Biomedical subjects

K Imai

Publications and source records attributed to K Imai.

At least 199 records · Page 11Linked to original sources

PubMed2001

Frequent alterations of the beta-catenin and TCF-4 genes, but not of the APC gene, in colon cancers with high-frequency microsatellite instability.

High-frequency microsatellite instability (MSI-H) due to defective DNA mismatch repair (MMR) is a characteristic of the majority of tumors from kindreds with hereditary nonpolyposis colorectal cancer (HNPCC) and a subset of sporadic cancers. To better understand the molecular characteristics of colon cancers with MSI-H, we analyzed these cancers for alterations of genes, such as APC, beta-catenin, and TCF-4 genes, involved in the Wnt signaling pathway. Following the National Cancer Institute (NCI) criteria, 385 unselected colon cancers were classified as follows: 50 (13%) MSI-H tumors, 36 (9%) low-frequency MSI (MSI-L) tumors, and 299 (78%) microsatellite stable (MSS) tumors. The frequency of APC mutations was significantly lower in MSI-H tumors (9 out of 50) than in MSI-L (12 out of 20) and MSS (66 out of 100) tumors (P = 0.0005 and P < 0.0001, respectively). In contrast, the frequency of exon 3 mutations in the beta-catenin gene was higher in MSI-H tumors (10 out of 50) than in MSI-L tumors (0 out of 30; P = 0.0110) and MSS tumors (3 out of 100; P = 0.0010). Frameshift mutations in a (A)9 tract of the TCF-4 gene were detected in 44% (22 out of 50) of MSI-H tumors, but not in any of the 20 MSI-L tumors or 40 MSS tumors. In total, 78% of MSI-H tumors and 84% of the remaining tumors had at least one alteration in APC, beta-catenin, or the TCF-4 genes. Although further analysis is needed to functionally characterize the consequences of each of these alterations on beta-catenin/TCF target gene expression, our results suggest that the activation of the Wnt signaling pathway plays a pivotal role in colon tumorigenesis, irrespective of MSI status.

Adenocarcinoma↗

PubMed2001

Characterization of soluble CD40 ligand released from human activated platelets.

We report here that soluble CD40 ligand (sCD40L) is released from human platelets when activated with collagen or thrombin. The sCD40L was detectable in the culture supernatants of platelets within 30 min after stimulation in vitro, and reached maximal levels in 3 h. The release was blocked by the metalloproteinase inhibitor, KB8301, indicating that the soluble CD40L is made by cleaving the membrane bound CD40L expressed on activated platelets. The sCD40L was undetectable in the supernatant of the activated platelets obtained from patients with X-linked hyper IgM syndrome (XHIM), who have defects in CD40L gene. Since sCD40L has been shown to have biologic function on the activation of vascular endothelial cells and B cells, these findings suggest that platelets play some roles in both inflammation and humoral immune response by releasing soluble CD40L.

Antibody Formation↗

PubMed2000

p53-inducible wip1 phosphatase mediates a negative feedback regulation of p38 MAPK-p53 signaling in response to UV radiation.

The stress-responsive p38 MAPK, when activated by genotoxic stresses such as UV radiation, enhances p53 activity by phosphorylation and leads to cell cycle arrest or apoptosis. Here we report that a member of the protein phosphatase type 2C family, Wip1, has a role in down-regulating p38-p53 signaling during the recovery phase of the damaged cells. Wip1 was originally identified as a gene whose expression is induced following gamma or UV radiation in a p53-dependent manner. We found that Wip1 is also inducible by other environmental stresses, such as anisomycin, H(2)O(2) and methyl methane sulfonate. UV-induction of Wip1 requires p38 activity in addition to the wild-type p53. Wip1 selectively inactivates p38 by specific dephosphorylation of its conserved threonine residue. Furthermore, Wip1 expression attenuates UV-induced p53 phosphorylation at Ser33 and Ser46, residues previously reported to be phosphorylated by p38. Wip1 expression also suppresses both p53-mediated transcription and apoptosis in response to UV radiation. These results suggest that p53-dependent expression of Wip1 mediates a negative feedback regulation of p38-p53 signaling and contributes to suppression of the UV-induced apoptosis.

