Abstract: Success in islet transplantation-based therapies for type 1 diabetes mellitus and an extreme shortage of pancreatic islets have motivated recent efforts to develop renewable sources of islet-replacement tissue. Although pancreatic progenitor cells hold a promising potential, only a few attempts have been made at the prospective isolation of pancreatic stem/progenitor cells, because of the lack of specific markers and the development of effective cell culture methods. We found that prominin1 (also known as CD133) recognized the undifferentiated epithelial cells, whereas platelet-derived growth factor receptor beta (PDGFRbeta) was expressed on the mesenchymal cells in the mouse embryonic pancreas. We then developed an isolation method for putative stem/progenitor cells by flow cytometric cell sorting and characterized their potential for differentiation to pancreatic tissue using both in vitro and in vivo protocols. Flow cytometry and the subsequent reverse transcription-polymerase chain reaction and microarray analysis revealed pancreatic epithelial progenitor cells to be highly enriched in the prominin1(high)PDGFRbeta(-) cell population. During in vivo differentiation, these cell populations were able to differentiate into endocrine, exocrine, and ductal tissues, including the formation of an insulin-producing cell cluster. We established the prospective isolation of putative pancreatic epithelial progenitor cells by sorting for prominin1 and PDGFRbeta. Since this strategy is based on the cell surface markers common to human and rodents, these findings may lead to the development of new strategies to derive transplantable islet-replacement tissues from human pancreatic stem/progenitor cells. Disclosure of potential conflicts of interest is found at the end of this article.
Abstract: Nodal signaling induces the formation of the endoderm and mesoderm during gastrulation. Nodal expression persists until the definitive endoderm progenitor has completely formed, and disappears thereafter. A tightly regulated Nodal expression system is essential for the differentiation of embryonic stem (ES) cells into distinct tissue lineages. On this basis, we established an ES cell differentiation system with the tetracycline-regulated expression of Nodal. The upregulated Nodal signaling pathway and its downstream transcriptional targets induced the specification of ES cells into definitive endoderm and mesoderm derivatives, and the subsequent downregulation of Nodal signaling promoted further maturation of the gut tube both in vitro and in vivo. Sustained expression of the Nodal gene inhibited the maturation of the definitive endoderm owing to persistent Oct3 and/or Oct4 expression and teratoma formation. Furthermore, quantitative single cell analysis by flow cytometry using CXCR4, VEGFR2 and PDGFR-alpha indicated that this protocol for definitive endoderm and mesoderm differentiation is superior to any other available protocol. Our findings also indicated that the Nodal or Nodal-related molecules secreted from Nodal-expressing ES cells could cause genetically unmanipulated ES cells to induce the expression of the Nodal signaling pathway and its downstream targets, which consequently leads to the differentiation of the ES cells into definitive endoderm and mesoderm. Our differentiation system, using tightly regulated Nodal expression, enabled us to investigate the mechanism of ES cell differentiation into definitive endoderm or mesoderm derivatives. Our findings also demonstrate that Nodal-expressing ES cells might be a source of highly active proteins that could be used for developing endoderm or mesoderm tissues in regenerative medicine.
Abstract: BACKGROUND: Success in islet-transplantation-based therapies for type 1 diabetes, coupled with a worldwide shortage of transplant-ready islets, has motivated efforts to develop renewable sources of islet-replacement tissue. Islets and neurons share features, including common developmental programs, and in some species brain neurons are the principal source of systemic insulin. METHODS AND FINDINGS: Here we show that brain-derived human neural progenitor cells, exposed to a series of signals that regulate in vivo pancreatic islet development, form clusters of glucose-responsive insulin-producing cells (IPCs). During in vitro differentiation of neural progenitor cells with this novel method, genes encoding essential known in vivo regulators of pancreatic islet development were expressed. Following transplantation into immunocompromised mice, IPCs released insulin C-peptide upon glucose challenge, remained differentiated, and did not form detectable tumors. CONCLUSION: Production of IPCs solely through extracellular factor modulation in the absence of genetic manipulations may promote strategies to derive transplantable islet-replacement tissues from human neural progenitor cells and other types of multipotent human stem cells.
