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Platelets circulate in the blood stream in a quiescent state but undergo explosive activation at sites of damage to the blood vessel wall, leading to formation of a haemostatic plug and cessation of bleeding. The quiescent state of the... more
Platelets circulate in the blood stream in a quiescent state but undergo explosive activation at sites of damage to the blood vessel wall, leading to formation of a haemostatic plug and cessation of bleeding. The quiescent state of the platelet is maintained in part by the inhibitory action of constitutively released nitric oxide from endothelial cells lining the blood vessels. Activation is brought about by contact with extracellular matrix proteins exposed at the site of tissue damage, notably collagen fibres, and is reinforced by release and generation of a large number of substances such as thrombin and thromboxanes. The remarkable number and diversity of these agents facilitates rapid formation of the platelet aggregate leading to arrest of bleeding. Activation of platelets within intact blood vessels, however, can lead to vascular occlusion with subsequent ischaemic tissue damage. In the most severe cases, this leads to myocardial infarction or stroke, two of the major causes of death in the Western world. The platelet, therefore, is a major target for therapeutic intervention in the prevention of thrombotic disease.
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To investigate the effect of diclofenac sodium and dexamethasone on cultured human Tenon's capsule fibroblasts. Two experiments were conducted. In the first experiment, fibroblasts were treated with either diclofenac sodium or... more
To investigate the effect of diclofenac sodium and dexamethasone on cultured human Tenon's capsule fibroblasts. Two experiments were conducted. In the first experiment, fibroblasts were treated with either diclofenac sodium or dexamethasone at different concentrations, and the cell growth was quantified by using Coulter counter and hexosaminidase methods at 1, 3, 5, and 7 days after adding the drugs. In the second experiment, the cells were treated with each drug for 24 hours and then the cultures were switched to a drug-free medium. The cell growth was quantified at day 7 after removing the drugs from the medium. In the first experiment, inhibition of fibroblast growth in a dose-dependent manner was observed from days 1 to 7 in the cultures treated with each drug. The inhibitory was more pronounced in the diclofenac treated cultures. The typical spindle-shaped fibroblasts treated with higher concentrations of the drugs became spherical cells. In the second experiment, inhibition was not observed when the cultures were switched to a drug-free medium. The spherical cells recovered to spindle-shaped cells and proliferated as normal cells. Our results have shown that diclofenac sodium and dexamethasone can significantly inhibit human Tenon's capsule fibroblast growth in a cell culture model. The inhibitory effect was not observed when the cultures were switched after 24 hours to a drug-free culture medium.
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In parallel, we measured the receptor binding affinities for epidermal growth factor-urogastrone (EGF-URO) and transforming growth factor-alpha (TGF-alpha) in cultured smooth muscle (GCM) and epithelial (GPC) cells derived from guinea pig... more
In parallel, we measured the receptor binding affinities for epidermal growth factor-urogastrone (EGF-URO) and transforming growth factor-alpha (TGF-alpha) in cultured smooth muscle (GCM) and epithelial (GPC) cells derived from guinea pig intestine. The relative order of binding affinities in the GCM cells was TGF-alpha > EGF-URO, in keeping with the relative order of biological potencies of these polypeptides in a guinea pig gastric circular muscle contractile bioassay. These data established by ligand binding criteria the presence of a TGF-alpha-preferring receptor in the guinea pig. In contrast, there was a reversed order of binding affinities (EGF-URO > TGF-alpha) for the polypeptides in GPC cells, in accord with an identical order of bioassay potencies previously observed in a guinea pig gastric longitudinal muscle contractile bioassay. Using a reverse transcription-polymerase chain reaction approach, we also cloned and sequenced putative EGF-URO receptor ligand binding d...
Research Interests: Molecular Biology, Biology, Molecular Pharmacology, Medicine, Humans, and 15 moreAnimals, Bioassay, Intestines, Epidermal Growth Factor, Epithelium, Epithelial cells, Guinea Pig, Receptor, Base Sequence, Neurosciences, Binding Site, Transforming Growth Factor, Molecular Sequence Data, Guinea pigs, and Pharmacology and pharmaceutical sciences
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Insulin Receptor Purification Julie D. Newman and Leonard C. Harrison 1. Introduction The insulin receptor is an integral membrane glycoprotein (Mr300 ... 50000 form with a consequent rapid decrease of kinase activity, in purified... more
Insulin Receptor Purification Julie D. Newman and Leonard C. Harrison 1. Introduction The insulin receptor is an integral membrane glycoprotein (Mr300 ... 50000 form with a consequent rapid decrease of kinase activity, in purified receptor preparations stored at 4 C (Kathuria et al ...
