Change of hepatitis B virus DNA and covalently closed circular DNA status in liver graft after liver transplantation.

Recurrence of hepatitis B virus (HBV) occurs despite prophylaxis, and covalently closed circular DNA (cccDNA) is thought to play a role owing to its resistance to prophylactic agents used. The aim of this study was to evaluate the changes of HBV DNA and cccDNA within the liver graft during liver transplantation (LT). Polymerase chain reaction (PCR) primers and probes were designed to measure total HBV DNA (tDNA) and cccDNA by real-time PCR.

Antitumor activity of ZD6474 in a metastatic orthotopic brain tumor model.

The objective of this study was to examine the antitumor effect of ZD6474, an orally available inhibitor of the vascular endothelial growth factor receptor-2 (VEGFR-2) and the epidermal growth factor receptor (EGFR), on tumor growth in an orthotopic metastatic brain tumor model. In order to determine the antitumor mechanism of ZD6474 treatment, in vitro and in vivo studies were performed. Human breast carcinoma cells (MDA-MB-435) were injected using direct intracranial (IC) inoculation (5x105 cells/100 µl) and internal carotid artery (ICA) injection (5x104 cells/100 µl) in Balb/c-nu female mice.

Stable expression of antisense Rb-1 RNA inhibits terminal differentiation of mouse myoblast C2 cells.

To determine the roles of the retinoblastoma gene (Rb-1) in skeletal muscle differentiation in vitro, we isolated C2 myoblasts stably expressing an antisense RNA directed to the 3'-untranslated region (3'UTR) of Rb-1 mRNA. The levels of Rb-1 mRNA and its product (pRb) in the clones transfected with antisense Rb were markedly decreased to 25-35% of those in the control clone. Cell growth of the clones was accelerated, especially in medium containing low concentrations of fetal calf serum.

Apoptosis was promoted at a nonpermissive temperature in DNA replication-defective temperature-sensitive mutants of mouse FM3A cells.

Apoptosis was promoted at the nonpermissive temperature in some temperature-sensitive (ts) mutant strains of mouse FM3A cells deficient in initiation of DNA replication. We examined expression of cell cycle regulation genes in the four ts mutant strains and found that two strains, tsFT107 and tsFT111, exhibited marked accumulation of p53 protein by a posttranscriptional mechanism at 16 h after temperature up-shift. These two strains also exhibited high levels of p21 mRNA expression, repression of cyclin A and D1 mRNAs, and obvious accumulation of underphosphorylated retinoblastoma protein.

Abnormalities in behavior, learning ability, and the cholinergic system induced by long-term ultraviolet A irradiation of mice.

Behavioral abnormalities, jumping reaction, increase in spontaneous activity abnormal violence, and lethargy were observed in long-term ultraviolet A (UVA)-irradiated hairy male Crj:CD-1 mice. The learning ability of 6- and 12-months UVA-irradiated mice was significantly reduced compared to un-irradiated age-matched mice. Acetylcholine levels, acetylcholinesterase and choline acetyltransferase activities in the whole brains were decreased in both of 6- and 12-month irradiated mice.

The effects of ultraviolet A and reactive oxygen species on the mRNA expression of 72-kDa type IV collagenase and its tissue inhibitor in cultured human dermal fibroblasts.

The effects of ultraviolet A (UVA) radiation and reactive oxygen species (ROS), generated with a xanthine and xanthine oxidase (XOD) system, on collagen enzymatic degradation involving the matrix metalloproteinase (MMP) and its tissue inhibitor of metalloproteinase (TIMP) were investigated using cultured human dermal fibroblasts. Total RNA was isolated and subjected to Northern blot analysis using cDNA clones for human interstitial collagenase (MMP-1), 72-kDa type IV collagenase (MMP-2) and TIMP-2. UVA irradiation resulted in an increase in MMP-1 mRNA up to 2.

Arrest of cell growth by necdin, a nuclear protein expressed in postmitotic neurons.

Necdin is a 325 amino acid residue protein localized to the nuclei of postmitotic neurons, which withdraw permanently from the cell cycle. To examine whether necdin confers the postmitotic phenotype, necdin cDNA was stably transfected into NIH3T3 cells, in which the protein was conditionally expressed using a eukaryotic lac repressor-operator expression system. When the transfectants were induced to express ectopic necdin, cell growth was arrested without appreciable reduction in cell viability.

