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Y Kono

Publications and source records attributed to Y Kono.

At least 91 records · Page 5Linked to original sources
PubMed1997

Extended primary culture of human hepatocytes in a collagen gel sandwich system.

To develop a strategy for extended primary culture of human hepatocytes, we placed human hepatocytes between two layers of collagen gel, called a "collagen gel sandwich." Maintenance of hepatocellular functions in this system was compared with that of identical hepatocyte preparations cultured on dry-collagen coated dishes or cocultured with rat liver epithelial cells. Human hepatocytes in a collagen gel sandwich (five separate cultures) survived for more than 4 wk, with the longest period of culture being 78 d. They maintained polygonal morphology with bile canaliculuslike structures and high levels of albumin secretion throughout the period of culture. In contrast, hepatocytes on dry-collagen became feature-less, and albumin secretion could not be detected after 14 d of culture. This loss of albumin secretion was partially recovered by overlaying one layer of collagen gel. Ethoxyresorufin O-deethylase activity, associated with cytochrome P450 1A2, was detected basally up to 29 d in collagen gel sandwich culture. These activities were induced four- to eightfold after induction with dibenz(a,h)anthracene. Cocultures also maintained basal activity up to 29 d. However, their inducibility was lower than that of hepatocytes in collagen gel sandwich. No ethoxyresorufin O-deethylase activity was detected in hepatocytes cultured on dry-collagen at 7 d. Thus, the collagen gel sandwich system preserves differentiated morphology and functions of human hepatocytes in primary culture for a prolonged period of time. This system is a promising model for studying human hepatocellular function, including protein synthesis and drug metabolism in vitro.

Adolescent↗
PubMed1997

High-pass resolution perimetry and a Humphrey Field Analyzer as indicators of glaucomatous optic disc abnormalities. A comparative study.

BACKGROUND: The "neural capacity (NC)" of high-pass resolution perimetry (HRP) may be correlated to the neuroretinal rim area of the optic disc. The authors compared HRP with conventional differential light sensitivity (DLS) perimetry in terms of correlation with glaucomatous optic disc changes. METHODS: Thirty-seven eyes of 37 normal-tension glaucoma (NTG) patients were examined. The authors used an Ophthimus system for HRP and a Humphrey Field Analyzer (HFA) for DLS perimetry. The total rim area and the area of temporal, superior, nasal, and inferior quadrants were determined using a confocal laser tomographic scanner. Quadrant NC (QNC) was calculated using the resolution thresholds corresponding to a given quadrant. Quadrant mean deviation (QMD) was calculated from the total deviations of HFA. RESULTS: In all eyes, NC and MD showed significant correlations with total rim area (Spearman rank correlation coefficient [rs] = 0.375, P < 0.05 for NC; rs = 0.336, P < 0.05 for MD). A significant correlation was observed in 21 eyes with early visual field changes (rs = 0.469, P < 0.05), but not in 16 eyes with advanced visual field changes. Both QNC and QMD showed significant correlations with inferior rim area (rs = 0.575, P < 0.01 for QNC; rs = 0.431, P < 0.01 for QMD). However, no significant correlations were found between QNC or QMD and superior rim area. CONCLUSIONS: The NC may relate to glaucomatous optic disc changes better than does MD in NTG, especially in eyes with early-stage visual field changes.

Adult↗
PubMed1997

Hepatitis B virus replication and viral antigen synthesis in hepatocyte lines derived from normal human liver.

Transient transfection and in vitro infection experiments were performed to characterize replication and antigen synthesis of the hepatitis B virus (HBV) in human hepatocyte lines HH29 and HHY41, derived from normal liver tissue. These liver cell lines are capable of supporting HBV replication and gene expression at levels similar to the human hepatoma cell line HuH-7. Strikingly, a very tight adhesion of HBV to the outer cell membrane of HH29 and HHY41 was observed under conditions that removed HBV to undetectable levels from HuH-7 hepatoma cells. However, no productive HBV infection could be established in these cells as determined by the absence of viral transcripts and de novo antigen synthesis. In conclusion, the human hepatocyte cell lines HH29 and HHY41 may be useful to study important aspects of late steps in the replication of HBV, but appear to lack certain cellular components that play a pivotal role during early steps of the viral life cycle.

Cell Line↗
PubMed1997

Gray scale second harmonic imaging of the liver: a preliminary animal study.

Gray scale second harmonic imaging (2.5 MHz/5.0 MHz) was evaluated in preliminary animal studies with a new ultrasound contrast agent (FS069). FS069 was administered intravenously in 10 rabbits (6 with normal liver, and 4 with implanted VX-2 tumors) and two woodchucks with hepatocellular carcinomas. The vasculature (including tumor vessels) and liver parenchyma were clearly enhanced at a low dosage (optimal dose was from 0.1 to 0.2 mL/kg) in all cases. Enhancement was reproducible and generally dose-dependent. Tumors were enhanced well during the early phase and tumor enhancement disappeared earlier than that of surrounding normal liver. Arterial phase and portal phase were easily distinguished and patterns of enhancement were diagnostic of the tumors. Gray scale second harmonic imaging is useful in the detection of hepatic tumors and in diagnosis of their hemodynamics.

Albumins↗
PubMed1997

Gray-scale second harmonic imaging of the liver with galactose-based microbubbles.

RATIONALE AND OBJECTIVES: The authors evaluate the efficacy of SHU 508A, a galactose-based contrast agent used in gray-scale second harmonic imaging in vitro and in vivo experiments. METHODS: A Toshiba prototype harmonic imaging system (2.5/5.0 MHz) was used with SHU 508A in a phantom experiment and to image the liver in five healthy rabbits and one rabbit that had VX-2 tumors in the liver. RESULTS: In the second harmonic imaging, most of the fundamental components of the backscattered echo were eliminated, and good images with high contrast were obtained in the phantom experiment. Liver parenchyma was enhanced clearly in all rabbits at 0.3 mL/kg, an effect that lasted for 90 seconds. The tumor, which was mostly necrotic, was depicted clearly as a negative enhanced area surrounded by enhanced healthy liver. CONCLUSIONS: Gray-scale second harmonic imaging is a promising new method for visualization of perfusion of organs.

Animals↗
PubMed1997

Insertions, duplications and substitutions in restricted gp90 regions of equine infectious anaemia virus during febrile episodes in an experimentally infected horse.

We have studied a horse which exhibited typical clinical signs of disease when experimentally infected with a non-adapted virulent strain of equine infectious anaemia virus (EIAV), designated V70. Five viruses (F1V, F2V, F3V, F4V and F5V) were recovered during periodic febrile episodes. Cross-neutralization tests revealed that all of these variants and the parental V70 were antigenically distinct. Sequencing of their full-length env gp90 genes and gp45 5' sequences revealed novel mutations at a limited number of nucleotide positions, consisting of insertions and duplications in the gp90 principal neutralizing domain (PND) in F1V, F3V and F5V. Parts or all of small units (6, 9 and 12 nucleotides) located just before the insertion site were used for the duplications. Furthermore, amino acid substitutions in the env PND and hypervariable region were also observed in all five viruses. These mutations may contribute to the generation of serial variants. Consequently, the full-length gp90 sequences showed close relationships between V70, F2V and F4V, and between F1V, F3V and F5V. In addition to the two domains (PND and hypervariable region), a comparison of these viruses with the reported env gp90 sequences revealed four additional variable domains, although these four domains were highly conserved among the five variants.

Amino Acid Sequence↗
PubMed1997

Absence of the mutant SOD1 in familial amyotrophic lateral sclerosis (FALS) with two base pair deletion in the SOD1 gene.

We determined the activity, content and mRNA of Cu/Zn superoxide dismutase (SOD1), and copper ion concentration in a Japanese pedigree of familial amyotrophic lateral sclerosis (FALS) having two basepair deletion in the 126th codon of the SOD1 gene. The activity and concentration of the SOD1 were low in red blood cells from the patients and the unaffected subjects with the SOD1 mutation. The SOD activity stain and Western blot analysis of the brain from one of the patients showed low SOD1 activity and the absence of the mutant SOD1. The mRNA due to the mutant SOD1 gene was, however, confirmed. Availability of the copper ions for oxidative catalytic DNA damage in the brain from the patient was 1.9-fold higher than those in the controls. We propose that the decrease of SOD1 activity and increased copper ions play a role in the neuronal death in this FALS.

Aged↗
PubMed1997

A new scoring system for comparing the results of high-pass resolution perimetry and differential light sensitivity perimetry in glaucoma patients.

