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Biomedical subjects

C Sato

Publications and source records attributed to C Sato.

At least 289 records · Page 16Linked to original sources
PubMed1988

Intermediate filament reconstitution in vitro. The role of phosphorylation on the assembly-disassembly of desmin.

Desmin, the myogenic intermediate filament protein, is a phosphoprotein containing phosphoserine, in vivo. The role of phosphorylation on assembly-disassembly and organization of the desmin filament has remained obscure. We report here on a stable and purified system which enables a biochemical examination of desmin filament assembly and disassembly. Using this in vitro system, we carried out stoichiometrical phosphorylations by purified protein kinases. The extent of polymerization-depolymerization was estimated using procedures related to centrifugation and electron microscopy. The evidence we obtained suggests that disassembly of the desmin filament and inhibition of the NaCl-dependent polymerization of the soluble desmin can reversibly occur with either cAMP-dependent or Ca2+-activated, phospholipid-dependent desmin phosphorylation.

Animals↗
PubMed1988

Loss of constitutional heterozygosity in colon carcinoma from patients with familial polyposis coli.

Recent studies have suggested a critical role of specific gene loss in several embryonic tumours and certain adult cancers. In retinoblastoma, hemizygosity or homozygosity of a recessive mutant allele results in the loss of normal gene product, and this seems to cause the manifestation of the disorder. Familial polyposis coli (FPC) is a human autosomal dominant trait characterized by numerous adenomatous polyps of the colon and rectum, and a high incidence of colon carcinoma. Karyotype analyses have failed to detect specific deletion or translocation. We report the use of polymorphic DNA markers to look for the somatic loss of heterozygosity at specific loci. Investigation of 38 tumours from 25 FPC patients, and 20 sporadic colon carcinomas from 19 patients, revealed frequent occurrence of allele loss on chromosome 22, with some additional losses on chromosomes 5, 6, 12q and 15. The FPC gene-linked DNA probe C11p11 also detected frequent allele loss in both familial and sporadic colon carcinomas but not in benign adenomas. These results suggest the possible involvement of more than one chromosomal locus in the development of familial and sporadic colon carcinomas.

Adenoma↗
PubMed1988

Rapid phosphorylation of MAP-2-related cytoplasmic and nuclear Mr 300,000 protein by serine kinases after growth stimulation in quiescent cells.

Antibody against brain microtubule-associated protein 2 (MAP-2) immunoprecipitated Mr 300,000 and 80,000 proteins of cultured fibroblasts and kidney cells. These proteins were not appreciably phosphorylated in quiescent cells, but were rapidly phosphorylated after growth stimulation by insulin, epidermal and fibroblast growth factors, transferrin, phorbol ester and diacylglycerol in the presence of Ca2+, in a manner similar to that of MAP-1-related Mr 350,000 protein (J. Cell Biol. 100, 748-753). A Ca2+ ionophore, which is known to make the quiescent cell competent but not to enter into the growth cycle, did not induce the phosphorylation. In a chase experiment, decay half lives of labeled phosphoproteins were 5 h for Mr 350,000 and 300,000 proteins, and 1.5 h for Mr 80,000 protein. On subcellular fractionation, phosphorylated Mr 350,000 and 300,000 proteins were detected first mainly in the cytoplasm and then in the nucleus, while Mr 80,000 phosphoprotein was consistently detected in the cytoplasm. The phosphorylation of these proteins occurred on serine residues after stimulation with various factors. Thus, the phosphorylation of cytoskeleton-associated Mr 350,000 and 300,000 proteins by serine kinases seems to be a common second process after growth stimulation and to link cytoplasmic and intranuclear events.

Animals↗
PubMed1988

Reversible cAMP-induced translocation of cytoskeleton-associated 300- to 350-kDa proteins from nucleus to cytoplasm.

