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1

Approach to a patient with elevated serum alkaline phosphatase.  

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Cholestasis develops either from a defect in bile synthesis, impairment in bile secretion, or obstruction to bile flow, and is characterized by an elevated serum alkaline phosphatase and gamma-glutamyltransferase disproportionate to elevation of aminotransferase enzymes. Key elements to the diagnostic workup include visualization of the biliary tree by cholangiography and evaluation of liver histology. The hope is that recent advances in understanding the genetic factors and immune mechanisms involved in the pathogenesis of cholestasis will lead to newer therapeutic interventions in the treatment of these diseases. PMID:22541695

Siddique, Asma; Kowdley, Kris V

2012-05-01

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Serum Alkaline Phosphatase Levels and Mortality of Chronic Hemodialysis Patients  

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Full Text Available Objective: Alkaline phosphatase (ALP is considered a biomarker of high bone turnover in hemodialysis (HD patients with secondary hyperparathyroidism. This study was conducted to determine whether high serum ALP levels are associated with increased all-cause mortality of HD patients. Patients and Methods: This was a retrospective cohort study conducted at a single center. The subjects were 195 patients on chronic HD therapy who were followed up for a 5 years, and relationships between their baseline data and outcomes were assessed statistically. The serum ALP level was used as the predictor, and the primary end point was all-cause mortality. Results: Based on the median serum ALP of 236 IU/L, the subjects were divided into a low-ALP group (<236 IU/l and a high-ALP group (?236 IU/l. The high-ALP group was older and had a longer dialysis vintage, lower serum phosphorus concentrations, and higher serum parathyroid hormone levels, and they also had lower serum albumin levels and higher C-reactive protein values. In a multivariate Cox model in which the baseline serum ALP levels were used adjusted for age, gender, HD vintage, comorbidity, bone metabolism parameters, and serum liver enzyme levels, each doubling of the serum ALP level was associated with a significant increase in the hazard of all-cause mortality (hazard ratio 10.70, 95% CI 1.53 - 74.24. Conclusion: High baseline serum ALP levels are associated with increased mortality of HD patients, independent of bone metabolism parameters and serum liver enzyme levels. ALP is a potential target for the treatment of HD patients.

Tetsuri Yamashita

2011-09-01

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Alkaline phosphatase bone isoenzyme and osteocalcin in the serum of hyperthyroid cats.  

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The effect of hyperthyroidism on serum markers for increased bone metabolism and turnover was evaluated in 36 cats with elevated serum levels of thyroxine and alkaline phosphatase. Serum was analyzed for total and ionized calcium and phosphorous. Alkaline phosphatase isoenzymes were separated by agarose gel electrophoresis and osteocalcin was measured by radioimmunoassay. Values for hyperthyroid cats were compared with those for healthy cats. Alkaline phosphatase bone isoenzyme was markedly increased in all 36 hyperthyroid cats. Osteocalcin was increased in 44% of the cats. There was no correlation among the magnitude of increase in alkaline phosphatase bone isoenzyme, osteocalcin, and serum thyroxine concentrations. Increased serum phosphorus was found in 35% of the cats. Total calcium was within the reference range in all cats, while 50% of the cats had reduced levels of serum ionized calcium. We conclude that hyperthyroid cats do have altered bone metabolism, although it is usually clinically insignificant. PMID:9111692

Archer, F J; Taylor, S M

1996-01-01

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Increased Serum Alkaline Phosphatase and Serum Phosphate as Predictors of Mortality after Stroke  

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Context: Serum Alkaline phosphatase (ALP) & phosphate are considered to be indicators of vascular calcification. Link between bone metabolism, vascular calcification, cardiovascular events have been well studied in chronic kidney disease and ischemic heart disease. Aims: To determine that increased serum phosphate and alkaline phosphatase are predictors of mortality rates and recurrent vascular events in stroke. Materials and Methods: Sixty patients admitted with acute stroke (ischemic & haemorrhagic) were included in the study. Their baseline clinical characteristics and biochemical parameters including serum ALP and phosphate were noted. All patients were followed up for a period of one year. The all- cause mortality, the mortality due to cardiovascular events and recurrent vascular events without death were noted during the follow up. Statistical analyses were done to look for any correlation between mortality and baseline levels of serum ALP and phosphate. Results: Of the 60 patients, 8 (13.3%) patients were lost for follow up. Fourteen (26.9%) patients died; of which 12 deaths were due to vascular causes and 2 deaths were due to non vascular causes. Increasing levels of serum ALP and phosphate correlated with all cause mortality and recurrent vascular events without death Conclusion: Serum ALP and phosphate prove to be cost effective prognostic indicator of mortality and recurrent vascular events in stroke. This finding has to be confirmed with studies including larger population. Further research on ALP inhibitors, Vitamin D analogues and phosphate binders to improve mortality in stroke population can be encouraged.

S, Pratibha; JB, Agadi

2014-01-01

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Comparative Effects of Lead on Serum, Liver and Brain High Molecular Weight Alkaline Phosphatase in Rats  

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Full Text Available The relationship between lead treatment and changes in the concentration of serum, liver and brain high and low molecular weight alkaline phosphatase has been investigated in this manuscript. Results obtained showed that every other day intrapritoneally injection of 39.5 ?mol kg-1 of lead as (Pb(CH3COO2.3H2O, in male rats for 2 consecutive weeks resulted in decreasing level of liver and brain alkaline phosphatase by 16.7 and 10.9%, respectively, whereas an elevation of serum enzyme activity by 28.4% was seen in comparison to untreated controls (p<0.05. Long-term exposure to 13.2 ?mol kg-1 of this salt, showed a statistically significant reduction in liver and brain levels of alkaline phosphatase by 18.7 and 13.2%, respectively and an increment in serum activity of the enzyme by 37.6% in compared to control group (p<0.05. Using gel filtration chromatography technique with sephacryl S300 showed that, in comparison to control groups, serum and liver homogenate from lead treated groups had a significant level of high molecular weight alkaline phosphatase, which might be considered as a potential biomarker for lead toxicity.

A.A. Moshtaghie

2006-01-01

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Serum calcium and phosphorus concentration and alkaline phosphatase activity in healthy children during growth and development  

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Full Text Available Introduction. Many changes happen during growth and development in an organism as a result of important hormone 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. Material and methods. 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. Results and conclusion. 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.

Savi? Ljiljana

2008-01-01

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Genome-wide association study on serum alkaline phosphatase levels in a Chinese population  

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Background: Serum alkaline phosphatase (ALP) is a complex phenotype influenced by both genetic and environmental factors. Recent Genome-Wide Association Studies (GWAS) have identified several loci affecting ALP levels; however, such studies in Chinese populations are limited. We performed a GWAS analyzing the association between 658,288 autosomal SNPs and serum ALP in 1,461 subjects, and replicated the top SNPs in an additional 8,830 healthy Chinese Han individuals. The interactions between s...

Li, Jun; Gui, Lixuan; Wu, Chen; He, Yunfeng; Zhou, Li; Guo, Huan; Yuan, Jing; Yang, Binyao; Dai, Xiayun; Deng, Qifei; Huang, Suli; Guan, Lei; Hu, Die; Deng, Siyun; Wang, Tian

2013-01-01

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

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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)

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Efficacy of pleural fluid alkaline phosphatase and its ratio to serum levels in distinguishing exudates from transudates  

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Full Text Available The objective of present study was to evaluate the efficacy of pleural fluid alkaline phosphatase and its ratio to serum levels to classify pleural fluids. A total of 80 patients were divided in transudates and exudates on the basis of extensive clinical, radiological and biochemical evaluation. The efficacy of pleural fluid alkaline phosphatase (P ALP and pleural fluid / serum alkaline phosphatase ratio (P/S ALP assessment along with that of Light?s criteria to accurately classify transudates and exudates were analyzed. Up to 89% transudates misclassified by Light?s criteria were correctly classified by pleural fluid alkaline phosphatase (P ALP and pleural fluid/serum alkaline phosphatase ratio (P/S ALP evaluation. Similarly 92% exudates misclassified by Light?s criteria were correctly classified by pleural fluid alkaline phosphatase (P ALP and pleural fluid/serum alkaline phosphatase ratio (P/S ALP evaluation. By applying a cut off value of 40.0 IU for P ALP, a sensitivity of 85% and specificity of 75% was found. For P/S ALP, applying a cut off value of 0.25 a sensitivity of 85% and specificity of 80% was found. Both P ALP and P/S ALP had a PPV of 92%. However, their respective NPV were 63% and 70%.

Gupta K

2004-01-01

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Serum bone specific alkaline phosphatase and urinary deoxypyridinoline in postmenopausal osteoporosis.  

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The objectives of this study were to: (i) evaluate the diagnostic sensitivity and specificity of the biochemical bone markers: serum total alkaline phosphatase (TALP), bone specific alkaline phosphatase (BSALP) and urinary deoxypyridinoline (Dpyr) in postmenopausal osteoporosis, (ii) compare the bone turnover of postmenopausal osteoporotic patients without and with hormone replacement therapy (HRT) against controls and (iii) identify the correlation between these bone markers and bone mineral density (BMD). We examined 42 postmenopausal women with BMD proven osteoporosis and 35 control subjects. Serum TALP, BSALP and urinary Dpyr were measured. All three biochemical bone markers showed comparable moderate diagnostic sensitivity but Dpyr had the highest diagnostic specificity. There were significantly higher serum TALP, BSALP and urinary Dpyr levels in non-HRT treated patients compared to controls (p<0.005, <0.0001 and <0.005 respectively). There were no significant differences in the levels of all three bone markers between HRT treated patients and control subjects. There was no significant correlation between TALP, BSALP or Dpyr and BMD in both controls and patients. In conclusion, the biochemical bone markers are not useful in diagnosis of postmenopausal osteoporosis but may have a role in monitoring progress and response to treatment. HRT treatment reduces bone turnover of postmenopausal osteoporosis. PMID:12166596

Nawawi, H M; Yazid, T N; Ismail, N M; Mohamad, A R; Nirwana, S I; Khalid, B A

2001-12-01

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Relationship between serum heat-stable alkaline phosphatase level and pregnancy  

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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

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Role of serum in the developmental expression of alkaline phosphatase in MC3T3-E1 osteoblasts.  

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MC3T3-E1 cells in culture exhibit a temporal sequence of development similar to in vivo bone formation. To examine whether the developmental expression of the osteoblast phenotype depends on serum derived factors, we compared the time-dependent expression of alkaline phosphatase (ALP)-a marker of osteoblastic maturation- in MC3T3-E1 cells grown in the presence of fetal bovine serum (FBS) or resin/charcoal-stripped (AXC) serum. ALP was assessed by measuring enzyme activity, immunoblotting, and Northern analysis. Growth of MC3T3-E1 cells in FBS resulted in the programmed upregulation of alkaline phosphatase (ALP) post-proliferatively during osteoblast differentiation. In the presence of complete serum, actively proliferating cells during the initial culture period expressed low ALP levels consistent with their designation as pre-osteoblasts, whereas postmitotic cultures upregulated ALP protein, message, and enzyme activity. In addition, undifferentiated early cultures of MC3T3-E1 cells were refractory to forskolin (FSK) stimulation of ALP, but became forskolin responsive following prolonged culture in FBS containing media. In contrast, MC3T3-E1 cells grown in AXC serum displayed limited growth and failed to show a time-dependent increase in alkaline phosphatase. Neither the addition of IGF-I to AXC serum to augment cell number or plating at high density restored the time-dependent upregulation of alkaline phosphatase. Cells incubated in AXC serum for 14 days, however, though expressing low alkaline phosphatase levels, maintained the capacity to upregulate ALP after FBS re-addition or forskolin activation of cAMP-dependent pathways. Such time-dependent acquisition of FSK responsiveness and serum stimulation of ALP expression only in mature osteoblasts indicate the possible presence of differentiation switches that impart competency for a subset of osteoblast developmental events that require complete serum for maximal expression. PMID:8126070

Yohay, D A; Zhang, J; Thrailkill, K M; Arthur, J M; Quarles, L D

1994-03-01

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Serum alkaline phosphatase isoenzymes in SD rats detected by polyacrylamide-gel disk electrophoresis.  

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Serum alkaline phosphatase (ALP) activity is frequently measured in toxicity studies. In the present study, we assessed the usefulness of a commercially available polyacrylamide-gel disk electrophoresis kit used in humans (AlkPhor System, Jokoh Co. Ltd., Tokyo, Japan) for identifying serum ALP isoenzymes in rats of the Sprague-Dawley strain (SD rats), which are commonly used in toxicity studies. We also examined age-related changes in serum ALP isoenzymes in SD rats. In order to identify the origin of each ALP isoenzyme, tissue ALP extracts from the liver, bone and small intestine (SI) and serum samples were treated with neuraminidase, antiintestinal ALP antibody, ALP inhibitor levamisole, and/or wheat germ agglutinin. It became clear that pretreatment of serum with neuraminidase is necessary for rat serum ALP isoenzyme analysis. The kit revealed that the main serum ALP isoenzymes in fasted 8-week-old intact rats were bone- and SI-derived and they tended to decrease with age. Serum liver-derived isoenzyme was slightly detected in both sexes of all ages examined, but it greatly increased in cholestasis model rats with bile-duct ligation, and rats of this model also had large molecular ALP detected in the stacking gel, suggesting hepatic damage. High-molecular intestinal ALP isoenzyme was slightly observed at the most cathodal side of the resolving gel. These results suggest that the present method is a useful tool for detecting serum ALP isoenzymes in SD rats and that concomitant levamisole inhibition with another gel is applicable for the evaluation of organ toxicity. PMID:22500783

Hatayama, Kazuhisa; Ichikawa, Yuko; Nishihara, Yoshito; Goto, Ken; Nakamura, Daichi; Wakita, Atsushi; Kobayashi, Jyunichi

2012-05-01

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Correlation Between Serum Level Parathormone, Alkaline Phosphatase, Calcium and Phosphorus of Patients Hemodialysis in Zahedan  

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Full Text Available Secondary hyperparathyroidism and its effects on bone tissue are among the most important complication of end-stage renal disease. In the present study, we investigated correlation between the serum parathormone level (PTH of hemodialysis men and women with calcium (Ca, phosphorus (Pi and alkaline phosphatase (ALP. We studied 30 chronic renal failure hemodialysis patients 16 men and 14 women, aged 22-66 years old (average 44 years old with dialysis duration of 5 months to 14 years. We measured the serum Ca., Pi and ALP in intervals of 3 months and serum PTH levels was measured in 3 month. Data analysis was performed using SPSS software. Our results showed that serum PTH and ALP levels were higher in women than in men (90% versus 70%, but abnormal range of serum Ca and Pi levels were higher in men then women (Ca : 8% versus 2%, Pi :58% versus 50%. Hemodialysis patients showed correlation between PTH and ALP (p<0.05, but the correlation of PTH with Ca and Pi levels was not statistically significant. No correlation was observed between PTH and ALP and Pi in men, however it was significant between PTH and Ca (p<0.08, r = - 0.63. The women showed correlation between PTH and ALP (p<0.05, but not between Ca and Pi levels. Based on the findings of this study, Secondary hyperparathyroidism and its effects on bone tissue were greater in women than men hemodialysis.

R. Saravani

2007-01-01

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Lectin affinity electrophoresis of serum alkaline phosphatase in metastasized breast cancer.  

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The use of serum alkaline phosphatase (ALP) isoenzymes as markers of breast cancer metastases and treatment efficacy has received little attention. Twenty-six breast cancer women (56+/-13 years, all post-menopausal) were prospectively evaluated during their first and third course of chemotherapy (4-week interval). Serum samples were analyzed for ALP isoenzymes (bone, liver, and intestine) using a lectin affinity electrophoresis kit (Hydragel 15 ISO-PAL, Sebia) adapted on a semi-automated Hydrasys system (Sebia). Results were compared with imaging techniques for the presence of metastases; bone ALP isoenzyme (B-ALP) results were compared with C-Terminal degradation products of type I collagen (S-CTX) (CrossLaps, IDS Nordic). Serum B-ALP, but not S-CTX, confirmed the presence of bone metastases (BM) (n=15) with 67/100% sensitivity/specificity (using a 69 UI/L ROC cut-off); ROC AUC was 0.806 (P=0.0004) (NS for S-CTX). Chemotherapy reduced serum B-ALP by 24% over 4 weeks (P=0.0012); there was no change for S-CTX. There was no specific clinical pattern for other ALP isoenzymes (liver and intestine). In conclusion, serum B-ALP, but not S-CTX, could help confirm the presence of BM in breast cancer patients. PMID:20087950

Le Bricon, Thierry; Gay-Bellile, Cécile; Cottu, Paul; Benlakehal, Mourad; Guillon, Hélène; Houzé, Pascal

2010-01-01

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Head and neck cancer. Relationship of the prechemotherapy serum alkaline phosphatase levels to response rate of induction chemotherapy.  

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Fifty-one patients with stage III or IV squamous cell carcinoma of the head and neck who received induction chemotherapy with cisplatin and bleomycin sulfate with and without high-dose methotrexate were studied. The relationship of the prechemotherapy levels of serum alkaline phosphatase, lactic dehydrogenase, SGOT, SGPT, BUN, creatinine, calcium, total protein, albumin, hemoglobin, uric acid, and bilirubin and the WBC and platelet counts was correlated with the response rate. The overall response rate was 65%. No notable relationship between any of the laboratory values and the response rate was found. In contrast to an earlier report, patients with a low alkaline phosphatase level responded as well as patients with an elevated serum alkaline phosphatase level. PMID:6172102

Coker, D D; Morris, D; Elias, E G; Didolkar, M S; Zentai, T A

1982-01-01

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Serum alkaline phosphatase isoenzymes in laboratory beagle dogs detected by polyacrylamide-gel disk electrophoresis.  

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Serum alkaline phosphatase (ALP) activity is frequently measured in toxicity studies. Itoh et al. (2002) reported that a commercially available polyacrylamide-gel (PAG) disk electrophoresis kit used in humans (AlkPhor System, Jokoh Co., Ltd., Tokyo, Japan) for identifying serum ALP isoenzymes was useful for veterinary clinicopathological diagnosis in mongrel dogs. In the present study, based on the report of Itoh et al. (2002), we tried to expand the application range of this kit to laboratory beagle dogs which are commonly used in toxicity studies. In order to identify the origin of each ALP isoenzyme, tissue ALP extracts from the liver, bone and small intestine and serum samples were treated with neuraminidase, anti-small intestinal ALP antibody, ALP inhibitor levamisole and/or wheat germ agglutinin (WGA). The main serum ALP isoenzymes in 5-month-old intact beagle dogs were bone-derived (bone and atypical ALP: corresponding to human variant bone ALP) and they tended to decrease with age. However, liver-derived ALP isoenzyme greatly increased in the serum of cholestasis model dogs. The cholestasis model dogs also had a large molecular ALP detected in the resolving gel. This ALP could be originated from intestinal ALP or corticosteroid-induced ALP (CALP), because the activity remained even after levamisole inhibition. CALP was observed in intact laboratory beagle dogs with individual differences. These results suggest that the present method is a useful tool for detecting serum ALP isoenzymes in laboratory beagle dogs and concomitant levamisole inhibition with another gel is applicable for the evaluation of organ toxicity. PMID:22008540

Hatayama, Kazuhisa; Nishihara, Yoshito; Kimura, Sayaka; Goto, Ken; Nakamura, Daichi; Wakita, Atsushi; Urasoko, Yoshinaka

2011-10-01

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ALP (Alkaline Phosphatase) Test  

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... of this website will be limited. Search Help? ALP Share this page: Was this page helpful? Also ... Panel ; Bone Markers ; Alkaline Phosphatase Isoenzymes; Bone Specific ALP At a Glance Test Sample The Test Common ...

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Pesticide-induced Changes in Serum Levels of Acid Phosphatase, Alkaline Phosphates and Glutamate Oxaloacetate Transaminase in Rats  

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Full Text Available Three insecticides (Monitor, Talstar and Benzenehexachloride {BHC}, orally administered at the doses of 0.06, 0.5 and 0.2 mg kg 1 body weight respectively for 21 days affected the body weight and biochemical environment of blood and liver in the rats. Talstar treatment resulted in an increase in the body weight while Monitor and BHC treatment reduced the body weight. After 21 days of the Monitor treatment, activities of acid phosphatases increased (55% and alkaline phosphates decreased (2% in the serum while slight inhibition was observed in the serum glutamate oxaloacetate transaminase activity. Benzenehexachloride treatment resulted in increased serum levels of acid phosphatase, alkaline phosphatase and glutamate oxaloacetate transaminase (42, 2 and 18% respectively. Talstar treatment decreased the activities of acid phosphatase and alkaline phosphatase (76 and 5%, respectively while glutamate oxaloacetate transaminase activity was increased (25%. These data suggest that chronic exposure to Monitor, Talstar and BHC causes hepatocyte necrosis and increases the liver enzyme synthesis.

Muhammad Nasim Khan

2003-01-01

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Persistent limb pain and raised serum alkaline phosphatase the earliest markers of subclinical hypovitaminosis D in Kashmir.  

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The present study was an attempt to assess the cause of persistent pain in lower limbs among children from Kashmir. The study was conducted on one hundred children attending Paediatric out-patient department of Sher-i-Kashmir Institute of Medical Sciences, Srinagar. All the children were in the age group of 5 to 14 years. They showed markedly raised levels of serum alkaline phosphatase, whereas serum phosphorus, serum calcium levels and antistreptolycin O-titres were normal in 93% cases. None of them had any rheumatic or rheumatoid pathology. Among 15 suspected clinical rickets only three were established radiologically. Dietary and socio-economic history revealed deficient vitamin D intake and less exposure to sun. It was hypothesized that sub-clinical vitamin D deficiency could be a major cause of persistent pain in lower limbs and raised serum alkaline phosphatase could be the earliest marker of vitamin D deficiency. It was confirmed by injecting single dose of vitamin D (3 lac I. U.) which relieved bone pain and lowered the levels of serum alkaline phosphatase to normal within 14 weeks of initiation of therapy. PMID:2620972

Masood, H; Narang, A P; Bhat, I A; Shah, G N

1989-01-01

 
 
 
 
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Alkaline phosphatase: an overview.  

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Alkaline phosphatase (ALP; E.C.3.I.3.1.) is an ubiquitous membrane-bound glycoprotein that catalyzes the hydrolysis of phosphate monoesters at basic pH values. Alkaline phosphatase is divided into four isozymes depending upon the site of tissue expression that are Intestinal ALP, Placental ALP, Germ cell ALP and tissue nonspecific alkaline phosphatase or liver/bone/kidney (L/B/K) ALP. The intestinal and placental ALP loci are located near the end of long arm of chromosome 2 and L/B/K ALP is located near the end of the short arm of chromosome 1. Although ALPs are present in many mammalian tissues and have been studied for the last several years still little is known about them. The bone isoenzyme may be involved in mammalian bone calcification and the intestinal isoenzyme is thought to play a role in the transport of phosphate into epithelial cells of the intestine. In this review, we tried to provide an overview about the various forms, structure and functions of alkaline phosphatase with special focus on liver/bone/kidney alkaline phosphatase. PMID:24966474

Sharma, Ujjawal; Pal, Deeksha; Prasad, Rajendra

2014-07-01

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Normalization of Serum Alkaline Phosphatase in Primary Sclerosing Cholangitis Associated with Ulcerative Colitis  

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Full Text Available Primary sclerosing cholangitis (PSC is commonly associated with ulcerative colitis (UC. PSC progresses independently of UC ultimately resulting in liver failure. There is no established medical treatment to improve the natural course of PSC. Normalization of serum alkaline phosphatase (ALP in early stage might delay the progress of PSC. A 20-year-old female, had a sudden attack of right hypochondralgia with high fever and abnormal liver function tests without elevation of bilirubin: ALP 478 IU/L, aspartate aminotransferase 360 IU/L, alanine aminotransferase 174 IU/L. Abnormal liver function tests returned to normal after the attacks. Morphological examinations initially indicated then confirmed a diagnosis of PSC. One month after displaying PSC symptoms administration of ursodeoxycholic acid was initiated. Similar attacks of cholangitis were repeated several times over the following two years. Even in the absence of these attacks, she always suffered postprandial hypochondralgia. There was no acute cholangitis in the year prior to the last hospitalization due to abdominal pain and bloody diarrhea. Findings were consistent with UC in the form of entire colitis. Sulfasalazine, metronidazole and semi-vegetarian diet (SVD were initiated. Metronidazole is routinely used in inflammatory bowel disease (IBD in our practice with the expectation of elimination of any potentially pathogenic bacteria. SVD was designed for IBD hoping to increase beneficial bacteria. A remission of UC was ascertained during hospitalization. Elevated ALP, in the absence of clinical cholangitis, was decreased to normal after the therapy for UC.

Mitsuro Chiba

2014-04-01

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Evaluation of serum sialic acid, heat stable alkaline phosphatase and fucose as markers of breast carcinoma.  

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Serum levels of total sialic acid (TSA), lipid bound sialic acid (LSA), heat stable alkaline phosphatase (HSAP) and fucose were measured in 39 patients with breast carcinoma, 14 patients with benign breast diseases and 35 healthy female individuals. Elevated levels of the four biomarkers in breast carcinoma were significant when compared with controls (p less than 0.001). Fucose levels were most sensitive (71.8%), while TSA levels were most specific (64.3%) for breast carcinoma. Sensitivity and specificity were 100% when combinations of LSA with fucose and TSA with HSAP were studied respectively. LSA was significantly elevated in infiltrating duct carcinoma patients compared with lobular carcinoma (p less than 0.001). TSA, HSAP and fucose also had lower mean values in lobular carcinoma as compared to infiltrating duct carcinoma. Increase in the levels of LSA and HSAP after surgical removal of the tumor in breast carcinoma occurred prior to the clinical evidence of the recurrence. The results indicate that the combination of the markers studied might be useful in breast cancer diagnosis and treatment monitoring. PMID:2382978

Patel, P S; Baxi, B R; Adhvaryu, S G; Balar, D B

1990-01-01

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Assessment of the serum levels of bone alkaline phosphatase with a new immunoradiometric assay in patients with metabolic bone disease  

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The authors measured serum bone alkaline phosphatase (B-ALP) with a new immunoradiometric assay (IRMA) in a large sample of healthy controls comprising 173 women and 180 men, 20-88 yr of age, and in patients with metabolic bone disease. Using serum samples from patients with liver disease and patients with Paget's disease with elevated total alkaline phosphatase (T-ALP) as a source of, respectively, liver and bone isoenyzmes, they determined a liver cross-reactivity of the IRMA of 16% that was confirmed by electrophoresis of the circulating alkaline phosphatase isoenzymes. The IRMA was linear for serial sample dilutions, the recovery ranged from 89-110%, and the intra- and interassay variations were below 7% and 9%, respectively. B-ALP increased linearly with age in both sexes, and the mean B-ALP serum levels were not significantly different for women and men (11.3 ± 4.8 ng/mL for women; 11.0 ± 4.0 ng/mL for men). The increase in B-ALP after the menopause was significantly higher than that in T-ALP (+77% vs. +24%; P<0.001). When the values of postmenopausal women were expressed as the SD from the mean of premenopausal women, the mean Z scores were 2.2± 1.8 for B-ALP and 0.9 ± 1.3 for T-ALP (P<0.001 between the two)

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Serum Proteins, Thyroid Hormones and Alkaline Phosphatase Concentrationsin Acute Experimental Trypanosoma congolense Infection in Yankasa Sheep Immunomodulated with Levamisole  

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Full Text Available Serum proteins, thyroid hormones and alkaline phosphatase concentrations were measured in Yankasa sheep experimentally infected with T. congolense. Parasitemia occurred in the T. congolense infected sheep immunomodulated with levamisole two days earlier than the infected group without immunodulation.Packed cell volume decreased significantly(p0.05 in the infected sheep with and without immunomodulation when compared to the controls throughout the period of the experiment. In general, levamisole administration did not appear to alter the infection when compared to the infected group without immunomodulation.

I.A. Lawal

2007-01-01

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Influence of radioprotective agents on the activities of isoenzymes of blood serum alkaline phosphatase in irradiated dogs  

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Changes of the total serum activity of alkaline phosphatase (AP) and its bone and intestinal isoenzymes were studied in dogs ?-irradiated by 3.0 Gy. The activities of AP isoenzymes were determined by means of a heat inactivation-inhibition method. After irradiation the serum AP activities were lower in general. In case of the bone isoenzyme the decrease was most pronounced. The changes of the total AP activity and of the intestinal isoenzyme were less distinct. The i.m. administration of the radioprotective mixture cystamine with mexamine (24 mg/kg + 4 mg/kg) before irradiation led only to a moderate reduction of the radiation-altered AP values in the dogs. The present results indicate that the radioprotective way used here renders only an unsatisfactory protection for large laboratory animals against ionizing radiation. (orig.)

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Skeletal alkaline phosphatase as a serum marker of bone metastases in the follow-up of patients with breast cancer.  

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Immunoradiometric determination of the bone isoenzyme of alkaline phosphatase with a method provided by Hybritech Inc., San Diego CA (USA) was carried out in 145 female patients, 97 of whom with radically operated breast cancer and 48 with benign mammary cysts, in order to evaluate the correlation of serum levels with the metabolic process of bone rearrangement in patients with bone metastases. This study shows that skeletal ALP, having high specificity (86.48%) and sensitivity (78.6%) for early progression (the average anticipation time compared to scintigraphic detection was 101 days) could represent a valid marker for bone metastases in association with mucinous markers in the follow-up of patients operated for breast cancer. In addition, dynamic serum determination of skeletal ALP could be a valid help in monitoring the efficacy of therapy in patients with bone progression. PMID:7629426

Reale, M G; Santini, D; Marchei, G G; Manna, A; Del Nero, A; Bianco, V; Marchei, P; Frati, L

1995-01-01

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Fluorimetric determination of alkaline phosphatase activity in human serum by use of a flow-through biosensor.  

Science.gov (United States)

A fluorimetric method for the determination of alkaline phosphatase activity based on the use of a flow-through biosensor is reported. The biochemical basis of the method is the hydrolysis of 4-methyl-umbelliferone-phosphate catalyzed by the analyte with fluorimetric monitoring of the 4-methyl-umbelliferone formed (lambda ex = 365 nm, lambda em = 445 nm). The enhancement of sensitivity achieved when the reaction product is retained on the support packed in the flow-cell makes the method suitable for determination of the analytes in serum samples after 1:50 dilution. The linear range is found to be between 0.1 and 20 U l-1, with relative standard deviation less than 2.2%. The use of this biosensor was tested by the determination of the analyte in human serum from healthy and sick individuals with excellent recoveries (94-103%). PMID:7765454

Sánchez-Cabezudo, M; Fernández-Romero, J M; Luque de Castro, M D

1994-09-30

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Association of alkaline phosphatase phenotypes with arthritides  

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Full Text Available Arthritides, a symmetrical polyarticular disease of the bone are a heterogenous group of disorders in which hereditary and environmental factors in combination with an altered immune response appear to play a causative and pathogenic role in its occurrence. Alkaline phosphatase (ALP is an enzyme found in all tissues, with particularly high concentrations of ALP observed in the liver, bile ducts, placenta, and bone.Alkaline phosphatase is an orthophosphoric monoester phosphohydrolase catalyzing the hydrolysis of organic esters at alkaline pH, indicating that alkaline phosphatase is involved in fundamental biological processes.1 The present study envisages on identifying the specific electromorphic association of alkaline phosphatase with arthritides. Phenotyping of serum samples was carried out by PAGE (Polyacrylamide gel electrophoresis following Davies (19642 protocol on 41 juvenile arthritis, 150 rheumatoid arthritis and 100 osteo arthritis apart from, 25 normal children and 100 adult healthy subjects. Phenotyping of alkaline phosphatase revealed an increase in preponderance of p+ and p++ phenotypes in juvenile, rheumatoid and osteo arthritic patients. However a significant association of these phenotypes was observed only with rheumatoid arthritis condition (c2:17.46. Similarly, a significant increase of p+ phenotypes in female rheumatoid arthritis patients was observed (c2:14.973, suggesting that the decrease in p° (tissue non specific synthesis/secretion of alkaline phosphatase could be associated with decreased mineralization and ossification process in arthritis condition.

Padmini A

2004-01-01

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Serum Bone Alkaline Phosphatase in Assessing Illness Severity of Infected Neonates in the Neonatal Intensive Care Unit  

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Full Text Available Background: Infections can influence bone metabolism of neonates, which may lead to changes in some bone metabolism biomarkers. The purpose of this study was to determine whether serum bone alkaline phosphatase (BALP, osteocalcin (OC and beta carboxy-terminal peptide of type I collagen (CTX, as specific biomarkers of bone metabolism, can be used to assess the severity of neonatal infections.Methods: Sixty-three neonates in the NICU were enrolled in this study. The neonates were divided into infected group (n=33 and non-infected group (n=30. The scores for neonatal acute physiology-perinatal extension II (SNAPPE-II were calculated and interleukin-6 (IL-6, procalcitonin (PCT, BALP, OC and CTX were measured among the neonates with or without infections, and among the infected neonates before and after treatment.Results: The serum BALP levels were lower in the infected group than that in the non-infected group (p<0.01. The serum BALP levels increased markedly in the infected neonates after treatment (p<0.01. The serum BALP levels were also inversely correlated with SNAPPE-II of infected neonates before and after treatment (r=-0.56, p<0.05; r=-0.37, p<0.05, respectively. In infected neonates, the differences between serum BALP levels before and after treatment were inversely correlated with those of IL-6 levels (p<0.05. There were no significant changes in the OC, CTX and PCT levels in the infected or non-infected group before and after treatment.Conclusion: Our data suggest that serum BALP level might be used as a marker for assessing the severity of illness in infected neonates.

Yaozong Zhang, Chenguang Xue, Tian Zhu, Xiaolan Du, Nan Su, Huabing Qi, Jing Yang, Yuan Shi, Lin Chen

2012-01-01

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ESTIMATION OF SERUM ALKALINE PHOSPHATASE, CHOLESTEROL, CALCIUM AND PHOSPHORUS DURING PRE-LA YING AND LAYING CONDITIONS IN DIFFERENT STRAINS OF CHICKENS  

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Full Text Available In order to estimate serum alkaline phosphatase, cholesterol, calcium and phosphorus during pre-laying and laying reproductive conditions, 60 hens of Desi, Fayoumi, Cross (Rhode Island Red X Fayoumi} and Nick Chick strains were maintained for one year. Five random blood samples from each strain were collected and analyzed during both pre egg laying and egg laying physiological conditions. It was observed that alkaline phosphatase activity increased significantly (P<0.05 during laying condition. Serum cholesterol remained unaffected by both the strain difference and laying condition. Serum calcium and phosphorus levels increased (P<0.05 during laying condition. The interaction of strain and stage of laying condition was found to exert significant (P<0.05 effect on serum calcium levels. The study showed that the availability of calcium and phosphorus in requisite quantities be provided in diet of laying hens to ensure sustained and quality egg production.

Bashir Mahmood Bhatti, Tanzeela Talat and Rozina Sardar

2002-04-01

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

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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 ?mol/L with the detection limit of 8.0 ?mol/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.

Wei Sun

2005-12-01

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Effect of One Period of Aerobic Exercise on Serum Levels of Alkaline Phosphatase and Osteocalcin in Patients with Type 2 Diabetes  

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Full Text Available Introduction: The purpose of this study was to investigate the effects of a ten week period of aerobic exercise training on serum alkaline phosphates and osteocalsin in type 2 diabetic patients. Methods: In a quasi-experimental trial study twenty one male patients with type 2 diabetes(40-50 years were randomly divided into exercise(n=11 and control(n=10 groups. The exercise group underwent a 10-week aerobic exercise program(three sessions per week, 45-60 minutes each session, at 50-65% of heart rate reserve. VO2max, BMI, fasting blood glucose and serum insulin, alkaline phosphatase and osteocalcin were measured at baseline and after exercise program. Results: Exercise program resulted in a significant increase in VO2max and a significant decrease in BMI, fasting blood glucose and HbA1c in exercise group; however, no significant changes were found in the insulin concentration, alkaline phosphatase and osteocalcin. The bone formation markers and other measured variables did not show significant change in control group. Conclusion: These results suggest that aerobic exercise leads to glycemic improvement in type 2 diabetic patients, but does not affect serum levels of alkaline phosphatase and osteocalcin.

Hossein-nezhad

2011-12-01

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Potentiometric assay for acid and alkaline phosphatase  

International Nuclear Information System (INIS)

Simple potentiometric kinetic assay for evaluation of acid and alkaline phosphatase activity has been developed. Enzymatically catalyzed hydrolysis of monofluorophosphate, the simplest inorganic compound containing P-F bond, has been investigated as the basis of the assays. Fluoride ions formed in the course of the hydrolysis of this specific substrate have been detected using conventional fluoride ion-selective electrode based on membrane made of lanthanum fluoride. The key analytical parameters necessary for sensitive and selective detection of both enzymes have been assessed. Maximal sensitivity of the assays was observed at monofluorophosphate concentration near 10-3 M. Maximal sensitivity of acid phosphatase assay was found at pH 6.0, but pH of 4.8 is recommended to eliminate effects from alkaline phosphatase. Optimal pH for alkaline phosphatase assay is 9.0. The utility of the developed substrate-sensor system for determination of acid and alkaline phosphatase activity in human serum has been demonstrated

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Serum levels of bone-specific alkaline phosphatase and procollagen type I carboxyterminal peptide in vitamin D deficiency.  

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Vitamin D deficiency in adults causes osteomalacia where there is a defect in bone mineralization resulting in an excess of unmineralised osteoid in the bone matrix. The aim of this study was to evaluate the markers of bone formation: total (TALP), bone-specific alkaline phosphatase (BSALP) and procollagen type I carboxyterminal peptide (PICP) in vitamin D deficiency. We studied 100 vitamin D deficient subjects and 82 gender-matched controls. Vitamin D deficiency was defined as serum 25-hydroxyvitamin D level of less than 7 ng/ml, and greater than 10 ng/ml for normal controls. Serum TALP assay was performed by a standard automated method, BSALP and PICP were measured by enzyme immunoassays (Metra Biosystems) and vitamin D by radioimmunoassay. There was significant difference in the TALP between female vitamin D deficient and control subjects (mean +/- sem = 99.8 +/- 8.2 vs 70.5 +/- 2.8 iu/l, p130 iu/l) was found in 20% (20/100) of the vitamin D deficient patients. There were no significant differences in BSALP or PICP between vitamin D deficient patients and gender-matched control subjects. There was no correlation between vitamin D and PICP in patients but in control subjects, a significant negative correlation (r= -0.431, p<0.0001) was found. In conclusion, although elevated TALP was observed in a minority of vitamin D deficient patients, it is a better marker than PICP. The lack of PICP response in vitamin D deficient subjects suggests the possibility of vitamin D deficiency leading to a block in osteoblast differentiation. PMID:12755282

Nawawi, H; Girgis, S I

2002-01-01

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Serum Proteins and Alkaline Phosphatase Levels in Patients with Tuberous Sclerosis  

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Six 4- to 37-year-old patients with tuberosis sclerosis (a chronic condition characterized by siezures, intercranial calcification, a reddish-yellow sebaceous glandular mass on the face, and frequent crises in early years), did not exhibit an elevation of the (alpha + beta) globulin fraction in their serum. (Author/MC)

Fischer, M. H.; And Others

1974-01-01

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Alkaline phosphatase and transaminase activity in rat liver and blood serum at delayed periods following external ?-irradiation combined with internal exposure to plutonium 239  

International Nuclear Information System (INIS)

A study was made of activity of alkaline phosphatase and alanine- and aspartate aminotransferase in rat liver and blood serum at remote times after external ?-irradiation combined with internal exposure to 239Pu nitrate delivered in two chronically effective doses. The radionuclide was shown to be mainly responsible for the changes observed in activity of the enzymes under study. The degree to which the changes were manifest depended upon dose of plutonium administered

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Pesticide-induced Changes in Serum Levels of Acid Phosphatase, Alkaline Phosphates and Glutamate Oxaloacetate Transaminase in Rats  

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Three insecticides (Monitor, Talstar and Benzenehexachloride {BHC}), orally administered at the doses of 0.06, 0.5 and 0.2 mg kg 1 body weight respectively for 21 days affected the body weight and biochemical environment of blood and liver in the rats. Talstar treatment resulted in an increase in the body weight while Monitor and BHC treatment reduced the body weight. After 21 days of the Monitor treatment, activities of acid phosphatases increased (55%) and alkaline phosphates dec...

Muhammad Nasim Khan; Tahira Sarwar

2003-01-01

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Isoenzimas de fosfatasa alcalina en el suero de pacientes con insuficiencia renal / Alkaline phosphatase isoenzymes in the serum of patients with renal insufficiency  

Scientific Electronic Library Online (English)

Full Text Available SciELO Spain | Language: Spanish Abstract in spanish Objetivo: estudiar la utilidad clínica de la determinación sérica de las isoenzimas de fosfatasa alcalina en pacientes con insuficiencia renal. Material y métodos: se midieron las isoenzimas de fosfatasa alcalina en un grupo de 58 pacientes: 22 con insuficiencia renal aguda (IRA) y 36 con fallo rena [...] l crónico (IRC) y sometidos a hemodiálisis, comparándose los resultados con los de una población de 30 adultos sanos. Las isoenzimas intestinal, ósea, hepática, macromolecular e intestinal variante se separaron por electroforesis sobre gel de agarosa, cuantificándose por densitometría. Resultados: la actividad total de fosfatasa alcalina se mostró significativamente aumentada en ambos grupos patológicos (p Abstract in english Objective: The aim of this study was to test the utility of serum alkaline phosphatase isoenzymes determination from patients with renal insufficiency. Material and methods: serum levels of alkaline phosphatase isoenzymes were determined in a group of 58 patients: 22 of them suffering acute renal in [...] sufficiency (ARI) and 36 with chronic renal failure (CRF) undergoing regular hemodialysis, results obtained were compared from a population of 30 healthy adults. Intestinal, bone, liver, macromolecular and intestinal variant isoenzymes, were separated by electrophoresis on agarose gel and quantified using a densitometer. Results: were found a significant increase the total alkaline phosphatase activity in both pathologic groups (p

Mª. R., Sánchez Navarro; Mª. E., Fernández-Conde; S., Blanco Martín; C., Samaniego.

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Alkaline phosphatase: Distinguishing between tuberculous and nontuberculous pleural effusion  

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Full Text Available Objectives: To evaluate the value of pleural fluid alkaline phosphatase and pleural fluid/serum alkaline phosphatase ratio for the purpose of differentiating tuberculous from nontuberculous pleural effusion. Materials and Methods: A total of 60 indoor patients, admitted to our hospital, having pleural effusion and suffering from varying etiologies, were included in this study. According to the final diagnosis, these 60 patients were divided into two groups: Tuberculous (30 and nontuberculous (30 pleural effusion. Results: The mean pleural alkaline phosphatase and pleural fluid/serum alkaline phosphatase ratio was significantly higher in tuberculous compared to nontuberculous pleural effusion. ( P < 0.0001. In receiver operating characteristic curve analysis, sensitivity and specificity values were 90% and 80% for a cut-off value of 71 IU/L for pleural alkaline phosphatase activity; and were 90% and 86.66% for a cut-off value of 0.51 for pleural fluid/serum alkaline phosphatase ratio. Conclusion: From this study it is concluded that alkaline phosphatase activity remains a useful test in differentiation of tuberculous from nontuberculous pleural effusion.

Jadhav Ashish

2009-01-01

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

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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 1?g 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.

Luiz Augusto de Souza

2011-12-01

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Sensitive fluorogenic substrate for alkaline phosphatase.  

Science.gov (United States)

Alkaline phosphatase serves both as a model enzyme for studies on the mechanism and kinetics of phosphomonoesterases and as a reporter in enzyme-linked immunosorbent assays (ELISAs) and other biochemical methods. The tight binding of the enzyme to its inorganic phosphate product leads to strong inhibition of catalysis and confounds measurements of alkaline phosphatase activity. We have developed an alkaline phosphatase substrate in which the fluorescence of rhodamine is triggered on P-O bond cleavage in a process mediated by a "trimethyl lock." Although this substrate requires a nonenzymatic second step to manifest fluorescence, we demonstrated that the enzymatic first step limits the rate of fluorogenesis. The substrate enables the catalytic activity of alkaline phosphatase to be measured with high sensitivity and accuracy. Its attributes are ideal for enzymatic assays of alkaline phosphatase for both basic research and biotechnological applications. PMID:21827735

Levine, Michael N; Raines, Ronald T

2011-11-15

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21 CFR 864.7660 - Leukocyte alkaline phosphatase test.  

Science.gov (United States)

... 2010-04-01 false Leukocyte alkaline phosphatase test. 864.7660 Section...and Packages § 864.7660 Leukocyte alkaline phosphatase test. (a) Identification. A leukocyte alkaline phosphatase test is a device used...

2010-04-01

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21 CFR 864.7660 - Leukocyte alkaline phosphatase test.  

Science.gov (United States)

...2010-04-01 false Leukocyte alkaline phosphatase test. 864.7660 Section... § 864.7660 Leukocyte alkaline phosphatase test. (a) Identification. A leukocyte alkaline phosphatase test is a device used...

2010-04-01

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Relationship between serum heat-stable alkaline phosphatase activity and blood pressure in patients with pre-eclampsia and eclampsia  

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Full Text Available Background : The objective of this study was to explore the relationship, if any, between theserum heat-stable alkaline phosphatase (HS-ALP activity and the blood pressure (BP of patients with pre-eclampsia and eclampsia. Method : The activity of HS-ALP was measured using the 4– nitrophenyl phosphate (4– NPP method after incubation at a high temperature of 65 0 C for exactly 30 minutes in one hundred normal pregnant women and in another one hundred with pre-eclampsia/eclampsia. The normal pregnant women were used as controls. The blood pressure (BP, systolic as well as diastolic was measured in each of the studied patient using desktop mercury sphygmomanometer. Results :In the patients with pre-eclampsia/eclampsia, it was found that the higher the systolic and diastolic BP, the higher is the activity of the HS-ALP. Conclusion : It can be concluded that the HS-ALP activity in patients with pre-eclampsia/eclampsia is positively related to the severity of the hypertension and therefore this could help in detecting early complication.

Aliyu I

2006-03-01

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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)

Rats were given a single exposure of various doses (0, 5, 50, 500, and 5000 rads) to local irradiation of 60Co ?-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)

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Serum proteins, trace metals and phosphatases in psoriasis  

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

Bhatnagar M

1994-01-01

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

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

Han Ju

2012-02-01

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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  

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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 months of GH treatment (p <0.0001). In addition, the molar ratio between IGF-I and IGFBP-3 increased significantlyfrom 0.22 to 0.33 after GH treatment (p <0.0001). Bone alkaline phosphatase increased significantly from 38.6 to 92.9 U/l during GH therapy in male patients (p <0.0001), whereas liver-derived alkaline phosphatase was unaltered by GH. In the females, the increase in bone alkaline phosphatase did not reach statistical significance (19.1 vs 40.0 U/l, p = 0.06). The GH-induced increase in bone alkaline phosphatase correlated significantly with the increase in serum IGFBP-3 (r = 0.46, p = 0.04) but not with the increase in serum IGF-I (p = 0.16).(ABSTRACT TRUNCATED AT 250 WORDS)

Juul, A; Pedersen, S A

1994-01-01

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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)

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)

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

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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 correlation between CXCR4 expression and serum ALP.Conclusion: CXCR4 siRNA/dextran-spermine nanoparticles appear to be highly effective, and may be suitable for further in vivo applications. Further research evaluation will be needed to determine the effect of CXCR4 silencing on serum ALP levels, which may be a useful marker to predict liver metastasis in colorectal cancer.Keywords: nanoparticles, cationized dextran, colorectal cancer, serum ALP enzyme, CXCR4, siRNA

Abedini F

2012-07-01

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Radiation inactivation of bovine intestinal alkaline phosphatase  

International Nuclear Information System (INIS)

The effects of 60CO ?-radiation on aqueous solutions of alkaline phosphatase have been studied. The primary radicals of water radiolysis, eaq-, OH. and H. all contribute to the observed inactivation with inactivating efficiencies of 0.019, 0.019 and 0.04, respectively; O2- also causes inactivation (efficiency = 0.014). The radical anions (SCN)2-, (Br)2-, and (I)2- cause inactivation at neutral pH and evidence is presented that cysteine and histidine residues are sites for radical anion reaction. Kinetic evidence suggests that inactivation is due to general denaturation of the protein, rather than destruction of the substrate binding site. Fractionation of the irradiated solution using FPLC following by analysis using fluorescence spectroscopy suggests that one process which leads to inactivation is the formation of alkaline phosphatase dimerized via tyrosine residues. (author)

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Radiation inactivation of bovine intestinal alkaline phosphatase  

Science.gov (United States)

The effects of 60Co ?-radiation on aqueous solutions of alkaline phosphatase have been studied. The primary radicals of water radiolysis, e -aq, OH· and H· all contribute to the observed inactivation with inactivating efficiencies of 0.019, 0.019 and 0.04, respectively; O -2 also causes inactivation (efficiency = 0.014). The radical anions (SCN) -2, (Br) -2 and (I) -2 cause inactivation at neutral pH and evidence is presented that cysteine and histidine residues are sites for radical anion reaction. Kinetic evidence suggests that inactivation is due to general denaturation of the protein, rather than destruction of the substrate binding site. Fractionation of the irradiated solution using FPLC following by analysis using fluorescence spectroscopy suggests that one process which leads to inactivation is the formation of alkaline phosphatase dimerized via tyrosine residues.

