Structure of the discoidin domain receptor 1 extracellular region bound to an inhibitory Fab fragment reveals features important for signaling

The discoidin domain receptors, DDR1 and DDR2, are constitutively dimeric receptor tyrosine kinases that are activated by triple-helical collagen. Aberrant DDR signaling contributes to several human pathologies, including many cancers. We have generated monoclonal antibodies (mAbs) that inhibit DDR1 signaling without interfering with collagen binding. The crystal structure of the monomeric DDR1 extracellular region bound to the Fab fragment of mAb 3E3 reveals that the collagen-binding discoidin (DS) domain is tightly associated with the following DS-like domain, which contains the epitopes of all mAbs. A conserved surface patch in the DS domain outside the collagen-binding site is shown to be required for signaling. Thus, the active conformation of the DDR1 dimer involves collagen-induced contacts between the DS domains, in addition to the previously identified association of transmembrane helices. The mAbs likely inhibit signaling by sterically blocking the extracellular association of DDR1 subunits.     

Effects of N-[N-(3, 5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT) on cell proliferation and apoptosis in Ishikawa endometrial cancer cells

Endometrial cancer is one of the most common gynecological malignancies in Japan, where the disease shows an increasing morbidity. However, surgical therapy remains the treatment of choice for endometrial cancers that tend to be insensitive to radiation therapy and chemotherapy. Therefore, novel therapeutic strategies are required. The Notch signaling pathway regulates embryogenesis and cellular development, but deregulated Notch signaling may contribute to tumorigenesis in several cancers. Moreover, γ-secretase inhibitors have been shown to be potent inhibitors of the Notch signaling pathway; they suppress cellular proliferation and induce apoptosis in several cancer cells. In the present study, we investigated the effect of N-[N-(3, 5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT, γ-secretase inhibitor) on the cell proliferation and apoptosis in Ishikawa endometrial cancer cells. Real-time PCR detected mRNA derived from NOTCH1 and HES1, which are target genes of the Notch signaling pathway, in Ishikawa endometrial cancer cells. After blocking Notch signaling, cellular proliferation decreased, accompanied by increased expression of p21 mRNA and decreased expression of the cyclin A protein. Furthermore, blockade of Notch signaling induced apoptosis. These results suggest that the Notch signaling pathway may be involved in cell proliferation through cell cycle regulation and apoptosis in Ishikawa endometrial cancer cells. Inhibition of the Notch signaling pathway by γ-secretase inhibitors is expected to be a potential target of novel therapeutic strategies for endometrial cancer.     

Expression and one-step purification of recombinant proteins using an alternative episomal vector for the expression of N-tagged heterologous proteins in Pichia pastoris

Here we report the construction of an alternative episomal vector, pBGP3, which allows the expression of heterologous proteins with N-terminal hexahistidine and myc-epitope tags in Pichia pastoris. To test the usefulness of pBGP3, four cellulases from termites were expressed. Production was confirmed by activity assays and Western blot using anti-c-Myc antibody. Purification was performed by single-step Ni(2+)-affinity chromatography, which confirmed the efficiency of pBGP3.     

Lectin-mediated resistance impairs plant virus infection at the cellular level

Plants possess a multilayered defense response, known as plant innate immunity, to infection by a wide variety of pathogens. Lectins, sugar binding proteins, play essential roles in the innate immunity of animal cells, but the role of lectins in plant defense is not clear. This study analyzed the resistance of certain Arabidopsis thaliana ecotypes to a potexvirus, plantago asiatica mosaic virus (PlAMV). Map-based positional cloning revealed that the lectin gene JACALIN-TYPE LECTIN REQUIRED FOR POTEXVIRUS RESISTANCE1 (JAX1) is responsible for the resistance. JAX1-mediated resistance did not show the properties of conventional resistance (R) protein-mediated resistance and was independent of plant defense hormone signaling. Heterologous expression of JAX1 in Nicotiana benthamiana showed that JAX1 interferes with infection by other tested potexviruses but not with plant viruses from different genera, indicating the broad but specific resistance to potexviruses conferred by JAX1. In contrast with the lectin gene RESTRICTED TEV MOVEMENT1, which inhibits the systemic movement of potyviruses, which are distantly related to potexviruses, JAX1 impairs the accumulation of PlAMV RNA at the cellular level. The existence of lectin genes that show a variety of levels of virus resistance, their targets, and their properties, which are distinct from those of known R genes, suggests the generality of lectin-mediated resistance in plant innate immunity.     