We previously reported that treatment of SV-3Y1 cells in an exponential growth state with 1 mM db-cAMP plus 1 mM theophylline induced reversible disappearance of nuclear dots stained by monoclonal anti-microtubule-associated protein (MAP)-1 antibody [T. Nakayama, K. Nishizawa, G. Kimura, and C. Sato (1986) Exp. Cell Res. 163, 246]. In the present study, we examined the relation between the intracellular localization and phosphorylation of 300- to 350-kDa proteins that are intracellular antigens for our anti-MAP-1 and -2 antibodies. Treatment with 1 mM db-cAMP plus 1 mM theophylline was found to result in a reversible decrease in immunofluorescent staining of the nucleus with polyclonal MAP-1 or -2 antibody, and a reversible increase in that of the cytoplasm. Simultaneous treatment with 2.5 microM colchicine, 2.5 microM colcemid, 20 microM putrescine, or 3 mM alpha-naphthyl phosphate in the presence of db-cAMP plus theophylline almost prevented this effect of db-cAMP plus theophylline. We examined the cytoplasmic and nuclear fractions by immunoperoxidase staining, immunoprecipitation, and 125I-protein A with anti-MAP-1 and -2 antibodies. Treatment with db-cAMP plus theophylline resulted in the increase of 300- to 350-kDa proteins in the cytoplasm and a decrease in the nucleus. This treatment also caused the dephosphorylation of 300- to 350-kDa proteins. The present research indicated that treatment with db-cAMP plus theophylline resulted in the reversible translocation of 300- to 350-kDa proteins from the nucleus to the cytoplasm accompanied by the dephosphorylation of these proteins.

Animals↗
PubMed1988

Chemical and viral transformation of cultured skin fibroblasts from patients with familial polyposis coli.

Treatment of skin fibroblasts from an FPC patient with 4NQO or MNNG followed by sequential passaging caused morphological changes of the cells, which showed characteristics of transformed cells such as a high frequency of colony formation in agarose, increased growth ability, and chromosomal abnormalities. This and other fibroblast lines from 5 of 12 FPC patients had an increased susceptibility to 4NQO cytotoxicity, which was caused by enhanced 4NQO-reductase activity rather than by reduced DNA repair. However, the susceptibility to cytotoxicity of MNNG and repair of MNNG-damaged DNA were normal in FPC cells. The tumor promoters TPA and DHTB enhanced the frequency of chemical transformation of the FPC fibroblasts, and protease inhibitors suppressed the promoter-enhanced transformation. The skin fibroblasts from many FPC patients exhibited increased susceptibility to transformation by murine sarcoma viruses. Analysis of the viral DNA and RNA after infection revealed that the increased susceptibility is determined at an early stage of transformation. Two out of 5 MNNG-transformed clones of FPC fibroblasts, isolated from agarose, had increased expression of c-Ki-ras or c-Ha-ras, and 4 of 4 MSV-transformed clones showed high expression of viral Ki-ras. These clones grew further after isolation from agarose, but were mortal and did not form tumors in nude mice. The present results suggest that additional changes in morphologically transformed FPC fibroblasts are required for malignant transformation.

4-Nitroquinoline-1-oxide↗
PubMed1988

Differential expression of carcinoembryonic antigen and nonspecific crossreacting antigen genes in human colon adenocarcinomas and normal colon mucosa.

The level of mRNA for carcinoembryonic antigen (CEA) and nonspecific crossreacting antigen (NCA) in human colon adenocarcinomas and normal colon mucosa was analyzed by Northern blot hybridization using as probes 32P-labeled CEA cDNA and synthetic oligodeoxyribonucleotides specific to CEA and NCA mRNA sequences. The major 3.5-kb mRNA and a minor 4.2-kb mRNA are shown to be CEA-specific and expressed in both tissues, albeit at slightly different degrees, suggesting that the expression of CEA is regulated posttranscriptionally. Another minor mRNA of 2.9 kb is NCA-specific and expressed predominantly in cancerous tissues, suggesting its usefulness as a marker for colon cancer.

