Filiz Ozcan

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is inactivated by S-sulfuration in vitro

Hydrogen sulfide (H2S) is produced enzymatically by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), as well as other enzymes in mammalian tissues. These discoveries have led to the crowning of H2S as yet another toxic gas that serves as a gasotransmitter like NO and CO. H2S is thought to exert its biological effects through its reaction with cysteine thiols in proteins, yielding sulfurated thiol (-SSH) derivatives. One of the first proteins shown to be modified by H2S was glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [1] where the S-sulfuration of the active site cysteine (Cys 152) resulted in ~7-fold increase in the activity of the enzyme. In the present study we have attempted to reproduce this result with no success. GAPDH in its reduced, or hydrogen peroxide, or glutathione disulfide, or nitrosonium oxidized forms was reacted with sulfide or polysulfides. Sulfide had no effect on reduced GAPDH activity, while polysulfides inhibited GAPDH to ~42% of control. S-sulfuration of GAPDH occurred at Cys 247 after sulfide treatment, Cys 156 and Cys 247 after polysulfide treatment. No evidence of S-sulfuration at active site Cys 152 was discovered. Both sulfide and polysulfide was able to restore the activity of glutathione disulfide oxidized GAPDH, but not to control untreated levels. Treatment of glutathione disulfide oxidized GAPDH with polysulfide also produced S-sulfuration of Cys 156. Treatment of a C156S mutant of GAPDH with sulfide and polysulfide resulted in S-sulfuration of Cys 152, which also caused a decrease and not an increase in enzymatic activity. Computational chemistry shows S-sulfuration of Cys 156 may affect the position of catalytic Cys 152, raising its pKa by 0.5, which may affect the nucleophilicity of Cys 152. The current study raises significant questions about the reported ability of H2S to activate GAPDH by the sulfuration of its active site thiol, and indicates that polysulfide is a stronger protein S-sulfurating agent than sulfide.

Neutral Sphingomyelinase Inhibition Alleviates Apoptosis, but not ER Stress, in Liver Ischemia–Reperfusion Injury

Abstract Previous studies have revealed the activation of neutral sphingomyelinase (N-SMase)/ceramide pathway in hepatic tissue following warm liver ischemia reperfusion (IR) injury. Excessive ceramide accumulation is known to potentiate apoptotic stimuli and a link between apoptosis and endoplasmic reticulum (ER) stress has been established in hepatic IR injury. Thus, this study determined the role of selective N-SMase inhibition on ER stress and apoptotic markers in a rat model of liver IR injury. Selective N-SMase inhibitor was administered via intraperitoneal injections. Liver IR injury was created by clamping blood vessels supplying the median and left lateral hepatic lobes for 60 min, followed by 60 min reperfusion. Levels of sphingmyelin and ceramide in liver tissue were determined by an optimized multiple reactions monitoring (MRM) method using ultrafast-liquid chromatography (UFLC) coupled with tandem mass spectrometry (MS/MS). Spingomyelin levels were significantly increased in all IR groups compared with controls. Treatment with a specific N-SMase inhibitor significantly decreased all measured ceramides in IR injury. A significant increase was observed in ER stress markers C/EBP-homologous protein (CHOP) and 78 kDa glucose-regulated protein (GRP78) in IR injury, which was not significantly altered by N-SMase inhibition. Inhibition of N-SMase caused a significant reduction in phospho-NF-kB levels, hepatic TUNEL staining, cytosolic cytochrome c, and caspase-3, -8, and -9 activities which were significantly increased in IR injury. Data herein confirm the role of ceramide in increased apoptotic cell death and highlight the protective effect of N-SMase inhibition in down-regulation of apoptotic stimuli responses occurring in hepatic IR injury.

