A microCT-based atlas of the central nervous system and midgut in sea spiders (Pycnogonida) sheds first light on evolutionary trends at the family level.

BACKGROUND: Pycnogonida (sea spiders) is the sister group of all other extant chelicerates (spiders, scorpions and relatives) and thus represents an important taxon to inform early chelicerate evolution. Notably, phylogenetic analyses have challenged traditional hypotheses on the relationships of the major pycnogonid lineages (families), indicating external morphological traits previously used to deduce inter-familial affinities to be highly homoplastic. This erodes some of the support for phylogenetic information content in external morphology and calls for the study of additional data classes to test and underpin in-group relationships advocated in molecular analyses. In this regard, pycnogonid internal anatomy remains largely unexplored and taxon coverage in the studies available is limited. RESULTS: Based on micro-computed X-ray tomography and 3D reconstruction, we created a comprehensive atlas of in-situ representations of the central nervous system and midgut layout in all pycnogonid families. Beyond that, immunolabeling for tubulin and synapsin was used to reveal selected details of ganglionic architecture. The ventral nerve cord consistently features an array of separate ganglia, but some lineages exhibit extended composite ganglia, due to neuromere fusion. Further, inter-ganglionic distances and ganglion positions relative to segment borders vary, with an anterior shift in several families. Intersegmental nerves target longitudinal muscles and are lacking if the latter are reduced. Across families, the midgut displays linear leg diverticula. In Pycnogonidae, however, complex multi-branching diverticula occur, which may be evolutionarily correlated with a reduction of the heart. CONCLUSIONS: Several gross neuroanatomical features are linked to external morphology, including intersegmental nerve reduction in concert with trunk segment fusion, or antero-posterior ganglion shifts in partial correlation to trunk elongation/compaction. Mapping on a recent phylogenomic phylogeny shows disjunct distributions of these traits. Other characters show no such dependency and help to underpin closer affinities in sub-branches of the pycnogonid tree, as exemplified by the tripartite subesophageal ganglion of Pycnogonidae and Rhynchothoracidae. Building on this gross anatomical atlas, future studies should now aim to leverage the full potential of neuroanatomy for phylogenetic interrogation by deciphering pycnogonid nervous system architecture in more detail, given that pioneering work on neuron subsets revealed complex character sets with unequivocal homologies across some families.

A Taxonomic List of Sea Spiders (Arthropoda, Pycnogonida) from Japanese Waters.

A literature-based list of sea spiders (pycnogonids) recorded in Japanese waters since 1891 is provided. The list includes a comprehensive collection of sampling records with geographical and bathymetric data. The present list reveals that 171 species of pycnogonids in 34 genera and 11 families have been recorded in Japanese waters. From the last comprehensive counting in 1991, 17 species have been added to the list of Japanese pycnogonid fauna over the past 30 years. The present species number nearly reaches the previously estimated species number (173 spp. in 2010). However, more species are expected in further collections from abyssal and even shallow waters.

Neostygarctus lovedeluxe n. sp. from the Miyako Islands, Japan: the first record of Neostygarctidae (Heterotardigrada: Arthrotardigrada) from the Pacific.

A new species of the previously monospecific marine tardigrade family Neostygarctidae is described. Neostygarctus lovedeluxe n. sp. was found from a submarine cave in Miyako Islands, Japan. This is the first record of Neostygarctidae from the Pacific. The new species is easily distinguished from the previously known N. acanthophorus by its number of dorsal spines, as N. lovedeluxe has two spines each on the three dorsal body plates in contrast to one in N. acanthophorus. Furthermore the morphology of the two clawed juvenile is reported for the first time in Neostygarctidae, providing new insights into the common sequence in some ontogenic traits.

Early development of the Japanese spiny oyster (Saccostrea kegaki): characterization of some genetic markers.

The phylum Mollusca is one of the major groups of Lophotrochozoa. Although mollusks exhibit great morphological diversity, only a few comparative embryological studies have been performed on this group. In the present study, to begin understanding the molecular development of the diverse morphology among mollusks, we observed early embryogenesis in a bivalve, the Japanese spiny oyster, Saccostrea kegaki. Although several studies have begun to reveal the genetic machinery for early development in gastropods, very little molecular information is available on bivalve embryogenesis. Thus, as a step toward identifying tissue-specific gene markers, we sequenced about 100 cDNA clones picked randomly from a gastrula-stage cDNA library. This basic information on bivalve embryology will be useful for further studies on the development and evolution of mollusks.

