Serotonin, ghrelin, and motilin gene/receptor/transporter polymorphisms in childhood functional constipation.

OBJECTIVE: Functional constipation is the most common form of constipation, and its exact aetiology is still unclear. However, it is known that deficiencies in hormonal factors cause constipation by changing physiological mechanisms. Motilin, ghrelin, serotonin acetylcholine, nitric oxide, and vasoactive intestinal polypeptide are factors that play a role in colon motility. There are a limited number of studies in the literature where hormone levels and gene polymorphisms of serotonin and motilin are examined. Our study aimed to investigate the role of motilin, ghrelin, and serotonin gene/receptor/transporter polymorphisms in constipation pathogenesis in patients diagnosed with functional constipation according to the Rome 4 criteria. METHODS: Sociodemographic data, symptom duration, accompanying findings, the presence of constipation in the family, Rome 4 criteria, and clinical findings according to Bristol scale of 200 cases (100 constipated patients and 100 healthy control) who applied to Istanbul Haseki Training and Research Hospital, Pediatric Gastroenterology Outpatient Clinic, between March and September 2019 (6-month period) were recorded. Polymorphisms of motilin-MLN (rs2281820), serotonin receptor-HTR3A (rs1062613), serotonin transporter-5-HTT (rs1042173), ghrelin-GHRL (rs27647), and ghrelin receptor-GHSR (rs572169) were detected by real-time PCR. RESULTS: There was no difference between the two groups in terms of sociodemographic characteristics. Notably, 40% of the constipated group had a family history of constipation. The number of patients who started to have constipation under 24 months was 78, and the number of patients who started to have constipation after 24 months was 22. There was no significant difference between constipation and control groups in terms of genotype and allele frequencies in MLN, HTR3A, 5-HTT, GHRL, and GHSR polymorphisms (p<0.05). Considering only the constipated group, the rates of gene polymorphism were similar among those with/without a positive family history of constipation, constipation onset age, those with/without fissures, those with/without skin tag, and those with type 1/type 2 stool types according to the Bristol stool scale. CONCLUSION: Our study results showed that gene polymorphisms of these three hormones may not be related to constipation in children.

Gastric Activity and Gut Peptides in Patients With Functional Dyspepsia: Postprandial Distress Syndrome Versus Epigastric Pain Syndrome.

GOALS: The goals of the study were to investigate in both postprandial distress syndrome (PDS) and epigastric pain syndrome (EPS) the gastric electrical activity and the gastric emptying (GE) time together with the circulating concentrations of motilin, somatostatin, corticotrophin-releasing factor, and neurotensin, and to establish whether the genetic variability in the neurotensin system genes differs between these 2 categories of functional dyspepsia (FD). BACKGROUND: The current FD classification is based on symptoms and it has been proven not to be completely satisfying because of a high degree of symptom overlap between subgroups. STUDY: Gastric electrical activity was evaluated by cutaneous electrogastrography: the GE time by C-octanoic acid breast test. Circulating concentrations of gut peptides were measured by a radioimmunoassay. NTS 479 A/G and NTSR1 rs6090453 SNPs were evaluated by PCR and endonuclease digestion. RESULTS: Fifty-four FD patients (50 female/4 male) were studied. Using a symptom questionnaire, 42 patients were classified as PDS and 12 as EPS, although an overlap between the symptom profiles of the 2 subgroups was recorded. The electrogastrographic parameters (the postprandial instability coefficient of dominant frequency, the dominant power, and the power ratio) were significantly different between the subgroups, whereas the GE time did not differ significantly. In addition, EPS was characterized by a different gut peptide profile compared with PDS. Finally, neurotensin polymorphism was shown to be associated with neurotensin levels. This evidence deserves further studies in consideration of an analgesic role of neurotensin. CONCLUSIONS: Analysis of gut peptide profiles could represent an interesting tool to enhance FD diagnosis and overcome limitations due to a distinction based solely on symptoms.

Therapeutic effects of Lactobacillus casei Qian treatment in activated carbon-induced constipated mice.