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As recently as five years ago, what one might term the “grind and bind era” of receptor studies was still in full bloom; and a large number of studies were focussed on determining receptor affinities and numbers under a variety of... more
As recently as five years ago, what one might term the “grind and bind era” of receptor studies was still in full bloom; and a large number of studies were focussed on determining receptor affinities and numbers under a variety of physiologic situations. It became clear from such studies that receptors are not static elements, fixed in the cell membrane at a single site. Rather, it is now fully appreciated that receptors for neurotransmitters, hormones and other messenger molecules are dynamic elements both in the plasma membrane and in the cytoplasmic compartment, as outlined in Fig. 1.
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Serine proteinases signal through proteinase-activated receptors (PARs). Our studies have implicated PAR 1 and PAR 4 (thrombin receptors) and PAR 2 (trypsin receptor) in human colon cancer growth. However, endogenous activators of PARs in... more
Serine proteinases signal through proteinase-activated receptors (PARs). Our studies have implicated PAR 1 and PAR 4 (thrombin receptors) and PAR 2 (trypsin receptor) in human colon cancer growth. However, endogenous activators of PARs in colon tumors are still unknown. Recently, members of the kallikrein-related peptidase (KLK) family have been shown to activate PARs in many cell systems. Thus, our aim was to determine if KLK14, a KLK family member, is expressed in colonic tumors and to see if this KLK can activate PARs in human colon cancer cells. The presence of KLK14 in colon cancer cell lines was assessed by RT-PCR, ELISA and immunofluorescence, and its expression in human colonic tissue was assessed by immunohistochemistry. The effects of recombinant KLK14 in parallel with PAR 1/2 agonists (SFLLRN-NH 2 , an activating peptide for both PAR 1 and PAR 2 ; TFLLR-NH 2, a selective activating peptide for PAR 1 and the selective PAR 2 agonists, 2-furoyl-LIGRLO-NH 2 ; SLIGRL-NH 2 ) were assessed for calcium flux, receptor internalization and ERK1/2 phosphorylation in a human colon cancer-derived HT29 cell line that expresses PAR 1 and PAR 2 . We found that: a) KLK14 mRNA (RT-PCR) was present in 16 out of 16 human colon cancer cell lines, b) KLK14 protein is present (ELISA) in HT29 conditioned media and other colon cancer cell lines; c) there was strong staining of KLK14 in the cytoplasmic compartment of HT29 cells and of human colonic tumors (immunofluorescence and immunohistochemistry; paraffin sections) d) KLK14 (0.1 µM) caused prompt increases in intracellular calcium ([Ca2+] i ) in HT29 cells (Microspectrofluorimetry with Fura 2/fluo-3). The KLK14-induced Ca 2+ -flux was abrogated after an initial challenge of the cells with SFLLRN-NH 2 (100 µM), which desensitizes PAR 1 and PAR 2 simultaneously. Desensitization with 2-furoyl-LIGRLO-NH 2 (10 µM) a potent PAR 2 agonist, attenuated most of the Ca 2+ flux induced by KLK14, whereas responses to TFLLR-NH 2, the PAR 1 agonist, were minimally affected. Consistently, a first challenge with thrombin did not affect KLK14-induced Ca 2+ flux. These results strongly suggest that KLK14 activates preferentially PAR 2 in HT29 cells. e) KLK14 initiates a dramatic loss of cell surface PAR 2 (microscopic analysis) due to its internalization and f) KLK14 induces a rapid (within 5-10 min) and significant ERK1/2 phosphorylation in HT29 cells. This KLK14-PAR signaling pathway may represent a novel therapeutic target for colon tumorigenesis. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4104.
