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Gastric contractions show two specific patterns in many species, migrating motor contractions (MMC) and postprandial contractions (PPCs), that occur in the fasted and fed states, respectively. In this study, we examined the role of somatostatin (SST) in gastric motility both in vivo and in vitro using the Asian house shrew (Suncus murinus). We performed in vivo recordings of gastric motility and in vitro organ bath experiments using S. murinus, which was recently established as a small laboratory animal for use in tests of gastrointestinal motility. SST (1.65 µg kg-1 min-1) was intravenously administered during phase II of MMC and PPCs. Next, the effect of SST on motilin-induced gastric contractions at phase I of MMC was measured. Cyclosomatostatin (CSST), an SST receptor antagonist, was administered at the peak of phase III of MMC. In addition, the effect of SST (10-11-10-9 M) on motilin-induced gastric contractions was evaluated using an organ bath experiment in vitro. In conscious, free-moving S. murinus, the administration of SST decreased the occurrence of the spontaneous phase II of MMC and PPCs. Pretreatment with SST and octreotide suppressed the induction of motilin-induced gastric contractions both in vivo and in vitro. Administration of CSST before the peak of spontaneous phase III contractions had no effect on gastric contractions. Endogenous SST is not involved in the regulation of gastric MMC and PPCs, but exogenous SST suppresses spontaneous gastric contractions. Thus, SST would be good for treating abnormal gastrointestinal motility disorders.

Ghrelin is a gut-derived peptide with several physiological functions, including feeding, gastrointestinal motility, and hormonal secretion. Recently, a host defense peptide, liver-expressed antimicrobial peptide-2 (LEAP2), was reported as an endogenous antagonist of growth hormone secretagogue receptor (GHS-R). The physiological relevance of the molecular LEAP2-GHS-R interaction in mammals has been explored; however, studies on non-mammals are limited. Here, we report the identification and functional characterization of ghrelin and its related molecules in Western clawed frog (Xenopus tropicalis), a known model organism. We first identified cDNA encoding X. tropicalis ghrelin and GHS-R. RT-qPCR revealed that ghrelin mRNA expression was most abundant in the stomach. GHS-R mRNA was widely distributed in the brain and peripheral tissues, and a relatively strong signal was observed in the stomach and intestine. In addition, LEAP2 was mainly expressed in intestinal tissues at higher levels than in the liver. In functional analysis, X. tropicalis ghrelin and human ghrelin induced intracellular Ca

Background The aim of this study was to investigate the fundamental mechanisms of colonic motility in the house musk suncus (Suncus murinus) as an established animal model of gut motility. Methods To measure gut motility in free-moving conscious suncus, strain gauge force transducers were implanted on the serosa of the colon and gastric body. Key results We recorded diurnal changes in colonic motility and observed the relationship between feeding and colonic motility. Giant migrating contractions (GMCs) of the colon were invariably detected during defecation and tended to increase during the dark period, thereby indicating that colonic motility has a circadian rhythm. Given that GMCs in the suncus were observed immediately after feeding during the dark period, we assume the occurrence of a gastrocolic reflex in suncus, similar to that observed in humans and dogs. We also examined the factors that regulate suncus GMCs. Intravenous administration of 5-HT (100 µg/kg), substance P (10 and 100 µg/kg), calcitonin gene-related peptide (10 µg/kg), and α2 adrenergic receptor antagonist yohimbine (0.5, 1, and 3 mg/kg) induced GMC-like contractions, as did intragastric and intracolonic administration of the transient receptor potential vanilloid 1 agonist, capsaicin (1 mg/kg). Conclusions & inferences These results indicate that the fundamental mechanisms of colonic motility in suncus are similar to those in humans and dogs, and we thus propose that suncus could serve as a novel small animal model for studying colonic motility.

Cholecystokinin (CCK) is a peptide hormone mainly secreted by small intestinal endocrine I-cells and functions as a regulator of gallbladder contraction, gastric emptying, gastrointestinal (GI) motility, and satiety. The cellular effects of CCK in these peripheral tissues are predominantly mediated via CCK-A receptors which are found in smooth muscles, enteric neurons, and vagal afferent neurons in humans and animal models. Although various functions of CCK have been reported to be neurally mediated, it can also stimulate contraction via the CCK receptor on the smooth muscle. However, the entire underlying neural and cellular mechanisms involved in CCK-induced GI contractions are not clearly understood. Here, we first determined the cDNA and amino acid sequences of CCK and CCK-A receptor along with the distributions of cck mRNA and CCK-producing cells in house musk shrew (Suncus murinus, the laboratory strain named as suncus) and examined the mechanism of CCK-induced contraction in the GI tract. Mature suncus CCK-8 was identical to other mammalian species tested here, and suncus CCK-A receptor presented high nucleotide and amino acid homology with that of human, dog, mouse, and rat, respectively. Suncus CCK mRNA and CCK-producing cells were found mainly in small intestine and colon. In the organ bath study, CCK-8 induced dose-dependent contractions in the suncus stomach, duodenum, and jejunum, and these contractions were inhibited by atropine and CCK-A receptor antagonist. These results suggest that CCK-8-induced contraction is mediated in the myenteric cholinergic neural network and that CCK-A receptor is partly responsible for CCK-8-induced contractions. This study indicates that suncus is a useful animal model to study the functions of CCK involved in GI motility.

