Two species of S-adenosylmethionine (S-Ado-Met) synthetase (EC 2.5.1.6) exist in rat liver cytosol and a distinct species of the enzyme exists in kidney cytosol. S-Ado-Met synthetases alpha and beta in rat liver cytosol have been partially purified about 200- and 80-fold, respectively. The apparent molecular weight estimated by gel filtration and the sedimentation coefficient are 210 000 and 9 S for S-Ado-Met synthetase alpha and 160 000 and 5.5 S for S-Ado Met synthetase beta. Both enzymes absolutely require Mg2+ and K+ for the activity and are completely inhibited by p-(chloromercuri)-benzoate. Kinetic studies indicate that S-Ado-Met synthetases alpha and beta exhibit negative cooperativity with low S0.5 (ligand concentration required for half-maximal velocity) for L-methionine (17 microM) and ATP (0.5 mM) and positive cooperativity with much higher S0.5 values (S0.5 (L-methionine) = 0.5 mM, S0.5 (ATP) = 2 mM), respectively. The cryoprotectants dimethyl sulfoxide and glycerol markedly lower the S0.5 values of S-Ado-Met synthetase beta without significant effect on Vmax. A single species of S-Ado-Met synthetase has been purified about 1000-fold from rat kidney cytosol. The kidney enzyme, termed S-Ado-Met synthetase gamma, has an apparent molecular weight of 190 000 and a sedimentation coefficient of 7.5 S and is resistant to the inhibition by p-(chloromercuri)benzoate. S-Ado-Met synthetase gamma exhibits slightly negative cooperativity with an apparent S0.5 value for L-methionine of 6 microM and for ATP of 70 microM.

Expression of mesothelin mRNA in PPJ was not strictly specific to PCa and was apt to be stronger in PCa than in IPMN. Quantitative detection of mesothelin mRNA in PPJ may have potential diagnostic implications for pancreatic tumors.

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VMP1 is a transmembrane protein located in the Golgi and endoplasmic reticulum. It is strongly and rapidly induced in pancreas during acute pancreatitis whereas it is hardly detectable in normal rats pancreas. Its overexpression promotes formation of intracellular vacuoles and cell death through apoptosis. Intracellular vacuolation is a cellular response to injury in the pancreas during pancreatitis to maintain function under stress. The vacuoles have been shown to contain both digestive and lysosomal enzymes suggesting an abnormal intracellular processing of newly synthesized enzymes in acute pancreatitis. Physiologically, digestive and lysosomal enzymes are synthesized in ribosomes attached to the rough endoplasmic reticulum. Vacuoles represent early morphological changes during apoptosis. The aim of this work was to examine the kinetics of VMP1 gene expression in spontaneous chronic pancreatitis in WBN/Kob rat.Four-week-old male WBN/Kob rats were fed a special breeding diet MB-3 to induce chronic pancreatitis. Sections were stained with hematoxylin and eosin for histological evaluation and vacuoles examination. VMP1 mRNA expression was determined by RT-PCR with a semi-quantitative analysis and in situ hybridization. TUNEL was used to detect cell apoptosis. Cytoplasmic vacuoles with different sizes were observed in the pancreas of the WBN/Kob rat from 4 to 24 weeks. The vacuoles increased in size and number at the onset of CP at 12 weeks. VMP1 mRNA expression began at 8 weeks, reached a peak at 12 weeks, decreased at 16 and 20 weeks, then disappeared at 24 weeks. It was showed VMP1 mRNA was located in acinar cells and absent in the islets of Langerhans, duct cells, inflammatory infiltrates, and other stromal cells by in situ hybridization analysis. The vacuoles formation was paralleled to VMP1 mRNA expression. The kinetics of VMP1 mRNA expression also corresponded to the apoptosis of acinar cells.Vacuolation is an early phenomenon proceeding cell death, and is related to apoptosis. We have shown, for the first time, that cytoplasmic vacuoles in acinar cells are increased during the course of CP in the WBN/Kob rat and that the number of vacuoles was most increased at the onset of the CP. Acinar cell vacuolation is possibly a general feature of the development of acute pancreatitis and therefore, may also have important pathophysiological implications for CP. VMP1 gene activation in chronic pancreatitis is part of the acinar cell response to aggression. In conclusion, we have revealed cytoplasmic vacuolization in CP of the WBN/Kob rat, and demonstrated that VMP1 mRNA expression is strongly induced at the onset of CP. VMP1 mRNA expression, relating to acinar cell apoptosis, may reflect important pathophysislogical changes in CP. Figure: A: VMP1 mRNA of 604 bp was expressed from 8 to 20 weeks. B: VMP1 mRNA expression began at 8 weeks reached its peak at 12 weeks decreased at 16 and 20 weeks.

The beta-form of S-adenosylmethionine synthetase among three isozymes has been purified from rat liver, and proven to be homogeneous. The molecular weight of this enzyme was estimated to be 100,000 by gel filtration on Sephacryl S-200, and the enzyme was shown to be composed of two subunits of 48,000 daltons. A rabbit antiserum against the normal rat liver beta-form of S-adenosylmethionine synthetase was used for immunochemical characterization. The alpha- and beta-forms of isozyme are immunochemically identical, but the antiserum did not react with the gamma-form from rat kidney.

