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Biomedical subjects

S Fujimoto

Publications and source records attributed to S Fujimoto.

At least 487 records · Page 27Linked to original sources

A reporter system using a flow cytometer to detect promoter/enhancer activity in lymphoid cell lines.

We have devised an experimental system using a flow cytometer to examine the promoter/enhancer activity of DNA fragments in human lymphoid cell lines. Murine CD8 alpha gene cDNA used as a reporter gene was inserted in the reporter constructs under the control of various promoter/enhancers. Furthermore, the Epstein-Barr virus (EBV) OriP, which supports a high transient expression, was also included in the reporter constructs. Cell lines expressing EBV nuclear antigen-1 (EBNA-1) were transfected with the reporter constructs by electroporation. The expression of the reporter gene was measured by a flow cytometric analysis. This experimental system is quite simple and may be especially useful for the analysis of transcriptional elements functioning in lymphocytes.

B-Lymphocytes↗

Enhancement of interferon-gamma-induced major histocompatibility complex class II gene expression by expressing an antisense RNA of poly(ADP-ribose) synthetase.

We studied the effect of poly(ADP-ribose) synthetase on the interferon-gamma (IFN-gamma)-inducible expression of major histocompatibility complex (MHC) class II molecules by expressing an antisense RNA of poly(ADP-ribose) synthetase. We constructed two expression plasmids capable of expressing an antisense RNA for poly(ADP-ribose) synthetase, carrying 0.7-kilobase long fragment of 5'-coding region (pAS-5') and full-length cDNA (pAS-FL) of poly(ADP-ribose) synthetase in an antisense orientation under control of metallothionein I promoter. We transfected the plasmid into human leukemia THP-1 cells and isolated transformants. Metal-inducible reduction in poly(ADP-ribose) synthetase activity was observed in two pAS-5'-transfected clones out of 72 neo-resistant clones examined, and metal-independent reduction in the activity was exhibited in pAS-FL-transfected clones. The antisense RNA was induced in a metal-dependent manner in the clones transfected with pAS-5', as judged by hybridizing with a sense riboprobe of the synthetase gene. The mRNA of the synthetase decreased 1 day after an addition of metal ions, and the synthetase activity of the transformants decreased by more than 90% 3 days after an addition of metal ions. Thus, we incubated the transformant clones in the presence of metal ions for 3 days and then treated them with IFN-gamma. The IFN-gamma-inducible expression of MHC class II molecules was amplified in the transformant clones, as judged by RNA blot analysis and flow cytometry. These results indicate that the decrease in poly(ADP-ribose) synthetase makes THP-1 cells favorable to induce MHC class II molecules by IFN-gamma.

Gene Expression↗

Role of mesenchymal cells in the neovascularization of the rabbit phallus.

The neovascularization of the rabbit phallus at ages between prenatal days 15 and 21 was investigated by light- and electron microscopy, computer-aided light microscopic reconstruction, and immunocytochemistry. The phalli are embedded by an abundance of mesenchymal cells, which are in contact with the neighboring ones or with the endothelial lining of growing capillaries. They often form solid cell cords that eventually make contact with the growing capillaries. The computer-aided reconstruction of the serial light micrographs reveals that these cell cords are involved in connecting the adjacent capillaries. The incorporation of such mesenchymal cell projections into the endothelial lining, occasionally conjugated with simple attachment devices, is frequently observed by transmission and scanning electron microscopy. The contact areas between the mesenchymal and endothelial cells show immunoreactions of fibronectin. These results indicate the successive transformation of mesenchymal cells to endothelial cells of the growing capillaries. As endothelial cells of the growing capillaries show mitotic proliferation, such vasoformative mesenchymal cells seem to be involved in the acceleration of the neocapillarization of the rabbit phallus. Fibronectin actively produced in the mesenchymal cells may participate in their migration and the mechanical linkage with the endothelial cells.

Animals↗

Identification of a molecular species in porcine ovarian luteal glutathione S-transferase and its hormonal regulation by pituitary gonadotropins.

