National Institute of Health Sciences

A specific assay has been developed for a blood-borne non-A, non-B hepatitis (NANBH) virus in which a polypeptide synthesized in recombinant yeast clones of the hepatitis C virus (HCV) is used to capture circulating viral antibodies. HCV antibodies were detected in six of seven human sera that were shown previously to transmit NANBH to chimpanzees. Assays of ten blood transfusions in the United States that resulted in chronic NANBH revealed that there was at least one positive blood donor in nine of these cases and that all ten recipients seroconverted during their illnesses. About 80 percent of chronic, post-transfusion NANBH (PT-NANBH) patients from Italy and Japan had circulating HCV antibody; a much lower frequency (15 percent) was observed in acute, resolving infections. In addition, 58 percent of NANBH patients from the United States with no identifiable source of parenteral exposure to the virus were also positive for HCV antibody. These data indicate that HCV is a major cause of NANBH throughout the world.

Plastic resin pellets (small granules 0.1-0.5 centimeters in diameter) are widely distributed in the ocean all over the world. They are an industrial raw material for the plastic industry and are unintentionally released to the environment both during manufacturing and transport. They are sometimes ingested by seabirds and other marine organisms, and their adverse effects on organisms are a concern. In the present study, PCBs, DDE, and nonylphenols (NP) were detected in polypropylene (PP) resin pellets collected from four Japanese coasts. Concentrations of PCBs (4-117 ng/g), DDE (0.16-3.1 ng/g), and NP (0.13-16 microg/g) varied among the sampling sites. These concentrations were comparable to those for suspended particles and bottom sediments collected from the same area as the pellets. Field adsorption experiments using PP virgin pellets demonstrated significant and steady increase in PCBs and DDE concentrations throughout the six-day experiment, indicating that the source of PCBs and DDE is ambient seawater and that adsorption to pellet surfaces is the mechanism of enrichment. The major source of NP in the marine PP resin pellets was thought to be plastic additives and/or their degradation products. Comparison of PCBs and DDE concentrations in mari