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In previous work, we identified two insulin receptor species, RI (KAV = 0.31) and RII (KAV = 0.53), that could be separated by gel filtration on Sepharose 6B. In the present study, we sought to establish that these two receptor species do... more
In previous work, we identified two insulin receptor species, RI (KAV = 0.31) and RII (KAV = 0.53), that could be separated by gel filtration on Sepharose 6B. In the present study, we sought to establish that these two receptor species do represent larger (RI) and smaller (RII) oligomeric forms of the receptor, rather than representing receptor species separated from each other by differential adsorption to the Sepharose matrix. Receptor solubilized from isolated human placenta membranes was purified by lectin–and insulin–agarose chromatography and was radiolabeled with carrier-free 125I. The labeled receptor was separated by Sepharose 6B gel filtration into two fractions (peak I, (KAV = 0.31; peak II. KAV = 0.53), was immunoprecipitated by anti-insulin receptor antibody, and was analysed by electrophoresis in nonreducing polyacrylamide slab gels. The auto-radiograms of the gels indicated that peak I (KAV = 0.31, RI receptor form) contained a number of receptor species of 240 000 da...
Research Interests: Chemistry, Chromatography, Kinetics, Medicine, Pregnancy, and 14 moreHumans, Insulin, Placenta, Female, Medical Physiology, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Receptor, Molecular weight, Size Exclusion Chromatography, Sepharose, Cell Membrane, Receptor Insulin, Pharmacology and pharmaceutical sciences, and Agarose
Oxytocin, like insulin, stimulates glucose oxidation in normal rat adipocytes. Fat cells from homozygous Brattleboro rats that exhibit diabetes insipidus (HoDI animals) and that have a normal number of oxytocin receptors, however, are... more
Oxytocin, like insulin, stimulates glucose oxidation in normal rat adipocytes. Fat cells from homozygous Brattleboro rats that exhibit diabetes insipidus (HoDI animals) and that have a normal number of oxytocin receptors, however, are unable to respond to oxytocin in terms of glucose oxidation. We now report that in adipocytes from HoDI animals that are responsive to insulin, oxytocin was also unable to stimulate lipogenesis. In contrast, oxytocin like insulin was able to inhibit epinephrine-stimulated lipolysis in adipocytes from HoDI animals. Thus, in HoDI adipocytes, the results indicate that the receptor–effector system is only partially defective.
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Circulating levels of oxytocin in the Brattleboro rat (homozygous diabetes insipidus strain, HoDI) are believed to be above normal.1+2 With many hormones, an excess leads to a decrease in tissue receptor number and/or res ~ o n s e . ~ .... more
Circulating levels of oxytocin in the Brattleboro rat (homozygous diabetes insipidus strain, HoDI) are believed to be above normal.1+2 With many hormones, an excess leads to a decrease in tissue receptor number and/or res ~ o n s e . ~ . A significantly lower number of receptors (one-seventh of normal) in the uterus of the HoDI rats together with unchanged uterine response to oxytocin has recently been reported.5 Glucose oxidation, which is stimulated by oxytocin in isolated fat cells of normal rats, does not occur in the adipocytes of HoDI rats, although these cells have unaltered oxytocin binding activity.fi Thus, epididymal adipocytes from HoDI rats are defective in coupling of oxytocinreceptor occupancy to the initiation of a response. This report describes some investigations into the source of the defect.
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We have evaluated factors, other than genetic, which might be related to the lack of an oxytocin-mediated insulinlike response (glucose oxidation; lipogenesis) in adipocytes from Brattleboro rats, homozygous for the diabetes insipidus... more
We have evaluated factors, other than genetic, which might be related to the lack of an oxytocin-mediated insulinlike response (glucose oxidation; lipogenesis) in adipocytes from Brattleboro rats, homozygous for the diabetes insipidus trait (HoDI rats). The manoeuvres used in an attempt to restore the glucoregulatory responses to oxytocin in HoDI cells (increased glucose in the fat pad digestion medium; increased calcium concentration in the oxidation assay; estrogen treatment; use of [1-14C]glucose as substrate; inclusion of adenosine in the assay medium; vasopressin replacement therapy) uniformly failed to result in oxytocin activation of HoDI adipocytes, in contrast, the contractile responses of estrogenized HoDI rat uteri were indistinguishable from those of estrogenized normal rats. We conclude that the nonresponsiveness of the Brattleboro adipocytes to the glucoregulatory actions of oxytocin is not due to factors related to the conditions of the bioassay. On the other hand, in...