Impaired IL-17 signaling pathway contributes to the increased collagen expression in scleroderma fibroblasts

Among IL-17 families, IL-17A and IL-17F share amino acid sequence similarity and bind to IL-17R type A. IL-17 signaling is implicated in the pathogenesis of various autoimmune diseases, but its role in the regulatory mechanism of extracellular matrix expression and its contribution to the phenotype of systemic sclerosis (SSc) both remain to be elucidated. This study revealed that IL-17A expression was significantly increased in the involved skin and sera of SSc patients, whereas the IL-17F levels did not increase. In contrast, the expression of IL-17R type A in SSc fibroblasts significantly decreased in comparison with that in normal fibroblasts, due to the intrinsic TGF-β1 activation in these cell types. Moreover, IL-17A, not IL-17F, reduced the protein expression of α1(I) collagen and connective tissue growth factor. miR-129-5p, one of the downregulated microRNAs in SSc fibroblasts, increased due to IL-17A and mediated the α1(I) collagen reduction. These results suggest that IL-17A signaling, not IL-17F, has an antifibrogenic effect via the upregulation of miR-129-5p and the downregulation of connective tissue growth factor and α1(I) collagen. IL-17A signaling is suppressed due to the downregulation of the receptor by the intrinsic activation of TGF-β1 in SSc fibroblasts, which may amplify the increased collagen accumulation and fibrosis characteristic of SSc. Increased IL-17A levels in the sera and involved skin of SSc may be due to negative feedback. Clarifying the novel regulatory mechanisms of fibrosis by the cytokine network consisting of TGF-β and IL-17A may lead to a new therapeutic approach for this disease.     

A simple method for in vivo measurement of implant rod three-dimensional geometry during scoliosis surgery

Scoliosis is defined as a spinal pathology characterized as a three-dimensional deformity of the spine combined with vertebral rotation. Treatment for severe scoliosis is achieved when the scoliotic spine is surgically corrected and fixed using implanted rods and screws. Several studies performed biomechanical modeling and corrective forces measurements of scoliosis correction. These studies were able to predict the clinical outcome and measured the corrective forces acting on screws, however, they were not able to measure the intraoperative three-dimensional geometry of the spinal rod. In effect, the results of biomechanical modeling might not be so realistic and the corrective forces during the surgical correction procedure were intra-operatively difficult to measure. Projective geometry has been shown to be successful in the reconstruction of a three-dimensional structure using a series of images obtained from different views. In this study, we propose a new method to measure the three-dimensional geometry of an implant rod using two cameras. The reconstruction method requires only a few parameters, the included angle θ between the two cameras, the actual length of the rod in mm, and the location of points for curve fitting. The implant rod utilized in spine surgery was used to evaluate the accuracy of the current method. The three-dimensional geometry of the rod was measured from the image obtained by a scanner and compared to the proposed method using two cameras. The mean error in the reconstruction measurements ranged from 0.32 to 0.45 mm. The method presented here demonstrated the possibility of intra-operatively measuring the three-dimensional geometry of spinal rod. The proposed method could be used in surgical procedures to better understand the biomechanics of scoliosis correction through real-time measurement of three-dimensional implant rod geometry in vivo.     

Hazardous components and health effects of atmospheric aerosol particles: reactive oxygen species, soot, polycyclic aromatic compounds and allergenic proteins

This review outlines recent advances in the investigation of the chemical properties, molecular interactions and health effects of hazardous compounds in atmospheric aerosols, in particular reactive oxygen species (ROS), soot, polycyclic aromatic compounds (PACs) and allergenic proteins. Epidemiological studies show correlations between air particulate matter and adverse health effects of air pollution including allergy, asthma, cardiovascular and respiratory diseases, but the causative relations and mechanisms of interaction on the molecular level are still unclear. ROS generated by photochemical and heterogeneous reactions in the atmosphere seem to play a key role in aerosol health effects and provide a direct link between atmospheric and physiological multiphase processes. Soot and PACs can trigger formation of ROS in vivo, leading to inflammation and cellular damage. PACs as well as allergenic proteins are efficiently oxygenated and nitrated upon exposure to ozone and nitrogen dioxide, which leads to an enhancement of their toxicity and allergenicity.     

Suppressors: Determinants of Specificity Produced by Plant Pathogens

Plant pathogens secrete suppressors that delay or prevent thehost defense responses, with resultant conditioning of hostcells such that they become susceptible even to avirulent ornon-pathogenic microorganisms. Suppressors have been characterizedas glycoproteins, glycopeptides, peptides and anionic and nonanionicglucans. A suppressor itself is non-toxic to plant cells and,thus, it can be distinguished from host-specific toxins producedby certain pathogens. Suppressors disturb fundamental functionsof host plasma membranes. For example, the suppressor from apea pathogen, Mycosphaerella pinodes, inhibits both the ATPaseactivity and polyphosphoinositide metabolism in pea plasma membranes,causing the temporary suppression of the signal-transductionpathway that leads to the expression of defense genes, whichencode key enzymes in the biosynthetic pathway to phytoalexin.In this review, evidence for the role of suppressors in thedetermination of plant host-parasite specificity is summarized. ³Present address: Plant Pathology Laboratory, School of Agriculture,Nagoya University, Chikusa, Nagoya, 464-01 Japan