Evaluation of seven alternative assays on the main ingredients in cosmetics as predictors of Draize eye irritation scores.

This study evaluates seven alternative assays carried out on the main ingredients in cosmetics to determine which battery is the best set and what is the best predictor of the maximal Draize rabbit eye irritation scores (MDESs). The assays consisted of the maximal primary Draize rabbit skin irritation scores (MDSSs), a cytotoxicity test on neutral red uptake using Chinese hamster lung cells (NR-EC(50)), a cytotoxicity test on MTT using normal skin fibroblasts (MTT-EC(50)), the hen's egg test-chorioallantoic membrane test using fertile chicken eggs (HET-CAM), a haemolysis test using red blood cells from Wistar rats (HC(50)), a protein denaturation test using haemoglobin from bovine (HDR), and pH. We tested 10% solutions of 24 tested chemicals, that is, 20 surfactants, three solvents and formaldehyde, to select from the assays a best set for prediction (x) and to obtain the best predictor [f(x)] based on the prediction sum of squares criterion.

Antiallergic constituents from oolong tea stem.

The antiallergic constituents of oolong tea stem were examined. The stem extracts inhibited the 48 h homologous passive cutaneous anaphylaxis (PCA) reactions or rats in a dose-dependent manner and showed the same extent of inhibitory activity as ketotifen. All antiallergic constituents from the stem were concentrated into chloroform and ethyl acetate fractions, when extracted by various solvents.

Alzheimer amyloid protein precursor enhances proliferation of neural stem cells from fetal rat brain.

We have investigated the effect of the Alzheimer amyloid protein precursor (APP) on the proliferation of neural stem cells. Two secretory forms of APP (sAPP770 and sAPP695, with and without the Kunitz-type serine protease inhibitor domain, respectively) were purified from conditioned media of COS-7 cells transfected with genetically modified APP cDNAs. Both secretory APPs promoted the growth of neural stem cells, and the effect of sAPP770 was greater than that of sAPP695.

Purification and characterization of D-xylose isomerase from Bifidobacterium adolescentis.

D-Xylose isomerase was purified to homogeneity from cell-free extracts of Bifidobacterium adolescentis by ammonium sulfate fractionation and chromatographies on DEAE-cellulose and Butyl-Toyopearl. The molecular weight of the purified enzyme was estimated to be 168,000 by gel filtration on TSKgel G-3000SW, and 53,000 on SDS-polyacrylamide gel electrophoresis. The optimum pH was around 7 and the enzyme was stable at pH 7-8.

The effect of reactive oxygen species on the biosynthesis of collagen and glycosaminoglycans in cultured human dermal fibroblasts.

The purpose of this study was to evaluate the possibility that the biological changes observed in connective tissue matrix components of photoaging skin may be induced by an alteration of biosynthesis in fibroblasts damaged by reactive oxygen species (ROS). We investigated the effect of ROS induced by xanthine and the xanthine oxidase system on the biosynthesis of connective tissue matrix components, collagen and glucosaminoglycans (GAGs) in cultured human dermal fibroblasts. ROS decreased collagen production and increased GAGs synthesis.

Protease inhibitors generate cytotoxic fragments from Alzheimer amyloid protein precursor in cDNA-transfected glioma cells.

A human glioma cell line (Bu-17) was stably transfected with full-length cDNA encoding beta/A4 amyloid protein precursor (APP). When the transfectants were treated with protease inhibitors (leupeptin, E-64, and antipain) and the lysosomotropic agent chloroquine, aberrantly processed fragments of APP having molecular sizes of 8-30 kDa were detected with an antibody against the carboxyl-terminal sequence of APP. Immunocytochemistry revealed that these fragments were localized in the lysosome-like organelles.

Effects of L-ascorbic acid on the mutagenicity of ethyl methanesulfonate in cultured mammalian cells.

The effects of L-ascorbic acid (AsA) on the mutations induced by ethyl methanesulfonate (EMS) were examined by means of the 6-thioguanine (6TG)-resistant mutation assay and chromosome aberration assay in cultured Chinese hamster V79 cells. When cells were treated with EMS at various concentrations in the presence of 100 micrograms/ml AsA, EMS-induced 6TG-resistant mutations were reduced about one third or one fourth. EMS-induced chromosome aberrations were also reduced by AsA.

