RESUMO
Molecular chaperones are presumed to associate with large secretory mucin glycoproteins during their synthesis in the endoplasmic reticulum (ER), but have not been identified to date. We decided to look for possible involvement of the chaperones calreticulin (CRT) and calnexin (CLN) during synthesis of two similar gastrointestinal mucins, MUC2 and MUC5AC. Pulse-chase labelling of MUC2 and MUC5AC with [(35)S]methionine/cysteine ([(35)S]Promix) was performed using LS180 and HT29/A1 colonic carcinoma cell lines and was followed by immunoprecipitation with anti-mucin and anti-chaperone antibodies. The precipitated labelled mucin precursors were analysed by SDS/PAGE and autoradiography. Using antibodies specific for each mucin, newly synthesized monomeric precursors of both MUC2 and MUC5AC were detected after a 15 min pulse and then disappeared as oligomers were formed during a 2 h chase period. Only homo-oligomers of MUC2 and MUC5AC were present in the cells. Using anti-CRT, the MUC2 monomeric precursor and oligomer were co-precipitated from both cell lines after a 15 min pulse and the oligomer less strongly after a 0.5 h chase, but there was little co-precipitation after a 2 h chase. At this time, MUC2 immunoprecipitated by anti-MUC2 was completely oligomerized and was endo-beta-N-acetylglucosaminidase-resistant, indicating that the mucin had reached the Golgi region. MUC2 co-precipitated with CRT at zero time and 0.5 h was endo-beta-N-acetylglucosaminidase-sensitive; therefore CRT must have associated with MUC2 in the ER. Treatment with tunicamycin (TUN) diminished the binding of MUC2 to CRT, suggesting a requirement for initial N-glycan addition during this process. Using anti-CLN, only a weak co-precipitation of MUC2, compared with that seen with anti-CRT, was detected in LS180 cells. In contrast with the findings for MUC2, there was no co-precipitation of MUC5AC with CRT or CLN from either cell line at the various time points. In conclusion, CRT and CLN appear to be involved in MUC2 synthesis at the stage of folding and oligomerization in the ER. Since no interaction of the chaperones with MUC5AC was detected at a similar stage of synthesis, these two structurally similar secretory mucins seem to have different chaperone requirements in the ER.
Assuntos
Adenocarcinoma/metabolismo , Proteínas de Ligação ao Cálcio/fisiologia , Neoplasias do Colo/metabolismo , Mucinas/biossíntese , Ribonucleoproteínas/fisiologia , Adenocarcinoma/patologia , Biopolímeros , Calnexina , Calreticulina , Neoplasias do Colo/patologia , Células HT29 , Humanos , Mucinas/metabolismo , Testes de Precipitina , Células Tumorais CultivadasRESUMO
The present study reveals that partial proteolytic degradation of rat Muc 2 mucin can occur rapidly even in the presence of a battery of proteinase inhibitors. During the initial steps of purification from homogenates of intestinal scrapings, degradation was rapid, causing release of the entire 118 kDa C-terminal glycopeptide and, as shown by N-terminal sequencing, a large (200 kDa) N-terminal glycopeptide fragment. Degradation could be prevented by adding 6 M guanidinium chloride provided that its presence was maintained throughout every step of purification. Even after purification, however, the mucin was still vulnerable to partial proteolysis unless it was stored in guanidinium chloride at -20 degrees C. These findings imply that a potent proteinase contaminant remains tightly bound to the mucin through every step of purification, or else that the mucin has autocatalytic properties. Because the C- and N-terminal regions of secretory mucins are required for their assembly into linear mucin polymers that form functional gels, our findings emphasize that extreme care is required to purify structurally intact mucin molecules. They also imply that the specific degradation steps described here are likely to occur rapidly after mucins are secreted into the intestinal lumen and come into contact with the products of sloughed cells.
