Biomarkers for human radiation exposure

There is a concern over the potential use of radioactive isotopes as a weapon of terror. The detonation of a radiation dispersal device, the so-called “dirty bomb” can lead to public panic. In order to estimate risks associated with radiation exposure, it is important to understand the biological effects of radiation exposure. Based on this knowledge, biomarkers to monitor potentially exposed populations after a radiological accident can be developed and would be extremely valuable for emergency response. While the traditional radiation exposure biomarkers based on cytogenetic assays serve as standard, the development of rapid and noninvasive tests for radiation exposure is needed. The genomics based knowledge is providing new avenues for investigation. The examination of gene expression after ionizing radiation exposure could serve as a potential molecular marker for biodosimetry. Microarray based studies are identifying new radiation responsive genes that could potentially be used as biomarkers of human exposure to radiation after an accident.     

Molecular detection of nematicidal crystalliferous Bacillus thuringiensis strains of Iran and evaluation of their toxicity on free-living and plant-parasitic nematodes

The characterization of nematode-effective strains and cry genes in the Iranian Bacillus thuringiensis (Bt) collection (70 isolates) is presented. Characterization was based on PCR analysis using 12 specific primers for cry5, cry6, cry12, cry13, cry14, and cry21 genes encoding proteins active against nematodes, crystal morphology, and protein band patterns as well as their nematicidal activity on root-knot nematode (Meloidogyne incognita) and two free-living nematodes (Chiloplacus tenuis and Acrobeloides enoplus). PCR results with primers for these genes showed that 22 isolates (31.5%) contain a minimum of one nematode-active cry gene. Strains containing the cry6 gene were the most abundant and represent 22.8% of the isolates. Bt strains harboring cry14 genes were also abundant (14.2%). cry21 and cry5 genes were less abundant, found in 4.2% and 2.8% of the strains, respectively. In total, six different nematode-active cry gene profiles were detected in this collection. Four isolates did not show the expected PCR product size for cry5, cry6, and cry21 genes; they might contain potentially novel insecticidal crystal protein genes. Twenty-two Bt isolates containing nematode-active cry genes were selected for preliminary bioassays on M. incognita. Based on these bioassays, four isolates were selected for detailed bioassays. Isolates YD5 and KON4 at 2 x 10(8) CFU/mL concentrations showed 77% and 81% toxicity on M. incognita, respectively. The free-living nematodes C. tenuis and A. enoplus were more susceptible and the highest mortality was observed within 48 h of incubation at all of the concentrations tested. Maximum mortality was recorded for isolates SN1 and KON4 at 2 x 10(8) CFU/mL concentrations and resulted in 68% and 77% adults deaths of C. tenuis and 68% and 72% for A. enoplus, respectively. Our results showed that PCR is a useful technique for toxicity prediction of nematicidal Bt isolates.     

Echinococcus granulosus: variability of the host-protective EG95 vaccine antigen in G6 and G7 genotypic variants

Cystic hydatid disease in humans is caused by the zoonotic parasite Echinococcus granulosus. As an aid to control transmission of the parasite, a vaccine has been produced for prevention of infection in the parasite’s natural animal intermediate hosts. The vaccine utilizes the recombinant oncosphere protein, EG95. An investigation into the genetic variability of EG95 was undertaken in this study to assess potential antigenic variability in E. granulosus with respect to this host-protective protein. Gene-specific PCR conditions were first established to preferentially amplify the EG95 vaccine-encoding gene (designated eg95-1) from the E. granulosus genome that also contains several other EG95-related genes. The optimized PCR conditions were used to amplify eg95-1 from several parasite isolates in order to determine the protein-coding sequence of the gene. An identical eg95-1 gene was amplified from parasites showing a G1 or G2 genotype of E. granulosus. However, from isolates having a G6 or G7 genotype, a gene was amplified which had substantial nucleotide substitutions (encoding amino acid substitutions) compared with the eg95 gene family members. The amino acid substitutions of EG95 in the G6/G7 genotypes may affect the antigenicity/efficacy of the EG95 recombinant antigen against parasites of these genotypes. These findings indicate that characterization of eg95 gene family members in other strains/isolates of E. granulosus may provide valuable information about the potential for the EG95 hydatid vaccine to be effective against E. granulosus strains other than the G1 genotype.     

Leishmania major: effects of proteophosphoglycan on reactive oxygen species, IL-12, IFN-gamma and IL-10 production in healthy individuals

Protozoan parasites of the genus Leishmania secrete a range of proteophosphoglycans (PPG) known to be important for successful colonization of Leishmania in the sandfly and for virulence in the mammalian host. PPGs are a large family of extensively glycosylated proteins with some unusual and unique features. In this study we purified PPG from culture supernatant of Leishmania major metacyclic promastigotes. In discontinuous SDS-PAGE, PPG could not enter the resolving gel but after mild acid hydrolysis several bands resolved. Agarose gel electrophoresis and immunoblot analysis using monoclonal antibody (WIC 79.3) indicated that the PPG preparation consisted of heterogeneous molecules. Compositional analysis showed that the PPG preparation contained 67% glycan, 28% protein and 5% phosphate. Additionally, the effect of PPG on reactive oxygen species (ROS) production and induction of IL-10, IL-12 and IFN-γ secretion by human peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals was investigated. The water-soluble secreted form of PPG at a concentration of 1 μg glycan/ml seems to be a potent inducer of ROS and IL-10 and to a lesser extent of IFN-γ and IL-12. Cytokines and ROS production was decreased in a dose-dependent manner as the concentration of PPG was increased to 100 μg glycan/ml.     