Hasan, N. M.; McCall, P. R.; Moore, J. S.; Power, D. M.

1994-03-01

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A double antibody radioimmunoassay specific for placental alkaline phosphatase  

International Nuclear Information System (INIS)

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.)

55

Negative modulation of alkaline phosphatase and creatine kinase by homobrassinolide  

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Full Text Available

Homobrassinolide is a plant hormone implicated in plant growth and development. Its effect on animal metabolism was less known to date. We have investigated its effect on the marker enzymes such as alkaline phosphatase and creatine kinase in selected rat tissues-brain, heart, liver, kidney, skeletal muscle and testis. Homobrassinolide was administered (66 and 330ng/ Kg body weight intradermally in male albino wistar strain rats and changes in alkaline phosphatase and creatine kinase activities were measured. An overall reduction in both the enzyme activities occurred within 2hr of administration with few exceptions. The reaction rate constants for the enzyme activities were in the order 10-7 mM/min for alkaline phosphatase and 10-3 mM/min for creatine kinase. Time course studies indicated a decrease in enzyme activities as a function of time. Elevated hemoglobin content correlated with rise in erythrocyte number. Blood glucose level decreased by a percentage of 15.7 and 21.7 compared to control with the administration of 10?g and 50?g homobrassinolide respectively. Serum cholesterol content showed 15% decrease and 25% increase compared to control following 10?g and 50?g homobrassinolide administration. We conclude that homobrassinolide inhibited both the enzymes in the tissues and produced erythrocytosis, leukocytosis and hypoglycemia, while cellular phosphorylation status remained principally affected by this oxysterol in rat. Even though the physiological and pathological significance of these observations is not clear, it is suggested that 28-HB enriched diets may not be appropriate for higher energy related work activities.

Keywords: Alkaline phosphatase; Creatine kinase; Homobrassinolide; Oxysterol; Phosphorylation; Rate constant.

G. Nirmal Kumar

2011-04-01

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Human placental alkaline phosphatase in liver and intestine  

International Nuclear Information System (INIS)

Three distinct forms of human alkaline phosphatase, presumably isozymes, are known, each apparently associated with a specific tissue. These are placental, intestinal, and liver (kidney and bone). The authors have used a specific immunoassay and HPLC to show that placental alkaline phosphatase is also present in extracts of liver and intestine in appreciable amounts

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Phosphatidylinositol anchor of HeLa cell alkaline phosphatase  

International Nuclear Information System (INIS)

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

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21 CFR 862.1050 - Alkaline phosphatase or isoenzymes test system.  

Science.gov (United States)

...2010-04-01 2010-04-01 false Alkaline phosphatase or isoenzymes test system...Chemistry Test Systems § 862.1050 Alkaline phosphatase or isoenzymes test system. (a) Identification. An alkaline phosphatase or isoenzymes test...

2010-04-01

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21 CFR 862.1050 - Alkaline phosphatase or isoenzymes test system.  

Science.gov (United States)

... 2010-04-01 false Alkaline phosphatase or isoenzymes test system...Systems § 862.1050 Alkaline phosphatase or isoenzymes test system. (a) Identification. An alkaline phosphatase or isoenzymes test...

2010-04-01

60

Alkaline phosphatase as a periodontal disease marker  

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Full Text Available Background: The potential of alkaline phosphatase (ALP as an important diagnostic marker of gingival crevicular fluid (GCF has been the subject to investigation since 1970. ALP is stored in specific granules and secretory vesicles of the neutrophils and is mainly released during their migration to the site of infection. It is also present in bacteria within dental plaque, osteoblasts and fibroblasts. It has, thus, become important to elucidate whether GCF levels of ALP are potential measures of the inflammatory activity occurring in the adjacent periodontal tissues. Objective: The aim of this study was to assess the total activity of ALP in the GCF collected from healthy sites, sites with gingivitis and with chronic adult periodontitis. An attempt was also made to establish the correlation of ALP activity with plaque index, gingival index, bleeding index and probing depth. Materials and Methods: A total of 18 patients were divided into three groups: viz., healthy sites, Group I; gingivitis, Group II; chronic periodontitis, Group III. Clinical parameters like plaque index, bleeding index, gingival index and probing depth were recorded. The ALP level in GCF of all three groups was determined by spectrophotometric analysis. Results: Total enzyme activity of ALP was significantly higher in periodontitis as compared with that in healthy and gingivitis sites, and was significantly and positively correlated with probing depth. Conclusion: ALP can be considered as a periodontal disease marker as it can distinguish between healthy and inflamed sites. However, to better define its capacity for periodontal diagnosis, additional longitudinal studies are required.

Malhotra Ranjan

2010-01-01

 
 
 
 
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Phosphorylation by Alkaline Phosphatase: Immobilization and Synthetic Potential  

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Full Text Available Phosphatases (AP, E.C. 3.1.3.1 are hydrolytic enzymes that naturally hydrolyse phosphomonoesters but in a so-called transphosphorylation reaction these enzymes are also able to transfer a phosphate group from phosphorylated compounds to alcoholic functions. This transphosphorylation catalysed by acid phosphatases using pyrophosphate as a phosphate donor has been studied in some detail. However, the acidic pH optimum of these enzymes limits some of their applications. The catalytic features of alkaline phosphatase are similar to the acid phosphatases and its alkaline pH optimum suggests a possible application of this enzyme in phosphorylation reactions which need to be carried out at higher pH. Here we explore the synthetic potential of bovine intestine alkaline phosphatase (AP in the phosphorylation of dihydroxyacetone (DHA and glycerol using pyrophosphate (PPi as phosphate donor. The phosphorylated compounds are intermediates in two multi-enzymatic cascade reactions for the synthesis of carbohydrates. The yields of dihydroxyacetone phosphate (DHAP and glycerol-1-phosphate at pH 8 (2.6 mM and 2.2 mM, respectively were comparable to the results obtained with the acid phosphatases at pH 4. Nevertheless, when the cascade reactions were carried out at pH 8, very low conversions were measured due to inactivation of the alkaline phosphatase by the product phosphate. To circumvent this inhibition, the alkaline phosphatase was immobilized on aldehyde-activated beads (Sepabeads EC-HA. The immobilization greatly diminished the inhibition by phosphate, and the immobilized alkaline phosphatase at pH 8 gave the same conversions in the cascade reaction starting from DHA as obtained with the acid phosphatase at pH 6. However, the immobilized enzyme was active for only one catalytic cycle and the beads could not be reused.

Lara Babich

2013-05-01

62

Negative modulation of alkaline phosphatase and creatine kinase by homobrassinolide  

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Homobrassinolide is a plant hormone implicated in plant growth and development. Its effect on animal metabolism was less known to date. We have investigated its effect on the marker enzymes such as alkaline phosphatase and creatine kinase in selected rat tissues-brain, heart, liver, kidney, skeletal muscle and testis. Homobrassinolide was administered (66 and 330ng/ Kg body weight) intradermally in male albino wistar strain rats and changes in alkaline phosphatase and creatine kinase...

Nirmal Kumar, G.; Lakshmy, S.; Srikumar, K.

2011-01-01

63

Overexpression of Human Bone Alkaline Phosphatase in Pichia Pastoris  

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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.

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

2000-01-01

64

Alkaline Phosphatase Expression/Activity and Multilineage Differentiation Potential are the Differences Between Fibroblasts and Orbital Fat-Derived Stem Cells - A Study in Animal Serum-Free Culture Conditions.  

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Human orbital fat tissues are a potential source to isolate stem cells for the development of regenerative medicine therapies. For future safe clinical application of these cells, it is critical to establish animal component-free culture conditions as well as to clearly define the stem cell population characteristics differentiating them from other cell types, such as fibroblasts. Therefore, the present study aimed to compare phenotypic and functional characteristics of orbital fat-derived stem cells (OFSCs) and fibroblasts resident in the eyelid skin in donor-matched samples grown in culture medium supplemented with pooled allogeneic human serum (HS) replacing fetal bovine serum (FBS). We first investigated the proliferative effects of OFSCs on HS, and then we compared the alkaline phosphatase (AP) expression and activity, immunophenotypic profile, and in vitro multilineage differentiation potential of OFSCs side-by-side with fibroblasts. The results showed that HS enhanced OFSCs proliferation without compromising their immunophenotype, AP activity, and osteogenic, adipogenic, and chondrogenic differentiation capacities. In contrast to OFSCs, the fibroblasts did not exhibit AP expression and activity and did not have multilineage differentiation potential. The results enabled us to successfully distinguish OFSCs from fibroblasts populations, suggesting that AP expression/activity and multilineage differentiation assays can be used reliably to discriminate mesenchymal stem cells from fibroblasts. Our findings also support the feasibility of pooled allogeneic HS as a safer and more effective alternative to FBS for clinical applications. PMID:24913281

Martins, Thaís Maria da Mata; de Paula, Ana Cláudia Chagas; Gomes, Dawidson Assis; Goes, Alfredo Miranda

2014-10-01

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Serum N-telopeptide of type I collagen and bone alkaline phosphatase and their relationship in patients with non-small cell lung carcinoma and bone metastases. Preliminary results.  

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Lung cancer represents the most common malignancy in Western countries and the presence of bone metastasis (BMs) may significantly worsen the prognosis. Several urinary and serum markers are altered in patients with BMs from non-small cell lung cancer (NSCLC). The aim of this study was to assess the usefulness of two serum markers of bone remodeling in patients with NSCLC and BMs. Thirty-five patients (24 men, 11 women, median age 63 years, range 51-72 years) with NSCLC were examined. There were 16 patients with confirmed BMs (Group A), and 19 age-matched (63.5±4.9 vs. 63.7±4.4 years; p=0.88) patients without BMs (Group B). Serum levels of bone resorption marker cross-linked amino-terminal telopeptide of type I collegen (NTx), and bone formation marker bone alkaline phosphatase (BAP) were measured in both groups by enzyme-linked immunosorbent assay. Both NTx (33.5±7.2 vs. 25.6±3.1 nM bone collagen equivalent, BCE/l) and BAP (51.7±6.0 vs. 40.7±7.3 U/l) serum levels were significantly (pnM BCE/l (TNx) and 50 U/l (BAP), the sensitivity was 56.2% and 37.5%, respectively (Odds ratio, OR=0.47, 95% confidence interval, CI 0.11-1.91, p=0.48), while the specificity was 89.5% and 84.2% (OR=0.62, 95% CI 0.09-4.26, p=0.50), respectively. No correlation was found between age and both NTx (R=-0.34, p=0.08) and BAP (R=-0.10, p=0.61) among patients with BMs. In conclusion, in patients with NSCLC and BMs both NTx and BAP are specific markers of bone remodeling, but their usefulness is limited in early diagnosis of metastatic disease. PMID:22110213

Lumachi, Franco; Marino, Filippo; Fanti, Giovanni; Chiara, Giordano B; Basso, Stefano M M

2011-11-01

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Evaluation of Testosterone and Alkaline Phosphatase Activity Changes in Epidydimis of Toxoplasma gondii Infected Rats  

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Full Text Available Objective: Toxoplasma gondii is a widespread protozoan parasite that infects a broad range of warm blooded animals and humans. The present study was investigated to evaluate testosterone, alkaline phosphatase activity and malondialdehyde in male rats experimentally infected by Toxoplasma gondii, RH strain.Material & Methods: Male Wistar rats (n=20 were allocated into two groups, group one (n=10 that received 0.6 cc tachyzoites of T. gondii intraperitoneally (I.P., and control group (n=10 that received nothing. Animals were kept in standard conditions. 30 days after inducing Toxoplasma infection, 5cc blood was collected for assessment of serum testosterone, alkaline phosphatase and malondialdehyde levels. Epididymis tissues of Rats in whole groups were removed and prepared for analysis.Results: Alkaline phosphatase, and Testosterone were significantly increased in group that was infected by T.gondii in comparison to control group (P0.05.Epididymis weights in toxoplasmosis group was significantly decreased in comparison to control group (P<0.05. Positive brown alkaline phosphatase were observed in epididym tissue of infected toxoplasma group in comparison to control group.Conclusion: This study showed that T. gondii has augmenter effects on alkaline phosphatase activity, testosterone and has harmful effect on epididymis tissue.

Fatemeh Afshari

2013-07-01

67

Characterization of Human Bone Alkaline Phosphatase in Pichia Pastoris  

Science.gov (United States)

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.

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

1999-01-01

68

Sensitive optical detection of alkaline phosphatase activity with quantum dots  

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

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

2014-01-15

69

Sensitive optical detection of alkaline phosphatase activity with quantum dots  

International Nuclear Information System (INIS)

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

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Alkaline Phosphatase from Venom of the Endoparasitoid Wasp, Pteromalus puparum  

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Using chromogenic substrates 5-bromo-4-chloro-3?-indolyl phosphate and nitro blue tetrazolium, alkaline phosphatase (ALPase) was histochemically detected in the venom apparatus of an endoparasitoid wasp, Pteromalus puparum L. (Hymenoptera: Pteromalidae). Ultrastructural observations demonstrated its presence in the secretory vesicles and nuclei of the venom gland secretory cells. Using p-nitrophenyl phosphate as substrate to measure enzyme activity, the venom ALPase was found to be temperat...

Zhu, Jia-ying; Yin Ye, Gong; Fang, Qi; Hu, Cui

2010-01-01

71

As fosfatases alcalinas, transaminases e gama-glutamil-transferase séricas em pacientes epilépticos tratados com carbamazepina The serum alkaline phosphatases, transaminases, and gamma-glutamil transferases in epileptic patients treated with carbamazepine  

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Full Text Available INTRODUÇÃO: A carbamazepina é a droga utilizada no tratamento de pacientes com epilepsia parcial (ou focal secundariamente generalizada. Apesar do uso terapêutico, este fármaco tem sido implicado no aumento das atividades séricas de algumas enzimas. Alguns autores descreveram valores de prevalência de 7,7%, 13% e 22% para aumento de atividade das fosfatases alcalinas séricas (FA ou EC 3.1.3.1. A divergência de resultados também foi encontrada para as atividades da gama-glutamil-transferase sérica (gama-glutamil transferase ou GGT ou EC 2.3.2.2. OBJETIVO: Assim, a meta desta pesquisa é determinar, dentre outros objetivos, a freqüência de alterações nas atividades das FA, GGT e transaminases (AST, aspartato-amino-transferase, EC 2.6.1.1; e ALT, alanina-amino-transferase, EC 2.6.1.2 de uma amostra de pacientes do ambulatório de epilepsia em Salvador, Bahia. MATERIAL E MÉTODOS: O desenho do estudo é descritivo do tipo série de casos, aprovado pelo Comitê de Ética local, no qual uma amostra de conveniência de 52 pacientes epilépticos de acompanhamento ambulatorial foi obtida sem interferência dos pesquisadores. Estes pacientes foram organizados por faixa etária de 12 a 30 e de 31 a 90 anos e, subdivididos por tempo de monoterapia com carbamazepina. As atividades séricas das enzimas GGT, FA, AST e ALT foram determinadas. RESULTADOS: As proporções de alterações por variáveis foram descritas: 42% para as FA, 18% para as GGT, 2% para as ALT e 12% para as AST, respectivamente. A faixa etária de 12 a 30 anos apresentou 56% de alterações nas FA enquanto que aquela de 31 a 90 anos, apenas 18%. CONCLUSÃO: Nós concluímos que as enzimas FA, GGT, AST e ALT apresentaram maiores freqüências de alterações de suas atividades naqueles pacientes com idade igual ou inferior a 30 anos, sendo que as FA apresentaram maiores valores.INTRODUCTION: Carbamazepine is the drug of choice used in the treatment of patients with partial (or focal epilepsy with secondary generalization. Despite its therapeutical use, this drug has been implicated in the increase of serum activities in some enzymes. Some authors have described prevalence values of 7.7%, 13%, and 22% for the increase of activity of serum alkaline phosphatases (AF or EC 3.1.3.1. A divergence in the results was also found for the activities of the serum g-glutamil transferase (gamma-glutamil transferase or GGT, or EC 2.3.2.2. OBJECTIVE: Hence, among other objectives, the aim of this research is to determine the frequency of sample alterations in serum enzymatic activities of AF, GGT and transaminases (AST, aspartate amino-transferase, EC 2.6.1.1; and ALT, alanine-amino-transferase, EC 2.6.1.2 in epilepsy ward patients in Salvador, Bahia. MATERIAL AND METHODS: The design of the study is descriptive and it is a case series type. It has been approved by the local Ethics Committee. In this study, a convenience sample of 52 epileptic patients who receive ambulatory care was obtained without interference by the researchers. These patients were divided according to age groups of 12 to 30 years and 31 to 90 years, which were then subdivided according to the period of monotherapy with carbamazepine. The serum activities of the enzymes GGT, AF, AST and ALT were determined. RESULTS: The ratios of alterations per variables were described: 42% for the FA, 18% for the GGT, 2% for the ALT, and 12% for the AST respectively. The age group of 12 to 30 years presented 56% of alterations in the AF while the group of 31 to 90 years presented only 18% of alterations. CONCLUSION: We conclude that the enzymes AF, GGT, AST, and ALT presented higher frequencies of alterations of their activities in those patients with age equal to or below 30 years, while the AF presented higher values.

Helder Jacobina Santos

2006-03-01

72

Trypanosoma rangeli: an alkaline ecto-phosphatase activity is involved with survival and growth of the parasite.  

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The aim of this work was to investigate whether an alkaline ecto-phosphatase activity is present in the surface of Trypanosoma rangeli. Intact short epimastigote forms were assayed for ecto-phosphatase activity to study kinetics and modulators using ?-glycerophosphate (?-GP) and p-nitrophenyl phosphate (pNPP) as substrates. Its role in parasite development and differentiation was also studied. Competition assays using different proportions of ?-GP and pNPP evidenced the existence of independent and non-interacting alkaline and acid phosphatases. Hydrolysis of ?-GP increased progressively with pH, whereas the opposite was evident using pNPP. The alkaline enzyme was inhibited by levamisole in a non-competitive fashion. The Ca(2+) present in the reaction medium was enough for full activity. Pretreatment with PI-PLC decreased the alkaline but not the acid phosphatase evidence that the former is catalyzed by a GPI-anchored enzyme, with potential intracellular signaling ability. ?-GP supported the growth and differentiation of T. rangeli to the same extent as high orthophosphate (Pi). Levamisole at the IC50 spared significantly parasite growth when ?-GP was the sole source of Pi and stopped it in the absence of ?-GP, indicating that the alkaline enzyme can utilize phosphate monoesters present in serum. These results demonstrate the existence of an alkaline ecto-phosphatase in T. rangeli with selective requirements and sensitivity to inhibitors that participates in key metabolic processes in the parasite life cycle. PMID:23994113

Dos-Santos, André L A; Dick, Claudia F; Silveira, Thaís S; Fonseca-de-Souza, André L; Meyer-Fernandes, José R

2013-10-01

73

Comparison of horseradish peroxidase and alkaline phosphatase-labelled antibodies in enzyme immunoassays.  

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The periodate method was found to be most effective for preparing horseradish peroxidase-sheep anti-human and horseradish peroxidase-donkey anti-mouse immunoglobulin (IgG) conjugates. The conjugates were improved by carrying out the oxidation of the enzyme at low pH. Anti-human and anti-mouse IgG-peroxidase conjugates (0.5 mg/mL IgG and 0.7 mg/mL IgG, respectively) were used at 1:15,000 and 1:8000 dilutions, respectively, in a sandwich ELISA to detect human and mouse IgG in buffer or in a growth medium containing 20% foetal calf serum. Using the peroxidase conjugates, it was possible to detect human and mouse IgG at concentrations as low as 1 ng/mL. The glutaraldehyde method was found to be much more effective than the periodate method for conjugating alkaline phosphatase to the antibodies. The optimum dilutions for anti/human and anti-mouse IgG-alkaline phosphatase conjugates (0.18 mg/mL IgG and 0.3 mg/mL IgG, respectively) in ELISA were 1:500 and 1:1000, respectively. The detection limit with alkaline phosphatase conjugates was 7 ng/ml for human IgG and 4 ng/ml for mouse IgG. PMID:3035992

Beyzavi, K; Hampton, S; Kwasowski, P; Fickling, S; Marks, V; Clift, R

1987-03-01

74

Specific Immunoassays for Placental Alkaline Phosphatase As a Tumor Marker  

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Human placental (hPLAP) and germ cell (PLAP-like) alkaline phosphatases are polymorphic and heat-stable enzymes. This study was designed to develop specific immunoassays for quantifying hPLAP and PLAP-like enzyme activity (EA) in sera of cancer patients, pregnant women, or smokers. Polyclonal sheep anti-hPLAP antibodies were purified by affinity chromatography with whole hPLAP protein (ICA-PLAP assay) or a synthetic peptide (aa 57–71) of hPLAP (ICA-PEP assay); the worki...

Stinghen, Se?rvio T.; Moura, Juliana F.; Zancanella, Patri?cia; Rodrigues, Giovanna A.; Pianovski, Mara A.; Lalli, Enzo; Arnold, Dodie L.; Minozzo, Joa?o C.; Callefe, Luis G.; Ribeiro, Raul C.; Figueiredo, Bonald C.

2006-01-01

75

ALKALINE PHOSPHATASE ACTIVITY AS A MARKER OF DOG SEMEN FREEZABILITY  

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

KOSINIAK-KAMYSZ K.

2007-01-01

76

ALKALINE PHOSPHATASE ACTIVITY AS A MARKER OF DOG SEMEN FREEZABILITY  

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

K. KOSINIAK-KAMYSZ

2013-12-01

77

Regan isoenzyme of alkaline phosphatase as a tumour marker for renal cell carcinoma.  

Science.gov (United States)

Alkaline phosphatase is an enzyme present in all tissues of the human body. Several isoforms of this enzyme have been described with different catalytic nature, stability and antigenic structure. Rises in the activity of alkaline phosphatase are recognised in various states including bone diseases, liver disease, pregnancy, hyperthyroidism and malignant processes. The Regan isoenzyme, a rare variant of placental alkaline phosphatase, has been identified circulating in association with various tumours. The reported case describes a rising Regan isoform of alkaline phosphatase concentrations that led to a new diagnosis of occult renal cell carcinoma and persistently elevated activity postoperatively signposting persistent or recurrent disease. PMID:24615345

Bukowczan, J; Pattman, S; Jenkinson, F; Quinton, R

2014-09-01

78

Specific immunoassays for placental alkaline phosphatase as a tumor marker.  

Science.gov (United States)

Human placental (hPLAP) and germ cell (PLAP-like) alkaline phosphatases are polymorphic and heat-stable enzymes. This study was designed to develop specific immunoassays for quantifying hPLAP and PLAP-like enzyme activity (EA) in sera of cancer patients, pregnant women, or smokers. Polyclonal sheep anti-hPLAP antibodies were purified by affinity chromatography with whole hPLAP protein (ICA-PLAP assay) or a synthetic peptide (aa 57-71) of hPLAP (ICA-PEP assay); the working range was 0.1-11 U/L and cutoff value was 0.2 U/L EA for nonsmokers. The intra- and interassay coefficients of variation were 3.7%-6.5% (ICA-PLAP assay) and 9.0%-9.9% (ICA-PEP assay). An insignificant cross-reactivity was noted for high levels of unheated intestinal alkaline phosphatase in ICA-PEP assay. A positive correlation between the regression of tumor size and EA was noted in a child with embryonal carcinoma. It can be concluded that ICA-PEP assay is more specific than ICA-PLAP, which is still useful to detect other PLAP/PLAP-like phenotypes. PMID:17489017

Stinghen, Sérvio T; Moura, Juliana F; Zancanella, Patrícia; Rodrigues, Giovanna A; Pianovski, Mara A; Lalli, Enzo; Arnold, Dodie L; Minozzo, João C; Callefe, Luis G; Ribeiro, Raul C; Figueiredo, Bonald C

2006-01-01

79

As fosfatases alcalinas, transaminases e gama-glutamil-transferase séricas em pacientes epilépticos tratados com carbamazepina / The serum alkaline phosphatases, transaminases, and gamma-glutamil transferases in epileptic patients treated with carbamazepine  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: Portuguese Abstract in portuguese INTRODUÇÃO: A carbamazepina é a droga utilizada no tratamento de pacientes com epilepsia parcial (ou focal) secundariamente generalizada. Apesar do uso terapêutico, este fármaco tem sido implicado no aumento das atividades séricas de algumas enzimas. Alguns autores descreveram valores de prevalência [...] de 7,7%, 13% e 22% para aumento de atividade das fosfatases alcalinas séricas (FA ou EC 3.1.3.1). A divergência de resultados também foi encontrada para as atividades da gama-glutamil-transferase sérica (gama-glutamil transferase ou GGT ou EC 2.3.2.2). OBJETIVO: Assim, a meta desta pesquisa é determinar, dentre outros objetivos, a freqüência de alterações nas atividades das FA, GGT e transaminases (AST, aspartato-amino-transferase, EC 2.6.1.1; e ALT, alanina-amino-transferase, EC 2.6.1.2) de uma amostra de pacientes do ambulatório de epilepsia em Salvador, Bahia. MATERIAL E MÉTODOS: O desenho do estudo é descritivo do tipo série de casos, aprovado pelo Comitê de Ética local, no qual uma amostra de conveniência de 52 pacientes epilépticos de acompanhamento ambulatorial foi obtida sem interferência dos pesquisadores. Estes pacientes foram organizados por faixa etária de 12 a 30 e de 31 a 90 anos e, subdivididos por tempo de monoterapia com carbamazepina. As atividades séricas das enzimas GGT, FA, AST e ALT foram determinadas. RESULTADOS: As proporções de alterações por variáveis foram descritas: 42% para as FA, 18% para as GGT, 2% para as ALT e 12% para as AST, respectivamente. A faixa etária de 12 a 30 anos apresentou 56% de alterações nas FA enquanto que aquela de 31 a 90 anos, apenas 18%. CONCLUSÃO: Nós concluímos que as enzimas FA, GGT, AST e ALT apresentaram maiores freqüências de alterações de suas atividades naqueles pacientes com idade igual ou inferior a 30 anos, sendo que as FA apresentaram maiores valores. Abstract in english INTRODUCTION: Carbamazepine is the drug of choice used in the treatment of patients with partial (or focal) epilepsy with secondary generalization. Despite its therapeutical use, this drug has been implicated in the increase of serum activities in some enzymes. Some authors have described prevalence [...] values of 7.7%, 13%, and 22% for the increase of activity of serum alkaline phosphatases (AF or EC 3.1.3.1). A divergence in the results was also found for the activities of the serum g-glutamil transferase (gamma-glutamil transferase or GGT, or EC 2.3.2.2). OBJECTIVE: Hence, among other objectives, the aim of this research is to determine the frequency of sample alterations in serum enzymatic activities of AF, GGT and transaminases (AST, aspartate amino-transferase, EC 2.6.1.1; and ALT, alanine-amino-transferase, EC 2.6.1.2) in epilepsy ward patients in Salvador, Bahia. MATERIAL AND METHODS: The design of the study is descriptive and it is a case series type. It has been approved by the local Ethics Committee. In this study, a convenience sample of 52 epileptic patients who receive ambulatory care was obtained without interference by the researchers. These patients were divided according to age groups of 12 to 30 years and 31 to 90 years, which were then subdivided according to the period of monotherapy with carbamazepine. The serum activities of the enzymes GGT, AF, AST and ALT were determined. RESULTS: The ratios of alterations per variables were described: 42% for the FA, 18% for the GGT, 2% for the ALT, and 12% for the AST respectively. The age group of 12 to 30 years presented 56% of alterations in the AF while the group of 31 to 90 years presented only 18% of alterations. CONCLUSION: We conclude that the enzymes AF, GGT, AST, and ALT presented higher frequencies of alterations of their activities in those patients with age equal to or below 30 years, while the AF presented higher values.

Helder Jacobina, Santos; Antonio de Souza, Andrade Filho; Olivia Lordelo, Sanches; Tiago, Spolador; Luís Erlon Araújo, Rodrigues.

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Study on alkaline and acid phosphatase activity in acute uranium intoxication  

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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.)

 
 
 
 
81

Intestinal alkaline phosphatase prevents metabolic syndrome in mice.  

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Metabolic syndrome comprises a cluster of related disorders that includes obesity, glucose intolerance, insulin resistance, dyslipidemia, and fatty liver. Recently, gut-derived chronic endotoxemia has been identified as a primary mediator for triggering the low-grade inflammation responsible for the development of metabolic syndrome. In the present study we examined the role of the small intestinal brush-border enzyme, intestinal alkaline phosphatase (IAP), in preventing a high-fat-diet-induced metabolic syndrome in mice. We found that both endogenous and orally supplemented IAP inhibits absorption of endotoxin (lipopolysaccharides) that occurs with dietary fat, and oral IAP supplementation prevents as well as reverses metabolic syndrome. Furthermore, IAP supplementation improves the lipid profile in mice fed a standard, low-fat chow diet. These results point to a potentially unique therapy against metabolic syndrome in at-risk humans. PMID:23569246

Kaliannan, Kanakaraju; Hamarneh, Sulaiman R; Economopoulos, Konstantinos P; Nasrin Alam, Sayeda; Moaven, Omeed; Patel, Palak; Malo, Nondita S; Ray, Madhury; Abtahi, Seyed M; Muhammad, Nur; Raychowdhury, Atri; Teshager, Abeba; Mohamed, Mussa M Rafat; Moss, Angela K; Ahmed, Rizwan; Hakimian, Shahrad; Narisawa, Sonoko; Millán, José Luis; Hohmann, Elizabeth; Warren, H Shaw; Bhan, Atul K; Malo, Madhu S; Hodin, Richard A

2013-04-23

82

Intestinal alkaline phosphatase prevents metabolic syndrome in mice  

Science.gov (United States)

Metabolic syndrome comprises a cluster of related disorders that includes obesity, glucose intolerance, insulin resistance, dyslipidemia, and fatty liver. Recently, gut-derived chronic endotoxemia has been identified as a primary mediator for triggering the low-grade inflammation responsible for the development of metabolic syndrome. In the present study we examined the role of the small intestinal brush-border enzyme, intestinal alkaline phosphatase (IAP), in preventing a high-fat-diet–induced metabolic syndrome in mice. We found that both endogenous and orally supplemented IAP inhibits absorption of endotoxin (lipopolysaccharides) that occurs with dietary fat, and oral IAP supplementation prevents as well as reverses metabolic syndrome. Furthermore, IAP supplementation improves the lipid profile in mice fed a standard, low-fat chow diet. These results point to a potentially unique therapy against metabolic syndrome in at-risk humans. PMID:23569246

Kaliannan, Kanakaraju; Hamarneh, Sulaiman R.; Economopoulos, Konstantinos P.; Nasrin Alam, Sayeda; Moaven, Omeed; Patel, Palak; Malo, Nondita S.; Ray, Madhury; Abtahi, Seyed M.; Muhammad, Nur; Raychowdhury, Atri; Teshager, Abeba; Mohamed, Mussa M. Rafat; Moss, Angela K.; Ahmed, Rizwan; Hakimian, Shahrad; Narisawa, Sonoko; Millán, José Luis; Hohmann, Elizabeth; Warren, H. Shaw; Bhan, Atul K.; Malo, Madhu S.; Hodin, Richard A.

2013-01-01

83

Purification and characterization of Ulva pertusa Kjellm alkaline phosphatase.  

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The activity of alkaline phosphatase (ALP, EC 3.1.3.1.) was found in seaweeds, including five kinds of green alga, eighteen kinds of red alga, and six kinds of brown alga, collected from the seaside of Dalian in China. The enzyme was purified 1230-fold from Ulva pertusa Kjellm. It had a specific activity of 48.6 U/mg protein and was proven to be homogeneous by SDS-PAGE with a subunit molecular mass of 19.5 kDa. The activity of ALP peaked at pH9.8, and was completely inhibited by DTT and partly by NBS. The Michaelis-Menten constant Km and the maximum reaction velocity Vmax, at pH 9.8 and 37 degrees C were 0.950 mM and 5.00 microM/min, respectively. PMID:12784882

Yang, Dong; Wang, Jingyun; Bao, Yongming; An, Lijia

2003-05-01

84

The influence of complexing pharmaceutical compositions on alkaline phosphatase  

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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.

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

2011-06-01

85

Inhibition of protein tyrosine phosphatase 1B and alkaline phosphatase by bis(maltolato)oxovanadium (IV).  

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Vanadate has been recognized as a specific and potent phosphatase inhibitor since its structure is similar to that of phosphate. In this study, we measured the inhibition of glutathione S-transferase-tagged protein tyrosine phosphatase 1B (GST-PTP1B) and alkaline phosphatase (ALP) by the insulin enhancing compounds, bis(maltolato)oxovanadium(IV) (BMOV). The results showed that the activity of GST-PTP1B was reversibly inhibited by solutions of BMOV with an IC(50) value of 0.86+/-0.02 microM. Steady state kinetic studies showed that inhibition of GST-PTP1B by BMOV was of a mixed competitive and noncompetitive type. In addition, incubation of GST-PTP1B with BMOV showed a time-dependent biphasic inactivation of the protein. On the other hand, the inhibitory behavior of BMOV on ALP activity was reversible and competitive with an IC(50) value of 32.1+/-0.6 microM. Incubation with BMOV did not show biphasic inactivation of ALP. The reversible inhibition of GST-PTP1B by BMOV is more potent than that of ALP, but solutions of BMOV inhibited both enzymes. This data support the suggestion that mechanisms for the inhibitory effects of BMOV on GST-PTP1B and ALP are very different. PMID:18728000

Li, Ming; Ding, Wenjun; Baruah, Bharat; Crans, Debbie C; Wang, Ruilin

2008-10-01

86

Effect of spironolactone on acid and alkaline phosphatase in the testes of albino rat  

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The biological activity of the mineralocorticoid antagonist, spironolactone depend upon its metabolism. In this study the effect of diuretic drug compound spironolactone on the acid and alkaline phosphatase in the testes of albino rat. The drug spironolactone was administered orally daily for 7th, 14th and 21st days at the dose of 100 mg/kg body weight. Among them, the side effects of drug are that the degradation of alkaline phosphatase in the testes; while acid phosphatase increased signifi...

Singh, P. K.; Singh, D. P.

2005-01-01

87

Alkaline phosphatase activity of water column fractions and seagrass in a tropical carbonate estuary, Florida Bay  

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Few phosphorus-depleted coastal ecosystems have been examined for their ability to hydrolyze phosphomonoesters. We examined seasonal (August 2006-April 2007) alkaline phosphatase activity in Florida Bay, a phosphorus-limited shallow estuary, using fluorescent substrate at low concentrations (?2.0 ?M). In situ dissolved inorganic and organic phosphorus levels and phosphomonoester concentrations were also determined. Water column alkaline phosphatase activity was partitioned into two particulate size fractions (>1.2 and 0.2-1.2 ?m) and freely dissolved enzymes (Thalassia testudinum. Our results indicate: (1) potential alkaline phosphatase activity in Florida Bay is high compared to other marine ecosystems, resulting in rapid phosphomonoester turnover times (˜2 h). (2) Water column alkaline phosphatase activity dominates, and is split equally between particulate and dissolved fractions. (3) Alkaline phosphatase activity was highest during cyanobacterial blooms, but not when normalized to chl a. These results suggest that dissolved, heterotrophic and autotrophic alkaline phosphatase activity is stimulated by phytoplankton blooms. (4) The dissolved alkaline phosphatase activity is relatively constant, while the particulate activity is seasonally and spatially dynamic, typically associated with phytoplankton blooms. (5) Phosphomonoester concentrations throughout the bay are low, even though potential hydrolysis rates are high. We propose that bioavailable dissolved organic P is hydrolyzed by dissolved and microbial alkaline phosphatase enzymes in Florida Bay. High alkaline phosphatase activity in the bay is also promoted by long hydraulic residence times. This background activity is primarily driven by carbon and phosphorus limitation of microorganisms, and regeneration of enzymes associated with cell lysis. Pulses of inorganic phosphorus and labile organic phosphorus and nitrogen may stimulate autotrophs, particularly cyanobacteria, which in turn promote biological activity that increase alkaline phosphatase activity of both autotrophs and heterotrophs in the bay.

Koch, Marguerite S.; Kletou, Demetris C.; Tursi, Rosanna

2009-08-01

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

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To know if alkaline phosphatase (AP) from schistosomes other than Schistosoma mansoni can be used as diagnostic marker for schistosomiasis in alkaline phosphatase immunocapture assay (APIA), we comparatively tested n-butanol extracts of adult worm membranes from a Venezuelan (JL) strain of S. mansoni (Ven/AWBE/Sm); a Cameroonian (EDEN) strain of Schistosoma intercalatum (Cam/AWBE/Si) and a Yemeni strain of Schistosoma haematobium (Yem/AWBE/Sh). APIA was evaluated with sera of patients from Venezuela, Senegal, and Gabon infected with S. mansoni, from Gabon infected with S. intercalatum or S. haematobium, from Chine infected with Schistosoma japonicum and from Cambodian patients infected with Schistosoma mekongi. Results indicate that 92.5% (37/40) of Venezuela sera, 75% (15/20) of Senegal sera, 39.5% (17/43) of S. haematobium sera, and 19.2% (5/26) S. intercalatum sera were APIA-positive with the Ven/AWBE/Sm preparation. APIA with the Cam/AWBE/Si preparation showed that 53.8% of S. intercalatum-positive sera had anti-AP antibodies, and 51.2%?S. haematobium-positive sera cross-immunocapturing the S. intercalatum AP. APIA performed with Yem/AWBE/Sh showed that 55.8%?S. haematobium sera were positive. Only two out of nine S. japonicum sera were APIA-positive with the Ven/AWBE/Sm and Cam/AWBE/Si, and no reaction was observed with Cambodian S. mekongi-positive sera. AP activity was shown to be present in all the schistosome species/strains studied. The use of APIA as a tool to explore the APs antigenicity and the presence of Schistosoma sp. infections through the detection of anti-Schistosoma sp. AP antibodies in a host, allowed us to demonstrate the antigenicity of APs of S. mansoni, S. intercalatum, and S. haematobium. PMID:24452918

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

2014-04-01

89

Paget’s Disease Of The Spine With Low Bone Alkaline Phosphatase  

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Full Text Available We present a case of 35-year-old man who was referred to us with a preliminary diagnosis of multiple spinal metastasis. Laboratory studies have shown a high serum alkaline phosphatase and a low bone alkaline phosphatase levels. Spinal magnetic resonance scans demonstrated involvement of T9, T11 and L3 vertebral bodies. The involved vertebrae appeared hypointense on T1- and hyperintense on T2-weighted images and a radionuclide bone scan has shown increased uptake in involved vertebral bodies. A transpedicular open biopsy was considered necessary for accurate diagnosis. Histopathologic evaluation of the specimen revealed typical “mosaic pattern” of Paget’s disease. After surgery, urinary deoxypridinoline and pridinoline levels were tested and were higher than normal. The patient was given oral doses of alendronate, 40 mg per day and follow-up magnetic resonance scans at 6 months and 2 years demonstrated improvement in the signal intensity of the involved vertebral bodies. This case not only shows that Paget’s disease can occur in the setting of low bone AP but also shows that the clinical improvement can be monitored by improvement in magnetic resonance signal.

Ferda CAGAVI

2005-06-01

90

A new substrate for alkaline phosphatase based on quercetin pentaphosphate.  

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We describe the characterization and application of quercetin pentaphosphate (QPP), a new fluorimetric substrate for the detection of alkaline phosphatase (ALP) activity. QPP exhibits major absorbance peaks at 260/410 nm and a strong fluorescence at ?ex/?em = 425/510 nm at alkaline pH. The product of enzymatic reaction between QPP and ALP has a strong absorbance peak at 324 nm with no fluorescence at the investigated wavelengths. The product generated from the enzymatic reaction was found to be proportional to ALP activity, and the ALP activity was monitored by the absorbance difference at 310 nm and 410 nm. The change in absorbance was found to be proportional to the ALP concentration with a linear detection range and a limit of detection of 0.01-16 U L(-1) and 0.766 U L(-1), respectively. The enzyme activity was also monitored by evaluating the change in fluorescence emission at 530 nm with a linear range of 0.01-8 U L(-1) and a detection limit of 0.062 U L(-1). Further, the validity of the new substrate for ALP in conjugated form was tested using Bacillus globigii spores as the model sample. A detection limit of 5998 spores per mL was obtained using QPP as the substrate. Unlike the parent compound, QPP substrate exhibits stability in solution for over three and half months and was stable under storage for over 12 months. The results obtained demonstrate the effectiveness of QPP for ALP and compare well with other fluorescent substrates, such as Fluorescein, Alexa Fluor and Cy5. PMID:25180235

Mwilu, Samuel K; Okello, Veronica A; Osonga, Francis J; Miller, Seth; Sadik, Omowunmi A

2014-11-01

91

Stabilization of bovine intestine alkaline phosphatase by sugars.  

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Bovine intestine alkaline phosphatase (BIALP) is widely used as a signaling enzyme in sensitive assays such as enzyme immunoassay. In this study, we evaluated the effects of sugars on the kinetic stability of BIALP in the hydrolysis of p-nitrophenylphosphate (pNPP). The temperatures reducing initial activity by 50% in a 30-min incubation, T(50), of BIALP with 1.0 M disaccharide (sucrose and trehalose) or 2.0 M monosaccharide (glucose and fructose) were 55.0-55.5 °C, 4.7-5.2 °C higher than without sugar (50.3±0.1 °C). The T(50) of BIALP increased to 58.4±0.3 °C when the trehalose concentration was from 1.0 to 1.5 M, but did not change when the glucose concentration was from 2.0 to 3.0 M. Thermodynamic analysis revealed that the stabilization of BIALP by sugars was driven by the increase in the enthalpy change of activation for thermal inactivation of BIALP. No sugars affected the k(cat) of BIALP in the hydrolysis of pNPP. These results suggest that not only trehalose, which is considered the most effective stabilizer of enzymes, but also sucrose, glucose, and fructose can be used as stabilizers of BIALP. PMID:22232245

Sekiguchi, Satoshi; Hashida, Yasuhiko; Yasukawa, Kiyoshi; Inouye, Kuniyo

2012-01-01

92

Alkaline phosphatase from venom of the endoparasitoid wasp, Pteromalus puparum.  

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Using chromogenic substrates 5-bromo-4-chloro-3'-indolyl phosphate and nitro blue tetrazolium, alkaline phosphatase (ALPase) was histochemically detected in the venom apparatus of an endoparasitoid wasp, Pteromalus puparum L. (Hymenoptera: Pteromalidae). Ultrastructural observations demonstrated its presence in the secretory vesicles and nuclei of the venom gland secretory cells. Using p-nitrophenyl phosphate as substrate to measure enzyme activity, the venom ALPase was found to be temperature dependent with bivalent cation effects. The full-length cDNA sequence of ALPase was amplified from the cDNA library of the venom apparatus of P. puparum, providing the first molecular characterization of ALPase in the venom of a parasitoid wasp. The cDNA consisted of 2645 bp with a 1623 bp open reading frame coding for 541 deduced amino acids with a predicted molecular mass of 59.83 kDa and pI of 6.98. Using multiple sequence alignment, the deduced amino acid sequence shared high identity to its counterparts from other insects. A signal peptide and a long conserved ALPase gene family signature sequence were observed. The amino acid sequence of this venom protein was characterized with different potential glycosylation, myristoylation, phosphorylation sites and metal ligand sites. The transcript of the ALPase gene was detected by RT-PCR in the venom apparatus with development related expression after adult wasp emergence, suggesting a possible correlation with the oviposition process. PMID:20575745

Zhu, Jia-Ying; Yin Ye, Gong; Fang, Qi; Hu, Cui

2010-01-01

93

Alkaline phosphatases of the mosquito, Culex tarsalis Coquillett.  

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Spectrophotometric and isoelectric focusing (IEF) electrophoretic characterization of the alkaline phosphatase (ALKP) of the mosquito, Culex tarsalis, are presented. With p-nitrophenylphosphate (Pnp) as substrate, ALKP was optimally active at 37 degrees C, pH 8.0, 30 mM MgCl2, Vmax was 35.8 mumoles/10 min and the Km was 5.7 mM, with no demonstrable requirement for Zn2+. The spectrophotometric enzyme(s) was stimulated by dithiothreitol, 2-mercaptoethanol, and poly-vinylpyrollidone (PVP); inhibited by NaF, several alternative cations (Ca2+, Ba2+, Fe2+, Cu2+), and EDTA. ALKP activity was cyclic during the 15 day post-adult emergence period of the study. No significant differences were noted between the specific activities of males and females. IEF electrophoresis revealed 6 ALKP isozymes detected with alpha-naphthylphosphate within the pH range 4.0-5.5, with a second group of 3 rather indistinct species in the pH 6.0-7.0 range. IEF ALKP isozymes were stimulated by Mg2+ and PVP and inhibited by EDTA (except ALKP5.0) and cysteine; partial inhibition with phenylalanine. IEF detection of ALKP activity with Pnp indicated that the majority of the activity was localized in the pH 4.0-5.5 range, in close agreement with the alpha-naphthylphosphate results. PMID:6467896

Houk, E J; Hardy, J L

1984-01-01

94

Lead Toxicity on Kinetic Behaviors of High and Low Molecular Weight Alkaline Phosphatase Isoenzymes of Rat, in vivo and in vitro Studies  

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The relationship between lead (Pb) toxicity and changes in the kinetic characteristics of serum, liver and brain high and low molecular weight alkaline phosphatase isoenzymes has been examined in this document. Alkaline phosphatase is a family of phosphomonoesterases that was measured in serum, liver and brain using paranitrophenol phosphate (pNPP) as substrate and 2-amino-2-methyl-1-propanol as buffer. Protein concentration was determined as described by Bradford. Results obtained showed tha...

Mirhashemi, S. M.; Ali-Asghar Moshtaghie; Ani, M.; Mohammad- Hossein Aarabi

2010-01-01

95

COMPARISON OF METHODS FOR ALKALINE PHOSPHATASE AND PEROXIDASE DETECTION IN MILK  

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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.

felipe Nael Seixas

2014-02-01

96

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

International Nuclear Information System (INIS)

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

97

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

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

Chung-Chiun Liu

2009-10-01

98

Activity and isoenzyme spectrum of alkaline phosphatase in liver and blood plasma of gamma-irradiated hen and chick embryos  

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Twelve day and 20 day-old embryos and one-day old chicks were gamma-irradiated with a single dose of 100 rad. On the 1st, 2nd and 72nd hours after treatment the alkaline phosphatase (AP) activity and isozyme spectrum in liver homogenates and blood plasma were determined. AP in the liver was demonstrated histochemically as well. The results show that the initial damage of the liver parenchyma by irradiation treatment is characterized by a slight decrease of liver AP activity and a marked increase in total plasma enzyme activity. The isozyme spectrum changes (increase of AP1 in the early period and increase of AP1 and AP5 in the later period) show that the initial liver parenchymal damage is followed by bile duct damage as well. Comparison of the results of biochemical and histochemical studies indicate the presence of direct correlation between serum and liver alkaline phosphatase activities. (A.B.)

