aeruginosa was incubated with diallyl trisulfide. Results indicated that the inhibition by garlic and diallyl trisulfide was significant. The 120-h 50 % effective concentrations of garlic and diallyl trisulfide (EC50) were 0.75 g L(-1) and 2.84 mg L(-1), respectively. Moreover, the inhibitory rate increased with increasing concentration and the growth of M. aeruginosa was inhibited by 90.0 % at the highest concentrations. We also show that the response of M. aeruginosa to stress could involve both impairment of the photosynthetic center PSII and alteration of APA. For example, at high garlic concentration (2.0 g L(-1)), Fv/Fm significantly decreased from 0.501 to 0.084 (p garlic as an environmentally friendly algicide.

  4. A missense mutation in the human liver/bone/kidney alkaline phosphatase gene causing a lethal form of hypophosphatasia

    International Nuclear Information System (INIS)

    Weiss, M.J.; Cole, D.E.C.; Ray, K.; Whyte, M.P.; Lafferty, M.A.; Mulivor, R.A.; Harris, H.

    1988-01-01

    Hypophosphatasia is an inherited disorder characterized by defective bone mineralization and a deficiency of serum and tissue liver/bone/kidney alkaline phosphatase (L/B/K ALP) activity. Clinical severity is variable, ranging from death in utero (due to severe rickets) to pathologic fractures first presenting in adult life. Affected siblings, however, are phenotypically similar. Severe forms of the disease are inherited in an autosomal recessive fashion; heterozygotes often show reduced serum ALP activity. The specific gene defects in hypophosphatasia are unknown but are thought to occur either at the L/B/K ALP locus or within another gene that regulates L/B/K ALP expression. The authors used the polymerase chain reaction to examine L/B/K ALP cDNA from a patient with a perinatal (lethal) form of the disease. They observed a guanine-to-adenine transition in nucleotide 711 of the cDNA that converts alanine-162 of the mature enzyme to threonine. The affected individual, whose parents are second cousins, is homozygous for the mutant allele. Introduction of this mutation into an otherwise normal cDNA by site-directed mutagenesis abolished the expression of active enzyme, demonstrating that a defect in the L/B/K ALP gene results in hypophosphatasia and that the enzyme is, therefore, essential for normal skeletal mineralization

  5. Gingival crevicular fluid alkaline phosphatase activity in relation to pubertal growth spurt and dental maturation: A multiple regression study

    Directory of Open Access Journals (Sweden)

    Perinetti, G.

    2016-04-01

    Full Text Available Introduction: The identification of the onset of the pubertal growth spurt has major clinical implications when dealing with orthodontic treatment in growing subjects. Aim: Through multivariate methods, this study evaluated possible relationships between the gingival crevicular fluid (GCF alkaline phosphatase (ALP activity and pubertal growth spurt and dentition phase. Materials and methods: One hundred healthy growing subjects (62 females, 38 males; mean age, 11.5±2.4 years were enrolled into this doubleblind, prospective, cross-sectional-design study. Phases of skeletal maturation (pre - pubertal, pubertal, post - pubertal was assessed using the cervical vertebral maturation method. Samples of GCF for the ALP activity determination were collected at the mesial and distal sites of the mandibular central incisors. The phases of the dentition were recorded as intermediate mixed, late mixed, or permanent. A multinomial multiple logistic regression model was used to assess relationships of the enzymatic activity to growth phases and dentition phases. Results: The GCF ALP activity was greater in the pubertal growth phase as compared to the pre - pubertal and post - pubertal growth phases. Significant adjusted odds ratios for the GCF ALP activity for the pre - pubertal and post - pubertal subjects, in relation to the pubertal group, were 0.76 and 0.84, respectively. No significant correlations were seen for the dentition phase. Conclusions: The GCF ALP activity is a valid candidate as a non - invasive biomarker for the identification of the pubertal growth spurt irrespective of the dentition phase.

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

    Science.gov (United States)

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

    2003-01-01

    The increase of n-6 polyunsaturated fatty acids (PUFA) in animal tissues has been proposed as a mechanism of lead (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 3 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 decreased triglycerides and increased 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.

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

    Science.gov (United States)

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

    2013-01-01

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

  8. Arginine Coordination in Enzymatic Phosphoryl Transfer: Evaluation of the Effect of Arg166 Mutations in Escherichia Coli Alkaline Phosphatase

    International Nuclear Information System (INIS)

    O'Brien, P.J.; Lassila, J.K.; Fenn, T.D.; Zalatan, J.G.; Herschlag, D.

    2008-01-01

    Arginine residues are commonly found in the active sites of enzymes catalyzing phosphoryl transfer reactions. Numerous site-directed mutagenesis experiments establish the importance of these residues for efficient catalysis, but their role in catalysis is not clear. To examine the role of arginine residues in the phosphoryl transfer reaction, we have measured the consequences of mutations to arginine 166 in Escherichia coli alkaline phosphatase on hydrolysis of ethyl phosphate, on individual reaction steps in the hydrolysis of the covalent enzyme-phosphoryl intermediate, and on thio substitution effects. The results show that the role of the arginine side chain extends beyond its positive charge, as the Arg166Lys mutant is as compromised in activity as Arg166Ser. Through measurement of individual reaction steps, we construct a free energy profile for the hydrolysis of the enzyme-phosphate intermediate. This analysis indicates that the arginine side chain strengthens binding by ∼3 kcal/mol and provides an additional 1-2 kcal/mol stabilization of the chemical transition state. A 2.1 (angstrom) X-ray diffraction structure of Arg166Ser AP is presented, which shows little difference in enzyme structure compared to the wild-type enzyme but shows a significant reorientation of the bound phosphate. Altogether, these results support a model in which the arginine contributes to catalysis through binding interactions and through additional transition state stabilization that may arise from complementarity of the guanidinum group to the geometry of the trigonal bipyramidal transition state

  9. Crevicular Alkaline Phosphatase Activity and Rate of Tooth Movement of Female Orthodontic Subjects under Different Continuous Force Applications

    Directory of Open Access Journals (Sweden)

    Rohaya Megat Abdul Wahab

    2013-01-01

    Full Text Available Purpose. This study is aimed to compare the effects of two different orthodontic forces on crevicular alkaline phosphatase activity, rate of tooth movement, and root resorption. Materials and Methods. Twelve female subjects of class II division 1 malocclusion participated. Maxillary canines with bonded fixed appliances acted as the tested teeth, while their antagonists with no appliances acted as the controls. Canine retraction was performed using nickel titanium coil spring that delivered forces of 100 gm or 150 gm to either side. Crevicular fluid was analyzed for ALP activity, and study models were casted to measure tooth movements. Root resorption was assessed using periapical radiographs before and after the force application. Results. ALP activity at the mesial sites peaked at week 1 for 150 gm group with significant differences when compared with the 100 gm group. Cumulative canine movements were significantly greater in the 150 gm force (2.10 ± 0.50 mm than in the 100 gm force (1.57 ± 0.44 mm. No root resorption was in the maxillary canines after retraction. Conclusions. A force of 150 gm produced faster tooth movements and higher ALP activity compared with the 100 gm group and had no detrimental effects such as root resorption.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Henk-Jan Prins

    2014-03-01

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

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Soung-Hoon Lee

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

  14. Engineered disulfide bonds increase active-site local stability and reduce catalytic activity of a cold-adapted alkaline phosphatase.

    Science.gov (United States)

    Asgeirsson, Bjarni; Adalbjörnsson, Björn Vidar; Gylfason, Gudjón Andri

    2007-06-01

    Alkaline phosphatase is an extracellular enzyme that is membrane-bound in eukaryotes but resides in the periplasmic space of bacteria. It normally carries four cysteine residues that form two disulfide bonds, for instance in the APs of Escherichia coli and vertebrates. An AP variant from a Vibrio sp. has only one cysteine residue. This cysteine is second next to the nucleophilic serine in the active site. We have individually modified seven residues to cysteine that are on two loops predicted to be within a 5 A radius. Four of them formed a disulfide bond to the endogenous cysteine. Thermal stability was monitored by circular dichroism and activity measurements. Global stability was similar to the wild-type enzyme. However, a significant increase in heat-stability was observed for the disulfide-containing variants using activity as a measure, together with a large reduction in catalytic rates (k(cat)) and a general decrease in Km values. The results suggest that a high degree of mobility near the active site and in the helix carrying the endogenous cysteine is essential for full catalytic efficiency in the cold-adapted AP.

  15. Carbon quantum dots-based recyclable real-time fluorescence assay for alkaline phosphatase with adenosine triphosphate as substrate.

    Science.gov (United States)

    Qian, Zhaosheng; Chai, Lujing; Tang, Cong; Huang, Yuanyuan; Chen, Jianrong; Feng, Hui

    2015-03-03

    A convenient, reliable, and highly sensitive real-time assay for alkaline phosphatase (ALP) activity in the continuous and recyclable way is established on the basis of aggregation and disaggregation of carbon quantum dots (CQDs) through the competitive assay approach. CQDs and adenosine triphosphate (ATP) were used as the fluorescent indicator and substrate for ALP activity assessment, respectively. Richness of carboxyl groups on the surface of CQDs enables their severe aggregation triggered by cerium ions, which results in effective fluorescence quenching. Under the catalytic hydrolysis of ALP, ATP can be rapidly transformed to phosphate ions. Stronger affinity of phosphate ions to cerium ions than carboxyl groups is taken advantage of to achieve fluorescence recovery induced by redispersion of CQDs in the presence of ALP and ATP. Quantitative evaluation of ALP activity in a broad range from 4.6 to 383.3 U/L with the detection limit of 1.4 U/L can be realized in this way, which endows the assay with high enough sensitivity for practical detection in human serum. The assay can be used in a recyclable way for more than three times since the generated product CePO4 as a precipitate can be easily removed from the standard assay system. This strategy broadens the sensing application of fluorescent CQDs with excellent biocompatibility and provides an example based on disaggregation in optical probe development.

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

    Science.gov (United States)

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

    2015-08-01

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

  17. Effect of carbonated hydroxyapatite incorporated advanced platelet rich fibrin intrasulcular injection on the alkaline phosphatase level during orthodontic relapse

    Science.gov (United States)

    Alhasyimi, Ananto Ali; Pudyani, Pinandi Sri; Asmara, Widya; Ana, Ika Dewi

    2018-02-01

    Nowadays, relapse in orthodontic treatment is considered very important because of high incidence of relapse after the treatment. Alkaline phosphatase (ALP) as a biomarker of bone formation will decrease in compression sites during relapse after orthodontic tooth movement. In this situation, manipulating alveolar bone remodeling to increase ALP level is considered one of the new strategies to prevent relapse properly. In the field of tissue engineering, in this study, carbonated hydroxyapatite (CHA) is expected to have the ability to incorporate advanced platelet rich fibrin (aPRF). Next, CHA will retain the aPRF containing various growth factors (GF) until it reaches into a specific targeted area, gradually degraded, and deliver the GF in a controlled manner to prevent relapse. Here, gingival crevicular fluid (GCF) of 45 samples (n=45) were collected and levels of ALP were analyzed using UV-Vis 6300 Spectrophotometer at 405 nm wavelength. We found that there is a significant difference of ALP levels (p<0.05) in GCF between treatments and control groups. ALP level was elevated significantly in CHA and CHA-aPRF groups at days 7 and 14 after debonding compared with the control groups. The peak level of ALP was observed at days 14 after debonding in groups C (0.789 ± 0.039 U/mg). Therefore, it can be concluded that the application of hydrogel CHA with controlled release manner incorporated aPRF enhances bone regeneration by increasing ALP level.

  18. Liver Fat, Hepatic Enzymes, Alkaline Phosphatase and the Risk of Incident Type 2 Diabetes: A Prospective Study of 132,377 Adults

    OpenAIRE

    Chen, Sean Chun-Chang; Tsai, Shan Pou; Jhao, Jing-Yun; Jiang, Wun-Kai; Tsao, Chwen Keng; Chang, Ly-Yun

    2017-01-01

    Previous studies have reported inconsistent results of the associations of alanine transaminase (ALT), aspartate transaminase (AST), gamma-glutamyltransferase (GGT) and alkaline phosphatase (ALP) with incident type 2 diabetes (diabetes hereafter). We aimed to resolve the controversy by taking nonalcoholic fatty liver disease (NAFLD) into account. The study population comprised 132,377 non-diabetic individuals (64,875 men and 67,502 women) aged 35?79 who had two or more health examinations dur...

  19. Comparison of Salivary pH, Buffering Capacity and Alkaline Phosphatase in Smokers and Healthy Non-Smokers: Retrospective cohort study

    OpenAIRE

    Ahmadi-Motamayel, Fatemeh; Falsafi, Parisa; Goodarzi, Mohammad T.; Poorolajal, Jalal

    2016-01-01

    Objectives: Saliva contains alkaline phosphatase (ALP)—a key intracellular enzyme related to destructive processes and cellular damage—and has buffering capacity (BC) against acids due to the presence of bicarbonate and phosphate ions. Smoking may have deleterious effects on the oral environment due to pH changes which can affect ALP activity. This study aimed to evaluate the salivary pH, BC and ALP activity of male smokers and healthy non-smokers. Methods: This retrospective cohort study ...

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

    International Nuclear Information System (INIS)

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

    2013-01-01

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

  1. Pst I restriction fragment length polymorphism of human placental alkaline phosphatase gene: Mendelian in segregation and localization of mutation site in the gene

    International Nuclear Information System (INIS)

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

    1988-01-01

    The pattern of inheritance of a Pst I restriction fragment length polymorphism (RFLP) of the human placental alkaline phosphatase gene was studied in nine nuclear families by Southern blot hybridization analysis of genomic DNA. The dimorphic RFLP is defined by the presence of allelic fragments 1.0 kilobase and 0.8 kilobase long. The results of this study show that the two alleles of the Pst I RFLP of the placental alkaline phosphatase gene segregate as codominant traits according to Mendelian expectations. For a polymorphism to be useful as a genetic marker the probability that an offspring is informative (PIC) must be at least 0.15. The allelic frequency of the 1.0-kilobase allele is 0.21, which correlates to a probability that an offspring is informative of 0.275 and is indicative of a useful polymorphism. By using probes derived from different regions of the placental alkaline phosphatase cDNA, the mutated Pst I site causing the RFLP was located in the penultimate intron 2497 base pairs downstream from the transcriptional initiation site

  2. Effects of tunicamycin, mannosamine, and other inhibitors of glycoprotein processing on skeletal alkaline phosphatase in human osteoblast-like cells.

    Science.gov (United States)

    Farley, J R; Magnusson, P

    2005-01-01

    Skeletal alkaline phosphatase (sALP) is a glycoprotein- approximately 20% carbohydrate by weight, with five presumptive sites for N-linked glycosylation, as well as a carboxy-terminal site for attachment of the glycolipid structure (glycosylphosphatidylinositol, GPI), which anchors sALP to the outer surface of osteoblasts. The current studies were intended to characterize the effects of inhibiting glycosylation and glycosyl-processing on the synthesis, plasma membrane attachment, cellular-extracellular distribution, and reaction kinetics of sALP in human osteosarcoma (SaOS-2) cells. sALP synthesis, glycosylation, and GPI-anchor attachment were assessed as total protein synthesis/immunospecific sALP synthesis, sialic acid content (i.e., wheat germ agglutinin precipitation), and insolubility (i.e., temperature-dependent phase-separation), respectively. sALP reaction kinetics were characterized by analysis of dose-dependent initial velocity data, with a phosphoryl substrate. The results of these studies revealed that the inhibition of either N-linked glycosylation or oligosaccharide synthesis for GPI-anchor addition could affect the synthesis and the distribution of sALP, but not the kinetics of the phosphatase reaction. Tunicamycin-which blocks N-linked glycosylation by inhibiting core oligosaccharide synthesis-decreased cell layer protein and the total amount of sALP in the cells, while increasing the relative level of sALP in the cell-conditioned culture medium (CM, i.e., the amount of sALP released). These effects were attributed to dose- and time-dependent decreases in sALP synthesis and N-linked glycosylation, and an increase in apoptotic cell death (P sALP specific activity, in the cells and in the CM; and (3) increases in the percentages of both anchorless and wheat germ agglutinin (WGA)-soluble sALP in the medium, but not in the cells (P sALP to the outside of the plasma membrane surface. Neither mannosammine nor tunicamycin had any effect on the reaction

  3. Variaciones de la enzima fosfatasa alcalina en la pulpa dental Variations of alkaline phosphatase enzyme in the dental pulp

    Directory of Open Access Journals (Sweden)

    Zoraida Pons Pinillos

    2005-08-01

    Full Text Available En las últimas décadas, numerosas investigaciones se han dedicado al estudio de los mecanismos potenciales implicados en el desarrollo de la caries dental y su prevención, sin embargo, a pesar de haber disminuido gradualmente el índice de caries en la población, son muchos los pacientes que necesitan tratarse la caries dental, tal es así que continuamente se están utilizando diferentes materiales en la búsqueda de aquel que ante una agresión a la pulpa, ayude a una respuesta biológica de la misma, conservando de esta forma su integridad. De ahí la importancia de la actividad de la fosfatasa alcalina de la pulpa en el proceso carioso, como una reacción ante el hidróxido de calcio que continuamente se está usando en toda la red docente-asistencial del país. Se seleccionaron 50 dientes monorradiculares, con pulpa viva y con caries de segundo, tercer y cuarto grado y 50 dientes sanos de pacientes de diferentes edades. Se extrajo la pulpa de cada diente y se realizaron improntas (3 por cada muestra, una de las cuales se procesó para obtener orientación morfológica, y las otras 2 para valorar la actividad de la fosfatasa alcalina. Para esto se utilizaron 2 métodos: el de calcio cobalto y el de alpha naftol fosfato de Gomori. Como resultado, se obtuvo que la pulpa tiene más actividad enzimática en caries profunda y que la edad del paciente no determina el aumento o disminución de dicha actividad.In the last decades, numerous investigations have been made on the study of potential mechanisms involved in the development of dental caries and their prevention. However, in spite of the gradual reduction of dental caries in the population, a lot of patients need to have their dental caries treated and different materials are continuously used searching for one that before the aggression to the pulp helps it to give a biological response, conserving this way its integrity. That's why the activity of the alkaline phosphatase of the pulp

  4. Comparison of Salivary pH, Buffering Capacity and Alkaline Phosphatase in Smokers and Healthy Non-Smokers

    Science.gov (United States)

    Ahmadi-Motamayel, Fatemeh; Falsafi, Parisa; Goodarzi, Mohammad T.; Poorolajal, Jalal

    2016-01-01

    Objectives: Saliva contains alkaline phosphatase (ALP)—a key intracellular enzyme related to destructive processes and cellular damage—and has buffering capacity (BC) against acids due to the presence of bicarbonate and phosphate ions. Smoking may have deleterious effects on the oral environment due to pH changes which can affect ALP activity. This study aimed to evaluate the salivary pH, BC and ALP activity of male smokers and healthy non-smokers. Methods: This retrospective cohort study took place between August 2012 and December 2013. A total of 251 healthy male non-smokers and 259 male smokers from Hamadan, Iran, were selected. Unstimulated whole saliva was collected from each participant and pH and BC were determined using a pH meter. Salivary enzymes were measured by spectrophotometric assay. Results: Mean salivary pH (7.42 ± 0.48 and 7.52 ± 0.43, respectively; P = 0.018) and BC (3.41 ± 0.54 and 4.17 ± 0.71; P = 0.001) was significantly lower in smokers compared to non-smokers. Mean ALP levels were 49.58 ± 23.33 IU/L among smokers and 55.11 ± 27.85 IU/L among non-smokers (P = 0.015). Conclusion: Significantly lower pH, BC and ALP levels were observed among smokers in comparison to a healthy control group. These salivary alterations could potentially be utilised as biochemical markers for the evaluation of oral tissue function and side-effects among smokers. Further longitudinal studies are recommended to evaluate the effects of smoking on salivary components. PMID:27606111

  5. Association between serum alkaline phosphatase and coronary artery calcification in a sample of primary cardiovascular prevention patients.

    Science.gov (United States)

    Panh, Loïc; Ruidavets, Jean Bernard; Rousseau, Hervé; Petermann, Antoine; Bongard, Vanina; Bérard, Emilie; Taraszkiewicz, Dorota; Lairez, Olivier; Galinier, Michel; Carrié, Didier; Ferrières, Jean

    2017-05-01

    A high level of serum alkaline phosphatase (ALP) is associated with an increased risk of mortality and myocardial infarction. ALP hydrolyses inorganic pyrophosphate, which is a strong inhibitor of calcium phosphate deposition. The aim of this study was to determine whether ALP is associated with the coronary artery calcium score (CACS). We examined the association of CACS, assessed by computed tomography scanning, and ALP, in 500 patients consecutively recruited, free of cardiovascular disease. The CACS were categorized into two groups: no calcification (CACS = 0) (n = 187) and with calcification (CACS>0) (n = 313). ALP activity was divided into three tertile groups: low ALP level (66 IU/L). The mean age was 60.9 ± 10.8 years, 49.6% of the patients were women. ALP ranged from 22 to 164 IU/L (mean 62.6 IU/L, SD 19.3). In univariate analysis, traditional cardiovascular risk factors, statin use (p = 0.001), and ALP (p = 0.001) were significantly associated with CACS. After adjusting for cardiovascular risk factors, only age (p = 0.001) and sex (p = 0.001) were independently associated with CACS. Compared to the tertile group with low levels of ALP, the intermediate tertile group [OR 2.11, 95% CI (1.12; 3.96), p = 0.02], as well as the high tertile group [OR 3.89, 95% CI (2.01; 7.54), p = 0.001)], was independently associated with CACS. In patients free of cardiovascular disease, high ALP levels are positively and independently associated with coronary artery calcification. The metabolic pathway of ALP and inorganic pyrophosphate could be a target for new therapies against vascular calcification. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Effect of Long-Term Hypodynamy on Alkaline Phosphatase Activity of Small Intestine in Japanese Quail Chicks

    Directory of Open Access Journals (Sweden)

    Ľ. Lenhardt

    2007-01-01

    Full Text Available The functional development of the small intestine was investigated in Japanese quail chicks subjected to simulated microgravity (hypodynamy on the second day after hatching and reared under these conditions to 63 days of age. On days 5, 7, 14, 21, 28, 35, 42, 56 and 63 the activity of brush-border-bound alkaline phosphatase (AP in the duodenum and jejunum were determined in experimental animals as well as in control quail chicks housed in a floor box during these periods. As compared with control quails the experimental animals displayed a significantly increased enzyme activity until day 42 in the duodenum and day 35 in the jejunum (P < 0.001 whereas in older quails no significant enzymatic differences between these groups was found. However, a decrease in food consumption due to a partial physical constraint cannot be excluded. Moreover, the results suggested that the activity of AP in the control birds did not change substantially during all the periods examined. In contrast, in older hypodynamy quail the AP activity significantly decreased in the duodenum on days 56 and 63 and in the jejunum on days 42, 56 and 63, respectively. These results indicate that a the enhanced intestinal function in early periods of life may reflect the higher sensitivity of small intestine to simulated weightlessness, b the decrease of the AP activity in older animals to the level of controls might be considered as a part of intestinal mechanisms involved in adaptation of quail chicks to long-term hypodynamy, c different activity of AP in the small intestine of Japanese quail may not have resulted only from hypodynamy but also due to decreased food intake.

  7. Micro-Droplet Detection Method for Measuring the Concentration of Alkaline Phosphatase-Labeled Nanoparticles in Fluorescence Microscopy

    Directory of Open Access Journals (Sweden)

    Rufeng Li

    2017-11-01

    Full Text Available This paper developed and evaluated a quantitative image analysis method to measure the concentration of the nanoparticles on which alkaline phosphatase (AP was immobilized. These AP-labeled nanoparticles are widely used as signal markers for tagging biomolecules at nanometer and sub-nanometer scales. The AP-labeled nanoparticle concentration measurement can then be directly used to quantitatively analyze the biomolecular concentration. Micro-droplets are mono-dispersed micro-reactors that can be used to encapsulate and detect AP-labeled nanoparticles. Micro-droplets include both empty micro-droplets and fluorescent micro-droplets, while fluorescent micro-droplets are generated from the fluorescence reaction between the APs adhering to a single nanoparticle and corresponding fluorogenic substrates within droplets. By detecting micro-droplets and calculating the proportion of fluorescent micro-droplets to the overall micro-droplets, we can calculate the AP-labeled nanoparticle concentration. The proposed micro-droplet detection method includes the following steps: (1 Gaussian filtering to remove the noise of overall fluorescent targets, (2 a contrast-limited, adaptive histogram equalization processing to enhance the contrast of weakly luminescent micro-droplets, (3 an red maximizing inter-class variance thresholding method (OTSU to segment the enhanced image for getting the binary map of the overall micro-droplets, (4 a circular Hough transform (CHT method to detect overall micro-droplets and (5 an intensity-mean-based thresholding segmentation method to extract the fluorescent micro-droplets. The experimental results of fluorescent micro-droplet images show that the average accuracy of our micro-droplet detection method is 0.9586; the average true positive rate is 0.9502; and the average false positive rate is 0.0073. The detection method can be successfully applied to measure AP-labeled nanoparticle concentration in fluorescence microscopy.

  8. Micro-Droplet Detection Method for Measuring the Concentration of Alkaline Phosphatase-Labeled Nanoparticles in Fluorescence Microscopy.

    Science.gov (United States)

    Li, Rufeng; Wang, Yibei; Xu, Hong; Fei, Baowei; Qin, Binjie

    2017-11-21

    This paper developed and evaluated a quantitative image analysis method to measure the concentration of the nanoparticles on which alkaline phosphatase (AP) was immobilized. These AP-labeled nanoparticles are widely used as signal markers for tagging biomolecules at nanometer and sub-nanometer scales. The AP-labeled nanoparticle concentration measurement can then be directly used to quantitatively analyze the biomolecular concentration. Micro-droplets are mono-dispersed micro-reactors that can be used to encapsulate and detect AP-labeled nanoparticles. Micro-droplets include both empty micro-droplets and fluorescent micro-droplets, while fluorescent micro-droplets are generated from the fluorescence reaction between the APs adhering to a single nanoparticle and corresponding fluorogenic substrates within droplets. By detecting micro-droplets and calculating the proportion of fluorescent micro-droplets to the overall micro-droplets, we can calculate the AP-labeled nanoparticle concentration. The proposed micro-droplet detection method includes the following steps: (1) Gaussian filtering to remove the noise of overall fluorescent targets, (2) a contrast-limited, adaptive histogram equalization processing to enhance the contrast of weakly luminescent micro-droplets, (3) an red maximizing inter-class variance thresholding method (OTSU) to segment the enhanced image for getting the binary map of the overall micro-droplets, (4) a circular Hough transform (CHT) method to detect overall micro-droplets and (5) an intensity-mean-based thresholding segmentation method to extract the fluorescent micro-droplets. The experimental results of fluorescent micro-droplet images show that the average accuracy of our micro-droplet detection method is 0.9586; the average true positive rate is 0.9502; and the average false positive rate is 0.0073. The detection method can be successfully applied to measure AP-labeled nanoparticle concentration in fluorescence microscopy.

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

    Science.gov (United States)

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

    2013-12-01

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

  10. Alkaline phosphatase role in bone marrow and spleen hemopoietic cells recovery after mouse whole-body irradiation

    International Nuclear Information System (INIS)

    Al Mouhamad, K.; Al Sheikh, F.

    2013-04-01

    Hematopoietic tissue is consisted of two distinctly different tissues, the first part is the hematopoietic stem cells and the second tissue is a mixture of many supportive cells which the most important one of them is alkaline phosphatase (ALP)-secreted-fibroblastic cells (FBCs). It was thought that FBCs play an important role in the hematopoiesis through ALP secretion. Our previous studies indicated that the ALP secretion in bone marrow (BM) increased after a whole mouse body irradiation when the BM cellular component is completely destroyed and, then it was decreased when the BM regain its cellular component. We performed some experiences to verify if there is any role to the ALP in the hematopoiesis. We irradiated three groups of mice to non-lethal dose, the first one was injected by Tetramizole (anti-ALP) 24 hours before irradiation, and the second was injected by Lisinopril (anti-hematopoiesis) 24 hours before irradiation and the third left without any injection. The fourth left as control. Many histological sections were taken from BM and spleen on 1, 3, 7 and 30 days after irradiation to perform ALP-histological detection. These experiences were repeated to count BM cells. ALP secretion level in the BM was reached the maximum 3 days after irradiation without any injection when the cell number was in minimum then, the level of ALP start to decrease and the cell number start to increase. ALP secretion delayed when the mice were injected by Tetramizole and BM cell population also delayed to return to its normal position. But, the ALP secretion increased directly after irradiation when the mice were injected by Lisinopril which, the ALP secretion, normally reached the maximum by the third day. These results may indicate a role to the ALP in BM and spleen hematopoietic cell recovery (author).

  11. Real-time fluorescence assay of alkaline phosphatase in living cells using boron-doped graphene quantum dots as fluorophores.

    Science.gov (United States)

    Chen, Li; Yang, Guancao; Wu, Ping; Cai, Chenxin

    2017-10-15

    This work reports a convenient and real-time assay of alkaline phosphatase (ALP) in living cells based on a fluorescence quench-recovery process at a physiological pH using the boron-doped graphene quantum dots (BGQDs) as fluorophore. The fluorescence of BGQDs is found to be effectively quenched by Ce 3+ ions because of the coordination of Ce 3+ ions with the carboxyl group of BGQDs. Upon addition of adenosine triphosphate (ATP) into the system, the quenched fluorescence can be recovered by the ALP-positive expressed cells (such as MCF-7 cells) due to the removal of Ce 3+ ions from BGQDs surface by phosphate ions, which are generated from ATP under catalytic hydrolysis of ALP that expressed in cells. The extent of fluorescence signal recovery depends on the level of ALP in cells, which establishes the basis of ALP assay in living cells. This approach can also be used for specific discrimination of the ALP expression levels in different type of cells and thus sensitive detection of those ALP-positive expressed cells (for example MCF-7 cells) at a very low abundance (10±5 cells mL -1 ). The advantages of this approach are that it has high sensitivity because of the significant suppression of the background due to the Ce 3+ ion quenching the fluorescence of BGQDs, and has the ability of avoiding false signals arising from the nonspecific adsorption of non-target proteins because it operates via a fluorescence quench-recovery process. In addition, it can be extended to other enzyme systems, such as ATP-related kinases. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2012-10-01

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

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

    Science.gov (United States)

    Wang, Shoubing; Xu, Ziran

    2016-01-01

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

  14. Analysis of human bone alkaline phosphatase isoforms: comparison of isoelectric focusing and ion-exchange high-performance liquid chromatography.

    Science.gov (United States)

    Sharp, Christopher A; Linder, Cecilia; Magnusson, Per

    2007-04-01

    Several isoforms of alkaline phosphatase (ALP) can be identified in human tissues and serum after separation by anion-exchange HPLC and isoelectric focusing (IEF). We purified four soluble bone ALP (BALP) isoforms (B/I, B1x, B1 and B2) from human SaOS-2 cells, determined their specific pI values by broad range IEF (pH 3.5-9.5), compared these with commercial preparations of bone, intestinal and liver ALPs and established the effects of neuraminidase and wheat germ lectin (WGA) on enzyme activity. Whilst the isoforms B1x (pI=4.48), B1 (pI=4.32) and B2 (pI=4.12) resolved as well-defined bands, B/I resolved as a complex (pI=4.85-6.84). Neuraminidase altered the migration of all BALP isoforms to pI=6.84 and abolished their binding to the anion-exchange matrix, but increased their enzymatic activities by 11-20%. WGA precipitated the BALP isoforms in IEF gels and the HPLC column and attenuated their enzymatic activities by 54-73%. IEF resolved the commercial BALP into 2 major bands (pI=4.41 and 4.55). Migration of BALP isoforms is similar in IEF and anion-exchange HPLC and dependent on sialic acid content. HPLC is preferable in smaller scale research applications where samples containing mixtures of BALP isoforms are analysed. Circulating liver ALP (pI=3.85) can be resolved from BALP by either method. IEF represents a simpler approach for routine purposes even though some overlapping of the isoforms may occur.

  15. Photoinduced electron transfer between Fe(III) and adenosine triphosphate-BODIPY conjugates: Application to alkaline-phosphatase-linked immunoassay.

    Science.gov (United States)

    Lin, Jia-Hui; Yang, Ya-Chun; Shih, Ya-Chen; Hung, Szu-Ying; Lu, Chi-Yu; Tseng, Wei-Lung

    2016-03-15

    Fluorescent boron dipyrromethene (BODIPY) analogs are often used as sensors for detecting various species because of their relatively high extinction coefficients, outstanding fluorescence quantum yields, photostability, and pH-independent fluorescence. However, there is little-to-no information in the literature that describes the use of BODIPY analogs for detecting alkaline phosphatase (ALP) activity and inhibition. This study discovered that the fluorescence of BODIPY-conjugated adenosine triphosphate (BODIPY-ATP) was quenched by Fe(III) ions through photoinduced electron transfer. The ALP-catalyzed hydrolysis of BODIPY-ATP resulted in the formation of BODIPY-adenosine and phosphate ions. The fluorescence of the generated BODIPY-adenosine was insensitive to the change in the concentration of Fe(III) ions. Thus, the Fe(III)-induced fluorescence quenching of BODIPY-ATP can be paired with its ALP-mediated dephosphorylation to design a turn-on fluorescence probe for ALP sensing. A method detection limit at a signal-to-noise ratio of 3 for ALP was estimated to be 0.02 units/L (~6 pM; 1 ng/mL). This probe was used for the screening of ALP inhibitors, including Na3VO4, imidazole, and arginine. Because ALP is widely used in enzyme-linked immunosorbent assays, the probe was coupled to an ALP-linked immunosorbent assay for the sensitive and selective detection of immunoglobulin G (IgG). The lowest detectable concentration for IgG in this system was 5 ng/mL. Compared with the use of 3,6-fluorescein diphosphate as a signal reporter in an ALP-linked immunosorbent assay, the proposed system provided comparable sensitivity, large linear range, and high stability over temperature and pH changes. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2016-01-01

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

  17. Fluoride therapy for osteoporosis: characterization of the skeletal response by serial measurements of serum alkaline phosphatase activity.

    Science.gov (United States)

    Farley, S M; Wergedal, J E; Smith, L C; Lundy, M W; Farley, J R; Baylink, D J

    1987-03-01

    Optimum use of fluoride therapy for osteoporosis requires a sensitive and convenient index of the skeletal response to fluoride. Since previous studies had shown that serum alkaline phosphatase activity (SALP) was increased in response to fluoride therapy, we examined serial measurements of SALP in 53 osteoporotics treated with 66 to 110 mg of sodium fluoride (NaF) for 12 to 91 months. SALP was increased in 87% of the subjects during therapy with fluoride. The increase in SALP was thought to reflect the osteogenic action of fluoride based on the findings that SALP correlated with both trabecular bone area (r = .81, P less than .001) and osteoid length (r = .67, P less than .01) in iliac crest biopsies, predicted increased bone density on spinal radiographs in response to fluoride therapy with an 87% accuracy, and predicted decreased back pain in response to fluoride with a 91% accuracy. In addition, the SALP response to fluoride was seen earlier than other therapeutic responses as indicated by the findings that the tau 1/2 for the SALP response (ie, time for 1/2 of the patients to show a significant response) was significantly less (1.2 +/- 0.3 yr) than that for the pain response (1.6 +/- 0.3 yr, P less than .05) or that for the radiographic response (3.7 +/- 0.5 yr, P less than .001). Although most patients responded to fluoride with an increase in SALP, evaluation of the kinetics of the SALP response to fluoride revealed marked interpatient variation.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. Dissolved phosphorus pools and alkaline phosphatase activity in the euphotic zone of the western North Pacific Ocean.

    Directory of Open Access Journals (Sweden)

    Masahiro eSuzumura

    2012-03-01

    Full Text Available We measured pools of dissolved phosphorus (P, including dissolved inorganic P (DIP, dissolved organic P (DOP and alkaline phosphatase (AP-hydrolyzable labile DOP (L-DOP, and kinetic parameters of AP activity (APA in the euphotic zone in the western North Pacific Ocean. Samples were collected from one coastal station in Sagami Bay, Japan, and three offshore stations between the North Pacific Subtropical Gyre (NPSG and the Kuroshio region. Although DIP concentrations in the euphotic zone at all stations were equally low, around the nominal method detection limit of 20 nmol L−1, chlorophyll a (Chl a concentrations were one order of magnitude greater at the coastal station. DOP was the dominant P pool, comprising 62–92% of total dissolved P at and above the Chl a maximum layer (CML. L-DOP represented 22–39% of the total DOP at the offshore stations, whereas it accounted for a much higher proportion (about 85% in the coastal surface layers. Significant correlations between maximum potential AP hydrolysis rates and DIP concentrations or bacterial cell abundance in the offshore euphotic zone suggest that major APA in the oligotrophic surface ocean is from bacterial activity and regulated largely by DIP availability. Although the range of maximum potential APA was comparable among the environmental conditions, the in situ hydrolysis rate of L-DOP in the coastal station was 10 times those in the offshore stations. L-DOP turnover time at the CML ranged from 4.5 d at the coastal station to 84.4 d in the NPSG. The ratio of the APA half saturation constant to the ambient L-DOP concentration decreased markedly from the NPSG to the coastal station. There were substantial differences in the rate end efficiency of DOP remineralization and its contribution as the potential P source between the low-phosphate/high biomass coastal ecosystem and the low-phosphate/low biomass oligotrophic ocean.

  19. Analysis of serum adenosine deaminase (ADA) and ADA1 and ADA2 isoenzyme activities in HIV positive and HIV-HBV co-infected patients.

    Science.gov (United States)

    Khodadadi, Iraj; Abdi, Mohammad; Ahmadi, Abbas; Wahedi, Mohammad Saleh; Menbari, Shahoo; Lahoorpour, Fariba; Rahbari, Rezgar

    2011-08-01

    To determine adenosine deaminase (ADA) activity as a possible diagnostic marker in HIV and HIV-HBV co-infected patients. Blood samples were collected from 72 healthy, 33 HIV positive and 30 HIV-HBV co-infected subjects. Blood CD4+ cell count was recorded and serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total ADA, and ADA1 and ADA2 isoenzyme activities were determined. Serum ALT, AST, total ADA and ADA2 isoenzyme activities were significantly higher in HIV positive and HIV-HBV co-infected groups compare to the control (pADA activities (R(2)=0.589, pADA was significantly increased in HIV and HIV-HBV co-infections. Therefore, because of its low cost and simplicity to perform, ADA activity might be considered as a useful diagnostic tool among the other markers in these diseases. Copyright © 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  20. Subcellular localisation and properties of histone phosphate phosphatase in human polymorphonuclear leukocytes: alterations in pregnancy and chronic granulocytic leukaemia and relationship to alkaline phosphatase

    International Nuclear Information System (INIS)

    Smith, G.P.; Peters, T.J.

    1981-01-01

    Using [ 32 P]histone as substrate, an assay for histone phosphate phosphatase was optimised for human polymorphonuclear leukocytes. Kinetic studies showed that the activity was optimal at pH 6.8, was stimulated by Mn 2+ and Co 2+ , and inhibited by sodium sulphite and zinc chloride. The apparent Ksub(m) of the enzyme for histone phosphate was 0.89 μmol/l. (Auth.)

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

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    Myriam Ermonval

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

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

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    Peifang Wang

    2014-08-01

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

  3. Inhibition of the gut enzyme intestinal alkaline phosphatase may explain how aspartame promotes glucose intolerance and obesity in mice.

    Science.gov (United States)

    Gul, Sarah S; Hamilton, A Rebecca L; Munoz, Alexander R; Phupitakphol, Tanit; Liu, Wei; Hyoju, Sanjiv K; Economopoulos, Konstantinos P; Morrison, Sara; Hu, Dong; Zhang, Weifeng; Gharedaghi, Mohammad Hadi; Huo, Haizhong; Hamarneh, Sulaiman R; Hodin, Richard A

    2017-01-01

    Diet soda consumption has not been associated with tangible weight loss. Aspartame (ASP) commonly substitutes sugar and one of its breakdown products is phenylalanine (PHE), a known inhibitor of intestinal alkaline phosphatase (IAP), a gut enzyme shown to prevent metabolic syndrome in mice. We hypothesized that ASP consumption might contribute to the development of metabolic syndrome based on PHE's inhibition of endogenous IAP. The design of the study was such that for the in vitro model, IAP was added to diet and regular soda, and IAP activity was measured. For the acute model, a closed bowel loop was created in mice. ASP or water was instilled into it and IAP activity was measured. For the chronic model, mice were fed chow or high-fat diet (HFD) with/without ASP in the drinking water for 18 weeks. The results were that for the in vitro study, IAP activity was lower (p < 0.05) in solutions containing ASP compared with controls. For the acute model, endogenous IAP activity was reduced by 50% in the ASP group compared with controls (0.2 ± 0.03 vs 0.4 ± 0.24) (p = 0.02). For the chronic model, mice in the HFD + ASP group gained more weight compared with the HFD + water group (48.1 ± 1.6 vs 42.4 ± 3.1, p = 0.0001). Significant difference in glucose intolerance between the HFD ± ASP groups (53 913 ± 4000.58 (mg·min)/dL vs 42 003.75 ± 5331.61 (mg·min)/dL, respectively, p = 0.02). Fasting glucose and serum tumor necrosis factor-alpha levels were significantly higher in the HFD + ASP group (1.23- and 0.87-fold increases, respectively, p = 0.006 and p = 0.01). In conclusion, endogenous IAP's protective effects in regard to the metabolic syndrome may be inhibited by PHE, a metabolite of ASP, perhaps explaining the lack of expected weight loss and metabolic improvements associated with diet drinks.

