RESUMEN
Pigment epithelium-derived factor (PEDF) is a glycoprotein with complex neuroprotective, anti-angiogenic, and anti-inflammatory properties, all of which could potentially be exploited as a therapeutic option for vascular complications in diabetes. We have previously shown that PEDF-derived synthetic peptide, P5-3 (FIFVLRD) has a comparable ability with full PEDF protein to inhibit rat corneal neovascularization induced by chemical cauterization. However, the effects of PEDF peptide on experimental diabetic nephropathy remain unknown. To address the issue, we modified P5-3 to stabilize and administered the modified peptide (d-Lys-d-Lys-d-Lys-Gln-d-Pro-P5-3-Cys-amide, 0.2 nmol/day) or vehicle to streptozotocin-induced diabetic rats (STZ-rats) intraperitoneally by an osmotic mini pump for 2 weeks. We further examined the effects of modified peptide on human proximal tubular cells. Renal PEDF expression was decreased in STZ-rats. Although the peptide administration did not affect blood glucose or blood pressure, it decreased urinary excretion levels of 8-hydroxy-2'-deoxyguanosine, an oxidative stress marker, and reduced plasminogen activator inhibitor-1 (PAI-1) gene expression, and suppressed glomerular expansion in the diabetic kidneys. High glucose or advanced glycation end products stimulated oxidative stress generation and PAI-1 gene expression in tubular cells, all of which were significantly suppressed by 10 nM modified P5-3 peptide. Our present study suggests that PEDF-derived synthetic modified peptide could protect against experimental diabetic nephropathy and inhibit tubular cell damage under diabetes-like conditions through its anti-oxidative properties. Supplementation of modified P5-3 peptide may be a novel therapeutic strategy for diabetic nephropathy.
Asunto(s)
Diabetes Mellitus Experimental/complicaciones , Nefropatías Diabéticas/prevención & control , Proteínas del Ojo/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Fragmentos de Péptidos/farmacología , Serpinas/metabolismo , Animales , Diabetes Mellitus Experimental/fisiopatología , Nefropatías Diabéticas/etiología , Humanos , Riñón/metabolismo , Masculino , Estrés Oxidativo , Ratas , Ratas WistarRESUMEN
Advanced glycation end products (AGEs) decrease adiponectin expression and suppress insulin signaling in cultured adipocytes through the interaction with a receptor for AGEs (RAGE) via oxidative stress generation. We have recently found that high-affinity DNA aptamer directed against AGE (AGE-aptamer) prevents the progression of experimental diabetic nephropathy by blocking the harmful actions of AGEs in the kidney. This study examined the effects of AGE-aptamer on adipocyte remodeling, AGE-RAGE-oxidative stress axis, and adiponectin expression in fructose-fed rats. Although AGE-aptamer treatment by an osmotic mini pump for 8 weeks did not affect serum insulin levels, it significantly decreased average fasting blood glucose and had a tendency to inhibit body weight gain in fructose-fed rats. Furthermore, AGE-aptamer significantly suppressed the increase in adipocyte size and prevented the elevation in AGEs, RAGE, and an oxidative stress marker, 8-hydroxydeoxyguanosine (8-OHdG), levels in adipose tissues of fructose-fed rats at 14-week-old, while it restored the decrease in adiponectin mRNA levels. Our present study suggests that AGE-aptamer could improve glycemic control and prevent adipocyte remodeling in fructose-fed rats partly by suppressing the AGE-RAGE-mediated oxidative stress generation. AGE-aptamer might be a novel therapeutic strategy for fructose-induced metabolic derangements.
