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Biomedical subjects

H Noguchi

Publications and source records attributed to H Noguchi.

At least 73 records · Page 4Linked to original sources
PubMed2001

Insertion of a suicide gene into an immortalized human hepatocyte cell line.

For developing a bioartificial liver (BAL) device, an attractive alternative to the primary human hepatocytes would be the use of highly differentiated immortalized human hepatocytes with a safeguard. To test the feasibility, the primary human hepatocytes were immortalized by a plasmid SV3neo encoding simian virus 40 large T antigen (SV40Tag) gene. A highly differentiated hepatocyte line OUMS-29 was established. A suicide gene of herpes simplex virus-thymidine kinase (HSV-TK) was retrovirally introduced into OUMS-29 cells as a safeguard for clinical application. One of the resulting HSV-TK-positive cell lines, OUMS-29/tk, grew in chemically defined serum-free medium with the gene expression of differentiated liver functions. OUMS-29/tk cells were 100 times more sensitive to ganciclovir compared with unmodified OUMS-29 cells in in vitro experiments. We have established a tightly regulated immortalized human hepatocyte cell line. Essentially unlimited availability of OUMS-29/tk cells may be clinically useful for BAL therapy.

Antiviral Agents↗
PubMed2001

Successful retroviral gene transfer of simian virus 40 T antigen and herpes simplex virus-thymidine kinase into human hepatocytes.

Current clinical reports have indicated that hepatocyte transplantation (HTX) could be used in patients with liver failure and in children with liver-based metabolic diseases. One of the major limiting factors of HTX is a serious shortage of donor livers for hepatocyte isolation. To address this issue, we immortalized adult human hepatocytes with a retroviral vector SSR#69 expressing the genes of simian virus 40 large T antigen and herpes simplex virus-thymidine kinase simultaneously. One of the resulting clones, NKNT-3, grew steadily in chemically defined serum-free medium without any obvious crisis and showed the gene expression of differentiated liver functions. Under the administration of 5 microM ganciclovir, NKNT-3 cells stopped proliferation and died in in vitro experiments. We have established a tightly regulated immortal human hepatocyte cell line. The cells could allow the need for immediate availability of consistent and functionally uniform cells in sufficient quantity and adequate quality.

Antigens, Polyomavirus Transforming↗
PubMed2001

Cre/loxP-based reversible immortalization of human hepatocytes.

An ideal alternative to the primary human hepatocytes for hepatocyte transplantation would be to use a clonal cell line that grows economically in culture and exhibits the characteristics of differentiated, nontransformed hepatocytes following transplantation. The purpose of the present studies was to establish a reversibly immortalized human hepatocyte cell line. Human hepatocytes were immortalized with a retroviral vector SSR#69 expressing simian virus 40 large T antigen (SV40Tag) gene flanked by a pair of loxP recombination targets. One of the resulting clones, NKNT-3, showed morphological characteristics of liver parenchymal cells and expressed the genes of differentiated liver functions. NKNT-3 cells offered unlimited availability. After an adenoviral delivery of Cre recombinase and subsequent differential selection, efficient removal of SV40Tag from NKNT-3 cells was performed. Here we represent that elimination of the retrovirally transferred SV40Tag gene can be excised by adenovirus-mediated site-specific recombination.

Cell Line↗
PubMed2001

Rapidly functional immobilization of immortalized human hepatocytes using cell adhesive GRGDS peptide-carrying cellulose microspheres.

With the development of biotechnology, hepatic support by a hybrid artificial liver (HAL) using hepatocytes has been given much attention. Because the availability of human livers is limited, we have established a tightly regulated immortal human hepatocyte cell line, NKNT-3, for developing HAL. Because high-density cell culture allows the compactness of the HAL device and its easy use under emergency circumstances, we have developed cell adhesive GRGDS peptide-containing cellulose microspheres (GRGDS/CMS). The GRGDS/CMS efficiently immobilized NKNT-3 cells within 24 h in a stirred suspension culture. Electron microscopic examinations demonstrated glycogen granules and well-developed endoplasmic reticulum and mitochondria in NKNT-3 cells attached to the GRGDS/CMS. The cells showed ammonia clearance activity, whereas HepG2-transformed human liver cells did not remove the loaded ammonia. An efficient adenoviral delivery of the lacZ reporter gene was performed in GRGDS/CMS-immobilized NKNT-3 cells. In this study we present rapid immobilization of NKNT-3 immortal human hepatocytes using cellulose microspheres carrying GRGDS peptides. These microspheres satisfied immediate preparation of NKNT-3 cells in sufficient quantity and of adequate quality.

