Forgot your password?
New user?
New Window Opens on the Secret Life of Microbes: Scientists Develop First Microbial Profiles of Ecosystems
CAS announces the release of SciFinder Scholar for Mac OS X
Press Releases
Press Releases
| Check out our New Publishers' Select for Free Articles |
Biochemical Pharmacology (v.83, #7)
DPP-4 inhibitors in the treatment of type 2 diabetes by Hélène Duez; Bertrand Cariou; Bart Staels (pp. 823-832).
Although being a primary objective in the management of type 2 diabetes, optimal glycaemic control is difficult to achieve and usually not maintained over time. Type 2 diabetes is a complex pathology, comprising altered insulin sensitivity and impaired insulin secretion. Recent advances in the understanding of the physiological functions of incretins and their degrading enzyme dipeptidyl-peptidase (DPP)-4 have led to the ‘discovery’ of a new class of oral anti-diabetic drugs. Several DPP-4 inhibitors (or gliptins) with different chemical structures are now available. These agents inhibit the degradation of the incretins glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) and hence potentiate glucose-dependent insulin secretion. DPP-4 inhibitors inhibit DPP-4 activity by almost 100% in vitro, maintaining a ≥80% inhibition throughout the treatment period in vivo, thus prolonging GLP-1 half-life, and significantly reducing HbA1c generally by −0.7 to 0.8% as well as fasting and post-prandial glycaemia. They are well-tolerated with no weight gain and few adverse effects, and, of particular interest, no increase in hypoglycaemic episodes. Although different by their chemical structure and pharmacokinetic properties, the DPP4 inhibitors currently available have proven similar glucose lowering efficacy.
Keywords: Dipeptidylpeptidase (DPP)-4 inhibitors; Gliptins; Glucagon-like peptide (GLP)-1; Glycaemic control; Type 2 diabetes
DPP-4 inhibitors in the treatment of type 2 diabetes by Hélène Duez; Bertrand Cariou; Bart Staels (pp. 823-832).
Although being a primary objective in the management of type 2 diabetes, optimal glycaemic control is difficult to achieve and usually not maintained over time. Type 2 diabetes is a complex pathology, comprising altered insulin sensitivity and impaired insulin secretion. Recent advances in the understanding of the physiological functions of incretins and their degrading enzyme dipeptidyl-peptidase (DPP)-4 have led to the ‘discovery’ of a new class of oral anti-diabetic drugs. Several DPP-4 inhibitors (or gliptins) with different chemical structures are now available. These agents inhibit the degradation of the incretins glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) and hence potentiate glucose-dependent insulin secretion. DPP-4 inhibitors inhibit DPP-4 activity by almost 100% in vitro, maintaining a ≥80% inhibition throughout the treatment period in vivo, thus prolonging GLP-1 half-life, and significantly reducing HbA1c generally by −0.7 to 0.8% as well as fasting and post-prandial glycaemia. They are well-tolerated with no weight gain and few adverse effects, and, of particular interest, no increase in hypoglycaemic episodes. Although different by their chemical structure and pharmacokinetic properties, the DPP4 inhibitors currently available have proven similar glucose lowering efficacy.
Keywords: Dipeptidylpeptidase (DPP)-4 inhibitors; Gliptins; Glucagon-like peptide (GLP)-1; Glycaemic control; Type 2 diabetes
Ah receptor- and Nrf2-gene battery members: Modulators of quinone-mediated oxidative and endoplasmic reticulum stress by Karl Walter Bock (pp. 833-838).
Quinones are ubiquitously present in mammals and their environment. They are involved in physiologic functions such as electron transport but are also toxic compounds. In particular, quinone–quinol redox cycles may lead to oxidative stress, and arylating quinones have been demonstrated to activate endoplasmic reticulum (ER) stress. To detoxify quinones coordinately regulated Ah receptor and Nrf2 gene batteries evolved. Two pathways are emphasized: (i) glutathione S-transferases, and (ii) NAD(P)H:quinone oxidoreductases NQO1 and NQO2 acting together with UDP-glucuronosyltransferases and sulfotransferases. Coupling between these enzymes may prevent oxidative and ER stress in a tissue-dependent manner, as discussed using benzo[a]pyrene detoxification in enterocytes, catecholestrogen metabolism in breast tissue and endometrium, and aminochromes in neurones and astrocytes. Possible consequences of chronic ER stress such as apoptosis and inflammation as well as therapeutic possibilities of modulating Ah receptor and Nrf2 are discussed. In conclusion, tight coupling of Ah receptor- and Nrf2-regulated enzymes may prevent quinone-mediated oxidative and ER stress.
Keywords: Abbreviations; AhR; Ah receptor; DME; drug-metabolizing enzyme; ER; endoplasmic reticulum; GSH; reduced glutathione; GST; glutathione S-transferase; NQO; NAD(P)Hquinone oxidoreductase; Nrf2; nuclear erythroid-related factor 2; ROS; reactive oxygen species; SULT; sulfotransferase; UGT; UDP-glucuronosyltransferaseQuinones; Ah receptor; Nrf2; Oxidative stress; Endoplasmic reticulum stress
Ah receptor- and Nrf2-gene battery members: Modulators of quinone-mediated oxidative and endoplasmic reticulum stress by Karl Walter Bock (pp. 833-838).
Quinones are ubiquitously present in mammals and their environment. They are involved in physiologic functions such as electron transport but are also toxic compounds. In particular, quinone–quinol redox cycles may lead to oxidative stress, and arylating quinones have been demonstrated to activate endoplasmic reticulum (ER) stress. To detoxify quinones coordinately regulated Ah receptor and Nrf2 gene batteries evolved. Two pathways are emphasized: (i) glutathione S-transferases, and (ii) NAD(P)H:quinone oxidoreductases NQO1 and NQO2 acting together with UDP-glucuronosyltransferases and sulfotransferases. Coupling between these enzymes may prevent oxidative and ER stress in a tissue-dependent manner, as discussed using benzo[a]pyrene detoxification in enterocytes, catecholestrogen metabolism in breast tissue and endometrium, and aminochromes in neurones and astrocytes. Possible consequences of chronic ER stress such as apoptosis and inflammation as well as therapeutic possibilities of modulating Ah receptor and Nrf2 are discussed. In conclusion, tight coupling of Ah receptor- and Nrf2-regulated enzymes may prevent quinone-mediated oxidative and ER stress.
Keywords: Abbreviations; AhR; Ah receptor; DME; drug-metabolizing enzyme; ER; endoplasmic reticulum; GSH; reduced glutathione; GST; glutathione S-transferase; NQO; NAD(P)Hquinone oxidoreductase; Nrf2; nuclear erythroid-related factor 2; ROS; reactive oxygen species; SULT; sulfotransferase; UGT; UDP-glucuronosyltransferaseQuinones; Ah receptor; Nrf2; Oxidative stress; Endoplasmic reticulum stress
Interactions of Bordetella pertussis adenylyl cyclase toxin CyaA with calmodulin mutants and calmodulin antagonists: Comparison with membranous adenylyl cyclase I by Dominik Schuler; Carolin Lübker; Gerald H. Lushington; Wei-Jen Tang; Yuequan Shen; Mark Richter; Roland Seifert (pp. 839-848).
We show that the adenylyl cyclase toxin CyaA interacts differentially with various calmodulin mutants and that calmidazolium inhibits CyaA directly.The adenylyl cyclase (AC) toxin CyaA from Bordetella pertussis constitutes an important virulence factor for the pathogenesis of whooping cough. CyaA is activated by calmodulin (CaM) and compromises host defense by excessive cAMP production. Hence, pharmacological modulation of the CyaA/CaM interaction could constitute a promising approach to treat whooping cough, provided that interactions of endogenous effector proteins with CaM are not affected. As a first step toward this ambitious goal we examined the interactions of CyaA with wild-type CaM and four CaM mutants in which most methionine residues were replaced by leucine residues and studied the effects of the CaM antagonists calmidazolium, trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). CyaA/CaM interaction was monitored by CaM-dependent fluorescence resonance energy transfer (FRET) between tryptophan residues in CyaA and 2′-(N-methylanthraniloyl)-3′-deoxy-adenosine 5′-triphosphate and catalytic activity. Comparison of the concentration/response curves of CaM and CaM mutants for FRET and catalysis revealed differences, suggesting a two-step activation mechanism of CyaA by CaM. Even in the absence of CaM, calmidazolium inhibited catalysis, and it did so according to a biphasic function. Trifluoperazine and W-7 did not inhibit FRET or catalysis. In contrast to CyaA, some CaM mutants were more efficacious than CaM at activating membranous AC isoform 1. The slope of CyaA activation by CaM was much steeper than of AC1 activation. Collectively, the two-step activation mechanism of CyaA by CaM offers opportunities for pharmacological intervention. The failure of classic CaM inhibitors to interfere with CyaA/CaM interactions and the different interactions of CaM mutants with CyaA and AC1 point to unique CyaA/CaM interactions.
