Biomedical subjects

M Matsushima

Publications and source records attributed to M Matsushima.

At least 91 records · Page 5Linked to original sources

PubMed1996

Mutational analysis of mismatch repair genes, hMLH1 and hMSH2, in sporadic endometrial carcinomas with microsatellite instability.

Microsatellite instability, monitored by replication error (RER), has been observed in both sporadic and hereditary types of endometrial carcinoma. In the hereditary tumors, this instability is considered to be caused by a germline defect in the DNA mismatch-repair system. We previously reported that nearly one-quarter of sporadic endometrial carcinomas examined revealed an RER-positive phenotype at multiple microsatellite loci. To investigate the role of genetic alterations of DNA mismatch-repair genes in sporadic endometrial carcinomas, we screened 18 RER(+) endometrial carcinomas for mutations of hMLH1 and hMSH2. Although we found no germline mutations, we detected two somatic mutations of hMLH1 in a single endometrial cancer; these two mutations had occurred on different alleles, suggesting that two separate mutational events had affected both copies of hMLH1 in this particular tumor. These data implied that mutations of hMLH1 or hMSH2 play limited roles in the development of sporadic endometrial carcinomas, and that the tumors with genetic instability might have alterations of other mismatch-repair genes, such as hPMS1 and hPMS2, or of unknown genes related to the mismatch-repair system.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Molecular cloning and mapping of a human cDNA (SC5DL) encoding a protein homologous to fungal sterol-C5-desaturase.

We have isolated a human cDNA clone homologous to fungal ERG3, a gene encoding sterol C-5 desaturase. The full nucleotide sequence of this human cDNA revealed a 708-bp open reading frame that encodes a 236-amino-acid polypeptide. The gene was expressed in all normal human tissues examined. We determined its location to chromosome 11q23.3 by fluorescence in situ hybridization.

Amino Acid Sequence↗

PubMed1996

A major improvement in the prognosis of individuals with IDDM in the past 30 years in Japan. The Diabetes Epidemiology Research International Study Group.

OBJECTIVE: To evaluate the time trends of mortality among individuals with IDDM in Japan. RESEARCH DESIGN AND METHODS: A historical prospective study of two independent population-based cohorts composed of individuals who were diagnosed between 1965 and 1969 (1960s cohort) and between 1975 and 1979 (1970s cohort), which included 286 IDDM patients (onset age < 18 years) for the 1960s cohort and 779 patients for the 1970s cohort, was performed. After 10 years of observation, mortality status and causes of deaths between the two cohorts were compared. RESULTS: The age-adjusted mortality rate per 100,000 person-years of the 1960s cohort was 754 (95% CI, 471-1,141); in contrast, that of the 1970s cohort was only 196 (95% CI, 107-329) (P < 0.001). The standardized mortality ratio of the 1960s cohort was 1,432 (95% CI, 898-2,161), and that of the 1970s cohort was 489 (95% CI, 267-821). Analyses of the causes of deaths revealed a marked decline in recent years in the number of deaths by acute complications and renal disease. CONCLUSIONS: A major decline in the mortality of diabetic children in Japan may be attributed to the dramatic changes in the quality of care and medical infrastructure that occurred after the mid-1970s.

Adolescent↗

PubMed1996

Expression of basic fibroblast growth factor mRNA in developing choroidal neovascularization.

PURPOSE: Basic fibroblast growth factor (bFGF) is an angiogenic peptide that may be important in the pathogenesis of choroidal neovascularization. We attempted to determine the transcription of the bFGF gene during the development of experimentally induced choroidal neovascularization. METHODS: Rat bFGF cDNA was inserted in the pBluescript to prepare antisense and sense riboprobes. Multiple krypton laser burns were applied to the posterior poles of the eyes of pigmented rats according to a protocol described for producing subretinal neovascularization in these animals. At intervals of up to 4 weeks after photocoagulation, the eyes were removed and cut into thin sections. The sections were subjected to histopathological analysis, cell proliferation study, or in situ hybridization with digoxigenin (DIG)-labeled single-strand riboprobes synthesized from rat bFGF cDNA. RESULTS: In normal adult rat retinas, bFGF mRNA expression was mainly observed in the ganglion cell layer and the inner nuclear layer. After laser photocoagulation, proliferation of RPE cells, fibroblast-like cells and cells in the choroid in the lesions were observed. Expression of bFGF mRNA was observed in the lesions 3 days to 2 weeks after laser treatment. Signals of bFGF mRNA were detected in the proliferating RPE-like cells, choroidal vascular endothelial cells and fibroblast-like cells, all of which are essential for neovascularization. However, bFGF mRNA expression was no longer detectable in these cells 4 weeks after photocoagulation. CONCLUSIONS: Our findings indicate that bFGF is normally transcribed in ganglion cells and the inner nuclear cell layer. During the neovascularization that followed laser photocoagulation, bFGF mRNA expression was detected within the laser lesions. It is thus probable that bFGF acts as a mediator in the neovascularization process.

