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1.  Distinct Neural Representation in the Dorsolateral, Dorsomedial, and Ventral Parts of the Striatum during Fixed- and Free-Choice Tasks 
The Journal of Neuroscience  2015;35(8):3499-3514.
The striatum is a major input site of the basal ganglia, which play an essential role in decision making. Previous studies have suggested that subareas of the striatum have distinct roles: the dorsolateral striatum (DLS) functions in habitual action, the dorsomedial striatum (DMS) in goal-directed actions, and the ventral striatum (VS) in motivation. To elucidate distinctive functions of subregions of the striatum in decision making, we systematically investigated information represented by phasically active neurons in DLS, DMS, and VS. Rats performed two types of choice tasks: fixed- and free-choice tasks. In both tasks, rats were required to perform nose poking to either the left or right hole after cue-tone presentation. A food pellet was delivered probabilistically depending on the presented cue and the selected action. The reward probability was fixed in fixed-choice task and varied in a block-wise manner in free-choice task. We found the following: (1) when rats began the tasks, a majority of VS neurons increased their firing rates and information regarding task type and state value was most strongly represented in VS; (2) during action selection, information of action and action values was most strongly represented in DMS; (3) action-command information (action representation before action selection) was stronger in the fixed-choice task than in the free-choice task in both DLS and DMS; and (4) action-command information was strongest in DLS, particularly when the same choice was repeated. We propose a hypothesis of hierarchical reinforcement learning in the basal ganglia to coherently explain these results.
PMCID: PMC4339358  PMID: 25716849
action value; basal ganglia; decision making; reinforcement learning; state value; striatum
2.  Condition interference in rats performing a choice task with switched variable- and fixed-reward conditions 
Because humans and animals encounter various situations, the ability to adaptively decide upon responses to any situation is essential. To date, however, decision processes and the underlying neural substrates have been investigated under specific conditions; thus, little is known about how various conditions influence one another in these processes. In this study, we designed a binary choice task with variable- and fixed-reward conditions and investigated neural activities of the prelimbic cortex and dorsomedial striatum in rats. Variable- and fixed-reward conditions induced flexible and inflexible behaviors, respectively; one of the two conditions was randomly assigned in each trial for testing the possibility of condition interference. Rats were successfully conditioned such that they could find the better reward holes of variable-reward-condition and fixed-reward-condition trials. A learning interference model, which updated expected rewards (i.e., values) used in variable-reward-condition trials on the basis of combined experiences of both conditions, better fit choice behaviors than conventional models which updated values in each condition independently. Thus, although rats distinguished the trial condition, they updated values in a condition-interference manner. Our electrophysiological study suggests that this interfering value-updating is mediated by the prelimbic cortex and dorsomedial striatum. First, some prelimbic cortical and striatal neurons represented the action-reward associations irrespective of trial conditions. Second, the striatal neurons kept tracking the values of variable-reward condition even in fixed-reward-condition trials, such that values were possibly interferingly updated even in the fixed-reward condition.
PMCID: PMC4327310  PMID: 25741231
habit; goal-directed; reinforcement learning; Q-learning; prefrontal cortex; striatum; task switching
3.  Novel Lysophospholipid Acyltransferase PLAT1 of Aurantiochytrium limacinum F26-b Responsible for Generation of Palmitate-Docosahexaenoate-Phosphatidylcholine and Phosphatidylethanolamine 
PLoS ONE  2014;9(8):e102377.
N-3 polyunsaturated fatty acids (PUFA), such as docosahexaenoic acid (DHA, 22:6n-3), have been reported to play roles in preventing cardiovascular diseases. The major source of DHA is fish oils but a recent increase in the global demand of DHA and decrease in fish stocks require a substitute. Thraustochytrids, unicellular marine protists belonging to the Chromista kingdom, can synthesize large amounts of DHA, and, thus, are expected to be an alternative to fish oils. DHA is found in the acyl chain(s) of phospholipids as well as triacylglycerols in thraustochytrids; however, how thraustochytrids incorporate DHA into phospholipids remains unknown. We report here a novel lysophospholipid acyltransferase (PLAT1), which is responsible for the generation of DHA-containing phosphatidylcholine and phosphatidylethanolamine in thraustochytrids. The PLAT1 gene, which was isolated from the genomic DNA of Aurantiochytrium limacinum F26-b, was expressed in Saccharomyces cerevisiae, and the FLAG-tagged recombinant enzyme was characterized after purification with anti-FLAG affinity gel. PLAT1 shows wide specificity for donor substrates as well as acceptor substrates in vitro, i.e, the enzyme can adopt lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine and lysophosphatidylinositol as acceptor substrates, and 15:0/16:0-CoA and DHA-CoA as donor substrates. In contrast to the in vitro experiment, only lysophosphatidylcholine acyltransferase and lysophosphatidylethanolamine acyltransferase activities were decreased in plat1-knockout mutants, resulting in a decrease of 16:0-DHA-phosphatidylcholine (PC) [PC(38∶6)] and 16:0-DHA-phosphatidylethanolamine (PE) [PE(38∶6)], which are two major DHA-containing phospholipids in A. limacinum F26-b. However, the amounts of other phospholipid species including DHA-DHA-PC [PC(44∶12)] and DHA-DHA-PE [PE(44∶12)] were almost the same in plat-knockout mutants and the wild-type. These results indicate that PLAT1 is the enzyme responsible for the generation of 16:0-DHA-PC and 16:0-DHA-PE in the thraustochytrid.
