I am a Chancellors Fellow at SoLS in the Molecular Biosciences Team and a Research Leader at the Woolcock Institute. I bring both industry (Pharma) and academic research experience from India and North America. My laboratory (Chronic Airways Disease Lab) focuses on discovering and identifying novel mechanisms that may act as first in class targets for treating chronic lung diseases such as asthma and COPD. We utilizes variety of translational model systems involving human airway cells from healthy and patient donors in conjunction with mouse models to test new therapies. One such process is autophagy; a highly conserved protective mechanism within the cell, which we have found to be dysregulated or impaired in asthma. We are trying to understand how this fundamental protective mechanism can be exploited to achieve clinical benefit in patients who do not respond to standard therapy while their lungs are constantly remodeled with chronic inflammation.
We are also interested in identifying novel and improving existing bronchodilators that target GPCRs to relax airways. I actively collaborate locally at UTS (SoLS, GSH, CHT), within Australia (Woolcock and Sydney Hospitals) and also with some of the leading researchers in North America, Europe and Asia.
- Chronic Lung Diseases (Asthma, COPD, IPF)
- New Targets and Disease Mechanisms
- Animal models of disease
- Airway Remodeling, Airway Hyperresponsiveness, Inflammation
- Autophagy, ER Stress, Mitochondrial Dysfunction
- GPCRs (bronchodilators), Calcium homeostatis
- Pharmacology of Airways Disease
- Respiratory Pathophysiology
- Human Physiology & Pharmacology
Chen, H., Li, G., Chan, Y.L., Chapman, D.G., Sukjamnong, S., Nguyen, T., Annissa, T., McGrath, K.C., Sharma, P. & Oliver, B.G. 2018, 'Maternal E-Cigarette Exposure in Mice Alters DNA Methylation and Lung Cytokine Expression in Offspring.', American journal of respiratory cell and molecular biology, vol. 58, no. 3, pp. 366-377.View/Download from: UTS OPUS or Publisher's site
E-cigarette usage is increasing, especially among the young, with both the general population and physicians perceiving them as a safe alternative to tobacco smoking. Worryingly, e-cigarettes are commonly used by pregnant women. As nicotine is known to adversely affect children in utero, we hypothesized that nicotine delivered via e-cigarettes would negatively affect lung development. To test this, we developed a mouse model of maternal e-vapor (nicotine and nicotine-free) exposure and investigated the impact on the growth and lung inflammation in both offspring and mothers. Female Balb/c mice were exposed to e-fluid vapor containing nicotine (18 mg/ml nicotine E-cigarette [E-cig18], equivalent to two cigarettes per treatment, twice daily,) or nicotine free (E-cig0 mg/ml) from 6 weeks before mating until pups weaned. Male offspring were studied at Postnatal Day (P) 1, P20, and at 13 weeks. The mothers were studied when the pups weaned. In the mothers' lungs, e-cigarette exposure with and without nicotine increased the proinflammatory cytokines IL-1, IL-6, and TNF-. In adult offspring, TNF- protein levels were increased in both E-cig18 and E-cig0 groups, whereas IL-1 was suppressed. This was accompanied by global changes in DNA methylation. In this study, we found that e-cigarette exposure during pregnancy adversely affected maternal and offspring lung health. As this occurred with both nicotine-free and nicotine-containing e-vapor, the effects are likely due to by-products of vaporization rather than nicotine.
Pan, S., Sharma, P., Shah, S. & Deshpande, D. 2017, 'Bitter Taste Receptor Agonists Alter Mitochondrial Function and Induce Autophagy in Airway Smooth Muscle Cells', American Journal of Physiology - Lung Cellular and Molecular Physiology, vol. 313, no. 1, pp. L154-L165.View/Download from: UTS OPUS or Publisher's site
Bitter taste receptor
agonists alter mitochondrial function and induce autophagy in airway
smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 313:
L154–L165, 2017. First published April 27, 2017; doi:10.1152/
ajplung.00106.2017.—Airway remodeling, including increased airway
smooth muscle (ASM) mass, is a hallmark feature of asthma and
COPD. We previously identified the expression of bitter taste receptors
(TAS2Rs) on human ASM cells and demonstrated that known
TAS2R agonists could promote ASM relaxation and bronchodilation
and inhibit mitogen-induced ASM growth. In this study, we explored
cellular mechanisms mediating the antimitogenic effect of TAS2R
agonists on human ASM cells. Pretreatment of ASM cells with
TAS2R agonists chloroquine and quinine resulted in inhibition of cell
survival, which was largely reversed by bafilomycin A1, an autophagy
inhibitor. Transmission electron microscope studies demonstrated the
presence of double-membrane autophagosomes and deformed mitochondria.
In ASM cells, TAS2R agonists decreased mitochondrial
membrane potential and increased mitochondrial ROS and mitochondrial
fragmentation. Inhibiting dynamin-like protein 1 (DLP1) reversed
TAS2R agonist-induced mitochondrial membrane potential
change and attenuated mitochondrial fragmentation and cell death.
Furthermore, the expression of mitochondrial protein BCL2/
adenovirus E1B 19-kDa protein-interacting protein 3 (Bnip3) and
mitochondrial localization of DLP1 were significantly upregulated by
TAS2R agonists. More importantly, inhibiting Bnip3 mitochondrial
localization by dominant-negative Bnip3 significantly attenuated cell
death induced by TAS2R agonist. Collectively the TAS2R agonists
chloroquine and quinine modulate mitochondrial structure and function,
resulting in ASM cell death. Furthermore, Bnip3 plays a central
role in TAS2R agonist-induced ASM functional changes via a mitochondrial
pathway. These findings further establish the cellular mechanisms
of antimitogenic effe...
Sharma, P., Yi, R., Nayak, A., Wang, N., Knight, M.J., Pan, S., Oliver, B. & Deshpande, D.A. 2017, 'Bitter Taste Receptor Agonists Mitigate Features of Allergic Asthma in Mice.', Scientific Reports, vol. 7, pp. 1-14.View/Download from: UTS OPUS or Publisher's site
Kota, A., Deshpande, D., Haghi, M., Oliver, B. & Sharma, P. 2017, 'Autophagy and airway fibrosis: Is there a link?', F1000 Research, vol. 6, no. 409, pp. 1-10.View/Download from: UTS OPUS or Publisher's site
In the past decade, an emerging process named 'autophagy has generated intense interest in many chronic lung diseases. Tissue remodeling and fibrosis is a common feature of many airway diseases, and current therapies do not prevent or reverse these structural changes. Autophagy has evolved as a conserved process for bulk degradation and recycling of cytoplasmic components to maintain basal cellular homeostasis and healthy organelle populations in the cell. Furthermore, autophagy serves as a cell survival mechanism and can also be induced by chemical and physical stress to the cell. Accumulating evidence demonstrates that autophagy plays an essential role in vital cellular processes, including tissue remodeling. This review will discuss some of the recent advancements made in understanding the role of this fundamental process in airway fibrosis with emphasis on airway remodeling, and how autophagy can be exploited as a target for airway remodeling in asthma and chronic obstructive pulmonary disease.
Sukjamnong, S., Chan, Y.L., Zakarya, R., Saad, S., Sharma, P., Santiyanont, R., Chen, H. & Oliver, B.G. 2017, 'Effect of long-term maternal smoking on the offspring's lung health.', American Journal of Physiology - Lung Cellular and Molecular Physiology, vol. 313, no. 2, pp. L416-L423.View/Download from: UTS OPUS or Publisher's site
Maternal smoking during pregnancy contributes to long-term health problems in offspring, especially respiratory disorders that can manifest in either childhood or adulthood. Receptors for advanced glycation end products (RAGE) are multiligand receptors abundantly localized in the lung, capable of responding to by-products of reactive oxygen species and proinflammatory responses. RAGE signaling is a key regulator of inflammation in cigarette smoking-related pulmonary diseases. However, the impact of maternal cigarette smoke exposure on lung RAGE signaling in the offspring is unclear. This study aims to investigate the effect of maternal cigarette smoke exposure (SE), as well as mitochondria-targeted antioxidant [mitoquinone mesylate (MitoQ)] treatment, during pregnancy on the RAGE-mediated signaling pathway in the lung of male offspring. Female Balb/c mice (8 wk) were divided into a sham group (exposed to air), an SE group (exposed to cigarette smoke), and an SE + MQ group (exposed to cigarette smoke with MitoQ supplement from mating). The lungs from male offspring were collected at 13 wk. RAGE and its downstream signaling, including nuclear factor-B and mitogen-activated protein kinase family consisting of extracellular signal-regulated kinase 1, ERK2, c-JUN NH2-terminal kinase (JNK), and phosphorylated JNK, in the lung were significantly increased in the SE offspring. Mitochondrial antioxidant manganese superoxide dismutase was reduced, whereas IL-1 and oxidative stress response nuclear factor (erythroid-derived 2)-like 2 were significantly increased in the SE offspring. Maternal MitoQ treatment normalized RAGE, IL-1, and Nrf-2 levels in the SE + MQ offspring. Maternal SE increased RAGE and its signaling elements associated with increased oxidative stress and inflammatory cytokines in offspring lungs, whereas maternal MitoQ treatment can partially normalize these changes.
Sharma, P., Panebra, A., Pera, T., Tiegs, B.C., Hershfeld, A., Kenyon, L.C. & Deshpande, D.A. 2016, 'Antimitogenic effect of bitter taste receptor agonists on airway smooth muscle cells.', American journal of physiology. Lung cellular and molecular physiology, vol. 310, no. 4, pp. L365-L376.View/Download from: UTS OPUS or Publisher's site
Airway remodeling is a hallmark feature of asthma and chronic obstructive pulmonary disease. Clinical studies and animal models have demonstrated increased airway smooth muscle (ASM) mass, and ASM thickness is correlated with severity of the disease. Current medications control inflammation and reverse airway obstruction effectively but have limited effect on remodeling. Recently we identified the expression of bitter taste receptors (TAS2R) on ASM cells, and activation with known TAS2R agonists resulted in ASM relaxation and bronchodilation. These studies suggest that TAS2R can be used as new therapeutic targets in the treatment of obstructive lung diseases. To further establish their effectiveness, in this study we aimed to determine the effects of TAS2R agonists on ASM growth and promitogenic signaling. Pretreatment of healthy and asthmatic human ASM cells with TAS2R agonists resulted in a dose-dependent inhibition of ASM proliferation. The antimitogenic effect of TAS2R ligands was not dependent on activation of protein kinase A, protein kinase C, or high/intermediate-conductance calcium-activated K(+) channels. Immunoblot analyses revealed that TAS2R agonists inhibit growth factor-activated protein kinase B phosphorylation without affecting the availability of phosphatidylinositol 3,4,5-trisphosphate, suggesting TAS2R agonists block signaling downstream of phosphatidylinositol 3-kinase. Furthermore, the antimitogenic effect of TAS2R agonists involved inhibition of induced transcription factors (activator protein-1, signal transducer and activator of transcription-3, E2 factor, nuclear factor of activated T cells) and inhibition of expression of multiple cell cycle regulatory genes, suggesting a direct inhibition of cell cycle progression. Collectively, these findings establish the antimitogenic effect of TAS2R agonists and identify a novel class of receptors and signaling pathways that can be targeted to reduce or prevent airway remodeling as well as bronchoc...
