RESUMEN
Amyloid formation has been most studied in the context of neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, as well as in amyloidosis. However, it is becoming increasingly clear that amyloid is also present in the healthy setting; for example nontoxic amyloid formation is important for melanin synthesis and in innate immunity. Furthermore, bacteria have mechanisms to produce functional amyloid structures with important roles in bacterial physiology and interaction with host cells. Here, we will discuss some novel aspects of fibril-forming proteins in humans and bacteria. First, the amyloid-forming properties of the antimicrobial peptide human defensin 6 (HD6) will be considered. Intriguingly, unlike other antimicrobial peptides, HD6 does not kill bacteria. However, recent data show that HD6 can form amyloid structures at the gut mucosa with strong affinity for bacterial surfaces. These so-called nanonets block bacterial invasion by entangling the bacteria in net-like structures. Next, the role of functional amyloid fibrils in human semen will be discussed. These fibrils were discovered through their property to enhance HIV infection but they may also have other yet unknown functions. Finally, the role of amyloid formation in bacteria will be reviewed. The recent finding that bacteria can make amyloid in a controlled fashion without toxic effects is of particular interest and may have implications for human disease. The role of amyloid in health and disease is beginning to be unravelled, and here, we will review some of the most recent findings in this exciting area.
Asunto(s)
Amiloide/biosíntesis , Bacterias/metabolismo , Mucosa Intestinal/microbiología , Infecciones Bacterianas/inmunología , Fenómenos Fisiológicos Bacterianos , Infecciones por VIH/transmisión , Humanos , Inmunidad Innata , Microbiota , Pliegue de Proteína , Semen/metabolismo , alfa-Defensinas/biosíntesis , alfa-Defensinas/inmunologíaRESUMEN
Insulin receptor complementary DNA has been cloned from an insulin-resistant patient with leprechaunism whose receptors exhibited multiple abnormalities in insulin binding. The patient is a compound heterozygote, having inherited two different mutant alleles of the insulin receptor gene. One allele contains a missense mutation encoding the substitution of glutamic acid for lysine at position 460 in the alpha subunit of the receptor. The second allele has a nonsense mutation causing premature chain termination after amino acid 671 in the alpha subunit, thereby deleting both the transmembrane and tyrosine kinase domains of the receptor. Interestingly, the father is heterozygous for this nonsense mutation and exhibits a moderate degree of insulin resistance. This raises the possibility that mutations in the insulin receptor gene may account for the insulin resistance in some patients with non-insulin-dependent diabetes mellitus.
Asunto(s)
Diabetes Mellitus Tipo 2/genética , Resistencia a la Insulina/genética , Receptor de Insulina/genética , Alelos , Secuencia de Bases , Línea Celular , Membrana Celular/metabolismo , Transformación Celular Viral , ADN/genética , Enfermedades del Sistema Endocrino/genética , Femenino , Amplificación de Genes , Trastornos del Crecimiento/genética , Herpesvirus Humano 4 , Heterocigoto , Humanos , Concentración de Iones de Hidrógeno , Insulina/sangre , Linfocitos/metabolismo , Monocitos/metabolismo , Mutación , Síndrome , TransfecciónRESUMEN
In the presence of 10 mM molybdate ion, we are able to detect the appearance of a [6,7-3H]-17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione-([3H]R5020) binding moiety in human prostatic cytosol which sedimented at approximately 8S in a glycerol density gradient. The specifically bound [3H]-R5020 was displaced by progesterone, tiramcinolone acetonide, and R5020 but not by cortisol, dihydrotestosterone, 17 beta-estradiol, or diethylstilbestrol. Specific binding of [3H]R5020 in the presence of molybdate ion was shown to saturate at 7 x 10(-10) M (n = 64 fmol/mg of protein). The optimum concentration of molybdate ion for enhancing specific binding of [3H]-R5020 was determined to be between 7 and 20 mM. Molybdate ion was also effective in stabilizing the 8S R5020-binding moiety in a preincubation for 16 hr at 0 degrees in the absence of added steroid.
