RESUMEN

OBJECTIVE: To examine the possible significance of homo- or heterozygous alpha-1-antitrypsin deficiency in the pathogenesis of aortic aneurysms (AA). DESIGN: Prospective investigation. SETTING: University hospital. PATIENTS: 300 controls representing the general population in our region of Southern Germany and 126 patients with aneurysmectomy and graft insertion. METHODS: The alpha-1-antitrypsin phenotype was determined by employing isoelectric focusing. Each patient was also evaluated for hypertension, lipometabolic dysfunction, smoking, hyperuricemia, and diabetes mellitus. MAIN OUTCOME MEASURES: The frequency and distribution of alpha-1-antitrypsin phenotypes and risk factors. RESULTS: 115 of 126 patients presented with one or several of the conventional risk factors: hypertension (61.5%), lipometabolic dysfunction (36.9%), smoking (58.4%), hyperuricemia (13.8%), or diabetes mellitus (6.9%). The following frequencies of alpha-1-antitrypsin phenotypes were determined: PiMM (82.5%), PiMV (4.7%), PiML (1.5%), PiMS (7.1%), PiSS (0.7%), PiMZ (3.0%). Indeed, when compared to the general population (control group) the percentage of the normal PiMM phenotypes was lower in the group of patients with AA (p<0.001). However, in our study this significant difference was not primarily due to the presence of patients homozygous or heterozygous for deficiency alleles PiMS, PiSS and PiMZ (p=0.0523) as has been previously reported, but rather to the high prevalence of the variants PiMV (p<0.005). CONCLUSIONS: Our study suggests that not only Pi-deficiency alleles, previously identified as being associated with AA, but also that Pi variants may play a pivotal role in the pathogenesis of AA.

Asunto(s)

Alelos , Aneurisma de la Aorta Abdominal/genética , Aneurisma de la Aorta Torácica/genética , Deficiencia de alfa 1-Antitripsina/genética , alfa 1-Antitripsina/genética , Adulto , Anciano , Anciano de 80 o más Años , Aneurisma de la Aorta Abdominal/etiología , Aneurisma de la Aorta Abdominal/cirugía , Aneurisma de la Aorta Torácica/etiología , Aneurisma de la Aorta Torácica/cirugía , Implantación de Prótesis Vascular , Electroforesis en Gel de Poliacrilamida , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Estudios Prospectivos , Factores de Riesgo , alfa 1-Antitripsina/metabolismo , Deficiencia de alfa 1-Antitripsina/complicaciones

RESUMEN

BACKGROUND AND STUDY AIMS: The flexibility required in academic endoscopy units is not provided by the available database systems. In a project involving substantial cooperation between endoscopists and computer scientists, we have developed an adaptable database, combined with a report generator embedded in the hospital's intranet. PATIENTS AND METHODS: Six workstations in different areas of the hospital were clustered with a UNIX operating system to implement multi-user capability and access control. A relational database was used to design an application appropriate to the specific needs of the endoscopy unit in a teaching hospital engaged in scientific research. Both the terminology used in standardized endoscopy nomenclature and a free text block facility were included. A graphical user interface was developed to assemble pertinent data, generate the reports, and supervise the database. RESULTS: A total of 4936 examinations including 2988 patients were entered consecutively during continuous routine operation of the system. Complete report generation required five minutes (median; range 1-9 minutes). Both structured items and free text were used in all the reports. Querying of the database was possible, concerning matters such as the need for repeated endoscopic therapy in acute gastrointestinal bleeding (4%), the search for Helicobacter pylori in appropriate patients (64%), the rate of accidental pancreatic duct visualization in endoscopic retrograde cholangiography (24%), and links between examinations and active trials (2%). Indicating improved report quality, the number and the diameter of esophageal varices in patients with varices were more frequently reported with the new report system than with previous typed reports (P<0.001). An anonymous questionnaire revealed that the readability of the computer-generated reports was better than that of the previous typewritten reports (P=0.01). CONCLUSIONS: This report describes the creation of a database application and a report generator meeting the needs of scientific and routine use, and the successful application of this system in an academic endoscopy unit.