In the present study, the therapeutic effects of Lactobacillus casei Qian (LC-Qian), the key microorganism in Tibetan yak yoghurt, on activated carbon-induced constipation were determined in vivo. ICR mice were treated with LC-Qian for nine days by oral administration. The body weight, defecation status, gastrointestinal transit and defecation time of mice were assessed, and the serum levels of motilin (MTL), gastrin (Gas), endothelin (ET), somatostatin (SS), acetylcholinesterase (AChE), substance P (SP) and vasoactive intestinal peptide (VIP) were further evaluated. Bisacodyl was used as the positive control. The time until the first black stool defecation following carbon intake of the normal, control, 100 mg/kg bisacodyl-treated, Lactobacillus bulgaricus (LB)-treated, LC-Qian (L)-and LC-Qian (H)-treated mice was 93, 231, 121, 194, 172 and 157 min, respectively. Following treatment with LC-Qian, the gastrointestinal transit was increased to 52.4% [LC-Qian (L)] and 65.8% [LC-Qian (H)], while that in the group treated with the common lactic acid bacteria of LB was 40.3%. The MTL, Gas, ET, AChE, SP and VIP serum levels were significantly increased and levels of SS were reduced in mice following LC-Qian treatment compared with those in the control mice (P<0.05). Reverse transcription quantitative polymerase chain reaction indicated that LC-Qian raised the c-Kit, GDNF as well as SCF mRNA expression levels and reduced the TRPV1 and NOS expression levels in tissue of the small intestine in mice. These results suggested that lactic acid bacteria prevent constipation in mice, among which LC-Qian was the most effective.

Identification and characterization of a motilin-like peptide and its receptor in teleost.

Although putative motilin receptor sequences have been reported in teleost, there is no proof for the existence of the motilin gene in teleost. In this study, we have identified a motilin-like gene in the genome of several fish species and cloned its cDNA sequence from zebrafish. The zebrafish motilin-like precursor shares very low amino acid (aa) identities with the previously reported motilin precursors. Processing of the zebrafish motilin-like precursor may generate a 17-aa C-terminal amidated mature peptide, the motilin-like peptide (motilin-LP). A putative zebrafish motilin receptor (MLNR) was also identified in zebrafish. In cultured eukaryotic cells transfected with the zebrafish MLNR, zebrafish motilin-LP could enhance both CRE-driven and SRE-driven promoter activities. Tissue distribution studies indicated that the zebrafish motilin-like gene is mainly expressed in the intestine and liver while the zebrafish MLNR gene is highly expressed in brain regions, suggesting that motilin-LP behaves like other gut hormones to regulate brain functions. These data suggest that the presence of a unique motilin/MNLR system in teleost.

Predictors of gastric myoelectrical activity in type 2 diabetes mellitus.

BACKGROUND: Previous studies have clearly demonstrated the delayed gastric emptying of solid meals in diabetics, whereas their gastric myoelectrical activity, which primarily determines gastric motility, has not yet been fully confirmed. GOALS: This study aimed to clarify the characteristics and potential predictors of gastric myoelectrical activity in type 2 diabetics. STUDY: Twenty-eight diabetics and 18 healthy controls participated. Duodenal biopsy sample was used for reverse transcription-polymerase chain reaction to evaluate cholecystokinin and motilin mRNA contents. Electrogastrography was performed before and after the test meal, and was assessed in terms of dominant frequency; dominant frequency instability coefficient; and the percentage of bradygastria, normogastria, and tachygastria. RESULTS: Over the entire recording period, dominant frequency was significantly lower, and dominant frequency instability coefficient and the percentage of bradygastria were significantly higher in diabetics than in controls. In diabetics, the multiple regression analysis demonstrated that dominant frequency instability coefficient and the percentage of tachygastria in the fasting period were dependent on fasting plasma glucose level and HbA1c, respectively. Moreover, dominant frequency over the entire period and the postprandial percentage of bradygastria were significantly associated with body mass index; the fasting percentage of bradygastria and postprandial dominant frequency instability coefficient were associated with fasting serum leptin level; the postprandial percentage of bradygastria was also associated with cholecystokinin mRNA content. CONCLUSIONS: Gastric myoelectrical activity in type 2 diabetics is impaired on dominant frequency, dominant frequency instability coefficient, and the percentage of bradygastria and predicted by body mass index, fasting serum leptin level, and cholecystokinin mRNA content besides the glycemic status.

[Expression of motilin and its precursor mRNA in normal parenchyma, benign and malignant tumors of human thyroid].

