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Heparin is a sulfated glycosaminoglycan (GAG), which contains N-acetylated or N-sulfated glucosamine (GlcN). Heparin, which is generally obtained from the healthy porcine intestines, is widely used as an anticoagulant during dialysis and treatments of thrombosis such as disseminated intravascular coagulation. Dermatan sulfate (DS) and chondroitin sulfate (CS), which are galactosamine (GalN)-containing GAGs, are major process-related impurities of heparin products. The varying DS and CS contents between heparin products can be responsible for the different anticoagulant activities of heparin. Therefore, a test to determine the concentrations of GalN-containing GAG is essential to ensure the quality and safety of heparin products. In this study, we developed a method for determination of relative content of GalN from GalN-containing GAG in heparin active pharmaceutical ingredients (APIs). The method validation and collaborative study with heparin manufacturers and suppliers showed that our method has enough specificity, sensitivity, linearity, repeatability, reproducibility, and recovery as the limiting test for GalN from GalN-containing GAGs. We believe that our method will be useful for ensuring quality, efficacy, and safety of pharmaceutical heparins. On July 30, 2010, the GalN limiting test based on our method was adopted in the heparin sodium monograph in the Japanese Pharmacopoeia., (Copyright © 2013 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

Heparin sodium and heparin calcium, which are widely used as anti-coagulants, are known to potentially contain the natural impurity dermatan sulfate (DS). Recently serious adverse events occurred in patients receiving heparin sodium in the US, and a contaminant oversulfated chondroitin sulfate (OSCS) was found to be a cause of the events. To ensure the quality and safety of pharmaceutical heparins, there is need of a physicochemical identification test that can discriminate heparin from the heparin-related substances as well as a sensitive purity test for OSCS. Recently, HPLC with a strong-anion exchange column was proposed as the methods for identifying heparin and determination of OSCS in heparin sodium. Although this method is convenient and easy to perform, the only column suitable for this purpose is the Dionex IonPac AS11-HC column. In this study, we developed alternative identification test and test for OSCS in both heparin sodium and heparin calcium using a weak anion-exchange column. The identification test allowed for separation of heparin from the impurity DS and contaminant OSCS in a shorter time. The purity test provided enough sensitivity, specificity, linearity, recovery and repeatability for OSCS. We believe that our methods will be useful for quality control of pharmaceutical heparins., (Copyright 2010 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.)

Percomorpha has been described as the "(unresolved) bush at the top" of the teleostean phylogenies and its intrarelationships are intrinsically difficult to solve because of its huge diversity (>15,000 spp.) and ill-defined higher taxa. Patterns of facial nerves, such as those of the ramus lateralis accessorius (RLA), have been considered as one of the candidate characters to delimit a monophyletic group within the percomorphs. Six families of the suborder Percoidei (Arripidae, Dichistiidae, Kyphosidae, Terapontidae, Kuhliidae, and Oplegnathidae) and suborder Stromateoidei (including six families) share the unique pattern 10 of RLA and it has been suggested that those fishes form a monophyletic group across the two perciform suborders. To evaluate the usefulness of the RLA pattern 10 as a phylogenetic marker within the percomorphs, we newly determined whole mitochondrial genome (mitogenome) sequences for the 13 species having RLA pattern 10 and their putatively, closely-related species (5 spp.). Unambiguously aligned sequences (14,263 bp) from those 18 species plus 50 percomrphs and two outgroups (total 70 species) were subjected to partitioned maximum likelihood and Bayesian analyses. The resulting trees clearly indicated that there were at least two independent origins of the unique facial nerve pattern: one in a common ancestor of Kyphosidae, Terapontidae, Kuhliidae, and Oplegnathidae and another one in that of the percoid Arripidae and Stromateoidei. Thus further detailed anatomical studies are needed to clarify the homology of this character between the two lineages. It should be noted that the latter two taxa (Arripidae and Stromateoidei) formed an unexpected, highly-supported monophyletic group together with Scombridae and possibly Chiasmodontidae and Bramidae, all lacking RLA pattern 10 (the former two are members of other perciform suborders Scombroidei and Trachinoidei, respectively). This novel, trans-subordinal clade has never been suggested by any morphological studies, although they share a common ecological characteristic, dwelling in the pelagic realm and often associated with long-distance migrations.