Research Interests: Endocrinology, Chemistry, Medicine, Adipose tissue, Oxytocin, and 15 moreAdenosine, Internal Medicine, Glucose, Insulin, Female, Animals, Drug Resistance, Male, Medical Physiology, Triglycerides, Rats, Oxidation-Reduction, Ovariectomized Rat, Uterine contraction, and Pharmacology and pharmaceutical sciences
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Research Interests: Genetics, Physiology, Western blotting, Signal Transduction, Cell line, and 14 moreHumans, Intestinal Mucosa, Polymerase Chain Reaction, Trypsin, Medical Physiology, Glycoproteins, Immunoprecipitation, High Pressure Liquid Chromatography, Caco-2 cells, Epithelial cells, Matrix Metalloproteinases, Enzyme Linked Immunosorbent Assay, Src family kinases, and Phosphatidylinositol 3-KINASE
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Research Interests: Immunology, Biology, Medicine, Extracellular Matrix, Dexamethasone, and 15 moreAllergy, Par, Humans, Mitogen Activated Protein Kinase, mRna expression levels, Epithelial cells, MMP, Matrix Metalloproteinases, Primary Culture, MAP Kinase, Enzymatic Activity, Clinical Allergy and Immunology, Matrix Metalloproteinase, DMEM, and Adrenal cortex hormones
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In the present study, we report the synthesis and biological evaluation of a series of new non-peptide PAR(1) mimetic receptor antagonists, based on conformational analysis of the S(42)FLLR(46) tethered ligand (TL) sequence of PAR(1).... more
In the present study, we report the synthesis and biological evaluation of a series of new non-peptide PAR(1) mimetic receptor antagonists, based on conformational analysis of the S(42)FLLR(46) tethered ligand (TL) sequence of PAR(1). These compounds incorporate the key pharmacophore groups in the TL sequence, guanidyl, amino and phenyl, which are essential for triggering receptor activity. Compounds 5 and 15 (50-100 microM) inhibited both TFLLR-amide (10 microM) and thrombin-mediated (0.5 and 1 U/ml; 5 and 10 microM) calcium signaling in a cultured human HEK cell assay.
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Shortly after the discovery of pressor activity in extracts of the posterior lobe of the pituitary gland (Oliver & Schafer 1895; Howell 1898) it was found that the addition of salt precipitated the active... more
Shortly after the discovery of pressor activity in extracts of the posterior lobe of the pituitary gland (Oliver & Schafer 1895; Howell 1898) it was found that the addition of salt precipitated the active material (Osborne & Vincent 1900). Dale (1906) demonstrated the presence of the oxytocic principle in similar extracts. This activity was later found to accompany the pressor activity in salt precipitates (Kamm et al . 1928). Some years later van Dyke and his co-workers purified the material isolated from fresh pituitary tissue by salt precipitation. It was found to be a protein with a molecular weight of approximately 30 000, that appeared to be homogeneous by physico-chemical criteria. The protein possessed oxytocic, pressor and antidiuretic activities in the same ratios as they occur in the intact bovine pituitary gland (van Dyke, Chow, Greep & Rothen 1942). At that time, this protein was considered to be the natural form of a hormone possessing oxytocic, pressor and antidiuretic activities. However, another view, first expressed by Dudley (1919), suggested that the activities were attributable to the presence of two separate polypeptides. Kamm et al . (1928) extended the work of Dudley and by solvent extraction isolated two active principles, pitocin and pitressin, with molecular weights less than 1000. The classical work of du Vigneaud showed that the pituitary posterior lobe owes its characteristic biological activities to the presence of the polypeptide hormones, oxytocin and vasopressin (du Vigneaud 1955). The relation between the hormones and the so-called van Dyke protein was elucidated in Fromageot's laboratory. It was found that in mild acid oxytocin and vasopressin were dissociated from an inactive protein, neurophysin (Acher, Chauvet & Olivry 1956; Acher & Fromageot 1957. ) The portein-hormone complex could be reconstituted from the components in neutal solution.
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The essential role of proteinases as regulatory digestive enzymes, recognized since the late 1800s, has been underscored by the discovery that more than 2% of the genome codes for proteinases and their inhibitors. Further, by the early... more
The essential role of proteinases as regulatory digestive enzymes, recognized since the late 1800s, has been underscored by the discovery that more than 2% of the genome codes for proteinases and their inhibitors. Further, by the early 1970s it was appreciated that in addition to their digestive actions, proteinases can affect cell function: (1) by the generation or degradation of peptide hormones and (2) by the direct regulation of signalling by receptors like the one for insulin. It was the discovery in the 1990s of the novel G-protein-coupled 'proteinase-activated receptor' (PAR) family that has caused a paradigm shift in the understanding of the way that proteinases can regulate cell signalling. This overview provides a perspective for the discovery of the PARs and my laboratory's role in (1) understanding the molecular pharmacology of these fascinating receptors and (2) identifying the potential pathophysiological roles that the PAR family can play in inflammatory d...