Asunto(s)

Redes de Comunicación de Computadores , Bases de Datos como Asunto , Endoscopía Gastrointestinal , Sistemas de Registros Médicos Computarizados , Programas Informáticos

RESUMEN

A potential role of homozygous or heterozygous alpha-1-antitrypsin deficiency alleles Pi*Z or Pi*S in the pathogenesis of aortic aneurysms has been debated in recently published papers. Therefore, we have determined the alpha-1-antitrypsin phenotype in 103 patients with aortic aneurysms using isoelectric focusing. The vast majority of patients (92.2%) had one or more of the established risk factors: hypertension (65.0%), lipometabolic dysfunction (34.9%), smoking (65.0%), hyperuricemia (16.5%) or diabetes mellitus (8.7%). In our patients, the deficiency alleles Pi*Z and Pi*S were more frequent than in the general population of our region, but these differences did not reach statistical significance (PiMS 6.7 versus 3.4%, PiMZ 3.8 versus 2.5%, PiSS 0,9 versus 0.2%). Furthermore, the patients with heterozygous or homozygous antitrypsin deficiency had similar patterns of risk factors to those of the patients with normal phenotypes. In one patient we found a heterozygous PiMZ antitrypsin deficiency associated with Marfan's syndrome. These data do not support the results of recently published studies of fewer cases that suggest a higher prevalence of antitrypsin deficiency alleles in patients with aortic aneurysms. A heterozygous alpha-1-antitrypsin deficiency as a cause or predisposing factor for the development of aortic aneurysms appears to be of little or no importance.

Asunto(s)

Aneurisma de la Aorta/genética , Heterocigoto , Deficiencia de alfa 1-Antitripsina , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Aneurisma de la Aorta/cirugía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Factores de Riesgo , alfa 1-Antitripsina/genética

RESUMEN

Dihydrolipoamide acyltransferase (E2), a catalytic and structural component of the three functional classes of multienzyme complexes that catalyze the oxidative decarboxylation of alpha-keto acids, forms the central core to which the other components attach. We have determined the structures of the truncated 60-mer core dihydrolipoamide acetyltransferase (tE2) of the Saccharomyces cerevisiae pyruvate dehydrogenase complex and complexes of the tE2 core associated with a truncated binding protein (tBP), intact binding protein (BP), and the BP associated with its dihydrolipoamide dehydrogenase (BP.E3). The tE2 core is a pentagonal dodecahedron consisting of 20 cone-shaped trimers interconnected by 30 bridges. Previous studies have given rise to the generally accepted belief that the other components are bound on the outside of the E2 scaffold. However, this investigation shows that the 12 large openings in the tE2 core permit the entrance of tBP, BP, and BP.E3 into a large central cavity where the BP component apparently binds near the tip of the tE2 trimer. The bone-shaped E3 molecule is anchored inside the central cavity through its interaction with BP. One end of E3 has its catalytic site within the surface of the scaffold for interaction with other external catalytic domains. Though tE2 has 60 potential binding sites, it binds only about 30 copies of tBP, 15 of BP, and 12 of BP.E3. Thus, E2 is unusual in that the stoichiometry and arrangement of the tBP, BP, and E3.BP components are determined by the geometric constraints of the underlying scaffold.

Asunto(s)

Complejo Piruvato Deshidrogenasa/ultraestructura , Saccharomyces cerevisiae/enzimología , Técnica de Fractura por Congelación , Modelos Moleculares , Conformación Proteica

RESUMEN

BACKGROUND: Although it has been shown that chronic administration of ursodeoxycholic acid increases gallbladder fasting and residual volume, it is unknown whether ursodeoxycholic acid exerts an acute effect on gallbladder volume. We therefore evaluated the effect of a single oral dose of ursodeoxycholic acid on gallbladder volume in healthy volunteers. METHODS: After the volunteers had fasted overnight, gallbladder volume was measured sonographically every 15 min for 5 h. Following a 1-h control period group I (n = 8) received ursodeoxycholic acid (1000 mg) orally with 100 ml of water, whereas group II (n = 8) received 100 ml of water (placebo) only. Gallbladder volumes were calculated, applying the sum-of-cylinders method. Serum levels of ursodeoxycholic acid were determined by gas chromatography at 1-h intervals. RESULTS: Gallbladder fasting volumes before ursodeoxycholic acid were similar in both groups (24.0 +/- 2.3 ml versus 25.4 +/- 3.3 ml; NS). After ingestion of ursodeoxycholic acid (group I) gallbladder volume increased rapidly, reaching 27.6 +/- 3.1 ml (p < 0.04) 1 h and 38.4 +/- 3.4 ml (p < 0.02) 4 h after ingestion of ursodeoxycholic acid. The individual gallbladder volumes after ingestion of ursodeoxycholic acid in group I increased to 146%-211% of pretreatment values. Ursodeoxycholic acid serum levels increased from 0.94 +/- 0.38 mumol/l to 10.51 +/- 1.36 mumol/l (p < 0.001) and correlated closely with gallbladder volumes (r = 0.80; p < 0.05). After ingestion of water only (group II) gallbladder volume decreased transiently from 15 min to 30 min after water intake and then remained at pretreatment values throughout the study period. CONCLUSION: Administration of a single oral dose of ursodeoxycholic acid causes a rapid increase in gallbladder volume, which reaches 163 +/- 10% of pretreatment volume at 4 h and is closely correlated with ursodeoxycholic acid serum levels.

