Activation of protein kinase A (cAMP-dependent protein kinase; PKA) triggers insulin secretion in... more Activation of protein kinase A (cAMP-dependent protein kinase; PKA) triggers insulin secretion in the β-cell. Adenylate cyclase toxin (ACT), a bacterial exotoxin with adenylate cyclase activity, and forskolin, an activator of adenylate cyclase, both dose-dependently increased insulin secretion in the presence, but not the absence, of glucose in insulin-secreting βTC3 cells. The stimulation of cAMP release by either agent was dose-dependent but glucose-independent. Omission of extracellular Ca2+ totally abolished the effects of ACT on insulin secretion and cytosolic cAMP accumulation. ACT and forskolin caused rapid and dramatic increases in cytosolic Ca2+, which were blocked by nifedipine and the omission of extracellular Ca2+. Omission of glucose completely blocked the effects of forskolin and partially blocked the effects of ACT on cytosolic Ca2+. PKA α, β and γ catalytic subunits (Cα, Cβ and Cγ respectively) were identified in βTC6 cells by confocal microscopy. Glucose and glucago...
Cognitive aids help clinicians manage critical events and have been shown to improve outcomes by ... more Cognitive aids help clinicians manage critical events and have been shown to improve outcomes by providing critical information at the point of care. Critical event guidelines, such as the Society of Pediatric Anesthesia's Critical Events Checklists described in this article, can be distributed globally via interactive smartphone apps. From October 1, 2013 to January 1, 2014, we performed an observational study to determine the global distribution and utilization patterns of the Pedi Crisis cognitive aid app that the Society for Pediatric Anesthesia developed. We analyzed distribution and utilization metrics of individuals using Pedi Crisis on iOS (Apple Inc., Cupertino, CA) devices worldwide. We used Google Analytics software (Google Inc., Mountain View, CA) to monitor users' app activity (eg, screen views, user sessions). The primary outcome measurement was the number of user-sessions and geographic locations of Pedi Crisis user sessions. Each user was defined by the use of a unique Apple ID on an iOS device. Google Analytics correlates session activity with geographic location based on local Internet service provider logs. Pedi Crisis had 1 252 active users (both new and returning) and 4 140 sessions across 108 countries during the 3-month study period. Returning users used the app longer and viewed significantly more screens that new users (mean screen views: new users 1.3 [standard deviation +/-1.09, 95% confidence interval 1.22-1.55]; returning users 7.6 [standard deviation +/-4.19, 95% confidence interval 6.73-8.39]P<.01) CONCLUSIONS: Pedi Crisis was used worldwide within days of its release and sustained utilization beyond initial publication. The proliferation of handheld electronic devices provides a unique opportunity for professional societies to improve the worldwide dissemination of guidelines and evidence-based cognitive aids.
D-Glucose is the major physiological stimulus for insulin secretion by pancreatic islets. Althoug... more D-Glucose is the major physiological stimulus for insulin secretion by pancreatic islets. Although the biochemical events involved in glucose-induced insulin secretion are incompletely understood (see McDaniel et al.' for review), it is generally accepted that an increase in intracellular Ca2+ is an early event. Recent studies by our laboratory have demonstrated that in normal islets the endoplasmic reticulum is directly involved in regulating intracellular Ca2+ levels.'