  4. Molecular basis of perinatal hypophosphatasia with tissue-nonspecific alkaline phosphatase bearing a conservative replacement of valine by alanine at position 406. Structural importance of the crown domain.

    Science.gov (United States)

    Numa, Natsuko; Ishida, Yoko; Nasu, Makiko; Sohda, Miwa; Misumi, Yoshio; Noda, Tadashi; Oda, Kimimitsu

    2008-06-01

    Hypophosphatasia, a congenital metabolic disease related to the tissue-nonspecific alkaline phosphatase gene (TNSALP), is characterized by reduced serum alkaline phosphatase levels and defective mineralization of hard tissues. A replacement of valine with alanine at position 406, located in the crown domain of TNSALP, was reported in a perinatal form of hypophosphatasia. To understand the molecular defect of the TNSALP (V406A) molecule, we examined this missense mutant protein in transiently transfected COS-1 cells and in stable CHO-K1 Tet-On cells. Compared with the wild-type enzyme, the mutant protein showed a markedly reduced alkaline phosphatase activity. This was not the result of defective transport and resultant degradation of TNSALP (V406A) in the endoplasmic reticulum, as the majority of newly synthesized TNSALP (V406A) was conveyed to the Golgi apparatus and incorporated into a cold detergent insoluble fraction (raft) at a rate similar to that of the wild-type TNSALP. TNSALP (V406A) consisted of a dimer, as judged by sucrose gradient centrifugation, suggestive of its proper folding and correct assembly, although this mutant showed increased susceptibility to digestion by trypsin or proteinase K. When purified as a glycosylphosphatidylinositol-anchorless soluble form, the mutant protein exhibited a remarkably lower Kcat/Km value compared with that of the wild-type TNSALP. Interestingly, leucine and isoleucine, but not phenylalanine, were able to substitute for valine, pointing to the indispensable role of residues with a longer aliphatic side chain at position 406 of TNSALP. Taken together, this particular mutation highlights the structural importance of the crown domain with respect to the catalytic function of TNSALP.

  5. Zinc and magnesium in the uterus of the pregnant and pseudopregnant mouse and the effects of Mg2+ ions on uterine alkaline phosphatase.

    Science.gov (United States)

    Buxton, L E; Murdoch, R N

    1981-01-01

    The levels of zinc and magnesium in the mouse uterus during early pregnancy and pseudopregnancy were determined using atomic absorption spectroscopy techniques. The total zinc and magnesium content of the uterus increased between days 5 and 12 of pregnancy and between days 5 and 9 of content of the pseudopregnancy when decidual cells were present. However, the metals were not accumulated at a rate sufficient to match increases in uterine weight and constant concentrations (micrograms of metals per gram wet weight ot tissue) were not maintained over the various reproductive stages studied. The accumulation of the metals was associated with the presence of decidual cells, and non-decidualized horns of pseudopregnant mice failed to increase their total content of zinc and magnesium between days 5 and 9. The magnesium content of each uterus was usually between 5- and 13-fold greater than the total zinc content. mg2+ in low concentration (0-2mM) stimulated both the pyrophosphatase and orthophosphatase activities of purified preparations of the mouse uterine metalloenzyme, alkaline phosphatase. Higher concentrations (up to 8 mM) of the cation decreased pyrophosphatase activity but did not alter orthophosphatase activity. Mg/+ was more effective, however, in increasing the orthophosphatase activity of the enzyme and its stimulating effects in this case were greater in carbonate-bicarbonate buffer than in glycine-NaOH buffer. Mg2+ did not significantly influence apparent Km values or the response of the enzyme to changes in temperature. Zn2+, however, was required to maintain the stability of alkaline phosphatase apoenzyme preparations. It was concluded that during normal pregnancy and pseudopregnancy zinc and magnesium would always be present in amounts considerably greater than those required to saturate alkaline phosphatase for full catalytic activity. Thus, while the metals exert major effects on the activity and stability of the enzyme in vitro, they may not be major

  6. Quantitative evaluation of the alkaline phosphatase activity in industrial and traditional dairy products supplied in Ahvaz as an indicator of pasteurization

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    M. Zarei

    2017-05-01

    Full Text Available Alkaline phosphatase is an indigenous milk enzyme and is probably, the most important indigenous milk enzyme from a dairy technology viewpoint which is used to determine the efficacy of the pasteurization process. The aim of this study was to assess the alkaline phosphatase activity of 200 samples of industrial and traditional yoghurt, ice cream and cheese, as well as raw and pasteurized milk samples. To achieve this purpose, p-nitrophenylphosphate was used as substrate and the amount of liberated p-nitrophenol was measured spectrophotometrically. The amount of liberated p-nitrophenol in all samples of raw milk was very high (6839±4070 µg/ml but in pasteurized milk samples, the amount was in the range of 0.75-52.96 µg/ml and 88% of the samples had less than 10 µg p-nitrophenol/ml, the maximum permissible limit of p-nitrophenol in pasteurized products. The amount of liberated p-nitrophenol was in the range of 5.68-1210 µg/ml and 2.61-18.22 µg/ml in traditional and industrial cheese samples, respectively and it was estimated at the range of 0.75-26.67 µg/ml and 0.71- 35.82 µg/ml for traditional and industrial ice cream samples, respectively. The lowest alkaline phosphatase activity was observed in both industrial and traditional yoghurt samples. Meanwhile, p-nitrophenol in 12% of industrial cheese, 44% of traditional cheese and 16% of both industrial and traditional ice cream samples was higher than 10 µg/ml which could be due to the inadequate pasteurization of the product or cross contamination with raw milk. The results of the present study showed a need for more strict attention in the pasteurization of milk and its products.

  7. Oxovanadium (iv) complexes with n/o- and o-donor ligands: their synthesis, characterization, semiempirical study and alkaline phosphatase activity (abstract)

    International Nuclear Information System (INIS)

    Munawar, K.S.; Ali, S.; Khan, A.N.

    2011-01-01

    Various N/O- and O-donor ligands and their oxovanadium complexes have been synthesized and characterized by different techniques such as FTIR, elemental analysis, thermogravimetery and conductometry. The IR data show the bidentate nature of the ligands and reveals hexa-coordinated geometry in the solid state which is also confirmed by semi-empirical study. Conductance measurements reveal the non-electrolytic nature of the complexes. These complexes have been checked for their alkaline phosphatase activity in the presence and absence of inhibitor which shows that by the addition of inhibitor the activity of enzyme decreases and at higher concentration it is completely inhibited. (author)

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

    DEFF Research Database (Denmark)

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

    2016-01-01

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

  9. Release of bacterial alkaline phosphatase in the rumen of cattle fed a feedlot bloat-provoking diet or a hay diet.

    Science.gov (United States)

    Cheng, K J; Hironaka, R; Costerton, J W

    1976-05-01

    Alkaline phosphatase (APase) was present in the bovine rumen in both cell-free and cell-associated states and levels of the enzyme varied with dietary regime. Reaction product deposition showed that the enzyme was associated with the mixed bacterial population. No enzyme was observed to be associated with protozoa. Trace activity of APase was also detected in the saliva. The presence of large amounts of APase in cell-free rumen fluid of cattle fed fine concentrate feed is believed to be due, in part, to the breakage of bacterial cells that occurs in the rumen.

  10. Joint influence of temperature and ions of metals on level of activity alkaline phosphatase the mucous membrane of intestines beluga, the starlet and their hybrid

    Directory of Open Access Journals (Sweden)

    D. A. Bednyakov

    2010-01-01

    Full Text Available In work joint influence of ions of bivalent metals (Mn, Fe, Co, Ni, Cu and Zn and temperatures on level of activity alkaline phosphatase mucous membrane beluga, starlet and their hybrid is shown. Dependence of response of enzyme on action of ions of metals according to their position in a periodic table of chemical elements is shown. The given dependence remains and at temperature change incubation, only at low temperatures the activating effect of metals being in the period beginning is maximum, and at high, is maximum inhibiting effect of metals being in the period end.

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

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    Nozawa Sérgio R.

    2002-01-01

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

  12. Assay format as a critical success factor for identification of novel inhibitor chemotypes of tissue-nonspecific alkaline phosphatase from high-throughput screening.

    Science.gov (United States)

    Chung, Thomas D Y; Sergienko, Eduard; Millán, José Luis

    2010-04-27

    The tissue-nonspecific alkaline phosphatase (TNAP) isozyme is centrally involved in the control of normal skeletal mineralization and pathophysiological abnormalities that lead to disease states such as hypophosphatasia, osteoarthritis, ankylosis and vascular calcification. TNAP acts in concert with the nucleoside triphosphate pyrophosphohydrolase-1 (NPP1) and the Ankylosis protein to regulate the extracellular concentrations of inorganic pyrophosphate (PP(i)), a potent inhibitor of mineralization. In this review we describe the serial development of two miniaturized high-throughput screens (HTS) for TNAP inhibitors that differ in both signal generation and detection formats, but more critically in the concentrations of a terminal alcohol acceptor used. These assay improvements allowed the rescue of the initially unsuccessful screening campaign against a large small molecule chemical library, but moreover enabled the discovery of several unique classes of molecules with distinct mechanisms of action and selectivity against the related placental (PLAP) and intestinal (IAP) alkaline phosphatase isozymes. This illustrates the underappreciated impact of the underlying fundamental assay configuration on screening success, beyond mere signal generation and detection formats.

  13. Profile of serum alkaline phosphatase after inoculation of mononuclear cells and bone morphogenetic protein in the repair of osteochondral defects in rabbits

    Directory of Open Access Journals (Sweden)

    Luiz Augusto de Souza

    2011-12-01

    Full Text Available In this study, serum alkaline phosphatase activity was measured in response to the repair of osteochondral defects in twenty-four New Zealand rabbits. The animals were divided into three groups: a control (GC, those treated with bone marrow mononuclear cells (GCM and those that received mononuclear cells with autologous bone morphogenetic protein (BMP + GCM. After exposing the trochlear groove of the left stifle joint, a wedge-shaped segment was removed. Later, the defect was filled with an osteochondral autograft preserved in 98% glycerin. For the GC group, only the bone graft was performed. For the GCM, in addition to the graft, 2x106 seed mononuclear cells were implanted. For the GCM + BMP, the same number of cells, associated with 1μg of bone morphogenetic protein, were intraarticularly administered. The osteoblastic response was measured by analyzing the serum alkaline phosphatase on day 0 (preoperative 3, 15, 30, and 45 after surgery, and by radiographic examinations. Analysis of variance in randomized blocks, factorial and Tukey’s test (p = 0.05 were made. The overall mean GCM was superior to the other groups and the highest rates were among the 15th and 45th days postoperatively. The discrepancy in values between individuals of the same group casts doubts on the veracity of the test.

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

    International Nuclear Information System (INIS)

    Watanabe, Kousuke; Emoto, Noriko; Sunohara, Mitsuhiro; Kawakami, Masanori; Kage, Hidenori; Nagase, Takahide; Ohishi, Nobuya; Takai, Daiya

    2010-01-01

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

  15. Esterase Isoenzyme Variants in Barley

    DEFF Research Database (Denmark)

    Hvid, S.; Nielsen, G.

    1977-01-01

    Gene symbols are proposed for 27 esterase isoenzyme alleles representing 10 loci in barley. Two new esterase loci, Est 9 and Est 10, each with an active and a silent allele, and three new alleles in previously described loci were found. A few chemical and physical characteristics of the different...... esterase isoenzyme systems were studied. The heat inactivation temperature differed for the isoenzymes coded by most of the loci, whereas the substrate and inhibitor specificity of the isoenzymes was less distinct. A possible relationship between some of the systems is discussed....

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

    International Nuclear Information System (INIS)

    Hu, Zu-Quan; Li, He-Ping; Zhang, Jing-Bo; Huang, Tao; Liu, Jin-Long; Xue, Sheng; Wu, Ai-Bo; Liao, Yu-Cai

    2013-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-02-18

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

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

    Science.gov (United States)

    Kechadi, Mohammed; Sotta, Bruno; Gamby, Jean

    2015-01-01

    This paper presents the use of polymer coated microelectrodes for the realtime conductivity monitoring in a microchannel photoablated through the polymer without contact. Based on this strategy, a small conductometry sensor has been developed to record in time conductivity variation when an enzymatic reaction occurs through the channel. The rate constant determination, k2, for the dephosphorylation of organic phosphate-alkaline phosphatase-superparamagnetic beads complex using chemically different substrates such as adenosine monoesterphosphate, adenosine diphosphate and adenosine triphosphate was taken as an example to demonstrate selectivity and sensivity of the detection scheme. The k2 value measured for each adenosine phosphate decreases from 39 to 30 s(-1) in proportion with the number (3, 2 and 1) of attached phosphate moiety, thus emphasizing the steric hindrance effect on kinetics. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. A Tenebrio molitor GPI-anchored alkaline phosphatase is involved in binding of Bacillus thuringiensis Cry3Aa to brush border membrane vesicles.

    Science.gov (United States)

    Zúñiga-Navarrete, Fernando; Gómez, Isabel; Peña, Guadalupe; Bravo, Alejandra; Soberón, Mario

    2013-03-01

    Bacillus thuringiensis Cry toxins recognizes their target cells in part by the binding to glycosyl-phosphatidyl-inositol (GPI) anchored proteins such as aminopeptidase-N (APN) or alkaline phosphatases (ALP). Treatment of Tenebrio molitor brush border membrane vesicles (BBMV) with phospholipase C that cleaves out GPI-anchored proteins from the membranes, showed that GPI-anchored proteins are involved in binding of Cry3Aa toxin to BBMV. A 68 kDa GPI-anchored ALP was shown to bind Cry3Aa by toxin overlay assays. The 68 kDa GPI-anchored ALP was preferentially expressed in early instar larvae in comparison to late instar larvae. Our work shows for the first time that GPI-anchored ALP is important for Cry3Aa binding to T. molitor BBMV suggesting that the mode of action of Cry toxins is conserved in different insect orders. Copyright © 2012 Elsevier Inc. All rights reserved.

  20. Copper(II) complexes of methimazole, an anti Grave's disease drug. Synthesis, characterization and its potential biological behavior as alkaline phosphatase inhibitor.

    Science.gov (United States)

    Urquiza, Nora M; Manca, Silvia G; Moyano, María A; Dellmans, Raquel Arrieta; Lezama, Luis; Rojo, Teófilo; Naso, Luciana G; Williams, Patricia A M; Ferrer, Evelina G

    2010-04-01

    Methimazole (MeimzH) is an anti-thyroid drug and the first choice for patients with Grave's disease. Two new copper(II) complexes of this drug: [Cu(MeimzH)(2)(NO(3))(2)]*0.5H(2)O and [Cu(MeimzH)(2)(H(2)O)(2)](NO(3))(2)*H(2)O were synthesized and characterized by elemental analysis, dissolution behavior, thermogravimetric analysis and UV-vis, diffuse reflectance, FTIR and EPR spectroscopies. As it is known that copper(II) cation can act as an inhibitor of alkaline phosphatase (ALP), the inhibitory effect of methimazole and its copper(II) complexes on ALP activity has also been investigated.

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

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Dong; Neoh, Koon Gee, E-mail: chenkg@nus.edu.sg; Kang, En-Tang

    2016-01-01

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

  2. Chitosan nanofiber scaffold improves bone healing via stimulating trabecular bone production due to upregulation of the Runx2/osteocalcin/alkaline phosphatase signaling pathway

    Science.gov (United States)

    Ho, Ming-Hua; Yao, Chih-Jung; Liao, Mei-Hsiu; Lin, Pei-I; Liu, Shing-Hwa; Chen, Ruei-Ming

    2015-01-01

    Osteoblasts play critical roles in bone formation. Our previous study showed that chitosan nanofibers can stimulate osteoblast proliferation and maturation. This translational study used an animal model of bone defects to evaluate the effects of chitosan nanofiber scaffolds on bone healing and the possible mechanisms. In this study, we produced uniform chitosan nanofibers with fiber diameters of approximately 200 nm. A bone defect was surgically created in the proximal femurs of male C57LB/6 mice, and then the left femur was implanted with chitosan nanofiber scaffolds for 21 days and compared with the right femur, which served as a control. Histological analyses revealed that implantation of chitosan nanofiber scaffolds did not lead to hepatotoxicity or nephrotoxicity. Instead, imaging analyses by X-ray transmission and microcomputed tomography showed that implantation of chitosan nanofiber scaffolds improved bone healing compared with the control group. In parallel, microcomputed tomography and bone histomorphometric assays further demonstrated augmentation of the production of new trabecular bone in the chitosan nanofiber-treated group. Furthermore, implantation of chitosan nanofiber scaffolds led to a significant increase in the trabecular bone thickness but a reduction in the trabecular parameter factor. As to the mechanisms, analysis by confocal microscopy showed that implantation of chitosan nanofiber scaffolds increased levels of Runt-related transcription factor 2 (Runx2), a key transcription factor that regulates osteogenesis, in the bone defect sites. Successively, amounts of alkaline phosphatase and osteocalcin, two typical biomarkers that can simulate bone maturation, were augmented following implantation of chitosan nanofiber scaffolds. Taken together, this translational study showed a beneficial effect of chitosan nanofiber scaffolds on bone healing through stimulating trabecular bone production due to upregulation of Runx2-mediated alkaline

  3. The effects of tissue-non-specific alkaline phosphatase gene therapy on craniosynostosis and craniofacial morphology in the FGFR2C342Y/+ mouse model of Crouzon craniosynostosis.

    Science.gov (United States)

    Wang, E; Nam, H K; Liu, J; Hatch, N E

    2015-04-01

    Craniosynostosis, the premature fusion of cranial bones, has traditionally been described as a disease of increased bone mineralization. However, multiple mouse models of craniosynostosis display craniosynostosis simultaneously with diminished cranial bone volume and/or density. We propose an alternative hypothesis that craniosynostosis results from abnormal tissue mineralization through the downregulation of tissue-non-specific alkaline phosphatase (TNAP) enzyme downstream of activating mutations in FGFRs. Neonatal Crouzon (FGFRC342Y/+) and wild-type (FGFR+/+) mice were injected with lentivirus to deliver a recombinant form of TNAP. Mice were sacrificed at 4 weeks postnatal. Serum was collected to test for alkaline phosphatase (AP), phosphorus, and calcium levels. Craniofacial bone fusion and morphology were assessed by micro-computed tomography. Injection with the TNAP lentivirus significantly increased serum AP levels (increased serum AP levels are indicative of efficient transduction and production of the recombinant protein), but results were variable and dependent upon viral lot and the litter of mice injected. Morphological analysis revealed craniofacial form differences for inferior surface (p=0.023) and cranial height (p=0.014) regions between TNAP lentivirus-injected and vehicle-injected Crouzon mice. With each unit increase in AP level, the odds of lambdoid suture fusion decreased by 84.2% and these results came close to statistical significance (p=0.068). These results suggest that TNAP deficiency may mediate FGFR2-associated craniosynostosis. Future studies should incorporate injection of recombinant TNAP protein, to avoid potential side effects and variable efficacy of lentiviral gene delivery. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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

    Science.gov (United States)

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

    2015-12-01

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

  5. Double epi-illumination microscopy with separate visualization of two antigens: a combination of epi-polarization for immunogold-silver staining and epi-fluorescence for alkaline phosphatase staining

    NARCIS (Netherlands)

    van der Loos, C. M.; Becker, A. E.

    1994-01-01

    We present a method for an epi-illumination immunohistochemical double staining approach. The method combines the use of an immuno-alkaline phosphatase technique and the immunogold-silver technique, visualized with epifluorescence and epi-polarization illumination, respectively. Out of six tested

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

    Directory of Open Access Journals (Sweden)

    Kala Mrinalini

    2005-12-01

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

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

    Science.gov (United States)

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

    2015-07-01

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

  8. Stimulation by parathyroid hormone of sup 45 Ca sup 2+ uptake in osteoblast-like cells: Possible involvement of alkaline phosphatase

    Energy Technology Data Exchange (ETDEWEB)

    Fukayama, S.; Tashjian, A.H. Jr. (Harvard School of Public Health, Boston, MA (USA))

    1990-04-01

    We have investigated the actions of human PTH (hPTH-(1-34)) on the association of 45Ca2+ with two human (SaOS-2 and MG-63) and two rat (ROS 17/2.8 and UMR-106) osteoblast-like cell types. In SaOS-2 cells, hPTH-(1-34) binds to specific membrane receptors to activate adenylate cyclase. Treatment of SaOS-2 cells with hPTH-(1-34) resulted in an increase in 45Ca2+ uptake, in a dose-dependent fashion, up to 2- to 4-fold above control values. The increase was first evident at 10 min and persisted for at least 30 min. Treatment with nimodipine, a calcium channel antagonist, was without effect on the stimulatory action of PTH. A similar enhancement of cell-associated 45Ca2+ was observed when the cells were incubated with vasoactive intestinal peptide, which acts via different receptors to activate adenylate cyclase in SaOS-2 cells. Treatment with (Bu)2cAMP also induced an increase in cell-associated 45Ca2+. Pretreatment of SaOS-2 cells with hPTH-(1-34) for 4 h, which induced homologous desensitization to a second challenge with the same peptide for stimulation of cAMP production, did not attenuate the further enhancement of cell-associated 45Ca2+ by a second treatment with hPTH-(1-34). We then examined a possible relationship between alkaline phosphatase (ALPase) and 45Ca2+ uptake. SaOS-2 cells contained high levels of alkaline phosphatase activity and continuously released the enzyme into the medium. Release was enhanced by treatment with hPTH-(1-34) for 10 min. Incubation of cells with levamisole (an inhibitor of the liver/bone/kidney type of ALPase) resulted in a rapid decrease in basal and PTH-stimulated 45Ca2+ uptake, while treatment with L-Phe-Gly-Gly was without effect. Treatment of the cells with ALPase (bovine kidney) enhanced 45Ca2+ uptake. In MG-63 cells, a stimulatory effect of hPTH-(1-34) on cell-associated 45Ca2+ was also observed; however, hPTH-(1-34) did not stimulate cAMP production in MG-63 cells.

  9. Circadian and longitudinal variation of serum C-telopeptide, osteocalcin, and skeletal alkaline phosphatase in C3H/HeJ mice.

    Science.gov (United States)

    Srivastava, A K; Bhattacharyya, S; Li, X; Mohan, S; Baylink, D J

    2001-10-01

    Inbred strains of mice are increasingly being used as an animal model to investigate skeletal disorders relevant to humans. In the bone field, one of the most convenient endpoints for evaluating genetic, physiological, or pharmaceutical perturbations is the use of biochemical markers. To apply biochemical markers in an effective manner, it is of key importance to establish the biological variation and appropriate sampling time. In this study, we evaluate two components: (i) circadian changes, and (ii) longitudinal variation for three serum markers, osteocalcin, C-telopeptide, and skeletal alkaline phosphatase (sALP), using 6-week-old C3H/HeJ (C3H) mice. To study circadian rhythms, the mice were randomly divided into eight groups of 15 mice each. Blood was collected at 3 h intervals, starting at 9:00 A.M. and continuing until 6:00 A.M. the next day. To determine whether circadian rhythm is intrinsically regulated or influenced by restricted food intake, it was also studied after a 12 h fasting period. Serum osteocalcin and C-telopeptide levels were measured by enzyme-linked immunoassay (ELISA) and skeletal alkaline phosphatase by a kinetic assay. The results demonstrated significant circadian variations in osteocalcin and C-telopeptide levels with a peak value between 0900 and 1200 h during daytime and a nadir between 15:00 and 18:00 h. The peak levels of C-telopeptide and osteocalcin were 26%-66% higher as compared with 24 h mean values. The pattern of the circadian variation of C-telopeptide and osteocalcin was similar in female and male animals and was not significantly affected by restricted food intake. The sALP levels were only marginally affected by the circadian rhythm. Longitudinal variations, expressed as coefficient of variation (CV), for osteocalcin, C-telopeptide, and sALP concentrations were 17%, 14%, and 16%, respectively. In addition, the longitudinal variations were not significantly influenced by the time of blood collection in sALP and osteocalcin

  10. pH-Dependent Binding of Chloride to a Marine Alkaline Phosphatase Affects the Catalysis, Active Site Stability, and Dimer Equilibrium.

    Science.gov (United States)

    Hjörleifsson, Jens G; Ásgeirsson, Bjarni

    2017-09-26

    The effect of ionic strength on enzyme activity and stability varies considerably between enzymes. Ionic strength is known to affect the catalytic activity of some alkaline phosphatases (APs), such as Escherichia coli AP, but how ions affect APs is debated. Here, we studied the effect of various ions on a cold-adapted AP from Vibrio splendidus (VAP). Previously, we have found that the active form of VAP is extremely unstable at low ionic strengths. Here we show that NaCl increased the activity and stability of VAP and that the effect was pH-dependent in the range of pH 7-10. The activity profile as a function of pH formed two maxima, indicating a possible conformational change. Bringing the pH from the neutral to the alkaline range was accompanied by a large increase in both the K i for inorganic phosphate (product inhibition) and the K M for p-nitrophenyl phosphate. The activity transitions observed as the pH was varied correlated with structural changes as monitored by tryptophan fluorescence. Thermal and urea-induced inactivation was shown to be accompanied by neither dissociation of the active site metal ions nor dimer dissociation. This would suggest that the inactivation involved subtle changes in active site conformation. Furthermore, the VAP dimer equilibrium was studied for the first time and shown to highly favor dimerization, which was dependent on pH and NaCl concentration. Taken together, the data support a model in which anions bind to some specific acceptor in the active site of VAP, resulting in great stabilization and catalytic rate enhancement, presumably through a different mechanism.

  11. Prokaryotic responses to ammonium and organic carbon reveal alternative CO2 fixation pathways and importance of alkaline phosphatase in the mesopelagic North Atlantic

    Directory of Open Access Journals (Sweden)

    Federico Baltar

    2016-10-01

    Full Text Available To decipher the response of mesopelagic prokaryotic communities to input of nutrients, we tracked changes in prokaryotic abundance, extracellular enzymatic activities, heterotrophic production, dark dissolved inorganic carbon (DIC fixation, community composition (16S rRNA sequencing and community gene expression (metatranscriptomics in 3 microcosm experiments with water from the mesopelagic North Atlantic. Responses in 3 different treatments amended with thiosulfate, ammonium or organic matter (i.e. pyruvate plus acetate were compared to unamended controls. The strongest stimulation was found in the organic matter enrichments, where all measured rates increased >10-fold. Strikingly, in the organic matter treatment, the dark DIC fixation rates —assumed to be related to autotrophic metabolisms— were equally stimulated as all the other heterotrophic-related parameters. This increase in DIC fixation rates was paralleled by an up-regulation of genes involved in DIC assimilation via anaplerotic pathways. Alkaline phosphatase was the metabolic rate most strongly stimulated and its activity seemed to be related to cross-activation by nonpartner histidine kinases, and/or the activation of genes involved in the regulation of elemental balance during catabolic processes. These findings suggest that episodic events such as strong sedimentation of organic matter into the mesopelagic might trigger rapid increases of originally rare members of the prokaryotic community, enhancing heterotrophic and autotrophic carbon uptake rates, ultimately affecting carbon cycling. Our experiments highlight a number of fairly unstudied microbial processes of potential importance in mesopelagic waters that require future attention.

  12. An elevated serum alkaline phosphatase level in hepatic metastases of grade 1 and 2 gastrointestinal neuroendocrine tumors is unusual and of prognostic value.

    Directory of Open Access Journals (Sweden)

    Maeva Andriantsoa

    Full Text Available In our clinical practice we have observed that despite a high hepatic metastatic tumor burden, serum alkaline phosphatase (AP levels are frequently normal in cases of metastatic neuroendocrine tumor (NET.We retrospectively reviewed the records of patients with grade 1 and 2 NETs with liver metastases but without bone metastases seen at our institution in 2013. In total, 49 patients were included (22 female, with a median age of 60 years (range: 28 to 84 years. The primary tumors were located in the duodenum/pancreas (n = 29, small bowel (n = 17 or colon/rectum (n = 3; 10 cases were grade 1 and 39 grade 2. Hepatic involvement was bulky, with more than 10 lesions in 23 patients and a tumor burden above 10% of the liver volume in 26 patients.Serum AP levels were elevated (≥ upper limit of normal (ULN in 16 patients. In multiparametric analysis, elevated serum AP levels were not associated with the primary site, grade, or number or volume of metastases. In multiparametric analysis, progression-free survival was only correlated with grade (p = 0.010 and AP level (p = 0.017.Serum AP levels are frequently normal in liver metastases from NET, even in the event of a major tumor burden, and the serum AP level can be of prognostic value.

  13. An elevated serum alkaline phosphatase level in hepatic metastases of grade 1 and 2 gastrointestinal neuroendocrine tumors is unusual and of prognostic value.

    Science.gov (United States)

    Andriantsoa, Maeva; Hoibian, Solene; Autret, Aurelie; Gilabert, Marine; Sarran, Anthony; Niccoli, Patricia; Raoul, Jean-Luc

    2017-01-01

    In our clinical practice we have observed that despite a high hepatic metastatic tumor burden, serum alkaline phosphatase (AP) levels are frequently normal in cases of metastatic neuroendocrine tumor (NET). We retrospectively reviewed the records of patients with grade 1 and 2 NETs with liver metastases but without bone metastases seen at our institution in 2013. In total, 49 patients were included (22 female), with a median age of 60 years (range: 28 to 84 years). The primary tumors were located in the duodenum/pancreas (n = 29), small bowel (n = 17) or colon/rectum (n = 3); 10 cases were grade 1 and 39 grade 2. Hepatic involvement was bulky, with more than 10 lesions in 23 patients and a tumor burden above 10% of the liver volume in 26 patients. Serum AP levels were elevated (≥ upper limit of normal (ULN)) in 16 patients. In multiparametric analysis, elevated serum AP levels were not associated with the primary site, grade, or number or volume of metastases. In multiparametric analysis, progression-free survival was only correlated with grade (p = 0.010) and AP level (p = 0.017). Serum AP levels are frequently normal in liver metastases from NET, even in the event of a major tumor burden, and the serum AP level can be of prognostic value.

  14. Midgut GPI-anchored proteins with alkaline phosphatase activity from the cotton boll weevil (Anthonomus grandis) are putative receptors for the Cry1B protein of Bacillus thuringiensis.

    Science.gov (United States)

    Martins, Erica Soares; Monnerat, Rose Gomes; Queiroz, Paulo Roberto; Dumas, Vinicius Fiuza; Braz, Shélida Vasconcelos; de Souza Aguiar, Raimundo Wagner; Gomes, Ana Cristina Menezes Mendes; Sánchez, Jorge; Bravo, Alejandra; Ribeiro, Bergmann Morais

    2010-02-01

    Cry toxins from Bacillus thuringiensis (Bt) are used for insect control. They interact with specific receptors located on the host cell surface and are activated by host proteases following receptor binding resulting in midgut epithelial cells lysis. In this work we had cloned, sequenced and expressed a cry1Ba toxin gene from the B thuringiensis S601 strain which was previously shown to be toxic to Anthonomus grandis, a cotton pest. The Cry1Ba6 protein expressed in an acrystaliferous B. thuringiensis strain was toxic to A. grandis in bioassays. The binding of Cry1Ba6 toxin to proteins located in the midgut brush border membrane of A. grandis was analyzed and we found that Cry1Ba6 binds to two proteins (62 and 65kDa) that showed alkaline phosphatase (ALP) activity. This work is the first report that shows the localization of Cry toxin receptors in the midgut cells of A. grandis. 2009. Published by Elsevier Ltd.

  15. Fluorescence turn-on detection of alkaline phosphatase activity based on controlled release of PEI-capped Cu nanoclusters from MnO2 nanosheets.

    Science.gov (United States)

    Zhang, Yunyi; Li, Yongxin; Zhang, Cuiyun; Zhang, Qingfeng; Huang, Xinan; Yang, Meiding; Shahzad, Sohail Anjum; Lo, Kenneth Kam-Wing; Yu, Cong; Jiang, Shichun

    2017-08-01

    A fluorescence turn-on assay for alkaline phosphatase (ALP) activity is developed through the controlled release of polyethyleneimine-capped copper nanoclusters (PEI-capped CuNCs) from the MnO 2 nanosheets. In an aqueous solution, the positively charged PEI-capped CuNCs could be adsorbed onto the surface of the negatively charged MnO 2 nanosheets. Such adsorption through favorable electrostatic interactions could efficiently quench the nanocluster fluorescence emission via resonance energy transfer from the PEI-capped CuNCs to the MnO 2 nanosheets. 2-Phospho-L-ascorbic acid (AAP) could be hydrolyzed to L-ascorbic acid (AA) in the presence of ALP. AA could reduce MnO 2 into Mn 2+ and trigger the disintegration of the MnO 2 nanosheets. As a result, the CuNCs were released and the quenched fluorescence was recovered efficiently. The detection strategy is simple, inexpensive, sensitive, selective, with low toxicity, and has better biocompatibility. The newly fabricated biosensor for ALP activity will potentially make it a robust candidate for numerous biological and biomedical applications.

  16. Synthesis of sulfadiazinyl acyl/aryl thiourea derivatives as calf intestinal alkaline phosphatase inhibitors, pharmacokinetic properties, lead optimization, Lineweaver-Burk plot evaluation and binding analysis.

    Science.gov (United States)

    Sajid-Ur-Rehman; Saeed, Aamer; Saddique, Gufran; Ali Channar, Pervaiz; Ali Larik, Fayaz; Abbas, Qamar; Hassan, Mubashir; Raza, Hussain; Fattah, Tanzeela Abdul; Seo, Sung-Yum

    2018-06-02

    To seek the new medicinal potential of sulfadiazine drug, the free amino group of sulfadiazine was exploited to obtain acyl/aryl thioureas using simple and straightforward protocol. Acyl/aryl thioureas are well recognized bioactive pharmacophore containing moieties. A new series (4a-4j) of sulfadiazine derived acyl/aryl thioureas was synthesized and characterized through spectroscopic and elemental analysis. The synthesized derivatives 4a-4j were subjected to calf intestinal alkaline phosphatase (CIAP) activity. The derivative 4a-4j showed better inhibition potential compared to standard monopotassium phosphate (MKP). The compound 4c exhibited higher potential in the series with IC 50 0.251 ± 0.012 µM (standard KH 2 PO 4 4.317 ± 0.201 µM). Lineweaver-Burk plots revealed that most potent derivative 4c inhibition CIAP via mixed type pathway. Pharmacological investigations showed that synthesized compounds 4a-4j obey Lipinsk's rule. ADMET parameters evaluation predicted that these molecule show significant lead like properties with minimum possible toxicity and can serve as templates in drug designing. The synthetic compounds show none mutagenic and irritant behavior. Molecular docking analysis showed that compound 4c interacts with Asp273, His317 and Arg166 amino acid residues. Copyright © 2018. Published by Elsevier Ltd.

  17. Oxidative Stress as Estimated by Gamma-Glutamyl Transferase Levels Amplifies the Alkaline Phosphatase-Dependent Risk for Mortality in ESKD Patients on Dialysis

    Directory of Open Access Journals (Sweden)

    Claudia Torino

    2016-01-01

    Full Text Available Alkaline phosphatase (Alk-Phos is a powerful predictor of death in patients with end-stage kidney disease (ESKD and oxidative stress is a strong inducer of Alk-Phos in various tissues. We tested the hypothesis that oxidative stress, as estimated by a robust marker of systemic oxidative stress like γ-Glutamyl-Transpeptidase (GGT levels, may interact with Alk-Phos in the high risk of death in a cohort of 993 ESKD patients maintained on chronic dialysis. In fully adjusted analyses the HR for mortality associated with Alk-Phos (50 IU/L increase was progressively higher across GGT quintiles, being minimal in patients in the first quintile (HR: 0.89, 95% CI: 0.77–1.03 and highest in the GGT fifth quintile (HR: 1.13, 95% CI: 1.03–1.2 (P for the effect modification = 0.02. These findings were fully confirmed in sensitivity analyses excluding patients with preexisting liver disease, excessive alcohol intake, or altered liver disease biomarkers. GGT amplifies the risk of death associated with high Alk-Phos levels in ESKD patients. This observation is compatible with the hypothesis that oxidative stress is a strong modifier of the adverse biological effects of high Alk-Phos in this population.

  18. Exploitation of phosphorescent labelling reagent of fullerol-fluorescein isothiocyanate and new method for the determination of trace alkaline phosphatase as well as forecast of human diseases

    International Nuclear Information System (INIS)

    Liu Jiaming; Huang Xiaomei; Liu Zhenbo; Lin Shaoqin; Li Feiming; Gao Fei; Li Zhiming; Zeng Liqing; Li Lianying; Ouyang Ying

    2009-01-01

    A new phosphorescent labelling reagent consisting of fullerol, fluorescein isothiocyanate and N,N-dimethylaniline (F-ol-(FITC) n -DMA) was developed. The mode of action is based on the reactivity of the active -OH group in F-ol with the -COOH group of FITC to form an F-ol-(FITC) n -DMA complex containing several FITC molecules. F-ol-(FITC) n -DMA increased the number of luminescent molecules in the biological target of WGA-AP-WGA-F-ol-(FITC) n -DMA (WGA and AP are wheat germ agglutinin and alkaline phosphatase, respectively) which improved the sensitivity using solid substrate room temperature phosphorimetry (SSRTP) detection. The proposed method provided high sensitivity and strong specificity for WGA-AP. The limit of detection (LD) was 0.15 ag AP spot -1 for F-ol and 0.097 ag AP spot -1 for FITC in F-ol-(FITC) n -DMA, which was lower than the method using single luminescent molecules of F-ol-DMA and FITC-DMA to label WGA (0.20 ag AP spot -1 for F-ol-DMA and 0.22 ag AP spot -1 for FITC-DMA). Results for the determination of AP in human serum were in good agreement with those obtained by enzyme-linked immunosorbent assay. The mechanism of F-ol-(FITC) n -DMA labelling of WGA was discussed.

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

    Science.gov (United States)

    Guha, Anirban; Gera, Sandeep; Sharma, Anshu

    2012-03-01

    Mastitis is a highly morbid disease that requires detection at the subclinical stage. Tropical countries like India mainly depend on milch buffaloes for milk. The present study was conducted to investigate whether the trace minerals viz. copper (Cu), iron (Fe), zinc (Zn), cobalt (Co) and manganese (Mn) and enzyme activity of lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in riverine buffalo milk can be used as an indicator of subclinical mastitis (SCM) with the aim of developing suitable diagnostic kit for SCM. Trace elements and enzyme activity in milk were estimated with Atomic absorption Spectrophotometer, GBC 932 plus and biochemical methods, respectively. Somatic cell count (SCC) was done microscopically. The cultural examination revealed Gram positive bacteria as the most prevalent etiological agent. A statistically significant (pnegative bacteria. The percent sensitivity, specificity and accuracy, predictive values and likelihood ratios were calculated taking bacterial culture examination and SCC≥2×10(5) 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.

  20. Detection of alkaline phosphatase in canine cells previously stained with Wright-Giemsa and its utility in differentiating osteosarcoma from other mesenchymal tumors.

    Science.gov (United States)

    Ryseff, Julia K; Bohn, Andrea A

    2012-09-01

    Osteosarcoma (OSA) is a common primary bone tumor in dogs. Demonstration of alkaline phosphatase (ALP) reactivity by tumor cells on unstained slides is useful in differentiating osteosarcoma from other types of sarcoma. However, unstained slides are not always available. The objectives of this study were to evaluate the diagnostic utility of detecting ALP expression in differentiating osteosarcoma from other sarcomas in dogs using cytologic material previously stained with Wright-Giemsa stain and to assess the sensitivity and specificity of ALP expression for diagnosing osteosarcoma using a specific protocol. Archived aspirates of histologically confirmed sarcomas in dogs that had been previously stained with Wright-Giemsa stain were treated with 5-bromo, 4-chloro, 3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) as a substrate for ALP. Cells were evaluated for expression of ALP after incubation with BCIP/NBT for 1 hour. Sensitivity and specificity of ALP expression for diagnosis of OSA were calculated. In samples from 83 dogs, cells from 15/17 OSAs and from 4/66 tumors other than OSA (amelanotic melanoma, gastrointestinal stromal tumor, collision tumor, and anaplastic sarcoma) expressed ALP. Sensitivity and specificity of ALP expression detected using BCIP/NBT substrate applied to cells previously stained with Wright-Giemsa stain for OSA were 88 and 94%, respectively. ALP expression detected using BCIP/NBT substrate applied to previously stained cells is useful in differentiating canine OSA from other mesenchymal neoplasms. © 2012 American Society for Veterinary Clinical Pathology.