Ammonia↗
PubMed2000

Enzymatic formation of unnatural aromatic polyketides by chalcone synthase.

Substrate specificity of recombinant chalcone synthase (CHS) from Scutellaria baicalensis (Labiatae) was investigated using chemically synthesized aromatic and aliphatic CoA esters. It was demonstrated for the first time that CHS converted benzoyl-CoA to phlorobenzophenone (2,4,6-trihydroxybenzophenone) along with pyrone by-products. On the other hand, phenylacetyl-CoA was enzymatically converted to an unnatural aromatic polyketide, phlorobenzylketone (2, 4,6-trihydroxyphenylbenzylketone), whose structure was finally confirmed by chemical synthesis. Furthermore, in agreement with earlier reports, S. baicalensis CHS also accepted aliphatic CoA esters, isovaleryl-CoA and isobutyryl-CoA, to produce phloroacylphenones. In contrast, hexanoyl-CoA only afforded pyrone derivatives without formation of a new aromatic ring. It was noteworthy that both aromatic and aliphatic CoA esters were accepted in the active site of the enzyme as a starter substrate for the complex condensation reaction. The low substrate specificity of CHS thus provided further insight into the structure and function of the enzyme.

Acyltransferases↗
PubMed2000

Inhibition of vertebrate squalene epoxidase by isoprenyl gallates and phenylalkyl gallates.

Galloy esters with 'substrate-like' isoprenoid or phenylalkyl side chains were newly synthesized and tested for the enzyme inhibition activities toward recombinant rat squalene epoxidase. Isoprenyl gallates (4-6) showed good inhibition (IC50 = 1.5 5.1 microM), as potent as previously reported substrate analogues, while phenylalkyl gallates (7-10) were moderate inhibitors of the enzyme (IC50 = 12-61 microM).

Animals↗
PubMed2000

Antioxidative galloyl esters as enzyme inhibitors of p-hydroxybenzoate hydroxylase.

Gallic acid and its esters were evaluated as enzyme inhibitors of recombinant p-hydroxybenzoate hydroxylase (PHBH), a NADPH-dependent flavin monooxygenase from Pseudomonas aeruginosa. n-Dodecyl gallate (DG) (IC(50)=16 microM) and (-)-epigallocatechin-3-O-gallate (EGCG) (IC(50)=16 microM), a major component of green tea polyphenols, showed the most potent inhibition, while product-like gallic acid did not inhibit the enzyme significantly (IC(50)>250 microM). Inhibition kinetics revealed that both DG and EGCG inhibited PHBH in a non-competitive manner (K(I)=18.1 and 14.0 microM, respectively). The enzyme inhibition was caused by specific binding of the antioxidative gallate to the enzyme, and by scavenging reactive oxygen species required for the monooxygenase reaction. Molecular modeling predicted that EGCG binds to the enzyme in the proximity of the FAD binding site via formation of three hydrogen bonds.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed2000

Are we inhibited? Renal insufficiency should not preclude the use of ACE inhibitors for patients with myocardial infarction and depressed left ventricular function.

