Impact of PDMS surface treatment in cell-mechanics applications.

As a widely used elastomer in cell mechanics studies, PDMS is exposed to a variety of surface treatments during cell culture preparation. Considering its viscoelastic nature in particular, effects of the aforementioned treatments on PDMS mechanical behaviour, especially at the relevant length scale of 100 µm, received limited attention. This is despite the fact that significant errors were reported in the quantification of cellular traction forces as a result of minute changes in PDMS mechanical properties. Hence, the effects of plasma oxidation, sterilization and incubation on PDMS modulus of elasticity, relaxation modulus and Poisson's ratio are studied here through tension and stress relaxation tests, with the results of the latter interpreted via the linear viscoelastic formulation. It is observed that although significant deviations from the properties of untreated PDMS are measured through this cycle of surface treatment, properties of untreated PDMS are almost recovered following incubation in cell medium. For example, the modulus of elasticity of treated PDMS was found to be 6% smaller than that of the untreated PDMS. The corresponding deviation was <3% and <1% for the relaxation modulus and time-averaged Poisson's ratio, respectively. The rate of change of the Poisson's ratio with time was also found to be reduced at the end of incubation process in cell medium. As a result, viscoelastic properties of untreated PDMS can safely be used within the error margins provided by this work.

Hitit Index to distinguish patients with and without Crimean-Congo hemorrhagic fever.

Crimean-Congo hemorrhagic fever (CCHF) is fatal. Therefore, it is very important to use an inexpensive, easily accessible, quick and accurate screening index based on clinical signs and laboratory parameters to identify patients suspected of having CCHF. Laboratory test results on the day of hospitalization for 268 inpatients suspected of having CCHF were used to calculate the laboratory section of the Hitit Index, while 65 of these were also monitored daily during their hospital stay to develop the clinical section of the Hitit Index. Two-hundred CCHF-negative outpatients were also evaluated. One-hundred and forty-nine inpatients were CCHF-positive and 119 inpatients were CCHF-negative. The Hitit Index is 5.6 - (5.3*lymphocyte) - (0.02*fibrinogen) - (12*direct bilirubin) + (0.04*AST) + (0.32*hematocrit) - (0.5*neutrophil) - (0.07*CKD-EPI) - (0.001*CK) ± conjunctival hyperemia (+1.5 in conjunctival hyperemia presence and -1.5 in conjunctival hyperemia absence). In 65 inpatients monitored daily, Hitit Index results for CCHF-positive and negative inpatients were 6.10(1.90-12.30) and -5.35(-8.83- -1.95), while CCHF-negative outpatients were -10.99(-15.64- -6.95) (P < 0.001), respectively. On hospitalization day, just one inpatient was false-negative in 27 CCHF-positive inpatients, while four were false-positive among 38 CCHF-negative inpatients using the Hitit Index. After 24 h, just one inpatient was diagnosed falsely among 27 CCHF-positive and 38 CCHF-negative inpatients, and there was no change after 48 h. Management of patients living in endemic regions suspected of having CCHF could be achieved within minutes using the Hitit Index. Patients with Hitit Index less than zero can be monitored as outpatients, while patients with Hitit Index results above zero must be hospitalized in infectious diseases wards. This study was not registered since it was retrospective.

Disorders of Lipid Metabolism in Chronic Kidney Disease.

Cardiovascular disease (CVD) is the leading cause of death in chronic kidney disease (CKD). One of the most important pathophysiological mechanisms for CVD in patients with CKD is the widespread and possibly accelerated formation of atherosclerotic plaques due to hyperlipidemia, uremic toxins, inflammation, oxidative stress, and endothelial dysfunction. Recent studies showed that the level of oxidized low-density lipoprotein cholesterol increases, and that high--density lipoprotein cholesterol dysfunction occurs as kidney function declines and inflammation becomes more prevalent. In this review, we aimed to discuss the effect of kidney dysfunction, oxidative stress, and inflammation on lipid -profile.

