Biomedical subjects

Y Fujiwara

Publications and source records attributed to Y Fujiwara.

At least 163 records · Page 9Linked to original sources

PubMed2000

Establishment of a visible peritoneal micrometastatic model from a gastric adenocarcinoma cell line by green fluorescent protein.

To establish a visible peritoneal micrometastatic model, an enhanced green fluorescent protein (EGFP) expressing plasmid vector was transfected into the gastric cancer cell line, MKN-45. Highly expressing EGFP cells were injected into the peritoneal cavity of nude mice. Then, the peritoneum and abdominal organs were harvested and observed on days 1, 4 and 7. Fluorescence stereomicroscopy identified peritoneal micrometastases that had been undetected by stereomicroscopy. Micrometastases as small as a single cell are detectable in this model. This peritoneal micrometastatic model should be a useful tool for research on metastasis of gastric cancer.

Adenocarcinoma↗

PubMed2000

Microsatellite analysis of melanoma lesions using (CA)13 oligonucleotides as an internal probe.

The accurate assessment of microsatellite loci on specific chromosome regions for loss of heterozygosity (LOH) is important to identify potential tumor suppressor gene locations and recently correlations to clinicopathology of tumors. Analysis of microsatellite markers usually requires performing polymerase chain reaction (PCR) with labeled primers. This often leads to spurious PCR products that make interpretations of specific PCR bands difficult. Assessment of LOH by radiolabeled PCR is not always easy to interperet when there are multiple bands present, multiple markers and specimens are being assessed, and in multiplex LOH PCR. We describe an approach to accurately verify PCR-based LOH in which labeled PCR primers are not needed to detect allele expression. Specificity is determined by using a digoxigenin-labeled oligonucleotide (CA)13 as an internal specific probe for hybridization. Because the majority of di-nucleotide microsatellite markers contain the sequence of (CA)n or (GT)n repeats, this (CA)n probe is highly versatile. Forty cutaneous melanoma biopsies from advanced stage patients were assessed using the oligonucleotide probe at five chromosome regions (1q, 6q, 9p, 10q, 11q). The LOH frequency in informative cases varied from 33% to 47% in which chromosome 6q was the highest followed closely by 11q. We observed a higher frequency of LOH in the 6q (47%) and 11q (41%) compared to previously reported studies using the probe technique. This new approach was also demonstrated to be efficient in multiplex-PCR to detect LOH in melanomas. Using the probe hybridization approach it was demonstrated that in advanced cutaneous melanomas LOH are quite frequently expressed on 5 different chromosome regions.

DNA Probes↗

PubMed2000

Regulation of hepatic high density lipoprotein binding protein, HB2, expression after administration of simvastatin to rabbits.

HB2 a candidate HDL receptor, is quite distinct from other HDL receptors in its structure. However, while changes in cellular cholesterol content, or a reduction in cholesterol biosynthesis accompany corresponding changes in HB2 expression, the level at which these changes occur have not been determined and the regulation and the function of HB2 remain uncertain. In order to further investigate the regulation of HB2, we administered simvastatin to rabbits to reduce cholesterol biosynthesis and follow changes in HB2 mRNA in various tissues. Six rabbits were given 15 mg/kg of simvastatin by oral administration daily and another six rabbits were given the same volume of saline as a control, for 21 days. They were then sacrificed to obtain samples of blood, liver, lung, jejunum and brain. Simvastatin reduced plasma total cholesterol by 47% and free cholesterol concentrations in liver and lung by 25 and 10%, respectively. Northern blot analysis showed that simvastatin lowered the expression of HB2 significantly in the liver and lung by 54% and 42% respectively but not in the jejunum or brain. These results support the findings of a previous study showing that HDL binding activity of both HB1 and HB2, which was determined by ligand blotting using HDL3 as a ligand, were reduced after administering cholesterol lowering agents. (Arteriosclerosis, 10: 1045-1050,1990). The present study suggests that simvastatin down-regulated HB2 at the transcriptional stage. Although the complete physiological function of HB2 is unclear, it appears to play some role in the cholesterol metabolism, warranting further studies to elucidate the nature of this interaction.