Asunto(s)
Adipocitos/patología , Aptámeros de Nucleótidos/administración & dosificación , Glucemia/análisis , Fructosa/administración & dosificación , Productos Finales de Glicación Avanzada/antagonistas & inhibidores , Receptor para Productos Finales de Glicación Avanzada/antagonistas & inhibidores , Adipocitos/efectos de los fármacos , Adiponectina/genética , Animales , Tamaño de la Célula/efectos de los fármacos , Dieta , Expresión Génica/efectos de los fármacos , Productos Finales de Glicación Avanzada/genética , Resistencia a la Insulina , Masculino , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Aumento de Peso/efectos de los fármacosRESUMEN
Colorectal decompression with a catheter was performed for evacuation of stool before definitive surgery in two patients with a persistent cloaca. Two newborn female infants with persistent cloaca received placement of a silicone balloon-tipped catheter in the rectum via the cloacal orifice under fluoroscopic guidance at the time of diagnosis. The length of the cloaca was 2 and 1.5 cm, respectively. The diameter of the catheter was matched to the patients' rectal size and the open end was wrapped in a diaper to allow continuous drainage of stool. The infants underwent bowel irrigation with warm saline thrice a day, at home. Total urogenital mobilization was carried out in the infants at the age of 7 and 8 months, respectively. Both infants had no abdominal distension, colorectal dilatation, or urinary tract infection while the catheter was in situ. The postoperative course was uneventful, except for minimal wound dehiscence in one patient. At present, both infants can void spontaneously without any urological problems. In infants with a persistent cloaca less than 3 cm long and normal urinary tract function, adequate evacuation of stool may be achieved by colorectal decompression with a catheter, thus avoiding the need for a colostomy.
Asunto(s)
Cloaca/anomalías , Procedimientos Quirúrgicos del Sistema Digestivo/métodos , Descompresión Quirúrgica , Femenino , Fluoroscopía , Humanos , Recién Nacido , Imagen por Resonancia Magnética , Uretra/cirugía , Vagina/cirugíaRESUMEN
This study determined the effect of the adenovirus E1A gene on nitric oxide (NO) production in alveolar epithelial (A549) cells. E1A-positive A549 cells (E1A transfectants), E1A-negative A549 cells (control transfectants) and untransfected A549 cells were placed in 96-well tissue culture plates. After stimulation with lipopolysaccharide (LPS) or cytokine mixture (CM), the biochemical reaction products of NO (nitrite and nitrate) in the culture medium were measured by chemiluminescence. The inducible (iNOS) and the endothelial (eNOS) isoforms of nitric oxide synthase (NOS) protein expression were examined by Western blotting. iNOS mRNA expression was examined by Northern blotting and RT-PCR. CM-induced NO production by E1A-positive A549 cells was significantly lower than that of E1A-negative cells (p < 0.0001). LPS stimulation failed to enhance NO production in both cell types. CM induced iNOS protein expression in E1A-negative A549 cells, but not in E1A-positive cells. eNOS protein expression was constitutive and was not affected by CM stimulation, LPS stimulation or E1A. CM induced iNOS mRNA expression in E1A-negative A549 cells, but not in E1A-positive cells. In conclusion, the adenovirus E1A gene suppressed NO production through transcriptional control of the iNOS gene in A549 cells. This inhibition of NO production may enable the virus to persist in human tissue, since NO is an antiviral effector of the innate immune system.
Asunto(s)
Proteínas E1A de Adenovirus/genética , Adenovirus Humanos/genética , Células Epiteliales/virología , Genes Virales , Óxido Nítrico/biosíntesis , Alveolos Pulmonares/citología , Proteínas E1A de Adenovirus/metabolismo , Adenovirus Humanos/metabolismo , Células Epiteliales/inmunología , Regulación de la Expresión Génica , Humanos , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , TransfecciónRESUMEN
Matrix metalloproteinase (MMP)-9 and tissue inhibitors of metalloproteinase (TIMP)-1 concentrations are increased in the sputum of asthma and chronic bronchitis patients, and are thought to be related to airflow obstruction. However, serum concentrations of these enzymes have not been clearly evaluated in patients with chronic obstructive pulmonary disease (COPD). The aim of this study was to examine the serum concentrations of these enzymes in COPD and asthmatic patients in order to determine their relationship with airway obstruction. Serum samples were obtained from 72 patients with COPD: 66 control subjects and 26 patients with asthma. Smoking histories of control subjects were matched with those of COPD patients. Serum concentrations of TIMP-1 and MMP-9 were determined by ELISA. The circulating TIMP-1 concentration was significantly higher in stable COPD patients than in control and asthmatic subjects, and was significantly negatively correlated with forced expiratory volume in one second/forced vital capacity in COPD patients. The molar ratio between MMP-9 and TIMP-1 was significantly lower in COPD patients than in control subjects. In patients with COPD, the serum TIMP-1 concentration was significantly increased during disease exacerbation. In conclusion, the current findings suggest that serum tissue inhibitors of metalloproteinase-1 concentration can be used as a serum marker of airway obstruction and exacerbation in chronic obstructive pulmonary disease patients.
