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Biomedical subjects

R Doi

Publications and source records attributed to R Doi.

At least 55 records · Page 3Linked to original sources
PubMed1998

Comparative study of 5' UTR and NS3R primers for the detection of GB virus C/hepatitis G virus RNA in Japanese.

AIMS/BACKGROUND: Many epidemiological studies of new hepatitis viruses, including GB virus C (GBV-C) and hepatitis G virus (HGV), have used polymerase chain reaction (PCR) primers designed for the third nonstructural region (NS3R). However, a homology study of GBV-C and HGV genomes revealed that the 5' untranslated region (5'UTR) was more conserved than NS3R. METHODS: We attempted to detect GBV-C/HGV using PCR primers corresponding to the 5' UTR, and compared its incidence to that derived from NS3R primers. Furthermore, PCR products amplified using the 5' UTR primers were sequenced and subjected to phylogenetic analysis. RESULTS: In patients with chronic hepatitis C, the prevalence of GBV-C/HGV by PCR with the NS3R and 5' UTR primers was 5.1% (4/78) and 17.9% (14/78), respectively, and in patients on hemodialysis, it was 0% (0/81) and 5.9% (5/85), respectively. We could not detect GBV-C/HGV in patients with non-A-C liver disease. The incidence of GBV-C/HGV by 5' UTR primers was higher than by NS3R primers. After DNA sequencing at 5' UTR, phylogenetic analysis showed two types of GBV-C/HGV, Jap and HGV types. CONCLUSION: 5' UTR primers proved highly sensitive for detection of GBV-C/HGV and were superior to the NS3R primers.

5' Untranslated Regions↗
PubMed1998

Pre-operative radio-chemotherapy enhances apoptotic cell death in oral squamous cell carcinoma.

The effect of pre-operative radio-chemotherapy (RCT) has been examined in a total of 15 oral squamous cell carcinomas (SCCs), in terms of apoptosis (cell loss) and proliferation. All the patients received pre-operative radiation at a dosage of 30 or 40 Gy, as well as anticancer agents including tagaful (FT), 5-fluorouracil (5-FU), bleomycin (BLM) and peplomycin (PEP). Surgical specimens were obtained before and after RCT, and serial sections were prepared for immunohistochemistry for p53 oncoprotein and Ki-67 antigen, as well as for terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL). TUNEL indices (TI; percentage of TUNEL-positive cells in the tumor cells) before and after RCT were 1.2+/-1.1 and 4.7+/-2.9 in the nine well-differentiated oral SCCs, and 1.0+/-0.7 and 3.9+/-2.1 in the six poorly differentiated SCCs, respectively. Similarly, Ki-67 indices (KI; percentage of Ki-67 antigen-positive cells in tumor cells) before and after RCT were 31.1+/-14.2 and 15.8+/-11.1 in the former, and 37.1+/-7.8 and 8.7+/- 13.4 in the latter, respectively. Thus, pre-operative RCT enhanced apoptotic cell death and abated proliferative activity significantly (P<0.05), regardless of histological differentiation. Enhancement of apoptosis was more prominent in the group treated with FT or 5-FU than with BLM or PEP. Oral SCC with >20% of nuclear p53-positive tumor cells was noted in six cases. Enhanced TI and abadement of KI did not differ among the p53-positive and -negative tumors.

Adult↗
PubMed1998

Adenoviral transduction efficiency partly correlates with expression levels of integrin alphavbeta5, but not alphavbeta3 in human gastric carcinoma cells.

Adenovirus has attracted much attention as a vector for gene therapy. Integrin alphavbeta3 and alphavbeta5 mediate adenovirus internalization into cells. We examined adenoviral transduction efficiency and expression of integrin alphavbeta3 alphavbeta5 in 9 human gastric carcinoma cell lines. The percentage of -gal-positive cells was more than 85% 3 days after infection with recombinant adenovirus carrying the bacterial LacZ gene (AxCALacZ) at a dose of 25 MOI in 7 cell lines and the transduction efficiency was 32 and 42% in HSC-39 and MKN-28 cells, respectively. Adenoviral transduction efficiency did not correlate with the histological cell types. Flow cytometric analysis revealed relatively high expression of integrin alphavbeta5 in MKN-28 and OCUM-2M cells, followed by MKN-1, MKN-7, MKN-45, MKN-74, TMK-1 and KATO-III cells. HSC-39 cells minimally expressed integrin alphavbeta5. On the other hand, integrin alphavbeta3 was expressed only in MKN-1 cells. These results suggest that the adenovirus vector might be useful for gene transduction into human gastric carcinoma cells and the transduction efficiency partly correlates with the expression levels of integrin alphavbeta5 but not integrin alphavbeta3.