Asunto(s)

Colagogos y Coleréticos/farmacología , Vesícula Biliar/efectos de los fármacos , Ácido Ursodesoxicólico/farmacología , Adulto , Colagogos y Coleréticos/sangre , Femenino , Vesícula Biliar/diagnóstico por imagen , Vaciamiento Vesicular/efectos de los fármacos , Humanos , Masculino , Factores de Tiempo , Ultrasonografía , Ácido Ursodesoxicólico/sangre

RESUMEN

Inhibitors of the HMG-CoA reductase have been shown to further reduce the biliary cholesterol saturation in patients treated with oral bile acids for cholesterol gallbladder stones. It was the aim of our study to evaluate the efficacy of simvastatin in addition to ursodeoxycholic acid in the dissolution of gallstone fragments after shock wave lithotripsy and adjuvant bile acid dissolution therapy. Eighteen patients with a single radiolucent gallbladder stone and a serum cholesterol of more than 250 mg/dl were randomly assigned to receive either ursodeoxycholic acid alone (750 mg per day, group A, n = 9) or in combination with simvastatin (20 mg per day, group B, n = 9) for the dissolution of the gallstone fragments generated by extracorporeal shock wave lithotripsy. The two groups were well matched regarding their baseline characteristics. At the primary end point of the study 6 months after lithotripsy, there was no difference between the groups in the rate of gallstone disappearance with 4 of 9 patients being stone free in each group. As evaluated by life table analysis, even further follow-up showed no significant difference between the groups (P = 0.8). In group B, serum cholesterol levels decreased by 22% at 3 months (P = 0.01 vs. baseline) and by 24% at six months (P = 0.02) during treatment while no significant change was observed in group A. With both regiments, no adverse effects were observed. While simvastatin added to ursodeoxycholic acid resulted in a decrease of elevated serum cholesterol levels in gallstone patients, it did not enhance stone disappearance after shock wave lithotripsy and adjuvant bile acid dissolution therapy.

Asunto(s)

Anticolesterolemiantes/administración & dosificación , Colagogos y Coleréticos/administración & dosificación , Colelitiasis/terapia , Litotricia , Lovastatina/análogos & derivados , Ácido Ursodesoxicólico/administración & dosificación , Anticolesterolemiantes/efectos adversos , Colagogos y Coleréticos/efectos adversos , Colesterol/sangre , Terapia Combinada , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Humanos , Lovastatina/administración & dosificación , Lovastatina/efectos adversos , Simvastatina , Ácido Ursodesoxicólico/efectos adversos

RESUMEN

To determine the rate and characteristics of gallstone recurrence after direct contact dissolution with methyl tert-butyl ether, 60 consecutive patients were followed for up to 4.5 years (median 2.2 years) after complete disappearance of all stone residues and debris and cessation of adjuvant bile acid therapy. Initial gallstones had been multiple in all but four patients. Twenty-eight of the 60 patients developed recurrent gallstones. The cumulative risk of gallstone recurrence (actuarial analysis) was 23 +/- 6%, 34 +/- 7%, 55 +/- 8%, and 70 +/- 9% at one, two, three, and four years, respectively. The recurrent stones were usually multiple and small (6 +/- 4 mm). Gallstone recurrence was associated with recurrent biliary pain in two patients, one of whom developed acute cholecystitis. Recurrent stones were cleared completely by bile acid medication with or without shock-wave lithotripsy in 61 +/- 15% of patients at one year (actuarial analysis). In conclusion, gallstone recurrence after successful contact dissolution of multiple stones with methyl tert-butyl ether has to be expected in a high percentage of patients. Most patients, however, remain free of biliary pain during long-term follow-up.

Asunto(s)

Colelitiasis/terapia , Éteres/uso terapéutico , Éteres Metílicos , Análisis Actuarial , Estudios de Cohortes , Éteres/administración & dosificación , Femenino , Humanos , Litotricia , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Recurrencia , Factores de Riesgo , Solventes/administración & dosificación

RESUMEN

Human herpesvirus-6 is a lymphotropic virus which infects susceptible individuals during the first year of life and usually causes life-long latency. In a variable percentage primary infections are followed by a short acute disease, exanthema subitum. Older individuals may suffer from infectious mononucleosis-like illnesses or from Kikuchi-Fujimoto's disease. In addition, there is a fairly wide spectrum of lymphoid and hematopoietic diseases or autoimmune disorders, which are associated with elevated titers of HHV-6 antibody, and from which replicating virus may be isolated. Such diseases include atypical polyclonal lymphoproliferation, Hodgkin's disease, chronic fatigue syndrome and systemic lupus erythematosus. The present article reviews the current knowledge of such associations.

