Both p16 and p15, encoded by genes located on chromosome 9p21, are inhibitors of cyclin-dependent kinases (CDK4/6) and upstream regulators of retinoblastoma protein (Rb) function. Methylation is an important mechanism for the inactivation of the p16 and p15 genes. To investigate whether these genes are inactivated by methylation of the 5'CpG islands of the genes, we examined 11 lymphoid cell lines and 18 primary lymphoid malignancies (malignant lymphoma and acute lymphoblastic leukemia) by Southern blotting analysis. The p16 gene was methylated in 3 of 11 lymphoid cell lines, whereas the p15 gene was methylated in 6 of 11 cell lines. Deletions of these genes occurred in 4 of 11 cell lines. T cell lineage leukemia cell lines tended to be deleted, and B cell lineage leukemia cell lines tended to be methylated frequently. In primary lymphoid malignancies, methylation of the p16 gene was rare in malignant lymphoma, but in acute lymphoblastic leukemia, the gene was methylated in 4 of 5 cases. Moreover, in one case, methylation of the p16 was not observed at diagnosis but was observed at relapse. In the only case of acute lymphoblastic leukemia in which we examined exon 1 of the p15, it was deleted. These results indicate that p16 and p15 gene methylation could be an important mechanism of inactivation of these genes in lvmnhoid malignancies.

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To study the role of heat shock protein 70 (hsp70) in apoptosis of B-chronic lymphocytic leukemia (B-CLL), We in vestigated intracellular hsp 70 and bcl-2 protein expression and sensitivity of B-CLL cells to apoptosisin vitroin comparison with normal lymphocytes and acute lymphoblastic leukemia (ALL) cells by flow cytometry. We found that intracellular hsp 70 expression was significantly higher (p<0.05) in B-CLL cells than in normal lymphocytes and ALL cells. However, intracellular bcl-2 protein expression was not significantly higher (p=0.0504) in B-CLL cells than in normal lymphocytes. In contrast, the sensitivity of B-CLL cells to apoptosis was higher than that of normal B lymphocytes or ALL cells. These results show the discrepancy between high expression of hsp70 and bcl-2 protein and high sensitivity of B-CLL cells to apoptosis. It is important to consider the role of hsp70 and bcl-2 protein in the pathogenesis of apoptosis in B-CLL.

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Recently, tree relationships of radical oxygen species (ROS) to aging and neurodegerative disorders, such as parkinsonism, have attracted interest. We examined the effects of iron as an ROS generator and the effects of antioxidants on aging in vivo in rats. Male Wistar rats aged 10, 15, and 20 months were used. The superoxide dismutase (SOD) activity and the content of phosphatidylcholine hydroperoxide (PC-OOH), a lipid peroxide, in rat brain were determined. The Mn-SOD activity was decreased in 20-month-old rats, but Cu, Zn-SOD activities in every age group were unchanged. The PC-OOH content was age-dependently increased. In rats given iron, the Cu, Zn-SOD activities increased in every age group, the increase in 10-month-old rats was much greater than that in 15- or 20-month-old rats. However, Mn-SOD activities were unchanged. Lipid peroxide content was unchanged. Ebselen, a glutathione peroxidase-mimetic compound, increased Mn-SOD activity. These results suggest that the ability to resist oxidative stress decreases with age.

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Age-related changes in proteins expressed in the cytosol of rat brain were investigated. When brains from 14-day-old embryos and 2-year-old rats were compared, we found more than 30 embryo-specific proteins expressed in embryonic rat brain. Of these proteins, we studied a protein, estimated with sodium dodecylsulfate-polyacrylamide gel electrophoresis to have a molecular weight of 40 kDa, which decreased with embryonic development and then disappeared within 2 weeks after birth. Partial amino acid sequences of the rat 40-kDa protein were identical to those of 40 kDa, the putative human leukocyte antigen (HLA) -DR-associated protein (PHAP) II. To amplify and clone the cDNA of the 40-kDa protein, reverse transcription polymerase chain reaction was carried out. At least three partial cDNA clones were obtained. The amino acid sequences deduced from these clones showed a high degree of homology with PHAPII, but a small nucleotide deletion was observed in two of them. These results indicate that the fetal rat-specific 40-kDa protein might be a rat homologue of human PHAPII and suggest that at least three minor isotypes are present in the brain. Although the function of PHAPII is unclear, the PHAPII homologue might play an important role in the development of the central nervous system in the rat.

