Biomedical subjects

N Sawada

Publications and source records attributed to N Sawada.

At least 127 records · Page 7Linked to original sources

PubMed1993

Monoclonal antibody 7H6 reacts with a novel tight junction-associated protein distinct from ZO-1, cingulin and ZO-2.

The tight junction is an essential element of the intercellular junctional complex; yet its protein composition is not fully understood. At present, only three proteins, ZO-1 (Stevenson, B. R., J. D. Siliciano, M. S. Mooseker, and D. A. Goodenough. 1986. J. Cell Biol. 103:755-766), cingulin (Citi, S., H. Sabanay, R. Jakes, B. Geiger, and J. Kendrick-Jones. 1988. Nature (Lond.). 333:272-275) and ZO-2 (Gumbiner, B., T. Lowenkopf, and D. Apatira. 1991. Proc. Natl. Acad. Sci. USA. 88:3460-3464) are known to be associated with the tight junction. We have generated a monoclonal antibody (7H6) against a bile canaliculus-rich membrane fraction prepared from rat liver. This 7H6 antigen was preferentially localized by immunofluorescence at the junctional complex regions of hepatocytes and other epithelia, and 7H6-affiliated gold particles were shown electron microscopically to localize at the periphery of tight junctions. Immunoblot analysis of a bile canaliculus-rich fraction of rat liver using 7H6, anti-ZO-1 antibody (R26.4C), and anti-cingulin antibody revealed that 7H6 reacted selectively with a 155-kD protein, whereas R26.4C reacted only with a 225-kD protein. Anti-cingulin antibody reacted solely with 140 and 108-kD proteins, indicating that the protein recognized by 7H6 is immunologically different from ZO-1 and cingulin. Immunoprecipitation of detergent extracts obtained from metabolically labeled MDCK cells with R26.4C coprecipitated a 160-kD protein, which corresponds to ZO-2, with ZO-1. However, 7H6 did not react with the 160-kD protein. These results strongly suggest that the 7H6 antibody recognizes a novel tight junction-associated protein different from ZO-1, cingulin and ZO-2.

Animals↗

PubMed1993

High sensitivity of LEC rats with chronic hepatitis to hepatocarcinogenesis: decreases in unscheduled and replicative DNA synthesis of the hepatocytes.

We carried out the following three experiments to clarify the mechanism of hepatocarcinogenesis in Long-Evans Cinnamon (LEC) rats. (1) Sensitivity to diethylnitrosamine (DEN): LEC rats (8 and 25 weeks old) without and with hepatitis and age-matched F344 rats were administered an intraperitoneal injection of a low dose of DEN. Eight weeks after the injection, the numbers of glutathione-S-transferase placental-form (GST-P)-positive foci in the 33-week-old LEC rat liver were significantly higher than those in the livers of the other three groups of rats. (2) Potential for unscheduled DNA synthesis (UDS): Isolated hepatocytes of 25-week-old LEC rats with chronic hepatitis showed about one-third the level of UDS induced by UV irradiation, as compared to that of age-matched F344 rats, while no significant difference was found between the UDS of isolated hepatocytes of 8-week-old LEC rats and age-matched F344 rats. (3) Potential for proliferation: Isolated hepatocytes from 8-week-old LEC rats responded well to epidermal growth factor (EGF) in culture, to almost the same degree as F344 rat hepatocytes, while a remarkable decrease in the responsiveness of hepatocytes isolated from 25-week-old LEC rats to EGF was found. These results suggested that LEC rat hepatocellular carcinoma could be naturally initiated after the onset of hepatitis by carcinogens contaminating food and the environment, probably due to the reduction of DNA repair activity, after which initiated hepatocytes selectively proliferate in response to growth stimuli endogenously produced as a result of continuous loss of hepatocytes (chronic hepatitis), because of a decrease in growth activity of non-initiated hepatocytes.

Animals↗

PubMed1993

Establishment and characterization of a simian virus 40-immortalized osteoblastic cell line from normal human bone.

We established a human osteoblastic cell line immortalized by simian virus 40 (SV40) in vitro, and designated it SV-HFO. Immunocytochemically, the cells were positive for SV40 large T-antigen, vimentin and osteocalcin, but negative for keratin and epithelial membrane antigen. The cells had characteristic morphologic and ultrastructural features of osteoblasts, produced alkaline phosphatase, and synthesized osteocalcin, the levels of which were elevated by treatment of the cells with 1a,25-dihydroxyvitamin D3. The cells proliferated and showed such osteoblastic properties even under serum-free conditions. The cells grew in soft agar, but did not form tumors when transplanted into athymic nude mice. Karyotypic analysis by the Q-banding technique showed that these cells were of human origin. The SV-HFO cell line is expected to serve as a suitable model for studying metabolism and carcinogenesis in human bone.

