PubMed Health⌕ Search

Biomedical subjects

R Takaki

Publications and source records attributed to R Takaki.

At least 73 records · Page 4Linked to original sources
PubMed1985

The relationship between molecular and cellular repair from potentially lethal damage induced by N-methyl-N'-nitro-N-nitrosoguanidine in Chinese hamster V79 cells.

The relationship between molecular and cellular repair from potentially lethal damage (PLD) induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was investigated in exponentially growing V79 Chinese hamster cells. We compared the repair processes by an alkaline sucrose sedimentation analysis and a colony formation assay. MNNG-treated cells were exposed to the conditioned medium (CM) from density-inhibited plateau-phase V79 cell cultures, as a post-treatment for the induction of PLD repair. When MNNG-treated cells were postincubated in CM, cell survival continuously increased for 18 h, and during this period, DNA replication was substantially suppressed. CM did not inhibit the rejoining of the single-strand breaks of parental DNA. Rather, parental DNA fragments sedimented more rapidly when postincubated in CM than in fresh medium. These data indicate that cellular recovery from MNNG-induced PLD increases in proportion to the resealing of MNNG-induced single-strand breaks of DNA during the suppression of DNA replication, suggesting that excision repair is involved in the PLD repair process.

Animals↗
PubMed1984

Chinese hamster ovary cell variants resistant to monensin, an ionophoric antibiotic. II. Growth requirement for insulin and altered insulin-receptor activity.

From the Chinese hamster ovary (CHO) cell, genetic variants (MonR-31 and MonR-32) relatively resistant to monensin, an ionophoric antibiotic, have been isolated. Growth of both MonR-31 and MonR-32 clones required higher doses of serum than CHO. Addition of insulin to media containing a low dose of serum restored full colony formation, but growth of MonR-31 or MonR-32 cells required more insulin than CHO cells. Specific binding of [125I]insulin was observed in these cell lines. The two MonR clones bound about one-half or less the [125I]insulin bound by CHO cells. Scatchard analysis for [125I]insulin binding at 4 degrees C and 37 degrees C showed altered number of binding sites, but not insulin affinity: The number of binding sites in the MonR cell was about a half or less that of the parental CHO cell. Down-regulation of insulin receptor was assayed when both CHO and MonR cells were incubated with 1 microgram/ml insulin. A 50-60% decrease in levels of insulin surface binding capacities was observed in CHO after exposure to insulin, whereas there was no decrease in MonR cell. The cellular uptake of 2-[3H]deoxyglucose into CHO cells was significantly enhanced in the presence of insulin, but only slight, if any, increase was observed in MonR cells.

Animals↗
PubMed1984

Establishment of glucagon-producing cells by cell hybridization.

Glucagon-producing cell lines were established by fusing pancreatic islet cells of adult hamster and 6-thioguanine-resistant hamster insulinoma cells. Under phase-contrast microscopy, the morphology of cultured hybrid cells was intermediate between those of the parental cells. The hybrid cells contained A-like granules, though few in number, and were stained with anti-glucagon antibody. The mode of chromosome number decreased to 78 or 79 by 3 mo after hybridization in comparison with the expected chromosome number of the heterokaryon of 104, and showed a minute decrease in 4 of 6 cell lines after 6 mo. The population doubling time ranged from 24 to 38 h, while that of parental insulinoma cells was 22.8 h. There was no correlation between the expression of cellular function and the stability of chromosome number or the length of population doubling time. The capacity of glucagon secretion was between that of the parental cells. The glucagon secreted into the medium, as assayed by the glucagon-specific antibody, was 0.6-2.5 ng/10(6) cells for 2 h, which was about 40% of total glucagon-like immunoreactivity secreted. Secretion of glucagon was not affected by high concentration of glucose, was markedly increased by theophylline, and was suppressed by exogenous insulin. All of the hybrid cells produced tumors on transplantation 6 mo after hybridization. The tumor-bearing hamsters exhibited high levels of plasma glucagon and blood glucose as well as a high level of serum insulin.

Adenoma, Islet Cell↗
PubMed1984

The role of thymic immunity and insulitis in the development of streptozocin-induced diabetes in mice.

