RESUMEN
BACKGROUND: Patients with Barrett's oesophagus (BO) are at risk of oesophageal adenocarcinoma. Because the pattern of mucosal mucins changes during neoplastic progression, it may serve as a marker of intraepithelial neoplasia. AIMS: To determine the expression pattern of mucins in neoplastic BO epithelium (high grade dysplasia) and correlate it with the expression of apoptosis markers Bax and Bcl-2. METHODS: Thirty seven patients with BO were studied: 16 without intraepithelial neoplasia, six with high grade intraepithelial neoplasia (HGN), and 15 with infiltrating adenocarcinoma. Biopsies were obtained from squamous epithelium, Barrett's epithelium, and (when present) foci of suspected HGN or adenocarcinoma. MUC1-4, MUC5AC, MUC5B, MUC6, Bax, and Bcl-2 mRNA were determined by semiquantitative RT-PCR. MUC2, MUC5AC, and MUC6 protein was determined by immunoblotting. RESULTS: Mucin expression varied between neoplastic progression stages in BO. Mucin mRNA levels were low in squamous epithelium, except for MUC4, and were at least four times higher in BO and HGN (p<0.001), but less so in adenocarcinoma. MUC4 expression was significantly lower in BO than in normal squamous epithelium, whereas in HGN and adenocarcinoma, levels were significantly higher than in BO (p = 0.037). The Bax:Bcl-2 ratio was increased in HGN compared with BO (p = 0.04). MUC2, MUC5AC, and MUC6 protein values correlated with mRNA data. CONCLUSIONS: Mucin expression varies during the development of oesophageal adenocarcinoma in BO. MUC4 could serve as a tumour marker in this process. In contrast to animal studies, upregulation of MUC4 in HGN is associated with increased apoptosis, suggesting that MUC4 plays a minor role in apoptosis regulation in BO.
Asunto(s)
Esófago de Barrett/metabolismo , Carcinoma in Situ/química , Neoplasias Esofágicas/química , Mucinas/análisis , Proteínas de Neoplasias/análisis , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Adenocarcinoma/química , Apoptosis/fisiología , Regulación Neoplásica de la Expresión Génica , Humanos , Mucina 4 , ARN Mensajero/análisis , ARN Neoplásico/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Proteína X Asociada a bcl-2RESUMEN
Rotavirus is the most important cause of viral gastroenteritis and dehydrating diarrhea in young children. Rotavirus nonstructural protein 4 (NSP4) is an enterotoxin that was identified as an important agent in symptomatic rotavirus infection. To identify cellular proteins that interact with NSP4, a two-hybrid technique with Saccharomyces cerevisiae was used. NSP4 cDNA, derived from the human rotavirus strain Wa, was cloned into the yeast shuttle vector pGBKT7. An intestinal cDNA library derived from Caco-2 cells cloned into the yeast shuttle vector pGAD10 was screened for proteins that interact with NSP4. Protein interactions were confirmed in vivo by coimmunoprecipitation and immunohistochemical colocalization. After two-hybrid library screening, we repeatedly isolated cDNAs encoding the extracellular matrix (ECM) protein laminin-beta3 (amino acids [aa] 274 to 878) and a cDNA encoding the ECM protein fibronectin (aa 1755 to 1884). Using deletion mutants of NSP4, we mapped the region of interaction with the ECM proteins between aa 87 and 145. Deletion analysis of laminin-beta3 indicated that the region comprising aa 726 to 875 of laminin-beta3 interacts with NSP4. Interaction of NSP4 with either laminin-beta3 or fibronectin was confirmed by coimmunoprecipitation. NSP4 was present in infected enterocytes and in the basement membrane (BM) of infected neonatal mice and colocalized with laminin-beta3, indicating a physiological interaction. In conclusion, two-hybrid screening with NSP4 yielded two potential target proteins, laminin-beta3 and fibronectin, interacting with the enterotoxin NSP4. The release of NSP4 from the basal side of infected epithelial cells and the subsequent binding to ECM proteins localized at the BM may signify a new mechanism by which rotavirus disease is established.
