Hamao Umezawa

The combined effects of bleomycin and sulfhydryl compounds on the thermal denaturation of DNA.

Abstract The reaction of bleomycin with DNA in vitro was shown to occur by a decrease in the melting temperature ( T m of DNA. This decrease of T m was observed when DNA was incubated with the antibiotic in the presence of a sulfhydryl compound such as 2-mercaptoethanol or dithiothreitol. It was also observed when DNA was incubated with 2-mercaptoethanol, dialyzed and thereafter bleomycin A 2 was added. It was not observed when DNA was incubated with bleomiycn A 2 alone and with the addition of 2-mercaptoethanol just before the determination of T m . The results indicate that, although 2-mercaptoethanol has no effect on T m , DNA must react with 2-mercaptoethanol since the T m of DNA after the reaction is decreased, as shown by the reaction with bleomycin A 2 . The effect of bleomycin A 2 on DNA in the presence of the sulfhydryl compound was diminished by the addition of Cu 2+ , Zn 2+ or Co 2+ . Bleomycin mixture or bleomycin A 2 in the copper-containing form does not significantly affect T m . Bleomycins and phleomycins are closely related peptide antibiotics, but phleomycin increased the T m of DNA irrespective of the presence of the sulfhydryl compound.

Stimulatory effect of Bestatin, a new specific inhibitor of aminopeptidases, on the blastogenesis of guinea pig lymphocytes

Abstract A potent protease-inhibitor of Actinomycetes origin, Bestatin. which is of dipeptide nature and inhibits aminopeptidase B and leucine-aminopeptidase competitively, strongly stimulates blastogenesis of small lymphocytes triggered with polyclonal mitogen. such as phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM) and lipopolysaccharide of Escherichiae coli (LPS), whereas it inhibits DNA synthesis of normal resting lymphocytes. The stimulatory effect is non-selective with respect to the category of small lymphocytes, i.e. T- and B-lymphocytes, but strikingly selective with respect to the stage of blastogenesis: the stimulation is greatest at a relatively early stage, diminishes as mitogen-activation proceeds, and is not appreciable at a later stage of lymphocyte blastogenesis. The pattern of Bestatin stimulation on lymphocyte blastogenesis is specific for the mitogen used: in T-lymphocyte activation with PHA or Con A, the stimulation first increases and then decreases with increase in mitogen concentrations, whereas in B-lymphocyte activation with LPS, with increasing concentrations of the mitogen, the stimulation increases to a plateau at approximately 100 μg/ml of mitogen. The optimum concentration of Bestatin was found to be approximately 50 μg/ml (0.16 mM) for either PHA or Con A activation, and 50 to 75 μg/ml for B-cell activation with LPS. Bestatin must remain in cultures of T- and B-lymphocytes with polyclonal mitogens for at least about 24 and 16 hr, respectively, to exert its stimulatory effect on blastogenesis. Biochemical results, together with those from autoradiographic analyses, indicate that Bestatin increases the number of blastoid-transformed lymphocytes with polyclonal stimulants. It is suggested that aminopeptidases, possibly located at the cell surface, may play a role in the control of lymphocyte activation during immune responses.

Inhibitory effects of phosphoramidon on neutral metalloendopeptidases and its application on affinity chromatography

Summary Phosphoramidon, a thermolysin inhibitor isolated from a culture filtrate of streptomyces, inhibited the other two neutral metalloendopeptidases [EC 3.4.24.4 group], and was shown to be a suitable ligand for affinity chromatography of these metalloendopeptidases.

Inhibition of RNA and DNA polymerase reactions by pluramycin A

Pluramycin A, isolated from the culture of Streptomyces pluricolorescens by Maeda et al (19561, is a basic antibiotic of orange needle crystals. The antitumor and antibacterial activity was reported by Takeuchi et al (1957). Lein et al (1962) observed phage induction of lysogenic bacteria by pluramycin. We investigated the activity of pluramycin A on macromolecular syntheses and observed that the antibiotic inhibits both protein and nucleic acids syntheses in the intact cells of bacteria. The effects were further investigated, using cell-free systems. The results are described in this communication. Protein synthesis was not significantly affected in bacterial cell-free systems but RNA and DNA polymerase reactions were markedly inhibited by the antibiotic, which was suggested to bind with DNA by the thermal transition curve. Effects on protein synthesis in cell-free systems from E. coli: Pluramycin A was observed to exhibit no significant effects on 14C-leucine incorporation into protein and polyuridylate-directed polyphenylalanine synthesis in cell-free systems obtained from exponentially growing cells of E. coli B. The method employed principally followed the one developed by Nirenberg and Matthaei (1961). The results are summarized in Tables 1 and 2.

Role of intramuscular enzymatic changes in the development of muscular weakness in rats with experimental allergic neuritis

We investigated the role of intramuscular enzymatic changes in the development of muscular weakness in rats suffering from experimental allergic neuritis. At an initial stage without apparent clinical symptoms, enzymatic changes of similar types occurred in the muscles of the forelimbs and hind limbs. At a later stage when the weakness appeared in the hind limb but not in the forelimb, dissociation of the pattern of the enzymatic changes occurred between the two limbs. Comparison of the intramuscular enzymatic changes between the two stages and between the two limbs suggested that the increased activities of aminopeptidases and endopeptidases play some important roles in the development of muscular weakness in this experimental model. Low molecular weight protease inhibitors may thus be worthy of a trial in this disease condition.