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Nedaplatin is an antitumor agent which is used to treat neural, cervical, lung, esophagus, and bladder cancer. To elucidate the mechanism of cell death induced by nedaplatin, we compared tumor cell lines of different lineages : glioblastoma (T98G), an ectodermal cell line, and promyelocytic leukemic cells (HL-60), a mesenchymal cell line. Nedaplatin induced apoptotic cell death (characterized by internucleosomal DNA cleavage of 180 to 200 base pair multiples) in HL-60 cells, but induced necrotic changes without induction of DNA laddering pattern in T98G cells. To examine the effect of nedaplatin on protease activities that are believed to be involved in apoptosis, activities of interleukin 1β converting enzyme (ICE) family proteases, ICE protease and apopain, were examined using synthetic substrates in place of endogenous substrate poly (ADP-ribose) polymerase. Nedaplatin activated apopain only in HL-60 cells but failed to activate ICE protease in either cell line. Immunoblot analysis with anti-bcl-2 antibody demonstrated that the 24-kDa protein was only expressed in the cytosol of nedaplatin-treated T98G cells. The present study demonstrates that nedaplatin triggers different signal transduction pathways for cell death induction among cells of different origin.

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We describe a rapid, convenient, simple, and inexpensive method for isolating pure DNA from agarose and polyacrylamide gels using cotton wool tubes. DNA can be recovered by centrifugation through cotton wool from gel slices. This method can be used to recover DNA fragments ranging in size from 193 by to 23, 130 by within 2 hours. The recovery rate of this method is 35 % to 50 %, when estimated for isolation of λ DNA-HindIII fragments. This method can be used to isolate DNA from gels with an agarose content of 0.5 % to 2.0 %, indicating that this method is dependent on gel concentration. Better yields were obtained with soft gels than with hard gels. We have also recovered 700-bp polymerase chain reaction products using cotton wool tubes from electrophoresis on both a 0.8 % agarose gel and a 6 % polyacrylamide gel, in which satisfactory yields of more than 50 % were obtained. The DNA recovered in this way is biologically active and can be used as a substrate for further experimental procedures without additional purification steps.

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We have analyzed the qualitative and quantitative changes in protein expression in rat brains during aging. To detect proteins transiently expressed in prenatal brain, protein maps of brain cytosol from different stages were compared. We found that more than 30 proteins are expressed specifically in embryonic brain. Among these proteins is a 33-kDa protein, p33, which was predominantly expressed in the prenatal stages and reduced in the early postnatal stage, was purified and partially characterized. Partial amino acid sequences of tryptic fragments of p33 showed that this protein had a sequence highly homologous with that of PCNA/cyclin. In addition, the p33 molecule also had unidentified sequences that showed no homology with any proteins on computer homology search. Western blot analysis using anti-phosphoamino acid antibodies showed that p33 reacted only with anti-phosphothreonine antibody. These results indicate that p33 is a novel fetal brain-specific protein having a PCNA/cyclin homologous sequence. We also believe that p33 might be a molecular species or a member of the PCNA/cyclin family involved in cell proliferation or differentiation or both of the fetal central nervous sytem on the basis of homology and phosphorylation of threonine residues.

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From May 1990 through April 1997, endoscopic retrograde cholangiopancreatography (ERCP) was performed in 1210 patients, of whom 652 were chosen as subjects of this study. We devised a new morphologic classification of the papilla of Vater. Forms of the protuberance of the major papilla were classified into three types : spherical type, flat type and irregular type. The opening of the pancreaticobiliary duct was classified into two types : spot type and elongated type. The epithelium around the opening was classified into two types : granular type and villous type. We studied these forms, separate openings of the pancreaticobiliary duct, the presence of oral protrusion and frenulum, presence of anomalous connections of the pancreaticobiliary ducts, abnormal adherence of the cystic duct, relation to cholangiopancreatic diseases in flat and irregular types. We conclude that a larger proportion of cases were of the elongated and villous types and were of separate openings in the flat and irregular types than the spherical type. All cases of the flat and irregular types tended to have oral protrusion and many cases of the irregular types did not have a frenulum. An anomalous connection of the pancreaticobiliary duct was noted in one case of the flat type, and abnormal adherence of the cystic duct was noted in no cases of either the flat or irregular type. An association with pancreas cancer was noted in cases of the flat and irregular types. Therefore, if an unusual form of the papilla of Vater is confirmed, it may be necessary to perform further examinations of the pancreaticobiliary tract such as computed tomography and magnetic resonance cholangiopancreatography.