99

The influence of ionizing radiation at the alkaline phosphatase activity and calcium 45 transport under hypothyroid conditions  

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The experiments with 2-month-old hypothyroid male rats (mercasolil 10 mg/kg per os during 21 days) have shown that total gamma-irradiation ( 0,5 Gy) caused phased changes of alkaline phosphatase - its reduction in the first dates of the investigation (3, 10 and 30 days), normal activity in a 90 days and repeated drop of the activity at the end of the experiment (180 days). In a 3 and 10 days after irradiation it was revealed the diminish of the calcium-accumulated ability of the duodenum mucous membrane in the hypothyroid rats. So it was concluded that combined action of radiation and mercasolil leaded to a decrease in calcium 45 transport in the duodenum and reduction in the activity of thermo labile isoenzyme of alkaline phosphatase in blood serum

100

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

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

Ishibe, M.; Rosier, R.N.; Puzas, J.E. (Department of Orthopaedics, University of Rochester, New York (United States))

1991-10-01

 
 
 
 
101

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

International Nuclear Information System (INIS)

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

102

The tillage effect on the soil acid and alkaline phosphatase activity  

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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 Ez?reni 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.

Maria-Magdalena Zamfirache

2011-12-01

103

Intestinal alkaline phosphatase inhibits the proinflammatory nucleotide uridine diphosphate.  

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Uridine diphosphate (UDP) is a proinflammatory nucleotide implicated in inflammatory bowel disease. Intestinal alkaline phosphatase (IAP) is a gut mucosal defense factor capable of inhibiting intestinal inflammation. We used the malachite green assay to show that IAP dephosphorylates UDP. To study the anti-inflammatory effect of IAP, UDP or other proinflammatory ligands (LPS, flagellin, Pam3Cys, or TNF-?) in the presence or absence of IAP were applied to cell cultures, and IL-8 was measured. UDP caused dose-dependent increase in IL-8 release by immune cells and two gut epithelial cell lines, and IAP treatment abrogated IL-8 release. Costimulation with UDP and other inflammatory ligands resulted in a synergistic increase in IL-8 release, which was prevented by IAP treatment. In vivo, UDP in the presence or absence of IAP was instilled into a small intestinal loop model in wild-type and IAP-knockout mice. Luminal contents were applied to cell culture, and cytokine levels were measured in culture supernatant and intestinal tissue. UDP-treated luminal contents induced more inflammation on target cells, with a greater inflammatory response to contents from IAP-KO mice treated with UDP than from WT mice. Additionally, UDP treatment increased TNF-? levels in intestinal tissue of IAP-KO mice, and cotreatment with IAP reduced inflammation to control levels. Taken together, these studies show that IAP prevents inflammation caused by UDP alone and in combination with other ligands, and the anti-inflammatory effect of IAP against UDP persists in mouse small intestine. The benefits of IAP in intestinal disease may be partly due to inhibition of the proinflammatory activity of UDP. PMID:23306083

Moss, Angela K; Hamarneh, Sulaiman R; Mohamed, Mussa M Rafat; Ramasamy, Sundaram; Yammine, Halim; Patel, Palak; Kaliannan, Kanakaraju; Alam, Sayeda N; Muhammad, Nur; Moaven, Omeed; Teshager, Abeba; Malo, Nondita S; Narisawa, Sonoko; Millán, José Luis; Warren, H Shaw; Hohmann, Elizabeth; Malo, Madhu S; Hodin, Richard A

2013-03-15

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Downscaling Alkaline Phosphatase Activity in a Subtropical Reservoir  

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

Tseng, Y.

2011-12-01

105

Radioimmunoassay for prostatic acid phosphatase in human serum. Methodologic aspects  

International Nuclear Information System (INIS)

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

106

Effects of sodium butyrate on human colonic adenocarcinoma cells. Induction of placental-like alkaline phosphatase.  

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We have examined the effects of the "differentiating agent," sodium butyrate, on the induction of alkaline phosphatase in human colonic tumor cell line LS174T. Culture of these cells in the presence of 2 mM butyrate caused this activity to increase from less than 0.0001 unit/mg of protein to greater than 0.7 unit/mg of protein over an 8-day period. This induction proceeded in a nonlinear fashion with a lag time of 2-3 days occurring before enzymatic activity began to rise. These increases in activity were accompanied by elevations in the content of a placental-like isozyme of alkaline phosphatase as demonstrated by "Western" immunoblots. Dome formation, indicative of differentiation in cultured cells, also required 3 days treatment with butyrate before becoming evident. The rate of biosynthesis of the enzyme, examined using metabolic labeling with L-[35S]methionine and immunoprecipitation, was found to increase continuously between days 2 and 6 of butyrate treatment. "Northern" blot analysis indicated that treatment of these cells with butyrate caused greater than 20-fold induction of a 2700-base mRNA that hybridized to a cDNA probe for placental alkaline phosphatase. The mRNA for alkaline phosphatase produced by these cells upon butyrate treatment was approximately 300-400 bases smaller than the mRNA for alkaline phosphatase found in placenta. Human small intestine also contained two mRNAs that hybridized relatively weakly with the placental alkaline phosphatase probe. These results indicate that a placental alkaline phosphatase-like protein and mRNA are induced by butyrate in LS174T cells with a time course consistent with cellular differentiation preceding induction. PMID:3805014

Gum, J R; Kam, W K; Byrd, J C; Hicks, J W; Sleisenger, M H; Kim, Y S

1987-01-25

107

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

International Nuclear Information System (INIS)

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)

108

CAN PREOPERATIVE SERUM ALKALINE PHOPHATASE AND SERUM CALCIUM BE USED AS A PREDICTORS OF POSTOPERATIVE TETANY  

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Full Text Available A total of 100 cases hyperthyroid patients were selected for this prospective study. The hyperthyoidism was confirmed by thyroid function tests and scintigraphy. Of these100 patients 60(60% patients had elevated serum alkaline phosphatase(ALPand decreased bone mineral density (BMD and 20(20% patients had normal ALP and normal BMD. All 100 patients underwent totalthyroidectomy. 20 patients with normal ALP did not show a shift in calcium levels and 80 patientswith elevated ALP showed shift in calcium levels (1 to 1.5 gm% 40 (40% patients with their calcium levels less than 9.5gm% and elevated ALP developed carpopedal spasm in postoperativeperiod. Hence we conclude that the combination of low normal calcium (< 9.5% and elevated ALP has a high predictive value for postoperative pedagogical spasm .

Chandrasekaran Maharajan

2012-11-01

109

Phosphorylation by alkaline phosphatase: immobilization and synthetic potential  

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Phosphatases (AP, E.C. 3.1.3.1) are hydrolytic enzymes that naturally hydrolyse phosphomonoesters but in a so-called transphosphorylation reaction these enzymes are also able to transfer a phosphate group from phosphorylated compounds to alcoholic functions. This transphosphorylation catalysed by acid phosphatases using pyrophosphate as a phosphate donor has been studied in some detail. However, the acidic pH optimum of these enzymes limits some of their applications. The catalytic feature...

Lara Babich; Peralta, Joana L. V. M.; Hartog, Aloysius F.; Ron Wever

2013-01-01

110

Alkaline phosphatase: a possible treatment for sepsis-associated acute kidney injury in critically ill patients.  

Science.gov (United States)

Acute kidney injury (AKI) is a common disease in the intensive care unit and accounts for high morbidity and mortality. Sepsis, the predominant cause of AKI in this setting, involves a complex pathogenesis in which renal inflammation and hypoxia are believed to play an important role. A new therapy should be aimed at targeting both these processes, and the enzyme alkaline phosphatase, with its dual mode of action, might be a promising candidate. First, alkaline phosphatase is able to reduce inflammation through dephosphorylation and thereby detoxification of endotoxin (lipopolysaccharide), which is an important mediator of sepsis. Second, adenosine triphosphate, released during cellular stress caused by inflammation and hypoxia, has detrimental effects but can be converted by alkaline phosphatase into adenosine with anti-inflammatory and tissue-protective effects. These postulated beneficial effects of alkaline phosphatase have been confirmed in animal experiments and two phase 2a clinical trials showing that kidney function improved in critically ill patients with sepsis-associated AKI. Because renal inflammation and hypoxia also are observed commonly in AKI induced by other causes, it would be of interest to investigate the therapeutic effect of alkaline phosphatase in these nephropathies as well. PMID:24462020

Peters, Esther; Heemskerk, Suzanne; Masereeuw, Rosalinde; Pickkers, Peter

2014-06-01

111

Control of placental alkaline phosphatase gene expression in HeLa cells: induction of synthesis by prednisolone and sodium butyrate  

International Nuclear Information System (INIS)

HeLa S3 cells produce an alkaline phosphatase indistinguishable from the enzyme from human term placenta. The phosphatase activity in these cells was induced by both prednisolone and sodium butyrate. Both agents stimulated de novo synthesis of the enzyme. The increase in phosphatase activity paralleled the increase in immunoactivity and biosynthesis of placental alkaline phosphatase. The fully processed phosphatase monomer in control, prednisolone-treated or butyrate-treated cells was a 64.5 K polypeptide, measured by both incorporation of L-[35S]methionine into enzyme protein and active-site labeling. The 64.5K polypeptide was formed by the incorporation of additional N-acetylneuraminic acid moieties to a precursor polypeptide of 61.5K. However, this biosynthetic pathway was identified only in butyrate-treated cells. In prednisolone-treated cells, the processing of 61.5K to 64.5K monomer was accelerated, and the presence of the 61.5 precursor could only be detected by either neuraminidase or monensin treatment. Phosphatase mRNA which comigrated with the term placental alkaline phosphatase mRNA of 2.7 kilobases was induced in the presence of either prednisolone or butyrate. Alkaline phosphatase mRNA is untreated HeLa S3 cells migrated slightly faster than the term placental alkaline phosphatase mRNA. Butyrate also induced a second still faster migrating alkaline phosphatase mRNA. Both prednisolone and butyrate increased the steady-statone and butyrate increased the steady-state levels of placental alkaline phosphatase mRNA. The data indicate that the increase in phosphatase mRNA by prednisolone and butyrate resulted in the induction of alkaline phosphatase activity and biosynthesis in HeLa S3 cells. Furthermore, both agents induced the expression of different alkaline phosphatase gene transcripts without altering its protein product

112

Identification of novel chromone based sulfonamides as highly potent and selective inhibitors of alkaline phosphatases.  

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A new series of structurally diverse chromone containing sulfonamides has been developed. Crystal structures of three representative compounds (2a, 3a and 4a) in the series are reported. All compounds were screened for their inhibitory potential against alkaline phosphatases (ALPs). Two main classes of ALP isozymes were selected for this study, the tissue non-specific alkaline phosphatase (TNALP) from bovine and porcine source and the tissue-specific intestinal alkaline phosphatases (IALPs) from bovine source. All sulfonamide compounds had a marked preference for IALP (K(i), up to 0.01 ± 0.001 ?M) over TNALPs. Kinetics studies of the compounds showed competitive mode of inhibition. Molecular docking studies were carried out in order to characterize the selective inhibition of the compounds. An additional interesting aspect of these chromone sulfonamides is their inhibitory activity against ecto-5'-nucleotidase enzyme. PMID:23831808

al-Rashida, Mariya; Raza, Rabia; Abbas, Ghulam; Shah, Muhammad Shakil; Kostakis, George E; Lecka, Joanna; Sévigny, Jean; Muddassar, Muhammad; Papatriantafyllopoulou, Constantina; Iqbal, Jamshed

2013-08-01

113

The Effect of Tamoxifen Treatment During 24 Months On Alkaline Phosphatase Concentration In Breast Cancer Patients  

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Full Text Available Determination of alkaline phosphatase (ALP isoenzyme activity is useful for the diagnosis and clinical evaluation of cancer patients, including breast cancer. 21 women were randomized to receive tamoxifen, probably the most widely used endocrine treatment for breast cancer worldwide, for 2 years, and 21 women constituted the control group. Control group were treated without Tamoxifen considering placebo effect. Alkaline phosphatase level was significantly lower in tamoxifen treated female breast cancer patients when compared with those of the treated patients with placebo. Results indicates that the tamoxifen decrease the level of ALP in first three months and then stabilize this level in the following months.

Betul Akcesme

2013-03-01

114

AVERRHOA CARAMBOLA (STAR FRUIT INDUCES HEPATIC ALKALINE PHOSPHATASE ACTIVITY IN RATS  

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Full Text Available The objective of the study is to examine the in vivo effect of star fruit juice at different storage conditions on the activity of alkaline phosphates in female Sprague Dawley (SD rats. A total of 20 healthy female rats weighing 180-200g were used for the experiment. All animals were divided into four groups with five animals per group (n=5. First control was served as control group. Second, third and forth groups were orally treated with a single dose daily of freshly prepared star fruit juice, juice stored for 1-hour and juice stored for 3-hour, respectively for 14 days. All animals were sacrificed at day-15 and various organs such as liver, kidney and heart were removed. All organs were homogenised and centrifuged to prepare cytosolic fraction as a source of alkaline phosphatase enzyme. Protein content of each organ was determined.  p-nitrophenol phosphate was used as a substrate to determine the activity of alkaline phosphatase using spectrophotometer. All results were analysed using Dunnett’s test. Based on the results obtained, a significant increase (P<0.01 of the activity of alkaline phosphatase in rat liver was observed in all star fruit juice treatment groups when compared to control rats. In conclusion, star fruit juice at different storage times was selectively induced the activity of alkaline phosphatase in rat liver but not in the heart and kidney.

Shamala F.

2011-01-01

115

Relation of salivary calcium, phosphorus and alkaline phosphatase with the incidence of dental caries in children  

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Full Text Available Aim: The purpose of this study was to assess possible relationship of Calcium, Phosphorus and Alkaline-phophatase levels in saliva with incidence of caries in child patients. Settings and Design: Children (n=75 attending Department of Pedodontics, St. Joseph Dental college, Eluru, with and without caries were categorized in to Group I: Consisting of 25 children with non-rampant caries, Group II: Consisting of 25 children with rampant caries, Group III: Consisting of 25 children without caries. (Control group. Materials and Methods: The samples of saliva were collected one week after oral prophylaxis. Unstimulated directly expectorated whole saliva samples were collected in clean, dry, sterilized glass bottles and fitted with proper rubber stoppers immediately. The samples were subjected to biochemical assay for estimation of calcium, phosphorus and alkaline phosphatase levels. Statistical analysis used: ANOVA. Results: The alkaline Phosphatase activity for rampant caries group was 18.66 K.A, and control group was 4.68 K.A. The values of alkaline phosphatase activity for minimal caries group was 6.16 KA. Conclusion: Saliva could reflect a caries risk situation was supported by the fact that alkaline phosphatase activity was very much significantly higher in caries prone groups.

Vijayaprasad K

2010-09-01

116

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

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

Sh Byranvand

2006-10-01

117

[Correlation between alkaline phosphatase activity and the degree of villi atrophy of the intestinal mucosa in children with glutenic enteropathy].  

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Eight children with primary and secondary enteropathy were examined, in whom predominantly advanced partial atrophy and subtotal atrophy of the villous apparatus were established. Enzymic histochemical study of the activity of alkaline phosphatase showed correlation with the degree of villous atrophy. Our results confirm that the alkaline phosphatase is an enzyme, indicating regenerative activity of enterocytes. PMID:2743932

Tsenova, V; Kovacheva, Iu

1989-01-01

118

Ectopic expression of alkaline phosphatase in proximal tubular brush border membrane of human renal cell carcinoma.  

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The present study was conducted to find out any alteration in the expression and activity of alkaline phosphatase in the brush border membrane (BBM) from renal cell carcinoma (RCC) in comparison to normal renal BBM. The specific activity of alkaline phosphatase was drastically reduced in homogenate as well as BBM from RCC kidney when compared to ALP activity in BBM of normal kidney. Kinetic studies revealed that diminished activity of alkaline phosphatase in BBM isolated from RCC was fraternized with decrease in maximal velocity (V(max)) and increase in affinity constant (K(m)) of the enzyme. SDS-PAGE studies showed that the BBM proteins having molecular weights ranging from 95 to 170 kDa were poorly expressed in RCC BBM in relative to normal kidney BBM. Incubation of SDS-PAGE gel with BCIP/NBT dye clearly showed that the expression of ALP in tumor renal BBM was markedly reduced as compared to normal kidney. Further, Western blot analysis using anti-alkaline phosphatase antibody also confirmed the reduced expression of ALP in tumor renal BBM. Lipid composition in reference to phospholipids, glycolipids and cholesterol in tumor renal BBM was altered to that of normal renal BBM, indicating alteration in membrane fluidity of tumor renal BBM. PMID:16081252

Prasad, R; Lambe, S; Kaler, P; Pathania, S; Kumar, S; Attri, S; Singh, S K

2005-09-25

119

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

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Free fatty acids released during intralumenal digestion of dietary fat must pass through the enterocyte brush border membrane before triacylglycerol reassembly and subsequent chylomicron delivery to the lymph system. In the present work fluorescent BODIPY fatty acid analogs were used to study 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 the brush border, and from work by others it is known that fat absorption is accompanied by a rise in serum AP and secretion of surfactant-like particles from enterocytes. We propose that these physiological processes may be triggered by the sequestering of dietary free fatty acids in lipid raft microdomains of the brush border.

Hansen, Gert H; Rasmussen, Karina

2011-01-01

120

Cord blood calcium, phosphate, magnesium, and alkaline phosphatase gestational age-specific reference intervals for preterm infants  

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Full Text Available Abstract Background The objective was to determine the influence of gestational age, maternal, and neonatal variables on reference intervals for cord blood bone minerals (calcium, phosphate, magnesium and related laboratory tests (alkaline phosphatase, and albumin-adjusted calcium, and to develop gestational age specific reference intervals based on infants without influential pathological conditions. Methods Cross-sectional study. 702 babies were identified as candidates for this study in a regional referral neonatal unit. After exclusions (for anomalies, asphyxia, maternal magnesium sulfate administration, and death, relationships were examined between cord blood serum laboratory analytes (calcium, phosphate, magnesium, alkaline phosphatase, and albumin-adjusted calcium with gestation age and also with maternal and neonatal variables using multiple linear regression. Infants with influential pathological conditions were omitted from the development of gestational age specific reference intervals for the following categories: 23-27, 28-31, 32-34, 35-36 and > 36 weeks. Results Among the 506 preterm and 54 terms infants included in the sample. Phosphate, magnesium, and alkaline phosphatase in cord blood serum decreased with gestational age, calcium increased with gestational age. Those who were triplets, small for gestational age, and those whose mother had pregnancy-induced hypertension were influential for most of the analytes. The reference ranges for the preterm infants ? 36 weeks were: phosphate 1.5 to 2.6 mmol/L (4.5 to 8.0 mg/dL, calcium: 2.1 to 3.1 mmol/L (8.3 to 12.4 mg/dL; albumin-adjusted calcium: 2.3 to 3.2 mmol/L (9.1 to 12.9 mg/dL; magnesium 0.6 to 1.0 mmol/L (1.4 to 2.3 mg/dL, and alkaline phosphatase 60 to 301 units/L. Conclusions These data suggest that gestational age, as well as potentially pathogenic maternal and neonatal variables should be considered in the development of reference intervals for preterm infants.

Lyon Andrew W

2011-08-01

 
 
 
 
121

21 CFR 862.1050 - Alkaline phosphatase or isoenzymes test system.  

Science.gov (United States)

...CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1050 Alkaline...group of enzymes with similar biological activity) in serum or plasma....

2010-04-01

122

Zinc and alkaline phosphatase in developing rat oral mucosa  

International Nuclear Information System (INIS)

Alkaline phosphates (AlkPase) of many different tissues and species have been shown to be zinc metalloenzymes. Specific regions of rat oral mucosa have a high activity of AlkPase. Combined autoradiography and enzyme histochemistry showed that they also retained injected radioactive zinc (65Zn). The AlkPase activity was inactivated by EDTA and reactivated with zinc. However, it could not be verified by polyacrylamide gel electrophoresis, combined with radioactivity measurements and enzyme analysis, that the 65Zn uptake of oral mucosa was incorporated in the AlkPase molecule

123

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

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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.

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

1999-01-01

124

Nucleotide sequence of the gene for alkaline phosphatase of Thermus caldophilus GK24 and characteristics of the deduced primary structure of the enzyme.  

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The gene encoding Thermus caldophilus GK24 (Tca) alkaline phosphatase was cloned into Escherichia coli. The primary structure of Tca alkaline phosphatase was deduced from its nucleotide sequence. The Tca alkaline phosphatase precursor, including the signal peptide sequence, was comprised of 501 amino acid residues. Its molecular mass was determined to be 54¿ omitted¿760 Da. On the alignment of the amino acid sequence, Tca alkaline phosphatase showed sequence homology with the microbial alkaline phosphatases, 20% identity with E. coli alkaline phosphatase and 22% Bacillus subtilis (Bsu) alkaline phosphatases. High sequence identity was observed in the regions containing the Ser-102 residue of the active site, the zinc and magnesium binding sites of E. coli alkaline phosphatase. Comparison of Tca alkaline phosphatase and E. coli alkaline phosphatase structures suggests that the reduced activity of the Tca alkaline phosphatase, in the presence of zinc, is directly involved in some of the different metal binding sites. Heat-stable Tca alkaline phosphatase activity was detected in E. coli YK537, harboring pJRAP. PMID:10556703

Park, T; Lee, J H; Kim, H K; Hoe, H S; Kwon, S T

1999-11-15

125

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

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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

Berta B Socarrás Ferrer

2001-12-01

126

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

Science.gov (United States)

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

Berezhetskyy, A.

2008-09-01

127

A Disposable Alkaline Phosphatase-Based Biosensor for Vanadium Chronoamperometric Determination  

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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 r...

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

2014-01-01

128

Immobilization of Alkaline Phosphatase on Microporous Nanofibrous Fibrin Scaffolds for Bone Tissue Engineering  

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Alkaline phosphatase (ALP) promotes bone formation by degrading inorganic pyrophosphate (PPi), an inhibitor of hydroxyapatite formation, and generating inorganic phosphate (Pi), an inducer of hydroxyapatite formation. Pi is a crucial molecule in differentiation and mineralization of osteoblasts. In this study, a method to immobilize ALP on fibrin scaffolds with tightly controllable pore size and pore interconnection was developed, and the biological properties of these scaffolds were characte...

Osathanon, Thanaphum; Giachelli, Cecilia M.; Somerman, Martha J.

2009-01-01

129

Evaluation of Testosterone and Alkaline Phosphatase Activity Changes in Epidydimis of Toxoplasma gondii Infected Rats  

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Objective: Toxoplasma gondii is a widespread protozoan parasite that infects a broad range of warm blooded animals and humans. The present study was investigated to evaluate testosterone, alkaline phosphatase activity and malondialdehyde in male rats experimentally infected by Toxoplasma gondii, RH strain.Material & Methods: Male Wistar rats (n=20) were allocated into two groups, group one (n=10) that received 0.6 cc tachyzoites of T. gondii intraperitoneally (I.P.), and control group (n=10) ...

Fatemeh Afshari; Amir Mahdi Imani; Sasan Najjari Asl; Farhang, Hossein H.; Khazar Ghasempour; Ezzatzadeh; Nava Ainechi

2013-01-01

130

The effect of acute inflammation on total alkaline phosphatase activity in dogs  

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The main purpose of this study was to investigate the effect of acute inflammation on total alkaline phosphatase (ALP) activity in dogs. In this study total ALP activity was determined in dogs with experimentally induced acute inflammation in order to characterize their potential value in this condition. For that, ALP concentrations were defined in plasmas from 9 mongrel male dogs (in an experimental group) and 6 mongrel male dogs (in a control group) at the age of 2 years and body weight 12-...

Zapryanova Dimitrinka

2013-01-01

131

Effect of MPG on the radiation induced changes in the intestinal activity of alkaline phosphatase in mice  

International Nuclear Information System (INIS)

Adult male Swiss mice were exposed to 250, 500 and 1000 R of gamma rays with or without a prior intraperitoneal injection of 20 mg/kg MPG. Alkaline phosphatase activity in the ileum was estimated at different post irradiation intervals. The changes in the enzyme activity was dose dependent. MPG reduced the radiation induced increase in the alkaline phosphatase activity of the intestine. (author)

132

Differential alkaline phosphatase responses of rat and human bone marrow derived mesenchymal stem cells to 45S5 bioactive glass  

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Bioactive glass is used as both a bone filler and as a coating on implants, and has been advocated as a potential osteogenic scaffold for tissue engineering. Rat derived mesenchymal stem cells (MSCs) show elevated levels of levels of alkaline phosphatase activity when grown on 45S5 bioactive glass as compared to standard tissue culture plastic. Similarly, exposure to the dissolution products of 45S5 elevates alkaline phosphatase activity and other osteogenic markers in these cells. We investi...

Reilly, Gwendolen C.; Radin, Shula; Chen, Andrew T.; Ducheyne, Paul

2007-01-01

133

The Study Of Serum Prostate Specific Antigen And Phosphatase Isoenzymes Activity As Diagnostic Parameters In Patients With Prostate Cancer In Nigeria  

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Full Text Available Serum activities of Acid Phosphatase (ACP and Prostatic Acid Phosphatase (PAP are still employed in most hospitals in Nigeria for the diagnosis of prostate cancer, because of lack of resources for prostate specific antigen (PSA assay. Serum PSA and activities of phosphatase isoenzymes ACP and PAP, Alkaline Phosphatase (ALP and Heat stable Alkaline Phosphatase (HSAP were studied in 71 apparently healthy male controls and 47 proven prostate cancer patients. There were statistically significant increases in the mean serum levels of PSA, PAP, ACP, ALP and HSAP in the prostate cancer patients compared to the controls (P<0.001. PSA level was increased above the cut-off level in 85.1% of patients, PAP in 66.0%, ACP in 57.5%, ALP in 34.0% and HSAP in 21.3% of cases. Serum levels of PSA, ACP and PAP were lower and of ALP and HSAP higher in patients with longer duration of the disease (P<0.05. The study confirms the relevance of PSA assay over ACP, PAP, ALP and HSAP in the diagnosis of prostate cancer patients. It highlights the need for the inclusion of PSA assay in hospitals for accurate diagnosis of prostatic carcinoma.

Igwe CU

2004-10-01

134

Lead Toxicity on Kinetic Behaviors of High and Low Molecular Weight Alkaline Phosphatase Isoenzymes of Rat, in vivo and in vitro Studies  

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Full Text Available The relationship between lead (Pb toxicity and changes in the kinetic characteristics of serum, liver and brain high and low molecular weight alkaline phosphatase isoenzymes has been examined in this document. Alkaline phosphatase is a family of phosphomonoesterases that was measured in serum, liver and brain using paranitrophenol phosphate (pNPP as substrate and 2-amino-2-methyl-1-propanol as buffer. Protein concentration was determined as described by Bradford. Results obtained showed that every other day intrapritoneally injection of 39.5 ?g kg-1 of lead as (Pb (CH3COO 2.3H2O, in male rats for 2 consecutive weeks resulted in decreasing level of liver and brain alkaline phosphatase by 16.7 and 10.9%, respectively, whereas an elevation of serum enzyme activity by 28.4% was seen in comparison to untreated controls (p<0.05. Long-term exposure to 13.2 ?g kg-1 of this salt, showed a statistically significant reduction in liver and brain levels of alkaline phosphatase by 18.7 and 13.2% respectively and an increment in serum activity of the enzyme by 37.6% in compared to control group (p<0.05. Using gel filtration chromatography technique with sephacryl S300 showed that, in comparison to control groups, serum and liver homogenate from lead treated groups had a significant level of high molecular weight alkaline phosphatase, which might be considered as a potential biomarker for lead toxicity. In vitro experiments showed that lead inhibited all the isoenzymes.

S.M. Mirhashemi

2010-01-01

135

An Alkaline Phosphatase Reporter Gene Assay for Induction of CYP3A4 In Vitro  

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Full Text Available CYP3A4 probably has the broadest catalytic activity of any cytochrome P450. It is a crucial task to test new drug candidates in a reliable system for their ability to induce expression of this enzyme. Firstly, a total of 300 bp core distal enhancer of CYP3A4 XREM region (-7972/-7673 were amplified from human genomic DNA. The PCR product was then ligated into a human secretory alkaline phosphatase cDNA-containing reporter vector (pSEAP2-1creating pX-SEAP2 plasmid. Secondly, 1143 bp of the CYP3A4 proximal promoter region (-1203/-61 was amplified from the genomic DNA and then ligated into pX-SEAP2 plasmid DNA (between XREM and alkaline phosphatase gene, creating pXP-SEAP2 plasmid. Reporter constructs were then co-transfected with an hPXR expression vector into human liver and intestinal cells in culture. Xenobiotic modulation of CYP3A4 promoter activity was determined by chemiluminescent secretory alkaline phosphatase assay. Significant CYP3A4 induction atthe transcriptional level using three different cell lines and four classical CYP3A4 inducers was observed. Transfection of reporter constructs in HepG2, HuH7 and Caco-2 cells, in general, produced similar pattern of induction by the same drugs with the exception ofphenobarbital. The results suggest that, carefully designed reporter gene systems can provide a useful in vitro approach for characterization of possible CYP3A4 inducers.

Hossein Hamzeiy

2006-01-01

136

Radiation-induced changes in liver and kidney alkaline phosphatase and esterase of mice  

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Whole-body exposure of mice to 2 Gy gamma radiation results in an increase in the specific and total activities of esterase and alkaline phosphatase found in the supernatant of liver and kidney. This increase developed at 1 h, reached a maximum at 24 h, and then declined at 48 h after irradiation. The increase in the activity of these enzymes was not accompanied by an increase in protein content. Electrophoretic analysis on acrylamide gel confirmed the results obtained by the quantitative biochemical analysis. An increase in the level and/or number of bands was observed postirradiation.

Auda, H.; Rashid, A.M.; Khaleel, A.H.; Nasser, M.J.

1987-09-01

137

Selective determination of cholesterol based on cholesterol oxidase-alkaline phosphatase bienzyme electrode.  

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A bienzyme electrode for the determination of cholesterol was prepared by the co-immobilization of cholesterol oxidase and alkaline phosphatase (ALP) on the carbon nanotubes modified electrode surface. The parameters influenced the performance of the bienzyme electrode such as the pH of the base solution; the concentration of DPP and the incubation time, were optimized. A linear calibration graph in the 0.05-2.0 mM concentration range with the sensitivity of 4.65 ?A mM(-1) was obtained. In addition, this work provides a new interferent-depleted method and universal protocol for the design of multi-enzyme biosensors. PMID:23019566

Zhang, Hongfang; Liu, Ruixiao; Zheng, Jianbin

2012-11-21

138

[Effect of insulin on the activity of bone alkaline phosphatase in culture].  

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Diabetes and osteoporosis are linked. The question remains, however, as to whether insulin has any direct effect on bone formation. To test this hypothesis we have measured, as a marker of osteoblast activity, alkaline phosphatase (ALP) released by rat limb intact bones incubated in the presence and in the absence of physiological concentration of insulin. The results indicate that insulin significantly (p less than 0.012) increases ALP by a mean value of 48% (from 5.4% to 215%) over matched controls. We conclude that insulin has a direct stimulatory effect on osteoblast activity, and that in the absence of this effect, as in diabetes, bone loss might occur. PMID:1815119

Rubinacci, A; Boniforti, F; Tessari, L

1991-01-01

139

The dermatophyte Trichophyton rubrum secretes an EDTA-sensitive alkaline phosphatase on high-phosphate medium  

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In this communication, we show that the growth of isolate H6 of the dermatophyte Trichophyton rubrum on non-buffered medium and under saturating phosphate conditions is dependent on the initial growth pH, with an apparent optimum at pH 4.0. In addition, irrespective of the initial growth pH, the pH of the medium alteredduring cultivation reaching values that ranged from 8.3 to 8.9. Furthermore, this isolate synthesized and secreted almost the same levels of an alkaline phosphatase with an app...

Ferreira-Nozawa Monica S.; Nozawa Sérgio R.; Martinez-Rossi Nilce M; Rossi Antonio

2003-01-01

140

Comparative study of alkaline phosphatase activity in lymphocytes, mitogen-induced blasts, lymphoblastoid cell lines, acute myeloid leukemia, and chronic lymphatic leukemia cells (N-alkaline phosphatase/thiophosphoric acid thioesters)  

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Alkaline phosphatase (orthophosphoricmonoester phosphohydrolase (alkaline pH optimum), EC 3.1.3.1) purified from a Burkitt lymphoma cell line (Daudi) and Moloney-virus-induced murine leukemia (YAC) showed unique catalytic properties in substrate specificity and inhibition by cysteamine-S-phosphate. It migrated on polyacrylamide gel electrophoresis in a single activity band. Alkaline phosphatase with similar properties was found in several human lymphoblastoid cell lines, in chronic lymphatic leukemic cells, in organs of leukemic mice, and in sera of patients with certain lymphoproliferative disorders. The unique kinetic properties of this enzyme were established using two kinds of substrate, namely, the monoesters of orthophosphoric acid (Type I) and the S-substituted monoesters of thiophosphoric acid (Type II). The enzyme catalyzed the hydrolysis of Type I, but failed to hydrolyze Type II. The enzyme probably interacts directly either in catalysis or in binding with the --P--O-- or --P--S-- bonds. Thus, in these respects it differs from the established enzymatic mechanism of other known alkaline phosphatases, and therefore can be considered a new enzyme. This enzyme was designated N-alkaline phosphatase. N-alkaline phosphatase was absent in untreated and mitogen-stimulated human blood lymphocytes, in murine lymph nodes, and pig thymus (even after mitogen stimulation), and in blast cells of acute myeloid leukemic patients. The appearance of N-alkaline phosphatase only in certain types of proliferating lymphocytes and in the sera of patients with lymphoproliferative disorders may be exploited as a cell marker and as a diagnostic tool.

Neumann, H. (Weizmann Inst. of Science, Rehovoth, Israel); Klein, E.; Hauck-Granoth, R.; Yachnin, S.; Ben-Bassat, H.

1976-05-01

 
 
 
 
141

Implication of BBM lipid composition and fluidity in mitigated alkaline phosphatase activity in renal cell carcinoma.  

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Previous study has documented reduced alkaline phosphatase (ALP) activity in brush border membrane (BBM) isolated from renal cell carcinoma (RCC). Diminished activity of ALP is associated with alteration in both increased K(m) as well as decreased V(max) of enzyme suggests that there may be a change in the conformation of enzyme as well as decreased number of ALP active molecules. The present study was conducted to find out any role of BBM lipid composition and its fluidity in diminished activity of alkaline phosphatase in renal cell carcinoma. Total phospholipids and glycolipids were significantly augmented in BBM from RCC as compared to control. Fractional analysis of total phospholipids revealed significantly increased phosphatidylethanolamine. Decreased fractions of sphingomyelin and phosphatidylinositol were observed. Cholesterol-to-total phospholipid molar ratios in tumor BBM was a significantly lower in tumor BBM. A significant reduction in polarization and microviscosity was found in BBM from RCC. Therefore, we conclude that alteration in membrane lipid composition and fluidity may play a substantial role in reduced activity of ALP in RCC. PMID:22810501

Sharma, Ujjawal; Singh, Shrawan Kumar; Pal, Deeksha; Khajuria, Ragini; Mandal, Arup Kumar; Prasad, Rajendra

2012-10-01

142

Inhibitors of tissue-nonspecific alkaline phosphatase: design, synthesis, kinetics, biomineralization and cellular tests.  

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Chronic kidney disease (CKD) is associated with numerous metabolic and endocrine disturbances, including abnormalities of calcium and phosphate metabolism and an inflammatory syndrome. The latter occurs early in the course of CKD and contributes to the development and progression of vascular calcification. A few therapeutic strategies are today contemplated to target vascular calcification in patients with CKD: vitamin K2, calcimimetics and phosphate binders. However, none has provided complete prevention of vascular calcification and there is an urgent need for alternate efficient treatments. Recent findings indicate that tissue-nonspecific alkaline phosphatase (TNAP) may represent a very promising drug target due to its participation in mineralization by vascular smooth muscle cells. We report the synthesis of four levamisole derivatives having better inhibition property on TNAP than levamisole. Their IC50, Ki and water solubility have been determined. We found that the four inhibitors bind to TNAP in an uncompetitive manner and are selective to TNAP. Indeed, they do not inhibit intestinal and placental alkaline phosphatases. Survival MTT tests on human MG-63 and Saos-2 osteoblast-like cells have been performed in the presence of inhibitors. All the inhibitors are not toxic at concentrations that block TNAP activity. Moreover, they are able to significantly reduce mineralization in MG63 and Saos-2 osteoblast-like cells, indicating that they are promising molecules to prevent vascular calcification. PMID:24183741

Debray, Julien; Chang, Lei; Marquès, Stéphanie; Pellet-Rostaing, Stéphane; Le Duy, Do; Mebarek, Saida; Buchet, René; Magne, David; Popowycz, Florence; Lemaire, Marc

2013-12-15

143

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

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

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

1997-01-01

144

Biocompatibility and Alkaline Phosphatase Activity of Phosphorylated Chitooligosaccharides on the Osteosarcoma MG63 Cell Line  

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Full Text Available Phosphorylated chitooligosaccharides (P-COS were prepared using a H3PO4, P2O5, Et3PO4 and hexanol solvent system. The P-COS were characterized by Fourier Transform Infrared Spectroscopy (FT-IR, Thermo gravimetric-Differential Thermal Analyzer (TG-DTA, 13C NMR, 31P NMR, X-ray diffraction analysis, solubility studies, biocompatibility and Alkaline Phosphatase Activity (ALP. The results reveal that phosphorylation occurred at the C3 and C6 position of OH groups and the C2 position of NH2 group. FT-IR confirmed no decomposition in pyranose ring in P-COS even with heating and treatment in acidic conditions. The amorphous nature of P-COS was confirmed by X-ray diffraction analysis. Further, the biocompatibility and alkaline phosphatase activity of P-COS were checked against the osteosarcoma MG63 cell line at different concentrations and no cytotoxicity was observed. After 12 h and 24 h of incubation, the ALP activity of P-COS was higher compared with the control group. These results suggest that P-COS is a biocompatible material and in future P-COS could open up a number of promising pharmaceutical and clinical applications to mankind.

Jayachandran Venkatesan

2010-10-01

145

High alkaline phosphatase activity of telangiectatic osteosarcoma (TOS) and its diagnostic significance.  

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Telangiectatic osteosarcoma (TOS) which occurred in the metaphysis of the right femoral bone in a 13-year-old female was reported. It showed osteolytic and cystic lesion without sclerotic change on roentgenogram and consisted histologically of various sized blood-filled spaces lined by layers of round to oval tumor cells in the thin fibrous septa. In some solid areas, a proliferation of atypical tumor cells with large prominent nucleoli was evident, embedded in the lace-like osteoid tissue. Mitotic cells were easily encountered. A large population of tumor cells revealed high alkaline phosphatase activity as well as 5'-nucleotidase activity, indicative of osteogenic cell origin. Ultrastructurally, they showed osteogenic characteristics of well-developed rough endoplasmic reticula, cytoplasmic microfibrils, and dense bodies, but not for those of endothelial cells. In this report, we suggest that alkaline phosphatase activity in biopsy and surgical specimens is useful for distinguishing TOS from other osteolytic bone tumors, with regard to its ontogenic discussion. PMID:3474863

Yoshida, H; Adachi, H; Naniwa, S; Yumoto, T; Morimoto, K; Furuse, K

1987-02-01

146

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

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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

147

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

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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 icesses in the chickens hatched from eggs irradiated before incubation. (Author)

148

Human alkaline phosphatase expression and secretion into chicken eggs after in vivo gene electroporation in the oviduct of laying hens.  

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In vivo gene electroporation was used to examine whether or not a recombinant protein is synthesized in the chicken oviduct and subsequently secreted into eggs. A plasmid DNA containing a secretion form of the human alkaline phosphatase gene was injected into mucosa of the chicken magnum. Immediately, in vivo gene electroporation was conducted. The human alkaline phosphatase activity in the oviduct mucosa increased and reached its peak at 2 days posttransfection, followed by a sharp decrease to a negligible level at 4 days posttransfection. In the egg white, the alkaline phosphatase activity showed a similar change to that in the magnum mucosa except for a delay of 4 days. The present results imply that in vivo gene electroporation method in the oviduct may serve as a rapid production system of recombinant proteins into chicken eggs. PMID:11890676

Takami, Hiroshi; Watanabe, Hisako; Ohmori, Yasushige; Park, Hyi-Man; Muramatsu, Tatsuo

2002-03-22

149

Intestinal alkaline phosphatase regulates protective surface microclimate pH in rat duodenum.  

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Regulation of localized extracellular pH (pH(o)) maintains normal organ function. An alkaline microclimate overlying the duodenal enterocyte brush border protects the mucosa from luminal acid. We hypothesized that intestinal alkaline phosphatase (IAP) regulates pH(o) due to pH-sensitive ATP hydrolysis as part of an ecto-purinergic pH regulatory system, comprised of cell-surface P2Y receptors and ATP-stimulated duodenal bicarbonate secretion (DBS). To test this hypothesis, we measured DBS in a perfused rat duodenal loop, examining the effect of the competitive alkaline phosphatase inhibitor glycerol phosphate (GP), the ecto-nucleoside triphosphate diphosphohydrolase inhibitor ARL67156, and exogenous nucleotides or P2 receptor agonists on DBS. Furthermore, we measured perfusate ATP concentration with a luciferin-luciferase bioassay. IAP inhibition increased DBS and luminal ATP output. Increased luminal ATP output was partially CFTR dependent, but was not due to cellular injury. Immunofluorescence localized the P2Y(1) receptor to the brush border membrane of duodenal villi. The P2Y(1) agonist 2-methylthio-ADP increased DBS, whereas the P2Y(1) antagonist MRS2179 reduced ATP- or GP-induced DBS. Acid perfusion augmented DBS and ATP release, further enhanced by the IAP inhibitor l-cysteine, and reduced by the exogenous ATPase apyrase. Furthermore, MRS2179 or the highly selective P2Y(1) antagonist MRS2500 co-perfused with acid induced epithelial injury, suggesting that IAP/ATP/P2Y signalling protects the mucosa from acid injury. Increased DBS augments IAP activity presumably by raising pH(o), increasing the rate of ATP degradation, decreasing ATP-mediated DBS, forming a negative feedback loop. The duodenal epithelial brush border IAP-P2Y-HCO(3-) surface microclimate pH regulatory system effectively protects the mucosa from acid injury. PMID:19451200

Mizumori, Misa; Ham, Maggie; Guth, Paul H; Engel, Eli; Kaunitz, Jonathan D; Akiba, Yasutada

2009-07-15

150

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

International Nuclear Information System (INIS)

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

151

Cloning and Expression of the Alkaline Phosphatase Gene from the Persian Type Culture Collection Escherichia coli K-12  

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Full Text Available The structural gene for alkaline phosphatase (phoA of E. coli K-12 strain obtained from the Persian type culture collection (PTCC 1268 was cloned into pTZ57R plasmid as cloning vector and pGEM-3Z plasmid as expression vector, respectively. The recombinant plasmids were confirmed by different restriction enzymes and determination of the nucleotide sequence. Protein expression was induced by isopropyl -D thiogalactopyranoside (IPTG and was analyzed using polyacrylamide gel electrophoresis (PAGE. The obtained results demonstrate a complete homology of the DNA sequence between the cloned alkaline phosphatase gene with the sequence present in the gene banks.

Hamid Mir Mohammad Sadeghi

2005-01-01

152

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

International Nuclear Information System (INIS)

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 Mg2+.

153

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

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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+}.

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

154

Expression, secretion and surface display of a human alkaline phosphatase by the ciliate Tetrahymena thermophila  

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Full Text Available Abstract Background Tetrahymena thermophila possesses many attributes that render it an attractive host for the expression of recombinant proteins. Surface proteins from the parasites Ichthyophthirius multifiliis and Plasmodium falciparum and avian influenza virus antigen H5N1 were displayed on the cell membrane of this ciliate. Furthermore, it has been demonstrated that T. thermophila is also able to produce a functional human DNase I. The present study investigates the heterologous expression of the functional human intestinal alkaline phosphatase (hiAP using T. thermophila and thereby presents a powerful tool for the optimization of the ciliate-based expression system. Results Functional and full length human intestinal alkaline phosphatase was expressed by T. thermophila using a codon-adapted gene containing the native signal-peptide and GPI (Glycosylphosphatidylinositol anchor attachment signal. HiAP activity in the cell extract of transformants suggested that the hiAP gene was successfully expressed. Furthermore, it was demonstrated that the enzyme was modified with N-glycosylation and localized on the surface membrane by the C-terminal GPI anchor. A C-terminally truncated version of hiAP lacking the GPI anchor signal peptide was secreted into the medium as an active enzyme. In a first approach to establish a high level expression system up to 14,000 U/liter were produced in a time frame of two days, which exceeds the production rate of other published expression systems for this enzyme. Conclusions With the expression of hiAP, not only a protein of commercial interest could be produced, but also a reporter enzyme that offers the possibility to analyze T. thermophila genes that play a role in the regulation of protein secretion. Additionally, the fact that ciliates do not secrete an endogenous alkaline phosphatase provides the possibility to use the truncated hiAP as a reporter enzyme, allowing the quantification of measures that will be necessary for further optimization of the host strains and the fermentation processes.

Herrmann Lutz

2011-01-01

155

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

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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…

Grunwald, Sandra K.; Krueger, Katherine J.

2008-01-01

156

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

International Nuclear Information System (INIS)

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

157

A disposable alkaline phosphatase-based biosensor for vanadium chronoamperometric determination.  

Science.gov (United States)

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

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

2014-01-01

158

Phosphate monoester hydrolysis by trinuclear alkaline phosphatase; DFT study of transition States and reaction mechanism.  

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Alkaline phosphatase (AP) is a trinuclear metalloenzyme that catalyzes the hydrolysis of a broad range of phosphate monoesters to form inorganic phosphate and alcohol (or phenol). In this paper, by using density functional theory with a model based on a crystal structure, the AP-catalyzed hydrolysis of phosphate monoesters is investigated by calculating two substrates, that is, methyl and p-nitrophenyl phosphates, which represent alkyl and aryl phosphates, respectively. The calculations confirm that the AP reaction employs a "ping-pong" mechanism involving two chemical displacement steps, that is, the displacement of the substrate leaving group by a Ser102 alkoxide and the hydrolysis of the phosphoseryl intermediate by a Zn2-bound hydroxide. Both displacement steps proceed via a concerted associative pathway no matter which substrate is used. Other mechanistic aspects are also studied. Comparison of our calculations with linear free energy relationships experiments shows good agreement. PMID:24683174

Chen, Shi-Lu; Liao, Rong-Zhen

2014-08-01

159

Alkaline Phosphatase and CD34 Reaction of Deciduous Teeth Pulp Stem Cells  

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Full Text Available Endothelial progenitor cells from the pulp of milk teeth were isolated for use in clinical applications and tissue engineering. Normal deciduous teeth from children of 7 to 8 years of age, which more than half the tooth root was extracted, were selected from the dental centre. Cells from enzyme treated pulps were cultured and cells resulting from the fifth and eight subculture were combined for cell surface marker determination experiments. Cells were positive for CD34 marker with a total of 99/45%, determined by flowcytometry. Cells also demonstrated alkaline phosphatase (ALP activity. From the developmental point of view, stem cells from the dental pulp seem to have derived from the neural crest, which our findings technically support this theory. In essence mobile progenitor cells from bone marrow of endothelial origin could also play a significant role in the derivation of dental pulp stem cells.