Motilin, a 22-amino-acid peptide produced in the upper small intestine, induces strong gastric contraction in fasted state. In many rodents, motilin and its cognate receptors exist as pseudogenes, which has delayed motilin research in the past decades. Recently, the house musk shrew (Suncus murinus) was developed as a useful model for studying motilin and gastrointestinal motility. However, due to a lack of motilin-producing cell lines and difficulties in culturing small intestinal cells, the regulatory mechanisms of motilin secretion and its messenger RNA (mRNA) transcription have remained largely unclear. In this study, we generated small intestinal organoids from S. murinus for the first time. Using methods similar to mouse organoid generation, we found crypt-like budding structures 3 days after isolating intestinal tissues. The organoids grew gradually with time. In addition, the generated organoids were able to be passaged and maintained for 6 months or longer. Motilin messenger RNA (mRNA) and immunopositive cells were observed in both S. murinus intestinal organoids and primary tissues. This is the first report of intestinal organoids in S. murinus, and our results suggest that S. murinus intestinal organoids could be useful for analyzing motilin secretion and transcription.

Motilin (MLN), a 22-amino-acid peptide hormone, is generally present in the mucosa of the upper gastrointestinal (GI) tract, mainly the duodenum of mammals, and it regulates GI motility, especially that related to interdigestive migrating contraction. However, MLN and its receptor are absent in mice and rats, and MLN does not cause any mechanical responses in the rat and mouse GI tracts. The guinea-pig is also a rodent, but expression of the MLN gene in the guinea-pig has been reported. In the present study, two guinea-pig MLNs, FIPIFTYSELRRTQEREQNKGL found in the Ensemble Genome Database (gpMLN-1) and FVPIFTYSELRRTQEREQNKRL reported by Xu et al. (2001) (gpMLN-2), were synthesized, and their biological activities were evaluated in the rabbit duodenum and guinea-pig GI tract in vitro. Both gpMLNs showed contractile activity in longitudinal muscle strips of the rabbit duodenum. The EC50 values of gpMLN-1 and gpMLN-2 were slightly higher than that of human MLN (hMLN), but the maximum contractions were as same as that of hMLN. Treatment with GM109 and hMLN-induced receptor desensitization decreased the contractile activity of both gpMLNs, indicating that the two gpMLN candidates are able to activate the MLN receptor (MLN-R) of the rabbit duodenum. In guinea-pig GI preparations, hMLN and gpMLNs did not show any mechanical responses in circular muscle strips from the gastric antrum or in longitudinal strips of the duodenum, ileum and colon although acetylcholine and 1,1-dimethyl-4-phenylpiperazinium (DMPP) caused definite mechanical responses. The DMPP-induced neural responses in the gastric circular muscle and ileal longitudinal muscles were not modified by gpMLN-1. Even in the gastric and ileal strips with intact mucosa, no mechanical responses were seen with either of the gpMLNs. Furthermore, RT-PCR using various primer sets failed to amplify the gpMLN-2 mRNA. In conclusion, gpMLNs including one that was already reported and the other that was newly found in a database were effective to the rabbit MLN-R, whereas they did not cause any contractions or modification of neural responses in the guinea-pig GI tract, indicating that the MLN system is vestigial and not functional in regulation of GI motility in the guinea-pig as well as in other rodents such as rats and mice.

