A potential approach for gene therapy targeting hepatoma using a liver-specific promoter on a retroviral vector.

Recent technological advances made in molecular biology and in vitro culture of human and other mammalian cells have led to broad medical and scientific acceptance of the feasibility of gene therapy for genetic diseases. Cancer might practically be one of the attractive targets for such therapy. For the treatment of cancer, it is important to manipulate the gene of interest such that it is expressed solely in cancer cells. We have developed a tissue-specific gene expression system, based on a tissue-specific promoter on a retroviral vector. A murine ecotropic retroviral vector was constructed in which the Escherichia coli beta-galactosidase gene served as a reporter; it was expressed under control of the albumin enhancer element and promoter. The tissue specificity of this vector was first assessed in vitro, and beta-galactosidase activity was detected exclusively in hepatoma cell lines. This recombinant retrovirus was injected directly into a subcutaneous tumor composed of transplantable murine MH-134 hepatoma cells, and expression of the gene was observed in vivo. Then this recombinant retrovirus was injected via the spleen or directly into the liver, resulting in the gene expression in dividing hepatocytes in partially hepatectomized mice, but not in nondividing hepatocytes in normal mice. Gene transfer specific to dividing hepatocytes and expression by means of retroviral vectors should possess high potential for selective elimination of hepatoma cells surrounded by nondividing normal hepatocytes.     

Genetic interpretation of enzyme variation in sexual and agamospermous taxa of Taraxacum sections Vulgaria and Mongolica

The inheritance of 15 enzymes, comprising at least 22 genetic loci, was investigated in crosses between sexual diploid individuals of Taraxacum sections Vulgaria and Mongolica. Patterns were consistent with simple Mendelian segregation. From the inheritance information isozyme phenotypes in agamospermous plants from natural populations were inferred. In some crosses part or all of the progeny originated from self-fertilization, sofar a very rare phenomenon in the sections Vulgaria and Mongolica. It is possible that the probability of self-fertilization increases after pollination by triploid pollen, affecting the cohabitation dynamics of the various ploidy components in mixed natural stands.     

Detection of H-2K mRNA in mouse 8-cell embryo by cDNA cloning

Mouse MHC class I gene expression in 8-cell embryo was examined by cDNA cloning. We constructed a cDNA library from 8-cell embryos of ICR mice and isolated a class I cDNA from 3.0 x 10(5) phage clones of the library. Sequencing analysis of this clone revealed it to include the cDNA fragment extending from the exon 6 of the cytoplasmic portion to 3' untranslated region 1 of H-2K gene. Qa, Tla or other embryonic class I cDNA have not been isolated in the library.     

Chromosomal genes essential for stable maintenance of the mini-F plasmid in Escherichia coli

We have isolated mutants of Escherichia coli which do not support stable maintenance of mini-F plasmids (delta ccd rep+ sop+). These host mutations, named hop, were classified into five linkage groups on the E. coli chromosome. Genetic analyses of these hop mutations by Hfr mating and P1 transduction showed their loci on the E. coli genetic map to be as follows: hopA in the gyrB-tnaA region, hopB in the bglB-oriC region, hopD between 8 and 15 min, and hopE in the argA-thyA region. Kinetics of stability of the sop+ and delta sop mini-F plasmids in these hop mutants suggest that the hopA mutants are defective in partitioning of mini-F rather than in plasmid replication. The hopB, hopC, and hopD mutants were partially defective in replication of mini-F. The physical structure of the plasmid DNA was normal in hopA, B, C, and D mutants. Large amounts of linear multimers of plasmid DNA accumulated in mutants of the fifth linkage group (hopE). None of the hop mutations in any linkage group affected the normal growth of cells.     

Pyridylamino sugar chain as an acceptor for galactosyltransferase

Pyridylamino asialo-agalacto-biantennary sugar chain (PA-acceptor), prepared from human alpha 1-acid glycoprotein, was incubated with bovine milk galactosyltransferase. Transfer of galactose residues to PA-acceptor was detected by HPLC analysis, and thus PA-acceptor was shown to be useful for galactosyltransferase assay. Moreover, three species of products, i.e. PA-acceptor monogalactosylated on the Man alpha 1-3 branch of the trimannosyl core, PA-acceptor monogalactosylated on the Man alpha 1-6 branch, and digalactosylated PA-acceptor, were separated and identified by reversed-phase HPLC, so we could simultaneously determine the branch specificity (the ratio of galactosylation on Man alpha 1-3 branch to that on Man alpha 1-6 branch) of the galactosyltransferase. We fractionated the bovine milk galactosyltransferase on a DEAE-5PW column and confirmed that there was a heterogeneity in this enzyme preparation. Each fraction was assayed for acceptor specificity (the ratio of the activity towards N-acetylglucosamine to that towards PA-acceptor) and branch specificity using the PA-acceptor. However, we could not detect differences in the specificities among the fractions. In addition, we found that alpha-lactalbumin stimulated the galactosyltransferase activity towards PA-acceptor.     