Glutathione S-transferase (GST) was purified to a homogeneous state from the cytosol fraction of porcine corpora lutea. The present study showed that the final enzyme preparation consisted of a single molecular species of hepatic GSTA1-1, which has been identified from its enzymatic properties, amino acid composition, N-terminal amino acid sequences, and immunological reactivity. Furthermore, it is suggested that ovarian GST is involved in steroidogenesis, especially in the step of progesterone synthesis, from the following results. (1) The final enzyme preparation had a significant delta 5-3-ketosteroid isomerase activity, which acts in steroid synthesis. (2) GST activity in the cultured porcine granulosa cells was remarkably increased in the luteinizing process, which was induced by addition of pituitary gonadotropins such as follicle-stimulating hormone (FSH) and luteinizing hormone (LH) to the culture system. (3) Changes of GST activity induced by FSH and LH were closely associated with progesterone production. (4) GST was detected within luteinizing and theca interna cells, but not in granulosa cells, by immunohistochemical staining of ovary tissues using anti-ovarian luteal-GST antibodies.

Amino Acid Sequence↗

Decreased E-cadherin expression in endometrial carcinoma is associated with tumor dedifferentiation and deep myometrial invasion.

Decreased E-cadherin expression in tumor cells has been suggested to promote tumor invasiveness. We examined E-cadherin expression in 30 cases of endometrial carcinoma by immunohistochemistry using a monoclonal antibody to E-cadherin and investigated its correlation with other histopathologic features of the tumor. We observed that: (1) E-cadherin expression decreased with loss of differentiation (P < 0.05); (2) E-cadherin expression was inversely correlated with depth of myometrial invasion (P < 0.05); (3) decreased E-cadherin expression was correlated with paraaortic node metastasis (P < 0.01); and (4) multivariate analysis comparing the depth of myometrial invasion to the pattern of E-cadherin expression, histologic grade, nuclear grade, and lymph-vascular space invasion showed that the depth of myometrial invasion was most strongly correlated with decreased E-cadherin expression (P < 0.005). These findings seem to be consistent with the concept that the dissociation of cancer cells due to decreased expression of E-cadherin facilitates invasion of tumor cells.

Analysis of Variance↗

Perinatal management of idiopathic thrombocytopenic purpura in pregnancy: risk factors for passive immune thrombocytopenia.

Thirty-nine pregnant women with idiopathic thrombocytopenic purpura (ITP) were studied in order to evaluate the influence of therapies for maternal ITP on fetal passive immune thrombocytopenia (PIT). Neonatal platelet counts were also compared with platelet counts, amount of PAIgG, and presence of circulating antiplatelet antibody in maternal blood. Eight of 41 neonates (19.5%) presented PIT without neonatal mortality. A higher incidence of PIT was observed in women with prior splenectomy than in women without splenectomy (66.7% vs 11.4%). Neither a therapeutic effect nor an increased risk of PIT was observed with steroids or gammaglobulin administration. No correlation was found between neonatal platelet counts and maternal platelet counts or maternal PAIgG, while positive cases for circulating antiplatelet antibody assay presented a higher incidence of PIT than negative cases. Additionally, a higher incidence of PIT was observed in women with a history of previous PIT than in women with a history of normal delivery. Prior splenectomy, presence of antiplatelet antibody in maternal blood, and a history of previous PIT seem to be risk factors for fetal PIT.

Cesarean Section↗

Serum levels of steroid sulfatase protein in gynecologic carcinomas.

Steroid sulfatase (STS) desulfates a number of 3 beta-hydroxysteroid sulfates, converting inactive steroid hormone to the active form. We have established an enzyme-linked immunosorbent assay (ELISA) of STS by using polyclonal antibody against STS purified from human placenta to measure the amount of the enzyme protein in sera. ELISA was performed by a 'Sandwich' method using a peroxidase conjugated anti-STS IgG Fab' fragment. A range of STS of 10-1,500 ng/ml in serum was assayed by this method. When the serum STS from the patients with gynecologic carcinomas was assayed by the ELISA, the level was significantly elevated in endometrial carcinoma (P < 0.05) and ovarian carcinoma (P < 0.01), respectively, as compared with that of normal healthy women.

Adult↗

Age-related changes in fibre number, fibre size, fibre type composition and adenosine triphosphatase activity in rat soleus muscle.