Biochemical and Biophysical Research Communications, Jun 18, 1998
Ca2+ channel expression and regulation of intracellular Ca2+ homeostasis were studied during reti... more Ca2+ channel expression and regulation of intracellular Ca2+ homeostasis were studied during retinoic acid (RA)-induced differentiation of the human teratocarcinoma cell line Ntera 2/C1.D1 (NT2- cells) into NT2N neurons, a unique model of human neurons in culture. The cytosolic Ca2+ level of undifferentiated NT2- cells was low (75 +/- 5 nM) and stable under basal conditions, and it was only marginally decreased (by 9%) upon removal of extracellular Ca2+. After 10 microM RA treatment, NT2- cells were irreversibly differentiated into a phenotype of neuron-like NT2N cells. Cytosolic Ca2+ level of NT2N neurons was higher (106 +/- 14 nM) than that of NT2- cells and spontaneously fluctuated (0.208 +/- 0.038 transients/min) under basal conditions. Although K+ increased 86Rb fluxes in both NT2- cells and NT2N neurons, it only increased cytosolic Ca2+ level in NT2N neurons. The K+-induced increase in cytosolic Ca2+ in NT2N neurons was antagonized by 0.1-10 microM nifedipine or verapamil, 5 microM omega-CgTx GVIA, but not by 1 microM omega-agatoxin IVA, 1 microM omega-agatoxin TK, 1 microM FTX-3.3, or 100 microM Ni+ implicating L- and N-type voltage-dependent Ca2+ channels. In L- and N-type channels, but not in P- and Q-types, mRNAs were expressed in NT2N neurons as well as NT2- cells. Quantitative analysis of L- and N-type Ca2+ protein levels showed major differences between NT2- cells and NT2N neurons. In NT2- cells, N-type Ca2+ channels were undetectable while L-type channels levels were fivefold lower compared to NT2N neurons. Our findings show that L- and N-type channels are expressed during differentiation of NT2- cells into neurons, and that these voltage-dependent Ca2+ channels have a major role in regulating intracellular Ca2+ homeostasis and neuronal excitability.
The novel islet-specific protein PANcreatic DERived Factor (PANDER; FAM3B) has been extensively c... more The novel islet-specific protein PANcreatic DERived Factor (PANDER; FAM3B) has been extensively characterized with respect to the -cell, and these studies suggest a potential function for PANDER in the regulation of glucose homeostasis. Little is known regarding PANDER in pancreatic ␣-cells, which are critically involved in maintaining euglycemia. Here we present the first report elucidating the expression and regulation of PANDER within the ␣-cell. Pander mRNA and protein are detected in ␣-cells, with primary localization to a glucagon-negative granular cytosolic compartment. PANDER secretion from ␣-cells is nutritionally and hormonally regulated by l-arginine and insulin, demonstrating similarities and differences with glucagon. Signaling via the insulin receptor (IR) through the PI3K and Akt/PKB node is required for insulin-stimulated PANDER release. The separate localization of PANDER and glucagon is consistent with their differential regulation, and the effect of insulin suggests a paracrine/endocrine effect on PANDER release. This provides further insight into the potential glucose-regulatory role of PANDER.
There has been a large amount of recent literature suggesting that omega-3 unsaturated fatty acid... more There has been a large amount of recent literature suggesting that omega-3 unsaturated fatty acids found in fish oils should be incorporated into the diet for the purpose of decreasing serum cholesterol levels. Inclusion of these fatty acids in the diet has been shown to decrease total serum cholesterol as well as low-density lipoprotein cholesterol. Some of these trials have been complicated by the fact that many of the subjects are afflicted with non-insulin-dependent diabetes mellitus. Unfortunately, the effects of omega-3 unsaturated fatty acids on insulin secretion have not been well characterized. In this study, we have examined the effect of a common omega-3 unsaturated fatty acid, eicosapentaenoic acid (C20:5), on insulin secretion. Using the beta-TC3 insulinoma cell line as a model system for studying insulin exocytosis, C20:5 selectively potentiated glucose-induced insulin secretion. At the same concentration at which it significantly increased glucose-induced insulin secretion, C20:5 did not affect glucose metabolism or intracellular free calcium concentrations. C20:5 also augmented potassium-induced insulin secretion. These data suggest that C20:5, an abundant omega-3 unsaturated fatty acid, acts to augment insulin secretion in a glucose-dependent manner.
BiP, immunoglobulin binding protein; [Ca 2ϩ ] i , intracellular Ca 2ϩ concentration; CMV, cytomeg... more BiP, immunoglobulin binding protein; [Ca 2ϩ ] i , intracellular Ca 2ϩ concentration; CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein; ER, endoplasmic reticulum; GRP78, glucose-regulated protein 78 kDa; IRS, insulin receptor substrate; KRB, Krebs-Ringer bicarbonate; SERCA, sarco(endo)plasmic reticulum Ca 2ϩ-ATPase; Syt3, synaptotagmin III. The symposium and the publication of this article have been made possible by an unrestricted educational grant from Servier, Paris.