Animals↗

PubMed2000

[A nationwide survey of anesthesia for laparoscopic and thoracoscopic surgeries].

This is the report the first nationwide survey of anesthetic management for laparoscopic and thoracoscopic surgeries. We mailed a questionnaire to anesthetists of 133 hospitals in Japan and 74 completed questionnaires were returned. The number of intra-abdominal and thoracic surgical procedures has been increasing. General anesthesia was used in all cases for endoscopic surgery. The double lumen tube was selected in 79% of patients with pneumothorax for the endobronchial intubation. Patients for the laparoscopic cholecystectomy (LC) were given various types of anesthetics. Thirty-two percent of patients who underwent LC was anesthetized with inhalation anesthetics combined with epidural anesthesia for the early ambulance and postoperative pain control. The maximum length of time for LC surgery was 12.5 hr. Complications related to laparoscopic surgery included bile duct injuries in 72 patients, postoperative bleeding in 32 patients, vascular injuries in 29 patients, pneumothorax in 26 patients, bronchial intubation in 17 patients, gas embolism in 11 patients, bowel injuries in 9 patients and postoperative ileus in 7 patients. Administering anesthesia for endoscopic procedures requires precise knowledge of the surgical procedures, physiologic changes and complications of the pneumoperitoneum, and one lung ventilation.

Anesthesia↗

PubMed2000

Up-regulation of cyclooxygenase-2 in squamous carcinogenesis of the esophagus.

Cyclooxygenase-2 (COX-2) is overexpressed in various types of human malignancies including squamous cell carcinomas (SCCs) of the esophagus, but little is known about COX-2 expression in premalignant esophageal squamous dysplasia. To elucidate the role of COX-2 in esophageal carcinogenesis, we examined the expression of this enzyme in normal squamous epithelium (n = 42), squamous dysplasia [high-grade dysplasia (HGD, n = 41; low-grade dysplasia (LGD, n = 33)]; carcinoma in situ (n = 16), mucosal invasive carcinoma (n = 18), and advanced SCC (n = 45). Immunohistochemistry showed a significantly high COX-2 expression in HGD compared with other lesions. The COX-2 score, an index determined by intensity and positivity of COX-2 staining (maximum 3.0), was 0.29 +/- 0.04 in normal esophagus, 1.75 +/- 0.11 in LGD, 2.89 +/- 0.05 in HGD, 2.17 +/-0.18 in CIS, 1.95 +/- 0.22 in mucosal invasive carcinoma, and 1.81 +/- 0.08 in advanced SCC. Results of reverse transcription-PCR assays confirmed those obtained by immunohistochemistry. COX-2 expression correlated with proliferation activity assessed by the proliferating cell nuclear antigen index in dysplastic lesions (P = 0.001) but not in SCCs. COX-2 expression in SCC did not correlate with various clinicopathological parameters including prognosis. Our results indicate that COX-2 is a sensitive marker for HGD and suggest that COX-2 may be involved in early stages of squamous carcinogenesis of the esophagus.

Antibodies, Monoclonal↗

PubMed2000

Helicobacter pylori: criminal or innocent bystander?

Most patients with peptic ulcer disease are infected with Helicobacter pylori. However, the infection may not be always causative for the disease. The prevalence of H. pylori infection is about 50% in general. Therefore, half of the patients with peptic ulcer could be infected with this microorganism just by chance. In such cases, H. pylori may do nothing for the ulcer, although it causes chronic active gastritis. This could be the reason for the existence of both H. pylori-positive nonrecurring ulcers and ulcers that recur even after cure of H. pylori infection. H. pylori-positive non-recurring ulcers should be included in non-H. pylori ulcer disease in addition to H. pylori-negative ulcers. NSAIDs are the major cause of H. pylori-negative ulcers, especially H. pylori-negative gastric ulcers. However, more than 10% of ulcers recurred even after cure of H. pylori infection in nonusers of NSAIDs in a 7-year follow-up study. In the same study, 46% of ulcers did not recur in patients in whom eradication of H. pylori failed. There are few (<5%) H. pylori-negative ulcers; about 10% of ulcers recur after cure of H. pylori infection, and about 40% of H. pylori-positive ulcers do not recur. These are all non-H. pylori ulcers, and they represent 30% of all ulcers, which is the general prevalence rate for nonulcer subjects. Therefore, H. pylori may be an innocent bystander in approximately one third of all ulcer patients.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2000

Genetic detection for micrometastasis in lymph node of biliary tract carcinoma.