Animals↗

PubMed1996

[Expression of basic fibroblast growth factor and its receptor in the process of wound healing of rat retina after laser photocoagulation].

We investigated the expression of mRNA of basic fibroblast growth factor (bFGF) and FGF receptor 1 in rat retina after laser photocoagulation using in situ hybridization method. Pigmented rats (Brown Norway strain) received weak photocoagulation by krypton laser (500 microns, 0.05 sec, 60 mW) in the posterior retina. On 1, 3, 5, 7, 14 days after laser photocoagulation, the rats were fixed by perfusion with phosphate-buffered 4% paraformaldehyde and the eyes were enucleated. The eyes were further fixed by immersion in the same fixative, then quickly frozen in liquid nitrogen and finally sectioned with a cryostat. In situ hybridization was performed on frozen sections with digoxigenin (DIG) labeled riboprobes synthesized from rat bFGF cDNA and FGF receptor 1 cDNA. In normal chorioretinal tissue, the signals of bFGF and FGF receptor 1 mRNA were seen in the ganglion cell layer and inner nuclear layer. On day 3 after photocoagulation, we observed expression of bFGF and FGF receptor 1 mRNA in the proliferating retinal pigment epithelial (RPE) cells and endothelial cells of choriocapillaris at the photocoagulated lesion. We also observed expression of bFGF mRNA in some macrophage-like cells. On day 14 after photocoagulation, these expressions had disappeared. Our results suggest that bFGF may be involved in the process of retinal wound healing after laser photocoagulation.

Animals↗

PubMed1996

FGF receptor 1 expression in experimental choroidal neovascularization.

Fibroblast growth factor (FGF) is an important factor in neovascularization. To clarify the role of FGF, we demonstrated the expression of FGF receptor 1 mRNA in experimentally induced choroidal neovascularization using in situ hybridization. Krypton laser photocoagulation was applied to the posterior retina of colored rats to induce choroidal neovascularization; the eyes were removed at various intervals after photocoagulation. Chorioretinal sections were made, and FGF receptor 1 cDNA fragments were used to make antisense and sense probes for in situ hybridization. In normal chorioretinal tissue, stain indicating FGF receptor 1 mRNA was observed in the ganglion cells and the inner nuclear layer. After photocoagulation, FGF receptor 1 mRNA appeared in the retinal pigment epithelial cells, in melanocytes of the choroid and in the choroidal blood vessel wall of the photocoagulation lesions. This suggests that FGF is involved in the process of neovascularization. Previous studies have reported that the capillary endothelial cells and retinal pigment epithelial cells produce basic FGF in vitro; FGF may affect the cells in an autocrine or paracrine manner.

Animals↗

PubMed1996

Gene expressions of basic fibroblast growth factor and its receptor in healing of rat retina after laser photocoagulation.

Basic fibroblast growth factor (bFGF) stimulates the mitogenesis of various cells and plays a key role in wound repair. Using in situ hybridization, we studied mRNA expressions of bFGF and one of its receptors, FGF receptor 1 (FGFR1), during wound repair of the rat retina after laser photocoagulation. Gene expressions of bFGF and FGFR1 were detected in the ganglion cell and inner nuclear layers of the normal adult rat retina. On day 3 following laser photocoagulation, proliferating retinal pigment epithelial (RPE) cells of the lesion showed intense gene expressions of bFGF and FGFR1. Macrophage-like cells that migrated into the lesion also showed gene expression of bFGF. These gene expressions decreased over time. The finding of elevated gene expressions of bFGF and FGFR1 after laser photocoagulation suggests the bFGF may be a factor in retinal wound repair.

Animals↗

PubMed1995

Effects of long-term omeprazole treatment on adult rat gastric mucosa--enhancement of the epithelial cell proliferation and suppression of its differentiation.