  1. Induction of rat alkaline phosphatase isozymes bearing a glycan-phosphatidylinositol anchor modified by in vivo treatment with a benzimidazole derivative linked to ethylbenzene.

    Science.gov (United States)

    Harada, T; Koyama, I; Sato, K; Komoda, T

    2000-10-01

    Serum alkaline phosphatase (ALP) is detected in soluble-form as a result of translocation from the membrane site by cleavage at the glycosyl-phosphatidylinositol moiety (GPI anchor). It is known that membrane-bound ALP (mALP) can be detected in serum in certain pathological and physiological conditions, and that it can be solubilized in vitro to soluble-ALP (sALP) by phosphatidylinositol-specific phospholipase C (PIPLC), phospholipase D, bile salt, detergent, etc. We observed a marked increase in ALP activity in the serum of rats given a benzimidazole derivative by gavage, and detected it as slow-migrating ALPs (SM-ALPs), which were mALP-like but resistant to PIPLC and n-butanol treatment on disc PAGE. On the other hand, ficin treatment made SM-ALPs shift to the sALP position. The molecular size of the SM-ALPs was smaller than that of sALP on sodium dodecyl sulphide-polyacrylamide slab-gel electrophoresis (SDS-PAGE), and immunoreactivity revealed the intestinal type. SM-ALPs were also detected in the duodenum and jejunum. The main sugar chain structure of SM-ALPs was the biantennary complex-type, which was coincided with intestinal sALP sugar chain. These results suggest that intestinal ALPs induced by the benzimidazole derivative were modified in their C-terminus or GPI anchor region and modification of this region may also participate in translocation into the bloodstream.

  2. Mediator 1 contributes to enamel mineralization as a coactivator for Notch1 signaling and stimulates transcription of the alkaline phosphatase gene.

    Science.gov (United States)

    Yoshizaki, Keigo; Hu, Lizhi; Nguyen, Thai; Sakai, Kiyoshi; Ishikawa, Masaki; Takahashi, Ichiro; Fukumoto, Satoshi; DenBesten, Pamela K; Bikle, Daniel D; Oda, Yuko; Yamada, Yoshihiko

    2017-08-18

    Tooth enamel is mineralized through the differentiation of multiple dental epithelia including ameloblasts and the stratum intermedium (SI), and this differentiation is controlled by several signaling pathways. Previously, we demonstrated that the transcriptional coactivator Mediator 1 (MED1) plays a critical role in enamel formation. For instance, conditional ablation of Med1 in dental epithelia causes functional changes in incisor-specific dental epithelial stem cells, resulting in mineralization defects in the adult incisors. However, the molecular mechanism by which Med1 deficiency causes these abnormalities is not clear. Here, we demonstrated that Med1 ablation causes early SI differentiation defects resulting in enamel hypoplasia of the Med1 -deficient molars. Med1 deletion prevented Notch1-mediated differentiation of the SI cells resulting in decreased alkaline phosphatase (ALPL), which is essential for mineralization. However, it does not affect the ability of ameloblasts to produce enamel matrix proteins. Using the dental epithelial SF2 cell line, we demonstrated that MED1 directly activates transcription of the Alpl gene through the stimulation of Notch1 signaling by forming a complex with cleaved Notch1-RBP-Jk on the Alpl promoter. These results suggest that MED1 may be essential for enamel matrix mineralization by serving as a coactivator for Notch1 signaling regulating transcription of the Alpl gene.

  3. The antioxidant effects of vitamin C on liver enzymes: aspartate aminotransferase, alanine aminotranferease, alkaline phosphatase and gamma-glutamyltransferase activities in rats under Paraquat insult

    Directory of Open Access Journals (Sweden)

    Benjamin Nnamdi Okolonkwo

    2013-06-01

    Full Text Available Paraquat (PQ is a bipyridylium herbicide; applied around trees in orchards and between crop rows to control broad-leaved and grassy weeds. Its oxidation results in the formation of superoxides which causes damage to cellular components. In this study, we determined the antioxidant effect vitamin C has on the liver enzymes [aspartate aminotransferase (SGOT, alanine aminotranferease (SGPT, alkaline phosphatase (ALP, and gamma-glutamyltransferase (GGT] of rats under this toxic insult. Male rats in groups (A, B, C and D were intraperitoneally injected with different sublethal increasing doses (0, 0.02, 0.04 and 0.06 g/kg body weigh of PQ respectively on monthly basis. Subsequently, the subgroups (A2, B2, C2 and D2 were given orally, 200 mg/L vitamin C, while the subgroups A1, B1, C1, and D1, received only water. Four animals per subgroup were decapitated on monthly basis and blood samples taken for enzyme assay. The parameters studied were - SGOT, SGPT, ALP and GGT - liver enzymes. The dose and time dependent PQ toxicity effect resulted in highly elevated Liver enzymes activities. The subgroups on vitamin C had significantly lower enzyme activities when compared to the same subgroups on only PQ insult. But the values were high when compared to the control subgroups (A1 and A2. These results were indication that vitamin C when given at moderate doses and maintained for a longer period could be a life saving adjunct to toxic insult.

  4. Antibody guided diagnosis and therapy of brain gliomas using radiolabeled monoclonal antibodies against epidermal growth factor receptor and placental alkaline phosphatase

    International Nuclear Information System (INIS)

    Kalofonos, H.P.; Pawlikowska, T.R.; Hemingway, A.

    1989-01-01

    Twenty-seven patients with brain glioma were scanned using 123 I-labeled monoclonal antibodies against epidermal growth factor receptor (EGFR1) or placental alkaline phosphatase (H17E2). Successful localization was achieved in 18 out of 27 patients. Eleven out of 27 patients were also studied using a nonspecific control antibody (11.4.1) of the same immunoglobulin subclass and observable tumor localization was also achieved in five patients. The specificity of targeting was assessed by comparing images obtained with specific and nonspecific antibodies and by examining tumor and normal tissue biopsies after dual antibody administration. Ten patients with recurrent grade III or IV glioma who showed good localization of radiolabeled antibody were treated with 40-140 mCi of 131 I-labeled antibody delivered to the tumor area intravenously (n = 5) or by infusion into the internal carotid artery (n = 5). Six patients showed clinical improvement lasting from 6 mo to 3 yr. One patient continues in remission (3 yr after therapy), but the other five who responded initially relapsed 6-9 mo after therapy and died. No major toxicity was attributable to antibody-guided irradiation. Targeted irradiation by monoclonal antibody may be clinically useful and should be explored further in the treatment of brain gliomas resistant to conventional forms of treatment

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

    Directory of Open Access Journals (Sweden)

    Keyhani doost Z

    2011-01-01

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

  6. Growth, extracellular alkaline phosphatase activity, and kinetic characteristic responses of the bloom-forming toxic cyanobacterium, Microcystis aeruginosa, to atmospheric particulate matter (PM2.5, PM2.5-10, and PM>10).

    Science.gov (United States)

    Xu, Ziran; Wang, Shoubing; Wang, Yuanan; Zhang, Jie

    2018-03-01

    Atmospheric particulate matter (APM), commonly seen and widely excited in environment, appears great enough to influence the biochemical processes in aquatic microorganisms and phytoplankton. Understanding the response of cyanobacteria to various factors is fundamental for eutrophication control. To clarify the response of cyanobacteria to APM, the effects of PM 2.5 , PM 2.5-10 , and PM >10 on Microcystis aeruginosa were researched. Variabilities in cell density, chlorophyll a, soluble protein, malondialdehyde, extracellular activity, and kinetic parameters of alkaline phosphatase were evaluated by lab-cultured experiments. Results showed that the PM 2.5 had a slight stimulation impact on the growth and enhanced both of the 48- and 72-h extracellular alkaline phosphatase activity (APA), the affinity of alkaline phosphatase for substrate, and the 72-h maximum enzymatic reaction velocity (V max ). Moreover, the stimulations in extracellular APA and V max enhanced with the increasing exposure concentrations. We also found there were no obvious distinctions on the effects of growth and alkaline phosphatase in M. aeruginosa between PM 2.5-10 and PM >10 exposure groups. Obviously, inhibitory effects on growth existed in 4.0 and 8.0 mg/L PM 2.5-10 and 8.0 mg/L PM >10 at 120 h. Furthermore, PM 2.5-10 and PM >10 exerted inhibitory effects on the extracellular APA during the 72-h exposure. Simultaneously, the V max was notably inhibited and the affinity of alkaline phosphatase for substrate was more inseparable compared with control in PM 2.5-10 and PM >10 treatments. Nevertheless, the inhibitors in extracellular APA and kinetic parameters were unrelated to PM 2.5-10 and PM >10 exposure concentrations. Two-way ANOVA results revealed that there were significant interactions between exposure concentration and diameter of APM on the 120-h cell density, soluble protein content, APA, and 72 h APA of M. aeruginosa. These results in our study would be meaningful to further

  7. Lineage analysis of the late otocyst stage mouse inner ear by transuterine microinjection of a retroviral vector encoding alkaline phosphatase and an oligonucleotide library.

    Directory of Open Access Journals (Sweden)

    Han Jiang

    Full Text Available The mammalian inner ear subserves the special senses of hearing and balance. The auditory and vestibular sensory epithelia consist of mechanically sensitive hair cells and associated supporting cells. Hearing loss and balance dysfunction are most frequently caused by compromise of hair cells and/or their innervating neurons. The development of gene- and cell-based therapeutics will benefit from a thorough understanding of the molecular basis of patterning and cell fate specification in the mammalian inner ear. This includes analyses of cell lineages and cell dispersals across anatomical boundaries (such as sensory versus nonsensory territories. The goal of this study was to conduct retroviral lineage analysis of the embryonic day 11.5(E11.5 mouse otic vesicle. A replication-defective retrovirus encoding human placental alkaline phosphatase (PLAP and a variable 24-bp oligonucleotide tag was microinjected into the E11.5 mouse otocyst. PLAP-positive cells were microdissected from cryostat sections of the postnatal inner ear and subjected to nested PCR. PLAP-positive cells sharing the same sequence tag were assumed to have arisen from a common progenitor and are clonally related. Thirty five multicellular clones consisting of an average of 3.4 cells per clone were identified in the auditory and vestibular sensory epithelia, ganglia, spiral limbus, and stria vascularis. Vestibular hair cells in the posterior crista were related to one another, their supporting cells, and nonsensory epithelial cells lining the ampulla. In the organ of Corti, outer hair cells were related to a supporting cell type and were tightly clustered. By contrast, spiral ganglion neurons, interdental cells, and Claudius' cells were related to cells of the same type and could be dispersed over hundreds of microns. These data contribute new information about the developmental potential of mammalian otic precursors in vivo.

  8. Comparison of Salivary pH, Buffering Capacity and Alkaline Phosphatase in Smokers and Healthy Non-Smokers: Retrospective cohort study.

    Science.gov (United States)

    Ahmadi-Motamayel, Fatemeh; Falsafi, Parisa; Goodarzi, Mohammad T; Poorolajal, Jalal

    2016-08-01

    Saliva contains alkaline phosphatase (ALP)-a key intracellular enzyme related to destructive processes and cellular damage-and has buffering capacity (BC) against acids due to the presence of bicarbonate and phosphate ions. Smoking may have deleterious effects on the oral environment due to pH changes which can affect ALP activity. This study aimed to evaluate the salivary pH, BC and ALP activity of male smokers and healthy non-smokers. This retrospective cohort study took place between August 2012 and December 2013. A total of 251 healthy male non-smokers and 259 male smokers from Hamadan, Iran, were selected. Unstimulated whole saliva was collected from each participant and pH and BC were determined using a pH meter. Salivary enzymes were measured by spectrophotometric assay. Mean salivary pH (7.42 ± 0.48 and 7.52 ± 0.43, respectively; P = 0.018) and BC (3.41 ± 0.54 and 4.17 ± 0.71; P = 0.001) was significantly lower in smokers compared to non-smokers. Mean ALP levels were 49.58 ± 23.33 IU/L among smokers and 55.11 ± 27.85 IU/L among non-smokers (P = 0.015). Significantly lower pH, BC and ALP levels were observed among smokers in comparison to a healthy control group. These salivary alterations could potentially be utilised as biochemical markers for the evaluation of oral tissue function and side-effects among smokers. Further longitudinal studies are recommended to evaluate the effects of smoking on salivary components.

  9. Comparison of Salivary pH, Buffering Capacity and Alkaline Phosphatase in Smokers and Healthy Non-Smokers; Retrospective cohort study

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    Fatemeh Ahmadi-Motamayel

    2016-08-01

    Full Text Available Objectives: Saliva contains alkaline phosphatase (ALP—a key intracellular enzyme related to destructive processes and cellular damage—and has buffering capacity (BC against acids due to the presence of bicarbonate and phosphate ions. Smoking may have deleterious effects on the oral environment due to pH changes which can affect ALP activity. This study aimed to evaluate the salivary pH, BC and ALP activity of male smokers and healthy non-smokers. Methods: This retrospective cohort study took place between August 2012 and December 2013. A total of 251 healthy male non-smokers and 259 male smokers from Hamadan, Iran, were selected. Unstimulated whole saliva was collected from each participant and pH and BC were determined using a pH meter. Salivary enzymes were measured by spectrophotometric assay. Results: Mean salivary pH (7.42 ± 0.48 and 7.52 ± 0.43, respectively; P = 0.018 and BC (3.41 ± 0.54 and 4.17 ± 0.71; P = 0.001 was significantly lower in smokers compared to non-smokers. Mean ALP levels were 49.58 ± 23.33 IU/L among smokers and 55.11 ± 27.85 IU/L among non-smokers (P = 0.015. Conclusion: Significantly lower pH, BC and ALP levels were observed among smokers in comparison to a healthy control group. These salivary alterations could potentially be utilised as biochemical markers for the evaluation of oral tissue function and side-effects among smokers. Further longitudinal studies are recommended to evaluate the effects of smoking on salivary components.

  10. Influence of culture medium supplementation of tobacco NT1 cell suspension cultures on the N-glycosylation of human secreted alkaline phosphatase.

    Science.gov (United States)

    Becerra-Arteaga, Alejandro; Shuler, Michael L

    2007-08-15

    We report for the first time that culture conditions, specifically culture medium supplementation with nucleotide-sugar precursors, can alter significantly the N-linked glycosylation of a recombinant protein in plant cell culture. Human secreted alkaline phosphatase produced in tobacco NT1 cell suspension cultures was used as a model system. Plant cell cultures were supplemented with ammonia (30 mM), galactose (1 mM) and glucosamine (10 mM) to improve the extent of N-linked glycosylation. The highest levels of cell density and active extracellular SEAP in supplemented cultures were on average 260 g/L and 0.21 U/mL, respectively, compared to 340 g/L and 0.4 U/mL in unsupplemented cultures. The glycosylation profile of SEAP produced in supplemented cultures was determined via electrospray ionization mass spectrometry with precursor ion scanning and compared to that of SEAP produced in unsupplemented cultures. In supplemented and unsupplemented cultures, two biantennary complex-type structures terminated with one or two N-acetylglucosamines and one paucimannosidic glycan structure comprised about 85% of the SEAP glycan pool. These three structures contained plant-specific xylose and fucose residues and their relative abundances were affected by each supplement. High mannose structures (6-9 mannose residues) accounted for the remaining 15% glycans in all cases. The highest proportion (approximately 66%) of a single complex-type biantennary glycan structure terminated in both antennae by N- acetylglucosamine was obtained with glucosamine supplementation versus only 6% in unsupplemented medium. This structure is amenable for in vitro modification to yield a more human-like glycan and could serve as a route to plant cell culture produced therapeutic glycoproteins. (c) 2007 Wiley Periodicals, Inc.

  11. Anti-idiotypic nanobody-alkaline phosphatase fusion proteins: Development of a one-step competitive enzyme immunoassay for fumonisin B{sub 1} detection in cereal

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    Shu, Mei [State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047 (China); Jiangxi-OAI Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047 (China); Xu, Yang, E-mail: xuyang@ncu.edu.cn [State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047 (China); Jiangxi-OAI Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047 (China); Liu, Xing [State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047 (China); College of Food Science and Technology, Hainan University, No. 58 Renmin Avenue, Haikou 570228 (China); Li, Yanping; He, Qinghua [Jiangxi-OAI Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047 (China); Tu, Zhui [State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047 (China); Fu, Jinheng [Jiangxi-OAI Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047 (China); Gee, Shirley J.; Hammock, Bruce D. [Department of Entomology and UCD Comprehensive Cancer Center, University of California, Davis, CA 95616 (United States)

    2016-06-14

    A rapid and sensitive one-step competitive enzyme immunoassay for the detection of FB{sub 1} was developed. The anti-idiotypic nanobody–alkaline phosphatase (Ab2β−Nb−AP) was validated by the AP enzyme activity and the properties of bounding to anti-FB1-mAb (3F11) through colorimetric and chemiluminescence analyses. The 50% inhibitory concentration and the detection limit (LOD) of colorimetric enzyme-linked immunosorbent assay (ELISA) for FB{sub 1} were 2.69 and 0.35 ng mL{sup −1}, respectively, with a linear range of 0.93–7.73 ng mL{sup −1}. The LOD of the chemiluminescence ELISA (CLIA) was 0.12 ng mL{sup −1}, and the IC{sub 50} was 0.89 ± 0.09 ng mL{sup −1} with a linear range of 0.29–2.68 ng mL{sup −1}. Compared with LC-MS/MS, the results of this assay indicated the reliability of the Ab2β−Nb−AP fusion protein based one-step competitive immunoassay for monitoring FB{sub 1} contamination in cereals. The Ab2β−Nb−AP fusion proteins have the potential to replace chemically-coupled probes in competitive enzyme immunoassay systems. - Highlights: • Ab2β−Nb−AP has the potential to replace chemically-coupled probes. • Ab2β−Nb−AP is homogeneous enzyme-labelled antigen can be prepared reproducibly. • We developed a green and rapid one-step competitive enzyme immunoassay. • The sensitivity of one-step CLIA was 9-folds higher than two-step ELISA.

  12. Anti-idiotypic nanobody-alkaline phosphatase fusion proteins: Development of a one-step competitive enzyme immunoassay for fumonisin B_1 detection in cereal

    International Nuclear Information System (INIS)

    Shu, Mei; Xu, Yang; Liu, Xing; Li, Yanping; He, Qinghua; Tu, Zhui; Fu, Jinheng; Gee, Shirley J.; Hammock, Bruce D.

    2016-01-01

    A rapid and sensitive one-step competitive enzyme immunoassay for the detection of FB_1 was developed. The anti-idiotypic nanobody–alkaline phosphatase (Ab2β−Nb−AP) was validated by the AP enzyme activity and the properties of bounding to anti-FB1-mAb (3F11) through colorimetric and chemiluminescence analyses. The 50% inhibitory concentration and the detection limit (LOD) of colorimetric enzyme-linked immunosorbent assay (ELISA) for FB_1 were 2.69 and 0.35 ng mL"−"1, respectively, with a linear range of 0.93–7.73 ng mL"−"1. The LOD of the chemiluminescence ELISA (CLIA) was 0.12 ng mL"−"1, and the IC_5_0 was 0.89 ± 0.09 ng mL"−"1 with a linear range of 0.29–2.68 ng mL"−"1. Compared with LC-MS/MS, the results of this assay indicated the reliability of the Ab2β−Nb−AP fusion protein based one-step competitive immunoassay for monitoring FB_1 contamination in cereals. The Ab2β−Nb−AP fusion proteins have the potential to replace chemically-coupled probes in competitive enzyme immunoassay systems. - Highlights: • Ab2β−Nb−AP has the potential to replace chemically-coupled probes. • Ab2β−Nb−AP is homogeneous enzyme-labelled antigen can be prepared reproducibly. • We developed a green and rapid one-step competitive enzyme immunoassay. • The sensitivity of one-step CLIA was 9-folds higher than two-step ELISA.

  13. Partial characterization and response under hyperregulating conditions of Na+-K+ ATPase and levamisole-sensitive alkaline phosphatase activities in chela muscle of the euryhaline crab Cyrtograpsus angulatus

    Directory of Open Access Journals (Sweden)

    Silvina Andrea Pinoni

    2008-03-01

    Full Text Available The occurrence, characteristics and response to changes in environmental salinity of Na+-K+ ATPase and levamisole-sensitive alkaline phosphatase (AP activities were studied in chela muscle of the euryhaline crab Cyrtograpsus angulatus. Chela muscle exhibited an Na+-K+ ATPase activity which was strongly dependent on ATP concentration, pH and temperature of the reaction mixture. Maximal activity was found at 1 mM ATP, 30-37°C and pH 7.4. Levamisole-sensitive AP activity was characterised at physiological pH 7.4 and at pH 8.0. I50 for levamisole-sensitive AP activity was 8.8 mM and 8.0 mM at pH 7.4 and 8.0, respectively. At both pH levels, levamisole-sensitive AP activity exhibited Michaelis-Menten kinetics (Km=3.451 mM and 6.906 mM at pH 7.4 and 8.0, respectively. Levamisole-sensitive AP activities were strongly affected by temperature, exhibiting a peak at 37ºC. In crabs acclimated to low salinity (10; hyperegulating conditions, Na+-K+ ATPase activity and levamisole-sensitive AP activity at the physiological pH were higher than in 35 psu (osmoconforming conditions. The response to low salinity suggests that both activities could be components of muscle regulatory mechanisms at the biochemical level secondary to hyperegulation of C. angulatus. The study of these activities under hyperegulating conditions contributes to a better understanding of the complexity of biochemical mechanisms underlying the adaptive process of euryhaline crabs.

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

    Science.gov (United States)

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

    2015-10-01

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

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

    Science.gov (United States)

    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. © 2015 Poultry Science Association Inc.

  16. Characterization of soluble and membrane-bound alkaline phosphatase in Nilaparvata lugens and their potential relation to development and insecticide resistance.

    Science.gov (United States)

    Wang, Zengxia; Liu, Shuhua; Yang, Baojun; Liu, Zewen

    2011-09-01

    Two forms (soluble and membrane-bound) of alkaline phosphatases (ALPs) were found in the brown planthopper, Nilaparvata lugens. In order to further study ALPs in N. lugens, two putative ALP genes (Nl-ALP1 and Nl-ALP2) were identified in this pest. Both Nl-ALP1 and Nl-ALP2 show approximately the same degree of sequence identity (40-50%) to other insect soluble and membrane-bound forms of ALP. Correlation of ALP activity and mRNA levels at different developmental stages, or following application of 20-hydroxyecdysone (20E) and insecticide fenvalerate, suggests that Nl-ALP1 and Nl-ALP2 might encode a soluble (sALP) and a membrane-bound ALP (mALP), respectively. Nl-ALP1-specific antibody Nl1-I detected only a specific band in soluble protein preparations and Nl-ALP2 specific antibody Nl2-I only detected a specific band in insoluble protein preparations, which provided conclusive linkages between Nl-ALP1 and a sALP and between Nl-ALP2 and a m ALP. Then, Nl-ALP1 was denoted as Nl-sALP for a sALP and Nl-ALP2 was denoted as Nl-mALP for a mALP. Only sALP activity and Nl-sALP mRNA level were induced by 20E and fenvalerate, which was confirmed by the density of specific band detected by Nl1-I in Sus strain with or without fenvalerate treatment. Additionally, the sALP activity, as well as Nl-sALP mRNA level, was significantly higher in a fenvalerate resistant population, compared with Sus strain. These results indicate that the sALP is more responsive to chemical stimulus, such as hormone and insecticide, and might play dual roles in development and insecticide tolerance. © 2011 Wiley Periodicals, Inc.

  17. Effects of 60Co gamma-ray local irradiation on rat liver on alkaline phosphatase, lactate dehydrogenase and catalase in the liver and serum

    International Nuclear Information System (INIS)

    Hishikawa-Itoh, Youko; Ayakawa, Yoshio; Miyata, Nobuki

    1980-01-01

    Rats were given a single exposure of various doses (0, 5, 50, 500, and 5000 rads) to local irradiation of 60 Co γ-ray on liver. Activities of alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and catalase in the serum and liver were measured at various time intervals after irradiation. These results were summarized as follows; 1. ALP activity in the serum had no effect on irradiation up to 500 rads, but in the case of 5000 rads irradiation exhibited a marked loss from 4 days after irradiation. ALP activity in the liver to 5000 rads exposure on 7 days after irradiation increased, on the other hand in the serum decreased, and the patterns of ALP activities in the liver and serum to the irradiation doses were opposite. 2. LDH activity in the serum by exposure to 5, 500 and 5000 rads increased at 4 days after irradiation, but at 7 days significantly decreased. LDH activity in the liver to the irradiation doses on 7 days after irradiation did not markedly change, but in the serum it tended to be low in inverse proportion to the irradiation doses. 3. Catalase activity in the serum to 50 and 500 rads exposure increased at 4 days after irradiation and decreased at 7 days, but to 5000 rads exposure it decreased in the course of time. Catalase activity in the liver and serum on 7 days after irradiation were inversely proportional to irradiation doses. It is difficult that catalase activity makes a index of clinical irradiation effects, because catalase activity decrease under the various conditions, such as cancer, anemia, infection of bacterias and so on. Since activities of ALP and LDH increase in almost disease, decrease of ALP activity and decrease following temporary increase of LDH activity by irradiation may be able to become a clinical indicator on irradiation effects. (author)

  18. Prognostic impact of alkaline phosphatase measured at time of presentation in patients undergoing primary percutaneous coronary intervention for ST-segment elevation myocardial infarction.

    Directory of Open Access Journals (Sweden)

    Pyung Chun Oh

    Full Text Available Serum alkaline phosphatase (ALP has been shown to be a prognostic factor in several subgroups of patients due to its promotion of vascular calcification. However, the prognostic impact of serum ALP level in ST-segment elevation myocardial infarction (STEMI patients with a relatively low calcification burden has not been determined. We aimed to investigate the association of ALP level measured at time of presentation on clinical outcomes in patients with STEMI requiring primary percutaneous coronary intervention (PCI.A total of 1178 patients with STEMI undergoing primary PCI between 2007 and 2014 were retrospectively enrolled from the INTERSTELLAR registry and classified into tertiles by ALP level (83 IU/L. The primary study outcome was a major adverse cardiac or cerebrovascular event (MACCE, defined as the composite of all-cause death, non-fatal myocardial infarction, non-fatal stroke, and ischemia-driven revascularization.Median follow-up duration was 25 months (interquartile range, 10-39 months. The incidence of MACCE significantly increased as ALP level increased, that is, for the 83 IU/L tertiles incidences were 8.7%, 11.7%, and 15.7%, respectively; p for trend = 0.003. After adjustment for potential confounders, the adjusted hazard ratios for MACCE in the middle and highest tertiles were 1.69 (95% CI 1.01-2.81 and 2.46 (95% CI 1.48-4.09, respectively, as compared with the lowest ALP tertile.Elevated ALP level at presentation, but within the higher limit of normal, was found to be independently associated with higher risk of MACCE after primary PCI in patients with STEMI.

  19. Alkaline Phosphatase in Stem Cells

    Czech Academy of Sciences Publication Activity Database

    Štefková, K.; Procházková, Jiřina; Pachernik, J.

    2015-01-01

    Roč. 2015, č. 2015 (2015) ISSN 1687-966X Institutional support: RVO:68081707 Keywords : PRIMORDIAL GERM-CELLS * P38 MAP KINASE * EMBRYONAL CARCINOMA-CELLS Subject RIV: BO - Biophysics Impact factor: 3.687, year: 2015

  20. Development of a single-chain variable fragment-alkaline phosphatase fusion protein and a sensitive direct competitive chemiluminescent enzyme immunoassay for detection of ractopamine in pork

    Energy Technology Data Exchange (ETDEWEB)

    Dong Jiexian; Li Zhenfeng; Lei Hongtao; Sun Yuanming [Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou 510642 (China); Ducancel, Frederic [CEA, iBiTec-S, Service de Pharmacologie et d' Immnoanalyse (SPI), CEA Saclay, F-91191 Gif sur Yvette (France); Xu Zhenlin [Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou 510642 (China); Boulain, Jean-Claude [CEA, iBiTec-S, Service de Pharmacologie et d' Immnoanalyse (SPI), CEA Saclay, F-91191 Gif sur Yvette (France); Yang Jinyi; Shen Yudong [Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou 510642 (China); Wang Hong, E-mail: gzwhongd@63.com [Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou 510642 (China)

    2012-07-29

    Graphical abstract: Detection model of dc-CLEIA based on anti-RAC scFv-AP fusion protein. Highlights: Black-Right-Pointing-Pointer The scFv-AP fusion protein against ractopamine (RAC) was produced. Black-Right-Pointing-Pointer A dc-CLEIA for RAC was developed based on the purified scFv-AP fusion protein. Black-Right-Pointing-Pointer The sensitivity of dc-CLEIA was 10 times as sensitive as dc-ELISA for RAC. Black-Right-Pointing-Pointer Recovery tests from pork samples were studied. Black-Right-Pointing-Pointer Good accuracy was obtained. - Abstract: A rapid, sensitive chemiluminescent enzyme immunoassay (CLEIA) for ractopamine (RAC) based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein was developed. The scFv gene was prepared by cloning the heavy- and light-chain variable region genes (V{sub H} and V{sub L}) from hybridoma cell line AC2, which secretes antibodies against RAC, and assembling V{sub H} and V{sub L} genes with a linker by means of splicing overlap extension polymerase chain reaction. The resulting scFv gene was inserted into the expression vector pLIP6/GN containing AP to produce the fusion protein in Escherichia coli strain BL21. The purified scFv-AP fusion protein was used to develop a direct competitive CLEIA (dcCLEIA) protocol for detection of RAC. The average concentration required for 50% inhibition of binding and the limit of detection of the assay were 0.25 {+-} 0.03 and 0.02 {+-} 0.004 ng mL{sup -1}, respectively, and the linear response range extended from 0.05 to 1.45 ng mL{sup -1}. The assay was 10 times as sensitive as the corresponding enzyme-linked immunosorbent assay based on the same fusion protein. Cross-reactivity studies showed that the fusion protein did not cross react with RAC analogs. DcCLEIA was used to analyze RAC spiked pork samples, and the validation was confirmed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS). The results showed a good correlation between

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

    Directory of Open Access Journals (Sweden)

    Abedini F

    2012-07-01

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

  2. Radioimmunoassay of creatine kinase isoenzymes in human serum: isoenzyme MM

    International Nuclear Information System (INIS)

    Van Steirteghem, A.C.; Zweig, M.H.; Schechter, A.N.

    1978-01-01

    Measurement of the mass concentration of serum enzymes by radioimmunoassay provides direct quantitation of specific isoenzymes and may be less subject to some of the limitations of traditional assay procedures for enzymes. We describe the development of a sensitive and specific radioimmunoassay for the muscle isoenzyme of creatine kinase, CK-MM, in human serum. CK-MM, purified from human skeletal muscle, was used to raise high-titer antisera and for iodination by the Chloramine T method. The radioimmunoassay required 50 μl of sample, utilized a double-antibody separation method, and was completed in 24 h. Cross reactivity with CK-BB was virtually zero, 3 to 17 percent with CK-MB. The mass concentration of CK-MM in the serum of healthy subjects ranged from 36 to 1668 μg/liter and correlated closely with total CK enzymatic activity. Serum concentrations of CK-MM from casually selected patients correlated less well with total CK enzymatic activity, suggesting the existence of other CK isoenzymes or the presence of inactive forms

  3. Interstrain polymorphisms of isoenzyme profiles and mitochondrial DNA fingerprints among seven strains assigned to Acanthamoeba polyphaga.

    Science.gov (United States)

    Kong, H H; Park, J H; Chung, D I

    1995-12-01

    Interstrain polymorphisms of isoenzyme profiles and mitochondrial (Mt) DNA fingerprints were observed among seven strains of Acanthamoeba isolated from different sources and morphologically assigned to A. polyphaga. Mt DNA fingerprints by eight restriction endonucleases (Bgl II, Sca I, Cla I, EcoR I, Xba I, Kpn I, Sal I, and Sst I) revealed considerable interstrain polymorphisms. Isoenzyme profiles revealed considerable interstrain polymorphisms for acid phosphatase, lactate dehydrogenase, and glucose-6-phosphate dehydrogenase while those for glucose phosphate isomerase, leucine aminopeptidase, and malate dehydrogenase showed similarity. Despite of the interstrain polymorphisms, the isoenzyme profiles and Mt DNA fingerprints of the strain Ap were found to be identical with those of the strain Jones. Mt DNA fingerprinting was found to be highly applicable for the strain identification, characterization, and differentiation.

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

    Science.gov (United States)

    Bertone, V; Tarantola, E; Ferrigno, A; Gringeri, E; Barni, S; Vairetti, M; Freitas, I

    2011-02-08

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

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

    Directory of Open Access Journals (Sweden)

    V. Bertone

    2011-02-01

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

  6. Influence of serum levels of alkaline phosphatase and gamma-glutamyl transpeptidase on the prognosis of patients with primary liver cancer undergoing radiofrequency ablation

    Directory of Open Access Journals (Sweden)

    ZHU Ge

    2016-12-01

    Full Text Available ObjectiveTo investigate the influence of serum levels of alkaline phosphatase (ALP and gamma-glutamyltransferase (GGT before treatment on the prognosis of patients with primary liver cancer undergoing radiofrequency ablation. MethodsA total of 165 patients with pathologically or clinically confirmed primary liver cancer who were admitted to Cancer Center of The First Hospital of Jilin University from October 2010 to June 2015 and treated with radiofrequency ablation were enrolled, and their preoperative clinical data were collected. The chi-square test was used for comparison of categorical data between groups, and the Kaplan-Meier method and Cox regression analysis were used to analyze the association of serum ALP and GGT levels before treatment with overall survival, progression-free survival, and clinical features. ResultsThere were significant differences in the 1-, 2-, and 5-year survival rates between the patients with normal (≤135 U/L or abnormal (>135 U/L serum ALP before treatment (91%/90%/35% vs 79%/68%/18%, P=0.01. There were also significant differences in the 1-, 2-, and 5-year survival rates between the patients with normal (≤45 U/L or abnormal (>45 U/L serum GGT before treatment (95%/85%/37% vs 87%/71%/21%, P<0.001. The serum levels of ALP (HR=1.766, 95%CI:1.068-2.921,P=0.027 and GGT (HR=2.312, 95%CI:1.367-3.912,P=0.002 before treatment were closely associated with the overall survival of patients with primary liver cancer after radiofrequency ablation and were independent prognostic factors. There were significant differences in the 1-, 2-, and 5-year progression-free survival rates between the patients with normal (≤135 U/L or abnormal (>135 U/L serum ALP before treatment (72%/52%/14% vs 50%/21%/3%, P<0.001; there were also significant differences in the 1-, 2-, and 5-year progression-free survival rates between the patients with normal (≤45 U/L or abnormal (>45 U/L serum GGT before treatment (81%/62%/18% vs

  7. iagnostic accuracy study comparing total alkaline phosphatase with intact parathyroid hormone 1-84 for the diagnosis of high turnover renal osteodystrophy in chronic renal failure on hemodialysis

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    Andrés Marcelo Rojas González

    2014-09-01

    Full Text Available INTRODUCTION High turnover renal osteodystrophy (HTRO is a highly prevalent complication in patients with chronic kidney disease and mineral bone disease (CKD-MBD, causing pain and significant fracture-associated morbidity and mortality. The diagnostic gold standard test is bone biopsy but there are other, more widely available screening tests such as 1-84 intact parathormone (1-84 iPTH and nonspecific markers such as total alkaline phosphatase (tALP. PURPOSE To determine the diagnostic value (ROC curve, predictive values and likelihood ratios of 1-84 iPTH and tALP for HTRO screening. METHODS A diagnostic accuracy study was performed on a sample of CKD-MDB patients, grouping them according to bone biopsy results and analyzing the results of the diagnostic tests as descriptive variables. RESULTS The study group comprised 188 patients with CKD-MDB, 36 of which had biopsy-confirmed HTRO (19.15%. The average age was 50.2 years in the biopsy group, and 53.4 years in the non-biopsy group (p=0.2385, most were male (63.8% and diabetic (80.5%. The mean time in dialysis was 5.02 years in the biopsy group, and 2.61 years for the non-biopsy group (p<0.001. The mean Kt/V was 1.44 in the biopsy group, and 1.40 in the non-biopsy group (p=0.5354. The mean tALP was 398.02 IU/L in the group with HTRO versus 141.76 IU/L in the group without HTRO (p<0.001. The best cut-off value for tALP was 300-350 IU/L with a near 80% post-test probability, but also with a 15-20% probability for HTRO if the test is negative. The mean 1-84 iPTH was 1248.01 pg/ml in the group with HTRO versus 350.76 pg/ml in the group without HTRO (p<0.001. The 1-84 iPTH cut-off reference value of 300 pg/ml was associated with a post-test probability of 30% for HTRO diagnosis and had a lower overall performance. The best cut-off value for iPTH 1-84 was 600 pg/ml with a post-test probability for HTRO of 70% if positive and less than 5% if the test results are negative. DISCUSSION Both markers show

  8. Aplicación del método inmunocitoquímico de la fosfatasa alcalina anti-fosfatasa alcalina para la clasificación inmunológica de las leucemias mieloides agudas Application of the alkaline phosphatase anti-alkaline phosphatase immunocytochemical method for the immunological classification of acute myeloid leukemias

    Directory of Open Access Journals (Sweden)

    Bertha B Socarrás Ferrer

    2006-04-01

    Full Text Available Se realizó el inmunofenotipaje celular de 30 pacientes con el diagnóstico de leucemia mieloide aguda por el método inmunocitoquímico fosfatasa alcalina anti-fosfatasa alcalina (APAAP introducido en nuestro laboratorio. Los marcadores estudiados fueron: CD3, CD13, CD15, CD19, CD33 y CD41. Para el estudio se utilizaron extendidos de médula ósea o sangre periférica fijados en acetona pura e incubados con el respectivo anticuerpo monoclonal. Posteriormente se añadió la inmunoglobulina anti ratón obtenida en conejo ( Linking y por último, el complejo APAAP. Los períodos de incubación fueron de 30 minutos y se realizaron lavados con solución amortiguadora entre cada uno de los pasos. La lectura de las láminas se realizó en microscopio óptico y se consideró positivo cuando el número de células marcadas era mayor o igual a 20 %. De los pacientes estudiados, el 93,3 % y el 90 %, respectivamente, expresaron antígenos pan mieloides CD13 y CD33; 16 de ellos expresaron el CD15 (53,3 %; 3 el CD19 (10 % y 2 el CD41 (6,6 %. Se concluyó que el método APAAP es rápido y de bajo costo y puede ser aplicado con confiabilidad en la clasificación inmunológica de las leucemias mieloides agudasThe cellular immunophenotyping of 30 patients with the diagnosis of acute myeloid leukemia was conducted by the alkaline phosphatase anti-alkaline phosphatase immunocytochemical method (APAAP introduced in our laboratory. The markers studied were: CD3, CD13, CD15, CD19, CD33 y CD41. Specimens of bone marrow or peripheral blood fixed in pure acetone and incubated with the respective monoclonal antibody were used for the study. Later on, the anti-mouse immunoglobulin obtained in rabbit (Linking was added and, finally, the APAAP complex. The incubation periods were of 30 minutes and lavages with buffer solution were carried out between one step and the other. The reading of the slides was performed on the optical microscope and it was considered positive when

  9. PURIFICATION AND CHARACTERISATION OF ALKALINE ...

    African Journals Online (AJOL)

    Prof. Adipala Ekwamu

    There was no clear decrease in the yield seen in the bands and the loss of enzyme was not observed with the gel analysis. It may ... The native gel results show clear distinct bands for the 3 alkaline phosphotase isoenzymes ..... British Medical.

  10. Digoxigenylated wheat germ agglutinin visualized with alkaline phosphatase-labeled anti-digoxigenin antibodies--a new, sensitive technique with the potential for single and double tracing of neuronal connections.

    Science.gov (United States)

    Veh, R W

    1991-01-02

    For double tracing experiments, wheat germ agglutinin (WGA) molecules labeled with two different haptens are desirable. In the present report the suitability of digoxigenylated WGA (DIG-WGA) for retrograde tracing was investigated. For this purpose the new tracer was pressure injected into rat brains and the transported DIG-WGA visualized via its digoxigenyl group with an alkaline phosphatase linked anti DIG antibody in permanently stained sections of high quality. With fixatives containing 2.5% glutaraldehyde only few positive cells were found. However, at milder fixation conditions (4% paraformaldehyde, 0.05% glutaraldehyde 0.2% picric acid, 30 min) retrogradely labeled cells were detected with a sensitivity comparable to tetramethylbenzidine protocols for conventional WGA-HRP (horseradish peroxidase) tracing. Preliminary experiments suggest excellent suitability for double labeling.