Flaxseed Protects Against Diabetes-Induced Glucotoxicity by Modulating Pentose Phosphate Pathway and Glutathione-Dependent Enzyme Activities in Rats.

This study investigated the effects of flaxseed (Linum usitatissimum L.) intake on general metabolism, pentose phosphate pathway (PPP) and glutathione-dependent enzymes in diabetic rats. Diabetes was induced by streptozotocin injection (40 mg/kg, i.p.) and the enzyme activities were determined spectrophotometrically. Diabetic and control rats were divided in two subgroups, one untreated, and one treated with flaxseed (0.714 g/kg body weight/day; orally) for 12 weeks. Flaxseed ameliorated decreased body weight (p < .05) and increased blood glucose (p < .001), triglyceride (p < .001), ALT (p < .001) and AST (p < .001) in diabetic rats. Diabetes resulted in increased glucose-6-phosphate dehydrogenase (G6PD) (p < .05) and decreased glutathione-S-transferase (GST) (p < .01), but unchanged 6-phosphogluconate dehydrogenase (6PGD) and glutathione reductase (GR) in the brain of rats. These alterations were partially improved by flaxseed in comparison to diabetic untreated group (p < .05). G6PD, 6PGD, GR were elevated (p < .001), while GST unchanged in the lung of diabetic untreated group compared to control. Flaxseed partially prevented the increase in 6PGD (p < .05) and GR (p < .01), but unaffected G6PD in the lung of diabetic rats. G6PD (p < .001), 6PGD (p < .05), GR (p < .001) were augmented, while GST showed a significant (p < .001) depletion in the pancreas of diabetic untreated rats compared to control. Diabetic alterations observed in pancreatic enzyme activities were significantly prevented by flaxseed. Furthermore, a remarkable decrease in 6PGD (p < .001) and an increase in G6PD (threefold of control) were found in the lens of diabetic untreated group that were completely prevented by flaxseed (p < .001). Flaxseed has beneficial effects against diabetes-induced glucotoxicity by modulating G6PD, 6PGD, GR and GST activities in tissues.

Stobadine protects rat kidney against ischaemia/reperfusion injury.

1. Ischaemia-reperfusion (I/R) injury, one of the main causes of acute renal failure, still needs satisfactory treatment for routine clinical application. Stobadine, a novel synthetic pyridoindole anti-oxidant, has the ability to reduce tissue injury induced by mechanisms involving reactive oxygen species during I/R. The aim of the present study was to determine the effects of stobadine on renal I/R injury. 2. Forty male Wistar rats were randomly divided into four groups as follows: sham, I/R, stobadine treated and I/R + stobadine treated. Stobadine (2 mg/kg, i.v.) was given intravenously to two groups of rats. The stobadine-treated group was treated with stobadine following sham operation before the abdominal wall was closed, whereas the I/R + stobadine group received stobadine at the beginning of reperfusion. Renal I/R was achieved by occluding the renal arteries bilaterally for 40 min, followed by 6 h reperfusion. Immediately thereafter, blood was drawn and tissue samples were harvested to assess: (i) serum levels of blood urea nitrogen and creatinine; (ii) serum and/or tissue levels of malondialdehyde (MDA), glutathione (GSH), glucose 6-phosphate dehydrogenase (G-6PD), 6-phosphogluconate dehydrogenase (6-PGD), glutathione reductase (GR) and glutathione peroxidase (GPx); (iii) renal morphology; and (iv) immunohistochemical staining for P-selectin. 3. Stobadine was able to significantly attenuate the renal dysfunction as a result of renal I/R injury. Ischaemia-reperfusion resulted in a significant increase in serum and kidney MDA levels and a decrease in serum and kidney GSH. Stobadine treatment at the beginning of reperfusion attenuated both the increased MDA levels and decreased GSH secondary to I/R injury. In addition, the decreased G-6PD activity observed after I/R was significantly attenuated by stobadine treatment. Stobadine did not alter 6-PGD activity after I/R. Neither GR nor GPx activity was significantly changed in the I/R alone or the I/R + stobadine groups compared with the sham group. In addition, stobadine decreased the morphological deterioration and high P-selectin immunoreactivity secondary to renal I/R injury. 4. A pyridoindole anti-oxidant, stobadine exerts a renal protective effect in renal I/R injury, which is probably due to its radical-scavenging and anti-oxidant activities.

