Tea polyphenols ameliorate fat storage induced by high-fat diet in Drosophila melanogaster

BackgroundPolyphenols in tea are considered beneficial to human health. However, many such claims of their bioactivity still require in vitro and in vivo evidence.ResultsUsing Drosophila melanogaster as a model multicellular organism, we assess the fat accumulation-suppressing effects of theaflavin (TF), a tea polyphenol; epitheaflagallin (ETG), which has an unknown function; and epigallocatechin gallate (EGCg), a prominent component of green tea. Dietary TF reduced the malondialdehyde accumulation related to a high-fat diet in adult flies. Other physiological and genetic responses induced by the high-fat diet, such as lipid accumulation in the fat body and expression of lipid metabolism-related genes, were ameliorated by the addition of TF, ETG, and EGCg, in some cases approaching respective levels without high-fat diet exposure. Continuous ingestion of the three polyphenols resulted in a shortened lifespan.ConclusionWe provide evidence in Drosophila that tea polyphenols have a fat accumulation-suppressing effect that has received recent attention. We also suggest that tea polyphenols can provide different desirable biological activities depending on their composition and the presence or absence of other chemical components.     

MitoFates: Improved Prediction of Mitochondrial Targeting Sequences and Their Cleavage Sites

Mitochondria provide numerous essential functions for cells and their dysfunction leads to a variety of diseases. Thus, obtaining a complete mitochondrial proteome should be a crucial step toward understanding the roles of mitochondria. Many mitochondrial proteins have been identified experimentally but a complete list is not yet available. To fill this gap, methods to computationally predict mitochondrial proteins from amino acid sequence have been developed and are widely used, but unfortunately, their accuracy is far from perfect. Here we describe MitoFates, an improved prediction method for cleavable N-terminal mitochondrial targeting signals (presequences) and their cleavage sites. MitoFates introduces novel sequence features including positively charged amphiphilicity, presequence motifs, and position weight matrices modeling the presequence cleavage sites. These features are combined with classical ones such as amino acid composition and physico-chemical properties as input to a standard support vector machine classifier. On independent test data, MitoFates attains better performance than existing predictors in both detection of presequences and in predicting their cleavage sites. We used MitoFates to look for undiscovered mitochondrial proteins from 42,217 human proteins (including isoforms such as alternative splicing or translation initiation variants). MitoFates predicts 1167 genes to have at least one isoform with a presequence. Five-hundred and eighty of these genes were not annotated as mitochondrial in either UniProt or Gene Ontology. Interestingly, these include candidate regulators of parkin translocation to damaged mitochondria, and also many genes with known disease mutations, suggesting that careful investigation of MitoFates predictions may be helpful in elucidating the role of mitochondria in health and disease. MitoFates is open source with a convenient web server publicly available.Mitochondria not only function as the provider of ATP but also play crucial roles in the metabolism of amino acids and lipids, the biosynthesis of iron-sulfur clusters, cell signaling pathways, and apoptosis in eukaryotic cells. Moreover, mitochondrial dysfunction has been implicated in a wide variety of medical conditions such as muscle and neurodegenerative disease, cardiovascular disease, diabetes, and cancer (1).Obtaining the complete proteome of mitochondria is an essential step toward fully understanding its role in health and disease. To this end, ∼900 (in yeast) and 1100 (in mouse) mitochondrial proteins have been identified by large-scale proteomics analyses (2, 3); and compiled with other relevant mitochondrial proteomics data in useful databases such as MitoCarta (3) and MitoMiner (4). However, these lists are probably not yet complete, and indeed fungi and animal mitochondria have been estimated to host ∼1000 and ∼1500 distinct proteins, respectively (5). Thus, many mitochondrial proteins seem to remain undiscovered even in model organisms. If high accuracy can be achieved, prediction of mitochondrial proteins from primary sequence can save time and effort by identifying promising novel candidate mitochondrial proteins.The vast majority of mitochondrial proteins are encoded in the nuclear genome and imported by translocator complexes in the mitochondrial membranes. These mitochondrial proteins can be classified into two groups based on the type of targeting signal they contain: an N-terminal cleavable targeting signal (presequence); or a noncleavable, internal targeting signal (6). A recent proteomic analysis of yeast estimated that ∼70% of mitochondrial proteins possess a presequence (7). Thus, improved prediction of presequences should contribute to detecting undiscovered mitochondrial proteins.Presequences reside in the first 10–90 N-terminal residues, exhibit a high composition of arginine and near absence of negatively charged residues (8, 9). Proteins containing such presequences are translocated by the TOM and TIM protein complexes in the outer and the inner membranes, respectively (6, 10, 11). Tom20 and Tom22 in the TOM complex are reported to initiate import of these proteins by recognizing presequence segments capable of forming a local amphiphilic α-helical structure with hydrophobic residues on one face and positively charged residues on the opposite face (6, 12, 13). Widely used prediction tools such as MitoProt, TargetP, Predotar, and TPpred2 were developed with these properties of presequences in mind (14–17).The cleavage of mitochondrial protein presequences is an important event implicated in efficient protein import (18) and disease (19). Upon import into mitochondria, most presequences are cleaved off by the heterodimer mitochondrial processing peptidase (MPP)¹ in the matrix, and some of them subsequently further cleaved by intermediate peptidases such as Oct1 (20) and the recently discovered Icp55 (7). Although methods exist to predict these cleavage sites, their accuracy leaves much room for improvement (7, 21). Because the correct primary sequence of mature proteins is a prerequisite for precise structural modeling, improving the accuracy of cleavage site prediction should be useful for planning protein crystallography experiments or other structural studies of mitochondrial proteins. Also, accurate in silico prediction of the mature N-termini of mitochondrial proteins could in principle be used to improve the identification of N-terminal peptides in shotgun proteomics.In this study, we describe MitoFates, a novel method for mitochondrial presequence and cleavage site prediction. MitoFates formulates presequence prediction as a binary classification problem, employing a standard support vector machine (SVM) classifier. Our contribution is the preparation of an updated data set incorporating some recent proteomic data; and the selection of classical and novel sequence features such as amino acid composition, physicochemical properties, a novel positive amphiphilicity score, novel presequence motifs, and refined position weight matrices (PWMs) modeling peptidase cleavage sites. On the task of discriminating between presequences and nonpresequences, MitoFates achieves a true positive rate of 76% at a false positive rate of only 1.7%, improving significantly on previous methods. Moreover, MitoFates predicts the position of cleavage sites with an error rate of only ∼29% versus ∼47% for the best previous method.To investigate the potential of MitoFates to reveal interesting candidate mitochondrial proteins, we looked for undiscovered mitochondrial proteins among 42,217 human proteins (including isoforms such as alternative splicing or translation initiation variants), and obtained 580 candidate undiscovered mitochondrial proteins. Open source software downloads and a convenient MitoFates web server is available at http://mitf.cbrc.jp/MitoFates/.     