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Using a guinea pig gastric longitudinal smooth muscle preparation, we have compared the contractile signaling pathways triggered by the thrombin receptor-activating peptide, TFLLR-NH2 (TF) and by epidermal growth factor-urogastrone (EGF).... more
Using a guinea pig gastric longitudinal smooth muscle preparation, we have compared the contractile signaling pathways triggered by the thrombin receptor-activating peptide, TFLLR-NH2 (TF) and by epidermal growth factor-urogastrone (EGF). In addition to inhibitors of tyrosine kinase [tyrphostin 47/AG213, genistein and the src-selective inhibitor CP118,556/PP1], cyclooxygenase (indomethacin, INDO) and diacylglycerol lipase (U57, 908), we also used the signal pathway probe inhibitors of mitogen-activated protein-kinase-kinase (MEK:PD98059), phosphatidylinositol 3'-kinase [PI3K: Wortmannin (WM) and LY294002], protein kinase C [PKC: GF109203X (GF)], and of the EGF-receptor kinase (PD153035). We found that in addition to the inhibition of both TF and EGF-stimulated contractions by the inhibitors of tyrosine kinase, cyclooxygenase and diacylglycerol lipase, the actions of TF and EGF were also attenuated by PD98059, WM/LY294002 and GF. However, PD153035 blocked only EGF-triggered contractions. The contractile actions of both TF and EGF were dependent on extracellular calcium. In contrast, the contractile action of arachidonic acid, via a presumed cyclooxygenase product that mediated the contractions caused by both TF and EGF, was not blocked by any of the signal pathway probe inhibitors. The contractile actions of both TF and EGF were accompanied by increases in tissue phosphotyrosyl proteins and an increase in tissue c-src kinase activity. We conclude that protease-activated receptor no. 1- (thrombin receptor) mediated contractions in the logitudial muscle, like EGF receptor-activated responses, require the influx of extracellular calcium and use parallel signal pathways upstream of the cyclooxygenase step, involving MEK, PI3K, kinase C and possibly cellular src. The TF-induced response did not involve trans-activation of the EGF receptor kinase; but the converse (i.e., trans-activation of protease-activated receptor no. 1 (thrombin receptor) by the EGF receptor kinase) could not be ruled out.
Research Interests: Calcium, Enzyme Inhibitors, Signal Transduction, Animals, Male, and 10 moreEpidermal Growth Factor, Muscle contraction, Protein Kinase Inhibitors, Arachidonic Acid, Quinazolines, Lipoprotein Lipase, Protein Kinase C, Protein tyrosine kinases, Guinea pigs, and Pharmacology and pharmaceutical sciences
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This commentary deals with the new observations that dendritic cell (DC) oxytocin receptors play a role in the inflammatory response generated in murine animal models of colitis. The overview provides a context of the discovery of... more
This commentary deals with the new observations that dendritic cell (DC) oxytocin receptors play a role in the inflammatory response generated in murine animal models of colitis. The overview provides a context of the discovery of oxytocin (OT), its chemical synthesis and the cell biology of its neurohypophysial synthesis and secretion. This perspective provides insight and raises questions to be answered related to the impact of OT in the gastrointestinal tract and to further the exploration of OT as a potentially locally synthesised regulator of intestinal inflammatory pathophysiology.
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In Homer’s 8th century BC Greek epic poem “Odyssey”, Odysseus, upon leaving town to do battle for an extended period, could not have done better than to leave his son, Telemachus, in the care of a trusted friend, Mentor. Thus,... more
In Homer’s 8th century BC Greek epic poem “Odyssey”, Odysseus, upon leaving town to do battle for an extended period, could not have done better than to leave his son, Telemachus, in the care of a trusted friend, Mentor. Thus, “mentorship” can be seen as a key process, whereby a more experienced individual takes on an advisor role for a less-experienced colleague.