Inhibition of neutral sphingomyelinase decreases elevated levels of nitrative and oxidative stress markers in liver ischemia–reperfusion injury

Oxidative stress and excessive nitric oxide production via induction of inducible nitric oxide synthase (NOS)-2 have been shown in the pathogenesis of liver ischemia–reperfusion (IR) injury. Neutral sphingomyelinase (N-SMase)/ceramide pathway can regulate NOS2 expression therefore this study determined the role of selective N-SMase inhibition on nitrative and oxidative stress markers following liver IR injury. Selective N-SMase inhibitor was administered via intraperitoneal injections. Liver IR injury was created by clamping blood vessels supplying the median and left lateral hepatic lobes for 60 min, followed by 60 min reperfusion. Nitrative and oxidative stress markers were determined by evaluating NOS2 expression, protein nitration, nitrite/nitrate levels, 4-hydroxynonenal (HNE) formation, protein carbonyl levels and xanthine oxidase/xanthine dehydrogenase (XO/XDH) activity. Levels of sphingmyelin and ceramide in liver tissue were determined by an optimized multiple reaction monitoring method using ultra-fast liquid chromatography coupled with tandem mass spectrometry (MS/MS). Spingomyelin levels were significantly increased in all IR groups compared to controls. Treatment with a specific N-SMase inhibitor significantly decreased all measured ceramides in IR injury. NOS2 expression, nitrite/nitrate levels and protein nitration were significantly greater in IR injury and decreased with N-SMase inhibition. Treatment with a selective N-SMase inhibitor significantly decreased HNE formation, protein carbonyl levels and the hepatic conversion of XO. Data confirm the role of nitrative and oxidative injury in IR and highlight the protective effect of selective N-SMase inhibition. Future studies evaluating agents blocking N-SMase activity can facilitate the development of treatment strategies to alleviate oxidative injury in liver I/R injury.

Increased Small Dense LDL and Decreased Paraoxonase Enzyme Activity Reveals Formation of an Atherogenic Risk in Streptozotocin-Induced Diabetic Guinea Pigs

This study aimed to investigate LDL subfraction distribution as well as serum cholesteryl ester transfer protein (CETP), lecithin-cholesterol acyltransferase (LCAT), and paraoxonase (PON1) activity in streptozotocin-induced diabetic guinea pigs. Materials/Methods. Guinea pigs were given a single intraperitoneal (ip) injection of streptozotocin (STZ) and animals having fasting blood glucose levels greater than 200 mg/dl, were considered diabetic. Protein levels of LCAT and CETP were determined via ELISA. Paraoxonase activity was measured kinetically by the formation of phenol while LDL subfraction analysis was done by disc polyacrylamide gel electrophoresis. Results. Plasma glucose and high-density lipoprotein (HDL) cholesterol were significantly increased while total cholesterol and LDL cholesterol were significantly decreased in diabetic guinea pigs compared to controls. LDL subfraction analysis revealed a significant decrease in nonatherogenic LDL-2 subfraction and a significant increase in atherogenic LDL-4 subfraction in diabetic guinea pigs compared to controls. Plasma CETP and PON1 levels were significantly decreased while LCAT showed no significant difference in diabetic guinea pigs compared to controls. Conclusion. Decreased non-atherogenic LDL-1, LDL-2 subfractions, increased small dense LDL-4 subfraction, and decreased PON1 activity, reveals formation of an atherogenic risk in diabetic guinea pigs. Decrease in CETP levels supports the observed increase in HDL cholesterol levels in diabetic guinea pigs.

Apelin-APJ system is responsible for stress-induced increase in atrial natriuretic peptide expression in rat heart

BACKGROUND The cardiovascular system is a primary target of stress and stress is the most important etiologic factor in cardiovascular diseases. Stressors increase expressions of atrial natriuretic peptide (ANP) and apelin in cardiac tissue. AIM The aim of the present study was to investigate whether stress-induced apelin has an effect on the expression of ANP in the right atrium of rat heart. METHODS The rats were divided into the control, stress and F13A+stress groups. In the stress and F13A+stress groups, the rats were subjected to water immersion and restraint stress (WIRS) for 6h. In the F13A+stress group, apelin receptor antagonist F13A, was injected intravenously immediately before application of WIRS. The plasma samples were obtained for the measurement of corticosterone and atrial natriuretic peptide. The atrial samples were used for immunohistochemistry and western blot analysis. RESULTS F13A administration prevented the rise of plasma corticosterone and ANP levels induced by WIRS. While WIRS application increased the expressions of apelin, HIF-1α and ANP in atrial tissue, while F13A prevented the stress-induced increase in the expression of HIF-1α and ANP. CONCLUSION Stress-induced apelin induces ANP expression in atrial tissue and may play a role in cardiovascular homeostasis by increasing ANP expression under WIRS conditions.

