Biomedical subjects
Publications and source records attributed to E Kobayashi.
50 open thymectomies were performed in adult rodents using intubation combined with a fibrin glue able to prevent hemorrhage and pulmonary air leakage. This method had a 100% success rate and lower mortality than the ordinal suction procedure. Although the conventional suction thymectomy has been widely used, the open thymectomy method would permit more complete thymectomies for immunological studies.
Methyl(1S)-1-bromomethyl-7-methyl-5-[(4-methylpiperazinyl)-carb onyloxy]- 3-[(5,6,7-trimethoxy-2-indolyl)-carbonyl]-1,2-dihydro-3H-pyrolo[3, 2-e] indole-8-carboxylate hydrobromide (KW-2189), a novel derivative of duocarmycin B2, was selected for extensive evaluation based on its improved antitumor activity, water solubility, and stability in the culture medium, as compared with duocarmycin B2. Although the in vitro cell growth-inhibitory activity of KW-2189 was less potent than that of duocarmycin B2, it significantly inhibited the growth of five murine solid tumors including Colon 26 adenocarcinoma, Colon 38 adenocarcinoma, and B16 melanoma in vivo. KW-2189 was also effective against murine P388 leukemia and L1210 leukemia not only by local administration (i.p.-i.p. system), but also by systemic administration (i.p.-i.v. or i.v.-i.v. system). The most remarkable feature of KW-2189 was its efficacy against various human xenografts, which was observed in 14 tumors among 16 tested tumors including drug-insensitive tumors by single i.v. administration. Tumor regression was observed in mice bearing LC-6 lung, St-4 and St-40 stomach, Li-7 liver, PAN-2 pancreas, and MX-1 breast carcinomas. In many cases, the activities of KW-2189 were more than those of clinically active agents, mitomycin C, Adriamycin, cisplatin, and cyclophosphamide. Delayed lethal toxicity, which was reported in mice treated with CC-1065 whose structure was similar to KW-2189, was not observed in mice treated with KW-2189. KW-2189 inhibited DNA synthesis more significantly than RNA or protein synthesis, although DNA strand breaks were not observed. KW-2189 was activated by porcine liver esterase, mouse liver homogenate or Hep G2 homogenate, and DU-86-DNA adducts were detected in KW-2189-treated HeLa S3 cells, suggesting that KW-2189 was converted to DU-86 in the cells. These results indicate that KW-2189 is an interesting candidate for further development as a novel antitumor agent.
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Exposure of cultured endothelial cells to endotoxin causes an increase in the amount of cellular heparan sulfate proteoglycan and a depletion of this molecule in the extracellular matrix. Concomitant with the decrease in the extracellular matrix is the appearance of a fraction of proteoglycan bearing altered carbohydrate moieties in the culture medium. beta-mercaptoethanol, mannitol, and dimethyl sulfoxide bring back to normal the structural properties of the proteoglycan in the medium and restore the matrix content in proteoglycan to a level comparable to that of control cells but do not affect the increase in the amount of proteoglycan on the cell. This "uncoupling" suggests that two independent chains of events underlie the synthetic and structural changes of the proteoglycan triggered by endotoxin in the endothelial cell.
Despite several technical improvements, orthotopic liver transplantation (OLT) in the rat remains the most difficult experimental microsurgical transplant to perform. The short anhepatic phase tolerated by the rat makes it difficult for beginners to perform the suprahepatic and portal vein anastomoses in the limited time available. To overcome this obstacle, we examined the ability of a portosystemic shunt, induced previously by subcutaneous transposition of the spleen (STS), to decompress the portal system during hepatic pedicle clamping and thus prolong the anhepatic phase of OLT in the rat. Effective drainage was shown by the ability of rats subjected to STS 3 weeks previously to survive portal pedicle clamping of 120 min, while normal rats all died after 90 min of clamping. We demonstrated that, in STS rats, OLT performed with an anhepatic phase of 1 hr had 100% survival at 1 week, while this procedure caused 100% death in normal rats. The ablation in STS rats of the drastic pH decrease and appearance of endotoxins seen in the portal blood of normal rats subjected to "OLT-like" clamping corroborated these results. This application of the STS model to the field of OLT has already proved in our laboratory to be extremely useful during the surgical learning phase of liver transplantation in the rat.
