Biomedical subjects
K Morishige
Publications and source records attributed to K Morishige.
Assignment of the murine inward rectifier potassium channel Irk2 (Kir2.2) gene to the central region of mouse chromosome 11.
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Transforming growth factor-alpha promotes tumor markers secretion from human ovarian cancers in vitro.
BACKGROUND: The regulatory mechanism of tumor markers secretion has not been well clarified. METHODS: Serum levels of CA 125 and tissue polypeptide antigen (TPA) from 17 patients with Stage III serous cystadenocarcinoma were measured prior to an initial surgical treatment. Epidermal growth factor receptor (EGFR) status was examined by an 125I-EGF binding assay in a human serous cystadenocarcinoma cell (SHIN-3) and in the 17 primary carcinomas. SHIN-3 cell and the EGFR-expressing primary cancer cells (n = 4) were cultured with or without various concentrations of transforming growth factor (TGF-alpha), a ligand for EGFR, and the CA 125 and TPA concentrations in the conditioned media were measured. RESULTS: EGFR was expressed in 12 primary carcinomas and in the SHIN-3 cell, and it was absent in the remaining 5 carcinomas. Pre-therapeutic serum CA 125 and TPA levels were significantly greater (P < 0.05) in patients with EGFR-expressing carcinomas (n = 5). These data suggest a possible involvement of EGFR in regulating these tumor markers secretion. TGF-alpha increased the CA 125 and TPA secretion from SHIN-3 cell. It also promoted the CA 125 secretion in 2 of 4 EGFR-expressing primary ovarian carcinoma specimens. CONCLUSIONS: These results suggest that a signal through the EGFR may be involved in regulating the CA 125 and TPA secretion from human ovarian carcinomas.
Differential distribution of classical inwardly rectifying potassium channel mRNAs in the brain: comparison of IRK2 with IRK1 and IRK3.
Distribution of IRK2 inwardly rectifying potassium channel mRNA in the mouse brain was studied using in situ hybridization histochemistry and compared with those of other classical inwardly rectifying potassium channel (IRK1 and IRK3) mRNAs. All these IRK channel mRNAs were detected in neurons, but not in glial cells. Their distribution patterns in the brain were, however, quite divergent: IRK2 mRNA was detected extremely high in granule cells of cerebellum, relatively high in motor trigeminal nucleus and moderate in olfactory bulb, piriform cortex, cerebral cortex, CA1 through CA3 regions of hippocampus, dentate gyrus and pontine nucleus. On the other hand, IRK1 mRNA was expressed throughout whole brain but in particular subsets of neurons, and IRK3 mRNA was in forebrain. Expression of these three IRK mRNAs overlapped in hippocampus, olfactory bulb, and cerebral cortex. This differential distribution of IRK mRNAs suggests that each of these channels has its specific function in regulation of the excitability of brain neurons.
A novel ATP-dependent inward rectifier potassium channel expressed predominantly in glial cells.
We have isolated a novel inward rectifier K+ channel predominantly expressed in glial cells of the central nervous system. Its amino acid sequence exhibited 53% identity with ROMK1 and approximately 40% identity with other inward rectifier K+ channels. Xenopus oocytes injected with cRNA derived from this clone expressed a K+ current, which showed classical inward rectifier K+ channel characteristics. Intracellular Mg.ATP was required to sustain channel activity in excised membrane patches, which is consistent with a Walker type-A ATP-binding domain on this clone. We designate this new clone as KAB-2 (the second type of inward rectifying K+ channel with an ATP-binding domain). In situ hybridization showed KAB-2 mRNA to be expressed predominantly in glial cells of the cerebellum and forebrain. This is the first description of the cloning of a glial cell inward rectifier potassium channel, which may be responsible for K+ buffering action of glial cells in the brain.
Leishmania donovani: pilot study for evaluation of therapeutic effects of inosine analogs against amastigotes in vitro and in vivo.
