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Biomedical subjects

A Abe

Publications and source records attributed to A Abe.

At least 19 recordsLinked to original sources
PubMed1999

Successful culture and sustainability in vivo of gene-modified human oral mucosal epithelium.

Human oral mucosal cells are an attractive site for tissue engineering because they are the most accessible cells in the body and easy to manipulate in vitro. They thus have possibilities for targeting by somatic gene therapy. We examined the efficiency of retrovirus-mediated gene transfer and the construction of mucosal epithelium in vivo. Human oral mucosal cells were transduced with a retroviral vector carrying the lacZ gene at high efficiency and constructed epithelium after G418 selection with 3T3 cells in vitro. The cultured oral mucosal epithelium membrane was then grafted onto immunodeficient mice. Beta-Gal expression was detected histochemically in vivo 5 weeks after grafting. Furthermore, we transduced factor IX cDNA into the mucosal epithelium membrane, and it was then transplanted into nude mice. Between 0.6 and 1.8 ng of human factor IX per milliliter was found in mouse plasma, and the production was continued for 23 days in vivo. These results confirmed that the oral mucosal epithelium is an ideal target tissue for gene therapy or tissue engineering.

Animals↗
PubMed1999

Distribution of 1,4-dioxane in relation to possible sources in the water environment.

The distribution of 1,4-dioxane was investigated in surface and groundwater in relation to possible sources. Water samples from river, coastal sea, and groundwater were collected and examined in Kanagawa Prefecture, Japan, from 1995 to 1998. Almost all samples were contaminated by 1,4-dioxane. On annual changes, the concentrations did not vary significantly in river water. However, some samples were still heavily polluted in groundwater. The highest correlation was between the concentration of 1,4-dioxane and 1,1,1-trichloroethane in the polluted groundwater samples. Soil and groundwater that was polluted by 1,1,1-trichloroethane in the past is still a possible source of 1,4-dioxane. In regard to a survey of other sources, effluents from the combined collection treatment from apartment houses, river basin sewage systems, and chemical plants were examined. The load unit of 1,4-dioxane from the treated effluent of domestic wastewater was assumed to be approximately 0.25 mg/person/day. This was derived from commonly used household chemicals, which contained 1,4-dioxane as a by-product. In river basin sewage systems, the concentrations in effluents varied significantly. High concentration inflows were probably not degraded and released. Although 50-60% of removal was recognized in acclimatized activated sludge from a chemical plant, 1,4-dioxane was easily desorbed with water from sludge.

Dioxanes↗
PubMed1999

Real-time detection system for quantification of hepatitis C virus genome.

BACKGROUND & AIMS: For diagnosis of hepatitis C virus infection and monitoring of viral load in patients, a highly sensitive and accurate hepatitis C virus quantification system is essential. METHODS: Hepatitis C virus genome was detected by real/time detection system using an ABI Prism 7700 sequence detector (Perkin Elmer Corp./Applied Biosystems, Foster City, CA). RESULTS: As few as 10 copies of the genome were detected, and the quantification range was between 10(1) and 10(8) copies (r > 0.99). This system was 10-100-fold more sensitive than an Amplicor monitor (Roche Diagnostic Systems, Branchburg, NJ). The coefficient of variation values for both intra-assay precision and interassay reproducibility of identifying the genome quantification ranged from 0.37% to 2.00% and 0.88% to 4.66%, respectively. The system could detect the genome in 98% of patients with chronic hepatitis, 95.8% of patients with liver cirrhosis, and 100% of patients with hepatocellular carcinoma who had the antibody to hepatitis C virus, but could not detect the genome in patients without the antibody. CONCLUSIONS: The establishment of a real-time detection system enables more accurate diagnosis of infection and monitoring of viral load in interferon-treated patients via quantification of viral genome.

Carcinoma, Hepatocellular↗
PubMed1999

Immune response of post-transplant peripheral lymphocytes against the patient pre-B cell line, NAGL-1.

