Biomedical subjects

M Emura

Publications and source records attributed to M Emura.

At least 37 records · Page 2Linked to original sources

PubMed1994

Neuroendocrine phenotype differentiation in a hamster lung epithelial cell line under low oxygen pressure or after transformation by diethylnitrosamine.

Oxygen pressure as low as 5% in the gas phase or 87 mmHg in the liquid phase induced various neuroendocrine cell (NEC) phenotypes in more than 80% of cells of a cloned fetal Syrian hamster lung epithelial cell line (M3E3/C3). Further, cells from a number of colonies transformed in an anchorage-independent manner by diethylnitrosamine (DEN) demonstrated a NEC phenotype. Since the cell line used is of a pluripotent stem cell type, both hypoxia and DEN appear to possess a potency for NEC phenotype induction.

Animals↗

PubMed1994

Comparison of biological effects of different polycyclic aromatic hydrocarbons in lung cells of hamster and rat in vitro.

The cytotoxicity and frequencies of transformation induced by 5 environmental polycyclic aromatic hydrocarbons (PAH) in hamster (M3E3/C3) and rat (WRB K3) lung cells were compared. Both cell strains investigated here retain major metabolic characteristics of the target cells in vivo and are thus able to effectively metabolize, i.e. activate, PAH. Cytotoxic effects of the carcinogen were determined in colony-forming assays and the PAH tested induced dose-dependent cytotoxic responses in the M3E3/C3 and WRB cells. They could then be classified into strong and weak cytotoxicity. Compared to the hamster cell system, the WRB cells were generally shown to be more sensitive. The transforming capacity of the compounds was determined by a soft agar colony formation assay detecting cells with anchorage independency (AI). All PAH investigated induced transformation to AI growth in both cell systems. The transforming activity of the PAH, relative to benzo[a]pyrene (B[a]P) as a reference substance, was determined to facilitate their ranking. This order of transforming potency appears to be similar to that observed in animal studies.

Animals↗

PubMed1994

Regulation of c-jun by lung carcinogens in Clara cells of hamsters.

In vitro differentiated hamster Clara cells were used to study the effects of lung carcinogens on the regulation of the c-jun oncogene. Northern blot analysis revealed a decrease in the expression of jun transcripts 24 h following the exposure of Clara cells to the direct acting forms of benzo[a]pyrene (BPDE*) or 5-methylchrysene (5MeCDE). To determine whether this decrease was mediated at the transcriptional level, we have used CAT reporter constructs driven by nested deletions of the 5' non-coding regulatory region of the c-jun oncogene. While BPDE was capable of activating certain regulatory domains of the c-jun promoter, this activation was not observed with either 5MeCDE or the less active lung carcinogens BADE or 6MeCDE. Analysis of enhancer elements identified the SP1 target site as a strong silencer after BPDE treatment. While positive regulatory element(s) mediating activation of c-jun by BPDE were localized within the promoter region up to -1639, further upstream sequences reduced this transcriptional activation. Thus, when the complete promoter region, up to -4500, was tested, no transcriptional activation was noted following BPDE treatment. These observations suggest that the regulation of c-jun in Clara cells exposed to potent lung carcinogens is mediated at the post-transcriptional level, possibly by reducing the stability and, in turn, the half life of c-jun mRNA. Overall, in contrast to the response of c-jun to numerous carcinogens and stress inducing agents noted in various other cell systems, our findings suggest the existence of a tissue-specific regulatory response for c-jun.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1994

[Two fibrinopulurent empyema cases--treatment with early mini-thoracotomy and transcatheter urokinase instillation].

We successfully treated two male patients with nontuberculous multiloculated empyemas by early mini-thoracotomy 3 and 6 weeks, respectively, after the onset of the disease. Transcatheter urokinase instillation facilitated drainage of a hemorrhagic loculated pleural collection in one patient after the operation. We believe that these treatments are less invasive and more effective than standard methods in providing early and complete resolution fibrinopurulent empyema in patients in whom this disorder might become chronic.

Aged↗

PubMed1993

Influence of culture passages on growth kinetics, xenobiotic metabolism, chromosomal stability and transformation in a clonal fetal hamster lung epithelial cell line.

