[The alliance of the parent, child and the nurse to challenge medical error]. / L'alliance parents-enfant-soignants à l'épreuve de l'erreur médicale.

A group composed of parents, nurses, and physicians involved in pediatric cancerology has reflected on medical errors within the Espace Éthique de l'Assistance publique-Hôpitaux de Paris. Based on narratives and qualitative analysis of histories and testimonies, this discussion aimed at exploring the causes, circumstances, and impacts of medical errors on the relations between these individuals. The study demonstrated that some circumstances actually promote medical errors, such as hard working conditions, mistrust, unreliable control procedures, not listening to parents, and caring for children in extreme situations of pain and suffering. Errors almost always result from the accumulation of several shortcomings. The tensions raised by a medical error can be overcome, provided that parents and caregivers trust each other from the onset of disease and that the medical errors are disclosed in a sincere way, whatever the medical consequences. The feelings raised by the painful experience of a medical error do not solely depend on the severity of the consequences, since seemingly benign errors may lead to long-term trauma, whereas severe errors, even those leading to death, do not necessarily breach trust. The keyword here is permanent vigilance. The capacity of caregivers to question their practice, from both a technical and ethical point of view, will determine their ability to learn from an error for the future. The depth and quality of this questioning, in the best of times encouraged by the institution, may also help children affected by a medical error and their family to move forward in their personal history, beyond such painful experiences.

Modulation of the voltage-gated sodium current in rat striatal neurons by DARPP-32, an inhibitor of protein phosphatase.

DARPP-32 is a cyclic adenosine monophosphate-regulated inhibitor of protein phosphatase 1, highly enriched in striatonigral neurons. Stimulation of dopamine D1 receptors increases phosphorylation of DARPP-32, whereas glutamate acting on N-methyl-D-aspartate receptors induces its dephosphorylation. Yet, to date, there is little direct evidence for the function of DARPP-32 in striatal neurons. Using a whole cell patch-clamp technique, we have studied the role of DARPP-32 in the regulation of voltage-gated sodium channels in rat striatal neurons maintained in primary culture. Injection of phospho-DARPP-32, but not of the unphosphorylated form, reduced the sodium current amplitude. This effect was similar to those induced by okadaic acid, with which there was no additivity and by tautomycin. Our results indicate that, in striatal neurons, sodium channels are under dynamic control by phosphorylation/dephosphorylation, and that phospho-DARPP-32 reduces sodium current by stabilizing a phosphorylated state of the channel or an associated regulatory protein. We propose that the DARPP-32-mediated modulation of sodium channels, via inhibition of phosphatase 1, contributes to the regulation of these channels by D1 receptors and other neurotransmitters which influence the state of phosphorylation of DARPP-32.

Dephosphorylation of Ser-137 in DARPP-32 by protein phosphatases 2A and 2C: different roles in vitro and in striatonigral neurons.

DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, Mr=32000) is highly expressed in striatonigral neurons in which its phosphorylation is regulated by several neurotransmitters including dopamine and glutamate. DARPP-32 becomes a potent inhibitor of protein phosphatase 1 when it is phosphorylated on Thr-34 by cAMP- or cGMP-dependent protein kinases. DARPP-32 is also phosphorylated on Ser-137 by protein kinase CK1 (CK1), in vitro and in vivo. This phosphorylation has an important regulatory role since it inhibits the dephosphorylation of Thr-34 by calcineurin in vitro and in striatonigral neurons. Here, we show that DARPP-32 phosphorylated by CK1 is a substrate in vitro for protein phosphatases 2A and 2C, but not protein phosphatase 1 or calcineurin. However, in substantia nigra slices, dephosphorylation of Ser-137 was markedly sensitive to decreased temperature, and not detectably affected by the presence of okadaic acid under conditions in which dephosphorylation of Thr-34 by protein phosphatase 2A was inhibited. These results suggest that, in neurons, phospho-Ser-137-DARPP-32 is dephosphorylated by protein phosphatase 2C, but not 2A. Thus, DARPP-32 appears to be a component of a regulatory cascade of phosphatases in which dephosphorylation of Ser-136 by protein phosphatase 2C facilitates dephosphorylation of Thr-34 by calcineurin, removing the cyclic nucleotide-induced inhibition of protein phosphatase 1.

Regulation of secretion of Alzheimer amyloid precursor protein by the mitogen-activated protein kinase cascade.

