Arginase II inhibition prevents nitrate tolerance

BACKGROUND AND PURPOSE Nitrate tolerance, the loss of vascular responsiveness with continued use of nitrates, remains incompletely understood and is a limitation of these therapeutic agents. Vascular superoxide, generated by uncoupled endothelial NOS (eNOS), may play a role. As arginase competes with eNOS for L-arginine and may exacerbate the production of reactive oxygen species (ROS), we hypothesized that arginase inhibition might reduce nitrate tolerance.

EXPERIMENTAL APPROACH Vasodilator responses were measured in aorta from C57Bl/6 and arginase II knockout (argII –/–) mice using myography. Uncoupling of eNOS, determined as eNOS monomer : dimer ratio, was assessed using low-temperature SDS-PAGE and ROS levels were measured using L-012 and lucigenin-enhanced chemiluminescence.

KEY RESULTS Repeated application of glyceryl trinitrate (GTN) on aorta isolated from C57Bl/6 mice produced a 32-fold rightward shift of the concentration–response curve. However this rightward shift (or resultant tolerance) was not observed in the presence of the arginase inhibitor (s)-(2-boronethyl)-L-cysteine HCl (BEC; 100 µM) nor in aorta isolated from argII –/– mice. Similar findings were obtained after inducing nitrate tolerance in vivo. Repeated administration of GTN in human umbilical vein endothelial cells induced uncoupling of eNOS from its dimeric state and increased ROS levels, which were reduced with arginase inhibition and exogenous L-arginine. Aortae from GTN tolerant C57Bl/6 mice exhibited increased arginase activity and ROS production, whereas vessels from argII –/– mice did not.

CONCLUSION AND IMPLICATIONS Arginase II removal prevents nitrate tolerance. This may be due to decreased uncoupling of eNOS and consequent ROS production.

Cigarette smoke-induced changes to alveolar macrophage phenotype and function are improved by treatment with procysteine

Defective efferocytosis may perpetuate inflammation in smokers with or without chronic obstructive pulmonary disease (COPD). Macrophages may phenotypically polarize to classically activated M1 (proinflammatory; regulation of antigen presentation) or alternatively activated M2 (poor antigen presentation; improved efferocytosis) markers. In bronchoalveolar lavage (BAL)–derived macrophages from control subjects and smoker/ex-smoker COPD subjects, we investigated M1 markers (antigen-presenting major histocompatibility complex [MHC] Classes I and II), complement receptors (CRs), the high-affinity Fc receptor involved with immunoglobulin binding for phagocytosis (Fc-gamma receptor, FcγR1), M2 markers (dendritic cell–specific intercellular adhesion molecule-grabbing nonintegrin [DC-SIGN] and arginase), and macrophage function (efferocytosis and proinflammatory cytokine production in response to LPS). The availability of glutathione (GSH) in BAL was assessed, because GSH is essential for both M1 function and efferocytosis. We used a murine model to investigate macrophage phenotype/function further in response to cigarette smoke. In lung tissue (disaggregated) and BAL, we investigated CRs, the available GSH, arginase, and efferocytosis. We further investigated the therapeutic effects of an oral administration of a GSH precursor, cysteine l-2-oxothiazolidine-4-carboxylic acid (procysteine). Significantly decreased efferocytosis, available GSH, and M1 antigen–presenting molecules were evident in both COPD groups, with increased DC-SIGN and production of proinflammatory cytokines. Increased CR-3 was evident in the current-smoker COPD group. In smoke-exposed mice, we found decreased efferocytosis (BAL and tissue) and available GSH, and increased arginase, CR-3, and CR-4. Treatment with procysteine significantly increased GSH, efferocytosis (BAL: control group, 26.2%; smoke-exposed group, 17.66%; procysteine + smoke-exposed group, 27.8%; tissue: control group, 35.9%; smoke-exposed group, 21.6%; procysteine + smoke-exposed group, 34.5%), and decreased CR-4 in lung tissue. Macrophages in COPD are of a mixed phenotype and function. The increased efferocytosis and availability of GSH in response to procysteine indicates that this treatment may be useful as adjunct therapy for improving macrophage function in COPD and in susceptible smokers.

Between-strand disulfides: Forbidden disulfides linking adjacent ß-Strands

Between-strand disulfides (BSDs) connect cysteine (Cys) residues across adjacent strands of β-sheets. There are four BSD types which can be found in regular β-structure: CSDs, which link residues immediately opposite each other in the β-structure (residues i and j); ETDs, which connect Cys out of register by one residue (i and j ± 1); BDDs, which join Cys at positions i and j ± 2; and BFDs, which link residues i and j ± 3. Formation of these disulfides was initially predicted to be forbidden, producing too much local strain in the protein fold. However, BSDs do exist in nature. Significantly, their high levels of strain allow them to be involved in redox processes under physiological conditions. Here we characterise BSD motifs found in the Protein Data Bank (PDB), discussing important intrinsic factors, such as the disulfide conformation and torsional strain, and extrinsic factors, such as the influence of the β-sheet environment on the disulfide and vice versa. We also discuss the biological importance of BSDs, including the prevalence of non-homologous examples in the PDB, the conservation of BSD motifs amongst related proteins (BSD clusters) and experimental evidence for BSD redox activity. For clusters of homologous BSDs we present detailed data of the disulfide properties and the variations of these properties amongst the “redundant” structures. Identification of disulfides with the potential to be involved in biological redox processes via the analysis of these data will provide important insights into the function and mechanism of BSD-containing proteins. Characterisation of thiol-based redox signalling pathways will lead to significant breakthroughs in understanding the molecular basis of oxidative stress and associated pathways, such as ageing and neurodegenerative diseases.

