Immunization with inactivated whole virus particle influenza virus vaccines improves the humoral response landscape in cynomolgus macaques.

Although antibody-inducing split virus vaccines (SV) are currently the most effective way to combat seasonal influenza, their efficacy can be modest, especially in immunologically-naïve individuals. We investigated immune responses towards inactivated whole influenza virus particle vaccine (WPV) formulations, predicated to be more immunogenic, in a non-human primate model, as an important step towards clinical testing in humans. Comprehensive analyses were used to capture 46 immune parameters to profile how WPV-induced responses differed to those elicited by antigenically-similar SV formulations. Naïve cynomolgus macaques vaccinated with either monovalent or quadrivalent WPV consistently induced stronger antibody responses and hemagglutination inhibition (HI) antibody titres against vaccine-matched viruses compared to SV formulations, while acute reactogenic effects were similar. Responses in WPV-primed animals were further increased by boosting with the same formulation, conversely to modest responses after priming and boosting with SV. 28-parameter multiplex bead array defined key antibody features and showed that while both WPV and SV induced elevated IgG responses against A/H1N1 nucleoprotein, only WPV increased IgG responses against A/H1N1 hemagglutinin (HA) and HA-Stem, and higher IgA responses to A/H1N1-HA after each vaccine dose. Antibodies to A/H1N1-HA and HA-Stem that could engage FcγR2a and FcγR3a were also present at higher levels after one dose of WPV compared to SV and remained elevated after the second dose. Furthermore, WPV-enhanced antibody responses were associated with higher frequencies of HA-specific B-cells and IFN-γ-producing CD4+ T-cell responses. Our data additionally demonstrate stronger boosting of HI titres by WPV following prior infection and support WPV administered as a priming dose irrespective of the follow up vaccine for the second dose. Our findings thus show that compared to SV vaccination, WPV-induced humoral responses are significantly increased in scope and magnitude, advocating WPV vaccination regimens for priming immunologically-naïve individuals and also in the event of a pandemic outbreak.

Conversion of Olmesartan to Olmesartan Medoxomil, A Prodrug that Improves Intestinal Absorption, Confers Substrate Recognition by OATP2B1.

PURPOSE: Olmesartan medoxomil (olmesartan-MX), an ester-type prodrug of the angiotensin II receptor blocker (ARB) olmesartan, is predominantly anionic at intestinal pH. Human organic anion transporting polypeptide 2B1 (OATP2B1) is expressed in the small intestine and is involved in the absorption of various acidic drugs. This study was designed to test the hypothesis that OATP2B1-mediated uptake contributes to the enhanced intestinal absorption of olmesartan-MX, even though olmesartan itself is not a substrate of OATP2B1. METHODS: Tetracycline-inducible human OATP2B1- and rat Oatp2b1-overexpressing HEK 293 cell lines (hOATP2B1/T-REx-293 and rOatp2b1/T-REx-293, respectively) were established to characterize OATP2B1-mediated uptake. Rat jejunal permeability was measured using Ussing chambers. ARBs were quantified by liquid chromatography-tandem mass spectrometry. RESULTS: Significant olmesartan-MX uptake was observed in hOATP2B1/T-REx-293 and rOatp2b1/T-REx-293 cells, whereas olmesartan uptake was undetectable or much lower than olmesartan-MX uptake, respectively. Furthermore, olmesartan-MX exhibited several-fold higher uptake in Caco-2 cells and greater permeability in rat jejunum compared to olmesartan. Olmesartan-MX uptake in hOATP2B1/T-REx-293 cells and in Caco-2 cells was significantly decreased by OATP2B1 substrates/inhibitors such as 1 mM estrone-3-sulfate, 100 µM rifamycin SV, and 100 µM fluvastatin. Rat Oatp2b1-mediated uptake and rat jejunal permeability of olmesartan-MX were significantly decreased by 50 µM naringin, an OATP2B1 inhibitor. Oral administration of olmesartan-MX with 50 µM naringin to rats significantly reduced the area under the plasma concentration-time curve of olmesartan to 76.9%. CONCLUSION: Olmesartan-MX is a substrate for OATP2B1, and the naringin-sensitive transport system contributes to the improved intestinal absorption of olmesartan-MX compared with its parent drug, olmesartan.

