Bovine fetuin is an inhibitor of insulin receptor tyrosine kinase

Fetuin has been identified earlier as the bovine homolog of the human plasma protein, alpha2-Heremans Schmid glycoprotein (alpha2-HSG). Although bovine fetuin shares over 70% amino acid sequence similarity with alpha2-HSG and rat fetuin, no common function(s) have been identified. We report that immunoaffinity purified bovine fetuin acts as an inhibitor of insulin receptor tyrosine kinase activity (IR-TKA) with half-maximal inhibition at 1.5 microM. In vitro, bovine fetuin (1.5 microM) blocked insulin-induced autophosphorylation of the human IR completely and the half-maximal inhibitory effect was observed at 0.5 microM. Incubation of HIRcB cells (rat1 fibroblasts transfected with wild-type human insulin receptor cDNA) with bovine fetuin (1.5 microM) inhibited insulin-induced tyrosine phosphorylation of the IR beta-subunit by 40%. In addition, bovine fetuin (2 microM) completely blocked insulin-stimulated DNA synthesis in H-35 rat hepatoma cells. Our results, together with earlier reports on rat fetuin and human alpha2-HSG, indicate a common IR-TK inhibitory function for fetuin homologs.     

Inhibition of experimental autoimmune encephalomyelitis by a tyrosine kinase inhibitor

Migration of lymphocytes from the blood into the brain is a critical event in the pathogenesis of experimental autoimmune encephalomyelitis. Lymphocyte adhesion to brain endothelium is the first step in lymphocyte entry into the central nervous system, leading subsequently to myelin damage and paralysis. In this paper we show that the tyrosine kinase inhibitor, tyrphostin AG490, prevents binding of freshly isolated mouse lymph node cells and of in vivo activated lymphocytes to endothelium of inflamed brain in Stamper-Woodruff adhesion assays. Moreover, AG490 inhibits adhesion of encephalitogenic T cell lines to purified ICAM-1 and VCAM-1, molecules implicated in T cell recruitment into the central nervous system. In contrast, 2-h treatment of T cell lines with high doses of tyrphostin AG490 have no effect on the viability, intracellular calcium elevation induced by Con A or TCR cross-linking, proliferation, or TNF production by Ag-stimulated T cell lines. Systemic administration of AG490 prevents the accumulation of leukocytes in the brain and the development of experimental autoimmune encephalomyelitis induced by proteolipid protein, peptide 139-151-specific T cell lines in SJL/J mice. Blood leukocytes isolated from mice treated with tyrphostin AG490 are less adhesive on purified very late Ag-4 ligands compared with adhesion of leukocytes from control animals. Our results suggest that inhibition of signaling pathways involved in lymphocyte adhesion may represent a novel therapeutic approach for demyelinating diseases.     

In vitro biological characterization and antiangiogenic effects of PD 166866, a selective inhibitor of the FGF-1 receptor tyrosine kinase

