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Biomedical subjects

K Matsubara

Publications and source records attributed to K Matsubara.

At least 235 records · Page 13Linked to original sources
PubMed1994

A new three-dimensional culture system for hepatocytes using reticulated polyurethane.

Poly-N-para-vinylbenzyl-lactonamide (PVLA)-coated reticulated polyurethane (PVLA-RPU) has been employed for the long-term maintenance of primary rat hepatocyte cultures. After 3 days of incubation of 2 x 10(7) hepatocytes/cm3 embedded in PVLA-RPU discs and kept in culture medium, most cells showed typical hepatocyte morphology, with some bile canaliculus-like intercellular spaces among the hepatocytes on examination with scanning and transmission electron microscopy. The cells were attached to the surface of the PVLA-RPU and formed multicellular spheroids in the reticulated pores. The hepatocytes maintained various liver-specific functions such as albumin secretion, ammonium metabolism, urea synthesis and gluconeogenesis, and they were viable. The liver-specific functions could be maintained for more than 1 month when the cells were kept in the rats' peritoneal cavities. This new system may be useful as a bioreactor for an artificial liver.

Albumins↗
PubMed1994

Identification of the heme compound copurified with deoxyribonucleic acid (DNA) from bloodstains, a major inhibitor of polymerase chain reaction (PCR) amplification.

The heme compound found in deoxyribonucleic acid (DNA) extracted from bloodstains, which is regarded as a major inhibitor of polymerase chain reaction (PCR), was characterized in comparison with alkaline and acid hematin, histidine and ammonia hemochromogens, and globin and serum albumin hemochromogens digested by proteinase K. Alkaline and acid hematin were almost completely removed by phenol/chloroform treatment and ethanol precipitation, so as not to be copurified with DNA from the specimens. Spectrophotometric results indicated that the contaminant was likely to be the product of proteinase K digestion of some heme-blood protein complex, which was not completely extracted by organic solvents and remained in the ethanol precipitates of DNA. The results of polyacrylamide gradient gel electrophoresis and intensity of the inhibition of PCR suggested that the ligand of the contaminant was a somewhat large molecule, resistant to the proteolysis by proteinase K. The addition of bovine serum albumin to the reaction mixture prevented the inhibition of PCR by the heme compounds, probably by binding to the heme. This showed that the inhibition was not due to the irreversible inactivation of the enzyme.

Base Sequence↗
PubMed1993

Transactivating function of integrated hepatitis B virus.

HBV DNAs are often found in integrated forms in human hepatocellular carcinoma (HCC). Discovery of a transactivation function coded by a limited region of the HBV genome has promoted us to survey our collection of HBV integrants with flanking cellular sequences, asking whether they might exhibit a transactivation function. In transient cotransfection assays using the HepG2 cell line, six out of the twelve integrants showed transactivation effects on the expression of cellular genes such as c-fos. These results strongly demonstrate that the transactivating effects of integrated HBV DNA are widely distributed, and some of these effects might be correlated to hepatocarcinogenesis.

Carcinoma, Hepatocellular↗
PubMed1993

cDNA analyses in the human genome project.

The ultimate goal of the human genome project is to decode all the genetic information carried in the genome. Towards this goal, the physical structure of the genome, as well as the functional aspects of the genome, must be understood. We initiated a cDNA project to collect the 'expression profiles' of all human genes, a database with which to describe which genes are expressed, and to what extent, in any given human cell at a particular time. Single-cycle sequencing of randomly selected members from a 3'-directed cDNA library is most appropriate for this purpose: the sequence data serve as a 'gene signature' to identify the expressing gene, and the frequency of appearance of the gene signature reflects the activity of the gene. The compiled data, which usually cover some 1000 sequencing results per sample, are referred to as an 'expression profile.' We applied this analysis to HepG2 (a cell line derived from a hepatocellular carcinoma), liver cells and lung cells. The expression profiles shed some light upon the unique features of gene expression in the cell or tissue tested. A comparison of the expression profiles among different cells has allowed active genes to be classified as housekeepers or those with cell-specific functions. A significant fraction of the abundantly expressed genes include those that are unique to the cell. In addition, the resulting collection of thousands of gene signatures is a useful source of probes for mapping and for isolating full-size cDNAs.