In 2005, the International Lipid Classification and Nomenclature Committee under the sponsorship of the LIPID MAPS Consortium developed and established a “Comprehensive Classification System for Lipids” based on well-defined chemical and biochemical principles and using an ontology that is extensible, flexible, and scalable. This classification system, which is compatible with contemporary databasing and informatics needs, has now been accepted internationally and widely adopted. In response to considerable attention and requests from lipid researchers from around the globe and in a variety of fields, the comprehensive classification system has undergone significant revisions over the last few years to more fully represent lipid structures from a wider variety of sources and to provide additional levels of detail as necessary. The details of this classification system are reviewed and updated and are presented here, along with revisions to its suggested nomenclature and structure-drawing recommendations for lipids. In 2005, the International Lipid Classification and Nomenclature Committee under the sponsorship of the LIPID MAPS Consortium developed and established a “Comprehensive Classification System for Lipids” based on well-defined chemical and biochemical principles and using an ontology that is extensible, flexible, and scalable. This classification system, which is compatible with contemporary databasing and informatics needs, has now been accepted internationally and widely adopted. In response to considerable attention and requests from lipid researchers from around the globe and in a variety of fields, the comprehensive classification system has undergone significant revisions over the last few years to more fully represent lipid structures from a wider variety of sources and to provide additional levels of detail as necessary. The details of this classification system are reviewed and updated and are presented here, along with revisions to its suggested nomenclature and structure-drawing recommendations for lipids. In an effort to support the growing field of lipidomics and establish the importance of lipids as a major class of biomolecules, the International Lipid Classification and Nomenclature Committee (ILCNC) developed a “Comprehensive Classification System for Lipids” that was published in 2005 (1Fahy E. Subramaniam S. Brown H.A. Glass C.K. Merrill Jr., A.H. Murphy R.C. Raetz C.R. Russell D.W. Seyama Y. Shaw W. al et A comprehensive classification system for lipids.J. Lipid Res. 2005; 46: 839-862Abstract Full Text Full Text PDF PubMed Scopus (1141) Google Scholar). For the purpose of classification, we define lipids as hydrophobic or amphipathic small molecules that may originate entirely or in part by carbanion-based condensations of thioesters (fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, and polyketides) and/or by carbocation-based condensations of isoprene units (prenol lipids and sterol lipids). The comprehensive classification system organizes lipids into these eight well-defined categories (Table 1) that cover eukaryotic and prokaryotic sources. It has been adopted internationally and widely accepted by the lipidomics community. The system is also available online on the LIPID MAPS (2Schmelzer K. Fahy E. Subramaniam S. Dennis E.A. The lipid maps initiative in lipidomics.Methods Enzymol. 2007; 432: 171-183Crossref PubMed Scopus (115) Google Scholar) website (http://www.lipidmaps.org). The comprehensive classification system has been under the guidance of the ILCNC, 3The ILCNC currently consists of Dr. Edward A. Dennis, Chair, (US), Dr. Robert C. Murphy (US), Dr. Masahiro Nishijima (Japan), Dr. Christian R. H. Raetz (US), Dr. Takao Shimizu (Japan), Dr. Friedrich Spener (Austria), Dr. Gerrit van Meer (The Netherlands), and Dr. Michael Wakelam (UK). Dr. Shankar Subramaniam serves as Informatics Advisor, and Dr. Eoin Fahy serves as Director. Meetings were held May 7, 2006 and May 4, 2008 in La Jolla, CA. which meets periodically to propose changes and updates to classification, nomenclature, and structural representation.TABLE 1Lipid categories of the comprehensive classification system and the number of structures in the LIPID MAPS databaseCategoryAbbreviationStructures in DatabaseFatty acylsFA2678GlycerolipidsGL3009GlycerophospholipidsGP1970SphingolipidsSP620Sterol LipidsST1744Prenol LipidsPR610SaccharolipidsSL11PolyketidesPK132 Open table in a new tab The initial version of the comprehensive classification system was more heavily focused on mammalian lipids, reflecting a bias toward the experimental interests of the LIPID MAPS Consortium (2Schmelzer K. Fahy E. Subramaniam S. Dennis E.A. The lipid maps initiative in lipidomics.Methods Enzymol. 2007; 432: 171-183Crossref PubMed Scopus (115) Google Scholar). However, due to considerable attention and requests from lipid researchers in a variety of fields, the classification system has now been extended to more fully represent lipid structures from nonmammalian sources, such as plants, bacteria, and fungi. For example, two new main classes (Glycosyldiradylglycerols and Glycosylmonoradylglycerols) have been added to the Glycerolipids category to accommodate key plant structural lipids, such as the sulfoquinovosyldiacylglycerols (3Norman H.A. Mischke C.F. Allen B. Vincent J.S. Semi-preparative isolation of plant sulfoquinovosyldiacylglycerols by solid phase extraction and HPLC procedures.J. Lipid Res. 1996; 37: 1372-1376Abstract Full Text PDF PubMed Google Scholar) found in chloroplasts. Also, the list of subclasses under the Sterols main class has been expanded to include a set of 15 different core structures (Ergosterols, Gorgosterols, Furostanols, etc.), which provide a structure-based classification of these molecules that span multiple phyla. Another key development has been the adoption of existing hierarchies (4Buckingham J. Dictionary of Natural Products on CD-ROM, Version 6.1. Chapman & Hall, London1998Crossref Google Scholar) for the Polyketide category and Prenol Lipids/Isoprenoids subclasses where the majority of these molecules are derived from natural product sources and have been studied intensively from a pharmaceutical and ecological standpoint. This in turn has necessitated the expansion of the number of existing classification levels (category, main class, and subclass) to accommodate an additional level of stratification in the case of the C10 to C30 isoprenoid subclasses that now contain entries at a fourth level of detail. The “LM_ID” identifier, whose format provides a systematic means of assigning a unique identification to each lipid molecule, has accordingly been expanded in length in these particular cases, with an additional two characters being used to describe the fourth level. A detailed overview of the changes and updates to the comprehensive classification system is presented below. As a consequence of adding an extra level of classification detail, the length of the LM_ID identifier was lengthened from 12 characters to 14 in cases where a lipid defined with four levels of classification is being described (Table 2). In this case, characters 9 and 10 specify the level-4 class. It should be emphasized that all lipids that do not require a fourth level of detail (i.e., the vast majority of them) still use a 12-digit LM_ID identifier.TABLE 2Format of LIPID MAPS identifier (LM_ID) in the comprehensive classification systemCharactersDescriptionExampleComments1–2Fixed “LM” designationLMAlways LM3–4Two-letter category codePROne of eight categories5–6Two-digit class code01—7–8Two-digit subclass code03“00” when no subclass9–10Two-digit fourth level code06Only used for lipids with four levelsLast four digitsUnique four-character identifier within subclass or within fourth level0002First two of the last four digits are letters in the case of the Glycosphingolipid subclasses Open table in a new tab In keeping with the theme of having a classification system dictated by molecular structure and function, the sterol lipid subclasses Phytosterols, Marine sterols, and Fungal sterols were retired because these refer to the lipid source (marine) or biological kingdom (plants and fungi). It is possible to identify a particular sterol in more than one of these three sources. These subclasses have been replaced by a new set of subclasses based on the carbon skeleton of the sterol core structure (Ergosterols, Gorgosterols, Furostanols, etc.). The details are outlined under the Sterol Lipids section below, and the complete description of this category can be found on the LIPID MAPS website 4Supplementary tables that provide the complete list of the classes, subclasses, and fourth class level (where applicable) of each of the eight categories of lipids are available on the LIPID MAPS website at http://www.lipidmaps.org. (http://www.lipidmaps.org). The natural products chemistry and medicinal chemistry literature describes tens of thousands of molecules that fall under the scope of lipids, based on their biosynthetic origin. In particular, isoprenoids and polyketides from diverse sources, such as plant, fungi, algae, bacteria, and marine invertebrates, are well documented and have been reviewed and classified in detail. The Dictionary of Natural Products (4Buckingham J. Dictionary of Natural Products on CD-ROM, Version 6.1. Chapman & Hall, London1998Crossref Google Scholar), a database available from Chapman and Hall/CRC (http://dnp.chemnetbase.com), has a classification hierarchy that covers polyketides and isoprenoids in depth. The LIPID MAPS comprehensive classification system has now incorporated some of these hierarchies relevant to natural products, with a view to covering both mammalian and nonmammalian lipids comprehensively. 3The ILCNC currently consists of Dr. Edward A. Dennis, Chair, (US), Dr. Robert C. Murphy (US), Dr. Masahiro Nishijima (Japan), Dr. Christian R. H. Raetz (US), Dr. Takao Shimizu (Japan), Dr. Friedrich Spener (Austria), Dr. Gerrit van Meer (The Netherlands), and Dr. Michael Wakelam (UK). Dr. Shankar Subramaniam serves as Informatics Advisor, and Dr. Eoin Fahy serves as Director. Meetings were held May 7, 2006 and May 4, 2008 in La Jolla, CA. It was recognized that additional levels of stratification were required to classify certain types of lipids and that the current three-level system of category/main class/subclass needed to be expanded. For example, in the Prenol Lipids category, 3The ILCNC currently consists of Dr. Edward A. Dennis, Chair, (US), Dr. Robert C. Murphy (US), Dr. Masahiro Nishijima (Japan), Dr. Christian R. H. Raetz (US), Dr. Takao Shimizu (Japan), Dr. Friedrich Spener (Austria), Dr. Gerrit van Meer (The Netherlands), and Dr. Michael Wakelam (UK). Dr. Shankar Subramaniam serves as Informatics Advisor, and Dr. Eoin Fahy serves as Director. Meetings were held May 7, 2006 and May 4, 2008 in La Jolla, CA. the Sesquiterpene C15 subclass contains ∼90 known variants based on their carbon skeletons (Bisabolanes, Germacranes, etc.). A fourth level of detail has been added to the LIPID MAPS comprehensive classification system to handle cases such as these. In response to worldwide interest in the comprehensive classification system for lipids, the scope has been expanded to cover lipids from nonmammalian sources, such as plants, bacteria, fungi, algae, and marine organisms. To accomplish this, several new lipid classes have been added, such as fatty acyl glycosides, glycosyldiradylglycerols, and various sterol skeletons. The Polyketide category has also been revised comprehensively. 3The ILCNC currently consists of Dr. Edward A. Dennis, Chair, (US), Dr. Robert C. Murphy (US), Dr. Masahiro Nishijima (Japan), Dr. Christian R. H. Raetz (US), Dr. Takao Shimizu (Japan), Dr. Friedrich Spener (Austria), Dr. Gerrit van Meer (The Netherlands), and Dr. Michael Wakelam (UK). Dr. Shankar Subramaniam serves as Informatics Advisor, and Dr. Eoin Fahy serves as Director. Meetings were held May 7, 2006 and May 4, 2008 in La Jolla, CA. The nomenclature of lipids falls into two main categories: systematic names and common or trivial names. The latter includes abbreviations that are a convenient way to define acyl/alkyl chains in acylglycerols, sphingolipids, and glycerophospholipids and synonyms such as “phosphatidyl” for “glycerophospho.” The generally accepted guidelines for lipid systematic names have been defined by the International Union of Pure and Applied Chemists and the International Union of Biochemistry and Molecular Biology (IUPAC-IUBMB) Commission on Biochemical Nomenclature (http://www.chem.qmul.ac.uk/iupac/) (5IUPAC-IUB Commission on Biochemical Nomenclature (CBN). The nomenclature of lipids (Recommendations 1976). 