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Biomedical subjects

E Hara

Publications and source records attributed to E Hara.

At least 19 recordsLinked to original sources
PubMed1995

Increase in expression levels of interferon-inducible genes in senescent human diploid fibroblasts and in SV40-transformed human fibroblasts with extended lifespan.

The normal human fibroblast line, TIG-3 which senesces at around 80 population doubling levels (PDLs), expressed interferon (IFN)-inducible genes such as 6-16, 2', 5'-oligoadenylate synthetase (2,5-A) and HLA B7 near the end of the proliferative lifespan. Other normal fibroblast line such as MRC-5 also expressed IFN-inducible genes when senesced. Clones transformed with SV40 T-antigen, which extended their proliferative lifespan by about 20-30 PDLs, also expressed IFN-inducible genes during their extended life. Anti-IFN-beta antibodies added in culture medium repressed the expression of IFN-inducible gene in both normal senescent and life-extended SV40-transformed cells. IFN-beta repressed DNA synthesis in normal TIG-3 and induced IFN-inducible genes in both normal and SV40-transformed TIG-3. Conditioned medium recovered from life-extended SV40-transformed cells contained IFN-beta, but not IFN-alpha, IFN-gamma or TNF-alpha and possessed an activity that inhibited DNA synthesis of young TIG-3. Addition of anti-IFN-beta antibodies into the medium enhanced the serum-induced DNA synthesis of near senescent (91% lifespan completed) TIG-3, while it neither induced DNA synthesis in fully senescent TIG-3 nor extended the proliferative lifespan of TIG-3. These results suggest that normal and SV40-transformed human fibroblasts increase expression of IFN-beta with increasing proliferative age especially near the end of their lifespan resulting in induction of IFN-inducible genes and possibly in growth repression.

Cell Line↗
PubMed1995

Postoperative membranous proliferation from the anterior capsulotomy margin onto the intraocular lens optic.

We performed slitlamp microscopy and specular microscopy in 15 patients who had cataract surgery to determine the clinical course of postoperative membranous proliferation from the anterior capsulotomy margin onto the intraocular lens optic. Membranous proliferation observed in 11 of the 15 eyes eventually resolved in 10 eyes. We speculate that lens epithelial cells were the origin of the membrane.

Aged↗
PubMed1995

Contrast visual acuities in cataract patients. III. Changes of contrast acuity profiles in normal and pathological eyes.

We compared pre- and postoperative visual acuities in 45 cataract patients without ocular pathology except lens opacity (group 1) and 20 patients with ocular pathologies and lens opacity (group 2) using Variable Contrast Visual Acuity Charts. Charts 1 and 4 have 90% contrast; chart 4 has white optotypes on a black background (reverse polarity), charts 2 and 3 have 15% and 2.5% contrast, respectively. The mean preoperative visual acuities of groups 1 and 2 measured with the high-contrast Landolt optotypes ranged from 0.02-0.80 and 0.22-0.40, respectively. Visual acuity improvements between the pre- and postoperative periods ranged from 1.37-1.61 and 0.52-1.24 octaves in groups 1 and 2, respectively, with the different charts. Visual acuity improvement was poorest with chart 3. The group 1 mean preoperative visual acuity measured with chart 2 was 1.11 octaves lower than with chart 1. In group 2, the visual acuity reduction was 1.81 octaves. The relation between the visual acuities measured with the high-contrast intermediate-contrast optotypes are important for predicting postoperative visual acuity improvement. An abnormal contrast acuity profile may indicate the presence of additional ocular pathologies.

Adult↗
PubMed1995

Suppression of mammary epithelial cell differentiation by the helix-loop-helix protein Id-1.