Structure-affinity relationship in the interactions of human organic anion transporter 1 with caffeine, theophylline, theobromine and their metabolites.

It is well known that human organic anion transporter 1 (hOAT1) transports many kinds of drugs, endogenous compounds, and toxins. However, little is known about the structure-affinity relationship. The aim of this study was to elucidate the structure-affinity relationship using a series of structurally related compounds that interact with hOAT1. Inhibitory effects of xanthine- and uric acid-related compounds on the transport of p-aminohippuric acid were examined using CHO-K1 cells stably expressing hOAT1. The order of potency for the inhibitory effects of xanthine-related compounds on PAH uptake was 1-methyl derivative>7-methyl derivative>3-methyl derivative falling dotsxanthine>1,3,7-trimethyl derivative (caffeine). The order of potency of the inhibition was 1,3,7-trimethyluric acid>1,3-dimethyluric acid>1,7-dimethyluric acid>1-methyluric acid>uric acid. A significant correlation between inhibitory potency and lipophilicity of the tested uric acid-related compounds was observed. The main determinant of the affinity of xanthine-related compounds is the position of the methyl group. On the other hand, lipophilicity is the main determinant of the affinity of uric acid-related compounds.

Uptake of dipeptide and beta-lactam antibiotics by the basolateral membrane vesicles prepared from rat kidney.

The transport of dipeptides and beta-lactam antibiotics across the rat renal basolateral membrane was examined. The initial uptake of glycylsarcosine and cefadroxil by rat renal basolateral membrane vesicles was inhibited by the presence of all the di- and tripeptides and beta-lactam antibiotics that were tested in this study. However, the uptake of both substrates was not inhibited by glycine, an amino acid. The initial uptake of zwitterionic beta-lactam antibiotics, cefadroxil, cephradine, and cephalexin, was stimulated by preloaded glycylsarcosine (countertransport effect). On the other hand, the uptake of dianionic beta-lactam antibiotics, ceftibuten and cefixime, was not affected. A concentration-dependent initial uptake of glycylsarcosine and cefadroxil suggested the existence of a carrier-mediated mechanism, whereas the transport of ceftibuten did not show any saturated uptake. The transporter that participates in the permeation of dipeptides and beta-lactam antibiotics across basolateral membranes showed lower affinity than did PEPT1 and PEPT2. This is the first study that showed an evidence for a peptide transporter, expressed in the rat renal basolateral membrane, that recognizes zwitterionic beta-lactam antibiotics using basolateral membrane vesicles isolated from normal rat kidney.

Ionic strength has a greater effect than does transmembrane electric potential difference on permeation of tryptamine and indoleacetic acid across Caco-2 cells.

The effects of transmembrane electric potential difference and ionic strength on the permeation of tryptamine and indoleacetic acid across a Caco-2 cell monolayer were examined. A decrease in the transmembrane electric potential difference caused by the addition of potassium ion to the transport buffer had no effect on the permeation rate of either compound. On the other hand, an increase in ionic strength resulted in a decrease in the permeation rate of tryptamine and an increase in the permeation rate of indoleacetic acid. The changes in the permeation rate with changes in the ionic strength were correlated with the membrane surface potential monitored by 1-anilino-8-naphthalenesulfonic acid (ANS), a fluorescent probe. We tested these effects using several other cationic and anionic compounds. These effects of ionic strength were found to be common to all drugs tested. The compound that showed a relatively lower permeation rate was given relatively stronger effect. The possibility of overestimation or underestimation caused by these effects should be considered when the permeation of an ionic compound is evaluated using a cell monolayer system.

Phenolsulfonphthalein transport by potential-sensitive urate transport system.

