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N Otaki

Publications and source records attributed to N Otaki.

At least 19 recordsLinked to original sources
PubMed2000

Rapid identification of metallothionein isoforms in liver cytosol fraction by capillary zone electrophoresis using EDTA.

Identification of metallothionein (MT) isoforms on capillary zone electrophoresis (CZE) analysis was studied using a linear polyacrylamide-coated capillary at pH 7.4 and EDTA. The CZE system was able to separate standard (purified and commercially available) MT specimens into their isoforms within 10 min. The peaks of MT-1 and MT-2 isoforms disappeared on addition of EDTA to the specimen, and the disappearance was shown to be time-dependent and dose-dependent, although the reason why the peaks decreased is still unclear. A heat-treated cytosol fraction prepared from Zn-injected mouse liver showed many major and minor peaks on CZE analysis. Two major peaks were identified to be MT-1 and MT-2, respectively, by co-injection with the purified MT isoforms. When EDTA was added to the cytosol fraction, the two major peaks, MT-1 and MT-2, and three other minor peaks disappeared time-dependently. Therefore, each MT isoform in the cytosol fraction can be identified by the addition of EDTA, also the peaks are identified by the corresponding migration times of purified MTs. Unknown substances like MT sub-isoforms may also be detected, although this question warrants clarification. From these results, it was concluded that the addition of EDTA is useful for identification of MT isoforms in cytosol fractions on CZE analysis.

Animals↗
PubMed1999

Identification of metallothionein isoforms on capillary zone electrophoresis by adding anti-metallothionein antibody.

The aim of this study was to identify metallothionein (MT) isoforms in mouse liver by using capillary zone electrophoresis (CZE). Purified MT-1 and MT-2 isoforms were completely separated by CZE using a polyacrylamide-coated tube at physiologic pH. There were two peaks in the cytosol fraction prepared from zinc-injected mouse liver, in which the migration times corresponded with those of purified MT-1 and MT-2 isoforms. When anti-MT monoclonal antibody was added with the purified MT-1 or MT-2 solution, the peaks decreased. Furthermore, the two peaks in the cytosol prepared from Zn-injected mouse liver decreased in a time-dependent manner from the electropherogram after the addition of the antibody. Therefore, those peaks were identified as MT-1 and MT-2 isoforms, respectively. In conclusion, the addition of anti-MT monoclonal antibody to the cytosol fraction of tissues is an effective method for identification of MT isoforms after separation using CZE.

Animals↗
PubMed1999

Acute biological effects of intratracheally instilled titanium dioxide whiskers compared with nonfibrous titanium dioxide and amosite in rats.

The dimensions of man-made mineral fiber whiskers are similar to those of some kinds of asbestos. Thus these mineral fibers raise the concern for potential health hazard for workers exposed in the occupational environments. This study was designed to define acute biological effects of intratracheally administered titanium dioxide whiskers (TO1) compared with nonfibrous titanium dioxide (TOP) and UICC amosite (Ams), and their relations to acute lung inflammation in rats. The observed geometric mean length (microm) and width (microm) and geometric standard deviation are: TO1(2.1[2.0], 0.14[1. 53]); Ams (4.3[3.3], 0.31[1.9]); and TOP (50 nm, 1-2 microm aggregates). Ten-week-old Wistar-Jcl male rats received a single tracheal injection of test materials at doses between 0.05 and 1.0 mg/rat. Control animals were injected with the same volume of saline. Lung tissue and bronchoalveolar lavage (BAL) fluid were collected from rats on days 1, 3, and 7 after administration. In the group injected with TO1, total protein, cytokine-induced neutrophil chemoattractant (CINC)/growth-regulated gene product (GRO), interleukin (IL) 1beta, and tumor necrosis factor (TNF) alpha increased on day 1. Subsequently, total elastolytic activity and fucose levels in BAL increased by day 3. All parameters, except for fucose in BAL, recovered to the normal levels. Animals in the Ams group showed increased total protein and CINC/GRO and decreased total elastolytic activity in a dose-dependent manner on day 1. The fucose level increased on day 3 in the Ams group. All parameters returned to their control levels on day 7. Animals in the TOP group did not show significant changes any of parameters during the experimental period. Gene expression of TNF-alpha and monocyte chemoattractant protein (MCP) 3 in the lung increased dose-dependently in the animals treated with the three materials. The mRNAs for eotaxin and MIP-1alpha were overexpressed in the lung of animals treated with Ams and TO1, while RANTES mRNA was overexpressed dose-dependently in the lung of animals treated with Ams on day 1. Onset of inflammatory response was more rapid in the Ams group than the TO1 group. Recovery of the fucose level in BAL was slower in the TO1 group than in the Ams group, though we observed similar histopathological changes in the lung of animals with TO1 or Ams. We conclude that whisker-induced acute biological effects in the lung may be related to the shape of the whiskers and not to their chemical composition or surface crystal structure, showing biological effects similar to those of UICC amosite.

