ZO-1 is required for protein kinase C gamma-driven disassembly of connexin 43

We have previously reported that protein kinase C gamma (PKC-gamma) is activated by phorbol-12-myristate-13-acetate (TPA) and that this causes PKC-gamma translocation to membranes and phosphorylation of the gap junction protein, connexin 43 (Cx43). This phosphorylation, on S368 of Cx43, causes disassembly of Cx43 out of cell junctional plaques resulting in the inhibition of dye transfer. The purpose of this study is to identify the specific role of zonula occludens protein-1 (ZO-1), a tight junction protein with recently established effects on gap junctions, in this PKC-gamma-driven Cx43 disassembly. For this purpose, ZO-1 levels in lens epithelial cells in culture were decreased by up to 70% using specific siRNA. The down-regulation of ZO-1 caused a stable interaction of PKC-gamma with Cx43 even without normal enzyme activation by TPA. However, after TPA activation of the PKC-gamma, the Cx43 did not disassemble out of plaques even though the PKC-gamma enzyme was activated and the Cx43 was phosphorylated on S368. Confocal microscopy demonstrated that the siRNA treatment caused a loss of ZO-1 from borders of large junctional Cx43 cell-to-cell plaques and resulted in the accumulation of Cx43 aggregates inside of cells. Loss of the specific "plaquetosome" arrangement of large Cx43 plaques surrounded by ZO-1 was accompanied by a complete loss of functional dye transfer. These results suggest that ZO-1 is required for Cx43 control, both for dye transfer, and, for the PKC-gamma-driven disassembly response.     

Poecillastrin D: a new cytotoxin of the chondropsin class from marine sponge Jaspis serpentina

Poecillastrin D (2) was isolated together with poecillastrin C (1) from the deep sea sponge, Japsis serpentina. Its structure was elucidated to be that of a macrolide lactam by spectroscopic methods. These compounds showed potent cytotoxicity against various tumor cell lines.     

Comparative profiling of serum glycoproteome by sequential purification of glycoproteins and 2-nitrobenzensulfenyl (NBS) stable isotope labeling: a new approach for the novel biomarker discovery for cancer

The recent progress in various proteomic technologies allows us to screen serum biomarker including carbohydrate antigens. However, only a limited number of proteins could be detected by current conventional methods such as shotgun proteomics, primarily because of the enormous concentration distribution of serum proteins and peptides. To circumvent this difficulty and isolate potential cancer-specific biomarkers for diagnosis and treatment, we established a new screening system consisting of the sequential steps of (1) immunodepletion of 6 high-abundance proteins, (2) targeted enrichment of glycoproteins by lectin column chromatography, and (3) the quantitative proteome analysis using 12C6- or 13C6-NBS (2-nitrobenzenesulfenyl) stable isotope labeling followed by MALDI-QIT-TOF mass spectrometric analysis. Through this systematic analysis for five serum samples derived from patients with lung adenocarcinoma, we identified as candidate biomarkers 34 serum glycoproteins that revealed significant difference in alpha1,6-fucosylation level between lung cancer and healthy control, clearly demonstrating that the carbohydrate-focused proteomics could allow for the detection of serum components with cancer-specific features. In addition, we developed a more simplified and practical technique, mass spectrometry-based glycan structure analysis and lectin blotting, in order to validate glycan structure of candidate biomarkers that could be applicable in clinical use. Our new glycoproteomic strategy will provide highly sensitive and quantitative profiling of specific glycan structures on multiple proteins, which should be useful for serum biomarker discovery.     

How did bonobos come to range south of the congo river? Reconsideration of the divergence of Pan paniscus from other Pan populations

While investigating the genetic structure in wild bonobos,¹ we realized that the widely accepted scenario positing that the Pleistocene appearance of the Congo River separated the common ancestor of chimpanzees (Pan troglodytes) and bonobos (P. paniscus) into two species is not supported by recent geographical knowledge about the formation of the Congo River. We explored the origin of bonobos using a broader biogeographical perspective by examining local faunas in the central African region. The submarine Congo River sediments and paleotopography of central Africa show that the Congo River has functioned as a geographical barrier for the last 34 million years. This evidence allows us to hypothesize that when the river was first formed, the ancestor of bonobos did not inhabit the current range of the species on the left bank of the Congo River but that, during rare times when the Congo River discharge decreased during the Pleistocene, one or more founder populations of ancestral Pan paniscus crossed the river to its left bank. The proposed scenario for formation of the Congo River and the corridor hypothesis for an ancestral bonobo population is key to understanding the distribution of great apes and their evolution.     

