Linear and cyclic synthetic peptides related to the main autophosphorylation site of the Src tyrosine kinases as substrates and inhibitors of Lyn †

Tyrosine protein kinases (TPKs) of the src family contain two major phosphoacceptor sites which are homologous to the Tyr 416 and Tyr 527 of pp60^c-src. The former represents the main autophosphorylation sites of these enzymes, and its phosphorylation correlates with increased kinase activity. It has previously been demonstrated that the Src-like tyrosine kinase expressed by the oncogene lyn displays a high affinity toward the heptapeptide H-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-OH, which reproduces the main autophosphorylation site of the Src family enzymes [Donella-Deana, A., Marin, O., Brunati, A.M. & Pinna, L.A. (1992) Eur. J. Biochem. 204 , 1159–1163]. Our study was addressed to the synthesis of some derivatives of this sequence in order to obtain both peptide substrates suitable for the detection of the Src-like tyrosine kinase activity and active site-directed inhibitors specific for this class of enzymes. For this purpose we synthesized by classical solution methods the heptapeptide and its dimeric form. Moreover, in order to improve the proteolytic resistance of these peptides we also synthesized their cyclic derivatives and their N-terminal acetylated and C-terminal amidated analogs. The correlation between the different structural properties induced by the modifications of the native sequence and the propensity of the peptides to act as Lyn substrates was examined. The kinetic data obtained indicate that the extent of the peptide phosphorylation varies considerably depending on the flexibility and length of the analogs. While the cyclization and the C-terminal amidation of the heptapeptide are detrimental for the Lyn activity, dimeric derivatives display very favourable kinetic constants. In particular the cyclic dimer is an especially suitable substrate for the tyrosine kinase and a powerful inhibitor of both the phosphorylation activity of Lyn and the enzyme autophosphorylation. © Munksgaard 1995.     

Synthetic Tyr-phospho and non-hydrolyzable phosphonopeptides as PTKs and TC-PTP inhibitors*

Tyrosine-specific protein kinases and phosphatases are important signal transducing enzymes in normal cellular growth and differentiation and have been implicated in the etiology of a number of human neoplastic processes. In order to develop agents which inhibit the function of these two classes of enzymes by interfering with the binding of their substrates, we synthesized analogs derived from the peptide EDNEYTA. This sequence reproduces the main autophosphorylation site of Src tyrosine kinases. In this work we report the synthesis, by classical solution methods, of the phosphotyrosyl peptide ED-NEYpTA as well as of three analogs in which the phosphotyrosine is replaced by a phosphinotyrosine and by two unnatural, non-hydrolyzable amino acids 4-phosphonomethyl-l -phenylalanine and 4-phosphono-l -phenylalanine. The Src peptide and its derivatives were tested as inhibitors of three non-receptor tyrosine kinases (Lyn, belonging to the Src family, CSK and PTK-IIB) and a non-receptor protein tyrosine phosphatase obtained from human T-cell (TC-PTP). The biomimetic analogues, which do not significantly affect the activity of CSK, PTK-IIB and TC-PTP, act:is efficient inhibitors on Lyn, influencing both the exogenous phosphorylation and, especially, its autophosphorylation. In particular, the Pphe derivative may provide a basis for the design of a class of inhibitors specific for Lyn and possibly Src tyrosine kinases, capable of being used in vivo and in vitro conditions. © Munksgaard 1995.     

Sleep deprivation impairs spatial working memory and reduces hippocampal AMPA receptor phosphorylation

Sleep is important for brain function and cognitive performance. Sleep deprivation (SD) may affect subsequent learning capacity and ability to form new memories, particularly in the case of hippocampus‐dependent tasks. In the present study we examined whether SD for 6 or 12 h during the normal resting phase prior to learning affects hippocampus‐dependent working memory in mice. In addition, we determined effects of SD on hippocampal glutamate α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptors and their regulatory pathways, which are crucially involved in working memory. After 12 h SD, but not yet after 6 h, spatial working memory in a novel arm recognition task was significantly impaired. This deficit was not likely due to stress as corticosterone levels after SD were not significantly different between groups. In parallel with the change in cognitive function, we found that 12 h SD significantly reduced hippocampal AMPA receptor phosphorylation at the GluR1‐S845 site, which is important for incorporation of the receptors into the membrane. SD did not affect protein levels of cyclic‐AMP‐dependent protein kinase A (PKA) or phosphatase calcineurin (CaN), which regulate GluR1 phosphorylation. However, SD did reduce the expression of the scaffolding molecule A‐kinase anchoring protein 150 (AKAP150), which binds and partly controls the actions of PKA and CaN. In conclusion, a relatively short SD during the normal resting phase may affect spatial working memory in mice by reducing hippocampal AMPA receptor function through a change in AKAP150 levels. Together, these findings provide further insight into the possible mechanism of SD‐induced hippocampal dysfunction and memory impairment.     