Adenoviridae↗
PubMed1997

Ischemia-reperfusion injury on the pancreas in rats: identification of acinar cell apoptosis.

An ischemia-reperfusion injury on the pancreas is involved in the pathophysiology of acute pancreatitis or tissue injuries after pancreas transplantation. On the other hand, recent studies have demonstrated that ischemia-reperfusion induces apoptosis in several organs such as kidney, heart, and brain. In the present study, we sought to characterize a pattern of injury during ischemia-reperfusion on the pancreas and determined whether ischemia-reperfusion on the pancreas causes the apoptotic process. Ischemia-reperfusion was induced by blocking the inferior splenic artery and removing the clamp in pentobarbital-anesthetized rats. Rats were sacrificed at 0-72 hr following a 60-min ischemia. Evans blue extravasation showed 3.5-fold increase at 2 hr after reperfusion, indicating a rapid increase of vascular permeability. Tissue myeloperoxidase activity, an index of neutrophil accumulation, significantly increased in a time-dependent manner until 48 hr after reperfusion. Histological analysis revealed the existences of interstitial cell infiltration and edema. DNA breaks of acinar cells were detected by gel electrophoresis and in situ nick end-labeling, and the numbers strikingly increased at 48 hr after reperfusion. Furthermore, Bax protein, an effector of apoptotic cell death, was expressed in acinar cells. The results indicate that an ischemia-reperfusion injury on the pancreas in rats resembles many features of acute pancreatitis. Apoptosis in acinar cells may be one of the specific features of the ischemia-reperfusion injury on the pancreas.

Amylases↗
PubMed1997

Detection of matrix metalloproteinase activity in human pancreatic cancer.

Destruction of the basement membrane (BM) is mandatory for tumor spread, and matrix metalloproteinases (MMPs) are known to be implicated in colon cancer invasion and metastasis by digesting type IV collagen, a main component of the BM. The current study analyzed the expression of MMP-2 and MMP-9 in pancreatic cancer tissues. Frozen specimens of pancreatic cancer (n = 10), a liver metastatic nodule from pancreatic cancer (n = 1), and normal pancreas (n = 3) were homogenized and analyzed by zymography. The activated form of MMP-9 (82 kDa) was detected in all of the normal and malignant tissues, while the activated form of MMP-2 (62 kDa) was detected in all of the pancreatic cancers and its metastatic tissue, but not in the normal pancreatic tissues. These results indicate that expression of the activated form of MMP-2 may be specific to pancreatic cancer, while that of MMP-9 may be unrelated to it.

Biomarkers, Tumor↗
PubMed1997

A hemizygous A to CC base change of the CHM gene causing choroideremia associated with pinealoma.

BACKGROUND: Although mutations of the CHM gene have been reported in the Caucasian patients with choroideremia, there have been no such reports in non-Caucasian patients. We analyzed the CHM gene in a Japanese patient with choroideremia associated with pinealoma. METHODS: The method for screening was a nonradioisotopic modification of single-strand conformation polymorphism (SSCP) analysis. The PCR products from the patient and the carrier were screened and directly sequenced using an automated DNA sequencer. The PCR product of the carrier was also subcloned into a vector and the subcloned products were sequenced. RESULTS: SSCP analysis showed an identical abnormal band shift in the patient and the carrier. Direct sequence analysis showed a hemizygous A to CC mutation at nucleotide 1608 of the CHM gene in the patient, suspected to result in the absence or truncation of the predicted CHM protein. The sequence using both the PCR product and the subcloned DNA of the carrier showed both wild-type and mutant bands indicating a heterozygote. CONCLUSION: The hemizygous mutation was detected in a patient and the heterozygous pattern in his mother, the carrier, suggesting that this mutation caused the disease.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Effects of structural modulation on biological activity of bombesin analogues with (E)-alkene bond.