  11. Experimental study on the usefulness of magnetotherapy in bone fractures (tibial osteotomy in the rat). Accumulation of 99 mTc MDP - tests of tensile strength - determination of alkaline phosphatase

    International Nuclear Information System (INIS)

    Sailer, R.

    1985-01-01

    Non-directional magnetic field therapy using a flux density of 60 G and a frequency of 25 Hz was carried out over 12 hours daily in rats in order to ascertain its influence on the healing process following osteotomy of the tibia with internal splint fixation of the fractured bone being carried out as an additional measure. The results thus achieved were compared to those seen in control animals, were no magnetotherapy was carried out, on the basis of scintiscan studies using 99 mTc MDP (degree of density in the callus formed around the fracture zone), the plasma levels of alkaline phosphatase and tests of tensile strength. The follow-up observations of the healing process were additionally based on radiological and histological evaluations of the animals. Beneficial effects of magnetotherapy on the healing process could not be confirmed with any statistical significance. (TRV) [de

  12. Towards the identification of alkaline phosphatase binding ligands in Li-Dan-Hua-Shi pills: A Box-Behnken design optimized affinity selection approach tandem with UHPLC-Q-TOF/MS analysis.

    Science.gov (United States)

    Tao, Yi; Huang, Surun; Gu, Xianghui; Li, Weidong; Cai, Baochang

    2018-05-30

    Alkaline phosphatase conjugated magnetic microspheres were synthesized via amide reaction, and employed as an effective adsorbent in affinity selection of binding ligands followed by UHPLC-Q-TOF/MS analysis. The analytical validity of the developed approach was evaluated under optimized conditions and the following figures of merit were obtained: linearity, 0.01-0.5 g L -1 with good determination coefficients (R 2  = 0.9992); limits of detection (LODs), 0.003 g L -1 ; and limits of quantitation (LOQ), 0.01 g L -1 . The precision (RSD%) of the proposed affinity selection approach was studied based on intra-day (0.8%) and inter-day (1.3%) precisions. Finally, the adsorbent was successfully applied to identification of binding ligands in Li-Dan-Hua-Shi pills and good recoveries were obtained in the range from 96.9 to 99.4% (RSDs 1.6-3.0%). Copyright © 2018 Elsevier B.V. All rights reserved.

  13. An evaluation of the effect of age and the peri-parturient period on bone metabolism in dairy cows as measured by serum bone-specific alkaline phosphatase activity and urinary deoxypyridinoline concentration.

    Science.gov (United States)

    Sato, Reiichiro; Onda, Ken; Kato, Hajime; Ochiai, Hideharu; Kawai, Kazuhiro; Iriki, Tsunenori; Kaneko, Kazuyuki; Yamazaki, Yukio; Wada, Yasunori

    2013-08-01

    Various biochemical markers help to evaluate the state of bone turnover in humans and could be used during the peri-parturient period in dairy cows when calcium (Ca) metabolism changes dramatically. To investigate this, the peri-partum characteristics of serum bone-specific alkaline phosphatase (BAP) and urinary deoxypyridinoline (DPD) were investigated. Both serum BAP activity and urinary DPD concentrations were increased and demonstrated wide variability in younger animals, and these findings were consistent with other bone turnover markers. Around the time of parturition, serum Ca concentration and serum BAP activity in multiparous cows were significantly lower than in primiparous cows, but urinary DPD concentration was unchanged. The use of BAP as a bone formation marker appears to be valuable for evaluating bone remodelling status in cows, but the specificity of the test needs to be confirmed. The DPD/BAP ratio around parturition demonstrated a clear difference in bone turnover status between the two parity groups with multiparous cows demonstrating increased signs of bone resorption compared with primiparous cows, corresponding to the Ca requirement for milk production. In future studies, the applicability of the ratio of bone resorption marker to bone formation marker should be evaluated for bone turnover assessment. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. IGF-1R Promotes Symmetric Self-Renewal and Migration of Alkaline Phosphatase+ Germ Stem Cells through HIF-2α-OCT4/CXCR4 Loop under Hypoxia.

    Science.gov (United States)

    Kuo, Yung-Che; Au, Heng-Kien; Hsu, Jue-Liang; Wang, Hsiao-Feng; Lee, Chiung-Ju; Peng, Syue-Wei; Lai, Ssu-Chuan; Wu, Yu-Chih; Ho, Hong-Nerng; Huang, Yen-Hua

    2018-02-13

    Hypoxia cooperates with endocrine signaling to maintain the symmetric self-renewal proliferation and migration of embryonic germline stem cells (GSCs). However, the lack of an appropriate in vitro cell model has dramatically hindered the understanding of the mechanism underlying this cooperation. Here, using a serum-free system, we demonstrated that hypoxia significantly induced the GSC mesenchymal transition, increased the expression levels of the pluripotent transcription factor OCT4 and migration-associated proteins (SDF-1, CXCR4, IGF-1, and IGF-1R), and activated the cellular expression and translocalization of the CXCR4-downstream proteins ARP3/pFAK. The underlying mechanism involved significant IGF-1/IGF-1R activation of OCT4/CXCR4 expression through HIF-2α regulation. Picropodophyllin-induced inhibition of IGF-1R phosphorylation significantly suppressed hypoxia-induced SDF-1/CXCR4 expression and cell migration. Furthermore, transactivation between IGF-1R and CXCR4 was involved. In summary, we demonstrated that niche hypoxia synergistically cooperates with its associated IGF-1R signaling to regulate the symmetric division (self-renewal proliferation) and cell migration of alkaline phosphatase-positive GSCs through HIF-2α-OCT4/CXCR4 during embryogenesis. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  15. Alkaline phosphatase and OCT-3/4 as useful markers for predicting susceptibility of human deciduous teeth-derived dental pulp cells to reprogramming factor-induced iPS cells.

    Science.gov (United States)

    Inada, Emi; Saitoh, Issei; Kubota, Naoko; Soda, Miki; Matsueda, Kazunari; Murakami, Tomoya; Sawami, Tadashi; Kagoshima, Akiko; Yamasaki, Youichi; Sato, Masahiro

    2017-11-01

    The aim of the present study was to prove that primary cells enriched with stem cells are more easily reprogrammed to generate induced pluripotent stem (iPS) cells than those with scarce numbers of stem cells. We surveyed the alkaline phosphatase (ALP) activity in five primarily-isolated human deciduous teeth-derived dental pulp cells (HDDPC) with cytochemical staining to examine the possible presence of stem cells. Next, the expression of stemness-specific factors, such as OCT(Octumer-binding transcription factor)3/4, NANOG, SOX2(SRY (sex determining region Y)-box 2), CD90, muscle segment homeodomain homeobox (MSX) 1, and MSX2, was assessed with a reverse transcription polymerase chain reaction method. Finally, these isolated HDDPC were transfected with plasmids carrying genes coding Yamanaka factors to determine whether these cells could be reprogrammed to generate iPS cells. Of the five primarily-isolated HDDPC, two (HDDPC-1 and -5) exhibited higher degrees of ALP activity. OCT-3/4 expression was also prominent in those two lines. Furthermore, these two lines proliferated faster than the other three lines. The transfection of HDDPC with Yamanaka factors resulted in the generation of iPS cells from HDDPC-1 and -5. The number of cells with the stemness property of HDDPC differs among individuals, which suggests that HDDPC showing an increased expression of both ALP and OCT-3/4 can be more easily reprogrammed to generate iPS cells after the forced expression of reprogramming factors. © 2016 John Wiley & Sons Australia, Ltd.

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

    Science.gov (United States)

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

    2015-01-01

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

  17. IGF-1R Promotes Symmetric Self-Renewal and Migration of Alkaline Phosphatase+ Germ Stem Cells through HIF-2α-OCT4/CXCR4 Loop under Hypoxia

    Directory of Open Access Journals (Sweden)

    Yung-Che Kuo

    2018-02-01

    Full Text Available Summary: Hypoxia cooperates with endocrine signaling to maintain the symmetric self-renewal proliferation and migration of embryonic germline stem cells (GSCs. However, the lack of an appropriate in vitro cell model has dramatically hindered the understanding of the mechanism underlying this cooperation. Here, using a serum-free system, we demonstrated that hypoxia significantly induced the GSC mesenchymal transition, increased the expression levels of the pluripotent transcription factor OCT4 and migration-associated proteins (SDF-1, CXCR4, IGF-1, and IGF-1R, and activated the cellular expression and translocalization of the CXCR4-downstream proteins ARP3/pFAK. The underlying mechanism involved significant IGF-1/IGF-1R activation of OCT4/CXCR4 expression through HIF-2α regulation. Picropodophyllin-induced inhibition of IGF-1R phosphorylation significantly suppressed hypoxia-induced SDF-1/CXCR4 expression and cell migration. Furthermore, transactivation between IGF-1R and CXCR4 was involved. In summary, we demonstrated that niche hypoxia synergistically cooperates with its associated IGF-1R signaling to regulate the symmetric division (self-renewal proliferation and cell migration of alkaline phosphatase-positive GSCs through HIF-2α-OCT4/CXCR4 during embryogenesis. : In this article, Huang and colleagues demonstrate that niche hypoxia promotes symmetric self-renewal proliferation and migration of PGC-like CD49f+AP+GSCs through IGF-IR regulation. Using a serum-free culture system, the crosstalk between IGF-1R and CXCR4 signaling was discovered. This work demonstrated that embryonic hypoxia synergistically cooperated with IGF-1R signaling to regulate the symmetric self-renewal and migration of PGC-like GSCs through a HIF-2α–OCT4/CXCR4 loop. Keywords: hypoxia, niche, germline stem cells, self-renewal, migration, IGF-1R, HIF-2α, OCT4, SDF-1, CXCR4

  18. Maternal antibiotic-induced early changes in microbial colonization selectively modulate colonic permeability and inducible heat shock proteins, and digesta concentrations of alkaline phosphatase and TLR-stimulants in swine offspring.

    Directory of Open Access Journals (Sweden)

    Marie-Edith Arnal

    Full Text Available Elevated intake of high energy diets is a risk factor for the development of metabolic diseases and obesity. High fat diets cause alterations in colonic microbiota composition and increase gut permeability to bacterial lipopolysaccharide, and subsequent low-grade chronic inflammation in mice. Chronic inflammatory bowel diseases are increasing worldwide and may involve alterations in microbiota-host dialog. Metabolic disorders appearing in later life are also suspected to reflect changes in early programming. However, how the latter affects the colon remains poorly studied. Here, we hypothesized that various components of colonic physiology, including permeability, ion exchange and protective inducible heat shock proteins (HSP are influenced in the short- and long-terms by early disturbances in microbial colonization. The hypothesis was tested in a swine model. Offspring were born to control mothers (n = 12 or mothers treated with the antibiotic (ATB amoxicillin around parturition (n = 11. Offspring were slaughtered between 14 and 42 days of age to study short-term effects. For long-term effects, young adult offspring from the same litters consumed a normal or a palm oil-enriched diet for 4 weeks between 140 and 169 days of age. ATB treatment transiently modified maternal fecal microbiota although the minor differences observed for offspring colonic microbiota were nonsignificant. In the short-term, consistently higher HSP27 and HSP70 levels and transiently increased horseradish peroxidase permeability in ATB offspring colon were observed. Importantly, long-term consequences included reduced colonic horseradish peroxidase permeability, and increased colonic digesta alkaline phosphatase (AP and TLR2- and TLR4-stimulant concentrations in rectal digesta in adult ATB offspring. Inducible HSP27 and HSP70 did not change. Interactions between early ATB treatment and later diet were noted for paracellular permeability and concentrations of colonic

  19. Morphologic, biometric, and isoenzyme characterization of Trichuris suis.

    Science.gov (United States)

    Oliveros, R; Cutillas, C; Arias, P; Guevara, D

    1998-06-01

    Trichuris suis isolates were collected from the cecum of Sus scrofa domestica (pig) and S. s. scrofa (wild boar). Morphology and biometry studies were carried out. Morphology studies showed the existence of typical caudal papillae in males of T. suis from wild boars, but no other difference was observed in the biometric parameters (total length, esophageal length, posterior-portion body length, and spicular length) of T. suis isolated from either host. Individual extracts were subjected to malate dehydrogenase (MDH), malic enzyme (ME), glucose 6-phosphate dehydrogenase (G6PD), lactate dehydrogenase (LDH), and superoxide dismutase (SOD) isoenzyme analysis following starch-gel electrophoresis, and the isoenzyme patterns were compared with those obtained from other species of trichurids. MDH, ME, G6PD, LDH, and SOD isoenzyme patterns were identical for T. suis from both hosts. MDH isoenzyme patterns were characterized by the presence of one cathodic isoenzyme. ME, G6PD, and LDH isoenzyme patterns indicated the presence of three phenotypes, whereas the SOD isoenzyme pattern showed only one phenotype characterized by the existence of two (anodic and cathodic) bands. Different LDH and SOD isoenzyme patterns observed for T. suis, T. ovis, and T. skrjabini confirm once more that isoenzyme patterns have potential as a diagnostic tool for differentiation of different species of Trichuris.

  20. Determination of trace alkaline phosphatase by affinity adsorption solid substrate room temperature phosphorimetry based on wheat germ agglutinin labeled with 8-quinolineboronic acid phosphorescent molecular switch and prediction of diseases

    Science.gov (United States)

    Liu, Jia-Ming; Gao, Hui; Li, Fei-Ming; Shi, Xiu-Mei; Lin, Chang-Qing; Lin, Li-Ping; Wang, Xin-Xing; Li, Zhi-Ming

    2010-09-01

    The 8-quinolineboronic acid phosphorescent molecular switch (abbreviated as PMS-8-QBA. Thereinto, 8-QBA is 8-quinolineboronic acid, and PMS is phosphorescent molecular switch) was found for the first time. PMS-8-QBA, which was in the "off" state, could only emit weak room temperature phosphorescence (RTP) on the acetyl cellulose membrane (ACM). However, PMS-8-QBA turned "on" automatically for its changed structure, causing that the RTP of 8-QBA in the system increased, after PMS-8-QBA-WGA (WGA is wheat germ agglutinin) was formed by reaction between -OH of PMS-8-QBA and -COOH of WGA. More interesting is that the -NH 2 of PMS-8-QBA-WGA could react with the -COOH of alkaline phosphatase (AP) to form the affinity adsorption (AA) product WGA-AP-WGA-8-QBA-PMS (containing -NH-CO- bond), which caused RTP of the system to greatly increase. Thus, affinity adsorption solid substrate room temperature phosphorimetry using PMS-8-QBA as labelling reagent (PMS-8-QBA-AA-SSRTP) for the determination of trace AP was established. The method had many advantages, such as high sensitivity (the detection limit (LD) was 2.5 zg spot -1. For sample volume of 0.40 μl spot -1, corresponding concentration was 6.2 × 10 -18 g ml -1), good selectivity (the allowed concentration of coexisting material was higher, when the relative error was ±5%), high accuracy (applied to detection of AP content in serum samples, the result was coincided with those obtained by enzyme-linked immunoassay), which was suitable for the detection of trace AP content in serum samples and the forecast of human diseases. Meanwhile, the mechanism of PMS-8-QBA-AASSRTP was discussed. The new field of analytical application and clinic diagnosis technique of molecule switch are exploited, based on the phosphorescence characteristic of PMS-8-QBA, the AA reaction between WGA and AP, as well as the relation between AP content and human diseases. The research results promote the development and interpenetrate among molecule

  1. Introducción del método inmunocitoquímico de la fosfatasa alcalina-antifosfatasa alcalina para la clasificación inmunológica de los Síndromes Linfo y Mieloproliferativos Agudos Introduction of the alkaline phosphatase-alkaline antiphosphatase immunocytochemical method for the immunological classification of the acute lympho-and myeloproliferative syndromes

    Directory of Open Access Journals (Sweden)

    Berta B Socarrás Ferrer

    2001-04-01

    Full Text Available Se realizó el inmunofenotipaje celular en 30 pacientes con el diagnóstico de síndromes linfo y mieloproliferativos agudos por el método inmunoenzimático fosfatasa alcalina-antifosfatasa alcalina (APAAp introducido en nuestro laboratorio. Los marcadores estudiados fueron: CD3, CD5, CD7, CD10, CD13, CD15, CD22, CD33, CD34 y CD41 mediante los anticuerpos monoclonales correspondientes, según cada caso. De las leucemias agudas, 16 resultaron ser leucemias linfoides (LLA (53,3 % y 12 mieloides (LMA (40 %. Entre las LLA, el 50 % fue del fenotipo B y del resto, 1 caso del tipo T (LLA-T (3,33 %. Un paciente se diagnosticó como leucemia aguda híbrida (LAH (3,33 % y el otro se clasificó como leucemia aguda indiferenciada (LAI (3,33 %. Se concluye que el APAAP es un método más rápido y tan eficaz como otros métodos enzimáticos para la clasificación inmunológica de los síndromes linfo y mieloproliferativosThe cellular immunophenotyping was carried out in 30 patients with the diagnosis of acute lympho- and myeloproliferative syndromes by the alkaline phosphatase-alkaline antiphosphatase immunoenzimatic method (APAA introduced in our laboratory. The CD3, CD5, CD7, CD10; CDl3, CDl5, CD22, CD33 and CD41 markers were studied by using the corresponding monoclonal antibodies, according to each case. Of the acute leukemias, 16 were lymphoid leukemias (ALL (53.3 % and 12 were myeloid leukemias (AML (40 %. Among the ALL, 50 % were phenotype B and of the rest, 1 case was type T (ALL-T (3.33 %. A patient was diagnosed acute hybrid leukemia (AHL (3.33 % and the other was classified as acute undifferentiated leukemia (AUL (3.33 %. It is concluded that the APAA is faster and as efficient as other enzimatic methods for the immunologic classification of the lympho- and myeloproliferative syndromes

  2. Radioimmunoassay of creatine kinase BB isoenzyme

    Energy Technology Data Exchange (ETDEWEB)

    Jianguo, Geng [Shanghai Medical Univ. (China). Zhongshan Hospital; and others

    1988-11-01

    A radioimmunoassay of creatine kinase BB isoenzyme (CK-BB) was developed by using CK-BB purified from human brain. The CK-BB antiserum was raised by immunizing rabbite and {sup 125}I-CK-BB iodinated with Bolton-Hunter reagent. The affinity constant was 3.0 x 10{sup 9} mol/L. No cross reactions with creatine kinase MM isoenzyme and neuron-specific enolase were found. The measuring range was 3.5 x 10{sup -8} {approx} 1.2 x 10{sup -5} mmol/L, the average recovery rate 97.5%, with the inter and intrassay CV 3.1% and 12%, respectively. The average serum CK-BB concentration in 83 normal persons was 1.5 x 10{sup -7} +- 8.1 x 10{sup -8} mmol/L, quite different from the values of acute myocardial infarction (5.2 x 10{sup -6} +- 1.2 x 10{sup -4} mmol/L, n = 28) and cerebral vascular accident (8.4 x 10{sup -4} +- 5.0 x 10{sup -4} mmol/L, n = 10).

  3. Radioimmunoassay of creatine kinase BB isoenzyme

    International Nuclear Information System (INIS)

    Geng Jianguo

    1988-01-01

    A radioimmunoassay of creatine kinase BB isoenzyme (CK-BB) was developed by using CK-BB purified from human brain. The CK-BB antiserum was raised by immunizing rabbite and 125 I-CK-BB iodinated with Bolton-Hunter reagent. The affinity constant was 3.0 x 10 9 mol/L. No cross reactions with creatine kinase MM isoenzyme and neuron-specific enolase were found. The measuring range was 3.5 x 10 -8 ∼ 1.2 x 10 -5 mmol/L, the average recovery rate 97.5%, with the inter and intrassay CV 3.1% and 12%, respectively. The average serum CK-BB concentration in 83 normal persons was 1.5 x 10 -7 +- 8.1 x 10 -8 mmol/L, quite different from the values of acute myocardial infarction (5.2 x 10 -6 +- 1.2 x 10 -4 mmol/L, n = 28) and cerebral vascular accident (8.4 x 10 -4 +- 5.0 x 10 -4 mmol/L, n = 10)

  4. Esterase Isoenzyme Profiles in Acute and Chronic Leukemias.

    Science.gov (United States)

    Drexler, H G; Gignac, S M; Hoffbrand, A V; Minowada, J

    1991-01-01

    Using isoelectric focusing (IEF) a number of carboxylic esterase isoenzymes (EC 3.1.1.1) with isoelectric points between pH 4.5-8.0 can be separated. One particular isoenzyme with an isoelectric point at about pH 6.0, the Mono-band, can be selectively and completely inhibited by sodium fluoride; this isoenzyme comprises a number of closely related subcomponents and may appear in more than one band on the gel. We analyzed the expression of typical esterase isoenzyme patterns in cells from a large panel of leukemias which were tested under identical conditions by IEF on horizontal thin-layer polyacrylamide gels with an ampholyte of pH 2-11. The 442 cases of acute and chronic myeloid and lymphoid leukemia (AML/AMMoL, CML/CMML, ALL, CLL) were classified according to clinical, morpho-cytochemical and immunophenotyping criteria. While bands between pH 4.5-5.5 appeared not to be specific for lineage or stage of differentiation, isoenzymes between pH 6.6-7.7 provided information on the type of leukemia involved. Seven typical isoenzyme patterns termed Mono1/Mono2 (fo monocyte-associated), My1/My2 (myeloid), Lym1/Lym2 (lymphoid) and Und (undifferentiated) could be discerned. Lym and Und patterns are characterized by fewer bands with a weaker staining intensity than Mono and My patterns. Nearly all cases of lymphoid leukemias (acute and chronic) expressed only Lym or Und esterase isoenzyme patterns, but no Mono or My patterns. Cases of acute or chronic myeloid and (myelo)monocytic leukemia showed strong isoenzyme staining displaying predominantly Mono or My isoenzyme patterns. The isoenzyme patterns found in CML in lymphoid or myeloid blast crisis corresponded to those seen in the respective acute leukemias, ALL or AML. The Mono-band was found in most cases of leukemias with monocytic elements (AMMoL 80%, CML 44%, CMML 100%), in the occasional case of CML-myeloid blast crisis or AML, but in none of the cases of ALL or CLL. This isoenzyme is a distinctive, specific marker for

  5. isoenzyme analysis of five endemic and one widespread kniphofia ...

    African Journals Online (AJOL)

    Preferred Customer

    ISOENZYME ANALYSIS OF FIVE ENDEMIC AND ONE WIDESPREAD ... plants. The over all mean inbreeding coefficient (F) was positive indicating slight deficiency in the number of ...... populations, indicates rather recent speciation.

  6. Influence of thorax irradiation on lactic dehydrogenase isoenzyme activity

    International Nuclear Information System (INIS)

    Valle, C.; Munnich, A.; Pasquier, C.

    The right hemi-thorax of rats was irradiated with 1200 and 3000 rads ( 60 Co) and blood samples were taken sequentially. The five lactic dehydrogenase (LDH) isoenzymes which have proved to be useful as biochemical indicators of acute pulmonary injury in other experimental animals (dogs), were assayed, after irradiation, as a function of time and as a functon of dose. There was no significant change in LDH isoenzyme activities after lung irradiation in rats [fr

  7. Relação tireóide-gônadas e níveis plasmáticos de fósforo, cálcio e fosfatase alcalina em ratas Relationship between thyroid, gonads and plasmatic levels of phosphorus, calcium and alkaline phosphatase in rats

    Directory of Open Access Journals (Sweden)

    R. Serakides

    2000-12-01

    Full Text Available A relação tireóide-gônadas-metabolismo ósseo foi estudada em ratas Wistar adultas, castradas ou intactas e mantidas em estado hipertireóideo ou eutireóideo por períodos de 30, 60 e 90 dias. Foram utilizadas como características do metabolismo ósseo o cálcio, o fósforo e a atividade da fosfatase alcalina plasmáticos, correlacionando-os com os valores de estrógeno, de progesterona e de T4 livre. Verificou-se que o hipogonadismo e o hipertireoidismo alteram as características plasmáticas do metabolismo ósseo. O hipertireoidismo induz hiperfosfatemia e hipocalcemia, o hipogonadismo tem pouca influência sobre o fósforo, mas potencializa a hiperfosfatemia e a hipocalcemia desencadeadas pelo hipertireoidismo. Com relação à fosfatase alcalina, conclui-se que o hipertireoidismo reduz o efeito do hipogonadismo sobre a atividade dessa enzima.The interrelation between thyroid, gonads and osseous metabolism was studied in either intact or castrated adult female rats kept under hyperthyroidism or euthyroidism for 30, 60, or 90 days. Plasmatic levels of phosphorus, calcium, and alkaline phosphatase were measured to assess the osseous metabolism. These characteristics were correlated to the levels of estrogen, progesterone, and free T4. Either hypogonadism or hyperthyroidism interfered with the plasmatic characteristics of osseous metabolism. Hyperphosphatemia and hypocalcemia were induced by hyperthyroidism, whereas the hypogonadism had little effect on the levels of phosphorus, but it had a potencialization effect on the hyperphosphatemia and hypocalcemia induced by hyperthyroidism. The effect of hypogonadism on the alkaline phosphatase activity was reduced by the hyperthyroidism.

  8. Isoenzymes performance of some rice varieties and their mutants

    International Nuclear Information System (INIS)

    Winarno, Ermin; Suliwarno, Ambyah; Ismachin, M.

    1992-01-01

    Isoenzymes performance of some rice varieties and their mutants. Genetics studies on alcohol dehydrogenase, malic enzyme, peroxidase, acid phosphase, and aminopeptidase isoenzymes were carried out on several groups of rice varieties and their mutant lines. The first groups consisted of Atomita I, Pelita I/1, A227/5, Mudgo, TN-1, and IR-26. The second group was Cisadane variety and its five mutants, namely OBS 18, OBS 208, OBS 297, OBS 306, and OBS 330. The third group was mutants line 627-10-3 and its mutants, namely 1063, 1066, 1067, 1076, and 1090. Isoenzymes extracts of the rice leaves were fractionated using polyacrylamide gel disc electrophoresis. The pattern of acid phosphate isoenzyme shows the specific character of rice mutants susceptible to brown plant hopper biotype 1. The gene(s) controlling malic enzyme in Cisadane's mutants is (are) estimated more resistant toward gamma irradiation than gene(s) responsible for controlling the other enzymes. Generally, the isoenzymes zymograms show that gene(s) controlling the mutants enzyme have undergone mutation. This case is shown by the changes of Rm value, as well as the amount and intensity of mutants bands. (authors). 7 refs., 7 figs

  9. Incorporation of carbohydrate residues into peroxidase isoenzymes in horseradish roots.

    Science.gov (United States)

    Lew, J Y; Shannon, L M

    1973-11-01

    Sliced root tissue of the horseradish plant (Armoracia rusticana), when incubated with mannose-U-(14)C, incorporated radioactivity into peroxidase isoenzymes. Over 90% of the radioactivity in the highly purified peroxidase isoenzymes was present in the neutral sugar residues of the molecule, i.e. fucose, arabinose, xylose, mannose. When the root slices were incubated simultaneously with leucine-4,5-(3)H and mannose-U-(14)C, cycloheximide strongly inhibited leucine incorporation into the peptide portion of peroxidase isoenzymes but had little effect on the incorporation of (14)C into the neutral sugars. These results indicated that synthesis of the peptide portion of peroxidase was completed before the monosaccharide residues were attached to the molecule. This temporal relationship between the synthesis of protein and the attachment of carbohydrate residues in the plant glycoprotein, horseradish peroxidase, appears to be similar to that reported for glycoprotein biosynthesis in many mammalian systems.

  10. Lactate dehydrogenase (LDH isoenzymes patterns in ocular tumours

    Directory of Open Access Journals (Sweden)

    Singh Rajendra

    1991-01-01

    Full Text Available Estimation of lactate dehydrogenase (LDH isoenzymes in the serum and aqueous humor was carried out in 15 cases of benign ocular tumour, 15 cases of malignant tumor and 15 normal cases. Cases of both sexes aged between 1 year and 75 years were included. LDH, isoenzymes specially LDH4 and LDH5 are higher and LDH1 and LDH2 lower in sera of patients with malignant tumor specially retinoblastoma as compared to benign tumor cases and control cases. LDH isoenzymes in aqueous humor are significantly higher and show a characteristic pattern in retinoblastoma cases, the concentration was presumably too low in the control, malignant tumor other than retinoblastoma and benign tumor cases as its fractionation was not possible.

  11. Serum creatine kinase isoenzymes in children with osteogenesis imperfecta.

    Science.gov (United States)

    D'Eufemia, P; Finocchiaro, R; Zambrano, A; Lodato, V; Celli, L; Finocchiaro, S; Persiani, P; Turchetti, A; Celli, M

    2017-01-01

    This study evaluates serum creatine kinase isoenzyme activity in children with osteogenesis imperfecta to determine its usefulness as a biochemical marker during treatment with bisphosphonate. The changes of creatine kinase (CK) isoenzyme activity during and after discontinuation therapy were observed. These results could be useful in addressing over-treatment risk prevention. The brain isoenzyme of creatine kinase (CKbb) is highly expressed in mature osteoclasts during osteoclastogenesis, thus plays an important role in bone resorption. We previously identified high serum CKbb levels in 18 children with osteogenesis imperfect (OI) type 1 treated for 1 year with bisphosphonate (neridronate). In the present study, serum CK isoenzymes were evaluated in the same children with continuous versus discontinued neridronate treatment over a further 2-year follow-up period. This study included 18 children with OI type 1, 12 with continued (group A) and 6 with ceased (group B) neridronate treatment. Auxological data, serum biochemical markers of bone metabolism, bone mineral density z-score, and serum total CK and isoenzyme activities were determined in both groups. Serum CKbb was progressively and significantly increased in group A (p < 0.004) but rapidly decreased to undetectable levels in group B. In both groups, the cardiac muscle creatine kinase isoenzyme (CKmb) showed a marked decrease, while serum C-terminal telopeptide (CTx) levels were almost unchanged. This study provides evidence of the cumulative effect of neridronate administration in increasing serum CKbb levels and the reversible effect after its discontinuation. This approach could be employed for verifying the usefulness of serum CKbb as a biochemical marker in patients receiving prolonged bisphosphonate treatment. Moreover, the decreased serum CKmb levels suggest a systemic effect of these drugs.

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

    Directory of Open Access Journals (Sweden)

    Babić Olivera B.

    2013-01-01

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

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

    Science.gov (United States)

    Coburn, S P; Schaltenbrand, W E

    1978-05-01

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

  14. The Alcohol Dehydrogenase Isoenzyme as a Potential Marker of Pancreatitis.

    Science.gov (United States)

    Jelski, Wojciech; Piechota, Joanna; Orywal, Karolina; Szmitkowski, Maciej

    2018-05-01

    Human pancreas parenchyma contains various alcohol dehydrogenase (ADH) isoenzymes and also possesses aldehyde dehydrogenase (ALDH) activity. The altered activities of ADH and ALDH in damaged pancreatic tissue in the course of pancreatitis are reflected in the human serum. The aim of this study was to investigate a potential role of ADH and ALDH as markers for acute (AP) and chronic pancreatitis (CP). Serum samples were collected for routine biochemical investigations from 75 patients suffering from acute pancreatitis and 70 patients with chronic pancreatitis. Fluorometric methods were used to measure the activity of class I and II ADH and ALDH activity. The total ADH activity and activity of class III and IV isoenzymes were measured by a photometric method. There was a significant increase in the activity of ADH III isoenzyme (15.06 mU/l and 14.62 mU/l vs. 11.82 mU/l; ppancreatitis or chronic pancreatitis compared to the control. The diagnostic sensitivity for ADH III was about 84%, specificity was 92 %, positive and negative predictive values were 93% and 87% respectively in acute pancreatitis. Area under the Receiver Operating Curve (ROC) curve for ADH III in AP and CP was 0.88 and 0.86 respectively. ADH III has a potential role as a marker of acute and chronic pancreatitis. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

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

    Science.gov (United States)

    Story, Sandra; Brigmon, Robin L

    2017-03-01

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

  16. Research on Phosphatases of Belladona Leaves and Their Purification

    Directory of Open Access Journals (Sweden)

    M. Khorsand

    1957-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Zhongyuan Ren

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

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

    DEFF Research Database (Denmark)

    Joner, E.J.; Jakobsen, I.

    1995-01-01

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

  19. Glutathione s-transferase isoenzymes in relation to their role in detoxification of xenobiotics

    NARCIS (Netherlands)

    Vos, R.M.E.

    1989-01-01

    The glutathione S-transferases (GST) are a family of isoenzymes serving a major part in the biotransformation of many reactive compounds. The isoenzymes from rat, man and mouse are divided into three classes, alpha, mu and pi, on the basis of similar structural and enzymatic

  20. The action of cobra venom phospholipase A2 isoenzymes towards intact human erythrocytes

    NARCIS (Netherlands)

    Roelofsen, B.; Sibenius Trip, M.; Verheij, H.M.; Zevenbergen, J.L.

    1980-01-01

    1. 1. Cobra venom phospholipase A2 from three different sources has been fractionated into different isoenzymes by DEAE ion-exchange chromatography. 2. 2. Treatment of intact human erythrocytes with the various isoenzymes revealed significant differences in the degree of phosphatidylcholine

  1. 21 CFR 862.1215 - Creatine phosphokinase/creatine kinase or isoenzymes test system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Creatine phosphokinase/creatine kinase or... Clinical Chemistry Test Systems § 862.1215 Creatine phosphokinase/creatine kinase or isoenzymes test system. (a) Identification. A creatine phosphokinase/creatine kinase or isoenzymes test system is a device...

  2. Creatine kinase radioimmunoassay and isoenzyme electrophoresis compared in the diagnosis of acute myocardial infarction

    International Nuclear Information System (INIS)

    Homburger, H.A.; Jacob, G.L.

    1980-01-01

    We compared, in 116 patients, the relative usefulness of results of tests for creatine kinase B-isoenzymes, as measured by radioimmunoassay, and the MB isoenzyme, as measured by electrophoresis, in diagnosis of acute myocardial infarction. The radioimmunoassay was specific for isoenzymes of creatine kinase containing the B subunit. All patients with acute transmural infarcts had positive test results by both techniques, but concentrations of B-isoenzymes were more frequently above normal than were MB bands in the case of patients with acute subendocardial infarcts and in the case of all patients with acute myocardial infarcts from whom sera were collected more than 24 h after onset of chest pain. Concentrations of B-isoenzymes also were increased, even when MB bands were not electrophoretically detectable in specimens from several patients without documented acute myocardial infarcts. These abnormal results presumably were caused by increased concentrations of the BB isoenzyme in serum. Accordingly, an increased concentration of B-isoenzymes had less diagnostic specificity and predictive value for acute myocardial infarction than did a detectable MB band. Results of isoenzyme electrophoresis were more reliable for establishing this diagnosis, but the results of radioimmunoassay were more reliable for excluding it in patients with chest pain as the primary symptom

  3. Cdc14 phosphatase

    DEFF Research Database (Denmark)

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

    2016-01-01

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

  4. Separation and analysis of arylsulfatase isoenzymes in body fluids of man

    International Nuclear Information System (INIS)

    Bostick, W.D.; Dinsmore, S.R.; Mrochek, J.E.; Waalkes, P.

    1978-01-01

    The isoenzymes were separated by anion exchange chromatography using polystyrene mini-columns packed with swollen fibrous DEAE-cellulose. The B isoenzyme was eluted in the void volume, and the A isoenzyme and the anionic inhibitors were retarded. Silver appears to be a more selective inhibitor for discriminating between sulfatase isoenzymes in mixtures, and may be used in dialyzed urine samples. Optical measurements were used to determine arylsulfatase activity in the column eluate fractions by monitoring the p-nitrocatechol reaction color at 510 nm. ASB isoenzyme activity has been found frequently to be above normal in the sera of patients with colorectal cancer and malignant melanoma. So the enzyme may be suitable as a biochemical marker used as a noninvasive measure of malignant activity. Although arylsulfatase activities were generally greater in urine samples than in serum samples, variable urinary excretion of enzyme and greatly increased concentration of endogenous inhibitors were noted. 17 figures; 7 tables; 40 references

  5. Changes in arginase isoenzymes pattern in human hepatocellular carcinoma

    International Nuclear Information System (INIS)

    Chrzanowska, Alicja; Krawczyk, Marek; Baranczyk-Kuzma, Anna

    2008-01-01

    Hepatocellular carcinoma (HCC) is one of the most common tumors worldwide affecting preferentially patients with liver cirrhosis. The studies were performed on tissues obtained during surgery from 50 patients with HCC, 40 with liver cirrhosis and 40 control livers. It was found that arginase activity in HCC was nearly 5- and 15-fold lower than in cirrhotic and normal livers, respectively. Isoenzymes AI (so-called liver-type arginase) and AII (extrahepatic arginase) were identified by Western blotting in all studied tissues, however the amount of AI, as well as the expression of AI-mRNA were lower in HCC, in comparison with normal liver, and those of AII were significantly higher. Since HCC is arginine-dependent, and arginine is essential for cells growth, the decrease of AI may preserve this amino acid within tumor cells. Concurrently, the rise of AII can increase the level of polyamines, compounds crucial for cells proliferation. Thus, both arginase isoenzymes seem to participate in liver cancerogenesis.

  6. Improved radioimmunoassay for creatine kinase isoenzymes in plasma

    International Nuclear Information System (INIS)

    Ritter, C.S.; Mumm, S.R.; Roberts, R.

    1981-01-01

    We describe convenient and relatively rapid procedures for purifying creatine kinase isoenzymes MM, BB, and MB, and their use in an improved radioimmunoassay for creatine kinase isoenzymes in plasma. The modifications include use of: (a) BB with a specific activity of 400 kU/G, which can be labeled with a specific radioactivity of 20 Ci/g; (b) albumin-free purified MB as inhibitor; (c) antiserum to MB creatine kinase; and (d) a second-antibody technique that necessitates only a 15-min incubation. The radioimmunoassay for MB has a sensitivity of 0.2 μg/L (80 mU/L) and a CV of <5%. Plasma MB average 22 (SD 12) μg/L in 200 normal subjects; 24 (SD 12) μg/L in 200 patients with chest pain without infarction; and 23 (SD 7) μg/L in 43 patients with renal disease, whether measured before or after dialysis. Peak values for plasma MB averaged 191 (SD 86) μg/L in 325 patients with documented myocardial infarction; BB was negligible. Extensive clinical experience indicates the radioimmunoassay to be suitably rapid, highly sensitive, and reliable as a diagnostic assay for MB on plasma

  7. The Effect of Direct Current Transthoracic Countershock on Human Myocardial Cells Evidenced by Creatine Kinase and Lactic Dehydrogenase Isoenzymes.

    Science.gov (United States)

    1986-05-01

    however, fractionation of these enzymes will identify their specific source. Plasma levels of CK isoenzymes (CKMB) and LDH isoenzymes ( LDHI ) are most...damage--inferred by isoenzyme levels of CKMB and/or LDHI in the serum above normal levels (see definitions of creatine kinase and lactic dehydrogenase

  8. Alginate-hydroxypropylcellulose hydrogel microbeads for alkaline phosphatase encapsulation.

    Science.gov (United States)

    Karewicz, A; Zasada, K; Bielska, D; Douglas, T E L; Jansen, J A; Leeuwenburgh, S C G; Nowakowska, M

    2014-01-01

    There is a growing interest in using proteins as therapeutics agents. Unfortunately, they suffer from limited stability and bioavailability. We aimed to develop a new delivery system for proteins. ALP, a model protein, was successfully encapsulated in the physically cross-linked sodium alginate/hydroxypropylcellulose (ALG-HPC) hydrogel microparticles. The obtained objects had regular, spherical shape and a diameter of ∼4 µm, as confirmed by optical microscopy and SEM analysis. The properties of the obtained microbeads could be controlled by temperature and additional coating or crosslinking procedures. The slow, sustained release of ALP in its active form with no initial burst effect was observed for chitosan-coated microspheres at pH = 7.4 and 37 °C. Activity of ALP released from ALG/HPC microspheres was confirmed by the occurance of effectively induced mineralization. SEM and AFM images revealed formation of the interpenetrated three-dimensional network of mineral, originating from the microbeads' surfaces. FTIR and XRD analyses confirmed formation of hydroxyapatite.

  9. Alginate-hydroxypropylcellulose hydrogel microbeads for alkaline phosphatase encapsulation

    NARCIS (Netherlands)

    Karewicz, A.; Zasada, K.; Bielska, D.; Douglas, T.E.L.; Jansen, J.A.; Leeuwenburgh, S.C.G.; Nowakowska, M.

    2014-01-01

    There is a growing interest in using proteins as therapeutics agents. Unfortunately, they suffer from limited stability and bioavailability. We aimed to develop a new delivery system for proteins. ALP, a model protein, was successfully encapsulated in the physically cross-linked sodium

  10. Lipid accumulation and alkaline phosphatase activity in human ...

    African Journals Online (AJOL)

    2012-10-03

    Oct 3, 2012 ... can be influenced by fat depot origin in in vitro models. For example, preadipocytes ... University of Pretoria and Waterfall City Hospital, Johannesburg4 ..... system, the identities of which are currently unknown. The ability of ...