Synthesis and conformational studies on peptides corresponding to a putative autophosphorylation site of abl TPK*

The transforming gene of Abelson murine leukaemia virus (v-abl) codes for a membrane-associated tyrosine-specific protein kinase (abl TPK). Analysis of the v-abl gene has shown that both the fibroblast-transforming and tyrosine-protein kinase activities reside within a minimal region encoding a protein of 43 kDa (p43^v-abl), which represents the most active, isolated form of this enzyme. Since the cellular substrates for p43 ^v-abl are yet to be identified, we synthesized by classical solution methods the octapeptide H-Gly-Asp-Thr-Tyr-Thr-Ala-His-Ala-OH, corresponding to the structural sequence of the main putative autophosphorylation site (Tyr 515) of the abl TPK, as well as some of its analogs modified in positions -2, -1, + 1 and + 3. The synthetic peptides were tested as substrates for the p43^v-abl. The kinetic data obtained indicate that the rates of their phosphorylation vary considerably depending on the sequence of the peptide, as expected. As a rule, no significant increment of the efficiency results from each substitution in the parent sequence. While the replacement of the two charged residues, namely Asp-2 and His-7, with neutral Ala is well tolerated, the substitution with amino acids bearing opposite charges is detrimental. The correlation between secondary structure of our synthetic octapeptides and their substrate recognition by p43^v-abl was studied using CD and fluorescence spectroscopy in 5 mM Tris, in 98% TFE/Tris and in 30 mM SDS solutions. The comparison of the spectroscopic data with the kinetic parameters does not confirm a close relationship between the conformational properties of these peptides and their enzymatic role.     

A New Nonfluoroscopic Navigation System to Guide Pulmonary Vein Isolation

Different techniques have been proposed to treat atrial fibrillation (AF) by catheter ablation. This study compares a new three-dimensional (3D) nonfluoroscopic navigation system with conventional fluoroscopy to guide pulmonary vein (PV) isolation. A total of 60 consecutive patients with paroxysmal or persistent AF were randomly assigned to 3D-guided ablation (group 1, n = 30), versus conventional fluoroscopy guidance ablation (group 2, n = 30). Complete PV isolation was achieved in both groups. The mean duration of fluoroscopy exposure (22 ± 8 vs 56 ± 10 minutes), and radiofrequency delivery (5 ± 1 vs 10 ± 3 minutes) were significantly shorter in group 1 than in group 2, P < 0.05 for both comparisons). The mean procedural time in group 1 was longer (225 ± 15 minutes) than in group 2 (156 ± 10 minutes, P < 0.05) due to the learning curve and time spent to generate the 3D maps. Over a mean follow-up of 7 ± 2 months, 6 patients (20%) in group 2 had AF recurrences compared to 3 patients (10%) in group 1 (ns). The new nonfluoroscopic 3D system allows a high-resolution reconstruction of the left atrium and PVs. It significantly reduces the mean radiofrequency delivery and fluoroscopy times as opposed to ablation performed under fluoroscopy guidance.     

Interruptions during nurses' drug rounds in surgical wards: observational study

Aims  The purpose of the study was to examine the frequency and perceived risk of interruptions to nurses during drug rounds in seven Italian surgical wards.
Background  Management of drug therapy is an integral part of the clinical role of nurses. Many errors are caused by interruptions to which the nurse is subjected during the drug rounds. However, the frequency of interruptions to nurses during drug rounds has not yet been documented.
Materials and methods  An observational study design was developed: (1) 56 randomized drug rounds, eight for each ward included, were observed and (2) 28 convenience samples of observed nurses were interviewed.
Results  The administration of a total of 945 medications was observed, an average of 2.2 per patient being treated: 298 interruptions were observed (one for every 3.2 drugs given). Ten different categories of interruption emerged. The highest risk of error as a result of interruption perceived by these nurses was related to the management of telephone calls.
Implications for nursing management  Many of the interruptions as a result of the organization of work could be avoided: one organizational priority should be to create a calm atmosphere for administering drugs. Ways to develop a calm atmosphere are discussed.     

Arrhythmia Detection in Single‐ and Dual‐Chamber Implantable Cardioverter Defibrillators: The More Leads,the Better?

The implantable cardioverter defibrillator (ICD) offers life-saving therapies for primary and secondary prevention of sudden cardiac death in high-risk patients. However, ICD detection algorithms consistently misclassify a substantial proportion of supraventricular rhythms, thus carrying the risk for inappropriate therapies. Although single-chamber ICD (Sc-ICD) discrimination tools have been reported to provide high specificity in rejecting sinus tachycardia and atrial fibrillation with a relatively low ventricular rate, accurate recognition of atrial fibrillation with faster ventricular rates, atrial tachycardias, atrial flutter, and some reentrant tachycardias is still an issue. Dual-chamber ICDs (Dc-ICDs) are supposed to overcome specificity issues by enhancing detection algorithms with information derived from the atrial and ventricular timing relationship. The initial promise of Dc-ICDs was to improve detection specificity without compromising sensitivity, and to translate this advantage over Sc-ICDs in a more selective use of aggressive therapies. Despite this solid background, superiority of Dc- over Sc-ICDs has never been convincingly demonstrated. The present review focuses on the efficacy of contemporary ICD arrhythmia discrimination tools and appraises the so far reported evidence supporting the superiority of Dc-ICDs in preventing inappropriate therapies.