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T Ikenoue

Publications and source records attributed to T Ikenoue.

At least 19 recordsLinked to original sources
PubMed2006

Absence of tyrosine kinase mutations in Japanese colorectal cancer patients.

Tyrosine kinases, which are important regulators of intracellular signal-transduction pathways, have mutated forms that are often associated with oncogenesis and are attractive targets for therapeutic intervention. Recently, systematic mutational analyses of tyrosine kinases revealed that a minimum of 30% of colorectal cancer contain at least one mutation in the tyrosine kinases. To further explore these mutations, we examined all reported mutations of NTRK3, FES, KDR, EPHA3, NTRK2, JAK1, PDGFRA, EPHA7, EPHA8, ERBB4, FGFR1, MLK4 and GUCY2F genes in the 24 colorectal cancer cell lines. Unexpectedly, among 24 colorectal cancer cell lines, only two cell lines (LoVo and CaR1) harbored mutation C1408T (R470C) in MLK4 gene. The mutation rate was extremely low compared to that previously reported. Therefore, we analyzed mutations in 46 colorectal cancer samples resected from the same number of Japanese patients. Surprisingly, none of the 46 samples contained any of the mutations reported. Based on our study, we advise that a more comprehensive tyrosine kinase gene mutation assay is necessary in the future.

Aged↗
PubMed2006

Absence of PIK3CA hotspot mutations in hepatocellular carcinoma in Japanese patients.

A recent study revealed that the p110alpha (PIK3CA), catalytic subunit of phosphatidylinositol 3-kinase (PI3K), is somatically mutated in many types of cancer. For example, PIK3CA is mutated in an estimated 35.6% of hepatocellular carcinoma (HCC) cases. To measure the frequency of PIK3CA hotspot mutations in Japanese HCC patients, exons 9 and 20 of the PIK3CA gene were sequenced in 47 clinical HCC samples. Contrary to expectations, no hotspot mutations were found any of the HCC samples. In addition, we found abnormally migrating waves near the end of exon 9 in the PCR chromatograms from 13 of the 47 samples. PCR amplification and subsequent cloning and sequencing revealed that these chromatograms contained two distinct sequences, the wild-type p110alpha sequence and a different sequence found on human chromosome 22q11.2, the Cat Eye Syndrome region, which contains a putative pseudogene of PIK3CA. These abnormally migrating waves were also found in noncancerous liver tissue, indicating that this was not a result of HCC-associated mutations. Therefore, it is likely that the percentage of hotspot mutations in the PIK3CA gene of Japanese HCC patients is lower than was previously reported.

Adult↗
PubMed2006

The hepatitis B virus X protein enhances AP-1 activation through interaction with Jab1.

Hepatitis B virus X protein (HBx) has many cellular functions and is a major factor in hepatitis and hepatocellular carcinoma caused by HBV infection. A proteomic approach was used to search for HBx-interacting proteins in order to elucidate the molecular mechanism of hepatocarcinogenesis. HBx was attached to myc and flag tags (MEF tags) and expressed in 293T cells; the protein complex formed within the cells was purified and characterized by mass spectrometry. COP9 signalosome (CSN) subunits 3 and 4 were subsequently identified as HBx-interacting proteins. In addition, CSN subunit 5, Jun activation domain-binding protein 1 (Jab1), was shown to be a novel cellular target of HBx. In vivo and in vitro interactions between HBx and Jab1 were confirmed by standard immunoprecipitation and GST pull-down assays. An analysis of HBx deletion constructs showed that amino acids 30-125 of HBx were responsible for binding to Jab1. Confocal laser microscopy demonstrated that HBx was mainly localized in the cytoplasm, while Jab1 was found mainly in the nucleus and partially in the cytoplasm, and that the two proteins colocalized in the cytoplasm. The cotransfection of HBx and Jab1 resulted in substantial activator protein 1 (AP-1) activation and knockdown of endogenous Jab1 attenuated AP-1 activation caused by HBx. In addition, the coexpression of HBx and Jab1 potentiated phosphorylation of JNK, leading to the subsequent phosphorylation of c-Jun, whereas the level of c-Jun and JNK phosphorylation induced by HBx was decreased in Jab1 knockdown cells. These results suggest that the interaction between HBx and Jab1 enhances HBx-mediated AP-1 activation.

