Several autors have described methods for the quantitative assay of 6-aminopenicillanic acid. Kato⁽¹⁾ extracted the penicillins with an organic solvent and determined the content of 6-aminopenicillanic acid (“penicillin nucleus”) in the fermented broth from the difference between the results of the iodometric assay of the extract and the original filtrate. Levitov ⁽²⁾ determined the content of the “penicillin nucleus” from the difference between the results of the iodometric and biological assays of broths. Wolf & Arnstein⁽³⁾ extracted penicillins with an organic solvent and after acylation of the water phase microbiologically determined the content of 6-aminopenicillanic acid. Batchelor et al.⁽⁴⁾ have described a chromatographic method for the identification of 6-aminopenicillanic acid based on its reaction with phenylacetylchloride in the presence of sodium bicarbonate resulting in the formation of benzylpenicillin. These authors mentioned the possibility of a quantitative determination of 6-aminopenicillanic acid from the sizes of the zones of inhibition around the biologically detected chromatograms.

We have published a quantitative method for the assay of penicillin and other antibiotics^(5,6) which we have now adapted also for the determination of 6-aminopenicillanic acid.

Besides a major antibiotic, kanamycin, a kanamycin-producing strain has been described by several authors to produce two or three active substances which were obtained by ion-exchange resin process. First, Umezawa et al. ⁽¹⁾ observed existence of kanamycin A and two other substances in their experiment of fractional precipitation of kanamycin reineckates, and they made paperchromatographic studies of active substances in the culture filtrate. Kanamycin showed Rf 0.21–0.26 by a paperchromatography using water-saturated butanol containing 2.0% p-toluenesulfonic acid, other two Rf 0 and Rf 0.37. The substance of Rf 0 had low toxicity and showed similar antibacterial spectrum as kanamycin A. But production of this substance was not constant. The substance of Rf 0.37 was described to be less effective against Mycobacterium 607 than kanamycin A.

Cron et al.⁽²⁾, Schmitz⁽³⁾ et al⁽⁴⁾, Gourevitch et al.⁽⁵⁾ and Umezawa et al.⁽⁵⁾, studied the substance of Rf 0.37 and it was named kanamycin B. According to these authors, kanamycin B was a water-soluble basic substance giving 2-deoxystreptamine, 3-glucosamine and other unidentified substances and its molecular weight was described to be 1,170. It has stronger inhibitory activity against microorganisms except against mycobacteria than kanamycin A. However, the latter is 2–4 times more active against mycobacteria than the former.

Thereafter, Rothrock et al.⁽⁶⁾ reported successful separation of kanamycins A and B by Dowex 1-x² resin chromatography. In this experiment, they determined electroconductivity and bacteriostatic activity of each fraction, and observed another active substance which was named kanamycin C.

Kanamycin B having higher bacteriostatic activity against test organisms than A confused results obtained by the cylinder plate method. Moreover, since as personally informed by Umezawa, kanamycin B had several times stronger toxicity than A, its percentage in the commercial products should be strictly controlled. Thus, differential assay of B from A was an important subject of our studies. We isolated kanamycin B, studied its chemical and biological properties, and established an useful method of determining B. In this paper, isolation, and properties of B are presented and the differential assay of B will be in next paper.

In the previous paper⁽¹⁾, we reported chemical and biological properties of kanamycin B. In that paper, we also described the necessity to establish a differential assay method of kanamycins A and B. After degradation by 40% sulfuric acid⁽²⁾, kanamycin A yielded a furfural-like substance, showing a specific absorption at 287 mμ⁽³⁾, and this substance was determined to be yielded from 6-glucosamine moiety of kanamycin A when treated with 4 N hydrochloric acid. On the other hand, kanamycin B having no 6-glucosamine did not show this property. Kanamycins A and B have been known to show an equal antimicrobial activity by agar diffusion assay. These properties were expected to be useful for the differential assay of these two substances. However, this method did not give a good reproducibility. Fluctuation of the results was considered to be due to difference of their dose-response relations.

Lamoy and Lannon⁽⁴⁾ reported that rates of diffusion of A and B were different and the result obtained by means of agar diffusion assay was invalid. They found, however, dose-response curves of A and B in turbidimetric assay were similar in the form using Staphylococcus aureus. Thus, they presented “two point turbidimetric” assay method.

The authors found a different characteristic between kanamycins A and B after acid degradation. Kanamycin B gave a microbiologically active substance after refluxed with 6 N hydrochloric acid for 30 to 60 minutes, but kanamycin A lost completely the activity after this treatment. This microbiological activity of B after the treatment could be easily determined by agar diffusion assay and gave an accurate value of B in a sample tested.

In this paper is presented a differential assay method of kanamycin B.

During the screening course of antibiotics from Streptomyces in this laboratory, a new crystalline antibiotic was isolated from a cultured broth that had been inoculated with strain A-59 of Streptomyces sp.

A new antibiotic, A-59 substance, is of sulfur-containing peptide nature, and highly active against Gram-positive bacteria.

Recently, several drugs are being clinically used as antifungal agents. In this connection, the development of drugs with wide and powerful antibiotic spectrum is expected .

Moldcidin B is a yellow needle crystalline pentane, which is produced from the Actinomyces No. 1068 and its variant strain. These strains are separated from the soil near Take hot spring, Fukushima Prefecture. The antibiotic spectrum of moldcidin B is wide. It is not only clinically used as ointment, tincture, or vaginal tablet for favus, trichomonas infection, etc., but also expected to be used for the internal application.

