Streptomyces sp. OA-6129, a soil actinomycete, was found to produce a new group of carbapenem antibiotics named OA-6129A, OA-6129B₁, OA-6129B₂ and OA-6129C. The taxonomy of the producer and the isolation and physicochemical properties of these new carbapenem compounds are described.

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The OA-6129 group of antibiotics, new carbapenem compounds, had relatively potent antimicrobial activities against Gram-positive and Gram-negative bacteria. Synergism of compound OA-6129A in combination with conventional β-lactam antibiotics was observed in antimicrobial activity against β-lactamase producers such as Proteus vulgaris GN 76 and Citrobacter freundii GN 346. The new carbapenem compounds were slightly superior to PS-5 in stability to kidney homogenates of various animal species.

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Following the discovery of new anthracycline antibiotics, auramycins A and B and sulfurmycins A and B, we found 10 minor analogues of auramycins and sulfurmycins, C, D, E, F and G, from the culture broth of a mutant strain of Streptomyces galilaeus OBB-111 and prepared 2 analogues as the chemical derivatives from auramycin G and sulfurmycin G. All analogues have a sugar moiety at C-7 position of the aglycones. These analogues exhibit activities against Gram-positive bacteria and P388 leukemia.

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Two strains of actinomycetes produce a new biologically active substance, designated FK-156. Examination of the morphological, cultural and physiological characteristics of the producing strains, No. C-353 and No. 6724, identified them as Streptomyces olivaceogriseus sp. nov. and Streptomyces violaceus, respectively.

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An interesting immunoactive peptide, FK-156, has been isolated from the fermentation broth of Streptomyces olivaceogriseus sp. nov. and Streptomyces violaceus. The compound was purified by column chromatography with activated carbon, ion exchange Sephadex and cellulose powder. FK-156, obtained as white powder, exhibits a wide variety of immunostimulatory activity in vivo and in vitro with experimental animals. Pretreatment of mice or rats with this peptide protected the animals against lethal infection with Escherichia coli and resulted in prolongation of life span of tumor bearing animals.

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FK-156, C₂₀H₃₃N₅O₁₁, mp 143-148°C (dec.) is a new immunostimulant produced by Streptomyces olivaceogriseus sp. nov. and S. violaceus. The structure has been elucidated to be D-Lac-L-Ala-γ-D-Glu-(L) meso-α, ε-A₂pm(L)-GlyOH on the basis of chemical and spectroscopic properties.

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For the structural confirmation of FK-156, two possible structures, 1 and its geometric isomer 2, were synthesized. Di-Z-meso-diaminopimelic acid (4) was converted into 14 via a sequence of reactions involving, as key steps, an enzyme-mediated asymmetric hydrolysis (6→7), followed by carbobenzyloxylation using a copper chelate procedure (7→8). Condensation of 14 and the appropriately protected lactoyl dipeptide 17 and removal of the protecting groups of the resulting 18 afforded 1. Protection of 7 to 22, followed by coupling to glycine via an azide method, gave 25. Derivatization of 25 to 29 and condensation with 17 gave 30, which was deprotected to yield 2. Compound 1 proved to be identical in all respects with the natural product.

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In searching for macromolecular antitumor antibiotics, a new screening method was developed that consisted of 1) a macromolecular antibiotic detecting system employing macromolecule permeable mutants of Escherichia coli, 2) a system to detect DNA-affecting antibiotics using DNA repair mutants, and 3) a mutagenicity detecting system, employing a valine resistance test. This new test system was applied to about 2, 900 kinds of culture fluids of Actinomycetes and consequently 15 samples were found which contained macromolecular antibiotics with DNA affecting properties.

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In searching for macromolecular antitumor antibiotics of microbial origin, 2, 875 kinds of Actinomycetes culture fluids were applied to a newly developed test system which consisted of antimicrobial assay using a macromolecule permeable mutant, DNA damage assay and mutagenicity test. As a result, 78 macromolecular antibiotics were found. Among them, 15 antibiotics precipitable with ammonium sulfate were macromolecular peptide antibiotics (protein antibiotics), of which molecular weight ranged from 10, 000 to 14, 000. Macromolecular peptide antibiotics AN-1, -5 and -15, termed type I antibiotics, showed stronger growth inhibitory effect on the uvrA and recA mutants, as compared to the effect on their parent, MP2. They also had mutagenic activity. AN-7, -9, -16, -18, -20, -22, -23, -25, and -26, termed type II, exhibited an increased inhibitory activity to a recA mutant but did not to an uvrA mutant. They all showed mutagenicity. AN-3, -11 and -13, type III antibiotics, gave similar influence on the DNA repair mutants, and on their parent, MP2. They had no mutagenic activity. Except for AN-11 and -13 of type III antibiotics, all antibiotics were inhibitory to the cell growth of a cancer cell, L1210.

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Gliovirin, a new antibiotic active against Pythium ultimum, has been isolated from Gliocladium virens. The structure of gliovirin was determined by NMR, mass spectral, and X-ray crystallographic analyses. Gliovirin may be derived from L, L-phenylalanine anhydride, which was also isolated from G. virens.

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¹³C NMR absorption for streptomycin (1), dihydrostreptomycin (2) and bluensomycin allow the structure of bluensomycin to be assigned as 3A rather than the alternative 3B.

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The preparations of 4-N-glycyllysinomicin and several 4-N-aminoacyl derivatives of compounds prepared from lysinomicin are presented. The new substances have lower antimicrobial activities than the original 4-N-unsubstituted lysinomicin derivatives. This result indicates that the structure-activity relationship observed with fortimicin A and fortimicin B does not apply to the lysinomicin derivatives studied.

