The role of the putative catalytic base in the phosphoryl transfer reaction in a protein kinase: first-principles calculations.

Protein kinases are important enzymes controlling the majority of cellular signaling events via a transfer of the gamma-phosphate of ATP to a target protein. Even after many years of study, the mechanism of this reaction is still poorly understood. Among many factors that may be responsible for the 1011-fold rate enhancement due to this enzyme, the role of the conserved aspartate (Asp166) has been given special consideration.

Nucleotide-induced conformational changes in the Saccharomyces cerevisiae SR protein kinase, Sky1p, revealed by X-ray crystallography.

Conformational changes are thought to play a key role in the function of active protein kinases, although little is known about how these changes relate to the mechanism of phosphorylation. Here we present four high-resolution structures of a single crystal form of Sky1p, a constitutively active serine kinase implicated in yeast RNA processing, each in a different state of nucleotide binding. By comparing the apoenzyme structure to the ADP- and ATP-bound Sky1p structures, we have revealed conformational changes caused by ATP binding or conversion from nucleotide reactant to product.

Design of a new peptidomimetic agonist for the melanocortin receptors based on the solution structure of the peptide ligand, Ac-Nle-cyclo[Asp-Pro-DPhe-Arg-Trp-Lys]-NH(2).

The solution structure of a potent melanocortin receptor agonist, Ac-Nle-cyclo[Asp-Pro-DPhe-Arg-Trp-Lys]-NH(2) (1) was calculated using distance restraints determined from 1H NMR spectroscopy. Eight of the lowest energy conformations from this study were used to identify non-peptide cores that mimic the spatial arrangement of the critical tripeptide region, DPhe-Arg-Trp, found in 1. From these studies, compound 2a, containing the cis-cyclohexyl core, was identified as a functional agonist of the melanocortin-4 receptor (MC4R) with an IC(50) and EC(50) below 10 nM.

Survival and normal neurological outcome after CPR with periodic Gz acceleration and vasopressin.

Background: We showed previously that whole body periodic acceleration along the spinal axis (pGz) is a novel method of cardiopulmonary resuscitation (CPR). The ultimate assessment of the value of any CPR technique is the neurological outcome after using such a technique. In this study, we determined the neurological outcome in pigs after prolonged pGz-CPR, with administration of vasopressin immediately prior to defibrillation.

Activation loop phosphorylation and catalysis in protein kinases: is there functional evidence for the autoinhibitor model?

Many protein kinases are activated strongly by the phosphorylation of a polypeptide region (activation loop) that lies outside the active-site cleft. Analysis of the X-ray crystallographic structures of the insulin receptor with the activation loop in the phosphorylated and dephosphorylated forms offers a testable model for the mechanism of activity regulation by the loop. In this model, the dephosphorylated activation loop can act as an autoinhibitor by blocking substrate access to the active site.

Noninvasive monitoring of cardiac output in human neonates and juvenile piglets by inductance cardiography (thoracocardiography).

Purpose: Thoracocardiography has been used in adult patients to noninvasively estimate changes in cardiac output (CO) by analysis of ventricular volume curves recorded by an inductive plethysmographic transducer encircling the chest at the level of the heart. The purpose of this study was to investigate the potential of thoracocardiography to monitor cardiac output in human neonates and in a small animal model, the juvenile piglet.

Materials And Methods: In 6 human premature neonates weighing 1.

Structural basis for peptide binding in protein kinase A. Role of glutamic acid 203 and tyrosine 204 in the peptide-positioning loop.

For optimal activity the catalytic subunit of cAMP-dependent protein kinase requires a phosphate on Thr-197. This phosphate anchors the activation loop in the proper conformation and contributes to catalytic efficiency by enhancing the phosphoryl transfer rate and increasing the affinity for ATP (1). The crystal structure of the catalytic subunit bound to ATP, and the inhibitor peptide, IP20, highlights the contacts made by the Thr-197 phosphate as well as the role adjacent residues play in contacting the substrate peptide.