Asunto(s)

Enfermedades Autoinmunes/virología , Síndrome de Fatiga Crónica/virología , Enfermedades Hematológicas/virología , Infecciones por Herpesviridae , Herpesvirus Humano 6/patogenicidad , Trastornos Linfoproliferativos/virología , Adolescente , Adulto , Anticuerpos Antivirales/sangre , Niño , ADN Viral/genética , Exantema Súbito/virología , Síndrome de Fatiga Crónica/epidemiología , Femenino , Alemania/epidemiología , Infecciones por Herpesviridae/epidemiología , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/patología , Infecciones por Herpesviridae/terapia , Herpesvirus Humano 6/inmunología , Herpesvirus Humano 6/aislamiento & purificación , Humanos , Lactante , Recién Nacido , Japón/epidemiología , Ganglios Linfáticos/patología , Trastornos Linfoproliferativos/epidemiología , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias/virología , Trasplante , Latencia del Virus

RESUMEN

After previous serological screening for Epstein-Barr virus (EBV), human herpesvirus-6 (HHV-6) and human cytomegalovirus (HCMV) showed elevated antibody titers against EBV and HHV-6 in more than 50% of patients with myelodysplasia and chronic myeloproliferative diseases, the present study was carried out in order to investigate viral antigen expression and distribution in bone marrow cells of these patients. Trephine biopsies were studied from 60 patients with myelodysplasia (MDS), 36 patients with chronic myelogenous leukemia (CML) and 18 patients with osteomyelofibrosis (PMF). Elevated anti-EBV EA titers were found in 62% of the MDS cases, in 33% of the CMLs and in 62% of the OMF patients. HHV-6 titers were elevated in 18% of the MDS cases, but in only one case each of CML and OMF. Antigen expression in bone marrow cells was even more frequent: EBV-EA was 76% in MDS cases, 77% in CML and 40% in OMF. HHV-6 p41 was observed in 47% of the MDS cases, in 54% of the CML cases and in 20% of the OMFs. In comparing these data with those from the literature and with our own studies in Hodgkin's disease, it is hypothesized that the reactivated herpesviruses may contribute to the pathogenesis of these hematopoietic disorders by interfering with the cytokine regulation of cell proliferation and differentiation.

Asunto(s)

Médula Ósea/virología , Infecciones por Herpesviridae/virología , Herpesvirus Humano 4/aislamiento & purificación , Herpesvirus Humano 6/aislamiento & purificación , Síndromes Mielodisplásicos/virología , Trastornos Mieloproliferativos/virología , Infecciones Tumorales por Virus/virología , Anticuerpos Antivirales/sangre , Antígenos Virales/análisis , Médula Ósea/patología , Cocarcinogénesis , Citocinas/fisiología , Células Madre Hematopoyéticas/virología , Infecciones por Herpesviridae/patología , Herpesvirus Humano 4/crecimiento & desarrollo , Herpesvirus Humano 4/inmunología , Herpesvirus Humano 4/patogenicidad , Herpesvirus Humano 6/crecimiento & desarrollo , Herpesvirus Humano 6/inmunología , Herpesvirus Humano 6/patogenicidad , Humanos , Modelos Biológicos , Síndromes Mielodisplásicos/sangre , Síndromes Mielodisplásicos/inmunología , Síndromes Mielodisplásicos/patología , Trastornos Mieloproliferativos/sangre , Trastornos Mieloproliferativos/inmunología , Trastornos Mieloproliferativos/patología , Infecciones Tumorales por Virus/patología , Activación Viral

RESUMEN

Fifty patients with various hyperplastic and malignant lymphoproliferative diseases were investigated for evidence of human herpesvirus-6 infection. Virus DNA and antigen expression was investigated in lymph node biopsies by in situ hybridization and immunohistology and was correlated with data of immunophenotyping. Supplemental immunoglobulin- and T cell receptor gene rearrangement studies were used to support the classification of the proliferative lymphoid lesion. Elevated numbers of cells carrying HHV-6 DNA and/or antigens were found in cases of Hodgkin's lymphoma and follicular center cell non-Hodgkin's lymphoma as well as atypical polyclonal lymphoproliferation (APL), yet not in reactive lymphoid hyperplasia and in most other lymphomas. Immunophenotyping showed that virus -infected cells were primarily lympho-histiocytic elements, less frequently Hodgkin's- and Reed-Sternberg cells, and not malignant B lymphocytes as in follicular center cell lymphomas. This suggests that the virus is rather not the causative oncogen in these cases, yet does not exclude a cocarcinogenic effect of it during the development and ths course of uncontrolled lymphoproliferation.