  11. Alkaline Phosphatase: MedlinePlus Lab Test Information

    Science.gov (United States)

    ... www.liverfoundation.org/abouttheliver/info/liverfunctiontests/ Centers for Disease Control and Prevention [Internet]. Atlanta: U.S. Department of Health and Human Services; Epstein-Barr Virus and Infectious Mononucleosis; [updated 2016 Sep 14; ...

  12. Kinetic Studies of Alkaline Phosphatase from the Liver of Agama ...

    African Journals Online (AJOL)

    group of catalytic proteins (enzymes) that have the ability to ... was determined by the method of Biuret (Gornall et al.,. 1949) using ... The result indicate that the enzyme has an optimum. pH of 9.9. ... Total protein (mg) Total activity. (x10-4).

  13. Activation of Rat Intestinal Alkaline Phosphatase by Taurine May be ...

    African Journals Online (AJOL)

    Dr. K.J. Umar

    kinetic analysis of L-phenylalanine was also investigated at 60 mM. ... six fatty acids. This part is the toxic .... The key experimental results obtained with the crude ALP extract ..... pre-treatment with rutin, an anti-inflammatory agent like taurine ...

  14. Kinetic studies of alkaline phosphatase extracted from rabbit (Lepus ...

    African Journals Online (AJOL)

    user

    activity, and the kinetic constants-maximum enzyme velocity (Vmax) and Michealis-Menten constant (Km) were evaluated. ... the enzyme a readily available parameter for diagnostic and research .... procedure while treatment means were separated by the least .... mammalian enzymes are responsible for this increase in ...

  15. Native myosin from adult rabbit skeletal muscle: isoenzymes and states of aggregation.

    Science.gov (United States)

    Morel, J E; D'hahan, N; Taouil, K; Francin, M; Aguilar, A; Dalbiez, J P; Merah, Z; Grussaute, H; Hilbert, B; Ollagnon, F; Selva, G; Piot, F

    1998-04-21

    The globular heads of skeletal muscle myosin have been shown to exist as isoenzymes S1 (A1) and S1 (A2), and there are also isoforms of the heavy chains. Using capillary electrophoresis, we found two dominant isoenzymes of the whole native myosin molecule, in agreement with what has previously been found by various techniques for native and nondenatured myosin from adult rabbits. Findings about possible states of aggregation of myosin and its heads are contradictory. By analytical ultracentrifugation, we confirmed the existence of a tail-tail dimer. By laser light scattering, we found a head-head dimer in the presence of MgATP. Capillary electrophoresis coupled with analytical ultracentrifugation and laser light scattering led us to refine these results. We found tail-tail dimers in a conventional buffer. We found tail-tail and head-head dimers in the presence of 0.5 mM MgATP and pure head-head dimers in the presence of 6 mM MgATP. All the dimers were homodimers. Naming the dominant isoenzymes of myosin a and b, we observed tail-tail dimers with isoenzyme a (TaTa) and with isoenzyme b (TbTb) and also head-head dimers with isoenzyme a (HaHa) and with isoenzyme b (HbHb).

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

    Science.gov (United States)

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

    1985-03-01

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

  17. Activity and isoenzyme composition of lipoxygenase of X-irradiated pea seeds

    International Nuclear Information System (INIS)

    Chepurenko, N.V.; Borisova, I.G.; Budnitskaya, E.V.

    1977-01-01

    Action of X-radiation, in vitro, on the isoenzyme composition and activity of lipoxygenase (E. C. 1.13.11.12), that catalyzes the process of peroxidation of unsaturated higher fatty acids, has been studied. Radiosensitivity of lipoxygenase isoenzymes has been found to vary. After doses above 10 krad, the postirradiation inactivation of lipoxygenase was due to a changed ratio of isoenzymes and their depolymerization; and it was accompanied by the appearance of a new protein fragment that had no specific lipoxygenase activity

  18. Ivermectin resistant and susceptible third-stage larvae of Haemonchus contortus: cholinesterase and phosphatase activities

    Directory of Open Access Journals (Sweden)

    Consuelo Giménez-Pardo

    2004-03-01

    Full Text Available Cholinesterase and acid phosphatase (AP, but not alkaline phosphatase activities, were detected in cytosolic and membrane-bound fractions of ivermectin resistant and susceptible Haemonchus contortus infective-stage larvae. Some differences in acetylcholinesterase activity of cytosolic fractions and in the AP activity of these fractions as well as in the response to AP inhibitors by membrane-bound fractions were detected. Data are discussed.

  19. Substrate specificity and pH dependence of homogeneous wheat germ acid phosphatase.

    Science.gov (United States)

    Van Etten, R L; Waymack, P P

    1991-08-01

    The broad substrate specificity of a homogeneous isoenzyme of wheat germ acid phosphatase (WGAP) was extensively investigated by chromatographic, electrophoretic, NMR, and kinetic procedures. WGAP exhibited no divalent metal ion requirement and was unaffected upon incubation with EDTA or o-phenanthroline. A comparison of two catalytically homogeneous isoenzymes revealed little difference in substrate specificity. The specificity of WGAP was established by determining the Michaelis constants for a wide variety of substrates. p-Nitrophenyl phosphate, pyrophosphate, tripolyphosphate, and ATP were preferred substrates while lesser activities were seen toward sugar phosphates, trimetaphosphate, phosphoproteins, and (much less) phosphodiesters. An extensive table of Km and Vmax values is given. The pathway for the hydrolysis of trimetaphosphate was examined by colorimetric and 31P NMR methods and it was found that linear tripolyphosphate is not a free intermediate in the enzymatic reaction. In contrast to literature reports, homogeneous wheat germ acid phosphatase exhibits no measurable carboxylesterase activity, nor does it hydrolyze phenyl phosphonothioate esters or phytic acid at significant rates.

  20. Research on Phosphatases of Belladona Leaves and Their Purification (Part 1

    Directory of Open Access Journals (Sweden)

    M. Khorsand

    1956-12-01

    Full Text Available Belladona leaves as well as all other studied leaves contains two distinct phosphatase fractions belonging respectively to types II and IIIi the major parts of these enzymes is extraetible by water. It was not possible to extract the non soluble fraction which is solidly retained by the cellular constituents. Phosphatase II does not differ from other phosphatnses of the same type. Whereas phosphatase III is distinetely different from enzymes of the same type of vegetal or animal origins. It is activated by bivalent metallic ions which are specific activators of the alkaline phcspbatnses: Mg-Zn-Ni and Co.

  1. Radioprotective effect of Panax ginseng on the phosphatases and lipid peroxidation level in testes of Swiss albino mice

    Energy Technology Data Exchange (ETDEWEB)

    Kumar M.; Sharma M.K.; Saxena P.S.; Kumar A. [Rajasthan Univ., Jaipur (India)

    2003-03-01

    The Panax ginseng has been used as traditional medicine for past several years among oriental people. The present investigation has been made to assess the radioprotective efficacy of ginseng root extract in the testicular enzymes of Swiss albino mice. The Swiss albino mice were divided into different groups. Ginseng treated group: The animals were administered 10 mg/kg body weight ginseng root extract intraperitoneal (i.p.). Radiation treated group: The animals were exposed to 8 Gy gamma radiation at the dose rate of 1.69 Gy/min at the distance of 80 cm. Combination group: Animals were administered ginseng extract continuously for 4 d and on 4th day they were irradiated to 8 Gy gamma radiation after 30 min of extract administration. The animals from above groups were autopsied on day 1, 3, 7, 14 and 30. Biochemical estimations of acid and alkaline phosphatases and Lipid peroxidation (LPO) in testes were done. In ginseng treated group acid and alkaline phosphatases activity and LPO level did not show any significant alteration. In irradiated animals there was a significant increase in acid phosphatase activity and LPO level. However, significant decline in alkaline phosphatase activity was observed. The treatment of ginseng before irradiation causes significant decrease in acid phosphatase and LPO level and significant increase in alkaline phosphatase activity. One of the cause of radiation damage is lipid peroxidation. Due to lipid peroxidation, lysosomal membrane permeability alters and thus results in release of hydrolytic enzymes. So, an increase in acid phosphatase was noticed after radiation treatment. The alkaline phosphatase activity is associated with membrane permeability and different stages of spermatogenesis. Due to membrane damage and depletion of germ cells of testes after irradiation the enzyme activity was decreased. Ginseng markedly inhibits lipid peroxidation. It acts in indirect fashion to protect radical processes by inhibition of initiation of

  2. Radioprotective effect of Panax ginseng on the phosphatases and lipid peroxidation level in testes of Swiss albino mice

    International Nuclear Information System (INIS)

    Kumar, M.; Sharma, M.K.; Saxena, P.S.; Kumar, A.

    2003-01-01

    The Panax ginseng has been used as traditional medicine for past several years among oriental people. The present investigation has been made to assess the radioprotective efficacy of ginseng root extract in the testicular enzymes of Swiss albino mice. The Swiss albino mice were divided into different groups. Ginseng treated group: The animals were administered 10 mg/kg body weight ginseng root extract intraperitoneal (i.p.). Radiation treated group: The animals were exposed to 8 Gy gamma radiation at the dose rate of 1.69 Gy/min at the distance of 80 cm. Combination group: Animals were administered ginseng extract continuously for 4 d and on 4th day they were irradiated to 8 Gy gamma radiation after 30 min of extract administration. The animals from above groups were autopsied on day 1, 3, 7, 14 and 30. Biochemical estimations of acid and alkaline phosphatases and Lipid peroxidation (LPO) in testes were done. In ginseng treated group acid and alkaline phosphatases activity and LPO level did not show any significant alteration. In irradiated animals there was a significant increase in acid phosphatase activity and LPO level. However, significant decline in alkaline phosphatase activity was observed. The treatment of ginseng before irradiation causes significant decrease in acid phosphatase and LPO level and significant increase in alkaline phosphatase activity. One of the cause of radiation damage is lipid peroxidation. Due to lipid peroxidation, lysosomal membrane permeability alters and thus results in release of hydrolytic enzymes. So, an increase in acid phosphatase was noticed after radiation treatment. The alkaline phosphatase activity is associated with membrane permeability and different stages of spermatogenesis. Due to membrane damage and depletion of germ cells of testes after irradiation the enzyme activity was decreased. Ginseng markedly inhibits lipid peroxidation. It acts in indirect fashion to protect radical processes by inhibition of initiation of

  3. Identification of 5α-reductase isoenzymes in canine skin.

    Science.gov (United States)

    Bernardi de Souza, Lucilene; Paradis, Manon; Zamberlam, Gustavo; Benoit-Biancamano, Marie-Odile; Price, Christopher

    2015-10-01

    Alopecia X in dogs is a noninflammatory alopecia that may be caused by a hormonal dysfunction. It may be similar to androgenic alopecia in men that is caused by the effect of dihydrotestosterone (DHT). The 5α-reductase isoenzymes, 5αR1 and 5αR2, and a recently described 5αR3, are responsible for the conversion of testosterone into DHT. However, which 5α-reductases are present in canine skin has not yet been described. The main objective of this study was to determine the pattern of expression of 5α-reductase genes in canine skin. Skin biopsies were obtained from healthy, intact young-mature beagles (three males, four females) at three anatomical sites normally affected by alopecia X (dorsal neck, back of thighs and base of tail) and two sites generally unaffected (dorsal head and ventral thorax). Prostate samples (n = 3) were collected as positive controls for 5α-reductase mRNA abundance measurement by real-time PCR. We detected mRNA encoding 5αR1 and 5αR3 but not 5αR2. There were no significant differences in 5αR1 and 5αR3 mRNA levels between the different anatomical sites, irrespective of gender (P > 0.05). Moreover, the mean mRNA abundance in each anatomical site did not differ between males and females (P > 0.05). To the best of the authors' knowledge, this is the first study demonstrating the expression of 5α-reductases in canine skin and the expression of 5αR3 in this tissue. These results may help to elucidate the pathogenesis of alopecia X and to determine more appropriate treatments for this disorder. © 2015 ESVD and ACVD.

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

    Directory of Open Access Journals (Sweden)

    Sosanka Protim SANDILYA

    2014-12-01

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

  5. Probing protein phosphatase substrate binding

    DEFF Research Database (Denmark)

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

    2012-01-01

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

  6. Protein tyrosine phosphatases: regulatory mechanisms.

    NARCIS (Netherlands)

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

    2008-01-01

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

  7. The effect of gamma irradiation on the formation of alpha-amylase isoenzymes in germinating wheat

    International Nuclear Information System (INIS)

    Machaiah, J.P.; Vakil, U.K.

    1979-01-01

    The biosynthesis of alpha-amylase during seedling growth commenced after a prolonged lag-period in wheat (cv. Vijay), irradiated at a high dose (200 krad). Also, a different requirement for exogenous gibberellins (GA) to stimulate the enzyme synthesis was noted in control and irradiated seeds. Further, the developmental patterns of three major isoenzymes of alpha-amylase (designated as α 1 , α 2 - and α 3 ) during germination were different. It was observed that α 1 -isoenzyme which appeared on the fourth day of germination of control seeds, was delayed in its development and was undetectable up to 4 days in samples irradiated with 200 krad. However, α 1 -isoenzyme appeared after 6 days or after 4 days in GA-treated samples in germinating seeds exposed to a high dose. These results suggested that two systems differing in their radiosensitivity and response to GA application were operating in germinating wheat for the synthesis of functional alpha-amylase molecules. (author)

  8. [Investigations on the distribution of serum LDH isoenzymes of patients with carcinoma laryngis (author's transl)].

    Science.gov (United States)

    T-Tomity, I; Takács, O

    1979-12-01

    The distribution of lactate dehydrogenase (LDH) isoenzymes in healthy blood donors and in patients suffering histological identified tumor (neoplasms) laryngis was investigated. The values of 110 healthy persons (controls) proved to be comparable with the average data described in literature. The LDH distribution pattern of 90 tumour affected persons showed significant differences comparing with controls. The alteration appears as absolute increase in LDH-1 (H4) isoenzyme parallel with progressive decrease in the hybrid enzyme rations containing M subunits (LDH-2, LDH-3, LDH-4), and the LDH-5 consisting of four M sununits was undetectable. The conclusions drawn from our investigations seem to be in agreement with Warburg's conception, supposing that in malignant tumours the aerob glycolysis increases. The determination of LDH isoenzyme distributions for diagnostic purposes in clinical practice is recommended.

  9. Glucose-6-phosphatase deficiency

    Directory of Open Access Journals (Sweden)

    Labrune Philippe

    2011-05-01

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

  10. Measurements of amylase isoenzymes in sera and saliva of patients after radiotherapy because of larynx carcinoma

    International Nuclear Information System (INIS)

    Tomasik, A.; Tarnawski, R.; Wierzynski, J.; Tarnawski, R.

    1994-01-01

    Serum and salivary alpha-amylase were measured for controls and patients with laryngeal carcinoma before and after localized irradiation including salivary glands. A significant increase in amylasemia was observed after irradiation. Alpha-amylase activity in saliva was decreased after irradiation but differences were not statistically significant due to the significant decrease of protein in saliva of irradiated group. An increase of salivary isoenzyme S activity was observed while pancreatic isoenzyme activity was not altered. This method allows easy differentiation of hyperamylasemia due to irradiation of parothyroid gland and disorders of the pancreas. Alpha-amylase activity measurements may detect metabolic changes in salivary glands after irradiation. (author)

  11. Radioimmunoassay of creatine kinase-B isoenzyme in human sera: results in patients with acute myocardial infarction

    International Nuclear Information System (INIS)

    Willerson, J.T.; Stone, M.J.; Ting, R.; Mukherjee, A.; Gomez-Sanchez, C.E.; Lewis, P.; Hersh, L.B.

    1977-01-01

    A radiommunoassay was developed to measure serum levels of the B isoenzyme of creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) (CPK) in order to evaluate the time course and frequency of MB isoenzyme elevation in patients with acute myocardial infarction. The method can identify as little as 0.2 ng of the B portion of the CPK-MB isoenzyme, does not significantly crossreact with CPK-MM isoenzyme, and is not affected by storage of serum at -20 0 . CPK isoenzyme containing B subunits was detected in 48 out of 51 sera from normal adults; serum levels in these individuals ranged between 1.2 and 12.5 ng/ml[mean +- SEM was 2.7 +- 0.30 ng/ml]. The mean serum level of CPK-B isoenzyme in a pool of sera obtained from 100 normal subjects was 2.9 +- 0.35 ng/ml; two patients with rhabdomyolysis that were studied had serum CPK-B isoenzyme levels of 2.5 and 3.5 ng/ml, respectively. In contrast, serum levels of the CPK-B isoenzyme were markedly elevated in sera from 18 patients with acute myocardial infarcts when obtained within 12 hr after hospital admission; the mean +- SEM concentration was 56 +- 7.8 ng/ml. We performed serial determinations on 14 patients with acute myocardial infarcts and demonstrated that maximal serum CPK-B levels occurred within the first 12 hr after admission and were lower thereafter. The serum concentration of B-containing CPK isoenzyme in 19 additional patients admitted with chest pain but without acute myocardial infarction was 3.4 +- 0.50 ng/ml. Thus, radioimmunoassay measurement of CPK-B isoenzyme appears to be a useful and sensitive test for the detection of acute myocardial infarcts in patients

  12. Parallel variation in isoenzyme and nitrogen fixation markers in a Rhizobium population

    DEFF Research Database (Denmark)

    Engvild, K.C.; Jensen, E.S.; Skøt, L.

    1990-01-01

    into the same 13 different groups. The largest group was represented 7 times according to isoenzymes and 8 times according to RFLP. This fixed non-random association of plasmid and chromosomal genotypes is consistent with a clonal population structure; it indicates limited exchange of plasmids under natural...

  13. Oronasal leishmaniasis caused by a parasite with an unusual isoenzyme profile

    DEFF Research Database (Denmark)

    Ibrahim, M.; Suliman, A.; Hashim, F.A.

    1997-01-01

    A 45-year-old Sudanese man from western Sudan presented with oronasal leishmaniasis of three years duration. He had no history of previous kala-azar or cutaneous leishmaniasis. The parasite isolated from the oral mucosa was characterized by isoenzymes using 12 enzymes and by polymerase chain...

  14. Phosphate-solubility and phosphatase activity in Gangetic alluvial soil as influenced by organophosphate insecticide residues.

    Science.gov (United States)

    Majumder, Shyam Prasad; Das, Amal Chandra

    2016-04-01

    An experiment was conducted under laboratory conditions to investigate the effect of four organophosphate insecticides, viz. monocrotophos, profenophos, quinalphos and triazophos at their field application rates (0.75, 1.0, 0.5 and 0.6 kg a.i.ha(-1), respectively), on the growth and activities of phosphate solubilizing microorganisms in relation to availability of insoluble phosphates in the Gangetic alluvial soil of West Bengal, India. The proliferation of phosphate solubilizing microorganisms was highly induced with profenophos (38.3%), while monocrotophos exerted maximum stimulation (20.8%) towards the solubility of insoluble phosphates in soil. The phosphatase activities of the soil (both acid phosphatase and alkaline phosphatase) were significantly increased due to the incorporation of the insecticides in general, and the augmentation was more pronounced with quinalphos (43.1%) followed by profenophos (27.6%) for acid phosphatase, and with monocrotophos (25.2%) followed by profenophos (16.1%) for alkaline phosphatase activity in soil. The total phosphorus was highly retained by triazophos (19.9%) followed by monocrotophos (16.5%), while incorporation of triazophos and quinalphos manifested greater availability of water soluble phosphorus in soil. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Phosphatases in Cancer : Shifting the balance

    NARCIS (Netherlands)

    E. Hoekstra (Elmer)

    2015-01-01

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

  16. Genetic structuring and differentiation of Echinococcus multilocularis in Slovakia assessed by sequencing and isoenzyme studies

    DEFF Research Database (Denmark)

    Snabel, V.; Miterpakova, M.; D'Amelio, S.

    2006-01-01

    Nucleotide sequencing of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene and isoenzyme analysis were used to survey the genetic variability in Echinococcus multilocularis populations from Slovakia. A sample of 12 isolates acquired from 10 different districts from red foxes exhibited......) in the CO1 fragment. These data, along with the recently gathered data from French isolates, are indicative of a genetically unique population occurring in Central and Western Europe. Electrophoretic examination of enzymes produced by 14 gene loci revealed intraspecific polymorphism only with the glucose...... between the species were obtained by isoenzyme analysis. Fixed genetic differences between the species were detected in the glucose-phosphate isomerase, esterase and aldolase systems, and partial differences were detected in four additional systems....

  17. Serum adenosine deaminase activity and its isoenzyme in patients treated for tuberculosis

    International Nuclear Information System (INIS)

    Rokayan, S.A

    2003-01-01

    Objective: Increased serum adenosine deaminase (ADA) activity, mainly associated with tuberculosis can also occur in a number of other diseases thus negatively affecting the diagnostic utility of ADA measurements in tuberculosis. The aim of the study was to determine whether or not the combined use of the activity of ADA, its isoenzymes and differential cell counts would provide a more efficient means of diagnosing tuberculosis than the use of ADA levels alone. Results: Data suggested significant (p 0.75) of ADA/sub 2/ADA was found to be better indicator of tuberculosis. Lymphocyte neutrophil ratio (L/N)> 0.69 gave additional benefit to increase the sensitivity and specificity for the use of ADA as marker in diagnosing tuberculosis. Conclusion: The combined use of activity of ADA, its isoenzymes and total and differential cell counts is a better indicator and gives better understanding to diagnose and evaluate tuberculosis and response to therapy. (author)

  18. Microcomputer Assisted Interpretative Reporting of Sequential Creatine Kinase (CK) and Lactate Dehydrogenase (LDH) Isoenzyme Determination

    Science.gov (United States)

    Talamo, Thomas S.; Losos, Frank J.; Mercer, Donald W.

    1984-01-01

    We have developed a microcomputer based system for interpretative reporting of creatine kinase (CK) and lactate dehydrogenase (LDH) isoenzyme studies. Patient demographic data and test results (total CK, CK-MB, LD-1, and LD-2) are entered manually through the keyboard. The test results are compared with normal range values and an interpretative report is generated. This report consists of all pertinent demographic information with a graphic display of up to 12 previous CK and LDH isoenzyme determinations. Diagnostic interpretative statements are printed beneath the graphic display following analysis of previously entered test results. The combination of graphic data display and interpretations based on analysis of up to 12 previous specimens provides useful and accurate information to the cardiologist.

  19. Prenatal diagnosis by isoenzymic differentiation of Treacher Collins' syndrome induced by retinoids in rats

    DEFF Research Database (Denmark)

    Granström, G; Kirkeby, S

    1990-01-01

    A series of branchial arch malformations was induced in 618 embryos from 72 pregnant rats by a single intraperitoneal injection of 10 mg/kg etretinate at 8.5 days of gestation. The litters developed several malformations, including microtia, low set and dorsally placed outer ears, defective middle...... branchial arches is accompanied by a pathological differentiation pattern that can be traced by determinations of isoenzymes in the branchial arches as well as in amniotic fluid....

  20. Does cytochrome P450 liver isoenzyme induction increase the risk of liver toxicity after paracetamol overdose?

    Directory of Open Access Journals (Sweden)

    Kalsi SS

    2011-10-01

    Full Text Available Sarbjeet S Kalsi1,2, David M Wood2–4, W Stephen Waring5, Paul I Dargan2–4 1Emergency Department, 2Clinical Toxicology, Guy's and St Thomas' NHS Foundation Trust, London; 3King's Health Partners, 4King's College London, London; 5York Teaching Hospital NHS Foundation Trust, York, UK Abstract: Paracetamol (acetaminophen, N-acetyl-p-aminophenol, 4-hydroxyacetanilide is the most common cause of acute liver failure in developed countries. There are a number of factors which potentially impact on the risk of an individual developing hepatotoxicity following an acute paracetamol overdose. These include the dose of paracetamol ingested, time to presentation, decreased liver glutathione, and induction of cytochrome P450 (CYP isoenzymes responsible for the metabolism of paracetamol to its toxic metabolite N-acetyl-p-benzoquinoneimine (NAPQI. In this paper, we review the currently published literature to determine whether induction of relevant CYP isoenzymes is a risk factor for hepatotoxicity in patients with acute paracetamol overdose. Animal and human in vitro studies have shown that the CYP isoenzyme responsible for the majority of human biotransformation of paracetamol to NAPQI is CYP2E1 at both therapeutic and toxic doses of paracetamol. Current UK treatment guidelines suggest that patients who use a number of drugs therapeutically should be treated as “high-risk” after paracetamol overdose. However, based on our review of the available literature, it appears that the only drugs for which there is evidence of the potential for an increased risk of hepatotoxicity associated with paracetamol overdose are phenobarbital, primidone, isoniazid, and perhaps St John's wort. There is no evidence that other drugs often quoted as increasing risk, such as carbamazepine, phenytoin, primidone, rifampicin, rifabutin, efavirenz, or nevirapine, should be considered risk factors for hepatotoxicity in patients presenting with acute paracetamol overdose. Keywords

  1. Uranium in alkaline rocks

    International Nuclear Information System (INIS)

    Murphy, M.; Wollenberg, H.; Strisower, B.; Bowman, H.; Flexser, S.; Carmichael, I.

    1978-04-01

    Geologic and geochemical criteria were developed for the occurrence of economic uranium deposits in alkaline igneous rocks. A literature search, a limited chemical analytical program, and visits to three prominent alkaline-rock localities (Ilimaussaq, Greenland; Pocos de Caldas, Brazil; and Powderhorn, Colorado) were made to establish criteria to determine if a site had some uranium resource potential. From the literature, four alkaline-intrusive occurrences of differing character were identified as type-localities for uranium mineralization, and the important aspects of these localities were described. These characteristics were used to categorize and evaluate U.S. occurrences. The literature search disclosed 69 U.S. sites, encompassing nepheline syenite, alkaline granite, and carbonatite. It was possible to compare two-thirds of these sites to the type localities. A ranking system identified ten of the sites as most likely to have uranium resource potential

  2. Uranium in alkaline rocks

    Energy Technology Data Exchange (ETDEWEB)

    Murphy, M.; Wollenberg, H.; Strisower, B.; Bowman, H.; Flexser, S.; Carmichael, I.

    1978-04-01

    Geologic and geochemical criteria were developed for the occurrence of economic uranium deposits in alkaline igneous rocks. A literature search, a limited chemical analytical program, and visits to three prominent alkaline-rock localities (Ilimaussaq, Greenland; Pocos de Caldas, Brazil; and Powderhorn, Colorado) were made to establish criteria to determine if a site had some uranium resource potential. From the literature, four alkaline-intrusive occurrences of differing character were identified as type-localities for uranium mineralization, and the important aspects of these localities were described. These characteristics were used to categorize and evaluate U.S. occurrences. The literature search disclosed 69 U.S. sites, encompassing nepheline syenite, alkaline granite, and carbonatite. It was possible to compare two-thirds of these sites to the type localities. A ranking system identified ten of the sites as most likely to have uranium resource potential.

  3. The expression of GST isoenzymes and p53 in non-small cell lung cancer.

    Directory of Open Access Journals (Sweden)

    MĂźzeyyen Ozhavzali

    2010-06-01

    Full Text Available This study investigated the immunohistochemical staining characteristics of glutathione-S-transferase alpha, pi, mu, theta and p53 in non-small cell lung carcinoma and normal lung tissue from 50 patients. The relationships between expressions of the Glutathione-S-transferase isoenzymes and some clinicopathological features were also examined. Expression of glutathione-S-transferase pi, mu, alpha, theta and p53 was assessed by immunohistochemistry for primary lung carcinomas of 50 patients from the Sanitarium Education and Research Hospital, Ankara lung cancer collection. The relationships between expression of the glutathione-S-transferase isoenzymes, p53 in normal and tumor tissue by Student T test and the clinicopathological data were also examined by Spearman Rank tests. When the normal and tumor tissue of these cases were compared according to their staining intensity and percentage of positive staining, glutathione-S-transferase alpha, pi, mu, theta expressions in tumor cells was significantly higher than normal cells (p<0.05. There was no significant difference in the expression of p53 between normal and tumor cells (p>0.05. When the immunohistochemical results of glutathione-S-transferase isoenzymes and p53 were correlated with the clinical parameters, there were no significant associations between glutathione-S-transferases and p53 expressions and tumor stage, tumor grade and smoking status (p>0.05.

  4. Extracellular phosphatases in a Mediterranean reservoir: seasonal, spatial and kinetic heterogeneity

    Czech Academy of Sciences Publication Activity Database

    Nedoma, Jiří; García, J.C.; Comerma, M.; Šimek, Karel; Armengol, J.

    2006-01-01

    Roč. 51, č. 7 (2006), s. 1264-1276 ISSN 0046-5070 R&D Projects: GA ČR(CZ) GA206/99/0028 Grant - others:SICST(ES) HID96-1374-CO2; ICST(ES) 1997SGR-122 Institutional research plan: CEZ:AV0Z60170517 Keywords : alkaline phosphatase * eutrophication * P limitation Subject RIV: DJ - Water Pollution ; Quality Impact factor: 2.502, year: 2006

  5. Alkaline earth metals

    International Nuclear Information System (INIS)

    Brown, Paul L.; Ekberg, Christian

    2016-01-01

    The beryllium ion has a relatively small ionic radius. As a consequence of this small size, its hydrolysis reactions begin to occur at a relatively low pH. To determine the stability and solubility constants, however, the Gibbs energy of the beryllium ion is required. In aqueous solution calcium, like the other alkaline earth metals, only exists as a divalent cation. The size of the alkaline earth cations increases with increasing atomic number, and the calcium ion is bigger than the magnesium ion. The hydrolysis of barium(II) is weaker than that of strontium(II) and also occurs in quite alkaline pH solutions, and similarly, only the species barium hydroxide has been detected. There is only a single experimental study on the hydrolysis of radium. As with the stability constant trend, it would be expected that the enthalpy of radium would be lower than that of barium due to the larger ionic radius.

  6. Advanced alkaline water electrolysis

    International Nuclear Information System (INIS)

    Marini, Stefania; Salvi, Paolo; Nelli, Paolo; Pesenti, Rachele; Villa, Marco; Berrettoni, Mario; Zangari, Giovanni; Kiros, Yohannes

    2012-01-01

    A short review on the fundamental and technological issues relevant to water electrolysis in alkaline and proton exchange membrane (PEM) devices is given. Due to price and limited availability of the platinum group metal (PGM) catalysts they currently employ, PEM electrolyzers have scant possibilities of being employed in large-scale hydrogen production. The importance and recent advancements in the development of catalysts without PGMs are poised to benefit more the field of alkaline electrolysis rather than that of PEM devices. This paper presents our original data which demonstrate that an advanced alkaline electrolyzer with performances rivaling those of PEM electrolyzers can be made without PGM and with catalysts of high stability and durability. Studies on the advantages/limitations of electrolyzers with different architectures do show how a judicious application of pressure differentials in a recirculating electrolyte scheme helps reduce mass transport limitations, increasing efficiency and power density.

  7. Expression and kinetic properties of a recombinant 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase isoenzyme of human liver.

    Science.gov (United States)

    Deyashiki, Y; Tamada, Y; Miyabe, Y; Nakanishi, M; Matsuura, K; Hara, A

    1995-08-01

    Human liver cytosol contains multiple forms of 3 alpha-hydroxysteroid dehydrogenase and dihydrodiol dehydrogenase with hydroxysteroid dehydrogenase activity, and multiple cDNAs for the enzymes have been cloned from human liver cDNA libraries. To understand the relationship of the multiple enzyme froms to the genes, a cDNA, which has been reported to code for an isoenzyme of human liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase, was expressed in Escherichia coli. The recombinant enzyme showed structural and functional properties almost identical to those of the isoenzyme purified from human liver. In addition, the recombinant isoenzyme efficiently reduced 5 alpha-dihydrotestosterone and 5 beta-dihydrocortisone, the known substrates of human liver 3 alpha-hydroxysteroid dehydrogenase and chlordecone reductase previously purified, which suggests that these human liver enzymes are identical. Furthermore, the steady-state kinetic data for NADP(+)-linked (S)-1-indanol oxidation by the recombinant isoenzyme were consistent with a sequential ordered mechanism in which NADP+ binds first. Phenolphthalein inhibited this isoenzyme much more potently than it did the other human liver dihydrodiol dehydrogenases, and was a competitive inhibitor (Ki = 20 nM) that bound to the enzyme-NADP+ complex.

  8. Protein kinase and phosphatase activities of thylakoid membranes

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  9. Upon Accounting for the Impact of Isoenzyme Loss, Gene Deletion Costs Anticorrelate with Their Evolutionary Rates.

    Directory of Open Access Journals (Sweden)

    Christopher Jacobs

    Full Text Available System-level metabolic network models enable the computation of growth and metabolic phenotypes from an organism's genome. In particular, flux balance approaches have been used to estimate the contribution of individual metabolic genes to organismal fitness, offering the opportunity to test whether such contributions carry information about the evolutionary pressure on the corresponding genes. Previous failure to identify the expected negative correlation between such computed gene-loss cost and sequence-derived evolutionary rates in Saccharomyces cerevisiae has been ascribed to a real biological gap between a gene's fitness contribution to an organism "here and now" and the same gene's historical importance as evidenced by its accumulated mutations over millions of years of evolution. Here we show that this negative correlation does exist, and can be exposed by revisiting a broadly employed assumption of flux balance models. In particular, we introduce a new metric that we call "function-loss cost", which estimates the cost of a gene loss event as the total potential functional impairment caused by that loss. This new metric displays significant negative correlation with evolutionary rate, across several thousand minimal environments. We demonstrate that the improvement gained using function-loss cost over gene-loss cost is explained by replacing the base assumption that isoenzymes provide unlimited capacity for backup with the assumption that isoenzymes are completely non-redundant. We further show that this change of the assumption regarding isoenzymes increases the recall of epistatic interactions predicted by the flux balance model at the cost of a reduction in the precision of the predictions. In addition to suggesting that the gene-to-reaction mapping in genome-scale flux balance models should be used with caution, our analysis provides new evidence that evolutionary gene importance captures much more than strict essentiality.

  10. Upon Accounting for the Impact of Isoenzyme Loss, Gene Deletion Costs Anticorrelate with Their Evolutionary Rates.

    Science.gov (United States)

    Jacobs, Christopher; Lambourne, Luke; Xia, Yu; Segrè, Daniel

    2017-01-01

    System-level metabolic network models enable the computation of growth and metabolic phenotypes from an organism's genome. In particular, flux balance approaches have been used to estimate the contribution of individual metabolic genes to organismal fitness, offering the opportunity to test whether such contributions carry information about the evolutionary pressure on the corresponding genes. Previous failure to identify the expected negative correlation between such computed gene-loss cost and sequence-derived evolutionary rates in Saccharomyces cerevisiae has been ascribed to a real biological gap between a gene's fitness contribution to an organism "here and now" and the same gene's historical importance as evidenced by its accumulated mutations over millions of years of evolution. Here we show that this negative correlation does exist, and can be exposed by revisiting a broadly employed assumption of flux balance models. In particular, we introduce a new metric that we call "function-loss cost", which estimates the cost of a gene loss event as the total potential functional impairment caused by that loss. This new metric displays significant negative correlation with evolutionary rate, across several thousand minimal environments. We demonstrate that the improvement gained using function-loss cost over gene-loss cost is explained by replacing the base assumption that isoenzymes provide unlimited capacity for backup with the assumption that isoenzymes are completely non-redundant. We further show that this change of the assumption regarding isoenzymes increases the recall of epistatic interactions predicted by the flux balance model at the cost of a reduction in the precision of the predictions. In addition to suggesting that the gene-to-reaction mapping in genome-scale flux balance models should be used with caution, our analysis provides new evidence that evolutionary gene importance captures much more than strict essentiality.

  11. THE PRESENCE OF 5 CYCLIC-NUCLEOTIDE PHOSPHODIESTERASE ISOENZYME ACTIVITIES IN BOVINE TRACHEAL SMOOTH-MUSCLE AND THE FUNCTIONAL-EFFECTS OF SELECTIVE INHIBITORS

    NARCIS (Netherlands)

    VANAMSTERDAM, RGM; DEBOER, J; TENBERGE, RE; NICHOLSON, CD; ZAAGSMA, J

    1991-01-01

    1 The profile of cyclic nucleotide phosphodiesterase (PDE) isoenzymes and the relaxant effects of isoenzyme selective inhibitors were examined in bovine tracheal smooth muscle. The compounds examined were the non-selective inhibitor 3-isobutyl-1-methylxanthine (IBMX), zaprinast (PDE V selective),

  12. Proposal for the Identification of Barley Varieties Based on the Genotypes for 2 Hordein and 39 Isoenzym Loci of 47 Reference Varieties

    DEFF Research Database (Denmark)

    Nielsen, G.; Johansen, Hanne Bay

    1986-01-01

    Fifty-nine spring and 7 winter barley varieties in lsquoThe Danish List of Varieties of Agricultural Crops, 1983/84rsquo were examined for variation at 39 isoenzyme and two hordein loci. Twenty-three isoenzyme loci had one allele only, and 16 loci had from two to five alleles. One hordein locus had...

  13. Immunohistochemical detection of cytochrome P450 isoenzymes in cultured human epidermal cells.

    Science.gov (United States)

    Van Pelt, F N; Meierink, Y J; Blaauboer, B J; Weterings, P J

    1990-12-01

    We used specific monoclonal antibodies (MAb) to human cytochrome P450 isoenzymes to determine the presence of these proteins in human epidermal cells. Two MAb (P450-5 and P450-8) recognize major forms of hepatic cytochrome P450 involved in biotransformation of xenobiotics. A third MAb, to cytochrome P450-9, is not fully characterized. The proteins were determined by the indirect immunoperoxidase technique after fixation with methanol and acetone. Biopsy materials for cultured keratinocytes, i.e., foreskin and hair follicles, contained the two major forms of cytochrome P450. In cultured keratinocytes derived from hair follicles the proteins were undetectable, whereas the keratinocytes derived from foreskin continued to express the two major forms of hepatic cytochrome P450. Cultured human fibroblasts and a human keratinocyte cell line (SVK14) showed staining similar to that of the foreskin keratinocytes. Cytochrome P450-9 was detectable only in human hepatocytes. The results indicate that, under the culture conditions applied, cultured human foreskin cells and the cell line SVK14 continue to express specific cytochrome P450 isoenzymes in culture, in contrast to hair follicle keratinocytes.

  14. Properties of purified cytosolic isoenzyme I of Cu,Zn-superoxide dismutase from Nicotiana plumbaginifolia leaves.

    Science.gov (United States)

    Ragusa, S; Cambria, M T; Scarpa, M; Di Paolo, M L; Falconi, M; Rigo, A; Cambria, A

    2001-11-01

    The isoenzyme I of cytosolic Cu,Zn-superoxide dismutase (SOD) from Nicotiana plumbaginifolia (tobacco) leaves has been purified to apparent homogeneity. The relative molecular mass of the native isoenzyme, determined by gel filtration chromatography, is about 33.2 kDa. SDS-polyacrylamide gel electrophoresis shows that the enzyme is composed of two equal subunits of 16.6 kDa The isolectric point, assayed by isoelectric focusing, in the pH range of 3.5-6.5, is 4.3. The enzyme stability was tested at different temperatures, pH, and concentration of inhibitors (KCN and H(2)O(2)). The catalytic constant (k(cat)) was 1.17 +/- 0.14 x 10(9) M(-1) s(-1) at pH 9.9 and 0.1 M ionic strength. The activation energy of the thermal denaturation process is 263 kJ mol(-1). The electrostatic surface potential of the modeled tobacco Cu,Zn-SOD I was calculated showing that the functional spatial network of charges on the protein surface has been maintained, independently of the amino acid substitution around the active sites. Copyright 2001 Academic Press.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-07-22

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

  16. Specific genetic deficiencies of the A and B isoenzymes of monoamine oxidase are characterized by distinct neurochemical and clinical phenotypes

    NARCIS (Netherlands)

    Lenders, J. W.; Eisenhofer, G.; Abeling, N. G.; Berger, W.; Murphy, D. L.; Konings, C. H.; Wagemakers, L. M.; Kopin, I. J.; Karoum, F.; van Gennip, A. H.; Brunner, H. G.

