Biomedical subjects
S Nakatsugawa
Publications and source records attributed to S Nakatsugawa.
Molecular cloning and characterization of a novel human gene (NESCA) which encodes a putative adapter protein containing SH3.
A full-length cDNA encoding a novel protein was isolated and sequenced from a human placental cDNA library. This cDNA consists of 1990 bp and has a predicted open reading frame encoding 433 amino acids. It possesses an Src homology 3 (SH3) motif, a leucine zipper motif and no catalytic domain, suggesting that it seems to be an adapter protein. PCR-based mapping with both a monochromosomal hybrid panel and radiation hybrid cell panels placed the gene to human chromosome 1q21-22.
Molecular cloning and characterization of a novel human gene (HERNA) which encodes a putative RNA-helicase.
A full-length cDNA encoding a novel protein was isolated and sequenced from a human hepatocellular cDNA library. This cDNA consists of 7037 base pairs and has a predicted open reading frame encoding 1924 amino acids. It possesses an RNA-helicase motif containing a DEXH-box in its amino-terminus and an RNase motif in the carboxy-terminus. From a striking homology to Caenorhabditis elegans K12H4.8, it might be a human homolog of the K12H4.8. PCR-based mapping with both a monochromosomal hybrid panel and radiation hybrid cell panels placed the gene to human chromosome 14q31 near the marker D14S605.
Molecular cloning and characterization of human MAWD, a novel protein containing WD-40 repeats frequently overexpressed in breast cancer.
A full-length cDNA clone encoding a novel protein containing WD-40 repeats, which were frequently involved in protein-protein interactions, was isolated and sequenced. This clone had a predicted open reading frame (ORF) encoding 350 amino acids possessing six repeats of WD-40 motif. It was most closely homologous to TRIP-1, a phosphorylation substrate of the transforming growth factor-beta type II receptor. In the process of characterizing the function of the new gene product, we found that overexpression of the gene seemed to activate mitogen-activated protein kinase and to promote anchorage-independent growth of the cells. Moreover, the gene product was frequently overexpressed in human tumor breast tissues compared with their normal breast tissues, suggesting that the gene might be involved in the tumor progression. Radiation hybrid mapping placed the gene into human chromosome 12q11-12 near the marker D12S1593.
Absence of correlation of MUC1 expression to malignant behavior of renal cell carcinoma in experimental systems.
A correlation between MUC1 expression in renal cell carcinomas (RCCs) and the clinical stages was previously demonstrated. To assess whether MUC1 expression is causally related to malignant tumor behavior, MUC1 cDNA was stably transfected into a renal carcinoma cell line SN12C that expresses trace levels of MUC1. MUC1 with sialylated carbohydrate chains was detected on the surface of transfected cells in two independent experiments. There was no correlation between MUC1 expression and in vitro growth and motility. In vivo growth of the transfectants at the site of orthotopic transplantation in nude mice was slower than mock transfected cells. Therefore, MUC1 alone did not seem to confer a malignant phenotype to RCCs.
Intercept-PCR, an improvement for elevating performance to find a new member of a certain gene family.
We have established a method by which the performance of reverse transcriptase coupled polymerase chain reaction (RT-PCR) for seeking a new gene is improved. The actual procedure is quite easy: it is only to add several specific oligonucleotides into the reaction mixture of the usual RT-PCR. To verify the effectiveness of this method is also easy: it is only to detect the PCR products in the preliminary experiment. The finding in the present study provides valuable information for gene cloning tactics.
Molecular cloning of macrophin, a human homologue of Drosophila kakapo with a close structural similarity to plectin and dystrophin.