PMCID: PMC4121067  PMID: 25090090
4.  Novel type of adenylyl cyclase participates in tabtoxinine-β-lactam-induced cell death and occurrence of wildfire disease in Nicotiana benthamiana 
Plant Signaling & Behavior  2014;9:e27420.
Tabtoxinine-β-lactam (TβL), a non-specific bacterial toxin, is produced by Pseudomonas syringae pv. tabaci, the causal agent of tobacco wildfire disease. TβL causes the plant cell death by the inhibiting glutamine synthetase, which leads to an abnormal accumulation of ammonium ions. To better understand the molecular mechanisms involved in TβL-induced cell death and necrotic wildfire lesions, we focused on adenylyl cyclase in Nicotiana benthamiana. We isolated the gene designated as NbAC (Nicotiana benthamiana adenylyl cyclase). Recombinant NbAC protein showed adenylyl cyclase activity in vitro. TβL-induced necrotic lesions were significantly suppressed in NbAC-silenced leaves compared with control plant leaves. However, the amount of ammonium ions was scarcely affected by NbAC-silencing. Furthermore, the silencing of NbAC also suppressed l-methionine sulfoximine-induced cell death without any changes in the amount of ammonium accumulated. When inoculated directly with P. syringae pv tabaci, NbAC-silenced plants showed reduced symptoms. These results suggest that NbAC might play an essential role in intracellular signal transduction during TβL-induced cell death and necrotic wildfire disease development.
PMCID: PMC4091384  PMID: 24398509
Nicotiana benthamiana; Adenylyl cyclase; cell death; tabtoxinine-β-lactam; virus-induced gene silencing
5.  Pharmacological Effects of JTT-551, a Novel Protein Tyrosine Phosphatase 1B Inhibitor, in Diet-Induced Obesity Mice 
Journal of Diabetes Research  2014;2014:680348.
Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of leptin signaling as well as insulin signaling. JTT-551 is a new PTP1B inhibitor, which is reported to improve glucose metabolism by enhancement of insulin signaling. We have evaluated an antiobesity effect of JTT-551 using diet-induced obesity (DIO) mice. A single administration of JTT-551 was provided to DIO mice with or without leptin, and DIO mice were given food containing JTT-551 for six weeks. A single administration of JTT-551 with leptin treatment enhanced the food inhibition and the signal transducer and activator of transcription 3 (STAT3) phosphorylation in hypothalamus. Moreover, chronic administration of JTT-551 showed an antiobesity effect and an improvement of glucose and lipid metabolism in DIO mice. JTT-551 shows an antiobesity effect possibly by enhancement of leptin signaling and could be useful in the treatment of type 2 diabetes and obesity.
PMCID: PMC4060493  PMID: 24987707
6.  JTT-130, a Novel Intestine-Specific Inhibitor of Microsomal Triglyceride Transfer Protein, Improves Hyperglycemia and Dyslipidemia Independent of Suppression of Food Intake in Diabetic Rats 
Journal of Diabetes Research  2014;2014:803832.
We investigated the effects of JTT-130 on glucose and lipid metabolism independent of the suppression of feeding by comparing with pair-fed animals. Male Zucker diabetic fatty (ZDF) rats were divided into control, JTT-130 treatment, and pair-fed groups. The rats were fed with a regular powdered diet with or without JTT-130 as a food admixture for 6 weeks. We compared the effects on glucose and lipid metabolism in JTT-130 treatment group with those in pair-fed group. Results. Hyperglycemia in ZDF rats was prevented in both JTT-130 treatment and pair-fed groups, but the prevention in pair-fed group became poor with time. Moreover, reduction in plasma cholesterol levels was observed only in JTT-130 treatment group. JTT-130 treatment group showed improved glucose tolerance at 5 weeks after treatment and significant elevation of portal glucagon-like peptide-1 (GLP-1) levels. The hepatic lipid content in JTT-130 treatment group was decreased as compared with pair-fed group. Furthermore, pancreatic protection effects, such as an increase in pancreatic weight and an elevation of insulin-positive area in islets, were observed after JTT-130 treatment. Conclusions. JTT-130 improves hyperglycemia and dyslipidemia via a mechanism independent of suppression of food intake, which is ascribed to an enhancement of GLP-1 secretion and a reduction of lipotoxicity.