Dissecting electrostatic interactions in Bacillus circulans xylanase through NMR-monitored pH titrations

NMR-monitored pH titration curves of proteins provide a rich source of structural and electrostatic information. Although relatively straightforward to measure, interpreting pH-dependent chemical shift changes to obtain site-specific acid dissociation constants (pK (A) values) is challenging. In order to analyze the biphasic titrations exhibited by the side chain (13)C(γ) nuclei of the nucleophilic Glu78 and general acid/base Glu172 in Bacillus circulans xylanase, we have revisited the formalism for the ionization equilibria of two coupled acidic residues. In general, fitting NMR-monitored pH titration curves for such a system will only yield the two macroscopic pK (A) values that reflect the combined effects of both deprotonation reactions. However, through the use of mutations complemented with ionic strength-dependent measurements, we are able to extract the four microscopic pK (Ai) values governing the branched acid/base equilibria of Glu78 and Glu172 in BcX. These data, confirmed through theoretical calculations, help explain the pH-dependent mechanism of this model GH11 xylanase by demonstrating that the kinetically determined pK (A) values and hence catalytic roles of these two residues result from their electrostatic coupling.     

Increase in Alphaproteobacteria in association with a polychaete,Capitella sp. I,in the organically enriched sediment

We conducted bioremediation experiments on the organically enriched sediment on the sea floor just below a fish farm, introducing artificially mass-cultured colonies of deposit-feeding polychaete, Capitella sp. I. To clarify the association between the Capitella and bacteria on the efficient decomposition of the organic matter in the sediment in the experiments, we tried to identify the bacteria that increased in the microbial community in the sediment with dense patches of the Capitella. The relationship between TOC and quinone content of the sediment as an indicator of the bacterial abundance was not clear, while a significant positive correlation was found between Capitella biomass and quinone content of the sediment. In particular, ubiquinone-10, which is present in members of the class Alphaproteobacteria, increased in the sediment with dense patches of the Capitella. We performed denaturing gradient gel electrophoresis (DGGE) analyses to identify the alphaproteobacterial species in the sediment with dense patches of the worm, using two DGGE fragments obtained from the sediment samples and one fragment from the worm body. The sequences of these DGGE fragments were closely related to the specific members of the Roseobacter clade. In the associated system with the Capitella and the bacteria in the organically enriched sediment, the decomposition of the organic matter may proceed rapidly. It is very likely that the Capitella works as a promoter of bacteria in the organically enriched sediment, and feeds the increased bacteria as one of the main foods, while the bacteria decompose the organic matter in the sediment with the assistance of the Capitella.     

A comprehensive peptidome profiling technology for the identification of early detection biomarkers for lung adenocarcinoma

The mass spectrometry-based peptidomics approaches have proven its usefulness in several areas such as the discovery of physiologically active peptides or biomarker candidates derived from various biological fluids including blood and cerebrospinal fluid. However, to identify biomarkers that are reproducible and clinically applicable, development of a novel technology, which enables rapid, sensitive, and quantitative analysis using hundreds of clinical specimens, has been eagerly awaited. Here we report an integrative peptidomic approach for identification of lung cancer-specific serum peptide biomarkers. It is based on the one-step effective enrichment of peptidome fractions (molecular weight of 1,000-5,000) with size exclusion chromatography in combination with the precise label-free quantification analysis of nano-LC/MS/MS data set using Expressionist proteome server platform. We applied this method to 92 serum samples well-managed with our SOP (standard operating procedure) (30 healthy controls and 62 lung adenocarcinoma patients), and quantitatively assessed the detected 3,537 peptide signals. Among them, 118 peptides showed significantly altered serum levels between the control and lung cancer groups (p<0.01 and fold change >5.0). Subsequently we identified peptide sequences by MS/MS analysis and further assessed the reproducibility of Expressionist-based quantification results and their diagnostic powers by MRM-based relative-quantification analysis for 96 independently prepared serum samples and found that APOA4 273-283, FIBA 5-16, and LBN 306-313 should be clinically useful biomarkers for both early detection and tumor staging of lung cancer. Our peptidome profiling technology can provide simple, high-throughput, and reliable quantification of a large number of clinical samples, which is applicable for diverse peptidome-targeting biomarker discoveries using any types of biological specimens.