We have determined the complete cDNA coding sequence of a novel cytoskeletal protein by the degenerative primer-mediated PCR strategy. This novel gene, named as macrophin (microfilament and actin filament cross-linker protein related to plectin and dystrophin, Accession No. AB029290), appears to be a human homologue of a Drosophila gene, kakapo, and shows close similarity to plectin and dystrophin on the search of BLAST homology-computed database. Comparison of the deduced protein sequences for macrophin and kakapo revealed that they were 66% similar, and both of them have an NH2-terminal actin-binding domain, a central rod region composed of spectrin-like repeats, and COOH-terminal Gas2-related region. The predicted sequences for macrophin are 5430 amino acids in length with a calculated molecular mass of 620 kDa, which is one of the largest size idenfied in human cytoskeletal proteins. High expression of macrophin was observed in brain, heart, lung, placenta, liver, kidney, and pancreas. In pancreas, we found that macrophin was specifically expressed in acinar cells than islet cells by in situ hybridization. By using radiation hybrid panel, we have mapped the macrophin gene to the chromosome 1p31-32.
Malignant and other properties of human colon carcinoma cells after suppression of sulfomucin production in vitro.
Although the loss of sulfomucins was known as an indicator of carcinogenesis and malignant progression of colonic epithelia, it was not known whether the loss was directly related to the malignant behavior of colon carcinoma cells. We have studied the biological properties of LS174T human colon carcinoma cells before and after suppression of sulfomucin production. Incorporation of [35S]-sulfate into high molecular weight mucins decreased after carcinoma cell treatment with 1.5% dimethylsulfoxide (DMSO) for 8 days. The amounts of sulfomucin determined using a sulfomucin-specific monoclonal antibody (mAb 91.9H), in Western blot and flowcytometric analyses, also decreased. In addition, the levels of MUC2 and MUC5B mucin gene expression measured by RT-PCR were reduced after DMSO-treatment, whereas the levels of MUC1, MUC5AC, and MUC6 mucin gene expression were not. The DMSO-treated cells were tested in vitro and in vivo for their properties. Differences were not detected in their anchorage-independent growth, anchorage-dependent growth, E-selectin-dependent cell adhesion or sensitivity to interleukin (IL)-2-activated lymphocyte cytolysis. When untreated or DMSO-treated LS174T cells were injected intrasplenically into nude mice, the treated cells lacking certain cell surface sulfomucins formed fewer metastatic colonies in the liver. These results suggest that the loss of sulfomucins by colonic epithelial cells during progression is not directly related to the enhanced malignant behavior.
Inhibitory effect of ND2001 on spontaneous multiple metastasis of NC 65 tumors derived from human renal cancer cells intradermally transplanted into nude mice.
The NC 65 tumor cell line derived from human renal cell carcinoma was selected from among nine human cell lines by determining the inhibition of invasion by ND2001 (sodium D-glucaro-delta-lactam) in vitro. The efficacy of this agent against these tumor cells was investigated in an experimental metastatic model of human tumors in vivo. Although ND2001 did not inhibit growth of NC 65 cells intradermally transplanted into male KSN mice (nu/nu), this agent inhibited multiple spontaneous metastasis.
The effects of static magnetic fields and X-rays on instability of microsatellite repetitive sequences.
To determine the genetic effect of static magnetic fields (SMF), which are not supposed to produce any significant DNA damage, we took advantage of DNA mismatch repair (MMR) deficient cells, in which all the errors produced during DNA replication are left uncorrected. We first established a simple and less labor-intensive method to analyze genetic changes in microsatellite repetitive sequences in the MMR-deficient cells. After exposure to a strong SMF (6.34T) for 24 h, both MMR deficient HCT116 cells and proficient HeLa S3 cells did not exhibit any significant effect on microsatellite changes. Moreover, when HCT116 cells were synchronized at the G1/S boundary by aphidicolin and exposed to SMF during the whole S-phase, no increase in microsatellite changes was either observed. In contrast, irradiation by a low dose X-ray (2Gy) significantly increased microsatellite changes in HCT116 cells. This suggested that exposure to strong SMF may not induce any significant level of genetic changes in microsatellite sequences.
[Increase of cellular fibrinolysis in human lung cancer cell line by radiation: relationship between urokinase-type plasminogen activator (uPA) and metastasis and invasion].