Alkaline Phosphatase↗

PubMed1993

Establishment of a novel monoclonal antibody, SE-1, which specifically reacts with rat hepatic sinusoidal endothelial cells.

A monoclonal antibody, SE-1, which specifically reacted with rat hepatic sinusoidal endothelial cells (HSE), was established with elutriator-separated HSE as immunogen. No crossreactivity was observed with other types of endothelial cells or non-endothelial cells. By immunoelectron microscopy, the antigen recognized by MAb SE-1 was localized to the membrane surface of HSE. Immunoblot analysis revealed that SE-1 recognized a single 45 KD band. Although the SE-1 antigen is not yet characterized, the above evidence strongly suggests that it is related to the specific function of HSE. Therefore, SE-1 may be a useful marker to study the role of HSE in various pathophysiological conditions of the liver.

Animals↗

PubMed1993

Human mammary epithelial cells undergo squamous differentiation in serum-free three-dimensional culture upon loss of growth activity.

Normal human mammary epithelial cells (HMEC) isolated from surgically resected breast tissues were cultured under serum-free conditions using MCDB 170 medium. With the increase in the number of passages, in particular after the 5th passage, the number of enlarged, flattened and vacuolated cells increased while cell proliferation decreased. The senescent cells occasionally had keratohyaline granules in the cytoplasm and were positive in immunohistochemistry for keratinizing squamous epithelium-specific cytokeratin 10. When HMEC were cultured between floating double-layered collagen gels, the cells lost growth activity, showed marked stratification, and became positive for carcinoembryonic antigen (CEA). The stratified cells underwent squamous differentiation and tonofilament bundles appeared around the nuclei. The stratification and squamous differentiation of HMEC were observed within seven days after transfer to the three-dimensional culture, regardless of the number of passages. These results indicate that the HMEC in vitro ultimately differentiate into squamous epithelia and also that there is a close relationship between the squamous-type differentiation and the loss of cell proliferation.

Breast↗

PubMed1993

Relationship between the expression of the gap junction protein and osteoblast phenotype in a human osteoblastic cell line during cell proliferation.

We examined i) the kinds of connexins, component proteins of gap junctions, that are expressed in osteoblasts and ii) the relationship between the expression of gap junctions and osteoblastic phenotype during cell proliferation and after the treatment with 1 alpha,25-dihydroxyvitamin D3. Human osteoblastic cells with (SV-HFO) or without (HFO) transformation by simian virus 40 and mouse osteoblast-like cells (MC3T3-E1) expressed connexin 43 (Cx43), but not Cx26 or Cx32, as revealed by Northern blot analysis and immunocytochemistry. The expression of Cx43 was significantly higher in SV-HFO cells in the confluent phase than in the proliferative phase. Similarly, the expression of alkaline phosphatase (ALP) and osteocalcin in SV-HFO cells in the confluent phase were higher than those in the proliferative phase. On the other hand, treatment of 1 alpha,25-dihydroxy-vitamin D3 did not change the expression of Cx43 in SV-HFO cells, but significantly induced the expression of ALP and osteocalcin. These results showed that the expression of gap junction protein in osteoblastic cells was coupled with cell differentiation in association with the expression of osteoblastic phenotype, but that the connexin expression is regulated in a way different from that of ALP and osteocalcin.

Alkaline Phosphatase↗

PubMed1993

[Cytotoxicity of anaerobic bacteria isolated from infected root canal].

Bacteria in the infected root canal play an important role in the progression of the periapical lesion. The purpose of the present study was to investigate the cytotoxic effects of bacteria isolated from an infected root canal on the periapical lesion. Four obligately anaerobic bacteria were isolated and their sonic extracts (SE) were prepared. The cytotoxic assay of each SE for MC3T3-E1 cell was used with respect to the morphological cell changes and [3H]-thymidine uptake. The following results were obtained: 1. Four obligately anaerobic bacteria were identified as follows: Porphyromonas asaccharolytica (P. a.), Fusobacterium nucleatum (F. n.), Eubacterium lentum (E. l.), and Peptostreptococcus micros (P. m.). 2. The protein concentrations which induced the morphological cell changes differed in each SE. The SE from P. m. caused cell abnormalities at 10 micrograms protein/ml, F. n. and E. l., at 30 micrograms protein/ml, and P. a., at 100 micrograms protein/ml. 3. The inhibition of the incorporation of [3H]-thymidine into DNA was concentration-dependent. The SE of P. a. showed less cytotoxicity than the others at lower concentrations; 3, 10, 30 micrograms protein/ml. The SE from F. n. had more cytotoxicity at higher concentrations; 100, 300 micrograms protein/ml. These results suggest that these bacterial sonic extracts have a potential to cause morphological cell changes and inhibit the cell proliferation in different concentrations.