Series Atherinomorpha, with its plentiful number of species and highly diversified ecological and morphological characters, is the most successful fish group at the surface layer of the ocean and many freshwater habitats, comprising 1552 species classified into three orders, six suborders, 21 families, and 193 genera. The group includes one of the most important research model organisms, the medaka (Oryzias latipes), together with diverse fishes with morphological, physiological, and ecological specializations, such as highly developed pectoral fins to glide, self-fertilization, and live-bearing. In this study, we examined the whole mitochondrial genomes (mitogenomes) from 17 species representing all of the three orders and six suborders within Atherinomorpha, with data from 70 additional percomorph species as ingroups, and two non-percomorph outgroup species. We subjected the unambiguously aligned mitogenome sequences to partitioned maximum likelihood and Bayesian phylogenetic analyses. The resulting phylogenies recovered a monophyletic Atherinomorpha within the Percomorpha, and demonstrated its phylogenetic affinity to the percomorph fishes (including cichlids) spawning demersal eggs with filaments. This study, further, provided the first molecular evidence for the monophyly of the respective atherinomorph orders (Atheriniformes, Beloniformes, and Cyprinodontiformes) with high posterior probabilities and mostly high bootstrap values, providing an important basis for the future studies on the phylogeny and evolution of this diverse group.

The fishes currently recognized as members of the order Gasterosteiformes (sticklebacks, pipefishes, and their relatives) number 278 species, classified into two suborders (Gasterosteoidei and Syngnathoidei), 11 families and 71 genera. Members of this group exhibit unique appearances, many of which are derived from armored bodies with bony plates in various forms. Although recent molecular phylogenetic studies have repeatedly questioned the monophyly of this order, none of the studies examined all of the representative families and the phylogenetic reality of the group has remained unclear. In this study, we examined whole mitochondrial genome (mitogenome) sequences from 13 gasterosteiform species representing all 11 families in the order, and subjected them to partitioned maximum likelihood and Bayesian analyses, with additional data from other percomorphs and outgroups (75 mitogenome sequences considered overall, including 10 newly determined). The resultant phylogenies indicated explicitly that previously recognized members of Gasterosteiformes had diverged basally within the Percomorpha into three different clades with the following subgroups: Syngnathoidei, Gasterosteoidei (minus Indostomidae), and Indostomidae. Monophyly of the order Gasterosteiformes and any combinations of the three subgroups were confidently rejected by statistical tests. Syngnathoidei (together with Dactylopteroidei) formed a monophyletic group, a sister-group relationship between Gasterosteoidei (minus Indostomidae) and Zoarcoidei was reconfirmed and Indostomidae was nested within the Synbranchiformes, rendering the latter group paraphyletic. Our study demonstrates a new perspective of gasterosteiform phylogeny, which will provide fundamental information for future studies of phylogeny, systematics, and evolution.

Tetraodontiformes includes approximately 350 species assigned to nine families, sharing several reduced morphological features of higher teleosts. The order has been accepted as a monophyletic group by many authors, although several alternative hypotheses exist regarding its phylogenetic position within the higher teleosts. To date, acanthuroids, zeiforms, and lophiiforms have been proposed as sister-groups of the tetraodontiforms. The monophyly and sister-group status was investigated using whole mitochondrial genome (mitogenome) sequences from 44 purposefully-chosen species (26 sequences newly-determined during the study) that fully represent the major tetraodontiform lineages plus all the groups that have been hypothesized as being close relatives. Partitioned Bayesian analyses were conducted with the three datasets that comprised concatenated nucleotide sequences from 13 protein-coding genes (with and without, or with RY-coding, 3rd codon positions), plus 22 transfer RNA and two ribosomal RNA genes. The resultant trees were well resolved and largely congruent, with most internal branches being supported by high posterior probabilities. Mitogenomic data strongly supported the monophyly of tetraodontiform fishes, placing them as a sister-group of either Lophiiformes plus Caproidei or Caproidei only. The sister-group relationship between Acanthuroidei and Tetraodontiformes was statistically rejected using Bayes factors. These results were confirmed by a reanalysis of the previously published nuclear RAG1 gene sequences using the Bayesian method. Within the Tetraodontiformes, however, monophylies of the three superfamilies were not recovered and further taxonomic sampling and subsequent efforts should clarify these relationships.

Cardinalfishes of the genus Apogon (Apogonidae) are one of the most speciose (>200 species) and numerically dominant fishes in coral reefs. Although the genus is divided into 10 subgenera, more than 70% of the species are included in the subgenus Ostorhinchus, most having either horizontal or vertical lines on the body. The phylogenetic relationship among 32 species of subgenus Ostorhinchus and 11 species of four other subgenera of Apogon, based on mitochondrially encoded 12S and 16S ribosomal genes and intervening tRNA(Val) gene, were investigated, using two species of the apogonid genus Fowleria as outgroups. The analyses demonstrated that Ostorhinchus (the most speciose subgenus) was polyphyletic, comprising at least three lineages, Ostorhinchus I, II, and III. Ostorhinchus I included two species, A. (O.) amboinensis and A. (O.) sangiensis, being a sister group to subgenus Zoramia. Ostorhinchus II and III included species with horizontal and vertical lines on the body, respectively. The respective monophylies of the latter two groups, together with a molecular clock calibration, indicated that in the evolutionary history of the genus, basic stripe patterns evolved first (more than 20 million years BP), with subsequent pattern diversification and modification.

