Sequence of an intestinal cDNA encoding human motilin precursor

A cDNA clone encoding the human motilin precursor was isolated from an intestinal library using synthetic oligonucleotide probes. The predicted amino acid sequence indicates that the motilin precursor consists of 115 amino acids and includes a 25-residue N-terminal signal peptide followed by the 22-amino-acid motilin sequence and a long, 68-residue C-terminal peptide. The amino acid sequence of human motilin predicted from the cDNA sequence is identical to its porcine counterpart, which has been determined by protein sequencing. Proteolytic processing of promotilin to motilin occurs at the sequence, Lys-Lys, this being the first reported instance of processing occurring at a pair of Lys residues. In other precursors it occurs at Lys-Arg, Arg-Arg, Arg, or very rarely Lys.     

Distribution and characterization of motilin receptors in the cat

We demonstrate binding of [¹²⁵I][Nle¹³-po]motilin to homogenates of cat gastric and small intestinal, but not to colonic smooth muscle tissue. The density was (B_max in fmol/mg protein): 0 (fundus); 12 ± 2 (corpus); 22 ± 3 (antrum); 55 ± 12 (duodenum); 44 ± 10 (jejunum); 17 ± 1 (ileum); 0 (colon). A significant (p < 0.05) difference was found between the dissociation constant for motilin in the stomach (pK_d = 8.84 ± 0.06) and in the small intestine (pK_d = 8.58 ± 0.08). The motilides erythromycin-A (EM-A), EM-523, and EM-A N-oxide displaced labeled [Nle¹³-po]motilin bound to cat duodenal receptor with potencies (pK_d) of 5.47 ± 0.23, 7.60 ± 0.24, and < 4.3, respectively. Studies with [Leu¹³-po]motilin fragments showed that the N-terminus of motilin interacts with the receptor. In the tissue bath, duodenal strips mounted in the longitudinal direction responded to motilin, EM-523, and EM-A (pEC₅₀: 8.29 ± 0.08; 7.12 ± 0.12; 5.99 ± 0.15). The compounds had a comparable intrinsic activity (83 ± 3%; 80 ± 5%; 82 ± 5% of the response to ACh), which was unaffected by atropine, TTX, hexamethonium, and zacopride but reduced by verapamil and calcium-free medium. Cat stomach and small intestine possess smooth muscle motilin receptors, which have comparable properties as those found in man and in rabbit.     

Sequence and characterization of cDNA encoding the motilin precursor from chicken, dog, cow and horse. Evidence of mosaic evolution in prepromotilin

Motilin is involved in the regulation of the fasting motility pattern in man and in dog, but may have a different role in other species. Immunoreactive motilin has been demonstrated in several species, but the sequence is mostly unknown. The aim of this study was to isolate and sequence the cDNA encoding the motilin precursor from several mammalian species and from chicken. Total RNA was isolated from the duodenal mucosa of the chicken, dog, cow and horse. In each case single stranded cDNA was synthesized. Motilin cDNA fragments were amplified by PCR, ligated into a plasmid and cloned. Clones which were positive after screening with an appropriate (32)P-labeled probe were sequenced. The 5'- and 3'-ends were determined by the rapid amplification of cDNA ends (RACE) method. Analysis of the cDNAs revealed an open reading frame coding for 115 (chicken and cow), or 117 (dog and horse) amino acids. It consists of a 25 amino acid signal peptide, motilin itself, and a 68 (chicken and cow) or 70 (dog and horse) amino acid motilin associated peptide (MAP). As in all motilin precursors already sequenced (man, monkey, pig and rabbit), an endoproteinase cleavage site is present at Lys(23)-Lys(24). Comparison of all known sequences shows considerable identity in amino acid and nucleotide sequence of the signal peptide and motilin. However, the MAPs differ not only in length but also, more strongly, in amino acid and nucleotide sequence. Our study demonstrates that the N- and C-terminal regions of the motilin precursor have evolved at different rates, which is evidence for 'mosaic evolution'.     