Keywords: Abbreviations; AC; adenylyl cyclase; AC1; membranous adenylyl cyclase isoform 1; CyaA; Bordetella pertussis; adenylyl cyclase toxin; CaM; calmodulin; CaM-WT; wild-type calmodulin; FRET; fluorescence resonance energy transfer; 2′-MANT-3′-d-ATP; 2′-(N-methylanthraniloyl)-3′-deoxy-adenosine 5′-triphosphate; PPI; protein-protein interaction; W-7; N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamideBordetella pertussis; Adenylyl cyclase; Calmodulin; Fluorescence spectroscopy; Calmodulin antagonists
Interactions of Bordetella pertussis adenylyl cyclase toxin CyaA with calmodulin mutants and calmodulin antagonists: Comparison with membranous adenylyl cyclase I by Dominik Schuler; Carolin Lübker; Gerald H. Lushington; Wei-Jen Tang; Yuequan Shen; Mark Richter; Roland Seifert (pp. 839-848).
We show that the adenylyl cyclase toxin CyaA interacts differentially with various calmodulin mutants and that calmidazolium inhibits CyaA directly.The adenylyl cyclase (AC) toxin CyaA from Bordetella pertussis constitutes an important virulence factor for the pathogenesis of whooping cough. CyaA is activated by calmodulin (CaM) and compromises host defense by excessive cAMP production. Hence, pharmacological modulation of the CyaA/CaM interaction could constitute a promising approach to treat whooping cough, provided that interactions of endogenous effector proteins with CaM are not affected. As a first step toward this ambitious goal we examined the interactions of CyaA with wild-type CaM and four CaM mutants in which most methionine residues were replaced by leucine residues and studied the effects of the CaM antagonists calmidazolium, trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). CyaA/CaM interaction was monitored by CaM-dependent fluorescence resonance energy transfer (FRET) between tryptophan residues in CyaA and 2′-(N-methylanthraniloyl)-3′-deoxy-adenosine 5′-triphosphate and catalytic activity. Comparison of the concentration/response curves of CaM and CaM mutants for FRET and catalysis revealed differences, suggesting a two-step activation mechanism of CyaA by CaM. Even in the absence of CaM, calmidazolium inhibited catalysis, and it did so according to a biphasic function. Trifluoperazine and W-7 did not inhibit FRET or catalysis. In contrast to CyaA, some CaM mutants were more efficacious than CaM at activating membranous AC isoform 1. The slope of CyaA activation by CaM was much steeper than of AC1 activation. Collectively, the two-step activation mechanism of CyaA by CaM offers opportunities for pharmacological intervention. The failure of classic CaM inhibitors to interfere with CyaA/CaM interactions and the different interactions of CaM mutants with CyaA and AC1 point to unique CyaA/CaM interactions.
Keywords: Abbreviations; AC; adenylyl cyclase; AC1; membranous adenylyl cyclase isoform 1; CyaA; Bordetella pertussis; adenylyl cyclase toxin; CaM; calmodulin; CaM-WT; wild-type calmodulin; FRET; fluorescence resonance energy transfer; 2′-MANT-3′-d-ATP; 2′-(N-methylanthraniloyl)-3′-deoxy-adenosine 5′-triphosphate; PPI; protein-protein interaction; W-7; N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamideBordetella pertussis; Adenylyl cyclase; Calmodulin; Fluorescence spectroscopy; Calmodulin antagonists
Sialylation of epidermal growth factor receptor regulates receptor activity and chemosensitivity to gefitinib in colon cancer cells by Jung-Jin Park; Jae Youn Yi; Yeung Bae Jin; Yoon-Jin Lee; Jae-Seon Lee; Yun-Sil Lee; Young-Gyu Ko; Minyoung Lee (pp. 849-857).
β-Galactoside α2,6-sialyltransferase (ST6Gal-I) has been shown to catalyze α2,6 sialylation of N-glycan, an action that is highly correlated with colon cancer progression and metastasis. We have recently demonstrated that ST6Gal-I-induced α2,6 sialylation is critical for adhesion and migration of colon cancer cells. Increase of α2,6 sialylation also contributes to radioresistance of colon cancer. A number of studies have focused on the involvement of sialylation in tumorigenesis, but the mechanism underlying ST6Gal-I-induced cancer progression and the identity of enzyme substrates has received scant research attention. To provide further support for the relevance of ST6Gal-I in the malignancy of colon cancer, we prepared and characterized a ST6Gal-I-knockdown SW480 colorectal carcinoma cell line. We found that inhibition of ST6Gal-I expression increased cell proliferation and tumor growth in vitro and in vivo. An examination of the effect of sialylation on epidermal growth factor receptor (EGFR) activity and downstream signaling, which are highly correlated with cell proliferation, showed that the loss of ST6Gal-I augmented EGF-induced EGFR phosphorylation and activation of extracellular signal-regulated kinase (ERK) in colon cancer cells. Moreover, ST6Gal-I induced sialylation of both wild type and mutant EGFR. These studies provide the first demonstration that ST6Gal-I induces EGFR sialylation in human colon cancer cell lines. Importantly, the anticancer effect of the EGFR kinase inhibitor, gefitinib, was increased in ST6Gal-I-deficient colon cancer cells. In contrast, overexpression of ST6Gal I decreased the cytotoxic effect of gefitinib. These results suggest that sialylation of the EGFR affects EGF-mediated cell growth and induces chemoresistance to gefitinib in colon cancer cells.
Keywords: ST6Gal I; EGFR; Colon cancer; Gefitinib; Sialylation
Sialylation of epidermal growth factor receptor regulates receptor activity and chemosensitivity to gefitinib in colon cancer cells by Jung-Jin Park; Jae Youn Yi; Yeung Bae Jin; Yoon-Jin Lee; Jae-Seon Lee; Yun-Sil Lee; Young-Gyu Ko; Minyoung Lee (pp. 849-857).
β-Galactoside α2,6-sialyltransferase (ST6Gal-I) has been shown to catalyze α2,6 sialylation of N-glycan, an action that is highly correlated with colon cancer progression and metastasis. We have recently demonstrated that ST6Gal-I-induced α2,6 sialylation is critical for adhesion and migration of colon cancer cells. Increase of α2,6 sialylation also contributes to radioresistance of colon cancer. A number of studies have focused on the involvement of sialylation in tumorigenesis, but the mechanism underlying ST6Gal-I-induced cancer progression and the identity of enzyme substrates has received scant research attention. To provide further support for the relevance of ST6Gal-I in the malignancy of colon cancer, we prepared and characterized a ST6Gal-I-knockdown SW480 colorectal carcinoma cell line. We found that inhibition of ST6Gal-I expression increased cell proliferation and tumor growth in vitro and in vivo. An examination of the effect of sialylation on epidermal growth factor receptor (EGFR) activity and downstream signaling, which are highly correlated with cell proliferation, showed that the loss of ST6Gal-I augmented EGF-induced EGFR phosphorylation and activation of extracellular signal-regulated kinase (ERK) in colon cancer cells. Moreover, ST6Gal-I induced sialylation of both wild type and mutant EGFR. These studies provide the first demonstration that ST6Gal-I induces EGFR sialylation in human colon cancer cell lines. Importantly, the anticancer effect of the EGFR kinase inhibitor, gefitinib, was increased in ST6Gal-I-deficient colon cancer cells. In contrast, overexpression of ST6Gal I decreased the cytotoxic effect of gefitinib. These results suggest that sialylation of the EGFR affects EGF-mediated cell growth and induces chemoresistance to gefitinib in colon cancer cells.
Keywords: ST6Gal I; EGFR; Colon cancer; Gefitinib; Sialylation
DNA demethylation increases sensitivity of neuroblastoma cells to chemotherapeutic drugs by Jessica Charlet; Michael Schnekenburger; Keith W. Brown; Marc Diederich (pp. 858-865).
Neuroblastoma is a common embryonal malignancy in which high-stage cases have a poor prognosis, often associated with resistance to chemotherapeutic drugs. DNA methylation alterations are frequent in neuroblastoma and can modulate sensitivity to chemotherapeutic drugs in other cancers, suggesting that manipulation of epigenetic modifications could provide novel treatment strategies for neuroblastoma. We evaluated neuroblastoma cell lines for DNA demethylation induced by 5-Aza-2′-deoxycytidine, using genome-wide and gene-specific assays. Cytotoxic effects of chemotherapeutic agents (cisplatin, doxorubicin and etoposide), with and without 5-Aza-2′-deoxycytidine, were determined by morphological and biochemical apoptosis assays. We observed that the extent of genome-wide DNA demethylation induced by 5-Aza-2′-deoxycytidine varied between cell lines and was associated with expression differences of genes involved in the uptake and metabolism of 5-Aza-2′-deoxycytidine. Treatment of neuroblastoma cells with a combination of chemotherapeutic drugs and 5-Aza-2′-deoxycytidine significantly increased the levels of apoptosis induced by cisplatin, doxorubicin and etoposide, compared to treatment with chemotherapeutic drugs alone. The variable demethylation of cell lines in response to 5-Aza-2′-deoxycytidine suggests that epigenetic modifiers need to be targeted to suitably susceptible tumours for maximum therapeutic benefit. Epigenetic modifiers, such as 5-Aza-2′-deoxycytidine, could be used in combination with chemotherapeutic drugs to enhance their cytotoxicity, providing more effective treatment options for chemoresistant neuroblastomas.