Through direct synthetic efforts, we discovered a small molecule that is a nanomolar inhibitor of the human fibroblast growth factor-1 receptor (FGFR) tyrosine kinase. PD 166866, a member of a new structural class of tyrosine kinase inhibitors, the 6-aryl-pyrido[2,3-d]pyrimidines, was identified by screening a compound library with assays that measure protein tyrosine kinase activity. PD 166866 inhibited human full-length FGFR-1 tyrosine kinase with an IC50 value of 52.4 +/- 0.1 nM and was further characterized as an ATP competitive inhibitor of the FGFR-1. In contrast, PD 166866 had no effect on c-Src, platelet-derived growth factor receptor-beta, epidermal growth factor receptor or insulin receptor tyrosine kinases or on mitogen-activated protein kinase, protein kinase C and CDK4 at concentrations as high as 50 microM. PD 166866 was a potent inhibitor of basic fibroblast growth factor (bFGF)-mediated receptor autophosphorylation in NIH 3T3 cells expressing endogenous FGFR-1 and in L6 cells overexpressing the human FGFR-1 tyrosine kinase, confirming a tyrosine kinase-mediated mechanism. PD 166866 also inhibited bFGF-induced tyrosine phosphorylation of the 44- and 42-kDa (ERK 1/2) mitogen-activated protein kinase isoforms in L6 cells, presumably via inhibition of bFGF-stimulated FGFR-1 tyrosine kinase activation. PD 166866 did not inhibit platelet-derived growth factor, epidermal growth factor or insulin-stimulated receptor autophosphorylation in vascular smooth muscle, A431 or NIHIR cells, respectively, further supporting its specificity for the FGFR-1. In addition, daily exposure of PD 166866 to L6 cells at concentrations from 1 to 100 nM resulted in a concentration-related inhibition of bFGF-stimulated cell growth for 8 consecutive days with an IC50 value of 24 nM. In contrast, PD 166866 had little effect on platelet-derived growth factor-BB-stimulated growth of L6 cells or serum-stimulated vascular smooth muscle cell proliferation. Finally, PD 166866 was found to be a potent inhibitor of microvessel outgrowth (angiogenesis) from cultured artery fragments of human placenta. These results highlight the discovery of PD 166866, a new nanomolar potent and selective small molecule inhibitor of the FGFR-1 tyrosine kinase with potential use as antiproliferative/antiangiogenic agent for such therapeutic targets as tumor growth and neovascularization of atherosclerotic plaques.     

In vivo pharmacology of MDMA and its enantiomers in rhesus monkeys.

The chiral nature of the MDMA molecule gives rise to two enantiomers, each of which is biologically active. This review attempts to cover the author's research into the in vivo effects of MDMA and its enantiomers, as well as other relevant publications which pertain to this topic. No particular differences between the capacities of racemic MDMA and its enantiomers to maintain behavior were noted, but antagonism of the 5-HT2A receptor produces a parallel rightward shift in the dose-effect function for the S(+)-enantiomer, but insurmountably reduces the reinforcing effects of R(-)-MDMA. Long-term self-administration of MDMA may lead to the development of chronic tolerance to the reinforcing effects of MDMA, but S(+)-MDMA is somewhat less susceptible to this effect than the racemate or the R(-)-enantiomer. Using PET neuroimaging, negligible occupancy at the dopamine transporter (DAT) was observed following administration of R(-)-MDMA, but reasonable DAT interaction was quantified following injection of S(+)-MDMA. The non-human primate studies reviewed herein caution that any results obtained in vivo with the MDMA enantiomers may not be particularly informative with regards to the racemate and vice versa. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Intramural delivery of a specific tyrosine kinase inhibitor with biodegradable stent suppresses the restenotic changes of the coronary artery in pigs in vivo

STUDY OBJECTIVE: Elevated pulmonary vascular resistance is a risk factor in heart transplantation and reversibility of high pulmonary vascular resistance is evaluated preoperatively in potential recipients using i.v. vasodilators or inhaled nitric oxide. Prostacyclin is a potent vasodilator, which when inhaled, has selective pulmonary vasodilatory properties. The aim of this study was to compare the central hemodynamic effects of inhaled prostacyclin with those of inhaled nitric oxide in heart transplant candidates. DESIGN: A pharmacodynamic comparative study. SETTING: Cardiothoracic ICU or laboratory for diagnostic heart catheterization at a university hospital. PATIENTS: Ten heart transplant candidates with elevated pulmonary vascular resistance (>200 dynes x s x cm(-5) and/or a transpulmonary pressure gradient > 10 mm Hg) were included in the study. INTERVENTIONS: Nitric oxide (40 ppm) and aerosolized prostacyclin (10 microg/mL) were administered by inhalation in two subsequent 10-min periods. Hemodynamic measurements preceded and followed inhalation of each agent. MEASUREMENTS AND RESULTS: Both inhaled nitric oxide and inhaled prostacyclin reduced mean pulmonary artery pressure (-7% vs -7%), pulmonary vascular resistance (-43% vs -49%), and the transpulmonary gradient (-44% vs -38%). With inhaled prostacyclin, an 11% increase in cardiac output was observed. Other hemodynamic variables, including the systemic BP, remained unaffected by each of the agents. CONCLUSIONS: Inhaled prostacyclin induces a selective pulmonary vasodilation that is comparable to the effect of inhaled nitric oxide. Major advantages with inhaled prostacyclin are its lack of toxic reactions and easy administration as compared with the potentially toxic nitric oxide requiring more complicated delivery systems.     

