Immunoaffinity profiling of tyrosine phosphorylation in cancer cells.

Tyrosine kinases play a prominent role in human cancer, yet the oncogenic signaling pathways driving cell proliferation and survival have been difficult to identify, in part because of the complexity of the pathways and in part because of low cellular levels of tyrosine phosphorylation. In general, global phosphoproteomic approaches reveal small numbers of peptides containing phosphotyrosine. We have developed a strategy that emphasizes the phosphotyrosine component of the phosphoproteome and identifies large numbers of tyrosine phosphorylation sites.

Sulfone COX-2 inhibitors increase susceptibility of human LDL and plasma to oxidative modification: comparison to sulfonamide COX-2 inhibitors and NSAIDs.

Clinical investigations have demonstrated a link between use of the sulfone cyclooxygenase-2 (COX-2) inhibitor, rofecoxib, and increased risk for atherothrombotic events. This increased risk was not observed for a sulfonamide COX-2 inhibitor (celecoxib), indicating a potential non-enzymatic mechanism for rofexocib. To test this hypothesis, we compared the independent effects of COX-2 inhibitors on human LDL oxidation, an important contributor to atherosclerotic cardiovascular disease.

Poly(ADP-Ribose) polymerase promotes cardiac remodeling, contractile failure, and translocation of apoptosis-inducing factor in a murine experimental model of aortic banding and heart failure.

Oxidant stress-induced activation of poly(ADP-ribose) polymerase (PARP) plays a role in the pathogenesis of various cardiovascular diseases. We have now investigated the role of PARP in the process of cardiac remodeling and heart failure in a mouse model of heart failure induced by transverse aortic constriction (banding). The catalytic activity of PARP was inhibited by the potent isoindolinone-based PARP inhibitor INO-1001 or by PARP-1 genetic deficiency.

Angiotensin II-mediated endothelial dysfunction: role of poly(ADP-ribose) polymerase activation.

Angiotensin II (AII) contributes to the pathogenesis of many cardiovascular disorders. Oxidant-mediated activation of poly(adenosine diphosphate-ribose) polymerase (PARP) plays a role in the development of endothelial dysfunction and the pathogenesis of various cardiovascular diseases. We have investigated whether activation of the nuclear enzyme PARP contributes to the development of AII-induced endothelial dysfunction.

Application of rapid, electrochemical Flash Titration to total acidity and alkalinity determinations in buffers, foods, and beverages.

A new technique (Flash Titration) for the determination of total acidity and total alkalinity was applied to food and beverage analysis. Requiring no liquid titrants, Flash Titration is a technique made possible through microfabrication of electrochemical components on a silicon chip. Acidic or basic titrant was generated electrochemically at a noble-metal electrode that intimately surrounded an ion-selective, field-effect transistor pH sensor.

Mitochondrial-to-nuclear translocation of apoptosis-inducing factor in cardiac myocytes during oxidant stress: potential role of poly(ADP-ribose) polymerase-1.

Objective: Oxidant stress-induced activation of poly(ADP-ribose) polymerase (PARP) plays a role in the pathogenesis of various cardiovascular diseases. We have now investigated the role of PARP in the death of cardiac myocytes in response to oxidant stress induced by hydrogen peroxide, with focus on the mitochondrial function.

Methods And Results: Using wild-type and PARP-1-deficient murine myocytes challenged with hydrogen peroxide, we found that mitochondrial respiration and mitochondrial membrane potential were better preserved in PARP-deficient myocytes and cellular NAD+ levels were maintained.

The efficacy of Herceptin therapies is influenced by the expression of other erbB receptors, their ligands and the activation of downstream signalling proteins.

ErbB2 and EGFR are attractive oncology therapeutic targets as their overexpression in tumors predicts a poorer clinical outcome in a variety of epithelial malignancies. However, clinical results with therapeutic compounds targeting these receptors have been mixed. Therefore, there is a need for improved predictive biomarkers for these targeted therapeutics.

Restoration of the endothelial function in the aortic rings of apolipoprotein E deficient mice by pharmacological inhibition of the nuclear enzyme poly(ADP-ribose) polymerase.

