J K Orak

Medical University of South Carolina, Charleston, SC, United States

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Publications (38)95.26 Total impact

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    ABSTRACT: BACKGROUND: The prevalence of hypertension and abnormal blood pressure (BP) patterns on 24-h ambulatory BP monitoring (ABPM) remains unknown in children with sickle cell disease (SCD). METHODS: Thirty-eight asymptomatic children with sickle cell disease (SCD) (12 HbSS receiving routine care, 13 HbSC, and 13 HbSS receiving chronic transfusion therapy) underwent 24-h ABPM. Average clinic BP, demographic and biochemical characteristics were collected. RESULTS: Median age was 13 years (range 11-16), body mass index (BMI) 19.1 kg/m(2) (range 18.2-21.1), and 50% were male. Seventeen subjects (43.6%) had ambulatory hypertension, while 4 (10.3%) were hypertensive based on their clinic BP. Mean systolic blood pressure (SBP) and diastolic blood pressure (DBP) dip were 8.3 ± 5.9% and 14.7 ± 7.6% respectively. Twenty-three subjects (59%) had impaired SBP dipping, 7 (18%) had impaired DBP dipping, and 5 (13%) had reversed dipping. Clinic and ABP classification were modestly correlated (rho = 0.38, P = 0.02). CONCLUSION: Abnormalities in ABP measurements and patterns in children with SCD are prevalent and require more attention from heath care providers. ABPM is a valuable tool in identifying masked hypertension and abnormalities in circadian BP.
    Pediatric Nephrology 08/2012; · 2.94 Impact Factor
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    ABSTRACT: While significant racial disparities in graft outcome persist among adult and pediatric kidney transplant recipients in the US, some international studies do not show these differences. The aim of this study is to examine predictors of graft outcomes and the impact of race in our pediatric kidney transplant cohort. Records of 109 pediatric kidney transplant recipients performed at our institution between 7/99 and 4/07 were studied. Patients were grouped based on race: African-American (AA) vs. non-AA. Fifty-five AA (12 ± 5 years) and 54 non-AA patients (11 ± 6 years) were studied. There were more females, pre-emptive transplants and living donors in the non-AAs. Survival analysis showed significantly higher rejection rates in AAs, P = 0.02, and lower unadjusted graft survival (P = 0.09). Cox Proportional Hazards Survival Regression Analysis revealed biopsy-proven acute rejection and delayed graft function contributed to worse graft survival, while pre-emptive transplantation had a favorable effect. Race was not an independent risk factor for decreased graft survival in the final model. In conclusion, our cohort showed several modifiable risk factors that can partially account for poorer graft survival in pediatric AA kidney transplant recipients.
    Saudi journal of kidney diseases and transplantation: an official publication of the Saudi Center for Organ Transplantation, Saudi Arabia 07/2012; 23(4):684-92.
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    ABSTRACT: There is paucity in the data examining the differences in mycophenolate mofetil (MMF) dosing and outcomes among pediatric kidney transplant recipients (PKTX) between races. The aims of this study were as follows (i) to assess whether higher doses of MMF are being utilized in African American (AA) PKTX (ii) to determine whether there is a correlation between MMF dose and outcomes between races, and (iii) to assess the adverse effects of MMF between races. This study analyzed 109 PKTX who received MMF between 7/99 and 5/08. Demographics were similar between groups. Fewer AAs received kidneys from living donors (18% vs. 44%), spent more time on dialysis (1.0 vs. 0.5 yr), and had more human leukocyte antigen mismatches (4 vs. 3). MMF doses among AA patients were higher throughout the study, with statistical differences at week 4, month 3, and month 18. AA patients had significantly higher acute rejection rates and trended toward poorer graft survival; infections, adverse events from MMF and post-transplant lymphoproliferative disease tended to be lower in the AA patients. AA PKTX received higher MMF doses within the first three yr post-transplant compared to their non-AA counterparts, yet demonstrate significantly more acute rejection episodes. Importantly, MMF caused fewer adverse events in AA patients, despite these patients receiving higher doses.