PMCID: PMC4033514  PMID: 24895641
7.  Pseudomonas-Derived Ceramidase Induces Production of Inflammatory Mediators from Human Keratinocytes via Sphingosine-1-Phosphate 
PLoS ONE  2014;9(2):e89402.
Ceramide is important for water retention and permeability barrier functions in the stratum corneum, and plays a key role in the pathogenesis of atopic dermatitis (AD). A Pseudomonas aeruginosa-derived neutral ceramidase (PaCDase) isolated from a patient with AD was shown to effectively degrade ceramide in the presence of Staphylococcus aureus-derived lipids or neutral detergents. However, the effect of ceramide metabolites on the functions of differentiating keratinocytes is poorly understood. We found that the ceramide metabolite sphingosine-1-phosphate (S1P) stimulated the production of inflammatory mediators such as TNF-α and IL-8 from three-dimensionally cultured human primary keratinocytes (termed “3D keratinocytes”), which form a stratum corneum. PaCDase alone did not affect TNF-α gene expression in 3D keratinocytes. In the presence of the detergent Triton X-100, which damages stratum corneum structure, PaCDase, but not heat-inactivated PaCDase or PaCDase-inactive mutant, induced the production of TNF-α, endothelin-1, and IL-8, indicating that this production was dependent on ceramidase activity. Among various ceramide metabolites, sphingosine and S1P enhanced the gene expression of TNF-α, endothelin-1, and IL-8. The PaCDase-enhanced expression of these genes was inhibited by a sphingosine kinase inhibitor and by an S1P receptor antagonist VPC 23019. The TNF-α-binding antibody infliximab suppressed the PaCDase-induced upregulation of IL-8, but not TNF-α, mRNA. PaCDase induced NF-κB p65 phosphorylation. The NF-κB inhibitor curcumin significantly inhibited PaCDase-induced expression of IL-8 and endothelin-1. VPC 23019 and infliximab inhibited PaCDase-induced NF-κB p65 phosphorylation and reduction in the protein level of the NF-κB inhibitor IκBα. Collectively, these findings suggest that (i) 3D keratinocytes produce S1P from sphingosine, which is produced through the hydrolysis of ceramide by PaCDase, (ii) S1P induces the production of TNF-α via S1P receptors, and (iii) released TNF-α stimulates the production of inflammatory mediators such as IL-8.
PMCID: PMC3934885  PMID: 24586752
8.  Correction: Role of Activation-Induced Cytidine Deaminase in the Development of Oral Squamous Cell Carcinoma 
PLoS ONE  2013;8(10):10.1371/annotation/2ce2152b-b063-432c-be3f-e2836f8198d8.
PMCID: PMC3813777
9.  Utilization of transposable element mPing as a novel genetic tool for modification of the stress response in rice 
Molecular Breeding  2013;32(3):505-516.
Transposable elements (TEs) are DNA fragments that have the ability to move from one chromosomal location to another. The insertion of TEs into gene-rich regions often affects changes in the expression of neighboring genes. Miniature Ping (mPing) is an active miniature inverted-repeat TE discovered in the rice genome. It has been found to show exceptionally active transposition in a few japonica rice varieties, including Gimbozu, where mPing insertion rendered adjacent genes stress-inducible. In the Gimbozu population, it is highly possible that several genes with modified expression profiles are segregating due to the de novo mPing insertions. In our study, we utilized a screening system for detecting de novo mPing insertions in the upstream region of target genes and evaluated the effect of mPing on the stress response of the target genes. Screening for 17 targeted genes revealed five genes with the mPing insertion in their promoters. In most cases, the alteration of gene expression was observed under stress conditions, and there was no change in the expression levels of those five genes under normal conditions. These results indicate that the mPing insertion can be used as a genetic tool to modify an expression pattern of a target gene under stress conditions without changing the expression profiles of those under natural conditions.
Electronic supplementary material
The online version of this article (doi:10.1007/s11032-013-9885-1) contains supplementary material, which is available to authorized users.