Secreted apoptosis-related protein (SARP) families are considered to counteract the oncogenic Wnt signaling pathway and inactivation of this gene may aid cancer development and progression. Recently, the aberrant methylation of SARP2 was detected frequently in pancreatic carcinoma (PCa) tissues, but not in normal pancreatic tissues.We evaluated the hypermethylation of SARP2 in pure pancreatic juices (PPJ) aspirated endoscopically from patients with PCa, intraductal papillary mucinous neoplasm of the pancreas (IPMN), chronic pancreatitis (CP), and a control group (C) who was consequently free of pancreatic disease by methylation-specific PCR (MSP) and real-time MSP.The incidence of the aberrant methylation of SARP2 using MSP was 79% (26/33) in the PPJ with PCa, and 85% (17/20) with IPMN. However, it was only 5% (1/19) in the PPJ with CP and 0% (0/10) in the PPJ of C, respectively. The incidences of aberrant methylation of SARP2 in the PPJ with PCa and IPMN were significantly higher than that in the PPJ with CP (p< 0.001, p< 0.001). Melting curve analysis by real-time MSP as shown in Figure revealed that the incidence of aberrant methylation of SARP2 in PPJ was 85% (28/33) with PCa, 82% (9/11) with the malignant group of IPMN, 56% (5/9) with the benign group of IPMN and 26% (5/19) with CP. In this analysis, there were significant differences between PCa and CP (p<0.001), and between the malignant group of IPMN and CP (p<0.005). In the quantitative analysis by real-time MSP with a suitable cut-off value, the incidences of aberrant methylation of SARP2 in the PPJ with PCa, the malignant group of IPMN, the benign group of IPMN and CP were 58 % (19/33), 55% (6/11), 33% (3/9) and 11% (2/19), respectively. The incidence of the aberrant methylation of SARP2 in the PPJ was significantly different between PCa and CP, and between the malignant group of IPMN and CP (p<0.005, p<0.05).These results suggest that promoter methylation of SARP2 in the PPJ may be a highly sensitive and useful marker for the detection of pancreatic neoplasms, including PCa and the malignant group of IPMN.

The tissue factor pathway inhibitor 2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor. Recently, the aberrant methylation of TFPI-2 was detected frequently in pancreatic carcinoma (PCa) tissues but not in normal pancreatic tissues. We analyzed the aberrant methylation of TFPI-2 in the pure pancreatic juice (PPJ) aspirated endoscopically from patients with various pancreatic diseases. Using the highly sensitive methylation-specific polymerase chain reaction (MSP) and quantitative MSP (Q-MSP) assay, we investigated the aberrant methylation of TFPI-2 in nine human PCa cell lines and in the PPJ from patients with PCa, intraductal papillary mucinous neoplasms (IPMN) and chronic pancreatitis (CP). The incidence of aberrant TFPI-2 methylation was seven (77.8%) of nine PCa cell lines by Q-MSP. In cell lines, the expression of TFPI-2 mRNA by quantitative reverse transcription-polymerase chain reaction showed an inverse correlation to the aberrant methylation of TFPI-2. The incidence of aberrant TFPI-2 methylation in the PPJ was 21 (58.3%) of 36 PCa patients, three (17.6%) of 17 IPMN and one (4.8%) of 21 CP by MSP assay. Using a suitable cut-off value of 2.5 according to the receiver operating characteristic curve, the incidence of aberrant TFPI-2 methylation in the PPJ by real-time MSP was 18 (62.1%) of 29 PCa patients, one (5.1%) of 17 IPMN and three (14.3%) of 21 CP, respectively. The incidence of quantitative TFPI-2 hypermethylation in the PPJ with PCa was significantly higher than that with IPMN (P < 0.001) or CP (P < 0.001). Moreover, the aberrant methylation rate of TFPI-2 in the PPJ was 100%, as observed (6/6) in the PCa patients with liver metastasis, and 86.7% (26/30) in stages IVa + IVb of PCa by Q-MSP assay. These results suggest that promoter methylation of TFPI-2 in the PPJ may be a useful marker in the diagnosis and progression of PCa using an endoscopically feasible approach. (Cancer Sci 2006; 97: 1267-1273) P ancreatic ductal adenocarcinoma has a poor prognosis with an overall 5-year survival rate ranging from 0.4%(1) to 4% (2) ; it is one of the top 10 causes of cancer death in the industrialized world.(3,4) Even in patients with PCa that underwent curative surgical resection, the 5-year survival rate was 21% among patients who received chemotherapy and 8% among patients who did not receive chemotherapy.(5) This is due both to the inherently aggressive biology of the disease and to its late diagnosis in most cases. Today, the diagnostic ability for fairly large PCa has improved markedly.(6) However, the number of patients with radical resection of PCa is still small and there are many cases in which differentiating a malignant lesion of the pancreas from a benign one is difficult. Although clinical doctors are able to use ultrasound sonography, computed tomography and endoscopic retrograde cholangio-pancreatography to characterize early pancreatic cancer from CP and IPMN, the diagnostic specificity is not sufficient to differentiate benign and malignant pancreatic tumors when the pancreatic tumor i...

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