Masanobu Kohsaka

FK-506, A NOVEL IMMUNOSUPPRESSANT ISOLATED FROM A STREPTOMYCES

The immuno-pharmacological profile of a novel immunosuppressive agent, FK-506 produced by a streptomycete, is presented here. We proceeded to test the effect of the agent on various in vitro immune systems. It showed that mixed lymphocyte reaction, cytotoxic T cell generation, the production of T cell-derived soluble mediators such as interleukin 2 (IL-2), interleukin 3 and gamma-interferon and the expression of the IL-2 receptor were suppressed by this agent. The IC50 values of FK-506 and ciclosporin (CS) in all tests were approximately 0.1 nM and 10 nM, respectively. Therefore, the novel agent, FK-506 suppressed in vitro immune systems at about hundred times lower concentration than CS.

NOCARDICIN A, A NEW MONOCYCLIC β-LACTAM ANTIBIOTIC

Nocardicin A is a new monocyclic beta-lactam antibiotic obtained from the fermentation broth of a strain of actinomycetes. The producing organism, strain WS 1571, was identified as Nocardia uniformis subsp. tsuyamanensis ATCC 21806. The antibiotic, obtained as colorless crystals, exhibits moderate in vitro antibacterial activity against a broad-spectrum of Gram-negative bacteria including Proteus and Pseudomonas. It has low toxicity in laboratory animals.

Prolongation of skin allograft survival in rats by a novel immunosuppressive agent, FK506

FK506, as immunosuppresant, was isolated from Streptomyces tsukubaensis. Intramuscular administration of FK506 (0.32 mg/kg or more) 5 days a week for two weeks after grafting prolonged the acdeptance time of F344 skin allograft to WKA rats. Similar results were obtained with cyclosporine at 32 mg/kg or more, but other immunosuppressives (i.e., prednisolone, azathioprine, and bredinin) gave only a marginal prolongation. The prolonging effect of FK506 was obtained in various donor-recipient combinations across a major or minor histocompatibility barrier. The agent also prolonged the acceptance time of mouse skin xenografts to rats. Furthermore, maintenance doses of 3.2 or 0.32 mg/kg twice a week after an initial 14-day treatment with the agent at 3.2 mg/kg gave graft survival as long as the treatment was continued for more than 120 days. Our findings show that FK506 has a potent immuno-suppressive effect in rate and suggest that the agent merits further investigation.

Cloning and nucleotide sequencing of new glutaryl 7-ACA and cephalosporin C acylase genes from Pseudomonas strains

Abstract We cloned a gene for the glutaryl 7-ACA acylase from Pseudomonas sp. A14 and genes for the cephalosporin C acylases from Pseudomonas diminuta N176 and V22 in Escherichia coli and determined their nucleotide sequences. Single open reading frames were recognized, which were composed of 2457 base pairs for the A14 gene and 2322 base pairs for the N176 and V22 genes. We also purified and characterized these enzymes from recombinant E. coli strains. Each enzyme was shown to be composed of two non-identical subunits. Determination of N-terminal amino acid sequences revealed that both subunits were encoded within an open reading frame, suggesting that both subunits of these enzymes are produced from a common precursor peptide. Comparison of amino acid sequences among these enzymes and other known glutaryl 7-ACA acylase and penicillin G acylase showed that there was 10 to 25% homology distributed heterogeneously along the sequences.

Immunosuppressive effect of FK506 on collagen-induced arthritis in rats

FK506, a new immunosuppressive agent, was given intramuscularly to rats for 12 days, starting on the day of type II collagen immunization. FK506 in doses of 0.32 mg/kg or more suppressed arthritis and also suppressed humoral and skin test response to type II collagen. FK506 suppressed arthritis only when given during the afferent limbs of immune response (0-4 days), whereas the drug was only marginally effective when treatment was started during the efferent limbs of immune response (7-11 days). FK506-induced immunosuppression continued and/or was maintained throughout the experiments (50 days). These rats immunized with type II collagen and treated with FK506 failed to develop arthritis even following a secondary immunization 50 days later but were fully capable of developing experimental allergic encephalomyelitis. This result suggest that FK506-treated rats develop specific unresponsiveness toward the type II collagen. It is concluded that FK506 is a strong immunosuppressive drug on collagen-induced arthritis.

A new antitumor antibiotic, FR-900482. II: Production, isolation, characterization and biological activity

FR-900482 is a new antitumor antibiotic produced by a new actinomyces named Streptomyces sandaensis No. 6897. It exhibits potent cytotoxic activity against various tumor cells in vitro. Furthermore, it has weak antimicrobial activity against some Gram-positive or Gram-negative bacteria.