Immunomodulatory action of the DNA methyltransferase inhibitor SGI-110 in epithelial ovarian cancer cells and xenografts

We aimed to determine the effect of SGI-110 on methylation and expression of the cancer testis antigens (CTAs) NY-ESO-1 and MAGE-A in epithelial ovarian cancer (EOC) cells in vitro and in vivo and to establish the impact of SGI-110 on expression of major histocompatibility (MHC) class I and Intracellular Adhesion Molecule 1 (ICAM-1) on EOC cells, and on recognition of EOC cells by NY-ESO-1-specific CD8+ T-cells. We also tested the impact of combined SGI-110 and NY-ESO-1-specific CD8+ T-cells on tumor growth and/or murine survival in a xenograft setting. EOC cells were treated with SGI-110 in vitro at various concentrations and as tumor xenografts with 3 distinct dose schedules. Effects on global methylation (using LINE-1), NY-ESO-1 and MAGE-A methylation, mRNA, and protein expression were determined and compared to controls. SGI-110 treated EOC cells were evaluated for expression of immune-modulatory genes using flow cytometry, and were co-cultured with NY-ESO-1 specific T-cell clones to determine immune recognition. In vivo administration of SGI-110 and CD8+ T-cells was performed to determine anti-tumor effects on EOC xenografts. SGI-110 treatment induced hypomethylation and CTA gene expression in a dose dependent manner both in vitro and in vivo, at levels generally superior to azacitidine or decitabine. SGI-110 enhanced the expression of MHC I and ICAM-1, and enhanced recognition of EOC cells by NY-ESO-1-specific CD8+ T-cells. Sequential SGI-110 and antigen-specific CD8+ cell treatment restricted EOC tumor growth and enhanced survival in a xenograft setting. SGI-110 is an effective hypomethylating agent and immune modulator and, thus, an attractive candidate for combination with CTA-directed vaccines in EOC.     

Effect of the benzylic structure of lignan on antioxidant activity

The effect of the benzylic structure of lignan on antioxidant activity was evaluated. Secoisolariciresinol (1) and 3,4-bis(4-hydroxy-3-methoxybenzyl)tetrahydrofuran (2), which have two secondary benzylic positions without oxygen, showed the highest antioxidant activity. Optically active verrucosin (4) was synthesized for the first time in this experiment.     

Control of shoot and root meristem function by cytokinin

Plant hormones regulate a variety of processes fundamental for growth and development. Recent studies have clearly shown that establishing adequate spatial and temporal distribution of hormones is central in the control of development. The activity of cytokinins (CKs) is essential to maintain undifferentiated cells in shoot apical meristem (SAM) and to promote cell differentiation in the root meristem (RAM). Detailed mechanisms how the gradient of CK activities is established in the meristem has begun to be elucidated.     