Cleavage of amyloid precursor protein (APP) by-secretase generates-amyloid (A), the major compone... more Cleavage of amyloid precursor protein (APP) by-secretase generates-amyloid (A), the major component of senile plaques in Alzheimer's disease. Cleavage of APP by R-secretase prevents A formation, producing nonamyloidogenic secreted APPs products. PKC-regulated APP R-secretase cleavage has been shown to involve tumor necrosis factor R (TNF-R) converting enzyme (TACE). To determine the location of APP cleavage, we examined PKC-regulated APPs secretion by examining cell surface versus intracellular APP in CHO cells stably expressing APP 695 (CHO695). We demonstrate that PKC regulates cell surface and intracellular APP cleavage. The majority of secreted APPs originates from the intracellular compartment, and PKC does not cause an increase in APP trafficking to the cell surface for cleavage. Therefore, intracellular APP regulated by PKC must be cleaved at an intracellular site. Experiments utilizing Brefeldin A suggest APP cleavage occurs at the Golgi or late in the secretory pathway. Experiments using TAPI, an inhibitor of TACE, demonstrate PKC-regulated APPs secretion from the cell surface is inhibited after pretreatment with TAPI, and APPs secretion from the intracellular pool is partially inhibited after pretreatment with TAPI. These findings suggest PKC-regulated APP cleavage occurs at multiple locations within the cell and both events appear to involve TACE.
Recent success in islet transplantation renews the hope for the complete cure of patients afflict... more Recent success in islet transplantation renews the hope for the complete cure of patients afflicted with Type 1 diabetes. However, in the Edmonton series, two to four pancreas donors were required to obtain a sufficient islet mass to reverse the diabetes of each patient. In view of the donor shortage, this represents a major obstacle preventing greater application of islet transplantation to diabetic patients. We hypothesised that increasing the expression of the insulin gene in transplanted islets would augment their capacity for insulin production, thereby allowing reversal of diabetes with a reduced islet mass. We used a replication defective adenovirus to deliver the human proinsulin gene (Ad-Ins) to isolated human islets. The function of Ad-Ins-transduced human islets was compared to islets transduced with a control vector (Ad-lacz). Ad-Ins-transduced islets produced two to three times more insulin than normal islets or those infected with Ad-lacz, as assessed by in vitro perif...
Cytokines have been shown to have dramatic effects on pancreatic islets and insulin-secreting bet... more Cytokines have been shown to have dramatic effects on pancreatic islets and insulin-secreting beta-cell lines. It is well established that cytokines such as interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and gamma-interferon (IFN-gamma) inhibit beta-cell function and are cytotoxic to human and rodent pancreatic islets in vitro. Despite the pleiotropic effects of cytokines on beta-cells, the specific signal transduction pathways and molecular events involved in beta-cell dysfunction remain largely unresolved. In this report, we have examined IL-1beta stimulation of c-Jun NH(2)-terminal kinase (JNK) activity in insulin-secreting clonal cell lines. We demonstrate that IL-1beta transiently activates 46- and 54-kDa isoforms of JNK in cultured RINm5F beta-cells. Furthermore, IL-1beta stimulation of JNK activity is specific, because TNF-alpha and IFN-gamma were without effect. Stable overexpression of JNK1 in RINm5F cells increased levels of activated JNK without af...
Ethylene glycol poisoning is an important toxicological problem in medical practice because early... more Ethylene glycol poisoning is an important toxicological problem in medical practice because early diagnosis and treatment can prevent considerable morbidity and mortality. When ingested in the form of antifreeze or other automotive products, ethylene glycol results in central nervous system depression, cardiopulmonary compromise, and renal insufficiency. Metabolism of ethylene glycol to organic acids is required for metabolic derangement and organ damage. Laboratory features of ethylene glycol poisoning include increased anion gap metabolic acidosis, increased osmolal gap, calcium oxalate crystalluria, and detectable ethylene glycol in serum. This Case Conference integrates discussion of the toxicokinetic and analytical variables that affect the laboratory diagnosis of ethylene glycol intoxication.
... Thromb Res 1994;74:515-22. 1.1. dhao-Hung H, Shwu-Yann W. The influence of time, temperature ... more ... Thromb Res 1994;74:515-22. 1.1. dhao-Hung H, Shwu-Yann W. The influence of time, temperature and packed ... centrations Interfere in Enzymatic Assay of Ethylene Gly-col, Anne F. Eder, Yvonne G. Dowdy, Jo Ann M. Gardiner, Bryan A. Wolf * and Leslie M. Shaw (Dept. ...