Asunto(s)
Enterotoxinas/metabolismo , Fibronectinas/metabolismo , Glicoproteínas/metabolismo , Laminina/metabolismo , Rotavirus/patogenicidad , Proteínas no Estructurales Virales/metabolismo , Animales , Sitios de Unión/genética , Células CACO-2 , ADN/genética , ADN Viral/genética , Enterotoxinas/química , Enterotoxinas/genética , Fibronectinas/química , Fibronectinas/genética , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Laminina/química , Laminina/genética , Ratones , Ratones Endogámicos BALB C , Estructura Terciaria de Proteína , Rotavirus/genética , Rotavirus/metabolismo , Infecciones por Rotavirus/etiología , Infecciones por Rotavirus/virología , Eliminación de Secuencia , Toxinas Biológicas , Técnicas del Sistema de Dos Híbridos , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genéticaRESUMEN
AIMS: Barrett's oesophagus constitutes metaplastic epithelium, often diagnosed by mucin histochemistry. We determined the mucins and trefoil factor family (TFF)-peptides that were expressed in Barrett's oesophagus, in order to study changes in protein expression in early stages of Barrett's oesophagus development. METHODS AND RESULTS: Biopsy specimens of 71 Barrett's oesophagus patients were collected, and sections were stained for secretory mucins by histochemistry. Immunohistochemistry was performed for secretory mucins (MUC2, MUC5AC, MUC5B, MUC6), TFFs (TFF1, TFF2, TFF3), and proliferation (Ki67). Protein expression in the tissue was measured semiquantitatively. MUC5AC and TFF1 showed high levels and strong colocalization in the surface epithelium, whereas MUC6, MUC5B and TFF3 were found in the deeper glandular structures. TFF2 was found in both surface and glandular epithelium. The co-ordinate expression patterns of these six markers were similar to gastric antrum epithelium. MUC2 expression was ubiquitously associated with goblet cells within intestinal metaplasia, occurring in 68% of patients, and was correlated with increasing proliferation in the epithelium. CONCLUSIONS: Virtually all cells in Barrett's oesophagus epithelium displayed a secretory phenotype, demonstrating a co-ordinate gastric-type MUC and TFF expression. When MUC2 expression was more pronounced, the expression patterns of the other MUCs and the TFFs were increasingly disturbed. MUC2 expression may constitute a marker for early change in the phenotype of Barrett's oesophagus as a precancerous lesion.
Asunto(s)
Esófago de Barrett/metabolismo , Biomarcadores de Tumor/metabolismo , Mucinas Gástricas/metabolismo , Mucinas/metabolismo , Proteínas Musculares , Neuropéptidos , Péptidos/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Esófago de Barrett/patología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Técnicas para Inmunoenzimas , Antígeno Ki-67/metabolismo , Masculino , Persona de Mediana Edad , Mucina 2 , Factor Trefoil-2 , Factor Trefoil-3RESUMEN
PURPOSE: This study evaluates the effects of enteral inulin on ileoanal pouch functioning by studying epithelial gene expression, cell turnover, and mucosal morphology. METHODS: Twenty patients with an ileoanal pouch received 24 g of inulin daily for three weeks, then a four-week wash-out period, and a placebo for three weeks. In this randomized, double-blind, crossover study, biopsy specimens of pouch mucosa were taken after each test period. Mucosal morphology, inflammation, epithelial proliferation, and cell death were assessed histologically. Expressions of proapoptotic and antiapoptotic regulators, intestinal fatty acid-binding protein, and mucin were quantified by Western blotting or enzyme-linked immunosorbent assay. The number of intestinal fatty acid-binding protein expressing cells was histologically assessed and a high iron diamine/Alcian blue staining was performed to discriminate between sulfated and nonsulfated acidic mucins. RESULTS: Inulin supplementation neither altered mucosal morphology nor influenced inflammation, epithelial cell proliferation, or cell death. The ratio between the proapoptotic and antiapoptotic regulators did not change after inulin supplementation. The number of intestinal fatty acid-binding protein-producing enterocytes and the intestinal fatty acid-binding protein expression level increased after inulin treatment, but did not reach statistical significance. The intestinal fatty acidbinding protein expression level correlated with the Pouchitis Disease Activity Index, which was at the brink of significance (P = 0.06). Mucin expression and the ratio between sulfated and nonsulfated acidic mucins were not altered by inulin supplementation. CONCLUSION: In this prospective study, inulin supplementation did not significantly alter pouch mucosal functioning because neither epithelial homeostasis nor epithelial gene expression was significantly altered by enteral inulin.
