Quantitative determination of ortho- and meta-tyrosine as biomarkers of protein oxidative damage in beta-thalassemia

Oxidative stress in thalassemia is caused by secondary iron overload and stems from blood transfusion and increased iron uptake. In this study, we hypothesized that levels of o- and m-tyrosine, products of hydroxyl radical attack on phenylalanine, would be elevated in beta-thalassemia (intermediate). This study represents the first report in which specific markers of protein oxidative damage have been quantified in thalassemia. We used GC/MS to assay o- and m-tyrosine at the femtomole level using only a few microliters of plasma. Levels of both markers were significantly higher in patients with beta-thalassemia than in controls and were positively correlated with serum ferritin, malondialdehyde, superoxide dismutase, glutathione peroxidase and glutathione. We conclude that o- and m-tyrosine are useful biomarkers of oxidative damage to proteins in thalassemia (intermediate) and may also be useful markers in other iron overload diseases. Positive correlations between o- and m-tyrosine levels and malondialdehyde as well as antioxidants such as superoxide dismutase, glutathione peroxidase and glutathione, are indicative of the broad impact of oxidative stress on blood plasma in thalassemia, with up-regulation of antioxidant proteins probably reflecting a homeostatic response to these increased stress levels.     

Differential Impact of Monsoon and Large Amplitude Internal Waves on Coral Reef Development in the Andaman Sea

The Andaman Sea and other macrotidal semi-enclosed tropical seas feature large amplitude internal waves (LAIW). Although LAIW induce strong fluctuations i.e. of temperature, pH, and nutrients, their influence on reef development is so far unknown. A better-known source of disturbance is the monsoon affecting corals due to turbulent mixing and sedimentation. Because in the Andaman Sea both, LAIW and monsoon, act from the same westerly direction their relative contribution to reef development is difficult to discern. Here, we explore the framework development in a number of offshore island locations subjected to differential LAIW- and SW-monsoon impact to address this open question. Cumulative negative temperature anomalies – a proxy for LAIW impact – explained a higher percentage of the variability in coral reef framework height, than sedimentation rates which resulted mainly from the monsoon. Temperature anomalies and sediment grain size provided the best correlation with framework height suggesting that so far neglected subsurface processes (LAIW) play a significant role in shaping coral reefs.     

Insight into the structural requirements of aminopyrimidine derivatives for good potency against both purified enzyme and whole cells of M. tuberculosis: combination of HQSAR,CoMSIA, and MD simulation studies

The Mycobacterium tuberculosis protein kinase B (PknB) is critical for growth and survival of M. tuberculosis within the host. The series of aminopyrimidine derivatives show impressive activity against PknB (IC₅₀ < .5 μM). However, most of them show weak or no cellular activity against M. tuberculosis (MIC > 63 μM). Consequently, the key structural features related to activity against of both PknB and M. tuberculosis need to be investigated. Here, two- and three-dimensional quantitative structure–activity relationship (2D and 3D QSAR) analyses combined with molecular dynamics (MD) simulations were employed with the aim to evaluate these key structural features of aminopyrimidine derivatives. Hologram quantitative structure–activity relationship (HQSAR) and CoMSIA models constructed from IC₅₀ and MIC values of aminopyrimidine compounds could establish the structural requirements for better activity against of both PknB and M. tuberculosis. The NH linker and the R₁ substituent of the template compound are not only crucial for the biological activity against PknB but also for the biological activity against M. tuberculosis. Moreover, the results obtained from MD simulations show that these moieties are the key fragments for binding of aminopyrimidine compounds in PknB. The combination of QSAR analysis and MD simulations helps us to provide a structural concept that could guide future design of PknB inhibitors with improved potency against both the purified enzyme and whole M. tuberculosis cells.     

Differential binding properties of B7-H1 and B7-DC to programmed death-1

Programmed death-1 (PD-1) is a negative regulatory receptor expressed on activated T and B cells. Two ligands for PD-1, B7-H1 (PD-L1) and B7-DC (PD-L2), have been identified, but their binding properties have not been characterized yet. In this study, we generated soluble Ig fusion proteins of these molecules and examined the kinetics and relative affinities of the interactions between B7-H1 or B7-DC and PD-1 by flow cytometry and surface plasmon resonance. The interaction of B7-DC/PD-1 exhibited a 2-6-fold higher affinity and had different association/dissociation kinetics compared with the interaction of B7-H1/PD-1. Our results suggest that the differential binding properties of B7-H1 and B7-DC may be responsible for differential contributions of these two PD-1 ligands to immune responses.     