In vivo association of Grb2 with pp116, a substrate of the T cell antigen receptor-activated protein tyrosine kinase

Numerous recent studies have implicated the src homology 2 and 3 domain-containing protein, Grb2, in coupling protein tyrosine kinase signaling pathways with the Ras signaling pathway. Ligation of the T cell antigen receptor results in the activation of both a PTK, and Ras; therefore, we investigated whether Grb2 may serve a similar function in T cells. Here we report that a GST/Grb2 fusion protein associates with several tyrosine phosphoproteins from lysates of T cell antigen receptor-stimulated Jurkat T cells. Two of these proteins, pp36 and pp116, bind to the Grb2 fusion protein with high affinity. Through the use of mutated Grb2 fusion proteins, we demonstrate that pp116 binds the amino-terminal src homology 3 domain of Grb2, the same domain of Grb2 thought to be primarily responsible for its interaction with SOS. We demonstrate further that pp116 associates with Grb2 in vivo, and we provide evidence that in the Jurkat T cell line Grb2 may exist complexed with either pp116 or with SOS.     

Crystal structure of an angiogenesis inhibitor bound to the FGF receptor tyrosine kinase domain

Angiogenesis, the sprouting of new blood vessels from pre-existing ones, is an essential physiological process in development, yet also plays a major role in the progression of human diseases such as diabetic retinopathy, atherosclerosis and cancer. The effects of the most potent angiogenic factors, vascular endothelial growth factor (VEGF), angiopoietin and fibroblast growth factor (FGF) are mediated through cell surface receptors that possess intrinsic protein tyrosine kinase activity. In this report, we describe a synthetic compound of the pyrido[2,3-d]pyrimidine class, designated PD 173074, that selectively inhibits the tyrosine kinase activities of the FGF and VEGF receptors. We show that systemic administration of PD 173074 in mice can effectively block angiogenesis induced by either FGF or VEGF with no apparent toxicity. To elucidate the determinants of selectivity, we have determined the crystal structure of PD 173074 in complex with the tyrosine kinase domain of FGF receptor 1 at 2.5 A resolution. A high degree of surface complementarity between PD 173074 and the hydrophobic, ATP-binding pocket of FGF receptor 1 underlies the potency and selectivity of this inhibitor. PD 173074 is thus a promising candidate for a therapeutic angiogenesis inhibitor to be used in the treatment of cancer and other diseases whose progression is dependent upon new blood vessel formation.     

The tyrosine kinase inhibitor CGP57148B selectively inhibits the growth of BCR-ABL-positive cells

The Philadelphia chromosome found in virtually all cases of chronic myeloid leukemia (CML) and in about one third of the cases of adult acute lymphoblastic leukemia is formed by a reciprocal translocation between chromosomes 9 and 22 that results in the fusion of BCR and ABL genetic sequences. This BCR-ABL hybrid gene codes for a fusion protein with deregulated tyrosine kinase activity that can apparently cause malignant transformation. CGP57148B, a 2-phenylaminopyrimidine derivative, has been shown to selectively inhibit the tyrosine kinase of ABL and BCR-ABL. We report here that this compound selectively suppresses the growth of colony-forming unit-granulocyte/macrophage (CFU-GM) and burst-forming unit-erythroid derived from CML over a 2-logarithmic dose range with a maximal differential effect at 1.0 micromol/L. However, almost all CML colonies that grow in the presence of 1.0 micromol/L CGP57148B are BCR-ABL-positive, which may reflect the fact that residual normal clonogenic myeloid precursors are infrequent in most patients with CML. We also studied the effects of CGP57148B on hematopoietic cell lines. Proliferation was suppressed in most of the BCR-ABL-positive lines; all five BCR-ABL-negative lines were unaffected. We conclude that this new agent may have significant therapeutic applications.     