Asunto(s)
Molibdeno/metabolismo , Próstata/metabolismo , Receptores de Progesterona/metabolismo , Unión Competitiva , Centrifugación por Gradiente de Densidad , Citosol/análisis , Humanos , Masculino , Promegestona/metabolismo , Próstata/ultraestructuraRESUMEN
BACKGROUND: Upper gastrointestinal tract intestinal metaplasia (IM) is termed Barrett's oesophagus (BO) or gastric intestinal metaplasia (GIM), depending on its location. BO and GIM are associated with chemical exposure resulting from gastro-oesophageal reflux and chronic Helicobacter pylori infection, respectively. Paneth cells (PCs), characterised by cytoplasmic eosinophilic granules, are found in a subset of IM at these sites, but histology may not accurately detect them. AIM: To determine human defensin 5 (HD5; an antimicrobial peptide produced by PCs) expression in BO and GIM, and to investigate its association with H pylori infection. METHODS: Endoscopic biopsies from 33 patients with BO and 51 with GIM, and control tissues, were examined by routine histology and for H pylori infection and HD5 mRNA and protein expression. RESULTS: In normal tissues, HD5 expression was specific for PCs in the small intestine. Five patients with BE and 42 with GIM expressed HD5, but few HD5 expressing cells in IM had the characteristic histological features of PCs. Most HD5 positive specimens were H pylori infected and most HD5 negative specimens were not infected. CONCLUSIONS: HD5 immunohistochemistry was often positive in IM when PCs were absent by conventional histology. Thus, HD5 immunohistochemistry may be superior to histology for identifying metaplastic PCs and distinguishing GIM from BO. The higher frequency of HD5 expression in GIM than in BO is associated with a higher frequency of H pylori infection, suggesting that in IM PCs may form part of the mucosal antibacterial response.
Asunto(s)
Esófago de Barrett/metabolismo , Defensinas/metabolismo , Mucosa Gástrica/metabolismo , Adulto , Anciano , Esófago de Barrett/microbiología , Western Blotting/métodos , Defensinas/genética , Defensinas/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Unión Esofagogástrica/metabolismo , Unión Esofagogástrica/patología , Femenino , Mucosa Gástrica/patología , Expresión Génica , Infecciones por Helicobacter/complicaciones , Infecciones por Helicobacter/metabolismo , Helicobacter pylori , Humanos , Masculino , Metaplasia/metabolismo , Metaplasia/microbiología , Persona de Mediana Edad , Células de Paneth/metabolismo , Células de Paneth/patología , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
The epithelial surface of the human small intestine is a barrier between the host and the microbial environment of the lumen. A human small intestine cDNA clone was found to encode a new member of the defensin family of antimicrobial peptides, named human defensin-6. Tissue expression of this mRNA is specific for the small intestine as determined by Northern blot analysis and polymerase chain reaction analysis. In situ hybridization demonstrated that human defensin-6 mRNA localizes to Paneth cells in the crypts of Lieberkühn. The finding of an abundant defensin mRNA in human Paneth cells supports the notion that these epithelial cells may play a key role in host defense of the human bowel. The results also strengthen the hypothesis that peptide-based host defenses are prevalent at mucosal surfaces in mammals.
Asunto(s)
Proteínas Sanguíneas/genética , Mucosa Intestinal/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Actividad Bactericida de la Sangre , Southern Blotting , Clonación Molecular , Secuencia de Consenso , ADN/genética , Defensinas , Disulfuros/química , Expresión Génica , Humanos , Hibridación in Situ , Mucosa Intestinal/citología , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , ARN Mensajero/genéticaRESUMEN
The cysteine-rich beta-defensin peptides are broad-spectrum bactericidal agents expressed in epithelial and myeloid tissues. The human beta-defensin-1 (hBD-1) gene maps adjacent to the human alpha-defensin cluster and is expressed in the respiratory, gastrointestinal and genitourinary tracts. Here, we characterize a mouse beta-defensin gene (mBD-1) which is: (1) closely related to hBD-1 both in sequence and gene organization; (2) expressed at high levels in the mouse kidney and at lower levels in brain, heart, lung, uterus, spleen, skeletal muscle, stomach, and small intestine; and (3) maps to mouse chromosome 8 at or near the location of the mouse alpha-defensin genes. These data indicate that mBD-1 is a close homolog of hBD-1, and suggest that analysis of its role in mouse host defense may provide significant insights into human epithelial innate immunity.
