Establishment of a highly differentiated immortalized adult human hepatocyte cell line by retroviral gene transfer.
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Adult↗
Biomedical subjects
N Kobayashi
Publications and source records attributed to N Kobayashi.
Establishment of a highly differentiated immortalized human hepatocyte cell line as a source of hepatic function in the bioartificial liver.
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Ammonia↗
Construction of a non-tumorigenic rat hepatocyte cell line for transplantation: reversal of hepatocyte immortalization by site-specific excision of the SV40 T antigen.
BACKGROUND/AIMS: Hepatocytes immortalized with a temperature-sensitive SV40 large T antigen (SV40Tag) function as well as primary hepatocytes following transplantation to reverse hepatic encephalopathy and improve survival in rodents with liver failure. The continued presence of SV40Tag in the conditionally immortalized hepatocytes may increase the risk of malignant tumor growth in transplant recipients. METHODS: We immortalized hepatocytes using a recombinant retrovirus containing the gene encoding SV40Tag flanked by loxP recombination target sites. Excision of SV40Tag from immortalized cells could then be accomplished by site-specific recombination with Cre-recombinase. RESULTS: Cells immortalized with this recombinant virus expressed SV40Tag and doubled in number every 48 h. After excision of the gene encoding SV40Tag with Cre-recombinase, cells stopped growing, DNA synthesis fell by 90%, and production of liver-specific mRNAs was either increased or became newly detectable. In addition, the morphology and epithelial cell polarity of the cells became more characteristic of differentiated hepatocytes. To determine their malignant potential, immortalized hepatocytes were transfected to express a second oncogene, activated H-ras. SV40Tag+/H-ras+-immortalized cells were capable of anchorage-independent growth and developed into tumors when injected in severe combined immunodeficiency mice. While Cre-recombinase delivery by recombinant adenovirus infection was not 100% efficient, when SV40Tag excision occurred anchorage-independent growth stopped and tumor formation in immunodeficient mice was abolished. Immortalized hepatocytes also contained the gene encoding herpes simplex virus thymidine kinase and treatment with ganciclovir produced complete regression of established tumors in mice. CONCLUSIONS: These studies extend previous work that indicates that a transplantable hepatocyte cell line could be developed for clinical use.
Animals↗
Rapid, real-time sampling of R-84760 in blood by in vivo microdialysis with tandem mass spectrometry.
A new technique involving rapid sampling of R-84760 in real-time was achieved using a combination of microdialysis (MD) and tandem mass spectrometry (MS/MS). After collecting the analyte in real-time by MD and separating it by MS/MS, the ion intensities are adapted to the data without any subsequent chromatographic separation or flow injection analysis. The R-84760 concentration was obtained from the plateau part of the ion intensities or the corrected values using an internal standard, after immersing the MD probe into the dialysis solution containing the drug for a definite time. Since contamination of the ion source was prevented by using an organic solvent for the perfused solution, it was possible to establish a stable analysis method. For an MD membrane of length 4 mm, the R-84760 concentration in saline was linear over the range 5-541.1 ng/ml (r2 = 0.9997). Moreover, the R-84760 concentration in rat whole blood was linear over the range 24.9-1868.9 ng/ml (r2 = 0.9993). As this method allowed the measurement of free drug concentration in rat blood, the analysis was also able to provide data needed for determining the protein-binding ratio. The protein-binding ratio obtained from the calibration curve in saline and rat whole blood was 87-90%, which was close to the result obtained by another analysis method. The concentration profile of R-84760 in blood as obtained by the MD-MS/MS method correlated well with the concentration profile in plasma, which was simultaneously monitored by LC-MS/MS.
Analgesics↗
Effects of imidapril on endothelin-1 and ACE gene expression in failing hearts of salt-sensitive hypertensive rats.