OBJECTIVE: To investigate the expression of motilin and its precursor mRNA in normal human thyroid. To compare the expression differences of motilin and it precursor mRNA between normal thyroid and intestines. To study the expression of motilin and its precursor mRNA in human thyroid tumors and their clinical implications. METHODS: RT-PCR, Southern blot and molecular cloning were used to detect motilin transcript expression in human thyroid and mucous membrane of small intestine. Real-time PCR and immunohistochemical techniques were used to quantify motilin precursor mRNA and motilin peptide in thyroid tissue samples including adenoma, medullary carcinoma, follicular carcinoma, papillary carcinoma and nodular goiter. RESULTS: (1) The expression of motilin and its precursor mRNA in normal human thyroid was primarily in the thyroid C cells. (2) RT-PCR and Southern blot showed that motilin mRNA expressed in human thyroid was identical to that expressed in duodenum with identical sequence deposited in NCBI Genbank of America. (3) Immunohistochemistry, Western blot research and real-time PCR studies showed that motilin and its precursor mRNA were expressed in normal and tumor tissues of human thyroid. Thyroid tumors (acidophilic adenoma, medullary carcinoma, follicular carcinoma, papillary carcinoma and nodular goiter) showed intense and diffuse immunostaining for motilin peptide. Moreover, the expression of motilin and its precursor mRNA in thyroid medullar carcinoma and acidophilic adenoma were significantly higher than those of normal thyroid tissue (P < 0.05). The expression in thyroid follicular and papillary carcinomas were significantly lower than those of normal thyroid tissue (P < 0.05). There was no difference of the expression between nodular goiter and normal thyroid tissue (P > 0.05). CONCLUSIONS: Motilin peptide and its precursor mRNA are expressed in C cells of human thyroid. The sequence of motilin is identical to that expressed in duodenum from NCBI Genbank of America. The expressions of both motilin and its precursor mRNA in thyroid medullary carcinoma and acidophilic adenoma are significantly increased. In contrast, their expressions in thyroid follicular and papillary carcinomas are significantly decreased. Motilin may regulate physiological functions of the thyroid through parafollicular cells. Motilin may be involved in the pathogenesis of medullary carcinoma and acidophilic adenoma of the thyroid.

Absence of motilin gene mutations in infantile hypertrophic pyloric stenosis.

BACKGROUND: Erythromycin treatment before 2 weeks of age has been shown to increase the risk of infantile hypertrophic pyloric stenosis (IHPS) up to 10 times. Erythromycin is a motilin agonist, a hormone that induces gastrointestinal contractions. The purpose of this study was to investigate if mutations in the motilin gene (MLN) cause IHPS or if the V15A polymorphism in MLN is associated with the disease. METHODS: The MLN was screened for mutations, and the V15A polymorphism was determined in a total of 57 patients with IHPS using polymerase chain reaction and DNA sequencing. The polymorphism genotype and allele frequencies among the patients were compared with 184 controls. RESULTS: We detected 3 different, not previously reported, MLN sequence variants in 4 patients. One of these variants results in an amino acid exchange in the motilin signal peptide (A25G). All 3 detected sequence variants were also found in controls or were not inherited with the disease. We found no significant association between the V15A polymorphism and the disease. CONCLUSIONS: We have excluded the MLN coding region as a major cause of IHPS. Future studies will evaluate the importance of this metabolic pathway in the pathogenesis of IHPS.

Ghrelin and a novel preproghrelin isoform are highly expressed in prostate cancer and ghrelin activates mitogen-activated protein kinase in prostate cancer.