Cell proliferation and differentiation are precisely coordinated during the development and maturation of the mammary gland, and this balance invariably is disrupted during carcinogenesis. Little is known about the cell-specific transcription factors that regulate these processes in the mammary gland. The mouse mammary epithelial cell line SCp2 grows well under standard culture conditions but arrests growth, forms alveolus-like structures, and expresses beta-casein, a differentiation marker, 4 to 5 days after exposure to basement membrane and lactogenic hormones (differentiation signals). We show that this differentiation entails a marked decline in the expression of Id-1, a helix-loop-helix (HLH) protein that inactivates basic HLH transcription factors in other cell types. SCp2 cells stably transfected with an Id-1 expression vector grew more rapidly than control cells under standard conditions, but in response to differentiation signals, they arrested growth and formed three-dimensional structures similar to those of control cells. Id-1-expressing cells did not, however, express beta-casein. Moreover, 8 to 10 days after receiving differentiation signals, they lost three-dimensional organization, invaded the basement membrane, and then resumed growth. SCp2 cells expressing an Id-1 antisense vector grew more slowly than controls; in response to differentiation signals, they remained stably growth arrested and fully differentiated, as did control cells. We suggest that Id-1 renders cells refractory to differentiation signals and receptive to growth signals by inactivating one or more basic HLH proteins that coordinate growth and differentiation in the mammary epithelium.

Animals↗
PubMed1994

Preparation of a subtractive cDNA library enriched in cDNAs which expressed at a high level in cultured senescent human fibroblasts.

Subtracted cDNA library was prepared by subtracting [cDNA from young growing SV40-transformed human fibroblasts] from [cDNA from growing SV40-transformed fibroblasts in extended lifespan]. Isolated cDNA clones which expressed at high level in life-extended transformed cells also expressed at high level in normal senescent fibroblasts but did at low level in growing and growth-arrested young cells. Neither fibronectin nor procollagen cDNA was isolated. This cDNA library is useful for isolation of senescent-specific cDNA species which express at high level in normal senescent cells but at low level in growing and growth-arrested young cells, avoiding growth-arrest-specific cDNAs.

Animals↗
PubMed1993

Identification of a universal B cell epitope on DNA topoisomerase I, an autoantigen associated with scleroderma.

OBJECTIVE: To investigate the distribution of B cell autoepitopes of human DNA topoisomerase I (topo I), an autoantigen associated with scleroderma. METHODS: A complementary DNA clone, T1B, was used to produce recombinant proteins of topo I as beta-galactosidase fusion proteins. Immunoreactivity to these fusion proteins was then tested in 35 anti-topo I-positive sera from patients with scleroderma, by immunoblotting, enzyme-linked immunosorbent assay, and double immunodiffusion. RESULTS: One epitope was found to be universally recognized by all sera tested. Thirty-two of the samples recognized multiple antigenic regions, but sera from the remaining 3 patients recognized only this universal epitope, and in longitudinal studies of 1 of these 3 patients, the serum recognized only this epitope for more than 2 years, even though multiple, potent, antigenic regions were found on topo I. CONCLUSION: Recognition of multiple epitopes in most patients suggests that the topo I molecule itself would drive the autoimmunity on topo I. However, antigen-driven autoimmunity could not explain the production of the monoreactive anti-topo I antibody seen in the 3 patients. We thus hypothesize that there is a process whereby recognition of the universal epitope by cross-reaction develops into antigen-driven autoimmunity.

Autoantibodies↗
PubMed1993

[Quantitative cytochemical analysis of T1 breast cancer].

The authors attempted to estimate the relationship between three biological parameters (nuclear DNA content, PCNA (proliferating cell nuclear antigen)/cyclin, HER-2/neu oncoprotein) and lymph node metastasis. We evaluated 37 breast cancers which were less than 2 cm in maximum dimension. Quantitative analysis was performed using a CAS 200 Image Analysis System, after Feulgen staining and immunochemical staining using anti-PCNA/cyclin monoclonal antibody and anti-HER-2/neu oncoprotein polyclonal antibody. In lymph node-negative cases 20.0% were aneuploid, while in lymph node-positive cases 58.8% were aneuploid. A total of 20.0% lymph node-negative cases were in the high proliferation group, as opposed to 52.9% of lymph node-positive cases. This analysis revealed a significant relationship between cell proliferation and lymph node metastasis. Analysis of the expression of HER-2/neu oncoprotein revealed no significant relationship between overexpression of HER-2/neu oncoprotein and lymph node metastasis, but the expression of HER-2/neu oncoprotein was significantly related to a shorter relapse-free survival.

Breast Neoplasms↗
PubMed1993

Review of laser treatment at Keio University Eye Clinic.