PMCID: PMC3782648  PMID: 24078785
Rice; Transposable element; Transposition; mPing; Gene expression; Reverse genetics
10.  Versatile Transformation System That Is Applicable to both Multiple Transgene Expression and Gene Targeting for Thraustochytrids 
A versatile transformation system for thraustochytrids, a promising producer for polyunsaturated fatty acids and fatty acid-derived fuels, was established. G418, hygromycin B, blasticidin, and zeocin inhibited the growth of thraustochytrids, indicating that multiple selectable marker genes could be used in the transformation system. A neomycin resistance gene (neor), driven with an ubiquitin or an EF-1α promoter-terminator from Thraustochytrium aureum ATCC 34304, was introduced into representatives of two thraustochytrid genera, Aurantiochytrium and Thraustochytrium. The neor marker was integrated into the chromosomal DNA by random recombination and then functionally translated into neor mRNA. Additionally, we confirmed that another two genera, Parietichytrium and Schizochytrium, could be transformed by the same method. By this method, the enhanced green fluorescent protein was functionally expressed in thraustochytrids. Meanwhile, T. aureum ATCC 34304 could be transformed by two 18S ribosomal DNA-targeting vectors, designed to cause single- or double-crossover homologous recombination. Finally, the fatty acid Δ5 desaturase gene was disrupted by double-crossover homologous recombination in T. aureum ATCC 34304, resulting in an increase of dihomo-γ-linolenic acid (C20:3n-6) and eicosatetraenoic acid (C20:4n-3), substrates for Δ5 desaturase, and a decrease of arachidonic acid (C20:4n-6) and eicosapentaenoic acid (C20:5n-3), products for the enzyme. These results clearly indicate that a versatile transformation system which could be applicable to both multiple transgene expression and gene targeting was established for thraustochytrids.
PMCID: PMC3346472  PMID: 22344656
11.  The Role of Fibroblasts in Complex Fractionated Electrograms During Persistent/Permanent Atrial Fibrillation 
Circulation Research  2011;110(2):275-284.
Electrogram-based catheter ablation, targeting complex fractionated atrial electrograms (CFAEs), is empirically known to be effective in halting persistent/permanent atrial fibrillation (AF). However, the mechanisms underlying CFAEs and electrogram-based ablation remain unclear.
Because atrial fibrosis is associated with persistent/permanent AF, we hypothesized that electrotonic interactions between atrial myocytes and fibroblasts play an important role in CFAE genesis and electrogram-based catheter ablation.
Methods and Results
We used a human atrial tissue model in heart failure and simulated propagation and spiral wave reentry with and without regionally proliferated fibroblasts. Coupling of fibroblasts to atrial myocytes resulted in shorter action potential duration, slower conduction velocity, and lower excitability. Consequently, heterogeneous fibroblast proliferation in the myocardial sheet resulted in frequent spiral wave breakups, and the bipolar electrograms recorded at the fibroblast proliferation area exhibited CFAEs. The simulations demonstrated that ablation targeting such fibroblast-derived CFAEs terminated AF, resulting from the ablation site transiently pinning the spiral wave and then pushing it out of the fibroblast proliferation area. CFAEs could not be attributed to collagen accumulation alone.
Fibroblast proliferation in atria might be responsible for the genesis of CFAEs during persistent/ permanent AF. Our findings could contribute to better understanding of the mechanisms underlying CFAE-targeted AF ablation.
PMCID: PMC3313658  PMID: 22179057
atrial fibrillation; complex fractionated atrial electrograms; catheter ablation; spiral wave reentry; fibroblasts
12.  Increase of Eicosapentaenoic Acid in Thraustochytrids through Thraustochytrid Ubiquitin Promoter-Driven Expression of a Fatty Acid Δ5 Desaturase Gene▿† 
Applied and Environmental Microbiology  2011;77(11):3870-3876.
Thraustochytrids, marine protists known to accumulate polyunsaturated fatty acids (PUFAs) in lipid droplets, are considered an alternative to fish oils as a source of PUFAs. The major fatty acids produced in thraustochytrids are palmitic acid (C16:0), n − 6 docosapentaenoic acid (DPA) (C22:5n − 6), and docosahexaenoic acid (DHA) (C22:6n − 3), with eicosapentaenoic acid (EPA) (C20:5n − 3) and arachidonic acid (AA) (C20:4n − 6) as minor constituents. We attempted here to alter the fatty acid composition of thraustochytrids through the expression of a fatty acid Δ5 desaturase gene driven by the thraustochytrid ubiquitin promoter. The gene was functionally expressed in Aurantiochytrium limacinum mh0186, increasing the amount of EPA converted from eicosatetraenoic acid (ETA) (C20:4n − 3) by the Δ5 desaturase. The levels of EPA and AA were also increased by 4.6- and 13.2-fold in the transgenic thraustochytrids compared to levels in the mock transfectants when ETA and dihomo-γ-linolenic acid (DGLA) (C20:3n − 6) were added to the culture at 0.1 mM. Interestingly, the amount of EPA in the transgenic thraustochytrids increased in proportion to the amount of ETA added to the culture up to 0.4 mM. The rates of conversion and accumulation of EPA were much higher in the thraustochytrids than in baker's yeasts when the desaturase gene was expressed with the respective promoters. This report describes for the first time the finding that an increase of EPA could be accomplished by introducing the Δ5 desaturase gene into thraustochytrids and indicates that molecular breeding of thraustochytrids is a promising strategy for generating beneficial PUFAs.