Effects of cadmium and zinc ions on purified lamb kidney cortex glucose-6-phosphate dehydrogenase activity.

Glucose-6-phosphate dehydrogenase (G-6-PD) is the first enzyme in the pentose phosphate pathway. Cadmium is a toxic heavy metal that inhibits several enzymes. Zinc is an essential metal but overdoses of zinc have toxic effects on enzyme activities. In this study G-6-PD from lamb kidney cortex was competitively inhibited by zinc both with respect to glucose-6-phosphate (G-6-P) and NADP+ with Ki values of 1.066 +/- 0.106 and 0.111 +/- 0.007 mM respectively whereas cadmium was a non-competitive inhibitor with respect to both G-6-P and NADP+ Ki values of 2.028 +/- 0.175 and 2.044 +/- 0.289 mM respectively.

Purification and kinetics of sheep kidney cortex glucose-6-phosphate dehydrogenase.

Glucose-6-phosphate dehydrogenase (G-6-PD) is one of the important enzymes, which is responsible for the production of NADPH and ribose-5-phosphate. NADPH is used for the biosynthetic reactions and protection of the cells from free radicals. We have investigated some properties and kinetic mechanism of the sheep kidney cortex G-6-PD. This enzyme has been purified 1,384-fold with a yield of 16.96% and had a specific activity of 27.69 U/mg protein. The purification procedure consists of 2', 5'-ADP-Sepharose 4B affinity chromatography after ultracentrifugation. The sheep kidney cortex G-6-PD was found to operate according to a Ping Pong Bi Bi mechanism. The kinetic parameters from sheep K(m) values for G-6-P and NADP(+) and V(m) were determined to be 0.041+/-0.0043 mM, 0.0147+/-0.001 mM and 28.23+/-0.86 microMol min(-1) mg protein(-1), respectively. The pH optimum was 7.4 and the optimum temperature was 45 degrees C. In our previous study we have found that lamb kidney cortex G-6-PD enzyme obeys 'Ordered Bi Bi' mechanism. We suggest that kinetic mechanism altered due to the aging since sheep G-6-PD uses a 'ping pong' mechanism.

Kinetic properties of glucose-6-phosphate dehydrogenase from lamb kidney cortex.

Glucose-6-phosphate dehydrogenase is the key regulatory enzyme of the pentose phosphate pathway and one of the products of this enzyme; NADPH has a critical role in the defence system against the free radicals. In this study, glucose-6-phosphate dehydrogenase from lamb kidney cortex kinetic properties is examined. The purification procedure is composed of two steps after ultracentrifugation for rapid and easy purification: 2', 5'-ADP Sepharose 4B affinity and DEAE Sepharose Fast Flow anion exchange chromatography. Previously, we used this procedure for the purification of glucose-6-phosphate dehydrogenase from bovine lens. The double reciprocal plots and product inhibition studies showed that the enzyme obeys 'Ordered Bi Bi' mechanism: K(m NADP+)K(m G-6-P) and K(i G-6-P) (dissociation constant of the enzyme--G-6-P complex) were found to be 0.018 +/- 0.002, 0.039 +/- 0.006 and 0.029 +/- 0.005 mM, respectively, by using nonlinear regression analysis. The enzyme was stable at 4 degrees C for a week.

Effects of vitamin E on microsomal Ca(2+) -ATPase activity and calcium levels in streptozotocin-induced diabetic rat kidney.