1977. Eur. J. Biochem. 79: 11–21; 1977. Hoppe-Seylers Z. Physiol. Chem. 358: 617–631; 1977. Lipids 12: 455–468; 1977. Mol. Cell. Biochem. 17: 157–171; 1978. Chem. Phys. Lipids 21: 159–173; 1978. J. Lipid Res. 19: 114–128; 1978. Biochem. J. 171: 21–35. (http://www.chem.qmul.ac.uk/iupac/lipid/).Google Scholar, 6I. U. P. A. C-I. U. B. Joint Commission on Biochemical Nomenclature (JCBN). Nomenclature of glycolipids. (Recommendations 1997) 2000. Adv. Carbohydr. Chem. Biochem. 55: 311–326; 1988. Carbohydr. Res. 312: 167–175; 1998. Eur. J. Biochem. 257: 293–298; 1999. Glycoconjugate J. 16:1–6; 1999. J. Mol. Biol. 286: 963–970; 1997. Pure Appl. Chem. 69: 2475–2487. (http://www.chem.qmul.ac.uk/iupac/misc/glylp.html)Google Scholar, 7I. U. P. A. C-I. U. B. Joint Commission on Biochemical Nomenclature (JCBN). Nomenclature of prenols. (Recommendations 1987) 1987. Eur. J. Biochem. 167: 181–184. (http://www.chem.qmul.ac.uk/iupac/misc/prenol.html)Google Scholar, 8I. U. P. A. C-I. U. B. Joint Commission on Biochemical Nomenclature (JCBN). Nomenclature of steroids (Recommendations 1989) 1989. Eur. J. Biochem. 186: 429–458. (http://www.chem.qmul.ac.uk/iupac/steroid/).Google Scholar). In response to several requests from knowledgeable lipid experts, abbreviations for Glycerophospholipid classes (see http://www.lipidmaps.org for GP category 3The ILCNC currently consists of Dr. Edward A. Dennis, Chair, (US), Dr. Robert C. Murphy (US), Dr. Masahiro Nishijima (Japan), Dr. Christian R. H. Raetz (US), Dr. Takao Shimizu (Japan), Dr. Friedrich Spener (Austria), Dr. Gerrit van Meer (The Netherlands), and Dr. Michael Wakelam (UK). Dr. Shankar Subramaniam serves as Informatics Advisor, and Dr. Eoin Fahy serves as Director. Meetings were held May 7, 2006 and May 4, 2008 in La Jolla, CA.) have been changed now in the comprehensive classification system to the more universally used two-letter “PC/PE/PS/PA/PI” format. Consequently, glycerophospholipids in the LIPID MAPS structure database and LIPID MAPS standards database as well as all the Glycerophospholipids drawing tools and mass spectrometry prediction tools have been updated to conform to this new abbreviation format (Table 3).TABLE 3Changes in abbreviations for Glycerophospholipids in the comprehensive classification systemClassSynonymOldNewGlycerophosphocholinesPhosphatidylcholinesGPChoPCaFor abbreviations of monoradyglycerophospholipids (lysophospholipids), LPX may be used, for example, LPC, LPE, LPA, etc.GlycerophosphoethanolaminesPhosphatidylethanolaminesGPEtnPEGlycerophosphoserinesPhosphatidylserinesGPSerPSGlycerophosphoglycerolsPhosphatidylglycerolsGPGroPGGlycerophosphoglycerophosphatesPhosphatidylglycerol phosphatesGPGroPPGPGlycerophosphoinositolsPhosphatidylinositolsGPInsPIGlycerophosphoinositol monophosphatesPhosphatidylinositol phosphatesGPInsPPIPGlycerophosphoinositol bis-phosphatesPhosphatidylinositol bis-phosphatesGPInsP2PIP2Glycerophosphoinositol For abbreviations of monoradyglycerophospholipids (lysophospholipids), LPX may be used, for example, LPC, LPE, LPA, Open table in a new tab The LIPID MAPS Consortium has considerable effort to establish guidelines for drawing lipid structures in a and and lipids are to which to the use of unique that more than the lipid community. the structure-drawing is the in molecular of lipids. However, classes of lipids well as for structure-drawing due to their A of structure-drawing tools has been developed and that of systematic and abbreviations E. Subramaniam S. LIPID MAPS online tools for lipid Res. 2007; PubMed Scopus Google Scholar). The structures may be and in a variety of of the structure-drawing tools for fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, and sterols are available in the section of the LIPID MAPS website (http://www.lipidmaps.org). of the structures are in the importance of these molecules in and is to have a database of lipids with a defined ontology that is extensible, flexible, and scalable. The ontology of lipids classification, nomenclature, structure and structural of all in the have developed a available comprehensive database of lipid structures of lipid molecules from existing and from the LIPID MAPS This database Fahy E. Brown A. Dennis E.A. Glass C.K. Merrill Jr., A.H. Murphy R.C. Raetz C.R. Russell D.W. al et LIPID MAPS structure Res. 2007; PubMed Scopus Google Scholar, E. R. A. J. Y. Subramaniam S. for lipidomics.Methods Enzymol. 2007; 432: PubMed Scopus Google Scholar), in to as the to and of lipid also contains systematic classification, nomenclature, and structure of lipids along with mass where than lipid molecules are now available on the LIPID MAPS and these have been adopted by the for as well as the of and database structures have been classified and to LIPID MAPS A number of different molecular such as and the and are and nomenclature of these molecules are also The database is and the include and structure-based the category, the subclasses and have been changed to and to accommodate The names of the fatty subclasses and have been to and A new acyl main class has been added to cover the number of found in bacteria, and marine of natural of fatty and PubMed Scopus Google Scholar, and of the natural PubMed Scopus Google Scholar). subclasses include acyl of and and The Glycerolipids category was to include two new main classes (Glycosyldiradylglycerols and Glycosylmonoradylglycerols) that contain key plant structural lipids, such as the found in chloroplasts. The existing subclasses were to the that is to the of on the for and of structures in the LIPID MAPS structure database have been For with two different two different structural are for with three different different of drawing all possible structural an is used as a A along with the number of possible is to the abbreviation and and a unique LM_ID is of this format is the The structure to the LM_ID on the LIPID MAPS website to the in the and the is to all in the are also cases within the and classes where are due to by certain of both the or to and where the of the at is by the acyl from the of to the at or can an In such cases when a is can be with a for example, or the is with a for example, It should be that the two-letter abbreviation or all possible types of lipid for example, having and The is by the for example, and the by the for example, The to the classes within the Glycerophospholipids In cases where is and is abbreviations such as and may be used, where the within refer to the number of and of all the For the Glycerophospholipids category, the subclass has been replaced by the more due to the that in are the in H.A. J.S. S. in and PubMed Scopus Google Scholar). updates have been for the Glycerophospholipid The lipids class has been replaced by the class to of with than As we have changed to two-letter abbreviations to describe glycerophospholipids in are abbreviations for all molecular of their These names to and are used widely in lipid as to systematic names. This format one or two chains where the structures of the chains are within is at the carbon of and the is at the In cases of molecules with at of the and of the at the the of is to the abbreviation and the abbreviation format is for molecules with at the carbon of the the of is to the and the structure is with In cases where is and is such as may be used to of and for all and are by an or identifier, as in and In the latter case, the an at of and a at the or may be with a in the for example, The “phosphatidyl” is used to refer to classes all types of chains and not acyl as was by guidelines (5IUPAC-IUB Commission on Biochemical Nomenclature (CBN). The nomenclature of lipids (Recommendations 1976). 1977. Eur. J. Biochem. 79: 11–21; 1977. Hoppe-Seylers Z. Physiol. Chem. 358: 617–631; 1977. Lipids 12: 455–468; 1977. Mol. Cell. Biochem. 17: 157–171; 1978. Chem. Phys. Lipids 21: 159–173; 1978. J. Lipid Res. 19: 114–128; 1978. Biochem. J. 171: 21–35. (http://www.chem.qmul.ac.uk/iupac/lipid/).Google Scholar). The classification is from that in the in this (2Schmelzer K. Fahy E. Subramaniam S. Dennis E.A. The lipid maps initiative in lipidomics.Methods Enzymol. 2007; 432: 171-183Crossref PubMed Scopus (115) Google Scholar). of is that for of the Glycosphingolipid subclasses, the structure of the is known the structure of the is In these cases, the last two digits of the LIPID MAPS LM_ID identifier are as to an and the and fourth last digits are a different two-letter identifier for unique within that For example, in the subclass the structure is an LM_ID of where the digits specify the unique and the digits a the has a and is a LM_ID of The Sterol lipids subclasses Phytosterols, Marine sterols, and Fungal sterols have been and replaced with a set of subclasses (Ergosterols, sterols, Gorgosterols, Furostanols, and that in the of their sterol core structures and cover multiple A. Sterols in marine Scopus Google Scholar, Phytosterols, and their in structural and Lipid Res. PubMed Scopus Google Scholar). The class has been with the and the class now includes and The class has been to the Prenol Lipids category the of the core structure is at with the of the of the Sterol Lipids The subclass of the Prenol lipids category has been added to the class. are a of that are to A. have in of and As the C10 to C30 isoprenoid subclasses now contain entries at a fourth level of detail. The LM_ID contain an extra two digits that specify the fourth level class, for example, the is an LM_ID of The class has been to the Prenol Lipids category the Sterol Lipids For the the main class acyl has been added to cover a variety of from plants, bacteria, and fungi. is the from the plant and from the of Scopus Google Scholar). It should be that this category covers structures in which fatty acyl/alkyl are to a lipids to a are found in their The category was revised and on the classification hierarchy used by the Dictionary of Natural Products (4Buckingham J. Dictionary of Natural Products on CD-ROM, Version 6.1. Chapman & Hall, London1998Crossref Google Scholar). are from bacteria, fungi, plants, and and have been heavily studied by natural products and for The new classification format provides a of the structural within this The toward classification of lipids is the of an ontology that is extensible, flexible, and scalable. be to and represent these molecules in a that is to databasing and The ILCNC the comprehensive classification system in 2005 and has been in and on a to considerable attention and requests from lipid researchers in a variety of fields, the classification system has been extended to more fully represent lipid structures from nonmammalian sources, such as plants, bacteria, and fungi. This system has been internationally accepted and is now widely used in and for The LIPID MAPS classification system has also been adopted by where hierarchies lipids, and have been and by the in format of the In an effort to LIPID MAPS lipid structures are now available on website where have been The classification system is available online where has been with an database of lipids. This in to as the and of lipid also contains systematic classification, nomenclature, and structure of lipids along with mass where structures have been classified and to LIPID MAPS The format of the LM_ID identifier (Table provides a systematic means of the classification hierarchy and assigning a unique identification to each lipid It also for the of new classification in the The database is and the include and structure-based This database is described in detail Fahy E. Brown A. Dennis E.A. Glass C.K. Merrill Jr., A.H. Murphy R.C. Raetz C.R. Russell D.W. al et LIPID MAPS structure Res. 2007; PubMed Scopus Google Scholar, E. R. A. J. Y. Subramaniam S. for lipidomics.Methods Enzymol. 2007; 432: PubMed Scopus Google Scholar). A of lipid structure-drawing tools in the section of the LIPID MAPS has been developed to structure with LIPID MAPS These tools are also of systematic names and detailed and databasing of lipid and has been to and database and to classify and LIPID MAPS These tools be expanded and as the scope of the classification system and over the The the of lipid researchers around the have and to attention in the Classification System for which to be to new and in the lipid The are also to the LIPID MAPS Consortium for their and to Dr. at the of for to this