The presence of regional lymph node metastasis is one of the most significant poor-prognosis factors in patients with biliary tract carcinoma. To establish a sensitive reverse transcription (RT)-PCR assay to detect micrometastases in lymph nodes of biliary tract carcinoma, we first investigated the optimal markers in biliary tract carcinoma. The expressions of the six candidates for a suitable RT-PCR marker [mammaglobin B, carcinoembryonic antigen (CEA), cytokeratin (CK) 20, prostate-specific antigen, and melanoma antigens (MAGE-1 and MAGE-3)] were evaluated in two bile duct cancer cell lines and human biliary tract carcinoma tissues. Of 32 carcinoma tissues, mammaglobin B, CEA, prostate-specific antigen, MAGE-1, MAGE-3, and CK 20 were expressed in 28 (88%), 26 (81%), 4 (13%), 5 (16%), 7 (22%), and 9 (28%), respectively. Mammaglobin B and CEA were considered to be good markers of the six candidates. We then examined 209 lymph nodes obtained from 15 patients with biliary tract carcinoma by RT-PCR assay using both mammaglobin B and CEA and compared the results with those of histological examination. All of 20 histologically positive lymph nodes for metastasis displayed the PCR product(s) of marker genes. Of 189 histologically negative nodes, 24 (13%) nodes expressed mammaglobin B and/or CEA mRNA, suggesting the presence of micrometastasis. Our findings suggest that mammaglobin B and CEA could be useful RT-PCR markers for the detection of lymph node micrometastases in biliary tract carcinomas. Our RT-PCR assay allows accurate clinical staging necessary for patient stratification with respect to adjuvant therapy after surgery.

Aged↗

PubMed2000

Cytoprotective effect of rabeprazole against ethanol-induced gastric mucosal damage: possible involvement of nitric oxide.

The cytoprotective effect of rabeprazole, a new proton pump inhibitor, against ethanol-induced gastric mucosal damage was investigated in rats. Rats received intraperitoneal injections of rabeprazole once only or once daily for 3 days. Subsequently, the rats were given 1 ml of absolute ethanol by oral intubation. Some rats given rabeprazole were treated with N-omega-nitro-L-arginine methyl ester (L-NAME) or indomethacin. Repeated administration of rabeprazole significantly inhibited ethanol-induced gastric mucosal damage, although single administration of this drug did not. Pretreatment with L-NAME abolished the cytoprotective effect of rabeprazole. This inhibitory effect of L-NAME was reversed by L-arginine but not by D-arginine. Pretreatment with indomethacin did not influence the cytoprotective effect of rabeprazole. These results suggest that repeated intraperitoneal administration of rabeprazole has a cytoprotective effect against ethanol-induced gastric mucosal damage and that this effect may be mediated via nitric oxide but not via prostaglandins.

2-Pyridinylmethylsulfinylbenzimidazoles↗

PubMed2000

CDC25B and p53 are independently implicated in radiation sensitivity for human esophageal cancers.