The genus Pseudolabrus comprises 11 species of marine nearshore fishes which are antitropically distributed: two species occur in East Asia, the remaining nine species being distributed in the Southern Hemisphere, mainly in the temperate Pacific. The distributions of their closely allied genera, collectively called "pseudolabrines" are, however, restricted to the Australia-New Zealand region. The molecular phylogeny of six of the 11 Pseudolabrus species from both Hemispheres and four of the five other pseudolabrine genera was reconstructed from nucleotide sequence data from mitochondrial DNA 12S rRNA, tRNAVal, and 16S rRNA genes. Both parsimony and Bayesian analyses were performed. Results are not consistent with a previous phylogenetic hypothesis based on osteological data, particularly in the relationship between Pseudolabrus and Notolabrus, indicating a probable need for reviewing the status of Notolabrus (or the delimitations of both Pseudolabrus and Notolabrus). The two Northern Hemisphere species of Pseudolabrus were monophyletic and nested deep into the clade of the Southern Hemisphere pseudolabrines, which indicates that both pseudolabrines and Pseudolabrus originated in the Southern Hemisphere. A dispersal rather than vicariance explanation for the antitropical distribution of Pseudolabrus is more parsimonious given the number of dispersal events, extinctions, and evolutionary adaptations required under the phylogeny. Based on molecular clock calibrations, the transequatorial divergence was suggested to be early to mid Pliocene at the earliest., (Copyright 2003 Elsevier Inc.)

A recent preliminary study using complete mitochondrial DNA sequences from 48 species of teleosts has suggested that higher teleostean phylogenies should be reinvestigated on the basis of more intensive taxonomic sampling. As a second step towards the resolution of higher teleostean phylogenies, which have been described as the "(unresolved) bush at the top of the tree," we reanalyzed their relationships using mitogenomic data from 100 purposefully chosen species that fully represented all of the higher teleostean orders, except for the Batrachoidiformes. Unweighted and weighted maximum parsimony analyses were conducted with the data set that comprised concatenated nucleotide sequences from 12 protein-coding genes (excluding 3rd codon positions) and 21 transfer RNA (tRNA) genes (stem regions only) from each species. The resultant trees were well resolved and largely congruent, with most internal branches being supported by high statistical values. All major, comprehensive groups above ordinal level as currently defined in higher teleosts (with the exception of the Neoteleostei and several monotypic groups), such as the Eurypterygii, Ctenosquamata, Acanthomorpha, Paracanthopterygii, Acanthopterygii, and Percomorpha, appeared to be nonmonophyletic in the present tree. Such incongruities largely resulted from differences in the placement and/or limits of the orders Ateleopodiformes, Lampridiformes, Polymixiiformes, Ophidiiformes, Lophiiformes, Beryciformes, Stephanoberyciformes, and Zeiformes, long-standing problematic taxa in systematic ichthyology. Of these, the resulting phylogenetic positions of the Ophidiiformes and Lophiiformes were totally unexpected, because, although they have consistently been considered relatively primitive groups within higher teleosts (Paracanthopterygii), they were confidently placed within a crown group of teleosts, herein called the Percomorpha. It should be noted that many unexpected, but highly supported relationships were found within the Percomorpha, being highly promising for the next investigative step towards resolution of this remarkably diversified group of teleosts.

