Sachin Shukla

EFFECTS OF ENDOSULFAN ON BIOENERGETIC PROPERTIES OF SKELETAL MUSCLE MITOCHONDRIA FROM THE FRESHWATER CATFISH (CLARIAS BATRACHUS)

Abstract The effects of a sublethal concentration of an organochlorine pesticide endosulfan on fish skeletal muscle mitochondria oxidizing isocitrate and succinate in vivo and in vitro were investigated. The endosulfan depressed significantly State 3 rates and RCR with succinate, whereas it prevented completely the oxidation of isocitrate. The CCCP-uncoupled rates and State 4 rates of succinate oxidation remained unaffected by endosulfan. The activity of masked ATPase was significantly increased in presence of endosulfan. There was a progressive stimulatory effect of endosulfan on mitochondrial ATPase. The skeletal muscle fibres of endosulfan exposed fish undergo ultrastructural changes that are characterized by deformation of the myofibrils and disappearance of mitochondria. Summarizing, it can be stated that endosulfan exerts an inhibitory effect on electron transport and affects ATP synthetase complex leading to an impairment in mitochondrial bioenergetics, which can be correlated with marked ultrastructural alterations in the skeletal muscle fibres of the fish.

Pax6 influences expression patterns of genes involved in neuro-degeneration

Background Pax6, a highly conserved multifunctional transcription factor, has been critical for neurogenesis and neuronal plasticity. It is presumed that if level of Pax6 approaches either low or null, critical genes responsible for maintaining functional status of neurons or glia would be modulated. Purpose Therefore, it has been intended to explore possibility of either direct or indirect influence of Pax6 in neurodegeneration. Methods The cell lines having origin of murine embryonic fibroblast (Pax6-non expressing, NIH3T3-cell line), murine neuroblastoma (Pax6-expressing brain-derived, Neuro-2a-cell line), and human glioblastoma-astrocytoma (U87MG) were cultured and maintained in a CO2 incubator at 37°C and 5% CO2 in DMEM containing 10% fetal bovine serum. The knockdown of endogenous Pax6 in Neuro-2a cells was achieved through siRNA based gene knock-down approach. The efficiency and validation of knock-down was done by real time PCR. The knock-down of Pax6 was successfully achieved. Results The levels of expression of transcripts of some of the proposed putative markers of neurodegeneration like Pax6, S100β, GFAP, BDNF, NGN2, p73α, p73δ, LDH, SOD, and Catalase were analyzed in Pax6 knockdown condition for analysis of role of Pax6 in neurodegeneration. Since the Pax6 has been proposed to bind to promoter sequences of catalase, and catalase suppresses TGFβ, relative lower levels of catalase in Neuro-2a and U-87MG as compared to NIH-3T3 indicates a possible progressive dominant negative impact of Pax6. However, presence of SOD and LDH indicates alternative protective mechanism. Conclusion Presence of BDNF and TGFβ indicates association between them in glioblastoma-astrocytoma. Therefore, Pax6 seems to be involved directly with p53 and TGFβ mediated pathways and indirectly with redox-sensitive pathway regulation. The neurodegenerative markers S100β, GFAP, BDNF, NGN2, p73α, p73δ, observed downregulated in Pax6 knockdown condition suggest Pax6-mediated regulation of these markers. Observations enlighten Pax6-mediated influences on cascades of genes involved in growth, differentiation and maturation of neurons and glia.

Impact of endosulfan on cytoplasmic and mitochondrial liver malate dehydrogenase from the freshwater catfish (Clarias batrachus).