PMCID: PMC3127612  PMID: 21478316
13.  Lipid rafts enriched in monosialylGb5Cer carrying the stage-specific embryonic antigen-4 epitope are involved in development of mouse preimplantation embryos at cleavage stage 
Lipid rafts enriched in glycosphingolipids (GSLs), cholesterol and signaling molecules play an essential role not only for signal transduction started by ligand binding, but for intracellular events such as organization of actin, intracellular traffic and cell polarity, but their functions in cleavage division of preimplantation embryos are not well known.
Here we show that monosialylGb5Cer (MSGb5Cer)-enriched raft domains are involved in development during the cleavage stage of mouse preimplantation embryos. MSGb5Cer preferentially localizes at the interfaces between blastomeres in mouse preimplantation embryos. Live-imaging analysis revealed that MSGb5Cer localizes in cleavage furrows during cytokinesis, and that by accumulating at the interfaces, it thickens them. Depletion of cholesterol from the cell membrane with methyl-beta-cyclodextrin (MbCD) reduced the expression of MSGb5Cer and stopped cleavage. Extensive accumulation of MSGb5Cer at the interfaces by cross-linking with anti-MSGb5Cer Mab (6E2) caused F-actin to aggregate at the interfaces and suppressed the localization of E-cadherin at the interfaces, which resulted in the cessation of cleavage. In addition, suppression of actin polymerization with cytochalasin D (CCD) decreased the accumulation of MSGb5Cer at the interfaces. In E-cadherin-targeted embryos, the MSGb5Cer-enriched raft membrane domains accumulated heterotopically.
These results indicate that MSGb5Cer-enriched raft membrane domains participate in cytokinesis in a close cooperation with the cortical actin network and the distribution of E-cadherin.
PMCID: PMC3089780  PMID: 21489308
14.  Mechanism of the anti-obesity effects induced by a novel Melanin-concentrating hormone 1-receptor antagonist in mice 
British Journal of Pharmacology  2009;159(2):374-383.
Background and purpose:
Melanin-concentrating hormone (MCH) is an orexigenic neuropeptide expressed in the lateral hypothalamus that is involved in feeding and body weight regulation. Intracerebroventricular infusion of a peptidic MCH1 receptor antagonist ameliorated obesity in murine models. Recently, small molecule MCH1 receptor antagonists have been developed and characterized for the treatment of obesity. However, little is known of the mechanism of the anti-obesity effects of MCH1 receptor antagonists.
Experimental approach:
To examine the mechanisms of action of the anti-obesity effect of MCH1 receptor antagonists more precisely, we conducted a pair-feeding study in mice with diet-induced obesity (DIO), chronically treated with an orally active and highly selective MCH1 receptor antagonist and examined changes in mRNA expression levels in liver, brown and white adipose tissues. We also assessed the acute effects of the MCH1 receptor antagonist in energy expenditure under thermoneutral conditions.
Key results:
Treatment with the MCH1 receptor antagonist at 30 mg·kg−1 for 1 month moderately suppressed feeding and significantly reduced body weight by 24%. In contrast, pair-feeding resulted in a smaller weight reduction of 10%. Treatment with the MCH1 receptor antagonist resulted in a higher body temperature compared with the pair-fed group. TaqMan and calorimetry data suggested that the MCH1 receptor antagonist also stimulated thermogenesis.
Conclusions and implications:
Our results indicate that an MCH1 receptor antagonist caused anti-obesity effects im mice by acting on both energy intake and energy expenditure.
PMCID: PMC2825359  PMID: 20015294
melanin-concentrating hormone; antagonist; pair-feeding; anti-obesity mechanism
15.  Threonine-5 at the N-terminus can modulate sarcolipin function in cardiac myocytes☆ 
Sarcolipin (SLN) has emerged as an important regulator of the atrial sarcoplasmic reticulum (SR) Ca2+ transport. The inhibitory effect of SLN on cardiac SR Ca2+ ATPase (SERCA) pump can be relieved by β-adrenergic stimulation, which indicates that SLN is a reversible inhibitor. However, the mechanism of this reversible regulation of SERCA pump by SLN is yet to be determined. In the current study using adult rat ventricular myocytes we provide evidence that the threonine 5 (T5) residue at the N-terminus of SLN which is conserved among various species, critically regulates the SLN function. Point mutation of T5→alanine exerts an inhibitory effect on myocyte contractility and calcium transients similar to that of wild-type SLN, whereas mutation of T5→glutamic acid which mimics the phosphorylation abolished the inhibitory function of SLN. Our results showed that T5 can be phosphorylated in vitro by calcium–calmodulin dependent protein kinase II (CaMKII). Blocking the CaMKII activity in WT-SLN overexpressing myocytes using autocamtide inhibitory peptide completely abolished the β-adrenergic response. Taken together, our data suggest that T5 is the key amino acid which modulates SLN function via phosphorylation/dephosphorylation mechanisms through CaMKII pathway.
