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K Mihara

Publications and source records attributed to K Mihara.

At least 109 records · Page 6Linked to original sources
PubMed1996

Mutation in p53 and de-regulation of p53-related gene expression in three human cell lines immortalized with 4-nitroquinoline 1-oxide or 60Co gamma rays.

In vitro models of malignant transformation of human cells may provide considerable insight into the mechanisms of multi-step carcinogenesis. It is well established that normal human cells must be immortalized before they can be malignantly transformed; however, they are stringently destined for aging and are rarely immortalized. The mechanism of cellular aging and immortalization is still unknown. We detected expression of only mutated p53 mRNA by direct sequencing of the reverse-transcribed mRNA in 3 human cell lines immortalized either with 4-nitroquinoline 1-oxide or with 60Co gamma rays. Consequently, only the mutated pS3 protein was expressed in each immortalized cell line. The expression of sdiI/p21 and mdm2, both of which are positively regulated by wild-type p53, was significantly down-regulated in the immortalized cell lines, resulting in over-expression of cdk2 and cdk4. Introduction of the sdiI/p21 gene into these cells was followed by a remarkable decrease in their ability to synthesize DNA. These results indicate that the p53 cascade may play an important role in the immortalization of human cells.

4-Nitroquinoline-1-oxide↗
PubMed1996

Reinitiation of protein translocation across the endoplasmic reticulum membrane for the topogenesis of multispanning membrane proteins.

The reinitiation of the translocation of the growing nascent chain across the endoplasmic reticulum membrane is essential for the topogenesis of multispanning membrane proteins. We investigated the requirements for the reinitiation process using model proteins in which systematically designed sequences were inserted after two preceding topogenic sequences, namely the N-terminal signal sequence (S) and stop transfer sequence (St). The model proteins were translated in vitro in the presence of rough microsomes, and the final topology of the proteins in the microsomal membrane was examined by proteolytic digestion. The structural requirements for S and the reinitiation sequence (R) overlapped to some extent, but substantial differences were noticed. When St and R were separated by a short cytoplasmic segment (58 amino acids), the efficiency of the reinitiation was not affected by the concentration of the signal recognition particle (SRP) in the translation system, even though the sequence inserted as R was an SRP-dependent signal sequence. However, when the cytoplasmic segment was longer (100 amino acids), the reinitiation efficiency was reduced, and the SRP improved the overall efficiency as well as impaired the accessibility of the processing site after the R to the signal peptidase.

Amino Acid Sequence↗
PubMed1996

Cytoplasmic chaperones determine the targeting pathway of precursor proteins to mitochondria.

Two ATP-dependent cytosolic chaperones, mitochondrial import stimulation factor (MSF) and hsp70, are known to be involved in the import of precursor proteins into mitochondria. Hsp70 generally recognizes unfolded proteins, while MSF specifically recognizes mitochondrial precursor proteins and targets them to mitochondria in a NEM-sensitive manner. Here we analyzed the relative contribution of these chaperones in the import process and confirmed that the precursor proteins are targeted to mitochondria via two distinct pathways: one requiring MSF and the other requiring hsp70. Both pathways depend on distinct proteinaceous components of the outer mitochondrial membrane. The MSF-dependent pathway is NEM-sensitive and requires the hydrolysis of extra-mitochondrial ATP for the release of MSF from the mitochondrial import receptor, whereas the hsp70-dependent pathway is NEM-sensitive and does not require extra-mitochondrial ATP. The NEM-insensitive, hsp70-dependent import became NEM-sensitive depending on the amount of MSF added. The relative importance of the two pathways appears to be determined by the affinities of MSF and hsp70 for the precursor proteins.

14-3-3 Proteins↗
PubMed1996

Persistence of hepatitis C virus RNA in established human hepatocellular carcinoma cell lines.