    Clinical Transplantation 07/2011; 25(4):534-40. · 1.63 Impact Factor
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    ABSTRACT: Previously, we and others documented that statins including-lovastatin (LOV) promote the differentiation of oligodendrocyte progenitor cells (OPCs) and remyelination in experimental autoimmune encephalomyelitis (EAE), an multiple sclerosis (MS) model. Conversely, some recent studies demonstrated that statins negatively influence oligodendrocyte (OL) differentiation in vitro and remyelination in a cuprizone-CNS demyelinating model. Therefore, herein, we first investigated the cause of impaired differentiation of OLs by statins in vitro settings. Our observations indicated that the depletion of cholesterol was detrimental to LOV treated OPCs under cholesterol/serum-deprived culture conditions similar to that were used in conflicting studies. However, the depletion of geranylgeranyl-pp under normal cholesterol homeostasis conditions enhanced the phenotypic commitment and differentiation of LOV-treated OPCs ascribed to inhibition of RhoA-Rho kinase. Interestingly, this effect of LOV was associated with increased activation and expression of both PPAR-γ and PTEN in OPCs as confirmed by various pharmacological and molecular based approaches. Furthermore, PTEN was involved in an inhibition of OPCs proliferation via PI3K-Akt inhibition and induction of cell cycle arrest at G1 phase, but without affecting their cell survival. These effects of LOV on OPCs in vitro were absent in the CNS of normal rats chronically treated with LOV concentrations used in EAE indicating that PPAR-γ induction in normal brain may be tightly regulated-providing evidences that statins are therapeutically safe for humans. Collectively, these data provide initial evidence that statin-mediated activation of the PPAR-γ-PTEN cascade participates in OL differentiation, thus suggesting new therapeutic-interventions for MS or related CNS-demyelinating diseases.
    Glia 11/2010; 58(14):1669-85. · 5.07 Impact Factor
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    ABSTRACT: Sickle cell disease (SCD) is associated with a large spectrum of renal abnormalities, one of which, microalbuminuria/proteinuria (MA/P), is a known predictor of end-stage renal disease. We studied 90 children with SCD (57% male; mean age 11.4 +/- 5.2 years) to determine the prevalence and examine clinical correlates of MA/P. The average of two spot urine microalbumin-to-creatinine samples obtained 6 months apart was recorded. Medical records were reviewed for demographic and biochemical data. Medication use, resting office blood pressures (BP), vaso-occlusive pain crises (VOC), and monthly transfusions were recorded. Fourteen children (15.5%) had MA/P. Hemoglobin (Hb) levels were significantly lower in the children with MA than in those without MA/P (8.8 +/- 1.1 vs. 9.8 +/- 1.4 g/dL, respectively) and were significantly correlated with MA (rho = 0.24, p = 0.03). Children with MA were more likely to have abnormal BP (p = 0.058), with 5/14 being hypertensive or pre-hypertensive. In a multivariate logistic regression model of MA, both Hb and BP classification remained in the final model. MA is a simple screening biomarker of early kidney injury in children with SCD. Larger studies to evaluate predictive factors of MA and the relationship to BP are needed.
    Pediatric Nephrology 08/2010; 25(8):1505-11. · 2.94 Impact Factor
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    ABSTRACT: To test the hypothesis that there is an increase in the incidence of childhood nephrolithiasis in the state of South Carolina. We analyzed demographic data from a statewide database on incidence of kidney stones from emergency department data and financial charges. Data were compared with population data from the US Census to control for population growth. There was a significant increase in the incidence of kidney stones in children between 1996 and 2007. The greatest rate of increase was seen in adolescents, pre-adolescents, and Caucasian children. Infants, toddlers, and African-American children did not show significantly increased incidence in the period. Girls show a growing predominance in our population. The amount of money charged for care of children with kidney stones has gone up >4-fold in our state. The incidence of kidney stone disease has risen dramatically in the state of South Carolina since 1996. Further studies investigating potential contributing factors are needed to prevent this costly and painful condition.