PURPOSE: There is evidence that the hormone ghrelin stimulates proliferation in the PC3 prostate cancer cell line although the underlying mechanism(s) remain to be determined. A novel, exon 3-deleted preproghrelin isoform has previously been detected in breast and prostate cancer cells; however, its characterization, expression, and potential function in prostate cancer tissues are unknown. EXPERIMENTAL DESIGN: Expression of ghrelin and exon 3-deleted preproghrelin was investigated in prostate cancer cell lines and tissues by reverse transcription-PCR and immunohistochemistry. Proliferation and apoptosis assays were done in the LNCaP prostate cancer cell line to determine if ghrelin stimulates proliferation and/or cell survival. Stimulation of mitogen-activated protein kinase (MAPK) pathway activation by ghrelin was determined in PC3 and LNCaP cells by immunoblotting with antibodies specific for phosphorylated MAPKs. RESULTS: Prostate cancer tissues display greater immunoreactivity for ghrelin and exon 3-deleted preproghrelin than normal prostate tissues, and prostate cancer cell lines secrete mature ghrelin into conditioned medium. Treatment with ghrelin (10 nmol/L), but not the unique COOH-terminal peptide derived from exon 3-deleted preproghrelin, stimulates proliferation in the LNCaP cells (45.0 +/- 1.7% above control, P < 0.01) and rapidly activates the extracellular signal-regulated kinase-1/2 MAPK pathway in both PC3 and LNCaP cell lines. Ghrelin, however, does not protect prostate cancer cells from apoptosis induced by actinomycin D (1 microg/mL). The MAPK inhibitors PD98059 and U0126 blocked ghrelin-induced MAPK activation, as well as proliferation, in both cell lines. CONCLUSIONS: These data suggest that these components of the ghrelin axis may have potential as novel biomarkers and/or adjunctive therapeutic targets for prostate cancer.

[Preproghrelin gene, ghrelin receptor and metabolic syndrome]. / Gen preproghreliny i receptor ghreliny a zespól metaboliczny.

Obesity is a multi-gene syndrome, expression of which is modulated not only by environmental factors but above all by a number of modified genes interacting with each other. Among candidate genes related to obesity phenotype is ghrelin gene. Ghrelin plays a significant role in feeding regulation and is the strongest stimulator of growth hormone secretion. Ghrelin acts by GHS1a receptor (growth hormone secretagogue receptor). Mutations in preproghrelin and ghrelin gene or ghrelin receptor gene could be responsible for low ghrelin levels observed in obese individuals. Among identificated mutations, two Arg51 Gln and Leu72Met are most often described and change amino-acid sequence of ghrelin (Arg51Gln) and preproghrelin (Leu72Met). Although no direct relationship between Arg51Gln mutation and obesity phenotype was found, it had been shown that carriers of Arg51Gln mutation had significantly decreased plasma ghrelin levels. Furthermore 51Gln allele carriers had higher prevalence of type 2 diabetes mellitus and hypertension than non-carriers. Met 72 carrier status is associated with higher serum IGF-1 levels and seems to be a protective factor against fat accumulation and cardiovascular complications of obesity. No evidence of relationship between ghrelin receptor gene polymorphisms and body mass regulation was found, however, until now there is no study on relationships between these polymorphisms and metabolic complications of obesity. The presence of genetic variants in ghrelin or GHS receptor gene could be responsible for impaired GH secretion in visceral type obesity and development of metabolic syndrome in some of obese subjects. On the other hand, some mutations in preproghrelin gene could be protective against metabolic syndrome.

Evidence for the presence of motilin, ghrelin, and the motilin and ghrelin receptor in neurons of the myenteric plexus.

Motilin, a 22-amino acid gastrointestinal peptide, and ghrelin, the natural ligand of the growth hormone secretagogue receptor, form a new group of structurally related peptides. Several lines of evidence suggest that motilin and ghrelin are involved in the control of gastrointestinal motility by the activation of receptors on enteric neurons. The aim of this study was to look for the existence of motilin, ghrelin, and their respective receptors in the myenteric plexus of the guinea pig. We used longitudinal muscle/myenteric plexus (LMMP) preparations and cultures of myenteric neurons of the guinea pig ileum, immunohistochemistry, and reverse transcriptase-polymerase chain reaction (RT-PCR). Most of the motilin-immunoreactive (IR; 72.8%) and motilin receptor-IR (68.9%) neurons were also positive for neuronal nitric oxide synthase (nNOS), 72.8% and 68.9%, few for choline acetyl transferase (ChAT), 11.4% and 11.9%, respectively. In contrast, ghrelin was mainly colocalized with ChAT (72.2%), and only 3.6% of ghrelin-positive cells showed nNOS-IR in the LMMP. Neither motilin nor the motilin receptor or ghrelin colocalized with calbindin. RT-PCR studies revealed motilin, ghrelin, and ghrelin receptor mRNA transcripts in LMMP preparations and in cultured myenteric neurons. In conclusion, this study, for the first time, provides direct evidence for the existence of motilin and ghrelin in myenteric neurons and suggests that both peptides may play a role in the activation of the enteric nervous system and hence in the regulation of gastrointestinal motility.