The inhibition of carbocyclic inosine (C-Ino), 3'-deoxyinosine (3'-dI), and 3'-fluoroinosine (3'-FI) to Leishmania donovani amastigotes was examined. J774.1 cells (a mouse macrophage line) were cultured in GIT medium with lipopolysaccharide and hemin and infected with the parasite. C-Ino (3 microM) completely inhibited and 3'-dI (30 microM) reduced to 40% the infection rate on Day 6 after infection. Pentostam (30 microM) resulted in a 38% infection rate. The therapeutic efficacies of nonentrapped free and liposome-entrapped inosine analogs were tested in mice infected with L. donovani. The mice were injected intravenously five times on alternate days, beginning 2 days after infection. Treatment with the nonentrapped free inosine analog of C-Ino (100 mg/kg), 3'-dI (100 mg/kg), or 3'-FI (50 mg/kg) resulted in an LDU that was 94, 68, or 73% lower, respectively, than the control values. Treatment with the corresponding entrapped inosine analog (10 mg/kg) caused decreases of 90, 69, or 68% LDU, respectively. The entrapped inosine analogs were inhibitory at doses one-fifth to one-tenth of the nonentrapped free inosine analogs. C-Ino had the strongest inhibitory effect among the three analogs tested in vitro and in vivo. Liposome-entrapped C-Ino had no severe side effects, although spleen weight increased. The agent may be useful as an anti-leishmanial drug.
Estrogen induces epidermal growth factor (EGF) receptor and its ligands in human fallopian tube: involvement of EGF but not transforming growth factor-alpha in estrogen-induced tubal cell growth in vitro.
We studied the estrogen-dependent expression of epidermal growth factor (EGF), transforming growth factor (TGF) alpha, and EGF receptor gene transcripts in human fallopian tubes in vivo and in vitro. Competitive polymerase chain reaction (PCR) was performed on the fallopian tube RNA samples from the postmenopausal women with or without estrogen replacement. Amounts of EGF, TGF alpha, and EGF receptors gene transcripts in the estrogen-treated group (n = 3) were significantly (P < 0.01) more than those in the untreated group (n = 3). Competitive PCR also showed that EGF, TGF alpha, and EGF receptor gene transcripts level in tubal cells were increased by estrogen in vitro: messenger RNA levels of these factors were significantly (P < 0.01, n = 3) increased in cells incubated with 10(-8) M estrogen compared with those in cells without estrogen treatment. We studied whether EGF and/or TGF alpha is involved in the estrogen-induced tubal cell growth in vitro. Estrogen enhanced the [3H]-thymidine incorporation into the cell in dose- and time-dependent manners in culture: estrogen treatment for more than 12 h significantly (P < 0.05) enhanced the [3H]-thymidine incorporation into the cell at 10(-8) M. The estrogen-induced cell growth was observed in association with the increase in EGF, TGF alpha, and EGF receptor messenger RNA levels by estrogen. If the EGF and/or TGF alpha is involved in the cell growth, then the estrogen-induced cell growth should be suppressed by blocking the action of EGF and/or TGF alpha. Therefore, we examined the effects of neutralizing monoclonal antibodies against EGF, TGF alpha, and EGF receptors. Anti-EGF antibody significantly reduced the estrogen-induced increase in [3H]-thymidine incorporation, whereas anti-TGF alpha antibody failed to show the effect. Anti-EGF receptor antibody showed a significant suppressive effect on the estrogen-induced increase in [3H]-thymidine incorporation. Moreover, the growth inhibitory effect by 1 microgram/ml anti-EGF was restored by 10(-8) M EGF but not by TGF alpha even at 10(-6) M. All these data suggest that estrogen induces EGF and TGF alpha/EGF receptors in the human fallopian tube and that EGF but not TGF alpha may be involved in the estrogen-induced human tubal cell growth in vitro.
[Follicle stimulating hormone (FSH)].