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The receptor binding and biological activity of epidermal growth factor-urogastrone (EGF) was characterized in the follicle-enclosed goldfish oocyte. The binding of 125I-labeled mouse EGF (mEGF) to goldfish ovarian membrane preparation... more
The receptor binding and biological activity of epidermal growth factor-urogastrone (EGF) was characterized in the follicle-enclosed goldfish oocyte. The binding of 125I-labeled mouse EGF (mEGF) to goldfish ovarian membrane preparation was peptide specific, saturable, reversible, and dependent on time and tissue concentration. Binding data analysis indicated the presence of a single class of high-affinity binding sites with an estimated equilibrium dissociation constant of 4.4 +/- 1.8 x 10(-10) M. The 125I-mEGF binding was displaced by unlabeled mEGF and by human recombinant transforming growth factor-alpha (hTGF-alpha). Both mEGF and hTGF-alpha were found to stimulate reinitiation of oocyte meiosis, as indicated by germinal vesicle breakdown (GVBD). Treatment with mEGF and hTGF-alpha stimulated GVBD from a basal level of 8.5 to approximately 30% with an estimated half-maximal effective dose for EGF of 5.80 +/- 0.82 + 10(-10) and for hTGF-alpha, 1.9 +/- 1.0 x 10(-10) M. Furthermore,...
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The small GTPase Ras-related C3 botulinum toxin substrate 1B (RAC1B) has been shown previously by RNA interference-mediated knockdown (KD) to function as a powerful inhibitor of transforming growth factor (TGF)-β1-induced cell migration... more
The small GTPase Ras-related C3 botulinum toxin substrate 1B (RAC1B) has been shown previously by RNA interference-mediated knockdown (KD) to function as a powerful inhibitor of transforming growth factor (TGF)-β1-induced cell migration and epithelial-mesenchymal transition in epithelial cells, but the underlying mechanism has remained enigmatic. Using pancreatic carcinoma cells, we show that both KD and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated knockout (KO) of RAC1B increased the expression of the TGF-β type I receptor ALK5 (activin receptor-like kinase 5), but this effect was more pronounced in CRISPR-KO cells. Of note, in KO, but not KD cells, ALK5 upregulation was associated with resensitization of TGFBR1 to induction by TGF-β1 stimulation. RAC1B KO also increased TGF-β1-induced C-terminal SMAD3 phosphorylation, SMAD3 transcriptional activity, growth inhibition, and cell migration. The KD of ALK5 expression by RNA interference or inactivat...
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We studied the potential roles for endogenous interleukin-1β (IL-1β) and for several signaling pathways in the spontaneous induction in vitro of inducible nitric oxide synthase (iNOS) in endothelium-denuded rat aorta rings. Added IL-1β... more
We studied the potential roles for endogenous interleukin-1β (IL-1β) and for several signaling pathways in the spontaneous induction in vitro of inducible nitric oxide synthase (iNOS) in endothelium-denuded rat aorta rings. Added IL-1β augmented, whereas the IL-1β receptor antagonist IL-1ra blocked, spontaneous iNOS induction. Furthermore, increases in IL-1β mRNA preceded those of iNOS mRNA. Mitogen-activated protein kinase kinase and phosphatidyl inositol 3′ kinase inhibition did not block iNOS induction, whereas nuclear factor κB inhibition did. The sarcoma virus tyrosine kinase (Src) family-selective inhibitor 4-amino-5(4-methylphenyl)-7-( t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) blocked the upregulation of IL-1β mRNA and the subsequent induction of iNOS but not the induction of iNOS stimulated by exogenously added IL-1β. In contrast, the non-Src inhibitors TP 47/AG 213 and genistein and the tyrosine phosphatase inhibitor vanadate did not affect the spontaneous upregulation of IL-...
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Inflammatory diseases of the gut are associated with altered electrolyte and water transport, leading to the development of diarrhea. Epithelially expressed aquaporins (AQPs) are downregulated in inflammation, although the mechanisms... more
Inflammatory diseases of the gut are associated with altered electrolyte and water transport, leading to the development of diarrhea. Epithelially expressed aquaporins (AQPs) are downregulated in inflammation, although the mechanisms involved are not known. We hypothesized that AQP3 expression in intestinal epithelial cells is altered in intestinal inflammation and that these changes are driven by tumor necrosis factor (TNF) Human colonic adenocarcinoma (HT-29) cells were treated with TNF to investigate signaling mechanisms in vitro. AQP3 expression was assessed by real-time PCR and radiolabeled glycerol uptake, with select inhibitors and a luciferase reporter construct used to further elucidate intracellular signaling. AQP3 expression was downregulated in HT-29 cells treated with TNF Luciferase reporter construct experiments revealed that TNF downregulated constitutive transcriptional activity of the AQP3 promoter, and inhibition of MEK/ERK and nuclear factor B (NF-B) signaling pre...