Animals↗

PubMed2000

Natural cytotoxic activity of peripheral-blood lymphocytes and cancer incidence: an 11-year follow-up study of a general population.

BACKGROUND: One of the most critical questions in immunosurveillance is whether differences between individuals with regards to natural immunological host defence can predict future development of cancer. Although this question has so far remained open, there are clear indications of significant roles of several naturally cytotoxic lymphocytes in preventing the development of cancer. We began a prospective cohort study among a Japanese general population in 1986, using various immunological and biochemical markers. METHODS: Natural cytotoxic activity of peripheral-blood mononuclear cells was assessed by isotope-release assay in 3625 residents of a Japanese population mostly older than 40 years of age, between 1986 and 1990. Immunological and biochemical markers were also measured, and participants were given a questionnaire on lifestyle. We did an 11-year follow-up survey of the cohort members looking at cancer incidence and death from all causes, and analysed the association between cytotoxic activity of peripheral-blood lymphocytes assessed at baseline and cancer incidence found in the subsequent follow-up. FINDINGS: 154 cancer cases were used in the analysis. When we categorised the cytotoxic activity of peripheral-blood lymphocytes by tertiles, age-adjusted relative risk of cancer incidence (all sites) was 0.72 (95% CI 0.45-1.16) for men with high cytotoxic activity, and 0.62 (0.38-1.03) for men with medium cytotoxic activity, taking the risk of those with low cytotoxic activity as reference. For women with high cytotoxic activity relative risk was 0.52 (0.28-0.95), and for those with medium cytotoxic activity 0.56 (0.31-1.01). For both sexes with high and medium cytotoxic activity risk was 0.63 (0.43-0.92) and 0.59 (0.40-0.87), respectively. INTERPRETATION: Our results indicate that medium and high cytotoxic activity of peripheral-blood lymphocytes is associated with reduced cancer risk, whereas low activity is associated with increased cancer risk suggesting a role for natural immunological host defence mechanisms against cancer.

Adult↗

PubMed2000

Enhancement of metastatic properties of pancreatic cancer cells by MUC1 gene encoding an anti-adhesion molecule.

MUC1 mucin expression has been shown to be associated clinicopathologically with metastasis and poor clinical outcome in a variety of tumors. To further investigate this finding experimentally, human pancreatic cancer S2-013 cells overexpressing MUC1 were used for spontaneous metastatic potential in nude mice. It was found that the number of lung metastases of MUC1 transfectants was significantly higher than that of control cells. To analyze the molecular mechanisms that underlie the increased metastatic activity, in vitro adhesion assays were performed. MUC1 mucin expression enhancedin vitro invasiveness and motility of S2-013 cells, and decreased the binding of S2-013 cells to type I collagen, Type IV collagen and laminin. Similar effects were not observed for cells expressing tandem repeat-deleted MUC1 cDNA. Adhesion properties were abolished by benzyl-alpha-GalNAc treatment, indicating that glycosylation of the extracellular domain of MUC1 was essential for these biological adhesive functions. Our data support the hypothesis that MUC1 expression contributes to the metastatic ability of pancreatic cancer cells.

Acetylgalactosamine↗

PubMed2000

Frequent hypermethylation of CpG islands and loss of expression of the 14-3-3 sigma gene in human hepatocellular carcinoma.