Transgenic mice overexpressing des-acyl ghrelin show small phenotype.

Ghrelin, a 28-amino acid acylated peptide, displays strong GH-releasing activity in concert with GHRH. The fatty acid modification of ghrelin is essential for the actions, and des-acyl ghrelin, which lacks the modification, has been assumed to be devoid of biological effects. Some recent reports, however, indicate that des-acyl ghrelin has effects on cell proliferation and survival. In the present study, we generated two lines of transgenic mice bearing the preproghrelin gene under the control of chicken beta-actin promoter. Transgenic mice overexpressed des-acyl ghrelin in a wide variety of tissues, and plasma des-acyl ghrelin levels reached 10- and 44-fold of those in control mice. They exhibited lower body weights and shorter nose-to-anus lengths, compared with control mice. The serum GH levels tended to be lower, and the serum IGF-I levels were significantly lower in both male and female transgenic mice than control mice. The responses of GH to administered GHRH were normal, whereas those to administered ghrelin were reduced, especially in female transgenic mice, compared with control mice. These data suggest that overexpressed des-acyl ghrelin may modulate the GH-IGF-I axis and result in small phenotype in transgenic mice.

Utilization of Escherichia coli outer-membrane endoprotease OmpT variants as processing enzymes for production of peptides from designer fusion proteins.

Escherichia coli outer-membrane endoprotease OmpT has suitable properties for processing fusion proteins to produce peptides and proteins. However, utilization of this protease for such production has been restricted due to its generally low cleavage efficiency at Arg (or Lys)-Xaa, where Xaa is a nonbasic N-terminal amino acid of a target polypeptide. The objective of this study was to generate a specific and efficient OmpT protease and to utilize it as a processing enzyme for producing various peptides and proteins by converting its substrate specificity. Since OmpT Asp(97) is proposed to interact with the P1' amino acid of its substrates, OmpT variants with variations at Asp(97) were constructed by replacing this amino acid with 19 natural amino acids to alter the cleavage specificity at Arg (P1)-Xaa (P1'). The variant OmpT that had a methionine at this position, but not the wild-type OmpT, efficiently cleaved a fusion protein containing the amino acid sequence -Arg-Arg-Arg-Ala-Arg downward arrow motilin, in which motilin is a model peptide with a phenylalanine at the N terminus. The OmpT variants with leucine and histidine at position 97 were useful in releasing human adrenocorticotropic hormone (1-24) (serine at the N terminus) and human calcitonin precursor (cysteine at the N terminus), respectively, from fusion proteins. Motilin was produced by this method and was purified up to 99.0% by two chromatographic steps; the yield was 160 mg/liter of culture. Our novel method in which the OmpT variants are used could be employed for production of various peptides and proteins.

Enhanced plasma ghrelin levels in rats with streptozotocin-induced diabetes.

Ghrelin is a novel gastrointestinal peptide that stimulates growth hormone secretion, food intake, and body weight gain. Increased ghrelin secretion has been reported in such negative energy states as starvation and low body weight. We investigated the dynamics of ghrelin in rats with streptozotocin-induced diabetes, because they present reduced body weight and hyperphagia. The plasma ghrelin levels and gastric preproghrelin mRNA expression levels of the diabetic rats increased significantly and their gastric ghrelin levels decreased significantly. Negative energy balance may enhance preproghrelin mRNA expression and ghrelin secretion into the bloodstream.

A variation in the ghrelin gene increases weight and decreases insulin secretion in tall, obese children.

Ghrelin is a recently recognized gut-brain peptide originally derived from the gastric mucosa. It stimulates growth hormone release, increases appetite and facilitates fat storage, and may interact with glucose metabolism. We studied the ghrelin gene in a group of 70 tall and obese children (mean age 9.4 year, Z body mass index [BMI] and Z height >3 and/or BMI percentile >99%). We found 10 single nucleotide polymorphisms. One common polymorphism of the ghrelin gene, which corresponds to an amino acid change in the tail of the prepro-ghrelin molecule, was significantly associated with children with a higher BMI (P = 0.001), and with lower insulin secretion during the first part of an oral glucose tolerance test (P = 0.05) although no difference in glucose levels was noted. This might suggest increased insulin sensitivity, although this is not supported by the lack of difference in fasting and 2 hour insulin levels; alternatively, this may be indicative of impaired first phase insulin secretion. These data suggest that variations in the ghrelin gene contribute to obesity in children and may modulate glucose-induced insulin secretion.