Administration, Inhalation↗
PubMed1996

Identification of metallothionein isoforms with capillary zone electrophoresis using a polyacrylamide-coated tube.

Metallothionein (MT) isoforms from various liver tissues were separated with capillary zone electrophoresis (CZE) using a polyacrylamide-coated tube at neutral pH. The electrophoresis was performed on MT-1 and MT-2 purified from mouse, rat, rabbit and human livers. The retention times of mouse and rat MT-1 coincided, while the retention times of rabbit and human MT-1 were longer. The retention times of MT-2 purified from the four sources were the same. MT-1 and MT-2 separated more definitely with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-Tris buffer (25 mM, pH 7.4) than with N-tris(hydroxymethyl)methyl-3-aminopropane sulfonic acid (TAPS)-Tris buffer (25 mM, pH 7.7) or with N-(2-acetamido)iminodiacetic acid (ADA)-Tris buffer (25 mM, pH 7.4). In addition, liver MT isoforms prepared from Zn- or Cd-administered mice could be separated.

Acrylic Resins↗
PubMed1996

Blocking effect of anti-mouse interleukin-6 monoclonal antibody and glucocorticoid receptor antagonist, RU38486, on metallothionein-inducing activity of serum from lipopolysaccharide-treated mice.

Although there is much evidence to suggest that lipopolysaccharide (LPS)-induced elevation of hepatic metallothionein (MT) contents is mediated by cytokines, the presence of MT-inducing activity in the serum of LPS-treated animals has not been examined. It was found that serum from LPS-treated mice stimulated MT induction in a hepatoma cell culture. The MT-inducing activity in serum was highest 2 h after LPS injection. Tumor necrosis factor and interleukin (IL)-6 levels in the serum were highest 1 and 2 h, respectively, after LPS injection. Anti-mouse IL-6 monoclonal antibody neutralized MT-inducing activity in serum obtained from mice 2 h after LPS injection. The MT-inducing activity in serum was blocked by the glucocorticoid antagonist, RU38486. A similar requirement for glucocorticoid was also observed in an IL-6-stimulated culture. These results show that the LPS-induced elevation of hepatic MT is mediated by IL-6, and the expression of the stimulating activity of IL-6 is dependent on the presence of glucocorticoid.

Analysis of Variance↗
PubMed1994

Localization of metallothionein in aged human brain.

The localization of metallothionein (MT), a small molecular weight heavy metal binding protein in aged human brain, was investigated by immunohistochemical techniques. The amount of MT and heavy metals (Zn, Cu) were also assayed by radioimmunoassay and atomic absorption spectrophotometry, respectively. Immunohistochemically, MT was found in the pia mater, ependymal cells, protoplasmic astrocytes nand glial processes neuropil of the gray matter and fibrous astrocytes of the white matter of the telencephalon, whereas oligodendroglia and microglia did not show any positive immunostaining for MT. Cytoplasm, glial processes and some nuclei of protoplasmic and fibrous astrocytes showed strong MT immunostaining. Vascular feet and adventitia were also positive for MT immunostaining. Moreover, the pia mater, astrocytes and ependyma in the diencephalon, mesencephalon, pons, medulla oblongata and spinal cord showed the same positive immunostaining for MT as the telencephalon. In the cerebellum, Bergmann's glia, protoplasmic astrocytes of the granular layer and fibrous astrocytes of the white matter showed strongly positive immunostaining for MT. According to the radioimmunoassay, the amount of MT in the brain was relatively high at about 39.3 +/- 9.5 micrograms/g wet weight; of the heavy metals in the brain, zinc and copper were detected.

