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M Fukasawa

Publications and source records attributed to M Fukasawa.

At least 19 recordsLinked to original sources
PubMed2006

Genome-wide profiling of promoter methylation in human.

DNA methylation in the promoter region of a gene is associated with a loss of that gene's expression and plays an important role in gene silencing. The inactivation of tumor-suppressor genes by aberrant methylation in the promoter region is well recognized in carcinogenesis. However, there has been little study in this area when it comes to genome-wide profiling of the promoter methylation. Here, we developed a genome-wide profiling method called Microarray-based Integrated Analysis of Methylation by Isoschizomers to analyse the DNA methylation of promoter regions of 8091 human genes. With this method, resistance to both the methylation-sensitive restriction enzyme HpaII and the methylation-insensitive isoschizomer MspI was compared between samples by using a microarray with promoter regions of the 8091 genes. The reliability of the difference in HpaII resistance was judged using the difference in MspI resistance. We demonstrated the utility of this method by finding epigenetic mutations in cancer. Aberrant hypermethylation is known to inactivate tumour suppressor genes. Using this method, we found that frequency of the aberrant promoter hypermethylation in cancer is higher than previously hypothesized. Aberrant hypomethylation is known to induce activation of oncogenes in cancer. Genome-wide analysis of hypomethylated promoter sequences in cancer demonstrated low CG/GC ratio of these sequences, suggesting that CpG-poor genes are sensitive to demethylation activity in cancer.

Adenocarcinoma↗
PubMed2006

Genomic imprinting in Dicer1-hypomorphic mice.

To address the function of RNA interference (RNAi) in transcriptional silencing in mammals, we analyzed genomic imprinting in Dicer1-hypomorphic mice, in which Dicer1 expression was significantly reduced. We did not observe any abnormality in the allelic expression of imprinted genes in these mice or their offspring, suggesting that reduced expression of Dicer1 did not significantly affect the maintenance and reprogramming of imprinting.

Animals↗
PubMed2005

Analysis of the susceptibility of CD57 T cells to CD3-mediated apoptosis.

After stimulation with anti-CD3 antibody in vitro, CD57(+) T cells showed a greater susceptibility to apoptosis than CD57(-)alphabetaT cell receptor (TCR)(+) T cells (regular alphabeta T cells). The apoptotic fraction of CD57(+) T cells showed an increased production of active caspase-3. An increase in both Fas expression and Fas-ligand (FasL) production was also observed in CD57(+) T cells, whereas the expression of survivin was suppressed in CD57(+) T cells compared to that of regular alphabeta T cells. CD57(+) T cells display a biased expansion of a few Vbeta T cell fractions in individuals, but such Vbeta T cells were not specifically susceptible to CD3-mediated apoptosis. The TCR expression level of CD57(+) T cells was much lower than that of regular T cells and anti-TCR antibody stimulation induced a smaller apoptotic proportion of CD57(+) T cells than did anti-CD3 antibody. Although the CD3epsilon expression levels were similar in both T cell subsets, the CD3zeta level of CD57(+) T cells was significantly higher than that of regular T cells. These results suggest that several apoptotic and anti-apoptotic molecules are involved in the CD3-induced apoptosis of CD57(+) T cells and raise the possibility that the imbalance in expression of the CD3epsilon and CD3zeta chains may also contribute to the susceptibility of CD57(+) T cells to undergo apoptosis.

Adult↗
PubMed2004

Preoperative radio/chemo-radiotherapy in combination with intraoperative radiotherapy for T3-4Nx rectal cancer.

AIMS: To analyse the results of a single institution experience of combined preoperative radio/chemo-radiotherapy and intraoperative electron-radiation therapy (IORT) for locally advanced rectal cancer and to compare the results with surgery alone retrospectively. METHODS: The study cohort comprised 99 patients with clinical T3-4NxM0 adenocarcinoma of the rectum who had received preoperative radio/chemo-radiotherapy, radical surgery, and IORT [Group I]. Until 1998, 67 patients were treated with radiation only [Group Ia], and after 1999, 32 patients were concurrently given tegafur and uracil (UFT) [Group Ib]. 68 patients with clinical T3-4NxM0 rectal cancer were treated with surgery alone [Group II]. RESULTS: The median follow-up was 67 months in Group I and 83 months in Group II. Local recurrence rate was 2% in Group I, which was significantly lower than 16% in Group II (p=0.002) Both disease-free survival and overall survival in Group I were significantly better than those in Group II (p=0.04, p=0.02, respectively). Sphincter preservation was possible in 78% in Group Ib, which was significantly more than 42% in Group Ia (p=0.002). CONCLUSIONS: The combined preoperative radio/chemo-radiotherapy and IORT for clinical T3-4Nx rectal cancer significantly reduces local recurrence and improves prognosis. Combination of preoperative radiotherapy and oral UFT improves the feasibility of sphincter-preservation.