Probing the nucleotide binding domain of the osmoregulator EnvZ using fluorescent nucleotide derivatives.

EnvZ is a histidine protein kinase important for osmoregulation in bacteria. While structural data are available for this enzyme, the nucleotide binding pocket is not well characterized. The ATP binding domain (EnvZB) was expressed, and its ability to bind nucleotide derivatives was assessed using equilbrium and stopped-flow fluorescence spectroscopy.

Phosphorylation driven motions in the COOH-terminal Src kinase, CSK, revealed through enhanced hydrogen-deuterium exchange and mass spectrometry (DXMS).

Previous kinetic studies demonstrated that nucleotide-derived conformational changes regulate function in the COOH-terminal Src kinase. We have employed enhanced methods of hydrogen-deuterium exchange-mass spectrometry (DXMS) to probe conformational changes on CSK in the absence and presence of nucleotides and thereby provide a structural framework for understanding phosphorylation-driven conformational changes. High quality peptic fragments covering approximately 63% of the entire CSK polypeptide were isolated using DXMS.

Mechanistic insights into Sky1p, a yeast homologue of the mammalian SR protein kinases.

The SRPK family is distinguished from typical eukaryotic protein kinases by several unique structural features recently elucidated by X-ray diffraction methods [Nolen et al. (2001) Nat. Struct.

3-D-conformal radiation therapy for pediatric giant cell tumors of the skull base.

Background: Giant cell tumors (GCT) of the base of skull are rare neoplasms. This report reviews the treatment of four pediatric patients presenting with aggressive giant cell tumor, using fractionated and combined, conformal proton and photon radiation therapy at Massachusetts General Hospital and Harvard Cyclotron Laboratory.

Patients And Methods: Three female patients and one adolescent male, ages 10-15 years, had undergone prior, extensive surgical resection(s) and were treated for either primary (two patients) or recurrent (two patients) disease.

Adolescent sexual assault: documentation of acute injuries using photo-colposcopy.

Objective: To document the frequency and types of genital injuries in adolescent women examined acutely following a sexual assault, and determine any historical correlates of injury.

Design: Retrospective chart review.

Setting: Sexual Assault Response Team services at a community hospital in an urban setting.

Comparison of megakaryopoiesis in vitro of paired peripheral blood progenitor cells and bone marrow harvested during remission in patients with acute myeloid leukaemia.

We have studied paired peripheral blood progenitor cells (PBPC) and bone marrow (BM) samples from 12 acute myeloid leukaemia (AML) patients following intensive chemotherapy, and assessed direct granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E), megakaryocyte CFU (CFU-Mk) numbers and the production of CD61+ (platelet glycoprotein IIIa) cells in suspension culture in response to various haemopoietic growth factor combinations. We found that CFU-GM and BFU-E numbers per 105 mononuclear cells were similar in both AML PBPC and BM harvests; CFU-Mk numbers, however, were significantly higher in PBPC than BM. In addition, the higher total white cell count of the PBPC harvests meant that PBPC have much higher numbers of total progenitors per collection.

Novel CPR with periodic Gz acceleration.

The effects of periodic Gz acceleration (pGz) on cardiovascular function and hemodynamics were determined in a pig model of acute cardiopulmonary resuscitation (CPR). The application of pGz (horizontal head-to-foot oscillations) at 2 Hz increased cardiac output in fibrillated animals proportional to the amplitude of the applied acceleration force that plateaued at 0.7 G.

Stereoselective Chelate-Controlled Addition of Grignard Reagents to Unsaturated Medium-Ring Heterocycles.

Various medium-ring heterocycles, bearing a C2-substituent that contains an accessible Lewis basic heteroatom, react with Grignard reagents with high levels of regio- and stereochemical control. The substrates can be prepared in the optically pure form by the Zr-catalyzed kinetic resolution; subsequent reaction with alkylmagnesium halides leads to the formation of optically pure alkylation products. The studies outlined herein probe the influence of the length and position of the heteroatom side chain on the facility and regio- and stereoselective outcome of the allylic substitution process.

