Efficient gene activation in mammalian cells by using recombinant adenovirus expressing site-specific Cre recombinase.

A recombinant adenovirus (Ad) expressing Cre recombinase derived from bacteriophage P1 was constructed. To assay the Cre activity in mammalian cells, another recombinant Ad bearing an on/off-switching reporter unit, where a LacZ-expression unit can be activated by the Cre-mediated excisional deletion of an interposed stuffer DNA, was also constructed. Co-infection experiments together with the Cre-expressing and the reporter recombinant Ads showed that the Cre-mediated switching of gene expression was detected in nearly 100% of cultured CV1, HeLa and Jurkat cells. These results suggest that the recombinant Ad efficiently expressed functional Cre and offers a basis for establishing a powerful on/off switching strategy of gene expression in cultured mammalian cells and presumably in transgenic animals. The method is also applicable to construction of recombinant Ad bearing a gene the expression of which is deleterious to propagation of recombinant Ad.     

Peroxidation of lipids and growth inhibition induced by UV-B irradiation

Cotyledons excised from dark-grown seedlings of cucumber (Cucumis sativus L.) were cultured in vitro under UV radiation at different wavelengths, obtained by passage of light through cut-off filters with different transmittance properties. Growth and the synthesis of chlorophyll (Chl) in cotyledons were inhibited and malondialdehyde was accumulated upon irradiation at wavelengths below 320 nm. Exogenous application of scavengers of free radicals reversed the growth inhibition induced by UV-B. Measurement of the fluorescence of Chl a suggested that electron transfer in photosystems was affected by UV-B irradiation. On the basis of these results, the involvement is postulated of active species of oxygen in damages to thylakoid membranes and the growth inhibition that are induced by UV-B irradiation.Abbreviations Chl chlorophyll - F_m maximal fluorescence (dark) - F_m maximal fluorescence (light) - F_v variable fluorescence (dark) - F_v variable fluorescence (light) - MDA malondialdehyde - O₂ Superoxide radical - PS photosystem - q_N non-photochemical quenching of fluorescence - q_P photochemical quenching of fluorescence - UV-B_BE biologically effective UV-B radiation - WL(T = 0.5) wavelength at which 50% transmittance occurs     

Block of acid secretion by amytal and its partial reversal by menadione with ascorbate in the gastric mucosa of the guinea-pig

The effect of amytal on energy metabolism and acid secretion in an isolated gastric mucosa of the guinea-pig were studied. Determination of adenine nucleotides, creatine phosphate, pyruvate and lactate in the gastric mucosa showed that amytal depressed the levels of ATP, creatine phosphate and energy charge with elevation of the AMP and pyruvate levels. This treatment inhibited concomitantly acid secretion and active chloride transport detected by short circuit current. The addition of menadione with ascorbate to the medium in the presence of amytal partially restored ATP and energy charge levels and also induced a partial recovery of acid secretion and active chloride transport. These results suggest that ATP is a direct energy donor for acid secretion in the gastric mucosa of the guinea-pig.     

Immuno-Localization of DEAD Family Proteins in Germ Line Cells of Xenopus Embryos.

In order to investigate whether a vasa -like protein is present in germ line cells of Xenopus , antibodies were produced which react specifically with synthetic oligopeptides of sequences from near the N- or C-termini or with one including the DEAD box of the Drosophila vasa protein.
Only the antibody against the oligopeptide including the DEAD box reacted strongly with germ plasm (GP) or with cytoplasm of germ line cells of Xenopus embryos by immunofluorescence microscopy. By immunoelectron microscopy, the antibody was demonstrated to react with the GP-specific structure, germinal granules, in cleaving embryos, and with their derivatives in the germ line cells of embryos at stages extending from gastrula to feeding tadpole. It also reacted with mitochondria not only in the GP and the germ line cells but also in somatic cells, and with myofibrils in muscle cells. By Western blotting, the antibody was shown to react with several bands of Mr 42–69 ± 10³ in protein samples from Xenopus embryos. In samples from Drosophila ovaries, it reacted with a Mr 71 ± 10³ band which was probably the vasa protein. This indicates the possibility that Xenopus embryos contain several DEAD family proteins. One of these is present on germinal granules, resembling the vasa protein on polar granules of Drosophila .     