The renin-angiotensin system and endothelin are important regulators of the cardiovascular system. Although increased production of endothelin-1 (ET-1) is reported in patients with heart failure, the detailed mechanism remains to be determined. To elucidate the relationship between the renin-angiotensin system and ET-1 in hypertensive heart failure, we evaluated the effects of long-term treatment with imidapril, an angiotensin converting enzyme (ACE) inhibitor, on preproET-1, endothelin A receptor (ETAR), and ACE mRNA expression in the left ventricle and evaluated these in relation to myocardial remodeling in the failing heart of Dahl salt-sensitive (DS) hypertensive rats fed a high salt diet. In DS rats fed an 8% NaCl diet after the age of 6 weeks, a stage of concentric left ventricular hypertrophy at 11 weeks (DSLVH) was followed by a distinct stage of left ventricular failure with chamber dilatation at 18 weeks (DSHF). Imidapril (DSHF-IM, n = 8, 1 mg/kg/day, subdepressor dose) or vehicle (DSHF-V, n = 8) was given from stage DSLVH to DSHF for 7 weeks, and age-matched (18 weeks) Dahl salt-resistant rats fed the same diet served as the control group (DR-C, n = 8). In both groups, blood pressure was similar and significantly higher than in DR-C. Markedly increased left ventricular end-diastolic diameter and reduced fractional shortening in DSHF-V was significantly ameliorated in DSHF-IM using transthoracic echocardiography. The preproET-1, ETAR, and ACE mRNA levels in the left ventricle were significantly increased in DSHF-V compared with DR-C, and significantly suppressed in DSHF-IM compared with DSHF-V. DSHF-V demonstrated a significant increase in the wall-to-lumen ratio and perivascular fibrosis in coronary arterioles, and myocardial fibrosis, with all these parameters being significantly improved by imidapril. In conclusion, myocardial remodeling and heart failure in DS rats fed a high salt diet were significantly ameliorated by a subdepressor dose of imidapril, which may be attributable to a decrease in ET-1 mRNA expression and angiotensin II in the left ventricle.
Angiotensin-Converting Enzyme Inhibitors↗
Effect of imidapril on myocardial remodeling in L-NAME-induced hypertensive rats is associated with gene expression of NOS and ACE mRNA.
Chronically administered N(omega)nitro-L-arginine methyl ester (L-NAME) produces vascular structural changes and fibrosis of the left ventricle (LV). However, very few studies have evaluated whether the beneficial effects of angiotensin-converting enzyme (ACE) inhibitors on these myocardial remodelings are associated with local gene expression of nitric oxide synthase (NOS) and ACE mRNA in the LV. Effects of long term treatment with imidapril, an ACE inhibitor, on gene expression of endothelial-cell NOS (eNOS) and ACE mRNA in the LV and its relation to myocardial remodeling in L-NAME-induced hypertensive rats were evaluated. Fifteen male Sprague-Dawley rats were given L-NAME (60 mg/ kg/day) in drinking water for 6 weeks to induce hypertension, and then treated with imidapril (L-NAME-I, n = 8, 1 mg/kg/day, subdepressor dose), or a vehicle (L-NAME-V, n = 7) for 4 weeks. Age-matched rats (C, n = 7) served as a control group. Blood pressure in L-NAME-V and L-NAME-I was similar and significantly higher than that in C. The level of eNOS mRNA in the LV was significantly decreased in L-NAME-V compared with C, and was significantly increased in L-NAME-I compared with C and L-NAME-V. The ACE mRNA and type I collagen mRNA expression levels were significantly increased in L-NAME-V compared with C, and significantly suppressed in L-NAME-I compared with L-NAME-V. L-NAME-V demonstrated a significant increase in wall-to-lumen ratio, perivascular fibrosis, and myocardial fibrosis. These changes in the microvasculature were improved significantly by imidapril. Myocardial remodeling in L-NAME-induced hypertensive rats was significantly ameliorated by a subdepressor dose of imidapril, which may be due to an increase in local eNOS mRNA expression and a decrease in angiotensin II in the LV.
Angiotensin-Converting Enzyme Inhibitors↗
A monoclonal antibody-based enzyme-linked immunosorbent assay of ursodeoxycholic acid 3-sulfates in human urine.
Sulfation of the 3-hydroxy group is assumed to be a major metabolic route of ursodeoxycholic acid (UDCA) which is used for treating various hepatobiliary diseases. We have developed a sensitive enzyme-linked immunosorbent assay (ELISA) for determining the total amount of nonamidated, glycine- and taurine-amidated ursodeoxycholic acid 3-sulfates (UDCA 3-Suls) using a newly established monoclonal antibody. In this study, 12 kinds of antibody-secreting hybridoma clones were generated by a fusion experiment between P3/NS1/1-Ag4-1 myeloma cells and the spleen cells from a BALB/c or an A/J mouse which had been immunized with a conjugate of nonamidated UDCA 3-Sul and bovine serum albumin. One of the monoclonal antibodies, Ba-10 (gamma2a, kappa), had suitable binding properties for clinical application, which was group-specific to the UDCA 3-Suls, and showed negligible cross-reactivities with various related bile acids including potentially interfering compounds, namely, the unconjugated UDCA, UDCA 7-N-acetylglucosaminide, the 3-sulfates of cholic acid, chenodeoxycholic acid and deoxycholic acid. The antibody Ba-10 allowed us to develop a sensitive competitive ELISA system whose measurable range was approximately 2-200 pg per assay. A serial dilution study indicated that the ELISA enables the direct measurement of the UDCA 3-Suls in human urine before and after the administration of exogenous UDCA. The daily urinary excretion rate of UDCA 3-Suls from healthy male volunteers (n = 5) was determined to be a mean of 131 +/- 61.2 (SD) microgram as the nonamidated UDCA 3-Sul equivalent.