As we have had the chance of obtaining moldcidin B, we have investigated its pharmacological action upon the isolated organs, respiration, blood pressure, and the corticosterone. The following experimental results have been made available to us.

Production of griseofulvin by some strains of the genus Penicillium was investigated. P. viridi-cyclopium Abe, P. brunneo-stoloniferum Abe, and P. nigricans var. sulfuratum Abe were compared with P. urticae known as griseofulvin-producing strain from the view point of industrial production of the antibiotic.

When these strains were surface cultured in a modified Czapek medium for 30 days at 28°C, they produced griseofulvin, and the strains of P. viridi-cyclopium were more potent than P. urticae. When P. viridi-cyclopium was incubated at 30°C for 2 weeks in the Czapek medium containing trace elements, production of the substance was 1,300–1,500 mcg/ml of culture filtrate, but in the medium without them the production was only about one-third of the medium containing them. A molasses medium containing cornsteep liquor gave a higher potency, 1,050 mcg/ml, at 1 week than in the Czapek medium, but the production was lower then after.

Ultraviolet absorption bands in ethanol solution of crude substance which was extracted from culture broth of the strain incubated in a molasses medium containing cornsteep liquor were observed at wave lengths of 233, 291, and 324 mμ, and were nearly identical with those of standard griseofulvin.

Griseofulvin is an antifungal antibiotic which was first isolated from the mycelium of Penicillium griseofulvum by Oxford, Raistrick & Simonart⁽¹⁾ in 1939. This substance is known to be active against fungal infections in plants, particularly Botrytis infections of lettuce and Alternaria blight of tomatoes.⁽²⁾ Recent reports^(3,4) revealed that griseofulvin has a high activity against infections of dermatophytic group-fungi, Trichophyton, Epidermophyton, and Microsporum, in man by oral medication.

The structure of griseofulvin was first established by Grove et al.⁽⁴⁾ in 1951. This substance has been produced by surface or submerged⁽⁵⁾ cultures of various species of the genus Penicillium: Penicillium griseofulvum Dierckx^(1,10), P. urticae Bainier⁽⁶⁾, P. patulum Bainier^(5,7), the above three are included in P. urticae by Raper and Thom⁽⁸⁾, P. janczewskii Zal(= P. nigricans (Bainier) Thom^(6,10,11), and P. raistrickii Smith⁽⁹⁾.

The present communication is concerned with preliminary investigation of the production of the antibiotic by several species of Penicillia which are preserved in the authors’ laboratory.

In a previous communication¹⁾ of this series, the oxidation of L-glutamic acid by the intact mycelium has been reported. In the present report (Part I), some results on the technical problem for manometric experiments which has not been investigated particularly up to the present, and on the culture condition for the study on L-amino acid oxidase and L-alanine-oxidizing enzyme by the intact mycelium will be reported.

As described in the previous paper²⁾, in the presence of diphosphopyridine nucleotide, DL-alanine was oxidized by the crude enzyme solution obtained from S. griseus, while in the presence of flavin adenine dinucleotide, DL-alanine and L-leucine were not oxidized by it. So, L-leucine and DL-alanine were used as the substrates for L-amino acid-oxidase and alanine oxidizing enzyme.

Krebs¹⁾, Zeller and Maritz^2,3), Knight⁴⁾, Green et al.⁵⁾, Usami and Sasaki⁶⁾ and Webster and Bernheim^7,8) demonstrated with bacteria, molds, and animal tissues that these microorganisms and animal tissues oxidized L-amino acids, and this oxidizing enzyme was flavin enzyme. On the other hand, in Streptomycetes, it was only investigated as to whether or not unique L-amino acid was utilized as the nutrient.

The mycelium suspension or the crude enzyme solution used by the author in the preceding studies^9,10), did not show high activity for L-leucine oxidation. It seems that the components of medium and age of mycelium are unsuitable. The various culture conditions on L-amino acid oxidation were investigated in Part I¹²⁾. Wiame et al.¹¹⁾ reported that L-alanine dehydrogenase of Bacillus subtilis was a diphosphopyridine nucleotide DPN-linked enzyme. As described in previous papers^10,12), it became clear that L-alanine was oxidized by alanine-oxidizing enzyme other than L-amino acid oxidase of S. griseus, and that it was also DPN-linked enzyme. Therefore, alanine oxidation was treated particularly. It is the purpose of this paper to report on the L-amino acid oxidase and alanine oxidizing enzyme activities of Streptomyces griseus.

Variotion, a new antifungal antibiotic clinically applied, has been improved by the research group of Nippon Kayaku Company. In the previous paper⁽¹⁾, the present authors reported on the pharmacology of variotin, especially on the skin irritability and effect on the autonomic nervous system. It was found that variotin caused no noticeable local reaction of skin by topical application, with almost no effect on hemogram and body weight. On the other hand, variotin was found to have a cholinergic action to the autonomic nervous system, the stress of which is somewhat lowered by a consecutive application. It was revealed also that variotin has an anesthetic action on muscle or anti-barium action at higher levels. It was concluded from such pharmacological action that the consecutive application of variotin causes less toxic effect, because patients are under the stress of infection.

Similar investigations with newly obtained variotin indicated a lowering of toxicity and an alleviation of pharmacological effect. The comparative study of variotin 1 (50 mg/ml) reported previously and variotin 2 (200 mg/ml) obtained newly is described in the present paper.

The present paper deals with the sensitization experiments on variotin, an antifungal antibiotic, using rabbits and guinea pigs