Between 1978 and 1992, 5310 eyes in 4317 cases were treated by laser photocoagulation at our clinic. Argon, krypton and dye lasers with slit lamp microscope were used 7556 times for 4446 eyes. Main indications included diabetic retinopathy, retinal vein occlusion, retinal break and glaucoma. For treatment of these diseases, argon blue-green and green laser were chosen in majority of cases. Macular diseases were treated most frequently by krypton laser. Endophotocoagulation by argon laser during vitreous surgery was performed in 399 eyes mainly with diabetic retinopathy, retinal detachment and retinal vein occlusion for 7 years. Treatment by Nd: YAG laser was done in 465 eyes for 5 years and more than 90% of cases were performed capsulotomy for after-cataract. Numbers of cases treated by any kinds of laser is increasing with the years.

Glaucoma↗
PubMed1992

Mechanism of induction of cellular DNA synthesis by the adenovirus E1A 12S cDNA product.

The mechanism of induction of DNA synthesis in quiescent rat 3Y1 cells by the adenovirus E1A gene was investigated using the 3Y1 derivative cell lines g12-21, gn12RB1, and gn12RB2. The g12-21 cells express the E1A 12S cDNA and the latter two cells express both the E1A 12S cDNA and the human retinoblastoma susceptibility (Rb) gene at different levels in response to dexamethasone (dex). The cDNA sequences of E1A-inducible cell cycle-dependent genes, clone 3 and clone 16, were isolated by differential screening of a cDNA library constructed from dex-treated g12-21 cells. The quiescent 3Y1 cells induced c-fos and c-myc expression within 2 h after serum stimulation and expressed clone 16 and clone 3 transiently at around 8 h before the onset of DNA synthesis (10 h). In contrast, the quiescent g12-21 cells treated with dex expressed a high level of E1A at 6 to 8 h after treatment and expressed clone 16 and clone 3 at around 8 h without stimulation of c-fos and c-myc expression, suggesting that E1A bypasses the cell cycle early in G1. The half-maximal rate of DNA synthesis was reached in a much shorter time in dex-treated g12-21 cells (12 h) than in serum-treated 3Y1 cells (18 h), suggesting that E1A also bypasses the cell cycle at the G1/S boundary. The gn12RB1 and gn12RB2 cells were unable to induce DNA synthesis in response to dex presumably due to lower levels of E1A expression, although gn12RB2 but not gn12RB1 cells could express clone 16 and clone 3. These results suggest that the level of E1A required for bypass at the G1/S boundary is higher than that required early in G1.

Adenovirus Early Proteins↗
PubMed1991

Cooperative effect of antisense-Rb and antisense-p53 oligomers on the extension of life span in human diploid fibroblasts, TIG-1.

Normal human diploid fibroblasts, TIG-1, which have a replicative life span of about 62 population doublings (PD), tended to senesce after about 50 PD with a gradual decrease in sensitivity to serum. Treatment of TIG-1 cells with the antisense-Rb oligomer, which completely depleted the retinoblastoma susceptibility gene product (RB), extended life span by about 10 PD. Treatment with the antisense-p53 oligomer alone had no effect; however, cotreatment with the antisense-Rb oligomer further potentiated the extension and the increased sensitivity to serum caused by the antisense-Rb oligomer alone, suggesting that p53 and RB function in separate, yet complementary pathways in signal transduction to senescence. The c-fos expression, which is presumed to be regulated negatively by RB, was not stimulated in partially senescent TIG-1 cells by treatment with the antisense-Rb oligomer.

Base Sequence↗
PubMed1991

Interaction of nuclear factors with the regulatory region of the N-myc gene during differentiation of human embryonal carcinoma cells.

The human embryonal carcinoma cell line, NEC14, can be induced to differentiate by the addition of 10(-2) M N,N'-hexamethylene-bis-acetamide (HMBA). During the early stage of HMBA-induced differentiation, the level of N-myc expression decreased steeply and transiently, and then quickly returned to its original level after reaching a minimal level at 18 h after addition of HMBA. Nuclear run-on experiments indicated that this transient decrease is regulated at the transcription start point. To investigate the mechanism of this down-regulation, the 5'-flanking region of the human N-myc gene was cloned and sequenced. Computer analysis of the sequence revealed high homology with the 5'-flanking region of the mouse N-myc gene, especially (greater than 80%) in the region of nt positions -1777 to -1732, nt positions -763 to -501 and nt positions -260 to + 1. The patterns of protein binding to the upstream region during the early stage of NEC14 cell differentiation were analyzed by gel retardation assay. The DNA fragments VIII and X, containing the sequences of nt positions -1437 to -1237 and nt positions -1863 to -1710, respectively, formed the DNA-protein complexes which were greatly reduced in quantity in the cell extract prepared 18 h after the addition of HMBA. This reduction, however, was not observed with an extract similarly prepared from the NEC14 derivative cell line, H10, expressing the N-myc gene constitutively. These results suggest a causal connection between the complex formation and the high-level transcription of the N-myc gene.

