Sonam Gupta

Role of reactive oxygen species in expulsion of Nippostrongylus brasiliensis from rats

To understand the mechanism for the expulsion of Nippostrongylus brasiliensis from rats, age-dependent variations in the metabolism of reactive oxygen species in the parasite and the host intestines were examined. N. brasiliensis showed an age-dependent increase in its susceptibility to xanthine-xanthine oxidase and t-butyl hydroperoxide generated oxidants as well as to H2O2. Protection obtained with several scavengers suggested that the worms were damaged by the combined action of oxidants generated by the in vitro systems employed. The level of superoxide dismutase in the nematode and its release into the surroundings exhibited a marked depression with advancement of age. No such alteration was, however, recorded for catalase and glutathione peroxidase. An appreciable decrease in the level of reduced glutathione in older N. brasiliensis appears to render them prone to oxidant attack. The rat intestines, on the other hand, exhibited an appreciable depression in catalase and a reduced glutathione content with progress of the infection. Vitamin E levels were elevated. The release of O2-. and H2O2 by the intestines was also found to be greater during later stages of the infection. The combined effect of the changes observed in N. brasiliensis and in the rat intestines may be at least partly responsible for expulsion of the nematode from the rats after day 10.

Reactive oxygen intermediates metabolizing enzymes in Ancylostoma ceylanicum and Nippostrongylus brasiliensis

Adult worms of Ancylostoma ceylanicum and Nippostronglyus brasiliensis were found to possess an active system for the detoxification of reactive oxygen intermediates. Xanthine oxidase, which is known to produce superoxide anion, was detected in both the nematode parasites in significant activities. Superoxide anion, thus produced, may quickly be eliminated by superoxide dismutase. Both parasites also exhibited the presence of catalase, peroxidase, and glutathione peroxidase for efficient removal of hydrogen peroxide. Glutathione reductase and glucose-6-phosphate dehydrogenase were, however, detected in low levels of activities. Endowment of A. ceylanicum and N. brasiliensis with these antioxidant enzymes, therefore, enables them to evade the hosts effector mechanism for their survival. Superoxide dismutase of both these nematodes showed marked inhibition by KCN and, hence, the enzyme appears to be of copper-zinc type.

Astrocyte activation and neurotoxicity: A study in different rat brain regions and in rat C6 astroglial cells.

The present study was conducted to investigate the effect of rotenone on astrocytes activation, their viability and its effect on neuronal death in different brain regions. Rotenone was injected in rat brain by intracerebroventricularly (bilateral) route at dose of 6 μg and 12 μg. In vitro C6 cells were treated with rotenone at concentration of 0.1, 0.25, 0.5, 1 and 2 μM. Rotenone administration to rat brain caused significant astrocytes activation in frontal cortex, cerebellum, cerebellar nucleus, substantia nigra, hypothalamus and hippocampus regions of the rat brain. Rotenone administration also led to significant degeneration of cells in all the studied regions along with altered nuclear morphology assessed by hematoxylin-eosin and cresyl violet staining. Histological staining showed the significantly decreased number of cells in all the studied regions except cerebellar nucleus in dose and time dependant manner. Rotenone administration in the rat brain also caused significant decrease in glutathione levels and augmented nitrite levels. In vitro treatment of rotenone to astrocytic C6 cells caused significantly increased expression of glial fibrillar acidic protein (GFAP) and decreased viability in dose and time dependent manner. Rotenone treatment to C6 cells exhibited significant generation of reactive oxygen species, augmented nitrite level, impaired mitochondrial activity, apoptotic chromatin condensation and DNA damage in comparison to control cells. Findings showed that oxidative stress play a considerable role in rotenone induced astrocyte death that was attenuated with co-treatment of antioxidant melatonin. In conclusion, results showed that rotenone caused significant astrocytes activation, altered nuclear morphology, biochemical alteration and apoptotic cell death in different rat brain regions. In vitro observations in C6 cells showed that rotenone treatment exhibited oxidative stress mediated apoptotic cell death, which was attenuated with co treatment of melatonin.

