A strain, which had been selected by the systematic screening of about 2,000 soil actinomycetes, was found to produce two antifungal substances. These substances were obtained in crystalline forms. One of them, the antifungal substance, No. 720-A, was studied in detail and confirmed to be identical with antimycin A discovered by Leben, Keitt, Dunchee, Scheneider and Strong⁽¹⁾⁽²⁾. It was also compared with virosin found by Nakazawa and others⁽³⁾, and antipiricullin found by Nakayama and others⁽⁴⁾. They were considered to be identical with antimycin A. Another antifungal substance, No. 720-B, produced by the same strain, was not obtained in a sufficient amount for the further studies. However, it was different from antimycin B described by Schneider, Tenner and Strong⁽⁵⁾ in the melting point.

In the present paper not only the production, purification and natures of the antifungal substances, No. 720-A and 720-B, but also characteristics of the strain are presented. In addition to the strain No. 2A-720 the authors isolated one more strain, No. 6A-369, belonging to the same Streptomyces. sp. These strains, No. 2A-720 and No. 6A-369, were compared with the virosin-producing strain. Nakazawa and others reported that among known species the virosin producing strain was resembling to Streptomyces olivochromogenus. Characters of the strains No. 2A-720 and No. 6A-369 were very resembling to the virosin-producing strain, and these strains were considered to belong to the same species. The authors, however, could not conclude that they belonged to S. olivochromogenus. Nakayama and others named their antifungal substance, antipiricullin, since it exhibited a strong inhibition to Piricularia oryzae, an organism causing a most serious disease of the rice plant. The antifungal substance, No. 720-A, was studied on their inhibitory effects on plant pathogenes, and the results were also briefly presented in this paper. The toxicity of the substance, No. 720-A, to flys was also described.

In the research for new antitumor substances produced by Streptomyces especially active against Ehrlich ascites carcinoma, the authors isolated a new strain No. 48-B-3 from the soil of Akihabara, Tokyo. This strain produces a substance which shows a specific activity against Ehrlich ascites carcinoma and Yoshida sarcoma. The authors obtained this substance in crude powder and designated it as Carzinocidin. In this paper, extraction and chemical and biological properties of this substance are reported.

In the previous paper⁽¹⁾, the extraction and some properties of the crude powder of carzinocidin which was produced by S. kitazawaensis and was effective for the destruction of Ehrlich ascites carcinoma and Yoshida sarcoma was described. In this paper, antitumor activities of this substance are reported.

The author and his coworkers reported that, under their test conditions, the emergence of streptomycin-resistance and isoniazid-resistance in the sensitive strain of Mycobacterium avium was prevented by the addition of relatively low concentrations of sulfathiazole, and the preventing effect of sulfathiazole was completely antagonized by the addition of paraaminobenzoic acid or folic acid⁽¹⁾⁽²⁾. In the present paper, the combined effect of sulfathiazole and streptomycin or isoniazid on the emergence of streptomycin-resistance or isoniazid-resistance in the sulfathiazole-resistant strain was observed.

The spontaneous mutation to drug-resistance in bacteria has been reported by many investigators (Luria and Delbruck⁽¹⁾, 1943; Oakberg and Luria⁽²⁾, 1947; Witkin⁽³⁾, 1947; Vennesland, et al.⁽⁴⁾ 1947; Yegian, et al.⁽⁵⁾, 1948; Demerec^(6,7), 1948, 1951; Newcombe, et al.^(8,9), 1949, 1951; Hsie, et al.⁽¹⁰⁾, 1950; Szybalski, et al.⁽¹¹⁾, 1953, and others) and reviewed by Luria⁽¹²⁾ (1947), Catcheside⁽¹³⁾ (1949), Braun⁽¹⁴⁾ (1953) and others. Mutation rates to drug-resistance were described by Oakberg and Luria⁽²⁾ (1947), Newcombe, et al.^(8,9), (1949, 1951), Hsie. et al.⁽¹⁰⁾ (1950), Demerec⁽⁷⁾ (1951), Szybalski, et al.⁽¹¹⁾ (1953), etc. Demerec⁽⁷⁾ (1951) estimated mutation rate to streptomycin-resistance in strain B of Escherichia coli and strain B/r, that is, a mutant of strain B resistant to radiation, and reported that the mutation rate was not appreciably affected by the strain used in tests. Szybalski and Bryson⁽¹¹⁾ (1953) estimated mutation rate to isoniazid-resistance and that to PAS-resistance in B. megaterium.

Concerning with Mycobacterium, naturally resistant forms of Mycobacterium tuberculosis have been isolated by Pyle⁽¹⁵⁾ (1947) directly from the sputa of patients who have not been treated with streptomycin, and the presence of naturally resistant mutants in stock cultures of H37Rv has been demonstrated by Vennesland, Ebert and Bloch⁽⁴⁾ (1947). A quantitative analysis of the resistance of Mycobacteria to streptomycin has been at first made by Yegian and Vanderlinde⁽⁵⁾ (1948), and the mutation rate to streptomycin-resistance has been reported by Hsie and Bryson⁽¹⁰⁾ (1950) only in Mycobacterium ranae.

However, it has been unknown whether the mutation rate of a drug-resistant mutant is the same as its parent strain or not. The present study is, therefore, concerning the mutation rate to streptomycin-resistance in a parent sensitive strain and its isoniazid-resistant mutant of Mycobacterium avium.

The authors have screened about thousand soil actinomycetes for their production of antitumor substances inhibiting ascites type of Ehrlich carcinoma of mice. The broth filtrate of about 5% of the strains, when 0.3 ml was daily intraperitoneally injected to mice bearing ascites type of Ehrlich carcinoma since the first day of the inoculation of one million tumor cells, inhibited the ascites increase and prolonged the survival period of mice. However, about 80% of these positive strains did not constantly produce antitumor substances thereafter, and the results in the further repeated tests fluctuated or were negative. Therefore, the percentage of the strains which produced constantly the antitumor substances in the repeated tests were 20% of the positive strains, that is, about 1% of the soil actinomycetes.

As it has been reported⁽¹⁾, the authors discovered an antitumor substance, sarkomycin,inhibiting Ehrlich carcinoma of mice⁽²⁾. Sarkomycin is an acidic substance. Therefore, the authors searched antitumor substances having basic characters. An antitumor subtance produced by one of the constantly positive strains was found to be basic, and the crude powder of this antitumor substance inhibited the ascites increase and prolonged the survival period, when 1.25 mcg was daily intraperitoneally injected to mice bearing ascites type of Ehrlich carcinoma. On the other hand, the mice tolerated the daily intraperitoneal injection of 250 mcg of this crude powder. The anti tumor substance was named pluramycin by the authors. It was further purified and it was confirmed that there were two kinds of pluramycins. One of them was dominating and was much stronger in the antitumor activity than the other. The authors named this main antitumor substance pluramycin A and another pluramycin B. Pluramycin A was purified and has been obtained in the crystalline state.

In the present paper the antitumor effect and toxicity of the crude power of pluramycins are presented. The antitumor effect and toxicity of pluramycin A crystal and the antitumor effect of the purified pluramycin B are also briefly described. The physical and chemical natures of pluramycins will be reported in another paper, but the antimicrobial effects of pluramycins are briefly recorded.

Actinomycin is the first antibiotic of streptomyces discovered by Waksman and others in 1940,⁽¹⁾ however, owing to the toxicity, the practical usefulness had not been considered, until Hackmann⁽²⁾ published the report concerning the antitumor effect of actinomycin C. Hackamnn confirmed the inhibitory effect of actinomycin C on various experimental animal tumors including Ehrlich mouse carcinoma, mouse carcinoma S37 and Walker rat carcinoma. Schulte and Lings⁽³⁾ made clinical studies on this actinomycin, and found that actinomycin C exhibited beneficial influences on lymphosarcomatosis, and its effect was rather superior to the effects of other treatments. According to their report, the effect on other malignant tumors was not conclusive.

Brockmann and others⁽⁴⁾ made extensive studies on chemistry of various actinomycins, and they confirmed the existences of various kinds of actinomycin. According to the streptomyces strains, three groups of actinomycins, A, B, and C, are produced, and in B there are B, and B2, and in C there are C₁, C₂, and C₃. Hackmann, Shulte, and Linz used actinomycin C containing C₁, C₂, and C₃ for the experiments. All the actinomycins consist of chromophore group and peptide. The chromophore group of all the actinomycins is considered to be the same, and each actinomycin is different in the constitution of the peptide part. The peptide parts of actinomycins, A, B₁, B₂, and C₁, consist of L-threonine, sarkosine, L-proline, D-valine and L-N-methyl valine. That of C₂ contains one more amino acid, D-alloisoleucine. C₃ is similar to C₂, but it does not contain D-valine.

Umezawa and others⁽⁵⁾ obtained an actinomycin from a strain which was classified to S. flavus. And this actinomycin was tested by Ukita and others (1949)⁽⁶⁾ on its effect on cells of Yoshida rat sarcoma. They briefly reported the distructive effects. Recently, Umezawa and others⁽⁷⁾ reported that the actinomycin inhibited the ascites increase and prolonged the survival period of mice bearing Ehrlich carcinoma. All the actinomycins are very similar in their antimicrobial effects and toxicities, and therefore, it can be expected that they are similar also in their anti tumor effects. Umezawa and others⁽⁸⁾ reported that they also obtained an actinomycin from a strain which was classified as S. flaveolus. This actinomycin was studied further by Endō. Among Brockmann’s actinomycins, it most resembled to A, though the identity was not conclusive. In this paper, it is called actinomycin J. The toxicity and the antitumor effects to Ehrlich mouse carcinoma and Yoshida rat sercoma was studied in detail. The author also made the similar studies on another actinomycin which had been also obtained by Umezawa and others. The results are presented in this paper.