Jha, A., Sharma, P., Anaparti, V., Ryu, M.H. & Halayko, A.J. 2015, 'A role for transient receptor potential ankyrin 1 cation channel (TRPA1) in airway hyper-responsiveness?', Canadian journal of physiology and pharmacology, vol. 93, no. 3, pp. 171-176.View/Download from: UTS OPUS or Publisher's site
Airway smooth muscle (ASM) contraction controls the airway caliber. Airway narrowing is exaggerated in obstructive lung diseases, such as asthma and chronic obstructive pulmonary disease (COPD). The mechanism by which ASM tone is dysregulated in disease is not clearly understood. Recent research on ion channels, particularly transient receptor potential cation channel, subfamily A, member 1 (TRPA1), is uncovering new understanding of altered airway function. TRPA1, a member of the TRP channel superfamily, is a chemo-sensitive cation channel that can be activated by a variety of external and internal stimuli, leading to the influx of Ca(2+). Functional TRPA1 channels have been identified in neuronal and non-neuronal tissues of the lung, including ASM. In the airways, these channels can regulate the release of mediators that are markers of airway inflammation in asthma and COPD. For, example, TRPA1 controls cigarette-smoke-induced inflammatory mediator release and Ca(2+) mobilization in vitro and in vivo, a response tied to disease pathology in COPD. Recent work has revealed that pharmacological or genetic inhibition of TRPA1 inhibits the allergen-induced airway inflammation in vitro and airway hyper-responsiveness (AHR) in vivo. Collectively, it appears that TRPA1 channels may be determinants of ASM contractility and local inflammation control, positioning them as part of novel mechanisms that control (patho)physiological function of airways and ASM.
Sharma, P., Jha, A., Stelmack, G.L., Detillieux, K., Basu, S., Klonisch, T., Unruh, H. & Halayko, A.J. 2015, 'Characterization of the dystrophin-glycoprotein complex in airway smooth muscle: role of -sarcoglycan in airway responsiveness.', Canadian journal of physiology and pharmacology, vol. 93, no. 3, pp. 195-202.View/Download from: UTS OPUS or Publisher's site
The dystrophin-glycoprotein complex (DGC) is an integral part of caveolae microdomains, and its interaction with caveolin-1 is essential for the phenotype and functional properties of airway smooth muscle (ASM). The sarcoglycan complex provides stability to the dystroglycan complex, but its role in ASM contraction and lung physiology in not understood. We tested whether -sarcoglycan (-SG), through its interaction with the DGC, is a determinant of ASM contraction ex vivo and airway mechanics in vivo. We measured methacholine (MCh)-induced isometric contraction and airway mechanics in -SG KO and wild-type mice. Last, we performed immunoblotting and transmission electron microscopy to assess DGC protein expression and the ultrastructural features of tracheal smooth muscle. Our results reveal an age-dependent reduction in the MCh-induced tracheal isometric force and significant reduction in airway resistance at high concentrations of MCh (50.0 mg/mL) in -SG KO mice. The changes in contraction and lung function correlated with decreased caveolin-1 and -dystroglycan abundance, as well as an age-dependent loss of caveolae in the cell membrane of tracheal smooth muscle in -SG KO mice. Collectively, these results confirm and extend understanding of a functional role for the DGC in the contractile properties of ASM and demonstrate that this results in altered lung function in vivo.
Boardman, C., Chachi, L., Gavrila, A., Keenan, C.R., Perry, M.M., Xia, Y.C., Meurs, H. & Sharma, P. 2014, 'Mechanisms of glucocorticoid action and insensitivity in airways disease.', Pulmonary Pharmacology and Therapeutics, vol. 29, no. 2, pp. 129-143.View/Download from: UTS OPUS or Publisher's site
Glucocorticoids are the mainstay for the treatment of chronic inflammatory diseases including asthma and chronic obstructive pulmonary disease (COPD). However, it has been recognized that glucocorticoids do not work well in certain patient populations suggesting reduced sensitivity. The ultimate biologic responses to glucocorticoids are determined by not only the concentration of glucocorticoids but also the differences between individuals in glucocorticoid sensitivity, which is influenced by multiple factors. Studies are emerging to understand these mechanisms in detail, which would help in increasing glucocorticoid sensitivity in patients with chronic airways disease. This review aims to highlight both classical and emerging concepts of the anti-inflammatory mechanisms of glucocorticoids and also review some novel strategies to overcome steroid insensitivity in airways disease.
Ghavami, S., Sharma, P., Yeganeh, B., Ojo, O.O., Jha, A., Mutawe, M.M., Kashani, H.H., Los, M.J., Klonisch, T., Unruh, H. & Halayko, A.J. 2014, 'Airway mesenchymal cell death by mevalonate cascade inhibition: integration of autophagy, unfolded protein response and apoptosis focusing on Bcl2 family proteins.', Biochimica et Biophysica Acta - Molecular Cell Research, vol. 1843, no. 7, pp. 1259-1271.View/Download from: UTS OPUS or Publisher's site
HMG-CoA reductase, the proximal rate-limiting enzyme in the mevalonate pathway, is inhibited by statins. Beyond their cholesterol lowering impact, statins have pleiotropic effects and their use is linked to improved lung health. We have shown that mevalonate cascade inhibition induces apoptosis and autophagy in cultured human airway mesenchymal cells. Here, we show that simvastatin also induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in these cells. We tested whether coordination of ER stress, autophagy and apoptosis determines survival or demise of human lung mesenchymal cells exposed to statin. We observed that simvastatin exposure activates UPR (activated transcription factor 4, activated transcription factor 6 and IRE1) and caspase-4 in primary human airway fibroblasts and smooth muscle cells. Exogenous mevalonate inhibited apoptosis, autophagy and UPR, but exogenous cholesterol was without impact, indicating that sterol intermediates are involved with mechanisms mediating statin effects. Caspase-4 inhibition decreased simvastatin-induced apoptosis, whereas inhibition of autophagy by ATG7 or ATG3 knockdown significantly increased cell death. In BAX(-/-)/BAK(-/-) murine embryonic fibroblasts, simvastatin-triggered apoptotic and UPR events were abrogated, but autophagy flux was increased leading to cell death via necrosis. Our data indicate that mevalonate cascade inhibition, likely associated with depletion of sterol intermediates, can lead to cell death via coordinated apoptosis, autophagy, and ER stress. The interplay between these pathways appears to be principally regulated by autophagy and Bcl-2-family pro-apoptotic proteins. These findings uncover multiple mechanisms of action of statins that could contribute to refining the use of such agent in treatment of lung disease.
Sharma, P., Basu, S., Mitchell, R.W., Stelmack, G.L., Anderson, J.E. & Halayko, A.J. 2014, 'Role of dystrophin in airway smooth muscle phenotype, contraction and lung function.', PLoS ONE, vol. 9, no. 7, pp. 1-13.View/Download from: UTS OPUS or Publisher's site
Dystrophin links the transmembrane dystrophin-glycoprotein complex to the actin cytoskeleton. We have shown that dystrophin-glycoprotein complex subunits are markers for airway smooth muscle phenotype maturation and together with caveolin-1, play an important role in calcium homeostasis. We tested if dystrophin affects phenotype maturation, tracheal contraction and lung physiology. We used dystrophin deficient Golden Retriever dogs (GRMD) and mdx mice vs healthy control animals in our approach. We found significant reduction of contractile protein markers: smooth muscle myosin heavy chain (smMHC) and calponin and reduced Ca2+ response to contractile agonist in dystrophin deficient cells. Immunocytochemistry revealed reduced stress fibers and number of smMHC positive cells in dystrophin-deficient cells, when compared to control. Immunoblot analysis of Akt1, GSK3 and mTOR phosphorylation further revealed that downstream PI3K signaling, which is essential for phenotype maturation, was suppressed in dystrophin deficient cell cultures. Tracheal rings from mdx mice showed significant reduction in the isometric contraction to methacholine (MCh) when compared to genetic control BL10ScSnJ mice (wild-type). In vivo lung function studies using a small animal ventilator revealed a significant reduction in peak airway resistance induced by maximum concentrations of inhaled MCh in mdx mice, while there was no change in other lung function parameters. These data show that the lack of dystrophin is associated with a concomitant suppression of ASM cell phenotype maturation in vitro, ASM contraction ex vivo and lung function in vivo, indicating that a linkage between the DGC and the actin cytoskeleton via dystrophin is a determinant of the phenotype and functional properties of ASM.
Yeganeh, B., Wiechec, E., Ande, S.R., Sharma, P., Moghadam, A.R., Post, M., Freed, D.H., Hashemi, M., Shojaei, S., Zeki, A.A. & Ghavami, S. 2014, 'Targeting the mevalonate cascade as a new therapeutic approach in heart disease, cancer and pulmonary disease.', Pharmacology and Therapeutics, vol. 143, no. 1, pp. 87-110.View/Download from: UTS OPUS or Publisher's site
The cholesterol biosynthesis pathway, also known as the mevalonate (MVA) pathway, is an essential cellular pathway that is involved in diverse cell functions. The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGCR) is the rate-limiting step in cholesterol biosynthesis and catalyzes the conversion of HMG-CoA to MVA. Given its role in cholesterol and isoprenoid biosynthesis, the regulation of HMGCR has been intensely investigated. Because all cells require a steady supply of MVA, both the sterol (i.e. cholesterol) and non-sterol (i.e. isoprenoid) products of MVA metabolism exert coordinated feedback regulation on HMGCR through different mechanisms. The proper functioning of HMGCR as the proximal enzyme in the MVA pathway is essential under both normal physiologic conditions and in many diseases given its role in cell cycle pathways and cell proliferation, cholesterol biosynthesis and metabolism, cell cytoskeletal dynamics and stability, cell membrane structure and fluidity, mitochondrial function, proliferation, and cell fate. The blockbuster statin drugs ('statins') directly bind to and inhibit HMGCR, and their use for the past thirty years has revolutionized the treatment of hypercholesterolemia and cardiovascular diseases, in particular coronary heart disease. Initially thought to exert their effects through cholesterol reduction, recent evidence indicates that statins also have pleiotropic immunomodulatory properties independent of cholesterol lowering. In this review we will focus on the therapeutic applications and mechanisms involved in the MVA cascade including Rho GTPase and Rho kinase (ROCK) signaling, statin inhibition of HMGCR, geranylgeranyltransferase (GGTase) inhibition, and farnesyltransferase (FTase) inhibition in cardiovascular disease, pulmonary diseases (e.g. asthma and chronic obstructive pulmonary disease (COPD)), and cancer.
Moodley, T., Wilson, S.M., Joshi, T., Rider, C.F., Sharma, P., Yan, D., Newton, R. & Giembycz, M.A. 2013, 'Phosphodiesterase 4 inhibitors augment the ability of formoterol to enhance glucocorticoid-dependent gene transcription in human airway epithelial cells: a novel mechanism for the clinical efficacy of roflumilast in severe chronic obstructive pulmonary disease.', Molecular pharmacology, vol. 83, no. 4, pp. 894-906.View/Download from: UTS OPUS or Publisher's site
Post-hoc analysis of two phase III clinical studies found that the phosphodiesterase 4 (PDE4) inhibitor, roflumilast, reduced exacerbation frequency in patients with severe chronic obstructive pulmonary disease (COPD) who were taking inhaled corticosteroids (ICS) concomitantly, whereas patients not taking ICS derived no such benefit. In contrast, in two different trials also performed in patients with severe COPD, roflumilast reduced exacerbation rates in the absence of ICS, indicating that PDE4 inhibition alone is sufficient for therapeutic activity to be realized. Given that roflumilast is recommended as an "add-on" medication to patients with severe disease who will inevitably be taking a long-acting 2-adrenoceptor agonist (LABA)/ICS combination therapy, we tested the hypothesis that roflumilast augments the ability of glucocorticoids to induce genes with anti-inflammatory activity. Using a glucocorticoid response element (GRE) luciferase reporter transfected into human airway epithelial cells [both bronchial epithelium + adenovirus 12 - SV40 hybrid (BEAS-2B) cells and primary cultures], roflumilast enhanced fluticasone propionate-induced GRE-dependent transcription. Roflumilast also produced a sinistral displacement of the concentration-response curves that described the augmentation of GRE-dependent transcription by the LABA formoterol. In BEAS-2B cells and primary airway epithelia, roflumilast interacted with formoterol in a positive cooperative manner to enhance the expression of several glucocorticoid-inducible genes that have anti-inflammatory potential. We suggest that the ability of roflumilast and formoterol to interact in this way supports the concept that these drugs together may impart clinical benefit beyond that achievable by an ICS alone, a PDE4 inhibitor alone, or an ICS/LABA combination therapy. Roflumilast may, therefore, be especially effective in patients with severe COPD.