Age Factors↗
PubMed1994

Metallothionein in human seminal plasma.

Metallothionein (MT) concentrations were measured in the seminal plasma of 4 fertile and 35 infertile men and in the hydrocele and spermatocele fluids. The relationship between MT content and sperm density, total number of sperm per ejaculate, sperm motility and abnormal form rates, leukocyte count and zinc levels in seminal plasma, as well as the relationship between MT and serum follicle-stimulating hormone, luteinizing hormone, testosterone, and prolactin were examined. MT was not detected in the hydrocele and spermatocele fluids. MT levels were related to zinc levels and to the leukocyte count in seminal plasma, but there was no correlation between MT and the other factors examined. This study supported previous findings that MT was secreted predominantly from the prostate and induced by inflammation of the prostate gland or seminal vesicles; the findings suggest that MT binds mainly to zinc and is one of the zinc-binding proteins in seminal plasma.

Adult↗
PubMed1994

Abnormal accumulation of copper-metallothionein in the liver and kidney of Long-Evans rats with a cinnamon-like coat color (LEC rats).

We determined the copper (Cu) and metallothionein (MT) concentrations in the liver and kidney supernatants of Long-Evans rats with a cinnamon-like coat color (LEC rats), and also measured the Cu and MT levels in the serum of these rats. Seven-week-old rats had abnormally high levels of both substances in the liver. The levels in the liver supernatant were over 80- and 16-fold higher, respectively, in LEC rats than in normal 7-week-old Wistar rats. LEC rats suffering from acute hepatitis or hepatoma had a much higher level of hepatic MT, but the Cu level was higher only in the liver of those with hepatoma. The serum levels of Cu and MT in LEC rats with acute hepatitis were more than 10-fold higher than those in normal LEC rats. These levels were decreased in the rats with chronic hepatitis or hepatoma. In the liver of LEC rats with hepatoma, the area of hepatocellular carcinoma and of noncancerous liver showed over twice higher Cu and MT levels than the area of cholangiofibrosis. The Sephadex G-75 elution profile from the liver supernatant of a normal LEC rat showed that the peak of Cu closely corresponded to that of MT recognized with anti-MT antiserum. The levels of Cu and MT in the kidney supernatant of LEC rats with acute hepatitis were more than 25-fold higher than in that of normal LEC rats. However, there were no marked increases in the levels in the kidney supernatant of LEC rats with chronic hepatitis or hepatoma.

Animals↗
PubMed1994

Induction and subcellular localization of metallothionein in regenerating rat liver.

A highly specific antiserum against rat liver metallothionein (MT) was raised in a Japanese white rabbit. Using this anti-MT antiserum, we found that MT was localized in the nuclei as well as in the cytoplasm of hepatocytes in newborn rats. Since it is known that these cells are growing actively, we suspected that there was a relationship between the localization of MT in cell nuclei and the cell proliferation. Therefore, the induction and subcellular localization of MT were examined in rat liver remaining after 70% removal. MT was induced in the remnant liver rapidly after the hepatectomy, its concentration being about 80-fold higher than that of the intact liver. Flow cytometric analysis revealed that MT was translocated into the nuclei from the cytoplasm of hepatocytes during liver regeneration after partial hepatectomy. The highest MT level in the nuclei was observed 24 h after hepatectomy. MT-stained positive nuclei were in S to G2M phases of the cell cycle of regenerating hepatocytes, and the nuclei in G1 phase were not stained with anti-MT antiserum. The increase in hepatic MT levels did not directly cause MT translocation into the nuclei. These results suggested that MT was a cell cycle-dependent, nuclear protein.

Animals↗
PubMed1993

Copper-metallothionein distribution in the liver of Long-Evans cinnamon rats: studies on immunohistochemical staining, metal determination, gel filtration and electron spin resonance spectroscopy.