CONTEXT: Angiotensin-converting enzyme (ACE) inhibitors have been shown to decrease mortality in patients with myocardial infarction and depressed left ventricular function, but physicians may be reluctant to prescribe ACE inhibitors to patients with concomitant renal insufficiency. OBJECTIVE: To evaluate whether patients with depressed left ventricular ejection fraction following acute myocardial infarction have a similar reduction in mortality from ACE inhibitors regardless of their renal function. DESIGN: Retrospective cohort study using medical record data. SETTING: All nonfederal acute care hospitals. PATIENTS: A cohort of 20,902 Medicare beneficiaries aged 65 years and older directly admitted to the hospital from February 1, 1994, through July 30, 1995, and with a documented left ventricular ejection fraction of less than 40% measured by echocardiography, radionuclide scintigraphy, or angiography following a confirmed acute myocardial infarction. MAIN OUTCOME MEASURES: One-year survival for patients who received or who did not receive an ACE inhibitor at hospital discharge, stratified by the patient's level of renal function. RESULTS: For the entire cohort, the receipt of an ACE inhibitor on hospital discharge was associated with greater 1-year survival (hazards ratio, 0.84; 95% confidence interval, 0.77-0.91) after adjusting for patient demographic characteristics, comorbidity, severity of illness (including left ventricular ejection fraction), and the receipt of other therapies. In stratified models, the receipt of an ACE inhibitor was associated with a 37% (16%-52%) lower mortality for patients who had poor renal function (serum creatinine level,<265 micromol/L [<3 mg/dL]) and a 16% (8%-23%) lower mortality for patients who had better renal function. Use of aspirin therapy attenuated the benefit of ACE inhibitors in patients with poor renal function. CONCLUSIONS: Moderate renal insufficiency should not be considered a contraindication to the use of ACE inhibitors in patients with depressed left ventricular ejection fraction following myocardial infarction. Use of aspirin therapy may attenuate the benefit of ACE inhibitors in patients with high serum creatinine levels; therefore, further studies are needed to determine whether treatment with aspirin, alternative antiplatelet agents, or anticoagulation is indicated for these patients.

Aged↗
PubMed2000

Isolation and characterization of the gene encoding mouse tax-responsive element-binding protein (TREB) 5.

TREB5/hXBP-1/HTF is a basic region leucine zipper protein which binds to a cyclic AMP responsive element (CRE)-like element in both human T-cell leukemia virus type 1 and human major histocompatibility complex (MHC) class II genes. To analyze the structure and transcription regulation of the TREB5 gene, we isolated the mouse TREB5 gene and cDNA. The mouse TREB5 gene contains five exons and four introns and spans approximately 5 kb. The deduced amino acid sequence of mouse TREB5 exhibited 77% and 94% homology to human and rat TREB5, respectively. The b-zip structure is completely conserved in mouse, rat and human. Southern blot analysis of the mouse genomic DNA demonstrated that positive bands exactly coincide with those expected from sequences of the cloned genes, indicating that the mouse TREB5 gene is present as a single copy. The transcription start site of the mouse TREB5 gene was mapped to -15 bp upstream from the ATG initiation codon. Promoter analysis of serial deletion mutants revealed that the -142 bp upstream region is the minimum sequence to promote mouse TREB5 gene expression and that the -1.0 kb upstream region is required for full promoter activity.

3T3 Cells↗
PubMed2000

Circadian component influences the photoperiodic induction of diapause in a drosophilid fly, Chymomyza costata.

The last-instar larvae of a drosophilid fly, Chymomyza costata enter diapause in response to the dark-phases longer than 9 h (Yoshida, T., Kimura, M.T., 1995. The photoperiodic clock in Chymomyza costata. Journal of Insect Physiology 41, 217-222). In order to switch the developmental programming of the sensitive larvae from continuous development to diapause, after they were transferred from the short (8 h) to the long (14 h) dark-phase, significantly less time (1-2 days) was required when the dark-phase was abruptly and asymmetrically extended into the evening, than when it was extended symmetrically into both morning and evening (2-3 days), or asymmetrically into the morning hours (4-6 days). Diapause was also induced in 40-70% of sensitive larvae that were reared under the gradually shortening light-phase (from 16 h to 2 h, by 1 h in each cycle), despite that the dark-phase remained constant and short (8 h). Larvae developed continuously, however, when reared under the gradually extending light-phase (from 16 h to 24 h) and a constantly short dark-phase. We interpret such results, with the help of the two-oscillator model of circadian rhythmicity (Pittendrigh, C.S., Daan, S., 1976. A functional analysis of circadian pacemakers in nocturnal rodents. V. Pacemaker structure: A clock for all seasons. Journal of Comparative Physiology A 106, 333-355), as indicating that two mutually coupled oscillators (evening and morning) differing in their entrainability may participate in measuring of the dark-phase duration. The levels of dopamine (DA) and serotonin (5-HT) in the larval CNS transiently increased (by up to 20%) after the dusk, while no apparent change was observed during the dawn. The dusk-related increase was observed also after the asymmetric extension of the dark-phase into evening, while the asymmetric extension into morning had no effect on the levels of the DA and 5-HT.