    The Journal of pediatrics 03/2010; 157(1):132-7. · 4.02 Impact Factor
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    ABSTRACT: Urinary bladder and renal dysfunction are secondary events associated with spinal cord injury (SCI) in humans. These secondary events not only compromise quality of life but also delay overall recovery from SCI pathophysiology. Furthermore, in experimental models the effects of SCI therapy on bladder and renal functions are generally not evaluated. In this study, we tested whether simvastatin improves bladder and renal functions in a rat model of experimental SCI. SCI was induced by controlled contusion of T9-T10 in adult female rats. Simvastatin (5 mg/Kg body weight) was administered at two hours after SCI and repeated every 24 hours until the end point. Simvastatin-treated SCI animals (simvastatin group) were compared with vehicle-treated SCI animals (vehicle group) in terms of the Basso Beattie Bresnahan score, tissue morphology, cell death, and bladder/renal functions. The urinary bladder of vehicle animals showed a 4.3-fold increase in size and a 9-fold increase in wet weight compared to sham animals. Following SCI, the urine to plasma osmolality ratio increased initially but decreased 1 week after SCI. Hematoxylin and eosin staining of bladder tissue showed transitional epithelial hyperplasia, degeneration of lamina propria, and enlargement of tunica adventia in addition to detrusor muscle hypertrophy. Rats treated with simvastatin for 14 days displayed remarkable recovery by showing decreased bladder size and maintenance of a normal urine/plasma osmolality ratio, in addition to improved locomotion. The muscularis layer of the bladder also regained its compact nature in simvastatin animals. Moreover, SCI-induced renal caspase-3 activity was significantly decreased in the simvastatin group indicating the ability of simvastatin to reduce the renal tubular apoptosis. Post-injury administration of simvastatin ameliorates bladder and renal dysfunction associated with SCI in rats.
    Journal of Inflammation 01/2010; 7:17. · 2.55 Impact Factor
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    ABSTRACT: This study examined the potential therapeutic effects of a combination therapy consisting of 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR) and N-acetyl cysteine (NAC) to attenuate ischemia-reperfusion (I/R) injury in a canine model of autologous renal transplantation. Male mongrel dogs (15-20 kg) underwent left nephrectomy followed by flushing and static preservation of the kidney in University of Wisconsin (UW) solution for 48 hr. The treatment group received AICAR (50 mg/kg) plus NAC (100 mg/kg) intravenously before the left nephrectomy. The compounds were added to the UW solution as well. All dogs underwent right nephrectomy 48 hr later followed by autotransplantation of the preserved left kidney. Treated dogs received a second dose of AICAR and NAC before implantation of the renal autograft. The treated dogs had excellent urine output posttransplant, with peak serum creatinine of 7.26 mg/dL on postoperative day (POD) 3 that normalized after 14 days. The control group were anuric and developed clinical symptoms of uremia on POD 1. Morphologic evaluation supported the protective effects of combination therapy. Immunohistochemical analysis revealed decrease of tumor necrosis factor-alpha, interferon-gamma, and inducible nitric oxide synthase; and TUNEL assay showed decreased apoptosis in the treated group. Combination therapy with AICAR and NAC attenuates renal I/R injury and improves the outcome of the transplanted kidney after prolonged cold preservation.
    Transplantation 10/2004; 78(5):654-9. · 3.78 Impact Factor
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    ABSTRACT: Renal ischemia is of great clinical interest because of its role in renal failure and renal graft rejection. The purpose of this study was to investigate the therapeutic effects of a combination therapy of: n-acetyl cysteine (NAC), a potent antioxidant, sodium nitroprusside (SNP), a nitric oxide donor and phosphormidon (P), an endothelin-1 converting enzyme inhibitor, on tissue protection against renal ischemia/reperfusion injury in the canine model. In this study, 15-20 kg male dogs were subjected to 90 minutes of warm unilateral renal ischemia after removal of one kidney and then divided into control, ischemia alone and treatment groups. Blood samples were collected from these dogs for measurement of kidney function tests and the kidneys were harvested at different time intervals for morphological evaluation, immunostaining and Tunnel Assay. Kidney function tests (serum creatinine and blood urea nitrogen [BUN]) showed a significant difference between untreated and treated groups. ** P <0.01, * P< 0.0001 for treated versus untreated. The protective effect of the combination therapy is also supported by light microscopic studies, immunostaining of renal tissue sections for pro-inflammatory cytokines (TNF-alpha and IFN-gamma), iNOS, and apoptosis by TUNEL assay. Our results suggest that pre-administration of a combination of NAC, SNP, and P attenuates renal ischemia/reperfusion injury. This has potential application in preservation of donor kidney for transplantation by protecting cells against free radical damage.