Assuntos
Mucosa Intestinal/metabolismo , Mucinas/metabolismo , Fragmentos de Peptídeos/metabolismo , Alquilação , Animais , Masculino , Mucina-2 , Mucinas/química , Fragmentos de Peptídeos/química , Ratos , Ratos WistarRESUMO
A 3' RACE technique was used to establish the nucleotide sequence encoding the C-terminal 379 amino acids of rat intestinal Muc3. Unlike the C-terminus of Muc2 and many secretory mucins, Muc3 contains two EGF motifs and a putative transmembrane domain. The mRNA for rat Muc3 is 7.5-8.0 kb.
Assuntos
Membrana Celular/metabolismo , Intestino Delgado/metabolismo , Mucinas/metabolismo , Proteínas de Neoplasias/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Fator de Crescimento Epidérmico/genética , Dados de Sequência Molecular , Mucina-3 , Mucinas/química , Mucinas/genética , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , RNA Mensageiro/genética , RatosRESUMO
The phorbol ester, phorbol 12-myristate 13-acetate (PMA), induces mucin secretion in the colonic tumor cell line T84 in a Ca(2+)-independent manner. To determine whether a specific protein kinase C (PKC) isoform is involved in colonic cells, we compared PMA-dependent mucin secretion by three human colonic tumor cell lines (T84, HT-29/A1, and LS 180) with the expression of PKC isoforms alpha, beta, delta, epsilon, and zeta, previously identified in human colon (L. A. Davidson, Y. H. Jiang, J. D. Derr, H. Aukema, J. R. Lupton, and R. S. Chapkin. Arch. Biochem. Biophys. 312:547-553, 1994). In each cell line PMA (10(-7) M) caused mucin secretion within 30 min. PMA-dependent mucin secretion was three to four times greater from HT-29/A1 and T84 cells than from LS 180 cells. All three-cell lines contained mRNA for PKC-alpha, PKC-epsilon, and PKC-zeta but not PKC-beta or -delta. Each cell line also expressed PKC-alpha, -epsilon, and -zeta protein. PKC-epsilon expression (mRNA and protein) was three to four times greater in HT-29/A1 and T84 cells than in LS 180 cells, correlating with PMA-responsive mucin secretion, whereas all cell lines contained similar levels of PKC-alpha mRNA and protein. When cells were stimulated by PMA, only PKC-epsilon was translocated from cytosol to membrane fractions early enough to stimulate mucin secretion. Because PKC-epsilon is also a Ca(2+)-independent isoform, it is likely to mediate mucin exocytosis in colonic cells.
Assuntos
Colo/metabolismo , Exocitose , Mucinas/metabolismo , Colo/enzimologia , Colo/patologia , Humanos , Imunoensaio , Isoenzimas/metabolismo , Isoenzimas/fisiologia , Reação em Cadeia da Polimerase , Proteína Quinase C/metabolismo , Proteína Quinase C/fisiologia , Proteína Quinase C-épsilon , Frações Subcelulares/enzimologia , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica , Células Tumorais CultivadasRESUMO
BACKGROUND & AIMS: Shwachman syndrome is an inherited condition with multisystemic abnormalities, including exocrine pancreatic dysfunction. The aim of this study was to evaluate the occurrence and progression of features in a large cohort of patients. METHODS: Clinical records of 25 patients with Shwachman syndrome were reviewed. RESULTS: Mean birth weight (2.92 +/- 0.51 kg) was at the 25th percentile. However, by 6 months of age, mean heights and weights were less than the 5th percentile. After 6 months of age, growth velocity was normal. Severe fat maldigestion due to pancreatic insufficiency was present in early life (fecal fat, 26% +/- 17% of fat intake; age, < 2 years). Serial assessment of exocrine pancreatic function showed persistent deficits of enzyme secretion, but 45% of patients showed moderate age-related improvements leading to pancreatic sufficiency. Neutropenia was the most common hematologic abnormality (88%), but leukopenia, thrombocytopenia, and anemia were also frequently encountered. Patients with hypoplasia of all three bone marrow cellular lines (n = 11) had the worst prognosis; 5 patients died, 2 of sepsis and 3 of acute myelogenous leukemia. Other findings included hepatomegaly and/or abnormal liver function test results and skeletal abnormalities. CONCLUSIONS: A wide and varied spectrum of phenotypic abnormalities among patients with Shwachman syndrome is described. Pancreatic acinar dysfunction is an invariable abnormality. Patients with severe bone marrow involvement may have a guarded prognosis.