Full length and protease domain activity of chikungunya virus nsP2 differ from other alphavirus nsP2 proteases in recognition of small peptide substrates

Alphavirus nsP2 proteins are multifunctional and essential for viral replication. The protease role of nsP2 is critical for virus replication as only the virus protease activity is used for processing of the viral non-structural polypeptide. Chikungunya virus is an emerging disease problem that is becoming a world-wide health issue. We have generated purified recombinant chikungunya virus nsP2 proteins, both full length and a truncated protease domain from the C-terminus of the nsP2 protein. Enzyme characterization shows that the protease domain alone has different properties compared with the full length nsP2 protease. We also show chikungunya nsP2 protease possesses different substrate specificity to the canonical alphavirus nsP2 polyprotein cleavage specificity. Moreover, the chikungunya nsP2 also appears to differ from other alphavirus nsP2 in its distinctive ability to recognize small peptide substrates.     

Inactivation of <Emphasis Type="Italic">Burkholderia pseudomallei</Emphasis><Emphasis Type="Italic">bsaQ</Emphasis> results in decreased invasion efficiency and delayed escape of bacteria from endocytic vesicles

Burkholderia pseudomallei, an infectious Gram-negative bacterium, is the causative pathogen of melioidosis. In the present study, a B. pseudomallei strain with mutation in the bsaQ gene, encoding a structural component of the type III secretion system (T3SS), was constructed. This bsaQ mutation caused a marked decrease in secretion of BopE effector and BipD translocator proteins into culture supernatant. The B. pseudomallei bsaQ mutant also exhibited decreased efficiencies of plaque formation, invasion into non-phagocytic cells and multinucleated giant cell (MNGC) development in a J774A.1 macrophage cell line. Co-localization of the bacteria and lysosome-associated membrane glycoprotein-1 (LAMP-1) containing vesicles suggested that defects in MNGC formation may result from the delayed ability of this B. pseudomallei mutant to escape from the vacuoles of macrophages. Veerachat Muangsombut and Supaporn Suparak contributed equally to this work.     

Analysis of the Prevalence,Secretion and Function of a Cell Cycle-Inhibiting Factor in the Melioidosis Pathogen Burkholderia pseudomallei

Enteropathogenic and enterohaemorrhagic Escherichia coli express a cell cycle-inhibiting factor (Cif), that is injected into host cells via a Type III secretion system (T3SS) leading to arrest of cell division, delayed apoptosis and cytoskeletal rearrangements. A homologue of Cif has been identified in Burkholderia pseudomallei (CHBP; Cif homologue in B. pseudomallei; BPSS1385), which shares catalytic activity, but its prevalence, secretion and function are ill-defined. Among 43 available B. pseudomallei genome sequences, 33 genomes (76.7%) harbor the gene encoding CHBP. Western blot analysis using antiserum raised to a synthetic CHBP peptide detected CHBP in 46.6% (7/15) of clinical B. pseudomallei isolates from the endemic area. Secretion of CHBP into bacterial culture supernatant could not be detected under conditions where a known effector (BopE) was secreted in a manner dependent on the Bsa T3SS. In contrast, CHBP could be detected in U937 cells infected with B. pseudomallei by immunofluorescence microscopy and Western blotting in a manner dependent on bsaQ. Unlike E. coli Cif, CHBP was localized within the cytoplasm of B. pseudomallei-infected cells. A B. pseudomallei chbP insertion mutant showed a significant reduction in cytotoxicity and plaque formation compared to the wild-type strain that could be restored by plasmid-mediated trans-complementation. However, there was no defect in actin-based motility or multinucleated giant cell formation by the chbP mutant. The data suggest that the level or timing of CHBP secretion differs from a known Bsa-secreted effector and that CHBP is required for selected virulence-associated phenotypes in vitro.