The persistence of the viral RNA of hepatitis C virus (HCV) was examined in 13 hepatocellular carcinoma (HCC) and two hepatoblastoma cell lines by reverse transcription followed by the polymerase chain reaction (RT-PCR). HCV RNA was detected in three HCC lines (JHH-1, JHH-4, and JHH-6) and negative-strand viral RNA was found in JHH-4, indicating that there is a putative replicative intermediate of HCV in JHH-4 cells. To rule out the possibility of contamination, the partial nucleotide sequences of HCV-specific PCR products of these three cell lines were determined. The clone from JHH-1 belonged to genotype 1 (1a or 1b), and the clones from JHH-4 and JHH-6 belonged to genotype 2b, but their sequences differed from each other. These cell lines may be useful for studies related to HCV.

Adolescent↗
PubMed1996

Two-dimensional electrophoretic studies on down-regulated intracellular transferrin in human fibroblasts immortalized by treatment with either 4-nitroquinoline 1-oxide or 60Co gamma rays.

The immortalization of human cells is an important early step of carcinogenesis. To investigate the mechanisms of immortalization, we established three immortalized human fibroblast cell lines by treatment with either 4-nitroquinoline 1-oxide (4NQO) or 60Co gama rays. Using two-dimensional gel electrophoresis (2-D PAGE), we identified proteins which are down-regulated in these immortalization cell lines. Their isoelectric points ranged between 5.5 and 6.3, and their molecular masses were approximately 80 kDa. The proteins were also decreased in another human fibroblast cell line immortalized with simian virus 40 (SV40) and two human cervical cell lines. The proteins were present in a cytoskeletal fraction, reacted with anti-transferrin antibody and some of them were phosphorylated. The proteins were identified as transferrin. Although the 2-D PAGE patterns of our proteins and serum transferrin of different origin were distinct from each other, their isoelectric points and molecular weights were similar. We implicate some functions of intracellular transferrin, other than an iron transporter, in immortalization of human cells and discuss a new aspect of transferrin metabolism in the cells.

4-Nitroquinoline-1-oxide↗
PubMed1996

Telomere elongation observed in immortalized human fibroblasts by treatment with 60Co gamma rays or 4-nitroquinoline 1-oxide.

Telomeres are the tandemly repeated (TTAGGG)n sequences that make up the structural and functional ends of all chromosomes in mammals. Many lines of evidence indicate that telomeres stabilize chromosomes, prevent aberrant recombination, and direct chromosome attachment to the nuclear membrane. Since DNA polymerase requires a labile primer to initiate unidirectional 5'-3' DNA synthesis, some bases at the 3' end of each template strand are not copied unless special mechanisms bypass this end-replication problem. To overcome this problem, most eukaryotic cells use telomerase, an enzyme that elongates telomeres. However, this enzyme has not been detected in normal human cells, and these cells lose telomeres with cell division. Cellular senescence might be the result of this loss. Thus, activation of telomerase seems to be critical for the immortalization of human cell lines. In addition, substantial evidence indicates that immortalization in itself is a rate-limiting step for the malignant transformation of human cells. We have treated normal human fibroblasts (AD387, KMS-6, and OUMS-24 lines) intermittently with either 60Co gamma rays or 4-nitroquinoline 1-oxide (4NQO) during serial subcultivations, and have obtained three immortalized cell lines, SUSM-1, KMST-6, and OUMS-24F. In KMS-6 and OUMS-24, the mean terminal restriction fragment length significantly decreased as the population-doubling level increased. The rate of telomere loss was 40 and 50 bp/population doubling in the KMS-6 and OUMS-24 cell lines, respectively. Once these normal cell lines were immortalized, their telomeres became elongated. Similar data were obtained for AD387 cells and their immortalized SUSM-1 cells. These results suggest that telomeres play a critical role in cellular senescence and in the immortalization processes of human cells.

4-Nitroquinoline-1-oxide↗
PubMed1996

Establishment and characterization of a human colon cancer cell line, OUMS-23, from a patient with familial adenomatous polyposis.