Wright, D., Sharma, P., Ryu, M.-.H., Rissé, P.-.A., Ngo, M., Maarsingh, H., Koziol-White, C., Jha, A., Halayko, A.J. & West, A.R. 2013, 'Models to study airway smooth muscle contraction in vivo, ex vivo and in vitro: implications in understanding asthma.', Pulmonary pharmacology & therapeutics, vol. 26, no. 1, pp. 24-36.View/Download from: UTS OPUS or Publisher's site
Asthma is a chronic obstructive airway disease characterised by airway hyperresponsiveness (AHR) and airway wall remodelling. The effector of airway narrowing is the contraction of airway smooth muscle (ASM), yet the question of whether an inherent or acquired dysfunction in ASM contractile function plays a significant role in the disease pathophysiology remains contentious. The difficulty in determining the role of ASM lies in limitations with the models used to assess contraction. In vivo models provide a fully integrated physiological response but ASM contraction cannot be directly measured. Ex vivo and in vitro models can provide more direct assessment of ASM contraction but the loss of factors that may modulate ASM responsiveness and AHR, including interaction between multiple cell types and disruption of the mechanical environment, precludes a complete understanding of the disease process. In this review we detail key advantages of common in vivo, ex vivo and in vitro models of ASM contraction, as well as emerging tissue engineered models of ASM and whole airways. We also highlight important findings from each model with respect to the pathophysiology of asthma.
Gerthoffer, W.T., Schaafsma, D., Sharma, P., Ghavami, S. & Halayko, A.J. 2012, 'Motility, Survival, and Proliferation', COMPREHENSIVE PHYSIOLOGY, vol. 2, no. 1, pp. 255-281.View/Download from: Publisher's site
Gerthoffer, W.T., Schaafsma, D., Sharma, P., Ghavami, S. & Halayko, A.J. 2012, 'Motility, survival, and proliferation.', Comprehensive Physiology, vol. 2, no. 1, pp. 255-281.View/Download from: UTS OPUS or Publisher's site
Airway smooth muscle has classically been of interest for its contractile response linked to bronchoconstriction. However, terminally differentiated smooth muscle cells are phenotypically plastic and have multifunctional capacity for proliferation, cellular hypertrophy, migration, and the synthesis of extracellular matrix and inflammatory mediators. These latter properties of airway smooth muscle are important in airway remodeling which is a structural alteration that compounds the impact of contractile responses on limiting airway conductance. In this overview, we describe the important signaling components and the functional evidence supporting a view of smooth muscle cells at the core of fibroproliferative remodeling of hollow organs. Signal transduction components and events are summarized that control the basic cellular processes of proliferation, cell survival, apoptosis, and cellular migration. We delineate known intracellular control mechanisms and suggest future areas of interest to pursue to more fully understand factors that regulate normal myocyte function and airway remodeling in obstructive lung diseases.
Ghavami, S., Yeganeh, B., Stelmack, G.L., Kashani, H.H., Sharma, P., Cunnington, R., Rattan, S., Bathe, K., Klonisch, T., Dixon, I.M.C., Freed, D.H. & Halayko, A.J. 2012, 'Apoptosis, autophagy and ER stress in mevalonate cascade inhibition-induced cell death of human atrial fibroblasts.', Cell death & disease, vol. 3, p. e330.View/Download from: UTS OPUS or Publisher's site
3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors (statins) are cholesterol-lowering drugs that exert other cellular effects and underlie their beneficial health effects, including those associated with myocardial remodeling. We recently demonstrated that statins induces apoptosis and autophagy in human lung mesenchymal cells. Here, we extend our knowledge showing that statins simultaneously induces activation of the apoptosis, autophagy and the unfolded protein response (UPR) in primary human atrial fibroblasts (hATF). Thus we tested the degree to which coordination exists between signaling from mitochondria, endoplasmic reticulum and lysosomes during response to simvastatin exposure. Pharmacologic blockade of the activation of ER-dependent cysteine-dependent aspartate-directed protease (caspase)-4 and lysosomal cathepsin-B and -L significantly decreased simvastatin-induced cell death. Simvastatin altered total abundance and the mitochondrial fraction of proapoptotic and antiapoptotic proteins, while c-Jun N-terminal kinase/stress-activated protein kinase mediated effects on B-cell lymphoma 2 expression. Chemical inhibition of autophagy flux with bafilomycin-A1 augmented simvastatin-induced caspase activation, UPR and cell death. In mouse embryonic fibroblasts that are deficient in autophagy protein 5 and refractory to autophagy induction, caspase-7 and UPR were hyper-induced upon treatment with simvastatin. These data demonstrate that mevalonate cascade inhibition-induced death of hATF manifests from a complex mechanism involving co-regulation of apoptosis, autophagy and UPR. Furthermore, autophagy has a crucial role in determining the extent of ER stress, UPR and permissiveness of hATF to cell death induced by statins.
Sharma, P., Ryu, M.H., Basu, S., Maltby, S.A., Yeganeh, B., Mutawe, M.M., Mitchell, R.W. & Halayko, A.J. 2012, 'Epithelium-dependent modulation of responsiveness of airways from caveolin-1 knockout mice is mediated through cyclooxygenase-2 and 5-lipoxygenase.', British journal of pharmacology, vol. 167, no. 3, pp. 548-560.View/Download from: UTS OPUS or Publisher's site
Acute silencing of caveolin-1 (Cav-1) modulates receptor-mediated contraction of airway smooth muscle. Moreover, COX-2- and 5-lipoxygenase (5-LO)-derived prostaglandin and leukotriene biosynthesis can influence smooth muscle reactivity. COX-2 half-life can be prolonged through association with Cav-1. We suggested that lack of Cav-1 modulated levels of COX-2 which in turn modulated tracheal contraction, when arachidonic acid signalling was disturbed by inhibition of COX-2.Using tracheal rings from Cav-1 knockout (KO) and wild-type mice (B6129SF2/J), we measured isometric contractions to methacholine and used PCR, immunoblotting and immunohistology to monitor expression of relevant proteins.Tracheal rings from Cav-1 KO and wild-type mice exhibited similar responses, but the COX-2 inhibitor, indomethacin, increased responses of tracheal rings from Cav-1 KO mice to methacholine. The phospholipase A inhibitor, eicosatetraynoic acid, which inhibits formation of both COX-2 and 5-LO metabolites, had no effect on wild-type or Cav-1 KO tissues. Indomethacin-mediated hyperreactivity was ablated by the LTD receptor antagonist (montelukast) and 5-LO inhibitor (zileuton). The potentiating effect of indomethacin on Cav-1 KO responses to methacholine was blocked by epithelial denudation. Immunoprecipitation showed that COX-2 binds Cav-1 in wild-type lungs. Immunoblotting and qPCR revealed elevated levels of COX-2 and 5-LO protein, but not COX-1, in Cav-1 KO tracheas, a feature that was prevented by removal of the epithelium.The indomethacin-induced hypercontractility observed in Cav-1 KO tracheas was linked to increased expression of COX-2 and 5-LO, which probably enhanced arachidonic acid shunting and generation of pro-contractile leukotrienes when COX-2 was inhibited.
Ghavami, S., Mutawe, M.M., Sharma, P., Yeganeh, B., McNeill, K.D., Klonisch, T., Unruh, H., Kashani, H.H., Schaafsma, D., Los, M. & Halayko, A.J. 2011, 'Mevalonate cascade regulation of airway mesenchymal cell autophagy and apoptosis: a dual role for p53.', PloS one, vol. 6, no. 1, p. e16523.View/Download from: UTS OPUS or Publisher's site
Statins inhibit the proximal steps of cholesterol biosynthesis, and are linked to health benefits in various conditions, including cancer and lung disease. We have previously investigated apoptotic pathways triggered by statins in airway mesenchymal cells, and identified reduced prenylation of small GTPases as a primary effector mechanism leading to p53-mediated cell death. Here, we extend our studies of statin-induced cell death by assessing endpoints of both apoptosis and autophagy, and investigating their interplay and coincident regulation. Using primary cultured human airway smooth muscle (HASM) and human airway fibroblasts (HAF), autophagy, and autophagosome formation and flux were assessed by transmission electron microscopy, cytochemistry (lysosome number and co-localization with LC3) and immunoblotting (LC3 lipidation and Atg12-5 complex formation). Chemical inhibition of autophagy increased simvastatin-induced caspase activation and cell death. Similarly, Atg5 silencing with shRNA, thus preventing Atg5-12 complex formation, increased pro-apoptotic effects of simvastatin. Simvastatin concomitantly increased p53-dependent expression of p53 up-regulated modulator of apoptosis (PUMA), NOXA, and damage-regulated autophagy modulator (DRAM). Notably both mevalonate cascade inhibition-induced autophagy and apoptosis were p53 dependent: simvastatin increased nuclear p53 accumulation, and both cyclic pifithrin- and p53 shRNAi partially inhibited NOXA, PUMA expression and caspase-3/7 cleavage (apoptosis) and DRAM expression, Atg5-12 complex formation, LC3 lipidation, and autophagosome formation (autophagy). Furthermore, the autophagy response is induced rapidly, significantly delaying apoptosis, suggesting the existence of a temporally coordinated p53 regulation network. These findings are relevant for the development of statin-based therapeutic approaches in obstructive airway disease.
Khanna, A., Raj, V.S., Tarai, B., Sood, R., Pareek, P.K., Upadhyay, D.J., Sharma, P., Rattan, A., Saini, K.S. & Singh, H. 2010, 'Emergence and molecular characterization of extensively drug-resistant Mycobacterium tuberculosis clinical isolates from the Delhi Region in India.', Antimicrobial agents and chemotherapy, vol. 54, no. 11, pp. 4789-4793.View/Download from: Publisher's site
We screened 194 Mycobacterium tuberculosis strains isolated from tuberculosis (TB) patients in Delhi and neighboring regions in India to identify the prevalence of extensive drug resistance (XDR) in clinical isolates. Among these, 104 isolates were found to be multidrug resistant (MDR), and 6 were identified as XDR isolates, which was later confirmed by antimicrobial susceptibility testing against the respective drug screening panel. Genotyping was carried out by amplifying and sequencing the following genes: rpoB (rifampin), katG (isoniazid), gyrA (fluoroquinolones), and rrs (amikacin, kanamycin, and capreomycin). Our analyses indicated that mutations at the hot spots of these genes were positively correlated with drug resistance in clinical isolates. The key mutation observed for rpoB was in the codon for amino acid position 531 (S531L), and other mutations were seen in the hot spot, including those encoding Q510P, L511H, D516V, and H526Y mutations. We identified S315T and R463L substitutions encoded in the katG locus. An S95T substitution encoded in the gyrA locus was the most common mutation observed in fluoroquinolone-resistant isolates. In addition, we saw D94G and D94N mutations encoded in the QRDR region. The 16S rRNA (rrs) gene encoded mainly the A1401G mutation and an additional mutation, G1484T, resulting in ribosomal modifications. Taken together, the data in this report clearly establish the presence of phenotypically distinct XDR strains in India by molecular profiling and further identify specific mutational hot spots within key genes of XDR-TB strains.