In vivo and ex vivo evaluation of the antithrombogenicity of human thrombomodulin immobilized biomaterials

Human thrombomodulin (hTM) is a newly described endothelial cell associated protein that functions as a potent natural anticoagulant by converting thrombin from a procoagulant protease to an anticoagulant. Focusing on the establishment of the practical evaluation of hTM immobilized materials, the activity of immobilized hTM was evaluated by in vivo and ex vivo blood contacting tests. As the basis for immobilization, regenerated cellulose films and hollow fibers were used. For the in vivo test, hTM immobilized cellular hollow fibers were implanted into dog blood vessels. Using hTM immobilized cellulose hollow fibers, a small scale dialyzer was assembled and its antithrombogenic activity was studied using human blood. As a result, it was revealed that the immobilized hTM still has co-enzymatic activity for activation of Protein C and anticoagulant activity. The coagulation time of the human blood passed through the hTM immobilized small dialyzer was effectively prolonged. It is expected that hTM immobilized cellulose should be a useful antithrombogenic biomaterial.     

In vivo evaluation of the Lillehei-Kaster heart valve prosthesis

Three decomposition products of Morazone (ingredient of the pharmaceutical preparation Rosimon-Neu) were observed following heat treatment in acid medium (hydrochloric or tartaric acid). These products were isolated by TLC and identified as bis-antipyryl-methane, phenmetrazine and 4-hydroxymethyl-antipyrine by mass spectrometry and IR-spectroscopy and 1H-nuclear magnetic resonance. Morazone and the metabolite phenmetrazine may be extracted from alkaline urine using chloroform, however acid hydrolysis (pH 1) of the urine before alkaline extraction will improve the sensitivity of detection of morazone by producing the metabolite phenmetrazine in addition to bis-antipyrylmethane. The metabolite 4-hydroxymethyl-antipyrine is barely detectable by TLC from alkaline extraction of urine.     

In vivo roles of receptor tyrosine kinases and cytokine receptors in early thymocyte development

The early phases of T-cell development require both cell-cell interactions and soluble factors provided by stromal cells within the thymic microenvironment. Still, the precise nature of the signals delivered in vivo by cytokines (resulting in survival, proliferation or differentiation) remains unclear. Recent studies using mice deficient in cytokines or in their receptors have helped to identify essential signaling pathways required for the development of intrathymic precursors to mature alpha beta and gamma delta T cells. In addition, cytokine requirements for the development of natural killer cells were revealed in such mutants. The results obtained demonstrate that the development of all classes of lymphocytes (natural killer, gamma delta T cells and alpha beta T cells) is cytokine dependent, but the specific requirements differ for each lineage.     

In vivo suppression of immune complex-induced alveolitis by secretory leukoproteinase inhibitor and tissue inhibitor of metalloproteinases 2

The pulmonary tree is exposed to neutrophil-derived serine proteinases and matrix metalloproteinases in inflammatory lung diseases, but the degree to which these enzymes participate in tissue injury remains undefined, as does the therapeutic utility of antiproteinase-based interventions. To address these issues, an in vivo rat model was examined in which the intrapulmonary deposition of immune complexes initiates a neutrophil-mediated acute alveolitis. In vitro studies demonstrated that rat neutrophils can release neutrophil elastase and cathepsin G as well as a neutrophil progelatinase, which was subsequently activated by either chlorinated oxidants or serine proteinases. Based on structural homologies that exist between rat and human neutrophil proteinases, rat neutrophil elastase and cathepsin G activities could be specifically regulated in vitro by recombinant human secretory leukoproteinase inhibitor, and rat neutrophil gelatinase activity proved sensitive to inhibition by recombinant human tissue inhibitor of metalloproteinases 2. When either of the recombinant antiproteinases were instilled intratracheally, in vivo lung damage as assessed by increased permeability or hemorrhage was significantly reduced. Furthermore, the coadministration of the serine and matrix metalloproteinase inhibitors almost completely prevented pulmonary damage while effecting only a modest decrease in neutrophil influx. These data support a critical role for neutrophil-derived proteinases in acute lung damage in vivo and identify recombinant human secretory leukoproteinase and recombinant human tissue inhibitor of metalloproteinases 2 as potentially efficacious interventions in inflammatory disease states.     