Adenocarcinoma↗
PubMed2001

A novel inhibitor of ceramide trafficking from the endoplasmic reticulum to the site of sphingomyelin synthesis.

Ceramide produced at the endoplasmic reticulum (ER) is transported to the lumen of the Golgi apparatus for conversion to sphingomyelin (SM). N-(3-Hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide (HPA-12) is a novel analog of ceramide. Metabolic labeling experiments showed that HPA-12 inhibits conversion of ceramide to SM, but not to glucosylceramide, in Chinese hamster ovary cells. Cultivation of cells with HPA-12 significantly reduced the content of SM. HPA-12 did not inhibit the activity of SM synthase. The inhibition of SM formation by HPA-12 was abrogated when the Golgi apparatus was made to merge with the ER by brefeldin A. Moreover, HPA-12 inhibited redistribution of a fluorescent analog of ceramide, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-d-erythro-sphingosine (C(5)-DMB-Cer), from intracellular membranes to the Golgi region. Among four stereoisomers of the drug, (1R,3S)-HPA-12, [corrected] which resembles natural ceramide stereochemically, was found to be the most active, although (1R,3S)-HPA-12 [corrected] did not affect ER-to-Golgi trafficking of protein. Interestingly, (1R,3S)-HPA-12 [corrected] inhibited conversion of ceramide to SM little in mutant cells defective in an ATP- and cytosol-dependent pathway of ceramide transport. These results indicated that (1R,3S)-HPA-12 [corrected] inhibits ceramide trafficking from the ER to the site of SM synthesis, possibly due to an antagonistic interaction with a ceramide-recognizing factor(s) involved in the ATP- and cytosol-dependent pathway.

Amides↗
PubMed2001

Small-sized adenocarcinoma of the lung. Cytologic characteristics and clinical behavior.

BACKGROUND: Although the cytologic characteristics of advanced adenocarcinomas of the lung have been described, the unique cytologic features of early adenocarcinomas have not been established. METHODS: Cytologic specimens from 193 patients with small-sized lung adenocarcinoma, up to 2 cm in dimension, were reviewed. Cytologic investigations were performed on sputum, bronchial brushings or washings, or fine-needle aspirates obtained from the patients preoperatively. The cytologic characteristics of early adenocarcinoma were confirmed with Image Processor for Analytical Pathology from Sumitomo Chemical Co., Ltd. RESULTS: Nuclear size, variations in nuclear size, appearance of nucleolus, and nuclear atypia of the adenocarcinoma cells were found to differ between the various histologic subtypes of adenocarcinoma. The nuclei in small but advanced adenocarcinoma were generally larger and showed more variation in size than those in early adenocarcinoma (localized bronchioloalveolar carcinoma). CONCLUSIONS: Surgically curable, early adenocarcinomas of the lung were found to have a unique cytologic appearance, including small nuclear size and slight variation in nuclear size. Using these unique characteristics, they can be distinguished easily from advanced adenocarcinomas.

Adenocarcinoma↗
PubMed2001

Double cancers in the common bile duct: molecular genetic findings with an analysis of LOH.

