Molecular identification of GHS-R and GPR38 in Suncus murinus.

We previously identified ghrelin and motilin genes in Suncus murinus (suncus), and also revealed that motilin induces phase III-like strong contractions in the suncus stomach in vivo, as observed in humans and dogs. Moreover, repeated migrating motor complexes were found in the gastrointestinal tract of suncus at regular 120-min intervals. We therefore proposed suncus as a small laboratory animal model for the study of gastrointestinal motility. In the present study, we identified growth hormone secretagogue receptor (GHS-R) and motilin receptor (GPR38) genes in the suncus. We also examined their tissue distribution throughout the body. The amino acids of suncus GHS-R and GPR38 showed high homology with those of other mammals and shared 42% amino acid identity. RT-PCR showed that both the receptors were expressed in the hypothalamus, medulla oblongata, pituitary gland and the nodose ganglion in the central nervous system. In addition, GHS-R mRNA expressions were detected throughout the stomach and intestine, whereas GPR38 was expressed in the gastric muscle layer, lower intestine, lungs, heart, and pituitary gland. These results suggest that ghrelin and motilin affect gut motility and energy metabolism via specific receptors expressed in the gastrointestinal tract and/or in the central nervous system of suncus.

Antagonistic effect of disulfide-rich peptide aptamers selected by cDNA display on interleukin-6-dependent cell proliferation.

Several engineered protein scaffolds have been developed recently to circumvent particular disadvantages of antibodies such as their large size and complex composition, low stability, and high production costs. We previously identified peptide aptamers containing one or two disulfide-bonds as an alternative ligand to the interleukin-6 receptor (IL-6R). Peptide aptamers (32 amino acids in length) were screened from a random peptide library by in vitro peptide selection using the evolutionary molecular engineering method "cDNA display". In this report, the antagonistic activity of the peptide aptamers were examined by an in vitro competition enzyme-linked immunosorbent assay (ELISA) and an IL-6-dependent cell proliferation assay. The results revealed that a disulfide-rich peptide aptamer inhibited IL-6-dependent cell proliferation with similar efficacy to an anti-IL-6R monoclonal antibody.

Coordination of motilin and ghrelin regulates the migrating motor complex of gastrointestinal motility in Suncus murinus.

Motilin and ghrelin are the gastrointestinal (GI) hormones released in a fasting state to stimulate the GI motility of the migrating motor complex (MMC). We focused on coordination of the ghrelin/motilin family in gastric contraction in vivo and in vitro using the house musk shrew (Suncus murinus), a ghrelin- and motilin-producing mammal. To measure the contractile activity of the stomach in vivo, we recorded GI contractions either in the free-moving conscious or anesthetized S. murinus and examined the effects of administration of motilin and/or ghrelin on spontaneous MMC in the fasting state. In the in vitro study, we also studied the coordinative effect of these hormones on the isolated stomach using an organ bath. In the fasting state, phase I, II, and III contractions were clearly recorded in the gastric body (as observed in humans and dogs). Intravenous infusion of ghrelin stimulated gastric contraction in the latter half of phase I and in the phase II in a dose-dependent manner. Continuous intravenous infusion of ghrelin antagonist (d-Lys3-GHRP6) significantly suppressed spontaneous phase II contractions and prolonged the time of occurrence of the peak of phase III contractions. However, intravenous infusion of motilin antagonist (MA-2029) did not inhibit phase II contractions but delayed the occurrence of phase III contractions of the MMC. In the in vitro study, even though a high dose of ghrelin did not stimulate contraction of stomach preparations, ghrelin administration (10(-10)-10(-7) M) with pretreatment of a low dose of motilin (10(-10) M) induced gastric contraction in a dose-dependent manner. Pretreatment with 10(-8) M ghrelin enhanced motilin-stimulated gastric contractions by 10 times. The interrelation of these peptides was also demonstrated in the anesthetized S. murinus. The results suggest that ghrelin is important for the phase II contraction and that coordination of motilin and ghrelin are necessary to initiate phase III contraction of the MMC.