The metabolic enhancer piracetam attenuates mitochondrion-specific endonuclease G translocation and oxidative DNA fragmentation

This study was performed to investigate the involvement of mitochondrion-specific endonuclease G in piracetam (P)-induced protective mechanisms. Studies have shown the antiapoptotic effects of piracetam but the mechanism of action of piracetam is still an enigma. To assess the involvement of endonuclease G in piracetam-induced protective effects, astrocyte glial cells were treated with lipopolysaccharide (LPS) and piracetam. LPS treatment caused significantly decreased viability, mitochondrial activity, oxidative stress, chromatin condensation, and DNA fragmentation, which were attenuated by piracetam cotreatment. Cotreatment of astrocytes with piracetam showed its significantly time-dependent absorption as observed with high-performance liquid chromatography. Astrocytes treated with piracetam alone showed enhanced mitochondrial membrane potential (MMP) in comparison to control astrocytes. However, in LPS-treated cells no significant alteration in MMP was observed in comparison to control cells. Protein and mRNA levels of the terminal executor of the caspase-mediated pathway, caspase-3, were not altered significantly in LPS or LPS + piracetam-treated astrocytes, whereas endonuclease G was significantly translocated to the nucleus in LPS-treated astrocytes. Piracetam cotreatment attenuated the LPS-induced endonuclease G translocation. In conclusion this study indicates that LPS treatment of astrocytes caused decreased viability, oxidative stress, mitochondrial dysfunction, chromatin condensation, DNA damage, and translocation of endonuclease G to the nucleus, which was inhibited by piracetam cotreatment, confirming that the mitochondrion-specific endonuclease G is one of the factors involved in piracetam-induced protective mechanisms.

The latex agglutination test: standardization and comparison with direct agglutination and dot-ELISA in the diagnosis of visceral leishmaniasis in India.

Laboratory diagnosis of visceral leishmaniasis (VL) is usually based on the detection of Leishmania amastigotes in samples of bone marrow or splenic aspirate obtained by invasive procedures. Serological tests serve as a useful adjunct and are especially valuable in early or highly immune cases where amastigotes may be too scanty to be seen easily. The direct agglutination test (DAT) is generally considered the most suitable of the four types of tests currently employed (IFAT, counter immuno-electrophoresis, ELISA and DAT). However, the latex agglutination test (LAT) was recently reported to be a rapid and sensitive screening tool for VL and one which could be carried out at the patients bedside. Further standardization and evaluation of LAT has now revealed that although it is comparable with DAT and dot-ELISA in terms of sensitivity it is far inferior because of cross-reactivity with other infections. This lack of specificity makes LAT unsuitable for routine diagnosis of VL even though it is rapid and sensitive. DAT still appears to be the best choice as a diagnostic tool, as it is very specific and does not require expensive equipment or reagents or much technical competence and the result can be visually interpreted. These merits make DAT very suitable for the diagnosis of VL in endemic areas of India.

6-Hydroxydopamine and lipopolysaccharides induced DNA damage in astrocytes: Involvement of nitric oxide and mitochondria

The present study was conducted to investigate the effect of the neurotoxins 6-hydroxydopamine and lipopolysaccharide on astrocytes. Rat astrocyte C6 cells were treated with different concentration of 6-hydroxydopamine (6-OHDA)/lipopolysaccharides (LPS) for 24 h. Both neurotoxins significantly decreased the viability of astrocytes, augmented the expression of inducible nitric oxide synthase (iNOS) and the astrocyte marker--glial fibrillar acidic protein. A significantly decreased mitochondrial dehydrogenase activity, mitochondrial membrane potential, augmented reactive oxygen species (ROS) level, caspase-3 mRNA level, chromatin condensation and DNA damage was observed in 6-OHDA/LPS treated astroglial cells. 6-OHDA/LPS treatment also caused the significantly increased expression of iNOS and nitrite level. Findings showed that 6-OHDA/LPS treatment caused mitochondrial dysfunction mediated death of astrocytes, which significantly involve the nitric oxide. Since we have observed significantly increased level of iNOS along with mitochondrial impairment and apoptotic cell death in astrocytes, therefore to validate the role of iNOS, the cells were co-treated with iNOS inhibitor aminoguanidine (AG, 100 μM). Co-treatment of AG significantly attenuated the 6-OHDA/LPS induced cell death, mitochondrial activity, augmented ROS level, chromatin condensation and DNA damage. GFAP and caspase-3 expression were also inhibited with co-treatment of AG, although the extent of inhibition was different in both experimental sets. In conclusion, the findings showed that iNOS mediated increased level of nitric oxide acts as a key regulatory molecule in 6-OHDA/LPS induced mitochondrial dysfunction, DNA damage and apoptotic death of astrocytes.