PMCID: PMC2843622  PMID: 19631655
SLN; Sarcolipin; SERCA; Ca2+ ATPase; Phosphorylation; Calcium calmodulin dependent protein kinase II; PLB; Phospholamban
16.  Effect of Food Restriction on Adipose Tissue in Spontaneously Diabetic Torii Fatty Rats 
Experimental Diabetes Research  2009;2009:715057.
Spontaneously Diabetic Torii-fa/fa (SDT fatty) rat is a new model of obese type 2 diabetes. SDT fatty rat exhibits obesity associated with hyperphagia. In this study, SDT fatty rats were subjected to pair-feeding with SDT-+/+ (SDT) rats from 6 to 22 weeks of age. The ratio of visceral fat weight to subcutaneous fat weight (V/S) decreased at 12 weeks of age in the pair-feeding rats. The intraperitoneal fat weight such as epididymal and retroperitoneal fat weight decreased, whereas mesenteric fat weight had no change. Cell size of the epididymal fat in the pair-feeding rats tended to decrease. Glucose oxidation level in epididymal fat in the pair-feeding rats at 12 weeks of age was recovered to a similar level with that in SDT rats. These results indicated that SDT fatty rat is a useful model to evaluate the functional or the morphological features in adipose tissue and develop a novel drug for antiobesity.
PMCID: PMC2729098  PMID: 19696902
17.  Primary Colonic Signet Ring Cell Carcinoma Presenting Carcinocythemia: An Autopsy Case 
Case Reports in Gastroenterology  2008;2(3):301-307.
Primary colorectal signet ring cell carcinoma (SRCC) is a rare but distinctive type of mucin-producing adenocarcinoma of the large intestine with still controversial clinicopathological features and prognosis. We encountered primary colonic SRCC in a 51-year-old Japanese man with extensive bone metastasis ultimately leading to carcinocythemia before the initiation of chemotherapy and surgical intervention. Three days before death, besides progressive disseminated intravascular coagulation that had been present on admission, hematological examination showed sudden leukocytosis with nonhematopoietic cells that subsequently turned out to be signet ring cells (SRCs). Carcinocythemia, the presence of circulating cancer cells in peripheral blood, is considered to be a rare but an ominous phenomenon occurring in the advanced stage of certain types of cancers, particularly mammary lobular carcinoma. It can be assumed that carcinoma cells lacking intercellular cohesiveness and polarized cell membrane organization, including SRCs as well as lobular carcinoma cells, can readily get access to the peripheral circulation; however, to our knowledge, this is the first report of primary colorectal SRCC that presented carcinocythemia. Extensive bone metastatic sites, in the present case, may have functioned as a reservoir of circulating SRCs.
PMCID: PMC3075188  PMID: 21490860
Signet ring cell carcinoma; Colon; Carcinocythemia; Disseminated intravascular coagulation
18.  Crystallization and preliminary X-ray analysis of the Pax6 paired domain bound to the Pax6 gene enhancer 
The mammalian Pax6 paired domain has been cocrystallizaed with a 25 bp DNA fragment of the Pax6 gene enhancer.
Pax6 is a member of the Pax family of transcription factors and is essential for eye development. Pax6 has two DNA-binding domains: the paired domain and the homeodomain. The Pax6 paired domain is involved in Pax6 gene autoregulation by binding to its enhancer. In this study, crystallization and preliminary X-ray diffraction analysis of the mammalian Pax6 paired domain in complex with the Pax6 gene enhancer was attempted. The Pax6 paired domain complexed with an optimized 25 bp DNA fragment was crystallized by the hanging-drop vapour-diffusion method. The crystal diffracted synchrotron radiation to 3.0/3.7 Å resolution and belongs to the monoclinic space group P21, with unit-cell parameters a = 62.21, b = 70.69, c = 176.03 Å, β = 90.54°. Diffraction data were collected to 3.7 Å resolution.
PMCID: PMC1978128  PMID: 16511221
Pax6 paired domain; Pax6 gene enhancer; Pax-family proteins
19.  Purification, crystallization and preliminary crystallographic characterization of the α2,6-sialyltransferase from Photobacterium sp. JT-ISH-224 
Crystallization of the α2,6-sialyltransferase from Photobacterium.
Sialyltransferases transfer sialic acid from cytidine-5-monophospho-N-acetylneuraminic acid (CMP-NeuAc) to the nonreducing termini of the oligosaccharyl structures of various glycoproteins and glycolipids. The newly cloned α2,6-sialyltransferase from Photobacterium sp. JT-ISH-224 (from the Vibrionaceae family) is composed of two domains: an unknown N-terminal domain and a catalytic C-terminal domain which shares significant homology with the Pasteurella multocida multifunctional sialyltransferase. The putative mature form of JT-ISH-224 α2,6-sialyltransferase was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method at 293 K. The crystal belonged to space group P3121 or P3221, with unit-cell parameters a = b = 90.29, c = 204.33 Å. X-ray diffraction data were collected to 2.5 Å resolution.