We have established a pre-B acute lymphoblastic leukemia (ALL) cell line, NAGL-1, from the bone marrow of a patient diagnosed with pre-B ALL. The patient has been disease-free for the 4 years since allogeneic bone marrow transplantation from her HLA-genotypically identical sister. NAGL-1 showed a pre-B cell phenotype (CD19+, CD10+, c mu+, s mu-) mostly identical to freshly isolated leukemic cells from the patient. This cell line strongly expressed HLA class I and HLA-DR molecules, as well as the costimulatory molecules CD54, CD40, and CD86. Cytotoxic T-lymphocyte (CTL) lines were generated by stimulating the donor-derived peripheral blood mononuclear cells with either irradiated leukemic cells or NAGL-1. Both CTL lines showed specific lysis against NAGL-1 in 51Cr release assays. Lytic activity was partially inhibited by anti-CD8 and anti-HLA class I monoclonal antibodies. Treatment of NAGL-1 with TNF-alpha increased its susceptibility to the CTL line. One CD8+ T cell clone derived from the CTL line killed both the patient phytohemagglutinin (PHA) blasts and NAGL-1 but not the donor PHA blasts, suggesting that the clone recognized the patient-specific minor antigen presented on both PHA blasts and NAGL-1. Utilization of leukemic cell lines could be a useful model for the development of CTL lines and clones for immunological study and potential immunotherapy.

Adolescent↗
PubMed1998

Two enteropathogenic Escherichia coli type III secreted proteins, EspA and EspB, are virulence factors.

Enteropathogenic Escherichia coli (EPEC) belongs to a family of related bacterial pathogens, including enterohemorrhagic Escherichia coli (EHEC) O157:H7 and other human and animal diarrheagenic pathogens that form attaching and effacing (A/E) lesions on host epithelial surfaces. Bacterial secreted Esp proteins and a type III secretion system are conserved among these pathogens and trigger host cell signal transduction pathways and cytoskeletal rearrangements, and mediate intimate bacterial adherence to epithelial cell surfaces in vitro. However, their role in pathogenesis is still unclear. To investigate the role of Esp proteins in disease, mutations in espA and espB were constructed in rabbit EPEC serotype O103 and infection characteristics were compared to that of the wild-type strain using histology, scanning and transmission electron microscopy, and confocal laser scanning microscopy in a weaned rabbit infection model. The virulence of EspA and EspB mutant strains was severely attenuated. Additionally, neither mutant strain formed A/E lesions, nor did either one cause cytoskeletal actin rearrangements beneath the attached bacteria in the rabbit intestine. Collectively, this study shows for the first time that the type III secreted proteins EspA and EspB are needed to form A/E lesions in vivo and are indeed virulence factors. It also confirms the role of A/E lesions in disease processes.

Actins↗
PubMed1998

Thyroid hormone regulates expression of shaker-related potassium channel mRNA in rat heart.

Effects of thyroid hormones on cardiac function or rhythm have been known; however, the mechanism is still unclear. In the present study examined were effects of triiodethyronine (T3) on voltage-gated potassium channel gene expression in rat heart since the potassium channels were presumed to modulate cardiac functions. The mRNA expression of five voltage-gated potassium channel gene alpha subunits (Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2) in heart was examined by ribonuclease protection assay in rats which were treated with T3 or propylthyouracil (PTU). All these genes except Kv1.4 mRNA were apparently expressed in the normal rat heart ventricle. Kv1.2 mRNA expression in ventricle was markedly suppressed by T3-treatment and enhanced by PTU-treatment. Interestingly, upregulation of Kv1.4 mRNA expression and downregulation of Kv1.5 mRNA expression were concomitantly induced in the ventricle by the PTU-treatment. In addition, the downregulation of the ventricular Kv1.5 mRNA expression induced by PTU was restored by T3 replacement. No changes of Kv2.1 and Kv4.2 mRNA expression were observed in the ventricles by the T3- or PTU-treatment. In heart atrium the same findings were observed. Kv1.4 mRNA expression, which was detectable in control rat atrium, also decreased significantly by T3-treatment. In contrast, no changes of Kv1.2, Kv1.4, and Kv1.5 mRNA expression in rat brains were induced by T3-treatment. These findings suggest that thyroid hormone specifically influences mRNA expression of Shaker-related potassium channel genes in rat hearts through a common T3 receptor-mediated regulation at a transcriptional level.