Gel-based proteomics of unilateral irradiated striatum after gamma knife surgery

Gamma knife surgery (GKS) is used for the treatment of various brain disorders. The biological effects of focal gamma ray irradiation on targeted or surrounding areas in the brain are not well-known. In the present study, we evaluated protein expression changes in the unilateral irradiated (60 Gy) striatum in rat. Striata of irradiated and control brains were dissected 16 h post-irradiation for analysis by large-format two-dimensional gel electrophoresis (2-DGE). In parallel, we also examined the un-targeted contralateral striatum over the control for potential changes in proteins patterns that may have occurred due to the effects of irradiation to the unilateral striatum. A total of 17 reproducible and differentially expressed silver nitrate-stained protein spots in the irradiated striatum was detected on 2-D gel. Their subsequent analysis by tandem mass spectrometry (nESI-LC-MS/MS) resulted in the identification of 13 nonredundant proteins. Interestingly, out of these 13 changed proteins, 2 proteins were also detected in the contralateral striatum. Some of the significantly changed proteins identified were creatine kinase, protein disulfide isomerase A3 precursor (PDA3), and peroxiredoxin 2 (Prx2). Western analysis with anti-PDA3 and anti-Prx2 antibodies revealed 4 and 2 cross-reacting protein spots on 2-D gel blots. Interestingly, after GKS, in the irradiated and un-irradiated striata, these spots showed a shift toward the acidic side, suggesting post-translational modifications. Taken together, these results indicate that unilateral irradiation during GKS triggers molecular changes in the bilateral striata.     

Dynamic aspects of extracellular loop region as a proton release pathway of bacteriorhodopsin studied by relaxation time measurements by solid state NMR

Local dynamics of interhelical loops in bacteriorhodopsin (bR), the extracellular BC, DE and FG, and cytoplasmic AB and CD loops, and helix B were determined on the basis of a variety of relaxation parameters for the resolved 13C and 15N signals of [1-13C]Tyr-, [15N]Pro- and [1-13C]Val-, [15N]Pro-labeled bR. Rotational echo double resonance (REDOR) filter experiments were used to assign [1-13C]Val-, [15N]Pro signals to the specific residues in bR. The previous assignments of [1-13C]Val-labeled peaks, 172.9 or 171.1 ppm, to Val69 were revised: the assignment of peak, 172.1 ppm, to Val69 was made in view of the additional information of conformation-dependent 15N chemical shifts of Pro bonded to Val in the presence of 13C-15N correlation, although no assignment of peak is feasible for 13C nuclei not bonded to Pro. 13C or 15N spin-lattice relaxation times (T1), spin-spin relaxation times under the condition of CP-MAS (T2), and cross relaxation times (TCH and TNH) for 13C and 15N nuclei and carbon or nitrogen-resolved, 1H spin-lattice relaxation times in the rotating flame (1H T1 rho) for the assigned signals were measured in [1-13C]Val-, [15N]Pro-bR. It turned out that V69-P70 in the BC loop in the extracellular side has a rigid beta-sheet in spite of longer loop and possesses large amplitude motions as revealed from 13C and 15N conformation-dependent chemical shifts and T1, T2, 1H T1 rho and cross relaxation times. In addition, breakage of the beta-sheet structure in the BC loop was seen in bacterio-opsin (bO) in the absence of retinal.     

Gene expression monitoring for gene discovery in models of peripheral and central nervous system differentiation,regeneration, and trauma

Gene expression monitoring using gene expression microarrays represents an extremely powerful technology for gene discovery in a variety of systems. We describe the results of seven experiments using Incyte GEM technology to compile a proprietary portfolio of data concerning differential gene expression in six different models of neuronal differentiation and regeneration, and recovery from injury or disease. Our first two experiments cataloged genes significantly up‐ or down‐regulated during two phases of the retinoic acid‐induced differentiation of the embryonal carcinoma line Ntera‐2. To identify genes involved in neuronal regeneration we performed three GEM experiments, which included changes in gene expression in rat dorsal root ganglia during the healing of experimentally injured sciatic nerve, in regenerating neonatal opossum spinal cord, and during lipopolysaccharide stimulation of primary cultures of rat Schwann cells. Finally we have monitored genes involved in the recovery phase of the inflammatory disease of the rat spinal cord, experimental allergic encephalomyelitis, as well as those responsible for protection from oxidative stress in a glutamate‐resistant rat hippocampal cell line. Analysis of the results of the approximately 70,000 data points collected is presented. J. Cell. Biochem. 80:171–180, 2000. © 2000 Wiley‐Liss, Inc.