Lysozyme is one of the most prominent antimicrobial peptides and has been identified from many mammalian species. However, this enzyme has not been studied in the order Insectivora, which includes the most primitive placental mammals. Here, we done the lysozyme cDNA from Suncus murinus (referred to as suncus, its laboratory name) and compare the predicted amino acid sequence to those from other mammalian species. Quantitative PCR analysis revealed a relatively higher expression of this gene in the spleen and gastrointestinal tract of suncus. The lysozyme-immunopositive (ip) cells were found mainly in the red pulp of the spleen and in the mucosa of the whole small intestine, including the follicle-associated epithelium and subepithelial dome of Peyer's patches. The lysozyme-ip cells in the small intestine were mostly distributed in the intestinal crypt, although lysozyme-expressing cells were found not only in the crypt but also in the villi. On the other hand, only a few lysozyme-ip cells were found in the villi and some granules showing intense fluorescence were located toward the lumen. As reported for other mammals, Ki67-ip cells were localized in the crypt and did not co-localize with the lysozyme-ip cells. Moreover, fasting induced a decrease in the mRNA levels of lysozyme in the intestine of suncus. In conclusion, we firstly identified the lysozyme mRNA sequence, clarified expression profile of lysozyme transcripts in suncus and found a unique distribution of lysozyme-producing cells in the suncus intestine.

Motilin, a peptide hormone consisting of 22 amino acid residues, was identified in the duodenum of pigs in the 1970s. It is known to induce gastrointestinal contractions during the interdigestive state in mammals. Although the motilin gene has been identified in various animal species, it has not been studied in amphibians. Here, we identified the motilin gene in the Japanese fire bellied newt (Cynops pyrrhogaster), and conducted an analysis of tissue distribution, morphological observations, and physiological experiments. The deduced mature newt motilin comprises 22 amino acid residues, like in mammals and birds. The C-terminus of the newt motilin showed high homology with motilin from other species compared to the N-terminus region, which is considered the bioactive site. Motilin mRNA expression in newts was abundant in the upper small intestine, with notably high motilin mRNA expression found in the pancreas. Motilin-producing cells were found in the mucosal layer of the upper small intestine and existed as two cell types: open-and closed-type cells. Motilin-producing cells in the pancreas were also found to produce insulin but not glucagon. Newt motilin stimulated gastric contractions but not in other parts of the intestines in vitro, and motilin-induced gastric contraction was significantly inhibited by treatment with atropine, a muscarinic acetylcholine receptor antagonist. These results indicate that motilin is also present in amphibians, and that its gastrointestinal contractile effects are conserved in mammals, birds, and amphibians. Additionally, we demonstrated for the first time the existence of pancreatic motilin, suggesting that newt motilin has an additional unknown physiological role.

Ghrelin is a multifunctional gut peptide with a unique structure, which is modified by a medium chain fatty acid at the third serine by ghrelin O-acyl transferase (GOAT). It is well known that the major source of plasma ghrelin is the stomach, but the transcriptional regulation of gastric ghrelin and GOAT is incompletely understood. Here, we studied the involvement of the nuclear receptors REV-ERBα and REV-ERBβ on ghrelin and GOAT gene expression in vivo and in vitro. Reverse-transcriptase polymerase chain reaction analysis showed that REV-ERBα and REV-ERBβ mRNAs were expressed in the stomach and a stomach-derived ghrelin cell line (SG-1 cells). In vivo experiments with mice revealed the circadian rhythm of ghrelin, GOAT, and REV-ERBs. The peak expression of ghrelin and GOAT mRNAs occurred at Zeitgeber time (ZT) 4, whereas that of REV-ERBα and REV-ERBβ was observed at ZT8 and ZT12, respectively. Treatment of SG-1 cells with SR9009, a REV-ERB agonist, led to a significant reduction in ghrelin and GOAT mRNA levels. Overexpression of REV-ERBα and REV-ERBβ decreased ghrelin and GOAT mRNA levels in SG-1 cells. In contrast, small-interfering RNA (siRNA)-mediated double-knockdown of REV-ERBα and REV-ERBβ in SG-1 cells led to the upregulation in the expression of ghrelin and GOAT mRNAs. These results suggest that REV-ERBs suppress ghrelin and GOAT mRNA expression.

Pyridoxine (PN), one of the vitamers of vitamin B6, plays an important role in the maintenance of epidermal function and is used to treat acne and rough skin. Clinical studies have revealed that PN deficiency causes skin problems such as seborrheic dermatitis and stomatitis. However, the detailed effects of PN and its mechanism of action in epidermal function are poorly understood. In this study, we examined the effects of PN on epidermal function in normal human epidermal keratinocytes and found that PN specifically causes an increase in the expression of profilaggrin mRNA, among marker genes of terminal epidermal differentiation. In addition, PN treatment caused an increase in the production of filaggrin protein in a concentration-dependent manner. Treatment with P2x purinoceptor antagonists, namely, pyridoxal phosphate-6-azo (benzene-2,4-disulfonic acid) tetrasodium salt hydrate and TNP-ATP hydrate, induced an increase in the filaggrin protein levels. Moreover, we showed that elevated filaggrin production induced upon PN treatment was suppressed by ATP (known as P2x purinoceptor agonist). This study is the first to report that PN causes an increase in filaggrin transcription and production, and these results suggest that PN-induced filaggrin production may be a useful target as a daily care component in atopic dermatitis, wherein filaggrin levels are specifically reduced.