    1996-01-01

    Monoamine oxidase (MAO) exists as two isoenzymes and plays a central role in the metabolism of monoamine neurotransmitters. In this study we compared the neurochemical phenotypes of previously described subjects with genetically determined selective lack of MAO-A or a lack of both MAO-A and MAO-B

  17. Role of creatine kinase isoenzymes on muscular and cardiorespiratory endurance: genetic and molecular evidence.

    Science.gov (United States)

    Echegaray, M; Rivera, M A

    2001-01-01

    The ability to perform well in activities that require muscular and cardiorespiratory endurance is a trait influenced, in a considerable part, by the genetic make-up of individuals. Early studies of performance and recent scans of the human genome have pointed at various candidate genes responsible for the heterogeneity of these phenotypes within the population. Among these are the genes for the various creatine kinase (CK) isoenzyme subunits. CK and phosphocreatine (PCr) form an important metabolic system for temporal and spatial energy buffering in cells with large variations in energy demand. The different CK isoenzyme subunits (CK-M and CK-B) are differentially expressed in the tissues of the body. Although CK-M is the predominant form in both skeletal and cardiac muscle, CK-B is expressed to a greater extent in heart than in skeletal muscle. Studies in humans and mice have shown that the expression of CK-B messenger RNA (mRNA) and the abundance and activity of the CK-MB dimer increase in response to cardiorespiratory endurance training. Increases in muscle tissue CK-B content can be energetically favourable because of its lower Michaelis constant (Km) for ADP. The activity of the mitochondrial isoform of CK (Scmit-CK) has also been significantly and positively correlated to oxidative capacity and to CK-MB activity in muscle. In mice where the CK-M gene has been knocked out, significant increases in fatigue resistance together with cellular adaptations increasing aerobic capacity have been observed. These observations have led to the notion that this enzyme may be responsible for fatigue under normal circumstances, most likely because of the local cell compartment increase in inorganic phosphate concentration. Studies where the Scmit-CK gene was knocked out have helped demonstrate that this isoenzyme is very important for the stimulation of aerobic respiration. Human studies of CK-M gene sequence variation have shown a significant association between a

  18. Intercropping Acacia mangium stimulates AMF colonization and soil phosphatase activity in Eucalyptus grandis

    Directory of Open Access Journals (Sweden)

    Daniel Bini

    Full Text Available ABSTRACT: Arbuscular mycorrhizal fungi (AMF are very important to plant nutrition, mostly in terms of acquisition of P and micronutrients. While Acacia mangium is closely associated with AMF throughout the whole cycle, Eucalyptus grandis presents this symbiosis primarily at the seedling stage. The aim of this study was to evaluate the dynamics of AMF in these two tree species in both pure and mixed plantations during the first 20 months after planting. We evaluated the abundance, richness and diversity of AMF spores, the rate of AMF mycorrhizal root colonization, enzymatic activity and soil and litter C, N and P. There was an increase in AMF root colonization of E. grandis when intercropped with A. mangium as well as an increase in the activity of acid and alkaline phosphatase in the presence of leguminous trees. AMF colonization and phosphatase activities were both involved in improvements in P cycling and P nutrition in soil. In addition, P cycling was favored in the intercropped plantation, which showed negative correlation with litter C/N and C/P ratios and positive correlation with soil acid phosphatase activity and soil N and P concentrations. Intercropping A. mangium and E. grandis maximized AMF root colonization of E. grandis and phosphatase activity in the soil, both of which accelerate P cycling and forest performance.

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

    Science.gov (United States)

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

    2017-12-01

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

  20. Beryllium and growth. III. The effect of beryllium on plant phosphatase

    Energy Technology Data Exchange (ETDEWEB)

    Hoagland, M B

    1952-01-01

    The purpose of the investigations was to correlate the apparent ability of beryllium to substitute for magnesium in plant growth with a specific biochemical effect of the metal. Through association with earlier work on beryllium inhibition of animal alkaline phosphatase, a study was made of the effect of beryllium and other metals upon the activity of a phosphatase derived from tomato leaves. Although only indirect evidence is available that this enzyme system was magnesium-activated, beryllium was found to inhibit reversibly the splitting of GP and ATP. Other metals were also found to be inhibitory but the ATP-ase inhibition - and especially the ratio of P split from GP to P split from ATP - was higher for beryllium than for any other metal studied. The significance of this finding in relation to energy metabolism, growth, and beryllium toxicity is discussed. 12 references, 5 figures, 2 tables.

  1. Two-site immunoradiometric assay for the MB isoenzyme of creatine kinase

    Energy Technology Data Exchange (ETDEWEB)

    Willson, V J.C.; Jones, H M; Thompson, R J [Cambridge Univ. (UK). Clinical School

    1981-06-18

    A two-site immunoradiometric assay for myocardial creatine kinase MB isoenzyme is described. The method utilizes immobilized anti-human creatine kinase BB antibodies and /sup 125/I-labelled anti-human creatine kinase MM antibodies and can specifically detect creatine kinase MB in the presence of approximately 1000-fold excess of creatine kinase MM or BB. Native kinase MB prepared from human heart and creatine kinase MB prepared by hybridisation of purified human creatine kinase MM and creatine kinase BB appeared to react identically in the assay. Serum estimations on patients with suspected myocardial infarction correlated with the presence of MB band on electrophoresis but preliminary results suggest that the two-site immunoradiometric assay may be more sensitive.

  2. A two-site immunoradiometric assay for the MB isoenzyme of creatine kinase

    International Nuclear Information System (INIS)

    Willson, V.J.C.; Jones, H.M.; Thompson, R.J.

    1981-01-01

    A two-site immunoradiometric assay for myocardial creatine kinase MB isoenzyme is described. The method utilizes immobilized anti-human creatine kinase BB antibodies and 125 I-labelled anti-human creatine kinase MM antibodies and can specifically detect creatine kinase MB in the presence of approximately 1000-fold excess of creatine kinase MM or BB. Native kinase MB prepared from human heart and creatine kinase MB prepared by hybridisation of purified human creatine kinase MM and creatine kinase BB appeared to react identically in the assay. Serum estimations on patients with suspected myocardial infarction correlated with the presence of MB band on electrophoresis but preliminary results suggest that the two-site immunoradiometric assay may be more sensitive. (Auth.)

  3. Alkalinity of the Mediterranean Sea

    OpenAIRE

    Schneider, Anke; Wallace, Douglas W.R.; Körtzinger, Arne

    2007-01-01

    Total alkalinity (AT) was measured during the Meteor 51/2 cruise, crossing the Mediterranean Sea from west to east. AT concentrations were high (∼2600 μmol kg−1) and alkalinity-salinity-correlations had negative intercepts. These results are explained by evaporation coupled with high freshwater AT inputs into coastal areas. Salinity adjustment of AT revealed excess alkalinity throughout the water column compared to mid-basin surface waters. Since Mediterranean waters are supersaturated with r...

  4. Leishmania isoenzyme polymorphisms in Ecuador: Relationships with geographic distribution and clinical presentation

    Science.gov (United States)

    Calvopina, Manuel; Armijos, Rodrigo X; Marco, Jorge D; Uezato, Hiroshi; Kato, Hirotomo; Gomez, Eduardo A; Korenaga, Masataka; Barroso, Paola A; Mimori, Tatsuyuki; Cooper, Philip J; Nonaka, Shigeo; Hashiguchi, Yoshihisa

    2006-01-01

    Background Determinants of the clinical presentation of the leishmaniases are poorly understood but Leishmania species and strain differences are important. To examine the relationship between clinical presentation, species and isoenzyme polymorphisms, 56 Leishmania isolates from distinct presentations of American tegumentary leishmaniasis (ATL) from Ecuador were analyzed. Methods Isolates were characterized by multilocus enzyme electrophoresis for polymorphisms of 11 isoenzymes. Patients were infected in four different ecologic regions: highland and lowland jungle of the Pacific coast, Amazonian lowlands and Andean highlands. Results Six Leishmania species constituting 21 zymodemes were identified: L. (Viannia) panamensis (21 isolates, 7 zymodemes), L. (V.) guyanensis (7 isolates, 4 zymodemes), L. (V.) braziliensis (5 isolates, 3 zymodemes), L. (Leishmania) mexicana (11 isolates, 4 zymodemes), L. (L.) amazonensis (10 isolates, 2 zymodemes) and L. (L.) major (2 isolates, 1 zymodeme). L. panamensis was the species most frequently identified in the Pacific region and was associated with several clinical variants of cutaneous disease (CL); eight cases of leishmaniasis recidiva cutis (LRC) found in the Pacific highlands were associated with 3 zymodemes of this species. Mucocutaneous leishmaniasis found only in the Amazonian focus was associated with 3 zymodemes of L. braziliensis. The papular variant of CL, Uta, found in the Andean highlands was related predominantly with a single zymodeme of L. mexicana. Conclusion Our data show a high degree of phenotypic variation within species, and some evidence for associations between specific variants of ATL (i.e. Uta and LRC) and specific Leishmania zymodemes. This study further defines the geographic distribution of Leishmania species and clinical variants of ATL in Ecuador. PMID:16968553

  5. Leishmania isoenzyme polymorphisms in Ecuador: Relationships with geographic distribution and clinical presentation

    Directory of Open Access Journals (Sweden)

    Mimori Tatsuyuki

    2006-09-01

    Full Text Available Abstract Background Determinants of the clinical presentation of the leishmaniases are poorly understood but Leishmania species and strain differences are important. To examine the relationship between clinical presentation, species and isoenzyme polymorphisms, 56 Leishmania isolates from distinct presentations of American tegumentary leishmaniasis (ATL from Ecuador were analyzed. Methods Isolates were characterized by multilocus enzyme electrophoresis for polymorphisms of 11 isoenzymes. Patients were infected in four different ecologic regions: highland and lowland jungle of the Pacific coast, Amazonian lowlands and Andean highlands. Results Six Leishmania species constituting 21 zymodemes were identified: L. (Viannia panamensis (21 isolates, 7 zymodemes, L. (V. guyanensis (7 isolates, 4 zymodemes, L. (V. braziliensis (5 isolates, 3 zymodemes, L. (Leishmania mexicana (11 isolates, 4 zymodemes, L. (L. amazonensis (10 isolates, 2 zymodemes and L. (L. major (2 isolates, 1 zymodeme. L. panamensis was the species most frequently identified in the Pacific region and was associated with several clinical variants of cutaneous disease (CL; eight cases of leishmaniasis recidiva cutis (LRC found in the Pacific highlands were associated with 3 zymodemes of this species. Mucocutaneous leishmaniasis found only in the Amazonian focus was associated with 3 zymodemes of L. braziliensis. The papular variant of CL, Uta, found in the Andean highlands was related predominantly with a single zymodeme of L. mexicana. Conclusion Our data show a high degree of phenotypic variation within species, and some evidence for associations between specific variants of ATL (i.e. Uta and LRC and specific Leishmania zymodemes. This study further defines the geographic distribution of Leishmania species and clinical variants of ATL in Ecuador.

  6. Phosphodiesterase isoenzyme families in human osteoarthritis chondrocytes – functional importance of phosphodiesterase 4

    Science.gov (United States)

    Tenor, Hermann; Hedbom, Erik; Häuselmann, Hans-Jörg; Schudt, Christian; Hatzelmann, Armin

    2002-01-01

    We studied whether selective inhibitors of cyclic nucleotide hydrolysing phosphodiesterase (PDE) isoenzymes influence IL-1β-induced nitric oxide (NO) release from human articular chondrocytes. In addition, the pattern of PDE isoenzymes contributing to cyclic nucleotide hydrolysis in human chondrocytes was characterized.Chondrocytes were isolated from human osteoarthritic cartilage and cultured in alginate beads. IL-1β-induced chondrocyte products (nitric oxide and prostaglandin E2) were measured in culture supernatants after 48 h incubation time. PDE activities were assessed in chondrocyte lysates. Inducible nitric oxide synthase (iNOS) and PDE4A-D proteins were detected by immunoblotting.The selective PDE4 inhibitors Piclamilast and Roflumilast partially attenuated IL-1β-induced NO production whereas selective inhibitors of PDE2 (EHNA), PDE3 (Motapizone) or PDE5 (Sildenafil) were inactive. Indomethacin reversed the reduction of IL-1β-induced NO by PDE4 inhibitors. It was shown that autocrine prostaglandin E2 (PGE2) enabled PDE4 inhibitors to reduce IL-1β-induced NO in this experimental setting.Major PDE4 and PDE1 activities were identified in chondrocyte lysates whereas only minor activities of PDE2, 3 and 5 were found. IL-1β and cyclic AMP-mimetics upregulated PDE4 activity and this was associated with an augmentation of PDE4B2 protein.Based on the view that nitric oxide contributes to cartilage degradation in osteoarthritis our study suggests that PDE4 inhibitors may have chondroprotective effects. PMID:11834608

  7. Alkaline fuel cells applications

    Science.gov (United States)

    Kordesch, Karl; Hacker, Viktor; Gsellmann, Josef; Cifrain, Martin; Faleschini, Gottfried; Enzinger, Peter; Fankhauser, Robert; Ortner, Markus; Muhr, Michael; Aronson, Robert R.

    On the world-wide automobile market technical developments are increasingly determined by the dramatic restriction on emissions as well as the regimentation of fuel consumption by legislation. Therefore there is an increasing chance of a completely new technology breakthrough if it offers new opportunities, meeting the requirements of resource preservation and emission restrictions. Fuel cell technology offers the possibility to excel in today's motive power techniques in terms of environmental compatibility, consumer's profit, costs of maintenance and efficiency. The key question is economy. This will be decided by the costs of fuel cell systems if they are to be used as power generators for future electric vehicles. The alkaline hydrogen-air fuel cell system with circulating KOH electrolyte and low-cost catalysed carbon electrodes could be a promising alternative. Based on the experiences of Kordesch [K. Kordesch, Brennstoffbatterien, Springer, Wien, 1984, ISBN 3-387-81819-7; K. Kordesch, City car with H 2-air fuel cell and lead-battery, SAE Paper No. 719015, 6th IECEC, 1971], who operated a city car hybrid vehicle on public roads for 3 years in the early 1970s, improved air electrodes plus new variations of the bipolar stack assembly developed in Graz are investigated. Primary fuel choice will be a major issue until such time as cost-effective, on-board hydrogen storage is developed. Ammonia is an interesting option. The whole system, ammonia dissociator plus alkaline fuel cell (AFC), is characterised by a simple design and high efficiency.

  8. Single-label kinase and phosphatase assays for tyrosine phosphorylation using nanosecond time-resolved fluorescence detection.

    Science.gov (United States)

    Sahoo, Harekrushna; Hennig, Andreas; Florea, Mara; Roth, Doris; Enderle, Thilo; Nau, Werner M

    2007-12-26

    The collision-induced fluorescence quenching of a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) by hydrogen atom abstraction from the tyrosine residue in peptide substrates was introduced as a single-labeling strategy to assay the activity of tyrosine kinases and phosphatases. The assays were tested for 12 different combinations of Dbo-labeled substrates and with the enzymes p60c-Src Src kinase, EGFR kinase, YOP protein tyrosine phosphatase, as well as acid and alkaline phosphatases, thereby demonstrating a broad application potential. The steady-state fluorescence changed by a factor of up to 7 in the course of the enzymatic reaction, which allowed for a sufficient sensitivity of continuous monitoring in steady-state experiments. The fluorescence lifetimes (and intensities) were found to be rather constant for the phosphotyrosine peptides (ca. 300 ns in aerated water), while those of the unphosphorylated peptides were as short as 40 ns (at pH 7) and 7 ns (at pH 13) as a result of intramolecular quenching. Owing to the exceptionally long fluorescence lifetime of Dbo, the assays were alternatively performed by using nanosecond time-resolved fluorescence (Nano-TRF) detection, which leads to an improved discrimination of background fluorescence and an increased sensitivity. The potential for inhibitor screening was demonstrated through the inhibition of acid and alkaline phosphatases by molybdate.

  9. Effect of Exogenous Phytase Addition on Soil Phosphatase Activities: a Fluorescence Spectroscopy Study.

    Science.gov (United States)

    Yang, Xiao-zhu; Chen, Zhen-hua; Zhang, Yu-lan; Chen, Li-jun

    2015-05-01

    The utilization of organic phosphorus (P) has directly or indirectly improved after exogenous phytase was added to soil. However, the mechanism by which exogenous phytase affected the soil phosphatases (phosphomonoesterase and phosphodiesterase) activities was not clear. The present work was aimed to study red soil, brown soil and cinnamon soil phosphomonoesterase (acid and alkaline) (AcP and AlP) and phosphodiesterase (PD) activities responding to the addition of exogenous phytase (1 g phytase/50 g air dry soil sample) based on the measurements performed via a fluorescence detection method combined with 96 microplates using a TECAN Infinite 200 Multi-Mode Microplate Reader. The results indicated that the acid phosphomonoesterase activity was significantly enhanced in red soil (p≤0. 01), while it was significantly reduced in cinnamon soil; alkaline phosphomonoesterase activity was significantly enhanced in cinnamon soil (p≤ 0. 01), while it was significantly reduced in red soil; phosphodiesterase activity was increased in three soils but it was significantly increased in brown soil (p≤0. 01) after the addition of exogenous phytase. The activities still remained strong after eight days in different soils, which indicated that exogenous phytase addition could be enhance soil phosphatases activities effectively. This effect was not only related to soil properties, such as pH and phosphorus forms, but might also be related to the excreted enzyme amount of the stimulating microorganism. Using fluorescence spectroscopy to study exogenous phytase addition influence on soil phosphatase activities was the first time at home and abroad. Compared with the conventional spectrophotometric method, the fluorescence microplate method is an accurate, fast and simple to use method to determine the relationships among the soil phosphatases activities.

  10. Crucial role of alkaline sphingomyelinase in sphingomyelin digestion: a study on enzyme knockout mice

    DEFF Research Database (Denmark)

    Zhang, Yao; Cheng, Yajun; Hansen, Gert H

    2011-01-01

    ) and KO mice were fed ³H-palmitic acid labeled SM together with milk SM by gavage. The lipids in intestinal content, intestinal tissues, serum, and liver were analyzed by TLC. In KO mice, nondigested ³H-SM in the intestinal content increased by 6-fold and the formation of ³H-ceramide decreased markedly....... The KO mice also showed significantly decreased radioactivity in liver and serum. Furthermore, alkaline phosphatase activity in the mucosa was reduced by 50% and histological comparison of two female littermates preliminarily suggested mucosal hypertrophy in KO mice. This study provides definite proof...... for crucial roles of alk-SMase in SM digestion and points to possible roles in regulating mucosal growth and alkaline phosphatase function....

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

    Science.gov (United States)

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

    2016-01-01

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

  12. Method of cleaning alkaline metal

    International Nuclear Information System (INIS)

    Kawakami, Yukio; Naito, Kesahiro; Iizawa, Katsuyuki; Nakasuji, Takashi

    1981-01-01

    Purpose: To prevent scattering of used sodium and aqueous alkaline solution when cleaning used sodium and metallic sodium adhering to equipment with an aqueous alkaline solution. Method: A sodium treating container is filled with an aqueous alkaline solution, and stainless steel gauze is sunk in the container. Equipment to be cleaned such as equipment with sodium adhering to it are retained under the gauze and are thus cleaned. On the other hand, the surface of the aqueous alkaline solution is covered with a fluid paraffin liquid covering material. Thus, the hydrogen produced by the reaction of the sodium and the aqueous alkaline solution will float up, pass through the liquid covering material and be discharged. The sodium will pass through the gauze and float upwardly while reacting with the aqueous alkaline solution in a partic ulate state to the boundary between the aqueous alkaline solution and up to the covering material, and thus the theratment reaction will continue. Thus, the cover material prevents the sodium and the aqueous alkaline solution from scattering. (Kamimura, M.)

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

    Directory of Open Access Journals (Sweden)

    Alceu Kunze

    2011-06-01

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

  14. Alkaline phosphatases in microbialites and bacterioplankton from Alchichica soda lake, Mexico

    Czech Academy of Sciences Publication Activity Database

    Valdespino-Castillo, P.M.; Alcantara-Hernandez, R.J.; Alcocer, J.; Merino-Ibarra, M.; Macek, Miroslav; Falcon, L.I.

    2014-01-01

    Roč. 90, č. 2 (2014), s. 504-519 ISSN 0168-6496 Institutional support: RVO:60077344 Keywords : dissolved organic phosphorus utilization * extracellular enzymes * microbial functional diversity Subject RIV: EE - Microbiology, Virology Impact factor: 3.568, year: 2014

  15. The Covalent Binding of Alkaline Phosphatase on Porous Supports and the Stability of the Immobilized Enzyme

    Science.gov (United States)

    1988-08-11

    and LiChrospher Si 4000 were obtained from Applied Science Laboratories, Inc. LiChrospher Si 100 was obtained from Alltech Assoc. The surface areas...Z U) w I < 0 The earliest attempt to take advantage of evanescent wave interactions in an optical waveguide to detect immunological reactions was made

  16. Enhanced Activity of Alkaline Phosphatases - Phytoplankton Response to Epilimnetic Phosphorus Depletion

    Czech Academy of Sciences Publication Activity Database

    Vrba, Jaroslav; Komárková, Jaroslava; Vyhnálek, Vojtěch

    1993-01-01

    Roč. 28, č. 6 (1993), s. 15-24 ISSN 0273-1223. [2nd International Conference on Reservoir Limnology and Water Quality. Č. Budějovice, 09.08.1992-14.08.1992] R&D Projects: GA AV ČR IA61706 Impact factor: 0.573, year: 1993

  17. An alkaline phosphatase transport mechanism in the pathogenesis of Alzheimer's disease and neurodegeneration

    NARCIS (Netherlands)

    Pike, Adrianne F; Kramer, Nynke I; Blaauboer, Bas J; Seinen, Willem; Brands, Ruud

    2015-01-01

    Systemic inflammation is associated with loss of blood-brain barrier integrity and neuroinflammation that lead to the exacerbation of neurodegenerative diseases. It is also associated specifically with the characteristic amyloid-β and tau pathologies of Alzheimer's disease. We have previously

  18. Phosphate solubilizing bacteria and alkaline phosphatase activity in coastal waters off Trivandrum

    Digital Repository Service at National Institute of Oceanography (India)

    Mamatha, S.S.; Gobika, A.; Janani, P.

    , Korea. Marine Pollution Bulletin. 62. pp. 2476–2482. 98 Journal of Coastal Environment Illmer, P. and Schinner, F. 1995. Solubilization of inorganic calcium phosphates solubilization mechanisms. Soil Biology and Biochemistry. 27. pp. 57...-solubilising microorganisms associated with the rhizosphere of mangroves in a semiarid coastal lagoon. Biology and Fertility of Soils. 30. 460-468. Wurl, O. 2009. Practical guidelines for the analysis of sea water. CRC Press, Boca Raton. pp. 143-178. Zohary, T...

  19. Intestinal alkaline phosphatase: selective endocytosis from the enterocyte brush border during fat absorption

    DEFF Research Database (Denmark)

    Hansen, Gert Helge; Niels-Christiansen, Lise-Lotte; Immerdal, Lissi

    2007-01-01

    explants. By immunofluorescence microscopy, fat absorption caused a translocation of IAP from the enterocyte brush border to the interior of the cell, whereas other brush-border enzymes were unaffected. By electron microscopy, the translocation occurred by a rapid (5 min) induction of endocytosis via...

  20. Relationship between thyroid, gonads and plasmatic levels of phosphorus, calcium and alkaline phosphatase in rats

    OpenAIRE

    Serakides, R.; Nunes, V.A.; Nascimento, E.F.; Silva, C.M.; Ribeiro, A.F.C.

    2000-01-01

    A relação tireóide-gônadas-metabolismo ósseo foi estudada em ratas Wistar adultas, castradas ou intactas e mantidas em estado hipertireóideo ou eutireóideo por períodos de 30, 60 e 90 dias. Foram utilizadas como características do metabolismo ósseo o cálcio, o fósforo e a atividade da fosfatase alcalina plasmáticos, correlacionando-os com os valores de estrógeno, de progesterona e de T4 livre. Verificou-se que o hipogonadismo e o hipertireoidismo alteram as características plasmáticas do meta...

  1. Spinal motor neuron neuroaxonal spheroids in chronic aluminum neurotoxicity contain phosphatase-resistant high molecular weight neurofilament (NFH).

    Science.gov (United States)

    Gaytan-Garcia, S; Kim, H; Strong, M J

    1996-04-15

    It has previously been shown that a single intracisternal inoculum of AlCl3 in young adult New Zealand white rabbits will induce a dose-dependent phosphatase resistance of high molecular weight neurofilament protein (NFH) that is proportionate to the extent of neurofilamentous inclusion formation (Strong and Jakowec, 1994). To determine if the potential for dissolution of aluminum-induced neurofilamentous inclusions was dependent on the degree of NFH phosphatase resistance, we have examined NFH phosphatase sensitivity in a reversible chronic model of aluminum neurotoxicity. Rabbits receiving repeated intracisternal inoculums of 100 microgram AlCl3 at 28 day intervals until day 267 develop spinal motor neuron perikaryal and neuroaxonal neurofilamentous aggregates in a stereotypic, dose-dependent fashion. In the rabbits receiving inoculums until day 156 with survival until day 267 without further aluminum exposure, neuroaxonal spheroids remained prominent while perikaryal inclusions largely resolved. Immunoreactivity to a monoclonal antibody recognizing phosphorylated NFH (SMI 31) was abolished in perikaryal aggregates at each time interval by dephosphorylation with bovine alkaline phosphatase. However, neuroaxonal spheroids maintained their immunoreactivity. Using time-course dephosphorylation studies of spinal cord homogenates, we observed a significant reduction in the rate of dephosphorylation of NFH following 267 days of AlCl3 exposure (P < 0.05). These observations suggest that neuroaxonal spheroids contain phosphatase-resistant NFH isoforms and that the potential for resolution of intraneuronal neurofilamentous inclusions correlates with the susceptibility of NF within these inclusions to enzymatic dephosphorylation.

  2. Bioprecipitation of uranium from alkaline waste solutions using recombinant Deinococcus radiodurans

    Energy Technology Data Exchange (ETDEWEB)

    Kulkarni, Sayali; Ballal, Anand; Apte, Shree Kumar, E-mail: aptesk@barc.gov.in

    2013-11-15

    Highlights: • Deinococcus radiodurans was genetically engineered to overexpress alkaline phosphatase (PhoK). • Deino-PhoK bioprecipitated U efficiently over a wide range of input U concentration. • A maximal loading of 10.7 g U/g of biomass at 10 mM input U was observed. • Radioresistance and U precipitation by Deino-PhoK remained unaffected by γ radiation. • Immobilization of Deino-PhoK facilitated easy separation of precipitated U. -- Abstract: Bioremediation of uranium (U) from alkaline waste solutions remains inadequately explored. We engineered the phoK gene (encoding a novel alkaline phosphatase, PhoK) from Sphingomonas sp. for overexpression in the radioresistant bacterium Deinococcus radiodurans. The recombinant strain thus obtained (Deino-PhoK) exhibited remarkably high alkaline phosphatase activity as evidenced by zymographic and enzyme activity assays. Deino-PhoK cells could efficiently precipitate uranium over a wide range of input U concentrations. At low uranyl concentrations (1 mM), the strain precipitated >90% of uranium within 2 h while a high loading capacity of around 10.7 g U/g of dry weight of cells was achieved at 10 mM U concentration. Uranium bioprecipitation by Deino-PhoK cells was not affected in the presence of Cs and Sr, commonly present in intermediate and low level liquid radioactive waste, or after exposure to very high doses of ionizing radiation. Transmission electron micrographs revealed the extracellular nature of bioprecipitated U, while X-ray diffraction and fluorescence analysis identified the precipitated uranyl phosphate species as chernikovite. When immobilized into calcium alginate beads, Deino-PhoK cells efficiently removed uranium, which remained trapped in beads, thus accomplishing physical separation of precipitated uranyl phosphate from solutions. The data demonstrate superior ability of Deino-PhoK, over earlier reported strains, in removal of uranium from alkaline solutions and its potential use in

  3. Study for identification of beneficial uses of Space (BUS). Volume 2: Technical report. Book 1: Development and business analysis of space processed isoenzymes

    Science.gov (United States)

    1975-01-01

    A separation method to provide reasonable yields of high specificity isoenzymes for the purpose of large scale, early clinical diagnosis of diseases and organic damage such as, myocardial infarction, hepatoma, muscular dystrophy, and infectous disorders is presented. Preliminary development plans are summarized. An analysis of required research and development and production resources is included. The costs of such resources and the potential profitability of a commercial space processing opportunity for electrophoretic separation of high specificity isoenzymes are reviewed.

  4. Severe Extracellular Matrix Abnormalities and Chondrodysplasia in Mice Lacking Collagen Prolyl 4-Hydroxylase Isoenzyme II in Combination with a Reduced Amount of Isoenzyme I.

    Science.gov (United States)

    Aro, Ellinoora; Salo, Antti M; Khatri, Richa; Finnilä, Mikko; Miinalainen, Ilkka; Sormunen, Raija; Pakkanen, Outi; Holster, Tiina; Soininen, Raija; Prein, Carina; Clausen-Schaumann, Hauke; Aszódi, Attila; Tuukkanen, Juha; Kivirikko, Kari I; Schipani, Ernestina; Myllyharju, Johanna

    2015-07-03

    Collagen prolyl 4-hydroxylases (C-P4H-I, C-P4H-II, and C-P4H-III) catalyze formation of 4-hydroxyproline residues required to form triple-helical collagen molecules. Vertebrate C-P4Hs are α2β2 tetramers differing in their catalytic α subunits. C-P4H-I is the major isoenzyme in most cells, and inactivation of its catalytic subunit (P4ha1(-/-)) leads to embryonic lethality in mouse, whereas P4ha1(+/-) mice have no abnormalities. To study the role of C-P4H-II, which predominates in chondrocytes, we generated P4ha2(-/-) mice. Surprisingly, they had no apparent phenotypic abnormalities. To assess possible functional complementarity, we established P4ha1(+/-);P4ha2(-/-) mice. They were smaller than their littermates, had moderate chondrodysplasia, and developed kyphosis. A transient inner cell death phenotype was detected in their developing growth plates. The columnar arrangement of proliferative chondrocytes was impaired, the amount of 4-hydroxyproline and the Tm of collagen II were reduced, and the extracellular matrix was softer in the growth plates of newborn P4ha1(+/-);P4ha2(-/-) mice. No signs of uncompensated ER stress were detected in the mutant growth plate chondrocytes. Some of these defects were also found in P4ha2(-/-) mice, although in a much milder form. Our data show that C-P4H-I can to a large extent compensate for the lack of C-P4H-II in proper endochondral bone development, but their combined partial and complete inactivation, respectively, leads to biomechanically impaired extracellular matrix, moderate chondrodysplasia, and kyphosis. Our mouse data suggest that inactivating mutations in human P4HA2 are not likely to lead to skeletal disorders, and a simultaneous decrease in P4HA1 function would most probably be required to generate such a disease phenotype. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Defining Starch Binding by Glucan Phosphatases

    DEFF Research Database (Denmark)

    Auger, Kyle; Raththagala, Madushi; Wilkens, Casper

    2015-01-01

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

  6. Mannose Phosphate Isomerase Isoenzymes in Plutella xylostella Support Common Genetic Bases of Resistance to Bacillus thuringiensis Toxins in Lepidopteran Species

    OpenAIRE

    Herrero, Salvador; Ferré, Juan; Escriche, Baltasar

    2001-01-01

    A strong correlation between two mannose phosphate isomerase (MPI) isoenzymes and resistance to Cry1A toxins from Bacillus thuringiensis has been found in a Plutella xylostella population. MPI linkage to Cry1A resistance had previously been reported for a Heliothis virescens population. The fact that the two populations share similar biochemical, genetic, and cross-resistance profiles of resistance suggests the occurrence of homologous resistance loci in both species.

  7. Effect of various synthetic dyes on the production of manganese-dependent peroxidase isoenzymes by immobilized Irpex lacteus

    Czech Academy of Sciences Publication Activity Database

    Šušla, Martin; Svobodová, Kateřina

    2008-01-01

    Roč. 24, - (2008), s. 225-230 ISSN 0959-3993 R&D Projects: GA ČR GP526/06/P102 Grant - others:CZ(CZ) G4 359/2007 Institutional research plan: CEZ:AV0Z50200510 Keywords : irpex lacteus * isoenzymes * manganese-dependent peroxidase Subject RIV: EE - Microbiology, Virology Impact factor: 0.945, year: 2008

  8. Rab11 is phosphorylated by classical and novel protein kinase C isoenzymes upon sustained phorbol ester activation.

    Science.gov (United States)

    Pavarotti, Martín; Capmany, Anahí; Vitale, Nicolas; Colombo, María Isabel; Damiani, María Teresa

    2012-02-01

    Rab11 is a small GTPase that controls diverse intracellular trafficking pathways. However, the molecular machinery that regulates the participation of Rab11 in those different transport events is poorly understood. In resting cells, Rab11 localizes at the endocytic recycling compartment (ERC), whereas the different protein kinase C (PKC) isoforms display a cytosolic distribution. Sustained phorbol ester stimulation induces the translocation of the classical PKCα and PKCβII isoenzymes to the ERC enriched in Rab11, and results in transferrin recycling inhibition. In contrast, novel PKCε and atypical PKCζ isoenzymes neither redistribute to the perinucleus nor modify transferrin recycling transport after phorbol ester stimulation. Although several Rabs have been shown to be phosphorylated, there is to date no evidence indicating Rab11 as a kinase substrate. In this report, we show that Rab11 appears phosphorylated in vivo in phorbol ester-stimulated cells. A bioinformatic analysis of Rab11 allowed us to identify several high-probability Ser/Thr kinase phosphorylation sites. Our results demonstrate that classical PKC (PKCα and PKCβII but not PKCβI) directly phosphorylate Rab11 in vitro. In addition, novel PKCε and PKCη but not PKCδ isoenzymes also phosphorylate Rab11. Mass spectrometry analysis revealed that Ser 177 is the Rab11 residue to be phosphorylated in vitro by either PKCβII or PKCε. In agreement, the phosphomimetic mutant, Rab11 S177D, retains transferrin at the ERC in the absence of phorbol-12-myristate-13-acetate stimulus. This report shows for the first time that Rab11 is differentially phosphorylated by distinct PKC isoenzymes and that this post-translational modification might be a regulatory mechanism of intracellular trafficking. Copyright © 2012 Soçiété Francaise des Microscopies and Société de Biologie Cellulaire de France.

  9. Evidence for phosphoprotein phosphatase in Streptomyces granaticolor

    Czech Academy of Sciences Publication Activity Database

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

    2000-01-01

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

  10. Enzyme kinetic characterization of protein tyrosine phosphatases

    DEFF Research Database (Denmark)

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

    2003-01-01

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

  11. Effects of fenspiride on human bronchial cyclic nucleotide phosphodiesterase isoenzymes: functional and biochemical study.

    Science.gov (United States)

    Cortijo, J; Naline, E; Ortiz, J L; Berto, L; Girard, V; Malbezin, M; Advenier, C; Morcillo, E J

    1998-01-02

    We have investigated the role of human bronchial cyclic nucleotide phosphodiesterases in the effects of fenspiride, a drug endowed with bronchodilator and anti-inflammatory properties. Functional studies on human isolated bronchi showed that fenspiride (10(-6)-3 x 10(-3) M, 30 min) induced a shift to the left of the concentration-response curves for isoprenaline and sodium nitroprusside with -logEC50 values of 4.1+/-0.1 (n = 7) and 3.5+/-0.2 (n = 8), respectively. Biochemical studies were carried out on three human bronchi in which separation of cyclic nucleotide phosphodiesterase isoenzymes was performed by ion exchange chromatography followed by determination of phosphodiesterase activity with a radioisotopic method. Phosphodiesterase 4 (cyclic AMP-specific) and phosphodiesterase 5 (cyclic GMP-specific) were the major phosphodiesterase isoforms present in the human bronchial tissue. The presence of phosphodiesterase 1 (Ca2+/calmodulin-stimulated), phosphodiesterase 2 (cyclic GMP-stimulated) and, in two cases, phosphodiesterase 3 (cyclic GMP-inhibited) was also identified. Fenspiride inhibited phosphodiesterase 4 and phosphodiesterase 3 activities with -logIC50 values of 4.16+/-0.09 and 3.44+/-0.12, respectively. Phosphodiesterase 5 activity was also inhibited with a -logIC50 value of approximately 3.8. Fenspiride (fenspiride is an effective inhibitor of both cyclic AMP and cyclic GMP hydrolytic activity in human bronchial tissues and this action may contribute to its airway effects.

  12. On the role of GAPDH isoenzymes during pentose fermentation in engineered Saccharomyces cerevisiae.

    Science.gov (United States)

    Linck, Annabell; Vu, Xuan-Khang; Essl, Christine; Hiesl, Charlotte; Boles, Eckhard; Oreb, Mislav

    2014-05-01

    In the metabolic network of the cell, many intermediary products are shared between different pathways. d-Glyceraldehyde-3-phosphate, a glycolytic intermediate, is a substrate of GAPDH but is also utilized by transaldolase and transketolase in the scrambling reactions of the nonoxidative pentose phosphate pathway. Recent efforts to engineer baker's yeast strains capable of utilizing pentose sugars present in plant biomass rely on increasing the carbon flux through this pathway. However, the competition between transaldolase and GAPDH for d-glyceraldehyde-3-phosphate produced in the first transketolase reaction compromises the carbon balance of the pathway, thereby limiting the product yield. Guided by the hypothesis that reduction in GAPDH activity would increase the availability of d-glyceraldehyde-3-phosphate for transaldolase and thereby improve ethanol production during fermentation of pentoses, we performed a comprehensive characterization of the three GAPDH isoenzymes in baker's yeast, Tdh1, Tdh2, and Tdh3 and analyzed the effect of their deletion on xylose utilization by engineered strains. Our data suggest that overexpression of transaldolase is a more promising strategy than reduction in GAPDH activity to increase the flux through the nonoxidative pentose phosphate pathway. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  13. Divergent lactate dehydrogenase isoenzyme profile in cellular compartments of primate forebrain structures.

    Science.gov (United States)

    Duka, Tetyana; Collins, Zachary; Anderson, Sarah M; Raghanti, Mary Ann; Ely, John J; Hof, Patrick R; Wildman, Derek E; Goodman, Morris; Grossman, Lawrence I; Sherwood, Chet C

    2017-07-01

    The compartmentalization and association of lactate dehydrogenase (LDH) with specific cellular structures (e.g., synaptosomal, sarcoplasmic or mitochondrial) may play an important role in brain energy metabolism. Our previous research revealed that LDH in the synaptosomal fraction shifts toward the aerobic isoforms (LDH-B) among the large-brained haplorhine primates compared to strepsirrhines. Here, we further analyzed the subcellular localization of LDH in primate forebrain structures using quantitative Western blotting and ELISA. We show that, in cytosolic and mitochondrial subfractions, LDH-B expression level was relatively elevated and LDH-A declined in haplorhines compared to strepsirrhines. LDH-B expression in mitochondrial fractions of the neocortex was preferentially increased, showing a particularly significant rise in the ratio of LDH-B to LDH-A in chimpanzees and humans. We also found a significant correlation between the protein levels of LDH-B in mitochondrial fractions from haplorhine neocortex and the synaptosomal LDH-B that suggests LDH isoforms shift from a predominance of A-subunits toward B-subunits as part of a system that spatially buffers dynamic energy requirements of brain cells. Our results indicate that there is differential subcellular compartmentalization of LDH isoenzymes that evolved among different primate lineages to meet the energy requirements in neocortical and striatal cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Phosphatase activity in Antarctica soil samples as a biosignature of extant life

    Science.gov (United States)

    Sato, Shuji; Itoh, Yuki; Takano, Yoshinori; Fukui, Manabu; Kaneko, Takeo; Kobayashi, Kensei

    Microbial activities have been detected in such extreme terrestrial environments as deep lithosphere, a submarine hydrothermal systems, stratosphere, and Antarctica. Microorganisms have adapted to such harsh environments by evolving their biomolecules. Some of these biomolecules such as enzymes might have different characteristics from those of organisms in ordinary environments. Many biosignatures (or biomarkers) have been proposed to detect microbial activities in such extreme environments. A number of techniques are proposed to evaluate biological activities in extreme environments including cultivation methods, assay of metabolism, and analysis of bioorganic compounds like amino acids and DNA. Enzyme activities are useful signature of extant life in extreme environments. Among many enzymes, phosphatase could be a good indicator of biological activities, since phosphate esters are essential for all the living terrestrial organisms. In addition, alkaline phosphatase is known as a typical zinc-containing metalloenzyme and quite stable in environments. We analyzed phosphatase activities in Antarctica soil samples to see whether they can be used as biosignatures for extant life. In addition, we characterized phosphatases extracted from the Antarctica soil samples, and compared with those obtained from other types of environments. Antarctica surface environments are quite severe environments for life since it is extremely cold and dry and exposed to strong UV and cosmic rays. We tried to evaluate biological activities in Antarctica by measuring phosphatase activities. Surface soil samples are obtained at the Sites 1-8 near Showa Base in Antarctica during the 47th Japan Antarctic exploration mission in 2005-6. Activities of acid phosphatase (ACP) and alkaline phosphatase (ALP) are measured spectrophotometrically after mixing the powdered sample and p-nitrophenyl phosphate solution (pH 6.5 for ACP, pH 8.0 for ALP). ALP was characterized after extraction from soils with

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

    Science.gov (United States)

    Okamura, Yasushi; Dixon, Jack E

    2011-02-01

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

  16. Clinical and preclinical treatment of urologic diseases with phosphodiesterase isoenzymes 5 inhibitors: an update

    Directory of Open Access Journals (Sweden)

    Wen-Hao Zhang

    2016-01-01

    Full Text Available Phosphodiesterase isoenzymes 5 inhibitors (PDE5-Is are the first-line therapy for erectile dysfunction (ED. The constant discoveries of nitric oxide (NO/cyclic guanosine monophosphate (cGMP cell-signaling pathway for smooth muscle (SM control in other urogenital tracts (UGTs make PDE5-Is promising pharmacologic agents against other benign urological diseases. This article reviews the literature and contains some previously unpublished data about characterizations and activities of PDE5 and its inhibitors in treating urological disorders. Scientific discoveries have improved our understanding of cell-signaling pathway in NO/cGMP-mediated SM relaxation in UGTs. Moreover, the clinical applications of PDE5-Is have been widely recognized. On-demand PDE5-Is are efficacious for most cases of ED, while daily-dosing and combination with testosterone are recommended for refractory cases. Soluble guanylate cyclase (sGC stimulators also have promising role in the management of severe ED conditions. PDE5-Is are also the first rehabilitation strategy for postoperation or postradiotherapy ED for prostate cancer patients. PDE5-Is, especially combined with α-adrenoceptor antagonists, are very effective for benign prostatic hyperplasia (BPH except on maximum urinary flow rate (Q max with tadalafil recently proved for BPH with/without ED. Furthermore, PDE5-Is are currently under various phases of clinical or preclinical researches with promising potential for other urinary and genital illnesses, such as priapism, premature ejaculation, urinary tract calculi, overactive bladder, Peyronie′s disease, and female sexual dysfunction. Inhibition of PDE5 is expected to be an effective strategy in treating benign urological diseases. However, further clinical studies and basic researches investigating mechanisms of PDE5-Is in disorders of UGTs are required.