Asunto(s)
Células Epiteliales/efectos de los fármacos , Regulación de la Expresión Génica , Inulina/farmacología , Proctocolectomía Restauradora , Adulto , Apoptosis , División Celular , Estudios Cruzados , Método Doble Ciego , Células Epiteliales/fisiología , Ácidos Grasos Volátiles/metabolismo , Femenino , Homeostasis , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/fisiología , Inulina/administración & dosificación , Masculino , Estudios ProspectivosRESUMEN
Proliferation, differentiation, and cell death were studied in small intestinal and colonic epithelia of rats after treatment with methotrexate. Days 1-2 after treatment were characterized by decreased proliferation, increased apoptosis, and decreased numbers and depths of small intestinal crypts in a proximal-to-distal decreasing gradient along the small intestine. The remaining crypt epithelium appeared flattened, except for Paneth cells, in which lysozyme protein and mRNA expression was increased. Regeneration through increased proliferation during days 3-4 coincided with villus atrophy, showing decreased numbers of villus enterocytes and decreased expression of the enterocyte-specific genes sucrase-isomaltase and carbamoyl phosphate synthase I. Remarkably, goblet cells were spared at villus tips and remained functional, displaying Muc2 and trefoil factor 3 expression. On days 8-10, all parameters had returned to normal in the whole small intestine. No methotrexate-induced changes were seen in epithelial morphology, proliferation, apoptosis, Muc2, and TFF3 immunostaining in the colon. The observed small intestinal sparing of Paneth cells and goblet cells following exposure to methotrexate is likely to contribute to epithelial defense during increased vulnerability of the intestinal epithelium.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Células Caliciformes/citología , Metotrexato/farmacología , Proteínas Musculares , Células de Paneth/citología , Animales , Apoptosis/efectos de los fármacos , Atrofia/inducido químicamente , Biomarcadores , Recuento de Células , Diferenciación Celular/fisiología , Expresión Génica/efectos de los fármacos , Etiquetado Corte-Fin in Situ , Neoplasias Intestinales/tratamiento farmacológico , Neoplasias Intestinales/patología , Intestinos/citología , Intestinos/efectos de los fármacos , Intestinos/fisiología , Masculino , Mucina 2 , Mucinas/genética , Péptidos , Proteínas/genética , ARN Mensajero/análisis , Ratas , Ratas Endogámicas , Regeneración/fisiología , Factor Trefoil-3RESUMEN
BACKGROUND: The bacterium Helicobacter pylori is able to adhere to and to colonise the human gastric epithelium, yet the primary gene product responsible as a receptor for its adherence has not been identified. AIMS: To investigate the expression of the gastric mucins MUC5AC and MUC6 in the gastric epithelium in relation to H pylori colonisation in order to examine their possible roles in the binding of H pylori. PATIENTS: Seventy two consecutive patients suspected of having H pylori infection. METHODS: MUC5AC, MUC6, and H pylori were detected in single sections of antral biopsy specimens using immunohistochemical triple staining. RESULTS: MUC5AC was expressed in the superficial epithelium and the upper part of the gastric pits. MUC6 expression was detected in the lower part of the gastric pits. The expression of both mucins in the epithelium was complementary. In each patient, there was a sharply delineated transition between MUC5AC and MUC6 producing cell populations. In all H pylori positive patients there was a striking colocalization of H pylori and MUC5AC; more than 99% of the bacteria were associated with either extracellular MUC5AC or the apical domain of MUC5AC producing cells. CONCLUSIONS: H pylori is very closely associated with extracellular MUC5AC and epithelial cells that produce MUC5AC. This indicates that MUC5AC, but not MUC6, plays a role in the adhesion of H pylori to the gastric mucosa.