PMCID: PMC2335162  PMID: 17671362
α2,6-sialyltransferase; Photobacterium sp. JT-ISH-224
20.  Regulation of the muscarinic K+ channel by extracellular ATP through membrane phosphatidylinositol 4,5-bisphosphate in guinea-pig atrial myocytes 
British Journal of Pharmacology  2005;145(2):156-165.
The present study was designed to examine the functional role of membrane phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in the regulation of the muscarinic K+ channel (IK,ACh) by extracellular ATP and adenosine in guinea-pig atrial myocytes, using the whole-cell patch-clamp method.Bath application of ATP in micromolar concentrations typically evoked a transient activation of IK,ACh; a rapid activation phase was consistently followed by a progressive decline even to the baseline level despite the continued presence of ATP. This progressive decline of IK,ACh was significantly attenuated either by blockade of phospholipase C (PLC) with compound 48/80 (100 μM) or by addition of PtdIns(4,5)P2 (50 μM) to the cell inside, suggesting that depletion of membrane PtdIns(4,5)P2 via PLC activation is mainly, if not totally, responsible for the progressive decline of IK,ACh during the presence of ATP.When atrial myocytes were exposed to wortmannin (50 μM) following ATP (50 μM) application to impair the resynthesis of PtdIns(4,5)P2, the activation of IK,ACh evoked by subsequently applied ATP (50 μM) was greatly reduced. Activation of IK,ACh by adenosine (100 μM) was partially reduced by pretreatment of atrial myocytes with ATP (100 μM) and was largely abolished by a further addition of wortmannin (50 μM) in the presence of ATP (100 μM). These results support the view that the activation of IK,ACh by ATP and adenosine depends on membrane PtdIns(4,5)P2 that is subject to reduction by extracellular ATP.The present study thus provides functional evidence to suggest that extracellular ATP activates PLC and thereby depletes membrane PtdIns(4,5)P2 that is critically involved in the activation process of IK,ACh by its agonists ATP and adenosine in guinea-pig atrial myocytes.
PMCID: PMC1576138  PMID: 15765102
IK,ACh; PtdIns(4,5)P2; ATP; P2Y receptor; wortmannin; PtdIns 4-kinase
21.  Evaluation of cardiac resynchronization therapy in drug-resistant idiopathic dilated cardiomyopathy by means of technetium-99m tetrofosmin electrocardiography-gated single-photon emission computed tomography 
The present case report describes a 72-year-old woman with drug-resistant heart failure. Cardiac resynchronization therapy was performed. Cardiac function was evaluated using a quantitative gated single-photon emission computed tomography (QGS) program with technetium-99m tetrofosmin. During atrial-right ventricular pacing, the left ventricular ejection fraction (LVEF) was 28%, end diastolic volume (EDV) was 141 mL and end systolic volume (ESV) was 101 mL. LVEF was 31%, EDV was 142 mL and ESV was 98 mL during dual chamber pacing. During atrial-left ventricular pacing, LVEF, EDV and ESV were 32%, 18 mL and 100 mL, respectively. Diastolic function was evaluated using Vcdiff software (Daiichi Radioisotope Laboratories Ltd, Japan). Cardiac resynchronization therapy may improve cardiac function, which was evaluated accurately and non-invasively by electrocardiography-gated single-photon emission computed tomography.
PMCID: PMC2716286  PMID: 19641716
Cardiac function; Cardiac resynchronization therapy; Quantitative gated SPECT
22.  Utilization of Ganglioside-Degrading Paenibacillus sp. Strain TS12 for Production of Glucosylceramide 
Applied and Environmental Microbiology  2002;68(11):5241-5248.
Gangliosides, sialic acid-containing glycosphingolipids, are membrane constituents of vertebrates and are known to have important roles in cellular differentiation, adhesion, and recognition. We report here the isolation of a bacterium capable of degrading gangliotetraose-series gangliosides and a new method for the production of glucosylceramide with this bacterium. GM1a ganglioside was found to be sequentially degraded by Paenibacillus sp. strain TS12, which was isolated from soil, as follows: GM1a → asialo GM1 → asialo GM2 → lactosylceramide → glucosylceramide. TS12 was found to produce a series of ganglioside-degrading enzymes, such as sialidases, β-galactosidases, and β-hexosaminidases. TS12 also produced β-glucosidases, but glucosylceramide was somewhat resistant to the bacterial enzyme under the conditions used. Taking advantage of the specificity, we developed a new method for the production of glucosylceramide using TS12 as a biocatalyst. The method involves the conversion of crude bovine brain gangliosides to glucosylceramide by coculture with TS12 and purification of the product by chromatography with Wakogel C-300 HG.
