Search

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.

Fundamental and clinical studies on a new yellow anthracycline antibiotic aclacinomycin A are reviewed. Favorable responses were seen in patients with acute leukemia refractory to daunomycin and adriamycin, malignant lymphoma, and breast, ovarian, lung, gastric, intestinal, and urinary bladder cancers by intravenous and intraperitoneal infusions or bladder instillation of aclacinomycin A alone in the phase II study.

Antibiotic research has been expanded to low molecular weight inhibitors of enzymes and immuno-modifiers, and continuous progress is being made in the screening, biosynthesis, and understandings of the mechanism of action of antibiotics, and the development of their useful derivatives.

We have been searching for enzyme inhibitors in culture filtrates of microbes and have found leupeptin, antipain, chymostatin, elastatinal, pepstatin, hydroxypepstatin, pepstanone and phosphoramidon as specific inhibitors of serine, thiol, carboxyl and metallo proteases. We found significant activities of aminopeptidases, phosphatase and esterase on surface membranes of various mammalian cells. We discovered bestatin, amastatin, forphenicine, esterastin and ebelactones A and B as specific inhibitors against these enzymes. These inhibitors were proved to bind to cells and modify immune responses. The usefulness of bestatin in cancer treatment has been suggested by clinical studies. It has been shown by several investigators that some endopeptidases such as Ca2+-activated neutral proteases and some other serine proteases may play important roles in muscular dystrophy. In addition to these endopeptidases, we found an abnormal increase in various enzyme activities in dystrophic mice and chickens. Especially, aminopeptidase activities are markedly increased. Moreover, its inhibitor bestatin became interesting on the aspects of its binding to cell surfaces. Bestatin and leupeptin which inhibit Ca2+-dependent protease showed some therapeutic effects against mouse dystrophy. Investigating enzyme activities in synovial fluid of patients with rheumatoid arthritis and osteoarthritis, we found increased activities of aminopeptidases, chymotrypsin-like enzyme, and phosphatase in rheumatoid arthritis but not in osteoarthritis. In chronic hemodialysis patients, RNase activity in serum is markedly elevated. Thus, enzyme inhibitors are increasing their potential usefulness in treatment of various diseases.

The present study was undertaken to elucidate further the enzymatic changes in dystrophic muscle using multivariate analysis. The activities of 14 kinds of enzymes, including 6 exopeptidases, 4 endopeptidases, beta-N-acetyl-D-glucosaminidase, phosphatase, esterase, and ribonuclease, were examined in forelimb and hindlimb muscles as well as in cardiac muscle of dystrophic mice and their controls. Two principal components identified from the enzymatic spectrum proved to be related especially to aminopeptidases and to serine proteinases, respectively. The enzymatic changes in forelimb muscle were very similar to those in hindlimb muscle when both were compared to those in cardiac muscle. The changes in aminopeptidases were unique to the limb muscles, whereas those of serine proteinases were unique to cardiac muscle of dystrophic mice. In the future, more attention should be focused on the role of exopeptidases in pathogenetic mechanisms of muscular dystrophy, because of the possibility that they play a major role in the initial stage of muscular dystrophy.

Activities of various hydrolytic enzymes were determined in rat organ homogenates and on the surface of cells from various sources, i.e., tumor cell strains, primary cultured cells, normal cells, and their transformants. Alanine, leucine, methionine, phenylalanine, and glycyl-proline aminopeptidases and esterase showed relatively high activities in all these organs and cells. In the kidney homogenate the aminopeptidase A activity was higher in other organs; i.e., the aminopeptidase A activity was lower than that of aminopeptidase B. Normal cells derived from kidneys showed the kidney-type pattern of amino-peptidases A and B on the surface of cells, whereas tumor cells from various origins were of another organ type. When cultured mouse fibroblast strain C3H2K and rat fibroblast strain 3Y1 cells were transformed by SV40 or by a ts A mutant and maintained at permissive temperature, aminopeptidase A activity was drastically decreased, and the ratio of aminopeptidase A to aminopeptidase B was reduced to the levels of tumor cells. If the ts A mutant-transformed cells were grown at the restrictive temperature, the ratio approached that of normal cells. In normal cells, however, cultivation at high or low temperature did not cause any change of the activities.

