Validation of the Calcineurin Phosphatase Assay

Koefoed‐Nielsen, Pernille; Karamperis, Nikolaos; Jørgensen, Kaj Anker

doi:10.1373/clinchem.2004.034066

Cited by 19 publications

(11 citation statements)

References 15 publications

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“…Using a method based on work done by Fruman et al [15] and with minor modifications, CaN activity was measured as the release of radiolabelled phosphate from a phosphorylated 19-amino acid peptide (Asp-Leu-Asp-Val-Pro-Ile-Pro-Gly-Arg-Phe-Asp-ArgArg-Val-Ser-Val-Ala-Ala-Glu). We recently published a detailed description and validation of the assay [16].…”

Section: Methodsmentioning

confidence: 99%

24‐h Monitoring of Calcineurin Phosphatase Activity in Healthy Subjects

Koefoed‐Nielsen¹,

Karamperis²,

Jørgensen³

2005

Scand J Immunol

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The calcineurin inhibitors cyclosporine and tacrolimus are the cornerstone immunosuppressants used in solid organ transplantation. Studies investigating calcineurin (CaN) activity in renal transplanted patients have been published, but basic properties of the enzyme activity in healthy subjects remain to be described. The aim of this study was to investigate whether CaN displays circadian variation or sex difference is present in healthy subjects. Twenty subjects had blood samples drawn every 4 h for a 24-h period. CaN activity was determined in whole blood as the release of 32 P from a phosphorylated peptide. Activity of the 32 P was quantitated by liquid scintillation and results converted to units CaN utilizing a calibration curve. We found no circadian variation in CaN activity and no difference between the two sexes. The clinical importance of these findings is that blood samples for calcineurin activity can be drawn without taking the exact time of day into consideration, but only considering the time of drug intake.

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Section: Methodsmentioning

confidence: 99%

24‐h Monitoring of Calcineurin Phosphatase Activity in Healthy Subjects

Koefoed‐Nielsen¹,

Karamperis²,

Jørgensen³

2005

Scand J Immunol

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“…Early mechanistic experiments that assessed Cn phosphatase activity involved the detection of free phosphate released from Cn-catalyzed hydrolysis of p-nitrophenylphosphate (110 ). When it became ap-preciated that the kinetic behavior of Cn toward small organic phosphates differs markedly from its behavior toward phosphorylated peptides and proteins (111 ), a method based on the radioactive detection of phosphate released from 32 P-labeled RII peptide, a fragment derived from the regulatory subunit of protein kinase A, came into use (112,113 ). A spectrophotometric assay of Cn activity based on the colorimetric detection of phosphate is currently available (114 ).…”

Section: Calcineurin Assaysmentioning

confidence: 99%

Molecular Diagnostics of Calcineurin-Related Pathologies

Musson

Cobbaert²,

Smit³

2012

Clinical Chemistry

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BACKGROUND The Ca2+-dependent protein phosphatase enzyme calcineurin (Cn) (protein phosphatase 3) is best known for its role as director of the adaptive immune response. One of its principal substrates is the nuclear factor of activated T cells (NFAT), which translocates to the nucleus after dephosphorylation to mediate gene transcription. Drugs targeting Cn (the Cn inhibitors tacrolimus and cyclosporin A) have revolutionized posttransplantation therapy in allograft recipients by considerably reducing rejection rates. CONTENT Owing primarily to intensive study of the side effects of the Cn inhibitors, the unique importance of Cn and Cn/NFAT signaling in the normal physiological processes of many other cell and tissue types is becoming more evident. During the last decade, it has become clear that an extensive and diverse array of clinical conditions can be traced back, at least in part, to a disturbed Cn-signaling axis. Hence, both diagnostics and therapeutic monitoring could benefit from a technique that conveniently reads out Cn/NFAT operative status. SUMMARY This review outlines the current knowledge on the pathologic conditions that have calcineurin as a common denominator and reports on the progress that has been made toward successfully applying Cn and Cn/NFAT activity markers in molecular diagnostics.

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“…The activity of the enzyme was measured as described by Fruman et al (1992), with modifications made by Koefoed-Nielsen et al (2004 …”

Section: Can Activitymentioning

confidence: 99%

Cyclosporin and tacrolimus impair insulin secretion and transcriptional regulation in INS‐1E beta‐cells

Øzbay

Smidt

Mortensen

et al. 2010

British J Pharmacology

131

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BACKGROUND AND PURPOSEIntroducing the calcineurin inhibitors cyclosporin (CsA) and tacrolimus (Tac) has improved the outcome of organ transplants, but complications such as new onset diabetes mellitus after transplantation (NODAT) decrease survival rates. EXPERIMENTAL APPROACHWe sought, in a beta-cell culture model, to elucidate the pathogenic mechanisms behind NODAT and the relative contribution of the calcineurin inhibitors. INS-1E cells were incubated at basal and stimulatory glucose concentrations, while exposed to pharmacologically relevant doses of CsA, Tac and vehicle for 6 or 24 h. RESULTSTac inhibited basal (P < 0.05), but not glucose-stimulated insulin secretion (GSIS) after 6 h of exposure. After 24 h, both agents inhibited basal and GSIS (P < 0.05). Calcineurin phosphatase activity was decreased by both drugs during all conditions. Apoptosis was only seen with CsA treatment, which also induced a slight suppression of calcineurin and insulin mRNA, as well as increased levels of the sterol receptor element binding protein (SREBP)-1c, a transcription factor thought to suppress genes essential for beta-cell function and induce insulin resistance. Expression levels of nuclear factor of activated T-cells (NFAT)-c1, -c2, -c3 and -c4 were not decreased notably by either drug. CONCLUSIONS AND IMPLICATIONSTac had acute inhibitory effects on basal insulin secretion, but prolonged exposure (24 h) to Tac or CsA revealed similar suppression of insulin secretion. These prolonged effects were mirrored by a total inhibition of calcineurin activity in beta-cells. CsA showed greater inhibition of beta-cell survival and transcriptional markers, essential for beta-cell function. AbbreviationsBax, Bcl-2 associated X protein; Bcl-2, B-cell leukaemia/lymphoma 2; CaN, calcineurin phosphatase; GSIS, glucose-stimulated insulin secretion; IRS-2, insulin receptor substrate 2; NFATc, nuclear factor of activated T-cells cytoplasmic; NODAT, new onset diabetes mellitus after transplantation; PDX1, pancreatic and duodenal homebox factor 1; SREBP-1c, sterol receptor element binding protein 1c

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Validation of the Calcineurin Phosphatase Assay

Cited by 19 publications

References 15 publications

24‐h Monitoring of Calcineurin Phosphatase Activity in Healthy Subjects

24‐h Monitoring of Calcineurin Phosphatase Activity in Healthy Subjects

Molecular Diagnostics of Calcineurin-Related Pathologies

Cyclosporin and tacrolimus impair insulin secretion and transcriptional regulation in INS‐1E beta‐cells

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