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Implantation and growth of epidermal growth factor (EGF) receptor expressing human ovarian cancer xenografts in nude mice is dependent on EGF.
BACKGROUND: The importance of epidermal growth factor (EGF) receptor-dependent growth has not been clarified for in vivo growth of primary human ovarian cancers. METHODS: Seventeen primary human ovarian cancer tissue samples were examined for the presence of EGF receptors by a 125I-EGF-binding study. Three groups of mice were inoculated with EGF receptor expressing and not-expressing cancer tissues. The groups were as follows: control group, Sx group (mice that underwent sialoadenectomy; EGF depleted mice), and Sx+EGF (EGF-replaced) group. The ability of the inoculated tissues to implant and grow then was studied. RESULTS: Of the 17 primary ovarian cancers, 12 expressed EGF receptors and 5 did not. Eight of 12 EGF-receptor expressing cancer tissues implanted and formed growing tumors in control animals. None implanted in the Sx animals. Epidermal growth factor receptor-expressing cancers implanted in Sx animals that received EGF administration. Two of five EGF receptor-negative ovarian cancers implanted and grew in both control and Sx animals. CONCLUSION: Growth of EGF receptor-expressing primary human ovarian cancers may be dependent on EGF in vivo.
Molecular cloning and functional expression of cDNA encoding a second class of inward rectifier potassium channels in the mouse brain.
We have cloned a second class of inward rectifier potassium channels, designated MB-IRK2, from a mouse brain cDNA library. The amino acid sequence of this clone shares 70% identity with the mouse IRK1. Xenopus oocytes injected with cRNA derived from MB-IRK2 expressed a K+ current, which showed inward rectifying channel characteristics similar to the MB-IRK1 current. In contrast to the MB-IRK1 current, however, the MB-IRK2 current exhibited significant inactivation during hyperpolarizing pulses. In patch clamp experiments with 140 mM K+ in the pipette, the single channel conductance of MB-IRK2 was 34.2 +/- 2.1 picosiemens (n = 5), a value significantly larger than that of MB-IRK1 (22.2 +/- 3.0 picosiemens, n = 5). Consistent with the whole cell current, the steady-state open probability (Po) of the MB-IRK2 channel decreased with hyperpolarization, whereas that of the MB-IRK1 remained constant. Northern blot analysis revealed the mRNA for MB-IRK2 to be expressed in forebrain, cerebellum, heart, kidney, and skeletal muscle. In the brain, the abundance of mRNA for MB-IRK2 was much higher in cerebellum than in forebrain and vice versa in the case of MB-IRK1. These results demonstrate that the IRK family is composed of multiple genes, which may play heterogenous functional roles in various organs, including the central nervous system.
Molecular cloning and functional expression of a novel brain-specific inward rectifier potassium channel.
We have cloned a novel brain-specific inward rectifier K+ channel from a mouse brain cDNA library and designated it MB-IRK3. The mouse brain cDNA library was screened using a fragment of the mouse macrophage inward rectifier K+ channel (IRK1) cDNA as a probe. The amino acid sequence of MB-IRK3 shares 61% and 64% identity to MB-IRK1 and RB-IRK2, respectively. Xenopus oocytes injected with cRNA derived from this clone expressed a potassium current which showed inward-rectifying channel characteristics similar to MB-IRK1 and RB-IRK2 currents, but distinct from ROMK1 or GIRK1 current. However, the single channel conductance of MB-IRK3 was approximately 10 pS with 140 mM extracellular K+, which was distinct from that of MB-IRK1 (20 pS). MB-IRK3 mRNA expressed specifically in the forebrain, which clearly differed from MB-IRK1 and RB-IRK2 mRNAs. These results indicate that members of the IRK family with distinct electrophysiological properties express differentially and may play heterogenous functional roles in brain functions.
Molecular cloning, functional expression and localization of a novel inward rectifier potassium channel in the rat brain.
