No abstract

A novel crystal protein that exhibited potent cytotoxicity against human leukemic T-cells was cloned from the Bacillus thuringiensis TK-E6 strain. The protein, designated as parasporin-2Ab (PS2Ab), was a polypeptide of 304 amino acid residues with a predicted molecular weight of 33,017. The deduced amino acid sequence of PS2Ab showed significant homology (84% identitiy) to parasporin-2Aa (PS2Aa) from the B. thuringiensis A1547 strain. Upon processing of PS2Ab with proteinase K, the active form of 29 kDa was produced. The activated PS2Ab showed potent cytotoxicity against MOLT-4 and Jurkat cells and the EC(50) values were estimated as 0.545 and 0.745 ng/mL, respectively. The cytotoxicity of PS2Ab was significantly higher than that of PS2Aa reported elsewhere. Although both cytotoxins were structurally related, it was thought that the minor differences found were responsible for the different cytotoxicities of PS2Ab and PS2Aa.

Cry46Ab from Bacillus thuringiensis TK-E6 is a new mosquitocidal toxin with aerolysin-type architecture, and has been shown that co-administration of Cry46Ab with other mosquitocidal Cry toxins results in synergistic toxicity against Culex pipiens Coquillett (Diptera: Culicidae) mosquito larvae. Cry46Ab therefore is expected to find use in improving the insecticidal activity of B. thuringiensis-based bioinsecticides. In the present study, the mode of action of Cry46Ab was explored by single-channel measurements of Cry46Ab channel-pores. The single-channel conductances of channel-pores formed in planar lipid bilayers by Cry46Ab were determined to be 31.8 ± 2.7 pS in 150 mM NaCl and 24.2 ± 0.7 pS in 150 mM CaCl2. Ion-selectivity measurements revealed that the channel-pores formed by Cry46Ab were cation selective. The permeability ratio of K + to Clwas approximately 4, and the preferences for cations were K + > Na + , K + > Ca 2+ , and Ca 2+ > Na +. A calcein release assay using liposomes suggested that Cry46Ab influences the integrity of membrane vesicles. Formation of cation-selective channel-pores has been observed with other insecticidal Cry toxins that have structures distinct from those of Cry46Ab; the capability of forming such pores may be a property required of insecticidal toxins.

To analyze the RSF1010‐specific priming mechanism, a library of randomly mutagenized ssiA sequences was constructed by chemical synthesis using mixed nucleotide phosphoramidites. Synthetic ssiA sequences with the single base‐substitutions were assayed for the SSI activity in E. coli JM109 expressing RepB' primase. It was demonstrated that the activity of ssiA was damaged markedly by single base‐substitutions within the possible stem‐loop structure and its 3'‐flanking region. It is conceivable that these domains are critical in recognition and primer synthesis by RepB' primase.

Human MDRI cDNA was introduced into the human cultured cells KB-3-l and Sclll-_osucchuronl?'ces pombe pmdl null mutant KN3. The drug sensitivity of KB-G2 and KN3/pgp, expressmg human P-glycoprotein, was exammed. KB-G2 was resistant to the peptlde antibiotics valinomycin and gramicidin D as well as having a typical multidrug resistance (MDR) phenotype.KN3/pgp was resistant to valinomycin and actinomycin D. but not to adriamycm.The ATP-hydrolysis-defiaent mutant did not confer KN3 resistance to these antiblotlcs. Human P-glycoprotein expressed in S. pombe seemed to lack N-glycosylation.The N-glycosylation-deficient mutant, however. conferred a typical MDR phenotype on KB-3-l. These results suggest that human P-glycoprotein fun&Ions as an efflux pump of vahnomycin and actinomycin D m the membrane of S. pomhe P-glycoprotein: