Biomedical subjects

K Sugasawa

Publications and source records attributed to K Sugasawa.

53 records · Page 3Linked to original sources

PubMed1994

Acidification of phagosomes and degradation of rod outer segments in rat retinal pigment epithelium.

PURPOSE: The authors investigated the phagocytic processes of the rod outer segments (ROS) in rat retinal pigment epithelium (RPE) cells, and the appearance of lysosomal enzymes, acidification, and degradation of the contents in the phagolysosomes. In particular, they examined the effect of bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, on the degradation of ROS in the RPE cells in vivo. METHODS: A lysosomal enzyme (cathepsin D), a lysosomal membrane protein (LGP107), and opsin were localized in the RPE cells by the immunogold electron microscopic technique. Bafilomycin A1 was injected into the vitreous, and acidification of the phagosomes was measured in vivo by injecting 3-[2,4-dinitroanilino]3'amino-N-methyldipropylamine (DAMP) in the vitreous and detecting the accumulation of DAMP in the phagolysosomes using anti-dinitrophenol antibody. RESULTS: Opsin was abundantly detected in phagosomes that did not contain cathepsin D, but the immunolabeling of opsin rapidly disappeared soon after the appearance of cathepsin D. By double staining with cathepsin D and DAMP, it was shown that the pH of the phagosomes dramatically decreased after fusion with lysosomes. When bafilomycin A1 was injected into the vitreous, many large phagolysosomes containing cathepsin D appeared in the RPE cells, in which the immunoreactivity of opsin was well preserved. CONCLUSIONS: Degradation of opsin and acidification proceeded almost parallel with the appearance of cathepsin D in the phagolysosomes. Bafilomycin A1 did not inhibit the fusion of phagosomes with lysosomes, but it increased intraphagosomal pH and markedly inhibited the degradation of ROS in the phagolysosomes. This result indicates that vacuolar-type H(+)-ATPase is essential for acidifying the lumen of phagolysosomes and subsequent protein degradation of ROS in the RPE cells.

Animals↗

PubMed1994

[Proteoglycan in Bruch's membrane of senescence accelerated mouse: localization and age-related changes].

We demonstrated the distribution of sulfated proteoglycans in Bruch's membrane of Senescence Accelerated Mouse histochemically and ultrastructurally using cuprolinic blue in conjunction with specific enzyme treatments and nitrous acid digestion. Two kinds of proteoglycan filaments were observed in the inner and outer collagenous layers, i.e., small collagen fibril-associated filaments (11 nm in average length), and large filaments (32 nm in average length). Intermediate size filaments (25 nm in average length) were seen in the basement membranes of the retinal pigment epithelium and choriocapillaris. Chondroitinase AC treatment eliminated the staining of filaments in the collagenous layers (chondroitin sulfate). Chondroitinase ABC treatment also eliminated the staining of filaments in the collagenous layers (chondroitin sulfate and dermatan sulfate). Nitrous acid eliminated the staining of filaments in both basement membranes (heparan sulfate). Proteoglycans containing chondroitin sulfate and dermatan sulfate were associated uniquely with collagen fibrils. Heparan sulfate proteoglycans were associated with the basement membranes of the pigment epithelium and choriocapillaris. With aging, the thickness of the basement membrane of the choriocapillaris and the staining of the filaments in the basement membranes of the pigment epithelium and choriocapillaris (heparan sulfate proteoglycans) increased. Collagen fibers became disarranged and the staining of both filaments in the collagenous layers decreased. The results of the staining characteristics probably reflect the aging of Bruch's membrane.

Aging↗

PubMed1993

Cell-free repair of UV-damaged simian virus 40 chromosomes in human cell extracts. I. Development of a cell-free system detecting excision repair of UV-irradiated SV40 chromosomes.