A basic peptide derived from human immunodeficiency virus (HIV)-1 Tat protein (positions 48–60) has been reported to have the ability to translocate through the cell membranes and accumulate in the nucleus, the characteristics of which are utilized for the delivery of exogenous proteins into cells. Based on the fluorescence microscopic observations of mouse macrophage RAW264.7 cells, we found that various arginine-rich peptides have a translocation activity very similar to Tat-(48–60). These included such peptides as the d-amino acid- and arginine-substituted Tat-(48–60), the RNA-binding peptides derived from virus proteins, such as HIV-1 Rev, and flock house virus coat proteins, and the DNA binding segments of leucine zipper proteins, such as cancer-related proteins c-Fos and c-Jun, and the yeast transcription factor GCN4. These segments have no specific primary and secondary structures in common except that they have several arginine residues in the sequences. Moreover, these peptides were able to be internalized even at 4 °C. These results strongly suggested the possible existence of a common internalization mechanism ubiquitous to arginine-rich peptides, which is not explained by a typical endocytosis. Using (Arg)n (n = 4–16) peptides, we also demonstrated that there would be an optimal number of arginine residues (n ∼ 8) for the efficient translocation. A basic peptide derived from human immunodeficiency virus (HIV)-1 Tat protein (positions 48–60) has been reported to have the ability to translocate through the cell membranes and accumulate in the nucleus, the characteristics of which are utilized for the delivery of exogenous proteins into cells. Based on the fluorescence microscopic observations of mouse macrophage RAW264.7 cells, we found that various arginine-rich peptides have a translocation activity very similar to Tat-(48–60). These included such peptides as the d-amino acid- and arginine-substituted Tat-(48–60), the RNA-binding peptides derived from virus proteins, such as HIV-1 Rev, and flock house virus coat proteins, and the DNA binding segments of leucine zipper proteins, such as cancer-related proteins c-Fos and c-Jun, and the yeast transcription factor GCN4. These segments have no specific primary and secondary structures in common except that they have several arginine residues in the sequences. Moreover, these peptides were able to be internalized even at 4 °C. These results strongly suggested the possible existence of a common internalization mechanism ubiquitous to arginine-rich peptides, which is not explained by a typical endocytosis. Using (Arg)n (n = 4–16) peptides, we also demonstrated that there would be an optimal number of arginine residues (n ∼ 8) for the efficient translocation. human immunodeficiency virus human T-cell lymphotrophic virus type-II brome mosaic virus flock house virus high performance liquid chromatography phosphate-buffered saline [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide N-(6-maleimidocaproyloxy)succinimide ester nuclear localization sequence Recently, methods have been developed for the delivery of exogenous proteins into living cells with the help of membrane-permeable carrier peptides such as HIV-11 Tat-(48–60) and Antennapedia-(43–58) (1Fawell S. Seery J. Daikh Y. Moore C. Chen L.L. Pepinsky B. Barsoum J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 664-668Crossref PubMed Scopus (1107) Google Scholar, 2Vives E. Brodin P. Lebleu B. J. Biol. Chem. 1997; 272: 16010-16017Abstract Full Text Full Text PDF PubMed Scopus (2064) Google Scholar, 3Nagahara H. Vocero-Akbani A.M. Snyder E.L. Ho A. Latham D.G. Lissy N.A. Becker-Hapak M. Ezhevsky S.A. Dowdy S.F. Nat. Med. 1998; 4: 1449-1452Crossref PubMed Scopus (886) Google Scholar, 4Schwarze S.R. Ho A. Vocero-Akbani A. Dowdy S.F. Science. 1999; 285: 1569-1572Crossref PubMed Scopus (2200) Google Scholar, 5Schwarze S.R. Dowdy S.F. Trends Pharmacol. Sci. 2000; 21: 45-48Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 6Derossi D. Joliot A.H. Chassaing G. Prochiantz A. J. Biol. Chem. 1994; 269: 10444-10450Abstract Full Text PDF PubMed Google Scholar, 7Derossi D. Calvet S. Trembleau A. Brunissen A. Chassaing G. Prochiantz A. J. Biol. Chem. 1996; 271: 18188-18193Abstract Full Text Full Text PDF PubMed Scopus (966) Google Scholar, 8Derossi D. Chassaing G. Prochiantz A. Trends Cell Biol. 1998; 8: 84-87Abstract Full Text PDF PubMed Scopus (665) Google Scholar, 9Lin Y.Z. Yao S. Veach R.A. Torgerson T.R. Hawiger J. J. Biol. Chem. 1995; 270: 14255-14258Abstract Full Text Full Text PDF PubMed Scopus (854) Google Scholar, 10Rojas M. Yao S. Lin Y.Z. J. Biol. Chem. 1996; 271: 27456-27461Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 11Rojas M. Donahue J.P. Tan Z. Lin Y.Z. Nat. Biotechnol. 1998; 16: 370-375Crossref PubMed Scopus (142) Google Scholar). By genetically or chemically hybridizing these carrier peptides, the efficient intracellular delivery of various oligopeptides and proteins was achieved. One of the most amazing examples is the Tat-β-galactosidase fusion protein (4Schwarze S.R. Ho A. Vocero-Akbani A. Dowdy S.F. Science. 1999; 285: 1569-1572Crossref PubMed Scopus (2200) Google Scholar), which has a molecular mass as high as 120 kDa. Intraperitoneal injection of the protein resulted in delivery of the protein with β-galactosidase activity to various tissues in mice, including the brain. The peptide-mediated approaches would allow the incorporation of peptides containing unnatural amino acids or nonpeptide molecules such as fluorescence probes. These methods would become powerful tools not only for therapeutic purposes as an alternative to gene delivery, but also for the understanding of the mechanisms behind fundamental cellular events, such as signal transduction and gene transcription. Besides the potential of Tat-(48–60) as a protein carrier, the internalization mechanism of the peptide attracted our interest. For example, Tat-(48–60) (GRKKRRQRRRPPQ) is a highly basic and hydrophilic peptide, which contains 6 arginine and 2 lysine residues in its 13 amino acid residues. However, the peptide was reported to be translocated through the cell membranes in 5 min at a concentration of 0.1 μm (2Vives E. Brodin P. Lebleu B. J. Biol. Chem. 1997; 272: 16010-16017Abstract Full Text Full Text PDF PubMed Scopus (2064) Google Scholar). Internalization of the peptide was not inhibited even at 4 °C. The peptide is less toxic to cells than other basic membrane-interacting agents. The above features suggested that the internalization mechanism of Tat-(48–60) was completely different from the typical transmembrane mechanisms reported so far. Questions arise as to whether such an efficient translocation is specific for Tat-(48–60) and Antennapedia-(43–58) peptides and what is the mechanism of the highly efficient internalization. Based on experiments using synthetic peptides, we suggest the possible presence of a very similar translocation mechanism to Tat-(48–60) present among the various arginine-rich peptides. We also suggest the possible existence of the optimum chain length of arginine peptides for the internalization. All the peptides used in this study were chemically synthesized by Fmoc (9-fluorenylmethyloxycarbonyl)-solid-phase peptide synthesis on a Rink amide resin as reported previously (12Futaki S. Ishikawa T. Niwa M. Kitagawa K. Yagami T. Bioorg. Med. Chem. 1997; 5: 1883-1891Crossref PubMed Scopus (31) Google Scholar). Fluorescent labeling of the peptides was conducted by the treatment with 1.5 eq of 5-maleimidofluorescein diacetate (Sigma) in dimethylformamide-methanol (1:2) for 3 h followed by reverse-phase HPLC purification. The fidelity of the products was ascertained by time-of-flight mass spectrometry. Carbonic anhydrase in phosphate-buffered saline (PBS) was simultaneously treated with fluorescein-5(6)-carboxamidocaproic acid N-hydroxysuccinimide ester (Sigma) andN-(6-maleimidocaproyloxy)succinimide ester (Dojin) (15 eq, each) at room temperature for 1 h to introduce the fluorescein and the maleimide function to the protein. After the removal of the unreacted reagents by gel-filtration on a Sephadex G-25 (Amersham Pharmacia Biotech) column, the cysteine of the respective arginine-rich peptides was allowed to react with the maleimide moiety on the above fluorescein-labeled protein at room temperature for 16 h, and then the unreacted peptides were removed by gel-filtration. Based on the molecular weight estimation by SDS-polyacrylamide gel electrophoresis, one or two molecules of basic peptides and fluorescein per protein were incorporated, respectively. Mouse macrophage RAW264.7 cells were maintained in RPMI 1640 medium with 10% heat-inactivated fetal bovine serum. Cells were grown on 60-mm dishes and incubated at 37 °C under 5% CO2 to ∼70% confluence. A subculture was performed every 3–4 days. For each assay, 4 × 104/ml cells were pelleted on a eight-well Lab-Tek-II chamber slide (Nalge Nunc) (250 μl/well) and cultured for 16 h. After complete adhesion, the culture medium was exchanged. The cells were incubated at 37 °C for 3 h with the fresh medium (250 μl) containing fluorescein-labeled peptides or proteins. The concentrations of the peptides and proteins were adjusted before addition to the cell based on their fluorescent intensity. Cells were washed three times with PBS, fixed with acetone-methanol (1:1) for 2 min at room temperature, washed three times with PBS again, and then mounted in fluorescent mounting medium containing 15 mmNaN3 (Dako). The distribution of fluorescein-labeled peptides was analyzed on a Zeiss Axioskop fluorescence microscope using a 100× oil immersion lens. Cells were grown, incubated with proteins, and fixed basically as described above. Cells were then treated with PBS containing 5 μm propidium iodide (200 μl) at room temperature for 30 min, washed four times with PBS, and mounted in glycerol:PBS (9:1) containing 1%p-phenylenediamine dihydrochloride. Data were obtained using a confocal scanning laser microscope MRC 1024 (Bio-Rad) equipped with a 60× oil immersion lens or LSM 510 (Zeiss) equipped with a 40× lens. The MTT assay was conducted basically in the same manner as reported previously (2Vives E. Brodin P. Lebleu B. J. Biol. Chem. 1997; 272: 16010-16017Abstract Full Text Full Text PDF PubMed Scopus (2064) Google Scholar). Cells (1 × 104/well) were cultured in 96-microtiter plates in RPMI 1640 medium with 10% heat-inactivated fetal bovine serum in the presence of peptides (HIV-1 Tat-(48–60): GRKKRRQRRRPPQ-amide; R9-Tat: GRRRRRRRRRPPQ-amide; HIV-1 Rev-(34–50): TRQARRNRRRRWRERQR-amide; FHV coat-(35–49): RRRRNRTRRNRRRVR-amide) at 10 or 100 μm. Cells were incubated at 37 °C under 5% CO2 for 24 h before addition of MTT (Sigma, 5 mg/ml in PBS) for 4 h. The precipitated MTT formazan was dissolved overnight in 0.04 n HCl in isopropanol (100 μl). The absorbance at 570 nm was then measured. Cell viability was expressed as the ratio of the A 570 of cells treated with peptide over the control samples. To obtain insight into the translocation mechanisms of the Tat-(48–60) peptide, Tat-(48–60), its d-amino acid-substituted analog (d-Tat) and arginine-substituted analog (R9-Tat), where residues corresponding to positions 49–57 were replaced with arginine, were synthesized (Fig.1 a). An extra cysteine amide was incorporated into the C terminus of each peptide for the fluorescent labeling. The peptides corresponding to nuclear localization sequences (NLS) derived from simian virus 40 (13) and nucleoplasmin (14Görlich D. Mattaj I.W. Science. 1996; 271: 1513-1518Crossref PubMed Scopus (1066) Google Scholar) were also synthesized as references. Treatment of the peptides with 5-maleimidofluorescein diacetate gave the corresponding fluorescein-labeled peptides. Internalization of the peptides was monitored by fluorescence microscopic observation after a 3-h incubation of the peptides with mouse macrophage RAW 264.7 cells at 37 °C. As a result, d-Tat and R9-Tat were internalized into the cell as efficiently as the Tat-(48–60) peptides, and localization into both the cytoplasm and nucleus was observed (Fig. 2). A similar internalization of the d-amino acid analog of Tat was reported by Huq et al. (15Huq I. Ping Y.-H. Tamilarasu N. Rana T.M. Biochemistry. 1999; 38: 5172-5177Crossref PubMed Scopus (44) Google Scholar) using a linear peptide corresponding to residues 37–72. These results would contradict the idea that a specific receptor may play a crucial role in the translocation of the Tat-(48–60) peptide. On the other hand, the simian virus 40-derived and nucleoplasmin-derived peptides showed a much lower degree of internalization. These NLS-derived peptides are rich in lysine. The above results suggested that arginine residues would play an important role in the translocation.Figure 2Translocation of the arginine-rich Tat-related peptides through the cell membranes. RAW264.7 cells were treated with fluorescein-labeled peptides derived from HIV-1 Tat-(48–60) (a), R9-Tat (b),d-Tat (c), and nucleoplasmin-NLS (d) (10 μm each) for 3 h.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Arginine-rich basic segments are used by a variety of RNA-binding proteins to recognize specific RNA structures (16Tan R. Frankel A.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5282-5286Crossref PubMed Scopus (177) Google Scholar). If arginine in the sequence plays an important role in the translocation, peptides corresponding to these RNA-binding segments may translocate through the cell membranes. To test this hypothesis, 10 arginine-rich RNA-binding peptides bearing a C-terminal Gly-Cys-amide (Fig. 1 b) were similarly prepared, fluorescein-labeled, and applied to the macrophage cells. To our surprise, all the peptides other than the human U2AF-(142–153) peptide translocated through the cell membranes and accumulated in the cytoplasm and nucleus (Fig. 3). As judged from the fluorescent intensity, the efficiency of incorporation into the cells showed a tendency to correspond to the number of arginine residues in the sequence. Internalization activity of the HIV-1 Rev-(34–50), FHV coat-(35–49), HTLV-II Rex-(4–16), and BMV Gag-(7–25) peptides, which have more than seven arginine residues in their sequences, were comparable with that of the Tat-(48–60) peptide. Fluorescence was observed in the cells as early as 5 min after the addition of these peptides (1 μm) to the medium. Less extensive internalization was observed in the case of the λ N-(1–22), φ21 N-(12–29), and yeast PRP6-(129–144) peptides that have five arginine residues in their sequences. The fluorescent intensity in the cells treated with the former peptides (0.1 μm) was judged not to be less than that in those treated with the latter peptides (10 μm). The P22 N-(14–30) and cowpea chlorotic mottle virus Gag-(7–25) peptides that have six arginine residues showed a moderate degree of translocation. HIV-1 Tat-(48–60) is reported to translocate through the cell membranes and accumulate in the nucleus, especially the nucleolus (2Vives E. Brodin P. Lebleu B. J. Biol. Chem. 1997; 272: 16010-16017Abstract Full Text Full Text PDF PubMed Scopus (2064) Google Scholar). A similar tendency was observed with the above peptides. Not only the RNA-binding peptides but also the DNA-binding peptides corresponding to the basic leucine zipper segments derived from cancer-related proteins, c-Fos and c-Jun, and the yeast transcription factor, GCN4, which were also rich in arginine (Fig. 1 c), were internalized into the cells with almost the same efficiency as that of Tat-(48–60) (Fig.4).Figure 4Translocation of DNA-binding peptides through the cell membranes. RAW264.7 cells were treated with the fluorescein-labeled peptides derived from human c-Jun-(252–279) (a) and yeast GCN4-(231–252) (b) (1 μm each) for 3 h.View Large Image Figure ViewerDownload Hi-res image Download (PPT) HIV-1 Tat-(48–60) was reported to induce little toxicity to HeLa cells (2Vives E. Brodin P. Lebleu B. J. Biol. Chem. 1997; 272: 16010-16017Abstract Full Text Full Text PDF PubMed Scopus (2064) Google Scholar). Using R9-Tat, HIV-1 Rev-(34–50), and FHV coat-(35–49) peptides as representatives of the above arginine-rich peptides, cytotoxicity of the peptides was investigated. Determined by the MTT assay, the above peptides did not show a significant cytotoxicity to the macrophage cells during the treatment with a peptide (10 μm) for 24 h. At 100 μm, cell viability of the cells treated with R9-Tat became 70%, whereas viability of those treated with other peptides as well as HIV-1 Tat-(48–60) was still greater than 95%. These results suggested that many of the arginine-rich peptides can be of low cytotoxicity as reported for the HIV-1 Tat-(48–60) peptide. The above experiments showed that a variety of arginine-rich RNA/DNA-binding peptides were able to translocate through the cell membranes. Little homology in these sequences was observed, except that they all have 5–11 arginine residues. Moreover, thed-amino acid substituted Rev-(34–50) peptide (1 μm) was internalized as efficiently as thel-peptide in 3 h not of the HIV-1 Tat-(48–60), R9-Tat, and FHV coat-(35–49) peptides in were of their not a significant secondary whereas the HIV-1 Rev-(34–50) peptide showed a typical of an peptide. The peptide, which was only internalized into the showed a very similar to that of the FHV coat-(35–49) peptide. These results were of the of even a common secondary in the membrane-permeable peptides. the cells were incubated with a peptide (1 μm) at 4 °C for 30 min, no significant in fluorescent intensity in the cell was observed using the HIV-1 Rev-(34–50), and FHV coat-(35–49) peptides These results suggested that typical so would not play a crucial role in the translocation of these arginine-rich of temperature on HIV-1 Rev-(34–50) peptide internalization. The cells were incubated with the peptide (1 μm) for 30 min at 4 °C or at 37 °C. the former the cells were at 4 °C for 1 h before addition of the peptide. All the were also conducted at 4 °C the of the Large Image Figure ViewerDownload Hi-res image Download (PPT) We on the whether the of arginine-rich peptides into the cells is or The cells were treated with the HIV-1 Rev-(34–50) peptide (1 μm) for 3 h, then the medium was with a fresh one not containing the peptide. The fluorescence intensity from the cells 1 h was almost comparable with or only less than that of the cells before the medium However, a in the fluorescence intensity was in the cells 6 h and complete of the fluorescence was observed 24 h To the above results were to the of the peptide from the cells, the medium was analyzed by an HPLC equipped with a fluorescence was at the corresponding to the were observed that at positions with those of the peptide treated with not we that the in fluorescence intensity of the cells resulted from the of the peptides, and not from the of the peptide. The whether the peptide a on the cell was also The above HIV-1 cells were 24 h and The cell number for the cells was comparable with that for the control cells peptide the cells were judged to to with little by the peptide. would be that the peptide in each of the cells cell significant in the intensity were not observed among the cells 6 h the of the which was to be h, a of cells have the 6 h. If the peptides would in one of the cells cell a in the fluorescence intensity be observed among the cells. However, study be to this To the of the above basic peptides as protein we basic Carbonic anhydrase was as a protein. anhydrase were using N-(6-maleimidocaproyloxy)succinimide ester as a R. H. M. M. Niwa M. S. H. 1998; PubMed Scopus Google Scholar) A fluorescein moiety was into the protein using the fluorescein-5(6)-carboxamidocaproic ester simultaneously with As judged from the SDS-polyacrylamide gel of the one to two molecules of the basic peptide and fluorescein moiety were into a of respectively. Carbonic anhydrase was into the cells with the help of the HIV-1 Rev-(34–50), FHV coat-(35–49), and R9-Tat peptides as efficiently as with the HIV-1 Tat-(48–60) peptide (Fig. of the in the and nucleus was also observed by fluorescence of the cells (Fig. microscopic of these demonstrated both and nuclear localization and not to the cellular membranes (Fig. On the other hand, fluorescein-labeled protein a carrier peptide was in a of the (Fig. suggested that the protein was in the and was not able to be into the was also into the cell with the help of these carrier peptides not The above strongly suggested the of arginine residues in the internalization. The possible existence of the internalization mechanism common in these arginine-rich peptides was also We then the of the number of arginine residues in the sequences. For peptides that are of residues of arginine were (Fig.1 To their C the Gly-Cys-amide was also for the fluorescein labeling. These results are in A. was on the translocation efficiency and intracellular localization among these peptides. showed low translocation and the internalization and in the is is that the degree of internalization as the chain length For the internalization of the peptide was not The same of was in the experiments using the of anhydrase with the arginine peptides (Fig. A similar tendency was observed on the protein delivery using and as the carrier Based on the confocal laser microscopic the anhydrase was efficiently internalized into the macrophage cells and in the nucleus was observed as was in the case of the HIV-1 Rev-(34–50) the to on the cell membranes after a 3-h incubation with the but significant in the nucleus was not this we have that not only Tat-(48–60) but also various arginine-rich peptides were able to translocate through the mouse macrophage membranes. These peptides the and arginine-substituted HIV-1 Tat-(48–60) RNA-binding peptides derived from proteins, such as HIV-1 Rev, HTLV-II BMV and FHV coat proteins, and the DNA-binding segments from c-Jun, and the proteins. a common or very similar mechanism for the internalization among these peptides. The mechanism is explained by by the peptides were internalized by the cell at 4 and there little homology both in the primary and secondary structures among these membrane-permeable peptides except that they have several arginine residues in the sequences. These results strongly suggest the possible presence of the common and internalization mechanisms among the arginine-rich basic peptides. As one more the features of the we have that the number of arginine residues has a significant on the of internalization and that there to be an optimal number of arginine residues for the internalization. still such efficient translocation is possible for the arginine-rich peptides. of arginine with B. S. D. Frankel A.D. Science. PubMed Scopus Google Scholar) or with such as B. 1997; PubMed Scopus Google Scholar) may be in the during the However, as was in the case of the is not to the mechanism only by of peptides on the membranes. Tat-(48–60) has been reported to various proteins into the cells not only into cultured cells but also into the various of a living mouse (4Schwarze S.R. Ho A. Vocero-Akbani A. Dowdy S.F. Science. 1999; 285: 1569-1572Crossref PubMed Scopus (2200) Google Scholar). As the arginine-rich peptides to have a similar ability as of proteins, study of the peptides may in peptides to specific cells by or with the help of other peptides. The results obtained not only on the possible presence of of ubiquitous transmembrane mechanisms for the arginine-rich peptides, but also on the of carrier molecules for the intracellular protein We are to S. and Y. and H. for