Assuntos
Anormalidades Múltiplas/fisiopatologia , Pancreatopatias/fisiopatologia , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Fenótipo , Prognóstico , SíndromeRESUMO
Mouse models for cystic fibrosis (CF) with no CFTR function (Cftr-/-) have the disadvantage that most animals die of intestinal obstruction shortly after weaning. The objective of this research was to extend the lifespan of CF mice and characterize their phenotype. Weanlings were placed on a nutrient liquid diet, and histologic and functional aspects of organs implicated in the disease were subsequently examined. Approximately 90% of Cftr-/- mice survived to 60 d, the majority beyond 100 d. Cftr-/- mice were underweight and had markedly abnormal intestinal histology. The intestinal epithelia did not respond to challenges with agents that raised intracellular cAMP, consistent with the absence of functional CFTR. No lesions or functional abnormalities were evident in the lungs. Liquid-fed Cftr-/- mice were infertile, although some males weaned to a solid diet were fertile before they died. Thus, we have succeeded in using dietary means to prolong the lives of Cftr-/- mice.
Assuntos
Fibrose Cística/genética , Genitália/patologia , Intestinos/patologia , Sistema Respiratório/patologia , Animais , Fibrose Cística/patologia , Dieta , Modelos Animais de Doenças , Feminino , Longevidade , Masculino , Camundongos , Camundongos Endogâmicos CFTR , Pâncreas/patologia , FenótipoRESUMO
Unlike most other mucins described to date, two intestinal mucins, rat MLP (rat Muc 2) and human MUC2 have a C-terminal tail that is enriched in cationic amino acids. The distribution of charge in each case resembles that of several well known heparin binding proteins. Peptides designated E20-14 and F13-15, corresponding to the C-terminal 14 amino acids of the two mucins, were synthesized and shown to bind 3 H-labelled heparin by a process that was saturable and mediated by strong electrostatic interactions, giving Kd values of 10 (-7) to 10 (-8) M. Using turbidometric analyses and native gel electrophoresis, we observed that peptide-heparin mixtures formed polydisperse aggregates that dissociated with a progressive increase in the concentration of heparin. Under certain conditions heparin protected the peptide from proteolysis by trypsin. Both heparin and dextran sulfate, the latter a highly sulfated synthetic polysaccharide, were potent inhibitors of 3 H-heparin binding to peptide E20-14, while less sulfated glycosaminoglycans were poorly- or non-inhibitory. Mucin in tissue dispersions and homogenates, or purified from rat intestine, did not bind to heparin, and failed to interact with an antibody specific for the peptide E20-14. Both mucin samples however, reacted with antibodies that recognize regions upstream of the C-terminal 14 amino acids. Immunofluorescent localization of E20-14 was confined to the basal perinuclear regions of goblet cells, whereas localization of an antibody to a flanking sequence on the N-terminal side of the C-tail, localized to mature mucin storage granules. These findings suggest that the heparin -binding C-tail of the mucin may be removed at an early stage of biosynthesis. Heparin-mucin complexes, if they form in vivo, are thus likely to be confined to the ER and/or Golgi compartments.