Decreased eicosapentaenoic acid levels in acne vulgaris reveals the presence of a proinflammatory state

This study aimed to determine circulating levels of polyunsaturated fatty acids (PUFAs), secretory phospholipase A2 (sPLA2), lipoprotein lipase (LPL) and measure circulating protein levels of angiopoietin-like protein 3 (ANGPTL3), ANGPTL4, cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) in patients with acne vulgaris. Serum from 21 control subjects and 31 acne vulgaris patients were evaluated for levels of arachidonic acid (AA, C20:4n- 6), dihomo-gamma-linolenic acid (DGLA, C20:3n-6), eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3). PUFA levels were determined by an optimized multiple reaction monitoring (MRM) method using ultra fast-liquid chromatography (UFLC) coupled with tandem mass spectrometry (MS/MS). Lipid profile, routine biochemical and hormone parameters were assayed by standard kit methods Serum EPA levels were significantly decreased while AA/EPA and DGLA/EPA ratio were significantly increased in acne vulgaris patients compared to controls. Serum levels of AA, DGLA and DHA showed no significant difference while activity of sPLA2 and LPL were significantly increased in acne vulgaris compared to controls. Results of this study reveal the presence of a proinflammatory state in acne vulgaris as shown by significantly decreased serum EPA levels and increased activity of sPLA2, AA/EPA and DGLA/EPA ratio. Increased LPL activity in the serum of acne vulgaris patients can be protective through its anti-dyslipidemic actions. This is the first study reporting altered EPA levels and increased sPLA2 activity in acne vulgaris and supports the use of omega-3 fatty acids as adjuvant treatment for acne patients.

Analysis of polyunsaturated fatty acids and the omega-6 inflammatory pathway in hepatic ischemia/re-perfusion injury

The aim of the present study was to assess omega-3 (n-3) and omega-6 (n-6) polyunsaturated fatty acids (PUFAs) in liver tissue and evaluate changes in the n-6-associated inflammatory pathway following liver ischemia/re-perfusion (IR) injury. Male Wistar rats which were allowed free access to standard rat chow were included in the study. Blood vessels supplying the median and left lateral hepatic lobes were occluded with an arterial clamp for 60 min, followed by 60 min of re-perfusion. Levels of arachidonic acid (AA, C20:4n-6), dihomo-gamma-linolenic acid (DGLA, C20:3n-6), eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3) in liver tissue were determined by an optimized multiple reaction monitoring method using ultra fast-liquid chromatography coupled with tandem mass spectrometry. Phospholipase A2 (PLA2), cyclooxygenase (COX) and prostaglandin E2 (PGE2) were measured in tissue samples to evaluate changes in the n-6 inflammatory pathway. Total histopathological score of cellular damage were significantly increased following hepatic IR injury. n-3 and n-6 PUFA levels were significantly increased in post-ischemic liver tissue compared to those in non-ischemic controls. No significant difference was observed in the AA/DHA and AA/EPA ratio in post-ischemic liver tissues compared with that in the control. Tissue activity of PLA2 and COX as well as PGE2 levels were significantly increased in post-ischemic liver tissues compared to those in non-ischemic controls. The results of the present study suggested that increased hydrolysis of fatty acids via PLA2 triggers the activity of COX and leads to increased PGE2 levels. Future studies evaluating agents which block the formation of eicosanoids derived from n-6 PUFAs may facilitate the development and application of treatment strategies in liver injury following IR.

Protective Effects of Erdosteine on Amikacin Induced Visual Evoked Potentials and Lipid Peroxidation Alterations

PURPOSE We aimed at investigating the effect of erdosteine administration on amikacin induced visual evoked potentials (VEPs) alterations in rats. METHODS For this purpose, forty male Wistar rats were divided into 4 groups: control, amikacin treated, erdosteine treated, and amikacin + erdosteine treated. Amikacin (600 mg/kg/day) was applied as a single dose of intramuscular injection for 14 days, and 10 mg/kg/day erdosteine was given by gastric gavage for the same period. We recorded all VEP components and measured plasma thiobarbituric acid reactive substance (TBARS) levels in all groups. RESULTS Amikacin increased the latencies of all VEP components (P1, N1, P2, N2, and P3) and elevated plasma TBARS levels compared with control and erdosteine treated rats (p < 0,01). However, prolonged latencies of VEP components in amikacin treated rats returned to control levels after erdostein administration. Treatment of amikacin and erdosteine together significantly decreased plasma TBARS levels (0.05 ± 0.018 nmol/g protein) compared with amikacin group (0.12 ± 0.038 nmol/g protein). CONCLUSIONS These results show that erdosteine has a protective effect on amikacin induced changes in the visual system.