Journal of chromatography. B, Biomedical applications, Jan 19, 1994
We describe a relatively simple and sensitive method to measure femtomole amounts of phosphatidic... more We describe a relatively simple and sensitive method to measure femtomole amounts of phosphatidic acid in cells. Phosphatidic acid was extracted from cells in the presence of 1-heptadecanoyl-2-heptadecanoyl-sn-glycero-3-phosphate as an internal standard, purified by two-dimensional thin-layer chromatography, and hydrolyzed to its constituent free fatty acids which were then derivatized to the corresponding pentafluorobenzyl esters. Pentafluorobenzyl esters of fatty acids were analyzed by gas chromatography with electron-capture detection. Long-chain fatty acids were resolved with excellent signal-to-noise ratios. Using heptadecanoic acid as an internal standard for quantitation, as little as 1 fmol of pentafluorobenzyl ester of stearic acid was detected with a linear response up to 10 pmol. Linear detector responses were obtained for all major classes of fatty acids. For phosphatidic acid measurement, the detection limit was at least 50 fmol thus achieving a 1000-fold increase in se...
A system of digitonin-permeabilized islet cells was developed to characterize Ca2+- and calmoduli... more A system of digitonin-permeabilized islet cells was developed to characterize Ca2+- and calmodulin-dependent protein phosphorylation further and to determine whether activation of this membrane-bound process was sufficient for initiation of Ca2+-stimulated insulin secretion. The efficacy of digitonin in permeabilizing the plasma membrane was assessed by Trypan Blue exclusion, by extracellular leakage of lactate dehydrogenase, and by permeability to [gamma-32P]ATP. This treatment did not detectably alter the ultrastructure of the permeabilized cells. Digitonin was equally effective when presented to islet cells that had been previously dispersed or directly to intact isolated islets. The Ca2+- and calmodulin-dependent phosphorylation of endogenous membrane-bound substrates could be demonstrated in the permeabilized cells incubated with [gamma-32P]ATP. This activity displayed characteristics that were similar to those described for the protein kinase measured in subcellular fractions ...
Activation of protein kinase A (cAMP-dependent protein kinase; PKA) triggers insulin secretion in... more Activation of protein kinase A (cAMP-dependent protein kinase; PKA) triggers insulin secretion in the β-cell. Adenylate cyclase toxin (ACT), a bacterial exotoxin with adenylate cyclase activity, and forskolin, an activator of adenylate cyclase, both dose-dependently increased insulin secretion in the presence, but not the absence, of glucose in insulin-secreting βTC3 cells. The stimulation of cAMP release by either agent was dose-dependent but glucose-independent. Omission of extracellular Ca2+ totally abolished the effects of ACT on insulin secretion and cytosolic cAMP accumulation. ACT and forskolin caused rapid and dramatic increases in cytosolic Ca2+, which were blocked by nifedipine and the omission of extracellular Ca2+. Omission of glucose completely blocked the effects of forskolin and partially blocked the effects of ACT on cytosolic Ca2+. PKA α, β and γ catalytic subunits (Cα, Cβ and Cγ respectively) were identified in βTC6 cells by confocal microscopy. Glucose and glucago...
Cognitive aids help clinicians manage critical events and have been shown to improve outcomes by ... more Cognitive aids help clinicians manage critical events and have been shown to improve outcomes by providing critical information at the point of care. Critical event guidelines, such as the Society of Pediatric Anesthesia's Critical Events Checklists described in this article, can be distributed globally via interactive smartphone apps. From October 1, 2013 to January 1, 2014, we performed an observational study to determine the global distribution and utilization patterns of the Pedi Crisis cognitive aid app that the Society for Pediatric Anesthesia developed. We analyzed distribution and utilization metrics of individuals using Pedi Crisis on iOS (Apple Inc., Cupertino, CA) devices worldwide. We used Google Analytics software (Google Inc., Mountain View, CA) to monitor users' app activity (eg, screen views, user sessions). The primary outcome measurement was the number of user-sessions and geographic locations of Pedi Crisis user sessions. Each user was defined by the use of a unique Apple ID on an iOS device. Google Analytics correlates session activity with geographic location based on local Internet service provider logs. Pedi Crisis had 1 252 active users (both new and returning) and 4 140 sessions across 108 countries during the 3-month study period. Returning users used the app longer and viewed significantly more screens that new users (mean screen views: new users 1.3 [standard deviation +/-1.09, 95% confidence interval 1.22-1.55]; returning users 7.6 [standard deviation +/-4.19, 95% confidence interval 6.73-8.39]P<.01) CONCLUSIONS: Pedi Crisis was used worldwide within days of its release and sustained utilization beyond initial publication. The proliferation of handheld electronic devices provides a unique opportunity for professional societies to improve the worldwide dissemination of guidelines and evidence-based cognitive aids.
