Browsing by Author "MacDonald, Justin A."

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    Calcium-independent smooth muscle contraction: a focus on zipper-interacting protein kinase (ZIPK)
    (2011) Moffat, Lori Dawn; Walsh, Michael P.; MacDonald, Justin A.
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    Exaggerated IL-15 and Altered Expression of foxp3+ Cell-Derived Cytokines Contribute to Enhanced Colitis in Nlrp3−/− Mice
    (2016-08-17) Hirota, Simon A.; Ueno, Aito; Tulk, Sarah E.; Becker, Helen M.; Schenck, L. Patrick; Potentier, Mireille S.; Li, Yan; Ghosh, Subrata; Muruve, Daniel A.; MacDonald, Justin A.; Beck, Paul L.
    The pathogenesis of Crohn’s disease (CD) involves defects in the innate immune system, impairing responses to microbes. Studies have revealed that mutations NLRP3 are associated with CD. We reported previously that Nlrp3−/− mice were more susceptible to colitis and exhibited reduced colonic IL-10 expression. In the current study, we sought to determine how the loss of NLRP3 might be altering the function of regulatory T cells, a major source of IL-10. Colitis was induced in wild-type (WT) and Nlrp3−/− mice by treatment with dextran sulphate sodium (DSS). Lamina propria (LP) cells were assessed by flow cytometry and cytokine expression was assessed. DSS-treated Nlrp3−/− mice exhibited increased numbers of colonic foxp3+ T cells that expressed significantly lower levels of IL-10 but increased IL-17. This was associated with increased expression of colonic IL-15 and increased surface expression of IL-15 on LP dendritic cells. Neutralizing IL-15 in Nlrp3−/− mice attenuated the severity of colitis, decreased the number of colonic foxp3+ cells, and reduced the colonic expression of IL-12p40 and IL-17. These data suggest that the NLRP3 inflammasome can regulate intestinal inflammation through noncanonical mechanisms, providing additional insight as to how NLRP3 variants may contribute to the pathogenesis of CD.
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    Improving the therapeutic potential of staphylokinase, a potent thrombolytic agent
    (2023-05-26) Baharian, Azin; Vogel, Hans J.; MacDonald, Justin A.; MacCallum, Justin
    Staphylokinase (Sak) is a small bacterial-derived protein (15.5 kDa) that can hydrolyze fibrin-rich blood clots by activating the conversion of plasminogen into plasmin. Several studies have shown the effectiveness of Sak as a thrombolytic agent that could be used to treat stroke or cardiac arrest. However, its short half-life in blood circulation and immunogenicity are the main issues that prevent Sak from clinical applications. Herein, we explore two modifications of Sak to improve its therapeutic potential. Covalently attaching PEG (polyethylene glycol) polymers to therapeutic proteins has been widely applied to improve their pharmacokinetic properties, resulting in several FDA-approved protein-based drugs. Although PEGylation of Sak has previously been investigated, the effects of PEGylation on the 3D structure of Sak have not been studied in detail. In the first part of this research project, site-specific PEGylation of the truncated version of Sak was performed in the immunogenic region of the protein, and three covalent PEGylated derivatives of the protein were prepared. NMR studies showed a slight structural perturbation upon PEGylation, mostly close to the PEGylation site, and a direct relationship between the hydrodynamic radius of the PEGylated protein and the PEG size. Together, the results suggest that PEG and Sak∆10 move relatively independently from each other. As a second approach, we introduce cyclic Sak (cyc-Sak), a novel form of staphylokinase with higher stability and improved plasminogen activation activity. Using an Ssp GyrB split intein, the N- and C-terminal ends of the linear Sak were connected by a peptide bond, rendering the protein into a cyclic form (cyc-Sak). This structural modification was generated at the protein expression level in Escherichia coli, and the cyclic protein could be purified by common chromatography techniques. Successful backbone cyclization was confirmed by NMR spectroscopy of the 13C,15N-labeled cyc-Sak and by chemical cleavage assays. Our studies show that mono-PEGylation and intein-mediated backbone cyclization of Sak are powerful strategies to improve its therapeutic potential. These approaches could be combined to facilitate the design of protein-based medications when the stability of therapeutic proteins is an obstacle to their clinical application.
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    Insights into the smoothelin like-1 gene promoter
    (2008) Mughal, Saad H.; MacDonald, Justin A.
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    Investigating the phosphorylation of ser695/thr696 and ser852/thr853 regions of smooth muscle myosin phosphatase targeting subunit
    (2012) Grassie, Michael; MacDonald, Justin A.
    MYPTl has proven to be a key regulator in smooth muscle contraction and relaxation through the mechanisms of Ca2+ sensitization and desensitization. The regulation of MYPTl is controlled through the phosphorylation of several key residues. Kinase assays were carried out to confirm which amino acid residues of MYPT 1 were targeted by PKA and PKG in vitro. The following residues were then examined for functional significance in vitro and in situ with the use of recombinant proteins and RT A smooth muscle. From our studies we demonstrate the first direct evidence of in situ dual phosphorylation of MYPTl at Ser695/Thr696 and Ser852/Thr853. Also, that pre­phosphorylation of Ser852 by PKA/PKG has an inhibitory effect on the ability of ROK to phosphorylate the neighboring inhibitory residue of Thr853, similar to the effect previously observed for the Ser695/Thr696 region. Furthermore, the pre-phosphorylation ofThr696 by ROK decreased the ability of PKA/PKG to phosphorylate Ser695 in vitro and in situ.