A human colon carcinoma cell line designated OUMS-23 has been established from metastatic pericardial fluid of a male familial adenomatous polyposis patient with colon cancer. Since 1984, the epithelial cells have been maintained in culture. Ultrastructural studies revealed the presence of numerous microvilli on the cell surface and desmosomes between the adjacent cells. The cells secreted carcinoembryonic antigen into the culture medium (15 ng/10(6) cells-1 24 h-1). The cells expressed heat-stable placental-type-like alkaline phosphatase, whereas the normal counterparts expressed tissue-unspecific alkaline phosphatase. Karyotypic analysis showed that the cell line was of human origin and that the chromosome number was broadly distributed between 53 and 118. Southern blot analysis of the APC gene revealed no abnormalities in OUMS-24 cells, while Northern blot analysis demonstrated that the expression of the gene was about one-half that of the normal human fibroblasts. No mutations at the "hot spots" of codons 12 and 61 of H-, K- and N-ras proto-oncogenes were detected in the cells. The cells could grow in soft agar at a cloning efficiency of 6.5%, and upon transplantation into nude mice the cells formed tumors, which were diagnosed as differentiated adenocarcinoma.

Adenomatous Polyposis Coli↗
PubMed1996

Possible inhibitory effect of diazepam on the metabolism of zotepine, an antipsychotic drug.

Effects of smoking and cytochrome P450 2C19 (CYP2C19) status on the single dose kinetics of zotepine and pharmacokinetic interaction between zotepine and diazepam were investigated. In 14 healthy volunteers, the pharmacokinetics of zotepine after a single oral 25 mg dose were compared between eight smokers and six non-smokers, or between seven extensive metabolizers (EMs) and seven poor metabolizers (PMs) of S-mephenytoin. There was no significant difference in any pharmacokinetic parameters between smokers and non-smokers, or between the EM and PM groups. In 17 patients treated with zotepine 80-340 mg/day, intra-individual changes in plasma concentrations of zotepine caused by coadministration of diazepam 10 mg/day for 2 weeks were examined. Plasma concentrations of zotepine were significantly increased after coadministration of diazepam (P < 0.05). Consequently, it is suggested that neither smoking nor CYP2C19 status affects the metabolism of zotepine. The elevation in plasma concentrations of zotepine after coadministration of diazepam may be a result of competitive inhibition of zotepine metabolism by diazepam via other isoenzyme than CYP2C19, e.g., CYP3A4.

Adult↗
PubMed1996

Interleukin-6 gene expression is preferentially restricted in VLA-5-MPC-1- immature but not in VLA-5+MPC-1+ mature myeloma cells.

Interleukin-6 (IL-6) is essential for the proliferation of myeloma cells, and IL-6 is considered to be produced from not only myeloma cells themselves but also microenvironments including stromal cells. To clarify which subpopulation of myeloma cells can produce IL-6, we examined IL-6 mRNA expression in immature and mature myeloma cells and normal plasma cells by RT-PCR. IL-6 mRNA expression was found in all (10/10) specimens of sorted VLA-5-MPC-1- immature myeloma cells and 27% (3/11) of VLA-5-MPC-1+ myeloma cells. On the contrary, no IL-6 mRNA was expressed in VLA-5+MPC-1+ mature myeloma cells (0/4) and CD19+CD56- normal plasma cells (0/5). IL-6R gene expression was detected in all normal and malignant plasma cells without exception. Therefore, these findings suggest that IL-6 production is preferentially restricted in immature not mature myeloma cells, and this may explain why immature myeloma cells show greater proliferative activity.

Base Sequence↗
PubMed1996

Cytoplasmic chaperones in precursor targeting to mitochondria: the role of MSF and hsp 70.

Despite extensive study since the early 1980s, the mechanism by which newly synthesized protein precursors are unfolded in the cytoplasm and targeted correctly to the mitochondrial surface prior to translocation through the mitochondrial membranes is understood poorly. Recently, an N-ethylmaleimide (NEM)-sensitive cytoplasmic factor called mitochondrial import stimulation factor (MSF), which catalyses the ATP-dependent unfolding of precursor proteins, was described. Unlike the more general chaperone proteins of the hsp70 families, MSF not only unfolds proteins but also targets the unfolded precursor proteins to the mitochondria. Here, Mihara and Omura summarize what is known about MSF and speculate on how it, and other cytoplasmic factors, may be involved in mitochondrial import.