The purpose of this study was to elucidate the transporter-mediated secretion systems for phenolsulfonphthalein in brush-border membranes. In human and rat renal brush-border membranes, a potential-sensitive transport system has been shown to be involved in the efflux of organic anions. The uptake of phenolsulfonphthalein into rat renal brush-border membrane vesicles was stimulated by an inside-positive membrane potential. This potential-sensitive uptake of phenolsulfonphthalein was inhibited by probenecid, pyrazinoate and urate. p-Aminohippurate had no effect on the potential-sensitive uptake of phenolsulfonphthalein. Moreover, urate competitively inhibited the uptake of phenolsulfonphthalein. On the other hand, the uptake of phenolsulfonphthalein was slightly increased in the presence of an outward Cl- gradient. These results suggest that phenolsulfonphthalein has high affinity for the potential-sensitive urate transport system but has low affinity for an anion exchanger.

Characterization of secretory intestinal transport of phenolsulfonphthalein.

It is known that secretory transport limits the oral bioavailability of certain drugs. However, there is little information on the secretion of anionic compounds in the intestine. Phenolsulfonphthalein (PSP) and p-aminohippuric acid (PAH) have been used widely as substrates for organic anion transport systems. PAH is transported in the secretory direction in the intestine. It is possible that PSP and PAH share the same transport system at the mucosal membrane. The purpose of this study was to characterize the transport system for PSP in the intestine. In the jejunum, the serosal-to-mucosal permeation rate of PSP was significantly reduced in an ATP-depleted condition, whereas a significant difference was not observed in the ileum. Some multidrug resistance-associated protein 2 (Mrp2) inhibitors inhibited PSP permeation in the jejunum. However, pravastatin, a substrate of Mrp2, did not inhibit the PSP permeation. The jejunal secretory transport of pravastatin was significantly reduced in an ATP-depleted condition and by addition of probenecid, but PSP did not affect the jejunal permeation of pravastatin. These results suggest that PSP is secreted into the intestinal lumen by Mrp2-like transporter and that two Mrp2 substrates, PSP and pravastatin, are likely to be transported by different transport systems at the mucosal membrane.

The use of an in vitro dissolution and absorption system to evaluate oral absorption of two weak bases in pH-independent controlled-release formulations.

The aim of this study was to compare the oral absorption of two weak bases including their pH-independent controlled-release preparations using an in vitro evaluation system. This system is able to simulate dissolution of drugs, pH change and permeation of drugs through the epithelial cell membrane in the gastrointestinal tract. Albendazole-polymers solid dispersion and pH-independent sustained-release granules of dipyridamole were prepared by using a solvent method. Elution profiles and predicted absorption of these preparations in gastric pH conditions similar to those in healthy subjects and patients with achlorhydria were compared with those of a physical mixture and commercial tablets. When a physical mixture or commercial tablets were used, the elution profile and predicted absorption of both albendazole and dipyridamole were extremely pH-dependent. On the other hand, when a solid dispersion and granules were used, elution and predicted absorption were not affected by changes in pH of the flowing solution in a drug-dissolving vessel. These results are in agreement with the results of our previous in vivo study using gastric acidity-controlled rabbits. Our results suggest that this in vitro system is useful for the evaluation of oral absorption of pH-independent controlled-release preparations.

Phylogenetic position of Loricifera inferred from nearly complete 18S and 28S rRNA gene sequences.

BACKGROUND: Loricifera is an enigmatic metazoan phylum; its morphology appeared to place it with Priapulida and Kinorhyncha in the group Scalidophora which, along with Nematoida (Nematoda and Nematomorpha), comprised the group Cycloneuralia. Scarce molecular data have suggested an alternative phylogenetic hypothesis, that the phylum Loricifera is a sister taxon to Nematomorpha, although the actual phylogenetic position of the phylum remains unclear. METHODS: Ecdysozoan phylogeny was reconstructed through maximum-likelihood (ML) and Bayesian inference (BI) analyses of nuclear 18S and 28S rRNA gene sequences from 60 species representing all eight ecdysozoan phyla, and including a newly collected loriciferan species. RESULTS: Ecdysozoa comprised two clades with high support values in both the ML and BI trees. One consisted of Priapulida and Kinorhyncha, and the other of Loricifera, Nematoida, and Panarthropoda (Tardigrada, Onychophora, and Arthropoda). The relationships between Loricifera, Nematoida, and Panarthropoda were not well resolved. CONCLUSIONS: Loricifera appears to be closely related to Nematoida and Panarthropoda, rather than grouping with Priapulida and Kinorhyncha, as had been suggested by previous studies. Thus, both Scalidophora and Cycloneuralia are a polyphyletic or paraphyletic groups. In addition, Loricifera and Nematomorpha did not emerge as sister groups.