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We investigated the effect of rewarming injury on the graft liver in rats. We extended the anhepatic phase to 40 minutes and the fixed rewarming time at the usual 15 minutes (group A, n=7) or at 35 minutes (group B, n=6) . Graft temperatures were measured at various times after transplantation. Survival, serum levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and hyaluronic acid, and histologic findings were compared between groups A and group B. The graft temperature 15 minutes after transplantation (22.7±0.7°C) was significantly lower than that after 3 5 minutes (27.7±0.8°C) . Survival was significantly lower in group A and serum levels of aspartate aminotransferase and alanine aminotransferase were significantly higher in group B. Histologic findings in group A were nearly normal, but severe damage to hepatocytes and sinusoids was observed in group B. We conclude that rewarming injures hepatocytes and sinusoids; therefore, rewarming injury affects graft viability. Furthermore, we speculate that cooling below 23°C during transplantation might be necessary for maintaining graft viability.

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The influence of the chloroform extract ofTripterygium wilfordiiHookf (TWH extract) on the macrophage phagocytic system was examined in BALB/c mice. TWH extract did not influence either the organ distribution of sheep red blood cells (SRBC) or the blood clearance of colloidal carbon when the extract was administered orally into mice at doses of 200 to 400 g/kg for six consecutive days before injection of SRBC and carbon. Hemagglutination titers against SRBC in the experimental and the control mice did not differ significantly even when 400 μg/kg of TWH extract was given six times before SRBC injection. However, when administered during the course of the response (day 0 to 5), TWH extract (200 μg/ kg/ day) markedly suppressed antibody formation. These results suggest that TWH extract may exert immunosuppressive action without inhibition of macrophage functions, such as antigen handling and processing activities.

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The effect of the chloroform extract ofTripterygium wilfordiiHookf (TWH) on inflammatory mediator production was examinedin vivoin BALB/c mice. Autoclaved Teflon chambers (30×10 mm) were implanted into the backs of BALB/c mice. After 10 days, 1 % zymosan A in a volume of 300 μl was injected into the chamber to induce local inflammatory responses. Exudate fluid in the chamber was withdrawn at various intervals after zymosan injection to measure the levels of inflammatory mediators, interleukin-1 β, tumor necrosis factor- α and prostaglandin E₂. After zymosan injection, levels of inflammatory mediators increased rapidly, reached maximums 1 to 2 days later, then slowly decreased to baseline levels. Oral treatment with TWH extract 2 hours before zymosan injection reduced in a dose-dependent manner concentrations of both interleukin-1 i and tumor necrosis factor- α in exudate fluid obtained from mice 24 hours after injection. However, TWH extract reduced prostaglandin E₂concentrations at only the highest dose used.

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An immunoelectron microscopic study using a preembedding double Immunostaining technique was applied to examine synaptic relations between catecholaminergic neurons and GABAergic neurons in the area postrema of the rat. As a supplement to our previous report¹⁾, both anti-dopamine- β -hydroxylase and anti-phenylethanolamine-N-methyltransferase antisera were used to identify the different catecholaminergic neurons. Similar to our previous study¹⁾, there were reciprocal synaptic relationships between GABAergic neurons and noradrenergic or adrenergic neurons. Although tyrosine-hydroxylase immunoreactive perikarya had often been observed postsynaptic to GABAergic axon terminals, neither dopamine-β-hydroxylase nor phenylethanolamine-N-methyltransferase immunoreactive perikarya were found to receive synapses from GABAergic axon terminals. Instead, dopa-mine-β-hydroxylase immunoreactive axon terminals were often found to make synapses on GABAergic perikarya, whereas no axo-somatic synapses were found between phenylethanolamine-N-methyltransferase immunoreactive neurons and GABAergic neurons.