Animals↗
Behavior of mono-, di-, and trihydroxyoctadecenoic acids during mashing and methods of controlling their production.
The behavior of mono-, di-, and trihydroxyoctadecenoic acids was investigated during laboratory-scale mashing under various conditions with a view to controlling their production. Using a malt in which the lipoxygenase activity was at only a trace level (less than 0.01 U/g) or starting the mashing at a higher temperature than that conventionally used (65 degrees C instead of 48 degrees C) significantly decreased the production of these hydroxy fatty acids. Lowering the pH of the mash to inhibit lipoxygenase activity and preventing O2 uptake by the mash using carbon dioxide were also effective in reducing the amounts of these acids produced during mashing. From the viewpoint of industrial-scale beer production, the prevention of O2 uptake by the mash was selected as an appropriate method for reducing oxidation during wort production without affecting the subsequent brewing process or the taste of the finished beer. After introducing oxidation prevention procedures, the content of trihydroxyoctadecenoic acids decreased by about 30% and the foam stability and taste were improved in commercial products brewed using less than 25% malts.
Journal Article↗
Effects of collateral vessel occlusion on oral glucose tolerance test in liver cirrhosis.
Alterations in carbohydrate metabolism associated with liver cirrhosis are characterized by a high serum insulin level and prolonged hyperglycemia on oral glucose tolerance test (OGTT). We measured plasma glucose, immunoreactive insulin (IRI), and C-peptide immunoreactivity (CPR) levels during a 75-g OGTT before and after varices obliteration in 10 cirrhotic patients with gastric varices. After obliteration, the indocyanine green retention rate was decreased and the portal flow velocity was increased. A significant decline in plasma glucose and IRI levels was also noted on OGTT. Moreover, the plasma glucose and IRI levels declined at 90 and 120 min in OGTT while they increased progressively by 120 min before obliteration. The levels of CPR were similar before and after treatment. These results indicate that decreased portal flow due to extrahepatic shunt and consequent impairment of insulin metabolism play a role in glucose intolerance observed in cirrhotic patients and that shunt occlusion improves glucose metabolism.
Adult↗
The effects of age and abnormal sperm count on the nondisjunction of spermatozoa.
PURPOSE: The effect of paternal age on the nondisjunction of sex chromosomes is controversial. Also, the prevalence of chromosomal anomalies in infertile patients is controversial, it has been reported that the sex chromosomal aneuploidy rate following treatment with intracytoplasmic sperm injection (ICSI) is higher than in naturally conceived pregnancies. We investigated the influence of paternal age and oligozoospermia on the nondisjunction of spermatozoa. METHODS: We determined the rate of aneuploidy for gonosomes and autosomes, using two-color fluorescence in situ hybridization (FISH) of the X and Y chromosomes and chromosomes 12 and 18 in 10 donors under 25 years of age who had a normal sperm count (> or = 20 x 10(6)/ml), 10 donors over the age of 39 years with idiopathic infertility and normozoospermia (> or = 20 x 10(6)/ml), and 5 oligozoospermic donors (< 20 x 10(6)/ml). RESULTS: There was no obvious relationship between increasing age and autosomal disomy (disomy 12 and disomy 18). Neither autosomal disomy nor diploidy was increased in any group. The frequency of X-, Y-, XX-, and YY-bearing sperm did not differ significantly among groups, but the frequency of XY-bearing sperm was significantly higher in the older infertile group than in the control donors. CONCLUSIONS: The incidence of nondisjunction of paternal sex chromosome in meiosis I was higher in older men with idiopathic infertility. The present results suggest that the risk of producing XXY fetuses is higher among men > 39 years of age with idiopathic infertility.
Adult↗
Recombining germline-derived CDR sequences for creating diverse single-framework antibody libraries.
We constructed a single-chain Fv antibody library that permits human complementarity-determining region (CDR) gene fragments of any germline to be incorporated combinatorially into the appropriate positions of the variable-region frameworks VH-DP47 and VL-DPL3. A library of 2 x 109 independent transformants was screened against haptens, peptides, carbohydrates, and proteins, and the selected antibody fragments exhibited dissociation constants in the subnanomolar range. The antibody genes in this library were built on a single master framework into which diverse CDRs were allowed to recombine. These CDRs were sampled from in vivo-processed gene sequences, thus potentially optimizing the levels of correctly folded and functional molecules, and resulting in a molecule exhibiting a lower computed immunogenicity compared to naive immunoglobulins. Using the modularized assembly process to incorporate foreign sequences into an immunoglobulin scaffold, it is possible to vary as many as six CDRs at the same time, creating genetic and functional variation in antibody molecules.