An ascidian RING finger gene is specifically expressed in a single cell of larval ocellus.

The ascidian nervous system is extremely simple, although the structure of it is comparable with the complex vertebrate nervous system. This simplicity makes the ascidian nervous system a good model to understand how the neuronal circuit is built up in the chordate nervous system. In order to study the formation of the neuronal circuit at the single cell level, molecular markers to characterize specific single cells are desired. In the present paper, we describe the gene expression pattern of CIGL: an ascidian homologue of Goliath, a Drosophila RING-finger gene. In the early embryonic stage, CiGl is expressed in the lateral part of the neural tube and in several peripheral nerve cells. Later in the larval stage, CiGl specifically marks ocellus: one of the pigment cells in the ascidian brain, which is involved in the photoreceptive system. CiGl will be useful to understand the differentiation mechanism of ocellus, and especially to test the model proposed by. In addition, the finding of this single cells specific gene expression pattern at a certain developmental stage encourages us to look for more genes which mark single cells, especially those that have not been well characterized.

Major role of organic anion transporters in the uptake of phenolsulfonphthalein in the kidney.

Phenolsulfonphthalein is used for testing renal function. However, its excretion mechanism has not been elucidated. The purpose of this study was therefore to elucidate the transporter-mediated excretion system for phenolsulfonphthalein. p-Aminohippuric acid, a substrate of rat organic anion transporter1 (rOat1), and cimetidine, a substrate of rOat3, reduced the urinary excretion of phenolsulfonphthalein. The uptake of phenolsulfonphthalein by kidney slices was found to consist of two components. The IC50 values of rOat1 substrates were higher than those of rOat3 substrates. In the presence of cimetidine, the Eadie-Hofstee plot gave a single straight line. The profile of the phenolsulfonphthalein uptake component in the presence of cimetidine was similar to that of the low-affinity component in the absence of cimetidine. We conclude that rOat1 and rOat3 are involved in the renal uptake of phenolsulfonphthalein and that phenolsulfonphthalein is a high-affinity substrate for rOat3 but is a relatively low-affinity substrate for rOat1.

Mechanism of active secretion of phenolsulfonphthalein in the liver via Mrp2 (abcc2), an organic anion transporter.

Phenolsulfonphthalein (PSP) has been selected as a model drug that is eliminated from both the kidney and liver in rats. Although the renal PSP transport system has been studied, few details of the biliary excretion of PSP have been reported. We investigated the biliary excretion system for PSP in rats. It has been reported that the biliary excretion of many organic anions from hepatocytes into bile is mediated by a primary active transporter, referred to as multidrug resistance-associated protein 2 (Mrp2/abcc2). The biliary excretion of PSP in SD rats was significantly decreased in the presence of Mrp2 inhibitors. The biliary excretion of PSP in Eisai hyperbilirubinemic rats (EHBR), hereditarily Mrp2-defective rats, was significantly lower than that in SD rats. Moreover, an efflux experiment using Caco-2 cells was carried out to confirm Mrp2-mediated PSP transport. Mrp2 inhibitors significantly decreased PSP efflux from Caco-2 cells. These results suggest that Mrp2 contributes to the biliary excretion of PSP in SD rats.

Comparison of the disposition behavior of organic anions in an animal model for Wilson's disease (Long-Evans Cinnamon rats) with that in normal Long-Evans Agouti rats.

Long-Evans Cinnamon (LEC) rats have an abnormality similar to that observed in Wilson's disease in humans and are therefore a good animal model for the study of Wilson's disease. LEC rats develop hereditary hepatitis and severe jaundice. Mutant animals with hyperbilirubinemia have been widely used as animal models for human diseases. Among these mutant animals, Eisai hyperbilirubinemic rats (EHBR) have defective biliary excretion of organic anions. Thus, biliary excretion of sulfobromophthalein (BSP) and urinary excretion of phenolsulfonphthalein (PSP) in LEC rats were compared with those in Long-Evans Agouti (LEA) rats. In LEC rats, the excretion of BSP, a multidrug resistance-associated protein 2 (Mrp2/Abcc2) substrate, was significantly decreased compared to that in LEA rats. It has been reported that the transport function for organic anions on the kidney is maintained in EHBR. However, the urinary excretion of PSP is impaired in LEC rats. It is possible that organic anion transporters responsible for the urinary excretion of PSP in LEA rats and EHBR are impaired in LEC rats. It is important to elucidate the relationship between organic anion secretion and Wilson's disease.

