Physical analysis of the genomes of hybrid phages between phage P1 and plasmid p15B

The genomes of three plaque-forming recombinant phages between phage P1 and plasmid p15B were characterized by restriction cleavage analysis and electron microscopic heteroduplex studies. The structure of all three P1-15 hybrid genomes differs from that of P1 DNA in the res mod region coding for restriction and modification systems EcoP15 and EcoP1, respectively. P1-15 hybrid 2 shows an additional major difference to P1 around the site of the residential IS1 element of P1 and it does not carry an IS1 in its genome.     

Sequence of the site-specific recombinase gene cin and of its substrates serving in the inversion of the C segment of bacteriophage P1.

Inversion of the 4.2-kb C segment flanked by 0.6-kb inverted repeats on the bacteriophage P1 genome is mediated by the P1-encoded site-specific cin recombinase. The cin gene lies adjacent to the C segment and the C inversion cross-over sites cixL and cixR are at the external ends of the inverted repeats. We have sequenced the DNA containing the cin gene and these cix sites. The cin structural gene consists of 561 nucleotides and terminates at the inverted repeat end where the cixL site is located. Only two nucleotides in the cixL region differ from those in the cixR and they are within the cin TAA stop codon. The cin promoter was localized by transposon mutagenesis within a 0.1-kb segment, which contains probable promoter sequences overlapping with a 'pseudo-cix' sequence cixPp. In a particular mutant, integration of an IS1-flanked transposon into the cin control region promoted weak expression of the cin gene. The cin and cix sequences show homology with corresponding, functionally related sequences for H inversion in Salmonella and with cross-over sites for G inversion in phage Mu. Based on a comparison of the DNA sequences and of the gene organizations, a possible evolutionary relationship between these three inversion systems and the possible significance of the cixPp sequence in the cin promoter are discussed.     

Mode of antitumor action of recombinant human tumor necrosis factor on the sarcoma Meth A transplanted in the mouse

The mode of antitumor action of rHu-TNF was elucidated in BALB/c mice bearing Meth A fibrosarcoma 7 days after transplantation with respect to time course, dose-response relationships and selectivity of the effects. The maximal cytotoxic effect on tumor cells revealed by inhibition of DNA synthesis and maximal lesional effect on tumor vasculature revealed by change in blood pool-size in the tissue were detected at 30 min and I h after administration of rHu-TNF, respectively. The dose-response relationship between cytotoxic and tumoricidal effects of rHu-TNF was irrespective of administration route. ED₅₀s of these antitumor effects afteri.v. administration of rHu-TNF were about 50 times as high as ED₅₀s afteri.t. administration. ED₅₀ ofi.t. given rHu-TNF for vascular effect was about 20 times as high as that for cytotoxicity while ED₅₀ ofi.v. rHu-TNF for vascular effect was only 2–3 times as high as that for cytotoxicity. The whole body autoradiographies with [¹²⁵I] HSA giveni.v. to see the blood influx into tumor tissue and [¹⁴C]thymidine given i.v. to see DNA synthesis in the whole body after administration of rHu-TNF revealed that the distribution of radioactivity was markedly changed in the tumor alone without any detectable change in other whole body tissues.In conclusion, thein vivo antitumor effect of rHu-TNF giveni.t. ori.v., appears to be exerted through the direct action on Meth A sarcoma rather than indirectly on tumor vasculature. Under present conditions, the effect of rHu-TNF in the whole body tissues seems rather selective on cells and vasculature of the tumor.     

Potential bile acid metabolites. 14. Hyocholic and muricholic acid stereoisomers

The complete set of the eight theoretically possible stereoisomeric 3,6,7-trihydroxy-5 beta-cholanic acids, four of which are new, related to hyocholic and muricholic acids were prepared from chenodeoxycholic acid. The principal reactions used were 1) cis-dihydroxylation of delta 6-compounds with osmium tetroxide/N-methylmorpholine N-oxide; 2) trans-dihydroxylation of 6 alpha, 7 alpha-epoxy compounds with boron trifluoride etherate in N,N-dimethyl-formamide; 3) inversion of equatorial 3 alpha-hydroxylated compounds to the corresponding 3 beta-epimers with diethyl azodicarboxylate/triphenylphosphine/formic acid; and 4) stereoselective reduction of 7-keto derivatives with zinc borohydride (or sodium borohydride) and by metallic potassium/tert-amyl alcohol.     

Bent DNA is needed for recombinational enhancer activity in the site-specific recombination system Cin of bacteriophage P1. The role of FIS protein

A series of recombinational enhancer mutants was constructed by manipulating the ClaI site between the two FIS binding sites of the Hin enhancer. These mutants include insertions from two to 12 base-pairs and two deletions of one or two base-pairs. Recombinational enhancer activity was found only with four mutants carrying either a four base-pair substitution, ten base-pair insertions or a one base-pair deletion, respectively; two other ten base-pair insertion mutants, however, were inactive, although FIS protein binding was unaffected. So, besides binding of FIS protein to its specific sites within the enhancer sequence and the correct helical positioning of these sites on the DNA, another criterion for enhancer activity must be fulfilled. DNA bending assays identify this requirement as a change of the enhancer DNA conformation, which FIS protein is able to induce and to stabilize. This conformational change of the DNA can be blocked by mutations in the central segment between the two FIS binding sites of the Hin enhancer. This sequence has special functions for the recombinational enhancer activity.     

A sulfated glucosylceramide from rat kidney

A novel sulfated glycosphingolipid containing a sulfated glucosyl residue was isolated from rat kidney and purified to homogeneity by column chromatographies with DEAE-Sephadex and silica beads. By compositional analyses, permethylation studies, one- and two-dimensional proton magnetic resonance spectroscopy, infrared spectroscopy, negative secondary ion mass spectrometry, solvolysis, and immunostaining on thin layer chromatogram, the structure of this glycolipid was proposed to be HSO3-3Glc beta 1-1Cer (where Cer is ceramide). The ceramide portion consisted of 4-D-hydroxysphinganine as the sole long chain base, and the fatty acid consisted of predominantly tetracosanoic acid, deduced from both composition analysis and negative secondary ion mass spectrometry. The yield of glucosyl sulfatide was about 5 nmol/g of tissue, being about three times as much as that of lactosylceramide sulfate.     

Purification of nitric oxide synthase from bovine brain: immunological characterization and tissue distribution.

Nitric oxide (NO) synthase (EC 1.14.23) was purified to homogeneity from bovine cerebrum. The molecular weight of NO synthase was estimated to be 150 kDa by both SDS/PAGE and gel filtration at high salt concentration. For activity, the enzyme required NADPH, Ca2+, calmodulin and tetrahydrobiopterin as cofactors. Rabbit polyclonal antibody to bovine brain NO synthase reacted with 150 kDa NO synthase in various bovine and rat organs, including the brain, pituitary and adrenal glands, but not with that in stimulated macrophages, indicating that there are at least two immunologically distinct NO synthases.