Animals↗
PubMed1998

Purification and characterization of 1-O-acylceramide synthase, a novel phospholipase A2 with transacylase activity.

A novel pathway for ceramide metabolism, 1-O-acylceramide formation, was previously reported (Abe, A., Shayman, J. A., and Radin, N. S. (1996) J. Biol. Chem. 271, 14383-14389). In this pathway a fatty acid in the sn-2 position of phosphatidylethanolamine or phosphatidylcholine is transferred to the 1-hydroxyl position of ceramide. An enzyme that catalyzes the esterification of N-acetylsphingosine was purified from the postmitochondrial supernatant of calf brain through consecutive steps, including ammonium sulfate fractionation, DEAE-Sephacel, phenyl-Sepharose, S-Sepharose, Sephadex G-75, concanavalin A-agarose, and heparin-Sepharose chromatography. The molecular mass of the enzyme was determined to be 40 kDa by gel filtration on Sephadex G-75. The enzyme bound to concanavalin A-agarose column was eluted with the buffer containing 500 mM alpha-methyl-D-mannopyranoside. Further purification by heparin-Sepharose chromatography resulted in separation of two peaks of enzyme activity. Coincidence between the transacylase activity and a stained protein of a molecular mass of 40 kDa was observed, as determined by SDS-polyacrylamide gel electrophoresis and recovery after separation over an acidic native gel. The second peak of activity from the heparin-Sepharose chromatography represented a purification of 193,000-fold. These results are consistent with the enzyme being a glycoprotein of a molecular mass of about 40 kDa with a single polypeptide chain. The purified enzyme had a pH optimum at pH 4.5. The divalent cations Ca2+ and Mg2+ enhanced but were not essential for the transacylase activity. Neither activation nor inactivation of the enzyme activity was observed in the presence of 2 mM ATP or 2 mM dithiothreitol. Preincubation of the enzyme with 1 mM N-ethylmaleimide, 1 mM phenylmethylsulfonyl fluoride, or 3.1 microM bromoenol lactone, a potent inhibitor of cytosolic Ca2+-independent phospholipase A2, had no significant effect on the enzyme activity. The enzyme activity was completely abolished in the presence of greater than 773 microM Triton X-100. Partial inhibition of the enzyme activity was observed in the presence of 10-100 microg/ml heparin. In the absence of N-acetylsphingosine, the enzyme acted as a phospholipase A2. These results strongly suggest that 1-O-acylceramide synthase is both a transacylase and a novel phospholipase A2.

Acetyltransferases↗
PubMed1998

The number of kringle IV repeats 3-10 is invariable in the human apo(a) gene.

The human apolipoprotein(a) (apo(a)) gene is a member of a family of related genes including plasminogen, apo(a)rg-B and apo(a)rg-C, which are clustered on chromosome 6q 2,7. Apo(a) contains ten different types of plasminogen-like kringle IV repeats (K-IV 1-10) one of which (K-IV 2) varies in number resulting in a remarkable size polymorphism of the protein. Sequence analysis of human apo(a) alleles and indirect evidence have suggested that K-IV 1 and K-IV 3-10 are each present once in individual alleles and that the 3' apo(a) region encompassing kringles IV 3-10, kringle V and the protease domain is invariable. To directly test this, we have constructed a restriction map of the apo(a) gene region from genomic DNA and from a yeast artificial chromosome (YAC) (K-IV 13) which contains the entire apo(a) gene. The presence of a 63 kb ClaI fragment encompassing kringles IV 3-10, kringle V and the protease domain and a 46 kb SwaI fragment, spanning kringles IV 5-10, kringle V and the protease domain was demonstrated by PFGE/Southern blotting in 30 unrelated subjects, who represented a range of apo(a) size alleles containing from 11 to 49 kringles. Our analysis demonstrates that the number of kringles IV 3-10 is invariable in the human apo(a) gene, suggesting that the 3'domain of Apo(a) is functionally important.

