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Biomedical subjects

K Fushimi

Publications and source records attributed to K Fushimi.

At least 37 records · Page 2Linked to original sources
PubMed1998

Establishment of a human fibroblast cell line producing tumor necrosis factor alpha (KMST-6/TNF) and growth inhibitory effects of its conditioned medium on malignant cells in culture.

To develop a new gene therapy model for cancer, a clonal cell line (KMST-6/TNF) which produces human tumor necrosis factor alpha (hTNF-alpha) has been developed by introducing hTNF-alpha cDNA into a human immortal fibroblast cell line (KMST-6). The conditioned medium (CM) of KMST-6/TNF cells inhibited the growth of various malignant human cell lines, but not that of normal human fibroblasts. Although the growth inhibitory effects of KMST-6/TNF CM were neutralized to a considerable degree by anti-TNF-alpha antibody, its inhibitory effects were more marked than the purified human natural TNF-alpha itself in the same units, suggesting that KMST-6/TNF CM contains some growth inhibitory substances other than TNF-alpha. However, interferons alpha, beta, and gamma were undetectable in the KMST-6/TNF CM.

Blotting, Northern↗
PubMed1998

Inhibition of pulmonary eosinophilia does not necessarily prevent the airway hyperresponsiveness induced by Sephadex beads.

BACKGROUND: The Lewis rat among highly inbred strains exhibits significant airway hyperresponsiveness (AHR) following intravenous administration of Sephadex G-200 (Sephadex). The aim of this study was to investigate the association of Sephadex-induced AHR with changes in airway inflammation in Lewis rats. METHODS: A suspension (0.5 mg/ml/rat) of Sephadex was intravenously administered to male Lewis rats on days 0, 2 and 5. Measurement of airway responsiveness to serotonin, bronchoalveolar lavage (BAL) and histological study were performed on day 2-11. RESULTS: Significant AHR induced by Sephadex was recognized on day2 (p < 0.05), and AHR reached a maximum on day 7 (p < 0.001). In the BAL study, eosinophils increased on day2 (p < 0.01) with a peak on day 5 (p < 0.05). In the histological study, we found Sephadex beads trapped in small arteries of the lung and granulomatous arteritis on day 2 or later. Pulmonary granulomas, horseshoe-shaped multinuclear giant cells, eosinophils and goblet cell hyperplasia were observed on day 2, and the degree became intense on day 5-7. GCC-AP0341 (10 mg/kg, i.p. x 3) inhibited the recruitment of eosinophils in BAL fluid and in lung tissue, but it did not inhibit AHR. The compound also inhibited pulmonary granulomas and goblet cell hyperplasia. CONCLUSION: The mechanism of Sephadex-induced AHR may not be directly associated with inflammatory changes such as recruitment of eosinophils, pulmonary granulomas and hyperplasia of goblet cells in rats.

Animals↗
PubMed1998

A novel angiotensin II-receptor antagonist, 606A, induces regression of cardiac hypertrophy, augments endothelium-dependent relaxation and improves renal function in stroke-prone spontaneously hypertensive rats.

It is well-known that cardiac hypertrophy and arterial and renal dysfunction are serious complications of hypertension. Therefore, we investigated the chronic effects of 606A (2-propyl-3-[2'(1H-tetrazole-5-yl)biphenyl-4-yl]methyl-5-acetyl-4,5,6,7- tetrahydro imidazo [4,5-c]pyridine-4-carboxylic acid disodium salt), a novel AT1-receptor antagonist, on these complications of hypertension in stroke-prone spontaneously hypertensive rats (SHRSP) using Wistar Kyoto rats (WKY) as the control. After 8 weeks treatment from 16 weeks of age with 606A by a subcutaneously implanted osmotic pump, cardiac function, cardiac weight, acetylcholine-induced endothelium-dependent relaxation in the isolated aorta and renal function were estimated. Furthermore, wall thickness of the left ventricle was studied morphologically. We found that 606A (0.3 mg, 1 mg and 3 mg/head/day) dose-dependently lowered blood pressure without any effects on heart rate in SHRSP. Long-term treatments with 606A significantly reduced cardiac weight, left ventricular wall thickness and left ventricular end diastolic pressure, whereas it did not affect cardiac contractility. Endothelium-dependent relaxation of the aorta was recovered, and total protein excretion as well as total protein excretion/creatinine excretion ratio was reduced to the level of WKY by the treatment. These results suggest that 606A not only has a hypotensive effect but also protects cardiac, renal and vascular tissues from complications of hypertension. Thus, 606A could be an useful drug for treatment of hypertension.