Involvement of glucose related energy crisis and endoplasmic reticulum stress: Insinuation of streptozotocin induced Alzheimer's like pathology

The present study was conducted to correlate the cellular and molecular alterations in Alzheimers pathology employing streptozotocin (STZ) induced experimental rat model. The STZ was administered in rat brain bilaterally by intracerebroventricular route using stereotaxic surgery followed by donepezil dosing. The Alzheimers related pathological marker like acetylcholinesterase (AChE) activity, tau phosphorylation and amyloid aggregation were observed after STZ administration. STZ treatment showed decreased glucose and glucose transporters (GLUT) level along with augmented level of calcium in both cortical and hippocampal regions of rat brain. Increased calcium level may correlate with endoplasmic reticulum (ER) stress and significantly increased expression of ER stress markers like GRP78, GADD and caspase-12 were observed in STZ treated rat brain. Cellular communication was also affected by STZ administration as observed by increased expression connexin 43. With this view the activation of astrocytes and microglia was also assessed and observed by augmented GFAP and cd11b expression which were partially inhibited with donepezil treatment. The significantly increased level of degenerating neurons, caspase-3 and DNA fragmentation was also observed in rat brain regions which were not inhibited with donepezil treatment and validating the clinical observations. In conclusion, study indicated the STZ induced occurrence of Alzheimers pathology. Further, STZ administration also caused depleted glucose level, inhibited mitochondrial activity, augmented calcium levels, ER stress, altered cellular communication and neuronal death which were partially attenuated with donepezil treatment.

On the potential of amino acids to support survival and energy status of Nippostrongylus brasiliensis in vitro.

Aerobically but not anaerobically, amino acids can sustain motility as well as glycogen and ATP levels of N. brasiliensis as effectively as glucose can. Proline is the most active amino acid and in combination with lysine, cysteine and phenylalanine, can completely replace glucose.

NADH oxidase and fumarate reductase of Ancylostoma ceylanicum

Ancylostoma ceylanicum, the hookworm parasite of cat, dog and man, was found to contain NADH and/or NADPH oxidase as well as fumarate reductase activities. Both the enzyme systems were predominantly located in the membranes of mitochondrial-rich preparations. The membranes also exhibited the presence of a reduced pyridine nucleotide transhydrogenase activity which transferred hydrogen from NADPH to NAD. Amongst respiratory inhibitors, rotenone (Site I inhibitor) markedly depressed both NADH oxidase and fumarate reductase while others, namely antimycin-A, KCN and azide, had a lesser effect.

Minocycline diminishes the rotenone induced neurotoxicity and glial activation via suppression of apoptosis, nitrite levels and oxidative stress

Graphical abstract Figure. No Caption available. HighlightsMinocyclin inhibits rotenone induced oxidative stress and increased nitrite levels.It inhibits augmented glial cell reactivity and DNA fragmentation.Minocycline inhibits the degenerative apoptotic neuronal death. ABSTRACT The study was conducted to evaluate the effect of minocycline against pesticide rotenone induced adverse effects in different rat brain regions. Assessment of oxidative stress, nitrite levels, degenerating neurons and level of cleaved caspase‐3 was done in frontal cortex, mid brain, hippocampus and striatum regions of rat brain. In addition the expression profile of neuronal (MAP2), astrocytes (GFAP) and microglia (cd11b) markers was done after treatments. Rotenone induced DNA fragmentation was also assessed in all studied rat brain regions by utilizing comet assay. Rotenone administration caused significantly decreased level of glutathione along with increased level of nitrite and lipid peroxidation. Significant oxidative and nitrosative stress was also observed after rotenone administration which was considerably inhibited in minocycline treated rats in time dependent manner. Fluorojade staining and levels of cleaved caspase 3 showed the degeneration of neurons and apoptosis respectively in studied rat brain regions which were further inhibited with minocycline treatment. Rotenone administration caused significantly increased reactivity of astrocytes, microglia and altered neuronal morphology in rat brain regions which was also partially restored with minocycline treatment. In conclusion, present study showed that minocycline treatment attenuated the rotenone induced oxidative stress, nitrite level, degeneration of neurons, augmented glial reactivity and apoptosis.