Keywords: Neuroblastoma; Chemotherapeutic drugs; MYCN; DNA methylation; 5-Aza-2′-deoxycytidine
DNA demethylation increases sensitivity of neuroblastoma cells to chemotherapeutic drugs by Jessica Charlet; Michael Schnekenburger; Keith W. Brown; Marc Diederich (pp. 858-865).
Neuroblastoma is a common embryonal malignancy in which high-stage cases have a poor prognosis, often associated with resistance to chemotherapeutic drugs. DNA methylation alterations are frequent in neuroblastoma and can modulate sensitivity to chemotherapeutic drugs in other cancers, suggesting that manipulation of epigenetic modifications could provide novel treatment strategies for neuroblastoma. We evaluated neuroblastoma cell lines for DNA demethylation induced by 5-Aza-2′-deoxycytidine, using genome-wide and gene-specific assays. Cytotoxic effects of chemotherapeutic agents (cisplatin, doxorubicin and etoposide), with and without 5-Aza-2′-deoxycytidine, were determined by morphological and biochemical apoptosis assays. We observed that the extent of genome-wide DNA demethylation induced by 5-Aza-2′-deoxycytidine varied between cell lines and was associated with expression differences of genes involved in the uptake and metabolism of 5-Aza-2′-deoxycytidine. Treatment of neuroblastoma cells with a combination of chemotherapeutic drugs and 5-Aza-2′-deoxycytidine significantly increased the levels of apoptosis induced by cisplatin, doxorubicin and etoposide, compared to treatment with chemotherapeutic drugs alone. The variable demethylation of cell lines in response to 5-Aza-2′-deoxycytidine suggests that epigenetic modifiers need to be targeted to suitably susceptible tumours for maximum therapeutic benefit. Epigenetic modifiers, such as 5-Aza-2′-deoxycytidine, could be used in combination with chemotherapeutic drugs to enhance their cytotoxicity, providing more effective treatment options for chemoresistant neuroblastomas.
Keywords: Neuroblastoma; Chemotherapeutic drugs; MYCN; DNA methylation; 5-Aza-2′-deoxycytidine
Small molecule inhibitors of arginyltransferase regulate arginylation-dependent protein degradation, cell motility, and angiogenesis by Sougata Saha; Junling Wang; Brian Buckley; Qingqing Wang; Brenda Lilly; Mikhail Chernov; Anna Kashina (pp. 866-873).
Posttranslational arginylation mediated by arginyltransferase (ATE1) is an emerging major regulator of embryogenesis and cell physiology. Impairments of ATE1 are implicated in congenital heart defects, obesity, cancer, and neurodegeneration making this enzyme an important therapeutic target, whose potential has been virtually unexplored. Here we report the development of a biochemical assay for identification of small molecule inhibitors of ATE1 and application of this assay to screen a library of 3280 compounds. Our screen identified two compounds which specifically affect ATE1-regulated processes in vivo, including tannic acid, which has been previously shown to inhibit protein degradation and angiogenesis and to act as a therapeutic agent in heart disease and cancer. Our data suggest that these actions of tannic acid are mediated by its direct effect on ATE1, which regulates protein degradation and angiogenesis in vivo.
Keywords: Abbreviations; Ate1; arginine transfer enzyme 1 or arginyltransferase 1; RRS; Arg-tRNA synthetase; BSA; bovine serum albumin; RGS4; regulator of G protein signaling 4; VEGF-A165; vascular endothelial growth factor A165; HUVEC; human umbilical vein endothelial cells; TRITC; retramethyl rhodamine isothiocyanate; CXCL12; C-X-C motif ligand 12; ECGS; endothelial cell growth supplement; bFGF; basic fibroblast growth factorATE1 inhibitors; Arginylation; tannic acid; Cell motility; Angiogenesis
Small molecule inhibitors of arginyltransferase regulate arginylation-dependent protein degradation, cell motility, and angiogenesis by Sougata Saha; Junling Wang; Brian Buckley; Qingqing Wang; Brenda Lilly; Mikhail Chernov; Anna Kashina (pp. 866-873).
Posttranslational arginylation mediated by arginyltransferase (ATE1) is an emerging major regulator of embryogenesis and cell physiology. Impairments of ATE1 are implicated in congenital heart defects, obesity, cancer, and neurodegeneration making this enzyme an important therapeutic target, whose potential has been virtually unexplored. Here we report the development of a biochemical assay for identification of small molecule inhibitors of ATE1 and application of this assay to screen a library of 3280 compounds. Our screen identified two compounds which specifically affect ATE1-regulated processes in vivo, including tannic acid, which has been previously shown to inhibit protein degradation and angiogenesis and to act as a therapeutic agent in heart disease and cancer. Our data suggest that these actions of tannic acid are mediated by its direct effect on ATE1, which regulates protein degradation and angiogenesis in vivo.
Keywords: Abbreviations; Ate1; arginine transfer enzyme 1 or arginyltransferase 1; RRS; Arg-tRNA synthetase; BSA; bovine serum albumin; RGS4; regulator of G protein signaling 4; VEGF-A165; vascular endothelial growth factor A165; HUVEC; human umbilical vein endothelial cells; TRITC; retramethyl rhodamine isothiocyanate; CXCL12; C-X-C motif ligand 12; ECGS; endothelial cell growth supplement; bFGF; basic fibroblast growth factorATE1 inhibitors; Arginylation; tannic acid; Cell motility; Angiogenesis
Reaction of platinum anticancer drugs and drug derivatives with a copper transporting protein, Atox1 by Maria E. Palm-Espling; Pernilla Wittung-Stafshede (pp. 874-881).
Platinum (Pt) containing anticancer drugs have been used in cancer treatment for several decades as they trigger cell death upon DNA binding. Pt-containing anticancer drugs and drug derivates with a variety of ligands around the Pt center (with Cisplatin being most well known) exist today in clinics and in clinical trials. However, a major drawback with these drugs is limited efficacy due to side reactions resulting in cell resistance. The cellular copper (Cu) transport pathway is proposed to be responsible for part of these side reactions through interactions with the Pt-containing drugs and possibly cellular export of Pt. The cytoplasmic Cu chaperone, Atox1, was recently found to bind Cisplatin in vitro and, when over-expressed in Escherichia coli, in vivo. Here we investigate how the chemical properties of six Pt-substances differentially affect binding, unfolding, and aggregation of Atox1 in vitro using near- and far-UV circular dichroism (CD) spectroscopy and SDS-PAGE. The results show that both ligand type and orientation dictate the interactions with Atox1. Only substances with two good leaving groups in cis-configuration result in near-UV CD changes that report on Cu–Pt interactions. The different substances promote Atox1 unfolding in a pattern that can be explained by ligand chemistry and geometry. Our work emphasize that ligands around the Pt-center have decisive roles in tuning protein interactions (prior to DNA binding) and therefore they also dictate the level of drug side effects and cellular resistance.
Keywords: Abbreviations; CD; circular dichroism; SDS; sodium dodecyl sulfate; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DTT; dithiothretiol; TCEP; Tris(2-carboxyethyl)phosphine; Ctr1; copper transporter 1; NMR; nuclear magnetic resonance; OCT; organic cation transporterAtox1; Cisplatin; Anticancer; Protein unfolding; Spectroscopy
Reaction of platinum anticancer drugs and drug derivatives with a copper transporting protein, Atox1 by Maria E. Palm-Espling; Pernilla Wittung-Stafshede (pp. 874-881).
Platinum (Pt) containing anticancer drugs have been used in cancer treatment for several decades as they trigger cell death upon DNA binding. Pt-containing anticancer drugs and drug derivates with a variety of ligands around the Pt center (with Cisplatin being most well known) exist today in clinics and in clinical trials. However, a major drawback with these drugs is limited efficacy due to side reactions resulting in cell resistance. The cellular copper (Cu) transport pathway is proposed to be responsible for part of these side reactions through interactions with the Pt-containing drugs and possibly cellular export of Pt. The cytoplasmic Cu chaperone, Atox1, was recently found to bind Cisplatin in vitro and, when over-expressed in Escherichia coli, in vivo. Here we investigate how the chemical properties of six Pt-substances differentially affect binding, unfolding, and aggregation of Atox1 in vitro using near- and far-UV circular dichroism (CD) spectroscopy and SDS-PAGE. The results show that both ligand type and orientation dictate the interactions with Atox1. Only substances with two good leaving groups in cis-configuration result in near-UV CD changes that report on Cu–Pt interactions. The different substances promote Atox1 unfolding in a pattern that can be explained by ligand chemistry and geometry. Our work emphasize that ligands around the Pt-center have decisive roles in tuning protein interactions (prior to DNA binding) and therefore they also dictate the level of drug side effects and cellular resistance.