M3E3/C3 is a clonal fetal hamster lung epithelial cell line which is used for studies of epithelial differentiation as well as for in vitro toxicologic tests. In this study growth kinetics, xenobiotic metabolism, chromosomal stability and transformation were investigated at increasing culture passage numbers up to 150. Cells of higher passages grew faster and reached higher cell densities than the cells of lower ones. As an indicator of xenobiotic metabolism we measured the activity of 7-ethoxycoumarin-deethylase (ECD), an enzyme belonging to the mixed function oxidase system. Up to passage number 100 the ECD activity strongly increased, followed by a slight decrease in additional passages. The chromosomal stability was assessed by the induction of micronuclei by benzo[a]pyrene (BaP) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). More micronuclei were always detected in cells of higher passages than of lower passages. The capability of cells to be transformed to anchorage independent growth by chemical carcinogens was examined using a soft agar test. After carcinogen exposure with BaP and MNNG, cells of higher passages showed higher transformation frequencies than cells of lower passages. Many cells at passage 150 exhibited an especially high soft agar growth even without carcinogen treatment and were therefore characterized as spontaneously transformed. These results show that metabolic and genetic characteristics of permanently growing cells differ remarkably depending on the culture passage. This has always to be considered when permanently growing cells are used for toxicological studies.

7-Alkoxycoumarin O-Dealkylase↗

PubMed1993

In vitro investigation on proliferative cystic keratinizing squamous lesions in the rat lung.

The proliferative cystic squamous cell lesion inducible by inhalation of particulate matter in the rat lung is characterized by the formation of keratin-filled cystic cavities of various sizes bordered by multiple layers of keratinizing squamous epithelial cells. The current investigation is primarily concerned with two points. One is whether the cells participating in this particular cystic configuration can recapitulate their specific in vivo morphogenetic behavior also in the in vitro circumstances. The other is whether these squamous epithelial cells are neoplastic in nature. Although the currently adopted cell culture system was two-dimensional, the specific morphogenetic pattern was reproduced in vitro in a corresponding manner by the squamous cells derived from the aforementioned rat lung lesions. Exposure of these cells to 12-O-tetradecanoyl-phorbol-13-acetate (TPA) at 100 ng/ml induced a marked polymorphism in them and also stimulated their keratinization. In soft agar culture, both TPA-exposed and unexposed cells developed colonies larger than 0.05 mm in diameter with an incidence of 0.73% and 1.12%, respectively. The lower incidence in TPA-exposed cultures can be interpreted in terms of TPA stimulation of keratinization. However, colonies larger than 0.1 mm in diameter were also developed by the TPA-exposed cells, indicating the presence of a cell population responsive to promotional effects of TPA. These results imply that the cells involved in the formation of cystic squamous epithelial cell lesions are already initiated and possess a potential for autonomous benign growth.

Animals↗

PubMed1993

In vitro induction of type II pneumocyte-related differentiation in a clonal fetal bronchiolo-alveolar epithelial cell line (M3E3/C3).