Inhibitory effects of basic drugs on the sodium-dependent transport of L-alanine via system B0 in the small intestine.

To elucidate the mechanism of the interaction of basic drugs with Na(+)-dependent L-alanine absorption from the small intestine, we investigated the effect of imipramine on the transport of L-alanine via system B(0), which is thought to be one of the main Na(+)-dependent systems for intestinal absorption of short-chain neutral amino acids, including L-alanine. The uptake of L-alanine by cells that express hATB(0) (human amino acid transporter B(0)) was inhibited in the presence of imipramine. The Eadie-Hofstee plot showed that the inhibition was not competitive with the substrate (L-alanine) but competitive with Na(+). When rat intestinal brush border membrane vesicles were used, several basic drugs had inhibitory effects. Inhibition was also observed in intestinal absorption evaluated by an in situ single-pass perfusion technique. However, the potencies of inhibition were different. The potency of inhibition was dependent on the lipophilicity of the drugs.

Liquid chromatographic method for the determination of ganciclovir and/or acyclovir in human plasma using pulsed amperometric detection.

We have developed a simple, rapid and highly sensitive method for determining plasma concentrations of ganciclovir and/or acyclovir by using reversed-phase chromatography followed by pulsed amperometric detection. A linear relationship between the amount of ganciclovir (0.05-10 microg/ml plasma) or acyclovir (0.1-20 microg/ml plasma) and peak height ratio was obtained. The relative standard deviations of all standard curves were greater than or equal to 0.999. The limits of detection for ganciclovir and acyclovir quantitation were 10 ng/ml and 50 ng/ml (signal/noise >3), respectively. Daily fluctuations of plasma standard curves (n=5) for the ganciclovir and acyclovir samples were small, with relative standard deviations (RSD) of 3.3 and 4.5% (n=5), respectively. The intra-assay precision for the ganciclovir and acyclovir samples were 6.9 (n=5) and 5.5% (n=5), respectively. Inter-assay precision of ganciclovir (n=3) and acyclovir (n=3) ranged from 2.6 to 6.8% and 3.5 to 5.0%, respectively. Using this method, the pharmacokinetics and removal of ganciclovir during continuous hemodiafiltration (CHDF) in a liver transplant recipient being treated for severe cytomegalovirus infection was investigated. The mean (+/-SD) ratio of ganciclovir concentrations at the inlet and outlet of the dialyzer (C(outlet)/C(inlet)) was 0.56+/-0.09. The areas under the curves of ganciclovir up to 12 h postdosing (AUC(0-->12)) at the inlet and outlet of the dialyzer were 12.54 microg h/ml and 7.16 microg h/ml, respectively. The ultrafiltrate of ganciclovir was 16.6 mg. The terminal elimination half-life (T(1/2)) of ganciclovir during CHDF was 3.6 h. These results demonstrate that CHDF effectively removes ganciclovir. Until formal guidelines have been established, ganciclovir or acyclovir dosage should be adjusted according to the results of monitoring of plasma drug concentration. The method described here is suitable for clinical monitoring of plasma ganciclovir or acyclovir levels in solid organ transplant recipients and for use in studies involving pharmacokinetics.

A new system for the prediction of drug absorption using a pH-controlled Caco-2 model: evaluation of pH-dependent soluble drug absorption and pH-related changes in absorption.