Adenocarcinoma↗
PubMed1988

Subcellular distribution of UDP-galactose:ceramide galactosyltransferase in rat brain oligodendroglia.

Oligodendrocytes isolated from 18-19-day-old rat brain were homogenized in 0.32 M sucrose. The homogenate was centrifuged at 100,000 g for 50 min in a gradient containing 0.8, 1.05, and 1.3 M sucrose. Three discrete bands were obtained at the interfaces 0.32-0.8 (F1), 0.8-1.05 (F2), and 1.05-1.3 M (F3). The distribution of UDP-galactose:ceramide galactosyltransferase (CgalT) activity in each fraction was measured using liposomes containing normal fatty acid-containing ceramides (NFA-CgalT activity) or 2-hydroxy fatty acid-containing ceramides (HFA-CgalT activity). Although detection of both CgalT activities was possible in all fractions, HFA-CgalT activity was enriched in F1 and F2 fractions, which also showed an enrichment of Golgi and endoplasmic reticulum markers, respectively. It is interesting that NFA-CgalT activity was significantly enriched in the F2 fraction. These results suggest that hydroxylated and nonhydroxylated galactocerebrosides may be synthesized at different intracellular locations.

Animals↗
PubMed1988

Stimulation of the phosphorylation of cytoskeletal 350-kDa and 300-kDa proteins by insulin-like growth factor-I, platelet-derived growth factor and phorbol ester in rat 3Y1 cells.

Insulin-like growth factor-I (IGF-I) stimulated the phosphorylation of cytoskeletal 350-kDa and 300-kDa proteins which were immunoprecipitated with antibodies against brain high molecular weight microtubule-associated proteins in quiescent rat 3Y1 cells. The data on the effective concentrations of IGF-I and 125I-labeled IGF-I binding indicated that type I IGF receptors mediate this IGF-I effect. Platelet-derived growth factor (PDGF) as well as phorbol ester (TPA) also stimulated the phosphorylation of these proteins. These proteins, whether immunoprecipitated from cells stimulated by insulin, IGF-I, TPA, PDGF, or epidermal growth factor, produced very similar phosphopeptide mapping patterns irrespective of the stimulant. The results suggest the possibility that these growth factors and phorbol esters may activate a common protein kinase which is responsible for the phosphorylation of the 350-kDa and 300-kDa proteins in cells.

Animals↗
PubMed1988

Clinical and etiological studies of chlamydial conjunctivitis in Sapporo, Japan.

Chlamydia trachomatis was isolated from 21.5% (61/284 cases) of the patients with conjunctivitis seen in an eye clinic in Sapporo, Japan, during a recent 4-year period. The frequency was the highest in newborn babies and the 20- to 29-year-old age group. The early incidence of neonatal conjunctivitis suggested that transmission had occurred at delivery. Isolation by tissue culture is the most sensitive method for detection of this condition but the Micro Trak direct fluorescence antibody test is more practical. The detection of a specific IgG antibody and IgA antibody against chlamydia in human sera and in tears showed that patients with chlamydial conjunctivitis had the antibody before the onset of conjunctivitis.

Adolescent↗
PubMed1987

Three subtypes of chronic schizophrenia identified using 11C-glucose positron emission tomography.

The authors used positron computed tomography (CT) and 11C-labeled glucose to measure brain glucose utilization in 20 chronic schizophrenic patients (18 men, 2 women, mean age 38) and 5 male control subjects (mean age 38). Positron emission tomography (PET) revealed at least three subtypes: hypofrontal (type A), hypoparietal (right-sided disturbance in right-handed and left-sided disturbance in left-handed patients) (type B), and normal (type C). The significant count reduction in the frontal lobe (Brodmann's area 10) in type A patients was 38%, while that in the parietal lobe (Brodmann's area 40) in type B patients was 26% in each lobe of the brain.

Adolescent↗
PubMed1987

Myelin galactolipid synthesis in different strains of mice.

