Studies on the subcellular localization and role of glutathione-insulin transhydrogenase in rat liver

₁₂₅I-insulin was shown to be internalized in vivo to a discrete population of low-density membranes (ligandosomes), distinct from the Golgi, endoplasmic reticulum, plasma membrane, and lysosomes. However, analytical subcellular fractionation shows that glutathione-insulin transhydrogenase is localized to the endoplasmic reticulum. Measurement of the specific enzyme activity of glutathione-insulin transhydrogenase showed no differences between normal, diabetic, and hyperinsulinaemic rats. These results suggest that glutathione-insulin transhydrogenase is not directly involved in the subceltular processing of receptor-bound internalized insulin.     

Ca2+-dependent Structural Changes in the B-cell Receptor CD23 Increase Its Affinity for Human Immunoglobulin E

Immunoglobulin E (IgE) antibodies play a fundamental role in allergic disease and are a target for therapeutic intervention. IgE functions principally through two receptors, FcϵRI and CD23 (FcϵRII). Minute amounts of allergen trigger mast cell or basophil degranulation by cross-linking IgE-bound FcϵRI, leading to an inflammatory response. The interaction between IgE and CD23 on B-cells regulates IgE synthesis. CD23 is unique among Ig receptors in that it belongs to the C-type (calcium-dependent) lectin-like superfamily. Although the interaction of CD23 with IgE is carbohydrate-independent, calcium has been reported to increase the affinity for IgE, but the structural basis for this activity has previously been unknown. We have determined the crystal structures of the human lectin-like head domain of CD23 in its Ca²⁺-free and Ca²⁺-bound forms, as well as the crystal structure of the Ca²⁺-bound head domain of CD23 in complex with a subfragment of IgE-Fc consisting of the dimer of Cϵ3 and Cϵ4 domains (Fcϵ3-4). Together with site-directed mutagenesis, the crystal structures of four Ca²⁺ ligand mutants, isothermal titration calorimetry, surface plasmon resonance, and stopped-flow analysis, we demonstrate that Ca²⁺ binds at the principal and evolutionarily conserved binding site in CD23. Ca²⁺ binding drives Pro-250, at the base of an IgE-binding loop (loop 4), from the trans to the cis configuration with a concomitant conformational change and ordering of residues in the loop. These Ca²⁺-induced structural changes in CD23 lead to additional interactions with IgE, a more entropically favorable interaction, and a 30-fold increase in affinity of a single head domain of CD23 for IgE. Taken together, these results suggest that binding of Ca²⁺ brings an extra degree of modulation to CD23 function.     

Mutations in DIIS5 and the DIIS4-S5 linker of Drosophila melanogaster sodium channel define binding domains for pyrethroids and DDT

Mutations in the DIIS4-S5 linker and DIIS5 have identified hotspots of pyrethroid and DDT interaction with the Drosophila para sodium channel. Wild-type and mutant channels were expressed in Xenopus oocytes and subjected to voltage-clamp analysis. Substitutions L914I, M918T, L925I, T929I and C933A decreased deltamethrin potency, M918T, L925I and T929I decreased permethrin potency and T929I, L925I and I936V decreased fenfluthrin potency. DDT potency was unaffected by M918T, but abolished by T929I and reduced by L925I, L932F and I936V, suggesting that DIIS5 contains at least part of the DDT binding domain. The data support a computer model of pyrethroid and DDT binding.     

Mycobacterial PE_PGRS proteins contain calcium-binding motifs with parallel beta-roll folds

The PE_PGRS family of proteins unique to mycobacteria is demonstrated to contain multiple calcium-binding and glycine-rich sequence motifs GGXGXD/NXUX. This sequence repeat constitutes a calcium-binding parallel beta-roll or parallel beta-helix structure and is found in RTX toxins secreted by many Gram-negative bacteria. It is predicted that the highly homologous PE PGRS proteins containing multiple copies of the nona-peptide motif could fold into similar calcium-binding structures. The implication of the predicted calcium-binding property of PE PGRS proteins in the light of macrophage-pathogen interaction and pathogenesis is presented.     

Effects of environmental air pollution on endogenous oxidative DNA damage in humans

Epidemiological studies conducted in metropolitan areas have demonstrated that exposure to environmental air pollution is associated with increases in mortality. Carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) are the major source of genotoxic activities of organic mixtures associated with respirable particulate matter, which is a constituent of environmental air pollution. In this study,we wanted to evaluate the relationship between exposure to these genotoxic compounds present in the air and endogenous oxidative DNA damage in three different human populations exposed to varying levels of environmental air pollution. As measures of oxidative DNA damage we have determined 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) by liquid chromatography–tandem mass spectrometry (LC–MS/MS) and cyclic pyrimidopurinone N-1,N² malondialdehyde-2′-deoxyguanosine (M₁dG) by the immunoslot blot assay from lymphocyte DNA of participating individuals. The level of endogenous oxidative DNA damage was significantly increased in individuals exposed to environmental air pollution compared to unexposed individuals from Kosice (8-oxodG adducts) and Sofia (M₁dG adducts). However, there was no significant difference in the level of endogenous oxidative DNA and exposure to environmental air pollution in individuals from Prague (8-oxodG and M₁dG adducts) and Kosice (M₁dG adducts). The average level of M₁dG adducts was significantly lower in unexposed and exposed individuals from Kosice compared to those from Prague and Sofia. The average level of 8-oxodG adducts was significantly higher in unexposed and exposed individuals from Kosice compared to those from Prague. A significant increasing trend according to the interaction of c-PAHs exposure and smoking status was observed in levels of 8-oxodG adducts in individuals from Kosice. However, no other relationship was observed for M₁dG and 8-oxodG adduct levels with regard to the smoking status and c-PAH exposure status of the individuals. The conclusion that can be made from this study is that environmental air pollution may alter the endogenous oxidative DNA damage levels in humans but the effect appears to be related to the country where the individuals reside. Genetic polymorphisms of the genes involved in metabolism and detoxification and also differences in the DNA repair capacity and antioxidant status of the individuals could be possible explanations for the variation observed in the level of endogenous oxidative DNA damage for the different populations.     