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Background : Although patients with inflammatory bowel disease (IBD) frequently exhibit various extraintestinal manifestations, pancreatic disorder is not generally considered as an extraintestinal complication of IBD. We investigated pancreatic dysfunction and imaging abnormalities in patients with IBD. We estimated serum and urinary pancreatic enzyme levels for evaluation of pancreatic function in 42 patients with IBD, including 29 patients with ulcerative colitis (UC) and 13 patients with Crohn's disease (CD) . In addition, some of the patients were examined by ultrasonography, abdominal computed tomography, and endoscopic retrograde pancreatography (ERP) for evaluation of pancreatic imaging. Elevation of serum or urinary pancreatic enzyme concentrations was observed in 23.8 % of patients, and abnormal pancreatic imaging, in 26.7 %. In 4 patients, the pancreatic enzyme concentrations decreased or normalized during remission of IBD. In one patient initial ERP showed irregularity of the main pancreatic duct, and follow-up ERP indicated normal appearances of the duct after remission of IBD. Our observations suggest that pancreatic dysfunction and imaging abnormalities develop in patients with IBD ; moreover, these may relate to the activity of disease and resolve when colonic inflammation is brought into remission.

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We studied the synergistic cytotoxicity of E1 Tor hemolysin (ETH, molecular mass ; 65 kDa) produced byVibrio choleraeO1 and live vibrios, such as classical 569B, E1 Tor N86, or Bengal O139 strain, to Intestine 407 cells of a human cell line. When Intestine 407 cells were incubated with live E1 Tor N86in vitro, the viability of Intestine 407 cells decreased, whereas the viability of Intestine 407 cells incubated with a classical 569B or Bengal O139 (10⁶cfu/ml) did not change. This finding suggests that live N86 are cytotoxic to Intestine 407 cells. We, however, detected cytotoxicity to Intestine 407 cells by live 569B, N86, or 0139 at 10⁶cfu/ml in the presence of a non-toxic dose of ETH (10 ng/ml) . The synergistic cytotoxicity by both ETH and live 569B was inhibited by dextran 4 with a molecular mass of 4 to 6 kDa. This result suggests that the pore formed by ETH may be required for synergistic cytotoxicity. The culture supernatant of 569B enhanced the cytotoxicity of ETH. Synergistic cytotoxicity was diminished by treatment of the culture supernatant with 0.4 % formalin or heating at 100°C or 56°C. These results imply that the proteineous factor (s) which cooperates with ETH is secreted by live 569B. Our findings of synergistic cytotoxicity by both live vibrio and ETH to human intestinal cells may contribute to the understanding of the pathogenesis of cholera, especially the role of ETH as a virulence factor.

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We examined the growth characteristics of dimethylhydrazine (DMH) -induced colon cancer in rats. Sprague-Dawley rats received 10 weekly s. c. injections of DMH at a dose of 40 mg/kg to induce colon cancer. At 22 weeks after the start of DMH treatment, barium enema was performed to visualize colon tumors. At five weeks, barium enema was repeated, and then an autopsy was performed. Tumor volume was calculated from both films and tumor doubling time was estimated. A total of 30 tumors were found at autopsy, which were fixed, sliced, and stained with PCNA to calculate the labeling index (LI) . The PCNA LI differed depending on the location of the tumor in thecolon. The PCNA LI of the upper area of the colon cancer (38.8±4.5%) was higher than that of the lower area in the same tumor (24.6±7.4%) (p<0.01) . In pathological studies, the PCNA LI did not correlate with histological type or with presence of lymph metast asis. PCNA LI also showed different regional staining in the normal mucosa in the colon, with a higher PCNA LI in the lower area in the colon (26. 9 ± 5.7 %) than in the upper area in the same location (13.5±6.1%) (p<0.01) . The upper area of colon tumors may therefore better reflect the growth characteristics of DMH-induced colon cancer in rats, because the tumor doubling time correlated well with the PCNA LI in this region.

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A case of mucinous cystadenoma of the appendix associated with acute total ulceretive colitis is reported. The pathogenesis of mucinous cystadenoma is discussed. To our knowledge, this is the first such case to be reported.

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