Fatemeh Abedini

2007-01-01

160

Kinetics of inactivation of Ulva pertusa Kjellm alkaline phosphatase by ethylenediaminetetraacetic acid disodium.  

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Ulva pertusa Kjellm alkaline phosphatase (EC 3.3.3.1) is a metalloenzyme, the active site of which contains a tight cluster of two zinc ions and one magnesium ion. The kinetic theory described by Tsou of the substrate reaction during irreversible inhibition of enzyme activity has been employed to study the kinetics of the course of inactivation of the enzyme by EDTA. The kinetics of the substrate reaction at different concentrations of the substrate p-nitrophenyl phosphate (PNPP) and inactivator EDTA indicated a complexing mechanism for inactivation by, and substrate competition with, EDTA at the active site. The inactivation kinetics are single phasic, showing that the initial formation of an enzyme-EDTA complex is a relative rapid reaction, following by a slow inactivation step that probably involves a conformational change of the enzyme. The presence of Zn2+ apparently stabilizes an active-site conformation required for enzyme activity. PMID:11916136

Yang, D; Wang, J; Peng, X; An, L

2001-10-01

 
 
 
 
161

A human monoclonal antibody specific to placental alkaline phosphatase, a marker of ovarian cancer.  

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Placental alkaline phosphatase (PLAP) is a promising ovarian cancer biomarker. Here, we describe the isolation, affinity-maturation and characterization of two fully human monoclonal antibodies (termed B10 and D9) able to bind to human PLAP with a dissociation constant (Kd) of 10 and 30 nM, respectively. The ability of B10 and D9 antibodies to recognize the native antigen was confirmed by Biacore analysis, FACS and immunofluorescence studies using ovarian cancer cell lines and freshly-frozen human tissues. A quantitative biodistribution study in nude mice revealed that the B10 antibody preferentially localizes to A431 tumors, following intravenous administration. Anti-PLAP antibodies may serve as a modular building blocks for the development of targeted therapeutic products, armed with cytotoxic drugs, radionuclides or cytokines as payloads. PMID:24247025

Ravenni, Niccolò; Weber, Marcel; Neri, Dario

2014-01-01

162

A Disposable Alkaline Phosphatase-Based Biosensor for Vanadium Chronoamperometric Determination  

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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.

Ana Lorena Alvarado-Gámez

2014-02-01

163

Reduced activity of alkaline phosphatase due to host-guest interactions with humic superstructures.  

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Nuclear Magnetic Resonance (NMR) spectroscopy was applied to directly study the interactions between the alkaline phosphatase enzyme (AP) and two different humic acids from a volcanic soil (HA-V) and a Lignite deposit (HA-L). Addition of humic matter to enzyme solutions caused signals broadening in (1)H-NMR spectra, and progressive decrease and increase of enzyme relaxation (T1 and T2) and correlation (?C) times, respectively. Spectroscopic changes were explained with formation of ever larger weakly-bound humic-enzyme complexes, whose translational and rotational motion was increasingly restricted. NMR diffusion experiments also showed that the AP diffusive properties were progressively reduced with formation of large humic-enzyme complexes. The more hydrophobic HA-L affected spectral changes more than the more hydrophilic HA-V. (1)H-NMR spectra also showed the effect of progressively greater humic-enzyme complexes on the hydrolysis of an enzyme substrate, the 4-nitrophenyl phosphate disodium salt hexahydrate (p-NPP). While AP catalysis concomitantly decreased NMR signals of p-NPP and increased those of nitrophenol, addition of humic matter progressively and significantly slowed down the rate of change for these signals. In agreement with the observed spectral changes, the AP catalytic activity was more largely inhibited by HA-L than by HA-V. Contrary to previous studies, in which humic-enzyme interactions were only indirectly assumed from changes in spectrophotometric behavior of enzyme substrates, the direct measurements of AP behavior by NMR spectroscopy indicated that humic materials formed weakly-bound host-guest complexes with alkaline phosphatase, and the enzyme catalytic activity was thereby significantly inhibited. These results suggest that the role of extracellular enzymes in soils may be considerably reduced when they come in contact with organic matter dissolved in the soil solution. PMID:23953249

Mazzei, Pierluigi; Oschkinat, Hartmut; Piccolo, Alessandro

2013-11-01

164

Effect of Zinc from Zinc Sulfate on Ewes` Weight, Milk Yield, Zn Concentrations in Serum and Serum Alkaline Phosphates Activity of Varamini Ewes  

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Full Text Available This experiment was conducted to investigate the effect of feeding supplemental zinc (zinc sulfate in different levels (0, 15 and 30 mg/kg on ewes weight, milk production, Zn concentrations in serum and serum alkaline phosphates activity. Thirty lactating Varaminni ewes were assigned to three experimental groups according to their live body weights, milk production and lambs sex in a completely randomized design. Ewes were fed a basal diet containing alfalfa, wheat straw, cottonseed meal, barley grain, wheat bran, cracked corn and vitamin-mineral supplements at 3.2% of BW to meet NRC requirements for protein, energy, macro minerals and micro minerals. The basal diet contained 15 mg/kg Zn and Zinc sulfate was added to the basal diet to supply 30 or 45 mg/kg of dietary zinc. Milk yielded, milk composition and ewes` weight was recorded at 7 and 21 days intervals respectively. Samples of the blood were taken three times (0, 35 and 64 for determination of Concentration of Zn, Cu and Fe, Na, K, Ca in serum. Also serum alkaline phosphates concentration of ewes was measured. Milk yield, milk composition and ewes` weight of ewes were not affected by supplemental zinc (p>0.05. Alkaline phosphatase concentration was increased with supplemental zinc linearly and this increase was significant (p<0.05. Blood mineral concentration was not affected by treatment (p>0.05.

A. Zali

2008-01-01

165

Immobilization of alkaline phosphatase on magnetic particles by site-specific and covalent cross-linking catalyzed by microbial transglutaminase.  

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Bacterial alkaline phosphatase (BAP) was site-specifically and covalently immobilized on magnetic particles (MPs) using the enzymatic reaction of microbial transglutaminase (MTG). Immobilization efficiency was affected by the chemical surface treatment of MPs and immobilized BAP exhibited more than 90% of the initial activity after 10 rounds of recycling. PMID:21398176

Moriyama, Kousuke; Sung, Kyunga; Goto, Masahiro; Kamiya, Noriho

2011-06-01

166

The effect of 50 kV X-ray irradiation on the alkaline phosphatase activity of growing rat bone  

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Alkaline phosphatase activity was decreased in tibial metaphysis of growing rats on the first day after 50 kV x-irradiation with 0.5-8.0 Gy. There were no differences in enzyme activity between the control and the irradiated metaphysis 30 days after irradiation. (author)

167

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

International Nuclear Information System (INIS)

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

168

Ratiometric Fluorescent Probe for Alkaline Phosphatase Based on Betaine-Modified Polyethylenimine via Excimer/Monomer Conversion.  

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Alkaline phosphatase (ALP) is an important diagnostic indicator for a number of human diseases since abnormal level of ALP is closely related to a variety of pathological processes; hence, the development of convenient and reliable assay methods for monitoring ALP is of great significance for medical sciences as well as biological diagnostics. Herein, we report the first ratiometric fluorescent sensing system for ALP. This sensing system consists of two components: the betaine-modified and positively charged polyethylenimine (PEI) and the negatively charged pyrene derivative containing one ALP-responsive phosphate group (Py-P, an aliphatic phosphate ester). In the absence of ALP, the two-component sensing system shows the excimer's emission of Py-P, since Py-P molecules complex with the positively charged polyelectrolyte via electrostatic interactions, leading to the formation of pyrene excimers. While in the presence of ALP, the phosphate moieties are cleaved from Py-P molecules due to the enzymatic reaction, thereby destroying the electrostatic interactions; as a result, the system displays the monomer emission of Py-P. This assay system is operable in aqueous media with a very low detection limit of 0.1 U/mL. The system is capable of detecting ALP in such biological fluid as serum, and this strategy may provide a new and effective approach for designing ratiometric sensing systems for detecting other biomolecules. PMID:25211600

Zheng, Fangyuan; Guo, Sihua; Zeng, Fang; Li, Jun; Wu, Shuizhu

2014-10-01

169

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

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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 polyphosphate species were detected by Raman and IR spectroscopy. The oxygen isotope data of the reactants and products will also be presented. The possibility that carbonate acts as an intermediate reagent, transferring the oxygen from water to phosphate in biological apatite mineral formation may explain why biological apatite exhibits a significant carbonate content, and how this mineral is formed with an insignificant hydroxyl content. 1 Kohn, M.J., and Cerling, T.E. Rev Mineral Geochem 2002 (48) 455 2 Kolodny, Y., Luz, B., Navon, O. Earth Planet Sci Lett 1983 (64) 398 3 Blake, R.E., O'Neil, J.R., Garcia, G.A. Geochim et Cosmochim Acta 1997 (61) 4411 4 Blake, R.E., Alt, J.C., and Martini, A.M. PNAS 2001 (98) 2148-2153 5 Liang, Y., and Blake, R.E. Geochim Cosmochim Acta 2009 (73) 3782) 6 Pasteris, J.D. et al. Biomaterials 2004 (35) 229 7 Omelon et al., PLoS ONE 2009 4(5), e5634

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

2011-12-01

170

The alkaline phosphatase PhoX is more widely distributed in marine bacteria than the classical PhoA.  

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Phosphorus (P) is a vital nutrient for all living organisms and may control the growth of bacteria in the ocean. Bacteria induce alkaline phosphatases when inorganic phosphate (P(i)) is insufficient to meet their P-requirements, and therefore bulk alkaline phosphatase activity measurements have been used to assess the P-status of microbial assemblages. In this study, the molecular basis of marine bacterial phosphatases and their potential role in the environment were investigated. We found that only a limited number of homologs to the classical Escherichia coli alkaline phosphatase (PhoA) were present in marine isolates in the Bacteroidetes and gamma-proteobacteria lineages. In contrast, PhoX, a recently described phosphatase, was widely distributed among diverse bacterial taxa, including Cyanobacteria, and frequently found in the marine metagenomic Global Ocean Survey database. These taxa included ecologically important groups such as Roseobacter and Trichodesmium. PhoX was induced solely upon P-starvation and accounted for approximately 90% of the phosphatase activity in the model marine bacterium Silicibacter pomeroyi. Analysis of the available transcriptomic datasets and their corresponding metagenomes indicated that PhoX is more abundant than PhoA in oligotrophic marine environments such as the North Pacific Subtropical Gyre. Those analyses also revealed that PhoA may be important when Bacteroidetes are abundant, such as in algal bloom episodes. However, PhoX appears to be much more widespread. Its identification as a gene that mediates organic P acquisition in ecologically important groups, and as a marker of P(i)-stress, constitutes an important step toward a better understanding of the marine P cycle. PMID:19212430

Sebastian, Marta; Ammerman, James W

2009-05-01

171

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

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

Wyszkowska, Jadwiga; Wyszkowski, Miros?aw

2010-01-01

172

Cytochemical localization of alkaline phosphatase and ouabain-sensitive K+-dependent p-nitrophenylphosphatase activities in brain capillaries of the newt.  

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The ultrastructural localization of alkaline phosphatase and K+-NPPase was investigated in brain capillaries of newt by a cytochemical study using whole brain perfusion. The alkaline phosphatase activity was present in both luminal and antiluminal membranes of the endothelial cells. By contrast, the K+-NPPase was located only in antiluminal membranes of the brain capillaries. This distinct enzymatic distribution suggested that the luminal and antiluminal membranes are functionally different. The role of alkaline phosphatase and K+-NPPase in the blood brain barrier is discussed. PMID:6097213

Franceschini, V; Del Grande, P; Ciani, F; Caniato, G; Minelli, G

1984-01-01

173

Intestinal alkaline phosphatase promotes gut bacterial growth by reducing the concentration of luminal nucleotide triphosphates.  

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The intestinal microbiota plays a pivotal role in maintaining human health and well-being. Previously, we have shown that mice deficient in the brush-border enzyme intestinal alkaline phosphatase (IAP) suffer from dysbiosis and that oral IAP supplementation normalizes the gut flora. Here we aimed to decipher the molecular mechanism by which IAP promotes bacterial growth. We used an isolated mouse intestinal loop model to directly examine the effect of exogenous IAP on the growth of specific intestinal bacterial species. We studied the effects of various IAP targets on the growth of stool aerobic and anaerobic bacteria as well as on a few specific gut organisms. We determined the effects of ATP and other nucleotides on bacterial growth. Furthermore, we examined the effects of IAP on reversing the inhibitory effects of nucleotides on bacterial growth. We have confirmed that local IAP bioactivity creates a luminal environment that promotes the growth of a wide range of commensal organisms. IAP promotes the growth of stool aerobic and anaerobic bacteria and appears to exert its growth promoting effects by inactivating (dephosphorylating) luminal ATP and other luminal nucleotide triphosphates. We observed that compared with wild-type mice, IAP-knockout mice have more ATP in their luminal contents, and exogenous IAP can reverse the ATP-mediated inhibition of bacterial growth in the isolated intestinal loop. In conclusion, IAP appears to promote the growth of intestinal commensal bacteria by inhibiting the concentration of luminal nucleotide triphosphates. PMID:24722905

Malo, Madhu S; Moaven, Omeed; Muhammad, Nur; Biswas, Brishti; Alam, Sayeda N; Economopoulos, Konstantinos P; Gul, Sarah Shireen; Hamarneh, Sulaiman R; Malo, Nondita S; Teshager, Abeba; Mohamed, Mussa M Rafat; Tao, Qingsong; Narisawa, Sonoko; Millán, José Luis; Hohmann, Elizabeth L; Warren, H Shaw; Robson, Simon C; Hodin, Richard A

2014-05-15

174

A folding study of Antarctic krill (Euphausia superba) alkaline phosphatase using denaturants.  

Science.gov (United States)

To gain insight into the structural and folding mechanisms of Antarctic krill alkaline phosphatase (ALP), the enzyme was properly purified by (NH4)2SO4 fractionation and by both Sephadex G-75 and DEAE anion exchange chromatography. The purified enzyme (62.6kDa; 2.62unit/mg) was unstable at temperatures exceeding 30°C. Denaturants, such as sodium dodecyl sulfate (SDS), guanidine HCl, and urea, were applied to evaluate the folding mechanism, including kinetics and thermodynamics, of krill ALP. Sodium dodecyl sulfate elicited no significant effect on ALP activity even at excessively high concentrations (300mM), whereas guanidine HCl and urea effectively inactivated the enzyme at concentrations of 2 and 3.5M, respectively. Kinetic studies showed that the enzymatic inhibition by guanidine HCl and urea represented a first-order reaction that was a monophasic unfolding process. This process was found to be associated with conformational changes without significant transient free-energy changes. Additionally, the overall structural changes occurred proximally to the active site pocket. Our study provides new insight into ALP of the Antarctic krill, which lives in extreme environmental conditions. PMID:25016161

Wang, Zhi-Jiang; Lee, Jinhyuk; Si, Yue-Xiu; Wang, Wei; Yang, Jun-Mo; Yin, Shang-Jun; Qian, Guo-Ying; Park, Yong-Doo

2014-09-01

175

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

Science.gov (United States)

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

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

2011-05-15

176

Effects of polyethylene glycol on bovine intestine alkaline phosphatase activity and stability.  

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In this study, we evaluated the effects of polyethylene glycol (PEG) on bovine intestine alkaline phosphatase (BIALP) activity and stability. In the hydrolysis of p-nitrophenylphosphate (pNPP) at pH 9.8 at 20 °C, the k(cat)/K(m) values of BIALP plus 5-15% w/v free PEG with molecular masses of 1, 2, 6, and 20 kDa (PEG1000, PEG2000, PEG6000, and PEG20000 respectively) were 120-140%, 180-300%, 130-170%, and 110-140% respectively of that of BIALP without free PEG (1.8 µM(-1) s(-1)), indicating that activation by PEG2000 was the highest. Unmodified BIALP plus 5% PEG2000 and BIALP pegylated with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine exhibited 1.3-fold higher activity on average than that of BIALP without free PEG under various conditions, including pH 7.0-10.0 and 20-65 °C. The temperatures reducing initial activity by 50% in 30-min incubation of unmodified BIALP plus 5% PEG2000 and pegylated BIALP were 51 and 47 °C respectively, similar to that of BIALP without free PEG (49 °C). These results indicate that the addition of PEG2000 and pegylation increase BIALP activity without affecting its stability, suggesting that they can be used in enzyme immunoassay with BIALP to increase sensitivity and rapidity. PMID:22056430

Sekiguchi, Satoshi; Yasukawa, Kiyoshi; Inouye, Kuniyo

2011-01-01

177

Enzymatic kinetic parameters for polyfluorinated alkyl phosphate hydrolysis by alkaline phosphatase.  

Science.gov (United States)

The hydrolysis kinetics of three polyfluorinated alkyl phosphate monoesters (monoPAPs), differing in fluorinated chain length, were measured using bovine intestinal alkaline phosphatase to catalyze the reaction. Kinetic values were also measured for analogous hydrogenated phosphate monoesters to elucidate the effects of the fluorinated chain on the rate of enzymatic hydrolysis. Michaelis constants (K(m)) were obtained by a competition kinetics technique in the presence of p-nitrophenyl phosphate (PNPP) using UV-vis spectroscopy. Compared with K(m) (PNPP), Michaelis constants for monoPAPs ranged from 0.9 to 2.1 compared with hydrogenated phosphates, which ranged from 4.0 to 13.0. Apparent bimolecular rate constants (k(cat)/K(m)) were determined by monitoring rates of product alcohol formation at low substrate concentrations using gas chromatography-mass spectrometry. The experimental values for k(cat)/K(m) averaged as 1.1 × 10(7) M(-1) s(-1) for monoPAPs compared with 3.8 × 10(5) M(-1) s(-1) for hexyl phosphate. This suggests that the electron-withdrawing nature of the fluorinated chain enhanced the alcohol leaving group ability. The results were used in a simple model to suggest that monoPAPs in a typical mammalian digestive tract would hydrolyze in approximately 100 s, supporting a previous study that showed its absence after a dosing study in rats. PMID:22714665

Jackson, Derek A; Mabury, Scott A

2012-09-01

178

Refined structures of placental alkaline phosphatase show a consistent pattern of interactions at the peripheral site.  

Science.gov (United States)

In order to gain deeper insights into the functional sites of human placental alkaline phosphatase, the structures of the enzyme with the putative regulators L-Phe, pNPP and 5'-AMP [Llinas et al. (2005), J. Mol. Biol. 350, 441-451] were re-refined. Significant variations in ligand positioning and identity were found compared with the previous report. The multiple corrections to the model improved the phases and the electron-density maps, allowing the modeling of omitted side chains and multiple disordered residues. These improvements led to a change in the position of L-Phe at the peripheral binding site, which appeared to be reversed. The structure with pNPP contained only p-nitrophenol in three distinct sites, while the structure with 5'-AMP contained the p-nitrophenyl group in two of the sites instead of 5'-AMP. Comparison of the re-refined models shows a consistent pattern of interactions at the peripheral site. PMID:20693656

Stec, Boguslaw; Cheltsov, Anton; Millán, José Luis

2010-08-01

179

Unfolding and inactivation of abalone (Haliotis diversicolor) alkaline phosphatase during denaturation by guanidine hydrochloride.  

Science.gov (United States)

Abalone, a kind of low poikilothermic invertebrate, is easily exposed to ocean environment stress. Since it is one of the important mariculture animals, the attention paid to the abalone study becomes increasing. Alkaline phosphatase (ALPase, EC 3.1.3.1) is a kind of zinc-contained metalloenzyme, which catalyzes the nonspecific hydrolysis of phosphate monoesters. Unfolding and inactivation of ALPase from abalone (Haliotis diversicolor) during denaturation by guanidine hydrochloride (GuHCl) of different concentrations has first been studied. The kinetic theory of the substrate reaction by enzyme was described by Tsou, which was applied to the study on ALPase's kinetic course of inactivation by GuHCl. The result showed that the inactivation of the enzyme by GuHCl was a slow, reversible reaction with fractional remaining activity. The microscopic rate constants were determined. The result, k(+0) > k'(+0), showed that the enzyme was protected by the substrate to a certain extent during guanidine denaturation. The changes of conformation of the enzyme in different concentrations of GuHCl have been studied by means of measuring the fluorescence spectra. The results showed that the inactivation occurred before the noticeable conformational changes of the enzyme molecule as a whole can be detected, which suggests that the active site of the enzyme has more flexibility than the whole enzyme molecule. These studies will facilitate the understanding of physiological and biochemical features of the H. diversicolor and will also help in the understanding of the abalone immune system. PMID:18931946

Liao, Jin Hua; Chen, Qing Xi; Zhang, Qian; Yang, Yi; Shi, Yan

2009-08-01

180

An enzyme immobilized microassay in capillary electrophoresis for characterization and inhibition studies of alkaline phosphatases.  

Science.gov (United States)

A simple and fast dynamically coated capillary electrophoretic method was developed for the characterization and inhibition studies of alkaline phosphatases(EC 3.1.3.1). An inside capillary enzymatic reaction was performed, and hydrolysis of the substrate 4-nitrophenylphosphate to 4-nitrophenol was measured. Fused-silica capillary surface was dynamically modified with polycationic polybrene coating. By reversal of the electroosmotic flow (EOF), analysis time was reduced up to 3 min as the anionic analytes were migrated in the same direction as the EOF. Furthermore, the sensitivity of the method was increased using electroinjection through high-field amplified injection. The baseline separation of 4-nitrophenylphosphate and 4-nitrophenol was achieved by employing 50 mM sodium phosphate as the running buffer (pH 8.5), 0.0025% polybrene, and a constant voltage of -15 kV, and the products were detected at 322 nm. Under the optimized conditions, a good separation with high efficiency was achieved. The new method was applied to study enzyme kinetics and inhibitor screening. K(m) and K(i) values obtained with the new CE method were compared well with the standard spectrophotometric method. Dynamic coating of fused-silica capillary gave fast and reproducible separation of substrate and product. The method can be easily optimized for inhibition studies of other isozymes. PMID:21439261

Iqbal, Jamshed

2011-07-15

 
 
 
 
181

Raft partitioning and dynamic behavior of human placental alkaline phosphatase in giant unilamellar vesicles.  

Science.gov (United States)

Much attention has recently been drawn to the hypothesis that cellular membranes organize in functionalized platforms called rafts, enriched in sphingolipids and cholesterol. The notion that glycosylphosphatidylinositol (GPI)-anchored proteins are strongly associated with rafts is based on their insolubility in nonionic detergents. However, detergent-based methodologies for identifying raft association are indirect and potentially prone to artifacts. On the other hand, rafts have proven to be difficult to visualize and investigate in living cells. A number of studies have demonstrated that model membranes provide a valuable tool for elucidating some of the raft properties. Here, we present a model membrane system based on domain-forming giant unilamellar vesicles (GUVs), in which the GPI-anchored protein, human placental alkaline phosphatase (PLAP), has been functionally reconstituted. Raft morphology, protein raft partitioning, and dynamic behavior have been characterized by fluorescence confocal microscopy and fluorescence correlation spectroscopy (FCS). Approximately 20-30% of PLAP associate with sphingomyelin-enriched domains. The affinity of PLAP for the liquid-ordered (l(o)) phase is compared to that of a nonraft protein, bacteriorhodopsin. Next, detergent extraction was carried out on PLAP-containing GUVs as a function of temperature, to relate the lipid and protein organization in distinct phases of the GUVs to the composition of detergent resistant membranes (DRMs). Finally, antibody-mediated cross-linking of PLAP induces a shift of its partition coefficient in favor of the l(o) phase. PMID:15895991

Kahya, Nicoletta; Brown, Deborah A; Schwille, Petra

2005-05-24

182

Alkaline phosphatase-positive cells isolated from human hearts have mesenchymal stem cell characteristics  

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Full Text Available Tissue-specific resident cells have been identified as a promising population of progenitor cells for cell-based therapies. We describe here the isolation from adult human hearts of tissue nonspecific alkaline phosphatase-positive cells (ALPL+ cells with mesenchymal stem cell (MSC characteristics. Samples from 24 adult cadaveric donors were obtained from a valve bank. Mean total ischemia time was 21.5 ± 9.1 hours. The success rate for the isolation of human heart-derived cells by the explant culture technique was 70% for the right auricle (14 of 20 trials and 33% for the right ventricle (7 of 21 trials. The total auricle-derived cell population (TAD was used for the purification of ALPL+ cells. TAD and ALPL+ cells expressed markers for MSC and pericytes. TAD cells and ALPL+ cells differentiated into adipocytes, osteoblasts and chondroblasts, and ALPL+ cells expressed markers of these three lineages more strongly than TAD cells, as shown by RT-PCR. This population therefore has a greater potential for differentiation into mesechymal lineages than TAD cells. Both cell populations express some markers of cardiac progenitors, such as GATA4, CD117 and VEGF. ALPL+ cells expressed troponin T and ABCG2, which are also markers of the cardiac lineage. Heart samples from tissue banks could be considered as sources of MSC with putative commitment towards cardiac lineages, even after prolonged ischemia times.

Alessandra Melo de Aguiar

2011-10-01

183

Electrochemical immunosensor of tumor necrosis factor ? based on alkaline phosphatase functionalized nanospheres.  

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A novel immunosensor for sensitive detection of tumor necrosis factor ? was reported. First of all, gold nanoparticles were uniformly assembled on the surface of poly (styrene-acrylic acid) nanospheres, which was used as the matrix to conjugate alkaline phosphatase (ALP). And then, the obtained composite was used as multi-enzyme functionalized label for immunoassay. Biocompatible polyaniline doped with poly (acrylic acid) was electro-polymerized at the glass carbon electrode to construct the matrix for the immobilization of antibody TNF-?. After the sandwich immunoreaction, the labeled ALP was used to hydrolyze ?-naphthyl phosphate to produce the electroactive ?-naphthol, which could be amperometrically detected. The results showed that the electrochemical signals were proportional to the logarithm of the antigen concentration in the range of 0.02-200.00 ng/mL with the detection limit of 0.01 ng/mL. The developed immunoassay showed high sensitivity, acceptable stability and reproducibility, which might have potentially broad applications in protein diagnostics and bioassay. PMID:20378330

Yin, Zhengzhi; Liu, Yan; Jiang, Li-Ping; Zhu, Jun-Jie

2011-01-15

184

Cloning and characterization of a cDNA coding for mouse placental alkaline phosphatase  

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Mouse alkaline phosphatase was partially purified from placenta. Data obtained by immunoblotting analysis suggested that the primary structure of this enzyme has a much greater homology to that of human and bovine liver ALPs than to the human placental isozyme. Therefore, a full-length cDNA encoding human liver-type ALP was used as a probe to isolate the mouse placental ALP cDNA. The cloned mouse cDNA is 2459 base pairs long and is composed of an open reading frame encoding a 524-amino acid polypeptide that contains a putative signal peptide of 17 amino acids. Homology at the amino acid level of the mouse placental ALP is 90% to the human liver isozyme but only 55% to the human placental counterpart. RNA blot hybridization results indicate that the mouse placental ALP is encoded by a gene identical to the gene expressed in mouse liver, kidney, and teratocarcinoma stem cells. This gene is therefore evolutionarily highly conserved in mouse and human

185

Purification and characterization of bone-specific alkaline phosphatase from a human osteosarcoma cell line.  

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Bone-specific alkaline phosphatase (bone ALP) levels are considered to reflect osteoblastic activity and can therefore be used as a marker of bone formation. However, bone ALP is difficult to distinguish from other ALP isoforms since the kidney, liver, and bone isoenzymes are encoded by the same gene and only differ because of post-translational modification of their carbohydrate side chains. The aim of this study was to purify and separate bone ALP which could be used to raise specific antisera against human bone ALP, from Saos-2, a human osteogenic sarcoma cell line. The procedure involved two steps. The first step, cultivation of 10(5) Saos-2 cells, yielded approximately 1 U ALP. Subsequent butanol extraction achieved 1.82-fold purification. For the second step, separating bone ALP, we used serial lectin affinity chromatography to distinguish between the carbohydrate side chains of the various ALP isoforms. A sample of the butanol extract was fractionated into three peaks (I, II, and III) by concanavalin A. Peaks II and III were subsequently identified as types IIa and IIIb bone ALP using pea lectin and wheat germ agglutinin columns, respectively. The specific activity of bone ALP was measured using commercial kits. Since bone ALP accounted for at least 84% of the total ALP activity after the final separation, this method appears more convenient and reproducible than others using bone or Pagetic sera. The bone ALP purified in this study could be used to raise monoclonal antibodies against bone-specific ALP. PMID:9405736

Nakayama, M; Gorai, I; Minaguchi, H; Rosenquist, C; Qvist, P

1998-01-01

186

Fluorimetric determination of alkaline phosphatase in solid and fluid dairy products.  

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A new assay has been developed for measuring residual alkaline phosphatase (ALP) activity in a wide variety of dairy products. The method proposed is simple, rapid and directly applicable to solid and liquid dairy samples. ALP in the test sample hydrolyzes a non fluorescent substrate, trifluoromethyl-beta-umbelliferone phosphate, to its highly fluorescent phenolate product. The assay is performed in a reverse micellar medium composed of mixed buffer (2-amino-2-methyl-1-propanol buffer pH 9.0 and borate buffer pH 9.0) in AOT/isooctane, at a temperature of 38 degrees C. Total test time is 450 s. Reaction rates are linear (except for butter) up to 8.5 and 11% (v/v) raw milk, for whole milk and chocolate milk, respectively. The detection limits are 0.04, 0.4 and 0.22% (v/v) raw milk, for whole milk, chocolate milk and butter, respectively. The precision of the fluorimetric method was assessed by repeated analysis of a pasteurized milk sample spiked with mixed herd raw milk. The accuracy of the method was evaluated by comparison with an official colorimetric assay using p-nitrophenylphosphate as ALP substrate. PMID:18968582

Fenoll, Jose; Jourquin, Gilles; Kauffmann, Jean-Michel

2002-04-01

187

Processing and maturation of carboxypeptidase Y and alkaline phosphatase in Schizosaccharomyces pombe.  

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Schizosaccharomyces pombe carboxypeptidase Y (CPY) is synthesized as a zymogen and transported into the vacuole where maturation and activation occurs. The 110-kDa S. pombe CPY precursor is processed twice and finally converted to a mature form consisting of polypeptides of approximately 19 and 32 kDa linked by a single disulfide bond. In Saccharomyces cerevisiae, maturation of CPY occurs mostly through the activity of vacuolar aspartyl protease Pep4p, whereas a Pep4p homolog has not been found in the S. pombe genome database. Based on analysis of protease-deficient mutants, we found that S. pombe CPY was not able to be processed or activated in isp6?psp3? double disruptants. Both Isp6p and Psp3p are subtilase-type serine proteases with related sequences. Moreover, alkaline phosphatase of S. pombe was found to be localized at the vacuolar membrane and was also unprocessed in isp6?psp3? double disruptants. Vacuolar localization of GFP-fused Isp6p and Psp3p was determined by fluorescence microscopy. These results suggest that the two serine proteases Isp6p and Psp3p are functional in the vacuole and are involved in proteolytic processing of vacuolar proteins. PMID:21153812

Mukaiyama, Hiroyuki; Iwaki, Tomoko; Idiris, Alimjan; Takegawa, Kaoru

2011-04-01

188

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

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Titania nanotubes (TNTs) were prepared by anodization on different substrates (titanium, Ti6Al4V and Ti6Al7Nb alloys) in ethylene glycol and glycerol. The influence of the applied potential and processing time on the nanotube diameter and length is analyzed. The as-formed nanotube layers are amorphous but they become crystalline when subjected to subsequent thermal treatment in air at 550°C; TNT layers grown on titanium and Ti6Al4V alloy substrates consist of anatase and rutile, while those grown on Ti6Al7Nb alloy consist only of anatase. The nanotube layers grown on Ti6Al7Nb alloy are less homogeneous, with supplementary islands of smaller diameter nanotubes, spread across the surface. Better adhesion and proliferation of osteoblasts was found for the nanotubes grown on all three substrates by comparison to an unprocessed titanium plate. The sensitivity towards bovine alkaline phosphatase was investigated mainly by electrochemical impedance spectroscopy in relation to the crystallinity, the diameter and the nature of the anodization electrolyte of the TNT/Ti samples. The measuring capacity of the annealed nanotubes of 50nm diameter grown in glycerol was demonstrated and the corresponding calibration curve was built for the concentration range of 0.005-0.1mg/mL. PMID:24582263

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

2014-04-01

189

In vitro investigation of the interaction between pentachlorophenol and alkaline phosphatase by spectroscopic methods  

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The interaction characteristics of alkaline phosphatase (ALP) with pentachlorophenol (PCP) were investigated using fluorescence, UV-vis absorption, and circular dichroism (CD) techniques. Results obtained from analysis of fluorescence intensity indicated that PCP has a strong ability to quench the intrinsic fluorescence of ALP through a static quenching procedure. The thermodynamic parameters ? H and ? S were observed to be -4.60 kJ mol -1 and 54.59 J mol -1 K -1, respectively, and the value of ? G was negative. These results indicate that the binding reactions were spontaneous, and both hydrophobic and electrostatic forces were involved in the interaction of PCP and ALP. Based on Forster's theory of non-radiation energy transfer, the binding distance, r, between the ALP and PCP was evaluated to be 2.50 nm and the critical distance R0 was 2.26 nm. The CD spectra results showed that the ?-helicity was decreased from 49.68% in native ALP to 47.28% in PCP-ALP systems, which indicate the secondary structure of ALP was changed slightly in the presence of PCP.

Liu, Qiongyu; Zhou, Peijiang; Chen, Yan

2012-02-01

190

Layer-specific activity of tissue non-specific alkaline phosphatase in the human neocortex.  

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The ectoenzyme tissue non-specific alkaline phosphatase (TNAP) is mostly known for its role in bone mineralization. However, in the severe form of hypophosphatasia, TNAP deficiency also results in epileptic seizures, suggesting a role of this enzyme in brain functions. Accordingly, TNAP activity was shown in the neuropil of the cerebral cortex in diverse mammalian species. However in spite of its clinical significance, the neuronal localization of TNAP has not been investigated in the human brain. By using enzyme histochemistry, we found an unprecedented pattern of TNAP activity appearing as an uninterrupted layer across diverse occipital-, frontal- and temporal lobe areas of the human cerebral cortex. This marked TNAP-active band was localized infragranulary in layer 5 as defined by quantitative comparisons on parallel sections stained by various techniques to reveal the laminar pattern. On the contrary, TNAP activity was localized in layer 4 of the primary visual and somatosensory cortices, which is consistent with earlier observations on other species. This result suggests that the expression of TNAP in the thalamo-recipient granular layer is an evolutionary conserved feature of the sensory cortex. The observations of the present study also suggest that diverse neurocognitive functions share a common cerebral cortical mechanism depending on TNAP activity in layer 5. In summary, the present data point on the distinctive role of layer 5 in cortical computation and neurological disorders caused by TNAP dysfunctions in the human brain. PMID:20977932

Négyessy, L; Xiao, J; Kántor, O; Kovács, G G; Palkovits, M; Dóczi, T P; Renaud, L; Baksa, G; Glasz, T; Ashaber, M; Barone, P; Fonta, C

2011-01-13

191

Gel chromatographic characterization of the hydrophobic interaction of glycosylphosphatidylinositol-alkaline phosphatase with detergents.  

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The interaction of a glycosylphosphatidylinositol (GPI) protein with different detergents was studied for the first time with a purified protein. Four differently hydrophobic fractions of GPI-alkaline phosphatase (GPI-AP) from calf intestine were used as model proteins. The mode of interaction was determined by investigating (i) the self-aggregation behaviour of the GPI-AP fractions, (ii) the interference of detergents with GPI-AP binding to octyl-Sepharose, and (iii) the elution of GPI-AP bound to octyl-Sepharose. It was shown that polyoxyethylene-type detergents surprisingly interact much stronger than n-octylglucoside with GPI-AP, which is in contrast to the known behaviour of GPI-proteins in natural membranes. Gel filtration chromatography of Triton X-100 at concentrations above the critical micellar concentration yields three different micelle species with apparent molecular weights of about 166, 54, and 16 kDa. GPI-AP fraction II, which is shown to bear only one anchor per dimer, does not bind to any of these micelles. We demonstrate that a complex is formed containing about 150 Triton X-100 molecules and about 4700 molecules of water per molecule of GPI-AP dimer. The experimental findings are in accordance with a simple geometrical model based on the physical data of fatty acids and the arrangement, mean size, and shape of Triton X-100 molecules. PMID:10746748

Gehrhardt, S; Blume, E; Cumme, G A; Bublitz, R; Rhode, H; Horn, A

2000-02-01

192

Identification and characterization of novel tissue-nonspecific alkaline phosphatase inhibitors with diverse modes of action.  

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Tissue-nonspecific alkaline phosphatase (TNAP) is a ubiquitous enzyme expressed at high levels in bone, liver, and kidney. It appears involved in dephosphorylation of numerous phosphate monoesters, but only 2 of them, pyrophosphate and pyridoxal phosphate, have yet been unequivocally documented. Discovery and characterization of other substrates could be considerably facilitated if specific and potent modulators of TNAP activity with various modes of action were available. Here, the authors describe in detail a high-throughput screening campaign to identify inhibitors of TNAP, performed within the Molecular Library Screening Center Network (MLSCN). A novel homogeneous luminescent TNAP assay was developed and optimized with respect to the enzyme and substrate concentrations, enabling identification of a large number of compounds overlooked by a conventional colorimetric assay. Several new chemical series were identified from screening the Molecular Libraries Small Molecule Repository (MLSMR) collection and demonstrated to have diverse selectivity and mode of inhibition profiles. The nanomolar potency of some of these scaffolds surpasses currently known inhibitors. This article provides an example of a success where the Roadmap Initiative collaborative model, sponsored by the National Institutes of Health, brought together a deep knowledge of target biology from a principal investigator's laboratory, a well-designed compound collection from the MLSMR, and an industrial-level screening facility and staff at the MLSCN center to identify pharmacologically active compounds, with outstanding selectivity data from a panel of more than 200 publicly accessible assays, through a high-throughput screen. PMID:19556612

Sergienko, Eduard; Su, Ying; Chan, Xochella; Brown, Brock; Hurder, Andrew; Narisawa, Sonoko; Millán, José Luis

2009-08-01

193

Probing the Origin of the Compromised Catalysis of E. coli Alkaline Phosphatase in its Promiscuous Sulfatase Reaction  

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The catalytic promiscuity of E. coli alkaline phosphatase (AP) and many other enzymes provides a unique opportunity to dissect the origin of enzymatic rate enhancements via a comparative approach. Here we use kinetic isotope effects (KIEs) to explore the origin of the 109-fold greater catalytic proficiency by AP for phosphate monoester hydrolysis relative to sulfate monoester hydrolysis. The primary 18O KIEs for the leaving group oxygen atoms in the AP-catalyzed hydrolysis of p-nitrophenyl ph...

Catrina, Irina; O Brien, Patrick J.; Purcell, Jamie; Nikolic-hughes, Ivana; Zalatan, Jesse G.; Hengge, Alvan C.; Herschlag, Daniel

2007-01-01

194

Kinetic Isotope Effects for Alkaline Phosphatase Reactions: Implications for the Role of Active Site Metal Ions in Catalysis  

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Enzyme-catalyzed phosphoryl transfer reactions have frequently been suggested to proceed through transition states that are altered from their solution counterparts, with the alterations presumably arising from interactions with active site functional groups. In particular, the phosphate monoester hydrolysis reaction catalyzed by Escherichia coli alkaline phosphatase (AP) has been the subject of intensive scrutiny. Recent linear free energy relationship (LFER) studies suggest that AP catalyze...

Zalatan, Jesse G.; Catrina, Irina; Mitchell, Rebecca; Grzyska, Piotr K.; O’brien, Patrick J.; Herschlag, Daniel; Hengge, Alvan C.

2007-01-01

195

Discovery and Validation of a Series of Aryl Sulfonamides as Selective Inhibitors of Tissue-Nonspecific Alkaline Phosphatase (TNAP)  

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We report the characterization and optimization of drug-like small molecule inhibitors of tissue-nonspecific alkaline phosphatase (TNAP), an enzyme critical for the regulation of extracellular matrix calcification during bone formation and growth. High-throughput screening (HTS) of a small molecule library led to the identification of arylsulfonamides as potent and selective inhibitors of TNAP. Critical structural requirements for activity were determined, and the compounds were subsequently ...

Dahl, Russell; Sergienko, Eduard A.; Mostofi, Yalda S.; Yang, Li; Su, Ying; Simao, Ana Maria; Narisawa, Sonoko; Brown, Brock; Mangravita-novo, Arianna; Vicchiarelli, Michael; Smith, Layton H.; O’neill, W. Charles; Milla?n, Jose? Luis; Cosford, Nicholas D. P.

2009-01-01

196

Investigations on vinylene carbonate. VI. Immobilization of alkaline phosphatase onto poly(vinylene carbonate)-jeffamine® hydrogel beads  

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Hydrogel-like polymers with reactive cyclic carbonate (CCA) groups have been obtained by the cross-linking reaction of poly(vinylene carbonate) (PVCA) with Jeffamines®. By this reaction, microspheric hydrogel beads with a high water content and a high concentration of reactive CCA groups were prepared. The beads were used as a matrix for the immobilization of the enzyme alkaline phosphatase (ALP) and showed a considerable capacity to couple ALP and a reasonable retention of activity for the ...

Chen, Guohua; Does, Leen; Bantjes, Adriaan

1993-01-01

197

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

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

1985-01-01

198

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

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

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

2006-07-01

199

Coupling technique of self-ordered ring and phosphorimetry for the determination of alkaline phosphatase and diseases prediction  

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Rhodamine S could emit strong and stable room temperature phosphorescence (RTP) on polyamide membrane (PAM) in the presence of heavy atom perturber Pb2+. When Rhodamine S-piperidine solution was dropped on PAM, the red (Rhod.S)n-P-SOR (Rhod.S, (Rhod.S)n, P and SOR refer to alizarin red S, multiple Rhod.S molecules, piperidine and self-ordered ring, respectively) formed on PAM, leading to the enhancement of room temperature phosphorimetry (RTP) intensity (Ip, 117.2) of (Rhod.S)n-P-SOR system, which was 2.4 times higher than that without SOR (Ip, 48.1). Wheat germ agglutinin (WGA) was labelled with (Rhod.S)n-P-SOR by the -NH- of Rhod.S reacting with the -COOH of WGA to form WGA-(Rhod.S)n-P-SOR. The formation of WGA-AP-WGA-(Rhod.S)n-P-SOR in the affinity adsorption (AA) reaction carried out between the -COOH of WGA in WGA-(Rhod.S)n-P-SOR and the -NH2 of alkaline phosphatase (AP) caused the RTP intensity (?Ip) of the WGA-AP-WGA-(Rhod.S)n-P-SOR system 7.8 times larger than that without (Rhod.S)n-P-SOR. Therefore, the coupling technique of SOR and solid substrate-room temperature phosphorimetry (SS-RTP) for the determination of trace AP has been established. This method possessed good selectivity, high sensitivity (Detection limit (L.D) was 3.4 × 10-16 g mL-1) and accuracy, and it has been applied to the determination of trace AP in human serum and the forecast of human diseases, and the results agreed well with those obtained by enzyme-linked immunoassay (ELISA). Besides, the mechanism of the coupling technique for the determination of AP was discussed.

Zhang, Li Hong; Zheng, Zhi Yong; Jiang, Shu-Lian; Cui, Ma-Lin; Jiao, Li; Lin, Xuan; Cai, Wen-Lian; Lin, Shao-Qin; liu, Jia-Ming

2012-11-01

200

Activity of alkaline and acidic phosphatase in glandular cells of uterine endometrium of puerperal ewes after exposure to polychlorinated biphenyls  

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Full Text Available The study is focused on the observation of alkaline and acidic phosphatase activity in the glandular cells of uterine endometrium in puerperal ewes after exposure to polychlorinated biphenyls. Ewes of Slovak merino breed (n=25 divided into 2 groups were included in the experiment. The animals in the experimental group (n=14 and control group (n=11 were euthanised on day 17, 25 and 34 postpartum. The ewes in the experimental group were given per os capsules of the chemical preparation Delor 105 of domestic proveniance containing polychlorinated biphenyls (PCB for a period of 30 days. This preparation is equivalent to the foreign preparation Aroclor 1254. A dose of 100 µg/kg of Delor 105 was given to the animals of the experimental group. These animals were euthanised on day 17 postpartum (n=4 i. e. 5 days from the end of a 30-day period of application; on day 25 postpartum (n=5 i.e. 17 days from the last application of PCB; on day 34 postpartum (n=5, which was equivalent to day 28 from the last application. The ewes from the control group were euthanised on day 17 (n=3, day 25 (n=4 and on day 34 (n-4 postpartum. When evaluating alkaline phosphatase (ALP activity in the glandular cells of the endometrium in the control group, a statistically significant increase (P<0.01 was observed on day 25 and on day 34 (P<0.001 compared to day 17 postpartum. No statistically significant differences in alkaline phosphatase (ALP activity were observed (P>0.05 in the experimental group. The mean values of its activity in the observed period were below the level of values of day 17 in the control group. Acidic phosphatase activity in the glandular cells of the ewes' endometrium showed a statistically conclusive increase between day 17 and day 25 as well as day 34 postpartum (P<0.001. Acidic phosphatase density in the experimental group of ewes showed no statistically marked change (P>0.05 at the observed intervals postpartum. The discussion is focused on PCB effect on the activity of alkaline and acidic phosphatase in the glandular cells of the endometrium of ewes in the puerperal period.

Valocky I.

2005-01-01

 
 
 
 
201

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

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

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

2000-08-01

202

Contribution of matrix vesicles and alkaline phosphatase to ectopic bone formation  

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

P., Ciancaglini; A.M.S., Simão; F.L., Camolezi; J.L., Millán; J.M., Pizauro.

2006-05-01

203

Effects of amines and aminoalcohols on bovine intestine alkaline phosphatase activity.  

Science.gov (United States)

Bovine intestine alkaline phosphatase (BIALP) is widely used as a signaling enzyme in sensitive assays such as enzyme immunoassay (EIA). In this study, we evaluated the effects of various aminoalcohols and amines on the activity of BIALP in the hydrolysis of p-nitrophenyl phosphate (pNPP) at pH 9.8, at 20 °C. The k(cat) values at 0.05 M diethanolamine, 0.1 M triethanolamine, and 0.2 M N-methylethanolamine were 190±10, 840±30, and 500±10 s(-1), respectively. The k(cat) values increased with increasing concentrations of diethanolamine, triethanolamine, and N-methylethanolamine and reached 1240±60, 1450±30, and 2250±80 s(-1), respectively, at 1.0M. On the other hand, the k(cat) values at 0.05-1.0M ethanolamine, ethylamine, methylamine, and dimethylamine were in the range of 100-600 s(-1). These results indicate that diethanolamine, triethanolamine and N-methylethanolamine highly activate BIALP and might be suitable as a dilution buffer of BIALP in EIA. Interestingly, the K(m) values increased with increasing concentrations of diethanolamine and N-methylethanolamine, but not triethanolamine: the K(m) value at 1.0M diethanolamine (0.83±0.15 mM) was 12-fold higher than that at 0.05M (0.07±0.01 mM), and that at 1.0M N-methylethanolamine (2.53±0.20 mM) was 14-fold higher than that at 0.2M (0.18±0.02 mM), while that at 1.0M triethanolamine (0.31±0.01 mM) was similar as that at 0.2M (0.25±0.01 mM), suggesting that the mechanisms of BIALP activation are different between the aminoalcohols. PMID:22112405

Sekiguchi, Satoshi; Hashida, Yasuhiko; Yasukawa, Kiyoshi; Inouye, Kuniyo

2011-07-10

204

Kinetic behaviour of calf intestinal alkaline phosphatase with pNPP.  