COP9 Signalosome Complex↗
PubMed2005

Association between endometriosis and genetic polymorphisms of the estradiol-synthesizing enzyme genes HSD17B1 and CYP19.

BACKGROUND: Endometriosis, an estrogen-dependent disease, is believed to be influenced by multiple genetic and environmental factors. Here, we evaluated whether the risk and severity of endometriosis are associated with polymorphisms in estradiol-synthesizing enzyme genes: the Ser312Gly polymorphism in 17-beta-hydroxysteroid dehydrogenase type 1 (HSD17B1) and the Arg264Cys polymorphism in cytochrome P450, subfamily XIX (CYP19). METHODS: All participants underwent diagnostic laparoscopy, and the stage of endometriosis was determined according to the Revised American Fertility Society classification. Of the 138 women enrolled, 59 had no endometriosis, 21 had stage I, 10 had stage II, 23 had stage III and 25 had stage IV. SNPs were discriminated by allele-specific oligonucleotide hybridization. RESULTS: Individuals having at least one A-allele (A/G or A/A genotype) of HSD17B1 showed a significantly increased risk of endometriosis (A/G genotype: adjusted OR, 3.06; 95%CI 1.21-7.74; A/A genotype: adjusted OR, 3.02; 95%CI 1.08-8.43). There was a significant trend associating A/G + A/A genotypes with severity of endometriosis (P for trend < 0.01). No statistically significant association was found for the CYP19 polymorphism. CONCLUSIONS: Evidence for association between the Ser312Gly polymorphism in HSD17B1 and endometriosis was found in a Japanese population. The A-allele of HSD17B1 appears to confer higher risk for endometriosis.

17-Hydroxysteroid Dehydrogenases↗
PubMed2002

Fibroblasts from the inner granulation tissue of the pseudocapsule in hips at revision arthroplasty induce osteoclast differentiation, as do stromal cells.

BACKGROUND: It has previously been shown that many osteoclast precursors are included in the granulation tissue within the pseudocapsule obtained at revision arthroplasty from hips with osteolysis. In vitro culture of only cells isolated from the granulation tissue has been previously shown to generate many mature osteoclasts. OBJECTIVE: To investigate the presence or otherwise of supporting cells, similar to stromal cells, which differentiate osteoclasts within the granulation tissue. METHODS: Cells isolated from the granulation tissue were cultured alone, and after four weeks fibroblast-like cells (granulation fibroblasts) remained. Rat non-adherent bone marrow cells (NA-BMCs) were co-cultured with the granulation fibroblasts with or without 1alpha,25(OH)2D3 (10(-8) M) or heat treated ROS 17/2.8 cell conditioned medium (ht ROSCM), or both. Multinucleated cells (MNCs), which formed, were assessed by biochemical and functional characterisation of osteoclasts. Receptor activator of NFkappaB ligand (RANKL) was investigated by immunohistochemistry. RESULTS: Co-culture of NA-BMCs and granulation fibroblasts caused the formation of tartrate resistant acid phosphatase (TRAP) positive MNCs, which had the calcitonin receptor (CTR), the Kat-1 antigen, which is specific to the surface of rat osteoclasts, and the ability to form pits in the presence of both 1alpha,25(OH)2D3 and ht ROSCM or in the presence of just ht ROSCM. RANKL was detected in fibroblast-like cells in the granulation tissue. CONCLUSION: These data suggest that granulation fibroblasts support osteoclast differentiation, as do osteoblasts/stromal cells, and may play a part in aseptic loosening.

Aged↗
PubMed2001

Altered expressions of glutamate transporter subtypes in rat model of neonatal cerebral hypoxia-ischemia.