Isolation of soil strains producing new cephalosporin acylases

Abstract Cephalosporin acylase is an industrially important enzyme which converts cephalosporins into 7-amino cephalosporanic acid (7-ACA), the starting material for most of clinically used cephalosporin derivatives. We surveyed soil strains for their glutaryl 7-ACA and cephalosporin C acylase activities. We obtained two new glutaryl 7-ACA acylase producers, Pseudomonas sp. A14 and Bacillus laterosporus J1 and two new cephalosporin C acylase producers, Pseudomonas sp. N176 and V22. Substrate profiles of A14 and J1 glutaryl 7-ACA acylases were quite different from that of the known glutaryl 7-ACA acylase GK16. While the deacylation rate of succinyl 7-ACA and adipyl 7-ACA by GK16 acylase was much lower than that of glutaryl 7-ACA, A14 acylase deacylated succinyl 7-ACA 13-fold faster than glutaryl 7-ACA and J1 acylase deacylated adipyl 7-ACA at a rate comparable to that for glutaryl 7-ACA. Meanwhile, cephalosporin C acylase producers N176 and V22 were shown to be different from the known cephalosporin C acylase producer SE83 in their abilities to reduce nitrate and to utilize several organic acids and sugars.

Immunosuppressive effect of FK506 on experimental allergic encephalomyelitis in rats

We investigated the effect of a new immunosuppressant, FK506, on the development of experimental allergic encephalomyelitis (EAE) in rats. EAE developed in 100% of rats immunized with myelin basic protein (MBP) in complete Freunds adjuvant. FK506 in doses of 1.0 mg/kg/day or more prevented the clinical signs of EAE for at least 50 days, when administered intramuscularly 5 days a week for 2 weeks starting on the day of immunization (days 0-4 and days 7-11), and a similar result was obtained, when the compound was given for 5 days (days 0-4). FK506, however, showed a significant but weak effectiveness when started from 7 days after immunization. Delayed-type hypersensitivity (DTH) to MBP developed before EAE, and anti-MBP antibody levels increased. Both humoral and cellular immune response to MBP were completely suppressed in rats treated with FK506. From these results, it is presumed that immunosuppression of cell-mediated immunity and/or humoral immunity by the treatment of FK506 actually causes the decreased incidence noted in the experiment for the development of EAE.

WS009 A and B, new endothelin receptor antagonists isolated from Streptomyces sp. no. 89009. I. Taxonomy, fermentation, isolation, physico-chemical properties and biological activities.

WS009 A and B novel endothelin receptor antagonists, have been isolated from the fermentation broth of Streptomyces sp. No. 89009. These antagonists were purified from the culture filtrate followed by Diaion SP-207, DEAE Toyopearl column chromatography and HPLC. WS009 A and B showed selective activity in an endothelin receptor binding assay with IC50 of 5.8 x 10(-6) M and 6.7 x 10(-7) M, respectively. On the basis of spectroscopic and chemical evidence, the structures of WS009 A and B have been established as 1 and 3, and are highly hydroxylated benz[a]anthraquinone chromophores.

A novel 7-β-(4-carboxybutanamido)-cephalosporanic acid acylase isolated from Pseudomonas strain C427 and its high-level production in Escherichia coli

Abstract We cloned the gene for 7-β-(4-carboxybutanamido)-cephalosporanic acid (GL-7ACA) acylase from Pseudomonas strain C427. The DNA sequence revealed an open reading frame of 2154 bp coding for 718 amino acid residues. The deduced amino acid sequence consists of 4 structural domains: (i) a signal peptide (positions 1–27), (ii) a small subunit of the acylase (positions 28–190), designated as α, (iii) a spacer peptide (positions 191–198), (iv) a large subunit (positions 199–718), designated as β. Plasmids were constructed to direct the synthesis of the acylase in Escherichia coli and the following results were obtained. The active acylase consists of two subunits which are processed from a single precursor protein, removing the spacer peptide during processing. A proportion of active acylase is secreted into the periplasm and the remainder is retained in the cytoplasm. The amount of precursor protein accumulated in the cytoplasm is greatly reduced when plasmids for the acylase lacking the signal sequence are expressed. Therefore, processing is independent of the translocation of the gene product through the cytoplasmic membrane, in contrast to the situation for penicillin G acylase. A high level of active enzyme production was achieved with a plasmid coding for an acylase in which the amino terminal sequence (positions 1–32) of native acylase is replaced by MFPTT.