Acetamides↗
PubMed1991

Subtractive cDNA cloning using oligo(dT)30-latex and PCR: isolation of cDNA clones specific to undifferentiated human embryonal carcinoma cells.

The human embryonal carcinoma cell line NEC14 can be induced to differentiate by the addition of 10(-2)M N,N'-hexamethylene-bis-acetamide (HMBA). A subtractive cDNA library specific to undifferentiated NEC14 cells was constructed using oligo(dT)30-Latex and polymerase chain reaction (PCR). The method was designed to improve the efficiency of subtraction and the enrichment of cDNA clones corresponding to low abundance mRNAs. The single strand of cDNA was made from mRNA prepared from the HMBA-treated NEC14 cells using an oligo(dT)30 primer covalently linked to Latex particles. After removal of the mRNA template by heat-denaturation and centrifugation, the subtractive hybridization was carried out between the cDNA-oligo(dT)30-Latex and mRNA from untreated NEC14 cells. Unhybridized mRNA collected by centrifugation was hybridized repeatedly to the cDNA-oligo(dT)30-Latex and subtractive mRNA was converted to cDNA. The subtractive cDNA was then amplified by PCR and cloned into pBluescript II KS-. The cDNA library thus constructed consisted of approximately 10,000 independent clones with cDNA inserts of 1.7 Kb on average. Differential hybridization of these transformants indicated that approximately 3% of them contained cDNA inserts specific to the undifferentiated EC cells, some of which were derived from low abundance mRNAs.

Acetamides↗
PubMed1991

Drug-induction decreases the mobility of cytochrome P-450 in rat liver microsomes: protein rotation study.

Effect of drug-induction on the rotation of cytochrome P-450 and on lipid fluidity in rat liver microsomes was examined. Rotational diffusion of cytochrome P-450 was examined by observing the decay of absorption anisotropy, r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. Analysis of r(t) was based on a "rotation-about-membrane normal" model. Microsomal lipid fluidity was measured by observing fluorescence anisotropy of DPH incorporated in the lipid bilayer. The absorption anisotropy decayed within 2 ms to a time-independent value. Rotational diffusion of cytochrome P-450 was dependent on the drug-induction with PB, MC, and PCB when compared with non-induced CON-microsomes. The observed values for the normalized time-independent anisotropy r(infinity)/r(0) are r(infinity)/r(0) = 0.41 (CON-microsomes), 0.54 (PB-microsomes), 0.52 (MC-microsomes), and 0.57 (PCB-microsomes). The average rotational relaxation time phi = 580-690 microseconds was almost unchanged over all microsomes presently examined. A significantly high value of r(infinity)/r(0) = 0.41-0.57 implies the co-existence of mobile and immobile populations of cytochrome P-450. Based on the assumption that the heme tilts about 55 degrees from the membrane plane for all species of P-450s besides P-450PB, 59% (CON-microsomes), 46% (PB-microsomes), 48% (MC-microsomes), and 43% (PCB-microsomes), respectively, of the cytochrome P-450 in microsomes is calculated to be mobile. Upon drug-induction the microsomal membrane was fluidized to some extent as judged by the steady-state fluorescence anisotropy of 0.156 for CON-microsomes and 0.139-0.148 for drug-induced microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

A transient decrease in N-myc expression and its biological role during differentiation of human embryonal carcinoma cells.