Germ-Line Mutation↗
Genetic mapping of a locus associated with bovine chronic interstitial nephritis to chromosome 1.
Chronic interstitial nephritis with diffuse zonal fibrosis (CINF) occurs in Japanese Black cattle (Wagyu) as an autosomal recessive disorder leading to death prior to puberty, first six months or a year of life. We performed a genome-wide scan using microsatellite markers in a Wagyu pedigree segregating for CINF and mapped the CINF locus to bovine chromosome 1. CINF was closest to microsatellites BM9019 and INRA49 (Z score = 12.0; P < 3.4 x 10(-10)).
Animals↗
Development of systemic sclerosis in a patient with systemic lupus erythematosus and topoisomerase I antibody.
We describe a patient with systemic lupus erythematosus (SLE) associated with topoisomerase I (topo I, Scl-70) antibody, a specific marker for systemic sclerosis (SSc). SSc patients who produce this antibody have severe cutaneous and visceral involvement, and eventually have a poor prognosis. It is rare to find this antibody in patients with other collagen diseases. Only four SLE patients have been reported in the English language literature who were topo I antibody-positive but had no clinical evidence of scleroderma. The serum of our patient with SLE had precipitating topo I antibody from the onset of his disease. Twelve years after the onset of SLE, he developed proximal scleroderma and pulmonary fibrosis. This case reconfirms the prognostic significance of topo I antibody as a predictive marker, and indicates that SLE patients with topo I antibody require careful follow-up for future development of scleroderma.
Adult↗
Transepithelial migration of activated eosinophils induces a decrease of E-cadherin expression in cultured human nasal epithelial cells.
BACKGROUND: The damage of respiratory epithelium in allergic diseases has a close correlation with the extent of eosinophil infiltration. It seems to be a good possibility that eosinophil infiltration could induce the changes in the expression of the epithelial cell adhesion molecules, which play a key role in the maintenance of structural and functional rigidity of epithelium. OBJECTIVE: We observed the expression of E-cadherin in cultured human nasal epithelial cells (HNECs) to study whether could it be affected by transepithelial migration of inflammatory cells, especially eosinophils. METHODS: In vitro study of the transmigration assay was designed using various types of inflammatory cells and HNEC monolayers. Various assays of each experimental group were done under the stimulation of interleukin-5 (IL-5) and/or platelet activating factor (PAF). Subsequently immunohistochemistry for E-cadherin was performed in the HNECs. The intensity of immunofluorescence of E-cadherin was quantified using confocal laser scanning microscopy (CLSM) system and compared before and after the transmigration. RESULTS: The mean intensity of immunofluorescence for E-cadherin decreased significantly after the transmigration of any types of inflammatory cells. Above all, the migration of eosinophils treated with IL-5 and PAF had an eminent effect on the decrease, whereas the degranulation extracts derived from eosinophils activated by IL-5 and secretory IgA (sIgA) did not affect the intensity. CONCLUSION: This work suggests that transepithelial migration of inflammatory cells can directly induce the decrease in epithelial E-cadherin expression. Furthermore, the most prominent change was induced by transmigration of activated eosinophils, which might be caused by some mechanisms independent of the eosinophil contents. The decrease in E-cadherin expression may trigger the damage of epithelial barrier, which contributes to the pathogenesis of allergic diseases.
Adolescent↗
cis-acting regulatory sequences for antitermination in the transcript of the Bacillus subtilis hut operon and histidine-dependent binding of HutP to the transcript containing the regulatory sequences.
The location of the cis-acting regulatory region for histidine-dependent antitermination of the Bacillus subtilis hut operon was determined. A secondary structure, whose sequences partially overlap with the downstream terminator, was found in the regulatory region of the hut transcript. Mutational analysis of the regulatory region showed that the secondary structure was required for histidine-dependent antitermination. An electrophoretic mobility-shift assay demonstrated that, in response to the presence of histidine and Mg2+, purified HutP bound hut RNA bearing putative secondary structure but not RNA lacking the potential to form putative secondary structure. Native gel electrophoresis showed that HutP existed as a hexamer. A filter-binding assay revealed that the concentration of histidine required for half-maximal binding of HutP to RNA was 3.1 mM and that the Kd for binding of HutP to RNA was approximately 0.56 microM in the presence of histidine. These results suggested that putative secondary structure in the regulatory region of hut mRNA could function as an antiterminator to inhibit the formation of the terminator structure and that HutP causes expression of the hut structural genes by binding to the putative antiterminator structure in response to the presence of histidine.