Asunto(s)
Enfermedad Pulmonar Obstructiva Crónica/enzimología , Inhibidor Tisular de Metaloproteinasa-1/sangre , Corticoesteroides/uso terapéutico , Anciano , Asma/sangre , Asma/diagnóstico , Asma/tratamiento farmacológico , Asma/etiología , Biomarcadores/sangre , Femenino , Humanos , Masculino , Metaloproteinasa 9 de la Matriz/sangre , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Enfermedad Pulmonar Obstructiva Crónica/etiología , Pruebas de Función Respiratoria , Fumar/efectos adversosRESUMEN
Insulin receptor (IR) and IGF-I receptor (IGF-IR) are structurally and functionally related and belong to the tyrosine kinase receptor family. In teleosti such as salmonids and turbot, occurrence of multiple IR and IGF-IR members has been reported, but the structures of a complete set of both IR and IGF-IR members in a single teleost species have not yet been characterized. In this study, we cloned and analysed four distinct cDNA clones for IR and IGF-IR members from the liver and kidney of the Japanese flounder (Paralichthys olivaceus). Deduced amino acid sequence analyses and phylogenetic analysis have revealed that two of them (fIR-1 and fIR-2) belong to IR members and the other two (fIGF-IR-1 and fIGF-IR-2) are IGF-IRs. fIR-1 and fIR-2 comprised 1369 and 1368 amino acid residues respectively, and fIGF-IR-1 and fIGF-IR-2 comprised 1412 and 1418 residues respectively. All the receptor proteins contained cysteine-rich domains in their alpha-subunits, and conserved each transmembrane and tyrosine kinase domains in their beta-subunits. The amino acid sequences of fIRs and fIGF-IRs showed more than 90% sequence identity with turbot IR and IGF-IR respectively. When compared with their mammalian homologues, fIGF-IR-1 and fIGF-IR-2 proteins contained large insertions at their C-termini, as was observed in the corresponding region of turbot IGF-IR. Occurrence of multiple species of mRNA for each IR and IGF-IR was suggested by Northern blot analyses. A ribonuclease protection assay revealed diverse expressions of four receptor mRNAs in a wide range of tissues including heart, liver, ovary, testis, brain, gill arch, kidney, skeletal muscle, intestine, stomach, spleen and eye of the flounder.
Asunto(s)
Lenguado/metabolismo , Factor I del Crecimiento Similar a la Insulina , Riñón/química , Hígado/química , Receptor de Insulina/genética , Receptores de Somatomedina/genética , Regiones no Traducidas 5' , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Clonación Molecular , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Isoformas de Proteínas/genética , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
As models of ion channel proteins and naturally occurring pore-forming peptides, we designed a series of Aib rich peptides [Ac-(Aib-Xxx-Aib-Ala)(5)-NH(2) (Xxx = Lys, Glu, Ser, and Gly: BXBA-20)] to investigate the effects of the side chains of the amino acid residues Lys, Glu, Ser, and Gly on the conformation and electrophysiological properties of ion channels. The conformation of peptides and their affinity for phospholipid membranes were evaluated by CD spectroscopy. Patch-clamp experiments revealed that all BXBA-20 peptides form ion channels in DPhPC bilayers exhibiting clearly resolved transitions between the open and closed states. The channel forming frequency was in the order BKBA-20>BEBA-20>BSBA-20>BGBA-20. In the case of BKBA-20 and BEBA-20, the self-assembled conductive oligomers expressed homogeneous and voltage-independent single channel conductances. In contrast, heterogeneous conductance was observed in BSBA-20 and BGBA-20 ion channels under similar experimental conditions. From these results, we conclude that peptides with a high degree of helical conformation, high amphipathicity, high affinity for lipid membranes, and self-associating characters in vesicles are most suitable for inducing ion channels with a high frequency of occurrence. Moreover, BEBA-20, BSBA-20, and BGBA-20 channels were cation-selective, whereas the BKBA-20 channel was non-selective.
