Application of L-DNA to the study of the specific DNA recognition mechanism of bleomycin.

The hexadeoxynucleotide analog, L-d(CGCGCG) composed of L-deoxyribose was synthesized and clearly shown to have the same conformation and dynamic properties with natural D-d(CGCGCG) except for chirality with CD spectra. This unnatural hexanucleotide was not cleaved by bleomycin, an antitumor DNA cleaving drug, but was able to bind to the DNA binding domain of bleomycin to a similar extent with the natural one. These results strongly suggest the importance of the other moiety than the DNA binding domain for the specific DNA recognition of bleomycin. Thus, L-oligonucleotides are useful for the study of DNA-drug interactions.     

Mechanism of Action of Showdomycin

Among the syntheses of DNA, RNA and protein in Escherichia coli cells, the DNA synthesis was found to be preferentially inhibited at lower concentrations of showdomycin. At such lower concentrations of this antibiotic, serious decreases in the synthesis of deoxycytidine phosphates and in de novo synthesis of deoxythymidine phosphates were found in parallel with the decrease in the synthesis of DNA, although the syntheses of other pyrimidine nucleotides were not significantly diminished. The salvage synthesis of deoxythymidine phosphates was very resistant to this antibiotic. The inhibitory action of this antibiotic on DNA synthesis could be reversed by the concomitant addition of a thiol compound or a nucleoside. When a nucleoside was added after the completion of the inhibition by showdomycin, the recovery of the DNA synthesis from the inhibition was detected only after the recovery of the syntheses of pyrimidine ribotides, pyrimidine deoxyribotides and RNA have become distinct.     

Mechanism of Action of Showdomycin

¹⁴C-Labelled showdomycin was rapidly taken up by Escherichia coli K-12 cells. The showdomycin uptake was highly temperature dependent, sensitive to azide and N-ethyl-maleimide, but was only partially inhibited by treatment with high concentration of iodoacetic acid.

The uptake of showdomycin was inhibited by a wide variety of nucleosides but not by purine and pyrimidine bases, nucleotides, ribose or ribose-5-phosphate. The inhibition of showdomycin uptake by adenosine was of a competitive type.

Since nucleosides inhibited the uptake of showdomycin but did not facilitate its efflux, they must play a role of inhibitors to the entry of the antibiotic into cells.

Removal of extracellular showdomycin by washing, or inhibition of its subsequent entry into cells by the addition of nucleosides or sulfhydryl compounds resulted in a rapid decrease in the intracellular level of the antibiotic during subsequent incubation.     

Biochemical Studies during the Development of the Chick Embryo

The devised method consists of the enzymatic hydrolysis, separation of deoxyribosides in the hydrolysate by paper chromatography and paper electrophoresis, and the estimation of the separated fractions with L. leichmannii. This method permits the determination of deoxyriboside composition of the smaller amounts of DNA or the related compounds with relatively higher accuracy even under the presence of some other compounds when nucleic acids and acid insoluble fractions of the chick embryo were analyzed.

The change of each deoxyriboside composition in acid insoluble fraction prepared from the 3 to 19 day old embryos investigated by the method. Among the major four deoxyribosides, the contents of deoxyguanosine and of deoxycytidine was nearly constant during the development of the embryo, whereas that of thymidine and of deoxyadenosine appeared to undergo the change slightly at the periods from 10 to 15 days incubation. It seems that this periods is also the most active time of the synthesis of DNA and of the changes of deoxyribosyl compounds in acid soluble fraction through the embryo growth.     

Some Properties and Characterization of Rice Seed Hemagglutinin

The phytohemagglutinin of rice seed has been purified by a sequence of steps involving fractionation with ammonium sulfate and successive chromatography on DEAE-and eMcellulose and finally gel filtration on Bio-Gel P-100. The purified rice seed hemagglutinin was shown to be homogeneous by electrophoresis on polyacrylamide gel and its molecular weight was 10,000, calculated from both the Ve/Vo value of gel filtration on Bio-Gel P-100 and the sum of the individual constituents (amino acids, sugars and metals). In addition to amino acid, the rice seed hemagglutinin contained 26.8% covalently bound carbohydrate which was identified and quantitated by gas chromatography of the acetylated alditols. Glucose was the predominant sugar with lesser amounts of glucosamine, xylose, and mannose also being present. And the rice seed hemagglutinin contained 1 g atom of calcium per molecule. The molecular weight of the rice seed hemagglutinin is smallest compared with some of phytohemagglutinins isolated from leguminous seeds and other plant sources. The rice seed hemagglutinin has the blastogenetic activity for human peripheral lymphocytes as well as Phaseolus vulgaris phytohemagglutinins or concanavalin A, jack bean hemagglutinin.     

Synthesis and evaluation of N-alkyl-S-[3-(piperidin-1-yl)propyl]isothioureas: High affinity and human/rat species-selective histamine H3 receptor antagonists

S-Alkyl-N-alkylisothiourea compounds containing various cyclic amines were synthesized in the search for novel nonimidazole histamine H₃ receptor (H₃R) antagonists. Among them, four N-alkyl S-[3-(piperidin-1-yl)propyl]isothioureas 18, 19, 22, and 23 were found to exhibit potent and selective H₃R antagonistic activities against in vitro human H₃R, but were inactive against in vitro human H₄R. Furthermore, three alkyl homologs 18–20 showed inactivity for histamine release in in vivo rat brain microdialysis, suggesting differences in antagonist affinities between species. In addition, in silico docking studies of N-[4-(4-chlorophenyl)butyl]-S-[3-piperidin-1-yl)propyl]isothiourea 19 and a shorter homolog 17 with human/rat H₃Rs revealed that structural differences between the antagonist-docking cavities of rat and human H₃Rs were likely caused by the Ala122/Val122 mutation.