Assuntos
Heparina/metabolismo , Mucosa Intestinal/metabolismo , Mucinas/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Imunofluorescência , Humanos , Técnicas In Vitro , Intestinos/química , Dados de Sequência Molecular , Estrutura Molecular , Mucina-2 , Mucinas/química , Mucinas/genética , Peptídeos/síntese química , Peptídeos/química , Polissacarídeos/metabolismo , Ligação Proteica , Conformação Proteica , RatosRESUMO
We have shown previously [McCool, Forstner and Forstner (1994) Biochem. J. 302, 111-118] using pulse-chase labelling of mucin with [3H]threonine that LS180 colonic tumour cells synthesize and secrete MUC2 without the addition of secretagogues. Treatment of the LS180 cells with monensin to disrupt Golgi function was also found to inhibit baseline secretion almost completely. In this paper we show that addition of nocodazole to inhibit microtubule assembly reduced baseline secretion by 53% over a 6 h chase period. In contrast, cytochalasin D did not affect the rate of unstimulated mucin synthesis or secretion, suggesting that baseline secretion is not influenced by disruption of actin microfilaments. In addition, regulated mucin secretion by LS180 cells was studied in response to carbachol, phorbol 12-myristate 13-acetate and A23187. Mucin released in response to secretagogues behaved identically on SDS/PAGE to that secreted under baseline conditions. T84 cells and the B6 subclone of the HT29 cell line responded in a similar manner to LS180 cells and secreted high-molecular-mass mucin which included MUC2 and behaved like LS180 mucin on SDS/PAGE. Neither monensin nor nocodazole significantly affected secretagogue-stimulated mucin secretion. Since these compounds inhibited secretion of labelled mucin under baseline conditions, mucin released by secretagogues must have come from a separate, unlabelled mucin pool in stored granules. Cytochalasin D, on the other hand, caused the release of small amounts of stored mucin, suggesting that actin microfilaments participate in regulated exocytosis. Thus two kinds of mucin secretion occur in LS180 cells. Unregulated secretion depends upon continuous transport of mucin granules from Golgi vesicles to the cell surface and does not utilize stored mucin, whereas regulated secretion involves the release of mucin from storage granules and is not affected by microtubule or Golgi disruption.
Assuntos
Adenocarcinoma/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias do Colo/metabolismo , Mucinas/metabolismo , Proteínas de Neoplasias/metabolismo , Biomarcadores Tumorais/biossíntese , Calcimicina/farmacologia , Carbacol/farmacologia , Tamanho Celular , Colchicina/farmacologia , Citocalasina D/farmacologia , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Humanos , Microscopia Eletrônica , Monensin/farmacologia , Mucina-2 , Mucinas/biossíntese , Proteínas de Neoplasias/biossíntese , Nocodazol/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais CultivadasRESUMO
We studied serial measurements of serum cationic trypsinogen in patients with cystic fibrosis to assess the predictability of changes in individuals and the value of longitudinal measurement in defining pancreatic status. Three hundred twenty-nine patients with cystic fibrosis, aged 3 days to 40 years, had serum levels of trypsinogen measured on 2 to 12 occasions for periods ranging from 1 week to 7 years. Patients were classified into three groups on the basis of 72-hour fecal fat studies performed at the time of diagnosis. Two hundred thirty-three patients had pancreatic insufficiency (PI), 78 had pancreatic sufficiency (PS), and 18 had PS at diagnosis but acquired PI during follow-up (PS-->PI). Infants with PI had greatly elevated serum trypsinogen levels that fell sharply in the first years of life, so that by age 7 years more than 95% had subnormal values; individual patient values followed a predictable course similar to previously reported cross-sectional age-related values. In patients with PS, serum trypsinogen levels generally remained within or above the normal range and, after age 10 years, were well above the upper limit for PI patients. Within-patient variance was significantly greater (p < 0.0001) in patients with PS than in those with PI who were older than 7 years of age. Changes in patients within PS-->PI generally followed the pattern seen in patients with PI, but values in older patients tended to be in the higher range. We concluded that serial measurement of serum trypsinogen is a valuable tool for monitoring the pancreatic status of patients with cystic fibrosis and PS.