Oxidant-mediated activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) plays a role in the development of endothelial dysfunction and the pathogenesis of various cardiovascular diseases. The aim of the current study was to investigate whether activation of PARP contributes to the development of endothelial dysfunction in the apolipoprotein E (ApoE) deficient mice. We tested whether PARP inhibition prevents the development of endothelial dysfunction and whether it restores function in vessels with established endothelial dysfunction.

Suppression of intestinal polyposis in Apcmin/+ mice by targeting the nitric oxide or poly(ADP-ribose) pathways.

Min mice have a germ-line nonsense mutation at codon 850 of the adenomatous polyposis coli (Apc) gene. These mice spontaneously develop multiple polyps in the small and large intestine at the age of 10-12 weeks. The aim of this study was to assess the role of reactive nitrogen species and poly(ADP-ribose) synthetase in tumorogenesis.

Poly(ADP-ribose) polymerase contributes to the development of myocardial infarction in diabetic rats and regulates the nuclear translocation of apoptosis-inducing factor.

Activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP)-1 by oxidant-mediated DNA damage is an important pathway of cell dysfunction and tissue injury during myocardial infarction. Because diabetes mellitus can substantially alter cellular signal transduction pathways, we have now investigated whether the PARP pathway also contributes to myocardial ischemia/reperfusion (MI/R) injury in diabetes mellitus in rodents. Myocardial ischemia/reperfusion in control and streptozotocin-diabetic rats was induced by transient ligation of the left anterior descending coronary artery.

2-(N-acyl) and 2-N-acyl-N(6)-substituted analogues of adenosine and their affinity at the human adenosine receptors.

A series of 2-(N-acyl) and 2-(N-acyl)-N(6)-alkyladenosine analogues have been synthesized from the intermediate 2-amino-6-chloroadenosine derivatives (2b and 7) and evaluated for their affinity at the human A(1), A(2A), and A(3) receptors. We found that 2-(N-acyl) derivatives of adenosine showed relatively low affinity at A(2A) and A(3) receptors, while the N(6)-cyclopentyl substituent in 4h and 4i induced high potency [A(1) (K(i))=20.7 and 31.

Protein interaction mapping on a functional shotgun sequence of Rickettsia sibirica.

Protein interaction maps can reveal novel pathways and functional complexes, allowing 'guilt by association' annotation of uncharacterized proteins. To address the need for large-scale protein interaction analyses, a bacterial two-hybrid system was coupled with a whole genome shotgun sequencing approach for microbial genome analysis. We report the first large-scale proteomics study using this system, integrating de novo genome sequencing with functional interaction mapping and annotation in a high-throughput format.

Poly(ADP-ribose) polymerase inhibition protect neurons and the white matter and regulates the translocation of apoptosis-inducing factor in stroke.

Focal cerebral ischemia activates the nuclear protein poly(ADP-ribose) polymerase (PARP). Apoptosis-inducing factor (AIF) is a flavoprotein that is normally confined to the mitochondria, but translocates to the nucleus, as shown by in vitro models of neuronal injury. Using INO-1001, a novel potent inhibitor of PARP, we determined the role of PARP activation in the process of AIF translocation in a rat model of focal cerebral ischemia.

Matrix metalloproteinase activation is an early event in doxorubicin-induced cardiotoxicity.

Matrix metalloproteinase (MMP) activation contributes to the development of various pathophysiological conditions, including dilated cardiomyopathy, congestive heart failure, and reperfusion injury. Increased oxidative and nitrosative stress have been implicated in the activation of MMPs and also in the cardiotoxicity of doxorubicin (DOX), a commonly used antitumor agent. Thus, we hypothesized that MMP activation occurs in DOX-induced cardiotoxicity.

The discovery and synthesis of novel adenosine substituted 2,3-dihydro-1H-isoindol-1-ones: potent inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1).

A series of novel 4-(N-acyl)-2,3-dihydro-1H-isoindol-1-ones have been prepared from methyl-3-nitro-2-methylbenzoate and linked through various spacers to the adenosine derivatives 11 and 12. We found that potent inhibition of poly(ADP-ribose)polymerase-1 (PARP-1) was achieved when isoindolinone was linked to adenosine by a spacer group of a specific length. Introduction of piperazine and succinyl linkers between the isoindolinone and adenosine core structures resulted in highly potent compounds 8a and 10b, which showed IC(50) values of 45 and 100 nM, respectively.

Recent advances in automated solid-phase carbohydrate synthesis: from screening to vaccines.