The aim of the present study is to investigate the differentiation of a cloned fetal Syrian hamster lung epithelial cell line, M3E3/C3, to assume morphological and biochemical features of Type II pneumocytes (phospholipid synthesis). The use of a soft agar overlay and a differentiation medium, based on RPMI 1640 combined with hormone supplements, increased the cellular content of phosphatidylcholine (PC) from 48.6% in the conventional culture without any of these factors (referred to as 'control') to 64.7% (p < 0.02). The other cell membrane-associated components, phosphatidylethanolamine (p < 0.05), sphingomyelin (p < 0.001), phosphatidylserine (n. s.), phosphatidic acid (p < 0.02) and phosphatidylinositol (p < 0.02) decreased. The content of phosphatidylglycerol showed no essential change (from 11.2% to 8.4%) and the content of disaturated phospholipids decreased from 32.0 to 23.4 micrograms/10(6) cells (p < 0.002). The phospholipid pattern of these differentiated cells is in rough accordance with that of primary isolated Type II pneumocytes. They incorporated 3H-choline over a period of four hours at a higher rate in the Type II pneumocyte-specific phospholipids, PC and dipalmitoyl-phosphatidylcholine (DPPC), than the undifferentiated control. The radiolabelling of PC and DPPC in the differentiated cells, after 3 hours of incubation with 3H-choline, was about 3.2-fold and 2.2-fold, respectively, higher than that in the control cells (p < 0.001). Intracytoplasmatic phospholipid granules were evident in the differentiated cells by light and fluorescence microscopy (modified Papanicolaou stain, Phosphin 3 R fluorescence). Furthermore, the differentiated cells had a high activity of alkaline phosphatase, whereas the control cells showed only little activity of this enzyme. Ultrastructurally, many concentric multilayered osmiophilic bodies, well developed Golgi apparatuses and many cytoplasmic protrusions comparable to microvilli, were detectable in the cuboidal shaped differentiated cells. The control cells remained wide and flattened on the plastic surface and produced a fibrillar extracellular matrix. In the simultaneously studied fetal lung fibroblasts none of these specific features were noted. These results indicate a specific differentiation capacity of the clonal fetal cell line, M3E3/C3, by closely resembling Type II pneumocytes.

Animals↗

PubMed1992

Bioassay of a tracheal smooth muscle-constricting factor released by respiratory epithelial cells.

Epithelium-derived factors of unknown identity have been proposed to modulate airway smooth muscle tone. We developed a novel sensitive bioassay system that allows serial perfusion of cultured respiratory epithelial cells and guinea pig trachea (GPT). GPT responses were assessed as diameter changes by computerized video microscopy (resolution, 15 microns). A permanent hamster lung epithelial cell line was grown on microcarrier beads and perfused in a cell column. When the outflow tubing from the epithelial cell column was connected to the inflow cannula, the detector GPT contracted, reaching 28 +/- 6% of the maximum methacholine (100 microM)-induced contraction (n = 12, P less than 0.001). Perfusion of the cell column with diclofenac (10 microM) or lysin-mono-acetylsalicylic acid (100 microM) abolished the GPT contraction, whereas selective perfusion of the detector GPT with either agent did not block the contraction. Analysis of the effluent of the epithelial cell column demonstrated a significant basal release of prostaglandins F2 alpha and E2 (PGF2 alpha and PGE2) and 6-ketoprostaglandin F1 alpha, whereas only marginal amounts of thromboxane B2 were detected. When given exogenously, PGF2 alpha, PGE2, PGI2, and U-46619 all contracted the GPT. It is concluded that lung epithelial cells can contract GPT by releasing a transferable factor. This factor is likely to be a constrictor cyclooxygenase product that is not produced in epithelium-denuded GPT.

Animals↗

PubMed1991

Secretory differentiation and cell type identification of a human fetal bronchial epithelial cell line (HFBE).

A human fetal bronchial epithelial cell line (HFBE) grew in an undifferentiated pattern under conventional culture conditions. Despite a somewhat fibroblastic shape the cells maintained immunoreactivity to cytokeratin, carcinoembryonic antigen and epithelial membrane antigen. When grown on a collagen gel in a growth-hormone-supplemented medium, their spindle shape became more conspicuous. With an additional supplement of vitamin A (6 micrograms/ml), most of the cells underwent differentiation by producing many bright inclusion bodies which proved to be strongly positive with periodic acid-Schiff and weakly positive with alcian blue staining. Electron microscopy revealed a well-developed rough endoplasmic reticulum, an enlarged Golgi apparatus and many highly electron-dense secretory granules resembling those of Clara cells. Biochemical analysis demonstrated that HFBE cells cultured on collagen gel with vitamin A secreted hyaluronic acid and neutral glycoproteins containing mainly N-linked glycoproteins whose glycans were of a complex type. A monoclonal antibody (SEC-41) generated against the neutral glycoproteins detected a glycoprotein of approximately 52 kDa in the spent culture medium of differentiated HFBE cells. This antibody also reacted with the intracytoplasmic secretory granules in these cells. When tested on frozen sections of lung tissue, the immunohistochemical reactivity of the SEC-41 antibody was confined to Clara cells, some type II pneumocytes in the adult lung, and respiratory epithelial cells in the fetal lung. Moreover, this antibody could detect secretory glycoprotein in broncho-alveolar lavages from two patients. This paper clearly demonstrates that cells derived from human fetal bronchial epithelium can be cultivated in an undifferentiated precursor state and, under appropriate culture conditions, can be stimulated to undergo differentiation into a Clara cell type.