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The hydrolysis of p-nitrophenyl phosphate (pNPP) by calf intestinal alkaline phosphatase (CIAP) was investigated with respect to kinetic parameters such as V(max), K(m) and K(cat) under varying pH, buffers, substrate concentration, temperature and period of incubation. Highest activity was obtained with Tris-HCl at pH 11, while in the case of glycine-NaOH buffer the peak activity was recorded at pH 9.5. The enzyme showed the following kinetic characteristics with pNPP in 50 mM Tris-HCl at pH 11 and 100 mM glycine-NaOH at pH 9.5 at an incubation temperature of 37 degrees C: V(max), 3.12 and 1.6 micromoles min(-1) unit(-1); K(m), 7.6 x 10(-4) M and 4 x 10(-4) M; and K(cat), 82.98 s(-1) and 42.55 s(-1), respectively. CIAP displayed a high temperature optimum of 45 degrees C at pH 11. The kinetic behaviour of the enzyme under different parameters suggested that the enzyme might undergo subtle conformational changes in response to the buffers displaying unique characteristics. Bioprecipitation of Cu2+ from 50 ppm of CuCl2 solution was studied where 64.3% of precipitation was obtained. P(i) generated from CIAP-mediated hydrolysis of pNPP was found to bind with copper and precipitated as copper-phosphate. Thus, CIAP could be used as a test candidate in bioremediation of heavy metals from industrial wastes through generation of metal-phosphate complexes. PMID:23617076

Chaudhuri, Gouri; Chatterjee, Saswata; Venu-Babu, P; Ramasamy, K; Thilagaraj, W Richard

2013-02-01

205

Computational modeling of the catalytic mechanism of human placental alkaline phosphatase (PLAP).  

Science.gov (United States)

Alkaline phosphatases (APs) catalyze the hydrolysis and transphosphorylation of phosphate monoesters. Quantum mechanical, molecular dynamics, and molecular docking techniques were applied to computationally model the catalytic mechanism of human placental AP (PLAP). Kinetic and thermodynamic evaluations were performed for each reaction step. The functional significances of the more important residues within the active site were analyzed. The role of the metal ion at the metal binding site M3 was also examined. The calculated activation and reaction energy and free energy values obtained suggested the nucleophilic attack of the Ser92 alkoxide on the phosphorus atom of the substrate would be the rate-limiting step of the catalytic hydrolysis of alkyl phosphate monoesters by PLAP. The reactivities of the wild-type M3-Mg enzyme and the M3-Zn protein were compared, and the main difference observed was a change in the coordination number of the M3 metal for the M3-Zn enzyme. This modification in the active site structure lowered the free energy profile for the second chemical step of the catalytic mechanism (hydrolysis of the covalent phosphoserine intermediate). Consequently, a greater stabilization of the phosphoseryl moiety resulted in a small increment in the activation free energy of the phosphoserine hydrolysis reaction. These computational results suggest that the activation of APs by magnesium at the M3 site is caused by the preference of Mg(2+) for octahedral coordination, which structurally stabilizes the active site into a catalytically most active conformation. The present theoretical results are in good agreement with previously reported experimental studies. PMID:21939286

Borosky, Gabriela L; Lin, Susana

2011-10-24

206

Alkaline phosphatase isoenzymes in mouse plasma detected by polyacrylamide-gel disk electrophoresis.  

Science.gov (United States)

Plasma alkaline phosphatase (ALP) activity is frequently measured in toxicity studies. In the present study, we assessed the usefulness of a commercially available polyacrylamide-gel (PAG) disk electrophoresis kit used in humans (AlkPhor System, Jokoh Co., Ltd., Tokyo, Japan) for identifying plasma ALP isoenzymes in mice of the Crlj:CD1 strain (ICR mice), which are commonly used in toxicity studies. We also examined age-related changes in plasma ALP isoenzymes in ICR mice. Electrophoresis was performed according to the manufacturer's instructions. In order to identify the origin of each ALP isoenzyme, in addition to plasma samples, tissue ALP extracts from the liver, bone and small intestine were treated with neuraminidase, anti-small intestinal ALP antibody, ALP inhibitor levamisole and/or wheat germ agglutinin (WGA). The kit revealed that main plasma ALP isoenzyme in intact ICR mice was bone-derived one, and it tended to decrease with age. On the other hand, liver-derived ALP isoenzyme greatly increased in plasma of cholestasis model mice induced by bile duct ligation. This model mouse had also a large molecular ALP detected in the stacking gel. This ALP was thought to be of intestinal origin because its activity remained even after levamisole inhibition. In addition, a minimum sample volume for sufficient resolution of plasma ALP isoenzymes was only 14µl. The results of this study suggest that the present method is a useful tool for detecting plasma ALP isoenzymes in mice and that pre-treatment of plasma with neuraminidase and concomitant levamisole inhibition with another gel is applicable for the evaluation of organ toxicity. PMID:21467748

Hatayama, Kazuhisa; Nishihara, Yoshito; Kimura, Sayaka; Goto, Ken; Nakamura, Daichi; Wakita, Atsushi; Urasoko, Yoshinaka

2011-04-01

207

Characterization of rat heart alkaline phosphatase isoenzymes and modulation of activity  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english Alkaline phosphatase (ALP) is important in calcification and its expression seems to be associated with the inflammatory process. We investigated the in vitro acute effects of compounds used for the prevention or treatment of cardiovascular diseases on total ALP activity from male Wistar rat heart h [...] omogenate. ALP activity was determined by quantifying, at 410 nm, the p-nitrophenol released from p-nitrophenylphosphate (substrate in Tris buffer, pH 10.4). Using specific inhibitors of ALP activity and the reverse transcription-polymerase chain reaction, we showed that the rat heart had high ALP activity (31.73 ± 3.43 nmol p-nitrophenol·mg protein-1·min-1): mainly tissue-nonspecific ALP but also tissue-specific intestinal ALP type II. Both ALP isoenzymes presented myocardial localization (striated pattern) by immunofluorescence. ALP was inhibited a) strongly by 0.5 mM levamisole, 2 mM theophylline and 2 mM aspirin (91, 77 and 84%, respectively) and b) less strongly by 2 mM L-phenylalanine, 100 mL polyphenol-rich beverages and 0.5 mM progesterone (24, 21 to 29 and 11%, respectively). ?-estradiol and caffeine (0.5 and 2 mM) had no effect; 0.5 mM simvastatin and 2 mM atenolol activated ALP (32 and 36%, respectively). Propranolol (2 mM) tended to activate ALP activity and corticosterone activated (18%) and inhibited (13%) (0.5 and 2 mM, respectively). We report, for the first time, that the rat heart expresses intestinal ALP type II and has high total ALP activity. ALP activity was inhibited by compounds used in the prevention of cardiovascular pathology. ALP manipulation in vivo may constitute an additional target for intervention in cardiovascular diseases.

A., Mota; P., Silva; D., Neves; C., Lemos; C., Calhau; D., Torres; F., Martel; H., Fraga; L., Ribeiro; M.N.M.P., Alçada; M.J., Pinho; M.R., Negrão; R., Pedrosa; S., Guerreiro; J.T., Guimarães; I., Azevedo; M.J., Martins.

2008-07-01

208

Characterization of rat heart alkaline phosphatase isoenzymes and modulation of activity  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english Alkaline phosphatase (ALP) is important in calcification and its expression seems to be associated with the inflammatory process. We investigated the in vitro acute effects of compounds used for the prevention or treatment of cardiovascular diseases on total ALP activity from male Wistar rat heart h [...] omogenate. ALP activity was determined by quantifying, at 410 nm, the p-nitrophenol released from p-nitrophenylphosphate (substrate in Tris buffer, pH 10.4). Using specific inhibitors of ALP activity and the reverse transcription-polymerase chain reaction, we showed that the rat heart had high ALP activity (31.73 ± 3.43 nmol p-nitrophenol·mg protein-1·min-1): mainly tissue-nonspecific ALP but also tissue-specific intestinal ALP type II. Both ALP isoenzymes presented myocardial localization (striated pattern) by immunofluorescence. ALP was inhibited a) strongly by 0.5 mM levamisole, 2 mM theophylline and 2 mM aspirin (91, 77 and 84%, respectively) and b) less strongly by 2 mM L-phenylalanine, 100 mL polyphenol-rich beverages and 0.5 mM progesterone (24, 21 to 29 and 11%, respectively). ?-estradiol and caffeine (0.5 and 2 mM) had no effect; 0.5 mM simvastatin and 2 mM atenolol activated ALP (32 and 36%, respectively). Propranolol (2 mM) tended to activate ALP activity and corticosterone activated (18%) and inhibited (13%) (0.5 and 2 mM, respectively). We report, for the first time, that the rat heart expresses intestinal ALP type II and has high total ALP activity. ALP activity was inhibited by compounds used in the prevention of cardiovascular pathology. ALP manipulation in vivo may constitute an additional target for intervention in cardiovascular diseases.

A., Mota; P., Silva; D., Neves; C., Lemos; C., Calhau; D., Torres; F., Martel; H., Fraga; L., Ribeiro; M.N.M.P., Alçada; M.J., Pinho; M.R., Negrão; R., Pedrosa; S., Guerreiro; J.T., Guimarães; I., Azevedo; M.J., Martins.

209

Noninvasive measurement of alkaline phosphatase activity in embryoid bodies and coculture spheroids with scanning electrochemical microscopy.  

Science.gov (United States)

Alkaline phosphatase (ALP) is an enzyme commonly used as an undifferentiated marker of embryonic stem cells (ESCs). Although noninvasive ALP detection has long been desired for stem cell research and in cell transplantation therapy, little progress has been made in developing such techniques. In this study, we propose a noninvasive evaluation method for detecting ALP activity in mouse embryoid bodies (mEBs) using scanning electrochemical microscopy (SECM). SECM has several advantages, including being noninvasive, nonlabeled, quantitative, and highly sensitive. First, we found that SECM-based ALP evaluation permits the comparison of ALP activity among mEBs of different sizes by monitoring the p-aminophenol (PAP) production rate in aqueous solution containing p-aminophenylphosphate (PAPP) normal to the surface area of each sample. Second, coculture spheroids, consisting of mEB and MCF-7 cells for the core and the concentric outer layer, respectively, were prepared as model samples showing heterogeneous ALP activities. The overall PAP production rate dramatically declined in the presence of the MCF-7 cell outer layer, which blocked the mass transfer of PAPP to inner mEB. This result indicated that the SECM response mainly originated from ALP located at the surface of the cellular aggregate, including mEBs and coculture spheroids. Third, taking advantage of the noninvasive nature of SECM, we examined the relevance of ALP activity and cardiomyocyte differentiation. Collectively, these results suggested that noninvasive SECM-based ALP activity normalized by the sample surface enables the selection of EBs with a higher potential to differentiate into cardiomyocytes, which can contribute toward various types of stem cell research. PMID:24053132

Arai, Toshiharu; Nishijo, Taku; Matsumae, Yoshiharu; Zhou, Yuanshu; Ino, Kosuke; Shiku, Hitoshi; Matsue, Tomokazu

2013-10-15

210

Characterization of rat heart alkaline phosphatase isoenzymes and modulation of activity  

Directory of Open Access Journals (Sweden)

Full Text Available Alkaline phosphatase (ALP is important in calcification and its expression seems to be associated with the inflammatory process. We investigated the in vitro acute effects of compounds used for the prevention or treatment of cardiovascular diseases on total ALP activity from male Wistar rat heart homogenate. ALP activity was determined by quantifying, at 410 nm, the p-nitrophenol released from p-nitrophenylphosphate (substrate in Tris buffer, pH 10.4. Using specific inhibitors of ALP activity and the reverse transcription-polymerase chain reaction, we showed that the rat heart had high ALP activity (31.73 ± 3.43 nmol p-nitrophenol·mg protein-1·min-1: mainly tissue-nonspecific ALP but also tissue-specific intestinal ALP type II. Both ALP isoenzymes presented myocardial localization (striated pattern by immunofluorescence. ALP was inhibited a strongly by 0.5 mM levamisole, 2 mM theophylline and 2 mM aspirin (91, 77 and 84%, respectively and b less strongly by 2 mM L-phenylalanine, 100 mL polyphenol-rich beverages and 0.5 mM progesterone (24, 21 to 29 and 11%, respectively. ?-estradiol and caffeine (0.5 and 2 mM had no effect; 0.5 mM simvastatin and 2 mM atenolol activated ALP (32 and 36%, respectively. Propranolol (2 mM tended to activate ALP activity and corticosterone activated (18% and inhibited (13% (0.5 and 2 mM, respectively. We report, for the first time, that the rat heart expresses intestinal ALP type II and has high total ALP activity. ALP activity was inhibited by compounds used in the prevention of cardiovascular pathology. ALP manipulation in vivo may constitute an additional target for intervention in cardiovascular diseases.

A. Mota

2008-07-01

211

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

Science.gov (United States)

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 ?-sheet core with 19 surrounding ?-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? 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 C atoms from completely buried residues, whereas that of VAP comprised 264 C atoms, which may maintain the stability of HaAP under low-salt conditions. These characteristics of HaAP may be responsible for its unique functional adaptation permitting activity over a wide range of salt concentrations. PMID:24598750

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

2014-01-01

212

Alkaline phosphatases contribute to uterine receptivity, implantation, decidualization, and defense against bacterial endotoxin in hamsters.  

Science.gov (United States)

Alkaline phosphatase (AP) activity has been demonstrated in the uterus of several species, but its importance in the uterus, in general and during pregnancy, is yet to be revealed. In this study, we focused on identifying AP isozyme types and their hormonal regulation, cell type, and event-specific expression and possible functions in the hamster uterus during the cycle and early pregnancy. Our RT-PCR and in situ hybridization studies demonstrated that among the known Akp2, Akp3, Akp5, and Akp6 murine AP isozyme genes, hamster uteri express only Akp2 and Akp6; both genes are co-expressed in luminal epithelial cells. Studies in cyclic and ovariectomized hamsters established that while progesterone (P?) is the major uterine Akp2 inducer, both P? and estrogen are strong Akp6 regulators. Studies in preimplantation uteri showed induction of both genes and the activity of their encoded isozymes in luminal epithelial cells during uterine receptivity. However, at the beginning of implantation, Akp2 showed reduced expression in luminal epithelial cells surrounding the implanted embryo. By contrast, expression of Akp6 and its isozyme was maintained in luminal epithelial cells adjacent to, but not away from, the implanted embryo. Following implantation, stromal transformation to decidua was associated with induced expressions of only Akp2 and its isozyme. We next demonstrated that uterine APs dephosphorylate and detoxify endotoxin lipopolysaccharide at their sites of production and activity. Taken together, our findings suggest that uterine APs contribute to uterine receptivity, implantation, and decidualization in addition to their role in protection of the uterus and pregnancy against bacterial infection. PMID:23929901

Lei, Wei; Nguyen, Heidi; Brown, Naoko; Ni, Hua; Kiffer-Moreira, Tina; Reese, Jeff; Millán, José Luis; Paria, Bibhash C

2013-01-01

213

Endotoxin- and ATP-neutralizing activity of alkaline phosphatase as a strategy to limit neuroinflammation  

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Full Text Available Abstract Background Alkaline phosphatase (AP is a ubiquitously expressed enzyme which can neutralize endotoxin as well as adenosine triphosphate (ATP, an endogenous danger signal released during brain injury. In this study we assessed a potential therapeutic role for AP in inhibiting neuroinflammation using three complementary approaches. Methods Mice were immunized to induce experimental autoimmune encephalomyelitis (EAE and treated with AP for seven days during different phases of disease. In addition, serological assays to determine AP activity, endotoxin levels and endotoxin-reactive antibodies were performed in a cohort of multiple sclerosis (MS patients and controls. Finally, the expression of AP and related enzymes CD39 and CD73 was investigated in brain tissue from MS patients and control subjects. Results AP administration during the priming phase, but not during later stages, of EAE significantly reduced neurological signs. This was accompanied by reduced proliferation of splenocytes to the immunogen, myelin oligodendrocyte glycoprotein peptide. In MS patients, AP activity and isoenzyme distribution were similar to controls. Although endotoxin-reactive IgM was reduced in primary-progressive MS patients, plasma endotoxin levels were not different between groups. Finally, unlike AP and CD73, CD39 was highly upregulated on microglia in white matter lesions of patients with MS. Conclusions Our findings demonstrate that: 1 pre-symptomatic AP treatment reduces neurological signs of EAE; 2 MS patients do not have altered circulating levels of AP or endotoxin; and 3 the expression of the AP-like enzyme CD39 is increased on microglia in white matter lesions of MS patients.

Huizinga Ruth

2012-12-01

214

Simultaneous demonstration of alkaline and acid phosphatase activity in bone, at bone-implant interfaces and at the epiphyseal growth plate in plastic-embedded undemineralized tissues.  

Science.gov (United States)

The aim of the present study was to determine if it was possible to detect in bone, at the epiphyseal growth plate and at the bone-implant interface, the presence of alkaline and acid phosphatases using Technovit 7200 VLC resin. In the plastic-embedded specimens it was possible to observe the simultaneous presence of acid and alkaline phosphatases at the epiphyseal growth plate in the presence of intra-articular implants. The morphology of the cells positive for the phosphatases was very clear, with no apparent diffusion of the reaction product and no sputter ground staining. PMID:9105594

Piattelli, A; Piattelli, M; Scarano, A

1997-04-01

215

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

Science.gov (United States)

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-flag and the sALP-FcD(10) isoforms faithfully mimic the biological properties of the human BALP isoforms in vivo validating the use of these recombinant enzymes in studies aimed at dissecting the pathophysiology and treating hypophosphatasia. PMID:19631305

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

2009-11-01

216

Evaluation of alkaline phosphatase levels and salivary hydroxyapatite ion activity in 6?12-year-old children with different caries severity  

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Full Text Available Introduction: Salivary calcium and phosphate levels might have a relationship with dental caries severity. Evaluation of different indexes such as alkaline phosphatase, which increases salivary phosphate levels, and hydroxyapatite ion activity, which reflects salivary calcium and phosphate levels, might help determine the risk of dental caries. The aim of the present study was to compare salivary alkaline phosphatase activity and hydroxyapatite ion activity index in 6?12-year-old children with different rates of dental caries. Materials and methods: In this descriptive-analytical study the teeth were examined in 152 children, aged 6?12, by a dentist and were placed into three experimental groups according to the dental caries severity as follows: group 1: severe dental caries (DMFT ? 6; group 2: moderate dental caries rate (1 < DMFT < 6; and group 3: mild dental caries rate (DMFT ? 1. After collection of saliva, salivary alkaline phosphatase levels and hydroxyapatite ion activity were measured for each sample. Data was analyzed using one-way ANOVA and a post hoc Tukey test (? = 0.05. Results: The means and standard deviations of alkaline phosphatase levels in groups 1 to 3 were 5.39 ± 2.96, 5.71 ± 3.68 and 5.83 ± 4.53 unit/dL, respectively. Hydroxyapatite ion activity rates were 25.80 ± 0.70, 28.28 ± 0.76 and 28.50 ± 0.56 in groups 1 to 3, respectively. The results of one-way ANOVA showed no significant differences between the experimental groups in alkaline phosphatase levels (p value = 0.830 and hydroxyapatite ion activity (p value = 0.065.Conclusion: According to the finding of the present study, alkaline phosphatase level and hydroxyapatite ion activity have no effect on tooth caries severity in 6?12-year-old children. Future studies are recommended. Key words: Alkaline phosphatase, Dental caries, Saliva

Loghman Rezaei- Soufi

2013-01-01

217

Use of an alkaline phosphatase-labeled synthetic oligonucleotide probe for detection of Campylobacter jejuni and Campylobacter coli.  

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A commercially available synthetic nucleic acid probe (SNAP) conjugated to alkaline phosphatase was compared with standard culture techniques for detecting Campylobacter species. The SNAP was able to detect either 5 ng of C. jejuni DNA or 10(5) CFU of bacteria. The SNAP could also detect DNA extracted from 10(5) CFU in mock-infected stool samples. The SNAP detected C. jejuni and C. coli but showed no reactivity with C. laridis, C. fetus subsp. fetus, C. fetus subsp. venerealis, C. fennelliae,...

Olive, D. M.; Johny, M.; Sethi, S. K.

1990-01-01

218

Amino acid substitutions at the subunit interface of dimeric Escherichia coli alkaline phosphatase cause reduced structural stability.  

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The consequences of amino acid substitutions at the dimer interface for the strength of the interactions between the monomers and for the catalytic function of the dimeric enzyme alkaline phosphatase from Escherichia coli have been investigated. The altered enzymes R10A, R10K, R24A, R24K, T59A, and R10A/R24A, which have amino acid substitutions at the dimer interface, were characterized using kinetic assays, ultracentrifugation, and transverse urea gradient gel electrophoresis. The kinetic da...

Martin, D. C.; Pastra-landis, S. C.; Kantrowitz, E. R.

1999-01-01

219

Identification and characterization of an Escherichia coli gene required for the formation of correctly folded alkaline phosphatase, a periplasmic enzyme.  

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Tn5 insertion mutations of Escherichia coli were isolated that impaired the formation of correctly folded alkaline phosphatase (PhoA) in the periplasm. The PhoA polypeptide synthesized in the mutants was translocated across the cytoplasmic membrane but not released into the periplasmic space. It was susceptible to degradation by proteases in vivo and in vitro. The wild-type counterpart of this gene (named ppfA) has been sequenced and shown to encode a periplasmic protein with a pair of potent...

Kamitani, S.; Akiyama, Y.; Ito, K.

1992-01-01

220

Combined influence of temperature and metal ions on the level of activity of alkaline phosphatase of intestinal mucosa of Acipenseridae  

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Full Text Available The combined influence of divalent metal ions (Mn, Fe, Co, Ni, Cu and Zn and temperature on the level of alkaline phosphatase activity of the mucous membrane of the Acipenseridae is shown. The dependence of the response of the enzyme to the action of metal ions according to their position in the periodic table of chemical elements is presented. This dependence is kept during the change of incubation temperature as well, but only at low temperatures the activating effect of metals is maximum in the early period at high temperatures the inhibitory effects of metals is maximum at the end of the period.

Bednyakov Dmitriy Andreevich

2012-11-01

 
 
 
 
221

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

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

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

2011-09-15

222

Differential expression of the bone and the liver tissue non-specific alkaline phosphatase isoforms in brain tissues.  

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The enzyme tissue non-specific alkaline phosphatase (TNAP) belongs to the ectophosphatase family. It is present in large amounts in bone in which it plays a role in mineralization but little is known about its function in other tissues. Arguments are accumulating for its involvement in the brain, in particular in view of the neurological symptoms accompanying human TNAP deficiencies. We have previously shown, by histochemistry, alkaline phosphatase (AP) activity in monkey brain vessels and parenchyma in which AP exhibits specific patterns. Here, we clearly attribute this activity to TNAP expression rather than to other APs in primates (human and marmoset) and in rodents (rat and mouse). We have not found any brain-specific transcripts but our data demonstrate that neuronal and endothelial cells exclusively express the bone TNAP transcript in all species tested, except in mouse neurons in which liver TNAP transcripts have also been detected. Moreover, we highlight the developmental regulation of TNAP expression; this also acts during neuronal differentiation. Our study should help to characterize the regulation of the expression of this ectophosphatase in various cell types of the central nervous system. PMID:21191615

Brun-Heath, Isabelle; Ermonval, Myriam; Chabrol, Elodie; Xiao, Jinsong; Palkovits, Miklós; Lyck, Ruth; Miller, Florence; Couraud, Pierre-Olivier; Mornet, Etienne; Fonta, Caroline

2011-03-01

223

Whole blood staining in suspension for nonspecific esterase and alkaline phosphatase analyzed with a Technicon H-1.  

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We adapted previously published methods for nonspecific esterase and alkaline phosphatase staining of white blood cells in suspension for use on a Technicon H-1 hematology analyzer. The objective was to develop a semiautomated method using whole blood that could be employed on a large scale for hematology laboratory applications, including toxicology studies, measurement of neutrophil left shift, and cytochemical classification of myeloid leukemias. The nonspecific esterase method uses the pararosaniline stain, generating the unstable substrate from two stable precursors. Whole blood is added to the substrate plus dye mix. Next, acid lysis and fixation steps destroy red cells and stabilize the monocyte staining. The alkaline phosphatase stain employs a stable naphthyl phosphate substrate and fast blue B coupling dye. The red cells are lysed with a pH 10.3 propanediol buffer, and the white blood cells are then stabilized with formalin fixation. For both methods the staining is performed off-line, and the sample is then diluted with propanediol to match the refractive index of the sheath on the H-1 analyzer, before aspiration into the direct cytometry port. A cytogram of scattered versus absorbed light is obtained. The number of cells staining and the intensity of the stain can be quantified from the cytogram. PMID:1693116

Ross, D W; Bishop, C; Henderson, A; Kaplow, L

1990-01-01

224

Perinatal undernutrition alters intestinal alkaline phosphatase and its main transcription factors KLF4 and Cdx1 in adult offspring fed a high-fat diet.  

Science.gov (United States)

Nutrient restriction during gestation and/or suckling is associated with an increased risk of developing inflammation, obesity and metabolic diseases in adulthood. However, the underlying mechanisms, including the role of the small intestine, are unclear. We hypothesized that intestinal adaptation to the diet in adulthood is modulated by perinatal nutrition. This hypothesis was tested using a split-plot design experiment with 20 controls and 20 intrauterine growth-retarded (IUGR) rats aged 240 days and randomly assigned to be fed a standard chow or a high-fat (HF) diet for 10 days. Jejunal tissue was collected at necropsy and analyzed for anatomy, digestive enzymes, goblet cells and mRNA levels. Cecal contents and blood serum were analyzed for alkaline phosphatase (AP). IUGR rats failed to adapt to HF by increasing AP activity in jejunal tissue and cecal content as observed in controls. mRNA levels of transcription factors KLF4 and Cdx1 were blunted in jejunal epithelial cell of IUGR rats fed HF. mRNA levels of TNF-? were lower in IUGR rats. They also displayed exacerbated aminopeptidase N response and reduced jejunal goblet cell density. Villus and crypt architecture and epithelial cell proliferation increased with HF in both control and IUGR rats. Serum AP tended to be lower, and serum levamisole inhibition-resistant AP fraction was lower, in IUGR than controls with HF. Serum fatty acids and triglycerides were higher in IUGR rats and higher with HF. In conclusion, the adult intestine adapts to an HF diet differentially depending on early nutrition, jejunal AP and transcription factors being blunted in IUGR individuals fed HF. PMID:22405696

Lallès, Jean-Paul; Orozco-Solís, Ricardo; Bolaños-Jiménez, Francisco; de Coppet, Pierre; Le Dréan, Gwénola; Segain, Jean-Pierre

2012-11-01

225

Multiple forms of human intestinal alkaline phosphatase: chemical and enzymatic properties, and circulating clearances of the fast- and slow-moving enzymes  

International Nuclear Information System (INIS)

Two forms of alkaline phosphatase orthophosphoric monoester phosphohydrolase (alkaline optimum, EC 3.1.3.1) have been purified from human small intestine by column chromatography on DEAE-cellulose and tyraminyl derivative affinity gel, and by preparative disc gel electrophoresis. Intestinal phosphatases were electrophoretically separated into two components, fast- and slow-moving enzymes, with apparent molecular weights of 140000 and 168000 and with subunit weights of 68000 and 80000, respectively. Organ distribution of injected 125I-labelled enzymes indicates that the desialylated hepatic enzyme was selectively distributed in liver, while the degalactosylated intestinal enzyme was incorporated into liver, lymph fluid, and small intestine. These results suggest that the pathway of circulating clearance of alkaline phosphatase has several routes. (Auth.)

226

Effect of vanadate, a potent alkaline phosphatase inhibitor, on 45Ca and 32P/sub i/ uptake by matrix vesicle-enriched fractions from chicken epiphyseal cartilage  

International Nuclear Information System (INIS)

Vanadate was tested in a hydroxyapatite-seeded ion uptake system to determine possible direct effects on mineral formation. The effect of vanadate on vesicle mineral ion uptake was complex; low dosages of vanadate (2-20 ?M) were stimulatory to Ca2+ uptake, but were inhibitory to P/sub i/. Higher dosages (>67 ?M) were inhibitory to both ions. The effect of vanadate on ion uptake was strongly influenced by the stage of vesicle loading; major effects were seen during the lag and early uptake phases, and minimal effects were seen in the terminal stages. Concentrations of vanadate highly inhibitory to vesicle ion uptake had minimal effects on ion accretion by a hydroxyapatite-seeded system. Inhibition of alkaline phosphatase activity by vanadate broadly paralleled inhibition of P/sub i/ and Ca2+ uptake; however, at low vanadate concentrations, inhibition of P/sub i/ uptake closely paralleled that of alkaline phosphatase. The data indicate that vanadate binds with high affinity to P/sub i/-loading sites, blocking initial P/sub i/ uptake. The close parallelism between inhibition of early P/sub i/ uptake and of alkaline phosphatase activity supports the concept that alkaline phosphatase is involved in P/sub i/ transport during the early stages of matrix vesicle ion loading. However, the fact that only about half of the P/sub i/ uptake was affected by vanadate, despite the progressive inhibition of alkaline phosphatase activity, indicates that alkaline phosphatase is not solely responsible for P/sub i/ uptake by the matrix vesicle-enriched fraction

227

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)

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 hydrogen bond donors within the active site, dominate in defining this reaction specificity. PMID:25299936

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

2014-11-01

228

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

Science.gov (United States)

A multivalent recognition and alkaline phosphatase (ALP)-responsive electrogenerated chemiluminescence (ECL) biosensor for cancer cell detection and in situ evaluation of cell surface glycan expression was developed on a poly(amidoamine) (PAMAM) dendrimer-conjugated, chemically reduced graphene oxide (rGO) electrode interface. In this strategy, the multivalency and high affinity of the cell-targeted aptamers on rGO provided a highly efficient cell recognition platform on the electrode. The ALP and concanavalin A (Con A) coated gold nanoparticles (Au NPs) nanoprobes allowed the ALP enzyme-catalyzed production of phenols that inhibited the ECL reaction of Ru(bpy)32+ on the rGO electrode interface, affording fast and highly sensitive ECL cytosensing and cell surface glycan evaluation. Combining the multivalent aptamer interface and ALP nanoprobes, the ECL cytosensor showed a detection limit of 38 CCRF-CEM cells per mL in human serum samples, broad dynamic range and excellent selectivity. In addition, the proposed biosensor provided a valuable insight into dynamic profiling of the expression of different glycans on cell surfaces, based on the carbohydrates recognized by lectins applied to the nanoprobes. This biosensor exhibits great promise in clinical diagnosis and drug screening.A multivalent recognition and alkaline phosphatase (ALP)-responsive electrogenerated chemiluminescence (ECL) biosensor for cancer cell detection and in situ evaluation of cell surface glycan expression was developed on a poly(amidoamine) (PAMAM) dendrimer-conjugated, chemically reduced graphene oxide (rGO) electrode interface. In this strategy, the multivalency and high affinity of the cell-targeted aptamers on rGO provided a highly efficient cell recognition platform on the electrode. The ALP and concanavalin A (Con A) coated gold nanoparticles (Au NPs) nanoprobes allowed the ALP enzyme-catalyzed production of phenols that inhibited the ECL reaction of Ru(bpy)32+ on the rGO electrode interface, affording fast and highly sensitive ECL cytosensing and cell surface glycan evaluation. Combining the multivalent aptamer interface and ALP nanoprobes, the ECL cytosensor showed a detection limit of 38 CCRF-CEM cells per mL in human serum samples, broad dynamic range and excellent selectivity. In addition, the proposed biosensor provided a valuable insight into dynamic profiling of the expression of different glycans on cell surfaces, based on the carbohydrates recognized by lectins applied to the nanoprobes. This biosensor exhibits great promise in clinical diagnosis and drug screening. Electronic supplementary information (ESI) available: CV and EIS during the electrode assembly, activity of the nanoprobes and the glycan-binding specificities of the lectins. See DOI: 10.1039/c4nr03053b

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

2014-09-01

229

Residual alkaline phosphatase activity in pasteurized milk heated at various temperatures--measurement with the fluorophos and Scharer rapid phosphatase tests.  

Science.gov (United States)

Milk containing three levels of milkfat (skim [0.5%], lowfat [2.0%], and whole [3.25%]) were heat treated at five different temperatures (59, 61, 63, 65, and 67 degrees C) using a laboratory scale, batch pasteurization method. Heated milk samples were removed at 5-min intervals, immediately cooled, and then assayed using the quantitative fluorometric method and the qualitative Scharer rapid test. Mean alkaline phosphatase (ALP) activity values as measured with the Fluorophos method decreased in all milk preparations as the time of sampling and temperature of heating increased. Samples representing the three fat levels and heat treated at 63 degrees C for 30 min, the minimum time/temperature allowed by the 1995 pasteurized milk ordinance (PMO), had ALP activity values <100 mU/liter. All values were below the 350 mU/liter standard for fluid milk products established by the Food and Drug Administration and cited in the 1995 PMO. Evaluation of the milks for adequacy of pasteurization with the Scharer rapid method indicated that those same milks were adequately pasteurized. PMID:9921835

Angelino, P D; Christen, G L; Penfield, M P; Beattie, S

1999-01-01

230

Investigating the kinetics of paramagnetic-beads linked alkaline phosphatase enzyme through microchannel resistance measurement in dielectric microchip.  

Science.gov (United States)

Real time monitoring of electrolyte resistance changes during hydrolysis of 4-nitrophenylphosphate (pNPP) by alkaline phosphatase (ALP) bound on paramagnetic-beads was performed into a small dielectric channel. The reaction kinetic fit with a non-competitive substrate-inhibition equation. Michaelis-Menten apparent constant, KM(app), was determined as 0.33±0.06mM and the maximum apparent rate, Vmax(app) as 98±5pMs(-1). The detection limits were 15fM for ALP and 0.75mM for pNPP. This miniaturized device constitutes a powerful tool for analysis of interaction between ligands. PMID:24613971

Faure, Mathilde; Sotta, Bruno; Gamby, Jean

2014-08-15

231

Discovery and validation of a series of aryl sulfonamides as selective inhibitors of tissue-nonspecific alkaline phosphatase (TNAP).  

Science.gov (United States)

We report the characterization and optimization of drug-like small molecule inhibitors of tissue-nonspecific alkaline phosphatase (TNAP), an enzyme critical for the regulation of extracellular matrix calcification during bone formation and growth. High-throughput screening (HTS) of a small molecule library led to the identification of arylsulfonamides as potent and selective inhibitors of TNAP. Critical structural requirements for activity were determined, and the compounds were subsequently profiled for in vitro activity and bioavailability parameters including metabolic stability and permeability. The plasma levels following subcutaneous administration of a member of the lead series in rat was determined, demonstrating the potential of these TNAP inhibitors as systemically active therapeutic agents to target various diseases involving soft tissue calcification. A representative member of the series was also characterized in mechanistic and kinetic studies. PMID:19821572

Dahl, Russell; Sergienko, Eduard A; Su, Ying; Mostofi, Yalda S; Yang, Li; Simao, Ana Maria; Narisawa, Sonoko; Brown, Brock; Mangravita-Novo, Arianna; Vicchiarelli, Michael; Smith, Layton H; O'Neill, W Charles; Millán, José Luis; Cosford, Nicholas D P

2009-11-12

232

Grazing-incidence small-angle X-ray scattering from alkaline phosphatase immobilized in atmospheric plasmapolymer coatings  

Science.gov (United States)

Grazing-incidence small-angle X-ray scattering (GISAXS) has been used to study proteins embedded in thin polymer films obtained by a new cold, atmospheric-pressure plasma technique. In order to test the efficiency of the technology, four samples of alkaline phosphatase incorporated in organic polymer coatings in different plasma conditions have been investigated. Data have been analysed in the framework of the distorted-wave Born approximation (DWBA), by using a new method for the simultaneous fitting of the two-dimensional diffuse scattering from each sample. As a result, protein film concentration and aggregation state as well as a set of parameters describing the polymer coatings have been obtained.

Ortore, M. G.; Sinibaldi, R.; Heyse, P.; Paulussen, S.; Bernstorff, S.; Sels, B.; Mariani, P.; Rustichelli, F.; Spinozzi, F.

2008-06-01

233

Enzymatic labeling of a single chain variable fragment of an antibody with alkaline phosphatase by microbial transglutaminase.  

Science.gov (United States)

Functional cross-linking of a single chain Fv fragment of anti-hen egg-white lysozyme antibody (scFv) and alkaline phosphatase (AP) was explored using microbial transglutaminase (MTG) from Streptomyces mobaraensis. A specific peptidyl linker for MTG was genetically fused to the N-terminus of each protein and the resultant proteins were obtained separately by bacterial expression. The recombinant peptide-tagged scFv and AP were site-specifically cross-linked by MTG through the extra peptidyl linkers in vitro, which mainly yielded the heterodimer (i.e., scFv-AP conjugate). The enzymatic cross-linking reaction had little influence on either the antigen-binding ability of the scFv moiety or the enzymatic activity of the AP moiety of the conjugate, allowing use within an enzyme-linked immunosorbent assay. The results obtained suggest that the enzymatic approach with MTG facilitates the posttranslational construction of functional fusion proteins. PMID:15112292

Takazawa, Takeshi; Kamiya, Noriho; Ueda, Hiroshi; Nagamune, Teruyuki

2004-05-20

234

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

DEFF Research Database (Denmark)

There are indications of a bone anabolic effect associated with bortezomib treatment. We present a study with long follow up, measuring bone specific alkaline phosphatase (bALP) for a year during and after treatment in an unselected cohort of myeloma patients treated with bortezomib, and assess factors of potential influence on the increase of bALP. Our main findings are that bALP increase is of short duration and declines significantly even during continued treatment with bortezomib. Only myeloma response was associated with a significant increase of bALP; whereas previous treatment with bortezomib, previous or concomitant treatment with zoledronic acid i.v., dose of bortezomib, line of treatment, or combination with other chemotherapy was not.

Plesner, Torben; Lund, Thomas

2012-01-01

235

A Ratiometric Fluorescent Probe Based on ESIPT and AIE Processes for Alkaline Phosphatase Activity Assay and Visualization in Living Cells.  

Science.gov (United States)

Alkaline phosphatase (ALP) activity is regarded as an important biomarker in medical diagnosis. A ratiometric fluorescent probe is developed based on a phosphorylated chalcone derivative for ALP activity assay and visualization in living cells. The probe is soluble in water and emits greenish-yellow in aqueous buffers. In the presence of ALP, the emission of probe changes to deep red gradually with ratiometric fluorescent response due to formation and aggregation of enzymatic product, whose fluorescence involves both excited-state intramolecular proton transfer and aggregation-induced emission processes. The linear ratiometric fluorescent response enables in vitro quantification of ALP activity in a range of 0-150 mU/mL with a detection limit of 0.15 mU/mL. The probe also shows excellent biocompatibility, which enables it to apply in ALP mapping in living cells. PMID:25208827

Song, Zhegang; Kwok, Ryan T K; Zhao, Engui; He, Zikai; Hong, Yuning; Lam, Jacky W Y; Liu, Bin; Tang, Ben Zhong

2014-10-01

236

Sex-dependent, zinc-induced dephosphorylation of phospholamban by tissue-nonspecific alkaline phosphatase in the cardiac sarcomere.  

Science.gov (United States)

We have previously reported that Zn(2+) infused into the coronary arteries of isolated rat hearts leads to the potent dephosphorylation of phospholamban (PLB) as well as a noticeable but less potent dephosphorylation of the ryanodine receptor 2. We hypothesized in the present study that a Zn(2+)-activated phosphatase is located in the vicinity of the sarcoplasmic reticulum (SR) where PLB and ryanodine receptor 2 reside. We report here the novel finding of tissue-nonspecific alkaline phosphatase (TNAP), a zinc-dependent enzyme, localized to the SR in the cardiac sarcomere of mouse myocardium. TNAP activity was enhanced by injection of Zn acetate into a tail vein before harvesting the heart and imaged using electron microscopy of electron dense deposits indicative of the hydrolysis of exogenous ?-glycerophosphate. TNAP activity was observed localized to the ends of the Z-line corresponding to SR and was qualitatively more visible in myocardium of males compared with females. Correspondingly, PLB phosphorylation status was potently reduced in myocardium of males injected with Zn acetate, whereas there was no apparent effect of Zn acetate injection on PLB phosphorylation in females. Surprisingly, Western blot analysis of TNAP content suggested a significantly lower TNAP content in males compared with females. These data suggest that TNAP plays a role in governing the phosphorylation status of calcium handling proteins in the SR. Furthermore, the content and activity of TNAP are differentially regulated between the sexes and thus may account for some sex differences in cardiopathologies associated with calcium handling. PMID:25015959

Wang, Yuan; Bishop, Nicole M; Taatjes, Douglas J; Narisawa, Sonoko; Millán, José Luis; Palmer, Bradley M

2014-09-15

237

Purification and Characterization of an Alkaline Phosphatase Induced by Phosphorus Starvation in Common Bean (Phaseolus vulgaris L.) Roots  

Scientific Electronic Library Online (English)

Full Text Available SciELO Mexico | Language: English Abstract in spanish Dos isoformas de fosfatasas obtenidas de raíz de frijol (Phaseolus vulgaris L.) mostraron un incremento en la actividad en respuesta a la deficiencia de fosfato. Una de ellas (APIII) se purificó a través de una cromatografía de intercambio iónico y una electroforesis preparativa. La masa molecular e [...] stimada para APIII fue de 35 kDa tanto por SDS-PAGE como por filtración molecular, sugiriendo que la enzima activa es monomérica. APIII se clasificó como una fosfatasa alcalina basada en sus requerimientos de pH 8 para catálisis. Esta enzima es activa sobre un amplio espectro de sustratos como polifosfato, glucose 1-fosfato y fosfoenolpiruvato, aunque muestra preferencia por pirofosfato. Su actividad se inhibe completamente por molibdato, vanadato y fosfato, aunque es inhibida parcialmente por fluoruro. Aún cuando los cationes divalentes no fueron escenciales para su actividad, la hidrólisis de pirofosfato se incrementó notablemente en presencia de Mg2+. Abstract in english 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 ma [...] ss 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 phosphoenol-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 pyrophosphatase activity of this enzyme, the hydrolysis of pyrophosphate increased substantially in the presence of Mg2+.

Lorena, Morales; Natalia, Gutiérrez; Vanessa, Maya; Carmen, Parra; Eleazar, Martínez-Barajas; Patricia, Coello.

2012-03-01

238

Purification and Characterization of an Alkaline Phosphatase Induced by Phosphorus Starvation in Common Bean (Phaseolus vulgaris L.) Roots  

Scientific Electronic Library Online (English)

Full Text Available SciELO Mexico | Language: English Abstract in spanish Dos isoformas de fosfatasas obtenidas de raíz de frijol (Phaseolus vulgaris L.) mostraron un incremento en la actividad en respuesta a la deficiencia de fosfato. Una de ellas (APIII) se purificó a través de una cromatografía de intercambio iónico y una electroforesis preparativa. La masa molecular e [...] stimada para APIII fue de 35 kDa tanto por SDS-PAGE como por filtración molecular, sugiriendo que la enzima activa es monomérica. APIII se clasificó como una fosfatasa alcalina basada en sus requerimientos de pH 8 para catálisis. Esta enzima es activa sobre un amplio espectro de sustratos como polifosfato, glucose 1-fosfato y fosfoenolpiruvato, aunque muestra preferencia por pirofosfato. Su actividad se inhibe completamente por molibdato, vanadato y fosfato, aunque es inhibida parcialmente por fluoruro. Aún cuando los cationes divalentes no fueron escenciales para su actividad, la hidrólisis de pirofosfato se incrementó notablemente en presencia de Mg2+. Abstract in english 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 ma [...] ss 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 phosphoenol-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 pyrophosphatase activity of this enzyme, the hydrolysis of pyrophosphate increased substantially in the presence of Mg2+.

Lorena, Morales; Natalia, Gutiérrez; Vanessa, Maya; Carmen, Parra; Eleazar, Martínez-Barajas; Patricia, Coello.

239

Light microscopical localization of enzymes by means of cerium-based methods. II. A new cerium-lead-technique for alkaline phosphatase.  

Science.gov (United States)

The use of cerium ions as a primary capture reagent in phosphatase histochemistry on the light and the electron microscopic level is a progress in the field of enzyme localization. Many influences of other captures (as of lead ions), e.g. enzyme inhibition, diffusion and other artefacts, are restricted when cerium-based methods are used. But the broader use of cerium is difficult, because cerium ions are at alkaline pH converted to the insoluble cerium hydroxide, which intensively precipitates in the incubation medium. This is an important disadvantage for a successful histochemical detection of alkaline phosphatase. The aim of this paper is to describe a new cerium-based method for the light microscopical detection of alkaline phosphatase, which is free from all these above mentioned problems. It is proposed a collidine buffer-sucrose containing medium, which holds cerium ions at pH = 9.0 in solution. The histochemical results of this method are excellent. The method is compared with a strontium based technique, the coupling azo dye technique for alkaline phosphatase as well as with in vitro and histochemical experiments with several chelator agents. The cerium-based collidine-sucrose technique is superior to all other procedures tested here and is recommended for a broader use. PMID:3933256

Halbhuber, K J; Zimmermann, N

1985-01-01

240

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

Science.gov (United States)

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

 
 
 
 
241

A sensitive dual colorimetric and fluorescence system for assaying the activity of alkaline phosphatase that relies on pyrophosphate inhibition of the peroxidase activity of copper ions.  

Science.gov (United States)

A novel and highly sensitive colorimetric and fluorescence assay for the accurate determination of alkaline phosphatase (ALP) activity has been developed. The assay takes advantage of the inhibition of the peroxidase activity of Cu(2+) ions caused by complexation with pyrophosphate (PPi), a natural substrate for ALP. This inhibition disappears when PPi undergoes ALP catalyzed hydrolysis to generate phosphate, which does not bind to Cu(2+) ions. Thus, ALP causes generation of uncomplexed Cu(2+) ions, which promote multiple oxidation reactions of Amplex UltraRed in the presence of hydrogen peroxide in conjunction with the production of intense fluorescence and colorimetric signals. By employing the fluorescence and colorimetric assay strategies, ALP can be detected at respective concentrations as low as 4.3 pM and 5.4 pM, detection limits that are much lower than those associated with previously described methods. The practical diagnostic capability of the assay system has been demonstrated by its use to detect ALP in human blood serum. PMID:25057515

Park, Ki Soo; Lee, Chang Yeol; Park, Hyun Gyu

2014-09-21

242

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)

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.

Grozdea Jean J

2002-01-01

243

Exchange potentials of phosphorus between sediments and water coupled to alkaline phosphatase activity and environmental factors in an oligo-mesotrophic reservoir.  