Keywords: Abbreviations; CD; circular dichroism; SDS; sodium dodecyl sulfate; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DTT; dithiothretiol; TCEP; Tris(2-carboxyethyl)phosphine; Ctr1; copper transporter 1; NMR; nuclear magnetic resonance; OCT; organic cation transporterAtox1; Cisplatin; Anticancer; Protein unfolding; Spectroscopy
Endothelial and neural factors functionally involved in the modulation of noradrenergic vasoconstriction in healthy pig internal mammary artery by Rosa María Pagán; Ana Cristina Martínez; Medardo Hernández; María Pilar Martínez; Albino García-Sacristán; Carlos Correa; Susana Novella; Carlos Hermenegildo; Dolores Prieto; Sara Benedito (pp. 882-892).
The role of endothelial and neural factors as modulators of neurogenic- and noradrenaline-induced vasoconstriction was examined in healthy pig internal mammary artery (IMA). Tetrodotoxin-, guanethidine-sensitive electrical field stimulation (EFS)-, and noradrenaline-elicited contractions were significantly diminished by prazosin ( n=8, P<0.001) and less so by rauwolscine, indicating functional α1- and α2-adrenoceptor-mediated noradrenergic innervation of the IMA. Endothelium removal reduced neurogenic ( n=8, P<0.01) but augmented noradrenaline responses ( n=8, P<0.01), suggesting the release of two endothelium-dependent factors with opposite effects. In the presence of endothelium, neurogenic and exogenous noradrenaline vasoconstrictions were enhanced byl-NOArg ( n=7, P<0.05 and P<0.01 respectively) and ODQ ( n=7, both P<0.05); in denuded arteries, nNOS inhibition with Nω-propyl-l-arginine increased neurogenic contraction ( n=7, P<0.05). Western blotting indicated the presence of neural and endothelial origin NO ( n=6, P<0.001). Tetraethylammonium ( n=9, P<0.001), iberiotoxin ( n=7, P<0.001) and 4-aminopyridine ( n=8, P<0.01) enhanced vasoconstrictions revealing a modulatory role of big conductance Ca2+-activated K+ (BKCa) and voltage-dependent K+ (Kv) channels in noradrenergic responses. Bosentan pretreatment ( n=8, P<0.05) suggested endothelin-1 as the inferred contractile neurogenic endothelial-dependent factor. Indomethacin-induced inhibition involved a muscular prostanoid ( n=9, P<0.05), functionally and immunologically localized, and derived from cyclooxygenase (COX)-1 and COX-2, as revealed by Western blots ( n=5, P=0.1267). Thus, noradrenergic IMA contractions are controlled by contractile prostanoid activation and endothelin-1 release, and offset by BKCa and Kv channels and neural and endothelial NO. These results help clarify the mechanisms of vasospasm in IMA, as the preferred vessel for coronary bypass.
Keywords: Internal mammary artery; Pig; Neurogenic contraction; Endothelium; Nitric oxide; Potassium channels
Endothelial and neural factors functionally involved in the modulation of noradrenergic vasoconstriction in healthy pig internal mammary artery by Rosa María Pagán; Ana Cristina Martínez; Medardo Hernández; María Pilar Martínez; Albino García-Sacristán; Carlos Correa; Susana Novella; Carlos Hermenegildo; Dolores Prieto; Sara Benedito (pp. 882-892).
The role of endothelial and neural factors as modulators of neurogenic- and noradrenaline-induced vasoconstriction was examined in healthy pig internal mammary artery (IMA). Tetrodotoxin-, guanethidine-sensitive electrical field stimulation (EFS)-, and noradrenaline-elicited contractions were significantly diminished by prazosin ( n=8, P<0.001) and less so by rauwolscine, indicating functional α1- and α2-adrenoceptor-mediated noradrenergic innervation of the IMA. Endothelium removal reduced neurogenic ( n=8, P<0.01) but augmented noradrenaline responses ( n=8, P<0.01), suggesting the release of two endothelium-dependent factors with opposite effects. In the presence of endothelium, neurogenic and exogenous noradrenaline vasoconstrictions were enhanced byl-NOArg ( n=7, P<0.05 and P<0.01 respectively) and ODQ ( n=7, both P<0.05); in denuded arteries, nNOS inhibition with Nω-propyl-l-arginine increased neurogenic contraction ( n=7, P<0.05). Western blotting indicated the presence of neural and endothelial origin NO ( n=6, P<0.001). Tetraethylammonium ( n=9, P<0.001), iberiotoxin ( n=7, P<0.001) and 4-aminopyridine ( n=8, P<0.01) enhanced vasoconstrictions revealing a modulatory role of big conductance Ca2+-activated K+ (BKCa) and voltage-dependent K+ (Kv) channels in noradrenergic responses. Bosentan pretreatment ( n=8, P<0.05) suggested endothelin-1 as the inferred contractile neurogenic endothelial-dependent factor. Indomethacin-induced inhibition involved a muscular prostanoid ( n=9, P<0.05), functionally and immunologically localized, and derived from cyclooxygenase (COX)-1 and COX-2, as revealed by Western blots ( n=5, P=0.1267). Thus, noradrenergic IMA contractions are controlled by contractile prostanoid activation and endothelin-1 release, and offset by BKCa and Kv channels and neural and endothelial NO. These results help clarify the mechanisms of vasospasm in IMA, as the preferred vessel for coronary bypass.
Keywords: Internal mammary artery; Pig; Neurogenic contraction; Endothelium; Nitric oxide; Potassium channels
Stereoisomers ginsenosides-20(S)-Rg3 and -20(R)-Rg3 differentially induce angiogenesis through peroxisome proliferator-activated receptor-gamma by Hoi-Hin Kwok; Guan-Lun Guo; Justin Kai-Chi Lau; Yuen-Kit Cheng; Jiang-Rong Wang; Zhi-Hong Jiang; Man-Hong Keung; Nai-Ki Mak; Patrick Ying-Kit Yue; Ricky Ngok-Shun Wong (pp. 893-902).
Ginsenosides are considered the major constituents that are responsible for most of the pharmacological actions of ginseng. However, some ginsenosides exist as stereoisomeric pairs, detailed and molecular exposition based on the structural differences of ginsenoside stereoisomers has not been emphasized in most studies. Here we explore the functional differences of ginsenoside Rg3 stereoisomers on angiogenesis. In this study, we demonstrated the distinctive differential angiogenic activities of 20(S)-Rg3 and 20(R)-Rg3 stereoisomers. 20(S)-Rg3 at micromolar concentration promotes human endothelial cells proliferation, migration and tube formation in vitro, as well as ex vivo endothelial sprouting. The effects induced by 20(S)-Rg3 are significantly more potent than 20(R)-Rg3. These effects are partially mediated through the activation of AKT/ERK-eNOS signaling pathways. Moreover, knockdown of peroxisome proliferator-activated receptor-gamma (PPARγ) by specific small interference RNA abolished the 20(S)-Rg3-induced angiogenesis, indicating that PPARγ is responsible for mediating the angiogenic activity of Rg3. Using reporter gene assay, the PPARγ agonist activity of 20(S)-Rg3 has been found 10-fold higher than that of 20(R)-Rg3. Computer modeling also revealed the differential binding is due to the chiral center of 20(S)-Rg3 can form a critical hydrogen bond with Tyr473 of PPARγ ligand binding domain. The present study elucidated the differential angiogenic effects of Rg3 stereoisomers by acting as agonist of PPARγ. The results shed light on the structural difference between two ginsenoside stereoisomers that can lead to significant differential physiological outcomes which should be carefully considered in the future development of ginsenoside-based therapeutics.
Keywords: Ginsenoside; Rg; 3; Stereoisomer; Angiogenesis; PPARγ
Stereoisomers ginsenosides-20(S)-Rg3 and -20(R)-Rg3 differentially induce angiogenesis through peroxisome proliferator-activated receptor-gamma by Hoi-Hin Kwok; Guan-Lun Guo; Justin Kai-Chi Lau; Yuen-Kit Cheng; Jiang-Rong Wang; Zhi-Hong Jiang; Man-Hong Keung; Nai-Ki Mak; Patrick Ying-Kit Yue; Ricky Ngok-Shun Wong (pp. 893-902).