Identification and expression of the motilin precursor in the guinea pig

Motilin has never been isolated from rodents, the most frequently used laboratory animals, despite several attempts. We have isolated and sequenced the motilin precursor from duodenal mucosa of guinea pig (GenBank accession number AF323752) and studied its expression in several tissues. The percent homology with human motilin is the lowest yet observed due to several unique substitutions in the C-terminal end. As expected, the precursor was present in the gut mucosa with the exception of the gastric corpus. It was also present in medulla oblongata, nucleus of the solitary tract, hypophysis, spinal cord, hypothalamus, and cerebellum but not in the cerebral cortex. For the first time we demonstrated motilin expression in the thyroid.     

The isolation and characterization of rabbit motilin precursor cDNA.

The cDNA sequence of rabbit motilin precursor has been determined. The predicted amino acid sequence indicates that the precursor consists of 133 amino acids and includes a 25 amino acid signal peptide followed by the 22 amino acid motilin sequence and an 86 amino acid motilin associated peptide (MAP). As in the human and porcine precursors, two lysine residues follow motilin in the rabbit sequence. Rabbit motilin shares 64% amino acid sequence identity with human and porcine motilin, and all amino acid substitutions represent conservative changes. Amino acid sequence alignments of the rabbit, human and porcine MAP sequences suggest three functional/structural motifs corresponding to a putative endoproteinase recognition site, a putative PEST site and a potential posttranslational processing recognition element.     

Characterization of complementary deoxyribonucleic acid for precursor of porcine motilin

Cloned cDNAs encoding the precursor protein for motilin and a novel peptide, motilin-associated peptide, were isolated from a library derived from porcine intestinal mucosa mRNA. Nucleotide sequence analysis predicts a precursor protein of 119 amino acids including a signal peptide in direct linkage with the 22 amino acid sequence for motilin, and a 70 amino acid peptide of unknown function. The putative bioactive moieties are separated by Lys-Lys, dibasic residues that serve as substrates for cleavage by proteolytic maturation enzymes in many polyprotein precursors. While there is an abundant literature detailing a spectrum of tissues and cell types which express motilin like immunoreactivity, analysis of mRNA derived from many of these tissues suggests that the mRNA for the mucosal motilin precursor is only transcribed in this tissue. The nature of the immunoreactive material in the central nervous system and other peripheral tissues remains to be determined.     

Alpha-melanotropin in the frog brain: regional distribution, quantification and subcellular distribution

The distribution of alpha-MSH containing neurons was studied by immunofluorescence in the brain of the frog Rana ridibunda. Most immunoreactive cell bodies were found in the ventral hypothalamic area. A rich network of fluorescent fibers was observed in the ventral infundibular region, coursing towards the preoptic area and the ventral telencephalon. Some fibers, directed backwards, project into median eminence. By means of a specific radioimmunoassay, the concentrations of alpha-MSH immunoreactive material has been determined in 10 different regions of the brain. The highest concentrations were observed in the infundibular and the preoptic regions. Using the immunogold technique, electron microscopy showed that immunostaining was restricted to 70-100 nm dense core vesicles in positive cell bodies and fibers. These results suggest that, in addition to well known hormonal (melanotropic) activity, alpha-MSH could play the role of a neurotransmitter in the frog brain.     

