Transient expression of Drosophila melanogaster deoxynucleoside kinase gene enhances cytotoxicity of nucleoside analogs

The Drosophila melanogaster deoxynucleoside kinase gene was introduced into HeLa cells with cationic lipids to allow its transient expression, and cytotoxic effects of several nucleoside analogs in the transfected cells were examined. Of the analogs tested, cytotoxicities of 1-beta-D-arabinofuranosylcytosine (araC), 5-fluorodeoxyuridine (FUdR), and 1-(2-deoxy-2-methylene-beta-D-erythro-pentofuranosyl)cytosine (DMDC) were increased by the deoxynucleoside kinase gene. These results suggest that the combination of the transient expression of the Drosophila deoxynucleoside kinase gene and these nucleoside analogs is a candidate for the suicide gene therapy.     

α7 mutants mimicking atypical motifs (YxxCC of loop-C, and E to H at −1′ in TM2) in the C. elegans LEV-8 subunit affect nicotinic acetylcholine receptor function

The ACR-8-like group of C. elegans nicotinic acetylcholine receptor (nAChR) subunits contain unusual motifs in the ACh binding site and in the −1′ position of transmembrane region two (TM2). Using site-directed mutagenesis (SDM) we have introduced these motifs into chicken α7 as it has not been possible to express C. elegans nAChR in vitro. Oocytes expressing α7 with the C. elegans binding motif show a reduced affinity and efficacy for both ACh and nicotine. The blocking action of the anthelmintic drug levamisole is reduced. The TM2 motif resulted in a non-functional receptor. We conclude that the TM2 motif profoundly restricts cation movement through the α7 channel but does not confer anion permeability. The altered form of the ACh binding motif is likely to result in a receptor with altered pharmacology, adding potential functional diversity at synapses in the nervous system and neuromuscular junctions of C. elegans.     

Structural characterization of the 16-kDa allergen, RA17, in rice seeds. Prediction of the secondary structure and identification of intramolecular disulfide bridges

The 16-kDa rice allergen, RA17, belonging to the alpha-amylase/trypsin inhibitor family was isolated from rice seed and structurally characterized by identifying cystine-containing peptides and predicting the secondary structure and hydrophobic regions. Eight peptides, which constitute three sets of cystine-containing peptides, were purified by HPLC from a thermolytic digest of RA17 and identified by their amino acid sequence and composition, indicating five intramolecular disulfide bridges: Cys34-Cys94, Cys26-(Cys50 or Cys51)-Cys110 and Cys12-(Cys62 or Cys64)-Cys122. Analyses of the CD spectrum and the Chou-Fasman prediction suggested that RA17 had some helical- and sheet-structure regions. Based on these experimental and predicted data, RA17 is proposed to be a globular molecule with a small hydrophobic core having folding restricted by five intramolecular disulfide bridges.     

Crystallographic studies on damaged DNAs. I. An N(6)-methoxyadenine residue forms a Watson-Crick pair with a cytosine residue in a B-DNA duplex

Oxyamines such as hydroxylamine and methoxylamine disturb DNA replication and act as potent mutagens, causing nucleotide transition from one purine to another or one pyrimidine to another. In order to investigate mismatch base-pairing in DNA damaged with oxyamines, a dodecamer with the sequence d(CGCGmo(6)AATCCGCG), where mo(6) A is 2'-deoxy-N(6)-methoxyadenosine, was synthesized and its crystal structure determined. No significant conformational changes are found between the present dodecamer and the original undamaged B-form dodecamer. Electron density maps clearly show that the mo(6)A residue forms a base-pair with a 2'-deoxycytidine residue through hydrogen bonds similar to a Watson-Crick G.C base-pair. For these hydrogen bonds to be made, N(6)-methoxyadenine must chemically take the imino form. The methoxylation thus enables the adenine base to mimic a guanine base. As a result, misincorporation of 2'-deoxycytidine instead of thymidine, or 2'-deoxyadenosine instead of 2'-deoxyguanosine, can occur in DNA replication.     

Cytogenetic mapping of 31 functional genes on chicken chromosomes by direct R-banding FISH

Using direct R-banding fluorescence in situ hybridization, we determined the location of 31 functional genes on chicken chromosomes. Replication R-banded chromosomes were obtained by synchronizing splenocyte cultures with excessive thymidine, followed by BrdU treatment. Thirty-one functional genes were directly localized to banded chicken chromosomes using genomic DNA and cDNA fragments as probes. The possibility of conserved linkage homology between chicken and human chromosomes was demonstrated for seven chicken chromosome regions (1p, 1q, 2q, 4p, 4q, and 5q).     

Kupffer cell-mediated down regulation of rat hepatic CMOAT/MRP2 gene expression

Lipopolysaccharides (LPS) induces intrahepatic cholestasis and canalicular multispecific organic anion transporter (CMOAT/MRP2) plays a central role in hepatic bilirubin transport. This study examined the role of Kupffer cell in LPS-induced cholestasis. Rats were injected intravenously with LPS. Kupffer cells were inactivated with gadolinium chloride (Gd). CMOAT/MRP2 mRNA expression was time- and dose-dependently decreased by LPS injection with a decrease in bile flow and an increase in serum bilirubin level. Gd pretreatment inhibited decrease in CMOAT/MRP2 mRNA and bile flow, and increase in serum bilirubin. Kupffer cell-conditioned medium decreased CMOAT/MRP2 expression. Addition of anti-IL-1 or anti-TNFalpha antibody restored CMOAT/MRP2 expression, whereas IL-1 and TNFalpha decreased the expression. MAP kinases were activated by addition of the conditioned medium, and addition of PD98059 or SB203580 restored CMOAT/MRP2 expression. These results suggest that LPS activates Kupffer cells to secrete IL-1 and TNFalpha, which in turn activate MAP kinases and decrease CMOAT/MRP2 expression.     

Closely Linked Polymorphic Markers for Determining the Autosomal Dominant Allele (Cy) in Rat Polycystic Kidney Disease

The Han:SPRD strain is an SD-background strainknown to be a model of polycystic kidney disease (PKD)expressed through an autosomal dominant gene (Cy).However, different genotypes of this strain cannot be identified in the neonatal period. First, toestablish an accurate method of determining thegenotypes (Cy/Cy, Cy/+, +/+) which cause differentdisease progressions, we used polymorphic markers on rat chromosome 5. PCR products of tissue DNAtemplated with D5Rat9 showed distinct patterns onelectrophoresis indicating three genotypes. Second, todetermine whether the same locus plays a major role inexpressing PKD, we performed linkage analyses in a [BN X(BN X Han:SPRD)F₁] backcross. Cy/Cy and Cy/+also caused PKD in a BN background. In this backcross,we discovered that D5Rat11 is located closer to the Cy locus than D5Mgh10, which is regardedas one of the closest loci. We conclude that D5Rat9 andD5Rat11 are useful markers for determining the presenceof the Cy allele, which is regarded as the gene responsible for PKD.