Asunto(s)
Adhesión Bacteriana/fisiología , Infecciones por Helicobacter/metabolismo , Helicobacter pylori/fisiología , Mucinas/metabolismo , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiología , Expresión Génica , Infecciones por Helicobacter/microbiología , Helicobacter pylori/aislamiento & purificación , Humanos , Inmunohistoquímica , Mucina 5AC , Mucina 6 , Estudios ProspectivosRESUMEN
BACKGROUND: Decreased synthesis of the predominant secretory human colonic mucin (MUC2) occurs during active ulcerative colitis. AIMS: To study possible alterations in mucin sulphation and mucin secretion, which could be the cause of decreased mucosal protection in ulcerative colitis. METHODS: Colonic biopsy specimens from patients with active ulcerative colitis, ulcerative colitis in remission, and controls were metabolically labelled with [35S]-amino acids or [35S]-sulphate, chase incubated and analysed by SDS-PAGE, followed by quantitation of mature [35S]-labelled MUC2. For quantitation of total MUC2, which includes non-radiolabelled and radiolabelled MUC2, dot blotting was performed, using a MUC2 monoclonal antibody. RESULTS: Between patient groups, no significant differences were found in [35S]-sulphate content of secreted MUC2 or in the secreted percentage of either [35S]-amino acid labelled MUC2 or total MUC2. During active ulcerative colitis, secretion of [35S]-sulphate labelled MUC2 was significantly increased twofold, whereas [35S]-sulphate incorporation into MUC2 was significantly reduced to half. CONCLUSIONS: During active ulcerative colitis, less MUC2 is secreted, because MUC2 synthesis is decreased while the secreted percentage of MUC2 is unaltered. Furthermore, sulphate content of secreted MUC2 is unaltered by a specific compensatory mechanism, because sulphated MUC2 is preferentially secreted while sulphate incorporation into MUC2 is reduced.
Asunto(s)
Colitis Ulcerosa/metabolismo , Mucinas/biosíntesis , Adolescente , Adulto , Anciano , Anticuerpos Monoclonales , Estudios de Casos y Controles , Niño , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Immunoblotting , Masculino , Persona de Mediana Edad , Mucina 2 , Mucinas/metabolismo , Radioisótopos de Azufre/metabolismoRESUMEN
To help us investigate the role of mucin in the protection of the colonic epithelium in the mouse, we aimed to identify the murine colonic mucin (MCM) and its encoding gene. We isolated MCM, raised an anti-MCM antiserum, and studied the biosynthesis of MCM in the gastrointestinal tract. Isolated MCM resembled other mucins in physicochemical properties. Anti-MCM recognized MCM as well as rat and human MUC2 on Western blots, interacting primarily with peptide epitopes, indicating that MCM was identical to murine Muc2. Using anti-MCM and previously characterized anti-human and anti-rat MUC2 antibodies, we identified a murine Muc2 precursor in the colon of approximately 600 kDa, which appeared similar in size to rat and human MUC2 precursors. Western blotting, immunoprecipitation of metabolically labeled mucins, and immunohistochemistry showed that murine Muc2 was expressed in the colon and the small intestine but was absent in the stomach. To independently identify murine Muc2, we cloned a cDNA fragment from murine colonic mRNA, encoding the 302 NH2-terminal amino acids of murine Muc2. The NH2 terminus of murine Muc2 showed 86 and 75% identity to the corresponding rat and human MUC2 peptide sequences, respectively. Northern blotting with a murine Muc2 cDNA probe showed hybridization to a very large mRNA, which was expressed highly in the colon and to some extend in the small intestine but was absent in the stomach. In situ hybridization showed that the murine Muc2 mRNA was confined to intestinal goblet cells. In conclusion, by two independent sets of experiments we identified murine Muc2, which appears homologous to rat and human MUC2. Because Muc2 is prominently expressed in the colon, it is most likely to be the predominant mucin in the colonic mucus layer.
Asunto(s)
Clonación Molecular , ADN Complementario/genética , Sistema Digestivo/metabolismo , Mucinas/genética , Mucinas/metabolismo , Secuencia de Aminoácidos , Animales , Colon/metabolismo , ADN Complementario/aislamiento & purificación , Femenino , Células Caliciformes/metabolismo , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Masculino , Ratones , Datos de Secuencia Molecular , Mucina 2 , Mucinas/aislamiento & purificación , ARN Mensajero/metabolismo , RatasRESUMEN
MUC-type mucins comprise a family of structurally related molecules, which are expressed in epithelia of the body that are in close contact with the milieu. Because of their large sizes and very complex structures, containing very extensive O-glycosylation, MUC-type mucins are difficult to study by conventional techniques. Many see MUC-type mucins as protective molecules; however, functional studies on the individual MUC-type mucins are very scarce. At present, essential steps in MUC research are to characterize the specific expression patterns of each MUC-type mucin in the body and to find methods to reliably quantify these MUC-type mucins. These aims can only be met at the level of the primary sequences of the MUC-type mucins, as the O-glycosylation even within one species of MUC-type mucin is not only very complex, but may also vary among individuals, organs, and cell types. We will discuss some recent advances in mucin research, particularly the identification of MUC precursor molecules in metabolic labeling experiments. We will try to define some strategic considerations in the study of the expression patterns of MUC-type mucins, which circumvent the complications caused by the very complex and heterogeneous O-glycosylation of the molecules.