The past year has witnessed significant advances in a new technology for the synthesis of complex carbohydrates. Solid-phase methods have been applied to the construction of previously inaccessible carbohydrates. Furthermore, the application of automated solid-phase carbohydrate synthesis is promising.

The adenosine A3 receptor agonist, N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide, is protective in two murine models of colitis.

This study evaluated the effects of the adenosine A(3) receptor agonist, N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA), in two murine models of colitis, the dextran sodium sulphate-induced colitis and the spontaneous colitis found in interleukin-10 gene deficient mice. IB-MECA was given orally twice a day at a dose of either 1 or 3 mg/kg/day. Evaluation of colon damage and inflammation was determined grossly (body weight, rectal bleeding) and biochemically (colon levels of myeloperoxidase, malondialdehyde, chemokines and cytokines).

Multiple pathways of peroxynitrite cytotoxicity.

Peroxynitrite is a reactive oxidant produced from nitric oxide (NO) and superoxide, which reacts with a variety of biomolecules including proteins, lipids and DNA. Peroxynitrite is produced by the body in response to a variety of toxicologically relevant molecules including environmental toxins. It is also produced by the body in response to environmental toxins, as well as in reperfusion injury and inflammation.

Part II: beneficial effects of the peroxynitrite decomposition catalyst FP15 in murine models of arthritis and colitis.

Background: Peroxynitrite is a reactive oxidant species produced from nitric oxide and superoxide, which has been indirectly implicated in the pathogenesis of many inflammatory conditions including arthritis and colitis. Here, using a novel peroxynitrite decomposition catalyst, FP15, we directly investigate the role of peroxynitrite in the pathogenesis of arthritis and colitis in rodent models.

Methods: Arthritis was induced in mice by intradermal collagen injection; incidence and severity of arthritis was monitored using a macroscopic scoring system.

Role of poly(ADP-ribose) polymerase activation in endotoxin-induced cardiac collapse in rodents.

Reactive oxygen and nitrogen species are overproduced in the cardiovascular system during circulatory shock. Oxidant-induced cell injury involves the activation of poly(ADP-ribose) polymerase (PARP). Using a dual approach of PARP-1 suppression, by genetic deletion or pharmacological inhibition with the new potent phenanthridinone PARP inhibitor PJ34 [the hydrochloride salt of N-(oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide], we studied whether the impaired cardiac function in endotoxic shock is dependent upon the PARP pathway.

NFkappaB1 (p50)-deficient mice are not susceptible to multiple low-dose streptozotocin-induced diabetes.

Insulin-dependent diabetes mellitus (IDDM) is a disease characterized by the autoimmune destruction of the pancreatic beta-cells, which requires the expression of a number of immune-related genes including major histocompatibility complex proteins, cytokines, chemokines, and cytotoxic enzymes, many of which are regulated by the transcription factor, NFkappaB. Inhibition of the entire NFkappaB family of transcription factors may be harmful, as these factors are involved in many normal physiological processes. However, identifying and targeting specific NFkappaB subunits critical for the pathogenesis of disease may prove to be valuable in designing new therapeutic strategies.

Activation of poly(ADP-ribose) polymerase contributes to the endothelial dysfunction associated with hypertension and aging.

Increased production of reactive oxygen and nitrogen species has recently been implicated in the pathogenesis of endothelial dysfunction associated with atherosclerosis, hypertension and aging. Oxidant induced cell injury triggers the activation of nuclear enzyme poly(ADP-ribose) polymerase (PARP), which in turn contributes to cardiac and vascular dysfunction in various pathophysiological conditions including diabetes, reperfusion injury and circulatory shock. Here we investigated the role of PARP activation in the pathogenesis of cardiac and endothelial dysfunction associated with atherosclerosis, hypertension and aging.

Endothelial dysfunction in aging animals: the role of poly(ADP-ribose) polymerase activation.

Recent work has demonstrated the production of reactive oxygen and nitrogen species in the vasculature of aging animals. Oxidant induced cell injury triggers the activation of nuclear enzyme poly(ADP ribose) polymerase (PARP) leading to endothelial dysfunction in various pathophysiological conditions (reperfusion, shock, diabetes). Here we studied whether the loss of endothelial function in aging rats is dependent upon the PARP pathway within the vasculature.