Real-time PCR quantification of precursor and mature microRNA

microRNAs (miRNAs) are challenging molecules to amplify by PCR because the miRNA precursor consists of a stable hairpin and the mature miRNA is roughly the size of a standard PCR primer. Despite these difficulties, successful real-time RT-PCR technologies have been developed to amplify and quantify both the precursor and mature microRNA. An overview of real-time PCR technologies developed by us to detect precursor and mature microRNAs is presented here. Protocols describe presentation of the data using relative (comparative C(T)) and absolute (standard curve) quantification. Real-time PCR assays were used to measure the time course of precursor and mature miR-155 expression in monocytes stimulated by lipopolysaccharide. Protocols are provided to configure the assays as low density PCR arrays for high throughput gene expression profiling. By profiling over 200 precursor and mature miRNAs in HL60 cells induced to differentiate with 12-O-tetradecanoylphorbol-13-acetate, it was possible to identify miRNAs who's processing is regulated during differentiation. Real-time PCR has become the gold standard of nucleic acid quantification due to the specificity and sensitivity of the PCR. Technological advancements have allowed for quantification of miRNA that is of comparable quality to more traditional RNAs.     

Anxiolytic effect of motilin and reversal with GM-109, a motilin antagonist, in mice

There have been few reports on the effects of the brain-gut peptide motilin on the central nervous system (CNS). We administered motilin intracerebroventricularly to mice and investigated the effect of motilin on anxiety using an elevated plus-maze. Motilin produced a significant decrease in anxiety with an inverted U-shaped dose response. To determine whether the anxiolytic effect of motilin was mediated via motilin receptors in the brain, the effect of GM-109, a novel motilin receptor antagonist, was investigated. GM-109 showed a significant and dose-dependent antagonism on the motilin-induced anxiolytic effect. GM-109 administered alone had no effect on anxiety. These results suggest that motilin receptors are present in the brain and may have a role in anxiety and emotion.     

Identification,distribution, and quantification of biominerals in a deciduous forest

Biomineralization is a common process in most vascular plants, but poorly investigated for trees. Although the presence of calcium oxalate and silica accumulation has been reported for some tree species, the chemical composition, abundance, and quantification of biominerals remain poorly documented. However, biominerals may play important physiological and structural roles in trees, especially in forest ecosystems, which are characterized by nutrient‐poor soils. In this context, our study aimed at investigating the morphology, distribution, and relative abundance of biominerals in the different vegetative compartments (foliage, branch, trunk, and root) of Fagus sylvatica L. and Acer pseudoplatanus L. using a combination of scanning electron microscopy and tomography analyses. Biomineral crystallochemistry was assessed by X‐ray diffraction and energy‐dispersive X‐ray analyses, while calcium, silicon, and oxalic acid were quantified in the compartments and at the forest scale. Our analyses revealed that biominerals occurred as crystals or coating layers mostly in bark and leaves and were identified as opal, whewellite, and complex biominerals. In both tree species, opal was mostly found in the external tissues of trunk, branch, and leaves, but also in the roots of beech. In the stand, opal represents around 170 kg/ha. Whewellite was found to suit to conductive tissues (i.e., axial phloem parenchyma, vascular bundles, vessel element) in all investigated compartments of the two tree species. The shape of whewellite was prismatic and druses in beech, and almost all described shapes were seen in sycamore maple. Notably, the amount of whewellite was strongly correlated with the total calcium in all investigated compartments whatever the tree species is, suggesting a biologic control of whewellite precipitation. The amount of whewellite in the aboveground biomass of Montiers forest was more important than that of opal and was around 1170 kg/ha. Therefore, biominerals contribute in a substantial way to the biogeochemical cycles of silicon and calcium.     

Sequence organization of the beta-globin mRNA precursor.

The sequence organization of the beta-globin mRNA precursor has been determined directly by analyzing the resistant fragments from the RNase A digestion of the precursor RNA-globin cDNA hybrid. Three fragments are obtained which proves that the beta-globin mRNA sequence in its precursor is split into three discontinuous segments. The two intervening sequences in the beta-globin gene are therefore transcribed and removed during mRNA maturation.     