    Journal of nephrology 01/2003; 16(1):63-74. · 2.02 Impact Factor
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    ABSTRACT: Renal ischemia is of clinical interest because of its role in renal failure and also renal graft rejection. To evaluate the effect of the combination of N-acetylcysteine (NAC), a potent antioxidant, sodium nitroprusside (SNP), a nitric oxide donor, and phosphoramidon (P), an endothelin converting enzyme inhibitor, on tissue protection against ischemia-reperfusion injury, we studied the biochemical and morphological changes due to 90 min of renal ischemia-reperfusion in the rat model. Ninety min of ischemia caused very severe injury and the animals could not survive after 4 days without any treatment. Whereas, animals in the treated groups survived i.e. the NAC group (25%), NAC + SNP group (43%) and in the NAC + SNP + P group (100%), 2 weeks after 90 min of ischemia. A significant increase in the serum levels of creatinine and urea nitrogen was shown in the untreated group and to a much lesser extent in the treated group, especially in the NAC + SNP + P group. The protective effect was also supported by light microscopic studies on renal tissue sections. We also measured the activities of antioxidant enzymes in tissue homogenates. With the exception of Mn-superoxide dismutase, the activities of antioxidant enzymes (catalase, glutathione peroxidase, CuZn-superoxide dismutase) were decreased in the untreated kidney. The administration of NAC alone and NAC + SNP protected against the loss of activities. Treatment with a combination of NAC, SNP and P showed a synergistic effect as evidenced by the best protection. These results suggest that pre-administration of a combination of antioxidant (NAC) with endothelin derived vasodilators (sodium nitroprusside and Phosphoramidon) attenuates renal ischemia-reperfusion injury, e.g. in donor kidney for transplantation, by protecting cells against free radical damage.
    Molecular and Cellular Biochemistry 12/2002; 240(1-2):9-17. · 2.33 Impact Factor
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    ABSTRACT: Ischemia followed by reperfusion has a number of clinically significant consequences. A number of pathophysiological processes appear to be involved in ischemia/reperfusion (I/R) injury. The mitogen activated protein kinases (MAPK) are integral components of the parallel MAP kinase cascades activated in response to a variety of cellular stress inducing ischemia/ATP depletion and inflammatory cytokines. Many studies suggest that members of the MAP kinase family in particular Jun N-terminal kinase (JNK) are activated in kidney following ischemia/reperfusion of this tissue. The present study underlines the therapeutic potential of the combination of N-acetyl cysteine (NAC), a potent antioxidant, sodium nitroprusside (SNP), a nitric oxide donor and phosphoramidon (P), an endothelin-1 converting enzyme inhibitor in ameliorating the MAPK induced damage during renal ischemia/reperfusion injury. Our previous results showed that 90 min of ischemia followed by reperfusion caused very severe injury and that the untreated animals had 100% mortality after the 3rd day whereas there was improved renal function and 100% survival of animals in the three drug combination treatment group. The present study, mainly on tissue sections, further supports the protection provided by the triple drug therapy. A higher degree of expression of all the three classes of MAPK, i.e. JNK, P38 MAP kinases and P-extracellular signal regulated kinases (ERKs) can be seen in kidneys subjected to ischemia/reperfusion insult. Pretreatment with a combination of N-acetyl cysteine, sodium nitroprusside, and phosphoramidon completely inhibits all three classes of MAPK and ameliorates AP-1 whereas individual or a combination of any two drugs is not as effective.