One purpose of this study was to develop a new system for the prediction of pH-dependent soluble drug absorption that takes into account the physiological condition of the gastrointestinal tract. Another purpose was to establish several models of different gastric acidities: a normal gastric acidity model, a low gastric acidity model (a model of achlorhydria), a temporarily elevated gastric acidity model (a model of a case in which an acidic drug was coadministered to temporarily elevate gastric acidity in the case of low gastric acidity), a weak antacid model (a model of a case in which a weak antacid drug, such as an H(2) receptor antagonist, was coadministered to temporarily elevate pH up to 6), and a strong antacid model (a model of a case in which a strong antacid drug, such as magnesium hydroxide, was coadministered to temporarily elevate pH up to 8.0). These models were used to evaluate variation in pH-related absorption in humans. Dipyridamole preparation (Persantin tablets) and glibenclamide preparation (Euglucon tablets), both poorly water-soluble and pH-dependent soluble drugs, were chosen as model drugs to determine whether absorption is altered by changes in levels of gastric acid. The extent of absorption of dipyridamole was remarkably lower when gastric pH was continuously elevated to 6.0, whereas it was increased when gastric pH temporarily decreased to 1.8. The extent of absorption of glibenclamide increased dramatically when gastric pH temporarily increased to 8.0, but did not change when gastric pH temporarily increased to 6.0. These results are consistent with reported results obtained in clinical studies. The results suggest that pH-related variations in absorption in humans can be accurately predicted using our new system.

Comparison of urinary excretion of phenolsulfonphthalein in an animal model for Wilson's disease (Long-Evans Cinnamon rats) with that in normal Wistar rats: involvement of primary active organic anion transporter.

PURPOSE: The aim of this study was to determine the cause of the decline in phenolsulfonphthalein (PSP) excretion in Long-Evans Cinnamon (LEC) rats. METHODS: The uptake of PSP into rat renal basolateral membrane vesicles (BLMV) was studied. Cyclosporin A (CYA) was used to modulate an ATP-dependent primary active transporter. PSP was intravenously injected into rats with or without CYA. The transcellular transport of PSP was examined by using primary cultured renal proximal tubule cells (PTC). RESULTS: No significant difference was found between the uptake of PSP into renal BLMV of Wistar rats and that into renal BLMV of LEC rats. In the presence of CYA, the urinary excretion and the plasma concentrations of PSP in Wistar rats were decreased and increased, respectively. In primary cultured renal PTC from Wistar rats, the basal-to-apical transport of PSP was greater than that in the opposite direction and the basal-to-apical transport of PSP was substantially reduced by the addition of CYA. However, CYA did not affect the basal-to-apical transport of PSP in PTC from LEC rats. CONCLUSIONS: The results suggest that PSP is transported by primary active organic anion transporter and that the activity level of this transporter is reduced in LEC rats.

An in vitro system for prediction of oral absorption of relatively water-soluble drugs and ester prodrugs.

We developed an in vitro system simulating the physiological condition in the gastrointestinal (GI) tract for prediction of oral absorption of relatively water-soluble drugs and ester prodrug pivampicillin. This evaluation system includes a drug-dissolving vessel (DDV, assumed stomach), a pH adjustment vessel (PAV, assumed intestine) and a side-by-side diffusion chamber that is mounted by a Caco-2 monolayer, which is grown on a polycarbonate filter, or by a rat intestine between the donor and receiver compartments. Our proposed system can accommodate large amounts of solid drugs, simulating a drastic pH change process in GI tract, that is, an orally administered solid drug is dissolved in the stomach (pH 1-2) and transferred to the intestine (pH 6), and that dissolution process can also be monitored. The optimal flow rates for our system are 0.35-1.10 ml/min. Using this system, cumulative permeations of eight relatively water-soluble drugs were compared, and these cumulative permeations indicated the ability of drug absorption in humans. Drugs that permeated across a Caco-2 monolayer at cumulative permeation of more than 0.03% or over 0.04% in rat intestine can be almost completely absorbed in humans. If the cumulative permeation across a Caco-2 monolayer is lower than 0.03% or below 0.04% in the rat intestine, there was a good linear correlation between cumulative permeation across a Caco-2 monolayer and oral absorption in humans, or between cumulative permeation across a rat intestine and oral absorption in humans. In the case of relatively water-soluble drugs, a good linear correlation was obtained between cumulative permeation across a Caco-2 monolayer and cumulative permeation across a rat intestine. This result indicates that it is possible to predict the oral absorption of a relatively water-soluble drug in humans based on the cumulative permeation of the drug across a Caco-2 monolayer and/or a rat intestine. The time course of permeation of the ester prodrug pivampicillin, which is metabolized in a Caco-2 monolayer or in a rat intestine, was also evaluated. It stated clearly that it is also possible to predict the oral absorption of pivampicillin in humans based on the cumulative permeation across a Caco-2 monolayer or rat intestine. Our newly developed system enables more kinds of oral preparations and also pH-dependent soluble drugs to be evaluated.