Calcium-bindings of wild type and mutant troponin Cs of Caenorhabditis elegans.

Apparent Ca(2+)-binding constant (K(app)) of Caenorhabditis elegans troponin C (CeTnC) was determined by a fluorescence titration method. The K(app) of the N-domain Ca(2+)-binding site of CeTnC was 7.9+/-1.6 x 10(5) M(-1) and that of the C-domain site was 1.2+/-0.6 x 10(6) M(-1), respectively. Mg(2+)-dependence of the K(app) showed that both Ca(2+)-binding sites did not bind competitively Mg(2+). The Ca(2+) dissociation rate constant (k(off)) of CeTnC was determined by the fluorescence stopped-flow method. The k(off) of the N-domain Ca(2+)-binding site of CeTnC was 703+/-208 s(-1) and that of the C-domain site was 286+/-33 s(-1), respectively. From these values we could calculate the Ca(2+)-binding rate constant (k(on)) as to be 5.6+/-2.8 x 10(8) M(-1) s(-1) for the N-domain site and 3.4+/-2.1 x 10(8) M(-1) s(-1) for the C-domain site, respectively. These results mean that all Ca(2+)-binding sites of CeTnC are low affinity, fast dissociating and Ca(2+)-specific sites. Evolutional function of TnC between vertebrate and invertebrate and biological functions of wild type and mutant CeTnCs are discussed.

Intracellular reduction of the cupric ion of bleomycin copper complex and transfer of the cuprous ion to a cellular protein.

The cupric ion of the bleomycin copper complex has been shown to be reduced and transferred to a cellular protein by the following mechanism: Bleomycin approximately Cu2+ leads to Bleomycin plus Cu+ X Cu+ plus M leads to M approximately Cu+. The intracellular reducing agents (X) are suggested to be sulfhydryl compounds, because their action is inhibited by N-ethylmaleimide. The active group of the cellular protein (M) that binds with the cuprous ion is suggested to be a sulfhydryl group. The action of the bleomycin copper complex in causing DNA fragmentation in cells can be explained by the mechanism presented in this paper. This mechanism in cells is also supported by the temperature dependency of the action of the bleomycin copper complex on cells.

A glyoxalase I inhibitor of a new structural type produced by Streptomyces.

Many streptomyces strains produced an inhibitor of crude glyoxalase prepared from rat liver which did not inhibit glyoxalase I prepared from yeast. Another inhibitor, C11H14O6, which inhibited glyoxalases prepared from both rat liver and yeast was obtained from a cultured broth of Streptomyces griseosproeus and crystallized. Preincubation of this inhibitor with reuduced glutathione increased its inhibitory activity, which suggested its reaction with reduced glutathione. It showed a strong inhibition of growth of HeLa cells and inhibition of Ehrlich ascites carcinoma by daily injection. It also showed weak inhibition of the solid type of Ehrlich carcinoma and prolonged the survival period of mice inoculated with L-1210 cells.

Biological studies on the degradation products of 3-[(S)-1'-phenylethylamino]propylaminobleomycin: a novel analog (pepleomycin).

Pepleomycin (PEP), 3-[(S)-1'-phenylethylamino]propylaminobleomycin has potent activity and is less pulmonary toxic than bleomycin (BLM). Biological activity and toxicity of the following degradation products of PEP have been studied in detail: the product of carbamoyl migration (ISO), the product of decarbamylation (DC), the product of ring closure of the side chain on the pyrimidine moiety (RC), the depyruvamide product (DP) and the product of an enzymatic inactivation (DA). These degradation products showed much lower activity than PEP in vitro: antimicrobial and anti-HeLa activities, inhibition of DNA synthesis in AH66 cells and the DNA strand cleavage. Acute toxicity and pulmonary toxicity were tested in mice. Results indicated much lower acute toxicity corresponding to the decreased in vitro activity when compared to PEP. DP and RC did not cause lung fibrosis in mice, while ISO and DC showed 1/2.6 and 1/5.7 degree of pulmonary toxicity, respectively, in comparison with PEP.

[Toxicological studies of pepleomycin sulfate. V. Short term intermittent toxicity in dogs (author's transl)].

The comparative studies in the short term intermittent toxicity of pepleomycin (NK631) and bleomycin (BLM) were performed on 10 beagle dogs of 13 approximately 15 months old. Two dogs per group were injected intravenously with NK631 and BLM in doses 5.0 and 2.5 mg/kg body weight every fourth day for 11 treatment. Two dogs served as control and were injected with saline solution. In the group of 5.0 mg/kg of NK631, one dog was sacrificed on day 37 of the treatment and another one dog died on day 33 of the treatment. All the dogs of other group survived until the end of the treatment. The toxicity to the hepatic and renal damage caused by NK631 was stronger than the BLM. On the contrary, the toxicity to lung and various mucocutaneous regions was weaker than the BLM. The grade of pulmonary fibrosis of NK631 was about 1/2 and 2/3 of the BLM in dose of 5 mg/kg and 2.5 mg/kg, respectively. Other toxicological changes such as anorexia and weight loss were almost similar to the BLM.

[Studies on organ distribution, absorption and excretion of pepleomycin sulfate (NK631) (author's transl)].

