摘要:

Recent progress in understanding the luminal biochemistry of regulated pancreatic exocrine secretion, including acid-base interactions between acinar and duct cells and pH-dependent processes that regulate membrane trafficking (endocytosis) at the apical plasma membrane, have led to the development of in vitro models of cystic fibrosis in the rat exocrine pancreas. Based on investigations in these model systems, a unifying hypothesis is presented that proposes that pancreatic dysfunction in cystic fibrosis occurs as a result of progressive acidification of the acinar and duct lumen, which leads to secondary defects in (i) apical trafficking of zymogen granule membranes and (ii) solubilization of secretory (pro)enzymes. By directly acidifying the pH of the acinar lumen in cholescystokinin-stimulated acini, the early cytological findings observed in cystic fibrosis, including (i) massive dilatation of the acinar lumen, (ii) decreased appearance of zymogen granules, (iii) loss of the apical pole of the acinar cell, and (iv) persistent aggregation of secretory (pro)enzymes released into the luminal space, have been reproduced in primary cultures of pancreatic tissue.

ISSN:0885-3177    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

Pancreatic adenocarcinoma involves activation of the Ki-rasoncogene, inactivation of the p53 tumor suppressor gene, and dysregulation of growth factors and perhaps metastasis genes. Ki-rasoncogene point mutations are known to be involved in pancreatic oncogenesis. The p53 tumor suppressor gene product plays a critical role in cell cycle regulation and also functions as a nuclear transcription factor. Point mutations in the p53 gene have been observed in a variety of malignancies. We determined the frequency of p53 protein overexpression and p53 point mutations in the conserved and nonconserved domains in pancreatic cancers as well as the coincidence of Ki-rasmutation in pancreatic ductal adenocarcinoma. Genomic DNA was isolated from 20 frozen pancreatic adenocarcinomas (14 primary, six metastases) along with six specimens of control pancreatic tissue and screened by single-strand conformation polymorphism (SSCP) analysis followed by direct genomic sequencing of SSCP variants. SSCP analysis was accomplished by incorporating32P-dCTP in 12 separate polymerase chain (PCR) amplifications covering the p53 coding exons 2–11. All mobility shifts on SSCP were subjected to direct genomic sequencing by the modified dideoxy method. Immuno-peroxidase (IP) staining was also done with a p53 monoclonal antibody. Ki-rascodon 12 mutational analysis was accomplished by incorporating32P-dCTP by polymerase chain reaction amplification utilizing mismatched primers, which create aBstN1 restriction endonuclease site spanning codon 12; the products were digested byBstN1 Polyacrylamide gel electrophoresis allowed distinction between wild-type and mutant Ki-ras. p53 mutations were found in 5 of 20 pancreatic cancers (three of 14 primary tumors, two of six metastatic tumors). Point mutations were observed in three of 14 primary tumors, and one of six metastases, while a 2-base pair duplication resulting in a premature stop codon in exon 5 was found in one metastatic tumor. Point mutations were noted in conserved domains (exons 4, 5, 8) and in the nonconserved domain (exon 10). IP staining revealed that eight of 14 of the primary tumors and two of six metastases exhibited moderate to strong nuclear staining (>30%), while no nuclear staining was evident in the controls. Ki-rascodon 12 mutations were found in 14 of 20 (70%) pancreatic cancers (nine of 14 primary tumors, five of six metastatic tumors) and none of the six controls. Fifty percent of the primary pancreatic tumors demonstrated moderate to strong nuclear staining. Extensive genetic analysis demonstrated mutations in 30% of the pancreatic cancers. One cancer had a nonsense mutation not detected by IP. Seven of 19 (37%) pancreatic cancers exhibited both Ki-raspoint mutation and p53 protein overexpression or mutation. Both genetic analysis and IP are required to characterize all p53 mutations in pancreatic cancer. Ki-rascodon 12 mutations and p53 protein overexpression are important steps in pancreatic oncogenesis.