The 14-3-3 sigma gene has been implicated in G2/M cell cycle arrest by p53. Frequent inactivation of the 14-3-3 sigma gene by hypermethylation of CpG islands has recently been reported in human breast carcinoma. The aim of this study was to examine the methylation status of CpG islands of the 14-3-3 sigma gene in hepatocellular carcinoma (HCC). The methylation status of the 14-3-3 sigma gene was evaluated in four normal liver tissues and 19 paired specimens of carcinoma and adjacent non-tumorous liver tissues using bisulfite-single strand conformation polymorphism (bisulfite-SSCP), a combination of sodium bisulfite modification and fluorescence-based polymerase chain reaction (PCR)-SSCP. The 14-3-3 sigma protein expression was examined by immunohistochemical staining. Hypermethylation of CpG islands of the 14-3-3 sigma gene was detected in 89% (17/19) of the HCC tissues but not in any of the four normal liver tissues. All of the 14 methylation-positive HCC samples analysed by immunohistochemistry showed loss of 14-3-3 sigma expression, while both of the methylation-negative HCC samples retained the expression, and a significant correlation was found between methylation and loss of expression. Lower levels of methylation were detected in adjacent non-tumorous liver tissues (6/16 in cirrhotic tissues and 1/3 in chronic hepatitis tissues), but the 14-3-3 sigma expression was retained in all of these tissues. In a methylation-positive HCC cell line, HLE, 5-aza-2'-deoxycytidine (5-aza-dC)-induced demethylation of CpG islands led to reactivation of gene expression, indicating that hypermethylation plays a causal role in inactivation of the 14-3-3 sigma gene in HCC. Hypermethylation and the resulting loss of expression of the 14-3-3 sigma gene corresponds to one of the most common abnormalities reported to date in HCC, suggesting their crucial role in the development and/or progression of HCC.

14-3-3 Proteins↗

PubMed2000

Ultramicroanalysis of reducing carbohydrates by capillary electrophoresis with laser-induced fluorescence detection as 7-nitro-2,1,3-benzoxadiazole-tagged N-methylglycamine derivatives.

A method for ultramicroanalysis of carbohydrates using capillary electrophoresis with laser-induced fluorescence detection was developed, based on precapillary conversion to 7-nitro-2,1, 3-benzoxadiazole (NBD)-tagged N-methylglycamines. Although the derivatization involves two-step reactions, i.e., reductive N-methylamination followed by condensation with NBD-F, they can be carried out in a one-pot fashion and proceed quantitatively within ca. 50 min in total. Since the reaction conditions are mild, it does not cause desialylation. The derivatives can be well separated by capillary electrophoresis and sensitively detected by argon laser-induced fluorescence. It allowed detection of monosaccharides of down to nanomolar concentrations for analytical sample solution, which correspond to the attomole injected amounts, and good linearity was observed over a wide range. It was also successfully applied to analysis of N-glycans in a microgram quantity of a glycoprotein. Studies on the cleanup of derivatized product are also described in relation to N-glycan analysis.

4-Chloro-7-nitrobenzofurazan↗

PubMed2000

d-Aspartate in a prolactin-secreting clonal strain of rat pituitary tumor cells (GH(3)).

d-Aspartate (d-Asp) is found in prolactin (PRL)-containing cells of the rat anterior pituitary gland [Lee et al., Brain Res. 838, 193-199, 1999]. In order to determine whether d-Asp is actually produced by the anterior pituitary gland and whether it plays a physiological role in PRL function, a PRL-secreting clonal strain of rat pituitary tumor cells (GH(3)) was employed in this study. HPLC analysis and immunocytochemical staining detected the presence and synthesis of d-Asp in the cytoplasm of these cells. In addition, thyrotropin-releasing hormone-stimulated PRL secretion was increased in a dose-dependent fashion by d-Asp from these cells. These results suggest that the anterior pituitary gland synthesizes d-Asp and that d-Asp acts as a messenger in this gland.

Animals↗

PubMed2000

A region of the sulfonylurea receptor critical for a modulation of ATP-sensitive K(+) channels by G-protein betagamma-subunits.

To determine the interaction site(s) of ATP-sensitive K(+) (K(ATP)) channels for G-proteins, sulfonylurea receptor (SUR2A or SUR1) and pore-forming (Kir6.2) subunits were reconstituted in the mammalian cell line, COS-7. Intracellular application of the G-protein betagamma2-subunits (G(betagamma)(2)) caused a reduction of ATP-induced inhibition of Kir6.2/SUR channel activities by lessening the ATP sensitivity of the channels. G(betagamma)(2) bound in vitro to both intracellular (loop-NBD) and C-terminal segments of SUR2A, each containing a nucleotide-binding domain (NBD). Furthermore, a single amino acid substitution in the loop-NBD of SUR (Arg656Ala in SUR2A or Arg665Ala in SUR1) abolished the G(betagamma)(2)-dependent alteration of the channel activities. These findings provide evidence that G(betagamma) modulates K(ATP) channels through a direct interaction with the loop-NBD of SUR.

