Distinct Distribution of Deoxynivalenol,Nivalenol, and Ergosterol in <Emphasis Type="Italic">Fusarium</Emphasis>-infected Japanese Soft Red Winter Wheat Milling Fractions

The occurrence of mycotoxins in small grain cereals and their retention in final products are serious concerns for food safety. Previously, we investigated the fate of deoxynivalenol and nivalenol in a Japanese soft red winter wheat cultivar during milling and we found that deoxynivalenol and/or nivalenol was readily distributed among flours for human consumption. In the present study, we analyzed the ergosterol concentrations in the milling fractions as an index of fungal biomass to elucidate the relationship between deoxynivalenol/nivalenol accumulation and fungal invasion into the grain, after the in-house validation of an analytical method for quantifying ergosterol in the resulting milling fractions (patent flour, low-grade flour, bran, and shorts). Using three samples with different levels of deoxynivalenol and/or nivalenol contamination, the contents of deoxynivalenol/nivalenol and ergosterol in the resulting milling fractions were evaluated. The concentration of ergosterol was always lowest in patent flour and highest in bran or shorts, indicating that most of the fungi is retained in the outer layers of grain (bran and shorts) even in highly contaminated grain. On the other hand, the concentrations of deoxynivalenol and nivalenol were similar in the low-grade and patent flours and only slightly lower than in the medium-level and high-level contaminated grains. Moreover, the percentage distribution of ergosterol was higher in bran than in other fractions in all cases, which differed from that of deoxynivalenol/nivalenol. This result indicates the diffusion of deoxynivalenol/nivalenol inside the grain that is independent of fungal invasion.     

Quantitative Proteomic Profiling Identifies DPYSL3 as Pancreatic Ductal Adenocarcinoma-Associated Molecule That Regulates Cell Adhesion and Migration by Stabilization of Focal Adhesion Complex

Elucidation of how pancreatic cancer cells give rise to distant metastasis is urgently needed in order to provide not only a better understanding of the underlying molecular mechanisms, but also to identify novel targets for greatly improved molecular diagnosis and therapeutic intervention. We employed combined proteomic technologies including mass spectrometry and isobaric tags for relative and absolute quantification peptide tagging to analyze protein profiles of surgically resected human pancreatic ductal adenocarcinoma tissues. We identified a protein, dihydropyrimidinase-like 3, as highly expressed in human pancreatic ductal adenocarcinoma tissues as well as pancreatic cancer cell lines. Characterization of the roles of dihydropyrimidinase-like 3 in relation to cancer cell adhesion and migration in vitro, and metastasis in vivo was performed using a series of functional analyses, including those employing multiple reaction monitoring proteomic analysis. Furthermore, dihydropyrimidinase-like 3 was found to interact with Ezrin, which has important roles in cell adhesion, motility, and invasion, while that interaction promoted stabilization of an adhesion complex consisting of Ezrin, c-Src, focal adhesion kinase, and Talin1. We also found that exogenous expression of dihydropyrimidinase-like 3 induced activating phosphorylation of Ezrin and c-Src, leading to up-regulation of the signaling pathway. Taken together, the present results indicate successful application of combined proteomic approaches to identify a novel key player, dihydropyrimidinase-like 3, in pancreatic ductal adenocarcinoma tumorigenesis, which may serve as an important biomarker and/or drug target to improve therapeutic strategies.     

Real-time detection of DNA hybridization on microarray using a CCD-based imaging system equipped with a rotated microlens array disk

This work describes a novel charge-coupled device (CCD)-based imaging system (MB Biochip Reader?) for real-time detection of DNA hybridization to DNA microarrays. The MB Biochip Reader? consisted of a laser light source (532 nm), a microlens array for generation of a multi-beam laser, and a CCD for 2-D signal imaging. The MB Biochip Reader? with a rotated microlens array, allowed large-field imaging (6.2 mm × 7.6 mm with 6.45 μm resolution) with fast time-resolution at 0.2 s without speckle noise. Furthermore, real-time detection of DNA hybridization, which is sufficient to obtain accurate data from tens of thousands of array element per field, was successfully performed without the need for laser scanning. The performance of the MB Biochip Reader? for DNA microarray imaging was similar to the commercially available photomultiplier tube (PMT)-based microarray scanner, ScanArray Lite. The system potentially could be applied toward real-time analysis in many other fluorescent techniques in addition to real-time DNA microarray analysis.     

Structural development of non-secosteroidal vitamin D receptor (VDR) ligands without any asymmetric carbon

Non-secosteroidal VDR ligands without any assymmetric carbon were designed and synthesized based on the structure of the previously reported non-secosteroidal VDR agonist LG190178. The VDR-agonistic activity of all synthesized compounds was evaluated, and 7b emerged as a potent agonist activity with an EC₅₀ value of 9.26?nM. Moreover, a docking simulation analysis was also performed to determine the binding mode of 7b with VDR-LBD.     

Deep Vascular Imaging in Wounds by Two-Photon Fluorescence Microscopy

Deep imaging within tissue (over 300 μm) at micrometer resolution has become possible with the advent of two-photon fluorescence microscopy (2PFM). The advantages of 2PFM have been used to interrogate endogenous and exogenous fluorophores in the skin. Herein, we employed the integrin (cell-adhesion proteins expressed by invading angiogenic blood vessels) targeting characteristics of a two-photon absorbing fluorescent probe to image new vasculature and fibroblasts up to ≈ 1600 μm within wound (neodermis)/granulation tissue in lesions made on the skin of mice. Reconstruction revealed three dimensional (3D) architecture of the vascular plexus forming at the regenerating wound tissue and the presence of a fibroblast bed surrounding the capillaries. Biologically crucial events, such as angiogenesis for wound healing, may be illustrated and analyzed in 3D on the whole organ level, providing novel tools for biomedical applications.     