The 14C activity of [14C]bleomycin bound to DNA in bleomycin-sensitive rat ascites hepatoma cells (AH-66) was 8.7 times higher than in resistant cells (AH-66F) when the cells were incubated with [14C]bleomycin. The difference in permeability to bleomycin was not significant; uptake of [14C]bleomycin by the sensitive cells was only 1.2 times larger than that by the resistant cells, and the radioactivity incorporated into the nuclei of sensitive cells was only 1.3-fold greater. The bleomycin-inactivating enzyme level in the resistant cells was 3.5 times higher than in the sensitive cells, indicating that the antibiotic incorporated into the resistent cells was reduced in DNA-binding activity to a large extent. The level of protein-free thiol compound in the sensitive cells was 1.8-fold higher than in the resistant cells, suggesting a possible enhancement of bleomycin action by intracellular thiol compound as is found in vitro. These factors probably affect the DNA strand scission and the sensitivity of cells to this antibiotic. Binding of [14C]bleomycin to DNA in vitro was studied in the presence and the absence of dithiothreitol. A large portion of the radioactivity bound in the presence of dithiothreitol was unstable to acid, but the acid-resistant binding was also enhanced by this thiol compound.

Affinity chromatography with an immobilized substrate proved to be a successful tool in the preparation of pure and undegraded cathepsin D (1). Immobilized reversible inhibitors are even more suitable for the isolation because the enzyme remains inactive during the affinity chromatography procedure. Pepstatin is known as a strong inhibitor of carboxyl proteases (2) and we wanted to make use of its inhibitory property for the isolation of cathepsin D. In this report, a method for isolation of cathepsin D is described using affinity chromatography on pepstatin-agarose.

The in vitro degradation of the new antitumor anthracycline antibiotic, aclacinomycin-A, was studied using rat liver homogenate. In the presence of NADH or NADPH, the glycosidic bond at C-7 position of aclacinomycin-A was reductively cleaved to produce 7-deoxyaklavinone and 7-deoxyaklavinone dimer, MA144 E1. Subcellular fractionation indicated that most of the enzyme activity was present in the microsomal fraction and required anaerobic condition and NADPH. The purified enzyme reduced the glycosidic metabolites, MA144 M1 and MA144 N1, as well as aclacinomycin-A. The optimum pH for the anthracycline glycoside reductase reaction using aclacinomycin-A as substrate was 7.4. The enzyme was sigmoidally saturated with aclacinomycin-A and showed the concentration of 1.2 x 10(-4) M required for half maximal activity, and Km value of 7.7 x 10(-5) M for NADPH. The degradative pathway of aclacinomycin-A and its glycosidic metabolites was discussed.

New anthracycline antibiotics have been isolated from the culture of Streptomyces galilaeus MA144-M1. Among 14 anthracycline compounds, aclacinomycin-A showed the strongest activity in inhibiting leukemia L-1210 and had lower toxicity than others. Antitumor activity of aclacinomycin-A against leukemia L-1210 and P-388, solid sarcoma-180, and lymphosarcoma 6C3HED was examined in comparison with adriamycin and daunomycin. Aclacinomycin-A showed the same degree of activity against leukemia L-1210 and P-388, when administered intraperitoneally, as daunomycin and somewhat less than adriamycin. In oral administration, aclacinomycin-A also exhibited a significant activity on leukemia L-1210. The degree of inhibition of the growth of sarcoma-180 and 6C3HED lymphosarcoma transplanted subcutaneously by aclacinomycin-A was almost the same as that of adriamycin and daunomycin, although the optimal dose was about twice more than adriamycin. Acute cardiotoxicity of aclacinomycin-A by a test using hamsters was more than 10 times lower than that of adriamycin.

Plasma levels of various hydrolytic enzymes in diabetic patients, as diagnosed by their blood glucose levels, were compared with those of control subjects. Most of the levels of the aminopeptidase activities examined were significantly increased, while those of angiotensin-converting enzyme (ACE) and esterase decreased, in diabetic patients. The results of multivariate analysis suggest that the increased aminopeptidase activities are related rather to secondary organ lesions than to the primary pancreatic lesion. It seems that the blood glucose level is not necessarily a good indicator of metabolic abnormalities underlying this pathological condition.