Apolipoproteins↗
PubMed1998

Docosahexaenoic/arachidonic acid omega-hydroxylation system and differentiation in the human colonic adenocarcinoma cell line, Caco-2.

The homogenate from Caco-2 cells of day 13 or 15 after subculturing had high omega-hydroxylation activity of docosahexaenoic acid (22:6(n-3)) or arachidonic acid (20:4(n-6)). Activity, maximal at pH 8.0, was inhibited in the presence of CO or metyrapone and in the absence of NADPH. Omega-hydroxylation activity of lauric acid in the homogenate was not detected. Apparent Michaelis constant (Km) values for 22:6(n-3) and 20:4(n-6) were found to be 4 and 7 microM. Omega-hydroxylation activity considerably increased with growth up to day 13 and then decreased until day 20 even though alkaline phosphatase (ALP) and leucine-aminopeptidase (LAP) activity increased with growth to day 20. Metyrapone in cultures caused omega-hydroxylation, ALP and LAP activity to decrease, while sodium butyrate dose-dependently increased that of omega-hydroxylation, ALP and an endogenous endonuclease and decreased lactate dehydrogenase (LDH) activity in culture medium. The omega-hydroxylation system thus appears quite likely to be associated with cytochrome P450, differentiation and/or apoptosis rather than cytotoxic cell death of Caco-2 cells. Substrate specificity, however, differed from that of human cytochrome P450 4A11.

Adenocarcinoma↗
PubMed1998

Surgical indication for congenital heart disease with extremely thickened media of small pulmonary arteries.

BACKGROUND: Nineteen patients (mean age, 7.6 months) with a percent wall thickness of more than 33% in the small pulmonary arteries were found to have extremely thickened media. Based on our findings, a criterion of operative indication is proposed. METHODS: The percentage of extremely thickened media of small pulmonary arteries for all pulmonary arteries was determined on microscopic lung sections and was introduced as an index for operative indication. RESULTS: Operative repair was performed in 16 patients: 9 died intraoperatively and 7 survived more than 12 months. In 4 of 5 patients that had pulmonary artery banding, medial hypertrophy remained despite pulmonary artery banding. Operative repair also had no positive effect. In operative and late deaths and in survivors without a decrease of pulmonary arterial pressure, the percentage of extremely thickened media of small pulmonary arteries was shown to be more than 10%, whereas in 5 survivors and 1 operative death with a significant postoperative decrease of pulmonary arterial pressure, the value was less than 7%. CONCLUSIONS: If a patient has less than 7% of small pulmonary arteries with extremely thickened media, operative repair is likely to be effective. When the value is higher than 10%, not only operative repair but also pulmonary artery banding cannot be recommended because of ineffectiveness and hazard.

Blood Pressure↗
PubMed1998

Inoperable pulmonary vascular disease in infants with congenital heart disease.

BACKGROUND: Among 120 infants less than 12 months of age who had lung biopsy and autopsy, 20 were inoperable because of severe irreversible pulmonary vascular disease. METHODS: The infants were classified into three groups. Group 1 comprised 6 patients who showed complete obstruction of the small pulmonary arterial lumen and atrophy of the peripheral arterial media and who were considered to have absolute operative contraindications. Group 2 comprised 6 patients who had no pathologic findings of absolute operative contraindication and had an index of pulmonary vascular disease of more than 2.2. They were isolated as having advanced plexogenic pulmonary arteriopathy. Group 3 comprised 8 patients who had extremely thickened media of small pulmonary arteries, with abnormally thickened media extending into the small peripheral arteries characterized by extremely narrow lumina and medial thickness exceeding luminal diameter. RESULTS: Six of the 9 patients in whom operative repair was abandoned on the basis of preoperative or intraoperative lung biopsy are still alive. Of the 11 patients who underwent operation without biopsy, none survived. CONCLUSIONS: Preoperative or intraoperative lung biopsy and assessment of arteriopathy based on the above criteria are recommended in all patients in whom fatal pulmonary vascular disease is suspected.

Atrophy↗
PubMed1998

Changes, with age, in the phospholipid content of the intestinal mucus layer of the newborn rabbit.

