LIPID PEROXIDATION AND ANTIOXIDANT STATUS: A COMPARISON BETWEEN MIDDLE AGED AND ELDERLY POPULATION

Objective : In recent years a large body of experimental research indicates that oxidative stress and antioxidant defenses are related to processes  such as aging and several diseases.Vitamins and antioxidant enzymes have a fundamental role in defending organisms from oxidative stress.The objective of present study was to compare age related oxidative stress in middle aged and older subjects. Design: The present hospital based nonintervention cross sectional study was designed to evaluate age related oxidative stress and its impact in 40 middle aged (35-55y) and 40 older subjects (60y)  by measuring Thiobarbituric Acid Reactive substances (TBARs) for lipid  peroxidation vis-à-vis antioxidant defense with estimatimation of plasma superoxide dismutase (SOD) and serum alpha-tocopherol concentration . Intervention: none Results: On the basis of the data obtained from our study it was evident that concentration of Thiobarbituric Acid reactive substances were significantly higher in older subjects, whereas enzymatic antioxidant serum superoxide dismutase and free radical scavenging alpha-tocopherol (vitamin E) were significantly lower in elderly age group than middle aged subjects. Conclusion :The present study provides some important information regarding age related oxidative stress in elderly population compared to middle aged subjects. Lipid peroxidation which refers to oxidative degradation of lipids and acts as an oxidative  stressor in the organism is significantly higher in elderly subjects but in so far as antioxidants are concerned we observe that they are significantly lower in elderly than middle aged subjects. Keywords: aging, antioxidant ,lipid peroxidation, oxidative stres

EFFECT OF SUB MAXIMAL EXERCISE ON ENDOTHELIUM-DERIVED NITRIC OXIDE CONCENTRATIONS IN HEALTHY YOUNG SUBJECTS.

Objective: To determine any possible contribution of 10 minutes single session sub maximal exercise on Nitric oxide level in healthy young population.Design: A descriptive observational study on forty healthy volunteers (20 male & 20 female) of 20-30 years designed to evaluate the level of nitric oxide after 10 minutes cycling.Intervention:  Young populations are allowed to exercise in a bicycle ergo meter for 10 minutes regular exercise under protocol YMCA Sub maximal Cycle Ergo meter Testing against a constant workload.  Main outcome measures: Serum Creatine Phosphokinase  and  Nitrate-Nitrite ratio (Surrogate marker of nitric oxide)  measured. Continuous heart rate monitoring is done from ECG system. Vo2 max calculated and energy expenditure for physical activity obtained from prediction equation after adjusting age, gender & body mass.Results: Comparison of Activity Energy Expenditure (AEE) before and after exercise done, (p value<0.0001)(n = 40).  Significant amount of  work done in this exercise. Increase in the serum creatine phosphokinase level (p value<0.0001). Serum Nitrate-Nitrite ratio (nitric oxide level) showed increase (p value 0.1113) in post exercise level.Conclusion: As far as energy expenditure concerned, significant muscular activity in both male & female showed insignificant endothelium derived Nitric oxide production. On the basis of the data obtained it is evident that in a single session sub maximal exercise beneficial effect of vasodilator Nitric oxide was not observed in studied  young population.Keywords: sub maximal exercise, activity energy expenditure, nitrate-nitrite ratio

GBM Derived Gangliosides Induce T Cell Apoptosis through Activation of the Caspase Cascade Involving Both the Extrinsic and the Intrinsic Pathway

<div><p>Previously we demonstrated that human glioblastoma cell lines induce apoptosis in peripheral blood T cells through partial involvement of secreted gangliosides. Here we show that GBM-derived gangliosides induce apoptosis through involvement of the TNF receptor and activation of the caspase cascade. Culturing T lymphocytes with GBM cell line derived gangliosides (10-20μg/ml) demonstrated increased ROS production as early as 18 hrs as indicated by increased uptake of the dye H₂DCFDA while western blotting demonstrated mitochondrial damage as evident by cleavage of Bid to t-Bid and by the release of cytochrome-c into the cytosol. Within 48-72 hrs apoptosis was evident by nuclear blebbing, trypan blue positivity and annexinV/7AAD staining. GBM-ganglioside induced activation of the effector caspase-3 along with both initiator caspases (-9 and -8) in T cells while both the caspase-8 and -9 inhibitors were equally effective in blocking apoptosis (60% protection) confirming the role of caspases in the apoptotic process. Ganglioside-induced T cell apoptosis did not involve production of TNF-α since anti-human TNFα antibody was unable to protect T cells from nuclear blebbing and subsequent cell death. However, confocal microscopy demonstrated co-localization of GM2 ganglioside with the TNF receptor and co-immunoprecipitation experiments showed recruitment of death domains FADD and TRADD with the TNF receptor post ganglioside treatment, suggesting direct interaction of gangliosides with the TNF receptor. Further confirmation of the interaction between GM2 and TNFR1 was obtained from confocal microscopy data with wild type and TNFR1 KO (TALEN mediated) Jurkat cells, which clearly demonstrated co-localization of GM2 and TNFR1 in the wild type cells but not in the TNFR1 KO clones. Thus, GBM-ganglioside can mediate T cell apoptosis by interacting with the TNF receptor followed by activation of both the extrinsic and the intrinsic pathway of caspases.</p></div

Involvement of mitochondria in GBM ganglioside mediated caspase activation.

