An insecticidal GroEL protein with chitin binding activity from Xenorhabdus nematophila

Xenorhabdus nematophila secretes insecticidal proteins to kill its larval prey. We have isolated an approximately 58-kDa GroEL homolog, secreted in the culture medium through outer membrane vesicles. The protein was orally insecticidal to the major crop pest Helicoverpa armigera with an LC50 of approximately 3.6 microg/g diet. For optimal insecticidal activity all three domains of the protein, apical, intermediate, and equatorial, were necessary. The apical domain alone was able to bind to the larval gut membranes and manifest low level insecticidal activity. At equimolar concentrations, the apical domain contained approximately one-third and the apical-intermediate domain approximately one-half bioactivity of that of the full-length protein. Interaction of the protein with the larval gut membrane was specifically inhibited by N-acetylglucosamine and chito-oligosaccharides. Treatment of the larval gut membranes with chitinase abolished protein binding. Based on the three-dimensional structural model, mutational analysis demonstrated that surface-exposed residues Thr-347 and Ser-356 in the apical domain were crucial for both binding to the gut epithelium and insecticidal activity. Double mutant T347A,S356A was 80% less toxic (p < 0.001) than the wild type protein. The GroEL homolog showed alpha-chitin binding activity with Kd approximately 0.64 microm and Bmax approximately 4.68 micromol/g chitin. The variation in chitin binding activity of the mutant proteins was in good agreement with membrane binding characteristics and insecticidal activity. The less toxic double mutant XnGroEL showed an approximately 8-fold increase of Kd in chitin binding assay. Our results demonstrate that X. nematophila secretes an insecticidal GroEL protein with chitin binding activity.     

Fluid shear stress synergizes with insulin-like growth factor-I (IGF-I) on osteoblast proliferation through integrin-dependent activation of IGF-I mitogenic signaling pathway

This study tested the hypothesis that shear stress interacts with the insulin-like growth factor-I (IGF-I) pathway to stimulate osteoblast proliferation. Human TE85 osteosarcoma cells were subjected to a steady shear stress of 20 dynes/cm(2) for 30 min followed by 24-h incubation with IGF-I (0-50 ng/ml). IGF-I increased proliferation dose-dependently (1.5-2.5-fold). Shear stress alone increased proliferation by 70%. The combination of shear stress and IGF-I stimulated proliferation (3.5- to 5.5-fold) much greater than the additive effects of each treatment alone, indicating a synergistic interaction. IGF-I dose-dependently increased the phosphorylation level of Erk1/2 by 1.2-5.3-fold and that of IGF-I receptor (IGF-IR) by 2-4-fold. Shear stress alone increased Erk1/2 and IGF-IR phosphorylation by 2-fold each. The combination treatment also resulted in synergistic enhancements in both Erk1/2 and IGF-IR phosphorylation (up to 12- and 8-fold, respectively). Shear stress altered IGF-IR binding only slightly, suggesting that the synergy occurred primarily at the post-ligand binding level. Recent studies have implicated a role for integrin in the regulation of IGF-IR phosphorylation and IGF-I signaling. To test whether the synergy involves integrin-dependent mechanisms, the effect of echistatin (a disintegrin) on proliferation in response to shear stress +/- IGF-I was measured. Echistatin reduced basal proliferation by approximately 60% and the shear stress-induced mitogenic response by approximately 20%. It completely abolished the mitogenic effect of IGF-I and that of the combination treatment. Shear stress also significantly reduced the amounts of co-immunoprecipitated SHP-2 and -1 with IGF-IR, suggesting that the synergy between shear stress and IGF-I in osteoblast proliferation involves integrin-dependent recruitment of SHP-2 and -1 away from IGF-IR.     

Protective effect of CardiPro against doxorubicin-induced cardiotoxicity in mice

The effect of CardiPro, a polyherbal formulation, with an antioxidant property, has been studied on doxorubicin (DXR)-induced cardiotoxicity in mice. CardiPro (150 mg/kg b.w., twice daily was administered orally for 7 weeks along with four equal injections (each containing 4.0 mg/kg b.w., DXR) intraperitoneally, once weekly (cumulative dose 16 mg/kg). After a 3-week post DXR treatment period, cardiotoxicity was assessed by noting mortality, volume of ascites, liver congestion, changes in heart weight, myocardial lipid peroxidation, antioxidant enzymes and histology of heart. DXR-treated animals showed higher mortality (50%) and more ascites. Myocardial SOD and glutathione peroxidase activity were decreased and lipid peroxidation was increased. Histology of heart of DXR-treated animals showed loss of myofibrils and focal cytoplasmic vacuolization. CardiPro significantly protected the mice from DXR-induced cardiotoxic effects as evidenced by lower mortality (25%), less ascites, myocardial lipid peroxidation, normalization of antioxidant enzymes and minimal damage to the heart histologically. Our data confirm the earlier reports that DXR cardiotoxicity is associated with the free radical-induced tissue damage. Administration of CardiPro, with an antioxidant property, protected the DXR-induced cardiotoxicity in mice.     

A New DNA‐Intercalative Cytotoxic Allylic Xanthone from Swertia corymbosa

Phytochemical investigation of the CHCl₃ fraction of Swertia corymbosa resulted in the isolation of a new 3‐allyl‐2,8‐dihydroxy‐1,6‐dimethoxy‐9H‐xanthen‐9‐one ( 1 ), along with four known xanthones, gentiacaulein ( 3 ), norswertianin ( 4 ), 1,3,6,8‐tetrahydroxyxanthone ( 5 ), and 1,3‐dihydroxyxanthone ( 6 ). Structure of compound 1 was elucidated with the aid of IR, UV, NMR, and MS data, and chemical transformation via new allyloxy xanthone derivative ( 2 ). Compounds 1 – 6 exhibited various levels of antioxidant and anti‐α‐glucosidase activities. Absorption and fluorescence spectroscopic studies on 1 – 6 indicated that these compounds could interact with calf thymus DNA (CT‐DNA) through intercalation and with bovine serum albumin (BSA) in a static quenching process. Compound 1 was found to be significantly cytotoxic against human cancer cell lines HeLa, HCT116, and AGS, and weakly active against normal NIH 3T3 cell line.     

Biomarkers in cancer screening,research and detection: present and future: a review

Biomarkers provide a powerful and dynamic approach to understanding the spectrum of malignancies with applications in observational and analytic epidemiology, randomized clinical trials, screening, diagnosis and prognosis. Defined as alterations in the constituents of tissues or body fluids, these markers offer a means for homogeneous classification of a disease and risk factor, and they can extend one's basic information about the underlying pathogenesis of disease. The goals in cancer research include finding biomarkers that can be used for the early detection of cancers, design individual therapies, and to identify underlying processes involved in the disease. Because so many myriad processes are involved in the diseased states, the goal is similar to ‘finding a needle in a haystack’. However, the development of many -omic technologies, such as genomics and proteomics, has allowed us to monitor a large number of key cellular pathways simultaneously. This has enabled the identification of biomarkers and signalling molecules associated with cell growth, cell death and cellular metabolism. These are also facilitating in monitoring the functional disturbance, molecular and cellular damage, and damage response. This brief review describes the development of biomarkers in cancer research and detection with emphasis on different proteomic tools for the identification and discovery of new biomarkers, different clinical assays to detect various biomarkers in different specimens, role of biomarkers in cancer screening and last but not the least, the challenges in this direction of cancer research.