Previous studies have indicated that the brains of DBA/2J (D2) mice have a more heavily myelinated CNS than those of C57BL/6J (B6) at postnatal days 17-21. However, the amount of myelin in the brains of F1 (B6 X D2) hybrids is even higher than in their parental strains. To investigate further factors involved in regulating myelinogenesis in these mice, we have focused on the synthesis of cerebrosides and sulfatides, galactolipids enriched in myelin. Brain slices from 14-, 17-, and 21-day-old D2, B6, and F1 mice were incubated with [3H]galactose and [35S]sulfate. After incubation, microsomes, myelin, and oligodendroglial cells were isolated, and the galactolipids were analyzed. At 21 days of age, the labeling of cerebrosides in F1 mice was higher than in D2 and B6 mice when the results were expressed as microsomal or myelin radioactivity per gram wet weight. At 14 and 17 days of age, the labeling of cerebrosides in F1 animals was similar to that in D2 mice and was considerably higher than that in B6 mice. The labeling of sulfatides in F1 animals was significantly higher than in the B6 parent at all ages studied, whereas it remained higher than that in the D2 parent only at 17 days of age. A similar relationship among the strains was observed when the synthesis of myelin galactolipids was estimated by measuring the in vitro activity of UDP-galactose:ceramide galactosyltransferase and 3'-phosphoadenylyl sulfate:galactosylceramide 3'-sulfotransferase. The results indicate that the increased accumulation of myelin galactolipids previously reported in the F1 mice is partially due to enhanced synthetic activity.

Aging↗
PubMed1987

Enhancement of pheomelanogenesis by L-dopa in the mouse melanocyte cell line, TM10, in vitro.

Cells of TM10, an established cell line, are melanocytes that contain equal amounts of eumelanin (black pigment) and pheomelanin (yellow pigment). The content of pheomelanin drastically increased when the cells were cultured in growth medium containing 0.2mM-L-dopa (L-dihydroxyphenylalanine), which is the common precursor for both eumelanogenesis and pheomelanogenesis. After this treatment, the amount of pheomelanin was 3.7-fold greater than that of control in TM10, whereas the amount of eumelanin changed very little. In contrast, 5-S-cysteinyl-dopa, which is the specific precursor for pheomelanogenesis downstream of L-dopa, did not cause preferential increase in pheomelanogenesis. Ultrastructural observations also confirmed these results; in 0.2mM-L-dopa, an increase in the number of pheomelanosomes was observed in the cytoplasm of TM10 cells. Our results also suggest that the L-dopa treatment results in a decrease in tyrosinase activity per melanosome.

Animals↗
PubMed1986

Characterization of microtubule-associated proteins isolated from bovine adrenal gland.

We investigated the biochemical characteristics of microtubule-associated proteins (MAPs) of both the adrenal medulla and the cortex. The major constituents of the adrenal MAPs isolated by the taxol-dependent procedure [Vallee, R. B. (1982) J. Cell. Biol. 92, 435-442] were several polypeptides in the high-molecular-mass region (high-Mr MAPs) and a 190000-Mr polypeptide (190-kDa MAP). In the cortex MAP fraction, the most prominent component was 190-kDa MAP, while the medulla MAP fraction was rich in high-Mr MAPs. Twice-cycled microtubule proteins prepared without taxol from the same sources also contained high-Mr MAPs and 190-kDa MAP. High-Mr MAPs contained protein species identical to MAP1 and MAP2 of mammalian brain as judged from electrophoretic mobility, heat-stability and immunoreactivity. 190-kDa MAP was classified as MAP subspecies distinct from high-Mr MAPs by several criteria. The MAP fractions had the ability to polymerize purified tubulin into microtubules, and the major MAP species (high-Mr MAPs and 190-kDa MAP) were found to cosediment with reconstituted microtubules. Tau factor, one of the major MAPs in the mammalian brain, appeared to be a minor species in the adrenal gland.

Adrenal Cortex↗
PubMed1986

Distinction of G0 cells from senescent cells in cultures of non-cycling human fetal lung fibroblasts by anti-MAP-1 monoclonal antibody staining.