Activation of protein kinase C (PKC) regulates the processing of Alzheimer amyloid precursor protein (APP) into its soluble form (sAPP) and amyloid beta-peptide (A beta). However, little is known about the intermediate steps between PKC activation and modulation of APP metabolism. Using a specific inhibitor of mitogen-activated protein (MAP) kinase kinase activation (PD 98059), as well as a dominant negative mutant of MAP kinase kinase, we show in various cell lines that stimulation of PKC by phorbol ester rapidly induces sAPP secretion through a mechanism involving activation of the MAP kinase cascade. In PC12-M1 cells, activation of MAP kinase by nerve growth factor was associated with stimulation of sAPP release. Conversely, M1 muscarinic receptor stimulation, which is known to act in part through a PKC-independent pathway, increased sAPP secretion mainly through a MAP kinase-independent pathway. A beta secretion and its regulation by PKC were not affected by PD 98059, supporting the concept of distinct secretory pathways for A beta and sAPP formation.

Characterization of the interaction between DARPP-32 and protein phosphatase 1 (PP-1): DARPP-32 peptides antagonize the interaction of PP-1 with binding proteins.

The catalytic subunit of PP-1 (PP-1C) is potently inhibited (IC50, approximately 1 nM) by DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, M(r) 32,000), inhibitor-1, and inhibitor-2. The NH2-terminal 50 amino acid residues of DARPP-32 and inhibitor-1 are similar, and phosphorylation of a common threonine residue (Thr-34/Thr-35) is necessary for inhibition of PP-1C. We have characterized further the interaction between DARPP-32 and PP-1C. Using synthetic peptides derived from the NH2-terminal region of DARPP-32, residues 6-11, RKKIQF, have been shown to be required for inhibition of PP-1C. Peptides containing this motif were able to antagonize the inhibition of PP-1C by phospho-DARPP-32 and phosphoinhibitor-1. The inhibition of PP-1C by inhibitor-2, but not by okadaic acid, microcystin, or calyculin A, was also attentuated by these antagonist peptides. These results together with results from other studies support a model in which two subdomains of phospho-DARPP-32 interact with PP-1C. The region encompassing phospho-Thr-34 appears to interact with the active site of the enzyme blocking enzyme activity. The region encompassing the RKKIQF motif binds to a domain of PP-1C removed from the active site. Amino acid sequence analysis indicates that basic and hydrophobic features of the RKKIQF motif are conserved in the binding domains of certain PP-1C targeting proteins, suggesting that interaction of inhibitor proteins and targeting proteins may be mutually exclusive.

Amyloid beta peptide formation in cell-free preparations. Regulation by protein kinase C, calmodulin, and calcineurin.

Amyloid beta peptide (Abeta) is a short peptide that is the major constituent of the amyloid plaques and cerebrovascular amyloid deposits found in Alzheimer's disease. The lack of availability of a cell-free system in which to study Abeta formation has limited our understanding of the molecular mechanisms involved in its production. We report here the reconstitution of such a cell-free system. The reconstituted Abeta formation was temperature-dependent and required ATP. Preincubation with purified protein kinase C (PKC) induced a pronounced inhibition of Abeta formation, similar to that observed in intact cells upon stimulation of PKC. The calmodulin antagonists W-7 and trifluoperazine inhibited Abeta formation and enhanced the action of PKC in both the cell-free system and intact cells. A role for the calcium/calmodulin-activated protein phosphatase calcineurin in the regulation of Abeta formation was demonstrated using a specific peptide inhibitor of calcineurin in vitro as well as cyclosporin A, a cell-permeant inhibitor of calcineurin, in intact cells. Our results suggest that a single substrate might mediate opposing actions of PKC and calcineurin in the regulation of Abeta formation.

Phosphorylation of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein, by casein kinase I in vitro and in vivo.

DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, M(r) = 32,000) is a potent inhibitor of protein phosphatase-1 when it is phosphorylated on Thr-34 by cAMP-dependent protein kinase. DARPP-32 is highly enriched in some specific cell populations such as striatonigral neurons and choroid plexus epithelial cells. Here we show that recombinant rat DARPP-32 is phosphorylated by casein kinase I on seryl residues to a stoichiometry of approximately 2 mol of phosphate/mol of protein. DARPP-32 is one of the best known substrates for casein kinase I (Km = 3.4 +/- 0.3 microM), whereas the homologous phosphatase-1 inhibitor, inhibitor-1, is not. Phosphorylation of DARPP-32 by casein kinase I does not alter its ability to inhibit protein phosphatase-1. Residues phosphorylated by casein kinase I were identified as Ser-137 and Ser-189 by site-directed mutagenesis and by protein sequencing. Ser-137 and the preceding stretch of 16-18 acidic residues are conserved in DARPP-32 among all species examined, whereas Ser-189 is not. Phosphorylation of Ser-137 induces an unusual increase in DARPP-32 electrophoretic mobility in polyacrylamide gels in the presence of SDS. In striatonigral neurons, DARPP-32 is phosphorylated on Ser-137 and the stoichiometry of phosphorylation on this residue in vivo appears to be higher in the substantia nigra (axon terminals) than in the striatum (soma and dendrites). These results indicate that casein kinase I is highly active in striatonigral neurons in which it may play important roles, including in protein phosphatase-1 modulation via phosphorylation of DARPP-32.