Doxycycline-inducible expression of SPARC/osteonectin/BM40 in MDA-MB-231 human breast cancer cells results in growth inhibition

SPARC (secreted protein acidic and rich in cysteine)/BM40/Osteonectin is a matricellular protein with multiple effects on cell behaviour. In vitro, its major known functions are anti-adhesive and anti-proliferative, and it is associated with tissue remodelling and cancer in vivo. SPARC is overexpressed in many cancers, including breast cancer, and the effects of SPARC seem to be cell type-specific. To study the effects of SPARC on breast cancer, we transfected SPARC into the MDA-MB-231 BAG, human breast cancer cell line using the Tet-On inducible system. By western analysis, we found low background levels in the MDA-MB-231 BAG and clone X parental cells, and prominent induction of SPARC protein expression after doxycycline treatment in SPARC transfected clones X5, X21, X24 and X75. Induction of SPARC expression did not affect cell morphology or adhesiveness to collagens type I and IV, but it slowed the rate of proliferation in adherent cultures. Cell cycle analysis showed that SPARC slowed the progression to S phase. Doxycycline induction of SPARC also slowed the rate of monolayer wound closure in the cultured wound healing assay. Thymidine inhibition of proliferation abrogated this effect, confirming that it was due to anti-proliferation rather than inhibition of migration. Consistent with this, we were unable to detect any differences in migration and Matrigel outgrowth analysis of doxycycline-stimulated cells. We conclude that SPARC is inhibitory to human breast cancer cell proliferation, and does not stimulate migration, in contrast to its stimulatory effects reported for melanoma (proliferation and migration) and glioma (migration) cells. Similar growth repression by SPARC has been reported for ovarian cancer cells, and this may be a common feature among carcinoma.

Neurobiology of schizophrenia spectrum disorders: The role of oxidative stress

Mitochondrial dysfunction and oxidative stress are increasingly implicated in the pathophysiology of schizophrenia. The brain is the body's highest energy consumer, and the glutathione system is the brain's dominant free radical scavenger. In the current paper, we review the evidence of central and peripheral nervous system anomalies in the oxidative defences of individuals with schizophrenia, principally involving the glutathione system. This is reflected by evidence of the manifold consequences of oxidative stress that include lipid peroxidation, protein carboxylation, DNA damage and apoptosis - all potentially part of the process of neuroprogression in the disorder. Importantly, oxidative stress is amenable to intervention. We consider the clinical potential of some possible interventions that help reduce oxidative stress, via augmentation of the glutathione system, particularly N-acetyl cysteine. We argue that a better understanding of the mechanisms and pathways underlying oxidative stress will assist in developing the therapeutic potential of this area.

Lovastatin for the adjunctive treatment of schizophrenia: a preliminary randomized double-blind placebo-controlled trial.

While statins target many of the pathways to neuroprogression in schizophrenia, the safety and efficacy of statins for treating schizophrenia has never been examined. This is an 8-week randomized double blind controlled clinical trial examining the efficacy and safety of adjunctive lovastatin (20 mg/day) treatment or placebo for people with schizophrenia. The baseline characteristics of the two groups were not different. Endpoint changes in Positive and Negative Syndrome Scale (PANSS) total and subscale scores did not differ between the two groups. However there was a significant difference between the doses of risperidone used in the two groups. The mean dose in the lovastatin and placebo groups were 4.8(1.8) and 3.4(1.4) mg/day, respectively (P<.03). No serious adverse events were reported. Slowness of movements, muscle rigidity, increased appetite, and decreased energy were the most common adverse effects, and these rates did not differ between the two groups. This study failed to demonstrate a benefit of lovastatin on symptoms of schizophrenia. This combination was well tolerated. However, a higher dosage of risperidone was used for treating the disorder in those taking concomitant lovastatin compared to placebo.