This article is concerned with the role of thymic immunity in the development of diabetes experimentally induced by multiple injections of subdiabetogenic doses of streptozocin (STZ). Euthymic +/+, +/nu, and athymic nu/nu mice of CD-1 and BALB/cAJcl origin were studied. Daily intraperitoneal (i.p.) injections of 30 mg/kg body wt of STZ for 5 consecutive days in CD-1 +/+ and +/nu mice resulted in hyperglycemia and mononuclear cell infiltrations of islets (insulitis). The CD-1 nu/nu mice developed neither insulitis nor hyperglycemia after the same treatment. In the nu/nu mice, when thymic immunity was restored by thymus grafting, both insulitis and hyperglycemia developed, thus demonstrating that thymic immunity was a prerequisite for the development of insulitis and hyperglycemia. There was a positive correlation among the degrees of thymic immunity, insulitis, and hyperglycemia in CD-1 +/nu, nu/nu with thymus grafts, and nu/nu mice, indicating that thymic immunity may amplify the diabetogenic effect of STZ by eliciting insulitis. In contrast, in BALB/cAJcl mice, a nonsusceptible strain to insulitis, no significant differences in plasma glucose levels were observed between the +/nu and nu/nu or between the nu/nu and thymus-grafted nu/nu mice. Furthermore, no significant difference was found in plasma testosterone levels between the +/nu and nu/nu mice of both CD-1 and BALB/cAJcl origin. In conclusion, our data indicate that thymic immunity enhances the diabetogenic effect of STZ by eliciting insulitis in susceptible mice.

Animals↗
PubMed1984

Cryopreservation of pancreatic islet cells.

Our study describes a useful procedure for cryopreservation of pancreatic islet cells. The pancreatic islets of adult hamsters were collected by collagenase digestion succeeded by gradient centrifugation and were dispersed by EDTA-Dispase treatment. The dispersed cells were suspended in medium consisting of 90% Dulbecco's modified Eagle's medium and 10% fetal bovine serum, supplemented with 10% dimethyl sulfoxide or glycerol. One milliliter each of the cell suspensions containing 10(6) cells was distributed into 2 ml polypropylene tubes, processed for freezing under six cooling conditions, and stored in liquid nitrogen (-196 degrees C). Of the various conditions tested, the cells suspended in dimethyl sulfoxide-containing medium and cooled in a program freezer at a rate of 0.5 degrees C/min down to -40 degrees C, succeeded by a rate of 5 degrees C/min down to -70 degrees C, resulted in the highest recovery rate of cells, 61.2% +/- 3.1%. This rate was comparable to those of several tissue culture cell lines frozen under similar conditions. Recovered cells preserved their morphologic characteristics under light and phase-contrast microscopy and formed cell sheets in culture. Response of insulin secretion to 3 mg/ml glucose appeared 6 hours after thawing, and the response to both 3 mg/ml glucose and 10 mmol/L theophylline was recovery to the same level as nonfrozen islet cells after 12 hours. The applicability of cryopreserved cells for the detection of islet cell surface antibody was demonstrated by the indirect method of immunofluorescence using islet cell surface antibody-positive human sera.

Animals↗
PubMed1983

Nonuniform distribution of aggregates in a suspension flowing around a solid body.

A flow of a highly concentrated latex suspension around a solid body in a thin layer between two parallel glass plates was observed by an optical microscope. A clear region, where suspended particles were rare, appeared near the rear stagnation point of the body. A model for explaining this phenomenon is proposed, in which the area of the clear region depends on an average size of aggregates of suspended particles. A possibility is suggested to use this phenomenon for measuring aggregate size in concentrated suspensions.

Erythrocyte Aggregation↗
PubMed1982

Production of anti-rat islet cell surface antibody in guinea pigs.