Abstract: BACKGROUND: We recently demonstrated that pancreatitis-associated ascitic fluid (PAAF) contains cytotoxic factor(s), inducing apoptosis in hepatocytes, and that PAAF induces hepatic adenosine triphosphate depletion, hepatocellular acidosis, and accumulation of hepatic intracellular sodium. Because ascitic fluid and serum from patients with hemorrhagic pancreatitis contain a lot of hematin, we aimed to test the hypothesis that hematin can induce hepatocellular injury, and then we compared its cytotoxicity with that of PAAF. METHODS: In vivo effects of intraperitoneal injection of hematin into the liver of healthy rats were evaluated with in situ nick-end labeling, blood biochemical analysis, and nuclear magnetic resonance spectroscopy. In vitro cytotoxic and apoptosis-inducing activities of hematin on rat primary culture hepatocytes were investigated with a cellular proliferation assay kit and DNA fragmentation enzyme-linked immunosorbent assay, respectively. Furthermore, PAAF was fractionated with Sephacryl S-300 gel column chromatography, and cytotoxic activities of its fractions on a human hepatoma cell line (HuH-7) were compared with those of hematin. RESULTS: Intraperitoneal injection of hematin into healthy rats caused apoptosis in the hepatocytes and elevated serum glutamate oxaloacetic transaminase and lactate dehydrogenase levels. Intraperitoneal injection of hematin also caused a significant decrease in the hepatic beta-adenosine triphosphate/inorganic phosphate ratio, severe hepatic intracellular acidosis, and a significant increase of hepatic intracellular sodium (Na(+)) concentration, similar to the effects of PAAF. In vitro, hematin decreased hepatocyte viability and increased the DNA fragmentation of hepatocytes, similar to the effects of 10% PAAF. Albumin reversed the cytotoxic effects of hematin and PAAF on HuH-7 cells nearly completely and partially, respectively. Fractionation of PAAF and hematin by gel column chromatography revealed that the first peak of cytotoxic activity of PAAF corresponded to that of hematin and that the cytotoxic activity was reversed by albumin nearly completely. CONCLUSIONS: These results suggest that hematin is one of the cytotoxic factors in PAAF that causes hepatocellular injury and that cellular injuries caused by hematin may be involved in the development of multiple organ failure associated with severe acute pancreatitis.
Abstract: The use of embryonic stem cells for cell-replacement therapy in diseases like diabetes mellitus requires methods to control the development of multipotent cells. We report that treatment of mouse embryonic stem cells with inhibitors of phosphoinositide 3-kinase, an essential intracellular signaling regulator, produced cells that resembled pancreatic beta cells in several ways. These cells aggregated in structures similar, but not identical, to pancreatic islets of Langerhans, produced insulin at levels far greater than previously reported, and displayed glucose-dependent insulin release in vitro. Transplantation of these cell aggregates increased circulating insulin levels, reduced weight loss, improved glycemic control, and completely rescued survival in mice with diabetes mellitus. Graft removal resulted in rapid relapse and death. Graft analysis revealed that transplanted insulin-producing cells remained differentiated, enlarged, and did not form detectable tumors. These results provide evidence that embryonic stem cells can serve as the source of insulin-producing replacement tissue in an experimental model of diabetes mellitus. Strategies for producing cells that can replace islet functions described here can be adapted for similar uses with human cells.
Abstract: BACKGROUND: We have reported that pancreatitis-associated ascitic fluid (PAAF) contains a cytotoxic factor(s) inducing apoptosis on renal tubular cells and hepatocytes. It has been suggested that elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) is associated with the development of cell damage and apoptosis. METHODS: To clarify the mechanism of hepatocellular injury in acute pancreatitis, the effect of PAAF on hepatocyte [Ca(2+)](i) was investigated. Primary cultures of rat hepatocytes were loaded with Fura-2/acetoxymethyl, and the changes of [Ca(2+)](i) were measured using spectrofluorometer. RESULTS: The baseline of hepatocyte [Ca(2+)](i) was 172 +/- 17 nM. [Ca(2+)](i) increased from 1 min after the addition of PAAF in a dose-dependent manner. Fractionation of PAAF revealed only one fraction (molecular weight >/= 5 x 10(4)) possessed both [Ca(2+)](i) elevation activity and cytotoxic activity. Neither 8-(N,N-diethyl-amino) octyl-3,4,5-trimethoxybenzoate (TMB-8) nor thapsigargin inhibited the PAAF-evoked [Ca(2+)](i) elevation. Chelation of extracellular Ca(2+) by ethylene glycol bis-(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) prevented the elevation of [Ca(2+)](i), but verapamil did not prevent it. Platelet-activating factor antagonist (TCV-309) blocked the PAAF-elicited [Ca(2+)](i) elevation. Pancreatitis-associated serum also increased hepatocyte [Ca(2+)](i). Moreover, PAAF increased [Ca(2+)](i) on Madin-Darby canine kidney cells in a dose-dependent manner. CONCLUSIONS: These results suggest that the dramatic elevation of hepatocyte [Ca(2+)](i) due to PAAF may be closely related to the hepatocellular injury in severe acute pancreatitis and that platelet-activating factor may play a pivotal role in increasing hepatocyte [Ca(2+)](i). Elevation of [Ca(2+)](i) in various cells may be involved in the mechanism of multiple organ failure in this disease.
Abstract: BACKGROUND: Apoptosis of hepatocytes has been reported to be involved in liver failure complicated with systemic manifestations such as endotoxemia. We hypothesized that hepatocyte apoptosis occurs in severe acute pancreatitis. METHODS: Induction of apoptosis was evaluated in the liver from rats with necrotizing pancreatitis. Apoptosis-inducing activity of the pancreatitis-associated ascitic fluid on hepatocytes was evaluated in vivo by intraperitoneal injection of the ascitic fluid and in vitro using rat primary hepatocyte culture. RESULTS: Apoptosis was detected in hepatocytes in the rats both with severe acute pancreatitis and with the intraperitoneal injection of the ascitic fluid by in situ nick-end labeling and DNA fragmentation. Apoptotic change and hepatic injury were ameliorated by administration of an interleukin-1 beta-converting enzyme inhibitor. The ascitic fluid exhibited cytocidal activity in rat primary hepatocyte culture via apoptosis, which was confirmed by DNA fragmentation, by cell cycle analysis, and by nuclear fragmentation. The neutralizing antibody for transforming growth factor-beta 1 partially blocked the apoptosis induction but the antibody to tumor necrosis factor-alpha had no effect. CONCLUSIONS: Apoptotic cell death occurs in hepatocytes in severe acute pancreatitis partially via transforming growth factor-beta 1 in the pancreatitis-associated ascitic fluid.