    Molecular and Cellular Biochemistry 12/2002; 240(1-2):19-29. · 2.33 Impact Factor
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    ABSTRACT: We report that endotoxin treatment results in decreased amounts of peroxisomes as well as changes in structure and function of peroxisomal membranes. Peroxisomes isolated from the liver of control and treated animals showed a marked decrease in total protein, but no significant alteration in the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) protein profile. However, the Western blot study of the peroxisomal beta-oxidation enzymes and catalase showed an increase in those enzymes in the peroxisomal peak of normal density in endotoxin-treated rats. Disintegration of peroxisomal membranes by carbonate treatment from endotoxin-treated liver and change in the fluidity of peroxisomal membranes suggests alterations in peroxisomal membrane structure. No such alterations were found in mitochondrial or microsomal membranes of endotoxin-treated livers. The lipid analysis of these organelles showed that the only organelle affected was the peroxisome, with a significant decrease in the phospholipid and cholesterol concentrations. To understand the mechanism of endotoxin-mediated alterations in peroxisomes, we studied the possible role of Kupffer cell secreted soluble factors (tumor necrosis factor alpha [TNF-alpha]) on the peroxisomal structure/function. Inactivation/elimination of Kupffer cells by gadolinium chloride before endotoxin treatment did not normalize the overall peroxisomal protein amount and the lipid composition of isolated peroxisomes. However, the levels of individual protein amount in remaining peroxisomes were normalized. Endotoxin also decreased peroxisomal beta-oxidation, and this was partially restored with gadolinium treatment. These results clearly show that peroxisomes are severely affected by endotoxin treatment and suggest that the damage to this organelle may contribute, at least in part, to endotoxin-induced hepatic cytotoxicity.
    Hepatology 03/2000; 31(2):446-55. · 12.00 Impact Factor
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    ABSTRACT: Reactive oxygen species (ROS; O2-, H2O2, and OH), normal by-products of cellular metabolic processes, are kept in control by antioxidant enzymes, such as catalase, glutathione peroxidase (GPX) and superoxide dismutases (SODs). To understand the role of antioxidant enzymatic defenses against ROS injury following ischemia-reperfusion, we examined the effect on kidney exposed to varying periods (30, 60 or 90 min) of ischemia followed by different periods of reperfusion. The enzymatic activities and protein levels of catalase, GPX, CuZnSOD and MnSOD were relatively unaffected at 30 min of ischemia followed by 0, 2 or 24 h reperfusion. However, 60 or 90 min of ischemia followed by 0, 2 or 24 h of reperfusion resulted in a decrease in activities and protein levels which paralleled the duration of ischemic injury. MnSOD activity tended to recover towards normal during reperfusion. Examination of the mRNA levels of these antioxidant enzymes demonstrated a severe decrease in mRNA levels of catalase and GPX at a time point of minimal ischemic injury (30 min of ischemia followed by reperfusion) suggesting that loss of mRNA of catalase and GPX may be the first markers of alterations in cellular redox in ischemia-reperfusion injury. Greater loss of mRNA for catalase, GPX and CuZnSOD was observed following longer periods (60 or 90 min) of ischemia. The mRNA for MnSOD was upregulated at all time points of ischemia-reperfusion injury. Actually, the greater decrease in mRNAs for catalase, GPX and CuZnSOD in the acute phase (within 24 h) subsequently showed a further decrease in these enzyme activities in the subacute phase (72 or 120 h after ischemia). These enzyme activities in the 30 min ischemia group, (but not in the 90 min group), already showed tendencies for normalization at 120 h after ischemia. To understand the molecular basis of the loss of mRNA of these antioxidant enzymes during ischemia-reperfusion injury, we examined the rate of transcription by nuclear run-on assays. The similar rates of transcription in control and kidney exposed to ischemia-reperfusion indicates that the loss of mRNA for catalase, GPX and CuZnSOD is possibly due to the increased rate of turnover of their mRNAs. These studies suggest that expression of antioxidant genes during ischemia-reperfusion are not coordinately expressed and that the differential loss of antioxidant enzymes may be the contributing factor(s) towards the heterogeneous renal tissue damage as a result of ischemia-reperfusion induced oxidative stress.