Anti-rat islet serum was prepared in guinea pigs by multiple subcutaneous inoculations of rat islets homogenates emulsified in complete Freund's adjuvant (CFA). The anti-rat islet serum was cytotoxic against rat spleen cells in the presence of complement and the nonspecific antibodies were observed with homogenates of rat livers and spleens. After absorption, the serum lost the cytotoxicity against the rat spleen cells yet showed specific cytotoxicity against the rat islet cells. The binding capacity of anti-rat islet antibody was determined by the indirect immunofluorescence test using FITC conjugated rabbit anti-guinea pig IgG serum. As the guinea pig anti-rat islet serum contained anti-insulin antibody, the role of this antibody in this cytotoxic activity and surface immunofluorescence was studied. However, the anti-insulin antibody used as the control showed neither cytotoxicity nor surface immunofluorescence. After neutralizing the anti-insulin antibody in the antiserum with insulin, the serum remained cytotoxic to the rat islet cells and a surface immunofluorescence appeared. These data show that specific anti-rat islet cell surface antibody can be produced in guinea pigs by multiple inoculations of rat islets homogenates with CFA.

Animals↗
PubMed1982

Differential effect of imidazole antibiotics on untransformed and virus-transformed rat cell lines.

The imidazole antimycotics clotrimazole and miconazole were tested on untransformed rat cell line 3Y1-B clone 1-6 (3Y1) and six transformed cell lines, which were independently isolated from 3Y1 or 3Y1-B clone 1 after infection with adenovirus 12 (AD-12) or with SV40, to determine their sensitivities to these drugs. The relative plating efficiency of three cell lines (T3, W4, and W5) transformed with AD-12 was reduced to 10(-1( of the initial value by clotrimazole (2 to 4 microgram/ml), whereas that of the parental cell line 3Y1 was reduced to 10(-1) of the initial value by clotrimazole (20 to 25 microgram/ml). By contrast, the differential effect of miconazole on 3Y1 and AD-12-transformed cell lines was found to be a factor of 2. The sensitivity of the SV40-transformed 3Y1 and the AD-12-transformed cell lines. The cellular sensitivity of untransformed 3Y1 cells to clotrimazole was significantly enhanced when exposed to various doses of the unsaturated fatty acid, linoleic acid. The untransformed and transformed cell lines showed sensitivities similar to the cytocidal activity of sterol-binding antimycotics, amphotericin B and filipin.

Amphotericin B↗
PubMed1981

Inhibition of DNA synthesis and cell cycle in Chinese hamster cells by sterol-binding polyene amphotericin B.

Amphotericin B, a sterol-binding polyene antibiotic, was found to inhibit DNA synthesis more than protein or RNA synthesis of asynchronous cultures of Chinese hamster V79 cells. DNA synthesis in the asynchronous V79 cells was inhibited to 40--60% of the control activity in the presence of 50 micrograms/ml amphotericin B. However, addition of 50 micrograms/ml of polyene immediately after to onset of DNA synthesis (early S phase) caused a drastic reduction of DNA synthesis (below 10--20% of the control in synchronized V79 cells, whereas the inhibition was much lessened when the polyene was added 1 h later (middle S phase). In contrast, there was no inhibition of DNA synthesis by amphotericin B in an amphotericin-B-resistant (AMBR) clone that was derived from V79. Flow microfluorometry analysis confirmed that a large number of asynchronous V79 cells were arrested in the G1 phase of the cell cycle when treated with lower dose of amphotericin B. A higher dose of the polyene antibiotic also accumulated cells at the G2 (or at both S and G2) phase as well as the G1 phase. Morphological studies by scanning electron microscope showed an increased number of V79 cells with decreased microvilli in V79 cells treated with amphotericin B.

Amphotericin B↗
PubMed1981

Isolation and primary culture of adult human hepatocytes. Ultrastructural and functional studies.

Biopsy tissue of adult human liver was gently dissociated with collagenase followed by Dispase. By repeated low g centrifugation, a large number of almost pure, viable hepatocytes was obtained. This is the first report of a successful procedure for obtaining adult human hepatocytes for study in tissue culture. The isolated cells have the typical morphology of liver parenchyma, and these characteristics persist throughout the period of culturing. Evidence of their function is indicated by albumin synthesis. This procedure is now being used to study human hepatocyte functions in vitro and the effects of a variety of agents including carcinogens and viruses.

Adult↗
PubMed1981

Mammary carcinoma induced by oral administration of ethyl methanesulphonate. Determination of some of the parameters affecting tumor induction.