BACKGROUND: Foodborne diseases are globally important, resulting in considerable morbidity and mortality. Parasitic diseases often result in high burdens of disease in low and middle income countries and are frequently transmitted to humans via contaminated food. This study presents the first estimates of the global and regional human disease burden of 10 helminth diseases and toxoplasmosis that may be attributed to contaminated food. METHODS AND FINDINGS: Data were abstracted from 16 systematic reviews or similar studies published between 2010 and 2015; from 5 disease data bases accessed in 2015; and from 79 reports, 73 of which have been published since 2000, 4 published between 1995 and 2000 and 2 published in 1986 and 1981. These included reports from national surveillance systems, journal articles, and national estimates of foodborne diseases. These data were used to estimate the number of infections, sequelae, deaths, and Disability Adjusted Life Years (DALYs), by age and region for 2010. These parasitic diseases, resulted in 48.4 million cases (95% Uncertainty intervals [UI] of 43.4-79.0 million) and 59,724 (95% UI 48,017-83,616) deaths annually resulting in 8.78 million (95% UI 7.62-12.51 million) DALYs. We estimated that 48% (95% UI 38%-56%) of cases of these parasitic diseases were foodborne, resulting in 76% (95% UI 65%-81%) of the DALYs attributable to these diseases. Overall, foodborne parasitic disease, excluding enteric protozoa, caused an estimated 23.2 million (95% UI 18.2-38.1 million) cases and 45,927 (95% UI 34,763-59,933) deaths annually resulting in an estimated 6.64 million (95% UI 5.61-8.41 million) DALYs. Foodborne Ascaris infection (12.3 million cases, 95% UI 8.29-22.0 million) and foodborne toxoplasmosis (10.3 million cases, 95% UI 7.40-14.9 million) were the most common foodborne parasitic diseases. Human cysticercosis with 2.78 million DALYs (95% UI 2.14-3.61 million), foodborne trematodosis with 2.02 million DALYs (95% UI 1.65-2.48 million) and foodborne toxoplasmosis with 825,000 DALYs (95% UI 561,000-1.26 million) resulted in the highest burdens in terms of DALYs, mainly due to years lived with disability. Foodborne enteric protozoa, reported elsewhere, resulted in an additional 67.2 million illnesses or 492,000 DALYs. Major limitations of our study include often substantial data gaps that had to be filled by imputation and suffer from the uncertainties that surround such models. Due to resource limitations it was also not possible to consider all potentially foodborne parasites (for example Trypanosoma cruzi). CONCLUSIONS: Parasites are frequently transmitted to humans through contaminated food. These estimates represent an important step forward in understanding the impact of foodborne diseases globally and regionally. The disease burden due to most foodborne parasites is highly focal and results in significant morbidity and mortality among vulnerable populations.