Ginsenosides are considered the major constituents that are responsible for most of the pharmacological actions of ginseng. However, some ginsenosides exist as stereoisomeric pairs, detailed and molecular exposition based on the structural differences of ginsenoside stereoisomers has not been emphasized in most studies. Here we explore the functional differences of ginsenoside Rg3 stereoisomers on angiogenesis. In this study, we demonstrated the distinctive differential angiogenic activities of 20(S)-Rg3 and 20(R)-Rg3 stereoisomers. 20(S)-Rg3 at micromolar concentration promotes human endothelial cells proliferation, migration and tube formation in vitro, as well as ex vivo endothelial sprouting. The effects induced by 20(S)-Rg3 are significantly more potent than 20(R)-Rg3. These effects are partially mediated through the activation of AKT/ERK-eNOS signaling pathways. Moreover, knockdown of peroxisome proliferator-activated receptor-gamma (PPARγ) by specific small interference RNA abolished the 20(S)-Rg3-induced angiogenesis, indicating that PPARγ is responsible for mediating the angiogenic activity of Rg3. Using reporter gene assay, the PPARγ agonist activity of 20(S)-Rg3 has been found 10-fold higher than that of 20(R)-Rg3. Computer modeling also revealed the differential binding is due to the chiral center of 20(S)-Rg3 can form a critical hydrogen bond with Tyr473 of PPARγ ligand binding domain. The present study elucidated the differential angiogenic effects of Rg3 stereoisomers by acting as agonist of PPARγ. The results shed light on the structural difference between two ginsenoside stereoisomers that can lead to significant differential physiological outcomes which should be carefully considered in the future development of ginsenoside-based therapeutics.
Keywords: Ginsenoside; Rg; 3; Stereoisomer; Angiogenesis; PPARγ
The impact of genetic polymorphisms of P2Y12, CYP3A5 and CYP2C19 on clopidogrel response variability in Iranian patients by Soha Namazi; Javad Kojuri; Andia Khalili; Negar Azarpira (pp. 903-908).
Clopidogrel is an inhibitor of platelet ADP P2Y12 receptors and currently used for prevention of stent thrombosis. Despite certain clinical benefit using this drug in patients undergoing percutaneous coronary intervention (PCI), some patients do not attain adequate antiplatelet effects. In this study, we investigated the role of three genetic factors ( P2Y12, CYP3A5, CYP2C19), demographic characteristics, and pathologic condition on clopidogrel response variability in Iranian patients after PCI.Patients who were candidate for elective PCI were enrolled in this study. All patients had received aspirin 80–325mg daily for ≥1 week before PCI. Blood samples were taken from patients at baseline, 2h after taking a 600-mg loading dose of clopidogrel, 24h and 30 days after PCI. Platelet aggregation was measured by turbidimetric aggregation assay with two different concentrations of ADP (5 and 20μM). CYP2C19*2(rs4244285), CYP2C19*3(rs4986893), CYP3A5 (A6986G), and P2Y12 (T744C) genotypings were performed by PCR-RFLP.One hundred and twelve patients were included in this study. Maximum clopidogrel non-responsiveness (25.90%) occurred at 2h after taking 600mg of the loading dose of clopidogrel. Although there were no significant associations between clopidogrel responsiveness and polymorphisms of CYP2C19, CYP3A5, and P2Y12 ( P>0.05), subjects who were CYP3A5 genotype expressor had a greater inhibition of platelet aggregation. No significant associations were observed between environmental factors and clopidogrel responsiveness ( P>0.05).Our results showed that P2Y12, CYP3A5, and CYP2C19 polymorphisms along with non-genetic factors were not responsible for the interindividual variability in response to clopidogrel in Iranian population.
Keywords: Clopidogrel; P2Y12; polymorphism; CYP3A5; polymorphism; CYP2C19; polymorphism; Post stent thrombosis
The impact of genetic polymorphisms of P2Y12, CYP3A5 and CYP2C19 on clopidogrel response variability in Iranian patients by Soha Namazi; Javad Kojuri; Andia Khalili; Negar Azarpira (pp. 903-908).
Clopidogrel is an inhibitor of platelet ADP P2Y12 receptors and currently used for prevention of stent thrombosis. Despite certain clinical benefit using this drug in patients undergoing percutaneous coronary intervention (PCI), some patients do not attain adequate antiplatelet effects. In this study, we investigated the role of three genetic factors ( P2Y12, CYP3A5, CYP2C19), demographic characteristics, and pathologic condition on clopidogrel response variability in Iranian patients after PCI.Patients who were candidate for elective PCI were enrolled in this study. All patients had received aspirin 80–325mg daily for ≥1 week before PCI. Blood samples were taken from patients at baseline, 2h after taking a 600-mg loading dose of clopidogrel, 24h and 30 days after PCI. Platelet aggregation was measured by turbidimetric aggregation assay with two different concentrations of ADP (5 and 20μM). CYP2C19*2(rs4244285), CYP2C19*3(rs4986893), CYP3A5 (A6986G), and P2Y12 (T744C) genotypings were performed by PCR-RFLP.One hundred and twelve patients were included in this study. Maximum clopidogrel non-responsiveness (25.90%) occurred at 2h after taking 600mg of the loading dose of clopidogrel. Although there were no significant associations between clopidogrel responsiveness and polymorphisms of CYP2C19, CYP3A5, and P2Y12 ( P>0.05), subjects who were CYP3A5 genotype expressor had a greater inhibition of platelet aggregation. No significant associations were observed between environmental factors and clopidogrel responsiveness ( P>0.05).Our results showed that P2Y12, CYP3A5, and CYP2C19 polymorphisms along with non-genetic factors were not responsible for the interindividual variability in response to clopidogrel in Iranian population.
Keywords: Clopidogrel; P2Y12; polymorphism; CYP3A5; polymorphism; CYP2C19; polymorphism; Post stent thrombosis
Purification and characterization of HSP-inducers from Eupatorium lindleyanum by Yasuhiro Yamashita; Tsuyoshi Ikeda; Minoru Matsuda; Daisuke Maji; Tatsuya Hoshino; Tohru Mizushima (pp. 909-922).
The expression of heat shock proteins (HSPs), particularly HSP70, provides resistance to stressors. We recently reported that ultraviolet (UV)-induced melanin production and skin damage were suppressed in transgenic mice expressing HSP70 and that an extract of Eupatorium lindleyanum induces the expression of HSP70 in cells. Here we report the purification of eupalinolide A and B (EA and EB) from E. lindleyanum, and describe their actions as HSP-inducers. EA and EB both induced the expression of HSP70 in cells at concentrations that did not significantly affect cell viability. Treatment of cells with EA or EB activated heat shock factor 1 (HSF1), while the artificial suppression of HSF1 expression diminished the EA- or EB-mediated induction of HSP70 expression. Furthermore, EB inhibited the interaction between HSF1 and HSP90, which is known to inhibit the activity of HSF1. These findings suggest that EA and EB induce the expression of HSP70 via the activation of HSF1 by inhibiting the interaction between HSF1 and HSP90. EA and EB both induced the expression of HSP70 synergistically with other stressors. Furthermore, pre-treatment of cells with EA or EB suppressed melanin production and stressor-induced apoptosis. These effects were suppressed by the artificial suppression of HSP70 expression. In vivo, the percutaneous administration of EB induced the expression of HSP70 and suppressed UVB radiation-induced damage, inflammatory responses and melanin production in the skin. These results suggest that EA and EB could be beneficial for use in cosmetics and medicines as a consequence of their inhibitory action on UV-induced skin damage and melanin production.
Keywords: Abbreviations; ANOVA; analysis of variance; DAPI; 4,6-diamidino-2-phenylindole dihydrochloride; DMEM; Dulbecco's modified Eagle's medium; FBS; fetal bovine serum; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; GGA; geranylgeranylacetone; HSF1; heat shock factor 1; HSP; heat shock protein; IBMX; 3-isobutyl-1-methylxanthine; MITF; microphthalmia-associated transcription factor; MPO; myeloperoxidase; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NF-κB; nuclear factor kappa B; UV; ultravioletHeat shock protein 70; Eupalinolide; Skin photoaging; Cell death; Melanogenesis
Purification and characterization of HSP-inducers from Eupatorium lindleyanum by Yasuhiro Yamashita; Tsuyoshi Ikeda; Minoru Matsuda; Daisuke Maji; Tatsuya Hoshino; Tohru Mizushima (pp. 909-922).
The expression of heat shock proteins (HSPs), particularly HSP70, provides resistance to stressors. We recently reported that ultraviolet (UV)-induced melanin production and skin damage were suppressed in transgenic mice expressing HSP70 and that an extract of Eupatorium lindleyanum induces the expression of HSP70 in cells. Here we report the purification of eupalinolide A and B (EA and EB) from E. lindleyanum, and describe their actions as HSP-inducers. EA and EB both induced the expression of HSP70 in cells at concentrations that did not significantly affect cell viability. Treatment of cells with EA or EB activated heat shock factor 1 (HSF1), while the artificial suppression of HSF1 expression diminished the EA- or EB-mediated induction of HSP70 expression. Furthermore, EB inhibited the interaction between HSF1 and HSP90, which is known to inhibit the activity of HSF1. These findings suggest that EA and EB induce the expression of HSP70 via the activation of HSF1 by inhibiting the interaction between HSF1 and HSP90. EA and EB both induced the expression of HSP70 synergistically with other stressors. Furthermore, pre-treatment of cells with EA or EB suppressed melanin production and stressor-induced apoptosis. These effects were suppressed by the artificial suppression of HSP70 expression. In vivo, the percutaneous administration of EB induced the expression of HSP70 and suppressed UVB radiation-induced damage, inflammatory responses and melanin production in the skin. These results suggest that EA and EB could be beneficial for use in cosmetics and medicines as a consequence of their inhibitory action on UV-induced skin damage and melanin production.