Asunto(s)

Antígenos Virales/análisis , Infecciones por Herpesviridae , Herpesvirus Humano 6/aislamiento & purificación , Hibridación in Situ , Trastornos Linfoproliferativos/virología , Cocarcinogénesis , ADN Viral/análisis , Infecciones por Herpesviridae/genética , Infecciones por Herpesviridae/patología , Herpesvirus Humano 6/genética , Herpesvirus Humano 6/inmunología , Enfermedad de Hodgkin/genética , Enfermedad de Hodgkin/patología , Enfermedad de Hodgkin/virología , Humanos , Hiperplasia , Inmunofenotipificación , Ganglios Linfáticos/patología , Ganglios Linfáticos/virología , Linfoma de Células B/genética , Linfoma de Células B/patología , Linfoma de Células B/virología , Trastornos Linfoproliferativos/genética , Trastornos Linfoproliferativos/patología , Proteínas de Neoplasias/análisis , Lesiones Precancerosas/patología , Lesiones Precancerosas/virología

RESUMEN

Non-specific interstitial pneumonia (NIP) occurs frequently in patients with HIV-infection. To elucidate the etiology of this pulmonary disorder, we searched for 13 different microorganisms in transbronchial biopsies from 15 patients with NIP, 15 patients with Pneumocystis carinii pneumonia (PCP) and 20 patients with lung diseases not related to HIV-infection using monoclonal antibodies and the APAAP- or PAP-technique for immunostaining. Chlamydia trachomatis and parainfluenza III were detected frequently and in great number. Adenovirus, influenza B, varicella zoster and cytomegalovirus were also found frequently, but not in great number. Measles virus, respiratory syncytial virus, influenza A and herpesviruses 1&2 were not found. Also not found were parainfluenza I, mycoplasma pneumoniae and coronavirus. In seven out of fifteen NIP patients at least one organism was shown, compared to nine out of fifteen patients with PCP and eight out of twenty patients in the control group.

Asunto(s)

Infecciones por Chlamydia/diagnóstico , Chlamydia trachomatis , Infecciones por VIH/complicaciones , Enfermedades Pulmonares Intersticiales/etiología , Virus de la Parainfluenza 3 Humana , Infecciones por Paramyxoviridae/diagnóstico , Infecciones Oportunistas Relacionadas con el SIDA/microbiología , Infecciones Oportunistas Relacionadas con el SIDA/virología , Infecciones por Adenoviridae/diagnóstico , Adulto , Anciano , Anticuerpos Antibacterianos , Anticuerpos Monoclonales , Anticuerpos Antivirales , Biopsia , Chlamydia trachomatis/inmunología , Femenino , VIH-1 , Humanos , Enfermedades Pulmonares Intersticiales/inmunología , Enfermedades Pulmonares Intersticiales/microbiología , Enfermedades Pulmonares Intersticiales/virología , Masculino , Persona de Mediana Edad , Virus de la Parainfluenza 3 Humana/inmunología , Neumonía por Pneumocystis/inmunología , Neumonía Viral/diagnóstico

RESUMEN

BACKGROUND/AIMS: The long-term outcome of nonoperative gallstone therapy depends on both absence of stones and absence of biliary pain. The aim of the present study was to determine the rate of stone recurrence and the rate of symptoms within 5 years after successful shock wave lithotripsy combined with bile acid therapy. METHODS: One hundred consecutive patients (single stones, n = 89; 2 or 3 stones, n = 11) were followed up for a median of 4.3 years after stone disappearance and discontinuation of bile acids. RESULTS: Twenty-three of the 100 patients developed recurrent stones. Calculated by actuarial analysis, the recurrence rate was 7% +/- 3%, 11% +/- 3%, 13% +/- 4%, 20% +/- 5%, and 31% +/- 7% (mean +/- SD) at 1, 2, 3, 4, and 5 years, respectively. The recurrent stones were small (6 +/- 5 mm) and were associated with recurrent biliary pain in 14 (61%) of the 23 patients. Repeated shock wave lithotripsy and/or bile acid medication resulted in stone disappearance in only 10 of 20 patients with recurrence. CONCLUSIONS: The long-term rate of stone recurrence after lithotripsy of primarily solitary gallbladder calculi is lower than expected from post-bile acid dissolution trials. Recurrence of stones frequently is associated with recurrence of biliary pain.