During the early 1950s when strongly resistant organisms had not yet appeared, the chemotherapy of most bacterial infections was assumed to be possible. However, tubercle bacilli soon became resistant to streptomycin and new active agents were required for the treatment of tuberculosis. At that time, Umezawa discovered the aminoglycoside antibiotic, kanamycin, in the search for new water-soluble basic antibiotics (H. Umezawa et al. 1957). Kanamycin was evaluated as an effective agent for the treatment of infections with resistant staphylococci and streptomycin-resistant tuberculosis, and later for the treatment of infections with resistant gram-negative bacteria. However, after widespread use of the antibiotic, in 1965 kanamycin-resistant strains appeared in patients, although at a frequency of less than 5%. Therefore, Umezawa undertook studies on the biochemical mechanisms of resistance to aminoglycoside antibiotics (H. Umezawa et al. 1967 a, b). The results of these studies suggested structures which would be active against resistant strains and many derivatives of aminoglycoside antibiotics were synthesized. Among these, 3’,4’-dideoxykanamycin B (dibekacin) (H. Umezawa et al. 1971) was useful in the treatment of infections with resistant gram-positive and -negative bacteria, including pseudomonas, and was marketed in 1975.

The author initiated the screening study of enzyme inhibitors produced by microorganisms in 1965, and found about 50 new microbial products. These small molecular enzyme inhibitors have various bioactivities, but no significant antimicrobial activity. The finding of these inhibitors indicates that microorganisms in nature have acquired the ability to produce various compounds that have no obvious function in the growth of microbial cells and that have widely varied chemical structures [12, 14]. Therefore, if an exact screening method can be established, it is reasonable to search for small molecular microbial products which can modulate immune responses.

In the last 11 years the authors have succeeded in isolating nearly 40 enzyme inhibitors of small molecular size from microbial origins. These inhibitors proved to be not only useful tools in analyses of homeostasis of living organisms but also promising agents for cancer chemotherapy. Leupeptin was originally isolated as an inhibitor against serine or thiol proteases such as trypsin, plasmin, papain and cathepsin B. And soon it was demonstrated that leupeptin suppressed chemical carcinogenesis in rats. Pepstatin has an extremely strong activity to inhibit pepsin and cathepsin D. It also inhibits ascites accumulation caused by neoplastic diseases. Bestatin is a specific inhibitor against aminopeptidase B and leucine aminopeptidase. The enzymes are located on the surface membrane in various kinds of cells including lymphocytes. Bestatin was shown to enhance not only blastogenesis of lymphocytes in vitro but also establishment of delayed-type hypersensitivity in vivo. Combined use of bestatin and other antitumor agents gave promising results in animal experiments. Studies on enzyme inhibitors have provided us a new approach to cancer chemotherapy.

We initiated the study of small molecular enzyme inhibitors produced by microorganisms and found about 50 new compounds. In this paper, the author reviewed our studies on inhibitors of various proteases and enzymes on the surface of animal cells. Small molecular inhibitors of cellular surface enzymes enhanced or suppressed immune response. Studies on kinin and its related areas are rapidly progressing as shown by papers presented in this symposium. Parallel to the progress in these areas, it will become possible to establish new screening methods and to find new compounds useful in the study of kinin and its related systems.

We performed a longitudinal study for 20 weeks on spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKR) to determine the relationship between peptide metabolism and the age-dependent increase in blood pressure. In both SHR and WKR, the plasma level of aminopeptidase A (AP-A) clearly showed an age-dependent decrease. The plasma level of aminopeptidase B paralleled that of AP-A in WKR, but such an age-dependency was not observed in SHR, thus showing a dissociation between the two aminopeptidases. With age in both strains, the level of angiotensin-converting enzyme tended to decrease, while that of kallikrein activity tended to increase. In addition to these findings, a multivariate study testing the relationship of blood pressure to these enzyme activities, as well as to plasma levels of angiotensin I and renin activity, suggested abnormalities in the networks of proteolytic enzymes and in the peptide metabolism surrounding the renin-angiotensin system in SHR. These abnormalities may play some important roles in pathophysiological mechanisms of hypertension in SHR.

The structures of bleomycins and of other bleomycin-phleomycin group of antibiotics were described. The activity of bleomycins and their derivatives in causing strand scission of SV40 viral DNA suggests that the beta-aminoalanine amide moiety and the carbamoyl group are involved in this reaction. More than one guanido group in the terminal amine of bleomycin-phleomycin group antibiotics caused irreversible renal toxicity in dogs. Pulmonary toxicity varied depending on the terminal amines. A bleomycin-inactivating enzyme which distributes widely in animal cells was shown to be a new aminopeptidase B which hydrolyzes beta-aminoalanine amide group. At least one of the reasons for activity against squamous cell carcinoma was shown to be due to the lower content of this enzyme. The inhibitor of this enzyme was synergistic to bleomycin in inhibiting growth of cells, thus suggesting the intracellular action of this enzyme. Selected for further study from the bleomycins containing various terminal amines, bleomycin 5033 which showed the same activity against squamous cell carcinoma in mouse skin as the bleomycin used at present and lower toxicity than the latter, and bleomycin A5196 which showed stronger activity and stronger toxicity but lower lung toxicity than the latter.