Science.gov (United States)

We investigated the exchange potentials of phosphates at the water-sediment interface together with in situ benthic-chamber fractionated alkaline phosphatase activity and bacteria estimates during September and October 1998 at two stations: station 1, which received immediately the urban inputs from the Taounate city, and station 2, located in the centre of the Sahela reservoir (Morocco). The results showed that low oxygenation enhanced both the bacterial abundance and the alkaline phosphatase activity. Size-fractionated (0.65-100 microm) bacteria attached to dead organic matter together with algae and zooplankton contributed strongly (78%) to the total alkaline phosphatase synthesis in the two sampled stations, suggesting that attachment to organic particles stimulated phosphatase activities. The appearance of anoxic conditions and the decrease of pH supported the dissolution of particulate phosphorus and the release of soluble reactive phosphorus. This latter, together with persisting discharges of organic matter, sewage, and olive mill waste will exacerbate the eutrophication of the reservoir. PMID:17531792

Mhamdi, Badre Alaoui; Azzouzi, Assia; Elloumi, Jannet; Ayadi, Habib; Mhamdi, Mohammed Alaoui; Aleya, Lotfi

2007-05-01

244

A comparison of salivary calcium, phosphate, and alkaline phosphatase in children with severe, moderate caries, and caries free in Tehran?s kindergartens  

Directory of Open Access Journals (Sweden)

Full Text Available The most common dental disease in childhood is dental caries. This study was carried out to recognize the components of saliva which are protective factors in children to evaluate and predict caries susceptible and caries resistant individuals. Unstimulated whole saliva was obtained from 75 children aged 3-5 years. They divided into three groups: decayed missing and filled teeth (dmft > 6 (severe caries, 1 < dmft < 6 (moderate caries, and dmft < 1 (caries free. Unstimulated whole saliva assayed by biochemical methods to determine salivary calcium, inorganic phosphate, and alkaline phosphatase. There was no significant changes in salivary calcium, alkaline to phosphate and alkaline phosphatase activity and their ratio with progress of caries ( P > 0.05 . Although the results showed that salivary phosphate and alkaline phosphatase in caries free group and calcium in the group with severe caries were somewhat more than those in other groups. Despite of the results of the present study, the relationship between salivary components and caries rate in children remains controversial. So more studies are necessary to achieve some practical criteria for predicting dental caries, recognition of susceptible persons, and finally prevention of caries in children.

Shahrabi M

2008-06-01

245

Stimulation by parathyroid hormone of 45Ca2+ uptake in osteoblast-like cells: Possible involvement of alkaline phosphatase  

International Nuclear Information System (INIS)

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

246

Comparison of Salivary Ion Activity Product for Hydroxyapatite (IPHA, Alkaline Phosphatase and Buffering Capacity of Adults According to Age and Caries Severity  

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Full Text Available Statement of Problem: Tooth caries is influenced by different biochemical characteristics of saliva. As hydroxyapatite is the main component of enamel, salivary ion activity product for hydroxyapatite (IPHA as well as alkaline phosphatase may be attributed to dental caries. Purpose: The aim of the present study was to compare salivary buffering capacity, alkaline phosphatase and IPHA of adults according to the dental caries and age. Materials and Method: One hundred and twenty 19 to 44 years old male individuals were divided into four groups according to the dental caries rate and age: group 1: 19-35 years old low dental caries (DMFT <5; group 2: 19-35 years old high dental caries (DMFT 5<; group 3: 35-44 years old low dental caries (DMFT <11 and 35-44 years old high dental caries (DMFT 11<. Five millilitre of unstimulated saliva was collected, and then buffering capacity, the level of alkaline phosphatase activity and IPHA was determined for each sample. Data was analyzed by soft ware SPSS using two-way ANOVA, Friedman and Mann-Whitney tests.Results: Mean and standard deviation of buffering capacity of group 1 to 4 was 2.66±0.54, 2.64±0.56, 2.70±0.70 and 2.26±0.82, respectively. The difference was not significance (p= 0. 305. Mean and standard deviation of alkaline phosphatase activity of group 1 to 4 was 5.82±2.91, 5.30±1.52, 4.77±1.82 and 4.55±1.61, respectively. There was no significant difference (p= 0.692. Mean and standard deviation of IPHA of group 1 to 4 was 29.39±0.61, 29.51±0.76, 29.14±0.56 and 29.75±0.75, respectively. The difference was significant (p= 0.049.Conclusion: Based on the results of the present study, buffering capacity and the level of alkaline phosphatase couldn’t affect dental caries, independently. However, the higher value of IPHA may be attributed to the higher dental caries rate. Ageing decreases alkaline phosphatase activity.

Vahedi M.

2012-12-01

247

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

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

Temelli Ozlem

2009-05-01

248

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

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

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

2007-05-01

249

Use of an alkaline phosphatase-labeled synthetic oligonucleotide probe for detection of Campylobacter jejuni and Campylobacter coli.  

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A commercially available synthetic nucleic acid probe (SNAP) conjugated to alkaline phosphatase was compared with standard culture techniques for detecting Campylobacter species. The SNAP was able to detect either 5 ng of C. jejuni DNA or 10(5) CFU of bacteria. The SNAP could also detect DNA extracted from 10(5) CFU in mock-infected stool samples. The SNAP detected C. jejuni and C. coli but showed no reactivity with C. laridis, C. fetus subsp. fetus, C. fetus subsp. venerealis, C. fennelliae, "C. upsaliensis," C. cinaedii, C. fecalis, C. hyointestinalis, C. mucosalis, or Helicobacter (Campylobacter) pylori. The SNAP also showed no cross-reactivity with other enteric pathogens. When applied to pure cultures, the SNAP detected 55 clinical isolates of C. jejuni and 11 clinical isolates of C. coli, with an accuracy of 100%. When applied directly to clinical specimens, the SNAP detected Campylobacter spp. in 19 of 23 culture-positive stool specimens (sensitivity, 82.6%; specificity, 100%). Pure cultures of the Campylobacter strains isolated from the four probe-negative, culture-positive stool specimens gave positive reactions with the SNAP. While the SNAP had excellent sensitivity and specificity for isolated bacterial colony isolates, the main limitation to the Campylobacter probe detection kit may be the sensitivity limit on direct detection of Campylobacter organisms in stools. PMID:2380381

Olive, D M; Johny, M; Sethi, S K

1990-07-01

250

Ultra-sensitive conductometric detection of heavy metals based on inhibition of alkaline phosphatase activity from Arthrospira platensis.  

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This study is based on the conductometric measurement of alkaline phosphatase activity (APA) from the cyanobacterium, Arthrospira platensis, called Spirulina. Cyanobacterium cells were directly immobilized, by physical adsorption, on the ceramic part of gold interdigitated transducers. This activity was inhibited in the presence of heavy metals and a variation of the local conductivity was measured after addition of the substrate. The Michaelis-Menten constant (Km) was evaluated to be 0.75 mM through a calibration curve of the substrate, disodium 4-nitrophenylphosphate p-nitrophenyl phosphate (pNPP). Inhibition of APA was observed with cadmium and mercury with a detection limit of 10(-20) M. The half maximal inhibitory concentration (IC50) was determined at 10(-19) M for Cd(2+) and 10(-17) M for Hg(2+), and the binding affinity of heavy metal (Ki) was equal to the IC50. On the sensor surface, scanning electron microscopy (SEM) images revealed a remarkable evolution of the cyanobacterium's external surface that was attributable to the first defense mechanism against toxic heavy metals in trace. This effect was also confirmed through the important increase of response time ?(90%) recorded for APA response towards the substrate pNPP after cell exposure to metallic cations. Lifetime of the Spirulina-based biosensor was estimated to be more than 25 days. PMID:23174485

Tekaya, Nadèje; Saiapina, Olga; Ben Ouada, Hatem; Lagarde, Florence; Ben Ouada, Hafedh; Jaffrezic-Renault, Nicole

2013-04-01

251

Kinetics of inhibition of rabbit intestine alkaline phosphatase by heteroligand peroxo complexes of vanadium(V) and tungsten(VI).  

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The present work was undertaken to examine and compare some biologically important properties of peroxo compounds of V(V) and W(VI) containing biogenic species as ancillary ligand. New anionic peroxovanadate(V) complex of the type Na[VO(O(2))(2)(triglycine)].3H(2)O (pV1) and a molecular peroxotungstate(VI) [WO(O(2))(2)(triglycine)].3H(2)O (pW1) were synthesized and characterized for the purpose and their stability in solution was ascertained. Studies on kinetics of inhibition of alkaline phosphatase activity by the newly synthesized compounds and series of dipeptide and amino acid containing peroxo complexes of vanadium and tungsten synthesized previously by us viz., Na[VO(O(2))(2)(gly-gly)(H(2)O)].H(2)O (gly-gly = glycyl-glycine), Na[VO(O(2))(2)(asn)].H(2)O (asn = asparagine), Na[VO(O(2))(2)(gln)].H(2)O (gln = glutamine), and [WO(O(2))(2)(gly-gly)(H(2)O)].3H(2)O, revealed that each of these species is a potent mixed-type inhibitor of the enzyme. Significant difference was noted between the peroxovanadium (pV) and peroxotungsten (pW) compounds in terms of their oxidant activity with reduced glutathione. PMID:19034394

Kalita, Diganta; Das, Siva Prasad; Islam, Nashreen S

2009-06-01

252

Kinetic isotope effects for alkaline phosphatase reactions: implications for the role of active-site metal ions in catalysis.  

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Enzyme-catalyzed phosphoryl transfer reactions have frequently been suggested to proceed through transition states that are altered from their solution counterparts, with the alterations presumably arising from interactions with active-site functional groups. In particular, the phosphate monoester hydrolysis reaction catalyzed by Escherichia coli alkaline phosphatase (AP) has been the subject of intensive scrutiny. Recent linear free energy relationship (LFER) studies suggest that AP catalyzes phosphate monoester hydrolysis through a loose transition state, similar to that in solution. To gain further insight into the nature of the transition state and active-site interactions, we have determined kinetic isotope effects (KIEs) for AP-catalyzed hydrolysis reactions with several phosphate monoester substrates. The LFER and KIE data together provide a consistent picture for the nature of the transition state for AP-catalyzed phosphate monoester hydrolysis and support previous models suggesting that the enzymatic transition state is similar to that in solution. Moreover, the KIE data provides unique information regarding specific interactions between the transition state and the active-site Zn2+ ions. These results provide strong support for a model in which electrostatic interactions between the bimetallo Zn2+ site and a nonbridging phosphate ester oxygen atom make a significant contribution to the large rate enhancement observed for AP-catalyzed phosphate monoester hydrolysis. PMID:17630738

Zalatan, Jesse G; Catrina, Irina; Mitchell, Rebecca; Grzyska, Piotr K; O'brien, Patrick J; Herschlag, Daniel; Hengge, Alvan C

2007-08-01

253

CONSUMPTION OF PHOSPHORUS AND PRODUCTION OF ALKALINE PHOSPHATASE DUR-ING GROWTH OF Streptomyces coelicolor IN A RICH MEDIUM  

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Full Text Available The growth of Strteptomyces coelicolor in a complex medium containing yeast extract, malt extract and glucose exhibited a biphasic mode of biomass accumulation. The first phase was associated with rapid biomass formation, phosphorus utilisation and lack of pigment production. The second stage was marked by slower growth and pigment formation. The demarcation between these phases appeared to result from the depletion of phosphorus in the media, which in turn allowed the red prodigiosin-like pigment to be expressed. Biomass formation appeared to involve the accumulation of phosphorus in the cells reaching a maximum level of 3.7 % of the dry weight. The depletion of phosphorus provoked the increase in alkaline phosphatase activity, which in turn produced a transient release of phosphorus into the medium, presumably from stored cellular phosphorus. Although glucose was consumed during growth, it did not constitute the only carbon source as the appearance of significant amounts of ammonium ions in the broth indicated deamination of amino compounds.

Ismini Nakouti and Glyn Hobbs

2012-02-01

254

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

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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 (?max) of 0.41 /day and a half saturation constant (Ks) of 0.71 ?M. In time-course experiments, APase was induced as dissolved inorganic phosphorus (DIP) concentrations fell below 0.83 ?M, 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.

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

2010-09-01

255

Identification of factors mediating the decrease of alkaline phosphatase activity caused by tension-force in periodontal ligament cells.  

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1. We examined the factors which mediate the decrease of alkaline phosphatase (ALP) activity in human peridontal ligament (PDL) cells in response to cyclic tension-force. 2. ALP activity in human PDL cells obtained from three donors in response to cyclic tension-force (24% elongation) was 43% lower than that of the corresponding control. 3. ALP activity was decreased by the addition of conditioned medium obtained from the culture of the cells exposed to tension-force. 4. The inhibitory effect of the conditioned medium on ALP activity was partially abolished in the presence of indomethacin (10(-6) M) and IL-1 beta antibody (10 ng/well). Moreover it was almost completely abolished in the presence of both indomethacin and IL-1 beta antibody. 5. Treatment of PDL cells with exogenous PGE2 or IL-1 beta for 24 hr caused a dose-dependent decrease in ALP activity. Treatment with both PGE2 (10(-8) M) and IL-1 beta (1.25 x 10(-10) M) together decreased ALP activity by 47% compared with the non-treated control. 6. These findings suggest that ALP activity in PDL cells was decreased in response to the cyclic tension-force and that the decrease in ALP activity was mainly mediated by PGE2 and IL-1 beta produced by PDL cells in response to cyclic tension-force. PMID:7875549

Yamaguchi, M; Shimizu, N

1994-10-01

256

In vitro response to alkaline phosphatase coatings immobilized onto titanium implants using electrospray deposition or polydopamine-assisted deposition.  

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Immobilization of biomolecules onto implant surfaces is one of the most straightforward strategies to control the interaction between an implant and its biological environment. Recently, it was shown that the enzyme alkaline phosphatase (ALP) could be efficiently immobilized onto titanium implants in a single step using polydopamine. We hypothesized that such polydopamine-ALP coatings can enhance the early attachment of cells and increase mineralization. Therefore, the current study aimed at immobilization of ALP onto titanium by means of either one- or two-step polydopamine-assisted immobilization or electrospray deposition, the comparative characterization of these experimental substrates and subsequent cell behavioral analysis using primary osteoblast-like cells. Uncoated titanium and ALP-free polydopamine coatings served as controls. Despite significant ALP surface activity and lower water contact for angles ALP-containing surface modifications, only marginal effects on early cell behavior (i.e., cell spreading) and osteogenic differentiation (i.e., proliferation, differentiation and mineralization) were observed in comparison to uncoated titanium. PMID:23640792

Nijhuis, Arnold W G; van den Beucken, Jeroen J J P; Jansen, John A; Leeuwenburgh, Sander C G

2014-04-01

257

The pharmacological role of phosphatases (acid and alkaline phosphomonoesterases) in snake venoms related to release of purines - a multitoxin.  

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Snake venom components, acting in concert in the prey, cause their immobilization and initiate digestion. To achieve this, several hydrolytic enzymes of snake venom have evolved to interfere in various physiological processes, which are well defined. However, hydrolytic enzymes such as phosphatases (acid and alkaline phosphomonoesterases) are less studied and their pharmacological role in venoms is not clearly defined. Also, they show overlapping substrate specificities and have other common biochemical properties causing uncertainty about their identity in venoms. The near-ubiquitous distribution of these enzymes in venoms, suggests a significant role for these enzymes in envenomation. It appears that these enzymes may play a central role in liberating purines (mainly adenosine) - a multitoxin and through the action of purines help in prey immobilization. However, apart from this, these enzymes could also possess other pharmacological activities as venom enzymes have been evolved to interfere in diverse physiological processes. This has not been verified by pharmacological studies using purified enzymes. Further research is needed to biologically characterize these enzymes in snake venoms, such that their role in venom is clearly established. PMID:21156030

Dhananjaya, Bhadrapura L; D'Souza, Cletus J M

2011-02-01

258

Lysosomal and alkaline phosphatase activity indicate macromolecule transport across the uterine epithelium in two viviparous skinks with complex placenta.  

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In addition to water and small inorganic ions, macromolecules traverse the uterine epithelium in viviparous skinks to be absorbed by the developing fetus. In some species of lizards with complex placenta, the paracellular pathway across the uterine epithelium becomes tighter and more highly regulated as gestation progresses, suggesting that the transcellular pathway may be an alternative route for molecules to travel across the epithelium. In this study, we identified an extensive formation of a lysosomal system in the apical region of uterine epithelial cells in the highly secretory omphaloplacental region of the skink placenta in two species from the Pseudemoia genus. We suggest that this lysosomal system assists apocrine secretion by digesting large macromolecules into smaller particles, allowing more effective transport across the plasma membrane of uterine epithelial cells. We also demonstrate alkaline phosphatase (AP) activity along the apical plasma membrane of uterine epithelial cells in the omphaloplacental region of skink uterus, an enzyme usually associated with active transport in secretory cells. Apocrine secretion, an extensive lysosomal system and AP activity, offer strong evidence that macromolecules are transported across uterine epithelium of the omphaloplacenta. Our study is the first to provide histochemical evidence of macromolecular transport across this region of the placenta in two species of skinks from the genus with the most complex placenta described in Australia. PMID:19422002

Biazik, Joanna M; Thompson, Michael B; Murphy, Christopher R

2009-12-15

259

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

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

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

2014-01-01

260

Activities of Aspartate Aminotransferase, Alanine Aminotransferase, Gamma-Glutamyltransferase, Alkaline Phosphatase in Plasma of Postpartum Holstein Cows  

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Full Text Available Depressed appetite and reduced Dry Matter Intake (DMI and feeding energy-dense diets for a long time around parturition of cows may lead to excessive lipid mobilization which causes the liver damage. This study was meant to determine the effects of postpartun enzymes metabolic status in Holstein cows. In this study, blood samples were during the whole experimental period, obtained from the jugular venepuncture from each animal on 1 week prepartum (week 1, days delivery (week 0 and 1st 9 weeks postpartum (week 1-9. They were analyzed for examining Aspartate aminotransferase (AST, Alanine Aminotransferase (ALT, Gamma-Glutamyltransferase (GGT, Alkaline Phosphatase (ALP activity. The resultes showed a higher activity of AST which was determined in the 1-3 weeks than other’s. ALT activity indicated a statistically significant increase from the 5-7 weeks of lactation and activity in the 7th week postpartum periods significally reached to the peak. GGT activity in the antepartum 1 week until delivery day was significally lower in comparison with the first to reach the 9th weeks postpartum. ALP activity in the delivery day and 6-8 weeks significant increased in process. Therefore, the AST, ALT, GGT and ALP of enzyme activity which could be used significantly change in the blood plasma of Holstein.

HuiFang Deng

2012-01-01

 
 
 
 
261

Characterization of recombinantly expressed rat and monkey intestinal alkaline phosphatases: in vitro studies and in vivo correlations.  

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Intestinal alkaline phosphatases (IALPs) are widely expressed in the brush border of epithelial cells of the intestinal mucosa. Although their physiologic role is unclear, they are very significant when it comes to the release of bioactive parent from orally dosed phosphate prodrugs. Such prodrugs can be resistant to cleavage by IALP, or alternatively undergo rapid cleavage leading to the release and precipitation of the less soluble parent. Because purified IALPs from preclinical species are not commercially available, and species differences have not been investigated to date, an effort was made to recombinantly express, purify, and characterize rat and cynomolgus monkey IALP (rIALP). Specifically, recombinant IALP (rIALP)-catalyzed cleavage of five prodrugs (fosphenytoin, clindamycin phosphate, dexamethasone phosphate, ritonavir phosphate, and ritonavir oxymethyl phosphate) was tested in vitro and parent exposure was assessed in vivo (rat only) following an oral dose of each prodrug. It was determined that the rate of phosphate cleavage in vitro varied widely; direct phosphates were more resistant to bioconversion, whereas faster conversion was observed with oxymethyl-linked prodrugs. Overall, the rat rIALP-derived data were qualitatively consistent with in vivo data; prodrugs that were readily cleaved in vitro rendered higher parent drug exposure in vivo. Of the five prodrugs tested, one (ritonavir phosphate) showed no conversion in vitro and no in vivo parent exposure. Finally, the apparent K(m) values obtained for fosphenytoin and clindamycin phosphate in vitro suggest that IALP is not likely to be saturated at therapeutic doses. PMID:23633529

Subramanian, Murali; Paruchury, Sundeep; Singh Gautam, Shashyendra; Pratap Singh, Sheelendra; Arla, Rambabu; Pahwa, Sonia; Jana, Snehasis; Katnapally, Prasannakumar; Yoganand, Vadari; Lakshmaiah, Basanth; Mazumder Tagore, Debarati; Ghosh, Kaushik; Marathe, Punit; Mandlekar, Sandhya

2013-07-01

262

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

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

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

2008-08-01

263

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

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

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

2003-01-01

264

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

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Alkaline phosphatases (APs) are widely distributed from microbes to humans and are involved in several important biological processes such as phosphate nutrition, signal transduction and pathogenesis. Alkaline phosphatases are also useful in various industrial applications and in recombinant DNA technology. A new AP enzyme from Sphingomonas sp. strain BSAR-1, termed PhoK, has been shown to be useful in uranium bioprecipitation. PhoK was expressed, purified and crystallized. The crystals belonged to space group P43212 or P41212, with unit-cell parameters a = b = 87.37, c = 168.16?Å, and contained one enzyme molecule in the asymmetric unit. Native diffraction data have been collected to 1.95?Å resolution at the ESRF. PMID:19724132

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

2009-01-01

265

Pyridoxamine-5-phosphate Enzyme-Linked Immune Mass Spectrometric Assay Substrate for Linear Absolute Quantification of Alkaline Phosphatase to the Yoctomole Range Applied to Prostate Specific Antigen.  

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

Florentinus-Mefailoski, Angelique; Marshall, John G

2014-11-01

266

Investigations on vinylene carbonate. V. Immobilization of alkaline phosphatase onto LDPE films cografted with vinylene carbonate and N-vinyl-N-methylacetamide  

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Low-density polyethylene (LDPE) films cografted with vinylene carbonate (VCA) and N-vinyl-N-methylacetamide (VIMA) were studied as a matrix for the immobilization of the enzyme alkaline phosphatase (ALP) either by direct fixation or by inserting spacers. When water-soluble alkyldiamines such as diaminoethylene, diaminobutane, diethylenetriamine, and diaminohexane were used as spacers between the matrix and the enzyme, the surface concentration (SC) of the active ALP coupled on the matrix was ...

Chen, Guohua; Does, Leen; Bantjes, Adriaan

1993-01-01

267

Distribution and properties of Ca2+-ATPase, phytase, and alkaline phosphatase in isolated enterocytes from normal and vitamin D-deficient rats.  

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The effects of vitamin D-deficiency and repletion on the distribution and activities of Ca2+-ATPase, phytase, and alkaline phosphatase in intact epithelial cells isolated from different regions of the villi and the crypts of the rat jejunum were studied. Similar distribution patterns of activities were found for the three enzymes. In all cases, the enzyme levels were the highest at the villus tip and gradually declined to low activities in the crypt. The Kms were very different between cells ...

Chan, S. D.; Atkins, D.

1983-01-01

268

Evidence of Associations Between Feto-Maternal Vitamin D Status, Cord Parathyroid Hormone and Bone-Specific Alkaline Phosphatase, and Newborn Whole Body Bone Mineral Content  

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In spite of a high prevalence of vitamin D inadequacy in pregnant women and neonates, relationships among vitamin D status (25(OH)D), parathyroid hormone (PTH), bone specific alkaline phosphatase (BALP), and whole body bone mineral content (WBBMC) in the newborn are poorly characterized. The purpose of the present study was to investigate the relationships between maternal and cord 25(OH)D, PTH, BALP, and WBBMC in newborns in a multiethnic population in Oakland, California and to evaluate the...

Loan, Marta D.; Gertz, Erik R.; Fung, Ellen B.; King, Janet C.; Dror, Daphna K.; Allen, Lindsay H.

2012-01-01

269

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  

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A new phosphorescent labelling reagent consisting of fullerol, fluorescein isothiocyanate and N,N-dimethylaniline (F-ol-(FITC){sub 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){sub n}-DMA complex containing several FITC molecules. F-ol-(FITC){sub n}-DMA increased the number of luminescent molecules in the biological target of WGA-AP-WGA-F-ol-(FITC){sub 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{sup -1} for F-ol and 0.097 ag AP spot{sup -1} for FITC in F-ol-(FITC){sub 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{sup -1} for F-ol-DMA and 0.22 ag AP spot{sup -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){sub n}-DMA labelling of WGA was discussed.

Liu Jiaming, E-mail: zzsyliujiaming@163.com [Department of Chemistry and Environmental Science, Zhangzhou Normal College, Zhangzhou 363000 (China); Huang Xiaomei [Department of Chemistry and Environmental Science, Zhangzhou Normal College, Zhangzhou 363000 (China); Department of Food and Biological Engineering, Zhangzhou Institute of Technology, Zhangzhou 363000 (China); Liu Zhenbo [Third Hospital of Xiamen, Xiamen 361100 (China); Lin Shaoqin [Department of Biochemistry, Fujian Education College, Fuzhou 350001 (China); Li Feiming; Gao Fei [Department of Chemistry and Environmental Science, Zhangzhou Normal College, Zhangzhou 363000 (China); Li Zhiming [Department of Food and Biological Engineering, Zhangzhou Institute of Technology, Zhangzhou 363000 (China); Zeng Liqing; Li Lianying; Ouyang Ying [Department of Chemistry and Environmental Science, Zhangzhou Normal College, Zhangzhou 363000 (China)

2009-08-26

270

Placental alkaline phosphatase activity and its relation to foetal growth and nutrition in appropriate and small for gestational age newborns at term.  

Science.gov (United States)

The placental alkaline phosphatase (PAP) activity progressively rises as pregnancy advances, possibly, because of its increasing synthesis by placental tissue. The present study examined the relationship between placental alkaline phosphatase activity and the biochemical indices of foetal nutrition (cord blood glucose, albumin) and growth (neonatal birth weight). Placental and umbilical cord blood samples were collected from 56 term deliveries 30 of them were appropriate for gestational age (AGA) and 26 were small for gestational age(SGA) and prepared for placental alkaline phosphatase assay, glucose and albumin estimations using standard procedures. The birth weights of the neonates at term were taken and recorded. Correlation analyses of the data obtained show significant positive relationships between PAP and cord blood glucose, albumin and birth weight in AGA newborn (r2 = 0.86, 0.71, 0.68 p<0.05) and (r2 = 0.69, 0.81, 0.73 p<0.05) in SGA newborn but no significant relationship with gestational age, also there was significant statistical difference between both groups in level of PAP, glucose and albumin. PMID:22435166

Mosbah, Amira A Abd El-Rahman; Abd-Ellatif, Nahla A Bahgat; Sorour, Ehab Ibrahim; El-Halaby, Alaa F

2011-12-01

271

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)

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

272

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

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

T. Raeisi

2014-07-01

273

Comparación del ultramicrométodo inmunocitoquímico (UMICIQ con el de la fosfatasa alcalina-anti fosfatasa alcalina (APAAP para la cuantificación de subpoblaciones linfocitarias T Comparison of the immunocytochemical ultramicromethod and the alkaline phosphatase - anti-alkaline phosphatase method for the quantification of T lymphocyte subsets  

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Full Text Available Se realizó un estudio comparativo entre el método inmunoenzimático que emplea el complejo fofatasa alcalina-anti fofatasa alcalina (APAAP y el ultramicrométodo inmunocitoquímico (UMICIQ utilizado para la detección de marcadores antigénicos y cuantificación de subpoblaciones de linfocitos T. Se estudiaron los antígenos celulares CD3, CD4 y CD8 en 30 individuos adultos supuestamente sanos. Al compararse los resultados por ambas técnicas, se encontraron diferencias estadísticamente significativas para una p A comparative study was conducted on the immunoenzymatic method based on the alkaline phosphatase - anti-alkaline phosphatase complex and the immunocytochemical ultramicromethod used for detecting antigen markers and for the quantification of T-lymphocyte subsets Cell antigens CD3, CD4 and CD8 from 30 apparently healthy adults were studied. When comparing the outcome of both techniques, statistically significant differences were found, (p< 0,05. It was concluded that although the diagnostic efficiency of both methods are similar, the alkaline phosphatase - anti-alkaline phosphatase method is quicker, more economical and less laborious than the immunocytochemical ultramicromethod, so it is recommended as a procedure of choice for these cell studies

Beatriz Socarrás Ferrer

2002-04-01

274

Bone morphogenetic protein-2-induced alkaline phosphatase expression is stimulated by Dlx5 and repressed by Msx2.  

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Alkaline phosphatase (ALP) is a widely accepted bone marker. Its expression is stimulated by bone morphogenetic protein (BMP)-2 treatment, the activation of BMP receptors and R-Smads, and the expression of Dlx5 and Runx2. However, how BMP-2 induces ALP expression is not clearly understood. We dissected the murine ALP promoter and found within it a Dlx5-binding cis-acting element by electrophoretic mobility shift assays and site-directed mutagenesis of the element. Dlx5 and the product of its target gene, Runx2, stimulated ALP promoter activity in an additive manner. However, because Dlx5 continued to stimulate ALP expression in Runx2(-/-) cells, the ALP stimulatory activity of Dlx5 is independent of Runx2. We also found that overexpression of Msx2 suppressed the mRNA level and enzyme activity of ALP that were induced by BMP-2 stimulation, and suppressed the Dlx5-stimulated ALP promoter activity by competing with Dlx5 for the cis-acting element in the ALP promoter. Moreover, Msx2 levels are constitutively high in C2C12 myogenic cells but decrease over time after BMP-2 treatment. This may explain why BMP-2 treatment of these cells results in immediate Dlx5 expression yet ALP expression commences only 1-2 days later. In other words, Msx2 in high levels counteracts initially the transcriptional activity of Dlx5 in low levels until a threshold Dlx5:Msx2 ratio is reached to the levels that allow the ALP stimulatory activity of Dlx5 to prevail. Thus, Dlx5 transactivates ALP expression, directly by binding to its cognate response element and/or indirectly by stimulating Runx2 expression, and Msx2 counteracts the direct transactivation of Dlx5. PMID:15383550

Kim, Youn-Jeong; Lee, Mi-Hye; Wozney, John M; Cho, Je-Yoel; Ryoo, Hyun-Mo

2004-12-01

275

The effects of culture conditions on the glycosylation of secreted human placental alkaline phosphatase produced in Chinese hamster ovary cells.  

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The effects of different culture conditions, suspension and microcarrier culture and temperature reduction on the structures of N-linked glycans attached to secreted human placental alkaline phosphatase (SEAP) were investigated for CHO cells grown in a controlled bioreactor. Both mass spectrometry and anion-exchange chromatography were used to probe the N-linked glycan structures and distribution. Complex-type glycans were the dominant structures with small amounts of high mannose glycans observed in suspension and reduced temperature cultures. Biantennary glycans were the most common structures detected by mass spectrometry, but triantennary and tetraantennary forms were also detected. The amount of sialic acid present was relatively low, approximately 0.4 mol sialic acid/mol SEAP for suspension cultures. Microcarrier cultures exhibited a decrease in productivity compared with suspension culture due to a decrease in both maximum viable cell density (15-20%) and specific productivity (30-50%). In contrast, a biphasic suspension culture in which the temperature was reduced at the beginning of the stationary phase from 37 to 33 degrees C, showed a 7% increase in maximum viable cell density, a 62% increase in integrated viable cell density, and a 133% increase in specific productivity, leading to greater than threefold increase in total productivity. Both microcarrier and reduced temperature cultures showed increased sialylation and decreased fucosylation when compared to suspension culture. Our results highlight the importance of glycoform analysis after process modification as even subtle changes (e.g., changing from one microcarrier to another) may affect glycan distributions. PMID:18553404

Nam, Jong Hyun; Zhang, Fuming; Ermonval, Myriam; Linhardt, Robert J; Sharfstein, Susan T

2008-08-15

276

Trapping the tetrahedral intermediate in the alkaline phosphatase reaction by substitution of the active site serine with threonine.  

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We report here the construction of a mutant version of Escherichia coli alkaline phosphatase (AP) in which the active site Ser was replaced by Thr (S102T), in order to investigate whether the enzyme can utilize Thr as the nucleophile and whether the rates of the critical steps in the mechanism are altered by the substitution. The mutant AP with Thr at position 102 exhibited an approximately 4000-fold decrease in k(cat) along with a small decrease in Km. The decrease in catalytic efficiency of approximately 2000-fold was a much smaller drop than that observed when Ala or Gly were substituted at position 102. The mechanism by which Thr can substitute for Ser in AP was further investigated by determining the X-ray structure of the S102T enzyme in the presence of the Pi (S102T_Pi), and after soaking the crystals with substrate (S102T_sub). In the S102T_Pi structure, the Pi was coordinated differently with its position shifted by 1.3 A compared to the structure of the wild-type enzyme in the presence of Pi. In the S102T_sub structure, a covalent Thr-Pi intermediate was observed, instead of the expected bound substrate. The stereochemistry of the phosphorus in the S102T_sub structure was inverted compared to the stereochemistry in the wild-type structure, as would be expected after the first step of a double in-line displacement mechanism. We conclude that the S102T mutation resulted in a shift in the rate-determining step in the mechanism allowing us to trap the covalent intermediate of the reaction in the crystal. PMID:17008720

Wang, Jie; Kantrowitz, Evan R

2006-10-01

277

Pyrophosphate inhibits mineralization of osteoblast cultures by binding to mineral, up-regulating osteopontin, and inhibiting alkaline phosphatase activity.  

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Inorganic pyrophosphate (PP(i)) produced by cells inhibits mineralization by binding to crystals. Its ubiquitous presence is thought to prevent "soft" tissues from mineralizing, whereas its degradation to P(i) in bones and teeth by tissue-nonspecific alkaline phosphatase (Tnap, Tnsalp, Alpl, Akp2) may facilitate crystal growth. Whereas the crystal binding properties of PP(i) are largely understood, less is known about its effects on osteoblast activity. We have used MC3T3-E1 osteoblast cultures to investigate the effect of PP(i) on osteoblast function and matrix mineralization. Mineralization in the cultures was dose-dependently inhibited by PP(i). This inhibition could be reversed by Tnap, but not if PP(i) was bound to mineral. PP(i) also led to increased levels of osteopontin (Opn) induced via the Erk1/2 and p38 MAPK signaling pathways. Opn regulation by PP(i) was also insensitive to foscarnet (an inhibitor of phosphate uptake) and levamisole (an inhibitor of Tnap enzymatic activity), suggesting that increased Opn levels did not result from changes in phosphate. Exogenous OPN inhibited mineralization, but dephosphorylation by Tnap reversed this effect, suggesting that OPN inhibits mineralization via its negatively charged phosphate residues and that like PP(i), hydrolysis by Tnap reduces its mineral inhibiting potency. Using enzyme kinetic studies, we have shown that PP(i) inhibits Tnap-mediated P(i) release from beta-glycerophosphate (a commonly used source of organic phosphate for culture mineralization studies) through a mixed type of inhibition. In summary, PP(i) prevents mineralization in MC3T3-E1 osteoblast cultures by at least three different mechanisms that include direct binding to growing crystals, induction of Opn expression, and inhibition of Tnap activity. PMID:17383965

Addison, William N; Azari, Fereshteh; Sørensen, Esben S; Kaartinen, Mari T; McKee, Marc D

2007-05-25

278

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

International Nuclear Information System (INIS)

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 mal transition state stabilization that may arise from complementarity of the guanidinum group to the geometry of the trigonal bipyramidal transition state

279

High sequence variability, diverse subcellular localizations, and ecological implications of alkaline phosphatase in dinoflagellates and other eukaryotic phytoplankton  

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Full Text Available Alkaline phosphatase (AP is a key enzyme for phytoplankton to utilize dissolved organic phosphorus (DOP when dissolved inorganic phosphorus is limited. While three major types of AP and their correspondingly diverse subcellular localization have been recognized in bacteria, little is known about AP in eukaryotic phytoplankton such as dinoflagellates. Here, we isolated a full-length AP cDNA from a latest-diverging dinoflagellate genus Alexandrium, and conducted comparative analyses with homologs from a relatively basal (Amphidinium carterae and late-diverging (Karenia brevis lineage of dinoflagellates as well as other eukaryotic algae. New data and previous studies indicate that AP is common in dinoflagellates and most other major eukaryotic groups of phytoplankton. AP sequences are more variable than many other genes studied in dinoflagellates, and are divergent among different eukaryotic phytoplankton lineages. Sequence comparison to the other characterized APs suggests that dinoflagellates and some other eukaryotic phytoplankton possess the putative AP as phoA type, but some other eukaryotic phytoplankton seem to have other types. Phylogenetic analyses based on AP amino acid sequences indicated that the “red-type” eukaryotic lineages formed a monophyletic group, suggesting a common origin of their APs. As different amino acid sequences have been found to predictably determine different spatial distribution in the cells, which may facilitate access to different pools of DOP, existing computational models were adopted to predict the subcellular localizations of putative AP in the three dinoflagellates and other eukaryotic phytoplankton. Results showed different subcellular localizations of APs in different dinoflagellates and other lineages. The linkage between AP sequence divergence, subcellular localization and ecological niche differentiation requires rigorous experimental verification, and this study now provides a framework for such a future effort.

SenjieLin

2012-07-01

280

High sequence variability, diverse subcellular localizations, and ecological implications of alkaline phosphatase in dinoflagellates and other eukaryotic phytoplankton.  

Science.gov (United States)

Alkaline phosphatase (AP) is a key enzyme for phytoplankton to utilize dissolved organic phosphorus (DOP) when dissolved inorganic phosphorus is limited. While three major types of AP and their correspondingly diverse subcellular localization have been recognized in bacteria, little is known about AP in eukaryotic phytoplankton such as dinoflagellates. Here, we isolated a full-length AP cDNA from a latest-diverging dinoflagellate genus Alexandrium, and conducted comparative analyses with homologs from a relatively basal (Amphidinium carterae) and late-diverging (Karenia brevis) lineage of dinoflagellates as well as other eukaryotic algae. New data and previous studies indicate that AP is common in dinoflagellates and most other major eukaryotic groups of phytoplankton. AP sequences are more variable than many other genes studied in dinoflagellates, and are divergent among different eukaryotic phytoplankton lineages. Sequence comparison to the other characterized APs suggests that dinoflagellates and some other eukaryotic phytoplankton possess the putative AP as phoA type, but some other eukaryotic phytoplankton seem to have other types. Phylogenetic analyses based on AP amino acid sequences indicated that the "red-type" eukaryotic lineages formed a monophyletic group, suggesting a common origin of their APs. As different amino acid sequences have been found to predictably determine different spatial distribution in the cells, which may facilitate access to different pools of DOP, existing computational models were adopted to predict the subcellular localizations of putative AP in the three dinoflagellates and other eukaryotic phytoplankton. Results showed different subcellular localizations of APs in different dinoflagellates and other lineages. The linkage between AP sequence divergence, subcellular localization, and ecological niche differentiation requires rigorous experimental verification, and this study now provides a framework for such a future effort. PMID:22783243

Lin, Xin; Zhang, Huan; Cui, Yudong; Lin, Senjie

2012-01-01

 
 
 
 
281

Interactive roles of CD73 and tissue nonspecific alkaline phosphatase in the renal vascular metabolism of 5'-AMP.  

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CD73 metabolizes extracellular 5'-AMP to adenosine; yet recent experiments in brain tissue suggest that CD73 is not required for the metabolism of 5'-AMP to adenosine because of tissue nonspecific alkaline phosphatase (TNAP), which like CD73 is a GPI-anchored ecto-enyzme with 5'-nucleotidase activity. Because adenosine importantly regulates renovascular function, we investigated whether both TNAP and CD73 are involved in the renovascular metabolism of 5'-AMP. To test this, we examined in isolated, perfused mouse kidneys the metabolism of 5'-AMP (applied to the lumen of the renal vasculature via intrarenal artery administration) to adenosine by measuring renal venous levels of 5'-AMP, adenosine, and inosine (adenosine metabolite) by mass spectrometry. In one study, we compared 5'-AMP metabolism in naive CD73+/+ (wild-type, n = 16) vs. CD73-/- (knockout, n = 16) kidneys; and in a second study, we compared 5'-AMP metabolism in CD73+/+ (n = 9) vs. CD73-/- (n = 8) kidneys pretreated with levamisole (1 mmol/l; TNAP inhibitor). In naive kidneys, 5'-AMP increased renal venous 5'-AMP, adenosine, and inosine, and these responses were similar in CD73+/+ vs. CD73-/- kidneys. Levamisole per se did not inhibit renovascular 5'-AMP metabolism; however, in the presence of levamisole, 5'-AMP increased renal venous 5'-AMP threefold more in CD73-/- vs. CD73+/+ kidneys and knockout of CD73 inhibited 5'-induced adenosine and inosine by 81 and 86%, respectively. TNAP mRNA, protein, and activity were similar in CD73+/+ vs. CD73-/- kidneys. In conclusion, CD73 and TNAP play interactive roles to metabolize luminally applied 5'-AMP in the renal vasculature such that inhibition of both is required to inhibit the production of adenosine. PMID:24990899

Jackson, Edwin K; Cheng, Dongmei; Verrier, Jonathan D; Janesko-Feldman, Keri; Kochanek, Patrick M

2014-09-15

282

Ovarian cancer stem-like cells show induced translineage-differentiation capacity and are suppressed by alkaline phosphatase inhibitor.  

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Spheroid formation is one property of stem cells-such as embryo-derived or neural stem cells-that has been used for the enrichment of cancer stem-like cells (CSLCs). However, it is unclear whether CSLC-derived spheroids are heterogeneous or whether they share common embryonic stemness properties. Understanding these features might lead to novel therapeutic approaches. Ovarian carcinoma is a deadly disease of women. We identified two types of spheroids (SR1 and SR2) from ovarian cancer cell lines and patients' specimens according to their morphology. Both types expressed stemness markers and could self-renew and initiate tumors when a low number of cells were used. Only SR1 could differentiate into multiple-lineage cell types under specific induction conditions. SR1 spheroids could differentiate to SR2 spheroids through epithelial-mesenchymal transition. Alkaline phosphatase (ALP) was highly expressed in SR1 spheroids, decreased in SR2 spheroids, and was absent in differentiated progenies in accordance with the loss of stemness properties. We verified that ALP can be a marker for ovarian CSLCs, and patients with greater ALP expression is related to advanced clinical stages and have a higher risk of recurrence and lower survival rate. The ALP inhibitor, levamisole, disrupted the self-renewal of ovarian CSLCs in vitro and tumor growth in vivo. In summary, this research provides a plastic ovarian cancer stem cell model and a new understanding of the cross-link between stem cells and cancers.This results show that ovarian CSLCs can be suppressed by levamisole. Our findings demonstrated that some ovarian CSLCs may restore ALP activity, and this suggests that inhibition of ALP activity may present a new opportunity for treatment of ovarian cancer. PMID:24280306

Liu, Kuei-Chun; Yo, Yi-Te; Huang, Rui-Lan; Wang, Yu-Chi; Liao, Yu-Ping; Huang, Tien-Shuo; Chao, Tai-Kuang; Lin, Chi-Kang; Weng, Shao-Ju; Ma, Kuo-Hsing; Chang, Cheng-Chang; Yu, Mu-Hsien; Lai, Hung-Cheng

2013-12-01

283

1-step versus 2-step immobilization of alkaline phosphatase and bone morphogenetic protein-2 onto implant surfaces using polydopamine.  

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Immobilization of biomolecules onto implant surfaces is highly relevant in many areas of biomaterial research. Recently, a 2-step immobilization procedure was developed for the facile conjugation of biomolecules onto various surfaces using self-polymerization of dopamine into polydopamine. In the current study, a 1-step polydopamine-based approach was applied for alkaline phosphatase (ALP) and bone morphogenetic protein-2 (BMP-2) immobilization, and compared to the conventional 2-step polydopamine-based immobilization and plain adsorption. To this end, ALP and BMP-2 were immobilized onto titanium and polytetrafluoroethylene (PTFE) substrates. The absolute quantity and biological activity of immobilized ALP were assessed quantitatively to compare the three types of immobilization. Plain adsorption of both ALP and BMP-2 was inferior to both polydopamine-based immobilization approaches. ALP was successfully immobilized onto titanium and PTFE surfaces via the 1-step approach, and the immobilized ALP retained its enzymatic activity. Using the 1-step approach, the amount of immobilized ALP was increased twofold to threefold compared to the conventional 2-step immobilization process. In contrast, more BMP-2 was immobilized using the conventional 2-step immobilization approach. Retention of ALP and BMP-2 was measured over a period of 4 weeks and was found to be similar for the 1-step and 2-step methods and far superior to the retention of adsorbed biomolecules due to the formation of covalent linkages between catechol moieties and immobilized proteins. The biological behavior of ALP and BMP-2 coatings immobilized using polydopamine (1- and 2-step) as well as adsorption was assessed by culturing rat bone marrow cells, which revealed that the cell responses to the various experimental groups were not statistically different. In conclusion, the 1-step polydopamine-based immobilization method was shown to be more efficient for immobilization of ALP, whereas the conventional 2-step method was shown to be more efficient for attachment of BMP-2 onto implant surfaces. PMID:23231507

Nijhuis, Arnold W G; van den Beucken, Jeroen J J P; Boerman, Otto C; Jansen, John A; Leeuwenburgh, Sander C G

2013-08-01

284

Immobilization of alkaline phosphatase on solid surface through self-assembled monolayer and by active-site protection.  

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Retaining biological activity of a protein after immobilization is an important issue and many studies reported to enhance the activity of proteins after immobilization. We recently developed a new immobilization method of enzyme using active-site protection and minimization of the cross-links between enzyme and surface with a DNA polymerase as a model system. In this study, we extended the new method to an enzyme with a small mono-substrate using alkaline phosphatase (AP) as another model system. A condition to apply the new method is that masking agents, in this case its own substrate needs to stay at the active-site of the enzyme to be immobilized in order to protect the active-site during the harsh immobilization process. This could be achieved by removal of essential divalent ion, Zn2+ that is required for full enzyme activity of AP from the masking solution while active-site of AP was protected with p-nitrophenyl phosphate (pNPP). Approximately 40% of the solution-phase activity was acquired with active-site protected immobilized AP. In addition to protection active-site of AP, the number of immobilization links was kinetically controlled. When the mole fraction of the activated carboxyl group of the linker molecule in self-assembled monolayer (SAM) of 12-mercaptododecanoic acid and 6-mercapto-1-ethanol was varied, 10% of 12-mercaptododecanoic acid gave the maximum enzyme activity. Approximately 51% increase in enzyme activity of the active-site protected AP was observed compared to that of the unprotected group. It was shown that the concept of active-site protection and kinetic control of the number of covalent immobilization bonds can be extended to enzymes with small mono-substrates. It opens the possibility of further extension of the new methods of active-site protection and kinetic control of immobilization bond to important enzymes used in research and industrial fields. PMID:24738440

Gao, En-Feng; Kang, Kyung Lhi; Kim, Jeong Hee

2014-06-01

285

Dissolved phosphorus pools and alkaline phosphatase activity in the euphotic zone of the Western north pacific ocean.  

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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?days at the coastal station to 84.4?days 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 and 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. PMID:22457661

Suzumura, Masahiro; Hashihama, Fuminori; Yamada, Namiha; Kinouchi, Shinko

2012-01-01

286

Red yeast rice stimulates osteoblast proliferation and increases alkaline phosphatase activity in MC3T3-E1 cells.  