D-Glucose is the major physiological stimulus for insulin secretion by pancreatic islets. Althoug... more D-Glucose is the major physiological stimulus for insulin secretion by pancreatic islets. Although the biochemical events involved in glucose-induced insulin secretion are incompletely understood (see McDaniel et al.' for review), it is generally accepted that an increase in intracellular Ca2+ is an early event. Recent studies by our laboratory have demonstrated that in normal islets the endoplasmic reticulum is directly involved in regulating intracellular Ca2+ levels.'
Biochemical and Biophysical Research Communications, Jun 18, 1998
Ca2+ channel expression and regulation of intracellular Ca2+ homeostasis were studied during reti... more Ca2+ channel expression and regulation of intracellular Ca2+ homeostasis were studied during retinoic acid (RA)-induced differentiation of the human teratocarcinoma cell line Ntera 2/C1.D1 (NT2- cells) into NT2N neurons, a unique model of human neurons in culture. The cytosolic Ca2+ level of undifferentiated NT2- cells was low (75 +/- 5 nM) and stable under basal conditions, and it was only marginally decreased (by 9%) upon removal of extracellular Ca2+. After 10 microM RA treatment, NT2- cells were irreversibly differentiated into a phenotype of neuron-like NT2N cells. Cytosolic Ca2+ level of NT2N neurons was higher (106 +/- 14 nM) than that of NT2- cells and spontaneously fluctuated (0.208 +/- 0.038 transients/min) under basal conditions. Although K+ increased 86Rb fluxes in both NT2- cells and NT2N neurons, it only increased cytosolic Ca2+ level in NT2N neurons. The K+-induced increase in cytosolic Ca2+ in NT2N neurons was antagonized by 0.1-10 microM nifedipine or verapamil, 5 microM omega-CgTx GVIA, but not by 1 microM omega-agatoxin IVA, 1 microM omega-agatoxin TK, 1 microM FTX-3.3, or 100 microM Ni+ implicating L- and N-type voltage-dependent Ca2+ channels. In L- and N-type channels, but not in P- and Q-types, mRNAs were expressed in NT2N neurons as well as NT2- cells. Quantitative analysis of L- and N-type Ca2+ protein levels showed major differences between NT2- cells and NT2N neurons. In NT2- cells, N-type Ca2+ channels were undetectable while L-type channels levels were fivefold lower compared to NT2N neurons. Our findings show that L- and N-type channels are expressed during differentiation of NT2- cells into neurons, and that these voltage-dependent Ca2+ channels have a major role in regulating intracellular Ca2+ homeostasis and neuronal excitability.
The novel islet-specific protein PANcreatic DERived Factor (PANDER; FAM3B) has been extensively c... more The novel islet-specific protein PANcreatic DERived Factor (PANDER; FAM3B) has been extensively characterized with respect to the -cell, and these studies suggest a potential function for PANDER in the regulation of glucose homeostasis. Little is known regarding PANDER in pancreatic ␣-cells, which are critically involved in maintaining euglycemia. Here we present the first report elucidating the expression and regulation of PANDER within the ␣-cell. Pander mRNA and protein are detected in ␣-cells, with primary localization to a glucagon-negative granular cytosolic compartment. PANDER secretion from ␣-cells is nutritionally and hormonally regulated by l-arginine and insulin, demonstrating similarities and differences with glucagon. Signaling via the insulin receptor (IR) through the PI3K and Akt/PKB node is required for insulin-stimulated PANDER release. The separate localization of PANDER and glucagon is consistent with their differential regulation, and the effect of insulin suggests a paracrine/endocrine effect on PANDER release. This provides further insight into the potential glucose-regulatory role of PANDER.