To study the aging of muscle fibres in red skeletal muscle, fibre number, fibre diameter and fibre type composition in the soleus muscle of male rats of 3, 12 and 24 months old were examined. The total number of muscle fibres remained unchanged, while average diameter increased slightly with increasing age. The staining intensity of myosin adenosine triphosphatase (ATPase) activity in the fibres decreased with advancing age. Therefore, observation on the basis of myosin ATPase histochemistry alone is not adequate to study the aging of muscle fibres. In the muscles of 24 month-old animals, four fibre types were recognized; 1) many (52%) type I-O fibres showing weak ATPase and succinate dehydrogenase (SDH) reactions with slight subsarcolemmal aggregates of diformazan (SAD); 2) some (33%) type M fibres showing weak ATPase and intense SDH reactions with marked SAD; 3) a few (12%) type O fibres showing weak ATPase and intense SDH reactions without SAD; and 4) very few (4%) type IIA fibres. Histochemical and morphometric results suggest that type I-O, type M and type O fibres are derived from type I, type I and type IIA fibres, respectively. Furthermore, no transitional fibres from type IIA to type I were observed. Therefore, age-related changes in fibre type composition in the muscle cannot be explained by the simple idea that most type IIA fibres are transformed into type I fibres.

Aging↗

Adoptive transfer of a Th2-like cell line prolongs MHC class II antigen disparate skin allograft survival in the mouse.

The MHC class II alloantigen-reactive CD4+ Th2-like cell line, HR2, was established. It was derived from the spleen cells of C57BL/6 (B6) mice immune to an MHC class II-disparate mutant mouse, B6.CH-2bm12 (bm12) which carries the I-Abm12 antigen. This cell line, HR2, secretes IL-4 and IL-10 in an antigen-specific manner, and can also proliferate in an autocrine manner through IL-4. To investigate the in vivo role of this Th2-like CD4+ T cell line in transplantation immunity, an adoptive cell transfer study using the skin allograft system was performed. Skins grafted from bm12 to B6 were rejected at 12.1 +/- 0.6 days in control B6 mice which received no treatment. Whereas skin graft survival was prolonged in B6 experimental mice which had received 2 x 10(7) HR2 cells, 75% of the grafts survived for > 40 days. Moreover, since skin grafts from fully allogeneic third party donor BALB/c mice were rejected at 12.0 +/- 0.4 days by B6 mice which had received the same number of HR2 cells, it was demonstrated that HR2 is involved in the regulation of bm12 skin graft rejection. Furthermore, cytotoxic T lymphocyte (CTL) activity by spleen cells from HR2 transferred B6 mice was not induced even by in vitro secondary stimulation, although CTL activity was induced well in spleen cells of control B6 mice which had rejected the graft. The underlying mechanism has not been fully clarified; however, these results demonstrate that Th2-like CD4+ T cells suppress allograft rejection.

Animals↗

Antigenic cross-reactivity between human T lymphotropic virus type I (HTLV-I) and retinal antigens recognized by T cells.

To investigate the relationship between uveitis and HTLV-I infection, we examined the cross-reactivity between HTLV-I antigens and retinal antigens recognized by T cells in B10.BR mice immunized with human HTLV-I-infected MT-2 cells. We found that T cells obtained from MT-2 immune mouse spleen responded not only to HTLV-I antigens but also to retinal antigens of various species. However, they did not respond to HTLV-I-negative lymphoid cell lines. Furthermore, established T cell lines from MT-2 immune spleen cells also responded to both HTLV-I and retinal antigens. The phenotype of the immune cells that responded to both HTLV-I and retinal antigens was CD4+, CD8-, and CD3+. The proliferative response of T cell lines to HTLV-I as well as various retinal antigens, was clearly blocked by addition of anti-CD3, anti-CD4, or anti-I-Ak MoAbs, but not by anti-CD8 antibody. The established T cell lines from HTLV-I immune spleen cells were all found to be CD3+, TCR beta +, CD4+, CD8- cells by flow cytometric analysis. These results indicate that an epitope of HTLV-I antigens is cross-reactive to an epitope of retinal antigens extracted from either human retinoblastoma or normal murine, rat, and bovine retinae at a T cell recognition level.

Animals↗

High-frequency S-layer protein variation in Campylobacter fetus revealed by sapA mutagenesis.

Campylobacter fetus utilizes paracrystalline surface (S-) layer proteins that confer complement resistance and that undergo antigenic variation to facilitate persistent mucosal colonization in ungulates. C. fetus possesses multiple homologues of sapA, each of which encode full-length S-layer proteins. Disruption of sapA by a gene targeting method (insertion of kanamycin (km) resistance) caused the loss of C. fetus cells bearing full-length S-layer proteins and their replacement by cells bearing a 50 kDa truncated protein that was not exported to the cell surface. After incubation of the mutants with serum, the survival rate was approximately 2 x 10(-2). Immunoblots of survivors showed that phenotypic reversion involving high-level production of full-length (98, 127 or 149 kDa) S-layer proteins had occurred. Revertants were serum resistant but caused approximately 10-fold less bacteraemia in orally challenged mice than did the wild-type strain. Southern hybridizations of the revertants showed rearrangement of sapA homologues and retention of the km marker. These results indicate that there exists high-frequency generation of C. fetus sapA antigenic variants, and that intracellular mechanisms acting at the level of DNA reciprocal recombination play key roles in this phenomenon.