To characterize fullerenes (C(60) and C(70)) as photosensitizers in biological systems, the generation of active oxygen species, through energy transfer (singlet oxygen (1)O(2)) and electron transfer (reduced active oxygen radicals such as superoxide anion radical O(2)(-)* and hydroxyl radical *OH), was studied by a combination of methods, including biochemical (DNA-cleavage assay in the presence of various scavengers of active oxygen species), physicochemical (EPR radical trapping and near-infrared spectrometry), and chemical methods (nitro blue tetrazolium (NBT) method). Whereas (1)O(2) was generated effectively by photoexcited C(60) in nonpolar solvents such as benzene and benzonitrile, we found that O(2)(-)* and *OH were produced instead of (1)O(2) in polar solvents such as water, especially in the presence of a physiological concentration of reductants including NADH. The above results, together with those of a DNA cleavage assay in the presence of various scavengers of specific active oxygen species, indicate that the active oxygen species primarily responsible for photoinduced DNA cleavage by C(60) under physiological conditions are reduced species such as O(2)(-)* and *OH.

Dietary and xenobiotic compounds can disrupt endocrine signaling, particularly of steroid receptors and sexual differentiation. Evidence is also mounting that implicates environmental agents in the growing epidemic of obesity. Despite a long-standing interest in such compounds, their identity has remained elusive. Here we show that the persistent and ubiquitous environmental contaminant, tributyltin chloride (TBT), induces the differentiation of adipocytes in vitro and increases adipose mass in vivo. TBT is a dual, nanomolar affinity ligand for both the retinoid X receptor (RXR) and the peroxisome proliferator-activated receptor gamma (PPARgamma). TBT promotes adipogenesis in the murine 3T3-L1 cell model and perturbs key regulators of adipogenesis and lipogenic pathways in vivo. Moreover, in utero exposure to TBT leads to strikingly elevated lipid accumulation in adipose depots, liver, and testis of neonate mice and results in increased epididymal adipose mass in adults. In the amphibian Xenopus laevis, ectopic adipocytes form in and around gonadal tissues after organotin, RXR, or PPARgamma ligand exposure. TBT represents, to our knowledge, the first example of an environmental endocrine disrupter that promotes adipogenesis through RXR and PPARgamma activation. Developmental or chronic lifetime exposure to organotins may therefore act as a chemical stressor for obesity and related disorders.