The specific bombesin receptor antagonist, (E)-alkene bombesin isostere (EABI-1), [D-Phe6,Leu13psi[(E)CH=CH]Leu14]bombesin(6-14) is a potent antagonist in terms of inhibition of bombesin-stimulated amylase release from rat pancreatic acini. This study examined the effects of EABI-1 (L-L diastereomer) and three novel bombesin analogues on amylase release in rat pancreatic acini. EABI-2 is a L-D diastereomer of EABI-1. EABI-3 is an analogue, of which leucine at position 13 of EABI-1 was replaced with valine. EABI-4 is a L-D diastereomer of EABI-3 (L-L). The order of agonist potency was EABI-2>EABI-3>EABI-4. EABI-1 showed no agonist activity at concentrations up to 100nM. On the other hand, all of four analogues had antagonist activity. The order of antagonist potency was EABI-1>EABI-3>EABI-4>EABI-2. EABI-1 was a complete antagonist, EABI-2 and EABI-3 were partial agonists, and EABI-4 had a weak agonist effect. The present study provides a useful information on the future development of peptide analogues for anticancer agents and biological tools for investigating actions of bombesin family peptides.

Amylases↗
PubMed1997

Calcium-evoked insulin release from insulinoma cells is mediated via calcium-sensing receptor.

BACKGROUND: We have previously reported that high extracellular calcium ([Ca2+]o) levels elicited rapid increases in the cytosolic free calcium ([Ca2+]i) and insulin release from human insulinoma cells. In this study we further investigated the mechanism for stimulus-secretion coupling of insulinoma cells exposed to high levels of [Ca2+]o. METHODS: Insulinoma tissues were surgically obtained for primary culture. The changes of [Ca2+]i level in response to various agents were monitored by fluorometry. Total RNA was extracted from tissues and subjected to reverse transcription-polymerase chain reaction (RT-PCR) with calcium-sensing receptor (CaR)-specific primers. PCR products were subcloned and sequenced. RESULTS: When [Ca2+]o level was elevated, [Ca2+]i in insulinoma cells was immediately increased. Application of neomycin abolished the increase in [Ca2+]i level, although extracellular nifedipine and lanthanum chloride did not affect it. The depletion of intracellular calcium stores with thapsigargin or carbachol eliminated the increase in [Ca2+]i level. RT-PCR analysis identified the 682 bp product, of which the sequence was identical to the corresponding regions of human parathyroid CaR. CONCLUSIONS: Intracellular Ca2+ release might be important in insulin release from insulinoma cells after exposing to high level of [Ca2+]o. CaR could be involved in this mechanism.

Adult↗
PubMed1997

Role of apoptosis in duct obstruction-induced pancreatic involution in rats.

It has been recognized in mammals that after pancreatic duct obstruction, acinar cells progressively disappear and pancreatic islets are preserved. Previous studies by electron microscopy have suggested the involvement of apoptosis in acinar cell deletion. In the present study, we employed molecular biological methods and investigated whether acinar cell deletion is due to apoptosis. In male Sprague-Dawley rats, pancreatic duct ligation was performed through a left paramedian incision. Pancreatic tissue was studied at each of the following intervals after ligation: 3, 6, 12, 18, and 24 h and 2, 3, 5, and 7 days. DNA fragmentation was determined by in situ labeling of DNA strand breaks on tissue sections [fluorescein-labeled terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling method (TUNEL)] and by electrophoretic detection of the fragments of extracted DNA. Tissue sections were also examined by hematoxylin/eosin staining and immunohistochemical staining of insulin. Pancreatic duct ligation induced acinar cell deletion by day 5. Pancreatic tissue from control rats demonstrated no TUNEL-positive nuclei. In contrast, acinar cells from rats 12 h to 5 days after duct ligation showed TUNEL-positive nuclei. The number of TUNEL-positive nuclei was maximum 2 days after duct ligation. Electrophoresis showed DNA fragmentation after duct ligation. Control rats showed a genomic DNA pattern. Islets were preserved throughout the experimental period in duct-ligated rats and control rats. The results suggest that apoptosis may be the dominant form of acinar cell death in the rat pancreatic duct ligation model.

Amylases↗
PubMed1997

Expression of Bcl-2 and PCNA in duct cells after pancreatic duct ligation in rats.