Ionized radiation leads to G1 arrest and apoptosis by a p53-dependent pathway and G2-M arrest through a p53-independent pathway. In this study, we evaluated the role of cell cycle-regulating molecules in the sensitivity of cancer cells for radiation therapy. Forty-seven patients with squamous cell carcinomas of the esophagus had undergone radiation therapy, followed by surgical resection. They were classified as sensitive to radiation (SR, 14 cases) with no residual tumor in the surgical specimen or as resistant to radiation (RR, 33 cases) with viable residual tumors. Their preradiation biopsy samples were immunohistochemically investigated for the expressions of cell cycle-related molecules, including p53, CDC25A, CDC25B, cyclin D1, cyclin B1, and Ki-67. p53 expression was negative in 71% (10 of 14) of SR and positive in 91% (30 of 33) of RR. The association was strong between high radiation sensitivity and negative p53 expression (P < 0.0001). CDC25B, which is not expressed in normal epithelium but is in the cytoplasm of esophageal cancers, was strongly expressed (2+) in 46% (6 of 14) of SR and in 6% (2 of 23) of RR. Thus, the sensitivity for radiation therapy was significantly correlated with CDC25B overexpression. With respect to CDC25A, cyclin D1, cyclin B1, and Ki-67, no statistically significant differences were found in their expressions between SR and RR tumors. p53 and CDC25B expressions showed no significant associations, and multivariate analysis revealed that both p53 and CDC25B are significant independent markers for predicting radiation sensitivity. CDC25B was revealed to be a novel predictor of radiation sensitivity in esophageal cancers. Because CDC25B is an oncogene, which affects G2-M progression, these results suggest the importance of a p53-independent G2-M checkpoint in radiation therapy.

Antimetabolites, Antineoplastic↗

PubMed1999

Characterization of DNA recognition by the human UV-damaged DNA-binding protein.

The UV-damaged DNA-binding (UV-DDB) protein is the major factor that binds DNA containing damage caused by UV radiation in mammalian cells. We have investigated the DNA recognition by this protein in vitro, using synthetic oligonucleotide duplexes and the protein purified from a HeLa cell extract. When a 32P-labeled 30-mer duplex containing the (6-4) photoproduct at a single site was used as a probe, only a single complex was detected in an electrophoretic mobility shift assay. It was demonstrated by Western blotting that both of the subunits (p48 and p127) were present in this complex. Electrophoretic mobility shift assays using various duplexes showed that the UV-DDB protein formed a specific, high affinity complex with the duplex containing an abasic site analog, in addition to the (6-4) photoproduct. By circular permutation analyses, these DNA duplexes were found to be bent at angles of 54 degrees and 57 degrees in the complexes with this protein. From the previously reported NMR studies and the fluorescence resonance energy transfer experiments in the present study, it can be concluded that the UV-DDB protein binds DNA that can be bent easily at the above angle.

Base Sequence↗

PubMed1999

Expression and genetic interaction of transcription factors GATA-2 and GATA-3 during development of the mouse central nervous system.

Here we examine the expression of transcription factors GATA-2 and GATA-3 during early stages of embryonic development in the central nervous system (CNS) of the mouse. GATA-2 is expressed as early as 9 dpc in the hindbrain, in ventral rhombomere 4, and transiently in ventral rhombomere 2 (r2). From 9.5 to 11.5 dpc, activation of the gene spreads to many sites of early neuronal differentiation, such as the olfactory bulbs, the pretectum, and the oculomotor nucleus in the midbrain, a thin stripe of cells lining the floor plate from the mesencephalon to the cervical spinal cord and a ventral column of cells spanning the neural tube from rostral hindbrain and including motor neuron as well as ventral interneuron precursors. GATA-3 is expressed in a pattern very similar to that of GATA-2. Distinguishing features are the lack of expression in r2 at 9 dpc and a slight delay in its activation. In addition, GATA-2 is activated in both the ventricular and the subventricular zones of the neural tube, whereas GATA-3 is restricted mainly to the subventricular zone. Expression analyses performed on GATA-2 -/- mouse embryos between E9.5 and 10.5 dpc established that: (i) the expression of GATA-3 in the developing CNS of the mouse embryo is dependent on the presence of GATA-2 and (ii) loss of GATA-2 leads to severe defects in neurogenesis, which strongly suggests that GATA-2 is involved, as in hematopoiesis, in the maintenance of the pool of ventral neuronal progenitors.

Animals↗

PubMed1999

Benzimidazolium triflate-activated synthesis of (6-4) photoproduct-containing oligonucleotides and its application.

