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.     

Membrane release and destabilization of Arabidopsis RIN4 following cleavage by Pseudomonas syringae AvrRpt2

The Arabidopsis RIN4 protein mediates interaction between the Pseudomonas syringae type III effector proteins AvrB, AvrRpm1, and AvrRpt2 and the Arabidopsis disease-resistance proteins RPM1 and RPS2. Confocal laser-scanning fluorescence microscopy following particle bombardment of tobacco leaf epidermal cells was used to examine the subcellular localization of fusions between GFP and RIN4 or several of its homologs and to examine the effects of cobombardment with AvrRpt2 or AvrRpml. This study showed that RIN4 was attached to the plasma membrane at its carboxyl terminus and that a carboxyl-terminal CCCFxFxxx prenylation or acylation (typically palmitoylation) motif, or both, was essential for this attachment. RIN4 was cleaved by AvrRpt2 at two PxFGxW motifs, one releasing a large portion of RIN4 from the plasma membrane and both exposing amino-terminal residues that destabilized the carboxyl-terminal cleavage products by targeting them for N-end ubiquitylation and proteasomal degradation. Plasma-membrane localization of RIN4 was not affected by AvrRpml. RIN4 was found to be part of a protein family comprising two full-length homologs and at least 11 short carboxyl-terminal homologs. Representatives of this family, comprising a full-length RIN4 homolog and two short carboxyl-terminal RIN4 homologs, were also attached to the plasma membrane and cleaved near their amino termini by AvrRpt2, but in contrast to RIN4, the major portions of these proteins remained on the plasma membrane. N-end degradation may play a minor role in RIN4 degradation but probably plays a major role in the degradation of RIN4 homologs and is, therefore, a major pathogenic consequence of AvrRpt2 cleavage.     

Two novel proteins in the mitochondrial outer membrane mediate beta-barrel protein assembly

Mitochondrial outer and inner membranes contain translocators that achieve protein translocation across and/or insertion into the membranes. Recent evidence has shown that mitochondrial beta-barrel protein assembly in the outer membrane requires specific translocator proteins in addition to the components of the general translocator complex in the outer membrane, the TOM40 complex. Here we report two novel mitochondrial outer membrane proteins in yeast, Tom13 and Tom38/Sam35, that mediate assembly of mitochondrial beta-barrel proteins, Tom40, and/or porin in the outer membrane. Depletion of Tom13 or Tom38/Sam35 affects assembly pathways of the beta-barrel proteins differently, suggesting that they mediate different steps of the complex assembly processes of beta-barrel proteins in the outer membrane.     

A comparison of the emission efficiency of four common green fluorescence dyes after internalization into cancer cells

In vivo optical imaging to enhance the detection of cancer during endoscopy or surgery requires a targeted fluorescent probe with high emission efficiency and high signal-to-background ratio. One strategy to accurately detect cancers is to have the fluorophore internalize within the cancer cells permitting nonbound fluorophores to be washed away or absorbed. The choice of fluorophores for this task must be carefully considered. For depth of penetration, near-infrared probes are ordinarily preferred but suffer from relatively low quantum efficiency. Although green fluorescent protein has been widely used to image tumors on internal organs in mice, green fluorescent probes are better suited for imaging the superficial tissues because of the short penetration distance of green light in tissue and the highly efficient production of signal. While the fluorescence properties of green fluorophores are well-known in vitro, less attention has been paid to their fluorescence once they are internalized within cells. In this study, the emission efficiency after cellular internalization of four common green fluorophores conjugated to avidin (Av-fluorescein, Av-Oregon green, Av-BODIPY-FL, and Av-rhodamine green) were compared after each conjugate was incubated with SHIN3 ovarian cancer cells. Using the lectin binding receptor system, the avidin-fluorophore conjugates were endocytosed, and their fluorescence was evaluated with fluorescence microscopy and flow cytometry. While fluorescein demonstrated the highest signal outside the cell, among the four fluorophores, internalized Av-rhodamine green emitted the most light from SHIN3 ovarian cancer cells both in vitro and in vivo. The internalized Av-rhodamine green complex appeared to localize to the endoplasmic vesicles. Thus, among the four common green fluorescent dyes, rhodamine green is the brightest green fluorescence probe after cellular internalization. This information could have implications for the design of tumor-targeted fluorescent probes that rely on cellular internalization for cancer detection.     

Purification and characterization of phospholipase B from Candida utilis

Phospholipase B (PLB) from the asporogenous yeast Candida utilis was purified to homogeneity from a culture broth. The apparent molecular mass was 90-110 kDa by SDS-PAGE. The enzyme had two pH optima, one acidic (pH 3.0) and the other alkaline (pH 7.5). At acidic pH the enzyme hydrolyzed all phospholipids tested without metal ions. On the other hand, the PLB showed substrate specificity and required metal ions for alkaline activity.The cDNA sequence of the PLB was analyzed by a combination of several PCR procedures. The PLB encoded a protein consisting of 643 amino acids. The amino acid sequence contained a lipase consensus sequence (GxSxG) and catalytic arginine and aspartic acid motifs which were identified as the catalytic triad in the PLB from Kluyveromyces lactis, suggesting that the catalytic mechanism of the PLB is similar to that of cytosolic phospholipase A(2) (cPLA(2)), found in mammalian tissues.     