Acetylcholine↗
PubMed1998

Difference in inhibitory effects of dexamethasone and cyclosporin A on Sephadex bead-induced airway hyperresponsiveness and inflammation in rats.

We investigated the effects of dexamethasone and cyclosporin A on Sephadex bead (Sephadex G-200, Sephadex)-induced airway hyperresponsiveness (AHR) and inflammation in rats. Sephadex (0.5 mg/animal) was intravenously administered on days 0, 2 and 5. Bronchoalveolar lavage, histological study and measurement of AHR were performed on day 7. Dexamethasone (0.1, 1 and 10 mg/kg, p.o. x 3) and cyclosporin A (0.1, 1 and 10 mg/kg, s.c. x 3) clearly inhibited the increase in eosinophils in bronchoalveolar lavage fluid after Sephadex injection. On histological study, pulmonary eosinophilia, granulomatous arteritis with horseshoe-shaped multinuclear giant cell formation and goblet hyperplasia were observed after Sephadex injection. Both dexamethasone (10 mg/kg x 3) and cyclosporin A (10 mg/kg x 3) inhibited these findings and an increase in eosinophil peroxidase in the lung. Dexamethasone dose-dependently inhibited AHR induced by Sephadex, and completely suppressed it at a dose of 1 mg/kg (x 3). Cyclosporin A, however, did not inhibit AHR even at a dose of 10 mg/kg ( x 3). These results show that there is a difference between dexamethasone and cyclosporin A in the inhibitory effect on Sephadex-induced AHR, and they suggest that eosinophils are not directly associated with the development of AHR after Sephadex injection.

Animals↗
PubMed1997

Phosphorylation of serine 256 is required for cAMP-dependent regulatory exocytosis of the aquaporin-2 water channel.

The aquaporin-2 (AQP2) vasopressin water channel is translocated to the apical membrane upon vasopressin stimulation. Phosphorylation of serine 256 of AQP2 by cAMP-dependent protein kinase has been shown, but its relation to vasopressin-regulated translocation has not been elucidated. To address this question, wild type (WT) AQP2 and a mutant with alanine in place of serine 256 of AQP2 (S256A) were expressed in LLC-PK1 cells by electroporation. Measurements by a stopped-flow light-scattering method revealed that the osmotic water permeability (Pf) of LLC-PK1 cells transfected with WT was 69.6 +/- 6.5 microm/s (24.8 +/- 2.2 microm/s for mock-transfected), and stimulation by 500 microM 8-(4-chlorophenylthio)-cAMP increased the Pf by 85 +/- 12%. When S256A AQP2 was transfected, the cAMP-dependent increase in the Pf was only 8 +/- 5%. After cAMP stimulation, the increase in surface expression of AQP2 determined by surface biotin labeling was 4 +/- 10%, significantly less than that for WT (88 +/- 5%). In addition, an in vivo [32P]orthophosphate labeling assay demonstrated significant phosphorylation of WT AQP2 and only minimal phosphorylation of S256A AQP2 in LLC-PK1 cells. Our results indicated that serine 256 of AQP2 is necessary for regulatory exocytosis and that cAMP-responsive redistribution of AQP2 may be regulated by phosphorylation of AQP2.