Pyridoxine (PN), one of the vitamers of vitamin B6, plays an important role in the maintenance of epidermal function and is used to treat acne and rough skin. Clinical studies have revealed that PN deficiency causes skin problems such as seborrheic dermatitis and stomatitis. However, the detailed effects of PN and its mechanism of action in epidermal function are poorly understood. In this study, we examined the effects of PN on epidermal function in normal human epidermal keratinocytes and found that PN specifically causes an increase in the expression of profilaggrin mRNA, among marker genes of terminal epidermal differentiation. In addition, PN treatment caused an increase in the production of filaggrin protein in a concentration-dependent manner. Treatment with P2x purinoceptor antagonists, namely, pyridoxal phosphate-6-azo (benzene-2,4-disulfonic acid) tetrasodium salt hydrate and TNP-ATP hydrate, induced an increase in the filaggrin protein levels. Moreover, we showed that elevated filaggrin production induced upon PN treatment was suppressed by ATP (known as P2x purinoceptor agonist). This study is the first to report that PN causes an increase in filaggrin transcription and production, and these results suggest that PN-induced filaggrin production may be a useful target as a daily care component in atopic dermatitis, wherein filaggrin levels are specifically reduced.

Under severe calorie restriction (CR), the ghrelin-growth hormone axis in mice is involved in the maintenance of plasma glucose levels. Ghrelin, a stomach-derived acylated peptide, is up-regulated by the sympathetic nerve in the negative energy status. Central corticotrophin-releasing factor receptor (CRF-R) signalling stimulates the sympathetic tone. The present study aimed to examine the effect of central CRF-R signalling on the maintenance of plasma glucose concentrations in severe calorie-restricted mice with the involvement of ghrelin. Intracerebroventricular injections of urocorin-1 and urocorin-2, which are natural ligands for CRF-R1 and CRF-R2, elevated plasma ghrelin concentrations and ghrelin elevation with an i.c.v. injection of urocorin-1 was cancelled by atenolol (β1 adrenergic receptor antagonist) administration. We then established a mice model of 60% CR and found that the administration of [d-Lys3]-GHRP-6 (a ghrelin receptor antagonist) in mice under 60% CR reduced the plasma glucose concentration more compared to the vehicle mice. Similarly, the atenolol injection in mice under 60% CR significantly reduced the plasma glucose concentration, which was rescued by the co-administration of ghrelin. An i.c.v. injection of the alpha helical CRH, a non-selective corticotrophin-releasing factor receptor antagonist, in mice under 60% CR significantly reduced the plasma glucose concentration, although the co-administration of α-helical CRH with ghrelin maintained plasma glucose levels. These results suggest that central CRF-R signalling is involved in the maintenance of plasma glucose levels in mice under severe CR via the sympathetic-ghrelin pathway.

Ghrelin is a unique fatty acid-modified peptide hormone produced in the stomach and has important roles in energy homeostasis and gastrointestinal motility. However, the medium-chain fatty acid source for ghrelin acyl-modification is not known. We found that a fat-free diet and the removal of intestinal microbiota did not decrease acyl-ghrelin production in the stomach or plasma acyl-ghrelin levels in mice. RT-PCR analysis showed that genes involving fatty acid synthesis, metabolism, and transport were expressed in pancreas-derived ghrelinoma (PG-1) cells. Treatment with an irreversible inhibitor of carnitine palmitoyltransferase-1 (CPT-1) strongly decreased acylated ghrelin levels but did not affect ghrelin or ghrelin o-acyl transferase (GOAT) mRNA levels in PG-1 cells. Our results suggest that the medium-chain fatty acid used for the acyl-modification of ghrelin is produced in ghrelin-producing cells themselves by β-oxidation of long-chain fatty acids provided from the circulation.

Gastric contractions exhibit characteristic motor patterns in the fasted state, known as migrating motor contractions (MMC). MMC consist of three periodically repeated phases (phase I, II and III) and are known to be regulated by hormones and the autonomic and enteric nervous systems. However, the central regulation of gastric contractions in the fasted state is not completely understood. Here, we have examined the central effects of motilin, ghrelin, γ-aminobutyric acid (GABA) and L-glutamate signaling on gastric MMC by using suncus (Suncus murinus) as an animal model, because of their similar gastric motor patterns to those observed in humans and dogs. Intracerebroventricular (i.c.v.) administration of motilin and ghrelin had no effect on phase I and II contractions, respectively. Conversely, i.c.v. administration of GABAA receptor antagonist, during phase I of the MMC, evoked phase II-like contractions and significantly increased the motility index (MI). This was compared with the i.c.v. administration of GABA which inhibited spontaneous phase II contractions with a significantly decreased MI. In addition, i.c.v. administration of L-glutamate during phase I also induced phase II-like irregular contractions with a significant increase in the MI. Taken together with previous findings, these results suggest that central GABAergic and glutamatergic signaling, with the coordination of both peripheral motilin and ghrelin, regulate phase II contractions of MMC in the fasted state.