Distribution of metallothionein (MT) and copper ion (Cu) in the liver of LEC (Long-Evans Cinnamon) rats was investigated to examine the relationship between Cu-MT induction and the development of hepatitis followed by hepatocellular carcinomas. Immunohistochemical studies on MT in the liver of LEC rats indicated that MT is accumulated in nuclei and cytosols. Both MT and Cu, estimated by radioimmunoassay and flameless atomic absorption spectrometry, respectively, in subcellular fractions of the liver were found to be concentrated highest in cytosols, followed by nuclei, mitochondria and microsomal fractions. Gel-filtration (Sephadex G-75) studies demonstrated that MT is induced as the Cu-MT form. Furthermore, the Cu-MT fragment purified by the gel-filtration contains the Cu(I)-MT form, as demonstrated by ESR (electron spin resonance) measurements at 77K. These results will be important for understanding the development of hepatitis in LEC rats.

Animals↗
PubMed1993

Induction of metallothionein by CdCl2 administration in rat prostate.

Metallothionein (MT) induction in the rat prostate gland was investigated by means of cadmium chloride administration. Ten-week-old male Wistar rats housed with cadmium free food were divided into three groups of six rats each and castrated. After seven days, 1 mg of testosterone propionate per rat was injected subcutaneously once a day until the end of the experiment. After three weeks, rats were injected daily for six days with a physiological saline, 0.3 mg/kg CdCl2, and 0.9 mg/kg CdCl2. MT concentration of the ventral and dorsal lobes was significantly increased in the three groups in proportion to the dose of CdCl2. MT content of the lateral lobe in three groups was also increased, but was not significantly different. Immunohistochemically, MT was induced mainly in the ventral lobe in the basal cells, and in the lateral and dorsal lobes in the epithelial cells. The weights of the prostatic lobes were similar in the three groups, and no histological change was identified. These results suggested that MT in the prostate was induced by cadmium administration, and that it may prevent cellular damage from harmful metals.

Animals↗
PubMed1993

Analysis of mammalian metallothionein isoforms by high-resolution SDS-gel electrophoresis.

Metallothioneins (MTs) are cysteine-rich heavy metal-binding proteins, whose possible functions are thought to be the protection against toxic metals as well as the regulation of essential metals. It is known that there are several MT isoforms, but the biological roles of the individual isoforms have not been elucidated. To facilitate the functional analysis of these isoforms, we improved an analytical method of MTs developed previously, which is based on a denaturing gel electrophoresis of chemically modified MTs. The established technique makes it possible not only to separate MT isoforms with a high resolution, but to estimate the levels of the individual isoforms by analyzing directly crude cell extracts. By this method, six MT isoforms were identified in the extracts of Cd-exposed human cells. It was also revealed that there is an apparent heterogeneity of the rat liver MT; five isoforms were identified in the liver extracts of Cd-injected rats. The present method will be useful in the functional analysis of the MT isoforms, as well as in a variety of aspects of the MT studies.

Animals↗
PubMed1992

Copper-metallothionein induction in the liver of LEC rats.

Recently, copper (Cu) was found to be unusually accumulated, suggesting the induction of metallothionein (MT) in the liver of LEC rats (Long-Evans rats with a cinnamon-like coat color), which develop spontaneous jaundice with hereditary hepatitis. Thus, the direct relationship between the unusual Cu accumulation and the induction of Cu-MT was investigated by giving LEC rats Cu-overloaded or Cu-deficient diets. Results based on the determinations of Cu and MT levels in several organs, as well as the gel-filtration profiles of the cytosols of liver homogenates, showed that dietary Cu induced Cu-MT and development of hepatic injury associated with jaundice.

Animals↗
PubMed1992

Unusual accumulation of copper related to induction of metallothionein in the liver of LEC rats.

Copper (Cu), iron (Fe), zinc (Zn) and manganese (Mn) levels in organs of LEC rats (Long-Evans rats with a cinnamon-like coat color), which develop spontaneous jaundice with hereditary hepatitis, were determined by instrumental neutron activation analysis method. Unusual accumulations of Cu in the liver of LEC rats were found, depending on the age of the animals, the metal concentration being more than approximately 20-40 times those of normal LEA rats (Long-Evans rats with an agouti coat color). Fe and Zn were also accumulated, in addition to Cu, significantly in the LEC rats. The unusual Cu accumulations in the liver of LEC rats were associated with the induction of metallothionein, estimated by radioimmunoassay method, in the liver of LEC rats, rather than that of superoxide dismutase, estimated by electron spin resonance -spin trapping method. These findings suggest that the unusual Cu accumulation in LEC rats is involved in the development of jaundice, hepatic injury and hepatocellular carcinoma.