It is well known that urokinase-type plasminogen activator (uPA) activates fibrinolysis of tumor cells and accelerates their metastasis and invasion. Human adenosquamous cell line, AOI cells, were stimulated to produce and accumulate of uPA by radiation. In AOI cells, there was relationship between uPA production and accumulation and the radiation doses. It was suggested that radiation had the possibility to accelerate the metastasis and invasion by increasing the production and accumulation of uPA from cancer cells.
Differential action on cancer and normal tissue by adrenochrome monoaminoguanidine methanesulfonate and cytochrome C combined with radiotherapy.
PURPOSE: The possibility that radioprotective effects on potent natural killer (NK) cells by adrenochrome monoaminoguanidine methanesulfonate+cytochrome C during radiotherapy (RT) for lung cancer might result in the radiosensitization of human lung cancer cells in vivo is examined. METHODS AND MATERIALS: Human lung cancer xenografts in the right hind legs of KSN mice (10 weeks old) were locally irradiated with 20 Gy of X ray. Adrenochrome monoaminogluanidine methanesulfonate (AMM) (10 mg/kg/day) and/or cytochrome C (CCC) (5 mg/kg/day) were given intraperitoneally immediately before or after RT, followed by daily administration for 4 days. Natural killer activities of host splenocytes were also tested with the standard 51Cr releasing assay with YAC-1 cells as target cells. In a clinical study, 65 patients with lung cancer were treated with more than 50 Gy of RT with or without combination with AMM+CCC, OK-432 or AMM+CCC+OK-432. Before and after RT, lymphocyte subsets in the peripheral blood were examined with dichromatic analysis using an Ortho Spectrum IIIFCM system and fluorescent MABs. In this study, the change in the absolute number of each subset was investigated. RESULTS: Adrenochrome monoaminoguanidine methanesulfonate+cytochrome C augumented NK activity in KSN nude mice, protected potent NK cells in patients with lung cancer against RT and sensitized the human lung cancer xenografts to RT. CONCLUSION: Adrenochrome monoaminoguanidine methanesulfonate+cytochrome C may have the potential as a differential modulator of radiosensitivity of normal tissues and of tumors.
Metabolic alterations in implanted human tumors after combined radiation and hyperthermia therapy measured by in vivo 31P MRS.
The bioenergetics of human lung tumors grown subcutaneously in KSN nude mice, were studied in vivo using 31P NMR spectroscopy up to 27 days following radiotherapy and/or hyperthermia. Six tumors were treated with radiation (20 Gy, single fraction) and hyperthermia (44 degrees C, 10 min). There was a significant increase in the ratio of inorganic phosphate to beta-nucleoside triphosphate (Pi/beta-NTP) 24 h after radiation plus hyperthermia (p < .01), but a significant decrease 6 days after the treatment (p < .05) relative to untreated controls. Furthermore, the combined therapy produce significant acidosis at 24 h post therapy followed by significant alkalosis at 6 days compared to no treatment. This biphasic pattern was also significant in comparison with the pretreatment values of Pi/beta-NTP and pH. The combined therapy produced not only tumor decline at 24 h indicated by increased Pi/beta-NTP ratio and acidic pH shift, but also metabolic activation of tumor cells at 6 days indicated by decreased Pi/beta-NTP ratio and alkalotic pH shift. The tumor blood flow estimated by hydrogen ion clearance curves were completely depleted at 24 h and fully recovered to pretreatment level at 6 days. Reasonable close negative correlation between the blood flow and Pi/beta-NTP ratio (r = -0.59, p < .01) indicated that the two contrasting physiological states were closely related to tumor perfusion status. The 31P spectra of tumors following the combined therapy were concluded to demonstrate additive physiological effects of hyperthermia and radiation.
Ionizing radiation-induced IL-1 alpha, IL-6 and GM-CSF production by human lung cancer cells.
A cell line derived from human lung cancer (AOI) was employed in the present study. A panel of cytokines were quantified by ELISA technique following cellular exposure to X-irradiation. Tremendous increase in the levels of both IL-1 alpha and IL-6 were observed, GM-CSF was also detected. A comparison of time kinetics of IL-1 alpha and IL-6 production was made with that of cell cycle progression which was determined by FCM BrdU/DNA bivariate analysis. No cell cycle specific changes were found. The biological implication of radiation-induced cytokine production was discussed.