Adult↗

PubMed1992

Evidence for fluid-phase pinocytosis of extrahepatic bile duct cells isolated from normal rats in culture.

In order to elucidate the physiological function of extrahepatic bile duct cells, we isolated epithelial cells from the rat extrahepatic bile duct by digesting resected segments of the extrahepatic bile duct with 0.15% trypsin in ice-cold Ca(2+)-free Hanks' balanced salt solution supplemented with 0.25 mM EDTA overnight. As a result, the epithelial cells were collected as aggregates and attached to culture dishes coated with type I collagen. Approximately 95% of the cells cultured for 24 hrs were found to be positive for gamma-glutamyl transpeptidase and cytokeratin-19, but negative for vimentin. These characteristics were identical to the features of rat extrahepatic biliary epithelial cells in situ. Ultrastructurally, the cells were long and columnar in configuration on the 2nd day in culture, and possessed numerous microvilli at the apical surface and well-developed junctional complexes at the lateral surface. These findings also indicate that the cells maintain an epithelial nature and are morphologically polarized. When the cells were exposed to a low dose of horseradish peroxidase (HRP) on the 2nd day in culture, which was followed by fixation and treatment with 3-3'-diaminobenzidine, HRP was found preferentially in the cytoplasmic vesicles near the apical surface. HRP was then observed in the intercellular spaces; however, the electron-dense tracer, ruthenium red, did not permeate into the intercellular spaces, and HRP was found in neither cytoplasms nor intercellular spaces when the cells were incubated in HRP-containing medium at 4 degrees C for 30 min. These results suggest that the extrahepatic bile duct epithelial cells are involved in the reabsorption of bile constituents.

Animals↗

PubMed1992

[Detection and identification of pathogenic bacteria by polymerase chain reaction with primers from DNA sequence of ribosomal RNA].

Applicability of the polymerase chain reaction method for identification of pathogenic bacteria was examined with the primers synthesized from the ribosomal RNA gene sequence containing both homologous and species-specific regions of bacterial species from Mycoplasma to Mycobacteria. Two out of the nine sets of promoters prepared, each covering about 650 nucleotides spanning from 16S RNA to 23S RNA regions, produced the corresponding DNA fragments from all the strains tested, and another set did so from all species but Mycoplasma. This method enabled one to detect and identify E. coli in a sample containing 2 x 10(2) CFU. The restriction enzyme patterns of the PCR products obtained with Hae-III, Hha-I, Mbo-I, Msp-I, Rsa-I and Taq-I were so characteristic as to differentiate one species from another. Ten strains of E. coli showed identical restriction patterns and 10 of S. aureus also showed identical patterns indicating that the restriction pattern is species-specific. The method may be applicable to detection and identification of a certain species bacteria which are suspected to be consealed in water or food samples, or clinical specimens, especially when the consealed bacterial genus or species can not be predicted.

Base Sequence↗

PubMed1991

Dealkylation rates of O6-alkyldeoxyguanosine, O4-alkylthymidine and related compounds in an alkyl-transfer system.

Bacterial O6-alkylguanine-DNA alkyltransferase (AGT) removes alkyl group from O6-alkylguanine and O4-alkylthymine residues in DNA, both of which are considered to be DNA damages most related to the induction of cancer and/or mutation. The repair process involves alkyl-transfer of an O-alkyl group to the active site of the enzyme, where an SH-group of cysteine residue plays the role of alkyl acceptor. In order to elucidate the chemical characteristics of substrates for this enzyme, dealkylation rates of O6-alkyldeoxyguanosine, O4-alkylthymidine and related compounds were measured using an alkyl-transfer system. Thiophenol-triethylamine system was employed as an alkyl acceptor and twenty-one O-alkyl compounds were tested. Dealkylation proceeded with pseudo first order kinetics. The half-life of O6-methyldeoxyguanosine (MedG) was 122 h and no remarkable dependence on N-9 substituents (H, CH3 and deoxyribose) was observed. A compound lacking 2-NH2 group underwent demethylation about three times faster than O6-methylguanines did, while, a compound lacking imidazole moiety underwent demethylation about 2.5 times more slowly. The half-life of O4-methylthymidine (MedT) was 38 h and no remarkable dependence on N-1 (H, CH3 and deoxyribose) and C-5 (H and CH3) substituents was observed. Deethylation proceeded much more slowly than demethylation. Substitution of selenophenol for thiophenol resulted in a 4.5 times faster MedG demethylation rate. Demethylation rates were moderately correlated with values for NMR chemical shift of CH3 group, an indicator of electron density, although the correlation curves of a series of MedG and MedT derivatives were quite different. This result suggests that some different rate-determining factors other than electron density are playing a role. These findings may be of help in resolving the details of the mechanisms of enzymic repair by bacterial and mammalian AGT.