Aclacinomycin, isolated from the culture of a Streptomyces, and 4'-O-tetrahydropyranyladriamycin, prepared by chemical derivation, exhibit significantly low cardiac toxicity and more effectiveness than does adriamycin. Pepleomycin, a new derivative of bleomycin, has 4-5 times lower pulmonary toxicity and more potent activity than the parent antibiotic. The future prospects of studies on antibiotics with potential usefulness in treatment of lung cancer are discussed.

An enzyme that inactivates Bleomycin (BLM) can be extracted from tissue. The activity of this enzyme varies markedly in different tissues. In the present paper, a method is described by which the activity of the BLM-inactivating enzyme in different organs and tissues of mice can be determined. The highest activity is found in liver; lower enzyme concentrations occur in the testis, spleen, lung and brain. In skin this enzyme is almost completely missing.

The in vitro metabolism of the antitumor anthracycline antibiotic, aclacinomycin-A, was studied using rat liver homogenate. In the presence of NADH or NADPH, aclacinomycin-A was converted to aclacinomycin-A analogs, MA144 M1 and MA144 N1, which were stereospecifically reduced at the keto group of the C-4''' position of L-cinerulose in aclacinomycin-A. Subcellular fractionation indicated that the production of MA144 M1, which was reduced to L-amicetose, was catalyzed by NADPH-dependent soluble cinerulose reductase I, and the production of MA144 N1, which was reduced to L-rhodinose, was catalyzed by NADPH-dependent soluble cinerulose reductase II and NADH-dependent microsomal cinerulose reductase. The properties of these three enzymes were studied. Soluble cinerulose reductase I which produces MA144 M1 showed a optimum pH at 6.3, Km values of 3.3 x 10(-4) M for aclacinomycin-A and 3.2 x 10(-5) M for NADPH. Soluble cinerulose reductase II which produces MA144 N1 showed a pH optimum at 6.3 and Km values of 2.0 x 10(-3) M for aclacinomycin-A and 4.0 x 10(-5) M for NADPH. All thesse reductases were sensitive to sulfhydryl reagents and were inhibited by vitamin K3. Microsomal cinerulose reductase showed sensitivity to diconmarol and ferrous ion. The main nondegradative pathways of aclacinomycin-A were discussed from these results.

S-2,3-Dicarboxyaziridine was found to be a potent competitive inhibitor (Ki = 0.08 microM) of fumarase from pig heart. The aziridine did not inactivate the enzyme or exhibit any observable substrate activity. It is likely that it functions as a transition state analogue mimicking the carbanion intermediate found in the normal catalytic reaction. The aziridine inhibited fumarate utilization in ruptured but not intact mitochondria.

In order to clarify the role of bleomycin functional groups in action mechanism, the metal-binding, dioxygen activation, and DNA cleavage of several synthetic analogues and biosynthetic intermediates of bleomycin have been investigated. The present results support that 1) the beta-aminoalaninepyrimidine-beta-hydroxyhistidine portion of the bleomycin molecule substantially participates in the Fe(II) and dioxygen interactions, 2) the transposition of the pyrimidine (or pyridine) and imidazole groups in the Fe(II)-coordination is essential for the effective binding and activation of molecular oxygen by the bleomycin ligands, and 3) the gulose-mannose moiety plays an important role as an environmental factor for the efficient dioxygen reduction and DNA cleavage, although the sugar portion does not contribute significantly to the nucleotide specificity in the DNA strand scission. Certain oligopeptides are able to mimic the metal-binding and dioxygen activation by bleomycin, but not induce the effective DNA cleavage. Probably, the bithiazole DNA interaction site of bleomycin delivers the iron/dioxygen chemistry to particularly the DNA (formula, see text) nucleotide sequences.

A new disorder of central myelination was found in black mutant hamsters showing trembling. The condition was inherited in an autosomal recessive mode. Histologically, a marked myelin deficiency was distributed throughout the central nervous system (CNS), and it was closely related to clinical manifestations observed in the mutants Ultrastructurally, abnormally thin myelin sheaths were observed in all axons with different diameters, and most axons below 1 micron in diameter were unmyelinated in the white matter of the spinal cord. There was no overt evidence of demyelination, but some myelin sheaths showed an aberrant configuration. The numbers of glial cells were not reduced, and there were no striking morphological abnormalities in them. It was suggested that there may be a dysfunction of oligodendrocytes resulting in retarded or defective myelination. This trembling hamsters may be a suitable animal model for the study of normal and abnormal myelinogenesis.