We report a 69-year-old man with double cancers in the common bile duct. One cancer was located between the superior and middle parts of the bile duct, while the other cancer was in the inferior part of the bile duct. Pylorus-preserving pancreatoduodenectomy was performed. There was no communication between the two cancers in either the mucosal layer or the subepithelial layer. On pathological examination, the upper cancer was diagnosed as poorly differentiated adenocarcinoma, while the lower one was found to be moderately differentiated adenocarcinoma. We analyzed loss of heterozygosity (LOH), using microsatellite markers on five chromosomal arms, in both the upper and the lower cancers. Both cancers showed common regions of LOH at 5q, 6q, 9p, 17p, and 18q, whereas the upper cancer showed one additional region of LOH at 8p, thus suggesting progression, due to the acquisition of the additional LOH, in the upper cancer. No LOH was observed in the region between the two cancers. The presence of one additional LOH in the upper cancer suggests that the upper cancer was a metastasis of the lower one.

Adenocarcinoma↗
PubMed2001

Expression of cholesteryl ester transfer protein in human atherosclerotic lesions and its implication in reverse cholesterol transport.

Reverse cholesterol transport (RCT) is the major protective system against atherosclerosis. In this system, cholesteryl ester transfer protein (CETP) is known to facilitate the transfer of neutral lipids between lipoproteins in plasma. We reported the pathophysiological significance of CETP by clinical studies with genetic CETP deficiency, showing that this protein plays a crucial role in the RCT system. However, information about the expression of this protein in the initial step of RCT, macrophages (Mphi) in the blood vessels, is still very limited. In the present study, we have performed immunohistochemical analyses on the expression of CETP in human atherosclerotic lesions. The immunoreactive mass of CETP was abundantly detected in foam cells in human aortic and coronary atherosclerotic lesions, but not in the normal arterial wall. A double immunostaining showed that the majority of CETP-positive foam cells were derived from Mphi and a minor population appeared to derive from smooth muscle cells. Transient transfection of CETP cDNA into COS-7 cells showed that high density lipoprotein (HDL)-mediated efflux of free cholesterol from the cells expressing CETP was much higher than that from mock-transfected cells, while uptake of HDL-lipids was not affected in cells transfected with CETP cDNA. Efflux of free cholesterol from the Mphi obtained from CETP deficiency was significantly decreased compared with that from normal subjects. These data indicate that CETP is expressed in Mphi in the atherosclerotic lesions and may possess an anti-atherogenic function to remove cholesterol from the cells, suggesting another role of CETP at the initial step of RCT.

Adult↗
PubMed2001

Clinical investigation of endothelin-1 and nitric oxide in patients with portal hypertension focusing on plasma levels and immunohistological staining of liver tissues.

Plasma concentrations of endothelin-1 (ET-1) and nitrite/nitrate of patients with portal hypertension were measured. Sixteen patients with liver cirrhosis (the LC group) and 14 patients with idiopathic portal hypertension (the IPH group) and 12 healthy subjects (normal controls) were included in this study. The peripheral venous plasma concentration of ET-1 was significantly higher in the LC group (6.69+/-2.44 pg/ml) than in the IPH group (3.07+/-0.84 pg/ml) and normal controls (1.79+/-0.36 pg/ml), while the value in the IPH group was also significantly higher than that in normal controls. The peripheral venous plasma concentration of nitrite/nitrate was significantly higher in the LC group (67.7+/-38.9 &mgr;Mol/l) than in the IPH group (32.3+/-24.4 &mgr;Mol/l) and normal controls (26.1+/-9.8 &mgr;Mol/l). Hepatic venous plasma concentrations of ET-1 and nitrite/nitrate were measured in 8 patients from the LC group and 10 patients from the IPH group. The plasma concentration of ET-1 in the hepatic vein was significantly higher than that in the peripheral vein in both the LC and the IPH groups. The plasma concentration of nitrite/nitrate in the hepatic vein was significantly higher than that in the peripheral vein in the LC group. We also investigated the localization of ET-1, endothelin receptor (ET receptor) and nitric oxide synthase (NOS) in the liver tissue of LC patients (n=10), IPH patients (n=10) and normal controls (n=10). The expressions of ET-1, ET A receptors, ET B receptors, and inducive NOS (iNOS) were detected in patients with LC, and the labeling index (LI) was significantly higher than that in patients with IPH and normal controls. The expressions of ET-1, ET A receptors, and ET B receptors were found in patients with IPH, and the LI was significantly higher than that in normal controls. The expression of endothelial NOS (eNOS) was scarce in both LC and IPH patients. From these results, overproduction of ET-1 in the liver was regarded as one of the causes of the high plasma concentration of ET-1 in patients with LC and IPH. One of the causes of the high plasma concentration of nitrite/nitrate in LC was considered to be overproduction of nitric oxide (NO) in the liver. And we suggested that ET-1 is at a relatively higher density than NO in the hepatic sinusoid in LC and IPH.