    Molecular and Cellular Biochemistry 03/2000; 205(1-2):1-11. · 2.33 Impact Factor
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    ABSTRACT: By using highly purified peroxisomes from rat liver, we have shown that peroxisomes contain manganese superoxide dismutase (MnSOD) activity and a 23 kDa protein immunoreactive with antibodies against purified mitochondrial MnSOD. Immunocytochemical studies have also revealed immunoreaction (immunogold) with MnSOD antibodies in mitochondria and peroxisomes. Studies of the intraperoxisomal localization of MnSOD have shown that in peroxisomes MnSOD is a component of the peroxisomal limiting membranes and dense core. Furthermore, the MnSOD level in peroxisomes was modulated by oxidative stress conditions such as ischemia-reperfusion or the treatment with ciprofibrate, a peroxisomal proliferator. These findings suggest that MnSOD in peroxisomes may play an important role in the dismutation of superoxide generated on the peroxisomal membrane for keeping the delicate balance of the redox state.
    Molecular and Cellular Biochemistry 08/1999; 197(1-2):7-12. · 2.33 Impact Factor
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    ABSTRACT: The activities of rat hepatic subcellular antioxidant enzymes were studied during hepatic ischemia/reperfusion. Ischemia was induced for 30 min (reversible ischemia) or 60 min (irreversible ischemia). Ischemia was followed by 2 or 24 h of reperfusion. Hepatocyte peroxisomal catalase enzyme activity decreased during 60 min of ischemia and declined further during reperfusion. Peroxisomes of normal density (d = 1.225 gram/ml) were observed in control tissues. However, 60 min of ischemia also produced a second peak of catalase specific activity in subcellular fractions corresponding to newly formed low density immature peroxisomes (d = 1.12 gram/ml). The second peak was also detectable after 30 min of ischemia followed by reperfusion for 2 or 24 h. Mitochondrial and microsomal fractions responded differently. MnSOD activity in mitochondria and microsomal fractions increased significantly (p < 0.05) after 30 min of ischemia, but decreased below control values following 60 min of ischemia and remained lower during reperfusion at 2 and 24 h in both organelle fractions. Conversely, mitochondrial and microsomal glutathione peroxidase (GPx) activity increased significantly (p < 0.001) after 60 min of ischemia and was sustained during 24 h of reperfusion. In the cytosolic fraction, a significant increase in CuZnSOD activity was noted following reperfusion in animals subjected to 30 min of ischemia, but 60 min of ischemia and 24 h of reperfusion resulted in decreased CuZnSOD activity. These studies suggest that the antioxidant enzymes of various subcellular compartments respond to ischemia/reperfusion in an organelle or compartment specific manner and that the regulation of antioxidant enzyme activity in peroxisomes may differ from that in mitochondria and microsomes. The compartmentalized changes in hepatic antioxidant enzyme activity may be crucial determinant of cell survival and function during ischemia/reperfusion. Finally, a progressive decline in the level of hepatic reduced glutathione (GSH) and concomitant increase in serum glutamate pyruvate transaminase (SGPT) activity also suggest that greater tissue damage and impairment of intracellular antioxidant activity occur with longer ischemia periods, and during reperfusion.
    Molecular and Cellular Biochemistry 11/1997; 176(1-2):337-47. · 2.33 Impact Factor
  • A K Singh, M K Gupta, J K Orak
    Annals of the New York Academy of Sciences 01/1997; 804:696-7. · 4.38 Impact Factor
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    ABSTRACT: The effect of endotoxin on antioxidant gene expression and antioxidant enzyme activity in homogenates of the heart, liver, and kidney from Sprague-Dawley rats was compared by quantitation of m-RNA and enzyme activities. Alterations in the message level for Cu-Zn superoxide dismutase (SOD), Mn SOD, and catalase varied with the tissue type, length of exposure to endotoxin, and dose of endotoxin. In general, endotoxin treatment reduced Cu-Zn SOD expression in the heart and liver, but had no noticeable effect in the kidney. Mn SOD message levels were increased in the heart and kidney but decreased in the liver. Catalase expression was reduced in the kidney and increased marginally in the heart and liver. With regard to enzyme activity, endotoxin treatment reduced Cu-Zn SOD activity in the heart, liver, and kidney. Mn SOD activity showed little change in the heart, but increased in the liver and, to a lesser extent, in the kidney. Catalase activity showed little change in the heart and kidney but was decreased at 12 h in the liver. The differing responses of tissues to the oxidant stress of endotoxin exposure should be considered when evaluating the effect of endotoxin on antioxidant enzymes.