Bleomycin (BLM) and pepleomycin (PEP) are two chemically related glycopeptide antitumor antibiotics which differ in their terminal residues only. Studying the growth-inhibitory potencies of BLM (clinical mixture), BLM-A2, BLM-B2 and PEP in the L5178y mouse lymphoma cell culture system, it was elucidated that the slopes of the dose-response curves at the ed 50 concentration (around 1 μg/ml) were steeper for PEP than for BLM. This result together with cytotoxicity determinations revealed a cytostatic action of PEP within a closer concentration range than BLM. Both drugs inhibit cell proliferation during S- and G2-phase. Given in combination, BLM and PEP inhibit cell proliferation in a highly significant synergistic way (FIC indexes: 0.25–0.46). This in vitro result, which might be of therapeutic importance, is correlated with differences on the molecular level. Determinations of the ratio between the number of single- and double-strand breaks in the DNA (the target molecule of the drugs) revealed a considerably lower value for DNA from BLM-treated cells (1.9:1) than for DNA from PEP-treated cells (13:1).

Numerous antitumor antibiotics have been discovered since the author initiated the screening study and reported the isolation of three antitumor antibiotics in 1953. During the last 15 years, there has been great progress in studies of the mechanisms of cytotoxic actions of antitumor compounds. These studies have shown that antitumor compounds bind or react with DNA, inhibit DNA or RNA synthesis, inhibit protein synthesis, interfere with membrane function, or interact with microtubules. The mechanisms of cytotoxic action and of therapeutic action have been studied for some antitumor antibiotics which have exhibited therapeutic effects on certain types of human tumors. It is possible that even those antitumor compounds which inhibit a life function common to normal and malignant cells can exhibit therapeutic action against certain types of human tumors. This therapeutic effect may occur if a tumor has a special characteristic, in a biochemical process or in its structure, in relation to the action of the antitumor compound: e.g., a tumor may lack a system to repair the damage caused by an antitumor compound; the antitumor compound may be distributed in a higher concentration in the tumor than in other tissues; the tumor may have a stronger activity to activate (or a weaker activity to inactivate) an antitumor compound than other tissues. Therefore, antitumor antibiotics which showed unique actions have been selected for clinical studies.

Thirty-four patients with cancer (30) or ARC (4) with severe T cell defect or imbalance persisting a long time after completion of any cytostatic treatment were treated by bestatin 30 mg/day 3 days per week during three weeks. The drug has no toxicity of any kind. Reassessment of T cell subsets after completion of bestatin therapy showed a significant improvement of the absolute number of CD4 cells in peripheral blood. CD8 subsets wether initially increased or decreased were modified towards normalisation but the modification reached statistical significance only in the subgroup with initial absolute defect of CD8 cells. CD4/CD8 ratio was significantly increased whether considering all cycles of therapy, or all those given to patients with initially high or normal CD8 subsets. Bestatin appears to have immunomodulating properties which might be useful in cancer patients.

Previous studies indicated the importance of hydrolytic enzymes in the pathogenesis of autoimmune diseases. In the present study, we examined the activities of such enzymes in various organs of the hybrids of the New Zealand Black and New Zealand White mouse (NZB/W mouse) as a laboratory model of human systemic lupus erythematosus. Of the 18 enzymatic activities tested, the activities of the post-proline-cleaving enzyme showed a particular behavior in the spleen of NZB/W mouse. The enzymatic activity progressively increased with age in contrast to the reverse tendency in the control. This phenomenon was not found in any organs other than the spleen. The activity of this enzyme showed a high level of correlation to that of proline-iminopeptidase in most organs tested from control animals. However, these correlations were almost completely absent in the spleen of NZB/W mice. This may suggest an important pathogenetic role for the post-proline-cleaving enzyme in immunological disturbances in this model animal.