Journal Article↗
PubMed2001

Telomerase activation and MAPK pathways in regenerating hepatocytes.

Although there have been many reports on the relationship between the activation of telomerase and carcinogenesis, the role of telomerase in normal cellular growth is still unclear. Recently, the telomerase upregulation during the process of liver regeneration has been reported, but the precise time course of its activity and factors contributing to the activation of telomerase have not yet been fully elucidated. In the present review, we demonstrate the relationship between the activation of the telomerase, the cell cycle progression and the growth-related signaling during the liver regeneration process using an in vivo mouse partial hepatectomy model. Moreover, the importance of the role of the MAPK pathways on the telomerase activity in regenerating hepatocytes is also displayed by using an in vitro culture model. In conclusion, the telomerase activity is upregulated before hepatocytes enter the S phase, and some growth factors such as EGF and HGF contribute to this process. The activation of the growth-related signaling pathways seems to play essential roles in the upregulation of the telomerase activity.

Animals↗
PubMed2000

Reduction of sphingomyelin level without accumulation of ceramide in Chinese hamster ovary cells affects detergent-resistant membrane domains and enhances cellular cholesterol efflux to methyl-beta -cyclodextrin.

We examined the effects of reduction of sphingomyelin level on cholesterol behavior in cells using 2 types of Chinese hamster ovary cell mutants deficient in sphingomyelin synthesis: LY-A strain defective in intracellular trafficking of ceramide for sphingomyelin synthesis, and LY-B strain defective in the enzyme catalyzing the initial step of sphingolipid biosynthesis. Although the sphingomyelin content in LY-A and LY-B cells was approximately 40 and approximately 15%, respectively, of the wild-type level without accumulation of ceramide, these mutant cells were almost identical in cholesterol content and also in plasma membrane cholesterol level to the wild-type cells. However, density gradient fractionation analysis of Triton X-100-treated lysates of cells prelabeled with [(3)H]cholesterol showed that the [(3)H]cholesterol level in the low-density floating fraction was lower in sphingomyelin-deficient cells than in wild-type cells. When cells were exposed to methyl-beta-cyclodextrin, cholesterol was more efficiently fluxed from sphingomyelin-deficient cells than wild-type cells. These results suggest that the steady state level of cholesterol at the plasma membrane is little affected by the sphingomyelin levels in Chinese hamster ovary cells, but that sphingomyelin levels play an important role in the retention of cholesterol in the plasma membrane against efflux to extracellular cholesterol-acceptors, due to interaction between sphingomyelin and cholesterol in detergent-resistant membrane domains.

Amphotericin B↗
PubMed2000

Reconstitution of ATP- and cytosol-dependent transport of de novo synthesized ceramide to the site of sphingomyelin synthesis in semi-intact cells.

Transport of ceramide synthesized at the endoplasmic reticulum to the Golgi compartment, where sphingomyelin (SM) synthase exists, was reconstituted within semi-intact Chinese hamster ovary cells. When [(3)H]ceramide that had been produced from [(3)H]sphingosine at 15 degrees C in perforated cells was chased at 37 degrees C, [(3)H]ceramide-to-[(3)H]SM conversion occurred in a cytosol-dependent manner. In various aspects (i.e. kinetics, ATP dependence, and temperature dependence), [(3)H]ceramide-to-[(3)H]SM conversion in perforated cells was consistent with that in intact cells. The cytosol from LY-A strain, a Chinese hamster ovary cell mutant defective in endoplasmic reticulum-to-Golgi transport of ceramide, did not support [(3)H]ceramide-to-[(3)H]SM conversion in perforated wild-type cells, whereas the wild-type cytosol rescued the conversion in perforated LY-A cells. Brefeldin A-treated cells, in which the endoplasmic reticulum and the Golgi apparatus were merged, no longer required cytosol for conversion of [(3)H]ceramide to [(3)H]SM. These results indicated that the assay of [(3)H]ceramide-to-[(3)H]SM conversion in semi-intact cells is a faithful in vitro assay for the activity of cytosol-dependent transport of ceramide and that LY-A cells are defective in a cytosolic factor involved in ceramide transport. In addition, conversion of [(3)H]ceramide to [(3)H]glucosylceramide in semi-intact cells was little dependent on cytosol, suggesting that ceramide reached the site of glucosylceramide synthesis by a cytosol-independent (or less dependent) pathway.