Asunto(s)

Colelitiasis/terapia , Litotricia , Adulto , Anciano , Ácido Quenodesoxicólico/uso terapéutico , Colelitiasis/complicaciones , Colelitiasis/fisiopatología , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Dolor/etiología , Probabilidad , Recurrencia , Ácido Ursodesoxicólico/uso terapéutico

Asunto(s)

Dolor Abdominal/etiología , Colestasis Intrahepática/genética , Cirrosis Hepática/genética , Fenotipo , Deficiencia de alfa 1-Antitripsina , Dolor Abdominal/genética , Adulto , Biopsia , Colestasis Intrahepática/complicaciones , Colestasis Intrahepática/patología , Humanos , Cuerpos de Inclusión/ultraestructura , Hígado/patología , Cirrosis Hepática/complicaciones , Cirrosis Hepática/patología , Masculino

RESUMEN

The present study was undertaken to determine if the cholecystokinin (CCK) receptor may be phosphorylated, and to gain insight into its regulation. For this, the ATP pool of rat pancreatic acini was prelabeled with 32P, and the cells were stimulated with various secretagogues. CCK receptors from treated cells were enriched by sequential fractionation to produce plasmalemma, and subsequent solubilization and lectin-affinity chromatography. This protocol detected a phosphorylated Mr = 85,000-95,000 plasma membrane glycoprotein with features similar to the CCK receptor. Phosphorylation of this protein occurred rapidly (less than 2 min) and in a concentration-dependent manner in response to CCK, and was inhibited by the CCK receptor antagonist L-364,718. Further evidence that this represented the CCK receptor included comigration of phosphorylated and CCK radioligand affinity-labeled proteins on sodium dodecyl sulfate-polyacrylamide gels, both in native forms and after endoglycosidase F deglycosylation, and the specific adsorption of the phosphoprotein to a CCK analogue affinity resin. Phosphorylation occurred predominantly on serine residues of the receptor protein. Phosphorylation of this protein was also enhanced in response to other secretagogues which, like CCK, stimulate a cascade leading to protein kinase C activation, and in response to direct activation of this enzyme by 12-O-tetradecanoylphorbol 13-acetate. Thus, the pancreatic CCK receptor is phosphorylated in a regulated manner, in response to both homologous and heterologous secretagogues, and to protein kinase C activation.

Asunto(s)

Páncreas/metabolismo , Receptores de Colecistoquinina/metabolismo , Sincalida/farmacología , Adenosina Trifosfato/metabolismo , Animales , Carbacol/farmacología , Fraccionamiento Celular , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Cinética , Masculino , Fosforilación , Proteína Quinasa C/metabolismo , Ratas , Ratas Endogámicas , Solubilidad , Acetato de Tetradecanoilforbol/farmacología

RESUMEN

To further define the structure of the pancreatic cholecystokinin (CCK) receptor and the topographical distance relationships between its subunits, we developed a series of monofunctional photoaffinity probes in which a fixed receptor-binding domain was separated from a photolabile nitrophenylacetamido group by defined lengths of a flexible spacer. The well-characterized CCK receptor radioligand 125I-D-Tyr-Gly-[(Nle28,31)CCK-26-33] provided the receptor-binding component of the probes, while the polymer poly(ethylene glycol) (2, 4, 7, and 10 monomer units long) was used as the spacer. The patterns of affinity labeling of rat pancreatic plasma membranes were examined as a function of spacer length. This ranged from 7.3 to 16.2 A, as calculated by root-mean-square end-to-end distances and validated experimentally by time-resolved fluorescence resonance energy transfer measurements. All probes in the series specifically labeled the Mr = 85,000-95,000 glycoprotein with Mr = 42,000 core, which has been proposed to contain the hormone recognition site. In addition, when the spacer length reached 16.2 A, membrane proteins of Mr = 80,000 and Mr = 40,000 were specifically labeled. The product of endo-beta-N-acetylglucosaminidase F digestion of the Mr = 80,000 protein was Mr = 65,000, similar to a protein previously identified in affinity labeling experiments using a CCK-33-based probe. These observations are consistent with the Mr = 85,000-95,000 pancreatic protein representing the hormone-binding subunit of the CCK receptor, while proteins of Mr = 80,000 and Mr = 40,000 may represent noncovalently associated subunits sited within 16.2 A of the binding domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Marcadores de Afinidad , Polietilenglicoles , Receptores de Colecistoquinina/análisis , Acetilglucosaminidasa , Animales , Cromatografía Líquida de Alta Presión , Fluorescencia , Glicosilación , Hidrólisis , Espectroscopía de Resonancia Magnética , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa , Peso Molecular , Páncreas/química , Ratas