Background: Findings from the Life Span Study (LSS) of the health effects of exposure to atomic bomb radiation have documented a strong dose-response relation between radiation exposure and breast cancer incidence., Purpose: We analyzed data from the LSS cohort to identify nonradiation risk factors for breast cancer and to determine whether these factors were independent of the effects of radiation on breast cancer occurrence., Methods: Breast cancer incidence was ascertained among a cohort of 22,200 residents of Hiroshima and Nagasaki, Japan, who had completed a mail survey between 1979 and 1981 to study nonradiation risk factors for disease. During the subsequent follow-up period (average 8.31 years), 161 cases of primary breast cancer were identified through population-based tumor registries in the two cities., Results: The risk of breast cancer was inversely related to age at menarche and weakly positive in relation to age at menopause and years of menstruation. A significant negative association of full-term pregnancy against breast cancer was observed, although the number of pregnancies beyond the first was not related to the rate of breast cancer in the cohort. Women having their first full-term pregnancy before age 30 were at decreased risk of breast cancer relative to older women, but there was no trend. A nonsignificant, positive trend in risk was associated with increasing weight and body mass (kg/m2). The risk of breast cancer among women with a history of estrogen use was 1.64 (95% confidence interval 1.02-2.64) and with diabetes 2.06 (95% confidence interval 1.27-3.34). It was not possible to distinguish among additive and multiplicative models of the joint association of radiation dose and various non-radiation-related exposures (age at menarche, full-term pregnancy, female hormone preparations) identified in this analysis., Conclusions: Nonradiation risk factors for breast cancer among Japanese atomic bomb survivors were consistent with those identified among other populations of women, although the prevalence of common risk factors was low. Reproductive factors and hormone use appear to act independently of radiation exposure on the risk of breast cancer among this population.

The binding of tropomyosin (Tm) to actin filaments was studied using the rotary shadowing technique. Electron micrographs showed, in addition to free Tm molecules, some Tm molecules bound to actin filaments at only one end creating "tethered molecules." The ratio of free and bound Tm was determined by comparing densities of free Tm molecules in micrographs with those in Tm alone specimens. At a 1 to 7 ratio of 17 nM chicken gizzard Tm (gTm) and 120 nM actin filaments, the sum of the tethered (7%) and the free was only 29% of total Tm. The missing Tm molecules are likely to be those bound to actin filaments lengthwise and not visualized by the technique. At a lower ratio of Tm (3 nM) to actin (120 nM), the majority of bound Tm appeared to be tethered. At a lower actin concentration but a higher ratio of Tm (12 nM) to actin (24 nM), 57% of bound Tm appeared to be tethered. The populations of free and tethered molecules were also affected by different salt and glycerol concentrations or type of Tm used: more with rabbit skeletal (sTm) than with gTm. The tethering indicates that the interaction of Tm molecules to actin filaments is stronger at one end than at the rest of the molecule. The bundling of actin filaments was observed under certain conditions (e.g., approximately 2 microM actin and approximately 0.5 microM sTm), suggesting that both ends of Tm molecules can bind to actin filaments but one end binds much more weakly than the other. All of these observations support the fact that free and tethered molecules are under rapid equilibrium and that incidental encounter of the ends of two tethered Tm molecules which are at the right positions, or of one tethered and a free molecule, would strengthen the remaining actin binding site(s), resulting in tight binding, thus, the tethered molecules represent a weak form of binding.

It has become accepted that deep-freeze-drying at or below -90 degrees C is necessary to preserve the structure of supramolecular assemblies such as actin filaments (AFs) for metal shadowing. This has kept the metal shadowing technique from widespread use in the study of proteins complexed with AFs because of the limited availability of the apparatus for deep-freeze-drying. I report here that adsorption to freshly cleaved mica, treatment with buffered uranyl acetate in glycerol solution, rinsing, and removal of liquid eliminate the need of freeze-drying to preserve the structure of AFs. This technique, in combination with metal shadowing, was applied to the study of AFs decorated with heavy meromyosin (HMM). It was observed that (1) when HMM molecules are associated with single AFs in the majority of cases only one head of each HMM molecule makes contact at the point furthest from the neck region; (2) binding of HMM causes bundling of AFs, probably by the two heads of each molecule binding different filaments; and (3) the binding of HMM to the bundled AFs appears to be more stable than that to a single AF. This method of specimen preparation requires no freeze-drying and is therefore easily applicable to other large protein complexes.

A method was devised to maintain a very low angle (2-3 degrees) during the metal casting of specimens for electron microscopy. With this modified rotary shadowing procedure the melting of myosin and tropomyosin (TM) was investigated. When protein solutions were sprayed on mica sheets and then heated to melt alpha-helices, myosin molecules did not show any sign of chain separation but appeared to have collapsed into loose clumps. A few molecules showed separation of the two chains at the light meromyosin-heavy meromyosin hinge region. Heating myosin in bulk solution at 65 degrees C before spraying caused extensive fusing of the myosin heads. In contrast, in the case of TM, separation of the chains appeared to occur at temperatures at which the unfolding of alpha-helices had been shown by circular dichroism. Dissolution of TM and myosin in 0.5% SDS followed by 150-fold dilution led to single chain species. This method capable of detecting single chain peptides of melting TM whose thickness is of the order of 1 nm may be applicable to the study of the structure of proteins previously not considered possible.