Ghrelin is abundantly produced in the stomach. Here, we found that glutamate decreased ghrelin expression and release in ghrelin-producing cells, and decreased levels of food intake and plasma acyl-ghrelin in mice. Treatment with siRNA of G protein-coupled receptor, family C, group 5, member B (GPRC5B) in ghrelin-producing cell lines completely blocked the effect of glutamate-induced ghrelin suppression. In addition, glutamate inhibited ghrelin release via the extracellular signal-regulated kinase (ERK) activity pathway, and stimulated CREB2 mRNA expression in ghrelin-producing cell lines. These results suggest that glutamate inhibits ghrelin release via ERK-CREB2 pathway. These results suggest that the GPRC5B-ERK-CREB2 pathway is involved in the inhibition of ghrelin expression and secretion in ghrelin cells.

Ghrelin, a peptide hormone produced in the stomach, has been known to be involved in the regulation of gastric contraction in humans and rodents. To elucidate the detailed mechanisms of ghrelin on gastric contractions, we used Suncus murinus, a recently established small animal model for gastrointestinal motility. S. murinus produces motilin, a family peptide of ghrelin, and its stomach anatomy and physiological patterns of gastric contractions, in fed and fasted states, are closely similar to humans. Ghrelin administration in phase II, and latter half of phase I, of the migrating motor contractions (MMC) enhanced gastric motility in S. murinus. In addition, we showed that ghrelin and motilin coordinately stimulated strong gastric contractions in vitro and in vivo. We also demonstrated that a pretreatment with a ghrelin antagonist, D-Lys3-GHRP6, inhibited the effects of motilin-induced gastric contractions, and a γ-aminobutyric acid (GABA) antagonist reversed this inhibition. Our results suggest that ghrelin is essential for motilin-induced gastric contractions and that ghrelin-mediated GABAergic neurons are involved in this neural pathway.

Molecular hydrogen (H2) showed protection against various kinds of oxidative-stress-related diseases. First, it was reported that the mechanism of therapeutic effects of H2was antioxidative effect due to inhibition of the most cytotoxic reactive oxygen species, hydroxy radical (•OH). However, after chronic administration of H2in drinking water, oxidative-stress-induced nerve injury is significantly attenuated even in the absence of H2. It suggests indirect signaling of H2and gastrointestinal tract is involved. Indirect effects of H2could be tested by giving H2water only before nerve injury, as preconditioning. For example, preconditioning of H2for certain a period (∼7 days) in Parkinson’s disease model mice shows significant neuroprotection. As the mechanism of indirect effect, H2in drinking water induces ghrelin production and release from the stomach via β1-adrenergic receptor stimulation. Released ghrelin circulates in the body, being transported across the blood–brain barrier, activates its receptor, growth-hormone secretagogue receptor. H2-induced upregulation of ghrelin mRNA is also shown in ghrelin-producing cell line, SG-1. These observations help with understanding the chronic effects of H2and raise intriguing preventive and therapeutic options using H2.

Neuromedin U (NMU) shows circadian expression in the rat pars tuberalis (PT), and is known to be suppressed by melatonin. Here we examined the involvement of adenosine in the regulation of Nmu expression. We found that the rat PT expressed adenosine receptor A2b and that an adenosine receptor agonist, NECA, stimulated Nmu expression in brain slice cultures. In vitro promoter assays revealed that NECA stimulated Nmu promoter activity via a cAMP response element (CRE) in the presence of adenosine receptor A2b. NECA also increased the levels of phosphorylated CRE-binding protein. These findings suggest that adenosine stimulates Nmu expression by activating the cAMP signaling pathway through adenosine receptor A2b in the rat PT. This is the first report to demonstrate that Nmu expression in the PT is regulated by adenosine, which acts as an intravital central metabolic signal, in addition to melatonin, which acts as an external photoperiodic environmental signal.