Obstruction of the pancreatic duct induces acinar cell deletion followed by duct proliferation and interstitial fibrosis. Apoptosis has been reported to be involved in the induction of acinar cell deletion after pancreatic duct ligation (PDL) in rats, however, the mechanism of pancreatic duct cell proliferation is still unknown. We hypothesized that Bcl-2 (antiapoptosis protein) and PCNA (cell cycle-related protein) could be involved in the mechanism of pancreatic duct cell proliferation after PDL. In PDL, rats, acinar cells decreased in number and disappeared completely after duct ligation and duct-lining cells increased in number and formed duct-tubular complexes. Immunohistochemical study showed that PCNA expression appeared in the ductules and centroacinar cells from early stages after duct ligation and that Bcl-2 expression in duct cells, which was faint in normal pancreas, increased significantly when acinar cells were diminishing. Western blotting demonstrated that Bcl-2 was detected as a single band at 26 kDa, and the intensity of Bcl-2 in PDL rats was approximately ninefold stronger than in normal pancreas. Expression of Bcl-2 and PCNA after pancreatic duct ligation may be related to the prevention of apoptosis and cell proliferation of pancreatic duct cells in rats.

Animals↗
PubMed1997

Role of neutrophils in cerulein-induced pancreatitis in rats: possible involvement of apoptosis.

We investigated the role of neutrophils and the involvement of apoptosis in cerulein-induced acute pancreatitis. Male Sprague-Dawley rats were divided into 2 groups. In the control group, acute pancreatitis was induced by subcutaneous injections of cerulein. In methotrexate-treated group, the rats received intraperitoneal injections of methotrexate to produce neutrophil depletion before the injections of cerulein. The rats were sacrificed at the indicated time points until 72 h after the first injection of cerulein. Neutrophil depletion ameliorated pancreatic edema and vacuole formation in acinar cells during the early stages of cerulein-induced acute pancreatitis. Electron microscopy, DNA gel electrophoresis and in situ nick end-labeling revealed the involvement of apoptosis in acinar cells in cerulein-induced acute pancreatitis. Furthermore, the number of apoptotic acinar cells in neutrophil-depleted rats showed an about 2-fold increase during the late stages when compared with those in the control rats. Our results suggest that neutrophil depletion in cerulein-induced pancreatitis leads to amelioration of pancreatic injury during the early stage, and enhancement of apoptosis by neutrophil depletion occurs during the late stage.

Acute Disease↗
PubMed1996

Normal and transforming Ras are differently regulated for posttranslational modifications.

Point mutation of the c-H-ras gene significantly increases cellular transforming activities of Ras. Since posttranslational modification and subsequent membrane localization are essential for the biological activities of Ras, we examined whether or not the mutation also affects these two factors. The normal (Gly(12)) or the transforming (Val(12)) c-H-ras gene was expressed in NIH3T3 cells using a metallothionein promoter. Expression of either type of Ras was efficiently induced by the cadmium treatment of these cells, and immunoprecipitation of metabolically labeled cell extracts revealed that both normal and transforming Ras were expressed as four differently migrating forms on SDS-polyacrylamide gels, two of which were slower migrating cytosolic precursors and the other two were faster migrating membrane-bound forms. There was no significant difference in half lives between normal and transforming Ras; however, posttranslational modification was quite different between the two types of Ras. Transforming Ras was processed and became membrane-bound forms much more efficiently than normal Ras. Interestingly, posttranslational modification and membrane localization of Ras was significantly inhibited when the c-myc oncogene was co-expressed with Ras. In contrast to the c-myc oncogene, expression of either wild type or mutant p53 did not affect the posttranslational modification of Ras, suggesting that the c-myc oncogene specifically impairs the posttranslational modification of Ras.

3T3 Cells↗
PubMed1996

Isolation of the mRNA-capping enzyme and ferric-reductase-related genes from Candida albicans.