The carcinogenic activity of ethyl methanesulphonate (EMS), an alkylating agent and a potent mutagen, was examined in Wistar King A and Sprague Dawley rats following oral administration. In the Wistar King A rats, mammary carcinomas were detected in all of the young female rats by the 32nd week after initiation of the experiment. To determine the optimal experimental conditions for the rapid and invariable induction of mammary carcinomas, the relationship among age, sex, strain of the rats and concentration and duration of EMS administration, and tumor production was investigated. The tumor incidence was higher in the younger female rats and the Wistar rats were more susceptible than Sprague-Dawley rats. Mammary carcinomas were also induced in the younger male rats. In addition, concommitant production of renal tumors occurred by the 40th week in young rats administered 10(-2) M or 2 x 10(-2) M of EMS solution, while renal and uterine tumors developed concommitantly in the older female rats given 3 x 10(-3) EMS by the 56th week. Histologically, the prevailing features of tumors were infiltrating ductal adenocarcinoma in the mammary glands, mesenchymal tumor in the kidney, and leiomyosarcoma in the uterus. Methyl methanesulphonate, an analogue of EMS, did not induce tumors in any organs.

Aging↗
PubMed1981

Adult human pancreatic endocrine cells in culture. Insulin and glucagon release and influence of cold storage.

Adult human pancreatic tissue was minced and digested with collagenase. Resulting cell clusters, including both exocrine and endocrine components, were suspended in the medium and inoculated into 35 mm Petri dishes. Viable cell clusters became spherical 4 or 5 hr after inoculation and attached to the bottom of the dish within 24 hr. They spread out to form a pavementlike epithelial cell sheet by the seventh day of culture. Insulin and glucagon release were determined on the culture day 3. Insulin release stimulated by 500 mg/dl glucose, 10 mM theophylline, and 200 micrograms/ml tolbutamide was increased 1.5- to 3.7-fold in comparison with that stimulated by 100 mg/dl glucose. A 20 mM arginine concentration did not affect insulin release. Glucagon release, stimulated by 10 mM theophylline and 20 mM arginine, showed 1.7- to 2.7-fold increase and was suppressed significantly by 500 mg/dl glucose. Tolbutamide did not affect glucagon release significantly. Ultrastructural studies demonstrated that islet cells were preserved excellently. Chopped pancreatic tissue could be stored at 4 degrees C for at least 12 hr, with retention of normal following subsequent in vitro culture of islet cells.

Adult↗
PubMed1980

Cultured skin fibroblasts in lipidoses. Enzymatic, histochemical, and ultrastructural relationship in Fabry's Tay-Sachs, and Sandhoff's diseases.

Enzymatic, histochemical, and ultrastructural studies were performed on cultured skin fibroblasts from patients with Fabry's disease, Tay-Sach's disease, and Sandhoff's disease and from their families (carriers). alpha-Galactosidase activity was deficient in the proband with Fabry's disease (lower in the homozygotes than in the heterozygote). Levels of hexosaminidase A in the patient with Tay-Sachs disease and hexosaminidase A and B in the patient with Sandhoff's disease were deficient and were lower in her mother than in the control subject. Lysosome-like structures were observed in cultured fibroblasts from the patients with each disease, as well as in the heterozygote with Fabry's disease and the carrier with Tay-Sach's disease. The amount of the accumulating arrays in the lysosome-like structures was related to low level of enzymatic activity.

Cells, Cultured↗
PubMed1979

Long-term culture of pancreatic islet cells with special reference to the beta-cell function.

Islet cells of adult rat pancreas, dissociated with EDTA-Dispase, were cultivated in Microtest wells for over 2 months. In our cultures, islet cells were free-floating and cohered with each other to reorganize histotypic aggregates resembling the nondissociated islets. Morphologically, excellent preservation of islet cells in the aggregates was confirmed during the culture using both light and electron microscopy. The function of islet beta-cells, as demonstrated by the synthesis and release of insulin, also was retained throughout the culture period. Islet beta-cells cultured for long periods exhibited better response to the short-term stimulation of theophylline than to a high concentration of glucose, as observed in the islets of fetal or newborn rat pancreas.

Animals↗