Gastric contractions exhibit characteristic motor patterns in the fasted state, known as migrating motor contractions (MMC). MMC consist of three periodically repeated phases (phase I, II and III) and are known to be regulated by hormones and the autonomic and enteric nervous systems. However, the central regulation of gastric contractions in the fasted state is not completely understood. Here, we have examined the central effects of motilin, ghrelin, γ-aminobutyric acid (GABA) and L-glutamate signaling on gastric MMC by using suncus (Suncus murinus) as an animal model, because of their similar gastric motor patterns to those observed in humans and dogs. Intracerebroventricular (i.c.v.) administration of motilin and ghrelin had no effect on phase I and II contractions, respectively. Conversely, i.c.v. administration of GABAA receptor antagonist, during phase I of the MMC, evoked phase II-like contractions and significantly increased the motility index (MI). This was compared with the i.c.v. administration of GABA which inhibited spontaneous phase II contractions with a significantly decreased MI. In addition, i.c.v. administration of L-glutamate during phase I also induced phase II-like irregular contractions with a significant increase in the MI. Taken together with previous findings, these results suggest that central GABAergic and glutamatergic signaling, with the coordination of both peripheral motilin and ghrelin, regulate phase II contractions of MMC in the fasted state.

Ghrelin was first isolated from human and rat as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R). In the present study, we determined the ghrelin cDNA sequence of the common marmoset (Callithrix jacchus), a small-bodied New World monkey, and investigated the distribution of ghrelin-producing cells in the gastrointestinal tract and localization profiles with somatostatin-producing cells. The marmoset ghrelin cDNA coding region was 354 base pairs, and showed high homology to that in human, rhesus monkey, and mouse. Marmoset ghrelin consists of 28 amino acids, and the N-terminal region is highly conserved as found in other mammalian species. Marmoset preproghrelin and mature ghrelin have 86.3% and 92.9% homology, respectively, to their human counterparts. Quantitative RT-PCR analysis showed that marmoset ghrelin mRNA is highly expressed in the stomach, but it is not detected in other tissues of the gastrointestinal tract. In addition, a large number of ghrelin mRNA-expressing cells and ghrelin-immunopositive cells were detected in the mucosal layer of the stomach, but not in the myenteric plexus. Moreover, all the ghrelin cells examined in the stomach were observed to be closed-type. Double staining showed that somatostatin-immunopositive cells were not co-localized with ghrelin-producing cells; however, a subset of somatostatin-immunopositive cells is directly adjacent to ghrelin-immunopositive cells. These findings suggest that the distribution of ghrelin cells in marmoset differs from that in rodents, and thus the marmoset may be a more useful model for the translational study of ghrelin in primates. In conclusion, we have clarified the expression and cell distribution of ghrelin in marmoset, which may represent a useful model in translational study.

The adenohypophysis is formed from the oral ectoderm and consists of the pars distalis (PD), pars intermedia, and pars tuberalis (PT). The mechanisms of PD development have been extensively studied, and the cellular differentiation of the PD is well understood. However, the morphogenesis and differentiation of the PT are still unclear, and the genes expressed during PT development remain largely unknown. We have explored genes specifically expressed in the PT during embryonic development and analyzed their spatiotemporal expression patterns. Microarray analysis of laser-captured PT and PD tissues obtained from chick embryos on embryonic day 10 (E10.0) has shown high expression of Cytokine-like 1 (CYTL1) and Gap junction protein alpha 5 (GJA5) genes in the PT. Detailed analysis of these spatiotemporal expression patterns during chick embryo development by in situ hybridization has revealed that CYTL1 mRNA first appears in the lateral head ectoderm and ventral head ectoderm at E1.5. The expression of CYTL1 moves into Rathke's pouch at E2.5 and is then localized in the PT primordium where it is continuously expressed until E12.0. GJA5 mRNA is transiently detected in the PT primordium from E6.0 to E12.0, whereas its expression is not detected in the PD during development. Thus, these genes might be involved in the regulation mechanisms of PT development and could be useful markers for PT development.

The migrating motor complex (MMC) is responsible for emptying the stomach during the interdigestive period, in preparation for the next meal. It is known that gastric phase III of MMC starts from the proximal stomach and propagates the contraction downwards. We hypothesized that a certain region of the stomach must be more responsive to motilin than others, and that motilin-induced strong gastric contractions propagate from that site. Stomachs of the Suncus or Asian house shrew, a small insectivorous mammal, were dissected and the fundus, proximal corpus, distal corpus, and antrum were examined to study the effect of motilin using an organ bath experiment. Motilin-induced contractions differed in different parts of the stomach. Only the proximal corpus induced gastric contraction even at motilin 10(-10) M, and strong contraction was induced by motilin 10(-9) M in all parts of the stomach. The GPR38 mRNA expression was also higher in the proximal corpus than in the other sections, and the lowest expression was observed in the antrum. GPR38 mRNA expression varied with low expression in the mucosal layer and high expression in the muscle layer. Additionally, motilin-induced contractions in each dissected part of the stomach were inhibited by tetrodotoxin and atropine pretreatment. These results suggest that motilin reactivity is not consistent throughout the stomach, and an area of the proximal corpus including the cardia is the most sensitive to motilin.