Combined mutagenicity of methyl methanesulfonate and ethyl methanesulfonate in Chinese hamster V79 cells.

The combined effects of methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) on the induction of 6-thioguanine (6TG)-resistant mutants and chromosome aberrations were examined in Chinese hamster V79 cells. Cells were simultaneously treated with EMS at a concentration of D20 and MMS at various concentrations for 3, 6 or 9 h. In other experiments cells were simultaneously treated with MMS at a concentration of D20 and EMS at various concentrations for 3, 6 or 9 h.

Repression of integrin beta 1 subunit expression by antisense RNA.

A quail cell line (QT6-c) was co-transfected with pTEX vector expressing RNA complementary to chicken integrin beta 1 subunit mRNA (Anti-Int) and pRSVneo vector by a calcium phosphate method. Transfectants showing reduced expression of quail integrin beta 1 subunit were selected with an immunoblot assay, and a few positive clones were examined in detail. Northern blot and immunoblot analyses revealed that the Anti-Int caused a clear reduction of the transcript encoding integrin beta 1 subunit depending on culture conditions.

Expression of chicken integrin beta 1 subunit in rat PC12 cells.

We transfected rat pheochromocytoma (PC12) cells with a cDNA encoding chicken integrin beta 1 subunit. The chicken integrin beta 1 subunit produced in stable transfectants associated with two major alpha subunits of rat integrins to form interspecific chimeric receptors. These receptors mediated cell spreading and initial neurite outgrowth on laminin as did corresponding endogenous integrins, although they were slightly less effective in inducing cell adhesion to laminin.

Cloning of the Membrane-Bound Aldehyde Dehydrogenase Gene of Acetobacter polyoxogenes and Improvement of Acetic Acid Production by Use of the Cloned Gene.

A genomic clone bank of Acetobacter polyoxogenes NBI1028 constructed in Escherichia coli by use of the expression vector pUC18 was screened with antibody raised against membrane-bound aldehyde dehydrogenase (ALDH; 75 kilodaltons [kDa]) from A. polyoxogenes NBI1028. A clone that synthesized a 41-kDa protein cross-reactive with anti-ALDH antibody was isolated.

Identification of secretory immunoglobulin A in human sweat and sweat glands.

Secretory immunoglobulin A (sIgA) plays an important role in local immune defense mechanisms. Although skin is always exposed to external antigens, the role of local immune defenses involving sIgA in the skin has not been adequately studied. In order to evaluate the presence of sIgA in sweat, we have measured the concentration of sIgA in human sweat by enzyme immunoassay and have localized the components of sIgA in the sweat glands of human axillary skin.

Urinary metabolites of N-nitrosodibutylamine in the rat: identification of N-acetyl-S-butyl-L-cysteine derivatives.

N-Acetyl-S-(butyl, 3-oxobutyl and 3-hydroxybutyl)-L-cysteines have been isolated and identified (as their methyl esters) from the urine of rats given N-nitrosodibutylamine (NDBA), N-nitrodibutylamine (NTDBA) and their corresponding alpha-acetoxy derivatives, N-nitroso-N-butyl(1-acetoxybutyl)amine and N-nitro-N-butyl(1-acetoxybutyl)amine, respectively. Greater amounts of these L-cysteine derivatives were detected in urine after administration of NDBA than of NTDBA. This suggests that the markedly different biological activities of NDBA and NTDBA might be due, in part, to a difference in their alkylating abilities in vivo.

Directly-acting mutagens formed from N-nitroso-N-(formylmethyl)alkylamines.

Directly-acting mutagens formed from N-nitroso-N-(formylmethyl)alkylamines (I) were isolated and identified as N-nitroso-N-alkyl-1-hydroxyimino-2-oxoethylamines (II). Their structures were elucidated on the basis of nuclear magnetic resonance spectra and confirmed by leading to their crystalline 2,4-dinitrophenylhydrazone. II (alkyl = ethyl and n-butyl) were strongly mutagenic to Salmonella typhimurium TA1535 and Escherichia coli WP2 hcr- without metabolic activation, while II with a tert-butyl group was not mutagenic.