Animals↗
PubMed1993

Aphidicolin potentiates apoptosis induced by arabinosyl nucleosides in human myeloid leukemia cell lines.

We investigated the effect of aphidicolin, an inhibitor of DNA polymerase alpha and delta, on the induction of apoptosis by arabinosyl nucleosides in a human promyelocytic leukemia cell line, HL-60. Pretreatment of HL-60 cells with aphidicolin (2 microM) significantly increased the number of morphologically apoptotic cells induced by 1-beta-D arabinofuranosylcytosine (ara-C) during 4 hr of incubation. This is consistent with the appearance of DNA fragmentation as determined quantitatively by diphenylamine or by agarose gel electrophoresis. The inhibition of cell growth on day 3 after drug exposure was correlated with the degree of apoptosis: Such synergistic interaction between aphidicolin and ara-C has also been observed in other human myeloid leukemia cell lines, U937 and KG-1. In addition, the induction of apoptosis by 9-beta-D arabinofuranosyladenine or 9-beta-D arabinofuranosylguanine is augmented by aphidicolin.

Aphidicolin↗
PubMed1993

Two-dimensional gel electrophoretograms of human chromosome specific restriction DNA fragments.

Human genomic DNA can be scanned by a two-dimensional gel electrophoresis of NotI cleaved, end-labelled DNA. This method, called Restriction Landmark Genome Scanning (RLGS) method yields about 2000 landmark spots in such a two-dimensional gel electrophoresis. To assign these spots to chromosomes, DNA from flow-sorted chromosome 3 (7.7 x 10(5) copies) and chromosome 4 (6.7 x 10(5) copies) were subjected to the same analysis. Ninety seven and 122 spots, respectively, were obtained in the electrophoretograms among which 16 spots were common to other chromosomes, and the others were unique to each chromosome. A method has been devised to assign these spots to the whole genomic profile. The ability to assign each spot to a chromosome will render this new mapping technology extremely useful, in which one can detect physical aberrations occurring in chromosomes, or it can be used as an auxiliary method in physical mapping of the human genome.

Autoradiography↗
PubMed1993

Specific expression of the pancreatic-secretory-trypsin-inhibitor (PSTI) gene in hepatocellular carcinoma.

Twenty hepatocellular carcinomas (HCC) were analyzed by Northern blotting to test the expression of pancreatic secretory trypsin inhibitor (PSTI). This gene was expressed in all HCCs, but not in other tumors, including mammary, thyroid, pulmonary and ovarian cancers. Some gastric and colonic cancers weakly expressed PSTI. Among cell lines examined in a similar manner, PSTI was expressed in all of 4 derived from hepatoma. On the other hand, among 15 cell lines derived from cancers other than hepatoma, only 3, derived from pancreatic, colonic and gastric cancers, weakly expressed PSTI. A CAT assay using a deletion set of the 5' region from the cloned PSTI gene has shown that in hepatoma cell lines, the expression of this gene is dependent on the presence of 2 regulatory regions that include an IL-6 responsive elements and an AP-I-binding site. However, in non-hepatoma cell lines, the 2 regulatory regions are not necessary for expression. The blood level of PSTI in 27 patients with HCC was significantly increased, and it was positively correlated with tumor size, suggesting that specific expression of PSTI in HCC causes this effect and that elevated blood level of PSTI without inflammation indicates the presence of HCC.

Adult↗
PubMed1993

Identification of the IL-6-responsive element in an acute-phase-responsive human pancreatic secretory trypsin inhibitor-encoding gene.

Pancreatic secretory trypsin inhibitor (PSTI) has been suggested to be an acute-phase reactant in humans and to be induced by inflammatory cytokines such as the interleukins IL-1 and IL-6. We report that PSTI is synthesized in hepatoma cells and that the gene expression is augmented by IL-6. The start points (tsp) for basal and augmented transcription are exactly the same as the tsp in normal pancreas. Analysis of the PSTI gene revealed that a 40-bp DNA fragment located between kb -3.84 and -3.80 carries the element responsible for both transcriptional activity and IL-6-induced gene expression. This 40-bp fragment contains TTGNNGNAATG, the consensus sequence for the NF-IL6-binding site, which is also known as the IL-6-responsive element that is conserved among various acute-phase genes. The basal activity was augmented by another sequence that lies between kb -4.0 and -3.9.