Assuntos
Fibrose Cística/sangue , Insuficiência Pancreática Exócrina/sangue , Tripsinogênio/sangue , Adolescente , Adulto , Envelhecimento/sangue , Criança , Pré-Escolar , Fibrose Cística/fisiopatologia , Progressão da Doença , Insuficiência Pancreática Exócrina/fisiopatologia , Fezes/química , Feminino , Humanos , Lactente , Recém-Nascido , Lipídeos/análise , Estudos Longitudinais , Masculino , Testes de Função Pancreática , PrognósticoRESUMO
Pulse-chase labelling experiments were performed using the mucin-producing colonic carcinoma cell line LS180. Cells were pulsed with [3H]threonine or [3H]glucosamine and chased in complete media without radiolabel for various lengths of time. From cell and media extracts obtained at each time point, mucin proteins were immunoprecipitated with specific anti-mucin antibodies and analysed by SDS/PAGE and fluorography. At short labelling times with [3H]threonine, without chase, a monomeric thiol-reduction-resistant mucin precursor of apparent molecular mass > 670 kDa was identified. The precursor, in contrast to oligomeric species, was not labelled by [3H]glucosamine but exhibited binding to Vicia villosa isolectin B4, suggesting the presence of some core GalNAc residues. Treatment with tunicamycin to inhibit N-glycosylation had no effect on the apparent mass of the precursor. Identity of the mucin antigen with MUC2 mucin was established by immunoprecipitation with antibodies specific for a MUC2 tandem repeat and C-terminal regions. With increasing chase time the precursor was replaced by thiol-reduction-sensitive mucin oligomers that reached peak intracellular radiolabelling with [3H]threonine by 2 h of chase, and then declined. Only oligomeric mucin was secreted into the medium. Secretion of [3H]threonine-labelled mucin was detectable after 2 h of chase and increased as the cytoplasmic mucin label declined. Monensin inhibited [3H]glucosamine incorporation, sialylation and baseline (non-regulated) mucin secretion without affecting initial [3H]threonine incorporation or oligomerization. Oligomerization and Golgi transport are therefore essential early steps in MUC2 mucin secretion. Oligomerization may follow some core O-glycosylation with GalNAc, but precedes elongation of oligosaccharide chains.
Assuntos
Mucosa Intestinal/metabolismo , Mucinas/biossíntese , Neoplasias do Colo , Eletroforese em Gel de Campo Pulsado , Glicosilação , Humanos , Intestinos/efeitos dos fármacos , Monensin/farmacologia , Mucina-2 , Mucinas/imunologia , Mucinas/metabolismo , Neuraminidase/farmacologia , Testes de Precipitina , Precursores de Proteínas/biossíntese , Células Tumorais CultivadasRESUMO
In the present report we describe the isolation and sequence of a partial cDNA (M2-798) for a rat intestinal mucin designated M2. A rat intestinal lambda ZAP II cDNA library was screened using a polyclonal antiserum which was prepared against deglycosylated high-molecular-mass glycopeptides of the purified mucin. Mucin cDNA clones were found to contain tandem repeats of 18 nt which encoded a threonine- and proline-rich peptide having a consensus sequence of TTTPDV. This is the same sequence reported recently by Gum, Hicks, Lagace, Byrd, Toribara, Siddiki, Fearney, Lamport and Kim [(1991) J. Biol. Chem. 266, 22733-22738] for a rat intestinal cDNA called RMUC 176. A novel feature present in the cDNA M2-798 is a 246 nt unique region at the 3' end which encodes a hydrophobic sequence of 82 amino acids. RNA blots probed with M2-798 cDNA produced a single hybridization band between 7.5 and 9.0 kb in rat small intestine and colon. An identical hybridization pattern was obtained with a PCR-generated cDNA probe corresponding solely to the unique hydrophobic region of M2-798, demonstrating that this region is encoded by the authentic M2 mRNA. Our data suggest that the unique region of M2 has the potential to be either a transmembrane region, or a domain which mediates hydrophobic interactions of the mucin with other molecules. Since we have previously reported another rat intestinal cDNA which encodes the C-terminus of a mucin-like peptide (MLP) [Xu, Wang, Huan, Cutz, Forstner and Forstner (1992) Biochem. J. 286, 335-338], we wished to discover whether M2 was encoded by the same gene. RNA blotting experiments with probes specific for M2 and MLP showed different mRNAs for each. The message for M2 (7.5-8.5 kb) was smaller than that for MLP (> 9.5 kb) and, unlike MLP, gave no signal in human colonic LS174T cells. The results of DNA blots probed with M2-798 and an MLP-probe suggest that M2 and MLP are likely to be single-copy genes. It would appear therefore that normal rat intestine, like human intestine, may express two different mucin genes.