Sharma, P. & Kumar, A. 2010, 'Immunoinformatics: Screening of potential T cell antigenic determinants in proteome of H1N1 swine influenza virus for virus epitope vaccine design', Journal of Proteomics and Bioinformatics, vol. 3, no. 9, pp. 275-278.View/Download from: Publisher's site
Influenza (H1N1) is a highly contagious respiratory pathogen that continues to evolve and threaten both veterinary and human public health. Presently existing vaccines against influenza (H1N1) are based on the generation of secondary response in form of neutralizing antibody primarily directed against surface proteins-hemagglutinin and neuraminidase. In this work, Propred and Propred I immunoinformatics tools have been used to predict the T cell epitopes from seven putative protein viz. Hemagglutinin, neuraminidase, polymerase PA, nucleocapsid protein, matrix protein, polymerase PB1, polymerase PB2 of influenza virus A/Minnesota/2009 (H1N1). Total of 15 epitopes were predicted for HLA class I and 14 epitopes for HLA class II molecules. These epitopes are found top scoring peptides and showed binding with maximum number of HLA alleles. The threshold percent taken in this analysis was 4% to select high affinity peptides by Propred and Propred I. The predicted epitopes may be served as a useful diagnostic reagent for evaluating T-cell responses in the context of natural infection and also might be helpful for designing of either a DNA vaccine or a subunit vaccine against H1N1 influenza. © 2010 Sharma P, et al.
Ghavami, S., Mutawe, M.M., Hauff, K., Stelmack, G.L., Schaafsma, D., Sharma, P., McNeill, K.D., Hynes, T.S., Kung, S.K., Unruh, H., Klonisch, T., Hatch, G.M., Los, M. & Halayko, A.J. 2010, 'Statin-triggered cell death in primary human lung mesenchymal cells involves p53-PUMA and release of Smac and Omi but not cytochrome c.', Biochimica et biophysica acta, vol. 1803, no. 4, pp. 452-467.View/Download from: Publisher's site
Statins inhibit 3-hydroxy-3-methyl-glutarylcoenzyme CoA (HMG-CoA) reductase, the proximal enzyme for cholesterol biosynthesis. They exhibit pleiotropic effects and are linked to health benefits for diseases including cancer and lung disease. Understanding their mechanism of action could point to new therapies, thus we investigated the response of primary cultured human airway mesenchymal cells, which play an effector role in asthma and chronic obstructive lung disease (COPD), to simvastatin exposure. Simvastatin induced apoptosis involving caspase-9, -3 and -7, but not caspase-8 in airway smooth muscle cells and fibroblasts. HMG-CoA inhibition did not alter cellular cholesterol content but did abrogate de novo cholesterol synthesis. Pro-apoptotic effects were prevented by exogenous mevalonate, geranylgeranyl pyrophosphate and farnesyl pyrophosphate, downstream products of HMG-CoA. Simvastatin increased expression of Bax, oligomerization of Bax and Bak, and expression of BH3-only p53-dependent genes, PUMA and NOXA. Inhibition of p53 and silencing of p53 unregulated modulator of apoptosis (PUMA) expression partly counteracted simvastatin-induced cell death, suggesting a role for p53-independent mechanisms. Simvastatin did not induce mitochondrial release of cytochrome c, but did promote release of inhibitor of apoptosis (IAP) proteins, Smac and Omi. Simvastatin also inhibited mitochondrial fission with the loss of mitochondrial Drp1, an essential component of mitochondrial fission machinery. Thus, simvastatin activates novel apoptosis pathways in lung mesenchymal cells involving p53, IAP inhibitor release, and disruption of mitochondrial fission.
Hashemi, M., Sharma, P., Eshraghi, M., Naderi, M., Moazeni-Roodi, A., Mehrabifar, H. & Taheri, M. 2010, 'Alpha-1 Antitrypsin: It's Role in Health and Disease.', Anti-Inflammatory and Anti-Allergy Agents in Medicinal Chemistry., vol. 9, no. 4, pp. 279-288.
Sharma, P., Ghavami, S., Stelmack, G.L., McNeill, K.D., Mutawe, M.M., Klonisch, T., Unruh, H. & Halayko, A.J. 2010, 'beta-Dystroglycan binds caveolin-1 in smooth muscle: a functional role in caveolae distribution and Ca2+ release.', Journal of cell science, vol. 123, no. Pt 18, pp. 3061-3070.View/Download from: Publisher's site
The dystrophin-glycoprotein complex (DGC) links the extracellular matrix and actin cytoskeleton. Caveolae form membrane arrays on smooth muscle cells; we investigated the mechanism for this organization. Caveolin-1 and beta-dystroglycan, the core transmembrane DGC subunit, colocalize in airway smooth muscle. Immunoprecipitation revealed the association of caveolin-1 with beta-dystroglycan. Disruption of actin filaments disordered caveolae arrays, reduced association of beta-dystroglycan and caveolin-1 to lipid rafts, and suppressed the sensitivity and responsiveness of methacholine-induced intracellular Ca2+ release. We generated novel human airway smooth muscle cell lines expressing shRNA to stably silence beta-dystroglycan expression. In these myocytes, caveolae arrays were disorganized, caveolae structural proteins caveolin-1 and PTRF/cavin were displaced, the signaling proteins PLCbeta1 and G(alphaq), which are required for receptor-mediated Ca2+ release, were absent from caveolae, and the sensitivity and responsiveness of methacholine-induced intracellular Ca2+ release, was diminished. These data reveal an interaction between caveolin-1 and beta-dystroglycan and demonstrate that this association, in concert with anchorage to the actin cytoskeleton, underpins the spatial organization and functional role of caveolae in receptor-mediated Ca2+ release, which is an essential initiator step in smooth muscle contraction.
Dastidar, S.G., Ray, A., Shirumalla, R., Rajagopal, D., Chaudhary, S., Nanda, K., Sharma, P., Seth, M.K., Balachandran, S., Gupta, N. & Palle, V. 2009, 'Pharmacology of a novel, orally active PDE4 inhibitor.', Pharmacology, vol. 83, no. 5, pp. 275-286.View/Download from: Publisher's site
BACKGROUND: Intracellular cyclic adenosine monophosphate (cAMP) in inflammatory cells and airway smooth muscle is critical to the modulation of inflammatory response generation. Phosphodiesterase 4 (PDE4), an enzyme that catalyzes cAMP degradation, is therefore being actively explored as a molecular target for the treatment of airway inflammation, particularly asthma and chronic obstructive pulmonary disease. The field has undergone major advances in optimizing generation of compounds with a safe therapeutic margin; however, most PDE4 inhibitors tested so far have unacceptable side effects, particularly nausea and vomiting. METHODS: We evaluated NIS-62949 in a wide range of in vitro and ex vivo cell-based assays to ascertain its anti-inflammatory potential. The compound was evaluated in murine models of lipopolysaccharide-induced endotoxemia and pulmonary neutrophilia. Parameters of airway inflammation, airway hyperreactivity and bronchoconstriction were evaluated in a guinea pig model of antigen-induced allergy. In order to assess the emetic potential, the compound was evaluated biochemically for binding to high-affinity rolipram-binding site. Subsequently, the compound was tested in a surrogate model for emesis, and the results obtained were correlated directly to tests conducted in a Beagle dog model. RESULTS: NIS-62949 is a potent, highly selective PDE4 inhibitor. The compound demonstrated potent ability to inhibit tumor necrosis factor-alpha release from human peripheral blood mononuclear cells, lymphocyte proliferation and cytokine production. The in vitro profile of NIS-62949 prompted further evaluation of the compound in vivo and the compound was found to be comparable to roflumilast in several experimental models of pulmonary inflammation. Importantly, NIS-62949 displayed a safer profile compared to roflumilast. CONCLUSIONS: Our results report the development of a promising, novel PDE4 inhibitor, NIS-62949, with a wider therapeutic window as compared to ...
Dastidar, S.G., Ray, A., Shirumalla, R., Rajgopal, D., Chaudhary, S., Nanda, K., Sharma, P., Seth, M.K., Balachandran, S., Gupta, N. & Palle, V. 2009, 'Pharmacology of a Novel, Orally Active PDE4 Inhibitor.', Pharmacology, vol. Pharmacology, no. 83 (5), pp. 275-286.View/Download from: UTS OPUS
Halayko, A.J. & Sharma, P. 2009, 'S100A8/A9 as a pro-inflammatory cytokine in obstructive airway disease via the multi-ligand receptor, RAGE', Anti-Inflammatory and Anti-Allergy Agents in Medicinal Chemistry, vol. 8, no. 4, pp. 306-317.View/Download from: Publisher's site
Asthma and chronic obstructive pulmonary disease (COPD) are persistent inflammatory conditions that have exhibited significantly increased prevalence in the past two decades. Though many current medications relieve the symptoms of obstructive airway disease, morbidity can still increase over time in individual patients. With particular respect to asthma, despite satisfactory control of symptoms in most patients with inhaled steroids, a sub-phenotype of subjects, representing 15% of all asthmatics, do not respond to steroids - these patients can exhibit severe asthma, which accounts for 50% of asthma health care costs. Moreover, inhaled steroids are not recommended as a sole therapy for COPD, and there is limited evidence for their effectiveness in preventing disease pathogenesis. Thus, it is important to better understand mechanisms for severe asthma and COPD and identify mediators released by cells, such as neutrophils, that are unresponsive to steroid therapy. This review focuses on the probable role of one the most abundant neutrophil proteins, called S100A8/A9, in asthma. S100A8/A9 is released in abundance in rheumatoid arthritis, inflammatory bowel disease and cancer, but there are no definitive studies on its role in obstructive airways disease. A primary receptor for S100A8/A9, which is uniquely expressed in high abundance in the lung, is the multi-ligand receptor for advanced glycated endproducts (RAGE) of the immunoglobin-like receptor family. RAGE participates in mediating fibroproliferative lung remodeling in idiopathic pulmonary fibrosis, and in bleomycin-exposed animal models. This review provides an overview of the S100A8/A9-RAGE axis, and discusses its potential in mediating chronic airway inflammation and tissue remodeling in asthma and COPD. © 2009 Bentham Science Publishers Ltd.
Hirota, J.A., Nguyen, T.T.B., Schaafsma, D., Sharma, P. & Tran, T. 2009, 'Airway smooth muscle in asthma: phenotype plasticity and function.', Pulmonary pharmacology & therapeutics, vol. 22, no. 5, pp. 370-378.View/Download from: Publisher's site
Clinical asthma is characterized by reversible airway obstruction which is commonly due to an exaggerated airway narrowing referred to as airway hyperresponsiveness (AHR). Although debate exists on the complex etiology of AHR, it is clear that airway smooth muscle (ASM) mediated airway narrowing is a major contributor to airway dysfunction. More importantly, it is now appreciated that smooth muscle is far from being a simple cell with only contractile ability properties. Rather, it is more versatile with the capacity to exhibit numerous cellular functions as it adapts to the microenvironment to which it is exposed. The emerging ability of individual smooth muscle cells to undergo changes in their phenotype (phenotype plasticity) and function (functional plasticity) in response to physiological and pathological cues is an important and active area of research. This article provides a brief review of the current knowledge and emerging concepts in the field of ASM phenotype and function both under healthy and asthmatic conditions.
Sharma, P. & Halayko, A.J. 2009, 'Emerging molecular targets for the treatment of asthma', Indian Journal of Biochemistry and Biophysics, vol. 46, no. 6, pp. 447-460.
Current therapeutic approaches for the treatment of asthma have limitations in their ability to target all the features of the disease. Indeed, existing pharmacological asthma therapies are based on decades old strategies that were developed prior to the rapid growth in knowledge stemming from cell and molecular biology in the past decade. Thus, there is an unmet need for developing new drugs to target these features along with improved efficacy and safety. In the present review, the limitations of prevalent pharmacological asthma therapy are discussed briefly, and some explanations are suggested as to why new therapeutic targets are required to treat asthma, and finally directions for novel asthma therapies are proposed.
Sharma, P. & Halayko, A.J. 2009, 'Emerging molecular targets for the treatment of asthma.', Indian journal of biochemistry & biophysics, vol. 46, no. 6, pp. 447-460.