Asymptomatic infant carriers of toxigenic Clostridium difficile are suggested to play a role in the transmission of C. difficile infection (CDI) in adults. However, the mode of C. difficile carriage in infants remains to be fully elucidated. We investigated longitudinal changes in carriage rates, counts, and strain types of toxigenic C. difficile in infants. Stools collected from 111 healthy infants in Belgium periodically from birth until the age of 6 months were examined by quantitative PCR targeting 16S rRNA and toxin genes. Toxigenic C. difficile was detected in 18 of 111 infants (16%) in the period up to the age of 6 months. The carriage rate of toxigenic C. difficile remained below 5% until the age of 3 months. The carriage rate increased to 13% 1 week after weaning (average age, 143 days) and reached 16% at the age of 6 months. Counts of toxigenic C. difficile bacteria ranged from 10 4 to 10 8 cells/g of stool. Notably, two infants retained >10 8 cells/g of stool for at least several weeks. Average counts in the 18 infants hovered around 10 7 cells/g of stool from the age of 3 days until the age of 6 months, showing no age-related trend. Genotyping of toxigenic C. difficile isolates from the 18 infants revealed that 11 infants each retained a particular monophyletic strain for at least a month. The genotype most frequently identified was the same as that frequently identified in symptomatic adult CDI patients. Thus, toxigenic C. difficile strains—potential causes of CDI in adults—colonized the infants' intestines. IMPORTANCE Our study provides longitudinal data on counts and strain types of toxigenic C. difficile in infants. We found that considerable numbers of toxigenic C. difficile bacteria colonized the infants' intestines. The results of strain typing suggest that toxigenic C. difficile carried by healthy infants could be potentially pathogenic to adults. These results and findings are informative not only for ecological studies but also for efforts to prevent or control the spread of CDI in adults.

Cytometry is a versatile and powerful method applicable to different fields, particularly pharmacology and biomedical studies. Based on the data obtained, cytometric studies are classified into high-throughput (HTP) or high-content screening (HCS) groups. However, assays combining the advantages of both are required to facilitate research. In this study, we developed a high-throughput system to profile cellular populations in terms of time- or dose-dependent responses to apoptotic stimulations because apoptotic inducers are potent anticancer drugs. We previously established assay systems involving protease to monitor live cells for apoptosis using tunable fluorescence resonance energy transfer (FRET)-based bioprobes. These assays can be used for microscopic analyses or fluorescence-activated cell sorting. In this study, we developed FRET-based bioprobes to detect the activity of the apoptotic markers caspase-3 and caspase-9 via changes in bioprobe fluorescence lifetimes using a flow cytometer for direct estimation of FRET efficiencies. Different patterns of changes in the fluorescence lifetimes of these markers during apoptosis were observed, indicating a relationship between discrete steps in the apoptosis process. The findings demonstrate the feasibility of evaluating collective cellular dynamics during apoptosis.

Motile cilia/flagella are essential for swimming and generating extracellular fluid flow in eukaryotes. Motile cilia harbor a 9+2 arrangement consisting of nine doublet microtubules with dynein arms at the periphery and a pair of singlet microtubules at the center (central pair). In the central system, the radial spoke has a T-shaped architecture and regulates the motility and motion pattern of cilia. Recent cryoelectron tomography data reveal three types of radial spokes (RS1, RS2, and RS3) in the 96 nm axoneme repeat unit; however, the molecular composition of the third radial spoke, RS3 is unknown. In human pathology, it is well known mutation of the radial spoke head-related genes causes primary ciliary dyskinesia (PCD) including respiratory defect and infertility. Here, we describe the role of the primary ciliary dyskinesia protein Rsph4a in the mouse motile cilia. Cryoelectron tomography reveals that the mouse trachea cilia harbor three types of radial spoke as with the other vertebrates and that all triplet spoke heads are lacking in the trachea cilia of Rsph4a-deficient mice. Furthermore, observation of ciliary movement and immunofluorescence analysis indicates that Rsph4a contributes to the generation of the planar beating of motile cilia by building the distal architecture of radial spokes in the trachea, the ependymal tissues, and the oviduct. Although detailed mechanism of RSs assembly remains unknown, our results suggest Rsph4a is a generic component of radial spoke heads, and could explain the severe phenotype of human PCD patients with RSPH4A mutation., Author summary Motile cilia are nanodevices driving extracellular fluid flow and are involved in human primary ciliary dyskinesia (PCD) including respiratory diseases, infertility, and laterality defect. Radial spoke (RS) is a T-shaped architecture inside of the axoneme of motile cilia and it regulates the motility and motion pattern of cilia. RS consists of the spoke head and the stalk, and the three-types of RS (RS1, RS2, RS3) exist in the axoneme of motile cilia. To date, it is well known mutation of the spoke head-related genes causes PCD. Among the spoke head-related genes, mutation of RSPH4A leads to most severe phenotype on the cilia ultrastructure in the PCD patients, but it remains unknown what determines the severe phenotype. Here, we show the role of the primary ciliary dyskinesia causal gene Rsph4a in the mouse motile cilia. We have found that Rsph4a plays a central role in all the three-types of RS assembly and is a generic component of spoke heads in the trachea, the ependymal tissues, and the oviduct in the mouse. Our results could explain the severe phenotype of human PCD patients with RSPH4A mutation.