<p>T cells were co-cultured with GBM derived gangliosides CCF52 and CCF4, and t-Bid induction was detected as shown in Fig 3A. Reduced fluorescence of DiOC₆ in T cells treated with U87 gangliosides (15μg/ml) for 48hrs, versus control cells is indicative of mitochondrial damage (Fig 3B). Evidence of mitochondrial damage was also observed in T cells exposed to GBM derived gangliosides as evidenced from mitochondrial cytochrome c release (Fig 3C). Time dependent induction of reactive oxygen species (ROS) was measured in purified T cells treated with 15μg/ml CCF52 and CCF4 derived gangliosides for 18hrs and 48hrs, as compared to media control for 48hrs, evidenced by H₂DCFDA staining and flow cytometric analysis (Fig 3D) (*p<0.05 vs Media, **p<0.01 vs Media, ***p<0.01 vs Media). ROS production is represented as the mean fluorescence intensity (MFI) of at least 3 independent experiments.</p

Human apoptosis proteome profiler array demonstrates ganglioside induced activation of pro-apoptotic and downregulation of anti-apoptotic proteins.

<p>Differential expression of pro- and anti-apoptotic proteins were examined in T cells cultured with CCF52 ganglioside (15μg/ml) for 48hrs using a human proteome profiler array kit (R&D Biosystems) as represented in Fig 4A and 4B. Fig 4C shows the entire apoptosis proteome profile array of T cells in presence or absence of CCF52 ganglioside. Data is representative of a single experiment out of two experiments done.</p

TALEN mediated targeted disruption of TNFR1 gene abolished the ganglioside GM2–TNFR1 interaction in Jurkat-T cells.

<p>Fig 7A shows schematic representation of TNFR1 specific TALEN pair. DNA sequence with black letters indicates TALEN target sequence against which TALEN pair has been designed, red letters represent spacer DNA sequences. TALEN modules are represented as yellow, red, green or blue boxes according to their base recognition specificity of A, T, G or C respectively. Large red box with overhanging 3 arrows indicates wild type Fok1 nuclease domain, shown in Fig 7A. Western immunoblotting was performed to check the expression level of TNFR1 transfected, G418 selected Jurkat-T cells vs wild type Jurkat-T cells. β-actin was used as loading control as shown in Fig 7B. Jurkat-T cells treated with GM2 or not for 10hrs were attached in poly-L-lysine coated coverslips and stained with GM2 specific and TNFR1 specific antibodies, counterstained with respective fluorescent tagged secondary antibodies and mounted on slides with Vectashield mounting media containing DAPI and assessed for co-localization of TNFR1 and GM2 under confocal microscope (Fig 7C). Both wild type and TNFR1 KO cells were treated with GM2 of not for 10hrs and processed as described above and visualized under confocal microscope as shown in Fig 7D. Scale bar represents 10μm.</p

Induction of T cell apoptosis by GBM derived gangliosides is mediated through caspase activation.

<p>Lysates from T cells treated with GBM derived gangliosides for 72hrs were resolved in a 12% SDS-PAGE and western immunoblot was performed to detect expression of caspases, as shown in Fig 2A. Fig 2B shows graphical representation demonstrating time dependent induction of caspases in purified T cells in response to CCF52 gangliosides for 18hrs, 48hrs & 72hrs as measured by staining the cells with fluorochrome labeled inhibitors of caspases (FLICA) (*p<0.05 vs Media, **p<0.01 vs Media, ***p<0.001 vs Media). Cells were acquired on a FACS-calibur multivariable flow cytometer and % caspase +ve T cells were analyzed using CellQuest 3.3 software. Representative density plot showing time dependent induction of caspases in T cells is shown in Fig 2C. Pre-treatment of T cells with inhibitors of caspases-3, -8, -9 (at 50μM each) and a pan caspase inhibitor (at 12.5μM) 2hrs prior to ganglioside (15μg/ml) treatment, significantly protected T cells from CCF52 ganglioside induced T cell death as evident by trypan blue exclusion in Fig 2D (*p<0.05 vs Media; **p<0.01 vs CCF52 ganglioside-15μg/ml; ***p<0.001 vs CCF52 ganglioside-15μg/ml), and by microscopic analysis of nuclear blebbing in Fig 2D (**p<0.01 vs Media; **p<0.01 vs CCF52 ganglioside-15μg/ml) as shown in Fig 2D. Data represents mean of at least 3 independent experiments unless mentioned otherwise.</p

Human apoptosis proteome profiler array demonstrates ganglioside induced activation of pro-apoptotic and downregulation of anti-apoptotic proteins.

<p>Differential expression of pro- and anti-apoptotic proteins were examined in T cells cultured with CCF52 ganglioside (15μg/ml) for 48hrs using a human proteome profiler array kit (R&D Biosystems) as represented in Fig 4A and 4B. Fig 4C shows the entire apoptosis proteome profile array of T cells in presence or absence of CCF52 ganglioside. Data is representative of a single experiment out of two experiments done.</p