Current therapeutic approaches for the treatment of asthma have limitations in their ability to target all the features of the disease. Indeed, existing pharmacological asthma therapies are based on decades old strategies that were developed prior to the rapid growth in knowledge stemming from cell and molecular biology in the past decade. Thus, there is an unmet need for developing new drugs to target these features along with improved efficacy and safety. In the present review, the limitations of prevalent pharmacological asthma therapy are discussed briefly, and some explanations are suggested as to why new therapeutic targets are required to treat asthma, and finally directions for novel asthma therapies are proposed.
Sharma, P., Tran, T., Stelmack, G.L., McNeill, K., Gosens, R., Mutawe, M.M., Unruh, H., Gerthoffer, W.T. & Halayko, A.J. 2008, 'Expression of the dystrophin-glycoprotein complex is a marker for human airway smooth muscle phenotype maturation.', American journal of physiology. Lung cellular and molecular physiology, vol. 294, no. 1, pp. L57-L68.View/Download from: Publisher's site
Airway smooth muscle (ASM) cells may contribute to asthma pathogenesis through their capacity to switch between a synthetic/proliferative and a contractile phenotype. The multimeric dystrophin-glycoprotein complex (DGC) spans the sarcolemma, linking the actin cytoskeleton and extracellular matrix. The DGC is expressed in smooth muscle tissue, but its functional role is not fully established. We tested whether contractile phenotype maturation of human ASM is associated with accumulation of DGC proteins. We compared subconfluent, serum-fed cultures and confluent cultures subjected to serum deprivation, which express a contractile phenotype. Western blotting confirmed that beta-dystroglycan, beta-, delta-, and epsilon-sarcoglycan, and dystrophin abundance increased six- to eightfold in association with smooth muscle myosin heavy chain (smMHC) and calponin accumulation during 4-day serum deprivation. Immunocytochemistry showed that the accumulation of DGC subunits was specifically localized to a subset of cells that exhibit robust staining for smMHC. Laminin competing peptide (YIGSR, 1 microM) and phosphatidylinositol 3-kinase (PI3K) inhibitors (20 microM LY-294002 or 100 nM wortmannin) abrogated the accumulation of smMHC, calponin, and DGC proteins. These studies demonstrate that the accumulation of DGC is an integral feature for phenotype maturation of human ASM cells. This provides a strong rationale for future studies investigating the role of the DGC in ASM smooth muscle physiology in health and disease.
Shirumalla, R., Sharma, P., Dastidar, S., Paliwal, J., Kakar, S., Varshney, B., Saini, G.S., Sattigeri, V., Salman, M. & Ray, A. 2008, 'Pharmacodynamic and pharmacokinetic characterization of RBx 7796: a novel 5-lipoxygenase inhibitor.', Inflammation Research., vol. 57, no. 3, pp. 135-143.View/Download from: UTS OPUS
RBx 7796, a 5-lipoxygenase inhibitor, was evaluated in in vivo efficacy models, in vitro ADME and in vivo pharmacokinetic models.
RBx 7796 was evaluated for inhibition of 5-lipoxygenase enzyme and release of LTB4 from isolated rat and human neutrophils. RBx 7796 was tested in allergic bronchoconstriction model in Balb/c mice and LPS induced airway hyperreactivity model in rats. RBx 7796 was evaluated for metabolic stability in liver microsomes and cytochrome P450 inhibition potential. Pharmacokinetic profile of RBx 7796 was also determined in rat and dog.
RBx 7796 inhibited 5-lipoxygenase enzyme and inhibited release of LTB4 from neutrophils. RBx 7796 also inhibited early and late airway reactivity following allergen challenge in mouse model. LPS induced increase in airway reactivity was blocked by RBx 7796. Compound was found to be stable in liver microsomes and devoid of major cytochrome P450 inhibition potential. The oral bioavailability of RBx 7796 in rat and dog was 83 % and 47 %, respectively. Following repeated daily administration, compound did not exhibit any sign of accumulation and/or tendency to induce its own metabolism.
The results suggest that RBx 7796 is an inhibitor of 5-lipoxygenase enzyme that is orally efficacious in two different models of airway reactivity. The molecule also demonstrated acceptable pharmacokinetic profile warranting further development.
Sharma, P., Schaafsma, D., halayko, A.J. & meurs, H. 2007, 'Insulin increases expression of contractile phenotypic markers in airway smooth muscle', Am J Physiol Cell Physiol., vol. 293, no. 1, pp. 429-439.
We have previously demonstrated that long-term exposure of bovine tracheal smooth muscle (BTSM) strips to insulin induces a functional hypercontractile phenotype. To elucidate molecular mechanisms by which insulin might induce maturation of contractile phenotype airway smooth muscle (ASM) cells, we investigated effects of insulin stimulation in serum-free primary BTSM cell cultures on protein accumulation of specific contractile phenotypic markers and on the abundance and stability of mRNA encoding these markers. In addition, we used microscopy to assess insulin effects on ASM cell morphology, phenotype, and induction of phosphatidylinositol (PI) 3-kinase signaling. It was demonstrated that protein and mRNA levels of smooth muscle-specific contractile phenotypic markers, including sm-myosin, are significantly increased after stimulation of cultured BTSM cells with insulin (1 microM) for 8 days compared with cells treated with serum-free media, whereas mRNA stability was unaffected. In addition, insulin treatment promoted the formation of large, elongate ASM cells, characterized by dramatic accumulation of contractile phenotype marker proteins and phosphorylated p70(S6K) (downstream target of PI 3-kinase associated with ASM maturation). Insulin effects on protein accumulation and cell morphology were abrogated by combined pretreatment with the Rho kinase inhibitor Y-27632 (1 microM) or the PI 3-kinase inhibitor LY-294002 (10 microM), indicating that insulin increases the expression of contractile phenotypic markers in BTSM in a Rho kinase- and PI 3-kinase-dependent fashion. In conclusion, insulin increases transcription and protein expression of contractile phenotypic markers in ASM. This could have important implications for the use of recently approved aerosolized insulin formulations in diabetes mellitus.
Sharma, P.K., Kuchumov, A.R., Chottard, G., Martin, P.D., Wall, J.S. & Vinogradov, S.N. 1996, 'The role of the dodecamer subunit in the dissociation and reassembly of the hexagonal bilayer structure of Lumbricus terrestris hemoglobin.', J Biol Chem, vol. 271, no. 15, pp. 8754-8762.
The dissociation of the approximately 3500-kDa hexagonal bilayer (HBL) hemoglobin (Hb) of Lumbricus terrestris upon exposure to Gdm salts, urea and the heteropolytungstates [SiW11O39]8- (SiW), [NaSb9W21O86]18- (SbW) and [BaAs4W40O140]27- (AsW) at neutral pH was followed by gel filtration, SDS-polyacrylamide gel electrophoresis, and scanning transmission electron microscopy. Elution curves were fitted to sums of exponentially modified gaussians to represent the peaks due to undissociated oxyHb, D (approximately 200 kDa), T+L (approximately 50 kDa), and M (approximately 25 kDa) (T = disulfide-bonded trimer of chains a c, M = chain d, and L = linker chains). OxyHb dissociation decreased in the order Gdm*SCN > Gdm.Cl > urea > Gdm.OAc and AsW > SbW > SiW. Scanning transmission electron microscopy mass mapping of D showed approximately 10-nm particles with masses of approximately 200 kDa, suggesting them to be dodecamers (a+b+c)3d3. OxyHb dissociations in urea and Gdm.Cl and at alkaline pH could be fitted only as sums of 3 exponentials. The time course of D was bell-shaped, indicating it was an intermediate. Dissociations in SiW and upon conversion to metHb showed only two phases. The kinetic heterogeneity may be due to oxyHb structural heterogeneity. Formation of D was spontaneous during HBL reassembly, which was minimal (
Krebs, A., Kuchumov, A.R., Sharma, P.K., Braswell, E.H., Zipper, P., Weber, R.E., Chottard, G. & Vinogradov, S.N. 1996, 'Molecular shape, dissociation, and oxygen binding of the dodecamer subunit of Lumbricus terrestris hemoglobin.', J Biol Chem, vol. 271, no. 31, pp. 18695-18704.
Small angle x-ray scattering of the 213-kDa dodecamer of Lumbricus terrestris Hb yielded radius of gyration = 3.74 +/- 0.01 nm, maximum diameter = 10.59 +/- 0.01 nm, and volume = 255 +/- 10 nm3, with no difference between the oxy and deoxy states. Sedimentation velocity studies indicate the dodecamer to have a spherical shape and concentration- and Ca2+-dependent equilibria with its constituent subunits, the disulfide-bonded trimer of chains a-c and chain d. Equilibrium sedimentation data were fitted best with a trimer-dodecamer model, ln K4 = 7 (association K in liters3/g3) at 1 degrees C and 4 at 25 degrees C, providing DeltaH = -20 kcal/mol and DeltaS = 4.4 eu/mol. Oxydodecamer dissociation at pH 8.0, in urea, GdmCl, heteropolytungstate K8[SiW11O39] and of metdodecamer at pH 7, was followed by gel filtration. Elution profiles were fitted with exponentially modified gaussians to represent the three peaks. Two exponentials were necessary to fit all the dissociations except in [SiW11O39]-8. Equilibrium oxygen binding measurements at pH 6.5-8. 5, provided P50 = 8.5, 11.5-11.9 and 11.9-13.5 torr, and n50 = 5.2-9. 5, 3.2-4.9, and 1.8-2.7 for blood, Hb, and dodecamer, respectively, at pH 7.5, 25 degrees C. P50 was decreased 3- and 2-fold in approximately 100 mM Ca2+ and Mg2+, respectively, with concomitant but smaller increases in cooperativity.
Imai, K., Sharma, P.K. & Vinogradov, S.N. 1996, 'Oxygen binding properties of Eudistylia vancouverii chlorocruorin and its dodecamer subunit.', Comp Biochem Physiol B Biochem Mol Biol, vol. 113, no. 3, pp. 613-618.
The oxygen equilibrium curves of the extracellular chlorocruorin of the polychaete Eudistylia vancouverii and its dodecamer subunit were determined over the pH range from 7.1 to 8.1 and in the presence and absence of 100 mM Mg2+. Although the chlorocruorin has a very low oxygen affinity, high cooperativity and a moderate alkaline Bohr effect (P50 = 145 mmHg, nmax = 6.9, phi = -0.44 at pH 7.1 and 25 degrees C), the dodecamer subunit has higher affinity and lower cooperativity and Bohr coefficient (P50 = 58 mmHg, nmax = 1.7, phi = -0.22). The presence of 100 mM Mg2+ increased the oxygen affinity and cooperativity of the chlorocruorin but had no significant effect on the dodecamer subunit. The oxygen binding properties of the chlorocruorin reassociated subsequent to complete dissociation in 4 M urea at neutral pH were similar to the properties of the dodecamer and not to those of the native chlorocruorin. Fitting to the Monod-Wyman-Changeux allosteric model gave KT of 0.0013 mmHg-1, Kr of 0.22 mmHg-1 and L(o) of 2.5 x 10(13) together with a size of the allosteric unit, N = 9.
Sharma, P., Ryves, W.J., Gordge, P.C., Evans, A.T., Shaun, N., Thomas, B., Parker, P.J. & Evans, F.J. 1995, 'Properties of a resiniferatoxin-stimulated, calcium inhibited but phosphatidylserine-dependent kinase, which is distinct from protein kinase C isotypes alpha, beta 1, gamma, delta, epsilon and eta.', The Journal of pharmacy and pharmacology, vol. 47, no. 4, pp. 297-306.