Abstract: BACKGROUND: The governance and power structure of the department of surgery depends to a large extent on the chairperson's decisions in Japanese medical schools. This paper reports the current collective opinions of surgery department chairpersons regarding the quality assessment of surgical faculty performance. METHODS: Surveyed were 78 chairpersons of general surgery departments from 72 Japanese medical schools. Chairpersons were questioned about administrative and organizational decision making: rank order requirements for full-time surgical faculties, coordination of staff for surgical operations, and performance outcome measures. RESULTS: In all, 68 (87%) chairpersons responded. When selecting surgical faculties, publishing competence (45%) and collaborative personality (44%) were the two foremost concerns of chairpersons. Teaching experience (0%) and board certification (2%) showed the lowest rate for the first priority among the 6 elements listed. The operator was mainly decided by the chairperson (63%) whereas the rest of the operative team members were decided by either the chairperson (28%), a specialty team (38%), or attending surgeons (32%). Thirty-three chairpersons (49%) of 68 respondents used the morbidity and mortality conference as the only available approach for assessing surgical performance on a regular basis, whereas the remaining half did not have routine outcome measures. CONCLUSIONS: The results of this study indicate that surgery department chairpersons deemed collaborative personality and publishing competence the two major requirements for candidates of surgical faculties. Although the morbidity and mortality conference is currently the only available approach for assessing surgical performance, the majority of chairpersons felt that outcome measures should be based on more objective and structured criteria.
Abstract: We previously reported that serum hepatocyte growth factor (HGF) levels are elevated in patients with acute pancreatitis and that pancreatitis-associated ascitic fluid (PAAF) contains cytotoxic factor(s) inducing apoptosis on Madin-Darby canine kidney (MDCK) cells. In this study, plasma HGF levels and HGF tissue distribution were investigated in rats with experimental acute pancreatitis, and the effects of HGF on the cytotoxic activity and apoptosis-inducing activity of PAAF also were examined. Plasma HGF levels were elevated in rats with two experimental pancreatitis models of different grades of severity. The degree of its elevation was correlated with the severity and the organ dysfunctions. In rats with severe pancreatitis, HGF protein and messenger RNA (mRNA) levels significantly increased in liver, kidney, and lung, which were injured organs. When anti-HGF neutralizing antibody was administered in severe pancreatitis, liver dysfunction worsened, and apoptotic cells increased in kidney. Recombinant HGF inhibited the cytocidal activity of PAAF on MDCK cells in a dose-dependent manner. Moreover, recombinant HGF prevented the apoptotic cell death (DNA fragmentation, nuclear fragmentation, and caspase-3 activation) induced by PAAF. These results suggest that HGF is produced in injured organs and may function as an organotrophic and antiapoptotic factor against the organ injuries in acute pancreatitis.
Abstract: BACKGROUND: The mechanism of acute pancreatitis-induced hepatocellular injury is unclear. We have observed hepatocyte apoptosis in rat acute necrotizing pancreatitis. These studies were designed to determine the mediator(s) responsible for hepatocyte apoptosis and to clarify the significance of macrophages as its source. METHODS: A rat sodium deoxycholate-induced pancreatitis model was used. Immunohistochemical studies for apoptosis-inducing mediators on hepatocytes were examined in the liver and on the peritoneal macrophages. The levels of transforming growth factor-beta1 (TGF-beta1) were also evaluated quantitatively with an enzyme-linked immunosorbent assay. Induction of apoptosis on the hepatocytes was evaluated by in situ nick-end labeling and tissue DNA fragmentation enzyme-linked immunosorbent assay. Finally, the effects of TGF-beta1 neutralization and macrophage depletion were examined. RESULTS: In the liver and the peritoneal macrophages, strong expression of TGF-beta1 was detected early in the course of pancreatitis. In sodium deoxycholate-induced pancreatitis, the levels of TGF-beta1 were also elevated in the plasma (9.2 +/- 0.8 ng/mL), in the pancreatitis-associated ascitic fluid (11.5 +/- 0.6 ng/mL), and in the liver homogenate (2.8 +/- 0.3 ng/g of liver tissue). Moreover, the amount of fragmented DNA of the liver with pancreatitis was 290% +/- 20% of that with a sham operation and serum alanine aminotransferase levels elevated to 248.2 +/- 67.0 IU/L. TGF-beta1 neutralization partly blocked the positive labeling on the nuclei of the hepatocytes, the elevation of the amounts of fragmented DNA (205% +/- 10% of sham operation), and the serum alanine aminotransferase level (144.2 +/- 14.9 IU/L). On the other hand, the macrophage depletion caused a marked decrease in the TGF-beta1 protein level in the plasma (4.8 +/- 1.2 ng/mL) or in the pancreatitis-associated ascitic fluid (8.0 +/- 1.0 ng/mL). Moreover, the macrophage depletion completely inhibited the elevation of the TGF-beta1 protein level in the liver homogenate (1.5 +/- 0.4 ng/g of liver tissue), and thereafter decreased the amounts of the positive labeling on the nuclei of the hepatocytes and decreased the amount of fragmented DNA (120% +/- 18% of sham operation) and the serum alanine aminotransferase elevation (119.2 +/- 24.2 IU/L). CONCLUSIONS: In a model of sodium deoxycholate-induced pancreatitis, macrophages are responsible for pancreatitis-induced hepatocellular injury by means of apoptosis, and macrophage-derived TGF-beta1 is one of the major factors inducing the hepatocyte apoptosis.