ATP-Binding Cassette Transporters↗

PubMed2000

An automatic analyzer for catecholamines and their 3-O-methyl metabolites using a micro coulometric flow cell as a postcolumn reactor for fluorogenic reaction.

A coulometric flow cell for a miniaturized LC system was developed. The cell was examined, as 3-O-methyl catecholamines were converted to their relative omicron-quinones for subsequent fluorometric and chemiluminescence detection. Its performance was evaluated in comparison with commercially available amperometric and coulometric detectors in terms of specification of the low dead volume and high conversion efficiency. The fully automated small-bore LC analyzer for simultaneous determination of catecholamines and their 3-O-methyl metabolites included precolumn pretreatment, column switching, column separation, postcolumn oxidative conversion, fluorometric derivatization, and chemiluminescence detection. The detection limits were 0.3-2.0 fmol for catecholamines and their 3-O-methyl metabolites. Because of the high sensitivity, the required volume of rat plasma sample was only 15 microL.

Animals↗

PubMed2000

Histone deacetylase inhibitors suppress IL-2-mediated gene expression prior to induction of apoptosis.

Histone deacetylase (HDAC) inhibitors can induce transcriptional activation of a number of genes and induce cellular differentiation as histone acetylation levels increase. Although these inhibitors induce apoptosis in several cell lines, the precise mechanism by which they do so remains obscure. This study shows that HDAC inhibitors, sodium butyrate and trichostatin A (TSA), abrogate interleukin (IL)-2-mediated gene expression in IL-2-dependent cells. The HDAC inhibitors readily induced apoptosis in IL-2-dependent ILT-Mat cells and BAF-B03 transfectants expressing the IL-2 receptor betac chain, whereas they induced far less apoptosis in cytokine-independent K562 cells. However, these inhibitors similarly increased acetylation levels of histones in both cells. Although histone hyperacetylation is believed to lead to transcriptional activation, the results showed an abrogation of IL-2-mediated induction of c-myc, bag-1, and LC-PTP gene expression. This observed abrogation of gene expression occurred prior to phosphatidylserine externalization, a process that occurs in early apoptotic cells. Considering the biologic role played by IL-2-mediated gene expression in cell survival, these data suggest that its abrogation may contribute to the apoptotic process induced by HDAC inhibitors. (Blood. 2000;96:1490-1495)

Animals↗

PubMed2000

Inactivation of the 14-3-3 sigma gene is associated with 5' CpG island hypermethylation in human cancers.

The cell cycle checkpoint plays an important role in maintaining the integrity of cells. Recently, one of the 14-3-3 protein family members, 14-3-3sigma, was shown to be regulated by p53 and to play a role in the G2-M-phase checkpoint. To determine whether 14-3-3sigma is inactivated in human cancers, the methylation status of the 5' region of 14-3-3sigma was investigated in a series of gastric, colorectal, and hepatocellular cancer cell lines. Of 22 cell lines examined, 6 showed aberrant methylation. The methylation status of 14-3-3sigma was found to be correlated with loss of expression, which was restored by 5-aza-2'-deoxycytidine treatment. Furthermore, normal G2 arrest after DNA damage was not demonstrated in the cell lines with methylation. In primary gastric cancers, 14-3-3sigma hypermethylation was observed frequently in 26 of 60 (43%) cases and observed more frequently in poorly differentiated adenocarcinomas (P = 0.0017). Our findings suggest that 14-3-3sigma is inactivated by aberrant methylation of the 5' region in various human cancers and that it might play an important role in the development of undifferentiated gastric cancers.