Targeted Metabolite Fingerprints of Thirteen Gambierdiscus, Five Coolia and Two Fukuyoa Species.

The genus Gambierdiscus produces an array of bioactive hydrophilic and lipophilic secondary metabolites that range in mode of action and toxicity. In this study, the metabolite fingerprint was mapped for thirteen Gambierdiscus, five Coolia and two Fukuyoa species (34 isolates) by assessing the production of 56 characterised secondary metabolites. Gambierdiscus polynesiensis was the only species to produce Pacific-ciguatoxin-3B (P-CTX3B), P-CTX3C, iso-P-CTX3B/C, P-CTX4A, P-CTX4B and iso-P-CTX4A/B. G. australes produced maitotoxin-1 (MTX-1) and MTX-5, G. cheloniae produced MTX-6 and G. honu produced MTX-7. Ubiquitous production of 44-methylgambierone was observed amongst all the Gambierdiscus isolates, with nine species also producing gambierone. Additional gambierone analogues, including anhydrogambierone (tentatively described herein), were also detected in all Gambierdiscus species, two Coolia and two Fukuyoa species. Gambieroxide was detected in G. lewisii and G. pacificus and gambieric acid A was detected in ten Gambierdiscus species, with G. australes (CAWD381) being the only isolate to produce gambieric acids A-D. This study has demonstrated that the isolates tested to date produce the known CTXs or MTXs, but not both, and highlighted several species that produced 'unknown' compounds displaying characteristics of cyclic polyethers, which will be the focus of future compound discovery efforts.

Transplacental Pharmacokinetic Model of Digoxin Based on Ex Vivo Human Placental Perfusion Study.

Digoxin is used as first-line therapy to treat fetal supraventricular tachycardia; however, because of the narrow therapeutic window, it is essential to estimate digoxin exposure in the fetus. The data from ex vivo human placental perfusion study are used to predict in vivo fetal exposure noninvasively, but the ex vivo fetal-to-maternal concentration (F:M) ratios observed in digoxin perfusion studies were much lower than those in vivo. In the present study, we developed a human transplacental pharmacokinetic model of digoxin using previously reported ex vivo human placental perfusion data. The model consists of maternal intervillous, fetal capillary, non-perfused tissue, and syncytiotrophoblast compartments, with multidrug resistance protein (MDR) 1 and influx transporter at the microvillous membrane (MVM) and influx and efflux transporters at the basal plasma membrane (BM). The model-predicted F:M ratio was 0.66, which is consistent with the mean in vivo value of 0.77 (95% confidence interval: 0.64-0.91). The time to achieve the steady state from the ex vivo perfusion study was estimated as 1,500 minutes, which is considerably longer than the reported ex vivo experimental durations, and this difference is considered to account for the inconsistency between ex vivo and in vivo F:M ratios. Reported digoxin concentrations in a drug-drug interaction study with MDR1 inhibitors quinidine and verapamil were consistent with the profiles simulated by our model incorporating inhibition of efflux transporter at the BM in addition to MVM. Our modeling and simulation approach should be a powerful tool to predict fetal exposure and DDIs in human placenta. SIGNIFICANCE STATEMENT: We developed a human transplacental pharmacokinetic model of digoxin based on ex vivo human placental perfusion studies in order to resolve inconsistencies between reported ex vivo and in vivo fetal-to-maternal concentration ratios. The model successfully predicted the in vivo fetal exposure to digoxin and the drug-drug interactions of digoxin and P-glycoprotein/multidrug resistance protein 1 inhibitors in human placenta.

Limited Impact of Murine Placental MDR1 on Fetal Exposure of Certain Drugs Explained by Bypass Transfer Between Adjacent Syncytiotrophoblast Layers.