Simian virus 40 minichromosomes were irradiated with ultraviolet light and used to study cell-free DNA repair reactions by soluble extracts of repair-proficient human cells. Damage-specific incorporation of labeled nucleotides was measured after linearization and electrophoresis of the viral DNA. Repair synthesis with irradiated chromosomes was dependent on the dose of ultraviolet light, but naked viral DNA irradiated at the same doses showed significantly higher template activity. The reactions absolutely required the presence of cell extract, but an extract from Chinese hamster cells was as active as those from human cells. The repair activity was apparently stimulated by the addition of unirradiated plasmid DNA, which did not affect the nucleosome density of the viral chromosomes. ATP and its regenerating system were required for the reaction, but total repair synthesis was little affected by exogenous addition of substrates for RNA synthesis and poly(ADP-ribosyl)ation. Most of the repaired viral chromosomal DNA was in the supercoiled, closed circular form, suggesting that excision repair reactions proceeded to the stage of ligation in the present cell-free system.

Animals↗

PubMed1993

Cell-free repair of UV-damaged simian virus 40 chromosomes in human cell extracts. II. Defective DNA repair synthesis by xeroderma pigmentosum cell extracts.

We have constructed a cell-free DNA repair system with UV-irradiated SV40 minichromosomes, as described in the accompanying paper (Sugasawa, K, Masutani, C., and Hanaoka, F. (1993) J. Biol. Chem 268, 9098-9104). In this study, we examined DNA repair synthesis by cell extracts from seven xeroderma pigmentosum (XP) complementation groups, A through G. DNA repair synthesis by XP cell extracts was lower than that with repair-proficient human 293 cell extract and did not increase to the level with the latter on increase in the amount of cell extract or the incubation time. The defects of XP cell extracts were complemented by addition of extracts from cells of different complementation groups, indicating that defective proteins in XP-A through G cells are directly involved in DNA repair. Addition of T4 endonuclease V, which is reported to complement defects of XP cells, stimulated DNA repair synthesis by the 293 cell extract, and also complemented the defects of all XP cell extracts. The XPAC gene product was shown to be involved in DNA repair synthesis using anti-xpac serum and xpac protein produced in Escherichia coli. Anti-xpac serum inhibited DNA repair synthesis by the 293 cell extract and xpac protein reversed the inhibition. Furthermore, xpac protein complemented the defects of extracts of two lines of XP-A cells (XP2OSSV and XP12ROSV) but had no effect on the reactions of extracts from cells of other complementation groups. These findings are consistent with previous results obtained in experiments with cells, indicating that our system is useful for analyzing the mechanisms of DNA excision repair in mammalian cells.

Cell Line↗

PubMed1993

[Fibronectin in the chorioretinal wound after laser photocoagulation].

Fibronectin (Fn) is a glycoprotein which mediates adhesion, extension, migration, and chemotaxis of the cells. We did a histochemical study of the time course of appearance and distribution of Fn in the chorioretinal wound after laser photocoagulation. In the normal chorioretinal tissue, Fn was detected in the internal limiting membrane, Bruch's membrane, and the endothelium of chorioretinal vessels. Shortly after laser photocoagulation, Fn appeared on Bruch's membrane and in the endothelium of choroidal vessels and choroidal stroma, and was prominent by one week after laser photocoagulation. Although Fn disappeared when wound healing was accomplished, it was still detected on Bruch's membrane one month after laser photocoagulation. These findings suggest that Fn is related to chorioretinal wound healing after laser photocoagulation.

Animals↗

PubMed1993

Abrogation of p53-mediated transactivation by SV40 large T antigen.

p53 is known to bind specifically to the 44-bp human DNA sequence in an immunoprecipitation assay. We show here that the transcription of the reporter CAT gene linked with the herpesvirus thymidine kinase (tk) promoter containing the 44-base sequence is enhanced by mouse wild-type but not mutant-type p53 in F9 and p53-null Saos-2 cells. The p53-mediated transactivation was dramatically abrogated by introduction of SV40 large T antigen (SVLT) in Saos-2 cells in which p53 was clearly associated with SVLT. Furthermore, the p53-SVLT complex did not bind to the 44-base sequence at all. Thus, SVLT sequesters the transactivation function of the wild-type p53 by inhibiting the binding of p53 to the 44-base sequence. This is good evidence to show 'loss of functions' in the product of a tumor-suppressor oncogene by a dominant oncogene product at a molecular level.