Asunto(s)
Proteínas Sanguíneas/genética , beta-Defensinas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Southern Blotting , Médula Ósea/metabolismo , Mapeo Cromosómico , Defensinas , Sistema Digestivo/metabolismo , Humanos , Riñón/metabolismo , Masculino , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Sistema Respiratorio/metabolismo , Mapeo Restrictivo , Distribución Tisular , Sistema Urogenital/metabolismoRESUMEN
We describe the isolation of naturally occurring human intestinal defensins HD-5 and HD-6 from ileal neobladder urine and ileal mucosa. Using an antibody-based detection assay, we found multiple N-terminally processed forms of HD-5. The predominant HD-5 forms in tissue were longer than those in neobladder urine (amino acid (aa) 23-94 and 29-94 versus aa 36-94, 56-94 and 63-94) suggesting that Paneth cells store prodefensin that is processed to mature defensin during or after degranulation. Search for mature HD-6 yielded aa 69-100 as the predominant form in both sources. The ileal neobladder is a promising model to study human Paneth cell secretion.
Asunto(s)
Proteínas Sanguíneas/orina , Íleon/metabolismo , Reservorios Urinarios Continentes , Secuencia de Aminoácidos , Proteínas Sanguíneas/química , Defensinas , Humanos , Íleon/trasplante , Datos de Secuencia Molecular , Células de Paneth/metabolismo , Fosfolipasas A/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
Antimicrobial peptides are a widespread component of host defense. We characterized the tissue distribution and cellular localization of expression of the magainin family of antimicrobial peptide genes in Xenopus laevis. Two genes from this family, magainin and PGLa, are expressed at high levels in the skin and throughout the gastrointestinal tract. Magainin and PGLa mRNAs are synthesized in the granular multinucleated cell (GMC) of the gastric mucosa, a cell shown previously to contain magainin and PGLa peptides by immunohistochemical methods. In addition, we have localized magainin and PGLa mRNAs to distinct cells of Xenopus small intestine. Further characterization of this large, granule-filled cell by electron microscopy demonstrates features in common with the Paneth cell of mammalian small intestine, previously identified as a site of expression of antimicrobial peptide genes of the defensin family in mouse and human. Our identification of granule-laden, eosinophilic intestinal cells in Xenopus as a site of magainin and PGLa antimicrobial peptide gene expression suggests that these cells are functional analogues of mammalian Paneth cells and further supports a conserved role of antimicrobial peptides in host defense of the vertebrate small intestine.
Asunto(s)
Antiinfecciosos , Péptidos Catiónicos Antimicrobianos , Mucosa Gástrica/citología , Mucosa Gástrica/metabolismo , Expresión Génica , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Biosíntesis de Péptidos , ARN Mensajero/biosíntesis , Animales , Elementos sin Sentido (Genética) , Northern Blotting , Hibridación in Situ , Mucosa Intestinal/citología , Mucosa Intestinal/ultraestructura , Intestino Delgado/citología , Intestino Delgado/ultraestructura , ARN Mensajero/análisis , Piel/metabolismo , Xenopus laevisRESUMEN
Magainins, antimicrobial peptides secreted by granular glands of frog skin, may be related to the high resistance to infections of this epithelial surface. The oral mucosa of healthy individuals is another tissue in which infection is not frequent, probably owing to the activity of potent salivary and mucosal defense mechanisms. To investigate if magainin-like factors are a component of these oral defense mechanisms, human and animal minor (mucosal) and major salivary glands were examined by immunohistochemistry, using a polyclonal rabbit anti-magainin antibody. Cryostat sections of (para) formaldehyde-fixed tissues were incubated with the antibody and then stained with fluorescein-complexed anti-rabbit IgG. Specific staining was observed in the apical portion of the cytoplasm of ductal epithelial cells of human submandibular and labial salivary glands. Diffuse staining was present in submandibular acinar cells. Bovine, rat, hamster, and mouse tissues were unreactive. The presence of magainin-like substances in human salivary gland duct cells is consistent with reports of the occurrence of other biologically active substances in salivary gland ducts.