  17. Purification of acidic phosphatase from mustard seedlings

    OpenAIRE

    sprotocols

    2014-01-01

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

  18. Enzymatic activity of proteases and its isoenzymes in fermentation process in cultivars of cocoa (Theobroma cacao L. produced in southern Bahia, Brazil

    Directory of Open Access Journals (Sweden)

    Luciane Santos SOUSA

    Full Text Available Abstract The fermentation of cocoa seeds envolves microbial processes and the action of enzymes. To identify the possible differences in the cocoa fermentation process, with regards to proteolysis, this study has the objective of determining protease activity (under predetermined conditions and its isoenzymes in two cocoa cultivars (PH-16 and HRT-1188 in different cocoa fermentation times, in addition to establishing the microbial load (molds and yeasts and aerobic mesophilic. Protease and its isoenzymes were extracted and partially purified and the enzymatic activities determined by spectrophotometry. The results showed that the proteases activity was higher at 66h of fermentation for both cultivars. When the isoenzymes activity was evaluated, the results demonstrated similar activity behavior for both cultivars, with regards to the isoenzymes aminopeptidase and carboxypeptidase, although the behavior of the endoprotease isoenzyme activity proved to be a little different for TSH-1188 cultivar. Concerning microbiological analyses, the results indicate that the period after molds and yeast counting reduction is consistent with the period of protease activity increase.

  19. The role of 11?-hydroxysteroid dehydrogenase type 1 and type2 isoenzymes on the pathogenesis of Cushing’s syndrome - doi:10.5020/18061230.2007.p104

    Directory of Open Access Journals (Sweden)

    Maria Betânia Pereira Toralles

    2012-01-01

    Full Text Available The action of glucocorticoids is modulated by isoenzymes 11?-hidroxiesteróide desidrogenases (11?-HSD type 1 and 2. The knowledge concerning these isoenzymes contribute to the understanding of the regulatory mechanisms involved in several disease processes of the Cushing’s syndrome, such as obesity, osteoporosis and hypertension. With the aim at describing the action of isoenzymes 11?-HSD type 1 and 2 in the Cushing’s syndrome, a literature review was done from 1990 - 2006 using the Medline data base, searching for the following key-words: Cushing’s syndrome, glucocorticoids, 11?-hydroxysteroid dehydrogenase, hypertension, osteoporosis and obesity. Review studies, meta-analysis and original articles were selected and chosen on the basis of methodological aspects and relevance. The exact mechanism by which cortisol increases blood pressure is not completely understood, but it involves, among others factors, changes in the sodium homeostasis. The conversion of cortisone to cortisol through expression of 11?-HSD1 induces the differentiation of preadipoctyes to mature adipoctyes and such patients develop an increase in visceral fat. The prevalence of osteoporosis in adult patients with Cushing’s syndrome is approximately 50% and glucocorticoids play a strong effect on the bone and calcium metabolism. The isoenzymes 11?-HSD1 and 11?-HSD2 have an important function in these several pathophysiology processes; however the isoenzymes action in the pathophysiology of the Cushing’s syndrome need to be more investigated.

  20. Misconceptions regarding basic thermodynamics and enzyme kinetics have led to erroneous conclusions regarding the metabolic importance of lactate dehydrogenase isoenzyme expression.

    Science.gov (United States)

    Bak, Lasse K; Schousboe, Arne

    2017-11-01

    Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate involving the coenzyme NAD + . Part of the foundation for the proposed shuttling of lactate from astrocytes to neurons during brain activation is the differential distribution of LDH isoenzymes between the two cell types. In this short review, we outline the basic kinetic properties of the LDH isoenzymes expressed in neurons and astrocytes, and argue that the distribution of LDH isoenzymes does not in any way govern directional flow of lactate between the two cellular compartments. The two main points are as follows. First, in line with the general concept of chemical catalysis, enzymes do not influence the thermodynamic equilibrium of a chemical reaction but merely the speed at which equilibrium is obtained. Thus, differential distribution of LDH isoenzymes with different kinetic parameters does not predict which cells are producing and which are consuming lactate. Second, the thermodynamic equilibrium of the reaction is toward the reduced substrate (i.e., lactate), which is reflected in the concentrations measured in brain tissue, suggesting that the reaction is at near-equilibrium at steady state. To conclude, the cellular distribution of LDH isoenzymes is of little if any consequence in determining any directional flow of lactate between neurons and astrocytes. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  1. Sample preparation in alkaline media

    International Nuclear Information System (INIS)

    Nobrega, Joaquim A.; Santos, Mirian C.; Sousa, Rafael A. de; Cadore, Solange; Barnes, Ramon M.; Tatro, Mark

    2006-01-01

    The use of tetramethylammonium hydroxide, tertiary amines and strongly alkaline reagents for sample treatment involving extraction and digestion procedures is discussed in this review. The preparation of slurries is also discussed. Based on literature data, alkaline media offer a good alternative for sample preparation involving an appreciable group of analytes in different types of samples. These reagents are also successfully employed in tailored speciation procedures wherein there is a critical dependence on maintenance of chemical forms. The effects of these reagents on measurements performed using spectroanalytical techniques are discussed. Several undesirable effects on transport and atomization processes necessitate use of the method of standard additions to obtain accurate results. It is also evident that alkaline media can improve the performance of techniques such as inductively coupled plasma mass spectrometry and accessories, such as autosamplers coupled to graphite furnace atomic absorption spectrometers

  2. The cytochrome P450 isoenzyme and some new opportunities for the prediction of negative drug interaction in vivo

    Directory of Open Access Journals (Sweden)

    Sychev DA

    2018-05-01

    Full Text Available Dmitrij A Sychev,1 Ghulam Md Ashraf,2 Andrey A Svistunov,3 Maksim L Maksimov,4 Vadim V Tarasov,3 Vladimir N Chubarev,3 Vitalij A Otdelenov,1 Natal’ja P Denisenko,1 George E Barreto,5,6 Gjumrakch Aliev7–9 1Russian Medical Academy of Postgraduate Education Studies, Moscow, Russia; 2King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia; 3Sechenov First Moscow State Medical University, Moscow, Russia; 4Branch Campus of the Federal State Budgetary Educational Institution of Further Professional Education «Russian Medical Academy of Continuous Professional Education» of the Ministry of Healthcare of the Russian Federation, Kazan State Medical Academy, Volga Region, Kazan, Russia; 5Departamento de Nutrición y Bioquímica, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá D.C., Colombia; 6Instituto de Ciencias Biomédicas, Universidad Autónoma de Chile, Santiago, Chile; 7GALLY International Biomedical Research Consulting LLC, San Antonio, TX, USA; 8School of Health Science and Healthcare Administration, University of Atlanta, Johns Creek, GA, USA; 9Institute of Physiologically Active Compounds Russian Academy of Sciences, Chernogolovka, Russia Abstract: Cytochrome (CYP 450 isoenzymes are the basic enzymes involved in Phase I biotransformation. The most important role in biotransformation belongs to CYP3A4, CYP2D6, CYP2C9, CYP2C19 and CYP1A2. Inhibition and induction of CYP isoenzymes caused by drugs are important and clinically relevant pharmacokinetic mechanisms of drug interaction. Investigation of the activity of CYP isoenzymes by using phenotyping methods (such as the determination of the concentration of specific substrates and metabolites in biological fluids during drug administration provides the prediction of negative side effects caused by drug interaction. In clinical practice, the process of phenotyping of CYP isoenzymes and some endogenous substrates in the ratio of cortisol to 6

  3. Structure, kinetic characterization and subcellular localization of the two ribulose 5-phosphate epimerase isoenzymes from Trypanosoma cruzi.

    Directory of Open Access Journals (Sweden)

    Soledad Natalia Gonzalez

    Full Text Available The enzyme of the pentose phosphate pathway (PPP ribulose-5-phosphate-epimerase (RPE is encoded by two genes present in the genome of Trypanosoma cruzi CL Brener clone: TcRPE1 and TcRPE2. Despite high sequence similarity at the amino acid residue level, the recombinant isoenzymes show a strikingly different kinetics. Whereas TcRPE2 follows a typical michaelian behavior, TcRPE1 shows a complex kinetic pattern, displaying a biphasic curve, suggesting the coexistence of -at least- two kinetically different molecular forms. Regarding the subcellular localization in epimastigotes, whereas TcRPE1 is a cytosolic enzyme, TcRPE2 is localized in glycosomes. To our knowledge, TcRPE2 is the first PPP isoenzyme that is exclusively localized in glycosomes. Over-expression of TcRPE1, but not of TcRPE2, significantly reduces the parasite doubling time in vitro, as compared with wild type epimastigotes. Both TcRPEs represent single domain proteins exhibiting the classical α/β TIM-barrel fold, as expected for enzymes with this activity. With regard to the architecture of the active site, all the important amino acid residues for catalysis -with the exception of M58- are also present in both TcRPEs models. The superimposition of the binding pocket of both isoenzyme models shows that they adopt essentially identical positions in the active site with a residue specific RMSD < 2Å, with the sole exception of S12, which displays a large deviation (residue specific RMSD: 11.07 Å. Studies on the quaternary arrangement of these isoenzymes reveal that both are present in a mixture of various oligomeric species made up of an even number of molecules, probably pointing to the dimer as their minimal functional unit. This multiplicity of oligomeric species has not been reported for any of the other RPEs studied so far and it might bear implications for the regulation of TcRPEs activity, although further investigation will be necessary to unravel the physiological

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

    Directory of Open Access Journals (Sweden)

    I. Lorenc-Kubis

    2015-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Lorenc-Kubis, I.; Morawiecka, B.

    1973-01-01

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

  6. Chromatographic separation and continuously referenced, on-line monitoring of creatine kinase isoenzymes by use of an immobilized-enzyme microreactor

    International Nuclear Information System (INIS)

    Denton, M.S.; Bostick, W.D.; Dinsmore, S.R.; Mrochek, J.E.

    1978-01-01

    We describe a new concept in continuously referenced monitoring of the isoenzyme activities of creatine kinase (EC 2.7.3.2) after liquid-chromatographic separation. After separation on a diethylaminoethyl-Sephacel column, the three isoenzymes of creatine kinase undergo a series of upled enzyme reactions, ultimately resulting in the formation of ultraviolet-detectable NADPH. A major advantage of this detection system is the immobilized-enzyme microreactor (2 x 17 mm), which may be removed and stored refrigerated when not in use. A split-stream configuration allows self-blanking of endogenous ultraviolet-absorbing constituents in authentic sera samples, which would otherwise make definitive diagnosis and quantitation difficult or impossible. This detection system is applicable to the automated analysis of creatine kinase isoenzymes in the clinical laboratory. 5 figures; 42 references

  7. Urinary N-acetyl-beta -D-glucosaminidase and its isoenzymes A & B in workers exposed to cadmium at cadmium plating

    Directory of Open Access Journals (Sweden)

    Rajan BK

    2007-07-01

    Full Text Available Abstract Objective The present study was carried out to determine the effect of cadmium exposure on Urinary N-acetyl-beta -D-glucosaminidase and its isoenzymes A and B in workers exposed at cadmium plating. Methods 50 subjects using cadmium during cadmium plating formed the study group. An equal number of age-sex matched subjects working in administrative section formed the control group. Urinary cadmium levels were determined by using a flameless atomic absorption spectrophotometer. Urinary N-acetyl-beta -D-glucosaminidase and its isoenzymes A and B were determined by using spectrophotmetric method. Results A significant increase of urinary total N-acetyl-beta -D-glucosaminidase and its isoenzymes A and B profiles were noted in study as compared to controls. The levels of urinary N-acetyl-beta -D-glucosaminidase and its isoenzymes A and B profiles were positively and significantly correlated with cadmium levels in urine. Multiple regression analysis was used to assess the effect of urinary cadmium or life style confounding factors (age, BMI, smoking and alcohol consumption on urinary N-acetyl-beta -D-glucosaminidase and its isoenzymes A and B. The analysis showed that the study subjects who had urine cadmium levels greater than 5 μg/g of creatinine, work duration >15 years, smoking and body mass index variables were significantly associated with urinary total N-acetyl-beta -D-glucosaminidase but not on isoenzymes A&B. Conclusion The results presented in this study shows that the increased levels of urinary N-acetyl-beta -D-glucosaminidase observed in cadmium-exposed workers could be used as biomarkers for suggesting preventive measure.

  8. Regulation of CD93 cell surface expression by protein kinase C isoenzymes.

    Science.gov (United States)

    Ikewaki, Nobunao; Kulski, Jerzy K; Inoko, Hidetoshi

    2006-01-01

    -lines. Whereas recombinant tumor necrosis factor-alpha (rTNF-alpha) slightly up-regulated CD93 expression on the U937 cells, recombinant interleukin-1beta (rIL-1beta), recombinant interleukin-2 (rIL-2), recombinant interferon-gamma (rIFN-gamma) and lipopolysaccharide (LPS) had no effect. Taken together, these findings indicate that the regulation of CD93 expression on these cells involves the PKC isoenzymes.

  9. Enzymatic Kinetic Properties of the Lactate Dehydrogenase Isoenzyme C4 of the Plateau Pika (Ochotona curzoniae

    Directory of Open Access Journals (Sweden)

    Yang Wang

    2016-01-01

    Full Text Available Testis-specific lactate dehydrogenase (LDH-C4 is one of the lactate dehydrogenase (LDH isozymes that catalyze the terminal reaction of pyruvate to lactate in the glycolytic pathway. LDH-C4 in mammals was previously thought to be expressed only in spermatozoa and testis and not in other tissues. Plateau pika (Ochotona curzoniae belongs to the genus Ochotona of the Ochotonidea family. It is a hypoxia-tolerant species living in remote mountain areas at altitudes of 3000–5000 m above sea level on the Qinghai-Tibet Plateau. Surprisingly, Ldh-c is expressed not only in its testis and sperm, but also in somatic tissues of plateau pika. To shed light on the function of LDH-C4 in somatic cells, Ldh-a, Ldh-b, and Ldh-c of plateau pika were subcloned into bacterial expression vectors. The pure enzymes of Lactate Dehydrogenase A4 (LDH-A4, Lactate Dehydrogenase B4 (LDH-B4, and LDH-C4 were prepared by a series of expression and purification processes, and the three enzymes were identified by the method of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE and native polyacrylamide gel electrophoresis (PAGE. The enzymatic kinetics properties of these enzymes were studied by Lineweaver-Burk double-reciprocal plots. The results showed the Michaelis constant (Km of LDH-C4 for pyruvate and lactate was 0.052 and 4.934 mmol/L, respectively, with an approximate 90 times higher affinity of LDH-C4 for pyruvate than for lactate. At relatively high concentrations of lactate, the inhibition constant (Ki of the LDH isoenzymes varied: LDH-A4 (Ki = 26.900 mmol/L, LDH-B4 (Ki = 23.800 mmol/L, and LDH-C4 (Ki = 65.500 mmol/L. These data suggest that inhibition of lactate by LDH-A4 and LDH-B4 were stronger than LDH-C4. In light of the enzymatic kinetics properties, we suggest that the plateau pika can reduce reliance on oxygen supply and enhance its adaptation to the hypoxic environments due to increased anaerobic glycolysis by LDH-C4.

  10. Zoonotic cutaneous leishmaniasis in Shiraz, Southern Iran: A molecular, isoenzyme and morphologic approach

    Directory of Open Access Journals (Sweden)

    Qasem Asgari

    2007-05-01

    Full Text Available

    BACKGROUND: Cutaneous leishmaniasis (CL with diverse clinical manifestations is prevalent and remains a major public health problem in Iran and its incidence has been doubled over the last decade. The present study is about the potential role of rodents in the epidemiology of CL in Kharameh district in Shiraz, Southern Iran.

    METHODS: From April 2004 to April 2005, a total of sixteen rodents were collected in live traps from the endemic area of CL in Kharameh district in Shiraz. Evans medium was used for culture. Specific polymerase chain reaction and isoenzyme electrophoresis methods were performed to characterize the parasite.

    RESULTS: The rodent species were Tatera indica. Three samples from Tatera indica were found positive (2 males and 1 female in Kafdehak and Sejel-Abad villages for L. major. Macrophages in the bone marrow of femoral bone were infected with the amastigote form of the parasite.

    CONCLUSIONS: It seems that T. indica is the reservoir host for CL in Kharameh (a district in Shiraz, Southern Iran. It was shown that the bone marrow of the rodents is the tissue of choice for light and ultrastructural studies of

  11. Alkaline resistant ceramics; Alkalimotstaandskraftiga keramer

    Energy Technology Data Exchange (ETDEWEB)

    Westberg, Stig-Bjoern [Vattenfall Utveckling AB, Aelvkarleby (Sweden)

    2001-02-01

    Despite durability in several environments, ceramics and refractories can not endure alkaline environments at high temperature. An example of such an environment is when burning biofuel in modern heat and power plants in which the demand for increasing efficiency results in higher combustion temperatures and content of alkaline substances in the flue gas. Some experiences of these environments has been gained from such vastly different equipment as regenerator chambers in the glass industry and MHD-generators. The grains of a ceramic material are usually bonded together by a glassy phase which despite it frequently being a minor constituent render the materials properties and limits its use at elevated temperature. The damage is usually caused by alkaline containing low-melting phases and the decrease of the viscosity of the bonding glass phase which is caused by the alkaline. The surfaces which are exposed to the flue gas in a modern power plant are not only exposed to the high temperature but also a corroding and eroding, particle containing, gas flow of high velocity. The use of conventional refractory products is limited to 1300-1350 deg C. Higher strength and fracture toughness as well as durability against gases, slag and melts at temperatures exceeding 1700 deg C are expected of the materials of the future. Continuous transport of corrosive compounds to the surface and corrosion products from the surface as well as a suitable environment for the corrosion to occur in are prerequisites for extensive corrosion to come about. The highest corrosion rate is therefore found in a temperature interval between the dew point and the melting point of the alkaline-constituent containing compound. It is therefore important that the corrosion resistance is sufficient in the environment in which alkaline containing melts or slag may appear. In environments such as these, even under normal circumstances durable ceramics, such as alumina and silicon carbide, are attacked

  12. Toxicokinetics of drugs of abuse: current knowledge of the isoenzymes involved in the human metabolism of tetrahydrocannabinol, cocaine, heroin, morphine, and codeine.

    Science.gov (United States)

    Maurer, Hans H; Sauer, Christoph; Theobald, Denis S

    2006-06-01

    This review summarizes the major metabolic pathways of the drugs of abuse, tetrahydrocannabinol, cocaine, heroin, morphine, and codeine, in humans including the involvement of isoenzymes. This knowledge may be important for predicting their possible interactions with other xenobiotics, understanding pharmaco-/toxicokinetic and pharmacogenetic variations, toxicological risk assessment, developing suitable toxicological analysis procedures, and finally for understanding certain pitfalls in drug testing. The detection times of these drugs and/or their metabolites in biological samples are summarized and the implications of the presented data on the possible interactions of drugs of abuse with other xenobiotics, ie, inhibition or induction of individual polymorphic and nonpolymorphic isoenzymes, discussed.

  13. UV-induced changes of structural and functional properties of blood lactatedehydrogenase isoenzymes in free sate and in the presence of serotonin

    International Nuclear Information System (INIS)

    Nakvasina, M.A.; Artyukhov, V.G.; Agisheva, N.V.

    1999-01-01

    Photoinduced changes in structural and functional properties of lactatedehydrogenase isoenzymes from human erythrocytes in free state and in the presence of serotonin have been studied by means of gel chromatography, electrophoresis, IR-spectrophotometry and by the method of definition of catalytic activity. UV radiation exposure induces photoinactivation of erythrocytes lactatedehydrogenase, while its inhibitory effect intensifies with the increase of radiation dose. Pronounced photoprotective effect of serotonin towards the molecules of erythrocytic lactatedehydrogenase isoenzymes has been discovered. It seems to be caused by formation of enzyme - biogenic amine complex affecting the secondary protein structure [ru

  14. Preliminary study on peroxidase isoenzyme detection and RAPD molecular verification for sweet pepper 87-2 carried by a recoverable satellite

    International Nuclear Information System (INIS)

    Liu Min; Li Jinguo; Wang Yalin; Zhang Zan; Xue Huai; Zhang Chunhua; Li Sherong

    1999-01-01

    The detection technology of peroxidase isoenzyme pattern and RAPD (Randomly Amplified Polymorphic DNA) analysis were used to space-flown sweet pepper variety of 87-2 carried by a recoverable satellite, and its correspondent ground control Longjiao-2. Determination of peroxidase isoenzyme revealed that the physiological activities of space-flown sweet pepper were significantly higher than those of its ground control. To detect the polymorphism of gene sequence and the difference in molecular level. 42 primers were used, among them, 38 primers amplified polymorphic products, and the amplified products from 5 primers of sweet pepper 87-2 differed from those of ground control

  15. Elevated CK-MB isoenzyme after exercise stress test and atrial pacing in patients with ischemic heart disease

    International Nuclear Information System (INIS)

    Marmor, A.T.; Klein, R.; Plich, M.; Groshar, D.; Schneeweiss, A.

    1988-01-01

    Using a highly sensitive monoclonal antibody kit for CK-MB, significant release of small amounts of CK-MB isoenzyme after exercise stress test was detected 4 to 6 h after induction of ischemia. This occurred in ten out of 15 patients with ischemic heart disease (66 percent) and in only one of the 18 healthy subjects (5.6 percent) serving as a control group. In five patients with coronary artery disease in whom atrial pacing was performed with simultaneous blood sampling from coronary sinus, a drastic elevation in CK-MB isoenzyme (from 2.04 +/- 2.06 ng/L to 10.88 +/- 6.9 ng/L; p less than 0.001) was detected within 10 to 30 min after induction of acute ischemia. A small but significant increase in total CK also was detected (from 21 +/- 12 IU/L to 52 +/- 14IU/L; p less than 0.01). These preliminary observations have to be further investigated in a larger group of patients before a definitive conclusion can be reached about the clinical significance of CK-MB release during exercise

  16. Macro creatine kinases: results of isoenzyme electrophoresis and differentiation of the immunoglobulin-bound type by radioassay

    International Nuclear Information System (INIS)

    Bohner, J.; Stein, W.; Steinhart, R.; Wurzburg, U.; Eggstein, M.

    1982-01-01

    In 2.9% of sera from 1253 unselected patients we detected two different types of macromolecular creatine kinases (CK;EC 2.7.3.2). One macro type was represented by immunoglobulin-linked CK; in sera containing macro CK-BB isoenzyme, 125 I-labeled CK-BB was bound with high affinity to the immunoglobulin fraction. Furthermore, during electrophoresis, macro CK-BB mostly migrated between CK-MB and CK-MM, and was fixed to Protein A from Staphlococcus aureus. We therefore propose radioelectrophoresis as a specific, highly sensitive, and simple method for detecting this type of macro CK. This form occurs predominantly in elderly women, is not correlated to any specific disease, and persists in blood over a long period of time. In contrast, a second type (macro-CK type 2) never bound radiolabeled CK isoenzymes, and was not adsorbed to protein A. Electrophoretic migration of this macro-CK type 2 was generally cathodic to CK-MM. We observed this type in severely ill patients, frequently those suffering from malignant tumors. Clinical observations and biochemical data suggest that macro-CK type 2 is of mitochondrial origin

  17. Cytological and isoenzyme analysis of the Bucay and Quevedo cytotypes of the onchocerciasis vector Simulium exiguum (Diptera: Simuliidae in Ecuador

    Directory of Open Access Journals (Sweden)

    M. Charalambous

    1993-03-01

    Full Text Available Four cytotypes of Simulium exiguum occur in Ecuador, where this morphospecies is the primary vector of onchocerciasis. In this paper, we give the first full description of the banding pattern of the larval polytene chromosomes of the Quevedo cytotypes differ from the chromosomal standard sequence (of the Cayapa cytotype by the fixed inversions IIL-5 and IIL-6. The Quevedo cytotype additionally differs from the standard and Bucay cytotypes by processing a differentiated X chromosome, wich is indicated by the inversion IIS-A. As the degree of reproductive isolation between the Bucay and Quevedo cytotypes has not yet been estabilished, they must be regarded as intraspecific variants of the same species. In fact, isoenzyme characterizations showed that the Bucay and Quevedo cytotypes are differentiated only to the extent expected of incipient species or geographical populations. Moreover, the sibiling species status previously given to the Bucay cytotype needs be reassessed, there being inadequate analysis from areas in Ecuador where Bucay occurs in sympatry with the standard Cayapa cytotype. No isoenzyme electromorphs were discovered that identified all or mostadult females of any one (cytotype-pure collection.

  18. Optical properties of alkaline earth borate glasses

    African Journals Online (AJOL)

    user

    ... devices; radiation shields, surgical lasers and their glass ceramic counter ... Alkaline earth oxides improve glass forming capability while heavy metal ... reports on optical properties of MO-B2O3 glasses containing alkaline earth oxides.

  19. Qualitative Carbohydrate Analysis using Alkaline Potassium ...

    Indian Academy of Sciences (India)

    IAS Admin

    CLASSROOM. 285. RESONANCE | March 2016. Qualitative Carbohydrate Analysis using Alkaline. Potassium Ferricyanide. Keywords. Alkaline potassium ferricyanide, qualitative ... Carbohydrates form a distinct class of organic compounds often .... Laboratory Techniques: A contemporary Approach, W B Saunders Com-.

  20. Trehalose 6-phosphate phosphatases of Pseudomonas aeruginosa.

    Science.gov (United States)

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

    2018-04-24

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

  1. Development of alkaline fuel cells.

    Energy Technology Data Exchange (ETDEWEB)

    Hibbs, Michael R.; Jenkins, Janelle E.; Alam, Todd Michael; Janarthanan, Rajeswari; Horan, James L.; Caire, Benjamin R.; Ziegler, Zachary C.; Herring, Andrew M.; Yang, Yuan; Zuo, Xiaobing; Robson, Michael H.; Artyushkova, Kateryna; Patterson, Wendy; Atanassov, Plamen Borissov

    2013-09-01

    This project focuses on the development and demonstration of anion exchange membrane (AEM) fuel cells for portable power applications. Novel polymeric anion exchange membranes and ionomers with high chemical stabilities were prepared characterized by researchers at Sandia National Laboratories. Durable, non-precious metal catalysts were prepared by Dr. Plamen Atanassovs research group at the University of New Mexico by utilizing an aerosol-based process to prepare templated nano-structures. Dr. Andy Herrings group at the Colorado School of Mines combined all of these materials to fabricate and test membrane electrode assemblies for single cell testing in a methanol-fueled alkaline system. The highest power density achieved in this study was 54 mW/cm2 which was 90% of the project target and the highest reported power density for a direct methanol alkaline fuel cell.

  2. Die Verwendung von Isoenzym-Polymorphismen - eine Herausforderungen bei der Züchtung neuer Baldriansorten (Valeriana officinalis L. s.l.

    Directory of Open Access Journals (Sweden)

    Penzkofer, Michael

    2014-09-01

    Full Text Available Mit Hilfe von Isoenzym-Polymorphismen sollte versucht werden, biochemische Unterschiede (Fingerprint zwischen verschiedenen Baldrianherkünften darzustellen, um im Anschluss diejenigen Individuen zu finden, die durch Kreuzung und nicht durch Selbstung entstanden sind. Bei Baldrian konnte für diese Methode kein Verfahrensprotokoll etabliert werden, womit auch eine Untersuchung von Kreuzungsnachkommen nicht erfolgte.

  3. Isoenzyme-specific up-regulation of glutathione transferase and aldo-keto reductase mRNA expression by dietary quercetin in rat liver.

    Science.gov (United States)

    Odbayar, Tseye-Oidov; Kimura, Toshinori; Tsushida, Tojiro; Ide, Takashi

    2009-05-01

    The impact of quercetin on the mRNA expression of hepatic enzymes involved in drug metabolism was evaluated with a DNA microarray and real-time PCR. Male Sprague-Dawley rats were fed an experimental diet containing either 0, 2.5, 5, 10, or 20 g/kg of quercetin for 15 days. The DNA microarray analysis of the gene expression profile in pooled RNA samples from rats fed diets containing 0, 5, and 20 g/kg of quercetin revealed genes of some isoenzymes of glutathione transferase (Gst) and aldo-keto reductase (Akr) to be activated by this flavonoid. Real-time PCR conducted with RNA samples from individual rats fed varying amounts of quercetin together with the microarray analysis showed that quercetin caused marked dose-dependent increases in the mRNA expression of Gsta3, Gstp1, and Gstt3. Some moderate increases were also noted in the mRNA expression of isoenzymes belonging to the Gstm class. Quercetin also dose-dependently increased the mRNA expression of Akr1b8 and Akr7a3. However, it did not affect the parameters of the other Gst and Akr isoenzymes. It is apparent that quercetin increases the mRNA expression of Gst and Akr involved in drug metabolism in an isoenzyme-specific manner. Inasmuch as Gst and Akr isoenzymes up-regulated in their gene expression are involved in the prevention and attenuation of cancer development, this consequence may account for the chemopreventive propensity of quercetin.

  4. Serum total and bone alkaline phosphatase and tartrate-resistant acid phosphatase activities for the assessment of bone fracture healing in dogs

    Directory of Open Access Journals (Sweden)

    C. Sousa

    2011-08-01

    Full Text Available O objetivo deste trabalho foi estudar o padrão de variação da atividade sérica da fosfatase alcalina total (tALP, da isoenzima óssea da fosfatase alcalina (BALP e da fosfatase ácida resistente ao tartarato (TRAP, assim como a variação da concentração dos minerais séricos durante o processo de cicatrização de fraturas ósseas no cão. A variação sérica destes marcadores do metabolismo ósseo foi avaliada em nove cães com fraturas diafisárias fechadas de ossos longos, submetidas a tratamento cirúrgico para osteosíntese. Durante o período pós-operatório, sete animais evoluíram no sentido de uma normal união óssea, sendo que dois deles desenvolveram um processo de não união óssea. Foram observados, relativamente à BALP, valores de actividade sérica mais elevados e com diferença estatística (P<0,05 no grupo de animais que evoluiu no sentido de uma normal união óssea, comparativamente ao grupo de animais que evoluiu no sentido do processo de não união. No grupo de animais que evoluiu para a completa união óssea foram, adicionalmente, observados valores diminuidos (P<0,05 da atividade sérica da TRAP, até ao dia 60 do período pós-operatório seguido de uma elevação estatisticamente significativa após este período. Em conclusão, os biomarcadores do metabolismo ósseo poderão vir a constituir um método auxiliar de diagnóstico na monitorização do processo de cicatrização de fracturas ósseas, possibilitando, a detecção precoce de complicações pós-operatórias.

  5. Effect of plaster cast immobilization on the turnover rates of soluble proteins and lactate dehydrogenase isoenzymes of rabbit M. soleus

    Energy Technology Data Exchange (ETDEWEB)

    Edes, I.; Dosa, E.; Sohar, I.; Guba, F. (Orvostudomanyi Egyetem, Szeged (Hungary). Biokemiai Tanszek)

    1982-01-01

    In atrophized muscle the decreases of the activity of LDH isoenzymes can be explained partly by a 15 per cent decrease of the enzyme synthesis and partly by a 25 per cent increase in catabolism. The quantities of the soluble proteins and LDH were measured after intravenously administered /sup 3/H-leucin incorporation, from the musculus soleus. LDH was isolated by means of affinity chromatography. Radioactivity was determined in a Packard Tri-Carb scintillation counter. The synthesis rate of soluble proteins barely changed during immobilization. In the atrophized muscle the decrease of the amount of soluble proteins could be almost exclusively interpreted in terms of a 25 per cent enchancement of degradative process. The accelerated catabolism is most probably due to the proteolytic enzymes activated by immobilization.

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

    Directory of Open Access Journals (Sweden)

    Kirstin eHobiger

    2015-02-01

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

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

    DEFF Research Database (Denmark)

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

    1995-01-01

    An experiment was set up to test the ability of arbuscular mycorrhizal (AM) roots and hyphae to produce extracellular phosphatases and to study the relationship between phosphatase activity and soil organic P (P-o). Non-mycorrhizal cucumber and cucumber in symbiosis with either of two mycorrhizal...... fungi were grown in a sandy loam-sand mixture in three-compartment pots. Plant roots were separated from two consecutively adjoining compartments, first by a 37 m mesh excluding roots and subsequently by a 0.45 m membrane excluding mycorrhizal hyphae. Soil from the two root-free compartments...... was sectioned in a freezing microtome and analyzed for extracellular acid (pH 5.2) and alkaline (pH 8.5) phosphatase activity as well as depletion of NaHCO-3-extractable inorganic P (P-i) and P-o. Roots and mycorrhizal hyphae depleted the soil of P-i but did not influence the concentration of P-o in spite...

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

    NARCIS (Netherlands)

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

    1995-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Beata Kuziemska

    2014-10-01

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

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

    Directory of Open Access Journals (Sweden)

    I. Lorenc-Kubis

    2015-01-01

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

  11. Inter- and intra-population genetic variability of introduced silkworm (Bombyx mori L.) strains raised in Bulgaria

    OpenAIRE

    Teodora Staykova

    2013-01-01

    The genetic variability of four populations belonging to two introduced silkworm strains (Bombyx mori L.) of various origins has been studied using isoenzymic analysis of six enzyme systems. Nonspecific esterases, phosphoglucomutase, malate dehydrogenase, acid phosphatase, alkaline phosphatase and hexokinase from different tissue of larvae 5th instar have been analysed using PAGE. Polymorphism in six from a total of nine loci has been found. Inter- and intra-population differences have been a...

  12. Progress of research on the influence of alkaline cation and alkaline solution on bentonite properties

    International Nuclear Information System (INIS)

    Ye Weimin; Zheng Zhenji; Chen Bao; Chen Yonggui

    2011-01-01

    Based on the previous laboratory studies and numerical simulation on bentonite in alkaline environments, the effects of alkaline cation and alkaline solution on mineral composition, microstructure, swelling capacity and hydraulic properties of bentonite are emphasized in this paper, temperature, pH values and concentration are discussed as main affecting factors. When bentonite is exposed to alkaline cation or alkaline solution, microstructure of bentonite will be changed due to the dissolution of montmorillonite and the formation of secondary minerals, which results in the decrease of swelling pressure. The amount of the reduction of swelling pressure depends on the concentration of alkaline solution. Temperature, polyvalent cation, salinity and concentration are the main factors affecting hydraulic properties of bentonite under alkaline conditions. Therefore, future research should focus on the mechanism of coupling effects of weak alkaline solutions on the mineral composition, microstructure, swelling capacity and hydraulic properties of bentonite under different temperatures and different pH values. (authors)

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

    DEFF Research Database (Denmark)

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

    1990-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Marie-Claude Gingras

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

  15. Alkaline azide mutagenicity in cowpea

    Energy Technology Data Exchange (ETDEWEB)

    Mahna, S K; Bhargava, Anubha; Mohan, Lalit [Cytogenetics and Mycology Laboratory, Department of Botany, Government College, Ajmer (India)

    1990-07-01

    Sodium azide is known as a potent mutagen in cereals and legumes. It is very effective in acidic medium in barley. Here an attempt is made to measure the effectiveness of sodium azide in alkaline medium (pH 7.4) on cowpea (Vigna unguiculata (L.) Walp., variety FS-68). Seeds pre-soaked in distilled water for 5 hours were treated with different concentrations (10{sup -6}, 10{sup -5}, 10{sup -4} and 10{sup -3}M) of sodium azide (NaN{sub 3}) for 4 hours at 28{+-} 2 deg. C. Bottles were intermittently shaken, then the seeds were thoroughly washed in running tap water and subsequently planted in pots. The treatment caused significant biological damage such as reduction in seed germination, length of root and shoot, number of nodules and pods per plant and morphological leaf variations. Morphological, as well as chlorophyll mutants, were detected in M{sub 2}.

  16. Alkaline azide mutagenicity in cowpea

    International Nuclear Information System (INIS)

    Mahna, S.K.; Bhargava, Anubha; Mohan, Lalit

    1990-01-01

    Sodium azide is known as a potent mutagen in cereals and legumes. It is very effective in acidic medium in barley. Here an attempt is made to measure the effectiveness of sodium azide in alkaline medium (pH 7.4) on cowpea (Vigna unguiculata (L.) Walp., variety FS-68). Seeds pre-soaked in distilled water for 5 hours were treated with different concentrations (10 -6 , 10 -5 , 10 -4 and 10 -3 M) of sodium azide (NaN 3 ) for 4 hours at 28± 2 deg. C. Bottles were intermittently shaken, then the seeds were thoroughly washed in running tap water and subsequently planted in pots. The treatment caused significant biological damage such as reduction in seed germination, length of root and shoot, number of nodules and pods per plant and morphological leaf variations. Morphological, as well as chlorophyll mutants, were detected in M 2

  17. Redox Regulation of Receptor Protein-Tyrosine Phosphatases

    NARCIS (Netherlands)

    Groen, A.J.

    2006-01-01

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

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

    Indian Academy of Sciences (India)

    MAMONI GARAI

    2017-09-19

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

  19. Novel Combinatorial Chemistry-Derived Inhibitors of Oncogenic Phosphatases

    National Research Council Canada - National Science Library

    Lazo, John

    1999-01-01

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

  20. catalysed oxidation of atenolol by alkaline permanganate

    Indian Academy of Sciences (India)

    Unknown

    Abstract. Kinetics of ruthenium (III) catalyzed oxidation of atenolol by permanganate in alkaline medium at constant ionic strength of 0⋅30 mol dm3 has been studied spectrophotometrically using a rapid kinetic accessory. Reaction between permanganate and atenolol in alkaline medium exhibits 1 : 8 stoichiometry.

  1. Effect of vanadium compounds on acid phosphatase activity

    OpenAIRE

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

    1996-01-01

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

  2. The pro-coagulant fibrinogenolytic serine protease isoenzymes purified from Daboia russelii russelii venom coagulate the blood through factor V activation: role of glycosylation on enzymatic activity.

    Directory of Open Access Journals (Sweden)

    Ashis K Mukherjee

    Full Text Available Proteases from Russell's viper venom (RVV induce a variety of toxic effects in victim. Therefore, four new RVV protease isoenzymes of molecular mass 32901.044 Da, 333631.179 Da, 333571.472 Da, and 34594.776 Da, were characterized in this study. The first 10 N-terminal residues of these serine protease isoenzymes showed significant sequence homology with N-terminal sequences of snake venom thrombin-like and factor V-activating serine proteases, which was reconfirmed by peptide mass fingerprinting analysis. These proteases were found to be different from previously reported factor V activators isolated from snake venoms. These proteases showed significantly different fibrinogenolytic, BAEE-esterase and plasma clotting activities but no fibrinolytic, TAME-esterase or amidolytic activity against the chromogenic substrate for trypsin, thrombin, plasmin and factor Xa. Their Km and Vmax values towards fibrinogen were determined in the range of 6.6 to 10.5 µM and 111.0 to 125.5 units/mg protein, respectively. On the basis of fibrinogen degradation pattern, they may be classified as A/B serine proteases isolated from snake venom. These proteases contain ∼ 42% to 44% of N-linked carbohydrates by mass whereas partially deglycosylated enzymes showed significantly less catalytic activity as compared to native enzymes. In vitro these protease isoenzymes induce blood coagulation through factor V activation, whereas in vivo they provoke dose-dependent defibrinogenation and anticoagulant activity in the mouse model. At a dose of 5 mg/kg, none of these protease isoenzymes were found to be lethal in mice or house geckos, suggesting therapeutic application of these anticoagulant peptides for the prevention of thrombosis.