We have cloned a novel inward rectifier potassium channel from a rat brain cDNA library and designated it RB-IRK2. The rat brain cDNA library was screened using a fragment of the mouse macrophage IRK1 cDNA as a probe. The amino acid sequence of RB-IRK2 shares 70%, 40% and 45% identity to mouse IRK1, rat ROMK1 and rat GIRK1, respectively. Xenopus oocytes injected with cRNA derived from RB-IRK2 expressed a potassium current which showed inward-rectifying channel characteristics similar to the IRK1 current, but distinct from the ROMK1 or the GIRK1 currents. However, the localization of RB-IRK2 mRNA in rat tissues, assessed by the Northern blot analysis, differed from that of mouse IRK1. These results indicate that the IRK family is composed of multiple genes, which express in different tissues and therefore may play heterogenous functional roles in various organs, including rat central nervous system.
Expression of epidermal growth factor and transforming growth factor-alpha in fallopian tube epithelium and their role in embryogenesis.
We studied the expression of epidermal growth factor (EGF) and transforming growth factor (TGF)-alpha in human fallopian tube epithelium at various menstrual stages. Immunohistochemical staining using anti-EGF and anti-TGF-alpha antibodies showed a specific staining in ampullary tube epithelium at late follicular and luteal stages but the staining was very weak at the early follicular stage. Quantitative reverse transcription and polymerase chain reaction (RT-PCR) using beta-actin mRNA as an internal standard revealed the menstrual-stage-specific expression of EGF and TGF-alpha gene transcripts: amounts of EGF and TGF-alpha mRNA relative to those of beta-actin were significantly higher at late follicular and luteal stages than at the early follicular stage. To clarify the biological role of these growth factors, mouse 2-cell embryos were cocultured with human fallopian tube epithelial cells with or without blocking the action of these growth factors. Cocultures significantly promoted blastocyst formation, but this promotive effect of the tubal epithelial cells was completely abolished by the addition of anti-EGF and/or anti-TGF-alpha monoclonal neutralizing antibodies to the coculture system. These results demonstrated that EGF and TGF-alpha were synthesized and expressed in fallopian tube epithelium at specific menstrual stages, and may be involved in early embryonic development.
Epidermal growth factor promotes adipogenesis of 3T3-L1 cell in vitro.
We have reported the importance of epidermal growth factor (EGF) for the induction of obesity in mice. In this study, we studied the effects of EGF on the induction of lipogenic enzymes and on the accumulation of triglyceride in a differentiated mouse adipocyte cell in vitro. Mouse 3T3-L1 preadipocytic cells differentiated into mature adipocytes after the differentiation procedure by insulin, dexamethasone, and methyl-isobutyl-xanthine. 125I-EGF binding studies in the differentiated 3T3-L1 cells showed specific 125I-EGF bindings, and they expressed gene transcripts for EGF receptors by reverse transcription and polymerase chain reaction at all differentiative stages examined. Although EGF showed inhibitory effects on the triglyceride accumulation when administered to the preadipocytic 3T3-L1 cells, EGF enhanced the adipogenesis in the differentiated cells in dose- and time-dependent manners. Administration of EGF at 0.1-1 nM from 4 days after the differentiation procedure for 10 days, significantly enhanced the acyl-Co A synthetase and lipoprotein lipase messenger RNA levels, both of which are rate-limiting enzymes to synthesize triglyceride in adipocytes. Moreover, 0.1-1 nM EGF increased the amounts of triglyceride accumulated in the cells, in proportion to the acyl-Co A synthetase and lipoprotein lipase messenger RNA levels. EGF rather failed the adipogenesis at 10 nM. Time course studies revealed that 1 nM EGF significantly increased the intracellular triglyceride levels from 4 through 16 days administration. These results suggest that EGF shows biphasic effects on adipocytes: although EGF inhibits preadipocytes differentiation into mature adipocytes, it promotes adipogenesis in the differentiated adipocytes.