ISSN:0885-3177    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

The present study was undertaken to detect K-raspoint mutations at codon 12 in pure pancreatic juice (PPJ) for the diagnosis of pancreatic cancer (PC) using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. PPJ was collected through a cannula under a duodenal fiberscope from 26 patients with PC and 32 patients with chronic pancreatitis (CP). DNA was extracted from PPJ and was used as the template for PCR. Analysis of PPJ by PCR-RFLP withBstNI revealed that the incidence of K-raspoint mutations at codon 12 was 81% (21/26) in patients with PC and 6% (2/32) in those with CP. With reference to the location of PC, the incidence of K-rasmutations was 79% (11/14) in the head, 86% (6/7) in the body, and 80% (4/5) in the tail of the pancreas. The incidence of K-rasmutants was 50% (1/2) in tumor size 1 (TS1; ≤2.0 cm in size), 71% (5/7) in TS2(2.1 to ≤4.0 cm), 89% (8/9) in TS3(4.1 to ≤6.0 cm), and 88% (7/8) in TS4(>6.1 cm). These results suggested that analysis of K-raspoint mutations at codon 12 in PPJ using the PCR-RFLP method is a promising new genetic test for the diagnosis of PC.

ISSN:0885-3177    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

The biochemical and pharmacological characteristics of specific binding sites for gastrin-releasing peptide (GRP) were investigated in normal exocrine pancreas and in an azaserine-induced pancreatic carcinoma in the rat, under similar experimental conditions. Cells from both types of tissues contained rapid, reversible, ternperature-dependent, and highly specific binding sites for GRP. Scatchard analysis of equilibrium data obtained with normal and tumor plasma membranes indicated a single class of high-affinity sites (KD= 0.42 ± 0.06 and 0.35 ± 0.05 nM, respectively), but the number of GRP receptors was significantly different (Bmax= 31 ± 4.5 and 189 ± 20 fmol/mg protein, respectively). Binding of125I-GRPl–27 was sensitive to GTP analogues, suggesting that the GRP receptor is functionally linked to a guanyl regulatory protein; however, the wheat germ agglutinin-agarose purified receptor had lost this G-protein activity. Cross-linking of125I-GRP1–27 either to normal and neoplastic cells or to crude membranes, solubilized membrane proteins, and partially purified receptors revealed the presence of a specific MW 75-kDa polypeptide. N-Glycanase treatment reduced this apparent MW to about 45 kDa. Together, these data suggest that normal and tumor pancreatic cells contain a specific GRP receptor that is expressed more on malignant pancreatic tissues.

ISSN:0885-3177    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

It is often difficult for pathologists to differentiate between benign and malignant islet cell tumors solely by histopathological criteria. The unequivocal evidence of malignancy is still the presence of metastases since cellular morphology or pleomorphism does not often render the biological clues of malignancy. Recently, nuclear DNA patterns have been reported for islet cell tumors using flow cytometry and image analysis to differentiate between benign and malignant tumors, yet conclusions are still controversial. With 28 surgically resected islet cell tumors, including 20 primary and eight metastatic tumors, nuclear DNA ploidy was analyzed and compared for the S-phase by image analysis and immunocytochemical staining for proliferating cell nuclear antigen (PCNA). Five additional autopsy cases were included for image analysis study. Small insulinomas (<5 cm) were all benign, whereas gastrinomas were more frequently malignant. One-half of all islet cell tumors (14 of 28) were diploid and S-phase values by image analysis did not correspond with the comparable data obtained by PCNA staining. With cumulative information on tumor size, hormone secretion, mitotic figures, and PCNA positivity, islet cell tumors can be classified as benign or malignant with confidence.