Full-length transcriptome analysis of human retina-derived cell lines ARPE-19 and Y79 using the vector-capping method.

PURPOSE. To collect an entire set of full-length cDNA clones derived from human retina-derived cell lines and to identify full-length transcripts for retinal preferentially expressed genes. METHODS. The full-length cDNA libraries were constructed from a retinoblastoma cell line, Y79, and a retinal pigment epithelium cell line, ARPE-19, using the vector-capping method, which generates a genuine full-length cDNA. By single-pass sequencing of the 5'-end of cDNA clones and subsequent mapping to the human genome, the authors determined their transcriptional start sites and annotated the cDNA clones. RESULTS. Of the 23,616 clones isolated from Y79-derived cDNA libraries, 19,229 full-length cDNA clones were identified and classified into 4808 genes, including genes of >10 kbp. Of the 7067 genes obtained from the Y79 and ARPE-19 libraries, the authors selected 72 genes that were preferentially expressed in the eye, of which 131 clones corresponding to 57 genes were fully sequenced. As a result, we discovered many variants that were produced by different transcriptional start sites, alternative splicing, and alternative polyadenylation. CONCLUSIONS. The bias-free, full-length cDNA libraries constructed using the vector-capping method were shown to be useful for collecting an entire set of full-length cDNA clones for these retinal cell lines. Full-length transcriptome analysis of these cDNA libraries revealed that there were, unexpectedly, many transcript variants for each gene, indicating that obtaining the full-length cDNA for each variant is indispensable for analyzing its function. The full-length cDNA clones (approximately 80,000 clones each for ARPE-19 and Y79) will be useful as a resource for investigating the human retina.

Noninvasive measurement of fetal augmentation index by fetal aortic diameter pulse and flow velocity waveforms.

OBJECTIVE: To study fetal systemic arterial stiffness in normal fetuses and compromised fetuses who had umbilical placental insufficiency (UPI). DESIGN: Prospective study. SETTING: University departments. SAMPLE: A total of 118 normal fetuses (21-40 weeks) and 55 fetuses (UPI group) with evidence of potential compromise (high umbilical artery pulsatility index). METHODS: A new real-time noninvasive measurement system based on a combined Doppler ultrasound and echo-tracking system was used as a measure of aortic/systemic arterial stiffness. The augmentation index (AI) of the fetal thoracic descending aorta was measured by using simultaneous measurements of diameter pulse and flow velocity waveforms. MAIN OUTCOME MEASURE: Augmentation index as a measure of stiffness. RESULTS: In normal fetuses, successful measurements for obtaining the AI were achieved in 103 of 118 fetuses. In the normal group, the AI, as well as placental resistance, decreased during the second trimester; in contrast, an increase in the AI was observed during the third trimester. Using the AI values from the normal group, the UPI group was divided into two subgroups: 29 fetuses with a normal AI and 26 fetuses with a high AI. The clinical outcome was significantly worse in the latter subgroup compared with the normal subgroup. CONCLUSIONS: The increase of afterload caused by a high umbilical placental resistance was associated with a decrease of aortic distensibility in the compromised fetuses, suggesting an alteration of aortic wall structure.

Directed evolution of a three-finger neurotoxin by using cDNA display yields antagonists as well as agonists of interleukin-6 receptor signaling.