We have separated a resiniferatoxin-stimulated histone-kinase activity from human neutrophils, elicited mouse macrophages and murine alveolar macrophages by hydroxyapatite chromatography. The assay conditions for resiniferatoxin kinase were optimized as part of this study and in the presence of phosphatidylserine but absence of Ca2+ the Ka for histone IIIs phosphorylation by resiniferatoxin was calculated as 16 nM. Using a phosphate gradient of 20-500 mM, peaks of protein kinase C activity could be washed from the hydroxyapatite column in 300 nM phosphate and resiniferatoxin kinase recovered in 500 mM phosphate. At the optimum concentration of 160 nM, the ability of resiniferatoxin to induce enzyme activity was compared with a range of phorbol esters all at the same concentration. These related compounds failed to activate resiniferatoxin kinase although they have previously been shown to activate protein kinase C isotypes. Similarly sn-1,2,-dioleoylglycerol and the potent irritant capsaicin at 30 microM failed to activate the kinase. A Scatchard analysis of [3H] phorbol dibutyrate binding produced a linear plot (Kd 41.6 nM; Bmax 11.6 fmol unit-1) and binding was inhibited by resiniferatoxin and 12-O-tetradecanoylphorbol-13-acetate (TPA), with resiniferatoxin 700 times more potent than TPA in this respect. A radiolabelled resiniferatoxin binding assay was also used to demonstrate specific binding of [3H]resiniferatoxin which could be inhibited by unlabelled compound. Resiniferatoxin kinase activity was shown to be distinct from the protein kinase C isotypes alpha, beta 1, gamma, delta and epsilon by means of immunological analysis and from the eta isotype, because that isotype was not stimulated by resiniferatoxin but was stimulated by TPA when a pseudosubstrate was used. In addition the resiniferatoxin-stimulated activity was inhibited in-vitro by the addition of Ca2+ (Ki 0.1-0.5 nM free Ca2+). Further purification of resiniferatoxin kinase by Superose chromatog...
Weber, R.E., Malte, H., Braswell, E.H., Oliver, R.W., Green, B.N., Sharma, P.K., Kuchumov, A. & Vinogradov, S.N. 1995, 'Mass spectrometric composition, molecular mass and oxygen binding of Macrobdella decora hemoglobin and its tetramer and monomer subunits.', J Mol Biol, vol. 251, no. 5, pp. 703-720.
The hexagonal bilayer hemoglobin (Hb) of the leech Macrobdella decora has an equilibrium sedimentation mass of 3544(+/- 80) kDa. Maximum entropy analysis of the electrospray ionization mass spectra of the Hb show three groups of peaks: two peaks of equal intensity at approximately 17 kDa, A (16,770.1 Da) and B (16,841.9 Da); three peaks at approximately 24 kDa, C (24,340.1 Da), D (24,398.6 Da) and E (24,420.0 Da) with relative intensities of 1:6:3, respectively; and three peaks of equal intensities at approximately 33 kDa, F (32,586.0 Da), G (32,714.5 Da) and H (32,849.9 Da). Although reduction with dithiothreitol does not affect the masses of peaks A through E, the approximately 33 kDa peaks give rise to four new peaks at approximately 16 kDa, P (16,052.2 Da), Q (16,537.3 Da), R (16,666.7 Da) and S (16,792.9 Da), indicating that F, G and H represent disulfide-bonded dimers of globin chains, P + Q, P + R and P + S, respectively. The relative intensities of the three groups of peaks are (A + B) to (C + D + E) to (F + G + H) = 0.39:0.26:0.32, and the globin to linker ratio 0.71:0.29 is in good agreement with the ratio 0.72:0.28 obtained by HPLC. The largest functional subunit obtained by dissociation at pH 7 in 4 M urea, is a subunit lacking linker chains with apparent mass 63(+/- 3) kDa. The equilibrium sedimentation profile of this subunit is fitted best as a monomer-dimer-tetramer equilibrium, with association constants K1,2 = 365 l g-1 and K1,4 = 8.1 x 10(5) l3 g-3. A model of the Hb consisting of a hexagonal bilayer of 36 tetramer and 42 linker subunits provides a total mass and globin to linker ratio closest to the experimental values. Equilibrium O2 binding measurements of the native Hb and its tetramer and monomer subunits were carried out over the pH range 6.6 to 8.0 at 10 and 25 degrees C, and in the absence and presence of Na+, Mg2+ and Ca2+. The Hb exhibits a moderately high O2 affinity, P50 = 4.4 torr at pH 7.5 and 25 degrees C, a high cooperativity (n...
Vinogradov, S.N. & Sharma, P.K. 1994, 'Preparation and characterization of invertebrate globin complexes.', Methods Enzymol, vol. 231, pp. 112-124.
Rashid, K.A., Haque, M., Siddiqi, A.H., Stern, M.S., Sharma, P.K., Vinogradov, S.N. & Walz, D.A. 1993, 'Purification and properties of the hemoglobins of the platyhelminth Isoparorchis hypselobagri (Trematoda: Isoparorchidae) and its host Wallagu attu (catfish).', Comp Biochem Physiol B, vol. 106, no. 4, pp. 993-998.
1. The hemoglobins of the trematode Isoparorchis hypselobagri and of its host Wallagu attu (catfish) were isolated and purified. 2. SDS-polyacrylamide gel electrophoresis showed both to consist of single, 15-17 kDa chains, having different electrophoretic mobilities. 3. Isoelectric focusing showed the trematode hemoglobin to be homogeneous with a pI of 4.2 and the host hemoglobin to consist of several components. 4. Gel filtration of freshly prepared trematode hemoglobin revealed one peak corresponding to M(r) approximately 17 kDa; gel filtration of a preparation which had been stored for 2-3 months demonstrated the presence of two peaks, whose elution volumes corresponded to M(r) of ca 35 and 17 kDa, respectively. 5. Reversed-phase chromatography of carboxymethylated 35 and 17 kDa peaks on a C8 column, gave a single peak a and two peaks b and c, respectively. 6. Edman degradation of peaks a, b and c obtained provided identical sequences of 27 amino acid residues for peaks a and c and another sequence differing at 10 of the 27 positions, for peak b. Edman degradation of the freshly prepared Isoparorchis hemoglobin provided the first 15 amino acid residues found for peaks a and c. The host hemoglobin gave an N-terminal sequence completely different from the trematode sequences. 7. Since gel filtration of the 35 and 17 kDa peaks showed no sign of an interconversion equilibrium, it appears that the 35 kDa peak and peak a represent a disulfide-bonded dimer of a monomer globin chain which shares the 27 N-terminal residues with chain c, the major monomer globin component of the 17 kDa peak.(ABSTRACT TRUNCATED AT 250 WORDS)
Gibson, Q.H., Regan, R., Olson, J.S., Carver, T.E., Dixon, B., Pohajdak, B., Sharma, P.K. & Vinogradov, S.N. 1993, 'Kinetics of ligand binding to Pseudoterranova decipiens and Ascaris suum hemoglobins and to Leu-29-->Tyr sperm whale myoglobin mutant.', J Biol Chem, vol. 268, no. 23, pp. 16993-16998.
The kinetics of binding of O2, CO, and NO to the octameric, two-domain hemoglobins of the parasitic nematodes Pseudoterranova decipiens and Ascaris suum were determined on nanosecond and picosecond time scales using flash photolysis. The two nematode hemoglobins have very similar kinetic properties. On the picosecond time scale, they exhibit an unusual behavior in showing a geminate reaction with oxygen that is biphasic and dependent on the flash intensity. The geminate reaction with NO is also faster and more complete than for sperm whale myoglobin; however, in contrast to the O2 reaction, it is homogeneous. In addition, the oxygen dissociation rate of P. decipiens hemoglobin, 0.0035 s-1, is as low as that of A. suum hemoglobin, 0.004 s-1 (Gibson, Q. H., and Smith, M. H. (1965) Proc. R. Soc. Lond. B Biol. Sci. 163, 206-214). A mutant of sperm whale myoglobin suggested by sequence alignment of the nematode hemoglobins, Leu-29-->Tyr, did not have kinetic properties similar to them.
Zafar, R.S., Weber, R.E., Sharma, P.K., Vinogradov, S.N. & Walz, D.A. 1993, 'Purification and characterization of recombinant polymeric hemoglobin P1 of Glycera dibranchiata.', Protein Expr Purif, vol. 4, no. 6, pp. 547-551.View/Download from: Publisher's site
The apoprotein of component P1 of the polymeric fraction of the intracellular hemoglobin of the marine polychaete Glycera dibranchiata has been expressed at a high level in Escherichia coli. The expressed globin was reconstituted with heme and purified. The N-terminal sequence of the recombinant P1 is identical to the cDNA-derived sequence of cloned P1 (Zafar et al., Biochem. Biophys. Acta, 1041, 117-123, 1990). Gel filtration, SDS-PAGE, optical spectra over the range 200-650 nm, and circular dichroism over the range 200-250 nm of the purified recombinant P1 were very similar to the polymeric fraction of native Glycera hemoglobin. The molar ellipticity at 222 nm provided an estimate of 77% for the alpha-helical content of the recombinant P1, in excellent agreement with that calculated from the crystal structure of Glycera monomeric component M-II. Although the oxygen binding affinity of the recombinant P1 is higher than that of the polymeric fraction of Glycera hemoglobin (3-4 torr vs 7-13 torr), which consists of at least six different single-chain hemoglobins, the Hill coefficient is lower (1.0-1.2 vs 1.2-1.4).
Gordge, P.C., Evans, A.T., Ryves, W.J., Sharma, P., Hassan, N.M., Aitken, A. & Evans, F.J. 1993, 'Molecular modelling of tumour-promoting and non-promoting diterpene esters and molecular co-ordinates of the potent irritant resiniferatoxin.', Cancer Lett, vol. 73, no. 2-3, pp. 77-84.
The lowest energy conformer of seventeen diterpenes, representing five different phorbol and daphnane diterpene nuclei, has been generated. TPA possessed the highest minimum free energy of these compounds; all other compounds possessed a lower minimum free energy. Compounds based on the resiniferonol nucleus possessed the lowest minimum free energy (9.4-16.6% of that of TPA). The molecular co-ordinates of the non-promoting but potent irritant resiniferatoxin (Rx) are also reported. These studies may be important in elucidation of the biochemical mechanisms of action of diterpene esters, including an understanding of the interactions of diterpene esters with the phorbol ester binding domain of the protein kinase C isoform family.
Merritt, J.E., Moores, K.E., Evans, A.T., Sharma, P., Evans, F.J. & MacPhee, C.H. 1993, 'Involvement of calcium in modulation of neutrophil function by phorbol esters that activate protein kinase C isotypes and related enzymes.', Biochem J, vol. 289 ( Pt 3), pp. 919-926.
In this study, the effects of a series of phorbol esters with different spectra of biological activities and different patterns of activation of the isoenzymes of protein kinase C (PKC) have been studied in human neutrophils. The aim was to gain more information on which isoenzymes of PKC are involved in neutrophil activation, specifically inhibition of fMet-Leu-Phe (fMLP)-stimulated bivalent cation influx and stimulation of O2-. release (either alone or potentiation of the response to fMLP). Prior addition of both phorbol 12-myristate 13-acetate (PMA) and sapintoxin A (SAPA) inhibited fMLP-stimulated Mn2+ influx. Higher concentrations of resiniferatoxin (RX) were also inhibitory, inhibition being more apparent at longer preincubation times. However, 12-deoxyphorbol 13-O-phenylacetate (DOPPA) showed only a slight inhibitory effect and required a prolonged preincubation. PMA, SAPA and RX, but not DOPPA, stimulated O2-. release by themselves. Lower concentrations of PMA, SAPA and RX, which were ineffective alone, considerably potentiated O2-. release stimulated by fMLP, whereas DOPPA had little or no effect. These results rule out a major role for PKC-delta (not activated by SAPA) and PKC-beta 1 (activated by DOPPA), but suggest the involvement of RX kinase in addition to PKC in the inhibition of fMLP-stimulated Mn2+ influx and potentiation of fMLP-stimulated O2-. release. However, when the cytosolic free Ca2+ concentration ([Ca2+]i) was elevated with the Ca2+ ionophore ionomycin, DOPPA was able to stimulate O2-. release, which probably reflects the known Ca2+ requirement for activation of PKC-beta 1 by DOPPA in vitro. The effects of the other phorbols were also enhanced when [Ca2+]i was elevated; all of the phorbols synergize, to variable extents, with Ca2+ to activate PKC in vitro. Enhancement of RX-stimulated O2- release by elevation of [Ca2+]i was unexpected, since RX kinase has been reported to be inhibited by high concentrations of Ca2+ in vitro. Finally, use...