PMCID: PMC129906  PMID: 12406710
23.  Molecular Cloning and Expression of Mn2+-Dependent Sphingomyelinase/Hemolysin of an Aquatic Bacterium, Pseudomonas sp. Strain TK4 
Journal of Bacteriology  2002;184(2):540-546.
We report here the molecular cloning and expression of a hemolytic sphingomyelinase from an aquatic bacterium, Pseudomonas sp. strain TK4. The sphingomyelinase gene was found to consist of 1,548 nucleotides encoding 516 amino acid residues. The recombinant 57.7-kDa enzyme hydrolyzed sphingomyelin but not phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, or phosphatidylethanolamine, indicating that the enzyme is a sphingomyelin-specific sphingomyelinase C. The hydrolysis of sphingomyelin by the enzyme was found to be most efficient at pH 8.0 and activated by Mn2+. The enzyme shows quite a broad specificity, i.e., it hydrolyzed 4-nitrobenz-2-oxa-1,3-diazole (NBD)-sphingomyelin with short-chain fatty acids and NBD-sphingosylphosphorylcholine, the latter being completely resistant to hydrolysis by any sphingomyelinase reported so far. Significant sequence similarities were found in sphingomyelinases from Bacillus cereus, Staphylococcus aureus, Listeria ivanovii, and Leptospira interrogans, as well as a hypothetical protein encoded in Chromobacterium violaceum, although the first three lacked one-third of the sequence corresponding to that from the C terminus of the TK4 enzyme. Interestingly, the deletion mutant of strain TK4 lacking 186 amino acids at the C-terminal end hydrolyzed sphingomyelin, whereas it lost all hemolytic activity, indicating that the C-terminal region of the TK4 enzyme is indispensable for the hemolytic activity.
PMCID: PMC139580  PMID: 11751833
24.  Ceramidase Activity in Bacterial Skin Flora as a Possible Cause of Ceramide Deficiency in Atopic Dermatitis 
A marked decrease in the content of ceramide has been reported in the horny layer of the epidermis in atopic dermatitis (AD). This decrease impairs the permeability barrier of the epidermis, resulting in the characteristic dry and easily antigen-permeable skin of AD, since ceramide serves as the major water-holding molecule in the extracellular space of the horny layer. On the other hand, the skin of such patients is frequently colonized by bacteria, most typically by Staphylococcus aureus, possessing genes such as those for sphingomyelinase, which are related to sphingolipid metabolism. We therefore tried to identify a possible correlation between the ceramide content and the bacterial flora obtained from the skin of 25 patients with AD versus that of 24 healthy subjects, using a thin-layer chromatographic assay of the sphingomyelin-associated enzyme activities secreted from the bacteria. The findings of the assay demonstrated that ceramidase, which breaks ceramide down into sphingosine and fatty acid, was secreted significantly more from the bacterial flora obtained from both the lesional and the nonlesional skin of patients with AD than from the skin of healthy subjects; sphingomyelinase, which breaks sphingomyelin down into ceramide and phosphorylcholine, was secreted from the bacterial flora obtained from all types of skin at similar levels for the patients with AD and the healthy controls. The finding that the skin of patients with AD is colonized by ceramidase-secreting bacteria thus suggests that microorganisms are related to the deficiency of ceramide in the horny layer of the epidermis, which increases the hypersensitivity of skin in AD patients by impairing the permeability barrier.
PMCID: PMC95668  PMID: 9874672
25.  Uncertainty in action-value estimation affects both action choice and learning rate of the choice behaviors of rats 
The European Journal of Neuroscience  2012;35(7):1180-1189.
The estimation of reward outcomes for action candidates is essential for decision making. In this study, we examined whether and how the uncertainty in reward outcome estimation affects the action choice and learning rate. We designed a choice task in which rats selected either the left-poking or right-poking hole and received a reward of a food pellet stochastically. The reward probabilities of the left and right holes were chosen from six settings (high, 100% vs. 66%; mid, 66% vs. 33%; low, 33% vs. 0% for the left vs. right holes, and the opposites) in every 20–549 trials. We used Bayesian Q-learning models to estimate the time course of the probability distribution of action values and tested if they better explain the behaviors of rats than standard Q-learning models that estimate only the mean of action values. Model comparison by cross-validation revealed that a Bayesian Q-learning model with an asymmetric update for reward and non-reward outcomes fit the choice time course of the rats best. In the action-choice equation of the Bayesian Q-learning model, the estimated coefficient for the variance of action value was positive, meaning that rats were uncertainty seeking. Further analysis of the Bayesian Q-learning model suggested that the uncertainty facilitated the effective learning rate. These results suggest that the rats consider uncertainty in action-value estimation and that they have an uncertainty-seeking action policy and uncertainty-dependent modulation of the effective learning rate.
PMCID: PMC3380560  PMID: 22487046
ambiguity; Bayesian inference; Bayesian Q-learning; neuroeconomics; reinforcement learning

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