Antigens, Polyomavirus Transforming↗

PubMed1992

Nonconservative segregation of parental nucleosomes during simian virus 40 chromosome replication in vitro.

Simian virus 40 chromosomes can be replicated in vitro with the same set of purified proteins required for the replication of naked DNA containing the viral origin. With these reconstituted systems, the fate of parental histones during replication was examined in vitro. The assembly of nucleosomes on replicating chromosomes was hardly affected by the presence of simultaneously replicating naked DNA competitor, suggesting that replication forks can traverse nucleosomes without the displacement of histones. Moreover, we demonstrate that the nascent nucleosomes were distributed almost equally between the leading and lagging strands. This distributive mode of nucleosome segregation favors the propagation of parental chromatin structures to both daughter cells, which can maintain cellular functions dictated by these structures during cell proliferation.

DNA Polymerase II↗

PubMed1992

Topoisomerase II plays an essential role as a swivelase in the late stage of SV40 chromosome replication in vitro.

The effects of topoisomerases I and II on the replication of SV40 DNA were examined using an in vitro replication system of purified proteins that constitutes the monopolymerase system. In the presence of the two topoisomerases, two distinct nascent DNAs were formed. One product arising from the replication of the leading template strand was approximately half the size of the template DNA, whereas the other product derived from the lagging template strand consisted of short DNAs. These products were synthesized from both SV40 naked DNA and SV40 chromosomes. For the replication of SV40 naked DNA, either topoisomerase I or II maintained replication fork movement and supported complete leading strand synthesis. When SV40 chromosomes were replicated with the same proteins, reactions containing only topoisomerase I produced shorter leading strands. However, mature size DNA products accumulated in reactions supplemented with topoisomerase II, as well as in reactions containing only topoisomerase II. In the presence of crude extracts of HeLa cells, VP-16, a specific inhibitor of topoisomerase II, blocked elongation of the nascent DNA during the replication of SV40 chromosomes. These results indicate that topoisomerase II plays a crucial role as a swivelase in the late stage of SV40 chromosome replication in vitro.

DNA Replication↗

PubMed1992

Complementation by a cloned human ubiquitin-activating enzyme E1 of the S-phase-arrested mouse FM3A cell mutant with thermolabile E1.

A temperature-sensitive growth mutant tsFS20 isolated from mouse FM3A cells was identified as a mutant with thermolabile ubiquitin-activating enzyme E1 by transfection with a full-length cDNA encoding the human E1 enzyme and cell-cell hybridization with an authentic E1 mutant ts85 previously isolated from FM3A cells. The resulting transformants produced thermoresistant E1 activity. Upon shift-up of temperature, asynchronously growing tsFS20 cells showed multiple points of cell-cycle arrest. At the nonpermissive temperature, tsFS20 cells that had been synchronized at the G1-S-phase progressed and accumulated in the mid-S-phase, as evidenced by the absence of G2-specific cdc2 kinase activity, while ts85 mutant cells, the widely used E1 mutant, reached the G2-phase and were arrested. Thus, the E1 mutation seemed to be involved in progression in the S-phase as well as in the G2-phase in the cell cycle. Degradation of short-lived abnormal proteins in tsFS20 cells was decreased to about 50% at the nonpermissive temperature, while the block was fully restored to the wild-type level in the transformant cells. Relevance of the unusually high incidence of the temperature-sensitive E1 mutation was discussed in terms of the E1 as a determinant of heat tolerance of cells.

Animals↗

PubMed1991

Replication of the simian virus 40 chromosome with purified proteins.

SV40 chromosomes prepared from infected CV-1 cells were replicated with the purified proteins of SV40 T antigen, HeLa DNA polymerase alpha-primase complex, single-stranded DNA-binding protein, and topoisomerases I and II, all of which have been shown to be essential for SV40 DNA replication in vitro. Replication started near the origin and proceeded bidirectionally. The maximum speed of replication fork movement was 200-300 nucleotides/min, which was similar to the rate of SV40 DNA replication with the same set of proteins. When replication products were digested with micrococcal nuclease, DNA fragments of 160-180 base pairs, which is the typical size of mononucleosomal DNA, were protected. This result indicates that replicated DNA was reconstructed into the nucleosome structure, complexed with parental histones.