Using a new standard, we compared the visual field defects detected by high-pass resolution perimetry (HRP) and differential light sensitivity (DLS) perimetry in 61 eyes of 61 open-angle glaucoma patients. Abnormal test points were determined using probability plot analysis of HRP and total deviation probability maps of the Humphrey Field Analyzer. To evaluate the agreement between the two tests, we developed a new scoring system. Thirty-three eyes, 23 eyes, and 5 eyes had good, fair, and poor agreement, respectively. In relation to Aulhorn's stage classification, the more advanced the glaucomatous stage, the more increased the percentages of good agreement. These results suggest that the distribution and severity of visual field defects demonstrated by HRP agree closely with those found using DLS perimetry in glaucomatous eyes, especially those with advanced visual field changes.

Adolescent↗
PubMed1997

In vivo dynamics of equine infectious anemia viruses emerging during febrile episodes: insertions/duplications at the principal neutralizing domain.

Equine infectious anemia virus (EIAV) is a good model for studying mechanisms generating escaped retrovirus variants. We previously sequenced the entire gp90-encoding region of 22 cDNA clones obtained from five antigenically distinct isolates (F1V to F5V) recovered during febrile episodes in horse 493 experimentally infected with the Japanese virulent EIAV strain V70. The results showed that the mutations occurred in the principal neutralizing domain (PND) by insertions/duplications. In this study, we further characterized the PND of virus isolates sequentially recovered during 22 febrile episodes in seven horses newly infected with V70 or one of the V70-derived variants. Sequencing of 70 cDNA clones derived from the 22 episodes confirmed the generation of various new viral quasispecies with insertions/duplications in the PND. Although the insertion/duplication sequences in a total of 92 cDNA clones were extensively heterogeneous, we hypothesized that all the insertions/duplications occurred during reverse transcription from viral genomic RNA to minus strand DNA. The insertion/duplication regions were derived from a part of the PND sequence, which consisted of five small units. These small units, some with various substitutions and/or deletions, were also generated, especially in regions with insertions/duplications. Of particular note was that all these virus variants, except for two cDNA variants, were generated by essentially four different duplication pathways. Thus, these results extend the significance of insertions/duplications in the PND to the novel generation of EIAV in vivo during febrile episodes.

Amino Acid Sequence↗
PubMed1997

Genetic variation of envelope gp90 gene of equine infectious anemia virus isolated from an experimentally infected horse.

Six strains of equine infectious anemia virus (EIAV) were recovered from febrile and non-febrile stages of a horse experimentally infected with the P337-V70 strain given once to a horse. The env gp90 genes of the isolates, the P337-V70 and P337-V26, avirulent virus derived from the P337-V70 strain, were sequenced. A comparison of the gp90 gene sequences revealed that amino acid variations among the viruses tested showed as high as 8.2 to 11.5%. In addition, the comparison also indicated that the isolates that recovered from the non-febrile stage were contained in nucleotide insertions in the principal neutralizing domain (PND) region. The insertions were arranged regularly with smaller segments. The nucleotide sequence of the P337-V26 gp90 gene was found to contain a six-nucleotides insertion and seven nucleotide substitutions outside the PND region, when compared with that of the P337-V70 strain.

Amino Acid Sequence↗
PubMed1997

Effects of cholestatic agents on the structure and function of bile canaliculi in neonatal rat hepatocytes in primary culture.

The effects of cytochalasin B and colchicine on the structure and function of bile canaliculi were studied in neonatal rat hepatocytes in primary culture. Cellular contacts of neonatal hepatocytes were not as tight as those of adult hepatocytes. There was no remarkable difference in the ultrastructure of bile canaliculi between neonatal and adult hepatocytes. Neonatal hepatocytes treated with cytochalasin B were round in shape and aggregated in groups of several cells. Actin filaments stained by rhodamine-phalloidin were disrupted and condensed at the cell periphery or around dilated bile canaliculi. Markedly-dilated bile canaliculi with less microvilli were observed by transmission electron microscopy while the secretory function of horseradish peroxidase, which was used as a marker for uptake, transport and secretion into bile canaliculi, were maintained. The lumen of dilated bile canaliculi was found close to the undersurfaces of hepatocytes by scanning electron microscopy after turning over the cultured cells. By colchicine treatment, the filamentous structure of microtubules in neonatal hepatocytes disappeared. The ultrastructure of the bile canaliculi was not affected by the treatment, but transport and secretion of horseradish peroxidase into bile canaliculi were inhibited. The development of strict cellular polarity in neonatal hepatocytes may be suppressed in neonatal hepatocytes; however, cholestatic agents which rearrange the cytoskeleton caused the same morphological or functional changes of bile canaliculi as in adult hepatocytes.