Selective processing of proalbumin determined by site-specific mutagenesis

Rat proalbumin is cleaved at the dibasic pair Arg-Arg and converted into a mature form with Glu at the NH2 terminus. In the present study site-directed mutagenesis of the albumin cDNA was designed to generate proalbumin variants in which Glu1 was substituted with various amino acid residues. The expression plasmids constructed were transfected into COS-1 cells, and the intracellular processing of proalbumins expressed was examined by labeling experiments. Substitution of Glu1----Ser allowed the expressed proalbumin to be processed as observed for the wild-type precursor. However, replacement of Glu1 with a hydrophobic residue (Val, Leu or Ile) resulted in no processing of proalbumin, despite retaining the same cleavage signal Arg-Arg as above. The results indicate that the residue at position 1 adjacent to the dibasic pair is also important for recognition by the proalbumin-processing enzyme.     

Callus regeneration from cotyledon protoplasts ofChamaecyparis obtusa (Hinoki cypress)

Summary Protoplasts were isolated from cotyledons of 1- to 1.5-mo.-old seedlings ofChamaecyparis obtusa using 1% driselase or 0.25% pectolyase Y-23 in combination with 1% cellulase RS in 0.6M mannitol solution. Cell division and colony formation were induced efficiently in liquid Murashige and Skoog’s (MS) medium containing 0.6M mannitol and 10 to 30 μM 2,4-dichlorophenoxyacetic acid or 1 μM of naphthaleneacegic acid at the cell density of 1 to 2×10³ ml. Continued callus proliferations was observed by transferring tissue to fresh medium of the same composition as the induction medium without mannitol. Campbell and Durzan’s medium and ammonium nitrate-free MS medium were less effective than MS medium. High concentration of benzyladenine (1 or 10 μM) was inhibitory to cell division.     

Some common properties of lectins from marine algae

Twelve kinds of lectins isolated from four species of marine algae, Boodlea coacta (Chlorophyta) and Hypnea japonica, Carpopeltis flabellata and Solieria robusta (Rhodophyta), were compared for their chemical and biological properties. These lectins were proteins or glycoproteins, similar to terrestrial plant lectins. However, unlike most terrestrial plant lectins, they had a small molecular size (4,200 to 25,000 daltons), were mostly monomeric, and had no affinity for monosaccharides. They strongly agglutinated trypsin-treated rabbit erythrocytes, and their activities commonly were inhibited by glycoproteins bearing N-glycans. From hemagglutination-inhibition tests with various glycoproteins and related compounds, it was found that B. coacta lectins recognize high-mannose N-glycans; H. japonica lectins complex N-glycans, and C. flabellata and S. robusta lectins recognize both types of N-glycans.     

Nucleotide sequence of Physarum polycephalum 5.8S rRNA gene and its flanking regions.

The nucleotide sequence of Physarum polycephalum 5.8S rRNA gene and its flanking regions has been determined. The homologies of the 5.8S rRNA sequence with those of Saccharomyces, Chlamydomonas and Xenopus were 56%, 50% and 52%, respectively. In spite of these relatively low homologies, its possible secondary structure was very similar to those of other species.     

Biosynthesis of triterpenoids from amino acids in Pisum sativum. The distribution of the radioactivity in squalene biosynthesized from radioisotopically labelled l-leucine and l-valine

Radioisotopically labelled l-leucine and l-valine were fed to Pisum sativum and incorporated into squalene and β-amyrin. Chemical degradation of the radioactive squalene revealed an equal distribution of the radioactivity in the isopentenyl pyrophosphate(IPP)-derived and the 3,3-dimethylallyl pyrophosphate(DMAPP)-derived moieties of the squalene molecule, unlike the unbalanced distribution in favour of the DMAPP-derived moiety of a monoterpenoid molecule biosynthesized from these amino acids by higher plants.