Glutamate transporters are essential for maintaining the extracellular levels of glutamate at synaptic clefts and are regulated developmentally in a subtype-specific manner. We investigated chronological changes of immunoreactivities for glial glutamate transporters GLAST and GLT-1 and a neuronal glutamate transporter, EAAC1, in postnatal 7-day-old rat neocortices and hippocampi at 12, 24, 48 and 72 h after hypoxia-ischemia. Glutamate transporter subtypes are differentially expressed in the ischemic core and the boundary area of the neonatal rat brain with hypoxia-ischemia. Expressions of these glutamate transporters decreased in the ischemic core at 12 h, then immunoreactivities for GLAST and GLT-1 were recovered at the hippocampus. This was accompanied by a GFAP-positive gliosis at 72 h, whereas these immunoreactivities were reduced at the neocortex in the ischemic core. Glial glutamate transporters, especially GLAST, were noted in some astrocytes appearing as apoptosis as well as shrunken pyramidal neurons mainly in the boundary area of the neocortex. Increased perikaryal expression of EAAC1 was associated with that of MAP2 at the border of the boundary area. These temporal and regional expressions of glutamate transporters may contribute towards understanding the excitotoxic cell death mechanism in hypoxic-ischemic encephalopathy during the perinatal period.

Amino Acid Transport System X-AG↗
PubMed2001

Systematic analysis of the TGF-beta-Smad signaling pathway in gastrointestinal cancer cells.

The transforming growth factor-beta (TGF-beta)-Smad signaling pathway has an important role in carcinogenesis. To study the frequency and mechanism of functional impairment of this pathway in human gastrointestinal cancers, we used a reporter assay to examine the response of 38 cell lines (11 colorectal, 9 pancreatic, 10 gastric, and 8 hepatic cancers) to TGF-beta. We then analyzed TGF-beta type II receptor (T beta RII) gene, immunoblots of Smad4, and restoration of the pathway by rescuing T beta R or Smad. We observed impaired signaling in 91% of colorectal, 67% of pancreatic, and 40% of gastric cancer cell lines, but in none of the hepatic cancer cells. We suggest that this pathway does not function as a tumor suppressor in hepatic carcinogenesis. The impairment is due to inactivation of T beta RII and Smad4 in colorectal and pancreatic cancers. However, because the signal was not recovered by rescuing T beta R or Smad genes in TGF-beta-response-defective gastric cancer cell lines, we suggest that novel molecules or mechanisms are involved in the impaired pathway in some gastric cancers.

Blotting, Western↗
PubMed2001

Reduced C-terminal Src kinase activity is correlated inversely with pp60(c-src) activity in colorectal carcinoma.

BACKGROUND: Specific kinase activity of the proto-oncogene product pp60(c-src) is reported to be elevated in patients with carcinoma of the colon, and a novel cytoplasmic protein-tyrosine kinase, C-terminal Src kinase (Csk), has been found to inactivate the members of the Src family protein-tyrosine kinase. In this study, Csk activity and pp60(c-src) activity were examined in colorectal tumors as well as in colon carcinoma cell lines. METHODS: Colorectal carcinoma tissue and adjacent nonneoplastic tissue from 24 patients, from 8 colon carcinoma cell lines, and from 1 normal colon cell line were used. The levels of pp60(c-src) and Csk in colorectal tissue and cell lines were analyzed by Western and/or Northern blot analysis, and their kinase activity levels were measured by in-gel kinase assay. RESULTS: In the samples from 24 patients with colorectal carcinoma, pp60(c-src) kinase activity and protein levels were increased by 7.8 +/- 0.55 and 2.6 +/- 0.13 times the control levels, respectively. Conversely, the Csk protein level and its kinase activity were reduced by 0.53 +/- 0.08 and 0.53 +/- 0.09 times the control levels, respectively. pp60(c-src) kinase activity was correlated inversely with Csk activity (correlation coefficient = -0.71; P < 0.0001). Of the cell lines, pp60(c-src) kinase activity and protein levels, respectively, were 7.4 +/- 1.22 and 1.86 +/- 0.28 times greater than normal control levels. Csk protein level and kinase activity, respectively, were 0.54 +/- 0.13 and 0.52 +/- 0.11 times less normal control levels and were correlated with mRNA amount. CONCLUSIONS: Csk mRNA, protein, and its kinase activity were reduced in colorectal carcinoma and were correlated with pp60(c-src) kinase activity level. The reduced activity of Csk may be involved in the transformation of a subset of colorectal carcinoma.

Aged↗
PubMed2001

Selective and long-term learning impairment following neonatal hypoxic-ischemic brain insult in rats.