Alanine↗
PubMed1997

Transformation of normal human fibroblasts into immortalized cells with the mutant p53 gene and X-rays.

In vitro cell transformation is a valuable approach for studying the mechanisms of multistep carcinogenesis of human cells. Since immortalization is an essential step for in vitro neoplastic transformation of human cells, this study addresses the question of whether mutant p53 contributes to the immortalization process of human cells. The mutant p53 gene (mp53: codon273Arg-His) was introduced into normal human fibroblasts (OUMS-24 line) and a G418-resistant clone, OUMS-24/P6 line, was obtained. This clone showed an extended life span and chromosome abnormalities, but senesced at the 79th population doubling level (PDL). When these cells were subjected to intermittent X-ray treatment, they became an immortalized cell line (OUMS-24/P6X). Although these immortalized cells showed chromosome abnormalities, they were not tumorigenic. On the other hand, normal OUMS-24 cells into which mp53 had not been introduced were not immortalized by the same X-ray treatment. These results indicate that introduction and expression of mp53 alone were not sufficient for immortalization of human cells, and that mutations of the remaining wild-type p53 or other genes may have been necessary for immortalization. In fact, no expression of the wild-type p53 was detected in the immortalized cells by RT-PCR. Expression of p21, which is located downstream of p53, was remarkably reduced in the immortalized cells, resulting in an increase in cdk2 and cdc2 kinase activity. These findings indicate that the p53-p21 cascade may play some role in the immortalization of human cells. On the other hand, there was no significant difference in expression of proteins such as Rb, p16, cdk4, cdk6, cyclin A and cyclin D1 between the normal and immortalized human fibroblasts.

Cell Transformation, Neoplastic↗
PubMed1997

Immortalization of mutant p53-transfected human fibroblasts by treatment with either 4-nitroquinoline 1-oxide or X-rays.

The study of in vitro cell transformation is valuable for understanding the multistep carcinogenesis of human cells. The difficulty in inducing neoplastic transformation of human cells by treatment with chemical or physical agents alone is due to the difficulty in immortalizing normal human cells. Thus, the immortalization step is critical for in vitro neoplastic transformation of human cells. We transfected a mutant p53 gene (mp53: codon 273Arg-His) into normal human fibroblasts and obtained two G418-resistant mp53-containing clones. These clones showed an extended life span but ultimately senesced. However, when they were treated with either 4-nitroquinoline 1-oxide or X-rays, they were immortalized. The immortalized cells showed both numerical and structural chromosome abnormalities, but they were not tumorigenic. The expression of mutant but not wild type p53 was detected in the immortalized cells by RT-PCR. Expression of p21, which is located downstream of p53, was remarkably reduced in the immortalized cells, resulting in increased cdk2 and cdc2 kinase activity. However, there was no significant difference between the normal and immortalized human cells in expression of another tumor suppressor gene, p16. These findings indicate that the p53-p21 cascade may play an important role in the immortalization of human cells.

4-Nitroquinoline-1-oxide↗
PubMed1997

Isolation of a gene encoding nodulin-like intrinsic protein of Escherichia coli.

Members of the membrane intrinsic protein (MIP) family are expressed in various organisms including plants, insects, and vertebrates. E. coli is known to have a MIP member gene, glycerol facilitator (G1pF). Here we report the isolation of E. coli gene encoding BniP, bacterial nodulin-like intrinsic protein. BniP encodes a 231 amino acid, 24 kDa protein with 42% amino acid identity to Nod26, 38% amino acid identity to AQP1, and 29% amino acid identity to G1pF. Analysis of deduced amino acid sequence predicted a hydrophobic protein with six membrane-spanning domains. Expression of BniP in Xenopus oocytes induced slight increase in osmotic water permeability, but not glycerol or ion permeability. Our results showed that BniP is a new member of the MIP channel-forming proteins of E. coli.