BACKGROUND: Directed evolution of biomolecules such as DNA, RNA and proteins containing high diversity has emerged as an effective method to obtain molecules for various purposes. In the recent past, proteins from non-immunoglobulins have attracted attention as they mimic antibodies with respect to binding potential and provide further potential advantages. In this regard, we have attempted to explore a three-finger neurotoxin protein (3F). 3F proteins are small (~7 kDa), structurally well defined, thermally stable and resistant to proteolysis that presents them as promising candidates for directed evolution. RESULTS: We have engineered a snake α-neurotoxin that belongs to the 3F family by randomizing the residues in the loops involved in binding with acetylcholine receptors and employing cDNA display to obtain modulators of interleukin-6 receptor (IL-6R). Selected candidates were highly specific for IL-6R with dissociation constants and IC50s in the nanomolar range. Antagonists as well as agonists were identified in an IL-6 dependent cell proliferation assay. Size minimization yielded peptides of about one-third the molecular mass of the original proteins, without significant loss of activities and, additionally, lead to the identification of the loops responsible for function. CONCLUSIONS: This study shows 3F protein is amenable to introduce amino acid changes in the loops that enable preparation of a high diversity library that can be utilized to obtain ligands against macromolecules. We believe this is the first report of protein engineering to convert a neurotoxin to receptor ligands other than the parent receptor, the identification of an agonist from non-immunoglobulin proteins, the construction of peptide mimic of IL-6, and the successful size reduction of a single-chain protein.

Identification of ghrelin in the house musk shrew (Suncus murinus): cDNA cloning, peptide purification and tissue distribution.

Ghrelin is the endogenous ligand for the growth hormone (GH) secretagogue receptor, and the sequence of ghrelin has been determined in many species from fish to mammals. In the present study, to reveal the production of ghrelin in the house musk shrew (Suncus murinus, order: Insectivora, suncus is used as a laboratory name), we determined the cDNA sequence and structure of suncus ghrelin and also demonstrated the ghrelin-producing cells in the gastrointestinal tract. Results of cDNA cloning and mass spectrometry analysis revealed that suncus ghrelin is composed of 18 or 26 amino acid residues and that the 3rd Ser was acylated mainly by n-octanoic acid. The 10 amino acids of the N-terminal region of suncus mature ghrelin were consistent with those of other mammals. Quantitative RT-PCR revealed that suncus ghrelin mRNA is highly expressed in the gastric corpus and pyloric antrum, and low expression levels were found in various tissues, including the intestinal tract. Ghrelin cells were found only in the corpus and antrum by immunohistochemistry and in situ hybridization, and most of the ghrelin cells were closed-type cells with relatively rich cytoplasm and scattered in the glandular body and base of the gastric mucosa. The density of ghrelin cells in the corpus was significantly greater than that in the antrum. The results of this study together with our recent results regarding motilin production in the suncus indicate that the suncus will be a useful model animal for study of physiological function of the motilin/ghrelin family.

House musk shrew (Suncus murinus, order: Insectivora) as a new model animal for motilin study.

Although many studies have demonstrated the action of motilin on migrating motor complex by using human subjects and relatively large animals, the precise physiological mechanisms of motilin remain obscure. One reason for the lack of progress in this research field is that large animals are generally not suitable for molecular-level study. To overcome this problem, in this study, we focused on the house musk shrew (Suncus murinus, order: Insectivora, suncus named as laboratory strain) as a small model animal, and we present here the results of motilin gene cloning and its availability for motilin study. The motilin gene has a high homology sequence with that of other mammals, including humans. Suncus motilin is predicted to exist as a 117-residue prepropeptide that undergoes proteolytic cleavage to form a 22-amino-acid mature peptide. The results of RT-PCR showed that motilin mRNA is highly expressed in the upper small intestine, and low levels of expression were found in many tissues. Morphological analysis revealed that suncus motilin-producing cells were present in the upper small intestinal mucosal layer but not in the myenteric plexus. Administration of suncus motilin to prepared muscle strips of rabbit duodenum showed almost the same contractile effect as that of human motilin. Moreover, suncus stomach preparations clearly responded to suncus or human motilin stimulation. To our knowledge, this is the first report that physiological active motilin was determined in small laboratory animals, and the results of this study suggest that suncus is a suitable model animal for studying the motilin-ghrelin family.

Primary structure, tissue distribution, and biological activity of chicken motilin receptor.