RESUMEN

The clinical suitability of conventional glyceromonooctanoin (GMOC) and ethylenediaminetetraacetic acid (EDTA) containing solvents for the dissolution of common bile duct stones is questionable. To improve the solvent-stone contact and the miscibility with bile, GMOC was hydrophilized by the addition of polyethyleneglycol-caprylglyceride, polyethyleneglycol-sorbitan-etheroleyl-ester, and polyethyleneglycol-sorbitanlauryl-ester (PEG-GMOC). This product was mixed with a bile acid-EDTA (BA-EDTA) solution in a ratio of 1:2 (v/v) for cholesterol solubilizing and calcium complexing capacities. To determine clinical efficiency, the new solvent was infused via a nasobiliary tube in 16 patients with endoscopically nonextractable common bile duct stones and compared with a group of 16 patients treated with an alternating GMOC/BA-EDTA regimen. Continuous perfusion with PEG-GMOC-BA-EDTA led to a total (12 patients) or partial (3 patients) disappearance of the stones within 2-15 days. Similarly, alternating GMOC and BA-EDTA treatment dissolved the stones in 12 patients. The average volume of PEG-GMOC-BA-EDTA infused contained only 27% of the GMOC applied during the alternating therapeutic regime. This reduction of the GMOC dose was associated with a significant reduction of adverse effects such as emesis, diarrhea and biliary pain. We concluded that GMOC is equally efficient in the new hydrophilized form but it is clearly superior as far as side effects are concerned. In all, this supports its clinical suitability for the dissolution treatment of common bile duct stones.

Asunto(s)

Ácidos y Sales Biliares/administración & dosificación , Ácido Edético/administración & dosificación , Cálculos Biliares/terapia , Glicéridos/administración & dosificación , Solventes , Anciano , Anciano de 80 o más Años , Caprilatos , Colangiografía , Esquema de Medicación , Emulsiones , Femenino , Cálculos Biliares/diagnóstico por imagen , Humanos , Masculino , Perfusión

RESUMEN

Cholecystokinin (CCK) receptors reside on a large number of cell types along the digestive tract and in the nervous system. A human neuroblastoma cell line (CHP212) has recently been described to express a type A receptor, with structural specificity similar to that on pancreatic acinar cells and gall bladder smooth muscle cells but different from the predominant type of binding site found in brain (type B). In this work, we have performed photoaffinity labeling and protease peptide mapping of the CHP212 receptor and have compared it to other type A CCK receptors. 125I-D-Tyr-Gly-[(Nle28,31,pNO2-Phe33)-CCK-26-33], a probe that possesses a photolabile residue at position 33 within the theoretical receptor-binding domain of this hormone, specifically labeled a Mr = 80,000-90,000 glycoprotein on this cell line, while labeling larger proteins (Mr = 85,000-95,000) on rat pancreas and human gall bladder. Deglycosylation with endo-beta-N-acetylglucosaminidase F yielded bands of Mr = 43,000 from CHP212 and gall bladder and Mr = 42,000 from pancreas. Peptide mapping of the deglycosylated bands using Staphylococcus aureus V8 protease demonstrated identical patterns in CHP212 and gall bladder and a similar but different pattern in pancreas. Thus, although possessing heterogeneity in their carbohydrate domains, CCK receptors on human neuroblastoma cells (CHP212) and human gall bladder smooth muscle cells have highly similar or identical protein cores. The core protein on another type A CCK receptor, from rat pancreas, appears to differ from these, likely representing molecular heterogeneity between species.

Asunto(s)

Neuroblastoma/metabolismo , Receptores de Colecistoquinina/análisis , Marcadores de Afinidad , Animales , Línea Celular , Vesícula Biliar/metabolismo , Glicosilación , Humanos , Proteínas de la Membrana/análisis , Páncreas/metabolismo , Péptido Hidrolasas , Mapeo Peptídico , Ratas

RESUMEN

Affinity labeling is a powerful method for biochemical characterization of hormone receptors, dependent on approximation of reactive groups on ligand and receptor. In this work, we have compared the efficiency of covalent labeling of the rat pancreatic cholecystokinin (CCK) receptor by decapeptide probes with differing photolabile moieties sited at their amino-terminus, mid-region, or carboxyl-terminus, or chemically crosslinkable via their amino-terminus. Each labeled the same M(r) = 85,000-95,000 plasma membrane glycoprotein with a protein core of M(r) = 42,000. Affinity labeling this band through the amino-terminus of the decapeptide, 125I-D-Tyr-Gly [(Nle28,31)CCK-26-33], was inefficient using bifunctional chemical reagents, m-maleimidobenzoyl-N-hydroxy-succinimide ester (0.07% of total incubated radioactivity, representing 0.4% of specifically bound counts) or disuccinimidyl suberate (0.02, 0.2%) or a photolabile carbene precursor (0.06, 0.2%). A benzophenone at this locus yielded more efficient labeling of this band (0.09, 11.8%), but high levels of nonspecific labeling. Probes attached through residues within the receptor-binding domain were particularly useful. Photolabile derivatives of phenylalanine at the carboxyl-terminus of this domain yielded better incorporation (4-nitro-Phe33: 0.23, 1.4%; 4-azido-Phe3: 0.67, 6.0%). A 6-nitro-Trp30 derivative in the middle of this domain gave similarly efficient labeling (0.08, 3.5%) despite being a less potent pancreatic secretagogue. These studies clearly demonstrate that the efficiency of covalent labeling of a receptor can be markedly affected by the nature and site of crosslinking chosen.