Acute-Phase Proteins↗
PubMed1993

Potential bioactivated neurotoxicants, N-methylated beta-carbolinium ions, are present in human brain.

Potential bioactivated neurotoxicants, 2-N-methyl-beta-carbolinium and 2,9-N,N'-dimethyl-beta-carbolinium ions, as well as N-methylation activities which form these charged species, were analyzed for the first time in the parietal association cortex and the substantia nigra of human brain using GC/MS and HPLC. The brains were taken during forensic autopsies from corpses without obvious degeneration of substantia nigra. In the cortex, 2-methyl-norharmanium ion (2-MeNH) and 2,9-dimethyl-norharmanium ion (2,9-Me2NH) were detected in almost all samples. 2-Methyl-harmanium ions (2-MeHA) and 2,9-dimethyl-harmanium ions (2,9-Me2HA) were detectable in only two samples. In substantia nigra samples pooled from 3 or 4 brains for analysis, 2-MeNH and 2,9-Me2NH levels were higher than those in the cortex, whereas 2-MeHA and 2,9-Me2HA were below detection limits. Their precursors, norharman (NH) and harman (HA), were also measured using HPLC/fluorescence detection. In both regions, NH and HA were present in almost all samples; levels of NH and HA were also significantly higher in the nigra than in the cortex. Using 9-methyl-NH and 2-MeNH as substrates, in vitro N-methylation of the 2[beta] and 9[indole] nitrogens toward beta-carbolines was measured both in the cortex and in the nigra. 2[beta]-N-Methylation activity was significantly higher than 9[indole]-N-methylation activity in both regions. Recent studies show that beta-carbolinium ions resemble the synthetic parkinsonian toxicant, MPP+, with respect to structure and neurotoxic activity. Such 'bioactivated' carbolinium ions could be endogenous causative factors in Parkinson's disease.

Brain Chemistry↗
PubMed1993

Transactivation of cellular promoters by an integrated hepatitis B virus DNA.

A new Hepatitis B virus(HBV) DNA integrant clone DA2-6, isolated from a human hepatocellular carcinoma(HCC) genomic library, was tested for its ability to transactivate expression of other genes. DA2-6 consists of 3.7 kb flanking cellular sequences and an integrated 2.8 kb HBV DNA which covers the region of preS, S, and the 3' truncated X. Using a chloramphenicol acetyltransferase (CAT) assay, a number of cellular and viral promoters were transactivated by DA2-6, and the spectrum of transactivational effect was the same as that by the wild type X gene of the virus. Deletion mutant analyses indicated that the transactivation function of DA2-6 is expressed by the region that encodes a truncated X-cell fusion product.

Amino Acid Sequence↗
PubMed1993

Differential effects of ceruletide on amphetamine-induced behaviors and regional dopamine release in the rat.

This study concerned the effects of ceruletide, a cholecystokinin (CCK)-related peptide, on amphetamine-stimulated behaviors (hyperlocomotion and stereotypy) and amphetamine-induced dopamine (DA) release from the striatum and the nucleus accumbens of the rat. Also, behavioral alterations due to ceruletide administration were compared with the change in DA release from these areas. Ceruletide 160 micrograms/kg s.c., attenuated hyperlocomotion induced by amphetamine, 1 mg/kg and 3 mg/kg s.c., but had no effect on amphetamine-induced stereotypy. Results from in vivo microdialysis experiments showed that s.c. administration of ceruletide caused a significant inhibition of the amphetamine-induced increase in DA release in the nucleus accumbens but not in the striatum. These neurochemical inhibitory effects of ceruletide disappeared completely with bilateral subdiaphragmatic vagotomy. However, infusion of 1 microM of ceruletide into the nucleus accumbens through the dialysis probe had no effect on amphetamine-induced DA release. These results suggest that the inhibitory effect of peripheral administration of ceruletide on amphetamine-induced hyperlocomotion is closely related to the change in DA release from the nucleus accumbens. In the nucleus accumbens, systemically administered ceruletide acts initially on the peripheral organs and influences the activity of DA terminals via an unknown path related to the vagus. Ceruletide had different actions on the dopaminergic system in the striatum and that in the nucleus accumbens.