Ghrelin is an appetite-stimulatory signal from stomach with structural resemblance to motilin.

BACKGROUND & AIMS: : Ghrelin, an endogenous ligand for growth hormone secretagogue receptor, was recently identified in the rat stomach. We examined the effects of the gastric peptide ghrelin on energy balance in association with leptin and vagal nerve activity. METHODS: : Food intake, oxygen consumption, gastric emptying, and hypothalamic neuropeptide Y (NPY) messenger RNA expression were measured after intra-third cerebroventricular or intraperitoneal injections of ghrelin in mice. The gastric vagal nerve activity was recorded after intravenous administration in rats. Gastric ghrelin gene expression was assessed by Northern blot analysis. Repeated coadministration of ghrelin and interleukin (IL)-1 beta was continued for 5 days. RESULTS: : Ghrelin exhibited gastroprokinetic activity with structural resemblance to motilin and potent orexigenic activity through action on the hypothalamic neuropeptide Y (NPY) and Y(1) receptor, which was lost after vagotomy. Ghrelin decreased gastric vagal afferent discharge in contrast to other anorexigenic peptides that increased the activity. Ghrelin gene expression in the stomach was increased by fasting and in ob/ob mice, and was decreased by administration of leptin and IL-1 beta. Peripherally administered ghrelin blocked IL-1 beta-induced anorexia and produced positive energy balance by promoting food intake and decreasing energy expenditure. CONCLUSIONS: : Ghrelin, which is negatively regulated by leptin and IL-1 beta, is secreted by the stomach and increases arcuate NPY expression, which in turn acts through Y(1) receptors to increase food intake and decrease energy expenditure. Gastric peptide ghrelin may thus function as part of the orexigenic pathway downstream from leptin and is a potential therapeutic target not only for obesity but also for anorexia and cachexia.

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.

Identification and characterization of a novel gastric peptide hormone: the motilin-related peptide.

BACKGROUND & AIMS: This study looked for new proteins with expression restricted to the gastric epithelium that may provide insight to the differentiation and function of the gastric unit. METHODS: A novel complementary DNA was isolated and sequenced, and its expression was examined in mouse tissues at both messenger RNA and protein levels. Subcellular localization was studied using immunoelectron microscopy. The posttraductional processing of the protein was analyzed in vitro by protein microsequencing and in vivo by Western blotting. RESULTS: We identified a novel protein that is mainly expressed by the secretory granules of the stomach enteroendocrine cells. This protein has sequence similarity with prepromotilin, the precursor of the motilin hormone and the motilin-associated peptide. As for the prepromotilin, a posttraductional maturation leads to a secreted peptide that is further cleaved at a dibasic site and gives rise to the motilin-related peptide (MTLRP) and MTLRP-associated peptide. CONCLUSIONS: We have identified and characterized a novel gene encoding the preproMTLRP protein. MTLRP presents similarity to motilin and is specifically expressed by enteroendocrine cells of the stomach and therefore represents a novel hormone.

Polymorphism of motilin gene in patients with Crohn's disease.

An increasing body of evidence supports the concept of genetic heterogeneity within inflammatory bowel disease (IBD). In this study, a polymorphism of the motilin gene, which determines an amino acid substitution in the motilin protein, has been investigated in IBD patients. Fifty patients with ulcerative colitis (UC), and 52 with Crohn's disease (CD) were investigated for anti-neutrophil cytoplasmatic antibodies (ANCA) and the polymorphism in the second exon of the motilin gene. Sixty unrelated blood donors served as controls. ANCA were found in 30% of UC and 13% of CD. In controls the DNA polymorphism identified two alleles (1 and 2) at a frequency of 42% and 58%, respectively. Patients with either UC or CD showed a slight increase in the frequency of allele 2 (69% and 60%, respectively; P > 0.05 vs controls). This allele was predominant in ANCA-positive CD patients (86%; P < 0.04) while in UC it did not differ. All ANCA-positive CD patients had the disease confined to the colon. A polymorphism of second exon of the motilin gene, leading to a protein variant, is significantly more frequent in the subset of ANCA-positive CD patients. This subgroup of patients appears to share peculiar genetic and clinical features.