1. Organ distribution of pepleomycin (NK631) in mice and rats was studied. NK631 was found at higher levels than bleomycin (BLM) in skin, lung, stomach, solid tumor, etc. in mice and rats. Furthermore NK631 was detected in the mesenteric and lumbar lymph node, esophagus and prostate in rats and also distributed at about twice as high levels as BLM in the AH109A hepatoma cell-metastasized lymph nodes. 2. For the elucidation of reason on low pulmonary toxicity of NK631 which is in spite of 1.5 times highly distribution in lung compared with BLM, inactivation of various BLMs by high molecular fraction of lung of mice and rats was determined. The order of inactivation rate of various BLMs in lung was as follows: BLM-M5196 greater than NK631 greater than BLM greater than BLM-HPE. There is an encouraging coincidence between index of pulmonary fibrosis in mice and inactivating rate in lung. 3. A comparative study on the serum level and urinary excretion of NK631 and BLM was performed in dogs. The blood level and urinary excretion rate of both drugs were almost similar. 4. The blood levels of NK631 were comparable to those of BLM in cancer patients.

[Safety evaluation of NK 631. Antigenicity, effect on delated hypersensitivity, irritative effect on eye mucous membrane and mutangenicity of pepleomycin (NK 631) (author's transl)].

1. Whether NK 631 is antigenic to guinea pigs and rabbits was studied by the methods of active and passive anaphylactic shock tests, Schultz-Dale reaction, passive cutaneous anaphylaxis, Ouchterlony, tanned red cell haemagglutination test and test according to the U.S. Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. However, none of the tests proved NK 631 to be antigenic. 2. The immunosuppressive effect of NK 631 was studied by delayed hypersensitivity to picryl chloride in normal and L-1210 tumor bearing mice. Therapeutic dosis of NK 631 was no immunosuppressed but toxic dosis of NK 631 was slightly decreased in ear thickness of delayed hypersensitivity. 3. The acute irritative effect of NK 631 and of bleomycin was studied by single instillation to the rabbit eye mucous membrane with 0.1 ml of either of 10, 33 and 100 mg/ml solution of the drugs in physiological saline. The irritative effect of NK 631 on the eye mucous membrane at each concentration was slightly severe than that of bleomycin at the same concentration. However, the manifestations were only mild to moderate dilatation of the conjunctival and nictating membrane blood vessels and eye mucous, and recovered or were mitigated 48 hours after the instillation. No severe changes such as corneal opacity, corneal desquamation, swelling and deaquamation of the conjunctival and nictating membrane were observed. The histopathological examination revealed no striking changes. 4. Mutagenicity of NK 631 and of bleomycin on Salmonella typhimurium strain TA 100 and TA 98 was studied. It was definitely shown that neither NK 631 nor bleomycin exerted any mutagenic action on either test strains.

[Studies on antitumor activities and pulmonary toxicity of pepleomycin sulfate (NK631) (author's transl)].

The antimicrobial and antitumor activities, and the pulmonary toxicity of pepleomycin (NK631) were studied in comparison with bleomycin (BLM). NK631 showed a broad antimicrobial spectrum against gram positive and gram negative bacteria equally to BLM, and its activity was about twice higher than BLM. NK631 showed higher activity on cultured HeLa S3 cells and higher antitumor effect on the transplanted tumors of Ehrlich solid carcinoma in mice, AH66 and AH66F ascites hepatoma in rats, and lower antitumor effect on Ehrlich ascites carcinoma in mice than BLM. Similarly to BLM, NK631 did not show satisfactory activity on L1210 leukemia in mice. NK631 showed marked effect on chemically induced squamous cell carcinoma, spontaneous lymph sarcoma of a dog, human and dog gastric cancer heterotransplanted in nude mice equally to BLM. Furthermore NK631 exhibited remarkably higher antitumor activity on lymph node metastasis of AH66 ascites hepatoma of rats and chemically induced gastric carcinoma of rats than BLM. Pulmonary toxicity of NK631 was low as 1/3 in incidence and 1/4 in grade of the BLM in old mice system. This trend was confirmed by chemical analysis of hydroxyproline in lung.

Typing of immunomodulators in terms of their effects on the electrophoretic pattern of serum proteins and antitumor combination therapy based on this typing.

Changes of serum proteins have recently received much attention in studies of immunomodulators. In this work, changes of serum proteins, especially LB, were studied by gel electrophoresis of sera after administration of 23 immunomodulators or antitumor agents. Fourteen of the 23 compounds increased the concentration of LB in the serum of normal ddY mice when injected once ip. Six compounds caused a very rapid (day 1) increase of LB, and 8 agents caused a slow increase (day 4 approximately day 10). On the basis of the results, these compounds were classified into type I (causing a rapid increase in LB; i.e., lipopolysaccharide, dextran sulfate and poly (I)-poly(C), type II (causing a slow increase in LB; i.g., lentinan, TAK and PS-K), and type O (causing no increase in LB; e.g., levamisole and bestatin). The antitumor activities of these three types of compounds in combination with lipopolysaccharide (type I) or lentinan (type II) were studied in an Ehrlich carcinoma-ddY mouse system. The results suggested that different types of compounds frequently showed synergistic antitumor activities. Typing of immuno-modulators and the antitumor activities of combinations of these compounds are discussed.

Combination antitumor therapy with lentinan and bacterial lipopolysaccharide against murine tumors.

The antitumor effects of lentinan and bacterial lipopolysaccharide (LPS) were studied. Combined treatment with lentinan and LPS was very effective against Ehrlich carcinoma in ddY mice, and the syngeneic mammary carcinoma MM46 in C3H/He mice. Studies on the effective doses and times of administration showed that a single injection of lentinan plus LPS 12 days after subcutaneous inoculation of these tumors caused complete regression in 60 approximately 90% of the animals; complete regression of MM46 carcinoma was seen in 83% of the animals.