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    Postnatal Impact of Bisphenol S Exposure on Oxidative Stress-Mediated Endothelial Dysfunction
    (2022-03-24) Easson, Sarah; Thompson, Jennifer; Patel, Vaibhav; MacDonald, Justin A.
    Bisphenols are synthetic polymers used in the production of household plastics; exposure to this compound has been associated with a multitude of adverse health outcomes. Bisphenol A (BPA) is the most well known and widely studied bisphenol. After several countries introduced regulatory bans on the sale and import of baby products containing BPA, manufacturers replaced BPA with structural analogues such as BPS and BPF. Although links between BPA exposure and reproductive, developmental, and metabolic diseases have been widely reported, few studies have investigated the impact of BPA on the vasculature and very little is known regarding the structural analogues that are increasingly replacing BPA. Bisphenols have been shown to increase the production of reactive oxygen species (ROS) in different cell types. Excess ROS in endothelial cells interacts with nitric oxide (NO), reducing is bioavailability. Therefore, BPS-induced ROS production in endothelial cells may impair NO-mediated vasodilation. Aim 1 of this study sought to determine whether BPS exposure decreases NO availability through the increased production of ROS. Findings demonstrated increased ROS production, decreased NO availability and uncoupling of endothelium NO synthase (eNOS) in human umbilical vein endothelial cells (HUVEC)exposed to BPS. RT-qPCR analysis determined that BPS reduced expression of antioxidant genes, while activity of the antioxidant was inhibited with BPS exposure. Aim 2 of this study determined whether postnatal exposure to BPS in C57BL/J6 mice impaired endothelium-dependent vasodilation in resistance vessels. Male and female mice were exposed to an environmentally relevant dose of BPS from the age of weaning (3 weeks) to early adulthood (12 weeks). Pressure myography evaluation of isolated mesenteric arteries demonstrated a reduced maximal response (Emax) to the NO-dependent dilator, methacholine, BPS-exposed male mice. Postnatal exposure in vivo had no measured effect on bodyweight, body composition, insulin sensitivity, or serum lipid peroxidation. In conclusion, BPS exposure reduced NO availability, leading to impaired endothelium-dependent dilation in the microvessels due toROS-mediated eNOS uncoupling. Therefore, exposure to BPS may contribute to the onset of vascular disease
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    Screening and Characterization of Antimicrobial Compounds and Material against Vibrio cholerae
    (2023-10-12) Kim, Haeun (Sara); MacDonald, Justin A.; Dong, Tao G.; DeVinney, Rebekah; Schriemer, David
    Antibiotic resistance (ABR) is a multisectoral global threat. With an increasing number of critical pathogens acquiring resistance against multiple classes of antibiotics, we face the terror of the post-antibiotic era. Vibrio cholerae, the cholera-causing pathogen, is no exception to the menace of multi-drug resistance. Strains from worldwide outbreaks have shown resistance to all antibiotics used to treat cholera, including ciprofloxacin. With cholera being prevalent in developing countries and regions affected by natural and anthropogenic disasters, an increase in fatalities is a rising concern in these areas with the number of effective antibiotics rapidly decreasing. This thesis explores the alternative approaches to treating cholera and controlling the spread of ABR pathogens in the environment. First, a library of 400 chemical compounds with antimicrobial activities, the Pathogen Box, is screened for compounds that exhibit killing or inhibiting activities against V. cholerae. Two compounds, MMV687807 and MMV675968, demonstrated bactericidal and bacteriostatic activity against the pathogen with high efficacy, respectively. RNA-seq analyses of V. cholerae treated with each compound revealed that their impact on multiple cellular functions, including carbon metabolism and iron homeostasis. Whole genome sequencing of spontaneous resistance mutants uncovered that activation of an efflux system via mutation to their regulator confers resistance to MMV687807. MMV675968 was identified to function similar to trimethoprim, targeting the dihydrofolate reductase of V. cholerae. This compound has the potential to be developed as a trimethoprim replacement with a minimum inhibitory concentration lower by 14-fold. Next, the antibacterial property of a newly synthesized cellulose aerogel cross-linked with different concentrations of silver/lignin nanoparticles (lignin only, 1Ag/L, 2Ag/L, and 3Ag/L) was studied in collaboration with Xiao He. Each aerogel was incubated with Gram-positive or Gram-negative pathogens, and surviving colonies were counted. Increase in killing efficiency was observed with increasing concentration of Ag/L nanoparticles. The cellulose aerogel with 3 Ag/L exhibited exceptional killing across all tested species: E. coli: > 99.99%, P. aeruginosa: > 99.9%, V. cholera: > 99.99%, S. aureus: > 99.99%, B. subtilis: > 97.4%. Through screening small molecules and exploring synthetic material cross-linked with nanoparticles, this thesis demonstrates the significance of developing new tools to inhibit bacterial pathogens and fight the spread of ABR.