Decreased Serum Levels of Sphingomyelins and Ceramides in Sickle Cell Disease Patients

Limited data are available on the serum levels of different sphingomyelin (CerPCho) and ceramide (CER) species in sickle-cell disease (SCD). This study was aimed at identifying the levels of C16-C24 CerPCho and C16-C24 CER in serum obtained from SCD patients and controls. Circulating levels of neutral sphingomyelinase (N-SMase) activity, ceramide-1-phosphate (C1P), and sphingosine-1-phosphate (S1P) were also determined. Blood was collected from 35 hemoglobin (Hb)A volunteers and 45 homozygous HbSS patients. Serum levels of C16-C24 CerPCho and C16-C24 CER were determined by an optimized multiple reaction monitoring (MRM) method using ultrafast liquid chromatography (UFLC) coupled with tandem mass spectrometry (MS/MS). Serum activity of N-SMase was assayed by standard kit methods, and C1P and S1P levels were determined by enzyme-linked immunosorbent assay. A significant decrease was observed in the serum levels of C18-C24 CerPCho in patients with SCD compared to controls. No significant difference was found in C16 CerPCho levels between the two groups. Very-long-chain C22-C24 CER were significantly decreased in SCD, while long-chain C16-C20 CER levels showed no significant difference between SCD patients and controls. Significant positive correlation was found between the serum total cholesterol levels and C18-C24 CerPCho and C22-C24 CER in SCD patients. Patients with SCD had significantly elevated serum activity of N-SMase as well as increased circulating levels of C1P and S1P compared to controls. The decrease in serum levels of C18-C24 CerPCho in patients with SCD was accompanied by decreased levels of C22-C24 CER. Future studies are needed to understand the role of decreased CerPCho and CER in the pathophysiology of SCD.

Effect of tauroursodeoxycholic acid on PUFA levels and inflammation in an animal and cell model of hepatic endoplasmic reticulum stress

The aim of this study was to evaluate hepatic polyunsaturated fatty acids (PUFAs) and inflammatory response in an animal and cell model of endoplasmic reticulum (ER) stress. Rats were divided into control, tunicamycin (TM)-treated, and TM + tauroursodeoxycholic acid (TUDCA)-treated groups. Hepatic ER stress was induced by TM and the ER stress inhibitor TUDCA was injected 30 min before induction of ER stress. Liver THLE-3 cells were treated with TM and TUDCA was administered in advance to decrease cytotoxic effects. Necroinflammation was evaluated in liver sections, while cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay kit. ER stress was confirmed by immunofluorescence and Western blot analysis of C/EBP-homologous protein and 78-kDa glucose-regulated protein. Arachidonic acid (C20:4n-6), dihomo-γ-linolenic acid (C20:3n-6), eicosapentaenoic acid (C20:5n-3), and docosahexaenoic acid (C22:6n-3) in liver tissue and THLE-3 cells were determined by liquid chromatography tandem mass spectrometry (LC-MS/MS). Phospholipase A2 (PLA2), cyclooxygenase (COX), and prostaglandin E2 (PGE2) were measured in tissue and cell samples. Hepatic ER stress was accomplished by TM and was alleviated by TUDCA. TM treatment significantly decreased PUFAs in both liver and THLE-3 cells compared to controls. PLA2, COX, and PGE2 levels were significantly increased in TM-treated rats and THLE-3 cells compared to controls. TUDCA leads to a partial restoration of liver PUFA levels and decreased PLA2, COX, and PGE2. This study reports decreased PUFA levels in ER stress and supports the use of omega-3 fatty acids in liver diseases demonstrating ER stress.