Keywords: Abbreviations; ANOVA; analysis of variance; DAPI; 4,6-diamidino-2-phenylindole dihydrochloride; DMEM; Dulbecco's modified Eagle's medium; FBS; fetal bovine serum; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; GGA; geranylgeranylacetone; HSF1; heat shock factor 1; HSP; heat shock protein; IBMX; 3-isobutyl-1-methylxanthine; MITF; microphthalmia-associated transcription factor; MPO; myeloperoxidase; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NF-κB; nuclear factor kappa B; UV; ultravioletHeat shock protein 70; Eupalinolide; Skin photoaging; Cell death; Melanogenesis
Effect of non-steroidal anti-inflammatory drugs and new fenamate analogues on TRPC4 and TRPC5 channels by Hongni Jiang; Bo Zeng; Gui-Lan Chen; David Bot; Sarah Eastmond; Sandra E. Elsenussi; Stephen L. Atkin; Andrew N. Boa; Shang-Zhong Xu (pp. 923-931).
Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used anti-inflammatory therapeutic agents, among which the fenamate analogues play important roles in regulating intracellular Ca2+ transient and ion channels. However, the effect of NSAIDs on TRPC4 and TRPC5 is still unknown. To understand the structure–activity of fenamate analogues on TRPC channels, we have synthesized a series of fenamate analogues and investigated their effects on TRPC4 and TRPC5 channels.Human TRPC4 and TRPC5 cDNAs in tetracycline-regulated vectors were transfected into HEK293 T-REx cells. The whole cell current and Ca2+ movement were recorded by patch clamp and calcium imaging, respectively.Flufenamic acid (FFA), mefenamic acid (MFA), niflumic acid (NFA) and diclofenac sodium (DFS) showed inhibition on TRPC4 and TRPC5 channels in a concentration-dependent manner. The potency was FFA>MFA>NFA>DFS. Modification of 2-phenylamino ring by substitution of the trifluoromethyl group in FFA withF,CH3,OCH3,OCH2CH3,COOH, andNO2 led to the changes in their channel blocking activity. However, 2-(2′-methoxy-5′-methylphenyl)aminobenzoic acid stimulated TRPC4 and TRPC5 channels. Selective COX1-3 inhibitors (aspirin, celecoxib, acetaminophen, and indomethacin) had no effect on the channels. Longer perfusion (>5min) with FFA (100μM) and MFA (100μM) caused a potentiation of TRPC4 and TRPC5 currents after their initial blocking effects that appeared to be partially mediated by the mitochondrial Ca2+ release.Our results suggest that fenamate analogues are direct modulators of TRPC4 and TRPC5 channels. The substitution pattern and conformation of the 2-phenylamino ring could alter their blocking activity, which is important for understanding fenamate pharmacology and new drug development targeting the TRPC channels.
Keywords: Non-steroidal anti-inflammatory drugs; Calcium channel; TRPC; Fenamate analogues; 2-Aminoethoxydiphenyl borate
Effect of non-steroidal anti-inflammatory drugs and new fenamate analogues on TRPC4 and TRPC5 channels by Hongni Jiang; Bo Zeng; Gui-Lan Chen; David Bot; Sarah Eastmond; Sandra E. Elsenussi; Stephen L. Atkin; Andrew N. Boa; Shang-Zhong Xu (pp. 923-931).
Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used anti-inflammatory therapeutic agents, among which the fenamate analogues play important roles in regulating intracellular Ca2+ transient and ion channels. However, the effect of NSAIDs on TRPC4 and TRPC5 is still unknown. To understand the structure–activity of fenamate analogues on TRPC channels, we have synthesized a series of fenamate analogues and investigated their effects on TRPC4 and TRPC5 channels.Human TRPC4 and TRPC5 cDNAs in tetracycline-regulated vectors were transfected into HEK293 T-REx cells. The whole cell current and Ca2+ movement were recorded by patch clamp and calcium imaging, respectively.Flufenamic acid (FFA), mefenamic acid (MFA), niflumic acid (NFA) and diclofenac sodium (DFS) showed inhibition on TRPC4 and TRPC5 channels in a concentration-dependent manner. The potency was FFA>MFA>NFA>DFS. Modification of 2-phenylamino ring by substitution of the trifluoromethyl group in FFA withF,CH3,OCH3,OCH2CH3,COOH, andNO2 led to the changes in their channel blocking activity. However, 2-(2′-methoxy-5′-methylphenyl)aminobenzoic acid stimulated TRPC4 and TRPC5 channels. Selective COX1-3 inhibitors (aspirin, celecoxib, acetaminophen, and indomethacin) had no effect on the channels. Longer perfusion (>5min) with FFA (100μM) and MFA (100μM) caused a potentiation of TRPC4 and TRPC5 currents after their initial blocking effects that appeared to be partially mediated by the mitochondrial Ca2+ release.Our results suggest that fenamate analogues are direct modulators of TRPC4 and TRPC5 channels. The substitution pattern and conformation of the 2-phenylamino ring could alter their blocking activity, which is important for understanding fenamate pharmacology and new drug development targeting the TRPC channels.
Keywords: Non-steroidal anti-inflammatory drugs; Calcium channel; TRPC; Fenamate analogues; 2-Aminoethoxydiphenyl borate
Interplay of sorbitol pathway of glucose metabolism, 12/15-lipoxygenase, and mitogen-activated protein kinases in the pathogenesis of diabetic peripheral neuropathy by Roman Stavniichuk; Hanna Shevalye; Hiroko Hirooka; Jerry L. Nadler; Irina G. Obrosova (pp. 932-940).
The interactions among multiple pathogenetic mechanisms of diabetic peripheral neuropathy largely remain unexplored. Increased activity of aldose reductase, the first enzyme of the sorbitol pathway, leads to accumulation of cytosolic Ca2+, essentially required for 12/15-lipoxygenase activation. The latter, in turn, causes oxidative–nitrosative stress, an important trigger of mitogen activated protein kinase (MAPK) phosphorylation. This study therefore evaluated the interplay of aldose reductase, 12/15-lipoxygenase, and MAPKs in diabetic peripheral neuropathy. In experiment 1, male control and streptozotocin-diabetic mice were maintained with or without the aldose reductase inhibitor fidarestat, 16mgkg−1d−1, for 12 weeks. In experiment 2, male control and streptozotocin-diabetic wild-type (C57Bl6/J) and 12/15-lipoxygenase-deficient mice were used. Fidarestat treatment did not affect diabetes-induced increase in glucose concentrations, but normalized sorbitol and fructose concentrations (enzymatic spectrofluorometric assays) as well as 12(S)-hydroxyeicosatetraenoic concentration (ELISA), a measure of 12/15-lipoxygenase activity, in the sciatic nerve and spinal cord. 12/15-lipoxygenase expression in these two tissues (Western blot analysis) as well as dorsal root ganglia (immunohistochemistry) was similarly elevated in untreated and fidarestat-treated diabetic mice. 12/15-Lipoxygenase gene deficiency prevented diabetes-associated p38 MAPK and ERK, but not SAPK/JNK, activation in the sciatic nerve (Western blot analysis) and all three MAPK activation in the dorsal root ganglia (immunohistochemistry). In contrast, spinal cord p38 MAPK, ERK, and SAPK/JNK were similarly activated in diabetic wild-type and 12/15-lipoxygenase−/− mice. These findings identify the nature and tissue specificity of interactions among three major mechanisms of diabetic peripheral neuropathy, and suggest that combination treatments, rather than monotherapies, can sometimes be an optimal choice for its management.
Keywords: Aldose reductase; Diabetic peripheral neuropathy; Dorsal root ganglion; 12/15-Lipoxygenase; MAPK; Sciatic nerve; Sorbitol pathway of glucose metabolism; Spinal cord; Streptozotocin-diabetic mouse
Interplay of sorbitol pathway of glucose metabolism, 12/15-lipoxygenase, and mitogen-activated protein kinases in the pathogenesis of diabetic peripheral neuropathy by Roman Stavniichuk; Hanna Shevalye; Hiroko Hirooka; Jerry L. Nadler; Irina G. Obrosova (pp. 932-940).