Hynes, A., Ryves, W.J., Gordge, P.C., Evans, A.T., Sharma, P. & Evans, F.J. 1993, 'EVIDENCE FOR ANONPKC RECEPTOR FOR RESINIFERATOXIN, A MEMBER OF THE PHORBOL ESTER FAMILY, IN STARCH ELICITED MURINE PERITONEAL MACROPHAGES', Pharmacy and Pharmacology Communications, vol. 45, no. 12 S, pp. 1102-1102.View/Download from: Publisher's site
Haque, M., Rashid, K.A., Stern, M.S., Sharma, P.K., Siddiqi, A.H., Vinogradov, S.N. & Walz, D.A. 1992, 'Comparison of the hemoglobins of the platyhelminths Gastrothylax crumenifer and Paramphistomum epiclitum (Trematoda: Paramphistomatidae).', Comp Biochem Physiol B, vol. 101, no. 4, pp. 673-676.
1. Gastrothylax crumenifer and Paramphistomum epiclitum parasitize the water buffalo Bubalus bubalis. 2. Gastrothylas hemoglobin consisted of two fractions of ca 30,000 and ca 18,000 by gel filtration. SDS-electrophoresis showed both to be single, ca 15,000 chains. 3. Paramphistomum hemoglobin was ca 16,000 by both gel filtration and SDS-electrophoresis. 4. Reversed-phase chromatography of carboxymethylated trematode and buffalo globins gave single peaks and two peaks, respectively. Although Paramphistomum hemoglobin provided and N-terminal sequence, Gastrothylax hemoglobin did not, suggesting blocked N-terminals. The buffalo sequences were found to be identical to the sequences of the alpha and beta chains of bovine hemoglobin. 5. Although Paramphistomum hemoglobin consists of only one chain, Gastrothylax hemoglobin consists either of one chain which aggregates to a dimer or of two different chains, only one of which aggregates to a dimer.
Gibson, Q.H., Bellelli, A., Regan, R., Sharma, P.K. & Vinogradov, S.N. 1992, 'Ligand binding by the chlorocruorin from Eudistylia vancouverii.', J Biol Chem, vol. 267, no. 17, pp. 11977-11981.
Carbon monoxide chlorocruorin from Eudistylia vancouverii shows three distinct first-order relaxations with rates of 2.9 x 10(9) s-1, 6.5 x 10(7) s-1, and 3.2 x 10(6) s-1 (geminate reactions) and three second-order relaxations with rates of 4.7 x 10(6) M-1 s-1, 7 x 10(5) M-1 s-1, and 7 x 10(4) M-1 s-1, when studied by flash photolysis. The amplitudes of the second-order reactions depend on the extent of photolysis. This may be due to relaxation from the liganded (R) to the unliganded (T) conformation following photolysis and suggests that the combination rates contribute to cooperativity. In a stopped-flow experiment only the slowest phase with a rate of 7 x 10(4) M-1 s-1 is observed. It is assigned to binding to the T-state protein. Fragments of the native protein containing 12 and 4 hemes react like the holoprotein suggesting that the tetramer is a major cooperative unit. Oxygen binding shows three geminate relaxations with rates of 2.5 x 10(10) s-1, 3.5 x 10(7) s-1, and 4.5 x 10(6) s-1, and two second-order rates of 1.5 x 10(7) M-1 s-1 and 1 x 10(6) M-1 s-1. The amplitudes of the second-order phases do not correlate with the extent of photolysis. The results with the two ligands are consistent with an allosteric transition fast enough to compete with a rebinding rate of 500 s-1 in the R to T direction (CO rebinding) but not fast enough to compete with oxygen rebinding. There is significant heterogeneity in the R-state kinetics, but the T-state reaction is homogeneous.
Sharma, P., Evans, A.T., Parker, P.J. & Evans, F.J. 1991, 'NADPH-oxidase activation by protein kinase C-isotypes.', Biochem Biophys Res Commun, vol. 177, no. 3, pp. 1033-1040.
The cell free activation of NADPH-Oxidase in membranes of mouse peritoneal macrophages by purified PKC-isotypes was investigated. Unstimulated intrinsic activity of PKC-isotypes showed little dependence on Ca2+ for activation of the oxidase. In the presence of TPA, the activation of the oxidase was greatly enhanced, and alpha-, and gamma-subtypes were strongly Ca2+ dependent in this system. Beta-, delta- and epsilon-subtypes were active both in the presence and absence of free Ca2+ ions. The results suggest that at resting Ca2+ levels certain PKC-isotypes can activate NADPH-oxidase.
Gibson, Q.H., Blackmore, R.S., Regan, R., Sharma, P.K. & Vinogradov, S.N. 1991, 'Ligand binding in a hierarchy of globin complexes. The hexagonal bilayer hemoglobin of Lumbricus terrestris and its heme-containing subunits.', J Biol Chem, vol. 266, no. 20, pp. 13097-13102.
The kinetics of CO and NO recombination with the giant approximately 3600-kDa hexagonal bilayer hemoglobin of Lumbricus terrestris and its subunits, the approximately 200-kDa dodecamer of globin chains (3 x chains (I + II + III + IV] (see preceding paper (Vinogradov S.N., Sharma, P.K., Qabor, A.N., Wall, J.S., Westrick, J.A., Simmons, J.H., and Gill, S.J. (1991) J. Biol. Chem. 266, 13091-13096], the 50-kDa disulfide-bonded trimer (chains II-IV), the monomer (chain I), and the approximately 30-kDa linker (chains VA, VB, and VI), were measured following photolysis over time scales ranging from picosecond to millisecond. CO recombination at 436 nm subsequent to excitation (9 ns) at 532 nm showed three phases covering a 100-fold range for the Hb, dodecamer, trimer, and linker protein. The proportion of the fast phase was 0.1-0.2 for the trimer, dodecamer, and Hb. The relative rates and amplitudes of the phases were not affected by changes in CO concentration or excitation intensity. The monomer showed a single phase with a rate of 2 x 10(6) M-1 s-1. The second-order reaction with NO showed two rates. The faster rate was 90 x 10(6) M-1 s-1 and accounted for approximately 0.7 of the reaction for all species except the monomer, where it accounted for the full reaction. The slower rate was 15 x 10(6) M-1 s-1 for all species except the monomer.
Sharma, P.K., Walz, D.A. & Vinogradov, S.N. 1991, 'Heterogeneity of the subunits of Lumbricus terrestris extracellular hemoglobin dissociated at alkaline pH.', Comp Biochem Physiol B, vol. 98, no. 1, pp. 47-51.
1. The gel filtration profiles of dissociated extracellular hemoglobin of Lumbricus terrestris obtained in 0.1 M borate buffers at pH greater than 9, using columns of Sephadex G100, Sephacryl S200 and Ultragel AcA 44, consist of at least two peaks A and B. 2. SDS-PAGE of several fractions across the complete elution profile demonstrates that only the fractions under the right hand portion of peak B are homogenous and consist of the monomer (M) subunit (Mr = 17,000). 3. The fractions under the first peak contain the remaining subunits, a disulfide-bonded trimer (T) subunit (Mr = 50,000) and of two subunits (D1 and D2) of ca 30,000. 4. Densitometry of the SDS-PAGE patterns suggests that the proportions of these subunits vary across the two peaks, implying that peak A does not consist of a complex of fixed stoichiometry of the T and D1 and D2 subunits. 5. Furthermore, the D1 and D2 subunits overlap the M subunit in the trough between peaks A and B and are present in the left hand portion of peak B, probably because of the self-association of the M subunit. 6. In addition, SDS-PAGE experiments with a single fraction of peak A, where the load and the duration of staining were varied, suggests that the relative proportions of the subunits are independent of these two variables.
Vinogradov, S.N., Sharma, P.K., Qabar, A.N., Wall, J.S., Westrick, J.A., Simmons, J.H. & Gill, S.J. 1991, 'A dodecamer of globin chains is the principal functional subunit of the extracellular hemoglobin of Lumbricus terrestris.', J Biol Chem, vol. 266, no. 20, pp. 13091-13096.
Repeated dissociation of the approximately 3600-kDa hexagonal bilayer extracellular hemoglobin of Lumbricus terrestris in 4 M urea followed by gel filtration at neutral pH produces a subunit that retains the oxygen affinity of the native molecule (approximately 12 torr), but only two-thirds of the cooperativity (nmax = 2.1 +/- 0.2 versus 3.3 +/- 0.3). The mass of this subunit was estimated to be 202 +/- 15 kDa by gel filtration and 202 +/- 26 kDa from mass measurements of unstained freeze-dried specimens by scanning transmission electron microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this subunit showed that it consists predominantly of the heme-containing subunits M (chain I, 17 kDa) and T (disulfide-bonded chains II-IV, 50 kDa). Mixing of subunits M and T isolated concurrently with the 200-kDa subunit resulted in partial association into particles that had a mass of 191 +/- 13 kDa determined by gel filtration and 200 +/- 38 kDa determined by scanning transmission electron microscopy and whose oxygen affinity and cooperativity were the same as those of the 200-kDa subunit. The results imply that the 200-kDa subunit is a dodecamer of globin chains, consisting of three copies each of subunits M and T (3 x chains (I + II + III + IV], in good agreement with the mass of 209 kDa calculated from the amino acid sequences of the four chains, and represents the largest functional subunit of Lumbricus hemoglobin. Twelve copies of this subunit would account for two-thirds of the total mass of the molecule, as suggested earlier (Vinogradov, S. N., Lugo, S. L., Mainwaring, M. G., Kapp, O. H., and Crewe, A. V. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8034-8038). The retention of only partial cooperativity by the 200-kDa subunit implies that full cooperativity is dependent on the presence of a complete hexagonal bilayer structure, wherein 12 200-kDa subunits are linked together by approximately 30-kDa heme-deficient chains.
Vinogradov, S.N., Sharma, P.K. & Walz, D.A. 1991, 'Iron and heme contents of the extracellular hemoglobins and chlorocruorins of annelids.', Comp Biochem Physiol B, vol. 98, no. 2-3, pp. 187-194.
1. A survey of the literature on the extracellular hemoglobins and chlorocruorins of over 30 species of annelids, covering the last 30 years, shows that the range of iron content is 0.211-0.265 wt.% (mean = 0.228 +/- 0.013, N = 28) and the range of the heme content is 1.83-3.64 wt.% (mean = 2.60 +/- 0.38, N = 29). 2. There is relatively little scatter in the values of the experimental iron contents and only one of the 28 values is clearly outside the standard deviation range. 3. The values of heme contents are much more scattered, with seven values, clearly outside the standard deviation limits. 4. The aberrant cases are discussed and it is noted that the mean heme content of 2.60 wt.% corresponds to an iron content of 0.236 wt.% in excellent agreement with the mean iron content of 0.228 wt.%. 5. This result suggests strongly that experimental values of iron and heme contents outside the ranges of 0.211-0.243 and 2.3-2.7 wt.%, respectively, corresponding to a minimum molecular mass outside the range 23,000-26,000, should be regarded with caution.
