Multimodality imaging approach for serial assessment of regional changes in lower extremity arteriogenesis and tissue perfusion in a porcine model of peripheral arterial disease.

Background: A standard quantitative imaging approach to evaluate peripheral arterial disease does not exist. Quantitative tools for evaluating arteriogenesis in vivo are not readily available, and the feasibility of monitoring serial regional changes in lower extremity perfusion has not been examined.

Methods And Results: Serial changes in lower extremity arteriogenesis and muscle perfusion were evaluated after femoral artery occlusion in a porcine model using single photon emission tomography (SPECT)/CT imaging with postmortem validation of in vivo findings using gamma counting, postmortem imaging, and histological analysis.

Different regulation modes of calcineurin regulatory subunit on its catalytic subunit with RII peptide and tau as substrates.

Calcineurin (CN) is a heterodimer of a catalytic subunit, calcineurin A (CNA), and a regulatory subunit (CNB). Here, we find that the mechanism by which CNB regulates CNA depends on the substrate involved. The regulation mechanism involving tau and its truncation segments is distinct from that involving RII peptide, and the efficiencies of CNA to dephosphorylate tau are constant regardless of whether CNB was present or not.

Tau binds both subunits of calcineurin, and binding is impaired by calmodulin.

Calcineurin, an important protein Ser/Thr phosphatase which acts on tau in vivo, is a heterodimer of a catalytic subunit, calcineurin A, and a regulatory subunit, calcineurin B, and is unique in being regulated by calmodulin. Here, we find that both subunits of calcineurin bind tau, and calmodulin interferes with the association between calcineurin and tau. The domains of both subunits of calcineurin and tau involved in binding are mapped.

Infusion of FK506, a specific inhibitor of calcineurin, induces potent tau hyperphosphorylation in mouse brain.

Calcineurin is a Ca2+/calmodulin-dependent protein phosphatase expressed at high levels in brain. Many electrophysiological and pharmacological findings have shown that calcineurin plays an important role in brain function. FK506 is always used as a specific calcineurin inhibitor in these researches.

The beta12-beta13 loop is a key regulatory element for the activity and properties of the catalytic domain of protein phosphatase 1 and 2B.

The molecular architectures of the catalytic core of protein phosphatase 1 (PP1) and protein phosphatase 2B (PP2B) are similar, and both contain a beta12-beta13 loop that consists of non-conserved residues. A truncation mutant containing the PP2B catalytic domain has previously been constructed in our laboratory, and designated CNAa. In this study, the PP1 catalytic subunit (PP1c) and CNAa, as well as mutants with the corresponding loops exchanged, were investigated using multiple substrates.

Inhibition of calcineurin by infusion of CsA causes hyperphosphorylation of tau and is accompanied by abnormal behavior in mice.

Calcineurin is a Ca2+/calmodulin-dependent phosphatase that dephosphorylates numerous substrates in different neuronal compartments. Genetic and pharmacological studies have provided insight into its involvement in the brain. Cyclosporin A (CsA) is used as a specific calcineurin inhibitor in many pharmacological experiments.

Effects of cyclosporin A, FK506 and rapamycin on calcineurin phosphatase activity in mouse brain.

Calcineurin is a Ca2+/calmodulin-dependent protein phosphatase expressed at high levels in brain. The immunosuppressive drugs cyclosporin A and FK506, but not rapamycin are specific inhibitors of calcineurin, the inhibitory effects of which have been elucidated in the immune system. Here by using these compounds as inhibitors, we assayed the enzyme in mouse brain after injection of 12.

Non-catalytic domains of subunit A negatively regulate the activity of calcineurin.

Calcineurin is composed of a catalytic subunit A (CNA) and a regulatory subunit B (CNB). In addition to the catalytic core, CNA further contains three non-catalytic domains--CNB binding domain (BBH), calmodulin binding domain (CBD), and autoinhibitory domain (AI). To investigate the effect of these three domains on the activity of CNA, we have constructed domain deletion mutants CNAa (catalytic domain only), CNAac (CNAa and CBD), and CNAaci (CNAa, CBD and AI).

Function and structure of recombinant single chain calcineurin.

Calcineurin (CN) is a Ca(2+)/calmodulin(CaM)-dependent serine/threonine protein phosphatase which is a heterodimer composed of a 61 kDa catalytic subunit (CNA) and a 19 kDa regulatory subunit (CNB). The enzyme is critical for several important intracellular signal-transducing pathways, including T-cell activation. Its crystal structure reveals that the C-terminal of CNB lies in close vicinity of the N-terminal of CNA and each end has a long arm not involved in the active site.