Alkylation↗

PubMed1991

Collagen-gel-embedded three-dimensional culture of human thyroid epithelial cells: comparison between the floating sandwich method and the dispersed embedding method.

Human thyroid epithelial cells were isolated from surgically resected human thyroid gland with collagenase and cultured for one week under EGF-supplemented conditions to allow them to proliferate. Then the cells were transferred to the following three-dimensional culture systems. One was a culture of isolated cells between floating double layers of collagen gel, designated the "floating sandwich method." The other was a culture of isolated cells mixed with collagen gel, designated the "dispersed embedding method." Many folliclelike structures with lumina of appreciable size were obtained by the former method. The cells cultured by the floating sandwich method exhibited a distinct polarity shown by the presence of numerous microvilli at the apical surface and close contact with collagen gels at the basal surface. On the other hand, only a few folliclelike structures were obtained by the dispersed embedding method, in which the folliclelike structures were small in size and the cells showed less distinct polarity than those observed in the floating sandwich method. Thus, the floating sandwich method appears to be suitable for studying the process and mechanism of in vitro organization of follicular structures by human thyroid epithelial cells.

Cell Division↗

PubMed1991

Formation of O6,7-dimethylguanine residues in calf thymus deoxyribonucleic acid treated with carcinogenic N-methyl-N-nitrosourea in vitro.

Treatment of calf thymus deoxyribonucleic acid (DNA) in vitro with a methylating carcinogen, N-methyl-N-nitrosourea (MNU), in phosphate buffer (pH 7.2) resulted in formation of O6,7-dimethylguanine residues in DNA besides the well-known methylated DNA adducts, 7-methylguanine, O6-methylguanine and 3-methyladenine. The product ratio (%) of O6,7-dimethylguanine versus 7-methylguanine was 0.32 after one MNU treatment. The significance of formation of O6,7-dimethylguanine residues in DNA is discussed briefly in relation to the carcinogenicity of MNU.

Animals↗

PubMed1990

Decreased activities of S-adenosylmethionine synthetase isozymes in hereditary hepatitis in Long-Evans rats.

Isozyme patterns of S-adenosylmethionine synthetase have been measured with or without dimethylsulfoxide in liver of LEC rat hereditary hepatitis. The activities of the alpha- and beta-forms are decreased with age after birth, and decreased to a half level of 36 weeks after birth. Concentration of S-adenosylmethionine in the liver is almost a half level of control rat. However, the activity of glycine- and tRNA-methyltransferases in the liver shows no significant change.

Animals↗

PubMed1990

The effect of choline and methionine deficiencies on the number and volume percentage of altered hepatic foci in the presence or absence of diethylnitrosamine initiation in rat liver.

The ability of methyl-deficient, amino-acid-defined diets to produce enzyme-altered foci was quantitatively determined in the livers of rats treated both with and without an initiating dose of diethylnitrosamine (DEN). Male weanling F-344 rats were fed a complete, amino-acid-defined diet for 1 week. They were then injected i.p. with a single dose of DEN (20 mg/kg body weight) and fed the complete diet for an additional week. Forty animals in each dose group were then maintained for 5-38 weeks on the complete diet (diet 1) or one of the three methyl-deficient diets customarily used in this laboratory: diet 2, devoid of methionine and choline; diet 3, devoid of methionine only; and diet 4, devoid of choline only. In diets 2 and 3, methionine was replaced by equimolar amounts of its metabolic precursor, DL-homocystine. Ten animals per group were killed 8, 12, 17, 24 and 41 weeks after DEN initiation. For 2 weeks prior to being killed, each group was maintained on the complete diet to minimize the histological abnormalities due to acute toxicity of the diets. Serial sections of the livers were obtained, stained sequentially for gamma-glutamyltranspeptidase, ATPase and glucose-6-phosphatase, and the quantitation of the focal lesions scored by these markers was carried out by quantitative stereology. The results indicated that, regardless of the enzyme marker(s) examined, there was a general correspondence between the volume and number of altered hepatic foci (AHF) formed and the previously described tumor-promoting activities of each diet. Thus, while all DEN-treated groups contained significant numbers of AHF 24 weeks after initiation, only the diet-2-fed animals displayed such foci at 8 weeks. Similarly, among the uninitiated rats, only those fed diet 2 exhibited the presence of AHF throughout the experimental period. Interestingly, the livers of uninitiated, choline-deficient rats showed a small number of AHF at 24 and 42 weeks; these foci were not observed at all in the corresponding DEN-untreated animals fed diet 3, deficient in methionine only. The results provide evidence that the carcinogenic effects of the methionine- and choline-deficient diet result more from its strongly promoting effect than from any initiating activity by the diet.