Journal Article↗
PubMed1996

No interaction between desipramine and bromperidol.

1. The authors studied the effects of coadministration of desipramine, which is a substrate of CYP2D6, on plasma concentrations of bromperidol and its reduced metabolite (reduced bromperidol). Clinical changes were also evaluated by the CGI and UKU. 2. The subjects were 13 schizophrenic inpatients receiving bromperidol 12.24 mg/day for 1.20 weeks. Desipramine 50 mg/day was coadministered for 1 week, and blood samplings and clinical ratings were performed before and after the coadministration. 3. Plasma concentrations of bromperidol and reduced bromperidol were measured by a HPLC method. 4. Desipramine coadministration did not affect plasma concentration of bromperidol (9.6 +/- 4.5 vs. 9.6 +/- 2.8 ng/ml) nor that of reduced bromperidol (2.8 +/- 2.5 vs. 2.8 +/- 2.1 ng/ml). 5. There was no significant change in the CGI scores nor UKU scores after desipramine coadministration. 6. The present study thus suggests that there is no interaction between desipramine and bromperidol.

Adult↗
PubMed1996

Identification of early plasma cells in peripheral blood and their clinical significance.

In the peripheral blood (PB) we detected so-called early plasma cells that might already be committed to entering the bone marrow (BM). By two-colour staining with FITC-anti-CD38 antibody, their intensity (CD38++) of expression of CD38 antigen was between that of germinal centre (GC) B cells (low expression (CD38+)) and that of BM plasma cells (high expression (CD38++)), and their phenotype was CD38++ CD19+ CD10- CD20- CD21+ CD24- CD39+ CD5- VLA-4+ VLA-5- MPC-1- without expression of surface membrane IgM (SmIgM). Morphological and immunological examination of the sorted cells confirmed that they were plasmacytoid cells with expression of cytoplasmic IgG (cIgG). Variations of these early plasma cells were examined in various diseases. In active systemic lupus erythematosus, bacterial septicaemia and liver cirrhosis, early plasma cell levels were significantly increased in PB, and after subsidence of such inflammation (inactive states) these cells returned to normal levels. In contrast, normal early plasma cells were significantly suppressed in myelomas, whilst normal or slightly increased numbers of early plasma cells was found in benign monoclonal gammopathy (BMG). In addition, the number of normal early plasma cells returned to a normal level in myeloma cases with complete responses. Therefore, early plasma cells were identified phenotypically, and an increase and decrease in these cells in PB may reflect mobilization and suppression, respectively, of activated B cells into BM plasma cells.

ADP-ribosyl Cyclase↗
PubMed1996

Effects of carbamazepine coadministration on plasma concentrations of trazodone and its active metabolite, m-chlorophenylpiperazine.

Effects of carbamazepine coadministration on plasma concentrations of trazodone and its active metabolite, m-chlorophenylpiperazine (m-CPP) were studied in six depressed patients treated with trazodone. The daily dose of trazodone was 150 mg in three cases and 300 mg in three cases. Carbamazepine, 400 mg/day, was coadministered for 4 weeks, and blood samples were taken before carbamazepine addition and at weekly intervals after the addition. Carbamazepine significantly (p < 0.01) decreased plasma concentrations of not only trazodone but also m-CPP at each week. On the average, plasma concentrations of trazodone and m-CPP at 4 weeks were 24 and 40% of the corresponding precarbamazepine values. This study thus suggests that carbamazepine coadministration decreases plasma concentrations of trazodone and m-CPP by inducing the metabolism of these compounds.

Anticonvulsants↗
PubMed1996

A method for rapid determination of zotepine by gas chromatography-mass spectrometry.