Linkages among three biochemical loci (Aco1, Ahd2, and Mup1) and four microsatellite loci (A8, Glut1, Jun, and Pnd) were determined to construct a linkage map of rat Chromosome (Chr) 5. Consequently, an extensive linkage map on rat Chr 5 was constructed with the following gene order: A8-Aco1-Mup1-Jun-Glut1-Ahd2-Pnd. In this linkage map, the Jun and A8 loci are newly placed, and two previously reported linkage groups on rat Chr 5 are connected by the Jun locus. The linkage map indicates an extensive linkage conservation between the loci on rat Chr 5 and those on mouse Chr 4.
Small bowel transplantation (SBT) is thought to be the only radical treatment for short bowel syndrome in childhood. It is very important that the length of the graft and the type of intestine be chosen carefully because this will determine the outcome of transplantation. This model of short bowel syndrome in the rat confirms that total intestinal resection results in malnutrition and failure to gain weight. After 10 cm of ileum was transplanted orthotopically in a syngeneic combination in rats with total intestinal resection, the animals gained weight. The authors determined that 10 cm of terminal ileum is the minimum length required for survival. Second, the immunologic basis of lethal graft-versus-host reaction (GVHR) as it relates to the intestinal graft length was also evaluated. Ileal grafts of 10 cm from LEW (RT1l) rats were implanted heterotopically into (LEW x BN)F1 rats. Ileal grafts of 10 or 40 cm were implanted from BN(RT1n) rats into (LEW x BN)F1 animals. A lethal GVHR always occurred after grafting a 10 cm ileum in the LEW/F1 combination, whereas only 17% of the BN/F1 recipients died of typical GVHR. However, in the latter combination, 67% lethal GVHR was induced when 40 cm of the intestinal graft was implanted. These results indicate that mesenteric lymph nodes are a major source of lethal GVHR, but gut-associated lymphoid tissue can also induce this.
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Effects of novobiocin on the induction of gamma-glutamyltranspeptidase(GGT)-positive foci, an early lesion occurring during hepatocarcinogenesis, after diethylnitrosamine(DEN) initiation were investigated in Fischer 344 rats. Animals were given DEN at a dose of 20 mg/kg b. w. followed by novobiocin at doses of 50, 100 and 200 mg/kg b. w. The latter two doses, but not 50 mg/kg b. w., significantly inhibited the development of GGT-positive foci, providing evidence of the possible involvement of mono(ADP-ribosyl)ation in the initiation phase of hepatocarcinogenesis in rats.
Effects of a lipophilic derivative of vitamin C, 2-O-octadecylascorbic acid (CV-3611), as well as its parent L-ascorbic acid (AscA), DL-alpha-tocopherol (alpha-T) and its hydrophilic derivative, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), on the number and size of gamma-glutamyltransferase (GGT)-positive putative preneoplastic lesions were examined and compared with their influences on 8-hydroxyguanine formation in DNA and 2-thiobarbituric acid-reacting substance generation in the livers of rats fed a choline-deficient, L-amino acid-defined (CDAA) diet for 12 weeks. A total of 90 male Fischer 344 rats, 6 weeks old, were divided into 18 groups each consisting of five rats. Group 1 received the CDAA diet alone; Groups 2, 3 and 4 received the CDAA diet containing respectively 0.01, 0.05 and 0.10% CV-3611; Groups 5-7, 8-10 and 11-13 similarly received the CDAA diet containing AscA, alpha-T and Trolox, respectively, at these same low, middle and high concentrations; Group 14 received a choline-supplemented, L-amino acid-defined (CSAA) diet alone; Groups 15-18 were given the CSAA diet containing CV-3611, AscA, alpha-T and Trolox, respectively, all at the 0.10% level. While all four vitamin derivatives exerted inhibitory effects on all four parameters, in each case dose-dependently, CV-3611 demonstrated the most pronounced effects. The present results indicated that lipophilic vitamin C derivatives may be particularly effective chemopreventive agents against CDAA diet-associated, oxidative stress-related hepatocarcinogenesis via its superior antioxidative properties.