Asunto(s)
Mucinas/análisis , Secuencia de Aminoácidos , Animales , Humanos , Mucinas/química , Mucinas/genéticaRESUMEN
BACKGROUND: In children, lactase and sucrase-isomaltase are essential intestinal glycohydrolases, and insufficiency of either enzyme causes diarrhea and malnutrition. Little is known about the regulation of lactase and sucrase-isomaltase expression in the duodenum during childhood. In this study, the mechanisms of regulation of duodenal expression of both enzymes were examined in a study population with ages ranging from 1 to 18 years. METHODS: Duodenal biopsy specimens from 60 white children were used to analyze tissue morphology and to quantify lactase and sucrase-isomaltase mRNA and protein. RESULTS: Among healthy subjects, high interindividual variability was noted in both mRNA and protein levels for lactase and sucrase-isomaltase. Lactase mRNA level per subject did not correlate with sucrase-isomaltase mRNA level and thus appeared independent. Both lactase and sucrase-isomaltase protein levels correlated significantly with their respective mRNA levels. For each enzyme, a significant inverse correlation was observed between the degree of villus atrophy and mRNA levels. Aging from 1 to 18 years did not result in significant changes in mRNA or protein levels of either enzyme. Immunostaining patterns within the duodenal epithelium for lactase differed from sucrase-isomaltase in adjacent sections, illustrating independent regulation at the cellular level. CONCLUSIONS: In the duodenum of white children, lactase and sucrase-isomaltase seem primarily regulated at the transcriptional level. The expression of each enzyme in the intestinal epithelium is regulated by an independent mechanism. Lactase and sucrase-isomaltase exhibit stable mRNA and protein levels in healthy children as they grow to adulthood. Mucosal damage affected levels of both enzymes negatively.
Asunto(s)
Duodeno/enzimología , Regulación Enzimológica de la Expresión Génica , Complejo Sacarasa-Isomaltasa/genética , beta-Galactosidasa/genética , Adolescente , Envejecimiento , Anticuerpos Monoclonales , Atrofia , Biopsia , Niño , Preescolar , Humanos , Inmunohistoquímica , Lactante , Mucosa Intestinal/enzimología , Mucosa Intestinal/patología , Lactasa , Estudios Prospectivos , ARN Mensajero/metabolismoRESUMEN
To elucidate the roles of human gallbladder mucin (HGBM), such as in gallstone formation and cytoprotection, it is essential to identify HGBM and study its expression. This was performed by metabolic labeling, Western blotting, immunohistochemistry, and RT-PCR. In a large number of individuals, antibodies against purified HGBM and against MUC5B detected a mucin precursor (approximately 470 kDa) in the gallbladder and colon, but not in the small intestine. In the gallbladder, Western blotting using specific anti-MUC5B antibodies showed that this mucin precursor represented an identical mucin, MUC5B. RT-PCR experiments demonstrated a similar tissue distribution pattern of MUC5B mRNA. Immunohistochemistry with anti-HGBM and anti-MUC5B showed staining in gallbladder epithelial cells and colonic goblet cells in the crypt base, but not in the small intestine; double labeling showed that HGBM was located in small granules within goblet cells, colocalizing to MUC2-containing goblet cells. Metabolic labeling demonstrated the secretion of mature MUC5B in the colon. Conclusively, MUC5B is identified as the prominent HGBM and is also expressed and secreted in the colon.