In the solid-phase synthesis of oligonucleotides containing the pyrimidine(6-4)pyrimidone photoproduct using a dinucleotide building block, considerable amounts of by-products were found as the chain length increased. The by-products were the major product when a 49mer was synthesized on a 40 nmol scale. It was assumed that these by-products were formed by the coupling of phosphoramidites with the N3 imino function of the 5' component of the (6-4) photoproduct. We examined imidazolium triflate and benzimidazolium triflate to find an alternative activator for DNA synthesis. Imidazolium triflate prevented by-product formation to some extent, but the coupling yields were low. Benzimidazolium triflate was comparable to tetrazole in coupling efficiency and reduced by-product formation to a great extent, without modification of the synthesizer program. The obtained 49mer was used to detect proteins that recognize UV-damaged DNA in HeLa cell extracts. Two major protein-DNA complexes were found when a 49mer duplex was used as probe, while a 30mer duplex failed to detect one of them. This application showed the usefulness of long chain 'damaged' oligonucleotides in biochemical studies.

Benzimidazoles↗

PubMed1999

Improved reporter strain for monitoring Cre recombinase-mediated DNA excisions in mice.

Effective use of conditional Cre recombinase-loxP gene modification requires Cre-expressing mouse strains with defined patterns of expression. To assess the in vivo functionality of Cre-expressing mice, we have engineered an improved reporter strain for monitoring Cre-mediated excisions. The beta-galactosidase-neomycin phosphotransferase fusion gene (betageo)-trapped ROSA26 locus was modified by gene targeting such that betageo is expressed only after Cre-mediated excision of loxP-flanked DNA sequences. betageo from the excised ROSA26 allele is expressed ubiquitously in embryos and adult mice. By mating the reporter strain with Cre-expressing transgenic mice, we have shown that the loxP-flanked ROSA26 allele is accessible to Cre during early embryogenesis, as well as in a specific hematopoietic lineage (T lymphocytes). This improved reporter strain should facilitate monitoring in vivo Cre-mediated excision events in a variety of experimental contexts.

Animals↗

PubMed1999

Possible mechanism for lead inhibition of vascular endothelial cell proliferation: a lower response to basic fibroblast growth factor through inhibition of heparan sulfate synthesis.

Although lead inhibits the proliferation of vascular endothelial cells, the mechanism has been incompletely understood. A lower response to basic fibroblast growth factor (bFGF) of growing bovine aortic endothelial cells after exposure to lead was investigated using a cell culture system in the present study. It was shown that lead significantly decreased the incorporation of [3H]thymidine into the acid-insoluble fraction of the cells but the inhibition disappeared in the presence of bFGF neutralizing antibody. Pretreatment with lead resulted in a reduction of the stimulation by exogenous bFGF on the [3H]thymidine incorporation. Lead decreased endogenous bFGF bound to cell surface heparan sulfate proteoglycans in a concentration-dependent manner but not the high affinity FGF receptor without a change of the accumulation within the cells. In spite of such a change in the endogenous bFGF distribution, the total amount of the growth factor synthesized was not significantly changed by lead. Although the binding of [125I]bFGF to heparan sulfate proteoglycans can be directly inhibited by lead, the inhibition was not so marked. On the other hand, lead markedly suppressed the incorporation of [35S]sulfate into heparan sulfate accumulated in the cell layer and the conditioned medium, suggesting that the metal inhibited the synthesis of the glycosaminoglycan in growing endothelial cells. Inhibition of the [3H]thymidine incorporation by lead was significantly restored by heparin. Since the binding of bFGF to its receptor is strongly promoted by heparan sulfate, the present data suggest that lead inhibits vascular endothelial cell proliferation by induction of a lower response to endogenous bFGF through a suppression of heparan sulfate synthesis.

Animals↗

PubMed1999

Lead inhibits the core protein synthesis of a large heparan sulfate proteoglycan perlecan by proliferating vascular endothelial cells in culture.