To identify and analyze genes functioning during reproductive cell formation in higher plants, cDNAs harboring the messages induced in meiotic prophase were isolated and characterized. A cDNA library constructed from microsporocytes in meiotic prophase of Lilium longiflorum was screened with a subtraction probe specific to meiotic prophase. Clones selected were classified into 18 groups by cross hybridization and partial sequencing. Northern blot analysis revealed that the transcripts corresponding to the respective cDNA groups began accumulating at the early stages of meiosis and exhibited clone-specific profiles during meiosis and the spore formation process. The amino acid sequences of the predicted gene products showed similarity with known gene products, e.g. heat shock proteins, serine proteases in Bacillus, and RAD 51 gene product in yeast. Half of the putative gene products had hydrophobic N-terminal regions, suggesting that they may function as signal peptides.
In the rat combination of DA (MHC haplotype RT1av1) donor into PVG(RT1c) recipient, liver grafts are not rejected and allow the acceptance of other organ grafts from the same donor strain. Here we show that an existing DA liver graft allows the acceptance of a DA small bowel graft in the PVG; the liver graft also prevented the graft-versus-host reaction normally associated with small bowel grafting. In addition, a liver graft could suppress the GVHR in F1 hybrid recipients of parental lymphoid cells, a classic GVHR model. GVHR suppression was immunologically specific and required that donor lymphocytes and liver be of the same strain. Besides their clinical implications, these results demonstrate the capacity of the liver for tolerance induction and suggest that it may play a physiological role in negative selection of T cells.
The expression of cripto gene product was examined immunohistochemically in 45 surgically resected pancreatic tumors, including 32 invasive ductal carcinomas, 4 intraductal papillary adenocarcinomas, 4 intraductal papillary adenomas, 2 mucinous cystadenomas, 2 islet cell tumors, and one solid and cystic tumor, and compared with that in 32 areas of accompanying chronic pancreatitis present in the cases of invasive ductal carcinomas and 5 non-tumorous areas of pancreas without pancreatitis. All pancreatic ductal tumors including adenomas and carcinomas showed positive staining with no difference in terms of staining intensity among intraductal tumors and invasive carcinomas with or without mucin hypersecretion. Islet cell tumors were positively stained but the solid and cystic tumor was negative. Duct epithelial cells and acinar cells were negative but islet cells were positive in the pancreas tissues without pancreatitis. Cells arranged in duct-like structures in areas of accompanying chronic pancreatitis were positively stained. The results suggest that cripto expression might be associated with a growth advantage of tumor cells and also with differentiation to form duct-like structures.
The present study was performed to assess the roles of hepatocellular oxidative damage to DNA and constituents other than DNA in rat liver carcinogenesis caused by a choline-deficient, L-amino acid-defined (CDAA) diet by examining the effects of the antioxidant N,N'-diphenyl-p-phenylenediamine (DPPD). The parameters used for cellular oxidative damage were the level of 8-hydroxy-guanine (8-OHGua) for DNA and that of 2-thiobarbituric acid-reacting substance (TBARS) for constituents other than DNA. A total of 40 male Fischer 344 rats, 6 weeks old, were fed the CDAA diet for 12 weeks with or without DPPD (0.05, 0.10 or 0.20%) or butylated hydroxytoluene (BHT, 0.25%). In the livers of the rats, the numbers and sizes of glutathione S-transferase (EC 2.5.1.18) placental form (GSTP)- and/or gamma-glutamyltransferase (GGT, EC 2.3.2.2)-positive lesions and levels of 8-OHGua and TBARS were determined. The GSTP-positive lesions of 0.08 mm2 or larger were all stained positively for GGT as well in cross-sectional area, whereas the smaller lesions were generally negative for GGT. DPPD and BHT reduced the size of the GSTP-positive lesions without affecting their total numbers. At the same time, they reduced TBARS generation without affecting 8-OHGua formation in DNA. The present results indicate that oxidative DNA damage (represented by 8-OHGua formation) and damage to constituents other than DNA (represented by TBARS generation) may play different roles in rat liver carcinogenesis caused by the CDAA diet; the former appears to be involved in the induction of phenotypically altered hepatocyte populations while the latter may be related to the growth of such populations.