BACKGROUND/PURPOSE: The high incidence of bacterial translocation in newborns is thought to be caused, in part, by the immaturity of the intestinal mucosal barrier. Recently, intestinal mucus phospholipids (PL) have been reported to be important factors in the function of this mucosal barrier. The aim of this study was to quantify changes, with age, in the intestinal mucus PL of the newborn rabbit. METHODS: Mucus was gently scraped from the small intestinal mucosal surface of rabbits of different ages (0, 7, 14, and 28 days old and adult; n = 6 for all groups). PL was extracted from the mucus and was separated by two-dimensional thin-layer chromatography. The isolated phospholipid spots were quantified for their phosphorus content. RESULTS: Total PL content of the mucus decreased significantly with age (day 0, 21+/-2; day 7, 16+/-4; day 14, 9+/-3; day 28, 2+/-1; adult, 1+/-1 micromol/g wet mucus; P = .0001). Phosphatidylcholine and phosphatidylethanolamine levels in the adult rabbits were significantly lower in comparison with the 0-, 7-, and 14-day-old pups (P < .05). In contrast, lysophosphatidylcholine and lysophosphatidylethanolamine were significantly higher in the 28-day-old and adult rabbits in comparison with the 0-, 7-, and 14-day-old pups (P < .05). Phosphatidylinositol + phosphatidylserine levels in 7-day-old rabbits was significantly higher compared with adult rabbits. There was no significant difference in the composition of sphingomyeline between groups. CONCLUSION: Significant changes in the content and composition of the intestinal mucus phospholipids were observed during the first month of life in rabbits.

Aging↗
PubMed1998

Recombinant adeno-associated virus-mediated gene transfer into human leukemia cell lines.

Adeno-associated virus (AAV)-based vector is a promising gene transfer vehicle by virtue of the characteristics of wild-type AAV:tropism to a wide range of human tissues and locus-specific integration at chromosome 19q13.3. To elucidate the nature of the recombinant AAV (rAAV), transduction of neomycin phosphotransferase enzyme gene (NeoR gene) into seven human leukemia cell lines was performed. Transduction efficiencies were assessed by colony formation assay and limiting dilution assay. The results suggested that both assays are comparable. Transduction efficiencies of the NeoR gene into K-562, MEG-O1, Raji, MOLT-3, HL-60, U937 and NKM-1 at a multiplicity of infection (MOI) of 0.1 were 0.27, 0.25, 0.015, 0.009, < 0.0025 and < 0.0025%, respectively. After purification and concentration of rAAV, 27% efficiency was observed in K562 at an MOI of 7 and a linear relationship between MOI and efficiency was confirmed, suggesting that this system may be useful for gene transduction into leukemia cells. Integration of the NeoR gene into the host genome was detected by Southern blotting analysis, which showed various sizes of digested fragments. A fluorescent in situ hybridization (FISH) study was carried out on 11 clones, in all of which the NeoR gene was integrated out of chromosome 19q13.3. In five of the clones, whole chromosome painting probes revealed that the integration sites were chromosomes 1q, 2q, 2q, 11p, 12p and 13q.

Dependovirus↗
PubMed1998

Polybrene increases the efficiency of gene transfer by lipofection.

Lipofection involves the introduction of foreign genetic information into mammalian cells through the use of lipophilic reagents that enhance cellular uptake of polynucleotides. Despite the use of currently optimized lipofection conditions, including the use of serum-depleted media, the efficiency of gene transfer is often low. We show here that, in a variety of cell lines, polybrene markedly enhances the efficiency of lipofection under standardized conditions and also compensates the serum-mediated inhibition of lipofection. Although the degree of the polybrene effect depends on the nature of the cell line, these results indicate that individually optimized concentrations of polybrene can be useful for increasing the efficiency of lipofectin-mediated gene transfer in vitro.

Animals↗
PubMed1998

A combination therapy with simvastatin and ursodeoxycholic acid is more effective for cholesterol gallstone dissolution than is ursodeoxycholic acid monotherapy.