PURPOSE: Multidrug resistance protein 1 (MDR1) is located at the interface between two syncytiotrophoblast layers in rodent placenta, and may influence fetal drug distribution. Here, we quantitatively compare the functional impact per single MDR1 molecule of MDR1 at the placental barrier and blood-brain barrier in mice. METHODS: MDR1A and MDR1B proteins were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Paclitaxel or digoxin was continuously administered to pregnant Mdr1a-/-/Mdr1b-/- or wild-type mice, and the drug concentrations in the maternal and fetal plasma and maternal brain were quantified by LC-MS/MS. RESULTS: MDR1A and MDR1B proteins are expressed in the membrane of mouse placental labyrinth, and total MDR1 at the placental barrier amounts to about 30% of that at the blood-brain barrier. The fetal-to-maternal plasma concentration ratio of digoxin was only marginally affected in Mdr1a-/-/Mdr1b-/- mice, while that of paclitaxel showed a several-fold increase. No such difference between the two drugs was found in the maternal brain distribution. The impact per single MDR1 molecule on the fetal distribution of digoxin was calculated to be much lower than that on the brain distribution, but this was not the case for paclitaxel. Our pharmacokinetic model indicates that the impact of placental MDR1 is inversely correlated to the ratio of permeability through gap junctions connecting the two syncytiotrophoblast layers to passive diffusion permeability. CONCLUSION: Our findings indicate that murine placental MDR1 has a minimal influence on the fetal concentration of certain substrates, such as digoxin, due to bypass transfer, probably via connexin26 gap junctions.

MicroRNA-126 suppresses the invasion of trophoblast-model JEG-3 cells by targeting LIN28A.

Inadequate trophoblast invasion and impaired trophoblast-induced vascular remodeling are features of preeclampsia. In this context, an angiogenesis-related microRNA, miR-126, is abnormally expressed in preeclampsia placentas, but its role in trophoblast development remains unclear. The purpose of this study was to investigate the roles of miR-126 in the proliferation, migration, and invasion processes of trophoblast cells using the human choriocarcinoma-derived JEG-3 cell line as a model. The mRNA expression profiling of JEG-3 cells with and without miR-126 overexpression, in combination with bioinformatics analysis, identified LIN28A as a putative target of miR-126. The results of real-time RT-PCR and luciferase assay were consistent with this idea. Overexpression of miR-126 in JEG-3 cells decreased the invasive ability of the cells without affecting proliferation or migration. The invasiveness of JEG-3 cells was significantly reduced to a similar extent by knockdown of LIN28A with siRNA and by miR-126-overexpression-induced downregulation of LIN28A, although the level of LIN28A protein was much lower in the siLIN28A-transfected cells. These results indicate that miR-126 suppresses JEG-3 cell invasion by targeting LIN28A, and suggest that miR-126-mediated downregulation of LIN28A might contribute to the onset/deterioration of preeclampsia.

Immune profiling of influenza-specific B- and T-cell responses in macaques using flow cytometry-based assays.

Influenza remains a significant global public health burden, despite substantial annual vaccination efforts against circulating virus strains. As a result, novel vaccine approaches are needed to generate long-lasting and universal broadly cross-reactive immunity against distinct influenza virus strains and subtypes. Several new vaccine candidates are currently under development and/or in clinical trials. The successful development of new vaccines requires testing in animal models, other than mice, which capture the complexity of the human immune system. Importantly, following vaccination or challenge, the assessment of adaptive immunity at the antigen-specific level is particularly informative. In this study, using peripheral blood mononuclear cells (PBMCs) from cynomolgus macaques, we describe detection methods and in-depth analyses of influenza virus-specific B cells by recombinant hemagglutinin probes and flow cytometry, as well as the detection of influenza virus-specific CD8+ and CD4+ T cells by stimulation with live influenza A virus and intracellular cytokine staining. We highlight the potential of these assays to be used with PBMCs from other macaque species, including rhesus macaques, pigtail macaques and African green monkeys. We also demonstrate the use of a human cytometric bead array kit in detecting inflammatory cytokines and chemokines from cynomolgus macaques to assess cytokine/chemokine milieu. Overall, the detection of influenza virus-specific B and T cells, together with inflammatory responses, as described in our study, provides useful insights for evaluating novel influenza vaccines. Our data deciphering immune responses toward influenza viruses can be also adapted to understanding immunity to other infections or vaccination approaches in macaque models.

Fluorouracil uptake in triple-negative breast cancer cells: Negligible contribution of equilibrative nucleoside transporters 1 and 2.