Motilin is a peptide hormone involved in gastrointestinal motility. GPR38, initially cloned as an orphan receptor, is now considered a specific receptor for motilin. Previously, molecular characterization of the motilin receptor had only been performed in mammalian and fish species. In this study, we cloned cDNA for chicken motilin receptor from the duodenum and characterized its primary structure, tissue distribution, and biological activity. The cDNA encoded 349 amino acids showing significant overall sequence identity to mammalian motilin receptors. Chicken motilin increased intracellular Ca2+ concentration in human embryonic kidney (HEK) 293 cells transiently expressing the recombinant chicken motilin receptor. Comparison of the cDNA sequence with the genomic sequence of chicken motilin receptor revealed that the chicken motilin receptor gene consists of two exons separated by an intron. Real-time PCR analysis showed that chicken motilin receptor mRNA is expressed in a wide range of tissues in 21-day-old chickens, with markedly high levels in the proventriculus, duodenum, and oviduct. The expression levels of the mRNA in the proventriculus and duodenum were highest just before hatching and rapidly decreased during post-hatch development. These results suggest that chicken motilin receptor is largely involved in gastrointestinal functions at pre- and post-hatch periods through an intracellular signaling pathway accompanied by an increase in Ca2+ levels.

Hypophyseal corticosteroids stimulate somatotrope differentiation in the embryonic chicken pituitary gland.

Although it is known that glucocorticoids induce differentiation of growth hormone (GH)-producing cells in rodents and birds, the effect of mineralocorticoids on GH mRNA expression and the origin of corticosteroids affecting somatotrope differentiation have not been elucidated. In this study, we therefore carried out experiments to determine the effect of mineralocorticoids on GH mRNA expression in the chicken anterior pituitary gland in vitro and to determine whether corticosteroids are synthesized in the chicken embryonic pituitary gland. In a pituitary culture experiment with E11 embryos, both corticosterone and aldosterone stimulated GH mRNA expression and increased the number of GH cells in both lobes of the pituitary gland in a dose-dependent manner. These effects of the corticosteroids were significantly reversed by pretreatment with mifepristone, a glucocorticoid receptor (GR) antagonist, or spironolactone, a mineralocorticoid receptor (MR) antagonist. Interestingly, an in vitro serum-free culture experiment with an E11 pituitary gland showed that the GH mRNA level spontaneously increased during cultivation for 2 days without any extra stimulation, and this increase in GH mRNA level was completely suppressed by metyrapone, a corticosterone-producing enzyme P450C11 inhibitor. Moreover, progesterone, the corticosterone precursor, also stimulated GH mRNA expression in the cultured chicken pituitary gland, and this effect was blocked by pretreatment with metyrapone. We also detected mRNA expression of enzymes of cytochrome P450 cholesterol side chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase1 (3beta-HSD1) in the developmental chicken pituitary gland from E14 and E18, respectively. These results suggest that mineralocorticoids as well as glucocorticoids can stimulate GH mRNA expression and that corticosteroids generated in the embryonic pituitary gland by intrinsic steroidogenic enzymes stimulate somatotrope differentiation.

Evaluation of oxidative stress during apoptosis and necrosis caused by D-galactosamine in rat liver.

Eighteen and twenty-four hours after intraperitoneal administration of D-galactosamine (1g/kg body weight) to rats, the activity of caspase-3-like protease in the liver increased significantly compared with that in the control group given saline. Histological examinations including the in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method found apoptotic hepatocytes 18 hr after the administration of D-galactosamine. Caspase-3 activity was barely detectable in the plasma of control rats, but increased significantly 24 hr after drug administration along with a dramatic increase in glutamate-oxaloacetate transaminase (GOT). These results indicated that D-galactosamine causes apoptosis in the liver by activating caspase-3, which is released to the plasma by secondary necrosis. The concentration of lipid hydroperoxides in the liver increased significantly 24 hr after D-galactosamine administration. In contrast, the concentration of vitamin C in the liver decreased significantly 18 and 24 hr after D-galactosamine administration. These results suggest that D-galactosamine induces severe oxidative stress in the liver, leading to extensive necrosis.