Abstract: To investigate impairment of cellular immunity in severe acute pancreatitis, alterations of peripheral lymphocytes in acute pancreatitis were examined. In 48 patients with severe acute pancreatitis, the mean peripheral lymphocyte count on admission was 959 + 105/mm3, and it was significantly decreased in the patients with subsequent infection (623 +/- 90/mm3 ) in comparison to those without infection (1084 +/- 135/mm(3)). According to an analysis of lymphocyte subsets, although both B and T lymphocytes were decreased in peripheral circulation in the patients with infection, it was primarily CD8-positive lymphocytes that decreased in these subsets. Cell cycle analysis of lymphocytes collected from these patients indicated that apoptotic changes occurred after 24 hours' incubation in lymphocytes from patients with severe pancreatitis but not in lymphocytes from healthy control subjects. In a rat model of experimental necrotizing pancreatitis, total peripheral lymphocytes and T lymphocytes were significantly decreased 5 hours after induction of pancreatitis. In severe pancreatitis, peripheral lymphocytes are eliminated from systemic circulation possibly as a result of apoptosis. It has been suggested that impairment of cellular immunity due to peripheral lymphocyte apoptosis is linked to the development of subsequent infectious complications in acute pancreatitis.
Abstract: The aim of this study was to assess the significance of peritoneal macrophage in inducing cytotoxicity in ascitic fluid associated with severe acute pancreatitis. The involvement of peritoneal macrophage was examined experimentally in rats by macrophage depletion with peritoneal lavage prior to the development of pancreatitis. More than 94% of the cellular components collected from peritoneal cavities by the lavage are macrophages. Although the ascitic fluid collected from the rats with necrotizing pancreatitis showed cytocidal effects via apoptosis on Madin-Darby canine kidney cells in a dose- and time-dependent manner, cytotoxicity or apoptosis-inducing activity almost disappeared from the ascitic fluid by the preceding peritoneal lavage. The ascitic fluid did not show significant differences by the lavage in osmolarity and in concentrations of albumin, bilirubin, amylase, and lipase. Although a slight reduction of tumor necrosis factor-alpha was noted with the lavage, tumor necrosis factor-alpha failed to induce apoptotic cell death in the cells, and the neutralization by antibody ameliorated neither cell death nor apoptosis. We conclude that peritoneal macrophages secrete apoptosis-inducing factor(s) into pancreatitis-associated ascitic fluid, other than tumor necrosis factor-alpha.
Abstract: BACKGROUND: Resection of the non-fibrotic pancreas is prone to postoperative pancreatic fistula because of well preserved exocrine secretions and easily crushed soft parenchyma. The purpose of this study was to evaluate ultrasonic dissection for division of the non-fibrotic pancreas in distal pancreatectomy. METHODS: All pancreata included in this study were soft on direct palpation and their main ducts had no dilatation, at least proximally from the transection line. Fifty-eight patients with gastric cancer or pancreatic disease were randomly assigned to the two groups. In the ultrasonic dissection (UD) group (n = 27), all pancreatic ducts were identified and ligated securely. The stump was left open without parenchymal suturing. In the conventional (CV) group (n = 31), the pancreas was cut with a knife and the stump was oversewn in mattress fashion. The main pancreatic duct was ligated in all patients in both groups. Pancreatic fistula was defined as a pancreatic fluid discharge for more than 7 days after operation diagnosed according to amylase concentration in the drainage fluid. RESULTS: In the UD group, approximately 20-30 tubes including a mean(s.d.) 5.2(0.8) (range 4-6) pancreatic ducts were skeletonized and ligated per patient. There were nine pancreatic fistulas (16 per cent); one in the UD group and eight in the CV group (P = 0.020). CONCLUSION: In distal pancreatectomy for the non-fibrotic pancreas, ultrasonic dissection without suture closure of the stump reduced the incidence of pancreatic fistula compared with conventional division and suture, in this randomized trial.