The Mesp1 gene encodes the basic HLH protein MesP1 which is expressed in the mesodermal cell lineage during early gastrulation. Disruption of the Mesp1 gene leads to aberrant heart morphogenesis, resulting in cardia bifida. In order to study the defects in Mesp1-expressing cells during gastrulation and in the specification of mesodermal cell lineages, we introduced a (beta)-galactosidase gene (lacZ) under the control of the Mesp1 promoter by homologous recombination. The early expression pattern revealed by (beta)-gal staining in heterozygous embryos was almost identical to that observed by whole mount in situ hybridization. However, the (beta)-gal activity was retained longer than the mRNA signal, which enabled us to follow cell migration during gastrulation. In heterozygous embryos, the Mesp1-expressing cells migrated out from the primitive streak and were incorporated into the head mesenchyme and heart field. In contrast, Mesp1-expressing cells in the homozygous deficient embryos stayed in the primitive streak for a longer period of time before departure. The expression of FLK-1, an early marker of endothelial cell precursors including heart precursors, also accumulated abnormally in the posterior region in Mesp1-deficient embryos. In addition, using the Cre-loxP site-specific recombination system, we could determine the lineage of the Mesp1-expressing cells. The first mesodermal cells that ingressed through the primitive streak were incorporated as the mesodermal component of the amnion, and the next mesodermal population mainly contributed to the myocardium of the heart tube but not to the endocardium. These results strongly suggest that MesP1 is expressed in the heart tube precursor cells and is required for mesodermal cells to depart from the primitive streak and to generate a single heart tube.

The initial microglial responses that occur after brain injury and in various neurological diseases are characterized by microglial accumulation in the affected sites of brain that results from the migration and proliferation of these cells. The early-phase signal responsible for this accumulation is likely to be transduced by rapidly diffusible factors. In this study, the possibility of ATP released from injured neurons and nerve terminals affecting cell motility was determined in rat primary cultured microglia. Extracellular ATP and ADP induced membrane ruffling and markedly enhanced chemokinesis in Boyden chamber assay. Further analyses using the Dunn chemotaxis chamber assay, which allows direct observation of cell movement, revealed that both ATP and ADP induced chemotaxis of microglia. The elimination of extracellular calcium or treatment with pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, suramin, or adenosine-3'-phosphate-5'-phosphosulfate did not inhibit ATP- or ADP-induced membrane ruffling, whereas AR-C69931MX or pertussis toxin treatments clearly did so. As an intracellular signaling molecule underlying these phenomena, the small G-protein Rac was activated by ATP and ADP stimulation, and its activation was also inhibited by pretreatment with pertussis toxin. These results strongly suggest that membrane ruffling and chemotaxis of microglia induced by ATP or ADP are mediated by G(i/o)-coupled P2Y receptors.

BACKGROUND: Once- or twice-weekly consumption of fish (or a small amount of fish intake) reduces the risk of coronary heart disease and sudden cardiac death in Western countries. It is uncertain whether a high frequency or large amount of fish intake, as is the case in Japan, further reduces the risk. METHODS AND RESULTS: To examine an association between high intake of fish and n3 polyunsaturated fatty acids and the risk of coronary heart disease, a total of 41,578 Japanese men and women aged 40 to 59 years who were free of prior diagnosis of cardiovascular disease and cancer and who completed a food frequency questionnaire were followed up from 1990-1992 to 2001. After 477,325 person-years of follow-up, 258 incident cases of coronary heart disease (198 definite and 23 probable myocardial infarctions and 37 sudden cardiac deaths) were documented, comprising 196 nonfatal and 62 fatal coronary events. The multivariable hazard ratios (HRs) and 95% confidence intervals in the highest (8 times per week, or median intake=180 g/d) versus lowest (once a week, or median intake=23 g/d) quintiles of fish intake were 0.63 (0.38 to 1.04) for total coronary heart disease, 0.44 (0.24 to 0.81) for definite myocardial infarction, and 1.14 (0.36 to 3.63) for sudden cardiac death. The reduced risk was primarily observed for nonfatal coronary events (HR=0.43 [0.23 to 0.81]) but not for fatal coronary events (HR=1.08 [0.42 to 2.76]). Strong inverse associations existed between dietary intake of n3 fatty acids and risk of definite myocardial infarction (HR=0.35 [0.18 to 0.66]) and nonfatal coronary events (HR=0.33 [0.17 to 0.63]). CONCLUSIONS: Compared with a modest fish intake of once a week or &20 g/d, a higher intake was associated with substantially reduced risk of coronary heart disease, primarily nonfatal cardiac events, among middle-aged persons.

The workshop was designed to present what is known about the production of micronuclei, what protocols are now accepted or proposed internationally, what new results have been obtained, and what new methods and protocols are likely to be forthcoming. This report is designed to convey the flavour of the workshop and to provide the essence of the new information. After the workshop an effort was made to determine what single protocol would satisfy the requirements set for the micronucleus test by as many regulatory agencies as possible. The result, reported here, includes the requirements of six regulatory authorities in Canada, the European Economic Community, the Organization for Economic Co-operation and Development, Japan, and the United States.

Neuropathic pain is an expression of pathological operation of the nervous system, which commonly results from nerve injury and is characterized by pain hypersensitivity to innocuous stimuli, a phenomenon known as tactile allodynia. The mechanisms by which nerve injury creates tactile allodynia have remained largely unknown. We report that the development of tactile allodynia following nerve injury requires activation of p38 mitogen-activated protein kinase (p38MAPK), a member of the MAPK family, in spinal microglia. We found that immunofluorescence and protein levels of the dually phosphorylated active form of p38MAPK (phospho-p38MAPK) were increased in the dorsal horn ipsilateral to spinal nerve injury. Interestingly, the phospho-p38MAPK immunofluorescence in the dorsal horn was found exclusively in microglia, but not in neurons or astrocytes. The level of phospho-p38MAPK immunofluorescence in individual microglial cells was much higher in the hyperactive phenotype in the ipsilateral dorsal horn than the resting one in the contralateral side. Intrathecal administration of the p38MAPK inhibitor, 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580), suppresses development of the nerve injury-induced tactile allodynia. Taken together, our results demonstrate that nerve injury-induced pain hypersensitivity depends on activation of the p38MAPK signaling pathway in hyperactive microglia in the dorsal horn following peripheral nerve injury.