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Aquaporin (AQP) 3 expression is altered in inflammatory bowel diseases, although the exact mechanisms regulating AQP abundance are unclear. Although interferon gamma (IFNγ) is centrally involved in intestinal inflammation, the effect of... more
Aquaporin (AQP) 3 expression is altered in inflammatory bowel diseases, although the exact mechanisms regulating AQP abundance are unclear. Although interferon gamma (IFNγ) is centrally involved in intestinal inflammation, the effect of this cytokine on AQP3 expression remains unknown. HT-29 human colonic epithelial cells were treated with IFNγ to assess AQP3 mRNA expression by real-time RT-PCR and functional protein expression through the uptake of radiolabelled glycerol. Transient knockdown of signal transducer and activator of transcription 1 (STAT1), STAT3, Sp1, and Sp3 were performed to determine the involvement of these transcription factors in the IFNγ-induced signalling cascade. AQP3 promoter regions involved in the response to IFNγ were assessed using a luciferase reporter system. Likewise, enteroids derived from human colonic biopsies were also treated with IFNγ to assess for changes in AQP3 mRNA expression. IFNγ decreased AQP3 mRNA expression in HT-29 cells in a time- and...
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We propose that in the microenvironment of inflammatory tissues, including tumours, extracellular proteinases can modulate cell signalling in part by regulating proteinaseactivated receptors (PARs). We have been exploring this mechanism... more
We propose that in the microenvironment of inflammatory tissues, including tumours, extracellular proteinases can modulate cell signalling in part by regulating proteinaseactivated receptors (PARs). We have been exploring this mechanism in a variety of inflammation and tumour-related settings that include tumour-derived cultured cells from prostate and bladder cancer, as well as immune inflammatory cells that are involved in the pathology of inflammatory diseases including multiple sclerosis. Our work shows that proteinase signalling via the PARs affects prostate and bladder cancer-derived tumour cell behaviour and can regulate calcium signalling in human T-cell and macrophage-related inflammatory cells as well as in murine splenocytes. Further, we find that the tumour-derived prostate cancer cells and immune-related cells (Jurkat, THP1, mouse splenocytes) can produce PAR-regulating proteinases (including kallikreins: KLKs), that can control tissue function by both a paracrine and a...
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While proteases are essential in gastrointestinal physiology, accumulating evidence indicates that dysregulated proteolysis plays a pivotal role in the pathophysiology of inflammatory bowel disease (IBD). Nonetheless, the identity of... more
While proteases are essential in gastrointestinal physiology, accumulating evidence indicates that dysregulated proteolysis plays a pivotal role in the pathophysiology of inflammatory bowel disease (IBD). Nonetheless, the identity of overactive proteases released by human colonic mucosa remains largely unknown. Studies of protease abundance have primarily investigated expression profiles, not taking into account their enzymatic activity. Herein we have used serine protease-targeted activity-based probes (ABPs) coupled with mass spectral analysis to identify active forms of proteases secreted by the colonic mucosa of healthy controls and IBD patients. Profiling of (Pro-Lys)-ABP bound proteases revealed that most of hyperactive proteases from IBD secretome are clustered at 28-kDa. We identified seven active proteases: the serine proteases cathepsin G, plasma kallikrein, plasmin, tryptase, chymotrypsin-like elastase 3 A, and thrombin and the aminopeptidase B. Only cathepsin G and throm...