The interactions among multiple pathogenetic mechanisms of diabetic peripheral neuropathy largely remain unexplored. Increased activity of aldose reductase, the first enzyme of the sorbitol pathway, leads to accumulation of cytosolic Ca2+, essentially required for 12/15-lipoxygenase activation. The latter, in turn, causes oxidative–nitrosative stress, an important trigger of mitogen activated protein kinase (MAPK) phosphorylation. This study therefore evaluated the interplay of aldose reductase, 12/15-lipoxygenase, and MAPKs in diabetic peripheral neuropathy. In experiment 1, male control and streptozotocin-diabetic mice were maintained with or without the aldose reductase inhibitor fidarestat, 16mgkg−1d−1, for 12 weeks. In experiment 2, male control and streptozotocin-diabetic wild-type (C57Bl6/J) and 12/15-lipoxygenase-deficient mice were used. Fidarestat treatment did not affect diabetes-induced increase in glucose concentrations, but normalized sorbitol and fructose concentrations (enzymatic spectrofluorometric assays) as well as 12(S)-hydroxyeicosatetraenoic concentration (ELISA), a measure of 12/15-lipoxygenase activity, in the sciatic nerve and spinal cord. 12/15-lipoxygenase expression in these two tissues (Western blot analysis) as well as dorsal root ganglia (immunohistochemistry) was similarly elevated in untreated and fidarestat-treated diabetic mice. 12/15-Lipoxygenase gene deficiency prevented diabetes-associated p38 MAPK and ERK, but not SAPK/JNK, activation in the sciatic nerve (Western blot analysis) and all three MAPK activation in the dorsal root ganglia (immunohistochemistry). In contrast, spinal cord p38 MAPK, ERK, and SAPK/JNK were similarly activated in diabetic wild-type and 12/15-lipoxygenase−/− mice. These findings identify the nature and tissue specificity of interactions among three major mechanisms of diabetic peripheral neuropathy, and suggest that combination treatments, rather than monotherapies, can sometimes be an optimal choice for its management.
Keywords: Aldose reductase; Diabetic peripheral neuropathy; Dorsal root ganglion; 12/15-Lipoxygenase; MAPK; Sciatic nerve; Sorbitol pathway of glucose metabolism; Spinal cord; Streptozotocin-diabetic mouse
The nicotine metabolite, cotinine, attenuates glutamate (NMDA) antagonist-related effects on the performance of the five choice serial reaction time task (5C-SRTT) in rats by Alvin V. Terry Jr; Jerry J. Buccafusco; R. Foster Schade; Leah Vandenhuerk; Patrick M. Callahan; Wayne D. Beck; Elizabeth J. Hutchings; James M. Chapman; Pei Li; Michael G. Bartlett (pp. 941-951).
Cotinine, the most predominant metabolite of nicotine in mammalian species, has a pharmacological half-life that greatly exceeds its precursor. However, until recently, relatively few studies had been conducted to systematically characterize the behavioral pharmacology of cotinine. Our previous work indicated that cotinine improves prepulse inhibition of the auditory startle response in rats in pharmacological impairment models and that it improves working memory in non-human primates. Here we tested the hypothesis that cotinine improves sustained attention in rats and attenuates behavioral alterations induced by the glutamate (NMDA) antagonist MK-801. The effects of acute subcutaneous (dose range 0.03–10.0mg/kg) and chronic oral administration (2.0mg/kg/day in drinking water) of cotinine were evaluated in fixed and variable stimulus duration (VSD) as well as variable intertrial interval (VITI) versions of a five choice serial reaction time task (5C-SRTT). The results indicated only subtle effects of acute cotinine (administered alone) on performance of the 5C-SRTT (e.g., decreases in timeout responses). However, depending on dose, acute treatment with cotinine attenuated MK-801-related impairments in accuracy and elevations in timeout responses, and it increased the number of completed trials. Moreover, chronic cotinine attenuated MK-801-related impairments in accuracy and it reduced premature and timeout responses when the demands of the task were increased (i.e., by presenting VSDs or VITIs in addition to administering MK-801). These data suggest that cotinine may represent a prototype for compounds that have therapeutic potential for neuropsychiatric disorders (i.e., by improving sustained attention and decreasing impulsive and compulsive behaviors), especially those characterized by glutamate receptor alterations.
Keywords: Attention; Schizophrenia; Impulsivity; Compulsivity; Nicotinic
The nicotine metabolite, cotinine, attenuates glutamate (NMDA) antagonist-related effects on the performance of the five choice serial reaction time task (5C-SRTT) in rats by Alvin V. Terry Jr; Jerry J. Buccafusco; R. Foster Schade; Leah Vandenhuerk; Patrick M. Callahan; Wayne D. Beck; Elizabeth J. Hutchings; James M. Chapman; Pei Li; Michael G. Bartlett (pp. 941-951).
Cotinine, the most predominant metabolite of nicotine in mammalian species, has a pharmacological half-life that greatly exceeds its precursor. However, until recently, relatively few studies had been conducted to systematically characterize the behavioral pharmacology of cotinine. Our previous work indicated that cotinine improves prepulse inhibition of the auditory startle response in rats in pharmacological impairment models and that it improves working memory in non-human primates. Here we tested the hypothesis that cotinine improves sustained attention in rats and attenuates behavioral alterations induced by the glutamate (NMDA) antagonist MK-801. The effects of acute subcutaneous (dose range 0.03–10.0mg/kg) and chronic oral administration (2.0mg/kg/day in drinking water) of cotinine were evaluated in fixed and variable stimulus duration (VSD) as well as variable intertrial interval (VITI) versions of a five choice serial reaction time task (5C-SRTT). The results indicated only subtle effects of acute cotinine (administered alone) on performance of the 5C-SRTT (e.g., decreases in timeout responses). However, depending on dose, acute treatment with cotinine attenuated MK-801-related impairments in accuracy and elevations in timeout responses, and it increased the number of completed trials. Moreover, chronic cotinine attenuated MK-801-related impairments in accuracy and it reduced premature and timeout responses when the demands of the task were increased (i.e., by presenting VSDs or VITIs in addition to administering MK-801). These data suggest that cotinine may represent a prototype for compounds that have therapeutic potential for neuropsychiatric disorders (i.e., by improving sustained attention and decreasing impulsive and compulsive behaviors), especially those characterized by glutamate receptor alterations.
Keywords: Attention; Schizophrenia; Impulsivity; Compulsivity; Nicotinic
Monohydroxylated metabolites of the K2 synthetic cannabinoid JWH-073 retain intermediate to high cannabinoid 1 receptor (CB1R) affinity and exhibit neutral antagonist to partial agonist activity by Lisa K. Brents; Anna Gallus-Zawada; Anna Radominska-Pandya; Tamara Vasiljevik; Thomas E. Prisinzano; William E. Fantegrossi; Jeffery H. Moran; Paul L. Prather (pp. 952-961).
K2 and several similar purported “incense products” spiked with synthetic cannabinoids are abused as cannabis substitutes. We hypothesized that metabolism of JWH-073, a prevalent cannabinoid found in K2, contributes to toxicity associated with K2 use. Competition receptor binding studies and G-protein activation assays, both performed by employing mouse brain homogenates, were used to determine the affinity and intrinsic activity, respectively, of potential monohydroxylated (M1, M3–M5) and monocarboxylated (M6) metabolites at cannabinoid 1 receptors (CB1Rs). Surprisingly, M1, M4 and M5 retain nanomolar affinity for CB1Rs, while M3 displays micromolar affinity and M6 does not bind to CB1Rs. JWH-073 displays equivalent efficacy to that of the CB1R full agonist CP-55,940, while M1, M3, and M5 act as CB1R partial agonists, and M4 shows little or no intrinsic activity. Further in vitro investigation by Schild analysis revealed that M4 acts as a competitive neutral CB1R antagonist ( Kb∼40nM). In agreement with in vitro studies, M4 also demonstrates CB1R antagonism in vivo by blunting cannabinoid-induced hypothermia in mice. Interestingly, M4 does not block agonist-mediated responses of other measures in the cannabinoid tetrad ( e.g., locomotor suppression, catalepsy or analgesia). Finally, also as predicted by in vitro results, M1 exhibits agonist activity in vivo by inducing significant hypothermia and suppression of locomotor activity in mice.In conclusion, the present study indicates that further work examining the physiological effects of synthetic cannabinoid metabolism is warranted. Such a complex mix of metabolically produced CB1R ligands may contribute to the adverse effect profile of JWH-073-containing products.
Keywords: Abbreviations; AAPCC; American Association of Poison Control Centers; BSA; bovine serum albumin; CB1R; cannabinoid 1 receptor; Δ; 9; -THC; delta-9-tetrahydrocannabinol; GDP; guanosine diphosphate; GTPγS; guanosine 5′-O-[gamma-thio]triphosphate; GPCR; G-protein coupled receptor; USDEA; United States Drug Enforcement Administration; USFDA; United States Food and Drug AdministrationK2/Spice; JWH-073; CB1; Cannabinoid; Synthetic cannabis; Obesity
Monohydroxylated metabolites of the K2 synthetic cannabinoid JWH-073 retain intermediate to high cannabinoid 1 receptor (CB1R) affinity and exhibit neutral antagonist to partial agonist activity by Lisa K. Brents; Anna Gallus-Zawada; Anna Radominska-Pandya; Tamara Vasiljevik; Thomas E. Prisinzano; William E. Fantegrossi; Jeffery H. Moran; Paul L. Prather (pp. 952-961).