There has been a large amount of recent literature suggesting that omega-3 unsaturated fatty acid... more There has been a large amount of recent literature suggesting that omega-3 unsaturated fatty acids found in fish oils should be incorporated into the diet for the purpose of decreasing serum cholesterol levels. Inclusion of these fatty acids in the diet has been shown to decrease total serum cholesterol as well as low-density lipoprotein cholesterol. Some of these trials have been complicated by the fact that many of the subjects are afflicted with non-insulin-dependent diabetes mellitus. Unfortunately, the effects of omega-3 unsaturated fatty acids on insulin secretion have not been well characterized. In this study, we have examined the effect of a common omega-3 unsaturated fatty acid, eicosapentaenoic acid (C20:5), on insulin secretion. Using the beta-TC3 insulinoma cell line as a model system for studying insulin exocytosis, C20:5 selectively potentiated glucose-induced insulin secretion. At the same concentration at which it significantly increased glucose-induced insulin secretion, C20:5 did not affect glucose metabolism or intracellular free calcium concentrations. C20:5 also augmented potassium-induced insulin secretion. These data suggest that C20:5, an abundant omega-3 unsaturated fatty acid, acts to augment insulin secretion in a glucose-dependent manner.
BiP, immunoglobulin binding protein; [Ca 2ϩ ] i , intracellular Ca 2ϩ concentration; CMV, cytomeg... more BiP, immunoglobulin binding protein; [Ca 2ϩ ] i , intracellular Ca 2ϩ concentration; CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein; ER, endoplasmic reticulum; GRP78, glucose-regulated protein 78 kDa; IRS, insulin receptor substrate; KRB, Krebs-Ringer bicarbonate; SERCA, sarco(endo)plasmic reticulum Ca 2ϩ-ATPase; Syt3, synaptotagmin III. The symposium and the publication of this article have been made possible by an unrestricted educational grant from Servier, Paris.
Cleavage of amyloid precursor protein (APP) by-secretase generates-amyloid (A), the major compone... more Cleavage of amyloid precursor protein (APP) by-secretase generates-amyloid (A), the major component of senile plaques in Alzheimer's disease. Cleavage of APP by R-secretase prevents A formation, producing nonamyloidogenic secreted APPs products. PKC-regulated APP R-secretase cleavage has been shown to involve tumor necrosis factor R (TNF-R) converting enzyme (TACE). To determine the location of APP cleavage, we examined PKC-regulated APPs secretion by examining cell surface versus intracellular APP in CHO cells stably expressing APP 695 (CHO695). We demonstrate that PKC regulates cell surface and intracellular APP cleavage. The majority of secreted APPs originates from the intracellular compartment, and PKC does not cause an increase in APP trafficking to the cell surface for cleavage. Therefore, intracellular APP regulated by PKC must be cleaved at an intracellular site. Experiments utilizing Brefeldin A suggest APP cleavage occurs at the Golgi or late in the secretory pathway. Experiments using TAPI, an inhibitor of TACE, demonstrate PKC-regulated APPs secretion from the cell surface is inhibited after pretreatment with TAPI, and APPs secretion from the intracellular pool is partially inhibited after pretreatment with TAPI. These findings suggest PKC-regulated APP cleavage occurs at multiple locations within the cell and both events appear to involve TACE.
Recent success in islet transplantation renews the hope for the complete cure of patients afflict... more Recent success in islet transplantation renews the hope for the complete cure of patients afflicted with Type 1 diabetes. However, in the Edmonton series, two to four pancreas donors were required to obtain a sufficient islet mass to reverse the diabetes of each patient. In view of the donor shortage, this represents a major obstacle preventing greater application of islet transplantation to diabetic patients. We hypothesised that increasing the expression of the insulin gene in transplanted islets would augment their capacity for insulin production, thereby allowing reversal of diabetes with a reduced islet mass. We used a replication defective adenovirus to deliver the human proinsulin gene (Ad-Ins) to isolated human islets. The function of Ad-Ins-transduced human islets was compared to islets transduced with a control vector (Ad-lacz). Ad-Ins-transduced islets produced two to three times more insulin than normal islets or those infected with Ad-lacz, as assessed by in vitro perif...