Bronchi↗

PubMed1991

Occurrence of sister-chromatid exchange and chromosomal aberration during vitamin A-induced cell differentiation in vitro.

Prompted by the recent growth in interest in the mechanisms of vitamin A (VA) action, we studied the effects of VA on the frequency of sister-chromatid exchange (SCE) and chromosome aberration (CA) in a culture system using a fetal Syrian hamster (female) pulmonary epithelial cell line (M3E3/C3). When manipulated by specific culture conditions, the cells in this system could be rendered competent for activation of polycyclic aromatic hydrocarbons. Cells induced to such a state were exposed to 0, 2, 8 and 24 micrograms/ml of VA for 4 days. The average frequency of SCE per metaphase increased from 1.64 at 0 micrograms/ml to 3.44 at 24 micrograms/ml with a moderate degree of dose dependence. In addition, the q-terminal area of X-chromosomes appears to be one of the most specifically vulnerable sites for SCE due to VA. The frequency of CA encompassing triradial, quadriradial, quinqueradial, ring and dicentric chromosomes also increased in a rather sigmoid fashion from 3.6% at 0 micrograms/ml to 14.8% at 24 micrograms/ml. Apart from the frequently demonstrated protective roles or otherwise less often encountered promotional effects of VA in the development of squamous metaplasia, neoplasia, neoplastic transformation or mutation, an alternative interpretation for the current results implies a possible relationship between SCE and CA caused by VA and cell differentiation and/or drug resistance mechanisms.

Animals↗

PubMed1991

Altered actin organization in an N-methyl-N'-nitro-N-nitrosoguanidine-transformed pulmonary epithelial cell line.

After in vitro-induced neoplastic transformation by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), altered actin organization in pulmonary epithelial cells was examined. A certain correlation was found between anchorage independency (AI), tumorigenicity in nude mice and altered actin organization. DNase I inhibition assay demonstrated significant loss of detergent-insoluble F-actin (stress fibers, p less than 0.02) and soluble F-actin (single filaments, p less than 0.0001) after AI transformation in comparison with untransformed cells, while the level of G-actin increased significantly (p less than 0.0001). At the same time, fluorescence and electron microscopy also revealed that after AI transformation there was a striking loss of stress fibers usually accompanied by reorganization of at least some of the lost stress fibers into F-actin aggregations. After s.c. implantation of AI-transformed cells into nude mice followed by recultivation of the developed tumors, DNase I-inhibition assay showed a significant increase (p less than 0.001) in the level of detergent-insoluble F-actin as compared with untransformed cells, but no significant difference in the amount of G-actin. However, most of this increased detergent-insoluble F-actin was in the form of aggregations as revealed by immunofluorescence and electron microscopy. This growth behaviour-dependent alteration in actin organization occurring after exposure to MNNG may be causally related to the progressive development of neoplastic phenotypes, although the biological significance of actin aggregation formation remains unclear. The results have also pointed out the importance of parallel investigations into both the biochemical and morphological statuses of actin, particularly when it may be regarded as an indicator of neoplastic transformation and malignancy.

Actins↗

PubMed1991

Induction and characterization of secretory differentiation in human fetal bronchial epithelial cell line (HFBE) cultured on collagen gel in growth hormone and vitamin A-supplemented medium.