Asunto(s)
Canales Iónicos/fisiología , Membrana Dobles de Lípidos/química , Péptidos/química , Ácidos Aminoisobutíricos/química , Ácidos Aminoisobutíricos/metabolismo , Dicroismo Circular , Diseño de Fármacos , Electrofisiología , Canales Iónicos/química , Membrana Dobles de Lípidos/metabolismo , Modelos Biológicos , Péptidos/síntesis química , Péptidos/metabolismo , Ingeniería de Proteínas , Estructura Secundaria de ProteínaRESUMEN
In diadromous and euryhaline teleosts, it has been established that prolactin (PRL) is a major hormone regulating the maintenance of water and electrolyte homeostasis by acting on its receptor (PRLR) expressed in the osmoregulatory organs. To investigate the major physiological role of PRL in a marine teleost, cDNA for the Japanese flounder (Paralichtys olivaceus) prolactin receptor (fPRLR) has been cloned and characterized. The predicted fPRLR is composed of 636 amino acids conserving common structural features, such as the WSXWS motif and box 1, that are observed in the members of the cytokine receptor superfamily. By Northern blot analysis, 3.5-kb transcripts for fPRLR were clearly detected in the gill, kidney, and intestine. By RNase protection assay, similarly high levels of mRNA expression were detected in these osmoregulatory organs and lower expression levels were seen in the brain for both males and females. Interestingly, a distinct expression level of fPRLR mRNA was observed in the testis, but not in the ovary. The present results suggest that PRL may play an important role in the control of water and electrolyte balance through PRLR expressed in the osmoregulatory organs in the marine teleost the Japanese flounder as well as in other teleosts. Furthermore, PRL may differentially regulate gonadal functions in males and females of Japanese flounder.
Asunto(s)
Lenguado/metabolismo , Expresión Génica , ARN Mensajero/análisis , ARN Mensajero/química , Receptores de Prolactina/genética , Equilibrio Hidroelectrolítico , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Southern Blotting , Química Encefálica , Secuencia Conservada , Femenino , Branquias/química , Intestinos/química , Riñón/química , Masculino , Datos de Secuencia Molecular , Especificidad de Órganos , Ovario/química , Filogenia , Receptores de Prolactina/química , Alineación de Secuencia , Testículo/química , Distribución TisularRESUMEN
The majority of CTL epitopes are derived from intracellular proteins that are degraded in the cytoplasm by proteasomes into peptides that are transported into the endoplasmic reticulum by the TAP complex. These peptides can be further processed into the optimal size (8-10 residues) for binding with nascent MHC class I molecules, generating complexes that are exported to the cell surface. Proteins or peptides containing CTL epitopes can be introduced into the cytoplasm of APCs by linking them to membrane-translocating Trojan carriers allowing their incorporation into the MHC class I Ag-processing pathway. The present findings suggest that these "Trojan" Ags can be transported into the endoplasmic reticulum in a TAP-independent way where they are processed and trimmed into CTL epitopes. Furthermore, processing of Trojan Ags can also occur in the trans-Golgi compartment, with the participation of the endopeptidase furin and possibly with the additional participation of a carboxypeptidase. We believe that these findings will be of value for the design of CTL-inducing vaccines for the treatment or prevention of infectious and malignant diseases.