Animals↗
PubMed1997

Sonographic detection of tumor blood flow using a new contrast agent in woodchuck hepatomas.

The aim of this study was to assess the ability of perfluoropropane-filled albumin microspheres to visualize tumor blood flow in woodchuck hepatomas. Ten tumors in five woodchucks with hepatomas were imaged before and after intravenous injection (dosages of 0.01 ml/kg to 2.0 ml/kg) of an agent using color Doppler sonography and gray scale ultrasonography. Both enhanced imaging modalities demonstrated blood flow around and within all tumors. Enhanced gray scale ultrasonography demonstrated sonographic "tumor blush" in seven tumors (70%). In conclusion, tumor blood flow in woodchucks was visualized more clearly using the agent on both color Doppler and gray scale ultrasonography.

Albumins↗
PubMed1996

Molecular cloning of manganese catalase from Lactobacillus plantarum.

A genomic clone encoding manganese-containing catalase has been isolated from lactic acid bacterium Lactobacillus plantarum, sequenced, and expressed in Escherichia coli cells with an inducible expression system. The primary structure of the enzyme deduced from the nucleotide sequence, that comprises 266 amino acid residues, showed no significant homology with that of any other proteins registered on the available data bases. No peptide motifs conserved among active sites of proteins including manganese-containing enzymes were found. The E. coli cells carrying an expression construct, in which the 5'-noncoding region of the manganese catalase gene was replaced with the lac promoter, highly induced a protein reacting with the antiserum to manganese catalase. The prediction of secondary structure from the deduced primary structure suggested that the L. plantarum manganese catalase, that is classified as a novel protein on the basis of its primary structure, has a main structural motif formed by four near parallel helices between which is the catalytic site manganese.

Amino Acid Sequence↗
PubMed1996

Modulation of the expression of liver-specific functions in novel human hepatocyte lines cultured in a collagen gel sandwich configuration.

Liver-specific functions were examined in two novel human hepatocyte lines established from normal liver tissue (HH29, HHY41) and one human hepatoma line (Hep G2), cultured either in a collagen gel sandwich configuration or on plain plastic dishes. At confluence, levels of albumin and alpha-fetoprotein mRNA were markedly increased in all lines cultured in the collagen gel sandwich over those on plastic, while levels of mRNA for mitochondrial elongation factor (EFTU), a ubiquitous enzyme, were equivalent. Secretion of albumin by cells cultured in the collagen gel sandwich was significantly increased in parallel with the abundance of albumin mRNA. Hepatocytes in the collagen gel sandwich configuration secreted alpha-fetoprotein of a higher molecular weight in addition to alpha-fetoprotein of standard molecular weight. The three-dimensional configuration of the collagen gel sandwich enhances the expression of liver-specific functions of human hepatocytes from continuous cell lines, probably by preserving proper cellular polarity in vitro.

Carcinoma, Hepatocellular↗
PubMed1996

Positive association between a DNA sequence variant in the serotonin 2A receptor gene and schizophrenia.

Sixty-two patients with schizophrenia and 96 normal controls were investigated for genetic association with restriction fragment length polymorphisms (RFLPs) in the serotonin receptor genes. A positive association between the serotonin 2A receptor gene (HTR2A) and schizophrenia was found, but not between schizophrenia and the serotonin 1A receptor gene. The positive association we report here would suggest that the DNA region with susceptibility to schizophrenia lies in the HTR2A on the long arm of chromosomes 13.

Adult↗
PubMed1996

Nested PCR for detection and typing of porcine reproductive and respiratory syndrome (PRRS) virus in pigs.

A nested polymerase chain reaction (PCR) was developed to detect porcine reproductive and respiratory syndrome (PRRS) virus. A common primer set for European and North American type isolates of PRRS virus was designed for reverse transcription PCR, and a specific primer set for each of the 2 type isolates was designed for nested PCR. The PCR that used a specific primer set detected the corresponding type of the virus at a level equivalent to 1 TCID50/100 microliters, but not the other type of isolates. Therefore, the method clearly differentiated the 2 types of virus from each other. The detection of PRRS virus by the nested PCR was as sensitive as virus isolation in cultures of porcine alveolar macrophages from infected pigs at the acute stages, and was more sensitive from pigs at the convalescent stages. The infecting virus type was determined by use of 2 specific primer sets even when virus isolation was negative in naturally infected pigs. It was concluded that the nested PCR is useful for diagnosis and typing of PRRS virus and studies of persistent infection by the virus.

Animals↗