Although microglia have long been considered as brain resident immune cells, increasing evidence suggests that they also have physiological roles in the development of the normal CNS. In this study, we found large numbers of activated microglia in the forebrain subventricular zone (SVZ) of the rat from P1 to P10. Pharmacological suppression of the activation, which produces a decrease in levels of a number of proinflammatory cytokines (i.e., IL-1β, IL-6, TNF-α, and IFN-γ) significantly inhibited neurogenesis and oligodendrogenesis in the SVZ. In vitro neurosphere assays reproduced the enhancement of neurogenesis and oligodendrogenesis by activated microglia and showed that the cytokines revealed the effects complementarily. These results suggest that activated microglia accumulate in the early postnatal SVZ and that they enhance neurogenesis and oligodendrogenesis via released cytokines.

Thirty-mer single-stranded oligonucleotides, with a sequence chosen from the known cDNA encoding the 64-kDa protein named Ag A or the MPB-70 protein of Mycobacterium bovis BCG and the human cellular proteins such as complement component 1 inhibitor and Ig rearranged lambda-chain, were used to dissect the capability to induce IFN and to augment NK cell activity of mouse spleen cells by coincubation in vitro. Three with the hexamer palindromic sequence as GACGTC were active, whereas two kinds of oligonucleotides with no palindrome were inactive. The oligonucleotides containing at least one of the different palindromic sequences showed no activity. When a portion of the sequence of the inactive oligonucleotides was substituted with either palindromic sequence of GACGTC, AGCGCT, or AACGTT, the oligonucleotide acquired the ability to augment NK activity. In contrast, the oligonucleotides substituted with another palindromic sequence such as ACCGGT was without effect. Furthermore, exchange of two neighboring mononucleotides within, but not outside, the active palindromic sequence destroyed the ability of the oligonucleotides to augment NK cell activity. Stimulation of spleen cells with the substituted oligonucleotide, A4a-AAC, induced production of significant amounts of IFN-alpha/beta and small amounts of IFN-gamma. Augmentation of NK activity of the cells by the oligonucleotide was ascribed to IFN-alpha/beta production. These results strongly suggest that the presence of the unique palindromic sequences, such as GACGTC, AGCGCT, and AACGTT, but not ACCGGT, is essential for the immunostimulatory activity of oligonucleotides.

Toxicogenomics focuses on assessing the safety of compounds using gene expression profiles. Gene expression signatures from large toxicogenomics databases are expected to perform better than small databases in identifying biomarkers for the prediction and evaluation of drug safety based on a compound's toxicological mechanisms in animal target organs. Over the past 10 years, the Japanese Toxicogenomics Project consortium (TGP) has been developing a large-scale toxicogenomics database consisting of data from 170 compounds (mostly drugs) with the aim of improving and enhancing drug safety assessment. Most of the data generated by the project (e.g. gene expression, pathology, lot number) are freely available to the public via Open TG-GATEs (Toxicogenomics Project-Genomics Assisted Toxicity Evaluation System). Here, we provide a comprehensive overview of the database, including both gene expression data and metadata, with a description of experimental conditions and procedures used to generate the database. Open TG-GATEs is available from http://toxico.nibio.go.jp/english/index.html.

The implementation of the ICH S7B and E14 guidelines has been successful in preventing the introduction of potentially torsadogenic drugs to the market, but it has also unduly constrained drug development by focusing on hERG block and QT prolongation as essential determinants of proarrhythmia risk. The Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative was established to develop a new paradigm for assessing proarrhythmic risk, building on the emergence of new technologies and an expanded understanding of torsadogenic mechanisms beyond hERG block. An international multi-disciplinary team of regulatory, industry and academic scientists are working together to develop and validate a set of predominantly nonclinical assays and methods that eliminate the need for the thorough-QT study and enable a more precise prediction of clinical proarrhythmia risk. The CiPA effort is led by a Steering Team that provides guidance, expertise and oversight to the various working groups and includes partners from US FDA, HESI, CSRC, SPS, EMA, Health Canada, Japan NIHS, and PMDA. The working groups address the three pillars of CiPA that evaluate drug effects on: 1) human ventricular ionic channel currents in heterologous expression systems, 2) in silico integration of cellular electrophysiologic effects based on ionic current effects, the ion channel effects, and 3) fully integrated biological systems (stem-cell-derived cardiac myocytes and the human ECG). This article provides an update on the progress of the initiative towards its target date of December 2017 for completing validation.

Acute myocardial infarction (AMI) is a common and lethal heart disease, and the recruitment of fibroblastic cells to the infarct region is essential for the cardiac healing process. Although stiffness of the extracellular matrix in the infarct myocardium is associated with cardiac healing, the molecular mechanism of cardiac healing is not fully understood. We show that periostin, which is a matricellular protein, is important for the cardiac healing process after AMI. The expression of periostin protein was abundant in the infarct border of human and mouse hearts with AMI. We generated periostin(-/-) mice and found no morphologically abnormal cardiomyocyte phenotypes; however, after AMI, cardiac healing was impaired in these mice, resulting in cardiac rupture as a consequence of reduced myocardial stiffness caused by a reduced number of alpha smooth muscle actin-positive cells, impaired collagen fibril formation, and decreased phosphorylation of FAK. These phenotypes were rescued by gene transfer of a spliced form of periostin. Moreover, the inhibition of FAK or alphav-integrin, which blocked the periostin-promoted cell migration, revealed that alphav-integrin, FAK, and Akt are involved in periostin signaling. Our novel findings show the effects of periostin on recruitment of activated fibroblasts through FAK-integrin signaling and on their collagen fibril formation specific to healing after AMI.

Antigen is thought to cross-link membrane-bound immunoglobulins (Igs) of B cells, causing proliferation and differentiation or the inhibition of growth. Protein tyrosine kinases are probably involved in signal transduction for cell proliferation and differentiation. The Src-like protein tyrosine kinase Lyn is expressed preferentially in B cells. The Lyn protein and its kinase activity could be coimmunoprecipitated with IgM from detergent lysates. Cross-linking of membrane-bound IgM induced a rapid increase in tyrosine phosphorylation of at least ten distinct proteins of B cells. Thus, Lyn is physically associated with membrane-bound IgM, and is suggested to participate in antigen-mediated signal transduction.

In the rabbit mesenteric arterial smooth muscle skinned by saponin, Ca2+ induced contraction in a concentration-dependent manner. Guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), a non-hydrolyzable GTP analogue, lowered the Ca2+ concentrations required for this contraction and increased the Ca2+ sensitivity of the skinned smooth muscle contraction. GTP gamma S alone did not induce the contraction in the absence of Ca2+. This GTP gamma S-enhanced Ca2+ sensitivity was completely abolished by an exoenzyme of Staphylococcus aureus, named EDIN, and an exoenzyme of Clostridium botulinum, named C3, both of which are known to ADP-ribosylate the rho p21 family that belongs to the ras p21-like small GTP-binding protein superfamily. The GTP gamma S-bound form of rhoA p21 overcame the inhibitory action of EDIN. smg p21B, another small GTP-binding protein, was inactive. EDIN ADP-ribosylated a protein, which was most likely to be rho p21, in the skinned smooth muscle. The GTP gamma S-bound form of rhoA p21, but not the GDP-bound form, substituted for GTP gamma S and enhanced the Ca2+ sensitivity of the skinned smooth muscle contraction. smg p21B was inactive. These results indicate that rhoA p21 is involved in the GTP gamma S-enhanced Ca2+ sensitivity of the smooth muscle contraction.

Preclinical toxicity studies have demonstrated that exposure of laboratory animals to liver enzyme inducers during preclinical safety assessment results in a signature of toxicological changes characterized by an increase in liver weight, hepatocellular hypertrophy, cell proliferation, and, frequently in long-term (life-time) studies, hepatocarcinogenesis. Recent advances over the last decade have revealed that for many xenobiotics, these changes may be induced through a common mechanism of action involving activation of the nuclear hormone receptors CAR, PXR, or PPARα. The generation of genetically engineered mice that express altered versions of these nuclear hormone receptors, together with other avenues of investigation, have now demonstrated that sensitivity to many of these effects is rodent-specific. These data are consistent with the available epidemiological and empirical human evidence and lend support to the scientific opinion that these changes have little relevance to man. The ESTP therefore convened an international panel of experts to debate the evidence in order to more clearly define for toxicologic pathologists what is considered adverse in the context of hepatocellular hypertrophy. The results of this workshop concluded that hepatomegaly as a consequence of hepatocellular hypertrophy without histologic or clinical pathology alterations indicative of liver toxicity was considered an adaptive and a non-adverse reaction. This conclusion should normally be reached by an integrative weight of evidence approach.