Vitamin E treatment has been found to be beneficial in preventing or reducing diabetic nephropathy. Increased tissue calcium and abnormal microsomal Ca(2+)-ATPase activity have been suggested as contributing factors in the development of diabetic nephropathy. This study was undertaken to test the hypothesis that vitamin E reduces lipid peroxidation and can prevent the abnormalities in microsomal Ca(2+)-ATPase activity and calcium levels in kidney of streptozotocin (STZ)-induced diabetic rats. Male rats were rendered diabetic by a single STZ injection (55 mg x kg(-1) i.p.). After diabetes was verified, diabetic and age-matched control rats were untreated or treated with vitamin E (400-500 IU kg(-1) x day(-1), orally) for 10 weeks. Ca(2+)-ATPase activity and lipid peroxidation (MDA) were determined spectrophotometrically. Blood glucose levels increased approximately five-fold (> 500 mg x dl(-1)) in untreated-diabetic rats but decreased to 340+/-27 mg x dl(-1) in the vitamin E treated-diabetic group. Kidney MDA levels did not significantly change in the diabetic state. However, vitamin E treatment markedly inhibited MDA levels in both control and diabetic animals. Ca(2+)-ATPase activity was 0.483+/-0.008 U l(-1) in the control group and significantly increased to 0.754+/-0.010 U l(-1) in the STZ-diabetic group (p < 0.001). Vitamin E treatment completely prevented the diabetes-induced increase in Ca(2+)-ATPase activity (0.307+/-0.025 U l(-1), p < 0.001) and also reduced the enzyme activity in normal control rats. STZ-diabetes resulted in approximately two-fold increase in total calcium content of kidney. Vitamin E treatment led to a significant reduction in kidney calcium levels of both control and diabetic animals (p < 0.001). Thus, vitamin E treatment can lower blood glucose and lipid peroxidation, which in turn prevents the abnormalities in kidney calcium metabolism of diabetic rats. This study describes a potential biochemical mechanism by which vitamin E supplementation may delay or inhibit the development of cellular damage and nephropathy in diabetes.

Pentose phosphate pathway, glutathione-dependent enzymes and antioxidant defense during oxidative stress in diabetic rodent brain and peripheral organs: effects of stobadine and vitamin E.

The aim of the present study was to investigate the effects of treatment with antioxidant stobadine (ST) on the activities of enzymes related with pentose phosphate pathway and glutathione-dependent metabolism and the other markers of oxidative stress in brain and peripheral organs of diabetic rats, and to compare the effects of ST treatment alone with the effects of treatments with another antioxidant vitamin E and ST plus vitamin E. Rats were made diabetic by the injection of streptozotocin (STZ; 55 mg/kg IP), and, 2 days later, some control and diabetic rats were left untreated or treated with ST (24.7 mg/kg/day, orally), vitamin E (400-500 U/kg/day, orally), or both substances together. In the brain, although 6-phosphogluconate dehydrogenase activity (6-PGD) did not change, glucose-6-phosphate dehydrogenase activity (G-6PD) was markedly increased in diabetic rats compared with controls; only combined treatment with ST and vitamin E produced a partial prevention on this alteration. The aorta G-6PD and 6-PGD of diabetic rats were 52% and 36% of control values, respectively. Neither single treatments with each antioxidant nor their combination altered the G-6PD and 6-PGD in aorta of diabetic rats. Glutathione peroxidase (GSHPx) activity was increased by STZ-diabetes in brain, heart, and kidney. In diabetic brain, vitamin E alone or combination with ST kept GSHPx at normal levels. Diabetes-induced stimulation in GSHPx did not decrease in response to the treatment with vitamin E in heart and kidney, but was greatly prevented by ST alone. The activity of glutathione reductase (GR) was decreased in brain and heart of diabetic rats. The treatment with each antioxidant or with a combination of both agents completely prevented this deficiency and resulted in further activation of GR in diabetic tissues. Glutathione S-transferase (GST) activity did not significantly change in diabetic brain and aorta. GST was stimulated by all treatment protocols in the brain of diabetic rats and was depressed in aorta of control rats. Catalase (CAT) was activated in diabetic heart but depressed in diabetic kidney. Diabetes-induced abnormalities in CAT activity did not respond to vitamin E alone in heart, was moderately ameliorated by the treatment with this vitamin in kidney, and was completely prevented by ST alone in both tissues. Superoxide dismutase (SOD) activity of brain and heart was unchanged by the diabetes but inhibited in diabetic kidney after the treatment ST alone or ST plus vitamin E. The lipid peroxidation (MDA) was increased in diabetic brain and heart. ST or vitamin E alone partly prevented diabetes-induced increase in MDA in brain and heart; however, antioxidant combination achieved a completely amelioration in MDA of these tissues of diabetic rats. Kidney MDA levels were similar in control and untreated diabetic animals. ST and vitamin E treatments, when applied separately or together, significantly reduced kidney MDA in both control and diabetic rats; and the combined effect of antioxidants was greater than that of each alone. These results are consistent with the degenerative role of hyperglycemia on cellular reducing equivalent homeostasis and antioxidant defense, and provide further evidence that pharmacological intervention of different antioxidants may have significant implications in the prevention of the prooxidant feature of diabetes and protects redox status of the cells.