Regulation of DNA-dependent protein kinase by the Lyn tyrosine kinase

The Src-like protein-tyrosine kinase Lyn is activated by ionizing radiation and certain other DNA-damaging agents, whereas the DNA-dependent protein kinase (DNA-PK), consisting of the catalytic subunits (DNA-PKcs) and Ku DNA-binding components, requires DNA double-stranded breaks for activation. Here we demonstrate that Lyn associates constitutively with DNA-PKcs. The SH3 domain of Lyn interacts directly with DNA-PKcs near a leucine zipper homology domain. We also show that Lyn phosphorylates DNA-PKcs but not Ku in vitro. The interaction between Lyn and DNA-PKcs inhibits DNA-PKcs activity and the ability of DNA-PKcs to form a complex with Ku/DNA. These results support the hypothesis that there are functional interactions between Lyn and DNA-PKcs in the response to DNA damage.     

Phosphorylation- and activation-independent association of the tyrosine kinase Syk and the tyrosine kinase substrates Cbl and Vav with tubulin in B-cells

Aggregation of the B-cell antigen receptor leads to the activation of the 72-kDa Syk protein-tyrosine kinase and the phosphorylation of tubulin on tyrosine. To explore the requirement of Syk catalytic activity for tubulin phosphorylation, tubulin was isolated from cytosolic fractions from anti-IgM-activated B-cells (DT40) that lacked endogenous Syk and immunoblotted with anti-phosphotyrosine antibodies. Tubulin was not tyrosine-phosphorylated in Syk- B-cells. Phosphorylation could be restored by the expression of wild-type, but not catalytically inactive, Syk. However, both catalytically inactive and wild-type Syk were capable of constitutive association with tubulin, indicating that tubulin phosphorylation is not required for this interaction. Anti-phosphotyrosine antibody immunoblotting of proteins adsorbed to colchicine-agarose revealed the presence of three major tubulin-associated phosphoproteins of 110, 90, and 74 kDa, the phosphorylation of which was dependent on Syk expression. The proteins of 110 and 90 kDa were identified as Cbl and Vav, two proto-oncogene products known to become prominently phosphorylated following receptor engagement. Both proteins were shown to be constitutively associated with tubulin.     

In vivo EPR: an effective new tool for studying pathophysiology, physiology and pharmacology

The development of spectrometers working at lower frequencies with improved resonators now permits the routine use of non-invasive EPR spectroscopy in vivo. The capabilities of EPR spectra to reflect environmental conditions, combined with the use of paramagnetic materials as selective non-toxic labels, has led to increasingly widespread and productive applications of the technique to complex problems involving physiology, pharmacology and pathophysiology. Some of the especially promising applications in which EPR techniques uniquely appear to provide valuable information are illustrated, including the measurement of oxygen and oxygen gradients, monitoring of the metabolism of xenobiotics, monitoring pharmacokinetics of drugs, measurement of perfusion, measurement of pH, recognition and labeling of receptors, and characterization of drug releasing systems.     

Phase I clinical trial of the flavonoid quercetin: pharmacokinetics and evidence for in vivo tyrosine kinase inhibition

In the present open study, the long-term safety, tolerability, and efficacy of citalopram in the treatment of elderly people with emotional disturbances were studied. One hundred twenty-three elderly patients with symptoms of depression-anxiety were included. Most of the patients (76%) were demented. Fifty-two patients completed a 12-month treatment. Irritability, depressed mood, anxiety, restlessness, and fear-panic were significantly reduced. The severity of illness from baseline to Month 9 was rated as significantly improved. The side effects were infrequent and mostly mild.     