Magnetization reversal in a Wiegand wire induces a pulse voltage in the pickup coil around the wire, called the Wiegand pulse. The Wiegand sensor features the Wiegand wire and the pickup coil. The amplitude and width of the Wiegand pulse are independent of the frequency of the magnetic-field change. The pulse is generated by the Wiegand sensor, which facilitates the use of the Wiegand sensor as a power supply for equipment without batteries. In order to meet the power consumption requirements, it is necessary to maximize the energy of the pulse signal from the Wiegand sensor, without changing the external field conditions. The distributions of the magnetic field generated from the applied magnet in air and in the Wiegand wire were simulated before the experiments. Simulation predicted an increase in the magnetic flux density through the center of the Wiegand wire. This study determined that the magnetic flux density through the center of the Wiegand wire, the position of the pickup coil, and the angle between the Wiegand sensor and the magnetic induction line were the main factors that affected the energy of a Wiegand pulse. The relationship between these factors and the energy of the Wiegand pulse were obtained.

The intestinal epithelial monolayer forms a mucosal barrier between the gut microbes and the host tissue. The mucosal barrier is composed of mucins and antimicrobial peptides and proteins (AMPs). Several animal studies have reported that Paneth cells, which occupy the base of intestinal crypts, play an important role in the intestinal innate immunity by producing AMPs, such as lysozyme, Reg3 lectins, α-defensins, and group IIA secretory phospholipase A2 (GIIA sPLA2). The house musk shrew (Suncus murinus) has only a few intestinal commensal bacteria and is reported to lack Paneth cells in the intestine. Although the expression of lysozyme was reported in the suncus intestine, the expression of other AMPs has not yet been reported. Therefore, the current study was focused on GIIA sPLA2 expression in Suncus murinus. GIIA sPLA2 mRNA was found to be most abundant in the spleen and also highly expressed in the intestine. Cells expressing GIIA sPLA2 mRNA were distributed not only in the crypt, but also in the villi. In addition, intragastric injection of lipopolysaccharide increased GIIA sPLA2 expression in the small intestine of suncus. These results suggest that suncus may host unique AMP-secreting cells in the intestine.

形態: カラー図版あり, Physical characteristics: Original contains color illustrations, Accepted: 2018-07-05, 資料番号: PA1910050000

Motilin was discovered in the 1970s as the most important hormone for stimulating strong gastric contractions; however, the mechanisms by which motilin causes gastric contraction are not clearly understood. Here, we determined the coordinated action of motilin and ghrelin on gastric motility during fasted and postprandial contractions by using house musk shrew (Suncus murinus; order: Insectivora, suncus named as the laboratory strain). Motilin-induced gastric contractions at phases I and II of the migrating motor complex were inhibited by pretreatment with (d-Lys3)-GHRP-6 (6 mg/kg/h), a ghrelin receptor antagonist. Administration of the motilin receptor antagonist MA-2029 (0.1 mg/kg) and/or (d-Lys3)-GHRP-6 (0.6 mg/kg) at the peak of phase III abolished the spontaneous gastric phase III contractions in vivo. Motilin did not stimulate gastric contractions in the postprandial state. However, in the presence of a low dose of ghrelin, motilin evoked phase III–like gastric contractions even in the postprandial state, and postprandial gastric emptying was accelerated. In addition, pretreatment with (d-Lys3)-GHRP-6 blocked the motilin-induced gastric contraction in vitro and in vivo, and a γ-aminobutyric acid (GABA) antagonist reversed this block in gastric contraction. These results indicate that blockade of the GABAergic pathway by ghrelin is essential for motilin-induced gastric contraction.