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Presentación de Antígeno , Epítopos de Linfocito T/metabolismo , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Secuencia de Aminoácidos , Animales , Presentación de Antígeno/efectos de los fármacos , Presentación de Antígeno/genética , Antígeno Carcinoembrionario/inmunología , Antígeno Carcinoembrionario/metabolismo , Línea Celular , Proteínas del Huevo/genética , Proteínas del Huevo/inmunología , Proteínas del Huevo/metabolismo , Epítopos de Linfocito T/inmunología , Productos del Gen tat/síntesis química , Productos del Gen tat/genética , Productos del Gen tat/inmunología , Productos del Gen tat/metabolismo , Vectores Genéticos/inmunología , Vectores Genéticos/metabolismo , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Líquido Intracelular/inmunología , Líquido Intracelular/metabolismo , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Ovalbúmina/genética , Ovalbúmina/inmunología , Ovalbúmina/metabolismo , Fragmentos de Péptidos/síntesis química , Inhibidores de Proteasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Procesamiento Proteico-Postraduccional/inmunología , Proteínas Recombinantes de Fusión/síntesis química , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T Citotóxicos/enzimología , Células Tumorales CultivadasRESUMEN
Response to genotoxic stress can be considered as a multistage process involving initiation of cell-cycle arrest and maintenance of arrest during DNA repair. Although maintenance of G2/M checkpoints is known to involve Chk1, Chk2/Rad53 and upstream components, the mechanisms involved in its initiation are less well defined. Here we report that p38 kinase has a critical role in the initiation of a G2 delay after ultraviolet radiation. Inhibition of p38 blocks the rapid initiation of this checkpoint in both human and murine cells after ultraviolet radiation. In vitro, p38 binds and phosphorylates Cdc25B at serines 309 and 361, and Cdc25C at serine 216; phosphorylation of these residues is required for binding to 14-3-3 proteins. In vivo, inhibition of p38 prevents both phosphorylation of Cdc25B at serine 309 and 14-3-3 binding after ultraviolet radiation, and mutation of this site is sufficient to inhibit the checkpoint initiation. In contrast, in vivo Cdc25C binding to 14-3-3 is not affected by p38 inhibition after ultraviolet radiation. We propose that regulation of Cdc25B phosphorylation by p38 is a critical event for initiating the G2/M checkpoint after ultraviolet radiation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Fase G2/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mitosis/fisiología , Fosfatasas cdc25/metabolismo , Proteínas 14-3-3 , Animales , Fase G2/genética , Células HeLa , Humanos , Ratones , Mitosis/genética , Índice Mitótico , Fosforilación , Unión Proteica , Serina/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Rayos Ultravioleta , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Human immunodeficiency virus (HIV)-specific helper T lymphocytes (HTL) play a key role in the immune control of HIV type 1 (HIV-1) infection, and as such are an important target of potential HIV-1 vaccines. In order to identify HTL epitopes in HIV-1 that might serve as vaccine targets, conserved HIV-1-derived peptides bearing an HLA-DR binding supermotif were tested for binding to a panel of the most representative HLA-DR molecules. Eleven highly cross-reactive binding peptides were identified: three in Gag and eight in Pol. Lymphoproliferative responses to this panel of peptides, as well as to the HIV-1 p24 and p66 proteins, were evaluated with a cohort of 31 HIV-1-infected patients. All 11 peptides were recognized by peripheral blood mononuclear cells from multiple HIV-infected donors. Many of the responsive HIV-infected subjects showed recognition of multiple peptides, indicating that HIV-1-specific T-helper responses may be broadly directed in certain individuals. A strong association existed between recognition of the parental recombinant HIV-1 protein and the corresponding HTL peptides, suggesting that these peptides represent epitopes that are processed and presented during the course of HIV-1 infection. Lastly, responses to the supermotif peptides were mediated by CD4(+) T cells and were restricted by major histocompatibility complex class II molecules. The epitopes described herein are potentially important components of HIV-1 therapeutic and prophylactic vaccines.