Three-dimensional structure of the tyrosine kinase c-Src

The structure of a large fragment of the c-Src tyrosine kinase, comprising the regulatory and kinase domains and the carboxy-terminal tall, has been determined at 1.7 A resolution in a closed, inactive state. Interactions among domains, stabilized by binding of the phosphorylated tail to the SH2 domain, lock the molecule in a conformation that simultaneously disrupts the kinase active site and sequesters the binding surfaces of the SH2 and SH3 domains. The structure shows how appropriate cellular signals, or transforming mutations in v-Src, could break these interactions to produce an open, active kinase.     

Structure of malhamensilipin A, an inhibitor of protein tyrosine kinase, from the cultured chrysophyte Poterioochromonas malhamensis

A new chlorosulfolipid, malhamensilpin A [1] was isolated from the cultured chrysophyte Poterioochromonas malhamensis. Malhamensilipin A was demonstrated to be a modest inhibitor of pp60v-src protein tyrosine kinase. The structure was determined by detailed spectral analysis to be a novel C24 hexachloro lipid containing a vinyl sulfate ester (2,11,12,13,15,16-hexachloro-14-hydroxy-n-tetracos-1E-enol-1-sulfa te).     

The tyrosine kinase inhibitor methyl 2,5-dihydroxycinnimate disrupts changes in the actin cytoskeleton required for neurite formation

In the current studies, we investigated the relationship between tyrosine phosphorylation and neurite formation. In SH-SY5Y neuroblastoma cells, the tyrosine kinase inhibitor methyl 2, 5-dihydroxycinnimate blocked neurite formation on laminin. This corresponded with inhibition of paxillin and focal adhesion kinase tyrosine phosphorylation as well as a disruption of actin filament organization and actin polymerization. This suggests that tyrosine phosphorylation helps direct changes in the actin cytoskeleton required for neurite formation.     

Tyrphostin AG957, a tyrosine kinase inhibitor with anti-BCR/ABL tyrosine kinase activity restores beta1 integrin-mediated adhesion and inhibitory signaling in chronic myelogenous leukemia hematopoietic progenitors

Abnormal beta1 integrin receptor function may contribute to the continuous proliferation and abnormal circulation of malignant hematopoietic progenitors in chronic myelogenous leukemia (CML). Previous studies suggest that abnormal integrin function in CML progenitors is related to the presence of the BCR/ABL oncogene. BCR/ABL may alter integrin function in CML by phosphorylating cytoskeletal and/or signaling proteins important for normal integrin function. We evaluated the effect of Tyrphostin AG957, a protein tyrosine kinase (PTK) inhibitor which has activity against the p210BCR/ABL kinase, on beta1 integrin function in CML progenitors. Incubation of CML marrow CD34+HLA-DR+ cells with Tyrphostin AG957 at concentrations that did not affect colony-forming cells (CFC) viability, but which partly inhibited p210BCR/ABL kinase activity, significantly increased CML CFC adhesion to stroma and alpha4beta1 and alpha5beta1 integrin binding fragments of fibronectin (FN). CML CFC proliferation, unlike that of normal CFC, is not inhibited following integrin receptor engagement with FN or anti-integrin antibodies. AG957 did not alter CML CFC proliferation by itself, but resulted in significant inhibition of CML CFC proliferation following integrin engagement. Another PTK inhibitor, Tyrphostin AG555, which does not have anti-p210BCR/ABL kinase activity, did not affect CML CFC adhesion or proliferation. Neither AG957 nor AG555 affected normal CFC adhesion or proliferation. In BCR/ABL expressing cells, AG957 partially inhibited phosphorylation of several proteins that are BCR/ABL PTK substrates and are involved in normal integrin signaling. These studies suggest that abnormal tyrosine phosphorylation may play an important role in defective integrin function in CML progenitors.