Amino Acid Sequence↗
PubMed1997

Induction of aquaporin 3 by corticosteroid in a human airway epithelial cell line.

Although aquaporin 3 (AQP3) is expressed in many tissues in the kidney, gastrointestinal tract, lung, and other organs, its physiological significance in the body still remains to be clarified. To determine whether AQP3 expression is regulated by dexamethasone in human airway epithelium, we studied mRNA expression, protein expression, and water permeability of the cell membrane in a human airway epithelial cell line (A549 cells). Expression of AQP3 mRNA and protein was studied by Northern blot analysis and immunoblot analysis, and osmotic water permeability (Pf) was measured by a stopped-flow light-scattering method. Expression of AQP3 mRNA and protein was detectable in A549 cells and was stimulated by dexamethasone. Pf in A549 cells after incubation with dexamethasone was approximately 2.5-fold greater than that without dexamethasone. Moreover, this dexamethasone-induced increase in Pf was inhibited after treatment with HgCl2. In conclusion, the present study shows that A549 cells express AQP3 and that dexamethasone upregulates the expression of AQP3 and increases the water permeability of the cell membrane. Dexamethasone-regulated AQP3 expression might be important in certain forms of pulmonary diseases accompanied by airway hypersecretion that are treated by corticosteroid administration.

Adenocarcinoma↗
PubMed1997

Alteration in water channel AQP-2 by removal of AVP stimulation in collecting duct cells of dehydrated rats.

Dehydration increased the expression of aquaporin of collecting duct (AQP-2) and translocated AQP-2 to the apical plasma membranes from cytoplasmic vesicles of collecting duct cells. We determined whether the abrupt decrease in circulating arginine vasopressin (AVP) by giving excess water affects the expression of AQP-2 mRNA and subcellular localization of AQP-2 in collecting duct cells of the dehydrated rats. The 72-h water deprivation increased plasma AVP levels to 3.1 pg/ml and the expression of AQP-2 mRNA by 336% in rats, which were concomitantly abolished by the 40 ml/kg oral water load. A 50% inhibition ofAQP-2 mRNA expression was obtained with 20 min after the forced water load. In immunohistochemistry and electron microscopy, the AQP-2 was manifestly present around the apical edge of collecting duct cells in the 72-h dehydrated rats. The AQP-2 was diffusely translocated into the cytoplasm 1 h after the forced water administration. These results indicate that AVP plays the on-off regulation of AQP-2 mRNA expression and that a majority of AQP-2 is regulated by the shuttle recycling in the collecting duct cells.

Animals↗
PubMed1997

Expression of AQP family in rat kidneys during development and maturation.

The mRNA expression and localization of the aquaporin (AQP) family in rat kidney were examined by ribonuclease protection assay and immunohistochemistry. AQP1, AQP2, AQP3, and AQP4 mRNA were hardly detectable in 16-day gestation fetuses. AQP1 mRNA was explosively expressed at 1 wk, keeping the level throughout life. AQP2 mRNA expression was apparently noticed in 18-day fetuses and was enhanced gradually with age to reach a plateau at 4 wk. AQP3 and AQP4 mRNA expression was significantly found at birth but was not changed remarkably thereafter. AQP2 protein appeared first at the apical side of collecting duct cells in 18-day fetuses. The staining intensity at the site increased with age, and basolateral staining was added in adult rats. AQP3 was distinctly demonstrated at the basolateral side of collecting duct cells after birth, and the staining intensity was almost stable throughout life. The progressive induction of AQP2 expression in the first 4 wk after birth is presumed to contribute to the maturation of urinary concentrating capacity during the kidney development.

Aging↗
PubMed1997

Immunolocalization and effect of dehydration on AQP3, a basolateral water channel of kidney collecting ducts.