Assuntos
Intestinos/química , Mucinas/genética , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Centrifugação com Gradiente de Concentração , DNA/química , DNA/isolamento & purificação , Retículo Endoplasmático/química , Glicosilação , Imuno-Histoquímica , Intestinos/ultraestrutura , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Peso Molecular , Mucinas/química , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Ratos , Sequências Repetitivas de Ácido NucleicoRESUMO
Expression of the gene for a rat intestinal mucin-like peptide (MLP) was studied by Northern-blot analyses of RNA prepared from a panel of rat tissues. Four probes (A-D) were constructed so as to span a 3.5 kb-long cDNA for rat MLP, and used for hybridization. Positive signals were obtained in intestine and colon, whereas lung, liver, stomach, submandibular gland and spleen were negative. The only transcript detected was approx. 9.5 kb in size. No mRNA splice variants were found. Hybridization in situ using probe B1, which corresponds to a cysteine-rich region near the C-terminus of MLP, confirmed that the gene for MLP is expressed by goblet cells of rat intestine and colon.
Assuntos
Mucosa Intestinal/metabolismo , Mucinas/biossíntese , Biossíntese Peptídica , Animais , Northern Blotting , DNA , Mucinas/genética , Hibridização de Ácido Nucleico , Peptídeos/genética , RNA Mensageiro/genética , RatosRESUMO
Patients with cystic fibrosis and pancreatic sufficiency were investigated for evidence of progressive pancreatic disease. From a cohort of 630 patients, 20 pancreatic-sufficient patients became pancreatic insufficient after an average duration of 5.6 y (range 0.6-20.6 y) from diagnosis. Among 54 patients documented to be pancreatic sufficient by direct pancreatic stimulation test, 47 remained pancreatic sufficient and seven developed pancreatic insufficiency. The patients who ultimately developed pancreatic insufficiency were younger and had greatly reduced outputs of enzyme, fluid, and electrolytes. Those who remained pancreatic sufficient showed enzyme secretion close to or within the non-cystic fibrosis control range. Twenty of these patients underwent a second pancreatic stimulation test after an average interval of 4 y (range 1.3-6.2 y). No significant alteration in enzyme, fluid, or electrolyte output was seen in the patients who remained pancreatic sufficient, but there was further reduction in enzyme and fluid output in the patients who developed pancreatic failure. In conclusion, the majority of pancreatic-sufficient patients with pancreatic enzyme secretion within the control range showed no deterioration of function over an extended time period. However, a small number of pancreatic-sufficient patients with reduced enzyme and fluid secretion are at risk of pancreatic failure.