Equilibrative nucleoside transporters (ENTs) 1 and 2 reportedly accept fluorouracil as a substrate. Here, we evaluated ENT1/2 expression at the messenger RNA (mRNA), protein, and functional levels in a panel of four triple-negative breast cancer (TNBC) cell lines, BT-549, Hs578T, MDA-MB-231, and MDA-MB-435, and we examined the relationship of the observed profiles to fluorouracil sensitivity. Nitrobenzylthioinosine (NBMPR) at 0.1 µM inhibits only ENT1, while dipyridamole at 10 µM or NBMPR at 100 µM inhibits both ENT1 and ENT2. We found that the uptake of [3 H]uridine, a typical substrate of ENT1 and ENT2, was decreased to approximately 40% by 0.1 µM NBMPR. At 100 µM, NBMPR almost completely blocked the saturable uptake of [3 H]uridine, but this does not imply a functional role of ENT2, because 10 µM dipyridamole showed similar inhibition to 0.1 µM NBMPR. Expression of ENT1 mRNA was almost 1 order of magnitude higher than that of ENT2 in all TNBC cell lines. Liquid chromatography-tandem mass spectrometry(LC-MS/MS) LC-MS/MS-based targeted protein quantification showed that ENT1 protein levels were in the range of 9.3-30 fmol/µg protein in plasma membrane fraction of TNBC cell lines, whereas ENT2 protein was below the detection limit. [3 H]Fluorouracil uptake was insensitive to 0.1 µM NBMPR and 10 µM dipyridamole, suggesting a negligible contribution of ENT1 and ENT2 to fluorouracil uptake. The levels of ENT1 mRNA, ENT1 protein, ENT2 mRNA, and ENT1-mediated [3 H]uridine uptake in the four TNBC cell lines showed no correlation with fluorouracil sensitivity. These results indicate that neither ENT1 nor ENT2 contributes significantly to the fluorouracil sensitivity of TNBC cell lines.

Development of a Pharmacokinetic Model of Transplacental Transfer of Metformin to Predict In Vivo Fetal Exposure.

Two types of systems are used in ex vivo human placental perfusion studies to predict fetal drug exposures, that is, closed systems with recirculation of the maternal and fetal buffer and open systems using a single-pass mode without recirculation. The in vivo fetal/maternal (F:M) ratio of metformin, a cationic drug that crosses the placenta, is consistent with that reported in an open system ex vivo but not with that in a closed system. In the present study, we aimed to develop a pharmacokinetic (PK) model of transplacental transfer of metformin to predict in vivo fetal exposure to metformin and to resolve the apparent inconsistency between open and closed ex vivo systems. The developed model shows that the difference between open and closed systems is due to the difference in the time required to achieve the steady state. The model-predicted F:M ratio (approx. 0.88) is consistent with reported in vivo values [mean (95% confidence interval): 1.10 (0.69-1.51)]. The model incorporates bidirectional transport via organic cation transporter 3 (OCT3) at the basal plasma membrane, and simulations indicate that the use of trimethoprim (an OCT3 inhibitor) to prevent microbial growth in the placenta ex vivo has a negligible effect on the overall maternal-to-fetal and fetal-to-maternal clearances. The model could successfully predict in vivo fetal exposure using ex vivo human placental perfusion data from both closed and open systems. This transplacental PK modeling approach is expected to be useful for evaluating human fetal exposures to other poorly permeable compounds, besides metformin. SIGNIFICANCE STATEMENT: We developed a pharmacokinetic model of transplacental transfer of metformin, used to treat gestational diabetes mellitus, in order to predict in vivo fetal exposure and resolve the discrepancy between reported findings in open and closed ex vivo perfusion systems. The discrepancy is due to a difference in the time required to reach the steady state. The model can predict in vivo fetal exposure using data from both closed and open systems.

Adverse effects of the coexistence of locomotive syndrome and sarcopenia on the walking ability and performance of activities of daily living in Japanese elderly females: a cross-sectional study.

[Purpose] The aim of this study was to investigate whether the coexistence of locomotive syndrome and sarcopenia is associated with the risk of fall or performance of activities of daily living in elderly females. [Participants and Methods] We categorized 112 Japanese elderly female participants under three groups: control, locomotive syndrome, and locomotive syndrome and co-existing sarcopenia. We compared the groups based on the mean scores of anthropometric and physical function measurements, the Timed Up and Go test, and the Tokyo Metropolitan Institute of Gerontology Index of Competence to evaluate activities of daily living. [Results] The Timed Up and Go test score significantly differed among the groups. The score of the Timed Up and Go test significantly correlated with scores of the skeletal muscle mass index, skeletal muscle strength grading, and usual gait speed. The total score of the Tokyo Metropolitan Institute of Gerontology Index of Competence significantly decreased in all the groups. [Conclusion] The coexistence of locomotive syndrome and sarcopenia is associated with an increased risk of fall and worsened performance of activities of daily living. Further, the risk of fall is associated with the skeletal muscle mass, skeletal muscle strength, and gait speed. It seems likely that, compared to sarcopenia, locomotive syndrome is more sensitive to lower limb dysfunctions.