ISSN:0885-3177    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

The mechanisms underlying the insulinotropic action of gastrin releasing peptide (GRP) were examined in normal mouse islets. GRP (100 nM) enhanced insulin secretion at glucose concentrations of ≥11.1 mM(p< 0.05) but only in the presence of extracellular Ca2+. The insulinotropic effect of the peptide studied during perifusion at 16.7 mMglucose was transient and vanished in time. GRP stimulated, transiently45Ca2+efflux from45Ca2+-prelabeled islets, both in the presence and in the absence of extracellular Ca2+(p< 0.05), suggesting that GRP releases Ca2+from intracellular stores. Similarly, GRP increased86Rb+efflux from86Rb+-prelabeled islets both in the presence and in the absence of extracellular Ca2+(p <0.001). In contrast to GRP-induced insulin secretion, the GRP-induced86Rb+efflux was sustained throughout the stimulation period, suggesting that increased K+conductance may be involved in the vanishing effect of GRP on insulin secretion. Furthermore, both inhibition of protein kinase C (PKC) by staurosporine (1–10 μM) and down-regulation of PKC activity by long-term incubation with the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate inhibited GRP-stimulated insulin secretion (p< 0.05). These results indicate that GRP activates PKC by an action involving liberation of Ca2+from Ca2+stores. Therefore, also the influence of GRP on phosphoinositide hydrolysis was studied by means of3H efflux from myo-[2-3H]inositol prelabeled islets. However, GRP did not stimulate the3H efflux. In contrast, GRP-stimulated insulin secretion was abolished by an inhibitor of phospholipase D, wortmannin (1 μM). The results suggest that GRP transiently potentiates glucose-induced insulin secretion by an action mediated by PKC activated by diacylglycerol formed through activation of phospholipase D. Simultaneously, an as yet unknown mechanism liberating Ca2+from intracellular stores is activated.

ISSN:0885-3177    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

To investigate the role of insulin in the potentiation effect of secretin and cholecystokinin (CCK) on pancreatic exocrine secretion, the pancreas was isolated from rats and perfused with modified Krebs-Henseleit solution containing glucose at three concentrations. Intraarterial glucose at concentrations of 2.5, 10, and 25 mMproduced modest but significant increases in both the pancreatic flow rate and the amylase output in a concentration-dependent manner. The mixture of secretin and CCK at concentrations of 18.5 and 14 pM, respectively, added to the glucose solutions augmented the pancreatic flow rate and amylase output in relation to the glucose concentration. In the streptozotocin-treated pancreas, the mixture of secretin and CCK failed to augment the pancreatic exocrine secretion unless exogenous insulin was added to the perfusate. Secretin markedly potentiated the CCK-induced amylase output when insulin was present in the circulation. However, CCK did not potentiate the secretin-induced flow rate even if insulin was present in the circulation. Insulin did not affect the actions of secretin alone but it potentiated the actions of CCK alone in both the pancreatic flow rate and the amylase output. It is concluded from the above results that insulin intensifies the combined actions of secretin and CCK in pancreatic exocrine secretion by potentiating the CCK action. Furthermore, in the presence of insulin, secretin is able to potentiate the pancreatic enzyme secretion stimulated by CCK.

ISSN:0885-3177    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

The amino acid consumption test (AACT) has been proposed as a simple tubeless test of pancreatic function, but studies of its diagnostic accuracy have produced conflicting results. Eighty-three consecutive patients with clinical suspicion of pancreatic disease under-went pancreatic stimulation for 1 h with an intravenous infusion of cerulein (50 ng/kg/h); the total plasma amino acid concentration was measured at — 15, 0, 30, 45, and 60 min of infusion. The maximal percentage decrease in plasma amino acid concentration during cerulein infusion was taken as an index of pancreatic function. In addition, patients had pancreatic function assessed with the pan-creolauryl test (PLT). Of the 83 patients studied, 24 were found to have chronic pancreatitis and four pancreatic cancer; the remaining 55 had various nonpancreatic digestive disorders. Pancreatic function, as assessed by the PLT, was impaired in 22 of the 28 patients with pancreatic diseases, and it was normal in all but four patients with nonpancreatic disorders. Cerulein infusion caused a decrease in total plasma amino acid concentration that was generally more pronounced in patients with nonpancreatic diseases (maximal percentage decrease: median, 14%; range, 4–28%) than in those with pancreatic diseases (maximal percentage decrease: median, 9%; range, 0–21%) (p< 0.001). Using a cutoff of a 14% amino acid decrease, the sensitivity of the AACT was 89% and the specificity 53% (diagnostic accuracy, 65%); with a cutoff of 12%, the sensitivity was 75% and the specificity 69% (diagnostic accuracy, 71%). The sensitivity of the PLT was 79% and the specificity 93% (diagnostic accuracy, 88%). The results indicate that the sensitivity of the AACT is relatively high, but they show that the specificity is low, making the test unsuitable for clinical use, at least in its present form.