Cytokines have been shown to have dramatic effects on pancreatic islets and insulin-secreting bet... more Cytokines have been shown to have dramatic effects on pancreatic islets and insulin-secreting beta-cell lines. It is well established that cytokines such as interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and gamma-interferon (IFN-gamma) inhibit beta-cell function and are cytotoxic to human and rodent pancreatic islets in vitro. Despite the pleiotropic effects of cytokines on beta-cells, the specific signal transduction pathways and molecular events involved in beta-cell dysfunction remain largely unresolved. In this report, we have examined IL-1beta stimulation of c-Jun NH(2)-terminal kinase (JNK) activity in insulin-secreting clonal cell lines. We demonstrate that IL-1beta transiently activates 46- and 54-kDa isoforms of JNK in cultured RINm5F beta-cells. Furthermore, IL-1beta stimulation of JNK activity is specific, because TNF-alpha and IFN-gamma were without effect. Stable overexpression of JNK1 in RINm5F cells increased levels of activated JNK without af...
Ethylene glycol poisoning is an important toxicological problem in medical practice because early... more Ethylene glycol poisoning is an important toxicological problem in medical practice because early diagnosis and treatment can prevent considerable morbidity and mortality. When ingested in the form of antifreeze or other automotive products, ethylene glycol results in central nervous system depression, cardiopulmonary compromise, and renal insufficiency. Metabolism of ethylene glycol to organic acids is required for metabolic derangement and organ damage. Laboratory features of ethylene glycol poisoning include increased anion gap metabolic acidosis, increased osmolal gap, calcium oxalate crystalluria, and detectable ethylene glycol in serum. This Case Conference integrates discussion of the toxicokinetic and analytical variables that affect the laboratory diagnosis of ethylene glycol intoxication.
... Thromb Res 1994;74:515-22. 1.1. dhao-Hung H, Shwu-Yann W. The influence of time, temperature ... more ... Thromb Res 1994;74:515-22. 1.1. dhao-Hung H, Shwu-Yann W. The influence of time, temperature and packed ... centrations Interfere in Enzymatic Assay of Ethylene Gly-col, Anne F. Eder, Yvonne G. Dowdy, Jo Ann M. Gardiner, Bryan A. Wolf * and Leslie M. Shaw (Dept. ...
Journal of chromatography. B, Biomedical applications, Jan 19, 1994
We describe a relatively simple and sensitive method to measure femtomole amounts of phosphatidic... more We describe a relatively simple and sensitive method to measure femtomole amounts of phosphatidic acid in cells. Phosphatidic acid was extracted from cells in the presence of 1-heptadecanoyl-2-heptadecanoyl-sn-glycero-3-phosphate as an internal standard, purified by two-dimensional thin-layer chromatography, and hydrolyzed to its constituent free fatty acids which were then derivatized to the corresponding pentafluorobenzyl esters. Pentafluorobenzyl esters of fatty acids were analyzed by gas chromatography with electron-capture detection. Long-chain fatty acids were resolved with excellent signal-to-noise ratios. Using heptadecanoic acid as an internal standard for quantitation, as little as 1 fmol of pentafluorobenzyl ester of stearic acid was detected with a linear response up to 10 pmol. Linear detector responses were obtained for all major classes of fatty acids. For phosphatidic acid measurement, the detection limit was at least 50 fmol thus achieving a 1000-fold increase in se...
A system of digitonin-permeabilized islet cells was developed to characterize Ca2+- and calmoduli... more A system of digitonin-permeabilized islet cells was developed to characterize Ca2+- and calmodulin-dependent protein phosphorylation further and to determine whether activation of this membrane-bound process was sufficient for initiation of Ca2+-stimulated insulin secretion. The efficacy of digitonin in permeabilizing the plasma membrane was assessed by Trypan Blue exclusion, by extracellular leakage of lactate dehydrogenase, and by permeability to [gamma-32P]ATP. This treatment did not detectably alter the ultrastructure of the permeabilized cells. Digitonin was equally effective when presented to islet cells that had been previously dispersed or directly to intact isolated islets. The Ca2+- and calmodulin-dependent phosphorylation of endogenous membrane-bound substrates could be demonstrated in the permeabilized cells incubated with [gamma-32P]ATP. This activity displayed characteristics that were similar to those described for the protein kinase measured in subcellular fractions ...
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