Asunto(s)

Marcadores de Afinidad , Reactivos de Enlaces Cruzados , Sondas Moleculares , Páncreas/metabolismo , Receptores de Colecistoquinina/análisis , Secuencia de Aminoácidos , Animales , Membrana Celular/metabolismo , Glicosilación , Masculino , Datos de Secuencia Molecular , Ratas , Ratas Sprague-Dawley

RESUMEN

Affinity-labeling probes with sites of cross-linking distributed along the ligand have been used to biochemically characterize the pancreatic cholecystokinin (CCK) receptor. Probes with photolabile sites spanning the receptor-binding domain have labeled a Mr = 85,000-95,000 plasma membrane protein, while a probe cross-linked via the amino terminus of CCK-33, far removed from the carboxyl-terminal receptor-binding domain, has labeled a distinct Mr = 80,000 protein. In this work, protease peptide mapping of the pancreatic proteins labeled by each of these probes has been performed to gain insight into the identities of the bands and to define domains of the labeled proteins. Photolabile decapeptide probes with sites of cross-linking at the amino terminus, mid region, and carboxyl terminus of the receptor-binding domain each labeled a Mr = 85,000-95,000 glycoprotein with a Mr = 42,000 core protein and similar Staphylococcus aureus V8 protease peptide maps. This confirms that each probe labels the same binding protein and the same domain of that protein. Serial slices through the broad labeled band were separately deglycosylated and protease-treated, demonstrating a single protein core with differential glycosylation. The CCK-33-based probe, however, labeled predominantly two proteins, one having similar sizes in its native and deglycosylated forms to that labeled by the decapeptide probes and a distinct Mr = 80,000 protein. Of note, the peptide map of the protein believed to be the same as that labeled by the shorter probes was different, suggesting that this probe labeled the binding subunit at a site distinct from that which was labeled by the short probes.

Asunto(s)

Marcadores de Afinidad/metabolismo , Páncreas/metabolismo , Receptores de Colecistoquinina/aislamiento & purificación , Marcadores de Afinidad/síntesis química , Animales , Membrana Celular/metabolismo , Masculino , Peso Molecular , Fragmentos de Péptidos/aislamiento & purificación , Mapeo Peptídico , Ratas , Ratas Endogámicas , Receptores de Colecistoquinina/metabolismo , Serina Endopeptidasas

RESUMEN

We report the preparation and characterization of a new type of intrinsic photoaffinity labeling probe, on the basis of the incorporation of a photolabile nitrotryptophan into a biologically relevant domain of a peptide. The model system used was the pancreatic cholecystokinin (CCK) receptor, previously affinity labeled with a variety of probes. Those studies have suggested that an Mr = 85,000-95,000 protein is more likely to be labeled as the site of covalent attachment approaches the receptor-binding domain of this hormone. Indeed, CCK has a Trp in the center of its receptor-binding region, and replacement of that residue with 6-nitrotryptophan resulted in a photolabile probe which affinity labeled the same Mr = 85,000-95,000 pancreatic membrane protein. This probe, 125I-D-Tyr-Gly-[(Nle28,31,6-NO2-Trp30)CCK-26-33], was synthesized by solid-phase and solution techniques and characterized by mass spectrometry. Following oxidative iodination, it was purified on HPLC to 2000 Ci/mmol. Binding to pancreatic membranes was rapid, temperature dependent, reversible, saturable, and specific and was with high affinity (Kd = 3 nM). While its binding affinity was only 3-fold lower than that of native CCK-8, this probe was 70-fold less potent than native hormone in stimulating amylase secretion (EC50 = 1 nM) and equally efficacious to native hormone. Despite the slight decrease in affinity, this probe demonstrated a high relative efficiency of covalent labeling of the Mr = 85,000-95,000 protein. This confirms that the Mr = 85,000-95,000 protein represents the hormone-binding subunit of the CCK receptor and demonstrates the utility of this type of photoaffinity labeling probe.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Páncreas/metabolismo , Receptores de Colecistoquinina/metabolismo , Marcadores de Afinidad , Animales , Sitios de Unión , Unión Competitiva , Colecistoquinina/metabolismo , Técnicas In Vitro , Masculino , Proteínas de la Membrana/metabolismo , Peso Molecular , Ratas , Triptófano/análogos & derivados