Regional blood flow during periodic acceleration.

Objective: To determine whether a motion platform that imparts noninvasive periodic acceleration (pGz) forces to the body causes systemic vasodilation and changes local organ blood flow.

Design: Prospective paired blocked design.

Setting: Medical center research laboratory.

Nucleotide release and associated conformational changes regulate function in the COOH-terminal Src kinase, Csk.

The COOH-terminal Src kinase (Csk) regulates a broad array of cellular processes via the specific phosphorylation and downregulation of Src family protein kinases. While Csk has been a topic for steady-state kinetic studies, the individual steps associated with substrate phosphorylation have not been investigated. To understand active-site phenomena, pre-steady-state and transient-state kinetic methods were applied to develop a catalytic pathway for substrate processing.

Electrostatic environment surrounding the activation loop phosphotyrosine in the oncoprotein v-Fps.

Autophosphorylation of Tyr-1073 in the activation loop of the oncoprotein v-Fps enhances the phosphoryl transfer reaction without influencing substrate, ATP, or metal ion binding affinities [Saylor, P., et al. (1998) Biochemistry 37, 17875-17881].

Stimulation of autologous proliferative and cytotoxic T-cell responses by "leukemic dendritic cells" derived from blast cells in acute myeloid leukemia.

Effective presentation of tumor antigens is fundamental to strategies aimed at enrolling the immune system in eradication of residual disease after conventional treatments. Myeloid malignancies provide a unique opportunity to derive dendritic cells (DCs), functioning antigen-presenting cells, from the malignant cells themselves. These may then co-express leukemic antigens together with appropriate secondary signals and be used to generate a specific, antileukemic immune response.

Induction of BIM, a proapoptotic BH3-only BCL-2 family member, is critical for neuronal apoptosis.

Sympathetic neuronal death induced by nerve growth factor (NGF) deprivation requires the macromolecular synthesis-dependent translocation of BAX from the cytosol to mitochondria and its subsequent integration into the mitochondrial outer membrane, followed by BAX-mediated cytochrome c (cyt c) release. The gene products triggering this process remain unknown. Here, we report that BIM, a member of the BH3-only proapoptotic subfamily of the BCL-2 protein family, is one such molecule.

Structural characterization of protein kinase A as a function of nucleotide binding. Hydrogen-deuterium exchange studies using matrix-assisted laser desorption ionization-time of flight mass spectrometry detection.

Transient state kinetic studies indicate that substrate phosphorylation in protein kinase A is partially rate-limited by conformational changes, some of which may be associated with nucleotide binding (Shaffer, J., and Adams, J. A.

Hypothalamic/pituitary function following high-dose conformal radiotherapy to the base of skull: demonstration of a dose-effect relationship using dose-volume histogram analysis.

Purpose: To evaluate the incidence and pattern of hypopituitarism from hypothalamic (HT) and pituitary gland (PG) damage following high-dose conformal fractionated proton-photon beam radiotherapy (PPRT) to the base of skull (BOS) region in adults. The relationship between dose, volume, and PG function is explored.

Methods And Materials: Between May 1982 to October 1997, 107 adults with non-PG and non-HT neoplasms (predominantly chordoma and chondrosarcomas) of the BOS were treated with PPRT after subtotal resection(s).

Evolution of a classification scale: medical evaluation of suspected child sexual abuse.

This article presents a revision of a system for classifying examination findings, laboratory findings, and children's statements and behaviors as to their possible relationship to sexual abuse. The revisions are based on published research studies and current recommendations from the American Academy of Pediatrics Committee on Child Abuse and Neglect, and the American Professional Society on the Abuse of Children. Part 1 of the classification system lists genital and anal findings that can be considered normal or nonrelated to abuse, nonspecific for abuse, concerning for abuse, and clear evidence of blunt force or penetrating trauma.