Site-directed mutagenesis of the alpha subunit of tryptophan synthase from Salmonella typhimurium

Site-specific mutagenesis has been used to prepare two mutant forms of the alpha subunit of tryptophan synthase from Salmonella typhimurium in which either cysteine-81 or cysteine-118 is replaced by a serine residue. These mutant proteins are potentially useful for x-ray crystallographic studies since a heavy metal binding site is specifically eliminated in each mutant. The purified mutant proteins are fully active in four reactions catalyzed by the wild type alpha 2 beta 2 complex of tryptophan synthase. However, the mutant alpha 2 beta 2 complexes dissociate more readily and are less heat-stable than the wild type alpha 2 beta 2 complex. Thus, cysteine-81 and cysteine-118 of the alpha subunit serve structural but not functional roles.     

1,25-Dihydroxyvitamin D stimulates sodium-dependent phosphate transport by renal outer cortical brush-border membrane vesicles by directly affecting membrane fluidity

We examined the effects of several forms of vitamin D added to renal brush-border membrane suspensions on phosphate and glucose transport and on membrane fluidity. The 1,25-D stimulated and the other vitamin D decreased phosphate uptake. In contrast, glucose uptake was not affected by the treatment of vitamin D. The 1,25-D resulted in a significant shift of the lower transition temperature in Arrhenius plots for phosphate, but not for glucose uptakes, from 15 degrees C to 11.5 degrees C. These data indicate that the 1,25-D may alter membrane fluidity, limited to the phosphate transporter, thus affecting the phosphate uptake.     

Isolation of endo A cDNA from mouse 8-cell stage embryos

To analyse the species of genes expressed in a cleavage stage mouse embryo, we have constructed a cDNA library containing 3.0 x 10(5) independent clones from about 2 x 10(3) embryos at the 8-cell stage of development. Endo A cDNA prepared from parietal yolk sac endoderm like PYS-2 cells was used to screen the library. Southern blot analyses using the endo A sequence as a probe and restriction mapping analyses revealed that four independent recombinants had been inserted endo A sequence. Sequencing data of these clones showed that endo A mRNA present in the 8-cell stage embryo is identical to that of parietal yolk sac endoderm cells.     

Comparison of arachidonic acid metabolism between myofibroblasts and fibroblasts isolated from rat palatal mucoperiosteum

Myofibroblasts were cultured successfully from experimental wound tissue in rat palatal mucoperiosteum. Arachidonic acid metabolizing activity in cultured myofibroblasts was compared with that in fibroblasts cultured from normal mucoperiosteum. Prostaglandins biosynthesized from [14C]arachidonic acid in cell-free homogenates of both myofibroblasts and fibroblasts were prostaglandins D2, E2 and F2 alpha, and the activity producing each prostaglandin was not significantly different between the myofibroblasts and the fibroblasts, whereas smooth muscle cells, which are histologically similar to myofibroblasts, produced mainly 6-ketoprostaglandin F1 alpha, and relatively small amounts of prostaglandin E2. The release of arachidonic acid from cells prelabeled with [14C]arachidonic acid was compared among three types of cell. The calcium ionophore A23187 strongly enhanced arachidonic acid release in all three cell types. Bradykinin, 5-hydroxytryptamine and prostaglandin F2 alpha affected the stimulation of arachidonic acid release in the fibroblasts but were less or not effective in the myofibroblasts and smooth muscle cells. In addition, prostaglandin E2 biosynthesized in response to several stimuli was measured by radioimmunoassay. The content of prostaglandin E2 correlated closely with arachidonic acid release. In this study, we showed homogeneity between the myofibroblasts and fibroblasts in prostaglandin synthesizing activity and similarity in response to various stimuli between the myofibroblasts and smooth muscle cells, from the standpoint of arachidonic acid metabolism.     

Sequence-specific modification of DNA by 6-hydroxybenzo[a]pyrene

6-Hydroxybenzo[a]pyrene cleaved phi X174 supercoiled DNA to open circular DNA in the presence of heavy metal ions. It induced an alkali-labile modification in DNA via an oxygen-radical-mediated reaction; the most frequent alkali-labile sites were on the 3' side of the pyrimidine residues of the pyrimidine cluster.