On staining with a monoclonal antibody raised against microtubule-associated protein-1 (MAP-1), dot-like structures were seen in the nuclei of interphase cells, but not in those of non-cycling G0-arrested cells. Dots were also not seen in the nuclei of non-cycling senescent human cells (IMR-90). A SV40-DNA-transformed subline of IMR-90 with a limited growth potential showed progressive decrease of cells with nuclei containing dots in the final stage of their lifespan. The dots appeared in G0-arrested IMR-90 cells when these cells were incubated in medium of high osmotic pressure for 3 min. In contrast, no dots appeared in senescent cells or X-ray-irradiated young cells when they were incubated in medium of high osmotic pressure. Thus irreversibly non-cycling cells could be distinguished from G0-phase cells on the level of whole cultures. The results suggest that senescent cells lose their division potential by entering an irreversible cell-cycle stage differing from G0.

Antibodies, Monoclonal↗
PubMed1986

Phosphorylated 350 kD protein in the nucleus as it is associated with cell transformation.

Protein kinases are thought to play a key role in signal transduction and oncogenesis, but little is known about the intranuclear phosphorylation events associated with transformation. Here we report on cell cycle-dependent phosphorylation of cytoskeleton-associated 350 kD protein and the regular interchange in its location between the nucleus and cytoplasm of normal cells. Persistent intranuclear location of the phosphorylated 350 kD protein was also found throughout the cell cycle in transformed cells, as detected by immunoprecipitation of 32P-phosphorylated 350 kD protein from isolated nuclei and immunofluorescent staining with a monoclonal antibody that recognized phosphorylated site of 350 kD protein. A conditional transformed phenotype induced by a temperature-sensitive (ts) viral oncogene or a transforming growth factor was also associated with the intranuclear presence of the phosphorylated 350 kD protein. Thus the 350 kD protein seems to be a target molecule of protein kinases that are stimulated directly or indirectly by growth factors or by oncogene products in the nucleus, and appears to be a new transformation-related nuclear antigen.

Animals↗
PubMed1986

Reversible cAMP-dependent change in nuclear localization of microtubule-associated protein-1 analogues.

Intranuclear immunofluorescent staining by monoclonal and polyclonal antibodies against microtubule-associated protein-1 (MAP-1) on SV-3Y1 cells disappeared when the cells were treated with 1 mM db-cAMP and 1 mM theophylline for 20-30 min at 37 degrees C. The nuclear dots of immunofluorescence disappeared and reappeared repeatedly on successive incubation of the cells with and without these drugs. The same phenomenon was induced by treatment of the cells with 6 mM theophylline or 6 mM papaverine which inhibits the cAMP-hydrolysing enzyme. The following results seem to support the hypothesis that cAMP-induced transfer of antigenic molecules from the nucleus to the cytoplasm is mediated by microtubules: Partial staining of the nucleus during the transitional period. Bright staining of the cytoplasm on treated cells in contrast to nuclear staining on control cells. Disappearance of the nuclear staining not only by the monoclonal antibody but also by the polyclonal antibody. Complete prevention of disappearance of nuclear dots induced by these drugs by pretreatment of the cells with colchicine (1 microgram/ml) or colcemid (1 microgram/ml).

Animals↗
PubMed1986

Structural organizations of replicon domains during DNA synthetic phase in the mammalian nucleus.

In mammalian cells, it has been shown that adjacent multiple DNA replicons, termed a replicon cluster or a replicon domain, are replicated coordinately in a defined temporal order during the DNA synthetic (S) phase. However, no intranuclear structure of this replicon domain has been revealed in the nucleus labelled with [3H]thymidine at the limited resolution level of autoradiography. By immunofluorescent staining with antibody against 5-bromodeoxyuridine (BrdU), we succeeded in detecting novel, intranuclear ring-like structures of replicating replicon domains that were organized temporarily during the S phase of mammalian cells with incorporated BrdU.

Animals↗