Evaluation of two-dimensional electronic portal imaging device using integrated images during volumetric modulated arc therapy for prostate cancer

BackgroundThe aim of the study was to evaluate analysis criteria for the identification of the presence of rectal gas during volumetric modulated arc therapy (VMAT) for prostate cancer patients by using electronic portal imaging device (EPID)-based in vivo dosimetry (IVD).Materials and methodsAll measurements were performed by determining the cumulative EPID images in an integrated acquisition mode and analyzed using PerFRACTION commercial software. Systematic setup errors were simulated by moving the anthropomorphic phantom in each translational and rotational direction. The inhomogeneity regions were also simulated by the I’mRT phantom attached to the Quasar phantom. The presence of small and large air cavities (12 and 48 cm³) was controlled by moving the Quasar phantom in several timings during VMAT. Sixteen prostate cancer patients received EPID-based IVD during VMAT.ResultsIn the phantom study, no systematic setup error was detected in the range that can happen in clinical (< 5-mm and < 3 degree). The pass rate of 2% dose difference (DD2%) in small and large air cavities was 98.74% and 79.05%, respectively, in the appearance of the air cavity after irradiation three quarter times. In the clinical study, some fractions caused a sharp decline in the DD2% pass rate. The proportion for DD2% < 90% was 13.4% of all fractions. Rectal gas was confirmed in 11.0% of fractions by acquiring kilo-voltage X-ray images after the treatment.ConclusionsOur results suggest that analysis criteria of 2% dose difference in EPID-based IVD was a suitable method for identification of rectal gas during VMAT for prostate cancer patients.     

Cyclin E2 is required for embryogenesis in Xenopus laevis

In mammalian cells, E-type cyclins (E1 and E2) are generally believed to be required for entry into S phase. However, in mice, cyclin E is largely dispensable for normal embryogenesis. Moreover, Drosophila cyclin E plays a critical role in cell fate determination in neural lineages independently of proliferation. Thus, the functions of cyclin E, particularly during early development, remain elusive. Here, we investigated the requirement for E-type cyclins during Xenopus embryogenesis. Although cyclin E1 has been reported as a maternal cyclin, inhibition of its translation in the embryo caused no serious defects. We isolated a Xenopus homologue of human cyclin E2, which was zygotically expressed. Sufficient inhibition of its expression led to death at late gastrula, while partial inhibition allowed survival. These observations indicate distinct roles for Xenopus cyclins E1 and E2, and an absolute requirement of cyclin E2 for Xenopus embryogenesis.     

Crystal structures of beta-amylase from Bacillus cereus var mycoides in complexes with substrate analogs and affinity-labeling reagents

The crystal structures of beta-amylase from Bacillus cereus var. mycoides in complexes with five inhibitors were solved. The inhibitors used were three substrate analogs, i.e. glucose, maltose (product), and a synthesized compound, O-alpha-D-glucopyranosyl-(1-->4)-O-alpha-D-glucopyranosyl-(1-->4)-D-xylopyranose (GGX), and two affinity-labeling reagents with an epoxy alkyl group at the reducing end of glucose. For all inhibitors, one molecule was bound at the active site cleft and the non-reducing end glucose of the four inhibitors except GGX was located at subsite 1, accompanied by a large conformational change of the flexible loop (residues 93-97), which covered the bound inhibitor. In addition, another molecule of maltose or GGX was bound about 30 A away from the active site. A large movement of residues 330 and 331 around subsite 3 was also observed upon the binding of GGX at subsites 3 to 5. Two affinity-labeling reagents, alpha-EPG and alpha-EBG, were covalently bound to a catalytic residue (Glu-172). A substrate recognition mechanism for the beta-amylase was discussed based on the modes of binding of these inhibitors in the active site cleft.     

Two carboxyl-terminal activation regions of Epstein-Barr virus latent membrane protein 1 activate NF-kappaB through distinct signaling pathways in fibroblast cell lines

Latent membrane protein 1 (LMP1), an Epstein-Barr virus transforming protein, is able to activate NF-kappaB through its carboxyl-terminal activation region 1 (CTAR1) and 2 (CTAR2), but the exact role of each domain is not fully understood. Here we show that LMP1 activates NF-kappaB in different NF-kappaB essential modulator (NEMO)-defective cell lines, but not in cells lacking both IkappaB kinase 1 (IKK1) and 2 (IKK2). Mutational studies reveal that CTAR1, but not CTAR2, mediates NEMO-independent NF-kappaB activation and that this process largely depends on IKK1. Retroviral expression of LMP1 mutants in cells lacking either functional NF-kappaB inducing kinase (NIK), NEMO, IKK1, or IKK2 further illustrates distinct signals from the two activation regions of LMP1 for persistent NF-kappaB activation. One originates in CTAR2, operates through the canonical NEMO-dependent pathway, and induces NFKB2 p100 production; the second signal originates in CTAR1, utilizes NIK and IKK1, and induces the processing of p100. Our results thus help clarify how two functional domains of LMP1 persistently activate NF-kappaB through distinct signaling pathways.