Amino Acid Sequence↗
A lipochito-oligosaccharide, Nod factor, induces transient calcium influx in soybean suspension-cultured cells.
Lipochito-oligosaccharides (Nod factors) produced by Rhizobium or Bradyrhizobium are the key signal molecules for eliciting nodulation in their corresponding host legumes. To elucidate the signal transduction events mediated by Nod factors, we investigated the effects of Nod factors on the cytosolic [Ca2+] of protoplasts prepared from roots and suspension-cultured cells of soybean (Glycine max and G. soja) using a fluorescent Ca2+ indicator, Fura-PE3. NodBj-V (C18:1, MeFuc), which is a major component of Nod factors produced by Bradyrhizobium japonicum, induces transient elevation of cytosolic [Ca2+] in the cells of soybean within a few minutes. This effect is specific to soybean cells and was not observed in the tobacco BY-2 cells. Furthermore, NodBj-V without MeFuc did not induce any cytosolic [Ca2+] elevation in soybean cells. Exclusion of Ca2+ from the medium, as well as pre-treatment of the cells with an external Ca2+ chelator or with a plasma membrane voltage-dependent Ca2+ channel inhibitor, suppressed the Nod factor-dependent cytosolic [Ca2+] elevation. These results indicate that transient Ca2+ influx from extracellular fluid is one of the earliest responses of soybean cells to NodBj-V (C18:1, MeFuc) in a host-specific manner.
Bradyrhizobium↗
Hepatocyte transplantation in rats with decompensated cirrhosis.
Hepatocyte transplantation improves the survival of laboratory animals with experimentally induced acute liver failure and the physiological abnormalities associated with liver-based metabolic deficiencies. The role of hepatocyte transplantation in treating decompensated liver cirrhosis, however, has not been studied in depth. To address this issue, cirrhosis was induced using phenobarbital and carbon tetrachloride (CCL(4)) and animals were studied only when evidence of liver failure did not improve when CCL(4) was held for 4 weeks. Animals received intrasplenic transplantation of syngeneic rat hepatocytes (G1); intraperitoneal transplantation of syngeneic rat hepatocytes (G2); intraperitoneal transplantation of a cellular homogenate of syngeneic rat hepatocytes (G3); intraperitoneal transplantation of syngeneic rat bone marrow cells (G4); or intrasplenic injection of Dulbecco's modified Eagle medium (DMEM) (G5). After transplantation, body weight and serum albumin levels deteriorated over time in all control (G2-G5) animals but did not deteriorate in animals receiving intrasplenic hepatocyte transplantation (G1) (P <.01). Prothrombin time (PT), total bilirubin, serum ammonia, and hepatic encephalopathy score were also significantly improved toward normal in animals receiving intrasplenic hepatocyte transplantation (P <. 01). More importantly, survival was prolonged after a single infusion of hepatocytes and a second infusion prolonged survival from 15 to 128 days (P <.01). Thus, hepatocyte transplantation can improve liver function and prolong the survival of rats with irreversible, decompensated cirrhosis and may be useful in the treatment of cirrhosis in humans.
Animals↗
Evaluation of tuned-aperture computed tomography depth discrimination for image series acquired variously with linear horizontal, linear vertical, and conical beam projection arrays.
OBJECTIVE: To compare depth discrimination by using tuned-aperture computed tomography (TACT) variously with linear horizontal, linear vertical, combined linear horizontal and linear vertical, and conical beam projection arrays. STUDY DESIGN: The first test object was a metallic mesh angled at 30 degrees to the surface of a computed dental radiography size No. 1 x-ray sensor. The second test object was a dry human mandible. The sensor was mounted on an optical bench constructed to permit free and precise geometrical settings for the horizontal and vertical angulations of the x-ray beam. The extent of blurring of horizontal and vertical wires in each TACT reconstructed image slice was observed for each of the tested beam projection arrays. RESULTS: With a linear horizontal beam projection array, it was not possible to determine the depth of structures parallel to the horizontal dimension, such as the mandibular canal. With a linear vertical beam projection, it was not possible to determine the depth of structures parallel to the vertical dimension. A conical array of beam projections was best suited to the task of depth discrimination of objects in all planes. The best second alternative was a combination of linear vertical and linear horizontal projections. CONCLUSIONS: Beam projection geometry is important for the accurate depth discrimination of TACT reconstructed images. A conical beam projection array is ideal.
Dental Alloys↗