Effects of long-term omeprazole treatment on the process of epithelial cell proliferation and differentiation in the adult rat gastric mucosa were investigated. Animals were treated with omeprazole (25 mg/kg body weight/day) for 28 days to induce anacidity in the stomach. The treatment induced a marked decrease in the number of chief cells in the gastric mucosa and at the same time an increase in that of immature pepsinogen-producing cells expressing class III mucin. This was accompanied by a decrease to 60% and 10% of the control values in the mucosal levels of pepsinogen and its mRNA, respectively. Moreover, the expression of cathepsin E in surface mucous cells was reduced. Cell proliferation studies revealed that the rate of bromodeoxyuridine-labeled cells was increased by omeprazole. The above-described changes were reversed by cessation of the treatment and they were not caused by the omeprazole-treatment at a dose which does not induce anacidity in the stomach. These results suggest that long-term omeprazole treatment reversibly increases the epithelial cell proliferation and suppresses its differentiation in the adult rate gastric mucosa probably by altering the acidic environment specific for the stomach.

Animals↗

PubMed1995

Structure of a glutathionylated human lysozyme: a folding intermediate mimic in the formation of a disulfide bond.

The three-dimensional structure of a mutant human lysozyme, C77A-a, in which the residue Cys77 is replaced by alanine, has been refined to an R value of 0.125 using 8230 reflections in the resolution range 10.0-1.8 A. It has been shown that C77A-a, in which the counterpart of Cys77 (Cys95) is modified with glutathione, has been shown to mimic an intermediate in the formation of the disulfide bond Cys77-Cys95 during the folding of human lysozyme [Hayano, Inaka, Otsu, Taniyama, Miki, Matsushima & Kikuchi (1993). FEBS Lett. 328, 203-208]. An earlier structure demonstrates that its overall structure is essentially identical to that of the wild-type protein and served as the starting model. The refined model includes atoms for all protein residues (1-130), 20 glutathione atoms and 113 water atoms. Further refinement shows more clearly the details of the protein, the bound glutathione molecule and solvent structure. However, the main-chain folding and the atomic thermal factors of the loop region from Thr70 to Leu79 were highly affected by the binding of the glutathione molecule, as compared with those of the wild-type protein. The bound glutathione shifted the main-chain atoms from Va174 to Ala77 by more than 6.0 A, and the temperature factors of the atoms in the loop region were quite high (more than 40 A(2)), indicating that the backbone conformation of this region is highly flexible and that the loop region is not folded in the specific conformation observed in the wild-type protein. These results strongly suggest that the loop structure in human lysozyme is folded later than the other regions of the protein in vivo, as observed in in vitro folding. Since the bound glutathione is efficiently and irreversibly dissociated by protein disulfide isomerase, the glutathione molecule may act as a protecting group to prevent the formation of an incorrect disulfide bond in the protein folding process in vivo.

Journal Article↗

PubMed1995

O-glycosylation of the Thr70 residue of cell-adhesive lysozyme in yeast.

The cell-adhesive protein Cys-RGD4 has been constructed using a yeast expression system by inserting the sequence Cys-Arg-Gly-Asp-Ser-Cys (CRGDSC) between Val74 and Asn75 of human lysozyme [Yamada, T., Uyeda, A., Kidera, A. & Kikuchi, M. (1994b) Biochemistry 33, 11678-11683]. The Cys74a, Arg74b, Gly74c, Asp74d, Ser74e, Cys74f-lysozyme mutant, purified from the yeast culture supernatant contained glycosylated variants, in addition to the unglycosylated form. Peptide mapping analyses suggested that the glycosylation occurred at the Thr70 residue in the Cys-RGD4 molecule. Electrospray ionization mass spectrometric analysis demonstrated the presence of two hexose residues in the major variant, and one, three, four, or five hexose residues in the minor variants. All of these hexose residues were identified as mannose by analysis of the oligosaccharide mixture obtained by mild alkaline treatment of the variants. No other glycosylation was observed, although the Cys-RGD4 molecule possesses a total of 12 threonine and serine residues. In addition, the Thr70 residue is not glycosylated in either native lysozyme or the Arg-Gly-Asp-Ser (RGDS)-inserted mutant, RGD4 [Yamada, T., Matsushima, M., Inaka, K., Ohkubo, T., Uyeda, A., Maeda, T., Titani, K., Sekiguchi, K. & Kikuchi, M. (1993) J. Biol. Chem. 268, 10588-10592]. Thus, this O-glycosylation seems to be specific for both the mutant lysozyme molecule and the site of the threonine residue. Structural analyses of these lysozymes by X-ray crystallography suggest that the conformation of the serine-containing or threonine-containing region can affect the specificity of yeast O-glycosylation.

Amino Acid Sequence↗

PubMed1995

Effect of CCK-B/gastrin receptor antagonist on pepsinogen-producing cells during omeprazole treatment.