The human embryonal carcinoma (EC) cell line, NEC14 can be induced to morphologically differentiate by the addition of 10(-2) M N,N'-hexamethylene-bis-acetamide (HMBA) in vitro. The expression of several cellular oncogenes (c-onc) in NEC14 cells was examined after induction of differentiation by HMBA. The level of N-myc expression was the highest in undifferentiated cells but decreased transiently to less than 1/10 of the original level shortly after the induction of differentiation. To investigate the role of the transient decrease in N-myc level on NEC14 cell differentiation, a chimeric human N-myc gene in which transcription is initiated at the human beta-actin gene promoter was constructed and introduced into NEC14 cells. Several transformants expressing the exogenous N-myc gene constitutively were established. These transformants showed 10- to 70-fold increases in plating efficiency and shorter population doubling times as compared with the parental NEC14 cells. The transformants were hard to induce, spontaneously differentiated cells on the periphery of cell clusters in culture, unlike parental NEC14 cells, and took longer for HMBA-induced morphological differentiation. The populations of the cells expressing HLA and SSEA-1 antigens increased from 10%-20% to nearly 100% in NEC14 cells after the induction of differentiation, while the populations expressing these antigens increased only to 50%-60% in one of the transformants, S11. The transformants gained an increased tumorigenic potential in nude mice, and the tumors produced consisted exclusively of EC stem cells. These results suggest that the additional expression of the exogenous N-myc gene (increased about two-fold) confers the more transformed state on the cells.

Antigens, Surface↗
PubMed1991

[Immunohistochemical Study of 3-methylcholanthrene inducible cytochrome P-450 in the stomach and liver--a guide of postoperative chemotherapy in gastric cancer].

Immunohistochemical determination of 3-methylcholanthrene (MC) inducible cytochrome P-450 (MC-P-450) was investigated in rat and human tissue, and its clinical availability was discussed. Induced by MC, MC-P-450 in rat liver and stomach was well stained immunohistochemically, showing clear contrast against control without induction. The staining intensity in the tissue was correlated with the amount of tissue MC-P-450 which was determined previously by electrophoretical and biochemical technics. By the same immunohistochemical method MC-P-450 in human liver and stomach was also detectable. The staining grade of MC-P-450 in human liver and stomach was different from each person. However, its intensities in liver and stomach in the same individual showed clear correlation with p less than 0.02. Since MC-P-450 in liver plays a major role in drug metabolism, the proof of correlation between staining degree of the resected stomach and hepatic tissue would provide useful clue in gastric cancer for postoperative administration of masked compounds activated by MC-P-450.

Animals↗
PubMed1990

A rapid and efficient cloning method with a solid-phase DNA probe: application for cloning the 5'-flanking region of the gene encoding human fibronectin.

A rapid and efficient gene-cloning method was developed using oligodeoxyribonucleotide probes (approx. 30-mer) immobilized by their 5' terminal to the gel (LiChrosphare Si1000; used for high-performance liquid chromatography). The method allowed us to concentrate DNA fragments having a particular base sequence in an eppendorf tube with very high efficiency within 2 h. For example, genomic DNA encoding the human fibronectin gene (fn) was concentrated by this method and cloned into lambda Charon4A vector. Screening of the concentrated genomic DNA library for the 5' region of the human fn gene showed that approx. 30% of 3000 recombinant phages tested were positive clones. Various factors affecting cloning efficiency were investigated.

Base Sequence↗
PubMed1990

Two-step transformation of rat 3Y1 cells by the adenovirus E1A and E1B genes.

The transformation of rodent cells by the adenovirus E1A and E1B genes was very efficient when these genes were physically linked. When they were cleaved, the transformation became very inefficient. To clarify this difference, the chimeric E1B genes in which either the adenovirus enhancer or the human beta-actin promoter was linked to the 5' side of the E1B gene were introduced into rat 3Y1 cells. The saturation density of these cell lines (eB or APrB) was similar to that of parental 3Y1 cells. When eB or APrB cell lines were supertransfected with the E1A gene, discrete dense foci were developed after 5-6 weeks, while the supertransfection of 3Y1 derivative cell lines, in which the enhancer-unlinked E1B gene was introduced, did not develop any dense foci. Analysis of the E1A and E1B transcripts in these cell lines indicated that the E1B gene is efficiently expressed in the presence of the E1A gene products if the enhancer is linked to the E1B gene and that an increased level of E1B proteins is required for an efficient expression of the E1A gene. These results indicated that E1A and E1B genes in separate pieces of DNA are capable of cooperatively transforming 3Y1 cells if appropriate cis-acting elements are attached and high-level expressions are achieved.

Actins↗