A systematic review and meta-analysis of behaviourally based psychological interventions and pharmacological interventions for trichotillomania

Background: Trichotillomania (hair-pulling disorder) is a debilitating and distressing disorder associated with great secrecy and shame. A lack of understanding regarding interventions for Trichotillomania contributes to poor routine outcomes for the disorder. Method: This systematic review and meta-analysis assessed the efficacy of behaviourally based psychological interventions and pharmacological interventions for trichotillomania compared to a range of control groups. Participants were adults who have been diagnosed with trichotillomania. A systematic search was conducted of the Cochrane library, EBSCOhost, MEDLINE before 1966, and Google Scholar for relevant randomised controlled trials. Results: Of the total 462 records identified, 12 studies were included in the quantitative synthesis, and nine studies were included in meta-analyses. Conclusions: Analyses revealed that-from medication approaches-fluoxetine was not found to be efficacious. However, N-acetyl cysteine, clomipramine, and olanzapine showed potential for the treatment of trichotillomania. Regarding psychotherapy, behaviour therapy showed superior efficacy when compared to a passive control group. However, when behaviour therapy was compared to an active control group (progressive muscle relaxation, supportive therapy), both conditions showed similar efficacy in treating trichotillomania. It was concluded that the psychological mechanisms in trichotillomania may be more complex than the behavioural model indicates. Implications and limitations are discussed.

Tunnelling current recognition through core-satellite gold nanoparticles for ultrasensitive detection of copper ions

We report a new method for ultrasensitive detection of Cu(2+), which is based on changes in the tunnelling recognition current across self-assembled core-satellite gold nanoparticles (GNPs) networks functionalised with amino acids (l-cysteine). The addition of copper ions induces the formation of GNP/l-cysteine/Cu(2+)/l-cysteine/GNP molecular junctions and generates a significant decrease in the resistance through the networks. The networks are ultrasensitive to over ten orders range of copper ion concentrations.

NDRG2 promotes myoblast proliferation and caspase 3/7 activities during differentiation, and attenuates hydrogen peroxide - but not palmitate-induced toxicity

The function of the stress-responsive N-myc downstream-regulated gene 2 (NDRG2) in the control of myoblast growth, and the amino acids contributing to its function, are not well characterized. Here, we investigated the effect of increased NDRG2 levels on the proliferation, differentiation and apoptosis in skeletal muscle cells under basal and stress conditions. NDRG2 overexpression increased C2C12 myoblast proliferation and the expression of positive cell cycle regulators, cdk2, cyclin B and cyclin D, and phosphorylation of Rb, while the serine/threonine-deficient NDRG2, 3A-NDRG2, had less effect. The onset of differentiation was enhanced by NDRG2 as determined through the myogenic regulatory factor expression profiles and myocyte fusion index. However, the overall level of differentiation in myotubes was not different. While NDRG2 up-regulated caspase 3/7 activities during differentiation, no increase in apoptosis was measured by TUNEL assay or through cleavage of caspase 3 and PARP proteins. During H2O2 treatment to induce oxidative stress, NDRG2 helped protect against the loss of proliferation and ER stress as measured by GRP78 expression with 3A-NDRG2 displaying less protection. NDRG2 also attenuated apoptosis by reducing cleavage of PARP and caspase 3 and expression of pro-apoptotic Bax while enhancing the pro-survival Bcl-2 and Bcl-xL levels. In contrast, Mcl-1 was not altered, and NDRG2 did not protect against palmitate-induced lipotoxicity. Our findings show that NDRG2 overexpression increases myoblast proliferation and caspase 3/7 activities without increasing overall differentiation. Furthermore, NDRG2 attenuates H2O2-induced oxidative stress and specific serine and threonine amino acid residues appear to contribute to its function in muscle cells.

The Glutathione System: a new drug target in neuroimmune disorders

Glutathione (GSH) has a crucial role in cellular signaling and antioxidant defenses either by reacting directly with reactive oxygen or nitrogen species or by acting as an essential cofactor for GSH S-transferases and glutathione peroxidases. GSH acting in concert with its dependent enzymes, known as the glutathione system, is responsible for the detoxification of reactive oxygen and nitrogen species (ROS/RNS) and electrophiles produced by xenobiotics. Adequate levels of GSH are essential for the optimal functioning of the immune system in general and T cell activation and differentiation in particular. GSH is a ubiquitous regulator of the cell cycle per se. GSH also has crucial functions in the brain as an antioxidant, neuromodulator, neurotransmitter, and enabler of neuron survival. Depletion of GSH leads to exacerbation of damage by oxidative and nitrosative stress; hypernitrosylation; increased levels of proinflammatory mediators and inflammatory potential; dysfunctions of intracellular signaling networks, e.g., p53, nuclear factor-κB, and Janus kinases; decreased cell proliferation and DNA synthesis; inactivation of complex I of the electron transport chain; activation of cytochrome c and the apoptotic machinery; blockade of the methionine cycle; and compromised epigenetic regulation of gene expression. As such, GSH depletion has marked consequences for the homeostatic control of the immune system, oxidative and nitrosative stress (O&NS) pathways, regulation of energy production, and mitochondrial survival as well. GSH depletion and concomitant increase in O&NS and mitochondrial dysfunctions play a role in the pathophysiology of diverse neuroimmune disorders, including depression, myalgic encephalomyelitis/chronic fatigue syndrome and Parkinson’s disease, suggesting that depleted GSH is an integral part of these diseases. Therapeutical interventions that aim to increase GSH concentrations in vivo include N-acetyl cysteine; Nrf-2 activation via hyperbaric oxygen therapy; dimethyl fumarate; phytochemicals, including curcumin, resveratrol, and cinnamon; and folate supplementation.