The present study investigated the effect of long-term treatment with omeprazole on pepsinogen-producing cells and examined whether the selective CCK-B/gastrin receptor antagonist was able to prevent the omeprazole-induced changes, if occurred, in rat stomach. Rats were treated with omeprazole and/or the CCK-B/gastrin receptor antagonist for 28 days. As a result, omeprazole markedly reduced mucosal pepsinogen activity and its mRNA concentration in rat stomach. Morphologically, in fundic glands omeprazole drastically decreased the proportion of mature chief cells and reciprocally increased that of immature chief cells which were positive for class III mucin. These effects of omeprazole were attenuated by an addition of the CCK-B/gastrin receptor antagonist. Our results suggest that omeprazole retards the differentiation of chief cells in fundic mucosa probably through hypergastrinemia in adult rat.

Animals↗

PubMed1995

Dissection of protein-carbohydrate interactions in mutant hen egg-white lysozyme complexes and their hydrolytic activity.

Trp62 in the binding subsite B of hen egg-white lysozyme shows general features often observed in protein-carbohydrate interactions including a stacking interaction and a hydrogen bonding network with water molecules. A previous report by our group showed that the perturbation of these interactions by substitution of Trp62 with tyrosine or phenylalanine affects the substrate binding modes and also enhances the hydrolytic activity. In order to elucidate the relationship between structural and functional changes of these protein-carbohydrate interactions, the Trp62Tyr and Trp62Phe mutants complexed with the substrate analogue, (GlcNAc)3, were analyzed at 1.8 A resolution by X-ray crystallography. The overall structures of the mutant enzymes are indistinguishable from that of the wild type enzyme. Although the wild-type enzyme binds (GlcNAc)3 in only one binding mode (A-B-C), the Trp62Tyr mutant binds (GlcNAc)3 in two binding modes (A-B-C, B-C-D) and the Trp62Phe mutant has an even weaker binding mode. The aromatic rings of Tyr62 and Phe62 maintain their interactions with the carbohydrate molecules, but make fewer stacking interactions with the GlcNAc in the B site than the wild-type enzyme does. The hydroxyl group of Tyr62 interacts weakly with a water molecule which mediates hydrogen bonding in the GlcNAc residues in the B and C sites. The C-6 hydroxyl group of the GlcNAc residue in the C site rotates around the C-5-C-6 bond to complete the hydrogen bond network in the Trp62Tyr mutant-(GlcNAc)3 complex. On the other hand, this hydrogen bonding network does not form in the Trp62Phe mutant-(GlcNAc)3. In addition to these structural studies, the kinetic parameters of the hydrolysis of 4-methylumbelliferyl N-acetyl-chitotriose, ((GlcNAc)3-MeU), have been determined in order to further characterize the enzymatic properties of these mutant lysozymes. This demonstrates that the modulation of the hydrogen bonding network, including the flexible part of the carbohydrate and water molecules and/or the slight reduction of stacking interaction in the B site, alters the binding mode toward the carbohydrate and induces an enhancement of the hydrolytic activity.

Acetylglucosamine↗

PubMed1995

Structure of a conformationally constrained Arg-Gly-Asp sequence inserted into human lysozyme.

To examine the effect of a conformational constraint introduced into the Arg-Gly-Asp (RGD) sequence on cell adhesion activity, we constructed a mutant protein by inserting an RGD-containing sequence flanked by two Cys residues between Val74 and Asn75 of human lysozyme. The CRGDSC-inserted lysozyme was expressed in yeast, purified, and designated as Cys-RGD4. Using baby hamster kidney cells, Cys-RGD4 was shown to possess even higher cell adhesion activity than that of the RGDS-inserted lysozyme, RGD4. The Cys-RGD4 protein was co-crystallized with a lysozyme inhibitor, tri-N-acetylchitotriose, and the three-dimensional structure was determined at 1.6-A resolution by x-ray crystallography. In contrast to RGD4, the inserted RGD-containing region of Cys-RGD4 was well defined. The structural analysis revealed that the two inserted Cys residues form a new disulfide bond in Cys-RGD4, as expected, and that the RGD region assumes a type II' beta-turn conformation of Gly-Asp with a hydrogen bond between the C = O of Arg and the H-N of Ser. In addition, it was confirmed that two more hydrogen bonds are present in the RGD region of the Cys-RGD4 lysozyme. These results suggest that the conformation of the RGD-containing region is rigid and stable in the Cys-RGD4 molecule and that the type II' beta-turn structure of RGD is essential for binding to integrins with high affinity.

Asparagine↗

PubMed1995