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Red yeast (Monascus purpureus) is used as a traditional hypocholesterolemic dietary food component in Asia due to its bioactive component, lovastatin. Recently, new evidence suggesting that the statins in red yeast enhance bone formation has been reported, but more research is still needed in order to support these claims of osteogenic effects. Therefore, in this study, we hypothesized that red yeast rice (in which red yeast is fermented) can improve osteogenic function through osteoblast cell proliferation and differentiation. We studied the effect of methanol extract of red yeast rice powder (RYRP) on osteoblast proliferation and differentiation by measuring mitochondrial enzyme activity and bone marker alkaline phosphatase (ALP) activity, respectively. Osteoblast-like MC3T3-E1 cells were cultured in various concentrations of RYRP methanol extract (0.001-1 mg/mL) during the osteoblast differentiation period (1, 5, 10, and 15 days). As measured by 3-[4,5-dimethylthiazol-2-y]-2,5-diphenyltetrazolium bromide assay, RYRP extracts stimulated cell proliferation during a 24-hour period, compared to cooked white rice powder extract. The most pronounced effect was observed at the concentration range between 0.075 and 0.1 mg/mL. This RYRP stimulatory effect for cell proliferation was observed during the whole osteogenic period. Cellular (synthesized) ALP activity was increased at a RYRP extract concentration of 0.075 mg/mL during 15 days of culture, but the medium (secreted) ALP activity did not show any significant change. This cellular ALP activity stimulation by RYRP extract was confirmed by the staining of ALP activity on cell matrix layers for matrix calcification. The results imply that RYRP extract may increase osteogenic effect by stimulating cell proliferation and ALP activity in osteoblastic cells. PMID:20797483

Cho, Young-Eun; Alcantara, Ethel; Kumaran, Santhy; Son, Kun-Ho; Sohn, Ho-Yong; Lee, Jong-Hwa; Choi, Chung-Sig; Ha, Tae-Youl; Kwun, In-Sook

2010-07-01

287

In vitro modulation of alkaline phosphatase activity of Saccharomyces cerevisiae grown in low or high phosphate medium  

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Full Text Available Our objective was to characterize the modulation of the activity of Saccharomyces cerevisiae alkaline phosphatases (ALPs by classic inhibitors of ALP activity, cholesterol and steroid hormones, in order to identify catalytic similarities between yeast and mammalian ALPs. S. cerevisiae expresses two ALPs, coded for by the PHO8 and PHO13 genes. The product of the PHO8 gene is repressible by Pi in the medium. ALP activity from yeast (grown in low or high phosphate medium homogenates was determined with p-nitrophenylphosphate as substrate, pH 10.4 (lPiALP or hPiALP, respectively. Activation of hPiALP was observed with 5 mM L-amino acids (L-homoarginine _ 186%, L-leucine _ 155% and L-phenylalanine - 168% and with 1 mM levamisole (122%; percentage values, in comparison to control, of recovered activity. EDTA (5 mM and vanadate (1 mM distinctly inhibited hPiALP (2 and 20%, respectively. L-homoarginine (5 mM had a lower activating effect on lPiALP (166% and was the strongest hPiALP activator. Corticosterone (5 mM inhibited hPiALP to 90%, but no effect was observed in low phosphate medium. Cholesterol, ß-estradiol and progesterone also had different effects on lPiALP and hPiALP. A concentration-dependent activation of lPiALP minus hPiALP was evident with all three compounds, most especially with ß-estradiol and cholesterol. These results do not allow us to identify similarities of the behavior of S. cerevisiae ALPs and any of the mammalian ALPs but allow us to raise the hypothesis of differential regulation of S. cerevisiae ALPs by L-homoarginine, ß-estradiol and cholesterol and of using these compounds to discriminate between S. cerevisiae lPiALP and hPiALP.

J Fernandes

2008-01-01

288

Site-directed mutagenesis maps interactions that enhance cognate and limit promiscuous catalysis by an alkaline phosphatase superfamily phosphodiesterase.  

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Catalytic promiscuity, an evolutionary concept, also provides a powerful tool for gaining mechanistic insights into enzymatic reactions. Members of the alkaline phosphatase (AP) superfamily are highly amenable to such investigation, with several members having been shown to exhibit promiscuous activity for the cognate reactions of other superfamily members. Previous work has shown that nucleotide pyrophosphatase/phosphodiesterase (NPP) exhibits a >10?-fold preference for the hydrolysis of phosphate diesters over phosphate monoesters, and that the reaction specificity is reduced 10³-fold when the size of the substituent on the transferred phosphoryl group of phosphate diester substrates is reduced to a methyl group. Here we show additional specificity contributions from the binding pocket for this substituent (herein termed the R' substituent) that account for an additional ~250-fold differential specificity with the minimal methyl substituent. Removal of four hydrophobic side chains suggested on the basis of structural inspection to interact favorably with R' substituents decreases phosphate diester reactivity 10?-fold with an optimal diester substrate (R' = 5'-deoxythymidine) and 50-fold with a minimal diester substrate (R' = CH?). These mutations also enhance the enzyme's promiscuous phosphate monoesterase activity by nearly an order of magnitude, an effect that is traced by mutation to the reduction of unfavorable interactions with the two residues closest to the nonbridging phosphoryl oxygen atoms. The quadruple R' pocket mutant exhibits the same activity toward phosphate diester and phosphate monoester substrates that have identical leaving groups, with substantial rate enhancements of ~10¹¹-fold. This observation suggests that the Zn²? bimetallo core of AP superfamily enzymes, which is equipotent in phosphate monoester and diester catalysis, has the potential to become specialized for the hydrolysis of each class of phosphate esters via addition of side chains that interact with the substrate atoms and substituents that project away from the Zn²? bimetallo core. PMID:24261692

Wiersma-Koch, Helen; Sunden, Fanny; Herschlag, Daniel

2013-12-23

289

Utilidad de la fosfatasa alcalina urinaria como marcador precoz de lesión tubular renal / Utility of urinary alkaline phosphatase as early marker of renal tubular failure  

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Full Text Available SciELO Argentina | Language: Spanish Abstract in spanish El objetivo de este estudio fue determinar la actividad de la fosfatasa alcalina urinaria (FALur) para evaluar precozmente lesión tubular y su utilidad diagnóstica. Los pacientes estudiados fueron: 20 Controles (C), 11 hipertensos (HTA), 23 diabéticos (DBT) y 34 con insuficiencia renal de diverso or [...] igen (IRDO). Se realizaron las determinaciones de: creatinina, clearence de creatinina (Jaffé cinético), FAL sérica y urinaria (cinético DGKC), microalbuminuria (inmunoturbidimétrico), proteiunuria (turbidimétrico), uroproteinograma, SDS-PAGE (al 12,5%) e isoenzimograma de FAL. La FAL sérica hallada fue normal, sin diferencia entre grupos y sin relación con el aumento de la FALur. El valor de corte recomendado para FALur fue de 8 UI/L. La FALur estuvo elevada en HTA e IRDO y normal en individuos con DBT. Los aumentos de FALur en IRDO se relacionaron con la lesión tubular estructural y en pacientes con HTA podrían relacionarse con alteración tubular precoz. Se propone la determinación de FALur para la detección temprana de lesión tubular ante falla renal establecida o en individuos con riesgo de desarrollarla, y se establece su utilidad en pacientes: - con DBT y HTA para seguimiento (junto a microalbuminuria y clearence de creatinina), - internados en riesgo de insuficiencia renal aguda: para orientar tratamientos, - con insuficiencia renal crónica: como indicador de lesión y pronóstico. Abstract in english The objective of this study was to determine the activity of urinary Alkaline Phosphatase (ALPur) to evaluate early tubular failure and its diagnostic usefulness. The patients studied were: 20 Controls (C), 11 with Hipertensión (HTA), 23 Diabetic (DBT) and 34 with renal Insufficiency of diverse orig [...] in (IRDO). The creatinine, creatinine clearence (kinetic Jaffé) serum and urinary ALP (kinetic DGKC), microalbuminuria (Immunoturbidimetric), proteiunuria (Turbidimetric), uroproteinogram, SDS-PAGE (12.5%) and ALP isoenzymes determinations were made. The results indicate that serum ALP was normal, without difference between groups, and no relation with the increase in ALPur. Recommended cut-off value of ALPur was 8 UI/L. ALPur was elevated in HTA and IRDO, and normal in DBT. Increases in ALPur in IRDO were related to the structural tubular injury, and those in HTA could be related to early tubular alteration. Determination of ALPur is proposed for early detection of tubular injury, before renal failure is established or when there is risk of developing it, establishing its usefulness in: - DBT and HTA patients: screening (together with microalbuminuria and creatinine clearence). - Hospitalized patients in risk of acute renal insufficiency: in order to orient treatments. - Patients with chronic renal insufficiency: as an indicator of injury and prognosis.

María Beatriz, Di Carlo; Alejandra Gabriela, Gomez; Leticia Bibiana, Madalena; María Laura, Facio; Marco Antonio, Pizzolato; Gustavo Alberto, Negri.

290

Utilidad de la fosfatasa alcalina urinaria como marcador precoz de lesión tubular renal Utility of urinary alkaline phosphatase as early marker of renal tubular failure  

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Full Text Available El objetivo de este estudio fue determinar la actividad de la fosfatasa alcalina urinaria (FALur para evaluar precozmente lesión tubular y su utilidad diagnóstica. Los pacientes estudiados fueron: 20 Controles (C, 11 hipertensos (HTA, 23 diabéticos (DBT y 34 con insuficiencia renal de diverso origen (IRDO. Se realizaron las determinaciones de: creatinina, clearence de creatinina (Jaffé cinético, FAL sérica y urinaria (cinético DGKC, microalbuminuria (inmunoturbidimétrico, proteiunuria (turbidimétrico, uroproteinograma, SDS-PAGE (al 12,5% e isoenzimograma de FAL. La FAL sérica hallada fue normal, sin diferencia entre grupos y sin relación con el aumento de la FALur. El valor de corte recomendado para FALur fue de 8 UI/L. La FALur estuvo elevada en HTA e IRDO y normal en individuos con DBT. Los aumentos de FALur en IRDO se relacionaron con la lesión tubular estructural y en pacientes con HTA podrían relacionarse con alteración tubular precoz. Se propone la determinación de FALur para la detección temprana de lesión tubular ante falla renal establecida o en individuos con riesgo de desarrollarla, y se establece su utilidad en pacientes: - con DBT y HTA para seguimiento (junto a microalbuminuria y clearence de creatinina, - internados en riesgo de insuficiencia renal aguda: para orientar tratamientos, - con insuficiencia renal crónica: como indicador de lesión y pronóstico.The objective of this study was to determine the activity of urinary Alkaline Phosphatase (ALPur to evaluate early tubular failure and its diagnostic usefulness. The patients studied were: 20 Controls (C, 11 with Hipertensión (HTA, 23 Diabetic (DBT and 34 with renal Insufficiency of diverse origin (IRDO. The creatinine, creatinine clearence (kinetic Jaffé serum and urinary ALP (kinetic DGKC, microalbuminuria (Immunoturbidimetric, proteiunuria (Turbidimetric, uroproteinogram, SDS-PAGE (12.5% and ALP isoenzymes determinations were made. The results indicate that serum ALP was normal, without difference between groups, and no relation with the increase in ALPur. Recommended cut-off value of ALPur was 8 UI/L. ALPur was elevated in HTA and IRDO, and normal in DBT. Increases in ALPur in IRDO were related to the structural tubular injury, and those in HTA could be related to early tubular alteration. Determination of ALPur is proposed for early detection of tubular injury, before renal failure is established or when there is risk of developing it, establishing its usefulness in: - DBT and HTA patients: screening (together with microalbuminuria and creatinine clearence. - Hospitalized patients in risk of acute renal insufficiency: in order to orient treatments. - Patients with chronic renal insufficiency: as an indicator of injury and prognosis.

María Beatriz Di Carlo

2007-09-01

291

Enzymatically mediated bioprecipitation of heavy metals from industrial wastes and single ion solutions by mammalian alkaline phosphatase.  

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The study was aimed at investigating the potential use of calf intestinal alkaline phosphatase (CIAP) enzyme in the removal of heavy metals (Cd(2+), Ni(2+), Co(2+) and Cr(3+/6+)) from single ion solutions as well as tannery and electroplating effluents. CIAP mediated bioremediation (white biotechnology) is a novel technique that is eco-friendly and cost effective unlike the conventional chemical technologies. Typical reactions containing the enzyme (CIAP) and p-nitrophenyl phosphate (pNPP) as substrate in Tris-HCl buffer (pH 8 and 11) and either single ion metal solutions (250 ppm and 1000 ppm) or effluents from tannery or electroplating industry were incubated at 37°C for 30 min, 60 min and 120 min. The inorganic phosphate (P(i)) generated due to catalytic breakdown of pNPP complexes free metal ions as metal-phosphate and the amount of metal precipitated was derived by estimating the reduction in the free metal ion present in the supernatant of reactions employing atomic absorption spectrophotometer (AAS). Better precipitation of metal was obtained at pH 11 than at pH 8 and between the two concentrations of different metals tested, an initial metal concentration of 250 ppm in the reaction gave more precipitation than with 1000 ppm. Experimental data showed that at pH 11, the percentage of removal of metal ions (for an initial concentration of 250 ppm) was in the following order: Cd(2+) (80.99%) > Ni(2+) (64.78%) > Cr(3+) > (46.15%) > Co(2+) (36.47%) > Cr(6+) (32.33%). The overall removal of Cr(3+) and Cr(6+) from tannery effluent was 32.77% and 37.39% respectively in 120 min at pH 11. Likewise, the overall removal of Cd(2+), Co(2+) and Ni(2+) from electroplating effluent was 50.42%, 13.93% and 38.64% respectively in 120 min at pH 11. The study demonstrates that bioprecipitation by CIAP may be a viable and environmental friendly method for clean-up of heavy metals from tannery and electroplating effluents. PMID:23030390

Chaudhuri, Gouri; Shah, Gaurav A; Dey, Pritam; S, Ganesh; Venu-Babu, P; Thilagaraj, W Richard

2013-01-01

292

In vitro modulation of alkaline phosphatase activity of Saccharomyces cerevisiae grown in low or high phosphate medium  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english Our objective was to characterize the modulation of the activity of Saccharomyces cerevisiae alkaline phosphatases (ALPs) by classic inhibitors of ALP activity, cholesterol and steroid hormones, in order to identify catalytic similarities between yeast and mammalian ALPs. S. cerevisiae expresses two [...] ALPs, coded for by the PHO8 and PHO13 genes. The product of the PHO8 gene is repressible by Pi in the medium. ALP activity from yeast (grown in low or high phosphate medium) homogenates was determined with p-nitrophenylphosphate as substrate, pH 10.4 (lPiALP or hPiALP, respectively). Activation of hPiALP was observed with 5 mM L-amino acids (L-homoarginine _ 186%, L-leucine _ 155% and L-phenylalanine - 168%) and with 1 mM levamisole (122%; percentage values, in comparison to control, of recovered activity). EDTA (5 mM) and vanadate (1 mM) distinctly inhibited hPiALP (2 and 20%, respectively). L-homoarginine (5 mM) had a lower activating effect on lPiALP (166%) and was the strongest hPiALP activator. Corticosterone (5 mM) inhibited hPiALP to 90%, but no effect was observed in low phosphate medium. Cholesterol, ß-estradiol and progesterone also had different effects on lPiALP and hPiALP. A concentration-dependent activation of lPiALP minus hPiALP was evident with all three compounds, most especially with ß-estradiol and cholesterol. These results do not allow us to identify similarities of the behavior of S. cerevisiae ALPs and any of the mammalian ALPs but allow us to raise the hypothesis of differential regulation of S. cerevisiae ALPs by L-homoarginine, ß-estradiol and cholesterol and of using these compounds to discriminate between S. cerevisiae lPiALP and hPiALP.

J, Fernandes; R, Amorim; I, Azevedo; M.J, Martins.

293

In vitro modulation of alkaline phosphatase activity of Saccharomyces cerevisiae grown in low or high phosphate medium  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english Our objective was to characterize the modulation of the activity of Saccharomyces cerevisiae alkaline phosphatases (ALPs) by classic inhibitors of ALP activity, cholesterol and steroid hormones, in order to identify catalytic similarities between yeast and mammalian ALPs. S. cerevisiae expresses two [...] ALPs, coded for by the PHO8 and PHO13 genes. The product of the PHO8 gene is repressible by Pi in the medium. ALP activity from yeast (grown in low or high phosphate medium) homogenates was determined with p-nitrophenylphosphate as substrate, pH 10.4 (lPiALP or hPiALP, respectively). Activation of hPiALP was observed with 5 mM L-amino acids (L-homoarginine _ 186%, L-leucine _ 155% and L-phenylalanine - 168%) and with 1 mM levamisole (122%; percentage values, in comparison to control, of recovered activity). EDTA (5 mM) and vanadate (1 mM) distinctly inhibited hPiALP (2 and 20%, respectively). L-homoarginine (5 mM) had a lower activating effect on lPiALP (166%) and was the strongest hPiALP activator. Corticosterone (5 mM) inhibited hPiALP to 90%, but no effect was observed in low phosphate medium. Cholesterol, ß-estradiol and progesterone also had different effects on lPiALP and hPiALP. A concentration-dependent activation of lPiALP minus hPiALP was evident with all three compounds, most especially with ß-estradiol and cholesterol. These results do not allow us to identify similarities of the behavior of S. cerevisiae ALPs and any of the mammalian ALPs but allow us to raise the hypothesis of differential regulation of S. cerevisiae ALPs by L-homoarginine, ß-estradiol and cholesterol and of using these compounds to discriminate between S. cerevisiae lPiALP and hPiALP.

J, Fernandes; R, Amorim; I, Azevedo; M.J, Martins.

2008-01-01

294

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)

Using [32P]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 Mn2+ and Co2+, and inhibited by sodium sulphite and zinc chloride. The apparent Ksub(m) of the enzyme for histone phosphate was 0.89 ?mol/l. (Auth.)

295

Changes in alkaline phosphatase levels in patients with prostate cancer receiving degarelix or leuprolide: results from a 12-month, comparative, phase III study.  

Science.gov (United States)

Study Type - Therapy (RCT) Level of Evidence 1b OBJECTIVE To compare the activity of degarelix, a new gonadotrophin-releasing hormone (GnRH) blocker, with leuprolide depot 7.5 mg in the control of total serum alkaline phosphatase (S-ALP) levels in patients with prostate cancer. PATIENTS AND METHODS In the randomized, phase III trial (CS21), patients with histologically confirmed prostate cancer (all stages), were randomized to one of three regimens: degarelix subcutaneous 240 mg for 1 month followed by monthly maintenance doses of 80 mg or 160 mg, or intramuscular leuprolide 7.5 mg/month. Patients receiving leuprolide could also receive antiandrogens for flare protection. We report exploratory S-ALP analyses from CS21, focusing on the comparison of degarelix 240/80 mg with leuprolide 7.5 mg, in line with the recent approvals of this dose by the USA Food and Drug Administration and the European Medicines Agency. RESULTS Overall, 610 patients were included, with a median age of 73 years and median prostate-specific antigen (PSA) level of 19.0 ng/mL. Baseline S-ALP levels were high in metastatic patients and highest in patients with metastatic disease and a haemoglobin level of degarelix but were maintained around baseline with leuprolide. The late rise in S-ALP seen with leuprolide was not apparent with degarelix. The pattern of S-ALP response was similar in patients with a baseline PSA level of > or =50 ng/mL. Between-treatment differences in patients with metastatic disease and those with a PSA level of > or =50 ng/mL were significant at day 364 (P = 0.014 and 0.007, respectively). CONCLUSION Patients with metastatic disease or those with PSA levels of > or =50 ng/mL at baseline had greater reductions in S-ALP levels with degarelix than with leuprolide. Patients in the degarelix group maintained S-ALP suppression throughout the study, in contrast to those in the leuprolide group. This suggests that degarelix might offer better S-ALP control than leuprolide and might prolong control of skeletal metastases, compared with GnRH agonists, over a 1-year treatment period. PMID:19912212

Schröder, Fritz H; Tombal, Bertrand; Miller, Kurt; Boccon-Gibod, Laurent; Shore, Neal D; Crawford, E David; Moul, Judd; Olesen, Tine Kold; Persson, Bo-Eric

2010-07-01

296

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

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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 measurement of 25-hydroxy vitamin-D is recommended in epileptic children taking anti-epileptic dugs. Supplemental vitamin-D administration in such patients may be helpful.

Keyhani doost Z

2011-01-01

297

The lipid raft-bound alkaline phosphatase activity increases and the level of transcripts remains unaffected in liver of merosin-deficient LAMA2dy mouse.  

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Alkaline phosphatase (AP) and other proteins add glycosylphosphatidylinositol (GPI) before addressing to raft domains of the cell membrane. Our previous report showing an increased density of lipid rafts in muscle of dystrophic Lama2dy mice prompted us to compare livers of normal (NL) and dystrophic mice (DL) for their levels of rafts. With this aim, hepatic rafts were isolated as Triton X-100 resistant membranes, and identified by their abundance of flotillin-2, alkaline phosphatase (AP) and other raft markers. The comparable abundance of cholesterol and flotillin-2 in rafts of NL and DL contrasted with the double AP activity both in rafts of DL and whole DL. The AP mRNA level was the same in NL and DL. Sedimentation analysis profiles revealed AP activity of NL distributed between dimeric (dAP) and monomeric AP (mAP), whose proportions and lectin-binding extent changed in DL. The increased AP activity and changed AP glycosylation in DL, the prevalence of mAP in NL and the enhanced stability of dAP in DL demonstrated the critical role that glycosylation and oligomerization play for AP catalysis. The higher AP activity of DL probably arises from dystrophy-associated changes in glycosyl transferases, which alter AP glycosylation and subunit folding with profitable effects for AP stability and catalysis. PMID:24680793

Montenegro, María Fernanda; Moral-Naranjo, María Teresa; Campoy, Francisco J; Muñoz-Delgado, Encarnación; Vidal, Cecilio J

2014-06-01

298

Integrating biotinylated polyalkylthiophene thin films with biological macromolecules: biosensing organophosphorus pesticides and metal ions with surface immobilized alkaline phosphatase utilizing chemiluminescence measurements  

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We describe a methodology for immobilizing the enzyme alkaline phosphatase onto a glass surface using a novel biotinylated copolymer poly (3-undecylthiophene-co-3- thiophenecarboxaldehyde) 6-biotinamido hexanohydrazide attached hydrophobically to silanized glass. The biotin-streptavidin protein interaction is used to carry out this immobilization. Alkaline phosphatase catalyzes the dephosphorylation of a class of macrocyclic compounds: including CSPD {chloro 3-[4-methoxy spiro(1,2 dioxetane-3-2-trichloro-(3.3.1.1)-decan]-4 yl}phenyl phosphate to a product species which emits energy by chemiluminescence. We can detect this chemiluminescence signal with a photomultiplier tube for both enzymatic catalysis in solution and the surface immobilized enzyme (streptavidin conjugate). This enzyme is inhibited by the organophosphorus class of pesticides as well as nerve agents. The enzyme is also inhibited by Be(II), Bi(III) as well as excess Zn(II), while the apoenzyme is reactivated by Zn(II). We demonstrate in this study that two representative organophosphorus pesticides inhibit the enzymatic production of chemiluminescent products. This is true for the enzyme conjugate both free in solution and immobilized. We can detect pesticides down to about 50 ppb for the enzyme in solution and 500 ppb for surface immobilized enzyme in a 100 (mu) l capillary. Detection of Zn(II) by apoenzyme reactivation occurs down to 3 ppb. Be(II) and Bi(III) are detected by inhibition down to 1 ppm.

Pande, Rajiv; Kamtekar, S.; Ayyagari, Madhu S. R.; Marx, Kenneth A.; Kumar, Jayant; Tripathy, Sukant K.; Kaplan, David L.

1995-05-01

299

Assay Format as a Critical Success Factor for Identification of Novel Inhibitor Chemotypes of Tissue-Nonspecific Alkaline Phosphatase from High-Throughput Screening  

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Full Text Available 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 (PPi, 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.

José Luis Millán

2010-04-01

300

2-Aminoethoxydiphenyl borate (2-APB) reduces alkaline phosphatase release, CD63 expression, F-actin polymerization and chemotaxis without affecting the phagocytosis activity in bovine neutrophils.  

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2-Aminoethoxydiphenyl borate (2-APB) interferes with the Ca(2+) influx and reduces the ROS production, gelatinase secretion and CD11b expression in bovine neutrophils. Moreover, it has been suggested that inhibition of the Ca(2+) channel involved in the store operated Ca(2+) entry (SOCE) is a potential target for the development of new anti-inflammatory drugs in cattle, however it is unknown whether 2-APB affects neutrophil functions associated with the innate immune response. This study describes the effect of 2-APB, a putative SOCE inhibitor, on alkaline phosphatase activity a marker of secretory vesicles, CD63 a marker for azurophil granules, F-actin polymerization and in vitro chemotaxis in bovine neutrophils stimulated with platelet-activating factor (PAF). Also, we evaluated the effect of 2-APB in the phagocytic activity against Escherichia coli and Staphylococcus aureus bioparticles. We observed that doses of 2-APB ?10 ?M significantly reduced alkaline phosphatase activity and in vitro chemotaxis, whereas concentrations of 2-APB ?50 ?M reduced CD63 expression and F-actin polymerization. Finally, we observed that 2-APB did not affect the phagocytic activity in neutrophils incubated with E. coli and S. aureus bioparticles. We concluded that inhibition of Ca(2+) influx could be a useful strategy to reduce inflammatory process in cattle. PMID:22226550

Conejeros, I; Velásquez, Z D; Carretta, M D; Alarcón, P; Hidalgo, M A; Burgos, R A

2012-01-15

 
 
 
 
301

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

International Nuclear Information System (INIS)

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

302

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

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

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

2010-08-27

303

Serum enzymes in thermal injury  

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Various metabolic and biological changes follow burn injury. Serum Thio-barbituric acid reactive substances (TBARS), transaminases, alkaline phosphatase and amylase were measured in 43 patients with thermal injury over the first 10 days of post burn period. No clear correlation between elevated serum enzymes except amylase and the burn size was observed on admission. Mean serum TBARS were significantly increased in the burn patients. Transaminases values increased till 5th day then declined o...

Bhagwat, V. R.; Subrahmanyam, M.; Pujari, K. N.

2007-01-01

304

Intestinal alkaline phosphatase activity as a molecular marker of enterotoxicity induced by single dose of 5-fluorouracil and protective role of orally administered glutamine  

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Full Text Available Background. One of the critical limitations for the administration of the chemotherapy is the toxicity affecting normal tissue. The main target organs for 5-fluorouracil (5-FU toxicity in humans and experimental animals are the gastrointestinal tract, bone marrow, and skin. The cytotoxic effects of antimetabolite chemotherapy are based on their role as substrates for the same transport processes and enzymes involved in anabolism and catabolism as the natural substrates. The main goal of our study was to analyze the dose-dependent antiproliferative effects of 5-FU on intestinal mucosa, enterotoxic potential of 5-FU in experimental animals and to test possible protective role of glutamine. Methods. In our study, we used Sprague Dawley rats. The control group of rats included 50 animals, while the groups where either 5-fluorouracil (5-FU alone or 5-FU and glutamine were administered included 200 animals. All experimental animals were further stratified according to the experimental model (25 animals in each of 8 experimental subgroups of animals. The 5-FU was administered by intraperitoneal application in single dose of 0, 100, 200, 300, and 400 mg of 5-FU per kg of body weight. Water solution of 1% glutamine was prepared daily and administered orally, in volume of 200 ml, for 7 days continuously, after the 7th day of 5-FU administration. Experimental animals were sacrificed 7 days after the administration of 5-FU. The isolation of enterocytes was performed according to the method of Kralovansky et al. In cell homogenate obtained by described method, we determined the protein content using the Biuret method and the DNA content using the Burton reagent. The activities of enzymes alkaline phosphatase (ALP, glutathione S-transferase (GST, glutathione reductase (GR, and glutathione peroxidase (GPX were determined by kinetic method. All paraffin samples of the small intestine were stained by haematoxiline and eosine(HE method. All the experiments were done in duplicate and analyzed by standard statistical methods. All the experiments were done in duplicate and analyzed by standard statistical methods. Results: Our results of enterotoxicity induced by intraperitonealy administered 5-FU showed statistically significant decrease of DNA content in small intestine samples of experimental animals, decrease in activity of intestinal alkaline phosphatase enzyme and the increase in glutathione-dependent enzymes. The glutamine supplementation reduced 5-FU intestinal toxicity. Conclusion: Intestinal alkaline phosphatase is a good marker of the dose-dependent enterotoxicity induced by 5-fluorouracil.

Bajin-Kati? Katica

2006-01-01

305

A Rapid Lateral Flow Immunoassay for the Detection of Tyrosine Phosphatase-Like Protein IA-2 Autoantibodies in Human Serum  

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Type 1 diabetes (T1D) results from the destruction of pancreatic insulin-producing beta cells and is strongly associated with the presence of islet autoantibodies. Autoantibodies to tyrosine phosphatase-like protein IA-2 (IA-2As) are considered to be highly predictive markers of T1D. We developed a novel lateral flow immunoassay (LFIA) based on a bridging format for the rapid detection of IA-2As in human serum samples. In this assay, one site of the IA-2As is bound to HA-tagged-IA-2, which is subsequently captured on the anti-HA-Tag antibody-coated test line on the strip. The other site of the IA-2As is bound to biotinylated IA-2, allowing the complex to be visualized using colloidal gold nanoparticle-conjugated streptavidin. For this study, 35 serum samples from T1D patients and 44 control sera from non-diabetic individuals were analyzed with our novel assay and the results were correlated with two IA-2A ELISAs. Among the 35 serum samples from T1D patients, the IA-2A LFIA, the in-house IA-2A ELISA and the commercial IA-2A ELISA identified as positive 21, 29 and 30 IA-2A-positive sera, respectively. The major advantages of the IA-2A LFIA are its rapidity and simplicity. PMID:25047039

Kikkas, Ingrid; Mallone, Roberto; Larger, Etienne; Volland, Herve; Morel, Nathalie

2014-01-01

306

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

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

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

2008-05-12

307

Nucleic acid-regulated perylene probe-induced gold nanoparticle aggregation: a new strategy for colorimetric sensing of alkaline phosphatase activity and inhibitor screening.  

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A positively charged perylene probe (probe 1) could induce aggregation of the gold nanoparticles (Au NPs). As a result, significant assay solution color changes were observed. A duplex DNA (DNA-1) could induce aggregation of the perylene probe. It was observed that DNA-1 could efficiently regulate the probe 1-induced Au NP aggregation. When probe 1 and DNA-1 were first mixed, DNA-1 induced aggregation of the perylene probe. Au NPs were subsequently added, and no induced aggregation of the Au NPs was observed. Thus the color of the assay solution remained to be red. The assay is quite sensitive; 200 pM DNA-1 could cause a clear solution color change. On the basis of this observation, a novel method for the detection of alkaline phosphatase (ALP) activity has been demonstrated. Our method does not require covalent immobilization of the nucleic acid, or the addition of an excess amount of salt. It is sensitive and convenient. PMID:24417549

Jiao, Huping; Chen, Jian; Li, Wenying; Wang, Fangyuan; Zhou, Huipeng; Li, Yongxin; Yu, Cong

2014-02-12

308

Variation of pH in lysed platelet concentrates influence proliferation and alkaline phosphatase activity in human osteoblast-like cells.  

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Activated platelets release a multifaceted blend of growth factors that has stimulatory effects on mesenchymal cells, both in vitro and in vivo, which imply beneficial effects on wound repair and tissue regeneration. Previous studies on fibroblast cultures have revealed that more potent growth factors, with respect to cell proliferation, are released in acidic preparations of lysed platelet concentrates in comparison with neutral and alkaline preparations. The current study was intended to investigate the influence of pH on lysed platelet concentrates with respect to release of growth factors, cell proliferation and alkaline phosphatase (ALP) activity in human osteoblast-like cells (hFOB 1.19). Cell proliferation was assessed with the MTT kit, ALP activity by conventional enzymatic reaction kinetics and growth factors platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) by enzyme-linked immunosorbent assays. Osteoblast-like cells were stimulated with lysed platelet concentrates preincubated at pH 4.4, 5.4, 7.4, and 7.6. A 3-13-fold increase of cell proliferation was found in comparison with controls and the most evident increase was observed with platelets activated at pH 5.4. The highest ALP activity was observed in preparations at pH 7.6. Platelets incubated in an acidic environment (pH 5.4) induced a higher proliferation compared with preincubation at neutral or alkaline pH and the level of PDGF was also found to be higher in acidic preincubations. The level of TGF-beta was, in contrast, lowest at pH 4.4. We suggest, based on these experimental findings, that acidic milieu influence platelets to release growth factors more potent to stimulate osteoblast proliferation than neutral and alkaline platelet preparations. Lysed platelet concentrates prepared at an alkaline pH might release additional components with stimulating effects resulting in other features than cell proliferation. This is the first report, to our knowledge, about a pH dependent stimulatory effect of lysed platelet concentrates on human osteoblast-like cell proliferation. Lysed platelet concentrates, preincubated in acidic or alkaline buffers, may benefit fracture healing, implant fixation and might also be advantageous in the treatment of wounds with platelet constituents; however, this has to be investigated in extended experimental and clinical settings. PMID:17365859

Wahlström, Ola; Linder, Cecilia; Kalén, Anders; Magnusson, Per

2007-03-01

309

Pretreatment of Rats with ?-tocopherol Alter Liver and Kidney Protein, Alkaline Phosphatase Activity and Phospholipid Profile after 24 Hour Intoxication with Cadmium  

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Full Text Available Cell membrane composition and fluidity are altered in diseases and previous reports suggest that membrane lipid is altered in heavy metal toxicity. This study was carried out to assess the effect of cadmium alone and its combination with different doses (75, 150 and 750 mg of ?-tocophenylacetate on the phospholipid profile and alkaline phosphatase activity in the kidney and liver of rats. The results obtained show that in these tissues, cadmium significantly (p<0.05 increased the levels of protein compared with the control, in the liver it significantly raised total lipid levels and decreased it in the kidney. Vitamin E in the form of ?-tocophenylacetate reversed these observations. Cadmium decreased alkaline phosphatase activity significantly (p<0.05 in both tissues; a trend that was counteracted by ?-tocophenylacetate supplementation in a dose dependent pattern. In both the liver and kidney, cadmium treatment reduced the levels of phosphatidylcholine (PC, phosphatidylethanolamine (PE and increased sphingomyelin (SGM. It raised the PC/PE and SGM/PC ratios, index of membrane fluidity. Vitamin E supplementation significantly reduced the SGM/PC ratios in a manner that appears to be dose related. In the liver the effect of the vitamin on the SGM/PC ratio range from about 400 to 300 to 800% in the 75, 150 and 750 mg supplemented groups, respectively. A similar trend was also observed in the kidney. We hypothesize that decreased membrane fluidity occasioned by increase in SGM/PC ratio occur in early cadmium toxicity and that vitamin E cushions the effect of cadmium by decreasing SGM/PC ratio.

G.E. Eriyamremu

2006-01-01

310

Validation of an analytical procedure for the determination of alkaline and alkaline- earth metals in mices´serum  

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Full Text Available The interaction between drugs or toxic substances with human body, and in particular those which transit natural barriers and get inside blood vessels, could to produce incorrect ions balance due to formation of aducts or for biological membrane damage and chemical alteration of ions in the surrounding. For this reason it is important to determine those elements in serum of test animals to establish experimental conditions for preclinic toxicological tests. In the present work, the results of design and validation of analytical procedures for the determination of sodium, potassium, calcium and magnesium from NMRI mouse’s serum using flame atomic absorption spectrometry. The procedures are recommended because they use a very little amount of serum sample, as well as their performance characteristics, like precision, trueness, sensibility and selectivity.

Alvarez M

2008-04-01

311

Establishment and characterization of a murine Gardner's osteosarcoma cell line (GOS/T): purification of bone-specific alkaline phosphatase (ALPase) and use of anti-ALPase as a diagnostic acid.  

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Gardner's murine osteosarcoma has been successfully cultured and maintained as a clonal cell line (GOS/T), capable of forming a tumor with neoplastic bone and osteoid tissue by inoculation of 1 x 10(7) cultured cells. Alkaline phosphatase (ALPase) level in the culture medium and the serum from the tumor-bearing mice increased significantly three times and 16 to 105 times, respectively. The bone-specific ALPase was purified by butanol extraction following gel-filtration through Sephadex G-200. The molecular weight of ALPase was determined as 420,000, which was three times 140,000, the molecular weight of a subunit. Immunofluorescence staining using anti-ALPase antiserum, which was made by inoculation of partially purified ALPase, revealed a positive reaction for GOS/T cell line and osteoblasts of the fetal mice. The data presented here suggest that the cell line of GOS/T can be regarded as an established cell line, and the immunological reaction with anti-ALPase antibody is a useful model system to study human osteosarcoma. PMID:3224688

Yumoto, T; Naniwa, S; Yoshida, H; Nahiro, J; Kasagi, N; Maeda, N

1988-01-01

312

Typing and subtyping of haptoglobin from native serum using disc gel electrophoresis in alkaline buffer: application to routine screening.  

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A method with which the six common phenotypes of human haptoglobin can be identified using unseparated serum is described. In contrast to other reported methods, both typing and subtyping of haptoglobin can be performed by polyacrylamide gel electrophoresis in alkaline buffer using 0.1-4.0 microliter of native serum with hemoglobin added. Haptoglobin-hemoglobin complexes are visualized by their peroxidase activity using benzidine and barium peroxide. This relatively inexpensive and fast method seems particularly well suited for the typing and subtyping of haptoglobin from minute amounts in large series of sera and other body fluids and thus may be useful in medical genetics and forensic medicine. PMID:6496936

Linke, R P

1984-08-15

313

Estado nutricional, consumo de lácteos y niveles séricos de calcio, fósforo y fosfatasas alcalinas en escolares de Mérida / Nutritional status, consumption of dairy products and levels sericos of calcium, phosphorus, and alkaline phosphatases in schoolchildren of Mérida  

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Full Text Available SciELO Venezuela | Language: Spanish Abstract in spanish Se realizó una investigación de Campo de Tipo Descriptiva Correlacional y de corte transversal para determinar el estado nutricional, consumo de lácteos y niveles séricos de calcio, fósforo, y fosfatasas alcalinas en escolares del 1er, 3er y 5to grado de la U.E "Rafael Antonio González" de la comuni [...] dad de Mesa Bolívar en el año 2007. La población estuvo conformada por la matricula escolar de 171 estudiantes. Se determinó la muestra con el método estratificado aleatorio simple, obteniéndose 47% de la matricula escolar, correspondiendo 80 niños distribuidos por grado: 21 niños en 1ero, 28 en 3ero y 31 en 5to, en edades comprendidas entre 6 a 12 años. Se determinó la cantidad y la frecuencia de consumo de productos lácteos para lo cual, se diseñó un cuestionario "ad hoc" contentivo de 10 ítems relacionados con la frecuencia de consumo, cantidad y tipo de lácteos. Se realizó evaluación nutricional a través de la Combinación de Indicadores (Peso para la Talla y Talla para la Edad) utilizando las tablas de Evaluación de la Organización Mundial de la Salud. Se determinaron los valores séricos de calcio, fósforo y fosfatasas alcalinas. Los escolares presentan 32,6% de malnutrición; tanto los niños (6-10 años y 11-12 años) como las niñas (8-12 años) presentaron un porcentaje de adecuación diario de calcio bajo (77,16%, 28,57% y 38,96%) respectivamente y 60% tienen hipocalcemia. Existe significancia estadística entre los niveles séricos de calcio y fósforo con el consumo diario promedio de calcio (p 0,05 y p 0,04). No hubo relación estadísticamente significativa entre el consumo de productos lácteos y el estado nutricional de los escolares. El estado nutricional de los escolares no depende del consumo diario de productos lácteos, sin embargo, dicho consumo si afecta los niveles séricos de calcio y fósforo. Abstract in english A cross-sectional descriptive correlational field research was conducted in order to determine the nutritional status, consumption of milk and serum levels of calcium, phosphorus, and alkaline phosphatase in students of 1st, 3rd and 5th grades of the "Rafael Antonio Gonzalez "school in Mesa Bolívar [...] in 2007. The population consisted of 171 students. We determined the sample with a simple random stratified method, yielding 47% of school enrollment, corresponding to 80 children distributed by grade: 21 children in 1st, 28 in 3rd, 31 in 5th, aged 6 to 12 years old. The amount and frequency of consumption of dairy products, with an "ad hoc" questionnaire designed containing 10 items related to the frequency of consumption, quantity and type of dairy product. Nutritional assessment was carried out by means of the combination of indicators (weight for height and height for age) using the tables of evaluation of the World Health Organization. Values were determined in serum calcium, phosphorus and alkaline phosphatase. The students had 32,6% of malnutrition, both boys (6-10 years and 11-12 years) and girls (8-12 years) had an adequate percentage of low calcium daily intake(77.16%, 28. 57% and 38.96%, respectively) and 60% had hypocalcemia. There is statistical significance between serum calcium and phosphorus with an average daily intake of calcium (p 0.05 and p 0.04). There was no statistically significant relationship between dairy products consumption and nutritional status of schoolchildren. The nutritional status of schoolchildren does not depend on daily consumption of dairy products, however, that consumption does affect serum calcium and phosphorus.

Lizbeth, Rojas; Gladys, Bastardo; Belquis, Sanz; G. Beatriz, Da Silva; Yurimay, Quintero de Rivas; Coromoto, Angarita; Maribel, Prada Briceño.

314

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

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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. Conclusion The results demonstrate the biochemical modulation of scFv binding. Also, the scFvs bound to the active site and denied the access to the substrate. The selection strategy could generate specific anti-enzyme antibodies to PLAP that can potentially be used for targeting, for modulating enzyme activity in in vitro and in vivo and as probes for the active site. This strategy also has a general application in selecting antibodies from combinatorial libraries to closely related molecules and conformations.

Kala Mrinalini

2005-12-01

315

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

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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.

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

1990-04-01

316

Role of sea water DIP and DOP in controlling bulk alkaline phosphatase activity in N.W. Mediterranean Sea (Toulon, France).  

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The regulation of alkaline phosphatase activity by dissolved inorganic (DIP) and organic phosphorus (DOP) and the contribution of DOP as phosphorus source were studied monthly in Toulon Bay (NW Mediterranean, France) in 2005-2006. The concentrations of DIP and DOP varied respectively from 0 to 0.185?M and from 0 to 0.329?M. The bulk activities (Vm, Km, Vm/Km) were measured using MUFP as substrate. Its high affinity component (Km: 0.05-1.00?M) was negatively correlated with the sum of the concentrations of DIP and DOP but not with these compounds taken independently. A negative correlation with DIP was found when the concentrations of DOP were lower than 0.08?M. A negative correlation with DOP was shown when the concentrations of DIP were lower than 0.05?M. This high affinity component can be considered as a valuable indicator for the potential utilization of the compounds which contribute to the intracellular phosphorus pool. PMID:22871673

Bogé, Gérard; Lespilette, Magali; Jamet, Dominique; Jamet, Jean Louis

2012-10-01

317

Differences in growth and alkaline phosphatase activity between Microcystis aeruginosa and Chlorella pyrenoidosa in response to media with different organic phosphorus  

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Full Text Available The growth of Microcystis aeruginosa and Chlorella pyrenoidosa in three dissolved organic phosphorus sources (glucose-1- phosphate, adenosine triphosphate, cyclic-adenosine monophosphate were studied in cultures separated by a dialysis membrane. Results showed that M. aeruginosa and C. pyrenoidosa could utilize those three forms of organic phosphorus, but their growth rates and cell abundances were low in comparison with those in the orthophosphate control. M. aeruginosa had a higher growth rate than C. pyrenoidosa in glucose-1-phosphate, and then became dominate in the separate cultures. In contrast, those two algal species didn’t show any significant differences in the growth rate and cell abundance in the medium with adenosine triphosphate and cyclicadenosine monophosphate. Alkaline phosphatase was an important enzyme for hydrolyzing glucose-1-phosphate, adenosine triphosphate and cyclic-adenosine monophosphate, the activity of which was positively correlated with the growth rate of algae. Considering the big proportion of glucose-1-phosphate in the Lake Taihu, the capability of M. aeruginosa to efficiently utilize this type of organic phosphorus source might be one of reason that why M. aeruginosa is the dominant species in this hyper-eutrophic lake.

Yang YU

2011-02-01

318

Stabilization of different types of transition states in a single enzyme active site: QM/MM analysis of enzymes in the alkaline phosphatase superfamily.  

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The first step for the hydrolysis of a phosphate monoester (pNPP(2-)) in enzymes of the alkaline phosphatase (AP) superfamily, R166S AP and wild-type NPP, is studied using QM/MM simulations based on an approximate density functional theory (SCC-DFTBPR) and a recently introduced QM/MM interaction Hamiltonian. The calculations suggest that similar loose transition states are involved in both enzymes, despite the fact that phosphate monoesters are the cognate substrates for AP but promiscuous substrates for NPP. The computed loose transition states are clearly different from the more synchronous ones previously calculated for diester reactions in the same AP enzymes. Therefore, our results explicitly support the proposal that AP enzymes are able to recognize and stabilize different types of transition states in a single active site. Analysis of the structural features of computed transition states indicates that the plastic nature of the bimetallic site plays a minor role in accommodating multiple types of transition states and that the high degree of solvent accessibility of the AP active site also contributes to its ability to stabilize diverse transition-state structures without the need of causing large structural distortions of the bimetallic motif. The binding mode of the leaving group in the transition state highlights that vanadate may not always be an ideal transition state analog for loose phosphoryl transfer transition states. PMID:23786365

Hou, Guanhua; Cui, Qiang

2013-07-17

319

Langmuir films from human placental membranes: preparation, rheology, transfer to alkylated glasses, and sigmoidal kinetics of alkaline phosphatase in the resultant Langmuir-Blodgett film.  

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In the present study, we studied the activity of human placental alkaline phosphatase (PLAP) constraint in a planar surface in controlled molecular packing conditions. For the first time, Langmuir films (LFs) were prepared by the spreading of purified placental membranes (PPM) on the air-water interface and their stability and rheological properties were studied. LFs exhibited a collapse pressure pi(C) = 48 mN/m, hysteresis during the compression-decompression cycle (C-D), indicating a plastic deformation, and a compressibility modulus (K) compatible with liquid-expanded phases. A phase transition point appeared at pi(T) = 28 mN/m and, following successive C-D, it moved toward lower surface areas and higher K, suggesting the lost of some non-PLAP proteins as components of vesicles that might protrude from the monolayer (confirmed by combining lipid/protein molar ratio analysis, PAGE-SDS and V(max)). LFs were transferred at 35 mN/m to alkylated glasses to obtain Langmuir-Blodgett films (LB(35)) the stability of which was confirmed by AFM. The kinetics of p-nitrophenyl phosphate (pNPP) hydrolysis at 37 degrees C catalyzed by PPM was Michaelian and exhibited the thermostability at 60 degrees C typical of PLAP. In LB(35), PLAP exhibited a sigmoidal kinetics which resembled the behavior of the partially metalated enzyme but might become from a cross-talk between protein and membrane structures. PMID:20033626

Clop, Eduardo M; Perillo, María A

2010-04-01

320

Effect of controlled redox potential and dissolved oxygen on the in vitro refolding of an E. coli alkaline phosphatase and chicken lysozyme.  

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The development of efficient purification strategies of recombinant active protein derived from inclusion bodies requires the knowledge of the effect of environmental variables, such as redox potential (RP) and dissolved oxygen tension (DOT), in order to control the protein folding process. However, that information is scarce and only few in vitro studies of the impact of such variables have been reported under constant controlled conditions. In this work, the effect of controlled RP and DOT on the refolding of E. coli alkaline phosphatase (AP) and chicken lysozyme (CL) enzymes were studied. Disulphide bonds of both enzymes were reduced in an instrumented vessel using 2-mercaptoethanol and nitrogen. In the latter case, guanidine hydrochloride was also used to denature the protein. Such conditions caused protein conformational changes, as determined by the intrinsic fluorescence spectra that correlated with a decrease on the activity in both cases. Reduced enzymes were then oxidized, under different constant and predetermined RP or DOT, by manipulating the gas composition in the vessel. Folding kinetics were followed as the recovery of enzyme activity. Results showed that the percentage of recovery and rate of increase of enzymatic activity directly depended on the RP and DOT. A higher folding efficiency was found under controlled DOT compared to controlled RP conditions. These results are useful for establishing protein folding strategies to improve the recovery of active protein from inclusion bodies. PMID:23608498

Meneses-Acosta, Angélica; Vizcaíno-Meza, Luis Rodolfo; Ayala-Castro, Hector G; Contreras, Martha A; Ortega-López, Jaime; Ramírez, Octavio T

2013-05-10

 
 
 
 
321

Chemical modification studies on alkaline phosphatase from pearl oyster (Pinctada fucata): a substrate reaction course analysis and involvement of essential arginine and lysine residues at the active site.  