Aging↗
PubMed1992

Metallothionein of prostatic tissues and fluids in rats and humans.

We analyzed metallothionein (MT) in rat prostates by gel filtration and radioimmunoassay. The concentration of MT in the prostate, kidney and liver of cadmium-induced rats was measured. The concentration of MT was also measured in normal prostate, benign prostatic hyperplasia, prostate cancer and the prostatic fluids from various prostatic diseases in humans. MT was detected in rat prostates by gel filtration and radioimmunoassay. The concentration of MT (micrograms/g wet tissue) was 0.3 +/- 0.1 (S.D.) in the ventral lobe, 30.4 +/- 24.0 in the lateral lobe, 5.2 +/- 0.9 in the dorsal lobe, 25.0 +/- 6.4 in the kidney and 2.0 +/- 1.5 in the liver of the rat control group. Change in MT content in CdCl2-induced organs increased quantitatively with the dose administered. The concentration of MT (micrograms/g wet tissue) in human prostate was 99.3 +/- 121.8 in the peripheral zone (PZ), 12.0 +/- 8.5 in the preprostatic region (PR), 7.3 +/- 3.1 in the central zone (CZ), 17.5 +/- 15.0 in benign hyperplastic nodules (A) and 4.2 +/- 0.5 in cancer tissue (CA). MT concentration in PZ was very high and that of CA, low (p less than 0.05). MT concentration in prostatic fluids (ng/mg protein) was 11.5 +/- 5.7 in normal patients, 3.8 +/- 2.3 in acute prostatitis, 6.5 +/- 3.7 in chronic prostatitis with pyuria, 39.6 +/- 3.9 in chronic prostatitis without pyuria and 16.9 +/- 3.0 in benign prostatic hyperplasia. We concluded that MT in the prostate is induced by heavy metals and secreted into prostatic fluid. Possibly, it is a marker of secretory function in the prostate.

Animals↗
PubMed1992

Immunohistochemical study of metallothionein in human seminal vesicles.

Metallothionein (MT) in human seminal vesicles was examined by use of the avidin-biotin-peroxidase complex method. Tissues were obtained from six patients with prostate cancer who underwent luteinizing hormone-releasing hormone agonist or estrogen therapy before radical prostatectomy (group 1) and from 18 patients without hormone therapy (three with prostate cancer, three with urinary bladder cancer, and twelve free of urogenital diseases at autopsy) (group 2). MT was localized in the cytoplasm and nuclei of epithelial cells and also in secretory products in the lumen. The epithelial cells lacked uniformity in immunoreaction; for instance, some stained strongly while others stained weakly. Smooth muscle cells were found to have positive immunoreaction, but other connective tissues had no immunoreaction. The number of strongly positive cells in group 1 was fewer than that in group 2 (not significant), and the secretory products in group 1 had no immunoreaction. These results suggest that MT is synthesized in the epithelial cells of the seminal vesicles and secreted into the fluids, and that the synthesis of MT is suppressed by the hormone therapy.

Aged↗
PubMed1991

Localization of metallothionein in nuclei of growing primary cultured adult rat hepatocytes.

In primary cultured adult rat hepatocytes stimulated by epidermal growth factor and insulin, dramatic changes in the subcellular distribution of metallothionein were clarified by indirect immunofluorescence using antisera specific for this protein. Metallothionein was detected only in the cytoplasm of cultured hepatocytes in the G0 and G1 phases, but was concentrated in the cell nuclei in the early S phase. The strongest staining pattern in the nuclei was observed 12 h after stimulation. Subsequently, the intensity of metallothionein staining in the nuclei decreased. These results suggest that primary cultured hepatocytes are suitable for examining the relation between subcellular localization of metallothionein and cell growth.

Animals↗