Abstract: BACKGROUND: Recently renal cell apoptosis has been reported in various disorders that result in renal failure. Thus we hypothesized that renal cell injury resulting from apoptosis is involved in renal failure with severe acute pancreatitis. METHODS: Renal cell apoptosis in kidneys harvested from rats with necrotizing pancreatitis was evaluated by in situ nick-end labeling. Ascitic fluid that had been collected 6 hours after development of pancreatitis was injected into the peritoneal cavities of healthy rats, and renal apoptosis was also evaluated. The apoptosis-inducing activity of the ascitic fluid was estimated in vitro with use of isolated rat renal tubules and the normal rat kidney cell line NRK52E by nuclear staining, cell cycle analysis, and DNA electrophoresis. RESULTS: Apoptosis was detected by in situ nick-end labeling on the renal tubules 6 hours after induction of pancreatitis in vivo. Similar tubular apoptosis was detected in the rats that had intraperitoneal injection of the ascitic fluid. In in vitro analyses the ascitic fluid induced nuclear and DNA fragmentation on the isolated renal tubules and promoted apoptosis on NRK52E cells in a time-dependent manner. CONCLUSIONS: Apoptotic cell death of renal tubules occurs in severe acute pancreatitis within several hours and is possibly involved in the mechanism of renal failure through undefined substance(s) in the ascitic fluid associated with pancreatitis.
Abstract: Hepatic dysfunction is one of the critical complications in acute pancreatitis but this mechanism is poorly understood. In patients with acute pancreatitis, hypoalbuminemia is often recognized, suggesting possible disturbance of hepatic transport of proteins and hepatic metabolites. The present study was undertaken to elucidate hepatic function in cerulein-induced pancreatitis from the viewpoint of intrahepatic vesicular transport. We examined the biliary excretion of lipopolysaccharide (LPS), whose pathway in the hepatocyte has been shown to be microtubule dependent. In vivo studies and ex vivo studies using isolated perfused rat liver (IPRL) showed that cumulative excretion of LPS in each period was significantly reduced, by 49 and 25%, respectively, compared with that in control rats. But studies of biliary secretion in vivo and ex vivo studies indicated statistical insignificance between the two groups. Moreover, biliary excretion of LPS was inhibited to 60% of the control by colchicine pretreatment, without affecting bile flow in IPRL, but gadolinium chloride had no effect. We conclude that transport of LPS across the hepatocyte from blood to bile is impaired in rats with cerulein-induced pancreatitis without being affected by Kupffer cell function. These results suggest that a disturbance of vesicular transport in hepatocyte may also occur in exocytosis and endocytosis via the sinusoidal membrane, cause impairment of hepatic transport of proteins and hepatic metabolites, and result in hepatic dysfunction in acute pancreatitis.
Abstract: Although a reduction in peripheral lymphocytes has been reported in clinical cases of acute pancreatitis, the thymic change remains still unknown. To investigate impairment of cellular immunity in acute pancreatitis, alterations of the thymus in rats with acute pancreatitis were examined experimentally. Male Wistar rats were used. Two groups with pancreatitis of different severity and a control group for each were established. The thymus was weighed and the number of thymocytes counted. Apoptosis in the thymus was examined by in situ nick-end labeling, DNA agarose gel electrophoresis, and cell cycle analysis using propidium iodide. Both thymus weight and number of thymocytes decreased significantly in the rats with necrotizing pancreatitis 20 h after induction of pancreatitis (P <0.02 vs sham operation). Neither thymus atrophy nor thymocyte reduction was observed in rats with edematous pancreatitis. In thymuses from rats with necrotizing pancreatitis, in situ nick-end labeling showed a significant increase in apoptotic changes of thymocytes, which was also confirmed by the stepladder pattern on agarose gel electrophoresis of the extracted DNA and by cell cycle analysis. It is concluded that thymus atrophy associated with apoptosis occurs in rats with necrotizing pancreatitis.
Abstract: This study was undertaken to elucidate the effect of the intravenous administration of ursodeoxycholic acid (UDCA) on endotoxemia in rats with obstructive jaundice from the viewpoint of the biliary excretion of lipopolysaccharide (LPS) through hepatocytes. In rats with obstructive jaundice, fluorescein isothiocyanate-labeled LPS was administered via the portal vein and then its biliary excretion was examined. A significant increase in the LPS excretion was thus noticed in UDCA-treated rats at a dose of 0.1 micromol/100 g body wt. per min. In place of UDCA, sodium taurocholate at the same dose also enhanced the biliary excretion of LPS. Secondly, we also examined whether or not UDCA protects against endotoxemia. In this experiment, nonlabeled LPS was administered via the portal vein and its peripheral concentration was then measured. In UDCA-treated rats, the endotoxin concentration was significantly lower. Finally, to elucidate the effect of UDCA on Kupffer cells, serum tumor necrosis factor (TNF-alpha) was measured. But UDCA had no effect on the TNF-alpha level. These findings thus demonstrate that the intravenous administration of UDCA protects against endotoxemia by enhancing the transport of LPS across the hepatocytes from blood to bile without affecting Kupffer cells, and that this biliary excretion of LPS is dependent on bile acids.