Transport of Pregabalin Via L-Type Amino Acid Transporter 1 (SLC7A5) in Human Brain Capillary Endothelial Cell Line.

PURPOSE: The anti-epileptic drug pregabalin crosses the blood-brain barrier (BBB) in spite of its low lipophilicity. This study was performed to determine whether L-type amino acid transporters (LAT1/SLC7A5 and LAT2/SLC7A8) contribute to the uptake of pregabalin. METHODS: Pregabalin uptake by LATs-transfected HEK293 cells or hCMEC/D3 cells, an in vitro human BBB model, was measured by LC-MS/MS analysis. Expression of LAT1 mRNA in hCMEC/D3 cells was determined by quantitative RT-PCR analysis. RESULTS: Overexpression of LAT1, but not LAT2, in HEK293 cells significantly increased the cellular uptake of pregabalin, and the LAT1-mediated uptake was saturable with a Km of 0.288 mM. LAT1-mediated amino acid uptake was inhibited specifically and almost completely in the presence of 1 mM pregabalin. The uptake of pregabalin by hCMEC/D3 cells was sodium-independent, saturable (Km = 0.854 mM), and strongly inhibited by large amino acids at 1 mM, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, a specific system L inhibitor, at 1 mM and by JPH203, a LAT1-selective inhibitor, at 10 µM. Pregabalin uptake in hCMEC/D3 cells was also decreased by 75% by the silencing of LAT1 gene using LAT1 siRNA. CONCLUSIONS: Our results indicate that LAT1, but not LAT2, recognizes pregabalin as a substrate. It is suggested that LAT1 mediates pregabalin transport at the BBB.

Examination of prezygotic and postzygotic isolating barriers in tropical Ulva (Ulvophyceae, Chlorophyta): evidence for ongoing speciation.

Phylogenetic clades based on DNA sequences such as the chloroplast rbcL gene and the nuclear ITS region are frequently used to delimit algal species. However, these molecular markers cannot accurately delimit boundaries among some Ulva species. Although Ulva reticulata and Ulva ohnoi occasionally bloom in tropical to warm-temperate regions and are clearly distinguishable by their reticulate or plain blade morphology, they have few or no sequence divergences in these molecular markers and form a monophyletic clade. In this study, to clarify the speciation and species delimitation in the U. reticulata-ohnoi complex clade, reproductive relationships among several sexual strains from the Philippines and Japan including offspring that originated from the type specimen of U. ohnoi were examined by culturing and hybridization in addition to the ITS-based analysis. As a result, both prezygotic and postzygotic reproductive isolation were revealed to occur between genetically perforated U. reticulata and imperforate U. ohnoi. They were also separated on the basis of sequence analysis of the ITS region. That strongly supports that the two taxa are independent biological species. Although no prezygotic barrier among the Philippine and Japanese strains of U. reticulata was observed, unexpectedly zoospores produced by hybrid sporophytes in some of their combinations mostly failed to develop, indicating partial formation of a postzygotic barrier despite a 0.2% divergence in the ITS sequence. These findings suggest speciation is still ongoing in U. reticulata.

Hypotaurine Is a Substrate of GABA Transporter Family Members GAT2/Slc6a13 and TAUT/Slc6a6.