Animals↗

PubMed1990

[Malignant pheochromocytoma presenting as an acute abdomen: a case report].

A case of right adrenal malignant pheochromocytoma was reported. The patient was a 16-year-old boy who complained of severe right side abdominal pain due to spontaneous hemorrhage into the retroperitoneal space. Right adrenalectomy was performed on Feb. 22, 1988 but the preoperative high serous catecholamine level did not drop to the normal level. Postoperatively bone and liver metastasis were detected by a 131I-MIBG scan. He was treated with a combination chemotherapeutic regimen consisting of cyclophosphamide, vincristine and dacarbazine in 2 repeated cycles but there was no effect. He died 5 months after the operation.

Abdomen, Acute↗

PubMed1989

Immortal epithelial cells of normal C3H mouse liver in culture: possible precursor populations for spontaneous hepatocellular carcinoma.

Long-term culture of primary hepatocytes derived from normal young male C3H/HeNJclMTV+ (C3H) and C57BL/6NJcl (C57) mice, respectively known for very high and low incidences of spontaneous hepatoma, resulted in development of multiple slowly growing epithelial colonies in the C3H case, the number of colonies being increased five-fold when 1.5 mM phenobarbital was added to the culture medium. On the other hand, the primary culture cells from C57 mouse liver gave rise to such epithelial colonies only very rarely, even with phenobarbital. Immunohistochemical investigation revealed alpha-fetoprotein and/or albumin production by the colony cells and ultrastructural analysis also revealed some hepatocytic features in them. Subculturing of individual colonies gave rise to cell lines which could be repeatedly passaged. Two of five lines implanted into athymic nude mice manifested tumorigenicity, the resultant neoplasms being diagnosed as a trabecular hepatocellular carcinoma and an adenocarcinoma. The experimental data suggest that the colony-forming immortal epithelial cells possibly represent early phase precursors of spontaneous mouse hepatocellular carcinomas. This culture system is expected to be useful for future elucidation of the mechanisms underlying spontaneous mouse hepatocarcinogenesis.

Animals↗

PubMed1989

H-ras gene is expressed at the G1 phase in primary cultures of hepatocytes.

The expression of c-H-ras and proliferating cell nuclear antigen (PCNA) in primary cultures of rat hepatocytes was determined in order to elucidate the relationship between the c-H-ras gene and the S phase of the cell cycle. In cells treated with EGF, elevation of c-H-ras expression was detected at the 22nd, 34th, 44th, and 54th h after plating, PCNA expression and DNA synthesis were detected at the 44th and 54th h. In cells without EGF treatment, only c-H-ras expression was detected at the 44th and 54th h. In our previous report, we showed that c-myc expression increased within several hours after plating, suggesting that isolated hepatocytes traverse from G0 to G1 under culture conditions, regardless of EGF treatment. These results clearly showed that the c-H-ras gene of adult rat hepatocytes was expressed in the mid-to-late G1 phase of the cell cycle as well as in the early S phase in primary culture.

Animals↗

PubMed1989

Hepatocytes from old rats retain responsiveness of c-myc expression to EGF in primary culture but do not enter S phase.

Responsiveness of rat hepatocytes from 22- to 24-month-old animals to growth stimulation was examined in primary culture by assay of replicative DNA synthesis and c-myc expression as parameters. Old rat hepatocytes showed very low DNA synthesis in response to EGF, rat sera, or hepatectomized rat sera, as compared to 2- to 3-month-old rat hepatocytes. However, c-myc expression was induced by EGF to a similar extent in cells from both old and young rats. These results suggest that the old cells can traverse from G0 to G1 phase of the cell cycle but they somehow are blocked from entering the S phase.

Aging↗