Experimental radiotherapy and metastasis of human lung cancer xenografts in nude mice.
Using a cell line derived from human lung cancer (AOI), we successfully established human xenografts in KSN nude mice, which showed high incidence of multiple spontaneous metastases. The highest incidence of metastasis in untreated hosts was observed in the spleen followed by the lungs and lymph nodes. The rate of metastasis reached 100% in the mice bearing large sized tumors, when metastasis to any organ or tissue was counted. Experimental radiotherapy caused remarkable redistribution of metastatic foci among different organs. Lung metastasis tended to decrease, while metastasis to the liver and the kidney was increased after radiotherapy. Radiation-induced production of cytokines was speculated to be responsible for such an alteration of metastasis pattern.
[Intravoxel incoherent motion (IVIM) imaging using an experimental MR unit with small bore].
IVIM images were constructed from a pair of spin echo (SE) sequences on a 2T MR unit with small bore. In the current study, two types of SE were used: the standard SE and the other sensitized to microscopic incoherent motion by adding motion probing gradient on either side of 180 degrees pulse. IVIM images of water and acetone phantoms were generated, and the diffusion coefficients obtained from these materials were consistent with the literature values. IVIM images of implanted human tumor in nude mouse showed relatively high apparent diffusion coefficient (2.0 x 10(-3) mm2/sec). However, in the sacrificed mouse, diffusion coefficient in the tumor was markedly decreased (0.5 x 10(-3) mm2/sec), which suggested the considerable contribution of perfusion to ADC. In conclusion, IVIM imaging was thought to be highly valuable because it can provide functional information about tumor perfusion.
[A waterborne outbreak of typhoid fever associated with a small drinking water supply system in Fuji city].
Fifteen cases of typhoid fever occurred in "HARADA" district Fuji City in Shizuoka Prefecture during the period of 1983 to 1985. Epidemiological and bacteriological investigations were carried out on the samples from a small water supply system, and drains of apartment houses in 1985. Water from a small river in the neighboring water supply system, and faecal specimens from people living in close proximity were investigated. The results obtained were as follows: 1) Salmonella typhi were isolated from 3/3 patients (1 pupil and 2 employees) in 1983, 3/5 patients (3 employees) in 1984, and from 4/7 patients (1 kindergartner, 1 pupil and 2 junior high school boys) in 1985. Phage type of these isolates were the same type "D1". 2) In the bacteriological survey on environment, S. typhi (phage type D1) were isolated from water of the water source of "HARADA" water supply in Fuji City with membrane filter methods, and from the sand source "HARADA" water supply with enrichment culture methods. Also, with an examination that tested 3,670 inhabitants, it was found that a carrier (phage type D1) was living near the "HARADA" water supply. 3) In the three selective media for the isolation of S. typhi, Bismuth sulfite agar (Difco) was the most sensitive, and detected 13 samples (61.9%) out of 21, from water source and sewage.
[A trial of ACNU and radiation therapy with sensitizing agents for malignant gliomas].
Twelve cases of malignant gliomas (anaplastic astrocytoma 4, glioblastoma 8, recurrent 3, primary 9) were treated with ACNU and radiation with sensitizing agents after the surgical removal of the tumor. BUdR, Vidarabine (Ara-A), Aciclovir (ACV) were applied for sensitizing agents. BUdR was administrated intraarterially prior to radiation (380 rad, two times a week), and Ara-A and ACV intravenously during and after the radiation. Total dosage of the radiation was 50-60 Grey for each case. All recurrent and eight primary patients died. The mean survival time of the recurrent patients was 17.7 months, while that of the primary patients was 13.4 months. One of the primary patient was glioblastoma and is still surviving more than 24 months by now. The complete response (CR) rate of the primary tumor patients observed by computerized tomography (CT) scan was 5/9. We can expect the availability of this trial for malignant gliomas because of high CR rate in primary tumor cases.