Impact of protein kinase CK2 on inhibitor of apoptosis proteins in prostate cancer cells

We have previously demonstrated that protein kinase CK2 is a potent suppressor of apoptosis in cells subjected to diverse mediators of apoptosis. The process of apoptosis involves a complex series of molecules localized in various cellular compartments. Among the various proteins that modulate apoptotic activity are inhibitors of apoptosis proteins (IAPs) which are elevated in cancers and have been proposed to block caspase activity. We have examined the impact of CK2 signal on these proteins in prostate cancer cells. Cellular IAPs demonstrate distinct localization and responsiveness to altered CK2 expression or activity in the cytoplasmic and nuclear matrix fractions. Modulation of cellular CK2 by various approaches impacts on cellular IAPs such that inhibition or downregulation of CK2 results in reduction in these proteins. Further, IAPs are also reduced when cells are treated with sub-optimal concentrations of chemical inhibitors of CK2 combined with low or sub-optimal levels of apoptosis-inducing agents (such as etoposide) suggesting that downregulation of CK2 sensitizes cells to induction of apoptosis which may be related to attenuation of IAPs. Decreased IAP protein levels in response to apoptotic agents such as TNFalpha or TRAIL were potently blocked upon forced overexpression of CK2 in cells. Together, our results suggest that one of the modes of CK2-mediated modulation of apoptotic activity is via its impact on cellular IAPs.     

Transitions between distinct compaction regimes in complexes of multivalent cationic lipids and DNA

Cationic lipids (CLs) have found widespread use as nonviral gene carriers (vectors), including applications in clinical trials of gene therapy. However, their observed transfection efficiencies (TEs) are inferior to those of viral vectors, providing a strong incentive for a detailed understanding of CL-DNA complex behavior. In recent systematic studies employing monovalent as well as newly synthesized multivalent lipids (MVLs), the membrane cationic charge density has been identified as a key parameter governing the TE of lamellar CL-DNA complexes. In this work, we use x-ray scattering and molecular simulations to investigate the structural properties of complexes containing MVLs. At low mole fraction of neutral lipids (NLs), Φ_NL, the complexes show dramatic DNA compaction, down to essentially close-packed DNA arrays with a DNA interaxial spacing d_DNA = 25 Å. A gradual increase in Φ_NL does not lead to a continuous increase in d_DNA as observed for DNA complexes of monovalent CLs. Instead, distinct spacing regimes exist, with sharp transitions between the regimes. Three packing states have been identified: 1), close packed, 2), condensed, but not close packed, with d_DNA = 27-28 Å, and 3), an expanded state, where d_DNA increases gradually with Φ_NL. Based on our experimental and computational results, we conclude that the DNA condensation is mediated by the multivalent cationic lipids, which assemble between the negatively charged DNA rods. Quite remarkably, the computational results show that the less tightly packed structure in regime 2 is thermodynamically more stable than the close packed structure in regime 1. Accordingly, the constant DNA spacing observed in regime 2 is attributed to lateral phase coexistence between this stable CL-DNA complex and neutral membranes. This finding may explain the reduced TE measured for such complexes: transfection involves endosomal escape and disassembly of the complex, and these processes are inhibited by the high thermodynamic stability. Our results, which demonstrate the existence of an inverse correlation between the stability and transfection activity of lamellar CL-DNA complexes are, therefore, consistent with a recently proposed model of cellular entry.     

The Toxoplasma gondii type-II NADH dehydrogenase TgNDH2-I is inhibited by 1-hydroxy-2-alkyl-4(1H)quinolones

The apicomplexan parasite Toxoplasma gondii does not possess complex I of the mitochondrial respiratory chain, but has two genes encoding rotenone-insensitive, non-proton pumping type-II NADH dehydrogenases (NDH2s). The absence of such "alternative" NADH dehydrogenases in the human host defines these enzymes as potential drug targets. TgNDH2-I and TgNDH2-II are constitutively expressed in tachyzoites and bradyzoites and are localized to the mitochondrion as shown by epitope tagging. Functional expression of TgNDH2-I in the yeast Yarrowia lipolytica as an internal enzyme, with the active site facing the mitochondrial matrix, permitted growth in the presence of the complex I inhibitor DQA. Bisubstrate kinetics of TgNDH2-I measured within Y. lipolytica mitochondrial membrane preparations were in accordance with a ping-pong mechanism. Using inhibition kinetics we demonstrate here that 1-hydroxy-2-alkyl-4(1)quinolones with long alkyl chains of C(12) (HDQ) and C(14) are high affinity inhibitors for TgNDH2-I, while compounds with shorter side chains (C(5) and C(6)) displayed significantly higher IC(50) values. The efficiency of the various quinolone derivatives to inhibit TgNDH2-I enzyme activity mirrors their inhibitory potency in vivo, suggesting that a long acyl site chain is critical for the inhibitory potential of these compounds.     

Phenolic constituents and genetic profile of Hypericum perforatum L. from India

The content of hypericins (hypericin and pseudohypericin), hyperforin, and flavonoids (rutin, hyperoside, quercitrin, and quercetin) and genetic profiles of eight accessions of Hypericum perforatum L., collected from different locations in India, have been determined. The secondary metabolite content was determined using a highly selective LC/MS/MS method. Pearson and Spearman's correlation coefficient were used to investigate the relationships between the secondary metabolites and a significant positive correlation was found between hypericin and pseudohypericin contents. Genetic profiling was undertaken using the random amplification of polymorphic DNA (RAPD) and single sequence repeat (SSR) methods. Among the 49 random primers used for the initial screening, only nine yielded polymorphic RAPD profiles. The SSR analysis shows that seven out of the 11 primers were polymorphic. There exists only a partial correlation between the chemical content and genetic profiling data among the accessions under study.