A novel phosphatidylcholine which contains pentadecanoic acid at sn-1 and docosahexaenoic acid at sn-2 in Schizochytrium sp. F26-b

Docosahexaenoic acid (DHA, 22:6n-3)-containing phospholipids are a ubiquitous component of the central nervous system and retina, however their physiological and pharmacological functions have not been fully elucidated. Here, we report a novel DHA-containing phosphatidylcholine (PC) in a marine single cell eukaryote, Schizochytrium sp. F26-b. Interestingly, 31.8% of all the fatty acid in F26-b is DHA, which is incorporated into triacylglycerols and various phospholipids. In phospholipids, DHA was found to make up about 50% of total fatty acid. To identify phospholipid species containing DHA, the fraction of phospholipids from strain F26-b was subjected to normal phase high-performance liquid chromatography (HPLC). It was found that DHA was incorporated into PC, lyso-PC, phosphatidylethanolamine, and phosphatidylinositol. The major DHA-containing phospholipid was PC in which 32.5% of the fatty acid was DHA. The structure of PC was analyzed further by phospholipase A2 treatment, fast atom bombardment mass spectrometry, and 1H- and 13C-NMR after purification of the PC with reverse phase HPLC. Collectively, it was clarified that the major PC contains pentadecanoic acid (C15:0) at sn-1 and DHA at sn-2; the systematic name of this novel PC is therefore "1-pentadecanoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine."     

Foot odor due to microbial metabolism and its control

To characterize foot odor, we analyzed its components by sensory tests, isolated microorganisms that produce it, and evaluated the mechanism of the occurrence of foot odor. As a result, foot odor was found to be derived from isovaleric acid, which is produced when Staphylococcus epidermidis, a resident species of the normal cutaneous microbial flora, degrades leucine present in sweat. In addition, Bacillus subtilis was detected in the plantar skin of subjects with strong foot odor, and this species was shown to be closely associated with increased foot odor. Therefore, we screened various naturally occurring substances and fragrant agents that inhibit microbial production of foot odor without disturbing the normal microbial flora of the human skin. As a result, we identified citral, citronellal, and geraniol as fragrant agents that inhibit the generation of isovaleric acid at low concentrations.     

N-terminal motifs in some plant disease resistance proteins function in membrane attachment and contribute to disease resistance

To investigate the role of N-terminal domains of plant disease resistance proteins in membrane targeting, the N termini of a number of Arabidopsis and flax disease resistance proteins were fused to green fluorescent protein (GFP) and the fusion proteins localized in planta using confocal microscopy. The N termini of the Arabidopsis RPP1-WsB and RPS5 resistance proteins and the PBS1 protein, which is required for RPS5 resistance, targeted GFP to the plasma membrane, and mutation of predicted myristoylation and potential palmitoylation sites resulted in a shift to nucleocytosolic localization. The N-terminal domain of the membrane-attached Arabidopsis RPS2 resistance protein was targeted incompletely to the plasma membrane. In contrast, the N-terminal domains of the Arabidopsis RPP1-WsA and flax L6 and M resistance proteins, which carry predicted signal anchors, were targeted to the endomembrane system, RPP1-WsA to the endoplasmic reticulum and the Golgi apparatus, L6 to the Golgi apparatus, and M to the tonoplast. Full-length L6 was also targeted to the Golgi apparatus. Site-directed mutagenesis of six nonconserved amino acid residues in the signal anchor domains of L6 and M was used to change the localization of the L6 N-terminal fusion protein to that of M and vice versa, showing that these residues control the targeting specificity of the signal anchor. Replacement of the signal anchor domain of L6 by that of M did not affect L6 protein accumulation or resistance against flax rust expressing AvrL567 but removal of the signal anchor domain reduced L6 protein accumulation and L6 resistance, suggesting that membrane attachment is required to stabilize the L6 protein.     

Functional analysis of an indole-diterpene gene cluster for lolitrem B biosynthesis in the grass endosymbiont Epichloë festucae

Epichloë festucae Fl1 in association with Lolium perenne synthesizes a diverse range of indole-diterpene bioprotective metabolites, including lolitrem B, a potent tremorgen. The ltm genes responsible for the synthesis of these metabolites are organized in three clusters at a single sub-telomeric locus in the genome of E. festucae. Here we resolve the genetic basis for the remarkable indole-diterpene diversity observed in planta by analyzing products that accumulate in associations containing ltm deletion mutants of E. festucae and in cells of Penicillium paxilli containing copies of these genes under the control of a P. paxilli biosynthetic gene promoter. We propose a biosynthetic scheme to account for this metabolic diversity.     

Inhibitory effect of sulphated polysaccharide porphyran on nitric oxide production in lipopolysaccharide-stimulated RAW264.7 macrophages

Porphyran, extracted from an edible red alga (Porphyra yezoensis), is a sulphated polysaccharide with a wide variety of biological activities including anti-tumour, antioxidant and immuno-modulating activities. In this study, we examined the effect of porphyran on nitric oxide (NO) production in mouse macrophage cell line RAW264.7 cells. Although no significant activity of porphyran to induce NO or tumour necrosis factor-α (TNF-α) production in RAW264.7 cells was observed at the concentration range tested (10-500 μg/ml), it was found for the first time that porphyran inhibited NO production and expression of inducible nitric oxide synthase (iNOS) in RAW264.7 cells stimulated with lipopolysaccharide (LPS). In the presence of 500 μg/ml porphyran, NO production and expression of iNOS in LPS-treated RAW264.7 cells were completely suppressed. On the other hand, porphyran showed only a marginal effect on the secretion of TNF-α from LPS-stimulated RAW264.7 cells. Electrophoretic mobility shift assay (EMSA) using infrared dye labelled oligonucleotide with nuclear factor-κB (NF-κB) consensus sequence suggested that porphyran inhibited the LPS-induced NF-κB activation. The LPS-inducible nuclear translocation of p65, and the phosphorylation and degradation of IκB-α were also inhibited by the pre-treatment with porphyran. Our results obtained in in vitro analysis suggest that porphyran suppresses NO production in LPS-stimulated macrophages by the blocking of NF-κB activation.