In vivo treatment with stobadine prevents lipid peroxidation, protein glycation and calcium overload but does not ameliorate Ca2+ -ATPase activity in heart and liver of streptozotocin-diabetic rats: comparison with vitamin E.

Hyperglycemia leads to excess production of reactive oxygen species (ROS), lipid peroxidation and protein glycation that may impair cellular calcium homeostasis and results in calcium sequestration and dysfunction in diabetic tissues. Stobadine (ST) is a pyridoindole antioxidant has been postulated as a new cardio- and neuroprotectant. This study was undertaken to test the hypothesis that the treatment with ST inhibits calcium accumulation, reduces lipid peroxidation and protein glycation and can change Ca2+,Mg2+-ATPase activity in diabetic animals. The effects of vitamin E treatment were also evaluated and compared with the effects of combined treatment with ST. Diabetes was induced by streptozotocin (STZ, 55 mg/kg i.p.). Some of diabetic rats and their age-matched controls were treated orally with a low dose of ST (24.7 mg/kg/day), vitamin E (400-500 IU/kg/day) or ST plus vitamin E for 10 weeks. ST and vitamin E separately produced, in a similar degree, reduction in diabetes-induced hyperglycemia. Each antioxidant alone significantly lowered the levels of plasma lipid peroxidation, cardiac and hepatic protein glycation in diabetic rats but vitamin E treatment was found to be more effective than ST treatment alone. Diabetes-induced increase in plasma triacylglycerol levels was not significantly altered by vitamin E treatment but markedly reduced by ST alone. The treatment with each antioxidant completely prevented calcium accumulation in diabetic heart and liver. Microsomal Ca2+,Mg2+-ATPase activity significantly decreased in both tissues of untreated diabetic rats. ST alone significantly increased microsomal Ca2+,Mg2+-ATPase activity in the heart of normal rats. However, neither treatment with ST nor vitamin E alone, nor their combination did change cardiac Ca2+,Mg2+-ATPase activity in diabetic heart. In normal rats, neither antioxidant had a significant effect on hepatic Ca2+,Mg2+-ATPase activity. Hepatic Ca2+,Mg2+-ATPase activity of diabetic rats was not changed by single treatment with ST, while vitamin E alone completely prevented diabetes-induced inhibition in microsomal Ca2+,Mg2+-ATPase activity in liver. Combined treatment with ST and vitamin E provided more benefits in the reduction of hyperglycemia and lipid peroxidation in diabetic animals. This study describes potential mechanisms on cellular effects of ST in the presence of diabetes-induced hyperglycemia that may delay or inhibit the development of diabetic complications. The use of ST together with vitamin E can better control hyperglycemia-induced oxidative stress.