Abstract: Surgical treatment for severe acute pancreatitis has not yet yielded satisfactory results. Several factors which might affect the surgical results of severe acute pancreatitis were analyzed in this study. The presence or absence of infection was not important as a factor determining the surgical results. The severity scores and some biochemical parameters such as CRP, IL-6, PMN-E, HGF seemed to be closely related to surgical results. It was likely that a significant decrease in lymphocyte counts in the blood on admission was closely related to the prognosis of the surgical patients. Timing and procedures for surgery should be more seriously considered in the treatment for patients with such poor general conditions.
Abstract: Serum levels of hepatocyte growth factor (HGF), C-reactive protein (CRP), and interleukin-6 (IL-6) were determined at the time of admission in 38 patients with acute pancreatitis. The clinical utility of HGF for the detection of severe pancreatitis and for predicting prognosis, bacterial infection (infected pancreatic necrosis or sepsis), and organ dysfunction (liver, kidney, and lung) during the clinical course of acute pancreatitis was compared with the clinical utility of CRP and IL-6 by analysis of receiver operator characteristic (ROC) curves. The optimum cutoff levels of HGF for severity, prognosis, infection, hepatic dysfunction, renal dysfunction, and respiratory dysfunction were 0.9, 1.1, 1.0, 1.1, 1.1, and 1.0 ng/ml, respectively. HGF was as useful as CRP and more useful than IL-6 for detection of severe pancreatitis and for predicting hepatic dysfunction. Moreover, HGF was more useful than CRP or IL-6 for predicting prognosis, renal dysfunction, and respiratory dysfunction. However, for predicting infection, CRP was more useful than HGF. These results suggest that serum HGF levels on admission may be a useful new clinical parameter for determining the prognosis of acute pancreatitis and that HGF may be closely related to the organ dysfunction of acute pancreatitis.
Abstract: We report herein the case of a 22-year old woman who underwent esophagectomy for corrosive esophageal stricture. This patient with mental disturbance swallowed a strong alkali in a suicide attempt and suffered from esophageal stenosis instead of steroid therapy. Because the stiffness and stricture of the esophagus proved to be refractory, transhiatal esophagectomy by blunt finger with gastroesophagostomy was performed four months after injury. Operative findings revealed severe stiffness and inflammation of the esophagus which had spread to the adjacent organs, especially the carina. As for pathological findings, most of the esophageal mucosa showed defects resulting from infiltration of inflammatory cells and capillary vascularization from the lamina propria to the extra-esophageal wall. The postoperative course was uneventful and perioperative psychological state had been stable leading to a decrease in tranquilizer dosage. This experience indicates that esophagectomy is the treatment of choice for such mental disturbed patients with severe stenosis.
Abstract: Sphingosine and ceramide, products of sphingomyelin hydrosis by sphingomyelinase, have recently been regarded as second messengers for cell biological actions. On the other hand, exocrine pancreas is a typical organ to perform regulatory secretion of digestive enzymes, depending upon extracellular signals. We investigated the effects of sphingosine or ceramide on amylase secretion from isolated rat pancreatic acini. Either sphingosine or cell-permeable ceramide inhibits CCK8- or carbachol-induced enzyme secretion from isolated rat pancreatic acini in a dose-dependent manner. Sphingosine or ceramide itself does not affect basal amylase secretion from the acini. Ceramide also inhibits NaF-induced amylase secretion, indicating that it acts post the activation of receptor-linked GTP-binding protein. In our experiments, ceramide inhibited Ca2+ ionophore-induced amylase secretion, but not phorbol ester-induced secretion. These results indicate that ceramide affects secretory processes post intracellular Ca2+ mobilization in the exocrine pancreas.
Abstract: Rab11 p24 is a Ras-like small guanosine triphosphate (GTP)-binding protein, and specific antibodies against it were newly developed to explore its function. Using the antibody, Rab11 p24 was shown to be abundant in rat pancreas as well as in most rat tissues. To explore the involvement of Rab11 p24 into the exocytotic process, the subcellular distribution of Rab11 p24 in rat pancreatic acini was evaluated also by use of the antibody. When the isolated acini were incubated with 1 x 10(-10) M cholecystokinin octapeptide (CCK-8) that induced the maximal stimulation, the amount of Rab11 p24 increased in the fractions of plasma membrane and zymogen granules, but decreased in the cytosol fraction. This redistribution was time-dependent and occurred within 1 min after the CCK-8 stimulation and reached a maximal level within 2 min after the stimulation. Moreover, a light microscopic immunolabeling technique on the isolated rat pancreatic acini also revealed that higher immunoreactivity with Rab11 p24 was observed over the zymogen granule membrane under CCK-8 stimulation. The present results indicate that Rab11 p24 is translocated from cytosol to the membrane fraction during stimulation with CCK-8 and suggest that Rab11 p24 is involved in the intracellular vesicular transport of isolated acini.
Abstract: We investigated the cytotoxicity on Madin-Darby canine kidney (MDCK) cells of pancreatitis-associated ascitic fluid (PAAF) collected from rats with experimental necrotizing pancreatitis. PAAF reduced viability of MDCK cells in a time- and dose-dependent manner. We detected DNA fragmentation on the PAAF-treated MDCK cells, indicating that the cytocidal action of PAAF is via apoptosis. From the results obtained, we conclude that PAAF contains factor(s) inducing apoptosis on MDCK cells, and we assume that apoptotic cell death is involved in the mechanism of organ failure in acute pancreatitis.