We characterized proteoglycans synthesized by growing cultured bovine aortic endothelial cells after exposure to lead. Lead significantly decreased the incorporation of both [3H]glucosamine and [35S]sulfate into glycosaminoglycans accumulated in the cell layer and the conditioned medium of the cells in a dose-dependent manner. Proteoglycans metabolically labeled with [35S]sulfate in the presence of lead were separated into heparan sulfate proteoglycans (HSPGs) and more highly charged chondroitin/dermatan sulfate proteoglycans by DEAE-Sephacel ion-exchange chromatography. It was found that lead markedly inhibited the synthesis of HSPGs. Sepharose CL-4B molecular sieve gel filtration showed that the marked decrease by lead occurred in the high molecular weight subclass of HSPGs. However, the length of heparan sulfate chains (approximately 50 kDa) was unchanged when analyzed by Sepharose CL-6B chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of core proteins showed that lead reduced the accumulation of a high molecular weight (approximately 400 kDa) HSPG core protein in the cell layer and the conditioned medium; the core protein was identified as a perlecan core by Western blot analysis. It is suggested that lead inhibits the synthesis of the perlecan core protein in growing endothelial cells without a change of heparan sulfate chain length. The present data support the hypothesis that inhibition of endothelial cell proliferation by lead may result from a lower response of the cells to endogenous basic fibroblast growth factor whose binding to the receptor is strongly promoted by heparan sulfate chains of perlecan.

Animals↗

PubMed1999

An antitumor promoter from Moringa oleifera Lam.

In the course of studies on the isolation of bioactive compounds from Philippine plants, the seeds of Moringa oleifera Lam. were examined and from the ethanol extract were isolated the new O-ethyl-4-(alpha-L-rhamnosyloxy)benzyl carbamate (1) together with seven known compounds, 4(alpha-L-rhamnosyloxy)-benzyl isothiocyanate (2), niazimicin (3), niazirin (4), beta-sitosterol (5), glycerol-1-(9-octadecanoate) (6), 3-O-(6'-O-oleoyl-beta-D-glucopyranosyl)-beta-sitosterol (7), and beta-sitosterol-3-O-beta-D-glucopyranoside (8). Four of the isolates (2, 3, 7, and 8), which were obtained in relatively good yields, were tested for their potential antitumor promoting activity using an in vitro assay which tested their inhibitory effects on Epstein-Barr virus-early antigen (EBV-EA) activation in Raji cells induced by the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). All the tested compounds showed inhibitory activity against EBV-EA activation, with compounds 2, 3 and 8 having shown very significant activities. Based on the in vitro results, niazimicin (3) was further subjected to in vivo test and found to have potent antitumor promoting activity in the two-stage carcinogenesis in mouse skin using 7,12-dimethylbenz(a)anthracene (DMBA) as initiator and TPA as tumor promoter. From these results, niazimicin (3) is proposed to be a potent chemo-preventive agent in chemical carcinogenesis.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1999

The effect of lithium on methamphetamine-induced regional Fos protein expression in the rat brain.

Lithium has been used widely for the treatment of manic states. Since amphetamines produce effects in humans similar to the symptoms of idiopathic mania, amphetamine administration to animals has been proposed as a model of this condition. To investigate the neurobiologic substrates of the antimanic effects of chronic lithium administration, we investigated its effects on methamphetamine-induced regional Fos protein expression in the rat brain. Chronic lithium administration (14 days; serum lithium concentration, 0.41+/-0.02 mEq/l) significantly reduced the number of neuronal nuclei showing immunoreactivity induced by methamphetamine (2mg/kg) in the prefrontal cortex, caudate/putamen, nucleus accumbens, and central nucleus of the amygdala. These results indicate the structural basis in CNS which is responsible for the antimanic effect of lithium.

Animals↗

PubMed1999

Intersections between blood cell development and leukemia genes.

Hematopoietic development is regulated in large part by transcription factors that control cell fate decisions and cellular differentiation. Several genes first discovered in the context of chromosomal translocations in leukemia also serve important functions in blood cell development. Gene-targeting experiments related to two of these factors, SCL/tal-1 and translocation-ets-leukemia (TEL), are reviewed here. SCL/tal-1, a T-cell basic helix-loop-helix oncoprotein, is required for the formation of all hematopoietic lineages. In addition, it is essential for angiogenesis in the yolk sac, indicating a dual function in blood and vessel development. TEL, an ets-related factor which is translocated to a variety of other genes in leukemias, is also required for proper angiogenesis in the yolk sac. Additional studies, however, demonstrate that TEL function is necessary for hematopoiesis to be established in the bone marrow microenvironment. These studies emphasize the intrinsic roles of leukemia-associated transcription factors in normal blood cell and vessel development.

Animals↗