Combinations of 1 -β- d -arabinofuranosylcytosine (araC), bleomycin (BLM) or adriamycin (ADM) with the dipeptide bestatin do not result in an enhanced in vitro cytotoxicity in the macrophage-free L 5178 y mouse lymphoma cell system. However, in macrophage-containing murine spleen lymphocytes bestatin causes a potentiating effect of the cytostatic drugs araC, BLM and ADM with respect to their potencies to inhibit DNA synthesis. In the presence of 1 μg bestatin/ ml , the ed 50 concentrations causing a 50% reduction of [ 3 H ]dThd incorporation were significantly lowered; 4.3 -fold in the studies with araC and BLM, and 1.8 -fold in the experiments with ADM. Bestatin, given alone, displays a stimulating effect on [ 5 H ]dThd incorporation into macrophage-containing lymphocyte cultures within the concentration range 0.1–10 μg/ml . In contrast to the bestatin-stimulated lymphocytes, ConA-stimulated as well as LPS-stimulated lymphocytes do not show a higher sensitivity to the selected drugs inhibiting DNA synthesis. These data should encourage the practical use of bestatin in combination with araC, BLM or ADM in cancer treatment.

Time course studies were done to reexamine the age-dependency of intramuscular enzymatic changes in dystrophic mice. Most of the activities of hydrolytic enzymes in dystrophic mice were elevated in comparison to the controls throughout the span of 8 weeks which was examined. In contrast, the activity of creatine kinase remained depressed throughout the same period. This tendency was similarly seen in the muscles of forelimb and hindlimb but not in heart muscle. The observations are compatible with the notion that the increased activities of hydrolytic enzymes are causally related to the destruction of muscular tissue, leading to the malfunction of contractile machinery in the dystrophic muscles.

Supersymmetry at finite temperature is studied in the framework of thermo field dynamics. A self-consistent loop-expansion method is presented and this expansion scheme is shown to satisfy the Ward-Takahashi relations for the vertex functions. It is pointed out that the zero-energy mode accompanied by supersymmetry breaking is shown to be a bound state of the superpair. This pole appears both in the fermion channel and the boson-fermion channel. The implication of the present analysis in the free case is also discussed.

Among a variety of anti-tumour agents tested, oxanosine and 5-azacytidine were found to be significantly more effective in inhibiting growth of rat kidney cells infected with a temperature-sensitive mutant of Rous sarcoma virus at a permissive temperature (33 degrees C) than at a non-permissive temperature (39 degrees C). These two nucleoside antibiotics were antagonistic to each other in cytotoxicity. They seem to share the same carrier-mediated membrane-transport system, because dipyridamole, a potent inhibitor of nucleoside transport, protected cells from the cytotoxicity of both drugs. Thymidine transport, which is twice as fast in cells at 33 degrees C as at 39 degrees C, was competitively inhibited by both drugs. Thus the differential toxicity of oxanosine and 5-azacytidine at the two temperatures is thought to be due to their increased transport via the thymidine-transport system, which is somehow under the influence of the active src-gene product.

Anticancer activity and pulmonary toxicity of bleomycin and peplomycin in mice were found to be dependent on administration schedules and routes. That is, at the same total dose of 50 mg/kg, the anticancer activity against Lewis lung carcinoma was most strongly exhibited by continuous infusion of peplomycin for 7 days among all schedules tested and decreased in the order of the continuous infusion greater than 4 times injections per day for 10 days greater than daily injection for 10 days greater than daily injection for 2 days. In contrast, the pulmonary toxicity occurred lowest in the continuous infusion and strongest in daily injection for 10 days among the schedules. With respect to administration routes, the anticancer activity and the pulmonary toxicity of them were strongly exhibited in the order of i.v. much greater than i.m. greater than s.c. greater than i.p., except that no route dependency was observed in the continuous infusion. The pulmonary toxicity of peplomycin was lower than that of bleomycin in every administration schedules and routes. From these results, the continuous infusion was concluded to be the best schedule to increase the anticancer activity and to reduce the pulmonary toxicity.

SUMMARY: To test the hypothesis that chloramphenicol production in Streptomyces venezuelae depends on the presence of a plasmid, mapping analysis was carried out by using eight markers in addition to chloramphenicol production and melanoid pigment formation. The sequence of the eight markers was determined on a circular linkage map as follows: -his-ade-str-leu-lys-met-ilv-pro-(his-). This sequence resulted in the frequency of quadruple crossover (q.c.o.) recombinants having the lowest value, 3·2 to 4·9%. However, the character of chloramphenicol non-production, which was obtained by incubating mycelia with acriflavin, was not located on this linkage map; more than 15% q.c.o. recombinants would have been required to explain the results. From these results and other tests, it is concluded that chloramphenicol production is controlled by a plasmid. This plasmid appeared to be non-transferable in conjugation.