Adenosine Triphosphate↗
PubMed2000

Different mechanisms between premitotic apoptosis and postmitotic apoptosis in X-irradiated U937 cells.

PURPOSE: Apoptosis is currently being evaluated for its importance as a pathway of radiation-induced cell death. However, the difference in the mechanisms between premitotic and postmitotic apoptosis following X-irradiation remains not well understood. We show here that the human monoblastoid cell line U937 can be induced to undergo these two different types of apoptosis. METHODS AND MATERIALS: U937 cells were irradiated at a dose of 5 or 20 Gy, and the DNA fragmentation rate was measured by both flow cytometric analysis and gel electrophoresis. Activation of caspase-3 was detected by Western blot analysis and fluorogenic assay using acetyl-Asp-Glu-Val-Asp-7-amino-4-methyl-coumarin (Ac-DEVD-AMC). Detection of mitochondrial transmembrane potential (DeltaPsi) was performed by using Rho123. Chasing of S-phase fraction following X-irradiation was performed after labeling with 5-bromo-2'-deoxyuridine (BrdU). Thymidine was used for synchronization of the cells. Inhibition of caspase-3 activity was achieved by Acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO). RESULTS: Time courses of the apoptotic rates, caspase activation, and DeltaPsi indicated that two different types of cell death were induced by the different X-ray doses. High-dose X-ray (20 Gy) induced a rapid and strong apoptosis, whereas low-dose X-ray (5 Gy) induced a slow and mild apoptosis. Cell-cycle analyses revealed that there was cell death before cell division in the former apoptosis but the cells must be dying after cell division in the latter apoptosis. By means of cell-cycle synchronization, the S-phase cells proved to be the most sensitive fraction to premitotic apoptosis, but an obvious difference in the susceptibility to cell death among the cell-cycle phases was not observed in postmitotic apoptosis. Ac-DEVD-CHO treatment effectively blocked caspase activity and premitotic apoptosis, but it failed to block postmitotic apoptosis. CONCLUSIONS: Irradiation of U937 cells at different X-ray doses induced two different types of apoptotic cell death, premitotic apoptosis and postmitotic apoptosis, which are characterized by the time course and cell-cycle specificity. Decision concerning these two types of apoptotic cell death may be made by the difference in the magnitude of cell damage following X-irradiation.

Apoptosis↗
PubMed2000

Neutral sphingomyelinase activity dependent on Mg2+ and anionic phospholipids in the intraerythrocytic malaria parasite Plasmodium falciparum.

Sphingolipid metabolism and metabolites are important in various cellular events in eukaryotes. However, little is known about their function in plasmodial parasites. Here we demonstrate that neutral sphingomyelinase (SMase) involved in the sphingomyelin (SM) catabolism is retained by the intraerythrocytic parasite Plasmodium falciparum. When assayed in a neutral pH buffer supplemented with Mg(2+) and phosphatidylserine, an activity for the release of the phosphocholine group from SM was detected in parasite-infected, but not in uninfected, erythrocyte ghosts. The SMase activity in the parasite-infected erythrocyte ghosts was enhanced markedly by anionic phospholipids including unsaturated but not saturated phosphatidylserine. Mn(2+) could not substitute for Mg(2+) to activate SMase in parasite-infected erythrocyte ghosts, whereas both Mn(2+) and Mg(2+) activated mammalian neutral SMase. The specific activity level of SMase was higher in isolated parasites than in infected erythrocyte ghosts; further fractionation of lysates of the isolated parasites showed that the activity was bound largely to the membrane fraction of the parasites. The plasmodial SMase seemed not to hydrolyse phosphatidylcholine or phosphatidylinositol. The plasmodial SMase, but not SM synthase, was sensitive to scyphostatin, an inhibitor of mammalian neutral SMase, indicating that the plasmodial activities for SM hydrolysis and SM synthesis are mediated by different catalysts. Our finding that the malaria parasites possess SMase activity might explain why the parasites seem to have an SM synthase activity but no activity to synthesize ceramide de novo.