Asunto(s)
Péptidos Catiónicos Antimicrobianos , Péptidos/inmunología , Glándulas Salivales/metabolismo , Glándula Submandibular/metabolismo , Proteínas de Xenopus , Adulto , Animales , Antimaláricos/inmunología , Antimaláricos/metabolismo , Bovinos , Niño , Cricetinae , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica/métodos , Magaininas , Masculino , Mesocricetus , Ratones , Ratones Endogámicos C57BL , Péptidos/metabolismo , Ratas , Ratas Endogámicas , Glándulas Salivales/citología , Glándulas Salivales Menores/citología , Glándulas Salivales Menores/metabolismo , Glándula Submandibular/citologíaRESUMEN
We have characterized a novel peptide-containing cell within the gastric mucosa of Xenopus laevis. The cell is a spherical, multinucleated syncytial structure containing a cytoplasmic space filled with dense rice-shaped granules, and is strikingly similar in morphology to the well-studied granular gland of the amphibian skin. Immunohistochemical and immunogold methods were used to demonstrate that several peptides previously isolated from the granular glands of the skin, including the antimicrobial peptides magainin and PGLa (a peptide with amino-terminal glycine and carboxy-terminal leucinamide), are also stored in granules present in these enteric cells. These data demonstrate that this enteric peptide-producing cell is strikingly similar both morphologically and biochemically to the granular gland, previously considered a highly specialized structure of the amphibian integument. This novel gastric mucosal cell, which we have designated a "granular multinucleated cell," is distinct in its morphology and its diversity of stored peptide products from other well-characterized peptide-containing cells in the vertebrate gastrointestinal tract.
Asunto(s)
Mucosa Gástrica/citología , Péptidos/análisis , Secuencia de Aminoácidos , Animales , Gránulos Citoplasmáticos/ultraestructura , Ensayo de Inmunoadsorción Enzimática , Mucosa Gástrica/metabolismo , Mucosa Gástrica/ultraestructura , Inmunohistoquímica/métodos , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/inmunología , Glándulas Sebáceas/citología , Fenómenos Fisiológicos de la Piel , Xenopus laevisRESUMEN
Metronidazole is effective for the treatment of acute pouchitis after ileal pouch-anal anastomosis, but it has not been directly compared with other antibiotics. This randomized clinical trial was designed to compare the effectiveness and side effects of ciprofloxacin and metronidazole for treating acute pouchitis. Acute pouchitis was defined as a score of 7 or higher on the 18-point Pouchitis Disease Activity Index (PDAI) and symptom duration of 4 weeks or less. Sixteen patients were randomized to a 2-week course of ciprofloxacin 1,000 mg/d (n = 7) or metronidazole 20 mg/kg/d (n = 9). Clinical symptoms, endoscopic findings, and histologic features were assessed before and after therapy. Both ciprofloxacin and metronidazole produced a significant reduction in the total PDAI score as well as in the symptom, endoscopy, and histology subscores. Ciprofloxacin lowered the PDAI score from 10.1+/-2.3 to 3.3+/-1.7 (p = 0.0001), whereas metronidazole reduced the PDAI score from 9.7+/-2.3 to 5.8+/-1.7 (p = 0.0002). There was a significantly greater reduction in the ciprofloxacin group than in the metronidazole group in terms of the total PDAI (6.9+/-1.2 versus 3.8+/-1.7; p = 0.002), symptom score (2.4+/-0.9 versus 1.3+/-0.9; p = 0.03), and endoscopic score (3.6+/-1.3 versus 1.9+/-1.5; p = 0.03). None of patients in the ciprofloxacin group experienced adverse effects, whereas three patients in the metronidazole group (33%) developed vomiting, dysgeusia, or transient peripheral neuropathy. Both ciprofloxacin and metronidazole are effective in treating acute pouchitis with significant reduction of the PDAI scores. Ciprofloxacin produces a greater reduction in the PDAI and a greater improvement in symptom and endoscopy scores, and is better tolerated than metronidazole. Ciprofloxacin should be considered as one of the first-line therapies for acute pouchitis.