Journal Article↗
PubMed2000

Potent and selective inhibition of squalene epoxidase by synthetic galloyl esters.

n-Alkyl esters (ethyl, octyl, dodecyl, and cetyl) of gallic acid were evaluated as enzyme inhibitors of recombinant rat squalene epoxidase (SE), a rate-limiting enzyme of cholesterol biogenesis. Dodecyl (6) (IC(50) = 0.061 microM) showed the most potent inhibition, which was far more potent than those of previously reported naturally occurring gallocatechins. Octyl gallate (5) (IC(50) = 0.83 microM) and cetyl gallate (7) (IC(50) = 0.59 microM) also showed good inhibition, while gallic acid (IC(50) = 73 microM) itself was not so active. In addition, chemically synthesized galloyl ester of cholesterol (9) (IC(50) = 3.9 microM), farnesol derivative (10) (IC(50) = 0.57 microM), and dodecyl galloyl amide (8) (IC(50) = 3.0 microM) were also potent inhibitors of SE. Inhibition kinetics revealed that dodecyl gallate inhibited SE in competitive (K(I) = 0.033 microM) and no-time-dependent manner. The potent inhibition of the flavin monooxygenase would be caused by specific binding to the enzyme, and by scavenging reactive oxygen species required for the epoxidation reaction.

Animals↗
PubMed2000

Green tea polyphenols: novel and potent inhibitors of squalene epoxidase.

The green tea gallocatechins, (-)-epigallocatechin-3-O-gallate (EGCG) (IC(50) = 0.69 microM), (-)-gallocatechin-3-O-gallate (GCG) (IC(50) = 0.67 microM), (-)-epicatechin-3-O-gallate (ECG) (IC(50) = 1.3 microM), and theasinensin A (IC(50) = 0.13 microM), were found to be potent and selective inhibitors of rat squalene epoxidase (SE), a rate-limiting enzyme of cholesterol biogenesis. On the other hand, flavan-3-ols without galloyl group at C-3 did not show significant enzyme inhibition. It was demonstrated for the first time that the cholesterol lowering effect of green tea may be attributed to their potent SE inhibition activities. Inhibition kinetics revealed that EGCG inhibited SE in noncompetitive (K(I) = 0.74 microM), and non-time-dependent manner. The potent enzyme inhibition would be caused by specific binding to the enzyme, and by scavenging reactive oxygen species required for the monooxygenase reaction.

Animals↗
PubMed2000

Prevention of acute liver failure in rats with reversibly immortalized human hepatocytes.

Because of a critical shortage in suitable organs, many patients with terminal liver disease die each year before liver transplantation can be performed. Transplantation of isolated hepatocytes has been proposed for the temporary metabolic support of patients awaiting liver transplantation or spontaneous reversion of their liver disease. A major limitation of this form of therapy is the present inability to isolate an adequate number of transplantable hepatocytes. A highly differentiated cell line, NKNT-3, was generated by retroviral transfer in normal primary adult human hepatocytes of an immortalizing gene that can be subsequently and completely excised by Cre/Lox site-specific recombination. When transplanted into the spleen of rats under transient immunosuppression, reversibly immortalized NKNT-3 cells provided life-saving metabolic support during acute liver failure induced by 90% hepatectomy.

Adult↗
PubMed2000

Transplantation of highly differentiated immortalized human hepatocytes to treat acute liver failure.

