Iqbal M. Choudhary

Characterization of Structural Features Controlling the Receptiveness of Empty Class II MHC Molecules

MHC class II molecules (MHC II) play a pivotal role in the cell-surface presentation of antigens for surveillance by T cells. Antigen loading takes place inside the cell in endosomal compartments and loss of the peptide ligand rapidly leads to the formation of a non-receptive state of the MHC molecule. Non-receptiveness hinders the efficient loading of new antigens onto the empty MHC II. However, the mechanisms driving the formation of the peptide inaccessible state are not well understood. Here, a combined approach of experimental site-directed mutagenesis and computational modeling is used to reveal structural features underlying “non-receptiveness.” Molecular dynamics simulations of the human MHC II HLA-DR1 suggest a straightening of the α-helix of the β1 domain during the transition from the open to the non-receptive state. The movement is mostly confined to a hinge region conserved in all known MHC molecules. This shift causes a narrowing of the two helices flanking the binding site and results in a closure, which is further stabilized by the formation of a critical hydrogen bond between residues αQ9 and βN82. Mutagenesis experiments confirmed that replacement of either one of the two residues by alanine renders the protein highly susceptible. Notably, loading enhancement was also observed when the mutated MHC II molecules were expressed on the surface of fibroblast cells. Altogether, structural features underlying the non-receptive state of empty HLA-DR1 identified by theoretical means and experiments revealed highly conserved residues critically involved in the receptiveness of MHC II. The atomic details of rearrangements of the peptide-binding groove upon peptide loss provide insight into structure and dynamics of empty MHC II molecules and may foster rational approaches to interfere with non-receptiveness. Manipulation of peptide loading efficiency for improved peptide vaccination strategies could be one of the applications profiting from the structural knowledge provided by this study.

Iridoid Glycoside from the Leaves of Clerodendrum volubile Beauv. Shows Potent Antioxidant Activity Against Oxidative Stress in Rat Brain and Hepatic Tissues

ABSTRACT Aim: This study aims at reporting the isolation, structure elucidation, and antioxidant potentials of ajugoside from C. volubile leaves in sodium nitroprusside (SNP)-induced oxidative stressed rat brain and hepatic tissues. Materials and Method: An iridoid monoterpene, ajugoside was isolated from the n-butanol fraction of C. volubile and evaluated for its antioxidant protective potential on brain and liver tissues of male Wister rats in an ex vivo model. Two molar concentrations (6.4 × 10−4 M and 1.28 × 10−3 M) of the metabolite and SNP were incubated with the tissues homogenate at 37°C for 2 hr prior to the test and assayed for catalase, superoxide dismutase (SOD) activities, and lipid peroxidation. α tocopherol (6.4 × 10−4 M) was used as standard. Results: Both molar concentrations exhibited high catalase activity in the tissues. However, 6.4 × 10−4 M ajugoside exhibited a very high SOD activity (liver: 96.45 and brain: 96.30%) and inhibition of lipid peroxidation (liver: 88.11 and brain: 93.27%) compared to the standard. 1.28 × 10−3 M ajugoside also exhibited good activities but lower than that of the standard and 6.4 × 10−4 M ajugoside. Discussion and Conclusion: Ajugoside showed potent antioxidant activities as evidenced by the synergistic high activities of SOD and catalase as well as inhibition of lipid peroxidation in the studied tissues.

A novel strategy for phosphopeptide enrichment using lanthanide phosphate co-precipitation

AbstractReversible phosphorylation of proteins is a common theme in the regulation of important cellular functions such as growth, metabolism, and differentiation. The comprehensive understanding of biological processes requires the characterization of protein phosphorylation at the molecular level. Although, the number of cellular phosphoproteins is relatively high, the phosphorylated residues themselves are generally of low abundance due to the sub-stoichiometric nature. However, low abundance of phosphopeptides and low degree of phosphorylation typically necessitates isolation and concentration of phosphopeptides prior to mass spectrometric analysis. In this study, we used trivalent lanthanide ions (LaCl3, CeCl3, EuCl3, TbCl3, HoCl3, ErCl3, and TmCl3) for phosphopeptide enrichment and cleaning-up. Due to their low solubility product, lanthanide ions form stable complexes with the phosphate groups of phosphopeptides and precipitate out of solution. In a further step, non-phosphorylated compounds can easily be removed by simple centrifugation and washing before mass spectrometric analysis using Matrix-assisted laser desorption/ionisation-time of flight. The precipitation method was applied for the isolation of phosphopeptides from standard proteins such as ovalbumin, α-casein, and β-casein. High enrichment of phosphopeptides could also be achieved for real samples such as fresh milk and egg white. The technology presented here represents an excellent and highly selective tool for phosphopeptide recovery; it is easily applicable and shows several advantages as compared with standard approaches such as TiO2 or IMAC. FigureHighly selective co-precipitation of phosphopeptides by trivalent lanthanide ions

Protection of CCl4-Induced Liver and Kidney Damage by Phenolic Compounds in Leaf Extracts of Cnestis ferruginea (de Candolle).

Background: The chemoprevention of chemically-induced hepatotoxicity is a crucial means of minimizing susceptibility to hepatic carcinogenesis and plants remain a rich source of anti-hepatotoxicants with antioxidant properties. Objective: The protective role of defatted-methanol (MECF) and ethyl acetate fractions (EF), obtained from Leaves of Cnestis ferruginea in rats induced with carbon tetrachloride (CCl4) toxicity was investigated. Materials and Methods: Adult male Wistar rats were orally administered MECF or EF (125 – 500 mg/kg bwt/5mL) or silymarin (25 mg/kg bwt/5 mL) separately for three days before intervention with an intraperitoneal dose of CCl4. Biomarkers of liver and kidney toxicity as well as Ca2+ regulation were evaluated. Results: Pre-treatment with MECF and EF significantly (P < 0.05) decreased the activities of serum alanine and aspartate aminotransferases, levels of urea, creatinine and cholesterol. A significantly (P < 0.05) enhanced Ca2+ -ATPase activity and lowered levels of membrane cholesterol: Phospholipid ratio were observed in liver microsomes of pre-treated as compared to CCl4 -only treated rats. Rat liver superoxide dismutase activity was enhanced by 125 mg/kg and 250 mg/kg of EF and MECF, while decreases were observed at 500 mg/kg. MECF and EF, like silymarin, attenuated CCl4 -induced hepatotoxicity, microsomal membrane Ca2+ -ATPase inactivation and renal dysfunction. Phytochemistry of MECF revealed the presence of anthraquinones, cardiac and flavone glycosides, tannins and trihydroxyl phenol. Conclusion: These findings suggest that the mechanism of hepatoprotection elicited by MECF and EF, involve its antioxidative properties and regulation of Ca2+ homeostasis.

Norditerpene and diterpene alkaloids from Consolidahohenackeri

Abstract Four diterpenoid alkaloids, two of them being the known compounds lycoctonineand ajaconine, were isolated from the aerial parts of Consolida hohenackeri. The two newcompounds were C19 alkaloids, hoheconsoline and consolinine. The structures of the compoundswere elucidated by 1-D and 2-D NMR techniques.