ISSN:0885-3177    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

Nitric oxide (NO) has been shown to play a significant role in inflammation. To clarify the role of NO in acute pancreatitis, we investigated the serum concentrations of NOx(NO2−plus NO3−) and tumor necrosis factor-α (TNF-α) and the grade of pancreatitis in cerulein-induced pancreatitis in mice pretreated with lipopolysac-charide (LPS) or not. LPS pretreatment aggravated the cerulein pancreatitis in association with a transient increase in serum TNF-α, which was followed by a gradual elevation of serum NOx. This elevation of serum NOxconcentration was inhibited by the NO synthase inhibitorNG-nitro-L-arginine (L-NNA). In addition, the activity of NADPH-diaphorase (NADPH-d), a marker for NO synthase, appeared in the peritoneal macrophages of LPS-pretreated mice after the induction of pancreatitis. No elevation of serum NOxor appearance of NADPH-d activity in peritoneal cells was found in mice without LPS pretreatment. Administration of L-NNA enhanced the elevation of pancreatitis-induced serum amylase in mice untreated with LPS, while L-NNA inhibited the elevation in LPS-pretreated mice. The effects of L-NNA were reversed by the administration of L-arginine but were not affected by D-arginine. These results suggested that (a) inflammatory cells may not be fully activated to produce excessive NO in uncomplicated edematous pancreatitis, and (b) edematous pancreatitis may be aggravated by excessively produced NO if bacterial infection is complicated and inflammatory cells are activated to express in-ducible NO synthase.

ISSN:0885-3177    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

Serum levels of human hepatocyte growth factor (HGF) were determined in 38 patients with acute pancreatitis by an enzyme-linked immunosorbent assay. The mean value of serum HGF levels on admission in the 38 patients was 1.69 ± 0.40 (SEM) ng/ml. In 35 patients, serum HGF levels were found to be positive (>0.39 ng/ ml), with an incidence of 92.1%. In 17 patients, they were >1.0 ng/ml, which was the cutoff value for fulminant hepatic failure. Serum HGF levels in the patients with severe acute pancreatitis (2.30 ± 0.61 ng/ml; mean ± SEM) were significantly higher than those in the patients with mild and moderate acute pancreatitis (0.63 ± 0.06 ng/ml). Sixteen of seventeen patients whose serum HGF levels were >1.0 ng/ml were evaluated as severe acute pancreatitis. Serum HGF levels were significantly elevated in the patients with higher Ranson scores, higher APACHE II scores, or higher computed tomography grades. Serum HGF levels in the patients with organ dysfunction (liver, kidney, or lung) were significantly higher than those in the patients without organ dysfunction. Moreover, serum HGF levels on admission in the nonsurvivors (3.17 ± 1.30 ng/ml) were significantly higher than those in the survivors (1.22 ± 0.33 ng/ml). The mortality rate of the patients showing serum HGF levels >2.0 ng/ml on admission was 50%. In the patients with a lethal outcome, the mean serum HGF level remained constantly >2.50 ng/ml during hospitalization. The serum HGF level reflected the clinical course of the disease rapidly and distinctly. Serum HGF levels increased with complications such as organ failure, infected pancreatic necrosis, and sepsis and decreased with successful intensive and surgical treatments. These results suggest that serum human HGF levels may reflect the severity, organ dysfunction, and prognosis in acute pancreatitis.

ISSN:0885-3177    

出版商:OVID    

年代:1996

数据来源: OVID