Dopamine- and cAMP-regulated phosphoprotein DARPP-32: phosphorylation of Ser-137 by casein kinase I inhibits dephosphorylation of Thr-34 by calcineurin.

Although protein phosphatases appear to be highly controlled in intact cells, relatively little is known about the physiological regulation of their activity. DARPP-32, a dopamine- and cAMP-regulated phosphoprotein of apparent M(r) 32,000, is phosphorylated in vitro by casein kinase I, casein kinase II, and cAMP-dependent protein kinase on sites phosphorylated in vivo. DARPP-32 phosphorylated on Thr-34 by cAMP-dependent protein kinase is a potent inhibitor of protein phosphatase 1 and an excellent substrate for calcineurin, a Ca2+/calmodulin-dependent protein phosphatase. Here we provide evidence, using both purified proteins and brain slices, that phosphorylation of DARPP-32 on Ser-137 by casein kinase I inhibits the dephosphorylation of Thr-34 by calcineurin. This inhibition occurs only when phospho-Ser-137 and phospho-Thr-34 are located on the same DARPP-32 molecule and is not dependent on the mode of activation of calcineurin. The results demonstrate that the inhibition is due to a modification in the properties of the substrate which alters its dephosphorylation rate. Thus, casein kinase I may play a physiological role in striatonigral neurons as a modulator of the regulation of protein phosphatase 1 via DARPP-32.

Mechanism of inhibition of protein phosphatase 1 by DARPP-32: studies with recombinant DARPP-32 and synthetic peptides.

The mechanism of inhibition of protein phosphatase-1 catalytic subunit (PP-1c) by recombinant DARPP-32 and synthetic peptides was studied. DARPP-32 was expressed in Escherichia coli as a non-fusion protein using a pEt-3a plasmid, purified to homogeneity and shown to have physicochemical properties similar to those of the protein purified from bovine brain. Recombinant DARPP-32 phosphorylated on threonine-34 by cAMP-dependent protein kinase inhibited PP-1c with an IC50 approximately 0.5 nM, comparable to that obtained with bovine DARPP-32. Non-phosphorylated DARPP-32, and mutated forms in which threonine-34 was replaced by an alanine or a glutamic acid, inhibited PP-1c with an IC50 approximately 1 microM. Surface plasmon resonance analysis showed binding of PP-1c to nonphospho- and phospho-DARPP-32-(8-38) synthetic peptides with apparent Kd values of 1.2 and 0.3 microM, respectively, supporting the existence of an interaction between non-phosphorylated DARPP-32 and PP-1c that is increased by phosphorylation of DARPP-32 at threonine-34. These results suggest a model in which DARPP-32 interacts with PP-1c by at least two low affinity sites, the combination of which is responsible for the high affinity (nM) inhibition.

Study of the conformation of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein, by fluorescence spectroscopy.

DARPP-32 is a potent inhibitor of protein phosphatase 1 when it is phosphorylated on Thr34 by cAMP-dependent protein kinase. DARPP-32 is also phosphorylated on Ser45 and Ser102 by casein kinase II, resulting in a facilitation of phosphorylation by cAMP-dependent protein kinase. We have studied the conformation of recombinant rat DARPP-32 by steady-state and time-resolved fluorescence. The steady-state emission spectra and quenching of the intrinsic (Trp163) and extrinsic fluorescence (acrylodan or lucifer yellow linked to Cys72) were consistent with a complete exposure of these residues to the aqueous environment. The intrinsic fluorescence of DARPP-32 was resolved into three decay components with lifetimes of 1, 3.4, and 7 ns, with the intermediate lifetime component giving the major contribution. The ratio between the amplitudes associated with the short and long decay constants was decreased upon denaturation. The rotational behavior of DARPP-32 measured by anisotropy decay revealed that Trp163 is located in a highly flexible peptide chain, whereas Cys72 is embedded in a more rigid environment. Phosphorylation by cAMP-dependent protein kinase did not alter any of the fluorescence parameters, whereas only minor effects were associated with casein kinase II phosphorylation. These findings indicate that DARPP-32 contains at least two distinct domains and that phosphorylation has no dramatic effects on its conformation.