We examined four different learning and memory tasks in rats which had been subjected to left carotid artery ligation followed by 2 h hypoxia (8% oxygen) when they were 7 days old. The examination began on the 4th week after insult and continued to 18 weeks post-insult. Compared with the control group, the hypoxic-ischemic group showed significant learning impairments in choice reaction time tasks relating to the attention process, and in plus-maze tasks and water maze tasks which examine long-term reference memory. In eight-arm radial maze tasks representing both short-term working memory and long-term reference memory, inferiority of the hypoxic-ischemic group was transient. Results of the sensorimotor test were normal in the hypoxic-ischemic group although slight flexion and twisting in the right forelimb was observed in 30% of the hypoxic-ischemic group when suspended by the tail. These abnormalities did not affect the results of learning tests. Findings of the study indicate that left-side brain damage produced by hypoxia-ischemia at 7 days of age resulted in selective and long-lasting learning and memory impairment.

Animals↗
PubMed2001

Basic fibroblast growth factor regulates expression of growth factors in rat epiphyseal chondrocytes.

Chondrocytes produce several local regulatory factors such as basic fibroblast growth factor (bFGF), transforming growth factor-beta (TGF-beta) and insulin-like growth factor-I (IGF-I). In this study, we examined the effect of bFGF on the expressions of both mRNA and protein of the growth factors synthesized by chondrocytes. Treatment of chondrocytes with bFGF (1-100 ng/ml) stimulated the mRNA expression of bFGF and TGF-beta up to 121-604% and 130-220% at 12 h compared with the controls, respectively. On the other hand, the treatment of chondrocytes with bFGF (1-100 ng/ml) suppressed IGF-I mRNA expression to 79-47% at 12 h compared with the controls. An enzyme-linked immunosorbent assay (ELISA) revealed that the treatment of chondrocytes with bFGF (1-100 ng/ml) also enhanced the production of TGF-beta proteins in the chondrocytes up to 299-508% at 24 h compared with controls. We conclude that bFGF influenced the local expression of growth factors by chondrocytes, suggesting autoregulation of growth factor expression during chondrogenesis.

Animals↗
PubMed2001

Immature osteoblastic cells express the pro-alpha2(XI) collagen gene during bone formation in vitro and in vivo.

Type XI collagen is predominantly found in cartilage. However, expression of the pro-alpha2(XI) collagen gene (COL11A2) has recently been detected in various non-cartilaginous tissues. We identified the differentiation stage at which COL11A2 was expressed in cultured fetal rat calvarial (FRC) cells and in rat femoral fracture calluses in order to investigate the involvement of COL11A2 during bone formation in vitro and in vivo. We also studied the alternative splicing of exons 6-8 in FRC cells and fracture calluses. In FRC cells, mineralized nodules stained with von Kossa stain were observed from day 9 after confluence. COL11A2 was highly expressed on days 0 and 5, but the expression levels were rapidly decreased on day 9 by Northern blot analysis. During rat femoral fracture repair, intramembranous ossification proceeded and newly formed woven bone was observed on the cortex on day 7 after fracture. In situ hybridization showed that COL11A2 signals were detected in osteoblastic cells in the newly formed woven bone. According to the maturation and remodeling of the woven bone into the trabecular bone, the distribution of the signal for COL11A2 mRNA was limited to the superficial osteoblastic cells of the newly formed trabecular bone. These results demonstrated that COL11A2 was expressed in relatively immature osteoblastic cells during bone formation in vitro and in vivo. RT-PCR showed that the shortest band corresponding to mRNA lacking exons 6-8 was clearly detected when using RNA from soft calluses. In contrast, the largest band corresponding to mRNA with exons 6-8 was predominant when using RNA from FRC cells or from hard calluses on days 7 and 14. These results indicate that the splicing pattern of exons 6-8 in osteoblastic cells is different from the pattern in chondrocytes.

Alternative Splicing↗
PubMed2001

Diagnosis and management of acute obstetrical DIC.