Asunto(s)
Colon/metabolismo , Vesícula Biliar/metabolismo , Mucinas/metabolismo , Western Blotting , Colon/citología , Humanos , Inmunohistoquímica , Mucina 2 , Mucina 5B , Mucinas/genética , Precursores de Proteínas/metabolismo , ARN Mensajero/metabolismoRESUMEN
To further clone the human gastric mucin MUC5AC cDNA, we screened a human gastric cDNA library with previously identified MUC5AC sequences. We obtained 32 independent clones encoding newly identified sequences comprising the entire N-terminal sequence of MUC5AC, up to 3024 bp upstream of the previously identified MUC5AC sequences. The N-terminus of MUC5AC shows high homology (43% identity) with the N-terminus of MUC2 and contains three domains homologous to the D-domains found in the pro-von Willebrand factor. Furthermore, the N-terminus of MUC5AC contains a putative leucine zipper motif not found in any other mucin identified so far. Moreover, a large central repetitive sequence was identified encoding approximately 2500 amino acids (7.5 kb). We were able to establish that the MUC5AC cDNA together with the previously identified 6.1 kb of MUC5AC cDNA sequence is about 16.6 kb, encoding 5525 amino acids. A model of the domain structure of MUC5AC is presented.
Asunto(s)
Cisteína/análisis , Leucina Zippers , Mucinas/genética , Secuencia de Aminoácidos , Southern Blotting , Clonación Molecular , ADN Complementario/análisis , Biblioteca de Genes , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mucina 5AC , Mucinas/química , Reacción en Cadena de la Polimerasa , Relación Estructura-ActividadRESUMEN
Mucins are synthesized and secreted by many epithelia. They are complex glycoproteins that offer cytoprotection. In their functional configuration, mucins form oligomers by a biosynthetic process that is poorly understood. A family of four human gastrointestinal mucin genes (MUC2, MUC5AC, MUC5B, and MUC6) is clustered to chromosome 11p15.5. To study oligomerization of these related mucins, we performed metabolic labeling experiments with [35S]amino acids in LS174T cells, and isolated mucin precursors by specific immunoprecipitations that were analyzed on SDS-PAGE. Each of the precursors of MUC2, MUC5AC, MUC5B, and MUC6 formed a single species of disulfide-linked homo-oligomer within 1 h after pulse labeling. Based on apparent molecular masses, these oligomeric precursors were most likely dimers. Inhibition of vesicular RER-to-Golgi transport, with brefeldin A and CCCP, did not affect the dimerization of MUC2 precursors, localizing dimerization to the RER. O-Glycosylation of MUC2 followed dimerization. Inhibition of N-glycosylation by tunicamycin retarded, but did not inhibit, dimerization, indicating that N-glycans play a role in efficient dimerization of MUC2 precursors. Based on sequence homology, the ability of MUC2, MUC5AC, MUC5B and MUC6 to dimerize most likely resides in their C-terminal domains. Thus, the RER-localized dimerization of secretory mucins likely proceeds by similar mechanisms, which is an essential step in the formation of the human gastrointestinal mucus-gels.
Asunto(s)
Mucinas/química , Mucinas/genética , Familia de Multigenes , Línea Celular , Cromosomas Humanos Par 11/genética , Dimerización , Retículo Endoplásmico Rugoso/química , Mucinas Gástricas/química , Mucinas Gástricas/genética , Mucosa Gástrica/química , Glicosilación , Humanos , Mucosa Intestinal/química , Mucina 5AC , Mucina 2 , Mucina 5B , Mucina 6 , Conformación Proteica , Precursores de Proteínas/química , Precursores de Proteínas/genéticaRESUMEN
The clinical importance of carbamoyl phosphate synthase I (CPSI) relates to its capacity to metabolize ammonia, because CPSI deficiencies cause lethal serum ammonia levels. Although some metabolic parameters concerning liver and intestinal CPSI have been reported, the extent to which enterocytes contribute to ammonia conversion remains unclear without a detailed description of its developmental and spatial expression patterns. Therefore, we determined the patterns of enterocytic CPSI mRNA and protein expression in human and rat intestine during embryonic and postnatal development, using in situ hybridization and immunohistochemistry. CPSI protein appeared during human embryogenesis in liver at 31-35 e. d. (embryonic days) before intestine (59 e.d.), whereas in rat CPSI detection in intestine (at 16 e.d.) preceded liver (20 e.d.). During all stages of development there was a good correlation between the expression of CPSI protein and mRNA in the intestinal epithelium. Strikingly, duodenal enterocytes in both species exhibited mosaic CPSI protein expression despite uniform CPSI mRNA expression in the epithelium and the presence of functional mitochondria in all epithelial cells. Unlike rat, CPSI in human embryos was expressed in liver before intestine. Although CPSI was primarily regulated at the transcriptional level, CPSI protein appeared mosaic in the duodenum of both species, possibly due to post-transcriptional regulation.