Asunto(s)
Antiinfecciosos/uso terapéutico , Ciprofloxacina/uso terapéutico , Metronidazol/uso terapéutico , Reservoritis/tratamiento farmacológico , Enfermedad Aguda , Adulto , Antiinfecciosos/administración & dosificación , Ciprofloxacina/administración & dosificación , Colitis Ulcerosa/cirugía , Femenino , Humanos , Masculino , Metronidazol/administración & dosificación , Reservoritis/patología , Índice de Severidad de la Enfermedad , Resultado del TratamientoAsunto(s)
Defensinas/inmunología , Sistema Digestivo/inmunología , Enfermedades Inflamatorias del Intestino/inmunología , Células de Paneth/inmunología , Defensinas/metabolismo , Regulación de la Expresión Génica , Humanos , Mucosa Intestinal/inmunología , Intestino Delgado/inmunología , Intestino Delgado/patología , Procesamiento Proteico-PostraduccionalAsunto(s)
Bronquios/ultraestructura , Fibrosis Quística/patología , Bronquios/química , Fraccionamiento Celular/métodos , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Epitelio/química , Epitelio/ultraestructura , Humanos , Proteínas de la Membrana/análisis , Proteínas de la Membrana/genéticaAsunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Inmunidad Innata/genética , Animales , Antiinfecciosos/química , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/genética , Defensinas/farmacología , Regulación de la Expresión Génica , Mamíferos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/farmacología , Relación Estructura-Actividad , VirulenciaAsunto(s)
Antiinfecciosos , Proteínas Sanguíneas/genética , Evolución Molecular , Animales , Antiinfecciosos/química , Proteínas Sanguíneas/química , Secuencia Conservada , Defensinas , Electroforesis en Gel de Agar , Humanos , Cinética , Desnaturalización de Ácido Nucleico , Hibridación de Ácido Nucleico/métodos , Oligodesoxirribonucleótidos/genética , Homología de Secuencia de Aminoácido , TemperaturaRESUMEN
Antimicrobial peptides are fundamental effector molecules of innate immunity, utilized in host defence by virtually all organisms studied. These gene-encoded peptides have direct antibiotic activity against a wide range of bacteria and other microbes. In humans and other mammals, defensins are a predominant class of such peptides. In the mammalian small intestine, Paneth cells, specialized secretory epithelial cells located at the base of the crypt invaginations lining the intestinal wall, produce defensins and other antibiotic proteins. Recent investigations in murine models provide compelling support for the hypothesis that enteric defensins play a pivotal role in defence from food- and water-borne pathogens in the intestinal lumen. Investigations by others indicate that intestinal commensal bacteria are key factors in the pathogenesis of IBD (inflammatory bowel disease) in genetically susceptible humans. Recent studies provide evidence that reduced expression of Paneth cell defensins may be a key factor in the pathogenesis of ileal Crohn's disease, a subgroup of IBD. Future studies to further define the function and regulation of Paneth cell defensins will enhance our understanding of normal small bowel physiology, and probably contribute to a better understanding of the pathogenesis of inflammatory and infectious diseases of the bowel. Such knowledge may provide new therapeutic targets and strategies.