ABSTRACT
Immunoglobulins are an important aspect of adaptive immune system and tetrameric IgM are the most prevalent immunoglobulins in Pisces.. In this study, we made an attempt to isolate and characterize immunoglobulins from the Afrcian cat fish, Clarias gariepinus (Burchell, 1822). The immunoglobulins were induced by immunization with BSA. Various methods such as ammonium sulphate precipitation, ion exchange chromatography (DEAE cellulose), gel permeation chromatography and affinity chromatography (Protein A & CNBr-activated agarose conjugated with BSA) were employed for purification of immunoglobulins. But for affinity chromatography involving BSA conjugated agarose, all other methods could purify immunoglobulins only partially, i.e., there was contamination of other proteins. Whereas with affinity chromatography, immunoglobulins could be isolated in purified form. Electrophoresis under denaturing condition resulted in one heavy and two light chain bands of molecular weights of 74.5 and 29.7 & 30.5 kDa, respectively. It resolved into single band on electrophoresis under native conditions. The molecular weight of immunoglobulin was estimated to be 890 kDa by gel filtration chromatography on Ultrogel AcA34. The immunoglobulin was further characterized by western blotting and MALDI-MS and N-terminal analysis. Rat anti-fish Ig generated against heavy chain showed cross reactivity with fish antibody raised against BSA or Aeromonas hydrophila.
ABSTRACT
Trichloroethylene (TCE) is major industrial pollutant that contaminate environment. Its exposure may lead to hepato-renal toxicity along with the cancer progression. Although extensive research is done on its toxicity, still not much is known about its genotoxic potential on humans in relation to genetic polymorphism. Cytochrome P450 (CYP P-450) and glutathione-S-transferases (GSTs) are important in cellular detoxification of TCE. Variations in gene sequences result in population specific regional genetic variations (polymorphism). Genotyping of CYP1A1, GSTM1, GSTT1 and GSTP1 polymorphism was performed in 220 normal and 97 solvent-exposed individuals from northern part of India using real time PCR, PCR and restriction digestion techniques. The parameters examined to study genotoxicity were chromosomal aberration (CA) and cytokinesis block micronucleus assay (CBMN) in lymphocyte culture in vitro. The observed average frequencies for GSTM1 (null) and GSTT1 (null) were 41, 22 and 12.7%, respectively in normal subjects whereas frequencies of CYP1A1/GSTP1 with (ile/ile) or (ile/val) or (val/val) were found to be 76.2/52, 21.4/42.1 and 2.4/5.9% respectively. It was further observed that the frequencies of above genes were found to be similar in solvent exposed groups. The distribution frequencies of GST genes, when compared with other reports from various regions of India show variations. In vitro TCE exposure (2, 4 and or 6 mM) did not show any significant genotoxic effect. TCE may be toxic due to its metabolite.