Assuntos
Fibrose Cística/fisiopatologia , Pâncreas/fisiopatologia , Adolescente , Adulto , Criança , Pré-Escolar , Fibrose Cística/complicações , Insuficiência Pancreática Exócrina/etiologia , Insuficiência Pancreática Exócrina/fisiopatologia , Feminino , Humanos , Lactente , Masculino , Testes de Função Pancreática , Fatores de TempoRESUMO
When subjected to thiol reduction, purified intestinal mucins have been shown to undergo a decrease in molecular mass and to liberate a 118-kDa glycopeptide (Roberton, A. M., Mantle, M., Fahim, R. E. F., Specian, R., Bennick, A., Kawagishi, S., Sherman, P., and Forstner, J. F. (1989) Biochem. J. 261, 637-647). The latter has been called a putative "link" component because it is assumed to be important for disulfide bond-mediated mucin polymerization. Controversy exists as to whether the putative link is an integral mucin component or a separate mucin-associated glycopeptide. In the present study both NH2-terminal and internal amino acid sequences of the 118-kDa glycopeptide of rat intestinal mucin were used to generate opposing oligonucleotide primers for polymerase chain reaction. A specific 1.2-kilobase (kb) product was obtained, from which a 0.5-kb HindIII fragment was used as a probe to screen a lambda ZAP II cDNA library of rat intestine. A 2.6-kb cDNA (designated MLP 2677) was sequenced and revealed an open reading frame of 2.5 kb encoding 837 amino acids. The deduced amino acid sequence showed that the putative link peptide is equivalent to the carboxyl-terminal 689 amino acids of a larger peptide. Northern blots revealed a mRNA size of approximately 9 kb. Computer searches revealed no sequence homology with other proteins, but similarities were seen in the alignment of cysteine residues in the link and in several domains of human von Willebrand factor, as well as cysteine-rich areas of bovine and porcine submaxillary mucins and a frog skin mucin designated FIM-B.1. In keeping with earlier demonstrations of the presence of mannose in the 118-kDa glycopeptide, there were several (13) consensus sequences for attachment of N-linked oligosaccharides within the link domain. Further sequencing of MLP 2677 in a direction 5' to the codon specifying the NH2-terminal proline of the link has revealed a coding region for 148 amino acids, including a unique 75-amino acid domain rich in cysteine and proline, and a region containing 4.5-variable tandem repeats (each 11-12 amino acids) rich in serine, threonine, and proline. The presence of mucin-like tandem repeats suggests that the entire cysteine-rich link peptide represents the carboxyl-terminal region (75.5 kDa) of a mucin-like peptide (MLP). The latter is estimated to have a molecular mass of approximately 300 kDa.
Assuntos
DNA/genética , Mucosa Intestinal/fisiologia , Mucinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Biblioteca Gênica , Glicopeptídeos/isolamento & purificação , Dados de Sequência Molecular , Peso Molecular , Mucinas/isolamento & purificação , Poli A/genética , Poli A/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro , Ratos , Mapeamento por Restrição , Homologia de Sequência do Ácido NucleicoRESUMO
The T84 colonic adenocarcinoma cell line, which has been used extensively as a model for studies of epithelial chloride secretion, also produces mucin and secretes it in culture. Electron microscopy of fixed sections of cultured cells, along with Immunogold labelling with an antibody to human small intestine (SI) mucin, revealed the presence of goblet-like cells with mucin-containing secretory granules. The mucin was of high molecular mass, had an amino acid composition similar to that of purified human SI and colonic mucins, and competed effectively with SI mucin for binding to the anti-(SI mucin) antibody. A sensitive solid-phase immunoassay specific for intestinal mucins was developed and used to measure mucin secretion by T84 cells. Cultures were treated for 30 min at 37 degrees C with a number of agents known to cause chloride secretion by T84 cell monolayers and the amount of mucin appearing in the medium was measured. Carbachol (1 mM), A23187 (10 microM), prostaglandin E1 (PGE1) (1 microM) and vasoactive intestinal polypeptide (VIP) (0.1 microM) all stimulated mucin release, but histamine (1 mM) had no effect. Whereas VIP is reported to stimulate chloride secretion more strongly than carbachol, it was less effective than carbachol in stimulating mucin secretion. Phorbol 12-myristate 13-acetate (PMA) (0.1-10 microM) also stimulated mucin release strongly, implicating a responsive protein-kinase C-dependent pathway. Additive secretory responses were obtained with combined stimulation by VIP (10 nM-1 microM) and carbachol (1 mM). Responses to stimulation with A23187 (1-10 microM) together with PMA (10 nM-10 microM) suggest that cytosolic Ca2+ concentration is a modulator of PMA activity.