14-3-3 Proteins↗

PubMed2000

Observation of a spin-flip M1 transition in 7(lambda)Li.

Using a large-acceptance germanium detector array (Hyperball), we have observed a spin-flip M1 gamma transition between the ground-state spin doublet of (7)(Lambda)Li (3/2(+)-->1/2(+)). The observed energy of 691.7+/-0.6(stat)+/-1.0(syst) keV provides crucial information on the strength of the spin-spin interaction between a Lambda and a nucleon. This is the first observation of well-identified hypernuclear gamma transitions using germanium detectors.

Journal Article↗

PubMed2000

Occurrence and determination of a natriuretic hormone, 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxy chroman, in rat plasma, urine, and bile.

The occurrence of a new natriuretic hormone, 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxy chroman (LLU-alpha, gamma-CEHC) in rat plasma was demonstrated and its concentration was determined using a coupled-column HPLC with a fluorometric derivatization with 4-N,N-dimethylaminosulfonyl-7-piperazino-2,1,3-benzoxadiazol e (DBD-PZ) followed by O-acetylation. The concentration of LLU-alpha was 328 +/- 113 nM in rat plasma (N = 5). LLU-alpha was found in not only urine, but also bile, suggesting an enterohepatic circulation in body. We also assigned the configuration at C-2 of LLU-alpha in these biological fluids as (S)-form by an HPLC with a chiral column. The LLU-alpha concentration decreased significantly by fasting for 3 days (P < 0.01). These results support the possibility that LLU-alpha is produced from gamma-tocopherol in diet via oxidative metabolism without racemization.

Animals↗

PubMed2000

Substitution of the heme binding module in hemoglobin alpha- and beta-subunits. Implication for different regulation mechanisms of the heme proximal structure between hemoglobin and myoglobin.

In our previous work, we demonstrated that the replacement of the "heme binding module," a segment from F1 to G5 site, in myoglobin with that of hemoglobin alpha-subunit converted the heme proximal structure of myoglobin into the alpha-subunit type (Inaba, K., Ishimori, K. and Morishima, I. (1998) J. Mol. Biol. 283, 311-327). To further examine the structural regulation by the heme binding module in hemoglobin, we synthesized the betaalpha(HBM)-subunit, in which the heme binding module (HBM) of hemoglobin beta-subunit was replaced by that of hemoglobin alpha-subunit. Based on the gel chromatography, the betaalpha(HBM)-subunit was preferentially associated with the alpha-subunit to form a heterotetramer, alpha(2)[betaalpha(HBM)(2)], just as is native beta-subunit. Deoxy-alpha(2)[betaalpha(HBM)(2)] tetramer exhibited the hyperfine-shifted NMR resonance from the proximal histidyl N(delta)H proton and the resonance Raman band from the Fe-His vibrational mode at the same positions as native hemoglobin. Also, NMR spectra of carbonmonoxy and cyanomet alpha(2)[betaalpha(HBM)(2)] tetramer were quite similar to those of native hemoglobin. Consequently, the heme environmental structure of the betaalpha(HBM)-subunit in tetrameric alpha(2)[betaalpha(HBM)(2)] was similar to that of the beta-subunit in native tetrameric Hb A, and the structural conversion by the module substitution was not clear in the hemoglobin subunits. The contrastive structural effects of the module substitution on myoglobin and hemoglobin subunits strongly suggest different regulation mechanisms of the heme proximal structure between these two globins. Whereas the heme proximal structure of monomeric myoglobin is simply determined by the amino acid sequence of the heme binding module, that of tetrameric hemoglobin appears to be closely coupled to the subunit interactions.

Chromatography, Gel↗

PubMed2000

Storage of lipid droplets in and production of extracellular matrix by hepatic stellate cells (vitamin A-storing cells) in Long-Evans cinnamon-like colored (LEC) rats.

