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Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Eighteen and twenty-four hours after intraperitoneal administration of d-galactosamine (1 g/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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]