UNLABELLED: Obstetrical disseminated intravascular coagulation (DIC) is usually a very acute, serious complication of pregnancy. The DIC diagnostic criteria in obstetrics (the obstetrical DIC score) help with making a prompt diagnosis and starting treatment early. These DIC diagnostic criteria, in which higher scores are given for clinical parameters than for laboratory parameters, have three components: (1) the underlying disease, (2) clinical symptoms, and (3) laboratory findings. It is justified that it is appropriate to initiate therapy for DIC when the obstetrical DIC score reaches 8 points or more before obtaining the results of coagulation tests. MANAGEMENT: (1) Control of the underlying disease: because prolongation of exposure to the triggering factors worsens DIC, it is important to eliminate the etiologic factors as rapidly as possible. Elimination of the cause of DIC can be easily performed in obstetrics, for example, by cesarean section. (2) Antithrombin (AT) therapy: AT monotherapy (1,500 to 3,000 units/day, 2 days) is preferably employed instead of heparin monotherapy or heparin-AT therapy because of the hemorrhagic side effects of heparin. (3) Synthetic serine protease inhibitors: continuous infusion ofgabexate mesilate (FOY) or nafamostat mesilate (FUT) is effective for DIC. Controlled multicenter trials showed a significant improvement not only in clinical response but also in platelet counts and prothrombin time (PT) in the AT group compared with the FOY group. (4) Activated protein C (APC) can inhibit thrombin generation and accelerate fibrinolytic activity. APC (5,000 to 10,000 units) is administered for 2 days in patients with placental abruption complicated by DIC. APC is a very safe, effective, and useful agent for the treatment of DIC.

Disseminated Intravascular Coagulation↗
PubMed2001

Long-term magnesium sulfate treatment as protection against hypoxic-ischemic brain injury in seven-day-old rats.

OBJECTIVE: Our purpose was to study the effects of long-term treatment with magnesium sulfate on hypoxic-ischemic brain damage in newborn rats. STUDY DESIGN: Seven-day-old rat pups (n = 120) were exposed to unilateral carotid artery ligation and 2 hours of hypoxia (8% oxygen in 92% nitrogen). Neuronal loss was evaluated in 4 groups. The loading dose group (n = 25) received a bolus injection of 270 mg/kg magnesium sulfate intraperitoneally. The maintenance group (n = 26) received 3 days of continuous infusion of magnesium sulfate with a micro-osmotic pump at a rate of 72 mg/kg per hour. The loading dose-plus-maintenance group (n = 23) received both. The control group (n = 46) did not receive magnesium. Seven days after the injury the pups were killed and the brains were removed for analysis. Severity of neuronal loss was evaluated in the cerebral cortex and hippocampus and compared among groups by chi2 test. RESULTS: Cyst formation resulting from necrosis was significantly decreased in the maintenance group (15%) and loading dose-plus-maintenance group (9%) but not in the loading dose group (52%) relative to the control group (46%). Severity of neuronal loss in the cerebral cortex and the hippocampus was also significantly improved in the maintenance group and the loading dose-plus-maintenance group but not in the loading dose group. Mortality rates were not different among the groups. Magnesium ion concentrations at 24 hours were significantly decreased from the preinfusion value of 0.49 +/- 0.01 mmol/L in the control group (0.34 +/- 0.03 mmol/L) but remained within normal ranges in the maintenance group (0.46 +/- 0.02 mmol/L with a pump infusing 72 mg/kg per hour and 0.56 +/- 0.05 mmol/L with a pump infusing 24 mg/kg hour). There was an inverse relationship between the maintenance dose and neuronal loss but not between the maintenance dose and mortality rate. CONCLUSION: Long-term magnesium administration had neuroprotective effects against hypoxia-ischemia in newborn rats.

Animals↗
PubMed2001

Effects of actinomycin D on fetal growth in rats.

OBJECTIVE: The goal of this study was to determine the effects of actinomycin D on fetal growth and prostaglandin dehydrogenase activity and to determine whether these effects could be obtained by administering extracts of fetal calf serum (SS-094). METHODS: Actinomycin D (7 microg/100 g body weight) was injected intraperitoneally on days 11 and 12 of pregnancy to induce growth restriction in rats. In another group, SS-094 was given (0.1 ml/100 g body weight) on days 12, 15, 17 and 19 of pregnancy, following the administration of actinomycin D. Placental prostaglandin dehydrogenase (PGDH) activity was measured on days 15 and 21 of pregnancy. Statistical analysis was performed by ANOVA and Dunnet's test. RESULTS: In the group injected with actinomycin D, fetal weight was significantly restricted (on day 15; p < 0.05; on day 21; p < 0.01), compared to the controls, and this restriction was successfully reversed by SS-094. PGDH activity in the placenta was significantly (p < 0.01) decreased in growth-restricted rats and remained low even after fetal weight recovered. CONCLUSIONS: Growth restriction in pregnant rats was successfully induced by actinomycin D, and SS-094 reversed this restriction. It does not seem that prostaglandin metabolism in the placenta is responsible for growth restriction.