Two models of pancreas transplantation were examined in the rat with a view to choosing one for regular use in functional experiments. A cuff technique applied to the renal vessels of the recipient was used. The histology and endocrine functioning of whole pancreas-duodenal transplant (Tx) plus bladder drainage (drainage model), and the pancreatic duct ligated segmental pancreas Tx (ligated model) were examined. Syngeneic operations were performed using either Lewis or Wistar rats. Streptozotocin (STZ) 60 mg/kg intravenously (i.v.) was used to render the recipient rats diabetic. A cuff technique was used with both models to anastomose the grafts to the renal vessels instead of the conventional technique of hand suturing to the abdominal vessels. This allows a shorter warm ischaemia time for the donor pancreas and leaves the systemic circulation intact leading to a high success rate for both techniques. Operation survival rates were 93% (n = 30) and 90% (n = 10) in the ligated and drainage models, respectively. The recipients in both groups were normoglycaemic for > 100 days. Histological examination revealed atrophic exocrine tissue early in the ligated group but only two from the drainage group showed exocrine atrophy at > 100 days. There was no statistical difference in i.v. glucose tolerance tests with both models showing a normal pattern. Thus, endocrine function remained independent of exocrine function. Both models are quicker than the conventional techniques. The duct ligation model was a simpler transplant to perform, suggesting that this should be the technique of choice in future experiments.
To demonstrate the possible role of foreign-body reaction to extravasated blood in provoking chronic cerebral vasospasm, talc (crystallized hydrous magnesium silicate) was injected as a non-biologic foreign material into the canine-cisterna cerebellomedullaris, and pathologic changes were followed. Angiographically, this cisternal talc injection induced delayed and prolonged constriction of the basilar artery, without any evidence of so-called early phase cerebral vasospasm that should occur shortly after an insult. Pathologically, around the spastic artery in the subarachnoid space with talc injection, the appearance of a moderate cellular migration coincided with cerebral vasoconstriction, which took place 2 days after the talc injection. In the spastic basilar arterial wall, marked constrictive and degenerative changes including myonecrosis and subintimal proliferation were induced by cisternal talc injection as early as on day 2, and the changes were progressive with time. These pathologic changes were extremely analogous to those of the human autopsy cases with chronic cerebral vasospasm (chronic VS) but more prominent than those observed in the experimental autologous blood-induced model. The present study demonstrated that a foreign-body reaction to talc alone could induce chronic VS in the absence of extravasated blood. Thus, it is possible to consider that inflammatory reactions to extravasated autologous blood in subarachnoid hemorrhage may give rise to chronic VS.
Location of the locus for a restriction fragment length polymorphism of N-ras-related sequences (NRAS2) on the rat chromosome 1 was determined by linkage analysis using a microsatellite locus (KAL) and a coat-color locus (C). The recombination frequencies between NRAS2 and C, NRAS2 and KAL, and C and KAL in 57 backcross progeny obtained from crosses of (WKS/Iar x IS/Iar) x WKS/Iar were 7.0 +/- 3.4%, 28 +/- 5.9%, and 23 +/- 5.6%, respectively. The order of the three loci on the rat chromosome 1 was determined as follows: KAL-C-NRAS2.
Co-Cr alloy is used more frequently than Ni-Cr alloy as a nonprecious alloy for cast plates in Japan. Titanium with good biocompatibility has often been contained in the composition of this Co-Cr alloy. However, since the melting temperature of Co-Cr alloy is very high, about 1300 degrees C, and it oxidizes easily, a vacuum-pressure casting machine capable of melting this alloy in a reduced atmosphere has recently been developed. In this study, using the vacuum-pressure casting machine, the possible effects of sectional area and the number of sprues attached to the cast plate wax pattern, as well as the alloy components, on the castability of three kinds of Co-Cr alloy containing titanium were examined. It was found that all of these parameters had significant effects on Co-Cr alloy castability.