Animals↗

Gestational age assessment in Japanese low birthweight infants.

We evaluated the validity of the Ballard scoring system for assessing gestational age in Japanese low birthweight infants. Infants included in this study were 116 neonates who were admitted to seven hospitals in Hokkaido. Gestational ages of all infants were prenatally established by ultrasonographic measurement of the crown-rump length performed between 8 and 12 weeks of gestation (clinical age). Of these, 74 infants were appropriate for gestational age (AGA) and the remaining were small for gestational age (SGA). Mean birthweight was 1703 g and 34% (39/116) were < 1500 g. The correlation coefficient for the total population was 0.93 (P < 0.001). Although mean Ballard age tended to overestimate clinical age, mean differences between Ballard age and clinical age did not exceed 0.6 weeks. Agreement within 2 weeks among all infants was 86.2%. Percentages of agreement within 2 weeks did not differ to a statistically significant degree between AGA and SGA. These results indicate that the Ballard method is of value in assessing gestational age in Japanese low birthweight infants.

Evaluation Studies as Topic↗

A comparison between the preoperative and operative findings of peritoneal cytology in patients with endometrial cancer.

Laparoscopy and hysteroscopy were simultaneously performed to examine the intraperitoneal and intrauterine spread of endometrial cancer prior to surgery. Peritoneal cytology (PC) was examined before and after hysteroscopy and at the time of laparotomy. To elucidate the mechanisms by which endometrial cancer cells spread into the peritoneal cavity, the changes in PC in sequential samples and the association between PC and other prognostic factors were examined. 1) Of the patients who had negative PC before hysteroscopy, 3 patients exhibited positive PC from their operation materials (12.5%). It was suggested that leakage of endometrial cancer cells into the peritoneal cavity was induced by hysteroscopy in 2 patients and by endometrial biopsy in one patient. 2) Of those patients with no intraperitoneal lesions, 3 patients had positive PC in the prehysteroscopy samples (11.1%). A statistically significant association was found between pelvic node metastasis and positive PC in the prehysteroscopy samples.

Endometrial Neoplasms↗

A study on the hemostatic effect of sodium alginate on uterocervical hemorrhage.

We examined a sodium alginate preparation (Alto) to determine its hemostatic effect, in comparison with other topical drugs, on hemorrhages in the uterocervical area. A total of 149 patients with hemorrhage from cervical erosion or after-treatment bleeding of the cervix uteri were subjects in this trial, and 84 (79%) of 106 patients treated with this preparation experienced a hemostatic effect. A significant positive correlation (p < 0.001) was noted between the degree of tissue adhesiveness and the hemostatic effect in all the drugs tested. As for the tissue adhesiveness, Alto was significantly more favorable (p < 0.001) than both Thrombin and Francetin T powder. Because Alto was softened by tissue moisture, its topical adhesiveness could be increased, which suggested that Alto was superior in hemostasis.

Adolescent↗

Cerebral palsy of cystic periventricular leukomalacia in low-birth-weight infants.

We studied ultrasound findings and neurodevelopmental outcome of 24 infants weighing 2500 g or less with cystic periventricular leukomalacia. Fourteen infants had symmetrical cysts in the parietal or occipital region (group 1) and 10 had non-symmetrical cysts (group 2). Each infant was followed for more than 4 years of age (mean 5 years and 7 months). Twenty out of 24 (83.3%) children developed cerebral palsy. All of group 1 had cerebral palsy (8 diplegia and 6 ataxic diplegia), while 6 (60%) in group 2 developed cerebral palsy (4 diplegia and 2 hemiplegia). There was a significant difference in the incidence of cerebral palsy and motor ability between the two groups. The size and site of the cyst did not predict cerebral palsy. The presence of symmetrical cysts in the parietal or occipital region is a highly reliable neurosonographic finding for predicting cerebral palsy.

Cerebral Palsy↗