K2 and several similar purported “incense products” spiked with synthetic cannabinoids are abused as cannabis substitutes. We hypothesized that metabolism of JWH-073, a prevalent cannabinoid found in K2, contributes to toxicity associated with K2 use. Competition receptor binding studies and G-protein activation assays, both performed by employing mouse brain homogenates, were used to determine the affinity and intrinsic activity, respectively, of potential monohydroxylated (M1, M3–M5) and monocarboxylated (M6) metabolites at cannabinoid 1 receptors (CB1Rs). Surprisingly, M1, M4 and M5 retain nanomolar affinity for CB1Rs, while M3 displays micromolar affinity and M6 does not bind to CB1Rs. JWH-073 displays equivalent efficacy to that of the CB1R full agonist CP-55,940, while M1, M3, and M5 act as CB1R partial agonists, and M4 shows little or no intrinsic activity. Further in vitro investigation by Schild analysis revealed that M4 acts as a competitive neutral CB1R antagonist ( Kb∼40nM). In agreement with in vitro studies, M4 also demonstrates CB1R antagonism in vivo by blunting cannabinoid-induced hypothermia in mice. Interestingly, M4 does not block agonist-mediated responses of other measures in the cannabinoid tetrad ( e.g., locomotor suppression, catalepsy or analgesia). Finally, also as predicted by in vitro results, M1 exhibits agonist activity in vivo by inducing significant hypothermia and suppression of locomotor activity in mice.In conclusion, the present study indicates that further work examining the physiological effects of synthetic cannabinoid metabolism is warranted. Such a complex mix of metabolically produced CB1R ligands may contribute to the adverse effect profile of JWH-073-containing products.
Keywords: Abbreviations; AAPCC; American Association of Poison Control Centers; BSA; bovine serum albumin; CB1R; cannabinoid 1 receptor; Δ; 9; -THC; delta-9-tetrahydrocannabinol; GDP; guanosine diphosphate; GTPγS; guanosine 5′-O-[gamma-thio]triphosphate; GPCR; G-protein coupled receptor; USDEA; United States Drug Enforcement Administration; USFDA; United States Food and Drug AdministrationK2/Spice; JWH-073; CB1; Cannabinoid; Synthetic cannabis; Obesity
pH dependence on functional activity of human and mouse flavin-containing monooxygenase 5 by Meike S. Motika; Jun Zhang; Erik C. Ralph; Mary A. Dwyer; John R. Cashman (pp. 962-968).
hFMO5 Structural Models. (A) Full-length hFMO5. (B) ShFMO5 Y228H variant. (C) hFMO6 D227K variant.Flavin-containing monooxygenase (FMO) 5 belongs to a family of enzymes that catalyze the oxygenation of nucleophilic N- and S-containing compounds. The FMO enzyme family consists of five forms (FMOs1–5) that share about 50–60% sequence identity to each other. A comparison of FMOs showed that the pH-dependence profile for functional activity of FMO5 differed significantly from that of other FMO enzymes. The objective of this study was to examine the pH-dependence of FMO5 to gain insight into the mechanism of action of FMO5. Recombinant mouse and human FMO5 (mFMO5 and hFMO5, respectively) were expressed as maltose-binding fusion proteins from Escherichia coli, purified with affinity chromatography, and examined for their N-oxygenation functional activity at different pH values. hFMO5 showed a broader range and greater functional activity from pH 6 to 11 compared to mFMO5. mFMO5 lost almost all functional activity at pH 6, while hFMO5 maintained almost normal enzyme activity. In order to identify the amino acid residues involved in the effects of pH on hFMO5 and mFMO5 functional enzyme activity, pH-studies in the range of pH 6–9 were done with chimeras of recombinant mouse and human FMO5 and variants of both. Results of these studies and molecular modeling showed that residues responsible for the differences in the pH profile between mFMO5 and hFMO5 were located at positions 227 and 228 of the enzyme. Further variants were made to investigate the role of these amino acids. The results of this study may help to explain the mechanism of FMO function.
Keywords: Human and mouse flavin-containing monooxygenase 5; pH-dependent enzyme activity; hFMO5 D227K; hFMO5 Y228H
pH dependence on functional activity of human and mouse flavin-containing monooxygenase 5 by Meike S. Motika; Jun Zhang; Erik C. Ralph; Mary A. Dwyer; John R. Cashman (pp. 962-968).
hFMO5 Structural Models. (A) Full-length hFMO5. (B) ShFMO5 Y228H variant. (C) hFMO6 D227K variant.Flavin-containing monooxygenase (FMO) 5 belongs to a family of enzymes that catalyze the oxygenation of nucleophilic N- and S-containing compounds. The FMO enzyme family consists of five forms (FMOs1–5) that share about 50–60% sequence identity to each other. A comparison of FMOs showed that the pH-dependence profile for functional activity of FMO5 differed significantly from that of other FMO enzymes. The objective of this study was to examine the pH-dependence of FMO5 to gain insight into the mechanism of action of FMO5. Recombinant mouse and human FMO5 (mFMO5 and hFMO5, respectively) were expressed as maltose-binding fusion proteins from Escherichia coli, purified with affinity chromatography, and examined for their N-oxygenation functional activity at different pH values. hFMO5 showed a broader range and greater functional activity from pH 6 to 11 compared to mFMO5. mFMO5 lost almost all functional activity at pH 6, while hFMO5 maintained almost normal enzyme activity. In order to identify the amino acid residues involved in the effects of pH on hFMO5 and mFMO5 functional enzyme activity, pH-studies in the range of pH 6–9 were done with chimeras of recombinant mouse and human FMO5 and variants of both. Results of these studies and molecular modeling showed that residues responsible for the differences in the pH profile between mFMO5 and hFMO5 were located at positions 227 and 228 of the enzyme. Further variants were made to investigate the role of these amino acids. The results of this study may help to explain the mechanism of FMO function.
Keywords: Human and mouse flavin-containing monooxygenase 5; pH-dependent enzyme activity; hFMO5 D227K; hFMO5 Y228H
Post-translational stabilization of thiopurine S-methyltransferase by S-adenosyl-l-methionine reveals regulation of TPMT*1 and *3C allozymes by Miha Milek; Alenka Smid; Riin Tamm; Natasa Karas Kuzelicki; Andres Metspalu; Irena Mlinaric-Rascan (pp. 969-976).
Thiopurine S-methyltransferase (TPMT; EC 2.1.1.67) plays a pivotal role in thiopurine treatment outcomes. However, little has been known about its intracellular regulation. Here, we describe the effect of fluctuations in physiological levels of S-adenosyl-l-methionine (SAM) and related metabolites on TPMT activity levels in cell lines and erythrocytes from healthy donors. We determined higher TPMT activity in wild-type TPMT*1/*1 individuals with high SAM concentrations ( n=96) compared to the low SAM level group ( n=19; P<0.001). These findings confirm the results of our in vitro studies, which demonstrated that the restriction ofl-methionine (Met) in cell growth media reversibly decreased TPMT activity and protein levels. Selective inhibition of distinct components of Met metabolism was used to demonstrate that SAM is implicitly responsible for direct post-translational TPMT stabilization. The greatest effect of SAM-mediated TPMT stabilization was observed in the case of wild-type TPMT*1 and variant *3C allozymes. In addition to TPMT genotyping, SAM may serve as an important biochemical marker in individualization of thiopurine therapy.
Keywords: S-adenosyl-; l; -methionine; Thiopurine S-methyltransferase; Pharmacogenetics; Post-translational; Stabilization; Individualization
Post-translational stabilization of thiopurine S-methyltransferase by S-adenosyl-l-methionine reveals regulation of TPMT*1 and *3C allozymes by Miha Milek; Alenka Smid; Riin Tamm; Natasa Karas Kuzelicki; Andres Metspalu; Irena Mlinaric-Rascan (pp. 969-976).
Thiopurine S-methyltransferase (TPMT; EC 2.1.1.67) plays a pivotal role in thiopurine treatment outcomes. However, little has been known about its intracellular regulation. Here, we describe the effect of fluctuations in physiological levels of S-adenosyl-l-methionine (SAM) and related metabolites on TPMT activity levels in cell lines and erythrocytes from healthy donors. We determined higher TPMT activity in wild-type TPMT*1/*1 individuals with high SAM concentrations ( n=96) compared to the low SAM level group ( n=19; P<0.001). These findings confirm the results of our in vitro studies, which demonstrated that the restriction ofl-methionine (Met) in cell growth media reversibly decreased TPMT activity and protein levels. Selective inhibition of distinct components of Met metabolism was used to demonstrate that SAM is implicitly responsible for direct post-translational TPMT stabilization. The greatest effect of SAM-mediated TPMT stabilization was observed in the case of wild-type TPMT*1 and variant *3C allozymes. In addition to TPMT genotyping, SAM may serve as an important biochemical marker in individualization of thiopurine therapy.
Keywords: S-adenosyl-; l; -methionine; Thiopurine S-methyltransferase; Pharmacogenetics; Post-translational; Stabilization; Individualization