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A study of the relative effectiveness of some eighteen natural and synthetically modified cytochalasins on the uptake of glucose by the Gram-positive bacterium Arthrobacter sialophilus showed that cytochalasins B, C or D and aspochalasins A, C or D were inactive natural congeners. The presence of an α, β-unsaturated carbonyl group in the macrolide moiety of these compounds with appropriate bioisosteric placement, as exemplified by cytochalasin A and aspochalasin B, are requisite molecular features. The transmembrane inhibitory index of active compounds was enhanced by increasing their lipophilicity. Thiol adducts of CA were around 20% as active in solute uptake inhibition as was the free drug. Radioactive 7-O-acetyl CA and its thiol adduct were each rapidly taken up by A. sialophilus and remained firmly bound to cellular components even after denaturant manipulations. These findings provide strong evidence for a stable association between CA and presumptive macromolecular receptors in transport and related processes.

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Cell-free extracts of Streptomyces clavuligerus convert δ-L-(α-aminoadipyl)-L-cysteinyl-D-valine (ACV) into an antibiotic product which is 30-50% penicillinase-insensitive. Thin-layer chromatography resolves this antibiotic product into one major penicillinase-sensitive component and one major and one minor penicillinase-resistant component. The major and minor penicillinase-resistant antibiotics co-chromatograph with deacetoxycephalosporin C and deacetylcephalosporin C, respectively. Ring expansion of a penicillin intermediate, as evidenced by the production of penicillinase-resistant antibiotic, shows an absolute requirement for α-ketoglutarate, while ATP, K⁺ and Mg²⁺ have lesser effects. Ring expansion activity is not sedimented by high speed centrifugation and is unaffected by membrane-disrupting treatments. Penicillin N and ACV (presumably via penicillin N) are the only substrates so far accepted by the ring expanding enzyme. New syntheses of penicillin N and isopenicillin N are described.

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4-Substituted 3-amino-5-hydroxybenzoic acids were tested in mutasynthesis experiments as potential starter-units for the biosynthesis of 3-substituted rifamycins. From our results it can be concluded that 3-substituents in rifamycin and other ansamycin chromophores must be introduced in a late biosynthetic step.

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The spontaneous mutant 18a derived from Streptomyces kasugaensis MB273 exhibited pleiotropic effect such as loss of aerial mycelium formation, aureothricin (AT) production, and of citrullin biosynthesis, as well as changes in plasmid; the mutant required cystine for production of aureothricin. An improved method of protoplast regeneration was applied to S. kasugaensis MB 273-18a and a regeneration efficiency of 90% or more was obtained. Sixty to ninety percent of the colonies regenerated from the 18a protoplasts exhibited reversion of the pleiotropic mutation in 18a. Moreover, of 13 regenerated strains which showed these drastic phenotypic variations, it was found that their plasmid types varied. These types could be divided into two groups; the RI type (5 strains) which contained a large amount of pSK2, a small amount of pSK3 and no pSK1, and the RII type (8 strains) in which no closed-circular DNA was detected. From these results, the following conclusions were obtained. First, plasmid curing in RII type strains and also the variation of plasmid copy in the RI type strains occurred as the result of protoplast regeneration. Second, the structural genes for biosynthesis of AT probably exist on chromosome. Third, regeneration of 18a protoplasts causes the reversion of pleiotropic mutation with high frequency. A working hypothesis was proposed to explain these complex phenomena.

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A plasmid, Rms425, mediating resistance to streptomycin(Sm) and mercury(Hg) was isolated from Pseudomonas cepacia GN11131 of clinical origin. Rms425 was transferred to Pseudomonas cepacia, Pseudomonas aeruginosa and Escherichia coli strains by transformation with purified DNA and by conjugation between isogenic strains of P. aeruginosa or of E. coil by mixing on membrane filters. The molecular weight of Rms425 was estimated to be about 32 megadaltons by electron microscopy and it was classified as incompatibility group P. Examining the incorporation of [γ-³²P] or [8-¹⁴C] from isotope-labelled ATP into Sm using the cell-free extracts from P. cepacia or E. coli strains harboring Rms425, we found that the Sm resistance conferred by Rms425 was due to the phosphorylation of the drug.

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A new derivative of mitomycin C (MMC), 7-N-(p-hydroxyphenyl)mitomycin C (M-83), had higher phage inactivation activity against phages φX174 and PM2 than MMC, and also higher DNA strand scission activity against their single- and double-stranded DNAs. M-83, at one third to one sixth concentration of MMC, showed the same level of phage inactivation and DNA strand scission activities. The mechanism of phage inactivation and DNA strand scission by M-83 were similar to those of MMC: (1) Reduction of M-83 was required for its action. (2) Oxygen radicals were involved in DNA strand scission, and metal ions possibly participated in the generation of oxygen radicals. (3) DNA strand scission was single strand scission, and dependent on temperature. The high DNA strand scission activity of M-83 is considered to reflect the rapid conversion to the active form.

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The electrophoretic mobility of phosphatidyl inositol liposomes at pH 7.4, 25°C, was reduced by aminoglycoside antibiotics, neamine and several polyamines in general accordance with the number of amino-groups on each molecule. There was good agreement between the relative position of the tested compounds on the mobility-concentration graph and available information about their relative mammalian toxicities in vivo. The slope of the graph for netilmicin was distinctively flat; a comparatively flat dose-response curve for netilmicin has been reported also from in vivo studies of nephrotoxicity. Investigation of a homologous series of α, ω, straight chain diaminoalkanes revealed that hydrophobicity did not contribute significantly to the observed interaction in this system. L-Lysine showed the weakest effect amongst all compounds tested, supporting the view that the overall positive charge on the molecule was the major determinant of the observed effect. Further structure-activity work is required to confirm whether this 'in vitro' test is predictive of the toxicity of aminoglycoside antibiotics in man.

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