Assuntos
Mucinas/biossíntese , Células Tumorais Cultivadas/metabolismo , Adenocarcinoma , Calcimicina/farmacologia , Carbacol/farmacologia , Linhagem Celular , Toxina da Cólera/farmacologia , Neoplasias do Colo , Fibrose Cística/metabolismo , Histamina/farmacologia , Humanos , Imunodifusão , Técnicas Imunoenzimáticas , Intestino Delgado/metabolismo , Microscopia Eletrônica , Mucinas/isolamento & purificação , Mucinas/metabolismo , Prostaglandinas/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/ultraestrutura , Peptídeo Intestinal Vasoativo/farmacologiaRESUMO
Four children who underwent extensive small-bowel resection in infancy developed recurrent iron deficiency anemia due to gastrointestinal bleeding between 4 and 12 years later. The initial resections were required for multiple ileal atresia (n = 2) and gastroschisis (n = 2). Three patients have had melena and one had persistently guaiac-positive stools. Three patients had protein-losing enteropathy, and in one there was persistent hypoalbuminemia. Colonoscopy identified circumferential ulcerative lesions at the surgical anastomosis (n = 2) and at laparotomy another patient had well-defined linear ulcers close to the surgical anastomosis. Histology demonstrated focal ulceration with chronic inflammation, but did not show granulomata, crypt abscesses, or malignancy. Multiple imaging procedures and gastroduodenoscopy failed to identify an alternative bleeding source. Medical therapy including iron, antacids, sucralfate, H2 antagonists, and cholestyramine was ineffective. Two patients have undergone anastomotic resection. One experienced symptomatic recurrence 4 months after surgery. Repeat colonoscopy found ulceration at the new anastomosis with similar histology. The other patient remains asymptomatic 7 months postsurgery. Recurrent gastrointestinal hemorrhage due to anastomotic ulceration, of unknown etiology, appears to be a late complication of small-bowel resection in infancy.
Assuntos
Anemia Hipocrômica/etiologia , Hemorragia Gastrointestinal/etiologia , Íleo/anormalidades , Atresia Intestinal/cirurgia , Enteropatias Perdedoras de Proteínas/etiologia , Músculos Abdominais/anormalidades , Músculos Abdominais/cirurgia , Anastomose Cirúrgica/efeitos adversos , Criança , Pré-Escolar , Doença Crônica , Feminino , Seguimentos , Humanos , Íleo/cirurgia , Lactente , Masculino , RecidivaRESUMO
Concentrated proteins in pancreatic juice could precipitate and plug pancreatic ducts, initiating pancreatic disease. In cystic fibrosis (CF), pancreatic fluid secretion is impaired due to defective anion transport and proteins are hyperconcentrated. To determine whether proteins in pancreatic secretions precipitate selectively or nonspecifically, duodenal secretions were obtained from subjects with and without cystic fibrosis, during pancreatic stimulation with cholecystokinin (CCK) and secretin, dialyzed against phosphate-buffered saline (PBS), and concentrated in stages by ultrafiltration. Precipitates obtained by centrifugation at 15,600 g for 10 min at 4 degrees C were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The only protein band regularly enriched in precipitates from 10 CF and 9 non-CF samples had an Mr of approximately 14,000. Monoclonal antibodies, raised independently in two laboratories against either pancreatic stone protein (PSP) or pancreatic thread protein (PTP), reacted with the Mr 14,000 protein(s). Differential extraction of PTP and PSP by 0.1N HCl and 10% sodium citrate produced Mr 14,000 products that reacted equally with each monoclonal antibody (MAB). Two-dimensional gel electrophoresis demonstrated two Mr 14,000 spots in each extract with pI's of 5.8 and 6.0. Each spot reacted equivalently with the MABs for PSP and PTP. PSP/PTP type proteins are sparingly soluble in pancreatic secretions and could contribute to protein plugging in pancreatic disease.