The mRNA-capping enzyme (mRNA 5'-guanylyltransferase) gene was cloned from a Candida albicans genomic DNA library by functional complementation of a Saccharomyces cerevisiae ceg1 delta null mutation. This gene, referred to as CGT1 (C. albicans guanylyltransferase 1), can encode a 52 kDa protein that is highly homologous to S. cerevisiae Ceg1p. CGT1 in a single-copy plasmid complemented the lethality of the S. cerevisiae ceg1 delta null mutation and, like S. cerevisiae Ceg1p, bacterially expressed Cgt1p was able to form a stable complex with the GMP moiety of GTP and to synthesize the cap structure in vitro, demonstrating that CGT1 is the C. albicans mRNA 5'-guanylyltransferase gene. CGT1 seemed to exist as a single copy in the C. albicans genome and was actively transcribed into mRNA. Another ORF was found in an opposite strand very close to the CGT1 locus. This gene shared significant sequence homology with S. cerevisiae FRE1, the gene encoding ferric reductase, and therefore was designated CFL1 (C. albicans ferric-reductase-like gene 1). Despite its sequence homology with S. cerevisiae FRE1, CFL1 mRNA was not induced by iron deprivation, and CFL1 did not complement the slow growth of a S. cerevisiae fre1 delta null mutant in the absence of iron, suggesting that CFL1 is functionally distinct from S. cerevisiae FRE1.

Amino Acid Sequence↗
PubMed1995

Effects of cyclosporine and tacrolimus (FK 506) on acute pancreatitis in mice.

OBJECTIVE: To use mice to examine the effects of cyclosporine and tacrolimus (FK 506) on two forms of acute pancreatitis often seen after clinical organ transplantation. METHODS AND DESIGN: In the first experiment, male CD-1 mice received cyclosporine (10 mg/kg), tacrolimus (0.32 mg/kg), or saline solution (control) subcutaneously once a day for 10 days. On the 11th day, acute edematous pancreatitis was induced by ceruletide (cerulein). In the second experiment, female ICR mice were fed with a choline-deficient, ethionine-supplemented (CDE) diet for 72 hours to induce necrotizing pancreatitis. After 30 hours on the CDE diet, the mice received cyclosporine (10 mg/kg), tacrolimus (0.32 mg/kg), or saline solution (control) subcutaneously twice daily for 3 days. RESULTS: The pancreatic dry-to-wet weight ratios after ceruletide injections significantly decreased in mice treated with cyclosporine but did not with tacrolimus. Cyclosporine also significantly increased serum amylase levels, but tacrolimus did not. Cyclosporine or tacrolimus alone did not produce pancreatitis. In the CDE diet groups there was a significant difference in survival among the cyclosporine-treated, the tacrolimus-treated, and the control groups. CONCLUSIONS: Cyclosporine or tacrolimus given alone does not induce acute pancreatitis. In contrast, cyclosporine can adversely affect the course of acute edematous pancreatitis, and both immunosuppressants may worsen the survival of mice with acute hemorrhagic necrotizing pancreatitis. This study also demonstrated that the deteriorating effect of tacrolimus is less potent than that of cyclosporine.

Acute Disease↗
PubMed1995

Effects of oncogenes on the resistance to cis-diamminedichloroplatinum(II) and metallothionein gene expression.

Transformation of NIH3T3 cells with the ras, the sis, or the neu oncogene rendered cells less susceptible to cis-diamminedichloroplatinum(II). Since resistance to cis-diamminedichloroplatinum(II) is reported to be associated with increased levels of metallothionein, we examined effects of these oncogenes on metallothionein gene expression. NIH3T3 cells were first transfected with the lacZ gene whose transcription is under the control of mouse metallothionein I promoter and then with the ras, the sis, or the neu oncogene. The ras and the sis oncogenes increased beta-galactosidase activities which were induced either by metal (cadmium and zinc) or by glucocorticoid (dexamethasone), whereas the neu oncogene repressed its activity. When SV40 early promoter was used instead of metallothionein I promoter for the lacZ gene transcription, the beta-galactosidase activities were not affected by metal, dexamethasone, or any of these oncogenes. This result was coincident with that of reverse transcription polymerase chain reaction that metal-induced MT I mRNA was only detected in the sis- or the ras-transformed cells, whereas any of these oncogenes did not affect the metal-induced transcription of the MT II gene. These results demonstrate that the ras and the sis oncogenes upregulate the metal- or glucocorticoid-induced transcription from metallothionein I promoter, but the neu oncogene negatively regulates it. Thus, resistance to the chemotherapeutic agent by oncogenic transformation is partly associated with the metallothionein gene expression, and MT I and MT II gene expressions are differently controlled by different oncogenes.

3T3 Cells↗