Asunto(s)
Epítopos de Linfocito T/inmunología , Antígenos VIH/inmunología , VIH-1/inmunología , Antígenos HLA-DR/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Secuencia de Aminoácidos , Presentación de Antígeno/inmunología , Linfocitos T CD4-Positivos/inmunología , División Celular , Reacciones Cruzadas , Mapeo Epitopo , Infecciones por VIH/inmunología , Humanos , Datos de Secuencia Molecular , Péptidos/inmunologíaRESUMEN
PURPOSE: The purpose of this study was to describe the time course, early postoperative changes, and morphologic features of normalization of the pylorus after pyloromyotomy for hypertrophic pyloric stenosis. METHODS: The subjects were 17 infants (9 boys, 8 girls) who underwent umbilical incision Ramstedt pyloromyotomy. The pyloric muscle mass was measured immediately before the operation and then at intervals from 3 days to 6 months after the operation using a 7.5-MHz ultrasound probe. RESULTS: In longitudinal section, the dorsal part of the pyloric muscle thickened transiently and then thinned to normal values by 5 months after the operation. It was 5.1 +/- 0.8 mm (mean +/- SD) preoperatively, increased to 6.0 +/- 0.3 mm by day 3 after the operation (P <.05), and thinned to 2.8 +/- 0.2 mm by 5 months after the operation. Concomitantly, the length of the pylorus gradually decreased (from 20.1 +/- 2.9 mm preoperatively to 16.9 +/- 2.7 mm by 3 days postoperatively [P <.05] and to less than 15 mm, by 4 months). In transverse section, the muscle normalized as in the longitudinal section. At the site of the incision it was 4.3 +/- 0.4 mm thick preoperatively, thickened to 4.6 +/- 0.4 mm by 3 days after the operation (P <.05), thinned to 2.1 +/- 0.9 mm by 7 days (P <.05), and then increased slightly, but always was less than 3.0 mm. Morphologically, in transverse section, the incised area looked like a wedge by 3 days after the operation. CONCLUSIONS: After pyloromyotomy for hypertrophic pyloric stenosis, there is an early transient increase in muscle thickness within the first few postoperative days followed by a slow decrease that reaches normal thickness (<3 mm) by 5 months. This decrease in thickness is accompanied by a gradual decrease in length to 75% of the preoperative value by 5 months. The morphologic features in this normalization are first a wedge (day 3), then a flat tire (days 7 and 14), and finally an elongated ring (5 months). J Pediatr Surg 36:582-586.
Asunto(s)
Procedimientos Quirúrgicos del Sistema Digestivo/métodos , Estenosis Pilórica/diagnóstico por imagen , Estenosis Pilórica/cirugía , Femenino , Estudios de Seguimiento , Motilidad Gastrointestinal/fisiología , Humanos , Hipertrofia/diagnóstico por imagen , Hipertrofia/cirugía , Lactante , Recién Nacido , Masculino , Monitoreo Fisiológico/métodos , Contracción Muscular/fisiología , Examen Físico , Periodo Posoperatorio , Cuidados Preoperatorios , Píloro/diagnóstico por imagen , Píloro/fisiología , Sensibilidad y Especificidad , Resultado del Tratamiento , UltrasonografíaRESUMEN
To characterize the sites in human p53 that become phosphorylated in response to DNA damage, we have developed polyclonal antibodies that recognize p53 only when it is phosphorylated at specific sites. Several attempts to generate an antibody to p53 phosphorylated at Ser(6) using a phosphoserine-containing peptide as an immunogen were unsuccessful; however, phosphorylation-specific antibodies were produced by using the phosphoserine mimetic, l-2-amino-4-phosphono-4, 4-difluorobutanoic acid (F(2)Pab), in place of phosphoserine. Fmoc-F(2)Pab was prepared by an improved synthesis and chemically incorporated using solid phase peptide synthesis. Affinity-purified antibodies elicited by immunizing rabbits with an F(2)Pab peptide coupled to keyhole limpet hemocyanin recognized a p53(1-39) peptide phosphorylated only at Ser(6) but not the unphosphorylated peptide or the same peptide phosphorylated at Ser(9), Ser(15), Ser(20), Ser(33), or Ser(37). Untreated A549 cells exhibited a background of constitutive phosphorylation at Ser(6) that increased approximately 10-fold upon exposure to either ionizing radiation or UV light. Similar results were obtained for Ser(9) using antibodies raised against a conventional phosphopeptide. Ser(9) was phosphorylated by casein kinase 1 in vitro in a phosphoserine 6-dependent manner. Our data identify two additional DNA damage-induced phosphorylations in human p53 and show that F(2)Pab-derivatized peptides can be used to develop phosphorylation site-specific polyclonal antibodies.
