Real-time observation of strand exchange reaction with high spatiotemporal resolution

RecA binds to single-stranded (ss) DNA to form?a helical filament that catalyzes strand exchange with a homologous double-stranded (ds) DNA. The study of strand exchange in ensemble assays is limited by the diffusion limited homology search process, which masks the subsequent strand exchange reaction. We developed a single-molecule fluorescence assay with a few base-pair and millisecond resolution that can separate initial docking from the subsequent propagation of joint molecule formation. Our data suggest that propagation occurs in 3?bp increments with destabilization of the incoming dsDNA and concomitant pairing with the reference ssDNA. Unexpectedly, we discovered the formation of?a dynamic complex between RecA and the displaced DNA that remains bound transiently after joint molecule formation. This finding could have important implications for the irreversibility of strand exchange. Our model for strand exchange links structural models of RecA to its catalytic function.     

Joint Modelling of Repeated Measures and Survival Time Data

In many clinical trials both repeated measures data and event history data are simultaneously observed from the same subject. These two types of responses are usually correlated, because they are from the same subject. In this article, we propose a joint model for the combined analysis of repeated measures data and event history data in the framework of hierarchical generalized linear models. The correlation between repeated measures and event time is modelled by introducing a shared random effect. The model parameters are estimated using the hierarchical‐likelihood approach. The proposed model is illustrated using a real data set for the renal transplant patients.     

Monitoring the antiviral effect of alpha interferon on individual cells

An infectious hepatitis C virus (HCV) cDNA clone (JFH1) was generated recently. However, quantitative analysis of HCV infection and observation of infected cells have proved to be difficult because the yield of HCV in cell cultures is fairly low. We generated infectious HCV clones containing the convenient reporters green fluorescent protein (GFP) and Renilla luciferase in the NS5a-coding sequence. The new viruses responded to antiviral agents in a dose-dependent manner. Responses of individual cells containing HCV to alpha interferon (IFN-alpha) were monitored using GFP-tagged HCV and time-lapse confocal microscopy. Marked variations in the response to IFN-alpha were observed among HCV-containing cells.     

Role of the cyclic lipopeptide massetolide A in biological control of Phytophthora infestans and in colonization of tomato plants by Pseudomonas fluorescens

Pseudomonas strains have shown promising results in biological control of late blight caused by Phytophthora infestans. However, the mechanism(s) and metabolites involved are in many cases poorly understood. Here, the role of the cyclic lipopeptide massetolide A of Pseudomonas fluorescens SS101 in biocontrol of tomato late blight was examined. Pseudomonas fluorescens SS101 was effective in preventing infection of tomato (Lycopersicon esculentum) leaves by P. infestans and significantly reduced the expansion of existing late blight lesions. Massetolide A was an important component of the activity of P. fluorescens SS101, since the massA-mutant was significantly less effective in biocontrol, and purified massetolide A provided significant control of P. infestans, both locally and systemically via induced resistance. Assays with nahG transgenic plants indicated that the systemic resistance response induced by SS101 or massetolide A was independent of salicylic acid signalling. Strain SS101 colonized the roots of tomato seedlings significantly better than its massA-mutant, indicating that massetolide A was an important trait in plant colonization. This study shows that the cyclic lipopeptide surfactant massetolide A is a metabolite with versatile functions in the ecology of P. fluorescens SS101 and in interactions with tomato plants and the late blight pathogen P. infestans.     

RGD-peptide presents anti-adhesive effect, but not direct pro-apoptotic effect on endothelial progenitor cells

Circulating endothelial progenitor cells (EPCs) contribute to neovascularization in tumor or ischemic tissues by multi-step events, including adhesion, migration, chemoattraction, and differentiation to endothelial cells. Anti-angiogenic RGD-peptides have been shown to directly induce apoptosis in human umbilical vein endothelial cells (HUVECs) and T cells. Here, we examined the effects of RGD-peptides on EPCs in terms of adhesive differentiation and apoptosis. When mononuclear cells (MNCs) isolated from human cord blood were cultured on fibronectin-coated plates for 7 days, RGD-peptide treatment decreased dose-dependently the number of adherent cells double positive for DiI-ac-LDL uptake and UEA-1 binding. The cells treated with RGD peptide were also stained less strongly by vWF or KDR antibody by immunofluorescence staining. Immobilization of the RGD-peptide promoted cell adhesion, but resulted in a deficiency in the development of ability of ac-LDL uptake and UEA-1 binding, showing an antagonistic effect. Accordingly, ex vivo-cultivated EPCs expressed integrin alpha5, alphav, beta1, beta3, and beta5, and antibodies to integrins alpha5, alphav, and beta1 decreased the number of adherent cells. However, viability of total MNCs containing early EPCs was not affected by RGD-peptide. In addition, neither an increase in apoptotic cell death nor a direct activation of caspase-3 by RGD-peptide was detected in ex vivo-cultivated EPCs, unlike in HUVECs. Interestingly, RGD-peptide rather enhanced Bcl-2 expression in ex vivo-cultivated EPCs and the EPCs themselves with a high Bcl-2/Bax ratio are comparatively resistant to apoptosis. Therefore, these results suggest that RGD-peptides may inhibit EPC differentiation by anti-adhesive effect, but not by a direct pro-apoptotic effect.     