LEC rats spontaneously develop hepatocellular carcinoma with cholangiofibrosis after chronic hepatitis, but the mechanism of development of the hepatic injury is not clear. To investigate the role of hepatic stellate cells in induction or suppression of hepatic fibrosis, we morphologically examined the liver of LEC rats. Accumulation of copper was analyzed by the Danscher-Timm's sulfide-silver method. Histopathological changes were evaluated by hematoxylin and eosin staining, and by Masson's trichrome method. Activated stellate cells were identified by immunostaining method for alpha-smooth muscle actin. Cytological alterations of the stellate cells were investigated by transmission electron microscopy. To evaluate the lipid content in the stellate cells, we analyzed the area of lipid droplets of the cells by morphometric analysis. Also for evaluation of the changes in the number of stellate cells, the numbers of nucleated stellate cells and parenchymal cells were counted and statistically analyzed. Hepatic parenchymal cells showed excessive accumulation of copper at 5 weeks of age. Submassive necrosis was observed at 19 weeks of age. The liver of LEC rats 1.5 years of age showed cholangiofibrosis and subcellular injury of hepatic parenchymal cells. However, no diffuse hepatic fibrosis was observed in the liver, and hepatic stellate cells around the regions of cholangiofibrosis were negative for alpha-smooth muscle actin. The area of lipid droplets of a stellate cell in the liver of LEC rats was 1.6 to 1.8 times as large as that of normal Wistar rats. The hepatic stellate cells did not participate in the accumulation of collagen fibers around themselves when the cells contained a large amount of vitamin A-lipid droplets, even though the development of hepatic lesions was in progress. Our present data are consistent with our previous hypothesis that there is an antagonistic relationship between the storage of vitamin A and the production of collagen in stellate cells.

Actins↗

PubMed2000

Transthyretin binds amyloid beta peptides, Abeta1-42 and Abeta1-40 to form complex in the autopsied human kidney - possible role of transthyretin for abeta sequestration.

The deposition of amyloid beta protein (Abeta), a proteolytic cleavage product of amyloid precursor protein (APP), is an invariable pathological feature of the Alzheimer's disease brain, while APP gene is widely expressed in all neuronal and non-neuronal tissues with the highest levels of expression in the brain, and kidney. To understand the role transthyretin (TTR) plays in the sequestration mechanism of Abeta in the kidney, we have investigated interactions of TTR with Abeta1-40 and Abeta1-42 molecules by an immunoprecipitation method, in vitro binding studies, and overlay assay. These in vivo and in vitro biochemical experiments showed that TTR bound Abeta1-42 preferentially, and Abeta1-40 only to a limited extent, to form TTR-monomer and -dimer-Abeta complexes in the normal human kidney. We provide new evidence supporting the hypothesis that TTR, an Abeta binding protein, plays an important role in the sequestration of Abeta and prevents amyloid formation in the kidney.

Amyloid beta-Peptides↗

PubMed2000

Development of an amino acid sequence and D/L-configuration determination method of peptide with a new fluorescence Edman reagent, 7-methylthio-4-(2,1,3-benzoxadiazolyl) isothiocyanate.

On the basis of the relationship between the fluorescence characteristics of the benzofurazan compounds and the Hammett constants (sigma p), a new fluorescence Edman reagent, 7-methylthio-4-(2,1,3-benzoxadiazolyl) isothiocyanate (MTBD-NCS) was designed and synthesized. MTBD-thiohydantoin (TH)-amino acid derivatives produced by the Edman sequencing method gave fluorescence, whereas other degradation byproducts such as MTBD-thiocarbamoyl (TC)- or carbamoyl (CA)-amino acids did not fluoresce. MTBD-NCS was applicable as an Edman sequencing reagent to the simultaneous determination of both the sequence and D/L-configuration of amino acids in peptides. Boron trifluoride (BF3) and HC1/methanol were adopted as the cyclization/cleavage and conversion reagents to suppress the amino acid residue racemization. The MTBD-TH-amino acids were separated on a reversed-phase column for amino acid sequencing, and their enantiomers were resolved on two types of polysaccharide-based chiral stationary phases for D/L-configuration determination. The method was successfully applied to the sequence and D/L-configuration determination of D-amino acid-containing peptide [D-Ala2]-deltorphin II.

Amino Acids↗