Asunto(s)
Daño del ADN , Serina/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Doxorrubicina/farmacología , Humanos , Oligopéptidos/farmacología , Fosforilación , Conejos , Radiación Ionizante , Células Tumorales Cultivadas , Rayos UltravioletaRESUMEN
OBJECTIVE AND DESIGN: This study was designed to determine if the hepatocyte growth factor (HGF)-Met system is involved in the repair process of inflamed mucosa of ulcerative colitis (UC) and in the development of UC-associated colorectal cancer. MATERIALS AND METHODS: HGF and c-met gene expressions were quantified in colonic mucosal specimens from healthy control subjects, patients with UC and patients with UC-associated colorectal cancer, using the competitive reverse transcription-polymerase chain reaction. Expression of HGF protein was determined by immunoblot analysis. Expression of c-Met protein was analyzed immunohistochemically. RESULTS: HGF and c-met gene expressions were increased in inflamed mucosa of UC, compared with control subjects. Gene expression of HGF was also increased in the surrounding inflamed mucosa of UC-associated cancers. In cases in which the HGF gene expression was increased, an apparent increase in protein levels of HGF in inflamed mucosa of UC were observed by immunoblot analysis. The c-met gene was overexpressed in UC-associated cancers and a high level of immunoreactivity of the c-Met protein was immunohistochemically detected within the cancer cells. CONCLUSION: We showed that HGF and c-met expression is increased in the inflamed mucosa of UC and that c-met is overexpressed in UC-associated colorectal cancers. These observations suggest HGF-Met system is involved in the repair process of the inflamed mucosa of UC and provide further support for the view that the inappropriate expressions of both HGF and c-met genes predispose to the development of colorectal cancer in patients with UC.
Asunto(s)
Colitis Ulcerosa/metabolismo , Expresión Génica , Factor de Crecimiento de Hepatocito/genética , Proteínas Proto-Oncogénicas c-met/genética , Adulto , Anciano , Colitis Ulcerosa/complicaciones , Colon/química , Neoplasias del Colon/química , Neoplasias del Colon/etiología , Femenino , Humanos , Immunoblotting , Inmunohistoquímica , Mucosa Intestinal/química , Masculino , Persona de Mediana Edad , Proteínas Proto-Oncogénicas c-met/análisis , Neoplasias del Recto/química , Neoplasias del Recto/etiología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The tumour suppressor p53 induces cellular senescence in response to oncogenic signals. p53 activity is modulated by protein stability and post-translational modification, including phosphorylation and acetylation. The mechanism of p53 activation by oncogenes remains largely unknown. Here we report that the tumour suppressor PML regulates the p53 response to oncogenic signals. We found that oncogenic Ras upregulates PML expression, and overexpression of PML induces senescence in a p53-dependent manner. p53 is acetylated at lysine 382 upon Ras expression, an event that is essential for its biological function. Ras induces re-localization of p53 and the CBP acetyltransferase within the PML nuclear bodies and induces the formation of a trimeric p53-PML-CBP complex. Lastly, Ras-induced p53 acetylation, p53-CBP complex stabilization and senescence are lost in PML-/- fibroblasts. Our data establish a link between PML and p53 and indicate that integrity of the PML bodies is required for p53 acetylation and senescence upon oncogene expression.