Aquaporin-3 (AQP3) is unique in its structure (lowest homology with other aquaporins) and in its function (significantly conductive to both small nonelectrolytes and water). However, there is a controversy among researchers on its water transport and induction by dehydration. We examined its localization and the effect of dehydration on its expression in the kidney, as well as its water channel activity when expressed in Xenopus oocytes. In vitro translation using reticulocyte lysate revealed that the size of rat AQP3 was 26 kDa, and the band shifted to around 31 kDa with microsomal fraction, which was sensitive to the digestion with N-glycosidase F. In Western blot analysis of rat kidney medulla, AQP3 appeared as a sharp band at 27 kDa and a broad band at 34-40 kDa. In immunohistochemistry, AQP3 was localized to principal cells and absent in intercalated cells in outer medulla. In inner medulla, AQP3 was restricted to inner medullary collecting duct (IMCD) cells. AQP3 was confined to the basolateral membrane of these cells. Although dehydration of rats for 2 days did not change the distribution pattern of AQP3 in IMCD cells, the dehydration increased AQP3 mRNA by twofold with slight increase of its protein level in kidney medulla. Finally, we confirmed its water channel activity when expressed in Xenopus oocytes. The human AQP3 stimulated osmotic water permeability by eightfold, which was inhibited by 0.3 mM mercury chloride by 34% and reversed by beta-mercaptoethanol. Our results indicate that AQP3 is a glycosylated protein and a mercury-sensitive water channel localized at the basolateral membrane of principal cells and IMCD cells, and its expression is induced by dehydration at both protein and mRNA level.

Animals↗
PubMed1997

Comparison of cellular characteristics between human hepatoma cell lines with wild-type p53 and those with mutant-type p53 gene.

Characteristics of human hepatoma cell lines with the wild-type p53 were compared with those of human hepatoma cell lines with the mutant-type p53. The p21 protein located downstream of p53 was expressed in cell lines with the wild-type p53 but was not expressed in cell lines with the mutant-type p53. As to other tumor suppressor genes such as p16 and p27, there was no difference in their expression between both types of cell lines. In addition, no marked difference was observed in the activities of CDK2 and CDK4 between cell lines with the wild-type and the mutant-type p53. Phosphorylated Rb protein was detected in all cell lines except the HLE line, indicating that this cell line may have a deletion of and/or a mutation of the Rb gene. These results indicate that abnormalities of tumor suppressor genes other than p53, p16, p27, and Rb may be involved in hepatocarcinogenesis. The population doubling time of the wild-type p53 cells was significantly longer than that of the mutant p53 cells. Neither type of cell line showed a specific chromosome distribution which would indicate karyotype instability. The cell lines expressing the wild-type p53 produced tumors at lower frequency than those with the mutant p53 gene. Although there was no significant difference in effects of TGF-beta 1, EGF, cholera toxin, and db-cAMP on cell growth between the two types of cells, all three cell lines with the wild-type p53 were resistant to cytotoxicity of TNF-alpha, while two of the three with the mutant p53 were very sensitive to its cytotoxic effects.

Animals↗
PubMed1997

Yeast functional assay of the p53 gene status in human cell lines maintained in our laboratory.

We used a yeast functional assay (functional analysis of separated alleles in yeast: FASAY) to determine the p53 gene status of human cell lines maintained in our laboratory. This assay enables the researcher to score wild-type p53 expression on the basis of the ability of expressed p53 to transactivate the reporter gene HIS 3 via the p53-responsive GAL 1 promoter in Saccharomyces cerevisiae. The cell lines examined were ten hepatoma, two hepatoblastoma, three in vitro immortalized fibroblast, two osteosarcoma, a chondrosarcoma, an ovarian teratocarcinoma and a colon cancer cell line. Out of 20 cell lines, 11 cell lines had mutations in both alleles of the p53 gene, and another 8 cell lines had no mutation in the p53 gene. Thus, 55% of the cell lines examined had mutations in the p53. Interestingly, PA-1 cells had both the normal and the mutant p53 alleles, showing that FASAY is a useful method for detecting the wild-type and mutated p53 genes simultaneously. As for the three liver cell lines harboring HBsAg, there was no relationship between their p53 gene status and the presence of HBsAg. Two cell lines were normal for p53 status, while the other had a mutation of the p53 gene.