Involvement of autocrine mechanism of transforming growth factor-beta in the functional differentiation of pregnant mouse mammary gland.
We studied the presence and possible role of the autocrine mechanism of transforming growth factor-beta (TGF-beta) in pregnant mouse mammary gland. Northern blot analysis revealed the expression of the TGF-beta 1 gene transcript at 2.5 kb in mammary epithelial cells isolated from virgin and mid-pregnant mice. The TGF-beta activity was higher in the conditioned medium from mid-pregnant mouse mammary explants than that from virgin explants by a bioassay system using mink lung epithelial cells. A binding study using [125I]TGF-beta 1 as a ligand showed that pregnant mouse mammary epithelial cells possessed a single class of high-affinity TGF-beta 1 binding sites (Kd = 28.0 pmol/l, 1.2 x 10(4) sites per cell). These results suggested the presence of a TGF-beta autocrine mechanism in pregnant mouse mammary epithelial cells. Next, we examined the effect of TGF-beta 1 on the functional differentiation of pregnant mouse mammary gland. Transforming growth factor-beta 1 inhibited alpha-lactalbumin production in cultured mammary explants from mid-pregnant mice in a dose-dependent manner without inhibiting DNA synthesis. All these results suggest that TGF-beta may play a role in regulating the functional differentiation of mouse mammary glands during pregnancy.
Molecular cloning, functional expression and localization of an inward rectifier potassium channel in the mouse brain.
We have cloned an inward-rectifier potassium channel from a mouse brain cDNA library, studied its distribution in the brain by in situ hybridization and determined the chromosomal localization of the gene. A mouse brain cDNA library was screened using a fragment of the mouse macrophage IRK1 cDNA as a probe. Two duplicate clones of approximately 5.5 kb were obtained. Xenopus ococytes injected with cRNA derived from the clone expressed a potassium channel with inwardly rectifying channel characteristics. The amino acid sequence of the clone was identical to that of IRK1 recently cloned from a mouse macrophage cell line. In situ hybridization study showed the mouse brain IRK1 to be generally distributed throughout the brain, but in particular subsets of neurons at high levels. The gene was placed in the distal region of mouse chromosome 11, which contains several uncloned neurological mutations. These results provide the first demonstration of the cloning and distribution of an inward rectifier potassium channel from the nervous system.
Structures of alkanes and alkanols adsorbed on graphite in solution: Comparison with scanning-tunneling-microscopy images.
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Autocrine growth mechanism by transforming growth factor (TGF)-beta 1 and TGF-beta 1-receptor regulation by epidermal growth factor in a human endometrial cancer cell line IK-90.
Transforming growth factor-beta 1 (TGF-beta 1) enhanced cell proliferation in a concentration-dependent manner in a human endometrial cancer cell line, IK-90. Scatchard analysis of TGF-beta 1 receptor in IK-90 cells, using 125I-TGF-beta 1 as a ligand, revealed the presence of a class of high-affinity TGF-beta 1 receptors (2,000 sites per cell, KD = 74pM). Moreover, IK-90 cells produced and secreted TGF-beta 1: TGF-beta 1 messenger RNA was detected at 2.5 and 4.0 kb by Northern-blot analysis using 32P-labeled TGF-beta 1 cDNA as a probe, and TGF-beta 1 activity in conditioned medium by the inhibition of 3H-thymidine uptake into CCl 64 mink lung epithelial cells. We investigated the regulation of TGF-beta 1 receptor by 4 kinds of growth factor: epidermal growth factor (EGF) but not TGF-beta 1, insulin or insulin-like growth factor-1 increased the level of TGF-beta 1 binding sites in a concentration- and time-dependent manner. These findings suggest that TGF-beta 1 may be a potential autocrine growth factor in a human endometrial cancer cell line IK-90 and that this autocrine mechanism may be affected by EGF.