Hypotaurine is a precursor of taurine and a physiological antioxidant that circulates in adult and fetal plasma. The purpose of the present study was to clarify whether hypotaurine is a substrate of Slc6a/gamma-aminobutyric acid (GABA) transporter family members. Radiolabeled hypotaurine was synthesized from radiolabeled cysteamine and 2-aminoethanethiol dioxygenase. The uptakes of [3H]GABA, [3H]taurine, and [14C]hypotaurine by HEK293 cells expressing mouse GAT1/Slc6a1, TAUT/Slc6a6, GAT3/Slc6a11, BGT1/Slc6a12, and GAT2/Slc6a13 were measured. TAUT and GAT2 showed strong [14C]hypotaurine uptake activity, while BGT1 showed moderate activity, and GAT1 and GAT3 showed slight but significant activity. Mouse TAUT and GAT2 both showed Michaelis constants of 11 µM for hypotaurine uptake. GAT2-expressing cells pretreated with hypotaurine showed resistance to H2O2-induced oxidative stress. These results suggest that under physiological conditions, TAUT and GAT2 would be major contributors to hypotaurine transfer across the plasma membrane, and that uptake of hypotaurine via GAT2 contributes to the cellular resistance to oxidative stress.

LAT1-Targeting Thermoresponsive Fluorescent Polymer Probes for Cancer Cell Imaging.

L-type amino acid transporter 1 (LAT1) is more highly expressed in cancer cells compared with normal cells. LAT1 targeting probes would therefore be a promising tool for cancer cell imaging. In this study, LAT1-targeting thermoresponsive fluorescent polymer probes based on poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) (P(NIPAAm-co-DMAAm)) were synthesized and their affinity for LAT1 was evaluated. The synthesized polymer probes interacted with LAT1 on HeLa cells, and inhibition of l-[³H]-leucine, one of the substrates for LAT1 uptake, was investigated. l-Tyrosine-conjugated P(NIPAAm-co-DMAAm) inhibited the uptake of l-[³H]-leucine, while P(NIPAAm-co-DMAAm) and l-phenylalanine-conjugated P(NIPAAm-co-DMAAm) did not. This result indicated that l-tyrosine-conjugated polymer has a high affinity for LAT1. The fluorescent polymer probes were prepared by modification of a terminal polymer group with fluorescein-5-maleimide (FL). Above the polymer transition temperature, cellular uptake of the polymer probes was observed because the polymers became hydrophobic, which enhanced the interaction with the cell membrane. Furthermore, quantitative analysis of the fluorescent probe using flow cytometry indicated that l-tyrosine-conjugated P(NIPAAm-co-DMAAm)-FL shows higher fluorescence intensity earlier than P(NIPAAm-co-DMAAm)-FL. The result suggested that cellular uptake was promoted by the LAT1 affinity site. The developed LAT1-targeting thermoresponsive fluorescent polymer probes are expected to be useful for cancer cell imaging.

The double edge to parasite escape: invasive host is less infected but more infectable.

Nonnative species that escape their native-range parasites may benefit not only from reduced infection pathology, but also from relaxed selection on costly immune defenses, promoting reallocation of resources toward growth or reproduction. However, benefits accruing from a reduction in defense could come at the cost of increased infection susceptibility. We conducted common garden studies of the shore crab Hemigrapsus sanguineus from highly parasitized native (Japan) populations and largely parasite-free invasive (USA) populations to test for differences in susceptibility to infection by native-range rhizocephalan parasites, and to explore differences in host resource allocation. Nonnative individuals showed at least 1.8 times greater susceptibility to infection than their native counterparts, and had reduced standing metabolic rates, suggesting that less of their energy was spent on physiological self-maintenance. Our results support an indirect advantage to parasite escape via the relaxation of costly physiological defenses. However, this advantage comes at the cost of heightened susceptibility, a trade-off of parasite escape that is seldom considered.

Contributions of system A subtypes to α-methylaminoisobutyric acid uptake by placental microvillous membranes of human and rat.

System A consists of three subtypes, sodium-coupled neutral amino acid transporter 1 (SNAT1), SNAT2, and SNAT4, which are all expressed in the placenta. The aim of this study was to evaluate the contributions of each of the three subtypes to total system A-mediated uptake in placental MVM of human and rat, using betaine and L-arginine as subtype-selective inhibitors of SNAT2 and SNAT4, respectively. Appropriate concentrations of betaine and L-arginine for subtype-selective inhibition in SNAT-overexpressing cells were identified. It was found that 10 mM betaine specifically and almost completely inhibited human and rat SNAT2-mediated [14C]α-methylaminoisobutyric acid ([14C]MeAIB) uptake, while 5 mM L-arginine specifically and completely inhibited [3H]glycine uptake via human SNAT4, as well as [14C]MeAIB uptake via rat SNAT4. In both human and rat placental MVM vesicles, sodium-dependent uptake of [14C]MeAIB was almost completely inhibited by 20 mM unlabeled MeAIB. L-Arginine (5 mM) partly inhibited the uptake in humans, but hardly affected that in rats. Betaine (10 mM) partly inhibited the uptake in rats, but hardly affected it in humans. These results suggest that SNAT1 is most likely the major contributor to system A-mediated MeAIB uptake by human and rat MVM vesicles and that the remaining uptake is mainly mediated by SNAT4 in humans and SNAT2 in rats. Thus, inhibition studies using betaine and L-arginine are useful to characterize the molecular mechanisms of system A-mediated transport.