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Alkaline phosphatases (ALP, EC 3.1.3.1) are ubiquitous enzymes found in most species. ALP from a pearl oyster, Pinctada fucata (PALP), is presumably involved in nacreous biomineralization processes. Here, chemical modification was used to investigate the involvement of basic residues in the catalytic activity of PALP. The Tsou's plot analysis indicated that the inactivation of PALP by 2,4,6-trinitrobenzenesulfonic acid (TNBS) and phenylglyoxal (PG) is dependent upon modification of one essential lysine and one essential arginine residue, respectively. Substrate reaction course analysis showed that the TNBS and PG inactivation of PALP followed pseudo-first-order kinetics and the second-order inactivation constants for the enzyme with or without substrate binding were determined. It was found that binding substrate slowed the PG inactivation whereas had little effect on TNBS inactivation. Protection experiments showed that substrates and competitive inhibitors provided significant protection against PG inactivation, and the modified enzyme lost its ability to bind the specific affinity column. However, the TNBS-induced inactivation could not be prevented in presence of substrates or competitive inhibitors, and the modified enzyme retained the ability to bind the affinity column. In a conclusion, an arginine residue involved in substrate binding and a lysine residue involved in catalysis were present at the active site of PALP. This study will facilitate to illustrate the role ALP plays in pearl formation and the mechanism involved. PMID:15833276

Chen, Hong-Tao; Xie, Li-Ping; Yu, Zhen-Yan; Xu, Guang-Rui; Zhang, Rong-Qing

2005-07-01

322

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

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

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

2014-01-01

323

Surface density as a significant parameter for the enzymatic activity of two forms of alkaline phosphatase immobilized on phospholipid Langmuir-Blodgett films.  

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Rat osseous plate alkaline phosphatase, a glycosylphosphatidylinositol (GPI)-anchored phosphomonohydrolase, was immobilized on Langmuir-Blodgett (LB) films. Enzyme solubilization either with polyoxyethylene-9-lauryl ether or with a glycosylphosphatidylinositol-specific phospholipase C resulted in a GPI-anchor-containing and a GPI-anchor-depleted form, respectively. Both forms were adsorbed on dimyristoylphosphatidic acid LB films and restricted to the outermost layer. The surface density and enzyme activity were determined using a quartz crystal microbalance and p-nitrophenylphosphatase activity, respectively. The detergent-solubilized form was co-spread with dimyristoylphosphatidic acid on the air/water interface and transferred to solid supports, providing an enzyme maximum surface density of 530 ng/cm2. Maximal phosphohydrolytic activity, corresponding to 43% of that observed in homogeneous medium, was obtained at a surface density of 179 ng/cm2. The phospholipase C-solubilized form was adsorbed directly from solution, reaching a maximum surface density of 1541 ng/cm2, although the phosphomonohydrolase activity was 10 times lower than that obtained for the anchor-containing form. The combined analysis of surface density and enzymatic activity suggests that the alignment of the protein molecules on the LB lipid films induced by the glycosylphosphatidylinositol anchor facilitates the access of the substrate to the active site. This access is hampered by increasing enzyme surface densities and depends on a specific orientation of the adsorbed enzyme. PMID:15158389

Caseli, Luciano; Furriel, Rosa Prazeres Melo; de Andrade, José Fernando; Leone, Francisco Assis; Zaniquelli, Maria Elisabete Darbello

2004-07-01

324

Expanding the spectrum of phenotypes associated with germline PIGA mutations: a child with developmental delay, accelerated linear growth, facial dysmorphisms, elevated alkaline phosphatase, and progressive CNS abnormalities.  

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Phosphatidyl inositol glycan (PIG) enzyme subclasses are involved in distinct steps of glycosyl phosphatidyl inositol anchor protein biosynthesis. Glycolsyl phosphatidyl inositol-anchored proteins have heterogeneous functions; they can function as enzymes, adhesion molecules, complement regulators and co-receptors in signal transduction pathways. Germline mutations in genes encoding different members of the PIG family result in diverse conditions with (severe) developmental delay, (neonatal) seizures, hypotonia, CNS abnormalities, growth abnormalities, and congenital abnormalities as hallmark features. The variability of clinical features resembles the typical diversity of other glycosylation pathway deficiencies such as the congenital disorders of glycosylation. Here, we report the first germline missense mutation in the PIGA gene associated with accelerated linear growth, obesity, central hypotonia, severe refractory epilepsy, cardiac anomalies, mild facial dysmorphic features, mildly elevated alkaline phosphatase levels, and CNS anomalies consisting of progressive cerebral atrophy, insufficient myelinization, and cortical MRI signal abnormalities. X-exome sequencing in the proband identified a c.278C>T (p.Pro93Leu) mutation in the PIGA gene. The mother and maternal grandmother were unaffected carriers and the mother showed 100% skewing of the X-chromosome harboring the mutation. These results together with the clinical similarity of the patient reported here and the previously reported patients with a germline nonsense mutation in PIGA support the determination that this mutation caused the phenotype in this family. PMID:24259184

van der Crabben, Saskia N; Harakalova, Magdalena; Brilstra, Eva H; van Berkestijn, Frédérique M C; Hofstede, Floris C; van Vught, Adrianus J; Cuppen, Edwin; Kloosterman, Wigard; Ploos van Amstel, Hans Kristian; van Haaften, Gijs; van Haelst, Mieke M

2014-01-01

325

Highly sensitive solid-phase immunoenzymometric assay for placental and placental-like alkaline phosphatases with a monoclonal antibody and monodisperse polymer particles.  

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We have used a mouse monoclonal antibody (H7) to placental alkaline phosphatase (PLAP, EC 3.1.3.1) in developing an immunoenzymometric assay for PLAP and PLAP-like enzymes. The antibody is bound to sheep anti-mouse IgG (Ab2) covalently coupled to tosylated shell-and-core light (1.07 g/cm3) monodisperse polymer particles. Adding the H7-Ab2-polymer particle suspension to a PLAP-containing sample gives maximal binding of the antigen within 10 min. PLAP and PLAP-like enzymes remain active and bound to the solid-phase throughout all assay manipulations, and can thus be saved for future testing. In testing the enzymes for inhibition by L-Phe, L-Phe-Gly-Gly, L-Leu, and L-homoarginine, the effect of all the inhibitors is fully reversible. The assay is highly versatile, and its sensitivity (routinely 0.05 micrograms/L) can be increased 1000-fold by adjusting the sample volume and incubation time (sample volume is irrelevant between 50 microL and 5 mL). We have measured the basal activities of PLAP in men and women and, by using enzyme inhibitors, have characterized it as corresponding to the PLAP-like phenotypes described in normal human testis. PMID:3880682

Millán, J L; Nustad, K; Nørgaard-Pedersen, B

1985-01-01

326

One-Step Detection of Aflatoxin-B(1) Using scFv-Alkaline Phosphatase-Fusion Selected from Human Phage Display Antibody Library  

DEFF Research Database (Denmark)

A unique human phage display library was used to successfully generate a scFv to the highly carcinogenic toxin aflatoxin B1. Such an antibody has major potential applications in therapy and diagnostics. To further exploit its analytical capacity, the scFv was genetically fused to alkaline phosphatase, thereby generating a novel and highly sensitive self-indicating reagent. The performance of this reagent was further characterized, demonstrating its efficacy. The sensitivity of scFv-AP fusion was three-fold better than that of the scFv form. The ability of this human library to generate antibodies to a small hapten was clearly demonstrated and this is linked to its intrinsic diversity, which exceeds many existing conventional human libraries. Our results indicate that demography may influence the diversity of the repertoire of the library in terms of its capacity to generate antibodies to specific targets. Equally, the approach demonstrated should also be applicable for other haptens and larger antigens.

Rangnoi, Kuntalee; Jaruseranee, Nanthnit

2011-01-01

327

Retinoic acid and tumour necrosis factor-alpha act in concert to control the level of alkaline phosphatase mRNA.  

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Retinoic acid has a specific role in cellular differentiation and is believed to act by regulating the transcription of specific genes. In the present work, evidence is provided to show that alkaline phosphatase (ALP) gene expression is mediated by retinoic acid in a model clonal cell line (UMR 201) derived from rat neonatal calvaria. These cells have the characteristics of relatively undifferentiated mesenchymal cells with a very low basal ALP activity which is dramatically increased by retinoic acid. Messenger RNA for ALP was clearly demonstrated when the cells were treated with 1 microM retinoic acid for 24 h. Recombinant human tumour necrosis factor-alpha (recombinant TNF-alpha) interacted with retinoic acid to potentiate the rise in ALP activity, although recombinant TNF-alpha alone had no effect. The potentiation of retinoic acid-induced ALP activity was correlated with an increased amount of mRNA for ALP with the combined treatment. By observing the rate of decay of mRNA for actin and ALP, we were able to demonstrate that the interaction between retinoic acid and recombinant TNF-alpha modulated the steady state of ALP mRNA. The mode of action of recombinant TNF-alpha may serve as a model for other paracrine regulators of cell function. PMID:2742744

Ng, K W; Hudson, P J; Power, B E; Manji, S S; Gummer, P R; Martin, T J

1989-07-01

328

Functional expression in insect cells of glycosylphosphatidylinositol-linked alkaline phosphatase from Aedes aegypti larval midgut: a Bacillus thuringiensis Cry4Ba toxin receptor.  

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Bacillus thuringiensis produces insecticidal crystal (Cry) proteins which bind to cell surface receptors on the brush border membrane of susceptible midgut larvae. The toxin-receptor interaction generates pores in midgut epithelial cells resulting in cell lysis. Here, a cDNA encoding membrane-bound alkaline phosphatase from Aedes aegypti (Aa-mALP) midgut larvae, based on the sequence identity hit to Bombyx mori membrane-bound ALP, was amplified by RT-PCR and transiently expressed in Spodoptera frugiperda (Sf9) insect cells as a 58-kDa membrane-bound protein via the baculovirus expression system and confirmed by digestion with phosphatidylinositol-specific phospholipase C and LC-MS/MS analysis. Immunolocalization results showed that Cry4Ba is able to bind to only Sf9 cells-expressing Aa-mALP. Moreover, these cells were shown to undergo cell lysis in the presence of 100 ?g/ml trypsin-treated toxin. Finally, trypan blue exclusion assay also demonstrated an increase in cell death in recombinant cells treated with Cry4Ba. Overall results indicated that Aa-mALP protein was responsible for mediating Cry4Ba toxicity against Sf9 cells, suggesting its role as a receptor for Cry4Ba toxin in A. aegypti mosquito larvae. PMID:21146607

Dechklar, Manasave; Tiewsiri, Kasorn; Angsuthanasombat, Chanan; Pootanakit, Kusol

2011-03-01

329

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

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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.

Benjamin Nnamdi Okolonkwo

2013-01-01

330

High frequencies of elevated alkaline phosphatase activity and rickets exist in extremely low birth weight infants despite current nutritional support  

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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

Parker Bruce R

2009-07-01

331

Short-term moderate hypothermia stimulates alkaline phosphatase activity and osteocalcin expression in osteoblasts by upregulating Runx2 and osterix in vitro.  

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Exposure of Normal Human Osteoblast cells (NHOst) to a period of hypothermia may interrupt their cellular functions, lead to changes in bone matrix and disrupt the balance between bone formation and resorption, resulting in bone loss or delayed fracture healing. To investigate this possibility, we exposed NHOst cells to moderate (35 °C) and severe (27 °C) hypothermia for 1, 12, 24 and 72 h. The effects of hypothermia with respect to cell cytoskeleton organization, metabolic activity and the expression of cold shock chaperone proteins, osteoblast transcription factors and functional markers, were examined. Our findings showed that prolonged moderate hypothermia retained the polymerization of the cytoskeletal components. NHOst cell metabolism was affected differently according to hypothermia severity. The osteoblast transcription factors Runx2 and osterix were necessary for the transcription and translation of bone matrix proteins, where alkaline phosphatase (Alp) activity and osteocalcin (OCN) bone protein were over expressed under hypothermic conditions. Consequently, bone mineralization was stimulated after exposure to moderate hypothermia for 1 week, indicating bone function was not impaired. The cold shock chaperone protein Rbm3 was significantly upregulated (p<0.001) during the cellular stress adaption under hypothermic conditions. We suggest that Rbm3 has a dual function: one as a chaperone protein that stabilizes mRNA transcripts and a second one in enhancing the transcription of Alp and Ocn genes. Our studies demonstrated that hypothermia permitted the in vitro maturation of NHOst cells probably through an osterix-dependent pathway. For that reason, we suggest that moderate hypothermia can be clinically applied to counteract heat production at the fracture site that delays fracture healing. PMID:24928274

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

2014-08-01

332

Genetic toggling of alkaline phosphatase folding reveals signal peptides for all major modes of transport across the inner membrane of bacteria.  

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Prediction of export pathway specificity in prokaryotes is a challenging endeavor due to the similar overall architecture of N-terminal signal peptides for the Sec-, SRP- (signal recognition particle), and Tat (twin arginine translocation)-dependent pathways. Thus, we sought to create a facile experimental strategy for unbiased discovery of pathway specificity conferred by N-terminal signals. Using a limited collection of Escherichia coli strains that allow protein oxidation in the cytoplasm or, conversely, disable protein oxidation in the periplasm, we were able to discriminate the specific mode of export for PhoA (alkaline phosphatase) fusions to signal peptides for all of the major modes of transport across the inner membrane (Sec, SRP, or Tat). Based on these findings, we developed a mini-Tn5 phoA approach to isolate pathway-specific export signals from libraries of random fusions between exported proteins and the phoA gene. Interestingly, we observed that reduced PhoA was exported in a Tat-independent manner when targeted for Tat export in the absence of the essential translocon component TatC. This suggests that initial docking to TatC serves as a key specificity determinant for Tat-specific routing of PhoA, and in its absence, substrates can be rerouted to the Sec pathway, provided they remain compatible with the Sec export mechanism. Finally, the utility of our approach was demonstrated by experimental verification that four secreted proteins from Mycobacterium tuberculosis carrying putative Tat signals are bona fide Tat substrates and thus represent potential Tat-dependent virulence factors in this important human pathogen. PMID:18819916

Marrichi, Matthew; Camacho, Luis; Russell, David G; DeLisa, Matthew P

2008-12-12

333

A signal "on" photoelectrochemical biosensor for assay of protein kinase activity and its inhibitor based on graphite-like carbon nitride, Phos-tag and alkaline phosphatase.  

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A highly sensitive and selective photoelectrochemical (PEC) biosensor is fabricated for the detection of protein kinase activity based on visible-light active graphite-like carbon nitride (g-C3N4) and the specific recognition utility of Phos-tag for protein kinase A (PKA)-induced phosphopeptides. For assembling the substrate peptides, g-C3N4 and gold nanoparticles (g-C3N4-AuNPs) complex is synthesized and characterized. When the immobilized peptides on g-C3N4-AuNPs modified ITO electrode are phosphorylated under PKA catalysis, they can be specifically identified and binded with biotin functionalized Phos-tag (Phos-tag-biotin) in the presence of Zn(2+). Then, through the specific interaction between biotin and avidin, avidin functionalized alkaline phosphatase (avidin-ALP) is further assembled to catalyze its substrate of l-ascorbic acid-2-phosphate trisodium salt (AAP) to produce electron donor of ascorbic acid (AA), resulting an increased photocurrent compared with the absence of phosphorylation event. Based on the specific identification effect of Phos-tag, the fabricated biosensor presents excellent selectivity for capturing the phosphorylated serine residues in the substrate peptides. With the good photoactivity of g-C3N4 and ALP-catalyzed signal amplification, the fabricated biosensor presents high sensitivity and low detection limit (0.015unit/mL, S/N=3) for PKA. The applicability of this PEC biosensor is further testified by the evaluation of PKA inhibition by HA-1077 with the IC50 value of 1.18?M. This new strategy is also successfully applied to detect the change of PKA activity in cancer cell lysate with and without drug stimulation. Therefore, the developed PEC method has great potential in screening of kinase inhibitors and highly sensitive detection of kinase activity. PMID:25286353

Yin, Huanshun; Sun, Bing; Dong, Linfeng; Li, Bingchen; Zhou, Yunlei; Ai, Shiyun

2015-02-15

334

Soluble ecto-5'-nucleotidase (5'-NT), alkaline phosphatase, and adenosine deaminase (ADA1) activities in neonatal blood favor elevated extracellular adenosine.  

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Extracellular adenosine, a key regulator of physiology and immune cell function that is found at elevated levels in neonatal blood, is generated by phosphohydrolysis of adenine nucleotides released from cells and catabolized by deamination to inosine. Generation of adenosine monophosphate (AMP) in blood is driven by cell-associated enzymes, whereas conversion of AMP to adenosine is largely mediated by soluble enzymes. The identities of the enzymes responsible for these activities in whole blood of neonates have been defined in this study and contrasted to adult blood. We demonstrate that soluble 5'-nucleotidase (5'-NT) and alkaline phosphatase (AP) mediate conversion of AMP to adenosine, whereas soluble adenosine deaminase (ADA) catabolizes adenosine to inosine. Newborn blood plasma demonstrates substantially higher adenosine-generating 5'-NT and AP activity and lower adenosine-metabolizing ADA activity than adult plasma. In addition to a role in soluble purine metabolism, abundant AP expressed on the surface of circulating neonatal neutrophils is the dominant AMPase on these cells. Plasma samples from infant observational cohorts reveal a relative plasma ADA deficiency at birth, followed by a gradual maturation of plasma ADA through infancy. The robust adenosine-generating capacity of neonates appears functionally relevant because supplementation with AMP inhibited whereas selective pharmacologic inhibition of 5'-NT enhanced Toll-like receptor-mediated TNF-? production in neonatal whole blood. Overall, we have characterized previously unrecognized age-dependent expression patterns of plasma purine-metabolizing enzymes that result in elevated plasma concentrations of anti-inflammatory adenosine in newborns. Targeted manipulation of purine-metabolizing enzymes may benefit this vulnerable population. PMID:23897810

Pettengill, Matthew; Robson, Simon; Tresenriter, Megan; Millán, José Luis; Usheva, Anny; Bingham, Taiese; Belderbos, Mirjam; Bergelson, Ilana; Burl, Sarah; Kampmann, Beate; Gelinas, Laura; Kollmann, Tobias; Bont, Louis; Levy, Ofer

2013-09-20

335

Evidence of Associations Between Feto-Maternal Vitamin D Status, Cord Parathyroid Hormone and Bone-Specific Alkaline Phosphatase, and Newborn Whole Body Bone Mineral Content  

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Full Text Available In spite of a high prevalence of vitamin D inadequacy in pregnant women and neonates, relationships among vitamin D status (25(OHD, parathyroid hormone (PTH, bone specific alkaline phosphatase (BALP, and whole body bone mineral content (WBBMC in the newborn are poorly characterized. The purpose of the present study was to investigate the relationships between maternal and cord 25(OHD, PTH, BALP, and WBBMC in newborns in a multiethnic population in Oakland, California and to evaluate the predictive value of the biochemical indices as indicators of WBBMC. Maternal and cord blood were collected from 80 mother-infant pairs and infant WBBMC was measured by dual energy X-ray absorptiometry 8–21 days post-birth. Cord PTH and BALP were each inversely correlated with infant WBBMC (r = ?0.28, p = 0.01 and r = ?0.26, p = 0.02 and with cord 25(OHD (r = ?0.24, p = 0.03 and r = ?0.34, p = 0.002, while cord 25(OHD and unadjusted or weight-adjusted WBBMC were not significantly correlated with one other. In multivariate regression modeling, infant WBBMC was most strongly predicted by infant weight ( p < 0.0001, while either PTH or BALP contributed modestly but significantly to the model (p = 0.006 and p = 0.03 respectively. Cord 25(OHD was not a significant predictor of infant WBBMC. This study provides evidence of associations between feto-maternal 25(OHD, cord PTH and BALP, and early infant WBBMC, though neither feto-maternal 25(OHD nor the measured biochemical indices were suitable indicators of WBBMC.

Marta D. Van Loan

2012-02-01

336

Chicken single-chain antibody fused to alkaline phosphatase detects Aspergillus pathogens and their presence in natural samples by direct sandwich enzyme-linked immunosorbent assay.  

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A sensitive and specific analytical method to detect ubiquitous aflatoxigenic Aspergillus pathogens is essential for monitoring and controlling aflatoxins. Four highly reactive chicken single-chain variable fragments (scFvs) against soluble cell wall proteins (SCWPs) from Aspergillus flavus were isolated by phage display. The scFv antibody AfSA4 displayed the highest activity toward both A. flavus and A. parasiticus and specifically recognized a surface target of their cell walls as revealed by immunofluorescence localization. Molecular modeling revealed a unique compact motif on the antibody surface mainly involving L-CDR2 and H-CDR3. As measured by surface plasmon resonance, AfSA4 fused to alkaline phosphatase had a higher binding capability and 6-fold higher affinity compared with AfSA4 alone. Immunoblot analyses showed that the fusion had good binding capacity to SCWP components from the two fungal species. Direct sandwich enzyme-linked immunosorbent assays with mouse antiaspergillus monoclonal antibody mAb2A8 generated in parallel as a capture antibody revealed that the detection limit of the two fungi was as low as 10(-3) ?g/mL, 1000-fold more sensitive than that reported previously (1 ?g/mL). The fusion protein was able to detect fungal concentrations below 1 ?g/g of maize and peanut grains in both artificially and naturally contaminated samples, with at least 10-fold more sensitivity than that reported (10 ?g/g) thus far. Thus, the fusion can be applied in rapid, simple, and specific diagnosis of Aspergillus contamination in field and stored food/feed commodities. PMID:24128348

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

2013-11-19

337

Cells cultured from the growing tip of red deer antler express alkaline phosphatase and proliferate in response to insulin-like growth factor-I.  

Science.gov (United States)

Deer antler growth provides a unique natural model of rapid and complete bone regeneration. In this study, the distal antler tips of male red deer (Cervus elaphus) were collected post-mortem during the annual growth period (April-August), and an in vitro system established for the culture of cells from three regions; the inner layer of the perichondrium, the reserve mesenchyme and the cartilage zone. Alkaline phosphatase (ALP) expression by cultured cells, as demonstrated by enzyme histochemistry and biochemical assay, reflected the stage of cellular differentiation. ALP activity was highest in cells cultured from the hypertrophic cartilage region (3.6 +/- 0.2 mumol/micrograms cell protein/minute), and lowest in undifferentiated mesenchymal cells (0.3 +/- 0.01 mumol/microgram cell protein/minute). ALP expression was lost with passage in culture. Levels of ALP activity in cultured cells correlated with the pattern and extent of enzyme expression in tissue sections as demonstrated by histochemical staining. Insulin-like growth factor (IGF)-I (10(-9)M-10(-7)M) was found to be mitogenic for cultured cells from all three zones as shown by increased incorporation of [3H]thymidine into DNA. These results demonstrate that cells from three different regions of the antler tip can be maintained in culture, and that antler cells share certain phenotypic characteristics of growth plate chondrocytes. These data provide further evidence of a role for IGF-1 in the regulation of antler growth. Antler regrowth is a potentially useful model for the study of the factors that regulate bone formation. PMID:7829985

Price, J S; Oyajobi, B O; Oreffo, R O; Russell, R G

1994-11-01

338

Effects of dietary vitamin E on mucosal maltase and alkaline phosphatase enzyme activities and on the amount of mucosal malonyldialdehyde in broiler chickens  

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Full Text Available The effects of dietary vitamin E levels on mucosal maltase and alkaline phosphatase (ALP enzyme activities and on the amount of mucosal malonyldialdehyde (MDA in broiler chickens were studied in the present study. One hundred and eighty of day old male broiler chicks (Ross 308 strain were randomly assigned into five groups, each with three replicates and 12 chicks in each replicate. Chickens in group A were fed corn-soy- based diet, while those in groups B, C, D and E were fed the same diet with 20, 60, 180, and 540 mg kg-1 vitamin E supplement (d-alpha tocopherol, respectively. Six birds were randomly chosen from each group, and were euthanized on days 10, 21, 32, and 42 of age. One segment of small intestine outset was homo-genized and mucosal ALP and maltase activity were measured. Moreover, mucosal lipid peroxidate amount was measured to reveal the impact of vitamin E on oxidative stress. Maltase activity was increased with the increase of vitamin E up to 60 mg kg-1 of diet while with further levels, it was decreased. Addition of 60 mg kg-1 of vitamin E to the diet significantly increased ALP enzyme activity (p ? 0.001. Addition of 540 mg kg-1 of vitamin E supplement to the diet led to the minimum amount of MDA at 32 days of age. It may be concluded that supplementation of broiler's diet with 60 mg kg-1of vitamin E can increase mucosal maltase and ALP enzyme activity.

Seyed Hamid Farrokhifar

2014-12-01

339

Cadherin, alkaline phosphatase, and aminopeptidase N as receptors of Cry11Ba toxin from Bacillus thuringiensis subsp. jegathesan in Aedes aegypti.  

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Cry11Ba is one of the most toxic proteins to mosquito larvae produced by Bacillus thuringiensis. It binds Aedes aegypti brush border membrane vesicles (BBMV) with high affinity, showing an apparent dissociation constant (K(d)) of 8.2 nM. We previously reported that an anticadherin antibody competes with Cry11Ba binding to BBMV, suggesting a possible role of cadherin as a toxin receptor. Here we provide evidence of specific cadherin repeat regions involved in this interaction. Using cadherin fragments as competitors, a C-terminal fragment which contains cadherin repeat 7 (CR7) to CR11 competed with Cry11Ba binding to BBMV. This binding was also efficiently competed by the CR9, CR10, and CR11 peptide fragments. Moreover, we show CR11 to be an important region of interaction with Cry11Ba toxin. An alkaline phosphatase (AaeALP1) and an aminopeptidase-N (AaeAPN1) also competed with Cry11Ba binding to Ae. aegypti BBMV. Finally, we found that Cry11Ba and Cry4Ba share binding sites. Synthetic peptides corresponding to loops ?8, ?2-?3 (loop 1), ?8-?9, and ?10-?11 (loop 3) of Cry4Ba compete with Cry11Ba binding to BBMV, suggesting Cry11Ba and Cry4Ba have common sites involved in binding Ae. aegypti BBMV. The data suggest that three different Ae. aegypti midgut proteins, i.e., cadherin, AaeALP1, and AaeAPN1, are involved in Cry11Ba binding to Ae. aegypti midgut brush border membranes. PMID:21037295

Likitvivatanavong, Supaporn; Chen, Jianwu; Bravo, Alejandra; Soberón, Mario; Gill, Sarjeet S

2011-01-01

340

Effect of crowding by dextrans and Ficolls on the rate of alkaline phosphatase-catalyzed hydrolysis: a size-dependent investigation.  

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The cell cytosol is crowded with macromolecules such as proteins, nucleic acids, and membranes. The consequences of such crowding remain unclear. How is the rate of a typical enzymatic reaction, involving a freely diffusing enzyme and substrate, affected by the presence of macromolecules of different sizes, shapes, and concentrations? Here, we mimic the cytosolic crowding in vitro, using dextrans and Ficolls, for the first time in a variety of sizes ranging from 15 to 500 kDa, in a concentration range 0-30% w/w. Alkaline phosphatase-catalyzed hydrolysis of p-nitrophenyl phosphate (PNPP) was chosen as the model reaction. A pronounced decrease in the rate with increase in fractional volume occupancy of dextran is observed for larger dextrans (200 and 500 kDa) in contrast to smaller dextrans (15-70 kDa). Our results indicate that, at 20% w/w, smaller dextrans (15-70 kDa) reduce the initial rate moderately (1.4- to 2.4-fold slowing), while larger dextrans (>200 kDa) slow the reaction considerably (>5-fold). Ficolls (70 and 400 kDa) slow the reaction moderately (1.3- to 2.3-fold). The influence of smaller dextrans was accounted by a combination of increase in viscosity as sensed by PNPP and a minor offsetting increase in enzyme activity due to crowding. Larger dextrans apparently reduce the frequency of enzyme substrate encounter. The reduced influence of Ficolls is attributed to their compact and quasispherical shape, much unlike the dextrans. PMID:16868935

Homchaudhuri, L; Sarma, Navanita; Swaminathan, Rajaram

2006-12-01

 
 
 
 
341

[In vitro study of the effect of bisphosphonates on mineralization induced by a composite material: poly 2(hydroxyethyl) methacrylate coupled with alkaline phosphatase].  

Science.gov (United States)

We have immobilized the mineralizing agent alkaline phosphatase (AlkP) in a hydrophilic polymer (poly 2(hydroxyéthyl) methacrylate) (pHEMA) in a copolymerization technique. Histochemical study on polymer sections revealed that AlkP has retained its biological activity. The image analysis of sections using a tessellation method showed a lognormal distribution of the area of the tiles surrounding AlkP particles thus confirming a homogeneous distribution of the enzyme in the polymer. Pellets of pHEMA-AlkP were incubated with a synthetic body fluid containing organic phosphates (beta-glycerophosphate). Mineral deposits with a rounded shape (calcospherites) were obtained in about 17 days. We have investigated the effects of three bisphosphonates (etidronate, alendronate and tiludronate) on this system. Bisphosphonates at a concentration of 10(-2) M totally inhibited AlkP in solution at a concentration of 10(-4) mg/ml. Inhibition has been reported being due to the chelation of a metal cofactor (Zn2+). Etidronate and alendronate appeared to inhibit the calcospherite deposition onto the pHEMA-AlkP material in a similar way. Both bisphosphonates possess three sites for mineral complexion. On the other hand, tiludronate having only two sites was associated with a reduced inhibitory effect on mineralization. When used in microgravity conditions, mineralization was impaired with etidronate and larger crystals were obtained with tiludronate. However, these effects were obtained in non-physiological conditions (a 20 degrees C temperature was used during the STS80 flight of the space shuttle). The pHEMA-AlkP material provides an interesting method to study the effects of pharmacological compounds and environmental factors on the bone and cartilage mineralization process. PMID:10923337

Filmon, R; Baslé, M F; Barbier, A; Chappard, D

2000-03-01

342

Poly(2-hydroxy ethyl methacrylate)-alkaline phosphatase: a composite biomaterial allowing in vitro studies of bisphosphonates on the mineralization process.  

Science.gov (United States)

We have immobilized the mineralizing agent alkaline phosphatase (AlkP) in a hydrophilic polymer: poly(2-hydroxy ethyl methacrylate) - (pHEMA) - in a copolymerization technique. Histochemical study on polymer sections revealed that AlkP has retained its enzymic activity. The image analysis of sections using a tessellation method showed a lognormal distribution of the area of the tiles surrounding AlkP particles, thus confirming a homogeneous distribution of the enzyme in the polymer. Pellets of pHEMA-AlkP were incubated with a synthetic body fluid containing organic phosphates (beta-glycerophosphate). Mineral deposits with a rounded shape (calcospherites) were obtained in about 17 days. We have investigated the effects of three bisphosphonic pharmacological compounds (etidronate, alendronate and tiludronate) on this system which mimics the mineralization process of cartilage and woven bone. Bisphosphonates at a concentration of 10(-2) M totally inhibited AlkP in solution at a concentration of 10(-4) mg/ml. Inhibition has been reported being due to the chelation of a metal cofactor (Zn2+). Etidronate and alendronate appeared to similarly inhibit the calcospherite deposition onto the pHEMA-AlkP material. Both bisphosphonates possess three sites for the mineral complexion by Ca chemisorbtion. On the other hand, tiludronate having only two sites, was associated with a reduced inhibitory effect on mineralization but larger crystals were obtained. The pHEMA-AlkP material contains an immobilized enzyme in a hydrogel and mimics the physiological conditions of matrix vesicles entrapped within the cartilage (or bone) matrix. It provides an interesting method to study the effects of pharmacological compounds on the mineralization process in bone and cartilage in a non cellular and protein-free model. PMID:11211096

Filmon, R; Baslé, M F; Barbier, A; Chappard, D

2000-01-01

343

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)

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 ?l 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 switch technique, lectin science and SSRTP.

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

344

Adsorption kinetics and dilatational rheological studies for the soluble and anchored forms of alkaline phosphatase at the air/water interface  

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Full Text Available SciELO Brazil | Language: English Abstract in portuguese Este trabalho apresenta aspectos de equilíbrio e dinâmicos da adsorção na interface ar/líquido de duas formas de fosfatase alcalina de placa óssea de ratos: DSAP, solubilizada com tensoativo não-iônico (C12E9), contendo uma âncora de glicosilfosfatidilinositol (GPI), e PLSAP, com a porção hidrofóbic [...] a da âncora clivada por fosfolipase-C. A tensão superficial dinâmica, gamadyn, e o módulo de elasticidade superficial dilatacional, épsilon, foram determinados para soluções de PLSAP, DSAP e C12E9 pelo método de oscilação harmônica e análise do formato da gota eixo-simétrica. Cinéticas de adsorção revelaram que DSAP adsorve trinta vezes mais rapidamente que PLSAP, apresentando um mínimo, e, para PLSAP, a tensão superficial cai continuamente. Para o sistema DSAP/C12E9, épsilon atinge um máximo na concentração crítica de agregação (CAC), mas para PLSAP, épsilon diminui continuamente com a concentração. Soluções de C12E9 apresentam épsilonmais elevados, decrescentes com a concentração. Um modelo, baseado na influência da âncora GPI, é proposto para explicar os resultados obtidos. Abstract in english This work presents equilibrium and dynamic aspects for the adsorption at the air/liquid interface of two rat osseous plate alkaline phosphatase forms: DSAP, solubilized by a surfactant, C12E9, and containing a glycosylphosphatidylinositol (GPI) anchor; and PLSAP, resulting from phospholipase-C cleav [...] age of the hydrophobic portion of the GPI anchor. Dynamic surface tension, gammadyn, and surface elasticity modulus, epsilon, were determined for PLSAP, DSAP and pure C12E9 solutions using harmonic oscillation and axisymmetric drop shape analysis Adsorption kinetics studies revealed that DSAP adsorbs thirty times faster than PLSAP, presenting a minimum in the curve. For DSAP/ C12E9 mixed system, e increases with concentration and a maximum appears at the critical aggregation concentration (CAC). For PLSAP, a continuous decreasing with concentration for gammadyn and epsilon was observed. For pure C12E9 solution, the elasticity modulus increases with concentration and epsilon values are higher when compared to the mixed system. A model based on the influence of the GPI anchor is proposed.

Luciano, Caseli; Douglas C., Masui; Rosa Prazeres M., Furriel; Francisco Assis, Leone; Maria Elisabete D., Zaniquelli.

2005-10-01

345

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)

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 the data of dc-CLEIA and HPLC-MS (R{sup 2} > 0.99), indicating that the assay was an efficient analytical method for monitoring food safety.

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

346

The dermatophyte Trichophyton rubrum secretes an EDTA-sensitive alkaline phosphatase on high-phosphate medium O dermatófito Trichophyton rubrum secreta uma fosfatase alcalina EDTA-sensível em meio contendo alta concentração de fosfato  

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In this communication, we show that the growth of isolate H6 of the dermatophyte Trichophyton rubrum on non-buffered medium and under saturating phosphate conditions is dependent on the initial growth pH, with an apparent optimum at pH 4.0. In addition, irrespective of the initial growth pH, the pH of the medium alteredduring cultivation reaching values that ranged from 8.3 to 8.9. Furthermore, this isolate synthesized and secreted almost the same levels of an alkaline phosphatase with an app...

Ferreira-nozawa, Monica S.; Nozawa, Se?rgio R.; Martinez-rossi, Nilce M.; Antonio Rossi

2003-01-01

347

Correlation of Serum Magnesium with Serum Parathormone in Regular Hemodialysis Patients  

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Full Text Available To consider the relationship of serum magnesium with the activity of the parathyroid gland in maintenance hemodialysis patients we designed a study to investigate the role of serum magnesium in regulating the parathyroid secretion. The study was conducted on patients undergoing maintenance hemodialysis treatment. Predialysis serum calcium, phosphorus, magnesium, alkaline phosphatase, intact serum PTH (iPTH, serum 25-hydroxy vitamin D (25-OH Vit D and plasma HCO3– were measured. The Urea Reduction Rate, duration and dosage of hemodialysis treatment were calculated also. In this study no significant correlation of serum magnesium with duration of hemodialysis treatment, alkaline phosphatase, plasma HCO3–, serum calcium and phosphorus patients were seen. In all patients a near significant inverse correlation of serum magnesium with iPTH (r = -0.30 , p = 0.079 was found, also a significant positive correlation of serum magnesium with serum 25-hydroxy vitamin D levels ( r = 0.40, p = 0.009 was seen. Earlier research concluded that some factors other than serum magnesium may be more important in the regulation of parahormone secretion in hemodialysis patients. A positive and strong association between serum magnesium with 25-hydroxy vitamin D level, needs to more attention to this aspect of hemodialysis patients.

Hamid Nasri

2006-01-01

348

Serum tartrate-resistant acid phosphatase 5b activity as a prognostic marker of survival in breast cancer with bone metastasis  

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Full Text Available Abstract Background Serum tartrate-resistant acid phosphatase 5b (TRACP 5b activity is a marker of osteoclast number and is elevated in breast cancer (BC patients with extensive bone metastasis, which might in turn reflect the tumour burden. We tested the hypothesis that baseline serum TRACP 5b activity and its interval change are potential prognostic markers of survival in BC patients with bone metastasis. Methods We analyzed the data from previous prospective studies. A total of 100 patients with newly diagnosed bone metastasis were included. Cox proportional regression model was used to evaluate the correlation between the overall survival time (OS and baseline serum TRACP 5b activity and its interval changes. The least significant change (LSC of TRACP 5b was calculated from data obtained from 15 patients with early BC. Results Estrogen receptor status (Hazard Ratio (HR = 0.397; p = 0.003 and visceral metastasis (HR = 0.492; p = 0.0045 were significantly correlated with OS. The OS was significantly shorter in those patients with higher baseline TRACP 5b activity based on a cut-off value to delineate the highest tertile (HR = 3.524; p p = 0.0015. Conclusions We found that TRACP 5b activity and its interval change after treatment bore a prognostic role in BC patients with bone metastasis and a high baseline serum TRACP 5b activity. Further prospective phase II study is necessary to confirm these results.

Liu Hsin-Yi

2010-04-01

349

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  

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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.

R. Serakides

2000-12-01

350

Estudio de la fosfatasa ácida y alcalina en suelos de la Región Pampeana Norte del área sojera argentina Study of acid and alkaline phosphatase in soils of the Pampean North Region from argentine soybean area  

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Full Text Available La transformación de los compuestos de fósforo orgánico (Po a fósforo inorgánico (Pi soluble, es denominada mineralización y es llevada a cabo por un grupo de enzimas conocidas como fosfatasas. En este trabajo, se estudió la actividad fosfatasa de cinco lotes de la región pampeana norte del área sojera argentina, mediante: la evaluación de la actividad fosfatasa del suelo, y el recuento de las comunidades bacterianas y fúngicas con esa actividad, y de esta manera se obtuvo información sobre el potencial de los mismos para movilizar el Po. Se determinó el número de bacterias aeróbicas heterotróficas cultivables (BAHC así como el de hongos cultivables (HC, además del número de productores de fosfatasas ácidas y alcalinas. El número de bacterias con actividad fosfatasa fue en promedio 6,85 10(5 UFC g-1 de suelo para la fosfatasa ácida; mientras que para la fosfatasa alcalina fue en promedio 5,80 10(5 UFC g-1 de suelo. En cambio, el valor medio de hongos con actividad fosfatasa ácida fue 1,78 10³ UFC g-1 de suelo y para la enzima alcalina 1,77 10³ UFC g-1 de suelo. No se encontraron diferencias estadísticamente significativas entre el número de bacterias y el de hongos con fosfatasa ácida o alcalina entre los distintos suelos. Por otro lado, el nivel de actividad de la fosfatasa alcalina osciló entre 5,72 y 15,5 mg p-nitrofenol kg-1 suelo h-1, mientras la ácida varió entre 27,4 y 105 mg p-nitrofenol kg-1 suelo h-1. Se encontraron diferencias significativas en la actividad enzimática entre los cinco suelos, siendo mayor la actividad ácida que la alcalina. Los resultados de este trabajo demostraron que los suelos estudiados presentan una actividad mineralizadora de fuentes de Po que coincide con otros trabajos de suelos cultivados y que el recuento de los organismos productores de fosfatasas complementa la información obtenida a partir de la determinación de la actividad fosfatasa del suelo. Mediante la utilización de ambos métodos, es posible estudiar la fosfatasa ácida y alcalina de un suelo y obtener información sobre el potencial del mismo para movilizar Po.Transformation of organic phosphorus (Po into soluble inorganic phosphorus (Pi is called mineralization and is carried out by phosphatase enzymes. The present research focuses on the study of the phosphatase activity of five soils from the soybean area of the Northern Pampean region, by evaluating the phosphatase activity in soil samples and the number of bacteria and fungi with that activity. Soil samples were collected and the total number and phosphatase activity of cultivated heterotrophic aerobic bacteria (CHAB and cultivated fungi (CF was assessed. No significant differences were observed in the numbers of CHAB and CH between the studied soils. The number of bacteria with acid phosphatase activity was 6.85 10(5 CFU g-1 soil, while alkaline activity was 5.80 10(5 CFU g-1 soil. In contrast, the number of fungi with acid phosphatase activity was 1.78 10³ CFU g-1 soil and with alkaline activity was 1.77 10³ CFU g-1 soil. No significant differences were observed in the number of bacteria and fungi with both enzymes. However, acid activity was higher than alkaline activity in soil samples. Alkaline phosphatase activity ranged from 5.72 to 15.5 mg p- nitrofenol kg-1 soil h-1 while acid activity varied from 27.4 to 10(5 mg p-nitrofenol kg-1 soil h-1. There were significant differences in phosphatase activity between the soybean soils. Our results show that the mineralization activities of Po sources are in agreement with other cultivated soils. On the other hand, the number of bacteria and fungi complements the information on soil phosphatase activity. Clearly, both methods allow the study of alkaline and acid phosphatase activity in soil and give information about the soil potential to mobilize Po.

Leticia Andrea Fernández

2008-07-01

351

Estudio de la fosfatasa ácida y alcalina en suelos de la Región Pampeana Norte del área sojera argentina / Study of acid and alkaline phosphatase in soils of the Pampean North Region from argentine soybean area  

Scientific Electronic Library Online (English)

Full Text Available SciELO Argentina | Language: Spanish Abstract in spanish La transformación de los compuestos de fósforo orgánico (Po) a fósforo inorgánico (Pi) soluble, es denominada mineralización y es llevada a cabo por un grupo de enzimas conocidas como fosfatasas. En este trabajo, se estudió la actividad fosfatasa de cinco lotes de la región pampeana norte del área s [...] ojera argentina, mediante: la evaluación de la actividad fosfatasa del suelo, y el recuento de las comunidades bacterianas y fúngicas con esa actividad, y de esta manera se obtuvo información sobre el potencial de los mismos para movilizar el Po. Se determinó el número de bacterias aeróbicas heterotróficas cultivables (BAHC) así como el de hongos cultivables (HC), además del número de productores de fosfatasas ácidas y alcalinas. El número de bacterias con actividad fosfatasa fue en promedio 6,85 10(5) UFC g-1 de suelo para la fosfatasa ácida; mientras que para la fosfatasa alcalina fue en promedio 5,80 10(5) UFC g-1 de suelo. En cambio, el valor medio de hongos con actividad fosfatasa ácida fue 1,78 10³ UFC g-1 de suelo y para la enzima alcalina 1,77 10³ UFC g-1 de suelo. No se encontraron diferencias estadísticamente significativas entre el número de bacterias y el de hongos con fosfatasa ácida o alcalina entre los distintos suelos. Por otro lado, el nivel de actividad de la fosfatasa alcalina osciló entre 5,72 y 15,5 mg p-nitrofenol kg-1 suelo h-1, mientras la ácida varió entre 27,4 y 105 mg p-nitrofenol kg-1 suelo h-1. Se encontraron diferencias significativas en la actividad enzimática entre los cinco suelos, siendo mayor la actividad ácida que la alcalina. Los resultados de este trabajo demostraron que los suelos estudiados presentan una actividad mineralizadora de fuentes de Po que coincide con otros trabajos de suelos cultivados y que el recuento de los organismos productores de fosfatasas complementa la información obtenida a partir de la determinación de la actividad fosfatasa del suelo. Mediante la utilización de ambos métodos, es posible estudiar la fosfatasa ácida y alcalina de un suelo y obtener información sobre el potencial del mismo para movilizar Po. Abstract in english Transformation of organic phosphorus (Po) into soluble inorganic phosphorus (Pi) is called mineralization and is carried out by phosphatase enzymes. The present research focuses on the study of the phosphatase activity of five soils from the soybean area of the Northern Pampean region, by evaluating [...] the phosphatase activity in soil samples and the number of bacteria and fungi with that activity. Soil samples were collected and the total number and phosphatase activity of cultivated heterotrophic aerobic bacteria (CHAB) and cultivated fungi (CF) was assessed. No significant differences were observed in the numbers of CHAB and CH between the studied soils. The number of bacteria with acid phosphatase activity was 6.85 10(5) CFU g-1 soil, while alkaline activity was 5.80 10(5) CFU g-1 soil. In contrast, the number of fungi with acid phosphatase activity was 1.78 10³ CFU g-1 soil and with alkaline activity was 1.77 10³ CFU g-1 soil. No significant differences were observed in the number of bacteria and fungi with both enzymes. However, acid activity was higher than alkaline activity in soil samples. Alkaline phosphatase activity ranged from 5.72 to 15.5 mg p- nitrofenol kg-1 soil h-1 while acid activity varied from 27.4 to 10(5) mg p-nitrofenol kg-1 soil h-1. There were significant differences in phosphatase activity between the soybean soils. Our results show that the mineralization activities of Po sources are in agreement with other cultivated soils. On the other hand, the number of bacteria and fungi complements the information on soil phosphatase activity. Clearly, both methods allow the study of alkaline and acid phosphatase activity in soil and give information about the soil potential to mobilize Po.

Leticia Andrea, Fernández; Marcelo Antonio, Sagardoy; Marisa Anahí, Gómez.

352

Immunocytochemical phenotyping of disseminated tumor cells in bone marrow by uPA receptor and CK18: investigation of sensitivity and specificity of an immunogold/alkaline phosphatase double staining protocol.  

Science.gov (United States)

Phenotyping of cytokeratin (CK)18-positive cells in bone marrow is gaining increasing importance for future prognostic screening of carcinoma patients. Urokinase-type plasminogen activator receptor (uPA-R) is one example of a potential aggressive marker for those cells. However, a valid and reliable double staining method is needed. Using monoclonal antibodies against uPA-R and CK18, we modified an immunogold/alkaline phosphatase double staining protocol. UPA-R/CK18-positive tumor cell controls exhibited black uPA-R staining in 15-80% of cases and red CK18 staining in almost 100% of tumor cells. Isotype- and cross-matched controls were completely negative. Bone marrow from healthy donors was always CK18-negative. Reproducibility of CK18-positive cell detection was estimated in a series of specimens from 61 gastric cancer patients comparatively stained with the single alkaline phosphatase-anti-alkaline phosphatase (APAAP) and our double staining method (10(6) bone marrow cells/patient). In four cases, double staining could not reproduce CK18-positive cells. In 34 cases it revealed fewer or equal numbers, and in 23 cases more CK18-positive cells than the APAAP method. Overall quantitative analysis of detected cell numbers (838 in APAAP, range 1-280 in 10(6); double staining 808, range 0-253) demonstrated relative reproducibility of APAAP results by double staining of 97%. Correlation of results between both methods was significant (p < 0.001, linear regression). Sensitivity of double staining tested in logarithmic tumor cell dilutions was one CK18-positive cell in 300,000. Specific uPA-R staining was seen on CK18-positive cells in bone marrow from 29 of 61 patients, and also on single surrounding bone marrow cells. To test the specificity of this staining, bone marrow cytospins from 10 patients without tumor disease were stained for uPA-R with the APAAP method. uPA-R expression was confirmed in all 10 cases, with a mean of 6.5% uPA-R-positive cells in 1000 bone marrow cells (SEM 1.2%). These results suggest that our double staining protocol is a sensitive, reproducible, and specific method for routine uPA-R phenotyping of disseminated CK18-positive cells in bone marrow of carcinoma patients. PMID:9016310

Allgayer, H; Heiss, M M; Riesenberg, R; Babic, R; Jauch, K W; Schildberg, F W

1997-02-01