Asunto(s)
Senescencia Celular/fisiología , Genes ras , Proteínas de Neoplasias/fisiología , Factores de Transcripción/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Acetilación , Animales , Núcleo Celular/metabolismo , Senescencia Celular/genética , Humanos , Lisina/metabolismo , Ratones , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiología , Proteína de la Leucemia Promielocítica , Transducción de Señal , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor , Regulación hacia ArribaRESUMEN
Growth hormone (GH) secreted from the pituitary is essential for postnatal growth in animals. GH exerts its actions by a direct effect on target organs and by stimulating insulin-like growth factor I (IGF-I) production. In the human pituitary, there is a naturally occurring variant protein which has a molecular mass of 20 kDa (20K hGH) besides the major 22 kDa hGH (22K hGH), but the physiological actions of 20K hGH are still poorly understood. In this study we have examined its effects on the IGF-I mRNA expression in the pro B-cell line Ba/F3 cells stably expressing hGH receptor (Ba/F3-hGHR). Ba/F3-hGHR cells were incubated for 2 h with a series of various concentrations (10 pM to approximately 10 nM) of 20K or 22K hGH. The IGF-I mRNA expression in the Ba/F3-hGHR cells was detected by the RT-PCR method. IGF-I gene expression was increased by 20K and 22K hGH stimulation, but not by PRL or IL-3 in the Ba/F3-hGHR. And this effect was not observed in parental Ba/F3 cells. Lower concentrations of 20K hGH more strongly induced IGF-I gene expression than 22K-hGH. These results suggest that 20K and 22K hGH stimulate the IGF-I gene expression in the Ba/F3-hGHR through hGH receptors, and that the stronger effect of 20K hGH than that of 22K hGH in enhancing the IGF-I gene expression may be correlated with a 20K hGH specific receptor dimerization mechanism.
Asunto(s)
Expresión Génica/efectos de los fármacos , Hormona de Crecimiento Humana/química , Hormona de Crecimiento Humana/farmacología , Factor I del Crecimiento Similar a la Insulina/genética , Línea Celular , Relación Dosis-Respuesta a Droga , Humanos , Peso Molecular , Concentración Osmolar , Isoformas de Proteínas/farmacología , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Previously, we identified and established the antigenicity of 17 CD8+ T cell epitopes from five P. falciparum Ags that are restricted by multiple common HLA class I alleles. Here, we report the identification of 11 peptides from the same Ags, cicumsporozoite protein, sporozoite surface protein 2, exported protein-1, and liver-stage Ag-1, that bind between at least five and up to 11 different HLA-DR molecules representative of the most common HLA-DR Ags worldwide. These peptides recall lymphoproliferative and cytokine responses in immune individuals experimentally immunized with radiation-attenuated Plasmodium falciparum sporozoites (irradiated sporozoites) or semi-immune individuals naturally exposed to malaria in Irian Jaya or Kenya. We establish that all peptides are recognized by individuals of each of the three populations, and that the frequency and magnitude of helper T lymphocyte responses to each peptide is influenced by the intensity of exposure to P. falciparum sporozoites. Mean frequencies of lymphoproliferative responses are 53.2% (irradiated sporozoites) vs 22.4% (Kenyan) vs 5.8% (Javanese), and mean frequencies of IFN-gamma responses are 66.3% (irradiated sporozoites) vs 27.3% (Kenyan) vs 8. 7% (Javanese). The identification of HLA class II degenerate T cell epitopes from P. falciparum validates our predictive strategy in a biologically relevant system and supports the potential for developing a broadly efficacious epitope-based vaccine against malaria focused on a limited number of peptide specificities.