Amphetamine↗
PubMed1993

Effect of X protein on transactivation of hepatitis B virus promoters and on viral replication.

The X gene product of hepatitis B virus (HBV) transactivates a wide variety of promoters, including four promoters on the HBV genome (Rossner, 1992, J. Med. Virol. 36, 101-117). We compared their transactivation efficiencies and investigated whether the spatial organization of the promoters with respect to other cis-acting elements might influence their activities. Eight reporter plasmid constructs containing the bacterial chloramphenicol acetyltransferase (CAT) gene were designed such that four had the isolated HBV promoters linked to the CAT gene. In the other four, the CAT gene was inserted downstream to each of the four promoters retained in context in the HBV genome. Cells of the human hepatoblastoma line HepG2 were transfected with each one of these reporters together with an effector plasmid, pRSVX, which allowed expression of X protein. All of these promoters could be stimulated by X protein by approximately 2- to 3.5-fold irrespective of their spatial context in the HBV genome. Mutational analysis of in-frame ATG codons in the X gene provides evidence that transactivator product(s) are produced by internal initiation of translation. Transfection of HepG2 cells with HBV genomes bearing a stop mutation in the X gene at codon 118 resulted in poor production of all viral components. Their syntheses were restored upon transfection of the wild-type X gene.

Base Sequence↗
PubMed1993

Morning increase in hemodynamic response to exercise in patients with angina pectoris.

The present study was conducted to determine whether or not there is diurnal variation in the hemodynamic responses to stimuli that increase myocardial oxygen demand, and the effects of such variation on electrocardiograms (ECG). Fifteen patients with angina pectoris, 17 patients with old myocardial infarction, and 8 healthy controls were examined in this study. Graded exercise stress testing was conducted in the supine position, once in the morning and once in the afternoon, using a bicycle ergometer. A standard 12-lead ECG was recorded before, immediately after, and 3, 5, and 10 min after the end of the exercise. The exercise ECG and blood pressure changes were compared among the groups and, within each group, the results after morning and afternoon exercise were compared. Hemodynamic responses, including heart rate, blood pressure, and the pressure-rate product, showed greater increases in the morning than in the afternoon in angina patients and controls, in association with greater depression of the electrocardiographic ST-segment. In contrast, patients with old myocardial infarction exhibited no difference in hemodynamic responses or the ST-pattern from morning to afternoon. The results suggest that diurnal variation of hemodynamic responses to increased oxygen demand may explain, at least partly, why myocardial ischemia of effort angina is more severe in the morning than in the afternoon.

Adult↗
PubMed1993

Detection and measurement of S-benzyl-N-acetylcysteine in urine of toluene sniffers using capillary gas chromatography.

We examined the urinary excretion of S-benzyl-N-acetylcysteine (SBAC) of toluene sniffers using capillary gas chromatography. SBAC was extracted from 10 ml urine with chloroform and backextracted into 1 M sodium bicarbonate solution. After acidification, the aqueous solution was reextracted with ethyl acetate, and then derivatized to its methyl ester (ME). The peak appearing in the gas chromatogram was identified as SBAC-ME by mass spectrometry. The calibration curve was constructed by plotting the peak height ratio of SBAC-ME and internal standard (S-phenethyl-N-acetylcysteine)-ME against analyte concentration using 10 ml toluene unexposed urine. It showed good linearity over the range of 0.05-3.0 mg/l (r = 0.99). We have applied this technique to urine samples from toluene sniffers. SBAC was detected in all urinary samples of sniffers (n = 30, 0.11-47.13 mg/l), but not at all in the urine of toluene unexposed subjects (n = 60). These results prove that SBAC is also formed from toluene by human metabolism, and detection of SBAC is considered a useful marker for inhalation of toluene.

Acetylcysteine↗