Abstract: We have previously purified smg GDP dissociation stimulator (GDS) from bovine brain and isolated its cDNA from a bovine brain cDNA library. smg GDS stimulates the GDP/GTP exchange reaction of a group of small GTP-binding proteins (G proteins), including at least c-Ki-ras p21, smg p21A, smg p21B, rhoA p21 and rhoB p21, by stimulating the dissociation of GDP from and the subsequent binding of GTP to each small G protein. In this study, we have isolated and sequenced the cDNA of smg GDS from a human brain cDNA library using the cloned bovine smg GDS cDNA. The cDNA has an open reading frame encoding a protein of 558 amino acids with a calculated Mr value of 61,122. Human smg GDS shares 93% nucleotide and 96% amino acid sequence homologies with bovine smg GDS. The isolated cDNA is expressed in Escherichia coli, and the encoded protein shows the physical and functional properties similar to those of bovine smg GDS.
Abstract: We have recently purified to near homogeneity the stimulatory GDP/GTP exchange protein for smg p21s (ras p21-like GTP-binding proteins) from bovine brain cytosol. This regulatory protein, named GDP dissociation stimulator (GDS), stimulates the GDP/GTP exchange reaction of smg p21s by stimulating the dissociation of GDP from and the subsequent binding of GTP to them. In this study, we have isolated and sequenced the cDNA of smg p21 GDS from a bovine brain cDNA library by using an oligonucleotide probe designed from the partial amino acid sequence of the purified smg p21 GDS. The cDNA has an open reading frame encoding a protein of 558 amino acids with a calculated Mr value of 61,066, similar to the Mr of 53,000 estimated for the purified smg p21 GDS by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose density gradient ultracentrifugation. The isolated cDNA is expressed in Escherichia coli, and the encoded protein exhibits smg p21 GDS activity. smg p21 GDS is overall hydrophilic, but there are several short hydrophobic regions. The smg p21 GDS mRNA is present in bovine brain and various rat tissues. smg p21 GDS has low amino acid sequence homology with the yeast CDC25 and SCD25 proteins, which may regulate the GDP/GTP exchange reaction of the yeast RAS2 protein, but not with ras p21 GTPase-activating protein, the inhibitory GDP/GTP exchange proteins (GDP dissociation inhibitor) for smg p25A and rho p21s, and the beta gamma subunits of heterotrimeric GTP-binding proteins such as Gs and Gi.
Abstract: We have recently found, by use of the rhoA p21 purified from bovine aortic smooth muscle, that it is similarly post-translationally processed as described for ras p21s: it is first geranylgeranylated at the cysteine residue in the C-terminal region followed by removal of the three C-terminal amino acids and the subsequent carboxyl methylation of the revealed C-terminal cysteine residue. In the present study, we investigated the function(s) of these post-translational modifications of the C-terminal region of rhoA p21 by use of the rhoA p21s purified from bovine aortic smooth muscle and rhoA p21-overexpressing Escherichia coli since the bacterial protein was not modified with a geranylgeranyl moiety. Bovine rhoA p21 bound to plasma membranes and phosphatidylserine-linked Affigel, but bacterial rhoA p21 did not bind to them. The inhibitory GDP/GTP exchange protein for rhoA p21, named GDP dissociation inhibitor (GDI), made a complex with the GDP-bound form of bovine rhoA p21 and thereby inhibited the dissociation of GDP from and the subsequent binding of GTP to it. However, rho GDI neither made a complex with the GDP-bound form of bacterial rhoA p21 nor affected these reactions of the bacterial protein. The stimulatory GDP/GTP exchange protein for rhoA p21, named GDP dissociation stimulator (GDS), stimulated the dissociation of GDP from bovine rhoA p21, but was inactive for the bacterial protein. In contrast, the GTPase activating protein for rhoA p21 is active not only for bovine rhoA p21 but also for the bacterial protein. These results suggest that the post-translational modifications of the C-terminal region of bovine rhoA p21, most presumably the geranylgeranylation, which are absent in bacterial rhoA p21, play important roles in its interaction with membranes and the stimulatory and inhibitory GDP/GTP exchange proteins but not with the GAP.
Abstract: We have isolated cDNA clones from a rat liver cDNA library that encode a ras p21-like small GTP-binding protein (24KG) which was purified from the microsomes-Golgi complex fraction of the rat liver. The cloning was accomplished using polymerase chain reaction amplified with a set of oligonucleotide primers which were designed from the partial amino acid sequences for 24KG. The cDNA contained an open reading frame encoding a 216 amino acid protein with a calculated Mr weight of 24,397. This Mr weight was similar to that of the purified 24KG estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The sequence analysis of 24KG revealed that a 24KG cDNA is the rat counterpart of a rab11 cDNA cloned from a Madin-Darby canine kidney cell cDNA library. The 1.0-kilobase 24KG mRNA corresponding to the isolated cDNA was also detected in various rat tissues, such as brain, testis, spleen, and heart.