Antigens, Polyomavirus Transforming↗

PubMed1991

Human ubiquitin-activating enzyme (E1): compensation for heat-labile mouse E1 and its gene localization on the X chromosome.

We have constructed interspecific somatic cell hybrids between a temperature-sensitive (ts) mutant cell line of mouse FM3A cells, ts85, that has a heat-labile ubiquitin-activating enzyme (E1) and a human diploid fibroblast cell line, IMR-90. A hybrid clone that could grow stably at a nonpermissive temperature (39 degrees C) was obtained. Segregation of the hybrid cells at a permissive temperature (33 degrees C) gave rise to temperature-sensitive clones. The electrophoresis of extracted histones and karyotype analysis of the segregants revealed a close correlation of the ability to grow at 39 degrees C, the presence of uH2A (ubiquitin-H2A semihistone) at 39 degrees C, and the presence of the human X chromosome. One of the hybrid clones that could grow at the nonpermissive temperature contained the X chromosome as the only human chromosome. The sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern of affinity-purified E1 showed that this hybrid clone contained both human and mouse type E1. Thus we conclude that the functional gene for human E1 is located on the X chromosome.

Animals↗

PubMed1990

Assembly of nascent DNA into nucleosome structures in simian virus 40 chromosomes by HeLa cell extract.

A soluble system was developed that could support DNA replication in simian virus 40 (SV40) chromosomes. DNA synthesis in this system required the presence of purified SV40 large tumor antigen, SV40 chromosomes prepared from virus-infected monkey cells, a crude extract from HeLa cells, and several low-molecular-weight components. In comparison to the replication of purified SV40 form I DNA, the rate of DNA synthesis was 15 to 20% in this system. DNA synthesis started near the replication origin of SV40 and proceeded bidirectionally in a semiconservative manner. Micrococcal nuclease digestion experiments revealed that the replicated DNA produced in this system became organized into a regularly spaced array of nucleosome core particles when an appropriate amount of purified HeLa core histones was added to the reaction mixture. SV40 form I DNA replicating under the same conditions was also assembled into nucleosomes, which were arranged in a rather dispersed manner and formed an aberrant chromatin structure.

Cell Line↗

PubMed1989

Heterogeneous assembly of nascent core histones to form nucleosomal histone octamers in mouse FM3A cells.

We investigated the assembly of newly synthesized histones into nucleosomal histone octamers using an isopycnic centrifugation analysis of these proteins of mouse FM3A cells that had been labeled by culture with heavy amino acids. Cross-linked histone octamers or non-cross-linked core histones were separated electrophoretically from other proteins on sodium dodecyl sulfate/polyacrylamide gels before being banded to equilibrium in cesium formate/guanidinium chloride density gradients. The density of core histones rapidly came to equilibrium after culture of cells in the medium containing heavy amino acids for a time as short as 30 min, indicating that the density of histone octamers in the density-labeled cells reflects the content of these new (dense) core histones relative to old (light) ones in the octamer assembly. Cross-linked histone octamers from these cells were of heterogeneous densities as judged from a broad band in a density gradient. The position and width of the band was essentially unaffected, though the peak position of the density distribution within the band moved progressively from a less to a more dense area, when the time of culture was prolonged from 2 h to 16 h. It is concluded, therefore, that new and old histones are assembled into histone octamers in heterogeneous ratios in contradiction to a conservative or semi-conservative assembly model and it is also suggested that incorporation into chromatin of new histones is not necessarily restricted to newly replicated chromatin regions.

Animals↗

PubMed1986

Characterization of the 100,000 dalton material produced by chromatin cross-linking reaction with dithio-bis(succinimidyl propionate).