The impact of a sublethal concentration of an organochlorine pesticide endosulfan on the activity, specific activity, electrophoretic patterns and kinetic properties of crude and purified forms of cytoplasmic malate dehydrogenase (cMDH) and mitochondrial malate dehydrogenase (mMDH) was evaluated in the liver of the freshwater catfish, Clarias batrachus. The endosulfan reduced significantly the activity and the specific activity of cMDH and mMDH, but had no effect on total cytoplasmic and mitochondrial protein contents. The inhibition produced by endosulfan was of mixed non-competitive-uncompetitive type (KiE > KiES) and of mixed competitive-non-competitive type (KiE < KiES) for crude cMDH and mMDH, respectively. The PAGE shows five distinct isoforms (C1 to C5) of cMDH and two isoforms (M1 and M2) of mMDH. The C5-isoform of liver cMDH is predominant and it corresponds to M2-isoform of mMDH. There are no endosulfan-associated differences in the relative charges of crude cMDH and mMDH as well as their purified isoforms, C5-cMDH and M2-mMDH. The relative molecular weights of the purified isoforms are not affected by endosulfan. The purified C5-cMDH and M2-mMDH of endosulfan-treated liver in vivo showed simple non-competitive (KiE = KiES) type of inhibition; whereas in vitro it was of uncompetitive (KiES) and mixed competitive-non-competitive (Ki < KiES) type for the two respective isoforms. G-1-P acts as an uncompetitive (KiES) inhibitor of C5-cMDH and mixed competitive-non-competitive (KiE < KiES) inhibitor of M2-mMDH of the control fish. The inhibitory pattern of G-1-P is modulated by endosulfan in case of C5-cMDH; whereas there is no alteration in case of M2-mMDH. Summarizing, it can be stated that endosulfan exerts an inhibitory effect on crude cMDH and mMDH in vivo, and their purified isoforms (C5-cMDH and M2-mMDH) in vivo as well as in vitro. The impact of endosulfan is mediated through enzyme-substrate-endosulfan (ES-END) complexing for cMDH and enzyme-endosulfan (E-END) complexing for mMDH.

Predictions on Impact of Missense Mutations on Structure Function Relationship of PAX6 and Its Alternatively Spliced Isoform PAX6(5a)

The PAX6 contains two DNA-binding domains, paired domain (PD), homeodomain (HD), and a transactivation domain (TD). Only the crystal structure of PD and the solution structure of HD of PAX6 are known. Mutations in PAX6 show variable penetrance, and expressivity of ocular and neural diseases, but the mechanism is poorly understood. Its alternatively spliced isoform PAX6(5a), is also required in a specific ratio for optimal functions. To understand impact of missense mutations on stability, and conformation of PAX6, whose functional analyses are described in PAX6 allelic variant database, were considered. Representative mutations like PAX6-L46R, -C52R, -V126D, -R128C, -R242T, -P375Q, -Q422R, -V256E, and -S259P from PD, HD, and TD of PAX6 were explored. The secondary structures were analyzed through PSIPRED, and relative solvent accessibilities (RSA) of the mutant and the wild type amino acid residues were compared through SABLE. The change in the contact residues and calculations of energy level were studied through SVMcon, MUpro, and FoldX, respectively. The 3D modeling was performed with the help of MODELLER and models were visualized in Chimera. Predictions suggest mutation induced alterations in local conformation or misfolding in DNA-binding domains of PAX6 and PAX6(5a). The predicted impact of mutations via secondary structure, changes in free energy, stability, conformation, and experimental reports on DNA-binding and transactivation, necessarily provides a strong background to explain structure-function relationship of PAX6 and PAX6(5a). However, because of their predictive nature, these findings need to be validated with other experimental evidences when structure of full length PAX6 is available.

Level of PAX5 in differential diagnosis of non-Hodgkin's lymphoma

Background & objectives: The PAX5, a paired box transcription factor and B-cell activator protein (BSAP), activates B-cell commitment genes and represses non-B-cell lineage genes. About 14 transcript variants of PAX5 have been observed in human. Any alteration in its expression pattern leads to lymphogenesis or associated diseases and carcinogenesis in non-lymphoid tissues. Its mechanisms of function in pathophysiology of non-Hodgkins lymphoma (NHL) are unclear. This study was intended to explore influence of PAX5 in cascade of NHL pathogenesis and diagnosis. Methods: Samples of 65 patients were evaluated by immunohistochemical staining for cellular localization of PAX5, CD19, CD3, cABL, p53, Ras and Raf and by TUNEL assay, RNA-isolation and reverse transcriptase (RT)-PCR, Western blot analysis, and lactate dehydrogenase (LDH) specific staining. Results: B-cell type NHL patients were positive for PAX5, p53, Ras, CD19, Raf and CD3. All of them showed TUNEL-positive cells. The differential expression pattern of PAX5, CD19, p53, CD3, ZAP70, HIF1α, Ras, Raf and MAPK (mitogen-activated protein kinase) at the levels of transcripts and proteins was observed. The LDH assay showed modulation of LDH4 and LDH5 isoforms in the lymph nodes of NHL patients. Interpretation & conclusions: The histological observations suggested that the patients represent diverse cases of NHL like mature B-cell type, mature T-cell type and high grade diffuse B-cell type NHL. The findings indicate that patients with NHL may also be analyzed for status of PAX5, CD19 and ZAP70, and their transcriptional and post-translational variants for the differential diagnosis of NHL and therapy.