  3. Isolation and isoenzyme characterization of Leishmania (Viannia braziliensis from a case of human cutaneous leishmaniasis in northeast centre of the state of São Paulo

    Directory of Open Access Journals (Sweden)

    MC Pinto

    2005-11-01

    Full Text Available The diagnosis of human cutaneous leishmaniasis in small towns is sometimes made without the species identification of the Leishmania, even in areas without previous epidemiological surveys. Here we report the isolation of a Leishmania strain from a patient of Rincão, state of São Paulo, that was identified by isoenzyme characterization as L. (Viannia braziliensis. Sand fly collections were made in the area where the patient live in order to investigate the likely vector species.

  4. Purkinje Cell Compartmentation in the Cerebellum of the Lysosomal Acid Phosphatase 2 Mutant Mouse (Nax - Naked-Ataxia Mutant Mouse)

    Science.gov (United States)

    Bailey, Karen; Rahimi Balaei, Maryam; Mannan, Ashraf; Del Bigio, Marc R.; Marzban, Hassan

    2014-01-01

    The Acp2 gene encodes the beta subunit of lysosomal acid phosphatase, which is an isoenzyme that hydrolyzes orthophosphoric monoesters. In mice, a spontaneous mutation in Acp2 results in severe cerebellar defects. These include a reduced size, abnormal lobulation, and an apparent anterior cerebellar disorder with an absent or hypoplastic vermis. Based on differential gene expression in the cerebellum, the mouse cerebellar cortex can normally be compartmentalized anteroposteriorly into four transverse zones and mediolaterally into parasagittal stripes. In this study, immunohistochemistry was performed using various Purkinje cell compartmentation markers to examine their expression patterns in the Acp2 mutant. Despite the abnormal lobulation and anterior cerebellar defects, zebrin II and PLCβ4 showed similar expression patterns in the nax mutant and wild type cerebellum. However, fewer stripes were found in the anterior zone of the nax mutant, which could be due to a lack of Purkinje cells or altered expression of the stripe markers. HSP25 expression was uniform in the central zone of the nax mutant cerebellum at around postnatal day (P) 18–19, suggesting that HSP25 immunonegative Purkinje cells are absent or delayed in stripe pattern expression compared to the wild type. HSP25 expression became heterogeneous around P22–23, with twice the number of parasagittal stripes in the nax mutant compared to the wild type. Aside from reduced size and cortical disorganization, both the posterior zone and nodular zone in the nax mutant appeared less abnormal than the rest of the cerebellum. From these results, it is evident that the anterior zone of the nax mutant cerebellum is the most severely affected, and this extends beyond the primary fissure into the rostral central zone/vermis. This suggests that ACP2 has critical roles in the development of the anterior cerebellum and it may regulate anterior and central zone compartmentation. PMID:24722417

  5. 130 kDa phosphatase from the liver of labeo rohita: isolation: purification and some kinetic properties

    International Nuclear Information System (INIS)

    Siddiqua, A.; Sherazi, M.; Shah, A.H.; Khan, A.R.; Khan, H.U.

    2009-01-01

    An isoenzyme of high molecular weight acid phosphatase (HM-ACP) from the live of fish rohu (Labeo Rohita) was isolated and purified to homogeneity. The enzyme had specific activity of 14.96 U/mg and a recovery of about 4%. The purification procedure included ammonium sulphate precipitation and series of chromatographic separations on SP-Sephadex C-50, CM-Cellulose and Sephacryl HR-200 columns. Nealry 500-folds purification was achieved. The molecular weight was estimated to be 120-130 kDa by polyacrylamide gel electrophoresis (PAGE) of native enzyme and 130 kDa by gel filtration on calibrated Sephadex G-100 column. sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) under reduced and non-reduced condition showed a band corresponding to 66 kDa confirming the dimeric nature of enzyme. para nitrophenyl phosphate and flavin mononucleotide were hydrolyzed effectively by the enzyme and found to be good substrates. Optimum temperature for the enzyme was 50 degree C and temperature stability was 0 degree-50 degree C. Similarly optimum ph for the enzyme was 5.4 and ph stability was 4.8-6.0. The K/sub m/ for the p-nitrophenyl phosphate was estimated to be 0.15 mM. The enzyme was competitively inhibited by the phosphate, vanadate, molybdate, tartrate, fluoride and pyridoxal-5-PO/sub 4/ while pyridoxamine-5-PO/sub 4/ showed poor inhibition. Metal ions such as Ag/sup +/, Cu/sup ++/ Zn/sup ++/ showed strong inhibition on the enzyme activity while other divalent ions like Mg/sup ++/, Mn/sup ++/ and Co/sup ++/ were found to be poor inhibitors. Modifiers like EDTA, methanol, ethanol, acetone and glycerol had no effect on the enzyme's activity. (author)

  6. Alkaline pH sensor molecules.

    Science.gov (United States)

    Murayama, Takashi; Maruyama, Ichiro N

    2015-11-01

    Animals can survive only within a narrow pH range. This requires continual monitoring of environmental and body-fluid pH. Although a variety of acidic pH sensor molecules have been reported, alkaline pH sensor function is not well understood. This Review describes neuronal alkaline pH sensors, grouped according to whether they monitor extracellular or intracellular alkaline pH. Extracellular sensors include the receptor-type guanylyl cyclase, the insulin receptor-related receptor, ligand-gated Cl- channels, connexin hemichannels, two-pore-domain K+ channels, and transient receptor potential (TRP) channels. Intracellular sensors include TRP channels and gap junction channels. Identification of molecular mechanisms underlying alkaline pH sensing is crucial for understanding how animals respond to environmental alkaline pH and how body-fluid pH is maintained within a narrow range. © 2015 Wiley Periodicals, Inc.

  7. The genes of all seven CYP3A isoenzymes identified in the equine genome are expressed in the airways of horses.

    Science.gov (United States)

    Tydén, E; Löfgren, M; Hakhverdyan, M; Tjälve, H; Larsson, P

    2013-08-01

    In the present study, we examined the gene expression of cytochrome P450 3A (CYP3A) isoenzymes in the tracheal and bronchial mucosa and in the lung of equines using TaqMan probes. The results show that all seven CYP3A isoforms identified in the equine genome, that is, CYP3A89, CYP3A93, CYP3A94, CYP3A95, CYP3A96, CYP3A97 and CYP3A129, are expressed in the airways of the investigated horses. Though in previous studies, CYP3A129 was found to be absent in equine intestinal mucosa and liver, this CYP3A isoform is expressed in the airways of horses. The gene expression of the CYP3A isoenzymes varied considerably between the individual horses studied. However, in most of the horses CYP3A89, CYP3A93, CYP3A96, CYP3A97 and CYP3A129 were expressed to a high extent, while CYP3A94 and CYP3A95 were expressed to a low extent in the different parts of the airways. The CYP3A isoenzymes present in the airways may play a role in the metabolic degradation of inhaled xenobiotics. In some instances, the metabolism may, however, result in bioactivation of the xenobiotics and subsequent tissue injury. © 2012 John Wiley & Sons Ltd.

  8. The Role of G22 A Adenosine Deaminase 1 Gene Polymorphism and the Activities of ADA Isoenzymes in Fertile and Infertile Men.

    Science.gov (United States)

    Fattahi, Amir; Khodadadi, Iraj; Amiri, Iraj; Latifi, Zeinab; Ghorbani, Marzieh; Tavilani, Heidar

    2015-10-01

    To evaluate frequency distribution of adenosine deaminase 1 (ADA1) G22 A alleles and genotypes in fertile and infertile men. In this study we evaluate frequency distribution of ADA1 G22 A alleles and genotypes in 200 fertile and 200 infertile men. The polymerase chain reaction-restriction fragment length polymorphism technique was used for determining ADA1 G22 A variants. In addition, ADA isoenzymes activities (ADA1 and ADA2) were measured using colorimetric method. The frequency of GG genotype was significantly higher and GA genotype was lower in infertile males compared with fertile men (P = .048 and P = .045, respectively). However, there was not any noticeable difference in allele distribution between groups (P >.05). Based on logistic regression analysis, the GA genotype has a protective role and can decrease the risk of male infertility 1.7 times (P = .046). There were significantly higher activities of ADAT and its isoenzymes in infertile males compared with fertile men (P ADA1 activity with GG genotype was higher than GA carriers in all population (P = .001). Our results revealed that the activity of ADA isoenzymes and distribution of ADA1 G22 A genotypes were different among fertile and infertile men and more likely the GA genotype, which had lower ADA1 activity and was higher in fertile men is a protective factor against infertility. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    NARCIS (Netherlands)

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

    2009-01-01

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

  10. Clonal structure of Trypanosoma cruzi Colombian strain (biodeme Type III: biological, isoenzymic and histopathological analysis of seven isolated clones

    Directory of Open Access Journals (Sweden)

    Camandaroba Edson Luiz Paes

    2001-01-01

    Full Text Available The clonal structure of the Colombian strain of Trypanosoma cruzi, biodeme Type III and zymodeme 1, was analyzed in order to characterize its populations and to establish its homogeneity or heterogeneity. Seven isolated clones presented the basic characteristics of Biodeme Type III, with the same patterns of parasitemic curves, tissue tropism to skeletal muscle and myocardium, high pathogenicity with extensive necrotic-inflammatory lesions from the 20th to 30th day of infection. The parental strain and its clones C1, C3, C4 and C6, determined the higher levels of parasitemia, 20 to 30 days of infection, with high mortality rate up to 30 days (79 to 100%; clones C2, C5 and C7 presented lower levels of parasitemia, with low mortality rates (7.6 to 23%. Isoenzymic patterns, characteristic of zymodeme 1, (Z1 were similar for the parental strain and its seven clones. Results point to a phenotypic homogeneity of the clones isolated from the Colombian strain and suggest the predominance of a principal clone, responsible for the biological behavior of the parental strain and clones.

  11. Soil properties influence kinetics of soil acid phosphatase in response to arsenic toxicity.

    Science.gov (United States)

    Wang, Ziquan; Tan, Xiangping; Lu, Guannan; Liu, Yanju; Naidu, Ravi; He, Wenxiang

    2018-01-01

    Soil phosphatase, which plays an important role in phosphorus cycling, is strongly inhibited by Arsenic (As). However, the inhibition mechanism in kinetics is not adequately investigated. In this study, we investigated the kinetic characteristics of soil acid phosphatase (ACP) in 14 soils with varied properties, and also explored how kinetic properties of soil ACP changed with different spiked As concentrations. The results showed that the Michaelis constant (K m ) and maximum reaction velocity (V max ) values of soil ACP ranged from 1.18 to 3.77mM and 0.025-0.133mMh -1 in uncontaminated soils. The kinetic parameters of soil ACP in different soils changed differently with As contamination. The K m remained unchanged and V max decreased with increase of As concentration in most acid and neutral soils, indicating a noncompetitive inhibition mechanism. However, in alkaline soils, the K m increased linearly and V max decreased with increase of As concentration, indicating a mixed inhibition mechanism that include competitive and noncompetitive. The competitive inhibition constant (K ic ) and noncompetitive inhibition constant (K iu ) varied among soils and ranged from 0.38 to 3.65mM and 0.84-7.43mM respectively. The inhibitory effect of As on soil ACP was mostly affected by soil organic matter and cation exchange capacity. Those factors influenced the combination of As with enzyme, which resulted in a difference of As toxicity to soil ACP. Catalytic efficiency (V max /K m ) of soil ACP was a sensitive kinetic parameter to assess the ecological risks of soil As contamination. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Alkaline pretreatment of Mexican pine residues for bioethanol ...

    African Journals Online (AJOL)

    Alkaline pretreatment of Mexican pine residues for bioethanol production. ... Keywords: Lignocellulosic biomass, alkaline pretreatment, enzymatic hydrolysis, fermentable sugars, fermentation. African Journal of Biotechnology Vol. 12(31), pp.

  13. High frequencies of elevated alkaline phosphatase activity and rickets exist in extremely low birth weight infants despite current nutritional support

    Science.gov (United States)

    Osteopenia and rickets are common among extremely low birth weight infants (ELBW, <1000 g birth weight) despite current practices of vitamin and mineral supplementation. Few data are available evaluating the usual course of markers of mineral status in this population. Our objectives in this study w...

  14. High frequencies of elevated alkaline phosphatase activity and rickets exist in extremely low birth weight infants despite current nutritional support

    Directory of Open Access Journals (Sweden)

    Parker Bruce R

    2009-07-01

    Full Text Available Abstract Background Osteopenia and rickets are common among extremely low birth weight infants (ELBW, Methods We evaluated all ELBW infants admitted to Texas Children's Hospital NICU in 2006 and 2007. Of 211 admissions, we excluded 98 patients who were admitted at >30 days of age or did not survive/stay for >6 weeks. Bone radiographs obtained in 32 infants were reviewed by a radiologist masked to laboratory values. Results In this cohort of 113 infants, P-APA was found to have a significant inverse relationship with BW, gestational age and serum phosphorus. In paired comparisons, P-APA of infants Conclusion Elevation of P-APA >600 IU/L was very common in ELBW infants. BW was significantly inversely related to both P-APA and radiologic rickets. No single value of P-APA was related to radiological findings of rickets. Given the very high risk of osteopenia and rickets among ELBW infants, we recommend consideration of early screening and early mineral supplementation, especially among infants

  15. Effect of Eight Weeks Forced Swimming Training with Methadone Supplementation on Aspartate Aminotransferase, Alanine Aminotransferase, and Alkaline Phosphatase of Rats

    Directory of Open Access Journals (Sweden)

    Seyed Ali Hoseini

    2016-12-01

    Full Text Available Background & Objective: Narcotics abuse can induce liver disorders; nevertheless, exercises improve liver disorders. The present research aimed to review the effect of eight weeks forced swimming training with methadone supplementation on liver enzymes of rats. Material & Method: In this experimental research, 48 rats were selected, and after one week adaptation to lab environment, they were randomly divided into four groups of 12 rats including (1 forced swimming training, (2 methadone supplementation, (3 forced swimming training with methadone supplementation, and (4 control. Groups 2 and 3 used 2 mg/kg methadone daily for 8 weeks. Also, groups 1 and 3 swam for 8 weeks, three sessions per week and each session for 30 minutes. For statistical analysis of data, one way ANOVA and Tukey post hoc tests were used (α≤0.05. Results: Findings showed that forced swimming training, methadone supplementation, and forced swimming training with methadone supplementation had no significant effect on AST (P=0.90 and ALT (P=0.99 enzymes; forced swimming training had significant effect on increase of ALP (P=0.001; also, forced swimming training, compared with methadone supplementation and combination of forced swimming training with methadone supplementation, had significant effect on increase of ALP (P=0.001. Conclusion: Accordingly, 8 weeks of forced swimming training with methadone has possibly no significant effect on liver enzymes.

  16. TMEM199 Deficiency Is a Disorder of Golgi Homeostasis Characterized by Elevated Aminotransferases, Alkaline Phosphatase, and Cholesterol and Abnormal Glycosylation

    NARCIS (Netherlands)

    Jansen, Jos C.; Timal, Sharita; van Scherpenzeel, Monique; Michelakakis, Helen; Vicogne, Dorothée; Ashikov, Angel; Moraitou, Marina; Hoischen, Alexander; Huijben, Karin; Steenbergen, Gerry; van den Boogert, Marjolein A. W.; Porta, Francesco; Calvo, Pier Luigi; Mavrikou, Mersyni; Cenacchi, Giovanna; van den Bogaart, Geert; Salomon, Jody; Holleboom, Adriaan G.; Rodenburg, Richard J.; Drenth, Joost P. H.; Huynen, Martijn A.; Wevers, Ron A.; Morava, Eva; Foulquier, François; Veltman, Joris A.; Lefeber, Dirk J.

    2016-01-01

    Congenital disorders of glycosylation (CDGs) form a genetically and clinically heterogeneous group of diseases with aberrant protein glycosylation as a hallmark. A subgroup of CDGs can be attributed to disturbed Golgi homeostasis. However, identification of pathogenic variants is seriously

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

    OpenAIRE

    Peifang Wang; Lingxiao Ren; Chao Wang; Jin Qian; Jun Hou

    2014-01-01

    Phosphorus (P) is an important limiting nutrient in aquatic ecosystems and knowledge of P cycling is fundamental for reducing harmful algae blooms and other negative effects in water. Despite their importance, the characteristics of P cycling under changing nutrient conditions in shallow lakes were poorly investigated. In this study, in situ incubation experiments were conducted in a natural riparian zone in the main diversion channel used for water transfer into Lake Taihu (Wangyu River). Va...

  18. Alkaline phosphatase: An appraisal of its critical role in c-limited deep-sea sediments of central Indian Basin

    Digital Repository Service at National Institute of Oceanography (India)

    Biche, S.U.; Das, A.; Mascarenhas-Pereira, M.B.L.; Nath, B.N.; Naik, Sonali; Helekar, S.; Sharma, R.; Valsangkar, A.B.; LokaBharathi, P.A.

    differences in redox status could govern differential response in ALP activity under similar hydrostatic pressure. Hydrothermal scavenging reactions striping soluble P from pore water might be widespread in CIB albeit at extreme low intensities. ALP might...

  19. Prognostic value of PINP, bone alkaline phosphatase, CTX-I, and YKL-40 in patients with metastatic prostate carcinoma

    DEFF Research Database (Denmark)

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

    2006-01-01

    %, BAP (55%), CTX-I (33%), and YKL-40 (43%). Univariate analysis showed an association to survival: PINP (HR = 1.6, P 4, P 4, P = 0.004). In multivariate Cox analysis performance status, WHO grade, Soloway score, PINP...

  20. Alkaline sorbent injection for mercury control

    Science.gov (United States)

    Madden, Deborah A.; Holmes, Michael J.

    2002-01-01

    A mercury removal system for removing mercury from combustion flue gases is provided in which alkaline sorbents at generally extremely low stoichiometric molar ratios of alkaline earth or an alkali metal to sulfur of less than 1.0 are injected into a power plant system at one or more locations to remove at least between about 40% and 60% of the mercury content from combustion flue gases. Small amounts of alkaline sorbents are injected into the flue gas stream at a relatively low rate. A particulate filter is used to remove mercury-containing particles downstream of each injection point used in the power plant system.

  1. Alkaline rocks and the occurrence of uranium

    International Nuclear Information System (INIS)

    Hambleton-Jones, B.B.; Toens, P.D.

    1980-10-01

    Many alkaline complexes contain uranium and other minerals in low concentrations and are regarded as constituting valuable potential reserves. Certain complex metallurgical problems, however, remain to be solved. Alkaline rocks occur in a number of forms and environments and it is noted that they are generated during periods of geological quiescence emplaced mainly in stable aseismic areas. Many occur along the extensions of oceanic transform faults beneath the continental crust and the application of this concept to areas not currently known to host alkaline complexes may prove useful in identifying potential target areas for prospecting operations [af

  2. Short-Term Effect of Vermicompost Application on Biological Properties of an Alkaline Soil with High Lime Content from Mediterranean Region of Turkey

    Science.gov (United States)

    Uz, Ilker; Tavali, Ismail Emrah

    2014-01-01

    This study was conducted to investigate direct short-term impact of vermicompost on some soil biological properties by monitoring changes after addition of vermicompost as compared to farmyard manure in an alkaline soil with high lime content from semiarid Mediterranean region of Turkey. For this purpose, mixtures of soil and organic fertilizers in different doses were incubated under greenhouse condition. Soil samples collected in regular intervals were analyzed for biological parameters including dehydrogenase, β-glucosidase, urease, alkaline phosphatase activities, and total number of aerobic mesophilic bacteria. Even though soil dehydrogenase activity appeared to be dose-independent based on overall evaluation, organic amendments were found to elevate dehydrogenase activity when sampling periods are evaluated individually. β-glucosidase, urease, alkaline phosphatase activity, and aerobic mesophilic bacterial numbers in vermicompost treatments fluctuated but remained significantly above the control. A slight but statistically significant difference was detected between organic amendments in terms of urease activity. Vermicompost appeared to more significantly increase bacterial number in soil. Clearly, vermicompost has a potential to be used as an alternative to farmyard manure to improve and maintain soil biological activity in alkaline calcareous soils from the Mediterranean region of Turkey. Further studies are needed to assess its full potential for these soils. PMID:25254238

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

    Directory of Open Access Journals (Sweden)

    Vasavada Abhay

    1993-01-01

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

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

    Science.gov (United States)

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

    2017-01-01

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

  5. Alkalinity in oil field waters - what alkalinity is and how it is measured

    International Nuclear Information System (INIS)

    Kaasa, B.; Oestvold, T.

    1996-01-01

    The alkalinity is an important parameter in the description of pH-behaviour, buffer capacity and scaling potentials in oil field waters. Although the alkalinity is widely used, it seems to be considerable confusion in connection with the concept. It is often used incorrectly and different authors define the concept in different ways. Several different methods for the determination of alkalinity can be found in the literature. This paper discusses the definition of alkalinity and how to use alkalinity in oil field waters to obtain data of importance for scale and pH predictions. There is also shown how a simple titration of oil field waters can give both the alkalinity and the content of organic acids in these waters. It is obvious from these findings that most of the methods used to day may give considerable errors when applied to oil field waters with high contents of organic acids. 8 refs., 8 figs., 5 tabs

  6. Optical properties of alkaline earth borate glasses

    African Journals Online (AJOL)

    user

    The alkaline earth borate glasses containing heavy metal oxides show good solubility of rare-earth ions. Glasses containing PbO exhibit low glass transition temperature (Tg) and high ..... These oxygen ions carry a partial negative charge and.

  7. Technetium recovery from high alkaline solution

    Energy Technology Data Exchange (ETDEWEB)

    Nash, Charles A.

    2016-07-12

    Disclosed are methods for recovering technetium from a highly alkaline solution. The highly alkaline solution can be a liquid waste solution from a nuclear waste processing system. Methods can include combining the solution with a reductant capable of reducing technetium at the high pH of the solution and adding to or forming in the solution an adsorbent capable of adsorbing the precipitated technetium at the high pH of the solution.

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

    Science.gov (United States)

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

    2018-03-29

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

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

    Science.gov (United States)

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

    2011-01-01

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

  10. Furocoumarins from grapefruit juice and their effect on human CYP 3A4 and CYP 1B1 isoenzymes.

    Science.gov (United States)

    Girennavar, Basavaraj; Poulose, Shibu M; Jayaprakasha, Guddadarangavvanahally K; Bhat, Narayan G; Patil, Bhimanagouda S

    2006-04-15

    Bioactive compounds present in grapefruit juice are known to increase the bioavailability of certain medications by acting as potent CYP 3A4 inhibitors. An efficient technique has been developed for isolation and purification of three furocoumarins. The isolated compounds have been tested for the inhibition of human CYP 1B1 isoform using specific substrates. Grapefruit juice was extracted with ethyl acetate (EtOAc) and the dried extract was loaded onto silica gel column chromatography. Further, column fractions were subjected to preparative HPLC to obtain three compounds. The purity of these compounds was analyzed by HPLC and structures were determined by NMR studies. The identified compounds, bergamottin, 6',7'-dihydroxybergamottin (DHB), and paradisin-A, were tested for their inhibitory effects on hydroxylase and O-dealkylase activities of human cytochrome P450 isoenzymes CYP 3A4 and CYP 1B1. Paradisin-A was found to be a potent CYP 3A4 inhibitor with an IC50 of 1.2 microM followed by DHB and bergamottin. All three compounds showed a substantial inhibitory effect on CYP 3A4 below 10 microM. Inhibitory effects on CYP 1B1 exhibited a greater variation due to the specificity of substrates. Paradisin A showed an IC50 of 3.56+/-0.12 microM for the ethoxy resorufin O-dealkylase (EROD) activity and 33.56+/-0.72 microM for the benzyloxy resorufin (BROD). DHB and bergamottin showed considerable variations for EROD and BROD activities with an IC50 of 7.17 microM and 13.86 microM, respectively.

  11. Theoretical study on the N-demethylation mechanism of theobromine catalyzed by P450 isoenzyme 1A2.

    Science.gov (United States)

    Tao, Jing; Kang, Yuan; Xue, Zhiyu; Wang, Yongting; Zhang, Yan; Chen, Qiu; Chen, Zeqin; Xue, Ying

    2015-09-01

    Theobromine, a widely consumed pharmacological active substance, can cause undesirable muscle stiffness, nausea and anorexia in high doses ingestion. The main N-demethylation metabolic mechanism of theobromine catalyzed by P450 isoenzyme 1A2 (CYP1A2) has been explored in this work using the unrestricted hybrid density functional method UB3LYP in conjunction with the LACVP(Fe)/6-31G (H, C, N, O, S, Cl) basis set. Single-point calculations including empirical dispersion corrections were carried out at the higher 6-311++G** basis set. Two N-demethylation pathways were characterized, i.e., 3-N and 7-N demethylations, which involve the initial N-methyl hydroxylation to form carbinolamines and the subsequent carbinolamines decomposition to yield monomethylxanthines and formaldehydes. Our results have shown that the rate-limiting N-methyl hydroxylation occurs via a hydrogen atom transfer (HAT) mechanism, which proceeds in a spin-selective mechanism (SSM) in the gas phase. The carbinolamines generated are prone to decomposition via the contiguous heteroatom-assisted proton-transfer. Strikingly, 3-N demethylation is more favorable than 7-N demethylation due to its lower free energy barrier and 7-methylxanthine therefore is the optimum product reported for the demethylation of theobromine catalyzed by CYP1A2, which are in good agreement with the experimental observation. This work has first revealed the detail N-demethylation mechanisms of theobromine at the theoretical level. It can offer more significant information for the metabolism of purine alkaloid. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Glutathione S-transferases in human renal cortex and neoplastic tissue: enzymatic activity, isoenzyme profile and immunohistochemical localization.

    Science.gov (United States)

    Rodilla, V; Benzie, A A; Veitch, J M; Murray, G I; Rowe, J D; Hawksworth, G M

    1998-05-01

    1. Glutathione S-transferase (GST) activity in the cytosol of renal cortex and tumours from eight men and eight women was measured using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. GST activities ranged from 685 to 2192 nmol/min/mg protein in cortex (median 1213) and from non-detectable (minimum 45) to 2424 nmol/min/mg protein in tumours (median 469). The activities in the tumours were lower than those in the normal cortices (p 0.05). 3. The age of the patients ranged from 42 to 81 years (median 62) and was not found to play a role in the levels of GST activity observed in cortex or in renal tumours from either sex. 4. Immunoblotting and immunohistochemical studies confirmed that GST-alpha was the predominant form expressed both in normal cortex and tumour and probably accounted for most of the GST activity present in these samples. GST-mu and GST-phi were expressed in both tumours and normal cortex and, while in some cases the level of expression in the cortices was higher than that found in the tumours, the reverse was also observed. Within the GST-mu class, GST M1/M2 was only detected in one sample (tumour), which showed the highest overall expression of GST-mu. GSTM3 was the predominant isoenzyme of the mu class in normal and tumour tissue, whereas GTM4 and GSTM5 were not detected. 5. These differences could have functional significance where xenobiotics or cytotoxic drugs are specific substrates for the different classes of GSTs.

  13. Effect of vanadium compounds on acid phosphatase activity.

    Science.gov (United States)

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

    1996-01-01

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

  14. Improved electrodes and gas impurity investigations on alkaline electrolysers

    DEFF Research Database (Denmark)

    Reissner, R.; Schiller, G.; Knoeri, T.

    Alkaline water electrolysis for hydrogenproduction is a well-established techniquebut some technological issues regarding thecoupling of alkaline water electrolysis andRenewable Energy Sources (RES) remain tobe improved.......Alkaline water electrolysis for hydrogenproduction is a well-established techniquebut some technological issues regarding thecoupling of alkaline water electrolysis andRenewable Energy Sources (RES) remain tobe improved....

  15. 200 kDa and 160 kDa neurofilament protein phosphatase resistance following in vivo aluminum chloride exposure.

    Science.gov (United States)

    Strong, M J; Jakowec, D M

    1994-01-01

    We have used time-course dephosphorylation experiments and two dimensional isoelectric focusing to assess the phosphorylation state of neurofilament (NF) proteins following the intracisternal inoculation of AlCl3. Littermates of New Zealand white rabbits, age 5-6 weeks, were inoculated with either 1000, 750, 500, 250 or 100 micrograms AlCl3 in 0.9% NaCl or 0.9% NaCl alone, killed 48 hours later and the NF-enriched cytoskeletal fraction isolated from the spinal cord. Neurofilamentous inclusions did not occur following inoculums of 100 or 250 micrograms AlCl3, but thereafter developed in increasing quantities in a dosage-dependent manner. Incubation of the NF-enriched fraction with E. Coli. alkaline phosphatase (enzyme: substrate 1:50) induced a replacement of the highly phosphorylated 200 kDa isoform of NFH with a more poorly phosphorylated 170 kDa isoform, confirmed by immunoblot analysis. This reaction was complete within 20 minutes with NF derived from NaCl, 100 or 250 micrograms AlCl3 inoculated rabbits and within 30 minutes for 500 micrograms AlCl3 inoculums. However, residual highly phosphorylated NFH isoforms persisted at 60 minutes for 750 micrograms inoculums and 90 minutes for that derived from 1000 micrograms AlCl3 inoculums. A similar inhibition of phosphatase activity was observed for NFM. Following two dimensional electrophoresis of the NF-enriched isolate, no alteration in the net phosphorylation state of individual NF subunit proteins was observed--regardless of the inoculum. These results demonstrate a dose-dependent induction of neurofilamentous inclusions in spinal motor neurons following intracisternal AlCl3 inoculation accompanied by increasing phosphatase resistance without a demonstrable alteration in NF net phosphorylation state.

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

    African Journals Online (AJOL)

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

  17. Phosphatases as an index of biotic contamination of dust

    NARCIS (Netherlands)

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

    1990-01-01

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

  18. Biocatalysis with Sol-Gel Encapsulated Acid Phosphatase

    Science.gov (United States)

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

    2010-01-01

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

  19. Anxious moments for the protein tyrosine phosphatase PTP1B

    OpenAIRE

    Krishnan, Navasona; Tonks, Nicholas K.

    2015-01-01

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

  20. WIP1 phosphatase as pharmacological target in cancer therapy

    Czech Academy of Sciences Publication Activity Database

    Pecháčková, Soňa; Burdová, Kamila; Macůrek, Libor

    2017-01-01

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

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

    DEFF Research Database (Denmark)

    Auger, Kyle; Raththagala, Madushi; Wilkens, Casper

    2016-01-01

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

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

    NARCIS (Netherlands)

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

    2014-01-01

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

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

    Czech Academy of Sciences Publication Activity Database

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

    2001-01-01

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

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

    African Journals Online (AJOL)

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

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

    Science.gov (United States)

    Kaur, Parvinder; Satyanarayana, T.

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

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

    Science.gov (United States)

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

    1975-01-01

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

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

    DEFF Research Database (Denmark)

    Petrone, Angiola; Battaglia, Fortunato; Wang, Cheng

    2003-01-01

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

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

    DEFF Research Database (Denmark)

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

    2006-01-01

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

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

    DEFF Research Database (Denmark)

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

    2006-01-01

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

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

    OpenAIRE

    Leis, J F; Kaplan, N O

    1982-01-01

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

  11. Pharmacological evaluation of the role of cyclooxygenase isoenzymes on the micturition reflex following experimental cystitis in rats

    Science.gov (United States)

    Lecci, Alessandro; Birder, Lori A; Meini, Stefania; Catalioto, Rose-Marie; Tramontana, Manuela; Giuliani, Sandro; Criscuoli, Marco; Maggi, Carlo A

    2000-01-01

    Prostanoids, generated from cyclooxygenase (COX) isoenzymes, play a role in the physiological function of the lower urinary tract and are important mediators of inflammatory hyperalgesia. The present work evaluates the effects of the COX-1/COX-2 inhibitor dexketoprofen as well as of a selective COX-2 inhibitor, NS-398, on urodynamic function following endotoxin (LPS) or cyclophosphamide (CYP)-induced inflammation of the urinary bladder. The application of arachidonic acid (330 μg rat−1) onto the serosal surface of the urinary bladder in control rats elicited bladder contractions which could be blocked in a dose-dependent manner by dexketoprofen (0.1–3 mg kg−1, i.v.) but not by NS-398 (0.2–6 mg kg−1, i.v.). Dexketoprofen (3 mg kg−1, i.v.) decreased the micturition frequency and increased the pressure threshold for triggering the micturition either when administered within 15 min or 3 h following surgery in control animals. NS-398 (6 mg kg−1, i.v.) decreased the micturition frequency and increased the pressure threshold when administered 3 h but not 15 min following surgery. Administration of LPS (2 mg kg−1, i.v., 90–120 min) increased both the micturition frequency and the pressure threshold for triggering the micturition reflex. Changes in urodynamic parameters induced by LPS were prevented by doses of either dexketoprofen (1 mg kg−1, i.v.) or NS-398 (2 mg kg−1, i.v.) which were ineffective in control animals. Pretreatment with CYP (150 mg kg−1, i.p., 48 h) increased the micturition frequency, pressure threshold, and the minimal intravesical pressure but decreased the mean amplitude of micturition contractions. In CYP-treated rats, dexketoprofen (1 mg kg−1, i.v.) or NS-398 (2 mg kg−1, i.v.) blocked the CYP-induced urodynamic changes with exception of the micturition contraction amplitude. These results indicate that COX-1 may be involved in modulating the threshold for activating the

  12. Assessing ocean alkalinity for carbon sequestration

    Science.gov (United States)

    Renforth, Phil; Henderson, Gideon

    2017-09-01

    Over the coming century humanity may need to find reservoirs to store several trillions of tons of carbon dioxide (CO2) emitted from fossil fuel combustion, which would otherwise cause dangerous climate change if it were left in the atmosphere. Carbon storage in the ocean as bicarbonate ions (by increasing ocean alkalinity) has received very little attention. Yet recent work suggests sufficient capacity to sequester copious quantities of CO2. It may be possible to sequester hundreds of billions to trillions of tons of C without surpassing postindustrial average carbonate saturation states in the surface ocean. When globally distributed, the impact of elevated alkalinity is potentially small and may help ameliorate the effects of ocean acidification. However, the local impact around addition sites may be more acute but is specific to the mineral and technology. The alkalinity of the ocean increases naturally because of rock weathering in which >1.5 mol of carbon are removed from the atmosphere for every mole of magnesium or calcium dissolved from silicate minerals (e.g., wollastonite, olivine, and anorthite) and 0.5 mol for carbonate minerals (e.g., calcite and dolomite). These processes are responsible for naturally sequestering 0.5 billion tons of CO2 per year. Alkalinity is reduced in the ocean through carbonate mineral precipitation, which is almost exclusively formed from biological activity. Most of the previous work on the biological response to changes in carbonate chemistry have focused on acidifying conditions. More research is required to understand carbonate precipitation at elevated alkalinity to constrain the longevity of carbon storage. A range of technologies have been proposed to increase ocean alkalinity (accelerated weathering of limestone, enhanced weathering, electrochemical promoted weathering, and ocean liming), the cost of which may be comparable to alternative carbon sequestration proposals (e.g., $20-100 tCO2-1). There are still many

  13. High temperature and pressure alkaline electrolysis

    DEFF Research Database (Denmark)

    Allebrod, Frank; Chatzichristodoulou, Christodoulos; Mogensen, Mogens Bjerg

    2013-01-01

    Alkaline electrolyzers have proven to operate reliable for decades on a large scale, but in order to become commercially attractive and compete against conventional technologies for hydrogen production, the production and investment costs have to be reduced. This may occur by increasing the opera......Alkaline electrolyzers have proven to operate reliable for decades on a large scale, but in order to become commercially attractive and compete against conventional technologies for hydrogen production, the production and investment costs have to be reduced. This may occur by increasing...

  14. Catalytic oxidation of soot over alkaline niobates

    International Nuclear Information System (INIS)

    Pecchi, G.; Cabrera, B.; Buljan, A.; Delgado, E.J.; Gordon, A.L.; Jimenez, R.

    2013-01-01

    Highlights: ► No previous reported studies about alkaline niobates as catalysts for soot oxidation. ► NaNbO 3 and KNbO 3 perovskite-type oxides show lower activation energy than other lanthanoid perovskite-type oxides. ► The alkaline niobate does not show deactivation by metal loss. - Abstract: The lack of studies in the current literature about the assessment of alkaline niobates as catalysts for soot oxidation has motivated this research. In this study, the synthesis, characterization and assessment of alkaline metal niobates as catalysts for soot combustion are reported. The solids MNbO 3 (M = Li, Na, K, Rb) are synthesized by a citrate method, calcined at 450 °C, 550 °C, 650 °C, 750 °C, and characterized by AAS, N 2 adsorption, XRD, O 2 -TPD, FTIR and SEM. All the alkaline niobates show catalytic activity for soot combustion, and the activity depends basically on the nature of the alkaline metal and the calcination temperature. The highest catalytic activity, expressed as the temperature at which combustion of carbon black occurs at the maximum rate, is shown by KNbO 3 calcined at 650 °C. At this calcination temperature, the catalytic activity follows an order dependent on the atomic number, namely: KNbO 3 > NaNbO 3 > LiNbO 3 . The RbNbO 3 solid do not follow this trend presumably due to the perovskite structure was not reached. The highest catalytic activity shown by of KNbO 3 , despite the lower apparent activation energy of NaNbO 3 , stress the importance of the metal nature and suggests the hypothesis that K + ions are the active sites for soot combustion. It must be pointed out that alkaline niobate subjected to consecutive soot combustion cycles does not show deactivation by metal loss, due to the stabilization of the alkaline metal inside the perovskite structure.

  15. Alkaline earth filled nickel skutterudite antimonide thermoelectrics

    Science.gov (United States)

    Singh, David Joseph

    2013-07-16

    A thermoelectric material including a body centered cubic filled skutterudite having the formula A.sub.xFe.sub.yNi.sub.zSb.sub.12, where A is an alkaline earth element, x is no more than approximately 1.0, and the sum of y and z is approximately equal to 4.0. The alkaline earth element includes guest atoms selected from the group consisting of Be, Mb, Ca, Sr, Ba, Ra and combinations thereof. The filled skutterudite is shown to have properties suitable for a wide variety of thermoelectric applications.

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

    Science.gov (United States)

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

    1996-06-01

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

  17. Mineralogical, petrological and geochemical aspects of alkaline and alkaline-carbonatite associations from Brazil

    Science.gov (United States)

    Morbidelli, L.; Gomes, C. B.; Beccaluva, L.; Brotzu, P.; Conte, A. M.; Ruberti, E.; Traversa, G.

    1995-12-01

    A general description of Mesozoic and Tertiary (Fortaleza) Brazilian alkaline and alkaline-carbonatite districts is presented with reference to mineralogy, petrology, geochemistry and geochronology. It mainly refers to scientific results obtained during the last decade by an Italo-Brazilian research team. Alkaline occurrences are distributed across Brazilian territory from the southern (Piratini, Rio Grande do Sul State) to the northeastern (Fortaleza, Ceará State) regions and are mainly concentrated along the borders of the Paraná Basin generally coinciding with important tectonic lineaments. The most noteworthy characteristics of these alkaline and alkaline-carbonatite suites are: (i) prevalence of intrusive forms; (ii) abundance of cumulate assemblages (minor dunites, frequent clinopyroxenites and members of the ijolite series) and (iii) abundance of evolved rock-types. Many data demonstrate that crystal fractionation was the main process responsible for magma evolution of all Brazilian alkaline rocks. A hypothesis is proposed for the genesis of carbonatite liquids by immiscibility processes. The incidence of REE and trace elements for different major groups of lithotypes, belonging both to carbonatite-bearing and carbonatite-free districts, are documented. Sr and preliminary Nd isotopic data are indicative of a mantle origin for the least evolved magmas of all the studied occurrences. Mantle source material and melting models for the generation of the Brazilian alkaline magma types are also discussed.

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

    Directory of Open Access Journals (Sweden)

    Irena Lorenc-Kubis

    2015-01-01

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

  19. Determination of Acidity and Alkalinity of Food Materials

    OpenAIRE

    三浦,芳助; 福永,祐子; 瀧川,裕里子; 津田,真美; 渡辺,陽子; 瀨山,一正

    2006-01-01

    The acidity and alkalinity of food materials in various menus was determined to clarify the influence of food on physiological functions. Menus mainly containing alkaline food materials (alkaline menu) and acid ones (acid menu) were compared. Determination of acidity and alkalinity was performed for each food material in the alkaline menu and acid menu, and acidity and alkalinity of one meal and a day's one were estimated. 1. Most of food materials in acid menu were assessed to be...

  20. The Alkaline Diet: Is There Evidence That an Alkaline pH Diet Benefits Health?

    Directory of Open Access Journals (Sweden)

    Gerry K. Schwalfenberg

    2012-01-01

    Full Text Available This review looks at the role of an alkaline diet in health. Pubmed was searched looking for articles on pH, potential renal acid loads, bone health, muscle, growth hormone, back pain, vitamin D and chemotherapy. Many books written in the lay literature on the alkaline diet were also reviewed and evaluated in light of the published medical literature. There may be some value in considering an alkaline diet in reducing morbidity and mortality from chronic diseases and further studies are warranted in this area of medicine.