Detection of phosphodiester adducts formed by the reaction of benzo[a]pyrene diol epoxide with 2'-deoxynucleotides using collision-induced dissociation electrospray ionization tandem mass spectrometry

In this study, we investigated the products formed following the reaction of benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (B[a]PDE) with 2′-deoxynucleoside 3′-monophosphates. The B[a]PDE plus 2′-deoxynucleotide reaction mixtures were purified using solid phase extraction (SPE) and subjected to HPLC with fluorescence detection. Fractions corresponding to reaction product peaks were collected and desalted using SPE prior to analysis for the presence of molecular ions corresponding to m/z 648, 632, 608 and 623 [M−H]⁻ consistent with B[a]PDE adducted (either on the base or phosphate group) 2′-deoxynucleotides of guanine, adenine, cytosine and thymine, respectively, using LC-ESI-MS/MS collision-induced dissociation (CID). Reaction products were identified having CID product ion spectra containing product ions at m/z 452, 436 and 412 [(B[a]Ptriol+base)−H]⁻, resulting from cleavage of the glycosidic bond between the 2′-deoxyribose and base, corresponding to B[a]PDE adducts of guanine, adenine and cytosine, respectively. Further reaction products were identified having unique CID product ion spectra characteristic of B[a]PDE adduct formation with the phosphate group of the 2′-deoxynucleotide. The presence of product ions at m/z 399 and 497 were observed for all four 2′-deoxynucleotides, corresponding to [(B[a]Ptriol+phosphate)−H]⁻ and [(2′-deoxyribose+phosphate+B[a]Ptriol)−H]⁻, respectively. In conclusion, this investigation provides the first direct evidence for the formation of phosphodiester adducts by B[a]PDE following reaction with 2′-deoxynucleotides.     

Conformational behavior of polypeptides derived through simultaneous global conservative site-directed mutagenesis of chymotrypsin inhibitor 2

The natural occurrence of conservative residue substitutions in proteins suggests that side-chain packing schemes in protein interiors can accommodate mutational replacements of residues by others of similar nature. To explore the extent to which such substitutions are tolerated, especially when introduced simultaneously and globally over the entire length of a polypeptide chain, we examined the conformational behavior of a model 65 residues-long protein, wild-type chymotrypsin inhibitor 2 (WTCI2), and two globally-mutated (GM) variants named GMCI2-1 and GMCI2-2, each incorporating 55 conservative residue substitutions. GMCI2-1, was soluble over a wide range of pH, and folded into a compact, spherical, monomer marked by (i) complete absence of surface hydrophobicity, (ii) a WTCI2-like betaII-type CD spectrum, (iii) high WTCI2-like thermal stability, and (d) 1D and 2D NMR spectra characteristic of folded protein structure. GMCI2-2 was insoluble over a wide range of pH, and could be solubilized only at pH 4.0, showing non-WTCI2-like far-UV CD spectra characterized by high helical content. These results tentatively indicate that polypeptides incorporating residues of identical nature at equivalent chain locations can show the potential to fold with similar characteristics. However, further detailed investigations would be required to determine whether indeed the structural fold of GMCI2-1 resembles that of WTCI2, and to evaluate the extent to which it does so.     

Heavy Metals Bioconcentration from Soil to Vegetables and Assessment of Health Risk Caused by Their Ingestion

The present study was undertaken to assess the non-carcinogenic human health risk of heavy metals through the ingestion of locally grown and commonly used vegetables viz. Raphanus sativus (root vegetable), Daucus carota (root vegetable), Benincasa hispida (fruit vegetable) and Brassica campestris leaves (leafy vegetable) in a semi-urbanized area of Haryana state, India. Heavy metal quantification of soil and vegetable samples was done using flame atomic absorption spectrophotometer. Lead, cadmium and nickel concentration in vegetable samples varied in range of 0.12–6.54 mg kg^?1, 0.02–0.67 mg kg^?1 and <0.05–0.41 mg kg^?1, respectively. Cadmium and lead concentration in some vegetable samples exceeded maximum permissible limit given by World Health Organization/Food and Agriculture Organization and Indian standards. Much higher concentrations of Pb (40–190.5 mg kg^?1), Cd (0.56–9.85 mg kg^-1) and Ni (3.21–45.87 mg kg^?1) were reported in corresponding vegetable fields’ soils. Correlation analysis revealed the formation of three primary clusters, i.e. Cu–Cd, Cd–Pb and Ni–Zn in vegetable fields’ soils further supported by cluster analysis and principal component analysis. Bioconcentration factor revealed that heavy metals’ uptake was more by leafy vegetable than root and fruit vegetables. Hazard index of all the vegetables was less than unity; thus, the ingestion of these vegetables is unlikely to pose health risks to the target population.