A rapid and sensitive method using solid-phase extraction and gas chromatography-mass spectrometry (GC-MS) has been developed for the determination of zotepine (ZTP), an atypical neuroleptic, in human plasma. The detection limit of ZTP was 1 microgram/L. Standard curves over the concentration range from 2.5 to 100 micrograms/L had a good linearity. Intraassay variability ranged from 2.2 to 3.3% and interassay variability from 3.5 to 6.6% at the concentration range of 5-75 micrograms/L. Our preliminary data of single-dose kinetics of ZTP by using this method suggested that the peak time and elimination half-life was much longer than previously reported, and that there appeared to be a second peak after 10-12 h of ZTP administration, indicating the possibility of the presence of enterohepatic recirculation.

Antipsychotic Agents↗
PubMed1996

Hallucinations after a therapeutic dose of benzodiazepine hypnotics with co-administration of erythromycin.

A case of repetitive hallucinations during treatment with a therapeutic dosage of triazolam (0.25 mg/day) and nitrazepam (5 mg/day) is presented. The patient suffered from acute pneumonia and chronic renal failure. Such non-psychotic symptoms as anxiety, tremor and depressed feeling were observed initially. However, after co-administration of erythromycin (600 mg/day), visual hallucinations and abnormal bodily sensations developed repeatedly after each administration of triazolam or nitrazepam. This report suggests that benzodiazepine hypnotics even at a therapeutic dosage with co-administration of erythromycin causes serious psychotic symptoms in vulnerable patients with physical complications.

Anti-Anxiety Agents↗
PubMed1996

Selective expression of mutated p53 in human cells immortalized with either 4-nitroquinoline 1-oxide or 60Co gamma rays.

Many studies on in vitro transformation of human cells indicate that the cells must be immortalized before they can be neoplastically transformed, indicating that immortalization is a critical step in multistep neoplastic transformation of human cells. We immortalized three human cell lines by repeated treatment with either 60Co gamma rays or a chemical carcinogen, 4-nitroquinoline 1-oxide, and found that all three immortalized cell lines have mutations in the tumor suppressor gene, p53. Direct sequencing of the reverse-transcribed mRNA and immunoprecipitation of p53 protein revealed that mutant p53 is selectively expressed in all the immortalized cell lines, whereas the genomic fragments of the immortalized cells contain wild-type and mutated p53 alleles. Although the mutated p53 is selectively expressed in the immortalized cells, expression of the wild-type p53 was induced by treatment of the cells with a hypomethylating reagent, 5-azacytidine, indicating that the wild-type p53 allele might be inactivated by hypermethylation of DNA. Actually, the entire genomic locus including the promoter region of p53 is hypermethylated in all the immortalized cell lines. Expression and phosphorylation of Rb was normal in these three cell lines. Thus, inactivation of both wild type p53 alleles and selective expression of mutated p53 seem to be key factors in the immortalization of human fibroblasts.

4-Nitroquinoline-1-oxide↗
PubMed1996

Immortalization of human cells and its mechanisms.

One of the contributions of in vitro neoplastic transformation studies on human cells is the confirmation that cells become malignant by a multistep process. In this review, we use the term "neoplastic transformation" to indicate that the cells become capable of forming a histologically malignant tumor, that is, one that invades and/or erodes normal tissue and is serially transplantable in a susceptible animal host. Phenotypically, the cells first become immortalized and then neoplastic. If normal human cells do not overcome aging by becoming immortalized, they cannot be neoplastically transformed. However, normal human cells are stringently destined to cellular aging and very rarely become immortalized by oncogenic DNA viruses (SV40, papillomavirus types 16 and 18, adenovirus types 5 and 12), chemicals, X-rays, or spontaneously. Once human cells get immortalized, they relatively easily become neoplastic. Evidence shows that immortalization is a critical step in the neoplastic transformation process of human cells. Therefore, investigation of the mechanisms of immortalization is indispensable in understanding the multistep carcinogenesis of human cells. Although the precise mechanisms of immortalization are unknown at present, data indicate that the p53 cascade appears to be important for the immortalization of human cells. The loss of functions of normal p53 may induce genomic instability, resulting in mutations of some other unknown genes that are more directly involved in the immortalization of human cells. These unknown genes, which are presumably located at chromosomes 1, 4, 6, 7, 9, 11, 13, 17, 18, and X are now under extensive investigation.

Cell Transformation, Neoplastic↗