Inhibitors of 3-hydroxy,3-methylglutaryl coenzyme A (HMG-CoA) reductase have been reported to decrease the cholesterol saturation index (CSI) in duodenal bile in humans and to prevent formation of cholesterol gallstones in animal studies. We performed a prospective study to evaluate the role of HMG-CoA reductase inhibitors as gallstone-dissolving agents. Fifty patients with radiolucent gallstones in a gallbladder opacifying at drip infusion cholecystography were treated with either 10 mg/day simvastatin plus 600 mg/day ursodeoxycholic acid (group 1, n=26) or 600 mg/day ursodeoxycholic acid alone (group 2, n=24) for 12 months. The ratio of solitary to multiple gallstone cases was 21:29. Plasma lipid levels were assessed and ultrasonographic examination of the gallbladder was performed at baseline and at 3-month intervals during treatment. Duodenal bile sampling was performed in five patients in each group at baseline and after 12 months of treatment. Plasma cholesterol decreased significantly in group 1 but not in group 2. In solitary gallstone cases, no significant difference in dissolution rates was observed between groups 1 (3 of 9, 33%) and 2 (4 of 12, 33%). In contrast, the dissolution rate in multiple gallstone cases was significantly higher in group 1 (12 of 17, 71%) than in group 2 (3 of 12, 25%) (p < 0.01). Bile cholesterol saturation index was significantly decreased (p < 0.01) but did not significantly differ between the two groups. These results suggest that combination therapy with simvastatin and ursodeoxycholic acid is more effective for cholesterol gallstone dissolution than ursodeoxycholic acid monotherapy in patients with multiple gallstones.

Bile↗
PubMed1998

Effect of an inhibitor of glucosylceramide synthesis on cultured human keratinocytes.

Glucosylceramide (GlcCer) is a major glycosphingolipid component of epidermis, which is thought to be related to the barrier function of skin permeability. However, the role of glycosphingolipids in keratinocyte growth and differentiation has not been fully clarified. It has been reported that D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), an inhibitor of GlcCer synthase (EC 2.4.1.80), depletes cells of glycosphingolipids. This inhibitor has been used as a tool for elucidating their functions. In the present study, the effect of PDMP on cultured normal human keratinocytes was investigated. The cells were treated with various concentrations of PDMP. Forty-eight hours later cell growth, thymidine incorporation, and lipid content were studied. The cell growth and the incorporation of thymidine into cells were inhibited by PDMP in a dose dependent manner. The synthesis of GlcCer was strongly inhibited by PDMP treatment, whereas no significant changes in ceramide level were observed. We concluded that GlcCer in epidermis may play an important role in regulating epidermal growth and suggested that PDMP may be beneficial for treating proliferative skin disorders in the future.

Adult↗
PubMed1998

[Experimental studies of polymorphonuclear leukocyte on mandibular bone infection model in rabbit].

Polymorphonuclear leukocytes (PMN) play important roles in the prevention of infection at an early stage. We studied the function of these leukocytes using rabbit models of mandibular bone infection to evaluate the conditions which could not be reproduced in human beings Streptococcus milleri NCTC7331 and Bacteroides fragillis NCTC9343 were inoculated into the mandibular bone of rabbits using the Satoh-Heimdahl method, to produce supposed multiple infection models. Rabbits inoculated with these bacteria were used as a test group, and animals with surgically induced inflammation were used as a control group. We compared the number of leukocytes, the function of PMN, and histopathologic findings. 1) The number of leukocytes increased after treatment, reached a perk on day 3, gradually diminished later, but remained slightly higher than the baseline level on day 7, with persistence of inflammation in both groups. 2) Adhesiveness, ability to migrate and NBT reduction, were accelerated in both groups. 3) These functions of PMN accelerated more in the test group because the bacteria inoculated induced stronger inflammatory reactions and activated a series of infection defense mechanisms in the hosts. 4) Histopathologic examination after treatment showed invasion of inflammatory cells, predominantly leukocytes, in both groups, but heavier and more extensive infiltration in the group treated with the bacteria. All measurements were higher in the test group than the control group. These results showed that in the test group, causative or accompanied microorganisms activated the host's infection defense mechanisms and accelerated the functioning of PMN at an acute stage of infection.

Animals↗