A type of secretory differentiation was induced and characterized in a human fetal bronchial epithelial cell line (HFBE), which was grown on a collagen substratum in a basal differentiative medium (BDM) containing growth hormones and with supplementation of various concentrations of vitamin A (VA). HFBE cells grown on a collagen gel in BDM with or without VA assumed a spindle shape with thick cytoplasm arranged in strands running parallel to each other. Under a phase-contrast microscope, cells cultured in the absence of VA possessed a small number of bright inclusion bodies, which proved to be positive to PAS and almost negative to alcian-blue (AB) staining. Electron microscopy revealed well-developed rough endoplasmic reticulum (rER), enlarged Golgi apparatus and a small number of high-density granules resembling serous or Clara cell granules. HFBE cells treated with VA at levels higher than 6 mu/ml showed a remarkable increase of the secretory granules and contained amorphous material in the rER. Addition of a low concentration of VA (6 ng/ml) only stimulated the growth of HFBE cells. In contrast, higher concentrations of VA significantly inhibited the growth and 3H-thymidine incorporation into DNA in a dose-dependent manner. HFBE cells cultured on collagen gel with VA secreted products with 2 different molecular weights into the medium. A high molecular weight-product, consisting of void volume fractions from a Bio-gel A 15-m column, was identified as hyaluronic acid based on the results obtained from the DEAE-ion exchange chromatography and specific enzymatic digestion. A low molecular weight-product fractionated on the A 15-m was tentatively identified as mainly neutral glycoproteins containing N-linked glycans. While the secretion of hyaluronic acid was inhibited by VA in a dose-dependent manner, the secretion of the neural glycoproteins was most enhanced by VA in the range from the physiological concentration of 600 ng/ml to 6 micrograms/ml. These biochemical data on the secretory products, together with the morphological findings, demonstrate that the HFBE cell line serves as a new model for investigating the cellular differentiation of human lung epithelium.

Bronchi↗

PubMed1991

Monoclonal antibodies against merokeratin from sheep's wool.

Four monoclonal antibodies designated as AMK-6, AMK-10, AMK-16 and AMK-27, were raised against merokeratin prepared from sheep's wool, and their cross-reactivity to epithelial cytokeratins were examined using immunoblot analysis and immunohistochemistry. AMK-16 which was specific to merokeratin in immunoblot analysis, demonstrated specific immunohistochemical staining of wool fibers of the sheep's skin. The other 3 monoclonal antibodies (AMK-6, -10, and -27) showed cross-reactivity towards epithelial cytokeratins with several molecular weights when examined by immunoblot analysis. However, each immunohistochemical staining of the sheep's skin with these monoclonal antibodies appeared to be cell type-specific. These results indicate that merokeratin and certain epithelial cytokeratins share many antigenic epitopes in common, and these monoclonal antibodies against merokeratin from sheep wool will be useful for the analysis of wool-forming cell differentiation.

Animals↗

PubMed1990

Unscheduled DNA synthesis and HPRT mutation in fetal Syrian hamster and human respiratory epithelial cells exposed to ethylnitrosourea.

We previously reported that the chromosomes of fetal Syrian hamster respiratory epithelial cells were less stable toward ethylnitrosourea (ENU) than those of comparable human cells. Following this, we compared the sensitivity of genetic materials of the same cell systems to the same mutagen in terms of unscheduled DNA synthesis (UDS) and mutation at HPRT locus (HPRT-). UDS occurred 5 (with 0.1 mg ENU/ml) to 7 (with 0.4 mg ENU/ml) times more frequently in the hamster cells than in the human cells. This much lower UDS frequency in human cells cannot be solely explained by the fact that the human cells possess only a moderately larger (1.6 to 2.9 times) size of intracellular deoxythymidine triphosphate (dTTP) pool than the hamster cells. This finding would thus indicate that the hamster cells actually carry out DNA repair, whether correct or aberrant, more often than the human cells. Moreover, HPRT- was also 5 (at 0.4 mg/ml) to 26 (at 0.8 mg/ml) times more frequent in the hamster cells than in the human cells. Therefore, the current results suggest that the DNA repair mechanisms of the hamster cells are less accurate and more unstable than those of the human cells. Our previous findings with regard to the chromosomal stability give support to this hypothesis.

Animals↗

PubMed1990

New functional cell-culture approach to pulmonary carcinogenesis and toxicology.