System A amino acid transporter SNAT2 shows subtype-specific affinity for betaine and hyperosmotic inducibility in placental trophoblasts.

Betaine uptake is induced by hypertonic stress in a placental trophoblast cell line, and involvement of amino acid transport system A was proposed. Here, we aimed to identify the subtype(s) of system A that mediates hypertonicity-induced betaine uptake. Measurement of [(14)C]betaine uptake by HEK293 cells transiently transfected with human or rat sodium-coupled neutral amino acid transporters (SNATs), SNAT1, SNAT2 and SNAT4 revealed that only human and rat SNAT2 have betaine uptake activity. The Michaelis constants (Km) of betaine uptake by human and rat SNAT2 were estimated to be 5.3 mM and 4.6 mM, respectively. Betaine exclusively inhibited the uptake activity of SNAT2 among the rat system A subtypes. We found that rat SNAT1, SNAT2 and SNAT4 were expressed at the mRNA level under isotonic conditions, while expression of SNAT2 and SNAT4 was induced by hypertonicity in TR-TBT 18d-1 cells. Western blot analyses revealed that SNAT2 expression on plasma membrane of TR-TBT 18d-1 cells was more potently induced by hypertonicity than that in total cell lysate. Immunocytochemistry confirmed the induction of SNAT2 expression in TR-TBT 18d-1 cells exposed to hypertonic conditions and indicated that SNAT2 was localized on the plasma membrane in these cells. Our results indicate that SNAT2 transports betaine, and that tonicity-sensitive SNAT2 expression may be involved in regulation of betaine concentration in placental trophoblasts.

Effect of an Introduced Phytoene Synthase Gene Expression on Carotenoid Biosynthesis in the Marine Diatom Phaeodactylum tricornutum.

Carotenoids exert beneficial effects on human health through their excellent antioxidant activity. To increase carotenoid productivity in the marine Pennales Phaeodactylum tricornutum, we genetically engineered the phytoene synthase gene (psy) to improve expression because RNA-sequencing analysis has suggested that the expression level of psy is lower than other enzyme-encoding genes that are involved in the carotenoid biosynthetic pathway. We isolated psy from P. tricornutum, and this gene was fused with the enhanced green fluorescent protein gene to detect psy expression. After transformation using the microparticle bombardment technique, we obtained several P. tricornutum transformants and confirmed psy expression in their plastids. We investigated the amounts of PSY mRNA and carotenoids, such as fucoxanthin and ß-carotene, at different growth phases. The introduction of psy increased the fucoxanthin content of a transformants by approximately 1.45-fold relative to the levels in the wild-type diatom. However, some transformants failed to show a significant increase in the carotenoid content relative to that of the wild-type diatom. We also found that the amount of PSY mRNA at log phase might contribute to the increase in carotenoids in the transformants at stationary phase.

Morphology of Gambierdiscus scabrosus sp. nov. (Gonyaulacales): a new epiphytic toxic dinoflagellate from coastal areas of Japan.

A new epiphytic dinoflagellate is described, G ambierdiscus scabrosus sp. nov., from tidal pools and rocky shores along the coastal areas of Japan. Cells are 63.2 ± 5.7 µm in depth, 58.2 ± 5.7 µm in width, and 37.3 ± 3.5 µm in length. The plate formula of G . scabrosus is Po, 4', 0a, 6'', 6c, ?s, 5''', 0p, and 2''''. Morphologically, G . scabrosus resembles G . belizeanus as follows: anterioposteriorly compressed cell shape, narrow 2'''' plate, and areolated surface. Despite this similarity, the cells of G . scabrosus can be distinguishable by the presence of the asymmetric shaped 3'' plate and the rectangular shaped 2' plate.