    Free Radical Biology and Medicine 02/1996; 21(4):533-40. · 5.27 Impact Factor
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    ABSTRACT: Plasma and urine concentrations of protein S were measured in five children with steroid-resistant nephrotic syndrome. It was found that plasma free protein S was reduced in three out of the five patients studied. Thus, acquired free protein S deficiency does occur in children with nephrotic syndrome and is one of many factors which may place them at risk for a thromboembolic event.
    Annals of clinical and laboratory science 01/1996; 26(3):279-82. · 0.88 Impact Factor
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    ABSTRACT: Earlier, we reported that rat liver peroxisomes contain Cu-Zn superoxide dismutase (J. Biol. Chem. 267, 6870), thereby suggesting a new antioxidant role for this organelle in free radical metabolism. In this study, we report for the first time that mammalian peroxisomes also contain glutathione peroxidase. Using highly purified rat liver peroxisomes isolated by Nycodenz gradient, we found that peroxisomes contain glutathione peroxidase which shows enzymatic activity with different substrates such as hydrogen peroxide, cumene hydroperoxide, and t-butyl hydroperoxide. This activity could be inhibited in vitro by mercaptosuccinate. Western blot analysis revealed that peroxisomes from control and ciprofibrate-treated livers show immunoreactive bands with antibodies raised against glutathione peroxidase. The intraperoxisomal distribution of glutathione peroxidase was investigated by using peroxisomal membrane and matrix proteins. The results revealed that glutathione peroxidase is a matrix enzyme. The presence of glutathione peroxidase in peroxisomes provides an alternate enzyme system responsible for the degradation of organic peroxides and the degradation of H2O2 under conditions in which catalase is inactivated (e.g., ischemia-reperfusion and endotoxemia). These findings suggest that glutathione peroxidase in peroxisomes may play a novel role in the cellular antioxidant responses to various oxidative stress conditions.
    Archives of Biochemistry and Biophysics 01/1995; 315(2):331-8. · 3.37 Impact Factor
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    ABSTRACT: To understand the mechanism of peroxisome proliferator-induced oxidative stress in non-mutagenic carcinogenesis, the effect of ciprofibrate, a peroxisome proliferator, on the activities and protein amounts of various antioxidant enzymes in different subcellular compartments was examined. Ciprofibrate treatment for short-term (3 weeks) as well as long-term (12 weeks) duration increased the total cellular catalase activity, whereas superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities were decreased significantly. Withdrawal of ciprofibrate from the diet did not normalize these activities. The observed decreases in total cellular SOD and GPX activities following ciprofibrate treatment were due to significant decreases in cytosolic CuZn SOD and GPX, whereas mitochondrial levels of Mn SOD and GPX were relatively unchanged. The peroxisomal CuZn SOD and GPX activities were increased significantly after both short- and long-term treatment, whereas catalase activity was reduced. Western blot analysis of cytoplasm for GPX and CuZn SOD showed a significant decrease in GPX and CuZn SOD proteins. Mitochondrial GPX protein was found to be slightly decreased, whereas Mn SOD protein levels did not show any significant change. The excessive production of H2O2 by oxidases and O2- by the cytochrome P450 enzyme system, along with the observed loss of antioxidant protection by loss of activities of catalase in peroxisomes and GPX and CuZn SOD in cytoplasm, may be the critical factors in peroxisomal proliferator-induced oxidative stress and initiation and promotion of carcinogenesis by this class of non-mutagenic agents. Both enzyme activities, as well as protein amounts of GPX and CuZn SOD, were higher in peroxisomes but lower in cytoplasm in ciprofibrate-treated liver as compared to control liver. The Mn SOD protein was decreased in peroxisomes, whereas mitochondrial Mn SOD was relatively unaffected in ciprofibrate-treated liver as compared to control. These observations suggest that the regulation of expression of peroxisomal CuZn SOD and Mn SOD is different from their counterparts in other cellular compartments.
    Carcinogenesis 10/1994; 15(9):1923-30. · 5.64 Impact Factor