Identification of cDNA encoding motilin related peptide/ghrelin precursor from dog fundus

A novel protein expressed by entero-endocrine cells of the mouse stomach was named prepromotilin Related Peptide (ppMTLRP) since it shares sequence similarities with the prepromotilin (Tomasetto et al.). The mouse ppMTLRP was found identical to the rat precursor of ghrelin (ppghrelin), an endogenous ligand specific for the Growth Hormone Secretagogue receptor identified from rat stomach (Kojima et al.). In the present study the cDNA encoding the dog counterpart of ppMTLRP/Ghrelin has been isolated and sequenced. The dog ppMTLRP/Ghrelin cDNA showed scores of respectively 80% and 75% homology with its human and mouse counterparts. By translation of the dog ppMTLRP/Ghrelin cDNA sequences, two ORFs could be deduced encoding either a 117 amino acid ppMTLRP/Ghrelin or the deleted Gln14 ppMTLRP/Ghrelin, as it was also known in mouse, rat and man. The dog ppMTLRP/Ghrelin shared 91% similarity and 78% identity, and 89% similarity and 78% identity with the human and mouse ppMTLRP/Ghrelin proteins respectively. The best score of homology was found in the MTLRP/Ghrelin sequence itself. Indeed the dog MTLRP/Ghrelin peptide shared 100% similarity and 93% identity, and 96% identity and similarity, with the human and mouse MTLRP/Ghrelin. Using Northern blot analysis to study dog ppMTLRP/Ghrelin gene expression on dog adult gut tissues, maximal expression level was found in the stomach fundus and corpus, and no expression could be detected in the stomach antrum nor in the duodenum, jejunum, ileum, colon or liver. In conclusion, we have identified ppMTLRP/Ghrelin from dog, and found that it is highly conserved with man, mouse or rat. The expression pattern along the gastro-intestinal tract is similar to the expression pattern previously described in mouse.     

Morphology, distribution, and density of sensory receptors in the glabrous skin of the cat rhinarium

The sensory organization of the cat rhinarium has been investigated. Individual rete pegs were found to contain a triad of receptors comprising free nerve endings ascending in the peg to terminate in close proximity to the skin surface, a basally situated layer of Merkel corpuscles, and an abundance of encapsulated receptors lying at the base and to one side of the rete peg. Neither the Merkel corpuscles nor the encapsulated receptors were evenly distributed. Merkel corpuscles were more abundant dorsally; ventrally they were fewer and asymmetrically arranged within individual rete pegs. The encapsulated corpuscles were more evenly distributed, but dorsally they were consistently present as encapsulated clusters of up to nine corpuscles.     

A novel HPLC procedure for detection and quantification of aminoacetone, a precursor of methylglyoxal, in biological samples

Increase in methylglyoxal is thought to be involved in different pathological conditions. Deamination of aminoacetone by semicarbazide-sensitive amine oxidase (SSAO) leads to production of methylglyoxal. We have synthesized aminoacetone and developed a novel HPLC procedure for its quantitative determination. The urinary excretion of aminoacetone is approximately 20-30 microg/mouse/day, and the concentration is about 0.5 microg/g in mouse liver and small intestine. SSAO inhibitor increases aminoacetone levels in both tissues and urines. Results confirm that aminoacetone is an endogenous substrate for SSAO. However, data also indicate that deamination is not the only catabolic pathway for aminoacetone.     

The motilin pharmacophore in CHO cells expressing the human motilin receptor

We performed a structure-activity study with the human motilin receptor, which was recently cloned from thyroid tissue. N-terminal fragments, Ala-analogs of motilin, and motilides were tested in a cell line that expresses the cloned human motilin receptor and apoaequorin. Full potency to induce calcium fluxes was obtained with N-terminal fragments of 14 amino acids. Motilin fragments 1-14 in which residues 1 (Phe), 4 (Ile), and 7 (Tyr) were replaced by Ala showed the largest reduction in potency. Only motilides with an enol configuration had markedly higher potencies compared to erythromycin A. The potencies to induce Ca(2+) fluxes correlated strongly with rabbit binding and contractility data, suggesting that the cloned receptor is indeed the motilin receptor, responsible for contractile effects. Conservation of the motilin pharmacophore in evolution indicates an important physiological role of motilin.