BACKGROUND: Temporary support of a damaged liver by a bioartificial liver (BAL) devise is a promising approach for the treatment of acute liver failure. Although human primary hepatocytes are an ideal source of hepatic function in BAL, shortage of human livers available for hepatocyte isolation is the limiting factor for the use of this modality. A clonal human hepatocyte cell line that can grow economically in culture and exhibit liver-specific functions should be an attractive solution to this problem. METHODS: To test this alternative, primary human fetal hepatocytes were immortalized using Simian virus 40 large T antigen. To investigate the potential of the immortalized cells for BAL, we transplanted the cells into the spleen of adult rats and performed a 90% hepatectomy 12 hr later. RESULTS: One of the cloned human liver cell lines, OUMS-29, showed highly differentiated liver functions. Intrasplenic transplanting of 20x10(6) OUMS-29 cells protected the animals from hyperammonemia and the associated hepatic encephalopathy. Survival was significantly prolonged in 90% of hepatectomized rats receiving OUMS-29 cells. CONCLUSIONS: A highly differentiated immortalized human hepatocyte cell line, OUMS-29, was able to provide metabolic support during acute liver failure induced by 90% hepatectomy in rats. Essentially unlimited availability of OUMS-29 cells may be clinically useful for BAL treatment.

Ammonia↗
PubMed2000

A malignant rhabdoid tumor of the kidney occurring concurrently with a brain tumor: report of a case.

Malignant rhabdoid tumor of the kidney (MRTK) is one of the most lethal neoplasms to occur in young infants. Cases of MRTK accompanying an embryonal tumor in the central nervous system have occasionally been described. We present herein an interesting case of MRTK that was clinically diagnosed preoperatively. A male infant aged 6 months with both a midline brain tumor and a renal neoplasm was transferred to our institution. Although roentgenographic evaluation suggested that the renal lesion was a Wilms' tumor, midkine (MK), a growth and differentiation factor characteristically present in the urine of patients with Wilms' tumor, was not detected. A preoperative diagnosis of MRTK was established based on the lack of urinary MK in addition to the typical clinical features of the young age and the concurrent brain tumor.

Brain Neoplasms↗
PubMed2000

Cervico-mediastinal bronchogenic cyst occurring in the prenatal period: report of a case.

We experienced a case of cervico-mediastinal bronchogenic cyst in which a cervical cystic mass was detected by prenatal ultrasonography. On prenatal ultrasound, a unilocular, well-defined and hypoechoic mass was detected in the fetal neck. The baby was born by a normal vaginal delivery at 40 weeks of gestation, and had no respiratory distress. Radiological investigations demonstrated a cyst in the cervico-mediastinal region, which displaced the trachea to the left. At the age of 32 days, an elective resection was easily performed through a right inferior collar incision after first aspirating the contents of the cyst. Prenatal sonography showing abnormal findings is effective for identifying cysts in the perinatal period and allows for the timely resection of such cysts before respiratory distress occurs.

Bronchogenic Cyst↗
PubMed2000

Intermittent administration of brain-derived neurotrophic factor ameliorates glucose metabolism in obese diabetic mice.

We have previously shown that brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, interacts with the endocrine system in obese diabetic mice, and systemic peripheral administration of BDNF regulates glucose metabolism in this model. Results from the present study show that the hypoglycemic effect induced by 2 weeks' daily administration of BDNF (20 mg/kg/d) to db/db mice lasts for several weeks after treatment cessation, irrespective of food reduction. On the other hand, the antidiabetic agent, metformin had no lasting effect. This duration of the BDNF hypoglycemic action prompted us to examine the efficacy of BDNF intermittent administration on glucose metabolism. BDNF administered once or twice per week (70 mg/kg/wk) to db/db mice for 3 weeks significantly reduced blood glucose concentrations and hemoglobin A(1c), (HbA(1c)) as compared with ad libitum-fed phosphate-buffered saline (PBS)-treated and pair-fed PBS-treated groups. This suggests that BDNF not only temporarily reduced blood glucose concentrations but also ameliorated systemic glucose balance in this obese diabetic mouse model during the experimental period. Our results indicate that BDNF could be a novel hypoglycemic agent with an exceptional ability to normalize glucose metabolism even with treatment as infrequently as once per week.

Animals↗