A chemical cross-linking reagent, dithio-bis(succinimidyl propionate), is known to be capable of cross-linking histones in nucleosomes so as to give one major product with the molecular weight of about 100,000. Because the product has been supposed to represent the cross-linked histone octamer, the reaction has been used for studying the movements of core histones in nucleosomes. However, the precise protein composition of the product has not been determined thus far, so that the use of the reaction was limited. We report here that the 100 kilodalton product is composed of the core histones, and does not contain significant amounts of any other proteins. Moreover, quantitative analysis of the content of each core histone confirmed that the four types of core histones participate in the product with an equal molar ratio. As one can specifically observe the behaviors of histone octamers with this reaction, it should be useful for research in various fields related to the dynamics and functions of the nucleosome.

Animals↗

PubMed1986

[Effects of MY-5116 on experimental animal models of type I-type IV allergic reactions and the formation of IgE antibody].

The effects of a new anti-allergic agent, MY-5116: isoamyl 5,6-dihydro-7,8-dimethyl-4,5-dioxo-4H-pyrano [3,2-c] quinoline-2-carboxylate, on experimental animal models of type I approximately type IV allergic reactions and the formation of IgE antibody were investigated. MY-5116 administered intraperitoneally at doses of 30 and 100 mg/kg suppressed significantly the homologous PCA in guinea pigs. MY-5116 administered intraperitoneally at the dose of 100 mg/kg also suppressed significantly the heterologous PCA in guinea pigs. MY-1250, the main active metabolite of MY-5116, showed no suppression on the Schultz-Dale reaction in guinea pigs, and MY-5116 showed no inhibition on the active systemic anaphylaxis in mice (type I). MY-5116 showed no inhibition on the reversed cutaneous anaphylaxis in rats (type II), the Forssman reaction in guinea pigs (type II), the Arthus reaction in mice (type III) and the delayed type hypersensitivity in mice (type IV). MY-5116 showed no suppression on the formation of IgE antibody in C3H/He and BALB/c mice and rats. From these results, it is concluded that MY-5116 selectively suppresses the experimental models of type I allergic reaction.

Anaphylaxis↗

PubMed1985

Rapid purification of nucleosome assembly protein (AP-I) and production of monoclonal antibodies against it.

A nucleosome assembly protein (AP-I) was purified approximately 50% from the cytosol of HeLa S3 cells by three purification steps. Using this protein fraction as an antigen, we established three stable hybridomas that secrete monoclonal antibodies specific for AP-I by the conventional method of cell fusion. Immunoblotting of the HeLa S3 cytosol, proved AP-I exists as a 58-kDa peptide in vivo, not as the 53-kDa peptide previously identified as active in nucleosome assembly (Ishimi, Y., et al., Eur. J. Biochem., 142, 431-439, 1984). An immunocytochemical study using the monoclonal antibody with the highest specificity against AP-I pin pointed the intranuclear localization of AP-I in HeLa S3 cells.

Animals↗

PubMed1984

Purification and initial characterization of a protein which facilitates assembly of nucleosome-like structure from mammalian cells.

A protein, which facilitates assembly of a nucleosome-like structure in vitro, was previously partially purified from mouse FM3A cells [Ishimi, Y. et al. (1983) J. Biochem. (Tokyo) 94, 735-744]. The protein has been purified to approximately 80% from FM3A cells by using histone-Sepharose column chromatography. It sedimented at 4.6 S and had a molecular mass of 53kDa. A preincubation of core histones with the 53-kDa peptide before DNA addition was necessary for the nucleosome assembly. The 53-kDa peptide bound to core histones and formed a 12-S complex. This complex contained stoichiometrical amounts of the 53-kDa peptide and core histones, and the core histones in this complex were composed of equal amounts of H2A, H2B, H3 and H4 histones. The nucleosomes were assembled by adding pBR322 DNA to the 12-S complex. When mononucleosome DNA and core histones were mixed in the presence of the 53-kDa peptide, formation of a 10.5-S complex was observed. The complex contained DNA and core histones in equal amounts, while no 53-kDa peptide was detected in the complex. From above results it is suggested that the 53-kDa peptide facilitates nucleosome assembly by mediating formation of histone octamer and transferring it to DNA. Rat antibody against the 53-kDa peptide did not bind to nucleoplasmin from Xenopus eggs. The relationship between the 53-kDa peptide and nucleoplasmin is discussed.

Animals↗