Stern, M.S., Vinogradov, S.N., Sharma, P.K., Ereifej, K. & Walz, D.A. 1990, 'Amino acid sequence of the monomer subunit of the giant extracellular hemoglobin of the aquatic oligochaete, Tubifex tubifex.', Eur J Biochem, vol. 194, no. 1, pp. 67-73.
The extracellular hemoglobin of the aquatic oligochaete Tubifex tubifex consists of four subunits: a monomer of 16.5 kDa, a disulfide-bonded trimer of about 50 kDa and at least two subunits of about 30 kDa. The complete amino acid sequence of the monomeric subunit was determined: it consists of 141 amino acid residues and has a molecular mass of 16,286 Da including a heme group. 39 residues (28%) were found to be identical with those in the corresponding positions in the monomeric globin chains from Lumbricus terrestris, Pheretima sieboldi, and Tylorrhynchus heterochaetus. Tubifex and Lumbricus are most similar, with 75 amino acid identities (53%). There are eight invariant residues amongst these monomeric globins and the intracellular monomeric globin of Glycera and the human beta-globin. The monomeric globin from Tubifex aligns best with those of group A, globins which have a Cys in their second position and an invariant Lys-Val-Lys at positions 9-11 [Gotoh et al. (1987) Biochem. J. 241, 441-445]. The two cysteine residues, at positions 2 and 131, appear to be disulfide-bonded.
Evans, A.T., Sharma, P., Ryves, W.J. & Evans, F.J. 1990, 'TPA and resiniferatoxin-mediated activation of NADPH-oxidase. A possible role for Rx-kinase augmentation of PKC.', FEBS Lett, vol. 267, no. 2, pp. 253-256.
The non-tumour promoting irritant, resiniferatoxin, was capable of activating the NADPH-oxidase respiratory burst of starch-elicited, but not resident mouse peritoneal macrophages in vitro. Unlike TPA, the response was synergised by incubation with zymosan. The Rx-stimulated NADPH-oxidase activity in a cell-free assay was selectively enhanced in the presence of exogenous Rx-kinase rather than PKC and in the absence of Ca2+. Since resiniferatoxin is a poor activator of PKC, it is probable that the Ca2(+)-independent Rx-kinase plays a role in activation of the macrophage respiratory burst following stimulation by zymosan.
Sharma, P. & Penney, D.G. 1990, 'Effects of ethanol in acute carbon monoxide poisoning.', Toxicology, vol. 62, no. 2, pp. 213-226.
The combined effects of ethyl alcohol (ETOH) intoxication and carbon monoxide (CO) poisoning were studied in the Levine-prepared rat. Infusion or injection of ETOH before and during 90 min of CO exposure to blood levels 2-4 times those considered legally drunk in humans, increased survival at 2400 ppm, and extended the tolerance time at 2400 ppm and 3000 ppm. CO exposure produced the usual hypothermia, hypotension and hemoconcentration; these responses were not altered by concurrent ETOH treatment. Blood ETOH concentration was increased in the presence of CO, and this was related to CO concentration. Although ETOH did not alter the average degree of hypoglycemia seen during the later stages of CO exposure, rats with the highest ETOH concentration tended to have the lowest blood glucose. ETOH increased the magnitude of the hyperglycemic rebound during recovery from exposure to both CO concentrations. Moreover, the magnitude of the recovery hyperglycemic rebound was directly related to the magnitude of the previous hypoglycemia, at both CO concentrations, with or without ETOH. Rats dying during exposure to both CO concentrations were severely hypoglycemic, whereas survivors maintained more or less normal blood glucose concentrations. The results suggest that the presence of ETOH during CO poisoning increases blood ETOH to higher than expected levels and provides a significant degree of survival protection.
Penney, D.G., Sharma, P., Sutariya, B.B. & Nallamothu, B.G. 1990, 'Development of hypoglycemia is associated with death during carbon monoxide poisoning', Journal of Critical Care, vol. 5, no. 3, pp. 169-179.View/Download from: Publisher's site
A significant fraction of carbon monoxide (CO) poisoning victims die before discovery or rescue. Although survivors usually show normal or elevated blood glucose, data from an animal model now suggest an association between low blood glucose and death during CO poisoning. Hypoglycemia observed during the later stages of CO poisoning in the rat was intensified by increased CO concentration (2,400 ppm, 2,700 ppm, 3,000 ppm) and by the infusion or injection of normal saline, both in survivors and in nonsurvivors, but glucose lowering was especially marked in the latter group. Mortality rate was similarly increased by these two conditions. Based on the change in blood glucose concentration early in CO poisoning, it should be possible to identify survivors and nonsurvivors. Survivors showed a hyperglycemic "rebound" 2 hours after CO. Nonsurvivors tended to maintain a higher body temperature and heart rate, and a relatively smaller decrease in blood pressure during CO exposure, presumably due to a higher metabolic rate. This is possibly a major factor responsible for the rapid lowering of blood glucose concentration. Concurrent administration of ethanol to two to four times the legal intoxication blood level in humans did not alter the severity of CO-induced hypoglycemia in either survivors or nonsurvivors, but did increase survival and exacerbated "rebound" hyperglycemia in survivors. Blood ethanol concentration is elevated by CO poisoning in a dose-dependent manner. The likely mechanisms) underlying CO-induced hypoglycemia, the complicating effect of concurrent ETOH intoxication, and their clinical relevance are discussed. © 1990.
Sharma, P., Evans, A.T. & Evans, F.J. 1990, 'CALCIUM INDEPENDENT, PHOSPHOLIPID DEPENDENT RESINIFERATOXINKINASE ACTIVATION OF NADPHOXIDASE IN A CELL FREE SYSTEM', Journal of Pharmacy and Pharmacology, vol. 42, no. 1 S, pp. 33P-33P.View/Download from: Publisher's site
Dubeck, J.K., Penney, D.G., Brown, T.R. & Sharma, P. 1989, 'Thyroxine treatment of neonatal rats suppresses normal and stress-stimulated heart cell hyperplasia and abolishes persistent cadiomegaly', Journal of Applied Cardiology, vol. 4, no. 4, pp. 195-205.
Kota, A., Xenaki, D., Deshpande, D., Oliver, B. & Sharma, P. 2017, 'ASK1 Inhibition Prevented Mitogen-Induced Human Airway Smooth Muscle Growth in Chronic Obstructive Pulmonary Disease', American Journal of Respiratory and Critical Care Medicine, American Thoracic Society.View/Download from: UTS OPUS
McAlinden, K., Chan, Y., Kota, A., Chen, H., Oliver, B. & Sharma, P. 2017, 'Maternal E-cigarette Vaping Enhances Development of Allergic Asthma In the Offspring', American Journal of Respiratory and Critical Care Medicine, American Thoracic Society.View/Download from: UTS OPUS
Rationale: E-cigarettes (eCig) are being considered as an alternative to quit cigarette smoking (CS) while their long-term safety and effect on lung patho-physiology are not known. Maternal eCig-vaping may be considered as a safer CS-replacement during pregnancy. Thus the effect of maternal eCig vaping needs further assessment, particularly the effect this has on offspring and development of allergic asthma later in life. Combining mouse models of maternal vaping and allergic asthma and human airway smooth muscle cells (ASM) in vitro we tested whether maternal eCig vaping enhances features of allergic asthma in the offspring.
Methods: Female BALB/c mice were vaped with either eCig vapour (± nicotine) or CS+eCig (+nicotine) or room air (control group). The eCig vaping was started prior to mating and continued during gestation and lactation while CS-exposure was used prior to mating and replaced with eCig during gestation and mating. The female offspring from these mothers were subjected to an ovalbumin (OVA)-induced allergic asthma model. 24 hours after the last aerosolized OVA or saline challenge, lung function measurements were performed using flexiVent (Scireq, Canada) to increasing concentration of methacholine (MCh). Airway inflammation was assessed by counting total immune cell influx in BAL fluid. Human ASM cells were treated with varying concentrations of eCig liquid condensate and key parameters of mitochondrial function were measured with a Seahorse XF analyzer.
Results: Repeated allergen-exposure induced Th2-driven inflammation in OVA-exposed mice, characterized by massive influx of leukocytes predominantly eosinophils (OVA: 3x105±8.3x104 vs Saline: 1.1x102±1x102) and to some extent neutrophils (OVA: 1.3x104±4.4x103 vs Saline: 1.3x102±1.1x102) into the airways. The effect of allergen on airway eosinophilia was significantly enhanced in the offsprings from eCig OVA (+Nic)-exposed mothers when compared with eCig OVA (-Nic) or CS+eCig animals. OVA-exposed ...
Morgan, W.A., Sharma, P., Kaler, B. & Bach, P.H. 1997, 'The modulation of protein kinase C by bile salts.', Biochem Soc Trans, England, p. 75S.
Evans, F.J., Parker, P.J., Olivier, A.R., Thomas, S., Ryves, W.J., Evans, A.T., Gordge, P. & Sharma, P. 1991, 'Phorbol ester activation of the isotypes of protein kinase C from bovine and rat brain.', Biochem Soc Trans, England, pp. 397-402.
Evans, A.T., Gordge, P.C., Sharma, P., Ryves, W.J. & Evans, F.J. 1991, 'Investigation of the tissue distribution of the calcium independent kinase, Rx kinase; which is distinct from known isozymes of protein kinase C.', Biochem Soc Trans, England, p. 157S.
Evans, A.T., Duggan, C., Gordge, P.C., Sharma, P. & Evans, F.J. 1991, 'A 20 KDA protein comprises the major portion of the phorbol ester stimulated platelet transferable aggregating substance (TAS) activity', Journal of Pharmacy and Pharmacology, Supplement, p. 10.
Sharma, P., Evans, A.T. & Evans, F.J. 1989, 'Selective diterpene esters display synergy with zymosan in stimulating the respiratory burst of mouse peritoneal macrophages', Journal of Pharmacy and Pharmacology, Supplement.
Sharma, P. 2017, 'Lung experts warn against legalising nicotine in e-cigarettes'.
Sharma, P., Yi, R., Nayak, A., Wang, N., Tang, F., Knight, M., Pan, S., Oliver, B. & Deshpande, D. 2017, 'Bitter Taste Receptor Agonists Mitigate Features of Allergic Asthma in Mice'.View/Download from: UTS OPUS
Asthma is characterized by airway inflammation, mucus secretion, remodeling and hyperresponsiveness (AHR). Recent research has established the bronchodilatory effect of bitter taste receptor (TAS2R) agonists in various models. Comprehensive pre-clinical studies aimed at establishing effectiveness of TAS2R agonists in disease models are lacking. Here we aimed to determine the effect of TAS2R agonists on features of asthma. Further, we elucidated a mechanism by which TAS2R agonists mitigate features of asthma. Asthma was induced in mice using intranasal house dust mite or aerosol ova-albumin challenge, and chloroquine or quinine were tested in both prophylactic and treatment models. Allergen challenge resulted in airway inflammation as evidenced by increased immune cells infiltration and release of cytokines and chemokines in the lungs, which were significantly attenuated in TAS2R agonists treated mice. TAS2R agonists attenuated features of airway remodeling including smooth muscle mass, extracellular matrix deposition and pro-fibrotic signaling, and also prevented mucus accumulation and development of AHR in mice. Mechanistic studies using human neutrophils demonstrated that inhibition of immune cell chemotaxis is a key mechanism by which TAS2R agonists blocked allergic airway inflammation and exerted anti-asthma effects. Our comprehensive studies establish the effectiveness of TAS2R agonists in mitigating multiple features of allergic asthma.
Dr. Andrew Halayko, University of Manitoba, Winnipeg, MB, Canada
Dr. Raymond Penn, Thomas Jefferson University, Philadelphia, PA, USA
Dr. Deepak Deshpande,Thomas Jefferson University, Philadelphia, PA, USA
Dr. Saeid Ghavami, University of Manitoba, Winnipeg, MB, Canada
NIPER, Guwahati, Assam, India