Proteinases are enzymes with established roles in physiological and pathological processes such as digestion and the homeostasis, destruction and repair of tissues. Over the past few years, the hormone-like properties of circulating... more
Proteinases are enzymes with established roles in physiological and pathological processes such as digestion and the homeostasis, destruction and repair of tissues. Over the past few years, the hormone-like properties of circulating proteinases have become increasingly appreciated. Some proteolytic enzymes trigger cell signalling via proteinase-activated receptors, a family of G protein-coupled receptors that have been implicated in inflammation and pain in inflammatory arthritis. Proteinases can also regulate ion flux owing to the cross-sensitization of transient receptor potential cation channel subfamily V members 1 and 4, which are associated with mechanosensing and pain. In this Review, the idea that proteinases have the potential to orchestrate inflammatory signals by interacting with receptors on cells within the synovial microenvironment of an inflamed joint is revisited in three arthritic diseases: osteoarthritis, spondyloarthritis and rheumatoid arthritis. Unanswered quest...
The essential role of proteinases as regulatory digestive enzymes, recognized since the late 1800s, has been underscored by the discovery that more than 2% of the genome codes for proteinases and their inhibitors. Further, by the early... more
The essential role of proteinases as regulatory digestive enzymes, recognized since the late 1800s, has been underscored by the discovery that more than 2% of the genome codes for proteinases and their inhibitors. Further, by the early 1970s it was appreciated that in addition to their digestive actions, proteinases can affect cell function: (1) by the generation or degradation of peptide hormones and (2) by the direct regulation of signalling by receptors like the one for insulin. It was the discovery in the 1990s of the novel G-protein-coupled ‘proteinase-activated receptor’ (PAR) family that has caused a paradigm shift in the understanding of the way that proteinases can regulate cell signalling. This overview provides a perspective for the discovery of the PARs and my laboratory’s role in (1) understanding the molecular pharmacology of these fascinating receptors and (2) identifying the potential pathophysiological roles that the PAR family can play in inflammatory disease. In t...
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On November 8, 2013, the Leaders in Medicine (LIM) program hosted the 5th Annual Research Symposium. Dr. Jerrold Ellner, Chief of the Infectious Diseases section at Boston Medical Centre and Professor of Medicine at Boston University... more
On November 8, 2013, the Leaders in Medicine (LIM) program hosted the 5th Annual Research Symposium. Dr. Jerrold Ellner, Chief of the Infectious Diseases section at Boston Medical Centre and Professor of Medicine at Boston University School of Medicine, was the keynote speaker and presented his lecture entitled “Tuberculosis – Past, Present and Future”. The LIM symposium gives a forum for LIM as well as non-LIM medical students to present their research work as either an oral or poster presentation. There were a total of 53 abstracts presented and five oral presentations. The symposium was attended by over 100 students and more than 30 staff members. The oral presentations included • Amrita Roy, Aboriginal identity, ethnic minority status, and prenatal depressive symptoms in a longitudinal pregnancy cohort study in Alberta. • David Nicholl, Obstructive sleep apnea treatment with continuous positive airway pressure decreases intraglomerular pressure and alters renal sensitivity to an...
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Structural cardiac remodeling after ischemic injury can induce a transition to heart failure from progressive loss of cardiac function. Cellular regenerative therapies are promising but face significant translational hurdles. Tissue... more
Structural cardiac remodeling after ischemic injury can induce a transition to heart failure from progressive loss of cardiac function. Cellular regenerative therapies are promising but face significant translational hurdles. Tissue extracellular matrix (ECM) holds the necessary environmental cues to stimulate cell-based endogenous myocardial repair pathways and promote adaptive remodeling toward functional recovery. Heart epicardium has emerged as an important anatomic niche for endogenous repair pathways including vasculogenesis and cardiogenesis. We show that acellular ECM scaffolds surgically implanted on the epicardium following myocardial infarction (MI) can attenuate structural cardiac remodeling and improve functional recovery. We assessed the efficacy of this strategy on post-MI functional recovery by comparing intact bioactive scaffolds with biologically inactivated ECM scaffolds. We confirm that bioactive properties within the acellular ECM biomaterial are essential for the observed functional benefits. We show that interaction of human cardiac fibroblasts with bioactive ECM can induce a robust cell-mediated vasculogenic paracrine response capable of functional blood vessel assembly. Fibroblast growth factor-2 is uncovered as a critical regulator of this novel bioinductive effect. Acellular bioactive ECM scaffolds surgically implanted on the epicardium post-MI can reprogram resident fibroblasts and stimulate adaptive pro-reparative pathways enhancing functional recovery. We introduce a novel surgical strategy for tissue repair that can be performed as an adjunct to conventional surgical revascularization with minimal translational challenges.