Alleles↗
PubMed1997

Enhancement of DNA-transfection frequency by X-rays.

This study was conducted to evaluate the frequency of DNA transfection into human cells following X-ray irradiation. We transfected plasmid DNA (pSV2neo) into human cells, HeLa and PA-1, by either calcium phosphate precipitation or the lipofection method immediately after irradiating the cells with various doses of X-rays. The transfection frequency was evaluated by counting the number of G418-resistant colonies. When circular plasmid DNA was used, irradiation up to a dose of 2 Gy dose-dependently increased the transfection frequency, which reached a maximum of 5 to 10-fold that of the control unirradiated cells. When linear plasmid DNA was used, the transfection frequency was 2 times higher than that of circular DNA. All five of the clones that were randomly chosen expressed the transfected neo gene. In addition, the pSV2neo gene was randomly integrated into the genomic DNA of each clone. These findings indicate that X-ray treatment can facilitate foreign DNA transfer into human cells and that radiation-induced DNA breaks may promote the insertion of foreign DNA into host DNA. The enhancement of DNA transfection with X-rays may be instrumental in practicing gene therapy.

Cell Line↗
PubMed1997

Water channel AQP1, 3, and 4 in the human peritoneum and peritoneal dialysate.

To clarify the mechanism of water transport driven by osmotic gradient through "ultrasmall pores" in the peritoneum, we tried to identify water channels in the peritoneum and cells in the peritoneal dialysate. Peritoneum was surgically excised from uremic patients at the insertion or removal of a catheter. Sediment was collected from 2 L of peritoneal dialysate by centrifugation at 1500 rpm. RNA was extracted and amplified by reverse transcription-polymerase chain reaction (RT-PCR). Contamination of reticulocytes was tested by the presence of ankyrin mRNA. Peritoneal tissue expressed aquaporin (AQP) 1, 3, and 4 (AQP1 > 3 > 4). Sediment of dialysate expressed mRNA of AQP1 and AQP3 (AQP1 > AQP3). The sample did not express ankyrin mRNA, indicating that the AQP1 in the sediment did not originate from reticulocytes. These data indicate that aquaporins are present in the peritoneum and might participate in water transport. Further quantitative analysis of aquaporin messages in the dialysate might clarify the pathogenesis of water removal failure.

Ankyrins↗
PubMed1996

Structure of aquaporin-2 vasopressin water channel.

Aquaporin-2 (AQP-2) is a vasopressin-regulated water channel in the kidney collecting duct. AQP-2 is selectively permeable to water molecule and is translocated between the apical membrane and subapical endosomes in response to vasopressin. To investigate the localization and structure of the aqueous pathway of the AQP-2 water channel, a series of site-directed mutants was constructed and functionally analyzed. Insertion of N-glycosylation reporter sequence into each hydrophilic loop (HL) indicated that AQP-2 has a six-membrane spanning topology and that insertional mutations in HL-2 or HL-5 do not alter water channel function. Mercury-sensitive site of AQP-2 is located near the second asparagine-proline-alanine (NPA) domain at cysteine 181, but not near the first NPA domain. Replacement of HL-3 or HL-4 with the corresponding part of Escherichia coli glycerol facilitator abolished water channel function without changing plasma membrane expression of the channel protein. Introduction of cysteine residues in His-122, Asn-123, Gly-154, Asp-155, or Asn-156 induced partial mercury sensitivity, and point mutations in asparagine 123 significantly altered water permeability. Our results implicate that the structure of AQP-2 is different from models previously proposed for AQP-1 and that HL-3 and HL-4 are closely located to the aqueous pathway.

Amino Acid Sequence↗