Analysis of Variance↗
PubMed2001

Insulin-meal interval and short-term glucose fluctuation in tightly controlled gestational diabetes mellitus.

OBJECTIVE: To study the effect of two insulin-meal intervals on short-term glucose fluctuations in tightly controlled gestational diabetes mellitus (GDM). METHODS: We performed a prospective and paired study in 11 Japanese GDM women requiring insulin for good glycemic control during the third trimester. The women were subjected to test two insulin-meal intervals: 15 min and 30 min. Both regimens were examined in each patient in random order, 2 days apart. Blood glucose was measured by an automated glucose monitor every 2 min. Short-term glucose fluctuations of the two observations were analyzed by two-way ANOVA for repeated measurements with a post hoc t test (p < 0.05). Data were expressed as mean +/- SD. RESULTS: Daily glucose profiles of the two groups showed that their glycemic controls on the days of observation were good and that the two glucose profile curves were superimposable. A transient decrease in glucose (nadir 62 +/- 6 mg/dl) was observed at 6-10 min of meal ingestion in the 30-min regimen, which was significantly different from the glucose fluctuations during the 15-min regimen. The 2-h postprandial glucose levels were similar in both experiments. CONCLUSIONS: In women with tightly controlled GDM during the third trimester, insulin-meal intervals of 15 min are beneficial when compared with 30-min intervals, in that they avoid preprandial hypoglycemia without increasing 2-h postprandial hyperglycemia.

Adult↗
PubMed2001

Frameshift mutations at mononucleotide repeats in RAD50 recombinational DNA repair gene in colorectal cancers with microsatellite instability.

To identify additional genes targeted for microsatellite instability (MSI), we search for human genes which contain mononucleotide repeats in their coding region, selected 7 genes (RAD50, DNA-PKcs, FLASH, Apaf-1, XPG, CtIP, and MLSN1), and analyzed frameshift mutations in them. Here we report that 60% (3 out of 5) of human colorectal cancer cell lines exhibiting a high frequency of MSI (MSI-H) and 46% (6 out of 13) of MSI-H primary colorectal tumors had mutations in the (A)9 repeat of RAD50 recombinational repair gene. In contrast, no frameshift mutations were found in any of the 5 MSI-negative colorectal cancer cell lines, 8 colorectal tumors exhibiting a low frequency of MSI (MSI-L), or 28 MSI-negative colorectal tumors. No mutations were found in the mononucleotide repeats of 6 other genes, even in MSI-H cancers. These results suggest that RAD50 frameshift mutations may play a role in the tumorigenesis of MSI-H colorectal cancers.

Colorectal Neoplasms↗
PubMed2001

Determination of Helicobacter pylori virulence by simple gene analysis of the cag pathogenicity island.

Nucleic acid amplification was performed for five loci in the cag pathogenicity island (PAI) of Helicobacter pylori (comprising cagA, the cagA promoter region, cagE, cagT, and the left end of cagII [LEC]), and gastric inflammation in patients was evaluated. Of 204 H. pylori isolates from Japanese patients (53 with peptic ulcer, 55 with gastric cancer, and 96 with chronic gastritis), 197 (96.6%) were positive for all five loci. Two isolates (1%) were negative for all five loci, and five isolates (2.4%) were positive for only cagA and LEC. These latter seven isolates were all from patients with mild chronic gastritis. Neutrophil infiltration in gastric mucosa was significantly milder in patients infected with partially or totally deleted-PAI strains than in those with intact-PAI strains. The cagE gene was a more accurate marker of an intact cag PAI than the cagA gene, and cagE seemed to be more useful in discriminating between H. pylori strains causing different rates of disease progression.

Adult↗