Abstract: 12-O-Tetradecanoylphorbol-13-acetate (TPA) activated the c-fos gene enhancer linked to the chloramphenicol acetyltransferase or luciferase reporter gene in the wild type PC-12 cells but not in the variant PC-12 cells that originated from the wild type cells. Transfection of the c-Ha-rasval12 complementary DNA (cDNA) or addition of dibutyryl cAMP to the wild type PC-12 cells as well as to the variant PC-12 cells activated the c-fos gene enhancer. Prolonged treatment of the wild type PC-12 cells with phorbol-12,13-dibutyrate caused down-regulation of protein kinase C. In these cells, TPA did not stimulate the c-fos gene enhancer any more, but transfection of the c-Ha-rasval12 cDNA still stimulated the c-fos gene enhancer to the same extent as induced in the control cells. Transfection of the c-Ha-rasval12 cDNA or addition of TPA to the wild type PC-12 cells stimulated the serum-response element but not the cAMP-response element. Dibutyryl cAMP stimulated both the serum-response element and the cAMP-response element in the wild type PC-12 cells. These results indicate that the c-Ha-rasval12 protein activates the serum-response element, but not the cAMP-response element in the c-fos gene enhancer, and that the signal pathway from the c-Ha-rasval12 protein to the c-fos serum-response element is independent of protein kinase C and cAMP-dependent protein kinase.
Abstract: In the present study, we have compared the mode of action of the v-abl protein in the regulation of gene expression with those of serine/threonine protein kinases such as protein kinase C, cyclic AMP-dependent protein kinase, and the activated c-raf protein, by measuring the transcriptional activity of the serum-response element, the 12-O-tetradecanoylphorbol-13-acetate (TPA)-response element, and the cyclic AMP-response element in NIH3T3 cells transfected with the v-abl gene. The results indicate that the v-abl protein stimulates the serum-response element and the TPA-response element, but not the cyclic AMP-response element, in a manner similar to that of the activated c-raf protein, but different from those of protein kinase C and cyclic AMP-dependent protein kinase.
Abstract: To compare the mode of action of ras p21 with those of protein kinases A and C in the regulation of gene expression in NIH/3T3 cells, we investigated the transcriptional activity of various enhancer/promoters and enhancer motifs in the cells transfected with the c-Ha-rasva112 complementary DNA (cDNA). The results indicate that the c-Ha-rasva112 protein stimulates the enhancer/promoters of the c-fos gene, the metallothionein IIA gene, the simian virus 40 (SV40) virus genome and the Rous sarcoma (RS) virus genome, and the serum-response element and the 12-O-tetradecanoylphorbol-13-acetate (TPA)-response element in a manner independent of protein kinases A and C in NIH/3T3 cells.
Abstract: We have recently purified to near homogeneity a novel type of regulatory protein for the rho proteins, ras p21-like small GTP-binding proteins, from bovine brain cytosol. This regulatory protein, named GDP dissociation inhibitor for the rho proteins (rho GDI), regulates the GDP/GTP exchange reaction of the rho proteins by inhibiting the dissociation of GDP from them, and the subsequent binding of GTP to them. In the present studies, we have isolated the cDNA of rho GDI from a bovine brain cDNA library using oligonucleotide probes designed from the partial amino acid sequences of the purified rho GDI and determined its complete nucleotide and deduced amino acid sequences. The cDNA contains an open reading frame encoding a protein of 204 amino acids with a calculated Mr value of 23,421. This Mr value is similar to those of the purified rho GDI estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose density gradient ultra-centrifugation, both of which are about 27,000. The rho GDI cDNA is expressed in Escherichia coli and COS7 cells and the encoded protein exhibits rho GDI activity. The 1.9-kilobase rho GDI mRNA corresponding to the isolated cDNA is detected in various rat tissues by Northern blot analysis. Hydropathy analysis indicates that rho GDI is overall hydrophilic except for one hydrophobic region. Computer homology search has revealed that rho GDI is a novel protein that does not share a high amino acid sequence homology with any known protein.
Abstract: Transfection of the cDNA encoding the activated c-raf-1 protein or addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) or dibutyryl cAMP to NIH/3T3 cells activated the c-fos gene enhancer linked to the chloramphenicol acetyltransferase or luciferase reporter gene. Prolonged treatment of NIH/3T3 cells with phorbol 12,13-dibutyrate caused down-regulation of protein kinase C. In these cells, addition of TPA did not stimulate the c-fos gene enhancer any more, but transfection of the c-raf-1 cDNA or addition of dibutyryl cAMP still stimulated the c-fos gene enhancer to the same extent as those induced in the control cells. Transfection of the c-raf-1 cDNA or addition of TPA to NIH/3T3 cells stimulated the serum response element and TPA response element but not the cAMP response element. In contrast, addition of dibutyryl cAMP to NIH/3T3 cells stimulated the cAMP response element but not the serum response element or TPA response element. These results indicate that the activated c-raf-1 protein stimulates the serum response element and TPA response element in a manner independent of protein kinase C and cAMP-dependent protein kinase. Since the c-fos gene enhancer has been shown to contain the serum response element and cAMP response element, it is most likely that the c-raf-1 protein is involved in the regulation of c-fos gene expression through the serum response element.