Amides↗
PubMed2000

Identification of the promoter region of human placental 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene.

The placenta-type isozyme of human 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (HP2K) is expressed in several tissues such as placenta, brain, testis, liver, kidney, skeletal muscle, and primary blood mononuclear cells. To better understand the regulation of HP2K gene expression, we isolated and characterized its genomic DNA, which includes the promoter region. The results of oligo-capping analysis indicate that the transcription start point (tsp) is an adenine residue 329 bp upstream of the translational start codon. DNA sequence analysis of this gene shows that the promoter region that contains the TATA box sequence and the 5'-UTR is different from the other known PFK-2/F2, 6BPase genes. In addition, its 5'-flanking and 5'-UTR both have G + C-rich sequences containing Sp1 binding sites. To identify the promoter/enhancer region of HP2K gene, we performed transfection analyses of human choriocarcinoma BeWo cells with HP2K promoter-luciferase constructs. These experiments identified a promoter region 164 bp upstream from the tsp and an enhancer region between -1265 and -1329 on the 5'-flanking sequences. We also showed that Sp1 sites were not essential for HP2K transcription. Following transfection, stimulation experiments with serum, progesterone and phorbol 12-myristate 13-acetate showed that only the construct with the enhancer containing putative early growth response-1 binding motif was responsive to serum. We propose that the transcription of HP2K is strictly controlled by tissue-specific factors even though its genomic DNA contains several transcriptional elements.

5' Untranslated Regions↗
PubMed2000

Inhibition of tumor cell growth by a specific 6-phosphofructo-2-kinase inhibitor, N-bromoacetylethanolamine phosphate, and its analogues.

The high rate of glycolysis despite the presence of oxygen and mitochondria in tumor cells implies an important role for this process in cell division. The rate of glycolysis is assumed to be dependent on the cellular concentration of fructose 2,6-bisphosphate, the concentration of which in turn depends on a bifunctional enzyme and the ratio of this enzyme's 6-phosphofructo-2-kinase versus its fructose 2,6-bisphosphatase activities. To prove the hypothesis that inhibition of glycolysis in tumor cells by 6-phosphofructo-2-kinase inhibitors would cause inhibition of tumor cell proliferation, ten N-bromoacetylethanolamine phosphate analogues were designed, synthesized, and tested. They were screened for their activities against various human tumor cell lines to study the effects of inhibition of glycolysis on cell proliferation. The relationship between the structure of these compounds and their inhibitory activity on cell proliferation was also discussed. It was found that the activity of N-(2-methoxyethyl)-bromoacetamide, N-(2-ethoxyethyl)-bromoacetamide, and N-(3-methoxypropyl)-bromoacetamide was comparable to that of the positive control AraC. These three inhibitors showed in vivo anticancer effects in P388 transplant BDF1 mice.

Animals↗
PubMed2000

[A case of ectopic prostatic tissue in urinary bladder and uretero-seminal vesicle fistula with ipsilateral aplastic kidney].

A case of ectopic prostatic tissue in urinary bladder and uretero-seminal vesicle fistula with ipsilateral aplastic kidney is reported. A 29-year-old man was admitted to our hospital for residual sensation and perineal pain. Ultrasound examination revealed a cystic change in the right side of urinary bladder. C.T. and M.R.I. revealed a defect of a right kidney and ipsilateral ureteral dilation and seminal vesicle swelling. A cystscopy under lumbar anesthesia revealed a edematous cystic change of the right side of trigone and defect of the right ureteral orifice. Trans urethral resection of the wall of urinary bladder was simultaneously performed. And the contents of the cyst were sperum and old red blood cells. A retrograde ureterograph showed blind-ending ureter at L5 level and seminal vesicle swelling. A resected bladder tissue contained prostatic tissue. After operation he was free from complaints. Uretero-seminal vesicle fistula with ipsilater aplastic kidney is rare disease. In addition a case complicated ectopic prostatic tissue in urinary bladder was not reported in Japanese medical literature.

Adult↗