Outgrowing endothelial progenitor-derived cells display high sensitivity to angiogenesis modulators and delayed senescence

Outgrowing endothelial progenitor-derived cells (EPDCs) originate from a novel hierarchy of endothelial progenitor cells. In this study, EPDCs isolated from human cord blood were examined for phenotype and functional features upon aging. Young or aged EPDCs were similar to human umbilical vein endothelial cells (HUVECs), in exhibiting typical endothelial phenotypes. However, EPDCs were more sensitive to angiogenesis inducers or inhibitors in proliferation and migration. In addition, EPDCs underwent senescence markedly slowly with sustained endothelial NO synthase expression and activation, and their ability to undergo capillary morphogenesis was retained throughout longterm culture. Thus, these results suggest that a homogenous population of EPDCs derived from clonogenic expansion may provide an effective vasculogenesis tool.     

Heat shock protein 27 is associated with irinotecan resistance in human colorectal cancer cells

Heat shock protein (Hsp) in tumor cells has been proposed to enhance their resistance to chemotherapeutic agents. In the present study, we investigated the influence of Hsp expression on the irinotecan resistance of human colorectal cancer cells. Among eight Hsp genes tested in this study, we confirmed that the expression of Hsp27 correlated with irinotecan resistance in colorectal cancer cells. Specific inhibition of Hsp27 expression using an antisense oliogodeoxynucleotide increased the irinotecan sensitivity. On the contrary, an overexpression of Hsp27 decreased the irinotecan sensitivity in colorectal cancer cells. Elevated expression of Hsp27 decreased caspase-3 activity in colorectal cancer cells. The expression level of Hsp27 determined by immunohistochemical analysis correlated with the clinical response to irinotecan in colorectal cancer patients. Hsp27 expression levels of irinotecan-non-responder (mean staining score, 6.28; proportion of high staining score, 64.2%) were significantly higher compared to those of irinotecan-responder (mean staining score, 3.16; proportion of high staining score, 33.3%) (P for t-test=0.045). These findings suggest that Hsp27 is involved in the irinotecan resistance of colorectal cancer cells possibly by reducing caspase-3 activity.     

NM23-H1 tumor suppressor physically interacts with serine-threonine kinase receptor-associated protein, a transforming growth factor-beta (TGF-beta) receptor-interacting protein, and negatively regulates TGF-beta signaling

NM23-H1 is a member of the NM23/NDP kinase gene family and a putative metastasis suppressor. Previously, a screen for NM23-H1-interacting proteins that could potentially modulate its activity identified serine-threonine kinase receptor-associated protein (STRAP), a transforming growth factor (TGF)-beta receptor-interacting protein. Through the use of cysteine to serine amino acid substitution mutants of NM23-H1 (C4S, C109S, and C145S) and STRAP (C152S, C270S, and C152S/C270S), we demonstrated that the association between these two proteins is dependent on Cys(145) of NM23-H1 and Cys(152) and Cys(270) of STRAP but did not appear to involve Cys(4) and Cys(109) of NM23-H1, suggesting that a disulfide linkage involving Cys(145) of NM23-H1 and Cys(152) or Cys(270) of STRAP mediates complex formation. The interaction was dependent on the presence of dithiothreitol or beta-mercaptoethanol but not H(2)O(2). Ectopic expression of wild-type NM23-H1, but not NM23-H1(C145S), negatively regulated TGF-beta signaling in a dose-dependent manner, enhanced stable association between the TGF-beta receptor and Smad7, and prevented nuclear translocation of Smad3. Similarly, wild-type NM23-H1 inhibited TGF-beta-induced apoptosis and growth inhibition, whereas NM23-H1(C145S) had no effect. Knockdown of NM23-H1 by small interfering RNA stimulated TGF-beta signaling. Coexpression of wild-type STRAP, but not STRAP(C152S/C270S), significantly stimulated NM23-H1-induced growth of HaCaT cells. These results suggest that the direct interaction of NM23-H1 and STRAP is important for the regulation of TGF-beta-dependent biological activity as well as NM23-H1 activity.