Modern pulmonary toxicology (including lung carcinogenesis) has, to assist its rapid development, constantly incorporated the knowledge obtained through cell and tissue-culture studies. While this has been carried out in rather a passive manner until quite recently, the currently necessary multi-disciplinary approach increasingly requires more active involvement of cell/tissue-culture techniques in this area. Our understanding in this regard is that one of such requirements is to establish a cell-culture system consisting of a single population of possible target cells for certain classes of hazardous inhalants. In addition, such target cells in culture should be able to function in a manner as closely resembling the situation in vivo as possible. In view of the culture techniques presently available, this requirement is probably too ideal to be met immediately. Nevertheless, efforts have been made in the last decade to achieve functioning cultures of Clara cells, type II pneumocytes or small mucus granule cells (SMGC), using undifferentiated cells obtained from animal and human fetuses. This attempt forms a sharp contrast to the usual approach, in that while the latter tries to keep the functions of adult cells in an already differentiated state, the former aims at inducing functional differentiation in undifferentiated cells by manipulating culture conditions. In carrying out these efforts, we have shown clear evidence that the type II pneumocytes and Clara cells induced in vitro are closely cognate and share a common precursor cell in culture, and that SMGC are at a pre-stage of differentiation to Clara cells. We have also shown an induced capacity for xenobiotic activation and conjugation in SMGC in culture. Our next plan is to prove similar activity (of mixed-function oxidase) in Clara cells and type II pneumocytes induced to differentiate in culture.

Animals↗

PubMed1990

Secretory differentiation of human fetal bronchial epithelial cells in culture. A study by histochemistry and electron microscopy.

Human fetal bronchial epithelial (HFBE) cells at 6-8 passages were cultivated on a collagen gel for 10 days. A basal differentiative medium (BDM), consisting of RPMI 1640 supplemented with hormones and growth factors, was employed. Histochemistry, scanning electron microscopy and transmission electron microscopy revealed that HFBE cells developed secretory granules when cultivated on collagen gel in BDM. They were electron-dense and stained positive for PAS but negative for alcian blue. On additional treatment with 8 micrograms/ml vitamin A (VA), the number of secretory granules was increased. Moreover, the HFBE cells lost their surface microvilli, and dilation of rough endoplasmic reticulum was more marked than in culture without VA.

Bronchi↗

PubMed1990

In vitro production of B cell growth factor and B cell differentiation factor by peripheral blood mononuclear cells and bronchoalveolar lavage T lymphocytes from patients with idiopathic pulmonary fibrosis.

The activation of B lymphocytes and formation of immune complexes have been suggested to play an important role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). To investigate the mechanisms of activation of B lymphocytes, we studied the production of B cell growth factor (BCGF) and B cell differentiation factor (BCDF) in patients with IPF and those with interstitial pneumonia associated with collagen vascular diseases (IP-CVD), in comparison with healthy controls. Culture supernatants of peripheral blood mononuclear cells from patients with IPF induced more IgM and IgA production by B lymphocytes than those from healthy controls, indicating a higher production of BCDF in the patients. Culture supernatants of T lymphocytes obtained from bronchoalveolar lavage fluids (BALF) of patients with IPF induced higher proliferation of B lymphocytes than those from healthy controls, indicating a higher production of BCGF. An increase in production of BCGF and BCDF was not observed in patients with IP-CVD. In the light of these results, it was suggested that there may be an imbalance in T lymphocyte subsets that release lymphokines like BCGF and BCDF in patients with IPF, and that the subsets may differ between blood and BALF. It remains to be elucidated whether the activation of B lymphocytes depending on T lymphocytes determines the development of disease in IPF.

Adult↗

PubMed1990

[Animal experiment and in vitro studies with inhaled environmental pollutants].

Risk assessment of inhaled toxicants is a critical treatise in inhalation toxicology. In particular, the extent of extrapolation from animal experiments to the human situation is frequently influenced by such factors as the method of application (Implantation, instillation or inhalation) and species-specific differences. This difficulty becomes especially conspicuous when the carcinogenicity is to be determined after the inhalation of, for example, diesel engine exhaust, coal oven flue gas, or cadmium compounds. Nowadays, in-vitro techniques are also available for successfully investigating the metabolism of hazardous compounds and thereby-induced DNA damage in animal and human airway epithelial cells; results gained through the employment of these techniques will, at all events, bear increasing weight in terms of the valuation and assessment of risks to humans.

Air Pollutants↗